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Expression of antagonism by lactic acid bacterium isolated from marcha: an ethnic fermented beverage of North-Eastern Himalayas.

Introduction

Lactic acid bacteria are gram positive, catalase negative, non sporulating bacteria that can ferment various carbohydrates to lactate. Lactic acid bacteria play an important role in food preservation and widely used as starter culture in food industry. They are well known for production of various antimicrobial compounds such as bacteriocin, organic acids, diacetly, acetoin, reuterin [1, 2] during fermentation. These compounds can be added to foods as food preservatives, additives and as self life extenders [3]. By definition, bacteriocins are ribosomally synthesized small proteins with bacteriocidal or bacteriostatic activity against genetically closely related species [4]. Bacteriocin from lactic acid bacteria are classified into four major groups based on structure and mode of action [5]. In general, bacteriocins from lactic acid bacteria are small cationic proteins (30-60 amino acid residues) with high isoelectric points and amphillic characteristics [6]. Traditional fermented foods are an integral part of the dietary culture of the various ethnic groups of people in Sikkim, north eastern region of India consisting of the Nepalese, Bhutias and Lepchas. There are various fermented food and beverage such as kinema (based on soya beans), gundruk (Brassica campestris leaves), sinki (radish, Raphanus sativus), mesu (bamboo shoots), churpi (milk), shel rod (rice preparation) and jnards (beers) that have constituted of about 20 percent of the basic diet for centuries [7]. These fermented products are traditionaly prepared by rural women using their indigenous knowledge and experiences [8]. Marcha is mixed dough inocula used extensively as starters for preparation of various indigenous alcoholic beverages in Sikkim [9].

The main concern in the food industry these days is to select suitable microorganism that can be used in the food industry in order to improve the quality, taste and safety of the processed food. During the past decade consumer have demanded to decrease the use of chemical additives in food processing because many of these chemicals have detrimental effect on health and regarded as carcinogenic [10]. Therefore, bacteriocin from lactic acid bacteria may offer novel approach in food preservation as they inhibit food spoilage and pathogenic bacteria and generally lactic acid bacteria itself regarded as safe. There are several reports that indicate bacteriocins from lactic acid bacteria are active against many food borne pathogens [11]. Several in vitro and in vivo experiments have revealed the antagonism effect of bacteriocin from lactic acid bacteria against major food borne pathogen such as Helicobacter pylori, Camphylobacter jejuni, E. coli, Staphylococcus aureus, Bacillus cereus, Listeria sp. [12, 13]. In this study we report bacteriocin from DM15, a strain of lactic acid bacteria isolated from Marcha, traditionally fermented beverage from Sikkim, northeast region of India. The antagonistic effect of bacteriocin from DM15 on other bacteria was determined.

Materials and Methods

Isolation of lactic acid bacteria

The dry samples of 100g marcha were collected aseptically in sterile falcon tubes from local markets of the Sikkim in India. Tubes were kept in an ice-box and transported to the laboratory for analyses. The isolation of lactic acid bacteria from marcha was done by dissolving 1g of marcha into 10 ml 0.85% sterile saline solution and was serially diluted up to 10-7 in the same diluents [14]. The dilutions were then plated on MRS agar plate (2% agar) by spreading and the plates were incubated at 30[degrees]C for 48 h. Randomly ten distinct colonies from [10.sup.-6] plates and ten distinct colonies from [10.sup.-7] plates were picked and inoculated into MRS medium for 24 h at 30[degrees]C. Purity of each single colony is checked by streaking again it into MRS agar plate and incubated for 24 h at 30[degrees]C and then sub-cultured in MRS broth.

Screening of the isolate for antagonistic activities

Screening of the isolates was done on the basis of extra cellular production of bacteriocin and its antagonistic activity against the indicator strain E. coli DH5a. The isolates were grown in DeMan, Rogosa and Sharpe (MRS) medium at 37[degrees]C without agitation for 18-20 h [15]. The cell free extract was obtained by centrifuging the bacterial culture at 10,000g for 15 min, followed by adjusting the pH of the solution to 6.0 by sterile 4N NaOH. The supernatant was then filtered through 0.2 [micro]m filter membrane and filtrate was used as crude bacteriocin against the indicator strain E. coli DH5a. The selected isolates was named as DM15 after discovers name, material name from where it has been isolated and its colony number. The isolate was maintained in MRS agar stab supplemented with 2% glucose as carbon source at 4[degrees]C. The growth of the selected isolate (OD at 600 nm) in MRS medium at 37[degrees]C and the pH drop due to the growth was recorded at every1h interval for 24 h.

Morphological, biochemical and physiological analysis of the isolate

After screening on the basis of antimicrobial activity, the isolate DM15 was characterized by biochemical and physiological studies. Pure colony of the isolate was obtained by streaking and colony morphology was observed by Gram staining with the help of light microscope. Endospore staining was carried out by heating the air dried bacterial smear with malachite green for 5 min, followed by rinsing with water and counterstaining with safranin for 30s.

Several biochemical tests were performed to ensure the nature of the isolate DM15 as lactic acid bacteria. The catalase activity was determined by transferring 500 [micro]l culture broth to micro centrifuge tube and then addition of few drops of 5% (v/v) [H.sub.2][O.sub.2] [14]. For this test E. coli was taken as positive control. Motility test and indole test were carried out by inoculating the culture into the motility indole lysine agar slant procured from Hi-Media Pvt. Ltd. The isolate was grown in triple sugar iron slant (Hi-media) in order to differentiate it from gram-negative enteric bacilli based on carbohydrate fermentation and the production of hydrogen sulfide. The TSI slant contains agar, a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate. The isolate was subjected to nitrate reduction test by nitrate agar slant (Hi-media) composed of large amounts of nitrate along with alpha-napthylamine and sulfanilic acid. E.coli was used as a positive control for motility indole lysine, TSI and nitrate test.

The growth of the isolate was determined in different growing condition, such as growth at different temperatures (10, 15, 45 and 50[degrees]C), at different initial medium pH (3.0 to 12.0), as well as their ability to grow in different salt (NaCl) concentrations (4-16%) to check their temp, pH and salt tolerance. The isolate was subjected to carbohydrate fermentation reactions using various carbohydrate sources by the method of Kandler and Weiss [16]. Fermentation was recorded by acid production which can be observed by a colour change of the phenol red indicator dye from red to yellow.

The isolate was also tested for susceptibility to several commercially available antibiotic octadisks (Hi-media Pvt.Ltd) using agar disk diffusion method. The discs were placed over the soft agar inoculated with 1% culture and incubated for 36-48 h at 30[degrees]C for observing the clear zone of inhibition around the discs [17]. The antibiotics used for this test were Amoxyclav, Cephalexin, Ciproflaxacin, Clindamycin, Cloxacillin, Co-Trimaxazole, Erythromycin, Tetracyclin, Ampicillin, Carbenicilllin, Cephatoxamine, Chloramphenicol, Co-Trimazine, Gentamicin, Norflaxacin, Oxacillin, Amikacin, Bacitracin, Novobiocin, Vancomycin, Penicillin-G, Tobramycin, Cephaloridine, Kanamycin, Linomycin, Methicillin, Oleandomycin, Nalidixic acid, Sulphamethoxazole, Cefactor, Cefixime, Piperacillin and Cefoperazone. Based on the measurement of the diameter of zone of inhibition around disks, the isolate was determined to be resistant, moderate or susceptible to the antibiotic [17].

Bacteriocin activity assay

The antagonistic activity of the screened isolate was determined by agar well diffusion assay [18]. The 50 [micro]l of crude bacteriocin was placed in the wells of soft agar (0.7%) inoculated with indicator strain E. coli DH5a over the base agar plate (1.8%). The plates were incubated at 37[degrees]C for 24 h and observed for clear zone of inhibition. The amount of bacteriocin production was calculated as arbitrary activity units. One arbitrary activity unit was defined as the reciprocal of the highest dilution showing a clear zone of growth inhibition. The bacteriocin activity of DM15 was also recorded after every 3 h up to 24 h. The protein concentration of the crude bacteriocin from DM15 was determined by Lowry method using Bovine serum albumin as standard [19].

Antimicrobial spectrum

The antimicrobial activity of the selected isolate DM15 was also detected against several Grams positive and Grams negative bacteria by agar well diffusion method and by agar spot method [20]. The indicator strains were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), IMTECH, Chandigarh, National Collection of Industrial Microorganism (NCIM), NCL, Pune and from the Agricultural Research Service Culture Collection (NRRL), U.S.A.

Purification of bacteriocin

The partial purification of crude bacteriocin was done by ammonium sulphate precipitation. Powder of ammonium sulfate was added slowly to the cell free supernatant to 20% saturation and then centrifuged 10,000g at 4[degrees]C for 20 mins. The supernatant was collected and was taken for carrying out 80% ammonium sulphate precipitation. The solution was stirred for 4-6 h at 4[degrees]C and then centrifuged at 10,000g at 4[degrees]C for 40 min [18]. The pellet was obtained and re suspended in 25 mM ammonium acetate buffer (pH-6.5) and dialyzed (MW cut off 3.5 kDa) against the same buffer. Serial dilutions of partially purified bacteriocin were carried out and the activity of partially purified bacteriocin was checked by inhibition zone of indicator strain E. coli DH5a after 24 h incubation.

Results

Morphological, biochemical and physiological analysis of the isolate

Gram staining and endospore staining of the isolate DM15 showed the purple and red colour respectively without the green spot in later, indicating the gram positive non spore forming bacteria (Table 1). The isolate DM15 was grown within the stab line in motility indole lysine agar slant and medium remains yellow (Table 1). The result indicated that the isolate can not utilize indole and did not posses decarboxylase enzyme that degrade the amino acid lysine provided in the medium (Table 1).

The strain DM15 was not able to utilize thiosulphate anion as a terminal electron acceptor, reducing it to sulphide and no production of H2S, as a result no visible black precipitation in the TSI medium although the isolate can ferment glucose, lactose and fructose (Table 1). There was no colour change observed in the nitrate test, hence it was proved that the isolate DM15 was not able to reduce nitrate to nitrite or another nitrogenous compound such as ammonia or nitrogen gas in the nitrate test (Table 1). Inability of any LAB strains to produce biogenic amines is a good indication for starter cultures [21]. The results of both physiological and biochemical tests are shown in Table 1. Growth of isolate DM15 at different initial medium pH starting from pH 3.0 and 12.0 was tested to determine the pH tolerance of the isolate. It was observed that the isolate can grow at pH-4.0 but can not grow above pH 9.0. The isolate grew well in between 10[degrees]C-40[degrees]C but no growth of the isolate was observed at temp 45[degrees]C and 50[degrees]C. The isolate was able to grow at 4% 6% and 10% NaCl concentration but not beyond 10% NaCl concentration.

The isolate DM15 could efficiently utilize cellibiose, maltose, lactose, fructose glucose and showed weak fermentation towards xylose, trehalose, mannitol (Table 2). The strain was not able to ferment arabinose, glycerol, sorbitol and naturally occurring deoxy sugar rhamnose. The isolate DM15 was resistant to norfloxacin and vancomycin (Table 3) which is characteristics of lactic acid bacteria [22]. Lactobacilli, Pediococci and Leuconostoc spp have been reported to have natural resistance to vancomycin, which is due to precense of D-alanine: D-alanin ligase related enzymes [23]. The data of antibiogram of DM15 was shown in Table 3. The strain DM15 was also resistant to cephalexin, ciprofloxacin, co-trimaxazole, ampicillin, co-trimazine, sulphamethaxole, amikacin, kanamycin, tobramycin and nalidixic acid. Some Lactobacilli have natural resistant to ciprofloxacin, sulphamethaxole, kanamycin and nalidixic acid [24]. The strain DM15 showed sensitivity towards erythromycin, bacitracin, penicillin, chloramphenicol, tetracycline, carbenicillin, cephaloridine and novobiocin.

Bacteriocin activity assay

Bacteriocin screening was performed by using agar spot method. Adjustment of the cell free supernatant to pH 6.0 with 2N NaOH prevented the inhibitory effect of lactic acid. The isolate showed maximum zone of inhibition towards E. coli DH5[alpha]. Hence E. coli DH5[alpha] was further used for quantitative determination of bacteriocin activity. Strain DM15 produced highest bacteriocin activity of 3200AU/ml in 18th hour in MRS medium at 37[degrees]C, as shown in Fig.1 along with the cell optical density reading and the pH profile of the isolate. The specific activity of the crude bacteriocin was 256 AU/mg which was high when compared to other bacteriocins against the same indicator strains [25].

[FIGURE 1 OMITTED]

Antimicrobial spectrum

The inhibitory activity of bacteriocin produced by DM15 was tested against gram positive and gram negative bacteria shown in Table 4. Some bacteriocins are highly specific and can only inhibit closely related species [26], but bacteriocin from DM15 was able to inhibit a variety of gram negative as well as gram positive species. The isolate DM15 showed highest sensitivity towards gram negative E. coli DH5[alpha] and gave clear zone of inhibition (> 15 mm). Bacteriocin from the isolate DM15 inhibit the growth of other food spoilage microorganism such as Staphylococcus epidermis, Alcaligenes feacalis, Staphylococcous aureus, Enterobacter aerogenes, and Bacillus cereus. The bacteriocin from the isolate DM15 was more potential than the bacteriocins from Pediococcus pentosaceus ACCEL and Pediococcus acidilactici LAB 5, that did not inhibit the growth of any Gram negative bacteria but were active against Gram positive Staphylococcus epidermis and Staphylococcous aureus [15, 27]

Purification of bacteriocin

Due to the proteinaceus nature, bacteriocin was precipitated by ammonium sulphate method. There was no zone of inhibition obtained with 20% ammonium sulphate purified fraction of bacteriocin against E. coli DH5[alpha]. With 80% saturation the clear zone was visible against the indicator strain E. coli DH5[alpha]. Similar results have been reported earlier where bacteriocins from lactic acid bacteria are precipitated within the range of 40-80% ammonium sulphate saturation [18, 28]. The purification table and bacteriocin activity are shown in Table 5. After 80% ammonium sulphate precipitation the bacteriocin from the isolate DM15 gave specific activity of 2480 AU/mg with 10 fold of purification and activity yield was 70%. The bacteriocin activity of partially purified bacteriocin from the isolate DM15 was 12500 AU/ml which were comparable to earlier report of aciodocin DSM 20079 from Lactobacillus aciodophillus DSM 20079 [25].

Discussion

The isolate was considered as lactic acid bacteria based on its gram positive reactions, nonmotility, absence of spore formation and catalase activity. The strain was able to grow at 10[degrees]C, 15[degrees]C, in pH 4 to pH 8 and 10% NaCl but no growth was observed at 45[degrees]C, in pH 10 and 12% NaCl. The isolate could not ferment arabinose, rhamnose, sorbitol and glycerol. The isolate was vancomycin and norfloxacin resistant like other lactic acid bacteria [22]. The bacteriocin from DM15 showed inhibition spectrum against gram positive as well as gram negative bacteria. The bacteriocin from the isolate DM15 showed clear zone of inhibition against gram positive bacteria such as Staphylococcous aureus, Staphylococcus epidermis, Bacillus cereus, Pediococcus pentosaceus like bacteriocin from other lactic acid bacteria. The bacteriocin from DM15 was also effectively inhibiting gram negative Alcaligenes feacalis, Enterobacter aerogenes unlike the bacteriocin from Pediococcus pentosaceus ACCEL and Lactobacillus acidophilus DSM 20079 [15, 25]. All these indicate a quite broad antibacterial spectrum of the bacteriocin from DM15. It showed highest sensitivity towards gram negative E. coli DH5a with bacteriocin activity of 3200AU/ml and specific activity of 256 AU/mg. The antagonistic activity of the isolate DM15 was because of bacteriocin was supported by the observation that it could be precipitated by 80% ammonium sulphate saturation and showed clear zone of inhibition against E.coli DH5[alpha]. The purification resulted in an approximate 10 fold increase in specific activity.

The present study demonstrated that the isolate DM15 produced bacteriocin in MRS medium at 37[degrees]C. The bacteriocin from DM15 appeared to be more potential than other bacteriocin from lactic acid bacteria. It gave clear zone inhibition against several food spoilage bacteria such as E. coli, Alcaligenes feacalis, Enterobacter aerogenes, Staphylococcous aureus, and Staphylococcus epidermis. The strong antagonistic activity of DM15 indicated that it could be used to control the growth of undesirable microorganism in food stuffs and considered as food preservative in the food industry.

Acknowledgement

This research work and Ms Deeplina Das (Senior Research Fellow) are supported by a project grant by the Council of Scientific and Industrial Research (CSIR), New Delhi, India, to AG.

References

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Deeplina Das and Arun Goyal *

Department of Biotechnology, Indian Institute of Technology, Guwahati-781 039, Assam, India

* Corresponding Author E-mail: arungoyl@iitg.ernet.in
Table 1: Biochemical and physiological properties of the isolate DM15.

Parameter DM15 Pediococcus E. coli
 pentosaceus

Gram Staining Gram positive Gram positive Gram negative
Cell morphology Small rod shape Cocci shape Rod shape
Colony White, small, white, smooth, Small circular
 circular circular (0.5-1mm in dia)
Characteristic (0.5-1 mm in (1-2 mm in dia)
 dia)
Catalase Test Negative Negative Positive
Endospore Negative Negative Negative
staining
Motility Test Non motile Non motile Motile
Indole Test Negative Negative Positive
Nitrate Test Negative Negative Positive
[H.sub.2]S Gas Negative Negative Positive
Production

Table 2: Carbohydrate fermentation profile of the isolate DM15.

S. No. Carbohydrate DM15 *

1 Xylose +
2 Trehalose +
3 Mellibiose ++
4 Galactose ++
5 Mannitol +
6 Sorbitol -
7 Raffinose ++
8 Cellobiose +++
9 Arabinose -
10 Fructose +++
11 Maltose +++
12 Lactose +++
13 Rhamnose -
14 Glucose +++
15 Sucrose ++
16 Glycerol -

* (+++): strongly positive, (++): fairly positive,
(+): weakly positive, (-): negative

Table 3: Antibiotic sensitivity of the isolate DM15.

S. Antibiotic Conc. DM15
No

1 Amoxyclav (Ac) 10 [micro]g M
2 Cephalexin (Cp) 10 [micro]g R
3 Ciprofloxacin (Cf) 10 [micro]g R
4 Clindamycin (Cd) 2 [micro]g S
5 Cloxacillin (Cx) 1 [micro]g M
6 Co-Trimaxazole (Co) 25 [micro]g R
7 Erythromycin (E) 15 [micro]g S
8 Tetracycline (T) 30 [micro]g S
9 Ampicillin (A) 10 [micro]g R
10 Carbenicillin (Cb) 100 [micro]g S
11 Cephotaxime (Ce) 30 [micro]g S
12 Chloramphenicol (C) 30 [micro]g S
13 Co-Trimazine (Cm) 25 [micro]g R
14 Gentamicin (G) 10 [micro]g M
15 Cefoperazone (Cs) 75 [micro]g S
16 Sulphamethoxazole (Sx) 50 [micro]g R
17 Norfloxacin (Nx) 10 [micro]g R
18 Oxacillin (Ox) 5 [micro]g M
19 Amikacin (Ak) 10 [micro]g R
20 Bacitracin (B) 10 U S
21 Novobiocin (Nv) 30 [micro]g S
22 Vancomycin (Va) 30 [micro]g R
23 Cephaloridine(Cr) 30 [micro]g S
24 Kanamycin (K) 30 [micro]g R
25 Lincomycin (L) 2 [micro]g S
26 Methicillin (M) 5 [micro]g M
27 Olaendomycin (OL) 15 [micro]g S
28 Penicillin G (P) 10 U S
29 Tobramycin(Tb) 10 [micro]g R
30 Nalidixic acid (Na) 30 [micro]g R
31 Cefexime (Cfx) 5 [micro]g R
32 Piperacillin (Pc) 100 [micro]g S

R - Resistant (0-0.1cm); M - Moderate (0.2-0.8 cm);
S - Sensitive (0.9-2.5 cm)

* Values in centimeter are the distance of zone of inhibition of
growth of microorganism

Table 4: Antibacterial spectrum of bacteriocin from DM15.

Test microorganism Medium Temp. Sensitivity *
 ([degrees]C)

Gram Positive
L. mesenteroides NRRL B-640 MRS 30 -
L. dextranicum NRRL B-1146 MRS 30 -
L. mesenteroides NRRL B-1149 MRS 30 -
L. citreum NRRL B-742 MRS 30 -
L. mesenteroides NRRL B-512 MRS 30 -
Pediococcus pentosaceus MRS 30 +
Bacillus cereus NCIM 2155 NB 30 +
Weissella confusa MRS 30 -
Staphylococcous aureus NB 30 ++
 MTCC 737
Staphylococcus epidermis NB 30 ++
 MTCC 6810

Gram Negative
E. coli DH5a LB 37 +++
Pseudomonas fluorescens NB 37 -
 NRRL B 1612
Klebsiella oxytoca MTCC 3030 NB 37 -
Alcaligenes feacalis NB 30 +
 MTCC 2952
Enterobacter aerogenes NB 30 +
 MTCC 7016

* (+++): zone>15 mm, (++): 10 mm < zone < 15 mm,
(+): 5mm < zone <10mm, (-): no zone of inhibition

Table 5: Analysis of bacteriocin activity of the isolate DM15.

DM15 Vol. Activity * Total Protein Total
 (ml) (AU/ml) Activity conc. protein
 (AU) (mg/ml)

Crude 50 3200 160000 12.5 625
(CFS)
80% AS 9 12500 112500 5.04 45.4
precipitation

DM15 Specific Yield Fold
 activity Purification
 (AU/mg)

Crude 256 - --
(CFS)
80% 2480 70 10
precipitation

* The bacteriocin activity was determined against indicator
strain E. coli DH5[alpha]

CFS: cell free supernatant

AS: Ammonium sulphate
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Author:Das, Deeplina; Goyal, Arun
Publication:International Journal of Biotechnology & Biochemistry
Date:Sep 1, 2011
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