Characterization of a bacteriocin from Pediococcus acidilactici PC and comparison of bacteriocin-producing strains using molecular typing procedures

The aim of this study was to isolate and characterize lactic acid bacteria (LAB) from naturally fermented pork sausages and test them for use as a probiotic starter culture in the production of fermented sausages. LAB (n=26) were isolated from natural fermented pork sausages. Isolates were identified using 16S rDNA or Internal Transcribed Spacer (ITS) region sequencing. After that, strains were characterized based on technological, functional and safety proprieties. A LAB strain was chosen and its survival was tested in simulated gastric juice and intestinal juice. Results indicate that Lactobacillus plantarum (n=15) was the predominant species in fermented sausage, followed by Enterococcus faecium (n=8), Lactobacillus brevis (n=1), Enterococcus durans (n=1) and Enterococcus hirae (n=1). L. plantarum BLS29 strain was selected because it was not able to produce CO2 and was able to multiply at temperatures ranging from 15 to 37°C, usually practiced during the sausage cure process. BLS29 showed better growth capacity than other isolated LABs when exposed to curing salts, and also demonstrated antimicrobial activity against foodborne pathogens. According to our findings, BLS29 can be a promising strain to be used as a probiotic starter culture for the production of fermented pork sausage.   Key words: Probiotics, Lactobacillus plantarum, fermented pork sausage.

BackgroundProbiotic bacteria can provide health benefits when delivered in functional foods. This study involved isolation of lactic acid bacteria (LAB) from traditionally dried and salted anchovy fish and characterization of their survival in simulated gastrointestinal digestion. Promising strains were used to prepare fermented fish sausages which were then evaluated for cytotoxicity activity against two cancer cell-lines, antidiabetic activity as determined by α-amylase and α-glucosidase inhibition, and antioxidant and proteolytic activities in vitro, as compared to non-fermented control sausages.ResultsOut of 85 LAB obtained, 13 isolates with high tolerance to simulated gastrointestinal digestion were obtained, which were identified as Enterococcus spp. Four E. faecium strains, one E. faecalis, and one E. durans were used separately to make fermented fish sausages. The α-amylase and α-glucosidase inhibition from fish sausages fermented by Enterococcus spp. ranged from 29.2 to 68.7% and 23.9 to 41.4%, respectively, during 21 days of storage. The cytotoxicity activities against Caco2 and MCF-7 cells of fish sausages fermented with Enterococcus spp. ranged from 18.0 to 24% and 13.9 to 27.9%, respectively. Cytotoxicity activities correlated positively with proteolysis and antioxidant activities, α-amylase and α-glucosidase inhibition activities, but negatively with the pH in fermented fish sausages. Strains also exhibited antimicrobial activity against foodborne pathogens and presented no significant concerns with regards to antibiotic resistance or virulence gene content.ConclusionsFish sausages fermented by potential probiotic isolates of Enterococcus spp. from dried fish had valuable health-promoting benefits compared with non-fermented control sausages.

Ethnic people of Iran consume variety of traditional fermented milk products including buttermilk made from ewe's milk. The purpose of this study was to isolate and characterize bacteriocin producing by Lactic acid bacteria from these products, and to exploit their potential as bio preservative. Ten strains of Lactic acid bacteria isolated from ewe's milk, traditional yoghurt and sour buttermilk from different areas in Azarbayjan-e-sharqi, Iran were screened for their ability to produce bacteriocin like inhibitory substances (BLIS). According to results, Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus brevis, Pediococcus acidilactici were shown to produce proteinaceous substances inhibitory against a number of Gram positive and negative bacteria including Staphylococcus aureus, Listeria monocytogenes and Salmonella enteritidis. The inhibitory activities of two Lactic acid bacteria (Lactobacillus paracasei and Pediococcus acidilactici isolated from ewe milk and buttermilk respectively) were unaffected by the action of pH neutralization and hydrogen peroxide while completely inhibited in the presence of proteolytic enzymes. The kinetic of bacteriocin like inhibitory substances against Staphylococcus aureus indicated a direct relationship between the growth rate and the amount of bacteriocin produced. The inhibitory activity of these lactic acid bacteria started in the early logarithmic phase and continued to the end of exponential phase. During ultrafiltration studies, bacteriocins produced by Pediococcus acidilactici, Lactobacillus paracasei were able to pass through the cellulose membranes with10 and 30 KDa. Titre of bacteriocins produced by Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus brevis were estimated 1600 AU/mL while the titre for bacteriocin which produced by Pediococcus acidilactici was calculated as 3200 AU/mL.

Lactic acid bacteria (LAB) found in Ethiopian traditional fermented foods and beverages have potential antagonistic effects against foodborne pathogens due to their capacity to produce various antimicrobial metabolites. This study evaluated the antagonistic activity of LAB isolated from Ethiopian traditional fermented foods and beverages against foodborne pathogens and characterized their antimicrobial substances. A total of 180 traditional fermented foods and beverages were collected, and the antagonistic activities of LAB were evaluated against selected foodborne pathogens. The effects of pH, temperature, enzymes, and food additives on the antagonistic effects of cell-free supernatant produced by LAB were investigated. LAB identification and characterization were conducted using an integrated phenotypic approach and MALDI TOF MS spectrum analysis, and data were analyzed using one-way ANOVA and Tukey post hoc analysis. A total of 956 LAB were isolated, of which seventeen (17 LAB) isolates of Pediococcus pentosaceus (Pc. pentosaceus (n = 7)), Pediococcus acidilactici (Pc. acidilactici (n = 2)), Enterococcus faecium (Ec. faecium (n = 6)), and Lactococcus lactis (Lc. lactis (n = 2)) were screened for antagonistic activity based on their ability to produce bacteriocins, probiotic activity, and preservative potential. Pc. pentosaceus JULABB16, Pc. pentosaceus JULABB01, and Ec. faecium JULABBr39 showed strong antagonistic activity against all pathogens, with mean inhibition zone diameters ranging from 23.50 to 35.50mm. Lc. lactis, Pc. pentosaceus, Pc. acidilactici, and Ec. faecium produced bioactive metabolites that were sensitive to proteolytic enzymes and capable of withstanding high temperatures (80-100°C) and acid concentrations (pH 2-10). The CFS produced by Lc. lactis, Pc. pentosaceus, Pc. acidilactici, and Ec. faecium showed the most impending antagonistic activity against all pathogens. The bioactive substances produced by LAB isolates had promising effects against food spoilage and pathogenic bacteria, making them potential natural food preservatives.

The ability of a commercial starter culture to perform a sausage fermentation was evaluated by culture –dependent and –independent methods. The starter culture, as well as the sausage during fermentation, was sampled and strains of lactic acid bacteria (LAB) and coagulase negative cocci (CNC) were isolated. After identification it was determined that Lactobacillus plantarum, contained in the inoculated starter, was the main LAB representative that conducted the transformation, while Staphylococcus xylosus, not declared in the label of the starter culture, was able to colonize the sausages studied. Molecular characterization of isolated Lb. plantarum and S. xylosus highlighted that the commercial mix contained several strains of the same species, and their behavior during the fermentation was different. Analysis of the nucleic acids extracted directly from the sausages confirmed the performance of Lb. plantarum, which was present and active throughout the fermentation, and highlighted the contribution of Lactobacillus curvatus. Introduction For fermented sausage production, the starter cultures are represented by mixtures of lactic acid bacteria (LAB) and coagulase-negative cocci (CNC). LAB are the main population responsible for the pH drop, followed by a second step in which CNC are neutralizing the organic acids produced by LAB, through production of peptides and aminoacids from their proteolytic activity. In addition, CNC are able to induce the release of various aromatic substances due to their capability to produce lipases (Montel et al., 1996). Recently, direct amplification of DNA and RNA by polymerase chain reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1993) showed very good applicability in monitoring the microbial ecology of Italian fermented sausages (Cocolin et al., 2001; Rantsiou et al., 2005). In this paper we wanted to validate the use of a commercial starter for the production of fermented sausages by molecular methods. DGGE was used to analyze PCR and RT-PCR products obtained from DNA and RNA extracted directly from the sample to monitor the bacterial dynamics during transformation. Moreover, LAB and CNC were isolated both from the starter culture and during fermentation and subsequently subjected to RAPD analysis to understand if the strains inoculated were able to conduct the fermentation process considered in the study. Materials and Methods Fermented sausages were prepared in a local meat factory using traditional techniques as previously described (Comi et al., 2005) and inoculated with 20 g starter culture containing S. carnosus and Lb. plantarum (Biostart SL1-200, Wiesby GMBH Co., Niebull, Germany). The ripening was conducted for 28 days. The fermented sausages were analyzed in triplicates at 0, 3, 5, 7, 14 and 28 days. Potentiometric measurements of pH were made using a pin electrode of a pH-meter (Radiometer Copenhagen pH M82, Cecchinato, Italy). The starter culture (20 g), of the same batch that was used in the production followed, was resuspended in 200 ml sterile water and left at room temperature for 30 min and serial dilutions were prepared in saline/peptone water (8 g/l NaCl, 1 g/l bacteriological peptone, Oxoid, Milan, Italy) and analyzed on MRS agar (Oxoid) incubated with a double layer at 30°C for 48 h and on Mannitol Salt Agar (MSA, Oxoid) incubated at 30°C for 48 h. Fifteen colonies of LAB were isolated from MRS agar and the same number of CNC were selected from MSA agar. The fermented sausages were analyzed to monitor the dynamic changes in the populations responsible for the ripening of fermented sausages, as well as their hygienic quality. In particular, 25 g of each sample were transferred into a sterile stomacher bag and 225 ml of saline/peptone water were added and mixed for 1 min and 30 s in a Stomacher machine (PBI, Italy). Further decimal dilutions were made and the following analyses were carried out on duplicate agar plates: a) total bacterial count (TBC) on Gelisate Agar (Oxoid) incubated for 48–72 h at 30°C; b) LAB on MRS agar; c) CNC on MSA (Oxoid);

[This corrects the article DOI: 10.3389/fmicb.2019.02705.].

A novel three-step combination of in vitro and ex vivo screening was established to massively screen host derived lactic acid bacteria (LAB) from the broiler chicken intestine with inhibitory activity against Escherichia coli. In a first step, a massive sample pool consisting of 7102 broiler-derived colonies from intestinal contents were established and sub-cultured. Supernatants thereof were incubated with an E. coli model strain to screen suitable isolates with inhibitory activity. A total of 76 isolates of interest were subsequently further studied based on either pH dependent or -independent activity in the second step of the assay. Here, in-depth growth inhibition of the E. coli model strain and the potential of isolates for lactic acid production as inhibitory substance were indexed for all isolates. Resulting scatter plots of both parameters revealed five isolates with exceptional inhibitory activity that were further studied under ex vivo condition in the third step of the assay. These isolates were taxonomically classified as strains of the species Lactobacillus agilis, Lactobacillus salivarius, and Pediococcus acidilactici. Samples from the broiler chicken intestine were inoculated with the Lactobacillus isolates and the E. coli model strain. After 8 and 24 h incubation, respectively, growth of the E. coli model strain was monitored by cultivation of the E. coli strain in antibiotic supplemented medium. By their superior inhibitory activity against the E. coli model strain, one L. agilis and one L. salivarius strain were selected and characterized for further application as probiotics in broiler chicken. Additionally, their antibiotic resistance patterns and resilience under gastric stress of isolates were also characterized. The results of this study demonstrate that the novel isolation procedure was able to efficiently and rapidly isolate and identify bacterial strains from a massive sample pool with inhibitory potential against specific types of bacteria (here E. coli). The introduction of the final ex vivo selection step additionally confirmed the inhibitory activity of the strains under conditions simulating the intestinal tract of the host. Furthermore, this method revealed a general potential for the isolation of antagonistic strains that active against other pathogenic bacteria with specific biomarker.

Processing lamb meat into fermented sausages can reduce the risk of spoilage and extend shelf life. Fermented sausages are commonly made using lactic acid bacteria (LAB), resulting in a product that is acidic and less firm. Therefore, it is necessary to add jack bean flour as a binding agent. This study aims to analyze physicochemical, microbiological and organoleptic characteristics of premium IPB lamb fermented sausages with varying proportions of jack bean flour (0% and 30%). The analyses included pH, water activity (aw), total acid, water, ash, fat, crude protein, carbohydrate, total LAB, E. coli, S. aureus, texture profile analysis (TPA), and sensory evaluation. The incorporation of jack bean flour into lamb meat fermented sausages can elevate the pH, springiness, chewiness, crude protein, and carbohydrate content of the fermented sausages. Furthermore, the addition of jack bean flour may reduce the total acid, aw, gumminess, water content, and bacteria (E. coli and S. aureus). The sensory aspects such as color in fermented sausage with addition of jack bean flour were preferred by the panelists. The addition of jack bean flour increased the total unsaturated fatty acids and essential amino acids in fermented sausages. The incorporation of jack bean flour aligns with the Indonesian National Standards for Meat Sausages 3820–2015 concerning moisture, fat, and protein content. This study suggests that incorporating lamb fermented sausage with 30% jack bean flour could result in significant benefits, including increased nutrition, enhanced sensory quality, improved texture, and extended storage life for fresh lamb products.

Many meat (or fish) products, obtained by the fermentation of meat originating from various animals by the flora that naturally contaminates it, are part of the human diet since millenaries. Historically, the use of bacteria as starters for the fermentation of meat, to produce dry sausages, was thus performed empirically through the endogenous micro-biota, then, by a volunteer addition of starters, often performed by back-slopping, without knowing precisely the microbial species involved. It is only since about 50 years that well defined bacterial cultures have been used as starters for the fermentation of dry sausages. Nowadays, the indigenous micro-biota of fermented meat products is well identified, and the literature is rich of reports on the identification of lactic acid bacteria (LAB) present in many traditional fermented products from various geographical origin, obtained without the addition of commercial starters (See Talon, Leroy, & Lebert, 2007, and references therein). The LAB species that are naturally present in those products and become dominant in the final processing steps essentially belong to Lactobacillus sakei, Lactobacillus curvatus, and Lactobacillus plantarum. These are also the three main species that are sold as starters for the fermentation of dry sausages, essentially in Europe, to which should be added the two other pediococci species Pediococcus pentosaceus and Pediococcus acidilactici (Hammes & Hertel, 1998). Since the last 20 years, many microbiologists have investigated the physiology of these LAB, in order to understand the mechanisms by which they contribute to the quality of the final product, and to improve their use. Molecular tools were therefore developed, leading to an increase of the knowledge about their genetics. More recently, the genomes of L. plantarum WCFS1 (Kleerebezem, et al, 2003), L. sakei 23K (Chaillou, et al., 2005), and P. pentosaceus ATCC25745 (Makarova, et al., 2006) were entirely sequenced, giving a general overview on the whole genetic repertoire of those bacteria. However, the description and analysis of all

Fermented meat products are defined as meat that is inoculated with a microbial starter culture during processing under controlled condition, or meat allowed to ferment by natural fermentation meat microbial flora to give desirable characteristics. Fermentation of meat is mainly by lactic acid bacteria (LAB). Their biochemical characteristics project them as sugar fermentative, catalase and nitration reduction negative bacteria. This report is based on a preliminary research of isolation and characterization of microbes in fermented meat products on selected Ghanaian markets. The work specifically seeks to isolate and identify both essential and pathogenic microbes in fermented meat. Identification of microbial species is important for consumer protection and food law enforcement. In this study, samples of fermented bush meats and fermented sausages from selected Ghanaian markets were analyzed for their microbial content and load. Isolated microbes in samples were identified as Lactic acid Bacteria species, Streptococci species, Staphylococci species and Micrococcus species. Lactic acid Bacteria species (LAB) and micrococci species are characterized as essentially non pathogenic bacteria species whiles streptococci species and staphylococci species are characterized as the pathogen species. Isolates obtained from fermented bush meat were identified as LAB, staphylococci and streptococci species whiles isolates of fermented sausage were also identified as LAB and micrococci species. The total variable count for fermented sausage on an average was 1.3× 105 cfu, for smoked fermented bush meat the average loads was 2× 105 cfu whiles for fresh fermented bush meat the load was 2.5× 106 cfu. Key words: Meat, bush meat, fermented meat products, food microbiology, food safety, Kumasi, Ghana.

Characterization of lactic acid bacteria isolated from infant faeces as potential probiotic starter cultures for fermented sausages

Profiling of autochthonous microbiota and characterization of the dominant lactic acid bacteria occurring in fermented fish sausages

Chapter 8 - Lactic acid bacteria in meat fermentation: Dry sausage safety and quality

To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects. Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14.4 and 12.4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin. Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products. Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.

SummaryThe aim of this study was to evaluate the bioconversion efficiency of rich in cellulose agro‐industrial by‐products such as wheat bran (WB), spent distiller's grain with solids (DGS), brewer's spent grain (BSG) and lupin (Lupinus angustifolius L.) wholemeal fraction (LF) to lactic acid (LA) using acid tolerant lactic acid bacteria (LAB) strains Lactobacillus sakei KTU05‐06, Pediococcus acidilactici KTU05‐7 and P. pentosaceus KTU05‐9. Carbohydrase preparation Depol™ 692L was used for the hydrolysis of non‐starch polysaccharides. Analysed raw materials were suitable substrates for LAB propagation and L‐lactic acid production. The lowest pH (3.6) was found in LF medium after 48 h fermentation with P. acidilactici and P. pentosaceus strains. The lowest pH (3.86) was measured in WB fermented with L. sakei, and in DGS and BSG (pH 3.8 and 3.9 respectively) fermented with P. acidilactici. The highest endoxylanase activity was excreted by the P. acidilactici and P. pentosaceus (84 and 69 XU g−1 respectively), and the highest α‐amylase activity was of L. sakei (255.6 AU g−1) after 24 h incubation in WB medium. The L‐lactic acid concentration of 86.11 g kg−1 was reached after the bioconversion of hydrolysed WB in combination with 48 h fermentation by P. pentosaceus KTU05‐9 strain. LA contents between 222 and 282 mg kg−1 was produced from lupin processing residues via fermentation using P. acidilactici and P. pentosaceus KTU05‐9 strains. The major challenge within the presented study is the viability of tested LAB in cereal waste media and effective LA production at a low pH (3.6–3.8).