Influence of different culture media on the antimicrobial activity of Pediococcus pentosaceus ST65ACC against Listeria monocytogenes.

Pediococcus pentosaceus ST65ACC is a bacteriocinogenic lactic acid bacteria (LAB) isolated from Brazilian artisanal cheese that is capable of inhibiting different food pathogens, mainly Listeria monocytogenes. The production of bacteriocins can be influenced by several growth conditions, such as temperature, pH, and medium composition. This study aimed to evaluate the effect of different culture media on the production of bacteriocins and antimicrobial activity of P. pentosaceus ST65ACC on L. monocytogenes Scott A. The strains were inoculated alone and in coculture in four different media: BHI broth, MRS broth, meat broth, and reconstituted skim milk (RSM) 10% (w/v). The culture media were then incubated at 37 °C for 96 h, and count analysis, pH measurement, and bacteriocin production were performed at 0, 24, 48, 72 and 96 h. L. monocytogenes was inhibited to nondetectable levels in coculture with P. pentosaceus ST65ACC in MRS broth within 96 h, consistent with the high production of bacteriocin throughout the analysis period (3,200-12,800 AU/mL). However, lower inhibitory activities of P. pentosaceus ST65ACC on L. monocytogenes Scott A were recorded in BHI, RSM, and meat broth, with low or no production of bacteriocins at the analyzed times. The composition of these culture media may have repressed the production and activity of bacteriocins and, consequently, the antagonist activity of P. pentosaceus ST65ACC on L. monocytogenes Scott A. The results showed that the antimicrobial activity was more effective in MRS broth, presenting greater production of bacteriocins and less variability when compared to the other media analyzed.

Development of a microencapsulated probiotic delivery system with whey, xanthan, and pectin.

Pediococcus pentosaceus is a lactic acid bacterium that has probiotic potential proven by studies. However, its viability can be affected by adverse conditions such as storage, heat stress, and even gastrointestinal passage. Thus, the aim of the present study was to microencapsulate and characterize microcapsules obtained by spray drying and produced only with whey powder (W) or whey powder combined with pectin (WP) or xanthan (WX) in the protection of P. pentosaceus P107. In the storage test at temperatures of - 20 °C and 4 °C, the most viable microcapsule was WP (whey powder and pectin), although WX (whey powder and xanthan) presented better stability at 25 °C. In addition, WX did not show stability to ensure probiotic potential (< 6 Log CFU mL-1) for 110 days and the microcapsule W (whey powder) maintained probiotic viability at the three temperatures (- 20 °C, 4 °C, and 25 °C) for 180 days. In the exposition to simulated gastrointestinal juice, the WX microcapsule showed the best results in all tested conditions, presenting high cellular viability. For the thermal resistance test, WP microcapsule was shown to be efficient in the protection of P. pentosaceus P107 cells. The Fourier transform infrared spectroscopy (FTIR) results showed that there was no chemical interaction between microcapsules of whey powder combined with xanthan or pectin. The three microcapsules produced were able to protect the cell viability of the microorganism, as well as the drying parameters were adequate for the microcapsules produced in this study.

Genomic Analyses of Pediococcus pentosaceus ST65ACC, a Bacteriocinogenic Strain Isolated from Artisanal Raw-Milk Cheese.

Pediococcus pentosaceus ST65ACC was obtained from a Brazilian artisanal cheese (BAC) and characterized as bacteriocinogenic. This strain presented beneficial properties in previous studies, indicating its potential as a probiotic candidate. In this study, we aimed to carry out a genetic characterization based on whole-genome sequencing (WGS), including taxonomy, biotechnological properties, bacteriocin clusters and safety-related genes. WGS was performed using the Illumina MiSeq platform and the genome was annotated with the Prokaryotic Genome Annotation (Prokka). P. pentosaceus ST65ACC taxonomy was investigated and bacteriocin genes clusters were identified by BAGEL4, metabolic pathways were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and safety-related genes were checked. P. pentosaceus ST65ACC had a total draft genome size of 1,933,194 bp with a GC content of 37.00%, and encoded 1950 protein coding sequences (CDSs), 6 rRNA, 55 tRNA, 1 tmRNA and no plasmids were detected. The analysis revealed absence of a CRISPR/Cas system, bacteriocin gene clusters for pediocin PA-1/AcH and penocin-A were identified. Genes related to beneficial properties, such as stress adaptation genes and adhesion genes, were identified. Furthermore, genes related to biogenic amines and virulence-related genes were not detected. Genes related to antibiotic resistance were identified, but not in prophage regions. Based on the obtained results, the beneficial potential of P. pentosaceus ST65ACC was confirmed, allowing its characterization as a potential probiotic candidate.

Effect of Polydextrose on the Growth of Pediococcus pentosaceus as Well as Lactic Acid and Bacteriocin-like Inhibitory Substances (BLIS) Production.

Pediococcus pentosaceus was cultivated in MRS medium supplemented or not with polydextrose under different conditions in order to evaluate its effect on cell growth, lactic acid and bacteriocin-like inhibitory substance (BLIS) production. Independent variables were pH (4.0, 5.0, 6.0), rotational speed (50, 100, 150 rpm), polydextrose concentration (0.5, 1.0, 1.5%) and temperature (25, 30, 35 °C), while cell concentration and productivity after 24 h, maximum specific growth rate, specific rate of substrate (glucose) consumption, volumetric and specific lactic acid productivities, yields of biomass and lactic acid on consumed substrate were the dependent. The maximum cell concentration (10.24 ± 0.16 gX L-1) and productivity (0.42 ± 0.01 gX L-1 h-1) were achieved at pH 6.0, 35 °C, 150 rpm using 1.5% polydextrose, while the maximum specific growth rate (0.99 ± 0.01 h-1) and yield of biomass (2.96 ± 0.34 gX gS-1) were achieved at the same pH and polydextrose concentration, but at 25 °C and 50 rpm. The specific substrate consumption rate (0.09 ± 0.02 gS gX-1 h-1) and the volumetric lactic acid productivity (0.44 ± 0.02 gP L-1 h-1) were maximized at pH 6.0, 35 °C, 50 rpm and 0.5% polydextrose. BLIS produced in this last run displayed the highest antibacterial activity against Escherichia coli, while the same activity was displayed against Enterococcus faecium using 1.5% polydextrose. These results appear to be quite promising in view of possible production of this BLIS as an antibacterial agent in the food industry.

Pan-genomic and comparative analysis of Pediococcus pentosaceus focused on the in silico assessment of pediocin-like bacteriocins.

Bacteriocins are antimicrobial peptides produced by different species of bacteria, especially the Gram-positive lactic acid bacteria (LAB). Pediococcus pentosaceus is widely applied in the industry and stands out as Bacteriocin-Like Inhibitory Substances (BLIS) producer known to inhibit pathogens commonly considered a concern in the food industries. This study aimed to perform in silico comparisons of P. pentosaceus genomes available in the public GenBank database focusing on their pediocin-like bacteriocins repertoire. The pan-genome analysis evidenced a temporal signal in the pattern of gene gain and loss, supporting the hypothesis that the complete genetic repertoire of this group of bacteria is still uncovered. Thirteen bacteriocin genes from Class II and III were predicted in the accessory genome. Four pediocin-like bacteriocins (54% of the detected bacteriocin repertoire) and their accompanying immunity genes are highlighted; penocin A, coagulin A, pediocin PA-1, and plantaricin 423. Additionally, in silico, modeling of the pediocin-like bacteriocins revealed different configurations of the helix motif compared to other physically determined pediocin-like structures. Comparative and phylogenomic analyses support the hypothesis that a dynamic mechanism of bacteriocin acquisition and purging is not dependent on the bacterial isolation source origin. Synteny analysis revealed that while coagulin A, pediocin PA-1, and Plantaricin 423 loci are associated with insertion sequences mainly from the IS30 family and are likely of plasmid origin, penocin A lies in a conserved chromosomal locus. The results presented here provide insights into the unique pediocin-like bacteriocin peptide fold, genomic diversity, and the evolution of the bacteriocin genetic repertoire of P. pentosaceus, shedding new insights into the role of these biomolecules for application in inhibiting bacterial pathogens, and suggesting that prospecting and sequencing new strains is still an alternative to mining for new probiotic compounds.

Evaluating the potential of Pediococcus pentosaceus as a biocontrol agent against tenuazonic acid-producing Alternaria alternata on livestock feeds.

AIMS: This study aims to demonstrate the potential of the lactic acid bacteria (LAB) Pediococcus pentosaceus LBM18 against the mycotoxin-producing Alternaria alternata TEF-1A and highlight its application as an effective grain silage inoculant to control mycotoxin contamination. METHODS AND RESULTS: The antifungal properties of Ped. pentosaceus lyophilized (PPL) were assessed by evaluating its effect on A. alternata TEF-1A grown in a corn silage-based medium, which included morphological changes by Scanning Electron Microscopy (SEM) observations, growth rate, conidia production assays, and inhibition of Tenuazonic acid (TeA) production by high-performance liquid chromatography (HPLC-MS/MS) analyses. Furthermore, TeA biosynthesis was monitored for changes at the molecular level by PKS gene expression. The growth and sporulation processes of A. alternata TEF-1A were affected by Ped. pentosaceus LBM18 in a concentration-dependent manner. Moreover, a significant inhibition of TeA production (74.3%) and the transcription level of the PKS gene (42.9%) was observed. CONCLUSIONS: Ped. pentosaceus is one of the promising LAB to be applied as an inoculant for corn silage preservation, aiming to inhibit mycotoxigenic fungi growth and their mycotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: Ped. pentosaceus could be used as an inoculant to reduce fungal and mycotoxins contamination in grain silage production.

Performance of lactating cows fed sugarcane silage treated with microbial additives

Background: No studies have been conducted evaluating sugarcane silage associated with both Lactobacillus plantarum and Pediococcus pentosaceus for lactating dairy cows. Objective: To evaluate diets containing different roughages with and without microbial inoculants on intake, digestibility and milk yield of medium-producing cows. Methods: A total of 15 Holstein cows distributed into a randomized block design were used. Dietary treatments were: 1) a corn silage-based diet (CS), 2) a fresh sugarcane-based diet (SC), 3) a sugarcane silage ensiled without inoculant (SS), 4) sugarcane silage ensiled with Lactobacillus buchneri (SSLB), and 5) sugarcane silage ensiled with Lactobacillus plantarum and Pediococcus pentosaceus (SSLP). Results: Digestible organic matter intake (DOMI) was lower (p<0.05) in cows fed SSLB (9.77 kg day-1) when compared with cows fed CS (13.29 kg day-1) and SSLP (12.42 kg day-1). Ensiling of sugarcane increased intake of neutral detergent fiber (NDF) compared to SC (mean of 6.00 kg day-1 versus 4.97 kg day-1; p<0.05). Dry matter digestibility was greater (p<0.05) in CS (77.80%) compared with diets based on sugarcane silage, whereas NDF digestibility was lower for treatments with sugarcane (p<0.05). Milk yield was similar among CS (27.99 kg), SC (25.59 kg), and silages with additives (25.47 and 27.07 for SSLB and SSLP, respectively). Cows fed CS produced more fat-corrected milk (25.89 kg) than those fed sugarcane-based diets (p<0.05). Conclusions: Fresh sugarcane or sugarcane silage with additives can be used as a roughage source for dairy cows producing up to 23.43 kg d-1 fat-corrected milk, considering the total diet is properly balanced.

Antecedentes: No existen estudios que evalúen la adición de Lactobacillus plantarum y Pediococcus pentosaceus en el ensilaje de caña de azúcar para vacas lactantes. Objetivo: Evaluar dietas con diferentes forrajes y ensilajes con y sin inoculantes microbianos sobre el consumo, digestibilidad y producción lactea de vacas de mediana producción. Metodología: Quince vacas Holstein se distribuyeron en un diseño de bloques al azar. Los tratamientos fueron: 1) dieta con ensilaje de maíz (CS), 2) dieta con caña de azúcar fresca (SC), 3) dieta con ensilaje de caña sin inoculantes (SS), 4) dieta con ensilaje de caña de azúcar ensilada con Lactobacillus buchneri (SSLB), y 5) dieta con ensilaje de caña de azúcar ensilada con Lactobacillus plantarum y Pediococcus pentosaceus (SSLP). Resultados: El consumo de materia orgánica digestible fue menor (p<0,05) en la dieta SSLB (9,77 kg day-1) en comparación con CS (13,29 kg dia-1) y SSLP (12,42 kg dia-1). El ensilaje de caña de azúcar promovió un mayor consumo de fibra detergente neutra (NDF) en comparación con SC (promedio de 6,00 kg dia-1 versus 4,97 kg dia-1; p<0,05). La digestibilidad de la materia seca fue mayor (p<0,05) para la dieta CS (77,80%) en comparación con las dietas con ensilaje de caña de azúcar, mientras que la digestibilidad de la NDF fue menor para las dietas a base de caña de azúcar (p<0,05). La producción de leche fue similar entre CS (27,99 kg), SC (25,59 kg) y ensilajes con aditivos (25,47 y 27,07 para SSLB y SSLP, respectivamente). Las vacas alimentadas con ensilaje de maíz produjeron más leche corregida por grasa (25,89 kg) que las alimentadas con dietas a base de caña de azúcar (p<0,05). Conclusión: La caña de azúcar fresca o ensilada con aditivos se puede utilizar como fuente de forraje para vacas que producen hasta 23,43 kg d-1 leche corregida por grasa, siempre que la dieta total esté equilibrada adecuadamente.

Antecedentes: Há carência de estudos avaliando a adição de Lactobacillus plantarum e Pediococcus pentosaceus na silagem de cana-de-açúcar para vacas em lactação. Objetivo: Avaliar dietas contendo diferentes forragens e silagens com e sem inoculantes microbianos sobre o consumo, digestibilidade e produção de leite de vacas de média produção de leite. Métodos: Quinze vacas Holandesas foram distribuídas em um delineamento em blocos casualizados. Os tratamentos foram: 1) dieta com silagem de milho (CS), 2) dieta com cana-de-açúcar fresca (SC), 3) dieta com silagem de cana ensilada sem inoculantes (SS), 4) dieta com silagem de cana-de-açúcar ensilada com Lactobacillus buchneri (SSLB), ou 5) dieta com silagem de cana- de-açúcar ensilada com Lactobacillus plantarum, and Pediococcus pentosaceus (SSLP). Resultados: O consumo de matéria orgânica digestível foi menor (p<0,05) na dieta SSLB (9,77 kg day-1) comparada com CS (13,29 kg dia-1) e SSLP (12,42 kg dia-1). A ensilagem da cana-de-açúcar promoveu maior consumo de fibra em detergente neutro (FDN) em comparação com SC (média de 6,00 kg dia-1 versus 4.97 kg dia-1; p<0.05). A digestibilidade da matéria seca foi maior (p<0,05) para a dieta CS (77,80%) comparada com as dietas com silagem de cana-de-açúcar, enquanto que a digestibilidade da FDN foi menor para as dietas baseadas em cana-de-açúcar (p<0,05). A produção de leite foi similar entre CS (27,99 kg), SC (25,59 kg) e silagens com aditivos (25,47 e 27,07 para SSLB e SSLP, respectivamente). Vacas alimentadas com silagem de milho produziram mais leite corrigido para gordura (25,89 kg) que aquelas alimentadas com dietas baseadas em cana-de-açúcar (p<0,05). Conclusão: A cana-de-açúcar fresca ou ensilada com aditivos pode ser utilizada como fonte volumosa para vacas produzindo até 23,43 kg d-1 leite corrigido para gordura, desde que a dieta total esteja apropriadamente balanceada.

Probiotic Properties and Proteomic Analysis of Pediococcus pentosaceus 1101.

Pediococcus pentosaceus 1101 was identified by using 16S rRNA and MALDI-Biotyper. The strain was exposed to conditions that resemble the gastrointestinal tract (GT) to evaluate its probiotic properties. That included the growth kinetics, proteolytic and inhibitory activities within a pH range, survival at low pH and in the presence of bile salts, antagonistic activity, cell-adhesion properties, and antibiotic resistance. The evaluation was followed by a genomic and proteomic analysis that involved the identification of proteins obtained under control and gastrointestinal conditions. The strain showed antagonistic activity against Gram-negative and Gram-positive bacteria, high resistance to acidity (87% logarithmic survival rate, pH 2) and bile salts (99% logarithmic survival rate, 0.5% w/v), and hydrophobic binding, as well as sensitivity to penicillin, amoxicillin, and chloramphenicol. On the other hand, P. pentosaceus 1101 has a genome size of 1.76 Mbp, with 1754 coding sequences, 55 rRNAs, and 33 tRNAs. The proteomic analysis showed that 120 proteins were involved in mechanisms in which the strain senses the effects of acid and bile salts. Moreover, the strain produces at least one lytic enzyme (N-acetylmuramoyl-L-alanine amidase; 32 kDa) that may be related to the antimicrobial activity. Therefore, proteins identified might be a key factor when it comes to the adaptation of P. pentosaceus 1101 into the GT and associated with its technological and probiotic properties.

Pediocin PA-1 production by Pediococcus pentosaceus ET34 using non-detoxified hemicellulose hydrolysate obtained from hydrothermal pretreatment of sugarcane bagasse.

Listeria monocytogenes is one of the foodborne pathogens of most concern for food safety. To limit its presence in foods, bacteriocins have been proposed as natural bio-preservatives. Herein, a bacteriocin was produced on hemicellulose hydrolysate of sugarcane bagasse by Pediococcus pentosaceous ET34, whose genome sequencing revealed an operon with 100% similarity to that of pediocin PA-1. ET34 grown on hydrolysate-containing medium led to an increase in the expression of PA-1 genes and a non-optimized purification step sequence resulted in a yield of 0.8 mg·L-1 of pure pediocin (purity > 95%). Culture conditions were optimized according to a central composite design using temperature and hydrolysate % as independent variables and validated in 3-L Erlenmeyers. Finally, a process for scaled-up implementation by sugar-ethanol industry was proposed, considering green chemistry and biorefinery concepts. This work stands up as an approach addressing a future proper sugarcane bagasse valorisation for pediocin production.

Isolation and Genetic Identification of Endophytic Lactic Acid Bacteria From the Amazonian Açai Fruits: Probiotics Features of Selected Strains and Their Potential to Inhibit Pathogens.

The açai palm (Euterpe oleracea) is native to the Amazon basin, a humid tropical forest. High levels of total mesophilic bacteria with high diversity have been consistently reported in açai fruits. As local consumers have few digestive problems, the results of the present study reveal the lactic acid bacteria (LAB) recovered from açai fruits with characteristics that suggest they are possible candidates for probiotics and antagonistic potential against pathogens for the first time. Açai fruits were sampled from five different locations in the Eastern Amazonia floodplains. Sixty-six isolates were recovered from fruits and tested for some probiotic characteristics following FAO/WHO guidelines. Approximately 65% of the isolates showed no catalase or oxidase activity, Gram-positive staining or cocci and bacilli cell morphology. Furthermore, 48% of the isolates demonstrated preliminary characteristics that suggest safety for use, as they presented no coagulase enzyme activity or gamma-hemolysis. These strains were identified as belonging to the genera Lactiplantibacillus and Pediococcus, and 32 strains also presented resistance to vancomycin, ciprofloxacin and streptomycin. In addition, 28 isolates showed a survival rate, expressed as log cycle reduction, higher than 0.9 under gastric conditions (pH 2). All strains tested positive in bile salts deconjugation tests and showed a survival rate higher than 0.8 in the presence of this salt. Regarding antimicrobial activity against pathogens, all strains were able to inhibit Salmonella Typhimurium (ATCC® 14028TM) and 97% were capable of inhibiting Escherichia coli (ATCC® 25922TM). Concerning the results of in vitro antagonistic assays, three isolates (B125, B135, and Z183 strains) were selected for antagonistic tests using açai juice contaminated with these two pathogens. All tested LAB strains were able to inhibit pathogen growth in açai juice. In summary, açai fruits are a potential source of LAB isolates to be investigated as probiotics.

Expression of coagulin A with low cytotoxic activity by Pediococcus pentosaceus ST65ACC isolated from raw milk cheese.

AIMS: We aimed to evaluate some specific conditions for growth of Pediococcus pentosaceus ST65ACC and its bacteriocin expression through ABC transporters; to purify the bacteriocin and determine its sequence; and to evaluate the cytotoxicity potential of the purified bacteriocin(s). METHODS AND RESULTS: The results presented for growth behaviour of P. pentosaceus ST65ACC showed that the bacterial growth was slightly influenced when cultured in MRS broth with different amounts of inoculum: 1, 2, 5 and 10%. The bacteriocin activity increased when 5 and 10% inocula were used. The carbon source (glucose) used in different amounts (1, 2, 3 or 4%) had no significant effect on growth and bacteriocin production. The studied strain P. pentosaceus ST65ACC was able to metabolize xylooligosaccharide (XOS) as the sole carbon source, resulting in the production of an antimicrobial peptide. The genes involved in the ABC transport system and sugar metabolism of P. pentosaceus ST65ACC were expressed at different levels. The bacteriocin produced by P. pentosaceus ST65ACC was partially purified by precipitation with ammonium sulphate (40% saturation), followed by reversed-phase liquid chromatography, resulting in the identification of an active bacteriocin. Tandem mass spectrometry was used to identify the partial sequence KYYGNGVTCGKHSCSVDWGK sharing high similarity to coagulin A. The semi-purified bacteriocin had low cytotoxicity based on estimated values for maximal nontoxic concentration (MNC) and cytotoxicity concentration (CC50 ). CONCLUSIONS: The bacteriocin produced by P. pentosaceus ST65ACC is similar to coagulin, with low cytotoxicity, strong antimicrobial activity and possible additional metabolite routes in the producer cell. In addition to MRS broth, bacteriocin was produced also in medium containing XOS (as the single carbon source). SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of evaluation of the role of ABC transporters in the expression of bacteriocin by P. pentosaceus, cultured in MRS and XOS.

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 48 h of fermentation (Xmax = 2.26 g/L), maximum specific growth rate (µmax = 0.180 h-1) and generation time (Tg = 3.84 h) were statistically coincident with those obtained in supplemented media. At pH 6.0 some variations occurred in these parameters between the control medium (Xmax = 2.68 g/L; µmax = 0.32 h-1; Tg = 2.17 h) and the above supplemented media (Xmax = 1.90, 2.52 and 1.86 g/L; µmax = 0.26, 0.33 and 0.32 h-1; Tg = 2.62, 2.06 and 2.11 h, respectively). Lactate production was remarkable at both pH values (13 and 16 g/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.

Bioprodução de pediocina por Pediococcus pentosaceus ET34 em resíduo agroindustrial hidrolisado bagaço de cana-de-açúcar / Bioproduction of Pediocin by Pediococcus pentosaceus ET34 in hydrolyzed sugar cane bagasse agroindustrial residue

Um dos grandes desafios nas indústrias alimentícia, farmacêutica e agropecuária é a busca por novos compostos para substituir os antibióticos. Como possíveis candidatos estão as bacteriocinas para serem utilizados paralelamente aos antibióticos ou até substituí-los. As bactérias ácido-láticas podem produzir substâncias inibitórias semelhantes às bacteriocinas (BLIS - bacteriocin-like inhibitory substances) que possuem efeito bacteriostático ou bactericida contra diferentes grupos de bactéria sendo largamente utilizadas como bioconservantes alimentares. Neste contexto, o objetivo deste trabalho foi o emprego de um resíduo agroindustrial, o hidrolisado de bagaço de cana-de-açúcar, como fonte de carbono em cultivos fermentativos para produção do BLIS pela cepa Pediococcus pentosaceus ET 34. Resultados revelaram que as células de ET34 foram capazes de crescer utilizando este resíduo agroindustrial como fonte de carbono e ensaios utilizando planejamento fatorial demonstraram que a agitação não influencia na produção de BLIS. Ao escalonar o cultivo em biorreatores, foi verificado que tanto o crescimento como a atividade antimicrobiana foram semelhantes aos obtidos em bancada com exceção da condição de 25% (v/v) de HBC (hidrolisado de bagaço de cana) que devido a maior viscosidade do meio, resultou em uma diminuição no crescimento e de produção de BLIS. O BLIS produzido por células de ET34 utilizando o HBC como fonte de carbono foi parcialmente purificado por sulfato de amônio e demonstrou atividade contra Listeria monocytogenes ATCC 934 e Salmonella enterica CECT 724. Desta maneira, pode-se concluir que Pediococcus pentosaceus ET34 foi capaz de crescer em meio contendo HBC como fonte de carbono produzindo BLIS em seu sobrenadante com ação frente a diferentes bactérias patogênicas. A possibilidade de utilizar uma fonte alternativa de carbono pode diminuir o custo de processo consideravelmente. Além disso, ensaios de planejamento fatorial, superfície de resposta e escalonamento em biorreator de bancada indicaram que concentrações baixas de HBC (5-15%, v/v) a 35 °C resultaram na maior produção de BLIS

The great challenge in the food, pharmaceutical and agricultural industries is the search for new compounds to replace antibiotics. Bacteriocins are possible candidates that can be used in parallel with the antibiotics or even to replace them. Lactic-acid bacteria can produce bacteriocin inhibitory substances (BLIS - bacteriocin-like inhibitory substances) that have a bacteriostatic or bactericidal effect against different bacterial species and are widely used as food bioconservatives. In this context, the aim of this work was to use of an agroindustrial waste, hydrolyzed sugarcane bagasse, as a carbon source in fermentative cultures for the production of BLIS by Pediococcus pentosaceus ET 34 strain. Results revealed that ET34 cells were able to grow using this agroindustrial residue as a carbon source, and trials using factorial design showed that agitation did not influence on the production of BLIS. When it was perform cultivation scale up in bioreactors, it was verified that both the growth and the antimicrobial activity were similar to those obtained in the workbench with the exception of the condition of 25% (v/v) of HBC (sugarcane bagasse hydrolyzate) that due to its higher viscosity, resulted in a decrease in growth and BLIS production. BLIS produced by ET34 cells using HBC as a carbon source that was partially purified by ammonium sulfate showed activity against Listeria monocytogenes ATCC 934 and Salmonella enterica CECT 724. Thus, it can be concluded that Pediococcus pentosaceus ET34 was able to grow in medium containing HBC as carbon source producing BLIS in its supernatant with action against different pathogenic bacteria. The possibility of using an alternative carbon source can greatly reduce the process cost. In addition, factorial design, response surface and scale up trials in bench bioreactors indicated that low concentrations of HBC (5-15% v/v) at 35 ºC resulted in higher BLIS production

Optimization of the yield of bacteriocin-like substance (BLIS) produced by Pediococcus pentosaceus and its application as food bioconservative / Otimização do rendimento de substância semelhante a bacteriocina (BLIS) produzido por Pediococcus pentosaceus e sua aplicação como bioconservante de alimentos

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria (LABs), which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. However, when this bacteriocin has not been completely characterized regarding its amino acid and the nucleotide sequences of the corresponding gene, the qualified term bacteriocin-like inhibitory substance (BLIS) is recommended. In order to increase the antimicrobial activity of bacteriocins, the ability of probiotics LABs, such as Pediococcus pentosaceus, to ferment different carbon and nitrogen sources has been studied. For the development of an improved culture medium, carbon and nitrogen sources must be considered as nutrients responsible for cell growth and bacteriocin production. The best condition, after 48 h of cultivation, for growth (3.420 g/L) and for BLIS production by Pediococcus pentosaceus ATCC 43200 was in Man, Rogosa and Sharp (MRS) culture medium supplemented with 1.5% peptone, initial pH 6.0 and under the following culture conditions: anaerobiosis, 30oC and agitation of 200 rpm. Compared with control (MRS without supplement), the growth of Pediococcus was significantly lower (1.995 g/L) as well as it reduced significantly its generation time from 2.05 h (control) to 1.28 h (MRS supplemented), a reduction of approximately 62.5%. Moreover, addiction of peptone to MRS medium promoted reduction of 4 h to the Pediococcus exponential phase onset. Regarding BLIS antimicrobial activity, addition of nitrogen source to MRS medium was also quite significant. Through the agar diffusion method, BLIS showed inhibition halos between 12.50 and 19.50 mm against LABs strains (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 and Carnobacterium piscicola CECT 4020). Against Listeria strains (Listeria innocua NCTC 111288 and Listeria seeligeri NCTC 11289), their antimicrobial activity was better detected in liquid medium assay, evaluating the minimal inhibitory concentration of 50%. BLIS was able to inhibit 60 and 100% of L. seeligeri and L. innocua, respectively, as well as, diluted 1x (v/v) in water was able to inhibit 100% growth of both Listeria. BLIS 17 showed also good results as food preservative when applied in ready-to-eat pork ham artificially contaminated with L. seeligeri in vacuum-package at 4oC during shelf life of 10 days. BLIS was able to maintain low Listeria multiplication, lower samples weight loss, low lipid peroxidation and good color parameters during samples storage. Results demonstrated the importance of optimizing the culture medium to increase microbial mass, to produce and to improve the activity of this antimicrobial molecule. Moreover, results also suggest the possible application of BLIS as a natural food preservative

Bacteriocinas são peptídeos produzidos por várias espécies de bactérias, especialmente bactérias ácido-láticas (BALs) e apresentam um amplo espectro de ação contra bactérias deteriorantes e patógenos de origem alimentar. Entretanto, quando estas bacteriocinas não foram completamente caracterizadas quanto a sequência de seus nucleotídeos e do seu gene correspondente, é recomendada a denominação de substância semelhante a bacteriocina (BLIS). Para aumentar a atividade antimicrobiana de bacteriocinas, a habilidade de BALs probióticas, como Pediococcus pentosaceus, em fermentar diferentes fontes de carbono e nitrogênio tem sido estudado. Para o desenvolvimento de um meio de cultura melhorado, fontes de carbono e nitrogênio devem ser consideradas como nutrientes responsáveis pelo crescimento celular e pela produção de bacteriocina. A melhor condição, após 48 h de cultivo, para o crescimento (3,420 g/L) e para a produção de BLIS por P. pentosaceus ATCC 43200 foi em meio de cultivo Man, Rogosa e Sharp (MRS) suplementado com 1,5% de peptona, pH inicial 6,0 e sob as seguintes condições de cultivo: anaerobiose, 30oC e agitação de 200 rpm. Comparado ao controle (MRS sem suplementação), o crescimento de Pediococcus foi significativamente menor (1,995 g/L) assim, como também, reduziu significativamente o tempo de geração de 2,05 h (controle) para 1,28 h (MRS suplementado), uma redução de aproximadamente 62,5%. Além disso, a adição de peptona ao meio MRS promoveu redução de 4 h para o início da fase exponencial de Pediococcus. Quanto a atividade antimicrobiana de BLIS, a adição de fonte de nitrogênio ao meio MRS também foi bastante significativa. Através do método ágar difusão, BLIS apresentou halos de inibição entre 12,50 a 19,50 mm contra cepas de BALs (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 e Carnobacterium piscicola CECT 4020). Contra cepas de Listeria (Listeria innocua NCTC 11288 e Listeria seeligeri NCTC 11289), a sua atividade inibitória foi melhor detectada em meio líquido, através da determinação da concentração mínima inibitória de 50%. BLIS sem diluição foi capaz de inibir 60 e 100% de L. seeligeri e L. innocua, 15 respectivamente, assim como, diluído 1x (v/v) em água foi capaz de inibir 100% o crescimento de ambas Listeria. BLIS também apresentou bons resultados como conservante de alimento quando aplicado em presunto contaminado artificialmente com L. seeligeri e armazenado a 4oC a vácuo por 10 dias. BLIS foi capaz de manter baixa a multiplicação de Listeria, menor perda de peso das amostras, baixa peroxidação lipídica e bons parâmetros de cor durante o armazenamento das amostras. Os resultados demonstraram a importância de se otimizar meio de cultivo tanto para o aumento da massa microbiana como para a produção e melhoramento da atividade desta molécula antimicrobiana. Além disso, os resultados também sugerem a possível aplicação de BLIS como conservante natural de alimentos

Stimulating Effects of Sucrose and Inulin on Growth, Lactate, and Bacteriocin Productions by Pediococcus pentosaceus.

Sucrose and inulin, when combined with glucose, behaved as stimulating agents of bacteriocin production by Pediococcus pentosaceus ATCC 43200. When such microbial strain was grown in glucose-based Man, Rogosa, and Sharpe (MRS) medium, without any additional supplement, it showed higher maximum cell concentration (2.68 ± 1.10 g/L) and longer generation time (2.17 ± 0.02 h), but lower specific growth rate (0.32 ± 0.01 h-1) than in the same medium supplemented with 1.0% of both ingredients (2.53 ± 1.10 g/L, 1.60 ± 0.05 h and 0.43 ± 0.02 h-1, respectively). Glucose replacement by sucrose or inulin almost completely suppressed growth, hence confirming that it is the preferred carbon source for this strain. Qualitatively, similar results were observed for lactate production, which was 59.8% higher in glucose-based medium. Enterococcus and Listeria strains were sensitive to bacteriocin, whose antimicrobial effect after 8 h increased from 120.25 ± 0.35 to 144.00 ± 1.41 or 171.00 ± 1.41 AU/mL when sucrose or inulin was added to the glucose-based MRS medium. Sucrose and inulin were also able to speed up P. pentosaceus growth in the exponential phase.

Isolation and Probiotic Potential of Lactobacillus Salivarius and Pediococcus Pentosaceus in Specific Pathogen Free Chickens

This study was conducted to isolate Lactobacillus salivarius and Pediococcus pentosaceus strains from cecal content and investigate their probiotic potential in specific pathogen free (SPF) chickens. L. salivarius and P. pentosaceus strains were isolated from the cecal content of SPF chickens and identified by 16s rDNA sequence analysis by BLAST analysis at the National Center for Biotechnology Information and phylogenetic analysis using DNAStar software. In an in vivo experiment, 180 7-day-old SPF chickens were randomly assigned into three groups. Group 1 served as a control that was fed a basal diet without probiotic supplementation, and groups 2 and 3 were fed the basal diets supplemented with L. salivarius and P. pentosaceus at 2×108 CFU/g, respectively. Body weight (BW), average daily gain (ADG), feed conversion ratio (FCR), dressing percentage (DP), and the apparent digestibility of crude protein (AD-CP) were calculated. We also determined meat color, fat content, shear force, water content and pH value of breast and thigh muscles; ammonia, urea nitrogen and uric acid content in plasma; fecal ammonia emission level and pH value; and Lactobacillus and Escherichia coli in ceca. Compared with the control group, L. salivarius and P. pentosaceus supplementation significantly increased BW, ADG, DP, AD-CP, fat content of meat, and the number of Lactobacillus in ceca (p 0.05), and decreased FCR, plasma ammonia content, fecal ammonia emission, and pH value and the number of E. coli in ceca (p 0.05). In the in vitro experiment, L. salivarius and P. pentosaceus treatments significantly decreased the ammonia content in medium compared with the control group without probiotic treatment (p 0.05). These results suggest that P. pentosaceus and L. salivarius strains show promising probiotic properties for improving growth, meat quality and microenvironment in chickens and decreasing ammonia content in the medium.(AU)

Isolation and Probiotic Potential of Lactobacillus Salivarius and Pediococcus Pentosaceus in Specific Pathogen Free Chickens

This study was conducted to isolate Lactobacillus salivarius and Pediococcus pentosaceus strains from cecal content and investigate their probiotic potential in specific pathogen free (SPF) chickens. L. salivarius and P. pentosaceus strains were isolated from the cecal content of SPF chickens and identified by 16s rDNA sequence analysis by BLAST analysis at the National Center for Biotechnology Information and phylogenetic analysis using DNAStar software. In an in vivo experiment, 180 7-day-old SPF chickens were randomly assigned into three groups. Group 1 served as a control that was fed a basal diet without probiotic supplementation, and groups 2 and 3 were fed the basal diets supplemented with L. salivarius and P. pentosaceus at 2×108 CFU/g, respectively. Body weight (BW), average daily gain (ADG), feed conversion ratio (FCR), dressing percentage (DP), and the apparent digestibility of crude protein (AD-CP) were calculated. We also determined meat color, fat content, shear force, water content and pH value of breast and thigh muscles; ammonia, urea nitrogen and uric acid content in plasma; fecal ammonia emission level and pH value; and Lactobacillus and Escherichia coli in ceca. Compared with the control group, L. salivarius and P. pentosaceus supplementation significantly increased BW, ADG, DP, AD-CP, fat content of meat, and the number of Lactobacillus in ceca (p 0.05), and decreased FCR, plasma ammonia content, fecal ammonia emission, and pH value and the number of E. coli in ceca (p 0.05). In the in vitro experiment, L. salivarius and P. pentosaceus treatments significantly decreased the ammonia content in medium compared with the control group without probiotic treatment (p 0.05). These results suggest that P. pentosaceus and L. salivarius strains show promising probiotic properties for improving growth, meat quality and microenvironment in chickens and decreasing ammonia content in the medium.

Molecular identification and evaluation of the probiotic ability of lacticacid bacteria from sow colostrum / Identificación molecular y evaluación de la capacidad probiótica de bacterias ácido lácticas aisladas del calostro de cerdas / Identificação molecular e avaliação da capacidade probiótica de bactérias ácido lácticas isoladas do colostro de porcas

Probiotics have become relevant in animal health, especially because of their ability to replace antibiotic growth promoters. This study assessed the probiotic ability of lactic acid bacteria (LAB) isolated from sow colostrum obtained from pig farms in the southwest of Antioquia, Colombia. Bacteria were identified by molecular methods. Colostrum (1 ml) was collected from 20 sows postpartum. Colostrum (0.1 ml) was plated on Man Rogosa and Sharpe agar (MRS) (Merck), and incubated anaerobically at 37 °C for 48h. Probiotic capacity was determined measuring growth at pH 3,0 and growth on 0.3% w/v ox bile salts. Hemolytic activity, antimicrobial activity against Salmonella typhimurium, and sensitivity to antibiotics commonly used in veterinary were also determined. Molecular identification was made by gen16S Ribosomal analysis method. Of all the strains tested, only BAL 1 and BAL3 showed probiotic ability to grow and withstand an acidic pH (3,0) and 0.3% w/v of ox bile. They had γ-hemolytic activity, were catalase-negative, and inhibited growth of Salmonella typhymurium with halos larger than 11 mm. They were sensitive to amoxicillin, penicillin, chloramphenicol and erythromycin, and were identified as Pediococcus pentosaceus. The existence of Pediococcus pentosaceus in sow colostrum and its probiotic ability was demonstrated.

Los probióticos han cobrado importancia en la salud animal, especialmente por la capacidad de reemplazar los antibióticos promotores de crecimiento. En este trabajo se evaluó la capacidad probiótica de algunas bacterias ácido lácticas (BAL) aisladas de leche calostro de cerdas en granjas del Valle de Aburrá (zona sur); las cuales fueron identificadas por métodos moleculares. Materiales y métodos: Se recolectó 1mL de calostro de 20. Se inocularon 0.1 mL de cada una de las muestras para su aislamiento sobre el agar Man Rogosa Sharpe (MRS) (Merck), incubándolas a 37 °C durante 48h en anaerobiosis. Se determinó la capacidad probiótica realizando las siguiente pruebas: tolerancia a un pH ácido de 3,0, crecimiento en bilis de buey 0,3%P/v, actividad hemolítica, actividad antimicrobiana frente a Salmonella tiphymurium, y sensibilidad a los antibióticos de uso común en veterinaria; y su identificación molecular se realizó por el método análisis del gen16S Ribosomal. De todas las cepas evaluadas solo dos mostraron tener capacidad probiótica al crecer y resistir a un pH ácido de 3,0 y 0,3% P/v de bilis de buey, con actividad γ-hemolitica, catalasa negativa, que inhibieron el crecimiento de Salmonella tiphymurium con halos mayores a 11mm sensibles a Amoxicilina, Penicilina, Cloranfenicol y Eritromicina, e identificadas como Pediococcus pentosaceus. Se demostró la existencia de Pediococcus pentosaceus con características probióticas en el calostro de cerdas.

Os probióticos tem aumentado sua importância na saúde animal, especialmente pela sua capacidade de ser utilizados ao invés dos antibióticos promotores de crescimento. Neste trabalho, avaliou-se a capacidade probiotica de algumas bactérias ácido lácticas (BAL) isoladas de leite de colostro de fêmeas suínas, as amostras foram obtidas de granjas localizadas em Sudeste antioqueño: as bactérias identificaram-se por métodos moleculares. Materiais e métodos: Coletou-se 1ml de colostro em 20 fêmeas suínas recém paridas, das cidades previamente descritas, cultivou-se em superfície 0,1 ml da amostra em ágar Man Rogosa e Sharpe (MRS) (Merck), e foram incubadas a 37 °C/48h em anaerobiose. Determinou-se a capacidade probiótica avaliando o crescimento dos isolados a pH de 3,0 e a sais biliares de boi 0.3%P/v, além, foi determinada a atividade hemolítica, atividade antimicrobiana frente a Salmonella tiphymurium e sensibilidade aos antibióticos de uso comum em veterinária. A identificação molecular realizou-se pelo método de análise do gene 16S Ribosomal. Resultados: de todas as cepas avaliadas só dois, BAL 1 e BAL3 mostraram ter capacidade probiótica ao crescer e resistir a um pH ácido de 3,0 e 0,3% P/v de bílis de boi, com atividade γ-hemolítica, catalase negativa, que inibiram o crescimento de Salmonella tiphymurium com anéis maiores a 11mm, sensíveis a Amoxicilina, Penicilina, Cloranfenicol e Eritromicina, e identificadas como Pediococcus pentosaceus. Conclusão: demostrou-se a existência de Pediococcus pentosaceus com características probióticas no colostro de fêmeas suínas.

Characterization of the phenolic fraction from Argentine wine and its effect on viability and polysaccharide production of Pediococcus pentosaceus.

OBJECTIVES: To qualitatively and quantitatively characterize a low molecular weight phenolic fraction (LMF) of Malbec wine from Cafayate, Argentina, and evaluate its effect on viability and exopolysaccharide production of Pediococcus pentosaceus 12p, a wine spoilage bacterium. RESULTS: The phenolic compounds detected were, in general, comparable to data previously reported but hydroxycinnamic acids were detected at higher concentrations than determined in other studies. Addition of LMF at identical concentrations present in wine or a four times concentrated LMF mixture to a synthetic wine-like medium produced a diminution in bacterial viability and exopolysaccharide production in the supernatant culture. Transmission electron microscopy revealed damage of bacterial cell integrity after 96 h of incubation only in the presence of four times concentrated LMF. CONCLUSION: This is the first time a low molecular weight phenolic fraction has been characterized in Cafayate wine and it has demonstrated a marked antimicrobial effect on an exopolysaccharide-producing wine spoilage bacterium.

Scale up of dextran production from a mutant of Pediococcus pentosaceus (SPAm) using optimized medium in a bioreactor

The mutant of Pediococcus pentosaceus (SPAm) produced earlier by UV-mutagenesis exhibiting higher dextransucrase activity as compared to wild-type was used. The generated mutant SPAm gave 12.2 mg/ml, a 20 percent higher dextran than wild-type. Response surface methodology was carried out for further enhancement of dextran production. To enhance dextran production by the mutant SPAm, Plackett-Burman Design and a 2² full factorial Central Composite Design was employed. After response optimization, the optimum concentration of sucrose and yeast extract was 5.115 percent (w/v) and 0.635 percent (w/v), respectively. The experimental values of dextran 36.0 mg/ml at flask level and 35.0 mg/ml at bioreactor level were in good agreement with the predicted value of 40.8 mg/ml. The increase in dextran production by the mutant SPAm using the optimized medium was 3 fold higher as compared to unoptimized medium.