WO2020207048A1 - 一种短小芽孢杆菌抗菌活性物质的制备方法及应用 - Google Patents

一种短小芽孢杆菌抗菌活性物质的制备方法及应用 Download PDF

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WO2020207048A1
WO2020207048A1 PCT/CN2019/126333 CN2019126333W WO2020207048A1 WO 2020207048 A1 WO2020207048 A1 WO 2020207048A1 CN 2019126333 W CN2019126333 W CN 2019126333W WO 2020207048 A1 WO2020207048 A1 WO 2020207048A1
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compound
ethyl acetate
preparation
antibacterial
petroleum ether
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French (fr)
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楚杰
刘可春
何秋霞
王莹
赵博
张轩铭
科洛米耶茨艾米丽娅
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山东省科学院生物研究所
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/02—Acyclic compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/04—Oxygen or sulfur attached to an aliphatic side-chain of a carbocyclic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aliphatically bound aldehyde or keto groups, or thio analogues thereof; Derivatives thereof, e.g. acetals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35—Ketones, e.g. benzophenone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/78—Separation; Purification; Stabilisation; Use of additives
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/524—Preservatives
    • 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
    • C12R2001/07—Bacillus

Definitions

  • the invention relates to a preparation method and application of an antibacterial active substance of Bacillus pumilus, and belongs to the field of microbial technology and application technology.
  • Antibacterial active substances have been a research hotspot at home and abroad in recent years, and can be widely used in biological feed, biological control, medicine, natural food preservatives, and cosmetics.
  • Bacillus can produce a variety of antibacterial substances, and its antibacterial mechanism is unique. It has an inhibitory effect on gram-negative bacteria, gram-positive bacteria, molds and yeasts. It has broad-spectrum antibacterial, high efficiency, safety, and resistance to resistance. Since Johnson et al. reported that Bacillus subtilis can produce antibacterial substances, institutions at home and abroad have successively isolated a variety of antibacterial substances from different Bacillus, including peptides, lipopeptides and antibacterial proteins.
  • Bacillus pumilus is a kind of Bacillus, which is widely found in nature.
  • the vegetative cells are rod-shaped when observed under a microscope, with a positive Gram reaction and can move.
  • the colony morphology is divided into two types: translucent and opaque.
  • Bacillus pumilus can secrete very active cellulase, lipase, xylanase and pectin lyase, etc., which is beneficial to the degradation of macromolecular substances. It can also produce antagonistic substances such as antibiotics and antibacterial proteins, with a wide range of bacteriostasis, and has an inhibitory effect on a variety of pathogens.
  • Chinese Patent Document CN103563991A discloses the formulation and preparation method of water dispersible granules of the rice blast biocontrol bacteria Bacillus pumilus fungicide.
  • the solid water dispersible granules contain the original powder of Bacillus pumilus TW strain (2 ⁇ 10 12 cfu/g) and auxiliary agents, which include carriers, dispersants, wetting agents, disintegrants, photoprotectants and adhesives.
  • Binding agent its processing method is to mix the active ingredient and the auxiliary agent probe in a certain proportion, and obtain it through airflow pulverization, granulation and drying.
  • Chinese patent document CN105494441A discloses a wettable powder of Bacillus pumilus containing a spore germination agent and a preparation method thereof.
  • the raw material of the wettable powder is composed of the following weight components: Bacillus pumilus fermentation broth 40- 70 parts; filler: 20-30 parts of bentonite; wetting agent: 1-4 parts of polyethylene glycol; dispersant: 0.2-1 part of sodium lauryl sulfate; spore germination agent: L-alanine 0.1-0.5 Parts, 0.1-0.5 parts of calcium 2,6-pyridinedicarboxylate; UV protective agent: 0.5-2 parts of carboxymethyl cellulose, 0.2-1 part of ⁇ -dextrin; drying protection agent: 1-4 parts of maltose, grain 0.2-1 part of sodium carboxylate.
  • Chinese patent document CN103205383A discloses a Bacillus pumilus (Bacillus pumilus) E14CGMCC No.6682.
  • the present invention also discloses a method for culturing Bacillus pumilus E14, which includes the steps of inoculating the Bacillus pumilus E14 strain into 2216E liquid medium, and culturing at 28°C and 150 rpm for 20 hours to obtain a bacterial solution.
  • the Bacillus pumilus E14 of the present invention or the bacterial liquid obtained by the method has the antibacterial use of inhibiting Vibrio harveyi, and can be used in the preparation of drugs for the cultivation of shrimp and crab, or as an inhibitor Bacteria drugs are added to shrimp and crab breeding feed.
  • Chinese patent document CN102965299A discloses a fermentation process of Bacillus pumilus LD-b1 and its use in the prevention and treatment of cucumber brown spot, cucumber gray mold, cucumber sclerotia, apple rot, eggplant fusarium wilt, wheat Application to Kub, black spot of cabbage and tomato gray mold.
  • Qu Fabin and others investigated the effects of the fermentation broth of Bacillus pumilus on tomato seed germination and seedling growth. The results showed that the fermentation broth of Bacillus pumilus significantly promoted tomato seed germination, root growth, and plant height and plant weight of seedlings. Among them, the 50-fold dilution has the most significant effect, and the effect of foliar spraying on the seedling application method is significantly better than root irrigation and seed soaking.
  • Chinese patent document CN105494441A discloses a wettable powder of Bacillus pumilus containing a spore germination agent and a preparation method thereof.
  • the raw material of the wettable powder is composed of the following weight components: Bacillus pumilus fermentation broth 40- 70 parts; filler: 20-30 parts of bentonite; wetting agent: 1-4 parts of polyethylene glycol; dispersant: 0.2-1 part of sodium lauryl sulfate; spore germination agent: L-alanine 0.1-0.5 Parts, 0.1-0.5 parts of calcium 2,6-pyridinedicarboxylate; UV protective agent: 0.5-2 parts of carboxymethyl cellulose, 0.2-1 part of ⁇ -dextrin; drying protection agent: 1-4 parts of maltose, grain 0.2-1 part of sodium carboxylate.
  • the present invention provides a preparation method and application of the antibacterial active substance of Bacillus pumilus.
  • step (1) The seed liquid prepared in step (1) is inoculated into the fermentation medium at a ratio of 3 to 5% by volume, and cultured at 28 to 32°C and 160 to 200 rpm for 45 to 50 hours to prepare a fermentation broth ;
  • the components per liter of the fermentation medium are as follows:
  • the seed culture medium is an LB broth culture medium, and the components per liter are as follows:
  • the method for preparing the antibacterial active substance derived from Bacillus pumilus is as follows:
  • step (iii) The concentrated and dried product obtained in step (ii) is separated by high performance liquid chromatography, and the antibacterial active substances obtained are the above-mentioned compound I, compound II, and compound III respectively.
  • the solid-liquid separation is centrifugation at 3500-4500 rpm for 25-35 minutes.
  • the concentration is 8-12 times the concentration of the original solution by using rotary evaporation.
  • the volume ratio of the concentrated solution to ethyl acetate is 1:1; preferably, the number of extractions is 2 to 4 times.
  • the silica gel column is a silica gel column with 200-300 mesh.
  • the different gradients of different solutions of petroleum ether, ethyl acetate, and methanol are specifically as follows, all of which are volume ratios:
  • the drying is a nitrogen blowing drying method.
  • the separation conditions of high performance liquid chromatography are as follows:
  • the preparation column adopts YMC-Pack SIL column, 250mm ⁇ 10mm; the mobile phase is 60% ethyl acetate and 40% n-butanol, wavelength 260nm, injection volume 5 ⁇ L, flow rate 2.0mL/min, column temperature 25°C, The pressure is 40 bar.
  • the present invention discloses for the first time three enol compounds with antibacterial activity.
  • This type of compound has a strong inhibitory effect on pathogenic bacteria, and is prepared by microbial fermentation with short production cycle, stable product and high content. It is widely used in the production of pesticides, medicines, health care products and cosmetics.
  • Figure 1 is a mass spectrometry detection result diagram of Compound I
  • Figure 2 is a diagram showing the result of hydrogen spectrum detection of compound I
  • Figure 3 is a diagram of the carbon spectrum detection result of compound I
  • Figure 4 is a mass spectrum detection result diagram of compound II
  • Figure 5 is a diagram showing the result of hydrogen spectrum detection of compound II
  • Figure 6 is a diagram of the carbon spectrum detection results of compound II.
  • Figure 7 is a mass spectrometry detection result diagram of compound III
  • Figure 8 is a diagram of the detection result of the hydrogen spectrum of compound III.
  • Figure 9 is a diagram of the carbon spectrum detection result of compound III.
  • Figure 10 is a photo of the experimental results of antibacterial activity of the concentrated dried product prepared in step (ii) of Example 3;
  • Figure 11 is a photo of the experimental results of the antibacterial activity of the three compounds prepared in Example 3.
  • Zone of inhibition of compound I 1. Zone of inhibition of compound I; 2. Zone of inhibition of compound II; 3. Zone of inhibition of compound III;
  • Figure 12 is a graph showing the antibacterial results of Compound I against different strains
  • Figure 13 is a graph showing the antibacterial results of compound II against different strains
  • Figure 14 is a graph showing the antibacterial results of compound III against different strains
  • Bacillus pumilus (Bacillus pumilus) BP Bacillus pumilus (Bacillus pumilus) BP, this strain has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC for short, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing) on March 21, 2019. Institute of Microbiology, Chinese Academy of Sciences), the deposit number is CGMCC NO.17424.
  • Bacillus pumilus BP-5 purchased from the China Collection of Microorganisms, number: CGMCC 1.8167.
  • CGMCC Bacillus pumilus BP
  • the biological characteristics of the strain it belongs to the genus Bacillus, the bacterial body is thin rod-shaped, and it is Gram-positive. It grows transparent on the LB slope and can produce antibacterial active ingredients.
  • Bacillus pumilus BP The cultivation method of Bacillus pumilus BP is as follows:
  • the seed medium is LB broth medium, and the components per liter are as follows:
  • step (2) Inoculate the seed liquid prepared in step (1) into the fermentation medium in a proportion of 3% by volume, and cultivate it at 30° C. and 180 rpm for 48 hours to prepare the fermentation liquid;
  • the components per liter of the fermentation medium are as follows:
  • the preparation method of antibacterial active substance derived from Bacillus pumilus BP the steps are as follows:
  • step (ii) Extract the concentrated solution obtained in step (ii) with ethyl acetate, the volume ratio of the concentrated solution to ethyl acetate is 1:1, take the extract phase, extract 4 times, combine the extract phases, concentrate, and then pass 200
  • step (iii) Separating the concentrated and dried product obtained in step (ii) by high performance liquid chromatography to obtain antibacterial active substances as the aforementioned compound I, compound II, and compound III;
  • the preparation column adopts YMC-Pack SIL column, 250mm ⁇ 10mmI.D, S-5 ⁇ m, 12nm, SL 12S-2510WT, Ser.No.114EA80134; mobile phase is 60% ethyl acetate (Eta-00060458HPLC) and 40% N-butanol (a-He-00010273HPLC), wavelength 260nm, injection volume 5 ⁇ L, flow rate 2.0mL/min, column temperature 25°C, pressure 40bar.
  • the preparation method of antibacterial active substance derived from Bacillus pumilus the steps are as follows:
  • step (ii) Extract the concentrated solution obtained in step (ii) with ethyl acetate.
  • step (iii) Separating the concentrated and dried product obtained in step (ii) by high performance liquid chromatography to obtain antibacterial active substances as the aforementioned compound I, compound II, and compound III;
  • the preparation column adopts YMC-Pack SIL column, 250mm ⁇ 10mm; the mobile phase is 60% ethyl acetate and 40% n-butanol, wavelength 260nm, injection volume 5 ⁇ L, flow rate 2.0mL/min, column temperature 25°C, The pressure is 40 bar.
  • BP-5 Bacillus pumilus
  • Antibacterial test method Determine the growth of the pathogenic bacteria by detecting the OD 600 of the pathogenic bacteria at different times to determine whether it has antibacterial effects. The specific steps are as follows:
  • the pathogenic bacteria liquid Before the test, prepare the pathogenic bacteria liquid to 10 5 cfu/mL, add 100 ⁇ L of the pathogenic bacteria liquid to each EP tube according to the test amount on the ultra-clean workbench, centrifuge at high speed for 1 min, remove the supernatant and retain the bacteria , And add 900 ⁇ L of fresh LB broth medium, mark and add 100 ⁇ L of the test sample correspondingly, and mix. At 0 hours and 15 hours, OD 600 was measured and the data was recorded. Each test was repeated 3 times.

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Abstract

提供了一株短小芽孢杆菌(Bacillus pumilus)BP，保藏编号CGMCC NO.17424。还提供了从该短小芽孢杆菌中提取出三种具有抗菌活性的脂肪醇类化合物及其用途。

Description

一种短小芽孢杆菌抗菌活性物质的制备方法及应用 技术领域
本发明涉及一种短小芽孢杆菌抗菌活性物质的制备方法及应用，属于微生物技术及应用技术领域。
背景技术
目前，畜禽养殖业抗生素的大量使用，导致耐药性产生及兽药残留，严重影响了畜产品品质，排泄残渣也导致土壤和水体污染。破坏了生态环境，直接危害人类健康。环境污染造成了人类生存环境的恶化，导致食品安全问题日益突出。
抗菌活性物质是近年来国内外的研究热点，可广泛应用于生物饲料、生防、医药、天然食品防腐剂、化妆品等方面。其中芽孢杆菌可产生多种抗菌物质，其抗菌机理独特，对革兰阴性菌、革兰阳性菌、霉菌及酵母菌均有抑制作用，具有广谱抗菌、高效、安全、不易产生耐药性、易降解等特点，自Johnson等报道枯草芽孢杆菌可以产生抑菌物质以来，国内外学者先后从不同芽孢杆菌中分离得到多种抑菌物质，包括多肽类、脂肽类及抑菌蛋白类等。
短小芽孢杆菌为芽孢杆菌属的一种，广泛存在于自然界中，显微镜下观察营养细胞为杆状，革兰氏反应阳性，能运动。菌落形态分为半透明状和不透明状两种。短小芽孢杆菌能够分泌活性很强的纤维素酶、脂肪酶、木聚糖酶和果胶裂解酶等，有利于降解大分子物质。还可以产生抗菌素及抗菌蛋白等拮抗性物质，抑菌范围广，对多种病原菌都有抑制作用。
目前，国内已有关于生产短小芽孢杆菌类产品的相关技术，如:中国专利文献CN103563991A(申请号201210271396.4)公开了水稻稻瘟病生防菌短短小芽孢杆菌杀菌剂水分散粒剂剂型和制备方法。所述固体的水分散粒剂包含短短小芽孢杆菌TW菌株原粉(2×10 12cfu/g)和助剂，助剂包括载体、分散剂、湿润剂、崩解剂、光保护剂和粘结剂；其加工方法是将有效成分和助剂探子一定比例混合，经气流粉碎、造粒、干燥获得。
中国专利文献CN105494441A(申请号200710021509.1)公开了一种含有芽孢萌发剂的短小芽孢杆菌的可湿性粉剂及其制备方法，该可湿性粉剂的原料由下列重量组份组成：短小芽孢杆菌发酵液40-70份；填料：膨润土20-30份；润湿剂：聚乙二醇1-4份；分散剂：十二烷基硫酸钠0.2-1份；芽孢萌发剂：L-丙氨酸0.1-0.5份，2，6-吡啶二羧酸钙0.1-0.5份；紫外保护剂：羧甲基纤维素0.5-2份，β-糊精0.2-1份；干燥保护剂：麦芽糖1-4份，谷氨酸钠0.2-1份。
中国专利文献CN103205383A(申请号201310128271.0)公开了一种短小芽孢杆菌(Bacillus pumilus)E14CGMCC No.6682。本发明还公开了短小芽孢杆菌E14的培养方法，其步骤：将短小芽孢杆菌E14菌株接种到2216E液体培养基中，28℃、150rpm培养20h，即得菌液。本发明所述的短小芽孢杆菌E14或所述的方法培养得到的菌液具备抑制哈维氏弧菌(Vibrio harveyi)的抑菌用途，可以用于虾蟹养殖药物的制备，或者用于作为抑菌药物添加到虾蟹养殖饲料中。
中国专利文献CN102965299A(申请号201210290374.2)公开了一株短小芽孢杆菌LD-b1 的发酵工艺及其在防治黄瓜褐斑病，黄瓜灰霉病，黄瓜菌核病，苹果腐烂病，茄子枯萎病，小麦赤毒病,白菜黑斑病和番茄灰霉病上的应用。曲发斌等考察了短小芽孢杆菌发酵液对番茄种子萌发及幼苗生长的影响，结果表明:短小芽孢杆菌发酵液对番茄种子萌发,根芽生长及幼苗的株高和株重具有明显的促进作用，其中以50倍稀释液的效果最为显著,而在幼苗施用方式上叶面喷施的效果明显优于灌根和浸种。
中国专利文献CN105494441A(申请号201510951106.4)公开了一种含有芽孢萌发剂的短小芽孢杆菌的可湿性粉剂及其制备方法，该可湿性粉剂的原料由下列重量组份组成：短小芽孢杆菌发酵液40-70份；填料：膨润土20-30份；润湿剂：聚乙二醇1-4份；分散剂：十二烷基硫酸钠0.2-1份；芽孢萌发剂：L-丙氨酸0.1-0.5份，2，6-吡啶二羧酸钙0.1-0.5份；紫外保护剂：羧甲基纤维素0.5-2份，β-糊精0.2-1份；干燥保护剂：麦芽糖1-4份，谷氨酸钠0.2-1份。
上述技术的关注点多放在防治植物病害和促进植物生长方向，并未对短小芽孢杆菌的活性物质进行进一步研究的相关报道。
发明内容
本发明针对现有技术的不足，提供了一种短小芽孢杆菌抗菌活性物质的制备方法及应用。
本发明技术方案如下：
一株短小芽孢杆菌(Bacillus pumilus)BP，该菌株已于2019年3月21日保存在中国微生物菌种保藏管理委员会普通微生物中心，地址：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，保藏编号CGMCC NO.17424。
上述短小芽孢杆菌的培养方法，步骤如下：
(1)将上述短小芽孢杆菌接种于种子培养基中，在35～38℃、160～200转/分的条件下，培养14～16小时，制得种子液；
(2)将步骤(1)制得的种子液按体积百分比3～5％的比例接种至发酵培养基中，在28～32℃，160～200转/分培养45～50h，制得发酵液；
所述发酵培养基，每升组份如下：
葡萄糖30.0g，K 2HPO 4×3H 2O 7.0g，KH 2PO 4 3.0g，(NH 4) 2SO 4 1.5g，柠檬酸钠·3H 2O 0.5g，MgSO 4·7H 2O 0.1g，pH7.0～7.2，加水定容至1L。
根据本发明优选的，所述步骤(1)中，种子培养基为LB肉汤培养基，每升组份如下：
蛋白胨10克，氯化钠10克，酵母膏5克，水定容至1000毫升。
来源于短小芽孢杆菌的抗菌活性物质，结构式分别如下所示：
化合物I，名称为：6,7-(α-羟基)二乙基十二烷，英文名称：6,7-(α-hydroxy)-diethyl-dodecane：
Figure PCTCN2019126333-appb-000001
化合物II，名称为：4,5-二丁基-1,2-(α-甲基)-环己烷二甲醇，英文名称：4,5-dibutyl-1,2-(α-methyl)-cyclohexanedimethanol：
Figure PCTCN2019126333-appb-000002
化合物III，名称为：4,5-二丁基-1,2-(α-甲基)-环己烷二甲酮，英文名称：4,5-dibutyl-1,2-(α-methyl)-cyclohexanedimethanol：
Figure PCTCN2019126333-appb-000003
上述来源于短小芽孢杆菌的抗菌活性物质的制备方法，步骤如下：
(i)将上述发酵液经固液分离，取上清液，经浓缩，制得浓缩液；
(ii)将步骤(ii)制得的浓缩液经乙酸乙酯萃取，取萃取相，浓缩后经硅胶柱层析分离，分别用石油醚、乙酸乙酯、甲醇不同溶液不同梯度洗脱，收集具有抗菌活性的石油醚/乙酸乙酯＝50:50洗脱液，浓缩，干燥，制得浓缩干燥物；
(iii)将步骤(ii)制得的浓缩干燥物经高效液相色谱分离，制得抗菌活性物质分别为上述化合物I、化合物II、化合物III。
根据本发明优选的，所述步骤(i)中，固液分离为在3500～4500转/分的条件下离心25～35分钟。
根据本发明优选的，所述步骤(i)中，浓缩为采用旋转蒸发，浓缩至原溶液浓度的8～12倍。
根据本发明优选的，所述步骤(ii)中，浓缩液与乙酸乙酯的体积比为1:1；优选的， 萃取次数为2～4次。
根据本发明优选的，所述步骤(ii)中，硅胶柱为200～300目的硅胶柱。
根据本发明优选的，所述步骤(ii)中，石油醚、乙酸乙酯、甲醇不同溶液不同梯度具体如下，均为体积比：
石油醚、石油醚/乙酸乙酯＝75:25、石油醚/乙酸乙酯＝50:50；石油醚/乙酸乙酯＝25:75；石油醚/乙酸乙酯＝0:100；乙酸乙酯/甲醇＝75:25；乙酸乙酯/甲醇＝50:50；乙酸乙酯/甲醇＝25:75，乙酸乙酯/甲醇＝0:100。
根据本发明优选的，所述步骤(ii)中，干燥为采用氮吹干燥方式。
根据本发明优选的，所述步骤(iii)中，高效液相色谱分离条件如下：
制备柱采用YMC-Pack SIL柱，250mm×10mm；流动相为60％的乙酸乙酯和40％的正丁醇，波长260nm，进样量5μL，流速2.0mL/min，柱温为25℃，压力为40bar。
上述具有抗菌活性的化合物I、化合物II、化合物III在制备抑制致病菌药物中的应用。
上述具有抗菌活性的化合物I、化合物II、化合物III在制备食品防腐剂中的应用。
上述具有抗菌活性的化合物I、化合物II、化合物III在制备抗菌化妆品中的应用。
上述具有抗菌活性的化合物I、化合物II、化合物III在制备抗菌保健品的应用。
上述具有抗菌活性的化合物I、化合物II、化合物III在制备农药中的应用。
上述具有抗菌活性的化合物I、化合物II、化合物III在制备抑菌口香糖中的应用。
有益效果
1、本发明首次发现了一株短小芽孢杆菌(Bacillus pumilus)BP，该菌株可以生成三种具有抗菌活性的脂肪醇类化合物，而现有短小芽孢杆菌中并未发现上述三种抗菌活性物质；
2、本发明首次公开了三种烯醇类具有抗菌活性的化合物，该类化合物具有很强的抑制致病菌的作用，且采用微生物发酵制备生产周期短、产物稳定、含量高，该化合物可广泛应用于农药、医药、保健品及化妆品生产中。
附图说明
图1为化合物I的质谱检测结果图；
图2为化合物I的氢谱检测结果图；
图3为化合物I的碳谱检测结果图；
图4为化合物II的质谱检测结果图；
图5为化合物II的氢谱检测结果图；
图6为化合物II的碳谱检测结果图；
图7为化合物III的质谱检测结果图；
图8为化合物III的氢谱检测结果图；
图9为化合物III的碳谱检测结果图；
图10为实施例3步骤(ii)制得的浓缩干燥物的抑菌活性实验结果照片；
图11为实施例3制得的三种化合物的抑菌活性实验结果照片；
其中：1、化合物I的抑菌圈；2、化合物II的抑菌圈；3、化合物III的抑菌圈；
图12为化合物I针对不同菌株的抑菌结果曲线图；
图13为化合物II针对不同菌株的抑菌结果曲线图；
图14为化合物III针对不同菌株的抑菌结果曲线图；
具体实施方式
为进一步解释说明本发明，下面将结合具体实施方式对本发明所述的技术方案进行更详细的介绍，但实施例仅用于说明，不对本发明有任何限制，基于本发明教导所作的任何替换或变换，均属于本发明的保护范围。
生物材料
短小芽孢杆菌(Bacillus pumilus)BP，该菌株已于2019年3月21日保存在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：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)，保藏编号为CGMCC NO.17424。
短小芽孢杆菌BP-5，购于中国微生物菌种保藏，编号：CGMCC 1.8167。
实施例1
一株短小芽孢杆菌(Bacillus pumilus)BP，该菌株已于2019年3月21日保存在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：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)，保藏编号为CGMCC NO.17424。
该菌株的生物学特征：为芽孢杆菌属，菌体细杆状，革兰氏阳性。在LB斜面上生长为透明状，能产生抗菌活性成分。
实施例2
短小芽孢杆菌短(Bacillus pumilus)BP的培养方法，步骤如下：
(1)将上述短小芽孢杆菌接种于种子培养基中，在37℃、180转/分的条件下，培养16小时，制得种子液；
所述种子培养基为LB肉汤培养基，每升组份如下：
蛋白胨10克，氯化钠10克，酵母膏5克，水定容至1000毫升；
(2)将步骤(1)制得的种子液按体积百分比3％的比例接种至发酵培养基中，在30℃，180转/分培养48h，制得发酵液；
所述发酵培养基，每升组份如下：
葡萄糖30.0g，K 2HPO 4×3H 2O 7.0g，KH 2PO 4 3.0g，(NH 4) 2SO 4 1.5g，柠檬酸钠·3H 2O 0.5g，MgSO 4·7H 2O 0.1g，pH7.0～7.2，加水定容至1L。
实施例3
来源于短小芽孢杆菌(Bacillus pumilus)BP的抗菌活性物质的制备方法，步骤如下：
(i)将实施例2制备的发酵液在3500转/分的条件下离心35分钟，取上清液，采用旋转蒸发，浓缩至原溶液浓度的8倍，制得浓缩液；
(ii)将步骤(ii)制得的浓缩液经乙酸乙酯萃取，浓缩液与乙酸乙酯的体积比为1:1，取萃取相，萃取4次，合并萃取相，浓缩，然后经200目的硅胶柱层析分离，分别用石油醚、乙酸乙酯、甲醇不同溶液不同梯度洗脱，收集具有抗菌活性的石油醚/乙酸乙酯＝50:50洗脱 液，浓缩，氮吹干燥，制得浓缩干燥物；
所述石油醚、乙酸乙酯、甲醇不同溶液不同梯度具体如下，均为体积比：石油醚、石油醚/乙酸乙酯＝75:25、石油醚/乙酸乙酯＝50:50；石油醚/乙酸乙酯＝25:75；石油醚/乙酸乙酯＝0:100；乙酸乙酯/甲醇＝75:25；乙酸乙酯/甲醇＝50:50；乙酸乙酯/甲醇＝25:75，乙酸乙酯/甲醇＝0:100；
采用琼脂扩散法方式检测大肠杆菌抑菌活性，如图10所示；
(iii)将步骤(ii)制得的浓缩干燥物经高效液相色谱分离，制得抗菌活性物质分别为上述化合物I、化合物II、化合物III；
所述高效液相色谱分离条件如下：
制备柱采用YMC-Pack SIL柱，250mm×10mmI.D，S-5μm,12nm，SL 12S-2510WT，Ser.No.114EA80134；流动相为60％的乙酸乙酯(Eta-00060458HPLC)和40％的正丁醇(a-He-00010273HPLC)，波长260nm，进样量5μL，流速2.0mL/min，柱温为25℃，压力为40bar。
分别对化合物I、化合物II、化合物III进行质谱、氢谱、碳谱检测，检测结果如图1-9所示；通过上述结果得出化合物I，名称为：6,7-(α-羟基)二乙基十二烷，英文名称：6,7-(α-hydroxy)-diethyl-dodecane，结构式如下：
Figure PCTCN2019126333-appb-000004
化合物II，名称为：4,5-二丁基-1,2-(α-甲基)-环己烷二甲醇，英文名称：4,5-dibutyl-1,2-(a-methyl)-cyclohexanedimethanol，结构式如下：
Figure PCTCN2019126333-appb-000005
化合物III，名称为：4,5-二丁基-1,2-(α-甲基)-环己烷二甲酮，英文名称：4,5-dibutyl-1,2-(a-methyl)-cyclohexanedimethanol，结构式如下：
Figure PCTCN2019126333-appb-000006
对上述三种化合物分别进行大肠杆菌的抑菌实验，实验化合物浓度分别为2.6mg/mL，3mg/mL，3.2mg/mL，根据实验进行稀释，结果如图11所示。
实施例4
来源于短小芽孢杆菌(Bacillus pumilus)的抗菌活性物质的制备方法，步骤如下：
(i)将实施例2制备的发酵液在4500转/分的条件下离心25分钟，取上清液，采用旋转蒸发，浓缩至原溶液浓度的12倍，制得浓缩液；
(ii)将步骤(ii)制得的浓缩液经乙酸乙酯萃取，浓缩液与乙酸乙酯的体积比为1:1，取萃取相，萃取2次，合并萃取相，浓缩，然后经200目的硅胶柱层析分离，分别用石油醚、乙酸乙酯、甲醇不同溶液不同梯度洗脱，收集具有抗菌活性的石油醚/乙酸乙酯＝50:50洗脱液，浓缩，氮吹干燥，制得浓缩干燥物；
所述石油醚、乙酸乙酯、甲醇不同溶液不同梯度具体如下，均为体积比：石油醚、石油醚/乙酸乙酯＝75:25、石油醚/乙酸乙酯＝50:50；石油醚/乙酸乙酯＝25:75；石油醚/乙酸乙酯＝0:100；乙酸乙酯/甲醇＝75:25；乙酸乙酯/甲醇＝50:50；乙酸乙酯/甲醇＝25:75，乙酸乙酯/甲醇＝0:100；
(iii)将步骤(ii)制得的浓缩干燥物经高效液相色谱分离，制得抗菌活性物质分别为上述化合物I、化合物II、化合物III；
所述高效液相色谱分离条件如下：
制备柱采用YMC-Pack SIL柱，250mm×10mm；流动相为60％的乙酸乙酯和40％的正丁醇，波长260nm，进样量5μL，流速2.0mL/min，柱温为25℃，压力为40bar。
对分离的三种化合物进行检测，结果同实施例3。
对比例1
采用现有的短小芽孢杆菌(BP－5)，购于中国微生物菌种保藏，编号：CGMCC1.8167。
按照实施例2-3的方法进行制备，结果未获得相关的化合物I～III(脂肪醇类化合物)。
实验例
抗菌试验方法：通过检测致病细菌不同时段的OD 600来测定该致病菌生长情况，判断是否具有抑菌效果。具体操作步骤如下：
试验前将致病菌液配制成10 5cfu/mL，在超净工作台上，根据试验用量，在每个EP管中添加100μL致病菌菌液，高速离心1min，去除上清保留菌体，并添加900μL新鲜LB 肉汤培养基，标记并对应添加100μL待测样品，混匀。分别在0小时和15小时，测定OD 600，记录数据。每组试验重复3次。
经检测，化合物I针对不同菌株的抑菌结果如图12所示；化合物II针对不同菌株的抑菌结果如图13所示；化合物III针对不同菌株的抑菌结果如图14所示。

Claims (10)

  1. 一株短小芽孢杆菌(Bacillus pumilus)BP，该菌株已于2019年3月21日保存在中国微生物菌种保藏管理委员会普通微生物中心，地址：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，保藏编号CGMCC NO.17424。
  2. 如权利要求1所述的短小芽孢杆菌的培养方法，其特征在于，步骤如下：
    (1)将上述短小芽孢杆菌接种于种子培养基中，在35～38℃、160～200转/分的条件下，培养14～16小时，制得种子液；
    (2)将步骤(1)制得的种子液按体积百分比3～5％的比例接种至发酵培养基中，在28～32℃，160～200转/分培养45～50h，制得发酵液；
    所述发酵培养基，每升组份如下：
    葡萄糖30.0g，K 2HPO 4×3H 2O 7.0g，KH 2PO 4 3.0g，(NH 4) 2SO 4 1.5g，柠檬酸钠·3H 2O 0.5g，MgSO 4·7H 2O 0.1g，pH7.0～7.2，加水定容至1L。
  3. 如权利要求2所述的培养方法，其特征在于，所述步骤(1)中，种子培养基为LB肉汤培养基，每升组份如下：
    蛋白胨10克，氯化钠10克，酵母膏5克，水定容至1000毫升。
  4. 来源于权利要求1所述短小芽孢杆菌(Bacillus pumilus)BP的抗菌活性物质，结构式分别如下所示：
    化合物I，名称为：6,7-(α-羟基)二乙基十二烷：
    Figure PCTCN2019126333-appb-100001
    化合物II，名称为：4,5-二丁基-1,2-(α-甲基)-环己烷二甲醇：
    Figure PCTCN2019126333-appb-100002
    化合物III，名称为：4,5-二丁基-1,2-(α-甲基)-环己烷二甲酮：
    Figure PCTCN2019126333-appb-100003
  5. 权利要求4所述来源于短小芽孢杆菌的抗菌活性物质的制备方法，其特征在于，步骤如下：
    (i)将上述发酵液经固液分离，取上清液，经浓缩，制得浓缩液；
    (ii)将步骤(ii)制得的浓缩液经乙酸乙酯萃取，取萃取相，浓缩后经硅胶柱层析分离，分别用石油醚、乙酸乙酯、甲醇不同溶液不同梯度洗脱，收集具有抗菌活性的石油醚/乙酸乙酯＝50:50洗脱液，浓缩，干燥，制得浓缩干燥物；
    (iii)将步骤(ii)制得的浓缩干燥物经高效液相色谱分离，制得抗菌活性物质分别为上述化合物I、化合物II、化合物III。
  6. 如权利要求5所述的制备方法，其特征在于，所述步骤(i)中，固液分离为在3500～4500转/分的条件下离心25～35分钟；
    优选的，所述步骤(i)中，浓缩为采用旋转蒸发，浓缩至原溶液浓度的8～12倍。
  7. 如权利要求5所述的制备方法，其特征在于，所述步骤(ii)中，浓缩液与乙酸乙酯的体积比为1:1；优选的，萃取次数为2～4次；
    优选的，所述步骤(ii)中，硅胶柱为200～300目的硅胶柱；
    优选的，所述步骤(ii)中，石油醚、乙酸乙酯、甲醇不同溶液不同梯度具体如下，均为体积比：
    石油醚、石油醚/乙酸乙酯＝75:25、石油醚/乙酸乙酯＝50:50；石油醚/乙酸乙酯＝25:75；石油醚/乙酸乙酯＝0:100；乙酸乙酯/甲醇＝75:25；乙酸乙酯/甲醇＝50:50；乙酸乙酯/甲醇＝25:75，乙酸乙酯/甲醇＝0:100；
    优选的，所述步骤(ii)中，干燥为采用氮吹干燥方式。
  8. 如权利要求5所述的制备方法，其特征在于，所述步骤(iii)中，高效液相色谱分离条件如下：
    制备柱采用YMC-Pack SIL柱，250mm×10mm；流动相为60％的乙酸乙酯和40％的正丁醇，波长260nm，进样量5μL，流速2.0mL/min，柱温为25℃，压力为40bar。
  9. 权利要求4所述具有抗菌活性的化合物I、化合物II、化合物III在制 备抑制致病菌药物中的应用；
    优选的，所述具有抗菌活性的化合物I、化合物II、化合物III在制备食品防腐剂中的应用。
  10. 权利要求4所述具有抗菌活性的化合物I、化合物II、化合物III在制备抗菌化妆品中的应用；
    优选的，所述具有抗菌活性的化合物I、化合物II、化合物III在制备抗菌保健品的应用；
    优选的，所述具有抗菌活性的化合物I、化合物II、化合物III在制备农药中的应用；
    优选的，所述具有抗菌活性的化合物I、化合物II、化合物III在制备抑菌口香糖中的应用。
PCT/CN2019/126333 2019-04-10 2019-12-18 一种短小芽孢杆菌抗菌活性物质的制备方法及应用 WO2020207048A1 (zh)

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