Genomic characterization and probiotic potential assessment of an exopolysaccharide-producing strain Pediococcus pentosaceus LL-07 isolated from fermented meat.

BACKGROUND: The genomic information available for Pediococcus pentosaceus is primarily derived from fermented fruits and vegetables, with less information available from fermented meat. P. pentosaceus LL-07, a strain isolated from fermented meat, has the capability of producing exopolysaccharides (EPS). To assess the probiotic attributes of P. pentosaceus LL-07, we conducted whole-genome sequencing (WGS) using the PacBio SequelIIe and Illumina MiSeq platforms, followed by in vitro experiments to explore its probiotic potential. RESULTS: The genome size of P. pentosaceus LL-07 is 1,782,685 bp, comprising a circular chromosome and a circular plasmid. Our investigation revealed the absence of a CRISPR/Cas system. Sugar fermentation experiments demonstrated the characteristics of carbohydrate metabolism. P. pentosaceus LL-07 contains an EPS synthesis gene cluster consisting of 13 genes, which is different from the currently known gene cluster structure. NO genes associated with hemolysis or toxin synthesis were detected. Additionally, eighty-six genes related to antibiotic resistance were identified but not present in the prophage, transposon or plasmid. In vitro experiments demonstrated that P. pentosaceus LL-07 was comparable to the reference strain P. pentosaceus ATCC25745 in terms of tolerance to artificial digestive juice and bile, autoaggregation and antioxidation, and provided corresponding genomic evidence. CONCLUSION: This study confirmed the safety and probiotic properties of P. pentosaceus LL-07 via complete genome and phenotype analysis, supporting its characterization as a potential probiotic candidate.

Risk management tool to define a corrective storage to enhance Salmonella inactivation in dry fermented sausages.

The resistance of Salmonella to the harsh conditions occurring in shelf-stable dry fermented sausages (DFS) poses a food safety challenge for producers. The present study aimed to model the behaviour of Salmonella in acid (with starter culture) and low-acid (without starter culture) DFS as a function of aw and storage temperature in order to build a decision supporting tool supporting the design of a corrective storage strategy to enhance the safety of DFS. Salmonella spp. were inoculated in the raw meat batter at ca. 6 Log cfu/g with a cocktail of 3 strains (CTC1003, CTC1022 and CTC1754) just before mixing with the other ingredients and additives. After stuffing, sausages were fermented and ripened following industrial processing conditions. Different drying-times were applied to obtain three batches with different aw (0.88, 0.90 and 0.93). Afterwards, DFS were stored at 4, 8, 15 and 25 °C for a maximum of three months and Salmonella spp. were periodically enumerated. The Weibull model was fitted to Log counts data to estimate inactivation kinetic parameters. The impact of temperature and aw on the primary inactivation parameters was evaluated using a polynomial equation. The results of the challenge tests showed that Salmonella spp. levels decreased during storage at all the assayed conditions, from 0.8 Log (in low-acid DFS at 4 °C) up to 6.5 Log (in acid DFS at 25 °C). The effect of both aw and temperature was statistically significant. Delta (δ) parameter decreased by decreasing aw and increasing temperature, while the shape (p) parameter ranged from above 1 (concave) at 10 °C to below 1 at 25 °C (convex). A common secondary model for the p parameter was obtained for each type of DFS, acid and low-acid, indicating that acidification during the production of DFS affected the time for the first Log reduction (δ) during the subsequent storage, but not the overall shape (p parameter) of the inactivation. The developed models covered representative of real conditions, such as Salmonella contamination in the raw materials and its adaptation to the harsh processing conditions. The good predictive performance shown when applying the models to independent data (i.e. up to 80% of the predictions within the 'Acceptable Simulation Zone' for acid sausages) makes them a suitable and reliable risk management tool to support manufacturers to assess and design a lethality treatment (i.e. corrective storage) to enhance the Salmonella inactivation in the product before DFS are released to the market.

In Vitro Assessment of Probiotic Potential of Saccharomyces Cerevisiae DABRP5 Isolated from Bollo Batter, a Traditional Goan Fermented Food.

Bollo is a traditional Goan fermented food in which coarse wheat/wheat and finger millet is leavened with toddy. We here isolated 42 yeast strains from Bollo batter. Initial screening of the isolates with probiotic properties yielded four yeast isolates (DABRP1, DABRP2, DABRP5 and DABRP12). These isolates exhibited tolerance to high bile salt concentration and acidic pH and resistance to various antibiotics, which indicated their probiotic nature. All these yeast isolates were identified as Saccharomyces cerevisiae through D1D2-LSU-rDNA sequencing. These yeast isolates also showed higher percent hydrophobicity towards chloroform followed by n-hexadecane and o-xylene indicating their mucosal surface-adhesive property. To evaluate the safety of the isolates for them to be called as generally recognized as safe, the pathogenic behavior of the isolates determined through the in vitro hemolysis assay and evaluation of DNase and gelatinase activities. None of the isolates exhibited hemolysis or produced DNase or gelatinase and thus were considered potentially safe. In terms of beneficial effects, the most potent isolate S. cerevisiae DABRP5 showed antibacterial activity against the test pathogens. It also showed excellent antioxidant activity with DPPH free radical scavenging activity of 68.85 ± 0.69%, anti-inflammatory activity with 60.39 ± 0.34% inhibition of protein denaturation, and antidiabetic activity with 71.75 ± 0.45% inhibition of α-amylase activity. The isolate produced α-amylase, lipase, and ß-galactosidase. The probiotic potential of the isolate S. cerevisiae DABRP5 was similar to that of the reference strain (Saccharomyces boulardii CNCM I-745) used in this study. The results thus indicate that yeast isolates from Bollo batter have probiotic potential.

Assessment of exopolysaccharides, bacteriocins and in vitro and in vivo hypocholesterolemic potential of some Egyptian Lactobacillus spp.

Lactobacilli probiotics have been suggested to reduce cholesterol with low side effects to host. Bacteriocins and exopolysaccharides (EPSs) production are two meaningful examples of functional applications of lactobacilli in the food industry. Eight Lactobacillus strains were isolated from some Egyptian fermented food and tested for their probiotic properties. Analysis of the monosaccharide composition by thin layer chromatography showed the presence of glucose, galactose and unknown sugar. The main functional groups of EPSs were elucidated by Fourier-Transform Infrared Spectroscopy. Their fermentation cultures displayed powerful antioxidant activities extending from 97.5 to 99%, 40-75% for their EPSs and free cells, respectively, and exhibited in vitro cholesterol downgrading from 48 to 82% and 72 to 91% after 48 and 120 h, respectively. Their EPSs showed good anticancer activities against carcinoma cells with low IC50 values for HCT-116, PC-3 and HepG-2 cells. To the best of our knowledge, there have been no previous reports on the potential of Lactobacillus EPSs activity against PC-3. The selected strains, L. plantarum KU985433 and L. rhamnosus KU985436 produced two different bacteriocins as detected by gel permeation chromatography with good antimicrobial activities. In vivo study demonstrated that feeding Westar rats with fermented milk exhibited greater cholesterol, LDL and blood triglyceride reduction for both strains. Whereas, HDL was increased by about 43 and 38%, respectively, and the atherogenic indices decreased.

Gas chromatography-mass spectrometry-based metabolomics analysis of metabolites in commercial and inoculated pickles.

BACKGROUND: Starter cultures are nowadays more and more used to make inoculated pickles (IPs), but it remains unclear whether there are differences in flavors between IPs and naturally fermented pickles. In this study 16 commercial pickles (CPs) produced by spontaneous fermentation method were purchased from markets in Sichuan province and Chongqing. Ten strains of three lactic acid bacteria species - Lactobacillus plantarum, Leuconostoc mesenteroides and Pediococcus ethanolidurans - were selected as single starter cultures to produce IPs. RESULTS: Differences in flavor components between the CPs and IPs were monitored using a combination of gas chromatography-mass spectrometry and multivariate statistical methods. Higher levels of nonvolatile substances such as glucose, fructose, tagatose, sucrose, lactic acid and mannitol were detected in most IPs than in the CPs. The values of flavor characteristics such as sweetness, umami and astringency, which were correlated positively with consumers' overall preferences for pickles, were higher in the IPs than in the CPs. Volatile compounds such as geranyl acetate, dimethyl trisulfide, eucalyptol and linalool were distinguished as the main compounds that contributed to the flavor characteristics of the CPs. In addition to dimethyl trisulfide, dimethyl disulfide was also an odor contributor to the IPs. CONCLUSIONS: The CPs and IPs had different flavor characteristics, especially in the composition and content of volatile components, and the inoculation method reflected some fermentation advantages, which could reduce the bitterness and increase umami and lead to a higher score of sensory preference. This will be helpful for industrial production. © 2020 Society of Chemical Industry.

Fermented foods in a global age: East meets West.

Fermented foods and alcoholic beverages have long been an important part of the human diet in nearly every culture on every continent. These foods are often well-preserved and serve as stable and significant sources of proteins, vitamins, minerals, and other nutrients. Despite these common features, however, many differences exist with respect to substrates and products and the types of microbes involved in the manufacture of fermented foods and beverages produced globally. In this review, we describe these differences and consider the influence of geography and industrialization on fermented foods manufacture. Whereas fermented foods produced in Europe, North America, Australia, and New Zealand usually depend on defined starter cultures, those made in Asia and Africa often rely on spontaneous fermentation. Likewise, in developing countries, fermented foods are not often commercially produced on an industrial scale. Although many fermented products rely on autochthonous microbes present in the raw material, for other products, the introduction of starter culture technology has led to greater consistency, safety, and quality. The diversity and function of microbes present in a wide range of fermented foods can now be examined in detail using molecular and other omic approaches. The nutritional value of fermented foods is now well-appreciated, especially in resource-poor regions where yoghurt and other fermented foods can improve public health and provide opportunities for economic development. Manufacturers of fermented foods, whether small or large, should follow Good Manufacturing Practices and have sustainable development goals. Ultimately, preferences for fermented foods and beverages depend on dietary habits of consumers, as well as regional agricultural conditions and availability of resources.

Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis.

The safety of microbial cultures utilized for consumption is vital for public health and should be thoroughly assessed. Although general aspects on the safety assessment of microbial cultures have been suggested, no methodological detail nor procedural guideline have been published. Herein, we propose a detailed protocol on microbial strain safety assessment via whole-genome sequence analysis. A starter culture employed in traditional fermented pork production, nham, namely Lactobacillus plantarum BCC9546, was used as an example. The strain's whole-genome was sequenced through several next-generation sequencing techniques. Incomplete plasmid information from the PacBio sequencing platform and shorter chromosome size from the hybrid Oxford Nanopore-Illumina platform were noted. The methods for 1) unambiguous species identification using 16S rRNA gene and average nucleotide identity, 2) determination of virulence factors and undesirable genes, 3) determination of antimicrobial resistance properties and their possibility of transfer, and 4) determination of antimicrobial drug production capability of the strain were provided in detail. Applicability of the search tools and limitations of databases were discussed. Finally, a procedural guideline for the safety assessment of microbial strains via whole-genome analysis was proposed.

Harnessing Microbes for Sustainable Development: Food Fermentation as a Tool for Improving the Nutritional Quality of Alternative Protein Sources.

In order to support the multiple levels of sustainable development, the nutritional quality of plant-based protein sources needs to be improved by food technological means. Microbial fermentation is an ancient food technology, utilizing dynamic populations of microorganisms and possessing a high potential to modify chemical composition and cell structures of plants and thus to remove undesirable compounds and to increase bioavailability of nutrients. In addition, fermentation can be used to improve food safety. In this review, the effects of fermentation on the protein digestibility and micronutrient availability in plant-derived raw materials are surveyed. The main focus is on the most important legume, cereal, and pseudocereal species (Cicer arietinum, Phaseolus vulgaris, Vicia faba, Lupinus angustifolius, Pisum sativum, Glycine max; Avena sativa, Secale cereale, Triticum aestivum, Triticum durum, Sorghum bicolor; and Chenopodium quinoa, respectively) of the agrifood sector. Furthermore, the current knowledge regarding the in vivo health effects of fermented foods is examined, and the critical points of fermentation technology from the health and food safety point of view are discussed.

Assessment of safety aspect and probiotic potential of autochthonous Enterococcus faecium strains isolated from spontaneous fermented sausage.

OBJECTIVE: The objectives of this research project were isolation, identification, and evaluation of the safety aspect and probiotics properties of 21 Enterococcus faecium strains isolated from sausages originated from southeastern Serbia. RESULTS: Analyzed E. faecium isolates showed tolerance to simulated gastrointestinal conditions. All the examined isolates grew well on media with 0.1% and 0.2% of phenol. None of the tested isolates were histamine-producers, while the synthesis of tyramine was observed for E. faecium sk8-1 and sk8-17. Full resistance to antibiotics was not observed for any examined isolate of E. faecium (penicillin, amoxicillin, and ofloxacin showed the effect on all tested isolates). An inhibition zone against examined pathogens was exhibited by all strains, with the largest inhibition zone against Pseudomonas spp., Proteus spp. and E. coli (12-30 mm/MIC values ranged from 0.5 to 12 mg mL-1). CONCLUSION: The results indicated that E. faecium isolates from spontaneously fermented sausage showed a potential for further investigation and possible application as probiotics.

Safety and Stability Assessment of Potential Probiotic Strains from Fermented Mango Brine Pickle.

Fermented foods are known to be rich source of valuable nutrients and probiotics. Previously, our study reported the isolation and characterization of eight potential probiotic strains from traditional mango brine pickle, which has been conventionally consumed for ages in raw form in Southern India. The present study reports on the safety assessment of these strains for the selection of prospective probiotic candidates. Hydrogen peroxide production, histidine decarboxylase activity (production of histamine), DNase activity, and presence of the virulence factor genes (assessed by PCR) were carried out to evaluate its safety. Bacillus licheniformis KT921419 and B. methylotrophicus KT921422 was found to show no adverse safety characteristics. These two strains were further assessed for their ability to survive in the native substrate (mango brine pickle) as single and mixed inoculums. Above strains maintained significant viability in mango brine pickle for a period of 6 months during storage at the room temperature. Results clearly proved the safety and stability of two of the potential probiotic strains, which can be further utilized in food applications under harsh conditions of high salt, low pH, and room temperature making these strains unique.

Assessment of the non-lactic acid bacteria microbiota in fresh cucumbers and commercially fermented cucumber pickles brined with 6% NaCl.

Limited documentation of the cucumber fermentation microbiome has impeded the understanding of the role of microbes on the quality of finished products. We characterized the microbiome of fresh and fermented cucumber samples using culture dependent and independent techniques, with an emphasis on the non-lactic acid bacteria (non-LAB) population. Insubstantial microbiome variations were observed among fresh cucumber types with Rhizobium (31.04%), Pseudomonas (14.08%), Pantoea (9.25%), Stenotrophomonas (6.83%), and Acinetobacter (6.5%) prevailing. The relative abundance of LAB remained below 0.4% and 4.0% on fresh cucumbers and day 3 of the fermentations brined with 6% sodium chloride, respectively. Fermentation cover brine samples collected on day 1 harbored Pseudomonas, Pantoea, Stenotrophomonas, Acinetobacter, Comamonas, Wautersiella, Microbacterium, Flavobacterium, Ochrobactrum and the Enterobacteriaceae, Citrobacter, Enterobacter and Kluyvera. Plate counts for presumptive Klebsiella and Pseudomonas from fermentation cover brine samples reached 2.80 ±â€¯0.36 and 2.78 ±â€¯0.83 log of CFU/mL, respectively, in 30% and 60% of the nine tanks scrutinized with selective media. Both genera were found in cover brine samples with pH values at 4.04 ±â€¯0.15. We aim at elucidating whether the low relative abundance of non-LAB in commercial cucumber fermentations, in particular Pseudomonas and Enterobacteriaceae, impacts the quality of fermented cucumbers.

Evaluation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for differentiation of Pichia kluyveri strains isolated from traditional fermentation processes.

RATIONALE: Non-Saccharomyces yeasts are widespread microorganisms that nowadays have gained importance for their ability to produce volatile compounds which in alcoholic beverages improve aromatic complexity and therefore the overall quality. Their rapid identification and differentiation in fermentation processes is vital for timely decision making. METHODS: A total of 19 strains of Pichia kluyveri isolated from mezcal, tejuino and cacao fermentations were analyzed with rep-PCR fingerprinting using the primer (GTG)5 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Microflex LT mass spectrometer using Biotyper 3.1 software (Bruker Daltonics). RESULTS: The comparative analysis between MS spectra and rep-PCR patterns obtained from these strains showed a high similarity between both methods. However, minimal differences between the obtained rep-PCR and MALDI-TOF MS clusters could be observed, especially by the presence and/or absence of one or more discriminating peaks even when they have similarities in their main spectra projection, observing that isolates from the same fermentative process were grouped into the same sub-cluster based on their MALDI-TOF MS profiles. CONCLUSIONS: The data shown suggests that MALDI-TOF MS is a promising alternative technique for rapid, reliable and cost-effective differentiation of native yeast strains isolated from different traditional fermented foods and beverages.

Antagonistic trait of Staphylococcus succinus strain AAS2 against uropathogens and assessment of its in vitro probiotic characteristics.

The desideratum aim of the present context was to isolate a promising antagonist probiotic bacterium from fermented food item as biocontrol agent against uropathogens. Among diversified isolates evaluated for antagonistic trait, Staphylococcus succinus strain AAS2 was found to be an auspicious candidate against urinary tract infection (UTI) causing bacterial pathogens, being the most active against Staphylococcus aureus with substantial activity of 352.5 ±â€¯5.4 AU/mL. Further, the in vitro probiotic attributes of strain AAS2 were assessed using systematic methodology. The isolate exhibited tolerance to acidic condition (up to pH 3.0) and simulated gastric juice (at pH 3.0) with fairly high survival logarithmic cell counts of 5.3 ±â€¯0.15 and 5.23 ±â€¯0.02 log cfu/mL, respectively. Additionally, strain AAS2 showed capability to resist 0.5% w/v bile salt too. It also revealed significant values of auto-aggregation (32.5 ±â€¯1.3-56.5 ±â€¯1.4%) and cell surface hydrophobicity (38.35 ±â€¯1.4%) properties. The isolate showed resistivity towards phenol (6.8 ±â€¯0.08 log cfu/mL) and lysozyme (58.6 ±â€¯1.6%). Further, the susceptibility trait of strain AAS2 to conventional antibiotics made this isolate a promising probiotic bacterium. Most importantly, the isolate depicted DPPH (2,2-Diphenyl-1-picrylhydrazyl) and hydroxyl radical scavenging activities in a concentration dependent manner, thereby exhibiting its propitious antioxidative properties. In a nutshell, the outcomes of this investigation divulge the plausible use of S. succinus strain AAS2 as biocontrol agent against uropathogens, and recommended further applications in pharmaceutics due to its pronounced probiotic traits.

Quality characteristics of sauerkraut fermented by using a Lactobacillus paracasei starter culture grown in tofu whey.

The quality parameters of sauerkraut fermented using Lactobacillus paracasei in terms of its lactic acid bacteria count, texture, colour and biochemical properties were studied. As a starter culture L. paracasei grown in tofu whey was used for sauerkraut fermentation. The experiments were planned using central composite rotatable design of response surface methodology for input variables - culture volume (ml), fermentation time (days) and salt concentration (g/100 g). The linear and interactive effect of variables on responses was understood by statistically significant (p < 0.01) second-order models. Amongst all the input variables culture volume was found to have an overwhelming effect over all the responses. There was a significant (p < 0.01) increase in the lactic acid bacteria count of finished product; it was less hard but there was a departure in colour from the traditional product. The optimized condition for sauerkraut fermentation in terms of culture volume (ml), fermentation time (day) and salt concentration (g/100 g) was 30 ml, 28 days and 1 g/100 g, respectively. It was also observed that phenolics content was better in starter culture sauerkraut over the one traditionally prepared.

Application of hydrolases and probiotic Pediococcus acidilactici BaltBio01 strain for cereal by-products conversion to bioproduct for food/feed.

The aim of this study was to apply the enzymatic treatment and fermentation by Pediococcus acidilactici BaltBio01 strain for industrial cereal by-products conversion to food/feed bioproducts with high amount of probiotic lactic acid bacteria (LAB). LAB propagated in potato media and spray-dried remained viable during 12 months (7.0 log10 cfu/g) of storage and was used as a starter for cereal by-products fermentation. The changes of microbial profile, biogenic amines (BAs), mycotoxins, lactic acid (L+/D-), lignans and alkylresorcinols (ARs) contents in fermented cereal by-product were analysed. Cereal by-products enzymatic hydrolysis before fermentation allows to obtain a higher count of LAB during fermentation. Fermentation with P. acidilactici reduce mycotoxins content in fermented cereal by-products. According to our results, P. acidilactici multiplied in potato juice could be used for cereal by-products fermentation, as a potential source to produce safer food/feed bioproduct with high amount of probiotic LAB for industrial production.

In vitro assessment of Pediococcus acidilactici Kp10 for its potential use in the food industry.

BACKGROUND: Selection of a microbial strain for the incorporation into food products requires in vitro and in vivo evaluations. A bacteriocin-producing lactic acid bacterium (LAB), Pediococcus acidilactici Kp10, isolated from a traditional dried curd was assessed in vitro for its beneficial properties as a potential probiotic and starter culture. The inhibitory spectra of the bacterial strain against different gram-positive and gram-negative bacteria, its cell surface hydrophobicity and resistance to phenol, its haemolytic, amylolytic and proteolytic activities, ability to produce acid and coagulate milk together with its enzymatic characteristics and adhesion property were all evaluated in vitro. RESULTS: P. acidilactici Kp10 was moderately tolerant to phenol and adhere to mammalian epithelial cells (Vero cells and ileal mucosal epithelium). The bacterium also exhibited antimicrobial activity against several gram-positive and gram-negative food-spoilage and food-borne pathogens such as Listeria monocytgenes ATCC 15313, Salmonella enterica ATCC 13311, Shigella sonnei ATCC 9290, Klebsiella oxytoca ATCC 13182, Enterobacter cloaca ATCC 35030 and Streptococcus pyogenes ATCC 12378. The absence of haemolytic activity and proteinase (trypsin) and the presence of a strong peptidase (leucine-arylamidase) and esterase-lipase (C4 and C8) were observed in this LAB strain. P. acidilactici Kp10 also produced acid, coagulated milk and has demonstrated proteolytic and amylolactic activities. CONCLUSION: The properties exhibited by P. acidilactici Kp10 suggested its potential application as probiotic and starter culture in the food industry.