The document discusses the microbiology of fermented foods like yogurt. It begins by describing the composition of milk and how heating milk and adding lactic acid bacteria cultures like Lactobacillus bulgaricus and Streptococcus thermophilus causes the milk proteins and sugars to ferment, producing yogurt. These bacteria grow symbiotically, with one species creating an environment for the other to thrive. The fermentation process turns milk sugar into lactic acid, causing the milk to thicken into a yogurt consistency. Precise temperature and time controls are needed during incubation to ensure the proper growth of bacteria and flavor development.

1. Microbiology Of Fermented Food
Production

2. Milk is
•Water
•Protein (casein and whey)
•Fat
•Sugar (lactose)
•Vitamins
• Minerals

3. Lactose
Lactic acid Bacteria
------------------------->
Acid causes casein (milk
protein) to denature and
hold water into a
semi-solid gel = yogurt
(Milk sugar)
How Does Milk Turn Into Yogurt?
Lactic Acid

4. Milk Yogurt
Bacteria produce acid
Casein protein micelles (bundles)
10-7 meters in diameter
Fat
globule
Acid causes Casein
bundles to fall apart
into separate casein
molecules.
These rebind to each
other in a network that
traps water.
=> makes a gel

5. Yogurt
Yogurt-like products have been made for millenia across Eastern Europe,
North Africa, Central Asia and India.
Contains bacteria that are “thermophilic” = heat loving

7. Terms
• Aerobic respiration
• Anaerobic respiration
• End product
• Fermentation
• Food fermentation
• Lactic acid
• Lactose
• Viscosity
• Yogurt

8. What is the composition of yogurt?
• Yogurt, a fermented dairy product whereby milk is inoculated
with bacteria cultures, is an example of a mixed pure culture
fermentation. That is, a controlled mixture of known cultures of
bacteria used in the fermentation process.
• A. Yogurt is the product of fermentation, a slow decomposition
process of organic substances induced by microorganisms or
enzymes. Food fermentation is the study of microbial activity,
usually anaerobic, on suitable substrates under controlled or
uncontrolled conditions. To produce yogurt, lactose, a compound
sugar found in milk known as lactin or milk sugar, is fermented by
two different species of bacteria: Lactobacillus and Streptococcus.

9. • B. Yogurt is commercially produced by adding to milk a 2–4%
nonfat dry milk powder that has been inoculated with a 5%
combination of Lactobacillus bulgaricus and Streptococcus
thermophilus (1:1 ratio). The milk mixture is then incubated at
45 degrees C for 3 to 6 hours. The product must be chilled
immediately.
• C. The fat content of yogurt varies from 0-3.5%; most yogurt is
low fat and contains 1–1.5% fat.
• D. Lactic acid, the end product of anaerobic metabolism of
glucose, provides the tart flavor of yogurt, as well as the
formation of a gel structure. The major flavor components of
yogurt are carbonyl compounds; among these, acetaldehyde is
the most important and gives the yogurt its green apple or
nutty flavor. Yogurt quality is based on color, appearance, body,
texture, and flavor.

10. • E. Ropy (slime-producing) lactic acid bacteria produce
polysaccharides that are released into the yogurt where they
increase viscosity and improve water retention. Viscosity is the
resistance of a fluid to flow. Yogurt has a high moisture content
of 82–86%.
• F. Milk SNF (solids-not-fat) content of yogurt varies from 9–
16%. SNF can be increased by adding milk powder, and by
other means. Increased SNF levels are needed to increase
protein content, which helps to increase product viscosity to
desired levels.
• G. Frozen yogurt is manufactured by mixing varied amounts of
fermented yogurt with ice milk containing sweeteners,
stabilizers, etc.

11. How are fermentation and anaerobic respiration needed to create
the yogurt product?
• A. Aerobic respiration is the total oxidative degradation of
glucose that must have oxygen to take place. Fermentation is
an example of anaerobic respiration, which takes place in the
absence of oxygen and consists essentially of the early stages
of aerobic respiration. During this type of respiration, glucose
is converted to a variety of end products, such as lactic acid.
• Since acids produce a sour taste, the result is a sour-tasting
dairy product. The increased acidity (lowered pH) of the milk
causes milk protein to coagulate and become more viscous.
The bacteria Streptococcus also produces other compounds
that have an effect on final flavor.

12. • B. Lactic acid bacteria are gram-positive, non-spore forming
bacteria that produce lactic acid as the major product of
fermentation. These bacteria are very important in pickling,
cheese making, fermented dairy products, and other
technologies.
• C. Milk proteins have the unique ability to curdle (form a gel).
Curdling is induced by proteolytic enzymes, lactic acid, heat,
and other means. Each milk gel has a solid structure consisting
of a protein matrix and other components. This gel matrix has
the ability to immobilize the liquid phase of milk. By modifying
this ability, it is possible to manufacture stable milk products
with a high water content (i.e. yogurt).

13. • D. Milk to be used in yogurt production is usually heated to
increase the total solids content in order to make a firm end
product. An end product is the final compound or substance
resulting from a chemical reaction. Heating causes the micelles
in the milk to interconnect in chains to form the gel matrix.
• E. Cooling of the yogurt after incubation (fermentation) stops
bacterial fermentation.
• F. If cows are receiving antibiotic treatments, their milk is
usually discarded for a specific period of time. Otherwise,
residual effects from the antibiotics in the milk could inhibit
the growth of desirable bacteria necessary for yogurt
production.

14. Making Yogurt in 4 Simple Steps
1. Start with Cow, Sheep, or Goat milk.
Casein before heat
pre-treatment:
Casein after heat pre-
treatment: Casein after acid:
2. Heat milk to 80 °C. Two purposes:
• destroy existing bacteria
• “condition” the proteins = begins the denaturing process
(a whey protein molecule binds to a casein molecule which disrupts the casein bundles
allowing them to make short branched micelle chains)
3. Cool milk to 40 °C and innoculate with bacteria
4. Incubate at 30 °C to 45 °C

19. Commercial Yogurt
Contains 2 species of bacteria specialized to grow well in milk (but can’t survive inside the
human body):
First,
Streptococcus
thermophilus is
more active, then
slows down when
acidity reaches
0.5%
Next, Lactobacillus
bulgaricus is more
acid tolerant and
takes over until acidity
>1%
These bacteria work in symbiosis. Each bacterium stimulates the growth of the other =>
acidifies the milk more rapidly than either partner on its own.

21. Fermentation Involves exposing the raw or starting
food materials to conditions that favor growth and
metabolism of specific and desirable microorganism.
As the desirable microorganisms grow, they utilize
some nutrients and produce some end products.
These end products, along with the unmetabolized
components of the starting materials, constitute the
fermented foods having desirable acceptance
qualities, many of which are due to the metabolic end
products.

22. Raw Or Starting Materials
A large number of raw materials from plant and animal sources are used to produce
fermented foods. These include:
Milk
Meat
Fish
Eggs
Vegetables
Fruits
Cereal grains
Lentils
Beans
Seeds

23. Microorganisms Used
Many species and strains of bacteria, yeasts and molds
are associated with fermentation of foods.
Depending on a product, fermentation may be
achieved by a single species and strain.
In most fermentations , a mixed population of several
bacterial species and strains , or even bacteria and
yeasts or bacteria and molds is involved.

24. When a fermentation process involves a
mixed population, the members should not
be antagonistic toward one another, rather,
they should preferably be synergistic.
Maximum growth of a desirable
microorganism and optimum fermentation
rate are dependent on environmental
parameters such as nutrients, temperature
of incubation, oxidation-reduction
potential and pH.

25. Fermentation Process
1- Natural Fermentation
Many raw materials used in fermentation contain both
desirable and associated microorganisms.
The conditions of incubation are set to favor rapid
growth of the desirable types and no or slow growth of
the associated types(many are undesirable).
A product produced by natural fermentation can have
some desirable aroma resulting from the metabolism of
the associated flora.

26. Because the natural microbial flora in the
raw materials may not always be the same,
It is difficult to produce a product with the
same characteristics over a long period of
time.
There is a chance of product failure
because of the growth of undesirable flora
and foodborne diseases by the
pathogens.

27. 3- Controlled Fermentation
The starting materials which may be heat-treated are
inoculated with a high population(one million cells/ml)
of a pure culture of single or mixed strains or species of
microorganism(starter culture).
Incubation conditions are set for the optimum growth
of the starter culture.
There is less chance of product failure and foodborne
diseases.
There may be no growth of desirable secondary flora,
as a result, a product may not have some delicate flavor
characteristics.

28. Fermented Dairy Products
Fermented dairy products can be broadly divided into two groups;
Fermented milk products
Cheeses
In fermented milk products, all milk constituents are retained in the
final products.
In cheeses, a large portion of milk constituents is removed in whey to
obtain the final product.

29. Milk Composition and Quality
The growth of desirable microorganisms and the quality of a fermented
dairy product are influenced by the composition and quality of milk
used in a fermentation process.
Cows milk contains approximately 3.2% protein, 4.8% lactose, 3.9%
lipids, 0.9% minerals, traces of vitamins and 87.2% water.
Casein is present as calcium caseinate at higher concentration than
albumin and globulin.
Lactose is the main carbohydrate, lipids are dispersed as globules of
different sizes in emulsion.

30. Minerals are present in solution and as colloid with casein.
Total solids: 12.8%.
The whey contains the water soluble components, some fat and water.
Milk contains natural antimicrobials; agglutinins and the lactoperoxidase
isothiocynate system.
The agglutinins can induce clumping of starter culture cells and slow their growth
and metabolism.
The lactoperoxidase-isothiocynate system can inhibit starter culture.
Antimicrobials can cause problems only when raw milk is used, because both are
destroyed by heating milk.

31. Milk can also contain antibiotics, either used in the feed
or used to treat animals for some infections such as
mastitis. Their presence can also affect the growth of
starter culture.
Some milk can contain heat stable proteases and
lipases produced by some psychrotrophic bacteria such
as Pseudomonas species during refrigerated storage of
raw milk before pasteurization. These enzymes remain
stable after heating and can cause product defect(low
yield of cheese, proteolysis, rancidity).
Before milk is used for fermentation, these aspects
should need to be considered.

32. Fermented Milk Products
Yogurt is made with Streptococcus thermophilus
and Lactobacillus delbrueckii subsp. Bulgaricus.
Buttermilk is made with lactobacillus spp. Without or
with Leuconostoc cremoris.
Acidophilus milk is made from Lactobacillus
acidophilus.

33. Microbiology Of Yogurt Fermentation
Plain yogurt has a semisolid mass due to coagulation of
milk(skim, low, or full fat) by starter culture bacteria.
It has a sharp acid taste with falvor similar to walnuts
and a smooth mouth feel.
The flavor is due to combined effect of acetaldehyde,
lactate,diacetyl and acetate.
About 90% of flavor is due to acetaldehyde.
Many types of yogurt are available in the market
e.g. plain Y., fruit Y., flavored and colored Y. and
sweetened Y.

34. Processing
Batch process for a low fat2%) plain yogurt
Homogenized milk (12% TS) plus stabilizer (1%), the stabilizer is added to give
desired gel structure.
Heating to 85C for 30 min, and cooled to 43.3C, heating helps to destroy vegetative
microbes and slightly destabilize casein for good gel formation.
Starter is added , incubated at 43.3C and pH 4.8 for 6h.
Quickly cooled to 29.4C for 30.min. to slow down further starter growth and acid
production especially by Lactobacillus spp., agitated and pumped to filler machine.
Packaged in containers, cooled by forced air to 4.4C to stop the growth of starter.
Held for 24h, pH drops to 4.3.

35. Starters
In a good product, the two starter species should be added at a Strep:Lacto ratio of
1:1, in the final product, the ratio should not exceed 3:2.
For balanced growth of the two species, the fermentation is conducted at 43.3C, at
this temperature both acid and flavor compounds are produced at the desired
level.
If the temperature is raised above 43.3C lactobacillus spp. Predominates, causing
more acid and less flavor production.
At temperture below 43.3C growth of Streptococcus spp. Is favored, forming a
product containing less acid and more flavor.

36. The two species show symbiotic growth while growing together
in milk.
Initially Streptococcus spp. Grows rapidly in the presence of
oxygen and produces formic acid and CO2.
The anaerobic condition , formic acid and CO2 stimulate growth
of lactobacillus spp. Which has good exoproteinase and
peptidase systems and produce amino acids and peptides from
milk proteins.

37. Some of the amino acids such as glycine, valine,
histidine, leucine and methionine are necessary for
good growth of Streptococcus spp. Which lacks
proteinase enzymes.
Streptococcus spp. Grows rapidly until pH drops to 5.5
at which time the growth of Streptococcus spp. slows
down.
However, growth of Lactobacillus spp. Continues fairly
rapidly until the temperature is reduced to 29.4C
following a drop in pH to 4.8.

40. Biochemistry
Lactose Metabolism
Both species have a constitutive β- galactosidase
system and lactose is hydrolyzed to glucose and
galactose.
Both species are homofermentative and produce
lactate from glucose by glycolysis.
Both species do not metabolize galactose, as a result,
galactose is excreted outside causing its accumulation
in yogurt.

41. Flavor Production
The major flavor in yogurt is acetaldehyde with some
diacetyl and acetate.
Acetaldehyde is produced from glucose via pyruvate by
Streptococcus spp. And from threonine by Lactobacillus
spp.
Formate Production
Formate(necessary for Lactobacillus growth) is produced
by Streptococcus thermophilus from pyruvate by the
action of formate lyase.
Pyruvate--------------Formate+ Acetate

42. Slime Formation(Glycan).
Β-galactosidase in some strains of streptococcus
thermophilus polymerizes glucose to produce
oligosaccharides and glycan, which may give a viscious
texture to yogurt.
Milk proteins in the presence of proteinases and
Lactobacillus yields peptides.
Excess accumulation of peptides , some of which cause
bitter flavor.
Peptides by the action of peptidases produces amino
acids necessary to Streptococcus, also threonine can be
used to produce flavor by Lactobacillus.

43. Microbial Problems
In plain yogurt, flavor problems can be
associated with the concentration of
acetaldehyde. A low concentration gives a
chalky and sour flavor. Too much
acetaldehyde gives a green flavor.
Too much diacetyl gives a buttery aroma.
Too much acid production during storage
are associated with bitter flavor.

44. Production of exopolysaccharides by
the starter can give a viscious and ropy
texture.
Growth of yeast during storage can also
produce fruity flavor especially in yogurt
containing fruits and nuts.
During long storage, molds can grow on
the surface.

46. Incubation Temperature
• 40-45 °C takes 2-3 hours
Produces a coarse protein network with thick strands give firmness but easily leak
whey (a process called syneresis - the separation of liquid from the gel)
• 30 °C takes 18 hours
Produces a finer more branched delicate network that holds the liquid whey

48. Mould spoilage in yoghurt
•Mould genera that can grow on yoghurt surface:
• Penicillium, Aspergillus, Mucor, Rhizopus, Alternaria, Monilia and
Absidia (Sørhaug, 2011)
•Moulds grow more slowly than yeast contaminants in yoghurts
•Fruit purees added to yoghurt – usually the main source of moulds
and yeasts
• - heat-resistant moulds often do not grow well at low temperatures
• - some genera (e.g., Mucor spp.) grow well at refrigeration
temperature
• Talaromyces spp. (relatively heat-resistant) may be present in fruit-
flavoured yoghurt (Sørhaug, 2011)

49. Mould growth in yoghurt

50. Penicillium spp. in yoghurt