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CN116083262A - Lactobacillus plantarum strain with aquatic pathogenic bacteria antagonistic property and preparation and application of preparation thereof - Google Patents

Lactobacillus plantarum strain with aquatic pathogenic bacteria antagonistic property and preparation and application of preparation thereof Download PDF

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CN116083262A
CN116083262A CN202210793310.8A CN202210793310A CN116083262A CN 116083262 A CN116083262 A CN 116083262A CN 202210793310 A CN202210793310 A CN 202210793310A CN 116083262 A CN116083262 A CN 116083262A
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lactobacillus plantarum
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高亮
缪凌鸿
于丹
林艳
孙梅
刘军锋
张海涛
陆丽婷
施大林
匡群
钱进
肖国娟
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Qinhuangdao Beidaihe New Area Marine And Fishery Bureau
Wuxi Zhongshui Fishery Medicine Co ltd
JIANGSU SUWEI MICROBIOLOGY RESEARCH CO LTD
Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences
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Wuxi Zhongshui Fishery Medicine Co ltd
JIANGSU SUWEI MICROBIOLOGY RESEARCH CO LTD
Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences
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Abstract

Lactobacillus plantarum strain with aquatic pathogenic bacteria antagonistic property and preparation and application of preparation thereof belong to the technical field of microorganisms. The invention relates to lactobacillus plantarumLactobacillus plantarum)The FF34 has wide carbon and nitrogen sources for fermentation, has amylase activity and can utilize starchy carbon sources. The nitrite is fast in degradation and has antagonism to various aquatic common pathogenic bacteria, and the FF34 single bacterial agent is obtained through preparation or the FF34 composite bacterial agent is prepared through mixed fermentation with various bacillus. The FF34 single microbial inoculant or the composite microbial inoculant is used for fermenting feed raw materials such as fish, shrimp and crab feed, cotton meal and the like, the nutrition components of the feed are obviously improved, and the obtained fermented feed is rich in probiotics, acid soluble protein and active small peptide, tanninThe content of anti-nutritional factors such as free gossypol, phytic acid and the like is reduced; FF34 microbial inoculum is added into the feed to promote the intestinal tissue growth of aquatic animals, improve liver function, improve antioxidant activity, improve the growth performance of the cultivated animals and reduce feed coefficient. The FF34 composite microbial inoculum is immobilized and then put into the culture water body, so that the water quality can be continuously improved.

Description

有水产病原菌拮抗特性的植物乳杆菌菌株及其制剂的制备及 应用Preparation and application of Lactobacillus plantarum strains with antagonistic properties against aquatic pathogens and their preparations

技术领域Technical Field

本发明涉及一种具有水产病原菌拮抗特性的植物乳杆菌菌株及其单菌制剂、其与芽孢杆菌的混合发酵培养方法及其复合菌剂的制备方法及应用,属于微生物技术领域。The invention relates to a plant lactobacillus strain with antagonistic properties against aquatic pathogens and a single bacterial preparation thereof, a mixed fermentation culture method of the plant lactobacillus strain and bacillus, and a preparation method and application of a composite bacterial preparation thereof, belonging to the technical field of microorganisms.

背景技术Background Art

植物乳杆菌是一类革兰氏阳性的乳杆菌,在自然界中广泛存在,是人体胃肠道益生菌群。研究表明,植物乳杆菌对人体具有多种有益功效,如免疫功能调节、慢性代谢性疾病调节、拮抗致病菌感染、调节肠道功能、调节精神和神经功能等。由于植物乳杆菌具有良好的生理特性及益生功能,植物乳杆菌在食品工业生产、医疗保健等领域被广泛应用。近年来,随着饲料禁抗政策的落实推进,鱼粉豆粕减量替代及代粮饲料开发的迫切需求,乳酸菌在动物养殖中的应用研究日益增多,植物乳杆菌作为国家微生物饲料添加剂目录品种,已从最初牛羊等反刍动物青贮饲料发酵应用,扩展至水产养殖领域的有益应用研究。目前研究报道植物乳杆菌在水产养殖中的有益作用主要表现为:(1)通过泼洒植物乳杆菌调节水质;(2)通过拌饵投喂或饲料发酵投喂,改善水产养殖动物胃肠道环境,抑制肠道有害菌的生长,促进饲料营养吸收,增强其对病原菌的抵抗力,促进水产养殖动物的生长。Lactobacillus plantarum is a type of Gram-positive lactobacillus that is widely found in nature and is a probiotic flora in the human gastrointestinal tract. Studies have shown that Lactobacillus plantarum has a variety of beneficial effects on the human body, such as regulating immune function, regulating chronic metabolic diseases, antagonizing pathogenic bacteria infection, regulating intestinal function, regulating mental and neurological functions, etc. Due to its good physiological characteristics and probiotic functions, Lactobacillus plantarum is widely used in food industry production, medical care and other fields. In recent years, with the implementation of the policy of banning antibiotics in feed, the urgent need to reduce the amount of fish meal and soybean meal and develop grain substitutes, the application research of lactic acid bacteria in animal breeding has increased. As a national microbial feed additive catalog variety, Lactobacillus plantarum has expanded from the initial fermentation application of silage for ruminants such as cattle and sheep to beneficial application research in the field of aquaculture. Current research reports show that the beneficial effects of Lactobacillus plantarum in aquaculture are mainly manifested in the following ways: (1) regulating water quality by spraying Lactobacillus plantarum; and (2) improving the gastrointestinal environment of aquatic animals, inhibiting the growth of harmful intestinal bacteria, promoting nutrient absorption of feed, enhancing their resistance to pathogens, and promoting the growth of aquatic animals by feeding them mixed with bait or fermenting feed.

目前乳酸菌在水产养殖生产实践中还没有得到大规模的应用,植物乳杆菌作为一种有益的乳酸菌在水产养殖中的应用也十分有限,其主要原因是(1)使用成本高。乳酸菌,包括植物乳杆菌在内,为严格厌氧菌或兼性厌氧菌,无芽孢,环境抗逆性差,易失活,菌种增殖生长时对营养要求高,氮源需求量大,高密度菌体培养所用碳源以糖类为主,无法利用小麦次粉等廉价的淀粉质农副产品。此外,大规模高密度纯种培养乳杆菌需要厌氧发酵设备和厌氧环境条件,培养成本高。与枯草芽孢杆菌等好氧菌剂相比,芽孢杆菌的好氧发酵采用通用好氧发酵罐,发酵系统成熟,发酵罐体积可达到50吨以上,而纯种乳杆菌发酵需要专一的厌氧系统,厌氧发酵罐规模小,无法达到好氧发酵的体积规模,因而乳杆菌纯种活菌制剂缺乏规模效应,制造费用高昂,使用成本高。(2)我国水产养殖种类繁多,养殖品种和地域环境复杂,能适用的植物乳杆菌优良菌株十分缺乏,有水产病原菌拮抗特性的菌株不仅来源少而且缺乏低成本规模化的培养方法,不能满足作为抗生素替代用菌剂的需求。(3)缺乏多功能的新型植物乳杆菌。目前水产养殖以高密度集约化的静水养殖模式为主,养殖密度高,投喂高蛋白饲料。由于养殖动物对饲料利用率低,如饲料蛋白的利用率仅在30%左右,饲料中大部分氮磷随着残饵和排泄物排入养殖水体,使养殖水体生态环境恶化,池塘自净与调节能力丧失,水体中氨氮、亚硝酸盐浓度超标,危害养殖动物健康,使其自身免疫能力下降,造成细菌性病害频发,给养殖生产带来极大危害。因此急需开发多功能的新型植物乳杆菌菌种,发挥降低亚硝酸盐等不良水质因子的净化调节功能,拮抗病原菌,提高饲料利用率,促进养殖动物生长。(4)目前鱼粉豆粕减量替代及代粮饲料开发的需求十分迫切,在借助益生菌的生长代谢来改善并提高棉粕、菜籽粕、桑树叶等植物蛋白资源的饲用营养价值方面,芽孢杆菌和乳酸菌均是优良的候选菌种。但目前应用于发酵饲料的菌种较为单一,且菌种之间的相容和协同作用较弱,对于肠道功能修复、水产动物炎症调控作用较差。(5)目前虽巳有植物乳杆菌发酵饲料或在饲料中添加植物乳杆菌能够改善水产养殖动物生长性能的研究报道,但植物乳杆菌对鱼类饲料营养利用和代谢影响等机理方面的研究缺乏,从而影响其推广应用。(6)植物乳杆菌为兼性厌氧菌,以泼洒方式施用时,一般植物乳杆菌对氧的耐受能力差,在含氧的养殖水体环境中极易失活,有效时间短,游离植物乳杆菌在池塘换水时易随水流失,需要重新添加,增加养殖成本。At present, lactic acid bacteria have not been widely used in aquaculture production practice. The application of Lactobacillus plantarum as a beneficial lactic acid bacteria in aquaculture is also very limited. The main reason is (1) high cost of use. Lactic acid bacteria, including Lactobacillus plantarum, are strict anaerobes or facultative anaerobes, without spores, poor environmental resistance, easy to inactivate, and have high nutritional requirements and large nitrogen source requirements during bacterial proliferation and growth. The carbon source used for high-density bacterial culture is mainly sugar, and cheap starchy agricultural and sideline products such as wheat flour cannot be used. In addition, large-scale high-density pure strain cultivation of lactobacillus requires anaerobic fermentation equipment and anaerobic environmental conditions, and the cultivation cost is high. Compared with aerobic bacterial agents such as Bacillus subtilis, the aerobic fermentation of Bacillus uses a general aerobic fermentation tank, the fermentation system is mature, and the volume of the fermentation tank can reach more than 50 tons, while the fermentation of pure strain lactobacillus requires a dedicated anaerobic system. The scale of anaerobic fermentation tanks is small and cannot reach the volume scale of aerobic fermentation. Therefore, pure strain live lactobacillus preparations lack scale effect, high manufacturing costs, and high cost of use. (2) There are many types of aquaculture in my country, and the aquaculture species and regional environment are complex. There is a great lack of suitable excellent strains of Lactobacillus plantarum. Strains with antagonistic properties against aquatic pathogens are not only scarce in source, but also lack low-cost and large-scale cultivation methods, which cannot meet the needs of antibiotic substitutes. (3) There is a lack of multifunctional new Lactobacillus plantarum. At present, aquaculture is mainly based on high-density intensive static water aquaculture, with high aquaculture density and high-protein feed. Due to the low feed utilization rate of farmed animals, such as the utilization rate of feed protein is only about 30%, most of the nitrogen and phosphorus in the feed are discharged into the aquaculture water body with leftover bait and excrement, which deteriorates the ecological environment of the aquaculture water body, loses the self-purification and regulation ability of the pond, and the concentration of ammonia nitrogen and nitrite in the water body exceeds the standard, which endangers the health of farmed animals, reduces their own immune ability, causes frequent bacterial diseases, and brings great harm to aquaculture production. Therefore, it is urgent to develop new multifunctional Lactobacillus plantarum strains to play the purification and regulation function of reducing nitrite and other adverse water quality factors, antagonize pathogens, improve feed utilization, and promote the growth of farmed animals. (4) At present, there is an urgent need to replace fish meal with soybean meal and develop feed substitutes. In terms of improving and enhancing the nutritional value of plant protein resources such as cottonseed meal, rapeseed meal, and mulberry leaves through the growth and metabolism of probiotics, Bacillus and lactic acid bacteria are both excellent candidate strains. However, the strains currently used in fermented feed are relatively single, and the compatibility and synergy between strains are weak, which has poor effects on intestinal function repair and inflammation regulation of aquatic animals. (5) Although there are reports that plant lactobacillus fermented feed or adding plant lactobacillus to feed can improve the growth performance of aquatic animals, there is a lack of research on the mechanism of plant lactobacillus' effects on fish feed nutrient utilization and metabolism, which affects its promotion and application. (6) Plant lactobacillus is a facultative anaerobic bacterium. When applied by spraying, plant lactobacillus generally has poor tolerance to oxygen and is easily inactivated in an oxygen-containing aquaculture water environment. The effective time is short, and free plant lactobacillus is easily lost with water when the pond water is changed, and needs to be added again, increasing the breeding cost.

发明内容Summary of the invention

本发明的目的是提供一种具有水产病原菌拮抗特性的植物乳杆菌菌株(Lactobacillus plantarum)FF34及其单菌培养方法、FF34与芽孢杆菌的复合菌混合培养方法、单菌菌剂及复合菌剂的制备方法及应用。The invention aims to provide a plant lactobacillus strain (Lactobacillus plantarum) FF34 with antagonistic properties against aquatic pathogens and a single-bacterium culture method thereof, a composite bacteria mixed culture method of FF34 and Bacillus, and a preparation method and application of a single-bacterium bacterial agent and a composite bacterial agent.

本发明植物乳杆菌FF34,兼性厌氧,对氧气有耐受能力,可发酵利用碳、氮源广泛,具有淀粉酶活性,能直接利用淀粉质碳源。植物乳杆菌FF34在缺氧条件下快速降解亚硝酸盐,对鳗利斯顿氏菌、爱德华氏菌、溶藻弧菌、嗜水气单胞菌及大肠杆菌等水产常见致病菌具有拮抗作用,其中对溶藻弧菌、爱德华氏菌、大肠杆菌和嗜水气单胞菌抑制效果显著。The plant lactobacillus FF34 of the present invention is facultatively anaerobic, has tolerance to oxygen, can ferment and utilize a wide range of carbon and nitrogen sources, has amylase activity, and can directly utilize starchy carbon sources. The plant lactobacillus FF34 rapidly degrades nitrite under anaerobic conditions, and has an antagonistic effect on common aquatic pathogens such as Listonella anguillarum, Edwardsiella, Vibrio alginolyticus, Aeromonas hydrophila, and Escherichia coli, among which the inhibitory effect on Vibrio alginolyticus, Edwardsiella, Escherichia coli, and Aeromonas hydrophila is remarkable.

通过菌种活化、固定化发酵培养和制剂制备获得FF34单菌水剂或粉剂;植物乳杆菌FF34与多种有益芽孢杆菌生物相容性好,如具有水产病原菌拮抗特性的枯草芽孢杆菌AB90008-15或解淀粉芽孢杆菌JSSW-LA或凝结芽孢杆菌JSSW-LA-07-1或酪酸梭状芽孢杆菌JSIM-MCB20040312或漳州芽孢杆菌JSSW-BP44,能够分别与这些芽孢杆菌之一进行协同发酵共培养,从而获得FF34复合菌剂。FF34 single bacteria aqueous solution or powder is obtained through strain activation, immobilized fermentation culture and preparation; Lactobacillus plantarum FF34 has good biocompatibility with a variety of beneficial Bacillus, such as Bacillus subtilis AB90008-15 or Bacillus amyloliquefaciens JSSW-LA or Bacillus coagulans JSSW-LA-07-1 or Clostridium butyricum JSIM-MCB20040312 or Zhangzhou Bacillus JSSW-BP44, which have antagonistic properties against aquatic pathogens, and can be synergistically fermented and co-cultured with one of these Bacillus to obtain FF34 composite bacterial agent.

植物乳杆菌FF34单菌菌剂或复合菌剂用于发酵棉粕、豆粕等饲料原料、桑树叶等新型树叶饲料原料及鱼虾蟹饲料,能够显著改善饲料原料的营养成分和品质,所得发酵饲料富含益生菌、酸溶蛋白及活性小肽,单宁、游离棉酚、植酸等抗营养因子含量降低;在基础日粮或高淀粉饲料中添加植物乳杆菌FF34单菌菌剂或复合菌剂,能够促进水产养殖动物的肠道组织生长,改善肝脏功能,抗氧化活性提高,显著改善水产养殖动物的生长性能,降低饲料系数。植物乳杆菌FF34复合菌剂通过轻石、活性碳或陶粒吸附固定化后,投放在养殖水体中,可降解水体的氨氮、亚硝酸盐、总磷、总氮含量,抑制水产病原菌的生长,持续改善养殖池塘水质。Lactobacillus plantarum FF34 single bacteria agent or compound agent is used to ferment feed raw materials such as cottonseed meal, soybean meal, new leaf feed raw materials such as mulberry leaves, and fish, shrimp and crab feed, which can significantly improve the nutritional content and quality of feed raw materials. The resulting fermented feed is rich in probiotics, acid-soluble proteins and active small peptides, and the content of anti-nutritional factors such as tannins, free cottonphenol, and phytic acid is reduced; adding Lactobacillus plantarum FF34 single bacteria agent or compound agent to the basic diet or high-starch feed can promote the growth of intestinal tissue of aquatic animals, improve liver function, increase antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce feed coefficient. After being adsorbed and fixed by pumice, activated carbon or ceramsite, Lactobacillus plantarum FF34 compound agent is placed in the aquaculture water body, which can degrade the ammonia nitrogen, nitrite, total phosphorus, and total nitrogen content of the water body, inhibit the growth of aquatic pathogens, and continuously improve the water quality of aquaculture ponds.

本发明的技术方案,一种植物乳杆菌FF34,分类命名为植物乳杆菌(lactobacillus plantarum),保藏于中国典型培养物保藏中心CCTCC,地址:中国武汉武汉大学,保藏编号CCTCC NO:M 20211221。The technical solution of the present invention is a Lactobacillus plantarum FF34, which is classified and named as Lactobacillus plantarum (lactobacillus plantarum), and is preserved in China Center for Type Culture Collection CCTCC, address: Wuhan University, Wuhan, China, and preservation number CCTCC NO: M 20211221.

本发明的植物乳杆菌菌株FF34,革兰氏阳性菌,镜检呈杆状,不产生芽孢,该菌株属于非严格厌氧菌,生长温度范围广,15~45℃均能生长。在MRS平板上厌氧培养48h,表面生长的菌落直径为1.0~3.0mm,圆形、表面光滑、湿润、不透明、乳白色,无色素产生。The plant lactobacillus strain FF34 of the present invention is a Gram-positive bacterium, which is rod-shaped under microscopic examination and does not produce spores. The strain belongs to a non-strict anaerobic bacterium and has a wide growth temperature range, and can grow at 15-45° C. After anaerobically culturing on an MRS plate for 48 hours, the diameter of the colony grown on the surface is 1.0-3.0 mm, and the colony is round, smooth, moist, opaque, milky white, and does not produce pigment.

植物乳杆菌FF34属于非严格厌氧菌,兼性厌氧,氧耐受,同型发酵糖类物质生成益生物质DL-乳酸,能够适应养殖池塘水体及底泥环境,可发酵利用碳、氮源广泛,具有淀粉酶活性,能直接利用淀粉质碳源。植物乳杆菌FF34在缺氧条件下快速降解亚硝酸盐,对鳗利斯顿氏菌、爱德华氏菌、溶藻弧菌、嗜水气单胞菌及大肠杆菌等水产常见致病菌具有拮抗作用,其中对溶藻弧菌、爱德华氏菌、大肠杆菌和嗜水气单胞菌抑制效果显著。Lactobacillus plantarum FF34 is a non-strict anaerobic bacterium, facultative anaerobic, oxygen-tolerant, homotypic fermentation of sugar substances to produce the beneficial substance DL-lactic acid, can adapt to the water and sediment environment of aquaculture ponds, can ferment and utilize a wide range of carbon and nitrogen sources, has amylase activity, and can directly utilize starch carbon sources. Lactobacillus plantarum FF34 rapidly degrades nitrite under anaerobic conditions and has antagonistic effects on common aquatic pathogens such as Listonella anguillarum, Edwardsiella, Vibrio alginolyticus, Aeromonas hydrophila, and Escherichia coli. Among them, the inhibitory effect on Vibrio alginolyticus, Edwardsiella, Escherichia coli, and Aeromonas hydrophila is significant.

植物乳杆菌FF34通过保藏菌种活化、发酵罐高密度固定化发酵培养及制剂制备获得其单菌水剂或粉剂;植物乳杆菌FF34还可以分别与具有水产病原菌拮抗特性的枯草芽孢杆菌AB90008-15或解淀粉芽孢杆菌JSSW-LA或凝结芽孢杆菌JSSW-LA-07-1或漳州芽孢杆菌JSSW-BP44或酪酸梭状芽孢杆菌JSIM-MCB20040312之一进行混合发酵共培养,获得功能复合菌剂。植物乳杆菌FF34单菌菌剂或复合菌剂用于发酵棉粕、豆粕等饲料原料、桑树叶等新型树叶饲料原料及鱼虾蟹饲料,能够显著改善饲料原料的营养成分,所得发酵饲料富含益生菌、酸溶蛋白及活性小肽,单宁、游离棉酚、植酸等抗营养因子含量降低;在基础日粮或高淀粉饲料中添加植物乳杆菌FF34单菌菌剂或复合菌剂,能够促进水产养殖动物肠道组织生长,改善肝脏功能,抗氧化活性提高,显著改善水产养殖动物的生长性能,降低饲料系数。植物乳杆菌FF34复合菌剂通过轻石、活性碳或陶粒吸附固定化后,投放在养殖水体中可降解水体的氨氮、亚硝酸盐、总磷、总氮含量,抑制水产病原菌的生长,持续改善养殖池塘水质。Lactobacillus plantarum FF34 is obtained by activation of preserved strains, high-density immobilized fermentation culture in a fermentation tank and preparation of preparations to obtain its single-bacteria aqueous solution or powder; Lactobacillus plantarum FF34 can also be mixed with Bacillus subtilis AB90008-15, Bacillus amyloliquefaciens JSSW-LA, Bacillus coagulans JSSW-LA-07-1, Bacillus Zhangzhou JSSW-BP44 or Clostridium butyricum JSIM-MCB20040312, which have antagonistic properties against aquatic pathogens, for co-culture to obtain a functional composite bacterial agent. Lactobacillus plantarum FF34 single bacteria agent or compound agent is used to ferment feed raw materials such as cottonseed meal, soybean meal, new leaf feed raw materials such as mulberry leaves, and fish, shrimp and crab feed, which can significantly improve the nutritional content of feed raw materials. The resulting fermented feed is rich in probiotics, acid-soluble proteins and active small peptides, and the content of anti-nutritional factors such as tannins, free cottonphenol, and phytic acid is reduced; adding Lactobacillus plantarum FF34 single bacteria agent or compound agent to the basic diet or high-starch feed can promote the growth of intestinal tissue of aquatic animals, improve liver function, increase antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce feed coefficient. After being adsorbed and fixed by pumice, activated carbon or ceramsite, Lactobacillus plantarum FF34 compound agent is placed in the aquaculture water body to degrade the ammonia nitrogen, nitrite, total phosphorus, and total nitrogen content of the water body, inhibit the growth of aquatic pathogens, and continuously improve the water quality of aquaculture ponds.

综上所述,植物乳杆菌FF34是一株能够改善饲料营养价值、提高水产养殖动物的生长性能,改善养殖池塘水质的多功能微生物菌株。In summary, Lactobacillus plantarum FF34 is a multifunctional microbial strain that can improve the nutritional value of feed, enhance the growth performance of aquaculture animals, and improve the water quality of aquaculture ponds.

所述植物乳杆菌FF34制备具有水产病原菌拮抗特性的单菌菌剂的方法,步骤如下:The method for preparing a single bacterial agent having antagonistic properties against aquatic pathogens by using Lactobacillus plantarum FF34 comprises the following steps:

(1)菌种活化:(1) Bacteria activation:

无菌开启植物乳杆菌FF34的冻干保藏菌种,接种于装有MRS肉汤的试管中,于30~35℃静置培养24~48h,然后转接于MRS肉汤三角瓶中,30~35℃培养活化20~48h;反复活化2~3次,镜检,计数,当菌体浓度达109CFU/mL时作为种子液;Aseptically open the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculate it into a test tube filled with MRS broth, culture it at 30-35°C for 24-48 hours, then transfer it to a MRS broth triangular bottle, culture it at 30-35°C for activation for 20-48 hours; repeatedly activate it 2-3 times, examine it under a microscope, count it, and use it as seed liquid when the bacterial concentration reaches 10 9 CFU/mL;

MRS肉汤培养基组成以g/L计:酪蛋白酶消化物10,牛肉膏粉10,酵母膏粉4,柠檬酸三铵2,乙酸钠5,硫酸镁0.2,硫酸锰0.05,磷酸氢二钾2,葡萄糖20,吐温-80 1.08,以蒸馏水定容配制,pH 5.7±0.2;MRS broth medium composition (g/L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2;

(2)固定化发酵培养:(2) Immobilized fermentation culture:

三角瓶种子培养液制备:将步骤(1)所得种子液以体积比1%~5%接种量接入装有固定化发酵培养基的3L三角瓶,装液量为体积比90%,于30~35℃静置培养24~40h,当菌体浓度达109CFU/mL时,作为三角瓶种子培养液;接入普通好氧发酵罐中进行一级、二级固定化发酵逐级放大扩培,即:3L三角瓶,再500L发酵罐,再10T发酵罐,发酵罐间移种以无菌空气压送方式完成接种;Preparation of triangular flask seed culture solution: The seed solution obtained in step (1) is inoculated at a volume ratio of 1% to 5% into a 3L triangular flask filled with immobilized fermentation medium, and the volume of the solution is 90% by volume, and is statically cultured at 30 to 35°C for 24 to 40 hours. When the bacterial concentration reaches 109 CFU/mL, it is used as the triangular flask seed culture solution; it is inoculated into ordinary aerobic fermentation tanks for primary and secondary immobilized fermentation and gradually expanded, i.e.: 3L triangular flask, then 500L fermentation tank, then 10T fermentation tank, and the inoculation is completed by transferring the seeds between fermentation tanks by sterile air pressure delivery;

一级固定化培养:将固定化培养种子液以体积比1%~5%接种量接入500L发酵罐中,发酵罐装液量为体积比80%~90%,无菌空气保持罐压至0.01~0.05Mpa,间歇搅拌,转速80~100r/min,30~35℃培养20~40h,pH为4.5~5.5时,结束培养,得到一级固定化培养液,其中植物乳杆菌FF34菌体被吸附固定在海藻酸钙+二氧化硅形成的凝胶小颗粒中,活菌体浓度≥5×109CFU/mL;Primary immobilized culture: inoculate the immobilized culture seed liquid in a volume ratio of 1% to 5% into a 500L fermenter, the volume of the fermenter is 80% to 90%, the tank pressure is maintained at 0.01 to 0.05Mpa with sterile air, stir intermittently, rotate at 80 to 100 r/min, culture at 30 to 35°C for 20 to 40 hours, and terminate the culture when the pH is 4.5 to 5.5 to obtain a primary immobilized culture liquid, in which the Lactobacillus plantarum FF34 cells are adsorbed and fixed in the gel particles formed by calcium alginate + silicon dioxide, and the concentration of live cells is ≥5×10 9 CFU/mL;

二级固定化培养:将一级固定化培养液用无菌空气压送,以体积比1%~5%接种量接入10T发酵罐中,发酵罐装液量为体积比70%~90%,无菌空气保持罐压至0.01~0.05Mpa,间歇搅拌,转速80~100r/min,30~38℃培养20~40h,当发酵液pH降至4.0~5.5,培养结束,得到二级固定化培养液,其中植物乳杆菌FF34菌体被吸附固定在海藻酸钙+二氧化硅形成的凝胶小颗粒中,菌体浓度≥5×109CFU/mL;Secondary immobilized culture: the primary immobilized culture solution is sent by sterile air pressure, and inoculated into a 10T fermenter at a volume ratio of 1% to 5%. The volume ratio of the fermenter is 70% to 90%. The sterile air is used to keep the tank pressure at 0.01 to 0.05 MPa, and the fermentation solution is stirred intermittently at a speed of 80 to 100 r/min and cultured at 30 to 38° C. for 20 to 40 hours. When the pH of the fermentation solution drops to 4.0 to 5.5, the culture is terminated to obtain a secondary immobilized culture solution, in which the Lactobacillus plantarum FF34 cells are adsorbed and fixed in the gel particles formed by calcium alginate + silicon dioxide, and the cell concentration is ≥5×10 9 CFU/mL;

固定化发酵培养基组成以g/L计:碳源10~40,氮源3~15,磷酸氢二钾0.2~5,硫酸镁0~0.2,硫酸锰0~0.05,碳酸钙5~40,海藻酸钠3~10,二氧化硅1~10,以自来水定容配制,pH 6.0~6.5,121℃灭菌30min;其中碳源包括小麦次粉、玉米淀粉、糊精、糖蜜、葡萄糖、果葡糖浆等碳源中的一种或几种组合。氮源包括鱼粉蛋白胨、胰蛋白胨、大豆蛋白胨、大豆分离蛋白、酵母膏、酵母粉、玉米浆、棉籽粕、菜籽粕、豆粕、花生粕及血粉等氮源中的一种或几种组合;The composition of the fixed fermentation medium is calculated in g/L: 10-40 carbon source, 3-15 nitrogen source, 0.2-5 potassium dihydrogen phosphate, 0-0.2 magnesium sulfate, 0-0.05 manganese sulfate, 5-40 calcium carbonate, 3-10 sodium alginate, 1-10 silicon dioxide, prepared with tap water, pH 6.0-6.5, sterilized at 121°C for 30 minutes; wherein the carbon source includes one or a combination of carbon sources such as wheat flour, corn starch, dextrin, molasses, glucose, fructose syrup, etc. The nitrogen source includes one or a combination of nitrogen sources such as fish meal peptone, tryptone, soy peptone, soy protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal, etc.;

(3)具有水产病原菌拮抗特性的植物乳杆菌FF34单菌菌剂的制备:(3) Preparation of a single bacterial agent of Lactobacillus plantarum FF34 with antagonistic properties against aquatic pathogens:

(A)植物乳杆菌FF34单菌水剂的制备:将步骤(2)所得二级固定化培养液进行灌装,获得植物乳杆菌FF34单菌水剂,菌液水剂中FF34活菌浓度≥5.0×109CFU/mL;(A) Preparation of Lactobacillus plantarum FF34 single bacteria aqueous solution: filling the secondary immobilized culture solution obtained in step (2) to obtain Lactobacillus plantarum FF34 single bacteria aqueous solution, wherein the concentration of live FF34 bacteria in the bacterial solution is ≥5.0×10 9 CFU/mL;

(B)植物乳杆菌FF34单菌粉剂的制备:将步骤(2)所得二级固定化培养液经离心收集湿菌体,按质量比为:湿菌体:碳酸钙:糊精:二氧化硅=1:0.1~2:0.5~2:0.01进行混合,经喷雾干燥或真空干燥得到植物乳杆菌FF34菌粉,菌粉中FF34活菌浓度不低于1×1010CFU/g;(B) Preparation of plant lactobacillus FF34 single bacterial powder: the secondary immobilized culture solution obtained in step (2) is centrifuged to collect wet bacterial cells, mixed in a mass ratio of wet bacterial cells: calcium carbonate: dextrin: silicon dioxide = 1:0.1-2:0.5-2:0.01, and spray-dried or vacuum-dried to obtain plant lactobacillus FF34 bacterial powder, wherein the live bacterial concentration of FF34 in the bacterial powder is not less than 1×10 10 CFU/g;

所述植物乳杆菌FF34与芽孢杆菌混合发酵共培养制备复合菌剂的方法,步骤如下:The method for preparing a composite bacterial agent by co-fermentation and co-culturing of Lactobacillus plantarum FF34 and Bacillus subtilis, comprises the following steps:

(1)与菌株FF34混合发酵培养的芽孢杆菌为:好氧芽孢杆菌、兼性好氧芽孢杆菌及厌氧芽孢杆菌,即具有水产病原菌拮抗特性的:(1) The Bacillus species mixed with strain FF34 for fermentation are: aerobic Bacillus species, facultative aerobic Bacillus species and anaerobic Bacillus species, i.e., those species that have antagonistic properties against aquatic pathogens:

枯草芽孢杆菌AB90008-15,其拮抗特性参见专利号ZL 201911117444.2;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:CGMCC NO.18684,保藏日期2019年10月15日。Bacillus subtilis AB90008-15, its antagonistic properties refer to patent number ZL 201911117444.2; deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, deposit number: CGMCC NO.18684, deposit date October 15, 2019.

或解淀粉芽孢杆菌JSSW-LA,其拮抗特性参见专利号ZL 201510733305.8;保藏于中国典型培养物保藏中心,保藏编号:CCTCC NO:M 2015602,保藏日期为2015年10月12日。Or Bacillus amyloliquefaciens JSSW-LA, its antagonistic properties refer to patent number ZL 201510733305.8; deposited in China Center for Type Culture Collection, deposit number: CCTCC NO:M 2015602, deposit date is October 12, 2015.

或凝结芽孢杆菌JSSW-LA-07-1,其拮抗特性参见专利ZL 201510057403.4,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:CGMCC NO.10182,保藏日期2014年12月15日。Or Bacillus coagulans JSSW-LA-07-1, its antagonistic properties refer to patent ZL 201510057403.4, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, deposit number: CGMCC NO.10182, deposit date December 15, 2014.

或酪酸梭状芽孢杆菌JSIM-MCB20040312,参见专利ZL201110083328.0,其抑菌性能参见论文“陈秋红等.益生菌酪酸菌CB-7的生物学特性研究[J].安徽农业科学,2011,39(10):5922~5925”,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CGMCC NO.1647,保藏日期2006年3月10日。Or Clostridium butyricum JSIM-MCB20040312, see patent ZL201110083328.0, its antibacterial performance see the paper "Chen Qiuhong et al. Study on the biological characteristics of probiotic Clostridium butyricum CB-7 [J]. Anhui Agricultural Science, 2011, 39(10): 5922-5925", deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, deposit number CGMCC NO.1647, deposit date March 10, 2006.

或漳州芽孢杆菌JSSW-BP44参见专利申请号202010066397.X,保藏于中国典型培养物保藏中心,保藏编号:CCTCC NO:2019989,保藏日期为2019年12月2日。Or Zhangzhou Bacillus JSSW-BP44, see patent application number 202010066397.X, deposited in China Center for Type Culture Collection, deposit number: CCTCC NO: 2019989, deposit date is December 2, 2019.

(2)FF34与上述芽孢杆菌混合发酵分两步进行:第一步发酵是将上述芽孢杆菌经斜面或三角瓶活化培养后,接种于混合发酵培养基中,接种量为体积比1%~10%,培养温度为30~37℃,培养24~48h,镜检菌体中芽孢形成,即第一步发酵结束;第二步发酵是先将第一步发酵所得发酵液用盐酸调整pH至5.0~5.4,然后接入活化后的植物乳杆菌FF34,接种量为体积比1%~10%,培养温度为30~35℃,间歇搅拌,流加浓度为20%(w/v)葡萄糖或30%碳酸钙或氨水,控制发酵液pH4.5~5.0,培养24h左右,获得FF34与芽孢杆菌混合发酵液,镜检可见乳杆菌与芽孢共存,混合发酵液中FF34活菌浓度的≥1.0×109CFU/mL,芽孢杆菌活菌浓度≥5.0×108CFU/mL,芽孢率≥90%;(2) The mixed fermentation of FF34 and the above-mentioned Bacillus was carried out in two steps: the first fermentation step was to activate the above-mentioned Bacillus by slant or triangular flask, and then inoculate it into the mixed fermentation medium, the inoculation amount was 1% to 10% by volume, the culture temperature was 30 to 37°C, and the culture was carried out for 24 to 48 hours. The first fermentation was completed by microscopic examination of the formation of spores in the bacteria; the second fermentation step was to first adjust the pH of the fermentation liquid obtained in the first fermentation to 5.0 to 5.4 with hydrochloric acid, and then inoculate the activated Lactobacillus plantarum FF34, the inoculation amount was 1% to 10% by volume, the culture temperature was 30 to 35°C, intermittent stirring was performed, and the concentration of 20% (w/v) glucose or 30% calcium carbonate or ammonia water was fed to control the pH of the fermentation liquid to 4.5 to 5.0, and the culture was carried out for about 24 hours to obtain a mixed fermentation liquid of FF34 and Bacillus. Microscopic examination showed that lactobacillus and spores coexisted, and the concentration of live FF34 in the mixed fermentation liquid was ≥1.0× 109 CFU/mL, Bacillus viable bacteria concentration ≥5.0×10 8 CFU/mL, spore rate ≥90%;

混合发酵培养基组成以g/L计:小麦次粉0~50,蛋白胨0.1~5,酵母膏0~5,乙酸钠0~3,玉米淀粉0~10,硫酸铵0~2,氯化钠0~5,碳酸钙0~5,磷酸二氢钾0~0.5,硫酸镁0~0.5,硫酸锰0~0.3,海藻酸钠0~5,自来水定容,pH 7.0,121℃灭菌30min;The composition of the mixed fermentation medium is in g/L: 0-50 wheat flour, 0.1-5 peptone, 0-5 yeast extract, 0-3 sodium acetate, 0-10 corn starch, 0-2 ammonium sulfate, 0-5 sodium chloride, 0-5 calcium carbonate, 0-0.5 potassium dihydrogen phosphate, 0-0.5 magnesium sulfate, 0-0.5 manganese sulfate, 0-0.3 sodium alginate, fixed to volume with tap water, pH 7.0, sterilized at 121°C for 30 min;

(3)植物乳杆菌FF34复合菌剂的制备:(3) Preparation of plant lactobacillus FF34 composite bacterial agent:

(A)将上述(2)步骤获得的植物乳杆菌FF34与芽孢杆菌混合发酵液直接进行灌装,获得植物乳杆菌FF34复合菌水剂,复合菌水剂中FF34活菌浓度≥1.0×109CFU/mL,芽孢杆菌活菌浓度≥5.0×108CFU/mL,芽孢率≥90%;(A) directly filling the mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus obtained in step (2) above to obtain a Lactobacillus plantarum FF34 composite bacterial solution, wherein the live bacteria concentration of FF34 in the composite bacterial solution is ≥1.0×10 9 CFU/mL, the live bacteria concentration of Bacillus subtilis is ≥5.0×10 8 CFU/mL, and the spore rate is ≥90%;

(B)将上述步骤(2)所得的植物乳杆菌FF34与芽孢杆菌混合发酵液经离心收集湿菌体,按质量比为:湿菌体:碳酸钙:二氧化硅=1:0.1~3:0.01进行混合,经喷雾干燥或真空干燥得到植物乳杆菌FF34复合菌剂粉剂,复合菌剂粉剂中FF34活菌浓度≥5×109CFU/g,芽孢杆菌活菌浓度≥5.0×109CFU/g。(B) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus obtained in the above step (2) is centrifuged to collect wet cells, mixed in a mass ratio of wet cells: calcium carbonate: silicon dioxide = 1:0.1 to 3:0.01, and spray-dried or vacuum-dried to obtain a Lactobacillus plantarum FF34 composite bacterial agent powder, wherein the live bacteria concentration of FF34 in the composite bacterial agent powder is ≥5×10 9 CFU/g, and the live bacteria concentration of Bacillus subtilis is ≥5.0×10 9 CFU/g.

所述上述步骤制备的植物乳杆菌FF34单菌菌剂及其复合菌剂的应用:Application of the plant lactobacillus FF34 single bacterial agent and its composite bacterial agent prepared in the above steps:

(1)植物乳杆菌FF34单菌菌剂或复合菌剂用于发酵鱼虾蟹饲料或棉粕、豆粕、桑树叶等饲料原料,植物乳杆菌FF34单菌菌剂或复合菌剂按质量百分比0.1%~5%与饲料或饲料原料混合,加水调节物料含水量25%~40%,28~35℃密闭发酵3~5天。上述饲料或饲料原料经植物乳杆菌FF34单菌或复合菌剂发酵后,无霉变,显著改善鱼虾蟹饲料或饲料原料的营养成分,所得发酵饲料富含益生菌、酸溶蛋白及活性小肽,单宁、游离棉酚、植酸等抗营养因子含量降低。(1) Lactobacillus plantarum FF34 single bacteria agent or composite bacteria agent is used to ferment fish, shrimp and crab feed or feed raw materials such as cottonseed meal, soybean meal, mulberry leaves, etc. The Lactobacillus plantarum FF34 single bacteria agent or composite bacteria agent is mixed with the feed or feed raw materials at a mass percentage of 0.1% to 5%, and water is added to adjust the moisture content of the material to 25% to 40%, and the material is sealed and fermented at 28 to 35° C. for 3 to 5 days. After the above-mentioned feed or feed raw materials are fermented by Lactobacillus plantarum FF34 single bacteria agent or composite bacteria agent, there is no mildew, and the nutritional components of fish, shrimp and crab feed or feed raw materials are significantly improved. The obtained fermented feed is rich in probiotics, acid-soluble proteins and active small peptides, and the content of anti-nutritional factors such as tannins, free cottony phenol, and phytic acid is reduced.

(2)在基础日粮或高淀粉饲料中按质量百分比0.05%~1%添加植物乳杆菌FF34单菌菌剂或复合菌剂,能够促进水产养殖动物的肠道组织生长,改善肝脏功能,提高抗氧化活性,显著改善水产养殖动物的生长性能,降低饲料系数。(2) Adding 0.05% to 1% by weight of Lactobacillus plantarum FF34 single bacteria agent or compound bacteria agent to the basic diet or high-starch feed can promote the growth of intestinal tissue of aquatic animals, improve liver function, enhance antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce the feed coefficient.

(3)植物乳杆菌FF34复合菌剂水剂与质量体积比2%~4%海藻酸钠混匀后,添加轻石、活性碳或陶粒浸润吸附1~4h后取出,再浸泡于2%~3%氯化钙溶液中,4~8℃固化交联1~10h,即制得植物乳杆菌FF34复合菌固定化菌球(块),菌球(块)中菌体浓度不低于5×108CFU/g。植物乳杆菌FF34复合菌固定化菌球(块)投放在养殖水体中,连续使用20天以上,,可以持续降解水体中的氨氮、亚硝酸盐、总磷、总氮的含量,抑制气单胞菌等水产病原菌的生长,持续改善养殖池塘水质。(3) After the plant lactobacillus FF34 composite bacterial agent is mixed with 2% to 4% sodium alginate by mass volume ratio, pumice, activated carbon or ceramsite is added for soaking and adsorption for 1 to 4 hours, then taken out and soaked in 2% to 3% calcium chloride solution, and cross-linked at 4 to 8°C for 1 to 10 hours to obtain the plant lactobacillus FF34 composite bacterial immobilized bacterial pellet (block), and the bacterial concentration in the bacterial pellet (block) is not less than 5×10 8 CFU/g. The plant lactobacillus FF34 composite bacterial immobilized bacterial pellet (block) is placed in the aquaculture water body and used continuously for more than 20 days, which can continuously degrade the content of ammonia nitrogen, nitrite, total phosphorus and total nitrogen in the water body, inhibit the growth of aquatic pathogens such as Aeromonas, and continuously improve the water quality of aquaculture ponds.

植物乳杆菌FF34抑菌性的测定:将致病菌与植物乳杆菌FF34菌液共同接入LB肉汤中,使培养基中FF34起始菌浓度为105~106CFU/mL,致病菌起始菌浓度为105~107CFU/mL,30℃静置培养24h,于0h、24h取样计数,观察FF34对致病菌的拮抗作用。结果显示,对鳗利斯顿氏菌、爱德华氏菌、溶藻弧菌、嗜水气单胞菌及大肠杆菌等水产常见致病菌具有拮抗作用,其中对溶藻弧菌、爱德华氏菌、大肠杆菌和嗜水气单胞菌抑制效果显著。Determination of the antibacterial activity of Lactobacillus plantarum FF34: The pathogenic bacteria and Lactobacillus plantarum FF34 were inoculated into LB broth, and the initial bacterial concentration of FF34 in the culture medium was 10 5 ~10 6 CFU/mL, and the initial bacterial concentration of pathogenic bacteria was 10 5 ~10 7 CFU/mL. The culture was statically cultured at 30℃ for 24 hours, and samples were taken and counted at 0h and 24h to observe the antagonistic effect of FF34 on pathogenic bacteria. The results showed that it had an antagonistic effect on common aquatic pathogens such as Listonella anguillarum, Edwardsiella, Vibrio alginolyticus, Aeromonas hydrophila and Escherichia coli, among which the inhibitory effect on Vibrio alginolyticus, Edwardsiella, Escherichia coli and Aeromonas hydrophila was significant.

植物乳杆菌FF34降解亚硝酸盐能力的测定:MRS肉汤培养基分装50mL/试管,与2mol/L亚硝酸钠溶液分别在121℃灭菌15min后,在每支装有MRS肉汤培养基的试管中分别添加2mol/L亚硝酸钠溶液0.1mL,,然后接种活化后的植物乳杆菌FF34菌悬液5mL,对照组接入5mL无菌蒸馏水,每组3个平行,35℃静置培养20h,其中0h、7h和20h取样,4000r/min离心10min,取上清液,用美国哈希试剂检测亚硝氮浓度。数据用SPSS18.0统计分析,全部结果均用平均值±标准误

Figure BDA0003731190040000061
表示,独立样本T检验比较检验各组间的差异。在厌氧条件下,20h内能够显著降解亚硝氮,降解率达到96%。Determination of the ability of Lactobacillus plantarum FF34 to degrade nitrite: MRS broth medium was divided into 50 mL/test tube, and sterilized with 2 mol/L sodium nitrite solution at 121°C for 15 min. Then, 0.1 mL of 2 mol/L sodium nitrite solution was added to each test tube containing MRS broth medium, and then 5 mL of activated Lactobacillus plantarum FF34 suspension was inoculated. The control group was inoculated with 5 mL of sterile distilled water. Each group had 3 parallels and was cultured at 35°C for 20 h. Samples were taken at 0 h, 7 h, and 20 h, and centrifuged at 4000 r/min for 10 min. The supernatant was taken and the nitrite nitrogen concentration was detected using the American Hach reagent. Data were statistically analyzed using SPSS18.0, and all results were expressed as mean ± standard error.
Figure BDA0003731190040000061
Indicates that the independent sample T test was used to compare the differences between the groups. Under anaerobic conditions, nitrite nitrogen can be significantly degraded within 20 hours, and the degradation rate reaches 96%.

本发明的有益效果:Beneficial effects of the present invention:

(1)一般乳杆菌(包括植物乳杆菌)营养要求复杂,缺乏淀粉酶活性,不仅需要葡萄糖、果糖或麦芽糖等单糖或二糖为碳源,而且需要氨基酸、肽、核酸衍生物、盐类、脂肪酸或脂肪酸脂类,且每个种都有特殊的营养要求,这种特性使得有益乳杆菌的规模化培养用原材料的来源受限,导致培养基成本增高。本发明植物乳杆菌FF34具有淀粉酶活性,能直接利用淀粉质碳水化合物,可利用的碳源、氮源广泛,这个特性区别于一般乳杆菌(包括一般植物乳杆菌),使得植物乳杆菌FF34能够利用的碳源从葡萄糖、果糖或麦芽糖等单糖或二糖扩大至淀粉质碳水化合物,从而使廉价的小麦次粉、棉粕、菜粕、豆粕等农副产品可以作为碳源、氮源直接用于FF34的增殖培养,大大降低培养基成本。(1) General lactobacillus (including plant lactobacillus) has complex nutritional requirements and lacks amylase activity. It not only needs monosaccharides or disaccharides such as glucose, fructose or maltose as carbon sources, but also needs amino acids, peptides, nucleic acid derivatives, salts, fatty acids or fatty acid lipids, and each species has special nutritional requirements. This characteristic limits the source of raw materials for large-scale cultivation of beneficial lactobacillus, resulting in increased culture medium cost. Plant lactobacillus FF34 of the present invention has amylase activity, can directly utilize starchy carbohydrates, and has a wide range of available carbon sources and nitrogen sources. This characteristic is different from general lactobacillus (including general plant lactobacillus), so that the carbon source that plant lactobacillus FF34 can utilize is expanded from monosaccharides or disaccharides such as glucose, fructose or maltose to starchy carbohydrates, thereby making cheap agricultural and sideline products such as wheat flour, cottonseed meal, rapeseed meal, soybean meal, etc. can be directly used as carbon sources and nitrogen sources for the proliferation and cultivation of FF34, greatly reducing the culture medium cost.

(2)植物乳杆菌FF34是一株兼性乳杆菌,对溶氧耐受性较好,不需要严格厌氧条件即能生长,这一特性使得采用普通好氧发酵罐大规模培养植物乳杆菌FF34成为可能,但好氧发酵罐罐压维持及种子罐与发酵罐之间的采用无菌空气压送移种方式,使得发酵培养过程中依然存在高溶氧的风险,从而抑制FF34的生长。本发明采用高密度低成本的固定化发酵扩培方法,在普通好氧发酵罐中,边发酵边海藻酸钠固定化包裹菌体的培养方法,利用植物乳杆菌FF34生长利用淀粉质或糖类产酸特性,溶解培养基中的碳酸钙释放游离钙离子,与培养基中海藻酸钠形成海藻酸钙凝胶,将菌体包裹在海藻酸钙凝胶小颗粒中,隔绝溶氧传递保护菌体的生长活性,从而获得高浓度的菌体,发酵液中植物乳杆菌FF34菌浓超过5×109CFU/mL;然后通过喷雾干燥获得FF34菌浓超过1×1010CFU/g的高浓度菌粉制剂,从而使得植物乳杆菌FF34水剂及菌粉制剂的使用成本进一步降低,有利于在畜禽渔生态养殖中推广应用植物乳杆菌FF34菌剂。(2) Lactobacillus plantarum FF34 is a facultative lactobacillus with good tolerance to dissolved oxygen and can grow without strict anaerobic conditions. This characteristic makes it possible to culture Lactobacillus plantarum FF34 on a large scale using ordinary aerobic fermenters. However, the maintenance of the tank pressure in the aerobic fermenter and the use of sterile air pressure to transfer the seeds between the seed tank and the fermenter still result in the risk of high dissolved oxygen during the fermentation process, thereby inhibiting the growth of FF34. The invention adopts a high-density and low-cost immobilized fermentation and expansion method, a cultivation method in which a bacterial body is immobilized and wrapped with sodium alginate while fermenting in a common aerobic fermentation tank, and utilizes the acid-producing property of plant lactobacillus FF34 by utilizing starch or sugar to dissolve calcium carbonate in a culture medium to release free calcium ions, and forms calcium alginate gel with sodium alginate in the culture medium, and wraps the bacterial body in small particles of the calcium alginate gel, isolates dissolved oxygen and transmits and protects the growth activity of the bacterial body, thereby obtaining high-concentration bacterial bodies, and the bacterial concentration of plant lactobacillus FF34 in the fermentation liquid exceeds 5×10 9 CFU/mL; then, a high-concentration bacterial powder preparation with a bacterial concentration of FF34 exceeding 1×10 10 CFU/g is obtained by spray drying, thereby further reducing the use cost of a plant lactobacillus FF34 aqueous agent and a bacterial powder preparation, and facilitating the popularization and application of the plant lactobacillus FF34 bacterial agent in ecological breeding of livestock, poultry and fishery.

(3)植物乳杆菌FF34与多种有益芽孢杆菌相容性好,能够进行多菌株混合发酵共培养。植物乳杆菌FF34的生长对营养要求低,可利用碳源和氮源广泛,可直接利用淀粉质碳水化合物。由于小麦次粉等淀粉质碳源是芽孢杆菌适宜的低廉碳源,因此植物乳杆菌FF34能够直接利用淀粉质碳源的特性,使得FF34能够与多种芽孢杆菌混合发酵培养,芽孢杆菌类型覆盖面广,涉及好氧芽孢杆菌、兼性好氧芽孢杆菌及厌氧芽孢杆菌,如具有水产病原菌拮抗特性的枯草芽孢杆菌AB90008-15(好氧菌)或解淀粉芽孢杆菌JSSW-LA(好氧菌)或凝结芽孢杆菌JSSW-LA-07-1(兼性好氧菌)或酪酸梭状芽孢杆菌JSIM-MCB20040312(厌氧菌)或漳州芽孢杆菌JSSW-BP44(兼性好氧菌)。FF34与芽孢杆菌在混合发酵培养基中共培养时,首先在这些芽孢杆菌形成芽孢后,通过调整pH,使芽孢处于休眠状态,然后继续接种FF34,充分利用发酵培养基中剩余的营养物质进行FF34增殖培养,最终获得含有高浓度的芽孢杆菌(芽孢率≥90%)+植物乳杆菌FF34的复合菌剂,降低了复合菌剂的发酵成本,减少了发酵废液排放。(3) Lactobacillus plantarum FF34 has good compatibility with a variety of beneficial Bacillus species and can be co-cultured with multiple strains for mixed fermentation. Lactobacillus plantarum FF34 has low nutritional requirements for growth, can utilize a wide range of carbon and nitrogen sources, and can directly utilize starchy carbohydrates. Since starchy carbon sources such as wheat bran are suitable and cheap carbon sources for Bacillus, the ability of Lactobacillus plantarum FF34 to directly utilize starchy carbon sources enables FF34 to be mixed and fermented with a variety of Bacillus. The types of Bacillus covered are wide, involving aerobic Bacillus, facultative aerobic Bacillus and anaerobic Bacillus, such as Bacillus subtilis AB90008-15 (aerobic bacteria) or Bacillus amyloliquefaciens JSSW-LA (aerobic bacteria) or Bacillus coagulans JSSW-LA-07-1 (facultative aerobic bacteria) or Clostridium butyricum JSIM-MCB20040312 (anaerobic bacteria) or Bacillus Zhangzhouensis JSSW-BP44 (facultative aerobic bacteria) with antagonistic properties to aquatic pathogens. When FF34 and Bacillus are co-cultured in a mixed fermentation medium, after these Bacillus form spores, the pH is adjusted to put the spores into a dormant state, and then FF34 is continuously inoculated to fully utilize the remaining nutrients in the fermentation medium for FF34 proliferation culture, and finally a composite bacterial agent containing a high concentration of Bacillus (spore rate ≥ 90%) + Lactobacillus plantarum FF34 is obtained, which reduces the fermentation cost of the composite bacterial agent and reduces the discharge of fermentation waste liquid.

(4)植物乳杆菌FF34对鳗利斯顿氏菌、爱德华氏菌、溶藻弧菌、嗜水气单胞菌及大肠杆菌等水产常见致病菌具有拮抗作用,其中对溶藻弧菌、爱德华氏菌、大肠杆菌和嗜水气单胞菌抑制效果显著。且植物乳杆菌FF34能够与枯草芽孢杆菌AB90008-15或解淀粉芽孢杆菌JSSW-LA或凝结芽孢杆菌JSSW-LA-07-1或酪酸梭状芽孢杆菌JSIM-MCB20040312等多种具有水产病原菌拮抗特性的芽孢杆菌进行混合发酵共培养,获得的复合菌剂则进一步丰富了菌剂拮抗水产病原菌种类,增强水产养殖动物病害防治的有效性,促进水产动物健康生长。(4) Lactobacillus plantarum FF34 has antagonistic effects on common aquatic pathogens such as Listonella anguillarum, Edwardsiella, Vibrio alginolyticus, Aeromonas hydrophila and Escherichia coli, among which the inhibitory effects on Vibrio alginolyticus, Edwardsiella, Escherichia coli and Aeromonas hydrophila are significant. In addition, Lactobacillus plantarum FF34 can be co-fermented with a variety of Bacillus with antagonistic properties against aquatic pathogens, such as Bacillus subtilis AB90008-15, Bacillus amyloliquefaciens JSSW-LA, Bacillus coagulans JSSW-LA-07-1 or Clostridium butyricum JSIM-MCB20040312, to obtain a composite bacterial agent that further enriches the types of aquatic pathogens that the bacterial agent can antagonize, enhances the effectiveness of aquatic animal disease prevention and control, and promotes the healthy growth of aquatic animals.

(5)植物乳杆菌FF34单菌或复合菌剂发酵饲料后,无霉变,发酵饲料富含益生菌,显著改善饲料营养成分,可溶性蛋白提高42.38%~116.31%,酸溶蛋白提高118.54%~218.53%,活性小肽提高42.14%~168.41%,并且植酸、单宁、游离棉酚等抗营养因子含量显著降低。本发明对于植物乳杆菌FF34对鱼类饲料营养利用和代谢影响等机理方面的初步研究显示:在基础日粮或高淀粉饲料中按质量百分比0.05%~1%添加植物乳杆菌FF34单菌菌剂或复合菌剂,能够促进水产养殖动物的肠道组织生长,改善肝脏功能,提高抗氧化活性,显著改善水产养殖动物的生长性能,降低饲料系数。(5) After the feed is fermented by Lactobacillus plantarum FF34 single bacteria or composite bacteria, there is no mildew, the fermented feed is rich in probiotics, the nutritional components of the feed are significantly improved, the soluble protein is increased by 42.38% to 116.31%, the acid-soluble protein is increased by 118.54% to 218.53%, the active small peptides are increased by 42.14% to 168.41%, and the content of anti-nutritional factors such as phytic acid, tannins, and free cottony phenol is significantly reduced. The preliminary study of the present invention on the mechanism of the influence of Lactobacillus plantarum FF34 on the nutrient utilization and metabolism of fish feed shows that adding Lactobacillus plantarum FF34 single bacteria or composite bacteria at a mass percentage of 0.05% to 1% in the basic diet or high-starch feed can promote the growth of intestinal tissue of aquatic animals, improve liver function, increase antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce the feed coefficient.

(6)植物乳杆菌FF34复合菌剂通过轻石、活性碳或陶粒吸附固定化后,投放在养殖水体中,与直接泼洒未固定化的菌剂相比,在含氧的养殖水体环境中固定化菌体不易失活,不易流失,有效时间超过20天,可持续降解水体的氨氮、亚硝酸盐、总磷、总氮含量,抑制水产病原菌的生长,改善养殖池塘水质。(6) The plant lactobacillus FF34 compound bacterial agent is immobilized by adsorption on pumice, activated carbon or expanded clay and then released into the aquaculture water. Compared with the direct spraying of unimmobilized bacterial agents, the immobilized bacteria are not easily inactivated or lost in the oxygen-containing aquaculture water environment. The effective time is more than 20 days. It can sustainably degrade the ammonia nitrogen, nitrite, total phosphorus and total nitrogen content in the water, inhibit the growth of aquatic pathogens, and improve the water quality of aquaculture ponds.

生物材料样品保藏Biomaterial sample storage

(1)植物乳杆菌FF34,分类命名为植物乳杆菌(lactobacillus plantarum),保藏于中国典型培养物保藏中心,简称CCTCC,地址:中国武汉武汉大学,保藏编号:CCTCC NO:M20211221,保藏日期2021年10月11日。(1) Lactobacillus plantarum FF34, classified as Lactobacillus plantarum, is deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M20211221, deposit date: October 11, 2021.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1植物乳杆菌及高淀粉添加对团头鲂幼鱼肝脏油红0染色切片。A图为15WM组(×200倍)的油红O染色切片,B图为35WM+LAB组(×200倍)的油红O染色切片,C图为35WM组(×200倍)的油红O染色切片。Figure 1 Effects of Lactobacillus plantarum and high starch supplementation on Oil Red O-stained sections of liver of young Megalobrama amblycephala. Figure A shows Oil Red O-stained sections of the 15WM group (×200 times), Figure B shows Oil Red O-stained sections of the 35WM+LAB group (×200 times), and Figure C shows Oil Red O-stained sections of the 35WM group (×200 times).

图2菌株FF-34基于16S rRNA基因序列比对结果以Enterococcus faecalis(Y18293.1)为外枝构建的Neighbor-Joining系统发育树。Fig. 2 Neighbor-Joining phylogenetic tree of strain FF-34 constructed based on 16S rRNA gene sequence alignment with Enterococcus faecalis (Y18293.1) as the outer branch.

具体实施方式DETAILED DESCRIPTION

实施例1:菌种的筛选Example 1: Screening of strains

在无锡市滨湖区马山镇河蟹养殖池塘采集池塘泥水,加入生理盐水,研磨制成匀浆液,取移液枪吸取0.1mL于MRS固体培养基上涂布25~35℃厌氧培养。根据菌落大小选取生长良好的菌落反复接种筛选,直至得到均匀的单个菌落,命名为FF34。The muddy water was collected from the crab breeding pond in Mashan Town, Binhu District, Wuxi City, and physiological saline was added to grind to make a homogenate. 0.1 mL was taken by a pipette and spread on MRS solid medium for anaerobic culture at 25-35°C. The colonies with good growth were selected according to the colony size and repeatedly inoculated and screened until a uniform single colony was obtained, which was named FF34.

实施例2:菌种鉴定Example 2: Bacterial species identification

(1)形态特征:植物乳杆菌菌株FF34,革兰氏染色为阳性,在MRS平板涂布后,35℃厌氧培养48h生长出的菌落凸起,表面光滑,细密,色白,不透明,直径1~3mm,MRS倾注培养的深层菌落呈透镜或菱形,平板底部菌落呈扁平圆形,显微镜(16×100)观察,菌体呈短杆状,两端呈圆形略方形态,成对或短链状排列,(0.9~1.2)μm×(2~4)μm,无鞭毛,能运动。(1) Morphological characteristics: Lactobacillus plantarum strain FF34 was Gram-positive. After being spread on an MRS plate and cultured anaerobically at 35°C for 48 h, the colonies grown were raised, smooth, dense, white, opaque, and 1 to 3 mm in diameter. The deep colonies in MRS pour-over culture were lens-shaped or diamond-shaped, and the colonies at the bottom of the plate were flat and round. Under a microscope (16×100), the bacteria were short rod-shaped with rounded and slightly square ends, arranged in pairs or short chains, (0.9 to 1.2) μm × (2 to 4) μm, without flagella, and able to move.

(2)生化特性:(2) Biochemical characteristics:

表1菌株FF-34生理生化特性–酶活、碳源氧化Table 1 Physiological and biochemical characteristics of strain FF-34 – enzyme activity, carbon source oxidation

Figure BDA0003731190040000081
Figure BDA0003731190040000081

Figure BDA0003731190040000091
Figure BDA0003731190040000091

+:阳性反应;-:阴性反应;W:弱阳性反应+: positive reaction; -: negative reaction; W: weak positive reaction

表2菌株FF-34生理生化特性–利用碳源产酸Table 2 Physiological and biochemical characteristics of strain FF-34 – Acid production using carbon sources

Figure BDA0003731190040000092
Figure BDA0003731190040000092

Figure BDA0003731190040000101
Figure BDA0003731190040000101

+:阳性反应;-:阴性反应;W:弱阳性反应+: positive reaction; -: negative reaction; W: weak positive reaction

(3)16S rRNA序列分析和系统发育树的构建:(3) 16S rRNA sequence analysis and phylogenetic tree construction:

植物乳杆菌菌株FF34的16S rRNA基因序列,如SEQ ID NO.1所示。The 16S rRNA gene sequence of Lactobacillus plantarum strain FF34 is shown in SEQ ID NO.1.

植物乳杆菌菌株FF34基因序列测序结果:将菌株所扩增的16S rRNA基因序列在NCBI通过Blast进行同源性检索,结果检索出为乳杆菌属的16S rRNA基因序列,采用邻接法构建菌株系统发育树,分离菌株在系统发育树上与植物乳杆菌Lactobacillus plantarumsubsp.plantarumATCC 14917(登录号:ACGZ01000098)属同一支。结合形态学和生理生化特征将所分离菌株鉴定为植物乳杆菌Lactobacillus plantarum(见图2)。Sequencing results of the gene sequence of Lactobacillus plantarum strain FF34: The 16S rRNA gene sequence amplified by the strain was searched for homology in NCBI by Blast, and the 16S rRNA gene sequence of the genus Lactobacillus was retrieved. The strain phylogenetic tree was constructed by the neighbor-joining method. The isolated strain belongs to the same branch as Lactobacillus plantarum subsp. plantarum ATCC 14917 (accession number: ACGZ01000098) on the phylogenetic tree. The isolated strain was identified as Lactobacillus plantarum by combining morphological and physiological and biochemical characteristics (see Figure 2).

实施例3:植物乳杆菌FF34对水产致病菌抑菌能力的测定Example 3: Determination of the antibacterial ability of Lactobacillus plantarum FF34 against aquatic pathogens

将致病菌与植物乳杆菌FF34菌液共同接入LB培养基中,30℃静置培养24h,于0h、24h取样计数,观察植物乳杆菌FF34对致病菌的拮抗作用。测定结果如表3所示:The pathogenic bacteria and the bacterial solution of Lactobacillus plantarum FF34 were inoculated into LB medium and cultured at 30°C for 24 hours. Samples were taken and counted at 0 hours and 24 hours to observe the antagonistic effect of Lactobacillus plantarum FF34 on the pathogenic bacteria. The results are shown in Table 3:

表3植物乳杆菌FF34与致病菌的共培养测定结果Table 3 Co-culture determination results of Lactobacillus plantarum FF34 and pathogenic bacteria

Figure BDA0003731190040000102
Figure BDA0003731190040000102

Figure BDA0003731190040000111
Figure BDA0003731190040000111

在共培养条件下,植物乳杆菌FF34对溶藻弧菌和爱德华氏菌的抑制能力最强,对大肠杆菌和嗜水气单胞菌次之,对鳗利斯顿氏菌抑制能力较弱。植物乳杆菌FF34在24h完全抑制了溶藻弧菌的生长,使得爱德华氏菌的增殖减少了约2个数量级,大肠杆菌和嗜水气单胞菌的增殖分别减少了一个数量级。Under co-culture conditions, Lactobacillus plantarum FF34 had the strongest inhibitory ability against Vibrio alginolyticus and Edwardsiella, followed by Escherichia coli and Aeromonas hydrophila, and had a weaker inhibitory ability against Listonella anguillarum. Lactobacillus plantarum FF34 completely inhibited the growth of Vibrio alginolyticus within 24 hours, reducing the proliferation of Edwardsiella by about 2 orders of magnitude, and the proliferation of Escherichia coli and Aeromonas hydrophila by one order of magnitude respectively.

实施例4:植物乳杆菌FF34降解亚硝酸盐能力的测定Example 4: Determination of the ability of Lactobacillus plantarum FF34 to degrade nitrite

MRS肉汤培养基分装50mL/试管,与2mol/L亚硝酸钠溶液分别在121℃灭菌15min后,在每支装有MRS肉汤培养基的试管中分别添加2mol/L亚硝酸钠溶液0.1mL,然后接种活化后的植物乳杆菌FF34菌悬液5mL,对照组接入5mL无菌蒸馏水,每组3个平行,35℃静置培养20h,其中0h、7h和20h取样,4000r/min离心10min,取上清液,用美国哈希试剂检测亚硝氮浓度。数据用SPSS18.0统计分析,全部结果均用平均值±标准误

Figure BDA0003731190040000112
表示,独立样本T检验比较检验各组间的差异。MRS broth medium was divided into 50mL/test tubes, sterilized with 2mol/L sodium nitrite solution at 121℃ for 15min, and then 0.1mL of 2mol/L sodium nitrite solution was added to each test tube containing MRS broth medium, and then 5mL of activated plant lactobacillus FF34 suspension was inoculated. The control group was connected with 5mL of sterile distilled water. Each group had 3 parallels and was cultured at 35℃ for 20h. Samples were taken at 0h, 7h and 20h, centrifuged at 4000r/min for 10min, and the supernatant was taken. The nitrite nitrogen concentration was detected with the American Hach reagent. The data were statistically analyzed using SPSS18.0, and all results were expressed as mean ± standard error.
Figure BDA0003731190040000112
Indicates that the independent sample T test was used to compare the differences among the groups.

表4结果显示植物乳杆菌FF34亚硝酸盐还原酶活性高,在厌氧条件下,能够快速降解亚硝氮,20h内降解极其显著,7h降解率82.30±0.50%,20h降解率达到96.00±0.01%。The results in Table 4 show that Lactobacillus plantarum FF34 has high nitrite reductase activity and can quickly degrade nitrite nitrogen under anaerobic conditions. The degradation is extremely significant within 20 hours, with a degradation rate of 82.30±0.50% in 7 hours and a degradation rate of 96.00±0.01% in 20 hours.

表4植物乳杆菌FF34降解亚硝氮能力Table 4 Nitrite degradation ability of Lactobacillus plantarum FF34

分组Grouping 对照组Control group FF34试验组FF34 test group 时间time 亚硝氮mg/LNitrite nitrogen mg/L 亚硝氮mg/LNitrite nitrogen mg/L 0h0h 135.22±2.34Aa 135.22±2.34 Aa 135.89±2.34Aa 135.89±2.34 Aa 7h7h 135.69±0.55Aa 135.69±0.55 Aa 24.05±0.99Bb 24.05±0.99 Bb 20h20h 134.31±0.20Aa 134.31±0.20 Aa 5.22±1.24Cb 5.22±1.24 Cb 20h亚硝氮降解率20h nitrite nitrogen degradation rate 1.12±1.37%b 1.12±1.37% b 96.00±0.01%a 96.00±0.01% a

注:同行不同小写字母表示差异显著(P<0.05),同列不同大写字母表示差异显著(P<0.05)。Note: Different lowercase letters in the same row indicate significant differences (P<0.05), and different capital letters in the same column indicate significant differences (P<0.05).

实施例5具有水产病原菌拮抗特性的植物乳杆菌FF34单菌制剂的制备Example 5 Preparation of a single bacterial preparation of Lactobacillus plantarum FF34 having antagonistic properties against aquatic pathogens

1)菌种活化:1) Bacteria activation:

无菌开启植物乳杆菌FF34的冻干保藏菌种,接种于装有MRS肉汤的试管中,于30~35℃静置培养24~48h,然后转接于MRS肉汤三角瓶中,30~35℃培养活化20~48h;反复活化2~3次,镜检,计数,当菌体浓度达109CFU/mL时作为种子液;Aseptically open the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculate it into a test tube filled with MRS broth, culture it at 30-35°C for 24-48 hours, then transfer it to a MRS broth triangular bottle, culture it at 30-35°C for activation for 20-48 hours; repeatedly activate it 2-3 times, examine it under a microscope, count it, and use it as seed liquid when the bacterial concentration reaches 10 9 CFU/mL;

MRS肉汤培养基组成以g/L计:酪蛋白酶消化物10,牛肉膏粉10,酵母膏粉4,柠檬酸三铵2,乙酸钠5,硫酸镁0.2,硫酸锰0.05,磷酸氢二钾2,葡萄糖20,吐温-80 1.08,以蒸馏水定容配制,pH 5.7±0.2;MRS broth medium composition (g/L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2;

(2)固定化发酵培养:(2) Immobilized fermentation culture:

三角瓶种子培养液制备:将步骤(1)所得种子液以体积比1%~5%接种量接入装有固定化发酵培养基的3L三角瓶,装液量为体积比90%,于30~35℃静置培养24~40h,当菌体浓度达109CFU/mL时,作为三角瓶种子培养液,接入普通好氧发酵罐中进行一级、二级固定化发酵逐级放大扩培,即:3L三角瓶,再500L发酵罐,再10T发酵罐,发酵罐间移种以无菌空气压送方式完成接种;Preparation of triangular flask seed culture solution: The seed solution obtained in step (1) is inoculated at a volume ratio of 1% to 5% into a 3L triangular flask filled with immobilized fermentation medium, and the volume of the solution is 90% by volume. It is cultured at 30 to 35°C for 24 to 40 hours. When the bacterial concentration reaches 109 CFU/mL, it is used as triangular flask seed culture solution and inoculated into ordinary aerobic fermentation tanks for primary and secondary immobilized fermentation and step-by-step expansion, i.e.: 3L triangular flask, then 500L fermentation tank, then 10T fermentation tank. The inoculation is completed by transferring the seeds between fermentation tanks by sterile air pressure delivery.

一级固定化培养:将固定化培养种子液以体积比1%~5%接种量接入500L发酵罐中,发酵罐装液量为体积比80%~90%,无菌空气保持罐压至0.01~0.05Mpa,间歇搅拌,转速80~100r/min,30~35℃培养20~40h,pH为4.5~5.5时,结束培养,得到一级固定化培养液,其中植物乳杆菌FF34菌体被吸附固定在海藻酸钙+二氧化硅形成的凝胶小颗粒中,活菌体浓度≥5×109CFU/mL;Primary immobilized culture: inoculate the immobilized culture seed liquid in a volume ratio of 1% to 5% into a 500L fermenter, the volume of the fermenter is 80% to 90%, the tank pressure is maintained at 0.01 to 0.05Mpa with sterile air, stir intermittently, rotate at 80 to 100 r/min, culture at 30 to 35°C for 20 to 40 hours, and terminate the culture when the pH is 4.5 to 5.5 to obtain a primary immobilized culture liquid, in which the Lactobacillus plantarum FF34 cells are adsorbed and fixed in the gel particles formed by calcium alginate + silicon dioxide, and the concentration of live cells is ≥5×10 9 CFU/mL;

二级固定化培养:将一级固定化培养液用无菌空气压送,以体积比1%~5%接种量接入10T发酵罐中,发酵罐装液量为体积比70%~90%,无菌空气保持罐压至0.01~0.05Mpa,间歇搅拌,转速80~100r/min,30~38℃培养20~40h,当发酵液pH降至4.0~5.5,培养结束,得到二级固定化培养液,其中植物乳杆菌FF34菌体被吸附固定在海藻酸钙+二氧化硅形成的凝胶小颗粒中,菌体浓度≥5×109CFU/mL;Secondary immobilized culture: the primary immobilized culture solution is sent by sterile air pressure, and inoculated into a 10T fermenter at a volume ratio of 1% to 5%. The volume ratio of the fermenter is 70% to 90%. The sterile air is used to keep the tank pressure at 0.01 to 0.05 MPa, and the fermentation solution is stirred intermittently at a speed of 80 to 100 r/min and cultured at 30 to 38° C. for 20 to 40 hours. When the pH of the fermentation solution drops to 4.0 to 5.5, the culture is terminated to obtain a secondary immobilized culture solution, in which the Lactobacillus plantarum FF34 cells are adsorbed and fixed in the gel particles formed by calcium alginate + silicon dioxide, and the cell concentration is ≥5×10 9 CFU/mL;

固定化发酵培养基组成以g/L计:碳源10~40,氮源3~15,磷酸氢二钾0.2~5,硫酸镁0~0.2,硫酸锰0~0.05,碳酸钙5~40,海藻酸钠3~10,二氧化硅1~10,以自来水定容配制,pH 6.0~6.5,121℃灭菌30min;其中碳源包括小麦次粉、玉米淀粉、糊精、糖蜜、葡萄糖、果葡糖浆等碳源中的一种或几种组合。氮源包括鱼粉蛋白胨、胰蛋白胨、大豆蛋白胨、大豆分离蛋白、酵母膏、酵母粉、玉米浆、棉籽粕、菜籽粕、豆粕、花生粕及血粉等氮源中的一种或几种组合。The composition of the fixed fermentation medium is calculated in g/L: 10-40 carbon source, 3-15 nitrogen source, 0.2-5 potassium dihydrogen phosphate, 0-0.2 magnesium sulfate, 0-0.05 manganese sulfate, 5-40 calcium carbonate, 3-10 sodium alginate, 1-10 silicon dioxide, prepared with tap water, pH 6.0-6.5, sterilized at 121°C for 30 minutes; wherein the carbon source includes one or a combination of carbon sources such as wheat flour, corn starch, dextrin, molasses, glucose, fructose syrup, etc. The nitrogen source includes one or a combination of nitrogen sources such as fish meal peptone, tryptone, soy peptone, soy protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal, etc.

(3)植物乳杆菌FF34单菌制剂的制备:(3) Preparation of plant lactobacillus FF34 single bacterial preparation:

(A)植物乳杆菌FF34单菌水剂的制备:将步骤(2)所得二级固定化培养液进行灌装,获得植物乳杆菌FF34水剂,菌液水剂中FF34活菌浓度≥5.0×109CFU/mL;(A) Preparation of Lactobacillus plantarum FF34 single bacterial solution: filling the secondary immobilized culture solution obtained in step (2) to obtain a Lactobacillus plantarum FF34 solution, wherein the concentration of live FF34 bacteria in the bacterial solution is ≥5.0×10 9 CFU/mL;

(B)植物乳杆菌FF34单菌粉剂的制备:将步骤(2)所得二级固定化培养液经离心收集湿菌体,按质量比为:湿菌体:碳酸钙:糊精:二氧化硅=1:0.1~2:0.5~2:0.01进行混合,经喷雾干燥或真空干燥得到植物乳杆菌FF34菌粉,菌粉中FF34活菌浓度不低于1×1010CFU/g。(B) Preparation of Lactobacillus plantarum FF34 single bacterial powder: The secondary immobilized culture solution obtained in step (2) is centrifuged to collect wet bacteria, and the mixture is mixed in a mass ratio of wet bacteria: calcium carbonate: dextrin: silicon dioxide = 1:0.1 to 2:0.5 to 2:0.01, and spray-dried or vacuum-dried to obtain Lactobacillus plantarum FF34 powder, wherein the concentration of live FF34 bacteria in the powder is not less than 1×10 10 CFU/g.

实施例6:植物乳杆菌FF34与酪酸梭状芽孢杆菌JSIM-MCB20040312(保藏号CGMCCNo.1647)混合发酵共培养及其复合菌剂制备Example 6: Mixed fermentation and co-cultivation of Lactobacillus plantarum FF34 and Clostridium butyricum JSIM-MCB20040312 (Accession No. CGMCC No. 1647) and preparation of composite bacterial agent

(1)菌种活化及发三角瓶种子液培养(1) Activation of bacterial strains and cultivation of triangular flask seed solution

(A)酪酸梭状芽孢杆菌JSIM-MCB 20040312的菌种活化及三角瓶种子液培养:(A) Activation of Clostridium butyricum JSIM-MCB 20040312 and culture of seed solution in triangular flask:

无菌开启酪酸梭状芽孢杆菌JSIM-MCB 20040312冻干保藏菌种,接入装有活化培养基的试管,进行静置厌氧培养,30~37℃培养18~24h,然后以体积比1%~10%的接种量转接于新鲜活化培养基,30~37℃培养20~24h,镜检,超过90%菌体形成芽孢时,即成熟,反复活化2~3次,种子菌悬液以体积比1%~10%接种量接入装有种子培养基的数个3L三角瓶中,三角瓶装液量为体积比80%~90%,30~37℃恒温培养箱中静置培养20~24h,镜检,超过90%菌体形成芽孢时,即作为三角瓶种子液;Aseptically opening the freeze-dried preserved strain of Clostridium butyricum JSIM-MCB 20040312, inoculating it with a test tube filled with an activation culture medium, and performing static anaerobic culture at 30-37° C. for 18-24 hours, then transferring it to a fresh activation culture medium at an inoculation amount of 1%-10% by volume, and culturing it at 30-37° C. for 20-24 hours. After microscopic examination, when more than 90% of the bacteria form spores, it is mature, and the activation is repeated 2-3 times. The seed bacterial suspension is inoculated with an inoculation amount of 1%-10% by volume into several 3L triangular flasks filled with seed culture medium, and the volume of the triangular flask is 80%-90% by volume. It is statically cultured in a constant temperature incubator at 30-37° C. for 20-24 hours, and after microscopic examination, when more than 90% of the bacteria form spores, it is used as triangular flask seed liquid;

酪酸梭状芽孢杆菌JSIM-MCB 20040312活化及种子培养基组成以g/L计:Clostridium butyricum JSIM-MCB 20040312 activation and seed culture medium composition (g/L):

酵母膏3~5,牛肉膏5~20,蛋白胨5~20,葡萄糖5~10,可溶性淀粉1~2,氯化钠5,乙酸钠3~5,盐酸半胱氨酸0.5~1,碳酸钙1~7,用去离子水配制,pH 6.0~7.5;Yeast extract 3-5, beef extract 5-20, peptone 5-20, glucose 5-10, soluble starch 1-2, sodium chloride 5, sodium acetate 3-5, cysteine hydrochloride 0.5-1, calcium carbonate 1-7, prepared with deionized water, pH 6.0-7.5;

(B)植物乳杆菌FF34的菌种活化及三角瓶种子液培养(B) Activation of Lactobacillus plantarum FF34 and culture of seed solution in triangular flask

无菌开启植物乳杆菌FF34的冻干保藏菌种,接种于装有MRS肉汤的试管中,于30~35℃静置培养24~48h,然后转接于MRS肉汤小三角瓶中,30~35℃培养活化20~48h;反复活化2~3次,镜检,计数,当菌体浓度109CFU/mL时作为种子液,以体积比1%~5%接种量接入装有MRS发酵培养基的数个3L三角瓶,装液量为体积比90%,于30~35℃静置培养24~40h,当菌体浓度达109CFU/mL时,作为三角瓶种子液;Aseptically opening the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculating it in a test tube filled with MRS broth, statically culturing it at 30-35°C for 24-48 hours, then transferring it to a small triangular bottle of MRS broth, culturing and activating it at 30-35°C for 20-48 hours; repeatedly activating it 2-3 times, microscopically inspecting and counting it, and when the bacterial concentration is 10 9 CFU/mL, using it as seed liquid, inoculating it in a volume ratio of 1%-5% inoculation amount into several 3L triangular bottles filled with MRS fermentation medium, the liquid volume is 90% by volume, statically culturing it at 30-35°C for 24-40 hours, and using it as triangular bottle seed liquid when the bacterial concentration reaches 10 9 CFU/mL;

MRS肉汤培养基组成以g/L计:酪蛋白酶消化物10,牛肉膏粉10,酵母膏粉4,柠檬酸三铵2,乙酸钠5,硫酸镁0.2,硫酸锰0.05,磷酸氢二钾2,葡萄糖20,吐温-80 1.08,以蒸馏水定容配制,pH 5.7±0.2;MRS broth medium composition (g/L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2;

(2)混合发酵共培养(2) Mixed fermentation and co-culture

FF34与酪酸梭状芽孢杆菌JSIM-MCB 20040312混合发酵分两步进行:第一步发酵:将上述(A)步骤获得的酪酸梭状芽孢杆菌JSIM-MCB 20040312发酵罐种子液以体积比1%~10%接入装有混合发酵培养基的500L发酵罐中,装液量70%~80%(V/V),30~37℃,静置培养24~48h,镜检菌体中芽孢形成,即第一步发酵结束;第二步发酵先将第一步发酵所得发酵液用盐酸调整pH至5.0~5.4,然后接入上述(B)步骤获得的植物乳杆菌FF34三角瓶种子液,接种量为体积比1%~10%,培养温度为30~35℃,间歇搅拌,流加浓度为20%(w/v)葡萄糖或30%碳酸钙或氨水,控制发酵液pH4.5~5.0,培养24h左右,获得FF34与芽孢杆菌混合发酵液,镜检可见乳杆菌与芽孢共存,混合发酵液中FF34活菌浓度的≥1.0×109CFU/mL,芽孢杆菌活菌浓度≥5.0×108CFU/mL,芽孢率≥90%;The mixed fermentation of FF34 and Clostridium butyricum JSIM-MCB 20040312 was carried out in two steps: the first fermentation step: the Clostridium butyricum JSIM-MCB obtained in step (A) above was The 20040312 fermentation tank seed liquid is inoculated into a 500L fermentation tank filled with mixed fermentation medium at a volume ratio of 1% to 10%, the liquid volume is 70% to 80% (V/V), and the temperature is 30 to 37°C. The mixture is cultured for 24 to 48 hours. The formation of spores in the bacteria is observed under a microscope, and the first fermentation is completed. In the second fermentation, the fermentation liquid obtained in the first fermentation is firstly adjusted to pH 5.0 to 5.4 with hydrochloric acid, and then the plant lactobacillus FF34 triangular flask seed liquid obtained in the above step (B) is inoculated with an inoculation volume ratio of 1% to 10%, the culture temperature is 30 to 35°C, the mixture is stirred intermittently, and the concentration of 20% (w/v) glucose or 30% calcium carbonate or ammonia water is fed, the pH of the fermentation liquid is controlled to 4.5 to 5.0, and the mixture is cultured for about 24 hours to obtain a mixed fermentation liquid of FF34 and bacillus. The coexistence of lactobacillus and spores can be seen under a microscope, and the concentration of live FF34 bacteria in the mixed fermentation liquid is ≥1.0×10 9 CFU/mL, Bacillus viable bacteria concentration ≥5.0×10 8 CFU/mL, spore rate ≥90%;

混合发酵培养基组成以g/L计:小麦次粉0~50,蛋白胨0.1~5,酵母膏0~5,乙酸钠0~3,玉米淀粉0~10,硫酸铵0~2,氯化钠0~5,碳酸钙0~5,磷酸二氢钾0~0.5,硫酸镁0~0.5,硫酸锰0~0.3,海藻酸钠0~5,自来水定容,pH 7.0,121℃灭菌30min;The composition of the mixed fermentation medium is in g/L: 0-50 wheat flour, 0.1-5 peptone, 0-5 yeast extract, 0-3 sodium acetate, 0-10 corn starch, 0-2 ammonium sulfate, 0-5 sodium chloride, 0-5 calcium carbonate, 0-0.5 potassium dihydrogen phosphate, 0-0.5 magnesium sulfate, 0-0.5 manganese sulfate, 0-0.3 sodium alginate, fixed to volume with tap water, pH 7.0, sterilized at 121°C for 30 min;

(3)植物乳杆菌FF34复合菌剂的制备:(3) Preparation of plant lactobacillus FF34 composite bacterial agent:

(A)将上述步骤(2)获得的植物乳杆菌FF34与酪酸梭状芽孢杆菌JSIM-MCB2004031混合发酵液直接进行灌装,获得植物乳杆菌FF34复合菌水剂,复合菌液水剂中FF34活菌浓度的≥1.0×109CFU/mL,芽孢杆菌活菌浓度≥5.0×108CFU/mL,芽孢率≥90%;(A) directly filling the mixed fermentation liquid of Lactobacillus plantarum FF34 and Clostridium butyricum JSIM-MCB2004031 obtained in the above step (2) to obtain a Lactobacillus plantarum FF34 composite bacterial liquid, wherein the live bacteria concentration of FF34 in the composite bacterial liquid is ≥1.0×10 9 CFU/mL, the live bacteria concentration of Bacillus sporogenes is ≥5.0×10 8 CFU/mL, and the spore rate is ≥90%;

(B)将上述步骤(2)所得的植物乳杆菌FF34与酪酸梭状芽孢杆菌JSIM-MCB2004031混合发酵液经离心收集湿菌体,按质量比为:湿菌体:碳酸钙:二氧化硅=1:0.1~3:0.01进行混合,经喷雾干燥或真空干燥得到植物乳杆菌FF34复合菌剂菌粉,菌粉中FF34活菌浓度≥5×109CFU/g,芽孢杆菌活菌浓度≥5.0×109CFU/g。(B) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Clostridium butyricum JSIM-MCB2004031 obtained in the above step (2) is centrifuged to collect wet cells, mixed in a mass ratio of wet cells: calcium carbonate: silicon dioxide = 1:0.1 to 3:0.01, and spray-dried or vacuum-dried to obtain Lactobacillus plantarum FF34 composite bacterial agent powder, wherein the live bacteria concentration of FF34 in the bacterial powder is ≥5×10 9 CFU/g, and the live bacteria concentration of Bacillus sp. is ≥5.0×10 9 CFU/g.

实施例7植物乳杆菌FF34与枯草芽孢杆菌AB90008-15(保藏号CGMCC NO.18684)混合发酵共培养及其复合菌剂制备Example 7 Mixed fermentation and co-cultivation of Lactobacillus plantarum FF34 and Bacillus subtilis AB90008-15 (Accession No. CGMCC NO.18684) and preparation of composite bacterial agent

(1)菌种活化及发三角瓶种子液培养(1) Activation of bacterial strains and cultivation of triangular flask seed solution

(A)枯草芽孢杆菌AB90008-15的菌种活化及种子制备:(A) Activation and seed preparation of Bacillus subtilis AB90008-15:

无菌开启枯草芽孢杆菌AB90008-15冻干管保藏菌种,接种于麸皮营养琼脂,于30~37℃培养24~48h,然后转接麸皮营养琼脂斜面,反复活化2~3次,镜检,当枯草芽孢杆菌90%以上菌体形成芽孢时,即为成熟,刮取菌苔,用无菌水制备菌悬液作为枯草芽孢杆菌种子液;Aseptically opening the Bacillus subtilis AB90008-15 freeze-dried tube preserved bacteria, inoculating it on bran nutrient agar, culturing it at 30-37° C. for 24-48 hours, then transferring it to the bran nutrient agar slant, repeatedly activating it 2-3 times, and inspecting it under a microscope. When more than 90% of the Bacillus subtilis bacteria form spores, it is mature, scraping the bacterial lawn, and preparing a bacterial suspension with sterile water as a Bacillus subtilis seed liquid;

麸皮营养琼脂斜面培养基组成以g/L计:蛋白胨10,牛肉膏3,NaCl 5,麸皮10,琼脂15-20,以蒸馏水定容配制,pH 7.0~7.2;The composition of the bran nutrient agar slant medium is in g/L: peptone 10, beef extract 3, NaCl 5, bran 10, agar 15-20, prepared with distilled water to a fixed volume, pH 7.0-7.2;

(B)植物乳杆菌FF34的菌种活化及三角瓶种子液培养(B) Activation of Lactobacillus plantarum FF34 and culture of seed solution in triangular flask

无菌开启植物乳杆菌FF34的冻干保藏菌种,接种于装有MRS肉汤的试管中,于30~35℃静置培养24~48h,然后转接于MRS肉汤小三角瓶中,30~35℃培养活化20~48h;反复活化2~3次,镜检,计数,当菌体浓度109CFU/mL时作为种子液,以体积比1%~5%接种量接入装有固定化发酵培养基的数个3L三角瓶,装液量为体积比90%,于30~35℃静置培养24~40h,当菌体浓度达109CFU/mL时,作为三角瓶种子液;Aseptically opening the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculating it in a test tube filled with MRS broth, statically culturing it at 30-35°C for 24-48 hours, then transferring it to a small triangular bottle of MRS broth, culturing and activating it at 30-35°C for 20-48 hours; repeatedly activating it 2-3 times, microscopically inspecting and counting it, using it as seed liquid when the bacterial concentration is 10 9 CFU/mL, inoculating it in a volume ratio of 1%-5% inoculation amount into several 3L triangular bottles filled with fixed fermentation medium, the liquid volume is 90% by volume, statically culturing it at 30-35°C for 24-40 hours, and using it as triangular bottle seed liquid when the bacterial concentration reaches 10 9 CFU/mL;

MRS肉汤培养基组成以g/L计:酪蛋白酶消化物10,牛肉膏粉10,酵母膏粉4,柠檬酸三铵2,乙酸钠5,硫酸镁0.2,硫酸锰0.05,磷酸氢二钾2,葡萄糖20,吐温-80 1.08,以蒸馏水定容配制,pH 5.7±0.2;MRS broth medium composition (g/L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, dipotassium hydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2;

固定化发酵培养基组成以g/L计:碳源10~40,氮源3~15,磷酸氢二钾0.2~5,硫酸镁0~0.2,硫酸锰0~0.05,碳酸钙5~40,海藻酸钠3~10,二氧化硅1~10,以自来水定容配制,pH 6.0~6.5,121℃灭菌30min;其中碳源包括小麦次粉、玉米淀粉、糊精、糖蜜、葡萄糖、果葡糖浆等碳源中的一种或几种组合。氮源包括鱼粉蛋白胨、胰蛋白胨、大豆蛋白胨、大豆分离蛋白、酵母膏、酵母粉、玉米浆、棉籽粕、菜籽粕、豆粕、花生粕及血粉等氮源中的一种或几种组合。The composition of the fixed fermentation medium is calculated in g/L: 10-40 carbon source, 3-15 nitrogen source, 0.2-5 potassium hydrogen phosphate, 0-0.2 magnesium sulfate, 0-0.05 manganese sulfate, 5-40 calcium carbonate, 3-10 sodium alginate, 1-10 silicon dioxide, prepared with tap water, pH 6.0-6.5, sterilized at 121°C for 30 minutes; wherein the carbon source includes one or a combination of carbon sources such as wheat flour, corn starch, dextrin, molasses, glucose, fructose syrup, etc. The nitrogen source includes one or a combination of nitrogen sources such as fish meal peptone, tryptone, soy peptone, soy protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal and blood meal, etc.

(2)混合发酵共培养(2) Mixed fermentation and co-culture

FF34与枯草芽孢杆菌AB90008-15混合发酵分两步进行:第一步发酵:将上述步骤(A)所得枯草芽孢杆菌种子菌悬液以体积比1%~10%接种量接入装有发酵培养基的500L发酵罐中进行一级种子罐培养,装液量为体积比50%~60%,通气量15m3/h,罐压0.05~0.1Mpa,转速为100~150r/min,30~37℃,通气培养24~48h,镜检菌体中芽孢形成,即第一步发酵结束;第二步发酵先将发酵液用盐酸调整pH至5.0~5.4,然后接入上述(B)步骤获得的植物乳杆菌FF34三角瓶种子液,接种量为体积比1%~10%,培养温度为30~35℃,间歇搅拌,流加浓度为20%(w/v)葡萄糖或30%碳酸钙或氨水,控制发酵液pH4.5~5.0,培养24h左右,获得FF34与芽孢杆菌混合发酵液,镜检可见乳杆菌与芽孢共存,混合发酵液中FF34活菌浓度的≥1.0×109CFU/mL,芽孢杆菌活菌浓度≥5.0×108CFU/mL,芽孢率≥90%;The mixed fermentation of FF34 and Bacillus subtilis AB90008-15 was carried out in two steps: the first fermentation step: the Bacillus subtilis seed suspension obtained in the above step (A) was inoculated at a volume ratio of 1% to 10% into a 500L fermentation tank filled with fermentation medium for primary seed tank culture, the liquid volume was 50% to 60% by volume, and the ventilation volume was 15m3 /h, tank pressure 0.05 ~ 0.1Mpa, speed 100 ~ 150r / min, 30 ~ 37 ℃, ventilation culture 24 ~ 48h, microscopic examination of spore formation in the bacteria, that is, the first fermentation is completed; the second fermentation is to first adjust the pH of the fermentation liquid to 5.0 ~ 5.4 with hydrochloric acid, and then access the plant lactobacillus FF34 triangular flask seed liquid obtained in the above step (B), the inoculation amount is 1% to 10% by volume, the culture temperature is 30 ~ 35 ℃, intermittent stirring, the flow concentration is 20% (w / v) glucose or 30% calcium carbonate or ammonia water, the pH of the fermentation liquid is controlled at 4.5 ~ 5.0, and the culture is about 24h to obtain a mixed fermentation liquid of FF34 and bacillus. Microscopic examination shows that lactobacillus and spores coexist, the live bacteria concentration of FF34 in the mixed fermentation liquid is ≥1.0 × 109 CFU / mL, the live bacteria concentration of bacillus is ≥5.0 × 108 CFU / mL, and the spore rate is ≥90%;

混合发酵培养基组成以g/L计:小麦次粉0~50,蛋白胨0.1~5,酵母膏0~5,乙酸钠0~3,玉米淀粉0~10,硫酸铵0~2,氯化钠0~5,碳酸钙0~5,磷酸二氢钾0~0.5,硫酸镁0~0.5,硫酸锰0~0.3,海藻酸钠0~5,自来水定容,pH 7.0,121℃灭菌30min;The composition of the mixed fermentation medium is in g/L: 0-50 wheat flour, 0.1-5 peptone, 0-5 yeast extract, 0-3 sodium acetate, 0-10 corn starch, 0-2 ammonium sulfate, 0-5 sodium chloride, 0-5 calcium carbonate, 0-0.5 potassium dihydrogen phosphate, 0-0.5 magnesium sulfate, 0-0.5 manganese sulfate, 0-0.3 sodium alginate, fixed to volume with tap water, pH 7.0, sterilized at 121°C for 30 min;

(3)植物乳杆菌FF34复合菌剂的制备:(3) Preparation of plant lactobacillus FF34 composite bacterial agent:

(A)将上述步骤(2)获得的植物乳杆菌FF34与枯草芽孢杆菌AB90008-15混合发酵液直接进行灌装,获得植物乳杆菌FF34复合菌水剂,复合菌液水剂中FF34活菌浓度的≥1.0×109CFU/mL,芽孢杆菌活菌浓度≥5.0×108CFU/mL,芽孢率≥90%;(A) directly filling the mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus subtilis AB90008-15 obtained in the above step (2) to obtain a Lactobacillus plantarum FF34 composite bacterial liquid, wherein the live bacteria concentration of FF34 in the composite bacterial liquid is ≥1.0×10 9 CFU/mL, the live bacteria concentration of Bacillus subtilis is ≥5.0×10 8 CFU/mL, and the spore rate is ≥90%;

(B)将上述步骤(2)所得的植物乳杆菌FF34与枯草芽孢杆菌AB9008-15混合发酵液经离心收集湿菌体,湿菌体:碳酸钙:二氧化硅=1:0.1~3:0.01进行混合,经喷雾干燥或真空干燥得到植物乳杆菌FF34复合菌剂菌粉,菌粉中FF34活菌浓度≥5×109CFU/g,芽孢杆菌活菌浓度≥5.0×109CFU/g。(B) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus subtilis AB9008-15 obtained in the above step (2) is centrifuged to collect wet cells, and the wet cells: calcium carbonate: silicon dioxide are mixed at a ratio of 1:0.1 to 3:0.01, and spray-dried or vacuum-dried to obtain Lactobacillus plantarum FF34 composite bacterial agent powder, wherein the live bacteria concentration of FF34 in the powder is ≥5×10 9 CFU/g, and the live bacteria concentration of Bacillus subtilis is ≥5.0×10 9 CFU/g.

实施例8:植物乳杆菌FF34复合菌剂发酵饲料结果Example 8: Results of fermentation of feed with Lactobacillus plantarum FF34 composite bacterial agent

将实施例7中制备的植物乳杆菌FF34复合菌剂粉剂,按质量百分比0.1~5%的添加量分别与青虾饲料、螃蟹饲料、龙虾饲料及棉粕混合后,加水调节物料含水量25%~40%,分别按1kg/袋分装体积容量为5L的发酵袋(市售),以不添加菌粉发酵的青虾饲料、螃蟹饲料、龙虾饲料及棉粕为对照,每组3个平行,在发酵袋中28~35℃密闭发酵3~5天,发酵结果如下表5,数据用SPSS18.0统计分析,全部结果均用平均值±标准误

Figure BDA0003731190040000161
表示,独立样本T检验比较各组间的差异。The Lactobacillus plantarum FF34 composite bacterial agent powder prepared in Example 7 was mixed with shrimp feed, crab feed, lobster feed and cottonseed meal in an addition amount of 0.1-5% by mass, and water was added to adjust the moisture content of the materials to 25%-40%. The materials were respectively packed into fermentation bags with a volume capacity of 5 L (commercially available) at 1 kg/bag. Shrimp feed, crab feed, lobster feed and cottonseed meal fermented without adding bacterial powder were used as controls. Three parallel groups were set and fermented in a closed fermentation bag at 28-35°C for 3-5 days. The fermentation results are shown in Table 5 below. The data were statistically analyzed using SPSS18.0. All results were expressed as mean ± standard error.
Figure BDA0003731190040000161
Indicates that the independent sample T test was used to compare the differences among the groups.

表5植物乳杆菌FF34复合菌剂虾蟹饲料的结果Table 5 Results of plant lactobacillus FF34 composite bacterial agent shrimp and crab feed

Figure BDA0003731190040000162
Figure BDA0003731190040000162

注:*表示差异显著(p<0.05)。Note: * indicates significant difference (p<0.05).

表5中结果显示,青虾饲料、螃蟹饲料、小龙虾饲料经植物乳杆菌FF34复合菌剂发酵后,发酵饲料富含有益乳酸菌和芽孢杆菌,饲料的营养价值提高显著,其中可溶性蛋白含量分别提高116.31%、92.57%、42.38%,酸溶蛋白含量218.53%、155.21%、118.41%,小肽含量分别提高45.20%、76.13%、168.41%。The results in Table 5 show that after the shrimp feed, crab feed and crayfish feed were fermented with the plantarum Lactobacillus FF34 composite bacterial agent, the fermented feed was rich in beneficial lactic acid bacteria and Bacillus, and the nutritional value of the feed was significantly improved. The soluble protein content increased by 116.31%, 92.57% and 42.38% respectively, the acid-soluble protein content increased by 218.53%, 155.21% and 118.41% respectively, and the small peptide content increased by 45.20%, 76.13% and 168.41% respectively.

表6植物乳杆菌FF34复合菌剂发酵棉粕的结果Table 6 Results of fermentation of cottonseed meal by Lactobacillus plantarum FF34 composite bacterial agent

Figure BDA0003731190040000163
Figure BDA0003731190040000163

Figure BDA0003731190040000171
Figure BDA0003731190040000171

注:*表示差异显著(p<0.05)。Note: * indicates significant difference (p<0.05).

表6结果显示:植物乳杆菌FF34复合菌剂发酵棉粕后,富含乳酸菌等益生菌,其中乳酸菌大量增殖,浓度高达109CFU/g,FF34复合菌剂发酵棉粕中芽孢杆菌可达107CFU/g。在发酵棉粕过程中,植物乳杆菌FF34复合菌剂抑制了霉菌的生长,杜绝了棉粕加水后产生的霉变。植物乳杆菌FF34复合菌发酵棉粕后,改善了棉粕的营养成分,显著提高了棉粕的粗蛋白、可溶蛋白、酸溶蛋白和活性小肽含量(p<0.05),可溶蛋白提高85.13%,酸溶蛋白提高155.42%,活性小肽提高42.14%,发酵棉粕虽然粗蛋白提高9.02%,但差异不显著(p>0.05)。此外,植物乳杆菌FF34复合菌剂发酵棉粕后,能显著降低棉粕中植酸、单宁及游离棉酚等抗营养因子的含量(p<0.05),植酸下降63.74%,单宁下降50.61%,游离棉酚下降54.17%。The results in Table 6 show that after the cottonseed meal was fermented by the plant lactobacillus FF34 compound bacteria agent, it was rich in probiotics such as lactic acid bacteria, among which lactic acid bacteria proliferated in large quantities, with a concentration as high as 10 9 CFU/g, and the bacillus in the cottonseed meal fermented by the FF34 compound bacteria agent could reach 10 7 CFU/g. In the process of fermenting cottonseed meal, the plant lactobacillus FF34 compound bacteria agent inhibited the growth of mold and prevented the mildew caused by the addition of water to the cottonseed meal. After the plant lactobacillus FF34 compound bacteria fermented cottonseed meal, the nutritional components of cottonseed meal were improved, and the crude protein, soluble protein, acid-soluble protein and active small peptide contents of cottonseed meal were significantly increased (p < 0.05), the soluble protein increased by 85.13%, the acid-soluble protein increased by 155.42%, and the active small peptide increased by 42.14%. Although the crude protein of the fermented cottonseed meal increased by 9.02%, the difference was not significant (p > 0.05). In addition, after cottonseed meal was fermented with the composite bacterial agent Lactobacillus plantarum FF34, the content of anti-nutritional factors such as phytic acid, tannins and free gossypol in cottonseed meal was significantly reduced (p < 0.05), with phytic acid decreasing by 63.74%, tannins decreasing by 50.61% and free gossypol decreasing by 54.17%.

实施例9:饲料中添加植物乳杆菌FF34对团头鲂幼鱼生长和饲料利用的作用Example 9: Effects of adding Lactobacillus plantarum FF34 to feed on growth and feed utilization of young Megalobrama amblycephala

在本实施例中,以鱼粉、豆粕、菜粕和棉粕为蛋白源,豆油为脂肪源,配制2组试验饲料,即常规对照饲料组(CN)和植物乳杆菌FF34添加组LAB,LAB组是在常规对照饲料(CN)中按质量百分比0.01%~1%添加实施例5制备的植物乳杆菌FF34单菌制剂,使得其中FF34菌浓为106cfu/g饲料。各组饲料中粗蛋白含量为32.5%、粗脂肪为6.2%、总能为17.4kJ/Kg。常规对照饲料组成为(质量百分比):鱼粉6.4%、豆粕25.4%、菜粕16.2%、棉粕15.3%、小麦淀粉16.6%、米糠5.7%、麸皮5.8%、豆油3.1%、磷酸二氢钙1.0%、预混料1.0%、维生素C0.5%、氯化胆碱0.4%、微晶纤维素2.1%、膨润土0.5%。In this example, fish meal, soybean meal, rapeseed meal and cottonseed meal were used as protein sources, and soybean oil was used as fat source to prepare two groups of test feeds, namely the conventional control feed group (CN) and the Lactobacillus plantarum FF34 added group LAB. The LAB group was prepared by adding the Lactobacillus plantarum FF34 single bacterial preparation prepared in Example 5 to the conventional control feed (CN) at a mass percentage of 0.01% to 1%, so that the FF34 bacterial concentration was 10 6 cfu/g feed. The crude protein content of each group of feed was 32.5%, the crude fat was 6.2%, and the gross energy was 17.4 kJ/Kg. The composition of the conventional control feed was (mass percentage): 6.4% fish meal, 25.4% soybean meal, 16.2% rapeseed meal, 15.3% cottonseed meal, 16.6% wheat starch, 5.7% rice bran, 5.8% bran, 3.1% soybean oil, 1.0% monocalcium phosphate, 1.0% premix, 0.5% vitamin C, 0.4% choline chloride, 2.1% microcrystalline cellulose, and 0.5% bentonite.

挑选120尾体质健康、规格均一的团头鲂幼鱼(初重13.5±0.5g)暂养一周后,随机分入6个室外网箱(1m×1m×1m),每个网箱20尾,分为2组,每组3个重复,每天表观饱食投喂试验饲料3次(8:00,12:00和17:00),为期8周。养殖期间水温保持28~31℃,溶氧≥7mg/L,氨氮≤0.1mg/L,pH7.3~7.8,采用自然光照周期(12L:12D),每周测定养殖水质情况。8周养殖试验结束后,禁食24h,测定生长指标、脏器指数和血液指标。120 healthy and uniform juveniles of Megalobrama amblycephala (initial weight 13.5±0.5g) were selected and kept for one week, and then randomly divided into 6 outdoor cages (1m×1m×1m), 20 in each cage, divided into 2 groups, 3 replicates in each group, and fed the experimental feed 3 times a day (8:00, 12:00 and 17:00) for 8 weeks. During the culture period, the water temperature was maintained at 28-31℃, dissolved oxygen ≥7mg/L, ammonia nitrogen ≤0.1mg/L, pH 7.3-7.8, and a natural light cycle (12L:12D) was used. The water quality of the culture was measured every week. After the 8-week culture experiment, the fish were fasted for 24h, and the growth index, organ index and blood index were measured.

生长性能指标计算如下:The growth performance index is calculated as follows:

增重率(WGR)(%)=100×(Wt-W0)/W0;Weight gain rate (WGR) (%) = 100 × (Wt-W0)/W0;

特定生长率(SGR)(%/d)=100×(ln Wt-ln W0)/t;Specific growth rate (SGR) (%/d) = 100 × (ln Wt-ln W0)/t;

饵料系数(FCR)=F/(Wt-W0);Feed coefficient (FCR) = F/(Wt-W0);

式中:W0为初始重(g);Wt为终末重(g);t为饲喂天数(d);F为摄食饲料总量(风干基础)(g)。Where: W0 is the initial weight (g); Wt is the final weight (g); t is the number of feeding days (d); F is the total amount of feed consumed (air-dried basis) (g).

血清血糖(GLU)、胆固醇(TC)、甘油三酯(TG)、天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、高密度脂蛋白(HDL)和低密度脂蛋白(LDL)含量使用深圳迈瑞全自动生化分析仪(BS-400Q2080,中国深圳)测定,所用的试剂盒均购自深圳迈瑞有限公司。CN对照组和LAB(植物乳杆菌试验组)数据采用SPSS18.0统计分析,全部结果均用平均值±标准误

Figure BDA0003731190040000181
表示,独立样本T检验比较各组间的差异,*表示两组差异显著,检验水平为P<0.05。结果如表7所示。Serum blood glucose (GLU), cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels were measured using Shenzhen Mindray automatic biochemical analyzer (BS-400Q2080, Shenzhen, China), and the kits used were purchased from Shenzhen Mindray Co., Ltd. The data of the CN control group and LAB (Lactobacillus plantarum test group) were statistically analyzed using SPSS18.0, and all results are expressed as mean ± standard error.
Figure BDA0003731190040000181
Indicates that the independent sample T test was used to compare the differences between the groups, and * indicates that the difference between the two groups was significant, with the test level being P < 0.05. The results are shown in Table 7.

表7饲料中添加植物乳杆菌FF34对团头鲂幼鱼生长和血液生理的影响Table 7 Effects of adding Lactobacillus plantarum FF34 to feed on growth and blood physiology of young bream

指标index CN(对照组)CN (control group) LAB(植物乳杆菌试验组)LAB (Lactobacillus plantarum test group) 生长性能指标Growth performance indicators WGR,%WGR, % 126.87±3.85126.87±3.85 155.48±2.69*155.48±2.69* SGR,%/dSGR, %/d 1.64±0.041.64±0.04 11.88±0.02*11.88±0.02* FCRFCR 2.18±0.062.18±0.06 11.89±0.05*11.89±0.05* 血清生化指标Serum biochemical indexes GLU,mmol/LGLU, mmol/L 9.93±0.569.93±0.56 7.14±0.70*7.14±0.70* LDL,mmol/LLDL, mmol/L 0.61±0.060.61±0.06 0.66±0.030.66±0.03 HDL,mmol/LHDL, mmol/L 1.04±0.091.04±0.09 0.64±0.02*0.64±0.02* TG,mmol/LTG, mmol/L 1.43±0.111.43±0.11 11.87±0.07*11.87±0.07* TC,mmol/LTC, mmol/L 5.83±0.205.83±0.20 7.03±0.17*7.03±0.17* ALT,mmol/LALT, mmol/L 4.74±0.374.74±0.37 2.78±0.27*2.78±0.27* AST,mmol/LAST, mmol/L 185.01±13.34185.01±13.34 142.32±6.81*142.32±6.81*

注:*表示差异显著(p<0.05)。Note: * indicates significant difference (p<0.05).

表7结果显示:添加106cfu/g的植物乳杆菌FF34显著提高了增重率(P<0.05)和特定生长率(P<0.05),显著降低了饲料系数(P<0.05),改善了团头鲂幼鱼对饲料的利用和生长性能。丙氨酸氨基转移酶和天冬氨酸氨基转移酶是反应肝脏代谢功能的指标,高密度脂蛋白能够输出胆固醇,促进胆固醇代谢。总胆固醇是合成肾上腺素、胆汁酸及维生素D等生理活性物质的重要原料。本应用实例结果说明,添加植物乳杆菌能够显著降低血液中血糖、丙氨酸转移酶、天冬氨酸氨基转移酶的含量(P<0.05),提示其具有改善肝脏代谢和解读功能,提高团头鲂血液生理健康。同时,总胆固醇等脂质物质的增加,也提示添加植物乳杆菌FF34能够促进鱼体脂质物质的代谢,为肝脏和机体的脂代谢提供前体物质。The results in Table 7 show that the addition of 10 6 cfu/g of Lactobacillus plantarum FF34 significantly increased the weight gain rate (P<0.05) and specific growth rate (P<0.05), significantly reduced the feed coefficient (P<0.05), and improved the utilization and growth performance of feed by young bream. Alanine aminotransferase and aspartate aminotransferase are indicators of liver metabolic function. High-density lipoprotein can export cholesterol and promote cholesterol metabolism. Total cholesterol is an important raw material for synthesizing physiologically active substances such as adrenaline, bile acid and vitamin D. The results of this application example show that the addition of Lactobacillus plantarum can significantly reduce the content of blood glucose, alanine transferase and aspartate aminotransferase in the blood (P<0.05), indicating that it has the function of improving liver metabolism and interpretation, and improving the blood physiological health of bream. At the same time, the increase in lipid substances such as total cholesterol also indicates that the addition of Lactobacillus plantarum FF34 can promote the metabolism of lipid substances in fish and provide precursor substances for lipid metabolism in the liver and the body.

实施例10:高淀粉饲料中添加植物乳杆菌FF34对团头鲂幼鱼高淀粉饲料利用和生长性能的影响Example 10: Effects of adding Lactobacillus plantarum FF34 to high-starch feed on high-starch feed utilization and growth performance of young bream

在本实施例中,以鱼粉、豆粕、菜粕和棉粕为蛋白源,豆油为脂肪源,配制2组试验饲料,即高淀粉对照饲料(HWM)组和高淀粉+植物乳杆菌FF34(HWM+LAB)组,在高淀粉对照饲料(HWM)中按质量百分比0.01%~1%添加实施例5制备的植物乳杆菌FF34单菌制剂即为(HWM+LAB)组,其中FF34菌浓为106cfu/g饲料。各组饲料中粗蛋白含量为32.5%、粗脂肪为6.2%、碳水化合物含量为43%、总能为17.4kJ/Kg。常规对照饲料组成为(质量百分比):鱼粉6.4%、豆粕21.6%、菜粕16.2%、棉粕15.3%、小麦淀粉33.2%、豆油3.9%、磷酸二氢钙1.0%、预混料1.0%、维生素C 0.5%、氯化胆碱0.4%、膨润土0.5%。In this embodiment, fish meal, soybean meal, rapeseed meal and cottonseed meal were used as protein sources, and soybean oil was used as fat source to prepare two groups of test feeds, namely, a high starch control feed (HWM) group and a high starch + Lactobacillus plantarum FF34 (HWM + LAB) group. The Lactobacillus plantarum FF34 single bacterial preparation prepared in Example 5 was added to the high starch control feed (HWM) at a mass percentage of 0.01% to 1%, namely the (HWM + LAB) group, wherein the FF34 bacterial concentration was 10 6 cfu/g feed. The crude protein content of each group of feed was 32.5%, the crude fat content was 6.2%, the carbohydrate content was 43%, and the gross energy was 17.4 kJ/Kg. The composition of the conventional control feed is (mass percentage): fish meal 6.4%, soybean meal 21.6%, rapeseed meal 16.2%, cottonseed meal 15.3%, wheat starch 33.2%, soybean oil 3.9%, monocalcium phosphate 1.0%, premix 1.0%, vitamin C 0.5%, choline chloride 0.4%, and bentonite 0.5%.

挑选120尾体质健康、规格均一的团头鲂幼鱼(初重13.5±0.5g)暂养一周后,随机分入6个室外网箱(1m×1m×1m),每个网箱20尾,分为2组,每组3个重复,每天表观饱食投喂试验饲料3次(8:00,12:00和17:00),,为期8周。养殖期间水温保持28~31℃,溶氧≥7mg/L,氨氮≤0.1mg/L,pH7..3~7.8,采用自然光照周期(12L:12D),每周测定养殖水质情况。8周养殖试验结束后,禁食24h,测定生长指标和脏器指数。120 healthy and uniform juveniles of Megalobrama amblycephala (initial weight 13.5±0.5g) were selected and kept for one week, and then randomly divided into 6 outdoor cages (1m×1m×1m), 20 in each cage, divided into 2 groups, 3 replicates in each group, and fed the experimental feed 3 times a day (8:00, 12:00 and 17:00) for 8 weeks. During the culture period, the water temperature was maintained at 28-31℃, dissolved oxygen ≥7mg/L, ammonia nitrogen ≤0.1mg/L, pH 7.3-7.8, and a natural light cycle (12L:12D) was used. The water quality of the culture was measured every week. After the 8-week culture experiment, the fish were fasted for 24h, and the growth index and organ index were measured.

生长性能指标计算如下:The growth performance index is calculated as follows:

增重率(WGR)(%)=100×(Wt-W0)/W0;Weight gain rate (WGR) (%) = 100 × (Wt-W0)/W0;

特定生长率(SGR)(%/d)=100×(ln Wt-ln W0)/t;Specific growth rate (SGR) (%/d) = 100 × (ln Wt-ln W0)/t;

饵料系数(FCR)=F/(Wt-W0);Feed coefficient (FCR) = F/(Wt-W0);

肠体比(VI)=100×Wi/Wb Intestine to body ratio (VI) = 100 × Wi / Wb

式中:W0为初始重(g);Wt为终末重(g);t为饲喂天数(d);F为摄食饲料总量(风干基础)(g);Wb为每尾鱼终末个体重(g);Wi为每尾鱼肠道重(g)。Where: W0 is the initial weight (g); Wt is the final weight (g); t is the number of feeding days (d); F is the total amount of feed ingested (air-dried basis) (g); Wb is the final individual weight of each fish (g); Wi is the intestinal weight of each fish (g).

在本实施例中,HWM(高淀粉对照饲料组)和HWM+LAB(高淀粉饲料+FF34试验组)数据采用SPSS18.0统计分析,全部结果均用平均值±标准误

Figure BDA0003731190040000191
Figure BDA0003731190040000192
表示,独立样本T检验比较各组间的差异,*表示两组差异显著,检验水平为P<0.05,结果如表8所示。In this example, the data of HWM (high starch control feed group) and HWM+LAB (high starch feed+FF34 test group) were statistically analyzed using SPSS18.0, and all results were expressed as mean ± standard error.
Figure BDA0003731190040000191
Figure BDA0003731190040000192
Indicates that the independent sample T test was used to compare the differences between the groups. * indicates that the difference between the two groups was significant, and the test level was P < 0.05. The results are shown in Table 8.

表8高淀粉饲料中添加植物乳杆菌FF34对团头鲂幼鱼生长和高淀粉饲料利用的影响Table 8 Effects of adding Lactobacillus plantarum FF34 to high-starch feed on growth and high-starch feed utilization of young bream

指标index HWM(高淀粉对照饲料组)HWM (high starch control diet group) HWM+LAB(高淀粉饲料+FF34试验组)HWM+LAB (high starch feed+FF34 test group) 末重,gFinal weight, g 38.26±0.6138.26±0.61 42.60±1.56*42.60±1.56* WGR,%WGR, % 139.59±7.50139.59±7.50 189.48±5.03*189.48±5.03* SGR,%/dSGR, %/d 1.74±0.061.74±0.06 2.12±0.03*2.12±0.03* FCRFCR 2.13±0.122.13±0.12 1.45±0.03*1.45±0.03* 肠体比Intestine to body ratio 3.02±0.223.02±0.22 3.75±0.18*3.75±0.18*

注:*表示差异显著(p<0.05)Note: * indicates significant difference (p<0.05)

团头鲂是我国大宗淡水鱼之一,对碳水化合物的耐受能力和利用能力相对较弱,饲料中过量碳水化合会引起鱼类的糖不耐受现象,导致生长受阻。表8结果显示,高淀粉饲料中添加植物乳杆菌FF34显著改善了团头鲂幼鱼的生长性能和饲料利用效率,其平均个体末重显著提高了11.3%(P<0.05),增重率显著提高了35.7%(P<0.05),特定生长率显著提高了21.8%(P<0.05),且饲料系数显著降低31.9%(P<0.05)。而且高淀粉饲料中添加植物乳杆菌FF34可提高肠体比指数,促进肠道生长发育,肠道和个体的比重显著提高了24.2%(P<0.05)。The bighead carp is one of the major freshwater fish in my country. Its tolerance and utilization of carbohydrates are relatively weak. Excessive carbohydrates in feed can cause sugar intolerance in fish, leading to growth retardation. The results in Table 8 show that the addition of Lactobacillus plantarum FF34 to high-starch feed significantly improved the growth performance and feed utilization efficiency of young bighead carp. Its average individual final weight increased significantly by 11.3% (P < 0.05), the weight gain rate increased significantly by 35.7% (P < 0.05), the specific growth rate increased significantly by 21.8% (P < 0.05), and the feed coefficient decreased significantly by 31.9% (P < 0.05). Moreover, the addition of Lactobacillus plantarum FF34 to high-starch feed can increase the intestinal body ratio index and promote intestinal growth and development. The ratio of the intestinal tract to the individual increased significantly by 24.2% (P < 0.05).

实施例11:高淀粉饲料中添加植物乳杆菌FF34对团头鲂幼鱼肝脏抗氧化的影响Example 11: Effect of adding Lactobacillus plantarum FF34 to high-starch feed on the antioxidant effect of the liver of young bream

在本实施例中,以鱼粉、豆粕、菜粕和棉粕为蛋白源,豆油为脂肪源,配制2组试验饲料,即高淀粉饲料(HCH)和高淀粉饲料+植物乳杆菌FF34(HCH+LAB),以高淀粉饲料(HCH)为对照组,在高淀粉饲料(HCH)中按质量百分比0.01%~1%添加实施例5制备的植物乳杆菌FF34单菌制剂即为(HCH+LAB)试验组,其中FF34菌浓为106cfu/g饲料。各试验组粗蛋白含量为33.4%、粗脂肪为6.5%、碳水化合物含量为47%。常规对照饲料组成为:鱼粉7%、豆粕23%、菜粕15%、棉粕12%、小麦淀粉36%、豆油4%、磷酸二氢钙1.0%、预混料1.0%、氯化胆碱0.5%、膨润土0.5%。In this embodiment, fish meal, soybean meal, rapeseed meal and cottonseed meal are used as protein sources, and soybean oil is used as fat source. Two groups of test feeds are prepared, namely high starch feed (HCH) and high starch feed + Lactobacillus plantarum FF34 (HCH + LAB). High starch feed (HCH) is used as the control group. The plant lactobacillus FF34 single bacterial preparation prepared in Example 5 is added to the high starch feed (HCH) at a mass percentage of 0.01% to 1%, which is the (HCH + LAB) test group, wherein the FF34 bacterial concentration is 10 6 cfu/g feed. The crude protein content of each test group is 33.4%, the crude fat content is 6.5%, and the carbohydrate content is 47%. The conventional control feed composition is: fish meal 7%, soybean meal 23%, rapeseed meal 15%, cottonseed meal 12%, wheat starch 36%, soybean oil 4%, calcium dihydrogen phosphate 1.0%, premix 1.0%, choline chloride 0.5%, bentonite 0.5%.

挑选120尾体质健康、规格均一的团头鲂幼鱼(初重13.1±0.4g)暂养一周后,随机分入6个室外网箱(1m×1m×1m),每个网箱20尾,分为2组,每组3个重复,每天表观饱食投喂试验饲料3次(8:00,12:00和17:00),,为期8周。养殖期间水温保持26~30℃,溶氧≥7mg/L,氨氮≤0.1mg/L,pH6..9~7.5,采用自然光照周期(12L:12D),每周测定养殖水质情况。8周养殖试验结束后,禁食24h,测定鱼体肝脏抗氧化指标。肝脏总抗氧化能力(TAOC)、过氧化氢酶(CAT)、总超氧化物歧化酶(SOD)、还原性谷胱甘肽(GSH)和丙二醛(MDA)含量测定采用南京建成生物工程研究所的试剂盒进行测定。120 healthy and uniform young bream (initial weight 13.1±0.4g) were selected and kept for one week, and then randomly divided into 6 outdoor cages (1m×1m×1m), 20 in each cage, divided into 2 groups, 3 replicates in each group, and fed with the test feed 3 times a day (8:00, 12:00 and 17:00) for 8 weeks. During the culture period, the water temperature was maintained at 26-30℃, dissolved oxygen ≥7mg/L, ammonia nitrogen ≤0.1mg/L, pH 6.9-7.5, and a natural light cycle (12L:12D) was used. The water quality of the culture was measured every week. After the 8-week culture experiment, the fish were fasted for 24h and the antioxidant index of the fish liver was measured. The total antioxidant capacity (TAOC), catalase (CAT), total superoxide dismutase (SOD), reduced glutathione (GSH) and malondialdehyde (MDA) contents of the liver were determined using the kits of Nanjing Jiancheng Bioengineering Institute.

在本实施例中,HCH(高淀粉饲料对照饲料组)和HCH+LAB(高淀粉饲料+植物乳杆菌FF34试验组)数据采用SPSS18.0统计分析,,全部结果均用平均值±标准误

Figure BDA0003731190040000201
表示,独立样本T检验比较各组间的差异,*表示两组差异显著,检验水平为P<0.05,结果如表9所示。In this example, the data of HCH (high starch feed control feed group) and HCH+LAB (high starch feed+Lactobacillus plantarum FF34 test group) were statistically analyzed using SPSS18.0, and all results were expressed as mean ± standard error.
Figure BDA0003731190040000201
Indicates that the independent sample T test was used to compare the differences between the groups. * indicates that the difference between the two groups was significant, and the test level was P < 0.05. The results are shown in Table 9.

表9高淀粉饲料中添加植物乳杆菌FF34对团头鲂幼鱼肝脏抗氧化的影响Table 9 Effects of adding Lactobacillus plantarum FF34 to high starch feed on liver antioxidant capacity of young bream

Figure BDA0003731190040000202
Figure BDA0003731190040000202

鱼类天生不耐受饲料中的高水平糖,饲料中过量的碳水化合物会降低鱼类生长性能和免疫力,出现糖代谢紊乱,表现出鱼类糖不耐受现象,并引起肝脏抗氧化能力减弱。导致肝脏抗氧化功能受损。表9结果显示:在高淀粉饲料中添加植物乳杆菌FF34显著提高了肝脏GSH、CAT和SOD水平(P<0.05),因此推测,植物乳杆菌FF34可以利用饲料中丰富的淀粉提高胆固醇水平来促进胆汁酸循环代谢,激活肝脏抗氧化能力,维持生长和健康。Fish are naturally intolerant to high levels of sugar in feed. Excessive carbohydrates in feed will reduce fish growth performance and immunity, cause sugar metabolism disorders, show fish sugar intolerance, and cause the liver's antioxidant capacity to weaken. This leads to impaired liver antioxidant function. The results in Table 9 show that adding Lactobacillus plantarum FF34 to high-starch feed significantly increased the levels of GSH, CAT and SOD in the liver (P < 0.05). Therefore, it is speculated that Lactobacillus plantarum FF34 can use the rich starch in the feed to increase cholesterol levels to promote bile acid circulation metabolism, activate liver antioxidant capacity, and maintain growth and health.

实施例12:高淀粉饲料中添加植物乳杆菌FF34对团头鲂幼鱼肌肉和肝脏脂肪沉积的影响Example 12: Effect of adding Lactobacillus plantarum FF34 to high-starch feed on fat deposition in muscle and liver of young bream

在本实施例中,以鱼粉、豆粕、菜粕和棉粕为蛋白源,豆油为脂肪源,配制3组试验饲料,即对照饲料(15WM)、高淀粉饲料(35WM)和高淀粉饲料+植物乳杆菌FF34(35WM+LAB),在高淀粉饲料(35WM)中按质量百分比0.01%~1%添加实施例5制备的植物乳杆菌FF34单菌制剂即为35WM+LAB试验组,其中FF34菌浓为106cfu/g饲料,饲料配方如表10。In this embodiment, fish meal, soybean meal, rapeseed meal and cottonseed meal were used as protein sources, and soybean oil was used as a fat source to prepare three groups of test feeds, namely, a control feed (15WM), a high starch feed (35WM) and a high starch feed + Lactobacillus plantarum FF34 (35WM+LAB). The Lactobacillus plantarum FF34 single bacterial preparation prepared in Example 5 was added to the high starch feed (35WM) at a mass percentage of 0.01% to 1%, namely the 35WM+LAB test group, wherein the FF34 bacterial concentration was 10 6 cfu/g feed, and the feed formula was as shown in Table 10.

表10本实施例饲料配方组成Table 10 Feed formula composition of this embodiment

Figure BDA0003731190040000211
Figure BDA0003731190040000211

挑选180尾体质健康、规格均一的团头鲂幼鱼(初重13.6±0.7g)暂养一周后,随机分入9个室外网箱(1m×1m×1m),每个网箱20尾,分为3组,每组3个重复,每天表观饱食投喂试验饲料3次(8:00,12:00和17:00),为期8周。养殖期间水温保持25~30℃,溶氧≥7mg/L,氨氮≤0.1mg/L,pH6.9~7.5,采用自然光照周期(12L:12D),每周测定养殖水质情况。8周养殖试验结束后,禁食24h,测定鱼体肌肉脂肪含量,分析肝脏中脂滴沉积。肌肉营养成分分析参照国标规定方法进行测定。采用常压干燥法105℃的烘箱中烘至恒重来计算干物质含量;采用凯氏定氮法(GB/T6432-1994)测定粗蛋白质含量;采用索氏抽提法(GB/T6433-1994)测定粗脂肪含量;采用560℃灼烧法(GB/T6438-1992)测定粗灰分含量。将固定于4%多聚甲醛中的肝脏组织块取出,制作肝脏冰冻切片,并采用油红O染色,用Image J软件分析油红O脂滴面积。本实施例中,15WM(对照饲料)、35WM(高淀粉饲料)和35WM+LAB(高淀粉饲料+植物乳杆菌FF34)之间数据比较采用单因素方差分析turkey’s检验,检验水平为P<0.05,结果如表11所示。180 healthy and uniform juveniles of Megalobrama amblycephala (initial weight 13.6±0.7g) were selected and kept for one week, and then randomly divided into 9 outdoor cages (1m×1m×1m), 20 in each cage, and divided into 3 groups, with 3 replicates in each group. The experimental feed was fed 3 times a day (8:00, 12:00 and 17:00) for 8 weeks. During the culture period, the water temperature was maintained at 25-30℃, dissolved oxygen ≥7mg/L, ammonia nitrogen ≤0.1mg/L, pH 6.9-7.5, and a natural light cycle (12L:12D) was used. The water quality of the culture was measured every week. After the 8-week culture experiment, the fish were fasted for 24h, the muscle fat content of the fish was measured, and the lipid droplet deposition in the liver was analyzed. The analysis of muscle nutrient components was determined according to the national standard method. The dry matter content was calculated by drying in an oven at 105°C under normal pressure until constant weight; the crude protein content was determined by the Kjeldahl method (GB/T6432-1994); the crude fat content was determined by the Soxhlet extraction method (GB/T6433-1994); and the crude ash content was determined by the 560°C burning method (GB/T6438-1992). The liver tissue blocks fixed in 4% paraformaldehyde were taken out, frozen liver sections were made, and stained with Oil Red O, and the Oil Red O lipid droplet area was analyzed using Image J software. In this embodiment, the data comparison between 15WM (control feed), 35WM (high starch feed) and 35WM+LAB (high starch feed+Lactobacillus plantarum FF34) was performed using a one-way ANOVA turkey's test, with a test level of P < 0.05, and the results are shown in Table 11.

鱼类由于天生不耐受饲料中的高水平糖,饲料中过量的碳水化合物会引起糖脂代谢紊乱,脂质在鱼体内过量沉积,导致氧化应激损伤和抗病力下降。图1中,肝组织切片中脂滴的油红O染色图片显示,摄食35WM组饲料的团头鲂肝组织脂滴沉积显著增加,而摄食的高淀粉饲料中添加植物乳杆菌(35WM+LAB)减少肝脏中脂滴面积。进一步分析团头鲂肌肉脂肪和肝脏脂滴面积(表11),发现与基础对照组相比(15WM),高淀粉组(35WM)显著增加了肌肉中粗脂肪量和肝脏脂滴沉积(P<0.05)。在高淀粉饲料中添加植物乳杆菌FF34(35WM+LAB)能够显著降低团头鲂鱼体肌肉粗脂肪含量(P<0.05),与对照组无显著差异;而35WM+LAB组肝脏脂滴沉积虽然高于对照组,但是显著低于高淀粉组(P<0.05)。Fish are naturally intolerant to high levels of sugar in feed. Excessive carbohydrates in feed can cause disorders of sugar and lipid metabolism, and excessive lipid deposition in the fish body, leading to oxidative stress damage and decreased disease resistance. In Figure 1, the Oil Red O staining of lipid droplets in liver tissue sections showed that the lipid droplet deposition in the liver tissue of the group of bream fed the 35WM group feed increased significantly, while the addition of Lactobacillus plantarum (35WM+LAB) to the high-starch feed reduced the area of lipid droplets in the liver. Further analysis of muscle fat and liver lipid droplet area of group of bream (Table 11) showed that compared with the basic control group (15WM), the high starch group (35WM) significantly increased the amount of crude fat in the muscle and the deposition of lipid droplets in the liver (P < 0.05). Adding Lactobacillus plantarum FF34 (35WM+LAB) to high-starch feed can significantly reduce the crude fat content in the body muscle of Megalobrama amblycephala (P<0.05), with no significant difference compared with the control group; although the liver lipid droplet deposition in the 35WM+LAB group was higher than that in the control group, it was significantly lower than that in the high-starch group (P<0.05).

图1植物乳杆菌及高淀粉添加对团头鲂幼鱼肝脏油红0染色切片。A图为15WM组(×200倍)的油红O染色切片,B图为35WM+LAB组(×200倍)的油红O染色切片,C图为35WM组(×200倍)的油红O染色切片。图中红色表示脂滴,蓝色表示细胞核。Figure 1 Effects of Lactobacillus plantarum and high starch supplementation on Oil Red O-stained sections of liver of young Megalobrama amblycephala. Figure A shows Oil Red O-stained sections of the 15WM group (×200 times), Figure B shows Oil Red O-stained sections of the 35WM+LAB group (×200 times), and Figure C shows Oil Red O-stained sections of the 35WM group (×200 times). Red in the figure indicates lipid droplets, and blue indicates cell nuclei.

表11高淀粉添加植物乳杆菌FF34对团头鲂幼鱼肌肉脂肪含量和肝脏脂滴沉积的影响Table 11 Effects of high starch supplementation with Lactobacillus plantarum FF34 on muscle fat content and liver lipid droplet deposition in juvenile Megalobrama amblycephala

分组Grouping 15WM15WM 35WM35WM 35WM+LAB35WM+LAB 肌肉脂肪含量,%Muscle fat content, % 4.88±0.61a 4.88±0.61 a 6.59±0.35b 6.59±0.35 b 4.75±0.48a 4.75±0.48 a 肝脏脂滴沉积,%Hepatic lipid droplet deposition, % 2.50±0.64a 2.50± 0.64a 28.48±3.01c 28.48±3.01 c 20.48±0.92b 20.48±0.92 b

注:同行小写字母相同表示差异不显著(p>0.05),同行不同小写字母表示差异显著(p<0.05)。Note: The same lowercase letters in the same row indicate no significant difference (p>0.05), and different lowercase letters in the same row indicate significant difference (p<0.05).

Claims (4)

1.一种植物乳杆菌FF34,分类命名为植物乳杆菌(lactobacillus plantarum),保藏于中国典型培养物保藏中心CCTCC,地址:中国 武汉 武汉大学,保藏编号CCTCC NO:M20211221,保藏日期为2021年10月11日。1. A Lactobacillus plantarum FF34, classified and named Lactobacillus plantarum , deposited in China Center for Type Culture Collection CCTCC, address: Wuhan University, Wuhan, China, with deposit number CCTCC NO: M20211221, and deposit date of October 11, 2021. 2.权利要求1所述菌株FF34制备具有水产病原菌拮抗特性的FF34单菌菌剂的方法,其特征在于步骤如下:2. A method for preparing a single bacterial agent having antagonistic properties against aquatic pathogens using the strain FF34 of claim 1, characterized in that the steps are as follows: (1)菌种活化:(1) Bacteria activation: 无菌开启植物乳杆菌FF34的冻干保藏菌种,接种于装有MRS肉汤的试管中,于30~35℃静置培养24~48h,然后转接于MRS肉汤三角瓶中,30~35℃培养活化20~48h;反复活化2~3次,镜检,计数,当菌体浓度达109CFU/mL时作为种子液;Aseptically open the freeze-dried preserved strain of Lactobacillus plantarum FF34, inoculate it into a test tube filled with MRS broth, culture it at 30-35°C for 24-48 hours, then transfer it to a MRS broth triangular bottle, culture it at 30-35°C for activation for 20-48 hours; repeatedly activate it 2-3 times, examine it under a microscope, count it, and use it as seed liquid when the bacterial concentration reaches 10 9 CFU/mL; MRS肉汤培养基组成以g/L计:酪蛋白酶消化物10,牛肉膏粉10,酵母膏粉4,柠檬酸三铵2,乙酸钠5,硫酸镁0.2,硫酸锰0.05,磷酸氢二钾2,葡萄糖20,吐温-80 1.08,以蒸馏水定容配制,pH 5.7±0.2;MRS broth medium composition (g/L): casein digest 10, beef extract powder 10, yeast extract powder 4, triammonium citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.05, potassium dihydrogen phosphate 2, glucose 20, Tween-80 1.08, made up to volume with distilled water, pH 5.7±0.2; (2)固定化发酵培养:(2) Immobilized fermentation culture: 三角瓶种子培养液制备:将步骤(1)所得种子液以体积比1%~5%接种量接入装有固定化发酵培养基的3L三角瓶,装液量为体积比90%,于30~35℃静置培养24~40h,当菌体浓度达109CFU/mL时,作为三角瓶种子培养液;Preparation of triangular flask seed culture solution: The seed solution obtained in step (1) is inoculated at a volume ratio of 1% to 5% into a 3L triangular flask filled with immobilized fermentation medium, and the volume is 90% by volume. The solution is cultured at 30 to 35°C for 24 to 40 hours. When the bacterial cell concentration reaches 10 9 CFU/mL, it is used as the triangular flask seed culture solution. 放大扩培:三角瓶种子培养液接入普通好氧发酵罐中进行一级、二级固定化发酵逐级放大扩培,即:3L三角瓶,再500L发酵罐,再10T发酵罐,发酵罐间移种以无菌空气压送方式完成接种;Scale-up and expansion: The seed culture solution in the triangular flask is connected to the common aerobic fermentation tank for primary and secondary fixed fermentation and scaled up step by step, i.e.: 3L triangular flask, then 500L fermentation tank, then 10T fermentation tank, and the inoculation is completed by sterile air pressure delivery between fermentation tanks; 一级固定化培养:将三角瓶种子培养液以体积比1%~5%接种量接入500L发酵罐中,发酵罐装液量为体积比80%~90%,无菌空气保持罐压至0.01~0.05Mpa,间歇搅拌,转速80~100r/min,30~35℃培养20~40h,pH为4.5~5.5时,结束培养,得到一级固定化培养液,其中植物乳杆菌FF34菌体被吸附固定在海藻酸钙+二氧化硅形成的凝胶小颗粒中,活菌体浓度≥5×109CFU/mL;Primary immobilized culture: the triangular flask seed culture solution is inoculated into a 500L fermenter at a volume ratio of 1% to 5%, the volume ratio of the fermenter is 80% to 90%, the tank pressure is kept at 0.01 to 0.05 MPa with sterile air, intermittent stirring, a speed of 80 to 100 r/min, and culture at 30 to 35°C for 20 to 40 hours. When the pH is 4.5 to 5.5, the culture is terminated to obtain a primary immobilized culture solution, in which the Lactobacillus plantarum FF34 bacteria are adsorbed and fixed in the small gel particles formed by calcium alginate + silica, and the concentration of live bacteria is ≥5×10 9 CFU/mL; 二级固定化培养:将一级固定化培养液用无菌空气压送,以体积比1%~5%接种量接入10T发酵罐中,发酵罐装液量为体积比70%~90%,无菌空气保持罐压至0.01~0.05Mpa,间歇搅拌,转速80~100r/min,30~38℃培养20~40h,当发酵液pH降至4.0~5.5,培养结束,得到二级固定化培养液,其中植物乳杆菌FF34菌体被吸附固定在海藻酸钙+二氧化硅形成的凝胶小颗粒中,菌体浓度≥5×109CFU/mL;Secondary immobilized culture: The primary immobilized culture solution is sent by sterile air pressure, and inoculated into a 10T fermenter at a volume ratio of 1% to 5%. The volume of the fermenter is 70% to 90%. The sterile air is used to keep the tank pressure at 0.01 to 0.05 MPa. Stir intermittently, rotate at 80 to 100 r/min, and culture at 30 to 38°C for 20 to 40 hours. When the pH of the fermentation solution drops to 4.0 to 5.5, the culture is terminated to obtain the secondary immobilized culture solution, in which the Lactobacillus plantarum FF34 cells are adsorbed and fixed in the small gel particles formed by calcium alginate + silica, and the cell concentration is ≥5×10 9 CFU/mL. 固定化发酵培养基组成以g/L计:碳源10~40,氮源3~15,磷酸氢二钾0.2~5,硫酸镁0~0.2,硫酸锰0~0.05,碳酸钙5~40,海藻酸钠3~10,二氧化硅1~10,以自来水定容配制,pH 6.0~6.5,121℃灭菌30min;其中碳源包括小麦次粉、玉米淀粉、糊精、糖蜜、葡萄糖、果葡糖浆中的一种或几种组合;氮源包括鱼粉蛋白胨、胰蛋白胨、大豆蛋白胨、大豆分离蛋白、酵母膏、酵母粉、玉米浆、棉籽粕、菜籽粕、豆粕、花生粕及血粉中的一种或几种组合;The composition of the fixed fermentation medium is calculated in g/L: 10-40 g of carbon source, 3-15 g of nitrogen source, 0.2-5 g of dipotassium hydrogen phosphate, 0-0.2 g of magnesium sulfate, 0-0.05 g of manganese sulfate, 5-40 g of calcium carbonate, 3-10 g of sodium alginate, and 1-10 g of silicon dioxide, which is prepared with tap water to a fixed volume, pH 6.0-6.5, and sterilized at 121°C for 30 min; wherein the carbon source comprises one or a combination of wheat flour, corn starch, dextrin, molasses, glucose, and fructose syrup; the nitrogen source comprises one or a combination of fish meal peptone, tryptone, soy peptone, soy protein isolate, yeast extract, yeast powder, corn steep liquor, cottonseed meal, rapeseed meal, soybean meal, peanut meal, and blood meal; (3)具有水产病原菌拮抗特性的植物乳杆菌FF34单菌菌剂的制备:(3) Preparation of Lactobacillus plantarum FF34 single bacterial agent with antagonistic properties against aquatic pathogens: (A)植物乳杆菌FF34单菌水剂的制备:将步骤(2)所得二级固定化培养液进行灌装,获得植物乳杆菌FF34单菌水剂,单菌水剂中FF34活菌浓度≥5.0×109 CFU/mL;(A) Preparation of Lactobacillus plantarum FF34 single-strain aqueous solution: filling the secondary immobilized culture solution obtained in step (2) to obtain a Lactobacillus plantarum FF34 single-strain aqueous solution, wherein the live bacteria concentration of FF34 in the single-strain aqueous solution is ≥5.0×10 9 CFU/mL; (B)植物乳杆菌FF34单菌粉剂的制备:将步骤(2)所得二级固定化培养液经离心收集湿菌体,按质量比为:湿菌体:碳酸钙:糊精:二氧化硅=1:0.1~2:0.5~2:0.01进行混合,经喷雾干燥或真空干燥得到植物乳杆菌FF34菌粉,菌粉中FF34活菌浓度不低于1×1010CFU/g。(B) Preparation of Lactobacillus plantarum FF34 single bacterial powder: The secondary immobilized culture solution obtained in step (2) is centrifuged to collect wet bacterial cells, which are mixed in a mass ratio of wet bacterial cells: calcium carbonate: dextrin: silicon dioxide = 1:0.1 to 2:0.5 to 2:0.01, and spray-dried or vacuum-dried to obtain Lactobacillus plantarum FF34 powder, wherein the concentration of live FF34 bacteria in the powder is not less than 1×10 10 CFU/g. 3.权利要求1所述菌株FF34与芽孢杆菌混合发酵培养及其复合菌剂的制备方法,其特征在于步骤如下:3. The method for preparing the composite bacterial agent by mixed fermentation culture of the strain FF34 and Bacillus as claimed in claim 1, characterized in that the steps are as follows: (1)与菌株FF34混合发酵培养的芽孢杆菌为:具有水产病原菌拮抗特性的枯草芽孢杆菌AB90008-15,或解淀粉芽孢杆菌JSSW-LA,或凝结芽孢杆菌JSSW-LA-07-1,或酪酸梭状芽孢杆菌JSIM-MCB20040312,或漳州芽孢杆菌JSSW-BP44之一;(1) The Bacillus cultured in mixed fermentation with strain FF34 is: Bacillus subtilis AB90008-15, or Bacillus amyloliquefaciens JSSW-LA, or Bacillus coagulans JSSW-LA-07-1, or Clostridium butyricum JSIM-MCB20040312, or Zhangzhou Bacillus JSSW-BP44, which have antagonistic properties against aquatic pathogens; (2)FF34与上述芽孢杆菌混合发酵:分两步进行,第一步发酵是将上述芽孢杆菌经斜面或三角瓶活化培养后,接种于混合发酵培养基中,接种量为体积比1%~10%,培养温度为30~37℃,培养24~48h,镜检菌体中芽孢形成,即第一步发酵结束;第二步发酵是先将第一步发酵所得发酵液用盐酸调整pH至5.0~5.4,然后接入活化后的植物乳杆菌FF34,接种量为体积比1%~10%,培养温度为30~35℃,间歇搅拌,期间流加w/v浓度为20%葡萄糖或30%碳酸钙或氨水,控制发酵液pH 4.5~5.0,培养24h,获得FF34与芽孢杆菌混合发酵液,镜检可见乳杆菌与芽孢共存,混合发酵液中FF34活菌浓度≥1.0×109 CFU/mL,芽孢杆菌活菌浓度≥5.0×108 CFU/mL,芽孢率≥90%;(2) Mixed fermentation of FF34 and the above-mentioned Bacillus: It is carried out in two steps. The first step of fermentation is to activate the above-mentioned Bacillus by slant or triangular flask and then inoculate it into the mixed fermentation medium. The inoculation amount is 1% to 10% by volume. The culture temperature is 30 to 37°C. The culture is carried out for 24 to 48 hours. The formation of spores in the bacteria is observed under a microscope, which means that the first step of fermentation is over. The second step of fermentation is to adjust the pH of the fermentation liquid obtained in the first step to 5.0 to 5.4 with hydrochloric acid, and then inoculate the activated Lactobacillus plantarum FF34. The inoculation amount is 1% to 10% by volume. The culture temperature is 30 to 35°C. Stirring is performed intermittently. During this period, 20% glucose or 30% calcium carbonate or ammonia water is added at a w/v concentration to control the pH of the fermentation liquid to 4.5 to 5.0. The culture is carried out for 24 hours to obtain a mixed fermentation liquid of FF34 and Bacillus. Microscopic examination shows that lactobacillus and spores coexist. The concentration of live FF34 in the mixed fermentation liquid is ≥1.0× 109 CFU/mL, Bacillus viable bacteria concentration ≥5.0×10 8 CFU/mL, spore rate ≥90%; 混合发酵培养基组成以g/L计:小麦次粉0~50,蛋白胨0.1~5,酵母膏0~5,乙酸钠0~3,玉米淀粉0~10,硫酸铵0~2,氯化钠0~5,碳酸钙0~5,磷酸二氢钾0~0.5,硫酸镁0~0.5,硫酸锰0~0.3,海藻酸钠0~5,自来水定容,pH 7.0,121℃灭菌30min;The composition of the mixed fermentation medium is in g/L: 0-50 wheat flour, 0.1-5 peptone, 0-5 yeast extract, 0-3 sodium acetate, 0-10 corn starch, 0-2 ammonium sulfate, 0-5 sodium chloride, 0-5 calcium carbonate, 0-0.5 potassium dihydrogen phosphate, 0-0.5 magnesium sulfate, 0-0.5 manganese sulfate, 0-0.3 sodium alginate, tap water to volume, pH 7.0, sterilized at 121°C for 30 min; 植物乳杆菌FF34复合菌剂的制备:Preparation of Lactobacillus plantarum FF34 composite bacterial agent: (A)将上述步骤(2)获得的植物乳杆菌FF34与芽孢杆菌混合发酵液直接进行灌装,获得植物乳杆菌FF34复合菌水剂,复合菌水剂中FF34活菌浓度≥1.0×109 CFU/mL,芽孢杆菌活菌浓度≥5.0×108 CFU/mL,芽孢率≥90%;(A) directly filling the mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus subtilis obtained in the above step (2) to obtain a Lactobacillus plantarum FF34 composite bacterial solution, wherein the live bacteria concentration of FF34 in the composite bacterial solution is ≥1.0×10 9 CFU/mL, the live bacteria concentration of Bacillus subtilis is ≥5.0×10 8 CFU/mL, and the spore rate is ≥90%; (B)将上述步骤(2)所得的植物乳杆菌FF34与芽孢杆菌混合发酵液经离心收集湿菌体,按质量比为:湿菌体:碳酸钙:二氧化硅=1:0.1~3:0.01进行混合,经喷雾干燥或真空干燥得到植物乳杆菌FF34复合菌粉剂,复合菌粉剂中FF34活菌浓度≥5×109CFU/g,芽孢杆菌活菌浓度≥5.0×109 CFU/g。(B) The mixed fermentation liquid of Lactobacillus plantarum FF34 and Bacillus obtained in the above step (2) is centrifuged to collect wet bacteria, mixed in a mass ratio of wet bacteria: calcium carbonate: silicon dioxide = 1:0.1 to 3:0.01, and spray-dried or vacuum-dried to obtain a Lactobacillus plantarum FF34 composite bacterial powder, wherein the live bacteria concentration of FF34 in the composite bacterial powder is ≥5×10 9 CFU/g, and the live bacteria concentration of Bacillus subtilis is ≥5.0×10 9 CFU/g. 4.权利要求2及3所述方法制备的植物乳杆菌FF34单菌菌剂及复合菌剂的应用,其特征在于:(1)植物乳杆菌FF34单菌菌剂或复合菌剂按质量百分比0.1%~5%的添加量用于发酵鱼虾蟹饲料及棉粕、菜籽粕饲料原料,桑树叶新型树叶饲料原料,能够显著改善鱼虾蟹饲料或饲料原料的营养成分,所得发酵饲料富含益生菌、酸溶蛋白及活性小肽,单宁、游离棉酚、植酸抗营养因子含量降低;(2)在基础日粮或高淀粉饲料中按质量百分比0.05%~1%添加植物乳杆菌FF34单菌菌剂或复合菌剂,能够促进水产养殖动物肠道组织生长,改善肝脏功能并提高抗氧化活性,显著改善水产养殖动物的生长性能,降低饲料系数;(3)植物乳杆菌FF34复合菌剂水剂与质量体积比2%~4%海藻酸钠混匀后,添加轻石、活性碳或陶粒浸润吸附1~4h后取出,再浸泡于2%~3%氯化钙溶液中,4~8℃固化交联1~10h,即制得植物乳杆菌FF34复合菌固定化菌球或块,菌球或块中菌体浓度不低于5×108CFU/g,植物乳杆菌FF34复合菌固定化菌球或块投放在养殖水体中,可降解水体的氨氮、亚硝酸盐、总磷、总氮含量,抑制水产病原菌的生长,持续改善养殖池塘水质。4. The application of the single bacterial agent of Lactobacillus plantarum FF34 and the composite bacterial agent prepared by the method described in claims 2 and 3, characterized in that: (1) the single bacterial agent of Lactobacillus plantarum FF34 or the composite bacterial agent is added in an amount of 0.1% to 5% by mass to ferment fish, shrimp and crab feed and cottonseed meal, rapeseed meal feed raw materials, and mulberry leaves as new leaf feed raw materials, which can significantly improve the nutritional components of fish, shrimp and crab feed or feed raw materials. The obtained fermented feed is rich in probiotics, acid-soluble proteins and active small peptides, and the content of tannins, free cottonpol and phytic acid anti-nutritional factors is reduced; (2) in the basal diet or high-starch feed, the single bacterial agent of Lactobacillus plantarum FF34 or the composite bacterial agent is added in an amount of 0.1% to 5% by mass to ferment fish, shrimp and crab feed and cottonseed meal, rapeseed meal feed raw materials, and mulberry leaves as new leaf feed raw materials. Adding 1% of Lactobacillus plantarum FF34 single bacteria agent or compound bacteria agent can promote the growth of intestinal tissue of aquatic animals, improve liver function and enhance antioxidant activity, significantly improve the growth performance of aquatic animals, and reduce feed coefficient; (3) After mixing Lactobacillus plantarum FF34 compound bacteria aqueous agent with 2% to 4% sodium alginate by mass volume ratio, add pumice, activated carbon or ceramsite to soak and adsorb for 1 to 4 hours, then take out, soak in 2% to 3% calcium chloride solution, and solidify and cross-link at 4 to 8°C for 1 to 10 hours to obtain Lactobacillus plantarum FF34 compound bacteria immobilized bacteria balls or blocks, the bacterial concentration in the bacteria balls or blocks is not less than 5×10 8 CFU/g, and the Lactobacillus plantarum FF34 compound bacteria immobilized bacteria balls or blocks are placed in the aquaculture water body, which can degrade the ammonia nitrogen, nitrite, total phosphorus and total nitrogen content of the water body, inhibit the growth of aquatic pathogens, and continuously improve the water quality of aquaculture ponds.
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