Isolation and characterization of Lactobacillus brevis C23 with ability to secrete antimicrobial substance for the inhibition of a foodborne pathogen Listeria monocytogenes ATCC 7644

Aims@#This study aims to isolate lactic acid bacteria (LAB) from various food sources to obtain a potent strain against Listeria monocytogenes. @*Methodology and results@#A total of 68 LAB isolates were selected to evaluate their antimicrobial activity against L. monocytogenes, a foodborne pathogen and a causative agent of listeriosis. The selected isolate was identified and characterized. The isolate C23 from cabbage showed the highest antimicrobial activity against L. monocytogenes ATCC 7644 with inhibition ability of 73.94%. The isolate was closely related to Lactobacillus brevis by 16S rRNA sequencing and subsequently deposited in GenBank with an accession number of MN880215, named as L. brevis C23. The cell free supernatant (CFS) of L. brevis C23 had high tolerance in low pH and was able to withstand up to 60 °C. The proteinaceous nature of the antimicrobial agent was also confirmed through the enzymatic test. The CFS was stable on different detergents as well as bile salts. Under transmission electron microscopy (TEM), the inhibitory effect of CFS against L. monocytogenes was proven by causing cell lysis.@*Conclusion, significance and impact of study@#Bacteriocin-like inhibitory substances (BLIS) of L. brevis C23 showed very promising potential in food industrial application.

Avaliação do efeito de cepas probióticas em biofilme de S. aureus sobre discos de titânio com superfície tratada / Assessment of probiotic bacterial strains effect on S. aureus biofilm on titanium discs with treated surfaces

Resumo Introdução As doenças peri-implantares apresentam como um dos principais fatores etiológicos o biofilme bacteriano, geralmente formado por uma microbiota semelhante à das doenças periodontais. Seu tratamento está centrado na descontaminação da superfície do implante e na remoção mecânica do biofilme, podendo ainda estar associado à administração de agentes antimicrobianos. Nesse sentido, tem sido cogitada a utilização de probióticos, que são microrganismos benéficos à saúde e que podem ter grande importância na cavidade oral, como coadjuvante no tratamento das peri-implantites. Objetivo Avaliar o efeito das cepas probióticas de Lactobacillus brevis e Bifidobacterium bifidum no crescimento do biofilme monoespécie de Staphylococcus aureus. Material e método Discos de titânio padronizados e com superfície tratada foram submersos em meio contendo caldo BHI e Staphylococcus aureus durante sete dias. Após esse período, o caldo foi retirado, os discos foram lavados e, então, introduzidos em um novo caldo BHI contendo as suspensões probióticas, sendo assim comparados a um grupo controle, sem probióticos. As amostras foram incubadas por 24h e então foram realizadas as diluições e a contagem das UFC (unidades formadoras de colônia) para Staphylococcus aureus. Resultado Após análise estatística dos dados, observou-se que a adição de ambos os probióticos resultaram em redução significativa (p<0,05) de UFC, quando comparados ao controle. Conclusão Conclui-se que os probióticos analisados (Lactobacillus brevis e Bifidobacterium bifidum) reduziram consideravelmente o crescimento do patógeno Staphylococcus aureus. Além disso, a cepa de Lactobacillus brevis apresentou efeito inibidor superior ao da cepa Bifidobacterium bifidum para ser utilizada como controle do biofilme bacteriano de Staphylococcus aureus.

Abstract Introduction One of the main etiological factors for peri-implant diseases is the bacterial biofilm, which usually features a similar microbiota to periodontal diseases. Its treatment focus on the decontamination of the implant surface and on the mechanical removal of biofilm, and it may also be associated to the administration of antimicrobial agents. Thus, the use of probiotics has been considered, since they feature beneficial microorganisms to health and may be of great importance for the oral cavity as an adjunct for the treatment of peri-implant diseases. Objective The aim of this in vitro study was to assess the effect of probiotic strains of Lactobacillus brevis and Bifidobacterium bifidum on the growth of single-species biofilm of Staphylococcus aureus. Material and method Standardized surface-treated titanium discs were submerged in a medium containing BHI broth and Staphylococcus aureus, for 7 days. After this period, the broth was removed, the discs were washed and, then, submerged in a new BHI broth containing probiotic suspensions and compared to a control group (with no probiotics). Samples were incubated for 24 hours and then the dilutions and CFU (colony-forming units) counting for Staphylococcus aureus were performed. Result Statistical analysis revealed that the addition of both probiotics resulted in a significant reduction (p<0,05) of CFU, when compared to the control group. Conclusion The assessed probiotics (Lactobacillus brevis and Bifidobacterium bifidum) considerably reduced Staphylococcus aureus growth. In addition, Lactobacillus brevis strain presented a superior inhibition effect than Bifidobacterium bifidum strain for Staphylococcus aureus bacterial biofilm control.

Increasing the thermostability of glutamate decarboxylase from Lactobacillus brevis by introducing proline / 生物工程学报

Glutamate decarboxylase, a unique pyridoxal 5'-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364P was significantly improved, the half-life time (t1/2) at 55 °C and the semi-inactivation temperature (T₅₀ ¹⁵) of the mutant G364P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.

The Predominant Lactic Acid Microorganisms and Proximate Composition of Spontaneously Fermented Gari and Fufu, Cassava Food Products

Aim: The present study is focused on determining if there are differences in the types of organisms responsible for spontaneous fermentation in two types of cassava food products, namely, fufu and gari, while also ensuring that the expected organoleptic properties associated with the fermentation process from this study location is reproducible. Study Design: A Complete Randomized Design (CRD) with three replications was adopted and used to test for significant differences between the two cassava products. Place and Duration of Study: The roots of two cassava varieties namely, TMS 97/0211 (white pulp) and TMS 97/2205 (yellow pulp) were obtained from the International Institute for Tropical Agriculture (IITA), Ibadan, and were processed at Ede, Nigeria between March and May 2016. Methodology: Using standardized spontaneous fermentation methods, the two varieties of cassava, were sampled eight hourly over a period of 5 days, for lactic acid bacteria and fungi. Samples were incubated anaerobically, representative microbial populations were enumerated and identified using standard microbiological protocols. Proximate analysis and sensory evaluations were conducted. Results: The results showed that the predominant lactic acid bacterial organisms were Lactobacillus brevisand L plantarum. On the other hand, the representative lactic acid fungal isolates were identified as Neurospora crassa, Aspergillus fumigatus and Saccharomyces spp. Investigation of succession organisms revealed differences between the dry cassava finished product, gari and the wet finished product, fufu. The fungal organisms were the predominant starter organisms found in gari, while, the predominant starter organisms found in fufu were the bacterial types. Conclusion: The present results show that in spite of the spontaneity of the fermentation process, the yellow cassava variety supports the growth and reproduction of similar fermentation organisms as the white variety. Furthermore, the prevailing microenvironment in the fermentation set up, that is, wet or dry is the most important factor in determining the predominating organisms in the fermentation process and the organoleptic and nutritional characteristics of the final product. Results from this study show that it is possible to reproduce the organoleptic and nutritional characteristics peculiar to this test location using the isolated lactic acid microorganisms.

Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer

ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

Attenuating effect of Lactobacillus brevis G101 on the MSG symptom complex in a double-blind, placebo-controlled study

BACKGROUND/OBJECTIVES: Lactobacillus brevis G101 suppresses the absorption of monosodium glutamate (MSG) from the intestine into the blood in mice. Therefore, the attenuating effect of orally administered G101 on monosodium glutamate (MSG) symptom complex was investigated in humans. MATERIALS/METHODS: Capsules (300 mg) containing Lactobacillus brevis G101 (1x1010 CFU/individual) or maltodextrin (placebo) was orally administered in 30 respondents with self-recognized monosodium glutamate (MSG) symptom complex for 5 days and the rice with black soybean sauce containing 6 g MSG (RBSM) was ingested 30 min after the final administration. Thereafter, the MSG symptom complex (rated on a 5-point scale: 1, none; 5, strong) was investigated in a double blind placebo controlled study. The intensity of the MSG symptom complex was significantly reduced in respondents of the G101 intake group (2.87 +/- 0.73) compared to that in those treated with the placebo (3.63 +/- 1.03) (P = 0.0016). Respondents in the placebo group exhibited more of the various major conditions of the MSG symptom complex than in the G101 intake group. Although there was no significant difference in the appearance time of the MSG symptom complex between subjects orally administered G101 and those administered the placebo, its disappearance in < 3 h was observed in 69.9% of subjects in the G101 treatment group and in 38.0% of subjects in the placebo group (P = 0.0841). CONCLUSIONS: Oral administration of Lactobacillus brevis G101 may be able to reduce the intensity of the MSG symptom complex.

Lactic acid production via cassava-flour- hydrolysate fermentation / Producción de ácido láctico vía fermentativa a partir de hidrolizado de harina de yuca

background: Lactic acid (LA) is a carboxylic acid widely used as preservative, acidulant, and/or flavouring in food industry; it is also used as a raw material for the production of lactate ester, propylene glycol, 2,3-pentanedione, propanoic acid, acrylic acid and acetaldehyde. In recent years, the demand for LA production has dramatically increased due to its application as a monomer for poly-lactic acid synthesis, a biodegradable polymer used as a plastic in many industrial applications. LA can be produced either by fermentation or chemical synthesis; the former route has received considerable interest, due to environmental concerns and the limited nature of petrochemical feedstocks; thus, 90% of LA produced worldwide is obtained by fermentation, this process comprises the bioconversion of a sugar solution (carbohydrates) into LA in the presence of a microorganism. Objectives: This work is aimed at studying the effect of pH control and culture media composition on the LA production using renewable sources from the agroindustry sector. Methods: A Lactobacillus brevis strain is used to perform lab scale experiments under aerobic and anaerobic conditions, using three different culture media compositions: a high nutritional content medium (MRS), as a reference, a low nutritional content medium with glucose as the only carbon source (GM), and a potential low nutritional content medium with cassava flour as carbon source (HY1). results: The higher LA production is accomplished under anaerobic conditions, 17.6 ± 0.1, 12.6 ± 0.2 y 13.6 ± 0.2 g LA/L, for MRS, GM and HY1 medium, respectively. The effect of pH on LA biosynthesis in a 5L bioreactor is also studied using the HY1 medium. For a fermentation time of 120 h, the highest LA concentration obtained was 24.3 ± 0.7g LA/L, productivity 0.20 g/L/h, YP/S 0.32g LA/g syrup, at pH 6.5...

Arginine specific aminopeptidase from Lactobacillus brevis

The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB) as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836) was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6). The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5 percent bile salts, the most important probiotic features.

Arginine specific aminopeptidase from Lactobacillus brevis

The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB) as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836) was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6). The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5 percent bile salts, the most important probiotic features.