Tannase activity by lactic acid bacteria isolated from grape must and wine

We examined a range of oenological lactic acid bacteria species and reference strains for their potential to degrade tannins. Bacterial tannase activity was checked by a spectrophotometric and a visual reading method. None of the strains belonging to the oenological species of the genus Lactobacillus, Leuconostoc, Oenococcus or Pediococcus were tannase producers, with the exception of Lactobacillus plantarum. All the L. plantarum strains analyzed were positive for tannase activity and their identities were reconfirmed by L. plantarum PCR-specific assay or by sequencing the 16S rDNA. Tannase activity could be considered an important criterion for the selection of malolactic starter cultures since it might confer advantages in the winemaking process by reducing astringency and haze in wine.     

Behavior of Listeria monocytogenes in pasteurized milk during fermentation with lactic acid bacteria

The behavior of Listeria monocytogenes in pasteurized milk during fermentation with starter and nonstarter lactic acid bacteria was investigated. Pasteurized milk was co-inoculated with approximately 10(4) CFU/ml of L. monocytogenes and 10(6) CFU/ml of Lactococcus lactis, Lactococcus cremoris, Lactobacillus plantarum, Lactobacillus bulgaricus, or Streptococcus thermophilus. Inoculated milks were incubated at 30 degrees C or 37 degrees C for 24 to 72 h. Listeria monocytogenes survived and also grew to some extent during incubation in the presence of all starter cultures; however, inhibition ranged from 83 to 100% based on maximum cell populations. During incubation with L. bulgaricus and L. plantarum, L. monocytogenes was completely inactivated after 20 h and 64 h of incubation at 37 degrees C and 30 degrees C, respectively. The pH of the fermenting milks declined steadily throughout the fermentation periods and was approximately 4.2 at the conclusion of the experimental period regardless both of the starter culture and pathogen combination or the temperature of incubation.     

Bacillus sphaericus: the highest bacterial tannase producer with potential for gallic acid synthesis

An indigenously isolated strain of Bacillus sphaericus was found to produce 1.21 IU/ml of tannase under unoptimized conditions. Optimizing the process one variable at a time resulted in the production of 7.6 IU/ml of tannase in 48 h in the presence of 1.5% tannic acid. A 9.26-fold increase in tannase production was achieved upon further optimization using response surface methodology (RSM), a statistical approach. This increase led to a production level of 11.2I U/ml in medium containing 2.0% tannic acid, 2.5% galactose, 0.25% ammonium chloride, and 0.1% MgSO(4) pH 6.0 incubated at 37°C and 100 rpm for 48 h with a 2.0% inoculum level. Scaling up tannase production in a 30-l bioreactor resulted in the production of 16.54 IU/ml after 36 h. Thus far, this tannase production is the highest reported in this bacterial strain. Partially purified tannase exhibited an optimum pH of 5.0 with activity in the pH range of 3 to 8; 50°C was the optimal temperature for activity. Efficient conversion of tannic acid to purified gallic acid (90.80%) was achieved through crystallization.     

Degradation of tannic acid by cell-free extracts of Lactobacillus plantarum

The ability of Lactobacillus plantarum CECT 748^T to degrade hydrolysable tannins was evaluated. Three commercial tannic acids were incubated in presence of cell-free extracts containing soluble proteins from L. plantarum. By HPLC analyses, almost a complete tannic acid degradation was observed in the three samples assayed. By using HPLC-DAD/ESI-MS, we partially determined the composition of tannic acid from Quercus infectoria galls. This tannic acid is a gallotannin mainly composed of monomers to tetramers of gallic acid. We studied the mechanism of its degradation by L. plantarum. The results obtained in this work indicated that L. plantarum degrades gallotannins by depolymerisation of high molecular weight tannins and a reduction of low molecular weight tannins. Gallic acid and pyrogallol were detected as final metabolic intermediates. Due to the potential health beneficial effects, the ability to degrade tannic acid is an interesting property in this food lactic acid bacteria.     

Response of a Lactobacillus plantarum human isolate to tannic acid challenge assessed by proteomic analyses

Scope : To gain insight on the mechanisms used by intestinal bacteria to adapt and resist the antimicrobial action of dietary tannins and identify targets for tannic acid in Lactobacillus plantarum. Methods and results : A proteomic analysis of an L. plantarum human isolate exposed to the tannic acid challenge was undertaken. Tannic acid targeted proteins involved in outstanding processes for bacterial stress resistance including cyclopropanation of membrane lipids, stress response at population scale and maintenance of cell shape. To respond to this aggression, tannic acid‐misfit cells of L. plantarum challenged with tannic acid reorganized their metabolic capacity to economize energy and express proteins involved in oxidative stress defense and cell wall biogenesis, indicating that the injury incurred by tannic acid was based on oxidative damage and disruption of the cell envelope. The induction of 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase, which is sensitive to changes in redox conditions and involved in ubiquinone biosynthesis in other bacteria, suggests for a tannic acid‐induced redox imbalance. Conclusion : The results reveal the adaptation of a gastrointestinal isolate of L. plantarum to tannic acid and identify antibacterial targets for this dietary compound. This provides the basis for the selection of tannin‐resistant microorganisms and their use to obtain health benefits from tannin‐containing diets.     

黑曲霉N5-5产单宁酶的酶学性质与固定化

本实验室自主分离的黑曲霉N5-5所产单宁酶已发现对没食子酸丙酯具有良好酶解效果。为了单宁酶的工业化应用,本次研究大批量培养单宁酶,探究其对单宁酸的酶解效果及酶的固定化效果。发酵采用先液体扩培后固体发酵的形式,提酶后利用陶瓷膜过滤技术纯化、浓缩酶液,并对比冷冻干燥和喷雾干燥两种不同干燥方式,还使用树脂载体对酶进行固定化。实验表明,纯化后单宁酶酶活达258.53 U/mL,可水解10%至50%浓度的单宁酸,其中30%以下底物浓度酶解效果较好;冷冻干燥对酶活影响不大而喷雾干燥使酶活降为174.02 U/mL;另外,单宁酶通过树脂载体固定化后,在实验条件下可重复使用至少4次。研究为提高黑曲霉N5-5发酵所产单宁酶的媒介效率、降低酶解反应成本、实现商业化生产建立了理论基础。     

Degradation of aflatoxin B(1) by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556(T)

Biological degradation of aflatoxin B(1) (AFB(1)) by Rhodococcus erythropolis was examined in liquid cultures and in cell-free extracts. Dramatic reduction of AFB(1) was observed during incubation in the presence of R. erythropolis cells (17% residual AFB(1) after 48 h and only 3-6% residual AFB(1) after 72 h). Cell-free extracts of four bacterial strains, R. erythropolis DSM 14,303, Nocardia corynebacterioides DSM 12,676, N. corynebacterioides DSM 20,151, and Mycobacterium fluoranthenivorans sp. nov. DSM 44,556(T) were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade AFB(1) was studied under different incubation conditions. Aflatoxin B(1) was effectively degraded by cell free extracts of all four bacterial strains. N. corynebacterioides DSM 12,676 (formerly erroneously classified as Flavobacterium aurantiacum) showed the lowest degradation ability (60%) after 24 h, while >90% degradation was observed with N. corynebacterioides DSM 20,151 over the same time. R. erythropolis and M. fluoranthenivorans sp. nov. DSM 44,556(T) have shown more than 90% degradation of AFB(1) within 4 h at 30 degrees C, whilst after 8 h AFB(1) was practicably not detectable. The high degradation rate and wide temperature range for degradation by R. erythropolis DSM 14,303 and M. fluoranthenivorans sp. nov. DSM 44,556(T) indicate potential for application in food and feed processing.     

东北传统发酵食品中高抗氧化活性植物乳杆菌的筛选及体外耐受性分析

为了获得高抗氧化活性植物乳杆菌,从东北传统发酵食品辣椒酱、臭豆腐、粘面子中筛选出75株植物乳杆菌。将75株植物乳杆菌分为无细胞上清液、完整细胞、无细胞提取物三个组分,以DPPH和ABTS+自由基清除率为指标对菌株进行筛选,分析不同指标间的相关性,并评价菌株耐酸、耐胆盐能力及对抗生素的敏感性。结果表明,23株菌株表现出较好的抗氧化活性,无细胞上清液对DPPH和ABTS自由基清除率均大于90%,完整细胞和无细胞提取物对这两种自由基清除率均大于30%。植物乳杆菌的三个组分在清除DPPH自由基中存在相关性,无细胞上清液在DPPH和ABTS+两种评价方法上存在极显著相关性。其中有5株植物乳杆菌(D2、H8、L20、L11和A2)在pH2.0环境中存活率均大于59%,在0.3%胆盐中的存活率均大于93%,对7种抗生素敏感性较强。因此,这5株植物乳杆菌对于开发抗氧化作用的功能食品具有潜在的应用价值。     

Asp.niger No.3液态发醇生产单宁麽酶条件的研究

研究不同碳源、氮源种类和比例及不同起始pH值等因素对黑曲霉生产单宁酶的影响;并考察了豆粕+NH4NO3分别与单宁、K2HPO4的交互作用。结果表明不同碳源、氮源种类和比例、不同起始pH值等因素对黑曲霉生产单宁酶的影响较大,单宁1.25,小麦粉0.35,豆粕0.67％,NH4NO30.05％,K2HPO0.035,pH6.0为产酶的适宜条件,最高酶活达到144.25U/100ml发酵液,酶比活为17.71。豆粕+NH4NO3、单宁、小麦粉对产酶有极显著影响;豆粕+NH4NO3与K2HPO4的交互作用极显著,与单宁的交互作用显著。本文还探索了菌丝生长与产酶的关系,结果表明黑曲霉产单宁酶属于生长偶联型。     

Protein Degradation by Lactobacillus plantarum and Lactobacillus casei in a Sausage Model System

ABSTRACT: :
The proteolytic activity of a starter culture involving Lactobacillus plantarum and Lactobacillus casei towards meat sarcoplasmic and myofibrillar proteins during the fermentation of a sausage-like system was studied. After 96 h of incubation the proteolytic system of L. plantarum CRL681 caused a degradation of both sarcoplasmic and myofibrillar proteins, whereas L. casei CRL705 showed a different affinity to meat proteins. The inoculation of both strains showed a higher activity toward sarcoplasmic fraction. These results correlated with a high rate of sarcoplasmic protein degradation observed in SDS-PAGE analysis. The generation of free amino acids as well as the pH drop at the end of the incubation period was maximal in presence of the mixed starter culture, thereby demonstrating the suitability of these strains to be used in the fermentation of meat products.     

Tyrosine and phenylalanine catabolism by Lactobacillus cheese flavor adjuncts

Bacterial metabolism of Tyr and Phe has been associated with the formation of aromatic compounds that impart barny-utensil and floral off-flavors in cheese. In an effort to identify possible mechanisms for the origin of these compounds in Cheddar cheese, we investigated Tyr and Phe catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under simulated Cheddar cheese-ripening (pH 5.2, 4% NaCl, 15 degrees C, no sugar) conditions. Enzyme assays of cell-free extracts indicated that L. casei strains catabolize Tyr and Phe by successive, constitutively expressed transamination and dehydrogenation reactions. Similar results were obtained with L. helveticus strains, except that the dehydrogenase enzymes were induced during incubation under cheese-ripening conditions. Micellar electrokinetic capillary chromatography of supernatants from L. casei and L. helveticus strains incubated under simulated cheese-ripening conditions confirmed that Tyr and Phe transamination and dehydrogenation pathways were active in both species and also showed these reactions were reversible. Major products of Tyr catabolism were phydroxy phenyl lactic acid and p-hydroxy phenyl acetic acid, while Phe degradation gave rise to phenyl lactic acid, phenyl acetic acid, and benzoic acid. However, some of these products were likely formed by nonenzymatic processes, since spontaneous chemical degradation of the Tyr intermediate p-hydroxy phenyl pyruvic acid produced p-hydroxy phenyl acetic acid, p-hydroxy phenyl propionic acid, and p-hydroxy benzaldehyde, while chemical degradation of the Phe intermediate phenyl pyruvic acid gave rise to phenyl acetic acid, benzoic acid, phenethanol, phenyl propionic acid, and benzaldehyde.     

Effect of adding an anaerobic fungal culture isolated from a wild blue bull (Boselophus tragocamelus) to rumen fluid from buffaloes on in vitro fibrolytic enzyme activity,fermentation and degradation of tannins and tannin‐containing Kachnar tree (Bauhinia variegata) leaves and wheat straw

The study investigated the effects of adding an anaerobic fungus (Piromyces sp FNG5; isolated from the faeces of a wild blue bull) to the rumen fluid of buffaloes consuming a basal diet of wheat straw and concentrates on in vitro enzyme activities, fermentation and degradation of tannins and tannin‐rich tree leaves and wheat straw. In experiment 1, strained rumen fluid was incubated for 24 and 48 h, in quadruplicate, with or without fungal culture using condensed tannin‐rich Bauhinia variegata leaves as substrates. In experiment 2, in vitro incubation medium containing wheat straw and different concentrations of added tannic acid (0–1.2 mg mL^?1) were incubated for 48 h, in quadruplicate, with strained buffalo rumen fluid with or without fungal culture. In experiment 3, tolerance of the fungal isolate to tannic acid was tested by estimating fungal growth in pure culture medium containing different concentrations (0–50 g L^?1) of tannic acid. In in vitro studies with Bauhinia variegata tree leaves, addition of the fungal isolate to buffalo strained rumen liquor resulted in significant (P < 0.01) increase in neutral detergent fibre (NDF) digestibility and activities of carboxymethyl cellulase (P < 0.05) and xylanase (P < 0.05) at 24 h fermentation. There was 12.35% increase (P < 0.01) in condensed tannin (CT) degradation on addition of the fungal isolate at 48 h fermentation. In in vitro studies with wheat straw, addition of the fungus caused an increase in apparent digestibility (P < 0.01), true digestibility (P < 0.05), NDF digestibility (P < 0.05), activities of carboxymethyl cellulase (P < 0.001), β‐glucosidase (P < 0.001), xylanase (P < 0.001), acetyl esterase (P < 0.001) and degradation of tannic acid (P < 0.05). Rumen liquor from buffaloes which had never been exposed to tannin‐containing diet had been found to have substantial inherent tannic acid‐degrading ability (degraded 55.3% of added tannic acid within 24 h of fermentation). The fungus could tolerate tannic acid concentration up to 20 g L^?1 in growth medium. The results of this study suggest that introduction of an anaerobic fungal isolate with superior lignocellulolytic activity isolated from the faeces of a wild herbivore may improve fibre digestion from tannin‐containing feeds and degradation of tannins in the rumen of buffaloes. Copyright © 2005 Society of Chemical Industry     

Effect of Tannic Acid on a Low Tannin African Sorghum Variety in Relation to Carbohydrate and Amylase

The effect of concentrations of 0-3% tannic acid on root growth, starch, soluble carbohydrate and alpha and beta amylase was determined during germination of a low tannin sorghum variety. At higher concentrations of tannic acid root growth was suppressed. Starch degradation and accumulation of soluble carbohydrates were reduced and both alpha and beta amylase synthesis was decreased. This suggests that tannins retard starch degradation indirectly by inhibiting the synthesis of starch hydrolytic enzymes during germination.     

单宁酶固定化及其降低蓝靛果汁口腔染色作用分析

为研究固定化单宁酶对催化蓝靛果单宁水解的影响,本实验对单宁微球固定单宁酶的最佳工艺条件、单宁酶固定化前后结构表征及在蓝靛果果汁水解的应用进行研究。结果表明,经响应面试验得到单宁酶固定化最佳工艺条件为酶质量浓度0.03?mg/mL、戊二醛质量分数0.10%、pH?5.4、固定化时间40?min,此条件下的酶活力回收率为71.78%,比活力为724.72?U/mg;扫描电镜图和红外图谱显示单宁酶很好地固定在单宁微球上。果汁中添加固定化单宁酶水解后,果汁中鞣花酸含量明显提高了29.12%。固定化酶添加量为4mg时,ΔE*ab为3.49;着色能力明显减弱。显微镜观察表明经过固定化酶处理后的果汁能明显降低对舌头黏膜的着色能力。     

具有α-葡萄糖苷酶抑制作用益生菌的筛选及特性分析

以实验室77 株益生菌为研究对象,从其菌体细胞代谢物（cell-free excretory supernatants,CFS）和细胞内容物（cell-free extracts,CFE）两方面分析菌株对α-葡萄糖苷酶的抑制活性;同时还从耐酸性、细胞黏附性等方面对具有α-葡萄糖苷酶抑制活性的菌株进行了益生特性评价;最后利用主成分分析进行综合性评价,以期筛选出具有α-葡萄糖苷酶抑制作用的益生菌。结果表明,77?株益生菌的CFE对α-葡萄糖苷酶没有抑制作用;而CFS对α-葡萄糖苷酶具有一定的抑制作用,抑制率为2.53%～15.76%。选取抑制率明显高于其他益生菌（编号为ST-2、1.1881、GS-3和BLP12）菌株进行益生特性的研究。其中ST-2表现出很高的耐酸性和细胞黏附性;GS-3在模拟消化液中有很强的耐受性等,各菌株特性不一。主成分分析表明菌株BLP12的综合性能最好：其对α-葡萄糖苷酶的抑制率可达15.10%;于pH?2.0孵育3?h后,存活率能达到71.04%;于2.0%的胆盐条件下孵育24?h,存活率为0.70%;依次经模拟唾液、胃液、肠液消化后,存活率仍能达到88.27%,但对HT-29细胞的黏附率较低,仅为1.93%,总体上菌株BLP12对体外模拟胃肠环境的适应性很强。该菌株经过16S基因序列鉴定为植物乳杆菌,可作为降糖益生菌株应用于降糖食品的开发。     

乳酸菌发酵大豆糖蜜生产乳酸及糖代谢变化

选用德氏乳杆菌保加利亚亚种KLDS1.8501、嗜酸乳杆菌KLDS1.0327、嗜酸乳杆菌ATCC11975、植物乳杆菌植物亚种CICC23168、干酪乳杆菌ATCC393、植物乳杆菌NAU322分别接种于大豆糖蜜,用高效液相色谱法测定乳酸的产生以及碳水化合物的利用情况,分析不同乳酸菌发酵大豆糖蜜生产乳酸能力及糖代谢能力。结果表明,在15 °Brix大豆糖蜜中,37?℃、pH?6.0条件下发酵24?h,植物乳杆菌植物亚种CICC23168的活细胞数达到6.66×109?CFU/mL,乳酸产生量为12.18?g/L,总糖消耗量为22.48?g/L,与其他菌株相比有明显优势。因此,植物乳杆菌植物亚种CICC23168是能利用大豆糖蜜发酵产乳酸的潜力菌株。     

Optimization of Tannase Production and Improvement of Nutritional Quality of Two Potential Low-Priced Plant Feedstuffs under Solid State Fermentation by Pichia kudriavzevii Isolated from Fish Gut

An autochthonous tannase yielding yeast strain Pichia kudriavzevii (GU939629), isolated from the gut of an Indian major carp (mrigal), Cirrhinus cirrhosus, has been used for extracellular tannase production and subsequent processing of two plant feedstuffs, Groundnut oil cake (GOC) and Pistia leaves (PL), under solid state fermentation (SSF). Of the two plant materials studied, GOC supported maximum tannase activity (0.82 ± 0.024 U/gds) degrading 94.1% of the initial tannin content, whereas SSF resulted in 0.68 ± 0.02 U/gds tannase activity and 89.1% tannin degradation through the use of PL as substrate. Following SSF for 15 d with optimized culture conditions, analysis of proximate composition revealed that there was significant increase (t-value significant at P < 0.05) in the contents of crude protein, lipid, minerals (Na, K, Ca, Mg, Zn, Fe, Cu, Mn, P), free amino acids and fatty acids; along with reduction in the contents of the other antinutritional factors, for example, crude fiber, phytic acid, and trypsin inhibitor. The results indicate that there is ample scope for further research to appraise potential application of gut microbiota for tannase production, as well as processing of low-cost plant feedstuffs for prospective use as feed ingredients for improved fish protein production.     

Activity of two histidine decarboxylases from Photobacterium phosphoreum at different temperatures, pHs, and NaCl concentrations

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. The authors reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases: constitutive and inducible enzymes. This article reports the effect of various growth and reaction conditions, such as temperature, pH, and NaCl concentration, on the activity of two histidine decarboxylases that were isolated and separated by gel chromatography from cell-free extracts of P. phosphoreum. The histidine decarboxylase activity of the cell-free extracts was highest in 7 degrees C culture; in 5% NaCl, culture growth was inhibited, and growth was best in the culture grown at pH 6.0. Moreover, percent activity of the constitutive and inducible enzymes was highest for the inducible enzyme in cultures grown at 7 degrees C and pH 7.5 and in 5% NaCl. The temperature and pH dependences of histidine decarboxylase differed between the constitutive and inducible enzymes; that is, the activity of histidine decarboxylases was optimum at 30 degrees C and pH 6.5 for the inducible enzyme and 40 degrees C and pH 6.0 for the constitutive enzyme. The differences in the temperature and pH dependences between the two enzymes extended the activity range of histidine decarboxylase under reaction conditions. On the other hand, histidine decarboxylase activity was optimum in 0% NaCl for the two enzymes. Additionally, the effects of reaction temperature, pH, and NaCl concentration on the constitutive enzyme activity of the cell-free extracts were almost the same as those on the whole histidine decarboxylase activity of the cell-free extracts, suggesting that the constitutive enzyme activity reflected the whole histidine decarboxylase activity.     

Expression of the catalase gene katA in starter culture Lactobacillus plantarum TISTR850 tolerates oxidative stress and reduces lipid oxidation in fermented meat product

The catalase gene katA of Lactobacillus sakei SR911 was cloned and expressed in Escherichia coli UM2 and Lactobacillus plantarum TISTR850 under strong lactococcal promoter P59 in E. coli-lactococcus expression vector pIL1020. The L. plantarum TISTR850 is a catalase-deficient strain isolated from local fermented meat product. The recombinant L. plantarum TISTR850 was shown to decompose hydrogen peroxide, and catalase activity approximately three times higher that of natural catalase-producing strain L. sakei SR911. The recombinant protein was also detected by in situ activity staining of the catalase enzyme. The recombinant L. plantarum TISTR850 did not accumulate hydrogen peroxide under glucose-limited aerobic conditions and remained viable after 60 h of incubation. The recombinant and host strain L. plantarum TISTR850 were used as starter cultures in the fermented meat product, and lipid oxidation was monitored over a 7-day storage at 20 degrees C determined as thiobarbituric acid-reactive substances (TBARS) value. The lipid oxidation level in the fermented meat product seeded with the catalase genetically modified starter culture L. plantarum TISTR850 was significantly lower than that of the natural catalase-deficient strain.     

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.