鱼露中组胺降解菌的分离筛选及耐受性

生物胺(biogenic amines)在某些食品尤其是发酵食品中广泛存在,具有一定的食用安全隐患.为获得用于鱼露等发酵食品的生物胺降解菌,从天然发酵鱼露中采用双层显色培养基法初步筛选出不产生物胺的菌株,再用高效液相色谱(HPLC)法进行复筛,得到一株具有高效组胺降解能力的菌株MZ5.经鉴定该菌株为库德毕赤酵母(Pic...     

四川泡菜中产γ-氨基丁酸微生物的系统发育与表达能力评估

为拓展产γ-氨基丁酸(γ-aminobutyric,GABA)微生物资源,以四川泡菜为分离源,从中分离产GABA的乳酸菌和酵母,并对其GABA表达能力进行评估。通过分离纯化,从四川泡菜中获得了338株乳酸菌和67株酵母。采用纸色谱测定,筛选获得12株乳酸菌和3株酵母菌具有产GABA的能力。生理生化和系统发育研究揭示12株乳酸菌分别被鉴定为Lactobacillus acidophilus(占比8.3%)、Lactobacillus fermentum(8.3%)、Lactobacillus kimchii(8.3%)、Lactobacillus suebicus(8.3%)、Lactobacillus brevis(16.7%)、Lactobacillus parafarraginis(8.3%)、Lactobacillus similis(8.3%)、Lactobacillus plantarum(25.2%)和Lactobacillus pentosus(8.3%);3株酵母中,2株被鉴定为Saccharomyces cerevisiae;1株被鉴定为Candida tanzawaensis。进一步采用高效液相色谱对菌株GABA的表达能力评估发现,乳酸菌Lactobacillus plantarum BC114和Lactobacillus brevis BC237发酵产GABA的能力较强,分别达1 720 mg/L和1 080 mg/L;酵母菌Saccharomyces cerevisiae JM037发酵产GABA的能力较强,达670 mg/L。     

Antioxidant activity and γ-aminobutyric acid (GABA) content in sea tangle fermented by Lactobacillus brevis BJ20 isolated from traditional fermented foods

GABA-producing lactic acid bacteria were isolated from kimchi and salt-fermented Jot-gal, which are traditional Korean fermented foods. The strain, BJ-20, isolated from salt-fermented Jot-gal (cod gut), possessed the highest GABA-producing ability in MRS broth with 1% monosodium glutamate (MSG), as determined by thin layer chromatography. The BJ-20 strain was identified as Lactobacillus brevis and designated as L. brevis BJ20. A sea tangle solution was fermented over 5 days to produce GABA using L. brevis BJ20. During fermentation, the GABA concentration dramatically increased, while the glutamic acid concentration decreased. This result indicates that the glutamic acid was converted to GABA by L. brevis BJ20 in the fermented sea tangle solution. Furthermore, the fermented solution exhibited strong antioxidant activities, such as DPPH scavenging, superoxide scavenging, and xanthine oxidase inhibition, which were higher than those of BHA as a positive control.     

ACE-inhibitory activity and gamma-aminobutyric acid content of fermented skim milk by <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> isolated from Xinjiang koumiss in China

Eighty-one strains of Lactobacillus were isolated from the koumiss collected in Xinjiang, China. The strains were cultivated in skim milk medium, ACE inhibitory activity and GABA concentrations in the culture supernatants were measured. Screening results revealed that ACE inhibitory activity of 16 strains was higher than 50% and two strains produced GABA. The Lactobacillus—ND01 strain produces both the high ACE inhibitory activity and GABA. The sequence of 16S rDNA of the Lactobacillus—ND01 showed 99% homology to L. helveticus. The first identification of the newly isolated Lactobacillus—ND01 strain which produces both high ACE inhibitory activity and GABA revealed differences from reported species in our study. The L. helveticus ND01 was resistant to acidic condition. The results suggest that L. helveticus ND01 showed good potential for application in the management of hypertension.     

Bacterial resistance after pulsed electric fields depending on the treatment medium pH

The objective was to evaluate and compare the pulsed electric field (PEF) resistance of four Gram-positive (Bacillus subtilis, Listeria monocytogenes, Lactobacillus plantarum, Staphylococcus aureus) and four Gram-negative (Escherichia coli, E. coli O157:H7, Salmonella serotype Senftenberg 775W, Yersinia enterocolitica) bacterial strains under the same treatment conditions. Microbial characteristics such as cell size, shape or type of the cell envelopes did not exert the expected influence on microbial PEF resistance. The most PEF resistant bacteria depended on the treatment medium pH. For instance, L. monocytogenes, which showed the highest PEF resistance at pH 7.0, was one of the most sensitive at pH 4.0. The most PEF resistant strains at pH 4.0 were the Gram-negatives E. coli O157:H7 and S. Senftenberg. A subsequent holding of PEF-treated cells in pH 4.0 for 2 h increased the degree of inactivation up to 4 extra Log₁₀ cycles depending on the bacterial strain investigated. Under these treatment conditions, the most PEF resistant bacterial strains were still the pathogens S. Senftenberg and E. coli O157:H7.

Industrial relevance

The design of appropriate food preservation processes by PEF requires the selection of an adequate target bacterial strain, which should correspond to the most PEF resistant microorganism contaminating food. This study indicates that the pH of the treatment medium plays an important role in determining this target bacterial strain. On the other hand, the combination of PEF and subsequent holding under acidic conditions has been proven to be an effective method in order to achieve a higher level of microbial inactivation.     

Production and properties of crude enterotoxin of Pseudomonas aeruginosa

Pseudomonas aeruginosa CTM-3 was found to be the most potentially enteroxigenic strain out of the 12 isolates recovered from milk, as a high fluid length ratio, i.e. F/L (1.1) in rabbit gut and a strong permeability response in rabbit skin (38.5 mm² necrotic zone) was obtained with this culture. No clear-cut relationship between the two tests was observed. Six of the ethidium bromide (300 μg/ml) cured variants of this culture completely lost their ability to produce enterotoxin indicating the possible involvement of a plasmid in enterotoxin synthesis. The crude enterotoxin from P. aeruginosa CTM-3 was completely inactivated in 15 s at 72°C. However, it was fairly stable at pH values in the range 4.5–7.5. Both pepsin and trypsin inactivated the enterotoxin activity at a concentration of 40 μg/ml. Organic acids, formalin and hydrogen peroxide had no significant effect on the enterotoxin activity. The need for further investigations with purified preparations is emphasized.     

Studies on screening of higher γ-aminobutyric acid-producing <Emphasis Type="Italic">Monascus</Emphasis> and optimization of fermentative parameters

γ-Aminobutyric acid (GABA), a hypotensive agent, can be produced by Monascus spp. Two hundred and fifteen GABA-producing Monascus strains were isolated from fermented bean curd and red-mold rice. The strain M6 with the highest production level of GABA (3.657 g/L) was isolated from fermented bean curd by using PDB with 0.5% monosodium glutamate (MSG) and identified as Monascus ruber based on morphological and ITS region sequence comparisons. The strain M6 was irradiated by UV to raise GABA production. The mutant strain of M6-13 had the highest GABA yield of 5.527 g/L, which was 1.5 times higher than that of the original strain M6. In addition, the strain M6-13 still had stable yield of GABA and no reverse mutation after sub-cultured for 15 generations. GABA production level of the mutant strain M6-13 increased to 7.826 g/L in submerged flasks culture by use of the orthogonal experiment method. Based on this, process analysis and optimization of GABA production of the strain M6-13 were studied in 3.7-L vessel fermentor and the maximum GABA yield reached 13.470 g/L.     

Biotransformation of monosodium glutamate to gamma-aminobutyric acid by isolated strain Lactobacillus brevis L-32 for potentiation of pentobarbital-induced sleep in mice

In the current study, we investigated the biotransformation of monosodium glutamate (MSG) to gamma-aminobutyric acid (GABA) by the growing and resting cells from an isolated bacterial strain, Lactobacillus brevis. This strain is a high GABA-producing strain that was identified and isolated from natural kimchi. We gathered the experiment results by design of response surface methodology (RSM) for optimum condition for GABA production and results indicated the optimum culture temperature (35°C) and culture time (58 h). Using resting cells from the same culture batch in the substrate-containing buffer, approximately 3.98 g/l of GABA was produced at a conversion rate of 65.6%. GABA-treated mice showed significantly increased sleep duration compared to that of a control group (p < 0.05) in the pentobarbital-induced sleep test using a hypnotic dose. These results suggest that biotransformed GABA could potentially be used a novel nutraceutical supplement for sleep.     

发酵鱼酱酸产GABA乳酸菌的分离筛选及发酵特性

从传统发酵鱼酱酸中筛选出产γ-氨基丁酸（γ-aminobutyric acid，GABA）的乳酸菌并分析其菌株发酵特性。通过薄层色谱法定性、Berthelot比色法定量获得产GABA菌株，并进行耐酸、耐胆盐、氨基酸脱羧酶活性、抑菌性、生长曲线及pH值、产酸速率等菌株发酵特性分析。结果表明：从分离自鱼酱酸不同发酵阶段的387 株乳酸菌中，获得15 株典型产GABA菌株，包括2 株食窦魏斯氏菌（Weissella cibaria）、1 株熊蜂魏斯氏菌（Weissella bombi）、11 株植物乳杆菌（Lactobacillus plantarum）和1 株戊糖乳杆菌（Lactobacillus pentosus）。15 株乳酸菌产GABA能力及发酵特性在主成分分析图上差异显著，其中食窦魏斯氏菌Y113产GABA量为0.239 mg/mL，高于其他菌株；植物乳杆菌Y279和Y64展现出较好的耐酸性、耐胆盐性、抑菌性、无氨基酸脱羧酶活性、生长速率及产酸速率快的特点。鉴于其优良的发酵特性、益生特性及产GABA能力，菌株Y279可作为鱼酱酸工业化、标准化生产的潜在优良菌株。     

Development of a novel method for feeding a mixture of -lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly- -3-hydroxybutyrate production

Fermentative production of poly- -3-hydroxybutyrate [P(3HB)] from a mixture of -lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l·h.     

A rapid and efficient method for directed screening of lipase-producing Burkholderia cepacia complex strains with organic solvent tolerance from rhizosphere

Lipase from Burkholderia cepacia strain is one of the most versatile biocatalysts and is used widely in many biotechnological application fields including detergent additives, the resolution of racemic compounds, etc. Based on the known whole genomic information of B. cepacia strain, both ampicillin and kanamycin were added to the TB-T medium to screen B. cepacia complex stains from rhizosphere soil samples. The selected colonies from the modified TB-T medium were then qualitatively determined the ability to produce extracellular lipase on the rhodamine B-olive oil agar plates. A total of 35 lipolytic pseudo-B. cepacia complex strains were isolated and the positive rate of lipolytic bacteria was 65%. Among them, 15 pseudo-B. cepacia complex strains showed tolerance to benzene, n-hexane and n-heptane at concentration of 10% (V/V) and were identified by the recA gene sequence. All of the 14 lipolytic bacteria were identified as B. cepacia complex strains except that the recA gene sequence of one lipolytic bacterium, strain ZMB009, was not obtained.     

Characterization of dextran produced from Leuconostoc citreum S5 strain isolated from Korean fermented vegetable

The production of dextran was optimized by a novel Leuconostoc citreum, which was isolated from Korean traditional fermented vegetable, Dongchimi. This strain was identified as Leuconostoc citreum S5 by a physiological analysis, API-kit analysis, 16-rDNA sequencing and RAPD-PCR. The type and production of dextrans were greatly affected by the sucrose concentration, food ingredients and fermentation time. In particular, the addition of skim milk and potato powder greatly increased the consistency of the culture broth after fermentation for 12 h at 30 °C, resulting in the maximum values after 72 h. The culture broth with the highest consistency, was obtained from the defined medium containing 20% sucrose, 1.5% skim milk and 0.5% potato powder, and it showed typical pseudoplastic behavior with a pH of 4.07, 1.12% titratable acidity, 472.5 Pa of the elasticity modulus (G′) and 85.0 Pa of viscosity modulus (G″). In addition, total dextran is composed of soluble dextran (33.2 g/L) and insoluble dextran (29.8 g/L). The conversion yield of sucrose was decreased by an increase, in the sucrose concentration in a defined medium, remaining sucrose and some of glucose and fructose. High-molecular weight dextran (2,000 kDa), as a minor fraction, was produced by increasing the sucrose. With the addition of food ingredients such as skim milk and potato powder, however, the conversion of sucrose is complete, showing only some fructose as a by-product. Low-molecular weight dextran (1,100 kDa) was only produced regardless of sucrose concentration.     

生物胺降解菌株的筛选与鉴定

从实验室保藏的不同来源乳酸菌菌株中筛选出具有生物胺降解能力的菌株,测定其在不同盐浓度、pH下的生长能力。结果表明,筛选出3株具有生物胺降解能力的菌株,经16SrDNA序列分析,菌株HM22鉴定为发酵乳杆菌(Lactobacillus fermentum),菌株HM24为植物乳杆菌(Lactobacillus plantarum),菌株YF10042为粪肠球菌(Enterococcus faecalis);其中植物乳杆菌HM24的降解能力最优,对色胺、苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺、精胺的降解率分别为(25.90±2.30)%,(35.75±2.71)%,(27.61±3.94)%,(25.91±3.76)%,(22.54±2.64)%,(34.55±1.90)%,(39.25±1.86)%,(55.66±7.08)%;当盐浓度为4%～10%时,3株菌株耐受力随盐浓度的增加而降低,;当pH为3.5～9.5时,3株菌株均可生长。     

Evaluation of novel lactose-positive and exopolysaccharide-producing strain of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> for fermented foods

A novel strain of lactic acid bacteria Pediococcus pentosaceus P 773 was isolated from spoiled beer and identified by means of 16S rDNA sequence analysis. The ability to assimilate lactose as a sole carbon source as a specific feature for this strain was detected and confirmed on dairy substrates. In the presence of sucrose containing substrates (sucrose, raffinose) this P. pentosaceus P 773 lactose-positive strain produced a complex of extracellular polysaccharides (Qp = 0.08 g/l/h) with a molecular mass about 2,000 kDa composed by glucose and fructose residues at a ratio 3:1, respectively. These exopolysaccharides were capable to stimulate the growth rate and biomass productivity of common constituent cultures of probiotic dairy starters (Bifidobacterium lactis, Lactobacillus acidophilus, Streptococcus thermophilus) as well as were assimilated as a sole carbon source by these strains. The present study confirmed the presence of lactose-positive and exopolysaccharide-producing strain of P. pentosaceus in natural environment which could be used as a starter culture to impart more functional attributes to fermented food.     

产γ-氨基丁酸乳酸菌的筛选及发酵过程研究

从不同泡菜中筛选到6株产γ-氨基丁酸(GABA)的乳酸菌,其中A号乳酸菌产量相对较高,GABA产量为1.261 g/L。A号菌株经16S rDNA鉴定为植物乳杆菌,初步命名为Lactobacillus plantarum WZ011。通过单因素和正交设计方法对其发酵培养基进行优化,得到最佳培养基成分(g/L):葡萄糖13,酵母膏5,谷氨酸钠12,盐酸吡哆醇0.15,无水乙酸钠2,MgSO4.7H2O 0.02,MnSO4.4H2O 0.001,FeSO4.7H2O 0.001,NaCl 0.001。Lactobacillus plantarum WZ011发酵动力学曲线表明GABA的发酵过程大致分为菌体生长与产物生成2个阶段。降低培养基的氮源含量和添加盐酸吡哆醇,谷氨酸钠的利用率提高至99%且GABA生产速率提高了2倍多。优化后GABA最高产量可达5.814 g/L,比优化前提高了79%,且提前了48 h进入GABA生产稳定期。     

Potential of a lactic acid bacterial starter culture with gamma-aminobutyric acid (GABA) activity for production of fermented sausage

The ability of lactic acid bacterial starter cultures to produce gamma-aminobutyric acid (GABA) during sausage fermentation was studied. Among 305 strains of lactic acid bacteria isolated from kimchi samples, 11 strains were selected as starter candidates based on the following criteria: growth speed, pH lowering ability, and biogenic amine productivity including GABA-producing activity. During in vitro tests, the Y8 (Lactobacillus brevis), O52, and KA20 strains produced 39.00 ± 1.36, 49.73 ± 3.80, and 64.59 ± 0.61 mg/kg of GABA, respectively. Interestingly, although isolate Y8 showed low productivity in vitro, the GABA content it produced during in situ tests (61.30 ± 2.61 mg/kg) was similar to that produced by isolate PM3 (L. brevis) used as positive control (69.64 ± 2.20 mg/kg). Therefore, isolate Y8 was selected as the best functional starter culture for the production of fermented sausage because it exhibited rapid growth, safety, and abundant GABA productivity.     

Biotransformation of (R)-(+)- and (S)-(−)-limonene to α-terpineol by Penicillium digitatum— investigation of the culture conditions

The biotransformation of (R)-(+)- and (S)-(−)-limonene by Penicillium digitatum was investigated. One strain of P. digitatum was able to convert (R)-(+)-limonene to pure (R)-(+)-α-terpineol in 8 h with a yield of up to 93%. It was found that (R)-(+)-limonene was converted much better into α-terpineol than (S)-(−)-limonene, and that no significant chemical conversion of the substrate occurred in control flasks at pH 3.5. The culture conditions involved such as the type and concentration of co-solvent applied and the sequential addition of substrate were investigated, taking into account some findings on the physical behaviour of the system. The highest bioconversion yields were obtained when the substrate was applied as a diluted solution in EtOH.     

一株产γ-氨基丁酸屎肠球菌的筛选和发酵条件优化及其益生特性分析

为拓宽产γ-氨基丁酸(γ-aminobutyric acid,GABA)微生物资源,以四川泡菜为分离源从中筛选高产GABA乳酸菌菌株,并对高产乳酸菌菌株进行生理生化、分子生物学鉴定,高产GABA发酵条件优化及其益生特性分析。结果表明:采用高效液相色谱对菌株产GABA能力分析发现菌株AB157的GABA产量最高为1.08 g/L。生理生化和16S rRNA鉴定菌株AB157为屎肠球菌(Enterococcus faecium)。通过单因素实验和响应面优化,确定屎肠球菌AB157的最佳培养条件为L-谷氨酸钠底物浓度5.2 g/L、发酵温度31 ℃、初始pH7、发酵时间70 h,在此条件下,屎肠球菌AB157的GABA产量达到1.60 g/L。屎肠球菌AB157的益生分析表明,其在pH2.0和3 g/L的胆盐环境中的存活率分别为39%和59%,胆固醇的脱除量为18.09 μg/mL,说明该菌株具有良好的耐酸和耐胆盐特性以及较强的降胆固醇能力,可作为潜在功能性乳酸菌资源进行后续研究开发。     

Reduction of soybean oligosaccharides and properties of alpha-D-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected] JK55

This study was undertaken to investigate the potential for reducing non-digestive oligosaccharides (NDO) in soy foods, as well as the influence of exogenous conditions on intracellular α-galactosidase (α-Gal) producing lactic acid bacteria. Two strains, Lactobacillus curvatus R08 and Leuconostoc mesenteriodes JK55, showed the highest levels of raffinose degrading activity at over 40 U mL⁻¹, and presented maximum activities during the stationary phase in a medium where raffinose was the only carbon source. Raffinose was the most effective inducer, followed by melibiose, and galactose; the enzymes were partially inhibited by fructose and sucrose. On the other hand, limited activity was observed in glucose. The strains displayed optimum activity levels at neutral pH and a 35–37 °C temperature range. The α-Gal activities of L. curvatus R08 and Leu. mesenteriodes JK55 were maintained at pH 6.5–10.0. The activity of the α-Gal enzyme was stable in a relatively broad range of temperatures from 0 to 40 °C for 3 h. In soymilk, Leu. mesenteriodes JK55 and L. curvatus R08 completely hydrolyzed the NDO after 18–24 h of fermentation. The abilities of L. curvatus R08 and Leu. mesenteriodes JK55 to degrade raffinose sugars and, particularly, to produce organic acids from sugar, could contribute to reductions in the anti-nutritional properties of soy, and to the accumulation of compounds with beneficial properties during food processing. Furthermore, this study provides the optimum conditions to induce α-Gal from these strains.     

Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium

A strictly anaerobic, mesophilic and chitinolytic bacterial strain, M-21, was isolated from a soil sample collected from Mie University campus and identified as Clostridium paraputrificum based on morphological and physiological characteristics, and 16S rRNA sequence analysis. C. paraputrificum M-21 utilized chitin and N-acetyl- -glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products. Hydrogen and carbon dioxide accounted for 65% and 35% of the gas evolved, respectively. The conditions for 1 l batch culture of C. paraputrificum, including pH of the medium, incubation temperature and agitation speed, were optimized for hydrogen production with GlcNAc as the carbon source. The bacterium grew rapidly on GlcNAc with a doubling time of around 30 min, and produced hydrogen gas with a yield of 1.9 mol H₂/mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45°C, agitation speed of 250 rpm, and working volume of 50% of the fermentor. The dry cell weight harvested from this culture was 2.0 g/l.