CN110607287A - 一种工业化利用杏鲍菇菌糠固体发酵生产漆酶和联产动物饲料的方法 - Google Patents
一种工业化利用杏鲍菇菌糠固体发酵生产漆酶和联产动物饲料的方法 Download PDFInfo
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Abstract
本发明公开了一种工业化利用杏鲍菇菌糠固体发酵生产漆酶和联产动物饲料的方法。本发明利用一种杏鲍菇菌糠为菌种和基料,辅以甘蔗渣、油茶饼粕和生姜渣为碳源,硝酸铵为氮源,混合无机盐为诱导物,在一定条件下经固体发酵生产漆酶和联产动物饲料的方法。本发明是利用杏鲍菇菌渣作为主要原料来工业化联产漆酶和动物饲料的方法,具有收率高、工艺条件温和、制备周期短,未使用蛋白胨等高价原料,成本低廉,操作简单,能够工业生产。本发明同时提供了一条能够高值化利用废弃生物质的有效途径。
Description
技术领域
本发明属于生物技术领域,具体涉及一种工业化利用杏鲍菇菌糠固体发酵生产漆酶和联产动物饲料的方法。
背景技术
食用菌菌糠,简称菌糠,又称菇渣、菌渣、下脚料等,是指食用菌出菇后的培养基废弃物,含有大量的食用菌菌丝残体、胞外酶、多糖、被生物酶降解的粗纤维和未被完全利用的木质素等成分,是优质的生物质资源。据中国食用菌协会统计,2017年我国食用菌总产量3712万吨,按食用菌平均生物转化率40%计算,菌渣总产量约5568万吨。由于食用菌栽培户缺乏相应处理技术、监管不力等,导致大量的菌渣并没有被合理地应用,而是被随意丢弃,造成生物质资源浪费、栽培环境和水体污染,同时也对人体健康产生危害。
食用菌在菌丝体生长和出菇过程中,会分泌降解木质纤维素的胞外酶,包括漆酶、木质素过氧化酶、纤维素酶和木聚糖酶等,其中以漆酶研究的最为广泛。漆酶是一类含铜离子的多酚类氧化酶。最早由日本科学家于紫胶漆树中发现,1894年被正式命名为漆酶。漆酶的作用底物广泛,可以利用酚类、胺类等多种化合物作为底物,最终将分子氧还原成水,不产生其他副产物,因而也被称为“绿色催化剂”,它在有毒物的降解方面、生物传感器的构建、造纸业等领域具有非常好的应用。并且漆酶具有很好的热稳定性,使得漆酶能在各种特殊领域得到重用,例如在功能高分子的合成和污水处理等领域。
真菌在生长过程中能合成漆酶,并在菌丝生长过程中分泌到胞外。如糙皮侧耳、刺芹侧耳、环柄侧耳等,但漆酶酶活普遍不高。发明专利申请CN201310712411.9“一种制备漆酶的方法”,其生产菌株为虎皮香菇,漆酶发酵酶活为462.59U/mL;发明专利申请CN200910083514.7“一种漆酶及其制备方法与专用生产菌株”,其生产菌株为血红密孔菌,漆酶发酵酶活为300U/mL;发明专利申请CN101942421B“一种竹黄菌发酵生产漆酶的方法”,漆酶发酵酶活为500U/mL;发明专利申请CN200810198678.X“一种产漆酶的灵芝菌株”,漆酶发酵酶活为8620U/mL,是目前酶活较高的菌株。以上均是利用真菌发酵得到漆酶,需要加入大量的新鲜培养基料,生产成本高。
发明内容
本发明的目的是针对现有技术的上述问题,提供一种工业化利用杏鲍菇菌糠固体发酵生产漆酶和联产动物饲料的方法。
本发明的技术方案概述如下:
一种工业化利用杏鲍菇菌糠固体发酵生产漆酶和联产动物饲料的方法,包括如下步骤:
将新鲜的杏鲍菇菌糠破碎,按照1Kg:1~2L的质量体积比加入新鲜培养基,在25~30℃条件下避光发酵3~7天,然后按1Kg:10L的质量体积比加入水,搅拌6h,过滤,过滤液即为漆酶粗酶液;滤渣按1Kg:1×106CFU的比列接种乳酸菌,混合均匀后装入厌氧发酵池,压实发酵7~14天,即得适合反刍动物食用的饲料。
所述新鲜培养基配方为:碳源7~16wt%,氮源8~15g/L,KH2PO4 0.2~0.6g/L,ZnSO4·7H2O0.05g/L,MgSO4·7H2O 0.03g/L,CuSO4·5H2O 0.2~0.6g/L,CaCl2·2H2O0.008g/L,MnSO4·H2O0.03~0.08g/L。
作为本发明方法的优选,所述的杏鲍菇菌糠是采摘菌棒3天内的新鲜菌糠。
作为本发明方法的优选,所述的培养基中的碳源为4~7wt%甘蔗渣、3~6wt%油茶饼粕、0~3wt%生姜渣的组合。
作为本发明方法的优选,所述培养基中的油茶饼粕为提取茶皂素之后的油茶饼粕。
作为本发明方法的优选,所述培养基中的生姜渣为提取生姜精油、姜辣素之后的生姜渣。
作为本发明方法的优选,所述的培养基中的氮源为10~12g/L的硝酸氨。
作为本发明方法的优选,所述的培养基配方为:碳源6wt%甘蔗渣和4wt%油茶饼粕、2wt%生姜渣的组合,氮源10g/L硝酸氨,KH2PO4 0.5g/L,ZnSO4·7H2O 0.05g/L,MgSO4·7H2O 0.03g/L,CuSO4·5H2O 0.4g/L,CaCl2·2H2O 0.008g/L,MnSO4·H2O 0.05g/L。
作为本发明方法的优选,所述的厌氧发酵时间为8~10天。
有益效果:
本发明利用一种杏鲍菇菌糠为菌种和基料,辅以甘蔗渣、油茶饼粕和生姜渣为碳源,硝酸铵为氮源,混合无机盐为诱导物,在一定温度下经固体发酵生产漆酶和联产动物饲料的方法。较之现有技术本发明具有以下显著进步:
1、利用杏鲍菇菌渣发酵生产漆酶并联产动物饲料,生产成本低,可延长杏鲍菇产业链,增加企业收入。
2、本方法所用的基料都为工业废弃生物质,有效利用了废弃的生物质资源,绿色环保。
3、本发明获得的漆酶的酶活高达812U/g,是杏鲍菇菌渣初始酶活21U/g的近40倍。
4、本方法是利用杏鲍菇菌渣生产漆酶之后再发酵制备动物饲料,可有效缩短发酵时间,同时提高饲料营养价值。
5、本发明方法利用杏鲍菇菌渣作为主要原料来工业化联产漆酶和动物饲料,收率高、工艺条件温和、制备周期短,未使用蛋白胨等高价原料,成本低廉,操作简单,适合工业化推广应用。
具体实施方式
下面结合具体实施例对本发明作进一步说明,但本发明不限于以下实施例。
实施例1
将采摘菌棒3天内的新鲜杏鲍菇菌糠10kg(采自湖南省宇秀生物科技有限公司,初始酶活力为21U/g,下同)在车间洁净区间破碎后70目筛,按照1Kg:1L的质量体积比加入培养基,在25℃条件下避光发酵7天,然后按1Kg:10L的质量体积比加入水,搅拌6h,过滤,过滤液即为漆酶粗酶液,固态发酵培养物的酶活为812U/g;滤渣按1Kg:1×106CFU的比列接种市售乳酸菌,混合均匀后装入厌氧发酵池,压实发酵8天,即得适合反刍动物食用的饲料。
培养基为:6wt%甘蔗渣和4wt%油茶饼粕和2wt%生姜渣的组合,10g/L硝酸氨,KH2PO4 0.5g/L,ZnSO4·7H2O 0.05g/L,MgSO4·7H2O 0.03g/L,CuSO4·5H2O 0.4g/L,CaCl2·2H2O 0.008g/L,MnSO4·H2O 0.05g/L。
表1杏鲍菇菌渣发酵前后的各指标测试值%n=3
实施例2
将采摘菌棒3天内的新鲜杏鲍菇菌糠10kg在在车间洁净区间破碎后过90目筛,按照1Kg:2L的质量体积比加入培养基,在28℃条件下避光发酵5天,然后按1Kg:10L的质量体积比加入水,搅拌6h,过滤,过滤液即为漆酶粗酶液,固态发酵培养物的酶活为675U/g。滤渣按1Kg:1×106CFU的比列接种市售乳酸菌,混合均匀后装入厌氧发酵池,压实发酵7天,即得适合反刍动物食用的饲料。
培养基为:碳源4wt%甘蔗渣和3wt%生姜渣的组合,氮源12(g/L)硝酸氨,KH2PO40.6(g/L),ZnSO4·7H2O 0.05(g/L),MgSO4·7H2O 0.03(g/L),CuSO4·5H2O 0.4(g/L),CaCl2·2H2O 0.008g/L),MnSO4·H2O 0.05(g/L)。
表2杏鲍菇菌渣发酵前后的各指标测试值%n=3
实施例3
将采摘菌棒3天内的新鲜杏鲍菇菌糠10kg在在车间洁净区间破碎后过80目筛,按照1Kg:1L的质量体积比加入培养基,在25℃条件下避光发酵7天,然后按1Kg:10L的质量体积比加入水,搅拌6h,过滤,过滤液即为漆酶粗酶液,固态发酵培养物的酶活为783U/g。滤渣按1Kg:1×106CFU的比列接种市售乳酸菌,混合均匀后装入厌氧发酵池,压实发酵9天,即得适合反刍动物食用的饲料。
培养基为:碳源5wt%(w/w)甘蔗渣、6wt%油茶饼粕和3wt%生姜渣的组合,氮源14(g/L)硝酸氨,KH2PO4 0.5(g/L),ZnSO4·7H2O 0.05(g/L),MgSO4·7H2O 0.03(g/L),CuSO4·5H2O 0.6(g/L),CaCl2·2H2O 0.008g/L),MnSO4·H2O 0.05(g/L)。
表3杏鲍菇菌渣发酵前后的各指标测试值%n=3
实施例4
将采摘菌棒3天内的新鲜杏鲍菇菌糠10kg在在车间洁净区间破碎后过70目筛,按照1Kg:1L的质量体积比加入培养基,在25℃条件下避光发酵7天,然后按1Kg:10L的质量体积比加入水,搅拌6h,过滤,过滤液即为漆酶粗酶液;固态发酵培养物的酶活为766U/g。滤渣按1Kg:1×106CFU的比列接种市售乳酸菌,混合均匀后装入厌氧发酵池,压实发酵10天,即得适合反刍动物食用的饲料。
培养基为:碳源2wt%甘蔗渣、6wt%油茶饼粕和3wt%生姜渣的组合,氮源11(g/L)硝酸氨,KH2PO4 0.3(g/L),ZnSO4·7H2O 0.05(g/L),MgSO4·7H2O 0.03(g/L),CuSO4·5H2O0.5(g/L),CaCl2·2H2O 0.008g/L),MnSO4·H2O 0.08(g/L)。
表4杏鲍菇菌渣发酵前后的各指标测试值%n=3
实施例5
将采摘菌棒3天内的新鲜杏鲍菇菌糠10kg在在车间洁净区间破碎后过70目筛,按照1Kg:1L的质量体积比加入培养基,在25℃条件下避光发酵7天,然后按1Kg:10L的质量体积比加入水,搅拌6h,过滤,过滤液即为漆酶粗酶液,固态发酵培养物的酶活为778U/g。滤渣按1Kg:1×106CFU的比列接种市售乳酸菌,混合均匀后装入厌氧发酵池,压实发酵8天,即得适合反刍动物食用的饲料。
培养基为:碳源6wt%甘蔗渣、6wt%油茶饼粕和1wt%生姜渣的组合,氮源11(g/L)硝酸氨,KH2PO4 0.3(g/L),ZnSO4·7H2O 0.05(g/L),MgSO4·7H2O 0.03(g/L),CuSO4·5H2O0.5(g/L),CaCl2·2H2O 0.008g/L),MnSO4·H2O 0.08(g/L)。
表5杏鲍菇菌渣发酵前后的各指标测试值%n=3
以上实施例中酶活测定方法如下:
(1)粗酶液漆酶酶活测定
以DMP为底物,3mL反应体系中含有10mmol·L-1DMP 0.5mL,20mmol·L-1pH值3.0磷酸-柠檬酸钠缓冲液2.4mL和0.1mL粗酶液,469nm下测定反应液1min内的吸光度变化。1个酶活力单位定义为在当前反应条件下每分钟氧化1μmol DMP的酶量。根据酶活力定义和比尔-朗伯定律,粗酶液酶活力计算公式为:
粗酶液酶活力
其中,ΔOD表示反应时间内吸光度的差值;V总表示反应体系总体积,单位:mL;n表示稀释倍数;Δt表示反应时间,单位:min,V酶表示反应体系中加入的粗酶液体积,单位:mL;ε为DMP氧化产物3,5,3’,5’-四甲氧基二苯基醌的摩尔消光系数,ε469=4.96×104L·mol-1·cm-1;b表示光在介质中通过的距离即比色皿宽度。
(2)发酵培养物含水率的测定
取1g发酵后的菌渣培养物放置于60℃烘箱,烘干至恒重,计算得到含水率β。
(3)固态发酵培养物的酶活力计算:取1g发酵后的菌渣培养物放置于60℃烘箱,烘干至恒重得干曲;以每克干曲含有的酶活力U/g表示固态发酵培养物的酶活。酶活计算公式为:
U/g湿曲=粗酶液酶活力×10
U/g干曲=U/g湿曲÷(1-β)
(4)实施例中,粗蛋白测定方法参照GB/T15673;粗灰分测定参照GB/T12532;粗纤维测定
参照GB/T5515;木质素测定参照GB/T20805。
Claims (8)
1.一种工业化利用杏鲍菇菌糠固体发酵生产漆酶和联产动物饲料的方法,其特征是包括如下步骤:
将新鲜的杏鲍菇菌糠破碎,按照1Kg:1~2L的质量体积比加入新鲜培养基,在25~30℃条件下避光发酵3~7天,然后按1 Kg:10L的质量体积比加入水,搅拌6 h,过滤,过滤液即为漆酶粗酶液;滤渣按1 Kg:1×106CFU的比列接种乳酸菌,混合均匀后装入厌氧发酵池,压实发酵7~14天,即得适合反刍动物食用的饲料;
所述新鲜培养基配方为:碳源7~16wt%,氮源8~15g/L, KH2PO4 0.2~0.6g/L, ZnSO4·7H2O 0.05g/L,MgSO4·7H2O 0.03g/L, CuSO4·5H2O 0.2~0.6g/L,CaCl2·2H2O 0.008g/L,MnSO4·H2O 0.03~0.08g/L。
2.根据权利要求1所述的方法,其特征在于所述的杏鲍菇菌糠是采摘菌棒3天内的新鲜菌糠。
3.根据权利要求1所述的方法,其特征在于所述的培养基中的碳源为4~7wt%甘蔗渣、3~6 wt %油茶饼粕及0~3 wt %生姜渣的组合。
4.根据权利要求3所述的方法,其特征在于所述培养基中的油茶饼粕为提取茶皂素之后的油茶饼粕。
5.根据权利要求3所述的方法,其特征在于所述培养基中的生姜渣为提取生姜精油、姜辣素之后的生姜渣。
6.根据权利要求1所述的方法,其特征在于所述的培养基中的氮源为10~12g/L的硝酸氨。
7.根据权利要求1-6中任一项所述的方法,其特征在于所述的培养基为:碳源6 wt %甘蔗渣和4 wt %油茶饼粕、2 wt %生姜渣的组合,氮源10g/L硝酸氨, KH2PO4 0.5g/L,ZnSO4·7H2O 0.05g/L,MgSO4·7H2O 0.03g/L,CuSO4·5H2O 0.4g/L,CaCl2·2H2O 0.008g/L,MnSO4·H2O 0.05g/L。
8.根据权利要求1所述的方法,其特征在于所述的厌氧发酵时间为8~10天。
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