WO2023279779A1 - 一株贝雷斯芽孢杆菌在水产品肝素提取中的应用 - Google Patents

一株贝雷斯芽孢杆菌在水产品肝素提取中的应用 Download PDF

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WO2023279779A1
WO2023279779A1 PCT/CN2022/082791 CN2022082791W WO2023279779A1 WO 2023279779 A1 WO2023279779 A1 WO 2023279779A1 CN 2022082791 W CN2022082791 W CN 2022082791W WO 2023279779 A1 WO2023279779 A1 WO 2023279779A1
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heparin
shellfish
bacillus velezensis
extraction
strain
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钟赛意
刘唤明
刘寿春
洪鹏志
邓楚津
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广东海洋大学
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  • the invention belongs to the technical field of microbes, and in particular relates to a strain of Bacillus velezensis HL-5 for improving the heparin extraction rate of aquatic products and its application.
  • Heparin is an acidic mucopolysaccharide composed of glucosamine, L-iduroside, N-acetylglucosamine and D-glucuronic acid alternately. It has a relatively high content of sulfate groups and is the highest known negative charge density. It has good anticoagulant effect and is the most widely used anticoagulant in clinical practice. In recent years, studies have also found that heparin has the functions of lowering blood fat, anti-inflammation, anti-atherosclerosis, anti-tumor, and inhibiting bacterial adhesion. It is one of the important mucopolysaccharide biochemical drugs.
  • heparin is derived from the small intestinal mucosa extracts of pigs, cattle, and sheep, and also exists in the blood vessel walls and lungs of animals.
  • the development is limited, and some scholars propose to find new resources of heparin from relatively clean marine organisms to replace terrestrial animal raw materials.
  • the ocean occupies more than 71% of the earth's surface area, and its huge biological bank and its unique environment have created its rich biological resources.
  • heparin can be obtained from various marine organisms such as bay scallops, various algae and marine bacteria, shrimps, mangrove crabs, tuna, mussels, and clams. Extract heparin.
  • Enzymatic hydrolysis is one of the commonly used methods to extract heparin.
  • Protease can break the glycopeptide bond between heparin and protein covalently, so as to separate heparin from heparin-protein complex, and also decompose miscellaneous proteins into small molecular peptides.
  • heparin/heparanoid is obtained by adjusting pH, heat denaturation and salting out to remove enzyme protein and degraded protein.
  • the patent CN201710419627.4 discloses a method for extracting heparin with a compound enzyme. Heparin is extracted by enzymolysis using a compound enzyme of alkaline protease and trypsin, and the extract is concentrated and filtered, then adsorbed with a resin, and then eluted Precipitate and dry to obtain crude heparin sodium, and then further refine the crude heparin sodium to obtain a high-purity heparin sodium product.
  • This method extracts heparin from the pig small intestine mucosa, which can improve the enzymatic hydrolysis efficiency and increase the production of heparin sodium. Crude yield.
  • the patent CN200910071972.9 discloses a method for extracting, separating and purifying heparin. Heparin is separated and purified from pig small intestine mucosa by protease hydrolysis, ultrasonic-assisted salting-out method, and ion-exchange resin adsorption. The heparin of this method The extraction rate reaches 230.81mg/kg, which is 40% higher than the traditional method.
  • the patent CN201811378238.2 discloses a shellfish heparin and its preparation method, which uses trypsin and papain to extract heparin from shellfish by enzymatic hydrolysis.
  • both pig small intestinal mucosa and shellfish contain a certain amount of fat combined with protein, and it is difficult to achieve a complete separation of fat and protein by the above methods.
  • the existing enzymatic hydrolysis method for extracting heparin only focuses on the enzymatic hydrolysis of protein, ignoring the enzymatic hydrolysis of fat. The presence of these fats prevents the protease from deeply enzymatically hydrolyzing the protein, thus making the proteolysis incomplete and affecting the extraction rate of heparinoids.
  • the purpose of the present invention is to solve the problem that the separation of protein and fat cannot be realized by adopting the existing technology, resulting in the low extraction rate and high cost of heparin prepared by the existing enzymatic hydrolysis method.
  • the inventors of this patent have screened out a Bacillus strain that can degrade fat and protein.
  • the Bacillus strain is Bacillus velezensis HL -5, preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO: 60403, and the preservation delivery date is July 2, 2018.
  • the preservation certificate For other preservation information about Bacillus velezensis HL-5, please refer to its preservation certificate.
  • Bacillus velezensis HL-5 is a bacillus producing lipase and protease, which can degrade fat and protein at low temperature.
  • the present invention claims the application of the Bacillus strain in the preparation of heparin, and the strain is Bacillus velezensis HL-5.
  • both land animals and marine organisms are sources of heparin extraction, and the Bacillus velezensis HL-5 isolated and identified in the present invention can be prepared from marine organisms or land animals under suitable conditions. described heparin.
  • the present invention elaborates the steps and methods for extracting the heparin from shellfish flesh (or tissue) in marine organisms . Based on the technical idea and enlightenment of the present invention, those skilled in the art can also use land animals as the source material of the heparin to obtain the heparin from the land animals by combining the existing technologies.
  • this patent provides a detailed description of inoculating the Bacillus velezensis HL-5 in shellfish pulp and preparing the heparin by fermentation.
  • the method for preparing the heparin by fermentation includes: cultivating the strain to obtain seed liquid; inoculating the seed liquid into shellfish pulp; cultivating, adding glucose to continue the cultivation and fermentation; after the fermentation, deactivating the enzyme , to obtain raw materials for preparing the heparin.
  • the method for preparing the heparin by fermentation includes: inoculating the strain in LB liquid medium, culturing at 15°C and 150 r/min for 24 hours to obtain seed liquid; collecting the bacterial cells in the seed liquid , inoculating the bacteria into the shellfish meat slurry, the volume ratio of the seed liquid to the shellfish meat slurry is 5-15%; cultivating at 15°C and 150r/min for 4-12h, adding glucose to continue the cultivation and fermentation for 30-40h The amount of glucose added is 0.2-5% of the mass of the shellfish meat slurry; after the fermentation is completed, the enzyme is inactivated in a boiling water bath for 10 minutes, cooled and centrifuged, and the supernatant is taken as the raw material for the heparin extraction and purification.
  • Bacillus velezensis HL-5 of the present invention is isolated from soil samples, screened on a tributyrin plate and a skim milk plate, and obtained as a bacillus producing lipase and protease.
  • the isolation, identification and performance verification of Bacillus velezensis HL-5 are described in detail in the examples of the present invention. What needs to be emphasized in this section is:
  • the primers for identifying the Bacillus velezensis HL-5 used bacterial universal primers 27F and 1492R; the PCR amplification system for identifying strains was: 1.5 ⁇ L of 27F, 1.5 ⁇ L of 1492R, 1.5 ⁇ L of MightyAmp DNA Polymerase, 2 ⁇ MightyAmp Buffer 30 ⁇ L, ddH 2 O 25.5 ⁇ L; PCR amplification reaction conditions: 98°C pre-denaturation for 2 minutes, 98°C for 10 seconds, 55°C for 15 seconds, 68°C for 90 seconds, 72°C for 10 minutes, from denaturation to the first The extension process was cycled 40 times.
  • Bacillus velezensis HL-5 utilizes the fat in the meat (tissue) as a carbon source, removes the fat in the meat (tissue), and realizes the separation of protein and fat.
  • the present invention proposes that Bacillus velezensis HL-5 is used to improve the efficiency of preparing the heparin from marine organisms, and further, to improve the efficiency of preparing the heparin from shellfish.
  • Bacillus velezensis HL-5 is used to improve the production of heparin from land animals. efficiency.
  • the present invention isolates and identifies a Bacillus velezensis HL-5, which enriches the strain resources for heparin preparation;
  • Bacillus velezensis HL-5 uses the fat in the shellfish as a carbon source, which can remove the fat in the shellfish, promote the deep enzymatic hydrolysis of protease and shellfish protein, and fully release heparin out, improve the extraction rate of shellfish heparin;
  • Aquatic products contain a certain amount of fat, which is combined with protein. The effective removal of fat facilitates subsequent separation and purification of heparin;
  • Bacillus velezensis HL-5 can also be used for the extraction of heparin from other aquatic products, and further used in the extraction of heparin from pigs, cattle, sheep and other animals. It has a wide range of applications and has explored a new type of heparin Heparin extraction method.
  • Figure 1 is a phylogenetic tree of Bacillus velezensis HL-5.
  • This example provides the screening and identification method of Bacillus velezensis HL-5.
  • the components of the enriched medium include tributyrin, ammonium sulfate and distilled water.
  • 50 mL of enrichment medium includes 100 uL of tributyrin, 0.5 g of ammonium sulfate, and 50 mL of distilled water, and is used after being sterilized at 121° C. for 20 minutes.
  • the preparation method of the tributyrin plate is as follows: take 2 mL of tributyrin, 10 g of peptone, 5 g of yeast powder, 10 g of NaCl, 20 g of agar, and 1000 mL of distilled water, mix thoroughly, and sterilize at 121° C. for 20 min.
  • the strains obtained from the primary screening were screened for protease production, that is, the strains selected by the primary screening on the plate were inoculated on the skim milk plate with a sterilized toothpick, marked, and cultured in an incubator at 15°C for 48 hours to observe the bacteria
  • the size of the hydrolysis circle around the body was evaluated, and the ability of the strain to hydrolyze tributyrin and skim milk powder was comprehensively evaluated, and the strain HL-5 was screened out.
  • this example provides an identification method for strain HL-5.
  • the identification of the strain used 27F and 1492R bacterial universal primers as forward and reverse primers, and MightyAmp DNA Polymerase was used for bacterial colony polymerase chain reaction (polymerase chain reaction, PCR) amplification.
  • PCR amplification system 1.5 ⁇ L of 27F, 1.5 ⁇ L of 1492R, 1.5 ⁇ L of MightyAmp DNA Polymerase, 30 ⁇ L of 2 ⁇ MightyAmp Buffer, 25.5 ⁇ L of ddH 2 O.
  • PCR reaction conditions 98°C (pre-denaturation) for 2min, 98°C (denaturation) for 10s, 55°C (refolding) for 15s, 68°C (extension) for 90s, 72°C for 10min, cycle 40 from denaturation to first extension Second-rate.
  • the PCR products were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing.
  • the sequencing results were searched by 16S rDNA database BLAST through NCBI, and the similarity between this strain and the strain Bacillus velezensis CR-502 was the highest, up to 99.78%. Some strains with a sequence similarity of 99% were selected, and the software Mega5.05 was used to make a phylogenetic tree using the Maximum likelihood method. The results are shown in Figure 1.
  • strain HL-5 is in the same branch as Bacillus velezensis CR-502 on the phylogenetic tree. Based on the above analysis, the strain HL-5 can be identified as Bacillus velezensis, and the classification is named (Bacillus velezensis) HL-5.
  • the nutrient agar plate and the nutrient agar slant medium described in this embodiment are prepared by using the prior art, and the present invention is not limited thereto. It should be noted that the enrichment medium and the tributyrin plate described in this embodiment are a preferred embodiment, and are not intended for the culture method of Bacillus velezensis HL-5. In particular, any Bacillus velezensis HL-5 obtained by culturing the medium described in this example or other equivalent replacement medium shall fall within the protection scope of the present invention.
  • This example presents the application of Bacillus velezensis HL-5 in the extraction of shellfish heparin, and explores the effect on the extraction rate of heparin by comparing the fermentation method with the enzymatic hydrolysis method of the prior art.
  • the enzymolysis solution was centrifuged after enzyme-inactivated cooling, the supernatant was added to 0.4 times the volume of ethanol, alcohol precipitation for 24 hours, the precipitate was redissolved with distilled water, and the insoluble matter was removed by centrifugation.
  • the (Bacillus velezensis) HL-5 preserved on the slant was inoculated in LB liquid medium, and cultured at 15°C and 150 r/min for 24 hours to obtain (Bacillus velezensis) HL-5 seed liquid, which was set aside.

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Abstract

提供了一株提高水产品肝素提取率的贝雷斯芽孢杆菌(Bacillus velezensis)HL-5的应用，贝雷斯芽孢杆菌(Bacillus velezensis)HL-5于2018年7月2日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO：60403。该菌株应用于贝类肝素的提取中，相对于传统的酶解方法，肝素提取率提高了10.55％～72.07％。贝雷斯芽孢杆菌(Bacillus velezensis)HL-5有较强的产脂肪酶与蛋白酶的能力，在水产品肝素的提取中有良好的应用前景。

Description

一株贝雷斯芽孢杆菌在水产品肝素提取中的应用 技术领域
本发明属于微生物技术领域，具体涉及一株提高水产品肝素提取率的贝雷斯芽孢杆菌(Bacillus velezensis)HL-5及其应用。
背景技术
肝素是由葡萄糖胺、L-艾杜糖醛苷、N-乙酰葡萄糖胺和D-葡萄糖醛酸等交替组成的一种酸性粘多糖，具有较高含量的硫酸基，是已知负电荷密度最高的生物大分子，具有良好的抗凝血作用，是临床使用最广泛的抗凝血剂。近年来，有研究还发现肝素具有降血脂、抗炎、抗动脉粥样硬化、抗肿瘤、抑制细菌黏附等作用，是重要的粘多糖类生化药物之一。
一般而言，肝素来源于猪、牛、羊的小肠黏膜提取物，也存在于动物的血管壁和肺之中，但由于受到肝素污染事件、疯牛病和宗教信仰的影响，陆地哺乳动物源肝素的开发受到限制，有学者提出从相对洁净的海洋生物中寻找肝素新资源以替代陆地动物原料。海洋占地球表面积的71％以上，庞大的生物库及其独特的环境，造就了其丰富的生物资源。近年来，从海洋生物中寻找肝素来源的替代原料的研究日益增多，已有报道从海湾扇贝、多种海藻和海洋细菌、虾、红树林蟹、金枪鱼、青口和文蛤等多种海洋生物中提取得到肝素。
酶解法是提取肝素常用的方法之一。蛋白酶能够破坏肝素与蛋白质共价结合的糖肽键，从而将肝素从肝素-蛋白复合物中分离出来， 也可将杂蛋白分解为小分子肽。最后通过调节pH，热变性和盐析除去酶蛋白和被降解的蛋白质制得肝素/类肝素。
例如，专利CN201710419627.4公开了一种复合酶提取肝素的方法，通过使用碱性蛋白酶和胰蛋白酶的复合酶进行酶解提取肝素，并将提取液浓缩过滤后，使用树脂进行吸附，然后洗脱沉淀，干燥得到肝素钠粗品，再将所制得的肝素钠粗品经进一步精制后，得到高纯度的肝素钠产品，该方法从猪小肠粘膜中提取肝素，能够提高酶解效率，同时提高肝素钠粗品的产率。还有，专利CN200910071972.9公开了一种肝素的提取、分离、纯化方法，通过蛋白酶水解法、超声波辅助盐析法、离子交换树脂吸附法从猪小肠粘膜中分离和纯化肝素，该方法的肝素提取率达到了230.81mg/kg，比传统方法提高了40％。另有，专利CN201811378238.2公开了一种贝类肝素及其制备方法，其采用胰蛋白酶和木瓜蛋白酶，通过酶解法从贝类中提取获得肝素。
需要指出的是，不论是猪小肠粘膜还是贝类，都含有一定量的与蛋白质结合在一起的脂肪，上述方法均难以实现脂肪与蛋白质的彻底分离。而现有的酶解法提取肝素只关注蛋白的酶解，忽略了脂肪的酶解。这些脂肪的存在阻止了蛋白酶对蛋白的深度酶解，从而使得蛋白酶解的不彻底，影响了类肝素的提取率。现有技术中也未见报道能够解决此问题的方法，因此本专利发明人针对该问题进行深入的试验研究具有重要的现实意义。
发明内容
本发明的目的在于解决采用现有技术无法实现蛋白与脂肪的分 离，造成现有酶解法制备肝素提取率较低、成本较高的问题。
基于本发明的上述意图，本专利的发明人筛选出一种可以实现降解脂肪与蛋白的芽孢杆菌菌株，通过生物形态和分子鉴定，所述芽孢杆菌菌株为贝雷斯芽孢杆菌(Bacillus velezensis)HL-5，保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO：60403，保藏送至日期为2018年7月2日。有关贝雷斯芽孢杆菌(Bacillus velezensis)HL-5的其它保藏信息参见其保藏存活证明。
本专利的发明人证实，贝雷斯芽孢杆菌(Bacillus velezensis)HL-5为产脂肪酶与蛋白酶的芽孢杆菌，可以在低温下降解脂肪与蛋白。基于这一发现，本发明请求保护该芽孢杆菌菌株在肝素制备中的应用，所述菌株为贝雷斯芽孢杆菌(Bacillus velezensis)HL-5。根据背景技术的记载，陆地动物和海洋生物均为肝素提取的来源，本发明分离鉴定的贝雷斯芽孢杆菌(Bacillus velezensis)HL-5在适宜条件下，可从海洋生物或陆地动物中制备所述肝素。作为贝雷斯芽孢杆菌(Bacillus velezensis)HL-5用于提取所述肝素的实施方式之一，本发明详细阐述了从海洋生物中的贝类肉体(或组织)提取所述肝素的步骤和方法。基于本发明的技术思路和启示，本领域普通技术人员结合现有技术，还可以将陆地动物作为所述肝素的来源物料，实现从陆地动物中获取所述肝素。
具体地，本专利给出了将所述贝雷斯芽孢杆菌(Bacillus velezensis)HL-5接种于贝类肉浆，采用发酵法制备所述肝素的详实描述。
进一步地，采用发酵法制备所述肝素的方法包括：培养所述菌种，得到种子液；向贝类肉浆中接种所述种子液；培养，加入葡萄糖继续培养发酵；发酵结束后，灭酶，得到制备所述肝素的原料。
更进一步地，采用发酵法制备所述肝素的方法包括：将所述菌株接种于LB液体培养基中，于15℃、150r/min培养24h，得到种子液；收集所述种子液中的菌体，向贝类肉浆液中接种所述菌体，所述种子液与贝类肉浆液的体积比为5～15％；15℃、150r/min培养4～12h，加入葡萄糖继续培养发酵30～40h，葡萄糖的加入量为贝类肉浆液质量的0.2～5％；发酵结束后，沸水浴灭酶10min，冷却后离心，取上清液作为所述肝素提取和纯化的原料。
本发明的贝雷斯芽孢杆菌(Bacillus velezensis)HL-5从土壤样品中分离得到，采用三丁酸甘油酯平板和脱脂牛奶平板筛选，得出其为一种产脂肪酶与蛋白酶的芽孢杆菌。有关贝雷斯芽孢杆菌(Bacillus velezensis)HL-5的分离鉴定和性能验证，本发明实施例中有详细的记载。本部分需要强调的是：
鉴定所述贝雷斯芽孢杆菌(Bacillus velezensis)HL-5的引物采用细菌通用引物27F和1492R；鉴定菌株的PCR扩增体系为：27F 1.5μL，1492R 1.5μL，MightyAmp DNA Polymerase 1.5μL，2×MightyAmp Buffer 30μL，ddH 2O 25.5μL；PCR扩增反应条件为：98℃预变性2min，98℃变性10s，55℃复性15s，68℃延伸90s，72℃延伸10min，从变性到第一次延伸过程循环40次。
本专利的发明人进一步揭示，贝雷斯芽孢杆菌(Bacillus  velezensis)HL-5将肉(组织)中的脂肪作为碳源加以利用，去除肉(组织)中的脂肪，实现蛋白与脂肪的分离，以促进蛋白酶与蛋白的深度酶解，从而使得肝素被充分释放出来，进一步提高肝素的提取率。由此，本发明提出，贝雷斯芽孢杆菌(Bacillus velezensis)HL-5用于提高从海洋生物中制备所述肝素的效率，进一步地，用于提高从贝类中制备所述肝素的效率。同理，基于本发明的技术思路和启示，本领域普通技术人员结合现有技术不难得出，贝雷斯芽孢杆菌(Bacillus velezensis)HL-5用于提高以陆地动物为物料制备所述肝素的效率。
与现有技术相比，本发明具有的有益效果或优点包括：
1)本发明分离鉴定了一种贝雷斯芽孢杆菌(Bacillus velezensis)HL-5，丰富了肝素制备的菌种资源；
2)贝雷斯芽孢杆菌(Bacillus velezensis)HL-5以贝肉中的脂肪作为碳源加以利用，能够去除贝肉中的脂肪，促进蛋白酶与贝肉蛋白的深度酶解，使得肝素被充分释放出来，提高贝类肝素的提取率；
3)水产品中含有一定量的脂肪，这些脂肪与蛋白质结合在一起。脂肪的有效去除，便于后续分离纯化肝素；
4)贝雷斯芽孢杆菌(Bacillus velezensis)HL-5还可用于其他水产品肝素的提取，更进一步的用于猪、牛、羊等动物的肝素提取，使用范围广，探索出一种新型的肝素提取方法。
附图说明
图1为贝雷斯芽孢杆菌(Bacillus velezensis)HL-5的系统发育树。
具体实施方式
下面，结合实施例对本发明的技术方案进行说明，但是，本发明并不限于下述的实施例。
实施例1
本实施例提供了所述贝雷斯芽孢杆菌(Bacillus velezensis)HL-5的筛选及其鉴定方法。
具体地，一种贝雷斯芽孢杆菌(Bacillus velezensis)HL-5的筛选方法，其过程如下：
(1)称取10g土壤样品放入250mL锥形瓶中，加入90mL无菌水，振摇混匀并在电炉上煮沸5min，取1mL于50mL富集培养基中，15℃、160r/min恒温振荡培养3天。所述富集培养基的组分包含三丁酸甘油酯、硫酸铵和蒸馏水。作为本发明优选的一种实施方式，50mL富集培养基包括三丁酸甘油酯100uL，硫酸铵0.5g，蒸馏水50mL，经121℃灭菌20min后使用。
(2)取富集之后的培养基按10倍稀释法进行稀释。取1mL富集之后的培养基到装有9mL无菌水的试管中进行稀释，分别稀释至10 -2、10 -3、10 -4、10 -5浓度梯度，取10 -3～10 -5浓度样液各100μL均匀涂布于三丁酸甘油酯平板中，每个梯度涂两个平板，于15℃培养箱中培养2天，挑取水解圈直径较大的菌落用已灭菌的牙签接种于三丁酸甘油酯平板，做好标记，于15℃下培养2天。
所述三丁酸甘油酯平板制备方法为：取三丁酸甘油酯2mL、蛋白胨10g、酵母粉5g、NaCl10g、琼脂20g、蒸馏水1000mL充分混匀，在121℃下灭菌20min。
(3)以水解圈直径和菌落直径比值为依据，挑取比值较大的菌落用平板划线法接种于营养琼脂平板，于15℃培养2天，挑取单菌落接种到营养琼脂斜面，于15℃培养箱中培养2天，再放到4℃冰箱内保存。
(4)将初筛得到的菌株进行产蛋白酶筛选，即将平板初筛挑选出来的菌株用已灭菌的牙签接种于脱脂牛奶平板，做好标记，分别于15℃培养箱中培养48h，观察菌体周围水解圈的大小，综合评价菌株水解三丁酸甘油酯与脱脂奶粉的能力，筛选出菌株HL-5。
具体地，本实施例提供了菌株HL-5的鉴定方法。该菌株的鉴定利用27F和1492R细菌通用引物作为正反向引物，利用MightyAmp DNA Polymerase进行细菌菌落聚合酶链式反应(polymerase chain reaction，PCR)扩增。
PCR扩增体系：27F1.5μL，1492R1.5μL，MightyAmp DNA Polymerase 1.5μL，2×MightyAmp Buffer 30μL，ddH 2O 25.5μL。
PCR反应条件：98℃(预变性)2min，98℃(变性)10s，55℃(复性)15s，68℃(延伸)90s，72℃延伸10min，从变性到第一次延伸这个过程循环40次。
将PCR产物送至生工生物工程(上海)股份有限公司测序。将测序结果，通过NCBI进行16S rDNA数据库BLAST检索，该菌株与菌株Bacillus velezensis CR-502的相似性最高，高达99.78％。选取序列相似度达99％的部分菌种，采用软件Mega5.05，Maximum likelihood法制作系统发育树，结果见图1。
由图1结果可知，菌株HL-5在系统发育树上与Bacillus velezensis CR-502在同一支系。综合以上分析，可鉴定该菌株HL-5为贝雷斯芽孢杆菌，分类命名为(Bacillus velezensis)HL-5。
本实施例中所述的营养琼脂平板和营养琼脂斜面培养基为采用现有技术配制，本发明并不做限定。需要说明的是，本实施例所述的富集培养基和三丁酸甘油酯平板是一种优选的实施方式，并不是对所述贝雷斯芽孢杆菌(Bacillus velezensis)HL-5培养方法的特别限定，凡是通过本实施例所述的培养基或其它等同替换的培养基培养得出贝雷斯芽孢杆菌(Bacillus velezensis)HL-5，均应落入本发明的保护范围。
实施例2
本实施例给出了贝雷斯芽孢杆菌(Bacillus velezensis)HL-5在贝类肝素提取方面的应用，并通过比较发酵法与现有技术的酶解法，探究对肝素提取率的影响。
(1)现有技术酶解法
将新鲜的菲律宾蛤蜊、贵妃蚌、海湾扇贝、象拔蚌、毛蚶、沙白贝、蛏子和钝缀锦蛤等贝类进行清洗，去壳后取全肉，加入蒸馏水(料液比为1:3)，置于高速组织捣碎机中匀浆。匀浆液在恒温水浴锅中55℃自溶5h，先后加入0.5％的2709碱性蛋白酶和木瓜蛋白酶进行酶解。酶解条件为：pH8，50℃，10h。
酶解液经灭酶冷却后离心，取上清液加入0.4倍体积的乙醇，醇沉24h，用蒸馏水对沉淀进行复溶，离心除去不溶物。取上清液加入 Sevag试剂(V氯仿：V正丁醇＝4:1)进行脱蛋白，离心，取上清液透析48h。样液浓缩后进行冷冻干燥处理，最终得8种贝类肝素粗品，并称量每种肝素粗品的质量。
(2)本发明发酵法
将斜面保藏的(Bacillus velezensis)HL-5接种于LB液体培养基中，于15℃、150r/min培养24h，得到(Bacillus velezensis)HL-5种子液，备用。
将新鲜的菲律宾蛤蜊、贵妃蚌、海湾扇贝、象拔蚌、毛蚶、沙白贝、蛏子和钝缀锦蛤等贝类进行清洗，去壳后取全肉，加入无菌水(料液比为1:3)，置于高速组织捣碎机中匀浆。
取匀浆液100mL加入到灭菌冷却后的三角瓶中；取(Bacillus velezensis)HL-5种子液8mL于8000r/min离心10min，收集菌体，并加8mL蒸馏水洗涤菌体，又于8000r/min离心10min，收集菌体；将收集到的菌体加入到含贝肉匀浆液的三角瓶中，于15℃、150r/min培养8h，然后再往三角瓶中加入2g葡萄糖继续培养30h。
发酵结束后沸水浴灭酶10min，冷却后离心(8000r/min、20min)，取上清液。上清液加入0.4倍体积的乙醇，醇沉24h，用蒸馏水对沉淀进行复溶，离心除去不溶物。取上清液加入Sevag试剂(V氯仿：V正丁醇＝4:1)进行脱蛋白，离心，取上清液透析48h。样液浓缩后进行冷冻干燥处理，最终得8种贝类肝素粗品，并称量每种肝素粗品的质量。
将以上通过酶解与发酵方法制备的各类贝类肝素粗品溶解于水 中。采用亚甲基蓝法测定肝素含量，以肝素钠为标准品，测定各种贝类肝素粗品中肝素含量，并计算各种贝类肝素的提取率。酶解法与发酵法提取的几种贝类肝素的提取率如表1所示。
表1，酶法与发酵法提取贝类肝素的提取率比对结果
Figure PCTCN2022082791-appb-000001
由表1可知，相对于酶解法而言，采用(Bacillus velezensis)HL-5发酵法明显提高了几种贝类肝素的提取率，其中提取率最高提高了72.70％，最低的提高了10.55％，平均提取率提高了27.80％，效果较为显著。
如上所述，即可较好地实现本发明，上述的实施例仅仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方 案做出的各种改变和改进，均应落入本发明确定的保护范围内。

Claims (3)

  1. 一株贝雷斯芽孢杆菌(Bacillus velezensis)HL-5，其特征在于，所述贝雷斯芽孢杆菌(Bacillus velezensis)HL-5保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO：60403。
  2. 权利要求1所述贝雷斯芽孢杆菌(Bacillus velezensis)HL-5在贝类肝素提取中的应用，其应用过程包括步骤：
    (1)将所述贝雷斯芽孢杆菌(Bacillus velezensis)HL-5接种于LB液体培养基中，于15℃、150r/min培养24h，得到种子液；
    (2)将贝类去壳后采全肉，将贝肉与无菌水按照质量比为1:0.5～1:10的比例匀浆，并将得到的贝肉匀浆液加入到无菌三角瓶中；
    (3)按照种子液：三角瓶中匀浆液的体积比为5％～15％的比例量取步骤(1)制备的贝雷斯芽孢杆菌(Bacillus velezensis)HL-5种子液，离心，收集菌体，然后再加入蒸馏水重新悬浮至原来的种子液体积，接着再离心收集菌体，并将第二次收集的菌体接种到步骤(2)中加入贝肉匀浆液的三角瓶中；
    (4)接种后的三角瓶于15℃、150r/min培养4～12h，然后再往三角瓶中加入葡萄糖继续培养30～40h，葡萄糖的加入量为贝肉匀浆液质量的0.2％～5％；
    (5)发酵结束后于沸水浴灭酶10min，冷却后离心取上清液作为后续肝素提取和纯化的原料。
  3. 根据权利要求2所述的应用，其特征在于，所述贝雷斯芽孢杆菌(Bacillus velezensis)HL-5用于提高贝类肝素的提取率。
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