Department of microbiology microbial food technology group a diploma in quality assurance in microbiology diploma

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  • Kefir is the self-carbonated fermented milk product with high nutritional status and therapeutic value.

  • It requires a special culture called kefir grains.

  • The grain consists of casein and gelatinous colonies of microorganisms, which live, in symbiosis.

  • The organism isolated is yeast such as Torula and Saccharomyces kefir and bacteria such as L.acidophilus, Streptococcus lactis and L.kefiranofaciens.

  • The yeast represents 5 – 10 % of the total micro flora.

  • The grains are irregular in shape, yellowish in color and insoluble in water.

  • Dried grains retain their activity for more than a year when stepped in milk the grains swell.

  • During fermentation process Lactobacillus sp. produces lactic acid and lacto fermenting yeast cell produce alcohol and CO2. All activities are controlled by incubation temperature.


  • Starter organisms produce risin, lactimine, streptocine is widely used in hospitals.

  • It is included in diets of patients suffering from intestinal diseases, anemia, metabolic disorders, hyper toxicity, and allergic diseases.

  • It is beneficial for the treatment of tuberculosis.

  • The product in diet reduces serum cholesterol level in infants.



  1. It is highly nutritious and suited as a supplement to local foods.

  2. Fermentation predigests several milk constituents, synthesizes water soluble vitamin of B complex and makes a nutritionally upgraded milk.

It is a fermented product made by using mesophilic starters.


Milk free from antibiotics & detergents with fat content of 0.5 – 1 % is homogenized at 150c

Heated at 900c – 13 mins & cooled to 230c.

Starter culture [Str. lactis, Str. cremoris – acid production, Str. diacetylactis, Leuconostoc citrovorum – aroma & flavor 1-2%].

Fermentation time is 16 – 20 hrs, acidity – 0.9%, mixed, cooled, bottled & stored at 50c.

Final product is viscous, drink with pleasing aroma, flavour.


  • Prepared from mare milk, which is inoculated with starter culture of 10 – 20%.

  • Cow milk or skimmed milk with 2.5% sucrose is used due to non-availability of mare milk.

  • Microflora includes Lactobacillus delbrueckeii ssp bulgaricus, L. acidophilus, Kluveromyces lactis.


  • Kumiss from mare milk is a good supplementary remedy for treatment of TB.



Butter is a concentrate of one of the 3 main constituents of milk ie., fat, proteins, lactose. The later 2 are present only in small proportion. Butterfat also contains the yellow coloring matter carotine and or its transformation products vitamin A and D.


Butter and moisture  16%

Milk fat  80%

Milk solids  2%

Butter making process:

1.Preparation of cream, pasteurization, cooling and starter addition:

Cream is produced by mechanical separation of unhomogenized whole milk. Cream is pasteurized between 88 – 930 c. It may be subjected to vacuum cooling to ripening temperature of 16-210 c and ripened with 4% of mixed starter culture having;

1.acid producers like Streptococci lactis /S.cremoris.

2.flavor producers like L.mesenteroides, S.diacetylactics.

The ripening may be in 2/3 stages to produce soft, firm butter.

2. Churning, washing and salting:

The cream is loaded for churning in machines. The machine has 3 sections;



3.working sections

The churning section consists of a horizontal cylinder and a rotating variable speed rotator/beater [0-1000 rpm] since churning lasts for 1-2 sec it is important to adjust the beater velocity to obtain optimum butter grain size.

The separating section consists of a horizontal cylinder. The first part of the cylinder is equipped with beaters for further treatment mixtures of butter grains and butter milk which is fed from the churning sections.

The second part of the cylinder is designed as a sieve for draining buttermilk. It is equipped with wire gauze, which retains even small butter grains.

The working section consists of inclined sections for transport of the butter. In the production of salted butter, a salt slurry [40-60%] is pumped into the first working section, in which it is worked into the butter before butter proceeds to the second working section. Any adjustment of butter moisture also takes place in the first working section. Water dosing is done automatically.

Quality of wash water:

The chill water used for washing butter granules is an important source of contamination of butter. The treatment of butter with wash water has 2 purposes:

  1. To wash away the free butter milk from the butter granules.

  2. To control the temperature of the granules for subsequent working process. The following organisms are known to infect butter through wash water.[P.putrifaciens, P.fluorescens, P.fragi, P.methicica]


Butter is packed either in bulk or in consumer’s size containers. Normally vegetable parchment is used to line butter boxes and also a wrapper for consumer packs. Polyethylene films replace parchment paper. Giving sodium propionate treatment can control mold growth.

Flavor of butter:

The flavor of butter is produced by the fermentation of citric acid by Leuconostoc and Streptococcus lactis. Citric acid is converted into pyruvate, co2 , acetic acid. Pyruvate is again metabolized to form CO2 and acetaldehyde. Acetaldehyde under neutral and acidic conditions forms acetic acid and ethanol. Under acidic conditions these products are further metabolized into diacetyl and acetyl methyl carbinol.


Cream separation [unhomogenised whole milk] was pasteurized at 88 – 930c, cooled at 16-210c.

Starter culture was added [Strep. lactis, Strep. cremoris – acid producers, Leuconostoc mesenteroides,

Strep. diacetylactis – flavor producers].

Churning [adding colour], Draining butter milk, Washing.

Adding salt [40-60%], Working [salt enters butter], Washing

Packing & storage at 50c.

Spoilage and defects:

Many of the defects of butter originate in the cream, from which it is made especially if the cream is held for several days. During this time lactic acid bacteria and other spoilage organisms may grow which may be followed by the growth of the molds, Geotrichum candidum.

Flavor defects:

The main defects developing in butter during storage are;

  1. Oxidative rancidity 2. Hydrolytic rancidity 3. Putrefactive taints

Growth of microorganisms in cream and in the milk from which it is separated may result in any of the following bad flavors.


Organism involved


Barny flavor
Surface taint/rabbito/putridity
Ester like flavor

Taste like cultured buttermilk due to souring of cream.
Resulting from lipolytic bacteria and mold.
Flavor similar to bakers yeast results from growth of yeast in cream or butter.

Produced by molds and Actinomycetes.

Lacking typical flavor Pseudomonas sp.
Produced by Streptococcus lactis
Intense old cream flavor caused by coliforms
P. fragi
Aeromonas hydrophila
Suggestive of metal caused by metal catalyzed oxidation.
Aromatic flavor[feeds eaten by cows]

Colour defects:

  1. Dark smoky discolouration Alternaria, Cladosporium

  2. green colouration  Penicillium

  3. Brown colouration  Alternaria

  4. Orange/yellow spots  Geotrichum

  5. Dry reddish pink area  Fusarium culmorum

  6. Pink colonies  yeast

Chemical defects:

1.Rancidity  lipase in cream

2.Tallowiness  oxidation of unsaturated fats catalyzed by copper and bacterial enzymes and favored by low pH, T0 , salt,air, ozone.

3.Fishiness  Trimethylamine is produced from lecithin.



Cheese making is a convenient way of converting fat & protein present in milk into a nutritious

product with good keeping qualities. Microorganisms play an important role in this process to provide texture & flavor to the product. It is one of earliest method of preserving milk solids. Cheese is a compressed fermented milk product.


Cheese can be classified into several types based on several criteria;

  1. Based on the firmness of cheese. [Moisture]

  2. Source of milk. [Cow, buffalo]

  3. Ripening. [Fungi/bacteria]

  4. Country of origin. [eg: cheddar  English, Roquefort  Southeast France]

  5. Content of fat. [Skim milk, full cream milk]

  6. Manufacturing process.

The basic procedure of manufacturing is same for all types of cheese. There are 4 major steps in the

Production of cheese.

  1. Control of the properties of milk.

  2. Coagulation

  3. Separation of whey & curd

  4. Cheese ripening

I. Control of the properties of milk:-

Good quality milk is more important for cheese making because it is not possible to pasteurize

Cheese milk intensively. The bacterial content of milk used for cheese making should be low because microorganisms growing in raw milk may develop unwanted flavour & enzyme. Some organisms survive pasteurization & cause fault in cheese. The number of psychrotrophs & thermoduric organism should be low. The physical, chemical & biological properties of the milk should be controlled.

Basic stages involved in cheese making:-


Standardization of milk is done to adjust for fat or to have a balance rate of fat & casein (1: 0.7).


The clarifier is an effective alternative for filtration for the removal of extraneous matter, leucocytes & Some bacteria. It is carried out at 32- 380 c Centrifugally.

  • Bactofugation:-

If centrifuged milk is passed through the unit the 2nd time about 90% of the remaining 10% bacteria

is removed. The sludge can be sterilized & reincorporated into the milk.

  • Homogenization:-

Milk is homogenized at low pressure the purpose is to reduce the whey exudation from the coagulum

to make cheese whiter & make promote fat hydrolysis.

  • Thermization:-

When raw milk must be stored for a few days using for cheese it is subjected to heat treatment 630 c

for 10-15 sec & cool to 50c prior to storing.

II. Coagulation:-

It is carried out by the use of any of the following methods:-

  • Use of lactic acid.

  • Addition of bacteria like lactobacillus sp. Or addition of milk clotting enzyme rennet.

  • Application of heat.

  • By the addition of salt.

  • Alteration of pH.

Among these only a few methods are applicable. The commonly used method is by the adjustment

of pH.

Coagulation by pH adjustment or Ripening of milk:-

This is achieved by heating the milk at around 30-33 0 C & adding the starter bacteria. The organisms grow using lactose as an energy source & converting it into lactic acid by a complex series of reaction by involving many different enzymes. Selected strains of lactic acid organisms are used which increase the acidity.

Functions of the starter:-

  • Ensures consistent acid development.

  • Aids rennet reaction & subsequent coagulation by the developed acidity.

  • Helps expulsion of whey from the curds.

  • Contributes to flavor & texture of cheese during ripening.

  • Suppresses the growth of undesirable organisms.

Microorganisms used for cheese making are;

Type Function

1. Strep. lactis Acid formation

2. Strep. cremoris Acid formation

3. Strep. diacetylactis Acid, gas & flavor production.

4. Strep. thermohiles Acid production in high scald cheese.

5. L. bulgaricus Acid production.

6. Strep. faecalis Acid & flavor in high scald cheese.

7. Propionibacterium shermanii Gas & flavor production.

Starters are used at concentration ranging from 0.5 to 2% of milk. The organism multiply during cheese making from about 10 7 CFU/ml in milk to around 10 9 cfu/gm of the curd. The growth gets checked at the salting cheese stage. All additives are added & mixed separately before rennet addition. To provide uniform colour to cheese annatto colour [alkaline extract from seeds of Bixa orelana] is added to get yellow tint. Calcium chloride at 0.01 to 0.03% of the milk is used to improve the firmness of the coagulation by rennet. The addition of 15gms of salt per 100kg of cheese milk prevents blowing (development of too much of gas in the cheese) caused by coliform bacteria or butyric acid or propionic acid bacteria.


After a mild increase in acidity of milk created by starter rennet extract is added to milk & uniformly distributed to effect coagulation of milk. The coagulation enzymes are,

Type Source of Enzymes

* Animal Calf (Chymosin or pepsin )

Pig (Pepsin)

* Bacteria B. Subtilis

B. Polymyra

B. mesenteroides

* Fungi Mucor meihei

M. pusilus

Endothica parasitica.

The enzymes act in 3 phases;

1. Primary / Enzymatic phase:-

It results in the conversion of one of the milk protein from a colloidal suspension to a fibrous network. This is done in the presence of calcium.

  1. Secondary/Clotting phase:-

The coagulation of the other function of enzymatic activity & coagulation can be achieved by an increase

in temperature or decrease in pH.

  1. Tertiary/ Proteolytic phase:-

Chymosin hydrolyze the milk protein to polypeptides. A part of polypeptides are broken down to

peptides & amino acids.

III. Separation of curd & whey:-

Separation can be done by mechanical means. Whey separation depends on temperature, pH & physical characteristics of the curd. Increased temperature enhances whey separation. Whey separating is carried out by the following methods:-

  • By cutting the curd & allowing the whey to flow.

  • By placing the curd in perforated containers & allowing the whey to drain through the perforations.

  • The curd can also be collected on a clean cloth & whey can be filtered out.

For cutting the curd, special knives are used for different sizes of cubes.


High scald cheese the temperature may be 52-58 0 C, in medium scald 30-42 0 C & in low scald around 30-35 0 C. During combined action of stirring & heat, lactic acid with the curd particle is formed by the starter organisms, embedded in cheese particles & curd cubes shrinks in size. When the desired development in the curd has reached whey is drained for texturing the curd

Draining the whey:-

The curd is allowed to settle, acidity measured, when it has reached desired level, the whey is run off until the compact mass of curd is formed in the vat.


It helps in uniform distribution of salt (1-2%) salt acts as a preservative & flavor enhancer.


The curd is filled in moulds & pressed. The degree of pressing & length of time various with the type of cheese.

Packaging & storing:-

Packaging protects  flavor contamination

  • Entry to external molecules.

  • Loss of moisture.

  • Enhances appearance.

  • Wax coating /plastic film for hard cheese.

  • Aluminium / plastic film for semi-hard cheese.

  • Maturing period is 2-24 months. The cheese is stored in increased T 0 in fermentation room & shifted to ripening room having lower temperature for the development of proteolysis, lipolysis, aroma & texture.

IV . Cheese ripening:-

It refers to the changes in the body to texture accompanied by the development of characteristic flavor typical to that of cheese. Flavor & aroma is produced by the action of microorganisms & enzymes which breakdown,

  • Carbohydrate producing lactic acid, acetic acid, Co2 & diacetyl.

  • H2O insoluble proteins to protease, peptons, peptides, amino acids, organic acids, NH3.

  • Fat to lower fatty acids, their esters & Ketones.

These changes are brought about by enzymes from,

-> Lactic acid bacteria in starter culture.

-> Miscellaneous non-starter bacteria in milk.

->Rennet & its substitutions used to coagulate the milk.

->Other microorganisms growing within or surface of cheese.

The cheesy flavor is mainly due to carboxyl, nitrogenous compound, fatty acids, sulphur compounds etc.

Manufacture of Cheddar cheese.

Raw milk

Pasteurize at 71 – 750C – 15 secs [pasteurization reduces the number of spoilage organisms & lactic acid bacteria & kills most pathogens]

Cool & incubate in cheese vat at 300c [add starter culture Lactococcus lactics ssp cremoris 1 – 1.5%]

[lactic acid fermentation develops]

Milk with 0.19 to 0.21 % lactic acid [rennet added].

[curd’s formation]

Cutting of curds.

[ whey released]

Scalding 38 – 400c & stirring [acid production continues without starter culture].

Cheddaring – Squeezing & Stretching the curd [acid production continues without starter growth giving a final lactic acid concentration of 0.6 – 0.8 % - primary metabolism].

Milling and salting [ salting prevents further starter activity, assists in preservation & adds flavor & further release of whey]

Moulding & pressing [further release of whey]


[secondary metabolism- Proteinase enzyme released from starter organism produce aminoacid , indole, sulphur compounds and phenol to enhance flavor. H2O2 and bacteriocin to inhibit pathogen and spoilage organisms]

[moisture content]

Soft cheese Semi-soft cheese Hard cheese Very hard cheese [ Very low

[50-80 %] [39-50%] [34 – 39%] moisture].

Eg: unripened cottage cheese

Ripened  camembert cheese

Salt cured  feta cheese

Eg: Ripened by moulds Roquefort cheese

Ripened by bacteria Brick, Gowda, Limburger

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