Chapter-2 (Food Tech) Food Microbiology

7/28/2019 Chapter-2 (Food Tech) Food Microbiology

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2. FOOD MICROBIOLOGYMORPHOLOGY AND STRUCTURE OF MICROORGANISMS IN FOODS

The microbial groups important in foods consist of several species and typesof bacteria, yeasts, molds, and viruses.

2.1. THE BACTERIAL CELL

Bacteria constitute a large domain of prokaryotic microorganisms.

Bacteria are unicellular, most ca. 0.51.0 2.010 mm in size, and have threemorphological forms: spherical (cocci), rod shaped (bacilli), and curved(comma)

They can form associations such as clusters, chains (two or more cells), otetrads. They can be motile or nonmotile.

Cytoplasmic materials are enclosed in a rigid wall on the surface and amembrane beneath the wall.

It also forms intrusions in the cytoplasm (mesosomes). The cytoplasmicmaterial is immobile and does not contain organelles enclosed in a separatemembrane. The ribosomes are 70S type and are dispersed in the cytoplasm.

The genetic materials (structural and plasmid DNA) are circular, not enclosedin nuclear membrane, and do not contain basic proteins such as histonesBoth gene transfer and genetic recombination occur, but do not involvegamete or zygote formation.

Cell division is by binary fission. Procaryotic cells can also have flagellacapsules, surface layer proteins, and pili for specific functions. Some alsoform endospores

On the basis of Gram-stain behavior, bacterial cells are grouped as Gram-negative or Gram-positive. Gram-negative cells have a complex cell walcontaining an outer membrane (OM) and a middle membrane (MM)

The OM is composed of lipopolysaccharides (LPS), lipoprotein (LP), and

phospholipids. Phospholipid molecules are arranged in a bilayer, with thehydrophobic part (fatty acids) inside and hydrophilic part (glycerol andphosphate) outside. LPS and LP molecules are embedded in the phospholipidlayer.

The OM has limited transport and barrier functions. The resistance of Gramnegative bacteria to many enzymes (lysozyme, which hydrolyzesmucopeptide), hydrophobic molecules (SDS and bile salts), and antibiotics(penicillin) is due to the barrier property of the OM. LPS molecules also haveantigenic properties.

Beneath the OM is the MM, composed of a thin layer of peptidoglycan ormucopeptide embedded in the periplasmic materials that contain severatypes of proteins.

Beneath the periplasmic materials is the plasma or inner membrane (IM),composed of a phospholipid bilayer in which many types of proteins areembedded.

Gram-positive cells have a thick cell wall composed of several layers ofmucopeptide (responsible for thick rigid structure) and two types of teichoicacids. Some species also have a layer over the cell surface, called surfacelayer protein (SLP).

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The wall teichoic acid molecules are linked to mucopeptide layers, and thelipoteichoic acid molecules are linked to both mucopeptide and cytoplasmicmembrane.

Teichoic acids are negatively charged (because of phosphate groups) andmay bind to or regulate the movement of cationic molecules in and out of thecell. Teichoic acids have antigenic properties and can be used to identifyGram-positive bacteria serologically. Because of the complexity in the

chemical composition of the cell wall, Gram-positive bacteria are consideredto have evolved before Gram-negative bacteria.

Summary of characteristics of typical bacterial cell structures

StructureFlagella

Function(s)Swimming movement

Predominant chemicalcompositionProtein

Pili

Sex pilusStabilizes mating bacteriaduring DNA transfer byconjugation

Protein

Common pili orfimbriae

Attachment to surfaces;protection againstphagotrophic engulfment

Protein

Capsules(includes"slime layers"andglycocalyx)

Attachment to surfaces;protection against phagocyticengulfment, occasionallykilling or digestion; reserve ofnutrients or protectionagainst desiccation

Usually polysaccharide;occasionallypolypeptide

Cell wall

Gram-positive

bacteria

Prevents osmotic lysis of cellprotoplast and confers rigidityand shape on cells

Peptidoglycan (murein)complexed with teichoicacids

Gram-negativebacteria

Peptidoglycan preventsosmotic lysis and confersrigidity and shape; outermembrane is permeabilitybarrier; associated LPS andproteins have variousfunctions

Peptidoglycan (murein)surrounded byphospholipid protein-lipopolysaccharide"outer membrane"

Plasmamembrane

Permeability barrier; transportof solutes; energy generation;location of numerous enzyme

systems

Phospholipid andprotein

RibosomesSites of translation (proteinsynthesis)

RNA and protein

InclusionsOften reserves of nutrients;additional specializedfunctions

Highly variable;carbohydrate, lipid,protein or inorganic

Chromosome Genetic material of cell DNA

Plasmid Extrachromosomal genetic DNA

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material

SHAPE AND SIZE OF BACTERIA

Most bacteria are 0.2 um in diameter and 2-8 um in length.

The three basic bacterial shapes are

coccus (spherical), bacillus (rod-shaped), and

spiral (twisted),

however pleomorphic bacteria can assume several shapes.

Arrangement of cocci

Cocci may be oval, elongated, or flattened on one side.

Cocci may remain attached after cell division. These group characteristicsare often used to help identify certain cocci.

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Cocci that remain in pairsafter dividing are calleddiplococci.

Cocci that remain in

chains after dividing arecalled streptococci.

Cocci that divide in twoplanes and remain ingroups of four are calledtetrads.

Cocci that divide in threeplanes and remain ingroups cube like groups ofeight are called sarcinae.

Cocci that divide inmultiple planes and formgrape like clusters orsheets are calledstaphylococci.

Bacilli

Since bacilli only divide across their short axis there are fewer groupings.

Bacillus is a shape (rod shaped) but there is also a genus of bacteria withthe name Bacillus. You wouldn't confuse the two, since you know therules for writing the genus and species names of organisms, right????

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Most bacilli appear as singlerods. Diplobacilli appear inpairs after division.

Streptobacilli appear inchains after division.

Some bacilli are so shortand fat that they look likecocci and are referred to as

coccobacilli.

Spiral bacteria

Spiral bacteria have one or more twists.

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Vibrios look like curvedrods.

Spirilla have a helicalshape and fairly rigidbodies.

Spirochetes have a helicalshape and flexible bodies.Spirochetes move bymeans of axial filaments,which look like flagellacontained beneath aflexible external sheath.

Other shapes

Stella are star-shaped.Haloarcula, a genus ofhalophilic archaea, arerectangular.

MAJOR BACTERIAL GROUPSA. Lactic Acid Bacteria

They are bacteria that produce relatively large quantities of lactic acid fromcarbohydrates.

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Species mainly from genera Lactococcus, Leuconostoc, Pediococcus,Lactobacillus, and Streptococcus thermophilus are included in this group.

B. Acetic Acid BacteriaThey are bacteria that produce acetic acid, such asAcetobacter aceti.

C. Propionic Acid BacteriaThey are bacteria that produce propionic acid and are used in dairyfermentation. Species such as Propionibacterium freudenreichii are includedin this group.

D. Butyric Acid BacteriaThey are bacteria that produce butyric acid in relatively large amounts. SomeClostridium spp. such as Clostridium butyricum are included in this group.

E. Proteolytic BacteriaThey are bacteria that can hydrolyze proteins because they produceextracellular proteinases. Species in genera Micrococcus, Staphylococcus,Bacillus, Clostridium, Pseudomonas, Alteromonas, Flavobacterium,

Alcaligenes, some in Enterobacteriaceae,and Brevibacterium are included in this group.

F. Lipolytic BacteriaThey are bacteria that are able to hydrolyze triglycerides because theyproduce extracellular lipases. Species in genera Micrococcus,Staphylococcus, Pseudomonas, Alteromonas, and Flavobacterium areincluded in this group.

G. Saccharolytic BacteriaThey are bacteria that are able to hydrolyze complex carbohydrates. Speciesin genera Bacillus, Clostridium, Aeromonas, Pseudomonas, and Enterobacterare included in this group.

H. Thermophilic BacteriaThey are bacteria that are able to grow at 50C and above. Species fromgenera Bacillus, Clostridium, Pediococcus, Streptococcus, and Lactobacillusare included in this group.

I. Psychrotrophic BacteriaThey are bacteria that are able to grow at refrigerated temperature (5C)Some species from Pseudomonas, Alteromonas, Alcaligenes, Flavobacterium,Serratia, Bacillus, Clostridium, Lactobacillus, Leuconostoc, Carnobacterium,Brochothrix,Listeria, Yersinia, andAeromonas are included in this group.

J. Thermoduric BacteriaThey are bacteria that are able to survive pasteurization temperaturetreatment. Some species from Micrococcus, Enterococcus, LactobacillusPediococcus, Bacillus (spores), and Clostridium (spores) are included in thisgroup.

K. Halotolerant Bacteria

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They are bacteria that are able to survive high salt concentrations (10%)Some species from Bacillus, Micrococcus, Staphylococcus, Pediococcus,Vibrio, and Corynebacterium are included in this group.

L. Aciduric BacteriaThey are bacteria that are able to survive at low pH (


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Species from Escherichia, Enterobacter, Citrobacter, and Klebsiella areincluded in this group. They are used as an index of sanitation.

U. Fecal ColiformsMainly Escherichia coli is included in this group. They are also used as anindex of sanitation.

V. Enteric PathogensPathogenic Salmonella, Shigella, Campylobacter, Yersinia, Escherichia, Vibrio,Listeria , hepatitis A, and others that can cause gastrointestinal infection areincluded inthis group.

IDENTIFICATION OF BACTERIA

1. Bacterial speciesa. Similar individuals:

i. A bacterial species is "a population of cells with similacharacteristics."

ii. Note that this definition is very different from how plant andanimal species are often defined, which usually involvessomething to do with sex.

b. Resemble fossil species:

i. Bacterial species resemble the way fossil species aredistinguished (i.e., phylogenetic species concept).

ii. Normally plant and especially animal species are defined as

populations of interbreeding or potentially interbreedingindividuals that don't breed with individuals of other, likedefined populations.

iii. For fossils, on the other hand, it cannot be determined whointerbred, or could have, with whom. Consequently, fossispecies are defined only in terms of character resemblance, justas are bacteria.

2. Bergey's manual

a. Bergey's manual is a guide to distinguishing bacterial species based onphenotypic differences between isolates.

3. Strain

a. A strain is a subset of a bacterial species differing from other bacteriaof the same species by some minor but identifiable difference.

b. A strain is "a population of organisms that descends from a singleorganism or pure culture isolate. Strains within a species may differslightly from one another in many ways." (p. 392, Prescott et al., 1996)

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c. Strains are often created in the laboratory by mutagenizingexisting strains or wild-type examples of bacterial species.

d. The term strain is also applicable to eucaryotic microorganisms , aswell as to viruses .

4. Type strain

a. "One strain of a species is designated as the type strain. It is usuallyone of the first strains studied and is often more fully characterizedthan other strains; however, it does not have to be the mosrepresentative member. Only those strains very similar to the typestrain are included in a species."

5. Serovar [serotype]

a. A serovaris a strain differentiated by serological means.

b. Individual strains ofSalmonella spp. are often distinguished anddistinguishable by serological means.

6. Biovar [biotype]

a. Biovars are strains that are differentiated by biochemical or other non-serological means.

7. Morphovar [morphotype]

a. A morphovaris a strain which is differentiated on the basis ofmorphological distinctions.

8. Isolate

a. An isolate is a pure culture derived from a heterogeneous, wild

population of microorganisms .

b. The term isolate is also applicable to eucaryotic microorganisms aswell as to viruses .

9. Classification

a. Placement of an organism within a scheme relating different types oorganisms, such as that presented in Woese's universal tree , is knowas classification.

b. Organisms are classified for scientific purposes.

10. Identification

a. Identification is the determination of whether an organism (or isolate inthe case of microorganisms ) should be placed within a group oorganisms known to fit within some classification scheme.

b. Organisms are identified for practical purposes, such as diagnosis odisease .

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c. Many identification techniques:

i. Many different criteria may be employed for identificationthough it is often desirable to employ the easiest techniquespossible.

ii. What techniques and tests may be necessary, however, dependon what organism is being identified and how much detail into

the organism's classification you are interested.

iii. Techniques include:

1. morphological identification

2. differential staining

3. use of differential media

4. serological methods

5. flow cytometry

6. phage typing

7. protein analysis

8. comparisons of nucleotide sequences

11. Morphological identification

a. A number of morphological characteristics are useful inbacterial identification. These include the presence or absence of:

i. endospores

ii. flagella

iii. glycocalyx

iv. etc.

b. Additional considerations include:

i. colony morphology

ii. cell shape

iii. cell size

iv. etc.

12. Serological methods [agglutination test, ELISA, Western blot]

a. Antibodies:

i. Serological methods employ antibodies and include:

1. agglutination tests93
http://www.mansfield.ohio-state.edu/~sabedon/biol3010.htm#flow_cytometryhttp://www.mansfield.ohio-state.edu/~sabedon/biol3010.htm#identificationhttp://www.mansfield.ohio-state.edu/~sabedon/biol3010.htm#identificationhttp://www.mansfield.ohio-state.edu/~sabedon/biol3010.htm#flow_cytometry


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2. ELISAs

3. Western blots

ii. It is antibody binding that all serological tests ultimately detect.

b. The basic premise behind all of these tests is that antibodies are highlyselective in terms of the proteins (or other cell structures) to which

they bind, to the point that they are able to distinguish the proteinscoming from one bacterial species among many species, or evenone strain among many strain.

13. Flow cytometry

a. Flow cytometryis a technique that can employ serologicamethods (but doesn't necessarily) that analyzes cells suspended in aliquid medium by light, electrical conductivity, or fluorescence as thecells individually pass through a small orifice.

14. Phage typing

a. Bacteriophage (or phage ) are viruses that infect bacteria .

b. Phage can be very specific in what bacteria they infect and the patternof infection by many phage may be employed inphage typing todistinguish bacterial species and strains.

15. Protein analysis [gel electrophoresis, SDS-PAGE, establishmentof clonality]

a. The size and other differences between proteins among differentorganisms may be determined very easily employing methods ofprotein separation using methods collectively known as gelelectrophoresis.

b. SDS-PAGE:

i. One popular technique goes by the name SDS-PAGE whichstands for sodium dodecyl sulfate-polyacrylamide gelelectrophoresis

ii. Note that another name for SDS is sodium lauryl sulfate, adetergent you will find in many shampoos.

c. Such methods are very good at detecting small differences betweenisolates and are especially good at establishing clonality.

16. Comparison of nucleotide sequences [Southern blot, nucleicacid hybridization, RFLP, DNA fingerprinting]

a. The actual sequence of bases (nucleotides) in the genome oforganisms may be inferred or actually determined (nucleotidesequencing) by a variety of methods.

b. Various methods of inference include:

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i. Southern blotting

ii. nucleic acid hybridization

iii. RFLP comparison (restriction fragment length polymorphism orDNA fingerprinting)

c. Another technique that is worth knowing about is PCR which stands for

polymerase chain reaction, a method of amplifying specific regions ofDNA found in an organisms genome by selectively catalyzing thereplication of those regions.

17. Most Probable Number Method (MPN)

The most probable number (MPN) is particularly useful for low concentrationsof organisms (


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negative results) and the next two higher dilutions (ex. a and b); if positive resultsoccur in higher unselected dilutions, shift each selection to the next higher dilution(ex. c). If there are still positive results in higher unselected dilutions, add thosehigher-dilution positive results to the results for the highest selected dilution (ex. d)If there were not enough higher dilutions tested to select three dilutions, then selecthe next lower dilutions (ex. e).

Case 2. No dilutions show all tubes positive. Select the 3 lowest dilutions (exf). If there arepositive results in higher unselected dilutions, add those higher-dilution positiveresults to the results for the highest selected dilution (ex. g).

2.2. YEASTS AND MOLDS

Both yeasts and molds are eucaryotic, but yeasts are unicellular whereasmolds are multicellular.

Eucaryotic cells are generally much larger (20 to 100 mm) than procaryotic

cells (1 to 10 mm). The cell wall does not have mucopeptide, is rigid, and is composed of

carbohydrates. The plasma membrane contains sterol. The cytoplasm is mobile (streaming

and contains organelles (mitochondria, vacuoles) that are membrane boundRibosomes are 80S type and attached to the endoplasmic reticulum.

The DNA is linear (chromosomes), contains histones, and is enclosed in anuclear membrane.

Cell division is by mitosis (i.e., asexual reproduction); sexual reproductionwhen it occurs, is by meiosis.

Molds are nonmotile, filamentous, and branched

The cell wall is composed of cellulose, chitin, or both. A mold (thallus) is composed of large numbers of filaments called hyphae.

An aggregate of hyphae is called mycelium. A hypha can be nonseptateseptate-uninucleate, or septate-multinucleate.

A hypha can be vegetative or reproductive. The reproductive hypha usuallyextends in the air and form exospores, either free (conidia) or in a sack(sporangium). Shape, size, and color of spores are used for taxonomicclassification.

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Yeasts are widely distributed in nature. The cells are oval, spherical, oelongated, about 530 210 mm in size They are nonmotile. The cell walcontains polysaccharides (glycans), proteins, and lipids. The wall can havescars, indicating the sites of budding.

The membrane is beneath the wall. The cytoplasm has a finely granulaappearance for ribosomes and organelles. The nucleus is well defined with anuclear membrane.

Molds are important in food because they can grow even in conditions inwhich many bacteria cannot grow, such as low pH, low water activity (Aw)and high osmotic pressure.

Many types of molds are found in foods. They are important spoilagemicroorganisms. Many strains also produce mycotoxins

Aspergillus. It is widely distributed and contains many species important in food

Members have septate hyphae and produce black-colored asexual sporeson conidia.

Many are xerophilic (able to grow in low Aw) and can grow in grainscausing spoilage. They are also involved in spoilage of foods such as jams,

cured ham, nuts, and fruits and vegetables (rot). Some species or strains produce mycotoxins (e.g., Aspergillus flavus

produces aflatoxin). Many species or strains are also used in food and food additive processing.

Asp. oryzae is used to hydrolyze starch by a-amylase in the production osake.Asp. nigeris used to process citric acid from sucrose and to produceenzymes such as b-galactosidase.

MAJOR FUNGI FOUND IN FOOD WITH MORPHOLOGY

Alternaria. Members are septate and form dark-colored spores on conidia.

They cause rot in tomatoes and rancid flavor in dairy products.

Some species or strains produce mycotoxins. Species: Alternaria tenuisFusarium.

Many types are associated with rot in citrus fruits, potatoes, and grainsThey form cottony growth and produce septate, sickle-shaped conidiaSpecies: Fusarium solani.

Geotrichum.

Members are septate and form rectangular arthrospores.

They grow, forming a yeastlike cottony, creamy colony.

They establish easily in equipment and often grow on dairy products(dairy mold). Species: Geotrichum candidum.

Mucor.

It is widely distributed. Members have nonseptate hyphae and producesporangiophores.

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They produce cottony colonies. Some species are used in foodfermentation and as a source of enzymes.

They cause spoilage of vegetables. Species: Mucor rouxii.

Penicillium.

It is widely distributed and contains many species.

Members have septate hyphae and form conidiophores on a blue-green

brush like conidia head. Some species are used in food production, such as Penicillium roqueforti

and Pen. camembertii in cheese.

Many species cause fungal rot in fruits andvegetables. They also causespoilage of grains, breads, and meat. Some strains produce mycotoxins(e.g., Ochratoxin A).

Rhizopus.

Hyphae are aseptate and form sporangiophores in sporangium.

They cause spoilage of many fruits and vegetables. Rhizopus stoloniferisthe common black bread mold.

Yeast genera:

Saccharomyces.

Cells are round, oval, or elongated. It is the most important genus andcontains heterogenous groups Saccharomyces cerevisiae variants areused in baking for leavening bread and in alcoholic fermentation.

They also cause spoilage of food, producing alcohol and CO2.

Pichia.

Cells are oval to cylindrical and form pellicles in beer, wine, and brine tocause spoilage. Some are also used in oriental food fermentation.

Species: Pichia membranaefaciens. Rhodotorula. They are pigment

forming yeasts and can cause discoloration of foods such as meat, fishand sauerkraut.

Species: Rhodotorula glutinis. Torulopsis. Cells are spherical to oval. Theycause spoilage of milk because they can ferment lactose (e.g., Torulopsisversatilis).

They also spoil fruit juice concentrates and acid foods.

Candida.

Many species spoil foods with high acid, salt, and sugar and form pellicleson the surface of liquids.

Some can cause rancidity in butter and dairy products (e.g., Candidalipolyticum).Zygosaccharomyces.

Cause spoilage of high-acid foods, such as sauces, ketchups, picklesmustards, mayonnaise, salad dressings, especially those with less acidand

o less salt and sugar (e.g.,Zygosaccharomyces bailii).

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2.3. MICROBIAL GROWTH IN FOOD

Foods, because they provide nutrients for us, also are excellent environmentsfor the growth of microorganisms.

Microbial growth is controlled by factors related to the food itself, or intrinsicfactors, and also to the environment where the food is being stored, or whatare described as extrinsic factors

The intrinsic or food-related factors include pH, moisture content, wateactivity or availability, oxidation-reduction potential, physical structure of thefood, available nutrients, and the possible presence of natural antimicrobiaagents.

Extrinsic or environmental factors include temperature, relative humiditygases (CO2, O2) present, and the types and numbers of microorganismspresent in the food.

2.4. INTRINSIC FACTORS and EXTRINSIC FACTORS

INTRINSIC FACTORS

Food composition is a critical intrinsic factor that influences microbial growthIf a food consists primarily of carbohydrates, spoilage does not result in majorodors.

Thus foods such as breads, jams, and some fruits first show spoilage byfungal growth.

In contrast, when foods contain large amounts of proteins and/or fats (forexample, meat and butter), spoilage can produce a variety of foul odors.

This proteolysis and anaerobic breakdown of proteins that yields foulsmelling amine compounds is called putrefaction

One major source of odor is the organic amine cadaverine (imagine the originof that name). Degradation of fats ruins food as well. For example, theproduction of short-chained fatty acids from fats renders butter rancid andunpleasant.

The pH of a food also is critical because a low pH favors the growth of yeastsand molds In neutral or alkaline pH foods, such as meats, bacteria are moredominant in spoilage and putrefaction.

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The presence and availability of water affect the ability of microorganisms tocolonize foods. Simply by drying a food, one can control or eliminate spoilageprocesses.

Water, even if present, can be made less available by adding solutes such assugar and salt.

Water availability is measured in terms of water activity (aw).

This represents the ratio of relative humidity of the air over a test solutioncompared with that of distilled water.

When large quantities of salt or sugar are added to food, mostmicroorganisms are dehydrated by the hypertonic conditions and cannogrow

Even under these adverse conditions, osmophilic and xerophilicmicroorganisms may spoil food. Osmophilic[Greek osmus, impulse, and

philein, to love] microorganisms grow best in or on media with a highosmotic concentration, whereas xerophilic[Greek xerosis, dry, and philein,to love] microorganisms prefer a low aw environment and may not growunder high aw conditions

The oxidation-reduction potential of a food also influences spoilage. Whenmeat products, especially broths, are cooked, they often have lower oxidation-reduction potentials.

These products with their readily available amino acids, peptides, and growth

factors are ideal media for the growth of anaerobes, including Clostridium The physical structure of a food also can affect the course and extent of

spoilage. The grinding and mixing of foods such as sausage and hamburgenot only increase the food surface area and alter cellular structure, but alsodistribute contaminating microorganisms throughout the food.

This can result in rapid spoilage if such foods are stored improperly.

Vegetables and fruits have outer skins (peels and rinds) that protect themfrom spoilage.

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Often spoilage microorganisms have specialized enzymes that help themweaken and penetrate protective peels and rinds, especially after the fruitsand vegetables have been bruised.

Many foods contain natural antimicrobial substances, including complexchemical inhibitors and enzymes.

Coumarins found in fruits and vegetables exhibit antimicrobial activity.

Cows milk and eggs also contain antimicrobial substances. Eggs are rich in

the enzyme lysozyme that can lyse the cell walls of contaminating gram-positive bacteria

Tabasco and other hot red pepper sauces apparently have particularlydesirable antimicrobial characteristics.

Herbs and spices often possess significant antimicrobial substancesgenerally fungi are more sensitive than most bacteria.

Sage and rosemary are two of the most antimicrobial spices.

Aldehydic and phenolic compounds are found in cinnamon, mustard, andoregano. These compounds inhibit microbial growth.

Other important inhibitors are garlic, which contains allicin, and cloves, whichhave eugenol.

Unfermented green and black teas also have well documented antimicrobiaproperties because of their polyphenol contents, which apparently arediminished when the teas are fermented.

Such teas are active against bacteria, viruses, and fungi and may haveanticancer properties.

EXTRINSIC FACTORS

Temperature and relative humidity are important extrinsic factors indetermining whether a food will spoil.

At higher relative humidities microbial growth is initiated more rapidly, evenat lower temperatures

When drier foods are placed in moist environments, moisture absorption can

occur on the food surface, eventually allowing microbial growth. The atmosphere in which the food is stored also is important. This is

especially true with shrink-packed foods because many plastic films allowoxygen diffusion, which results in increased growth of surface associatedmicroorganisms.

Excess CO2 can decrease the solution pH, inhibiting microbial growth. Storingmeat in a high CO2 atmosphere inhibits gram-negative bacteria, resulting ina population dominated by the lactobacilli.

The observation that food storage atmosphere is important has led to thedevelopment modified atmosphere packaging(MAP).

By the use of modern shrink-wrap materials and vacuum technology, it is

possible to package foods with controlled atmospheres. With a carbon dioxide content of 60% or greater in the atmosphere

surrounding a food, spoilage fungi will not grow, even if low levels of oxygenare present.

Some oxygen is kept because if all the oxygen is removed, thepsychrophileClostridiumgasigenescan grow.

This organism can produce gases in 14 days at 2C, which leads to swollenfood packages.

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Such MAP procedures, while assisting in controlling spoilage, also cause ashift in the general structure of the microbial community, from gramnegativeto gram-positive organisms.

2.5. MICROBIAL GROWTH

1. LAG PHASE

o Cells are adjusting to environment.

o Cells are synthesizing needed macromolecules.

2. LOG PHASE OR EXPONENTIAL PHASE

o Cells are undergoing binary fission.

3. STATIONARY PHASE

o Growth rate slows down.

o Some cells die.

o Due to depletion of nutrients and/or accumulation waste.

4. DEATH PHASE

o Cells die.

2.6. SERIAL DILUTION

Viable Plate Count Sample serially diluted and dilutions plated

Count multiplied by dilution factor

Technique assumes that 1 cell will give rise to 1 colony (colony forming uni

(CFU)

Counts ONLY living (viable) organisms

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2.7. MICROBIAL SPOILAGE

Microbial growth in foods can lead to visible changes, including a variety ofcolors caused by spoilage organisms

Meat and dairy products, with their high nutritional value and the presence ofeasily usable carbohydrates, fats, and proteins, provide ideal environmentsfor microbial spoilage.

Proteolysis and putrefaction are typical results of microbial spoilage of suchhigh-protein materials.

2.8. SPOILAGE OF MILK

Unpasteurized milk undergoes a predictable four-step succession duringspoilage;1) Acid production by Lactococcus lactissubsp. Lactisis followed by additiona

acid production associated with the growth of more acid toleranorganisms such as Lactobacillus.

2) At this point yeasts and molds become dominant and degrade theaccumulated lactic acid, and the acidity gradually decreases.

3) Eventually protein-digesting bacteria become active, resulting in a putridodor and bitter flavor.

4) The milk, originally opaque, can eventually become clear In comparisonwith meat and dairy products

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2.9. SPOILAGE OF FRUITS AND VEGETABLES:

Most fruits and vegetables have a much lower protein and fat content andundergo a different kind of spoilage.

Readily degradable carbohydrates favor vegetable spoilage by bacteriaespecially bacteria that cause Soft rots, such as Erwiniacarotovora, whichproduces hydrolytic enzymes.

The high oxidation-reduction potential and lack of reduced conditions permits

aerobes and facultative anaerobes to contribute to the decompositionprocesses.

Bacteria do not seem important in the initial spoilage of whole fruits; insteadsuch spoilage often is initiated by molds.

These organisms have enzymes that contribute to the weakening andpenetration of the protective outer skin.

Food spoilage problems occur with minimally processed, concentrated frozencitrus products. These are prepared with little or no heat treatment, andmajor spoilage can be caused by Lactobacillus and Leuconostocspp., whichproduce diacetyl-butter flavors.

Saccharomyces and Candida can also spoil juices.

Concentrated juice has a decreased water activity (aw _ 0.8 to 0.83), andwhen kept frozen at about _9C, the juices can be stored for long periods.

However, when concentrated juices are diluted with water that containsspoilage

Molds are a special problem for tomatoes. Even the slightest bruising of thetomato skin, exposing the interior, will result in rapid fungal growthFrequently observed genera include Alternaria, Cladosporium,Fusarium,andStemphylium.

This growth affects the quality of tomato products, including tomato juicesand ketchups.

Molds can rapidly grow on grains and corn when these products are heldunder moist conditions

Infection of grains by the ascomycete Claviceps purpura causes ergotism, atoxic condition. Hallucinogenic alkaloids produced by this fungus can lead toaltered behavior, abortion, and death if infected grains are eaten.

2.10. FUNGAL TOXINS:a) Alfatoxin

Fungus-derived carcinogens include the aflatoxins and fumonisins.

Aflatoxinsare produced most commonly in moist grains and nut products.

Aflatoxins were discovered in 1960, when 100,000 turkey poults died fromeating fungus-infested peanut meal.

Aspergillusflavuswas found in the infected peanut meal, together with

alcohol-extractable toxins termed aflatoxins. These flat-ringed planar compounds intercalate with the cells nucleic acids

and act as frameshift mutagens and carcinogens.

This occurs primarily in the liver, where they are converted to unstablederivatives

Aflatoxins B1 and B2, after ingestion by lactating animals, will be modified inthe animal body to yield the aflatoxins M1 and M2,

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If cattle consume aflatoxin-contaminated feeds, these also can appear in milkand dairy products.

The aflatoxins are potent hepatocarcinogens, which have been linked toeffects on immunocompetence, growth, and disease resistance in livestockand laboratory animals

b) Fumonisins:

More recently discovered fungal contaminants of corn are thefumonisins, These are produced by Fusariummonili forme and cause

leukoencephalomalacia in horses, pulmonary edema in pigs, and esophageacancer in humans.

The fumonisins function by disrupting the synthesis and metabolism osphingolipids, important biochemically active compounds, which influence awide variety of cell functions.

The fumonisins inhibit ceramide synthase, a key enzyme for the proper use ofatty subtances in the cell. Thus it is extremely important to store corn andcorn products under dry conditions where these fungi cannot develop.

OTHER FUNGAL TOXINS

Algal toxins contaminate fish and thus affect the health of marine animalshigher in the food chain; they also can contaminate shellfish and fin fishwhich are later consumed by humans.

Most toxins are produced by dinoflagellates, but some diatoms also are toxic.

Major human diseases that result from algal toxins in marine products includeamnesic, diarrhetic, and neurotoxic shellfish poisoning

These toxins are known to cause peripheral neurological system effectsoften in less than one hour after ingestion.

2.11. FOOD BORNE DISEASES

Recent estimates indicate that Norwalk-like viruses, Campylobacter jejuni and

Salmonella are the major causes of food-borne diseases. In addition, Escherichia coli O157:H7 and Listeria are important food-related

pathogens.

There are two primary types of food-related diseases:

food-borne infections

food intoxications.

All these food-borne diseases are associated with poor hygienic practicesWhether by water or food transmission, the fecal-oral route is maintained, withthe food providing the vital link between hosts. Fomites, such as sink faucetsdrinking cups, and cutting boards, also play a role in the maintenance of thefecal-oral route of contamination.

Afood-borne infection

involves the ingestion of the pathogen, followed bygrowth in the host, including tissue invasion and/or the release of toxins.

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SALMONELLOSIS

Salmonellosis results from ingestion of a variety ofSalmonella

serovarsparticularly typhimurium and enteritidis Gastroenteritis is the disease of most concern in relation to foods such as

meats, poultry, and eggs, and the onset of symptoms occurs after anincubation time as short as 8 hours.

Salmonella infection can arise from contamination by workers in foodprocessing plants and restaurants, as well in canning processes

Campylobacter jejuniis considered a leading cause of acute bacteriagastroenteritis in humans and can affect persons of all ages.

This important pathogen is often transmitted by uncooked or poorly cookedpoultry productsoften occurs when kitchen utensils and containers are usedforchicken preparation and then for salads. Contamination with asfew as 10

viable Campylobacter jejuni cells can lead to the onset of diarrheaCampylobacter jejuni also is transmitted by raw milk,and the organism hasbeen found on various red meats.

LISTEROSIS

Listeriosis, is caused by Listeria monocytogenes

The outbreak was traced to pinhole leaks in the heat exchangers of apasteurizing unit.

The leaks allowed incoming raw milk to contaminate the pasteurized milkbefore production of the cheese.

Listeria is difficult to work with because an extended incubation of samples is

required for growth and detection.

ESCHERICHIA COLI

Escherichia coli is now recognized as an important food-borne diseaseorganism. Enteropathogenic, enteroinvasive, and enterotoxigenic types cancause diarrhea. E. coli O157:H7 with its specific somatic (O) and flagellar (H)antigens is thought to have acquired enterohemorrhagic genes from Shigellaincluding the genes for shiga like vero cytotoxins.

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Enterohemorrhagic E. coli has been found in meat products such ashamburger and salami, in unpasteurized fruit drinks, on fruits and vegetablesand in untreated well water.

Prevention of food contamination by E. coli O157:H7 is essential from thetime of production until consumption.

Hygiene must be monitored carefully in larger-volume slaughterhouses wherecontact of meat with fecal material can occur.

Even fruits and vegetables should be handled with care because diseaseoutbreaks have been caused by imported produce.

It may be possible to reduce this threat by destroying the pathogen withgamma irradiation, a food preservation method under consideration for wideruse.

PRIONS:

An infectious agent of increasing worldwide concern with respect to foodsafety is a prion that causes the poorly understood new variant Creutzfeldt

Jakob Disease (vCJD).

This is one of a group of progressively degenerative neuronal diseasestermed transmissible spongiformencephalopathies (TSEs), and is associatedwith beef cattle.

It is often called the mad cow disease.

Sprouts:

Care must be taken especially when the seeds are germinated, because anewly germinating seed releases organic matter and creates aspermosphere that stimulates microbial growth in a way similar to thatwhich occurs in the rhizosphere.

Contaminated alfalfa, beans, watercress, mungbean, mustard, and soybeansprouts can be major sources of typhoid and cholera.

Shellfish and finfish also present major concerns. Raw sewage cancontaminate shellfish-growing areas; in addition, waterborne pathogens suchas Vibrio are more prevalent in the water column during the warm months

Viruses also can be a problem. Oysters are filter feeders that process severaliters of water per day, leading to the potential concentration of at least 100types of enteric viruses. Reverse transcriptase PCR

(RT-PCR) can be used to detect RNA viruses in oysters based on the presenceof their nucleic acids.

FOOD-BORNE INTOXICATIONS

Intoxication produces symptoms shortly after the food is consumedbecause growth of the disease-causing microorganism is not required.

1) Toxins produced by Staphylococcus: Most Staphylococcus aureu sstrains cause a staphylococcal enteritis related to the synthesis oextracellular toxins

The main reservoir ofS. aureusis the human nasal cavity.

Frequently S. aureus is transmitted to a persons hands and thenis introduced into food during preparation.

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Growth and enterotoxin production usually occur whencontaminated foods are held at room temperature for severahours.

2) Toxin Produced by Spore forming Bacteria:Three gram-positive rodsare known to cause food intoxications: Clostridium botulinum, C.

perfringens, and Bacillus cereus

Clostridium perfringensfood poisoning is one of the more widespread food

intoxications. These microorganisms, which produce exotoxins, must grow tolevels of approximately 106 bacteria per gram or higher in a food to causedisease. At least 108 bacteria must be ingested.

They are common inhabitants of soil, water, food, spices, and the intestinatract.

Upon ingestion the cells sporulate in the intestine. The enterotoxin is asporespecific protein and is produced during the sporulation processEnterotoxin can be detected in the feces of affected individuals.

Clostridium perfringens food poisoning is common and occurs after meatproducts are heated, which results in O2 depletion. If the foods are cooledslowly, growth of the microorganism can occur. At 45C, enterotoxin can bedetected 3 hours after growth is initiated.

Onset of the symptomswatery diarrhea, nausea, and abdominal crampsusually occurs in about 8 to 16 hours.

Bacillus cereusalso is of concern in starchy foods. It can cause two distincttypes of illnesses depending on the type of toxin produced: an emetic illnesscharacterized by nausea and vomiting with an incubation time of 1 to 6hours, and a diarrheal type, with an incubation of 4 to 16 hours.

The emetic type is often associated with boiled or fried rice, while thediarrheal type is associated with a wider range of foods.

2.12. FERMENTED FOOD PRODUCTSThe major fermentations used in food microbiology are the lactic, propionic, andethanolic fermentations

Fermented MilksThese fermentations are carried out by mesophilic, thermophilic, and therapeuticlactic acid bacteria, as well as by yeasts and molds

Mesophilic- Buttermilk Production

Mesophilic milk fermentations result from similar manufacturingtechniques, in which acid produced through microbial activity causesprotein denaturation.

To carry out the process, one usually inoculates milk with the desired

starter culture incubates it at optimum temperature (approximately 20 to30C), and then stops microbial growth by cooling. Lactobacillus spp. AndLactococcuslactiscultures are used for aroma and acid production.

The organism Lactococcus lactis subsp. Diacetilactis converts milk citrateto diacetyl, which gives a special buttery flavor to the finished product.

The use of these microorganisms with skim milk produces culturedbuttermilk

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Thermophilic- Yogurt Production

In addition to mesophilic milk fermentations, thermophilic fermentationscan be carried out at temperatures around 45C.

An important example is yogurt production. Yogurt is made using aspecial starter culture in which two major bacteria are present in a 1:1ratio: S. thermophilus and L. bulgaricus.

With these organisms growing in concert, acid is produced by

Streptococcus, and aroma components are formed by the Lactobacillus. Freshly prepared yogurt contains 109 bacteria per gram.

Therapeutic- Acidophilus milk

Fermented milks may have beneficial therapeutic effects. Acidophilusmilk is produced by using Lactobacillus acidophilus.

L. acidophilus may modify the microbial flora in the lower intestine, thusimproving general health, and it often is used as a dietary adjunct.

Many microorganisms in fermented dairy products stabilize the bowemicroflora, and some appear to have antimicrobial properties.

They may involve minimizing lactose intolerance, lowering serum

cholesterol, and possibly exhibiting anticancer activity. Severalactobacilli have antitumor compounds in their cell walls.

Another interesting group used in milk fermentations are thebifidobacteria.

The genus Bifidobacteriumcontains irregular, nonsporing, gram-positiverods that may be club-shaped or forked at the end

Bifidobacteria are nonmotile, anaerobic, and ferment lactose and othesugars to acetic and lactic acids.

Bifidobacteria are thought to help maintain the normal intestinal balancewhile improving lactose tolerance; to possess antitumorigenic activityand to reduce serum cholesterol levels

In addition, some believe that they promote calcium absorption and the

synthesis of B-complex vitamins.

KEFIR

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Yeast-lactic fermentations include kefir, a product with an ethanoconcentration of up to 2%.

Kefir products tend to be foamy and frothy, due to active carbon dioxideproduction. This process is based on the use of kefir grains as aninoculum.

These are coagulated lumps of casein that contain yeasts, lactic acidbacteria, and acetic acid bacteria

In this fermentation, the grains are used to inoculate the fresh milk andthen recovered at the end of the fermentation.

Cheese Production

Cheese is one of the oldest human foods and is thought to have beendeveloped approximately 8,000 years ago.

About 2,000 distinct varieties of cheese are produced throughout theworld, representing approximately 20 general types

Often cheeses are classified based on texture or hardness as softcheeses (cottage, cream, Brie), semisoft cheeses (Muenster, Limburgerblue), hard cheeses (cheddar, Colby, Swiss), or very hard cheeses(Parmesan).

All cheese results from a lactic acid fermentation of milk, which results incoagulation of milk proteins and formation of a curd.

Rennin, an enzyme from calf stomachs, but now produced by geneticallyengineered microorganisms, can also be used to promote curd formation.

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After the curd is formed, it is heated and pressed to remove the watery partof the milk or whey, salted, and then usually ripened

The cheese curd can be packaged for ripening with or without additionamicroorganisms.

Cheese curd inoculation is used in the manufacture of Roquefort and bluecheese.

In this case Penicillium roqueforti spores are added to the curds just before

the final cheese processing. Sometimes the surface of an already formed cheese is inoculated at the start

of ripening; for example, Camembert cheese is inoculated with spores ofPenicillium camemberti. The final hardness of the cheese is partially afunction of the length of ripening.

Soft cheeses are ripened for only about 1 to 5 months, whereas hard cheesesneed 3 to 12 months, and very hard cheeses like Parmesan require 12 to 16months ripening.

The ripening process also is critical for Swiss cheese. Gas production byPropionibacterium contributes to final flavor development and hole or eyeformation in this cheese.

Some cheeses are soaked in brine to stimulate the development of specificfungi and bacteria; Limburger is one such cheese.

Production of Alcoholic Beverages

A variety of plants that contain adequate carbohydrates can be used toproduce alcoholic beverages. When carbohydrates are available in readilyfermentable form, the fermentation can be started immediately.

For example, grapes are crushed to release the juice or must, which can beallowed to ferment without further delay.

The must also can be sterilized by pasteurization or the use of sulfur dioxideand then the desired microbial culture added.

In contrast, before cereals and other starchy materials can be used assubstrates for the production of alcohol, their complex carbohydrates mustbe hydrolyzed.

They are mixed with water and incubated in a process called mashing.

The insoluble material is then removed to yield the wort, a clear liquidcontaining fermentable sugars and other simple molecules.

Much of the art of beer and ale production involves the controlled hydrolysisof protein and carbohydrates to provide the desired body and flavor of thefinal product.

Wines and Champagnes

Wine production, or the science ofenology [Greek oinos, wine, and ology,the science of], starts with the collection of grapes, continues with theicrushing and the separation of the liquid (must) before fermentation, andconcludes with a variety of storage and aging steps

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All grapes have white juices.

To make a red wine from ared grape, the grape skins areallowed to remain in contactwith the must beforefermentation to release theirskin-coloring components.

Wines can be created by usingthe natural grape skinmicroorganisms. This naturalmixture of bacteria and yeastsgives unpredictablefermentation results.

To avoid such problems, onecan treat the fresh must witha sulfur dioxide fumigant andadd a desired strain of Saccharomyces cerevisiaeorS. ellipsoideus.

After inoculation the juice isfermented for 3 to 5 days attemperatures varyingbetween 20 and28C.

Depending on the alcoholtolerance of the yeast strain,the final product may contain10 to 18% alcohol.

Clearing and development offlavor occur during the agingprocess.

A critical part of wine makinginvolves the choice of whetherto produce a dry (noremaining free sugar) or asweeter (varying amounts of free sugar) wine.

This can be controlled by regulating the initial must sugar concentration.

With higher levels of sugar, alcohol will accumulate and inhibit thefermentation before the sugar can be completely used, thus producing asweeter wine.

During final fermentation in the aging process, flavoring compoundsaccumulate and influence the bouquet of the wine.

Microbial growth during the fermentation process produces sediments, which

are removed during racking. Racking can be carried out at the time the fermented wine is transferred to

bottles or casks for aging or even after the wine is placed in bottles. Many processing variations can be used during wine production.

The wine can be distilled to make a burned wine or brandy.Acetobacterand Gluconobactercan be allowed to oxidize the ethanol to acetic acid andform a wine vinegar.

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In the past an acetic acid generator was used to recirculate the wine over abed of wood chips, where the desired microorganisms developed as a surfacegrowth. Today the process is carried out in large aerobic submerged culturesunder much more controlled conditions.

Natural champagnes are produced by continuing the fermentation in bottlesto produce a naturally sparkling wine.

Sediments that remain are collected in the necks of inverted champagne

bottles after the bottles have been carefully turned. The necks of the bottlesare then frozen and the corks removed to disgorge the accumulatedsediments.

The bottles are refilled with clear champagne from another disgorged bottleand the product is ready for final packaging and labeling.

Beers and Ales

Beer and ale production uses cereal grainssuch as barley, wheat, and rice. Thecomplex starches and proteins in thesegrains must be changed to a more readily

usable mixture of simpler carbohydratesand amino acids.

This process, involves germination of thebarley grains and activation of theirenzymes to produce a malt.

The malt is then mixed with water and thedesired grains, and the mixture istransferred to the mash tun or cask inorder to hydrolyze the starch to usablecarbohydrates.

Once this process is completed, the mashis heated with hops (dried flowers of thefemale vine Humuluslupulis), which wereoriginally added to the mash to inhibitspoilage microorganisms.

The hops also provide flavor and assist inclarification of the wort.

In this heating step the hydrolyticenzymes are inactivated and the wort canbe pitchedinoculatedwith the desiredyeast.

Most beers are fermented with bottomyeasts, related to Saccharomyces

carlsbergensis, which settle at the bottomof the fermentation vat.

The beer flavor also is influenced by theproduction of small amounts of glyceroland acetic acid. Bottom yeasts producebeer with a pH of 4.1 to 4.2 and requiring7 to 12 days of fermentation

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With a top yeast, such as Saccharomyces cerevisiae, the pH is lowered to 3.8to produce ales. Freshly fermented (green) beers are aged or lagered, andwhen they are bottled, CO2 is usually added.

Beer can be pasteurized at 140F or higher or sterilized by passage throughmembrane filters to minimize flavor changes.

In many places there is increased interest in specialty beers.

Local Braumeisters develop unique products with special brewing

techniques and ingredients.

Distilled Spirits

Distilled spirits are produced by anextension of beer productionprocesses. The fermented liquid isboiled, and the volatile componentsare condensed to yield a product witha higher alcohol content than beer.

Rye and bourbon are examples ofwhiskeys.

Rye whiskey must contain at least 51%rye grain, and bourbon must contain atleast 51% corn.

Scotch whiskey is made primarily ofbarley.

Usually a sour mash is used; themash is inoculated with ahomolactic(lactic acid is the major fermentation

product) bacterium such asLactobacillus delbrueckii, which canlower the mash pH to around 3.8 in 6to 10 hours.

This limits the development ofundesirable organisms.

Vodka and grain alcohols are alsoproduced by distillation.

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Gin is vodka to which resinous flavoring agentsoften juniper berrieshavebeen added to provide a unique aroma and flavor.

Sauerkraut:

Sauerkraut or sour cabbage, is produced from wilted, shredded cabbage

Usually the mixed microbial community of the cabbage is used.

A concentration of 2.2 to 2.8% sodium chloride restricts the growth of gram-negative bacteria while favoring the development of the lactic acid bacteria.

The primary microorganisms contributing to this product are Leuconostocmesenteroides and Lactobacillus plantarum.

A predictable microbial succession occurs in sauerkrauts development.

The activities of the lactic acid-producing cocci usually cease when the acidcontent reaches 0.7 to 1.0%. At this point Lactobacillus plantarum andLactobacillus brevis continue to function.

The final acidity is generally 1.6 to 1.8, with lactic acid comprising 1.0 to1.3% of the total acid in a satisfactory product.

Documents

Chapter-2 (Food Tech) Food Microbiology