血红蛋白降解菌的分离筛选与鉴定

为了从环境中筛选高效降解血红蛋白的菌株,采用鲜血琼脂平板,以产生溶血圈比值大小为判定标准,从南京某屠宰场下水道中分离到一株具有较强降解血红蛋白能力的菌株NJM4.经形态学观察、生理生化测定和16S rRNA基因分析(GenBank登陆号为EU234500),初步认为该菌株为短小芽孢杆菌(Bacillus pumilus).该菌株在血红蛋白溶液中可将其逐渐降解为小分子的多肽.在血红蛋白发酵培养基中于37℃、120 r/min培养24 h,降解率达53.64 %.     

降解大豆胰蛋白酶抑制剂的短小芽孢杆菌菌株及其胞外蛋白的鉴定

本研究以大豆胰蛋白酶抑制剂为唯一碳源,从豆类内部筛选获得3株具有降解大豆胰蛋白酶抑制剂的细菌。通过对菌株的16Sr DNA基因的分析,3株细菌分别被鉴定为枯草芽孢杆菌LZ013-1、短小芽孢杆菌LZ013-2和类芽孢杆菌LZ013-3。进一步研究发现LZ013-2菌株的上清液具有高效降解大豆胰蛋白酶抑制剂的活性,2h可将胰蛋白酶抑制剂抑制率降低73.60%。将LZ013-1和LZ013-2菌株应用于豆粕发酵中,两株芽孢杆菌均能高效降解胰蛋白酶抑制剂。原始豆粕的胰蛋白酶抑制剂活性为22.26 mg/g,经过LZ013-1和LZ013-2菌株的液态发酵后分别降低为0.50 mg/g和0.63 mg/g,经过固态发酵后分别降低为1.06 mg/g和1.03 mg/g。通过硫酸铵分级沉淀和凝胶柱层析,分离LZ013-2菌株发酵上清液中具有降解活性的蛋白质,并采用质谱鉴定分离得到的蛋白质,筛选并鉴定出2种活性蛋白,分别为肽酶S8(Uniprot ID:A0A2T0DB16)和肽酶M84(Uniprot ID:A8FIH7)。     

屠宰畜禽血液血红蛋白(HB)及其脱色技术研究进展

畜禽血液含有丰富的营养物质,尤其是蛋白质,其含量可与瘦肉相媲美,它是一种优质的动物蛋白资源.本文就血液的营养价值着手,介绍了血红蛋白的分子结构及含量,概括了几种脱色方法的特点、应用和研究进展,以及酶法水解脱色的研究状况.     

Degradation of polysaccharides and non-digestible oligosaccharides by Bacillus subtilis and Bacillus pumilus isolated from Soumbala, a fermented African locust bean (Parkia biglobosa) food Condiment

In order to select starter cultures for controlled fermentation of African locust beans, Bacillus subtilis and B. pumilus from Soumbala were investigated for their ability to degrade arabinogalactan, stachyose, raffinose and sucrose—the main polysaccharides and oligosaccharides in African locust bean. The methods used were liquefaction of gels of galactomannan for screening, pH measurement and High Performance Anion Exhange Chromatography – Pulsed Amperometric Detection (HPAEC–PAD) for detailed degradation study of the carbohydrates. For B. subtilis isolates, liquefaction of galactomannan gels was observed and HPAEC–PAD showed that arabinogalactan and stachyose were partially degraded while raffinose was fully degraded after 48 h of fermentation. Melibiose, fructose and traces of galactose were detected as residual sugars. For B. pumilus isolates much weaker degradation of the carbohydrates was observed. Two isolates of B. subtilis showed strong ability to degrade the oligosaccharides and they are seen as promising starter cultures for fermentation of African locust bean.     

富硒短小芽孢杆菌D1-019的筛选与鉴定

将无机硒耐受性筛选和“红硒法”筛选相结合,经过4 轮筛选,从前期分离的119 株来源于鸭食的菌株中,筛选到1 株富硒菌株D1-019。基于菌落形态观察、革兰氏染色、16S rDNA序列分析和系统发育树分析,D1-019菌株被鉴定为益生菌短小芽孢杆菌。采用正交试验设计方法,获得的最佳富硒条件为培养基初始pH 5,30 ℃培养48 h,培养6 h时加入质量浓度为20 μg/mL的亚硒酸钠。在该富硒条件下,短小芽孢杆菌D1-019对培养基中亚硒酸钠的转化率为100%,菌体有机硒含量为（1 327±113）mg/kg,优于已报道富硒微生物。结果表明,短小芽孢杆菌D1-019有潜力开发成为一个可应用于食品领域的新富硒益生菌源。     

提高蜡状芽孢杆菌(B.cereus)降解畜禽血红蛋白能力研究

本文选用蜡状芽孢杆菌(B.cereus)作为出发菌株,对其进行连续驯化诱导和定向诱变处理,以提高畜禽血红蛋白的降解能力.结果表明:蜡状芽孢杆菌连续驯化诱导后,其产蛋白酶活力、可溶性蛋白质、游离氨基酸含量及Hb降解率分别从出发菌株的483.81 U/ml、2.69%、1.44mg/100ml、40.25%提高到631.22U/ml、3.04%、19.6mg/100ml、50.84%.采用HNO2(0.001%,120s)和UV(15W,25cm,60s)进行连续定向诱变处理五次,所获得的第五代诱变菌株,在Hb发酵培养液中,其产蛋白酶活力、可溶性蛋白质、游离氨基酸含量及Hb降解率分别为1233.15mg/100ml、3.52%、24.lmg/100ml、51.83%,与出发菌株相比,降解畜禽血红蛋白的各项指标都有较明显的提高.     

短小芽孢杆菌漆酶基因的克隆表达及重组漆酶降解黄曲霉毒素M1研究

为明确短小芽孢杆菌CotA漆酶的酶学性质,探究其在黄曲霉毒素M1降解中的应用潜力。经分析,短小芽孢杆菌E-1-1-1中cotA基因全长1 486 bp,编码495 个氨基酸,无信号肽序列,分子质量约为58 kDa。该重组漆酶最适反应温度为40 ℃,最适pH 4.0;Km为3.01 mmol/L,Vmax为0.795 mmol/（L·min）;金属离子和化学试剂会对酶活力造成不同程度影响,K＋、Na＋、乙醇和十二烷基硫酸钠对该酶有明显的抑制作用,抑制率分别为31.0%、13.6%、64.5%和100%,而Mn2＋和Zn2＋则表现出明显的促进作用,酶活力分别提高了33.0%和23.9%;该酶与牛奶中的黄曲霉毒素M1反应72 h后,黄曲霉毒素M1降解率为54.3%,毒性降低9.1%。结果表明,短小芽孢杆菌CotA漆酶作为黄曲霉毒素M1降解酶具有巨大的应用潜力。     

Genotyping of starter cultures of Bacillus subtilis and Bacillus pumilus for fermentation of African locust bean (Parkia biglobosa) to produce Soumbala

Bacillus spp. are the predominant microorganisms in fermented African locust bean called Soumbala in Burkina Faso. Ten strains selected as potential starter cultures were characterised by PCR amplification of the16S-23S rDNA intergenic transcribed spacer (ITS-PCR), restriction fragment length polymorphism of the ITS-PCR (ITS-PCR RFLP), pulsed field gel electrophoresis (PFGE) and sequencing of the 968-1401 region of the 16S rDNA. In previous studies, the isolates were identified by phenotyping as Bacillus subtilis and Bacillus pumilus. The phenotyping was repeated as a reference in the present study.The ITS-PCR and ITS-PCR RLFP allowed a typing at species level. The PFGE was more discriminative and allowed a typing at strain level. Full agreement with the phenotyping was observed in all cases. The sequencing of the 16S rDNA allowed the identification at species level with an identity from 97% to 100% comparing the sequences to those from the GenBank databases. The desired cultures of B. subtilis and B. pumilus from African locust bean fermentation were distinguished by ITS-PCR and ITS-PCR RLFP from Bacillus cereus and Bacillus sphaericus which sometimes occur in the beginning of the fermentation.     

生物酶法酶解猪血红蛋白 (Hb)的研究

以猪血红蛋白的水解率、氮溶解指数、总氮收率及血红素含量为指标,选择温度、pH、酶底物浓度比([E]/[S])、底物浓度([S])为试验因素,选用L9(3+)正交表对其水解条件进行探讨.结果表明其最佳水解条件为最初pH7.5,温度为55℃,酶底物浓度比(E/S)8000U/g,底物浓度6%,水解时间8h.     

Bacillus pumilus CN8菌株降解猪血Hb酶的分离纯化及酶学性质研究

Bacillus pumilus CN8菌株的发酵液,经离心分离得到粗酶液,再经硫酸铵盐析、透析,DEAE-Cellulose-52离子交换层析等步骤获得电泳纯的中性蛋白酶。得到比活达686.66U/mg的酶蛋白,纯化倍数为13.2,回收率为35.0%。SDS-PAGE测得其分子量大约为97000Da。该酶的最适作用pH为7.4,最适反应温度为42℃,在pH7.0~8.5范围内较稳定,在30~45℃比较稳定。经酶学性质测定,K+、Mg2+对酶活力具有保护作用,甘油对酶活具有抑制作用。