INTRODUCTION:

The term probiotic was described as growth promoting factors produced by microorganisms. The word "probiotic" is derived from the Greek word 'probios' meaning 'for life' and has had several different meanings over the years. The probiotics are microorganisms or substances that contribute to intestinal microbial balance^[1]. The probiotics used as a live microbial feed supplement, which beneficially affects the host animal by improving intestinal microbial balance^[2].

Several probiotic bacteria produce a variety of antimicrobial compounds (e.g., hydrogen peroxide, nitric oxide, bacteriocins) that may enhance their ability to compete against other gastrointestinal microbes and which could potentially inhibit pathogenic (disease-causing) bacteria^[3].

Traditionally, bacteriocin production is an important measure in selection of probiotic strain. The earlier studies have demonstrated impact of bacteriocin production on the ability of strain to compete within the GI tract.

Bacteriocins are biomolecules produced by microorganisms and have narrow as well as broad spectrum of activity. The bacteriocin family includes a diversity of proteins in terms of size, microbial target, mode of action, release, and immunity mechanisms and can be divided into two main groups such as those produced by Gram-negative and Gram-positive bacteria^[4,5]. Bacteriocins are significant in medicine because they are made by non-pathogenic bacteria that normally colonize human body. A variety of probiotic bacteria are used as potential therapeutic agents. These include lactic acid bacteria^[6], Bifidobacteria^[7], Saccharomyces^[8], and streptococci^[9]. The probiotic bacteria are non-pathogenic and non-toxic. In addition, PB must survive the transition to target niche and then persist, serving to protect the host against infection by pathogenic microorganisms^[10] .This study deals with Isolation and biochemical and microbiological characterization of Bacteriocin producing Lactic Acid Bacteria from Diary Effluent.

MATERIALS AND METHODS:

Materials

All chemicals used were of analytical grade procured from Strata gene, SRL and Sigma. Media components were purchased from Hi Media Laboratories.

Sample collection and Serial dilution

The sample was collected from Sangam Diary Effluent. The company was located in the Vadlamudi, Guntur (Dt), Andhra Pradesh, India. The effluent sample 1 ml was added to 10 ml of sterile distilled water. Mix vigorously and take 1 ml from this tube, add to another tube containing 9 ml of sterile water to get the dilution of 10^-1.From this tube 1ml was taken and added to another tube containing 9 ml of sterile water to get the dilution of 10^-2. This serial dilution procedure was repeated up to 10^-9dilutions. From each dilution 0.1 ml was placed on to MRS agar medium by spread plate method and plates were incubated at 37°C for 24h to allow microbial growth.

Preparation of Pure Cultures

Colonies with different morphology were selected and pure cultures was developed using MRS agar medium by streak plate method. The medium was prepared by autoclaving at 121°C for 15 min at 15 lbs. The medium was then poured into Petri plates and was allowed to solidify. The selected colonies were streaked onto agar plates and the plates were incubated at 37°C for 24 h in an inverted position.

Indicator Strains

The Indicator strains namely Staphylococcus aureus (3103), Streptococcus sps (9724), Bacillus subtilis (1305), Klebsiella pneumonia (10309), Escherichia coli (9537), Enterobacter aerogenes (8558), Proteus vulgaris (7299) were used to check Bacteriocin activity and was collected from Microbial type culture collection, IMTECH, Chandigarh.

Screening of Bacteriocin Producing Bacteria

Agar Well Diffusion Method0

The antimicrobial activity for selected colonies was identified by agar well diffusion method against all indicator strains under sterile aerobic conditions. The inhibitory activity against Staphylococcus aureus, Streptococcus sps, Bacillus subtilis, Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, Proteus vulgaris were tested on nutrient agar media by inoculating with 100 µl of test sample. A sterile cork borer of diameter 7.0 mm was used to cut uniform wells in the agar plates. The isolated bacteria were grown in MRS broth at 37°C for 18-24h. After incubation the cells were removed by centrifugation at 10,000 rpm at 4°C for 15 min. The cell free supernatant was collected and it is adjusted to pH 6.5 to 7.0 with 1N NaOH and filter sterilized through 0.22 µm membranes. Each well was filled with 100 µl of this filter-sterilized supernatant. The plates were incubated at 37°C for 24 h. After 24h of incubation the antimicrobial activity was determined by measuring diameter of inhibition zone around the wells. The isolated bacteria showing widest zone of inhibition against indicator microorganisms was selected for further studies^[11].

Morphological and Biochemical characterization of the isolates

The isolates were monitored for morphological and biochemical characterization according to Bergey’s Manual of Determinative Bacteriology^[12]. Morphological features were identified by growing cultures on MRS agar media and Gram staining was performed for each isolate. Different Biochemical tests were carried out includes IMVIC tests, catalase test, starch hydrolysis gelatin hydrolysis, H₂S production test, urease test, oxidase test and carbohydrate fermentation of various sugars^[13].

Growth Curve for Isolated Strains

MRS medium was prepared for strains and sterilized by autoclaving at 121°C for 15 min. After cooling, 1% inoculum was added to the broth and incubated. For every 2hrs time interval optical density at 600 nm was measured using UV-Visible spectrophotometer and the values are tabulated to plot the growth curve. The optical density was measured up to 48 hours.

Characterization of Crude Bacteriocin

Effect of Temperature

MRS medium was prepared for the strains and sterilized by autoclaving at 121°C, 15lbs pressure. After cooling 1% inoculum was added to broth and incubated for 24h at 37°C. After incubation, culture was centrifuged at 10,000 rpm, 4°C for 15 min. The cell free supernatant was adjusted to different temperatures 25ºC, 35ºC, 45ºC, 55ºC, 65ºC, 75ºC, 85ºC, 95ºC, 100ºC, and 121°C for 30 min. The bacteriocin activity was checked after heat treatment by well diffusion method ^[14].

Effect of pH

The cell free supernatant was adjusted to pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 with Hydrochloric Acid (1N HCl) and Sodium Hydroxide (1N NaOH) and then assayed for bacteriocin activity using agar well diffusion method.

Antibiotic Resistance

MRS agar medium was inoculated with isolated strains and lawn was prepared on the surface of agar plate. The antibiotic discs Amikacin, Ampicillin and Chloramphenicol were placed on the surface of agar plate and pressed. Allow the plates to diffuse antibiotics into the medium for 1hr. Incubate the plates for overnight and observe the zone of inhibition.

Acid and bile salt tolerance

The procedures of Tambekar and Bhutada (2010) were used to determine acid and bile salt tolerance. The isolated strains were inoculated into the MRS medium of varying pH i.e 2, 2.5 and 3 as well as MRS medium of varying concentrations of bile salts 0.5, 1.0, 1.5 and 2.0% and incubated at 37ºC for 48h. Then 0.1ml inoculums was added to MRS medium by pour plate method and incubated at 37ºC for 48h. The growth of isolated strains on agar plates designate isolates as acid and bile salt tolerance^[15].

RESULTS AND DISCUSSIONS:

Isolation of Bacteriocin Producing Bacteria

The bacterial isolates around ten colonies with different morphology were isolated on MRS agar medium. The four bacterial colonies showed catalase negative and were selected for screening bacteriocin production. Pure cultures were developed for these strains and preserved at 4°C for further studies.

Table 1: Bacteriocin activity (Zone of inhibition) against indicator strains for 24hrs

Bacteriocin activity (Zone of inhibition in mm) after 24h
Name of the organism	Strain 3	Strain 5
Escherichia coli (MTCC 9537)	17	22
Staphylococcus aureus (MTCC 3103)	14	20
Streptococcus sps (MTCC 9724)	8	12
Klebsiella pneumoniae (MTCC 10309)	9	15
Proteus vulgaris (MTCC 7299)	24	18
Enterobacter aerogenes (MTCC 8558)	8	14
Bacillus subtilis (MTCC 1305)	----	----

Screening of bacteriocin activity by Agar Well Diffusion Method

Among 4 colonies 2 strains showed bacteriocin activity and were named as strain 3 and strain 5. These two colonies showed strong inhibition against Escherichia coli (MTCC 9537), Staphylococcus aureus (MTCC 3103) and Proteus vulgaris (MTCC 7299) where as medium inhibition against Streptococcus sps (MTCC 9724), Klebsiella pneumonia (MTCC 10309) and Enterobacter aerogenes (MTCC 8558). There is no effect was found on Bacillus subtilis (MTCC 1305). The inhibition zones in mm were depicted in (Table 1). Among these two strains, strain 5 has more inhibitory activity on the strains E. coli, S. aureus and P. vulgaris and was represented in the figure 1.

Morphological and Biochemical characterization of strains 3 and 5

Strain 3 and 5 were bacilli in shape and both strains are Gram positive. The strain 3 is white transparent in color where as strain 5 is pink in color. These are represented in Table 2. Strains 3 and 5 showed positive for Methyl red test, Starch hydrolysis, and Gelatin hydrolysis and negative for Indole, Citrate Utilization Test, Voges-Proskauer Test, Catalase, Oxidase, Hydrogen Sulfide Test and Urease Test. The images were represented in the figures 2 and 3. In Carbohydrate fermentation Strains 3 and 5 showed negative for glucose, fructose, sucrose, maltose and lactose. In the case of dextrose fermentation strain 3 is positive for both acid and gas producer, where as strain 5 is only the acid producer. The images were represented in the figure 4.

Table 2: Morphological and Biochemical characteristics of strain 3 and 5

	Strain 3	Strain 5
Morphological features
Gram staining	Gram positive	Gram negative
Shape	Bacilli	Bacilli
Color	White transparent	Pink
Biochemical tests
Indole Production	Negative	Negative
Methyl Red	Positive	Positive
Voges-Proskauer	Negative	Negative
Citrate Utilisation	Negative	Negative
Catalase	Negative	Negative
Starch Hydrolysis	Positive	Positive
Gelatin Hydrolysis	Positive	Positive
H₂S Test	Negative	Negative
Urease Test	Negative	Negative
Oxidase test
Carbohydrate fermentation
Maltose	Negative	Negative
Sucrose	Negative	Negative
Lactose	Negative	Negative
Fructose	Negative	Negative
Dextrose	Positive, Acid and gas producer	Positive, Acid producer
Glucose	Negative	Negative

Figure 1: Bacteriocin activity of starins 3 and 5 against E. coli (MTCC 9537), S. aureus (MTCC 3103), Streptococcus sps (MTCC 9724) K. pneumonia (MTCC 10309), P. vulgaris (MTCC 7299), E. aerogenes (MTCC 8558) and B. subtilis (MTCC 1305)

Among the two strains bacteriocin produced by strain 5 showed more activity compared with 3. The effect of pH on bacteriocin activity was measured in terms of inhibition zones. In figure 7(a) and 7(b) the bacteriocin activity was high on E.coli for the two strains. For the two strains maximum activity was observed from 6-7 range of pH. With the increase of pH the antimicrobial activity was decreased. Based on these results neutral pH was suitable for bacteriocin activity.

Antibiotic resistance

The two isolated strains are resistant to amikacin, ampicillin and chloramphenicol. Thus, the resistance indicates that if isolated probiotics induced in patients treated with antibiotic therapy may be helpful in faster recovery of the patients due to rapid establishment of desirable microbial flora.

Acid and Bile salt tolerance

The isolates have showed good acid and bile salt tolerance when they are incubated at varying pH and bile salts concentration. The probiotic potential is its ability to resist acidic pH and bile salts. When probiotics reaches the stomach and transit through intestine they must first survive in acidic pH as low as 2 and the bile salt concentration at 2%. This is a pre requisite for colonization and metabolic activity of the probiotic organisms. Thus they reach intestine and helps in maintenance of the microbial flora.

CONCLUSION:

The maximum bacteriocin activity was observed at 30-50°C range of temperature for isolates 3 and 5 and beyond this temperature no activity was found. Isolate 3 and 5 were bacilli in shape and gram positive. Isolate 5 is pink in color and acid producer where as 3 is transparent in nature, and it produces both acid and gas. The maximum bacteriocin activity was found at neutral pH and the isolates also confirm probiotics activity.

REFERENCES:

1. Parker, R.B (1974). Probiotics, the other half of the antibiotic story. Animal Nutrition and health, 29, 4-8.

2. Fuller (1989). Probiotics in man and animals. Journal of Applied Bacteriology. 66, 365-378.

3. Indira, M, John Babu D, Chaitanya M, and Vidya P Kodali (2015). Isolation and characterization of bacteriocin producing lactic acid bacteria from curd International Journal of Chem. Tech Research 8(1), 388-396, 2015.

4. Gordon, E.A (2006). The diversity of bacteriocins in gram negative bacteria. In bacteriocins: Ecology and Evolution (Berlin, Springer), pp 5-18.

5. Heng, NC, Burtenshaw GA, Jack RW, and Tagg JR (2007). Ubericin A, a class IIa bacteriocin produced by streptococcus uberis. Applied Environment Microbiology. 73,7763-7766.

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8. Czerucka, T. Piche, P. Rampal (2007). Review article: Yeast as probiotics – Saccharomyces boulardii. Alimentary Pharmacology and Therapeutics, 26, 767-778.

9. Meurman J.H, Stamatova, I (2007). Probiotics: contributions to oral health. Oral Diseases 13(5),443-51.

10. Klaenhammer, T.R, Kullen, M.J (1995). Selection and design of probiotics. International journal of food microbiology 15, 45-57.

11. Tagg J.R, Mc Given A.R (1971), Assay system for Bacteriocins. Applied Microbiology, 21, 943.

12. Noel, R. Krieg, John G. Holt (1984). Bergey’s manual of systemic bacteriology. William and Wilkins, Baltimore and London.

13. Cappucino, G and Sherman N. Microbiology: A Laboratory manual 5^th edition.

14. Ravi,M Prabhu, M, Subramanyam, M. (2011). Isolation of bacteriocin producing bacteria from mango pulp and its antimicrobial activity. Journal of Microbiology and Biotechnology Research 1(2), 54-63.

15. Tambekar, D.H. and S.A. Bhutada, (2010). An evaluation of probiotic potential of Lactobacillus sp. from milk of domestic animals and commercial available probiotic preparations in prevention of enteric bacterial infections. Recent Research in Science and Technology, 2, 82-88.

16. Aslam, M Shahid, M., (2011). Purification and characterization of bacteriocin isolated from S. themophilus. African Journal of Microbiology Research.5, 2642-2648.

Received on 30.09.2015 Modified on 12.10.2015

Research J. Pharm. and Tech. 8(11): Nov., 2015; Page 1560-1565

DOI: 10.5958/0974-360X.2015.00278.4


Starch hyhdrolysis	Gelatin hydrolysis	Hydrogen sulfide test	Urease test	Oxidase test


CATALEAST TEST