Antimicrobial activity of some lactic acid bacteria isolated from local environment in Egypt

1 Department of Food Microbiology, National Research Center, Dokki, Cairo, Egypt. 2 Agricultural Microbiology Department, Faculty of Agriculture, Cairo University, Giza, Egypt. 3 Unidad Mixta de Investigación en Endocrinología, Nutrición y Dietética Clínica, Universitat de ValènciaInstituto de Investigación Sanitaria La Fe, Valencia, Spain. 4 Gastrolab, Grupo de Ciencias de la Alimentación Basada en la Evidencia y Experimentación (CiAlBEx), Instituto de Ciencias de los Materiales, Parque Científico, Universitat de València. Paterna, Spain.


INTRODUCTION
Antimicrobial agents are of enormous value for combating infectious diseases.Their efficacy has been threatened by possible spread of the phenomena of the "microbial resistance" since many microorganisms start to gain resistance to the antibiotics (Teuber et al., 1999).Searching for natural alternatives to the antibiotics is now a goal of many researchers.They have been tried not to use in treating gastrointestinal disease only, but also to use in food industry as natural antimicrobial substances that many have a bacteriostatic or bacteriocidal effect against many food borne pathogens (Soomro et al., 2002;Oyetayo et al., 2003;Savadogo et al., 2004).*Corresponding author.E-mail: jose.soriano@uv.esAuthor(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Immune modulation by probiotics was recognized as an important aspect of host gut health (Papadimitriou et al., 2015).It has been suggested that the health benefits of some probiotics used in functional foods and pharmaceutical preparations are due to the capacity of these microorganisms to stimulate the host immune system (Perdigon et al., 2002).
Lactic acid bacteria (LAB), which is natural occurred in our environment, may act as source for such nature antimicrobial compounds.This antimicrobial effect of LAB has been appreciated by man for more than 1000 years ago and has enabled him to extend the shelf life of many foods through fermentation processes since it can inhibit the growth of such microorganisms including spoilage and pathogenic bacteria (Okereke et al., 2012;Aslim et al., 2005).This effect may have resulted from the action of organic acids, hydrogen peroxide, diacetyle and/or a compound(s) called bacteriocin(s) (Gonzalez et al., 2010;Oyetayo et al., 2003;Savadogo et al., 2004).Bacteriocins are low molecular-mass peptides or protein with a bacteriostatic or bacteriocidal mode of action in particular against closely related species (Deraz et al, 2005).These bacteriocins may contribute to the competitiveness of the producing strain in the environmental product ecosystem (Caplice and Fitzgerald, 1999).An arid variety of LAB, that produce antimicrobial substance such as hydrogen peroxide, CO 2 , diacetyl, acetaldehyde, D-isomers of amino acids, reuterin and bacteriocins have already been isolated from many fermented products.This includes naturally fermented sausages (Aslim et al., 2005), fermented milk and dairy products (Morea et al., 1999) and silage which is stored fermented vegetable product (Ennahar et al., 1996).
The aims of this study were to screen the antimicrobial activities of what so-called wild strains of LAB isolated from different sources of our environment including raw and fermented milk, cheeses, other dairy products, sausage and silage.Thus, the potential of these isolates by new antagonistic metabolites, other than acids and hydrogen peroxide, to inhibit food borne pathogens as well as food spoilage bacteria was evaluated.

Samples
In this study, fifty-two samples were collected from different locations in Cairo and Giza governorates.The collected samples included four samples each of raw sheep, goat and buffalo's milk, five samples of yoghurt (zabady), four samples of naturally acidified milk (Rayeb), five samples each of Kareish and Domiati cheese (soft type), seven samples of Ras cheese (hard type), five samples of ice cream, four samples of naturally fermented sausage, two samples of naturally fermented silage as well as three samples of fresh plants.Samples were immediate placed in ice box and transferred to the Laboratory where they were subjected to the microbiological analysis in the day of purchasing.In exception instances, the samples were stored at refrigerator temperature for up to a maximum of 24 h before analysis.

Cultures and media
The indicator organism strains were obtained from the stock cultures of the Agricultural Research Centre in Giza including Escherichia coli 0157: H7 ATCC 6933, Bacillus cereus ATCC 33018, Staphylococcus aureus ATCC 20231, Salmonella typhimurium ATCC 14028, and Pseudomonas aeruginosa ATCC 9027.Listeria monocytogenes V7 strain serotype 1 (milk isolate), was obtained from the Department of Food Science from University of Wisconsin-Madison (USA).All strains were routinely maintained by sub culturing once a week in tryptone soya broth/agar and stored at 4°C until use.The purity of all strains was checked before each use.All medium were purchased to Oxoid (Madrid, Spain).MRS agar/broth was used for isolation/cultivation of lactobacilli from the examined samples; however M17 agar/broth were used for isolation/cultivation of lactococci.Tryptone soya broth was used for cultivation and propagation of all the tested bacterial strains.Preservation and propagation of all species of LAB was done using litmus milk.

Isolation
Twenty-five grams of each sample was aseptically transferred to plastic bag and homogenized in 225 ml of sterile buffered peptone water (BPW).Ten-fold dilutions of homogenates were prepared and inoculated onto plates of MRS and M17 agar for isolation of lactobacilli and lactococci, respectively.MRS plates were incubated in anaerobic condition using CO2 generated kits (Anaerobe-Gen, Oxoid) and incubated at 37°C, while M17 plates were incubated in normal aerobic conditions at 32°C.All plates were incubated for period of three to five days.Characteristic colonies of LAB were counted and expressed as colony forming units (cfu/g or ml).Three to five of typical isolates from each sample were picked up, grown, purified and maintained at MRS slants for future grouping.

Grouping
The isolates of LAB were propagated twice in MRS broth at 37°C.Then inoculums of 16 hours old grown onto MRS agar were mainly identified, according to Bergey's Manual (2009), for their morphological characteristics, including the cell form and their cellular arrangement, Gram-stain reaction (Brock et al., 1994), catalase production (Herrero et al., 1996) as well as their ability to produce gas from glucose (Kandler and Weiss, 1986).The grouped isolates were then kept in litmus milk, supplemented with 1% glycerol, for future identification.

Culture supernatant
The isolates of LAB were propagated overnight (16 h) in 100 ml MRS broth (pH 6.8).A cell free solution was obtained by centrifugation of culture at 5000 rpm at 4°C for 20 min, followed by filtration of supernatant through sterilized filter (0.2 µm, Millipore).To avoid the effect of the produced organic acids, the obtained cellfree supernatant was neutralized to 6.5-7.0 using 1 N sodium hydroxide.Also, the effect of hydrogen peroxide was eliminated by the addition of 5 mg.Catalase per each one ml of the supernatant and left at 25°C for 30 min.The obtained treated supernatants were tested for their antimicrobial activities against the indicators bacteria.

Proteinaceous nature treatments
The proteinaceous nature of each cell-free supernatant fluid was evaluated by heat sensitivity test as well as treating with some proteolytic enzymes.Supernatants were heated for 10 min at 85°C and the loose in their activities was evaluated by the agar diffusion assay method.The same was true after treating the supernatant with various proteolytic enzymes including trypsin and αchymotrypsin.In this case each enzyme was dissolved in sterile 4 ml phosphate buffer pH 7 and added to the cell free culture supernatants fluid to obtain a final concentration of 1 mg/ml (Bonade et al., 2001).Control samples contained only buffer solution were done.The samples with and without enzymes were incubated for one hour at 37°C.

Detection of the antimicrobial activity
The antimicrobial activity, of seventy-eight isolates of LAB against the reference strains, was determined by the agar diffusion method (Settanni and Corsetti, 2008).One ml culture of the activated indicator strain(s) (10 4 to 10 5 cell/ml) was inoculated into 20 ml of Mueller-Hinton agar (Becton Dickinson, USA) and poured in Petri dishes.After solidification of the agar, wells of 5 mm diameter were cut in, and 50 µm of the treated supernatant fluid, from a culture of strain under test, was added to each well.Dishes were stored for 2 h in refrigerator followed by incubation for 24 h at 37°C.The diameters of the inhibition zones were measured (Rammelsberg and Radler, 1990).

RESULTS
A total of fifty-two samples of food or feed products were investigated for their load of LAB.Maximum and minimum counts obtained on media of MRS and M17 agar plates are given in Table 1 which demonstrated that high LAB population ranged from 10 9 to 10 7 densities were detected in fermented milk products including Rayeb and yoghurt milk followed by raw goat's milk and cheeses (Ras, Domiati and Karish) and ended by buffalo and sheep raw milk.Samples of feed products reported populations ranged from 10 6 to 10 3 at surface of greenplants and silage however the lowest counts of LAB ranged from 10 5 to 10 3 were detected in sausage and icecream.The bacteriostatic or bacteriocidal effect of cellfree extracts of LAB that either picked up from MRS or M17 plates, against the tested pathogenic microorganisms expressed by the detected clear zones of inhibition, are listed in Table 1.In our study, 347 LAB were isolated from food and feed products and picked from both the used types of media, were found to have this action of inhibition.In this case, their effect was based on the action of metabolites of LAB (bacteriocins proteinaceous synthesizing substances produced by such strain, as well as a combination of antimicrobial substances such as hydrogen peroxide and organic acids).After elimination of the effect of acidity and hydrogen peroxide of these extracts, the number of the effective extracts / isolates was reduced to 78.The source from which these isolates have been isolated is shown in Table 1.Of these isolates, 19 strains were spherical however 59 were rods/coco bacilli in shape or in their cell form.The lowest counts of LAB detected in ice-cream samples could be explained by the fact that the ice-cream mixtures have been previously exposed to high temperatures, during the pasteurization process of the product.Results in our study indicate that, all the selected 347 isolates of LAB were Gram-positive and catalase-negative, 127 isolates were spherical however, and the other 220 isolates were rods/coco bacilli in shape.All the isolates were not able to produce gas from glucose.The only exception was two strains, rods, gave non-significant amount of gas.
The source of these isolates as well as their efficacy and spectrum of action based on the action of bacteriocins like substances, expressed as diameters of inhibition against the indicator microorganisms are shown in Table 2.A wide range of the reported inhibition zones ranged from 6 to 12 mm, for all the tested 78 LAB isolates, against the various tested indicators was obtained.The biggest diameter (12 mm) was obtained using the extract of isolates S33 which have been isolated from Domiati cheese, against the indicator P. aeruginosa however, the smallest diameter (6 mm) was obtained with the extracts of many isolates originated from all the investigated products against all the different tested pathogenic indicators.According to the Table 2, bacteriocins produced by LAB isolates were effective against Gram-negative indicators than Gram-positive.Where, out of the 78 bacteriocin cell-free extracts, S 52, S 33 and S 13 extracts were effective against Gramnegative indicators including E. coli 0157:H7, S. typhimurium and P. aeruginosa respectively, while only S 32, S 75 and S 7 extracts were effective against S. aureus, L. monocytogenes V7 and B. cereus, respectively.This phenomena may depend on the species of the bacterial indicators used in this work and also may provide insight into other mechanisms that may happen in the bacterial cell wall and contribute to these antimicrobial effects.Other explanation could be the functional capacities of the bacteriocin itself which could be explained by specific-specific mechanisms of action.Although the numbers of bacteriocins, produced by LAB isolates, effective against Gram-negative indicators were more than that those effective against Gram-positive, but it is found that when the individual extract was effective in the same time against both Gram-positive and Gramnegative, the Gram-positive bacteria were more susceptible to the bacteriocin than did Gram-negative.For example as is shown in Table 2, the extract of strain S3, isolated from sausage, gave inhibition zone of 7 mm against S. typhimurium, however it gave 10, 9 and 8 mm inhibition zone diameter against B. cereus, S. aureus and L. monocytogenes, respectively.
The bacteriocin-producing (bacteriocin-like substances) strains (78 isolates) were subjected to identification according to their morphological, cultural, physiological and biochemical characteristics as described in Bergey's Manual of Systematic Bacteriology, 2009.Of the identified 78 LAB isolates, 19 isolates were found to belong to genus Lactococcus however the other 59 isolates were referred to genus Lactobacillus.The representative species of the total cocci were identified as 3 strains Lactococcus lactis sub sp.Lactis and 16 strains Streptococcus thermophilus.On the other hand, the identified strains belonging to genus Lactobacillus were 13 strains of Lactobacillus jensenii, 12 strains Lactobacillus acidophilus, 7 strains Lactobacillus casei sub sp.rhamnosus, 6 strains Lactobacillus plantarum, 6 strains Lactobacillus helveticus, 6 strains Lactobacillus gasseri, 5 strains Lactobacillus reuteri, 3 strains Lactobacillus delbrueckii sub sp.bulgaricus and one strain of L. casei sub sp.casei (Table 3).

DISCUSSION
The presence of high counts of LAB detected in cheese and fermented milk possibly due to the natural fermentation processes and/or the addition of lactic starters during the processing of such products (Ayad et al., 2004;Tamine and Robinson, 2007).Fluctuated numbers of LAB detected in different types of the investigated raw milk may be depended on the methods used in handling and storage of this product as well as the hygienic status of the animal that produced such milk (Ayad et al., 2004).The environmental stress may explain the moderate counts of LAB, detected in the feed samples including surface of green plants (Ranong, 1999).These low LAB counts found in sausage samples may be due to the use of sausage spices and preservatives that might be added during processing (Giuseppe et al., 2005).The antimicrobial effect of these 78 isolates has not been changed after heating at 85°C for 10 min; however this effect completely inactivated after treatment with the proteolytic enzymes indicating that these inhibitory compounds, produced by such isolates, are proteinaceous nature which consider the main characteristic of any bacteriocin (Herreros et al., 2005).
According to our Table 2, similar results have been reported by many investigators in different countries (Savadogo et al., 2004).The extent by which Grampositive and Gram-negative indicator organisms were susceptible to the bacteriocin-like substances is presented in Table 2. Savadogo et al. (2004) reported that action of the bacteriocins may explain by the interaction with lipoteichoic acids, which are absent in Gram-negative bacteria, and these molecules would play the role of sites needed to produce the bacteriocinal effect.This last observation is in accordance of other reports and studies which stated that some bacteriocins produced by Gram-positive bacteria, regardless of its source, have a broad spectrum of activity against Grampositive microorganisms than did with Gram-negative (Savadogo et al., 2004).Others reported that bacteriocins from normal inhabitant of gastrointestinal tract of humans and animals have antagonistic actions against wide range of Gram-positive and in some instances for also Gram-negative bacteria.Lima and Filho (2005) found that 265 out of 474 LAB produced bacteriocins which demonstrated activity against many Gram-positive organisms however the only Gram-negative susceptible organism was Salmonella spp.We believe that further understanding of the molecular foundation of the relationship between microbes (bacteriocin-producer)microbes (inhibited-organism) should/may explain or at least clarify these findings.
Isolation of such LAB species from foods was reported by many investigators in different countries.In Morocco Benkerroum et al. (2000) isolated a bacteriocin-producing L. lactis sub sp.Lactis strain from a soft white cheese effective against L. monocytogenes.In Senegal, Diop et al. (2007) isolated 220 LAB strains from traditional fermented foods.Twelve of them were bacteriocinproducing isolates of which two isolates were identified as L. lactis subsp lactis and Enterococcus faecium.In Nigeria, Adetunji and Adegoke (2007) identified L. plantarum and Lactobacillus brevis as the most dominant bacteriocin-producing LAB strains isolated from West African soft cheese however Lactococcus lactis, streptococcus lactis and Lactobacillus fermentum were less dominant.In France, Ennahar et al. (1996) identified six bacteriocin-producing LAB isolates from cheese including three Lactococcus strains, two Enterococcus strains and one Lactobacillus strain which was active against Listeria organism.In Belgium, Enan et al. (1996) isolated one strain of L. plantarum, from dry sausage, exhibiting an inhibitory activity against L. monocytogenes, B. cereus, Clostridium perfringens and Clostridium sporogenes.In Brazil, Moreno et al. (2000) isolated 15 strains of L. lactis (14 Lactococcus Lactis subsp.lactis and one L. lactis subsp cremoris) from local dairy products that were able to produce bacteriocins inhibited Listeria monocytogenes and S. aureus. in Turkey, Erdogrul and Erbilir (2006) isolated two strains from foods

Table 1 .
Maximum and minimum counts of LAB/g using MRS and M17 agar plates.
Number between brackets is the numbers of the analyzed samples.a No. of isolates that produce proteinaceous inhibitive substance against one or more of the tested indicator organisms.

Table 2 .
Inhibition of various indicator organisms by bacteriocin-like substances produced by isolated LAB.