枯草芽孢杆菌中性蛋白酶的研究进展

中性蛋白酶因作用条件温和广泛应用于食品、医药以及饲料等领域。枯草芽孢杆菌为肠道益生菌,且具有较强的蛋白酶活性 而受到极大关注。该文对枯草芽孢杆菌中性蛋白酶的特性与应用、提高枯草芽孢杆菌中性蛋白酶活力的途径以及枯草芽孢杆菌中性 蛋白酶的分离纯化方法进行了综述,并对存在的问题进行了分析与展望,旨在为枯草芽孢杆菌产中性蛋白酶的深入研究提供参考。     

Silage review: Recent advances and future uses of silage additives

Additives have been available for enhancing silage preservation for decades. This review covers research studies published since 2000 that have investigated the efficacy of silage additives. The review has been divided into 6 categories of additives: homofermentative lactic acid bacteria (LAB), obligate heterofermentative LAB, combination inoculants containing obligate heterofermentative LAB plus homofermentative LAB, other inoculants, chemicals, and enzymes. The homofermentative LAB rapidly decrease pH and increase lactic acid relative to other fermentation products, although a meta-analysis indicated no reduction in pH in corn, sorghum, and sugarcane silages relative to untreated silages. These additives resulted in higher milk production according to the meta-analysis by mechanisms that are still unclear. Lactobacillus buchneri is the dominant species used in obligate heterofermentative LAB silage additives. It slowly converts lactic acid to acetic acid and 1,2-propanediol during silo storage, improving aerobic stability while having no effect on animal productivity. Current research is focused on finding other species in the Lb. buchneri group capable of producing more rapid improvements in aerobic stability. Combination inoculants aim to provide the aerobic stability benefits of Lb. buchneri with the silage fermentation efficiency and animal productivity benefits of homofermentative LAB. Research indicates that these products are improving aerobic stability, but feeding studies are not yet sufficient to make conclusions about effects on animal performance. Novel non-LAB species have been studied as potential silage inoculants. Streptococcus bovis is a potential starter species within a homofermentative LAB inoculant. Propionibacterium and Bacillus species offer improved aerobic stability in some cases. Some yeast research has focused on inhibiting molds and other detrimental silage microorganisms, whereas other yeast research suggests that it may be possible to apply a direct-fed microbial strain at ensiling, have it survive ensiling, and multiply during feed out. Chemical additives traditionally have fallen in 2 groups. Formic acid causes direct acidification, suppressing clostridia and other undesired bacteria and improving protein preservation during ensiling. On the other hand, sorbic, benzoic, propionic, and acetic acids improve silage aerobic stability at feed out through direct inhibition of yeasts and molds. Current research has focused on various combinations of these chemicals to improve both aerobic stability and animal productivity. Enzyme additives have been added to forage primarily to breakdown plant cell walls at ensiling to improve silage fermentation by providing sugars for the LAB and to enhance the nutritive value of silage by increasing the digestibility of cell walls. Cellulase or hemicellulase mixtures have been more successful at the former than the latter. A new approach focused on Lb. buchneri producing ferulic acid esterase has also had mixed success in improving the efficiency of silage digestion. Another new enzyme approach is the application of proteases to corn silage to improve starch digestibility, but more research is needed to determine the feasibility. Future silage additives are expected to directly inhibit clostridia and other detrimental microorganisms, mitigate high mycotoxin levels on harvested forages during ensiling, enhance aerobic stability, improve cell wall digestibility, increase the efficiency of utilization of silage nitrogen by cattle, and increase the availability of starch to cattle.     

生物基高效脱墨剂的制备及应用研究

采用廉价及可再生的脂松香、椰子油酸、棕榈酸为原料,制备生物基脱墨剂,并应用于废纸脱墨。采用FT-IR对原料进行了表征,以正交试验法优化了生物基脱墨剂的制备工艺,探讨了生物基脱墨剂的最佳用量及脱墨性能,并与传统脱墨剂做脱墨性能对比。FT-IR结果表明,生物质原料中既有饱和的脂肪酸也有不饱和的脂肪酸。生物基脱墨剂优化的制备条件为时间30min、温度90℃、m（脂松香）：m（椰子油酸）：m（棕榈酸）为2.5：3.5：4.0、NaOH溶液的质量分数40%。生物基脱墨剂是一种高效脱墨剂,最适加入量为0.20%;在加入量为0.20%,浮选温度为40℃,浮选时纸浆质量分数1.0%,浮选pH值为9,浮选时间为20min时,脱墨后废纸浆白度可达58.67%（ISO）,残余油墨值达274.42mm²/m²,油墨去除率达57.81%;实际应用结果表明生物基脱墨剂的脱墨性能优于传统的S型和天小脱墨剂。     

黑曲霉N5-5单宁酶的纯化及酶学性质测定

采用中空纤维膜超滤和葡聚糖凝胶层析相结合的方法对黑曲霉N5-5单宁酶进行纯化,然后对纯酶性质进行测定。结果显示,黑曲霉N5-5单宁酶用该方法纯化后,可纯化近20倍,酶活力可回收23.30%。对纯酶作十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,可知黑曲霉N5-5单宁酶为分子质量64.2 k D的单肽链蛋白。纯酶的酶促反应最适温度为45℃,且在25~45℃范围内热稳定性良好;酶促反应最适p H值为5.0,且在p H 5.0~5.5范围内酸碱稳定性良好。另外,反应动力学测定结果表明,该酶对底物没食子酸丙酯的米氏常数K_m为0.916 mmol/L,最大反应速率vmax为0.877 mmol/(L·min)。     

高糖胁迫下槲皮素对酿酒酵母胞内损伤的保护作用与机制

以酿酒酵母S288c为模型,分析高糖胁迫下槲皮素对其胞内损伤的保护作用及机制。结果表明:与对照相比,高糖胁迫不影响酵母胞内活性氧(reactive oxygen species,ROS)水平,但显著降低了胞内酶比活力(P0.05);槲皮素处理后,与对照组和高糖组相比,酿酒酵母胞内ROS水平、超氧化物歧化酶和过氧化氢酶活力均显著下降(P0.05),而过氧化物酶(peroxidase,POD)比活力极显著升高(P0.01),说明POD比活力对高糖耐受性反应更为灵敏,可作为衡量高糖胁迫应激机制的重要生理指标,槲皮素可通过调节胞内POD比活力来提高机体的抗氧化能力。另外,实时荧光定量聚合酶链式反应结果表明高质量浓度葡萄糖显著抑制了酵母中GPD2和SUC2的表达水平(P0.05),并极显著提高了HXT1的表达水平(P0.01),而对GUT1的表达影响不显著;槲皮素处理后,高糖胁迫下酵母中GPD2、SUC2和HXT1的表达水平显著提高(P0.05),而GUT1无显著变化,说明槲皮素可能通过高渗透甘油途径、菊糖水解途径和己糖转运途径等来促进葡萄糖的分解代谢,从而达到保护机体细胞免受伤害的作用。结果表明槲皮素对高糖诱导的酿酒酵母胞内损伤具有保护作用,其作用机制可能与自身的抗氧化作用以及利用调节机体内高渗透甘油途径与糖的分解和转运途径存在一定的关联性。     

壳聚糖没食子酸衍生物酶法催化条件优化及抗氧化活性和细胞毒性

利用漆酶、辣根过氧化物酶在均相和非均相反应体系中催化壳聚糖与没食子酸反应,以增强壳聚糖的抗氧化性。研究酶种类、反应体系p H值、反应温度、反应时间、酶用量和底物的质量比等因素对产物接枝率的影响。利用单因素试验和正交试验确定的最适反应条件为反应p H 4.5、反应温度25℃、反应时间5 h、没食子酸与壳聚糖的质量比3∶1、漆酶用量4 U,此条件下衍生物的接枝率为65.2%。对衍生物分别进行体外抗氧化活性和细胞毒性检测,结果表明,在相同添加量的情况下,衍生物的抗氧化性显著高于未改性的壳聚糖,而且产物无细胞毒性。     

温度对银耳胞外酶活力及营养品质特性的影响

研究不同温度对银耳栽培过程中生长发育各阶段的7种胞外酶活力和银耳品质特性的影响;采用相关性分析和通径分析2种数据分析方法,探讨各胞外酶平均酶活力与银耳品质的关系,揭示各胞外酶对银耳品质的相对重要性,为银耳工厂化高质、高产栽培提供可靠理论依据。结果表明,在适宜的温度范围内,同种胞外酶活力变化规律相似,不同胞外酶活力变化具有明显的阶段性;对比研究发现,在栽培温度(23.0±0.5)℃时,成熟银耳的干生物学效率、多糖含量和蛋白质含量在7个温度组中最高,分别为35.67%、21.51%和8.53%。相关性分析表明,所测胞外酶的平均酶活力(除羧甲基纤维素酶外)与干生物学效率和多糖含量呈正相关性,蛋白质含量与7种胞外酶平均酶活力均呈正相关性;进一步通径分析发现,蛋白酶是影响银耳营养品质的关键胞外酶。综上所述,在栽培温度(23.0±0.5)℃时,基质中主要胞外酶具有高活力,银耳的营养品质最高,可将此温度作为银耳工厂化栽培的指导温度。     

响应面试验优化东北黄豆酱制曲工艺

以黄豆和面粉为主要原料,采用混合菌种制曲,以蛋白酶、淀粉酶的活性为评价指标,利用Box-Behnken响应面试验考察不同工艺条件对豆酱曲酶活的影响。试验结果表明,采用添加熟面粉的方式,湿豆与面粉质量比10∶4、接种量0.07%、制曲时间48 h,在此最佳制曲工艺条件下,蛋白酶酶活为1 049.83 U/g,淀粉酶酶活为781.29 U/g。     

酸浓度和pH值对浓香型大曲酯化酶催化活力的影响

研究了己酸、乳酸、丁酸、乙酸的浓度以及pH值对浓香型大曲酯化酶催化合成四种酯（己酸乙酯、乳酸乙酯、丁酸乙酯、乙酸乙酯）的影响。结果表明,大曲酯化酶催化合成四种酯的最适酸质量浓度分别为己酸8 g/L,乳酸1 g/L,丁酸9 g/L,乙酸12 g/L;在同一酸浓度条件下,大曲酯化酶催化合成己酸乙酯、丁酸乙酯、乙酸乙酯最适pH值均为4.5,乳酸乙酯最适pH值为6.0;大曲酯化酶催化相同物质的量浓度混合酸的试验结果表明,己酸乙酯的合成以酯化酶催化反应为主,而乳酸乙酯和丁酸乙酯的合成以化学反应为主。     

产纤溶酶菌株L21的鉴定与发酵条件研究

利用纤维蛋白平板法从海参体内肠道中筛选到一株产纤溶酶活力较高的菌株L21,通过16S rDNA序列测定比对分析,初步鉴定菌株L21为交替假单胞菌（Pseudoalteromonas sp.）。采用单因素试验、Plackett-Burman试验及响应面法对菌株L21的发酵条件进行优化,结果表明,菌株L21最佳发酵工艺条件为NaCl 1 g/L,CaCl2 1.03 g/L,MgSO4 0.8 g/L,初始pH 7.40。在此工艺条件下,菌株L21纤溶酶活力达到399.86 U/mL,较优化前（86.09 U/mL）提高了4.64倍。体外溶血实验结果表明该菌株具有作为安全性溶栓剂的潜力。