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The aim of this study was to formulate a Selenium (Se)-bioenriched fermented beverage using selenized lactic acid bacteria (LAB) with desirable sensory attributes and shelf-life. The fruit-origin strains Lactiplantibacillus paraplantarum CRL 2051 and Fructobacillus tropaeoli CRL 2034 were grown in MRS-fructose with 5 mg/L Se before inoculation. Then, the selenized strains were inoculated separately or together in a fruit juice and cowmilk beverage and allowed to ferment at 30 °C for 14 h. During microbial growth, the strains accumulated 62.8-93.5 µg/L of total Se, with 32.7-47.8 µg/L composed of the amino acids selenocysteine (SeCys), and 6.1-12.7 µg/L of selenomethionine (SeMet). The beverages fermented by L. paraplantarum CRL 2051 alone and by the mixed culture showed the highest levels of general acceptance and best sensory attributes. The latter fermented beverage exhibited high microbial resistance to cold storage after 52 days and to gastrointestinal tract conditions as well as an acceptable sensory shelf-life of 42 days. For the first time, microbial selenization previous to food fermentation successfully allowed Se fortification and the formulation of a functional Se-enriched beverage with desirable sensory properties and shelf-life. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05984-4.
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The present study focused on the antibacterial and antibiofilm activity of novel lactic acid bacterial (LAB) strains isolated from the healthy human volunteers of different age groups and their consortium (LABCON), against the enteropathogenic bacteria. From the study, methanolic extract of LAB isolates and their consortia were found to have promising antibacterial activity and antibiofilm activity against Escherichia coli (ATCC 35218) and Staphylococcus aureus (ATCC 25923). The antimicrobial compounds including the DL-3 phenyllactic acid, DL-p-hydroxyphenyllactic acid, and Succinic acid produced by the LAB could be considered to inhibit the growth and biofilm formation by E. coli (ATCC 35218) and S. aureus (ATCC 25923). Detailed insight into the antibiofilm activity could also be demonstrated by Confocal Raman microscopy attached with AFM and Fluorescent microscope. From the results of the study, the consortium LABCON was superior in antimicrobial and antibiofilm activity and can be considered to have promising application in infection control.
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Probiogenomics can provide important insights bout probiotic candidate bacteria. This study aimed to perform an in-depth genomic characterization of the probiotic candidate Lactiplantibacillus plantarum CNPC024 to investigate its probiosis mechanisms, identify metabolic pathways that might benefit the host, and improve the safety assessment for this strain to be effectively used as a probiotic. After whole-genome sequencing in Illumina MiSeq platform, the de novo genome assembly resulted in a 3.2 Mb draft genome. According to the Average Nucleotide Identity (ANI) analysis with 46 randomly validated probiotic LAB belonging to the Lactobacillaceae family, the strain showed a 99% nucleotide identity with other L. plantarum probiotic species. We identified a set of determinants conferring tolerance to bile salts and low pH conditions, as well as temperature, oxidative and osmotic stressors via the glutathione-glutaredoxin system (Grxs). As a ßgalactosidaseproducing strain, it has the potential to be used in fermented dairy products for lactose-intolerant individuals. There were no significant hits for transferable antibiotic-resistance genes. We also identified gene clusters associated with production of bacteriocins (plantaricins E, F and K). Lastly, we detected metabolic pathways associated with the production of tryptophan-derived metabolites that could potentially modulate the host's immune responses.
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In scenarios where yeast and bacterial cells coexist, it is of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared, mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries, and analyzed using bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter Counter (at 1:1 and 100:1 ratios). We observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter Counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (p > .05). In contrast, the Coulter Counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (p < .05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasn't possible due to an overlap between yeast cell debris and bacterial cells. We conclude that each method has limitations, advantages, and disadvantages. ONE-SENTENCE SUMMARY: This study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision.
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Microbiología Industrial , Saccharomyces cerevisiae , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/citología , Microbiología Industrial/métodos , Citometría de Flujo/métodos , Recuento de Colonia Microbiana/métodos , Carga Bacteriana/métodos , Saccharum/microbiología , Microscopía/métodosRESUMEN
This review aims to provide an overview of artisanal Mexican cheeses microbiota focused on microbiological quality and safety, as well as native Lactic acid Bacteria (LAB) diversity. For the search, key words of artisanal Mexican cheeses varieties was carried out through several online databases and original articles were screened and data about populations of indicator microorganisms, presence of pathogens, and native LAB identified were extracted. Several artisanal Mexican cheeses exceeded the permissible limit established in Mexican regulation (NOM-243-SSA1-2010) for indicator microorganisms, as well as in some types of cheese, the presence of pathogens was confirmed. However, other varieties of artisanal Mexican cheeses possess unique physicochemical characteristics, and during their manufacturing particular steps are used that contribute to ensuring their quality and safety. Additionally, strains able to control the growth of pathogenic and spoilage bacteria are part of the microbiota of some artisanal Mexican cheeses. About native LAB diversity, it is composed by species of Lactobacillus, Enterococcus, Streptococcus, Leuconostoc, Weisella, Lactococcus, Pediococus, Aerococus, Carnobacterium, Tetragenococus, among others genera. Otherwise, artisanal Mexican cheeses represent an important source of specific LAB with several approaches within human health because they showed potential for the development of functional foods, nutraceutical, and bioprotective cultures.
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Queso , Microbiología de Alimentos , Lactobacillales , Queso/microbiología , Lactobacillales/aislamiento & purificación , México , Biodiversidad , MicrobiotaRESUMEN
This study investigated the health-functional properties of a lactic fermented pomegranate juice (FPJ) enriched with pomegranate seed oil (FPJO) by using the fruit-origin strain Lactiplantibacillus paraplantarum CRL 2051 (FPJO-CRL2051). For this aim, the in vitro human antiplatelet aggregation effect and antioxidant activities were determined in the fermented juices while in vivo studies using high-fat-diet (HFD) C57BL/6 mice fed with a high-fat diet or pomegranate fermented juices for 8 weeks were performed. A high anti-platelet aggregation activity for FPJO-CRL2051 was determined. The formulated juice was administered to C57BL/6 HFD mice over 8 weeks, which showed a significant decrease in triglycerides, LDL-C, and pro-inflammatory cytokines levels. The FPJO-CRL2051 administration was effective in ameliorating liver damage caused by HFD, reducing fat accumulation and oxidative biomarkers, and improving the liver fatty acid profile by incorporation of conjugated fatty acids. This study shows the significance of lactic fermentation in developing novel fermented plant-based beverages with enhanced functional activities with a circular economy approach for the prevention of metabolic disorders.
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BACKGROUND: Consumption of pseudocereal-based foods decreased in phytate concentration can provide better nutrition concerning mineral bioavailability. This study aimed to evaluate the mineral bioavailability of quinoa sourdough-based snacks in a murine model. The mice were divided into five groups. One group was fed with basal snacks; three control groups received quinoa-based snacks made from non-fermented dough, dough without inoculum, and chemically acidified dough; and the test group (GF) received quinoa snacks elaborated from sourdough fermented by a phytase-positive strain, Lactiplantibacillus plantarum CRL 1964. Food intake, body weight, and mineral concentration in blood and organs (liver, kidney, and femur) were determined. RESULTS: Food consumption increased during the feeding period and had the highest (16.2-24.5%) consumption in the GF group. Body weight also increased during the 6-weeks of trial. The GF group showed higher (6.0-10.2%) body weight compared with the other groups from the fifth week. The concentrations of iron, zinc, calcium, magnesium, and phosphorus in blood, iron and phosphorus in the liver, manganese and magnesium in the kidney, and calcium and phosphorus in the femur increased significantly (1.1-2.7-fold) in the GF group compared to the control groups. CONCLUSION: The diet that includes quinoa snacks elaborated with sourdough fermented by phytase-positive strain L. plantarum CRL 1964 increased the concentrations of minerals in the blood, liver, kidney, and femur of mice, counteracting the antinutritional effects of phytate. This study demonstrates that the diminution in phytate content and the consequent biofortification in minerals are a suitable tool for producing novel foods. © 2024 Society of Chemical Industry.
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The traditional Mexican fermented beverage pulque has been considered a healthy product for treating gastrointestinal disorders. Lactic acid bacteria (LAB) have been identified as one of the most abundant microbial groups during pulque fermentation. As traditional pulque is consumed directly from the fermentation vessel, the naturally associated LABs are ingested, reaching the consumer's small intestine alive, suggesting their potential probiotic capability. In this contribution, we assayed the probiotic potential of the strain of Lactiplantibacillus plantarum LB1_P46 isolated from pulque produced in Huitzilac, Morelos State, Mexico. The characterization included resistance to acid pH (3.5) and exposure to bile salts at 37 °C; the assay of the hemolytic activity and antibiotic resistance profiling; the functional traits of cholesterol reduction and ß-galactosidase activity; and several cell surface properties, indicating that this LAB possesses probiotic properties comparable to other LAB. Additionally, this L. plantarum showed significance in in vitro antimicrobial activity against several Gram-negative and Gram-positive bacteria and in vivo preventive anti-infective capability against Salmonella in a BALB/c mouse model. Several functional traits and probiotic activities assayed were correlated with the corresponding enzymes encoded in the complete genome of the strain. The genome mining for bacteriocins led to the identification of several bacteriocins and a ribosomally synthesized and post-translationally modified peptide encoding for the plantaricin EF. Results indicated that L. plantarum LB1_P46 is a promising probiotic LAB for preparing functional non-dairy and dairy beverages.
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Amid the current environmental crisis caused by plastic accumulation, one of the proposed solutions to manage this problem is using biodegradable polymers. However, the impact of adding biodegradable polymers to the well-established circular economy of recyclable polymers, such as HDPE, has not been fully considered. Therefore, there is a need to reconsider the way we consume, dispose of, and manage biodegradable polymers after use. This study evaluates the effect of varying the contents of a biodegradable polymer, taking poly(lactic acid) (PLA) as a model biodegradable polymer, on the thermal and mechanical properties of HDPE. The study highlights the importance of identifying and disposing of biodegradable polymers to avoid mixtures with HDPE, in order not to affect mechanical performance when considering reprocessing and a new life cycle of this conventional polymer.
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Microorganisms with the ability to modulate the immune system (immunobiotics) have shown to interact with different pattern recognition receptors (PRRs) expressed in nonimmune and immune cells and exert beneficial effects on host's health maintenance and promotion. Suitable assay systems are necessary for an efficient and rapid screening of potential immunobiotic strains. More than a decade of research has allowed us to develop efficient in vitro models based on porcine receptors and cells (porcine immunoassay systems) to study the immunomodulatory effects of lactic acid bacteria (LAB). In addition, detailed studies of model immunobiotic LAB strains with proved abilities to improve immune health in humans (Lactobacillus rhamnosus CRL1505) or pigs (Lactobacillus jensenii TL2937) allowed us to select the most suitable biomarkers that have to be evaluated in those porcine immunoassay systems. Our in vitro models, utilizing transfectant cells expressing PRRs along with an established porcine intestinal epitheliocyte (PIE) cell line, have proven to be valuable tools for immunobiotic selection and for gaining insights into the molecular mechanisms responsible for their beneficial effects.
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Lactobacillales , Animales , Porcinos , Inmunoensayo/métodos , Lactobacillales/inmunología , Probióticos , Línea Celular , Humanos , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Reconocimiento de Patrones/inmunología , Lactobacillus/inmunologíaRESUMEN
Fermenting fruit juices with lactic acid bacteria (LAB) is a sustainable method to enhance fruit harvests and extend shelf life. This study focused on blackberries, rich in antioxidants with proven health benefits. In this research, we examined the effects of fermentation (48 h at 37 °C) at 28 days on whey-supplemented (WH, 1:1) blackberry juice (BJ) inoculated with two LAB mixtures. Consortium 1 (BJWH/C1) included Levilactobacillus brevis, Lactiplantibacillus plantarum, and Pediococcus acidilactici, while consortium 2 (BJWH/C2) comprised Lacticaseibacillus casei and Lacticaseibacillus rhamnosus. All of the strains were previously isolated from aguamiel, pulque, and fermented milk. Throughout fermentation and storage, several parameters were evaluated, including pH, lactic acid production, viscosity, stability, reducing sugars, color, total phenolic content, anthocyanins, and antioxidant capacity. Both consortia showed a significant increase in LAB count (29-38%) after 16 h. Sample BJWH/C2 demonstrated the best kinetic characteristics, with high regression coefficients (R2 = 0.97), indicating a strong relationship between lactic acid, pH, and fermentation/storage time. Despite some fluctuations during storage, the minimum LAB count remained at 9.8 log CFU/mL, and lactic acid content increased by 95%, with good storage stability. Notably, sample BJWH/C2 increased the total phenolic content during storage. These findings suggest that adding whey enhances biomass and preserves physicochemical properties during storage.
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Antioxidantes , Fermentación , Jugos de Frutas y Vegetales , Lactobacillales , Suero Lácteo , Antioxidantes/metabolismo , Suero Lácteo/metabolismo , Suero Lácteo/química , Suero Lácteo/microbiología , Jugos de Frutas y Vegetales/microbiología , Jugos de Frutas y Vegetales/análisis , Lactobacillales/metabolismo , Rubus/química , Rubus/metabolismo , Rubus/microbiología , Almacenamiento de Alimentos/métodos , Concentración de Iones de Hidrógeno , Consorcios Microbianos/fisiología , Ácido Láctico/metabolismoRESUMEN
Background and Aim: The feeding and sanitary conditions significantly influence the productivity of farm animals. This study aimed to assess the impact of a lactic acid-producing microbial additive on broiler chicken productivity. Materials and Methods: A 42-day experimental period utilized 120 1-day-old Cobb 500 chicks with an average weight of 46 g. In groups of 30 each, the chicks were randomly assigned to four experimental designs. The following treatments were assessed: T1 without intervention (control), T2 with bacitracin at a concentration of 0.5 g/L, T3 with a 5% probiotic mixture (PM), and T4 with a 7.5% PM. The birds were fed the commercial balanced feed without anticoccidials daily, while vaccines were administered according to the recommended biosecurity plan by the commercial house. Drinking water was treated with PM containing lactobacilli, yeasts, and short-chain organic acids. Result: In T4, a 7.5% PM resulted in a final weight of 2361.2 g (p < 0.05), a total weight gain of 1412.8 g (p < 0.05), and improved feed efficiency with a feed conversion of 2.00 (p < 0.05), during which feed intake was lower than in the other groups. Conclusion: Microbial additives with lactic acid activity are a cost-effective and feasible solution for broiler chicken productivity.
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Alterations of the microbiota-gut-brain axis has been associated with intestinal and neuronal inflammation in Parkinson's disease (PD). The aim of this work was to study some mechanisms associated with the neuroprotective effect of a combination (MIX) of lactic acid bacteria (LAB) composed by Lactiplantibacillus plantarum CRL2130 (riboflavin overproducing strain), Streptococcus thermophilus CRL808 (folate producer strain), and CRL807 (immunomodulatory strain) in cell cultures and in a chronic model of parkinsonism induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in aged mice, and under levodopa-benserazide treatment. In vitro, N2a differentiated neurons were exposed to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and treated with intracellular bacterial extracts or with conditioned media from BV-2 cells exposed to the bacterial extracts. In vivo, motor skills, tyrosine hydrolase (TH) in brain and cytokine concentrations in serum and in brain were evaluated. The study of the faecal microbiota and the histology of the small intestine was also performed. The results showed that the neuroprotective effect associated with LAB MIX administration did not interfere with levodopa-benserazide treatment. This effect could be associated with the antioxidant and immunomodulatory potential of the LAB selected in the MIX, and was associated with the significant improvement in the motor tests and a higher number of TH + cells in the brain. In addition, LAB MIX administration was associated with modulation of the immune response. LAB administration decreased intestinal damage with an increase in the villus length /crypt depth ratio. Finally, the administration of the LAB MIX in combination with levodopa-benserazide treatment was able to partially revert the intestinal dysbiosis observed in the model, showing greater similarity to the profiles of healthy controls, and highlighting the increase in the Lactobacillaceae family. Different mechanisms of action would be related to the protective effect of the selected LAB combination which has the potential to be evaluated as an adjuvant for conventional PD therapies.
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Benserazida , Levodopa , Ratones Endogámicos C57BL , Fármacos Neuroprotectores , Trastornos Parkinsonianos , Animales , Levodopa/farmacología , Benserazida/farmacología , Benserazida/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Fármacos Neuroprotectores/farmacología , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/metabolismo , Masculino , Ratones , Combinación de Medicamentos , Microbioma Gastrointestinal/efectos de los fármacos , Modelos Animales de Enfermedad , Lactobacillales , Probióticos/uso terapéutico , Antiparkinsonianos/farmacología , Antiparkinsonianos/uso terapéutico , Streptococcus thermophilus/efectos de los fármacosRESUMEN
Bile acids (BAs) are the main endogenous modulators of the composition and metabolic activity of the intestinal microbiota. In the present work, the effect of conjugated (glycodeoxycholic, glycocholic, taurodeoxycholic, taurocholic acids) and free BAs [cholic acid (CA) and deoxycholic acid (DCA)] on the survival, biological molecules, and structural and surface properties of two potential probiotic lactic acid bacteria (LAB) was evaluated. For this, viability assays, Raman spectroscopy, scanning electron microscopy (SEM), and zeta potential (ZP) measurements were employed. Our results evidenced that free BAs were more toxic than conjugates, with CA being significantly more harmful than deoxycholic acid (DCA). RAMAN studies show that BAs modify the bands corresponding to proteins, lipids, carbohydrates, and DNA. SEM showed that BAs cause surface distortions with depressions and fold formation, as well as incomplete cell division. DCA was the one that least altered the ZP of bacteria when compared to CA and taurodeoxycholic acid, with gradual changes towards more positive values. In general, the magnitude of these effects was different according to the BA and its concentration, being more evident in the presence of CA, even at low concentrations, which would explain its greater inhibitory effect. This work provides solid evidence on the effects of BAs on LAB that will allow for the development of strategies by which to modulate the composition of the microbiota positively.
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The organic acids produced by lactic acid bacteria (LAB) during the fermentation of sourdoughs have the ability to reduce the growth of different molds. However, this ability depends on the LAB used. For this reason, in this study, the proportions of different LAB were optimized to obtain aqueous extracts (AEs) from sourdough to reduce fungal growth in vitro, control the acetic acid concentration, and obtain a specific lactic to acetic acid ratio. In addition, the optimized mixtures were used to formulate partially baked bread (PBB) and evaluate the mold growth and bread quality during refrigerated storage. Using a simplex-lattice mixture design, various combinations of Lactiplantibacillus plantarum, Lacticaseibacillus casei, and Lactobacillus acidophilus were evaluated for their ability to produce organic acids and inhibit mold growth. The mixture containing only Lpb. plantarum significantly reduced the growth rates and extended the lag time of Penicillium chrysogenum and P. corylophilum compared with the control. The AEs' pH values ranged from 3.50 to 3.04. Organic acid analysis revealed that using Lpb. plantarum yielded higher acetic acid concentrations than when using mixed LAB. This suggests that LAB-specific interactions significantly influence organic acid production during fermentation. The reduced radial growth rates and extended lag times for both molds compared to the control confirmed the antifungal properties of the AEs from the sourdoughs. Statistical analyses of the mixture design using polynomial models demonstrated a good fit for the analyzed responses. Two optimized LAB mixtures were identified that maximized mold lag time, targeted the desired acetic acid concentration, and balanced the lactic to acetic acid ratio. The addition of sourdough with optimized LAB mixtures to PBB resulted in a longer shelf life (21 days) and adequately maintained product quality characteristics during storage. PBB was subjected to complete baking and sensory evaluation. The overall acceptability was slightly higher in the control without sourdough (7.50), followed by bread formulated with the optimized sourdoughs (ranging from 6.78 to 7.10), but the difference was not statistically significant (p > 0.05). The sensory analysis results indicated that the optimization was used to successfully formulate a sourdough bread with a sensory profile closely resembling that of a nonsupplemented one. The designed LAB mixtures can effectively enhance sourdough bread's antifungal properties and quality, providing a promising approach for extending bread shelf life while maintaining desirable sensory attributes.
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Staphylococcus aureus is a well-known pathogen capable of producing enterotoxins during bacterial growth in contaminated food, and the ingestion of such preformed toxins is one of the major causes of food poisoning around the world. Nowadays 33 staphylococcal enterotoxins (SEs) and SE-like toxins have been described, but nearly 95% of confirmed foodborne outbreaks are attributed to classical enterotoxins SEA, SEB, SEC, SED, and SEE. The natural habitat of S. aureus includes the skin and mucous membranes of both humans and animals, allowing the contamination of milk, its derivatives, and the processing facilities. S. aureus is well known for the ability to form biofilms in food processing environments, which contributes to its persistence and cross-contamination in food. The biocontrol of S. aureus in foods by lactic acid bacteria (LAB) and their bacteriocins has been studied for many years. Recently, LAB and their metabolites have also been explored for controlling S. aureus biofilms. LAB are used in fermented foods since in ancient times and nowadays characterized strains (or their purified bacteriocin) can be intentionally added to prolong food shelf-life and to control the growth of potentially pathogenic bacteria. Regarding the use of these microorganism and their metabolites (such as organic acids and bacteriocins) to prevent biofilm development or for biofilm removal, it is possible to conclude that a complex network behind the antagonistic activity remains poorly understood at the molecular level. The use of approaches that allow the characterization of these interactions is necessary to enhance our understanding of the mechanisms that govern the inhibitory activity of LAB against S. aureus biofilms in food processing environments.
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Biopelículas , Enterotoxinas , Microbiología de Alimentos , Staphylococcus aureus , Staphylococcus aureus/fisiología , Enterotoxinas/metabolismo , Humanos , Animales , Lactobacillales/fisiología , Lactobacillales/metabolismo , Industria Lechera , Bacteriocinas/metabolismo , Contaminación de Alimentos/prevención & control , Leche/microbiologíaRESUMEN
Poly(lactic-acid) (PLA) is a biodegradable polymer widely used as a packaging material. Its monomer, lactic acid, and its derivatives have been used in the food, cosmetic, and chemical industries. The accumulation of PLA residues leads to the development of green degrading methodologies, such as enzymatic degradation. This work evaluates the potential use of three cutinolytic enzymes codified in the Aspergillus nidulans genome to achieve this goal. The results are compared with those obtained with proteinase K from Tritirachium album, which has been reported as a PLA-hydrolyzing enzyme. The results show that all three cutinases act on the polymer, but ANCUT 1 releases the highest amount of lactic acid (25.86 mM). Different reaction conditions assayed later led to double the released lactic acid. A decrease in weight (45.96%) was also observed. The enzyme showed activity both on poly L lactic acid and on poly D lactic acid. Therefore, this cutinase offers the potential to rapidly degrade these package residues, and preliminary data show that this is feasible.
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Introduction: Levilactobacillus brevis CRL 2013, a plant-derived lactic acid bacterium (LAB) with immunomodulatory properties, has emerged as an efficient producer of γ-aminobutyric acid (GABA). Notably, not all LAB possess the ability to produce GABA, highlighting the importance of specific genetic and environmental conditions for GABA synthesis. This study aimed to elucidate the intriguing GABA-producing machinery of L. brevis CRL 2013 and support its potential for safe application through comprehensive genome analysis. Methods: A comprehensive genome analysis of L. brevis CRL 2013 was performed to identify the presence of antibiotic resistance genes, virulence markers, and genes associated with the glutamate decarboxylase system, which is essential for GABA biosynthesis. Then, an optimized chemically defined culture medium (CDM) was supplemented with monosodium glutamate (MSG) and yeast extract (YE) to analyze their influence on GABA production. Proteomic and transcriptional analyses were conducted to assess changes in protein and gene expression related to GABA production. Results: The absence of antibiotic resistance genes and virulence markers in the genome of L. brevis CRL 2013 supports its safety for potential probiotic applications. Genes encoding the glutamate decarboxylase system, including two gad genes (gadA and gadB) and the glutamate antiporter gene (gadC), were identified. The gadB gene is located adjacent to gadC, while gadA resides separately on the chromosome. The transcriptional regulator gadR was found upstream of gadC, with transcriptional analyses demonstrating cotranscription of gadR with gadC. Although MSG supplementation alone did not activate GABA synthesis, the addition of YE significantly enhanced GABA production in the optimized CDM containing glutamate. Proteomic analysis revealed minimal differences between MSG-supplemented and non-supplemented CDM cultures, whereas YE supplementation resulted in significant proteomic changes, including upregulation of GadB. Transcriptional analysis confirmed increased expression of gadB and gadR upon YE supplementation, supporting its role in activating GABA production. Conclusion: These findings provide valuable insights into the influence of nutrient composition on GABA production. Furthermore, they unveil the potential of L. brevis CRL 2013 as a safe, nonpathogenic strain with valuable biotechnological traits which can be further leveraged for its probiotic potential in the food industry.
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AIMS: This study aimed to prospect and isolate lactic acid bacteria (LAB) from an artisanal cheese production environment, to assess their safety, and to explore their bacteriocinogenic potential against Listeria monocytogenes. METHODS AND RESULTS: Samples were collected from surfaces of an artisanal-cheese production facility and after rep-PCR and 16S rRNA sequencing analysis, selected strains were identified as to be belonging to Lactococcus garvieae (1 strain) and Enterococcus faecium (14 isolates, grouped into three clusters) associated with different environments (worktables, cheese mold, ripening wooden shelves). All of them presented bacteriocinogenic potential against L. monocytogenes ATCC 7644 and were confirmed as safe (γ-hemolytic, not presenting antibiotic resistance, no mucus degradation properties, and no proteolytic or gelatinase enzyme activity). Additionally, cell growth, acidification and bacteriocins production kinetics, bacteriocin stability in relation to different temperatures, pH, and chemicals were evaluated. According to performed PCR analysis all studied strains generated positive evidence for the presence of entA and entP genes (for production of enterocins A and enterocins P, respectively). However, pediocin PA-1 associated gene was recorded only in DNA obtained from E. faecium ST02JL and Lc. garvieae ST04JL. CONCLUSIONS: It is worth considering the application of these safe LAB or their bacteriocins in situ as an alternative means of controlling L. monocytogenes in cheese production environments, either alone or in combination with other antimicrobials.