jb prajapati 080315
TRANSCRIPT
J.B. PrajapatiPrincipal & Dean
SMC College of Dairy Science, Anand Agricultural University,
Anand–388 110 (Gujarat)
Probiotics for Geriatric Population
JBP-YIMPSF-080315 1
Global ageing…..
2000-2030: Adults worldwide >65 years of age to double from 420 million to 973 million
JBP-YIMPSF-080315 2
The greying of India…
Improvement in health care
Improved living
standards
improvement in
socioeconomic status
JBP-YIMPSF-080315 3
Consequences of ageing…
Loss of physiological functions
Loss of physical and metal faculties, weakness of health
Increased vulnerability to diseases
Chronic, disabling and multiple health problems
Great discomfort to elderly
Distress to family
Clinical, social and economical problems
Martinez et al, 2014; National Academy of Sciences, 2012JBP-YIMPSF-080315 4
Age related changes in the organ system…..1
Organ system Effects of aging
Body composition Progressive reduction in total body water and leanbody massIncrease in body fat
Cardiac andperipheral vascularsystem
Heart changes (stiffening, reduced muscle strength)Reduction in the intrinsic heart rate, Atherosclerosis and loss of elasticity of vessel walls
Musculoskeletal Loss of muscle tissueOsteoarthritisOsteoporosis
Central nervoussystem
Increased sensitivityDecreased blood flowDecline in receptors and pathways (fewer brain cellsand connections)
JBP-YIMPSF-080315 5
Age related changes in the organ system….2
Organ system Effects of aging
Gastrointestinal Decreased secretion of hydrochloric acid and pepsinDysfunction in GI motilityDecreased GI blood flowReduction in liver volume and blood flow
Immune system Decreased immunity to diseasesGreater susceptibility to infections
Respiratory Vital capacity may decline with age, Increased rigidity of chest wallReduced thorax muscle strength and endurance
Sensory Visual impairment, thickening and yellowing of thelens of the eyeHearing impairment,Decline in the ability to taste and smell
JBP-YIMPSF-080315 6
Ageing associated with multiple illnesses and physical ailments and majority of these chronic conditions defy cure
Hence…..Need of the hour..
Encouraging healthy ageing through diet which can also overcome malnutrition
a preventive or alternative therapy which is milder with less of adverse effects
JBP-YIMPSF-080315 8
MICROBES IN HEALTHY AGEING
JBP-YIMPSF-080315 9
Combating putrefaction in the gut by hygiene, diet and biologicals
Elderly gut microbiota different than that of younger adults
high inter-individual variation in microbiota composition in elderly
JBP-YIMPSF-080315 11
Bacteriodetes
Bacteriodetes
Firmucutes
Firmucutes
The solution…
Improve microbial balance of the intestinal tract of elderly
Providing healthier nutrition
Developing elderly specific functional foods
JBP-YIMPSF-080315 12
Probiotics in geriatric health-emerging evidences Probiotics have been recommended by WHO as an adjunctive therapy for
nutritional deficiency (Kurniawan and Simadibrata, 2011)
The potential role of probiotics on the gut-brain axis is an emerging study area particularly important in relation to neurodegerative diseases of elderly (Martinez, et al, 2014).
Results of some previous studies……..
JBP-YIMPSF-080315 14
Selected studies in elderly showing effect of probiotics on gut microbiota and the immune system.
Strain Product Assay design1;n2
Age(years)
Effect3 Reference
Bifidobacteriumlactis HN019
Dehydrated sachets mixed with low fat milk
Xover;30 Median 69
↑T helper cell (CD4), activated T lymphocytes (CD 25) and NK cells ↑ ex vivo phagocytic capacity of mononuclear and polymorphonucleaer phagocytes and the tumoricidal activity of NK
Gill et al., 2001
Lactobacillus casei (Shirota)
Probiotic dairy product
DBRPC;124 61±7.3 Improved the mood of those whose mood was initially poorNo improvement of frequency of defecation
Benton et al., 2006
JBP-YIMPSF-080315 15
B. lactis HN019 Mixture with skim milk
DBRPC;4 groups of 20
>60 ↑bifidobacteria, lactobacilli and enterococci↓Enterobacteria
Ahmed et al., 2007
Lactobacillus delbrueckii subsp. bulgaricus B481
Capsule with dehydrated probiotics
DBRPC;61 >85 ↑NK cells↑immune risl parameters↓proinflammatory cytokine IL-B↑ antimicrobial peptide β defensins02(hBD-2)
Moro Garcia et al., 2012
BifidobacteriumlongumBB536
Enteral tube feeding
DBRPC;hospitalized 45
81.7±8.1
↑Bifidobacterium ↑ IgA after influenza vaccination (A/H1N1, A/H3N2 and B)↑ NK cell activity(in subjects with lower NK cell activity)
Akatsuet al., 2012
Lactobacillus plantarum CECT 7315 and 7316
Capsule with dehydrated probiotics
DBRPC;nursing home 60
65-85 ↑ response to influenza vaccination (↑ influenza specific IgA and IgG)
Bosch et al., 2012
JBP-YIMPSF-080315 16
Bifidobacteriuminfantis CCUG 52486, B. longumSp 0713, Lactobacillus rhamnosus GG and Lactobacillus casei (Shirota)
Fermemented milk
In vitro PBMC; 16
65-76 Each single probiotic enhanced NK activity with B. infantis effect was influenced by ageing In the youngest ↑Bifidobacterium , ↑IFN-ϒ(not by LGG)↑IL 6 production in the older and B. infantis was the most anti inflammatory
You and Yaqoub, 2012
B. longum BB536
Enteral tube feeding
DBRPC (two trials);Hospitalized B3/123
65-102 Regularised bowel movements with lower input. No other differences
Kondo et al., 2013
B. longum Bar 33 and Lactobacillus helveticus Bar 13
biscuits DBRPC;32
71-88 ↓ opportunistic pathogens Clostridium cluster XI, Clostridium difficle. Clostridium perfringes, Enterococcus faecium and Campylobacter
Rampelliet al., 2013
1Type of Intervention Assay; Xover=cross over assay; DBRPC=double blind randomized placebo control2n=number of subjects3NK=natural killer cells; Il=ineterleukin A/G; IFN-ϒ=interferon-gamma
JBP-YIMPSF-080315 17
Selected studies in elderly showing the effect of synbiotics on gut microbiota and the immune system
Treatment Product Assay design2;n3
Age(years)
Effect4 References
B. bifidum BB-02, B. lactisBL01 and inulin
6 g chicory inulin (Rafilose) plus capsule with capsules with dehydrated probiotics per day
DBRPC;18 >62 ↑bifidobacteriaand lactobacilli
Bartosch et al., 2005
B. longum2C (DSM 14579) and 46 (DSM 14583) vs. B. animalis BB12 and oatmeal
Fermented oat meal
DBRPC;169 Avg 84-3
↑Bifidobacterium species in all samples correlated to↓ TNF –α and IL-10
Ouwehand et al., 2008
JBP-YIMPSF-080315 18
B. longum 46 and B. longum2C and oatmeal
fermented oat meal
DBRPC;66
84±8 ↑Bifidobacteriumcalanufatum, B. bifidumand Bifidobacteriumbreve
Lahtinen et al., 2009
L. rhamnosus GG and FOS
250 g/d commercial yoghurt with LGG and 2.4% scFOS (Actilight)
DBRPC; 12 women constipation, nursing home
76-90 No increase of bifidobacteria↓presence of LGG in faeces of elderly froup than in younger adults↑evaluation number in the elderly, probably due to the presence of FOS
Granata et al., 2013
JBP-YIMPSF-080315 19
Work done at AAU
JBP-YIMPSF-080315 20
Metagenomic and Clinical investigation of synbiotic fermented dairy product containing probiotic
Lactobacillus helveticus MTCC 5463 in geriatric volunteers
21
PARTNERS: Dairy Microbiology Department, Anand Agricultural University, Anand
PI : Dr JB Prajapati, Professor, Dairy MicrobiologyCo-PI : Dr Vijendra Mishra, Associate Prof., Dairy Microbiology Mrs Suja Senan, Asst. Prof., Dairy Microbiology Mrs Sreeja V. Asst. Prof., Dairy Microbiology
Animal Biotechnology Department, Anand Agricultural University, AnandCo-PI : Dr CG Joshi, Prof., Animal Biotechnology
HM Patel Centre for Medical Care & EducationPramukh Swami Medical College, Karamsad-Gujarat
Co-PI : Dr Himanshu Pandya, Prof., Medicine Dr Sunil Trivedi, Prof of Microbiology Dr Uday S Singh, Prof of Community Medicine Dr Rupal Patel, Asst. Prof., Microbiology Dr Manisha Gohel, Asst. Prof. Community Medicine
Dr Ajay Pathak, Statistical Expert
Vidya Dairy, AnandCo-PI : Dr HK Desai, Managing Director
JBP-YIMPSF-080315
22
Objectives
Selection of functional synbiotic dairy product
To conduct feeding trial in old age subjects and validate health benefits
Study changes in composition and biochemical activities of gastrointestinal microflora
Metagenomic analysis of gut microflora composition and metabolic pathways.
To manufacture product on pilot scale and survey its acceptability
Aim of the study
to clinically investigate the influence of an indigenous probiotic culture, fed through well accepted synbiotic dairy food, in geriatric volunteers and carry out metagenomic analysis for gut microflora.
JBP-YIMPSF-080315
Selection of synbiotic product
0
2
4
6
8
10
0day 7day 14day 21day 28day
Honey
Carrot
Oat
Musali
Storage period in days
Overall acceptability of synbiotic lassi products during storage at 5 � 2 � c
Se
nso
ry sc
ore
8.5
9
9.5
10
10.5
11
0 7 14 21 28
Honey
Carrot
Oat
Musali
Storage period in days
Lo
g
cfu/m
l
Changes in lactobacilli count (log cfu/ml) of synbiotic lassiproducts during storage at 5 � 2 � c
Changes in lactobacilli count of synbiotic products during storage at 5 ± 2 °c
Overall acceptability of synbiotic lassi during storage at 5 ± 2 °c
JBP-YIMPSF-080315 23
Methodology
24
Representation of the study design and sample collection
JBP-YIMPSF-080315
Intervention- probiotic fermented milk supplemented with honey
Data collection….. The changes in composition and biochemical activities of gastrointestinal
microflora were studied on the basis of their population in faecal sampes and facecal enzyme activity.
Collection of blood for Haematological, lipid, and immunological parameters.
Metagenomic analysis of gut microflora composition was studied by using semi conductor based amplicon sequencing on Ion Torrent PGM sequencer.
The metagenomic data obtained were analysed using MG-RAST and QIIME followed by STAMP statistical analysis.
The product was prepared on pilot scale at Vidya Dairy and survey of its acceptability in the geriatrics is conducted.
26JBP-YIMPSF-080315
Quantification of gut flora by traditional plating
JBP-YIMPSF-080315 27
T0 T30 P0 P308.00
8.20
8.40
8.60
8.80
9.00
9.20
9.40
8.47
9.19 9.179.10
Series1
Changes in Lactobacilli count
Quantification of MTCC 5463 by RT-PCR method
a standard curve for the strain Lb. helveticus MTCC 5463 is obtained. The number of cells of lactobacilli in the target samples was determined by comparing the Ct values obtained to the standard curve.
28
1 2 3 4 5 6 70
5
10
15
20
25
30
35
f(x) = − 3.44038138809637 x + 36.9876775955033R² = 0.994054720282937
Standard curve for lactobacilli
Log no. cells/ml
Thre
shol
d cy
cle
(Ct)
JBP-YIMPSF-080315
Levels of L. helveticusMTCC 5463 in faecaes of the elderly volunteers
The strain was not detected in any of the subjects in Group A or B before active test feeding
A statistically significant increase in the fecal amounts of strain confirmed the ability of the strain to colonize the human gut when delivered in a fermented drink.
29
T0T30
P0P30
0
1
2
3
4
5
6
7
8
0
7.77751367776294
5.938915834661446.15106445963725
7.91826054724741
Group A
Group B
Time period (30days interval)
Lo
g G
ene
Co
pie
s/g
of
feac
al m
atte
r
T0=Before probiotic FeedingT30= After probiotic FeedingP0= Before placebo feedingP30= After placebo feeding
Group A-Started with test product, Group B – started with placebo product.
JBP-YIMPSF-080315
Feacalβ-glucuronidase activity
The mean β-glucuronidase activity was reduced in test group from 1.40 to 0.73 (Microgram/min/mg of protein) while in case of placebo group, no effect on enzyme activity was observed.
Enzyme β-glucuronidase activity in the faeces of all subjects in the probiotic group, were highly significant (p = 0.00029) while in case of placebo group it showed non-significant difference (p= 0.4082).
30
T0 T30 P0 P300.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.41
0.73
1.461.52
Time period (30 day interval)
Mic
rog
ram
/min
/mg
ofp
rote
in
T0=Before probiotic Feeding, T30= After probiotic FeedingP0= Before placebo feeding, P30= After placebo feeding
JBP-YIMPSF-080315
Effect on serum calcium and creatinine
Characteristics
Probiotic group Placebo
N T0 T30P-value N P0 P30
P-value
Heamoglobin (13-17 g/dl) 58
12.41±1.52
12.40±1.39
0.89 6512.29±1.
7112.17±1.
710.08
Heamatocrit (36-53%) 57
38.89±3.45
39.57±3.13
0.00 5939.88±3.
4039.61±3.
060.24
Calcium (8.6-10.2mg/dl)
628.45±0.6
19.36±0.4
5<0.001 69
9.56±0.68
8.65±0.76
<0.001
31
Group comparison of hematological parameters and calcium
Serum calcium level was significantly improved in probiotic Lassi Group (p<0.001).
The means of probiotic as well as placebo groups of volunteers for haemoglobulin and serum creatinine were not significant before and after intervention
JBP-YIMPSF-080315
Effect on the Immunological parameters (TNF-α, IL2, IFN–γ and IgG or IgM levels)
32
Immunological parameters
Placebo group Probiotic group
P value
Subjects with normal or abnormal value* (as defined for each parameter) before dietary supplement(a)
Subjects with Significant benefit observed
(b)
% Correction
(b/a X100)
Subjects with normal or abnormal value* (as defined) before dietary supplement(c)
Subjects with Significant benefit observed
(d)
% Correction
(d/c X100)
TNF-α## 34* 01 2.9% 44* 12 27.2% 0.011
IFN–γ ** 00 00 00 03 03 100% NA
IL 2** 53 02 3.77% 55 11 20% 0.016
IgG** 58 03 5.1% 58 00 00 0.24#
IgM** 58 00 00 58 00 00 NA# Fisher’s Exact Test* Any value reported beyond 25% of the upper/lower limit of the normal range has been taken as abnormal value.##A moderate increase of TNF-α level from normal value to maximum three times than the upper limit of normal range; as well as, a decrease from abnormally high value (more than ten times higher than the upper limit of the normal range) to at least half of the baseline abnormal level.** A moderate increase of levels from sub-optimal or normal level to at least double of the upper limit of normal range
Salient findings
A significant immunomodulatory effect on the TNF-α and IL2 levels for the benefit of the subjects among probiotic group in comparison to placebo group.
There was however no significant beneficiary effect found on IFN–γ, IgG or IgM levels.
33JBP-YIMPSF-080315
Paired t test results for lipid profile parameters in 2 groups of human subjects
34
Characteristics Probiotic group Placebo
N T0 T30P-value
N P0 P30P-value
Serum cholesterol (130-220mg/dl)
54 161.67±41.05158.09±
42.630.12 68
174.32±49.99
167.09±43.11
<0.001
triglyceride (upto 170mg/dl)
60 103.77±49.84104.00±56.4
30.96 69
116.38±71.01
108.58±70.74
0.03
HDL (30-68mg/dl)
62 46.21±12.46 47.08±13.97 0.24 6949.67±15.9
748.77±12.9
80.34
LDL(100-129mg/dl)
55 98.48±37.12 92.93±35.79 0.01 5388.93±38.3
784.56±31.1
30.09
VLDL(upto 38mg/dl)
61 21.63±12.04 21.34±11.97 0.74 6923.28±14.2
021.71±14.1
20.03
TC/HDL(upto 5.0)
62 3.91±1.22 3.74±1.20 <0.001 69 3.77±1.33 3.65±1.23 0.04
LDL/HDL(upto 3.5)
62 2.37±0.96 2.21±0.91 <0.001 69 2.23±1.01 2.13±0.96 0.04
Metagenomic analysis of gut microflora composition and metabolic pathways
The metagenomic study of faecal microflora of geriatric volunteers revealed that they were dominated by Firmicutes (50%), Acintobacteria (20%) and Proteobacteria (10%).
Changes in the phylum composition after probiotic feeding included a 7% increase in Firmicutes, 1.5 % rise in Actinobacteria and 1.9% increase in Proteobacteria.
Proteobacteria were higher in non responders than in responders.
The STAMP analysis revealed that among responders and non responders the chief genera of Firmicutes that showed significant difference wereLactobacillus, Clostridium, Eubacterium, and Blautia (q< 0.002) while the genera of Proteobacteria included Shigella, Escherichia, Burkholderia and Camphylobacter (q-value<0.002).
35JBP-YIMPSF-080315
To summarize… Gut microbiota in elderly was shown to be strongly influenced by diet.
Faecal Lactobacilli count increased and their presence also helped in reducing faecal β-glucuronidas activity.
A significant immunomodulatory effect on the TNF-α and IL2 levels for the benefit of the subjects among treated group in comparison to placebo group was observed.
The metagenomic study revealed that the faecal samples of geriatric volunteers were dominated by Firmicutes (50%), Acintobacteria (20%) and Proteobacteria (10%).
Changes in the phylum composition after probiotic feeding included a 7% increase in Firmicutes, 1.5 % rise in Actinobacteria and 1.9% increase in Proteobacteria
JBP-YIMPSF-080315 38
To summarize…
Very few probiotic intervention studies in India.
Knowledge regarding gut microbiome of elderly of different geographical regions of India is required.
Efforts should be put to better understand bacterial shifts in gut microbiome during a probiotic therapy in geriatric populations
JBP-YIMPSF-080315 39