Inhibitiory Activity of Lactic Acid Bacteria against Hazardous Microbes

One hundred of lactic cultures were evaluated for their ability to inhibit hazardous microbes, such as Salmonella enteritidis, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, and Bacillus cereus by agar well diffusion method. None of them showed inhibitory halo against S. enteritidis, while 27 strains showed inhibitory activity against S. typhimurium, 6 against E. coli, 9 against ampicillin resistant E. coli, 31 against L. monocytogens, 10 against B. cereus. pH of the culture does not explain for the inhibitory activity except against B. cereus. A neutralized culture from corn silage showed highest inhibitory activity against S. typhimurium, and the size of inhibitory halo was same as 10 ug/mL of ampicillin. The culture was identified to be Lactobacillus buchneri on the basis of biochemical characteristics and utilization of substrates. Using the culture as probiotics could be expected to reduce antibiotics for animal feeding.


INTRODUCTION
Probiotics are viable bacterial cell preparation or foods containing viable bacterial cultures or components of bacterial cells that have beneficial effects on the health of the host (Delley et al., 1990;Hertel et al., 1991;Ehrmann et al., 1992;Ehrmann et al., 1994;Charteris et al., 1997).Many of these probiotics are lactic acid bacteria (Lee et al., 1999).The pioneering evidence of the competitive exclusion concept of lactic acid bacteria was obtained from poultry chickens by Nurmi and Rantala (1973).The newely hatched birds do not obtain the normal gut flora of the adult, owing to modern management methods.Since normal flora is lacking, the intestines of the birds are easily colonized by pathogens, most often by Salmonella or Coliforms.When the chickens were inoculated just after birth with the caecal contents of an adult bird, the frequency of Salmonella infections was radically reduced and the number of Salmonella needed to colonize the caeca of the birds increased.The specific role of lactic acid bacteria as a probiotic has been extensively discussed by Juven et al. (1991).Lactic acid bacteria produce many kinds of metabolites which might affect the other microbes in the gut.Lactic acid produced both by homolactic and heterolactic strains reduce pH of the luminal contents, which is most obvious in the stomach of neonatal piglets (Cranwell et al., 1976).Moreover, acetic acid and hydrogen peroxide produced by lactic acid bacteria are inhibitory against coliforms, salmonella, and clostridia in vitro (Nousiainen, 1993).
Lactic acid bacteria play an essential role in the majority of food fermentations, and a wide variety of strains are routinely employed as starter cultures in the manufacture of dairy, meat, vegetable and bakery products.One of the most important contributions of these microorganisms is the extended shelf life in the fermented product by comparison to that of the raw substance.Growth of spoilage and pathogenic bacteria in these foods is inhibited due to competition for nutrients and the presence of starter-derived inhibitors such as lactic acid, hydrogen peroxide and bacteriocins (Ray and Daeschel, 1992).Currently, artificial chemical preservatives are employed to limit the number of microorganisms capable of growing within foods, but increasing consumer awareness of potential health risks associated with some of these substances has led researchers to examine the possibility of using bacteriocins produced by lactic acid bacteria as biopreseratives (Abee, 1995).However, the inhibitory activity of bacteriocins is confined to Gram-positive bacteria and inhibition of Gramnegatives by them has not been demonstrated.
The aim of this study was to screen lactic bacterial cultures having inhibitory activity against hazardous bacteria for the development of animal probitics and food biopreservatives.

Microorganisms and media
The Lactobacilli cultures were obtained from ATCC and KTCC, and isolated from silages and dairy products.Isolation from silages and dairy products was carried out by incubating the samples in MRS agar containing sodium azide for 2 days.Acid producing, Gram positive, and rod cultures were presumed as Lactobacillus organisms.Acid production was measured by pH, and shape of the cell was examined using light microscopy (×1,500) (Clinigold, USA) after Gram-staining.Identification of Lactobacillus species was conducted by API 50CHL kit (bioMerieux, France) and the software (APILAB Plus Ver. 3.3.3;bioMerieux,France).S. enteritidis IFO 3313, S. typhimurium IFO 12529, E. coli F4 and F5, L. monocytogenes ATCC 15313, and B. cereus ATCC 11778 were maintained by subcultured in tryptic soy broth (TSB) (Difco).E. coli F4 and F5 were donated by Lab. of Veterinary Medicine, Seoul National University.

Inhibitory activity of lactic acid bacteria
Agar well diffusion method was modified from that of Tagg and McGIVEN (1971).Petri dishes with metal borer of 8 mm diameter are poured with tryptic soy agar (TSA) and inoculated by testing strain.By removing borer after solidification, the base of each hole was sealed with a drop (0.05 mL) of melted TSA, and then either 100 µl of lactic   culture or neutralized lactic culture filtrate was added to the well.The inoculated plates are incubated at 37°C for 1 day after overnight preincubation at 4°C to allow for diffusion of the inhibiting factors.10, 50, 100, and 500 µg/mL of ampicillin and nisin (Sigma, USA) were used as control.

RESULTS AND DISCUSSION
The inhibitory activity of ampicillin and nisin against S. enteritidis IFO 3313, S. typhimurium IFO 12529, E.coli F4 and F5, L. monocytogenes ATCC 15313, and B. cereus ATCC 11778 by agar well diffusion method according to concentration is given in Figure 1.Nisin showed inhibitory activity only against L. monocytogens, while ampicillin has inhibitory activity against all of them except L. monocytognes and E. coli F5.Although B. cereus generally shows high resistance to ampicillin, colistin and polymyxin (Bernhard et al., 1981), the type of antibiotic and the concentration affecting vegetative growth of Bacillus strains may differ (Russell, 1982;Ochi and Freese, 1983).
Among those hundred strains of lactic acid bacteria, no strain was found to have inhibitory activity against S. enteritidis.Human illness caused by infection with Salmonella enterica serovar Enteritidis (S. enteritidis) increased worldwide beginning as early as the mid-1970s and, by 1990, this serovar displaced Salmonella enterica serovar Typhimurium (S. typhimurium) as the primary cause of Salmonellosis in the world (Baumler et al., 2000;Anonymous, 2001).It can be assumed that inhibition of S. enteritidis by lactic acid bacteria seems have little possibility, but possible in case of S. typhimurium.22 strains of LAB cultures and 6 strains of neutralized LAB cultures showed inhibitory activity against S. typhimurium (Figure 2).One strain showed inhibitory activity in both culture and neutralized culture.Among those strains, C-11, CSII-12-1, and CSII-12-2 showed higher inhibitory activity than that of others.C-11 strain isolated from rye silage was identified to be L. buchineri by API test (Table 1), CSII-12-1 and CSII-12-2 isolated from corn silage were also (Table 2).C-11 and CSII-12-2 could not utilize lactose and galactose.Inhibitory activity of the neutralized cultures might from bacteriocin produced by the LAB because the inhibitory activity of acid would be eliminated and that of hydrogen peroxide would not be made by neutralization.Antibiotic activity of bacteriocin could be affected by pH (Liu and Hansen, 1990).Purification of the bacteriocin is under studying.5 strains of LAB cultures and 2 strains of neutralized LAB cultures showed inhibitory activity against E. coli F4 (Figure 3).One strain showed inhibitory activity in both culture and neutralized culture.But all of them were weak and less than 5 mm. 4 strains of LAB cultures and 5 strains of neutralized LAB cultures showed inhibitory activity against E. coli F5 (Figure 4).Because E. coli F5 weren't inhibited by ampicillin, further study is required about the inhibiting agent of the neutralized cultures.Especially, neutralized CSII-12-1 culture showed inhibitory activity against E. coli F5 as well as S. typhimurium.26 strains of LAB cultures and 6 strains of neutralized LAB cultures showed inhibitory activity against L. monocytogenes (Figure 5).It was thought that not many strains showed bacteriocin activity comparing the report of Aroutcheva et al. (2001).Accroding to the report, approximately 80% of the isolated lactobacilli from vaginal specimens produced bacteriocin that inhibited growth of 4 strains of Gardnerella vaginalis.However, not all strains of G. vaginalis could be inhibited by lactobacilli-producing bacteriocin.It was noted that under pH 4 of the cultures could inhibit Bacillus cereus, and no neutralized strain could (Figure 6).Role of probiotics in health and diseases is well summarized in 'Handbook of probiotics' (Lee et al., 1999).Orally administered yoghurt bacteria could protect against S. typhimurium infection in Male Sprague-Dawley rat.Intraperitoneal administration of L. casei strain Shirota could protect against L. monocytogenes in male BALB/c mice.It can be inferred from the results, the screened lactic acid bacteria might be useful as a probiotics for protection against hazardous microbes infection with the experimental results in animal model.
In conclusion, inhibitory activity of one hundred lactic

Figure 2 .
Figure 2. Inhibitory activity of lactic acid bacterial cultures against Salmonella typhimurium.Line: pH of the culture, red stick: antimicrobial activity of lactic cultures, yellow stick: antimicrobial activity of neutralized lactic cultures, photograph: inhibitory halo of the neutralized CSII12-1 and CSII-2 culture against S. typhimurium.

Figure 3 .Figure 4 .Figure 5 .
Figure 3. Inhibitory activity of lactic acid bacterial cultures against Escherichia coli F4.Line: pH of the culture, red stick: antimicrobial activity of lactic cultures, yellow stick: antimicrobial activity of neutralized lactic cultures Figure 4. Inhibitory activity of lactic acid bacterial cultures against Escherichia coli F5.Line: pH of the culture, red stick: antimicrobial activity of lactic cultures, Yellow stick: antimicrobial activity of neutralized lactic cultures.

Figure 6 .
Figure 6.Inhibitory activity of lactic acid bacterial cultures against Bacillus cereus.Red stick: Antimicrobial activity of lactic cultures.

Table 1 .
Utilization of carbohydrate substrates of the C-11 Glycerol