Preliminary Research on Preparing Procedure of Milk-clotting Enzyme from Mucor pusillus
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摘要: 以食用菌菌糠和豆渣1∶1(w/w)混合物作为微小毛霉固体发酵培养基,探讨微小毛霉凝乳酶粉末制剂的制备工艺。制备过程包括:三角瓶固体发酵、加水提取、Al2(SO4)3絮凝、微滤除菌、超滤浓缩、室温醇沉、40℃真空干燥等7个步骤。结果显示:凝乳酶在水溶液中的热变性为52℃左右,而在70%含水酒精中的变性温度则降至35℃左右。酶液酒精浓度达到70%时对凝乳酶醇沉效果最好,其酶活回收率可达77.24%。本试验最终得到纯度为65.8%的粗酶粉末制剂,酶分离过程的回收率为69.1%,单位质量的培养基转化成凝乳酶粗酶的效率为4 975 SUg-1。研究还表明,与常规制备工艺所采用的低温醇析和真空冷冻干燥的苛刻条件相比,在室温醇析和40℃真空干燥等相对简陋条件下制备微小毛霉凝乳酶,仍可获得较高的酶活回收率。Abstract: In this research, dried soymilk residue and spent culture substrate of Flammlina velutip were mixed at the ratio of 1∶1(w/w) as the culture medium for Mucor pusillus to generate milk-clotting enzyme (rennet). The process of preparing crude milk-clotting enzyme powder was investigated. The procedure was divided in 7 steps including solid-state fermentation in Erlemeyer flask, water extraction, flocculation with Al2(SO4)3, microfiltration to remove fungi, ultrafiltration to concentrate enzyme, ethanol precipitation under room temperature, vacuum drying at 40℃. Results showed that the temperature for inactivation of rennet was around 52℃ in water solution, while lowered it to 35℃ in ethanol solution. The ethanol concentration for the best effect in rennet precipitation was 70%, which recovered rennet activity of 77.24%. The final product obtained was in crude enzyme powder with its purity in rennet for 65.8% and the recovering efficiency of filtrated enzyme for 69.1%. The biotransformation of rennet in crude enzyme was 4 975 SU per gram of culture medium. Comparing conventional procedure in low-temperature precipitation and freeze-drying, results indicated that the milk-clotting enzyme of Mucor pusillus could be recovered in certain extent of rennet activity even with room-temperature precipitation and 40℃ vacuum drying.
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Key words:
- soymilk residue /
- Mucor pusillus /
- milk-clotting enzyme /
- enzymatic preparation
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[1] [2] 司方,王明力.大豆豆渣的综合利用研究进展[J].贵州农业科学,2008,36(6):154-156. [3] [4] 王加启,于建国.GB/T5009.5-2003饲料分析与检验[M].北京:中国计量出版社,2003:46-51. [5]
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