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Extraction of bacteriocin and study of its antigonastic assay.

Introduction

Bacteriocins are antimicrobial proteinaceous substances with a narrow spectrum of activity than antibiotics. They are defined are ribosomally synthesized, secreted, bactericidal peptides. Lactic acid bacteria bacteriocins are biologically active proteins or protein complexes that act bactericidally. Lactic acid bacteria (LAB) are widely used for the fermentation and preservation of a wide range of milk, meat and vegetable food. Lactic acid bacteria (LAB) can be found as human flora in the mouth, intestine and vagina. LAB has been a subject of intense interest with respect to production of growth inhibition antimicrobial compounds. The antimicrobial compounds produced by these bacteria include organic acids, like lactic acid, hydrogen peroxide and diacetyl (Daeschel, M.A) that play an essential role in ensuring the safety and extending the shelf life of these products. These acids render a low ecological pH, which can interfere with the growth of surrounding microorganisms. The production of lactic acid by LAB is known to prevent the growth of spoilage or pathogenic bacteria and thus is a natural method of extending the shelf life of food. Additionally, some lactic acid bacteria of the group produce hydrogen peroxide (oxidizing agent) and bacteriocins, which are antimicrobial substances. The bacteriocins of lactic acid-fermenting bacteria are well studied because of the commercial use of these bacteria in the food industry for making dairy products such as cheese. Many wild-type bacterial strains produce bacteriocins, which in addition to acting as inhibitors, can also function as signalling molecules. This may provide a competitive advantage to the producing strain in colonisation or competition in the gastrointestinal tract (GIT). If bacteriocins have a controlling influence on gutmicroflora they could be used as tools to influence the fate of commensal (resident) or pathogenic (Disease-causing) bacteria (such as Clostridium difficile) in the GIT

Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. There are several large categories of bacteriocin which are only phenomenologically related. These include the bacteriocins from gram-positive bacteria, the colicins [4], the microcins, and the bacteriocins from Archaea. The bacteriocins from E. coli are called colicins (formerly called 'colicines,' meaning 'coli killers'). They are the longest studied bacteriocins. They are a diverse group of bacteriocins and do not include all the bacteriocins produced by E. coli. For example the bacteriocins produced by Staphylococcus warneri, are called as warnerin (5) or warnericin. In fact, one of the oldest known so-called colicins was called colicin V and is now know as microcin V. It is much smaller and produced and secreted in a different manner than the classic colicins. The bacteriocins of lactic acid-fermenting bacteria are called lantibiotics. This naming system is problematic for a number of reasons. First, naming bacteriocins by what they putatively kill would be more accurate if their killing spectrum were contiguous with genus or species designations. The bacteriocins frequently possess spectra that exceed the bounds of their named taxa and almost never kill the majority of the taxa for which they are named. Further, the original naming is generally derived not from the sensitive strain the bacteriocin kills, but instead the organism that produces the bacteriocin. This makes the use of this naming system a problematic basis for theory; thus the alternative classification systems.

A number of bacteriocins are produced by L. plantarum. These include plantaricin B, plantaricin BN, plantaricin A, plantaricin C, plantaricin S and T, plantaricin, F, plantaricin C19 and SA6 and other unnamed bacteriocins. However, with the exception of plantaricin A, information on the genetic and biochemical characteristics of L. plantarum bacteriocins is still scant.

Materials and Methods

Isolation of lactic acid bacteria

Isolation of lactic acid bacteria using food sources such as curd was carried out. MRS medium (DeMan, Rogosa and Sharpe) was used for isolation of Lacticacid bacteria. After several identification tests (Gram test, monochrome test, catalase test, motility test etc) and subculturing, lactic acid bacteria were obtained.(for results see table 1)

Extraction of crude bacteriocin

These were inoculated in MRS broth. After centrifugating the broth (having LAB), the obtained lysate was subjected to purification by using ammonium sulfate precipitation method. Alternatively purification may also be done by column chromatography or HPLC. Assay of extracted and purified bacteriocin was carried out using well diffusion method on common indicator strains such as E. coli, Bacillus spp.

Protocol for purification of bacteriocin

The crude bacteriocin an extra cellular protein was isolated by using following protocol. The broth was separated from the cells by centrifugation at 10,000 rpm for 15 minutes. The cell free supernatant obtained was subjected to further purification by ammonium sulphate precipitation. The ammonium sulphate precipitation was carried out at 20% and 40% and the supernatant and pellet obtained at 40% were assayed for their bacteriocin activity.

Ammonium sulphate pecipitation

Ammonium sulphate precipitation at 0-20% and 20-40% was carried out as follows:

For 0-20% precipitation:The broth was centrifuged at 6000g for 20-25min. The CFS (cell free supernatant) was collected and the pellet containing the cells was discarded. To the CFS, ammonium sulphate was added to reach the required saturation level. (20%) To get 20%saturation, ammonium sulphate was added to the cell free supernatant at the concentration of 114g/l.

The solution was stirred on magnetic stirrer for 15-20 Minutes. The solution was centrifuged at 10,000g for 15-20min. Supernatant was taken for carrying out 40% ammonium Sulphate precipitation. Pellet was discarded. For 40% saturation the concentration of ammonium sulphate added was 123g/l.

Assay of bacteriocin

The purified sample was then subjected to assay. 30%MRS agar plates were inoculated with test organism (E.coli). These plates were allowed to cool and wells were prepared. Now the purified sample of bacteriocin along with control was poured in the wells. The plates were then incubated to observe the zone of inhibition. An alternative method to demonstrate their production, stab inoculation of multiple strains on separate multiple nutrient agar Petri dishes can be done followed by incubation at 30 [degrees]C for 24 h. Subsequent overlaying of each plate with one of the strains (in soft agar), and incubation at 30 [degrees]C for 24 h. After this process, the presence of bacteriocins can be inferred if there are zones of growth inhibition around stabs.

Results

Isolation of lactic acid bacteria

Streilized plates of MRS (DeMan, Rogosa, Sharpe) agar plates were spread with 0.1ml portion of [10.sup.-3] dilution, and were incubated at 37[degrees]C for 48hrs under anaerobic conditions. After several subcultures isolated colonies were obtained. These were identified by various tests. The obtained colony characteristics were compared with the standard strain of Lactic acid bacteria. The colony characteristics of the isolated colony is mentioned in table 1.

Extraction and assay of bacteriocin

Baceriocin was extracted from Lactic acid bacteria. The action of bacteriocin was checked on the indicator organism(E.coli). Antimicrobial activity of the extracted and purified bacteriocin was checked on the available indicator organism (E.coli). Clear and distinct zones of inhibition were seen on the plates. For 20% ammonium sulphate precipitate (purified bacteriocin), small zone of inhibition was obtained against E.coli. Corresponding measurements of zones of inhibition are mentioned in table 2.

A small zone of inhibition was obtained against E.coli for 20% ammonium sulphate precipitated pellet. Similarly the purified sample of bacteriocin by 40% ammonium sulphate precipitation was assayed against the available indicator organism. So again the zones of inhibition were seen. The measurements of zone of inhibition are mentioned in table 3.

A prominent zone of inhibition was obtained against E.coli for 40% ammonium sulphate supernatant.

Discussion

Lactic acid bacteria are widely used for the fermentation and preservative of a wide range of milk, meat and vegetable foods. Many researches have carried out on the physical and chemical characteristics of bacteriocins and their stability under different conditions. Most of the bacteriocins are plasmid encoded (Davey, 1984; Dufour et al., 1991) and hence the gene coding bacteriocin are being sequenced ,manipulated for enhancement of bacteriocin production. As the genetic determinents for bacteriocin synthesis, are located in plasmid so bacteriocin genes can be conveniently , transmitted to different starter cultures of Lactic acid bacteria. (Ross et al., 1999). In our present investigation study, we have carried out extraction of bacteriocin from lactic acid bacteria and the antagonistic activity of the extract against the test organism was carried out and it was found to be positive.

A bacteriocin nisin, best understood and most thoroughly characterized bacteriocin. Nisin is non-toxic to humans and animals (Hurst, 1981). In US, the FDA gave nisin a GRAS status (Generally Regarded As Safe) in 1980 (Febderal register, 1988). But nisin is too expensive to be used as an additive in cattle feed and hence research is being carried out to obtain cheaper and stable bacteriocin produced by Lactic acid bacteria.

References

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Noopur. M. Sapatnekar (1), Sucheta N. Patil (2) and B.A. Aglave (3)

(1), (3) Department of Biotechnology, Institute of Life Sciences, HPT Arts and RYK Science College, Nasik-5

(2) Department of Biotechnology, K.T.H.M. College, Nasik-2

(3) Correspondence author
Table 1: Colony characteristics of the isolated colonies.

1. Catalase test Negative

2. Monochrome staining Rod shaped colonies

3. Gram staining Gram positive

4. Motility Non-motile

5. Colony characteristics Whitish circular, pin pointed colonies
 were obtained.

Table 2: Assay of bacteriocin (20% ammonium sulphate precipitation).

Well no. Contents of Volume (in Result Zone
 the well. microlitre) measurement
 (cm)

1 Assay buffer 120 No zone of 0.0
 inhibition
2 20%sulphate 120 Prominent zone 1.4
 supernatant of inhibition

3 20%pellet 120 Zone observed 1.2

Table 3: Assay of bacteriocin (40% ammonium sulphate precipitation).

Well Contents of Volume Result Zone
No. the well (microlitre) measurement(cm)

1 Assay buffer 120 No zone of 0.0
 inhibotion

2 40%ammonium 120 Prominent 1.8
 sulphate zone of
 supernatant inhibition

3 40%pellet 120 Zone observed 1.6
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Author:Sapatnekar, Noopur. M.; Patil, Sucheta N.; Aglave, B.A.
Publication:International Journal of Biotechnology & Biochemistry
Date:Dec 1, 2010
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