Sky Journal of Microbiology Research Vol. 1(7), pp. 59 - 67, September, 2013 Available online http://www.skyjournals.org/SJMR ISSN 2315-876X ©2013 Sky Journals

Full Length Research Paper

A novel innate approach for better augmentation of Lactobacillus sp from fermented product using herbal extract

Divakar V2., Murugalatha N. Kannan1*, Mohan kumar A3 and Banu Raviganesh4 1,2

Department of Microbiology, Hindusthan College of Arts and Science, coimbatore 641028, Tamilnadu, India. 3Department of Zoology, Chikkanna Government Arts College, Tirupur 641602, Tamilnadu, India

4Department of Biotechnology, Hindusthan College of Arts and Science, coimbatore, Tamilnadu, India.

Accepted 2 August, 2013

Development of Lactobacillus can be very well carried on with MRS medium. In the present study the growth of Lactobacillus is compared and much quantified with natural herbal extracts like Aloe vera, Neem and Tulsi in combination with MRS medium. Medium supplemented with neem supported Lactobacillus and it showed better antimicrobial activity. The genomic characterization was not mutated by these herbal supplements. Key words: Lactobacillus, herbal extracts, antimicrobial activity.

INTRODUCTION Lactic acid bacteria (LAB) are characterized as gram-positive, usually non-motile, non-sporulating bacteria that produce lactic acid as a major or sole product of their fermentative metabolism. Optimal growth temperature occurred between 28 to 42°C (strain dependent) and a pH range 3.0 to 8.0 and up to 0.5% NaCl. Lactobacillus, also called Doderlein's bacillus, are a major part of the lactic acid bacteria group. They are named as such because most of its members convert lactose and other sugars to lactic acid.

Various amino acids, vitamins and minerals are essential for Lactobacillus growth (Kandler, 1983). Accordingly, they are commonly associated with nutritious environments like foods, decaying material and the mucosal surfaces of the gastrointestinal and urogenital tract (Kandler, 1983) where they enhance the host protection against pathogens (Havenaar et al., 1992).

Lactobacillus has been used for many centuries in food fermentation processes. Some Lactobacillus species are used for the production of yogurt, cheese, sauerkraut, pickles, beer, wine, cider, kimchi, cocoa and other fermented foods, as well as animal feeds, such as silage. *Corresponding author. Email: navikal123@gmail.com.

Sourdough bread is made using a "starter culture”, which is a symbiotic culture of yeast and lactic acid bacteria growing in a water and flour medium. Lactobacilli, especially L. casei and L. brevis, are some of the most common beer spoilage organisms. The species operate by lowering the pH of the fermenting substance by creating the lactic acid, neutralizing it to the desired extent. Some strains of Lactobacillus species and other lactic acid bacteria may possess potential therapeutic properties including anti-inflammatory and anti-cancer activities, as well as other features of interest. Lactobacilli can also be used to restore particular physiological balance such as in the vaginal eco-system. Their role is (Kandler, 1983) to physically protect the vaginal epithelium by building a thick layer separating the epithelium from pathogens, (Havenaar et al., 1992) to physiologically keep the balance of the vaginal ecosystem in maintaining the pH at 4.5, and (Kandler and Weiss, 1986) generating hydrogen peroxide against pathogens. Lactobacilli are highly tolerant to low pH and can easily maintain low pH and protect the vaginal eco-system from Gram-negative and Gram-positive bacteria.

Lactic acid bacteria (LAB) have been used successfully, with few adverse effects, to prevent antibiotic associated diarrhea, to treat acute infantile diarrhea and recurrent Clostridium difficult disease and to treat various diarrheal illness. The antagonistic property

60 Sky. J. Microbiol. Res. is attributed to the lowered pH, the undissociated acids and production of other primary and secondary antimicrobial metabolites produced by LAB. The metabolites produced by the fermentation process, except the volatile ones, are kept in the foods and result in growth inhibition of food spoilage or poisoning bacteria and detoxification of noxious compounds of plant origin. The primary antimicrobial effect exerted by LAB is the production of lactic acid and reduction of pH. In addition, LAB produce various antimicrobial compounds, which can be classified as low-molecular-mass (LMM) compounds such as hydrogen peroxide (H2O2), carbon dioxide (CO2), diacetyl (2,3-butanedione), uncharacterized compounds, and high-molecular-mass (HMM) compounds like bacteriocin. All of these can antagonize the growth of some spoilage and pathogenic bacteria in foods.

In view of their commercial importance, a complete characterization of their genome and further genetic manipulation may have great potential for the improvement of these microorganisms. However, compared with that of Lactococcus lactis, the genetic knowledge of lactobacilli is sparse and the availability of genetic systems for its study is limited. Natural plasmids of Lactobacilli may play a significant role in vector development techniques that are the basis for applied genetics. Resident plasmids are abundant in Lactobacillus strains. Although some industrially significant characteristics such as metabolic functions, restriction/modification systems, and drug resistance are plasmid DNA encoded, most of these plasmids remain cryptic.

Lactic acid bacteria (LAB), used for centuries by man to preserve food, produce a wide variety of antagonistic compounds, including lactic acid, hydrogen peroxide and bacteriocins. Bacteriocins are antimicrobial peptides that are bactericidal toward bacteria taxonomically close to the producer (Kandler, 1983). The bacteriocins produced by LAB have been extensively studied (Havenaar et al., 1992) and classified into three main groups (Kandler and Weiss, 1986): (I) lantibiotics, small peptides (<5 kDa), which are characterized by the presence of lanthionine and/or methyllanthionine residues in the polypeptide; (II) nonlantibiotic, low-molecularweight (<10 kDa), heat-stable peptides; and (III) nonlantibiotic, large (>30 kDa), heat-labile peptides. Class II bacteriocins can be subdivided into (IIA) Listeriaactive peptides, (IIB) two peptide bacteriocins, (IIC) Sec-dependent bacteriocins, and (IID) class II bacteriocins that do not belong to the other subgroups.

Freeze-drying is commonly used for the long-term preservation and storage of microorganisms in stock collections as well as for the production of starter cultures for the food industry. The choice of an appropriate suspending medium is of primary importance to increase the survival rate of the lactic acid bacteria (LAB) during and after freeze-drying although the success of

the process also depends on several factors such as growth phase, extent of drying, rehydration, suspension medium, cryoprotectors etc., During freezing or freeze drying, cellular damage may occur, resulting in a mixed population containing unharmed cells and dead cells as well as those sublethally injured.

Damage may not lead directly to death since in a suitable environment the injured cells may repair and regain normal functions. Information on the requirements for recovery from sub lethal injury is important from the standpoint of food microbiology and culture collections. LAB can also be preserved for short-term storage. The techniques used will be considered under the following headings:

i.) Short-term maintenance for daily or weekly use. Rich, undefined media such as MRS, LAPTg, M17, or Elliker broths are commonly used. ii.) Long-term preservation, as in a culture collection, where immediate access is less important, but maintenance of the characteristics of the species and strains is the primary objective. This projects aims to formulate a variety of media to facilitate the enhanced growth of Lactobacillus isolated from commercially available grape wine. Using readily available herbal components like Neem extract, Tulsi extract and Aloe Vera extract have been supplemented as an additional nutrient source to the basal medium used for the cultivation of Lactobacillus species. Differences in the growth patterns are analyzed and species characterization was done using molecular genetic techniques.

MATERIALS AND METHODS Collection of samples Wine samples were collected from the local area shops in Coimbatore, Tamilnadu. Immediately after collection, the samples were stored aseptically in low temperature (4°C) refrigerator to protect from contamination and deterioration. Wine samples were serially diluted in peptone medium and incubated at 23°C for 30 min. before plating by which 50% of recovery of LAB was increased. Diluted samples were plated onto De Man Rogosa Sharpe (MRS) medium for Lactobacillus sp isolation and incubated at 37

ºC or 48

- 72 h. Well-isolated colonies with typical characteristics namely pure white, small (2 - 3 mm in diameter) with entire margins were picked from each plate and transferred to MRS broth.

Identification of lactic acid bacteria Identification of Lactobacillus was performed according to their morphological, cultural, physiological and

biochemical characteristics (Kandler and Weiss, 1986 and Sharpe et al., 1979): Gram reaction, production of catalase, carbohydrate fermentation patterns, growth at 15 and 45°C in the lactobacilli De Man Rogosa and Sharpe (MRS) broth as described by Bergey’s Manual of systematic Bacteriology (Kandler and Weiss, 1986), methyl red and Voges-Proskauer test in MRVP medium, nitrate reduction in nitrate broth, indole production in tryptone broth. Purified cultures were maintained at -20°C in MRS broth with 10% glycerol and enriched in MRS broth incubated at 37°C for 24 h. The isolates were designated as WL1, WL2. WL3, WL4 and WL5. The identified genus Lactobacillus species was further classified to the species level based on their ability to ferment sugars (Singh and Rakesh Roshan Sharma, 2009). Media for cultivation Substrate A modified method of Gonzalez (Gonzalenz and Kunka, 1987.) was carried out in which Aloe vera, Tulsi and Neem leaves were cut into steak sand than were macerated in a blender, filtered and were used as substrates in the MRS medium. 75% of the Aloe vera, Tulsi, and Neem extract previously adjusted to pH 6.5 was added to the MRS broth and autoclaved for 118°C/10 min to prevent thermal degradation. MRS was considered as control. 2 ml of the culture isolates were added to the medium and incubated at 37°C for 24 h. The microbial growth were determined at 0, 24, 48 h by spectrophotometric method. Detection of inhibitory activity Agar spot assay test Lactic acid bacterial isolates were cultured in 5 ml of MRS broth at 30°C for 16 h. Aliquots (2 μl) of the culture were spotted onto agar plates containing 10 ml of MRS medium. After 18 h at 30°C, the plates were overlaid with 5 ml of the appropriate soft agar (1% agar) inoculated with the cell suspension of the indicator strain Lactobacillus acidophilus at a final concentration of 10

5

CFU/ml (Kilic et al., 1996). The plates were incubated at 37°C for 24 to 72 h, depending on the growth of the indicator strain and the appearance of inhibitory zones were observed. Inhibition was scored positive if the zone was wider than 2 mm in diameter. Agar-well diffusion assay The strains that were selected as potential bacteriocin

Divakar et al. 61 producers were grown in MRS broth at 37°C for 48 h. Cells were separated by centrifugation at 5000 rpm for 10 min at room temperature. Around 6 mm diameter wells were made on preinoculated agar media and each well was inoculated with 100 μl of culture supernatant of bacteriocin producing Lactobacillus strains after neutralization with NaOH (Toba et al., 1991). Inhibitory activity was performed against certain Gram positive and Gram negative organisms: Bacillus amyloliquifaciens (MTCC 1270), Bacillus cereus (MTCC 1272), Bacillus mycoides (MTCC 645), Lactobacillus acidophilus (MTCC 447), Lactobacillus delbrueckii sub sp. lactis (MTCC 911), Lactococcus lactis sub sp. lactis (MTCC 440), Streptococcus faecalis (MTCC 459), Staphylococcus aureus (MTCC 740), Klebsiella pneumoniae (MTCC 3384), Proteus vulgaris (MTCC 744), Pseudomonas aeruginosa (MTCC 647) and Salmonella typhi (MTCC 531). Inhibition zones around the wells were measured and recorded. Genomic DNA isolation Genomic DNA was isolated by phenol chloroform method from the isolates grown in Aloevera, Tulsi and Neem added MRS medium and in MRS medium alone to detect whether the herbal extracts have modified the genomic nature which might affect the genotypic characterization. RESULTS Isolation of lactic acid bacteria Totally 10 wine samples were collected from different places of Coimbatore and were processed for isolation of Lactobacilli. Among the processed samples 5 colonies with typical characteristics namely pure white, small (2 - 3 mm diameter) with entire margins were picked and were subjected to morphological and biochemical characteristics. Identification of lactic acid bacteria All the 5 isolates reacted positively to gram staining under a light microscope. Lactobacilli are generally long rods some times they are short rods, coccoid. It happens that cells of coccoid form strains, were not able to show their growth at 45°C. Few isolates from buffalo and goat were able to utilize citrate indicating the ingress of citrate into a cell with an aid of citrate permease. Lactobacillus sp do not possess flagella and do not create endospores, nitrates are not reduced, gelatin is not liquefied, indole is not produced, acidic and non acidic end products are not produced and are catalase negative (Table 1).

The identified genus Lactobacillus sp was further classified to the species level. Strains were able to

62 Sky. J. Microbiol. Res.

Table 1. Biochemical characterization of Lactobacillus species isolated from Wine samples.

S.N

o

Iso

late

s

Mo

rph

olo

gy

Gra

m R

ea

cti

on

Mo

tili

ty Growth

Ind

ole

MR

VP

Cit

rate

Ca

tala

se

Nit

rate

Re

du

cti

on

Ge

lati

n

15⁰C 45⁰C

1 WL1 Rod + - + - - - - - - - - 2 WL2 Rod + - + - - - - - - - - 3 WL3 Rod + - + - - - - - - - - 4 WL4 Rod + - + - - - - - - - - 5 WL5 Rod + - + - - - - - - - -

Table 2. Phenotypic profile of Lactobacillus species isolated from Wine samples.

S.

No

Iso

late

s

Ara

bin

os

e

Cell

ob

iose

La

cto

se

Ma

nn

os

e

Rib

os

e

Me

lezit

ose

Ma

nn

ito

l

Me

lib

ios

e

Sa

lic

in

So

rbit

ol

Xy

los

e

Raff

ino

se

Tre

ha

lose

Rh

am

no

se

Pro

ba

ble

Ide

nti

ty

1 WL1 - + + - + + - + - - + - + Lb.buchneri

2 WL2 + + - + - - + - + + + - + + Lb.acidophillus

3 WL3 - + + + - + + - - - - - + - Lb.viridescens

4 WL4 + + + + - - + + + + - + + + Lb.animalis

5 WL5 - + + + + + + + + - + + + Lb.plantarum

ferment sugars at different percentages which were much significant for identification of the species. Among ten different sugars, sucrose was fermented by all the isolated strains. Trehalose was utilized by 94% of the isolates and 60 - 80% of the isolates fermented all other sugars. The differentiating characters of Lactobacillus sp are given in Table 2 and based on the above characters it was concluded that Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus viridescens, Lactobacillus animalis and Lactobacillus buchneri were found in wine sample. Evaluation of different concentration of herbal extracts

Different natural herbal filtrates from Aloevera, Neem, Tulsi were added as substrates to the MRS medium and the growth enhancement of the isolates were studied. The MRS medium supported by Tulsi enhanced the growth of the isolates well when compared to the other two extracts. L. plantarum grew well in the medium containing Tulsi and Neem. Tulsi medium and Neem medium also supported the L. animalis enhancing their growth at a higher rate than other other organisms (Figure 1, 2 and 3). The highest percentage of growth of

the isolates were shown in the medium containing Tulsi than other three herbal extracts (Figure 4) Detection of inhibitory activity Agar spot assay test The culture supernatants from the Lactobacillus species were tested for antibacterial activity against the same group of lactobacilli. All the five isolates have shown clear zone of inhibition against the indicator organism and they were selected as potential bacteriocin producers. Agar well diffusion assay The culture supernatant obtained from 5 bacteriocin producer strains were tested for antibacterial activity against both Gram positive and Gram negative bacteria like L. acidophilus (MTCC 447), B. cereus (MTCC 1272), B. mycoides (MTCC 645), E. coli (MTCC 1680), K. pneumoniae (MTCC 3384), S. aureus (MTCC 740), S. faecalis (MTCC 459) and P. aeruginosa (MTCC 647). Bacteriocins obtained from the isolates showed inhibitory activity against their own group and against certain pathogens. Tulsi seeded medium has survived superior

Divakar et al. 63

0

0.2

0.4

0.6

0.8

1O

D V

alu

e

Growth of Lactobacillus isolates in Aloevera supplemented medium

0 hr

24 hrs

48 hrs

Figure 1. Growth of Lactobacillus species in Aloevera – MRS medium.

0

0.5

1

1.5

2

OD

va

lue

Growth of Lactobacillus in Tulsi supplemented medim

0 hr

24 hrs

48 hrs

Figure 2. Growth of Lactobacillus species in Tulsi – MRS medium.

64 Sky. J. Microbiol. Res.

00.20.40.60.8

1

1.2O

D v

alu

e

Growth of Lactobacillus in Neem supplemented medium

0 hr

24 hrs

48 hrs

Figure 3. Growth of Lactobacillus species in Neem – MRS medium.

for bacteriocin production medium than Aloevera and Neem. Among all isolates WL3 and WL5 ie L. viridescens and L. plantarum showed wide range of inhibition against their own group and certain gram positive and gram negative organisms. Antimicrobial compounds from WL5 showed the highest inhibitory activity against E. coli, B. mycoides and S. faecalis. B. cereus, K. pneumonia and P. aeruginosa were resistant to the antimicrobial compounds produced by WL1, WL2 and WL4 grown in medium containing all the herbal extracts. Genomic DNA isolation Genomic DNA was isolated by Phenol chloroform method from the isolates grown in Aloevera, Tulsi and Neem added MRS medium and in MRS medium. The bands were observed and there was no difference in the bands of Lactobacillus species isolated from the MRS medium with herbal extracts (Plate 1). Discussion

Wine is a popular and important beverage that accompanies and enhances a wide range of European and Mediterranean-style cuisines, from the simple and traditional to the most sophisticated and complex. Wine is important in cuisine not just for its value as a beverage, but as a flavor agent, primarily in stocks and braising,

since its acidity lends balance to rich savory or sweet dishes.

Epidemiological studies have consistently demonstrated that moderate consumption of alcohol and wine is statistically associated with a decrease in death due to cardiovascular events such as heart failure (Lindberg et al., 2008). Isolation of lactic acid bacteria Wine samples were collected from local areas of Coimbatore and processed for isolation of LAB. The microbial colonies were counted in wine samples by standard plate count. From the tested samples five bacterial cultures were isolated to draw conclusion about the resident lactobacilli. A Gram-positive rod was isolated from a commercial grape wine undergoing a sluggish/stuck alcoholic fermentation (Charles et al., 2000). Identification of lactic acid bacteria The isolates were classified into the genera Lactobacillus based on their morphological and biochemical characters (Sharpe et al., 1979) (Tables 1 and 2). All the isolates of Lactobacillus sp were able to grow at 15°C and all of the isolates were not able to grow at 45°C. De Man et al. (1960) stated that Lactobacilli are generally isolated on rich media such as MRS, which is routinely used for the

Divakar et al. 65

Plate 1: Genomic DNA Isolation of bacteriocin producing Lactobacillus species from

herbal added MRS medium

Plate 1. Genomic DNA Isolation of bacteriocin producing Lactobacillus species from herbal added MRS medium.

isolation and counting of Lactobacilli for most fermented food products. Kandler and Weiss (1986) have classified Lactobacillus sp isolates from temperate regions according to their morphology, physiology and molecular characteristics. Few strains were able to utilize citrate and were found to be non motile, catalase, indole, VP negative, nitrates are not reduced and gelatin was not liquefied. Sphelhaug and Hardlender (1989) reported that MRS agar was suitable for bacteriocin assay of lactobacilli. Coppola et al. (2000) studied the morphological characters of lactobacilli from raw milk, natural whey starter and cheese. Lactic acid bacteria were examined from South African grape and wine samples remained unidentified. The identification of these isolates was performed by BLAST and phylogenetic analyses of 16S rDNA gene sequences, which indicated that the isolates belonged to Lactobacillus florum (Mtshali et al., 2012).

Azizpour (2009) stated that LAB isolated from rainbow trout of west Azarbaijan, Iran were Gram positive, catalase positive bacilli, were able to grow at 15 and 45°C. The species identified showed 80% or more similarity to the MTCC type cultures and showed variations in their sugar fermentation pattern.

The predominant Lactobacillus species was further classified to the species level. The differentiating characteristic of Lactobacillus species is given in Tables 1 and 2. Lactobacillus plantarum, Lactobacillus buchneri, Lactobacills acidophilus, Lactobacillus viridescens and Lactobacillus animalis were found in wine samples. The species most frequently isolated were L. vermiforme (24

strains) and L. casei subsp. casei (32 strains). 24 of the strains of L. vermiforme, three strains of L. buchneri, one strain of L. plantarum and two strains of L. casei subsp. casei were isolated from spoiled fortified wine which contained 22% (vol/vol) ethanol.

Evaluation of different concentration of herbal extracts Aloevera, Neem, Tulsi herbal were added as substrates to the MRS medium and the growth enhancement of the isolates were studied. Almost all the herbal extracts supported the growth of the isolates. Tulsi and neem added MRS supported heavy growth of L. plantarum. Tulsi medium and Neem medium also supported the L. animalis enhancing their growth at a higher rate than other other organisms (Figure 1, 2 and 3). The highest percentages of growth of the isolates were shown in the medium containing Tulsi than other three herbal extracts (Figure 4). Aloe vera juice (Aloevera) as a primary substrate fermentation crops for high concentration of viable cells of two active probiotic bacteria: Lactobacillus plantarum (NCIMB 11718) and Lactobacillus casei (NRRL-1445). The higher viable counts were obtained using pure Aloevera juice as fermentation broth for both strains (1 x 10

9 CUF/mL for L. plantarum and 1 x 10

11

CUF/m L. para, L. casei) being the bacterial cultures growth rates in Aloe vera similar to those obtained on MRS medium (µ = 2.85 and µ = 2.71 h-1 for L. plantarum and L. casei respectively).

66 Sky. J. Microbiol. Res.

0102030405060708090

100

MRS &Aloe Vera

MRS & Tulsi MRS & Neem MRS

Pe

rce

nta

ge

Growth of Lactobacillus isolates

Figure 4. Growth of Lactobacillus species in herbal added MRS medium.

Detection of inhibitory activity of bacteriocin producing Lactobacillus species Agar spot assay In this study in vitro assay was carried to characterize the antimicrobial potential of the culture supernatant to inhibit some pathogenic bacteria. Hundred LAB isolates were screened for bacteriocin producers by Agar spot assay test (Kilic et al., 1996). Isolates inhibiting the indicator organism by clear zone of inhibition were selected as bacteriocin producers. Total inhibition diameter was calculated for each LAB strain as the sum of the inhibition diameter against the indicator strain. The results of the Agar spot assay showed that 100% of the isolates were able to inhibit the indicator organism by producing bacteriocin. Nowroozil et al. (2004) has stated that antibacterial activities were done by an agar spot in which only 14.3% of strains made known to produce bacteriocin. Agar well diffusion assay The inhibitory spectrum of the cell free supernatant fluid against variety of Gram positive and Gram negative pathogens was widely varied. Among the bacteriocins tested, almost all the bacteriocin producing strains had a broader host range. It was observed that all the seven bacteriocins had an inhibitory effect on B. mycoides, E. coli, S. faecalis and S. aureus. However, K. pneumonia, P. aeruginosa and B. cereus were resistant to WL1, WL2 and WL4. Researchers have reported that naturally occurring antimicrobials produced by certain bacteria are

effective at controlling undesirable microorganisms in cottage cheese (Weber and Broich, 1986 and Tortorello et al., 1991). An expanded host range has been noted recently for a number of Lactobacillus bacteriocins which kills E. faecalis, Listeria monocytogenes (Klaenhammer, 1993). Toba et al. (1991) determined bacteriocins in L. gasseri, L. acidophilus JCM 1132 and L. acidophilus LAPT 1060 strains from infant feces active against other Lactobacillus strains. Bacteriocins form the pores in the membrane of sensitive cells and deplete the transmembrane potential and/or the pH gradient, resulting in the leakage of cellular materials (Chikindas et al., 1993 and McAuliffe et al., 2001). The inhibitory effect was assumed to be due to bacteriocin and not due to H2O2 since there was no oxidizing effect on bacterial cells which will destroy the basic molecular structure of cell proteins (Zsolt Zalan et al., 2005).

Genomic DNA isolation The technique for chromosomal DNA isolation circumvents the need for phenol-chloroform extractions and cesium chloride gradients. Isolated DNA is consistently greater than 25 kb in size and can be used directly for sub cloning, polymerase chain reaction amplification, restriction digestions and library construction (Ulrich and Hughes, 2001).

In this study it highlights the importance of using an herbal plants and where necessary multiple bioassays covering the entire spectrum of activities that can provide a reliable evaluation of the biological efficacy of herbal plants.

REFERENCES Azizpour K (2009). Biochemical characterization of Lactobacillus

isolated from rainbow trout (Oncorhynchus mykiss) of west Azarbaijn, Iran. Res. J. of Biol. Sci., 4(3): 324 - 326.

Charles G, Edwards I, Matthew D, Collins Paul A, Ana VR (2000). Lactobacillus nagelii sp. nov., an organism isolated from a partially fermented wine. Int. J. of Syst. and Evol. Microbiol., 50: 699 – 702.

Chikindas ML, Garcia-Garcera MJ, Driessen AJ, Ledeboer AM, Nissen-Meyer J, Nes IF, Abee T, Konings WN, Venema G (1993). Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells. Appl. Environ. Microbiol., 59: 3577–3584.

Coppola R, Nanni, Iorizzo M, Sorrentino A, Sorrentino E, Chiavari C, Grazia L (2000). Microbiological characteristics of Parimigiano Reggiano cheese during the cheese making and the first months of the ripening. Lait., 80: 479-490.

De Man JC, Rogosa M, Sharpe ME (1960). A medium for the cultivation of lactobacilli. J. of Appl. Bacteriol., 23: 130-135.

Gonzalenz F, Blair S. Kunka (1987). Plasmid associated bacteriocin production and sucrose fermentation in Pediococcus acidilactici. App. Env. Microbiol. 53(10): 2534 -2538.

Havenaar R, Ten Brink B, Huis in’t Veld JHJ (1992). Selection of strains for probiotics use. In: Fuller, R (Ed.), Probiotics: the scientific basis. (1st Edn.), London, Chapman and Hall. pp: 209-224.

Kandler O (1983). Carbohydrate metabolism in Lactic acid Bacteria. Antonie Van Leeuwenhoek. J. Microbiol. Serol., 49: 209-224.

Kandler O, Weiss N (1986). Genus Lactobacillus beijerinck 1901. In Bergey

’s Manual of systematic Bacteriology, 2:1209-1234.

Kilic AO, Pavlova SI, Ma W, Tao L (1996). Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of phages isolated from yogurt. Appl. Environ. Microbiol., 62: 2111-2116.

Klaenhammer TR (1993). Genetics of bacteriocins produced by lactic acid bacteria. FEMS. Microbiol. Rev., 12: 39-86.

Lindberg Matthew L, Ezra A. Amsterdam (2008). "Alcohol, wine, and cardiovascular health". Clinical Cardiol., 31 (8): 347–51).

McAuliffe O, Ross RP, Hill C (2001). Lantibiotics: Structure, biosynthesis and mode of action. FEMS Microbiol. Rev., 25: 285–308.

Mtshali PS, Divol B, Du Toit M (2012). Identification and characterization of Lactobacillus florum strains isolated from South African grape and wine samples. Int. J. Food Microbiol., 153(1-2):106-13.

Nowroozi J, Mirzaii M, Norouzi M (2004). Study of Lactobacillus as Probiotic bacteria. Iranian. J. Publ. Health., 33(2): 1 -7.

Sharpe ME, Fryer TF, Smith DG (1979). Identification of Lactic acid Bacteria. In: Identification methods for Microbiologists, E.M. Gibbs, F.A. Skinner (Eds), London: Academic Press, pp. 233-259.

Divakar et al. 67 Singh GP, Rakesh RS (2009). Dominating species of Lactobacilli and

Leuconostoc present among the lactic acid bacteria of milk of different cattle’s. Asian J. Exp. Sci., 23 (1): 173-179.

Sphelhaug SR, Hardlender SK (1989). Initiation of food born bacterial pathogens by bacteriocins from Lactococcus lactis and Pediococcus spentosaceous. J. Food Prot., 52: 856-862.

Toba T, Yoshioka E, Itoh T (1991). Acidophilucin A, a new heat-labile bacteriocin produced by Lactobacillus acidophilus LAPT 1060. Lett. Appl. Microbiol., 12: 106-108.

Tortorello ML, Best S, Batt CA, Woolf HD, Bencher J (1991). Extending the shelf life of cottage cheese. Identification of spoilage flora and their control using food grade preservatives. Cult. Dairy. Prod. J., 26: 8-9.

Ulrich RL, Hughes TA (2001). A rapid procedure for isolating chromosomal DNA from Lactobacillus species and other Gram-positive bacteria. Lett. Appl. Microbiol., 32(1):52-6.

Weber GH, Broich WA (1986). Shelf life extension of cultured dairy foods. Cult. Dairy. Prod. J., 21: 19-2.

Zsolt Z, Edina N, Agnes B, Anna H (2005). Influence of Growth Medium on Hydrogen Peroxide and bacteriocin production of Lactobacillus strains. Food Technol. Biotechnol., 43(3): 219-225.