pradipjntu.files.wordpress.com · Web viewThe whey proteins constitute about 18 percent of the protein content of milk. Casein, a protein found only in milk, contains all of the essential

INDEX

Chapter Content Page No

1. ABSRACT 5

2. Objectives and Introduction 6

3. Materials and Methods 17

4. Results and Discussions

39

5 Conclusion 44

6 References 46

1

ISOLATION OF MICROORGANISMS WITH LIPOLATIC ACTIVITY FROM DAIRY PRODUCTS

Abstract : Lipolytic bacteria are the heterogenous group of bacteria, which produce

lipases, which catalyze the hydrolysis of fats to fatty acids and glycerol.

Microorganisms, may be involved in the oxidation of fats, auto oxidation is

common. Many of the proteolytic bacteria are also lipolytic. The main source for

isolation of lipolytic microorganisms is butter and other dairy products. The

Microbiological quality of butter depends upon the quality of cream and sanitary

conditions used in the processing. Many psychrotrophic bacteria, molds and

yeasts are lipase producers. The main Lipolytic bacteria are pseudomonas

florescence, alcaligens, staphylococcus, seratia, micrococcus, bacillus,

clostridium, coliforms, enterococcus . The main mold is Geotrichum candidum and

yeast is candida species.

Lipases (lipolytic enzymes) are traditionally added to cows' milk to

produce cheese such as Feta, Romano, Kefalotyri, and Parmesan which are

traditionally made from goats' or sheeps' milk. That's because goats' and sheep's

milk, especially goats' milk, have more natural lipase than cows' milk.

Commercial lipases are commonly extracted from kid goats. Enzymes from the

bacteria showing high lipolytic activity may be commercialized

2

OBJECTIVES AND

INTRODUCTION

3

ISOLATION OF MICROORGANISMS WITH LIPOLYTIC

ACTIVITY

FROM DAIRY PRODUCTS (MILK)

OBJECTIVES: 1. Isolation of lipolytic microorganisms from Dairy products.

2. Screening of the isolates with high lipolytic activity. 3. Identification of the isolates with high lipolytic activity.

INTRODUCTION :

Milk contains about 87 percent water and 13 percent solids. The fat

portion of the milk contains fat soluble vitamins. The solids other than fat include

proteins, carbohydrates, water soluble vitamins, and minerals. These nutrients in

milk help make it nature’s most nearly perfect food.

Milk products contain high quality proteins. The whey proteins constitute

about 18 percent of the protein content of milk. Casein, a protein found only in

milk, contains all of the essential amino acids. It accounts for 82 percent of the

total proteins in milk and is used as a standard for evaluating protein of other

foods. Protein is needed to build and repair body tissues and to form antibodies

which circulate in the blood and help fight infection.

Milk contains the following nutrients: calcium, phosphorus, magnesium,

and potassium. The calcium found in milk is readily absorbed by the body.

Phosphorus plays a role in calcium absorption and utilization. Phosphorus is

needed in the proper ratio to calcium to form bone.

Milk and milk products like cheese, yogurt, and frozen dairy desserts are

the main source of calcium contributing about three-quarters of the calcium in the

food supply. Milk provides these two minerals in approximately the same ratio as

found in bone. Milk is also a significant source of riboflavin (vitamin B2) which

helps promote healthy skin and eyes, as well as vitamins A and D,B-12, and also

of riboflavin, calcium, phosphorus, magnesium, potassium, and zinc.Calcium is

important from a public-health perspective, because current calcium intakes by

many consumers are not sufficient for them to attain optimal peak bone mass and

4

to prevent age-related loss of bone, leading to osteoporosis. Bone mass peaks

around age 30, usually remains stable in the 30's, and commonly begins a decline

in the 40's that accelerates around age 50. Recent research also indicates that

adequate calcium intake is one key to achieving optimal blood pressure.

Milk is one of the widely consumed nutrient food and also it is an

excellent Culture medium for the growth and reproduction of micro

organisms. Microbial growth can be controlled by cooling the milk. Most micro-

organisms reproduce slowly in colder environments. Cooling milk also slows

chemical deterioration. The temperature of freshly drawn milk is about 38°C.

Bacteria multiply very rapidly in warm milk and milk sours rapidly if held at

these temperatures. If the milk is not cooled and is stored in the shade at an

average air temperature of 16°C, the temperature of the milk will only have fallen

to 28°C after 3 hours. Cooling the milk with running water will reduce the

temperature to 16°C after 1 hour. At this temperature bacterial growth will be

reduced and enzyme activity retarded. Thus, milk will preserved for longer

period if cooled.

Natural souring of milk may be advantageous: for example, in smallholder

butter-making, the acid developed assists in the extraction of fat during churning.

The low pH retards growth of lipolytic and proteolytic bacteria and therefore

protects the fat and protein in the milk. The acidity of the milk also inhibits the

growth of pathogens. It does not, however, retard the growth of moulds.

Naturally soured milk is used to make many products, e.g. irgo, yoghurt,

sour cream, ripened buttermilk and cheese. These products provide ways of

preserving milk and are also pleasant to consume. They are produced by the

action of fermentative bacteria on lactose and are more readily digested than fresh

milk.

The initial microflora of raw milk reflects directly microbial

contamination during production. The microflora in milk when it leaves the farm

is determined by the temperature to which it has been cooled and the temperature

at which it has been stored. The initial bacterial count of milk may range from less

than 1000 cells/ml to 106/ml. High counts (more than 105/ml) are evidence of

5

poor production hygiene. Rapid tests are available for estimating the bacterial

quality of milk.

The first and most universal change effected in milk is its souring. So

universal is this phenomenon that it is generally regarded as an inevitable change

which can not be avoided,The phenomenon is well understood. It is due to the

action of certain of the milk bacteria upon the milk sugar which converts it into

lactic acid, and this acid gives the sour taste and curdles the milk. After this acid

is produced in small quantity its presence proves deleterious to the growth of the

bacteria, and further bacterial growth is checked. After souring, therefore, the

milk for some time does not ordinarily undergo any further changes.

Milk souring has been commonly regarded as a single phenomenon, alike

in all cases. When it was first studied by bacteriologists it was thought to be due

in all cases to a single species of micro-organism which was discovered to be

commonly present and named Bacillus acidilactic. Such balanced diet milk

becomes contaminated with several types of micro organisms which originate

form soil , water, skin and the air of animals , utensils , from the milk handlers.

The number of species of bacteria which have been found to sour milk has

increased until something over a hundred are known to have this power.

These different species do not affect the milk in the same way. All

produce some acid, but they differ in the kind and the amount of acid, and

especially in the other changes which are effected at the same time that the milk is

soured, so that the resulting soured milk is quite variable. The spoilage of milk

due to production of heat resistance proteolytic enzymes degrades casein.

The spoilage of milk results in the production of many off- flavours

which are characterized as fruity , musty , bitter, rancid , putrid. Bacteria may

be classified according to their optimum temperature for growth and heat

resistance .

6

The bacteria encountered in milk are of the following 4 temperature types

1)Psychrophilic

2)Mesophilic

3)Thermophilic

4) Thermoduric

A) Many psychotropic bacteria are lipase producers. The main

Psychrotrophic bacteria are pseudomonas florescence, alcaligens, staphylococcus,

serratia, micrococcus, coliforms, enterococcus, Achromobacter, Vibrio,

Flavobacterium and Alcaligenes, They arc killed in the pasteurization process,

but are sometimes found in pasteurized milk.

B) The most important mesophilic bacteria are streptococci, lactobacilli and

coliforms, which produce acid and gas and off flavours. They are killed in the

pasteurization process

C) Thermophilic bacteria grow well at the temperature used in

pasteurization, specially when the low temperature holding method is followed.

Thermophilic bacteria develop best at 55-650C with minimum and maximum of

400C and 800C respectively. Most thermophilic forms are found in two genera,

Bacillus and Clostridium .

Ex: Bacillus stearothermophilus is an example of this type.

D) Thermoduric bacteria survive pasteurization in considerable numbers but

do not grow at pasteurization temperatures. Since they are not killed by

pasteurization, they may contaminate the containers. As a result of the faulty cleaning

of the containers, the subsequent batches of milk processed through the same

containers will become heavily contamined. Microbacterium lacticum, Micrococcus

luteus, Streptococcus thermophiles, Bacillus subtilis exemplify this category.The

main mold is Geotrichum candidum and yeast is candida species.

Sachharomycetaceae is the producer of lipases. Saccharomyces cerevisiae ,

Debaryomyces hasenii , Debaryomyces kleockeri and lipomyces starkeyi ,

cryptococcaceae , candida antarctica , C.deformans, C.rugosa and C.lipolytica

Candida parapsilosis, candida valida, Debramyces vanriji , Dedramyces hansenii,

7

Kluyveromyces marxianus , pichia burtonii , pichia kluyveri , Geotrichum

fermentans are producers of Lipases . The strain Hansenula anomala has the

highest lipolytic activity .

Copra and Cocca Butter may be spoiled by molds.During cold storage

after milk collection, psychrotrophic bacterial populations dominate the microflora,

and their extracellular enzymes, mainly proteases and lipases, contribute to the

spoilage of dairy products .

Milk pasteurization Pasteurization is the most common process used to destroy bacteria in

milk. In pasteurization, the milk is heated to a temperature sufficient to kill

pathogenic bacteria, but well below its boiling point. This also kills many non-

pathogenic organisms and thereby extends the storage stability of the milk.

Numerous time/temperature combinations are recommended but the

most usual is 72°C for 15 seconds followed by rapid cooling to below 10°C. This is

normally referred to as High Temperature Short Time (HTST) treatment. It is carried

out as a continuous process using a plate heat-exchanger to heat the milk and a

holding section to ensure that the milk is completely pasteurised.

Milk is normally pasteurized prior to sale as liquid milk. Pasteurisation

is used to reduce the microbial counts in milk for cheese-making, and cream is

pasteurised prior to tempering for butter-making in some factories.

Batch pasteurization is used where milk quantities are too small to

justify the use of a plate heat-exchanger. In batch pasteurisation, fixed quantities of

milk are heated to 63°C and held at this temperature for 30 minutes. The milk is then

cooled to 5°C and packed. The lower temperature used for batch pasteurisation

means that a longer time is required to complete the process—30 minutes at 63°C,

compared with 15 seconds a 72°C.

Effects of pasteurisation on milk Pasteurisation reduces the cream layer, since some of the fat globule

membrane constituents are denatured. This inhibits clustering of the fat globules and

consequently reduces the extent of creaming. However, pasteurisation does not

reduce the fat content of milk. Pasteurisation has little effect on the nutritive value of

milk. The major nutrients are not altered. There is some loss of vitamin C and B

8

group vitamins, but this is insignificant. The process kills many fermentative

organisms as well as pathogens. Micro-organisms that survive pasteurisation are

putrefactive. Although pasteurised milk has a storage stability of 2 to 3 days,

subsequent deterioration is cause by putrefactive organisms. Thus, pasteurised milk

will putrefy rather than develop acidity. In rural milk processing, many processes

depend on the development of acidity, and hence pasteurisation may not be

appropriate.

Milk sterilization In pasteurization, milk receives mild heat treatment to reduce the

number of bacteria present. In sterilisation, milk is subjected to severe heat treatment

that ensures almost complete destruction of the microbial population. The product is

then said to be commercially sterile. Time/temperature treatments of above 100°C for

15 to 40 minutes are used. The product has a longer shelf life than pasteurised milk.

Another method of sterilisation is ultra-heat treatment, or UHT. In this system, milk

is heated under pressure to about 140°C for 4 seconds. The product is virtually

sterile. However, it retains more of the properties of fresh milk than conventionally

sterilised milk.

Triglycerides are Tri- esters of glycerol and three fatty acids . They

are charceterized as either fats ( lipids that are solid at room temperature ) or

oils( lipids that are liquid at room temperature ), and are common components of

foods. Other types of lipids in foods include the fattyacid mono and di esters of

glycerol, termed mono glycerides and di glycerides , respectively . These are usually

generated as intermediates in the break down of fats and oils Tri glycerides are

Lipolytic bacteria are the heterogenous group of bacteria, which produce lipases,

which catalyze the hydrolysis of fats to fatty acids and glycerol. Triglycerides have

very low water solubilities , while the solubilities of mono and Di glycerides can

be greater . Hydrolysis of the ester bonds of the Tri , Di , and Mono glycerides

(lipolysis) Liberates free fatty acids ( FFA) . In food systems , such lipolysis is

usually catalyzed by enzymes , generally by the group of enzymes known as

lipases .

Lipases are defined as those enzymes capable of hydrolyzing the

carboxylic acid easterbonds of water – insoluble substrates . The biological role of

9

lipases is to initiate the metabolism of fats and oils by reducing them to readily

metabolized free fatty acids and glycerol .Humans readily detect the shorter chain

length fatty acids , up to about 10 carbons in length , by smell or taste . In some

cases, example : dairy products , it is often desirable that some or all sizes of

these shorter free fatty acids be released by lipolysis of endogenous lipid , or the

added during processing . They confer characteristic falvor or fragnence .

Fermented sausausages are also have lipolytic activity . Longer chain fatty acids ,

particularly those containing double bonds , will oxidize their lipolytic release from

a glyceride .

Some hydrolysis of the fats and oils in foods is non-microbial in

origin , the result of spontaneous lipid hydrolysis and the action of lipases that are

naturally present many food material. Fatty acid oxidation can also generate

undesirable favours . In some cases, oxidation and or the actions of endogeneous

lipases can play a larger role In spoilage than do microbial lipases . How ever ,

lipase production is a wide spread trait of bacteria , yeasts and molds. This ability

to produce lipase does not always result in lipolytic damage . Since the synthesis or

activity of the enzyme may be inhibited by components of the food or by the

conditions on incubation .

Lipases can be significant contributors to product deterioration . In

addition to lipases which act on triglycerides , micro organisms can produce other

lipid – hydrolyzing enzymes. Chief among these are the phospholipases , which

convert Phospho lipids , the primary components of cell membranes, to FFA ,

Lyso phosphatides , Mono and Di glycerides , Glycero phosphotides and simplar

materials . The presence of a Phospho lipase can stimulate the activity of a lipase .

For example, Phospholipase –C from Bacillus cereus or pseudomonas flurosence

enhances the lipolytic activities of both milk lipoprotein lipase and commercial

Rhizopus lipase . Micro organism that produce glycosidic enzymes can in

conjunction with bacterial proteases . Degrade milk membranes and then by expose

the milk lipids to lipases . Thus , the glycosidases can contribute indirectly to

lipolytic activity.

The glycosidases of pseudomonas fluroscences , in contrast to the

phosphorlipase-C and lipase produced by that organism are completely inactivated

10

by Milk pasteurization temperatures and therefore , would not be expected to play a

role in Protein traiting lipase activity in pasteurized or other wise similarly heated

samples. By the use of special plating media micro organisms that produce lipase

can be enumerated . Such enumartion as not usually performed on a protein basis.

Food manufactures and processors analyse for lipolytic micro organisms only when

a problem arises .

Determination of the number of lipolytic micro organisms Present in

a food sample can reveal the food processor whether the particular lipid – related

problem is microbial or non- microbial in origin . The fatty parts of food made up

of fats and oils. The fats and oils themselves are subject more often to chemical

than to microbial spoilage. Besides the fatty glycerides, natural fats and oils

usually contains small amounts of fatty acids and glycerol , other liquid alcohols

and sterols , Hydrocarons, proteins and Nitrogenous Compounds , phophatides ,

caroteinoid pigments . The chief types of spoilage result from hydrolysis ,

oxidation .

Flavour reversion : Flavor reversion is defined as the appearance of object is on able

flavors from less oxidation than is needed to produce rancidity .Oils that contain

lenolenic acid , fish oils , vegetable oils . Butter fat and Meat fats become “

tallowy” as the result of oxidation but butter fat is called rancid well only

hydrolysis of fatty acids and glycerol has taken place.

Some of the pigments produced by micro organisms are fat soluble

and therefore, can diffuse into fat , producing discoloration , ranging through

yellow, red, purple and brown . stamping-Ink discoloration of MEAT fat caused by

Yellow pigment micro cocci and bacilli . The fat- soluble pigment is an

Oxidation-Reduction indicator that changes from Yellow- green – blue and finally to

purple as Fat becomes more oxidized by the peroxides formed bythe bacteria ,

yellow , pink , red fat-soluble pigments may be produced by various bacteria,

yeasts and molds .Many of the proteolytic bacteria are also lipolytic. The main

source for isolation of lipolytic microorganisms is butter and other dairy

products.Yeasts and yeast like fungi are specific group of micro organisms on

various substrates . It is assumed that yeasts and yeast like fungi can adopt to

11

substrates rich in fat under conditions of anthropogenic impact . This

characteristic has become urgent due to utilization of industrial waste. The main

index of their activity is excreted lipolytic enzymes. Microorganisms, may be

involved in the oxidation of fats, auto oxidation is common. The Microbiological

quality of butter depends upon the quality of cream and sanitary conditions used in

the processing.

BIOCHEMICAL ACTIVITIES : :

If allowed to stand under condition that permit bacterial growth, raw

milk of a good sanitary quality will rapidly undergo a series of chemical changes.

The principal change is lactose fermentation to lactic acid. This change is brought

about by acid uric lactic organisms, especially Strepotococcus lactis and certain

lactobacilli. These include two distinct biochemical types, homo-and

heterofermentative. In homofermentation lactic acid is the major product of lactose

fermentation. Hetero fermentative organisms, however, produce lactic, acetic,

propionic, and some other acids, and some alcohols and gases such as CO2 and H2

Organisms continue to form lactic acid until the concentration of acid is itself too

great for the organisms to remain live.

Microbacteria, micrococci, coliforn18, etc. also ferment lactose to

lactic acid and other products. Many Clostriifiul1J species and, some yeasts such as

Torula lactic, and Torula cremoris ferment lactose with acid and gas production.

As the acidity continues to increase and reaches a pH of 4.7, it eventually causes a

precipitation of casein. Organisms capable of metabolizing lactic and other acids

develop especially acid uric, yeasts and moulds. The acidity of milk is diminished

and the alkaline products of protein decomposition such as amines, ammonia and the

like are produced.

This is accomplished by many species of the genera Bacillus, Clostridium,

Pseudomonas,Proteus and numerous other forms.

The action of microorganisms does not involve fat as readily as it does lactose and

protein. Lipolysis results from the action of lipase produced by bacteria such as

Pseudomonas, Achromobacter and by some yeasts and moulds. Fat is hydrolysed to

glycerol and fatty acids. Some of the fatty acids, for example, butyric and caproic

acid give milk products, distinctive and usually rancid, odours and flavours.

12

Several microorganisms also bring about certain objection able changes in the milk

which may not be deleterious to health. Rapines in milk is sometimes encountered.

The milk become ropy or slimy and may be pulled out into long threads. It is

produced by several organisms but the most important species is Alcaligenes

viscolactis. A rapid fermentation of lactose in milk is sometimes observed and is

known as stormy fermentation. This is brought about by Clostridium perfringens.

The curd become torn to shreds by the vigorous fermentation and gas production.

Several organisms have been isolated from milk which impart brilliant colours.

Pseudomonas syncyanea imparts blue colour, pseudomonas synxantha yellow

colour and Serratia marcescens red colour to the milk. From the review of the

literature it was Observed the importance of the analysis. In day to day human.

Bacterial types commonly associated with milk.

Pseudomonas SpoilageBrucella PathogenicEnterobacteriaceae Pathogenic and spoilage StaphylococciStaphylococcus aureus PathogenicStreptococcusS. agalactiae PathogenicS. thermophilus Acid fermentationS. lactis Acid fermentationS. lactis-diacetyllatic Flavour productionS. cremoris Acid fermentationLeuconostoc lactis Acid fermentationBacillus cereus Spoilage LactobacillusL. lactis Acid productionL. bulgaricus Acid productionL. acidophilus Acid productionPropionibacterium Acid productionMycobacterium tuberculosis Pathogenic

13

MATERIALS

AND METHODS

14

OBJECTIVE :

1) The present study was carried out to enumerate and identify lipolytic

Microorganisms from Milk samples collected from milk collection centres.

GENERAL METHODS: :

1) Lipolytic activity can be measured by a clear zone formation around

Each colony due to hydrolysis of Tributyrin as a substrate in the

lipase reagent when colonies grown on Tributyrin agar .

2) Lipolytic activity can be measured by a Greenish blue zone formation

around colonies due to lipase producing micro organisms present in the

Sample when grown on butter fat medium .

3) Production of lipase from bacteria can be tested by the following methods.

1) Growth on Egg – Yolk Agar 2) Tween- Hydrolysis

3) Inoculation on to a nutrient agar medium containing a lipid.

MATERIALS REQUIRED :

1) Butter fat agar medium (PH- 7.8) , 2) Ringer solution - full strength ,

2) Ringer solution - quarter strength , 4) CUSO4 Standard solution ,

5) Hot water bath , 6) Sterile 500ml beaker , 7) Sterile dilution botteles

8) Sterile 1ml, 10ml , 25ml pipettes , 9) sterile spreader

10)2 Milk samples collected from milk Local Dairy Units and named as

Sample-1 , sample-2.

PRINCIPLE l : The development of greenish blue zone around the growth /

colony is due to insoluble copper salts of fatty acids set free on lipolysis by

lipase producing micro organisms .

15

MEDIA AND MATERIALS REQUIRED :

1)Butter fat agar medium preparation :

Butter fat : 5.0 gm

Yeast extract : 3.0 gm

Peptone : 5.0 gm

Agar : 15.0 gm

Distilled Water : 1000ml

Milk sample 1 : 5ml

Milk sample 2 : 5ml

Dissolved the constituents in Distilled water and sterilize at 1210 c for 10 min .

2) Ringer Solution Full strength :

Nacl : 9.0 Kg

Kcl : 0.42 gm

Anhydrous cacl2 : 0.48 gm

Sodium Bicaronate : 0.20 gm

Dis.H20 : 1000ml

Dissolved the ingredients in Distilled water . Dispensed in flasks and

sterile at 1210c for 10 min. In the present work we measured the constituents for

125 ml .

3)Ringer Solution – Quarter strength :

1 Part of Full strength Ringer’s solution and 3 parts of Distilled water .

Autoclaved at 1210c for 10 min. In Present work we Prepared Ringer Full

strength solution 32 ml and mixed with 168 ml Autoclaved at 1210c for 10 min.

16

4)Cuso4 Solution ( Aqueous standard Solution):

Nacl - 58 .44 gm

Kcl - 74.55 gm

CaCl2 - 110.99 gm

NaHCO3 - 84.01 gm

Distilled Water - 1000 ml

Dissolve all the ingredients in required amount and preserve in amber

Coloured bottle in dark environment .

PROTOCOL : 1) Prepared butterfat agar medium plates 24 hrs before the start of the

experiment.

2) Warmed the Ringer’s solution at 40-450c for 15 min in a hot water bath .

3) Milk sample was taken in a sterile beaker and warmed at 40-450c for 15 min .

4) Pipetted 25 ml of gently mixed , melted Milk samples in to 125ml of warm

(400 c) Ringer’s solution in a dilution bottle and shacked the mixture well .

5) Prepared sterile dilutions of the above mixture in Quarter strength Ringer’s

solution .

6)Poured 1ml (or) 0.1 ml two milk samples from the different dilutions

(10-4 to 10-6) on butter fat agar plates.

7) Spreaded the suspension over the media to ensure uniform distribution of

Cells On the Butter fat agar medium to get isolated colonies.

8) Incubated the inverted plates at 21± 10c for 3 days and 32±10c for 2 days.

9) After incubation the plates were observed for the appearance of colonies

of micro organisms.

10) The plates were flooded with saturated CUSO4 Solution for 10-15 min.

11) The CUSO4 Solution was drained and the agar plate was kept under

17

running tap water to remove the excess CUSO4 Solution.

1) COLONY MORPHOLOGY :

The number of lipolytic organisms per gram was estimated by standard

formula Dilution factor (1: 10 ) :

No . Of Lipolytic Organisms / gram ═ No. of Colonies × Dilution Factor

Volume of sample added

Dilution of the sample was made by adding 1ml of the sample to 9ml

of Distilled water to make a sample solution of 1:10 .

OBSERVATION RESULTS :

Sample-1 :

1) The No. of colonies was counted 10-4 in all the 6 plates kept for analysis .

Plate No 1( LP1) : 74 Colonies

Plate No.2 (LP2) : 70 Colonies

Plate No.3(LP3) : 68 Colonies

Plate No.4(LP4) : 76Colonies


Plate No.6 (LP6) : 72Colonies

Average ═ 74 + 70+ 68+ 76 +80+72 ═ 440 ═ 73

6

2) The No. of colonies was counted 10-5 in all the 6 plates kept for analysis .





18



Average ═ 78 + 78 + 75+ 75+82+72 ═ 460 ═ 76

6 6

3)The No. of colonies was counted 10-6 in all the 6 plates kept for analysis .

Plate No 1( LP1) : 76Colonies






Average ═ 76 + 70 + 72+ 74 +76+76 ═ 444 ═ 74

6 6

The number of colonies in the dilutions of 10-4 to 10-6 was counted

73, 76, 74 respectively .

Sample -2 :








Average ═ 44 + 30+ 35+ 42 +48+43 ═ 242 ═ 40

6 6



19






Average ═ 48 + 41 + 35+ 42+46+42 ═ 254 ═ 42

6 6


Plate No 1( LP1) : 76Colonies






Average ═ 48 + 41 + 44+ 42 +46+45 ═ 266 ═ 44

6 6

The number of colonies in the dilutions of 10-4 to 10-6 was counted

40, 42, 44 respectively .

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2)COLONY MORPHOLOGY :

Sample : 1

The colonies were large to very large , gray-white, blistery and dry in appearance

Sample: 2:

The colonies were Small , Creamy white , round , discrete colonies

21

3)OBSERVATION OF ZONE FORMATION:

After adding CUSO4 Solution the colonies appears blue in colour due to

insoluble copper salts of fatty acids set free on lipolysis by lipase producing

micro organisms . The washed plates were observed for the presence of zones

around the Colonies. The presence of greenish- Blue zones around the colonies

was Observed . This indicates the presence of the lipase production by the

micro organisms present in the butter sample .

GRAM STAINING TECHNIQUE :

The test was originally developed by Christian Gram in 1884, but was

modified by Hucker in 1921. The modified procedure provided greater reagent

stability and better differentiation of organisms.

The Gram stain is used to classify bacteria on the basis of their forms,

sizes, cellular morphologies, and Gram reactions; in a clinical microbiology

laboratory, it is additionally a critical test for the rapid presumptive diagnosis of

infectious agents and serves to assess the quality of clinical specimens.

Gram stain permits the separation of all bacteria into two large groups,

those which retain the primary dye (gram-positive) and those that take the color of

the counterstain (gram-negative).

The primary dye is crystal violet and the secondary dye is usually either

safranin O or basic fuchsin. Some of the moreCommon

formulations include: saturated crystal violet (approximately 1%), Hucker’s

crystal violet, and 2% alcoholic crystal violet.

PROTOCOL :

1. Deparaffinize sample and hydrate to distilled water

2. Place slides of staining rack

3. Add 1ml of crystal violet solution to the sample. Alternativley gentian

violet can be used

4. Add 250ul of 5% sodium biocarbonate

5. Gently mix solutions for one minute

6. Rinse with water

7. Cover sample with Gram's iodine solution and incubate for 1 minute

8. Rinse with water

22

9. Blot with filter paper until dry

10. Incubate breifly in acetone. This step should be short as longer incubation

will cause some gram positives to become gram negative. The time of

incubation is usually determined by the decolorization of the background

tissue. Repeat if necessary.

11. Cover sample with basic fuchsin solution for 3 minutes

12. Rinse with water

13. Blot excess water with filter paper

14. Dip slide in acetone

15. Dip slide in 0.1% picric acid in acetone

16. Rinse in acetone

17. Rinse in 50% acetone - 50% histolene

18. Rinse in 100% histolene. Repeat if necessary

19. Mount slide

Gram positive bacteria stain blue and gram negative bacteria stain red.

PROCEDURE :

1)The gram-positive cell envelope consists of a thick layer of peptidoglycan

embedded with techoic acids and a plasma membrane comprised of phospholipids

with integral membrane proteins traversing the bilayer.

2)The cells are flooded with crystal violet dye. Crystal violet is a water-soluble,

basic dye. In solution, basic dyes produce dye particles with positive charges

(cations). Sometimes the crystal violet dye particle is abbreviated CV+.

3)The individual crystal violet ions penetrate the thick peptidoglycan layer of the

cell as well as the plasma membrane, making their way through the matrix created

by the crosslinking of polysaccharides and proteins within the peptidoglycan layer.

4)Gram's iodine solution is added. This solution consists of a mixture of iodine

and potassium iodide. The active constituent in this solution is the iodide ion or an

iodine-iodine complex.

5) Like the crystal violet dye particles, the iodide ions are also able to penetrate

the thick peptidoglycan layer of the cell. Here, the iodide ions mix with the

crystal violet dye particles that were added in the previous step.

23

6) The crystal violet and iodide ions react, forming a crystal violet-iodine complex.

This complex is insoluble in water and produces particles much larger than either

the iodide ions or the crystal violet ions individually.

7) The alcohol/acetone mixture displaces water in the peptidoglycan layer,

resulting in dehydration. This loss of water causes the thick peptidoglycan layer to

shrink, tightening the matrix created by the crosslinking of polysaccharides and

proteins.

8) It is important to know that this decolorizing step is a critical step in the Gram

stain protocol. Exposure to the alcohol for too long can cause cells that are gram-

positive to lose too much of the dye complex due to damage to the peptidoglycan

layer. These cells will not appear gram-positive when the staining procedure is

complete.

9) The counterstain, normally safranin, is added. Like crystal violet, safranin is a

weakly water-soluble, basic dye that produces cationic stain particles in solution

that bind negatively charged moieties such as the techoic acids, peptides and

phospholipid heads within the envelope and in the cytoplasm.

10) Safranin, because of its small size, is able to penetrate the dehydrated

peptidoglycan layer and bind to negatively charged moieties. Because the safranin

is much lighter in color than the crystal violet-iodine complex.

11) When viewed under a microscope, gram-positive cells appear purple due to the

crystal violet-iodine complex retained inside.

12) The gram-negative cell envelope consists of a thin layer of peptidoglycan

surrounded by two phospholipid membranes, one interior and one exterior.

Polysaccharide chains are bound to the phosphate heads of the outer membrane to

form lipopolysaccharides. Both the membranes contain integral membrane

proteins. Place cursor over each membrane for ID.

13) The cells are flooded with crystal violet dye. Crystal violet is a water-soluble,

basic dye. In solution, basic dyes produce dye particles with positive charges

(cations). Sometimes the crystal violet dye particle is abbreviated CV+.

14) The individual crystal violet ions penetrate the thin peptidoglycan layer of the

cell as well as the plasma membrane, making their way through the matrix created

by the crosslinking of polysaccharides and proteins within the peptidoglycan layer.

24

15) Gram's iodine solution is added. This solution consists of a mixture of iodine









18) A decolorizing solution, normally consisting of a mixture of ethyl alcohol and

acetone, is added. Numerous variations of the decolorizing solution formula are

used in labs.

19) The alcohol/acetone mixture displaces water in the peptidoglycan layer,

resulting in dehydration. This loss of water causes the thin peptidoglycan layer to

shrink slightly, tightening the matrix created by the crosslinking of

polysaccharides and proteins. The alcohol/acetone mixture also disrupts and

dissolves the outer membrane, exposing the peptidoglycan layer to the

environment.

20) Safranin, because of its small size, is able to penetrate the dehydrated

peptidoglycan layer and bind to negatively charged moieties. Because

safranin is the only stain present, the cells will have a pink or red Colour.

GRAM’S STAINING :

Sample-1 :

Rod shaped cells was observed with purple colour . Therefore it was identified as

Gram positive bacteria .

Sample-2 :

Rod shaped cells was Observed with purple colour . Therefore it was also

identified As Gram positive bacteria .

25

BIOCHEMICAL TESTS – PROCEDURES 1.CITRATE UTILIZATION:

This test is one of several techniques used to assist in the identification of

enterobacteria. The test is based on the ability of an organism to use citrate as its

only source of carbon and ammonia as its only source of nitrogen.

PRINCIPLE :

The test organism is cultured in a medium which contains sodium citrate,

an ammonium salt, and the indicator bromo-thymol blue.Growth in the medium is

shown by turbidity and a change in color of the indicator from light green to blue,

due to the alkaline reaction, following citrate utilization.

REQUIREMENTS: Koser’s citrate medium:

Formula and preparation:Oxoid dehydrated medium

Grams per litreSodium ammonium phosphate 1.5Potassium dihydrogen phosphate 1.0Magnesium sulphate 0.2Sodium citrate 2.5Bromothymol blue 0.016

The medium is used at a concentration of 0.52 grams in every 100ml of

distilled water. Prepare the medium as per the instructions in the kit. Distribute

in 3ml amounts in small screw cap bottles or tubes. Sterilize by autoclaving (with

caps loosened) at 121oC for 15 minutes .When cool, tighten the container tops.

Date the medium and give it a batch number.

QUALITY CONTROL :

pH of medium: This should be within the range pH 6.6-7.0 at room temperature.

Performance: Test the medium by inoculating it with bacterial species of known

positive and negative citrate activity

Storage: Store in a cool dark place.

26

Shelf life: Up to 2 years providing there is no change in the volume or appearance

of the medium to suggest contamination or an alteration of pH.

INOCULATION :

Use a straight wire to inoculate the medium to prevent any carry over of

nutrients from the test culture .A light inoculum must be used, and the bottle caps

should be loosened during incubation.

METHOD:

1. Using a sterile straight wire, inoculate 3-4ml of sterile Koser’s citrate

medium with a broth culture of the test organism. Care must be taken not

to contaminate the medium with carbon particles, such as from a

frequently flamed wire.

2. Incubate the inoculated broth at 35-37oC for up to 4 days, checking daily

for growth.

RESULTS :

Turbidity and blue color………….Positive test (Citrate utilized)

No Growth……………………….Negative test (Citrate not utilized)

CONTROLS :

Positive citrate control: Klebsiella pneumoniae.

Negative citrate control: Escherichia coli

3)HYDROGEN SULPHIDE PRODUCTION :

The detection of hydrogen sulphide gas is used mainly to assist in the

identification of enterobacteria and occasionally to differentiate other bacteria

such as Bacteroides and Brucella species. Hydrogen sulphide is produced when

sulphur containing amino acids are decomposed.The technique used to detect the

release of hydrogen sulphide gas must not be too sensitive other wise it will detect

the small places of hydrogen sulphide produced by most enterobacteria.

27

USE OF KLIGLER IRON AGAR(KIA) TO DETECT HYDROGEN

SULPHIDE

This medium is suitable for detecting H2S production by enterobacteria.H2S is

detected by the ferric citrate contained in the medium.

LEAD ACETATE PAPER TO DETECT HYDROGEN SULPHIDE

When a sensitive technique for detecting H2S production is required,the lead

acetate paper test is recommended.

1. Inoculate a tube or bottle of sterile peptone water or nutrient broth with the

test organism.

2. Insert a lead acetate paper strip in the neck of the bottle tube above the

medium and stopper well.

3. Incubate the inoculated medium at 35-37oC and examine daily for a

blackening of the lower part of the strip.

RESULTS :

Blackening……………………………Positive test (H2S produced)

No blackening………………………. Negative test ( No H2S is produced)

4)INDOLE TEST :

Testing for indole production is important in the identification of

enterobacteria. Most strains of E.coli, P.vulgaris, P.rettgeri, M.morganii, and

Providencia species break down the amino acid tryptophan with the release of

indole.

PRINCIPLE :

The test organism is cultured in the medium which contains tryptophan.

Indole production is detected by Kovac’s or Ehrlich’s reagent which contains 4

(p)-dimethylaminobenzaldehyde. This reacts with the indole to produce a red

colored compound.

28

In the following method the use of the combined motility Indole Urea

(MIU) is described. A Kovac’s reagent paper strip is inserted in the neck of the

tube and indole production is indicated by a reddening of the strip. Indole is a

volatile substance (easily vaporized). The tube must be well stoppered during

incubation.

The indole test can also be carried out by culturing the organism in

tryptone water or peptone water containing tryptophan, and detecting indole

production by adding Kovac’s or Ehrlich’s reagent to an18-24 h culture.

REQUIREMENTS:

1. MOTILITY INDOLE UREA MEDIUM (MIU ):

MIU semi solid medium is used to differentiate enterobacteria species by

their motility, urease and indole reactions.

Formula and Preparation…………… To make 1 litre of MIU base medium

Tryptone………………………………………………………….30g

(or pancreatic digest of casein)

Potassium dihydrogen Phosphate…………………………… 1g

Sodium Chloride………………………………………………….5g

Agar…………………………………………………………… 4g

Phenol red 2.5g/l (0.25%)……………………………………… 2ml

Distilled water………………………………………………… 1 litre

Mix the dry ingredients in the water and heat to 100oC to dissolve the

chemicals (place the flask in a container of boiling water).

Allow to cool to 50-55oC and then add the phenol red solution. Mix well.

Dispense in 95ml amounts in screw-cap bottles. Sterilize by autoclaving

(with caps loosened) at 121oC for 15 minutes. When the medium has

cooled, tighten the bottle caps.

2. KOVAC’S REAGENT STRIPS

METHOD :

29

Using a sterile straight wire, inoculate 5 ml of sterile MIU medium with a

smooth colony of the test organism.

Place an indole paper strip in the neck of the MIU tube above the medium,

and stopper the tube. Incubate at 35-37 oC overnight.

Examine for indole production by looking for a reddening of the lower

part of the strip.

RESULTS:

Reddening of strip…………………………Positive test (Indole produced)No red color……………………………… .Negative test ( No Indole produced)

5) VOGES - PROSKAUER TEST :

This test is occasionally used to assist in the differentiation of

enterobacteria. K.pneumoniea, Vibrio cholerae and some strains of Enterobacter,

ferment glucose with the production of acetylmethylcarbinol ( acetoin ) which can

be detected by an oxidation reaction.

PRINCIPLE:

The test organism is cultured in a glucose phosphate peptone water for 48

hrs. sodium hydroxide and a small amount of creatine are then added. Under

alkaline conditions and exposure to the air, the acetoin produced from the

fermentation of the glucose is oxidized to diacetyl which forms a pink compound

with the creatine.

REQUIREMENTS :

1. GLUCOSE PHOSPHATE PEPTONE WATER :

Glucose phosphate peptone water is a fluid medium used in the Vogues-

Proskauer test and Methyl Red test.

Formula and Preparation --------- To make about 50 bottles

Peptone………………………………………….0.5g

Glucose (dextrose)…………………………… 0.5g

Di-Potassium hydrogen phosphate……… 0.5g

Distilled Water………………………………….100ml.

30

Dissolve the peptone and phosphate salt in the water by steaming. When

cool, filter, and adjust the pH to 7.5.

Add the glucose and mix well.

Dispense the medium in 2 ml amounts in small screw-cap tubes or bottles.

Sterilize by autoclaving (with caps loosened) at 115oC for 10 minutes.

When cool, tighten the container tops.

3. Sodium hydroxide, 400g/l.

4. Creatine powder.

METHOD ;

1. Inoculate 2 ml of sterile glucose phosphate peptone water with the test

organism. Incubate at 35-37 oC for 48 hrs.

2. Add a very small amount (knife point) of creatine and mix.

3. Add about 3 ml of the sodium hydroxide reagent and shake well. The

sodium hydroxide reagent is corrosive, therefore handle with care and do

not mouth- pipette.

4. Remove the bottle cap, and leave for 1 hour at room temperature. Look for

the slow development of a pink-red color.

RESULTS:

Pink-red color……………………… Positive test (Acetoin produced)

No pink-red color……………………Negative test (No acetoin produced)

6) NITRATE REDUCTION TEST :

This test is used to differentiate members of the Enterobacteriaceae that

produce the enzyme nitrate reductase, from gram negative bacteria that do not

produce the enzyme. The test is also helpful in differentiating Mycobacterium

species.

PRINCIPLE :

A heavy inoculum of the test organism is incubated in a broth containing

nitrate. After 4 hours, the broth is tested for the reduction of nitrate to nitrite by

adding sulphanilic acid reagent. If nitrite is present, the acid reagent is diazotized

and forms a pink-red compound with alpha-naphthylamine.

31

When nitrite is not detected it is necessary to test whether the organism

has reduced the nitrate beyond nitrite. This is done indirectly by checking whether

the broth still contains nitrate. Zinc dust is added which will convert any nitrate to

nitrite. If no nitrite is detected when the zinc dust is added, it can be assumed that

the entire nitrate has been reduced beyond nitrite to nitrogen gas or ammonia by a

nitrate reducing organism.

REQUIREMENTS:

1. NITRATE BROTH :

Nitrate broth can be prepared by adding 0.1 gram of potassium nitrate to

100 ml of peptone water.

The medium is used at a concentration of 0.9 grams in every 100 ml of

distilled water.

Sterilize by autoclaving (with loose caps) at 121oC for 15 minutes. When

the medium has cooled, stopper tightly.

1. Sulphanilic acid reagent

2. Alpha- naphthalamine reagent.

3. Zinc dust.

METHOD:

1. Inoculate 0.5 ml of sterile nitrate broth with a heavy growth of the test

organism.

2. Incubate at 35-37oC for 4 hours.

3. Add 1 drop of sulphanilic acid reagent and 1 drop of alpha- naphthylamine

reagent.

4. Shake to mix and look for a red color.

RESULTS:

Red color………………………………Positive test(Nitrate reduced)

32

If no red color is produced, add a very small amount (knife point) of zinc

dust powder. Look again for a red color and interpret as follows:

Red color…………………………Negative test(No reduction of nitrate)

No red color………………………… Positive test (Nitrate reduced)

7) UREASE TEST

Testing for urease enzyme activity is important in differentiating

enterobacteria. Proteus strains are strong urease producers. Y. enterocolitica also

shows urease activity (weekly at 35-37C). Salmonellae and shigellae do not

produce urease.

PRINCIPLE

The test organism is cultured in a medium which contains urea and the

indicator phenol red. If the strain is urease-producing, the enzyme will break

down the urea (by hydrolysis) to give ammonia and carbon dioxide. With the

release of ammonia, the medium becomes alkaline as shown by a change in color

of the indicator to red-pink. The method described is that which uses the

combined motility indole urea (MIU) medium.

REQUIREMENTS :

Motility indole urea (MIU) medium:

MIU semi solid medium is used to differentiate enterobacteria species by

their motility, urease and indole reactions.

Formula and PreparationTo make 1 litre of MIU base mediumTryptone………………………………………………………… 30g(Or pancreatic digest of casein)Potassium dihydrogen Phosphate…………………………… 1gSodium Chloride………………………………………………… 5gAgar……………………………………………………………….4gPhenol red 2.5g/l (0.25%)……………………………………… 2mlDistilled water……………………………………………………1 litre

33

Mix the dry ingredients in the water and heat to 100oC to dissolve the

chemicals (place the flask in a container of boiling water).

Allow to cool to 50-55oC and then add the phenol red solution. Mix well.

Dispense in 95ml amounts in screw-cap bottles. Sterilize by autoclaving

(with caps loosened) at 121oC for 15 minutes. When the medium has

cooled, tighten the bottle caps.

METHOD:

1. Using a sterile straight wire, inoculate a tube of sterile MIU medium with

a smooth colony of the test organism. When inoculating KIA medium at

the same time, inoculate the KIA tube first and then stab the MIU

medium.

2. Place an indole paper strip in the neck of the MIU tube above the medium.

Stopper the tube and incubate at 35-37o C overnight.

3. Examine for urease production by looking for a red-pink color in the

medium.

RESULTS:

Red-pink color…………………Positive test(Urease produced)

No red-pink color………………Negative test (No urease produced)

8) OXIDASE TEST :

The oxidase test is used to assist in the identification of Pseudomonas, Neisseria,

Vibrio, and Pastuerella species, all of which produce oxidase.

PRINCIPLE:

A piece of filter paper is soaked with a few drops of oxidase reagent. A

colony of the test organism is then smeared on the filter paper. If the organism is

oxidase producing, the phenyldiamine in the reagent will be oxidized to a deep

purple color.Occasionally the test is performed by flooding the culture plate with

oxidase reagent but this technique is not recommended for routine use because the

reagent rapidly kills bacteria. It can be useful, however, when attempting to

isolate N.gonorrhoeae colonies from mixed cultures in the absence of selected

34

medium.The oxidase positive colonies must be removed and subcultured within

30 seconds of flooding the plate.

REQUIREMENTS :

1. Oxidase reagent (freshly prepared)

This is a 10 g/l solution of tetra methyl-p-phenylenediamineihydrochloride.

METHOD :

1. Place a piece of filter paper in a clean Petri dish and add 2 or 3 drops of freshly prepared oxidase reagent.

2. Using a piece of stick or glass rod (not an oxidized wire loop), remove a colony of the test organism, and smear it on the filter paper.

3. Look for the development of a blue-purple color within a few seconds.

RESULT:

Blue-purple color………………… Positive test (Within 10 seconds) Oxidase

produced.

No blue-purple color……………… Negative test (Within 10 seconds) No oxidase

produced

9) CATALASE TEST:

This test is used to differentiate those bacteria that produce the enzyme

catalase, such as staphylococci, from non-catalase producing bacteria such as

streptococci.

PRINCIPLE :

Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and

water.An organism is tested for catalase production by bringing it into contact

with hydrogen peroxide. Bubbles of oxygen are released if the organism is a

catalase producer. The culture should not be more than 24 hours old.

35

Care must be taken if testing an organism cultured on a medium containing blood

because catalase is present in red cells. If any of the blood agar is removed with

the colony, a false positive reaction will occur. It is usually recommended,

therefore that catalase testing be performed from a blood-free culture medium

such as nutrient agar.

REQUIREMENTS:

Hydrogen peroxide, 3% H2O2 -------- (10 volume solution)

METHOD:

Pour 2-3ml of the hydrogen peroxide solution into a test tube.

Using a sterile wooden stick or a glass rod, remove a good growth of the

test organism and immerse it in the hydrogen peroxide solution.

Look for immediate bubbling.

RESULTS:

Active bubbling……………………… Positive test (Catalase produced)

No release of bubbles………………… Negative test (No catalase produced)

36

results AND DISCUSSIONS

37

RESULTS:

BIOCHEMICAL CHARACTERISTICS :

S.NO Characteristics Sample -1 Sample-2 Positive Negative

1 Motility + _ NA NA

2 Gram stain Gram-Positive

rods

Gram

Positive rods

NA NA

3 Growth onButter

Fat Agar

Small,Creamy

white round ,

discrete colonies

Small,Creamy

white round ,

discrete

colonies

NA NA

4 Catalase Bacillus Lactobacillus Sample -1 Sample -2

5 Oxidase Bacillus Lactobacillus Sample-1 Sample-2

6 Indole Bacillus Lactobacillus ------ Sample-1

Sample-2

7 Methyl Red Bacillus Lactobacillus Sample-1

Sample-2

---------------

8 Voges Proskauer Bacillus Lactobacillus Sample- 1

Sample-2

---------------

9 Citrate Bacillus Lactobacillus Sample-1 Sample-2

10 Urease Bacillus Lactobacillus Sample-1 Sample-2

1 Glucose Bacillus Lactobacillus Sample-1

Sample-2

2 Galactose Bacillus Lactobacillus Sample-1

Sample-2

3 Xylose Bacillus Lactobacillus Sample-1

Sample-2

4 Sucrose Bacillus Lactobacillus Sample-1

Sample-2

38

DISCUSSION: Pathogenic organisms of both bovine and human origin have been isolated

from milk. Milk, therefore, can serve as a carrier of diseases. Many serious

epidemics were caused by the consumption of such products before this fact was

clearly recognized. However, this became less common as milk sanitation has

improved and pasteurization is being more widely practised.

The disease organisms present in milk may be derived from (1) diseased

animals or (2) persons collecting and handling milk: Thus the danger is due to the

inoculum and not to the growth of organisms in the milk.

The health of animal is an important factor. Several diseases of cattle

including staphylococcal and streptococcal infections, tuberculosis, brucellosis,

Salmonellosis, Q fever and Foot and mouth disease may be transmitted to man.

The organisms causing these diseases may get into the milk either directly from the

udder, or indirectly from infected body discharges, which may drop, splash, or be

blown into the milk. Some of the important diseases of human origin that have

been transmitted by milk are (1) typhoid fever (2) diphtheria, (3) scarlet fever,

(4) dysentery (5) septic sore throat and (6) poliomyelitis.

It is also possible for humans to infect animals. For example, mastitis may

be caused by a variety of organisms, including Staphylococcus aureus. The

infecting organism, in some cases, has been traced to humans.The organisms exist

in milk as dormant spores which, unless the milk is subjected to the action of

certain physical and chemical stimuli such as heat, cold, or the action of alkalis,

remain un germinated.

MILK FERMENTATION:

Raw milk produced under normal conditions develops acidity. It has long

been recognised that highly acid milk does not putrefy. Therefore, allowing milk

to develop acidity naturally preserves the other milk constituents. Bacteria in

milk are responsible for acid development. They produce acid by the anaerobic

breakdown of milk carbohydrate—lactose—to lactic acid and other organic acids.

Anaerobic breakdown of carbohydrate to organic acids or alcohols is called

fermentation.A number of sugar fermentations are recognized in milk. They can

39

be either homofermentative, with one end product, or heterofermentative, with

more than one end product.

1. Streptococci and Lactobacilli., 2. Propionibacteria. , 3. Yeasts – Candida and

Torula. 4. Coliform bacteria. are the organisms responsible for fermentation .

40

The lactic acid fermentation is the most important one in milk and is

central to many processes. Propionic fermentation is a mixed-acid fermentation

and is used in the manufacture of Swiss cheese varieties. Alcohol fermentation

can be used to prepare certain fermented milks and also to make ethyl alcohol

from whey.

The coliform gassy fermentation is an example of a spoilage fermentation.

Large numbers of coliform bacteria in milk indicates poor hygiene. The coliform

gassy fermentation disrupts lactic acid fermentation, and also causes spoilage in

cheese.

The factors that affect microbial growth also affect milk fermentation.

Fermentation rates will generally parallel the microbial growth curve up to the

stationary phase. The type of fermentation obtained will depend on the numbers

and types of bacteria in the milk, storage temperature and the presence or absence

of inhibitory substances.

The desired fermentations can be obtained by temperature manipulation or

by adding a selected culture of micro-organisms—starter—to pasteurised or

sterilised milk. In smallholder milk processing, traces of milk from previous

batches are often used to provide `starter' for subsequent batches. Other sources

include the container and additives such as cereal grains.

The fermentation will be established once the organisms dominate the medium

and will continue until either the substrate is depleted or the end product

accumulates. In milk, accumulation of end product usually arrests the

fermentation. For example, accumulation of lactic acid reduces milk pH to below

4.5, which inhibits the growth of most micro-organisms, including lactic-acid

producers. The fermentation then slows and finally stops.

41

CONCLUSIONS

42

CONCLUSION :

Bacteria are contaminants of all fresh foods. In order to avoid excessive

spoilage, various measures can be employed to kill bacteria or to retard bacterial

growth. These include keeping foods cold (or frozen), boiling (as is done for

canned foods), salting (pickling), dehydrating (as in beef jerky), and adding anti-

bacterial preservatives. In the particular case of milk, pasteurization combined

with refrigeration is the most common technique used. Pasteurization does not kill

all the bacteria (or spores) in milk, but does eliminate most of the pathogenic

bacteria that have been historically associated with milk, such as tuberculosis,

brucellosis, and typhoid. Pasteurization was first developed in order to kill these

pathogens, but it was soon discovered that this process also improved the keeping

quality of the milk without sacrificing the taste. Pasteurization can be

accomplished by heating milk to 63-650 C for 30 minutes or to 71° c for 15

seconds (flash pasteurization) followed by rapid cooling. Flash pasteurization is

the most common technique used. Pasteurization does not prevent spoilage, but it

reduces the bacterial population so that spoilage occurs more slowly. Milk can be

essentially sterilized by Ultra High Temperature (UHT) pasteurization in which it

is heated to a higher temperature than is used for normal pasteurization, but just

for a few seconds (149° C for 6 to 9 seconds). This milk can be stored for several

months at room temperature without spoiling. There are a variety of bacteria that

can be present in raw milk due to improper Collection preservation and

maintenance .

From the results data it was observed that the good hygienic conditions

maintained during collection of milk will minimize the microbial colony count

and the Lipolytic activity of the bacteria.

43

REFERENCES

44

References :

Alatosavva .M. and Alato ssava ,T. (2006) Phenotypic characterization of raw milk associated psychrotrophic bacteria . Microbiol.

APHA (1978) . American public health Association in standard methods for examination of dairy products (Ed.Elmer ,H.Marth .Washington ,D.C)

APHA, 1980. Compendium of methods for examination of foods. 12th Edition of American Public Health Association Inc., Washington, D.C.

Austain, B. (1988). Methods in Aquatic Bacteriology. A Wiley -Interscience Publication . 222-231.

Braun, P. and K. Fehlhaber, 2002. Combined w effect of temperature, a and pH on enzymatic a c tiv ity of spoi lage causing bacte ria .Milchwissenschaft, 57: 134-136. 23. Santos, E. de S., E.de Carvalho and L.R. de Abrew, 1999 .

Psych ro troph ic bacte ria : consequences of their presence in milk and cheeses. Boletim de Sociedade Braisileria de Ciencia e Technologia de Alimentos, 33: 129-138.

Brock, T., (1986). introduction: an overview of the thermophiles. in: Brock, T. thermophiles: general, molecular, and applied microbiology, John Wiley and Sons, New York, 2-15.

Buchanan , R.E. and N.E.Gibbons , 1974 , Bergey’s manual of determination bacteriology .5th Edn. The Williams and Wilkins company , Baltimore, USA.

Campos, L and Felix, C. R. (1995). Purification and Characterization of a Glucoamylase from Humicola grisea. Applied Environmental Microbiology.61. 2436-2438.

Canigova, M. and E. Benczova, 2001. The microflora changes of raw milk during its refrigerated storage. Acta Fytotechnica et Zootechnica, 4: 104-106.

Celestino, E.L., M. Iyer and H. Roginski, 1996. The effects of refrigerated storage on the quality of raw milk. Australian J. Dairy Tech., 51: 59-63.

45

Cempirkova, R., 2002. Psychrotrophic vs. total bacterial counts in bulk milk samples. Vet. Med. Czech., 47: 227-233.

Cappuccino, J.G. and N. Sherman, 1999. Microbiology: A laboratory manual . Benjamin/Cummings Publ. Company, New York., pp: 477.

Dicks LM, Silvester M, Lawson PA, Collins MD (2000). Lactobacillus fornicalis sp. nov., isolated from the posterior fornix of the human vagina.

Dogan,B.and Brook ,K.J. (2003) Genetic diversity and spoilage potentials among pseudomonaas sp. Isolated from fluid milk products and dairy processing plants. Appl.Environ.Microbiol., 69: 130-138.

Doolittle, W. F. (1999). Phylogenetic classification and the universal tree. Science, Vol. 284. 2124-2128.

Fairbairn, D.J. and B.A. Law, 1986. Proteinases of psychrotrophic bacteria: their production, properties, effects and control. J. Dairy Res.,

53: 139-177.

Fujio, Y. and Kume, S. (1991). Characteristics of highly thermostable neutral protease produced from Bacillus stearotherrtmophilus. World Journal of Microbiology and Biotechnology.7. 12-16

Geetha, R. and V. Prasad, 2001. Studies on the performance of cultures on lactic acid bacteria in lactose hydrolysed buffalo skim milk. Cheiron,

30: 81-84

Gopi,H., Parthiban M. and Dhana lakshmi ,B.(2001). Bacteriological quality of private brands of milk in chennai city .Cheiron ,30: 106-107.

Harrigan.W.F. and Mc.Cance , M.E(1976). Laboratory method in food and beverage Microbiology, Academic press, London.

Hisotsuyanagi, K. (1979). Stepwise introduction of regulatory genes stimulating production of α -amylase into Bacillus subtilis: Construction of α-amylase extrahyper producing strain. Agric boil chem. 43:2343-2349.

Harris ,P.L.Cuppett,L. and Buller man, L.B(1990) A technique for comparison of isolation of lipolytic bacteria .J. Food prot. 53: 176-177.

Holm, C., Jespen , L.Larsen, M. and Jespersen.L,(2004). Predominant microflora of down graded Danish bulk tank milk .J. Dairy Sci. 87 : 1151-1157.

46

Khali. A, M. salim and A.K. Sallal, (1998). Enumeration of thermotolerant bacteria from recreational thermal ponds in Jordan", Cytobios. 57-63.

Kohlmann, K.L., S.S. Nielsen, L.R. Steenson and M.R. Ladisch, 1991. Production or proteases by psychrotrophic microorganisms. J. Dairy Sci.,

74: 3275-3283

Kuzin , A.A.Gudkov , A.V. and Shergin , N.A(1992). Contamination of raw milk by psychrotrophs. Molochnaya promyshlennost, 5: 31-34 .

Lutz Fisher et al. (1995). Purification and characterization of thermotolerant β-Galactosidases from thermomyces lanuginose. Applied and Environmental Microbiology.61. 1497-1501.

Mac, Faddin., J. F, (1980). biochemical tests for Identification of medical bacteria "2nd ed., Waverly Press Inc. U.S.A. 50-53.

Marshall ,R.T(1993) Standard methods for the examination of dairy products , 16th Ed. American Public Health Association , Washington,D.C.

Matta, H. and V. Punj, 1996. Isolation and identification of proteolytic psychrotrophic spore forming bacteria from milk. Indian J. Dairy Sci.,49: 695-699

Prakash, M., V. Karthikeyan and N. Karmegam, 2006. Determination of total counts of psychrotrophic bacteria and coliforms in milk samples. J. Ecotox. Environ. Monit., 16: 291-296.

Priest, f., Good, M. and Todd, C. (1988). Anumerical classification of the genus bacillus. Journal of general microbiology. Vol.134.184.

Putz, M. and H. Gierl, 1991. Investigation of total bacterial count and of selected bacteria in different milk samples. Bayerisches Kandwirtschaftliches Jahrbuch, 68: 515-520.

Rahman, A.H. and H.A. Aait, 1988. Incidence and level of occurrences of proteolytic microorganisms in some selected foods. Ass. Vet. Medical J., 19: 72-78.

Saleha A.A.(1992) Psychrotrophic bacteria in pasteurized milk in Malaysia . Proceedings of the 3rd World Congress On Food borne infections and Intoxications , Berlin, Germany , 16-19 June 1992:1.pp190-192.

47

Sorhaug, T. and L. Stepaniak, 1997. Psychrotrophs and their enzymes in milk and dairy products: quality aspects. Trends Food Sci.Technol.,

8: 35-41.

Snedecor G.W .and Cochran E.G.(1989).Statistical Methods .8th Edn. The Iowa state university press, Ames ,Iowa, USA.

Tachibena, Y., et al. (1999). Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a Thermococcus sp. Applied Environmental Microbiology.65. 1991-1997.

Tapan, K et al (1998). Oxidation of Methyl-Substituted Naphthalenes: Pathway in a Versatile Sphingomonas Paucimobilis Strain. Applied and Environmental Microbiology. 64. 1884-1889.

The Ohio State University Food Science and Technology Department. Lactic Acid Bacteria - Carbohydrate and Protein Metabolism.

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pradipjntu.files.wordpress.com · Web viewThe whey proteins constitute about 18 percent of the protein content of milk. Casein, a protein found only in milk, contains all of the essential

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