CN112553284A - 一种自然共生的混合培养物降解粗饲料生产柠檬酸的方法 - Google Patents
一种自然共生的混合培养物降解粗饲料生产柠檬酸的方法 Download PDFInfo
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Abstract
本发明涉及生物技术可再生能源领域,具体为一种自然共生的混合培养物降解粗饲料生产柠檬酸的方法,所述的混合培养物YakQH5由厌氧真菌(Neocallimastix frontalis)和甲烷菌(Methanobrevibacter gottschalkii)组成,于2020年3月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.19299,所述的混合培养物YakQH5能够分别降解15种粗饲料,并产生大量柠檬酸,尤其以苜蓿为底物时,柠檬酸产量高达46.0mM,在发酵过程中添加复合抗生素,还可以防止混合培养物在发酵过程中不受细菌污染,进一步提高厌氧发酵效率,本发明所述混合培养物YakQH5具有重要的工业应用价值。
Description
技术领域
本发明涉及生物技术可再生能源领域,具体为一种自然共生的混合培养物降解粗饲料生产柠檬酸的方法。
背景技术
粗饲料是指天然水分含量在60%以下,干物质中粗纤维含量等于或高于18%,并以风干物形式饲喂的饲料,如牧草、农作物秸秆、树叶、酒糟和秕壳等。粗饲料的主要成分是木质纤维素,纤维含量高,蛋白质、矿物质含低,适口性差,畜禽消化率低,从而限制了它的应用。这些粗饲料每年产生的数量非常巨大,如小麦秸秆、玉米秸秆、水稻秸秆、燕麦秸秆、大豆秸秆、黄豆秸秆、洋芋秸秆、苜蓿、麸皮、酒糟、番茄渣、小麦壳等,它们作为能源物质仍然没有得到合理开发利用。目前主要采用生物学手段,即采用能分泌高效木质纤维素降解酶类的微生物来降解和利用大量粗饲料。但目前有效提高粗饲料的木质纤维素利用率、实现木质纤维素的生物转化的微生物菌种资源却极端缺乏。如何建立高效生物转化技术、实现木质纤维素的生物转化是解决巨大粗饲料资源的根本出路。
牦牛(Bos grunniens)是生活在海拔最高处,耐粗饲、耐严寒的,能适应高寒缺氧的草食性反刍动物。青藏高原是牦牛主产区。我国牦牛饲养以放牧为主,牦牛瘤胃内栖息着大量独特的,复杂多样的微生物群落协同降解低质野牧草和冷季干枯草为牦牛提供生存能量和营养物质,使牦牛瘤胃成为高效降解木质纤维素的天然厌氧发酵罐。牦牛瘤胃内存在着自然共生的厌氧真菌和甲烷菌共培养物,它们能够分泌高活性木质纤维素降解酶降解野生牧草和干枯牧草为牦牛提供生长的营养,因此从牦牛瘤胃分离自然的厌氧真菌和甲烷菌共培养物应用于降解廉价木质纤维素底物生产乙酸和甲烷等代谢终产物在工业应用方面具有重要意义。
柠檬酸(化学名称:2-羟基-均丙三羧酸)是一种重要的有机酸,无色晶体,常含一分子结晶水。在化工和纺织业、食品业、环保业、禽畜生产业、化妆业和医药等行业具有极多的用途。1784年C.W.舍勒首先通过在水果榨汁中加石灰乳形成柠檬酸钙沉淀的方法制取柠檬酸。中国用发酵法制取柠檬酸以1942年汤腾汉等报告为最早。1952年陈声等开始用黑曲霉浅盘发酵制取柠檬酸。1965年进行了生产100t甜菜糖蜜原料浅盘发酵制取柠檬酸的中间试验,并于1968年投入生产。1966年后,天津市工业微生物研究所、上海市工业微生物研究所相继开展用黑曲霉进行薯干粉原料深层发酵柠檬酸的试验研究,并获得成功,从而确定了中国柠檬酸生产的这一主要工艺路线,其工艺简单,不需添加营养盐,产率高,是中国独特的先进工艺。中国石油发酵柠檬酸的研究较早。1970年,天津、上海、沈阳等地研究单位利用解脂假丝酵母进行石蜡油发酵生产柠檬酸的试验。1979年徐子渊等筛选出对氟乙酸敏感的变异株解脂假丝酵母,显著地提高了石油发酵柠檬酸的产率。目前随着高产菌株的应用和新技术的不断开拓,柠檬酸发酵和提取收率都有明显提高,柠檬酸工业发展突飞猛进。
发明人在攻读博士期间首次研究了放牧牦牛瘤胃厌氧真菌和甲烷菌共培养物分别以小麦秸秆、玉米秸秆和水稻秸秆为底物进行厌氧发酵(魏亚琴.牦牛瘤胃厌氧真菌与甲烷菌共培养物的多样性及其纤维降解特性研究[D].2016.),通过检测产气量、多糖水解酶活性、各种酯酶活性、干物质降解率、酚酸释放量、甲烷和乙酸产量来评估厌氧真菌和甲烷菌共培养物降解秸秆功效,其中厌氧真菌和甲烷菌共培养物N.frontalis Yak16+M.ruminantium,Piromyces Yak18+M.ruminantium和O.joyonii Yak7+M.ruminantium分别降解三种秸秆所产生的乙酸和甲烷的产量很高,但是柠檬酸的产量极低,所以没有在博士论文中列出柠檬酸的数据。迄今为止,国内外没有关于厌氧真菌,或者厌氧真菌和甲烷菌共培养物降解秸秆等粗饲料产生柠檬酸的报道。
发明内容
针对上述技术问题,本发明的目的是提供一种自然共生的混合培养物YakQH5降解粗饲料生产柠檬酸的方法,所述的混合培养物YakQH5由厌氧真菌(Neocallimastixfrontalis)和甲烷菌(Methanobrevibacter gottschalkii)组成,所述的方法包括如下步骤:
(1)混合培养物YakQH5菌剂的制备:以10%v/v接种量向以小麦秸秆为底物的厌氧培养基中接入混合培养物YakQH5,加入复合抗生素厌氧培养即得到高活力的菌剂;
(2)发酵降解粗饲料生产柠檬酸:吸取步骤(1)制备的菌剂,按10%v/v接种量分别接入以1%w/v粗饲料为底物的厌氧培养基中,同时加入1%v/v复合抗生素,培养。
优选地,所述的混合培养物YakQH5于2020年3月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.19299,保藏地址为:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,电话:010-64807355。分离自青藏高原的海南州星海镇的全放牧牦牛瘤胃内容物中。
优选地,所述的步骤(1)中厌氧培养的温度为39℃,时间为72小时;所述的步骤(2)中厌氧培养的温度为39℃,时间为5天。
优选地,所述的复合抗生素为青霉素钠和硫酸链霉素,在厌氧培养基中的浓度分别为1600IU/mL和2000IU/mL。
优选地,厌氧培养基配方为:酵母膏1.0g,蛋白胨1.0g,NaHCO3 7.0g,刃天青1.0g/L1mL,L-半胱氨酸盐酸盐1.7g,晨饲前采集瘤胃液8000×g,4℃离心20min后的上清170mL,盐溶液Ⅰ82.5mL,盐溶液II 16.5mL,蒸馏水定容至1000mL。
优选地,所述的盐溶液I配制步骤如下:NaCl 6g,(NH4)2SO4 3g,KH2PO4 3g,CaCl2·2H2O 0.4g,MgSO4·2H2O 0.6g,蒸馏水定容至1000mL。
优选地,所述的盐溶液II配制步骤如下:4g K2HPO4,蒸馏水定容至1000mL。
优选地,所述的步骤(2)分别加入各种底物后除氧,充入二氧化碳,高温高压灭菌。
优选地,所述的步骤(2)加入的底物分别为小麦秸秆、胡麻秸秆、洋芋秸秆、棉花秸秆、大豆秸秆、红豆草秸秆、芦苇秆、剑麻、麸皮、瓜子壳、花生壳、小麦壳、滤纸、番茄渣和苜蓿中的一种或几种。
优选地,所述的步骤(2)加入的底物为苜蓿。
本发明的有益效果是:①本发明中采用的厌氧真菌(Neocallimastix frontalis)和甲烷菌(Methanobrevibacter gottschalkii)的混合培养物YakQH5是从牦牛瘤胃液中分离获得的,牦牛瘤胃中的微生物协同降解低质野草为牦牛提供生存必需的营养物质,使牦牛适应青藏高原的严酷环境而生存。长期的自然选择和进化使牦牛瘤胃成为一个高效木质纤维素降解酶系统,与人工混合的厌氧真菌和甲烷菌共培养物相比,牦牛瘤胃自然存在的厌氧真菌和甲烷菌共培养物具有高效降解木质纤维素的显著优势。②采用混合培养物YakQH5厌氧发酵降解小麦秸秆、胡麻秸秆、洋芋秸秆、棉花秸秆、大豆秸秆、红豆草秸秆、芦苇秆、剑麻、麸皮、瓜子壳、花生壳、小麦壳、滤纸、番茄渣和苜蓿共15种粗饲料,其中降解苜蓿(即以苜蓿为底物)产生的柠檬酸浓度为46.0mM,显著高于其它底物。③在发酵过程中添加复合抗生素,可防止共培养物体系不受细菌污染,提高厌氧发酵效率。④同时,本发明中采用的共培养物可以通过保藏在体外存活传代,便于推广,为生产提供了极大的方便。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中所使用的培养基如下:
厌氧培养基配方:酵母膏1.0g,蛋白胨1.0g,NaHCO3 7.0g,刃天青(1.0g/L)1mL,L-半胱氨酸盐酸盐1.7g,晨饲前采集瘤胃液8000×g,4℃离心20min后的上清170mL,盐溶液I165mL,盐溶液II 165mL,蒸馏水定容至1000mL。
盐溶液I配制步骤如下:NaCl 6g,(NH4)2SO4 3g,KH2PO4 3g,CaCl2·2H2O 0.4g,MgSO4·2H2O 0.6g,蒸馏水定容至1000mL。
盐溶液II配制步骤如下:4g K2HPO4,蒸馏水定容至1000mL。
传代培养基:在厌氧培养基中添加1%w/v的粉碎风干的小麦秸秆。然后除氧后灭菌。
除氧方法:厌氧管或厌氧瓶通过针头接入带有真空泵和高纯CO2的抽气装置进行培养基除氧。首先真空泵抽出管中气体达到负压时培养基颜色改变,然后充入高纯CO2。每管抽气充气3次,其中第1次约15min,其余二次每次5min,最后1次充气后用无菌株针再放气使得厌氧管内外压平衡,并于121℃高温高压湿热灭菌20min备用。
厌氧发酵罐又称厌氧发酵瓶,是在一个密闭罐体内完成料液的发酵、沼气产生的过程,主要用来满足微生物的生活条件,使它们在合适的环境中生活,以达到发酵旺盛,产气量高的目的。
实施例一、混合培养物YakQH5菌剂的制备
吸取1mL混合培养物YakQH5接种到亨氏厌氧管中的9mL以风干粉碎的小麦秸秆为底物的厌氧培养基中,同时加入0.1mL复合抗生素(1600IU/mL青霉素和2000IU/mL硫酸链霉素),39℃厌氧培养72h,即达到生长高峰,此时发酵液为高活力菌剂。
实施例二、混合培养物YakQH5发酵生产柠檬酸
在100mL体积厌氧发酵瓶中盛45mL液体基本培养基,分别以0.5g粉碎后的风干的小麦秸秆、胡麻秸秆、洋芋秸秆、棉花秸秆、大豆秸秆、红豆草秸秆、芦苇秆、剑麻、麸皮、瓜子壳、花生壳、小麦壳、滤纸、番茄渣和苜蓿共15种粗饲料作为底物。除氧。灭菌。把传代培养72h的混合培养物YakQH5用无菌注射器分别吸取5mL接种到上述加有各个粗饲料的厌氧培养基中,同时加入0.5mL复合抗生素(1600IU/mL青霉素和2000IU/mL硫酸链霉素),39℃厌氧培养5天。共设置3个平行实验,隔24h测定厌氧瓶中的柠檬酸浓度。通过液相色谱法测定柠檬酸:
使用液相色谱仪(LC2030 PLUS,岛津,日本),配置有Acclaim 120 C18 3um柱(2.1mm×150mm),检测波长210nm,柱温25℃,紫外检测器温度40℃,流动相A:磷酸钾水溶液(pH=2),流动相B:乙腈,流速0.5mL/min,0分钟99%A—1%B,20分钟80%A—20%B,进样量10μL。实验结果显示,共培养物(N.frontalis+M.gottschalkii)YakQH5分别降解15种粗饲料的同时产生高浓度柠檬酸,以苜蓿为底物所产柠檬酸产量显著高于其它底物,具体结果如下表:
表1混合培养物YakQH5分别降解15种粗饲料所产柠檬酸的产量
注:—,代表未测出。a,b,c,d表示统计学差异性(p<0.05)
实验结果如表1所示,混合培养物YakQH5在5天培养期内分别降解15种粗饲料产生柠檬酸产量达到的最高值分别为:以小麦秸秆为底物柠檬酸产量达到32.8mM,以胡麻秸秆为底物发酵产柠檬酸产量达到28.2mM,以洋芋秸秆为底物发酵产柠檬酸产量达到31.0mM,以棉花秸秆为底物发酵产柠檬酸的产量达到20.0mM,以大豆秸秆为底物发酵产柠檬酸的产量达到27.1mM,以红豆草秸秆为底物发酵产柠檬酸的产量达到40.1mM,以芦苇秆为底物发酵产柠檬酸的产量达到24.6mM,以剑麻为底物发酵产柠檬酸的产量达到27.8mM,以麸皮为底物发酵产柠檬酸的产量达到16.3mM,以小麦壳为底物发酵产柠檬酸的产量达到20.7mM,以滤纸为底物发酵产柠檬酸的产量达到21.4mM,以番茄渣为底物发酵产柠檬酸的产量达到11.5mM,以苜蓿为底物发酵产柠檬酸的产量达到46.0mM。
通过以上实施例我们可以看到,牦牛瘤胃自然共生的混合培养物YakQH5分别降解15种粗饲料的同时产生大量柠檬酸,尤其是降解苜蓿所产柠檬酸产量达到最高值46.0mM,具有重要的工业应用价值。
Claims (8)
1.一种自然共生的混合培养物YakQH5降解粗饲料生产柠檬酸的方法,其特征在于,所述的混合培养物YakQH5由厌氧真菌(Neocallimastix frontalis)和甲烷菌(Methanobrevibacter gottschalkii)组成,所述的方法包括如下步骤:
(1)混合培养物YakQH5菌剂的制备:以10%v/v接种量向以小麦秸秆为底物的厌氧培养基中接入混合培养物YakQH5,加入复合抗生素,厌氧培养即得到高活力的菌剂;
(2)发酵降解粗饲料生产柠檬酸:吸取步骤(1)制备的菌剂,按10%v/v接种量分别接入以1%w/v粗饲料为底物的厌氧培养基中,同时加入1%v/v复合抗生素,培养。
2.如权利要求1所述的方法,其特征在于,所述的混合培养物YakQH5于2020年3月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.19299,保藏地址为:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,电话:010-64807355。
3.如权利要求1所述的方法,其特征在于,所述的步骤(1)中厌氧培养的温度为39℃,时间为72小时;所述的步骤(2)中厌氧培养的温度为39℃,时间为120小时。
4.如权利要求1所述的方法,其特征在于,所述的复合抗生素为青霉素钠和硫酸链霉素,在厌氧培养基中的浓度分别为1600IU/mL和2000IU/mL。
5.如权利要求1所述的方法,其特征在于,厌氧培养基配方为:酵母膏1.0g,蛋白胨1.0g,NaHCO3 7.0g,刃天青1.0g/L 1mL,L-半胱氨酸盐酸盐1.7g,晨饲前采集瘤胃液8000×g,4℃离心20min后的上清170mL,盐溶液Ⅰ82.5mL,盐溶液II 16.5mL,蒸馏水定容至1000mL;所述的盐溶液I配制步骤如下:NaCl 6g,(NH4)2SO4 3g,KH2PO4 3g,CaCl2·2H2O 0.4g,MgSO4·2H2O 0.6g,蒸馏水定容至1000mL;所述的盐溶液II配制步骤如下:4g K2HPO4,蒸馏水定容至1000mL。
6.如权利要求1所述的方法,其特征在于,所述的步骤(2)分别加入各种底物后除氧,充入二氧化碳,高温高压灭菌。
7.如权利要求1所述的方法,其特征在于,所述的步骤(2)加入的底物为小麦秸秆、胡麻秸秆、洋芋秸秆、棉花秸秆、大豆秸秆、红豆草秸秆、芦苇秆、剑麻、麸皮、瓜子壳、花生壳、小麦壳、滤纸、番茄渣和苜蓿中的一种或几种。
8.如权利要求7所述的方法,其特征在于,所述的步骤(2)加入的底物为苜蓿。
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WO2022143581A1 (zh) * | 2020-12-30 | 2022-07-07 | 甘肃省科学院生物研究所 | 一种自然共生的混合培养物降解粗饲料生产柠檬酸的方法 |
CN115968965A (zh) * | 2023-01-06 | 2023-04-18 | 甘肃省科学院生物研究所 | 牦牛瘤胃厌氧真菌和甲烷菌自然混合培养物发酵液在改善西门塔尔牛肉品质中的应用 |
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CN115968965A (zh) * | 2023-01-06 | 2023-04-18 | 甘肃省科学院生物研究所 | 牦牛瘤胃厌氧真菌和甲烷菌自然混合培养物发酵液在改善西门塔尔牛肉品质中的应用 |
CN115968965B (zh) * | 2023-01-06 | 2024-06-14 | 甘肃省科学院生物研究所 | 牦牛瘤胃厌氧真菌和甲烷菌自然混合培养物发酵液在改善西门塔尔牛肉品质中的应用 |
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