Characterization of Lactic Acid Bacteria from Dry Sausages

Biotechnology & Biotechnological Equipment ISSN: 1310-2818 (Print) 1314-3530 (Online) Journal homepage: https://www.tandfonline.com/loi/tbeq20 Characterization of Lactic Acid Bacteria from Dry Sausages S. Stoyanovski, V. Chipeva, S. G. Dimov, Sv. Danova, I. Dimitrova, L. Yocheva, S. Antonova-Nikolova & I. Ivanova To cite this article: S. Stoyanovski, V. Chipeva, S. G. Dimov, Sv. Danova, I. Dimitrova, L. Yocheva, S. Antonova-Nikolova & I. Ivanova (2009) Characterization of Lactic Acid Bacteria from Dry Sausages, Biotechnology & Biotechnological Equipment, 23:sup1, 870-873, DOI: 10.1080/13102818.2009.10818560 To link to this article: https://doi.org/10.1080/13102818.2009.10818560 © 2009 Taylor and Francis Group, LLC Published online: 15 Apr 2014. Submit your article to this journal Article views: 143 View related articles Citing articles: 5 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tbeq20 CHARACTERIZATION OF LACTIC ACID BACTERIA FROM DRY SAUSAGES S. Stoyanovski1, V. Chipeva2, S. G. Dimov3, Sv. Danova4, I. Dimitrova1, L. Yocheva5, S. Antonova-Nikolova1, and I. Ivanova1 Sofia University “St. Kliment Ohridski”, Faculty of Biology, Dept. of General and Industrial Microbiology 1 NBIMCC 2 Sofia University “St.Kliment Ohridski”, Faculty of Biology, Department of Genetics3 Bulgarian Academy of Science, Institute of Microbiology Acad. Angel Bonchev 4 Sofia University “St.Kliment Ohridski” Faculty of Medicine 5 Correspondence to: Prof. Iskra Ivanova E-mail: [email protected] ABSTRACT Eight genera (Lactobacillus, Leuconostoc, Staphylococcus, Enterococcus, Lactococcus, Micrococcus, Streptococcus) are most commonly used meat starter cultures. The Lactobacilli associated with meat fermentation and the efforts to accurate classification and identification of them are becoming more important as various isolates of LAB become more commonly used as starter cultures. The present work deals with characterization of the microflora associated with naturally fermented Bulgarian sausages (Lukanka) and sausages (Lukanka) with defined starter culture produced by “Tandem“. More then 200 strains of genera Lactobacillus were isolated during the different stages of the fermentation process of the naturally fermented sausages. The morphological, cultural and physiological and biochemical analysis were performed. In such a system such as meat with a wide variety of complex substrates serving as sources of peptoses, organic acid and other fermentable compounds different end products are formed. Research on the production of lactic acid, determination of pH, screening for producers of antimicrobials was performed. Keyword: lactobacilli, dry sausages, bacteriocins Introduction Dry and semi dry sausages represent the largest category of fermented meat products. Traditionally, dry sausages acquired their particular sensory characteristics from exposure to salt, indigenous gram positive microorganisms such as LAB, staphylococci, micrococci. Lactic acid bacteria (LAB) have been used in fermentations to preserve the nutritive qualities of various foods. The lactobacilli associated with meat fermentations are members of the genus Lactobacillus. most common lactobacilli found in dry sausages .However, attempts to identify lactobacilli isolated from meat are usually less than successful because most of documented descriptions and schemes of identification are based on isolates from other food sources .A series of investigation on atypical lactic acid streptococci isolated from fermented meats resulted in their identification as Lb.sakei and Lb.curvatus (1). These species outnumbered the typical lactobacilli, identified as Lactobacillus delbrueckii XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY subsp.lactis, Lb.brevis, Lb.farciminis, Lb.buchneri, Lb.plantarum, Lb.curvatus, Lb.alimentarius ,Weissella virdescens, unspecified leuconostocs, and pediococci 1,000fold and were typically psychrotrophylic and less acid tolerant.(1,2) Efforts to accurate classification and identification of them are becoming more important as various isolates of LAB become more commonly used as starter cultures. Bacteriocins, produced by LAB have attracted a great interest in food industry due to their applicational potentiality in food preservation. It was documented that such species as Lb.sake, Lb.curvatus, Lactobacillus plantarum produce bacteriocins which may found broader application in starter cultures for meat fermentation in the future. The present work deals with characterization of the microflora associated with naturally fermented bulgarian sausages (Lukanka) and sausages (Lukanka) with defined starter culture produced by “Tandem“. More then 200 strains were isolated during the different stages of the fermentation process of the naturally fermented sausages. In this paper, we 870 BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE report on the screening of antimicrobial compounds of 7 lactic acid bacteria isolated from a small scale facility producing traditional dry sausages and as well their taxonomic determination. DigiGenius system (Syngene) TABLE 2 RAPD oligonucleotide primers L9 and L10 Materials and methods The 7 LAB strains considered of in this study were isolated from a small-scale facility producing dry sausage “lukanka”. The strains with numbers SM3, SM6, SM7, SM9, SM11, SM12 and SM28 were used. All the used strains were identified to belong to genera Lactobacillus. Initial identification of all the strains was performed by API 50CHL system (BioMerieux, France), according to the manufacturer’s instructions. The isolates were subcultured twice (10% inoculum, 24 hours, 30 ˚C) in 15 ml MRS broth and kept frozen at –20 ˚C in MRS supplemented with 10% glycerol. The isolates were tested against L.innocua F (ENITIAA, Ecole Nationale des Ingénieurs des Techniques des Industries Agricoles et Alimentaires, Nantes, France) and E.coli NBIMCC 3398. An agar diffusion assay was used for the detection of the antagonistic activity (7). Total DNA from strains SM3, SM6, SM7, SM9, SM11 and SM12, was isolated with GeneElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich) according to the manufacturer. GS-PCR was performed according to (3,4) using two Lactobacillus–specific primers sets (Table 1). TABLE 1 Genus-specific PCR with Lab0677F/Lact71R primers set (Pane A) and LactoF/LactoR primers set (Pane B). 100 bp ladder was used as molecular weight marker Lab0677F 5-'CTCCATGTGTAGCGGTG-3' Lact71R 5'-TCAAAACTAAACAAAGTTTC-3 LactoF 5'-TGGAAACAGRTGCTAATACCG-3' LactoR 5'-GTCCATTGTGGAAGATTCCC-3' Moura et al. Byun et al. L9 GCAGCCGG Sohier et al. L10 AGTCAGCCAC Tynkkynen et al. Characterization of the antimicrobial compounds. Isolates exhibiting antagonistic activity against pathogenic microorganisms were investigated for their antimicrobial compounds. These isolates were grown overnight at 30 ˚C in 10 ml MRS broth. A cell free solution was obtained by centrifugation (15min at 4 ˚C). The samples were adjusted to pH 6.5 to rule out acid inhibition. Sensitivity to heat of the antibacterial compounds was investigated by treatment the culture supernatant in water bath at 80 ˚C for 10 min and for 1 hour. The residual activity was determined by agar diffusion assay. Sensitivity to proteolytic enzymes of the antibacterial compounds was investigated by addition of trypsin and proteinase K at final concentration of 1mg to the culture supernatants. The samples were incubated for 3 hours and the residual activity determined by agar diffusion method. Results and discussion Lactic acid bacteria originally isolated from traditional sausages are probably the best candidates for improving the microbiological safety of these foods, because they are well adapted to the conditions in the sausages and should therefore be more competitive that LAB from other sources. LAB strains were screened for exhibition of antagonistic activities against indicator microorganisms as E.coli and L.innocua. Results were presented on fig.1a and 1b and table 3. Therefore, characterizing the antibacterial compounds responsible for the inhibition of these pathogens was of interest. RAPD PCR was performed according to (5) and (6) with oligonucleotide primers L9 and L10 respectively (Table 2). The results were analyzed with GeneTools v. 4.00 software (Syngene). The amplification was analyzed on 1,2 % agarose gel with TAE buffer system in Hoeffer HE 33 electrophoresis system (GE Healthcare). The gels were visualized with ethidium bromide and documented with BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE 871 Fig. 1a Antibacterial activity against E.coli XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY Fig. 2a Antibacterial activity against L.innocua F Fig. 1b Antibacterial activity against L.innocua Fig. 2b Antibacterial activity against E.coli TABLE 3 Effect of enzymes and heat treatment on inhibitory activity of Lactobacillus strains, cell-free supernatants Strain SM3 SM6 SM7 SM9 SM11 SM12 SM28 Activity against L.innocua (mm sterile zone) control Temperature Trypsin /proteinase control K 9 17 12 12 10 18 12 18 12 12 12 18 12 18 12 11 11 18 12 20 Activity against E.coli (mm sterile zone) Temperature Trypsin /proteinase K 18 17 17 16 17 16 - Regarding the proteinaceous nature, the thermostability at 80˚C for 10 minutes of the antibacterial compounds of strains SM3, SM7, SM11 and A28 and their residual activities, it was concluded that these antimicrobials were bacteriocin like. The most promising strain from the examined ones seemed to be strain SM28. Its antimicrobial activity against E.coli is higher in comparison with the other strains. These antibacterial compounds may constitute interesting weapons to fight against biofilm growth and implementation of undesirable microorganisms on the processing surfaces. The next task of study was the affiliation of the strains. The genus affiliation of the strains investigated was determined by the Lactobacilllus-specific primers sets (3,4) Fig. 3a and 3b. Fig. 3a Genus-specific PCR with Lab0677F/Lact71R primers set. 100 bp ladder was used as molecular weight marker Fig. 3b Genus-specific PCR with LactoF/LactoR primers set. 100 bp ladder was used as molecular weight marker XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY 872 BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE The electrophoretic patterns obtained with the RAPD-PCR were subjected to UPGMA analysis with GeneTools v. 4.00 software, and dendrograms reflecting the phylogenetic relatedness of the investigated strains were constructed. Fig. 4a RAPD analysis with L9 primer UPGMA analysis of the obtained RAPD band patterns with L9 primer Fig. 4 b RAPD analysis with L10 primer UPGMA analysis of the obtained RAPD band patterns with L10 primer. The results from API test indicate that strain SM7 belong to species Lactobacillus pentosus 99 % and strain SM9 belong to species Lactobacillus brevis 96.7 % respectively It is the first report on bacteriocin-like activities of strains that occur within the microbial ecosystem of dry bulgarian sausage “lukanka” produced from a small scale facility and their preliminary taxonomic determination. REFERENCES 1. Hammes, W. P., A. Bantleon, and S. Min (1990). FEMSMicrobiol.Lett.87:165-174 2. Michael P. Doyle and Larry R. Beuichael (2007) Food Microbiology, Chapter 37,795-815, Third edition, ASM Press Washington BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE In both cases, with L9 and L10 oligomer primers, clustering in two concordant groups was obtained, the first group comprising strains SM3, SM7, SM9 and SM11 and the second one strains SM6 and SM12 (Fig.4a and 4b). 3. Byun, R., Nadkarni. M.A., Chhour. K.L., Martin. F.E., Jacques. N.A. and Hunter. N. (2004). J. Clin. Microbiol. 42 (7): 3128-3136 4. Moura, P., Simões, F., Gírio, F., Loureiro-Dias, M.C. and Esteves, M.P. (2007). J Basic Microbiol 47:148-157 5. Sohier D, Coulon J, Lonvaud-Funel A. (1999). Int J Syst Bacteriol. 49(3):1075-1081 6. Tynkkynen S, Satokari R, Saarela M, MattilaSandholm T, Saxelin M. (1999). Appl Environ Microbiol. 65(9):3908-3914. 7. Bertrand-Harb, C., Ivanova, I. V., Dalgalarrondo, M. and Haertle, T. (2003). Int. Dairy J. 13, 39-45. 873 XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY