Transcriptome Analysis Reveals the Role of Sucrose in the Production of Latilactobacillus sakei L3 Exopolysaccharide.

Latilactobacillus (L.) sakei is a species of lactic acid bacteria (LAB) mostly studied according to its application in food fermentation. Previously, L. sakei L3 was isolated by our laboratory and possessed the capability of high exopolysaccharide (EPS) yield during sucrose-added fermentation. However, the understanding of sucrose promoting EPS production is still limited. Here, we analyzed the growth characteristics of L. sakei L3 and alterations of its transcriptional profiles during sucrose-added fermentation. The results showed that L. sakei L3 could survive between pH 4.0 and pH 9.0, tolerant to NaCl (<10%, w/v) and urea (<6%, w/v). Meanwhile, transcriptomic analysis showed that a total of 426 differentially expressed genes and eight non-coding RNAs were identified. Genes associated with sucrose metabolism were significantly induced, so L. sakei L3 increased the utilization of sucrose to produce EPS, while genes related to uridine monophosphate (UMP), fatty acids and folate synthetic pathways were significantly inhibited, indicating that L. sakei L3 decreased self-growth, substance and energy metabolism to satisfy EPS production. Overall, transcriptome analysis provided valuable insights into the mechanisms by which L. sakei L3 utilizes sucrose for EPS biosynthesis. The study provided a theoretical foundation for the further application of functional EPS in the food industry.

Production and SERS characterization of bacteriocin-like inhibitory substances by latilactobacillus sakei in whey permeate powder: exploring natural antibacterial potential.

Bacteriocins are antimicrobial compounds that have awakened interest across several industries due to their effectiveness. However, their large-scale production often becomes unfeasible on an industrial scale, primarily because of high process costs. Addressing this challenge, this work analyzes the potential of using low-cost whey permeate powder, without any supplementation, to produce bacteriocin-like inhibitory substances (BLIS) through the fermentation of Latilactobacillus sakei. For this purpose, different concentrations of whey permeate powder (55.15 gL-1, 41.3 gL-1 and 27.5 gL-1) were used. The ability of L. sakei to produce BLIS was evaluated, as well as the potential of crude cell-free supernatant to act as a preservative. Raman spectroscopy and surface-enhanced Raman scattering (SERS) provided detailed insights into the composition and changes occurring during fermentation. SERS, in particular, enhanced peak definition significantly, allowing for the identification of key components, such as lactose, proteins, and phenylalanine, which are crucial in understanding the fermentation process and BLIS characteristics. The results revealed that the concentration of 55.15 gL-1 of whey permeate powder, in flasks without agitation and a culture temperature of 32.5 °C, presented the highest biological activity of BLIS, reaching 99% of inhibition of Escherichia coli and Staphylococcus aureus with minimum inhibitory concentration of 36-45%, respectively. BLIS production began within 60 h of cultivation and was associated with class II bacteriocins. The results demonstrate a promising approach for producing BLIS in an economical and environmentally sustainable manner, with potential implications for various industries.

A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.

We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

Antioxidant and Antibacterial Activities of Nano-probiotics Versus Free Probiotics Against Gastrointestinal Pathogenic Bacteria.

Antibiotic-resistant pathogenic bacteria and the oxidative stress related to their infections are dangerous health problems. Finding new safe, effective antibacterial and antioxidant agents is an urgent global need. Probiotics are a strong candidate for possible antibacterial and antioxidant agents. The delivery of these probiotics without any effect on gastrointestinal digestion is the most important point for their application. The encapsulation of the probiotics on nanoparticles or other supports is a well-known method for the safe delivery of the probiotics. Little information is known about the effect of the probiotic encapsulation on its antibacterial and antioxidant activity. The present study tried to investigate the effect of probiotic encapsulation on nano-chitosan on its antioxidant activity and antibacterial activity against some pathogenic bacteria. We encapsulated some known probiotic species on nano-chitosan and investigated the antibacterial activity of the nano-probiotics and free probiotics against gastrointestinal pathogenic bacteria. The antioxidant characters of the free and encapsulated probiotics were investigated in terms of DPPH radicle scavenging activity, ferric ion chelating activity, hydroxyl radicle scavenging activity, superoxide anion radicle scavenging activity, and anti-lipid peroxidation activity. Results showed the superiority of the encapsulated probiotics as antibacterial and antioxidant agents over the free ones. The encapsulation improved the antibacterial activity of Sporolactobacillus laevolacticus against Bacteroides fragilis by 134% compared to the free one. Also, significantly, the encapsulation increased the hydroxyl radicle scavenging activity of Enterococcus faecium by about 180% compared to the free one. Nano-chitosan encapsulation synergistically increased the antioxidant and antibacterial activity of the studied probiotics. This can be promising for controlling pathogenic bacteria. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01140-2.

Heat-Treated Paraprobiotic Latilactobacillus sakei KU15041 and Latilactobacillus curvatus KU15003 Show an Antioxidant and Immunostimulatory Effect.

The lactic acid bacteria, including Latilactobacillus sakei and Latilactobacillus curvatus, have been widely studied for their preventive and therapeutic effects. In this study, the underlying mechanism of action for the antioxidant and immunostimulatory effects of two strains of heat-treated paraprobiotics was examined. Heat-treated L. sakei KU15041 and L. curvatus KU15003 showed higher radical scavenging activity in both the 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assays than the commercial probiotic strain LGG. In addition, treatment with these two strains exhibited immunostimulatory effects in RAW 264.7 macrophages, with L. curvatus KU15003 showing a slightly higher effect. Additionally, they promoted phagocytosis and NO production in RAW 264.7 cells without any cytotoxicity. Moreover, the expression of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 was upregulated. These strains resulted in an increased expression of inducible nitric oxide synthase and cyclooxygenase-2. Moreover, the nuclear factor-κB and mitogen-activated protein kinase signaling pathways were stimulated by these strains. These findings suggest the potential of using L. sakei KU15041 and L. curvatus KU15003 in food or by themselves as probiotics with antioxidant and immune-enhancing properties.

Spotlight on autochthonous microbiota, morpho-textural characteristics, and volatilome of a traditional Polish cold-smoked raw sausage.

The aim of the present study was to obtain information on the bacterial diversity of traditional Polish cold-smoked raw sausages (Kielbasa polska wedzona) manufactured by two artisanal producers using different selective growth media and a metataxonomic analysis. Physico-chemical and morpho-textural characteristics were also carried out, together with Microextraction-Gas Chromatography/Mass Spectrometry (HS-SPMEGC/MS) to study the volatile organic compounds (VOCs). The results overall obtained allowed a picture of the microbiota, the morpho-textural characteristics, and the volatilome of traditional Polish cold-smoked raw sausages (Kielbasa polska wedzona) to be drawn for the first time. In more detail, viable counting revealed active populations of presumptive lactobacilli, enterococci, coagulase-negative cocci, and a few spoilage microorganisms typically occurring in raw meat products. The metataxonomic analysis revealed the dominance of Latilactobacillus sakei occurring with a relative frequency between 77% and 89%. Pediococcus pentosaceus, Weissella hellenica, and Leuconostoc carnosum were detected among the minority taxa. In the sausages herein studied, no histamine levels of concern were detected. The Principal Component Analysis (PCA) performed on the Amplicon Sequence Variants (ASVs) did not show significant differences in the microbiota composition among producers. The HS-SPMEGC/MS analysis allowed the detection and identification of more than 90 volatile components belonging to ten main classes, namely: aldehydes, ketones, esters and acetates, acids, alcohols, phenols, furans, sulphur compounds, terpenoids, and benzene derivatives. The detected VOCs originated from spices, smoke, and microbial metabolism. The PCA of volatile compounds allowed differences between the sausage samples of the two producers to be identified.

Heat-Killed Latilactobacillus sakei CNSC001WB and Lactobacillus pentosus WB693 Have an Anti-inflammatory Effect on LPS-Stimulated RAW 264.7 Cells.

Excessive inflammatory results, such as those seen in rheumatoid arthritis and cardiovascular diseases, are known to cause various complications. Therefore, we aimed to investigate whether heat-killed Latilactobacillus sakei CNS001WB and Lactobacillus pentosus WB693 can prevent inflammatory reactions. When LPS-stimulated RAW 264.7 cells were handled with either heat-killed Lact. sakei CNSC001WB or Lact. pentosus WB693, the production of nitric oxide reduced. Furthermore, the expression of cyclooxygenase (COX)-2 and proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and IL-6, was suppressed. The expression of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), which play important roles in inflammatory diseases, especially arthritis, was also reduced. Moreover, these strains inhibited nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, which activate various cytokines and inflammatory mediators. Additionally, heat-killed Lact. sakei CNSC001WB and Lact. pentosus WB693 inhibited the reactive oxygen species (ROS) production. Based on these results, we concluded that heat-killed Lact. sakei CNSC001WB and Lact. pentosus WB693 sufficiently inhibited the inflammatory response and may have anti-inflammatory potential.

The Effect of Ophthalmic and Systemic Formulations of Latilactobacillus sakei on Clinical and Immunological Outcomes of Patients With Dry Eye Disease: A Factorial, Randomized, Placebo-controlled, and Triple-masking Clinical Trial.

Dry eye disease (DED) is one of the most prevalent eye diseases. This study aimed to evaluate the efficacy and safety of Latilactobacillus sakei (L. sakei) either as an ophthalmic bacterial lysate (drops, no live organism) or as an oral probiotic (capsules) on immunological and clinical outcomes of patients with DED. This study was a randomized, placebo-controlled, triple-masking clinical trial with four parallel arms. Patients were randomly assigned in a 2x2 factorial design combining active vs placebo capsules and active vs placebo eye drops in a 1:1x1:1 ratio. The ophthalmic drops are approved for use in the European Union as a medical device (CE registration code 0425-MED-004235). A total of 40 patients were evaluated. DED signs and symptoms decreased significantly by using active drops compared to placebo, as measured by the Ocular Surface Disease Index (OSDI), Tear Break-up Time (TBUT), and Schirmer I tests (all p<0.0001). Conversely, neither active capsules nor their interaction effect with active drops achieved significance vs placebo. There was also a significant decrease in the tear levels of IL-6 (p=0.0007), TNFα (p<0.0001), and IFNγ (p<0.0001) in patients receiving active drops. Intake of both active products (drops and capsules) was well tolerated. Postbiotic ophthalmic formulation containing L. sakei lysate significantly improved the signs and symptoms of DED and suppressed ocular surface inflammatory response. Conversely, oral intake of L.sakei as a probiotic capsule had no effect in these patients (ClinicalTrials.gov: NCT04938908).

Phylogenomic analysis in Latilactobacillus sakei by using polymorphisms detected by next-generation sequencing.

Latilactobacillus sakei is a lactic acid bacterium used to produce a wide range of fermented food products. To understand their characteristics and adaptability to various nutrient sources, we applied strain-specific, nucleotide-concatenated (SSC) sequences to the phylogenetic analysis of 32 L. sakei strains isolated from various locations and products. SSC sequences were developed by concatenating the polymorphisms detected by whole-genome sequencing. This enabled us to use sufficient polymorphisms and avoid the bias caused by selecting partial sequences, such as that in core genome and multi-locus sequence typing. SSC sequence-based analysis revealed that the phylogenetic relations for L. sakei are based on the different nutrition sources rather than geographical distance.

Latilactobacillus sakei Wikim0066 Protects Skin through MMP Regulation on UVB-Irradiated In Vitro and In Vivo Model.

Ultraviolet (UV) B exposure induces wrinkle formation, collagen fiber breakdown, and transepidermal water loss (TEWL). UVB irradiation induces the expression of mitogen-activated protein kinase (MAPK), activator protein 1 (AP-1), and nuclear factor kappa B (NF-κB), which affect the expression of matrix metalloproteinases (MMP). We confirmed the effects of Latilactobacillus sakei wikim0066 (wikim0066) on UVB-irradiated Hs68 cells and HR-1 hairless mice cells. wikim0066 restored the production of type I procollagen by regulating the expression of MMP-1 and -3, MAPK, AP-1, and NF-κB in UVB-irradiated Hs68 cells and HR-1 mice. Oral administration of wikim0066 alleviates wrinkle formation, epidermal thickness, and TEWL in UVB-irradiated HR-1 hairless mice. These results indicated that wikim0066 has the potential to prevent UVB-induced wrinkle formation.

Antioxidant Effects and Probiotic Properties of Latilactobacillus sakei MS103 Isolated from Sweet Pickled Garlic.

Fermented vegetable-based foods, renowned for their unique flavors and human health benefits, contain probiotic organisms with reported in vitro antioxidative effects. This study investigates the probiotic properties of Latilactobacillus sakei MS103 (L. sakei MS103) and its antioxidant activities using an in vitro oxidative stress model based on the hydrogen peroxide (H2O2)-induced oxidative damage of RAW 264.7 cells. L. sakei MS103 exhibited tolerance to extreme conditions (bile salts, low pH, lysozyme, H2O2), antibiotic sensitivity, and auto-aggregation ability. Moreover, L. sakei MS103 co-aggregated with pathogenic Porphyromonas gingivalis cells, inhibited P. gingivalis-induced biofilm formation, and exhibited robust hydrophobic and electrostatic properties that enabled it to strongly bind to gingival epithelial cells and HT-29 cells for enhanced antioxidant effects. Additionally, L. sakei MS103 exhibited other antioxidant properties, including ion-chelating capability and the ability to effectively scavenge superoxide anion free radicals, hydroxyl, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, and 2,2-diphenyl-1-picrylhydrazyl. Furthermore, the addition of live or heat-killed L. sakei MS103 cells to H2O2-exposed RAW 264.7 cells alleviated oxidative stress, as reflected by reduced malondialdehyde levels, increased glutathione levels, and the up-regulated expression of four antioxidant-related genes (gshR2, gshR4, Gpx, and npx). These findings highlight L. sakei MS103 as a potential probiotic capable of inhibiting activities of P. gingivalis pathogenic bacteria and mitigating oxidative stress.

Intraspecific difference of Latilactobacillus sakei in inflammatory bowel diseases: Insights into potential mechanisms through comparative genomics and metabolomics analyses.

Inflammatory bowel diseases (IBDs) are chronic inflammatory diseases of the gastrointestinal tract that have become a global health burden. Studies have revealed that Latilactobacillus sakei can effectively alleviate various immune diseases, including colitis, rheumatoid arthritis, and metabolic disorders. Here, we obtained 72 strains of L. sakei from 120 fermentation and fecal samples across China. In total, 16 strains from different sources were initially screened in an in vitro Caco-2 model induced by dextran sulfate sodium. Subsequently, six strains (four exhibiting effectiveness and two exhibiting ineffectiveness) were selected for further validation in an in vivo colitis mouse model. The results demonstrated that L. sakei strains exhibited varying degrees of amelioration of the colitis disease process. Notably, L. sakei CCFM1267, the most effective strain, significantly restored colon length and tight-junction protein expression, and reduced the levels of cytokines and associated inflammatory enzymes. Moreover, L. sakei CCFM1267 upregulated the abundance of Enterorhabdus, Alloprevotella, and Roseburia, leading to increased levels of acetic acid and propionic acid. Conversely, the other four strains (L. sakei QJSSZ1L4, QJSSZ4L10, QGZZYRHMT1L6, and QGZZYRHMT2L6) only exhibited a partial remission effect, while L. sakei QJSNT1L10 displayed minimal impact. Therefore, L. sakei CCFM1267 and QJSNT1L10 were selected for further exploration of the mechanisms underlying their differential mitigating effects. Comparative genomics analysis revealed significant variations between the two strains, particularly in genes associated with carbohydrate-active enzymes, such as the glycoside hydrolase family, which potentially contribute to the diverse profiles of short-chain fatty acids in vivo. Additionally, metabolome analysis demonstrated that acetylcholine and indole-3-acetic acid were the main differentiating metabolites of the two strains. Therefore, the strains of L. sakei exhibited varying degrees of effectiveness in alleviating IBD-related symptoms, and the possible reasons for these variations were attributed to discrepancies in the carbohydrate-active enzymes and metabolites among the strains.

Distribution, inducibility, and characterisation of prophages in Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria (LAB) are used as starters in a wide variety of food fermentations. While the number of reports of phages infecting other LAB steadily increased over the years, information about phage associated with Latilactobacillus sakei, a frequently used meat starter, remains scarce. RESULTS: In this study, a predictive genomic analysis of 43 Latilactobacillus sakei genomes revealed the presence of 26 intact, eleven questionable and 52 incomplete prophage sequences across all analysed genomes with a range of one to five predicted prophage sequences per strain. Screening 24 sakei strains for inducible prophages by utilising UV light or mitomycin C, we identified seven lysogenic strains showing lysis after induction during subsequent growth monitoring. Electron microscopic analysis revealed fully assembled virions in the purified lysates of four samples, thus confirming successful prophage induction. All virions featured icosahedral, isomeric heads and long, most likely non-contractile tails indicating siphoviruses. By performing phylogenetic analyses with various marker genes as well as full prophage sequences, we displayed a remarkably high diversity of prophages, that share a similar gene module organisation and six different chromosomal integration sites were identified. By sequencing viral DNA purified from lysates of Latilactobacillus sakei TMW 1.46, we demonstrate that simultaneous induction of multiple prophages is possible. CONCLUSIONS: With this work, we not only provide data about the incidence of prophages harboured by the meat starter Latilactobacillus sakei, we also demonstrated their potential to impact growth of their host after induction, as well as forming seemingly fully assembled virions.

Evaluation of metabolic activities and probiotic characteristics of two Latilactobacillus sakei strains isolated from pastirma.

The current study was carried out to investigate metabolic activities and main probiotic characteristics of two Latilactobacillus sakei strains (8.P1 and 28.P2) isolated from pastirma, a highly seasoned, air-dried cured beef. Both strains showed antimicrobial activity against important foodborne pathogens like Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa and so forth. For the characterization of antimicrobial activity, the effect of various enzymes, temperature and pH were tested. The results of the tests demonstrated that the antimicrobial activity of strains was based on the production of protein-structured compounds such as bacteriocin or bacteriocin like peptides. In metabolic activity studies, amounts of the lactic acid, proteolytic activity and hydrogen peroxide produced by the 8.P1 and 28.P2 were found to range between 16.09 and 17.32 mg/mL, 0.24 and 0.04 mg/mL and 0.98 and 0.04 µg/mL, respectively. It was also observed that neither strain could produce exopolysaccharide. Both strains were found susceptible to vancomycin, chloramphenicol, gentamicin, tetracycline, and netilmicin sulfate. When the strains are evaluated with respect to their probiotic potential, 28.P2 could tolerate acidic conditions, but 8.P1 showed sensitivity. The survival rate of the strains in the simulated gastric juice and their adhesion abilities were found suitable to stay alive in the gastrointestinal tract and to proliferate in the intestine. The evaluation of all the features of both strains demonstrated that both strains had the potential to be used as a protective culture. In addition, it was observed that 8.P1 and 28.P2 were more suitable as a starter culture and a probiotic candidate respectively.

Control of Listeria monocytogenes in chicken dry-fermented sausages with bioprotective starter culture and high-pressure processing.

Listeria monocytogenes is one of the most relevant pathogens for ready-to-eat food, being a challenge for the food industry to comply with microbiological criteria. The aim of the work was to assess the behavior of L. monocytogenes in two types of chicken-based dry-fermented sausages during the fermentation and ripening, with or without a bioprotective starter culture (Latilactobacillus sakei CTC494). To complement the challenge testing approach, simulations with different predictive models were performed to better understand the role of contributing factors. The impact of post-processing strategies, such as high-pressure processing and/or corrective storage was assessed. The chicken meat was inoculated with a cocktail of three L. monocytogenes strains, mixed with other ingredients/additives and stuffed into small (snack-type) or medium (fuet-type) casings. Snack-type was fermented (22°C/3 days) and ripened (14°C/7 days), while fuet-type was ripened (13°C/16 days). At the end of ripening, HPP (600 MPa/5 min) and/or corrective storage (4 or 15°C/7 days) were applied. The suitability of HPP after fermentation was evaluated in the snack-type sausages. Pathogen growth (>3 Log10) was observed only during the fermentation of the snack type without a starter. The bioprotective starter prevented the growth of L. monocytogenes in the snack-type sausages and enhanced the inactivation (1.55 Log10) in fuet-type sausages, which could be related to the higher lactic acid production and consequent decrease of pH, but also the production of the antilisterial bacteriocin sakacin k. The gamma concept model allowed us to identify the main factors controlling the L. monocytogenes' growth, i.e., the temperature during the early stages and a w at the end of the production process. The earlier acidification linked with the addition of starter culture made the interaction with the other factors (undissociated lactic acid, a w and temperature) to be the growth-preventing determinants. High-pressure processing only caused a significant reduction of L. monocytogenes in snack-type, which showed higher a w . The application of HPP after fermentation did not offer a relevant advantage in terms of efficacy. Corrective storage did not promote further pathogen inactivation. The findings of the work will guide the food industry to apply effective strategies (e.g., fermentation temperature and bioprotective starter cultures) to control L. monocytogenes in chicken dry-fermented sausages.

Latilactobacillus sakei: a candidate probiotic with a key role in food fermentations and health promotion.

Latilactobacillus sakei is used extensively in industrial production and food fermentations. The species is primarily derived from fermented meat and vegetable products and is also found in human feces. Genomics and metabolomics have revealed unique metabolic pathways in L. sakei and molecular mechanisms underlying its competitive advantages in different habitats, which are mostly attributed to its flexible carbohydrate metabolism, cold tolerance, acid and salt tolerance, ability to cope with oxygen changes, and heme uptake. In recent years, probiotic effects of L. sakei and its metabolites have been identified, including the ability to effectively alleviate metabolic syndrome, inflammatory bowel disease, and atopic dermatitis. This review summarizes the genomic and metabolic characteristics of L. sakei and its metabolites and describes their applications, laying a foundation for their expanded use across the food and healthcare industries.

Unfolding microbiota and volatile organic compounds of Portuguese Painho de Porco Preto fermented sausages.

In the present study, bacterial and fungal diversity, as well as volatile profiles, of ready-to-eat Portuguese Painho de Porco Preto fermented sausages manufactured by two artisanal producers in the districts of Beja (producer A) and Evora (producer B) were studied. To this end, different selective growth media and a metataxonomic analysis were combined with Headspace Solid-Phase Microextraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS) analysis. The results of the microbiological viable counts revealed active microbial populations of lactic acid bacteria (up to 8 Log cfu g-1), coagulase negative cocci (up to 6 Log cfu g-1), and eumycetes (up to 6 Log cfu g-1). Bacterial populations were characterized by high relative frequencies of Latilactobacillus sakei (up to 72%), together with Weissella and Staphylococcus equorum. The mycobiota was mainly dominated by Debaryomyces hansenii (up to 55% of the relative frequency) and Kurtzmaniella zeylanoides (up to 24% of the relative frequency). Unexpected species as Wickerhamomyces subpelliculosus and Zygosaccharomyces rouxii were also detected. HS-SPME-GC/MS analysis allowed to identify a complex volatile profile, showing 159 volatile organic compounds (VOCs). VOCs belonged to twelve classes, such as aldehydes, ketones and lactones, esters and acetates, alcohols, terpenoids, sulfur compounds, aliphatic hydrocarbons, aromatic hydrocarbons, nitrogen compounds, acids, furans and pyrans, and phenols. The analysis of VOCs composition provided evidence that samples from the two producers (A and B) were different, as confirmed by the Principal Component Analysis. Hence, it is likely that the raw materials used, as well as variations related with the empirical practice of the butchers, strongly influenced the final product. The results obtained in the present study represent a further advancement in the knowledge on the biodiversity and VOCs composition of Portuguese fermented sausages. To better understand the interactions occurring between the autochthonous microorganisms and the meat batter in the Painho de Porco Preto fermented sausage, microbial and VOCs dynamics must be further deepened throughout the production process.

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

The use of fish flesh to produce fermented sausages is uncommon, especially in European countries where fermented sausages are mainly obtained using mammalian meat. In the present study, the microbiota naturally occurring in novel fermented fish sausages, handcrafted using marine fish species caught in the Mediterranean Sea, was studied. To this end, fish sausages were subjected to physico-chemical analyses (including histamine quantification). Microbiological traits of sausages were studied via viable counting and metataxonomic analysis. Sausages were also subjected to the detection of genes encoding histidine decarboxylase of both Gram-positive (hdcA) and -negative (hdc) bacteria. The results of histamine quantification showed different contents among fish sausage samples. Moreover, the presence of the hdcA gene was below the detection limit in all the samples, whereas the hdc gene was detected only in samples from batch 2, characterized by high levels of Enterobacteriaceae. In the analysed samples, viable lactic acid bacteria, coagulase-negative staphylococci, and eumycetes were detected. Bacterial composition displayed the highest frequency of Latilactobacillus sakei, whereas eumycetic composition displayed the highest frequency of Kurtzmaniella zeylanoides. In order to select potential adjunct cultures for product improvement, 60 lactic acid bacteria (22 isolates of L. sakei and 38 of Latilactobacillus curvatus) were isolated from sausage samples and characterized for: i) the presence of the hdcA gene; ii) the production of exopolysaccharides (EPS); iii) the presence of genes involved in the production of EPS; iv) the production of bacteriocins against Listeria innocua. None of the isolates tested positive for the presence of the hdcA gene. Moreover, 39 out of 60 isolates showed the formation of mucoid colonies, thus attesting the production of EPS. Interestingly, 56 out of 60 isolates were positive for the gene epsD/E, whereas 37 out of 60 isolates were positive for the gene epsA, all these genes encoding the production of heteropolysaccharides. Of note, the EPS production capability and the absence of hdcA gene could represent a starting point for future selection of the isolates as autochthonous adjunct cultures to improve texture, sensory traits and safety of the fermented fish sausages under study. None of the L. sakei or L. curvatus isolates exerted a bactericidal effect against L. innocua.

Unravelling microbial populations and volatile organic compounds of artisan fermented liver sausages manufactured in Central Italy.

The aim of the present study was to obtain information on the occurrence of bacteria and eumycetes in ready-to-eat fermented liver sausages manufactured by 20 artisan producers located in the Marche Region (Italy). To this end, culture-dependent analyses and metataxonomic sequencing were carried out. Physico-chemical parameters and volatilome of the fermented liver sausages were also studied. Finally, the presence of hepatitis E virus (HEV) was also assessed via real-time-RT-(q)PCR assays. Active microbial populations mainly represented by lactic acid bacteria, enterococci, coagulase-negative cocci, and eumycetes were detected. Enterobacteriaceae, Pseudomonadaceae, and sulfite-reducing anaerobes were not detected in most of the samples. Latilactobacillus sakei dominated in all the analyzed samples, reaching abundances up to 80%. Staphylococcus xylosus and Staphylococcus equorum were also detected. Among minority bacterial taxa, Weissella spp., Leuconostoc spp., Macrococcus caseolyticus, Brochothrix thermosphacta, Staphylococcus succinus, Lactobacillus coryniformis, Lactiplantibacillus plantarum, Lactococcus garviae, Psychrobacter spp., and Carnobacterium viridans were detected. The mycobiota was mainly composed by Debaryomyces hansenii that was present in all samples at the highest frequency. Among minority fungal taxa, Aspergillus spp., Penicillium spp., Kurtzmaniella zeylanoides, Candida spp., Yamadazyma spp., Scopulariopsis spp., Yarrowia spp., and Starmerella spp. were detected. Interestingly, associations between some taxa and some physico-chemical parameters were also discovered. The absence of HEV in all the samples attested a high level of safety. Finally, most of the VOCs detected in the analyzed fermented liver sausages belonged to six classes as: terpenoids, aldehydes, ketones, alcohols, esters, and acids. Nitrogen compounds, sulfur compounds, phenols, hydrocarbons, lactones, furans, and aromatic hydrocarbons were also identified. Several significant relationships were observed between mycobiota and VOCs.

Impact of different sugars and glycosyltransferases on the assertiveness of Latilactobacillus sakei in raw sausage fermentations.

Latilactobacillus sakei comprises a biodiversity of strains, which display different assertiveness upon their application as starter cultures in raw sausage fermentation. While the assertiveness of winning partner strains has been referred to competitive exclusion based on genomic settings enabling occupation of multiple niches of the sausage habitat, single strain assertiveness of L. sakei remained unexplained. In this study we assessed the impact of the expression of a glycosyltransferase enabling the production of a glucan from sucrose to the assertiveness of L. sakei TMW 1.411, which expresses a plasmid-encoded glycosyltransferase (gtf). In a sausage fermentation model wild type L. sakei TMW 1.411 and its plasmid-cured mutant were employed in competition with each other and with other Latilactobacillus sakei strains. To differentiate any effects resulting from general sugar utilization from those of glucan formation, the experiments were carried out with glucose, fructose, and sucrose, respectively. It was shown that the type of sugar affects the individual strains behaviour, and that the wild type was more competitive than the mutant in the presence of any of these sugars. In direct competition between wild type and mutant, a clear competitive advantage could also be demonstrated for the strain possessing the plasmid with the glycosyltransferase. Since this competitive advantage was observed with all sugars, not just sucrose, and Gtf expression has been shown as independent of the employed sugar, it is suggested that possession of the gtf gene-carrying plasmid confers a competitive advantage. It appears that the Gtf contributes to competitive exclusion and the establishment of colonization resistance, to a larger extent by an adhesive functionality of the Gtf on the cellular surface than by the production of glucan. Hence, gtf genes can be used as a possible additional marker for the selection of assertive L. sakei starter strains in sausage fermentation.