可降解咖啡碱菌株的筛选与鉴定

目的：筛选能够降解咖啡碱的细菌,以期在该毒素的生物脱毒中得到应用。方法：以咖啡碱作为唯一碳源和氮源进行咖啡碱降解菌株的初筛,之后将初筛的10 株菌通过液体培养基咖啡碱降解实验最终筛选出降解能力最强的1 株菌。结果：筛选出的HZ-1 菌株在48h 即可将咖啡碱完全降解,对目标菌株细胞形态、生理生化以及16SrDNA 进行鉴定,确定该菌株为Pseudomonas lutea,属于假单胞菌属。结论：利用咖啡碱作为唯一碳源和氮源从土壤中筛选出降解咖啡碱良好的菌株,为脱除咖啡碱提供了微生物菌种,填补了国内空白。     

葡萄中碳黑曲霉的分离及其产生赭曲霉毒素A的研究

碳黑曲霉(Aspergillus carbonarius)是葡萄中产生赭曲霉毒素A(Ochratoxin A,OTA)的重要菌株.采集烟台赤霞珠(Cabernet sauvignon)葡萄,接种于孟加拉红培养基,从中分离到7株黑曲霉群(Aspergillus section black group)真菌,其中3株鉴定属于碳黑曲霉种,占黑色曲霉的43.8%.此3菌株分别接种在粮粒培养基上,静置培养,全部产生OTA,最高浓度达到1300μg/kg,而且,其中2株碳黑曲霉菌株在可可浆培养基上产生荧光,而从葡萄样品中未检出OTA.     

游离棉酚脱除菌株的分离鉴定及体内定植效果研究

该研究从藜麦茎叶和番茄杆样品中筛选分离出高效游离棉酚脱除菌株,为棉籽粕生物脱毒提供优良菌株。测定筛选菌株的游离棉酚脱除能力及产蛋白酶能力,采用人工胃液、人工肠液耐受实验,抗生素敏感实验及表面特性实验研究筛选菌株在体内定植能力,并通过形态学观察、生理生化试验及16S rDNA序列分析对筛选菌株进行鉴定。结果表明,筛选出1株高效游离棉酚脱除芽孢杆菌,编号为S-77,其游离棉酚脱除率为81.30%,产蛋白酶酶活为767.27 U/g。菌株S-77被鉴定为东洋芽孢杆菌（Bacillus toyonensis）,具有耐人工胃液、肠液效果及较好黏附于宿主肠壁细胞的能力,对抗生素敏感,可用于棉籽粕生物脱毒。     

不同条件对炭黑曲霉产赭曲霉毒素A能力的影响

为了解一株分离自新疆葡萄园的炭黑曲霉菌株在不同培养条件下产生赭曲霉毒素A(OTA)的能力,试验采用孟加拉红培养基,利用高效液相-荧光法对OTA进行定量检测,在单因素试验的基础上,采用响应面法优化产毒条件。结果表明,对该菌株产生OTA的条件影响从大到小依次为葡萄糖质量浓度、乙醇体积分数、SO2质量浓度。最优产毒条件:在温度28℃条件下,葡萄糖质量浓度92.26g/L、SO2质量浓度8.26mg/L、乙醇体积分数0.05%。在此条件下,OTA产量为7.427ng/mL。     

中国南方稻谷主产区黑曲霉菌群的流行特征及产毒能力研究

本研究从我国南方十省份代表性粮库中采集稻谷样品,根据形态学特征筛选初步鉴别出黑曲霉群（Aspergillus niger aggregate）菌株,进一步依据ITS和CaM序列特征在种水平上进行鉴定;采用ELISA、HPLC-FLD方法,结合OTA生物合成基因鉴定方法联合确证黑曲霉群中分离株的产OTA能力。结果表明,从稻谷样品中共筛选出107株,划分为4个种：黑曲霉（A. niger）、百岁兰曲霉（A. welwitschiae）、塔宾曲霉（A. tubingensis）和新黑曲霉（A. neoniger）。其中百岁兰曲霉和黑曲霉在广西广东一带流行率较高,塔宾曲霉的流行率随纬度的升高而升高,说明地理环境因素对我国南方地区仓储稻谷中黑曲霉群流行特征的影响较大。虽然黑曲霉群菌株产OTA测定中ELISA检测出7株菌假阳性,但进一步采用HPLC-FLD结合OTA生物合成基因鉴定方法确证107株黑曲霉群菌株均不产生OTA。本研究为我国稻谷的安全储藏和霉菌针对性防控提供了数据支撑。     

黄曲霉毒素B1 降解菌株的分离鉴定、发酵条件的 优化及活性组分分析

为筛选出一株高效降解黄曲霉毒素B1（AFB1）的菌株，选取土壤为菌株来源，进行富集培养，经过以香豆素为唯一碳源的初筛和添加AFB1的复筛后，对筛选得到的各菌株进行16S rDNA鉴定，比对其测序结果，选取降解AFB1最高的菌株，探究发酵时间、发酵温度、初始pH、接种量、培养基对菌株降解AFB1的影响规律，通过正交试验优化发酵条件，并对降解方式进行初步探索。结果表明：经过初筛和复筛得到7株能够降解AFB1的菌株，经鉴定均为伯克霍尔德菌属，其中Burkholderia sp. D6的AFB1降解率最高；最优发酵条件为以LB培养基为发酵培养基、发酵时间84 h、发酵温度37 ℃、初始pH 7.0、接种量10%，在此条件下Burkholderia sp. D6对AFB1的降解率为（87.91±2.32)%；初步判断降解AFB1的活性组分是蛋白质或者酶。综上，通过筛选得到了一种能够高效降解AFB1的菌株，蛋白质或酶参与了AFB1的降解。     

宜春明月山富硒土壤中耐硒酵母菌株的筛选与鉴定

从宜春明月山毛竹林采集的富硒土壤中,筛选出耐硒酵母菌株并进行鉴定。采用PDA培养基、营养琼脂培养基、马丁氏培养基筛选出耐亚硒酸钠浓度较高的酵母菌株,根据菌株形态学特征和26S rDNA基因序列分析,对其进行分类鉴定。从土壤中分离得到7株耐硒菌株,其中2株具有典型酵母菌特征的菌株,经鉴定结果表明:菌株A₁为Saitozymapodzolica,菌株P为Cyberlindnerasaturnus。得到2株耐硒酵母菌株,丰富了耐硒酵母资源。     

玉米赤霉烯酮脱毒菌株的筛选及脱毒机理初探

为筛选高效脱除玉米赤霉烯酮(Zearalenone,ZEN)的脱毒菌株,利用96孔板高通量筛选法,从135份赤霉病高发区域土壤样品中成功筛选出具有较强ZEN清除作用的两株芽孢杆菌Fu2-3和Fu41-3。经16S rRNA序列分析,菌株Fu2-3和Fu41-3分别为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和特基拉芽孢杆菌(Bacillus tequilensis)。对其降解机理的初步研究表明,两株菌对ZEN均具有一定程度的吸附作用,但主要脱毒作用仍为生物的降解作用。BLYES法研究结果表明,菌株Fu41-3的脱毒体系中仍具有较大雌激素毒性,菌株Fu2-3脱毒体系无雌激素响应,该菌可将ZEN转化为雌激素含量极低的代谢产物,脱毒效果更佳。筛选获得了两株ZEN降解芽孢杆菌,为ZEN的生物脱毒提供了一定的理论依据。     

耐酸耐胆盐益生乳酸菌的筛选与鉴定

为了筛选出人源性的耐酸、耐胆盐的益生菌,利用含胆盐(0.2%)的5种选择性培养基(TPY、BHI、MRS、SL、乳酸杆菌选择性琼脂培养基),以溴甲酚紫为酸性指示剂,从婴儿粪便中初步筛选出42株耐胆盐的乳酸菌。根据菌株在pH5.0,4.0,3.5的MRS液体培养基中培养时OD值的变化情况,筛选出3株具有较好酸耐受性的菌株。然后通过平板菌落计数方法测定筛选出的3株菌在pH 3.0条件下0~3h存活率的变化情况,筛选出对酸和胆盐耐受能力最强且最稳定的菌株经16SrDNA分子生物学鉴定为Lactobacillus plantarum。该菌能够作为潜在的益生菌菌株用于后期深入地挖掘其益生功能。     

发酵乳杆菌SM-7的筛选及对小鼠降胆固醇作用

筛选1 株具有高效降胆固醇活性的乳酸菌菌株并研究其降胆固醇的特性。采用分离自新疆酸马奶的10 株乳酸菌,通过益生活性的测定以及降胆固醇实验筛选得到1 株具有高效降胆固醇的益生菌株SM-7,经过鉴定为发酵乳杆菌。该菌株在液体培养基中的胆固醇脱除率为66.82%,用该菌液饲喂小鼠4 周,研究其对小鼠血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)以及体质量和肝、肾质量的影响。结果显示,实验组小鼠的血清TC、TG、LDL-C 含量均显著低于高脂模型组(P ＜ 0.01),而HDL-C 含量没有显著差异(P ＞ 0.05)。这表明发酵乳杆菌SM-7 对实验性高脂血症小鼠血清有显著的降胆固醇作用。     

Reduction of Ochratoxin A Levels in White Wine by Yeast Treatments

This paper describes the abilities of 21 yeast strains isolated from six different wine‐grapes of Turkey to bind ochratoxin A (OTA). Viable (10⁸ CFU/mL) and heat‐treated yeast cells were incubated both in phosphate‐buffered saline (PBS) and white wine containing 10 ng OTA per mL for 4 h at 25°C. After centrifugation, the concentration of OTA was measured in the supernatant fraction using a high‐performance liquid chromatography system coupled with fluorescence detector. The adsorption abilities of OTA by viable yeast strains within 4 h ranged from 1.96 to 26.11% in PBS. On the other hand, a slight decrease was observed in the percentage of OTA removal by yeast strains in white wine when compared to their activity in PBS. The addition of yeasts at 10⁸ CFU/mL resulted in a reduction to a maximum of 21.40% in white wine, with respect to the control. Among the yeasts, Candida famata D7 was found to be the most efficient binder to OTA both in spiked white wine and PBS. In addition, dead yeast cells can potentially be used for removing OTA (a maximum of 30.45%) from white wine.     

Environmental factors and weak organic acid interactions have differential effects on control of growth and ochratoxin A production by Penicillium verrucosum isolates in bread

Studies were conducted to examine the effect of water activity (a(w), 0.97, 0.95, 0.93), pH (4.5, 6.0) and preservative type and concentration (potassium sorbate (KS), calcium propionate (CaP), 300 and 3000 ppm) on colonisation and ochratoxin A (OTA) production by three strains of Penicillium verrucosum on bread analogues. At 25 degrees C, colony extension from a point source was similar for all three strains at the different environmental treatments examined, with optimum growth at 0.97 and 0.96 a(w) and pH 6. Temporal OTA production over 36 days varied significantly with strain, pH and a(w) level. Maximum OTA was produced at approximately 28-36 days for two of the strains with an optimum at 0.93 a(w) at pH 6.0. One strain produced 75-80 microg g(-1) analogue under these conditions. However, at pH 4.5, optimum OTA was produced at 0.95 a(w) after a similar time period on bread analogues, with maximum amounts of about 25-35 microg g(-1) bread. KS and CaP incorporation inhibited growth completely at pH 4.5 and 3000 ppm at all three a(w) levels. However, at suboptimal concentration, little growth inhibition occurred. At pH 6, even with 3000 ppm, growth of all three strains occurred. Where effective inhibition of growth was achieved (3000 ppm/pH 4.5, all a(w) levels), no OTA was detected. However, when 300 ppm was used, up to 7 microg g(-1) was detected in bread analogues at pH 4.5. At pH 6, OTA was produced at all treatment conditions by all three strains, although preservative-treated bread had less present. Statistical analysis showed that many of the one-, two- and three-way interactions had a significant effect on growth and OTA production. These results suggest that there is a significant risk from OTA contamination if lower concentrations of existing preservatives are used in wheat-based bakery products contaminated by spoilage mycotoxigenic moulds such as P. verrucosum.     

Study of the effect of water activity and temperature on ochratoxin A production by Aspergillus carbonarius

The effect of water activity (aw) (0.78-0.99) and temperature (15 and 30 degrees C) on growth and production of ochratoxin A (OTA) of six Aspergillus carbonarius strains was studied in two culture media: Czapek yeast autolysate (CYA) agar and yeast extract sucrose (YES) agar, during a period of 30 days. The strains were selected to include different sources and different reported abilities to produce OTA and were characterized by RAPD and ITS-5.8S rDNA sequencing. CYA showed to be better culture medium than YES for OTA production in the isolates tested. OTA concentration was higher at 15 degrees C than at 30 degrees C. At 30 degrees C, ranges for OTA production were more restrictive than those for growth. OTA was produced from 0.86, 0.90 or 0.94 aw depending on the strain. At 15 degrees C, growth and OTA production were detected only in the 0.94-0.99 aw range. The molecular study performed showed that five of the strains were conspecific and no correlation was found between molecular data and the OTA production level or origin. The remaining strain had never been able to produce OTA and will probably represent a new species in the Aspergillus section Nigri. Our results show that A. carbonarius is able to grow and produce OTA in a wide range of water activities at both high and low temperatures.     

水牛乳中高产胞外多糖乳酸菌的筛选与鉴定

从生水牛乳中筛选出两株高产胞外多糖（EPS）的乳酸菌株LB2、LB7,经MRS肉汤培养基发酵后,菌液中的胞外多糖（EPS）产量分别达135 mg/L、148 mg/L,通过形态学、生理生化特征、API细菌鉴定系统、16S rRNA序列分析,鉴定出菌株LB2为植物乳杆菌（Lactobacillus plantarum）,LB7为类肠膜魏斯氏菌（Weissella paramesenteroides）。将其应用到水牛乳酸奶的发酵中,结果表明,植物乳杆菌LB2和类肠膜魏斯氏菌LB7均可用来发酵水牛乳酸奶,且能有效增加酸奶的黏度和胞外多糖产量,其黏度和EPS产量分别为4 050 mPa·s和149 mg/L。     

Quality parameters and RAPD-PCR differentiation of commercial baker's yeast and hybrid strains

Baker's yeast, Saccharomyces cerevisiae, is a key component in bread baking. Total of 12 commercial baker's yeast and 2 hybrid strains were compared using traditional quality parameters. Total of 5 strains with high leavening power and the 2 hybrid strains were selected and evaluated for their alpha-amylase, maltase, glucoamylase enzymes, and compared using random amplified polymorphic DNA (RAPD). The results revealed that all selected yeast strains have a low level of alpha-amylase and a high level of maltase and glucoamylase enzymes. Meanwhile, the Egyptian yeast strain (EY) had the highest content of alpha-amylase and maltase enzymes followed by the hybrid YH strain. The EY and YH strains have the highest content of glucoamylase enzyme almost with the same level. The RAPD banding patterns showed a wide variation among commercial yeast and hybrid strains. The closely related Egyptian yeast strains (EY and AL) demonstrated close similarity of their genotypes. The 2 hybrid strains were clustered to Turkish and European strains in 1 group. The authors conclude that the identification of strains and hybrids using RAPD technique was useful in determining their genetic relationship. These results can be useful not only for the basic research, but also for the quality control in baking factories.     

Inhibition of A. carbonarius growth and reduction of ochratoxin A by bacteria and yeast composites of technological importance in culture media and beverages

Five composites of yeast and six of bacterial isolates from fermented products were studied, in order to assess their ability to inhibit Aspergillus carbonarius growth and reduce OTA concentration in culture media and beverages. The antagonistic effect of the above composites against A. carbonarius growth was studied in synthetic grape medium of pH 3.5 and a_w 0.98, 0.95, 0.92 after incubation at 25 °C. Different combinations of initial inocula of bacteria or yeast composites and fungi were used (10² cfu/mL vs 10⁵ spores/mL; 10⁵ cfu/mL vs 10² spores/mL; and 10⁵ cfu/mL vs 10⁵ spores/mL). Regarding the OTA reduction experiment, 10³ and 10⁷ cfu/mL of the bacteria and yeast composites were inoculated in liquid media of different pH (3.0, 4.0, 5.0, and 6.1 or 6.5) and initial OTA concentration (50 and 100 μg/L) and incubated at 30 °C. Moreover, grape juice, red wine, and beer were supplemented with 100 μg/L of OTA and inoculated with composites of 16 yeasts (16YM) and 29 bacterial (29BM) strains (10⁷ cfu/mL) to estimate the kinetics of OTA reduction at 25 °C for 5 days. Fungal inhibition and OTA reduction were calculated in comparison to control samples. None of the bacterial composites inhibited A. carbonarius growth. The high inoculum of yeast composites (10⁵ cfu/mL) showed more efficient fungal inhibition compared to cell density of 10² cfu/mL. All yeast composites showed higher OTA reduction (up to 65%) compared to bacteria (2-25%), at all studied assays. The maximum OTA reduction was obtained at pH 3.0 by almost all yeast composites. For all studied beverages the decrease in OTA concentration was higher by yeasts (16YM) compared to bacteria (29BM). The highest OTA reduction was observed in grape juice (ca 32%) followed by wine (ca 22%), and beer (ca 12%). The present findings may assist in the control of A. carbonarius growth and OTA production in fermented foodstuffs by the use of proper strains of technological importance.     

Removal of ochratoxin A by wine Saccharomyces cerevisiae strains

The aim of this work was to examine two wine strains of Saccharomyces cerevisiae (Syrena LOCK 0201 and Malaga LOCK 0173 strains) and thermally inactivated biomass of bakery yeast (BS strain) for their ability to remove ochratoxin A (OTA) from model YPG, white grape GM, and blackcurrant BM media. The media was initially contaminated by 1 μg/mL OTA. The influence of OTA on yeast growth parameters, kinetic of fermentation, and amount of ethanol, glycerol, and acids were determined. It was found that both yeast strains were able to decrease the toxin amount in YPG, GM, and BM media. Strain Malaga LOCK 0173 was able to remove 82.8 and 10.7 % ochratoxin A from grape and blackcurrant medium, respectively. In case of Syrena LOCK 0201 strain, the OTA reduction was higher: 85.1 % for grape and 65.2 % for blackcurrant media. From 54.1 to 64.4 % of initial ochratoxin A concentration was removed after the contaminated wine treatment by thermally inactivated baker’s yeast strain (BS) cells. The elongation of lag phase in contaminated YPG medium compared on toxin-free medium was noted. In white grape and blackcurrant medium, the differences between the final cell number, fermentation rate, moreover the ethanol, glycerol, and acids production in the medium with OTA and the control were not statistically significant. The results showed that the application of selected strains of yeasts in winemaking involving raw material contaminated with OTA might reduce the toxin contamination as well as the health risk related to human exposure to this toxin. Moreover, the application of heat-inactivated yeast’s biomass for toxin adsorption gives new possibilities in oenology.     

Autoinducer-2 activity of gram-negative foodborne pathogenic bacteria and its influence on biofilm formation

ABSTRACT:  This study evaluated whether autoinducer-2 (AI-2) activity would be associated with biofilm formation by Salmonella and Escherichia coli O157:H7 strains on food contact surfaces. In study I, a Salmonella Typhimurium DT104 strain and an E . coli O157:H7 strain, both AI-2 positive, were individually inoculated into 50 mL of Luria–Bertani (LB) or LB + 0.5% glucose (LBG) broth, without or with stainless steel or polypropylene ( Salmonella ) coupons. At 0, 14 ( Salmonella ), 24, 48, and 72 h of storage (25 °C), cells in suspension and detached cells from the coupons, obtained by vortexing, were enumerated on tryptic soy agar. In study II, a Salmonella Thompson AI-2-positive strain and an AI-2-negative strain, and an E . coli O157:H7 AI-2-positive strain and an AI-2-negative strain were inoculated into LB broth with stainless steel coupons. Cells were enumerated as in study I. In both studies, AI-2 activity was determined in cell-free supernatants. Cell numbers of S . Typhimurium DT104 on biofilms were higher ( P < 0.05) in LB than those in LBG, while the E . coli O157:H7 strain showed no difference ( P ≥ 0.05) in biofilm cell counts between LB and LBG after storage for 72 h. Both S . Typhimurium DT104 and E . coli O157:H7 strains produced higher ( P < 0.05) AI-2 activity in LBG than LB cell suspensions. Cell counts of AI-2-positive and-negative S . Thompson and E . coli O157:H7 strains were not different ( P ≥ 0.05) within suspensions or coupons (study II). The results indicated that, under the conditions of this study, AI-2 activity of the pathogen strains tested may not have a major influence on biofilm formation on food contact surfaces, which was similar between AI-2-positive and -negative strains.     

酒曲中产凝乳酶微生物菌株的分离筛选及鉴定

针对酒曲中的微生物进行分离纯化,得到11株细菌和2株真菌,并采用酪蛋白平板法和Arima时间法筛选出了1株产凝乳酶的细菌菌株编号为LB-51。通过形态学观察、生理生化实验和16S rDNA序列分析鉴定该菌株为解淀粉芽孢杆菌,将该产凝乳酶菌株命名为解淀粉芽孢杆菌GSBa-1。该菌株在液体LB培养基中发酵72 h产凝乳酶的凝乳活力为(431.53±15.89)SU/mL,蛋白水解活力为(5.05±0.59)U/mL,所产凝乳酶凝乳活力高而蛋白水解活力低,凝乳酶粗酶单位酶活力为1.54×10~5SU/g。解淀粉芽孢杆菌GSBa-1是分离筛选自酒曲中的一株高产凝乳酶细菌,因此其来源安全,可作为工业化候选菌株进一步研究开发。     

Effect of pH on ochratoxin A production by Aspergillus niger aggregate species

The effect of pH (2-10) on growth and ochratoxin A (OTA) production by 12 Aspergillus niger aggregate strains was studied in two culture media: Czapek yeast autolysate agar (CYA) and yeast extract sucrose agar (YES), over 30 days. The strains were selected to include different sources, different reported abilities to produce OTA and different ITS-5.8S rDNA RFLP patterns. YES was a better culture medium than CYA for OTA production. In this medium, OTA was produced from pH 2 or 3 to 10 depending on the strain. The results show the ability of A. niger aggregate strains not only to grow, but also to produce OTA over a wide pH range. The results will lead to a better understanding of the role of A. niger aggregate strains in the OTA contamination of several food commodities.