microbes involved in food feermentation

1. SEMESTER-IV
PAPER-3 (USMB-403) OPTION: A UNIT: I: 1a
Microorganism used in food fermentation
1. Discuss the criteria used to select a microorganism for beneficial purposes in foods
⮚ Beneficial microorganisms are used in foods in several ways. These include actively growing
microbial cells, nongrowing microbial cells, and metabolic by-products and cellular components
of microorganisms.
⮚ An example of the use of growing microbial cells is the conversion of milk to yogurt by
bacteria.
⮚ Nongrowing cells of some bacteria are used to increase shelf life of refrigerated raw milk or raw
meat.
⮚ Many by-products, such as lactic acid, acetic acid, some essential amino acids, and bacteriocins
produced by different microorganisms, are used in many foods.
⮚ Finally, microbial cellular components, such as single-cell proteins (SCPs), dextran, cellulose,
and many enzymes, are used in food for different purposes.
⮚ These microorganisms or their by-products or cellular components have to be safe, food grade,
and approved by regulatory agencies.
⮚ When the microbial cells are used in such a way that they are consumed live with the food (as in
yogurt), it is very important that they and their metabolites have no detrimental effect on the
health of the consumers.
⮚ When a by-product (such as an amino acid) or a cellular component (such as an enzyme) is used
in a food, the microorganisms producing it have to be regulated and approved, and the
by-product and cellular component have to be safe.
⮚ If a food-grade microorganism is genetically modified, its use in food has to be approved,
especially if the genetic material used is obtained from a different source or is synthesized. Thus,
the microorganisms used for these purposes have to meet some commercial and regulatory
criteria.
2. Discuss microbiology of fermented foods
⮚ Food fermentation involves a process in which raw materials are converted to fermented foods
by the growth and metabolic activities of the desirable microorganisms.
⮚ The microorganisms utilize some components present in the raw materials as substrates to
generate energy and cellular components, to increase in population, and to produce many usable
by-products (also called end products) that are excreted in the environment. The unused
components of the raw materials and the microbial by-products (and sometimes microbial cells)
together constitute fermented foods.
⮚ The raw materials can be milk, meat, fish, vegetables, fruits, cereal grains, seeds, and beans,
fermented individually or in combination. Worldwide, more than 3500 types of fermented foods
are produced.
⮚ Many ethnic types are produced and used in small localities by small groups of people. Many of
the fermented foods consumed currently have been produced and consumed by humans for
thousands of years.
⮚ The old city civilizations, dating as far back as 3000 to 5000 B.C. in the Indus Valley,
Mesopotamia, and Egypt, developed exceptional skills in the production of fermented foods
from milk, fruits, cereal grains, and vegetables.
⮚ The process not only produced new foods but also helped preserve the excess of raw materials
both of plant and animal origins.

2. ⮚ The basic principles developed by these ancient civilizations are used even today to produce
many types of fermented foods by a process known as natural fermentation.
● In this method, either the desirable microbial population naturally present in the raw
materials or some products containing the desirable microbes from a previous
fermentation (called back slopping), are added to the raw materials.
● Then the fermentation conditions are set so as to favor growth of the desirable types but
prevent or retard growth of undesirable types that could be present in the raw materials.
⮚ In another type of fermentation, called controlled or pure culture fermentation, the
microorganisms associated with fermentation of a food are first purified from the food,
identified, and maintained in the laboratory.
● When required for the fermentation of a specific food, the microbial species associated
with this fermentation are grown in large volume in the laboratory and then added to the
raw materials in very high numbers.
● Then the fermentation conditions are set such that these microorganisms grow
preferentially to produce a desired product.
● Characteristics of some of are discussed here.
⮚ The microorganisms used in fermentations, in controlled fermentation, are also referred to as
starter cultures.
⮚ Many of these microbial species are present in raw materials that are naturally fermented, along
with other associated microorganisms, some of which may contribute to the desirable
characteristics of the products.
3. List the genera that are now included in the group of lactic acid bacteria.
⮚ At present, bacterial species from 12 genera are included in a group designated as lactic acid
bacteria because of their ability to metabolize relatively large amounts of lactic acids from
carbohydrates.2–4
⮚ The genera include
⮚ Lactococcus ⮚ Streptococcus ⮚ Aerococcus ⮚ Carnobacterium
⮚ Leuconostoc, ⮚ Lactobacillus ⮚ Vagococcus ⮚ Weissella
⮚ Pediococcus ⮚ Enterococcus ⮚ Tetragenococcus, ⮚ Oenococcus
⮚ Many of the genera have been created recently from previously existing genera and include one
or a few species.
⮚ For example, Lactococcus and Enterococcus were previously classified as Streptococcus Group
N and Group D, respectively.
⮚ Vagococcus is indistinguishable from Lactococcus, except that these bacteria are motile.
Weissella and Oenococcus are separated from Leoconostoc.
⮚ Tetragenococcus includes a single species that was previously included with Pediococcus
(Pediococcus halophilus).
⮚ Carnobacterium was created to include a few species that were previously in genus
Lactobacillus and are obligatory heterofermentative.

3. ⮚ However, species from the first five genera, i.e., Lactococcus, Leuconostoc, Pediococcus,
Streptococcus, and Lactobacillus, are used as starter cultures in food fermentation and are
discussed here.
⮚ The status ofothers, except Tetragenococcus halophilus and Oenococcus oeni, with respect to
use in food, is not clear at present.
A. Lactococcus
⮚ This genus includes several species, but only one species, Lactococcus lactic, has been widely
used in dairy fermentation.
⮚ It has three subspecies (ssp.), ssp. lactis, ssp. cremoris, and ssp. hordniae, but only the first two
are used in dairy fermentation.
⮚ The biovar Lac. lactis ssp. lactis biovar diacetylactis is also used in dairy fermentation.
● The cells are ovoid, ca. 0.5 to 1.0 m in diameter, present in pairs or short chains, nonmotile,
nonsporulating, and facultative anaerobic to microaerophilic
● In general, they grow well between 20 and 30 0
C, but do not grow in 6.5% NaCl or at pH 9.6. In
a suitable broth they can produce ca. 1% L(+)-lactic acid and reduce the pH to ca. 4.5.
● Subsp. cremoris can be differentiated from subsp. Lactis by its inability to grow at 40ºC, in 4%
NaCl, ferment ribose, and hydrolyze arginine to produce NH3. Biovar diacetylactis, as
compared with others, produces larger amounts of CO2 and diacetyl from citrate.
● They are generally capable of hydrolyzing lactose and casein. They also ferment galactose,
sucrose, and maltose.
● Natural habitats are green vegetation, silage, the diary environment, and raw milk.
B. Streptococcus
⮚ Only one species, Streptococcus thermophilus, has been used in dairy fermentation.
⮚ A change in designation to Str. salivarius ssp. thermophilus was suggested but not made.
⮚ They are used in dairy fermentation.
● The Gram-positive cells are spherical to ovoid, 0.7 to 0.9 m in diameter, and exist in
pairs to long chains.
● The cells grow well at 37 to 40ºC, but can also grow at 52ºC. They are facultative
anaerobes and in glucose broth can reduce the pH to 4.0 and produce L(+)-lactic
acid.
● They ferment fructose, mannose, and lactose, but generally not galactose and sucrose.
Cells survive 60ºC for 30 min. Their natural habitat is unknown, although they are found
in milk.
C. Leuconostoc
⮚ The Gram-positive cells are spherical to lenticular, arranged in pairs or in chains, nonmotile,
nonsporulating, catalase negative, and facultative anaerobes.
⮚ The species grow well between 20 and 30ºC, with a range of 1 to 37ºC.
⮚ Glucose is fermented to D(–)-lactic acid, CO2, ethanol, or acetic acid, with the pH reduced to
4.5 to 5.0. The species grow in milk but may not curdle.
⮚ Also, arginine is not hydrolyzed.
⮚ Many form dextran while growing on sucrose.
⮚ Citrate is utilized to produce diacetyl and CO2.
⮚ Some species can survive 60ºC for 30 min.
⮚ Leuconostoc species are found in plants, vegetables, silage, milk and some milk products, and
raw and processed meats.

4. ⮚ At present, five species are known: Leuconostoc mesenteroides, Leu. paramesenteroides, Leu.
lactis, Leu. carnosum, and Leu. gelidum.
⮚ Leu. mesenteroides has three subspecies: subsp. mesenteroides, ssp. dextranicum, and ssp.
cremoris. Leu. mesenteroides ssp. cremoris and Leu. lactis are used in some dairy and vegetable
fermentations.
⮚ Many of these species, particularly Leu. carnosum and Leu. gelidum, have been associated with
spoilage of refrigerated vacuum-packaged meat products.5
⮚ Leuconostocs are morphologically heterogenous and may contain genetically diverse groups of
bacteria.
⮚ Recently, two new genera have been created from it: Weisella and Oenococcus. Oen. oeni is
found in wine and related habitant and is used for malolactic fermentation in wine.
D. Pediococcus
⮚ The cells are spherical and form tetrads, but they can be present in pairs. Single cells or chains
are absent.
⮚ They are Gram-positive, nonmotile, nonsporulating, facultative anaerobes.
⮚ They grow well between 25 and 40􀁲C; some species grow at 50􀁲C.
⮚ They ferment glucose to L(+)- or DL-lactic acid, some species reducing the pH to 3.6.
⮚ Depending on the species, they can ferment sucrose, arabinose, ribose, and xylose. Lactose is
not generally fermented, especially in milk, and milk is not curdled.2 Some strains may have
weak lactose-hydrolyzing capability, especially in broth containing lactose as a carbohydrate
source.
⮚ Depending on the species, they are found in plants, vegetables, silage, beer, milk, and fermented
vegetables, meats, and fish.
⮚ The genus has seven to eight species, of which Ped. pentosaceus and Ped. acidilactici are used
in vegetables, meat, cereal, and other types of fermented foods.
⮚ They have also been implicated in ripening and flavor production of some cheeses as secondary
cultures. These two species are difficult to differentiate, but compared with Ped. acidilactici,
Ped. pentosaceus ferments maltose, does not grow at 50ºC, and is killed at 70ºC in 5 min.2 Ped.
halophilus, used in fermentation of high-salt products, is now classified as Tet. halophilus.
4. How are the species in genus Lactobacillus (the basis) divided into groups? List two
species from each group.
⮚ The genus Lactobacillus includes a heterogenous group of Gram-positive, rodshaped, usually
nonmotile, nonsporulating, facultative anaerobic species that vary widely morphologically and
in growth and metabolic characteristics.
⮚ Cells vary from very short (almost coccoid) to very long rods, slender or moderately thick, often
bent, and can be present as single cells or in short to long chains.
⮚ While growing on glucose, depending on a species, they produce either only lactic acid [L(+),
D(–), or DL] or a mixture of lactic acid, ethanol, acetic acid, and CO2.
⮚ Some also produce diacetyl. Many species utilize lactose, sucrose, fructose, or galactose, and
some species can ferment pentoses. Growth temperature can vary from 1 to 50ºC, but most that
are used as starter cultures in controlled fermentation of foods grow well from 25 to 40ºC.
Several species involved in natural fermentation of some foods at low temperature can grow
well from 10 to 25ºC.
⮚ While growing in a metabolizable carbohydrate, depending on a species, the pH can be reduced
between 3.5 and 5.0.

5. ⮚ They are distributed widely and can be found in plants; vegetables; grains; seeds; raw and
processed milk and milk products; raw, processed, and fermented meat products; and fermented
vegetables; some are found in the digestive tract of humans, animals, and birds.
⮚ Many have been associated with spoilage of foods. Among the large number of species, some
have been used in controlled fermentation (dairy, meat, vegetables, and cereal), some are known
to be associated with natural fermentation of foods, a few are consumed live for their beneficial
effect on intestinal health, and some others have an undesirable effect on foods.
⮚ On the basis of their metabolic patterns of hexoses and pentoses , the species have been divided
into three groups :
● Those in Group I ferment hexoses (and disaccharides such as lactose and sucrose) to
produce mainly lactic acids and do not ferment pentoses (such as ribose, xylose, or
arabinose).
● Those in Group II, depending on the carbohydrates and the amounts available, either
producenmainly lactic acid, or a mixture of lactic, acetic, and formic acids, ethanol, and
CO2.
● Group III species ferment carbohydrates to a mixture of lactate, acetate, ethanol, and
CO2.
⮚ The three Lactobacillus delbrueckii subspecies are used in the fermentation of dairy products,
such as some cheeses and yogurt.
⮚ They grow well at 45ºC and ferment lactose to produce large amounts of D(–) lactic acid.
β-galactosidase in these subspecies is constitutive.
⮚ Lab. acidophilus and Lab. reuteri are considered beneficial intestinal microbes (probiotic) and
present in the small intestine.
⮚ Lab. Acidophilus is used to produce fermented dairy products and also either added to
pasteurizedmilk or made into tablets and capsules for consumption as probiotics. It metabolizes
lactose, and produces large amounts of D(–)-lactic acid.
⮚ However, in Lab. acidophilus, β-galactosidase is generally inducible.
⮚ Lab. helveticus is used to make some cheeses and ferment lactose to lactic acid (DL). Lab. casei
ssp. casei is used in some fermented dairy products.
⮚ It ferments lactose and produces L(+)-lactic acid.
⮚ Some strains are also used as probiotic bacteria. Strains of Lab. casei ssp. rhamnosus (also
called Lab. rhamnosus) are now used as a probiotic bacterium. Some strains of Lab.
johnsonii are also used in probiotics.
⮚ Lab. plantarum is used in vegetable and meat fermentation. It produces DL-lactic acid. Lab.
curvatus and Lab. sake can grow at low temperatures (2 to 4ºC) and ferment vegetable and meat
products. Lab. sake is used to ferment sake wine.
⮚ Lab. kefir is important in the fermentation of kefir, an ethnic fermented sour milk.
⮚ Lab. sanfrancisco is associated with other microorganisms in the fermentation of San Francisco
sourdough bread.
⮚ Lab. viridescens, Lab. curvatus, and Lab sake are associated with spoilage of refrigerated meat
products.
F. Oenococcus
⮚ Oen. oeni, previously designated as Leu. oeni, has the general characteristics of Leuconostoc
spp. It is found in the winery environment.
⮚ It is sometimes used to accelerate malolactic fermentation in wine. The cells transport malate in
wine and metabolize it to lactic acid and CO2. This process reduces the acidity of wine.

6. 5. OTHER STARTER CULTURES
A. Bifidobacterium
⮚ They are morphologically similar to some Lactobacillus spp. and were previously included in
the genus Lactobacillus.
⮚ The cells are Gram-positive, rods of various shapes and sizes, present as single cells or in chain
of different sizes.
⮚ They are nonsporeforming, nonmotile, and anaerobic, although some can tolerate O2 in the
presence of CO2.
⮚ The species grow optimally at 37 to 41ºC, with a growth temperature range of 25 to 45ºC.
⮚ They usually do not grow at a pH above 8.0 or below 4.5.
⮚ They ferment glucose to produce lactic and acetic acid in a 2:3 molar ratio without producing
CO2, and also ferment lactose, galactose, and some pentoses.
⮚ They have been isolated from feces of humans, animals, and birds and are considered beneficial
for the normal health of the digestive tract.
⮚ They are present in large numbers in the feces of infants within 2 to 3 d after birth, and usually
present in high numbers in breast-fed babies.
⮚ They are usually found in the large intestine.
⮚ Many species of this genus have been isolated from the feces of humans and animals.
⮚ Some of these include Bifidovacterium bifidum, Bif. longum, Bif. brevis, Bif. infantis, and Bif.
adolescentis.
⮚ All have been isolated in humans; however, some species are more prevalent in infants than in
adults.
⮚ of these species have been added to dairy products to supply live cells in high numbers to restore
and maintain intestinal health in humans.
B. Propionibacterium
⮚ The genus includes species in the classical or dairy propionibacterium group and the cutaneous
or acne propionibacterium group.
⮚ , only the diary group is discussed. The cells are Gram-positive, pleomorphic thick rods 1 to 1.5
m in length, and occur in single cells, pairs, or short chains with different configurations.
⮚ They are nonmotile, nonsporulating, anaerobic (can also tolerate air), catalase positive, and
ferment glucose to produce large amounts of propionic acid and acetic acid.
⮚ They also, depending on the species, ferment lactose, sucrose, fructose, galactose, and some
pentoses. They grow optimally at 30 to 37ºC. Some species form pigments.
⮚ They have been isolated from raw milk, some types of cheeses, dairy products, and silage.
⮚ At present, four species of diary propionibacterium are included in the genus:
Propionibacterium freudenreichii, Pro. jensenii, Pro. thoenii, and Pro. acidipropionici.
⮚ All four are associated with natural fermentation of Swiss-type cheeses, but Pro. freudenreichii
has been used as a starter culture in controlled fermentation.
C. Brevibacterium
⮚ The genus contains a mixture of coryniform bacterial species, some of which have important
applications in cheese production and other industrial fermentations. Brevibacterium linens is
used in cheese ripening as it has extracellular proteases.
⮚ The cells are nonmotile, Gram-positive, and capable of growing in high salt and wide pH ranges.

7. D. Acetobacter
⮚ A species in this genus, Ace. aceti, is used to produce acetic acid from alcohol.2 The cells are
Gram-negative; aerobic; rods (0.5 to 1.5 m); occurring as single cells, pairs, or chains; and can
be motile or nonmotile.
⮚ They are obligate aerobes, catalase positive, and oxidize ethanol to acetic acid and lactic acid to
CO2 and H2O. They grow well from 25 to 30ºC.
⮚ They are found naturally in fruits, sake, palm wine, cider, beer, sugar cane juice, tea fungus, and
soil. Some species synthesize large amounts of cellulose
6. In yeast fermentation of different foods and beverages, only one species is used. Name the
species, and discuss how one species can be effective in so many fermentation processes.
Discuss the characteristics of bottom and top yeasts. Name a species that is now used
commercially to produce β-galactosidase (lactase)
Yeasts raise, ferment, carbonate, and otherwise transform a wide variety of agricultural products.
⮚ Bread uses the carbon dioxide produced by the yeast to leaven, or raise, it.
⮚ This fermentation takes some time, so those in a hurry (e.g., the Israelites during their exodus
from Egypt) eat unleavened bread.
⮚ The production of beer is more complex and involves several steps.
⮚ Enzymes must be made, and they must break down starch to fermentable carbohydrates before
the carbohydrates can be fermented to alcohol.
⮚ Since wine grapes contain simple sugars that are easily fermented, wine making is in some ways
simpler than beer making, but in other ways, due to the complexity of grapes and their
chemistry, it is more complex.
⮚ Vinegar production is in theory very easy, since it involves only a single oxidation step.
⮚ In reality, getting enough oxygen into the fermenting liquid can be quite challenging.

8. Many yeasts are important in food, but most are involved with the spoilage of food and mycotoxin
production (by molds). Several are, however, used in food bioprocessing. At present, genetic
improvements are being made to improve their desirable characteristics.
Yeasts
⮚ Among the many types of yeasts, only a few have been associated with fermentation of foods
and alcohol, production of enzymes for use in food, production of SCPs, and as additives to
impart desirable flavor in some foods.
⮚ The most important genus and species used is Saccharomyces cerevisiae.It has been used to
leaven bread and produce beer, wine, distilled liquors, and industrial alcohol; produce invertase
(enzyme); and flavor some foods (soups).
⮚ However, many strains have been developed to suit specific needs.
⮚ The cells are round, oval, or elongated.
● They multiply by multipolar budding or by conjugation and formation of ascospores.
● The strains are generally grouped as bottom yeasts or top yeasts.
● Top yeasts grow very rapidly at 20ºC, producing alcohol and CO2. They also form
clumps that, because of rapid CO2 production, float at the surface.
● In contrast, bottom yeasts grow better at 10 to 15ºC, produce CO2 slowly (also grow
slow), do not clump, and thus settle at the bottom.
⮚ Top yeasts and bottom yeasts are used according to the need of a particular fermentation
process. Candida utilis has been used to produce SCPs.
⮚ It is a false yeast (Fungi imperfecti) and reproduces by budding (not by conjugation). The cells
are oval to elongated and form hyphae with large numbers of budding cells. They are also
involved in food spoilage.
⮚ Kluyveromyces marxianus and Klu. marxianus var. lactis can hydrolyze lactose and have been
associated with natural fermentation, along with other yeasts and lactic acid bacteria, of
alcoholic dairy products such as kefir.
⮚ They have also been associated with spoilage of some dairy products.
⮚ At present, they are used to produce β-galactosidase (lactase) for use commercially to
hydrolyze lactose. The enzyme is now used to produce low-lactose milk.
7. How are molds used in different ways in food? Name two species and list their uses. What
precautions are needed while using a mold strain in food fermentation?
Many molds are important in food, but most are involved with the spoilage of food and mycotoxin
production (by molds). Several are, however, used in food bioprocessing. At present, genetic
improvements are being made to improve their desirable characteristics.
⮚ Although most molds are associated with food spoilage and many form mycotoxins while
growing in foods, other species and strains are used in processing of foods and to produce
additives and enzymes for use in foods.
⮚ In general, molds are multicellular, filamentous fungi.
● The filaments (hyphae) can be septate or nonseptate and have nuclei.
● They divide by elongation at the tip of a hypha (vegetative reproduction) or by forming
sexual or asexual spores on a spore-bearing body.
⮚ Among many genera, several species from genera Aspergillus and Penicillium, and a few from
Rhizopus and Mucor, have been used for beneficial purposes in food.

9. ⮚ Strains to be used for such purposes should not produce mycotoxins.
⮚ It is difficult to identify a nonmycotoxin-producer strain in the case of natural fermentation, but
should be an important consideration in the selection of strains for use in controlled
fermentation.
⮚ Aspergillus oryzae is used in fermentation of several oriental foods, such as sake, soy sauce, and
miso. It is also used as a source of some food enzymes. Asp. Niger is used to produce citric acid
and gluconic acid from sucrose.
⮚ It is also used as a source of the enzymes pectinase and amylase. Penicillium roquefortii is used
for ripening of Roquefort, Gorgonzola, and blue cheeses.
⮚ Some strains can produce the neurotoxin roquefortin.
⮚ In the selection and development of strains for use in cheese, this aspect needs careful
consideration.
⮚ Pen. camembertii is used in Camembert cheese and Pen. caseicolum is used in Brie cheese.
They are also used to produce the enzyme glucose oxidase.
SEMESTER-IV
PAPER-3 (USMB-403) OPTION: A UNIT: I: 1b
Microbiology of fermented food
Microorganisms have long played a major role in the production of food and beverages.
Traditional fermented foodstuffs include:
1 alcoholic beverages, especially beers, wines and distilled spirits, which are derived from sugars and
starches;
2 dairy products, particularly cheeses, yoghurt, sour cream and kefir;
3 fish and meat products, such as fish sauce and fermented sausages; and
4 plant products, notably cereal-based breads and fermented rice products; along with fermented
fruits, vegetables and legumes, including preserved olives and gherkins, sauerkraut, soy sauce, tofu,
fermented cassava, cocoa and coffee beans.
● Many of these products originally evolved as a means of food preservation.
● The stabilizing microbial activity may result in lower water activity, modified pH, generation of
inhibitory compounds (alcohol, bacteriocins, etc.) and removal of nutrients readily utilized by
potential spoilage organisms.
● Importantly, besides providing long-term stability, these fermentation processes also generate
desirable flavour, aroma and texture.
⮚ The role of microorganisms in this field is now even more diverse. Microbial biomass is used as
novel food, such as single cell protein products, speciality mushrooms and probiotic
preparations; numerous microbial fermentation products are also incorporated as food additives
and supplements.

10. ⮚ In addition, microbial enzymes are utilized extensively as food processing aids, and some may
be added to animal feed to improve its nutritional value
Dairy fermentations
⮚ Milk from any mammal, but particularly from cow, horse, sheep, goat and buffalo, may be used
to produce fermented dairy products such as cheese, butter, sour cream, kefir and yoghurt.
⮚ Their production has traditionally been a means of preservation, which is accomplished through
changes in water content, and acid and bacteriocin formation. The final products also have
enhanced texture, flavour and aroma.
⮚ More recently popularized dairy products include fermented ‘health’ drinks and foods, referred
to as probiotics. These products contain live bacteria such as Lactobacillus acidophilus and
Bifidobacterium species, which are alleged to improve the functioning of the gut and stabilize its
microflora.
⮚ The microorganisms used in the production of fermented dairy products are :
● Primarily lactic acid bacteria, which are naturally present in milk.
● Other organisms, notably filamentous fungi, may be involved in later maturation
processes of some cheeses.
⮚ Starter cultures, often containing specific mixtures of lactic acid bacteria, are now
predominantly used to inoculate pasteurized, rather than raw milk.
⮚
⮚ Use of pasteurized milk not only reduces the possibility of growth of potential pathogens,but
also lowers the activity of certain milk enzymes that can affect some dairy fermentations. Basic
manufacturing processes are very similar for many of these dairy products.
⮚ Fermentation generates lactic acid, which modifies milk proteins, and forms flavour and aroma
compounds, notably diacetyl, which imparts a buttery flavour.
⮚ The lactic acid bacteria used as starter cultures are somewhat prone to infection by destructive
bacteriophages. Attempts to overcome these problems involve the use, wherever possible, of
aseptic techniques and the introduction of phage-resistant bacterial strains.
Butter production
● There are two main types of butter, sweet cream butter and cultured butter, but only the latter
involve the use of microorganisms.
● Cultured butter is usually prepared from pasteurized cream ripened with bacteria for 24– 48h
prior to churning.
● Lactococcus lactis ssp. Diacetylactis and Leuconostoc citrovorum are often used which produce
acid and flavour compounds, particularly diacetyl.
Yoghurt production
⮚ Yoghurts are traditional sour milk preparations that have become major dairy products.

11. ⮚ There are several variants, but commercial forms are usually prepared from whole milk which
may be supplemented with protein such as skim milk powder. This helps to form the
protein-gel structure of the product.
● These raw materials are heated and then cooled prior to inoculation; the heating is
necessary, otherwise later protein coagulation does not produce a smooth gel.
● Inoculation involves a mixed starter culture containing thermophilic strains of
Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus in a of ratio 1 :
1.
● The former produces mainly acid, whereas the latter generates more organoleptic
compounds, particularly acetaldehyde.
● Their proteolytic enzymes and extracellular polymers also aid protein-gel formation.
● Yoghurt can be pasteurized to improve storage-life or remain ‘live’, the latter reputedly
having probiotic qualities.
Cheese production
⮚ The bulk of world cheese production, which is now over 1010
kg/annum, is from 5x1010
L of
cow’s milk. Cheese making essentially involves concentration of milk fat and protein by
removing water.
⮚ However, there are numerous different types of cheese with widely varying texture and flavour.
Textures range from soft through to very hard, and flavours vary from mild to very strong.
The vast range is due to differences in:
1 the type of milk used;
2 the diet of the milk producer, which in turn is influenced by the local soil, vegetation and climatic
factors;
3 the particular strain of microorganisms used for inoculation at each phase of production, including
starter and ripening stages, which contributes different organoleptic compounds and modifies the
texture;
4 the processing methods employed; and
5 the environmental conditions during ripening, particularly temperature and humidity.
There is no standard method of cheese making, as limitless variations exist for all stages of the process
and a vast quantity is still produced with little or no reference to the underlying science. However, for
most cheeses, the process basically involves:
1 pretreatment of raw milk;
2 formation of solid curd;
3 removal of the liquid whey from the curd;
4 curd processing; and
5 ripening and ageing.
⮚ First, raw milk is checked for various chemical and microbiological quality parameters and then
pasteurized.
⮚ The production of coagulated milk proteins or curd is then achieved by the activities of lactic
acid bacteria, such as Lactococcus lactis, L. cremoris and Streptococcus thermophilus.
⮚ These bacteria have the ability to lower the pH through the fermentation of lactose to lactic acid,
which facilitates protein coagulation. They also influence the flavour of the final product by

12. producing specific flavour and aroma compounds, and perform essential proteolysis and
lipolysis in later maturation.
⮚ A mixed starter culture is often used, consisting of several strains of these mesophilic or
thermophilic streptococci and lactobacilli, which may be prepared in heattreated milk or
whey-based media.
⮚ Use of defined starter cultures reduces batch-to-batch variations in both production time and
levels of acid generated.

13. ⮚ An inoculums of 0.5–2.0% (v/v) is added and the fermentation is performed at around 32°C for
10–75 min. For some cheeses this may be further controlled by heat treatment at 55°C, which
inhibits mesophiles and promotes the of thermophiles.
⮚ Thus, the initial selection of suitable microbial strains, the amount of starter culture used, the
length of preripening and the incubation temperature are important in creating many subtle
differences in the final colour, flavour and aroma.

14. ⮚ Curd formation may be promoted, in all but soft, unripened cheeses such as cottage and cream
cheeses, by the addition of specific proteolytic enzymes.
⮚ Traditionally, rennin (chymosin, aspartic protease EC 3.4.23.4) is used, which is prepared in a
crude form from the stomach, abomasum, of veal calves and is referred to as rennet.
⮚ Due to a shortage of available calf chymosin and the requirement for so-called ‘vegetarian’
cheeses, fungal proteases with similar properties to calf chymosin are now also employed. In
addition, the calf chymosin gene has been introduced into several microorganisms for the
commercial production of recombinant enzyme
⮚ The milk component primarily involved in curd formation is the protein casein, which is a
mixture of α-1, α-2, β and κ caseins. κ casein is important in maintaining the colloidal
stability of milk proteins.
⮚ Addition of rennin results in the removal of surface glycopeptide from the k casein.
Consequently, the casein becomes unstable and aggregates in the presence of calcium ions to
form a gel.
⮚ As the gel forms it entraps fats and ultimately forms white creamy lumps, referred to as curd.
⮚ Precipitated curd is soft but can be readily separated from the liquid whey by holding the
mixture in cheese cloth.
⮚ Semi-dried curd that remains in the cheese cloth is usually salted and other ingredients may be
added, such as colouring agents, herbs, or a further microbial inoculum. It is then pressed and
placed into a shaping mould or cut into blocks.
⮚ For many cheeses this is followed by a period of ripening or ageing to develop the final flavour
and texture.
⮚ Ripening involves the modification of proteins and fats by microbial and milk proteases
and lipases that remain in the young cheese. Some countries allow acceleration of flavour
development through the addition of commercial enzyme preparations.
⮚ Lysozyme may also be added in the manufacture of hard-cooked Emmental, ouda and
Gruyère-type cheeses to prevent growth of the spoilage organism, Clostridium tyrobutyricum,
which otherwise would be troublesome in the later stages of ripening.
⮚ In addition, the curd may have been inoculated with a bacterial or fungal culture before ripening.
For example, Propionibacterium freundenreichii ssp. shermanii is used for the production of
Swisstype cheeses, e.g. Emmental and Gruyère. As these bacteria grow they modify the flavour
and generate gas bubbles that result in holes or eyes within the cheese.
⮚ Internally mould-ripened blue-veined cheeses, including Danish Blue, Gorgonzola, Roquefort
and Stilton, primarily use Penicillium roqueforti.

15. ⮚ Traditional manufacture relies on the natural development of the mould that originates from
spore populations that become established in the local cheese-making environment.
⮚ Large-scale production now uses spore inocula, which are mixed into the curd before the
cheeses are pressed and formed. Young cheeses are usually punctured with stainless steel rods to
promote fungal growth by increasing oxygen levels within the cheese and then stored under
controlled humidity at around 9°C.
⮚ Cheeses are ripened for up to a year during which they develop the characteristic blue veins, and
the aroma and flavour that is due to methyl ketones such as 2- heptanone.
⮚ Camembert-type and other surface-ripened cheeses use Penicillium camemberti, which
originates naturally from the environment, or the surface of the cheese may be sprayed with a
spore inoculum.
⮚ The mould grows on the surface of the cheese for 1–6 months to produce the characteristic white
crust or rind. At the same time, its hydrolytic enzymes are secreted into the cheese where they
modify the flavour and texture.
DEVELOPMENTS IN CHEESE MAKING

16. ⮚ Bacteriophage infections of starter cultures have been a major problem in cheese and other dairy
fermentations. The use of phage inhibitory media, defined strains, the rotation of strains and
phage-insensitive bacterial strains has improved the situation.
⮚ However, phage infections continue to cause problems within the industry. Attempts are being
made to harness the natural defences of the bacteria, such as inhibition of bacteriophage
adsorption to the bacterium and blocking of phage nucleic acid penetration.
⮚ The genes for these phage resistance factors appear to be plasmid encoded and resistance
plasmids may be exploited to improve the ability of industrial strains to resist phage attack.
⮚ In lactic acid bacteria, the genes for many of the key enzymes involved in lactose and casein
metabolism are not chromosomal, but plasmid-borne and prone to being lost. The mapping of
the plasmids and subsequent isolation of these genes has allowed them to be transferred to the
chromosome, providing strains with greater stability.
⮚ Such techniques may also lead to the introduction of genes from other lactic acid bacteria and
heterologous genes. These may provide additional properties, especially the ability to produce
extra organoleptic compounds, remove off-flavours, speed ripening and destroy microbial
contaminants.
⮚ Alternate methods of introducing additional enzymes, and antimicrobial agents to prevent
spoilage include the use of encapsulation technology. The enzyme or agent can be contained
within liposomes (microscopic phospholipid spheres) and added, probably most conveniently
during curd processing.
BREAD
⮚ As ancient societies became more sophisticated, their cereal food progressed from porridges and
gruels to unleavened flatbreads and, finally, to breads leavened with yeast. This occurred as
early as 2700 B.C.E., as evidenced by archeological remains of Egyptian baking ovens
⮚ In those early times, bakeries were often attached to breweries so that the yeast by-product of
brewing (Saccharomyces cerevisiae) could be used in bread making. Today, specialized strains
of yeast are used for each purpose.
⮚ Use of yeast to leaven cereal products dates back over 4000 years. Their primary function is to
generate carbon dioxide, which can be controlled by the quantity of yeast added, level of
fermentable sugars present and the temperature.
⮚ The rising of bread relies on the fact that wheat and several related cereal grains contain gluten
proteins. Glutens contribute to the final flavor of bread and have the characteristic of forming
long molecular strings when they are ‘kneaded’.
⮚ These bind the bread together and have important elastic properties, allowing the formation of
dough.
⮚ Bread dough traps the carbon dioxide generated by the baker’s yeast (normally specific strains
of S. cerevisiae ) and rises due to the pressure of the carbon dioxide build-up.
⮚ The yeast also add flavours, alcohol and acids. Importantly, it helps mature or chemically
modify the gluten to promote even expansion of the dough and gas retention during baking.

17. ⮚ On baking, the bread protein is denatured and, along with the starch, forms the typical open
crumb texture.
⮚ Bread dough containing more than 20% rye flour must be acidified to produce acceptable bread.
This is due to its lack of gluten, which requires other components to bind the necessary water
(e.g. pentosans).
⮚ They do this much better at lower pH and the acidic conditions also inhibit unwanted amylase
activity. Acidification may be achieved by adding citric acid or by sour-dough fermentation; the
latter also contributes additional flavour.
⮚ The dough may be fermented by natural microflora of the dough ingredients.
⮚ Initially, Gram-negative enteric bacteria are involved, followed by lactic acid bacteria that
reduce the pH to 3.6–3.9.
⮚ Alternatively, a portion of dough from a previous batch, predominantly containing a mixture of
lactic acid bacteria, may be used as an inoculum.
⮚ More recently, single strain commercial starter cultures of heterofermentative lactic acid bacteria
have become available for this purpose.
.
Prodecudre
● The first step in bread making is to mix flour, sugar (as a fermentable carbohydrate), fat (for
texture), salt, and other ingredients.
● The yeast Saccharomyces cerevisiae (baker’s yeast) is added in the form of dried powder, block,
or cream at 1 to 6% on a weight basis per weight of flour.
● The yeast’s main role in bread production is to produce the carbon dioxide that makes the bread
rise.
● However, the yeast also produces amylases that break down starch to the more fermentable
glucose.
● Water is added, and the dough is kneaded so that gluten protein in the flour stretches and the
dough forms a viscoelastic (i.e., both viscous and elastic) mass.
● The bread is fermented or “proofed” at 28 to 32°C for several hours.
● The dough is then remixed (or “punched” in home usage) to evenly distribute the gas cells,
portioned into loaves, and fermented again so that the loaves double in volume.
● During the baking at around 200°C for 30 min, carbon dioxide expands to increase the bread
volume by 40%.
● The gas is captured by the protein as it “sets” by heat denaturation.
● For sourdough breads, a stable mixture of heterofermentative lactic acid bacteria (LAB) is also
added, at levels of 107 to 109 colony-forming units (CFU)/g.
● The Lactobacillus species, primarily Lactobacillus sanfranciscensis, add a characteristic “bite”
to the bread, improve texture, and prevent spoilage.
● When sourdough breads are made by artisans, the inoculum is usually made using some of the
last batch.
● The sequential transfer of the cultures can propagate the starter culture for decades.
SOYA BEAN FERMENTATIONS
⮚ Fermented soya bean products are a major part of the diet in south-east Asia, where they are
largely substitutes for fermented dairy products. Some of these foods are also becoming popular
in the west.
⮚ There are several different solid, paste and liquid products such as tempeh, miso and soy sauces,
respectively.

18. ⮚ Tempeh production involves fermentation of cooked whole or dehulled soya beans by Rhizopus
species, R. oigosporus often being the preferred organism in commercial processes. The
resulting cake-like product can be cut into cubes and fried or cooked with other ingredients.
⮚ Soy sauces are used as condiments, or colouring and flavouring agents.
⮚ There are many different types, but their manufacture involves three basic stages. The first stage,
referred to as koji, is a solid substrate aerobic fermentation of cooked soya beans or steamed
defatted soya flakes, along with wheat flour or rice. Normally, a mixture of strains of Aspergillus
oryzae is used and at 25–30°C the hydrolysis of constituent starch, protein and pectins is
accomplished in 2–3 days.
⮚ Moromi is the second stage and involves an anaerobic fermentation of the liquid slurry that
results from the addition of brine 24% (w/v) sodium chloride solution) to the koji.
⮚ During this phase, the activities of Pediococcus halophilus initially acidify and prevent spoilage.
Subsequently, Candida species or Zygosaccharomyces rouxii perform an alcoholic fermentation
and produce additional flavour compounds such as furanones.
⮚ The product is traditionally matured for 6–9 months to develop the full flavour before the liquid
is filtered, pasteurized and finally bottled.
COFFEE, COCOAAND TEA FERMENTATIONS
COFFEE
⮚ Berries of the coffee plant (Coffea arabica and Coffea canephora) are processed to obtain the
bean from within by either a dry or wet process.
⮚ The wet process, which generally produces better quality beans, involves the activities of
indigenous bacteria, filamentous fungi and yeasts.
⮚ The pulpy mucilaginous material that surrounds the beans is removed by the action of
endogenous plant enzymes, pectinolytic bacteria and fungi.
⮚ This is followed by an acid fermentation by lactic acid bacteria, primarily Leuconostoc
mesenteroides and Lactobacillus brevis.
⮚ The residual pulp is washed from the beans, which are then dried, hulled and roasted.
COCOA
⮚ Cocoa production involves similar microbial activities, but these are rather more important than
for coffee.
⮚ The pods of Theobroma cacao, which contain the beans, are opened and the mucilaginous
material is removed by fermentation.
⮚ Initially there is an alcoholic fermentation performed by a mixture of yeasts, notably Candida,
Hansenula, Pichia and Saccharomyces.
⮚ This is followed by an increase in the activity of lactic acid bacteria, particularly Lactobacillus
plantarum and Lactobacillus fermentum.
⮚ Finally, acetic acid bacteria from the genera Acetobacter and Gluconobacter predominate and
oxidize the ethanol to acetic acid.
TEA
⮚ Tea is prepared from the leaves of Camellia sinensis.
⮚ Although their preparation is referred to as a fermentation, production of fermented black tea
and partially fermented teas does not involve microorganisms.
⮚ Endogenous plant enzyme activities are responsible for the changes within the leaves following
loss of moisture, withering and leaf maceration.
IDLI

19. ⮚ Idli is a popular fermented breakfast and hospital food which has been eaten in South India for
many years.
⮚ It is prepared from rice grains and the seeds of the leguminous mung grain, Phaeseolus mungo,
or from black beans, Vigna mungo, which are also known as dahl.
⮚ When the material contains Bengal grain, Circer orientium, the product is known as khaman.
⮚ It has a spongy texture and a pleasant sour taste due to the lactic acid in the food. It is often
embellished with flavoring ingredients such as cashew nuts, pepper and ginger.
Production of Idli
⮚ The seeds of the dahl (black gram) are soaked in water for 1-3 hours to soften them and to
facilitate decortication, after which the seeds are mixed and pounded with rice in a proportion of
three parts of the beans and one of rice.
⮚ The mixture is allowed to ferment overnight (20-22 hours). In the traditional system the
fermentation is spontaneous and the mixture is leavened up to approximately 2 or 3 times.
⮚ The organisms involved in the acidification have been identified as Streptococcus faecalis, and
Pediococcus spp.
⮚ The leavening is brought about by Leuconostoc mesenteroides, although the yeasts, Torulopsis
candida and Trichosporon pulluloma have also been found in traditional Idli. The fermented
batter is steamed and served hot.
⮚ Idli is highly nutritious, being rich in nicotinic acid, thiamine, riboflavin, and methionine.