Discovery Science • RESEARCH • FOOD SCIENCE

Shah et al. Bulging Effect: Impact of thermophiles in pasteurized milk pouches, Discovery Sci., 2013, 4(12), 34-39, www.discovery.org.in www.discovery.org.in/ds.htm © 2013 Discovery Publication. All Rights Reserved

Page

34

Shah BP1, Shah DR2, Shah RR3, ChauhanDivyesh4☼, ChauhanParesh5

1. Dean & Principal, S.M.C College of Dairy Science, Anand Agricultural UniversityCampus, Anand, Gujarat, India 2. Managing Director,Vidya Dairy, Anand Agricultural University Campus, Anand, Gujarat, India 3. Assistant Professor, ADIT Engineering college, Vallabh Vidyanagar, Gujarat, India 4. Microbiologist, Vidya Dairy, Anand Agricultural University Campus, Anand, Gujarat, India 5. Chemist, Vidya Dairy, Anand Agricultural University Campus, Anand, Gujarat, India ☼Corresponding author: Divyesh Chauhan, Microbiologist, Vidya Dairy, Anand Agricultural University Campus, Anand, Gujarat, India. Mail: dchauhan3116@yahoo.com, Mobile No.(+91)08140350847 Received 16 March; accepted 10 May; published online 01 June; printed 16 June 2013

ABSTRACT A study was undertaken to check the protagonist possessions of thermophilic bacteria in defect development under prolonged incubation of packed milk pouches at 55oC temperature for 36 hours. Total thermophilic count, Total Mesophilic count, Coliform, Yeast and Mold along with MBRT tests were conducted at the time interval of on hour. The bell shaped graphical growth pattern was examined for the parameters including total thermophilic count and total mesophilic count. The counts initially increased up to 25 hours and gradually reduced at the end of 36 hours. Total viable cells also affect MBRT time resulting in to “U” shapedgraphical pattern examined for the MBRT test. Coliforms were not detected in initial hours but examined its presence in mid hours between 6th to 20th hours, which were gradually reduced and vanished due to accumulation of acid and gas resulting in to imbalance of pH. It was also observed that fungi did not tolerate 55oC temperature and disappeared gradually due to lack of adaptation resulting in to impotent to cultivate at 55oC temperature after 5 hours. Pure, partial or combined influence of total microbial activity at 55oC temperature in pasteurized milk leads to development of numerous sensory defects including acidic flavour, bitter taste, inexpressive and unpleasant flavour along with physical defects like gassy curd formation and bulging effect on packaging materials progressively. Key Words: Pasteurized milk, High temperature, Thermophilic Bacteria, Microbial Activity, Defects development, Bulging Effect. Abbreviations: C-Centigrade, MBRT- Methylene Blue Reduction Test, O-R- Oxidation Reduction, CFU-Colony Forming Unit, ml- Milliliter

1. INTRODUCTION Milk is a major part of food consumption and plays a protuberant role in routine diet throughout the world. The milk provides a high nutritious substrate that can support a wide variety of bacteria and other microbes for their growth and reproduction (Philips et al. 1990).The hygiene control of fluid milk is necessary to provide the public with safe, wholesome and high quality beverage. It involves many careful and controlled steps, irrespective of whether the end result is fluid milk, cream or any other of the numerous milk products. It is concerned with such matters as the health of the cow, the health of farmers, the condition of the farm, the milking operation, delivery of milk to the dairy plant, processing control of the milk and eventually delivery to the home. Proper Pasteurization destroys major possible pathogens and reduces the number of viable organisms in the milk (Cole et al.1955). If proper cleaning and sanitizing protocols are not followed for pasteurizing equipment, an increase in certain types of organisms may possible, resulting in to a build-up of milk solids in the equipment. This is particularly true in long hold pasteurization method, where one pasteurizer may be used several times in a day for pasteurizing different batches of milk without being cleaned between two batches. If the original raw milk contains such types of organisms that are resistant to heat or capable of growth at pasteurization temperature, the chance of this type

of bacteria buildup in the equipment is increased. This type of bacteria belongs to thermophilic or thermoduric strains. Thermophile is a group of organisms that thrives temperature between the ranges of 50 oC TO 70 oC temperature. Unlike other types of bacteria thermophiles can survive at much hotter temperature, where other bacteria would be damaged and sometimes killed if exposed to same temperature. Thermophilic organisms having the set of proteins and membrane properties which retains unusually stable at the extreme high temperature. As a prerequisite for their survival, thermophiles contain enzymes that can function at high temperature.

RESEARCH • FOOD SCIENCE Discovery Science, Volume 4, Number 12, June 2013

Science Bulging Effect: Impact of thermophiles in pasteurized milk pouches

ISS

N 2

278

– 54

85

E

ISSN

227

8 –

5477

discovery

Comparison: It is a performance of valuation or assessment of effects side by side in command to realize to what degree they are related or diverse. It is may be used to convey out resemblances or alterations amongst two things of the identical type typically to determine crucial features or sense either systematically or otherwise. Content: The amount of things described here can be write or speak about in a form of book, an article, a programme or speech, etc.

Pasteurization: Pasteurization refers to the process of heating every particle of milk to at least 63 oC for 30 minutes or 72oC for 15 seconds, in approved and properly operated equipment. After Pasteurization the milk is immediately cooled to 5oC or below.

Thermophile: Thermophile is an organism — a type of extremophile - that thrives at relatively high temperatures, between 45 and 122 °C (113 and 252 °F).

Discovery Science • RESEARCH • FOOD SCIENCE

Shah et al. Bulging Effect: Impact of thermophiles in pasteurized milk pouches, Discovery Sci., 2013, 4(12), 34-39, www.discovery.org.in www.discovery.org.in/ds.htm © 2013 Discovery Publication. All Rights Reserved

Page

35

2. SCOPE OF THE STUDY The objectto carry out this study is to enumerate thermophilic bacteria, their protagonist possessions in defects development and Influence of high temperature (55oC) on O-R potential (Eh value) of methylene blue dye in pasteurized milk under prolonged incubation at 55oC temperature. An attempt was also made to study the impact

of high temperature (55oC) on the growth pattern of coliform, total mesophilic count and Total fungal load.

3. MATERIALS & APPARATUS -Sterile Petridishes (Dishes-Culture, Petri, BOROSIL). -Sterile 2.2 ml Pipettes (Graduated BOROSIL, 7056).

Table 1 Thermophilic count of pasteurized

Hour MBRT (Hours.)

Total Thermophilic Count (cfu/ml)

Coliform Count

(cfu/ml)

Total Mesophilic

Count (cfu/ml)

Total Yeast & mold Count

(cfu/ml) Sensory Analysis/

Organoleptic Taste. Bulging Effect /Condition of Milk Pouch.

0 Hour 7:00 6,00 0 8,000 7 Fresh & Pleasant Flavour O.K. 1stHour 6:50 7,00 0 7,000 6 Fresh & Pleasant Flavour O.K. 2nd Hour 6:50 1,200 0 9,000 3 Fresh & Pleasant Flavour O.K. 3rdHour 6:20 2,000 0 10,000 1 Fresh & Pleasant Flavour O.K. 4th Hour 6:15 4,600 0 12,000 2 Fresh & Pleasant Flavour O.K. 5thHour 6:05 7,600 0 10,000 0 Fresh & Pleasant Flavour O.K. 6thHour 5:55 11,300 3 9,000 0 Fresh & Pleasant Flavour O.K. 7th Hour 6:10 13,200 5 11,000 0 No Freshness O.K. 8thHour 5:45 16,500 7 12,000 0 No Freshness O.K. 9thHour 5:30 18,900 11 14,000 0 No Freshness O.K. 10thHour 5:35 36,000 17 17,000 0 Slight Acidic O.K. 11th Hour 5:40 68,000 19 21,000 0 Slight Acidic O.K. 12thHour 5:20 1,20,000 11 27,000 0 Slight Acidic O.K. 13th Hour 5:10 1,98,000 10 36,000 0 Acidic O.K. 14thHour 5:05 2,81,000 9 47,000 0 Acidic Slightly Bulged 15thHour 4:50 3,70,000 11 59,000 0 Highly Acidic Slightly Bulged 16th Hour 4:45 7,40,000 12 67,000 0 Highly Acidic Slightly Bulged

17thHour 4:00 14,10,000 8 71,000 0 Curdle and Unpleasant Flavour Slightly Bulged

18thHour 3:00 15,20,000 6 1,12,000 0 Curdle and Unpleasant Flavour Slightly Bulged

19th Hour 2:45 30,00,000 5 1,17,000 0 Curdle and Unpleasant Flavour Slightly Bulged

20thHour 2:20 61,00,000 1 2,11,000 0 Curdle + Bitter + Unpleasant Flavour Highly Bulged

21stHour 2:00 1,07,00,000 0 3,00,000 0 Gassy Curd + Bitter + Unpleasant Flavour Highly Bulged

22nd Hour 2:00 2,07,00,000 0 6,80,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour Highly Bulged

23rdHour 1:45 4,00,00,000 0 9,10,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour Highly Bulged

24thHour 1:30 7,00,00,000 0 10,20,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour Highly Bulged

25thHour 1:40 6,00,00,000 0 17,20,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour Highly Bulged

Table 1 Thermophilic count of pasteurized (Continues)

Hour MBRT (Hours.)

Total Thermophilic Count (cfu/ml)

Coliform Count

(cfu/ml)

Total Mesophilic

Count (cfu/ml)

Total Yeast & mold Count

(cfu/ml) Sensory Analysis/

Organoleptic Taste.

Bulging Effect /Condition of Milk Pouch.

26th Hour 1:35 2,89,,00,000 0 13,00,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

27thHour 1:40 2,01,00,000 0 11,00,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

28th Hour 2:05 1,15,00,000 0 5,00,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

29th Hour 2:40 1,09,00,000 0 2,00,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

30thHour 4:00 60,00,000 0 60,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

31stHour 5:00 40,00,000 0 64,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

32nd Hour 6:30 13,00,000 0 49,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

33rdHour 8:00 7,00,000 0 47,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

34thHour 8:15 4,00,000 0 31,000 0 Gassy Curd + Bitter +Putrid Extremely Bulged

35thHour 10:00 1,00,000 0 39,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

36th Hour 13:00 70,000 0 45,000 0 Gassy Curd + Bitter +Putrid + Unpleasant Flavour

Extremely Bulged

Discovery Science • RESEARCH • FOOD SCIENCE

Shah et al. Bulging Effect: Impact of thermophiles in pasteurized milk pouches, Discovery Sci., 2013, 4(12), 34-39, www.discovery.org.in www.discovery.org.in/ds.htm © 2013 Discovery Publication. All Rights Reserved

Page

36

-Violet Red Bile Agar(M049, Himedia):- Prepared according to Supplier’s direction. - Potato Dextrose Agar(M096, Himedia):-Prepared according to Supplier’s direction. -Standard Plate Count Agar (M091A, Himedia):-Prepared according to Supplier’s direction. -Agar-Agar Powder -Standard Gram’s Reagent Kit (K001, Himedia). -Standard tubes for Methylene Blue Reduction Test (Tubes, 15 X 150, 9800, BOROSIL) -Phosphate Buffer: The buffer was prepared by dissolving 34 grams of Potassium Dihydrogen Orthophosphate (Qualigens, 13405) in 1000 ml of distilled water with final pH 7.2 used as the stock phosphate buffer and further diluted it by taking 1.25 ml of stock solution of phosphate buffer in 1000 ml of distilled water in volumetric flask (Flask Volumetric, BOROSIL,5640). 9.2 ml of same buffer distributed in test tubes (15 x 150 mm) and stoppered with non-absorbent cotton followed by autoclave at 121 oC for 15 lbs for 15 minutes. -Methylene Blue Dye: The Dye solution was prepared according to supplier’s direction (PROLABO, 330733). The dye solution was placed in amber colour bottle in a dark and cool place to avoid photoreaction.

4. METHODOLOGY 4.1. Collection and sampling of Pasteurized Milk Samples A total 36 pouches of Standard Pasteurized Milk(Fat:4.5 %, SNF:8.5 %, Pack size:-200ml) of one particular lot was selected for the study purpose from the cold storage(Temperature 5.0oC) of Vidya Dairy, Anand, Gujarat, India. Samples were brought to microbiological laboratory for the evaluation of microbial quality characteristics under prolonged holding of pasteurized milk at 55oC temperature. The samples were stored in atypical type of incubator (Figure 1) and removed for a few minutes for sampling at the interval of1hour. The samples were analyzed at each hour for different parameters including thermophilic count, total mesophilic count, coliform count, total fungal count, methylene blue reduction test, examination of physical defects along with sensory evaluation.

4.2. Enumeration of Thermophilic Counts From the collected sample, 1 ml of pasteurized milk transferred in 9 ml of phosphate buffer to make primary dilution (10-1). Then a series up to 10-6 dilution was prepared by transferring primarydilution (1ml) into test tube containing sterile phosphate buffer (9ml) to obtain 10-2dilution and repeating the operations with sterile diluents(9 ml) using the 10-2dilution and further dilutions to obtain 10-3, 10-4,10-5and 10-6 dilutions. Samples (1 ml) of 10-1,10-2 10-3,10-4,10-5 and 10-6were transferred in to petridishes(in duplicate) through sterile pipettes(2.2 ml) and warmed(42oC±0.5oC) sterile plate count agar(15 ml, Agar concentration 2.0 %) was evenly mixed with the inoculum.The plates were allowed to get solidified at 25oC for 10 minutes followed by incubation at 55oC ±0.5oC for 48 hours.Parallel to these plates, control plates were also run without inoculum to check sterility of Plate Count Agar medium. The petridishes having 30-300 colonies were selected to enumerate thermophilic counts.

4.3. Enumeration of Mesophiles, Coliforms and Fungal Count Same sampling and dilution plan implemented as 4.2 for the parameters includingtotal mesophiles count, coliforms and total fungal count. Standard protocol was employed for cultivation and enumeration of coliform count using Violet Red Bile Agar, total mesophilic count using Plate Count Agar (Agar concentration 1.5%) and for total fungal load using Potato Dextrose Agar. The plates were allowed to get solidified at 25oC for 10 minutes. Petridishes containing Violet Red Bile Agar and Plate Count Agar were incubated at 37oC±0.5oC for 48 hours whereas petridishes containing

Potato Dextrose Agar were incubated at 25 ±0.5oC for 6 days. Parallel to these plates, control plates of respective media were also run without inoculum to check sterility of respective medium.

4.4. Methylene Blue Reduction Test In pre-sterilized test tube (15 x 150 mm) 10ml of milk sample was taken followed by addition of 1.0 ml of methylene blue dye. The air tight rubber cocks were placed at the opening of the test tube to avoid entrance of atmospheric oxygen. Tubes were gently inverted three times for even distribution of dye content in milk. The tubes were placed in water bath at 55oC ±0.5oC. At the interval of half an hour, each tube was examined for discoloration of dye.

5. RESULTS & DISCUSSION Thermophilic count of pasteurized milk was evaluated and results are presented in Figure 1. A wide distinction was observed in thermophilic count upon prolonged incubation at 55oC ±0.5oC for 48 hours. At 0 hour, the thermophilic load was 600 cfu/ml which was extended up to 4,600 after 4 hours upon incubation at the 55oC ±0.5oC temperatures (Table 1). The drastic increase count was observed in between 10 hours 36,000 cfu/ml to 24 hours7, 00, 00,000 cfu/ml. Freshness and pleasant flavour of pasteurized milk was partially inexpressivebetween 7 to 9 hours but after 10 hours, some defects of acidic flavour, gassy curd (Figure 3), clot formation (Figure 4) and bulging effect (Figure 5) were examined. The pleasant flavour of milk was majorly deteriorated between 17 to 26hours as would be expected merely by looking at the plate counts of Thermophiles. The general trend in most samples examined was for the count to become great by 6-25 hours but after 25 hours the counts were gradually decrease up to 70,000 cfu/ml after 36 hours (Graph 1).

Thermoduric organisms in milk are referred to be the heat resistant Micrococci and Microbacteria. However, the spore forming bacteria are truly heat resistant organisms, and on rare occasions these have been the cause of high count in pasteurized products. These spore forming organisms can cause spoilage of pasteurized milk. When this type of spoilage occur the milk usually becomes putrid. The spore forming bacteria are present in soil, dust; grains etc. and easily contaminate the raw milk if proper sanitary and milking practices are not followed on the farm. The presence of spore forming bacteria is indicated by the formation of gassy curd in the sample (Figure 3 & 4).The presence of these bacteria in milk is undesirable and is indicative of the presence of dirt and dust in pasteurized milk . Almost all of the thermophilic aerobic spore forming bacteria can produce more or less acid from a wide variety of carbohydrates and related compounds which is present in the pasteurized milk.The most conspicuous conversion of nitrogenous compound such as casein, whey protein and peptide molecules present in the milkare effected by thermophilic aerobic spore forming bacteria which may leads to proteolysis, ammonification, and nitrification. The quite early investigation of thermophilicbacteria, it was found that hydrolytic enzymes resistant to high temperature could be separated from the culture filtrates of thermophilic bacteria. Certain bacteria i.e. Bacillus stereothermophilus contains malic dehydrogenase, cytochrome oxidase, cytochrome b, cytochrome c, succinooxidase, aldolase and aspyrase which may give thermo stability to an organisms (Georgic et al.1951). Thermophiles can be discriminated from mesophiles from genomic features. For example, the Guanine : Cytocine content levels in the coding regions of some signatures genes were consistently identified as correlated with the temperature range condition when the association analysis was applied to Mesophilic and thermophilic organisms regardless of their phylogeny, oxygen requirement, salinity, or habitat conditions. DNA molecules of thermophiles are not denatured at high temperature due to high content of Guanine: Cytocine which are inter-connected with each other by triple hydrogen bonding. The thermostability is also based on protein’s composition and sequence of amino acid, which determines

CFU: The cell or aggregate of cells which gives rise to a single colony into the plate count technique. Abbreviation: CFU (colony forming unit).

Coliform: Coliforms are Gram-negative, rod-shaped facultatively anaerobic bacteria. Identification criteria used are production of gas from glucose (and other sugars) and fermentation of lactose to acid and gas within 48 h at 37ºC.

Discovery Science • RESEARCH • FOOD SCIENCE

Shah et al. Bulging Effect: Impact of thermophiles in pasteurized milk pouches, Discovery Sci., 2013, 4(12), 34-39, www.discovery.org.in www.discovery.org.in/ds.htm © 2013 Discovery Publication. All Rights Reserved

Page

37

the strength of bonds that form within protein molecule and thus regulate its three dimensional structure, covalent disulphide bond, hydrogen and other weak bond, prevents denaturation of protein (Patel at al. 2004).

Influence of Thermophilic bacteria on Methylene Blue Reduction Time is presented in the Figure 2. It was witnessed that under the condition of incubation when milk pouch samples preserved at 55oC ±0.5oC, the MBRT was 7:00 at 0 hour which was gradually reduced up to 1:30 hours at 24hours. But after 24 hours the MBRT timing is further raised to 13 hours at the end of 36 hours. The “U” shaped graphical pattern for Methylene Blue Reduction Test was

examined (Graph 2.) in pasteurized milkwhich may be due to typical growth pattern and growth curve. In initial phase MBRT was higher which was gradually decrease due to accumulation of metabolites including mixed acid and gas formation resulting in to pH imbalance laid to bacterial state in decline phase. Due to admission of microbes in decline phase, duration of MBRT was further increased due to deficiency of viable bacteria in sample which generally utilizes oxygen. It was also assumed that such type of pattern in MBRT may be the result of pure, partial or combined effect of microflora which affects Eh value (Oxidation-Reduction Potential) of Methylene Blue dye.

Figure 1 Incubation of Pasteurized milk at 55o C (Bulging Effect)

Figure 2 MBRT (Initial & After)

Figure 3 Gas formation in MBRT Test

Figure 4 Physical defect: Curd Formation

Figure 5 Bulging Effect (Right to Left), Pouch No.1: None, Pouch No.2: High Bulging, Pouch No.3: Slight Bulging

Discovery Science • RESEARCH • FOOD SCIENCE

Shah et al. Bulging Effect: Impact of thermophiles in pasteurized milk pouches, Discovery Sci., 2013, 4(12), 34-39, www.discovery.org.in www.discovery.org.in/ds.htm © 2013 Discovery Publication. All Rights Reserved

Page

38

Coliform counts in pasteurized milk, occasionally used as an index of milk quality are expected to increase with the standard plate count. High coliform counts confirm the poor sanitation practices. The genera comprises Escherichia coli, Enterobacter aerogenes, Klebsiella spp. Relevant to coliform load, it was inspected that on 0 hour, coliform count was not

detected but gradually they were raised up to 3 cfu/ml at the end of 6 hours which was further raised to 19 cfu/ml at 11 hours. But after 11 hours, coliform counts were gradually reducedto 0 cfu/ml at the end of 36hours (Graph 3). Similar trend was scrutinized for total mesophilic count which was 8,000 cfu/ml at 0 hour which extended up to 17,20,000 cfu/ml at the end of 25 hours but gradually decreased up to 45,000 cfu/ml at the end of 36 hours (Graph 4). In the case of yeast and mold Count, at 0 hour counts were 7 cfu/ml which were not further raised due to lack of adaptation of fungal cells at high temperature (Graph 5). High temperature causes rupture of hydrogen bonds in protein and DNA. This result in to denaturation of proteins and melting of DNA due to breakage of hydrogen bonding between Guanine: Cytocine as well Adenine: Thymine. Cell membrane is also disrupted at high temperature resulting in to death or inhibition of organisms including psychrophiles, fungi and certain groups of mesophiles. It is the clear cut influence of high temperature on metabolic and enzymatic regulations of total microflora.

7. CONCLUSION From the results, certain evidence came out which exhibits the influence of higher temperature (55oC ±0.5oC) on the quality of pasteurized milk. The thermophilic load is increased upon prolonged holding of pasteurized milk at the 55oC ±0.5oCtemperatures. Impact of thermophiles and total microbes present in pasteurized milk on O-R potential gives a graphical trend of “U” shape due to particular growth pattern and growth curve. Bell shaped graphical trend was examined for total thermophilic count, coliform count and

Graph 2 Influence of Thermophilic Bacteria on Methylene Blue Reduction Test

Graph 1 Hour wise growth pattern Total Thermophilic bacteria in Pasteurized Milk

Graph 3 Hour wisegrowth pattern of Total Coliform bacteria in Pasteurized Milk

Graph 4 Hour wise growth pattern of Total Coliform bacteria in Pasteurized Milk

Graph 5 Hour wise growth pattern of Total Yeast and Mold in Pasteurized Milk

Discovery Science • RESEARCH • FOOD SCIENCE

Shah et al. Bulging Effect: Impact of thermophiles in pasteurized milk pouches, Discovery Sci., 2013, 4(12), 34-39, www.discovery.org.in www.discovery.org.in/ds.htm © 2013 Discovery Publication. All Rights Reserved

Page

39

total Mesophilic count under prolonged incubation of milk pouches at 55oC ±0.5oC. Longer storage of pasteurized milk i.e. 36 hours at higher temperature may result in to development of physical defects like acidic flavour, gassy

curd clot formationand bulging effect. It may be the purely, partially or combined effect of total microbial load present in pasteurized milk.

SUMMARY OF RESEARCH The study point out the effect of thermophilic bacteria on the quality of pasteurized milk upon extended incubation at 55oC temperature for 36 hours. The thermophilic bacteria and other microbial flora which can resist and reproduce at 55 oC leads accumulation of certain metabolites resulting in to undesirable changes in pasteurized milk i.e. development of off flavours and off taste including bitter, putrid along with physical defects including gassy curd, clot formation and bulging effect in packedpasteurized milk pouches. FUTURE ISSUES From the results it can be deduced that extended storage of pasteurized milk at 55oC temperature may raisenumerous defects. The climatic condition of India, especially in summer season the ambient temperature remains nearer to 42 oC during noon period, any deviation in cold chain during dispatch activity may leads to economic losses of productivity which may result in to flood of consumer’s complaints. The knowledge relevant to presence and numbers of thermophilic bacteria is useful to determine dispatch activity, cold chain maintenance and distribution strategies of pasteurized milk in market. DISCLOSURE STATEMENT The experimental work described here was successful completed under the financial assistance of Vidya Dairy, Anand Agricultural University Campus, Anand-388 001, Gujarat, India. PHOTO GALLERY To the casual spectator, photographs displayed in this research work may appear to be magnitude work. To exhibit photographs at this degree requires a network of solid support and it was delivered by Sunny Kharwa. We are indebted grateful to him for his unusual aptitude and boost us with his special attention and care. ACKNOWLEDGEMENT Much thanks to our colleagues Bhaskar Trivedi, Harshad Oza and Nikunj Patel for their constructive criticism, valuable suggestions and their assistance as active listener, Quick thinker and win-win negotiator.

REFERENCES

1. Phillps J, Grffith M. Pasteurized Dairy Products: Constraints imposed by environmental contamination from environmental sources. Advances in Environmental Science and Technology. 1990, 387-456

2. Cole C. Food Processing. 1955, 16, 54

3. Georgi C, Militzer W. The existence of a cell granule in thermophilic bacterium. Bilo.Me.1951, 76, 598-601

RELATED RESOURCES

1. American Public Health Association, 1978, 7, 107-113 2. Patel R, Patel K. Experimental Microbiology. 2004, 1 , 129-

130 3. Marsh C, Militzer W. Thermal Enzymes V Properties of

malic dehydrogenase. Arch. Biochem. Biophys. 1952, 36, 269-275

4. Militzer W, Burans L. Thermal Stability of Pyruvic oxidase. Fedration Proc. 1952, 11, 260

5. Militzer W, Tuttles L. Thermal enzymes III. Arch. Biochem. Biophys. 1951, 31, 416-423