Production of -aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods

Lactobacillus strains that accumulated γ-aminobutyric acid (GABA) in culture medium were screened to determine strains with high GABA-producing ability. One strain, NFRI 7415, which was isolated from a Japanese traditional fermented fish (funa-sushi), showed the highest GABA-producing ability among the screened strains. Identification tests (i.e., 16S rDNA sequencing and sugar assimilation ability) indicated that NFRI 7415 belongs to Lb. paracasei. The GABA production was further improved by the addition of pyridoxal phosphate to the culture medium and pH regulation of culture medium at pH 5.0. Under optimal cultivation conditions, strain NFRI 7415 produced GABA at a concentration of 302 mm when the glutamate concentration in the culture medium was 500 mm.     

乳酸菌联合酿酒酵母发酵对龙须菜多糖结构特征及抗氧化性的影响

为明确微生物发酵对龙须菜浆液多糖体外抗氧化活性的改善效果,以期为龙须菜的高值化利用提供理论参考。采用乳酸菌副干酪乳杆菌亚种RP38与酿酒酵母JJ4复合发酵龙须菜浆液,利用色谱及光谱分析技术解析了龙须菜多糖的结构变化,考察了发酵前后浆液多糖的自由基清除能力。研究结果表明,龙须菜经发酵后浆液多糖质量分数由0.30%提高至0.79%,其中硫酸酯多糖质量分数由0.03%提高至0.12%。多糖的凝胶渗透色谱及红外光谱研究结果表明,微生物发酵不改变龙须菜多糖的官能基团,但可降解龙须菜多糖分子质量,进而使其结构暴露出更多的硫酸酯基团。核磁共振扫描发现,硫酸酯多糖的硫酸酯主要分布在半乳糖的C-6位,微生物发酵并不改变硫酸酯在糖链上的分布位置,但可以提高硫酸酯的含量。进一步研究发现,发酵后的龙须菜浆液多糖的羟自由基清除能力提高了5.33%,超氧阴离子自由基清除能力提高了32.12%。龙须菜浆液发酵后多糖抗氧化活性提高可能与生成更强给氢能力的小分子多糖以及硫酸酯基团暴露量增多有关。     

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion, which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and R1 stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS R1 also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10–24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS R1 and with the increases in Bacteroides in cultures with both microbial EPS (R1 and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids.     

Fermentation of rice using amylolytic Bifidobacterium

Twenty-two amylolytic bifidobacteria grown in BHI-starch media were compared for the amylase activity of the intra- and extra-cellular enzymes. The activity of the cells grown in the liquid medium differed considerably. Among the strains tested B. adolescentis Int57 and B. adolescentis ZS8 exhibited higher activities than others. In rice medium containing 0.05% -cysteine·HCl and 0.2% yeast extract, the amylolytic Bifidobacterium strains grew considerably better than non-amylolytic Bifidobacterium strains. B. adolescentis Int57, which showed highest growth and amylase activity in the rice medium, was chosen and rice fermentation was investigated. The titratable acidity (TA) reached 2.43 and pH decreased to 4.4 after 24 h fermentation. The relative ratio of acetic acid to lactic acid gradually decreased during fermentation. The concentration of reducing sugar and amylase activity gradually increased and reached 14 mg maltose equivalent/ml and 35 mU/ml min, respectively, in 24 h. The accumulated reducing sugar was mostly maltotriose. The layer-separation of fermented product was stabilized by the addition of 1% gelatin. It was suggested that amylolytic bifidobacteria may be used for the production of fermented rice products.     

Effects of curing sodium nitrite additive and natural meat fat on growth control of Listeria monocytogenes by the bacteriocin-producing Lactobacillus curvatus strain CWBI-B28

In realistic model meat systems, the separate and combined effects of fat content and sodium nitrite on the antilisterial activity of the bacteriocin of Lactobacillus curvatus CWBI-B28 were studied. In laboratory fermentations where Listeria monocytogenes was co-cultured at 4 °C with bacteriocin-producing CWBI-B28 in lean pork meat (fat content: 13%) without added nitrite, a strong antilisterial effect was observed after one week. The effect was maintained for an additional week, after which a slight and very gradual rebound was observed. Both added nitrite (20 ppm) and a high-fat content (43%) were found to antagonise this antilisterial effect, the Listeria cfu count reached after six weeks being 200 times as high in high-fat meat with added nitrite than in lean meat without nitrite. This antagonism could not be attributed to slower growth of the bacteriocin-producing strain, since CWBI-B28 grew optimally in fat-rich meat with 20 ppm sodium nitrite. Bacteriocin activity was also measured in the samples. The observed activity levels are discussed in relation to the degree of antilisterial protection conferred.     

Phytate degradation by human gut isolated Bifidobacterium pseudocatenulatum ATCC27919 and its probiotic potential

The growing awareness of the relationship between diet and health has led to an increasing demand for food products that support health above and beyond providing basic nutrition. Probiotics are live organisms present in foods, which yield health benefits related to their interactions with the gastrointestinal tract. Phytases are a subgroup of phosphatases that catalyse the desphosphorylation of phytate, which reduces its negative impact on mineral bioavailability, and generates lower inositol phosphates. The aims of this investigation were to (i) study the ability of the probiotic candidate Bifidobacterium pseudocatenulatum to degrade phytate in synthetic medium, to (ii) identify the lower inositol phosphates generated, to (iii) study its survival under conditions mimicking gastrointestinal passage and finally to (iv) assess adhesion of the bacteria to Caco-2 cells. The first steps of InsP₆ degradation by B. pseudocatenulatum phytate-degrading enzyme/s were preferentially initiated at the DL-6-position and 5-position of the myo-inositol ring. It suggests that the main InsP₆ degradation pathway by B. pseudocatenulatum by sequential removal of phosphate groups was D/L-Ins(1,2,3,4,5)P₅ or D/L-Ins(1,2,3,4,6)P₅; D/L-Ins(1,2,3,4)P₄; to finally Ins(1,2,3)P₃ and D/L-Ins(1,2,4)P₃/D/L-Ins(1,3,4)P₃. This human strain also showed a notable tolerance to bile as well as a selective adhesion capacity (adhesion to control surfaces was zero), to human intestinal Caco-2 cells comparable to the commercial probiotic B. lactis. The phytate-degrading activity constitutes a novel metabolic trait which could contribute to the improvement of mineral absorption in the intestine as a nutritional probiotic feature with potential trophic effect in human gut.     

番茄发酵液中乳酸杆菌的分离鉴定及对模拟胃肠液耐受性研究

为获得具有较好生长能力及胃肠耐受性的果蔬发酵剂,作者以自然发酵60 d的番茄液为对象,利用形态学及16S rDNA基因序列分析等方法,从中分离鉴定出3株乳酸杆菌（分别命名为L1、L2、L3）,并研究其生长特性及对模拟胃肠环境的耐受能力。其中,L1和L3属于植物乳杆菌属（Lactiplantibacillus plantarum）;L2为乳杆菌属（Lactobacillus brevis）,3株菌的最适生长pH值均为6.6,最适生长温度为30~38 ℃。耐受性研究发现,L1和L3具有更强的高温及高盐耐受性,L2具有更好的胆盐耐受性;3株乳酸杆菌尤其是L3对人工胃液环境具有良好的耐受能力,存活率均高于96%。在人工肠液环境下3株菌的存活率超过90%,尤其L1表现出更好的稳定性。结果表明,作者分离得到的3株乳杆菌均具有良好的生长特性和胃、肠耐受特性,本研究结果可为果蔬发酵剂的研制及其发酵制品的开发提供参考。     

Compositions, antioxidant and antimicrobial activities of Helichrysum (Asteraceae) species collected from Turkey

The methanolic extracts of 16 Helichrysum species were investigated for their in vitro antioxidant, radical scavenging and antimicrobial activities. All the extracts showed strong antioxidant and radical scavenging activity. The highest total antioxidant capacity as ascorbic acid equivalents (AAE) of 194.64 mg/g dry extract was obtained for Helichrysum noeanum in the phosphomolybdenum assay. The highest IC₅₀ value (7.95 μg/ml) was observed for the extract of Helichrysum stoechas subsp. barellieri in the DPPH assay. The total phenolic contents of the extracts ranged from 66.74 to 160.63 mg gallic acid equivalents (GAE)/g dry extract. The major component present in the extracts was identified as chlorogenic acid followed by apigenin-7-glucoside and apigenin by HPLC analysis. All the extracts showed significant antimicrobial activity against microorganisms containing 13 bacteria and two yeasts in the agar diffusion method.     

Hydrolysis of β-glucosyl ester linkage of p-hydroxybenzoyl β- -glucose, a chemically synthesized glucoside, by β-glucosidases

To investigate the hydrolysis of glucosyl esters by β-glucosidase, p-hydroxybenzoyl β- -glucose (pHBG) was chemically synthesized. The hydrolytic activity of some β-glucosidases for pHBG was compared to that for p-nitrophenyl β-glucoside (pNPG). The Clavibacter michiganense and Flavobacterium johnsonae enzymes could hydrolyze pHBG and steviol glycosides which are natural glucosyl esters. The commercial β-glucosidase originating from Caldocellum saccharolyticum also hydrolyzes pHBG despite having no activity for steviol glycosides. The β-glucosidase from Aspergillus niger cleaved the glucosyl ester linkage much more weakly than the glucosidic linkage. The pH-activity profile for the hydrolysis of pHBG was similar to that of pNPG by the C. saccharolyticum β-glucosidase. The similar profiles for these substrates suggested that the active site for the glucosyl ester chemically resembles that for glucoside with respect to catalysis. Kinetic analysis of the C. saccharolyticum β-glucosidase for mixed substrates of pHBG and pNPG showed that the hydrolysis of pHBG competed with that of pNPG. This result indicated that there is only one active site for both the glucosyl ester and glucoside. Mass spectroscopic analysis of the hydrolysates of pHBG in H₂¹⁸O suggested that β-glucosidase hydrolyzes glucosyl esters between the anomeric carbon of glucose and the carbonyl oxygen, not between the carbonyl carbon and the carbonyl oxygen.     

Optimization of bioactive compounds extraction from jabuticaba (Myrciaria cauliflora) skins assisted by high pressure CO2

Important bioactive compounds from Brazilian jabuticaba skins were effectively extracted by High Pressure Carbon Dioxide Assisted Extraction (HPCDAE). Statistical design was used to optimize the extraction variables: extraction pressure (65–135 bar), temperature (40–80 °C) and volume ratio of solid–liquid mixture/pressurized CO₂ (RS‐L/CO₂(%) (20–80%). The analysis performed to predict the optimum values for the extraction variables, in order to obtain the condition that result in an extract with high anthocyanin (2.2 ± 0.3 mg cyanidin-3-glucoside/g dry skins) and phenolic compound contents (13 ± 1 mg gallic acid equivalents/g dry skins), gave as best conditions, 117 bar extraction pressure, 80 °C extraction temperature and 20% volume ratio of solid–liquid mixture/pressurized CO₂ (RS‐L/CO₂(%)). Compared to Pressurized Liquid Extraction (PLE) and to control experiment the experimental results obtained using optimum HPCD Assisted-Extraction conditions were much more effective and faster in extracting total anthocyanins and phenolic compounds.Industrial relevance: Industrially, there is an increasing demand for faster extraction procedures with reduced organic solvent consumption to lower pollution burden. HPCD Assisted-Extraction combines the advantages of enhanced mass transfer rates increasing secondary metabolite diffusion from the vegetable matrix into the environmentally friendly solvent extraction. High pressure extraction methods, such as HPCD Assisted-Extraction, have other advantages that should be considered, such as the fact that native enzymes, which degrade phenolic compounds, are inhibited by extraction pressure increasing and CO₂ addition, and that processed vegetable materials do not require additional sterilization steps.     

Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.     

Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

Fumigant properties of physical preparations from mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) beetle for stored grain insects

Vapors released from foliage of mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) Beetle, through a patented process, were hypothesized to have an insecticidal time of action (24 h or less after time of exposure) similar to the fumigant methyl bromide. Patented preparations were more effective from plants harvested from a relatively wet site in mid to late summer (5 July to 11 September). Bioassays with the lesser grain borer, Rhyzopertha dominica (F.), 0–3 days after adult emergence indicated an LT₅₀ of 7.0±1.2 h for the volatiles generated from only 30 mg dry processed plant material (=0.56 mg active ingredients) per ml headspace. Hatching of eggs of the Indian meal moth, Plodia interpunctella (Hübner), was completely suppressed when exposed 4–20 h after oviposition to a concentration of 7 mg processed plant material per ml headspace (=0.14 mg active ingredients) in a container that allowed passive diffusion and from which the terpenes disappeared by 48 h. Adult red flour beetle, Tribolium castaneum (Herbst), had an LT₅₀ of 40.7±1.2 h when exposed to 29 mg processed plant material per ml headspace. Gas chromatography/mass spectrometry (GC/MS) analyses of the headspace above this processed plant material revealed five major peaks, all non-chlorinated and non-brominated. The two main volatiles, 1,8-cineole and camphor, occurred initially in a mean ratio of 1:3.2, gradually shifting to 1:2.4 over 24 h. The μg/ml headspace of each detectable compound in a sealed container was followed intensely (0.25, 1, 2, 12, 24, 48, and 72 h) for 72 h and at less frequent intervals for 60 days. The active compounds released by the plant material in a closed, but not airtight container, were no longer detectable after 24 h based on GC/MS analysis. Fumigative studies with the same ratio of the two main compounds generated synthetically indicated that embryos of P. interpunctella and adults of R. dominica were as sensitive to the synthetic mixture as they were to the processed plant material. Although one could apply the precise commercial terpenes in the same ratio, the plant material provides a natural formulation that is conveniently diluted (formulated) to levels safe for handling. Therefore, this preparation method and plant material shows good potential as an alternative to methyl bromide for protection of stored grain, commodity, and space fumigations. No residues are detectable in the headspace of aerated commodity, milled product, or in fumigated space.     

双歧杆菌生理生化特性及调控大鼠肠道菌群、脂质代谢功能的研究进展

肠道微生态是人和动物体内最为复杂、最为主要的微生态系统。双歧杆菌是肠道微生态存在的一种益生菌,在防治肥胖、代谢综合征等多个疾病领域呈现出潜在应用前景,成为食品补充剂、保健食品等多个领域研究的热点。双歧杆菌能够通过调节饮食结构与能量摄入,肠道菌群微环境、微生物-肠-脑轴及肠道免疫等途径调控大鼠肠道菌群,保护肠道黏膜屏障,并能通过对碳水化合物利用途径、抵抗炎症应激途径、调节脂肪分解途径、基因途径、胆汁酸盐共沉淀与菌体同化胆固醇途径等调控脂质代谢,同时肠道菌群与脂质代谢之间具有双向调节作用。但双歧杆菌对肠道菌群、脂质代谢的效果,因应用领域、配方、添加数量而异,仍需要更深度控制研究和临床验证来确立配方、添加数量等。且应用于食品领域时,可能通过与宿主自身肠道菌群交流遗传物质途径,导致肠道耐药基因转移或耐药病原菌传播。本文就双歧杆菌调控大鼠肠道菌群及脂质代谢多种途径和机制进行综述,并对相关问题进行展望,以期为双歧杆菌相关领域研究提供思路。     

Effects of different probiotic strains of Lactobacillus and Bifidobacterium on bacterial translocation and liver injury in an acute liver injury model

Septic complications represent frequent causes of morbidity in liver diseases and following hepatic operations. Most infections are caused by the individual own intestinal microflora. The intestinal microflora composition is important in physiological and pathophysiological processes in the human gastrointestinal tract, but their influence on liver in different situations is unclear. We therefore studied the effect of different Lactobacillus strains and a Bifidobacterium strain on the extent of liver injury, bacterial translocation and intestinal microflora in an acute liver injury model.Sprague–Dawley rats were divided into five groups: acute liver injury control, acute liver injury+B. animalis NM2, acute liver injury+L. acidophilus NM1, acute liver injury+L. rhamnosus ATCC 53103, and acute liver injury+L. rhamnosus DSM 6594 and L. plantarum DSM 9843. The bacteria were administered rectally daily for 8 days. Liver injury was induced on the 8th day by intraperitoneal injection of -galactosamine (1.1 g/kg BW). Samples were collected 24 h after the liver injury. Liver enzymes and bilirubin serum levels, bacterial translocation (to arterial and portal blood, liver and mesenteric lymph nodes (MLNs)), and intestinal microflora were evaluated.L. acidophilus NM1; L. rhamnosus ATCC 53103, and L. rhamnosus DSM 6594+L. plantarum DSM 9843 decreased bacterial translocation compared to the liver injury control group. B. animalis NM2 increased bacterial translocation to the mesenteric lymph nodes. The levels of alanine aminotransferase (ALAT) were significantly lower in the L. acidophilus, L. rhamnosus ATCC 53103, L. rhamnosus DSM 6594+L. plantarum DSM 9843 groups compared to the liver injury group. The L. rhamnosus and L. rhamnosus+L. plantarum groups significantly reduced ALAT levels compared to the B. animalis group. All administered bacteria decreased the Enterobacteriaceae count in the cecum and colon.Administration of different lactobacilli and a Bifidobacterium strain in an acute liver injury rat model, has shown different effects on bacterial translocation and hepatocellular damage. L. acidophilus, L. rhamnosus, and L. rhamnosus+L. plantarum reduced bacterial translocation and hepatocellular damage. B. animalis NM2 increased bacterial translocation to the mesenteric lymph nodes and did not affect hepatocellular damage.     

Heat-induced aggregation of β-lactoglobulin A and B with α-lactalbumin

β-Lactoglobulin A and β-lactoglobulin B were heated at 75°C in the absence and presence of α-lactalbumin, and the aggregation products were characterized by size exclusion chromatography in combination with multi-angle laser light scattering and electrophoretic techniques. α-Lactalbumin did not form aggregates when heated alone, but in admixture with β-lactoglobulin it was incorporated into both the disulphide-bonded and the hydrophobically associated aggregates as well as forming α-lactalbumin dimers and other oligomers. The presence of α-lactalbumin diminished the proportion of smaller aggregates and increased the number of very large aggregates within both variant protein mixtures. In the presence of α-lactalbumin, β-lactoglobulin A was converted into a series of disulphide-bonded and the hydrophobically associated aggregates more slowly, but with a greater proportion of hydrophobically associated aggregates, than β-lactoglobulin B. These patterns are similar to that when β-lactoglobulin A or B are heated on their own. These and other results indicate that the mechanism of aggregation of α-lactalbumin/β-lactoglobulin mixtures is governed by β-lactoglobulin.