紫外线诱变选育高产豆豉纤溶酶菌株及其产酶条件优化

该研究以分离自豆豉样品中产豆豉纤溶酶的枯草芽孢杆菌（Bacillus subtilis）DC-1为出发菌株,采用紫外线诱变选育高产豆豉纤溶酶且稳定遗传的菌株,并以豆豉纤溶酶活力为评价指标,通过单因素试验及正交试验对其发酵条件进行优化。结果表明,筛选出一株高产豆豉纤溶酶活力且稳定遗传的诱变菌株DC-V5,其最优发酵条件为：接种量3%、装液量70 m L/250 m L、发酵温度34℃、初始pH 6.5。在此优化发酵条件下,诱变菌株DC-V5产豆豉纤溶酶活力最高达到（451.26±11.09）IU/m L,是优化前出发菌株DC-1的1.74倍。     

根霉产纤溶酶液体发酵培养基的优化

通过单因素,均匀设计和正交试验,从碳氮比、无机氮源、胰蛋白胨、pH值方面优化了根霉液体发酵产生纤溶酶的培养基组成.试验结果表明,试验范围内菌株液体发酵产纤溶酶的适宜培养基组成为:麸皮水5.6%,豆粕水解液5.3%,二者体积比为2∶3,磷酸氢二铵0.4%,硝酸钠0.5%,pH值自然.优化培养基的纤溶酶酶活力为140.00U/mL.     

豆豉纤溶酶分离及其特性的研究

采用硫酸铵分级沉淀、SephadexG-100凝胶过滤层析,从豆豉粗酶液中提取纤溶酶。分离后的纤溶酶样品在SDS-PAGE电泳中有一个活性峰呈单一条带,分子量为35kDa,活性回收率为22.5%,纯化倍数为11.7;另一活性峰呈三条带,需进一步分离。特性研究表明:在pH7.0左右,温度40℃时,酶活力最高;该纤溶酶不耐热,其纤溶活性可被Mn2+、Mg2+以及Na2SO3激活,Ca2+以及乙醇、明胶对酶活力具有较强的抑制作用;NaCl浓度低于10%,酶活力能够基本保持稳定。     

β-葡萄糖苷酶冻干保护剂的研究

β-葡萄糖苷酶参与生物体的糖代谢,对维持生物体正常生理功能起着重要作用。采用单因素试验和正交试验,对β-葡萄糖苷酶真空冷冻干燥保护剂配方进行筛选和优化。得到冻干保护剂最佳配方为乳糖6%、蔗糖7%、海藻糖7%,甘露醇2%。在此条件下,经过真空冷冻干燥,β-葡萄糖苷酶的残余酶活可达80.25%。     

豆豉纤溶酶活性检测及其纯化研究

通过琼脂糖-纤维蛋白平板法测定收集到的豆豉中纤溶酶的活性,实验结果显示,采集于贵州南明蔡家关的豆豉样品DC-H8的纤溶酶活性最高,达到了285.759IU/mL.以该豆豉样品为研究对象,对其中的纤溶酶进行了纯化研究,实验确定的最佳纯化方案为:取一定体积的豆豉纤溶酶粗酶液,在低温(4℃)下,加入4％的活性炭粉末脱色35min;在分段盐析中,首先选择15％饱和度的硫酸铵除去杂蛋白,再用75％饱和度的硫酸铵使纤溶酶充分沉淀分离;最后用葡聚糖凝胶G-50层析分离纯化纤溶酶,收集具有较高纤溶活性的组分,得到了纯化的酶液.最终得到的豆豉纤溶酶酶液纯化倍数达到了27.74倍,活性回收率69.7％,比活力达到了13946.1IU/mg.     

豆豉营养评价及其粗纤溶酶活性检测

试验检测了收集到的不同豆豉的蛋白质、水分、总糖含量等,了解各种豆豉的营养价值;并通过琼脂糖-纤维蛋白平板法测定了各种豆豉纤溶酶的活性,分析了影响豆豉纤溶酶粗酶液活性的因素。结果显示,豆豉样品DC-H8的营养价值较高;试验确定豆豉纤溶酶粗酶液的提取方法:取一定量的豆豉,保持豆豉整粒,加5倍体积的生理盐水,于4℃浸提16h,离心、过滤后测定酶活,豆豉样品DC-H8的纤溶酶活性最高,为285.759 IU/mL。     

黑曲霉乳糖酶冻干保护剂的研究

针对乳糖酶在冻干过程中的失活问题,研究了几种保护剂对乳糖酶真空冷冻干燥过程中的保护作用,使用单因素筛选和正交试验方法,确定了乳糖酶保护剂最优配方为1%甘露醇、2%乳糖、7%可溶性淀粉,对乳糖酶真空冷冻干燥过程中具有显著保护作用,残余酶活为73.55%。     

正交旋转组合设计优化豆豉纤溶酶Subtilisin FS33的培养条件

从中国传统豆豉中分离筛选出一株高产纤溶酶菌株Bacillus subtilis DC33,并研究营养和环境因素条件对 DC33产酶的影响.在单因素水平试验基础上,选择对 DC33产纤溶酶影响较大的聚蛋白胨、半乳糖、硫酸镁、硫酸锂和吐温 80五因素,通过正交旋转组合设计得到DC33产酶活力与营养因素水平的回归方程,极值分析得DC33优化发酵培养基(g/L):聚蛋白胨22,半乳糖1.56,硫酸镁0.5,硫酸锂0.01,K2HPO4 2.0,NaH2PO4 1.0,CaCO3 2.0,吐温80 0.33 mL.在此条件下,DC33产纤溶酶达到780 U/mL,较优化前提高86.7%.     

豆豉纤溶酶载纳米脂质体的制备与表征

为提高豆豉纤溶酶口服生物利用度,建立了豆豉纤溶酶载纳米脂质体系统,并对其表征进行评价。采用硫酸铵梯度法制备的豆豉纤溶酶纳米脂质体的包封率为(52.74±4.24)%,粒径(72.04±31.2)nm,平均Zeta电位-44.2 mV,PdI 0.237。试验结果表明:豆豉纤溶酶纳米脂质体在体外稳定性好,在人工肠液中的释放符合一级动力学释放规律,豆豉纤溶酶在12 h后释放接近完全,无突释现象。豆豉纤溶酶及载酶纳米脂质体肠吸收液酶活性测定结果表明:豆豉纤溶酶及其载酶纳米脂质体在小肠均有吸收,吸收后仍具有纤溶活性。纳米脂质体可有效促进豆豉纤溶酶的吸收。     

产纤溶酶菌株的分离和鉴定及纤溶酶基因在酿酒酵母中的表达

目的:从豆豉中分离具有强纤溶活性的菌株,实现纤溶酶基因在酿酒酵母中的表达.方法:通过纤维蛋白平板法从豆豉中分离具有强纤溶活性的菌株,结合形态特征、生理生化特性和16S rDNA序列分析鉴定该菌株;通过PCR扩增豆豉纤溶酶基因,构建pYES2-DFE重组质粒,转化酿酒酵母H158感受态细胞,经β-半乳糖诱导表达后,用纤雏蛋白平板法分别检测发酵液上清和菌体破碎上清的纤溶酶活性.结果:鉴定该菌株为枯草芽孢杆菌(Bacillus subtilis);活性检测结果是酿酒酵母发酵液上清具有纤溶酶活性,而菌体破碎上清没有活性.结论:豆豉纤溶酶基因在酿酒酵母中实现了分泌表达.     

不同奶茶体系共存物对大豆分离蛋白性质的影响

为解决大豆分离蛋白在奶茶体系中稳定性较差的问题,本文探究了奶茶体系中共存物(酪蛋白、单甘脂、奶油、茶多酚、糖类、盐类、奶粉、红茶粉)对大豆分离蛋白乳化性(乳化活性和乳化稳定性)的影响。并通过乳状液ζ-电位和粒径的测量,探讨奶茶体系共存物与大豆分离蛋白相互作用及对其乳化性的影响规律。结果表明:在酪蛋白、茶多酚、乳糖添加量分别为0.2%、0.06%、0.1%时大豆分离蛋白乳化活性最小;在单甘脂、卡拉胶、氯化钠、脱脂奶粉添加量分别为0.15%、0.02%、0.02%、2%时大豆分离蛋白乳化稳定性最大;在乳糖添加量为0.1%时大豆分离蛋白乳化稳定性最小;在酪蛋白、茶多酚、乳糖添加量分别为0.2%、0.06%、0.1%时乳状液ζ-电位绝对值最小;在单甘脂、卡拉胶、碳酸镁添加量分别为0.15%、0.02%、0.01%时乳状液ζ-电位绝对值最大;在柠檬酸钠添加量为0.02%时,乳状液平均粒径最大,在碳酸镁、脱脂奶粉添加量分别为0.01%、2%时,乳状液平均粒径最小。奶茶体系中不同的共存物影响了乳状液的双电层结构,进而改变了大豆分离蛋白的乳化性。     

A Modelling study on skimmed milk lactose hydrolysis and β‐galactosidase stability using three reactor types

The hydrolysis of lactose in skimmed milk and β‐galactosidase inactivation studies were carried out in three different devices – bioreactor, sonicator and homogeniser – to evaluate the performance of such reactors that have different operational systems. The experiments were carried out using β‐galactosidase produced from Kluyveromyces marxianus lactis. At the optimum process conditions obtained from the experiments performed in bioreactor, sonicator and homogeniser, 89%, 90% and 54% of lactose were hydrolysed and the enzyme lost its activity by 14%, 13% and 24%, respectively, at the end of the processing time of 30 min. The commercial milk lactose content (1 g/L lactose) was reached at 60 min for bioreactor and sonicator. After evaluation of the data, it was found that the kinetics of hydrolysis and enzyme inactivation could be represented by a first‐order kinetic model and a single‐step non‐first‐order enzyme inactivation kinetic model, respectively, for all process conditions applied. The activation energy for hydrolysis reaction and the enzymatic inactivation energy values were also calculated.     

Commercial Mineral Enhanced Dairy By‐Products as Sodium Replacers,Antioxidants and Calcium Fortifiers in Sausages

Sausages are perceived as high in Na and with a too high Na:K ratio. Frankfurter type sausages are regarded as important contributors of sodium in the diet and thereby of health risks. Surplus products from the dairy industry are various mineral powders enriched in either potassium, calcium, or phosphate and include various amounts of lactose. Sausages were produced at 3 sodium levels (equivalent to 13, 15, and 17 g NaCl/kg sausage) using 4 different milk ingredients (a dried skimmed milk powder, a calcium enriched milk powder, a potassium enriched powder, and a lactose enriched powder). The sausages with added calcium and potassium enriched milk powders resulted in the hardest sausages when compared at the same sodium level. Milk mineral addition also produced whiter and less red sausages. No effect on rancidity after 6 wk at chill (4 °C) storage was observed by adding milk minerals, when compared with adding dried skimmed milk powder. A significant advantage of using these milk minerals in sausages is that the Na:K ratio can be reduced from an unhealthy (in this study 36) to a far healthier ratio ( ? 2) with limited or no taste changes. High additions of milk calcium (6 g/kg), where Ca‐phosphates prevail, added as milk mineral, had no influence on sensory bitterness or aftertaste as typically observed for CaCl₂ additions. Ca additions to sausages are presently presumed to be an advantage with respect to human nutrition.     

鼠李糖乳杆菌冻干保护剂的研究

以菌种冻干后存活率为指标,研究糖类、蛋白质类、多元醇类对鼠李糖乳杆菌的冻干保护作用。实验结果表明,在脱脂乳、甘油、海藻糖、蔗糖、葡萄糖、乳糖、糊精、抗坏血酸、谷氨酸钠、麦芽糖10种物质中保护效果较好的物质分别为脱脂乳、海藻糖、甘油。将它们确定为单因素进行响应面分析,响应面优化结果为:脱脂乳11.12%、海藻糖4.87、甘油2.96%。菌种冻干后的细胞成活率能达到96.48%±1.27%,与理论预测值接近。     

Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media

Viability, efficacy against Penicillium expansum on Golden Delicious apples, and storage stability of freeze-dried Candida sake strain CPA-1 were studied. The effect of several protective agents and rehydration media was investigated in the freeze drying of C. sake. Skimmed milk at 10% concentration was a good rehydration medium for all protectants tested. In general, good viability results were obtained when the same solution was used as a protectant and as a rehydration medium. The best survival was obtained when C. sake cells were protected with 10% lactose + 10% skimmed milk and rehydrated with skimmed milk (85% viability). The potential for biocontrol of the best freeze-dried treatments against P. expansum on apples was compared with that of fresh cells. Freeze-dried treatments at 1 x 10(7) CFU/ml reduced the incidence of decay by 45 to 66%. The best biocontrol effect was obtained with cells that had been freeze dried using 10% lactose + 10% skimmed milk as a protectant and 1% peptone as a rehydration medium, with a 66% reduction in rot incidence. However, in all treatments, the efficacy of freeze-dried cells was significantly lower than fresh cells. The stability of freeze-dried samples decreased during storage and was influenced by storage temperature. In the best treatment, storage of C. sake cells for 60 days at 4 degrees C resulte in final concentrations of 2.5 x 10(8) CFU/ml, which was a 10-fold reduction in relation to the initial starting concentration of cells prior to freeze drying.     

中草药复配乳蛋白ACE抑制肽的试验研究

为了将具有降血压功效的药食同源中草药与具有降血压活性的牛奶蛋白酶解液复配,以完成一款具有辅助降压功效的功能性牛奶的实验研究提供理论依据和技术参数。试验以ACE抑制率为指标,优化胰蛋白酶酶解脱脂乳的最佳工艺,并从15味中草药中筛选出具有较高ACE抑制活性的提取液,再将优化所得酶解液和筛选出的中草药提取液进行复配,获得高ACE抑制率的活性功能乳组方。结果表明:胰蛋白酶酶解脱脂乳的最佳酶解工艺为底物质量浓度7 g/100 mL、酶解时间6 h、加酶量5%、酶解温度31℃、pH6.9,脱脂乳酶解液的ACE抑制率为58.51%;筛选得出山楂、决明子的ACE抑制率较高,分别为50.88%、45.34%;且当山楂提取液和决明子提取液混合时ACE抑制活性提高,两者为2:1时,ACE抑制率较高为52.52%;酶解液和中草药提取液(山楂提取液:决明子提取液为2:1)按1:1进行复配,ACE抑制率最高为59.09%,对研究具有辅助调节血压效应的保健功能性乳制品具有理论和实践探讨意义。     

Induction of apoptosis in HL-60 cells by skimmed milk digested with a proteolytic enzyme from the yeast Saccharomyces cerevisiae

Bovine skimmed milk digested with cell-free extract of the yeast Saccharomyces cerevisiae was found to exhibit proliferation inhibition activity towards human leukemia (HL-60) cells. The optimum pH for digestion of skimmed milk and production of the proliferation inhibition factor was pH 4.8. Nondigested skimmed milk exhibited little suppressive effect on the proliferation of HL-60 cells. An active enzyme involved in the production of cell proliferation inhibitory materials from skimmed milk was purified from the cell-free extract of S. cerevisiae by a series of column chromatographies: DEAE-Sephacel, D-tryptophan methyl ester-Sepharose 4B, Hiload Superdex G-200 and HPLC Mono Q. The homogeneous purified enzyme and exhibited a molecular mass of 33 kDa in sodium dodeceyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was identified as protease B by N-terminal amino acid sequence analysis. Bovine skimmed milk digested with purified protease B was found to inhibit proliferation activity of HL-60 cells most strongly when digestion was conducted at pH 4.8. The cell proliferation inhibition activity induced by digested skimmed milk was shown to be due to the induction of apoptosis, demonstrated by the formation of apoptotic bodies and fragmentation of DNA in treated cells. The proliferation inhibition factors produced were recovered in the soluble fraction of 92% ethanol, suggesting that the factors were hydrophilic low molecular mass substances derived from skimmed milk.     

乳酸菌菌种的简便分离和培养

嗜热链球菌和保加利亚杆菌是酸乳制品的常用菌种,因其有共生关系,较难分离。用特殊的培养基如M17和MRS酸化培养基可以将此共生苗分离。本文介绍的培养基同样能起到分离的作用,且成分简单,取材方便。     

Kinetic and thermodynamic study of thermal inactivation of the antimicrobial peptide P34 in milk

The knowledge on thermal inactivation of biopreservatives in a food matrix is essential to allow their proper utilisation in food industry, enabling the reduction of heating times and optimisation of heating temperatures. In this work, thermal inactivation of the antimicrobial peptide P34 in skimmed and fat milk was kinetically investigated within the temperature range of 90–120 °C. The inactivation kinetic follows a first-order reaction with k-values between 0.071 and 0.007 min⁻¹ in skimmed milk, and 0.1346 and 0.0119 min⁻¹ in fat milk. At high temperatures, peptide P34 was less resistant in fat milk, with a significant decrease in residual activity as compared with skimmed milk. At temperatures below 110 °C, the fat globules seem to have protective effect to the peptide P34. Results suggest that peptide P34 is heat stable in milk with activation energy of 90 kJ mol⁻¹ in skimmed milk and 136 kJ mol⁻¹ in fat milk.     

Effects of inulin and sugar levels on the viability of yogurt and probiotic bacteria and the physical and sensory characteristics in probiotic ice-cream

Ice-cream containing probiotic bacteria was produced by mixing fortified milk fermented with probiotic strains with ice-cream mixes with different sugar concentrations (15, 18, 21% (w/w)). Cultures were grown (37 °C, 12 h) in UHT skimmed milk supplemented with or without inulin addition (1% and 2%). The fermented milk was added to the ice-cream mix to a level of 10% w/w.