Expression and characterization of a multicopper oxidase from Lactobacillus fermentum / 生物工程学报

Biogenic amines (BAs) are low molecular weight organic compounds that present in fermented foods. Large amount of ingested biogenic amines can cause allergy or significant symptoms. Reduction of BAs by enzymatic reaction in fermented foods is one of the most efficient methods for removal of biohazard compounds and assurance food safety. In this study, the multicopper oxidase (MCO) gene in the genome of Lactobacillus fermentum was successfully cloned in Escherichia coli BL21 and expressed at 484 U/L. The recombinant MCO was purified by the immobilized metal affinity chromatography method. The optimal reaction temperature and pH for this enzyme was detected to be 50 °C and 3.5. The Km and Vmax values of the recombinant MCO was determined to be 1.30 mmol/L and 7.67×10⁻² mmol/(L·min). Moreover, this MCO dramatically degrades histamine and tyramine by 51.6% and 40.9%, and can degrade other BAs including tryptamine, phenylethylamine, putrescine, cadaverine and spermidine, and was found to be tolerant to 18% (W/V) NaCl. The recombinant MCO is also capable of degrading BAs in soy sauce. The degradation rate of total BAs in soy sauce reaches 10.6% though a relatively low level of enzyme (500 U/L) is used. Multicopper oxidase has the potential to degrade biogenic amines in fermented foods, which lays a foundation for the further application of this kind of food enzymes.

Modelo de quesería artesanal bajo un signo distintivo en el Caribe colombiano: caso Atlántico

Introducción. Se presenta una propuesta de signo distintivo para la producción y comercialización de queso costeño de alto impacto en el departamento del Atlántico y en la región Caribe colombiana. Objetivo. Proponer a los productores de queso costeño, un modelo de producción y comercialización del producto, que siente las bases para convertir la quesería artesanal en un instrumento de desarrollo regional, a partir de la fabricación de un producto inocuo de base biotecnológica. Materiales y métodos. Estudio de tipo descriptivo abordado desde los aspectos socio-cultural y biotecnológico, para la identificación de valores colectivos relacionados con tradición quesera, preferencias de producción, asociatividad, cumplimiento de normas legales, signo distintivo y uso de cultivos lácticos autóctonos en sus procesos productivos para incorporar características del queso típico de la región. Resultados. Mediante el desarrollo de un signo distintivo es posible no solo el posicionamiento del queso costeño en el mercado como producto de alto valor, sino dejar atrás la indiferenciación actual propia de un producto básico, y lograr la reducción de riesgo de contaminación, teniendo en cuenta el valor agregado que representan los inóculos microbianos como innovación biotecnológica en la calidad del producto. Conclusión. El modelo presentado convenció al productor de que bajo el signo distintivo su producto tiene mayor valor agregado y, en consecuencia, podrá obtener un precio mayor al actual.

Introduction. A distinctive sign proposal is presented for high-impact production and marketing of coast cheese in the state of Atlántico and in the Colombian Caribbean region. Objective. To propose coast cheese producers a production and marketing model of the product; a model which lays the foundation to covert craft cheese factories into a regional development instrument from the manufacture of a biotechnological-based innocuous product. Materials and methods. Descriptive research addressed from social-cultural and biotechnological aspects for the identification of collective values associated to cheese-making tradition, production preferences, association capacity, compliance with legal regulations, distinctive sign, and use of native dairy cultures in their production processes in order to incorporate characteristics of the typical cheese of the region. Results. Through the development of a distinctive sign, it is possible not only to position the coast cheese in the market as a high-value product but also to left behind the present non-differentiation typical of a basic product and to achieve the decrease of contamination risks, bearing into account the value added brought by microbial inoculation as biotechnological innovation in the product quality. Conclusion. The model shown has convinced the producers that under the distinctive sign their product has better value added and then they would be able to gain a higher price.

Introdução. Se apresenta uma proposta de signo distintivo para a produção e comercialização de queijo litorâneo de alto impacto no departamento do Atlântico e na região do Caribe colombiano. Objetivo. Propor aos produtores de queijo litorâneo, um modelo de produção e comercialização do produto, que sente as bases para converter a indústria do queijo artesanal num instrumento de desenvolvimento regional, a partir da fabricação de um produto inócuo de base biotecnológica. Materiais e métodos. Estudo de tipo descritivo abordado desde os aspectos sociocultural e biotecnológico, para a identificação de valores coletivos relacionados com a tradição da indústria do queijo, preferências de produção, associatividade, cumprimento de normas legais, signo distintivo e uso de cultivos lácteos autóctones nos seus processos produtivos para incorporar características do queijo típico da região. Resultados. Mediante o desenvolvimento de um signo distintivo é possível não só o posicionamento do queijo litorâneo no mercado como produto de alto valor, senão deixar atrás a indiferença atual própria de um produto básico, e conseguir a redução do risco de contaminação, tendo em conta o valor agregado que representam os inócuos microbianos como inovação biotecnológica na qualidade do produto. Conclusão. O modelo apresentado convenceu ao produtor de que sob o signo distintivo seu produto tem maior valor agregado e, por consequência, poderá obter um preço maior ao atual.

Effects of Lactobacillus fermentum on the Inhibition of Cytoxan on Human Colorectal Cancer HCT116 Cells / 中国药房

OBJECTIVE:To study the effects of Lactobacillus fermentum(Lb-f)on inhibition of cytoxan(CTX) on HCT116 cells of human colorectal cancer in vitro and in vivo. METHODS:HCT116 cells were cultured in vitro. MTT assay was used to in-vestigate the effects of 4,2,1μg/ml CTX and combined with 10μg/ml Lb-f on the survival rate of HCT116 cells. HCT116 cell xe-nograft tumor nude mice model was induced to investigate the effects of 100,50,25 mg/kg CTX and combined with 180 mg/kg Lb-f on tumor volume,tumor weight,relative tumor inhibition rate,the sensitization effect of chemotherapy (by q value),the number of peripheral blood leucocyte and platelet. RESULTS:Compared with CTX alone,CTX combined with Lb-f had no signifi-cant effect on survival rate of HCT116,relative inhibition rate of tumor volume in nude mice, relative inhibition rate of tumor weight and q value (P>0.05),but increased the number of peripheral blood leucocyte and platelet (P<0.05). CONCLUSIONS:No synergistic effects of Lb-f is found on the inhibition of CTX on the growth of HCT116 in vitro and in vivo;Lb-f can inhibit the decrease of peripheral blood leucocyte and platelet of HCT116 cell xenograft tumor nude mice induced by CTX.

Probiotic characterization of lactic acid bacteria isolated from infants feces and its application for the expression of green fluorescent protein

Aims: In this study, lactic acid bacteria (LAB) were isolated from 42 healthy infants and determined for probiotic properties. Twelve LAB isolates with potential probiotic properties were selected and screened for their feasibility of heterologous protein expression by selection of erythromycin sensitive isolates. Methodology and results: One of eleven erythromycin-sensitive LAB isolates identified and designated as Lactobacillus fermentum 47-7 was able to acquire and stable maintain the Escherichia coli-Lactobacillus shuttle vector, pRCEID-LC13.9. Further electrotransformation of L. fermentum 47-7 with the recombinant pLC13.9:LDH-PRO1:GFPuv containing green fluorescent protein (GFP) gene found that recombinant L. fermentum can express GFP. Conclusion, significance and impact of study: The probiotic L. fermentum isolate can be used as host for expression of heterologous proteins and could possibly be further developed as the alternate oral delivery system for various biomolecules for biotechnological application.

Assessment of Infant Formula and Follow-on Formula Supplemented with Lactobacillus fermentum CECT5716.

The Norwegian Scientific Commitee for Food Safety (VKM) has appointed a working group of experts to answer a request from the Norwegian Food Safety Authority regarding risk assessment of Lactobacillus fermentum CECT5716 in infant formula intended for use from birth (0 month) and in infant formulae and follow-on formulae intended for children after the age of 6 month. The mandate of this risk assessment was not to evaluate the health claims related to the products as such health claims are assessed by EFSA. However, in the EFSA opinions so far (November 2009), a cause and effect relationship has not been established between the consumption of L. fermentum CECT5716 containing products and the claimed effects. In the assessment of L. fermentum CECT5716 by the expert committee in EFSA, the panel considered that the strain is sufficiently characterized. We are not aware of any data indicating that this strain has been the cause of human disease. Its potential toxicity and ability to translocate has been studied in adult mice after oral administration of doses 10 000 times greater than those normally consumed by humans without observing any bacteremia or translocation to spleen or liver. However, strains of Lactobacillus have from time to time been isolated from human blood cultures and may therefore, although seldom, translocate. L. fermentum CECT5716 is fully susceptible to all antibiotics that are recommended by the Panel on additive and products or substances used in animal feed (FEEDAP) of EFSA and is considered safe with respect to absence of antimicrobial resistance genes. DNA-sequence data do not imply the presence of any gene(s) coding for toxin production, but confirming in vitro-studies are missing. The FBO refers to two randomized clinical studies in healthy infants of 1-6months of age with respect to possible safety aspects, concluding that the L. fermentum CECT5716 was well tolerated and safe for the groups examined. However, data concerning safety aspects specifically concerned with new-borns, or for immunocompromised infants are lacking. The FBO has submitted some data regarding possible long-term adverse effects of giving the strain daily as a “monoculture” over a prolonged period of time. These data are not sufficient to draw any conclusion regarding long-term safety of the strain. It is supposed that the early composition of the human gastro-intestinal tract microbiota can have long-lasting functional effects. If that is the case, a daily supply of a “monoculture” of a single, specific strain such as L. fermentum CECT 5716, in large quantities over a prolonged period of time to age groups where the intestinal flora is still developing may therefore have unknown, but possible long-lasting adverse effects.

Antimicrobial Properties of Purified Bacteriocins Produced from Lactobacillus casei and Lactobacillus fermentum against Selected Pathogenic Microorganisms.

Aims: (1) To isolate bacteriocin samples produced from Lactobacillus using natural fermented foods which include: palm-wine, milk, locust beans, fufu (white solid food made from cassava), ogi (known as pap) and dairy fermented product (Yogurt); (2) extraction and purification of these bacteriocin samples using centrifugation and ammonium sulphate respectively and removal of impurities using dialysis. (3) to confirm the production of bacteriocin by performing antimicrobial assay against some selected pathogenic microorganisms. (4) to examine the effects of pH, heat and storage stability as well as biopreservative efficiency of the bacteriocin samples in pap, kunu (made from millet) and fresh orange juice; (5) to investigate the effect of viable antibiotics on the growth of isolates. Study Design: Data were analyzed using the statistical software package SPSS version 16.0 and standard errors of mean (SEM) for all the graphs plotted were represented with error bars while their characterization was designed using reciprocal of the highest dilution (2n) that resulted in the inhibition of the indicator lawn. Thus, the arbitrary units (AU) of the bacteriocin activity per milliliter (AU/ml) were defined as 2n× 1000/ 10μl. Place and Duration of Study: Microbiology Laboratory, Sacred heart Hospital, Abeokuta and Department of Microbiology, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria, between March 2010 and November 2012. Methodology: Bacteriocins, otherwise known as the antimicrobial compounds were produced from the Lactobacillus strains and then isolated from Nigerian fermented foods which include: palm wine, milk, yoghurt, locust beans, ogi and fufu. These foods were isolated using de Mann Rogosa and Sharpe medium. The isolated microorganisms (L. fermentum and L.casei) were identified phenotypically after isolation. Bacteriocins were extracted and purified from the Lactobacillus strains by centrifugation, followed by ammonium sulphate precipitation and dialysis. The antimicrobial activities of the crude bacteriocins were tested against nine selected pathogenic clinical isolates collected from the University College Hospital, Ibadan. The tested isolates were Shigella dysenteriae, Shigella flexneri, Enteropathogenic Escherichia coli type 1, Enteropathogenic Escherichia coli type 2, Enterohaemorrhagic E. coli type 3, Salmonella typhi, Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. Results: The bacteriocins of Lactobacillus fermentum and Lactobacillus casei showed a broad range of activities and had higher significant effect (P< 0.05) on the selected pathogenic microorganisms. The effects of pH on the bacteriocins were active in range of 2 to 6. Bacteriocins produced by Lactobacillus casei were stable at 800C for 15 minutes while bacteriocins produced by Lactobacillus fermentum were stable at 1000C for 15 minutes. It was observed that these bacteriocins can be stored between -200C and 40C and they had significant difference on the selected pathogenic microorganisms (P<0.05). The preservative activities of the bacteriocins tested on different foods showed that the bacteriocin of Lactobacillus fermentum had maximum reduction on bacterial population. Lactobacillus fermentum and Lactobacillus casei isolates were resistant to erythromycin of 70% and 100% for cotrimoxazole, ciprofloxacin, augmentin and amoxicillin. Conclusion: This study showed that bacteriocins from fermented foods could be used as an effective control for pathogenic microorganisms as they were able to exhibit antimicrobial activity against the test organisms when investigated for bacteriocin production and when characterized.

Chemical inhibition of the contaminant Lactobacillus fermentum from distilleries producing fuel bioethanol

The purpose of this study was to determine the Minimum Inhibitory Concentration (MIC) of pure or mixed chemicals for Saccharomyces cerevisiae and Lactobacillus fermentum in the samples isolated from distilleries with serious bacterial contamination problems. The biocides, which showed the best results were: 3,4,4' trichlorocarbanilide (TCC), tested at pH 4.0 (MIC = 3.12 mg/l), TCC with benzethonium chloride (CBe) at pH 6.0 (MIC = 3.12 mg/l) and TCC mixed with benzalkonium chloride (CBa) at pH 6.0 (MIC = 1.53 mg /l). If CBa was used in sugar cane milling in 1:1 ratio with TCC, a 8 times reduction of CBa was possible. This formulation also should be tested in fermentation steps since it was more difficult for the bacterium to develop resistance to biocide. There was no inhibition of S. cerevisiae and there were only antibiotics as an option to bacterial control of fuel ethanol fermentation by S. cerevisiae.

In vitro antagonistic growth effects of Lactobacillus fermentum and Lactobacillus salivarius and their fermentative broth on periodontal pathogens

As lactobacilli possess an antagonistic growth property, these bacteria may be beneficial as bioprotective agents for infection control. However, whether the antagonistic growth effects are attributed to the lactobacilli themselves or their fermentative broth remains unclear. The antagonistic growth effects of Lactobacillus salivarius and Lactobacillus fermentum as well as their fermentative broth were thus tested using both disc agar diffusion test and broth dilution method, and their effects on periodontal pathogens, including Streptococcus mutans, Streptococcus sanguis, and Porphyromonas gingivalisin vitro at different concentrations and for different time periods were also compared. Both Lactobacillus salivarius and Lactobacillus fermentum and their concentrated fermentative broth were shown to inhibit significantly the growth of Streptococcus mutans, Streptococcus sanguis, and Porphyromonas gingivalis, althoughdifferent inhibitory effects were observed for different pathogens. The higher the counts of lactobacilli and the higher the folds of concentrated fermentative broth, the stronger the inhibitory effects are observed. The inhibitory effect is demonstrated to be dose-dependent. Moreover, for the lactobacilli themselves, Lactobacillus fermentum showed stronger inhibitory effects than Lactobacillus salivarius. However, the fermentative broth of Lactobacillus fermentum showed weaker inhibitory effects than that of Lactobacillus salivarius. These data suggested that lactobacilli and their fermentative broth exhibit antagonistic growth activity, and consumption of probiotics or their broth containing lactobacilli may benefit oral health.

Bacteriocins produced by L fermentum and L acidophilus can inhibit cephalosporin resistant E coli

Reemerging infections occur due to resistant bacteria. Such infections create restrictions for clinicians and microbiologists in drug selection. Such problems demand new strategies for solution. Use of bacteriocins for this purpose may be fruitful. In the present research work, the inhibitory effects of bactericins on cephalosporin resistant Escherichia coli are used as model system for the control of antibiotic resistant pathogenic bacteria. Cephalosporin resistant Escherichia coli strain was isolated from pus by using conventional methodology. For bacteriocin production, Lactobacilli strains were selected by using selective media. Out of seventy two strains isolated from yogurt, fecal materials of human, chick, parrot and cat, only two strains (strain 45 and strain 52) were found to produce bacteriocins having antimicrobial potential against cephalosporin resistant Escherichia coli. Biochemical characterization showed that strain 45 belonged to group of Lactobacillus fermentum and strain 52 to Lactobacillus acidophilus. Both strains showed maximum growth at 25ºC and 35ºC respectively. Suitable pH was 5.5 and 6.0 for Lactobacillus fermentum and Lactobacillus acidophilus respectively. Bacteriocins produced by both strains were found stable at 50, 75 and 100ºC for 60min. Function of bacteriocin was also not disturbed due to change in pH. These findings suggest that bacteriocin produced by Lactobacillus fermentum and Lactobacillus acidophilus can be used for the infection control of cephalosporin resistant Escherichia coli.

An experimental study of casein phosphopeptide-amorphic calcium phosphate on the growth of Lactobacillus fermentum / 实用口腔医学杂志

To investigate the effect of casein phosphopeptide-amorphic calcium phosphate(CPP-ACP) on the growth of Lactobacillus fermentum and to evaluate the possibility of CPP-ACP as a biological anticariogenic agent. Lactobacillus fermentum was inoculated in BHI with different concentrations of CPP-ACP and grew in anaerobic condition at 37 ℃ for 48 h. Used MTT method to evaluate the effects of CPP-ACP on the growth of Lactobacillus fermentum. CPP-ACP inhibited the growth of Lactobacillus fermentum effectively. With the concentration of CPP-ACP increasing, the values of A(550 nm) decreased. It was found that CPP-ACP had inhibitory effect on the growth of Lactobacillus fermentum, the inhibitory effect increased with CPP-ACP concentration.

Antibacterial and antioxidant activities of acid and bile resistant strains of lactobacillus fermentum isolated from Miang

Miang is a kind of traditional fermented tea leaves, widely consumed in northern Thailand as a snack. It contains several kinds of Lactobacilli spp. The aim of this study was to isolate strains of Lactobacillus fermentum from miang and to investigate their antibacterial and antioxidant activities. The agar spot and well assays were used for determination of antibacterial power. The antibacterial mechanism was investigated by cell morphologic change under scanning electron microscope (SEM). Antioxidant activity was studied by means of free radical scavenging and ferric reducing power assays. The acid and bile screening tests indicated that L. fermentum FTL2311 and L. fermentum FTL10BR presented antibacterial activity against several pathogenic bacteria: Listeria monocytogenes DMST 17303, Salmonella Typhi DMST 5784, Shigella sonnei DMST 561 (ATCC 11060)and Staphylococcus aureus subsp. aureus DMST 6512 (ATCC 6538Ptm). The results from SEM suggested that the antibacterial action was due to the destruction of cell membrane which consequently caused the pathogenic cell shrinking or cracking. The antioxidant study suggested that both L. fermentum FTL2311 and L. fermentum FTL10BR strains could liberate certain substances that possessed antioxidant activity expressed as trolox equivalent antioxidant capacity (TEAC) and equivalent concentration (EC) values for free radical scavenging and reducing mechanisms, respectively. The supernatant of L. fermentum FTL2311 broth revealed TEAC and EC values of 22.54±0.12 and 20.63±0.17 µM.mg-1 respectively, whereas that of L. fermentum FTL10BR yielded TEAC and EC values of 24.09±0.12 and 21.26±0.17 µM.mg-1 respectively. These two strains isolated from miang present high potential as promising health-promoting probiotics.