Production Of Bacteriocin-like Inhibitory Substances Research Articles

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 (55.15gL-1, 41.3gL-1 and 27.5gL-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 55.15gL-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 60h 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.

Enhancing food preservation with postbiotic metabolites γ-aminobutyric acid (GABA) and bacteriocin-like inhibitory substances (BLIS) produced by Lactobacillus brevis C23 co-cultures in plant-based medium

Lactic acid bacteria (LAB) can produce γ-aminobutyric acid (GABA) with antioxidant properties and sedative effects when it binds to the GABA receptor in the human brain. LAB can also produce bacteriocin-like inhibitory substances (BLIS) with antimicrobial capabilities during carbohydrate fermentation. GABA and BLIS are natural compounds with potential health benefits and food preservation properties. Lactobacillus brevis C23 was co-cultured with three different LABs as inducers, which produced the highest GABA content and BLIS activity. They were cultured in various plant-based media to obtain an edible and better-tasting final product over commercially available media like MRS broth. A coconut-based medium with additives was optimized using response surface methodology (RSM) to increase GABA and BLIS production. The optimized medium for maximum GABA production (3.22 ± 0.01 mg/mL) and BLIS activity (84.40 ± 0.44%) was a 5.5% coconut medium containing 0.23% glucose, 1.44% Tween 20, 0.48% L-glutamic acid, and 0.02% pyridoxine. Due to the presence of GABA, the cell-free supernatant (CFS) as a postbiotic showed higher antioxidant activity than other food preservatives like nisin and potassium sorbate. Finally, microbiological tests on food samples showed that the postbiotic was more effective than other preservatives at combating the growth of LAB, molds and coliform bacteria, making it a possible food preservative.

Partial characterization of a bacteriocin-like inhibitory substance produced by Streptococcus equinus C6I9, a bovine rumen isolate.

The present study aimed to evaluate the production and the factors that influence the antimicrobial activity of a bacteriocin-like inhibitory substance (BLIS) produced by Streptococcus equinus C6I9, a bovine rumen isolate. The antagonist activity was evaluated by the spot-on-lawn assay. Antimicrobial activity of the crude extracts obtained using NaCl acidic solution (pH 2, 100 mM) was evaluated along the bacterial growth and in growth medium containing different carbon and nitrogen sources, by agar diffusion method. Crude extract was evaluated regarding temperature stability, storage conditions, and pH sensitivity. S. equinus C619 inhibited both Gram-positive and Gram-negative strains. The crude extract was sensitive to proteinase K, and showed great stability to different temperatures and pH (2 to 12). The presence of meat extract (1.5 g/L) and sucrose (8 g/L) resulted in higher production of BLIS by S. equinus C6I9. The BLIS molecular mass was determined as approximately 3.5 kDa. The results obtained in this study evidenced the production of a broad spectrum activity, thermo- and pH stable BLIS by the ruminal isolate S. equinus C6I9, important features for its biotechnological application.

Influence of growth parameters on bacteriocin-like inhibitory substances (BLIS) production by lactic acid bacteria.

This study aimed to investigate the possible production of bacteriocin-like inhibitory substances (BLIS) and optimize it by Lactobacillus sakei and combined Lactobacillus curvatus/Pediococcus acidilactici, lactic acid bacteria used for the meat fermentation. The effect of growth parameters on BLIS production was evaluated by measuring the antibacterial activity of the cell-free supernatant of these bacteria. The stability of the cell-free supernatant of combined L.curvatus/P.acidilactici bacteria was also examined after heat treatment and pH variations. Results showed highest BLIS production by L.curvatus/P.acidilactici was at growth temperatures of 28°C and pH 5, while the optimum condition for production of BLIS by L.sakei was 24°C and pH 6.5. The BLIS production by L. curvatus/P. acidilactici bacteria was greatly influenced by carbon and nitrogen sources. The antimicrobial activity of BLIS in supplemented De Man, Rosa, and Sharpe broth (MRS) was 2044.44 AU/mL, 4.84-fold greater than unsupplemented MRS. None of the carbon sources used in this study affected the BLIS production by the L.sakei, while pea peptone enhanced the antimicrobial activity and showed a value of 188.89 AU/mL, 1.47-fold greater than unsupplemented MRS. The characterization of the cell-free supernatant of L. curvatus/P. acidilactici bacteria showed the heat stability and activity of BLIS at pH 2-10.

Evaluation of inhibitory compounds produced by bacteria isolated from a hydrogen-producing bioreactor during the self-fermentation of wheat straw.

The objective of this study was to evaluate the inhibitory activity of compounds secreted by bacteria isolated from a hydrogen-producing bioreactor to understand how these microorganisms interact in this community. In vitro inhibitory assays were performed using samples secreted by bacteria subject to different treatments to determine if their inhibitory effect was due to organic acids, non-proteinaceous compounds or bacteriocin-like inhibitory substances (BLIS). Bacterial isolated were suppressed 43%, 30% and 27% by neutralized, precipitated and non-neutralized cell-free supernatants, respectively. Non-hydrogen producers (non-H2 P) lactic acid bacteria (LAB) (Lactobacillus plantarum LB1, Lactobacillus pentosus LB7, Pediococcus acidilactici LB4) and hydrogen producers (H2 P) LAB (Enterococcus faecium F) were inhibited by the production of organic acids, non-proteinaceous compounds and BLIS. Meanwhile, the obligate anaerobe H2 P (Clostridium beijerinckii B) inhibited by the production of non-proteinaceous compounds and BLIS. The presence of BLIS was confirmed when proteolytic enzymes affected the inhibitory activity of secreted proteins in values ranging from 20% to 42%. The BLIS produced by L. plantarum LB1, P. acidilactici LB4, L. pentosus LB7 and E. faecium F showed molecular masses of ~11, 25, 20 and 11 kDa, respectively. It was demonstrated antagonistic interactions between Lactobacillus-Enterococcus and Pediococcus-Enterococcus species, generated by the secretion of organic acids, non-proteinaceous compounds and BLIS. We report the interactions between LAB isolated from hydrogen-producing bioreactors. These interactions might impact the dynamics of the microbial population during hydrogen generation. Our work lays a foundation for strategies that allow controlling bacteria that can affect hydrogen production.

Bacteriocin-Like Inhibitory Substances in Staphylococci of Different Origins and Species With Activity Against Relevant Pathogens.

Bacteriocins are antimicrobial peptides with relevance in the modulation of human and animal microbiota that have gained interest in biomedical and biotechnological applications. In this study, the production of bacteriocin-like inhibitory substances (BLIS) was tested among a collection of 890 staphylococci of different origins (humans, animals, food, and the environment) and species, both coagulase-positive (CoPS, 238 isolates of 3 species) and coagulase-negative staphylococci (CoNS, 652 isolates of 26 species). Of the 890 staphylococci, 60 (6.7%) showed antimicrobial activity by the spot-on-lawn method against at least one of the 25 indicator bacteria tested. BLIS-producer (BLIS+) isolates were detected in 8.8% of CoPS and 6.0% of CoNS. The staphylococcal species with the highest percentages of BLIS+ isolates were S. chromogenes (38.5%), S. pseudintermedius (26.7%), and S. warneri (23.1%). The production of BLIS was more frequently detected among isolates of pets, wild animals, and food. Moreover, 13 BLIS+ isolates showed wide antimicrobial activiy spectrum, and 7 of these isolates (of species S. aureus, S. pseudintermedius, S. sciuri, and S. hominis) demonstrated antimicrobial activity against more than 70% of the indicator bacteria tested. The genetic characterization (by PCR and sequencing) of the 60 BLIS+ isolates revealed the detection of (a) 11 CoNS and CoPS isolates carrying putative lantibiotic-like genes; (b) 3 S. pseudintermedius isolates harboring the genes of BacSp222 bacteriocin; and (c) 2 S. chromogenes isolates that presented the gene of a putative cyclic bacteriocin (uberolysin-like), being the first report in this CoNS species. Antimicrobial susceptibility testing was performed in BLIS+ isolates and one-third of the CoNS isolates showed susceptibility to all antibiotics tested, which also lacked the virulence genes studied. These BLIS+ CoNS are good candidates for further characterization studies.

Bacteriocin-like inhibitory substances production by Enterococcus faecium 135 in co-culture with Ligilactobacillus salivarius and Limosilactobacillus reuteri.

The use of lactic acid bacteria (LAB) and probiotic cultures in the breeding of animals such as poultry and swine are quite common. It is known that those strains can produce bacteriocins when grown in pure culture. However, the production of bacteriocin using co-culture of microorganisms has not been much studied so far. The present study contributes with innovation in this area by embracing the production of bacteriocin-like inhibitory substances (BLIS) by a newly isolated strain of Enterococcus faecium 135. Additionally, the co-cultivation of this strain with Ligilactobacillus salivarius and Limosilactobacillus reuteri was also investigated. The antimicrobial activity of the produced BLIS was evaluated against Listeria monocytogenes, Listeria innocua, Salmonella enterica, and Salmonella enterica serovar Typhimurium using two methods: turbidimetric and agar diffusion. In addition, the presence of enterocin genes was also evaluated. The BLIS produced showed a bacteriostatic effect against the bio-indicator strains, and the highest antimicrobial activities expressed by arbitrary units per mL (AU/mL) were obtained against L. monocytogenes in monoculture (12,800 AU/mL), followed by the co-culture of E. faecium with Limosilactobacillus reuteri (400 AU/mL). After concentration with ammonium sulfate, the antimicrobial activity raised to 25,600 AU/mL. Assays to determine the proteinaceous nature of the BLIS showed susceptibility to trypsin and antimicrobial activity until 90°C. Finally, analysis of the presence of structural genes of enterocins revealed that four enterocin genes were present in E. faecium 135. These results suggest that BLIS produced by E. faecium 135 has potential to be a bacteriocin and, after purification, could potentially be used as an antimicrobial agent in animal breeding.

Extractive Fermentation for Recovery of Bacteriocin-Like Inhibitory Substances Derived from Lactococcus lactis Gh1 Using PEG2000/Dextran T500 Aqueous Two-Phase System

This work aimed to optimize the parameters affecting partitioning of a bacteriocin-like inhibitory substances (BLIS) from Lactococcus lactis Gh1 in extractive fermentation using polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS). This system was developed for the simultaneous cell cultivation and downstream processing of BLIS. Results showed that the molecular weight of PEG, PEG concentration, and dextran T500 affect the partition coefficient (K), purification factor (PF), and yield of BLIS partitioning. ATPS composed of 10% (w/w) PEG2000 and 8% (w/w) dextran T500, provided the greatest conditions for the extractive BLIS production. The K (1.00 ± 0.16), PF (2.92 ± 0.37) and yield (77.24 ± 2.81%) were increased at selected orbital speed (200 rpm) and pH (pH 7). Sustainable growth of the cells in the bioreactor and repeated fermentation up to the eighth extractive batch were observed during the scale up process, ensuring a continuous production and purification of BLIS. Hence, the simplicity and effectiveness of ATPS in the purification of BLIS were proven in this study.

Fermentation strategies for improving the production of bacteriocin‐like inhibitory substances by Lactobacillus brevis C23 with nutrient supplementation, pH, and temperature variations

In this study, fermentation strategies were carried out for improving the production of bacteriocin-like inhibitory substances by Lactobacillus brevis C23 with nutrient supplementation, pH, and temperature variations, as well as fed-batch cultivation. The BLIS activity was highest (91.52%) in the MRS medium supplemented with 1.5% (w/v) lactose and 1% (w/v) meat extract. pH 5 augmented both the cell biomass and BLIS activity, but incubation temperature only affected growth at 37°C. Fed-batch cultivation strategy that utilized meat extract feeding would positively affect cell growth, whereas BLIS activity was enhanced by lactose feeding. The highest BLIS activity from L. brevis C23 was obtained at the 24 hr mark by feeding the culture medium with lactose (77.73%). The BLIS production of L. brevis C23 fed with meat extract followed a similar trend to lactose (74.20%). Increased BLIS activity in the final solution would reduce the downstream step such as concentrating the purified product. Novelty impact statement Synergic effect of lactose and meat extract enhanced BLIS activity (91.52%) of CFS by Lactobacillus brevis C23 as compared to the control (69.29%) Temperature at 40℃ did not affect the BLIS activity, but it inhibited the cell growth. Fed-batch cultivation strategy that utilized meat extract feeding would positively affect cell growth, whereas BLIS activity was enhanced by lactose feeding.

In vitro Characterization of Enterococcus faecium strain LCM08 as a probiotic

Background: Under the present situations, where health improvement has become the major concern, food supplements, augmenting health is a matter of interest. Presently, the concept of "functional foods" possessing medicinal attributes has been reborn as probiotics. Acid-tolerant and Gram-positive Lactic acid bacteria (LAB), being the major class of probiotics, possess health-promoting characteristics, provide better health with a robust immune system, and treat various digestive and neurological disorders. Hence, the aim of the present study was the isolation and identification of LAB.
 Methodology: The LAB were isolated from various sources, including food products and clinical samples. Enterococcus faecium LCM08 being the most potent isolate tested for probiotic traits, including viability under acidic conditions, biological concentrations of bile salts and enzymes.
 Results: A total of fifty-four strains were isolated, of which 7.2% showed broad-spectrum inhibition against the tested pathogens. Amongst them, Enterococcus faecium LCM08 owing to its broad-spectrum antagonism, when tested for its ability to withstand acidic conditions, displayed 63.58% viability and 72.19% survivability in the presence of bile salts. Moreover, strain sustained in the presence of digestive enzymes including protease (94.65%), trypsin (93.57%) and pancreatin (75.0%) as well as exhibited growth at temperatures ranging between 25˚C and 30˚C where the maximum viable counts recorded as 95% and 96.4%. The strain also produced antimicrobial metabolites at neutral pH. However, elevated temperatures (45˚C-50˚C) inhibited the production of bacteriocin-like inhibitory substances.
 Conclusion: The preliminary investigations revealed that Enterococcus faecium LCM08 possesses the key attributes pre-requisite for a probiotic candidate.

Evaluation of the Estimation Capability of Response Surface Methodology and Artificial Neural Network for the Optimization of Bacteriocin-Like Inhibitory Substances Production by Lactococcus lactis Gh1.

Bacteriocin-like inhibitory substances (BLIS) produced by Lactococcus lactis Gh1 had shown antimicrobial activity against Listeria monocytogenes ATCC 15313. Brain Heart Infusion (BHI) broth is used for the cultivation and enumeration of lactic acid bacteria, but there is a need to improve the current medium composition for enhancement of BLIS production, and one of the approaches is to model the optimization process and identify the most appropriate medium formulation. Response surface methodology (RSM) and artificial neural network (ANN) models were employed in this study. In medium optimization, ANN (R2 = 0.98) methodology provided better estimation point and data fitting as compared to RSM (R2 = 0.79). In ANN, the optimal medium consisted of 35.38 g/L soytone, 16 g/L fructose, 3.25 g/L sodium chloride (NaCl) and 5.40 g/L disodium phosphate (Na2HPO4). BLIS production in optimal medium (717.13 ± 0.76 AU/mL) was about 1.40-fold higher than that obtained in nonoptimised (520.56 ± 3.37 AU/mL) medium. BLIS production was further improved by about 1.18 times higher in 2 L stirred tank bioreactor (787.40 ± 1.30 AU/mL) as compared to that obtained in 250 mL shake flask (665.28 ± 14.22 AU/mL) using the optimised medium.

Influence of Culture Conditions and Medium Compositions on the Production of Bacteriocin-Like Inhibitory Substances by Lactococcus lactis Gh1.

Antibacterial peptides or bacteriocins produced by many strains of lactic acid bacteria have been used as food preservatives for many years without any known adverse effects. Bacteriocin titres can be modified by altering the physiological and nutritional factors of the producing bacterium to improve the production in terms of yield and productivity. The effects of culture conditions (initial pH, inoculum age and inoculum size) and medium compositions (organic and inorganic nitrogen sources; carbon sources) were assessed for the production of bacteriocin-like inhibitory substances (BLIS) by Lactococcus lactis Gh1 in shake flask cultures. An inoculum of the mid-exponential phase culture at 1% (v/v) was the optimal age and size, while initial pH of culture media at alkaline and acidic state did not show a significant impact on BLIS secretion. Organic nitrogen sources were more favourable for BLIS production compared to inorganic sources. Production of BLIS by L. lactis Gh1 in soytone was 1.28-times higher as compared to that of organic nitrogen sources ((NH4)2SO4). The highest cell concentration (XmX = 0.69 ± 0.026 g·L−1) and specific growth rate (μmax = 0.14 h−1) were also observed in cultivation using soytone. By replacing carbon sources with fructose, BLIS production was increased up to 34.94% compared to BHI medium, which gave the biomass cell concentration and specific growth rate of 0.66 ± 0.002 g·L−1 and 0.11 h−1, respectively. It can be concluded that the fermentation factors have pronounced influences on the growth of L. lactis Gh1 and BLIS production. Results from this study could be used for subsequent application in process design and optimisation for improving BLIS production by L. lactis Gh1 at larger scale.

Biological control of Erwinia mallotivora, the causal agent of papaya dieback disease by indigenous seed-borne endophytic lactic acid bacteria consortium.

Dieback disease caused by Erwinia mallotivora is a major threat to papaya plantation in Malaysia. The current study was conducted to evaluate the potential of endophytic lactic acid bacteria (LAB) isolated from papaya seeds for disease suppression of papaya dieback. Two hundred and thirty isolates were screened against E. mallotivora BT-MARDI, and the inhibitory activity of the isolates against the pathogen was ranging from 11.7–23.7 mm inhibition zones. The synergistic experiments revealed that combination of W. cibaria PPKSD19 and Lactococcus lactis subsp. lactis PPSSD39 increased antibacterial activity against the pathogen. The antibacterial activity was partially due to the production of bacteriocin-like inhibitory substances (BLIS). The nursery experiment confirmed that the application of bacterial consortium W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 significantly reduced disease severity to 19% and increased biocontrol efficacy to 69% of infected papaya plants after 18 days of treatment. This study showed that W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 are potential candidate as biocontrol agents against papaya dieback disease.

Improvement of Lactobacillus plantarum for the enhanced production of bacteriocin like inhibitory substance using combinatorial approach

Abstract Over the last few decades, emerging cases of chemical additives and food borne pathogens have enforced the demand of natural antimicrobial compounds. Bacteriocins are one of the natural alternatives to the chemical food preservatives. The research on new and improved bacteriocins is in full swing due to some limitations of commercially available bacteriocins. The aim of the study is to improve the indigenously isolated strain for the enhanced production of bacteriocin like inhibitory substance (BLIS) using combinatorial approach. In the current study, the efficiency of BLIS produced by Lactobacillus plantarum KIBGE-IB45 was improved by applying a combinatorial strategies. Primarily, production of BLIS was optimized using one factor at a time approach. To further enhance the potential of L. plantarum, chemical mutagens were used followed by statistical optimization using multivariate approach. The results revealed that synergistic effect of chemical mutagen and a sequential statistical optimization have significantly enhanced the production of BLIS at 35 °C in MRS medium with an initial pH 9.0 after 16 h of incubation. Response surface methodology revealed 2.0 fold increased in the production of BLIS. The ability of BLIS to inhibit the Listeria monocytogenes ATCC 7644 has enforced its application as a natural preservative to resolve the uncontrolled issues of food industry.

Molecular characterization of Bacillus, lactic acid bacteria and yeast as potential probiotic isolated from fermented food

Abstract Spontaneous fermentation or traditional method of preserving indigenous foods using several microorganisms, is frequently practiced in the marginal world. Fermented seeds and milk are mostly consumed in the form of food condiments and desserts in Africa, Asia and others parts of world. Our previous studies deal with the production of Bacteriocin-Like Inhibitory Substances by Bacillus from fermented food and quality of fermented milk consumed in Burkina Faso. The rep-PCR and sequencing were used to characterize thirty eights strains isolated various fermented foods from Burkina Faso. Phylogenetic tree were constructed by the neighbour-joining method based on 16S or 26S rRNA genes sequences using MEGA X. Based on colonies characteristics and cells morphology, biochemical tests and gene sequencing, the isolates were identified as Bacillus cereus sensu lato (13), Bacillus pumilus group (03) with one strain (LCG1) presumed LAB was identified as Bacillus australimaris or Bacillus pumilus by 16S rRNA sequencing, Enterococcus durans (03), Lactobacillus paracasei (03), Lactobacillus plantarum (04), Leuconostoc pseudomesenteroides (01), Saccharomyces cerevisiae (04), Kluyveromyces marxianus (01), Candida tropicalis (01), Pichia kudriavzevii (01), Clavispora lusitaniae (02), Rhodotorula mucilaginosa (01) and, Cyberlindnera fabianii (01). Several microorganisms with potential technological interest are housed in fermented foods from Burkina Faso. These microorganisms are responsible for the fermentation of food through their enzymatic activity, leading to production of fermented food with desirable organoleptic characteristics, improved food safety, the enrichment of nutrients and the promotion of health of consumers.

Effects of pH and sugar supplements on bacteriocin-like inhibitory substance production by Pediococcus pentosaceus.

To improve bacteriocin-like inhibitory substance (BLIS) production by Pediococcus pentosaceus ATCC 43200, the influence of pH as well as the addition of sugars-either prebiotic (inulin) or not (sucrose)-on its metabolism were investigated. This strain was grown at pH 5.0 or 6.0 either in glucose-based MRS medium (control) or after addition of 0.5, 1.0 or 1.5% (w/w) sucrose and inulin (GSI-MRS) in the same percentages. In the control medium at pH 5.0, cell mass concentration after 48h of fermentation (Xmax = 2.26g/L), maximum specific growth rate (µmax = 0.180h-1) and generation time (Tg = 3.84h) were statistically coincident with those obtained in supplemented media. At pH 6.0 some variations occurred in these parameters between the control medium (Xmax = 2.68g/L; µmax = 0.32h-1; Tg = 2.17h) and the above supplemented media (Xmax = 1.90, 2.52 and 1.86g/L; µmax = 0.26, 0.33 and 0.32h-1; Tg = 2.62, 2.06 and 2.11h, respectively). Lactate production was remarkable at both pH values (13 and 16g/L) and improved in all supplemented media, being 34 and 54% higher than in their respective control media, regardless of the concentration of these ingredients. Cell-free supernatant of the fermented control medium at pH 5.0 displayed an antimicrobial activity against Enterococcus 101 5.3% higher than that at pH 6.0 and even 20% higher than those of all supplemented media, regardless of the concentration of supplements. BLIS production was favored either at pH 5.0 or in the absence of any additional supplements, which were able, instead, to stimulate growth and lactate production by P. pentosaceus.

Isolation and Characterization of Lactic Acid Bacteria Producing Bacteriocin like Inhibitory Substance (BLIS) from “Gappal”, a Dairy Product from Burkina Faso

Indigenous fermented foods are known for their nutritional and functional properties but they are often spoiled by pathogenic bacteria that can constitute a food safety problem. “Gappal” is a no-thermal treat food based on millet dough and milk and its production conditions can constitute a food safety problem. The aim of this study was to screen and identify LAB producing Bacteriocin-like inhibitory substances using a matrix similar to “Gappal”. The detection of potential BLIS was first performed using overlaid method after enrichment of samples in whey and millet dough. The isolates demonstrating inhibiting area were preselected, purified and tested for the presence of antibacterial properties using their neutralized cell-free culture supernatant and subsequently treated with catalase in combination with protease, pepsin or trypsin. The antimicrobial effect of two isolates (Gbf48 and Gbf50) after growth on MRS broth over 12 h at 30˚C were active against E. faecalis ATCC 19433, M. luteus ATCC 49732, S. aureus ATCC 2523, L. monocytogenes, B. megaterium, B. sphaericus and B. cereus with an activity of 2560 AU/mL. The 16S RNA gene sequencing identification indicated that these isolates are Pediococcus acidilactici. Gbf 48 and Gbf 50 could be used to improve preservative factors for a controlled fermentation of non thermal treatment fermented food for their potential of acidification adds to BLIS production.

Stability of Bacteriocin-Like Inhibitory Substance (BLIS) Produced by Pediococcus acidilactici kp10 at Different Extreme Conditions.

Nowadays, bacteriocin industry has substantially grown replacing the role of chemical preservatives in enhancing shelf-life and safety of food. The progress in bacteriocin study has been supported by the emerging of consumer demand on the applications of natural food preservatives. Since food is a complex ecosystem, the characteristics of bacteriocin determine the effectiveness of their incorporation into the food products. Among four commercial media (M17 broth, MRS broth, tryptic soy broth, and nutrient broth) tested, the highest growth of Pediococcus acidilactici kp10 and bacteriocin-like-inhibitory substance (BLIS) production were obtained in the cultivation with M17. BLIS production was found to be a growth associated process where the production was increased concomitantly with the growth of producing strain, P. acidilactici kp10. The antimicrobial property of BLIS against three indicator microorganisms (Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus) remained stable upon heating at 100°C but not detectable at 121°C. The BLIS activity was also observed to be stable and active at a wide pH range (pH 2 to pH 7). The BLIS activity remained constant at -20°C and -80°C for 1 month of storage. However, the activity dropped after 3 and 6 months of storage at 4°C, -20°C, and -80°C with more than 80% reduction. The ability of bacteriocin from P. acidilactici kp10 to inhibit food-borne pathogens while remaining stable and active at extreme pH and temperature is of potential interest for future applications in food preservatives.

Identification of Streptococcus gallolyticus subsp. gallolyticus (Biotype I) Competence-Stimulating Peptide Pheromone.

Streptococcus gallolyticus subsp. gallolyticus, a member of the group D streptococci, is normally found in the bovine rumen and human gut. It is an opportunistic pathogen that was recently determined to be a bacterial driver of colorectal cancer, in addition to causing other diseases, such as infective endocarditis, bacteremia, neonatal meningitis, and septicemia. As an emerging pathogen, not much is known about this bacterium, its virulence mechanisms, or its virulence regulatory pathways. Previous studies suggest that S. gallolyticus subsp. gallolyticus uses a ComRS pathway, one of many Streptococcus quorum-sensing circuitries, for competence. However, thus far, the ubiquitous ComABCDE pathway has not been studied, nor has its regulatory role in S. gallolyticus subsp. gallolyticus We therefore sought to study the S. gallolyticus subsp. gallolyticus ComABCDE quorum-sensing pathway and have identified its peptide pheromone, which is termed the competence-stimulating peptide (CSP). We further determined that this peptide regulates the production of bacteriocin-like inhibitory substances (BLISs), a phenotype that has been linked with the ComABCDE pathway in both Streptococcus pneumoniae and Streptococcus mutans Our data show that S. gallolyticus subsp. gallolyticus TX20005 produces a 21-mer CSP signal, which differs from CSP signals of other Streptococcus species in that its active form begins three residues after the double-glycine leader signal of the ComC precursor peptide. Additionally, our data suggest that this peptide might not be related to competence induction, as opposed to CSP signaling peptides in other Streptococcus species. This study provides the first evidence that S. gallolyticus subsp. gallolyticus utilizes quorum sensing to eliminate competitors, presenting a potential pathway to target this emerging human pathogen.IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus is an emerging human pathogen known as a causative agent of infective endocarditis, and recently, of colorectal cancer. In this work, we revealed a functional quorum-sensing circuitry in S. gallolyticus subsp. gallolyticus, including the identification of the central signaling peptide pheromone, competence-stimulating peptide (CSP), and the regulatory role of this circuitry in the production of bacteriocin-like inhibitory substances (BLISs). This work uncovered a mechanism by which this bacterium outcompetes other bacterial species and thus provides a potential tool to study this opportunistic pathogen.

Optimization of biosurfactant and bacteriocin-like inhibitory substance (BLIS) production by Lactococcus lactis CECT-4434 from agroindustrial waste

The use of low-cost substrates and wastes as fermentative media is a simple strategy for overcoming excessive production costs of bioactive molecules and to make the process more profitable. In this context, the aim of this work was to optimize the production of biosurfactant by Lactococcus lactis CECT-4434 from whey and vinasse from industrial sources. For this purpose, a 24 central composite design (CCD) was proposed to maximize biosurfactant production. Besides, the ability of Lc. lactis to synthesize bacteriocin-like inhibitory substance (BLIS) was also evaluated, therefore contributing to the profitability of the biotechnological process. Results showed that whey at 15% positively influenced biosurfactant production (on average 8.9 mg/L), reducing surface tension by about 18.1 mN/m. Molecular composition of the biosurfactant was determined by Fourier transform infrared spectroscopy and nuclear magnetic resonance which confirmed the presence of carbohydrates, lipids and proteins, defining this molecule as a glycolipopeptide. Regarding the BLIS synthesis, the highest concentration of sucrose (2.0%) and the lowest vinasse level (1.0%) reduced the production of BLIS identified by the decrease in antimicrobial activity (mean inhibition zone of 10.7 mm). Conversely, the lowest sucrose concentration and highest vinasse level exerted the strongest inhibition, with a mean inhibition zone of 12.2 mm against Staphylococcus aureus CECT-239.