Antibacterial effects of supernatant of lactic acid bacteria isolated from different Dough’s in Gorgan city in north of Iran

This study was conducted with the aim of isolation of Lactic Acid Bacteria (LAB) from dough and their effects on the main pathogenic bacteria in intestine. Morphological, cultural and biochemical characteristics were employed to identify lactic acid bacteria, isolated from dough in different areas in Gorgan city, Iran. From 13 traditional dough and 2 industrial dough samples a total of 35 isolates were isolated, 32 isolates from local dough and 3 isolates from industrial dough. 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. Introduction Interest in microorganisms occurring in foods is primarily due to the biotechnological potential of new bacterial species and strains [1]. Lactic acid bacteria (LAB) are widely distributed in nature and occur naturally as indigenous microflora in raw milk, drinking yoghurt, etc. Lactic acid bacteria are a group of gram-positive bacteria including the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus and play an important role in many food fermentation processes. The lactic acid fermentation has long been known and applied by humans for making different food stuffs. In addition, they strongly determine the flavour, texture and frequently, the nutritional value of food and feed products [2-4]. In fermented foods, LAB display numerous antimicrobial activities. This is mainly due to the production of organic acids and other compounds, such as bacteriocins and antifungal peptides [5]. Several studies have shown the inhibitory activities of numbers of LAB such as Lactobacillus brevis isolated from Turkish dairy products [6] and Lactobacillus acidophilus isolated from Iranian yoghurt against Staphylococcus aureus [7]. According to reports, it appears that Middle East is the origin of fermented dairy products mainly yoghurt [8]. In Iran, a number of traditional dairy products are consumed of which yoghurt, well known, as Mast is one of the most popular fermented milk products. While, traditionally made sour buttermilk especially made from ewe milk is more common in rural areas of the country [9]. It is a well-established fact that the composition of LAB in these traditional dairy products is varied and inconstant. In Iran, there are different kinds of traditional dairy products which are produced from cow, camel, sheep and goat milk such as drinking yoghurt, yoghurt, kashk, gharaghooroot, cheese, etc. In comparison with the commercial species, composition of lactic acid bacteria is more varied and inconstant in these products. The main objective of present study was to investigate LAB of traditional dough, which might provide important information regarding its probiotic potential and its utilization in the future. Material and methods Dough samples During the spring of 2014, a total of 15 dough (13 traditional and 2 industrial dough 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 [10] 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 [11]. 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 Correspondence to: Zeinab Rashti, Department of Food Science and Technology, Islamic Azad University, Azadshahr Branch, Iran, E-mail: rashti.zeinab@gmail.com


Introduction
Interest in microorganisms occurring in foods is primarily due to the biotechnological potential of new bacterial species and strains [1]. Lactic acid bacteria (LAB) are widely distributed in nature and occur naturally as indigenous microflora in raw milk, drinking yoghurt, etc. Lactic acid bacteria are a group of gram-positive bacteria including the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus and play an important role in many food fermentation processes. The lactic acid fermentation has long been known and applied by humans for making different food stuffs. In addition, they strongly determine the flavour, texture and frequently, the nutritional value of food and feed products [2][3][4]. In fermented foods, LAB display numerous antimicrobial activities. This is mainly due to the production of organic acids and other compounds, such as bacteriocins and antifungal peptides [5]. Several studies have shown the inhibitory activities of numbers of LAB such as Lactobacillus brevis isolated from Turkish dairy products [6] and Lactobacillus acidophilus isolated from Iranian yoghurt against Staphylococcus aureus [7].
According to reports, it appears that Middle East is the origin of fermented dairy products mainly yoghurt [8]. In Iran, a number of traditional dairy products are consumed of which yoghurt, well known, as Mast is one of the most popular fermented milk products. While, traditionally made sour buttermilk especially made from ewe milk is more common in rural areas of the country [9].
It is a well-established fact that the composition of LAB in these traditional dairy products is varied and inconstant. In Iran, there are different kinds of traditional dairy products which are produced from cow, camel, sheep and goat milk such as drinking yoghurt, yoghurt, kashk, gharaghooroot, cheese, etc. In comparison with the commercial species, composition of lactic acid bacteria is more varied and inconstant in these products. The main objective of present study was to investigate LAB of traditional dough, which might provide important information regarding its probiotic potential and its utilization in the future.

Dough samples
During the spring of 2014, a total of 15 dough (13 traditional and 2 industrial dough 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 [10] 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 [11]. 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.
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 dough samples 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 2h at 4ºC prior to incubation at 37ºC for 24h. 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 15 dough samples (13 traditional and 2 industrial dough samples) were collected from Gorgan city (Table 1).
Also average number of microbial count in MRS Agar is the maximum amount in water buffalo samples (6/7 × 10 8 CFU/ml) and minimum amount in cow samples (1/8 × 10 8 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 [12]. 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 [13].

Isolation and identification of lactic acid bacteria
35 species of lactic acid bacteria were isolated and identified from 15 samples of dough that the types and numbers of these bacteria shown in Tables 4 and 5, according to type of dough.
The highest frequency of lactic acid bacteria isolated from the dough samples, is belongs to the species include Lactobacillus casei, Lactobacillus acidophilus and Lactobacillus Delbrueckii. It is shown the number and percentage of different species of lactic acid bacteria isolated from dough samples, in Figure 1.
According to Table 5, the maximum number of lactic acid bacteria is in sheep and the minimum number is in camel dough. Any kind of lactic acid bacteria have been isolated from camels dough due to high yeast.

Antagonistic effect of LAB
Lactic acid bacteria isolated from dough samples showed antagonistic activity against pathogenic bacteria with the production of the antimicrobial compound. The results are shown in Table  6. Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus rhamnosus, isolated from sheep dough, inhibit the growth of all pathogenic bacteria. Lactobacillus casei has the highest antibacterial activity against pathogenic bacteria.
Lactobacillus acidophilus isolated from sheep and goat dough, have no antimicrobial effect on pathogenic bacteria. So in total, the lactic acid bacteria isolated from sheep dough, produce greater antimicrobial compounds against pathogenic bacteria.

NO.
Type Number of samples   Table 3. Average counts of lactic acid bacteria in dough samples in MRS Agar in Gorgan city. Kazemi et al, 2010, in the same study found the opposite result and Lactobacillus acidophilus showed the highest inhibitory effect with an inhibition zone diameter of 14 mm [14].

Conclusion
The fermented dairy products can be possibly a good source of potential probiotic organisms. In Iran, a number of researchers have reported the isolation of LAB from dairy products like butter, kashk and cheese [15]. However, there are some fermented dairy products in Iran, which have yet not been evaluated for their health benefit, mainly their probiotic properties. Lactic acid bacteria display numerous antimicrobial activities in fermented foods. This is mainly due to the production of organic acids, but also of other compounds, such as ethanol, H 2 O 2 , diacetyl, reuterin and bacteriocins. Several bacteriocins with industrial potential have been purified and characterized. Application of bacteriocin-producing starter cultures in fermented