Since ancient ages milk and milk products have been used by the human population. During the past few decades, with regard to living standards, diet, hygiene and usage of antibiotics, developing countries have been changing fast. Society considers the emotional and cultural importance of milk, the very first food of mammals including humans. Milk has been considered as nature’s most perfect food for humans since they have been habituated with it since birth. An important constituent of a balanced diet for human beings includes milk and dairy products, which have long been recognized as these products provide a wide range of essential nutrients.

Milk and milk products serve as a prime habitat to an immense variety of microorganisms and their presence in milk leads to the large-scale variations in attributes of milk like odor, taste and quality of milk. Adulteration in milk and dairy products by the presence of microorganisms arises from inside of udder, outside of the udder, by vendors and equipment which is used for storage to transport to long distances. In the udder of a mammal, milk is a sterile substance but as soon as milk passes out of the teat of the cow, it is inoculated by normal flora of the animal. The origin of pathogenic microorganisms present in milk may be from a cow, or it may be in humans, and in some cases it may be transmitted by both.

Pathogenic bacteria in raw milk are also involved in the spoilage of milk and these pathogens are predominantly resulting from unclean conditions and poor handling. These pathogenic microorganisms in raw milk are responsible for many milk-borne illnesses like brucellosis, typhoid fever and tuberculosis especially in developing countries. Drinking milk arises most of the milk-borne disorders that are caused by Listeria monocytogenes, Salmonella sp., Campylobacter sp., Staphylococcus aureus, Yersinia sp., Escherichia coli and Clostridium botulinum.

Boiling of milk in developing countries is a common practice and it increases the shelf life of milk as well as enhances the taste of the milk. [1] reported that when raw milk was exposed to boiling for 15 seconds, there is a noticeable decrease in total bacterial load. In colloidal forms in milk, lipids and proteins have a shielding role in microorganisms against heat treatments given to kill microorganisms, consequently some microorganisms are expected to survive in milk even after boiling.

Pasteurization is defined as a thermal process used to reduce health risks from pathogenic microorganisms present in milk and also to lengthen shelf life of the product. To treat different cases of mastitis various antibiotics are used. To cure diseases of cattle, antibiotics are usually used. The excessive use of these antibiotics has made antibiotic treatment ineffective and has managed to develop multiple antibiotic resistances among microorganisms causing diseases. Milk serves as an efficient vehicle for transmission of diseases to humans and approximately 90% of dairy-related diseases in human beings arise from unhygienic milk products. There are regulations that require proper hygienic handling of milk and its Pasteurization to protect public health against milk-borne infections.

Material and methods:

Isolation of bacterial colony

20 ml milk sample was equally divided into two parts in sterile test tubes each containing 10 ml. One part of the sample (10 ml) was boiled for 15 minutes at 100° C to prepare boiled Pasteurized milk. Both boiled and un-boiled Pasteurized milk samples were serially diluted from 10^-1 to 10^-10 and plated. The plates were then incubated at 37°C for 24 hours. After 24 hours the CFU value was then calculated. The single colonies formed in the spread plate were taken to prepare a pure culture of these microorganisms.

Staining

The pure cultures were taken for Gram staining to detect the Gram characteristics of the isolated microorganisms.

The pure cultures were taken for endospore staining also to ensure the presence of spore-forming bacteria in milk. 72 hours old pure cultures grown without glucose medium, were taken for this staining process in order to get more number of endospores.

The pure cultures were tested for catalase activity. This test was done in a clean, dry and grease-free glass slide by observing the evolution of oxygen bubbles. The organisms that showed the bubble formation is considered positive for the presence of catalase enzyme, that is catalase positive.

The pure cultures were used for the oxidase test. A small piece of filter paper was soaked in 1% Kovacs Oxidase reagent and dried. A well-isolated colony from a fresh bacterial plate was picked by using an inoculating loop and rub onto treated filter paper. Then observed for the colour changes.

Conclusions:

The isolated pure cultures were screened by their colony colour, colony formation, colony elevation, and colony margin. The Gram characteristics and shape of these microorganisms also studied to ensure the screening process. Endospore staining of these microorganisms showed the thermostability of these organisms and the ability to form spores in adverse conditions.

The catalase test ensures the presence of hydrogen peroxidase enzyme in the microorganisms. So the microorganisms showed positive results might be from these genera- (Staphylococci, Micrococci, Listeria, Corynebacteriumdiphtheriae, Burkholderiacepacia, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia), Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus, and Rhodococcus).

The Oxidase test result confirms that the positive microorganisms are from Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella, Pasteurella, Moraxella, Helicobacter pylori, Legionella genera and the negative organisms are from Enterobacteriaceae genera.

This study presents a thorough microbiological study of boiled and un-boiled Pasteurized milk. Though it is a preliminary study, but ensures a positive contribution in the study of milk microbiology.

Funding and Acknowledgement:

The study was supported by Department of Higher Education, Science and Technology and Biotechnology, Government of West Bengal, India (BT/ST/P/S&T/1G-09/2017).The authors would like to take the opportunity to thank the Principal, Dr. Rajyasri Neogy and Department of Microbiology, Vijaygarh Jyotish Ray College for providing all sorts of support for conducting the work.

References:

[1]. Ahmed M.M. Metwally, Nadia M.A.Dabiza, Wagih I.El-Kholy and Zeinab I. Sadek. The Effect of Boiling on Milk Microbial Contents and Quality. Journal of American Science 2011;7(2):110-114.

[2]. Muhammad Shoaib, IqraMuzammil, Muhammad Hammad, Zeeshan Ahmad Bhutta and IshratYaseen. A Mini-Review on Commonly used Biochemical Tests for Identification of Bacteria. International Journal of Research Publications 2708-3578.

[3]. Nada K. Alharbiand Albandary Nasser Alsaloom. Characterization of Lactic Bacteria Isolated from Raw Milk and Their Antibacterial Activity against Bacteria as the Cause of Clinical Bovine Mastitis. Journal of food quality. vol. 2021ID 6466645

[4]. Nazia Afrin*, S.M. Rokon-Ud-Doula and Rasheda Yasmin Shilpi. Detection of pathogenic bacteria from raw, pasteurized and UHT milk available in the local market of Gazipur District. Jahangirnagar University J. Biol. Sci. 7(2): 13-19, 2018

[5]. NazishMazhar Ali, Khadija Sarwar, Syed Abdullah Mazhar, IramLiaqat, SaiqaAndleeb, BushraMazhar and BushraKalim. Effect of medicinal plants, Heavy metals and antibiotics against pathogenic bacteria isolated from raw, Boiled and pasteurized milk. Pak. J. Pharm. Sci., Vol.30 No.6, November 2017, pp.2173-2182

[6]. Qinyuan Li, Xiu Chen, Yi jiang and Chenglinjiang. Cultural,physiological and biochemical identification of Actinobacteria. Actinobacteria basics and biotechnological applications. Doi: 10.5772/61462

[7]. Samia Afrin, Md. Rezwanul Habib, Mohammad Ashiqul Islam and Md. Harun-ur-Rashid. Physical, chemical and microbiological qualities of dahi collected from Bogra and Mymensingh district of Bangladesh. Asian Australas. J. Biosci. Biotechnol. 2016, 1 (1), 134-140

[8]. Svetlana Ferreira Lima, Marcela Lucas de Souza Bicalho and Rodrigo CarvalhoBicalho. Evaluation of milk sample fractions for characterization of milk microbiota from healthy and clinical mastitis cows. PLoS ONE13(3):e0193671

[9]. T.C. Venkateswarulu, K. Abraham Peele, S. Krupanidhi, K. Prakash Narayana Reddy, M. Indira,A. Ranga Rao, R. Bharath Kumar, K. VidyaPrabhakar. Biochemical and molecular characterization of lactase producing bacterium isolated from dairy effluent. Journal of king saud university – 32 (2020) 1581-1585

[10]. Tassadaq Hussain, AneelaRoohi, ShehzadMunir, Iftikhar Ahmed, Jafar Khan, Veronique Edel-Hermann, Kil Yong Kim and Muhammad Anees. Biochemical characterization and identification of bacterial strains isolated from drinking water sources of Kohat, Pakistan. African Journal of Microbiology Research. Vol. 7(16), pp. 1579-1590

[11]. Xueru Wang, Yang Liu and Jun Sun. Physiological and Biochemical Characterization of Isolated Bacteria from a Coccolithophore Chrysotiladentata (Prymnesiophyceae) Culture. Diversity 2022, 14, 2.