Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Bio prospecting of Riboflavin producing bacteria from different riboflavin enriched food sources

Riboflavin is an essential, water-soluble vitamin (B2) and a component of basic cellular metabolism. The aim of the present study is to isolate and characterize riboflavin producing bacteria from different food sources. Ten different riboflavin enriched food sources were collected from Vellore district. Totally 72 bacterial strains were isolated and cultured on nutrient agar plates. Out of these, 43 strains were identified as riboflavin producers. Isolated bacterial strains HDS27, HDS07, HDS14, HDS18, HDS38 and HDS54 isolated from milk, mushroom, spinach, lamb kidney, beef liver and mackerel fish were found to be potent riboflavin producers. Based on morphological, biochemical and molecular characterization, the potent strains were identified as Lactobacillus plantarum (HDS27), Bacillus cereus (HDS07), Delftia tsuruhatensis (HDS14), Citrobacter freundii (HDS18), Enterobacter cloacae (HDS38) and Bacillus cereus (HDS54). The selected potent isolates HDS27 from milk and HDS07 from mushroom showed a maximum riboflavin production of 3.69 mg/L and 2.9mg/L respectively. The present study explores the riboflavin producing novel bacteria from different food sources. This is the first report that the Enterobacter cloacae isolated from beef liver, Delftia tsuruhatensis from spinach and Citrobacter freundii from lamb kidney has the ability to produce riboflavin. These potent strains could be a better starter for substituting the conventional bacteria for large scale production of riboflavin in industry.

Antibacterial activity of Lactobacillus plantarum BS25, Pediococcus acidilactici S3 crude, and partially-purified cell-free supernatants against methicillinsusceptible and methicillin-resistant Staphylococcus aureus strains

Background@#The rising public health threat brought about by antibiotic resistance, such as of Staphylococcus aureus, opened doors of opportunities for natural products research to explore novel antimicrobial agents. @*Objective@#This study aimed to determine the antimicrobial activity of cell-free supernatants from Lactobacillus plantarum BS25 and Pediococcus acidilactici S3 against Staphylococcus aureus (ATCC# 25923) and methicillin-resistant S. aureus (ATCC# 33591). @*Methodology@#Cell-free supernatants (CFS) of Lactobacillus plantarum BS25 and Pediococcus acidilactici S3, isolated from fermented rice-fish mixture balao-balao and fermented spicy sausage longganisa, respectively, were tested against methicillin-susceptible (MSSA, ATCC 25923) and methicillin-resistant (MRSA, ATCC 33591) Staphylococcus aureus strains for antibacterial activity using the resazurin assay. @*Results@#Both BS25 and S3 CFS showed high activities against MSSA and partial inhibition against MRSA. Proteinaceous components of the CFS were extracted using ammonium sulfate precipitation with BS25 and S3 exhibited low activities against MSSA but partial inhibition was observed against MRSA. Other small molecules were extracted from the CFS through liquid-liquid extraction using ethyl acetate and tested in 100, 250, 500, 750, and 1000 ppm concentrations. The 1000-ppm concentrations of the BS25 and S3 ethyl acetate extracts achieved the highest antibacterial activity against MSSA and MRSA. @*Conclusion@#This study showed that the crude cell-free supernatants, ammonium sulfate precipitates, and ethyl acetate extracts of BS25 and S3 CFS exhibited potential in inhibiting Gram-positive MSSA and MRSA. However, the partially-purified samples require relatively high concentrations in order to produce significant inhibition activities and therefore require further purification.

Probiotic supplementation affects IGF-1 and leptin levels in Nile tilapia hepatopancreatic tissue / Suplementação com probiótico afeta os níveis de IGF-1 e leptina no tecido hepatopancreático de tilápias-do-nilo

This work aimed to assess the effect of the probiotic strain, Lactobacillus plantarum, on the levels of leptin, IGF-1 and their receptors on the hepatopancreatic tissues of Nile tilapia (Oreochromis niloticus) and then correlate fish growth performance and gut microbiological parameters. Fish juveniles (±23g) were reared in a recirculation system with constant aeration and temperature (25°C). They were distributed into six polyethylene tanks (45L) and fed twice a day at 5% of the tank biomass with the respective diets: control (commercial diet without probiotic) and supplemented with L. plantarum inoculum (1 x 108 CFU mL-1), both in triplicate. After 30 days of feeding, L. plantarum-fed fishes showed greater weekly growth rate, final weight, and feed conversion rate, in addition to higher count of lactic-acid bacteria and lower count of pathogenic bacteria in the intestinal tract, when compared to the control group. The immunostaining intensity for IGF-1 and leptin hormones was lower after L. plantarum supplementation than in the control group, with no change in the level for receptors. This reduction could implicate important changes in fish metabolism and homeostasis.(AU)

O presente trabalho teve como objetivo avaliar o efeito da cepa probiótica Lactobacillus plantarum sobre os níveis de leptina, IGF-1 e seus receptores no tecido hepatopancreático de tilápia-do-nilo (Oreochromis niloticus) e correlacionar com o desempenho zootécnico e os parâmetros microbiológicos intestinais dos peixes. Juvenis de tilápia-do-nilo (±23g) foram distribuídos em seis tanques de polietileno (45L) conectados a um sistema de recirculação, com aeração e temperatura constantes (25°C). Os peixes foram alimentados duas vezes ao dia, a 5% da biomassa do tanque, com as respectivas dietas: controle (dieta comercial sem probiótico) e suplementada com L. plantarum (1 x 108 UFC mL-1), ambas em triplicata. Após 30 dias de cultivo, os peixes alimentados com L. plantarum apresentaram maiores ganho de peso semanal, peso final e conversão alimentar, bem como maior contagem de bactérias ácido-láticas e menor contagem de bactérias patogênicas no trato intestinal das tilápias alimentadas com dieta probiótica, em comparação ao grupo controle. A intensidade da imunomarcação para os hormônios IGF-1 e leptina foi menor com a suplementação de L. plantarum do que no grupo controle, sem alterar os níveis de seus receptores. Essa redução pode implicar mudanças importantes no metabolismo e na homeostase dos peixes.(AU)

Characterization of plantaricin IIA-1A5 biosynthesized by Lactobacillus plantarum IIA-1A5 in corn steep liquor based medium

Aims@#To characterize the plantaricin IIA-1A5 crude extract that biosynthesized by Lactobacillus plantarum IIA-1A5 using corn steep liquor (CSL) based medium. @*Methodology and results@#Lactobacillus plantarum IIA-1A5 was grown in several media containing different components including corn steep liquor (CSL), molasses and MRS (de Man Rogosa Sharpe) as control medium for 24 h at 37 °C. Antibacterial activities of the cell-free supernatant were expressed as diameter of inhibition zones observed by paper disc method. The results showed that CSL medium produced cell-free supernatant of L. plantarum IIA-1A5 with significantly higher antibacterial activity againts Staphylococcus aureus ATCC 25923 (9.81 mm), Lactobacillus monocytogenes ATCC 7644 (9.61 mm), Bacillus cereus (8.97 mm) and Escherichia coli ATCC 25922 (9.23 mm) were not significantly different compared to control MRS broth media (9.59 mm). CSL medium added only with 3% yeast extract and Tween 80 produced supernatant which showed similar antibacterial activity either to 10% molasses or control medium (Medium K and B). The CSL medium was considered more efficient and low cost, therefore this medium was selected for production and characterization of plantaricin IIA-1A5 crude extract. Further characterization performed by SDS PAGE analysis showed that crude plantaricin had molecular weight of approximately 9.9 kDa, higher than that produced in control medium (8.0 kDa). However, both plantaricins were categorized under the same class for small bacteriocin (class II). This study also revealed the plantaricin IIA-1A5 produced in CSL medium was stable to heat and pH and not significantly different compared to control MRS broth media. The antibacterial activity of plantaricin IIA-1A5 crude extract against S. aureus ATCC 25923 (10.09 mm) was not significantly different with 1000 ppm sodium benzoate (9.70 mm) and 300 ppm sodium nitrite (9.82 mm).@*Conclusion, significance and impact of study@#The CSL medium produced cell-free supernatant of L. plantarum IIA 1A5 had significant antibacterial activity characterization againts S. aureus ATCC 25923, L. monocytogenes ATCC 7644, B. cereus and E. coli ATCC 25922. Comparison of the inhibition activity of plantaricin IIA-1A5 crude extract against pathogen with synthetic preservatives indicated that plantaricin IIA-1A5 crude extract have the potency to replace synthetic preservatives. CSL based medium is potential to be used for low-cost plantaricin IIA-1A5 production.

Immunogenicity of engineered Lactobacillus plantarum expressing porcine epidemic diarrhea virus S1 gene / 生物工程学报

To investigate whether the engineered Lactobacillus plantarum expressing the porcine epidemic diarrhea virus (PEDV) S1 gene can protect animals against PEDV, guinea pigs were fed with recombinant L. plantarum containing plasmid PVE5523-S1, with a dose of 2×10⁸ CFU/piece, three times a day, at 14 days intervals. Guinea pigs fed with wild type L. plantarum and the engineered L. plantarum containing empty plasmid pVE5523 were used as negative controls. For positive control, another group of guinea pigs were injected with live vaccine for porcine epidemic diarrhea and porcine infectious gastroenteritis (HB08+ZJ08) by intramuscular injection, with a dose of 0.2 mL/piece, three times a day, at 14 days intervals. Blood samples were collected from the hearts of the four groups of guinea pigs at 0 d, 7 d, 14 d, 24 d, 31 d, 41 d and 48 d, respectively, and serum samples were isolated for antibody detection and neutralization test analysis by enzyme-linked immunosorbent assay (ELISA). The spleens of guinea pigs were also aseptically collected to perform spleen cells proliferation assay. The results showed that the engineered bacteria could stimulate the production of secretory antibody sIgA and specific neutralizing antibody, and stimulate the increase of IL-4 and IFN-γ, as well as the proliferation of spleen cells. These results indicated that the engineered L. plantarum containing PEDV S1 induced specific immunity toward PEDV in guinea pigs, which laid a foundation for subsequent oral vaccine development.

Microencapsulation of Lactobacillus plantarum 299v and its storage in kuini juice

Aims@#Probiotics are living microorganism, when administrated in sufficient quantity can exert beneficial effect to the host. This study focused on the microencapsulation by co-extrusion to increase the viability of Lactobacillus plantarum 299v (Lp299v) in gastrointestinal conditions, and its storage stability in kuini juice at refrigerated (4 °C) and ambient temperature (25 °C). @*Methodology and results@#Lp99v was encapsulated with 1.5% w/v sodium alginate and chitosan coating (0.1% w/v) and yielded a microencapsulation efficiency of 97.71%. The Lp299v microbeads produced were spherical in shape and exhibited a mean microbeads size of 618.75 ± 25.85 µm. Acid and bile tolerance of both free and encapsulated Lp299v were tested in simulated gastric juice (SGJ) for 2 h and in simulated intestinal juice (SIJ) for 4 h, respectively. The encapsulated Lp299v maintained above 108 CFU/mL after exposure to artificial gastrointestinal juice, whereas a significant loss of viability was observed in the free cells. The storage stability of encapsulated Lp299v in kuini juice was determined during 4 weeks of storage at 4 °C and 25 °C. Results showed that encapsulated Lp299v was capable to remain viable (107 CFU/mL) for at least 4 weeks in a refrigerated condition. However, free Lp299v did not survived under both refrigerated and ambient temperature as the storage period extended. @*Conclusion, significance and impact of study@#Lp299v entrapped in chitosan-coated alginate microbeads produced by co-extrusion method is able to enhance the viability of Lp299v above the minimum recommended level in harsh environment (gastrointestinal conditions and low pH of kuini juice).

Efecto antifúngico y antimicotoxigénico de Lactobacillus plantarum CRL 778 a diferentes valores de actividad de agua / Antifungal and antimycotoxigenic effect of Lactobacillus plantarum CRL 778 at different water activity values

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi with high impact Lactic acid bacteria; in food safety due to its toxicity. In the last decade, the presence of OTA was widely reported in different foods. In this study, the ability of Lactobacillus (L.) plantarum CRL 778 to control growth and OTA production by Aspergillus (A.) niger 13D strain, at different water activity (a w) values (0.955, 0.964, 0.971, 0.982, and 0.995) was determined in vitro. Both parame ters were significantly (p<0.05) reduced by the lactobacilli and the effect depended on a w. Greatest growth rate inhibition (46.9%) was obtained at a w = 0.995, which is the most suitable value for growth and production of antifungal metabolites (lactic acid, acetic acid, phenyllac-tic and hydroxyl-phenyllactic acids) by L. plantarum CRL 778. Besides, morphological changes and inhibition of melanin synthesis were observed in colonies of A. niger 13D in presence of L. plantarum CRL 778 at a w ranged between 0.971 and 0.995. In addition, maximum reduction (90%) of OTA production took place at a w = 0.971, while inhibition of fungi growth was more evident at a w =0.995. These findings suggest that L. plantarum CRL 778 could be used for control of ochratoxigenic fungal growth and OTA contamination in different fermented foods with a w values between 0.971 and 0.995.

Ocratoxina A (OTA) es una micotoxina producida por hongos filamentosos con un alto impacto en la seguridad alimentaria debido a su toxicidad. En la última década se ha reportado ampliamente a nivel mundial, la presencia de OTA en diversos alimentos. En este estudio se evaluó in vitro, la capacidad de Lactobacillus (L.) plantarum CRL 778 de controlar el crecimiento y la producción de OTA por Aspergillus (A.) niger 13D, a diferentes valores de actividad de agua (a w): 0.955, 0.964, 0.971,0.982 y 0.995). La cepa láctica redujo significativamente (p <0.05) ambos parámetros, siendo el efecto dependiente del valor de a w. La mayor inhibición del crecimiento (46.9%) se obtuvo a a w =0.995, valor más adecuado para el crecimiento y producción de metabolitos antifúngicos (ácido láctico, ácido acético, ácidos fenil-láctico e hidroxi-fenil láctico) por la cepa láctica. Además, se observaron cambios morfológicos en las colonias de A. niger 13D, crecidas en presencia de L. plantarum CRL 778 a valores de a w de 0.971 y 0.995. El porcentaje máximo de reducción en la producción de OTA (90%) por la cepa láctica se observó a un valor de a w = 0.971, mientras la inhibición del crecimiento fúngico fue mayor cuando a w = 0.995. Estos hallazgos sugieren que L. plantarum CRL 778 podría emplearse para el control de la contaminación por hongos ocratoxigénicos en alimentos con valores de aw comprendidos entre 0.971-0.995.

Efeito da oligofrutose (FOS) sobre a capacidade de acidificação da cultura nativa Lactobacillus plantarum CNPC 003 isolada ou em co-cultura com Streptococcus thermophilus em leite de cabra / Influence of oligofrutose (FOS) on the acidification capacity of the indigenous culture Lactobacillus plantarum CNPC 003 solely or in coculture with Streptococcus thermophilus in goat milk

Objetivou-se analisar o comportamento fermentativo da cultura Lactobacillus plantarum CNPC 003 em leite de cabra em quatro tratamentos por 6 horas: T1 – L. plantarum CNPC 003; T2 – L. plantarum + oliogofrutose (FOS); T3 – L. plantarum + Streptococcus thermophilus; T4 – com L. plantarum + S. thermophilus + FOS. Os tratamentos estudados diferiram significativamente entre si em relação à acidez e ao pH após 6h de fermentação (p<0,05), tendo sido verificada uma influência positiva do uso de S. thermophilus e da adição de FOS sobre esses parâmetros. Do mesmo modo, a presença de FOS melhorou a viabilidade dos microrganismos estudados após 6h de fermentação. A utilização de S. thermophilus e de FOS é uma alternativa viável para o uso em leites fermentados contendo a cepa nativa L. plantarum CNPC 003.

Lactobacillus plantarum as a malolactic starter culture in winemaking: a new (old) player?

Malolactic fermentation (MLF) is a process in winemaking responsible for the conversion of L-malic acid to L-lactic acid and CO2, which reduces the total acidity, improves the biological stability, and modifies the aroma profile of wine. MLF takes place during or after alcoholic fermentation and is carried out by one or more species of lactic acid bacteria (LAB), which are either present in grapes and cellars or inoculated with malolactic starters during the winemaking process. Although the main bacterium among LAB used in commercial starter cultures for MLF has traditionally been Oenococcus oeni, in the last decade, Lactobacillus plantarum has also been reported as a malolactic starter, and many works have shown that this species can survive and even grow under harsh conditions of wine (i.e., high ethanol content and low pH values). Furthermore, it has been proved that some strains of L. plantarum are able to conduct MLF just as efficiently as O. oeni. In addition, L. plantarum exhibits a more diverse enzymatic profile than O. oeni, which could play an important role in the modification of the wine aroma profile. This enzymatic diversity allows obtaining several starter cultures composed of different L. plantarum biotypes, which could result in distinctive wines. In this context, this review focuses on showing the relevance of L. plantarum as a MLF starter culture in winemaking.

Histopathological and ultrastructural findings induced by heat-inactivated Lactobacillus plantarum and the culture supernatant on the intestinal mucosa of piglets: an ex vivo approach / Achados histopatológicos e ultraestruturais induzidos por Lactobacillus plantarum inativado pelo calor na mucosa intestinal de leitões: estudo ex-vivo

In the present study, histological, morphometrical and ultrastructural analysis were performed to investigate intestinal mucosa changes in piglets jejunal explants exposed to two concentration of heat-inactivated Lactobacillus plantarum and their respective culture supernatants. Jejunal explants were incubated for 4 hours in DMEM culture medium with a) only culture medium (control group), b) heat-inactivated Lactobacillus plantarum strain1 - LP1 (1.1 x 108CFU/ml), c) heat-inactivated Lactobacillus plantarum strain2 - LP2 (2.0 x 109CFU/ml), d) heat-inactivated Lactobacillus plantarum strain1 culture supernatant (CS1), and e) heat-inactivated Lactobacillus plantarum strain2 culture supernatant (CS2). Explants exposed to heat-inactivated L. plantarum strain 1 and 2 showed multifocal to difuse villi atrophy, villi apical necrosis and enterocyte flattening. Morphological assessment revealed similar results with bacterial adhesion to mucus and intestinal epithelial cells and, morphometric analysis showed a decreased villi height compared to the control group. Alterations in explants treated with the culture supernatant of both strains include mild villi atrophy and mild enterocyte apical necrosis. Morphological assesment reveled numerous well delineated villi and, morphometric analysis showed a significant increase in villi height compared to the control group. In general, exposure to the culture supernatants improved the intestinal morphology.(AU)

No presente estudo, foram realizadas análises histológica, morfométrica e ultraestrutural para investigar as alterações da mucosa intestinal em explantes jejunais de leitões expostos a duas cepas e concentrações de Lactobacillus plantarum inativado pelo calor e seus sobrenadantes de cultura. Os explantes jejunais foram incubados durante quatro horas, em meio de cultura DMEM com: a) meio de cultura (grupo controle); b) Lactobacillus plantarum, cepa 1 - LP1 (1,1 x 108CFU/mL); c) Lactobacillus plantarum, LP2 (2,0 x 109CFU/mL); d) sobrenadante da cultura do Lactobacillus plantarum, cepa 1 (SC1); e e) sobrenadante da cultura do Lactobacillus plantarum, cepa 2 (SC2). Os explantes expostos às cepas 1 e 2 do L. plantarum inativado pelo calor mostraram atrofia difusa de vilosidades, necrose apical das vilosidades e achatamento de enterócitos. A avaliação morfológica revelou resultados semelhantes, com adesão bacteriana ao muco e às células epiteliais intestinais, e a análise morfométrica mostrou uma diminuição da altura das vilosidades em relação ao grupo controle. Alterações nos explantes tratados com o sobrenadante da cultura de ambas as cepas caracterizaram-se por atrofia leve das vilosidades e necrose apical leve dos enterócitos. A avaliação morfológica revelou vilosidades bem delineadas, e a análise morfométrica mostrou um aumento significativo na altura das vilosidades em comparação ao grupo controle. Em geral, a exposição aos sobrenadantes da cultura melhora a morfologia intestinal.(AU)

Dietary effect of Lactobacillus plantarum CJLP55 isolated from kimchi on skin pH and its related biomarker levels in adult subjects / 한국영양학회지

PURPOSE: The skin pH is maintained by epidermal lactate, free fatty acids (FFAs), and free amino acids (FAAs). As a significant determinant of skin health, the skin pH is increased (less acidic) under abnormal and aged skin conditions. In a search for dietary alternatives that would promote an acidic skin pH, this study investigated the dietary effects of Lactobacillus plantarum CJLP55 isolated from Korean kimchi on the skin pH, and epidermal levels of lactate, FFAs, and FAAs in adult subjects. METHODS: Seventy eight subjects (mean age 24.9 ± 0.5 years, range 19 ~ 37 years) were assigned randomly to ingest CJLP55, Lactobacillus strain from kimchi, (n = 39, CJLP group) or placebo supplements (n = 39, placebo group) for 12 weeks in a double-blind, placebo-controlled trial. Skin pH and epidermal levels of lactate, FFAs and FFAs were assessed at 0, 6 and 12 weeks. RESULTS: Although significant decreases in skin pH were observed in both the CJLP and placebo groups at 6 weeks, the skin pH was decreased significantly only in the CJLP group at 12 weeks. In parallel, the epidermal level of lactate in the CJLP group was also increased by 25.6% at 12 weeks. On the other hand, the epidermal level of FAAs were not altered in the CJLP and placebo groups, but the epidermal level of total FFAs, including palmitic acid and stearic acid, was lower in the CJLP group than in the placebo group over 12 weeks. The changes in the other FFAs, such as palmitoleic acid and oleic acid, were similar in the CJLP and placebo groups over 12 weeks. CONCLUSION: Overall, a dietary supplement of CJLP55 promotes acidic skin pH with a selective increase in epidermal lactate in adult subjects.

Seleção e aplicação de bactérias láticas produtoras de folato para obtenção de um leite fermentado bioenriquecido e avaliação de biodisponibilidade do folato produzido / Screening and application of folate-producing lactic acid bacteria for production of bioenriched fermented milk and evaluation of the bioavaliability of the produced folate

A deficiência de folato é um problema de saúde pública que vem sendo combatido através da fortificação obrigatória de ácido fólico. Entretanto, a ingestão excessiva pode causar efeitos colaterais indesejados. A forma natural da vitamina produzida por algumas cepas de bactérias láticas pode ser uma alternativa segura para aumentar a ingestão de folato pela população. Este trabalho visou selecionar as melhores cepas de BAL produtoras de folato, identificar as melhores condições para a produção da vitamina e avaliar a biodisponibilidade do folato produzido pelas cepas no leite fermentado, empregando-se testes em modelos animais. O estudo foi desenvolvido com cinco cepas de Streptococcus thermophilus (34v, 170v, 268v, 361v e 341 pc) e um cepa de Lactobacillus plantarum (16cv), todas boas produtoras de folato. As condições de produção e a combinação de cepas que resultaram na melhor produção folato foram utilizadas para produzir um leite biofermentado (BFM), avaliando-se a biodisponibilidade da vitamina produzida empregando-se um modelo animal de depleção/repleção, com camundongos Balb/c submetidos a 14 dias de depleção, seguido de 21 dias de repleção da vitamina. Após os 35 dias, os animais foram sacrificados, retirando-se sangue, rins, fígado, baço e intestino para quantificação de folato e avaliação histológica da mucosa intestinal. Todas as quantificações de folato foram feitas pelo método microbiológico. A melhor combinação de cepas (St. Thermophilus 34v + Lb. Plantarum 16cv) resultou na produção de 300 ng/mL de folato, sendo a temperatura de 42ºC melhor que 37ºC para a produção da vitamina. A produção de folato no leite na fermentação em pH controlado 6,0 foi 35% superior à observada no leite fermentado em pH livre. As concentrações de folato no baço e glóbulos vermelhos foram mais altas nos camundongos que ingeriram leite fermentado bioenriquecido (BFM) em relação aos demais grupos de animais. A relação vilosidade/cripta nos camundongos dos grupos BFM foi igual à observada nos animais que receberam leite suplementado com ácido fólico. Houve aumento da hemoglobina, hematócrito e hemácias nos camundongos que ingeriram BFM, evidenciando a bioeficácia do folato produzido. Esses resultados indicam que a produção de folato por bactérias láticas selecionadas em produtos fermentados pode ser uma boa alternativa para aumentar a ingestão de vitamina B9 pela população

A deficiência de folato é um problema de saúde pública que vem sendo combatido através da fortificação obrigatória de ácido fólico. Entretanto, a ingestão excessiva pode causar efeitos colaterais indesejados. A forma natural da vitamina produzida por algumas cepas de bactérias láticas pode ser uma alternativa segura para aumentar a ingestão de folato pela população. Este trabalho visou selecionar as melhores cepas de BAL produtoras de folato, identificar as melhores condições para a produção da vitamina e avaliar a biodisponibilidade do folato produzido pelas cepas no leite fermentado, empregando-se testes em modelos animais. O estudo foi desenvolvido com cinco cepas de Streptococcus thermophilus (34v, 170v, 268v, 361v e 341 pc) e um cepa de Lactobacillus plantarum (16cv), todas boas produtoras de folato. As condições de produção e a combinação de cepas que resultaram na melhor produção folato foram utilizadas para produzir um leite biofermentado (BFM), avaliando-se a biodisponibilidade da vitamina produzida empregando-se um modelo animal de depleção/repleção, com camundongos Balb/c submetidos a 14 dias de depleção, seguido de 21 dias de repleção da vitamina. Após os 35 dias, os animais foram sacrificados, retirando-se sangue, rins, fígado, baço e intestino para quantificação de folato e avaliação histológica da mucosa intestinal. Todas as quantificações de folato foram feitas pelo método microbiológico. A melhor combinação de cepas (St. Thermophilus 34v + Lb. Plantarum 16cv) resultou na produção de 300 ng/mL de folato, sendo a temperatura de 42ºC melhor que 37ºC para a produção da vitamina. A produção de folato no leite na fermentação em pH controlado 6,0 foi 35% superior à observada no leite fermentado em pH livre. As concentrações de folato no baço e glóbulos vermelhos foram mais altas nos camundongos que ingeriram leite fermentado bioenriquecido (BFM) em relação aos demais grupos de animais. A relação vilosidade/cripta nos camundongos dos grupos BFM foi igual à observada nos animais que receberam leite suplementado com ácido fólico. Houve aumento da hemoglobina, hematócrito e hemácias nos camundongos que ingeriram BFM, evidenciando a bioeficácia do folato produzido. Esses resultados indicam que a produção de folato por bactérias láticas selecionadas em produtos fermentados pode ser uma boa alternativa para aumentar a ingestão de vitamina B9 pela população

Characteristics of Vegetable Juice Fermented with Lactobacillus plantarum MKHA15 and Leuconostoc mesenteroids MKSR

The objective of this study was to develop fermented vegetable juices that possess antidiabetic and antioxidant activities. Lactobacillus plantarum MKHA15 (MKHA15) and Leuconostoc mesenteroides MKSR (MKSR) were applied to ferment onion, cabbage, and tomato juices at 37℃ and 30℃ for 72 h, respectively, and their functionality was tested using the 12 h hour-fermented juice by MKHA15, and 48 h hour-fermented juice by MKSR. Inhibition of α-glucosidase activity was observed in all fermented juices. The onion juice fermented by MKHA15 showed significantly higher α-glucosidase inhibition activity compared to other juices. All juices showed more than 70% inhibition of α-amylase activity. The DPPH radical scavenging activity of onion juice fermented by MKSR showed significantly lower activity than cabbage and tomato juices; however, no difference was observed between the types of starter cultures. The SOD-like activity of cabbage juice fermented by MKSR was the highest among the fermented juices. The juices fermented by MKHA15 showed higher reducing power than those by MKSR. Therefore, we believe that cabbage, onion and tomato juice fermented by MKHA15 and MKSR would be useful in probiotic juices, as they possess antidiabetic and antioxidant activities.

Supplementation of Fermented Barley Extracts with Lactobacillus Plantarum dy-1 Inhibits Obesity via a UCP1-dependent Mechanism / 生物医学与环境科学（英文）

OBJECTIVE@#We aimed to explore how fermented barley extracts with Lactobacillus plantarum dy-1 (LFBE) affected the browning in adipocytes and obese rats.@*METHODS@#In vitro, 3T3-L1 cells were induced by LFBE, raw barley extraction (RBE) and polyphenol compounds (PC) from LFBE to evaluate the adipocyte differentiation. In vivo, obese SD rats induced by high fat diet (HFD) were randomly divided into three groups treated with oral gavage: (a) normal control diet with distilled water, (b) HFD with distilled water, (c) HFD with 800 mg LFBE/kg body weight (bw).@*RESULTS@#In vitro, LFBE and the PC in the extraction significantly inhibited adipogenesis and potentiated browning of 3T3-L1 preadipocytes, rather than RBE. In vivo, we observed remarkable decreases in the body weight, serum lipid levels, white adipose tissue (WAT) weights and cell sizes of brown adipose tissues (BAT) in the LFBE group after 10 weeks. LFBE group could gain more mass of interscapular BAT (IBAT) and promote the dehydrogenase activity in the mitochondria. And LFBE may potentiate process of the IBAT thermogenesis and epididymis adipose tissue (EAT) browning via activating the uncoupling protein 1 (UCP1)-dependent mechanism to suppress the obesity.@*CONCLUSION@#These results demonstrated that LFBE decreased obesity partly by increasing the BAT mass and the energy expenditure by activating BAT thermogenesis and WAT browning in a UCP1-dependent mechanism.

Construction and application of mCherry red fluorescent protein fusion expression system in lactic acid bacteria / 生物工程学报

In order to enrich the library of domestic research about new red fluorescent marker in lactic acid bacteria (LAB), we described a new fusion expression system in Lactobacillus plantarum WCFS1 based on the pSIP vector. This system contained red fluorescent protein mCherry as a marker and bile salt hydrolase gene (bsh) as a reporter gene. Moreover, in this study, four different promoters (PsppA, PldhL, P32 and PslpA) were used to regulate the expression of the fusion protein mCherry-BSH, completing the inducible and constitutive expression in lactic acid bacteria. The recombinant protein mCherry-BSH presented activity of red fluorescence and bile salt hydrolase (BSH). The successful construction of the fusion expression system in LAB using a red fluorescent protein mCherry provides favorable conditions for the distribution, intestinal colonization and survival rate of lactic acid bacteria, so as to reveal the function mechanism of its probiotic characteristics; and the system also could lay the foundation for researches on protein expression, cellular localization and properties identification of active protein in lactic acid bacteria.

Potencial efeito preventivo do Lactobacillus acidophilus e Lactobacillus plantarum em pacientes com pólipos ou câncer colorretal / Potential preventive effect of lactobacillus acidophilus and lactobacillus plantarum in patients with polyps or colorectal câncer

ABSTRACT BACKGROUND: Colorectal cancer is one of the major causes of death worldwide. Many studies have been done on the biology of its formation as well as its treatment in recent years. One of the factors involved in the formation or treatment of this malignancy can be attributed to the microbial flora in the intestine. OBJECTIVE: This study investigate the potential preventive effect of Lactobacillus acidophilus and Lactobacillus plantarum in patients with polyps or colorectal cancer (CRC). METHODS: A total of 77 samples were selected in the form of three groups including individuals suffering from CRC, polyps and healthy subjects. Genomic DNA of fecal specimens and standard strains were extracted and amplified employing primers targeting of the 16S rRNA gene for initial detection. Absolute Real Time PCR quantification was used to determine the copy of the bacterial expression per gram of feces. RESULTS: No significant difference were observed between age and gender in the mentioned groups (P=0.06). The average copy number of Lactobacillus acidophilus shows Significant difference between the healthy group and those with polyps (P<0.0001), the healthy group and those with colorectal cancer (P<0.0001), as well as those with polyps and the colorectal cancer patients (P<0.0001). CONCLUSION: These results may indicate that taking Lactobacillus acidophilus in people with a family history of CRC and people with polyps may be a way of preventing, treating or reducing the severity of CRC.

RESUMO CONTEXTO: O câncer colorretal é uma das principais causas de morte em todo o mundo. Muitos estudos têm sido feitos sobre a biologia de sua formação, bem como o seu tratamento nos últimos anos. Um dos fatores envolvidos na formação ou no tratamento desta malignidade pode ser atribuído à flora microbiana no intestino. OBJETIVO: Este estudo investigou o potencial efeito preventivo de Lactobacillus acidophilus e Lactobacillus plantarum em pacientes com pólipos ou câncer colorretal (CCR). MÉTODOS: Um total de 77 amostras foram selecionadas e três grupos foram formados, a saber, indivíduos portadores de CCR, pólipos e indivíduos saudáveis. O DNA genomico de espécimes fecais e de amostras padrão foi extraído e amplificado empregando primers que focalizaram o gene do rRNA 16S para a deteção inicial. A quantificação do PCR em tempo real absoluto foi utilizada para determinar a cópia da expressão bacteriana por grama de fezes. RESULTADOS: Não foram observadas diferenças significativas entre idade e sexo nos grupos citados (P=0,06). O número médio de cópias de Lactobacillus acidophilus mostra diferença significativa entre o grupo saudável e aqueles com pólipos (P<0,0001), o grupo saudável e aqueles com câncer colorretal (P<0,0001), bem como aqueles com pólipos e câncer colorretal pacientes (P<0,0001). CONCLUSÃO: Estes resultados podem indicar que a ingestão de Lactobacillus acidophilus em pessoas com antecedentes familiares de CCR e pessoas com pólipos pode ser uma forma de prevenir, tratar ou reduzir a gravidade da CCR.

Combined effect of bacteriocin produced by Lactobacillus plantarum ST8SH and vancomycin, propolis or EDTA for controlling biofilm development by Listeria monocytogenes / Efecto combinado de la bacteriocina producida por Lactobacillus plantarum ST8SH y la vancomicina, el própolis o el EDTA para controlar el desarrollo del biofilm producido por Listeria monocytogenes

The Listeria monocytogenes strains selected in the present study exhibited similar behavior in biofilm formation, independently of the tested conditions (bacteriocin from L. plantarum ST8SH, vancomycin, propolis (a natural antimicrobial product) and EDTA (chelating agent)), individual or in associations. The individual application of vancomycin had better inhibitory activity than that of propolis and EDTA; however, the association of the previously mentioned antimicrobial agents with bacteriocins resulted in better performance. However, when we compared the effects of vancomycin, propolis and EDTA, we could clearly observe that the combined application of bacteriocin and vancomycin was more effective than the combination of bacteriocin and propolis, and bacteriocin and EDTA. Considering the current need to reduce the use of antimicrobials and chemical substances in food processing, propolis can represent an alternative to improve the inhibitory effect of bacteriocins against L. monocytogenes biofilm formation, based on the obtained results. In general, high concentrations of bacteriocin produced by L. plantarum ST8SH were more effective in biofilm inhibition, and similar results were observed for vancomycin and propolis; however, all tested EDTA concentrations had similar effect on biofilm formation.

Las cepas de Listeria monocytogenes seleccionadas en el presente estudio presentaron comportamientos similares en la formación de biofilms, independientemente de los tratamientos a las que fueron sometidas (bacteriocina de Lactobacillus plantarum ST8SH, vancomicina, própolis (produto natural antimicrobiano) y EDTA (agentes quelante)), individual o en combinaciones. La aplicación individual de vancomicina presentó una mejor actividad inhibitoria frente a las aplicaciones individuales de própolis y de EDTA; sin embargo, la combinación de estos agentes antimicrobianos con las bacteriocinas resultó en un mejor desempeño. Se observó claramente que la aplicación combinada de bacteriocina y vancomicina fue más efectiva para controlar el desarrollo de biofilm en comparación con la combinación de la bacteriocina y el própolis o de la combinación de la bacteriocina y el EDTA. Considerando la necesidad actual de reducir el uso de sustancias antimicrobianas y químicas en el procesamiento de alimentos y sobre la base de los resultados obtenidos, se puede afirmar que el própolis representa una alternativa para mejorar el efecto inhibitorio de bacteriocinas contra la formación de biofilm de L. monocytogenes. En general, altas concentraciones de la bacteriocina producida por L. plantarum ST8SH fueron más eficaces en la inhibición del biofilm, y se observaron resultados similares para la vancomicina y el própolis; sin embargo, todas las concentraciones de EDTA evaluadas tuvieron un efecto similar en la formación de biofilm.

Action time and feed frequency of Lactobacillus plantarum for Nile tilapia / Tempo de ação e frequência alimentar do Lactobacillus plantarum para tilápia-do-nilo

O objetivo desta pesquisa foi avaliar o tempo de ação do Lactobacillus plantarum no trato intestinal, assim como em qual frequência esse probiótico deve ser oferecido para tilápia-do-nilo (Oreochomis niloticus). Quarenta e oito tilápias foram alimentadas com ou sem probiótico, durante 14 dias. Após esse período, os peixes foram esviscerados, e amostras do trato intestinal foram semeadas em ágar TCBS, cetrimide e MRS. Esse procedimento foi repetido dois, quatro e seis dias após o 14º dia. Depois disso, 252 tilápias foram divididas em quatro tratamentos, peixes alimentados com probiótico suplementado em 100%, 50%, 25% e 0% (controle) das alimentações. Após nove semanas, os índices zootécnicos foram avaliados. No dia zero, verificou-se maior concentração de bactérias ácido-lácticas e menor concentração de Vibrios spp. e de Pseudomonas spp. no trato intestinal dos alevinos alimentados com probiótico. Peixes alimentados com 100% e 50% das alimentações suplementadas com probiótico apresentaram melhores índices zootécnicos que os peixes alimentados com 25% de suplementação e sem suplementação. O L. plantarum atuou por quatro dias após a última alimentação, e, para obter os melhores índices, essa cepa deve ser suplementada em 50% das alimentações por dia para tilápia-do-nilo (O. niloticus).(AU)

Screening and characterization of lactic acid bacterial strains that produce fermented milk and reduce cholesterol levels

ABSTRACT Objective To screen for and characterize lactic acid bacteria strains with the ability to produce fermented milk and reduce cholesterol levels. Methods The strains were isolated from traditional fermented milk in China. In vitro and in vivo evaluation of cholesterol-reduction were used to identify and verify strains of interest. Characteristics were analyzed using spectrophotometry and plate counting assays. Results The isolate HLX37 consistently produced fermented milk with strong cholesterol-reducing properties was identified as Lactobacillus plantarum (accession number: KR105940) and was thus selected for further study. The cholesterol reduction by strain HLX37 was 45.84%. The isolates were acid-tolerant at pH 2.5 and bile-tolerant at 0.5% (w/v) in simulated gastric juice (pH 2.5) for 2 h and in simulated intestinal fluid (pH 8.0) for 3 h. The auto-aggregation rate increased to 87.74% after 24 h, while the co-aggregation with Escherichia coli DH5 was 27.76%. Strain HLX37 was intrinsically resistant to antibiotics such as penicillin, tobramycin, kanamycin, streptomycin, vancomycin and amikacin. Compared with rats in the model hyperlipidemia group, the total cholesterol content in the serum and the liver as well as the atherogenic index of rats in the viable fermented milk group significantly decreased by 23.33%, 32.37% and 40.23%, respectively. Fewer fat vacuoles and other lesions in liver tissue were present in both the inactivated and viable fermented milk groups compared to the model group. Conclusion These studies indicate that strain HLX37 of L. plantarum demonstrates probiotic potential, potential for use as a candidate for commercial use for promoting health.