Isolation and characterisation of Lactobacillus species from sheep milk

 Isolation and characterization of Lactobacillus species from sheep milk

Abhinandan Patil ^{1, 4-5}, John Disouza ^{1, 7}, Archana Dhavalshankh ³, Chandrashekhar Mote ⁶, Shivaji Pawar ^{1, 2}

¹ Centre for Interdisciplinary Research, D. Y. Patil University, Kolhapur, (MS). India

² Centre for Innovative and Applied Research, Anekant Education Society, Baramati, (MS). India

³ Department of Pharmacology, D.Y.P.M.C, Kolhapur (MS). India

⁴ Chate Group of Education, Kolhapur (MS). India

⁵ Life Inspiration social foundation, Kolhapur (MS). India

⁶ Department of Veterinary Pathology KNP College of Veternary Science, Shirwal, (MS), India 

⁷ Tatyasaheb Kore College of Pharmacy, Warananagar (MS). India

* Correspondence author: Shivaji Pawar

Email: shpawar1946@gmail.com

Abstract

The microbiota present in the human gut plays an vital role in the maintaining the normal health of the individual. These microbes are called as probiotics. Lactobacillus is the one of the probiotics assisting the digestion of the food and assimilation of the nutrition. The Lactobacillus are also found in the milk of milking animals. The discovery and investigation of these microbes’ activities will help us to understand the pharmacological activities exhibited by these microbes. The basic techniques of isolation using the selective media and characterisation studies will help to understand the nature of these microbes ex. Gram staining, arginine hydrolysis test etc.

Keywords: Lactobacillus, Probiotics, Milk, Gram staining. ­

1.      Introduction

The use of the microbes as functional food has explored its use in medical science. Probiotics are discovered from the different natural sources from time to time as the functional food [1–5,5]. Lactobacillus is found as the most preferred genera in this direction with its utility in the dairy and allied sciences [6]. The most difficult task to use these microbes as the functional food is studying the growth parameters along with the genomic analysis. The common part observed in the case of probiotics from the same genera are its differences in physiological and biochemical characterizations [7].

Fig. 1 represents the general plating method used for the isolation of lactic acid bacteria on artificial Lactobacillus selective media by taking sheep milk samples.

Fig.1. Isolation of the lactic acid bacteria on the a) NRCLA and b) MRS media from sheep milk samples

1. Isolation of lactic acid bacteria from sheep milk in selective media

180 milk samples were collected from the Indian sheep’s breed from local places of Kolhapur, Sangli and Admapur areas of Maharashtra. The samples (50 ml) collected were stored at 5 °C until use. For bacterial enumeration, milk samples (1 ml) were kept at -78 °C in 15% glycerol before use. MRS (de Man, Rogosa & Sharpe) and NRCLA (Neutral Red Chalk Lactose Agar) broths and agar media Lactobacillus selective media were obtained as a gift sample from the Siffin Pharma, Germany [8]. MRS media was prepared by autoclaving 6.5 g MRS agar media in 100 ml distilled water, while NRCLA media was prepared by taking 5.1 g NRCLA agar media in 100 ml distilled water. The samples were inoculated on both MRS and NRCLA media by four quadrant streaking method and were incubated for a period of 48 h in a micro-aerophilic condition. After incubation, the individual colonies found on the NRCLA media were sub-cultured on MRS media and transferred into sterile MRS broth mediums. The purification of individually selected colonies were again carried out by the streak plate technique with the serial dilution method [8]. The isolated colonies were again kept at -78°C in 15% glycerol before use and were evaluated for their biochemical analysis.

 

 

 

 

 

Table 1. MRS media composition

Sr. no	Media ingredients	g/l
1	Proteose peptone	10.0
2	Beef extract	10.0
3	Yeast extract	5.0
4	Dextrose	20.0
5	Polysorbate 80	1.0
6	Ammonium citrate	2.0
7	Sodium acetate	5.0
8	Magnesium sulphate	0.1
9	Manganese sulphate	0.05
10	Dipotassium phosphate	2.0
Agar pH (25 oC) - 6.5 g/l (approximately)
5.5 g/100ml distilled water at 15 lbs pressure (121 oC)

 

 

 

 

 

 

 

 

 

 

Table 2. NRCLA media composition

Sr. No	Media ingredient	g/l
1	Peptic digest of animal tissue	3.0
2	Beef extract	3.0
3	Yeast extract	3.0
4	Lactose	10.0
5	Calcium carbonate	15.0
6	Neutral red	0.05
7	Agar	15.0
pH adjusted to 6.8 at 25°C

Table 3. MRS broth composition

Sr. no	Media ingredients	g/l
1.	Peptone	10.0
2.	Lab-lemco powder	8.0
3.	Yeast extract	4.0
4.	Glucose	20.0
5.	Sorbitan mono-oleate	1.0 (ml)
6.	Tri-mmonium citrate	2.0
7.	Sodium acetate	5.0
8.	Magnesium sulphate	0.2
9.	Manganese sulphate	0.05
10.	Dipotassium phosphate	2.0
pH (25 oC) - 6.5 g/l (approximately)
5.2 g/100ml distilled water at 15 lbs pressure (121 oC)

 

2 Conventional lab techniques for analysis of LAB

a) Gram Staining

The Gram staining of the isolates was determined by light microscopy using Gram staining reagents. It is known that LABs are gram-positive [9]. This means that these cultures will produce blue-violet color for Gram-positive bacteria and vice-versa. The cultures were grown in MRS media at 37 °C for 24 h under micro-aerophilic conditions. Fresh cultures were used for gram staining. After incubation, the cultures were aseptically transferred into 1.5 ml of eppendorf tubes and centrifuged for 3 min at 9000 rpm. The cells were resuspended in sterile water by removing the supernatant. L. acidophilus from NCIM was used as positive control and E. coli was used as the negative control.

b) Catalase test

Catalase is an enzyme released by the microbes during the metabolic process. This enzyme act on hydrogen peroxide breaking it into water and oxygen and producing the gas bubbles. The release of the gas bubbles during the test indicates the presence of catalase enzyme.

2H₂O₂→ 2 H₂O + O₂  ------------------------------------------------ 1 (Equation)

           The catalase test was carried out on the isolates to see their reactions to catalase. To do this, two methods can be performed. 18 h incubated cultures of isolates were grown on MRS agar at room temperature. Furthermore, for the catalase test fresh liquid cultures of LAB were used in which 3% hydrogen peroxide solution was added to 1 ml of cultures [10].

c) Gas production from glucose

This test determines the hetero-fermentative and homo-fermentative nature of the isolates by the release of CO₂ production from glucose. The overnight 1% cultures of the isolates were inoculated in MRS broths lacking citrate into the inverted Durham tubes. These cultures were further incubated for 48 h at 37 °C. The production of the CO₂ gas in Durham tubes indicates the presence of the glucose [8,11].

d) Growth at different temperatures

This test uses the bromecresol purple as an indicator in the freshly prepared MRS media. 50 μl overnight cultures of inoculum were added into 5 ml tube of modified MRS media and incubated for 7 days at 20 °C, 30 °C, 40, and 50 °C. During these incubation time, the change of the color from purple to yellow of the cells at different temperatures were observed [8,12]. L.acidophilus from NCIM was used as a positive control.

e) Arginine hydrolysis test

The arginine MRS modified medium and the Nessler reagent was used to view ammonia release from arginine. The freshly prepared 1% culture of the isolates was added into the MRS of 5 ml tubes containing 0.3% of L-arginine hydrochloride. The tubes were further incubated for 18 hours at 37 °C. After incubation, 50 μl of cultures were observed against the white background.  50 μl of the Nessler reagent was pipetted into the cultures and the change in the color was observed. The positive reaction was indicated by a bright orange color, while the yellow color determines the negative reaction. For the negative control, arginine free MRS was used [13].

Results and Discussion

1. Physiological and biochemical identification of LAB

All the isolates were subjected to Gram staining and they were examined under a light microscope (100X magnification). All the strains show blue-purple color staining, except E. coli which is used as a negative control reference. Hence all the isolated strains are found Gram-positive bacteria (Fig. 3.3. A-C), while E. coli shows pink color as it is Gram-negative bacteria (Fig. 3.3.D).

Fig. 2. Gram staining a) Sample A, b) Sample b, c) L. acidophilus (positive control) and d) E. coli (negative control) (100X)

The isolated Lactobacillus were long and rod-shaped. Isolates were tested for catalase activity. All isolates are catalase negative, as none of them given catalase activity. All strains show no gas production hence are homo-fermentative in nature (Fig. 3.4).

Fig. 3. Homofermentative nature observed in case of all Lactobacillus strain by Durham tube method

Another criterion for the identification of the isolates was the study of growth pattern at different temperatures. From the results of 7 days observation, all of the isolates show maximum growth between 35 °C ~ 37 °C. However, significantly very less growth is observed at 20 °C and 50 °C (Fig. 3.5).

Fig. 4. Stress heat tolerance of Lactobacillus at various temperature

Arginine hydrolysis test was used as another step to follow the identification procedure. The isolates which gave the bright orange are found in producing ammonia from arginine. The yellow color indicated negative arginine hydrolysis. According to this test, both isolated strains produced ammonia from arginine.

 

References

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