CN113564058A - 一种利用食品废弃物促进粗毛革孔菌生产漆酶的方法 - Google Patents
一种利用食品废弃物促进粗毛革孔菌生产漆酶的方法 Download PDFInfo
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Abstract
本发明公开了一种利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,该方法以食品废弃物作为发酵基质,为粗毛革孔菌提供碳源、氮源以及生物诱导因子,促进菌丝生长和漆酶诱导表达,主要包括以下步骤:(1)食品加工废弃物的预处理;(2)粗毛革孔菌种子液的准备;(3)液体产酶培养基的制备;(4)液态发酵生产漆酶。本发明提供的利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,产酶效率高,发酵周期短,成本低廉,无需额外添加任何化学诱导剂,同时有利于促进食品废弃物的资源化利用,具有明显的经济效益和社会效益。
Description
技术领域
本发明属于微生物酶学技术领域,具体涉及一种利用食品废弃物促进粗毛革孔菌生产漆酶的方法。
背景技术
白腐真菌是漆酶的主要来源之一,菌株分泌漆酶的类型有组成型和诱导型两种。真菌漆酶的表达与发酵条件、诱导条件等多种因素相关,因而可以通过优化培养基组成及培养条件来提高漆酶的产量。在真菌发酵过程中,适量添加诱导剂对提高漆酶的表达具有显著作用。常见的化学诱导剂除了硫酸铜,还有愈创木酚、2,6-二甲基苯酚、对羟基苯甲醛等芳香族诱导剂,但这些诱导剂大多为环境污染物,具有一定的毒性,不符合绿色环保的生产理念。相比于芳香型化学诱导剂和金属离子型诱导剂,生物诱导剂凭借来源丰富、成本低廉、安全无毒等优势,具有极大的开发潜力。因此,一些天然生物诱导剂受到越来越多的关注,如富含木质纤维素的农林废弃物(如秸秆、木屑、甘蔗渣、竹粉、树叶、葡萄藤、棉籽壳等)已被发现有利于促进真菌漆酶的表达,它们除了可以提供碳源、氮源外,还可能含有某些诱导真菌表达漆酶的成分。麦麸、菌渣、酒糟、花生壳、果皮、茶渣等食品废弃物的生物诱导作用也引起我们的关注。其中,果皮类原料营养成分较为丰富,且含有丰富的黄酮类化合物,被认为是较为优良的真菌产酶基质原料和生物诱导剂。以食品生产、加工和利用过程中产生的废弃物作为发酵基质,一方面可以减少大量食品废弃物带来的环境压力,另一方面也可以代替商品碳源和氮源,节约漆酶的生产成本,促进漆酶的工业化生产。目前,发展低成本、高效的发酵策略对漆酶的生产有着十分重要的意义。
发明内容
本发明的目的是针对目前利用真菌发酵产漆酶存在的生产水平低、发酵周期长以及生产成本高昂等问题,提供一种以食品废弃物为发酵基质促进粗毛革孔菌诱导表达漆酶的方法,促进真菌漆酶的规模化生产和产业化应用。
本发明通过以下技术方案予以实现:
一种利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,以食品废弃物作为液态发酵基质,为粗毛革孔菌提供碳源、氮源以及生物诱导因子,无需额外添加任何化学诱导剂。
所述方法具体步骤如下:
(1)食品废弃物的预处理:将食品废弃物原料在60-80℃下烘干至含水量低于8-10%,粉碎,过60-80目筛,制成粉状原料,密封保存;
(2)种子液的准备:从粗毛革孔菌保藏斜面挑取一环菌丝接入无菌的、装有50mL液体PDA培养基和磁力搅拌子的150mL三角瓶中,28℃、120rpm摇瓶培养直至瓶内长满菌丝球,采用磁力搅拌将菌丝球打散,将菌液倒入无菌50mL离心管中,4℃下10000rpm离心20min,将上清液倒掉,称量菌丝体湿重。按照菌丝体∶无菌水=1∶3(w/v)的比例加入无菌水,即配成菌丝种子液(0.25g/g);
(3)液体产酶培养基的制备:称量2.0-3.0g食品废弃物粉状原料加入到250mL锥形瓶中,再加入100mL含有3.0g/L磷酸二氢钾、1.5g/L七水硫酸镁、0.5g/L无水氯化钙,pH为5.0的无机盐培养液,121℃灭菌20min;
(4)液态发酵生产漆酶:按照1-5%(v/v)的接种量将步骤(2)制得的种子液接入产酶培养基中,培养温度为28-30℃,摇床转速为110-130rpm,培养时间为5-7天。
作为本发明的进一步改进,所述产酶培养基的碳源是柚皮粉、脐橙皮粉、脐橙果渣和罗汉果渣中的一种或多种按照任意配比的组合,氮源是麸皮粉或菜籽粕粉等农副产品中的一种;碳源优选柚皮粉,氮源优选麸皮粉。
作为本发明的进一步改进,所述的液体产酶培养基的碳氮比为16∶1-20∶1,优选16∶1。
与现有技术相比,本发明有益效果包括:
(1)本发明采用食品废弃物作为液态发酵基质生产真菌漆酶,代替葡萄糖、牛肉膏等商用碳源和氮源,减少漆酶的生产成本,同时实现了食品废弃物资源化利用,化废为宝,减少环境污染。
(2)本发明中食品废弃物中含有木质纤维素、微量元素及黄酮类物质,可为菌丝生长提供营养,促进菌丝体的生长和诱导漆酶的表达,提高产酶效率,缩短发酵周期,且无需再额外添加任何化学诱导剂。
附图说明
图1为不同碳源对粗毛革孔菌产漆酶的影响;
图2为不同氮源对粗毛革孔菌产漆酶的影响;
图3为不同碳氮比对粗毛革孔菌产漆酶的影响。
具体实施方式
下面结合具体实施例和附图,对本发明作进一步地说明。
以下实施例中采用的菌株均为中国典型培养物保藏中心保藏的粗毛革孔菌NCULAC F1(保藏编号为CCTCC No.M2021731)。
实施例1不同碳源对粗毛革孔菌产漆酶的影响
以商品碳源葡萄糖为对照,探讨不同果皮果渣原料作为碳源对粗毛革孔菌诱导产漆酶的影响,包括如下步骤:
(1)食品废弃物的预处理:将各种果皮果渣废弃物原料在60℃下烘干至含水量低于10%,粉碎,过60目筛,密封保存;
(2)种子液的准备:从粗毛革孔菌的保藏斜面挑取一环菌丝接入无菌的、装有50mL液体PDA培养基和一颗磁力搅拌子的150mL三角瓶中,28℃、120rpm摇瓶培养直至瓶内长满菌丝球,采用磁力搅拌将菌丝球打散,将菌液倒入无菌50mL离心管中,4℃下10000rpm离心20min,将上清液倒掉,称量菌丝体湿重。按照菌丝体∶无菌水=1∶3(w/v)的比例加入无菌水,即配成菌丝种子液(0.25g/g);
(3)液体产酶培养基的制备:分别称量2.0g葡萄糖、柚皮粉、脐橙皮粉、脐橙皮粉+脐橙果渣和罗汉果渣作为碳源加入到250mL锥形瓶中,以0.5g酒石酸铵为氮源,再加入100mL含有3.0g/L磷酸二氢钾、1.5g/L七水硫酸镁、0.5g/L无水氯化钙,pH为5.0的无机盐培养液,121℃灭菌20min;
(4)液态发酵生产漆酶:按照3%(v/v)的接种量将步骤(2)制得的种子液接入产酶培养基中,培养温度为28-30℃,摇床转速为120rpm,培养时间为7天。
结果如图1所示,所有处理组均获得了较高的酶活,而对照组几乎未检测到酶活,说明食品废弃物原料对粗毛革孔菌漆酶的表达具有很好的诱导效果,同时能够代替葡萄糖作为碳源,为菌丝的生长提供营养来源。
实施例2不同氮源对粗毛革孔菌产漆酶的影响
采用实施例1的方法,优选柚皮粉为碳源,设计以麸皮粉、菜籽粕粉、蛋白胨、酵母浸粉、硫酸铵、酒石酸铵为氮源,考察不同来源的氮源对粗毛革孔菌产漆酶的影响。
结果如图2所示,麸皮粉对粗毛革孔菌发酵产漆酶的诱导效果最佳,在发酵5天获得最大漆酶活力,达5865U/L,明显高于其他处理组,其次是菜籽粕粉,其余商品氮源组的酶活均较低。麸皮粉和菜籽粕粉的诱导效果均明显高于其他商品氮源,这表明食品废弃物原料代替商用氮源以促进漆酶生产是非常可行的,可大大降低生产成本,提高产酶效率。
实施例3不同碳氮比对粗毛革孔菌产漆酶的影响
采用实施例1的方法,优选柚皮粉为碳源,麸皮粉为氮源,考察不同碳氮比对粗毛革孔菌产漆酶的影响。
根据JY/T 017-1996方法,采用元素分析仪测定柚皮粉和麸皮粉的总碳、总氮含量(结果如表1所示),调节柚皮粉和麸皮粉的添加量,使混合物料的碳氮比分别为12∶1、16∶1、20∶1、24∶1、28∶1、32∶1。
表1柚皮粉和麸皮粉的总碳、总氮组分情况
总碳(%) | 总氮(%) | 碳氮比 | |
柚皮粉 | 41.35 | 1.00 | 41.35 |
麸皮粉 | 40.37 | 3.30 | 12.23 |
结果如图3所示,培养基碳氮比为16∶1-20∶1时,粗毛革孔菌产漆酶活力明显提升,其中,碳氮比为16∶1时,粗毛革孔菌产漆酶活力最高,达12000U/L。
以上所述仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形、改进及替代,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (6)
1.一种利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,其特征在于:所述方法以食品废弃物作为液态发酵基质,为粗毛革孔菌提供碳源、氮源以及生物诱导因子,无需额外添加化学诱导剂。
2.根据权利要求1所述的利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,其特征在于,包括如下步骤:
(1)食品废弃物的预处理:将食品废弃物原料在60-80℃下烘干至含水量低于8-10%,粉碎,过60-80目筛,制成粉状原料,密封保存;
(2)种子液的准备:从粗毛革孔菌保藏斜面挑取一环菌丝接入无菌的、装有50mL液体PDA培养基和磁力搅拌子的150mL三角瓶中,28℃、120rpm摇瓶培养直至瓶内长满菌丝球,采用磁力搅拌将菌丝球打散,将菌液倒入无菌50mL离心管中,4℃下4000-10000rpm离心10-20min,将上清液倒掉,称量菌丝体湿重;按照菌丝体∶无菌水=1∶3(w/v)的比例加入无菌水,即配成0.25g/g菌丝种子液;
(3)液体产酶培养基的制备:称量2.0-3.0g步骤(1)处理后的食品废弃物粉状原料加入到250mL锥形瓶中,再加入100mL含有3.0g/L磷酸二氢钾、1.5g/L七水硫酸镁、0.5g/L无水氯化钙,pH为5.0的无机盐培养液,121℃灭菌20min;
(4)液态发酵生产漆酶:按照1-5%(v/v)的接种量将步骤(2)制得的种子液接入产酶培养基中,培养温度为28-30℃,摇床转速为110-130rpm,培养时间为5-7天。
3.根据权利要求书2所述的利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,其特征在于:所述产酶培养基的碳源是柚皮粉、脐橙皮粉、脐橙果渣和罗汉果渣中的一种或多种按照任意配比的组合,所述产酶培养基的氮源是麸皮粉或菜籽粕粉中的一种。
4.根据权利要求书3所述的利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,其特征在于:所述产酶培养基的碳源为柚皮粉,所述产酶培养基的氮源为麸皮粉。
5.根据权利要求书2所述的利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,其特征在于:所述产酶培养基的碳氮比为12∶1-24∶1。
6.根据权利要求书5所述的利用食品废弃物诱导粗毛革孔菌表达漆酶的方法,其特征在于:所述产酶培养基的碳氮比为16∶1。
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