Investigation of Antagonistic Effects of Isolated Lactic Acid Bacteria from Different Cheeses of Gorgan City against Main Intestinal Pathogenic Bacteria

Lactic Acid Bacteria (LAB) have been shown to elicit positive health effects particularly in humans. Competitive exclusion of pathogens is one of the most important beneficial health claims of probiotic bacteria. This study was conducted with the aim of isolation of Lactic Acid Bacteria from different Cheeses and their effects on the main pathogenic bacteria in intestine which might provide important information regarding its probiotic potential and its utilization in the future. Morphological, cultural and biochemical characteristics were employed to identify lactic acid bacteria, isolated from different Cheeses in different areas in Gorgan city, Iran. From 9 traditional Cheese and 2 industrial Cheese samples a total of 38 isolates were isolated, 36 isolates from local Cheese and 2 isolates from industrial Cheese. The results showed that Lactobacillus casei has the highest frequency. Also this species showed antagonistic activity against pathogens including Escherichia coli, Staphylococcus aureus, Bacillus cereus and Citrobacter freundii, with an inhibition zone diameter of 17 mm. Investigation of Antagonistic Effects of Isolated Lactic Acid Bacteria from Different Cheeses of Gorgan City against Main Intestinal Pathogenic Bacteria


Introduction
Now-a-days consumers are concerned about the synthetic chemicals used as preservatives in food, and there is resulting trend towards less processed food. These untreated foods can harbour dangerous pathogens which can multiply under refrigeration and without oxygen. A solution to this dilemma is the use of antimicrobial metabolites of fermentative microorganisms [1]. Many antimicrobial chemicals have been in use for a long time without any known adverse effects. Many of the organic compounds which have stirred interest are antimicrobial metabolites of bacteria used to produce, or associated with fermented foods.
In fermentation, the raw materials are converted by microorganisms (bacteria, yeast and molds) to products that have acceptable qualities of food. In common fermented products such as yogurt, lactic acid is produced by the starter culture bacteria to prevent the growth of undesirable microorganisms [2]. Food fermentations have a great economic value and it has been accepted that these products contribute in improving human health. LAB have contributed in the increased volume of fermented foods worldwide especially in foods containing probiotics or health promoting bacteria [1].
Lactic acid bacteria (LAB) are a group of gram-positive bacteria including the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus. The general description of the bacteria included in the group is gram-positive, non-spore forming, cocci or rods, which produce lactic acid as the major end product during the fermentation of carbohydrates [3].
These bacteria are the major component of the starters used in fermentation, especially for dairy products, and some of them are also natural components of the gastrointestinal microflora [4].
Lactic acid bacteria have been used in the production of foods, especially fermented foods because they can produce several compounds that contribute to taste, smell, color, and texture of the foods. In addition, they can produce antimicrobial substances including bacteriocins that have ability to inhibit pathogenic and food spoilage bacteria [3]. The antagonistic activity of such bacteria is known to inhibit a large number of enteric and urinary pathogenic bacteria [5]. The effect of probiotic LAB on competitive exclusion of pathogens has been demonstrated with human mucosal material in vitro, and in vivo in chickens and pigs [6]. Enteric bacterial pathogens must adhere to and penetrate the protective intestinal mucus layer in order to reach and invade the enterocytes and cause clinical infection [7]. The present investigation reports isolation and characterization of LAB species having potential application in the local dairy industry such as chesses fermentation, source of probiotics and microbial contaminants reducing agents of the product.

Cheese samples
During the spring of 2014, a total of 11 cheeses (9 traditional and 2 industrial cheese samples) were collected from Gorgan city, Iran. The samples were collected in sterile universal tubes and kept cool until they could be taken to the laboratory, where they were kept at 4ºC for further use.

Isolation of lactic acid bacteria
The samples were aseptically weighted and homogenazied. From each sample, a 1:10 dilution was subsequently made using peptone water followed by making a 10 fold serial dilution. 1 ml from each dilution was then subcultured, in duplicate, into the Plate Count Agar and MRS agars (Merck, Germany) used for isolating LAB [8] and were incubated anaerobically using the Gas Pack system (Merck Anaerocult type A) at 37ºC for 2 days.

Identification of the bacterial strains
All strains were initially tested for gram reaction, catalase production and spore formation [9]. Colonies were characterized on MRS Broth and M17 Broth. Strains with gram positive and catalase negative reactions were finally used for further identification. Growth at different temperatures (10ºC and 45ºC) for 2 days, growth in the presence of 6.5% NaCl, were considered to identify the strains.
All strains were also tested for fermentation of L-arabinose, D-xylose, galactose, D-fructose, sorbitol, lactose, mannitol, saccharose, rhaminose and mannose. Staphylococcus aureus (PTCC 1431), Escherichia coli (PTCC 1399), Bacillus cereus (ATCC 1252) and Citrobacter freundii (1600) were used as indicator culture. As mentioned earlier, the culture broths of both the producer and indicator strains were adjusted to McFarland Index 0.5 (1.5 × 10 8 ) to use. The growth of Lactic acid bacteria strains isolated from cheeses at 37°C was visually confirmed by the changes in turbidity of Muller Hinton Agar or BHI after 24h of incubation.
The surface of Muller Hinton Agar plates were evenly streaked with selected indicator strains, with a sterile cotton swab. The culture broth of the producer strains (100 ul) were poured into the wells (7 mm) made in these agar plates with a sterile borer. All plates were stored for 2 h at 4ºC prior to incubation at 37ºC for 24 h. The antimicrobial activity was recorded as appearance of clear zone around the wells and the zone diameter (Resistance: 7 mm<zone, Inter mediate: 8-9 mm<zone, Sensitive: 10 mm<zone) measured in millimeter. All tests were run in duplicate.

Results and Discussion
During the spring of 2014, a total of 11 cheese samples (9 traditional and 2 industrial cheese samples) were collected from Gorgan city ( Table 1).

Microbial count results
The results showed that the average number of microbial count in Plate count agar is the maximum amount in sheep samples (5/4 × 10 7 CFU/gr) and minimum amount in water buffalo samples (1 × 10 4 CFU/ml) ( Table 2). Kiai et al. [10] have reported in previous studies, the microbial count in traditional yogurt in Golestan province between CFU/ml 7/4 × 10 9 and 3/92 × 10 9 CFU/ml. Also average number of microbial count in MRS Agar is the maximum amount in cow samples (3/3 × 10 7 CFU/ml) and minimum amount in goat samples (1/3 × 10 7 CFU/ml) (Table 3), that has been reported in previous studies on local yogurts, between CFU/ml 3/6 × 10 9 and 7/2 × 10 9 CFU/ml [10]. The daily intake of 10 8 to 10 9 live bacteria, is the minimum acceptable value, therefore daily intake of 100 grams of probiotic product containing up to 1 × 10 6 -5 × 10 8 Cfu live bacteria per gram of product, can provide the optimum desired [11].

Results of isolation and identification of lactic acid bacteria
38 species of lactic acid bacteria were isolated and identified from 11 samples of cheese that the types and numbers of these bacteria shown in Tables 4 and 5, according to type of cheese.     Table 5, the maximum number of lactic acid bacteria is in sheep and the minimum number is in Industrial cheese. The highest frequency of lactic acid bacteria isolated from the cheese samples, is belongs to the species include Lactobacillus casei, Streptococcus lactis and Enterococcus faecium. It is shown the number and percentage of different species of lactic acid bacteria isolated from cheese samples ( Figure 1).

Results of antagonistic effect of LAB
Lactic acid bacteria isolated from cheese samples showed antagonistic activity against pathogenic bacteria with the production of the antimicrobial compound. The results are shown in Table 6. Lactobacillus casei, Lactobacillus acidophilus, isolated from sheep cheese, inhibit the growth of all pathogenic bacteria. Lactobacillus casei has the highest antibacterial activity against pathogenic bacteria.
Enterococcus faecium isolated from cow and goat cheese and also Streptococcus thermophilus and Streptococcus lactis isolated from sheep cheese, have no antimicrobial effect on pathogenic bacteria. So in total, the lactic acid bacteria isolated from sheep cheese, produce greater antimicrobial compounds against pathogenic bacteria [12], in the same study found the similar result and Lactobacillus acidophilus showed the highest inhibitory effect with an inhibition zone diameter of 14 mm.

Conclusion
As we enter in the new millennium, people are aware that for spending a healthy life style diet play a major role in preventing diseases and promoting health. Therefore there is an increasing trend for foods containing probiotic cultures. Probiotics are viable organisms and supportive substances that improve intestinal microbial balance, such as Lactobacillus acidophilus and bioactive proteins [13]. Antimicrobial effect exerted by the LAB is mainly due to acid production, hydrogen peroxide, fatty acids, aldehydes and other compounds [14]. The empirical evidence that for many years, linked the use of fermented dairy products such as cheese and milk with the promotion of intestinal health is today well supported by modern science. Commercial lactic acid bacterial products have been one of the major health related foods in the world. When a LAB is used as probiotic, it should be safe and possess some basic characteristics, such as the tolerance to acid, ability to adhere to human intestinal epithelium and having antagonistic activity against bacterial pathogens.
This study proved the presence of viable LAB micro flora in cheese samples in Gorgan city. Overall results of this research suggests that the selected LAB strains isolated might be appear to possess probiotic potential, and hence could be exploited further for their use in fermented dairy products.  Table 6: Results in inhibition of lactic acid bacteria isolated from cheeses against pathogenic bacteria.