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Food Biotechnology

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Title: Food Biotechnology


1
Food Biotechnology
  • Dr. Tarek ElbashitiFood Microbiology 2

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.

3
  • 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.

4
  • 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.

5
  • 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.

6
  • Obligate psychrophiles capable of growth at or
    near 0C but not at 20C. Such organisms usually
    have a maximum growth temperature of 1517C.
  • Psychrotrophic organisms are capable of growth at
    or near 0C but exhibit optimum growth at
    approximately 25C and are frequently unable to
    grow at 30C.
  • Mesophiles exhibit growth from 2045C with an
    optimum growth temperature usually in the range
    of 3035C.
  • Thermophiles exhibit growth in the range of
    4565C.
  • Hyperthermophiles are organisms from oceanic
    thermal vents and hot springs that are restricted
    to growth temperatures from 70120C.
    Hyperthermophiles have not yet been isolated from
    foods.

7
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.

8
  • Producing bakers 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.

9
  • The bakers 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.

10
  • 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).

11
YEAST CELLS
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13
  • 2. Molds
  • Molds are classified into four classes.
  • 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).

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15
  • 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.

16
  • 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.

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  • 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.

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  • 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.

21
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).

22
  • There are three general metabolic groups of
    bacteria
  • 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.

23
  • 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 O157H7.

24
  • 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.

25
  • METABOLIC CONTROL FOR ENHANCED METABOLITE
  • PRODUCTION
  • 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.

26
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
  • nucleotide deletions,
  • (2) base-pair substitutions, and
  • (3) gene duplications.

27
  • 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.

28
  • 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.

29
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.

30
  • 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.

31
  • 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.

32
  • 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 7.510 NaCl.
  • NaCl at a level of 7.5 is notably inhibitory to
    most Gram-positive and Gram-negative bacteria.

33
  • 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 3.55.5)
    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.

34
  • 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 5565C 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 20C and resulting development of
    psychrotrophic organisms that grow optimally at
    about 25C and which are also capable of growth
    at 2C.

35
  • By incubating the plates at 10C, and then
    replica-plating the resulting colonies to sets of
    plates incubated at 10 and 20C, one can readily
    identify and isolate obligately psychrophilic
    fishery bacteria, capable of growth from 017C
    but unable to grow at 20C.

36
  • 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 glucosemineral
    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.

37
  • 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 45) 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.

38
  • 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.

39
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.

40
  • 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.
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