李氏禾内生细菌Bacillus cereus J01吸附Cr^(3+)的研究

为考察李氏禾内生细菌蜡样芽孢杆菌Bacillus cereus J01菌株吸附Cr^(3+)的性能,以蜡样芽孢杆菌Bacillus cereus J01失活菌体作为生物吸附剂,采用单因素实验方法考察pH、温度、Cr^(3+)初始浓度、吸附剂用量、吸附时间等条件对Cr^(3+)吸附性能的影响,分析其等温吸附过程、动力学过程及热力学过程,并采用红外光谱方法对吸附机理进行初步分析。结果表明:(1)在50 mL反应体系中,当pH值为6、温度为40℃、Cr^(3+)初始浓度为150 mg·L^(-1)、吸附剂投加量为0.2 g、吸附时间为12 h时,蜡样芽孢杆菌Bacillus cereus J01失活菌体对Cr^(3+)的吸附性能达到最佳,其平衡吸附量和Cr^(3+)去除率分别为34.30 mg·g^(-1)、91.60%。(2)等温吸附过程分析结果显示,朗格缪尔吸附等温模型能更好地模拟蜡样芽孢杆菌Bacillus cereus J01失活菌体对Cr^(3+)的吸附,其吸附过程更倾向于单分子层吸附。(3)吸附过程动力学分析结果显示,蜡样芽孢杆菌Bacillus cereus J01失活菌体对Cr^(3+)的吸附更符合准二级动力学速率方程。(4)吸附过程热力学分析结果显示,在40℃下,吸附过程的△G、△H和△S分别为-2.609 kJ·mol^(-1)、61.792 kJ·mol^(-1)和206.11 J·mol^(-1),该温度下吸附过程是自发过程。(5)红外光谱分析结果显示,蜡样芽孢杆菌Bacillus cereus J01失活菌体对Cr^(3+)的吸附可能通过细胞成分中的氨基、羟基和羰基起作用。该研究结果表明该菌的失活菌体对Cr^(3+)具有较强的吸附性能,并在铬污染的环境治理中具有较好的应用潜力。

Lactobacillus rhamnosus GR-1 attenuates foodborne Bacillus cereus-induced NLRP3 inflammasome activity in bovine mammary epithelial cells by protecting intercellular tight junctions

Background:Bacillus cereus is an important pathogen that causes human food poisoning,specifically diarrhea and vomiting.B.cereus can also induce mastitis in dairy cows and has a strong survival ability in milk,as it cannot be inactivated by high-temperature short-time pasteurization.Therefore,B.cereus can enter the market through pasteurized milk and other dairy products,imposing enormous hidden dangers on food safety and human health.Results:In this study,B.cereus 2101(BC)was isolated from milk samples of cows with mastitis.BC grew rapidly with strong hemolysis,making it difficult to prevent mastitis and ensure food security.MAC-T cells were treated with BC and/or Lactobacillus rhamnosus GR-1(LGR-1).Pretreatment with LGR-1 protected the integrity of tight junctions and the expression of zonula occludens-1(ZO-1)and occludin destroyed by BC.Furthermore,LGR-1 pretreatment reduced the expression of NOD-like receptor family member pyrin domain-containing protein 3(NLRP3),caspase recruitment and activation domain(ASC),Caspase-1 p20,gasdermin D(GSDMD)p30,inflammatory factors(interleukin(IL)-1βand IL-18),and cell death induced by BC.Moreover,LGR-1 pretreatment reduced NLRP3 inflammasome activity and increased expressions of ZO-1 and occludin induced by lipopolysaccharides(LPS)+ATP stimulation.MAC-T cells were transfected with NLRP3 si RNA or MCC950 and/or treated with BC and/or LGR-1.NLRP3-si RNA transfection and MCC950 attenuated BC-induced NLRP3 inflammasome activity.Expression of inflammatory cytokines and cell death suggested that the inflammatory pathway might play an important role in the induction of the NLRP3 inflammasome by BC and the protection of LGR-1.Conclusions:These results suggest that LGR-1 might be a probiotic alternative to antibiotics and could be administered to prevent mastitis in dairy cows,thus ensuring food security.

响应面法模拟优化蜡样芽孢杆菌W-3菌株产木聚糖酶发酵条件

【目的】应用响应面法对产木聚糖酶的工艺进行优化,为进一步大规模生产提供参考依据。【方法】通过单因素试验分别考察碳源、氮源、pH、温度、接种量和转速6个因素对蜡样芽孢杆菌W-3产木聚糖酶的影响。通过Design Expert 10.0.3软件中的Box-Behnken进行响应面优化设计。通过对小麦麸皮、氯化铵、pH、温度、接种量和转速进行不同组合,系统地优化了发酵条件。利用二次响应面回归方法建立酶产量与各因素之间的数学模型,并通过回归分析确定各因素对酶产量的影响程度。【结果】小麦麸皮添加量、氯化铵添加量、pH和温度是影响发酵的主效应因素,单因子优化显示,蜡样芽孢杆菌W-3发酵产木聚糖酶条件为小麦麸皮35 g/L、氯化铵5 g/L、pH 8.0、温度70℃、接种量2%、转速210 r/min。在单因子优化的基础上进一步应用响应面优化,找到了更优的发酵参数,经过优化后的发酵条件为小麦麸皮33.0 g/L、氯化铵5.1 g/L、pH 8.3、温度73℃、接种量2%、转速210 r/min,该条件下蜡样芽孢杆菌W3发酵产生的木聚糖酶的酶活为523.24 U/mL。【结论】本试验通过响应面法优化了蜡样芽孢杆菌W-3发酵条件,在33.0 g/L小麦麸皮、5.1 g/L氯化铵、pH 8.3、73℃、2%接种量、210 r/min条件下,可获得较高的木聚糖酶的酶活(523.24 U/mL),比优化之前(225.53 U/mL)提高了2.32倍。

BIOSYNTHESIS AND THERMAL PROPERTIES OF POLY(3-HYDROXYBUTYRATE-co-3-HYDROXYVALERATE)WITH LARGE VARIETY OF HYDROXYVALERATE CONTENTS BY BACILLUS CEREUS

Biosynthesis and thermal properties of poly（3-hydroxybutyrate-co-3-hydroxyvalerate） （PHBV） with different HV （hydrovalerate） content produced by a Bacillus cereus strain were investigated. A large variety of HV contents （up to about 90 mol%） of PHBV could be produced by this strain. Combined nitrogen sources containing both yeast extract and ammonium sulphate were better for cell growth and polyhydroxyalkanoates （PHA） production than either yeast extract or ammonium sulphate alone. Propionic acid is more favorable for the production of HV content than that of valeric acid. Finally, thermal properties of PHBV produced by this strain are found close to the results of other groups.

产木聚糖酶蜡芽孢杆菌W-3发酵麦麸对蛋鸡生产性能、蛋品质和血液生化指标的影响

为探讨产木聚糖酶蜡芽孢杆菌W-3(Bacillus Cereus W-3)发酵麦麸替代部分玉米对海兰褐商品蛋鸡蛋品质和血液生化指标的影响。将420只30周龄海兰褐蛋鸡随机分为4个处理,每处理7个重复,每重复15只,对照组饲喂基础日粮,试验组用B.cereus W-3发酵麦麸分别替代基础日粮中5%、10%和15%的玉米。预饲期1周,试验期8周。结果表明,与对照组相比,B.cereus W-3发酵麦麸替代蛋鸡饲粮15%玉米显著降低采食量(P<0.05),替5%~15%代玉米时显著提高鸡蛋哈夫单位(P<0.05),替代10%或15%玉米显著提高血浆白蛋白含量(P<0.05)。4个处理组间其余产蛋性能、蛋品质和血液生理生化指标均无显著差异(P>0.05)。本研究表明,B.cereus W-3发酵麦麸替代海兰褐蛋鸡饲粮中5%~15%玉米,不影响产蛋性能、蛋品质和血液生理生化指标,并具有降低饲粮成本、改善蛋品质和蛋鸡免疫力的潜力。

基于商用芭蕉芋RS3型抗性淀粉碳源的菌株筛选、分离与功能性评价

目的筛选、鉴定和功能性评价优势酵解商用芭蕉芋RS3型抗性淀粉的健康人体肠道菌,为商用芭蕉芋RS3型抗性淀粉合生元制剂提供高价值菌种。方法采用商用芭蕉芋RS3型抗性淀粉改良BHI固体培养基,筛选并鉴定优势酵解肠道菌,用DPPH自由基清除实验和ABTS自由基清除实验考察菌株抗氧化能力,用溶血性实验和药敏实验评价其安全性。结果菌株1-6和菌株S1能够发酵商用芭蕉芋RS3型抗性淀粉并显著降低体系pH值(P<0.05),经鉴定均为蜡样芽孢杆菌。两种菌株无细胞提取上清液对DPPH自由基清除率分别79.63%和51.56%,对ABTS自由基清除率分别为63.79%和39.39%。两种菌株均无溶血性且对抗菌药物敏感。结论菌株1-6与菌株S1具有抗氧化潜力且安全性高,可作为健康食品市场应用的高价值候选菌株。

Comparative evaluation of physico-chemical characteristics of biopolyesters P（3HB） and P（3HB-co-3HV） produced by endophytic Bacillus cereus RCL 02

BACKGROUND： Bacteria endogenously residing within the plant tissues have attracted significant attention for production of biopolyester, polyhydroxyalkanoates （PHAs）. Bacillus cereus RCL 02 （MCC 3436）, a leaf endophyte of oleaginous plant Ricinus communis L. accumulates 81% poly（3-hydroxybutyrate） [P（3HB）] of its cell dry biomass when grown in mineral salts （MS） medium. METHODS： The copolymer production efficiency of B. cereus RCL 02 was evaluated in valeric acid supplemented MS medium under biphasic cultivation condition. The copolymer so produced has been compared with the P（3HB） isolated from RCL 02 in terms of thermal, mechanical and chemical properties. RESULTS： Valeric acid supplementation as co-substrate in the medium has led to the production of copolymer of 3- hydroxybutyrate （3HB） and 3-hydroxyvalerate （3HV） [P（3HB-co-3HV）] with 14.6 mol% 3HV. The identity of the polymers has been confirmed by X-ray diffraction （XRD） analysis, Fourier transform infrared （FTIR） and nuclear magnetic resonance （NMR） spectroscopic studies. Thermogravimetric analysis （TGA） revealed that P（3HB） and P（3HB-co-3HV） films degraded at 278.66℃ and 273.49℃, respectively. The P（3HB-co-3HV） showed lower melting temperature （165.03℃） compared to P （3HB） （170.74℃） according to differential scanning calorimetry （DSC）. Incorporation of 3HV monomers decreased the tensile strength （21.52 MPa）, tensile modulus （0.93 GPa）, storage modulus （E＇） （0.99 GPa） and increased % elongation at break （12.2%） of the copolyester. However, P（3HB） showed better barrier properties with lower water vapor transmission rate （WVTR） of 0.55 g-mil/100 in2/24 h. CONCLUSION： These findings emphasized exploration of endophytic bacterial strain （RCL 02） to produce biodegradable polyesters which might have significant potential for industrial application.

Synthesis of scl-poly （3-hydroxyalkanoates） by Bacillus cereus found in freshwater, from monosaccharides and disaccharides

BACKGROUND： Polyhydroxyalkanoates are a good substitute for synthetic plastic because they are highly biocompatible, ecofriendly, and biodegradable. Bacteria in freshwater bodies such as rivers, tube wells, and canals are exposed to alternating high and low concentrations of substrates that induce PHA production. METHODS： Fresh water samples were collected for isolation of bacterial strains. Screening of PHA in bacterial cells was performed with Sudan and Nile Red staining. Extracted PHA was characterized by FTIR. RESULTS： In this study, nine bacterial isolates were selected for PHA production on the basis ofphenotypic screening. Their ability to accumulate PHAs was determined using different monosaccharides and disaccharides. Two bacterial isolates Bacillus cereus T1 （KY746353） and Bacillus cereus R3 （KY746354） produced PHAs. Optimal growth of the bacterial strain （T1） was observed in the presence of glucose, followed by maximum production of PHAs （63% PHAs） during the logarithmic phase of growth. B. cereus R3 （KY746354） accumulated 60% PHAs by dry cell weight. CONCLUSION： PHA accumulation was relatively less with fructose, but both strains showed increased production （up to 50%） with sucrose. The polymer produced was characterized by Fourier-transform infrared spectroscopy （FTIR）, which showed that the compound contains short-chain PHAs.

蜡样芽胞杆菌GXBC-3三个普鲁兰酶基因的表达及其酶学特性

探索获得优良的新型普鲁兰酶基因,丰富普鲁兰酶理论,对实现普鲁兰酶国产化具有重要意义。分析GenBank数据库中蜡样芽胞杆菌假定Ⅰ型、Ⅱ型普鲁兰酶基因序列,从实验室保藏的蜡样芽胞杆菌Bacilluscereus GXBC-3中克隆得到3个普鲁兰酶基因pulA、pulB、pulC,并分别导入大肠杆菌进行胞内诱导表达。纯化重组酶酶学性质研究表明重组酶PulA能水解α-l,6-和α-l,4-糖苷键,为Ⅱ型普鲁兰酶,以普鲁兰糖为底物时,最适反应温度及pH分别为40℃和6.5,比活力为32.89 U/mg;以可溶性淀粉为底物时,最适反应温度及pH分别为50℃和7.0,比活力为25.71 U/mg。重组酶PulB和PulC二者均只能水解α-l,6-糖苷键,为I型普鲁兰酶,以普鲁兰糖为底物时,其最适反应温度及pH分别为45℃、7.0和45℃、6.5,比活力分别为228.54 U/mg和229.65 U/mg。

三重微滴数字PCR定量检测即食米粉中致病菌方法研究

目的建立三重微滴数字PCR(ddPCR)方法同时定量检测即食食品中的沙门菌、蜡样芽胞杆菌和单核细胞增生李斯特菌。方法选择以沙门菌ttrA/ttrC、蜡样芽胞杆菌essC、单核细胞增生李斯特菌侵袭相关内肽酶基因等3个单拷贝基因对应的3对引物探针,采用实时荧光定量PCR验证引物/探针特异性后,建立三重ddPCR方法同时定量检测3种致病菌的拷贝数。结果该方法的线性范围分别为:沙门菌25~22687 copies/20μL;蜡样芽胞杆菌19~15620 copies/20μL;单核细胞增生李斯特菌18~23373 copies/20μL,线性相关因子r^(2)≥0.999,6个浓度3次重复测定3种菌的相对标准偏差(RSD)≤12%,重复性好,对于上述菌株的最低检出限分别为6、3和7 copies/20μL;采用已建立的ddPCR方法和平板计数方法对模拟染菌米粉样品进行检测,两种方法测定值结果RSD小于9%,结果一致性较好。结论本研究建立的三重ddPCR同时定量检测即食食品中沙门菌、蜡样芽胞杆菌和单核细胞增生李斯特菌的方法与平板计数法相比,更快速、灵敏,结果准确。