枯草芽孢杆菌产D-阿洛酮糖3-差向异构酶的发酵优化

D--阿洛酮糖是D--果糖在C-3位的差向异构体,由于较低的热量和与蔗糖相似的甜度,D-阿洛酮糖成为一个具有发展前景的功能性甜味剂。D--阿洛酮糖 3-差向异构酶（D--psicose 3-epimerase,EC 5.1.3.30,DPEase）能够催化D--果糖生产D--阿洛酮糖,这种生物酶法制备的功能性甜味剂D--阿洛酮糖由于简单的纯化步骤和高产物浓度等优点受到关注。该研究对1株前期构建的枯草芽孢杆菌Bacillus subtilis 1A751/pUB-P43dpe-dal产DPEase进行了3 L发酵罐水平培养,通过优化溶解氧（DO）、pH、温度、初始碳源质量浓度确定最适发酵条件为：DO为30%、pH 6.0、温度37 ℃、初始葡萄糖质量浓度15 g/L,最高发酵酶活达78.3 U/mL。在此基础上进行补料发酵优化,得到最适补料条件为：在发酵5 h进行补料,碳源补料速率8.0 g/（L·h）,在此条件下,发酵9 h全细胞酶活高达123.0 U/mL。此发酵策略为扩大工业化生产提供了参考。     

来源于生孢噬纤维菌的新型β-葡萄糖苷酶SmBgl3A的性质研究

通过表达水平分析比较,从生孢噬纤维菌Sporocytophaga sp. CX11基因组中筛选出在菌株降解纤维二糖过程中起关键作用的β-葡萄糖苷酶基因SmBgl3A。该基因含有2 283 bp碱基对,编码760个氨基酸,其编码蛋白质与来源于Bacteroides ovatus的β-葡萄糖苷酶BoGH3B同源性为46.76 %。将该基因在大肠杆菌中进行异源表达,粗酶液经Ni-NTA亲和层析一步纯化得到电泳级纯度的酶蛋白SmBgl3A,重组蛋白质相对分子质量与理论值（81.2×10⁴）一致。酶学性质表征结果表明,SmBgl3A最适温度和pH分别为35 ℃和6.5;pH在5.0~7.0、温度在40 ℃以下时,稳定性较好,同时有着较好的NaCl耐受性。SmBgl3A的最适底物为pNPG,以pNPG为底物时的比活力为14.74 U/mg;K_m为10.88 mmol/L,k_cat为46.72 s< sup > -1。SmBgl3A作为一种新型的、酶学性质优良的β-葡萄糖苷酶,进一步扩展了β-葡萄糖苷酶的种类,为该酶在纤维素降解、食品工业等领域的应用奠定基础。     

来源于Anoxybacillus sp. SK3-4普鲁兰酶PulASK的固定化及酶学性质研究

普鲁兰酶在淀粉加工、多糖制备以及啤酒酿造等领域有着广泛应用。然而,该酶游离下催化产物需分离纯化和本身不可再生限制了其工业应用。磁性纳米材料具有可重复使用、磁回收性质和比表面积大等特点,有助于解决游离酶的工业化应用难题。作者将源于LAnoxybacillus sp. SK3-4的普鲁兰酶PulASK固定在纳米磁性材料Fe₃O₄@Histidine上以制备固定化酶复合体：Fe₃O₄@Histidine/PulASK,测定固定化酶的酶学性质和动力学参数。结果显示,与游离酶PulASK相比,固定化酶Fe₃O₄@Histidine/PulASK的最适反应温度由60 ℃提高到65 ℃,最适pH与游离PulASK相同;在60 ℃,pH 6.0下孵育7 h,固定化酶残余酶活为62%,而游离酶的仅为30%。分析酶动力学数据可知,在60 ℃,pH 6.0的情况下,游离酶PulASK的K_m为4.7 mmol/L,为Fe₃O₄@Histidine/PulASK的1.47倍;Fe₃O₄@Histidine/PulASK的k_cat值是350 s^-1,与PulASK的k_cat值相当,Fe₃O₄@Histidine/PulASK的k_cat/K_m是PulASK的1.57倍;并且Fe₃O₄@Histidine/PulASK在重复使用9次后,活力仍保持50%以上。与PulASK相比,Fe₃O₄@Histidine/PulASK具有良好的热稳定性且可重复使用,具有潜在应用于食品工业的价值。     

极耐热α-L-鼠李糖苷酶的性质及其在异槲皮素制备中的应用研究

α-L-鼠李糖苷酶可特异性切除糖苷类化合物上连接的α-L-鼠李糖基,已被广泛用于食品、医药等工业领域。作者旨在获得耐受高温的α-L-鼠李糖苷酶,提高工业生产的效率和降低使用成本。首先对嗜热菌Sulfolobus islandicus来源的α-L-鼠李糖苷酶基因进行克隆及大肠杆菌的异源表达,随后测定了系列酶学性质,接着优化了该酶制备异槲皮素的酶法制备工艺。结果表明：重组酶SisRha在90 ℃下酶活力最佳,具有极好的热稳定性,其在80 ℃下孵育120 min后酶活力几乎没有损失;最适反应pH为5.5,在pH 4.5~7.0时,pH稳定性良好;重组酶SisRha对人工底物对硝基苯酚鼠李糖糖苷（pNPR）的K_m值为（0.15±0.03） mmol/L,V_max值为（6.26±0.51） U/mg,kcat值为（10.48±0.86） s-1;对底物pNPR、芦丁、朝藿定C、柚皮苷、橘皮苷和淫羊藿苷的比活力分别为25.06、11.89、6.74、3.14、2.77、0.42 U/mg。重组酶SisRha转化芦丁生成异槲皮素的适宜工艺为：用酶量0.4 U/mL ,在85 ℃、pH 5.0的条件下反应1 h,可将2 mmol/L芦丁几乎全部转化,摩尔转化率高达98.56%。本研究丰富了现有的α-L-鼠李糖苷酶资源,为嗜热菌来源的α-L-鼠李糖苷酶的研究奠定了基础,同时提供了一种高温转化芦丁制备异槲皮素的方法。     

太行菊黄酮对α-葡萄糖苷酶活性的抑制作用

为研究太行菊醇提物不同极性萃取相中黄酮对α-葡萄糖苷酶活性的影响及其抑制作用的动力学特征,采用萃取法制得太行菊黄酮的乙酸乙酯相、正丁醇相、残余水相和石油醚相,使用体外（PNPG） 法建立抑制α-葡萄糖苷酶活性的筛选模型,通过酶促反应动力学绘制Lineweaver-Burk曲线,分析太行菊黄酮对α-葡萄糖苷酶作用的抑制类型。太行菊不同极性萃取物对α-葡萄糖苷酶呈现不同程度的抑制作用,而且抑制活性与黄酮的质量浓度之间呈现明显的剂量效应关系。其中乙酸乙酯相和正丁醇相中黄酮质量分数及α-葡萄糖苷酶抑制活性均显著高于其他萃取相,且高于阿卡波糖,而残余水相和石油醚相中黄酮对α-葡萄糖苷酶的抑制活性低于阿卡波糖。酶抑制活性乙酸乙酯（IC₅₀=1.01 mg/mL）<正丁醇（IC₅₀=1.25 mg/mL）<阿卡波糖（IC₅₀=1.47 mg/mL）<残余水相（IC₅₀=1.77 mg/mL）<石油醚（IC₅₀=2.12 mg/mL）。酶抑制动力学研究发现,乙酸乙酯相和正丁醇相中太行菊黄酮对α-葡萄糖苷酶是混合型抑制类型中竞争与非竞争关系;残余水相和石油醚相中太行菊黄酮对α-葡萄糖苷酶是竞争与反竞争的混合抑制类型。太行菊醇提物不同极性萃取相黄酮均具有一定的α-葡萄糖苷酶的抑制活性,在开发成辅助降血糖的保健食品或药品方面具有良好的潜力,有望将太行菊醇提物乙酸乙酯相和正丁醇相黄酮开发为新型α-葡萄糖苷酶抑制剂。     

米根霉乳酸脱氢酶的特性研究

乳酸脱氢酶(LDH)是米根霉发酵生产L-乳酸中催化丙酮酸转化成L-乳酸过程的关键酶。本研究从米根霉As3.819中初步分离出乳酸脱氢酶,并结合发酵条件对其酶学特性进行了研究。结果表明,该乳酸脱氢酶的最适反应pH为7.4,最适催化温度为30～50℃。Mg2+、Ca2+对该酶有激活作用,K+、Zn2+对该酶有抑制作用。以NADH和丙酮酸为底物的米氏常数分别为7.22×10-4mol/L和1.24×10-3mol/L。     

D-核糖发酵液电渗析脱盐及工艺优化

D--核糖发酵液中残存着大量的无机离子,如不去除会影响后续D--核糖的纯化。目前工业上常采用离子交换树脂脱盐,随着料液含盐量的增加,其脱盐成本也成比例增加,并产生大量的废水,既不经济也不环保。为明确电渗析脱盐是否可行、具体脱盐成本及最佳的脱盐工艺,以D-D-核糖发酵液的滤液为原料,采用均相阳离子交换膜CMX和阴离子交换膜AMX组成的小试设备LANRAN-40-2040,进行了电渗析法脱盐研究。结果表明,将脱盐率控制在54%,产品收率达到97.68%,但当进一步提高脱盐率时,会有一定量的D--核糖渗透到电渗析浓缩室中,且随着运行时间的增加,浓缩室的D--核糖质量浓度增加较快。采用电渗析联合离子交换树脂脱盐工艺比全树脂脱盐工艺产品收率提高了1.9%,节约一半的离子交换树脂及酸碱用量,并减少一半的废水排放,因此为D--核糖的工业生产提供较好的参考。     

贝莱斯芽孢杆菌角蛋白酶的克隆表达、纯化及酶学性质

为了获得更好的羽毛粉降解酶，本文从贝莱斯芽孢杆菌基因组中筛选到一个角蛋白酶基因（baker1），对其克隆并在大肠杆菌BL21（DE3）中异源表达，最后对重组角蛋白酶（BaKer1）进行了分离纯化、表征及应用研究。实验结果表明，BaKer1的最适反应pH为9.5，最适反应温度为55℃，在中温和碱性条件下具有较好的稳定性。1 mmol/L Ca²⁺对BaKer1有显著地促进作用，5 mmol/L Mn²⁺对BaKer1有轻微地抑制作用，其他金属离子对BaKer1影响不大。PMSF对BaKer1的活性具有显著抑制作用，表明BaKer1是典型的丝氨酸蛋白酶。分别以羽毛粉、偶氮酪蛋白、酪蛋白为底物测定了BaKer1的动力学参数，K_m值分别为5.06、1.09和1.39 mmol/L，k_cat/K_m值分别为1 058.24、2 536.54和3 733.79·(s·mmol/L)。BaKer1处理羽毛粉，能达到酸水解效果的 33.51%，说明它在羽毛粉降解过程中具有潜在的应用价值。     

辐照方式对南酸枣糕品质及货架期的影响

目的：选择最佳辐照方式及辐照剂量处理南酸枣糕。方法：分别采用0～10 kGy梯度剂量⁶⁰Co-γ射线和电子束辐照处理南酸枣糕,测定其微生物负载、蛋白质、脂肪、总糖、L（+）-抗坏血酸、有机酸、pH值及感官品质等指标以及在常温贮藏期间品质的变化。结果：⁶⁰Co-γ射线和电子束辐照对南酸枣糕杀菌作用明显,4 kGy辐照后样品中微生物限量指标菌（菌落总数、大肠菌群、霉菌）和致病菌限量指标菌（沙门氏菌、金黄色葡萄球菌）均未检出。不超过10 kGy的⁶⁰Co-γ射线和电子束辐照对南酸枣糕中蛋白质、脂肪、总糖、有机酸（以柠檬酸为主）、pH值及感官品质无显著影响,但辐照后南酸枣糕中L（+）-抗坏血酸含量与对照组相比显著降低（P＜0.05）,与辐照剂量呈负相关,且⁶⁰Co-γ射线辐照对其影响效应大于电子束辐照。设定4～10 kGy剂量⁶⁰Co-γ射线和电子束辐照处理南酸枣糕批量产品,在常温下贮藏0～360 d,产品的微生物限量指标菌（菌落总数、大肠菌群、霉菌）均<10 CFU/g。南酸枣糕的气味、滋味和组织状态无明显变化,但随着贮藏时间延长,其色泽会由于发生褐变而加深,贮藏270 d尚可接受。结论：⁶⁰Co-γ射线和电子束辐照是南酸枣糕杀菌贮存的有效方法,推荐南酸枣糕辐照杀菌的工艺剂量为4～10 kGy。     

具有极高热稳定性的β-（2，6）果聚糖蔗糖酶及其应用

挖掘具有高热稳定性的β-（2，6）果聚糖蔗糖酶（levansucrase，LS），并应用果聚糖蔗糖酶进行β-（2，6）果聚糖的高效合成。以分子动力学模拟的方式筛选出具有潜在高热稳定性的β-（2，6）果聚糖蔗糖酶。将目的酶在大肠杆菌Escherichia coli BL21（DE3）中进行重组及诱导表达，并通过镍柱亲和层析进行纯化。在不同温度下进行保温实验以验证LS的热稳定性，并通过条件优化获得产物的高转化率。Cedi-LS在65 ℃时显示出最高活性，远高于已鉴定的其他LS。同时，在45 ℃下保温72 h，重组酶Cedi-LS能够保留90%以上的相对活性；在55 ℃下保温60 h，其保留活性仍可达到初始活性的60%以上，表现出优异的热稳定性。在反应过程中，Cedi-LS可同时生成低聚糖、低相对分子质量的β-（2，6）果聚糖和高相对分子质量的β-（2，6）果聚糖。当温度从65 ℃降至35 ℃时，Cedi-LS倾向于产生HMW-levan，其相对分子质量可达8.4×10⁶。在pH 5.5和35 ℃的条件下，以质量分数30%的蔗糖为底物，加酶量定20 μg/mL，蔗糖转化为β-（2，6）果聚糖的平衡转化率为41.4%。作者鉴定了一种具有高热稳定性的LS，并且这种LS能够高效生产一系列具有不同相对分子质量的果聚糖。     

D-阿洛酮糖酶法生产的研究进展

稀有单糖D-阿洛酮糖是D-果糖的C-3差向异构体,主要通过D-塔格糖3-差向异构酶或D-阿洛酮糖-3-差向异构酶对D-果糖进行异构化获得。D-阿洛酮糖不仅可以作为食品添加剂和膳食补充剂,而且具有改善胰岛素抵抗、增强抗氧化和降血糖控制等多种生理功能。因此,D-阿洛酮糖作为传统高能量糖如蔗糖、果糖等的健康替代品,具有重要的研究价值。作者综述了D-阿洛酮糖的理化性质、来源、体内代谢、生理功能、应用和最新生产技术,讨论了D-阿洛酮糖生产中存在的问题及解决方案。针对低废物生成、低能耗、高糖产量等特点,提出了一种绿色、可循环利用的D-阿洛酮糖生产工艺技术。     

石榴籽油在D-半乳糖诱导的衰老小鼠体内的抗氧化作用

研究了石榴籽油(PGSO)在D-半乳糖诱导的衰老小鼠体内的抗氧化作用。将60只昆明小鼠随机分为6组:健康组、模型组、维生素E(V_E)阳性对照组以及PGSO低、中、高剂量组,持续给药45 d后,测定各组小鼠的体重,分析PGSO对小鼠肝、肾、脑与血清中抗氧化系统的作用;检测各组小鼠肝、肾中G6PD的活性和NADPH含量。结果表明:与模型组比较,PGSO可拮抗小鼠体重的减轻,降低肝、肾、脑及血清中MDA含量,增加GSH含量,提高T-AOC活性以及抗氧化酶SOD和GSH-Px的活性;且PGSO各剂量组小鼠肝和肾中G6PD的活性均增强,NADPH含量均增加。研究表明PGSO对D-半乳糖诱导的衰老小鼠体内的氧化应激具有明显的拮抗作用。     

植物乳杆菌发酵桑葚酵素工艺优化及质量评价

目的:解决新鲜桑葚难以保存的问题，将新鲜桑葚制成桑葚酵素。方法:以新鲜桑葚汁为原料，植物乳杆菌为生产菌种，总酚含量为评价指标，通过单因素试验结合响应面试验优化了植物乳杆菌桑葚酵素的发酵工艺，并对桑葚酵素的理化、微生物及感官质量指标进行评价。结果:优化后发酵工艺条件为发酵时间40 h、发酵温度32℃、接种量25%,所制得的植物乳杆菌桑葚酵素的总酚含量达(43.48±0.67)μg/mL,是未经发酵的桑葚汁的1.62倍，可溶性固体物含量为5.36%,pH值为4.08±0.01,微生物指标满足国家标准；桑葚酵素呈紫红、色泽均匀，具有浓郁的桑葚果香和发酵的香味、无异味，酸味柔和、风味好，有光泽、无杂质及沉淀。结论:经植物乳杆菌发酵制得桑葚酵素的过程有生物活性物质产生，有利于提高桑葚酵素质量。     

鸡源大肠埃希氏菌mcr-1基因携带及分析

目的 了解在农业农村部禁止使用多黏菌素作为动物促生长使用后四川部分地区鸡源大肠埃希氏菌（E.coli）mcr-1基因的携带情况,为制定进一步防控措施提供依据。方法 采集四川部分地区市场售卖点肉鸡直肠拭子,用含有多黏菌素（终浓度4 μg/mL）的EC肉汤增菌接种含多黏菌素（终浓度4 μg/mL）的麦康凯平板,挑取可疑菌落,采用PCR方法鉴定菌株并检测mcr-1基因;微量肉汤稀释法测定mcr-1基因阳性菌株对临床常见抗菌药物耐药情况。脉冲场凝胶电泳（PFGE）对mcr-1基因阳性菌株进行同源分析。耐药基因质粒结合实验验证mcr-1基因传播途径。结果 从70份肉鸡样本中的13份检出mcr-1基因阳性大肠埃希氏菌,检出率18.57%（13/70）,对实验的13种抗生素,除13株mcr-1阳性菌株对头孢西丁有12株敏感以外,对其他抗生素都表现出不同程度的耐药,其中四环素和甲氧苄啶/磺胺甲恶唑耐药率最高,达到了100%（13/13）;其次是氨苄西林和氯霉素,耐药率为84.62%（11/13）。PFGE显示13株mcr-1阳性大肠埃希氏菌分属13个不同的型别;质粒结合实验显示mcr-1基因能够通过质粒传播。结论 mcr-1基因在鸡大肠内大肠杆菌中检测率比较高,且鸡大肠中mcr-1阳性大肠埃希氏菌的耐药情况比较严重。     

Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll

The aim of this study was to design a thermal treatment(s) for pork luncheon roll, which would destroy Bacillus cereus and Clostridium perfringens vegetative cells and spores. B. cereus and C. perfringens vegetative and spore cocktails were used to inoculate luncheon meat. Samples were subjected to different temperatures and removal times. The decimal-reduction times (D-values) were calculated by linear regression analysis (D = -1/slope of a plot of log surviving cells versus time). The log(10) of the resulting D-values were plotted against their corresponding temperatures to calculate (-1/slope of the curve) the thermal resistance (z-values) of each cocktail. The D-values for vegetative cells ranged from 1 min (60 degrees C) to 33.2 min (50 degrees C) for B. cereus and from 0.9 min (65 degrees C) to 16.3 min (55 degrees C) for C. perfringens. The D-values for B. cereus spores ranged from 2.0 min (95 degrees C) to 32.1 min (85 degrees C) and from 2.2 min (100 degrees C) to 34.2 min (90 degrees C) for C. perfringens. The z-values were calculated to be 6.6 and 8.5 degrees C for B. cereus vegetative and spores, respectively, and 7.8 and 8.4 degrees C for C. perfringens vegetative cells and spores, respectively. The D-values of B. cereus and C. perfringens suggest that a mild cook of 70 degrees C for 12s and 1.3 min would achieve a 6 log reduction of B. cereus and C. perfringens vegetative cells, respectively. The equivalent reduction of B. cereus and C. perfringens spores would require the pork luncheon meat to be heated for 36 s at 105 and 110 degrees C, respectively. The results of this study provide the thermal inactivation data necessary to design a cooking protocol for pork luncheon roll that would inactivate B. cereus and C. perfringens vegetative cells and spores. The data may also be used in future risk assessment studies.     

Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes

The effects of different concentrations of Zataria multiflora Boiss. essential oil (EO: 0, 5, 15 and 30 μl 100 ml⁻¹) and nisin (N: 0, 0.25 and 0.5 μg ml⁻¹), temperatures (T: 25 and 8 °C), and storage times (up to 21 days) on growth of Salmonella typhimurium and Staphylococcus aureus in a commercial barley soup were evaluated in a factorial design study. The growth of S. typhimurium was significantly (P < 0.05) decreased by EO concentrations and their combinations with N concentrations at 8 °C. For S. aureus, the viable count was significantly (P < 0.05) inhibited by EO and N concentrations and their combinations, incubated at both storage temperatures. The mechanism of the antimicrobial action of EO, N, and their combinations against cell membranes of the tested organisms were also studied by measurement of the release of cell constituents and by the electronic microscopy observations of the cells. The significant increase of the cell constituents’ release of both organisms was observed as a result of treatments with EO and EO in combination with N. Electronic microscopy observations revealed that the cell membranes of S. typhimurium treated by EO and EO in combination with N were significantly damaged, while cells treated with only N looked similar to untreated cells. The electron micrographs of treated cells of S. aureus with EO, N, and their combination also showed important morphological damages and disrupted membranes.     

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 °C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 °C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 ± 0.2 log CFU g⁻¹ to less than detectable limit (<1.0 log CFU g⁻¹).     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.