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.

Alternative culture medium design for biomass production of autochthonous meat starter Latilactobacillus sakei sp. sakei ACU-2.

The autochthonous strain Latilactobacillus sakei sp. sakei ACU-2 was selected as a meat starter culture for dry sausage production. Transferring this strain from laboratory scale to industry requires an increase in biomass production, while lowering process costs. In this study, a combination of techniques was applied in order to optimize the culture medium composition to enhance biomass production of L. sakei ACU-2. One variable at a time experiments, Plackett-Burman design, and mixture design were performed to fulfill the strain nutritional requirements. Eventually, the optimized formulation contained 19.46 g/L yeast extract; 8.28 g/L whey protein concentrate; 2.26 g/L soy peptone; 30 g/L cerelose; 1 g/L Tween 80; 5 g/L sodium acetate; 0.2 g/L magnesium sulfate and 0.05 g/L manganese sulfate. When L. sakei ACU-2 was cultivated in a bioreactor using the alternative medium, an enhancement of 75.5% of biomass production was achieved, in comparison to its growth in the commercial de Man, Rogosa, and Sharpe medium. Furthermore, a reduction of 62-86% of the cost was also attained. These results support a promising large-scale application of the designed medium for high biomass yields of the starter culture at minor costs.

A New Bacteriocin from Latilactobacillus sakei: In vitro and In situ Application.

AIMS AND BACKGROUND: Natural preservatives are a viable alternative to replace chemical preservatives that have potential toxicity and carcinogenic effects. OBJECTIVE: To prove the effectiveness in increasing the microbiological stability of Minas Frescal cheese with the addition of a bacteriocin obtained from Latilactobacillus sakei as a natural preservative. METHODS: A new broad-spectrum bacteriocin was evaluated for its functional activity in vitro and in situ when applied in the formulation of Minas Frescal cheese. A commercial bacteriocin was used as a positive control. RESULTS: The inhibitory action of the bacteriocin studied was confirmed, with a reduction of 42.86% in the count of coagulase-positive Staphylococcus in relation to the negative control, at the end of the 30 days of study. For the group of thermotolerant coliforms, the bacteriocin studied showed greater efficiency than the commercial preservative. In vitro analyzes showed the inhibitory action of bacteriocin, above 87% inhibition against S. aureus, E. coli and Salmonela enteritidis, and approximately 90% against Listeria monocytogenes. CONCLUSION: It was concluded that the bacteriocin produced by the Latilactobacillus sakei strain has great potential for application in foods such as Minas Frescal cheese.

Development of Active Packaging Based on Agar-Agar Incorporated with Bacteriocin of Lactobacillus sakei.

In the search for new biodegradable materials and greater microbiological safety and stability of perishable food products, this study aimed to develop a bioplastic antibacterial film incorporating bacteriocin for application in commercial curd cheese and monitoring of microbiological stability. Films with good handling characteristics as well as physical, barrier, and mechanical properties were obtained. Regarding the antibacterial activity, the microbial reduction was demonstrated in a food matrix, obtaining a reduction of 3 logarithmic cycles for the group of coagulase positive staphylococci and from 1100 to <3.00 MPN/g in the analysis of thermotolerant coliforms. Therefore, the film presented food barrier characteristics with the external environment and adequate migration of the antibacterial compound to the product, contributing to the reduction of contamination of a food with high initial microbial load.

New Active Packaging Based on Biopolymeric Mixture Added with Bacteriocin as Active Compound.

The objective of this work was to develop a chitosan/agar-agar bioplastic film incorporated with bacteriocin that presents active potential when used as food packaging. The formulation of the film solution was determined from an experimental design, through the optimization using the desirability function. After establishing the concentrations of the biopolymers and the plasticizer, the purified bacteriocin extract of Lactobacillus sakei was added, which acts as an antibacterial agent. The films were characterized through physical, chemical, mechanical, barrier, and microbiological analyses. The mechanical properties and water vapor permeability were not altered by the addition of the extract. The swelling property decreased with the addition of the extract and the solubility increased, however, the film remained intact when in contact with the food, thus allowing an efficient barrier. Visible light protection was improved by increased opacity and antibacterial capacity was effective. When used as Minas Frescal cream cheese packaging, it contributed to the increase of microbiological stability, showing a reduction of 2.62 log UFC/g, contributing a gradual release of the active compound into the food during the storage time. The film had an active capacity that could be used as a barrier to the food, allowing it to be safely packaged.

Studies on host-foodborne bacteria in intestinal three-dimensional cell culture model indicate possible mechanisms of interaction.

Spheroids of intestinal cells (Caco-2) were used to evaluate the adhesion/invasion ability of Listeria monocytogenes (pathogen) and Lactobacillus sakei 1 (potential probiotic). Besides, transcriptomic analyses of Caco-2 cells in three dimensional cultures were done, with the aim of revealing possible host-foodborne bacteria interactions. Result of adhesion assay for L. monocytogenes in Caco-2 spheroids was 22.86 ± 0.33%, but it was stimulated in acidic pH (4.5) and by the presence of 2% sucrose (respectively, 32.56 ± 1.35% and 33.25 ± 1.26%). Conversely, the invasion rate of L. monocytogenes was lower at pH 4.5, in comparison with non-stressed controls (18.89 ± 1.05% and 58.65 ± 0.30%, respectively). L. sakei 1 adhered to Caco-2 tridimensional cell culture (27.30 ± 2.64%), with no invasiveness. There were 19 and 21 genes down and upregulated, respectively, in tridimensional Caco-2 cells, upon infection with L. monocytogenes, which involved immunity, apoptosis; cytoprotective responses, cell signalling-regulatory pathways. It was evidenced despite activation or deactivation of several pathways in intestinal cells to counteract infection, the pathogen was able to hijack many host defense mechanisms. On the other hand, the probiotic candidate L. sakei 1 was correlated with decreased transcription of two genes in Caco-2 cells, though it stimulated the expression of 14 others, with diverse roles in immunity, apoptosis, cytoprotective response and cell signalling-regulatory pathways. Our data suggest the use of tridimensional cell culture to mimic the intestinal epithelium is a good model for gathering broad information on the putative mechanisms of interaction between host and bacteria of importance for food safety, which can serve as a basis for further in-depth investigation.

Control of pathogens in fresh pork sausage by inclusion of Lactobacillus sakei BAS0117.

A pork sausage was produced with low sodium content (1.64%) to which Lactobacillus sakei was added with the aim of developing a meat pork sausage for cooking and having technological, organoleptic, and hygienic advantages. The lactic acid bacteria (LAB) L. sakei, Lactococcus sp., and Pediococcus pentosaceus were submitted to extreme pH, temperature, and NaCl conditions. Lactobacillus sakei was used in pork sausage because of its resistance to different culture conditions and its antimicrobial potential. The food-borne pathogens Listeria monocytogenes Scott A, Enterococcus faecalis, and Staphylococcus aureus were used as indicator microorganisms to evaluate the antimicrobial activity of selected LAB strains. Salmonella enterica serotype Choleraesuis is a common pathogen of pigs. To the raw sausage product containing L. sakei and nonpathogenic endogenous microbiota, we added about >104 and <105 CFU/g of S. enterica serotype Choleraesuis to evaluate the inhibitory potential of L. sakei towards this pathogen. Salmonella Choleraesuis was inhibited in the presence of L. sakei over 7 days of storage of the meat product (about 3.0 log cycles reduction). Lactobacillus sakei significantly increased inhibition when compared with the nonfermented sausage. Thus, L. sakei BAS0117 played an important role as an additional hurdle in the fermented meat pork sausage during storage.

A peptidomic approach of meat protein degradation in a low-sodium fermented sausage model using autochthonous starter cultures.

Fermented sausage technology is currently compromised in decreasing the addition of NaCl. Use of starter cultures with peptidogenic potential could be a valuable strategy that can mask or hide off flavors produced by the use of NaCl substituents. In the present work, the peptidogenic potential of four lactic acid bacteria species was evaluated in a low-sodium beaker sausage (BS) model. Using a peptidomic approach, a total of 86 low molecular weight (LMW) peptides were accurately identified, mostly derived from myofibrillar proteins, especially actin, which generated 53 peptides. The BS inoculated with L. curvatus CRL705 generated 56 LMW peptides, followed by Enterococcus (E.) mundtii CRL35 with 43 peptides. In addition, BS inoculated with Lactobacillus (L.) plantarum and with L. sakei produced higher amino acid amounts over time as compared to the rest of BS models, highlighting the importance of both, time and sample effect on the overall free amino acid generation. The presence of each LAB strain in BS models generated a unique profile of small peptides and amino acids that could serve as a distinctive biochemical trait to differentiate specific fermented products. According to these results, E. mundtii and L. sakei, which are compatible between them, are proposed as the most efficiently adapted to low-sodium conditions. The use of selected strains during the processing of low-sodium fermented sausages could have a positive effect on the production of small peptides and free amino acids.

Characterization of multiple antilisterial peptides produced by sakacin P-producing Lactobacillus sakei subsp. sakei 2a.

Antimicrobial compounds produced by lactic acid bacteria can be explored as natural food biopreservatives. In a previous report, the main antimicrobial compounds produced by the Brazilian meat isolate Lactobacillus sakei subsp. sakei 2a, i.e., bacteriocin sakacin P and two ribosomal peptides (P2 and P3) active against Listeria monocytogenes, were described. In this study, we report the spectrum of activity, molecular mass, structural identity and mechanism of action of additional six antilisterial peptides produced by Lb. sakei 2a, detected in a 24 h-culture in MRS broth submitted to acid treatment (pH 1.5) and proper fractionation and purification steps for obtention of free and cell-bound proteins. The six peptides presented similarity to different ribosomal proteins of Lb. sakei subsp sakei 23K and the molecular masses varied from 4.6 to 11.0 kDa. All peptides were capable to increase the efflux of ATP and decrease the membrane potential in Listeria monocytogenes. The activity of a pool of the obtained antilisterial compounds [enriched active fraction (EAF)] against Listeria monocytogenes in a food model (meat gravy) during refrigerated storage (4 °C) for 10 days was also tested and results indicated that the populations of L. monocytogenes in the food model containing the acid extract remained lower than those at time 0-day, evidencing that the acid extract of a culture of Lb. sakei 2a is a good technological alternative for the control of growth of L. monocytogenes in foods.

Proteomic and genetics insights on the response of the bacteriocinogenic Lactobacillus sakei CRL1862 during biofilm formation on stainless steel surface at 10°C.

Some lactic acid bacteria have the ability to form biofilms on food-industry surfaces and this property could be used to control food pathogens colonization. Lactobacillus sakei CR1862 was selected considering its bacteriocinogenic nature and ability to adhere to abiotic surfaces at low temperatures. In this study, the proteome of L. sakei CRL1862 grown either under biofilm on stainless steel surface and planktonic modes of growth at 10°C, was investigated. Using two-dimensional gel electrophoresis, 29 out of 43 statistically significant spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ten proteins resulted up-regulated whereas 16 were down-regulated during biofilm formation. Differentially expressed proteins were found to belong to carbohydrate, nucleotide, aminoacid and lipid metabolisms as well as translation, peptide hydrolysis, cell envelope/cell wall biosynthesis, adaption to atypical conditions and protein secretion. Some proteins related to carbohydrate and nucleotide metabolisms, translation and peptide degradation were overexpressed whereas those associated to stress conditions were synthesized in lower amounts. It seems that conditions for biofilm development would not imply a stressful environment for L. sakei CRL1862 cells, directing its growth strategy towards glycolytic flux regulation and reinforcing protein synthesis. In addition, L. sakei CRL1862 showed to harbor nine out of ten assayed genes involved in biofilm formation and protein anchoring. By applying qRT-PCR analysis, four of these genes showed to be up regulated, srtA2 being the most remarkable. The results of this study contribute to the knowledge of the physiology of L. sakei CRL1862 growing in biofilm on a characteristic food contact surface. The use of this strain as green biocide preventing L. monocytogenes post-processing contamination on industrial surfaces may be considered.

Antimicrobial activity of whey protein films supplemented with Lactobacillus sakei cell-free supernatant on fresh beef.

The aim of this work was to evaluate the antimicrobial activity of whey protein isolate (WPI) films supplemented with Lactobacillus sakei NRRL B-1917 cell-free supernatant on beef inoculated with Escherichia coli ATCC 25922 or Listeria monocytogenes Scott A; additionally, sensory evaluation was performed on wrapped beef cubes. Supernatant concentrates were obtained from Lb. sakei cultures in MRS broth after centrifugation, filtering, and freeze-drying. Films were prepared with WPI (3% w/w), alginate (0.625% w/w), rehydrated supernatant (18 mg/ml), and glycerol. Films were used to wrap beef cubes inoculated with ≈103 CFU/g E. coli or L. monocytogenes. Sensory evaluation was carried out on grilled beef wrapped or not with the studied antimicrobial films. During refrigerated storage, antimicrobial films reduced 1.4 log10 CFU/g of L. monocytogenes after 120 h, while E. coli decreased 2.3 log10 CFU/g after 36 h. Grilled beef wrapped with antimicrobial film was well accepted by panelists, besides scores evidenced no significant differences (p > 0.05) between wrapped and unwrapped beef.

Control of Listeria monocytogenes biofilms on industrial surfaces by the bacteriocin-producing Lactobacillus sakei CRL1862.

The effect of the bacteriocin-producing Lactobacillus sakei CRL1862 and its bacteriocin in the control of Listeria biofilm formation on industrial surfaces at 10°C was investigated. A screening among different Listeria species was performed allowing selecting L. monocytogenes FBUNT for its use as a biofilm producer on stainless steel (SS) and polytetrafluoroe-thylene (PTFE) surfaces. Three conditions were simulated to evaluate the ability of the bacteriocinogenic strain to displace, exclude and compete pathogen biofilm formation. Lactobacillus sakei CRL1862 effectively inhibited biofilm formation by L. monocytogenes FBUNT through the three assayed mechanisms, pathogen inhibition being more efficient on PTFE than on SS surface. Moreover, co-culture of L. monocytogenes FBUNT with the bacteriocin-producer displayed the highest efficacy reducing the pathogen by 5.54 ± 0.12 and 4.52 ± 0.01 on PTFE and SS, respectively. Industrially, the pre-treatment with L. sakei CRL1862 or its bacteriocin (exclusion) constitutes the most realistic way to prevent pathogen biofilm settlement. The use of bacteriocins and/or the bacteriocin-producer strain represents a safe and environmentally-friendly sanitation method to mitigate post-processing food contamination.

Evaluation of an autochthonous starter culture on the production of a traditional dry fermented sausage from Chaco (Argentina) at a small-scale facility.

The performance of a mixed starter culture, SAS-1, comprised of the autochthonous strains Lactobacillus sakei ACU-2 and Staphylococcus vitulinus ACU-10, was evaluated into the production process of a traditional dry sausage. Microbiological, physicochemical and sensory analyses were carried out to accomplish this goal. Results showed an improvement in performance through the introduction of SAS-1; adding mixed starter culture rapidly decreased pH, inhibited the growth of contaminant microorganisms and enhanced the beneficial ones, diminished TBARS, and highlighted color and aroma attributes. However, most influential organoleptic descriptors among consumer acceptance were not affected by the addition of the starter. This starter culture would represent a valuable tool to improve the homogeneity of artisanal manufacture of this traditional food.