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Isolation of Pediococcus Acidilactici Kp10 with Ability to Secrete

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Isolation of Pediococcus Acidilactici Kp10 with Ability to Secrete Abbasiliasi et al. BMC Microbiology 2012, 12:260 http://www.biomedcentral.com/1471-2180/12/260 RESEARCH ARTICLE Open Access Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry Sahar Abbasiliasi1, Joo Shun Tan4, Tengku Azmi Tengku Ibrahim2, Ramakrishnan Nagasundara Ramanan3, Faezeh Vakhshiteh4, Shuhaimi Mustafa5, Tau Chuan Ling6, Raha Abdul Rahim7 and Arbakariya B Ariff1* Abstract Background: Lactic acid bacteria (LAB) can be isolated from traditional milk products. LAB that secrete substances that inhibit pathogenic bacteria and are resistant to acid, bile, and pepsin but not vancomycin may have potential in food applications. Results: LAB isolated from a range of traditional fermented products were screened for the production of bacteriocin-like inhibitory substances. A total of 222 LAB strains were isolated from fermented milk products in the form of fresh curds, dried curds, and ghara (a traditional flavor enhancer prepared from whey), and fermented cocoa bean. Eleven LAB isolates that produced antimicrobial substances were identified as Lactococcus lactis, Lactobacillus plantarum,andPediococcus acidilactici strains by biochemical methods and 16S rDNA gene sequencing. Of these, the cell-free supernatant of Kp10 (P. acidilactici) most strongly inhibited Listeria monocytogenes. Further analysis identified the antimicrobial substance produced by Kp10 as proteinaceous in nature and active over a wide pH range. Kp10 (P. acidilactici) was found to be catalase- negative, able to produce β-galactosidase, resistant to bile salts (0.3%) and acidic conditions (pH 3), and susceptible to most antibiotics. Conclusion: Traditionally prepared fermented milk products are good sources of LAB with characteristics suitable for industrial applications. The isolate Kp10 (P. acidilactici) shows potential for the production of probiotic and functional foods. Keywords: Lactic acid bacteria, Pediococcus acidilactici, Bacteriocin-like inhibitory substance, Listeria monocytogenes, Fermentation, Identification Background designated as generally regarded as safe (GRAS) micro- The screening of microorganisms isolated from naturally organisms [2]. occurring processes is the most common method of LAB are normally found in nutrient-rich environments obtaining strains useful for industrial applications. This and are able to grow in most raw foods. These bacteria holds true for lactic acid bacteria (LAB), which are used are fastidious and require fermentable carbohydrates, worldwide to produce a variety of fermented foods [1]. amino acids, fatty acids, salts, and vitamins for growth Because LAB have been used in food production for [3]. Because of their metabolic properties, LAB play an centuries without posing any health risks, they are important role in the food industry, contributing signifi- cantly to flavor, texture, and frequently the nutritional * Correspondence: [email protected] value of foods [4]. 1Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor 43400 Because of the rapid rise and spread of multi-resistant UPM, Malaysia bacterial pathogens, new methods are needed to combat Full list of author information is available at the end of the article © 2012 Abbasiliasi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbasiliasi et al. BMC Microbiology 2012, 12:260 Page 2 of 12 http://www.biomedcentral.com/1471-2180/12/260 infection. Antibiotics are widely used to prevent the Results spread of pathogenic bacteria; however, many antibiotics Isolation of BLIS-producing strains are broad-spectrum drugs that kill bacterial species in- A total of 222 LAB strains were isolated from nine test discriminately [5]. Bacteriocins have a relatively narrow samples (Table 1). After preliminary identification, 11 of spectrum of killing activity, and some can be consid- these strains were found to produce antimicrobial sub- ered pathogen-specific designer drugs. Given the di- stances. All 11 isolates were gram-positive, catalase- versity of bacteriocins produced in nature, it may be negative, and able to ferment glucose and produce acid. arelativelysimpletasktoidentifybacteriocinseffect- Both cocci and bacilli were identified. The isolates Kp8 ive against specific human pathogens [5]. In addition, and Kp10 showed the highest antimicrobial activity bacteriocin use may reduce the need for chemical (888.56 AU/mL). additives in food and minimize the intensity of food pro- cessing techniques, contributing to the production of Characterization of isolates with API 50 CHL more healthful foods [6]. The carbohydrate fermentation patterns of the 11 iso- In recent years, attention has been focused on LAB lates were determined by using the API 50 CHL micro- from different sources that produce bacteriocins that identification system (Table 2). The isolates Gh1, C22, are considered safe as food biopreservatives and can and C13 were able to hydrolyze ribose, d-xylose, galact- be degraded by gastrointestinal proteases [7]. These ose, glucose, fructose, mannose, n-acetyl-glucosamine, probiotic compounds have been used in a variety of amygdalin, esculin, arbutin, salicin, cellobiose, maltose, industrial applications relevant to both human and lactose, trehalose, starch, gentiobiose, and gluconate. animal health without producing side effects. There is However, mannitol and sucrose were hydrolyzed by Gh1 an ongoing need to identify new strains with useful but not by C22 or C13. The isolates Kp8 and Kp10 were characteristics. Therefore, the main objective of this able to hydrolyze glycerol, l-arabinose, ribose, d-xylose, study was to isolate and characterize LAB that pro- galactose, glucose, fructose, mannose, mannitol, n- duce bacteriocin-like inhibitory substances (BLIS) acetyl-glucosamine, esculin, salicin, cellobiose, gentio- from traditionally prepared milk products (e.g., fresh biose, and d-tagatose. The isolates Com4, Pak1, Com5, curds, dried curds, and ghara) and locally fermented C6, C7, and Pak7 were able to hydrolyze, ribose, galact- cocoa beans. These fermented products do not use ose, glucose, fructose, mannose, mannitol, n-acetyl-glu- starter cultures; fermentation is the result of wild cosamine, amygdalin, arbutin, esculin, salicin, cellobiose, flora present in the surrounding environment. Wild maltose, lactose, melibiose, sucrose, trehalose, melezi- LAB strains represent a natural reservoir of strains tose, and gentiobiose but differed in their ability to not exposed to any industrial selection and are poten- metabolize glycerol, sorbose, rhamnose, sorbitol, α-me- tial probiotics and bacteriocin producers [8]. In this thyl-d-mannoside, α-methyl-d-glucoside, raffinose, tura- study we identified and characterized LAB strains that nose, d-tagatose, l-fucose, d-arabitol, and gluconate. To produce high BLIS levels for possible applications in identify the isolates, their carbohydrate metabolism pat- the food industry. terns were analyzed using the API database (Table 3). Table 1 Morphological, biochemical characteristics and antimicrobial activity of LAB isolates Fresh curds Dried curds Ghara Fermented cocoa beans Pg Cam Pak Ky Kp Sat Kbo Gh1 C Cam4 Cam5 Pak1 Pak7 Kp8 Kp10 C6 C7 C13 C22 No. of LAB isolates 10 8 26 20 20 40 40 10 48 (culturedinMRSandM17) No. of isolates showing 02 202 001 4 antimicrobial activity Cell morphology ND Bacilli Bacilli ND Cocci ND ND Cocci Bacilli Bacilli Cocci Cocci Gram stain reaction ND + + ND + ND ND + + Catalase activity ND - - ND - ND ND - - Glucose fermentation ND + + ND + ND ND + + Activity (AU/mL) against ND 276.51c 276.51c 26.78 a 26.78a ND 888.56 d 888.56d ND ND 115.21b 26.78a 26.78a 26.78a 26.78a L. monocytogenes ATCC15313 Positive reaction (+), negative reaction (−), not detected (ND). Values with different superscript letters (a, b, c, d) are significantly different. Abbasiliasi http://www.biomedcentral.com/1471-2180/12/260 Table 2 Biochemical profiles of LAB isolates et al. BMC Microbiology LAB isolates Gh1 Com4 Pak1 Kp8 Com5 Kp10 C22 C6 C7 C13 Pak7 Gh1 Com4 Pak1 Kp8 Com5 Kp10 C22 C6 C7 C13 Pak7 0 Control - - - - - - - - - - - 1 Glycerol - + + + + + - - + - + 26 Salicin + + + + + + + + + + + 2 Erythritol - - - - - - - - - - - 27 Celiobiose + + + + + + + + + + + 2012, 3 D-Arabinose - - - - - - - - - - - 28 Maltose + + + - + - + + + + + 4 L- Arabinose - - - + - + - - - - - 29 Lactose + + + - + - + + + + + 12 :260 5 Ribose + + + + + + + + + + + 30 Melibiose - + + - + - - + + - + 6 D-Xylose + - - + - + + - + + - 31 Sucrose + + + - + - - + + - + 7 L-Xylose - - - - - - - - - - - 32 Trehalose + + + - + - + + + + + 8 Adonitol - - - - - - - - - - - 33 Inulin - - - - - - - - - - - 9 ß-Methyl-D-Xyloside - - - - - - - - - - - 34 Melezitose - + + - + - - + + - + 10 Galactose + + + + + + + + + + + 35 Raffinose - + - - + - - + + - - 11 Glucose + + + + + + + + + + + 36 Starch + - - - - - + - - + - 12 Fructose + + + + + + + + + + + 37 Glycogen - - - - - - - - - - - 13 Mannose + + + + + + + + + + + 38 Xylitol - - - - - - - - - - - 14 Sorbose - - - - - - - + + - - 39 Gentibiose + + + + + + + + + + + 15 Rhamnose - - - - - - - - + - - 40 Turanose - - - - - - - + + - - 16 Dulcitol - - - - - - - - - -
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