Abstract: A study was carried out to identify Lactic Acid Bacteria (LAB) isolated from milk and milk products. One hundred and twenty four isolates were Gram-positive, were identified to genus level using morphological, physiological and biochemical tests. Three genera of lactic acid bacteria were identified and there were Lactobacillus, Lactococcus and Leuconostoc from these genera. Seventy six isolates were selected after showed antibacterial activity against the sensitive strain Lactococcus lactis subsp. lactis MTCC3038. Of these 25 isolates were showed high antibacterial activity. These 25 isolates were selected for identification to species level using physiological and biochemical test and compared as Bergey’s manual of systematic bacteriology. The isolate, which showed strongest activity, was identified as Lactococcus lactis subsp. cremoris using characters of biochemical tests. It was given the strain number as Lactococcus lactis CCSUB94. It produced bacteriocin which was active against closely related lactic acid bacteria as well as a wide range of food pathogens including Listeria monocytogenes, Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Salmonella typhi.
INTRODUCTION
The bacteriocin from food grade lactic acid bacteria (LAB) appear to qualify as ideal food biopreservative primarily because they have proven non-toxic to humans, do not alter the nutritional properties, effective at low concentration, active under refrigerated storage and can be used even in the preservation of foods by the use of bacteriocins produced by lactic acid bacteria. Bacteriocins, as defined by Tagg et al. (1976) are proteinaceous compounds produced by bacteria that exhibit a bactericidal mode of action against related as well as unrelated organisms. These bacteriocins were initially demonstrated in E. coli (Gratia , 1925) and later in Gram- positive bacteria (Tagg et al., 1976).
Bacteriocins produced by lactic acid bacteria have been the focus of many investigations because of their particular importance in the dairy industry. Lactic acid bacteria isolated mainly from fresh vegetables and from the skin of animals, (Lilliana and Stouvenel, 2006) therefore it is believed that their presence in milk is due to contamination source. Its presence in human being or animals is accidental because of they are not normally found in a significant number in excrement or soil (Roissart and Luquet, 1994). Their most important habitat is in untreated milk, fermented milk and cheeses. Lactococcus lactis subsp. lactis, either in pure form or associated with other micro-organisms is the mesophilic strain most commonly used as a starter culture for lactic products, thus they fulfill an irecplaceable role in ensuring the structure, taste, conservation and healthfulness of these products (Jensen and Hammer, 1993; Salminen and Von Wright, 1993; Roissart and Luquet, 1994; Boonmee et al., 2004; Ziadi et al., 2005; Do-Won et al., 2006).
The antagonistic activity of lactic acid strains isolated from milk and milk product was performed. As a result of these screening several strains were found to produce bacteriocin like substances. The starin identified by physiological and biochemical tests. Lactococcus lactis subsp. lactis MTCC3038 was used a positive control.
Here we report the characterization of a bacteriocin produced by Lactococcus lactis CCSUB94 with wide inhibitory spectrum including a group of foodborne pathogens.
MATERIALS AND METHODS
Isolation of Lab Strains
We screened LAB starins from a total number of 124 sample of food products
and different milk and milk products comprising cheese-22, curd-15, buffalo
milk-34, cow milk-15, goat milk-15, pasteurized milk-10, sweets-8 (burfi,mava-ladoo,ghevar),
collected from various cattle yard, dairy and sweet shops in Meerut region,
India.
Twenty five milliliter sample were collected in sterile 100 mL capacity sample bottles after taking appropriate precautions. The sample was processed immediately for isolation of the Lactic Acid Bacteria (LAB). LAB was isolated by different methods using MRS medium (peptone-10 g, beef extract-10 g, yeast extract-5 g, glucose-20 g, disodium hydrogen phosphate-2 g, sodium acetate-5 g, triammonium citrate-2 g, MgSO4. 7 H2O-0.02 g, MnSO4.4H2O-0.02 g, Tween 80-1 mL, agar-15 g, made to 1000 mL with distilled water, pH 6.5) (de Man et al., 1960).
Dillution Method
Ten grams of curd, cream, milk samples and jam were aseptically transferred
to 90 mL of sterile physiological saline (0.9% NaCl) (warmed to 45°C for
cream) and mixed well.
Cheese samples were prepared by transferring 10 g of aseptically weighed sample to 100 mL sterile 2% sodium citrate solution at 45 to 50°C and homogenized for 3 min.
In all the cases serial dilution were subsequently prepared in sterile physiological saline. One milliliter sample by appropriate dilutions were plated out on MRS medium. The plates were incubated at 37°C for 24 h. After 24 h typical colonies were picked up randomly and transferred to MRS broth (de Man, et al., 1960) for microscopic examination and gas production subsequently.
One hundered milliliter of milk sample was taken in glass and with 2 g of curd sample incubated at 37°C and the sampling was done after every 1 h. sample was refrigerated at 4°C. Each sample was then centrifuged at 15000 rpm for 30 min at 4°C in the high-speed refrigerated centrifuge (Sigma 2K15). The supernatant was then filtered through sterile membrane filter of 0.45 μm pore size (Millipore). Cell free filtrate thus obtained was stored in vials, at -20°C. Supernatant was used for screening for bacteriocin production.
The pellet was then streaked with sterized loop on MRS plates for isolation of LAB.
Identification of Isolates to Genus Level
The isolates were identified to genus level by Gram staining (Harrigan and
McCance, 1993), observing growth at 10, 15 and 45°C, temperature and in
saline MRS broth (4 and 6.5% NaCl w/v) was determined by visual turbidity after
72 h incubation. Other tests were catalase test (Harrigan and McCance, 1993),
oxidase test (Collins and Lyne, 1987), nitrate reductase test, gas production
from glucose was assessed in Sugar Basal Medium (SBM) broth containg 2% (w/v)
glucose dispensed in test tubes containing inverted Durham tubes. The inoculated
tubes were examinated for the production of gas after 3 days’s incubation.
The salt tolerance test was done using MRS broth containing 6.5 (w/v) NaCl with
incubation time of 4 days at 37°C.
Identification of Selected Isolates to Species Level
Carbohydrate (glucose, sucrose, reffinose, fructose) fermentation test was
done, as above. Arginine deamination was detected in SBM supplemented with 1%
(w/v) arginine monochloride, 0.3% (w/v) Bacto-agar and 0.01% (w/v) phenol red,
pH 7.2. After inoculation the medium was incubated at 37°C for 3 days. Arginine
hydrolysis was observed by the culture turning yellow.
Maintenance of Isolates
Pure cultures were stored frozen at -20 °C in 30 % sterile skimmed milk
in small vials of 2 mL.
Screening for Bacteriocin-producing Strains
The LAB cultures isolated in this study were screened for their antagonistic
activity against bacteriocin sensitive strain MTC3038, by using Yang et al.
(1992) method.
Bacteriocin Activity Assay
Aliquots from culture of producer strain were adjusted to pH-3.0 with 0.5
M HCl to avoid the adsorption of molecules of bacteriocin can to the producer
cell surfaces. Thereafter centrifuged at 15000 rpm for 30 min at 4°C. The
supernatants containing overall anti-bacterial activity (bacteriocin extract)
were adjusted to pH 6.0 and frozen until further use.
Antimicrobial Activity
Antibacterial activity of bacteriocin extracts was determined by a photometric
bioassay method (Cabo et al., 1999) using L. lactis subsp.
lactis MTCC3038 as the target organism. Bacteriocin extracts were diluted
in sterile distilled water. (This step eliminated the need to correct the pH
of bacteriocin extracts). The diluted bacteriocin extracts (2.5 mL) was added
into sterile culture tubes. Each tube was inoculated with 2.5 mL of culture
of target sensitive strain (dilution to an absorbance of 0.3 at 700 nm with
sterile buffered MRS broth pH 6.3).
Controls consisting of three culture tubes in which the diluted bacteriocin extracts was substituted by sterile distilled water. The tubes were incubated for 6 h at 37°C.
Growth inhibition was measured spectophtometrically (Systronics UV-VIS double-beam spectrophotometer 2201) at 700 nm. Bacteriocin activity was defined as the amount of bacteriocin needed to obtain 50% growth inhibition (lethal dose = 50) of the indicator strain and was expressed in bacteriocin units (BU mL-1).
Test Organisms
Antibacterial activity of bacteriocin was tested against some pathogenic
and nonpathogenic bacteria like Lactococcus lactis subsp. lactis MTCC3038,
Bacillus polymyxa, Bacillus subtilis MTCC441, Salmonella typhi
MTCC734, Shigella sonnei MTCC2957, Escherichia coli MTCC119,
Staphylococcus aureus MTCC96, Clostridium perfringens MTCC450,
Enterobaceter faecalis MTCC439, Enterobaceter aerogenes MTCC111,
Streptococcus pneumoniae MTCC1935, Pseudomonas aeruginosa MTCC2581
and Listeria monocytogenes MTCC657. These cultures were collected from
the Institute of Microbial Technology (IMTECH), Chandigarh India and were maintained
in Department of Microbiology in 30% sterile skimmed milk in small vials of
2 mL, at - 20°C.
Inhibitory activity titres against the indicator bacteria were determined by agar diffusion well assay (Varadaraj et al., 1993). Bacteriocin activity is calculated by AU mL-1 (Highest dilution that showed a distinct zone of inhibition X 20 (1 000 μL/50 μL).
AU mL-1 = highest dilution that showed clear well-defined zone of inhibition x 1000 μL/Volume (μL) used in the well.
RESULTS
Isolation of LAB
From milk and milk products 76 bacteriocin producing strains were isolate.
Strain number CCSUB94 showed highest antibacterial activity against test strain
Lactococcus lactis subsp. lactis MTCC3038. we selected for detail
study.
Identification of Isolates to Genus Level
Seventy six isolates were identified compared to the Bergey’s manual
of systematic bacteriology (Holt et al., 1994). Twenty four isolates
were rod shaped and could be identified as Lactobacillus. The ability to produce
gas was an important characteristics for distinguishing the Leuconostoc
(Garvie, 1984). Twenty isolates belonged to the genus Leuconostoc, 32
isolates belonged to the genus Lactococcus.
Identification of Isolates to Species Level
Table 1 and 2 showed that 25 isolates
of LAB were showed higher antibacterial activity. These selected for identification
to species level with biochemical tests. Nine belonged to the species Lactococcus
lactis subsp. lactis, 6 belonged to the species Lactococcus lactis
subsp. cremoris, 2 belonged to L. raffinolactis and 2 belonged
to the species Lactobacillus casai, 4 L. acidophilus and two belonged
to Lactobacillus leichmanhii.
| Table 1: | Identity of isolated lactic acid bacteria |
| Note: Lc. = Lactococcus; Lb. = Lactobacillus | |
| Table 2: | Antibacterial activity of bacteriocin produced by Lactococcus lactis CCSUB94 against different test organisms |
| Data represents average of three trials. Antibacterial activity was determined by agar well diffusion method. Formation of a well-defined inhibitory zone around the well containing the test sample was measured. (-) and (+) signs indicate no inhibition or inhibition growth. R: Resistant; S: Sensitive | |
DISCUSSION
During the present study, 101 milk and milk products samples were collected from which 124 LAB were isolated. These included 67% Lactococcus sp., 29% Lactobacillus sp. and 19% Leuconostoc which suggest that raw milk harbour highest number of Lactococcus sp. followed by Lactobacilli. Dave and Prajapati (1994) and Leisner et al. (1999) also isolated the nisin producing Lactococcus lactis subsp. lactis from raw milk. These isolates were screened for their antagonistic activity and only one isolate (Lactococcus sp. CCSUB94) was selected for detailed studies. As per the characteristics described by Collins and Lyne (1983) the isolated strain was identified. The strain was Gram-positive cocci in pairs or small chains. The strain was found to be catalase negative, therefore, couldn’t be the species of Staphylococcus or Micrococcus, which are catalase positive. The optimum temperature for growth of the organism is 35 to 38°C, therefore, it can not be a member of the pathogenic group of Streptococci i.e., Streptococcus lancefield group A, B, C, D. viridans group or Streptococcus pneumonia. The organism is lactose positive therefore, may not be an Enterococci, which are lactose negative. Taking the above facts into consideration, it can be concluded that the organism is a species of Lactococcus. The organism was further identified at species and subspecies level according to the Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). Based upon carbohydrate fermentation profile and other physiological standards suggested by Shaw and Harding (1984) and Schillinger (1990), strain was classified as Lactococcus lactis CCSUB94.
CONCLUSIONS
The bacteriocin produced by Lactococcus lactis CCSUB94 was assayed by agar well diffusion method and bacteriocin activity was measured in terms of AU mL-1. The highest dilution that gave a define zone of growth inhibition was used to calculate AU mL-1. Mode of action of bacteriocin produced by Lactococcus lactis CCSUB94 was tested and the behavior of the bacteriocin produced by isolated strain was considered as bactericidal.