study of diversity in the micro flora of different types of milk · milk is a water-based emulsion...

`International Journal of All Research Education and Scientific Methods (IJARESM), ISSN: 2455-6211

Volume 8, Issue 10, October-2020, Impact Factor: 7.429, Available online at: www.ijaresm.com

IJARESM Publication, India >>>> www.ijaresm.com Page 256

Study of diversity in the micro flora of different types of

milk

N.S. Raja1*, G. Aravinda Lochan

2

1,2 Department of Genetic Engineering SRMIST Kattankulathur, Tamilnadu, India

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ABSTRACT

Raw milk consists of heterogeneous microorganisms having the capacity to colonize the GIT either permanently or

transiently. These bacteria may engage in generating a healthy immune system. This form the indication for

carrying out our research work on the characterization of microflora in different milk types. Native buffalo milk

was collected in sterile conditions and commercially available pasteurized milk from Arokya, and Thirumala brands

were also used for the study. Spread plates of different milk samples were prepared in nutrient media. Quadrant

streaking method was used to obtain singles colonies. These single colonies cultured in LB medium for conducting

biochemical tests. Single colonies obtained from the quadrant plate were tested with colony PCR method using

universal 16sRNA sequencing primers. The amplified product was sequenced using sanger sequencing method. The

results obtained were analyzed using BLAST and presence of pseudomonas aeruginosa was confirmed in the milk of

buffalo which is due to improper hygienic condition of animal, surrounding and vessel.

Keyword: microflora, milk, bacteria, BLAST, PCR, gastro intestinal tract

INTRODUCTION

The food we eat and the microbes present in it influence the constitution of the gut microflora in the lower areas of the

GIT[1]. The modernized, clean living conditions have drastically changed the way the body responds to common antigens found in the environment[2]. Milk is a water-based emulsion of fat globules that contains dissolved carbohydrates, protein,

and minerals[3]. The pH of milk ranges from 6.4 to 6.8and it changes over time[4]. Milk from other bovines and non-

bovine mammals varies in composition, but has a similar pH [5]. From an early age, milk happens to be one of the key

foods to a growing child. Milk consumption aid increased ability in satiating hunger[6], promoting muscle growth[7],

reducing body fat[8], promoting stronger bones[9]and also act as an alternative source for calcium and vitamin D[10].

Studies have now shown that children consuming raw milk are less likely to develop immune disorders[11].Do Thi Kim

Lien et. al conducted one study and concluded that consumption of milk has benefited the children by decreasing the

chances of underweight and stunting and drastically improve the quality of life in childrens of rural Vietnam[12].

Raw milk consists of a variety of microorganisms having the capacity to colonize the GIT either permanently or

transiently[13]. Since, these bacteria may also engage in the development of a healthy immune system[14], this forms the

rationale for carrying out our research on the characterization of microflora in different milk types. The source for microflora in milk is developed form the microorganisms present within the adder glands of the animal, on the skin surface

of the adder and the vessel used to collect milk for storing and distributing the milk, even the surrounding play an important

role for the development microflora that is seen in the milk[15].

While the milk collected is stored under cold conditions certain P. sychrotrophic bacteria dominate the flora event though

its optimal temperature is higher. It was also demonstrated that these extracellular enzymes can endure pasteurization at

72°C for 15 sec and extreme temperature processing at 138°C for 2 sec or 149°C for 10 sec [16-18]. These not only tend to

spoil the milk by producing proteases and lipases but also have the capacity to infect the host as well as the

consumer[19].Considering all these factors responsible for the generation of microflora in milk we were motivated to

examine and characterize the microflora of various types of milk available in Potheri locality by obtaining bacterial

colonies from spread and quadrant plating on LB medium followed by biochemical test and 16sRNA PCR method. To identify the strain obtained from the milk samples and analyzed using BLAST, 16sRNA products were then given for

sequencing.



IJARESM Publication, India >>>> www.ijaresm.com 57

MATERIALS AND METHODS

Materials and sample collections:

Milk samples from buffalos were collected from Urpakkam and other pasteurized milk samples like AROKYA,

TIRUMALA were brought from the nearby retailer store in Potheri. While collecting the milk samples certain steps were

taken to maintain the sterility. The steps involve boiling the container with hot water that is used to collect the milk for 10-15 minutes before collecting the milk, using thermal flask to avoid milk spoiling due to the heat while transportation, usage

of gloves while collecting milk sample, usage of paraffin tapes to avoid interception of new microorganisms into the milk

sample. The milk samples were then transferred into sterile falcon tube inside the air flow chamber and sealed the cap with

paraffin tape.

Experimental methods:

Culturing:

Spread plates where prepared in nutrient agar plates by serial diluting the milk samples that where collected. Dilutions of 3,

5, 7and 10 were plated for each of the milk sample using air flow chamber and kept for overnight incubation at 37°C. The

selected colonies were subjected to quadrant streaking under air flow chamber and kept for overnight incubation at 37°C.

Here no selective media was use to ensure that all the microorganisms that are present in milk are obtained. Then the single

colonies were isolated from the quadrant plates using 200µl tip was kept into a test tube containing lb broth which is autoclaved. The tube was allowed to incubate for overnight at 37°C for the culture to grow.

CHARACTERIZATION AND EVALUATION:

Characterization studies were performed using the culture grown in LB broth. Catalase reactivity test was performed by

placing a drop of the LB cultures of different colonies (that were selected from quadrant plates.) on a slide to which few

drops of hydrogen peroxide was added and the results were noted. Grams staining test was performed using the gram

staining Hi-media kit and the results were noted.

PCR amplificationstudy:

The selected colonies from the quadrant plate where taken in an ependoff tube containing 20 µl of sterile distilled water inside air flow chamber. Then the tube was subjected to heating using water bath for 1 minute followed by centrifugation at

5000rpm for 5 minutes. Then the supernatant was taken as template for the colony PCR. The master mix was prepared for

50 µl containing the following Taq-.25 µl, buffer-5 µl, dNTP- 2 µl, sterile distilled water-39.75 µl, 16sRNA universal

forward primer-1 µl, 16sRNA universal reverse primer- 1 µl, template-1 µl. Amplification of the PCR were carried out in

the thermocycler by setting the conditions: 94°C for 5 mins, 35 cycles of 94°C for 1 minute, 59°C for 45 seconds, 72°C for

1minute and 72° C for 7 mins. The PCR products were separated on 1% agarose gel in 0.5x TBE buffer, stained with

ethidium bromide and the gel images were captured by using a gel documentation system (gel doc).

16s rRNA Sequencing and BLAST studies:

After PCR analysis the products were tested for sequencing using 16s rRNA sequencing and BLAST studies and finally the

sequence was confirmed by sanger sequencing.

RESULTS AND DISCUSSIONS

Culturing and separation of microbes:

The current research work mainly deals with the assessment of diversified microflora present in different sources of milks

obtained from local area. The microbial composition and analysis of various dairy products including raw milk, cheese and,

butter is topic of great interest and ample amount of work has also been done by several researchers[20]. In this research

work we have assessed the milk from three different sources including Arokya milk, Tirumala milk and buffalo milk.

The culturing was performed on spread plates containing nutrient agar by serial dilution of milk samples as described in

experimental section. The images of spread plates are shown in figure 1. The colonies of microorganisms can be visible in

all types of milk obtained from local market. Tirumala milk had shown very crowded and compacted microbial colonies (See Fig 1. B) as compared to other milks.




Figure 1:1 Image of spread plates of A) Arokya milk B) Tirumala milk andC) Buffalo milk

Then the single colonies were isolated from the quadrant plates using 200µl tip was kept into a test tube containing lb broth

which is autoclaved. The tube was allowed to incubate for overnight at 37°C for the culture to grow

Figure 2: Quadrant plates A)Arokya milk colony1,B) Arokya milk colony2,C) Arokya milk colony3,D)Tirumala

milk colony4, E)Buffalo milk colony5.

Figure2 shows the quadrant plates with colonies of microorganisms. In this way microorganisms are separated in the form

of various colonies from the local source of milk. In all plates the growth of microbial colonies were prominently observed.

Table 1 shows the details of bacterial colonies selected in this study.

Table 1: Bacterial colonies selection

S.NO Sample name NO. of colonies selected

1 Arokya milk sample 3

2 Tirumala milk sample 1

3 Buffalo milk sample 1




Catalase test for differentiation of microbes:

The differentiation of isolated microorganisms was carried out using different tests and techniques like catalase test, gram

staining, PCR amplification, 16s rRNA Sequencing.The catalase test is widely used in microbiology for differentiation of

catalase positive micrococcus and staphylococcal microbes from catalase negative streptococci species.The degradation of

hydrogen peroxide into oxygen and water is mediated by the enzyme catalase. When a small inoculum is inserted into

hydrogen peroxide, the presence of the enzyme in a bacterial isolate is apparent and the gradual formation of oxygen bubbles takes place[21-22]. It has been observed that colonies from 1 to 4 showed catalase negative bacteria while colony

five had catalase positive bacteria.

Figure 3: Catalase test Colonies 1-4 showed –ve for the test and colony5 showed +ve for the test

Gram staining:

A typical technique used to separate two large classes of bacteria based on their distinct constituents of the cell wall is gram

staining. By painting these cells red or violet, the Gram stain technique differentiates between Gram positive and

Gramnegative types [23].

Figure .4 Gram Staining results from the above observation Colonies 1-4 seem to be gram +ve and colony 5seem to

be gram –ve bacteria




In gram staining also first four colonies were found to be gram positive and fifth was observed as gram negative bacteria.

The differentiation was made on the basis of staining observed in cell wall.So the milk obtained from buffalo showed the

presence of gram negative and catalase positive bacteria based on the studies of gram and catalase testing respectively.

Table 2 shows the comparative results of microbial analysis.

Table 2: Tests conducted and results

Colony name Catalase test Gram test PCR

results

Species identified in

sequencing

Colony1 -ve +ve +ve Could be certain species of

lactobacillus from Catalase test

and Gram test but the exact

stain could not be predicted due

to sequencing

Colony2 -ve +ve +ve

Colony3 -ve +ve +ve

Colony4 -ve +ve +ve

Colony5 +ve -ve +ve peudomonas aeruginosa

PCR amplification, 16s rRNA Sequencing and BLAST studies: The selective amplification of DNA or RNA targets using the polymerase chain reaction is PCR amplification. With

repetitive thermal cycling, PCR facilitates the amplification of DNAsequencing in an exponential way. PCR enables several

millions of copies of DNA to be produced using heating and cooling cycles[24]. The detailed methodology is explained in

experimental section.

Figure .5 Colony PCR amplification gel imageL9-L13 Milk sample PCR products, L13-10 bands are seen and no

band seen in L9. L9- Arokya milk colony1, L10- Arokya milk colony2, L11- Arokya milk colony3, L12- Tirumala

milk colony4, L13- Buffalo milk colony5.

PCR products were separated on 1% agarose gel in 0.5x TBE buffer, stained with ethidium bromide and the gel images were captured by using a gel documentation system (gel doc).As can be seen from figure 5, there were no band was visible

in L9 and at rest of the places clear bands are visible and those were compared with 16sRNA universal forward primer band

obtained at L1.The colony PCR product that was amplified was sent for 16s rDNA sequencing. The sequencing obtained

was saved in fasta format. BlASTin NCBI was used to identify and analyze the species.




Table 3: 16s rRNA Sequencing results.

16s rRNA study confirmed that the sequence of microbes present in colony 5 was 99% matched with pseudomonas

aeruginosa and further it was verified with BLAST analysis and chromatogram imaging as shown in Figure 6 and 7.

Figure 6: BLAST results of colony 5 showing species of pseudomonas Aeruginosa

Figure 7: Chromatogram image after sanger sequencing for pseudomonas aeruginosa

Colony Microflora found

Colony5 The sequence 99% matched

with pseudomonas aeruginosa.

Colony4 Sample degraded







Sanger sequencing is a DNA sequencing process based on the selective integration by DNA polymerase of chain-

terminating dideoxynucleotides during in vitro DNA replication. In this study the sanger sequencing was done for colony 5

microbes which was confirmed as pseudomonas aeruginosa.G. Kaban et al. conducted study and identified lactic acid

bacteria and catalase positive cocci using catalase test and identified the microorganisms’ using such chemical test[25].

Similarly, S.Vaseekaran et al also identified thermostable α amylase producing bacterias using catalase test[26]. These studies confirmed that the chemical test also serves the purpose of identification of bacterias. So, in our study we have also

identified four colonies using chemical tests.

CONCLUSION

The study confirmed that colony 5 isolated form buffalo milk showed 99% matching genetic sequence to that

ofpseudomonas aeruginosawhich is agram negative and catalase positive bacteria. This microorganism is resistant to

number of antibiotics and main root cause of many infectious diseases.The presence of this harmful m microorganism may

be due to the improper hygienic condition of animal, surrounding and vessel.By this we can conclude that improper

maintenance of the cattle and the animal may be more dangerous, thereforeregular inspections of these cattle would help in

avoiding the spread of such harmful bacteria.

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study of diversity in the micro flora of different types of milk · milk is a water-based emulsion...

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