Isolation and Biochemical Characterization of Probiotic Lactobacillus Species Isolated from Curd Samples of Southern Regions of Vellore, Tamil Nadu

 

Harsha Achanta, Suneetha V*

School of Biosciences & Technology, VIT University, Vellore 632014, India

 

ABSTRACT:

Probiotics are viable economically useful microorganisms, when consumed with fermented products like yoghurt or dietary supplements, confer healthy benefits on the human beings. Dairy products are rich in Lactic acid bacteria (LAB). Dairy products especially curd and yoghurt are the major sources of probiotics, when included as part of daily diet will help in promoting good health. Probiotics must deliver beneficial results on the host without any toxic effects while being able to survive in the gut conditions of the host. Probiotics isolated from same species as that are available in the host are more sensible and effective. In our study, we have isolated lactobacillus species from different curd samples collected from the following places in Vellore -Old katpadi, VG rawnagar, Brahmapuram, Kangeyanellur, Sathuvacheri. The isolate was morphologically characterized using different staining techniques like simple staining, gram staining endospore staining and scanning electron microscope (SEM). The biochemical characterization was carried out using the standard biochemical tests namely Indole test, Catalase test and Citrate utilization test. Then the growth curve of the isolate was determined using the Calorimeter. In our study the morphological aspects of isolated bacteria are analyzed for further validation for probiotic nature of these isolates.

 

 

 

 

 

INTRODUCTION:

Probiotics are live useful microorganisms, when consumed with fermented products like yoghurt or dietary supplements, confer healthy benefits on the host. (1) Probiotics are rich in microorganisms belonging to genera Lactobacillus, Bifidobacterium and Streptococcus. (1,2) Lactic acid bacteria (LAB) microorganisms play an important role in boosting the immune system and support proper maintenance and functioning of intestines. (3, 4, 5)

 

Dairy products especially yoghurt is the major source of probiotics, when included as part of daily diet will help in promoting good health. Along with vitamins, calcium, minerals, and protein obtained from milk products other healthful properties of fermentation-derived peptides and butyric acid are also found in some dairy products. Application of probiotics: strengthens immune system and improves resistance power, reduction of lactose intolerance, improves bowel movements, prevention of diarrhea, reduction in the risk of colon cancer, reduction in cholesterol level and improves general health. LAB is the most common bacteria as probiotics. Strains of Lactobacillus vary from each type of fermented food. Gastrointestinal tract and vagina are two major body parts where Lactobacillus is found. There are more than 50 species of Lactobacilli. Lactobacillus has been used for treating and preventing a wide variety of diseases and conditions (6,7,8,). As there is huge geographical diversity in probiotic bacteria available in curd samples there is so much scope to study them and characterize them to use these bacteria for different purposes to promote healthier living of human beings. In this study our main aim is to isolate Lactobacillus colony from different curd samples and evaluate the probiotic potential. Characterization of isolated bacteria was done using different tests.

 

Figure 1: Collection of samples

 

MATERIALS AND METHODS:

Source of Lactobacillus:

Curd samples were collected from different regions of Vellore, Tamil Nadu. The experiments were conducted at VIT, Vellore University. Samples were transported and stored in aseptic conditions at 4°C to prevent contamination and spoilage. Pour-plate method was used for the screening and isolation of bacteria. The places from which the samples were collected (9, 10, 11)

1.      V.G. RAWNAGAR

2.      BRAHMAPURAM

3.      OLD KATPADI

 

Media used:

Lactobacillus MRS Agar (From Hi Media) contains protease peptone 10.00 g/l, beef extract 10.00 g/l, yeast extract 5.00 g/l, dextrose 20.00 g/l, polysorbate 80 1.00 g/l, ammonium citrate 2.00 g/l, sodium acetate 5.00 g/l, magnesium sulphate 0.10 g/l, manganese sulphate      0.05 g/l.

+

Figure 2: Bacterial colonies from the curd sample in pour plate method

 

Figure 3: Colony formed by streak plate method

 

Reagents used:

Several reagents used during the course of this research are as follows: ethanol (both 70% and 90%), hydrogen peroxide, bromothymol blue indicator, NaOH, Folin’s reagent, sodium carbonate (Na₂CO₃), Kovac’s reagent, phosphate buffer (prepared from 1M Sodium dihydrogen phosphate (NaH₂PO₄) and 1M Disodium hydrogen phosphate (Na₂HPO₄), potassium ferry cyanide, trichloro acetic acid, ferric chloride, ascorbic acid, peptone, iodine solution, safrannin red indicator, Conc. H₂SO₄, HCl, NaCl, Starch, Bile salt, sucrose, glucose, mannitol, phenol red indicator, potassium nitrate, Simmons’s citrate agar, 1% trypton broth. (12-15)

 

METHODS:

Screening and Isolation of Bacteria:

For this first we collected curd samples from different regions of Vellore and stored it in aspectic conditions for our study. MRS media was prepared for the selection and isolation of Lactobacillus. 1 ml of well mixed curd sample was enriched in 9ml of MRS broth for 24 hours at 37°C. The enriched samples were inoculated with pour plate technique and incubated for 48 hours at 37°C. We repeated this technique to obtain pure culture. Then we have isolated the colony on petri plates by streak plate method (16,17,18,19,20) and identified it. By repetition of this method we got pure bacterial colonies. In streak plate method, we took 5.535gm MRS agar in conical flask with distilled water and 100ml of MRS agar was prepared. The conical flasks and petri plates were autoclaved for 15 to 20 minutes at 121°C with 15 psi pressure. MRS agar plates were steaked with the bacterial culture broth which was incubated for 24 hours at 37^oC. Plates were observed for presence of colonies. For morphological study, microscopic examination was done after Gram staining. (21-25)

 

Staining for microscopic examination:

Simple staining with standard protocol was used to increasing contrast so that morphology, size, and arrangement of organisms can be determined. Gram staining helps to differentiate bacterial species based on the presence of high levels of peptidoglycan, and physical properties of their cell walls. Bacteria are classified into two large groups i.e. Gram positive and Gram-negative based on this staining. Lactobacilli are Gram positive bacteria (26,27,28). To confirm the purity of isolation of Lactobacilli, colony from each isolated sample is stained using standard protocol and observed under 20X and 40X magnification of the microscope. Endospore staining was done using standard protocol to identify any endospore forming bacteria from the isolated samples.

 

Figure 4:  Simple staining

 

Figure 5:  Gram staining

 

Figure 6: Endospore staining

Growth kinetics studies for isolated strain:

The medium for bacterial growth was prepared in LB Broth. To 100ml of distilled water 5.51 grams of MRS media was added. Then it was mixed well and the media was sterilized for 15 min (121ºC, 15 psi). The medium was cooled to room temperature, inoculated with the bacterial culture and then incubated for 48hrs. O.D value was measured for 48 hours at a time interval of 1hour. Different growth phases were determined. Graph was plotted between numbers of cells (O.D value) Vs time at room temperature. Specific growth rate is defined as the increase in the cell mass per unit time. The specific growth rate is commonly given by the symbol, µ, and the most common units are in reciprocal of time. In this study, µ is calculated by taking slope in the exponential phase. (29.30)

 

Biochemical test:

Indole test:

Detecting the presence of the enzyme tryptophanas which hydrolyzes tryptophan into indole, pyruvic acid and ammonia. Tube of tryptone broth is inoculated with small amount of pure culture.10g of peptone was dissolved in 1% of distilled water. It was sterilized in the autoclave at 15 lb pressure and 121°C for 15 min temperature. The tryptone broth was inoculated with different strain of bacteria and one tube was kept as an un-inoculated comparative control. The inoculated and control tubes were incubated at 35°C for 48 hrs. After 48 hrs of incubation, 1 ml of Kovac’s reagent was added to each tube including control. The tubes were kept for shaking gently at intervals of 10-15 minutes; tubes were allowed to stand to permit the reagent to come to the top. The development of a cherry red colour in the top layer of the tube inoculated with bacteria indicates a possible test for the indole production. The absence of red colour in the tubes inoculated indicates an indole negative test.

 

Figure 7: No change in color indicates negative test for indole test

 

Table 1: Plate count and CFU of different sample Biochemical testing:

Strain name	Colony number	CFU unit/ml
SH I	67	2.7 * 108
SH II	49	2.9 * 108
SH III	44	4.4 * 108
SH IV	53	5.3 * 108

 

Catalase test:

Catalase test was done to test the formation of air bubble due to the presence of oxygen produced by the aerobic organism. Hydrogen peroxide is lethal for the organism. To test this, we added H₂O₂ to test tube. Using a wooden applicator stick, a small amount of organism from a well-isolated 18- to 24-hour colony was collect and place into the test tube. Tube was placed against a dark background and was observe for immediate bubble formation (O₂ + water = bubbles) at the end of the wooden applicator stick. Positive reactions are evident by immediate effervescence (bubble formation). Using a magnifying glass or microscope weak positive reactions were observed. No bubble formation (no catalase enzyme to hydrolyse the hydrogen peroxide) represents a catalase-negative reaction.

 

Figure 8: Absence of air bubble indicates negative test for catalase test

 

Citrate Utilization Test:

Simmons’s citrate agar tests for the ability of an organism to use citrate as its sole source of carbon. This media contains a pH indicator called bromthymol blue. Citrase is produced by some bacteria that breakdown the citrate to oxaloacetic acid and acetic acid, these product latter convert to pyrucic acid. This test is performed by inoculating the microorganism into an organic synthetic media, Simmon’s citrate agar where sodium citrate is the only source of carbon and energy. When citric acid is metabolized, it generates CO2 which combine with sodium and water to form sodium carbonate as an alkaline product. The agar media changes from green to blue at an alkaline pH.

 

Figure 9: Blue color appears in test indicates positive test for citrate utilization

 

CO₂ + Na⁺ + H₂O                        Na₂CO₃

 

NaCl Tolerance:

For the determination of NaCl tolerance of isolated lactobacillus 10 test tube containing MRS broth were adjusted with different concentration (1-10%) of NaCl. After sterilization, each test tube was incubated with 1 % (v/v) fresh overnight culture of Lactobacillus and incubated at 37⁰C for 24 hours. After 24 hour of incubation their growth were determined by observing their turbidity. Maximum growth was indicated as double positive sign (++) and normal growth by single positive sign (+) and no growth by negative sign (-).

 

Figure 10: Clear zone around colony indicates positive test for the amylase production

 

Table 2: Results of biochemical test NaCl tolerance test

Test	Sh	Sh	Sh	sh
Indole	-ve	-ve	-ve	-ve
Catalase test	-ve	-ve	-ve	-ve
Citrate utilization test	+ve	+ve	+ve	+ve

The isolated lactobacillus from curd could tolerate 1-9 % NaCl.

 

Scanning Electron Microscope:

It is a type of electron microscope that produces images of the test samples when scanned with a specific focused beam of electrons. The electrons interact with atoms in the sample, producing different signals that contain information about the test sample's composition. The electron beam is basically scanned in a raster scan pattern and the beam's position is combined with the detected signal to produce a n appropriate image. SEM can achieve resolution better than one nanometer. Specimens can be observed in both high and low vacuum, in wet conditions, and at different elevated temperatures. The most common SEM mode is detection of secondary electrons emitted by atoms excited by the electron beam. The number of secondary electrons that can be detected is generally dependent on other things like topography.

 

RESULTS AND DISCUSSION:

Isolation of Bacteria:

First we collected curd samples from different regions of Vellore. Bacterial colony was isolated from the different region curd sample by the pour plate technique and a pure culture was obtained by the streak plate technique. First pour plate technique was done to isolate the number of colony and then the streak plate technique was done to get the pure culture. We screened the four bacterial isolates from different regions of Vellore.

 

Table 3: Tolerance to NaCl of isolated Lactobacilli

Concentration of NaCl %	SH I	SH II	SH III	SH IV
1	++	++	++	++
2	++	++	++	++
3	+	++	+	++
4	+	+	+	+
5	+	+	+	+
6	+	+	+	+
7	+	+	+	+
8	+	+	+	+
9	+	-	-	-
10	-	-	-	-

 

 

Control

 

 

Figure 11: After incubation color changed but air did not produce which indicates the positive test for the acid but –ve for air bubble.

 

Staining of Bacteria:

Simple Staining:

This test is useful in classifying two distinct types of bacteria based on the structural difference of their cell walls. By this method we can identify the bacterial species by shape, size and based on spore formation. By this method we got the characteristic of bacteria is rod shaped bacteria which is the characteristic of bacillus species. A Gram stain negative cell would lose the colour immediately from a decolourising agent, but a Gram stain positive cell would retain the dye, and still appear purple. The decolourising agent is made up of ethanol, but can sometimes be made up of a mixture of acetone and alcohol. By this test we got the purple colour colony which means it is gram positive bacteria. Staining procedure used is the Schaeffer-Fulton. This procedure uses Malachite Green as the primary stain for the spores. This stain cannot be decolorized from the spores once they are removed from the heat, however, water can remove the Malachite Green from the vegetative cells, making them again colorless. The vegetative cell is counterstained with Safranin. This causes the surrounding sporangium to appear red/pink. So, endospore forming bacteria shows pink vegetative cell with the green color spore but non-endospore forming bacteria shows only pink color vegetative cell. In this staining, only red color vegetative cell appeared so this bacterial culture is non-endospore forming cell.

 

 

Figure 12: SEM analysis

 

Figure 13: SEM analysis

 

 

 

Growth Kinetics:

From the OD measurement at 580 nm the bacterial growth kinetics for the pure culture was established. We took readings up to 42 hours. Different culture strains show different time period for the lag phage, exponential phage and stationary phage. After inoculation, these cells grow and divide increasingly rapidly as they acclimatize to the culture conditions. This acclimatization period, which lasts for 6-15 hour for different strain, is called the lag phase. Once adapted to the conditions, the rate of cell division accelerates and increase in the number of cells in the culture is logarithmic. This period lasts for 7 to 25 hour in different strain, and is called the exponential growth phase. Cell division rate then slows as light penetration through the culture and/or nutrients become limiting.

 

CONCLUSION:

This research study envisages the Lactobacillus a probiotic, from Vellore culinary curd samples.

 

ACKNOWLEDGEMENTS:

We sincerely like to express our gratitude to Dr G. Viswanathan, founder and Hon’ble Chancellor, VIT University, Vellore, for his constant support and encouragement. We want to express special thanks to Dr Sekar Viswanathan, Mr. Sankar Viswanathan and Mr. G.V. Selvam for their constant motivation and help.

 

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