CN114437985A - A strain of Enterobacter aerogenes and its application in the synthesis of microbial polysaccharides - Google Patents
A strain of Enterobacter aerogenes and its application in the synthesis of microbial polysaccharidesDownload PDFInfo
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明具体涉及一株产气肠杆菌及其在合成微生物多糖中的应用，属于微生物学、生物工程技术和化工技术领域。The invention specifically relates to a strain of Enterobacter aerogenes and its application in synthesizing microbial polysaccharide, belonging to the fields of microbiology, bioengineering technology and chemical technology.
背景技术Background technique
多糖既可以在工业上被用作增稠剂、稳定剂、凝胶剂和去污剂等，也可以被作为抗肿瘤、抗氧化或具有益生元功能的生物活性因子。多糖来源广泛，细菌、真菌、藻类和植物都可以合成种类各异的多糖。尽管多糖来源众多，但市售多糖仍以藻类和高等植物来源的多糖为主。这些生物聚合物通过直接从生物质中提取获得，并可进行化学水解或发酵以获得能够聚合的最小分子。Polysaccharides can be used industrially as thickeners, stabilizers, gelling agents and detergents, etc., and can also be used as anti-tumor, antioxidant or bioactive factors with prebiotic functions. Polysaccharides come from a wide range of sources, and bacteria, fungi, algae and plants can synthesize different types of polysaccharides. Although there are many sources of polysaccharides, the commercially available polysaccharides are still dominated by polysaccharides from algae and higher plants. These biopolymers are obtained by direct extraction from biomass and can undergo chemical hydrolysis or fermentation to obtain the smallest molecules that can polymerize.
随着生物技术的高速发展，微生物多糖的功能研究也日益完善。来自细菌、酵母和霉菌等微生物的多糖，代表着一个未开发的市场。多糖的微生物合成和积累通常发生在微生物的生长阶段之后。微生物产生的多糖根据其在细胞中的位置可分为三大类：(1)胞质多糖，为细胞提供碳和能量来源；(2)组成细胞壁的多糖，包括肽聚糖和脂多糖等；(3)以胶囊或生物膜形式渗出到细胞外环境的多糖，称为胞外多糖(EPSs)。EPSs分为两组：同多糖和杂多糖。同型多糖由单一类型的单糖组成，如右旋糖酐或左旋聚糖。杂多糖由多种单糖组成，如具有复杂结构，通常在细胞内以重复单元形式合成的黄原胶等。杂多糖构成了细菌EPSs的大部分。EPSs生物合成可分为三个主要步骤：(1)碳基质的同化，(2)多糖的细胞内合成和(3)细胞外EPSs渗出。EPSs帮助细胞发挥各种功能，如防止竞争等生物胁迫，以及来自温度、光照强度或pH值的非生物胁迫。对于嗜酸或嗜热物种和古细菌，EPSs可帮助菌株适应极端条件。With the rapid development of biotechnology, the research on the function of microbial polysaccharides has become increasingly perfect. Polysaccharides from microorganisms such as bacteria, yeasts and moulds represent an untapped market. Microbial synthesis and accumulation of polysaccharides generally occurs after the growth phase of the microorganism. The polysaccharides produced by microorganisms can be divided into three categories according to their location in the cell: (1) cytoplasmic polysaccharides, which provide carbon and energy sources for cells; (2) polysaccharides that make up cell walls, including peptidoglycan and lipopolysaccharide, etc.; (3) Polysaccharides exuded to the extracellular environment in the form of capsules or biofilms are called extracellular polysaccharides (EPSs). EPSs are divided into two groups: homopolysaccharides and heteropolysaccharides. Homopolysaccharides consist of a single type of monosaccharide, such as dextran or levan. Heteropolysaccharides are composed of a variety of monosaccharides, such as xanthan gum, which has a complex structure and is usually synthesized in the form of repeating units in cells. Heteropolysaccharides constitute the majority of bacterial EPSs. EPSs biosynthesis can be divided into three main steps: (1) assimilation of the carbon matrix, (2) intracellular synthesis of polysaccharides and (3) extracellular exudation of EPSs. EPSs help cells perform various functions, such as protection from biotic stresses such as competition, as well as abiotic stresses from temperature, light intensity or pH. For acidophilic or thermophilic species and archaea, EPSs help strains adapt to extreme conditions.
目前大多数的微生物多糖合成均以糖基原料为底物。未见有使用产气肠杆菌在非糖发酵培养基中大量合成微生物多糖的报道。At present, most microbial polysaccharide synthesis is based on sugar-based raw materials. There is no report on the use of Enterobacter aerogenes to synthesize microbial polysaccharides in non-sugar fermentation medium.
发明内容SUMMARY OF THE INVENTION
本发明的目的是开发一株在非糖培养基中合成胞外多糖的产气肠杆菌Enterobacter aerogenes NJ1023。The purpose of the present invention is to develop a strain Enterobacter aerogenes NJ1023 which synthesizes extracellular polysaccharide in non-sugar medium.
本发明另外一个目的是提供上述产气肠杆菌的应用。Another object of the present invention is to provide the application of the above-mentioned Enterobacter aerogenes.
为解决上述问题，本发明采取的技术方案如下：In order to solve the above-mentioned problems, the technical scheme that the present invention takes is as follows:
本发明从石化炼制工厂附近含油污的土壤中筛选得到一株产气肠杆菌，其分类命名为产气肠杆菌NJ1023(Enterobacter aerogenes NJ1023)，已保藏于中国典型培养物保藏中心(简称CCTCC)，保藏地址：湖北省武汉市洪山区八一路武汉大学中国典型培养物保藏中心，邮编：430072，保藏编号为CCTCC M 20211243，保藏日期为2021年10月8日。In the present invention, a strain of Enterobacter aerogenes is obtained by screening the oily soil near the petrochemical refinery plant, and the strain is named Enterobacter aerogenes NJ1023 (Enterobacter aerogenes NJ1023), which has been preserved in the China Type Culture Collection (CCTCC for short) , deposit address: Wuhan University Chinese Type Culture Collection Center, Bayi Road, Hongshan District, Wuhan City, Hubei Province, zip code: 430072, deposit number CCTCC M 20211243, deposit date is October 8, 2021.
所述产气肠杆菌NJ1023的菌株形态特征：革兰氏阴性菌，无芽孢，菌体大小长约1.0-3.0μm、宽0.5-1.0μm；菌落呈圆形、凸起、灰白色。Morphological characteristics of the strain of Enterobacter aerogenes NJ1023: Gram-negative bacteria, no spores, the size of the cell is about 1.0-3.0 μm in length and 0.5-1.0 μm in width; the colony is round, convex and gray-white.
所述产气肠杆菌NJ1023在发酵生产微生物胞外多糖中的应用。The application of the Enterobacter aerogenes NJ1023 in the fermentation production of microbial extracellular polysaccharide.
具体步骤为：The specific steps are:
S1.菌株活化：将产气肠杆菌NJ1023接种于斜面培养基，静置培养，30-35℃培养48-72h；进一步优选地，34℃培养72h；S1. Strain activation: inoculate Enterobacter aerogenes NJ1023 on a slant medium, culture at rest, and culture at 30-35°C for 48-72 hours; more preferably, culture at 34°C for 72 hours;
S2.液体种子培养：取活化好的菌种，无菌条件下接种于种子液的摇瓶中，振荡培养，得到发酵种子液；培养温度为30-35℃，摇瓶转速为200-250rpm，培养时间为48-72h；进一步优选地，培养温度为34℃，摇瓶转速为220rpm，培养时间为48h；S2. Liquid seed culture: take the activated strain, inoculate it into the shake flask of the seed liquid under aseptic conditions, and shake the culture to obtain the fermented seed liquid; The culture time is 48-72h; further preferably, the culture temperature is 34°C, the rotation speed of the shake flask is 220rpm, and the culture time is 48h;
S3.胞外多糖发酵培养：将发酵种子液在无菌条件下以1％-8％(v/v)的接种量，接种于发酵培养基中，振荡培养，当发酵液中微生物多糖浓度不再上升时，停止发酵，得到含有胞外多糖的发酵液；培养温度为24-28℃，转速50-150rpm，pH 6-8，培养时间48-72h。进一步优选地，培养温度为27℃，摇瓶转速为100rpm，pH 7，培养时间为48h。S3. Extracellular polysaccharide fermentation culture: the fermented seed liquid is inoculated into the fermentation medium with an inoculum amount of 1%-8% (v/v) under aseptic conditions, and the culture is shaken. When the concentration of microbial polysaccharides in the fermentation liquid is not When it rises again, the fermentation is stopped to obtain a fermentation broth containing extracellular polysaccharides; the culture temperature is 24-28° C., the rotation speed is 50-150 rpm, the pH is 6-8, and the culture time is 48-72 h. Further preferably, the culture temperature is 27°C, the rotation speed of the shake flask is 100 rpm, the pH is 7, and the culture time is 48h.
斜面培养基为蛋白胨5～15g/L，酵母粉2～8g/L，氯化钠8～18g/L，琼脂粉15～30g/L，溶剂为水，pH值自然；更优选地，斜面培养基为蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，琼脂粉20g/L，溶剂为水，pH值自然；The slant medium is peptone 5～15g/L, yeast powder 2～8g/L, sodium chloride 8～18g/L, agar powder 15～30g/L, the solvent is water, and the pH value is natural; more preferably, the slant culture The base is peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, agar powder 20g/L, the solvent is water, and the pH value is natural;
种子液培养基为酵母粉5-15g/L，牛肉膏5-10g/L，磷酸二氢钾2-5g/L，溶剂为水，摇瓶装液量为20％；培养基pH值6.8-7.2，优选为7.0；液体种子培养基可以不外加碳源，也可以添加葡萄糖、甘油等微生物常用碳源。The seed liquid medium is yeast powder 5-15g/L, beef extract 5-10g/L, potassium dihydrogen phosphate 2-5g/L, the solvent is water, and the volume of the shake bottle is 20%; the pH value of the medium is 6.8-7.2 , preferably 7.0; the liquid seed medium may not be supplemented with a carbon source, or a common carbon source for microorganisms such as glucose and glycerol may be added.
发酵培养基为牛肉膏3-10g/L，蛋白胨10-15g/L，NaCl 2-8g/L，溶剂为水，装液量为40％；发酵培养基可以不外加碳源，也可以添加葡萄糖、甘油等微生物常用碳源。葡萄糖使用量在0-60g/L，甘油使用量在0-40g/L。The fermentation medium is beef extract 3-10g/L, peptone 10-15g/L, NaCl 2-8g/L, the solvent is water, and the liquid filling volume is 40%; the fermentation medium can be no carbon source or glucose can be added , glycerol and other microorganisms commonly used carbon sources. The amount of glucose used is 0-60g/L, and the amount of glycerol used is 0-40g/L.
微生物胞外多糖的提取：Extraction of microbial exopolysaccharides:
(1)取上述含有胞外多糖的发酵液，离心除去菌体，上清液旋转蒸发浓缩后加入2-5倍体积的无水乙醇，离心取沉淀，沉淀用乙醇洗涤，再高速离心收集沉淀，沉淀经恒温干燥后可获得微生物多糖粗品；(1) get the above-mentioned fermented liquid containing extracellular polysaccharide, centrifuge to remove thalline, add dehydrated alcohol of 2-5 times volume after the supernatant is concentrated by rotary evaporation, centrifuge to get the precipitation, wash the precipitation with ethanol, and then collect the precipitation by high-speed centrifugation , after the precipitation is dried at constant temperature, the crude microbial polysaccharide can be obtained;
(2)将上述步骤(1)的多糖粗品溶解于超纯水中，调pH至7.0-8.0，加入质量为多糖1％-4％的胰蛋白酶，50-60℃水解1-2h后，调pH至5.0-6.0，添加质量为多糖质量1‰-4‰的木瓜蛋白酶，60-70℃水解2-4h后，于100℃水浴加热3-6min，以终止酶反应；(2) Dissolve the crude polysaccharide product of the above step (1) in ultrapure water, adjust the pH to 7.0-8.0, add trypsin with a mass of 1%-4% polysaccharide, hydrolyze at 50-60°C for 1-2h, adjust the pH to 7.0-8.0 pH to 5.0-6.0, add papain with a mass of 1‰-4‰ of the polysaccharide mass, hydrolyze at 60-70°C for 2-4h, and heat it in a water bath at 100°C for 3-6min to terminate the enzymatic reaction;
(3)将步骤(2)所得蛋白酶处理液调pH后加入三氯乙酸，搅拌，离心取上清；所得清液加入Sevag试剂，充分震荡脱去蛋白，制得多糖溶液；(3) adding trichloroacetic acid after adjusting the pH of the protease treatment solution obtained in step (2), stirring, and centrifuging to obtain the supernatant; adding the Sevag reagent to the obtained supernatant, and fully shaking to remove the protein to prepare a polysaccharide solution;
(4)将步骤(3)所得多糖溶液用氨水调为碱性，滴加氧化剂，至溶液为浅黄色，保温1-2h，然后用草酸中和，超纯水透析后冷冻干燥得多糖。(4) The polysaccharide solution obtained in step (3) was adjusted to be alkaline with ammonia water, and an oxidant was added dropwise until the solution was light yellow, kept for 1-2 h, then neutralized with oxalic acid, and the polysaccharide was freeze-dried after ultrapure water dialysis.
本发明的一个实施例中制备得到的微生物多糖经过鉴定，具有如下性质：The microbial polysaccharide prepared in one embodiment of the present invention has been identified and has the following properties:
分析测定胞外多糖的分子量：产气肠杆菌NJ1023合成的胞外多糖的分子量为2.7×103Da。Analysis and determination of the molecular weight of exopolysaccharide: The molecular weight of exopolysaccharide synthesized by Enterobacter aerogenes NJ1023 was 2.7×103 Da.
分析胞外多糖的单糖组成：产气肠杆菌NJ1023合成的胞外多糖是由木糖、葡萄糖、半乳糖和N-乙酰氨基葡萄糖四种单糖组成。此四种单糖的摩尔比例为0.27:4.52:1.74:0.2。此胞外多糖的组成单糖种类和摩尔比例，与现有微生物胞外多糖的组成方式都不相同。在化学组成上，产气肠杆菌NJ1023合成的胞外多糖极具创新性。Analysis of the monosaccharide composition of exopolysaccharide: The exopolysaccharide synthesized by Enterobacter aerogenes NJ1023 is composed of four monosaccharides: xylose, glucose, galactose and N-acetylglucosamine. The molar ratio of these four monosaccharides is 0.27:4.52:1.74:0.2. In addition, the monosaccharide species and molar ratio of the composition of the extracellular polysaccharide are different from the composition mode of the existing microbial exopolysaccharide. In terms of chemical composition, the extracellular polysaccharide synthesized by Enterobacter aerogenes NJ1023 is very innovative.
对胞外多糖样品进行傅里叶红外光谱分析，光谱如图1所示。样品在3233、2967、1650、1234、1082cm-1处存在多糖的特征吸收峰。其中在3233cm-1位置附近的峰是由于O-H的伸缩振动，为糖类物质的特征峰，2967cm-1处有一个较小的峰，这是由于多糖中C-H的伸缩振动造成的，这两个特征峰的出现可以确定该物质为多糖。1650cm-1的峰可能是酰胺结构的C＝O伸缩振动吸收峰。1234cm-1处的吸收峰是羧基中的O-H发生振动产生的，说明胞外多糖中存在糖醛酸残基，胞外多糖中含有3.4％(w/w)糖醛酸。Fourier transform infrared spectroscopy was performed on the exopolysaccharide samples, and the spectra are shown in Figure 1. The samples have characteristic absorption peaks of polysaccharides at 3233, 2967, 1650, 1234 and 1082 cm-1 . The peak near 3233cm-1 is due to the stretching vibration of OH, which is a characteristic peak of sugars, and there is a smaller peak at 2967cm-1 , which is caused by the stretching vibration of CH in polysaccharides. The appearance of characteristic peaks can confirm that the substance is a polysaccharide. The peak at 1650 cm-1 may be the C=O stretching vibration absorption peak of the amide structure. The absorption peak at 1234cm-1 is generated by the vibration of OH in the carboxyl group, indicating that there are uronic acid residues in the exopolysaccharide, and the exopolysaccharide contains 3.4% (w/w) uronic acid.
有益效果：Beneficial effects:
本发明首次筛选得到一株能在非糖培养基中合成胞外多糖，且在非糖培养基中胞外多糖合成量达到212mg/g的微生物菌株-产气肠杆菌Enterobacter aerogenes NJ1023。该菌株合成胞外多糖的单糖组成及摩尔比例都与现有报道不同。这对于微生物多糖的生产和拓展应用具有十分重要的社会与经济意义。For the first time, the present invention obtains a microbial strain Enterobacter aerogenes NJ1023 which can synthesize extracellular polysaccharide in non-sugar medium and the amount of extracellular polysaccharide in non-sugar medium reaches 212 mg/g. The monosaccharide composition and molar ratio of the extracellular polysaccharide synthesized by this strain are different from the existing reports. This is of great social and economic significance for the production and expansion of microbial polysaccharides.
附图说明Description of drawings
图1产气肠杆菌NJ1023合成胞外多糖的红外光谱图。Fig. 1 Infrared spectrum of exopolysaccharide synthesized by Enterobacter aerogenes NJ1023.
具体实施方式Detailed ways
根据下列实施例可以更好的理解本发明，然而本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。The present invention can be better understood according to the following examples, but those skilled in the art can easily understand that the content described in the examples is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims. invention.
实施例1：产气肠杆菌Enterobacter aerogenes NJ1023的分离筛选。Example 1: Isolation and screening of Enterobacter aerogenes NJ1023.
该实施例所用培养基的组成如下：The composition of the culture medium used in this example is as follows:
(1)富集培养基：葡萄糖20g/L，酵母粉5g/L，磷酸氢二钾5g/L，溶剂为水，pH值7.5；(1) enrichment medium: glucose 20g/L, yeast powder 5g/L, dipotassium hydrogen phosphate 5g/L, solvent is water, pH value is 7.5;
(2)固体平板培养基：葡萄糖20g/L，酵母粉5g/L，磷酸氢二钾5g/L，硫酸镁0.5g/L，琼脂粉20g/L，溶剂为水，pH值调至7.5；(2) Solid plate medium: glucose 20g/L, yeast powder 5g/L, dipotassium hydrogen phosphate 5g/L, magnesium sulfate 0.5g/L, agar powder 20g/L, the solvent is water, and the pH value is adjusted to 7.5;
(3)多糖发酵培养基：牛肉膏3g/L，蛋白胨10g/L，NaCl 5g/L，溶剂为水，pH为7.5。(3) Polysaccharide fermentation medium: beef extract 3 g/L, peptone 10 g/L, NaCl 5 g/L, water as solvent, pH 7.5.
(4)该实施例的具体操作过程如下：(4) The concrete operation process of this embodiment is as follows:
筛选微生物多糖产生菌步骤如下：从36份扬子石化炼制工厂附近土壤中各取2g分别接入装有富集培养基的三角摇瓶中，装液量为100mL/500mL，在30℃、150rpm条件下富集培养48h。取3mL培养液转接到相同的液体富集培养基中在相同条件下进行第二次富集培养，培养48h，再反复1次，即富集3次。在无菌条件下，将第三次富集的培养液稀释至10-5和10-6，各取200μL涂布于固体平板培养基上，30℃培养48h。根据菌落表面湿润粘稠的程度，对在培养基表面生长出的菌落分离纯化；每一株被纯化的菌株再接种到多糖发酵培养基中，在30℃，150rpm条件下培养48h，然后测定每一株菌的多糖产量，编号为NJ 1023的菌株合成胞外多糖量最高，101mg/g胞外多糖。The steps for screening microbial polysaccharide-producing bacteria are as follows: 2 g of each of the 36 soils near the Yangzi Petrochemical refinery were put into a conical flask with enriched medium, and the liquid volume was 100 mL/500 mL. Condition for enrichment culture for 48h. Take 3 mL of culture medium and transfer it to the same liquid enrichment medium for the second enrichment culture under the same conditions, culture for 48h, and repeat once again, that is, enrichment 3 times. Under sterile conditions, the third enriched culture medium was diluted to 10-5 and 10-6 , 200 μL of each was spread on solid plate medium, and cultured at 30° C. for 48 hours. The colonies grown on the surface of the medium were separated and purified according to the degree of the colony surface wet and viscous; each purified strain was then inoculated into the polysaccharide fermentation medium, and cultured at 30°C and 150rpm for 48h, and then each strain was measured. The polysaccharide yield of one strain, the strain numbered NJ 1023 synthesized the highest amount of exopolysaccharide, 101 mg/g exopolysaccharide.
对NJ 1023进行菌落和种属鉴定：NJ 1023为革兰氏阴性菌，无芽孢。菌体大小长约1.0-3.0μm、宽0.5-1.0μm。菌落呈圆形、凸起、灰白色。通过16S rDNA测序可以得到该菌株属于产气肠杆菌(Enterobacter aerogenes)。Colony and species identification of NJ 1023: NJ 1023 is a Gram-negative bacteria without spores. The size of the cells is about 1.0-3.0 μm long and 0.5-1.0 μm wide. Colonies were round, raised, and gray-white. Through 16S rDNA sequencing, it can be obtained that the strain belongs to Enterobacter aerogenes.
将该菌株命名为产气肠杆菌NJ1023，保藏于中国典型培养物保藏中心(简称CCTCC)，保藏编号为CCTCC M 20211243，保藏日期为2021年10月8日。The strain was named Enterobacter aerogenes NJ1023, and was deposited in the China Center for Type Culture Collection (CCTCC for short) with the deposit number CCTCC M 20211243 and the deposit date was October 8, 2021.
实施例2：产气肠杆菌Enterobacter aerogenes NJ1023的生长条件Example 2: Growth conditions of Enterobacter aerogenes NJ1023
本实施例说明非糖培养基对菌株生长的影响。This example illustrates the effect of non-sugar medium on the growth of the strain.
菌株活化：将产气肠杆菌NJ1023接种于斜面培养基，静置培养。斜面培养基为蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，琼脂粉20g/L，溶剂为水，pH值自然；30℃培养72h。Strain activation: Enterobacter aerogenes NJ1023 was inoculated into the slant medium and cultured at rest. The slant medium was peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, agar powder 20g/L, water as solvent, and natural pH value; cultured at 30°C for 72h.
液体种子培养：取活化好的菌种，无菌条件下接种于种子液的摇瓶中，振荡培养，得到发酵种子液。种子液培养基为酵母粉5g/L，牛肉膏5g/L，磷酸二氢钾5g/L，溶剂为水，摇瓶装液量为20％；培养基pH值为7.0；培养温度为34℃；摇瓶转速为220rpm；培养时间为48h。获得的OD600为3.65。Liquid seed culture: take the activated strain, inoculate it in a shake flask of the seed liquid under aseptic conditions, and shake it to cultivate to obtain a fermented seed liquid. The seed liquid medium is 5g/L of yeast powder, 5g/L of beef extract, 5g/L of potassium dihydrogen phosphate, the solvent is water, and the volume of the shake bottle is 20%; the pH value of the medium is 7.0; the culture temperature is 34°C; The rotating speed of the shake flask was 220 rpm; the incubation time was 48 h. TheOD600 obtained was 3.65.
实施例3：产气肠杆菌Enterobacter aerogenes NJ1023的生长条件Example 3: Growth conditions of Enterobacter aerogenes NJ1023
本实施例说明葡萄糖培养基对菌株生长的影响。This example illustrates the effect of glucose medium on strain growth.
菌株活化：将产气肠杆菌NJ1023接种于斜面培养基，静置培养。斜面培养基为蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，琼脂粉20g/L，溶剂为水，pH值自然；34℃培养48h。Strain activation: Enterobacter aerogenes NJ1023 was inoculated into the slant medium and cultured at rest. The slant medium was peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, agar powder 20g/L, water as solvent, natural pH value; cultured at 34°C for 48h.
液体种子培养：取活化好的菌种，无菌条件下接种于种子液的摇瓶中，振荡培养，得到发酵种子液。种子液培养基为葡萄糖60g/L，酵母粉5g/L，牛肉膏5g/L，磷酸二氢钾5g/L，溶剂为水，摇瓶装液量为20％；培养基pH值为7.0；培养温度为34℃；摇瓶转速为220rpm；培养时间为48h，获得的OD600为5.88。Liquid seed culture: take the activated strain, inoculate it in a shake flask of the seed liquid under aseptic conditions, and shake it to cultivate to obtain a fermented seed liquid. The seed liquid medium is glucose 60g/L, yeast powder 5g/L, beef extract 5g/L, potassium dihydrogen phosphate 5g/L, the solvent is water, and the volume of the shake bottle is 20%; the pH of the medium is 7.0; The temperature was 34°C; the rotation speed of the shake flask was 220 rpm; the incubation time was 48 h, and the obtained OD600 was 5.88.
实施例4产气肠杆菌Enterobacter aerogenes NJ1023合成胞外多糖Example 4 Enterobacter aerogenes NJ1023 synthesizes extracellular polysaccharide
本实施例说明非糖培养基对菌株合成胞外多糖的影响。This example illustrates the effect of non-sugar medium on the synthesis of extracellular polysaccharides by strains.
菌株活化：将产气肠杆菌NJ1023接种于斜面培养基，静置培养。斜面培养基为蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，琼脂粉20g/L，溶剂为水，pH值自然；34℃培养48h。Strain activation: Enterobacter aerogenes NJ1023 was inoculated into the slant medium and cultured at rest. The slant medium was peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, agar powder 20g/L, water as solvent, natural pH value; cultured at 34°C for 48h.
液体种子培养：取活化好的菌种，无菌条件下接种于种子液的摇瓶中，振荡培养，得到发酵种子液。种子液培养基为酵母粉5g/L，牛肉膏5g/L，磷酸二氢钾5g/L，溶剂为水，摇瓶装液量为20％；培养基pH值为7.0；培养温度为34℃；摇瓶转速为220rpm；培养时间为48h，获得的OD600为3.65。Liquid seed culture: take the activated strain, inoculate it in a shake flask of the seed liquid under aseptic conditions, and shake it to cultivate to obtain a fermented seed liquid. The seed liquid medium is yeast powder 5g/L, beef extract 5g/L, potassium dihydrogen phosphate 5g/L, the solvent is water, and the volume of the shake bottle is 20%; the pH value of the medium is 7.0; the culture temperature is 34°C; The rotation speed of the shake flask was 220 rpm; the incubation time was 48 h, and the obtained OD600 was 3.65.
胞外多糖发酵培养：将发酵种子液在无菌条件下以4％(v/v)的接种量，接种于发酵培养基中，振荡培养，当发酵液中微生物多糖浓度不再上升时，停止发酵，得到含有胞外多糖的发酵液。发酵培养基为牛肉膏3g/L，蛋白胨10g/L，NaCl 5g/L，溶剂为水，装液量为40％，培养温度为27℃，转速100rpm，pH 7，培养时间48h，获得212mg/g胞外多糖。Fermentation culture of extracellular polysaccharide: the fermented seed liquid is inoculated into the fermentation medium with an inoculation amount of 4% (v/v) under aseptic conditions, and the culture is shaken. When the concentration of microbial polysaccharide in the fermentation liquid no longer rises, stop Fermentation to obtain a fermentation broth containing extracellular polysaccharides. Fermentation medium is beef extract 3g/L, peptone 10g/L, NaCl 5g/L, solvent is water, liquid filling volume is 40%, culture temperature is 27°C, rotation speed 100rpm, pH 7, culture time 48h, obtain 212mg/L g exopolysaccharides.
微生物胞外多糖的提取：Extraction of microbial exopolysaccharides:
(1)取上述含有胞外多糖的发酵液，离心除去菌体，上清液在60℃条件下，旋转蒸发浓缩为原体积的1/4后加入2-5倍体积的无水乙醇，离心取沉淀，沉淀用75％乙醇洗涤，再高速离心收集沉淀，沉淀经恒温干燥后可获得微生物多糖粗品；(1) Get the above-mentioned fermentation broth containing extracellular polysaccharide, remove the bacterial cells by centrifugation, the supernatant liquid is concentrated to 1/4 of the original volume by rotary evaporation under the condition of 60 ° C, and then add 2-5 times the volume of absolute ethanol, and centrifuge Take the precipitate, wash the precipitate with 75% ethanol, collect the precipitate by high-speed centrifugation, and obtain the crude microbial polysaccharide after the precipitate is dried at a constant temperature;
(2)将上述步骤(1)的多糖粗品溶解于超纯水中，用Na2CO3调pH至7.0-8.0，加入质量为多糖1％-4％的胰蛋白酶，50-60℃水解1-2h后，用草酸调pH至5.0-6.0，添加质量为多糖质量1‰-4‰的木瓜蛋白酶，60-70℃水解2-4h后，于100℃水浴加热3-6min，以终止酶反应；(2) Dissolve the crude polysaccharide product of the above step (1) in ultrapure water, adjust the pH to 7.0-8.0 with Na2 CO3 , add trypsin with a mass of 1%-4% polysaccharide, and hydrolyze 1 at 50-60° C. After -2h, adjust the pH to 5.0-6.0 with oxalic acid, add papain with a mass of 1‰-4‰ of the polysaccharide mass, hydrolyze at 60-70°C for 2-4h, and heat it in a water bath at 100°C for 3-6min to terminate the enzymatic reaction ;
(3)将步骤(2)所得蛋白酶处理液用草酸调pH至7.0，加入2％三氯乙酸，搅拌，11000rpm离心10-15min，取上清；(3) adjusting the pH of the protease treatment solution obtained in step (2) to 7.0 with oxalic acid, adding 2% trichloroacetic acid, stirring, centrifuging at 11000 rpm for 10-15 min, and taking the supernatant;
(4)将步骤(3)所得清液加入1/3体积的Sevag试剂，充分震荡脱去蛋白，制得多糖溶液；(4) adding the Sevag reagent of 1/3 volume to the clear liquid obtained in step (3), fully shaking to remove the protein, to prepare a polysaccharide solution;
(5)将步骤(4)所得多糖溶液用氨水调pH值至8.0，在50℃下滴加30％的H2O2，至溶液为浅黄色，保温1-2h，然后用草酸中和pH至7.0；超纯水透析2d后，冷冻干燥得多糖。(5) Adjust the pH value of the polysaccharide solution obtained in step (4) to 8.0 with ammonia water, add 30% H2 O2 dropwise at 50° C. until the solution is light yellow, keep warm for 1-2 hours, and then neutralize the pH with oxalic acid to 7.0; after dialysis with ultrapure water for 2 days, polysaccharide was freeze-dried.
胞外多糖结构分析Exopolysaccharide Structural Analysis
(1)分子量分析(1) Molecular weight analysis
采用段腊梅(段腊梅，苹果梨渣多糖结构表征及其与乳清蛋白相互作用研究[D].渤海大学，2021)报道的分子量测定方法分析胞外多糖的分子量。该胞外多糖的分子量为2.7×103Da。The molecular weight of exopolysaccharides was analyzed by the molecular weight determination method reported by Duan Lamei (Duan Lamei, Polysaccharide structure characterization of apple pear pomace and its interaction with whey protein [D]. Bohai University, 2021). The molecular weight of the exopolysaccharide was 2.7×103 Da.
(1)单糖组成(1) Monosaccharide composition
采用段腊梅(段腊梅，苹果梨渣多糖结构表征及其与乳清蛋白相互作用研究[D].渤海大学，2021)报道的单糖组成测定方法分析胞外多糖的单糖组成。产气肠杆菌NJ1023合成的胞外多糖是由木糖、葡萄糖、半乳糖和N-乙酰氨基葡萄糖四种单糖组成。此四种单糖的摩尔比例为0.27:4.52:1.74:0.2。此胞外多糖的组成单糖种类和摩尔比例，与现有微生物胞外多糖的组成方式都不相同。在化学组成上，产气肠杆菌NJ1023合成的胞外多糖极具创新性。The monosaccharide composition of exopolysaccharides was analyzed by the monosaccharide composition determination method reported by Duan Lamei (Duan Lamei, Polysaccharide structure characterization of apple pear pomace and its interaction with whey protein [D]. Bohai University, 2021). The exopolysaccharide synthesized by Enterobacter aerogenes NJ1023 is composed of four monosaccharides, xylose, glucose, galactose and N-acetylglucosamine. The molar ratio of these four monosaccharides is 0.27:4.52:1.74:0.2. In addition, the monosaccharide species and molar ratio of the composition of the extracellular polysaccharide are different from the composition mode of the existing microbial exopolysaccharide. In terms of chemical composition, the extracellular polysaccharide synthesized by Enterobacter aerogenes NJ1023 is very innovative.
(2)活性基团(2) Active group
对胞外多糖样品进行傅里叶红外光谱分析，光谱如图1所示。样品在3233、2967、1650、1234、1082cm-1处存在多糖的特征吸收峰。其中在3233cm-1位置附近的峰是由于O-H的伸缩振动，为糖类物质的特征峰，2967cm-1处有一个较小的峰，这是由于多糖中C-H的伸缩振动造成的，这两个特征峰的出现可以确定该物质为多糖。1650cm-1的峰可能是酰胺结构的C＝O伸缩振动吸收峰。1234cm-1处的吸收峰是羧基中的O-H发生振动产生的，说明胞外多糖中存在糖醛酸残基。通过咔唑-硫酸比色法(周鸿立等，玉米须多糖中糖醛酸的含量测定2014)测定胞外多糖中含有3.4％(w/w)糖醛酸。1200-900cm-1范围内的特征峰主要是由于糖环的振动吸收，与C-O-H的伸缩振动和糖环的C-O-C的伸缩振动有关。在1082cm-1的峰是吡喃环的变形振动，表明胞外多糖可能为吡喃型多糖。Fourier transform infrared spectroscopy was performed on the exopolysaccharide samples, and the spectra are shown in Figure 1. The samples have characteristic absorption peaks of polysaccharides at 3233, 2967, 1650, 1234 and 1082 cm-1 . The peak near 3233cm-1 is due to the stretching vibration of OH, which is a characteristic peak of sugars, and there is a smaller peak at 2967cm-1 , which is caused by the stretching vibration of CH in polysaccharides. The appearance of characteristic peaks can confirm that the substance is a polysaccharide. The peak at 1650 cm-1 may be the C=O stretching vibration absorption peak of the amide structure. The absorption peak at 1234cm-1 is generated by the vibration of OH in the carboxyl group, indicating that there are uronic acid residues in the exopolysaccharide. The exopolysaccharide contained 3.4% (w/w) uronic acid by carbazole-sulfuric acid colorimetry (Zhou Hongli et al., Determination of uronic acid content in corn silk polysaccharide 2014). The characteristic peaks in the range of 1200-900 cm-1 are mainly due to the vibrational absorption of the sugar ring, which is related to the stretching vibration of COH and the stretching vibration of COC of the sugar ring. The peak at 1082cm-1 is the deformation vibration of the pyran ring, indicating that the exopolysaccharide may be a pyran-type polysaccharide.
实施例5产气肠杆菌Enterobacter aerogenes NJ1023合成胞外多糖Example 5 Enterobacter aerogenes NJ1023 synthesizes extracellular polysaccharide
本实施例说明葡萄糖培养基对菌株合成胞外多糖的影响。This example illustrates the effect of glucose medium on the synthesis of extracellular polysaccharides by strains.
菌株活化：将产气肠杆菌NJ1023接种于斜面培养基，静置培养。斜面培养基为蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，琼脂粉20g/L，溶剂为水，pH值自然；34℃培养48h。Strain activation: Enterobacter aerogenes NJ1023 was inoculated into the slant medium and cultured at rest. The slant medium was peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, agar powder 20g/L, water as solvent, natural pH value; cultured at 34°C for 48h.
液体种子培养：取活化好的菌种，无菌条件下接种于种子液的摇瓶中，振荡培养，得到发酵种子液。种子液培养基为葡萄糖60g/L，酵母粉5g/L，牛肉膏5g/L，磷酸二氢钾5g/L，溶剂为水，摇瓶装液量为20％；培养基pH值为7.0；培养温度为34℃；摇瓶转速为220rpm；培养时间为48h，获得的OD600为5.88。Liquid seed culture: take the activated strain, inoculate it in a shake flask of the seed liquid under aseptic conditions, and shake it to cultivate to obtain a fermented seed liquid. The seed liquid medium is glucose 60g/L, yeast powder 5g/L, beef extract 5g/L, potassium dihydrogen phosphate 5g/L, the solvent is water, and the volume of the shake bottle is 20%; the pH of the medium is 7.0; The temperature was 34°C; the rotation speed of the shake flask was 220 rpm; the incubation time was 48 h, and the obtained OD600 was 5.88.
胞外多糖发酵培养：将发酵种子液在无菌条件下以4％(v/v)的接种量，接种于发酵培养基中，振荡培养，当发酵液中微生物多糖浓度不再上升时，停止发酵，得到含有胞外多糖的发酵液。发酵培养基为葡萄糖30g/L，牛肉膏3g/L，蛋白胨10g/L，NaCl 5g/L，溶剂为水，装液量为40％，培养温度为27℃，转速100rpm，pH 7，培养时间48h，获得266mg/g胞外多糖。Fermentation culture of extracellular polysaccharide: the fermented seed liquid is inoculated into the fermentation medium with an inoculation amount of 4% (v/v) under aseptic conditions, and the culture is shaken. When the concentration of microbial polysaccharide in the fermentation liquid no longer rises, stop Fermentation to obtain a fermentation broth containing extracellular polysaccharides. The fermentation medium is glucose 30g/L, beef extract 3g/L, peptone 10g/L, NaCl 5g/L, the solvent is water, the liquid filling volume is 40%, the culture temperature is 27°C, the rotation speed is 100rpm, the pH is 7, and the culture time 48h, 266mg/g exopolysaccharide was obtained.
实施例6产气肠杆菌Enterobacter aerogenes NJ1023合成胞外多糖Example 6 Enterobacter aerogenes NJ1023 synthesizes extracellular polysaccharide
本实施例说明甘油培养基对菌株合成胞外多糖的影响。This example illustrates the effect of glycerol medium on the synthesis of extracellular polysaccharides by strains.
菌株活化：将产气肠杆菌NJ1023接种于斜面培养基，静置培养。斜面培养基为蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，琼脂粉20g/L，溶剂为水，pH值自然；34℃培养48h。Strain activation: Enterobacter aerogenes NJ1023 was inoculated into the slant medium and cultured at rest. The slant medium was peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, agar powder 20g/L, water as solvent, natural pH value; cultured at 34°C for 48h.
液体种子培养：取活化好的菌种，无菌条件下接种于种子液的摇瓶中，振荡培养，得到发酵种子液。种子液培养基为甘油60g/L，酵母粉5g/L，牛肉膏5g/L，磷酸二氢钾5g/L，溶剂为水，摇瓶装液量为20％；培养基pH值为7.0；培养温度为34℃；摇瓶转速为220rpm；培养时间为48h，获得的OD600为6.12。胞外多糖发酵培养：将发酵种子液在无菌条件下以4％(v/v)的接种量，接种于发酵培养基中，振荡培养，当发酵液中微生物多糖浓度不再上升时，停止发酵，得到含有胞外多糖的发酵液。发酵培养基为甘油30g/L，牛肉膏3g/L，蛋白胨10g/L，NaCl 5g/L，溶剂为水，装液量为40％，培养温度为27℃，转速100rpm，pH 7，培养时间48h，获得284mg/g胞外多糖。Liquid seed culture: take the activated strain, inoculate it in a shake flask of the seed liquid under aseptic conditions, and shake it to cultivate to obtain a fermented seed liquid. The seed liquid medium is 60g/L of glycerol, 5g/L of yeast powder, 5g/L of beef extract, 5g/L of potassium dihydrogen phosphate, the solvent is water, and the volume of the shake bottle is 20%; the pH value of the medium is 7.0; The temperature was 34°C; the rotation speed of the shake flask was 220 rpm; the incubation time was 48 h, and the obtained OD600 was 6.12. Fermentation culture of extracellular polysaccharide: the fermented seed liquid is inoculated into the fermentation medium with an inoculation amount of 4% (v/v) under aseptic conditions, and the culture is shaken. When the concentration of microbial polysaccharide in the fermentation liquid no longer rises, stop Fermentation to obtain a fermentation broth containing extracellular polysaccharides. Fermentation medium is glycerol 30g/L, beef extract 3g/L, peptone 10g/L, NaCl 5g/L, solvent is water, liquid filling volume is 40%, culture temperature is 27°C, rotation speed 100rpm, pH 7, culture time 48h, 284mg/g exopolysaccharide was obtained.
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| CN114437985B (en) | 2023-04-11 |
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