Food Biotechnology

Dr. Tarek ElbashitiFood Microbiology 2

• Food biotechnology integrates biochemistry, chemistry, microbiology, and chemical engineering for the enhanced production of food products.

• The application of microbiology to food systems encompasses methods involved in the assessment of microbial food safety and the use of microorganisms for the production of foods and beverages, food products, food additives.

• Microorganisms involved either directly or indirectly with food systems include bacteria, molds, yeasts, and algae.

Applications of Microbiology to Foods• Ancient Egyptians used fermentation to produce

beer and convert grape juice to wine.• Also, the aerobic conversion of the alcohol in

wine to the acetic acid of vinegar, and the leavening of bread.

• The present practices of using, for example, pectinases for enhanced release of fruit juices from tissue and amylases for the enzymatic modification of starches, are examples involving the indirect application of microorganisms to foods and food components.

• The production of xanthan gum by the plant pathogenic bacterium Xanthomonas campestris for use as a viscosity agent in beverages and semisolid food products is an example of the use of an originally undesirable organism for the production of a desirable food and beverage additive.

• The use of the mold Aspergillus niger to produce high yields of citric acid as a food and beverage acidulant was established in the 1920s and is a classic example of an initial surface culture process that was eventually converted to a submerged aerated process with the use of mutants.

The Nature of Microorganisms• Microscopic organisms are presently divided into

three major groups: (1) Eubacteria (bacteria), which lack a discernible

nucleus and mitochondria; (2) Archaebacteria (bacteria), which also lack a

discernible nucleus and mitochondria; and (3) Eukaryotes (yeasts, molds, algae, and

protozoa), which possess both a clearly discernible nucleus and mitochondria, plus filamentous structures known as endothelial reticulum.

• All microorganisms are allocated to a specific group with respect to growth temperature.

• Obligate psychrophiles capable of growth at or near 0°C but not at 20°C. Such organisms usually have a maximum growth temperature of 15–17°C.

• Psychrotrophic organisms are capable of growth at or near 0°C but exhibit optimum growth at approximately 25°C and are frequently unable to grow at 30°C.

• Mesophiles exhibit growth from 20–45°C with an optimum growth temperature usually in the range of 30–35°C.

• Thermophiles exhibit growth in the range of 45–65°C.

• Hyperthermophiles are organisms from oceanic thermal vents and hot springs that are restricted to growth temperatures from 70–120°C. Hyperthermophiles have not yet been isolated from foods.

FUNGI

1. Yeasts• Yeasts can be divided into two metabolic

groups: facultative anaerobes and obligate aerobes.

• The facultative anaerobes are capable of anaerobic growth and fermentative conversion of sugars to ethyl alcohol, CO2, and cell mass, in addition to the aerobic conversion of sugars to CO2 and H2O, and much higher yields of cell mass.

• Producing baker’s yeast using sucrose derived from molasses requires vigorous aeration of the culture medium so that a maximum amount of carbon flows to cell mass production and not to ethyl alcohol formation.

• Vigorous aeration of S. cerevisiae strains in the presence of an abundant level of carbohydrate (about 3%) results in the metabolic dominance of fermentation and is known as the crabtree effect.

• This in turn results in a significant level of ethyl alcohol and a notably reduced level of cell mass.

• The baker’s yeast industry is able to overcome the crabtree effect using incremental feeding which involves the pulsed addition of molasses to aerated culture tanks, so that at no time does the residual level of sucrose rise above 0.0001%.

• Thus there is no feedback repression of mitochondria formation caused by elevated levels of sucrose.

• All yeasts are capable of utilizing glucose. • The utilization of other sugars depends on the

species; the spectrum of sugars used constitutes a major criterion for the identity of yeasts.

• All yeasts are capable of utilizing ammonium sulfate as a sole source of nitrogen.

• Very few yeasts are capable of utilizing nitrate as a sole nitrogen source.

• Among ascospore-producing yeasts, the number (1, 4, or 8) and shape of ascospores (spherical, oval, kidney, hat, saturn, needle) in asci constitutes an additional major criterion for genus and species identity.

• Most yeasts divide by budding; however, members of the strongly fermentative yeast genus Schizosaccharomyces divide solely by transverse fission (Figure 1.2).

YEAST CELLS

2. Molds• Molds are classified into four classes. a. The Phycomycetes do not have complete

cross walls in their hyphae and therefore exhibit unidirectional protoplasmic streaming (coenocytic movement) or flow throughout their hyphae.

• Phycomycetes also possess the unifying characteristic of producing aerially borne asexual fruiting structures known as sporangia, with internal sporangiospores borne on a bulblike structure referred to as the columella (Figure 1.3).

• Some, but not all, Phycomycetes produce a sexual spore, known as a zygospore, derived from the fusion of opposite mating types which occurs freely in culture media (Figure 1.3).

• Color and the microscopic orientation and appearance of these structures are used to establish genera and species.

b. The class Ascomycetes houses fungi (both yeasts and molds) that produce the sexual ascospore.

• Molds in this class have complete cross walls in their hyphae and therefore do not exhibit protoplasmic streaming.

• All ascomycete molds produce characteristic conidiospores, which occur in chains or clusters.

• The characteristic blue-green coloration of members of the genus Penicillium (Figure 1.4) is due to the coloration of the long chains of conidiospores borne by all members of this genus.

• The characteristic coloration (yellow, brown, green) of various species of the genus Aspergillus (Figure 1.4) is also due to the coloration of the conidiospores.

• The major criteria for the establishment of genus and species of this class are the visual coloration of the mass of growth in conjunction with the microscopic appearance and three dimensional orientation of the hyphae and conidiospores.

• A major distinction between ascomycete yeasts and molds is derived from the fact that yeasts produce “naked” asci and frequently contain four and sometimes eight ascospores, depending on the species.

• The asci of yeasts occur free in the medium, whereas most ascomycete molds produce asci with internal ascospores inside a fruiting structure known as a cleistothecium (completely closed) or as a perithecium (open at one end) (Figure 1.4).

C. The class Fungi imperfecti (Figure 1.5), otherwise known as Deuteromycetes, is essentially identical to the Ascomycetes (hyphal crosswalls are present and conidiospores are produced) except that the sexual ascospore is not produced.

d. The class Basidiomycetes houses molds and yeastlike organisms that produce the sexual basidiospore; many also produce conidiospores.

• Other basidiomycetes produce budding yeastlike cells, which can result in confusing such isolates with true yeasts.

• The commercial use of molds in various food systems usually involves the harvesting of the asexual sporangiospores or conidiospores for use as inoculum.

• This allows the density of the inoculum to be based on the precise density or number of spores per unit of volume, which can be readily determined by microscopic count.

• The use of mycelial mass as an inoculum is more difficult with respect to directly determining the quantity of the cell mass in the inoculum volume, for obvious physical reasons.

Bacteria

• Bacteria are now classified into two major groups, the Eubacteria and the Archaebacteria (which were formerly grouped under the Protista).

• The majority of bacteria involved with food systems are Eubacteria.

• The Archaebacteria presently house the unique halobacteria, which are obligate halophiles and can cause the red stainting of salted fish.

• All bacteria fall into two convenient groups, those that stain purple with the Gram stain (Gram-positive) and those that stain red with the Gram stain (Gram-negative).

• There are three general metabolic groups of bacteria:

(1) obligate aerobes, (2) facultative anaerobes, and (3) obligate anaerobes. • Representative members of each of

these groups are found among both the Gram-positive and Gram-negative bacteria.

Serotypes:• Serotyping involves the production of antibodies

following the injection of a suitable mammal with the microorganism or a specific extract of the organism.

• If an organism is nonflagellated then serotyping will be based on the somatic antigens.

• If the organism is flagellated then serotyping may also be based on the flagella antigens.

• Three antigenic sites are recognized: somatic (O) (German “Ohne”) or body, flagella (H) (German “Hauch”) or motility, and K (German “Kapsel”), e.g., Escherichia coli O157:H7.

• The O antigens are comprised of the O polysaccharides that are on the surface and are heat stable.

• The K and H antigens are heat labile. • With whole bacterial cells, agglutination

methods are used. • With soluble antigens such as toxins,

precipitin or gel diffusion assays are used.

METABOLIC CONTROL FOR ENHANCED METABOLITEPRODUCTION:• A number of microbial processes in the production of

various food additives involve limiting one or more critical nutrients.

• The submerged production of citric acid by A. niger involves limiting both iron and phosphate to achieve maximum yields.

• The production and excretion of maximum amounts of glutamic acid by Corynebacterium glutamicum is dependent on cell permeability.

• Increased permeability can be achieved through biotin deficiency, through oleic acid deficiency in oleic acid auxotrophs, through the addition of saturated fatty acids or penicillin, or by glycerol deficiency in glycerol auxotrophs.

MUTAGENESIS FOR OVERPRODUCTION OF METABOLITES

• Increased yields of microbially produced food additives can often be achieved by the selection of overproducing mutants.

• Such desirable mutants will frequently be produced spontaneously or by a mutagenic agent.

• There are three fundamental types of mutational events:

(1) nucleotide deletions,(2) base-pair substitutions, and (3) gene duplications.

• Mutants that result from large deletions have the greatest stability.

• Mutants exhibiting high levels of reversion to wild-type cells are usually derived from base-pair substitution mutations.

• Alkylating agents are among the most potent direct-acting mutagenic agents.

• A variety of methods has been developed for the production of mutants.

• The frequently used mutagen N-methyl N-nitrosoguanidine (nitrosoguanidine or NTG) functions by forming a methyl group adduct to guanine, and results in multiple mutations.

• Ethyl methane sulfonate is less lethal and usually results in single mutations.

• Although yielding large numbers of mutants, such agents usually result in mutations with significant rates of reversion to wild type.

• Exposure of cells to ultraviolet (UV) irradiation results in approximately equal numbers of both deletions and base-pair substitutions.

• UV irradiation will therefore yield a higher percent of more stable mutants (derived from frame shift mutations) and is the preferred method for mutagenesis.

SELECTIVE CULTIVATION

1. Selective Enrichment• Microorganisms of public health significance

associated with foods are usually present as a small minority of the total microbial population.

• For both detection and quantitation of such microorganisms, selective enrichment cultivation is usually undertaken.

• Selective enrichment involves conditions of cultivation that favor the development of the target organism over that of the usual majority of extraneous microorganisms.

• Selective agents are often derived from the environment in which such organisms of public health significance are found.

• Most culture media for selective enrichment of E. coli make use of the fact that the organism is a common inhabitant of the intestinal tract of mammals and hence is normally in contact with surface-active agents such as bile salts, which are frequently used as selective agents for the organism.

• Incubation temperature has been used successfully with selective chemical agents for the selective cultivation and enumeration of certain bacteria.

• Selective enrichment of pathogenic vibrios such as Vibrio vulnificus and Vibrio parahaemolyticus makes use of the fact that they are marine organisms and hence are quite tolerant of the somewhat alkaline pH of seawater, which is usually about 7.8.

• This tolerance toward an alkaline growth environment extends to a pH of 8.5 to 8.7, which is the usual initial pH range used for enrichment cultivation of these organisms.

• The natural habitat for Staphylococcus aureus is the human skin.

• The level of sodium chloride on the skin surface during physical exertion under warm weather conditions is often at or near saturation, when one considers the process of evaporation.

• S. aureus, as would be expected, exhibits a significant level of salt tolerance, resulting in the use of enrichment and isolation media for the organism containing 7.5–10% NaCl.

• NaCl at a level of 7.5% is notably inhibitory to most Gram-positive and Gram-negative bacteria.

• A wide variety of antibiotics is now added to media for selective isolation of bacterial pathogens from foods.

• Yeasts and molds are closely associated with acid fruits.

• The use of acidified culture media (pH 3.5–5.5) is a long established and highly effective method of inhibiting most bacteria, while allowing unrestricted growth of yeasts and molds.

• The successful use of selective enrichment media is predicated on cultivation and enumeration of undamaged cells.

2. Use of Incubation Temperature for Selective Isolation

• Temperature alone can be a highly selective mechanism for the selective isolation of specific groups of organisms.

• Incubation at 55–65°C will ensure the sole development of obligately thermophilic organisms such as Bacillus stearothermophilus.

• Determination of the number of bacteria on fish generally involves incubation of culture plates at 20°C and resulting development of psychrotrophic organisms that grow optimally at about 25°C and which are also capable of growth at 2°C.

• By incubating the plates at 10°C, and then replica-plating the resulting colonies to sets of plates incubated at 10 and 20°C, one can readily identify and isolate obligately psychrophilic fishery bacteria, capable of growth from 0–17°C but unable to grow at 20°C.

3. The Use of Mineral Salts Media for the Isolation of Unique Carbon Sources Utilizing Microorganisms

• The isolation of microorganisms capable of utilizing unique carbon sources is greatly facilitated if the organism being sought is capable of growth in a strictly glucose–mineral salts medium.

• Unique carbon and energy sources such as methanol, cholesterol, and naphthalene, can then be added as sole carbon sources with the assurance that only organisms capable of attacking these unique substrates will grow.

4. Selective Isolation as a Result of Sequential Biochemical Activity:

• The production of vinegar represents a unique sequence of environmental and biochemical events.

• The sugars in a fruit juice are first fermented anaerobically by yeasts to ethanol, which is then subjected to vigorous aeration, resulting in the oxidative conversion of ethyl alcohol to acetic acid (usually 4–5%) by resident acetic acid bacteria.

• Ethanol as an intermediate product will sustain the growth of fewer microorganisms than will glucose because it contains less energy than glucose and is therefore more restrictive with respect to microorganisms capable of attacking it.

• Acetic acid contains even less energy than ethanol, and, at a level of 4%, results in complete microbial preservation or stability in the absence of oxygen.

• The methane fermentation, which is frequently used to digest solid waste materials, involves the initial anaerobic formation of intermediate metabolic products such as ethanol, and lower fatty acids such as acetic acid and butyric acid, by members of the genus Clostridium and various facultative anaerobes.

• These metabolic intermediates are then converted anaerobically to gaseous methane by methane bacteria with the result that the initial solid matter is converted to a gaseous product.

ELECTROSTIMULATION OF METABOLITE PRODUCTION

• A notably innovative approach toward increasing the microbial production of an end product is electrostimulation.

• Electrical stimulation of microbial metabolite production is based on the use of an artificial electron carrier such as neutral red, allowing an electric current to indirectly supply the electron-driving force needed to generate a proton-motive force for energy conservation and the electrons needed for growth and end product production.

• The system involves the use of an electrochemical bioreactor utilizing an anodic chamber and a cathodic chamber divided by a cation selective membrane.

• The cathodic chamber is used as the fermentation vessel.

• The neutral red serves as a reduced electron carrier and migrates from the anode to the cathode where it enters the cells and couples to a suitable redox enzyme system which accepts the electrons.

• Electrostimulation has been used to increase L-glutamic acid production by Brevibacterium flavum, and shows promise with other fermentation processes.