WO2022143581A1 - 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 PDF

Info

Publication number
WO2022143581A1
WO2022143581A1 PCT/CN2021/141840 CN2021141840W WO2022143581A1 WO 2022143581 A1 WO2022143581 A1 WO 2022143581A1 CN 2021141840 W CN2021141840 W CN 2021141840W WO 2022143581 A1 WO2022143581 A1 WO 2022143581A1
Authority
WO
WIPO (PCT)
Prior art keywords
citric acid
straw
mixed culture
anaerobic
yakqh5
Prior art date
Application number
PCT/CN2021/141840
Other languages
English (en)
French (fr)
Inventor
魏亚琴
王治业
赵疆
Original Assignee
甘肃省科学院生物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 甘肃省科学院生物研究所 filed Critical 甘肃省科学院生物研究所
Priority to US17/789,228 priority Critical patent/US20240011060A1/en
Publication of WO2022143581A1 publication Critical patent/WO2022143581A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/48—Tricarboxylic acids, e.g. citric acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/26—Methylomonas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/59—Biological synthesis; Biological purification

Definitions

  • the invention relates to the field of biotechnology renewable energy, in particular to a method for producing citric acid by degrading roughage by a natural symbiotic mixed culture.
  • Roughage refers to feeds with natural moisture content below 60%, crude fiber content equal to or higher than 18% in dry matter, and fed in the form of air-dried matter, such as forage grass, crop straw, leaves, distiller's grains and husks.
  • the main component of roughage is lignocellulose, which has high fiber content, low protein and mineral content, poor palatability, and low digestibility of livestock and poultry, thus limiting its application.
  • the amount of these roughages produced every year is very large, such as wheat straw, corn straw, rice straw, oat straw, soybean straw, soybean straw, potato straw, alfalfa, bran, distiller's grains, tomato pomace, wheat husk, etc., which are still used as energy materials. Not properly exploited.
  • Yak (Bos grunniens) is a herbivorous ruminant that lives at the highest altitude, is resistant to roughage and severe cold, and can adapt to high cold and hypoxia.
  • the Qinghai-Tibet Plateau is the main producing area of yak.
  • the feeding of yak in my country is mainly based on grazing.
  • the synergistic degradation of low-quality wild forage grass and dry grass in the cold season provides yak with survival energy and nutrients, making yak rumen a highly efficient degradable wood fiber.
  • Citric acid (chemical name: 2-hydroxy-m-propanetricarboxylic acid) is an important organic acid, colorless crystal, often containing a molecule of crystal water. It has many uses in chemical and textile industry, food industry, environmental protection industry, livestock production industry, cosmetic industry and pharmaceutical industry. In 1784, C.W. Scheler first prepared citric acid by adding lime milk to the fruit juice to form calcium citrate precipitation. In 1942, Tang Tenghan et al. reported that citric acid was produced by fermentation method in China. In 1952, Chen Sheng et al. began to ferment citric acid with Aspergillus niger.
  • the inventor first studied the co-culture of grazing yak rumen anaerobic fungi and methanogens using wheat straw, corn straw and rice straw as substrates for anaerobic fermentation (Wei Yaqin. Yaqin rumen anaerobic fungi and methanogens co-cultured) for the first time.
  • Research on the diversity of cultures and their fiber degradation characteristics [D].2016.) by detecting gas production, polysaccharide hydrolase activity, various esterase activities, dry matter degradation rate, phenolic acid release, methane and acetic acid production.
  • the object of the present invention is to provide a kind of natural symbiotic mixed culture YakQH5 degrading roughage to produce citric acid method, and described mixed culture YakQH5 is made up of anaerobic fungi (Neocallimastixfrontalis) and methane bacteria (Methanobrevibacter gottschalkii) , the method includes the following steps:
  • the mixed culture YakQH5 was preserved in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on March 9, 2020, and the preservation number is CGMCC No. 19299, and the preservation address is: 1 Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences, Tel: 010-64807355. It was isolated from the rumen contents of whole-grazing yak in Xinghai Town, Hainan Prefecture, on the Qinghai-Tibet Plateau.
  • the temperature of the anaerobic cultivation in the step (1) is 39°C and the time is 72 hours; the temperature of the anaerobic cultivation in the step (2) is 39°C and the time is 5 days.
  • the compound antibiotics are sodium penicillin and streptomycin sulfate, and the concentrations in the anaerobic medium are 1600 IU/mL and 2000 IU/mL, respectively.
  • the formula of the anaerobic medium is: yeast extract 1.0g, peptone 1.0g, NaHCO 3 7.0g, resazurin 1.0g/L1mL, L-cysteine hydrochloride 1.7g, and rumen juice collected before morning feeding After centrifugation at 8000 ⁇ g for 20 min at 4°C, 170 mL of supernatant, 82.5 mL of salt solution I, 16.5 mL of salt solution II, and distilled water were made up to 1000 mL.
  • salt solution I preparation steps are as follows: NaCl 6g, (NH 4 ) 2 SO 4 3g, KH 2 PO 4 3g, CaCl 2 2H 2 O 0.4g, MgSO 4 2H 2 O 0.6g, distilled water Make up to 1000mL.
  • the preparation step of the salt solution II is as follows: 4g K 2 HPO 4 , dilute to 1000mL with distilled water.
  • step (2) after adding various substrates, oxygen is removed, carbon dioxide is charged, and high temperature and high pressure sterilization is performed.
  • the substrates added in the step (2) are respectively wheat straw, flax straw, artichoke straw, cotton straw, soybean straw, red bean straw, reed straw, sisal, bran, melon seed shell, peanut shell, One or more of wheat husk, filter paper, tomato pomace and alfalfa.
  • the substrate added in the step (2) is alfalfa.
  • the beneficial effects of the present invention are: (1) The mixed culture YakQH5 of anaerobic fungi (Neocallimastix frontalis) and methane bacteria (Methanobrevibacter gottschalkii) adopted in the present invention is isolated from yak rumen juice, and the synergistic degradation of microorganisms in the yak rumen is low. Weeds provide the yak with essential nutrients for survival, enabling the yak to adapt to the harsh environment of the Qinghai-Tibet Plateau to survive. Long-term natural selection and evolution have made the yak rumen a highly efficient lignocellulose-degrading enzyme system.
  • co-cultures of anaerobic fungi and methanogens naturally present in yak rumen have Significant advantages of efficient degradation of lignocellulose.
  • Anaerobic medium formula yeast extract 1.0g, peptone 1.0g, NaHCO 3 7.0g, resazurin (1.0g/L) 1mL, L-cysteine hydrochloride 1.7g, rumen juice collected before morning feeding 8000 ⁇ g, 170 mL of supernatant after centrifugation at 4°C for 20 min, 165 mL of salt solution I, 165 mL of salt solution II, and distilled water to dilute to 1000 mL.
  • the salt solution I preparation steps are as follows: NaCl 6g, (NH 4 ) 2 SO 4 3g, KH 2 PO 4 3g, CaCl 2 2H 2 O 0.4g, MgSO 4 2H 2 O 0.6g, distilled water to 1000mL.
  • salt solution II The preparation steps of salt solution II are as follows: 4g K 2 HPO 4 , dilute to 1000mL with distilled water.
  • Passaging medium 1% w/v ground and air-dried wheat straw was added to the anaerobic medium. Then sterilize after deoxygenation.
  • Deoxygenation method The anaerobic tube or anaerobic bottle is connected to an aspirating device with a vacuum pump and high-purity CO 2 through a needle to deoxygenate the medium.
  • the color of the medium changes when the gas in the vacuum pump evacuates the tube to a negative pressure, and then is filled with high-purity CO 2 .
  • Each tube is pumped and inflated 3 times, the first time is about 15 minutes, and the other two times are 5 minutes each time.
  • use a strain-free needle to deflate again to balance the internal and external pressure of the anaerobic tube, and quench it at 121 °C high temperature, high pressure and humid heat.
  • Bacteria 20min standby.
  • Anaerobic fermentation tank also known as anaerobic fermentation bottle, is a process in which the fermentation of feed liquid and the production of biogas are completed in a closed tank. It is mainly used to meet the living conditions of microorganisms, so that they can live in a suitable environment to achieve vigorous fermentation. , the purpose of high gas production.
  • Embodiment one the preparation of mixed culture YakQH5 bacterial agent
  • Embodiment two, mixed culture YakQH5 fermentation produces citric acid
  • the mixed culture YakQH5 subcultured for 72h was inoculated into the above-mentioned anaerobic medium with each roughage with a sterile syringe, and 0.5mL of compound antibiotics (1600IU/mL penicillin and 2000IU/mL streptomycin sulfate) were added simultaneously. , 39 °C anaerobic culture for 5 days. A total of 3 parallel experiments were set up, and the citric acid concentration in the anaerobic flask was measured every 24 hours. Determination of citric acid by liquid chromatography:
  • Table 1 Yields of citric acid produced by degrading 15 kinds of roughages by YakQH5 mixed culture
  • the experimental results are shown in Table 1.
  • the mixed culture YakQH5 degraded 15 kinds of forages to produce the highest citric acid yields: the yield of citric acid reached 32.8 mM with wheat straw as the substrate, and the yield of citric acid reached 32.8 mM with flax straw.
  • the yield of citric acid produced by fermentation as the substrate reached 28.2 mM
  • the yield of citric acid produced by the fermentation of artichoke straw as the substrate reached 31.0 mM
  • the yield of citric acid produced by the fermentation of cotton straw as the substrate reached 20.0 mM
  • the fermentation of soybean straw as the substrate reached 20.0 mM.
  • the yield of citric acid reached 27.1mM, the yield of citric acid produced by fermentation with red bean straw as substrate reached 40.1mM, the yield of citric acid produced by fermentation of reed straw as substrate reached 24.6mM, and the yield of citric acid produced by fermentation with sisal as substrate reached 24.6mM.
  • the yield of citric acid reached 27.8 mM, the yield of citric acid produced by fermentation with bran as substrate reached 16.3 mM, the yield of citric acid produced by fermentation of wheat husk as substrate reached 20.7 mM, and the yield of citric acid produced by fermentation with filter paper as substrate reached 20.7 mM.
  • the yield reached 21.4 mM, the yield of citric acid produced by fermentation with tomato pomace as substrate reached 11.5 mM, and the yield of citric acid produced by fermentation of alfalfa as substrate reached 46.0 mM.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

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

[根据细则26改正10.01.2022]　耗牛瘤胃厌氧真菌和甲烷菌共培养物降解粗饲料生产柠檬酸的方法 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，NaHCO 3 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，(NH 4)2 SO 4 3g，KH 2 PO 4 3g，CaCl 2·2H 2 O 0.4g，MgSO 4·2H 2 O 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 K 2 HPO 4，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，NaHCO 3 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，(NH 4)2 SO 4 3g，KH 2 PO 4 3g，CaCl 2 ·2H 2 O 0.4g，MgSO 4·2H 2 O 0.6g，蒸馏水定容至1000mL。
盐溶液II配制步骤如下：4g K 2 HPO 4，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CO 2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CO 2。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(LC2030PLUS，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 (2)

  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由厌氧真菌和甲烷菌组成，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；
    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；
    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；
    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/m；
    厌氧培养基配方为：酵母膏1.0g，蛋白胨1.0g，NaHCO 37.0g，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Ⅰ 82.5mL，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；
    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；
    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、小麦壳、滤纸、番茄渣和苜蓿中的一种或几种。
  2. 如权利要求1所述的方法，其特征在于，所述的步骤(2)加入的底物为苜蓿。
PCT/CN2021/141840 2020-12-30 2021-12-28 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 WO2022143581A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/789,228 US20240011060A1 (en) 2020-12-30 2021-12-28 Method For Producing Citric Acid by Degrading Roughages with the Rumen Fungus-methanogen Co-culture from Qinghai Yaks

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011616690.5 2020-12-30
CN202011616690.5A CN112553284B (zh) 2020-12-30 2020-12-30 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

Publications (1)

Publication Number Publication Date
WO2022143581A1 true WO2022143581A1 (zh) 2022-07-07

Family

ID=75034857

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/141840 WO2022143581A1 (zh) 2020-12-30 2021-12-28 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

Country Status (3)

Country Link
US (1) US20240011060A1 (zh)
CN (1) CN112553284B (zh)
WO (1) WO2022143581A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN116172130A (zh) * 2023-01-18 2023-05-30 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牦牛肉品质中的应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112553284B (zh) * 2020-12-30 2021-12-14 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
CN114164188B (zh) * 2021-11-26 2022-09-02 甘肃省科学院生物研究所 牦牛瘤胃厌氧真菌和甲烷菌共培养物降解芦苇秆生产漆酶的方法及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010124147A1 (en) * 2009-04-22 2010-10-28 Qteros, Inc. Compositions and methods for methane production
CN106978355A (zh) * 2017-03-16 2017-07-25 甘肃省科学院生物研究所 一种厌氧真菌和甲烷菌的共培养物及在奶牛饲用天然酶中的应用
CN112553284A (zh) * 2020-12-30 2021-03-26 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

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107130001B (zh) * 2017-04-06 2019-05-03 甘肃省科学院生物研究所 一种共培养物及其发酵小麦秸秆产甲烷的方法
CN106987572B (zh) * 2017-04-06 2019-08-30 甘肃省科学院生物研究所 一种厌氧发酵玉米秸秆生产木聚糖酶的方法
CN107326060B (zh) * 2017-04-06 2019-06-11 甘肃省科学院生物研究所 一种厌氧发酵玉米秸秆生产乙酸的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010124147A1 (en) * 2009-04-22 2010-10-28 Qteros, Inc. Compositions and methods for methane production
CN106978355A (zh) * 2017-03-16 2017-07-25 甘肃省科学院生物研究所 一种厌氧真菌和甲烷菌的共培养物及在奶牛饲用天然酶中的应用
CN112553284A (zh) * 2020-12-30 2021-03-26 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

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHENG YAN FEN, JIN WEI, MAO SHENG YONG, ZHU WEI-YUN: "Production of Citrate by Anaerobic Fungi in the Presence of Co-culture Methanogens as Revealed by 1H NMR Spectrometry", ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES, vol. 26, no. 10, 31 October 2013 (2013-10-31), KR , pages 1416 - 1423, XP055948697, ISSN: 1011-2367, DOI: 10.5713/ajas.2013.13134 *
LI YUQI, SUN MEIZHOU, LI YUANFEI, CHENG YANFEN, ZHU WEIYUN: "Co-cultured methanogen improved the metabolism in the hydrogenosome of anaerobic fungus as revealed by gas chromatography-mass spectrometry analysis", ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES, vol. 33, no. 12, 1 December 2020 (2020-12-01), KR , pages 1948 - 1956, XP055948701, ISSN: 1011-2367, DOI: 10.5713/ajas.19.0649 *
LI, YUANFEI ET AL.: "Advance in the Co-culture of Anaerobic Fungi and Methanogens", ACTA MICROBIOLOGICA SINICA, vol. 61, no. 1, 2021, pages 1 - 12, XP055948818 *
LI, YUANFEI: "Investigation of the Effect of Methanogens on the Metabolism of Anaerobic Fungi by Co-culture Technique", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 15 July 2020 (2020-07-15), pages 1 - 164, XP055948814, ISSN: 1674-022X *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN116172130A (zh) * 2023-01-18 2023-05-30 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牦牛肉品质中的应用

Also Published As

Publication number Publication date
CN112553284A (zh) 2021-03-26
CN112553284B (zh) 2021-12-14
US20240011060A1 (en) 2024-01-11

Similar Documents

Publication Publication Date Title
WO2022143581A1 (zh) 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
CN107365718B (zh) 巨大芽孢杆菌myb3及其在秸秆发酵饲料中的应用
CN102076860A (zh) 制备酵母菌生物质的方法
CN106834141B (zh) 一种厌氧真菌及用其发酵水稻秸秆生产甲酸的方法
CN106834140B (zh) 一种厌氧真菌及用其发酵小麦秸秆生产乙醇的方法
CN106811438B (zh) 一种秸秆降解酸化菌剂及其制备方法
CN114672469B (zh) 一种犏牛瘤胃自然共培养物发酵粗饲料生产漆酶的方法
CN107535671A (zh) 提高瘤胃蛋白利用率的微生物发酵黄酒糟饲料及制备方法
CN103614323B (zh) 一种解淀粉芽孢杆菌的培养基及应用
CN114164188B (zh) 牦牛瘤胃厌氧真菌和甲烷菌共培养物降解芦苇秆生产漆酶的方法及应用
CN106987572B (zh) 一种厌氧发酵玉米秸秆生产木聚糖酶的方法
CN104140990A (zh) 一种以菜粕为原料液态发酵生产伊枯草菌素的方法
CN112725314B (zh) 一种自然共生的混合培养物发酵粗饲料生产内切葡聚糖酶的方法
CN107446868B (zh) 一株甲基营养型芽孢杆菌及其降解羽毛产寡肽的应用
CN110042072A (zh) 一种降解黄曲霉毒素b1的菌株及其应用
CN107326060B (zh) 一种厌氧发酵玉米秸秆生产乙酸的方法
CN102382771B (zh) 一种β-葡萄糖苷酶产生菌及利用该菌转化制备京尼平的方法
CN101580853A (zh) 鹅源草酸青霉发酵秸杆技术
CN106609250B (zh) 一种发酵中药渣有机肥菌剂的制备方法及其应用
CN114410486B (zh) 一株米曲霉菌株及其在饲用蛋白开发方面的应用
CN116083405A (zh) 一种酒糟降解酶制剂及菌酶协同生产单细胞蛋白的方法
CN116004399A (zh) 一株粗糙脉孢菌dz01菌株及其在发酵杜仲叶饲料中的应用
CN110592047B (zh) 一种梨囊鞭菌发酵秸秆生产阿魏酸酯酶的新方法和应用
CN108588135B (zh) 一种餐厨垃圾与废油脂生物柴油副产物粗甘油联合发酵生产乳酸的方法
CN110592048B (zh) 一种梨囊鞭菌发酵秸秆生产乙酰酯酶的方法和应用

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 17789228

Country of ref document: US

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21914317

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21914317

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 21914317

Country of ref document: EP

Kind code of ref document: A1