B.cereus MBL13-U产牛骨胶原蛋白酶工艺优化
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摘要
为了获得能够高效降解牛骨胶原蛋白的新型胶原蛋白酶,以蜡样芽孢杆菌(Bacillus cereus MBL13-U)为出发菌种,以胶原蛋白酶活力为检测指标,对影响发酵的4个因素(菌种接种量、发酵温度、p H、发酵时间)进行单因素试验。通过四元二次正交旋转组合试验,确定了B.cereus MBL13-U菌株发酵制备胶原蛋白酶的最佳工艺。当菌种接种量4%(体积分数)、发酵温度35℃、初始p H6.4、发酵时间46 h时,胶原蛋白酶活力达(92.31±1.13)U/m L。将其作用于牛骨胶原蛋白,通过扫描电子显微镜对酶解过程中的牛骨胶原蛋白进行结构分析。分析结果表明:酶解破坏了牛骨胶原蛋白原本完整的表面结构,使其所含的Ⅰ型胶原蛋白更多地暴露在表面,加快水解。
To obtain the new collagenase which could efficiently degrade bovine bone collagen,the Bacillus cereus MBL13-U was used as the starting strain,and the collagenase activity was used as an indicator.The single factor experiment was performed on four factors(inoculation amount of strain,fermentation temperature,initial p H,and fermentation time),respectively.Through four-factor quadric orthogonal rotation experiment,the optimum process of fermentation for production of bovine bone collagenase by B.cereus MBL13-U was acquired.When inoculation amount of strain was 4%(volume fraction),fermentation temperature was 35 ℃,initial p H was 6.4 and fermentation time was 46 h,the activity of collagenase was(92.31 ± 1.13) U/mL.Then bovine bone collagen was disposed by the collagenase.The structural properties of bovine bone collagen in enzymolysis were characterized by scanning electron microscope.The results show that the intact surface structure of bovine bone collagen is destroyed by the enzymolysis,so the type I collagen is more exposed to the surface and the hydrolysis is faster.
To obtain the new collagenase which could efficiently degrade bovine bone collagen,the Bacillus cereus MBL13-U was used as the starting strain,and the collagenase activity was used as an indicator.The single factor experiment was performed on four factors(inoculation amount of strain,fermentation temperature,initial p H,and fermentation time),respectively.Through four-factor quadric orthogonal rotation experiment,the optimum process of fermentation for production of bovine bone collagenase by B.cereus MBL13-U was acquired.When inoculation amount of strain was 4%(volume fraction),fermentation temperature was 35 ℃,initial p H was 6.4 and fermentation time was 46 h,the activity of collagenase was(92.31 ± 1.13) U/mL.Then bovine bone collagen was disposed by the collagenase.The structural properties of bovine bone collagen in enzymolysis were characterized by scanning electron microscope.The results show that the intact surface structure of bovine bone collagen is destroyed by the enzymolysis,so the type I collagen is more exposed to the surface and the hydrolysis is faster.
引文
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[3]孙蓓,王龙刚.畜禽骨的综合利用现状及发展前景[J].中国调味品,2011,36(4):1-4.
[4]刁静静,孔保华,陈洪生.骨蛋白水解物的功能特性及抗氧化性的研究进展[J].肉类研究,2007(6):26-29.
[5]GELSE K,PSCHL E,AIGNER T.Collagens-structure,function,and biosynthesis[J].Advanced drug delivery reviews,2003,55(12):1531-1546.
[6]HOU H,LI B,ZHANG Z,et al.Moisture absorption and retention properties,and activity in alleviating skin photodamage of collagen polypeptide from marine fish skin[J].Food chemistry,2012,135(3):1432-1439.
[7]ZHANG Y,MA L,CAI L,et al.Effect of combined ultrasonic and alkali pretreatment on enzymatic preparation of angiotensin converting enzyme(ACE)inhibitory peptides from native collagenous materials[J].Ultrasonics sonochemistry,2017,36:88-94.
[8]齐景凯,张玉芬.牛骨酶解工艺及其酶解液中游离氨基酸的分析研究[J].中国食品添加剂,2013(1):109-115.
[9]蔡丽华.牛骨酶解制备血管紧张素转换酶抑制肽的研究[D].武汉:华中农业大学,2010.
[10]李思思,宋诗清,肖作兵,等.脂肪酶预处理对牛骨蛋白酶解液的影响[J].中国食品学报,2016,16(4):130-136.
[11]李陈.一种新的短小芽孢杆菌胶原蛋白酶的分离、纯化及酶学性质研究[D].雅安:四川农业大学,2008.
[12]杨南南,李会,吴燕,等.Gibberella intermedia WX12产特异性糖苷酶的发酵工艺优化[J].食品与生物技术学报,2016,35(10):1028-1034.
[13]应晨,阮川芬,汤逸飞,等.内生产酶溶杆菌R-2-1发酵工艺优化[J].浙江农业学报,2015,27(2):220-228.
[14]孟娜,魏胜华,郑长龙.黑曲霉发酵产橙皮苷酶的工艺优化[J].生物技术,2011,21(2):77-80.
[15]任佳明,李付丽,吴鑫颖,等.紫色红曲霉FBKL3.0018液态发酵产酯化酶的研究[J].中国酿造,2016,35(7):69-73.