WO2018054059A1 - 一种应用解淀粉芽孢杆菌生产生物表面活性素的方法 - Google Patents

一种应用解淀粉芽孢杆菌生产生物表面活性素的方法 Download PDF

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WO2018054059A1
WO2018054059A1 PCT/CN2017/082424 CN2017082424W WO2018054059A1 WO 2018054059 A1 WO2018054059 A1 WO 2018054059A1 CN 2017082424 W CN2017082424 W CN 2017082424W WO 2018054059 A1 WO2018054059 A1 WO 2018054059A1
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bacillus amyloliquefaciens
strain
ammonium
biosurfactant
fermentation medium
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徐岩
吴群
郅岩
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江南大学
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    • C12R2001/07—Bacillus

Definitions

  • the invention relates to a method for producing biosurfactant by using Bacillus amyloliquefaciens, belonging to the technical field of industrial microorganisms.
  • the biosurfactant is a ring of long-chain fatty acids (C 12 -C 17 ) and 7-amino acid polypeptides linked by lactone bonds synthesized by Bacillus subtilis via the non-ribosomal pathway (NRPS).
  • NRPS non-ribosomal pathway
  • Lipopeptide which has antibacterial, antiviral, antitumor, thrombolytic, cholesterol-lowering medicinal effects and superior surface active functions.
  • the broad-spectrum antibacterial effect of biosurfactant is expected to make up for the ubiquitous antibiotic resistance problem.
  • the antiviral and anti-tumor functions of biosurfactant also provide some options for the design of new drugs, while biosurfactants are superior.
  • the surface active function (capable of reducing the surface tension of water molecules from 72mN/M to 27mN/M) has broad application prospects in cosmetics, pharmaceuticals, emulsifiers, moisturizers and fine chemicals.
  • the output value of the world surfactant industry in 2006 was more than 20 billion US dollars, of which North America, Western Europe and China accounted for more than 70% of the market.
  • biosurfactant has shown broad application value and market prospects. Therefore, the development of microbial strains and production processes capable of efficiently synthesizing surfactin are of great significance for the industrial production and popularization of surfactin.
  • biosurfactant-producing bacteria mainly adopts the combination of mutation breeding and random screening. Although the biosurfactant yield of the mutagenized strain is improved, it is still due to low yield and poor strain stability. Can not be applied to industrial production, screening wild-type high-yield strains is still the primary task to achieve industrial production of biosurfactants.
  • Bacillus subtilis Bacillus amyloliquefaciens
  • Bacillus membrane formation pathway is activated, the synthesis of biosurfactin is feedback-inhibited, and the metabolic synthesis of the surfactant is also terminated.
  • Bacillus in nature can achieve the goal of coping with environmental stress and gaining competitive advantage by forming a biofilm, so its biosurfactin synthesis ability is weak, and only the dose that induces biofilm formation can be synthesized.
  • biosurfactant surfactin has antibiotic activity, and through the non-specific attack on the phospholipid bilayer of the cell membrane, cytoplasmic leakage is formed, thereby achieving the purpose of killing microorganisms. This is another reason why most Bacillus cannot over-synthesize surfactin.
  • the original yield of Bacillus wilfordii is generally 100-200mg/L.
  • the yield of biosurfactant can be increased to some extent after fermentation medium and fermentation conditions, it is necessary to continue to increase the yield of biosurfactant. Expensive special fermentation equipment is required, such as the application of a fermentation-membrane extraction coupling reactor, and the highest yield of biosurfactant of Bacillus subtilis is about 6.5 g/L.
  • the invention first provides an ultra-efficient synthetic biosurfactant surfactin Bacillus amyloliquefaciens MT45, which was deposited on June 6, 2016 at the General Microbiology Center of the China Microbial Culture Collection Management Committee, with the preservation number CGMCC NO.12593, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China. Institute of Microbiology, Chinese Academy of Sciences.
  • the colony morphology of the B. amyloliquefaciens MT45 strain is as follows: the colony is round and translucent, the surface of the colony is smooth and wrinkle-free, and no biofilm is formed, and the colony is slightly convex in the center, and the cells are rod-shaped and spore-forming under the microscope. Gram stain positive.
  • the invention also provides a method for producing biosurfactant by using the above B. amyloliquefaciens MT45, comprising the following steps:
  • the seed culture solution obtained in the step (1) is inoculated in a fermentation medium at a dose of 1% to 5%, and cultured at 25 to 37 ° C for 30 to 55 hours.
  • the seed medium formulation is glucose 2%, yeast powder 1%, sodium chloride 0.5%, natural pH.
  • the fermentation medium has a carbon source of 10-100 g/L, a nitrogen source of 1-10 g/L, a peptone of 0.1-1.5 g/L, and a potassium dihydrogen phosphate of 2-10 g/ L, disodium hydrogen phosphate 2-10g / L, magnesium sulfate 0-0.2g / L;
  • the carbon source includes one of sucrose, glucose, fructose, mannose, chitosan, glycerin, soluble starch, dextrin Or several;
  • the nitrogen source comprises one or more of ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium carbonate, ammonium hydrogencarbonate.
  • the carbon source of the fermentation medium is sucrose, glucose, fructose, or glycerol.
  • the carbon source of the fermentation medium is glucose or sucrose.
  • the nitrogen source of the fermentation medium is ammonium nitrate, ammonium carbonate or ammonium sulfate.
  • the fermentation medium is formulated by a carbon source of 10-30 g/L, a nitrogen source of 2-6 g/L, a peptone of 0.1-1.5 g/L, and a potassium dihydrogen phosphate of 2-10 g/ L, disodium hydrogen phosphate 2-10 g / L, magnesium sulfate 0-0.2 g / L.
  • the fermentation medium is formulated by sucrose or glucose 10-30 g/L, ammonium nitrate or sodium carbonate or ammonium sulfate 2-6 g/L, peptone 0.1-1.5 g/L, phosphoric acid. Potassium dihydrogen 2-10g / L, disodium hydrogen phosphate 2-10g / L, magnesium sulfate 0.01-0.2g / L.
  • the fermentation medium has a formulation of 30 g/L of sucrose, 6 g/L of ammonium nitrate, 0.5 g/L of peptone, 2-10 g/L of potassium dihydrogen phosphate, and disodium hydrogen phosphate 2 -10 g/L, magnesium sulfate 0.18 g/L.
  • the fermentation medium has a formulation of sucrose 10 g/L, ammonium nitrate 6 g/L, peptone 1.5 g/L, potassium dihydrogen phosphate 2 g/L, and disodium hydrogen phosphate 2 g/L. .
  • the fermentation medium has a formulation of 30 g/L of sucrose, 6 g/L of ammonium nitrate, 0.5 g/L of peptone, 2-10 g/L of potassium dihydrogen phosphate, and disodium hydrogen phosphate 2 -10 g/L, magnesium sulfate 0.18 g/L.
  • the fermentation medium has a formulation of 10 g/L of sucrose, 6 g/L of nitrogen source, 1.5 g/L of peptone, 0.14 g/L of zinc sulfate, and 4 g/L of potassium dihydrogen phosphate.
  • step (1) picks a B. amyloliquefaciens CGMCC NO. 12593 pure seed colony, is seeded in a seed culture medium, and is cultured at a temperature of 37 ° C and a shaking speed of 200 rpm for 24 hours to obtain a seed. Culture medium.
  • the invention provides a novel strain of Bacillus amyloliquefaciens MT45, which is capable of super-synthesis of biosurfactant by using an inorganic salt medium.
  • the biosynthesis of the strain is synchronized with the growth of the cells, and the product (surfactin) synthesis is synchronized with the growth of the strain without lag period.
  • the strain does not form a biofilm, avoids product feedback inhibition, and the strain is highly resistant to the product.
  • the culture method provided by the invention adopts a preferred ratio of a preferred carbon source and a nitrogen source and a carbon nitrogen source, and can synthesize and produce 9.43 g/L of biosurfactant at a maximum of 55 h, which can meet the requirements of industrial large-scale production and has a huge Industrial application prospects.
  • Bacillus amyloliquefaciens has been deposited with the General Microbiology Center of the General Microorganisms Collection and Management Committee of China on June 6, 2016. The deposit number is CGMCC No.12593, and the deposit address is Beichen West Road, Chaoyang District, Beijing. No. 3 Institute No. 3 Institute of Microbiology, Chinese Academy of Sciences.
  • Figure 1 shows the colony morphology (A) and micrograph (B) of B. amyloliquefaciens MT45 on nutrient agar plates.
  • Figure 2 is a phylogenetic tree of B. amyloliquefaciens MT45 based on 16s rRNA sequence alignment.
  • FIG. 3 is a growth curve of Bacillus amyloliquefaciens MT45 (A) and a model microorganism Bacillus subtilis ATCC 6051 (B) in a fermentation medium and a synthetic curve of biosurfactant.
  • composition of the activated culture solution is: 1%-5% of glucose, 1%-5% of yeast powder, 0.3%-1.0% of sodium chloride, water balance, pH 6.5-7.0.
  • the solid medium composition is: 1-10 mM potassium phosphate, 50-100 mM Mops, 2-6 mM magnesium chloride, 0.1-1 mM calcium chloride, 10-80 ⁇ M manganese chloride, 10-80 ⁇ M ferric chloride, 2-6 ⁇ M zinc chloride, 2-6 ⁇ M thiamine, 0.1-1% glycerol, 0.1-1% glutamic acid, 1%-2% agar.
  • Nutrient medium peptone 2%, yeast powder 1%, glucose 0.5%, pH 7.5.
  • Seed medium glucose 2%, yeast powder 1%, sodium chloride 0.5%, natural pH.
  • the fermentation medium carbon source 10-100g/L, nitrogen source 1-10g/L, peptone 0.1-1.5g/L, potassium dihydrogen phosphate 2-10g/L, disodium hydrogen phosphate 2-10g/L, Magnesium sulfate 0.01-0.1 g / L.
  • the sample was collected from the Chinese liquor brewing high temperature Daqu, and 5 g of the sample was dissolved in 10 ml of a 1% by mass sodium chloride solution, vortexed and mixed, and then 1 mL was inoculated into the activated medium at a temperature of 50-60 ° C, 200 rpm. It is activated for 24 to 36 hours under the condition of rotation speed to prepare a mature activated bacterial liquid.
  • the mature activated bacterial solution was taken and diluted with a 1% by mass sodium chloride solution.
  • a dilution with a dilution gradient of 10 -2 -10 -6 was selected and applied sequentially to a solid medium plate.
  • the culture was carried out for 48-72 h at a temperature of 30 ° C and a humidity of 40% - 60%; a large number of single colonies were obtained.
  • the biosurfactant content in the fermentation broth was detected, and finally a pure culture of the high-produced surfactant active peptide was obtained, which was numbered MT45.
  • Determination of biosurfactant in fermentation broth Accurate quantification of biosurfactants by ultra performance liquid chromatography.
  • the waters BEH C18 column was used, and the mobile phases were (A) 0.1% aqueous formic acid and (B) HPLC grade methanol, elution conditions of 30%-70% methanol for 5 min, 70%-100% methanol for 1 min. Samples were detected at 210 nm using a diode array detector. After integrating the obtained chromatographic peaks, the insertion method was used to compare with the standard curve to obtain the exact content of the surfactant.
  • Example 3 The strains obtained by screening in Example 1 were subjected to strain identification.
  • the strain numbered MT45 obtained in Example 1 was subjected to strain identification.
  • the B. amyloliquefaciens MT45 strain forms round and translucent colonies on the solid plate medium; the surface of the colony is smooth and wrinkle-free, and no biofilm is represented by the fold colonies; the colony has slight protrusions. Under the microscope, the cells were rod-shaped, forming spores, and Gram staining was positive (Fig. 1).
  • the MT45 was determined to be Bacillus amyloliquefaciens (Fig. 2).
  • amyloliquefaciens MT45 was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on June 6, 2016, and the deposit number is CGMCC No. 12593.
  • the fermentation medium was inoculated with a seeding amount of 5%, and the fermentation medium was composed of glucose 10 g/L, peptone 1.5 g/L, and phosphoric acid. Potassium hydrogen chloride 2g / L, disodium hydrogen phosphate 2g / L, magnesium sulfate 0.18g / L.
  • the fermentation was carried out at 37 ° C and 200 rpm using a liquid volume of 100 mL in a 500 ml triangular flask. Timed sampling was used to determine the amount of cell growth and biosurfactant production during fermentation. The results are shown in Fig. 3.
  • the fermentation of Bacillus amyloliquefaciens MT45 to 55 h can synthesize 3.0 g/L of biosurfactant.
  • Bacillus amyloliquefaciens MT45 can synthesize biosurfactant with high efficiency, and the highest yield can reach 50% of biomass; the high concentration of biosurfactant synthesized during culture also shows the high tolerance of strain MT45 to the product. Compared with the model strain Bacillus subtilis ATCC6051, biosurfactant synthesis was carried out simultaneously with the growth of the cells, and the product synthesis had no lag phase. These characteristics are beneficial to shorten the fermentation cycle of B. amyloliquefaciens MT45, produce more target products, and facilitate the industrial application of MT45 strain.
  • Example 5 Utilization of Bacillus amyloliquefaciens MT45 on a carbon source.
  • Bacillus amyloliquefaciens MT45 can produce various biosurfactants using various sugars as carbon sources, mainly sucrose, glucose, fructose, mannose, chitosan, glycerin, soluble starch, dextrin.
  • the other components of the fermentation medium were: peptone 0.5 g/L, potassium dihydrogen phosphate 2.7 g/L, disodium hydrogen phosphate 5 g/L, and magnesium sulfate 0.5 g/L.
  • the concentration of the carbon source was 10 g/L.
  • the 250 mL Erlenmeyer flask was loaded with 50 mL, the inoculum amount was 2%, and the culture conditions were 37 ° C, 200 rpm. The results are shown in Table 1.
  • Example 6 Utilization of Ammonium Bacteria by Bacillus amyloliquefaciens MT45.
  • Bacillus amyloliquefaciens MT45 can utilize various inorganic ammonium salts to produce biosurfactants, mainly ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium carbonate and ammonium hydrogencarbonate.
  • the other components of the fermentation medium are: sucrose 20g / L, peptone 0.5g / L, Potassium dihydrogen phosphate 2.7 g / L, disodium hydrogen phosphate 5 g / L, magnesium sulfate 0.5 g / L.
  • the concentration of the nitrogen source was 8 g/L.
  • the 250 mL Erlenmeyer flask was loaded with 50 mL, the inoculum amount was 2%, and the culture conditions were 37 ° C, 200 rpm. The results are shown in Table 2.
  • Example 7 Bacillus amyloliquefaciens MT45 Production of Biosurfactants Using Different Fermentation Media
  • LB medium peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L.
  • the crude biosurfactant obtained in Example 7 was purified by a method of preparing a liquid phase.
  • a C18 column was used, and the mobile phase was 0.1% aqueous formic acid and methanol, and the elution conditions were linear elution (30%-70% methanol 60 min).
  • the UV detector was set to detect a wavelength of 210 nm.
  • the eluted fraction between 40.5 and 53.5 minutes was collected, and the eluted fraction was subjected to rotary evaporation to remove the solvent to obtain a pure biosurfactant (Fig. 4).

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Abstract

一种应用解淀粉芽孢杆菌生产生物表面活性素的方法，属于工业微生物技术领域。应用筛选得到的解淀粉芽孢杆菌CGMCC No.12593合成生物表面活性素(surfactin)，产量高达9.43g/L，并且发酵过程具有生物表面活性素合成与菌株生长同步且无延滞期的特点。所述菌株及发酵方法将在生物表面活性素的工业生产中发挥重要作用，应用前景广阔。

Description

一种应用解淀粉芽孢杆菌生产生物表面活性素的方法 技术领域
本发明涉及一种应用解淀粉芽孢杆菌生产生物表面活性素的方法，属于工业微生物技术领域。
背景技术
生物表面活性素，即surfactin，是枯草芽孢杆菌经非核糖体途径(NRPS)合成的由长链脂肪酸(C12-C17)和含7个氨基酸的多肽经内酯键连接起来的一种环状脂肽，具有抗菌、抗病毒、抗肿瘤、溶血栓、降胆固醇的药用功效以及优越的表面活性功能。生物表面活性素的广谱抗菌功效有望弥补现今普遍存在的抗生素耐药性难题，生物表面活性素的抗病毒和抗肿瘤功能也为新型药物设计提供了一些可选方案，而生物表面活性素优越的表面活性功能(能够将水分子的表面张力从72mN/M降低至27mN/M)使其在化妆品，医药，乳化剂，保湿剂和精细化工中有着广泛的应用前景。据统计，2006年世界表面活性剂产业的产值在200亿美元以上，其中北美，西欧和中国占据了70％以上的市场份额。生物表面活性素作为一种优越的生物表面活性剂，显示出广阔的应用价值和市场前景。因此，开发能够高效合成surfactin的微生物菌株及生产工艺对于surfactin的工业化生产和普及有重要意义。
目前，生物表面活性素产生菌的选育主要采用诱变育种和随机筛选相结合的方法，虽然诱变菌株的生物表面活性素产量有所提高，但仍由于产率过低、菌株稳定性差而无法应用于工业生产，筛选野生型高产菌株仍然是实现生物表面活性素工业化生产的首要任务。
据文献报道，芽孢杆菌(枯草芽孢杆菌，解淀粉芽孢杆菌)在菌体生长过程中仅有部分细胞能合成生物表面活性素，继而诱发形成生物菌膜(biofilm)。芽孢杆菌菌膜形成途径被激活后，生物表面活性素的合成受到反馈抑制，继而表面活性素的代谢合成也随之结束。自然界中的芽孢杆菌通过形成生物菌膜以达到应对环境压力，获取竞争优势的目的，因而其生物表面活性素的合成能力都较弱，仅能合成诱导生物膜形成的剂量。此外，生物表面活性素surfactin具有抗生素活性，通过非特异性攻击细胞膜的磷脂双分子层，形成胞浆渗漏，从而达到杀灭微生物的目的。这也是大多数芽孢杆菌不能过量合成surfactin的另一原因。野生型芽孢杆菌的原始产量一般为100-200mg/L，虽然经发酵培养基及发酵条件后，生物表面活性素的产量能达到一定程度的提高，要想继续提高生物表面活性素的产率则需要昂贵的特殊发酵设备，如应用发酵-膜提取偶联反应器，枯草芽孢杆菌的生物表面活性素最高产量约为6.5g/L。昂贵的生产成本是生物表面活性素仍不能商业化生产的主要因素。因此，获得高产生 物表面活性素的天然高产菌株，提高生产效率，降低生产成本，仍然是生物表面活性素商业化应用的根本解决途径。
发明内容
本发明首先提供一株超高效合成生物表面活性素surfactin的解淀粉芽孢杆菌(Bacillusamyloliquefaciens)MT45，该菌株已于2016年6月6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.12593，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。
所述的解淀粉芽孢杆菌MT45菌株的菌落形态表现为：菌落圆形、半透明，菌落表面光滑无褶皱、不形成生物菌膜，菌落中央微微凸起，显微镜下细胞呈杆状、形成芽孢，革兰氏染色阳性。
本发明还提供了一种应用上述解淀粉芽孢杆菌MT45生产生物表面活性素的方法，包括以下步骤：
(1)挑取解淀粉芽孢杆菌CGMCC NO.12593纯种菌落，活化得到种子培养液；
(2)将步骤(1)得到的种子培养液按照1％-5％的接种量接种于发酵培养基中，并在25-37℃条件下培养30-55h。
在本发明的一种实施方式中，所述种子培养基配方为葡萄糖2％、酵母粉1％、氯化钠0.5％，自然pH。
在本发明的一种实施方式中，所述发酵培养基的配方为碳源10-100g/L，氮源1-10g/L，蛋白胨0.1-1.5g/L，磷酸二氢钾2-10g/L，磷酸氢二钠2-10g/L，硫酸镁0-0.2g/L；所述碳源包括蔗糖、葡萄糖、果糖、甘露糖、壳聚糖、甘油、可溶性淀粉、糊精中的一种或几种；所述氮源包括硝酸铵，硫酸铵，氯化铵，碳酸铵，碳酸氢铵中的一种或几种。
在本发明的一种实施方式中，所述发酵培养基的碳源为蔗糖、葡萄糖、果糖、或甘油。
在本发明的一种实施方式中，所述发酵培养基的碳源为葡萄糖或蔗糖。
在本发明的一种实施方式中，所述发酵培养基的氮源为硝酸铵、碳酸铵或硫酸铵。
在本发明的一种实施方式中，所述发酵培养基的配方为碳源10-30g/L，氮源2-6g/L，蛋白胨0.1-1.5g/L，磷酸二氢钾2-10g/L，磷酸氢二钠2-10g/L，硫酸镁0-0.2g/L。
在本发明的一种实施方式中，所述发酵培养基的配方为蔗糖或葡萄糖10-30g/L，硝酸铵或碳酸安或硫酸铵2-6g/L，蛋白胨0.1-1.5g/L，磷酸二氢钾2-10g/L，磷酸氢二钠2-10g/L，硫酸镁0.01-0.2g/L。
在本发明的一种实施方式中，所述发酵培养基的配方为蔗糖30g/L，硝酸铵6g/L，蛋白胨0.5g/L，磷酸二氢钾2-10g/L，磷酸氢二钠2-10g/L，硫酸镁0.18g/L。
在本发明的一种实施方式中，所述发酵培养基的配方为蔗糖10g/L，硝酸铵6g/L，蛋白胨1.5g/L，磷酸二氢钾2g/L，磷酸氢二钠2g/L。
在本发明的一种实施方式中，所述发酵培养基的配方为蔗糖30g/L，硝酸铵6g/L，蛋白胨0.5g/L，磷酸二氢钾2-10g/L，磷酸氢二钠2-10g/L，硫酸镁0.18g/L。
在本发明的一种实施方式中，所述发酵培养基的配方为蔗糖10g/L，氮源6g/L，蛋白胨1.5g/L，硫酸锌0.14g/L，磷酸二氢钾4g/L，磷酸氢二钠2g/L，硫酸镁0.18g/L。
在本发明的一种实施方式中，步骤(1)挑取解淀粉芽孢杆菌CGMCC NO.12593纯种菌落，种于种子培养基，在温度37℃，摇床转速为200rpm条件下培养24h得到种子培养液。
本发明具有以下有益效果：
本发明提供一种新型解淀粉芽孢杆菌MT45菌株，该菌株能够利用无机盐培养基超强合成生物表面活性素。所述菌株生物表面活性素的合成与菌体生长同步，具有产物(surfactin)合成与菌株生长同步且无延滞期，菌株不形成生物菌膜、避免产物反馈抑制，菌株对产物耐受性强的特点。本发明提供的培养方法采用优选的碳源和氮源及碳氮源优选的配比，发酵55h能最高合成生产9.43g/L的生物表面活性素、，能够满足工业大规模生产要求，具有巨大的工业应用前景。
生物材料保藏
解淀粉芽孢杆菌(Bacillus amyloliquefaciens)，已于2016年6月6日保藏在中国普通微生物菌种保藏管理委员会普通微生物中心，其保藏编号为CGMCC No.12593，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。
附图说明
图1为解淀粉芽孢杆菌MT45在营养琼脂平板上的菌落形态(A)和显微镜图片(B)。
图2为基于16s rRNA序列比对的解淀粉芽孢杆菌MT45系统进化树。
图3为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)MT45(A)和模式微生物枯草芽孢杆菌(Bacillus subtilis)ATCC6051(B)在发酵培养基中的生长曲线和生物表面活性素的合成曲线。
图4解淀粉芽孢杆菌MT45产生物表面活性素的纯化。
具体实施方式
活化培养液的组成为：以质量百分配比计，葡萄糖1％-5％、酵母粉1％-5％、氯化钠0.3％-1.0％，水余量；pH6.5-7.0。
固体培养基组成为：1-10mM磷酸钾，50-100mM Mops，2-6mM氯化镁，0.1-1mM氯化钙，10-80μM氯化锰，10-80μM氯化铁，2-6μM氯化锌，2-6μM硫胺素，0.1-1％甘油，0.1-1％谷氨酸，1％-2％琼脂。
营养培养基：蛋白胨2％，酵母粉1％，葡萄糖0.5％，pH 7.5。
种子培养基：葡萄糖2％、酵母粉1％、氯化钠0.5％，自然pH。
所述发酵培养基：碳源10-100g/L，氮源1-10g/L，蛋白胨0.1-1.5g/L，磷酸二氢钾2-10g/L，磷酸氢二钠2-10g/L，硫酸镁0.01-0.1g/L。
实施例1菌株筛选
从中国白酒酿造高温大曲中采集样品，取5g样品溶解于10ml的质量百分比1％的氯化钠溶液中，漩涡震荡混匀后吸取1mL接种于活化培养液中，在温度50-60℃，200rpm转速条件下活化24-36h，制成成熟活化菌液。
取成熟活化菌液，加入质量百分比为1％的氯化钠溶液进行稀释。选择稀释梯度为10-2-10-6的稀释液，依次涂布于固体培养基平板上。在温度为30℃，湿度为40％-60％条件下培养48-72h；获得大量单菌落。将获得的单菌落接种至营养培养基发酵培养后，检测发酵液中的生物表面活性素含量，最终得到一株高产生物表面活性素的纯培养物，编号为MT45。
实施例2生物表面活性素的测定方法
生物表面活性素的提取：将发酵液8000rpm，4℃，离心10min。发酵上清液用6mol/L的盐酸调pH 2.0，放在4℃冰箱静置2小时。然后，12000rpm，4℃，离心30min，获得沉淀。将沉淀用甲醇萃取后获得到生物表面活性素粗产品。
发酵液中生物表面活性素的测定方法：采用超高效液相色谱法对生物表面活性素进行准确定量。采用waters BEH C18色谱柱，流动相分别为(A)0.1％甲酸水溶液和(B)HPLC级甲醇，洗脱条件为30％-70％甲醇5min，70％-100％甲醇1min。采用二极管阵列检测器，在210nm检测样品。将得到的色谱峰积分后，采用插入法同标准曲线比对，得到表面活性素的准确含量。
实施例3将实施例1筛选得到的菌株进行菌种鉴定。
将实施例1获得的编号为MT45的菌株进行菌种鉴定。
解淀粉芽孢杆菌MT45菌株在固体平板培养基上形成圆形、半透明菌落；菌落表面光滑无褶皱，不形成以褶皱菌落为代表的生物菌膜；菌落中央微微凸起。显微镜下细胞呈杆状、形成芽孢，革兰氏染色阳性(图1)。
对菌株16r rRNA基因序列进行测序(SEQ ID NO.1)，并和数据库比对，进化树分析， 确定MT45为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)(图2)。
解淀粉芽孢杆菌MT45于2016年6月6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，保藏号为CGMCC No.12593。
实施例4解淀粉芽孢杆菌MT45的发酵过程
将实施例3鉴定得到的解淀粉芽孢杆菌MT45菌株在种子培养基中活化后，以5％的接种量接种发酵培养基，发酵培养基组成为葡萄糖10g/L，蛋白胨1.5g/L，磷酸二氢钾2g/L，磷酸氢二钠2g/L，硫酸镁0.18g/L。采用500ml三角瓶装液量100mL，在37℃，200rpm条件下进行发酵。定时取样测定细胞生长量和发酵过程中的生物表面活性素产量。结果如图3，解淀粉芽孢杆菌MT45发酵至55h能最高合成3.0g/L的生物表面活性素。
解淀粉芽孢杆菌MT45能超高效合成生物表面活性素，最高产量可达生物量的50％；培养过程中合成的高浓度生物表面活性素也体现了菌株MT45对产物的高耐受性。且与模式菌株枯草芽孢杆菌(Bacillussubtilis)ATCC6051相比，生物表面活性素合成与菌体生长同步进行，产物合成无延滞期。这些特性有利于缩短解淀粉芽孢杆菌MT45发酵周期，产生更多的目标产物，有利于MT45菌株的工业化应用。
实施例5解淀粉芽孢杆菌MT45对碳源的利用。
解淀粉芽孢杆菌MT45可以利用各种糖作为碳源来生产生物表面活性剂，主要有蔗糖、葡萄糖、果糖、甘露糖、壳聚糖、甘油、可溶性淀粉、糊精。发酵培养基的其它组成为：蛋白胨0.5g/L，磷酸二氢钾2.7g/L，磷酸氢二钠5g/L，硫酸镁0.5g/L。碳源的浓度为10g/L。250mL的锥形瓶的装样量为50mL，接种量为2％，培养条件为37℃，200rpm。结果见表1。
表1解淀粉芽孢杆菌MT45对碳源的利用
Figure PCTCN2017082424-appb-000001
实施例6解淀粉芽孢杆菌MT45对铵盐的利用。
解淀粉芽孢杆菌MT45可以利用各种无机铵盐来生产生物表面活性剂，主要有硝酸铵，硫酸铵，氯化铵，碳酸铵，碳酸氢铵。发酵培养基的其它组成为：蔗糖20g/L，蛋白胨0.5g/L， 磷酸二氢钾2.7g/L，磷酸氢二钠5g/L，硫酸镁0.5g/L。氮源的浓度为8g/L。250mL的锥形瓶的装样量为50mL，接种量为2％，培养条件为37℃，200rpm。结果见表2。
表2解淀粉芽孢杆菌MT45对铵盐的利用
Figure PCTCN2017082424-appb-000002
实施例7解淀粉芽孢杆菌MT45利用不同发酵培养基生产生物表面活性素
将解淀粉芽孢杆菌MT45接种于LB培养基中，37℃，200rpm下培养过夜。将种子液按照2％的接种量转接于发酵培养基中，37℃，200rpm下培养48h。不同组分的发酵培养基的生物表面活性剂的产量如表3
表3解淀粉芽孢杆菌MT45利用不同培养基发酵生产生物表面活性素
Figure PCTCN2017082424-appb-000003
其中，LB培养基：蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L。
生物表面活性素的提取：将发酵液8000rpm，4℃，离心10min。发酵上清液用6mol/L的盐酸调pH 2.0，放在4℃冰箱静置2小时。然后，12000rpm，4℃，离心30min，获得沉淀。将沉淀用甲醇萃取后获得到生物表面活性素粗产品。
实施例8解淀粉芽孢杆菌MT45产生物表面活性素的纯品制备
将实施例7中得到的生物表面活性素粗产品采用制备液相的方法进行纯化。采用C18色谱柱柱，流动相为0.1％甲酸水溶液和甲醇，洗脱条件为线性洗脱(30％-70％甲醇60min)。设置紫外检测器检测波长为210nm。收集40.5-53.5分钟之间的洗脱组分，洗脱组分经旋转蒸发仪旋蒸去除溶剂，得到生物表面活性素纯品(附图4)。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人， 在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2017082424-appb-000004
Figure PCTCN2017082424-appb-000005
Figure PCTCN2017082424-appb-000006

Claims (10)

  1. 一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens)，已于2016年6月6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.12593，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。
  2. 根据权利要求1所述的一株解淀粉芽孢杆菌，其特征在于，所述菌株外在表现为菌落圆形、半透明，菌落表面光滑无褶皱、不形成生物菌膜，菌落中央微微凸起，显微镜下细胞呈杆状、形成芽孢，革兰氏染色阳性；菌株生长与生物表面活性素合成同步，无延滞期；菌株发酵过程中不形成生物菌膜，有利于产物超高效合成。
  3. 一种应用权利要求1所述解淀粉芽孢杆菌CGMCC NO.12593生产生物表面活性素的方法，其特征在于，包括以下步骤：
    (1)挑取解淀粉芽孢杆菌CGMCC NO.12593纯种菌落，活化得到种子培养液；
    (2)将步骤(1)得到的种子培养液按照1％-5％的接种量接种于发酵培养基中，并在25-37℃条件下培养30-55h；所述发酵培养基的配方为碳源10-100g/L，氮源1-10g/L，蛋白胨0.1-1.5g/L，磷酸二氢钾2-10g/L，磷酸氢二钠2-10g/L，硫酸镁0-0.2g/L；所述碳源包括蔗糖、葡萄糖、果糖、甘露糖、壳聚糖、甘油、可溶性淀粉、糊精中的一种或几种；所述氮源包括硝酸铵，硫酸铵，氯化铵，碳酸铵，碳酸氢铵中的一种或几种。
  4. 根据权利要求3所述的方法，其特征在于，所述种子培养基配方为葡萄糖2％、酵母粉1％、氯化钠0.5％，自然pH。
  5. 根据权利要求3所述的方法，其特征在于，所述发酵培养基的碳源为葡萄糖或蔗糖。
  6. 根据权利要求3或5所述的方法，其特征在于，所述发酵培养基的氮源为硝酸铵、碳酸铵或硫酸铵。
  7. 根据权利要求3所述的方法，其特征在于，所述发酵培养基的配方为碳源10-30g/L，氮源2-6g/L，蛋白胨0.1-1.5g/L，磷酸二氢钾2-10g/L，磷酸氢二钠2-10g/L，硫酸镁0.01-0.2g/L。
  8. 权利要求1所述解淀粉芽孢杆菌CGMCC NO.12593在制备具有抗菌、抗病毒、抗肿瘤、溶血栓、降胆固醇的药物中的应用。
  9. 权利要求1所述解淀粉芽孢杆菌CGMCC NO.12593在制备表面活性剂中的应用。
  10. 权利要求1所述解淀粉芽孢杆菌CGMCC NO.12593在制备乳化剂中的应用。
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