Lactic Acid Bacteria Lactobacillus Plantarum Research Articles

Lactobacillus plantarum passage through an oro-gastro-intestinal tract simulator: Carrier matrix effect and transcriptional analysis of genes associated to stress and probiosis

Dietary probiotics should reach the intestine viable and in high numbers; therefore, they should tolerate the stress associated to the gastro-intestinal (GI) environment. Indeed, all along the different GI sections, probiotics are challenged by several sources of stress, including low pH, bile and digestive enzymes. Bacterial cells are equipped with various defense mechanisms to allow survival in hostile environments. The food matrix used to deliver beneficial bacteria may contribute to their probiotic action, e.g. by enhancing survival to stress and gut colonization. In this study, the survival of the lactic acid bacterium Lactobacillus plantarum WCFS1, a model probiotic strain, was examined in a human oro-gastric-intestinal (OGI) in vitro system, using different carrier matrices to compare protective and buffering properties. Higher survival was observed in complex and/or nutrient-rich matrices, and when potential prebiotics were added. The molecular response of L. plantarum to the OGI transit was analyzed by studying the transcriptional levels of genes involved in stress response and probiosis. The OGI steps of higher mortality corresponded to greater induction of stress genes, thus implying their involvement in adaptation to the gut environment. Plantaricins were significantly upregulated all along the different OGI sections; adhesion genes were mainly induced by gastric environment.

INFLUENCE OF LACTOBACILLUS PLANTARUM ONU 87 AND AUTOLYSATE OF BACTERIA ERWINIA CAROTOVORA ZM1 ON INFECTIVITY OF SOFT ROT DISEASE PATHOGENS

Influence of lactic acid bacteria Lactobacillus plantarum ONU 87 and autolysate of Erwinia carotovora ZM1 cells containing bacteriophages and macromolecular bacteriocins on infectivity of ten Erwinia carotovora strains (soft rot disease pathogens) has been studied. Infectious process has been modeled on potato tubers (Solanum tuberosum L.), carrot roots (Daucus carota L.) and tomato plantlets (Lycopersicon esculentum Mill.). The level of inhibition of infective activity by separately used Lactobacillus plantarum ONU 87 and Erwinia carotovora ZM1 autolysate depends on the origin of active substance and test-object. Coeffect of Lactobacillus plantarum ONU 87 and cell autolysate of Erwinia carotovora ZM1 results in complete inhibition of infectivity of phytopathogenic erwinias on all the test-objects.

Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45d did not differ from those of the control Caciocavallo Pugliese cheese at 60d of ripening. In contrast, the cheese made with AC at 45d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.

Fermentation by Amylolytic Lactic Acid Bacteria and Consequences for Starch Digestibility of Plantain, Breadfruit, and Sweet Potato Flours

The potential of tropical starchy plants such as plantain (Musa paradisiaca), breadfruit (Artocarpus communis), and sweet potato (Ipomoea batatas) for the development of new fermented foods was investigated by exploiting the capacity of some lactic acid bacteria to hydrolyze starch. The amylolytic lactic acid bacteria (ALAB) Lactobacillus plantarum A6 and Lactobacillus fermentum Ogi E1 were able to change the consistency of thick sticky gelatinized slurries of these starchy fruits and tubers into semiliquid to liquid products. Consequently, a decrease in apparent viscosity and an increase in Bostwick flow were observed. These changes and the production of maltooligosaccharides confirmed starch hydrolysis. Sucrose in sweet potato was not fermented by strain A6 and poorly fermented by strain Ogi E1, suggesting possible inhibition of sucrose fermentation. In all 3 starchy plants, rapidly digestible starch (RDS) was higher than slowly digestible starch (SDS) and resistant starch (RS) represented between 17% and 30% dry matter (DM). The digestibility of plantain was not affected by fermentation, whereas the RDS content of breadfruit and sweet potato decreased and the RS content increased after fermentation. The characteristics resulting from different combinations of gluten free starchy plants (plantain, breadfruit, sweet potato) and amylolytic lactic acid bacteria (ALAB) offer opportunities to develop new functional fermented beverages, mainly for breadfruit and sweet potato, after further investigation of their formulation, sensory attributes, nutritional, and prebiotic characteristics.

ANTIMICROBIAL ACTIVITY OF BACTERIOCIN FROM INDIGENOUS Lactobacillus plantarum 2C12 AND ITS APPLICATION ON BEEF MEATBALL AS BIOPRESERVATIVE

One purpose of food preservation is to extend the shelf life of foods. Biological preservations canbe conducted by adding antimicrobial substances, such as bacteriocin produced by lactic acid bacteriaand has been characterized as biopreservatives. The aims of this research were to evaluate antimicrobialactivity of bacteriocin produced by indigenous lactic acid bacteria Lactobacillus plantarum 2C12isolated from local beef and to study the quality of beef meatball with 0.3% bacteriocin asbiopreservative at different storage times (0, 3, and 6 days) in cold temperature (4oC), compared to 0.3%nitrite and control (without preservative). The results showed that bacteriocin from L. plantarum 2C12could inhibit pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and SalmonellaTyphimurium. Bacteriocin was effective as well as nitrite as biopreservatives of meatballs by inhibitingthe growth of total microbes and E. coli. The addition of bacteriocin did not lead the physical andnutritional changes in the meatballs. The quality of meatball with bacteriocin treatment conformed withIndonesia National Standard of meatball.

CcpA and three newly identified proteins are involved in biofilm development in Lactobacillus plantarum

The aim of this study was to identify genes involved in biofilm development in the probiotic lactic acid bacterium Lactobacillus plantarum. The ability of L. plantarum LM3 and of some derivative mutant strains to form biofilm has been investigated. Biofilm microtitre plate assays showed that L. plantarum LM3-2, carrying a null mutation in the ccpA gene, coding the CcpA master regulator, was partially impaired in biofilm production compared to wild type (LM3). Moreover, we found three genes in the L. plantarum genome, hereby named flmA, flmB, and flmC, whose deduced amino acid sequences show significant identity with the Streptococcus mutans BrpA (biofilm regulatory protein A). We investigated the role of FlmA, FlmB, and FlmC in biofilm formation by isolating strains carrying null mutations in the corresponding genes. Our results suggest involvement of the Flm proteins in biofilm development. Moreover, transcriptional studies show that expression of flmA, flmB, and flmC is under the control of CcpA. These results, together with the reduced ability of LM3-2 (ccpA1) to form biofilm, strongly suggest a positive role of the master regulator CcpA in biofilm development.

Microbial populations during maize storage in Cameroon

Key microbiota of maize kernels from two farms in Cameroon were characterised at harvest and during five months of drying / storage in firewood kitchens. Moulds populations shifted from Fusarium sp. to Penicillium and Aspergillus during drying, and aflatoxigenic moulds were absent. Lactic acid bacteria ( Lactobacillus plantarum , Leuconostoc citreum ) were only present earlier on, and Enterobacteriaceae became dominant. Common yeasts were Candida quercitrusa (early storage) and Meyerozyma guilliermondii (late storage). Strains of L. plantarum and M. guilliermondii are known to inhibit mould growth and could be used in an energy-efficient system for moist-storage of maize kernels. Keywords: Maize, mycotoxins, mould, yeast, lactic acid bacteria, biocontrol

Yeast mannan structure necessary for co-aggregation with Lactobacillus plantarum ML11-11

Lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and yeast Saccharomyces cerevisiae form significant mixed-species biofilm with direct cell–cell contact. Co-aggregation of L. plantarum ML11-11 and S. cerevisiae cells, mediated by the interaction between surface protein(s) on L. plantarum ML11-11 cells and surface mannan of S. cerevisiae cells, contributes significantly to mixed-species biofilm formation. In this study, co-aggregation activities of yeast mutants that were deleted of genes related to mannan biosynthesis were investigated to clarify the mannan structures essential for interaction with L. plantarum ML11-11. Among the 12 deletion mutants which had various incomplete mannan structures, only the mnn2 mutant lost the co-aggregation activity. In the mnn2 mutant, the gene coding the activity of attaching first branching mannose residue to mannan main chain is deleted and therefore the mnn2 mutant has unbranched mannan. From this result, it is clarified that the specific structure, consisted of mannan main chain to which are attached side chains containing one or more mannose residues, is critical for co-aggregation with L. plantarum ML11-11.

Biological activity of Streptomyces noursei against ochratoxin A producing Aspergillus niger

Aspergillus niger isolated from some common medicinal and herbal plants used in Egypt, was found to be producer for ochratoxin A. This research investigate the biological control of ochratoxin A production by A. niger, using some bacterial isolates belonging to true bacteria (Bacillus pumilus), Lactic acid bacteria (Lactobacillus acidophilus, Lactobacillus bulgaricus and Lactobacillus plantarum) and Actinomycete isolates numbered 1, 2, 3, 4, 5, 6 and 7, that proved variable antagonistic activity against A. niger. The Actinomycete isolate number 3 was the most effective and identified as Streptomyces noursei. The optimal conditions for antifungal production by S. noursei were performed on starch nitrate medium at pH 7 after 7 days of growth at 30°C. The active antifungal substance(s) were separated, semi purified and characterized as a protein substance. The semi purified substance inhibited spores formation, thinned the conidiophores and deformed both strigmata and conidial heads.

Preliminary X-ray analysis of twinned crystals of the Q88Y25_Lacpl esterase from Lactobacillus plantarum WCFS1.

Q88Y25_Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxylesterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-terminally His(6)-tagged Q88Y25_Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His(6)-tagged Q88Y25_Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 Å resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 Å.

Utilization of Echium amoenum Extract as a Growth Medium for the Production of Organic Acids by Selected Lactic Acid Bacteria

Gol-gavzaban (Echium amoenum Fisch. & Mey.) is an indigenous herbal plant, related to the Boraginaceae family, cultivated historically in Iran. In this study, the suitability of Echium extract as a raw material for production of fermented juice by four strains of lactic acid bacteria (Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii) was examined. Echium extract was inoculated with these bacteria and incubated at 30 °C for 24 h. Changes in microbial population, pH, acidity, sugars, and organic acids metabolism were measured during the fermentation process. Results showed that all of the selected bacteria were capable to grow well in Echium extract without any supplementation. They all metabolized sugars including glucose, fructose and sucrose simultaneously. L. paracasei showed more affinity to sugar consumption at almost 45.2%, 38%, and 21.6% of initial glucose, fructose, and sucrose concentration, respectively. Lactic acid was produced immediately after the fermentation started. L. plantarum, L. delbrueckii, and L. acidophilus produced 6.8, 6.6, and 6.4 g/l lactic acid, respectively, which were significantly higher than that produced by L. paracasei (5 g/l). On the other hand in the case of acetic acid, L. paracasei produced significantly greater quantities in comparison with other strains (4.48 g/l). It was proved that E. amoenum was a desirable media for the production of some organic acids by all these bacterial strains.

Factors influencing the production of volatile phenols by wine lactic acid bacteria

This work aimed to evaluate the effect of certain factors on the production of volatile phenols from the metabolism of p-coumaric acid by lactic acid bacteria (LAB) ( Lactobacillus plantarum, L. collinoides and Pediococcus pentosaceus). The studied factors were: pH, l-malic acid concentration, glucose and fructose concentrations and aerobic/anaerobic conditions. It was found that, in the pH range of 3.5 to 4.5, the higher the pH the greater the production of volatile phenols. This behaviour is correlated with the effect of pH on bacterial growth. Increasing levels of l-malic acid in the medium diminished the production of 4-vinylphenol (4VP) and stimulated the production of 4-ethylphenol (4EP) by L. plantarum NCFB 1752 and L. collinoides ESB 99. The conversion of 4VP into 4EP by the activity of the vinylphenol reductase may be advantageous to the cells in the presence of l-malic acid, presumably due to the generation of NAD +, a cofactor required by the malolactic enzyme. Relatively high levels of glucose (20 g/L) led to an almost exclusive production of 4VP by L. plantarum NCFB 1752, while at low concentrations (≤ 5 g/L), 4EP is mainly or solely produced. Part of the glucose may be diverted to the production of mannitol as an alternative pathway to regenerate NAD +. This is corroborated by the experiments done with fructose, a compound that can be used as an electron acceptor by some bacteria becoming reduced to mannitol. In anaerobiosis, the reduction of 4VP into 4EP is clearly favoured, which is consistent with the need to increase the availability of NAD + in these conditions. This study shows that the amount and the ratio 4VP/4EP produced by LAB are greatly affected by certain environmental and medium composition factors. The behaviour of the bacteria seems to be driven by the intracellular NAD +/NADH balance.

Seaweed (Ulva reticulata) Based Fermented Marine Silage Feed Preparation under Controlled Conditions for Penaeus monodon Larval Development

Fermentation of seaweed to produce larval feed was developed using an in situ fermentor. Marine single cell detritus (MSCD), a seaweed based fermented product is an ideal material for feeding shrimp larvae. This paper deals with the development of protocol for the preparation of MSCD using the fermentor. Ulva reticulata, a green seaweed, was chosen as the base material and the enzyme Cellulase was used for the degradation of seaweeds into single cell units. Fermentation of Ulva sp was carried out by a consortium comprising a lactic acid bacterium Lactobacillus plantarum (LAB) and the yeast of food grade Saccharomyces cerevisiae (grape source). Potato powder a sugar substrate and soya powder a nitrogen substrate were also added. The process of fermentation was monitored continuously by estimating the lactic acid concentration, pH and also by the odour. The microbial propagation pattern for a period of 50 days has also been observed to understand the product's keeping quality.

Genomic diversity and versatility of Lactobacillus plantarum, a natural metabolic engineer

In the past decade it has become clear that the lactic acid bacterium Lactobacillus plantarum occupies a diverse range of environmental niches and has an enormous diversity in phenotypic properties, metabolic capacity and industrial applications. In this review, we describe how genome sequencing, comparative genome hybridization and comparative genomics has provided insight into the underlying genomic diversity and versatility of L. plantarum. One of the main features appears to be genomic life-style islands consisting of numerous functional gene cassettes, in particular for carbohydrates utilization, which can be acquired, shuffled, substituted or deleted in response to niche requirements. In this sense, L. plantarum can be considered a “natural metabolic engineer”.

Comparison of Lactic Acid Bacteria Fermentation with Acid Treatments for Chitosan Production from Shrimp Waste

The traditional procedure for chitosan production involves use of a strong acid (HCl) for demineralization of chitin. This study reports application of a mixed culture of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus lactis) fermentation in demineralization of chitin for chitosan production from shrimp waste. Chitosan produced from shrimp waste with lactic acid bacteria fermentation at 30°C for 72 h or 1 M lactic acid treatment at room temperature for 2 h followed by alkaline treatments (0.5 M NaOH at 25°C for 4 h followed by 12.5 M NaOH at 70°C for 10 h) contained very low protein (0.8–1.1%) and ash (<0.01%) contents with the same solubility (100%) as those of chitosan produced with hydrochloric acid treatment. Lactic acid bacteria fermentation and lactic acid treatment may be utilized in chitosan production from shrimp waste to reduce use of strong chemicals.

Sensitivity of sheep intestinal lactic acid bacteria to secondary compounds extracted from Acacia saligna leaves

The sensitivity of the lactic acid bacteria (LAB) Lactobacillus plantarum and Enterococcus faecium), isolated from sheep faeces, to secondary compounds extracted from Acacia saligna leaves was investigated. Secondary compounds (SC) extracted from A. saligna foliage were: total phenolics (TP), saponins (SP), alkaloids (AK), a methanol extract (ME) and an aqueous extract (AF). All SC were examined with and without PEG for inhibitory effects on L. plantarum, E. faecium and a LAB mixture ( L. plantarum + E. faecium 1:1). The highest sensitivity ( i.e., inhibition zone) occurred in L. plantarum to all SC, whereas E. faecium was more resistant. All LAB were more sensitive to TP than to other SC, as inhibition zones ranged from 7 to 26 mm 2 for AK and TP in L. plantarum, from 5 to 15 mm 2 for the AF and ME in E. faecium, and from 5 to 14 mm 2 for AK and TP in the LAB mixture. Adding SC to the bacterial medium reduced (P<0.05) growth and lactic acid production. Within each bacterial species, growth, biomass and lactic acid production were lowest (P<0.05) when TP extract was added to cultures of L. plantarum and the LAB mixture, and when ME was added to cultures of E. faecium. Combining both LAB strains increased biomass production with all SC. Resistant colonies increased (P<0.001) with TP addition in L. plantarum, and with TP, SP and AK in E. faecium. PEG decreased inhibitory effects of SC and increased bacterial growth and production of lactic acid and biomass, probably due to its ability to form insoluble complexes with SC. Response to PEG was stronger against TP than other SC, especially L. plantarum and the LAB mixture. Overall, L. plantarum was more susceptible than E. faecium or the LAB mixture to SC, and had a higher response to PEG addition, which reduced toxicity in both bacterial strains. Lactic acid bacteria would be affected to a different extent by SC reaching the small intestine, whereas PEG may neutralize inhibitory effects of SC on these bacterial species.

Kinetic study of high pressure microbial and enzyme inactivation and selection of pasteurisation conditions for Valencia Orange Juice

SummaryKeeping quality of fresh orange juice is highly dependent on pectinolytic enzyme activity and the growth of spoilage microorganisms. The inactivation kinetics of indigenous pectin methylesterase (PME) and of the two more pressure resistant species of spoilage lactic acid bacteria (LAB) Lactobacillus plantarum and L. brevis in freshly squeezed Valencia orange juice under high hydrostatic pressure (100–500 MPa) combined with moderate temperature (20–40 °C) was investigated. PME inactivation followed first order kinetics with a residual PME activity (15%) at all pressure–temperature combinations used. The values of activation energy and activation volume were estimated at each pressure and at each temperature, respectively. Values of 90 kJ/mol and −30 mL/mol at reference pressure of 300 MPa and reference temperature of 35 °C were estimated respectively. The corresponding zT and zP values of LAB inactivation were also estimated at all conditions tested. Values of 19.5 °C and 95 MPa at reference pressure of 300 MPa and reference temperature of 30 °C were estimated respectively for L. plantarum, while the corresponding values for L. brevis were 40 °C and 82 MPa, respectively, at the same reference conditions. Pressure and temperature were found to act synergistically both for PME and LAB inactivation. The PME and LAB inactivation rate constants were expressed as functions of the temperature and pressure process conditions. These functions allow the determination of the pressure/temperature conditions that achieve the target enzyme and microbial inactivation at a selected processing time. The process conditions of 350 MPa at 35 °C for 2 min are proposed as effective for Valencia orange juice cold pasteurisation.

균상재배 느타리버섯부산물 생균발효사료의 반추동물 조사료원으로서의 가치 평가

본 연구는 균상재배 느타리버섯부산물(SMS)의 반추동물 조사료원으로서의 가치를 평가하기 위하여 in situ 반추위 소실율 및 면양을 이용한 in vivo 영양소 소화율, 체내 질소이용성 및 섭취행동 패턴을 평가하였다. SMS에 당밀 5% (w/w, 건물기준), 유산균(Lactobacillus plantarum) 0.5% (v/w) 그리고 효모 (Saccharomyces cerevisiae) 0.5% (v/w)를 첨가하여 30일간 혐기발효 시킨 후 반추위캐뉼라가 장착된 평균체중 620 kg Holstein 육우 2두를 이용하여 in situ 소실율을 평가하였다. 면양대사실험은 평균체중 48kg의 숫 면양 6두 이용하여 <TEX>$3{\times}3$</TEX> 라틴방각법으로 하였다. 대조구는 볏짚 100%를, 처리구는 볏짚의 25%와 50%를 생균발효 SMS로 각각 대체 급여하였다. 균상재배 버섯부산물의 반추위 in situ DM 및 NDF 이용성은 생균발효처리에 의해 개선되었다(P<0.001). 생균발효 SMS를 볏짚의 25% 및 50% 대체하여 면양에게 급여하였을 때, 단백질과 섬유소(NDF, ADF, CF) 소화율은 감소하였으나 (P<0.05), 조회분 소화율은 증가하였으며(P<0.001), 면양체내 단백질 축척량은 차이가 없었다(P>0.05). 볏짚을 생균발효 SMS로 대체 급여함에 따라 면양의 반추시간은 평균 28% 감소하였다(P<0.05). 생균발효 SMS는 볏짚의 76% 수준의 에너지가를 보였다. 결과적으로, 균상재배 느타리버섯부산물은 유지목적의 반추동물 조사료원으로서 보다 적합한 것으로 사료되었다. An in situ ruminal disappearance trial and an in vivo sheep metabolism trial were conducted to evaluate the nutritional value of spent mushroom substrate (SMS, originated from Pleurotus osteratus bed-type cultivation). The raw SMS was ensiled (ESMS) for 30 days with 5% (w/w, DM basis) molasses, 0.5% (v/w) yeast (Saccharomyces cerevisiae) and 0.5% (v/w) lactic acid bacteria (Lactobacillus plantarum). Two ruminally cannulated Holsteins (average BW 620 kg) were used to evaluate in situ disappearance. Six sheeps (average BW 48 kg) were fed, in <TEX>$3{\times}3$</TEX> Latin square design, rice straw alone (Control), 25% (ESMS-25) and 50% (ESMS-50) of rice straw were replaced with ensiled SMS. For an in situ trial, ruminal DM and neutral detergent fiber (NDF) disappearance of SMS were increased after ensiling (P<0.05). For a sheep trial, protein and fiber (NDF, acid detergent fiber, crude fiber) digestibilities were decreased (P<0.05), crude ash digestibility was increased (P<0.001), and nitrogen retention was not affected (P>0.05) as rice straw was replaced with ensiled SMS. Ruminating time was decreased by an average of 28% by feeding ensiled SMS (P<0.05). Ensiled SMS (Bed-type cultivation) had 76% of energy value of rice straw. Consequently, ensiled SMS (Bed-type cultivation, 100% cotton waste) could be used as a roughage source appropriate for maintenance type rations for ruminants.

Characterization of a Nitroreductase with Selective Nitroreduction Properties in the Food and Intestinal Lactic Acid Bacterium Lactobacillus plantarum WCFS1

Nitroreductases reduce nitroaromatic compounds and other oxidants in living organisms, having interesting implications in environmental and human health. A putative nitrobenzoate reductase encoding gene (lp_0050) was recently annotated in the completed DNA sequence of lactic acid bacterium Lactobacillus plantarum WCFS1 strain. In this research, this L. plantarum gene was cloned and expressed, and the corresponding protein (PnbA) was biochemically characterized. This L. plantarum PnbA reductase is a 216 amino acid residue FMN-flavoprotein, which exhibits 23% identity with Pseudomonas putida and Ralstonia eutropha nitroreductases and <11% identity with those from enterobacteria such as E. cloacae . This reductase also showed 32-43% identity (65-72% similarity) to predicted PnbA proteins from other lactic acid bacteria. It utilized a wide range of electron acceptors including dichlorophenolindophenol (DCPIP), nitroblue tetrazolium (NBT), ferricyanide, and quinones (menadione, benzoquinone), but not pyridinium cations (paraquat and N-methyl-beta-carbolines), and it was inhibited by dicoumarol and diphenyliodonium. HPLC-MS and spectroscopic data showed that it specifically catalyzed the reduction of the 4-nitroaromatic group to the corresponding hydroxylamine in the presence of NAD(P)H. Kinetics parameters (V(max) and K(m)) showed a higher efficiency for the reduction of 2,4-dinitrobenzoate than for the reduction of 4-nitrobenzoate. It was chemoselective for the reduction of 4-nitrobenzoates, being unable to reduce other nitroaromatics. Then, L. plantarum PnbA reductase might be more specific than other microbial nitroreductases that reduce a wider range of nitroaromatic compounds. The physiological and functional role of nitroreductases remain unknown; however, their presence in lactic acid bacteria widely occurring in foods and the human intestinal tract should be of further interest.

Microbial and chemical properties of aji-no-susu, a traditional fermented fish with rice product in the Noto Peninsula, Japan

Aji-no-susu is a Japanese fermented fish product prepared from salted horse mackerel Trachurus japonicus, and cooked rice. We studied the organic acid and free amino acid contents and microflora in 12 aji-no-susu products to clarify their features as a lactic-acid-fermented food. Salinity of the samples was approximately 7.0% (rice portion) and 6.0% (fish portion) (w/w). Water activity was approximately 0.9, and pH was approximately 4.4 and lower. In the rice portions, lactic acid content was very high (57 mg/g sample). The predominant amino acids were alanine (2.3 mg/g rice portion) and lysine (2.1 mg/g). In the case of long-fermented (4 and 12 months) aji-no-susu, a high content of gamma-aminobutyric acid (GABA, 1.5 and 1.4 mg/g) was detected. Total viable counts in rice and fish portions were 7.7 and 7.4 log colony-forming units (cfu)/g, respectively. The number of lactobacilli in the rice and fish portions was 7.3 and 7.1 log cfu/g, respectively. Yeasts were detected in eight samples. Furthermore, acid-tolerant lactic acid bacteria (LAB) (Lactobacillus plantarum), GABA-producing LAB (Lactobacillus sp.), and halophilic or halo-tolerant yeast (Debaryomyces hansenii) were isolated and identified. Results in this study indicate that aji-no-susu is a typical traditional lactic-acid-fermented fish product.