effects of the human...

LECTURES IN

PROBIOTICS

physiologic & physiopathologic

effects of the human microbiota

GÖRAN MOLIN

Lectures in Probiotics ©Göran Molin 2013

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Lectures in

probiotics

Physiologic and physiopathologic effects

of the human microbiota

GÖRAN MOLIN

Lund 2013


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Copying is prohibited

Second edition

© Copyright: Göran Molin, 2012, 2013


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Contents

Preface - 11

I. Fundamentals – 13

Intention - 15

Probiotics - 15

Definitions - 15

Safety - 16

Who come up with the idea? - 16

Prebiotics - 18

Synbiotics - 19

Bacteria - 19

Classification and identification - 21

Definitions - 21

Taxonomy in historical perspective – 21

Essence theory - 21

Biological variation - 22

Evolution - 23

Operative taxonomic units - 24

Pure cultures – 24

Phenotypic characteristics – 25

Direct gene identification - 25

Hierarchical ranks - 27

Strain - 28

Type and pathogenic E. coli – 29

Typing - 29

E. coli - 29

RAPD-typing - 30

Species – 30

Definition - 30

DNA:DNA homology - 31

16S rRNA similarity - 32

Ribotyping - 32

Base pair composition - 33

Classification of genus and higher ranks – 34

Genus – 34

Phenetic and phylogenetic taxonomy - 34

Phylum – 35

Formalities – 36

To conclude - 36

References - 38

II. Dietary bacteria - 41

Intention - 43

Consumption of lactobacilli - 43

Lactic acid preservation - 44


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Lactic acid bacteria - 46

What is a LAB - 46

Lactococcus - 47

Streptococcus - 47

Lactobacilli - 48

Leuconostoc - 48

Weissella - 48

Oenococcus – 48

Pediococcus - 48

Lactobacillus - 49

Bifidobacteria - 50

Two adverse genera wrongly regarded as LAB - 51

Enterococcus - 51

Carnobacterium - 51

Products and processes - 51

Simple technique - 51

Ethiopian kocho - 52

Sour salmon buried in the ground - 53

Fermentation in sealskin bags – 53

Plant material - 53

Fermented auk - 54

Salted gherkins - 54

Wine - 55

Lactic acid fermented capers - 56

Sauerkraut – 57

Korean kimchi - 58

Green olives in brine - 59

Nigerian ogi - 60

Tanzanian togwa - 61

Sour milk - 62

Yoghurt - 62

Filmjölk - 63

Kefir - 63

Antimicrobial properties of milk - 63

Cheese - 64

Lactic acid fermented sausages - 66

Lactobacillus spp. often used as probiotics - 68

Probiotic dose - 68

Lactobacillus casei and Lactobacillus paracasei - 69

Lactobacillus rhamnosus - 70

Lactobacillus acidophilus - 70

Lactobacillus johnsonii - 71

Lactobacillus salivarius - 71

Lactobacillus fermentum - 71

Lactobacillus reuteri - 71

Lactobacillus plantarum - 72

Phenolics – 73

ProViva: a probiotic food product - 74

To conclude - 76

References - 77


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III. Gut microbiota (The bacterial flora of the gut) - 83

Intention - 85

Physiological and physiopathological effects - 85

Bacterial load of the digestive tract - 87

Viable count - 87

The bacterial core - 87

The bacterial flora - 88

Overview - 88

Mouth - 88

Stomach - 90

Jejunum - 90

Ileum - 90

Large intestine - 91

Taxonomic considerations - 92

Clostridium - 92

Bacteroides - 92

Adverse bacteria in the human microbiota - 93

A disturbed microbiota - 93

Lipopolysaccharides (LPS) - 94

Bacterial interaction - 95

Immune system - 95

The intestinal mucous membrane (mucosa) - 96

Barrier - 96

Leakage - 97

Variety and diversity - 99

Mammalians - 99

Mother and child axis - 99

The first bacteria - 99

Bacterial vaginosis - 101

Starting with E. coli - 101

Atopic eczema and bacterial diversity - 103

Old age – 105

Dietary factors affecting the gut microbiota - 106

To consider - 107

References - 108

IV. Effects - 115

Intentions - 117

Probiotic bacteria - 117

How to prove health beneficial effects – 119

Evidence based - 119

Koch’s postulates - 121

Medicine versus nutrition - 122

Immune modulation - 124

Virus - 125

Rotavirus diarrhoea - 125

Winter vomiting disease - 126

Common cold – 126

Bacterial balance and C. difficile - 127


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Functional bowel disorders - 129

Dysfunctions without clear pathogenic explanations - 129

Irritable bowel syndrome - 130

Definition - 130

Treatment with B. animalis DN-173010 - 131

Treatment with B. infantis 35624 - 131

Treatment with L. plantarum 299v – 131

299v in freeze-dried preparation - 131

Inflammatory bowel diseases (IBD) - 133

Chronic inflammation - 133

Crohn’s disease - 133

Autoimmune disease - 133

Failures with probiotics - 134

Ulcerative colitis - 134

Inflammation driven by the microbiota - 134

Probiotic treatment - 135

Animal models against intestinal inflammation - 136

Animal models are needed - 136

Dextran sulphate sodium induced colitis - 137

Methotrexate induced enterocolitis - 137

Liver injuries - 139

Fibrosis and cirrhosis - 139

Minimal hepatic encephalopathy (MHE) - 140

Alcohol-related liver disease - 140

Non-Alcoholic Fatty Liver Disease (NAFLD) - 141

Accumulation of fat and inflammation - 141

Microbiota - 142

Probiotics - 142

Animal model for liver injury - 143

Patients in intensive-care - 145

Systemic inflammation - 145

Acute pancreatitis - 145

Metabolic syndrome - 147

Low-grade inflammation - 147

Overweight and obesity - 148

High energy yielding microbiota - 148

Pro-inflammatory microbiota - 150

To consider - 151

References - 153

Appendix A: Inflammation - 165

Defence reaction - 167

Acute, chronic and subclinical inflammation - 168

Immunoactive cells and components - 168

Gut-associated lymphoid tissue - 168

Macrophages – 169

Toll-like receptors - 169

NOD-like receptors - 169

Microfold cells – 170

Dendritic cells and intraepithelial lymphocytes - 170


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IgA antibodies – 170

References - 170

Appendix B: Oxidative stress – 173

Reactive oxidative radicals and antioxidants - 175

Oxygen free radicals - 175

Antioxidative reactions - 176

References -177

Index - 179


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Preface

The university as an abstract idea has a long historical tradition which has been

extremely successful in terms of societal progress. A distinguishing mark for this

concept is that the university is a place where newly acquired knowledge, or at least

knowledge that is considered to be the most well informed one for the time being, is

mediated by those who brought it to light (scholars) to those who search for it

(students). The short textbook you now have before your eyes originates from a

university-course labelled “Probiotics” (comprising 7.5 university credits) which is

given at the Faculty of Engineering at Lund University. The book covers in text, a series

of PowerPoint illustrated lectures that are given in blocks during the course.

I who wrote this have since 1976 when I took my PhD been involved in microbiological

research and from the late 1980ies primarily in probiotics and the gut microbiota.

During this time up to today I constantly have been involved in explaining my scientific

activities in the context of the results of other scientists to students, colleagues,

journalists, decision-makers and myself. Thus, I think I can say with some confidence

that I have experience of scientific learning (me) and teaching (others) which hopefully

is reflected in this short textbook. However, to be perfectly honest to you reader, the

focus of this book is set on the scientific subject-content and not on teaching methods

per se.

This can of course be seen as a major weakness in these days when so much effort is put

into the mission of teaching pedagogics to scholars. There seem to be a consensus

between university bureaucrats, politicians and many students that scholars are lousy

teachers without the necessary pedagogical knowledge. The Faculty of Engineering at

Lund University has therefore set out to change this undesired situation and make it

obligatory for scholars to attend specific courses where they are taught by distinguished

consultants in pedagogy about, for example, “constructivist-driven” teaching and

“constructive alignment” (for an overview see J. B. Biggs, Teaching for quality learning

at university. Buckingham: Open University Press, 1999). Constructive alignment is to

have coherence between assessment, teaching strategies and intended learning outcomes

in an educational programme, something that can sound evident and easy in theory but

it is for certain not that easy to implement in reality. But even the theory “constructive

alignment” as such has been harshly criticized by other scientists in pedagogy, for

example by Loretta M. Jervis and Les Jervis (What is the constructivism in constructive

alignment? Bioscience Education Journal 6: 2005.

http://www.bioscience.heacademy.ac.uk/journal/vol6/beej-6-5.aspx) and they state

amongst other things that “alignment and learning outcomes are the death of originality

and serendipity”. In the abstract of their paper they summarize and cite P. Bourdieu

(Science of Science and Reflexivity. Cambridge, UK: Polity Press, 2004) “We would

identify our perspective not as that of constructivists, but rather as realists, accepting

that – science is a construction, but one in which discoveries are irreducible to the

construction and social conditions, which made them possible”. Dear reader, do you

understand? I don’t. I have major short-comings when it comes to the science of

pedagogics, but on the other hand, I also lack insight in, for example, the sciences of

mathematics and theology and my only excuse for this outrageous negligence is that my


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own scientific field actually is microbiology and not pedagogy, mathematics or

theology. When it comes to the hard core of learning and teaching, my opinion is that

the most important factors are the quality of the content that is taught or learned and the

willingness of the scholar to teach and the student to learn, not so much how and in

what form the package of knowledge is given. Unfortunately, it will always be difficult

to evaluate the value of the knowledge package given/received at a course as its value

not necessarily is seen immediately but can come into use in someone’s mind long after

the course has been ended.

A scholar that was arguing for his form of “school” in his particular field of subject with

major success because he had absolute means for proving that he was right and his

opponent was wrong was the samurai Miyamoto Musashi that lived in Japan 1584

to1645. During Musashi’s lifetime new towns emerged in Japan and numerous Kendo

schools were founded. The different schools learned out different combat techniques

and strategies. Musashi early began his studies in ”the way of the sword” and won 60

single combats before he reached the age of 29, and when he settled down at the age of

50 he had participated in 6 wars. Beside his studies in single combat and war strategy

Miyamoto Musashi become a painter, priest and scholar. He lived the last years of his

life in a cave and wrote a short textbook "Go rin no sho” (A book of five rings) to his

pupil Teruo Nobuyuki. The book is about martial art and deals with both the strategy of

warfare and the methods of single combat. The book is however not a thesis on strategy,

but a guide for students who want to learn strategy, and actually this book is about

pedagogy in a rather pragmatic way. Musashi states that there are many different

schools in how to be successful in combat. The different schools are arguing about

different weapon designs, movements and attitudes. But these different forms of combat

are of minor consequence, he says. The conclusive and most important thing is

according to Musashi:”When you take up a sword, you must feel intent on cutting the

enemy”,”the one purpose of all is to cut the enemy”, and “you should only be concerned

with killing the enemy”. The weapon is not crucial, “you can win with a long weapon,

and you can also win with a short weapon”. In other words, the outward form is of small

consequence. It is the content, the achievement that matters. Musashi proved his point

by going from Kendo school to Kendo school, duelling and killing his opponents, and

around 1612 he even stopped using a real sword in the fights but used a wooden sword

instead, in order to prove the superiority of his art, and that the type of instrument was

unimportant as long as it fitted its purpose. He stated “my school is no school”.

I think it is important to remember in science as well as in learning/teaching that when

you set out to do something, be clear of the goal. The form, for example, “constructive

alignment” in pedagogy and what type of technique (apparatus) that is used in a

scientific experiment are just means to reach the goal, not the goal itself. So when you

take up a course you must feel intent on learning, the one purpose of all is to learn. You

can like or dislike the teacher, PowerPoint-presentations, lectures or textbook but if you

make that a major concern you will lose track on the final goal to achieve knowledge.

On the other hand, Musashi also said “do nothing that is of no use”. So, don’t attend

useless courses and don’t perform useless scientific experiments. The trick however, is

to know what is useless and what is not. If you can do that distinction, you have reached

a deeper understanding or should we call it enlightment.

Lund / Göran Molin


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I. Fundamentals


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Intention

The educational intention with Lecture-block I – Fundamentals – is to define the

concept of probiotics and put it into its scientific framework of biology in general and

bacteriology in particular. The term “probiotics” comprises bacteria of many different

sorts that are used for a specific purpose, and these bacteria interact not only with the

mammalian body but also with the highly complex community of different sorts of

bacteria occupying the ecological niche of the mammalian digestive tract. Thus, it is

meaningless to discuss and try to reach a deeper understanding of probiotics and the

interactions of the gut microbiota without knowledge of different bacterial taxa and the

major concepts of bacterial taxonomy. This learning-block labelled Fundamentals will

provide the reader with necessary tools for mental handling of the bacterial world, i.e. it

will give basic facts of bacteriological thinking. Furthermore, in an attempt to provide a

basis for more comprehensive understanding of the taxonomic tools, short notices are

given of the historical growth of the biological thought in the scientific community.

However, if you already are well oriented in biology and bacteriology you can regard

most parts of this block as a brief rehearsal.

Probiotics

Definitions

Originally, probiotics meant organisms or substances, which contribute to intestinal

microbial balance, in contrast to antibiotics that counteract microbial activity. However,

currently a widely accepted definition is that “probiotics are live microorganisms which

when administrated in adequate amounts confer a health benefit on the host” (Anon,

2002). In other words, the designation probiotics are referring to a function, and not to a

taxonomic unit. Lactobacillus paracasei and Bifidobacterium animalis are examples of

bacterial species where specific strains frequently are used commercially as probiotics.

Strain is bacterial cells with identical genome, and each bacterial species includes a large but unknown number of different strains

L. paracasei or Lactobacillus casei, (the taxonomists have disagreed in the past about

the nomenclature of these two species) are, for example, used as probiotics in the two

different products, Actimel (Danone) and Yakult (Yakult) while B. animalis, often

called Bifidobacterium lactis, is used in Activia (Danone). The different strains of these

three commercial products are on the packages labelled ‘defensis’, ‘Shirota’ and

‘bifidus regularis’, respectively. These three probiotic food-products are marketed

world-wide and are examples of major commercial successes.

When the word “probiotics” first was mentioned in scientific literature, it was used as

label for substances secreted by one microorganism to stimulate another (Lilley and

Stillwell, 1965). Later “probiotics” became organisms or substances, which contributed


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to intestinal microbial balance (Parker, 1974) or “probiotics” are live microbial feed

supplements, which beneficially affects the host animal by improving its intestinal

microbial balance” (Fuller, 1989). An imbalance of the bacterial flora is sometimes

referred to as dysbiosis or dysbacteriosis.

A more recent definition of probiotics is that it is “living microorganisms, which upon

ingestion in certain numbers, exert health benefits beyond inherent basic nutrition”

(Schaafsma, 1996), and a more specified example is “probiotics are microbial dietary

adjuvants that beneficially affect the host physiology by modulating mucosal and

systemic immunity, as well as improving nutritional and microbial balance in the

intestine” (Naidu, 1999). The most frequently used definition today is linked to a

definition first launched by an international working group, i.e. “probiotics are live

microorganisms which when administrated in adequate amounts confer a health benefit

on the host” (Joint FAO/WHO Working group report on drafting guidelines for the

evaluation of probiotics in food, London, Ontario, Canada, April 30 and May 1, 2002).

There seems to day to be consensus about the meaning of the term probiotics. Thus

“probiotics are living microorganisms with health beneficial effects when administered

to the body”. Furthermore, it is more or less understood that these “health beneficial

effects” should have been proved scientifically. So far, the term probiotics is not

regulated by food legislation.

Probiotics are living microorganisms with scientifically proved health beneficial effects when administered to the body.

Safety

Health promotion is the leading theme in the probiotic concept which implies that there

should be no adverse effects linked to an intake of probiotics. Primarily, probiotics

should be seen as a food component and not as a medicine. Side effects can be accepted

by medicines provided that the beneficial effects are important enough, but as a health-

beneficial food component probiotics should be free from all type of pathogenic

activities. Thus, probiotics should not be dangerous to ingest for any category of

consumer, not even if they are very young or very old, or if they are

immunocompromised. In fact, the bacteria given as probiotics should be reasonably safe

even if they by some reason or another would enter into the circulation (blood stream).

It is also important that the probiotic strain don’t have qualifications for promoting

spread of antibiotic resistance or other unwanted features. Probiotics should not be

prone to function as vector for mobile genes.

Who came up with the idea?

When Louis Pasteur and Robert Koch in the second half of the 19th Century discovered

bacteria and demonstrated that bacteria both spoil food and give rise to diseases, the

emerging food industry started to look upon bacteria as a major problem. The obvious


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solution was to eradicate all bacteria from food, for example, by heat treatment under

the axiom that “a good bug is a dead bug”. This is still a popular strategy, used by

today’s food industry to reduce, not only the bacterial hazards, but also to prevent

unexpected quality changes during storage.

However, there were early voices claiming that bacteria could be beneficial. One of

these was Elie Metchnikoff (1845-1916) who was a professor at the University of Paris

and won the Nobel prize 1908 for the discovery of phagocytosis. Metchnikoff argued

that certain components of the bacterial flora of the gut could be beneficial to the health

by counteracting bacteria with physiopathological effects (1901 in British Medical

Journal; 1907 in the book, The Prolongation of Life. Heinemann, London). Metchnikoff

suggested that the living bacteria in lactic acid fermented milk, as for example in

yoghurt, could have health beneficial effects after ingestion (Metchnikoff, 1907). This

view later underpinned the international launch of yoghurt, and up to recently, milk has

been the main carrier of probiotics.

Metchnikoff´s theory was that:”the intestinal flora not only influence the outcome of an

infection by a pathogen, but is also responsible for the insidiously chronic toxaemia that

hastens atrophy and ageing” (1901, British Medical Journal). Metchnikoff suggested:

"the introduction of this Bulgarian clotted milk into our diet may counteract, or at least

diminish, the injurious effect of the intestinal flora”, and a dairy in Paris launched

“yoghurt” for the first time outside Balkan, a sour milk product fermented with “la

Lactobacilline” à la Metchnikoff”, with a culture consisting of Streptococcus

thermophilus and Lactobacillus delbrueckii subspecies (subsp.) bulgaricus. The

fermentation with strains of these two taxa define yoghurt in many countries, e.g. Food

and Drug Administration (FDA) in United States of America (USA) defines yoghurt as

”a coagulated milk obtained by lactic acid fermentation, due to Lactobacillus

bulgaricus and Streptococcus thermophilus” (current taxonomy has reclassified L.

bulgaricus to L. bulgaricus delbrueckii subspecies bulgaricus).

It was later argued that the traditional yoghurt-culture had poor survival in the passage

of the acid condition of the stomach. In the 1930thies Lactobacillus acidophilus was

suggested in USA as a suitable complement to the yoghurt culture as L. acidophilus was

claimed to have higher resistance to low pH. In Japan during the 1930thies,

Lactobacillus casei strain Shirota was launched (the L. casei strain Shirota has later

been reclassified as Lactobacillus paracasei). The strain Shirota was isolated by the

scientist Minoru Shirota and is now the probiotic ingredient of the international product

Yakult. Another strain of L. paracasei, i.e. L. paracasei defencis (= DN-114001; also

called “immunitas” in the past) is used in the milk-based product Actimel (Danone).

In Sweden, the probiotic strain Lactobacillus plantarum 299v was launched 1994. L.

plantarum 299v was included in different fruits drinks with the brand name ProViva,

but is now marketed both in fruit drinks and freeze-dried in capsules.

Examples on some other commercially available Lactobacillus strains used as

probiotics are L. paracasei F19, L. rhamnosus GG, L. johnsonii La1 (Lj1), L. reuteri

SD2112, L. acidophilus DDS-1 and L. acidophilus NCFM.

In the 1950thies the idea to use Bifidobacterium strains as probiotics come up. The

background was that Bifidobacterium spp. (spp. stands for species in plural) to a high

degree seemed to dominate the faeces flora of breast-fed infants. The genus


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Bifidobacterium is from a phylogenetic perspective very different from Lactobacillus

and most probably Bifidobacterium perform quite different actions in the human gastro-

intestinal tract. In contrast to Lactobacillus, Bifidobacterium can grow without vitamins

and growth-factors and use inorganic nitrogen (ammonia) as the sole nitrogen source.

The genus Bifidobacterium was described for the first time 1900 by Tissier.

Bifidobacterium spp. frequently discussed in connection to probiotics are

Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis (which

currently have been reclassified as Bifidobacterium longum biovariant infantis), and

Bifidobacterium animalis and Bifidobacterium lactis (more correctly B. lactis should be

named Bifidobacterium animalis subspecies lactis).

The strain B. animalis BB12 is the most well-known probiotic strain of

Bifidobacterium. BB12 is often designated as B. lactis.

Prebiotics

In analogy with probiotics, the term “prebiotics” was invented by Gibson and Roberfoid

(1995), and aiming on dietary fibres that stimulated the multiplication of beneficial

bacteria in the gut. The conditions for a food component to be called prebiotics are that

(i) the substrate must not be hydrolysed or absorbed in the stomach or small intestine,

(ii) the substrate must be selective for beneficial bacteria in the colon by encouraging

the growth/metabolism of these organisms, and (iii) the substrate will alter the

microbiota to a healthy composition by inducing beneficial luminal/systemic effects

within the host.

Prebiotics are non digestible food ingredients that selectively stimulate the growth and/or activity of health beneficial components of the colonic microbiota.

The beauty of the prebiotic concept is that it utilise microorganisms already present in

the gut. It is much easier from a practical point of view to incorporate a dietary fibre in a

food product than living microorganisms, and dietary fibres are much easier to store for

prolonged times than living microorganisms.

The prebiotic concept also has its pitfalls: (i) if no beneficial bacteria are present in the

digestive tract, beneficial bacteria can’t be stimulated; (ii) are prebiotics selective

enough, i.e. do they only stimulate growth of beneficial bacteria or could they under

some circumstances also stimulate growth of adverse bacteria?

Dietary fibres in general are supposed to reach colon and there they are fermented by

the microbiota and converted into mainly butyric acid, propionic acid, acetic acid and

carbon dioxide. Butyric acid and propionic acid are general regarded to have beneficial

effects on the metabolism, and especially butyric acid is an important energy source for

the colonic epithelial cells.

Hypothetically, if a certain dietary fibre that is supposed to have prebiotic effects (i.e.

stimulate bifidobacteria and lactobacilli in colon after administration) also results in an


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increased amount of butyric acid in the faeces (colon content) that must be regarded as

especially good as butyric acid is beneficial for the condition of the intestinal mucosa.

On the other hand, neither bifidobacteria nor lactobacilli are able to produce butyric acid

so this increase in butyric acid is not a marker for an increased content of lactobacilli

and bifidobacteria. But it can be a marker for an increase in Clostridium species (spp.)

as many clostridia are efficient producers of butyric acid. Many clostridia are also

harmless and some might even be beneficial but there is also Clostridium spp. that are

efficient producers of hydrogen sulphide (H2S) which is a compound with cell-toxic

effects. Thus, some Clostridium spp. can be harmful for the colonic mucosa, and a high

concentration of butyric acid in colon is no guarantee for a healthy colonic environment.

A major problem with prebiotics is to certify that only beneficial bacteria are stimulated

and not adverse ones.

Usually an increased amount of Bifidobacterium and lactic acid bacteria as

Lactobacillus in faeces after consumption of a dietary fibre is taken as proof of the

prebiotic capacity of the tested fibre.

Synbiotics

Supplementation with prebiotics or probiotics is both meant to affect the bacterial flora

(microbiota) of the gastro-intestinal tract in a beneficial way and impose physiological

health effects in the gut. Prebiotics suffer from the drawback that there can always be a

chance that they stimulate adverse components of the microbiota as well as the positive

ones, and if no positive bacteria are present in the gut, they can of course not be

stimulated.

Problems with probiotics can be that they have difficulties to establish themselves in

competition with the resident bacterial flora of the host, and the fact that neither

lactobacilli nor bifidobacteria are able to produce butyric acid, and hardly any

significant amounts of propionic acid. Hence, the obvious strategy to minimize the

hurdles would be to combine prebiotics with probiotics, a combination that sometimes

is called “synbiotics”. Synbiotics are mixtures of probiotics and prebiotics.

It should however be pointed out that probiotics not only have effects in the large

bowel. Probiotics also exercise immunological and other physiological effects on its

way down the digestive tract by direct contact with epithelial and immunological cells

(starting already in the mouth).

Bacteria

The gut harbours at least 1014

living bacteria, and this bacterial flora (microbiota)

represents more than 9 000 000 bacterial genes while the human body is only

represented by about 23 000 genes (Yang et al. 2009). In other words, the human

microbiota has a huge potential of different abilities that can be adjusted accordingly to


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the existing conditions. Bacteria are functionally important for mammalian health by

protecting the host from colonization of pathogens and providing nutritionally

advantages, for example, by breaking down indigestible polysaccharides to short-chain

fatty acids and by producing vitamin K. It is also clear that specific members of the

digestive microbiota directly affect mucosal immunity (Feng and Elson 2011; Atarashi

et al. 2011). Furthermore, certain components of the microbiota can have effects on

systemic immunology and on the barrier function of the gastro-intestinal mucosa.

Bacteria are obviously important for us, and a fair question then arises, what sort of

organisms are actually these “bacteria”? An answer to the question can be that bacteria

are small ancient life-form. They are found as individual cells or cells aggregated

together as clumps. Bacteria have a single circular DNA chromosome that is found

within the cytoplasm. Bacteria don’t have a nucleus, and they lack intracellular

organelles.

Bacteria are an ancient domain, of small, usually unicellular organisms.

So far no human science or techniques have been able to create a living cell out of

cellular constituents. Thus, the living organism is so much more than its known

molecular building stones. An organism can be defined as an individual composed of

mutually dependent parts constituted for sub-serving vital processes. Some organisms

exist as a singular cell (unicellular organisms), which usually applies to bacteria.

One ordinary bacterial cell enclosed in one ml of water correspond to a concentration of

about one ppt (ppt = parts per trillion, i.e. 1 per 1012

), i.e. bacteria are very small

creatures. On the other hand, the numbers of bacteria in a certain ecological niche can

be very high, as it is in colon of humans and animals. Bacteria are one of the three

domains of organisms. The others are Archaea and Eucarya. Eucarya includes

protozoa, algae, fungi, slime fungi, plants, mosses and animals. Archaea are organisms

that on the surface look just as Bacteria. They are small and unicellular. However, there

are major differences under the surface, e.g. Archaea have C20-diethers in the cell

membranes. C20-diethers are present in 3.8 billion (109) years old sediments so initially

Archaea were supposed to be the oldest living form on earth. But it was soon concluded

that also traces of Bacteria could be found from the same time period. In other words,

bacteria have been around for long time, much longer than multi-cell eukaryotes.

Bacteria also have had plenty of time to evolve in different directions, and therefore the

phylogenetic differences between different sorts of bacteria can be huge.

Now and then it is stated that the bacterial flora of the gut is adapted to the human host.

But is it so? It seems more likely that human and other mammalian beings are adapted

to bacteria, considering the fact that bacteria have been around on earth in about 4x109

years while our own species, Homo sapiens doesn’t seem to have existed in its present

form for much more than 200 000 years. A key-question is then, what sorts of bacteria

have we been adapted to? Or put it in another way, what sorts of bacteria were present

in the dawn of mankind (or in the dawn of our mammalian predecessors) and started to

interact with us? An answer to that question will most probably also answer, what sort

of bacteria that are most beneficial for our well-being.


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Classification and identification

Definitions

The diversity of life is dealt within the academic discipline “systematics”, also called

“taxonomy”, even if taxonomy in the strict sense is the science of “classification” (the

division into groups according to similarity/relationship) while systematics includes all

aspects of the field, i.e. besides classification also such activities as identification (the

comparison of an unknown object with known ones) and the naming of organisms. It is

important to distinguish between classification and identification.

Systematics is the science about organism’s sort and diversity, and about all types of relationships between them. Taxonomy [from the Greek táxis (order, formation), and nómos (law)] is the science of classification. Classification is the division into groups according to similarity/relationship. Identification is the comparison of an unknown object with known ones.

Organisms can be classified in many different ways, and one way to do it is to divide

bacteria after how they utilize energy, i.e. organisms that are able to synthesise their

own organic material from inorganic molecules are called “autotrophes” while

organisms that utilise complex organic molecules for biomass synthesis and energy

generation are called “heterotrophes”, and then are the autotrophes subdivided into

photoautotrophes (use light as energy source) and chemoautotrophes (oxidase inorganic

molecules as NH3, H2, S or Fe).

Organisms can also be classified in accordance to their relation to oxygen. If they grow

in the presence of the oxygen in air, they are called aerobes, i.e. they have respiratory

energy metabolism. But if they are unable to grow in the presence of oxygen they are

called anaerobes, i.e. they have no respiratory energy metabolism. There are also

organisms that are said to be microaerophils, i.e. the organism can handle small

amounts of oxygen, or facultative anaerobes, i.e. aerobic organisms that can switch to

anaerobic metabolism when needed. The general anaerobic strategy to generate energy is

by fermentation which is an anaerobe oxidation of organic molecules where the role as terminal

electron acceptor is taken by other organic molecules.

In aerobic respiration, oxygen is utilised as the terminal electron acceptor (hydrogen acceptor).

However, some aerobic organisms can also run anaerobic respiration. In that case, oxygen is not

used as terminal electron acceptor. Instead can compounds as, for example, nitrate, sulphate or

carbon dioxide be used as terminal electron acceptor. To classify bacteria in accordance to

energy utilization or respiration is a superficial and actually artificial classification

without much relevance for the evolutionary relationships between organisms.

Taxonomy in historical perspective

Essence theory. The way of looking at biologic classification has changed through

History. The antique, Greek so called “essence theory” stated that all organisms can be


22

defined by their “essence” (a unique indivisible character). By the use of dichotomic

division of the different essences of organisms in nature, a classification system could

be established. This classification goes downward from the highest rank down to the

lowest one, sensu stricto. The principle was launched by Plato but Aristotle was the first

one to apply it for living organisms (Figure 1).

Figure 1. The antique, Greek essence theory states that all organisms can be defined by their “essence” (a unique indivisible character) and classification can be performed by dividing things into two (dichotomic classification).

A functional condition for dichotomic classification is that taxa (taxonomic groups) on

all hierarchical levels are “natural”. Natural classification means classification

according to the true and only valid classification system. This postulates that there is a

distinct essence for all taxa, on all hierarchical levels. However, this is not true for

organisms according to the current scientific knowledge. On the other hand, the

dichotomic system works well for identification purposes when a classification already

has been set up.

Biological variation. Much later a botanical classification system was set up by Andrea

Cesalpino (1583) where different plants were grouped together according to similarity

and then certain critical characters (essences) were pointed out as markers for the

different groups. These essences/markers were then used in a dichotomic identification

system.

The general problem to handle the biological variation within the theory of essence

became clear 1686 when John Ray suggested a sexual definition of the species, i.e.

parents that together can have off-springs belong to the same species. However, just a

sexual definition is not enough to identify a specific species in nature, and it is almost

impossible to point out single morphologic characteristics that coincides the sexual

definition. The obvious solution is to combine different morphological criteria when

defining a taxon (taxonomic unit). This was suggested by the French botanist, Pierre

Magnol in 1692. Another French botanist Michel Adanson questioned the dichotomic,

“downwards” classification 1764. He introduced an “upwards” classification where the

base was the species, and different species were lumped together into genera, and

genera into families etc. He also used a combination of different characters when he

Organism

Plant Animal

Tree Herb

Deciduous

tree

Conifer

long needles short needles

sensu stricto sensu stricto


23

described the species. Unfortunately, the ideas of Adanson were almost completely

neglected by his contemporaries, much due to the impact of the Swedish botanist Carl

Linnaeus (1707-1778; or Carl von Linné as his name become when he was raised to

nobility 1761).

Carl Linnaeus believed in the theory of essence and his classification system, “the

sexual system” of Linnaeus was much more premature than the classification systems

presented by John Ray and Michel Adanson. However, Linnaeus was good in marketing

his system and in spite of the shortcomings of the system he managed to intuitively

classify plants in a successful way. Many of the genera defined by Linnaeus in “Flora

Svecia” (1755) are still valid. Linnaeus arranged organisms in a system of different

hierarchical levels in accordance with the one we use today. Moreover, he introduced

uniform nomenclature-rules, i.e. to name the species by the genus name and a species

epithet, e.g. Rosa canina (dog-rose), Sus scrofa (pig) and Escherichia coli (coli-

bacterium). Carl Linnaeus launched his ideas: 1735 in “Systema naturae” and 1753 in

“Species plantarum”.

Evolution. The first scientist to suggest biological evolution was the French zoologist

Jean-Baptiste Lamarck. He presented his ideas in “Philosophie zoologique” (1809), but

without gaining much hearing for the idea. Maybe the lack of sympathy was because he

failed to give any good explanation for the evolution. Lamarck proposed that evolution

is caused by inherited experience which few believed in at the time, especially as the

general idea for the time was that Earth only was 6000 year old (calculated from the

Bible), i.e. too small time span to allow an evolutionary development.

However, recently it has been clear that in some cases it seems that environmental

experiences actually can be inherited. New findings in “epigenetics” agree to a certain

degree with the ideas of Lamarck. Inheritable but reversible changes in the DNA can

occur and those changes are independent of the sequence of nucleotides. Such changes

are, for example, DNA-methylation and modification of histones (proteins on the

chromosome). Thus, environmental factors that a pregnant mother and her foetus are

exposed for can induce traces in the DNA. An interesting question is if the bacterial

flora of the gut can have epigenetic effects, e.g. of relevance for the immune system?

That would mean that the bacterial flora of the mother could imprint immunological

characteristics in the off-spring.

All credit for the evolutionary idea went to Charles Darwin that 1859 published the

book “On the origin of species by means of natural selection”. Geologists had by then

proved that the Earth actually was by far older than expected earlier. Simplified, the

theory of Darwin consists of two theses, (i) successive (gradual) development and (ii)

natural selection. The theory of natural selection gives an explanation to the evolution.

The theory of natural selection was later further developed in the so called “Synthetic

theory” which postulates that (i) individuals in a population have genomic differences,

and (ii) individuals (genomes) with the most efficient reproduction will dominate the

population.

Individuals with the most efficient reproduction are not necessarily the best individuals

from a human perspective. It is important to hold in mind that the theory of evolution is

not a “natural law”. The theory of evolution cannot be used for broadcasting future

evolution. It can only give an explanation to what already has happen. However, the


24

theory of evolution is a well established scientific hypothesis with major impact on the

biological thought.

The concept Phylogenetic taxonomy which is much in use today was originally

launched by the German zoologist Willi Hennig in his book “Grunzüge einer Theorie

der Phylogenetischen Systematik” (1950). In this book he criticised the mixed phenetic

(classifying according to similarity; see below) and evolutionary view of taxonomy and

he wanted to purify the evolutionary way of thinking. Hennig made up taxonomic

working-rules based strictly on evolutionary principles. He introduced the terms

“phylogenetic” and “cladogram” (clados is branch in Greek). The latter is a diagram

(tree) of phylogenetic relationships between taxa.

Operative taxonomic units

Pure cultures

When characterizing bacteria, it is not individual cells that are characterized. It is

populations of living cells in a pure culture, or genes amplified to multiple copies, for

example, by “polymerase chain reaction” (PCR) that are studied. When a pure culture is

studied for taxonomic purposes it can be characterised with both phenotypic and

genotypic methods (Figure 2).

Figure 2. Traditional procedure for bacteriological characterization used for classification or identification.


25

Phenotypic characteristics

The phenotypic characteristics are equal to the expressed characteristics of an organism,

or in other words, the characteristics that are visualised in reality. This is in contrast to

the genomic characteristics, i.e. the genotype, which are the structure, or the

construction of the genome.

Examples of different types of phenotypic characteristics are morphological,

physiological, biochemical, structural and immunological ones. Phenotypic

characteristics are crucial as they reflect the reality (what the organism actually can do)

but they can from a taxonomic stand point easily be misinterpreted. Morphology is

especially unreliable for bacteriological classification, but can be usable for

identification. In fact, traditional phenotypic features have been the most used way to

perform bacteriological classification and identification in the past.

Examples of phenotypic key tests often used for identification are the Gram reaction

(characteristics of the cell wall), morphology (for example, cell form in the microscope

as rods or cocci, and colony appearance on an agar plate), fermentation ability of

different carbohydrates, the oxidase reaction (“oxidase” is in this respect actually

aiming at cytochrome c and cytochrome oxidase that is the same as cytochrome a), the

catalase reaction, mobility, and growth under different specialised conditions (for

example, on Rogosa agar plates).

The Gram-staining was developed by Christian Gram (1884) and has proven to be an

important phenotypic character that is reflected in the phylogenetic relationships

between bacteria. Most members of the phyla Firmicutes and Actinobacteria are gram-

positive while all other phyla are gram-negative. In short, the cell wall of gram-positive

bacteria have one layer of cell membranes and a thick layer of peptido-glucane, while

the cell wall of Gram-negative bacteria have two layers of cell membranes and, in

between, a layer of peptido-glucane. Gram-staining is technically tricky to perform, and

an alternative way is to treat a colony with concentrated KOH and record if the cell-

walls break. Gram-negative bacteria are subjected to lysis. The KOH-method was

described by Gregersen (1978).

Many gram-negative bacteria have lipopolysaccharides (LPS) in the outer membranes

of the cell wall. LPS is regarded as an endotoxin and will activate the human immune

system if it comes in contact with it. Many types of bacterial LPS are strongly

proinflammatory, for example, LPS from E. coli. If LPS comes into the circulation

(blood stream), it causes immunological reactions that depending on concentration can

result in fever (in concentrations of nanograms), fall in blood pressure, internal

bleeding, coagulation of the blood and irreversible chock (death).

Direct gene identification

Direct gene identification is a way to avoid the bottleneck of culturing bacteria in pure

cultures in the laboratory when they shall be classified or identified. Instead all bacterial

genes in a sample are extracted and then identified (or classified). Direct gene

identification has up to now mostly been directed towards the 16S ribosomal RNA

(rRNA) gene but, lately, so called “shotgun” Sanger sequencing or massively parallel


26

pyrosequencing have also been used in an attempt to obtain unbiased samples of all

genes of a community (Eisen, 2007). The term “metagenomics” is frequently used as a

label for studies where more or less all the genetic material is recovered from

environmental samples and identified directly (Handelsman et al. 1998).

Biological taxonomy in general, and bacteriological taxonomy in particular, has during

the last decades been more and more built up around the molecules of ribosomal RNA

(rRNA), or in fact, the gene controlling the structure of the rRNA (rDNA) molecule.

rRNA is a relatively preserved molecule that only slowly has been changed through the

evolution and can thus be compared between different kinds of organisms in order to

trace the path of evolution. There are three kinds of rRNA in bacteria: 5S rRNA (120

nucleotides), 16S rRNA (1 540 nucleotides), and 23S rRNA (2 900 nucleotides).

Currently some of an golden standard for direct gene identification of the dominating

taxa in an environmental sample is to (i) extract all the 16S rRNA genes from the

sample (for example, faeces), (ii) PCR-amplify all the 16S rRNA genes with universal

primers, (iii) clone the amplified genes into Escherichia coli, (iv) PCR-amplify (PCR

stands for polymerase chain reaction) the cloned 16S rRNA gene, (v) sequence the

cloned 16S rRNA gene, and (vi) and identify the sequenced gene by compare it with

known gene-sequences using a data base (Figure 3).

Figure 3. A schematic procedure for direct 16S rRNA gene identification by PCR-amplification, cloning and sequencing.


27

Hierarchical ranks

There are plenty of different methods for studying the genome in bacteria and to use

these methods in bacterial systematic (classification, identification and typing), e.g.

gene sequencing where especially the 16S ribosomal RNA (rRNA) gene has been

targeted, DNA:DNA-homology, restriction fragment length polymorphisms (RFLP) of

the 16S rRNA gene (Ribotyping), temporal temperature gradient gel electrophoresis

(TTGE), randomly amplified polymorphic DNA (RAPD) and restriction endonuclease

analysis (REA). Different methods are differently useful at different taxonomic ranks,

i.e. different hierarchical levels in taxonomy (Figure 4).

Figure 4. The different hierarchical levels (taxonomic ranks) used in bacterial taxonomy.

The operative taxonomic unit (OTU) for bacterial classification or identification

(bacterial taxonomy) is, either a pure culture originating from the multiplication of a

single cell by culturing (isolate), or a gene sequence. The procedure for identification is

to compare the unknown OTU with known ones, and when it then comes to

classification, also with other unknown OTUs. Closely related, singular sequences or

isolates (pure cultures) form a species; closely related species form a genus, and so on

(Figure 4). According to this and the nomenclature rules of bacterial systematic, the

different hierarchical designations of three strains belonging to three different phyla are

exemplified in Table 1.

Table 1. Taxonomic position of three different strains belonging to three different phyla ( T after the strain number marks that the strain is the type strain for the species).

Phylum Proteobacteria Firmicutes Actinobacteria

Class Gammaproteobacteria Bacilli Actinobacteria Order Enterobacteriales Lactobacillales Bifidobacteriales

Family Enterobacteriaceae Lactobacillaceae Bifidobacteriaceae Genus Escherichia Lactobacillus Bifidobacterium

Species Escherichia coli L. plantarum Bifidobacterium animalis Strain E. coli ATCC 11775

T L. plantarum 299v B. animalis BB12


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Strain

The strain is the lowest hierarchical level bacterial systematic. Theoretically, the strain

is the same as a clone of cells, i.e. a population of bacterial cells with identical genome.

A prerequisite for the strain concept is that it must be possible to separate the individual

strain from other similar cells of the same species.

Strain (= clone) = a population of genetically identical cells

In practice it will be the quality of the method used to show genomic differences on a

low hierarchical level that will define the strain. The better the resolution capacity of the

method is the more precise will the strain definition become. The absolute method will

be to sequence all genomic material in the cell (plasmids and other mobile units

included). A more pragmatic method is to use “restriction endonuclease analysis” of the

total genome (REA). REA is schematically depicted in Figure 5.

Figure 5. Principle of restriction endonuclease analysis (REA) of the total chromosomal DNA, with the use of frequently cutting endonucleases and conventional agarose electrophoresis.

It is important to keep in mind that the bacterial strain theoretically should be a

population of genetically identical cells, but more pragmatic, the bacterial strain is a

taxonomic unit where the cell population is seen as almost identical individuals by a

method with a reasonable high capacity to resolve genomic differences. Thus, the

definition of a strain is dependent on what method that is used for the identification. For

example, pulsed field gel electrophoresis (PFGE) of chromosomal DNA digested with

restriction endonuclease has been somewhat of a golden standard for strain

identification/classification in clinical microbiology. However, PFGE is a technique for

the separation of relatively large pieces of DNA so the used restriction endonucleases

must cut the chromosomal DNA in relatively large fragments, i.e. PFGE has less

capacity than REA to visualise small differences in the genome, because in REA

frequently cutting restriction endonucleases can be used. On the other hand, PFGE is

from a technical point of view less complicated to run.


29

The strain identity is given after the species epithet. There are no generally accepted

rules of how the strain identity should be given. Mostly it is given as a combination of

letters followed by a number, but it can sometimes be given as a name (especially in

commercial contexts). If the strain has been deposited in an international culture

collection, the strain identity will be given as capital letter notation linked to the name

of the culture collection, followed by a serial number. Examples of international culture

collections and their letter notations are, for example, American Type Culture

Collection (ATCC), Deutsche Sammlung von Mikroorganismen (DSM), Czech

Collection of Microorganisms (CCM) and Culture Collection University of Göteborg

(CCUG).

When a new bacterial species is described, a so called “type strain” must be sent in for

deposition in a recognised international culture collection, wherefrom it later can be

purchased. Then, the strain will get a number but it will also be recognised as the type

strain of that particular species, i.e. it will serve as a living identity key for the species.

To mark the type-strain identity, a T is put directly after the strain number, for example,

the type strain of the species Escherichia coli is Escherichia coli ATCC 11775T if it is

purchased from the American type culture collection. If the same strain instead is

purchased from Deutsche Sammlung von Mikroorganismen, the name of the type strain

is Escherichia coli DSM 30083T. The two differently labelled type strains are supposed

to be identical.

Type and pathogenic E. coli

Typing. Below the taxonomic rank of species and subspecies, but above the level of

strain, the concept of “type” is often used, especially in the clinical bacteriology and in

bacterial epidemiology. This is a concept that traditionally has been linked to pathogens

and virulence factors or how the immune system reacts towards different bacterial

populations of a certain species. That is, the typing-system is closely connected to the

method used for defining the type. Different methods or different target characteristics

of the bacteria give different typing-systems. Sometimes, the type can be close to the

strain, but mostly a certain type can include numerous different strains but with some

important characteristics in common.

E. coli. As an example, enterovirulent E. coli (EEC) are divided in at least four

subgroups or types of EEC that can cause gastro-enteritis in humans.

Enterotoxigenic E. coli (ETEC) produces a heat-stable toxin (st) and a heat-labile toxin

(lt). ETEC can cause gastro-enteritis (“traveller’s diarrhoea”), resulting in watery

diarrhoea and abdominal pain. It is a typical tourist’s disease. The infective dose is 108

to 1010

CFU, i.e. a quite high dose is needed for infection.

Enteropathogenic E. coli (EPEC) is defined as E. coli belonging to serogroups

epidemiolologically implicated as pathogens but whose virulence mechanism is

unrelated to the excretion of typical E. coli toxins. EPEC cause “infantile diarrhoea” and

is one of the most frequent causes to diarrhoea in children in underdeveloped areas. The

infective dose is low in children but above 106 CFU in adults. The mortality rate is up to

50% in children in underdeveloped countries without access to health care.


30

Enteroinvasive E. coli (EIEC) can invade the epithelial cells of the intestine. EIEC

cause “bacillary dysentery” which often is mistaken for dysentery caused by Shigella.

Blood and mucous appear in the stool. The acute infection can be followed by

haemolytic uremic syndrome where the functions of the kidneys are failing. The

infective dose is low and even ten CFU can be enough to cause disease.

Enterohemorrhagic E. coli (EHEC) can produce verotoxins (VT) which is a shiga-like

toxin of two kinds, vt1 and vt2. EHEC mostly belongs to the E. coli serotype O157:H7

(explanation below). EHEC can cause diarrhoea (initially watery but becomes grossly

bloody) and severe cramping (abdominal pain). The acute infection can be followed by

haemolytic uremic syndrome. The infective dose is very low and can be even lower than

10 CFU. EHEC adhere to the colon mucosa, but it doesn’t invade (infect) the tissue.

EHEC produce toxins: The verotoxins (shiga-like toxins I and II) adhere to receptors

that are especially frequent in the capillaries of the kidneys. It can lead to HUS

(haemolytic uremic syndrome), i.e. the kidneys stop to function. HUS mostly hit

children and elderly. EHEC enterocolitis can also lead to TTP (thrombotic

thrombocytopenic purple) where the blood capillaries are blocked. TTP mostly hit

middle aged individuals. E. coli serotype O157:H7 is the most common serotype

amongst EHEC-strains.

E. coli strains are divided into different serotypes. The serotypes are in this case based

on three fundamental antigens, O, K and H. O-antigens are based on a polysaccharide

moiety, associated to the outer membrane. There are more than 170 different O-groups

of E. coli. K-antigens are based on polysaccharides that are part of the cell capsule.

There are three different K groups. H antigens are part of the flagella, i.e. only motile

strains can be H-typed. There are more than 50 H groups.

It can be pointed out that non-pathogenic E. coli often is present in high numbers in the

human gastro-intestinal tract, but even if they are non-pathogenic and normally don’t

cause infections, they must be regarded as a potential hazard. At high numbers also the

non-pathogenic E. coli can exercise adverse effects, e.g. having a pro-inflammatory

effect on the host.

RAPD-typing. A convenient method to type the genome of isolates is the method of

randomly amplified polymorphic DNA (RAPD). This PCR-based method is easily

performed and can relatively quickly be run for large numbers of isolates. A draw-back

with the method is the low reproducibility between different PCR-machines and

laboratories. Depending on the actual species and laboratory protocol a RAPD-type can

be more or less close to the strain-level.

In short the procedure for RAPD is the following. (i) PCR is run on a crude cell extract

of an isolate (or purified DNA) at semi-stringent conditions using a few-mere primer.

(ii) Electrophoresis is used for showing fragment size of the amplified DNA. The

obtained band pattern on the electrophoresis gel (or an image of it) can be used as a

genomic fingerprint of the tested isolate.

Species

Definition. It is often said that there are between 500 to 1000 different bacterial species

in the gut (Sekirov et al. 2010). However, many of these species are present in low


31

numbers. The number of dominating species, making up 99% of total amount of

bacteria, is lower but the exact number can differ widely between individuals,

depending on the extent of diversity.

The total number of known bacterial species is for the time being about 6500, i.e. the

number of bacterial species that have been formally described and validated. These

bacterial species are distributed between 1500 different genera (about).

The species is by tradition the basic unit of systematics. In older days (for example in

the days of Carl Linnaeus), the species was regarded as a creation of God and the

scientist just discovered it. The current opinion is that the species is constructed by the

scientific community by the use of classification. This becomes especially evident in

bacteriology but is perhaps less obvious in zoology. The species concept differs also

between different biological disciplines. An example of species definition of animals is

the following by Mayr (1982): “A species is a reproductive community of populations

reproductively isolated from others that occupies a specific niche in nature”.

A more general, phylogenetically based species definition is: “A species is an

irreducible cluster of organisms, within which there is a parental pattern of ancestry and

descent, and which is diagnostically distinct from other such clusters” (Cracraft, 1987).

The zoological species-concept of, for example, Mayr (1982) seems fairly clear, but the

species concept becomes much more muddled when involving organisms with

vegetative reproduction. This is a problem that became especially pronounced in several

botanical cases. For example, the plant genus Taraxacum (dandelion;”maskros” in

Swedish) is divided into 500 to1000 different species (the exact number is dependent on

who the scientist is that has made the division); Rubus (blackberry;”björnbär” in

Swedish) is divided into 90 to 4000 species, and Hieracium (hawkweed;”fibbla” in

Swedish) into 2000-10 000 different species.

A consensus definition of species is that species are populations of organisms that have a high level of genetic similarity.

The genomic span between different populations (and strains) of bacteria within the

same species is much wider than for the botanical or zoological species. Historically

this depends on the fact that the morphology of bacteria are much less varied then for

plants and animals, and that the current bacterial species definition is based on a

methodology resulting in less homogenous species.

DNA:DNA homology. The generally accepted standard-method for species definition

in bacteriology is DNA:DNA-hybridization of the total genomic DNA. Unfortunately,

this method is a bit unreliable from a technical point of view. The method is work-

intensive and it can only be used for pair wise comparison. DNA:DNA-hybridization

can also be used for identification by the use of probes with DNA-sequences which are

known to be unique for a certain species or genus.

The DNA:DNA hybridization homology of different bacterial strains (or isolates) has to

be performed par wise. It is generally accepted that strains with a relative ratio of

binding of ≥70% DNA:DNA-homology at optimal and stringent re-association

temperatures are regarded to belong to the same species. Optimal temperature is a


32

temperature 25oC below the melting point of the DNA, and stringent temperature is the

temperature 15oC below the melting point of the DNA.

Genomic species concept of bacteria: Strains with a relative ratio of binding of ≥70% DNA:DNA-homology at optimal and stringent re-association temperature are regarded to be the same species.

20% to 30% DNA:DNA homology have sometimes been used to define a "mini-genus”,

e.g. in the family Enterobacteriaceae. However, homology values in the range 20-30%

are unreliable, and homology values below 20% are meaningless due to methodological

short-comings of the method.

It must be stressed that the bacterial species represents a much wider biological

variation than the zoological and botanical species. This can be demonstrated by the fact

that the 70% DNA:DNA-homology that defines the bacterial species does not work on

animals, e.g. the DNA:DNA-homology between a human and a Chimpanzee is close to

100%; man:Gibon about 90%; man:Capuchin about 80%; man:Lemur about 50%

Besides of providing the bacterial species with too wide lines of demarcation (too high

phylogenetic variation within the species), the DNA:DNA homology has

methodological short-comings. It is also highly impractical to just have results of

comparisons in pairs, and not having a final, absolute value for each strain that could be

stored in a data base and re-used when new strains are added into the species. The

DNA:DNA homology concept works in the way that every new strain has to be tested

on the laboratory against the type strain and all other strains that could be of interest.

Most probably the current DNA:DNA-homology concept will be exchanged in the

future with a better method for species definition.

16S rRNA-similarity. Today, 16S rRNA gene sequence similarity is often used to give

a tentatively species definition, but this method is unable to differentiate between

closely related species, i.e. the resolution capacity 16S rRNA gene sequence

determination is too low for species definition. Even if the similarity in 16S rRNA gene

is 100% between a test-strain and the types strain of a particular species (similarity

coefficient = 1.00), it is not certain that the isolate belongs to the species of the type

strain. It can belong to a closely related species. It has been suggested that a similarity

in 16S rRNA gene of >97% indicates closely related species (Stackebrandt and Goebel

1994). On the other hand, it has been statistically shown that there is only 50% chance

for two 16S rRNA genes to belong to the same species even if the similarity is >99.8%

(Keswanit and Whitman 2001).

Ribotyping. A more precise and reliable method than DNA:DNA-homology to use at

the levels of species and subspecies is the Restriction fragment length polymorphisms

(RFLP) of the 16S rRNA gene. When the method is used for the 16S rRNA gene the

method is called ribotyping. The principle of ribotyping is schematically outlined in

Figure 6.


33

Figure 6. Schematically procedure for ribotyping, or restriction fragment length polymorphisms (RFLP) of the 16S rRNA gene.

Ribotyping is pointing out the number of 16S rRNA operons in the genome and give a

fingerprint that reflects the distribution of these operons. The phylogenetic relationships

are in this way fairly well reflected in the so called ribotype pattern.

Ribotyping is more easily performed, and more reproducible than DNA:DNA

hybridisation. Also a larger number of strains can be compared between each other and

not only pair-wise, i.e. the electrophoretic band pattern can numerically be compared

between each other and old patterns can be compared with more newly processed

patterns.

Some genomically homogenous species can be directly identified by ribotyping.

However, more heterogeneous species form, with ribotyping, several subgroups below

the species level, i.e. the species includes several different ribotypes. In other words, if

ribotyping should be the golden standard for species definition it would lead to more

bacterial species, bur less heterogeneous ones.

Base pair composition of DNA. A description of a new species or genus must include

a base pair composition of the DNA-nucleotides, i.e. the mol % guanine (G) + cytosine

(C) compared to the total amount of nucleotides in the genome.

It is important to understand that base pair composition (G+C ratio) can’t be used for

identification. It can only be used for classification. Thus, if two strains (or isolates)

have identical G+C ratio they are not necessarily similar. In contrast, if two strains have

different G+C ratio, they are truly different. The magnitude of the difference in G+C

ratios between taxa is important.

The G+C ratio within the whole domain Bacteria reaches from 22% (Clostridium

putrefaciens) up to 77% (e.g. Aurbacterium and Cellulomonas). This can be compared

with birds and mammalians that have a range of 40-44% and 32-46% (vertebrates 36-

46%; invertebrates 32-42%), respectively.


34

The variation of the G+C ratio between bacterial strains within a well defined species

should not exceed 3 mol %, and the G+C ratio within an “authentic” bacterial genus

should not exceed 10 mol %.

The aberrations sp. or spp. after a genus name stands for one unnamed species or several unnamed species, respectively. For example, Escherichia sp. (sp. = species [one single species]), and Escherichia spp. (spp. = species [several species]).

Classification of genus and higher

ranks

Genus

Species with close phylogenetic relationships are gathered into genus, and ideally the

variation in G+C ratio within an “authentic” genus should not exceed 10 mol %. The

sequence of the 16S rRNA gene can be used for classifying or identifying on the genus

level and on all higher taxonomic ranks, i.e. from genus to phylum (Figure 3).

Strains with ≥97% similarity in the 16S rRNA gene can be regarded with reasonably certainty to be related to the same genus.

16S rRNA gene sequencing gives bacteriological systematics its backbone while

DNA:DNA-hybridisation defines the species.

Phenetic and phylogenetic taxonomy

Gene sequencing has lead to a bacterial taxonomy based on phylogeny and with the

intention that the classification into different taxonomic hierarchies truly reflects the

evolution. However, evolutionary connections are not always the same as apparent

similarity. Classification based on similarity is called phenetic taxonomy, and before the

success of gene technological methodology, phenetic taxonomy was the norm, even if it

was assumed to reflect the path of evolution, i.e. the phylogeny.

The terms “phenetic” and “phylogenetic” should not be mixed up with “phenotype”

(characteristics that are expressed and made visual by the organism) and genotype

(structure of the genome).

Phenetic taxonomy is relationships according to similarity. Phylogenetic taxonomy is relationships according to joint evolution.

A bacteriological example on the difference between phenetic and phylogenetic

taxonomy: Lactobacillus and Bifidobacterium are two phenetically similar genera, e.g.

both are gram-positive rods, ferment sugars to lactic acid and acetic acid, and they live


35

in the human digestive tract. However, Lactobacillus and Bifidobacterium are

phylogenetically very different and belong to different phyla (divisions), i.e. Firmicutes

and Actinobacteria, respectively.

Phylum

The number of bacterial species gradually increases as new ones are defined, and along

with the increasing number of different species also the number of different taxa at

higher hierarchical ranks increases, and so do the number of the highest hierarchical

ranks (phylum or as it is called in more traditional terminology, division). Different

bacterial phyla are shown in Table 2. Actinobacteria (gram-positive), Bacteroidetes

(gram-negative), Firmicutes (gram-positive), Fusobacteria (gram-negative),

Proteobacteria (Gram-negative) and Verrucomicrobia (gram-negative) are examples on

phyla (plural for phylum) frequently occurring in the human digestive tract.

Table 2. Different bacterial phyla (divisions).

Most gram-negative, food associated bacteria as, for example, the genera Pseudomonas,

Psychrobacter, Aeromonas and the family Enterobacteriaceae belong to the

Proteobacteria (the class Gammaproteobacteria).

Most gram-positive bacteria associated to food as, for example, the genera

Lactobacillus, Bacillus, Clostridium, Staphylococcus, Enterococcus and Lactococcus

belong to the phylum Firmicutes (Gram-positive bacteria with low guanine + cytosine

ratio of the DNA [G+C <55%]). Firmicutes occupies a dominating position in the

digestive tract, typically represented by genera as Clostridium, Ruminococcus and

Eubacterium. However, the gram-positive genus Bifidobacterium belongs to the phylum

Actinobacteria (gram-positive bacteria with a high guanine + cytosine content [G+C

>50%]).


36

Formalities

The official forum for the publication of new bacterial taxa from subspecies to phylum

is the International Journal of Systematic and Evolutionary Microbiology. This journal

publishes research papers and establishes novel bacterial names. New names are

summarised in a notification list. Each monthly issue also contains a compilation of

validated new names (the validation list), i.e. names and descriptions of taxa that have

been previously published in other scientific journals or books are listed in the

International Journal of Systematic and Evolutionary Microbiology, provided the

descriptions and naming have been performed correctly. Publications relating to new

bacterial taxa, and validation of descriptions published elsewhere requires that a type

strain of the taxon in question have been deposited in two recognised public strain

collections in different countries.

The ICSP's International Code of Nomenclature of Prokaryotes states that all bacterial

new names must be published or indexed in the International Journal of Systematic and

Evolutionary Microbiology to be deemed valid. So far the journal has officially

validated around 6500 species and 1500 genera. It was estimated in 2004 that over 300

new names had been published but not validated, i.e. these names are so far not valid.

All validly published names in bacterial systematic, with the original references,

together with the hierarchical structure for bacterial taxa from phylum to species can be

seen on a website with the title, LPSN; List of prokaryotic names with standing in

nomenclature. J.P. Euzéby: http://www.bacterio.cict.fr/index.html

To conclude

Bacteria make up a huge domain of widely diverse organisms with access to far more

metabolic means than mammalian cells. As human beings, we eat bacteria either as

unintentional dietary compounds or as probiotics, and furthermore, we harbour a dense

and diverse community of bacteria in our digestive tract and these bacteria interact in

different way with the dietary compounds we ingest, and with our body. Different

bacteria are doing different things in accordance with their inherent nature and the

prevailing environmental circumstances. In order to try to understand what is going on

in our bodies we must obtain knowledge about the different sort of bacteria, and

particularly about those that are used as probiotics and those that are resident in the

human digestive tract in high amounts. In the learning-block Fundamentals several

aspects of bacteria have been dealt with in a relatively condensed way, i.e. those are the

aspects that the author believes to be an important background for the understanding of

the subsequent learning-blocks. However, certain things can be more important to hold

in mind than others, and below follow some points of importance to think a bit extra on:

Catch 1. The term “probiotics” does not refer to any specific kind of taxa. “Probiotics”

is not a taxonomic unit. There are different definitions for probiotics but today the term

is used in the meaning that probiotics are living microorganisms with health beneficial

effects when administered to the body. The fact that probiotics is supposed to have

http://www.bacterio.cict.fr/index.html


37

health effect imply that this health effect also has to be proven scientifically. Some

strains of relatively few species of Lactobacillus and Bifidobacterium are the ones

mostly used commercially as probiotics. Both Lactobacillus and Bifidobacterium

include many different species and the phylogenetic difference between these two

genera is huge.

The probiotic concept should not be mixed-up with the concept of prebiotics.

Catch 2. There are many different bacteria around and when dealing with them, we

must know their characteristics, i.e. how they perform in different situations. In order to

get such information, we need know their names as the taxonomic name is the key to

the collected scientific knowledge of the bacterium in question. In search for the name,

we can isolate living bacteria from the sample and study them in pure cultures or we can

isolate bacterial genes directly from the sample and determine the nucleotide sequence.

Both strategies can be used for either identification or classification.

It is important to be aware of the logic difference between identification and

classification. The difference may appear self-evident, but nevertheless identification

and classification have often been confused, not the least in the history of science.

Classification is a purely scientific activity with the aim to characterize unknown

organisms and clarify their evolutionary (phylogenetic) relationships with known

(already described and ordered) organisms, or the order of nature if you so like. The

evolutionary relationships between organisms are visualised by the different

hierarchical levels in taxonomy where the ranks of genus and species are crucial, and

reflected in the scientific (taxonomic) name.

In the probiotic concept, the strain definition is important, because scientific attempt to

prove health-effects of probiotics will be performed on the strain. Health-effects can

vary a lot between different strains of the same species.

Catch 3. Traditionally, bacterial taxonomy has been based on phenotypical

characteristics (phenotypes), i.e. what the bacterial cell looks like in the microscope and

what metabolic or other features it can express in pure cultures. This makes sense as it is

these expressed characteristics that you primarily want to know about when trying to

figure out the role of bacteria in an environmental system, such as the digestive tract of

the body. On the other hand, the comparison of phenotypic features and ordering

different groups of bacteria according to similarity in phenotypic characteristics can

muddle the true phylogenetic (evolutionary) relationships between different bacteria as

phylogenetic distant taxa can have several easily measured phenotypic characteristics in

common, for example the genera Lactobacillus and Bifidobacterium that both are gram-

positive rods producing lactic acid during fermentation of glucose. However, there can

be other less easily visualised phenotypic features that are strikingly different, in this

case for example the metabolic pathway used for the production of lactic acid from

glucose. The true differences and similarities (when all possible features are included)

are better reflected by the evolutionary (phylogenetic) relationships between the

different bacteria. It is difficult to trace the path of evolution by bacterial phenotypes but

considerably more successful to do it by comparing genomic nucleotide sequences and

the gene of 16S rRNA has been proved to be especially useful for tracing the phylogeny

of bacteria, i.e. to build the bacterial taxonomy.


38

References

Atarashi, K., Tanoue, T., Shima, T., Imaoka, A. et al. (2011). Induction of colonic

regulatory T cells by indigenous Clostridium species. Science 331: 337-341.

Anon (2002). Joint FAO/WHO Working Group Report. Guidelines for the Evaluation

of Probiotics in Food. London, Ontario, Canada.

CraCraft, J. (1987). Species concepts and the ontology of evolution. In Biology and

Philosophy 2. Philpapers Online research in philosophy.

http://philpapers.org/rec/CRASCA

Eisen, J.A. (2007). Environmental shotgun sequencing: its potential and challenges for

studying the hidden world of microbes. PLoS Biol. 5: e82.

Feng, T. and Elson, C.O. (2011). Adaptive immunity in the host-microbiota dialog.

Mucosal Immunol. 4: 15-21.

Fuller, R. (1989). Probiotics in man and animals. Journal of Applied Bacteriology 66:

365-368.

Gibson, G.R. & Roberfoid, M.B. (1995). Dietary modulation of the human colonic

microbiota: introducing the concept of prebiotics. Journal of Nutrition 125:1401-1412.

Gregersen, T. (1978). Rapid method for distinction of gram-negative from gram-

positive bacteria. European Journal of Applied Microbiology and Biotechnology, 5,

123-127.

Handelsman, J.; Rondon, MR.; Brady, S.F.; Clardy, J.; Goodman, R.M. (1989).

Molecular biological access to the chemistry of unknown soil microbes: a new frontier

for natural products. Chem. Biol. 5: 245–249.

Keswanit, J. & Whitman, W.B. (2001). Relationship of 16S rRNA sequence similarity

to DNA hybridization in prokaryotes. Int J System Evol Microbiol; 51: 667-78.

Lilley, D.M. & Stillwell, R.H. (1965). Probiotics: growth promoting factors produced

by microorganisms. Science 147:747-748.

Mayr, E. (1982). The growth of the biological thought. Harvard University Press,

Cambridge, Massachusetts.

Naidu, A.S., Bidlack, W.R. & Clemens, R.A. (1999). Probiotic spectra of lactic acid

bacteria (LAB). Crit. Rev. Food Sci. Nutr. 38:13-126.

Parker, R.B. (1974). Probiotics, the other half of the antibiotic story. Animal Nutr.

Health 29:4-8.

Schaafsma G. (1996). Significance of probiotics in human diets. SOMED, XXIst

International Congress on Microbial Ecology and Disease, Paris 23-30 October.

Abstract no. 117, p 38.

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Sekirov, I., Russell, S.L., Antunes, L.C.M., Finlay, B.B. (2010). Gut microbiota in

health and disease. Physiol. Rev. 90: 859-904.

Stackebrandt E, Goebel B. (1994). Taxonomic note: a place for DNA-DNA

reassociation and 16S rRNA sequence analysis in the present species definition in

bacteriology. Int J Bacteriol 44: 846-849.

Yang, X., Xie, L., Li, Y. & Wei, C. (2009). More than 9,000,000 unique genes in

human gut bacterial community: Estimating gene numbers inside a human body. PLoS

ONE 4: e6074


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41

II. Dietary bacteria


42


43

Intention

The educational intention with Lecture-block II – Dietary bacteria – is to provide

information of harmless bacteria that have been consumed in high numbers by humans

for considerable length of time, and of food products that by tradition have been based

on the activity of these bacteria. In principle, all the foods described below could be

matrixes for probiotics if a probiotic strain able to multiply in the product is used as

starter culture. The traditional fermented foods are empirically developed food-concepts

that have proved their value for generations of humans. It is the author’s believes that

research and development (R&D) managers can use this knowledge successfully in the

development of new probiotic foods. Probiotics can be marketed as more or less pure

bacteria in form of capsules, tablets or powders, but in a combination of actively

metabolising bacteria and nutritionally beneficial food components the strength of the

probiotic concept can be enhanced.

Consumption of lactobacilli

Traditional, so called lactic acid fermented foods normally contain high numbers of live

bacteria (“lactic acid bacteria”), but can also contain dietary fibres (some with prebiotic

effects) and compounds as flavonoids, vitamin E and vitamin C, able to protect from

oxygen free radicals (antioxidants). Both prebiotics and antioxidants can protect and

support the live bacteria in the food, but can also add to the health beneficial effects of

the probiotics. Hence, the traditional lactic acid fermented foods can be a good starting

point for product development of probiotic foods with health benefits (probiotic

functional foods).

Consumption of live lactic acid bacteria (LAB) included in lactic acid fermented foods

has been a regular part of the food intake of humans for a long time. In fact, there are

archaeological indications that humans have used this technique from the beginning of

time. This technique was presumably invented 1.5 million years ago by the early

humanoids (Figure 7).

Humans have obviously consumed large numbers of live LAB for a long time, and

presumably those LAB associated to plant material were consumed before those

associated to milk. Lactic acid fermentation is the simplest way to preserve food, and if

it is made correctly, presumably also the safest way. Before the Industrial Revolution,

lactic acid fermentation was applied just as much in Europe as it still is in rural areas of

Africa, South America, and several parts of Asia. In fact, it could very well be that the

human digestive tract evolved to adapt to a more or less daily supply of live LAB. This

supply ceased in industrialized countries during the twentieth century, which eventually

may have lead to gastro intestinal (GI) problems, and maybe also to immunologically

dependant dysfunctions.


44

Figure 7. A suggested time scale for human developments, showing when the technique of lactic acid fermentation probably came into use.

In Sweden, as in the rest of North Europe, lactic acid fermented vegetables have been

widely consumed up to modern times. For example, a Swedish cookery book from 1755

carefully describes procedures how to make sauerkraut, fermented spinach and

fermented sorrel (“Cajsa Wargs kokbok”). However, when the emerging food industry

of Europe gradually took over the food production and discovered new technical means

to process and preserve food in the nine-teen century, the significance of lactic acid

fermentation decreased. Furthermore, Louis Pasteur proved that milk turned sour by the

activity of bacteria (1857) and Robert Koch showed that cholera was caused by bacteria

(1883). Thus, living bacteria in food were looked upon with suspicion by both the

Industry and the Society. So with the best of intentions of saving consumers from

pathogenic bacteria, and also to control the storage stability of the products, the food

industry started to systematically kill all microorganisms in food.

Lactic acid preservation

The general principle of lactic acid fermentation is that a raw-material containing

carbohydrates is stored under conditions restricting the access of air (oxygen). Bacteria

and yeast that contaminate the raw-material will spontaneously multiply, the oxygen

tension in the material will decrease, carbon dioxide will accumulate, the pH will

decrease, and in the end, the product will be completely dominated by LAB (and

occasionally also by yeast).

The final pH in lactic acid fermented products usually is between 3.5 and 4.0, i.e.

growth of most bacteria is inhibited by the low pH. However, the product can be


45

covered by moulds on the surface if the surface is exposed for air (oxygen). Yeast can

also grow in the product and affect the eating quality in positive or negative direction

depending on the type of product. Yeast produces ethanol by fermentation of

carbohydrates which contributes to the preservation.

Generally the lowest pH for growth in food is for: Bacteria >3.0 Moulds >1.6 Yeasts >1.4

The pH-susceptibility for different bacterial taxa frequently found in foods increases in

the order Pseudomonas (most sensitive) < Enterobacteriaceae < Bacillus <

Enterococcus < Leuconostoc < Pediococcus < Lactobacillus (highest resistance against

low pH).

Lactic acid bacteria as Leuconostoc, Pediococcus and Lactobacillus have inhibitory

effects on several other bacteria, primarily on gram-negative bacteria as, for example,

Enterobacteriaceae, Pseudomonas and Aeromonas, but also some gram-positive taxa as

Clostridium and Listeria. Examples on taxa that occasionally can be a problem in lactic

acid fermented foods if the pH isn’t low enough are Bacillus cereus and Enterococcus.

Different inhibitory factors can be produced by lactic acid bacteria (LAB) and released

into the food environment, e.g. (i) carboxylic acids, (ii) hydrogen peroxide, (iii) nitrogen

oxide, and (iv) ”bacteriocins”. The carboxylic acids are mostly lactic acid and acetic

acid which lower the pH, but carboxylic acids have apart from the pH-effect,

antimicrobial affects by themselves, and especially in un-dissociated form, i.e. at low

pH.

Many LAB over-produce hydrogen peroxide (H2O2) if they are exposed to oxygen.

Hydrogen peroxide has powerful oxidation capacity and therefore possesses strong

antimicrobial effects. Just as hydrogen peroxide, nitrogen oxide (NO) also has strong

oxidative properties which are deleterious to microorganisms. NO is produced by the

limited number of LAB that are able to break-down the amino acid arginin.

Finally, bacteriocins are compounds, often peptides (but not always), with antibiotic

effects. Most bacteriocins produced by LAB are active against a relatively narrow

spectrum of closely related organisms. Different bacteriocins are produced by different

species and strains. A well-known bacteriocin is nisin that is produced by Lactococcus.

Nisin has antimicrobial effects against a relatively wide range of different bacterial taxa.

Nisin is produced by the starter culture in the Swedish sour milk “filmjölk”

(Lactococcus lactis strains).


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Lactic acid bacteria

What is a LAB?

The organisms performing the conversion of carbohydrates to carboxylic acids in

fermented foods, mainly lactic acid, are by tradition called Lactic acid bacteria (LAB).

The term “lactic acid bacteria” was used early by food microbiologists, and 1919 the

Danish bacteriologist Orla-Jensen tried to define key-features of LAB. He said “the true

lactic acid bacteria form a large natural group of non-motile, non-spore-formers, Gram-

positive cocci and rods that at fermentation of sugar mainly produce lactic acid”.

Followers have added new key-features to the list and these criteria are frequently cited

in textbooks. Thus, a typical list of determinative criteria for the description of LAB are

that they should be: (i) Gram-positive, (ii) able to ferment sugars to mainly lactic acid,

(iii) catalase negative, (iv) non-motile, (v) unable to form endospores (a endospore is a

spore formed within the cell), (vi) able to grow at low pH (<5.5), and (vii) non-

pathogenic. However, it must be stressed that these key-criteria are of poor relevance as

LAB isn’t a “natural group” (natural taxonomic unit) as Orla Jensen assumed. When

dealing with LAB, it is to be preferred to refer to the actual genera and/or species in

question. Unfortunately, based on this old fashioned, artificial taxonomy, different

systematically defined taxa have been included in the group LAB. But, in reality some

of these taxa as for example Bifidobacterium and Enterococcus are not supposed to

dominate in lactic acid fermented foods, either they can’t be found (Bifidobacterium) or

they can ruin the edibility (Enterococcus). Thus, LAB is not a systematically defined

group based on evolutionary relationships as the old bacteriologists assumed, currently

it is a term used by food microbiologist when they refer to bacteria responsible for the

fermentation of lactic acid fermented foods.

LAB is a functional group and applies to bacteria that are harmless to both food quality and human health, and that occurs spontaneously in high numbers in traditional lactic acid fermented foods.

From the systematic point of view, LAB means a relatively wide variety of bacterial

groups. How many genera and species that should be included into the LAB-concept

depend much on how many different types of food that are included and how strict the

quality requirements are set for these foods. For example, the higher the eating quality

is for a lactic acid fermented food product, the fewer types of bacteria are generally

involved in the final fermentation. In a product of poorer quality, all types of more or

less unwanted organisms can be present in the final product and in fairly high numbers,

but those organisms should of course not be regarded as LAB just because they happen

to contaminate and multiply in a lactic acid fermented product.

The only absolute condition for the organisms involved in lactic acid fermentation of

food must be that they produce lactic acid and that they are harmless to consume in high

numbers, even for consumers with underlying sicknesses that weaken their

immunological defence. The taxa frequently occurring in high numbers in traditional,

and spontaneously fermented lactic acid fermented foods, are species of the genera

Lactobacillus, Pediococcus, Weissella, Leuconostoc, Oenococcus, Lactococcus, and the

species Streptococcus thermophilus and closely related species to S. thermophilus.


47

The genera Lactobacillus and Pediococcus belong to the family Lactobacillaceae which

also includes the relatively new genera Paralactobacillus and Sharpea. They can all be

included in the trivial expression "lactobacilli”.

Leuconostoc, Weissella and Oenococcus belong to the family Leuconostocaceae

together with the genus Fructobacillus.

Lactococcus and S. thermophilus have from the phylogenetic point of view relatively

little in common with Lactobacillaceae and Leuconostocaceae even if they all are

included in the order Lactobacillales. Thus, they are members of the same general

branch of evolution. The order Lactobacillales belongs to the phylum (division)

Firmicutes with the characteristics in common of being gram-positive and having a low

ratio of guanine and cytosine in their genome.

The functional concept, LACTIC ACID BACTERIA (LAB), includes primarily the following taxa: Lactococcus Lactobacillus Leuconostoc Oenococcus Pediococcus Weissella Streptococcus thermophilus

Bifidobacterium, Enterococcus and Carnobacterium are now and then wrongly

regarded as LAB. However, Bifidobacterium is a health promoting genus and are

frequently used as probiotics. In contrast, Enterococcus and Carnobacterium are

dubious from a health perspective and should be avoided in foods.

Lactococcus

The genus Lactococcus which includes 9 different species ferments carbohydrates to

lactic acid. Lactococcus possesses superoxide-dismutase and menaquinone, i.e. systems

for efficient utilization of oxygen in the metabolism. Lactococcus and particularly the

species Lactococcus lactis is traditionally used as starter culture in the Swedish sour

milk product ”Filmjölk”, and in most types of European cheeses. Phylogenetically,

Lactococcus is, as mentioned, only distantly related to Lactobacillus.

Streptococcus

The species S. thermophilus forms together with three other Streptococcus species, a

taxonomic unit that is relatively distantly related to most other Streptococcus species. S.

thermophilus ferments carbohydrates to lactic acid, and is traditionally included in the

starter culture for yoghurt. S. thermophilus grows rapidly in milk at temperatures

around 45oC. In nature, S. thermophilus is found associated to mucous membranes of

animals. The high growth temperature of S. thermophilus could give a hint that they are

more connected to birds than humans as birds generally has a relatively high body

temperature (around 40oC).


48

The genus Streptococcus includes more than 90 different species, and several

Streptococcus spp. are pathogenic, e.g. Streptococcus pyogenes. Many Streptococcus

species are associated to the mucous membranes of man and animals. Streptococcus

pyogenes (Group A streptococci = GAS) is a notorious pathogen and cause septic

throat, scarlet fever and other pyogenic and septicemic infections. The infective dose

can be less than 1000 colony forming units (CFU), and the incubation period is 1–3

days. Symptoms are sore throat, pain on swallowing, tonsillitis, high fever, headache,

nausea, vomiting, malaise and rhinorhea. On the other hand, S. thermophilus and those

Streptococcus spp. closely related to S. thermophilus are non-pathogenic and could be

regarded as LAB.

Lactobacilli

The genera Lactobacillus, Pediococcus, Leuconostoc, Weissella and Oenococcus have a

relatively close phylogenetic relationship. They can all be included in the more trivial

expression “lactobacilli”.

Leuconostoc. The genus Leuconostoc includes 25 species. Leuconostoc are coccoid

cells but occasionally rod-like. Leuconostoc ferments carbohydrates to lactic acid,

always with a heterofermentative product pattern, i.e. they ferment glucose to lactic

acid, ethanol, carbon dioxide, and depending on circumstances, also to acetic acid.

Leuconostoc is spontaneously occurring in high numbers in lactic acid fermented milk,

and is often dominating the initial fermentation of plant material. In nature, Leuconostoc

is probably often associated to insects and snails. The species Leuconostoc

mesenteroides has been isolated from human intestinal mucosa.

Weissella. The genus Weissella includes13 species. Weissella appears in the microscope

in the forms of rods or cocci. Weissella ferments carbohydrates to lactic acid, and

always with a heterofermentative product pattern. Weissella is spontaneously occurring

in high numbers in many lactic acid fermented foods, and it can be found associated to

human mucous membranes.

Oenococcus. The genus Oenococcus includes two species, Oenococcus oeni and

Oenococcus kitaharae. Oenococcus is a coccoid or ellipsoidal cell, fermenting

carbohydrates to lactic acid, always with a heterofermentative product pattern.

Oenococcus is spontaneously occurring in high numbers in wine and beer. Oenococcus

can grow at low pH and in 10% ethanol.

Pediococcus. The genus Pediococcus includes 15 species. Pediococcus-cells appear

under the microscope as cocci in pair or in tetrahedrons. Pediococcus ferments

carbohydrates to lactic acid, and is commercially used as starter culture in smoked,

fermented sausages. Pediococcus is often isolated from fermented vegetables and

cereals, and from beer. Pediococcus can be regarded as facultatively heterofermentative.

Phylogentically, Pediococcus is closely related to Lactobacillus. In fact the different

Pediococcus spp. are in phylogentic trees (cladograms) mixed with different

Lactobacillus spp. Pediococcus and Lactobacillus have by tradition been regarded as

different genera due to the easily recognised differences in cell morphology.


49

Lactobacillus. The genus Lactobacillus includes more than 90 different species and

subspecies. Lactobacillus-cells are rod-formed, and ferments carbohydrates to lactic

acid. Lactobacillus is spontaneously dominating most traditional lactic acid fermented,

foods. Lactobacillus is associated to mucous membranes of the oral cavity, gastro-

intestinal tract and vagina of animals and man.

Food microbiologists have since long divided the different Lactobacillus spp. into two

functional groups based on what end-products that they form during fermentation of

hexoses, i.e. during growth on, for example, glucose without access to oxygen. If lactic

acid is the only end-product from glucose, the lactobacilli are said to be

homofermentative (catabolism through the fructose-1,6-biphosphate pathway), but if

also carbon dioxide and ethanol and/or acetic acid is formed together with the lactic acid

the lactobacilli are said to be heterofermentative. The word fermentation stands for

growth/catabolism in absence of oxygen.

There is also some homofermentative Lactobacillus spp. that are able to ferment

pentoses. Fermentation of pentoses yields lactic acid and acetic acid. Some

homofermentative Lactobacillus spp, can also ferment malic acid to lactic acid and

carbon dioxide, and/or, ferment citrate to diacetyl, acetoin and carbon dioxide. These

basically homofermentative lactobacilli are said to be facultatively heterofermentative

and the true heterofermentative lactobacilli are then said to be obligatory

heterofermentative.

Homofermentative Lactobacillus: glucose >>>>> lactic acid Heterofermentative Lactobacillus: glucose >>>>> lactic acid, CO2, ethanol (acetic acid)

Some homofermentative Lactobacillus are labelled facultatively heterofermentative because they can ferment: pentoses >>>>>>> lactic acid + acetic acid malic acid >>>>>> lactic acid + CO2 citrate >>>>>>>>> diacetyl + acetoin + CO2

Facultatively heterofermentative lactobacilli growing on glucose in the presence of

oxygen (aerobic conditions) produce beside lactic acid also acetic acid, diacetyl and

acetoin.

The division of lactobacilli into the three functional groups: (i) the obligatory

homofermentatives, (ii) the facultatively heterofermentatives and (iii) the obligatory

heterofermentatives is of pragmatic relevance for food technologists as the catabolic

end-products can affect the food characteristics (quality). Carbon dioxide can be a food

technological problem, especially in connection with packed products and too much

acetic acid in the product can give the product a sticky taste. Ethanol has it’s specific

qualities, unwanted in some products but most wanted in others.

Phylogenetically, Lactobacillus is a heterogeneous genus with a guanine + cytosine

(G+C) mol% content varying in the interval 32-53%. This range is about twice as large

as normally accepted for a well defined genus. The type species of Lactobacillus is

Lactobacillus delbrueckii, which is an obligatory homofermentative organism. The mol

% G+C for L. delbrueckii is 49-51. Paradoxically, the mol % G+C for some of the other

well known obligatory homofermentatives, such as Lactobacillus acidophilus,

Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri are only 32-


50

37%, 35-38%, 35-37% and 33-35%, respectively, i.e. much lower than that of the type

species.

Taxonomic efforts to understand the phylogenetic relationships within the genus

Lactobacillus have been directed towards comparative analysis of primarily the 16S

ribosomal ribonucleic acid (rRNA) gene (rDNA) sequences (Schleifer and Ludwig

1995; Vasques et al. 2004), but later also towards an extended numbers of other genes

(Makarovaa et al. 2006; Zhang et al. 2011), and based on whole-genome alignments

(Canchaya et al. 2006). These analyses show an extreme divergence of the

Lactobacillus genome and support the recognition of new subgeneric divisions, i.e. the

genus Lactobacillus can be divided into several genomic groups on the genus-level.

Furthermore, several of the facultatively heterofermentative Lactobacillus spp. are

closely related to Pediococcus spp.

The Lactobacillus spp. have been divided phylogenetically into three groups that are not

altogether in agreement with the traditionally, phenotypically-based, subgroups

(fermentation-groups). Thus, many of the obligatory homofermentatives (for example,

L. delbrueckii, L. acidophilus, L. crispatus, L .jensenii and L. gasseri) form one group.

This rDNA-group has been called the “L. delbrueckii-group”. The second group,

including Lactobacillus spp. of all three fermentation-groups and also some

Pediococcus spp., has been called the “L. casei group”. And the third group, the

“Leuconostoc group” included Leuconostoc spp., some obligatory heterofermentative

Lactobacillus spp. and Weissella spp. (Schleifer and Ludwig 1995).

However, Lactobacillus can be further subdivided into at least six different groups, i.e.

(i) the L. acidophilus group (L. delbrueckii is linked to this group but is somewhat

atypical), (ii) the L. salivarius group, (iii) the L. reuteri group, (iv) the L. brevis group,

(v) the L. plantarum group and (vi) the L. casei group.

Bifidobacteria

The genus Bifidobacterium includes more than 45 different species of bifidobacteria.

Bifidobacterium ferments carbohydrates to acetic acid and lactic acid and follows a

unique catabolic pathway. Also, Bifidobacterium assimilates inorganic nitrogen

(ammonium) for growth and biosynthesis. Bifidobacterium is sensitive to oxygen.

Bifidobacterium is usually present in high numbers in the ileum and colon.

Bifidobacterium does not usually occur spontaneously in lactic acid fermented foods.

Bifidobacterium has an optimum pH for growth at 6.5-7.0, and the genus doesn’t

usually grow below pH 4.5.

Phylogenetically, Bifidobacterium is totally different from both Lactobacillus and

Lactococcus. Bifidobacterium belongs to the phylum Actinobacteria while

Lactobacillus and Lactococcus belong to Firmicutes.

Bifidobacterium has a high guanine + cytosine mol % (55-66%).


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Two adverse genera sometimes wrongly called LAB

Enterococcus. The genus Enterococcus includes about 40 species. It is a tough gram-

positive bacterium that frequently is present in high number in faeces. Enterococcus can

occur spontaneously in different kinds of foods, for example in cheese, and now and

then are non-pathogenic strains of Enterococcus faecium even included in sour-milk

products and cheese with the claim of being probiotic or beneficial for the taste,

respectively.

On the other hand, Enterococcus is frequently causing urinary tract infections.

Enterococcus is easily acquiring antibiotic resistance, and it is frequently causing sepsis

in hospitalised patients. Even enterococcal food poisoning may occur due to production

of enterotoxin if Enterococcus is allowed to grow to high numbers (>107 CFU per g) in

the food. Symptoms are then diarrhoea, abdominal cramps, vomiting and fever. The

duration of the symptoms is 2 to 36 hours.

Carnobacterium. The genus Carnobacterium that today includes around 10 species was

formed 1987 after a discovery that some former Lactobacillus species (“Lactobacillus

carnis”; “Lactobacillus piscicola” and “Lactobacillus divergens”) didn’t belong to the

genus Lactobacillus (Collins et al. 1987). Carnobacterium is rod formed, and ferments

carbohydrates to lactic acid, but can’t grow at pH 5.5 or lower. Carnobacterium is

frequently found spontaneously growing to high numbers on vacuum-packed meat and

meat products (or in other types of packages with modified atmosphere).

Carnobacterium can produce biogenic amines and has a relatively close phylogenetic

relationship with Enterococcus.

Products and processes

Simple technique

The reason for letting food undergo lactic acid fermentation can vary between different

applications. The primary goal with the lactic acid fermentation can be to (i) increase

the shelf-life and microbial safety of the product; (ii) increase the control and the safety

of a food processing step; (iii) improve the taste and the consistency of the product; (iv)

achieve health beneficial effects.

An ancient and simple technique for fermentation with the prime goal to keep the food

from spoiling is to dig a hole in the ground, fill in the raw food material and put a cover

on top, preferably with some weights in order to press out as much air as possible.

The same technique, to use a hole in the ground and with the main purpose to prevent

spoilage was up to modern time used for the preservation of forage-plants in Sweden.

Today the farmers normally do not use pits in the ground, they use silo-tanks or large

(usually white) plastic bags. However, for example, a large producer of “organic” milk


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in Skåne has gone back to the pit-concept, but in a more modern form, i.e. huge basins

of concrete. Generally it takes 1-2 weeks for the fermentation of the forage-plants, for

example grass, and for the pH to drop to 3.9-4.2 in the silage.

Sometimes starter cultures are used to control the forage fermentation (Pediococcus spp.

and/or Lactobacillus spp.) or the raw-materials are pre-treated with formic acid (to

lower the pH and select for lactic acid bacteria). But today, usually large bundles of

tightly pressed forage are individually wrapped and sealed in plastic foil, and no starter

cultures are added.

Ethiopian kocho

Ensete ventricosum is a perennial, banana like, starchy root crop which grows in

Ethiopia at altitudes of 1500 to 3000 m above sea level. The height of the plant when

harvested can reach 6 to 8 m (6-8 years after planting). It is a leading staple crop for

about 10 million Ethiopian inhabitants. The plant is traditionally processed as follows

(Figure 8):

The pseudo stem and corm are pulverized with a long wooden pestle, and pounded into

a pulp from which the fibres are removed. The remaining scrapings, the pulp and the

inner corm, are kneaded together, rolled into balls and wrapped in fresh enset leaves.

The leave-wrapped packages with fresh enset mash are packed in a pit in the ground

that has been completely lined with leaves, and the packages are left for pre-

fermentation for two to five days. After this pre-fermentation, the packages are opened

and the mash is once again mixed and thoroughly kneaded, rolled into balls and

wrapped in new fresh enset leaves. The new packages are pressed into the pit. Some of

the waste mash and cellulosic material from the production is put on top to create a

cover, and stones are put on top of this cover. The aim is to limit the access of air into

the pit. The major fermentation takes about 2 weeks at a temperature 15-18oC, but the

kocho can be left in the closed pit for 6 months up to years (in the colder regions of

Ethiopia). It can be pointed out that this is the same temperature interval that is used for

the fermentation of sauerkraut in Europe.

In the colder regions of Ethiopia, the general belief is that the quality of the kocho

improves with storage time. In the warmer regions, it gets too sour and becomes

discoloured if it is stored too long. Kocho is mainly baked into bread or cooked and

eaten alone or in combination with various indigenous foods. High quality kocho or

variants of kocho are also eaten unheated and are considered by the public to posses

beneficial health effects.

In the beginning of the kocho-fermentation (first days), the fermentation is typically

dominated by Leuconostoc mesenteroides, but after approximately a week Lactobacillus

spp. reach similar numbers. After two weeks both Lactobacillus and Leuconostoc are

present in high numbers (about 5x109 CFU per g). The viable count of Lactobacillus

remains high for months, while the count of Leuconostoc declines. The same sequential

pattern for Leuconostoc and Lactobacillus has been described for both sauerkraut and

salted gherkins. A controlling factor can be the pH. Leuconostoc is less resistant than

Lactobacillus to low pH, and the species Lactobacillus plantarum is especially hardy at

low pH. The dominating LAB in kocho bought in markets in Ethiopia are often


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Lactobacillus plantarum, Weissella minor (earlier classified as Leuconostoc) and

Pediococcus pentosaceus (Gashe 1987; Nigatu 1998).

Figure 8. Flow scheme of the production of Ethiopian kocho.

Sour salmon buried in the ground

The technique of digging a hole in the ground was in the old days also used in the North

of Sweden for the preservation of salmon; the so called “gravad lax” (buried salmon, in

Swedish). During the summer, fresh-caught salmon were salted and buried in the sandy

river banks at the mouth of rivers, and left for spontaneous fermentation. The sour

salmon could then be fetched for consumption during the winter season.

Fermentation in sealskin bags

Plant material. One example of the importance of lactic acid fermented plant material

for indigenous living people is constituted by the Tschuktscer people living in Siberia

on the Tschuktsch peninsula along the seashore of the North polar sea. The Tschuktscer

people were described by the Swedish explorer A.E. Nordenskiöld during his expedition

around Asia in his voyages through the North-East Passage (1878-1880). At this time,

the Tschuktscer was a primordial society of hunters and fishermen, and a major

component in their diet was lactic acid fermented plant material. During the summer-

months they collected different kinds of plant material, such as leafs from osier (Salix)

and the plant Rhodiola. After being picked, the plant material was pressed into sealskin


54

bags that were sealed and left for spontaneous fermentation during the summer months.

During the autumn, the content froze in the form of the out-stretched bags. The frozen

mass was cut in pieces and eaten as it was, or together with meat or fish, or it could also

be used in hot soups. Nordenskiöld speculated that the observed consumption of lactic

acid fermented plant material could mimic the human diet during the Stone Age.

Fermented Auk. Fermentation in sealskin bags of whole birds of the species Alle alle

(Little auk; in Swedish “Alekung”) results in the traditional Greenlandic food kiviak.

The auks are caught in the spring stuffed in a hollowed-out body of a seal with around

300 to 500 auk birds per seal-pouch. The seal-pouch is sewn up, sealed with fat, put

under a pile of rocks on the permafrost ground and left to ferment for 3 to 18 months. In

older days the fermented auk birds become a food resource during the winter.

Salted gherkins

Cucumbers (Cucumis sativis) contain a higher proportion of water and, hence are highly

susceptible to microbial spoilage. Field-cultivated cucumbers for the food industry are

harvested during a short season in the autumn and are used mainly for the production of

different sorts of pickles. Only a relatively small proportion is marketed as salted

gherkins, the traditional lactic acid fermented product. In Sweden, the production of

salted gherkins to be used in the food industry as raw material for pickles, is around

2000 tons per year, but can be up to 7000 tons depending on the harvest. The production

in, for example, the USA is at least 100 times as high.

The cucumbers used for the lactic acid fermentation often become heavily contaminated

by microorganisms from the soil in the field. A typical aerobic viable count of bacteria

can be 5x106 colony forming units (CFU) per g cucumber; the viable count of

Enterobacteriaceae can typically be 106 CFU/g while the count of lactobacilli mostly is

fairly low, for example, 5x103 CFU/g. Thus, the odds for a successful preservation of

the cucumbers by spontaneous lactic acid fermentation apparently seem to be quite low.

But surprisingly enough, by the use of some salt (NaCl) it is feasible to control the

fermentation and get a safe product with long shelf-life (Figure 9).

In Sweden, the cucumbers are put into a brine to achieve a final concentration of 5%

NaCl. Traditionally this occurs in open containers of wood holding 25 ton cucumber

each, and situated outdoors. After fermentation the container is covered with a tarpaulin.

In warmer countries, the salt concentration is usually higher during the fermentation (up

to 8% NaCl), and after the completed fermentation, the salt concentration is increased

from 8% to 16% NaCl to ensure a long shelf-life of the product. In this way, salted

gherkins can be stored for at least one year.

Malic acid fermentation: malic acid >>>>>> lactic acid + CO2

Under the conditions that the salt concentration in the brine isn’t too high, the first LAB

to increase to a dominating position are Leuconostoc, and as the fermentation proceeds

they will be succeeded by Lactobacillus; generally the same succession as has been seen

in Ethiopian kocho and in European and American sauerkraut. A typical bacterial

species found in high numbers at the end of fermentation is L. plantarum, both in salted

gherkins and in sauerkraut. However, when used as a starter culture, L. plantarum can

cause gas pockets of carbon dioxide in the cucumbers. This gas vacuoles are caused by


55

so called malic acid fermentation, i.e. malic acid is catabolised to lactic acid and carbon

dioxide.

Figure 9. Flow scheme of the traditional production of salted gherkins. The sugar in the cucumbers consists mainly of glucose and fructose.

Wine

Red wines undergo malic acid fermentation which is performed by lactobacilli after the

yeast had produced the alcohol. In wine-fermentation, it is of course a requirement that

(i) yeasts (Saccharomyces) convert sugar into alcohol, but in red wine it is also

important that (ii) malic acid is converted to lactic acid and carbon dioxide which is

done by lactobacilli, and (iii) the conversion of different phenolic compounds derived

from the grapes, e.g. polyphenoles as tannins. This polyphenol conversion is also

performed by lactobacilli (Figure 10).

The malolactic fermentation affects the taste and the conversion of polyphenoles affects

especially the colour of the wine but also the taste. The polyphenoles in young red wine

(900-1400 mg per litre wine) can consist of, for example: 70% anthocyanins, 15%

flavanols, 6% hydroxybenzoic acid and derivates, 3.8% phenolic alcohols, 3.6%

flavonols, 1.1% hydroxycinnamic acid and derivates, and 0.5% stilbenes (Garcia-Ruiz

et al. 2008).

It has been shown in a small randomized, crossover controlled intervention study on

humans that a moderate intake of red wine affected the composition of the bacterial

flora of the gut (measured in faecal samples), and decreased the blood pressure and the

concentration of triglycerides, total cholesterol, HDL cholesterol and C-reactive


56

proteins (CRC) in the blood. The decrease was linked to an increase in bifidobacteria

(Queipo-Ortuno et al. 2012). It was shown that it was the polyphenols in the wine that

was the active component, not the alcohol.

Figure 10. Production-scheme for wine. The process-steps are as described by Pérez-Serradilla and Luque de Castro (2008).

Lactic acid fermented capers (caper berries)

Caper berries are the fruits of Capparis spp. (mainly Capparis spinosa L.). Capparis is

a Mediterranean shrub cultivated for its buds and fruits. The main producers of

fermented caper berries are Mediterranean countries, especially Greece, Italy, Turkey,

Morocco and Spain. The fermentation is often done by traditional, manual procedures.

The fruits are collected from the shrubs during June and/or July and immersed in tap

water, where the fermentation takes place spontaneously for approximately 5 to 7 days

at a temperature range of 23oC to 43

oC (Figure 11). After the fermentation, the capers

are placed in brine to a final NaCl-concentration of around 10% (w/v). The fermentation

can take place in vats (for example, 150 litres) and the pH fall below 4, and typically

stop around 3.3.

The total aerobic count of the harvested berries immersed in water can be around 1000

CFU per ml and after the fermentation, the total count (and the lactobacilli count) goes

up to 108 CFU per ml. Typically the bacterial flora after the fermentation is dominated


57

by L. plantarum (around 50% of cases), but also by, for example, Pediococcus

pentosaceus, Lactobacillus brevis, Lactobacillus fermentum and Lactobacillus pentosus

can occur in high numbers (Pulido et al. 2005).

Figure 11. Production-scheme for the lactic acid fermentation of Capers (caper berries).

Sauerkraut

In a Swedish cookery book from 1755, the procedure to make sauerkraut is described.

Sauerkraut should be made according to the following description: “The bottom of a

tight barrel is covered with the white cabbage leaves and then it is filled with good

measure of finely chopped white cabbage which gradually is pressed down with a

wooden pole until the cabbage is immersed in juice. Into the barrel also a few barberry

berries, some salt, a few roasted peas and dill where the seed have been carefully

removed are thrown in. Some caraway is put on the top and then all is covered with

cabbage leaves, and a barrel lid that can go down into the barrel is put on top and

pressed down into the barrel with heavy stones. The barrel is left for fermentation in a

room that is neither too hot nor too cold. When the fermentation is ended the stones are

removed and a lid put on the barrel that is stored as cold as possible” (Cajsa Wargs

Kokbok, 1755).

Sauerkraut has since long a reputation of being healthy. This is reflected by the fact that

the English captain James Cook during his voyage around the world (1768-1780) forced

his crew to eat sauerkraut. James Cook became famous not only for his geographic

discoveries but also for his extraordinary record of high survival of the seamen on his

ships.

One example of current industrial sauerkraut production has been described from

Wisconsin (USA) where the fermentation takes place in cement tanks of about 100 m3

volume (Plengvidhya et al. 2007). The fermentation is carried out with 2.3 % NaCl

(final equilibrated concentration). The salt is added by a dry salting process using


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shredded cabbage. The cabbage is manually spread in the tanks, covered with plastic

sheeting, and initially weighted down with water on top of the plastic sheeting (Figure

12). The fermentation temperature is not controlled, but it is typically around 18oC.

Glucose and fructose are the primary fermentable sugars in the cabbage and are from

the start present in concentrations between 1.5% (w/w) and 2.2%.

Figure 12. Industrial sauerkraut fermentation in Wisconsin (USA) as described by Plengvidhya et al. (2007).

The fermentation is run for about 14 days and during this time, pH drops to 3.4-3.7,

lactic acid and acetic acid are produced to concentrations of about 130 and 50 mM,

respectively (Plengvidhya et al. 2007). Originally cabbage has a total viable count of

about 5 x 106 CFU/g and a count of Enterobacteriaceae of about 10

6 CFU/g. During the

first week of fermentation, there is a rapid decrease in Enterobacteriaceae and an

increase in LAB. Initially Leuconostoc mesenteroides is the dominating species of LAB

but already after 3-7 days different species of Lactobacillus (but also some Weissella

and Pediococcus) reach substantial numbers. After 14 days, the sauerkraut is heavily

dominated by L. plantarum (80-100% of the bacterial flora; Plengvidhya et al. 2007).

Korean kimchi

A popular lactic acid fermented product in Korea is kimchi. Kimchi is processed in

similar ways as sauerkraut, but it is based on different kind of vegetables and spices.

There are hundreds of different varieties of kimchi in Korea but major components are

Chinese cabbage and radish, and often used spices are red pepper, garlic, green onion

and ginger. The ingredients are salted to 2% to 3% NaCl and usually fermented for 2 to

3 days at temperatures around 20oC. In the beginning of the fermentation Leuconostoc

is the dominating taxa in the product but gradually replaced by Lactobacillus, Weissella

and Pediococcus. The load of bacteria in the final product is 108-10

9 CFU LAB per

gram, and the pH is usually between 3.6 and 4.5. Kimchi seems to be increasingly

known outside Korea, and may be a food concept with international potential.


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Green olives in brine

The lactic acid fermentation of green olives (Spanish-style) starts with a pre-treatment

with lye (1.3-2.6 [w/w] of NaOH) for 5 to 7 hours to hydrolyze and remove some of the

bitter tasting phenolics (mostly ortho-diphenols and their glucosides). Then, the olives

are put in a brine solution and subjected to spontaneous lactic acid fermentation (Figure

13). After the fermentation is finished, the salt in the brine is increased to 8% (w/w) to

ensure the keeping qualities of the olives for extended storage periods

L. plantarum is normally found in high numbers at the end of the fermentation. They are

thought to be coexisting with a yeast flora. The yeast is believed to release B-vitamins

that are utilized by the lactobacilli.

From a nutritional perspective it is important to note that lactic acid fermentation often

reduces phytates and tannins which are compounds that might reduce the bioavailability

of essential minerals as iron, zinc and calcium.

Phytates are often reduced in lactic acid fermented products mainly due to enzymes

(phytases) present in the plant material. The plant-derived phytases become activated by

the decreasing pH. However, also yeast growing together with the lactobacilli produces

phytases, and hence can brake-down phytates.

In contrast, tannins can be digested by some LAB. L. plantarum and the genomically

related species, L. paraplantarum and L. pentosus are able to brake-up tannins, and they

can also digest flavonoids and phenolic acids. Pediococcus normally lack tannases but

can break-up flavonoids.

Figure 13. Flow scheme of traditional production of green olives in brine.


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Nigerian ogi

Ogi is a traditional lactic acid fermented cereal-based product from Nigeria. It is used as

a weaning food to children (gruel), but also widely consumed by adults as porridge, for

example, at breakfast, or as a cooked stiff gel (agidi) eaten together with stews, soups or

fried bean-cakes (Figure 14).

Ogi can be made from maize, sorghum or millet, but most popular is that made from

maize (Zea mays) and sorghum (Sorghum bicolor or Sorghum dabar). Maize is

frequently used for the ogi-gel (agidi) while the red sorghum (Sorghum bicolor) often is

preferred in weaning food. Red sorghum generally gives gruel of lower viscosity than

maize, which is advantageous when the consumers are young children, as the bulking

effect of maize will reduce the intake.

Figure 14. Flow scheme of the traditional wet-milling procedure for ogi production.

Ogi is traditionally produced by a work-intensive procedure. The cereal grains are

cleaned and steeped in water for 1-3 days, where the first spontaneous fermentation

occurs. After the water is poured off, the grains are wet-milled and wet-sieved through a

cloth or a fine wire-mesh. The pomace, mostly consisting of hulls, is discarded and can

be used as animal feed. The remaining flour suspension is left for sedimentation for 1-3

days (Figure 14). During this step, spontaneous lactic acid fermentation occurs. When

the ogi is sour enough, the supernatant is decanted and the flour cake is stirred with


61

boiling water or with the decanted supernatant to form gruel or porridge. The ogi can

also be cooked in water into a thick gel (agidi) that traditionally is put in leaf packages.

Paradoxically in view of the fact that red sorghum is preferred in weaning food, the

content of tannins is high in red sorghum, and tannins can react with proteins which

make them indigestible in the gut. However, the protein digestibility of high-tannin

sorghum is significantly improved by the lactic acid fermentation. Interestingly, L.

plantarum are able to degrade tannins and L. plantarum are frequently a dominate part

of the bacterial flora in ogi. Furthermore, it has been shown that L. plantarum can be

used as a single-strain starter for producing high quality ogi. Also Weissella confusa

seems frequently be present in high numbers in spontaneously ferment ogi.

Tanzanian togwa

Togwa is a cereal-based, lactic acid fermented beverage, consumed in Tanzania as

refreshment, and given to infants as a weaning food. Togwa is mostly made from flour

of maize (Zea mays), sorghum (Sorghum bicolour) or finger millet (Eleusine coracana).

Rice (Oryza sativa) and cassava (Manihot esculenta) flour or mixtures of cassava and

cereals are also used in some areas. Sorghum-based togwa seems to be preferred by

many consumers. The different steps in the preparation of togwa are summarized in

Figure 15 (Kingamkono et al. 1999).

Figure 15. Flow-scheme of the production of Tanzanian togwa.

The flour for making togwa is mixed with water (1 part flour and 9 parts of water) and

the gruel is cooked for 10 to 20 minutes, and then cooled down to about 35oC. At his


62

point 10% (v/v) old togwa is mixed into the gruel (back slopping), together with 5%

(w/v) flour made of malted grains.

Malt flour is made from sorghum or millet. The malt-flour is prepared by soaking the

grains for 12 h, then the grains are drain-dried, spread out and on, for example, green

leaves, trays or jute mats. The grains are covered with wet cloths and allowed to

germinate at about 30oC for 3 to 6 days. After sun-drying and milling of the germinated

grains, the malt flour is ready for use.

Heat treated gruel, malt flour and old togwa (used as a starter culture) are mixed, and

left for 9 h to 24 h for lactic acid fermentation. Togwa has a pH of 3.2 to 4.0, and often

contains high levels of L. plantarum. It has been shown that togwa made by using L.

plantarum as single-strain starter culture, equals spontaneously fermented togwa in

eating quality (Kingamkono et al. 1999).

In spite of the fact that togwa often is produced under poor hygienic conditions, where

inferior water quality and the malting procedures expose the product to considerable

hygienic obstacles, togwa is not known for causing food-borne outbreaks of disease

(Kingamkono et al. 1999). On the contrary, togwa has been shown to lower the

incidence of enteropathogenes in the faeces of children, and it has been shown to

improve the condition of the intestinal mucosa in children with acute diarrhoea

(Kingamkono et al. 1999).

Sour-milk

Yoghurt. Yoghurt is according to Food and Drug Administration (FDA) in United

States of America ”a coagulated milk obtained by lactic acid fermentation, due to

Lactobacillus bulgaricus (L. delbrueckii subspecies bulgaricus) and Streptococcus

thermophilus”. The different production-steps for yoghurt are shown in Figure 16.

Figure 16. Flow-scheme for the production of yoghurt.


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The high initial heating temperature of the milk will kill-off contaminating bacteria, but

will also help to improve the consistency of the final product (make it thicker).

It should be noticed that the fermentation temperature of yoghurt is relatively high (40-

45oC). A consequence of the high fermentation temperature is that the production time

can be kept short. This is an advantage from an industrial perspective. It also follows

from the high fermentation temperature that the starter culture must be able to grow at

such a high temperature.

The high temperature will have effects on the flavour development in the product.

Examples of aroma compounds formed in yoghurt during fermentation are acetaldehyde

(2-40 parts per million, ppm), acetone (1-4 ppm), acetoin (2-4 ppm) and diacetyl (up to

10 ppm).

Filmjölk. The Swedish sour-milk “filmjölk” is produced in similar manner as yoghurt

(Figure 16). The major differences are (i) the lower fermentation temperature (19-22oC)

and thereby the longer fermentation time (18-20 h), and (ii) the use of another type of

starter culture, i.e. a mixed starter culture of Lactococcus lactis and Leuconostoc. The

typical starter culture for filmjölk is not only a mixture of the species Lactococcus lactis

subspecies lactis, Lactococcus lactis subspecies cremoris, Leuconostoc mesenteroides

subspspecies cremoris and Leuconostoc lactis, but also a complex mixture of different,

undefined strains of each species. The same type of starter culture is mostly used in the

production of Swedish semi-hard cheese.

The sugars in the milk (lactose [4.8%], glucose and galactose) are fermented to lactic

acid by mainly Lactococcus lactis. Milk also contains citrate (1.8 g per litre) and the

citrate is catabolised by the Leuconostoc strains and some specific strains of

Lactococcus lactis (previously designated Lactococcus diacetolactis) to acetic acid,

carbon dioxide and diacetyl. Diacetyl is then partly converted to acetoin. The diacetyl

provides Filmjölk with much of its characteristic taste, a taste distinctly different from

yoghurt.

Kefir. Kefir is a fermented milk beverage that has been fermented by a mixture of

yeast’s and lactic acid bacteria. These organisms are established in the so called “kefir

grain” which is a structure that spontaneously is formed in the fermenting milk. The

kefir grain consists of polysaccharides, proteins, yeast cells and bacteria. Lactobacillus

kefir and Lactobacillus kefiranofaciens are examples of organisms producing the

polysaccharides of the kefir grain. Lactic acid, ethanol and carbon dioxide (together

with smaller amounts of diacetyl and acetoin) are produced during the fermentation.

Antimicrobial properties of milk. Milk contains natural components with

antimicrobial effects. For example, antibodies directed against bacteria or bacterial

components, a low iron content combined with an efficient complex-binder of iron

(lactoferrin) and the enzyme lactoperoxidase (Figure 17).

There is about 30 mg lactoperoxidase per ml of milk. When this enzyme comes in

contact with hydrogen peroxide, the hydrogen peroxide is reduced with the help of

electrons donated from thiocyanate (natural component in milk; up to 5 ppm) (Figure

17). The intermediary, instable, highly oxidative products of thiocyanate (HOSCN) are

strongly bacteriocidic (kills bacteria). The thiocyanates are especially active against

gram-negative bacteria, such as Proteobacteria. Macrophages generate hydrogen


64

peroxide to kill bacteria and the presence of lactoperoxidase enhances the killing

efficiency. Hydrogen peroxide can also be produced by many lactic acid bacteria.

Figure 17. The lactoperoxidase reaction in milk. HOSCN is strongly bacteriocidic.

Cheese

More than 50 different cheese varieties were made in Sweden in the year of 2001. The

oldest and most popular varieties are Herrgårdsost, Prästost, Västerbotten, Svecia and

Hushållsost. Examples of internationally well-known, semi-hard cheeses are Cheddar

(British), Emmentaler (Swish) and Jarlsberg (Norwegian). Cheese consumption is

increasing internationally, mainly due to the increased use of cheese as a food

ingredient.

The manufacture of cheese comprises a chain of operations, which concentrates and

preserves the milk by fermentation, dehydration and salting. The principal steps in the

manufacture of Swedish semi-hard cheese are shown in Figure 18. However, it should

be emphasised that each cheese variety has its own traditional specific manufacturing

protocol.

The raw milk for cheese manufacturing is separated and its microflora is reduced by

pasteurisation (70 oC to 73

oC for 15s to 20 s). Bactufugation or microfiltration can also

be used to remove bacterial spores. The reduction of the contaminating microorganisms

is crucial, as the risk of potential pathogens and product spoilers in the final cheese must

be kept to a minimum. It is also important that large-scale, reproducible, cheese

production takes place under strictly controlled conditions in order to maintain a

controlled quality. Common quality-reducing bacteria are Clostridium tyrobutyricum,

which cause excessive gas formation and also cause off-flavour by production of butyric

acid. C. tyrobutyricum can form endospores.

The milk is fermented by a starter culture. The starter cultures in Swedish, semi-hard

cheese, are usually undefined, i.e. the included strains are not defined. This type of

culture is called “mesophilic LD culture” with mixed species. LD stands for L lactic

acid and D lactic acid. Both forms of lactic acids are produced by the starter culture.


65

The acid producers in the culture are mainly Lactococcus lactis subspecies lactis and

Lactococcus lactis subspecies cremoris, which catabolise lactose to lactic acid. The

flavour producers are primarily Lactococcus lactis subsp. lactis biovariant diacetylactis

and Leuconostoc mesenteroides subsp. cremoris, which metabolise citrate to carbon

dioxide, diacetyl, acetoin and 2,3 butanediol. The starter culture usually contains an

unknown number of different strains of each species. This is regarded as a protective

action against the treat of phage infections. Bacteriphages can kill the bacteria of the

starter culture but bacteriphages are strain specific so a mixture of strains certify the

function of the starter even if a phage-infection should take place.

Figure 18. Flow scheme of the main steps in the manufacturing of semi-hard cheese. The “cooking” is usually between 38oC and 40oC during 20 to 40 min. The whole procedure to the step of salt brining takes 1 to 1.5 day. The ripening usually proceeds at about 12oC for 3 to 12 months.

The rate at which lactic acid is produced is one of the factors controlling the moisture

content of the cheese and it is regulated by the starter culture. The final pH of the cheese

is controlled by water addition, i.e. by indirectly controlling the amount of lactose in the

cheese. The type of starter and the “cooking” temperature also affect pH. Mesophilic

starter cultures have optimal growth temperatures around 26oC and do not multiply at

temperatures above 38-40oC. The cheese should be depleted of lactose and citrate after

about one week after the initiation of fermentation.

Coagulation is accomplished by the addition of calf rennet (“löpe” in Swedish), which

mainly consists of the enzyme chymosin. This enzyme cleaves the -casein that is

responsible for the micelle stability in milk and this in combination with added CaCl2,

coagulates milk. When the coagulation is complete the gel is cut into cubes and

converted into curd grains through the combined action of stirring, heating and

decreasing pH. Moisture control is of major importance for the final characteristics of


66

the cheese. The curd grains are collected in moulds, pressed to shape and then brine

salted (Figure 18).

The above described sequential unit operations set the conditions for the subsequent

maturation during which the sensory characteristics of the cheese develop. Swedish

semi-hard cheese are consumed as “mild” (“mild” same word as in Swedish) after

approximately 3 months of ripening and as “very mature” (“stark” in Swedish) after

about 12 months of ripening.

During the process, the starter culture reaches about 109 CFU per g after 1-2 days, but

then during ripening, gradually decline in numbers. Instead there is a spontaneous

growth of contaminating bacteria in the cheese during the ripening period. These

bacteria are called “non-starter lactic acid bacteria” and consist in Scandinavian semi-

hard cheeses (made with pasteurised milk) mainly of Lactobacillus paracasei. In

Mediterranean cheeses also Lactobacillus plantarum is occurring frequently, especially

in cheeses made with unpasteurised milk. Non-starter lactic acid bacteria reach a

number of about 106 to 10

8 CFU per g cheese after about 5 months. This spontaneous

growing Lactobacillus affect the flavour development during ripening, sometimes in a

positive direction, but sometimes in a negative way, all depending on the characteristics

of the dominating strain. Both L. paracasei and L. plantarum are frequently found in the

human digestive tract.

Lactic acid fermented sausages

There is different kind of lactic acid fermented sausages with different processing times

(Table 3). It is the drying of sausages that takes time, and the time is reflecting the

dryness of the product.

There are two major categories of lactic acid fermented sausages: (i) “semidry”

sausages with a water content of 40-60% and a water activity of 0.93-0.98, and (ii)

“dry” sausages with a water activity of 20-40% and a water activity of 0.80-0.90. The

water activity, aW = p/p0, where, p = steam pressure over the product, and, p0 = steam

pressure over pure water. The water activity over pure water is 1.0, and the relative

humidity (RH) in per cent, in the gas phase, is the same as aW x 100.

Table 3. Different types of high-quality lactic acid fermented sausages.


67

Examples on lowest aw -limit of growth for some different groups of bacteria are: 0.97

for Pseudomonas; 0.95 for Enterobacteriaceae; 0.93 for Lactobacillus; 0.85 for

Staphylococcus.

For bacteria in general, the lowest water activity of growth in food is mostly 0.93 (but

the extremes can grow down to a pH of 0.85), while the corresponding lowest water

activity for yeast is 0.85 (extremes, down to 0.80), and for moulds 0.75 (extremes down

to 0.60).

Lactic acid fermentation occurs spontaneously in the beginning of cold smoking or at

the start of a drying process. Gradually the pH of the product decreases down to

somewhere between 4.2 and 4.7 (Figure 19).

Figure 19. Flow diagram of the main steps in the manufacture of lactic acid fermented sausage (in Swedish “kallrökt korv”).

The bacterial load of the raw material after mixing is often between 105 CFU and 10

8

CFU per g meat mixture. The starter culture usually gives the mixture a concentration of

106 CFU starter bacteria per g meat mixture (Figure 19).

Examples of taxa used as starter cultures for lactic acid fermented sausages are:

Pediococcus pentosaceus, especially at higher fermentation temperatures (40-45oC), and

L. plantarum or Lactobacillus sake that are used mostly at cold smoking at temperatures

around 30oC and below.

A strain of the genus Staphylococcus is sometimes used for improving the colour of the

cold smoked products. The rationale behind this is that Staphylococcus produces

catalase, and catalase catalyses the break-down of hydrogen peroxide that can be


68

produced by Lactobacillus growing in the product. Hydrogen peroxide can react with

the meat pigment yielding green-greyish spots in the product.

Lactobacillus often used as probiotics

Probiotic dose

Humans ingest bacteria together with food, both unintentionally and on purpose. The

unintentionally eaten bacteria could have adverse effects on the health if the food is

contaminated with pathogenic bacteria, or if the food is contaminated with spoilage

bacteria that perhaps not are directly pathogenic but can exercise negative health effects

when eaten in high numbers (food close to spoilage). However, also beneficial bacteria,

“dietary bacteria” can be eaten unintentionally when fermented foods are consumed.

Especially lactic acid fermented foods can contain high amounts of live bacteria and

bacteria of the same species that are used for probiotics, i.e. intentionally eaten bacteria.

But, the amount of live bacteria that can be ingested by traditional fermented products

depends much on the type of product, e.g. a traditional yoghurt may contain up to 108

to109 CFU per ml and a consumption of 100 ml will give a final dose of 10

10 to10

11

CFU of LAB while cheese may contain around 107 CFU per g and a consumption of 20

g will in this case yield a dose of 2x108 CFU. However, it should be pointed out that

many commercially available, traditionally fermented products today are heat-treated

after the fermentation in order to prevent uncontrolled fermentation during storage, i.e.

no living LAB are left in the product.

Generally, a daily dose of probiotics ought to be around 109 CFU (preferably 10

10 CFU)

to show effects. There is no upper limit in numbers. The higher the daily intake is the

better are the chances to obtain desired effects. It can be pointed out that the human

digestive tract contains at least 1014

CFU, and a dose of 109 CFU only makes up 0.001%

of the total number of bacteria in the human gut.

The commercially most frequently used food-carrier for probiotics is yoghurt or

yoghurt-like products. Some examples of these milk-based products are: Actimel

(Danone), Yakult (Yakult), Lc1 (Nestlé), Gefilac (Valio), Cultura (Arla) and Verum

hälsoyoghurt (Norrmejerier). These types of milk-based products usually contain 105-

108 CFU of the probiotic Lactobacillus-strain per ml product. The numbers is to a high

degree dependant on the hardiness of the particular strain.

Bifidobacterium are generally more difficult to keep alive in products, and the viable

count is low in many products. It seems as Bifidobacterium animalis is a hardier species

than the most other Bifidobacterium spp., and unusually tough against exposure to

oxygen (for being a bifidobacteria). Especially hardy against storage is the strain

Bifidobacterium animalis BB12.

Examples of Lactobacillus spp. frequently used as probiotics are Lactobacillus casei,

Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus acidophilus,


69

Lactobacillus johnsonii, Lactobacillus salivarius, L. fermentum, Lactobacillus reuteri

and Lactobacillus plantarum. Phylogenetically, L. casei, L. paracasei and L. rhamnosus

are related, and L. acidophilus is related to L. johnsonii.

L. salivarius, L. fermentum, L. reuteri and L. plantarum are neither especially closely

related to the other mentioned Lactobacillus spp. nor to each other. They represent

different phylogenetic subgroups within the genus Lactobacillus. Most of these

Lactobacillus species are also frequently found in the human digestive tract (Figure 20).

Figure 20. Lactobacillus species frequently occurring in the human gut and in some cases, also frequently found in traditional lactic acid fermented products.

Lactobacillus casei and Lactobacillus paracasei

The species Lactobacillus casei (casei, of cheese) was described and given its name

1916 by a Danish bacteriologist, Orla-Jensen. However, the original type strain was lost

and a new type strain (neotype strain) was selected by Hansen and Lessel (1971).

Unfortunately, the neotype of L. casei had relatively low similarity to most L. casei-like

strains isolated from, for example, cheese. Because of this, a new species named L.

paracasei was described based on atypical L. casei strains found in foods (Collins et al.

1989). But some scientist objected to the description of L. paracasei and argued for

keeping the original classification of L. casei (with several subspecies), and they

assigned a new neotype strain for L. casei that was more similar to environmental

isolates than the neotype strain of Hansen and Lessel from 1971. This proposal was later

turned down by the Judicial Commission of the International Committee on Systematics

of Bacteria, deciding that L. paracasei should remain (Tindall 2008). The consequence

of this has been that the majority of strains, before labelled L. casei, today should be


70

named L. paracasei. True L. casei strains seem to be relatively rare in the environment,

at least in foods and in the gastro-intestinal tract.

L. paracasei grow spontaneous during lactic acid fermentation of dairy products, and

mostly constitute the dominating flora in cheese, especially in semi hard cheese made

from pasteurised milk (106 – 10

8, CFU per g cheese). L. paracasei are also frequently

found in the human digestive tract. L. paracasei is a facultatively heterofermentative

lactobacilli.

The Yakult culture (Yakult, Japan), “Lactobacillus casei Shirota” was isolated 1930 by

the Japanese scientist Minoru Shirota. This strain should be designated Lactobacillus

paracasei. Also the strain “L. casei defencis” (= DN-114001; also called “immunitas”

in the past) that is included in the yoghurt-like product Actimel (Danone, France)

should be designated Lactobacillus paracasei. A third example on a L. paracasei strain

commercially used as probiotics is Lactobacillus paracasei F19 that mostly is included

in different yoghurt-like products (Arla, Denmark).

Lactobacillus rhamnosus

Lactobacillus rhamnosus was originally classified and named L. casei subspecies

rhamnosus (Hansen 1968), but later reclassified and named L. rhamnosus (Collins et al.

1989). L. rhamnosus has been used as probiotics since the 1970s. Especially one strain,

L. rhamnosus GG (Valio, Finland) has been used a lot, all over the word. GG stands for

Gorbach and Goldin which were the two scientists that originally isolated and patented

the strain. In the beginning, this strain was wrongly identified as L. acidophilus. L.

rhamnosus GG differ from the majorities of L. rhamnosus strains by lacking the ability

to ferment lactose.

L. rhamnosus is facultatively heterofermentative, and grows spontaneously and quickly

during lactic acid fermentation of milk. L. rhamnosus are frequently found in the gastro-

intestinal tract of humans, and especially in breast fed babies. Some strains have now

and then been accused for causing infections, but the evidences for this are weak.

Lactobacillus acidophilus

Lactobacillus acidophilus was described by Moro (1900) and has been used in lactic

acid fermented milk as probiotics since the 1930s. L. acidophilus has in the past often

been mixed up with other obligatory homofermentative Lactobacillus species as

Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum,

Lactobacillus gasseri, Lactobacillus jensenii or Lactobacillus johnsonii (Fujisawa et al.

1992).

L. acidophilus grows poorly in milk if not additional growth factors are added, e.g. in

the form of yeast extract. L. acidophilus is not normally found in spontaneously

fermented food products, but is frequently found in the gastro-intestinal tract of man

and animals.


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Lactobacillus johnsonii

Lactobacillus johnsonii was described by Fujisawa et al. (1992). L. johnsonii produces

hydrogen peroxide (H2O2) when exposed to oxygen, and is often a dominating part of

the resident microbiota in vagina, but can also be found in the digestive tract of humans

and animals. L. johnsonii is not usually found in spontaneously fermented food

products.

The strain L. johnsonii La1 (= NCC533; Nestlé) that has been used in the probiotic

product LC1, was originally identified as L. acidophilus.

Lactobacillus salivarius

L. salivarius was described by Rogosa (1953). It is traditionally regarded as obligatory

homofermentative. However, the species contains both homofermentative and

facultatively heterofermentative strains. Most strains produce L-lactic acid from

available hexoses by homofermentation. Heterofermentative end-products (lactic acid,

acetic acid and ethanol) are produced from hexoses by some strains that also can

ferment ribose. L. salivarius is frequently isolated from the mammalian digestive tract

A relatively well characterized strain with probiotic impact is Lactobacillus salivarius

UCC118 (University College Cork, Ireland). This strain is facultatively

heterofermentative.

Lactobacillus fermentum

Lactobacillus fermentum was described by Beijerink (1901). L. fermentum is an

obligatory heterofermentative Lactobacillus. L. fermentum has, compared to many other

Lactobacillus spp., an unusual high mol % guanine+cytosine of the DNA (52-54%),

which clearly points out that L. fermentum is phylogenetically very different from most

other Lactobacillus spp. On the other hand, the type species of the genus Lactobacillus,

Lactobacillus delbrueckii also has a relatively high mol % guanine + cytosine of the

DNA, i.e. 49-51%.

L. fermentum is frequently found in high numbers in spontaneously fermented foods,

and in the digestive tract of humans and animals.

Examples of commercially available strains of L. fermentum with probiotic claims are

L. fermentum PCC (Probiomics Ltd, Australia), and L. fermentum ME-3 (University of

Turku, Estonia).

Lactobacillus reuteri

Lactobacillus reuteri is an obligatory heterofermentative Lactobacillus that was

described by Kandler et al. (1980; Anon 1982). Many Lactobacillus reuteri strains

produce the anti-microbial substance, reuterin (b-hydroxy-propionaldehyde) which is an

inter-mediate in the glycerol dissimilation (Axelsson et al. 1989).


72

L. reuteri does not normally grow spontaneously in lactic acid fermented foods, and has

poor storage stability, presumably due to low oxygen tolerance. L. reuteri is frequently

found in the digestive tract of humans and animals.

Different strains of L. reuteri are marketed as probiotics (BioGaia AB, Sweden); one is

L. reuteri ATCC 55730. The company also market strains named “Protectis” and

“Prodentis”, but it is unclear if these are different names for ATCC 55730.

Lactobacillus plantarum

Lactobacillus plantarum (plantarum means “of plants”) was described by Orla-Jensen

(1919) and Bergey et al. (1923). L. plantarum frequently occurs spontaneously, in high

numbers, in most lactic acid fermented foods, especially when the foods are based on

plant material, for example, in brined olives (Fernández Gonzalez et al.1993), capers

(caper berries; Pulido et al. 2005), sauerkraut (Dedicatoria et al.1981; Plengvidhya et al.

2007), salted gherkins (McDonald et al. 1993), sour-dough (Lönner and Ahrné 1995),

Nigerian ogi (made from maize or sorghum) (Johansson 1995a), Ethiopian kocho (made

from starch from Ensete ventricosum) (Gashe 1985; Nigatu 1998), Ethiopian sour-

dough made out of tef (Eragrostis tef) (Gashe 1987; Nigatu 1998) and cassava

(Oyewole and Odunfa 1990; Moorthy and Mathew 1998). Furthermore, L. plantarum

occurs in grape juice and wine (Vaquero et al. 2004). Thus, it is obvious that individuals

consuming lactic acid fermented products of plant origin also consume large amounts of

L. plantarum. And in view of this, it is not surprising that L. plantarum frequently

occurs on the human mucosa of the digestive tract, from the mouth to the rectum (Molin

et al. 1993; Ahrné et al. 1998).

L. plantarum have a high tolerance to low pH (Daeschel and Nes 1995). The fact that L.

plantarum frequently predominate in spontaneously, lactic acid fermented foods where

the pH usually is below 4.0, and also survive the passage through the acid conditions of

the human stomach (Johansson et al. 1993), points to its high resistance to acid

conditions. L. plantarum can ferment many different carbohydrates and has a relatively

large genome, indicating that L. plantarum has abilities to adapt in many different

environments (Kleerebezem et al. 2003).

L. plantarum has a growth requirement for manganese and can accumulate high

intercellular levels of manganese (Archibald and Fridovich 1981b). Manganese provides

a defence for L. plantarum against oxygen toxicity by the reduction of oxygen radicals

to hydrogen peroxide (Archibald and Fridovich 1981a). The produced H2O2 can then be

converted to O2 and water by manganese cofactored pseudocatalase (Kono and

Fridovich 1983a, 1983b).

L. plantarum and the genomically related species, Lactobacillus paraplantarum and

Lactobacillus pentosus are able to brake-up tannins (Osawa et al. 2000; Vaquero et al.

2004), and can metabolise flavonoids and phenolic acids (Barthelmebs et al. 2000;

Barthelmebs et al. 2001).This is a relatively rare ability among LAB. However, also

Pediococcus can metabolise flavonoids, even if Pediococcus seems to lack the ability to

brake-up tannins.

A well known, and commercially available probiotic strain in many countries is L.

plantarum 299v (=DSM 9843) that originally has been isolated from human intestinal


73

mucosa (Probi AB, Lund, Sweden). A probiotic strain that scientifically also is well

characterized is L. plantarum WCFS1 (= NCIMB 8826).

Phenolics

Plants synthesise a complex variety of phenolics. There are polyphenoles as tannins,

lignans and lignins, and polyphenols only containing a two-phenolic ring structures as

flavonoids and stilbenes. There are also phenolic acids, for example gallic acid and

ellagic acids. In the plant, the phenolics have many physiological functions but they can

also protect the plant from parasites. Often they are difficult to digest, both for animals

and microbes, and they can possess powerful antimicrobial activities. Polyphenols are

well-known for their anti-nutritional effects in obstructing the absorption of iron and

reducing the availability of aminoacids which in nutritional terms must be seen as

negative, however, polyphenols may also slow down the up-take of sugars and lipids

which in situations of overeating and gormandizing must be regarded as advantageous.

A potential health beneficial ability of phenolics is their anti-oxidative properties, i.e.

the scavenging of electrons from oxygen free radicals. Flavanoids are often powerful

antioxidants, and especially powerful are the anthocyanines, present in for example

darkly coloured fruits, for example blueberry, blackcurrant, blackberry and red grapes.

L. plantarum and closely related species as L. pentosus and L. paraplantarum can split

up tannins into flavanoids, and flavanoids into phenolic acids. Pediococcus spp. don’t

normally posses tannase and are then unable to split up tannins into flavanoids, but

some pediococci can convert flavanoids into phenolic acids.

In their native form, many polyphenols can’t be absorbed in the small intestine and

instead they go to colon where they can be converted by the intestinal bacterial flora to

compounds that more easily are absorbed. An example of such a group of polyphenols

which are hard to digest is the anthocyanins. These compounds are especially difficult

to brake-up and are not absorbed in the gastro-intestinal tract in their original form. But

if they have been converted by the bacterial flora to compounds as phenyl valeric acids,

phenyl propionic acids, phenyl acetic acids and benzoic acid derivates, they are

absorbed and are transported to the liver (Scalbert et al. 2002; Figure 21). Phenolics

reaching the liver and other organs can have: (i) antioxidative capacity per se, but they

can also mediate (ii) metabolic effects of anti-inflammatory consequences. Phenolic

metabolites that are released from the liver are further transported via the systemic

circulation through the body and finally to the kidneys where they are discharged in the

urine.

Many polyphenols can be regarded as dietary fibres. They are indigestible and reach the

colon where they can be split by colonic bacteria (Figure 21). Polyphenols can even

have prebiotic effects as they can possess antimicrobial effects that affect different

bacterial groups differently. Especially some phenolic metabolites as, for example,

benzoic acid derivates have powerful antimicrobial effects.


74

Epidemiological studies points at strong correlations between high consumption of

fruits and vegetables and decreased risk for cardiovascular diseases and cancer.

Furthermore, animal models prove causal connections between the consumption of

fruits and diseases as type 2 diabetes and cancer. An interesting question is if the

protective capacity of fruits and vegetables are dependent on the composition of the

bacterial gut-flora, i.e. if the presence of certain, polyphenole-converting taxa in the gut

enhance the disease-preventive effects of fruits and vegetables? This question remains

to be answered.

Figure 21. Polyphenols digested in colon and phenolic metabolites transported through the portal blood to the liver.

ProViva: a probiotic food product

A commercially successful probiotic food product with the brand name ProViva was

developed and launched in Sweden 1992. ProViva was from the beginning and until

2011 produced and marketed by Skånemejerier (Malmö). Today ProViva is marketed

and produced by ProViva AB (Lunnarp, Sweden), with Danone as the majority

shareholder. In USA, products of the same concept are marketing by NextFoods under

the brand name GoodBelly. However, the holder of the rights to the probiotic strain, L.

plantarum 299v included in these products, and the ones granting the quality of the

active probiotic strain (L.plantarum 299v) which is used as starter culture in ProViva is

the public company Probi AB (Lund, Sweden).

ProViva is based on a relatively unorthodox design-concept, and probably is this design

one reason for the commercial success. The base in ProViva is a lactic acid fermented


75

oatmeal beverage (gruel) that is fermented with L. plantarum 299v (Figure 22), and this

gruel is then mixed into different fruit drinks. During the lactic acid fermentation L.

plantarum 299v multiplies in the oatmeal gruel and reaches about 109 CFU of per ml

gruel. The lactic acid fermented oatmeal gruel is an integral part of ProViva, where 5%

fermented oatmeal gruel is mixed with different types of fruit drinks, e.g. rose hip,

strawberry, blueberry and blackcurrant. In the final product (ProViva), there is about 5 x

107 CFU of L. plantarum 299v per ml of fruit drink. The product is refrigerated.

Figure 22. Flow scheme from 1992 of the production of lactic acid fermented oatmeal gruel to be used in ProViva.

The viscosity of the oatmeal gruel in Figure 22 is during the process lowered by a

supplement of malt flour (malted barley). The supplement of malt in combination with

the heat treatment and the gradual decrease in pH during the lactic acid fermentation

results in a decreased viscosity. The fermented oatmeal beverage was originally

intended as a base for a nutritional formula for enteral feeding (nasogastric feeding),

where a low viscosity and high energy liquid are prerequisites. Without added malt

flour, the oat meal gruel with the stated concentration of flour (18.5%; w/w) will form a

thick porridge impossible to administer through a thin tube. The decrease in viscosity is

presumably in large part due to degradation of starch. Malt is rich in amylases. There is

also an increased solubility of β-glucans, and if higher amounts of malt are used, or

extra malt flour is added after the heat treatment, there is also a substantial reduction in

total amount of β-glucans. However, the β-glucans are considered valuable as they are

believed to delay intestinal absorption and beneficially affect cholesterol and glucose

metabolism. The process as it is seen in Figure 22 does only cause a relatively small, if

any, reduction of the total content of β-glucans even if the viscosity is significantly

affected.


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To conclude

So far relatively few species of mostly the two genera Lactobacillus and

Bifidobacterium have been used commercially as probiotics. A few strains of L.

paracasei, L. plantarum, L. reuteri and B. animalis have been used successfully in

different foods and food supplements in an international scale. Considering the vast

amount of untried species and strains there seems to be a huge unexploited potential in

finding new probiotic strains.

In a safety perspective, it can be wise to keep the selection of potential probiotic strains

from such species that have been eaten by humans for long time and in high numbers,

i.e. the species dominating the flora of dietary bacteria in traditionally fermented foods.

These dietary bacteria are dominated by lactic acid bacteria (LAB). It is important to

consider the fact that among the LAB there are many different taxonomic unities

(genera, species and strains) with highly different characteristics, and that the human

body respond differently on these different characteristics. The human immune system

recognises the differences.

By comparing the different examples of traditional lactic acid fermented foods given in

this lecture-block certain similarities can be perceived (i) in how to control of the

fermentation and (ii) in the spontaneous succession of different sorts of LAB during the

process. It becomes obvious that certain LAB species occurs irrespectively of the

geographical location of the fermentation. An important controlling factor for the

selection of what sorts of LAB that will occur in high numbers in the fermentation is the

type of raw material that are fermented, cereals and vegetables give rise to a certain

profile of LAB, while milk give another, and meat a third. Also the fermentation

temperature may influence the LAB-profile in the product. Important factors for the

actual selection of LAB in the fermentation, is the restricted access to air and the falling

pH along with the fermentation. The restriction to air means primarily a shortage of

oxygen, but equally important is the increased concentrations of carbon dioxide that are

produced by the bacteria and gradually enriched in an enclosed environment. Also

frequently used for controlling the fermentation is salt (NaCl).

LAB that are found in high numbers in fermented products are by obvious reasons

thriving fairly well and have good survival in the product, i.e. these taxa can favourably

be combined with these food products. Using a probiotic strain in a food environment

that it is suited for also means that the probiotic bacterium can be added to the food as

starter culture and then by its own ability multiply to high numbers. The commercial

advantage with this is that it is cheaper than adding freeze dried bacteria to the product

in its final concentration. A functional advantage in letting the probiotics multiply in the

carrier (food) is that the probiotics presumably are more metabolic active when eaten. A

draw-back can be that the actively growing probiotics in a negative way may change the

eating quality of the food, for example, by producing carbon dioxide and acetic acid as

heterofermentative LAB tend to do. On the other hand, the probiotics may by

interaction with food components increase the health benefits and nutritional value of

the food product. From the point of view of efficiency, a high number of active

probiotics in the product is desirable. In other words, there are several aspects to take

into account when probiotics are mixed into foods.


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The Swedish product ProViva can be taken as example of a modern probiotic food

concept that basically is designed in a similar way as a traditional lactic acid fermented

food product, i.e. the fermented oatmeal gruel of ProViva has much in common with the

Tanzanian togwa. Furthermore, the probiotic strain of ProViva belongs to the species L.

plantarum which frequently is dominating in traditional fermented foods based on

cereals and other raw materials of plant origin. In other words, the probiotic strain can

be used as starter culture in the product and by its own account multiply up to high

numbers in the product.

An interesting and relatively new field in Nutrition is the physiological effects of

phenolics in, for example, fruits and tea. Polyphenols are to high degree passing

undigested through the small intestine but can be digested by some bacterial taxa in

ileum and colon and the phenolic metabolites can then be absorbed. However, it is a

limited number of taxa in the gut-microbiota that are able to digest polyphenols and if

those taxa should be absent, the host would not be able to absorb much phenolic

metabolites and will of course neither be able to make full use of the antioxidative and

anti-inflammatory properties of the phenolics. In such individuals, it would be

especially interesting to combine intake of polyphenol-rich food with probiotics able to

digest polyphenols. However, only a few LAB have this ability but L. plantarum is one

of them. L. plantarum 299v in ProViva has a certain ability to handle the content of

phenolic compounds in fruits. This can improve the survival of the bacterium in a fruit

drink, but the ability to digest polyphenols can also lead to absorption of phenolic

metabolites with physiological capacities in the digestive tract of the consumer.

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III. Gut microbiota The bacterial flora of the gut


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Intention

The educational intention with Lecture-block III – Gut microbiota – is to provide an

overview of the complex community of bacteria living in the digestive tract of humans

and how the composition of this microbiota (bacterial flora) is affected by different

factors. The intention is also to point at the two major targets (or hot spots) for the

interaction of the different components of the microbiota with the body, i.e. the mucosal

barrier function and the immune system. Crucial scientific questions are to sort out

which components of the microbiota that have physiological effects (beneficial for the

well-being) for the host and which ones that have physiopathological (adverse) effects,

and the mechanisms behind these different interactions. For the time being this is to a

high degree a huge white area but some conclusions can nevertheless be drawn,

especially when it comes to the obviously harmful taxa. This is reflected in learning-

block III.

A prerequisite for a relevant discussion around the role of the diverse microbiota in the

human body is access to a sound bacterial taxonomy where the natural taxa of the

microbiota have been made clear and can be used as keys in the search for a deeper

understanding of the role of the microbiota in health and disease.

Physiological and physiopatholgical

effects

Humans ingest bacteria with the food, but humans also harbour huge amounts of

resident bacteria (domestic bacteria) on the mucous membranes, from the moth to the

rectum, and also in the lumen of the digestive tract. Additionally, females harbour dense

populations of bacteria in the vagina. Both ingested bacteria and resident bacteria can,

depending of sort, have different effects on the human body. The bacteria can have (i)

physiological effects, i.e. effects that are consistent for normal functioning of the body,

or they can have (ii) physiopathological effects, i.e. adverse effects that impose

functional changes associated with injury and disease. In other words, some resident

bacteria of the digestive tract and the vagina may aggravate injuries or dysfunctions

while others may work the other way around and suppress or even have a healing effect

on injuries and dysfunctions (Figure 23).

The original idea of probiotics is that by ingesting wholesome bacteria (probiotics) the

beneficial bacteria of the GI-tract can be promoted, and the balance between beneficial

bacteria (suppressors) and adverse ones (aggravators) can be altered in an advantageous

direction for the human health (Figure 23). However, probiotics can do more than

affecting the balance of the microbiota. Thus, besides affecting (i) the composition of

the resident microbiota on the mucosa and in lumen, probiotics can affect (ii) the

epithelial cells by direct cell-to-cell contact, (iii) the immune system by contact with


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immune-competent cells (macrophages, dendritic cells, B-lymphocytes, and T-

lymphocytes found in Peyer's patches and other gut-associated lymphoid tissue) and

antibodies, and (iv) the digestion by interaction with lumen-constituents (originating

from both the food and the body). On the other hand, direct cell to cell contact with the

epithelial cells and with immune-competent cells, together with interaction with lumen-

constituents are activities that can be valid not only for probiotics but also for all the

different bacterial components making up the microbiota. Thus, by a direct intervention

towards the composition of the microbiota, ingestion with probiotics may indirectly,

through changes in the composition of the microbiota, lead complex physiological

effects.

Figure 23. Schematic representation of the hypothesis that certain bacteria in the gut have physiological effects and may suppress injuries as, for example, inflammation (suppressors), while certain bacteria tend have physiopathological effects and may aggravate injuries (aggravators).

The bacterial flora of the digestive tract is mostly complex and this even if only those

taxa present in a dominating position are taken into account. So far, the relevance for

physiological or physiopathological potential is still unknown for the majority of the

taxa making up the microbiota of the digestive tract. Exceptions are bacteria that belong

to taxa with well-known pathogenic, or taxa with probiotic, potentials. These groups can

with some certainty be judge as adverse or as beneficial for the health, respectively.

Strains of certain species of Lactobacillus and Bifidobacterium have in intervention

studies in animals and humans proved to possess beneficial health effects, and these two

genera are generally regarded as beneficial components of the bacterial flora in the

digestive tract. Lactobacillus is perhaps more prominent in the small intestine while

Bifidobacterium more abundant in the large intestine. There is also some causal

evidence of health beneficial effects of Faecalibacterium prausnitzii (Sokol et al. 2008).


87

When it comes to adverse components of the bacterial flora of the gut, two adverse

species often abundantly found in the digestive tract are Escherichia coli and

Bacteroides fragilis. Many strains of these two species are well-known pathogens. Even

if the pathogenic strains not usually are a normal part of the resident flora, the non-

pathogenic strains of these species have so much in common with their pathogenic

relatives that they, in high numbers, are likely to impose a negative stress on the host.

Bacterial load of the digestive tract

Viable count

The human microbiota of the digestive tract starts already in the mouth, which harbours

a viable count of 108–10

10 colony forming units (CFU) of resident bacteria per g saliva.

These bacteria are constantly fed to the GI-channel by the swallowing reflex. The

numbers are reduced in the stomach (around 103 CFU/g gastric juice) due to the low

pH, and the numbers remain low in the duodenum and the jejunum (102–10

4 CFU/g

content) where there is a high intestinal motility and the flow of gastric juices are strong

(bile and enzymes originating from pancreas). Then, the bacterial concentration

increases steeply in the ileum and colon to around 1010

CFU/g content in the ileum and

1010

–1012

CFU/g content in the colon. These are extremely high densities of living

bacteria, and such high concentrations can’t be found anywhere in nature outside the

distal part of the digestive tract of mammalians.

The bacteria in the digestive tract are of different kinds and, traditionally, attempts to

identify them have been done by pure-culture technique. In pure-culture technique

isolates are cultured at the laboratory and both phenotypic and genotypic characteristics

of the bacterial population can be studied. Currently however, methods are more

directed towards direct gene-identification. Mostly the identification is targeting the 16S

ribosomal RNA (rRNA) gene but, lately, so called shotgun Sanger sequencing, or

massively parallel pyrosequencing have also been used in an attempt to obtain unbiased

samples of all microbial genes of a community (Eisen 2007). The entirety of the

microbial genes belonging to the microbiota is called the microbiome.

The term “metagenomics” is frequently used as a label for studies where more or less all

the genetic material is recovered and identified directly from environmental samples

(Handelsman et al. 1998). For example, metagenomics was used on 124 samples of

faeces from different individuals, and each one of the individuals was shown to harbour

at least 160 prevalent bacterial species (Qin et al. 2010).

The bacterial core

Some species of the gut microbiota are found in many individuals and some are only

found in a few persons. In an attempt to establish the existence of a phylogenetic “core”

of the microbiota common for a majority of individuals, Tap et al. (2009) obtained more


88

than ten-thousand 16S rRNA gene sequences by PCR-amplification and cloning from

faeces of 17 individuals. Three-thousand-one-hundred-eighty operational taxonomic

units (OTUs) were detected, but most of these only appeared in a few individuals, and

only 2.1% of the OTUs were present in more than 50% of the individuals. On the other

hand, most of the OTUs belonged to the phyla Firmicutes (about 80%), Bacteroidetes

(about 20%), Actinobacteria (about 3%), Proteobacteria (1%) and Verrucomicrobia

(0.1%). Consequently, when bacteria are identified on higher hierarchical levels of

taxonomy such as phylum (division) and class, the individual differences between

persons appear to be smaller, while the differences between habitats within the same

individual are more pronounced. For example, there is a significant difference in the

composition of the microbiota between the oral cavity and rectum (Costello et al. 2009),

and between jejunum and colon within the same individual (Wang et al. 2005).

Furthermore, the general profile of the gut-microbiota of an individual seems to be

reasonably stable over time (Costello et al. 2009).

Faecalibacterium, included in Firmicutes, is an example of a genus present in high

levels in most individuals (Stearns et al. 2011). Faecalibacterium is an efficient

producer of butyric acid and is presumably high levels of Faecalibacterium in the gut

have physiological and health beneficial effects.

The bacterial flora

Overview. Most data of the microbiota of the human gut are founded on faecal samples.

Faecal samples reasonably well reflect the colonic flora but it doesn’t tell much about

the bacterial flora of the small intestine. Frequently dominating genera in the human

digestive tract are summarised in Table 4. Generally in humans and other mammalians

the most frequently occurring and dominating phyla of bacteria are Firmicutes (gram-

positive) and Bacteroidetes (gram-negative). Other frequently occurring phyla are

Proteobacteria (gram-negative), Actinobacteria (gram-positive), Verrucomicrobia

(gram-negative), Fusobacteria (atypical gram-negative) and Spirochaetes (gram-

negative). More recently reported phyla are Tenericutes and Synergistetes that can

occur in lower frequencies (Koren et al. 2012).

Table 4 reflects genera within the different phyla that frequently can be found in high

numbers in the human digestive tract.

Mouth. The bacterial diversity seems to be higher in the mouth than in any other part of

the digestive tract (Stearns et al. 2011). The dominating phyla in the oral cavity are

Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria, Bacteroidetes and

Spirochaetes (Huyghe et al. 2008; Lazarevic et al. 2009; Stearns et al. 2011).

Frequently identified genera are Streptococcus, Prevotella, Veillonella, Neisseria,

Haemophilus, Rothia, Porphyromonas and Fusobacterium (Nasidze et al. 2009). The

most frequent genus of all in the saliva is Streptococcus.

There is a high bacterial diversity in the mouth and huge differences between people,

but mostly there seem to be relatively minor geographic differences. However, different

members of the family Enterobacteriaceae can vary significantly in frequency between

different geographic locations (Nasidze et al. 2009).


89

Table 4. Taxa dominating the bacterial microbiota of the digestive tract (1,2).

Phyla/Division Class Family Genus Gram(3)

Actinobacteria Actinobacteria Micrococcaceae Rothia * +

Actinobacteria Actinobacteria Bifidobacteriaceae Bifidobacterium +

Firmicutes Bacilli Streptoccaceae Streptococcus +

Firmicutes Bacilli Lactobacillaceae Lactobacillus +

Firmicutes Bacilli Enterococcaceae Enterococcus +

Firmicutes Negativicutes Veillonellaceae Veillonella (−)

Firmicutes Negativicutes Veillonellaceae Dialiser (−)

Firmicutes Clostridia unclassified Mogibacterium * +

Firmicutes Clostridia Peptostreptococcaceae Peptostreptococcus * +

Firmicutes Clostridia Lachnospiraceae Coprococcus +

Firmicutes Clostridia Lachnospiraceae Dorea +

Firmicutes Clostridia Lachnospiraceae Roseburia (−)

Firmicutes Clostridia Lachnospiraceae Butyrivibrio (−)

Firmicutes Clostridia Ruminococcaceae Ruminococcus +

Firmicutes Clostridia Ruminococcaceae Faecalibacterium +

Firmicutes Clostridia Ruminococcaceae Anaerotruncus +

Firmicutes Clostridia Ruminococcaceae Subdoligranulum +

Firmicutes Clostridia Clostridiaceae Clostridium +

Firmicutes Clostridia Clostridiaceae Blautia +

Firmicutes Clostridia Eubacteriaceae Eubacterium +

Firmicutes Clostridia unclassified Collinsella +

Firmicutes Erysipelotrichia Erysipelotrichaceae Holdemania +

Proteobacteria Betaproteobacteria Alcaligenaceae Sutterella -

Proteobacteria Betaproteobacteria Neisseriaceae Neisseria -

Proteobacteria Deltaproteobacteria Desulfovibrionaceae Bilophila -

Proteobacteria Gammaproteobacteria Pasteurellaceae Haemophilus * -

Proteobacteria Gammaproteobacteria Enterobacteriaceae Enterobacter * -

Proteobacteria Gammaproteobacteria Enterobacteriaceae Serratia * -

Proteobacteria Gammaproteobacteria Enterobacteriaceae Escherichia -

Proteobacteria Gammaproteobacteria Enterobacteriaceae Klebsiella -

Proteobacteria Gammaproteobacteria Moraxellaceae Acinetobacter -

Proteobacteria Gammaproteobacteria Pseudomonadaseae Pseudomonas * -

Proteobacteria Gammaproteobacteria Cardiobacteriaceae Cardiobacterium -

Bacteroidetes Bacteroidia Prevotellaceae Prevotella * -

Bacteroidetes Bacteroidia Porphyromonadaceae Porphyromonas * -

Bacteroidetes Bacteroidia Porphyromonadaceae Parabacteroides -

Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides -

Bacteroidetes Bacteroidia Rikenellaceae Alistipes -

Fusobacteria Fusobacteria Fusobacteriaceae Fusobacterium -

Spirochaetae Spirochaetes Brachyspiraceae Brachyspira -

Verrucomicrobia Verrucomicrobiae Verrucomicrobiaceae Akkermansia -


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Notes for Table 4. (1) Data from Pettersson et al. (2003), Wang et al. (2005), Hayashi et al. (2005), Bik et al. (2006), Lazarevic et al. (2009), Li et al. (2009), Nasidze et al. (2009), Turnbaugh et al. (2009) and Qin et al. (2010).

(2) Genus identification has been made by direct gene identification, mostly of the 16S rRNA gene by cloning and sequencing. (3) Gram = Gram-reaction. Negative gram-reaction within parenthesis means that the reaction is negative or variable. It has been shown for Butyrivibrio fibrisolvens that the negative gram-reaction is due to a thin cell wall and that the cell wall has Gram-positive characteristics (Cheng et al. 1977). Presumably, this is also the case for the other Butyrivibrio spp. and perhaps also for other Firmicutes with gram-negative reaction, i.e., they presumably do not contain lipopolysaccharides (LPS) and are usually associated with a gram-positive cell wall. * Taxa typically found dominating in the upper GI tract (mouth to jejunum) but mostly much less pronounced in the distal GI tract (ileum to rectum).

Stomach. The stomach is a relatively harsh environment for bacteria and due to the low

viable counts found there, it can always be debated whether these bacteria are resident

or transient. On the other hand, Helicobacter pylori that is the causative agent of gastric

ulcers, is definitely a resident bacteria of the stomach.

An adult person produces about two litres of gastric juice daily and the pH in lumen is

below 2 under fasting conditions, but close to the epithelial cells due to the mucus layer

pH is conderably higher, i.e.5 to 6. Resident or transient, the most frequently occurring

phyla in the stomach are Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and

Fusobacteria. Frequently occurring genera are Helicobacter, Streptococcus, Prevotella,

Neisseria, Haemophilus and Porphyromonas (Bik et al. 2006; Li et al. 2009).

Jejunum. The mucosal microbiota of jejunum is typically dominated by the phylum

Firmicutes, and to a lesser extent by Proteobacteria, Bacteroidetes, Fusobacteria and

Actinobacteria (Wang et al. 2005). Streptococcus is often the most abundant genus with

species as, for example, Streptococcus mitis, Streptococcus salivarius, Streptococcus

oralis, Streptococcus parasanguinis and Streptococcus anginosus. Other Firmicutes

than Streptococcus can, for example, be Veillonella, Mogibacterium and

Peptostreptococcus (Wang et al. 2005). Sometimes Lactobacillus can be found in a

dominating position in the jejunum (Hayashi et al. 2005).

Gammaproteobacteria that can be expected to be found are Haemophilus, Escherichia,

Acinetobacter and Pseudomonas (Wang et al. 2005). The Bacteroidetes can typically be

represented by Prevotella.

Ileum. The mucosal microbiota of ileum is typically dominated by the phyla of

Firmicutes and Bacteroidetes, but also Verrucomicrobia, Proteobacteria and

Fusobacteria can be expected to be found (Wang et al. 2005). Firmicutes is in ileum, in


91

contrast to jejunum, usually dominated by different, so called, Clostridium clusters

(defined by Collins et al. 1994), including genera as Clostridium, Coprococcus, Dorea,

Ruminococcus, Roseburia, Faecalibacterium and Dialiser (Wang et al. 2005).

Occasionally, also the amount of Lactobacillus can be relatively high in ileum (Hayashi

et al. 2005).

The Bacteroidetes in ileum is mostly of the genus Bacteroides, and example of often

abundant species are Bacteroides thetaiotaomicron, Bacteroides vulgatus and

Bacteroides uniformis (Wang et al. 2005).

Large intestine. In colon and rectum, the mucosal microbiota is mostly dominated by

the two phyla Firmicutes and Bacteroidetetes. Firmicutes is often represented by

different species included in the so called “Clostridium clusters XIVa” (defined by

Collins et al. 1994) which contains species as, for example, Eubacterium halii,

Eubacterium eligens, Dorea formicigenerans, Ruminococcus lactaris, Ruminococcus

gnavus, Ruminococcus torques, Ruminococcus obeum, Clostridium leptum, Clostridium

symbiosum, Clostridium boltei and Roseburia intestinalis (to mention some). But there

will most probably also be species of the “Clostridium cluster IV” , for example

Faecalibacterium prausnitzii and Clostridium orbiscindens; “Clostridium cluster IX” ,

for example Dialister invisus; “Clostridium cluster XIVb”, for example Clostridium

lactatifermentans (Wang et al. 2005; Zhang et al. 2009; Turnbaugh et al. 2009; Qin et

al. 2010).

The phylum Bacteroidetetes can be represented by the more or less frequently occurring

species Bacteroides vulgatus, Bacteroides thetaiotaomicron, Bacteroides ovatus,

Bacteroides stercoris, Bacteroides caccae, Bacteroides putredinis, Bacteroides merdae

Parabacteroides distasonis (former Bacteroides distasonis), Roseburia intestinalis,

Alistipes putredinis (former Bacteroides putredensis), Bacteroides uniformis and

Bacteroides fragilis (Wang et al. 2005; Zhang et al. 2009; Turnbaugh et al. 2009; Qin et

al. 2010).

Less frequent but still present in the large intestine are normally also the phyla

Verrucomicrobia, for example the species Akkermansia muciniphila; Proteobacteria,

for example E. coli, Acinetobacter johnsonii, Sutterella wadsworthensis and Neisseria

subflava; Actinobacteria, for example Bifidobacterium longum; Fusobacteria, for

example, Fusobacterium varium (Wang et al. 2005; Zhang et al. 2009; Turnbaugh et al.

2009; Qin et al. 2010).

The microbiota is mostly heterogeneous and differs widely between individuals with

regard to both composition and diversity. However, now and then a person without

clinical symptoms can be grossly dominated by E. coli (Pettersson et al. (2003).

Opportunistic pathogens that frequently can be found in high numbers in the large

intestine of individuals without obvious clinical symptoms are Bacteroides fragilis,

Escherichia coli, Bilophila wadsworthia and Sutterella wadsworthensis. Less frequently

found opportunistic pathogens, found in relatively high amounts of the large intestine

are Acinetobacter baumannii, Brachyspira aalborgi, Cardiobacterium hominis,

Clostridium perfringens, Klebsiella pneumoniae and Veillonella parvula (Pettersson et

al. 2003).


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Taxonomic considerations

Clostridium. The so called Clostridium clusters of Collins et al. (1994) include species

from different genera, for example, Clostridium symbiosum, Eubacterium

formicigenerans, Roseburia intestinalis, Ruminococcus obeum, Clostridium

aminophilum, Eubacterium halii, Clostridium oroticum, Ruminococcus torques,

Faecalibacterium prausnitzi, Clostridium leptum, Oscillospira quilliermondii,

Clostridium orbiscindens, Dialister invisus, Propionispira arboris, Clostridium

aminobutyricum, Peptostreptococcus anaerobius and Clostridium lactatifermentas. All

are designated to the phylum Firmicutes (Firmicutes consists for the time being of 6

different classes, 9 orders and 36 families). The system to divide Clostridium and

related genera in different so called Clostridium-clusters, where each cluster is on the

hierarchical level of class, was introduced by Collins et al. (1994). Phylogenetical

classification of bacteria based on the 16S rRNA gene was at that time a new thing, and

doing so, it becomes obvious that many Clostridium spp. were misclassified. Collins et

al. (1994) divided the different Clostridium spp. and related taxa into “Clostridium-

clusters” on a hierarchical class-level and labelled the different groups Clostridium

cluster I through XVIII.

The old definition of the genus Clostridium that now has been shown to be artificial was

based on phenotypic traits, i.e. the genus Clostridium was defined as strictly anaerobic

rods that were able to form endospores. The fact that the genus Clostridium is

heterogeneous is also reflected by the wide range of guanine+cytosine mol% of 22 to

55% which is a far too wide span to represent a natural genus. The pathogenic species

Clostridium perfringens and Clostridium botulinum belong to the Clostridium-cluster I

of Collins et al. (1994), and Clostridium difficile belongs to Clostridium-cluster X1.

Generally clostridia often produce acetate and butyrate, and sometimes propionate in

colon.

Bacteroides. Bacteria classified to the phylum Bacteroidetes consists often when found

in ileum and colon of the gram-negative genus Bacteroides. The phylum Bacteroidetes

includes for the time being 3 different classes, 3 orders and 15 families. The genus

Bacteroides includes a lot of different species but some of the more, frequently

occurring ones in the digestive tract are B. vulgatus, B. uniformis, B. thetaiotaomicron,

Bacteroides ovatus, B. stercoris, B. caccae, B. putredinis, B. merdae, B. fragilis, B.

intestinalis, B. pectinophilus, B. finegoldii, B. eggerthii, B. capillosus, B. dorei and B.

xylanisolvens.

The most aggressive Bacteroides species and the one that most frequently is involved in

secondary infections of the abdominal is Bacteroides fragilis. Some B. fragilis strains

can produce toxins. Those toxinogenic strains have been suggested to be involved in the

initiation of colorectal cancer. Furthermore, it has been shown that patients suffering

from inflammatory bowel disease (IBD) have a higher proportion of B. fragilis on the

intestinal mucosa than healthy individuals (Swidsinski et al. 2005).

B. fragilis is the type species of the genus Bacteroides. Closely related species to B.

fagilis are B. caccae, B. distasonis, B. eggerthii, B. merdae, B. ovatus, B. stercoris, B.

thetaiotaomicron, B. uniformis and B.vulgatus. Bacteroides often produce acetate and

butyrate in the digestive tract, and sometimes also propionate.


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Adverse bacteria in the microbiota

A disturbed microbiota

For some chronic diseases, the pathologic agent might be a disturbed microbiota

(dysbiosis) rather than an individual organism, and this presumably means a decreased

bacterial diversity and/or different degrees of overgrowth by more aggressive fractions

of resident bacteria, i.e., bacteria inducing an inflammatory response by the immune

system. Species that are well-known to include pathogenic or opportunistically

pathogenic strains, and that also have been found as a substantial part of the gut

microbiota of apparently healthy individuals, are E. coli and B. fragilis. Increased

proportions of E. coli and B. fragilis in the gut have been linked to inflammatory bowel

disease (IBD) (Kleessen et al. 2002; Swidsinski et al. 2005; Wang et al. 2007).

Which components of the bacterial flora that have health beneficial effects and which

components that have adverse effects, is are interesting questions. True pathogens as,

for example, Salmonella and Shigella, are of course highly damaging to the body and

the immune system of the body responds violently, and try to counteract the offenders

with a strong inflammation. However, these true pathogens are not normally part of

healthy individuals gut microbiota (true pathogens seldom are). On the other hand,

bacteria not being clearly pathogenic but phylogenetically related to pathogens can be

part of a normal resident microbiota, and these non-pathogenic relatives may share

adverse characteristics with their pathogenic counterparts. This makes them more likely

to exercise adverse effects when present in high numbers on the mucosa.

Examples of opportunistically pathogenic species that can occur as a substantial part of

the resident microbiota are E. coli, Klebsiella pneumonia (both belonging to the class

Gammaproteobacteria), Sutterella wadsworthensis (Betaproteobacteria), Bilophila

wadswothia (Deltaproteobacteria), Fusobacterium varium (Fusobacteria), Bacteroides

fragilis (Bacteroidetes), Prevotella melaninogenica (Bacteroidetes), Vellionella parvula

(Firmicutes), Streptococcus pneumonia (Firmicutes), Streptococcus anginosus

(Firmicutes) and Clostridium perferingens (Firmicutes).

An attempt to look for correlation between systemic inflammation and faecal microbiota

showed that about 10% of the total variability of the microbiota was related to the pro-

inflammatory cytokines IL-6 and IL-8 (Biagi et al. 2010). All taxa that showed a

slightly positive correlation with either IL-6 or IL-8 belonged to the phylum

Proteobacteria (Biagi et al. 2010).

IL stands for “interleukin” which is a cytokine. IL-6 initiates production of acute phase proteins and takes part in finish-off the acute inflammation. IL-6 is produced by macrophages and T-cells. IL-8 activates white blood cells to pass out from the blood vessels and is produced by macrophages and epithelial cells after stimulation of bacteria or bacterial products. “Acute phase reaction” is the response of the “acute phase proteins” in inflammation. The concentration of acute-phase proteins in plasma either increase or decrease in inflammation.


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Lipopolysaccharides

Gram-negative bacteria contain lipopolysaccharide (LPS) as the major constituent in the

outer leaflet of the outer cell membrane. LPS contains large, variable regions of

polysaccharides and oligosaccharides and a relatively conserved lipid region (lipid A),

which is the endotoxic and biologically active moiety. The interaction of LPS with

macrophages results in the release of pro-inflammatory cytokines such as TNF-alpha,

IL-6, and IL-1, and can lead to endotoxic shock, which is an often fatal outcome of

sepsis.

TNF (tumour necrosis factor) alpha is a pro-inflammatory cytokine that is produced by monocytes/macrophages and activated T-cells. IL-1 (interleukin-1) is a group of three pro-inflammatory polypeptides (IL-1-α, IL-1β and IL-1Ra) involved in immune regulation and inflammatory response.

The gram-reaction of different taxa relevant for the digestive tract is a factor of

importance as gram-negative bacteria can be expected to contain LPS (Table 4). For

example, both the facultatively aerobic E. coli and the strictly anaerobic B. fragilis

contain LPS, but the chemical structure is somewhat different between the two species.

The mammalian immune system reacts differently towards the different LPS types

(Lindberg et al. 1990). The endotoxic activity of LPS of B. fragilis is relatively low

compared with LPS from E. coli and other Enterobacteriaceae (Poxton et al. 1995), but,

nevertheless, LPS from Bacteroides is a potent stimulator of the innate immune system

(Berezowa et al. 2009). The immune response to LPS can differ also between LPS from

different species of Bacteroides (Kasper et al. 1977; Berezowa et al. 2009).

Gram-negatives that typically contaminate food, and therefore are ingested on a more or

less regular basis and sometimes in high quantities, are Gammaproteobacteria, e.g. the

families Enterobacteriaceae and Pseudomonadaceae. However, different diet

components can also affect the gut microbiota, e.g. a high-fat diet can increase the

proportion of gram-negatives in the gut but also increase the leakage of LPS through the

intestinal barrier out into the body (Cani et al. 2007). It is not known why gram-

negative components of the microbiota should be stimulated by a fat-rich diet, or why

the barrier function of the mucosa should decrease. However, one speculation could be

that a fat-rich diet increases the amount of bile in the gut, and bile has strong

antimicrobial effects, but some taxa have higher resistance against bile than others, e.g.,

Enterobacteriaceae and Bacteroides that are known for their comparably high bile

resistance. Furthermore, bile is a powerful detergent which might have effects on the

permeability of the intestinal mucosa and, thus, mediates an increased leakage of LPS.

It should be stressed that it is not only gram-negatives and LPS that can induce

inflammation; other cell components and metabolites can be involved, and there are also

several gram-positive pathogenic and opportunistic pathogenic bacteria that can induce

inflammation (González-Navajas et al. 2008). One example of the latter is

Enterococcus, which is frequently found as a contaminant in foods, and Enterococcus is

also frequently found in the GI-tract.

It should also be borne in mind that, in combination, different taxa of the microbiota,

can enhance each other’s adverse effects. This has been demonstrated in animal models,

e.g. in a rat models for intra-abdominal sepsis that cause a two-phase disease process

consistent with intra-abdominal sepsis in humans: It was shown that a combination of


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obligate anaerobes such as B. fragilis or Fusobacterium varium and facultative aerobes

such as E. coli or Enterococcus faecalis cause early peritonitis and mortality, and

abscess development (Onderdonk 2005). In this case, E. coli was necessary for the

mortality, and a combination of E. coli and B. fragilis was needed for the abscess

development (Onderdonk et al. 1976; Onderdonk 2005).

Bacterial interaction

Immune system

There are complex relationships between the intestinal immune system and the resident

microbiota of the digestive tract. It is crucial for the epithelial cells and the mucosal

immune system to distinguish between pathogenic and non-pathogenic agents. Intestinal

epithelial cells are capable of detecting bacterial antigens and initiating and regulating

both innate and adaptive immune responses. Signals from bacteria can be transmitted to

adjacent immune cells such as macrophages, dendritic cells and lymphocytes through

molecules expressed on the epithelial cell surface, e.g. Toll-like receptors (TLRs).

TRLs are so named because of their similarity to a receptor first identified in the fruit fly Drosophila melanogaster by German scientists, a protein coded by the Toll-gene (“toll” means fantastic in German). At least ten types of human TLRs are known. TLRs are expressed in most tissues in healthy adult individuals.

Interaction of TLRs and bacterial molecular patterns results in activation of an

intracellular signalling cascade, up-regulation of inflammatory genes, production of pro-

inflammatory cytokines and interferons, and recruitment of blood cells other than

lymphocytes (myeloid cells). Interaction with TLRs also stimulates expression of co-

stimulatory molecules required to induce an adaptive immune response of antigen

presenting cells.

The presence of bacteria with inflammation-inducing capabilities in the digestive tract

can cause an increased inflammatory tone. The inflammation can be local in the mucosa

but can also involve the whole body (systemic). Systemic inflammation over prolonged

time increases the risk for many diseases, e.g. heart and cardio-vascular diseases,

diabetes type 2, non alcoholic fatty liver disease (NAFLD) and obesity. Inflammation in

digestive tissue over prolonged periods increases the risk for cancer.

The immune system consists of different types of white blood cells and proteins. The white blood cells are of different kind:

Monocytes are big white blood cells in the blood stream. Monocytes are produced by stem-cells in the bone marrow.

Macrophages are white blood cells within tissue. Macrophages are converted monocytes, and they perform phagocytosis.


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Dendritic cells are also converted monocytes. They are messengers between the innate and adaptive immune systems. Dendritic cells present antigens.

There are three major types of lymphocytes, T-cells, B cells and NK cells. T-cells are lymphocytes where the binding capacity to body-components has been evaluated by a selection process in thymus.

B cells are lymphocytes that produce antibodies, present antigens and can develop into memory cells. They are essential component of the adaptive immune system.

NK cells are cytotoxic lymphocytes. They are large granular lymphocytes, and are a major component of the innate immune system.

Neutrophil granulocytes (neutrophil or polymorphonuclear neutrophil = PMN) are the most abundant type of white blood cells and form an essential part of the innate immune system. Neutrophils are highly motile, and phagocytic. They express and release cytokines, and have a key-role in the defence against invading pathogens. Neutrophils can disarm and kill many types of microbes.

Eosinophil granulocytes (eosinophils) are white blood cells that produce granule proteins, reactive oxygen species, lipid mediators, enzymes, growth factors and cytokines. They are active against multicellular parasites and virus, and control mechanisms associated with allergy and asthma.

Basophil granulocytes (basophils) are white blood cells that are susceptible to staining by base dyes and contain large cytoplasmic granules. They appear in many inflammatory reactions, particularly in allergy. Basophils contain heparin (prevents blood from clotting) and histamine (promotes blood flow to tissues).

Mast cells (mastocytes) are immune cells resident in several types of tissue and contain many granules rich in histamine and heparin. They are intimately involved in wound healing and the defence against pathogens. Mast cells play a role in allergy and anaphylaxis (hypersensitivity).

The intestinal mucous membrane (mucosa)

Barrier. The mucosa of the digestive tract functions as the local defence barrier that

prevents the invasion and systemic spread of bacteria and other unwanted compounds,

such as LPS. The mucosa consists of a single layer of epithelial cells, and confers

selective permeability through either a route straight through the epithelial cells

(transcellular) or between them (paracellular). The transfer of small molecules, for

example short-chain fatty acids, amino acids, electrolytes and sugars are transporter-

mediated through cells while medium-sized hydrophilic compounds are transferred

paracellularly. Protein-sized molecules are normally prohibited from paracellular

transport (Groschwitz and Hogan 2009; Keita and Söderholm 2010). The paracellular

barrier function is brought about by complex structures of proteins supervising and

selectively blocking the entry to the narrow gap between the epithelial cells. These gap-

structures are called the “tight junctions” or in other words, the tight junction is the

closely associated area between two cells whose membranes join together. The tight

junctions are responsible for the paracellular, barrier function (Shen et al. 2009). The

tight junctions also take part in the transfer of external stimuli to the epithelial cells,


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governing the proliferation and differentiation of the epithelial cells (González-Mariscal

et al. 2008). Examples of tight junction proteins are occludin and claudin with an intra-

cellular connection to the zonulin.

The epithelial tissue in the mucosa of the digestive tract produces mucin that clings

tightly to the epithelial surface, but mucin also is released into the lumen. The mucin

layer of the mucosa protects the epithelial cells against aggressive bacteria and

unfavourable substances. Depending on sort of bacterium, bacterial interaction can

stimulate the mucin production of the mucosa, e.g. some Lactobacillus can stimulate

mucin production when they adhere to epithelial cells of the mucosa. The mucin

production of the mucosa is also increased when the mucosa is attacked and become

inflamed.

There are bacterial groups in the microbiota of the digestive tract with ability to brake-

down and digest mucin, e.g. Akkermansia muciniphila and Ruminococcus torques.

Mucins are high molecular weight, heavily glycosylated proteins that most bacteria are

unable to brake-down. A key characteristic of mucins is their ability to form gels and

bind to pathogens, and thus the mucins become a part of the defence system of the

body. The genus Akkermansia which belongs to the phylum Verrucomicrobia can be

found in varying numbers in most adult individuals. Akkermansia seems to be a marker

for an intestinal system in ecological balance, i.e. low number is an indication of a

disturbed ecology. It is unclear if Akkermansia has any health beneficial effects on its

own account. On the other hand, no taxa of the phylum Verrucomicrobia are known to

be pathogenic.

Leakage. Under certain conditions, intestinal barrier function can be impaired or

overwhelmed, allowing bacteria and LPS within the digestive tract to reach systemic

organs and tissues, a process termed bacterial translocation, i.e. translocation is the

passage of viable bacteria through the epithelial mucosa into lamina propria and then to

the mesenteric lymph nodes and possibly other tissue (Berg and Garlington, 1979). Also

parts from microorganisms or products of microorganisms are sometimes included in

the expression “translocation”, for example bacterial components as LPS. The status of

the mucosa is critical for several categories of patients, for example, those (i) at risk for

multiple organic failure; (ii) undergoing chemo or radiotherapy; (iii) with HIV; (iv)

undergoing abdominal surgery; (v) undergoing transplantation.

Bacterial translocation is the passage of viable bacteria through the epithelial mucosa into the lamina propria and then to the mesenteric lymph nodes and possibly other tissues

Furthermore, there are a variety of human diseases in which it has been suggested that

abnormal permeability of the mucosa is important. These diseases include autoimmune

diseases, e.g. type 1 diabetes, celiac disease, multiple sclerosis, rheumatological

diseases and Crohn’s disease. But also in atopic dermatitis and irritable bowel syndrome

(IBS) it seems as the permeability of the intestinal mucosa is increased.

In contrast, bacterial translocation is not necessarily occurring only in connection with

trauma and disease, there is evidence that portal vein endotoxaemia of gut origin in

minute amounts is a normal physiological phenomenon (Jacob et al. 1977; Nolan 1981).

During normal conditions, this low-grade endotoxaemia of gut origin is rapidly cleared

by the cells of the reticuloendothelial system of the liver (Mathison and Ulevitch 1979;

Ruiter et al. 1981). Through the portal blood flow draining the digestive tract, intestinal


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bacteria and bacterial products, such as LPS, reach the liver and the parenchymal cells

(hepatocytes) and the non-parenchymal cells (encompassing endothelial cells, Kupffer

cells, hepatic stellate cells and Pit cells; Pit cells are liver-specific natural killer cells),

help to sustain normal physiology and homeostasis, and participate in systemic, as well

as in local inflammation and immune response (Ishibashi et al. 2009).

High amounts of LPS-synthesizing bacteria (gram-negative bacteria) in the gut can

cause low-grade systemic inflammation by a continuous leakage (translocation) of LPS

to the liver (Figure 24). A high proportion of E. coli in the human gut microbiota is not

unusual in persons living in urban societies, and high loads of E. coli is most likely

something that ought to be avoided from a health perspective.

Figure 24. Translocated bacteria and bacterial components (e.g. lipopolysaccharides) will by the portal blood come to the liver that will respond with inflammation in order to eliminate them.

It has been shown in human adults that there is a link between metabolic diseases and

bacterial populations in the gut (Larsen et al. 2010). In a study including 36 male adults

with a broad range of age and body-mass indices (BMIs), among which 18 subjects

were diagnosed with type 2 diabetes, the proportions of the class Clostridia were

significantly less in the diabetic group compared to the control group, while the class

Beta-proteobacteria was highly enriched in diabetic subjects compared to the non-

diabetic ones (Larsen et al. 2010). Similar results were found in a study involving 345

Chinese individuals, i.e. patients with type two diabetes had a certain degree of

unbalanced microbiota (dysbiosis) compared to healthy individuals (Qin et al. 2012).

The abundance of clostridial taxa, including genera as Faecalibacterium and Roseburia

was decreased in the diabetes patients, and the abundance of opportunistic pathogens as

Bacteroides caccae, Eggerthella lenta and E. coli was increased (Qin et al. 2012).


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Generally, more and more evidence are gathered pointing in the direction that the many

members of the different classes of Proteobacteria are negative for the health and

making up harmful parts of the resident microbiota of the human digestive tract. Hence,

it is an obvious opportunity to use probiotics and other dietary regimes for counteracting

Proteobacteria in the digestive tract and get beneficial, long-term health effects as a

result.

Variety and diversity

Mammalians

Inheritance and diet are in the long run the two most important factors for influencing

the composition the gut microbiota of an individual. When the bacterial composition of

106 faecal samples, from 60 different mammalians (including humans) was compared,

it was concluded that both the diet and phylogeny influenced the bacterial diversity (Ley

et al. 2008). The bacterial diversity was highest in herbivorous animals (plant eating

animals) and lowest in carnivorous animals (meat eating animals). The bacterial

diversity of omnivorous animals (eating all kind of foods) was in between that of the

strictly plant-eating and the meat-eating ones. It was also obvious from this study that

the microbiota between different kinds of animals was surprisingly similar when

compared at the hierarchical level of phylum (division). The major variations are seen at

lower hierarchical levels, where variations between individuals, even individuals from

the same geographical area, eating the same type of diet, sometimes can be larger than

that between different mammalian species.

No statistically significant overall difference in diversity could be seen between Korean

and American adults, even if significant differences in the composition of the bacterial

flora could be seen (Lee et al. 2011). However, there were no differences between

African Americans and European Americans suggesting the importance of the diet for

the composition of the microbiota. Interestingly, within each country (South Korea and

USA, respectively) the differences in composition of the microbiota were significantly

greater for individuals in different families than those within the same family (Lee et al.

2011).

Mother and child axis

The first bacteria. Adult humans carry about 1 kg bacterial biomass in the digestive

tract. In fact, the bacterial density of the human large bowel is as high as it can be in

nature, and from a bacterial perspective, humans and other mammalians can be

considered as mobile, high-efficiency fermenters, producing bacteria at a high rate, and

spreading them around. The “baby-fermenter” is inoculated with its first bacteria at

birth, and due to the fact that the gut-microbiota participates in shaping and tuning of

the immune system of the individual, the pioneer microbiota of an infant can be of


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special importance as it can be of consequence for the future health of the individual.

The foetus has during the pregnancy only indirectly come in contact with bacteria

through the blood-bound immune system of the mother. But during birth the new-borne

infant becomes directly exposed to the mother’s microbiota, and this contact usually

continues during the upbringing of the child. Thus, normally a child inherits the

microbiota of its mother.

At birth, the baby become contaminated with bacteria originating mainly from the

mother’s (i) vagina, (ii) rectum and (iii) skin, and as soon as the mother starting to

cuddle the baby, (iv) also from the mouth, and if breast fed (v) from the nipples and

milk-canals of the mothers breasts. It seems as the factor overruling all other factors in

determining the composition of the microbiota is the link to the mother (Turnbaugh et

al. 2009). However it is not clear if it is the genetic inheritance from the mother or if it

is the actual direct transfer of the microbiota from the mother that is most important for

this establishment of the microbiota of an individual. The bacterial exchange with the

mother can also continue as a gradual long-term effect when the child lives in close

contact with the mother, also after weaning, and chairing diet with the mother. The

bacterial diversity of the child’s gut microbiota is generally increasing with age up to

adulthood (Yatsunenko et al. 2012).

Anyway, the first live bacteria the child will meet at a normal birth will be the vaginal

bacteria of the mother. In view of the fact that a healthy vagina normally are nearly

totally dominated by lactobacilli or bifidobacteria and that breast milk contains

components with prebiotic effects, i.e. breast milk stimulates the growth of lactobacilli

and bifidobacteria in the gut, it seems reasonable to assume that lactobacilli and

bifidobacteria somehow are meant to be present in high numbers in breast-fed children

and to be amongst the first ones to come in direct contact with the immune system of

the baby. The lactobacilli that most frequently dominating the vagina of fertile women

are homofermentative species of Lactobacillus, such as Lactobacillus gasseri,

Lactobacillus crispatus, Lactobacillus iners, Lactobacillus johnsonii and Lactobacillus

jensenii (Vásquez et al. 2002; Ravel et al. 2011). Some women are dominated by

Bifidobacterium or a combination of lactobacilli and bifidobacteria. The pH of the

vagina normally is in the range 4-5 and the bacterial population has a density of about

108 cells per ml of vaginal secretion (Ravel et al. 2011). Usually one or two strains of

Lactobacillus are dominating in the individual woman.

The lactobacilli-flora in vagina differs often somewhat from that of the digestive tract.

All the above mentioned Lactobacillus spp. typical for the vagina can also occur in the

digestive tract, but more frequently occurring in the digestive tract are Lactobacillus

rhamnosus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus reuteri,

Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus acidophilus, Weissella

confusa and Leuconostoc mesenteroides. Not surprisingly, lactobacilli in levels of at

least 108

16S rRNA gene copies per gram faeces can be found one to two days after

birth in all naturally born infants (neonates; Karlsson et al. 2011). However,

bifidobacteria were after the same time only occurring in around 20% of the children.

The microbiota was mostly dominated by a one to three genera, and even if lactobacilli

were present in reasonably high numbers in all neonates, the faecal flora could be

dominated by another genera (higher copy numbers), e.g. by Escherichia (30% of the

neonates) and Bacteroides (14% of the neonates; Karlsson et al. 2011). Measurements

from the mucosa of the small intestine instead of faeces had probably given samples


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with higher abundance of Lactobacillus as lactobacilli generally are more dominating

on the mucosa and also higher up in the digestive tract.

The occurrence of different bacteria in the faecal microbiota of children, not only in

Sweden but also in Italy and England, and this time one week after birth, showed that

bifidobacteria now could be found in more children (65%), but the occurrence of

lactobacilli was lower (12%) (Adlerberth et al. 2007). Staphylococci were found in all

children, and also the potentially pathogenic one, Staphylococcus aureus could be found

(in 17% of the children), and E. coli was frequently isolated (42%). Hence, an obvious

question to ask is: why so few lactobacilli and bifidobacteria and why so many adverse

bacteria in European children? One answer may be that many mothers either have a

corrupted microbiota in the vagina or in the digestive tract, or both.

Bacterial vaginosis. It is widely recognised that the vaginal bacterial flora can be

disturbed, leading to the syndrome of bacterial vaginosis. Actually, bacterial vaginosis

is defined as loss of lactobacilli-dominance in the vagina and a foul smell. Bacterial

vaginosis increases the risk for (i) urinary tract infection, (ii) miss-carriage, and (iii)

preterm delivery. Bacterial vaginosis can also increase the risk for fungal infections.

Examples of bacteria dominating in vagina at bacterial vaginosis are Prevotella,

Atopobium, Eggerthella, Gardnerella, Megasphaera, Leptotrichia and Sneathia (Thies

et al. 2007; Ferris et al. 2007; Fredricks et al. 2007; Ravel et al. 2011). These bacteria

often originate from colon and some of them are included in the class “Clostridium

cluster XI” and the phyla Fusobacteria and Bacteroidetes, but also members of

Enterobacteriaceae can be involved. At least Enterobacteriaceae and Prevotella can be

characterised as pro-inflammatory.

In the United States of America the prevalence of bacterial vaginosis 2001-2004

(National Health and Nutrition Examination Survey Data) was 29% of U.S. fertile

women (14 – 49 years of age) (Allsworth and Peipert 2007). Interestingly, bacterial

vaginosis has been associated with a high-fat diet (Neggers et al. 2007), and it was

concluded that “increased dietary fat intake is associated with increased risk of bacterial

vaginosis” (Neggers et al. 2007).

Probiotics for the treatment of bacterial vaginosis has proven to be effective for certain

probiotic strains (Falagas et al. 2007). There are clinical trials that show that oral

administration of L. acidophilus or L. rhamnosus GR-1 and L. fermentum RC-14 for

two months resulted in the cure of bacterial vaginosis. However, there are also reported

trials that didn’t found any significant differences in the cure rate (Falagas et al. 2007).

Starting with E. coli. If a mother has a disturbed microbiota in the digestive tract

and/or in the vagina, this corrupted microbiota can be inherit by the baby. One example:

the development of the faecal microbiota in a naturally born, Swedish baby was one

week after birth totally colonised with E. coli, i.e. this baby started life with a relatively

aggressive microbiota (Figure 25). Presumably, the mother of the child harboured a

somewhat twisted microbiota at the time of birth.

It is obvious that a young child have a much less diverse microbiota than an adult

individual, but already after weaning the microbiota becomes more complex and after

one year it starts to resemble the adult microbiota (Figure 25). However, the particular

baby of Figure 25 had a tough introduction to bacteria as it started with a more or less

pure E. coli microbiota, and then after two months the E. coli was supplemented with


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Bacteroides fragilis. Both E. coli and B. fragilis possess LPS and must be regarded as

pro-inflammatory bacteria. Further, these two species include true pathogenic strains.

Thus, there is an obvious risk that this child gets an elevated tone of inflammation

which might have negative health-consequences later in life.

Figure 25. Dominating bacterial flora in faeces of a breast fed infant, one week of age, two months of age and still breast fed, and one year of age (not breast fed any longer). The bacterial flora was identified by direct analysis of 16S rRNA genes by PCR-amplification, cloning and sequencing. Data have been taken from Wang et al. (2004).


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A high amount of E. coli in newborns can originate from the mother. It has been shown

in rats that a high amount of E. coli in the gut of the rat dam (rat mother) can increase

the systemic inflammatory tone in her off-springs (Fåk et al. 2007). Pregnant rat dams

were administered either broad-spectrum antibiotics, three days prior to parturition, or

E. coli in the drinking water one week before parturition and during 14 d of lactation.

Both procedures resulted in an increased level of E. coli in faeces. It was shown in both

groups of rat dams with increased proportion of E. coli gave birth to pups with elevated

systemic inflammation, i.e. they had increased haptoglobin levels in the blood (Fåk, et

al. 2007).

In contrast, if pregnant and lactating rat dams were administered L. plantarum 299v in

the drinking water until their pups had reached 14 d of age, no increase in haptoglobin

occurred. Instead, the gut-growth was stimulated and the barrier function of the gut was

improved (Fåk et al. 2008).

Haptoglobin (Hp) is an acute phase protein that binds free haemoglobin (released from erythrocytes), and thereby inhibits oxidative stress. The Hp-haemoglobin complex is removed by the reticuloendothelial system (RES), mostly in spleen.

RES (reticuloendothelial system) is part of the immune system, i.e. phagocytic cells, primarily monocytes and macrophages, accumulated in lymph nodes and spleen. Also the Kupffer cells are included in RES.

C-reactive protein (CRP) is an acute phase protein. CRP binds to certain bacteria and to the surface of dead or dying cells in order to activate macrophages and the complement system.

The complement system is a biochemical cascade that helps antibodies to clear pathogens from the organism. It is part of the innate immune system that is not adaptable and does not change over the course of an individual's lifetime.

Atopic eczema and bacterial diversity

In a study of the faecal microbiota of 35 infants from Göteborg (Sweden), London

(England) and Rome (Italy), the microbiota was followed from the first week after birth

to one year of age (Wang et al. 2008). The microbiota of one of these children is

depicted in Figure 25. Naturally, the composition varied within wide limits between

different children, but in the particular child showed in Figure 25, E. coli was

dominating in the start of life (one week of age) and then the microbiota gradually

developed towards a higher diversity, and a composition relatively typical also for adult

individuals (after 1 year). Unfortunately, this specific child developed atopic eczema

(diagnosis at 18 month of age). Atopic eczema is an inflammation of the skin that

usually starts in early childhood (most children grow out of it). “Atopic” describes

individuals with allergic tendencies, and individuals getting atopic eczema have an

increased risk of developing other atopic conditions such as asthma and hay fever later

in life.

The negative effect of E. coli for the risk of getting atopic eczema has been

demonstrated in an epidemiological study where more than 900 infants were followed

from one month to two years of age. It was demonstrated that the presence of E. coli at


104

one month of age was associated with a higher risk of developing eczema at the age of

two years, and this risk was increased with increasing numbers of E. coli (Penders et al.

2007). Also, colonisation with Clostridium difficile was associated with a higher risk of

developing eczema (Penders et al. 2007).

High presence of E. coli and C. difficile indicates an unbalanced (biased) gut-microbiota

(dysbiosis), and it seems as early overgrowth with E. coli can be a contributing factor to

the atopic eczema. Overgrowth and an unbalanced microbiota mean that fewer

organisms dominate the flora, i.e. the bacterial diversity has decreased to a lower value.

It was shown in the 35 children from Göteborg, London and Rome (see above) that

infants with low bacterial diversity at one week of age got atopic eczema in a higher

frequency after 18 months than infants with high bacterial diversity at one week of age

(Wang et al. 2008). The diversity was in this case measured with a method called

Terminal Restriction Fragment Length Polymorphis (T-RFLP). T-RFLP is

schematically shown in Figure 26. This method that is directed towards the 16S rRNA

genes in a bacterial community is well suited for measuring bacterial diversity in

samples of complex bacterial communities.

A low bacterial diversity in the digestive tract is a negative factor by itself,

irrespectively of what groups of bacteria that are dominating the flora (Wang et al.

2008). Interestingly, it has been shown that the bacterial diversity in faeces of children

from a rural African village in Burkina Faso was higher than in faeces from European,

urbanized, children (De Filippoa et al. 2010). The diet and living conditions in the rural

African village are presumably more similar to that of ancient human settlements than

that of the current European community. Furthermore, the European children had a

higher proportion of Enterobacteriaceae in the gut than the African ones. Similar results

were seen in both children and healthy adults from rural Malawi (Afica) when they were

compared with subjects from metropolitan United States of America, i.e. the diversity

was lower for the American subjects (Yatsunenko et al. 2012).

It has been shown that administration of the probiotic strain L. paracasei F19 can

reduce eczema in weaning infants (West 2008). One-hundred-seventy-nine infants, four

month of age, were randomized to a daily intake of L. paracasei F19 (dose 108 CFU per

day) for 9 months. The probiotic treatment reduced the risk for eczema with 50% and

enhanced the Th1/Th2 ratio. It was concluded that “the reduction of eczema might be

explained by probiotic effects on both T cell-mediated immune responses and

reinforced gut microbial function” (West 2008).

T- helper cells (Th-cells) are a sub-group of lymphocytes that are involved in establishing and maximizing the capabilities of the immune system. They have no cytotoxic or phagocytic activity. Proliferating Th-cells differentiate into two major subtypes, Th1 and Th2. The different subtypes produce different cytokines, and affect different lymphocytes.

When it comes to probiotics and diversity, an obvious question is: Eating a single

probiotic strain, doesn’t that lower the bacterial diversity in the gut? And perhaps

somewhat unexpected the answer is no, not necessarily. It has been shown in adults,

eating the single strain L. plantarum 299v that the bacterial diversity on the rectal

mucosa actually was increased by the probiotic administration (Karlsson et al. 2010).

Thus, consumption of probiotics can be a strategy to favour a more diverse intestinal

microbiota. On the other hand, this is presumably not true for all types of probiotics. An

explanation to the increased diversity may be that L. plantarum 299v improved the


105

environment of the digestive tract in such a way that it opens up for the coexistence of

bacterial groups that had been suppressed by a suboptimal environment.

Figure 26. Principle of the PCR-based method Terminal Restriction Fragment Length Polymorphis (T-RFLP). The target gene for the analysis is the 16S rRNA gene. The method that normally reflects the dominating bacterial taxa at the hierarchical family-level is suitable for evaluation of bacterial diversity.

Old age

Age as such seems only to have an influence on the composition of the human

microbiota in very young or in very old individuals, i.e. no conclusive differences in the

gut microbiota could be seen in humans between 25 years and 78 years of age (Biagi et

al. 2010). However, humans between 99 and 104 years old were shown to have lower

diversity and an increased proportion of Proteobacteria compared to subjects between

25 and 78 years (Biagi et al. 2010). This shift in microbiota in these extremely old

individuals is probably due to a failing health-status. The gut microbiota in older people

correlates with health status, and the diversity is lower in hospitalized individuals than

in community dwellers (Claesson et al. 2012).


106

On the other hand, the ageing process is known to adversely affect the immune system

(Goodwin 1995; Kalache 1999), so perhaps an active intervention with probiotics can

be fruitful. For example, thirty healthy elderly volunteers were given a dietary

supplement of a probiotic drink containing Bifidobacterium lactis for three weeks.

Several markers for the function of the immune system were improved by the treatment.

Thus, the proportion of mononuclear leukocytes staining positively for CD3+ (T

lymphocytes), CD4+ (MHC II–restricted T cells), CD25+, and CD56+ (NK cells) as

well as the phagocytic capacity of mononuclear and polymorphonuclear phagocytes and

the tumoricidal activity of NK cells increased significantly in blood after the probiotic

administration. The greatest relative increase in immune function occurred in

individuals with poor immune response before the intervention (Gill et al. 2001).

Dietary factors affecting the gut

microbiota

One purpose of eating probiotics is to affect the bacterial flora of the digestive tract in a

beneficial direction, i.e. to increase the proportion of beneficial bacteria and decrease

the amount of adverse bacteria. For the time being, lactobacilli, bifidobacteria,

Faecalibacterium and maybe Ruminococcus are seen as beneficial components of the

gut-microbiota while opportunistic pathogens and pro-inflammatory bacteria are

regarded as adverse. Additionally, a high bacterial diversity in the gut is beneficial for

the health while a low diversity is linked to physiopathological effects.

It is clear that diet can affect both the composition of the gut microbiota (Claesson et al.

2012) and the diversity (Wu et al. 2011). The abundance of singular taxa can be

detected within 24 hours after initiating a new diet, but more thorough changes of the

microbiota needs a long-term dietary regime (Wu et al. 2011; Claesson et. al 2012).

A well-known negative factor is antibiotics that decrease diversity and promote

overgrowth of adverse bacteria. A dietary factor with negative effects is fat that in high-

fat diets decrease diversity and promote pro-inflammatory bacteria.

Dietary factors with beneficial effects on the bacterial flora are, besides probiotics and

lactic acid fermented foods with high numbers of live lactobacilli, dietary fibres in

general, but especially those with more substantial prebiotic effects as, for example,

inulin and fructo-oligosaccharides. There are also dietary sources of polyphenols that

possess prebiotic effects, e.g. in green tea and in red wine.

The knowledge of what singular dietary compounds that actually can affect the bacterial

flora of the digestive tract in positive or in negative directions is still highly limited. It is

however clear that a varied diet with a high content of fibres and a low content of fat

over time increase the diversity of the microbiota of the digestive tract (Claesson et al.

2012). The negative effects of an adverse microbiota (low diversity and an increased

proportion of proinflammatory taxa) are less obviously seen in younger and middle


107

aged adults, but become more clearly demonstrated in older individuals (Claesson et al.

2012).

To consider

It is important for a relevant discussion around the role of the microbiota in the human

body to be aware of the different bacterial taxa that are involved, and a prerequisite for

being able to take part in such a discussion is to know these taxa by their scientific

names. Making the microbiota more apprehensible, it is fruitful to have knowledge

about the evolutionary relationships between the different taxa because phylogenetic

relationships reflect much of the characteristics different taxa may share (or not share)

with each other. Phyla as Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and

Verrucomicrobia are seen frequently whenever the human gut microbiota is mapped

and especially Firmicutes and Bacteroidetes are often making up major parts of the

colonic microbiota. Firmicutes represents many different genera as for example,

Streptococcus, Clostridium, Ruminococcus, Eubacterium, Faecalibacterium and

Veillonella. Streptococcus is more prominent in the mouth and the upper part of the

small intestine (jejunum) and the others more prominent in the lower part of the small

intestine (ileum) and in colon. Bacteroidetes is mostly represented by Prevotella and

Porphyromonas in the mouth and by Bacteroides in the colon.

E. coli belongs to the family Enterobacteriaceae which is a family included in the class

of Gamma-proteobacteria in the phylum Proteobacteria. The load of E. coli differs

widely between individuals, and it can also change with time. Overgrowth of E. coli is a

negative factor for the health status and can lead to increased permeability of the

mucous membrane and to low-grade systemic inflammation.

Bifidobacterium belong to the phylum Actinobacteria and can reach high colonic loads,

especially in young individuals. Bifidobacterium is a positive factor for the health

status. Gens from Verrucomicobia are often seen both in human samples and in samples

from the environment, but relatively few of these genes can be identified to known

species. The reason is that the bacteria these genes belong to often are difficult to grow

in pure cultures in the Laboratory. An exception is Akkermansia which is known for its

ability to digest mucin. Akkermansia is found in the intestine of both humans and other

mammalians and seems to be a marker for an intestinal ecology in balance. No known

pathogens belong to the phylum Verrucomicrobia.

It is recommended that the Table 4 is studied in some detail.

The effects of the microbiota on human homeostasis (homeostasis is the ability or inclination of an organism to maintain internal equilibrium by adjusting its physiological

process) became most clear in neonates (babies) where the bacterial flora consists of just

a few taxa and the immune system of the very young individual is having its first direct

experiences of bacteria. In other words, the pioneer gut microbiota can affect the health-

status of the growing individual but may also have consequences for the health status

later in life. Obviously, there is a close relationship between a mother and her baby and


108

it is the mother that primarily contaminates the new borne baby with her own

microbiota. Hence, a mother with a disturbed intestinal microbiota and/or vaginosis can

transfer a pioneer microbiota with negative consequences for the baby.

Low-bacterial diversity, an overgrowth of E. coli, and/or a high proportion of

Bacteroides fragilis can have negative consequences for health status of the human host,

while high diversity and high proportions of Lactobacillus and Bifidobacterium are

beneficial factors. Both E. coli and B. fragilis are gram-negative bacteria with

lipopolysaccharides (LPS) associated to the cell wall. LPS and especially LPS from E.

coli and related taxa in the family Enterobacteriaceae have strong pro-inflammatory

capacity.

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IV. Effects


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Intention

The Lecture-block IV “Effects” is regarding to the text volume, the far largest block.

Lecture-block IV is divided into different subparts, first an introductionary one, then

different aspects of probiotic effects on the digestive tract, including the upper

respiratory tract, and finally, more systemic effects involving liver, pancreas, and effects

on dysfunctions associated with the metabolic syndrome.

The prime intention with block IV is to give an account of different health beneficial

effects of probiotics and the gut microbiota on different functions and organs in the

human body. Attempts are also made to give reasonable explanations to the observed

effects. Though, it should be held in mind that probiotics and the influence of the gut

microbiota on the body are subjects that until relatively recently either was minimized

or derided by the medical and nutritional establishment. An increasingly number of new

scientific reports are gradually changing the opinion towards acceptance but still the

knowledge about effects and the more detailed explanations for many of these observed

effects are still insufficient. The field of probiotics is scientifically complicated due to

the biological complexity. Just the vast numbers of different kinds of living bacteria, all

with different characteristics that are involved, make simplified generalisations difficult.

For the time being there is a wealth of suggested effects of probiotics and of the gut

microbiota on human homeostasis and dysfunctions, but the suggestions are mostly

based on few or even solitary scientific reports. Many observations come from animal

studies, and the majority of the reported human studies include relatively few

individuals, i.e. the clinical trials are small compared with studies evaluating effects of

pharmaceuticals. In other words, there is often no general consensus in the scientific

community for many of the reported effects. This lack of generally accepted truths can

lead to confusion. Lecture-block IV should not be regarded as a complete review of all

available reports in the area, neither as a testimony of basic, generally accepted

knowledge. Lecture-block IV provides in parts, short accounts of selected scientific

studies that either points on interesting observations or on reports judged as being good

evidence for a certain effect.

Another important aim of lecture-block IV is to give examples how different health

effects can be scientifically visualised, e.g. design of studies, markers of health and

disease and end-points for animal and human studies. Presented facts are to a high

degree given with references so the reader will be able to find and make her/his own

judgement of the relevance of the reported effects.

Probiotic bacteria

Genera such as Lactobacillus and Bifidobacterium have been identified as beneficial to

the human health. Popular species to be used commercially as probiotics are, for

example, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus acidophilus,


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Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus

plantarum, Bifidobacterium longum and Bifidobacterium animalis. However, it should

be held in mind that the phylogenetic difference is extremely wide between the two

genera Lactobacillus and Bifidobacterium as they belong to different phyla, but there

can also be considerable differences between different Lactobacillus species, e.g.

between L. acidophilus, L. fermentum, L. reuteri and L. plantarum. Even within

different strains of the same species, the genomic differences can be considerable due to

the wide limits of the bacterial species concept. Consequently, due to major genetic

differences between different probiotic strains, it is to be expected that both the human

body and the gut microbiota will respond differently to different probiotic strains. This

also stresses the fact that it is of crucial importance to know both the species name and

the strain identity of probiotics.

Originally, “probiotics” meant organisms or substances that contribute to intestinal

microbial balance, in contrast to antibiotics that counteract microbial activity (Lilley and

Stillwell 1965; Parker 1974). However, the currently widely accepted definition is that

probiotics are live microorganisms which when administrated in adequate amounts

confer a health benefit on the host. Thus, the traditional idea, which is reflected in the

early definitions of probiotics, presumes a balance between beneficial and adverse

bacteria in the gut and as long as the beneficial bacteria are present in adequate numbers

they strengthen gut-health, while excessive growth of adverse bacteria creates problems.

Consumption of probiotics affects this balance in a positive direction by increasing the

amount of beneficial bacteria in the gut, and hence, affects the balance in a beneficial

direction. However, this indirect way of affecting the human body through the

composition of the gut microbiota is not the only option for probiotics to affect the

body. Ingested bacteria will pass through the digestive tract and hit the lining of

epithelial cells covering an estimated area of about 250 m2. The ingested bacteria will

also come into contact with immune-cells, i.e. macrophages, dendritic cells, B-

lymphocytes, and T-lymphocytes found in Peyer's patches and other gut-associated

lymphoid tissue (GALT). These interactions between ingested probiotics, presumably

starting already in the mouth, carry on through the system as the probiotics pass down

the gastro-intestinal (GI) tract.

Ingested bacteria can trigger major effects also when passing through the upper, lowly

populated parts of the GI-tract. For example, it has been shown in healthy volunteers

that exposure to the probiotic strain L. plantarum WCFS1 in duodenum

(“tolvfingertarmen” in Swedish; duodenum is situated directly after the stomach) that an

exposure for L. plantarum WCFS1 during a relatively short time (i) regulated epithelial

tight junction proteins (Karczewski et al. 2010), and (ii) induced immune responses

(van Baarlena et al. 2009). In the former case, it was shown that the scaffold protein

ZO-1 and the transmembrane protein occludin were significantly increased in the

vicinity of the tight-junction structures, which leads to an improved mucosal barrier

function of the mucosa (Karczewski et al. 2010).

Concerning the immune response, L. plantarum WCFS1 caused a response in the

duodenal mucosa of healthy volunteers, and it was shown that the gene expression

induced in the intestinal tissue by live L. plantarum WCFS1 correlated with that of

immune tolerance via the NFkappa-B (NF-κB) pathways (van Baarlena et al. 2009).

Interestingly, heat-killed and live L. plantarum cells induced different expression

profiles in the epithelial cells.


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NF-κB stands for “nuclear factor kappa-light-chain-enhancer of activated B cells”. NF-κB is a protein complex that controls the transcription of DNA. NF-κB plays a key role in regulating the immune response to infections. Incorrect regulation of NF-κB is linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development.

L. plantarum 299v has been shown to increase mucin (MUC2 and MUC3) expression

level in epithelial cells originating from colon (HT-29 cells; Mack et al. 1999; Mack et

al. 2003). Mucins make up the slimy surface of the mucosa and protect the mucosa from

aggressive microorganisms and unwanted compounds.

Considering the different ways that probiotics can affect the body, one of the most

important outcomes for health is that probiotics can improve the condition of the

mucosa and thus strengthen the epithelial barrier function of the mucosa. A decreased

barrier function of the mucosa and, consequently, an increased leakage through the

mucosa is a problem linked to many different diseases and dysfunctions.

It should be held in mind that probiotics are living organisms and as such they can

exercise several fundamentally different actions on different target-functions in the

body.

Probiotics can: 1) Modulate the immune response, 2) strengthen the tight-junctions between the epithelial cells in the mucous membrane, 3) stimulate the epithelial cells of the mucous membrane to produce more mucin, 4) decrease the proportion of adverse bacteria in the gut, 5) improve the digestion, by for example, increasing the absorption of iron and other essential metals, and breaking down indigestible carbohydrates and polyphenols.

All probiotic strains are not able to fulfil all sorts of missions. The capacity of different

strains for acting on the different target-function can vary. A rational for combining

different probiotic strains in a mixture could be that they are differently good in

affecting the different function, e.g. one strain can be especially good in modulating the

immune system through dendritic cells and T-cells while another can have a strong

influence on the epithelial barrier function of the mucosa.

How to prove beneficial health effects

Evidence based

Probiotics are living microorganisms with scientifically demonstrated health beneficial

effects when administered to the body. A crucial question is then how these health

beneficial effects should be proved? A prerequisite for the Natural sciences is that they

should be evidence-based. Evidence-based means that rules for proving an explanation


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should be set up before-hand and then followed. It is not in line with the idea of Natural

science to have a preconceived explanation and then search for proofs to support this

explanation and in the process neglecting data speaking against it. Furthermore, it is

important in evidence-based science that any trained scientist should come to the same

conclusion when mimicking the study and following the rules that have been set up.

In evidence based medicine, evidences obtained from different types of studies are

ranked differently. Highest value in proving the effect of a certain treatment in medicine

is the (i) “randomized, placebo-controlled trial (often abridged, RCT)”, (ii) secondly

ranked is the “cohort study” and then following in order: (iii) “case control study”, (iv)

“case report”, (v) “expert opinion”, (vi) “animal models” and (vii) “in vitro studies”. It

may seem odd that expert opinions are ranked higher than animal studies. However, this

is medicine where the object always is the patient and the expert in this case is a trained

and experienced physician that during his daily work comes in contacts with a lot of

patients and clinical situations, i.e. the expert opinion is based on empirically gained

knowledge from observations of the reality. In many other fields of Natural sciences as

for example nutrition, a well-designed animal study must be regarded to have a much

higher explanation-value than the opinion of an individual scientist in nutrition or an

individual nutritionist.

In the expression “randomized, double blind, placebo-controlled trial”: “double blind”

imply that all subjects in the study get a product where they are unaware if it contains

the active compound or not, i.e. the administrated products are coded and not only the

study-subjects but also the operators of the study, and the ones performing the analyses

are unaware of which individuals that receive the active product or which ones that

receive the placebo. Placebo is the seemingly similar product without the active

compound). “Randomised” implies that test persons at random are divided into different

test groups, i.e. it is not allowed to select certain persons for certain test groups.

Sometimes so called “cross over” is applied on RCTs, meaning that after a certain run-

time of the study, the volunteers that got the placebo instead get the active product, and

vice versa.

In a so called open-label trials, both the researchers and participants know which

treatment that is being administered. Open-label trials may be appropriate for comparing

two, very similar treatments to determine which one that is most effective. An open-

label trial may still be randomized.

A cohort is a group of subjects who share a common characteristic or experience within

a defined period. A “cohort study” (sometimes also called “panel study”) is an

observational study. Cohort studies can either be conducted prospectively (in the future)

or retrospectively (looking backwards) from archived records.

A “case-control study” identifies factors that may contribute to a condition by comparing

subjects who have that condition (the cases), with subjects who are without the studied

condition, but otherwise similar (the controls).

“Case report” is used in medicine to describe, in detail the symptoms, signs, diagnosis,

treatment, and follow-up of an individual patient.


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All trials have an endpoint (end-point) or a primary outcome, i.e. a mark of termination

or completion. In clinical or nutritional trials, the endpoint refers to the sign that defines

the final outcome of the trial.

Evidence-based medicine can be defined as the integration of high quality research-

evidence with clinical expertise and patient values. The concept of evidence based

medicine has been transferred to the field of nutrition, i.e. evidence-based nutrition.

Thus also in nutrition, including probiotics, the randomized, placebo-controlled trial

(RCT) has the greatest weight of proof, because RCT is the only design that permits

strong causal (implying an effect) inference (the act of conclude from evidence).

Koch’s postulates

The classical way to prove the pathogenic ability of an organism and link it to a

particular disease is to follow the different steps in the so called Koch’s postulates that

were set up by Robert Koch in 1884:

1) The microorganism can always be found in association with a particular disease, but not in healthy individuals. 2) The microorganism can be grown in the laboratory in pure culture. 3) This pure culture will produce the same disease when inoculated into a new, susceptible individual. 4) The microorganism can be recovered from the infected, diseased individual and grown again in pure culture in the Laboratory.

Should the postulates of Koch be converted for proving the health beneficial effect of a

probiotic organism it may be:

1) The microorganism can be found in association with healthy individuals.

2) The microorganism can be grown in the Laboratory in pure culture.

3) This pure culture will exercise health beneficial effects when placed into a new

subject.

4) The microorganism can be recovered from the inoculated subject and grown again in

pure culture in the Laboratory.

However, there are some general difficulties to apply the modified Koch’s postulates on

probiotics: One obstacle is to isolate and identify the probiotic strain from the digestive

tract where a wide range of similar, related organisms may be present. This requires a

method where individual bacterial strains can be identified and distinguished from other

bacterial strains of the same species.

In contrast to true pathogenic organisms which only are found in diseased individuals,

probiotic bacteria are not necessarily limited to healthy individuals. They may very well

be present in diseased individuals where the probiotic bacteria do not managed to cure

the disease.

And last but not least, from a medical point of view it is relatively easy to measure how

a patient is cured from a disease, or getting a disease after being infected with a

pathogenic agent. It is much more difficult to prove improvement of health in non


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diseased individuals, or the prevention of future diseases, by the consumption of a

specific nutritional compound.

Medicine versus nutrition

In medicine, the endpoint is a disease or at least a dysfunction with a diagnosis and the

treatment aim to counteract the disease or dysfunction. If the treatment involves a

substance administrated orally, it means that the subject will be eating a drug. Usually, a

drug will be a defined and powerful substance that is active in low concentrations.

Hence, the effects of the drug can be noted after relatively short time of treatment and if

a randomised placebo-controlled trial (RCT) is to be designed for proving the

efficiency, the individuals in the placebo group will never come in contact with active

compound.

This is in sharp contrast to the situation in nutrition, where the endpoints are

improvement of health-status or prevention of future dysfunctions and diseases.

Furthermore, the test substance (the active food component) should preferably be tested

in primarily healthy individuals. Food components that are supposed to be wholesome

without side effects are usually less powerful than drugs and the health-improving or ill-

preventing effects are often not seen at first, but need prolonged exposure periods to be

made visual. Furthermore, if a RCT is set up, it is in nutrition often difficult to have a

placebo group where the individuals never come in contact with the active compound as

the is a dietary compound, and test persons in the placebo group can’t stop eating, and

must include essential components in the diet. The problem becomes especially

troublesome if the study should be run over long time-periods.

Nutritional factors can affect the health status from birth to death. However, when a

disease has emerged, it is the responsibility of Medicine to mitigate developed

dysfunctions. This is exemplified in Figure 27. Nutritional knowledge and dietary

means can be applied to prevent the metabolic syndrome, and to a certain degree

Nutrition can also contribute by suppressing symptoms of the metabolic syndrome.

However, if the dysfunctions became substantial or if a disease related to the metabolic

syndrome emerges, it becomes a medical problem. The proof of efficiency of drugs

against diseases, in this case for example, non-alcoholic fatty liver disease, obesity, type

2 diabetes and/or cardiovascular diseases, are preferably achieved through RTCs

(Figure 27).

Drugs are powerful, active at low concentrations and show effects after relatively short

exposure time. In contrast, if we believe that supplement of a certain dietary compound,

let’s say multivitamins, through life decrease the risks for developing high blood

pressure in the age of 55, how should that be evaluated by a RCT? It becomes too

complicated, too expensive and ethically totally impossible to have a RCT going for

years and where the placebo group are denied important food components as, for

example, vitamins. What then must be done instead of a RCT is to set up a cohort study.

This in order to see if there are any negative correlation between the consumption of the

dietary compound (in this case multivitamins) and the disease or dysfunction (in this

case high blood pressure). If such correlation is found it remains to be proved that the

consumption of multivitamin also is the actual cause for the blood pressure reducing


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effect, and not just a consequence caused by some other factor than the vitamins.

However, such causal evidence can be collected with the use of animal models. On the

other hand, this proof of concept is weaker than a RCT, but in many cases is the only

feasible way. With this in mind, results from short-time RCT within the field of

nutrition that in spite of all the muddling circumstances actually points at significant

improvements in health status should not be easily dismissed, especially if several

studies point in the same direction.

Figure 27. An individual can through the years acquire the metabolic syndrome, which increases the risk for diseases as non alcoholic fatty-liver disease, type 2 diabetes and cardiovascular diseases. Nutritional factors can have long-term effects and decrease the risk of developing the metabolic syndrome, and they can mitigate the symptoms of the metabolic syndrome which lower the risk for developing disease. The diseases are treated with drugs within the scientific field of Medicine. The effects of drugs are preferably proved in randomised, double blind, placebo controlled studies (RCTs). The long-term effect of dietary components is difficult to study with RTC, instead Cohort studies and animal models have to be used for proving causal effects.

Probiotics are dietary supplements and probiotics are primarily aiming to improve the

health-status and prevent dysfunction and diseases. A general problem in evaluating the

probiotic effect in RCT is that the same bacterial strain or strains with similar effects as

the test-strain can be spontaneously present in some of the test subjects without any

administration of the active product. The probiotic strain or similar strains can also

occur spontaneously in lactic acid fermented foods that the subject might be eating

during the study time, or test persons can be contaminated with the test-strain from

other individuals by direct contact. Furthermore, it is impossible in a RCT aiming to test

a specific probiotic strain to get rid of the disturbing influence of the test person’s own

microbiota of resident bacteria, and it is extremely difficult to exclude all influence of

environmental bacteria contaminating the test person during the study time.


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Immune modulation

Probiotics can affect the immune system which can be seen by following changing

levels of different immunological agents in blood and tissue, and these agents can be

used as markers for the function of the immune system. One key factor in the immune

system is the T-regulatory cells which are involved in the regulation of immune

response, maintaining immunological self-tolerance and immune homeostasis, and also

the control of autoimmunity and cancer surveillance. T cells can be characterised by the

expression of FoxP3 (fork head box P3) which is a protein with a comprehensive

function for regulatory T cells. The number of regulatory T cells and especially those

expressing FoxP3 is important for describing different types of diseased states.

Consequently, immune markers as, for example, the regulatory T cells are highly

interesting to follow in clinical trials with probiotics in order to give an explanation to

observed effects on dysfunctional or diseased conditions or prevention of a diseases.

The use of immunological markers in order to explain a studied effect on a dysfunction

is exemplified in the following study where it was shown that administration of the

probiotic strain L. paracasei F19 reduced eczema in weaning infants (West 2008). One-

hundred-seventy-nine infants, four month of age, were randomized to a daily intake of

L. paracasei F19 (dose 108 CFU per day) for 9 months. The probiotic treatment reduced

the risk for eczema with 50% and enhanced the ratio of two phenotypes of T-helper

cells (Th), i.e. the ratio of Th1 and Th2. It was concluded that “the reduction of eczema

might be explained by probiotic effects on both T cell-mediated immune responses and

reinforced gut microbial function” (West 2008). In conclusion, the authors saw a

substantial preventive effect on a disease and could provide an explanation to the effect

by following critical immunological markers in form of T-helper cells.

T-helper cells (Th) are a sub-group of lymphocytes involved in establishing and maximizing the capabilities of the immune system. Th-cells have no cytotoxic or phagocytic activity. Proliferating Th-cells differentiate into two major subtypes, Th1 and Th2. The different types produce different cytokines, and affect different lymphocytes.

In contrast to the above referred study, many human studies on probiotic effects only

demonstrate effects on immunological markers without link to effects on a particular

dysfunction of the body, i.e. the change of the immunological marker become the end-

point of the study without direct connection to a dysfunction or a disease.

This can be exemplified by a study where the following claim was given as a conclusion

of the study “Milk fermented with yoghurt cultures plus L. casei DN-114001 was able

to modulate the number of lymphocytes and CD56 cells in subjects under academic

examination stress” (Marcos et al. 2004). The study was a prospective, randomized,

controlled and parallel one, where university students were allocated to one of two

groups, receiving during 6 weeks (3 weeks prior to, as well as the 3-week duration of

the examination period) either: a glass (200 ml) of semi-skimmed milk each day

(control group, n=63) or two 100 ml portions per day (treatment group, n=73) of

fermented milk (Actimel, Danone) containing Lactobacillus delbrueckii subspecies

bulgaricus (107 CFU/ml) and Streptococcus thermophilus (10

8 CFU/ ml) and

Lactobacillus casei (paracasei) DN-114001 (108 CFU/ml). Anxiety and immunological


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measurements were monitored at the baseline (start of treatment) and at the end of study

(6 weeks later at the end of the examination period). The anxiety would have been a

nice end-point in this study but unfortunately no significant effects were reached in

anxiety. Among several measured blood parameters, significant changes were only seen

for the number of lymphocytes and CD56 (Marcos et al. 2004). The practical relevance

for the health status by this change in immune parameters remains unclear. The immune

system is highly complex with a waste number of different markers that often are

related to each other and where a comprehensive and full knowledge of function and

interplay is lacking, or at least highly muddled.

CD stands for “clusters of differentiation”. CD56 positive cell means a T-cell with the membrane protein CD56 on the surface. CD56 is the same as NCAM (Neural Cell Adhesion Molecule) which is a binding glycoprotein expressed on the surface of neurons, glia, skeletal muscle and natural killer cells. NCAM has been implicated in cell-cell adhesion, neurite outgrowth, synaptic plasticity, and learning and memory.

In general, immunological markers are insufficient end-points. On the other hand,

combined with endpoints revealing undisputable health improvements, immunological

markers can be helpful to explain causes and mechanisms. Good endpoints in the study

referred to above had been if the students drinking the product have had better results in

the exam or less anxiety during the exam-period.

Virus

Rotavirus diarrhoea

Probiotics can have effects against viral diarrhoea, which primarily have been shown for

rotavirus gastroenteritis (Huang et al., 2002). Thus, probiotics do not only targeting

components of the bacterial flora of the digestive tract, but also viruses in the small

bowel. Rotavirus is one of several viruses that cause the type of infections that in every

day talk are referred to as “stomach flu”. Rotavirus gastroenteritis is usually an easily

managed disease in childhood, but nevertheless, worldwide more than 500 000 children

younger than 5 years die each year due to rotavirus infection, as a result of dehydration

due to the vomiting and diarrhoea which is a consequence of the disease.

The name rotavirus refers to the wheel-like appearance of the virus that is a double-

stranded RNA-genome. Rotavirus is the major cause of viral gastroenteritis in infants

and young children. The infection is spread by the faecal-oral route, and rotavirus

infects the epithelial cells lining the small intestine with an incubation period of 1-2

days. The duration is 4-7 days, and the symptoms are vomiting, diarrhoea, fever and

pain, and as said, rotavirus gastroenteritis can lead to severe dehydration in small

children.


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Especially, the probiotic strain L. rhamnosus GG (Valio, Finland) has proved to have

effects towards the symptoms of rotavirus diarrhoea. L. rhamnosus GG mitigates the

symptoms and reduces the duration of rotavirus gastroenteritis (Marteau et al. 2001). It

seems that L. rhamnosus GG activates the unspecific immunological defence during the

infection, and increases the level of specific antibodies in the following-up period. Thus,

it appears as the probiotic effect against rotavirus primarily is an effect mediated by the

immune system. However, also probiotic derived effects on the mucosa (improved

mucosal status) can contribute to a higher resistance against the infection and decreased

symptoms of the disease.

Winter vomiting disease

In perspective of the effects of probiotics against rotavirus gastroenteritis, a relevant

question is: Do probiotics also help against winter vomiting disease (calicivirus

gastroenteritis)? Calicivirus gastroenteritis, also called winter vomiting disease or

“stomach flu” is caused by a RNA virus of the genus Norovirus and family

Caliciviridae. Calisivirus is preferably transmitted in institutional settings and by

contaminated food and water. Clinical features of calicivirus gastroenteritis are

diarrhoea and vomiting, generally without fever, after an incubation period of 2-3 days

(the duration is one to eleven days). No specific therapy exists, but rehydration can be

mitigated by replacing fluid losses and correcting electrolyte disturbances.

The question remains: Do probiotics help against winter vomiting disease? And not

much to an answer can currently be find in the literature. In one open cased-controlled

study on elderly in a service facility in Japan, it was concluded that during a treatment

period of one month during an outburst of norovirus gastroenteritis no significant

difference in the incidence of norovirus gastroenteritis occurred between a treatment

group consuming Lactobacillus casei Shirota in milk (39 individuals) and a non-

administered control group (38 individuals) (Nagata et al. 2011). On the other hand, the

mean duration of fever, here defined as a body temperature above 37oC, after the onset

of gastroenteritis was 1.5 days in the probiotic group and 2.9 days in the non-treated

group, i.e. the probiotic treatment reduced the duration of fever with almost 50%.

Common cold

Common cold, or just cold, is a viral upper respiratory tract infection (also called acute

viral rhinopharyngitis or acute coryza) that can be caused by several different viruses,

i.e. rhinoviruses (in about 30–50% of cases), coronaviruses (10–15%) and influenza

virus (5–15%), but common cold can also be caused by other viruses as human

parainfluenza viruses, human respiratory syncytial virus, adenoviruses, enteroviruses or

metapneumovirus. Cold is the most frequent, human infectious disease. Adults have on

average 2 to 4 infections per year and children have 6 to12 infections per year. Due to

the fact that there are so many different types of viruses and that the viruses tend to

mutate, it is impossible to become immune to all colds.

Without specifying which viruses that have been active and responsible for the

infection, consumption of probiotics has in some cases been proved helpful in


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mitigating common cold (de Vresea et al. 2005; de Vresea et al. 2006; Berggren et al.

2011).

There were two double blind, randomized, controlled trials on common cold, using a

probiotic study-product in form of tablets containing Lactobacillus gasseri PA 16/8,

Bifidobacterium longum SP 07/3 and Bifidobacterium bifidum MF 20/5 (Merck,

Consumer Health Care). Together the three strains made up a dose of 5x107 CFU (per

tablet). In the first study, 454 healthy volunteers (age 18–67) consumed 1 tablet per day

for 3 months (de Vresea et al. 2005), and in the second study 479 healthy volunteers

(age 18–67) consumed 1 tablet per day for 3 to 5 months (de Vresea et al. 2006). Both

studies resulted in shorter common cold episodes and reduced severity of symptoms.

However, the intake of the study product had no effect on the incidence of common

cold.

In contrast, the study of Berggren et al. (2011) showed a reduction in the incidence of

common cold episodes. Also, the number of days with symptoms was reduced together

with a reduction of the “total symptom score” and a reduction of the pharyngeal

symptoms. In this study, the two probiotic strains L. plantarum HEAL9 and L.

paracasei 8700:2 were used in a dose of 109 CFU (Probi AB). The bacteria were in

form of a lyophilised powder in malto-dextrine, packet in sachets. The placebo

contained malto-dextrine without bacteria (Berggren et al. 2010). 272 healthy

volunteers (age 18 to 65) were eating the product (a sachet per day) for 12 weeks. The

product is sold on the Swedish market both as food supplements (tablets with the brand

name “Probi frisk”). The probiotic strains have also been included in a food product

where the bacteria are mixed in fruit juices under the brand name “Friscus”

(Skånemejerier, Malmö). However, this product was dropped from the Swedish market

after some years. A somewhat similar product with the same probiotic strains was

launched 2012 in Australia as a breakfast juice (www.goldencircle.com.au).

The traditional view on probiotics is that it is beneficial for the gut. In the case of

common cold however, it is beneficial for the upper respiratory tract. On the other hand,

the common cold viruses infect the mucous membranes even if they are situated in the

upper respiratory tract instead of the intestines, and probiotics administrated orally will

for certain reach the mouth and throat, and presumably also, indirectly to some extent

reach the mucous membranes of the nose. And, if probiotics can be beneficial for the

condition of the mucosa in the gut and for the immune system, it is perhaps not so

surprising that it also can have effects on the mucosa of the upper respiratory tract.

Bacterial balance and C. difficile

An example of application of probiotics that is well in line with the original idea that

probiotics can improve the balance of the bacterial flora of the gut is when probiotic

therapy successfully has been used against Clostridium difficile. C. difficile (belongs to

Clostridium cluster XI; class Clostidia) can cause colonic inflammation. C. difficile is

an anaerobic, spore forming rod that frequently is found in minor (and then harmless)

http://www.goldencircle.com.au/


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amounts in colon of healthy individuals, but can cause colitis when it multiply to high

amounts.

Antibiotics can disturb the bacterial balance of the gut which leads to “antibiotic

associated diarrhoea”. Antibiotic associated diarrhoea (also called pseudomembranous

colitis) is an infection in the colon caused by an unfavourable change in the gut-

microbiota due to the antibiotic treatment. C. difficile is the major cause of antibiotic

associated diarrhoea (C. difficile colitis). The incidence of antibiotic associated

diarrhoea in hospitalized patients is reported to be 3-29 % and C. difficile accounts for

20-60 % of these diarrhoeas (Wiström et al. 2001; McFarland 1993). Over time there

has been an increase in the incidence of C. difficile diarrhoea (McFarland 1993; Pépin et

al. 2003; Ricciardi et al. 2007). Furthermore, to make thing worse, a highly virulent C.

difficile type (ribotype 027) with increased morbidity (morbidity is the incidence of ill

health) and mortality (mortality is the incidence of death in a population) has emerged

(McDonald et al. 2005; Loo et al. 2005). C. difficile produces several virulence factors,

i.e. enterotoxin, cytotoxin and an inhibitor for bowel motility. Clinical symptoms range

from mild diarrhoea to toxic megacolon which in turn may lead to bowel perforation. C.

difficile associated diarrhoea is usually treated with the antibiotics metronidazole or

vancomycin.

In several studies and with treatment by different probiotic organisms, probiotics have

been proved effective against antibiotic associated diarrhoea and C. difficile induced

diarrhoea, (D’Souza 2002). Especially, the preventing effect is obvious (Hickson et al.

2007; Klarin et al. 2008). In the study of Hickson et al. (2007), 100 g product (active

product in form of Actimel, Danone, or a placebo product) were given twice per day

during antibiotic treatment of 135 hospital patients (mean age 74 years) and one week

after termination of the antibiotic treatment. The product (Actimel) contained L. casei

(L. paracasei) DN-114001, (108 CFU/ml Actimel), Streptococcus thermophilus (10

8

CFU/ml Actimel) and L. delbrueckii subspecies bulgaricus (107

CFU/ml Actimel). Only

seven out of 57 (12%) of the patients in the probiotic group developed diarrhoea

associated with the use of antibiotics, compared with 19 out of 56 (34%) in the placebo

group. No one in the probiotic group and 9 out of 53 (17%) in the placebo group had

diarrhoea caused by C. difficile. The conclusion was that Actimel can reduce the

incidence of antibiotic associated diarrhoea and C. difficile associated colitis (Hickson et

al. 2007). This is an interesting study as it is relatively large, and with clear and useful

endpoints. The study is also performed with a commercially available product.

In a study of Klarin et al. (2008), 100 ml of a treatment product containing L. plantarum

299v in an oatmeal gruel (109 CFU/ml) was given as enteral feeding (a thin tube

through the nose) twice per day for the first 2 days and then 50 ml per day to 22 patients

in intensive care (mean age 65 years). A corresponding control group got the oat meal

gruel without probiotics. Colonisation with C. difficile was detected in 19% of the

controls but in none of the patients treated with L. plantarum. The conclusion was that

enteral administration of L. plantarum 299v to critically ill patients treated with

antibiotics reduced the colonisation with C. difficile (Klarin et al. 2008). Drawbacks

with the study are that it is small and that the endpoint just is the presence of C. difficile,

not prevention from colitis caused by C. difficile. Interesting is however that this study

was performed in severely sick patients, and no side effects of the massive

administration of L. plantarum 299v could be noticed in this vulnerable category of

patients.


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The ability of L. plantarum 299v to prevent from antibiotic-associated diarrhoea and

presence of C. diffficile-toxin in faeces was evaluated in a randomized, double-blind,

placebo-controlled study on 163 patients treated for infections at an infectious diseases

clinic (Lönnermark et al. 2010). The patients were either administrated 200 ml per day

of L. plantarum 299v in ProViva (blueberry; 5x107 CFU per ml) or the same amount of

a placebo without probiotics. The treatment ran during the antibiotic treatment and for

seven days after termination of the antibiotic treatment. The primary outcome of the

study was negative, i.e. no statistically significant changes in the proportion of patients

developing diarrhoea or with C. difficile-toxin in faeces could be seen. However, the

overall risk of developing loose or watery stools was decreased by the probiotic

treatment, and furthermore, the risk of developing nausea was also decreased

(Lönnermark et al. 2010).

Even if it is almost consensus in the scientific community about certain advantages of

probiotics in connection to antibiotic therapy, there are also critical voices pointing at

studies where no effects have been found. For example, in a huge randomised, double-

blind, placebo-controlled study where 1470 inpatients received a probiotic mixture of L.

acidophilus NCIMB 30157, L. acidophilus NCIMB 30156, Bifidobacterium bifidum

NCIMB 30153 and Bifidobacterium lactis NCIMB 30172 during antibiotic therapy

while 1471 patients received placebo (Allen, et al. 2013). The author’s conclusion of

the study is as follows “We identified no evidence that lactobacilli and bifidobacteria

was effective in prevention of antibiotic-associated diarrhoea or Clostridium difficile

diarrhoea”, i.e. no outcomes of the study were fulfilled. This appears at least on the

surface as a highly reliable study, not the least for the inclusion of so many patients. But

the study has some major flaws: 1) Formulation of the conclusion reveals

bacteriological ignorance of the authors. They seem to believe that two strains of L.

acidophilus and two strains of Bifidobacterium generally can represent all available

species and strains of “lactobacilli” and “bifidobacteria”. The study has of course only

shown the lack of effect of a mixture of the four tested strains. 2) The time schedule for

probiotic treatment and antibiotic treatment in relation to the evaluation of disease is

unclear. The timing between antibiotic therapy, probiotic treatment and evaluation of

the outcome of the treatment is absolutely crucial for the reliability of the study. 3) The

compliance has been low, e.g. it appears as 25% of patients have got less than 2/3 of the

probiotic dose. 4) The frequency of C. difficile diarrhoea was low in the study, i.e. only

12 patients in the probiotic group got C. difficile diarrhoea (0.8%) and 18 patients in the

placebo group (1.2%): Thus, even if many patients were included, the number of

diseased patients was low which makes reliable evaluation of the effects of probiotic

treatment uncertain. Nevertheless, the probiotic treatment reduced the C. difficile cases

with 33% which shouldn’t be neglected.

Functional bowel disorders

Dysfunctions without clear pathogenic explanations

Examples of common transient symptoms of intestinal disorders are bloating and

flatulence, abdominal pain, diarrhoea and constipation. However, if these conditions are

of more chronic nature (they have occurred for at least 6 months, and they are present


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for more than 3 days per month during a period of 3 months) the medical diagnosis

becomes “functional bowel disorder” (FBD). FBD is then subdivided into irritable

bowel syndrome (IBS), functional bloating, functional constipation, and functional

diarrhoea. These different gut associated conditions are solely diagnosed by their

symptoms and may be caused by different reasons, or according to medical thermo

logy, they can have multiple aetiologies (Longstreth et al. 2006). The criteria of FBD

and IBS are consensus criteria that gradually have been worked out by the “Rome

foundation” which is an independent organization that provides support for activities

designed to create scientific data and educational information to assist in the diagnosis

and treatment of functional gastrointestinal disorders (FGIDs). The “Rome criteria”

have been evolving from the first set of criteria issued in 1989 (The Rome Guidelines

for IBS) through the Rome Classification System for FGIDs (1990), or Rome-1, the

Rome I Criteria for IBS (1992) and the FGIDs (1994), the Rome II Criteria for IBS

(1999) and the FGIDs (1999) to the more recent Rome III Criteria (2006).

Due to the fact that functional bowel disorder (FBD) is a frequently occurring

phenomenon in urban populations and that it cause discomfort in otherwise healthy

citizens, makes these individuals an interesting target-group for the probiotic market. If

a company want to make a more general claim that a certain probiotic food-product is

good for the gut health this claim can be backed up by studies in FBD-persons. FBD is a

diagnosis of discomfort and, formally, not a disease. This makes it possible to use data

from FBD-studies as argument in connection to functional foods without risking to be

accused by the authorities to market a pharmaceutical drug (which must be registered as

such and is then not allowed in the food segment).

Irritable bowel syndrome

Definition. Irritable bowel syndrome (IBS) is a functional bowel disorder in which

abdominal pain or discomfort is associated with defecation or a change in bowel habit,

and with features of disordered defecation. IBS is a commonly occurring syndrome of

disorders of unknown cause. The absence of strict pathogenic features has for long

made IBS to a disease without a proper diagnosis. Attempts have been made to develop

criteria for a positive diagnosis of IBS (Manning et al. 1978; Thompson et al. 1992).

IBS is a chronic relapsing condition that perhaps occurs in most adults at some point in

their lives. Symptoms begin before the age of 35 in 50% of patients, and 40% of

patients are aged 35-50 (Maxvell et al. 1997). IBS was in the 1990:s found in 18% of

the adult population in the Bristol area of United Kingdom (Heaton et al. 1992).

Throughout the world, about 10%–20% of adults and adolescents have symptoms

consistent with IBS. In most countries more females then men suffer from IBS.

IBS is currently defined according to the so called “Rome-III-criteria” which is a system

that has been developed to classify the functional gastrointestinal disorders based on

clinical symptoms. The Rome III criteria for IBS are as follows: Recurrent abdominal

pain or discomfort at least 3 days/month in 3 months associated with two or more of the

following:

1) improvement with defecation,

2) onset associated with a change in frequency of stool,

3) onset associated with a change in form (appearance) of stool.


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Treatment with Bifidobacterium animalis DN-173010. Effect of a fermented milk

containing Bifidobacterium animalis DN-173010 (Activia, Danone) on the health-

related quality of life and on symptoms of irritable bowel syndrome (IBS) in adults in

primary care has been shown in a randomized, double-blind, controlled trial (Guyonnet

et al. 2007). 274 adults with constipation-predominant IBS consumed 125 g Activia per

day (dose: 1010

CFU Bifidobacterium animalis) for 6 weeks. The study showed a

beneficial effect on discomfort (score) and increased stool frequency in those subjects

that had less than 3 stools per week at the start of the treatment (Guyonnet et al. 2007).

On the package and in advertisements, the company (Danone) calls the strain

Bifidobacterium animalis DN-173010 for “BIFIDUS REGULARIS” and claims that

Activia “help regulate the digestive system” and “help with slow intestinal transit”.

Treatment with Bifidobacterium infantis 35624. Treatment with Bifidobacterium

infantis 35624 alleviated symptoms in IBS. With the aim to compare the response of

symptoms in IBS, and to compare the symptom-response with cytokine ratios, 77

individuals with IBS were randomized to receive either Lactobacillus salivarius

UCC4331 or Bifidobacterium infantis 35624, each in a dose of 1010

CFU in a malted

milk drink for 8 weeks (or the malted milk drink alone as placebo)(O’Mahony et al.

2005). The symptoms of IBS were recorded and blood was sampled for estimation of

peripheral blood mononuclear cell release of the cytokines interleukin (IL)-10 and IL-12

were performed at the beginning and at the end of the treatment. Symptoms connected

to abdominal pain/discomfort, bloating/distension, and bowel movement difficulty were

reduced during treatment with B. infantis 35624. At start, patients with IBS

demonstrated an abnormal ratio of IL-10 to IL-12, indicative of a proinflammatory, Th-

1 state. This ratio was normalized by treatment with B. infantis 35624. Hence, B.

infantis 35624 alleviates symptoms in IBS, and this symptomatic response was

associated with a normalization of the ratio of an anti-inflammatory cytokine (IL-10) to

a proinflammatory cytokine (IL-12) (O’Mahony et al. 2005). In contrast, the tested L.

salivarius strain of O’Mahony et al. (2005) did not showed any substantial effect

against IBS.

Treatment with Lactobacillus plantarum 299v in fruit drink. Lactobacillus

plantarum 299v has in a couple of studies been shown to counteract IBS. L. plantarum

299v in the fruit drink ProViva (ProViva AB, Lunnarp, Sweden) was administrated to

patients with IBS in two, double blinded, placebo controlled studies, one in Poland

(Niedzielin et al. 2001) and one in Sweden (Nobaek et al. 2000). In both studies the

patients were divided into two groups, one was given L. plantarum 299v in ProViva

rosehip (active product) and the other a similar rosehip drink without L. plantarum 299v

(placebo). In the Swedish study, patients with slight to moderate symptoms, mainly

bloating and pain, were included (Nobaek et al. 2000) while the Polish study requited

patients that besides bloating and pain also had problems with irregularity in defecation

and stool consistency (Niedzielin et al. 2001).

The results of the Polish study were that the magnitude of several of the experienced

IBS symptoms decreased in the L. plantarum group, and a higher proportion of the

patients became free from symptoms in the treatment group than in the placebo group

(Niedzielin et al. 2001). In the Swedish study, L. plantarum 299v significantly

decreased the subjectively experienced bloating during the treatment period (Nobaek et

al. 2000). Pain was significantly reduced in both the treatment-group and in the

placebo-group, but the decrease was more rapid and more pronounced in the L.


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plantarum group. Twelve months after the treatment, the patients given L. plantarum

299v in the study, still experienced a better overall gastrointestinal function than the

patients that had got the placebo (Nobaek et al. 2000).

The bloating and pain experienced by IBS-patient might be due to abnormal colonic

fermentation giving rise to an excess of gas production, especially of hydrogen (King et

al. 1998). In a small randomised placebo controlled study on L. plantarum 299v in

ProViva, the gas production and composition of different gases were measured after 4

weeks consumption. However, no difference was seen between the placebo and the

treatment group (Sen et al. 2002). On the other hand, if the patients were provoked by

consuming 20 g lactulose, the hydrogen in the breath was significantly decreased in the

group pre-treated with L. plantarum 299v. Thus, probably the composition of the

intestinal microbiota had been changed in a favourable way by the probiotic treatment.

It should be pointed out that the study of Sen et al. (2002) was performed with a cross-

over design that in this case might disfavour differences between the groups.

Furthermore, in a randomized, placebo controlled, double blinded study in perfectly

healthy volunteers that consumed L. plantarum 299v in ProViva (1010

CFU/day for 3

weeks) experienced a decrease in flatulence during the treatment period (Johansson et

al. 1998). At the same time, the total level of carboxylic acids in faeces increased due to

an increase in the concentration of acetic acid and propionic acid (Johansson et al.

1998). The carboxyl acids are produced by the gut microbiota, and this change in acid

composition points at significant changes in the composition of the microbiota. L.

plantarum 299v are not known to be able to produce propionic acid. The increased

concentration of especially propionic acid must be regarded as beneficial from a health-

perspective. Propionic acid is utilized as an energy source by the epithelial cells of the

intestine. Short-chain fatty acids are in fact the major energy source of the colonic

epithelial cells. An increased level of short-chain fatty acids in the lumen is therefore

beneficial for the condition of the mucosa. Moreover, absorbed propionic acid comes

via the portal blood to the liver where it can have positive effects on both the lipid

metabolism and has anti-inflammatory effects in the liver.

The original health claim on the package of ProViva was that it “mitigates abdominal

gas production”, a claim that was approved by the Swedish authorities after an

assessment of an expert-group. This was before the European Union (EU) decided to set

up European rules for health claims for foods. ProViva with L. plantarum 299v was the

first probiotic product that proved to have effects against IBS.

Treatment with L. plantarum 299v in freeze-dried preparation: Freeze-dried L.

plantarum 299v in a capsule was given to subjects between 18-70 years with IBS in a

double blind, placebo controlled, parallel-designed study (Ducrotté et al. 2012). In total

214 IBS patients were recruited to the study by general practitioners in four clinical

centres in India. The test product contained 1010

CFU per capsule in potato starch while

the placebo product just contained potato starch. Patients consumed one capsule per day

for 4 weeks. The primary endpoint of the study was improvement of the frequency of

abdominal pain episodes, and secondary endpoints were changes in severity of

abdominal pain, changes in frequency and severity of abdominal bloating and in feeling

of incomplete rectal emptying. L. plantarum 299v significantly decreased both pain

severity and daily frequency of pain episodes. Similar results were obtained for

bloating. The conclusion of the authors were that “a 4 week treatment with L. plantarum


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299v provided effective symptom relief, particularly of abdominal pain and bloating, in

IBS patients fulfilling the Rome II criteria” (Ducrotté et al. 2012).

Inflammatory bowel diseases

Chronic inflammation

Inflammatory bowel disease (IBD) is a chronic inflammation along the gastro-intestinal

(GI) tract. It can be limited to the large bowel (ulcerative colitis) or it can be situated

anywhere along the GI-tract (Crohn’s disease). Ulcerative colitis is a relatively

superficial ulcerative inflammation, while Crohn’s disease is a transmural,

granulomatous inflammation. There are also several other forms of IBD, but they are

occurring less frequently and they are in some cases not so well defined. Examples of

other forms of IBD than ulcerative colitis and Crohn’s disease are collagenous colitis,

lymphocytic colitis, ischemic colitis, diversion colitis, Behçet's syndrome and

indeterminate colitis. IBD is thought to be due to an abnormal and uncontrolled immune

response to normally occurring constitutes of the intestine. The aetiology of IBD is

unknown. Microbial agents appear to be involved in the pathogenesis of IBD, and

intestinal bacteria seem to be an important factor in development and chronicity. Hence,

there is a complex interaction between bacteria, mucosa and immune system but this

interaction is far from clear.

Probiotics may have a potential for mitigating inflammation in IBD patients. For

example, it was shown in twenty IBD patients (15 with Crohn’s disease and 5 with

ulcerative colitis) that ingestion of Lactobacillus rhamnosus GR-1 and L. reuteri RC-14

in yoghurt for 30 days was associated with significant anti-inflammatory effects, or at

least changes in different immunological markers that pointed at a suppressed

inflammation (Baroja et al. 2007).

Crohn’s disease

Autoimmune disease. Crohn’s disease was first described 1932 and is regarded as an

autoimmune disease. It usually hits at 15-25 of age (prevalence, 30-50 individuals per

100 000), and can appear anywhere in the digestive tract, from mouth to anus. Though,

it often starts in terminal ileum. The exact cause of Crohn’s disease is unknown. The

idea is that it is a combination of environmental factors and genetic predisposition that

coincides, and results in a malfunction in the innate immune systems where chronic

inflammation being caused by adaptive immunity that tries to compensate for the

reduced function of the innate immune system. There is no known cure for Crohn’s

disease. Treatment is restricted to controlling symptoms, maintaining remission and

preventing relapse, and here can perhaps probiotics be of some help, for example by

counteract pro-inflammatory bacteria in the digestive tract.


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It is clear that the microbiota of the digestive tract can aggravate the inflammation,

especially certain aggressive components of the microbiota as Escherichia coli and

Bacteroides fragilis. For example, the immune response of the human epithelial cells in

intestinal mucosal samples taken from patients with Crohn's disease was seen to differ

considerably when the mucosa was exposed to Escherichia coli (strain ATCC 35345) or

Lactobacillus casei (strain DN-114001) (Llopis et al. 2009): An exposure to L. casei

decreased the secretion of TNF-alpha, IFN-gamma, IL-2, IL-6, IL-8, and CXCL1, and

down regulated expression of IL-8, IL-6, and CXCL1 in the human tissue while, in

contrast, an exposure to E. coli up-regulated the expression of all the cytokines (Llopis

et al. 2009). Live L. casei counteracted the pro-inflammatory effects of E. coli on

inflamed mucosa by specific down-regulation of key pro-inflammatory mediators.

Pro-inflammatory cytokines (cytokine = interleukin, IL): TNF (tumour necrosis factor) alpha is produced by monocytes/macrophages and activated T-cells. IFN (interferon) gamma is a key cytokine in chronic inflammation (originally, proteins that increase the resistance of body cells against viruses). IL-2 stimulates the growth of T-cells (and is also produced by T-cells). IL-6 initiates production of acute phase proteins and takes part in finish-off the acute inflammation (produced by macrophages and T-cells). IL-8 activates white blood cells to pass out from the blood vessel (produced by macrophages and epithelial cells after stimulation of bacteria or bacterial products). CXC-chemokines binds to neutrophlic granulocytes and have similar effects as IL-8.

Failures with probiotics. In a small study including only 11 patients with Crohn's

disease, the patients received L. rhamnosus GG for six months and they were also given

antibiotics one week before the treatment started. The endpoint of the study was

sustained remission, but no significant difference in median time to relapse was

observed between placebo and treatment group (Schultz et al. 2004).

In another study, children with Crohn's disease in remission were given L. rhamnosus

GG in addition to standard therapy in order to try to prolong the remission, but no

prolongation was obtained (Bousvaros et al. 2005). Neither was any effect reached by

administration of L. johnsonii LA1 to patients with Crohn's disease after surgical

resection in order to keep-up the remission (Marteau et al. 2006; Van Gossum et al.

2007) or by administration of L. rhamnosus GG to adult Crohn's patients (Prantera et

al. 2002). Hence, it seems that neither L. rhamnosus GG nor L. johnsonii LA1 are the

best choices to give to patients with Crohn's disease.

Ulcerative colitis

Inflammation driven by the microbiota. Ulcerative colitis causes ulcers, or open sores

in colon, and gives diarrhoea mixed with blood (prevalence is 10-100 per 100000

persons). Ulcerative colitis starts from below and moves upwards in colon. It usually

debuts at 20-40 years of age. Ulcerative colitis increases the risk for malignant

transformation, i.e. colorectal cancer. Ulcerative colitis has no known cause, but

presumably it has a genetic component to susceptibility that can be triggered by

environmental factors. Ulcerative colitis is often treated as an autoimmune disease, but


135

there is no consensus about ulcerative colitis being an autoimmune disease. Ulcerative

colitis is characterized by periods of remission marked by episodes of clinical relapse

caused by acute colonic and/or rectal inflammation. Treatment is primarily aimed at

reducing inflammation during relapse and secondarily at prolonging the time spent in

remission of clinical symptoms.

During the acute phase of inflammation, macrophages, neutrophils and eosinophils are

infiltrating the lamina propria of the colonic mucosa. Aggregating neutrophils lead to

the formation of abscesses (Asakura et al. 1999). Activated dendritic cells and

macrophages secrete cytokines that trigger and differentiate T cells, and activating the

adaptive immune response.

Patients with ulcerative colitis seem to have a higher numbers of bacteria associated to

the mucosa than healthy persons. The intestinal microbiota in patients with active

ulcerative colitis has been shown to be less diverse than in healthy subjects (Nishikawa

et al. 2009). Enterobacteriaceae might be involved in the pathogenesis of ulcerative

colitis. High proportions of Enterobacteriaceae (E. coli and Enterobacter) and of

Bacteroides fragilis, together with substantial amount of Pseudomonas aeruginosa,

Haemophilus parainfluenzae and Clostridium difficile were found on the inflamed

colonic mucosa taken during surgery from a 12-year-old girl suffering from acute

ulcerative colitis (Wang et al. 2007).

Hydrogen sulphide has been implicated in the pathogenesis of ulcerative colitis (Pitcher

et al. 2000), and sulphate-reducing bacteria have received attention due to their ability

to reduce sulphate to sulphide as a by-product of their respiration. Hydrogen sulphide is

cell toxic and freely permeable to cell membranes and inhibits butyrate oxidation in

colonocytes (Rowan et al. 2009).

The number of lactobacilli seems to be relatively low at active ulcerative colitis (Fabia

et al. 1993). However, lactobacilli were predominantly detected in inactive patients, and

lactobacilli were suggested to have a role in the induction of remission (Andoh et al.

2007). It has been hypothesized that the changing condition in the intestine may

influence the amount of Lactobacillus and the sort of Lactobacillus (Zoetendal et al.

2002; Zhang et al. 2007).

Probiotic treatment. Probiotics has been given to patients suffering from ulcerative

colitis, and it seems as some efficiency has been reached, both in intervention and

maintenance therapy. A meta-analysis to evaluate the induction of remission and

maintenance of probiotic therapy was accomplished by Sang et al. (2010) on thirteen

randomized, controlled studies. It was concluded that probiotics were more effective

than placebo in maintaining remission.

A probiotic mixture of strains of L. casei, L. plantarum, L. acidophilus, L. delbrueckii

subspecies bulgaricus, Bifidobacterium longum, Bifidobacterium breve,

Bifidobacterium infantis and Streptococcus thermophilus (the mixture is labelled

VSL#3) has been used for treatment of mild-to-moderate active ulcerative colitis (Sood

et al. 2009). Six weeks of probiotic treatment resulted in a significantly higher

percentage of patients with more than 50 % improvement in “ulcerative colitis disease

activity index” score, and after 12 weeks, significantly more patients achieved remission

(Sood et al. 2009).


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Several species and strains of bacteria with claimed probiotic potential have been used

in clinical trials, e.g. the a mixture VSL#3 (including L. casei, L. plantarum, L.

acidophilus, L. delbrueckii subspecies bulgaricus, Bifidobacterium longum,

Bifidobacterium breve, Bifidobacterium infantis and Streptococcus thermophilus)

(Bibiloni et al. 2005; Sood et al.2009), a mixture of L. rhamnosus and L. reuteri, L.

rhamnosus GG (Zocco et al. 2006), B. breve strain Yakult and B. bifidum strain Yakult

(Kato et al. 2004) and L. acidophilus (Baroja et al. 2007). Probiotics have shown effects

in treatment of active mild-to-moderate ulcerative colitis by decreasing clinical activity

index, preventing relapse, and induction of remission.

Also, bifidobacteria has been used against ulcerative colitis: Improvements of

histological scores and increases in faecal butyrate, propionate and short-chain fatty acid

concentrations were registered after administration of bifidobacteria (Kato et al. 2004).

Consumption of three strains of Bifidobacterium (brand name BIFICO) by patients with

ulcerative colitis induced depressed activation of the transcriptional factor NF-kappa-B,

decreased expressions of TNF-alfa and IL-1β, while the expression of IL-10 was

elevated, which indicates a anti-inflammatory action by the probiotic treatment (Cui et

al. 2004).

A combination of inulin-oligofructose with prebiotic properties and a strain of

Bifidobacterium longum with probiotic properties were given daily for one month to

patients with active ulcerative colitis (2x1011

CFU of B. longum and 6 g fructo-

oligosaccharide/inulin mix, brand name Synergy 1, Orafti, Tienen, Belgium) (Furrie et

al. 2005). The trial that was double-blind, randomised and placebo controlled and

involved 18 patients with active ulcerative colitis. Nine patients were assigned to the

test group and nine to the placebo group. Rectal biopsies revealed that the test group

had reduced inflammation and regeneration of epithelial tissue compared to the control.

Thus, short term treatment with a bifidobacteria and inulin-oligofructose of active

ulcerative colitis resulted in improvement of the full clinical appearance of chronic

inflammation in patients. These results are convincing (Furrie et al. 2005). However, it

should be held in mind that this is a pilot study that needs to be repeated in a larger scale

for verification of the results.

Animal models for intestinal inflammation

Animal models are needed. Animal models are helpful means for comparing the

causal effects between different probiotic strains in vivo, and for clarifying mechanisms

of action and certifying safety. Mice and rats are the two most frequently used animal

species for in vivo tests. The interpretation of test results from animals can never be

directly transferred into human settings, and the interpretation must always be done with

considerably caution. However, an animal model is always much closer to the

biological reality than in vitro models.

The effect of probiotics on inflammation in the intestine can be studied in many

different types of animal models. An example of a well-established and frequently used

model is the dextran sulphate sodium (DSS) induced colitis, and another example, less

used model, is to induce enterocolitis with methotrexate. Below follow examples of

how these two models have been used in order to evaluate effects of probiotics.


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Dextran sulphate sodium (DSS) induced colitis. DSS is a non-genotoxic sulphated

polysaccharide used to induce experimental chronic colitis (Cooper et al. 1993). The

precise mechanism by which DSS induces colonic inflammation is unknown.

Administration of DSS through the oral route results in an acute inflammation in colon

(colitis), with lesion resembling those of human ulcerative colitis, symptomatically and

histologically.

This model applied in rats was, for example, used by Osman et al. (2004) for evaluating

and comparing the ability of different strains of Lactobacillus and Bifidobacterium to

counteract colitis. Rats were in this case fed probiotics daily for 7 days before the start

of the DSS-administration, and the probiotic feeding continued during the DSS-

administration that went on for 10 days. 5% DSS dissolved in water was given by

orogastric tube (can also be given in the drinking water or in the food). The health status

of the animal were recorded by a Disease Activity Index calculated on body weight

change, stool consistency and bleeding from anus. This index that reflects the severity

of the colitis is an obvious primal end-point of the model (severity of disease), but

preferably also different inflammatory markers and histological examination of the

colonic mucosa can be added to the examination scheme.

It was seen in the particular study of Osman et al (2004), where 3 different

Lactobacillus strains and two different Bifidobacterium strain were compared for their

efficiency to mitigate colonic inflammation (colitis), that three of the test-strains

reduced inflammation and severity of the colitis. The tested strain of L. paracasei and

the test-strain of L. gasseri, did not show any effect on the disease activity index. Best

in this comparison was Bifidobacterium longum subspecies infantis DSM 15158,

followed by L. plantarum 299v.

The DSS concept works well in both mice and rats. The major reason to choice rats

before mice is that the former are larger which makes surgery and the taking of

specimens more easy, and also bigger samples can be taken, for example, more blood.

Methotrexate induced enterocolitis. Methotrexate is a drug used in treatment of

cancer, autoimmune diseases and as an abortifacient (a substance that induces abortion)

in the induction of medical abortions. Methotrexate acts by inhibiting the metabolism of

folic acid. Methotrexate can also cause inflammation in the intestinal mucosa which is a

negative side effect when it is used in therapy, but an effect that is utilised in the

enterocolitis animal model. Thus, the animal gets methotrexate in a daily dose that

induces and maintains a mucosal inflammation (Mao et al. 1996a). This inflammation

will engage both the small and the large bowel (enterocolitis).

Methotrexate induced enterocolitis in rats was used as model in one of the first animal

studies that clearly demonstrated the potential of probiotics to mitigate intestinal

inflammation (Mao et al. 1996a). In this particular study it was shown in rats with

enterocolitis that the probiotic strain L. plantarum 299v mitigated the mucosal

inflammation induced by the chemotherapy (methotrexate). This could be measured in

different ways: The most obvious way is to compare the decrease in body weight

between the treatment group and a control group where the percentage of body weight

loss after 3 days was almost four times higher in the control than the probiotic treatment

group (Mao et al. 1996a).


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A marker for inflammation in the mucosa is myeloperoxidase (MPO) and the more

severe the inflammation becomes the higher the MPO concentration will be. MPO

catalyse the formation of hypochlorous acid from hydrogen peroxide (see formula of

reaction on the right) (Figure 28). The HOCl molecule is instable and the formed

hypochlorite ion is highly reactive, and it is also cytotoxic. Hypochorite is used by the

neutrophils to kill bacteria and other pathogens, and high numbers of active neutrophils

will result in elevated levels of myeloperoxidase. Thus, myeloperoxidase becomes a

marker for infiltration of neutrophils in the tissue, i.e. inflammation in the tissue. The

MPO level in both ileal and colonic mucosa of the methotrexate treated rats was

significantly decreased by L. plantarum 299v (Mao et al. 1996a).

Figure 28. Myeloperoxidase (MPO) catalysing the conversion of hydrogen peroxide to the highly bacteriocidal hypochlorite.

MPO is a peroxidase enzyme that is most abundantly present in neutrophil granulocytes. MPO has a haem pigment, which gives a green colour to secretions rich in neutrophils. HOCl is cytotoxic, and is used by the neutrophils to kill bacteria and other pathogens.

It was also shown by Mao et al. (1996a) that healthy rats had a viable count of

lactobacilli on the ileal mucosa of about 108 CFU per g mucosal tissue while the count

of Enterobacteriaceae was considerably lower, i.e. around 104 CFU per g mucosal

tissue. However, in the enterocolitic rats, the Enterobacteriaceae count increased and

reached the same level as that for lactobacilli (108 CFU per g mucosal tissue).

Treatment with L. plantarum 299v suppressed the increase in Enterobacteriaceae.

Increased levels of Enterobacteriaceae means also elevated levels of

lipopolysaccharides (LPS), which can increase the leakage of LPS through the mucosal

barrier, especially if the mucosal barrier function has been decreased due to

inflammation. It was seen in the methotrexate model that compared to the enterocolite-

control the concentration of LPS in blood decreased by the treatment with L. plantarum

299v.

One important factor for the defence in the gut is IgA antibodies secreted out in the

lumen. IgA antibodies bind to the cell surface of bacteria and block their attachment to

the mucosa, and mitigate their penetration of the mucosa. In the methotrexate induced

enterocolitis model it was demonstrated that secretory IgA antibodies in ileum and


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colon was depressed by the inflammation and that treatment with L. plantarum 299v

increased the amount of IgA antibodies compared to the enterocolite-control (Mao et

al. 1996b).

15-20% of the antibodies in blood are IgA. IgA antibodies are the dominating antibodies in secretion, as slime, saliva, tears, milk and sweat. IgA antibodies bind to bacteria outside the mucosa and prohibit binding to epithelial cells.

Liver injuries

Fibrosis and cirrhosis

The gut and the liver are closely connected. A well functioning link between the gut and

the liver is dependent on both an intact intestine and a liver in balance with respect to

immunologic response and metabolism of endogenous and exogenous compounds. The

liver is an important site for bacterial phagocytosis and clearance as it contains the

largest population of tissue macrophages.

Macrophages in the liver are called Kupffer cells.

Chronic liver injury is associated with the development of fibrosis, since repeated and

continuous cellular damage of liver cells leads to the activation of hepatic stellate cells

and their production of extracellular matrix proteins. An advanced stage of hepatic

fibrosis is cirrhosis, in which functional liver tissue is largely replaced by scar tissue and

regenerating nodules. Cirrhosis is mostly caused by alcoholism, hepatitis B and C, and

fatty liver disease.

Fibrosis can promote bacterial overgrowth, weakened barrier function through restricted

intestinal motility, decreased bile acid production, and decreased nutrient absorption and

availability. Disturbances of the gut-microbiota are prevalent in patients with chronic

liver disease. Patients with chronic liver disease can be expected to suffer from

intestinal bacterial overgrowth and increased bacterial translocation, in combination

with failure of immune defence mechanisms, and predisposition to bacterial infections.

Thus, clinical studies suggest an increased translocation of enteric bacteria to mesenteric

lymph nodes in cirrhotic patients, together with an enhanced intestinal permeability, and

endotoxins in portal and systemic circulation. Also, ultrastructural abnormalities in the

epithelial layer of the small intestine and a decreased gut barrier function can be seen in

patients with cirrhosis (Such et al. 2002; Zuckerman et al. 2004). Consequently, an

impaired gut barrier function might contribute to the progression of chronic liver

damage. There is also a strict relationship between altered intestinal permeability and

portal hypertension, i.e. high blood pressure in the portal vein (Xu et al. 2002).

An adverse gut microbiota seems to be able to induce liver disease by means of

increased portal delivery of lipopolysaccharides (LPS) and other endotoxins, which

leads to activation of the stationary macrophages in the liver (Kuppfer cells) and


140

induction of production of “transforming growth factor beta”, and subsequent activation

of hepatic stellate cells. Stellate cell is a star shaped nerve cell.

Minimal hepatic encephalopathy

Minimal hepatic encephalopathy (MHE) is a disorder in patients with cirrhosis, and a

disorder that seriously can impair daily functioning and quality of life. Increased level

of ammonia in the blood is most probably a key factor in the pathogenesis.

Treatment for 30 days with a probiotic mixture consisting of four different strains of the

species Pediococcus pentosaceus, Leuconostoc mesenteroides, L. paracasei and L.

plantarum along with dietary fibres was administrated to MHE-patients (Liu et al.

2004). The patients had unusually high faecal loads of E. coli and Staphylococcus spp.,

and the probiotic supplementation with the preparation of probiotics and fibres led to

reduction of the viable count of E. coli and Staphylococcus, but also to a reduction of

Fusobacterium (Liu et al. 2004). Furthermore, the treatment led to decreased ammonia

levels in the blood, together with a reduction in the circulating levels of endotoxins (Liu

et al. 2004). Also the concentrations of serum bilirubin and alanine aminotransferase

(ALT) were decreased compared to pre-treatment values (Liu et al. 2004).

Alterations of the intestinal flora, improvement of the clinical status and lowered blood

ammonia levels by the ingestion of probiotics have also been seen without fibre-

supplementation, i.e. for a strain of L. acidophilus (Macbeth et al. 1965; Read et al.

1966) and for a “probiotic yoghurt” (Bajaj et al. 2008).

Alanine aminotransferase (ALT) is an indicator for the damage of liver cells (hepatocytes). Aspartate aminotransferase (AST) is an indicator for the damage of liver cells, but AST can also be found in the heart and different muscle tissues. Bilirubin is a product from destroyed red blood cells that will increase if the liver is failing, and is a measure of hepatic transport function and the severity of jaundice (icterus).

Alcohol-related liver disease

Chronic ethanol consumption causes changes in the liver, e.g. fatty liver, inflammation

and cirrhosis. Cirrhosis is characterized by replacement of liver tissue by fibrosis, scar

tissue and regenerative nodules. Inflammation associated to accumulated fat in the liver

is called steatohepatitis and is characterised by infiltration of monocytes, macrophages,

neutrophils, and lymphocytes, occurring as a consequence of activation of inflammatory

mediators. During alcoholic steatohepatitis, serum TNF-α, IL-6, and IL-8 levels are

increased and correlate with markers for the acute-phase response, liver function, and

clinical outcome (McClain et al. 1999).

Alcoholic steatohepatitis also lead to an increase in the load of gut-bacteria and a

change in the composition of the microbiota, which may result in overgrowth,

endotoxaemia, and increased bacterial production of acetaldehyde from ethanol.

Acetaldehyde is more toxic than ethanol, and an increased concentration of

acetaldehyde can accentuate the liver damage. Moreover, ethanol and acetaldehyde


141

affect intracellular signal-transduction pathways leading to the disruption of epithelial

tight junctions, which increases the paracellular permeability of macromolecules.

Hence, an impaired gut barrier function might be a contributing factor in the progression

of chronic liver damage.

Beneficial effects of probiotics on mild alcoholic induced hepatitis has been reported in

an open-label, prospective, randomized, clinical trial with analyses before treatment

(“baseline”) and after treatment (Kirpich et al. 2008). The aim with this study was to

clarify the effects of probiotics on the gut microbiota, and alcohol-induced liver injury.

Patients (66 adult Russian male) with the diagnosis alcoholic psychosis were

randomized to receive Bifidobacterium bifidum (no strain label are given) and L.

plantarum 8PA3 for 5 days (32 patients), versus standard therapy alone (abstinence plus

vitamins; 34 patients). The average daily volume of alcohol consumed 1 to 2 weeks

before the recruitment was approximately 750 ml per day of Russian vodka (40%

ethanol). Stool cultures and measurements of liver enzymes were performed at baseline

and again after termination of therapy. Results were compared between groups and with

24 healthy, matched controls who did not consume alcohol. The treatment resulted in

reduction of the levels of alanine aminotransferase (ALT), aspartate aminotransferase

(AST), gamma glutamyl transpeptidase, lactate dehydrogenase, and total bilirubin in the

blood. Also the faecal microbiota was affected by the alcohol consumption, i.e. the level

of lactobacilli and bifidobacteria were lower in the alcoholic patients than the non-

alcoholic control group (Kirpich et al. 2008).

In another open-label trial, 12 patients with alcoholic cirrhosis received L. casei Shirota

three times daily for 4 weeks (Stadlbauer et al. 2008). Data were compared with 13

healthy subjects and 8 cirrhotic patients who did not receive probiotics. Neutrophil

oxidative burst, phagocytosis, toll-like-receptor (TLR) expression, plasma-cytokines

and ex vivo endotoxin-stimulated cytokine production were measured.

The baseline for neutrophil phagocytic capacity in the cirrhotic patients was

significantly lower compared to the healthy controls, but was normalized at the end of

the study by the probiotic treatment. TLR2, TLR4 and TLR9 were over-expressed on

the surface of neutrophils in the cirrhotic patients compared to the healthy controls, but

normalised by the treatment (Stadlbauer et al. 2008). Normalization of the expression of

TLR4 is especially interesting in view of the fact that TLRs detects lipopolysaccharides

(LPS).

Oxidative burst (respiratory burst) is the rapid release of reactive oxygen species (superoxide radical and hydrogen peroxide) from cells as neutrophils and monocytes when they come into contact with microorganisms. Toll-like receptors (TLRs) are a class of protein s that play a key role in the innate immune system. TRL receptor 4 (TRL4) detects lipopolysaccharides and activates the innate immune system (TLR4 = CD284).

Non-Alcoholic Fatty Liver Disease

Accumulation of fat and liver inflammation. Non-Alcoholic Fatty Liver Disease

(NAFLD) is an inflammation in the liver due to accumulation of fat, when the fat

accumulation not is due to excessive alcohol consumption. NAFLD is related to insulin


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resistance and the metabolic syndrome. NAFLD is a comprehensive term for a chain of

conditions with successively increasing severity, i.e. steatosis, steatohepatitis (non-

alcoholic steatohepatitis; NASH), liver fibrosis, cirrhosis with failing liver function, and

hepatocellular carcinoma (carcinoma is any malignant cancer that arises from epithelial

cell).

Steatohepatitis is characterized by inflammation and with concurrent fat accumulation

in the liver. NAFLD is strongly linked to obesity and type 2 diabetes. The mildest form

of NAFLD is steatosis (fat accumulating in the liver). Hence, NAFLD comprises a

spectrum of diseases ranging from simple steatosis to non-alcoholic steatohepatitis

(NASH), fibrosis and cirrhosis. In its initial phase, the healthy liver becomes steatoic

mainly as a consequence of peripheral resistance to insulin. The insulin resistance

increases the transport of fatty acids from adipose tissue to the liver. Steatosis renders

hepatocytes susceptible to further obstacles. Once steatosis is established there are

several factors that can lead to an inflammatory process with hepatocellular

degeneration and fibrosis as result, such negative factors are LPS leaking out from the

intestinal microbiota to the liver, ethanol, oxidative stress and cytokines aggravating

dysfunction of liver cells (Medina et al. 2004).

NAFLD is associated with a number of diseases such as obesity, type 2 diabetes,

hyperlipidaemia (too much fat in the blood), coeliac disease, exposure to different

medications and environmental toxins, total parenteral nutrition and surgical procedures

as for example bypass of jejunum or ileum and other operations in the digestive tract

(Younossi et al. 2002; Lirussi et al. 2007). About 30% of the adult population in USA

and about 25% in Italy suffer from NAFLD (Bellentani and Marino 2009).

Microbiota. An endogenous factor that may contribute to the pathogenesis of NAFLD

is the microbiota of the digestive tract. Hepatic oxidative stress may be increased by

enhanced endogenous production of ethanol. It has been shown that obese female

NASH patients have higher levels of ethanol in the breath (Nair et al. 2001). This may

be caused by small intestinal bacterial overgrowth, which has been shown in patients

with non-alcoholic steatohepatitis (Wigg et al. 2001). Gram-negative bacteria in the gut

may also affect hepatic oxidative stress through release LPS, leading to production of

inflammatory cytokines by stimulation of luminal epithelial cells and Kupffer cells.

Kupffer cells are the main producers of TNF-α, a central mediator in the pathogenesis

(development) of NASH (Pessayre et al. 2002).

Probiotics. It can be speculated whether probiotics might counteract the development of

NAFLD, for example, by replacing aggravating bacteria in the digestive tract, which in

turn can decrease the production of proinflammatory cytokines like TNF-α. An

alternative could be that the probiotic bacteria might improve the epithelial barrier

function and thereby avoid exposure beyond the normal limit of LPS and ethanol to the

liver. However, despite the rationale for the possible therapeutic role of probiotics, no

placebo controlled trials have been performed so far in patients with NAFLD/NASH

(Lirussi et al. 2007).

Therapeutic effect of probiotics on different liver diseases, including a small group of

NAFLD patients, was shown in a prospective study (Loguercio et al. 2005). A mixture

of probiotic strains (VSL#3) containing strains of L. casei (should presumably be L.

paracasei), L. plantarum, L. acidophilus, L. delbrueckii subsp. bulgaricus, B. longum,

B. brevis, B .infantis and Streptococcus thermophilus were given to patients with (i)


143

non-alcoholic fatty liver disease (NAFLD; 22 patients); (ii) alcoholic liver cirrhosis

(AC; 20 patients) and (iii) hepatitis C virus infection (HCV; 36 patients) in a dose of

totally 5x1011

CFU per day for 3 months. Different markers of the liver function,

oxidative stress and inflammation were measured before the start of the probiotic

treatment and after 3 months of treatment. Aspartate aminotransferase (AST), alanine

aminotransferase (ALT) and bilirubin decreased in all patient categories. In the NAFLD

and the AC patients the probiotics decreased the oxidative stress (the markers

malondialdehyde and 4-hydroxynonenal was decreased) as well as in the AC patients

also the inflammatory markers were improved (TNF-alfa, IL-6 and IL-10) (Loguercio et

al. 2005).

Malondialdehyde (MDA = CH2(CHO)2 ) is a marker for oxidative stress. Reactive oxygen species degrade poly-unsaturated lipids, forming MDA, which cause toxic stress in cells and form covalent protein adducts.

Animal model for liver injury

The ability of probiotics to block translocation from the intestines out into the body

(primarily to the liver) can be followed and scrutinised in different type of animal

models. One way to do this is to start the translocation of bacteria from the gut by

harming the liver. For example, this can be done by starting an inflammation in the liver

by an intraperitoneal injection of D-galactosamine (Adawi et al. 1997). When the liver

become inflamed the barrier capacity of the gut mucosa is weakened and bacteria in the

gut start to leak out into the body. Translocating bacteria and components from bacteria

will through the portal blood reach the liver and aggravate the liver inflammation.

(Figure 29).

Figure 29. A vicious circle of weakening barrier function of the gut mucosa due to liver inflammation leads to successively increasing liver injury.


144

Using the liver injury model in rats, it has been demonstrated that a pre-treatment with

different strains of Lactobacillus or Bifidobacterium can decrease the permeability of

the mucosa (i.e. increase the barrier effect of the mucosa) and, hence, the number of

intestinal bacteria that are able to translocate to the liver can be decreased, which in turn

results in a less inflamed liver. Thus, some probiotics can break the vicious circle

depicted in Figure 29 by improving the barrier function of the intestinal mucosa.

However, the capacity to do this differs between different species and strains of both

Lactobacillus and Bifidobacterium. It is obvious that different probiotic strains are

differently good in increasing the barrier function of the mucosa in the digestive tract.

L. plantarum 299v and Bifidobacterium longum subspecies infantis CURE19 have in

this rat model shown to decrease the translocation of gut derived bacteria to the liver

(Figure 30), and at the same time they reduced the concentration of alanine

aminotransferase (ALT), asparagines aminotransferase (AST) and bilirubin in the blood

(Adawi et al. 1997; Osman et al. 2005). AST and ALT concentration in blood are

frequently used as markers for liver injury in both humans and animals. The

concentration of these enzymes in the blood increases when the liver is injured.

Figure 30. Rats were orally administered different strains of Lactobacillus and Bifidobacterium, twice daily for 8 days, before injection with D-galactosamine that started an inflammation in the liver. The bright red column to the left shows translocation of intestinal bacteria to the liver in animals that had not been pre-treated with lactobacilli or bifidobacteria (liver injury control); *marks a statistically significant decrease compared to the liver injury control (P<0.05). Data are taken from Osman et al. (2005).

The animal model used to obtain the results of Figure 30 is a well established and is a

model that is relatively easy to run for studying bacterial translocation from the

intestines. The model can preferably be used to test the effects different dietary

compounds, for example different probiotic strains, on the barrier function of the

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intestinal mucosa. The status of the mucosa can be differently affected by different

dietary components.

Patients in intensive-care

Systemic inflammation

Intensive-care medicine (critical-care medicine) is a discipline dealing with the support

of life maintenance and organ support in patients who are critically ill. It is obvious that

there are many factors to watch out for in these situations, but one factor of relevance

for probiotics is the problem with a leaky gut leading to an exadurated inflammation

that can be of danger for the patient.

A decrease in systemic inflammation by administration of L. plantarum 299v has been

seen in critically ill patients in the form of a decreased level of IL-6 in the blood

(McNaught et al. 2005). Thus, 103 critically ill patients were randomised to receive L.

plantarum 299v in ProViva (strawberry) in addition to conventional therapy. The

median value of the daily volume of ProViva was 213 ml, corresponding to a dose of

about 1010

CFU of L. plantarum 299v per day. The median for the duration of intake

was 9 days. It was found that enteral administration of L. plantarum 299v to critically ill

patients was associated with a late attenuation of the systemic inflammatory response

(decreased levels of IL-6; McNaught et al. 2005).

Acute pancreatitis

Pancreatitis is in its acute form a severe inflammatory condition. In pancreatitis,

pancreatic enzymes are activated and attack tissue which leads to ischemia and

reperfusion. This condition generates oxygen free radicals that mediate inflammation

and tissue injury. The resulting systemic inflammation weakens the barrier function of

the intestinal mucosa, and gut bacteria can start to translocate into the body. This

becomes a vicious cycle that can result in multi-organ failure and/or sepsis.

Pancreatitis is mostly trigged by gall-stones or alcohol. 15-20% of the patients with

acute pancreatitis develop severe acute pancreatitis, with a mortality rate of 9-27%. It is

assumed that enteral administration of probiotics (tube feeding) can help the patients by

decreasing the bacterial translocation and eventually suppress the systemic

inflammation. This has been shown in an animal model for acute pancreatitis where the

rats were treated with L. plantarum 299v (Mangiante et al. 2001), but also in patients

with acute pancreatitis (Olah et al. 2002; Qin et al. 2008). The referred human studies

were both performed with L. plantarum as probiotics but with different strains (Olah et

al. 2002; Qin et al. 2008).

In contrast, in a randomised, double-blind, placebo-controlled trial over the eventual

probiotic prophylaxis in severe acute pancreatitis, a mixture of strains of other species


146

than L. plantarum was used (Besselink et al. 2008). The mixture with the brand name

Ecologic 641 (Winsclowe Bio Industries, Amsterdam, Netherlands) consisted of strains

of L. acidophilus, L. casei (presumably it should be L. paracasei), L. salivarius,

Lactococcus lactis, Bifidobacterium bifidum and Bifidobacterium lactis (= B. animalis).

The outcome of this study was negative, and nine patients in the probiotic group

developed bowel ischemia (eight with fatal outcome), compared with none in the

placebo group (Besselink et al. 2008).

In the study of Besselink et al. (2008), describing the failure of using the bacterial

mixture Ecologic 641 for helping patients with acute pancreatitis, the explanation of the

authors was that the administrated bacteria may have increased the oxygen metabolism

in the mucosa, and hence, the production of oxygen free radicals which caused the local

inflammation leading to ischemia.

Ischemia (= restriction in blood supply) results in tissue damage because of lack of oxygen and nutrients and the build-up of metabolic waste.

Oxygen free radicals: O2-

. HO2

.

. OH H2O2

If this assumption is correct, it would be of interest to know if this effect was caused by

a certain strain (or strains) of the mixture or if all strains were to blame. Of the species

included into the study by Besselink et al. (2008), Lactococcus lactis is the most likely

one to consume oxygen due to the fact that this species has an efficient aerobic

metabolism. However, some Lactobacillus strains and especially strains of certain

species (for example L. acidophilus and L. crispatus) are well known producers of

hydrogen peroxide. Often these strains lack catalase or other means to convert hydrogen

peroxide to water which results in an accumulation of hydrogen peroxide in the

environment of the bacteria. This could be beneficial in some situation, for example

fighting of E. coli on a vaginal mucosa, but can be a negative feature in a critically ill

patient at risk for an over-reacting inflammatory response. In this situation, it is

beneficial if the probiotic strain have an efficient means to convert the hydrogen

peroxide to water, for example the mangane-pseudocatalese of L. plantarum.

Bifidobacterium bifidum is the most unlikely one of the strains in the Ecologic 641

mixture to produce oxygen free radicals because this is a strictly anaerobic organism,

unable to metabolise oxygen. The failed clinical trial of Besselink et al. (2008), stress

the importance of using well defined bacterial strains with well-known characteristics in

probiotic applications. For example, if a probiotic strain should be given to critically ill

patients, the strain must have documented anti-inflammatory capacity in vivo. In other

words, the strain must be carefully evaluated in animal inflammation-models before it is

given to severely sick humans. In this case, the ability of the different strains to cause

oxidative stress and inflammation ought to have been more carefully evaluated

beforehand.

It can be noted that the two successful studies to counteract side effects of acute

pancreatitis (Olah et al. 2002; Qin et al. 2008) both used a single strain of L. plantarum

while this species was absent in the mixture of strains called Ecologic 641 in the study

of Besselink et al. (2008).


147

Metabolic syndrome

Low-grade inflammation

The metabolic syndrome is a combination of disorders that increase the risk of

developing cardiovascular disease and type-2 diabetes. Factors contributing to the

syndrome are increased triglycerides in the blood, decreased HDL cholesterol in the

blood, increased blood pressure, increasing fasting plasma glucose and central obesity.

The metabolic syndrome is characterised by a systemic, low-grade inflammation and

increased insulin resistance.

Lipopolysaccharides (LPS) leaking out into the body from the gram-negative part of the

intestinal microbiota may be the triggering factor for the low-grade inflammation

(“subclinical inflammation”) (Pendyala et al. 2012). A low-grade inflammation means

that there is a slight elevation of different inflammatory markers in the blood compared

to the normal levels.

Oral administration of probiotics can be a strategy to improve the gut barrier function

and suppress gram-negative bacteria in the digestive tract. A decreased level of

lactobacilli in the gut of elderly persons has been shown to be linked to the count of

white blood cells, blood glucose and content of oxidised low-density lipoprotein (ox-

LDL), all risk markers in the pathogenesis of inflammation, metabolic syndrome and

cardiovascular disease (Mikelsaar et al. 2010).

The ability of many Lactobacillus strains to counteract, for example, E. coli is well

known, and the ability of certain probiotic strains, for example, L. plantarum 299v, to

mitigate bacterial translocation from the gut has been proved in rats (Adawi et al. 1997;

Kasravi et al. 1997; Adawi et al. 1999; Wang et al. 2001; Osman et al. 2005) but it has

also been tentatively shown in humans (McNaught et al. 2005; Klarin et al. 2008).

Furthermore, it has been demonstrated in healthy humans that L. plantarum WCSF1

increased the relocation of the proteins occludin and ZO-1 into the tight junction area

between duodenal epithelial cells which ought to result in a tighter mucosa (Karczewski

et al. 2010).

Type-2 diabetes and obesity are characterised by a low-grade inflammation but it is still

unclear if the inflammation is the cause of the condition or just a consequence of it.

The microbiota of the digestive tract is important in relation to inflammation, and a

favourable influence on the composition of the gut microbiota by, for example,

administration of probiotics can be a strategy to mitigate inflammation. As been said

before, ingesting probiotics can affect the composition of the resident gut microbiota,

but probiotics may also have more direct effects on the immune system and the

permeability of the mucosa. The better the barrier function of the mucosa is the smaller

is the risk of translocation of pro-inflammatory bacteria and pro-inflammatory

components of bacterial origin.

In a small, doubled blind, randomised placebo controlled study it was shown in male

volunteers with a slightly elevated cholesterol level in the blood, that a dose of 2x1010

L.

plantarum 299v per day for 6 weeks (400 ml per day of ProViva rosehip), decreased the


148

concentrations of cholesterol and fibrinogen in serum (Bukowska et al. 1998). The

placebo in this study was a corresponding fruit drink without probiotics.

The same probiotic rosehip drink (ProViva) as in the study of Bukowska et al. (1998),

were given in the same amount and during the same length of time to “healthy” smokers

(Naruszewicz et al. 2002). Due to the smoking, smokers suffer from a systemic low-

grade inflammation. The treatment-product was compared with a placebo in form of a

rosehip-drink without probiotics. The treatment with L. plantarum 299v for 6 weeks

decreased the levels of fibrinogen, F2-isoprostanes and IL-6 in the blood.

Fibrinogen is an acute phase protein that is produced in the liver at inflammation. Fibrinogen is a marker for inflammation, and it is an independent risk factor for thrombosis. IL-6 is a pro-inflammatory cytokine that regulates the production of fibrinogen. F2-isoprostanes are biochemical markers of lipid peroxidation and oxidative stress.

The treatment with L. plantarum 299v for 6 weeks also decreased the systolic blood

pressure and serum-concentration of leptin in the smokers (Naruszewicz et al. 2002).

Leptin is produced by fat tissue and in smaller amounts by other peripheral organs, and

there seems to be a close relation between fastening insulin concentrations, leptin and

systolic blood pressure. Primarily leptin is a marker for fat-tissue (amount of fat in the

body) but it can also be regarded as a marker for inflammation. Thus, the finding of

Naruszewicz et al. (2002) that probiotic treatment for 6 weeks significantly lowered a

marker for body fat (leptin) was a quite new and remarkable one.

Leptin (from the Greek “leptos”; meaning thin) came to attention when the obesity gene and its product leptin were discovered. Leptin is a hormone-like messenger protein encoded by the obese (ob) gene. Leptin is primarily produced by adipose tissue and in smaller amounts by other peripheral organs. It is a messenger substance with a complex signalling pattern, interacting with many organs and also signalling to the immune system. Leptin levels in the blood are increased at food intake, infections, fever and systemic inflammation, but are decreased at starvation, weight loss and anorexia nervosa (La Cava et al. 2004).

Overweight and obesity

High-energy yielding microbiota. The human intestinal microbiota contains around

1011

colony forming units (CFU) of bacteria per g colonic content, i.e. the gut contains

at least 1014

live bacterial cells that make up about 1 kg of biomass. These bacteria,

centrally located in the body, interact with the host and with food constituents in the gut,

and they can, for example, be important for how much energy a human can utilise from

food.

It has been shown in mice that the gastro-intestinal microbiota was essential for the

processing of dietary polysaccharides (Bäckhed et al. 2004). Furthermore, it was shown

that in adult germ-free mice, inoculated and colonized with a normal mouse gut

microbiota produce a 60% increase in body fat content within 14 days (Bäckhed et al.

2004). However, this “bacterial organ” the microbiota, does also exercise physiological

effects and some components of the microbiota can have physiopathological effects.

Thus, the germ free mice that were colonized with a normal gut microbiota, not only

showed an increased accumulation of fat, they also got increased insulin resistance,


149

increased metabolic rate (oxygen consumption) and increased leptin levels (Bäckhed et

al. 2004).

In a follow-up study, the microbiota of male leptin-deficient mice (lacking the gene for

leptin) was compared with siblings with intact leptin gene (Ley et al. 2005). It was

shown that leptin-deficient mice consumed more feed and became heavier, and got

higher epididymal fat-pad weights (the epididymis is part of the male reproductive

system and the associated fat pad can be used as marker for fatness). The fat, heavy

mice showed a reduction in the proportion of bacterial taxa belonging to the phylum

Bacteroidetes and a proportional increase in taxa of Firmicutes (Ley et al. 2005). The

conclusion of the authors was that bacteria of the phylum Firmicutes were linked to

obesity.

To test the findings in mice, the same group of scientists performed a study in faeces

from obese and lean humans (Ley et al. 2006). Here it was shown that the relative

proportion of Bacteroidetes in comparison with Firmicutes was lower in obese humans

than in lean humans and that the proportion of Bacteroidetes increased with time in

obese individuals put on a low-calorie diet. In this study (Ley et al. 2006), an increasing

abundance of Bacteroidetes correlated with percentage loss of body weight (Ley et al.

2006). Furthermore, the same group of scientists presented results demonstrating that

germ-free mice that were inoculated with a microbiota rich in Firmicutes exhibited

higher percentage-increase in body fat over 2 weeks than mice colonized with a

Bacteroidetes-rich flora (Turnbaugh et al. 2006). The mice receiving a Firmicutes-rich

microbiota got that from obese, leptin-deficient mice, while the mice inoculated with the

Bacteroidetes-rich microbiota got that from lean, leptin-intact mice (Turnbaugh et al.

2006). The authors speculated that “the bacterial flora of the gut may be a new

therapeutic target for people suffering from obesity”.

On the other hand, the above findings about the GI-microbiota and obesity raise at least

three questions:

1) Are the observed differences in microbiota really the cause of obesity? The obesity

can just as well have been a marker of a more efficient and better functioning ecological

system.

2) Is the high proportion of Firmicutes unique for obese individuals?

3) The genus Lactobacillus belongs to the phylum of Firmicutes, does that mean that

administration of Lactobacillus as probiotics and in lactic acid fermented foods leads to

overweight and obesity?

The first question is a difficult one and for the time being, there is no clear answer to it,

even if a guess can be that the Firmicutes-rich microbiota is a somewhat healthier one

than the Bacteroidetes-rich microbiota, and therefore will lead to better appetite growth

and fat accumulation. But, the two following questions are easily answered, i.e. both

answers are negative: Bacteria of the phylum of Firmicutes are not unique for obese

individuals, and you will not be fat by eating Lactobacillus.

It should be hold in mind that Firmicutes includes a wide spectrum of different

organisms, i.e. the phylum Firmicutes includes six different classes, nine different

orders, 36 different families, more than 240 different genera and more than 1500

different species. Some species are well-known pathogens as Bacillus anthracis,

Streptococcus pyogenes, Listeria monocytogenes and Clostridium difficile, causing

specific and very different diseases, and some are well-known dietary bacteria safely


150

ingested with lactic acid fermented food, for example, Lactobacillus and Lactococcus.

Pathogens and lactobacilli have very different effects on the human body, and it is

misleading to place them under the same umbrella. It has even been demonstrated that

rats given a so called “western diet” (a diet rich in sugar and fat) for 6 months (a

relatively long time in the life span of a rat) come out somewhat leaner and in better

condition when the diet had been supplemented with the probiotic strain L. plantarum

HEAL19 (Karlsson et al. 2011).

Pro-inflammatory microbiota. An alternative explanation to how the GI-microbiota

can affect fattening has been launched by Cani et al. (2007). It is an established fact that

type-2 diabetes and obesity are characterized by insulin resistance and low-grade

inflammation. Obese individuals have elevated levels of inflammatory markers such as

CRP and Il-6 in the blood, but also the concentration of leptin is increased. Leptin is a

pro-inflammatory messenger to macrophages and T-cells, and leptin can amongst other

things be regarded as a marker for inflammation. Cani et al. (2007) showed that

lipopolysaccharides (LPS) originating from the microbiota in the GI-tract (gram-

negative bacteria) were the triggering factor for inflammation and obesity in mice, and a

high-fat diet increased the LPS-concentration in the blood, and also the proportion of

LPS-containing bacteria in the gut. It was proposed on the basis of experiments in mice

that changes in the gut microbiota towards a microbiota with a higher proportion of

LPS-producing taxa (for example from the phyla Proteobacteria and Bacteroidetes) can

be caused by a high-fat diet. This can in turn and perhaps also in combination with the

high-fat diet, causes endotoxemia (LPS in the blood) which induces systemic

inflammation. The inflammation will initiate a process leading to fat accumulation that

overtime can lead to overweight, obesity and type-2 diabetes (Cani et al 2008; Figure

31).

Figure 31. A leaky gut mucosa leads to translocation of pathological agents, e.g. lipopolysaccharides (LPS), and the consequence is inflammation. Long-term inflammation increases the risk for many diseases and may trigger fat accumulation which can lead to overweight and obesity.

A high fat diet has also been shown to increase the level of LPS in human blood

originating from bacteria in the gut (Pendyala et al. 2012). Then of course a reasonable

question would be: Why should fat in the diet stimulate elevated levels of LPS in the


151

blood? However, it is not fully clarified why gram-negative components of the

microbiota seem to be stimulated by a fat-rich diet, or/and why the barrier function of

the mucosa seems to decrease. A speculation could be that a fat-rich diet increases the

amount of bile in the gut, and bile has strong antimicrobial effects but some taxa have

higher resistance against bile than others, e.g. Enterobacteriaceae and Bacteroides are

known for their high bile resistance. Furthermore, bile is a powerful detergent which in

excess might have effects on the permeability of the mucosa and mediate an increased

leakage of LPS.

Nevertheless, there are indications that the composition of the gut microbiota in the long

run can have effects on the metabolic syndrome and associated disease, and fat persons

have been shown to have a less diverse gut microbiota than lean individuals (Lee et al.

2011), and obese and overweight preschool children harboured a higher proportion of

Enterobacteriaceae in the gut than children of normal weight (Karlsson et al. 2012).

Furthermore, when the faecal microbiota of 169 obese Danish individuals were

compared with that of 123 non-obese ones, it was seen that individuals with low number

of bacterial genes had more overall adiposity, higher insulin resistance and

dyslipidaemia and higher inflammatory status (Le Chatelier et al. 2013). The number of

bacterial genes is here taken as a measure of the “richness” of the microbiota (richness

is a straight forward, but somewhat coarse measure of diversity). The low-diversity

microbiota was characterized by the genera Bacteroides, Parabacteroides,

Ruminococcus, Campylobacter, Dialister, Porphyromonas, Staphylococcus and

Anaerostipes while the high-diversity microbiota was characterized by

Faecalibacterium, Bifidobacterium, Lactobacillus, Butyrivibrio, Alistipes, Akkermansia,

Coprococcus and Methanobrevibacter (Methanobrevibacter belongs to the kingdom

Archaea) (Le Chatelier et al. 2013).

In conclusion, there ought to be a potential for probiotics against these dysfunctions,

especially probiotics with abilities to strengthen the barrier effect of the GI mucosa and

to mitigate inflammation. Disorders in the lipid metabolism can cause hypertension, and

hypertension is often linked to hypercholesterolemia. There have been probiotics on the

market with the claim of affect blood cholesterol. For example, yoghurt supplemented

with L. acidophilus and B. longum increased the beneficial HDL cholesterol (Kiessling

et al. 2002), and the blood pressure could be decreased in elderly hypertensive subjects

by intake of a sour-milk fermented with Lactobacillus helveticus together with the yeast

Saccharomyces cerevisiae (Hata et al. 1996).

To consider

Probiotics can improve the status of the mucosa and strengthen the mucosal barrier

effect. Thus, probiotics can (i) modulate the response of the immune system; (ii)

strengthen the tight-junctions of the epithelial cell in the mucous membrane; (iii)

stimulate the epithelial cells of the mucous membrane to produce more mucin; (iv)

decrease the proportion of adverse bacteria in the gut; (v) improve the digestion.

Different probiotic strains may have different capacity to fulfil different missions.


152

It is difficult and requires a lot of resources to prove an effect of a drug against a

specific disease, but it is even more difficult to prove health beneficial effects of dietary

compounds in non-diseased subjects. In the latter it is often the prevention of a disease

that shall be proved, and randomised placebo controlled studies ought to be run for long

time periods to prove the preventative effect. However, when it comes to testing of

dietary compounds, it may be impossible to run placebo controlled trial, as the running

time will be too long and it may be impossible to provide an acceptable placebo. Instead

cohort studies backed up with animal studies to prove the causal connection can be an

accessible way. On the other hand, if the dietary compound to be tested is powerful

enough to give short time effects, and the test-compound can be excluded from the

placebo, it is feasible to apply placebo controlled studies for the testing of dietary

compounds, e.g. the effect of a probiotic strain. The value of evidence of such short

time studies is much dependant on the quality of the endpoints of the study. The best

endpoints are those of improvement or prevention of dysfunctions. The value of

evidence for improvement of biomarkers is much weaker and the relevance of

biomarkers can always be called in question. However some biomarkers are more

established and reliable than others. For example, cholesterol in the blood is a well

established biomarker in the metabolic syndrome for predicting the risk for

cardiovascular diseases. There are many immune-related compounds that at least

theoretically can be used as biomarkers for the status of the immune system and as such

giving hints about the health-status. However, there are often different opinions in the

scientific community about how they should be interpreted.

There are good evidences from randomised, placebo controlled studies that some

probiotics can mitigate antibiotic associated diarrhoea, and colitis due to C. difficile. It

is also well documented that some probiotic strain can mitigate rotavirus enterocolitis.

These are diseases and they are frequently occurring in certain groups of populations,

i.e. outbreaks can be fortelled and placebo controlled studies can be set up during the

right seasons and in subjects with increased risk for the disease. Thus, the effect of the

probiotic treatment can be measured with relevant endpoints, as the duration of the

disease and severity of symptoms.

It can be noted that probiotics can act both against pathogenic bacteria and pathogenic

virus, and be active both in the small intestine (rotavirus infects in the small intestine)

and in the large intestine (C. difficille infects the large intestine). And there is also

reliable placebo controlled studies, showing that some probiotics are able to counteract

common colds in the upper respiratory tract, i.e. virus infecting the mucosa in the

mouth, nose and throat. The reason for this broad working ability of probiotics is

presumably that the probiotics can improve the status of the mucosa and/or upgrade the

efficiency of the immune system. Both protection functions can be expected to have

relevance for the power of resistance against mucosal infections, irrespectively of

location and sort of infectious agent.

Functional bowel disorders are frequently occurring problems in urbanised populations.

A disorder as the irritable bowel syndrome (IBS) is not linked to a specified disease.

There is convincing evidence in form of randomised, placebo controlled studies that

different sort of probiotics can mitigate the symptoms of IBS, particularly pain and

bloating. When it comes to inflammatory bowel diseases (IBD), probiotics might have a

role in mitigate symptoms, but are not likely to affect the actual disease. The

inflammation in IBD is enhanced by unfavourable changes in the gut microbiota


153

towards a higher proportion of more aggressively pro-inflammatory bacteria. Probiotics

can counteract these pro-inflammatory bacteria and can presumably have a somewhat

inflammation suppressing effect on the immune system. Attempts to suppress symptoms

in IBD patients have been more promising in ulcerative colitis than in Crohn’s disease.

From animal models it is clear that probiotics can improve the barrier effect of the

mucosa at severe intestinal inflammation and at liver injury. In liver injury, it is

especially important to block translocation of gut bacteria as those, through the portal

blood will reach the liver and enhance the inflammation in the liver. Unfortunately,

there are for the time being not much human evidences for the effect of probiotics on

liver status. Otherwise this seems to be a promising field for future probiotic research

and developments.

There are interesting evidence for the capability of probiotics to decrease markers for

systemic low-grade inflammation. This is presumably, in some way or another

dependent on decreased leakage of bacteria or bacterial components through the

intestinal mucosa. It has also been pointed out, mainly based on animal models that the

starting point of overweight and obesity can be an increased leakage of proinflammatory

bacteria and their components through the GI-mucosa, leading to low-grade systemic

inflammation. The systemic inflammation can in turn lead to the metabolic syndrome,

obesity, type-2 diabetes and cardiovascular diseases. A high proportion of

Proteobacteria (for example E. coli and other taxa of the family Enterobacteriaceae) in

the gut microbiota is suggested to be the driving force of such a development.

Proteobacteria are generally proinflammatory due to the association of immune-

aggravating lipopolysaccharides (LPS) in the cell wall.

Concerning overweight and obesity, it has been pointed out that different compositions

of the gut microbiota are differently efficient to harvest energy from indigestible

polysaccharides. A high ratio of Firmicutes to Bacteroidetes in the gut microbiota has

been suggested to be correlated with overweight and obesity. However, it seems not

likely that this is the cause of the fat accumulation. Probably a high proportion of

Firmicutes in the gut microbiota is a marker for a good intestinal ecology, and hence, an

efficient energy-generating system.

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Appendix A:

Inflammation


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167

Defence

Inflammation is a defence-reaction of the body against an injury. The word

inflammation originates from the Latin “inflammatio” which means fire. Inflammation

is traditionally characterised by redness, swelling, pain, heat and impaired body

functions. Redness and heat are caused by increased blood flow, swelling by

accumulation of fluid, and pain by the swelling, but also by release of compounds

giving rise to nerve signals. Impaired functions can be caused by different reasons but,

in a certain analogy to fire, inflammation is devastating in order to clear away harmful

agents and so to say prepare the ground for healing.

Inflammation can be triggered off by both internal and external factors. Powerful

triggers for inflammation are the presence of microorganisms in sites where they do not

belong. Microorganisms contain structures alien to the body. For example, bacteria have

cell walls in contrast to human cells and the bacterial cell wall includes lots of

compounds that are alien to the human body. Cells and molecules involved in the

inflammatory defence system react immediately against alien elements that become

danger signals to the body. Inflammation can also be triggered by injuries to body tissue

and breakage of body-cells. When the cells are damaged, compounds that are normally

hidden within the cells are released and become danger signals.

All forms of immune reactions will lead to activation of the inflammatory defence

system. Consequently, inflammation can be started by infections, decomposition of

body tissue by trauma and autoimmunity or allergy. In autoimmunity the specific

immune system attacks body cells and tissue and trigger the inflammation, and in

allergy the inflammation is provoked by the specific immune system being activated

against different types of harmless compounds in the environment.

Inflammation is initiated by cells already present in the tissue, for example, resident

macrophages, dendritic cells and mast cells. Danger signals trigger these cells into

activation, and inflammatory mediators are released, which starts the process

responsible for the clinical signs of inflammation.

The process of inflammation proceeds along the following stages:

1) Blood vessels are expanded which lead to increased blood flow.

2) The permeability of the wall of the blood vessels is increased, which results in an

outflow of fluid and plasma proteins into the tissue.

3) White blood cells are recruited from the blood circulation to the tissue.

When the process of inflammation has been initiated, it will proceed until the source of

the inflammation has been erased and the process of healing can start. However, if the

cause of the inflammation cannot be eliminated, the inflammation will continue, and

then it will often vary in intensity over time.


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Acute, chronic and subclinical

In acute inflammation, there will be an accumulation of neutrophils (neutophil

granulocytes) in the inflamed tissue, while in chronic inflammation there will be an

accumulation of lymphocytes, macrophages and plasma cells in the tissue and these will

also infiltrate the connecting tissue. In an allergic reaction, however, there will be a

rapid accumulation of eosinophils (eosinophil granulocytes) and T-lymphocytes, and

sometimes also neutrophils (Mölne and Wold 2007). Acute inflammation is typically

caused by an infection, but death of human cells or decomposition of cancer tumours

will lead to acute inflammation. Chronic inflammation can, for example, be caused by

autoimmune diseases and reactions against foreign elements.

In an acute inflammatory response, the concentration of acute phase proteins such as C

reactive protein (CRC) and serum amyloid A protein (SAA) can increase steeply and

rise to 10 000-fold above the normal value (Pepys and Baltz 1983). However, different

markers for acute inflammation can also be monitored more closely for minor

deviations from normality. This type of slight elevation from the norm can be called

“low-grade inflammation”, or “subclinical inflammation”. Consequently, in this type of

condition the sharp short-term fluctuations of inflammatory markers are ignored;

instead, long-term systemic concentrations of the markers are taken into account,

especially if they correlate with more obvious risk factors such as, for example, blood

cholesterol and blood pressure. Low-grade systemic inflammation is associated with an

increased risk of cardiovascular disease (Ridker 2003). Obese individuals have higher

CRP levels than subjects of normal weight (Visser et al. 199; Piéroni et al. 2003).

Immunoactive cells and components

Gut-associated lymphoid tissue

The gut is associated with several types of lymphoid organs that collectively are

referred to as gut-associated lymphoid tissue (GALT). GALT is the largest collection of

lymphoid tissues in the body and consists of organised lymphoid tissues comprising

mesenteric lymph nodes, Payer´s patches, isolated lymphoid follicles, and

cryptopatches, as well as diffusely scattered lymphocytes and dendritic cells in the

lamina propria and intestinal epithelium. Some of them, such as Payer’s patches and the

isolated lymphoid follicles, are within the intestinal mucosa itself. In addition, intestinal

lymph drains into the mesenteric lymph nodes, which constitute a key checkpoint to

determine the anatomical location of tolerogenic or inflammatory responses.


169

Macrophages

Macrophages have different major functions in inflammation, i.e. (i) antigen

presentation, (ii) phagocytosis and (iii) immune-modulation through production of

various cytokines and growth factors. Monocytes/macrophages produce a wide range of

biologically active molecules participating in both beneficial and detrimental outcomes

of inflammatory reactions. They are also able to phagocytose and destroy infectious

agents. Therefore, monocytes/macrophages play a critical role in initiation,

maintenance, and resolution of inflammation. Macrophages form varying phenotypes

depending on what signals they encounter. Different subsets of macrophages express

different patterns of chemokines, surface markers and metabolic enzymes.

Toll-like receptors

Intestinal epithelial cells are capable of detecting bacterial antigens and initiating and

regulating both innate and adaptive immune responses. Signals from bacteria can be

transmitted to adjacent immune cells such as macrophages, dendritic cells and

lymphocytes through molecules expressed on the epithelial cell surface, for example, by

Toll-like receptors (TLRs) (Cario et al. 2002). TLRs alert the immune system to the

presence of highly conserved microbial antigens often termed “pathogen-associated

molecular patterns” (PAMPs) present on most microorganisms. Examples of PAMPs

include LPS, peptidoglycan, flagellin, and microbial nucleic acids. At least ten types of

human TLRs are known.

NOD-like receptors

Besides the TLRs there is another family of membrane-bound receptors for detection of

proteins called NOD-like receptors or “nucleotide-binding domain, leucine-rich repeat

containing” proteins (NLRs). The best characterised members are NOD1 and NOD2,

but more than twenty different NLRs have been identified. NRLs are located in the

cytoplasm and are involved in the detection of bacterial PAMPs that enter the body-cell.

NRLs are especially important in tissues where TLRs only are expressed at low levels

(Philpott et al. 2001). This is the case in the epithelial cells of the GI-tract where the

cells are in constant contact with the microbiota, and the expression of TLRs must be

down-regulated in order to avoid over-stimulation. On the other hand, if these epithelial

gut-cells become infected with invasive bacteria or bacteria interacting directly with the

plasma membrane, they will come into contact with NLRs and defence mechanisms can

be activated (Girardin et al. 2001). NLRs are also involved in sensing other endogenous

warning signals which will result in the activation of inflammatory signalling pathways,

such as nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases

(MAPKs). Both NOD1 and NOD2 recognise peptidoglycan moieties found in bacteria.

NOD1 can sense peptidoglycan moieties containing meso-diaminopimelic acid, which

primarily are associated to gram-negative bacteria. NOD2 senses the muramyl dipeptide

motif that can be found in a wider range of bacteria (Girardin et al. 2003; Hasegawa et

al. 2006). The ability of NRLs to regulate, for example, nuclear factor-kappa B (NF-κB)

signalling and interleukin-1-beta (IL-1β) production, indicates that they are important

for the pathogenesis of inflammatory human diseases, such as Crohn’s disease.


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Microfold cells

The follicle-associated epithelium, which covers Peyer’s patches (organized lymphoid

tissue, measuring a few centimetres in length) is located along the small intestine (about

30) and is particularly abundant in the ileum. The epithelium harbours shorter villi that

contain a type of specialised cells called microfold cells (M cells). M cells have

numerous microfolds on the epithelial side and are specialised in capturing antigens,

apoptotic epithelial cells or bacteria from the lumen, and transport them to Peyer’s

patches for sampling by dendritic cells or for destruction by macrophages (Newberry

and Lorenz, 2005).

Dendritic cells and intra-epithelial lymphocytes

Dendritic cells may present antigen locally to T cells, migrate to T cell zones or to

mesenteric lymph nodes, or interact with memory B cells (Pickard et al. 2004). Both

pathogenic and non-pathogenic bacteria can also enter the mucosal tissue through

lamina propria associated dendritic cells, which extend their dendrites through epithelial

cell tight junctions (Rescigno et al. 2001).

Also, intra-epithelial lymphocytes located in the epithelium might recognise microbial

antigens (Cheroutre 2004). In addition to intestinal epithelial cells, the epithelium

includes specialised cells such as goblet cells, which secrete the protective mucus layer

limiting the contact between bacteria and epithelial cells, and Paneth cells, which reside

in the crypts of the small intestine and secrete bactericidal peptides (Cash et al. 2006).

IgA antibodies

Secretory IgA is the predominant class of immunoglobulin found in intestinal

secretions. It is produced by plasma cells residing in the lamina propria and is

transported to the lumen by the polyimmunoglobulin receptor. IgA molecules contribute

to specific immunity by capturing antigens, thereby inhibiting mucosal penetration

(Cerutti and Rescigno 2008).

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173

Appendix B

Oxidative stress


174


175

Reactive oxygen species and antioxidants

Oxidative stress is caused by an excess of oxygen free radicals. Oxidative stress can

damage tissue, DNA, proteins and lipids and when injuring the body cells, the body

responds with inflammation. The most cell-damaging effect of reactive oxygen species

(ROS) is presumably lipid peroxidation of polyunsaturated fatty acids and

phospholipids in the cell membranes. The peroxidation changes structure and

permeability of the cell membrane. Oxidative stress can initiate, but also enhance

inflammation.

Oxidation is a free radical chain reaction, and so called “reactive oxygen species”

(ROS) include not only oxygen free radicals but also some nonradical derivates as

hydrogen peroxide (H2O2) and hyperchlorous acid (HOCL). In the cell, ROS are

involved in the energy generation, and in synthesis, cell growth and cell maintenance.

ROS are also used by immune cells in connection to phagocytosis to kill the alien

microorganism. Immune cells as neutrophils and macrophages can produce large

amounts of oxygen free radicals in order to kill the phagocyted microorganisms.

Inflammation leads to overproduction of ROS (Halliwell 1997).

The human body possesses antioxidant system in protection from the adverse effects of

ROS. There are antioxidative enzymes as, for example, superoxide dismutase, catalase,

glutathione peroxidise and glutathione reductase. There are also a nonenzymatic

defence of antioxidants, e.g. glutathione, alfa-topopherol, ascorbic acid and iron-binding

proteins. An antioxidant can be defined as a substance that, when present at low

concentration compared to that of an oxidizable substrate, delay or prevent its oxidation

(Shahidi and Zhong 2010).

Important dietary antioxidants are polyphenols of plant origin, carotenoids and vitamins

C, E and A. Antioxidants can exert different mode of inhibitory effects against

oxidation. They can scavenge free radicals, chelate metal ions, inactivate ROS and

inhibit pro-oxidative enzymes. Most polyphenols breaks the chain reaction of oxidation

and neutralize free radical by donating a hydrogen atom. The formed antioxidant radical

is stabilized by delocalization of the unpaired electron around the phenol ring to form

stable resonance hybrids (Shahidi et al. 1992).

Oxygen free radicals

Oxygen metabolism leads to production of oxygen free radicals, i.e.

O2- . HO2

.

. OH H2O2

Oxygen free radicals are reactive and cause cell damage if they are not disarmed.

The superoxide radical is formed when a single electron is transferred from a substrate

to the oxygen molecule:


176

In water, O2-

. is converted to HO2

. (proteonated) and

. OH (the hydroxyl radical). HO2

.

and . OH are the most reactive and harmful forms of oxygen free radicals. However, the

conversion from the superoxide form must be catalysed by a metal, preferably iron or

copper (Halliwell 1997):

Hydrogen peroxide (H2O2) is formed spontaneously:

Antioxidative reaction

Organisms living in environments exposed to oxygen and metabolising oxygen have

active defence system for taking care of the oxygen free radicals. For example, they can

possess superoxide dismutase, catalase or peroxidise:

Strictly anaerobic bacteria as Bifidobacterium lack these enzymes and are also unable to

use oxygen in their metabolism. They are also sensitive for exposure to oxygen.

More microaerophilic than anaerobic bacteria as Lactobacillus are sometimes, at least to

a minor extent able to utilize oxygen in the metabolism. However, this ability differs

between species. Species as L. reuteri and L. acidophilus are usually relatively sensitive


177

to oxygen while species as L. plantarum, L. rhamnosus and L. paracasei are more

oxygen resistant.

Lactobacillus never possesses superoxide dismutase. Superoxide dismutase is typically

found in truly aerobic organisms. For example, Lactococcus have superoxide dismutase.

L. plantarum use instead manganese for the protection against oxygen free radicals. In

fact L. plantarum is quite unique in the biological world due to the fact that L.

plantarum can grow and multiply in the complete absence of iron. L. plantarum does

not have any iron containing enzymes which is most unusual in biology. This may be an

advantage in the protection from oxygen free radicals, i.e. the conversion of the

superoxide radical to a hydroxyl radical is efficiently catalysed by iron, and

in the absence of iron less hydroxyl radicals are formed.

There is also Lactobacillus species that possess NADH-oxidase and NADH peroxidise

for taking care of the oxygen free radicals.

References

Halliwell, B. (1997). Antioxidants and human disease: a general introduction. Nutr. Rev.

55: S44-S52.

Shahidi, F., Janitha, P.K. and Wanasundara, P.D. (1992). Phenolic antioxidants. Crit.

Rev. Food. Sci Nutr. 32: 67-103.

Shahidi, F. and Zhong, Y. (2010). Lipid oxidation and improving the oxidative stability.

Chem. Soc. Rev. 39: 4067-4079.


178


179

Index

abscess

acetaldehyde

acetic acid

acetoin

acetone

Acidobacteria

Acinetobacter

A. baumannii

A. johnsonii

Actimel

Actinobacteria

Activia

acute

acute phase protein

Adanson

aerobe

Aeromonas

age

Agidi

aggravator

Akkermansia

A. muciniphila

Alanine aminotransferase (ALT)

Alcaligenaceae

Ale

Alistipes

A. putredinis (former Bacteroides putredinis)

allergy

ALT

ammonium

amyloid A protein (SAA)

anaerobe

Anaerotruncus

animal model

antibody

antioxidant

application

Aquifcae

Archaea

Aristotle

aspartate aminotransferase (AST)

AST

ATCC (American type culture collection)

Atobium


180

Atopic

Auk

Aurbacteruim

butyric acid

essence

autoimmunity

autotrophe

Bacilli

Bacillus

B. cereus

B. anthracis

Bacteria

bacterial flora

bacteriocin

Bacteroidia

Bacteroides

B. caccae

B. capillosus

B. dorei

B. eggerthii

B. finegoldii

B. fragilis

B. intestinalis

B. pectinophilus

B. putredinis

B. merdae

B. ovatus

B. stercoris

B. thetaiotaomicron

B. vulgatus

B. xylanisolvens

Bacteroidetes

Bactufugation

balance

barrier

baseline

base pair composition

basophile granulocyte (basophile)

B-cell

Behçet’s syndrome

benzoic acid

beta-glucan

Betaproteobacteria

Bifidobacteriaceae

Bifidobacteriales

Bifidobacterium

B. animalis

B. animalis BB12

B. animalis DN-173010

B. bifidum


181

B. bifidum MF 20/3

B. breve

B. infantis

B. infantis 35624

B. longum

B. longum SP 07/3

B. longum subspecies infantis DSM 15158

B. longum subspecies infantis CURE19

B. lactis

Bifico

Bifidus Regularis

bile

bilirubin

Bilophila

B. wadsworthia

blackberry

blackcurrant

Blautia

blood pressure

blueberry

B-lymphocyte

Brachyspira

B. aalborgi

Brachyspiraceae

brest

B-vitamin

butanediol

Butyrivibrio

cabbage

calcium

Caliciviridae

calicivirus

cancer

caper berries

Capparis

carbon dioxide

carboxylic acid

carcinoma

Cardiobacteriaceae

Cardiobacterium

Carnobacterium

C. divergens

C. hominis

C. piscicola

cassava

CCM (Czech collection of microorganisms)

CCUG (culture collection University of Göteborg)

CD (cluster of different ion)


182

CD56

CD284

Cellulomonas

Cesalpino

cheese

Cheddar

Chemoautotroph

chemokine

chemotherapy

child

Chlamydiae

Chlorobi

Chloroflexi

cholera

cholesterol

Chorizos

chronic

Chrysiogenetes

cirrhosis

citrate

cladogram

claudin

class

classification

coagulation

coeliac disease

cold

colitis

collagenous colitis

colon

Coprococcus

core

corona virus

Cracraft

clone

cloning

Clostridia

Clostridiaceae

Clostridium

C. aminobutyricum

C. aminophilum

C. boltei

C. botulinum

C. difficile

C. lactatifermentans

C. leptum

C. orbiscindens

C. oroticum

C. perfringens

C. putrefaciens


183

C. symbiosum

C. tyrobutyricum

Collinsella

complement system

C-reactive protein (CRP)

Critical-care

Crohn’s disease

cucumber

Cucumis sativis

Cytokine

Cytotoxic

CXC

Deferribacteres

Deinococcus-Thermus

Deltaproteobacteria

dendric cell

dermatitis

Desulfovibrionaceae

D-galactosamine

diabetes

diacetyl

Dialiser

D. invisus

diarrhoea

Dictyoglomi

DNA

DNA:DNA-homology

DNA:DNA-hybridization

domain

Dorea

D. formicigenerans

Dose

DSS (dextran sulphate sodium)

dysbacteriosis

dysbiosis

eczema

EEC (enterovirulent E. coli)

Eggerthella

E. lenta

EIEC (enteroinvasive E. coli)

EHEC (enterohemorrhagic E. coli)

Eleusine coacana

ellagic acid

empire

endotoxaemia

endotoxin

end-point

Ensete ventricosum

Enterobacter

Enterobacteriaceae


184

Enterobacteriales

Enterococcus

E. faecium

E. faecalis

enteropathogen

enterotoxin

eosinophil granulocyte (eosinophil)

EPEC (enteropathogenic E. coli)

epigenetic

epithelial

Erysipelotrichia

Erysipelotrichaceae

Escherichia

E. coli

ETEC (enterotoxigenetic E. coli)

ethanol

Eubacteriaceae

Eubacterium

Eubacterium

E. eligens

E. formicigenerans

E. halii

Eucarya

Euzéby

facultatively heterofermentative

Faecalibacterium

F. prausnitzii

Family

FBD (functional bowel disorder)

FDA (Food and drug administration)

fermentation

fever

fibrinogen

Fibrobacteres

fibrosis

filmjölk

Firmicutes

flavanol

flavonoid

flavonol

folic acid

forage

FoxP3 (fork head box P3)

F2-isoprostane

Friscus

fructo-oligosaccharides

Fusobacteria

F. varium

Fusobacteriaceae

Fusobacterium


185

gallic acid

gall-stone

GALT

Gammaproteobacteria

GAS

gastro-intestinal (GI)

G+C-ratio

Gemmatimonadetes

genus

goblet cell

Gram

Gram-reaction

Gut-associated lymphoid tissue (GALT)

Haemophilus

H. parainfluenzae

H-antigen

Haptoglbin

HDL-cholesterol

Helicobacter pylori

Hennig

hepatic stellate cell

hepatitis

hepatocyte

heterofermentative

heterotrophe

Hieracium

histone

Holdemania

Homofermentative

HT-29 cell

HUS (haemolytic uremic syndrome)

hydrogen

hydrogen peroxide

hydrogen sulphide

hydroxybezoic acid

hydroxyl radical

hyperlipidaemia

hypochlorite

IBD (inflammatory bowel disease)

IBS (irritable bowel syndrome)

icterus

IgA

IFN-gamma

IL-1

IL1-beta

IL-2

IL-4

IL-6

IL-8

IL-10


186

IL-12

IL-13

Ileum

immune-modulation

immune system

infant

influenza virus

intensive-care

interferon

interleukin (IL)

inulin

iron

ischemia

ischemic colitis

isolate

jaundice

jejunum

K-antigen

Kimchi

Kiviak

Klebsiella

K. pneumoniae

Koch

Kocho

kupffer cell

lactic acid

LAB (lactic acid bacteria)

Lachnospiraceae

Lactobacillaceae

Lactobacillales

Lactobacillus

L. acidophilus

L. amylovorus

L. brevis

L. bulgaricus

L. casei

L. casei Shirota

L. casei defencis

L. casei DN-11401

L. crispatus

L. delbrueckii

L. delbrueckii subspecies bulgaricus

L. fermentum

L. fermentum ME-3

L. fermentum RC-14

L. helveticus

L. gallinarum

L. gasseri

L. gasseri PA 16/8

L. jensenii


187

L. johnsonii

L. johnsonii La1 (Lj1)

L. kefir

L. kefiranofaciens

L. paracasei

L. paracasei F19

L. paracasei 8700:2

L. pararplantarum

L. pentosus

L. plantarum

L. plantarum 299v

L. plantarum WCFS1

L. plantarum HEAL9

L. rhamnosus

L. rhamnosus GG

L. rhamnosus GR-1

L. reuteri

L. reuteri RC-14

L. sake

L. salivarius

L. salivarius UCC118

L. salivarius UCC4331

Lactococcus

L. lactis subspecies lactis

L. lactis subspecies cremoris

L. diacetolactis

lactoferrin

lactoperoxidase

lactulose

Lamarck

lamina propria

leakage

leptin

Leptotrichia

Lenthisphaerae

Leuconostoc

L. mesenteroides subspecies mesenteroides

L. mesenteroides subspecies cremoris

L. lactis

Lignin

lignin

Linnaeus

Linné

lipoteichoic acid

Listeria

L. monocytogenes

liver

low-grade

LPS (lipopolysaccharide)

LPSN (List of prokaryotic names with standing in nomenclature)


188

lumen

lymphocyte

lymphocytic colitis

lymphoid

macrophage

Magnol

maize

malic acid

malt

mammalian

manganese

Manihot esculenta

mast cell

Mayr

M-cell

Medvurst

Megasphaera

meta-analysis

metabolic syndrome

mesophilic LD culture

metagenomics

Metchnikoff

methotrexate

microaerophile

microbiota

Micrococcaceae

millet

minimal hepatic encephalopathy (MHE)

Mogibacterium

monocyte

Moraxellaceae

morbidity

morphology

mortality

mother

mould

MPO (myeloperoxidase)

MUC

mucin

mucous membrane

multiple organic failure

myeloid cell

myeloperoxidase

NaCl

NADH-oxidase

NADH-peroxidase

NAFLD (non-alcoholic fatty liver disease)

NCAM (neural cell adhesion molecule)

Negativicutes

Neisseria


189

N. subflava

Neisseriaceae

neutrophil granulocyte (neutrophil)

NFkappa-B

nitrogen oxide (NO)

Nitrospira

NK cell

NOD-like

non-alcoholic-fatty-liver-disease (NAFLD)

non-starter lactic acid bacteria

Nordenskiöld

Norovirus

nucleotide-binding

O-antigen

oats

oatmeal

occludin

Oenococcus

O. oeni

O.kitaharae

Ogi

oligosaccharide

olive

open-label

opportunistic

order

Oryza sativa

Oscillospira quilliermondii

OTU (operational taxonomic unit)

Overgrowth

overweight

oxidase

oxidative burst

oxygen free radical

PAMP

pancreatitis

paneth cell

Parabacteroides

P. distasonis (former Bacteroides distasonis)

paracellular

Pasteur

Pasteurellaceae

Pathogen-associated molecular patterns (PAMP)

pathogenic

PCR (polymerase chain reaction)

Pediococcus

P. pentosaceus

Pentose

peptidoglycan

Peptostreptococcaceae


190

Peptostreptococcus

P. anaerobius

peritonitis

permeability

Peyer’s patch

PFGE (pulse field gel electrophoresis)

pH

phagocytic

phagocytosis

phenetic

phenolic acid

phenyl acetic acid

phenyl propionic acid

phenyl valeric acid

photoautotroph

phylum (pl. phyla)

phylogenetic

physiological

physiopathological

phytate

pit cell

placebo

Planctomycetes

plasma cell

polyphenols

polysaccharide

Porphyromonadaceae

Porphyromonas

prebiotics

Prevotella

P. melaninogenica

Prevotellaceae

Propionispira arbores

Porphyromonadaceae

Porphyromonas

portal blood

probiotics

Proteobacteria

pseudocatalase

Proviva

Probi frisk

Pseudomonas

P. aeruginosa

Pseudomonadaseae

Psychrobacter

pure culture

radical

radiotherapy

RAPD (randomly amplified polymorphic DNA)

Ray


191

REA (restriction endonuclease analysis)

rectum

relative humidity

rennet

resident

respiration

reticuloendothelia

reticuloendothelial system (RES)

RFLP (restriction fragment length polymorphisms)

rhinovirus

Rhodiola

ribotype

ribotyping

rice

Rikenellaceae

rRNA (ribosomal RNA)

RNA

Rome

Roseburia

R. intestinalis

rotavirus

Rothia

Rubus

Ruminococcaceae

Ruminicoccus

R. gnavus

R. lactaris

R. obeum

R. torques

SAA

Saccharomyces cerevisiae

salami

Salix

Salmonella

Sanger sequencing

sauerkraut

sausage

Saccharomyces

safety

salmon

scaffold protein

scarlet fever

Schinkenplockwurst

secretary IgA

semidry

sensu strict

septic throat

sequencing

serotype

Serratia


192

Shigella

Shirota

Siberia

silage

Sirochaetes

smoking

Sneathia

sorghum

Sorghum

species

spp. (sp. = one species; spp. = several species)

Spirochaetae

Spirochaetes

Staphylococcus

S. aureus

Steatohepatitis

steatosis

stilbene

stomach

stomach flue

strain

Streptoccaceae

Streptococcus

S. anginosus

S. mitis

S. oralis

S. parasanguinis

S. pneumonia

S. pyogenes

S. salivarius

S. thermophilus

subclinic

Subdoligranulum

superoxide

suppressor

surgery

Sutterella

S. wadsworthensis

synbiotics

Synergisetes

systematic

systolic

tannin

Taraxacum

Taxonomy

T-cell

Th

T-helper cell (Th-cell)

Thermodesulfobacteria

Thermomicrobia


193

Thermotogae

Tight junction

thiocyanate

T-lymphocyte

TLR2

TLR4

TLR9

Toll-like receptor (TLR)

TGF-beta

TNF-alpha

Togwa

transcellular

translocation

transmembrane protein

transplantation

trauma

T-RFLP

Tschuktsch

TTGE (temporal temperature gradient gel electrophoresis)

Tschuktscer

TTP (thrombotic thrombocytopenic purple)

Tumour necrosis factor (TNF)

typing

type

type strain

ulcerative colitis

vagina

vaginosis

Veillonella

V. parvula

Veillonellaceae

Verrucomicrobia

Verrucomicrobiae

Verrucomicrobiaceae

Verrucomicrobium

virus

vomit

VSL#3

Warg

water activity

weaning

Weissella

W. minor

W. confusa

white blood cell

wine

winter vomiting disease

Yakult

yeast

yoghurt


194

Zea mays

zinc

ZO-1

zonulin


195


196

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