Original Article APPLIED FOOD BIOTECHNOLOGY, 2016, 3 (1): 43-51 pISSN: 2345-5357 Journal homepage: www.journals.sbmu.ac.ir/afb eISSN: 2423-4214 In Vitro characterization of Lactococcus lactis strains Isolated from Iranian Traditional Dairy Products as a Potential Probiotic Fatemeh Nejati 1, Tobias A. Oelschlaeger 2 1. Department of Food Science, Agriculture Faculty, Shahrekord Branch, Islamic Azad University, P.O. Box166, Shahrekord, Iran 2. Institut für Molekulare Infektionsbiologie, University of Wurzburg, Germany Abstract Article Information Few studies have been reported regarding probiotic properties of Lactococcus Article history: lactis strains although they are extensively used as starter cultures in the Received 24 Oct 2015 production of dairy products. In this study 8 wild isolates of Lactococcus lactis Revised 3 Nov 2015 were evaluated in vitro with regard to resistance to simulated gastric and Accepted 2 Dec 2015 intestinal juices, adherence ability to Caco-2 cells and HT29-MTX-E12 cell Keywords: lines, anti-microbial activity, hydrophobicity and antibiotic susceptibility. The Cell lines adherence, results revealed that all isolates had better survival after exposure to simulated Gastrointestinal tract stresses, gastrointestinal tract stresses in comparison to control probiotic Lactobacillus Lactococcus lactis, rhamnosus GG. Regarding adherence efficiency, almost all isolates exhibited Probiotic similar adherence with control. Three isolates showed antibacterial activity against Gram-positive pathogens (Staphylococcus aureus and Listeria Correspondence to: monocytogenes) through spot-agar method. Almost all isolates (seven out of Fatemeh Nejati eight) showed similar hydrophobicity to control probiotic. Regarding to Department of Food Science, Agriculture Faculty, Shahrekord antibiotic resistance, all isolates were susceptible to gentamicin, ampicillin, Branch, Islamic Azad University, ciprofloxacin, erythromycin, tetracycline, penicillin, kanamycin and P.O. Box:166. Shahrekord, Iran nitrofurantoin. Although, further investigations are necessary, it was concluded Tel: +98 -38-33361093 that strains derived from raw milk and home-made dairy products could be a Fax: +98-38-33361093 remarkable reservoir for identification of new potential probiotic strains. E-mail: [email protected] 1. Introduction Functional foods containing safe microorganisms ococci and lactococci [4,5], and non-LAB, such as convey beneficial effects, named probiotics, have Escherichia coli [6]. According to this, new bacterial attracted much attention in the last decade. Probiotics isolates originating from food environment with superior are defined as live microorganisms which after cons- probiotic characteristics have been reported frequently umption in adequate numbers can confer health in recent years. benefits to the host [1]. Lactococcus lactis is usually used as starter in Several well-known probiotic strains are of manufacturing different dairy products. However, in commercial interest and currently used in the recent years, several interesting evidences show L. production of different functional foods and in dietary lactis strains, alone or in combination with other supplements in the form of capsules and tablets. probiotics, have the potential to be beneficial to human Lactobacilli and Bifidobacteria as autochthonous health and considered as probiotic [7-9]. inhabitants of human’s gastrointestinal tract have been There are many different characteristics expected extensively subjected in probiotic characterization ass- from a candidate probiotic strain, for example safety, ays. However, probiotic characteristics have been rep- presenting antimicrobial activity, surviving in harsh orted for other food-derived lactobacilli [2,3], other conditions of upper part of human GIT, e.g. low pH and members of lactic acid bacteria (LAB), such as enter- the presence of bile salts, and the ability to adhere to the 43 Evaluation of Lactococcus lactis as probiotic intestinal cells and mucus and interact correctly with incubated overnight at 32°C. After that, cells were gastrointestinal epithelia cells in order to confer any harvested, washed twice with 0.85 %w v-1 NaCl and re- health effect [10-12]. So, in order to present a strain as suspended in 500 µL of the same solution. 100 µL of probiotic, extensively deep analyses are usually nec- bacterial suspensions were added to 900 µL of simulated essary to investigate its viability and interaction with the gastric or intestinal juice, so that initial populations human host. ranged from 8.2 to 9.0 log CFU ml-1 and incubated at The objective of this study was to test a number of 37°C. Samples were withdrawn after 90 and 180 min LAB isolates that had been identified as L. lactis for from simulated gastric and intestinal juice, respectively. probiotic characteristics via in vitro experiments. These In order to determine viable cells, plate counts with M17 strains have been isolated from raw milk and home- agar were done at time 0 and after incubation [10]. made dairy products (cheese and butter) [13]. The parameters examined to determine likely probiotic 2.3. Adhesion to epithelial intestinal cell lines features of the isolates included the viability after 2.3.1 Cell cultures exposing the bacteria in vitro to GIT conditions, test for Caco-2 and HT29-MTX-E12 (E12) cells were grown adhesion to Caco-2 and HT29-MTX-E12 cells, antimic- in Dulbecco’s modified Eagle Medium (DMEM) (56°C robial activity, cell surface hydrophobicity and antibiotic for 30 min) containing 10% v/v inactivated fetal bovine susceptibility. serum (PAA, Colbe, Germany) and without antibiotics. Both cell lines were cultured at 37°C in an atmosphere 2. Materials and Methods of 5 % v/v CO2 in a CO2 incubator. The experiments 2.1 Bacterial strains and growth conditions were performed in 24-well tissue culture plates. Caco-2 The 8 wild type bacterial strains were isolated from cells were seeded at a concentration of 4 × 105 cells/well milk and traditional cheese and butter in Iran (the data and used after 24 h incubation when confluent growth have been described in our previous study) [13]. Strains was achieved (6.2 × 105 cells/well). HT29-MTX-E12 used in the current study were identified as L. lactis, (E12) cells were seeded at a concentration of 6 × 104 including AS1, SPT2, GC10, JP51, FK23, JP32, AS2 cells/well and were used after 7 days when they had and DC103 isolates by 16s rRNA sequencing. M17 produced mucus. The culture medium (1 mL cell-1) was medium (Oxoid, Frankfurt, Germany) supplemented replaced by fresh medium at days 3, 5 and 6 after with 5% lactose, was used for cultivation of all isolates. seeding. For both cell lines, 1 h before adding the Overnight incubation at 32°C was used for culturing of bacterial suspension DMEM was replaced by fresh isolates. The commercial probiotic strains Lactobacillus medium. Mucus production after 7 days was confirmed rhamnosus GG (ATCC 53103) was used as a positive by microscopic evaluation of stained cells. For this control (Ardeypharm GmbH, Herdecke, Germany(. purpose E12 were seeded and cultivated on Nunc Lab- Pathogenic indicator strains were Entrotoxigenic Tek Chamber Slides (Thermo Scientific) and stained by Escherichia coli (ETEC) strain H10407 (O78:H11), Periodic acid-Schiff [14]. Escherichia coli (UPEC) strain 536 (O6:K15:H31), Salmonella enterica serovar typhimuirum SL1344, 2.3.2 Adhesion assay Shigella flexneri M90T, Listeria monocytogenes EGD Bacterial overnight cultures (18 h) incubated under and Staphylococcus aureus Cowan 1 (ATCC 12598). S. appropriate conditions were used in this assay. Two aureus and L. monocytogenes were grown in tryptic soy hundred microliters of each bacterial overnight culture broth and brain heart infusion, respectively. Other were used to inoculate 5 ml DMEM containing 10% strains were cultured in Luria Bertani broth. All strains fetal bovine serum and incubated 1 h at 37°C. After that, ° were grown overnight at 37 C. 25 µL of each culture were added to wells of a 24 well plate containing the confluent monolayer of Caco-2 or 2.2. Resistance to simulated gastric and small E12 cells and 1 mL DMEM (to reach the final concen- intestine juices tration of 1-2 × 106 CFU of bacteria per well). After Simulated gastric juice was prepared by dissolving 3 incubation for 90 min at 37 C and 5 %v/v CO2, non-adh- g l-1 pepsin (P7000 Sigma-Aldrich, Germany) in sterile erent bacteria were removed by washing the cells two salt solution (125 mM NaCl, 7 mM KCl, 45 mM times with 1 mL of phosphate buffer solution. Then, the NaHCO3, pH= 3.0), and pH adjusted to 2.5 with 0.1 N cell monolayer was lysed by addition of 1 ml pre- HCl using gentle mixing. Great attention needed to be warmed filter-sterilized 0.1% Triton X-100 and shacked paid in order to prevent denaturation of pepsin. for 15 min at room temperature. The number of colony Simulated small intestinal juice was prepared by forming units was determined after dilution and plating dissolving 3 g l-1 ox-gall (Fluca 70168, Germany) and 1 on M17-agar. Assays were carried out triplicate indep- g l-1 pancreatin (P3292Sigma-Aldrich, Germany) in endently and in duplicate each time for each isolate. sterile salt solution (45 mM NaCl), and pH adjusted to 8.0 by 0.1 M NaOH. Both solutions were prepared 2.4. Hydrophobicity of bacterial strains freshly and filter-sterilized through a 0.22 µm mem- Bacterial hydrophobicity was determined by the n- brane. Survival under simulated gastric and small hexadecane test according to Lukic et al. [15] with slight intestinal conditions was determined as previously desc- modification. Briefly, 500 µL of 18 h cultures were ribed [10]. Five milliliters of each bacterial culture were washed once with 0.85 %w v-1 NaCl and re-suspended 44 Appl Food Biotechnol, Vol. 3, No. 1 (2016) Nejati and Oelschlaeger in the same buffer to achieve OD600= 0.4-0.5. In the All isolates were included in antibiotic susceptibility following, 500 µL of n-hexadecane (Merck, Germany) tests against a selection of nine antibiotics including was added to 2.5 mL of bacterial suspension.