WO2021031360A1 - Complex microbial inoculant for improving adhesion of clostridium butyricum in intestinal tracts of eels - Google Patents

Complex microbial inoculant for improving adhesion of clostridium butyricum in intestinal tracts of eels Download PDF

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WO2021031360A1
WO2021031360A1 PCT/CN2019/115468 CN2019115468W WO2021031360A1 WO 2021031360 A1 WO2021031360 A1 WO 2021031360A1 CN 2019115468 W CN2019115468 W CN 2019115468W WO 2021031360 A1 WO2021031360 A1 WO 2021031360A1
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clostridium butyricum
bacillus
adhesion
intestinal tracts
microbial inoculant
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吴少坤
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集美大学
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    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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  • the invention relates to the field of aquaculture, in particular to a composite microbial agent for improving the adhesion of Clostridium butyricum in the intestines of eels.
  • Clostridium butyricum, or butyricum is often used as a probiotic to prevent and treat diseases such as diarrhea and enteritis in humans and animals caused by various factors.
  • the bacterium mainly exists in the soil, human and animal intestines. It is Gram-positive, anaerobic and produces spores. It is generally regarded as a safe probiotic.
  • the long-term use of antibiotics has caused negative effects such as residues and the production of resistant strains.
  • General antibiotics can kill all microorganisms including beneficial resident bacteria in the intestinal tract, resulting in imbalance of the micro-ecological environment in the intestinal tract, loss of the barrier, and exposure of the intestinal membrane, which can easily cause cell damage and reduce the ability to resist pathogenic bacteria and harmful substances.
  • Adding probiotics is beneficial to restore the intestinal microecological balance, establish a biological barrier, promote the repair of intestinal membranes, enhance immunity, improve the digestive environment, and inhibit the proliferation of harmful bacteria.
  • probiotics can adhere to the intestinal mucosa is considered to be a prerequisite for intestinal colonization. It is also the key to probiotics staying and multiplying in the host's intestines for a long time to play a long-lasting probiotic effect. Therefore, whether they have the ability to adhere is probiotics An important criterion for strain screening. Secondly, the in vitro antibacterial properties of probiotics are also important reasons for their functions. They usually produce antibacterial metabolites, such as organic acids, hydrogen peroxide, etc., the secretion of bacteriocins, and the competition for intestinal adhesion. Immune defense, etc.
  • the technical problem to be solved by the present invention is to provide a composite microbial agent that improves the adhesion of Clostridium butyricum in the intestines of eels, improves the colonization ability of Clostridium butyricum in the intestines, and is beneficial for probiotics in the intestines of the host. Stay in the tract for a long time, multiply, and play a long-lasting probiotic effect.
  • the present invention is realized as follows:
  • a composite bacterial agent for improving the adhesion of Clostridium butyricum in the intestinal tract of an eel comprising Clostridium butyricum and Bacillus gliocladium, and live bacteria of Clostridium butyricum and Bacillus gliocladium
  • the number ratio is 1:1-100.
  • the concentration of the viable bacteria of Clostridium butyricum and Bacillus glaciens is (1-10) ⁇ 108 CFU/mL independently.
  • the invention utilizes Bacillus glacius to secrete a stable mucus-like substance (ie capsule) with a certain thickness outside the cell wall.
  • a large number of capsules are attached to the surface of Clostridium butyricum, which improves the adhesion of Clostridium butyricum to the intestinal tract.
  • the ability of epithelial cells; that is, the ability of clostridium butyricum to colonize in the intestine is improved, which is conducive to probiotics staying and multiplying in the host's intestine for a long time, and exerting a lasting probiotic effect.
  • the present invention relates to a composite bacterial agent for improving the adhesion of Clostridium butyricum in the intestinal tract of an eel.
  • the composite bacterial agent includes Clostridium butyricum and Bacillus glaciens, and the Clostridium butyricum and Bacillus glaci The ratio of viable bacteria number is 1:1-100.
  • the ratio of the number of viable bacteria between Clostridium butyricum and Bacillus glaciens is 1:1.
  • the concentration of the viable bacteria of Clostridium butyricum and Bacillus glaciens is independently (1-10) ⁇ 108 CFU/mL.
  • Clostridium butyricum was isolated from the intestine of eel. Bacillus mucilaginosus was provided by Changke Biotechnology Company.
  • Cultivation Inoculate the cell suspension on a well culture plate, the bottom wall of the culture plate is coated with type I collagen, and counted by trypan blue staining.
  • the inoculation concentration per well is 104.
  • the total volume of high-sugar culture medium is 1 mL, containing 5% FBS. , 0.01mg/L epidermal growth factor (EGF), 200U/L insulin, 100mg/L amoxicillin, 100mg/L streptomycin and 100,000 U/L gentamicin. Incubate at 28°C, 5% CO2 incubator.
  • EGF epidermal growth factor
  • the optimal culture time is 90 minutes, the transfer of the original culture medium is better. At this time, a large amount of intestinal epithelium is not adhered and suspended in the original culture medium.
  • Bacterial adhesion rate (%) (Concentration of bacteria released after Trion treatment/Concentration of bacteria added) ⁇ 100
  • the primary cultured cells were digested with 0.25% trypsin to prepare a cell suspension. Mix 20 ⁇ L of the suspension with an equal amount of 2% trypan blue solution, drop it into a cell counting plate, and count the unstained living cells. Dilute the amount of viable cells to a final concentration of 1x105 cells/mL. Use DMEM as the cell diluent.
  • the cells are seeded in a 24-well culture plate at 28°C and 5% CO2 in an incubator. After the cells are covered with the bottom wall (about 3 -5d), remove the culture solution and add 0.5ml of new culture solution.
  • the first group was added with 500 ⁇ L of Clostridium butyricum (1x108CFU/mL)
  • the second group was added with 500 ⁇ L of Clostridium butyricum (1x108CFU/mL) and 500 ⁇ L of Bacillus glia (1x108CFU/mL)
  • the third group was added with 500 ⁇ L Clostridium butyricum (1x108CFU/mL) and 5ml of Bacillus glaci (1x109CFU/mL), each group repeated 12 holes, and continued to culture for 90min. Rinse gently with Hank's solution 3 times, and remove the rinse solution to remove unadhered bacteria.
  • the average adhesion rate of the first group was 8.1%
  • the average adhesion rate of the second group was 13.1%
  • the average adhesion rate of the third group was 12.3%.
  • the invention utilizes Bacillus glacius to secrete a stable mucus-like substance (ie capsule) with a certain thickness outside the cell wall.
  • a large number of capsules are attached to the surface of Clostridium butyricum, which improves the adhesion of Clostridium butyricum to the intestinal tract.
  • the ability of epithelial cells; that is, the ability of clostridium butyricum to colonize in the intestine is improved, which is conducive to probiotics staying and multiplying in the host's intestine for a long time, and exerting a lasting probiotic effect.

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Abstract

A complex microbial inoculant for improving the adhesion of clostridium butyricum in intestinal tracts of eels, the complex microbial inoculant comprising clostridium butyricum and bacillus mucilaginosus, wherein the viable count ratio of the clostridium butyricum to the bacillus mucilaginosus is 1:1-100; and the live bacterium concentrations of the clostridium butyricum and the bacillus mucilaginosus are independently (1-10)x108CFU/mL. According to the microbial inoculant, the colonization capability of clostridium butyricum in intestinal tracts is improved, the long-time staying and propagation of probiotics in host intestinal tracts are facilitated, and a lasting probiotic effect is achieved.

Description

一种提高丁酸梭菌在鳗鲡肠道的粘附性的复合菌剂A composite microbial agent for improving the adhesion of Clostridium butyricum in the intestine of eel 技术领域Technical field
本发明涉及水产养殖领域，具体涉及一种提高丁酸梭菌在鳗鲡肠道的粘附性的复合菌剂。The invention relates to the field of aquaculture, in particular to a composite microbial agent for improving the adhesion of Clostridium butyricum in the intestines of eels.
背景技术Background technique
丁酸梭菌，即酪酸菌，常作为益生菌用来防治由于多种因素引起的人和动物的腹泻、肠炎等疾病。该菌主要存在于土壤、人和动物肠道内，革兰氏阳性，厌养产芽孢，通常被认为是安全的益生菌。抗生素的长期使用，造成了残留、耐药菌株产生等负面影响。一般抗生素能够杀死肠道内包括有益常驻菌的所有微生物，致使肠道内微生态环境失衡，屏障消失，肠豁膜暴露，易引起细胞损伤，降低对致病菌及有害物质的抵御能力。添加益生菌有利于恢复肠道微生态平衡，建立生物屏障，促进肠勃膜修复，增强免疫，改善消化环境，抑制有害菌增殖。Clostridium butyricum, or butyricum, is often used as a probiotic to prevent and treat diseases such as diarrhea and enteritis in humans and animals caused by various factors. The bacterium mainly exists in the soil, human and animal intestines. It is Gram-positive, anaerobic and produces spores. It is generally regarded as a safe probiotic. The long-term use of antibiotics has caused negative effects such as residues and the production of resistant strains. General antibiotics can kill all microorganisms including beneficial resident bacteria in the intestinal tract, resulting in imbalance of the micro-ecological environment in the intestinal tract, loss of the barrier, and exposure of the intestinal membrane, which can easily cause cell damage and reduce the ability to resist pathogenic bacteria and harmful substances. Adding probiotics is beneficial to restore the intestinal microecological balance, establish a biological barrier, promote the repair of intestinal membranes, enhance immunity, improve the digestive environment, and inhibit the proliferation of harmful bacteria.
益生菌能否粘附于肠道粘膜，被认为是肠道定植的前提条件，也是益生菌在宿主肠道内长时间逗留、繁殖，发挥持久益生作用的关键，因此是否具有粘附能力是益生菌菌种筛选的一个重要标准。其次，益生菌体外抗菌特性也是其发挥功能的重要原因，通常表现为产生具有抗菌性代谢物质，如有机酸，过氧化氢等，细菌素的分泌，肠道粘附的占位竞争，促进免疫防御等。Whether probiotics can adhere to the intestinal mucosa is considered to be a prerequisite for intestinal colonization. It is also the key to probiotics staying and multiplying in the host's intestines for a long time to play a long-lasting probiotic effect. Therefore, whether they have the ability to adhere is probiotics An important criterion for strain screening. Secondly, the in vitro antibacterial properties of probiotics are also important reasons for their functions. They usually produce antibacterial metabolites, such as organic acids, hydrogen peroxide, etc., the secretion of bacteriocins, and the competition for intestinal adhesion. Immune defense, etc.
发明概述Summary of the invention
技术问题technical problem
本发明要解决的技术问题，在于提供一种提高丁酸梭菌在鳗鲡肠道的粘附性的复合菌剂，提高了丁酸梭菌在肠道定植的能力，有利于益生菌在宿主肠道内长时间逗留、繁殖，以及发挥持久益生作用。The technical problem to be solved by the present invention is to provide a composite microbial agent that improves the adhesion of Clostridium butyricum in the intestines of eels, improves the colonization ability of Clostridium butyricum in the intestines, and is beneficial for probiotics in the intestines of the host. Stay in the tract for a long time, multiply, and play a long-lasting probiotic effect.
问题的解决方案The solution to the problem
技术解决方案Technical solutions
本发明是这样实现的：The present invention is realized as follows:
一种提高丁酸梭菌在鳗鲡肠道的粘附性的复合菌剂，所述复合菌剂包括丁酸梭菌和胶质芽孢杆菌，所述丁酸梭菌和胶质芽孢杆菌的活菌数比为1∶1-100。A composite bacterial agent for improving the adhesion of Clostridium butyricum in the intestinal tract of an eel, the composite bacterial agent comprising Clostridium butyricum and Bacillus gliocladium, and live bacteria of Clostridium butyricum and Bacillus gliocladium The number ratio is 1:1-100.
进一步地，所述丁酸梭菌和胶质芽孢杆菌的活菌数比为1∶1。Further, the ratio of the number of viable bacteria of Clostridium butyricum and Bacillus glaciens is 1:1.
进一步地，所述丁酸梭菌和胶质芽孢杆菌的活菌浓度独立的为(1-10)x108CFU/mL。Further, the concentration of the viable bacteria of Clostridium butyricum and Bacillus glaciens is (1-10)×108 CFU/mL independently.
发明的有益效果The beneficial effects of the invention
有益效果Beneficial effect
本发明具有如下优点：The present invention has the following advantages:
本发明利用胶质芽孢杆菌分泌一种于细胞壁外具有一定厚度的稳定粘液状物质(即荚膜)，大量的荚膜附着在丁酸梭菌表面，提高了丁酸梭菌粘附于肠道上皮细胞的能力；即提高了丁酸梭菌在肠道定植的能力，有利于益生菌在宿主肠道内长时间逗留、繁殖，以及发挥持久益生作用。The invention utilizes Bacillus glacius to secrete a stable mucus-like substance (ie capsule) with a certain thickness outside the cell wall. A large number of capsules are attached to the surface of Clostridium butyricum, which improves the adhesion of Clostridium butyricum to the intestinal tract. The ability of epithelial cells; that is, the ability of clostridium butyricum to colonize in the intestine is improved, which is conducive to probiotics staying and multiplying in the host's intestine for a long time, and exerting a lasting probiotic effect.
发明实施例Invention embodiment
本发明的实施方式Embodiments of the invention
本发明涉及一种提高丁酸梭菌在鳗鲡肠道的粘附性的复合菌剂，所述复合菌剂包括丁酸梭菌和胶质芽孢杆菌，所述丁酸梭菌和胶质芽孢杆菌的活菌数比为1∶1-100。The present invention relates to a composite bacterial agent for improving the adhesion of Clostridium butyricum in the intestinal tract of an eel. The composite bacterial agent includes Clostridium butyricum and Bacillus glaciens, and the Clostridium butyricum and Bacillus glaci The ratio of viable bacteria number is 1:1-100.
较优的，所述丁酸梭菌和胶质芽孢杆菌的活菌数比为1∶1。Preferably, the ratio of the number of viable bacteria between Clostridium butyricum and Bacillus glaciens is 1:1.
所述丁酸梭菌和胶质芽孢杆菌的活菌浓度独立的为(1-10)x108CFU/mL。The concentration of the viable bacteria of Clostridium butyricum and Bacillus glaciens is independently (1-10)×108 CFU/mL.
以下结合具体实施例对本发明作进一步的说明。The present invention will be further described below in conjunction with specific embodiments.
实施例Example
(1)实验菌的制备，分别将丁酸梭菌和胶质芽孢杆菌培养24h后，1000r/min离心5min使用培养DMEM培养液，均将菌液调整为1x108CFU/ml备用。(1) Preparation of experimental bacteria. After culturing Clostridium butyricum and Bacillus glioblastus for 24 hours, the culture solution was centrifuged at 1000 r/min for 5 minutes, and the bacterial solution was adjusted to 1x108 CFU/ml for use.
丁酸梭菌从鳗鲡肠道中分离得到。胶质芽孢杆菌由昶科生物科技公司提供。Clostridium butyricum was isolated from the intestine of eel. Bacillus mucilaginosus was provided by Changke Biotechnology Company.
(2)肠道上皮细胞前处理与分离：鳗鲡饲养于含大量抗生素液(阿莫西林、链酶素、庆大霉素)随后禁食数天。头部敲打致死，阿莫西林、链酶素、庆大霉素的水体中，放入70％乙醇中消毒1分钟。取出中段肠道，剪除肠系膜，纵行剪开肠壁，用D-Hank’s液，其中含200mg/L阿莫西林、200mg/L链霉素和20万U/L 庆大霉素，冲洗数次，直到洗去肠道内异物以及黏液，再将肠段浸泡于上述抗生素液数分钟，取出冲洗数次。将肠壁剪碎，加入溶于D-Hank’s液的0.2g/L胶原蛋白酶I、0.02％EDTA和0.2g/L胶原蛋白酶组成的酶消化液，28℃水浴中振荡消化20min，吸管反复吹打直到光镜下出现大量细胞及细胞团，加入DMEM(含5％胎牛血清)终止消化，1000r/min，离心5min，弃上清液，加入含抗生素的制得细胞悬液，处理过程中尽量缩短时间。(2) Pretreatment and separation of intestinal epithelial cells: eels are fed in a solution containing a lot of antibiotics (amoxicillin, streptavidin, gentamicin) and then fasted for several days. The head was beaten to death, and the amoxicillin, streptavidin, and gentamicin water were sterilized in 70% ethanol for 1 minute. Take out the middle intestine, cut off the mesentery, cut open the intestinal wall longitudinally, use D-Hank's solution, which contains 200mg/L amoxicillin, 200mg/L streptomycin and 200,000 U/L gentamicin, rinse several times , Until the foreign bodies and mucus in the intestine are washed away, and then soak the intestine segment in the above antibiotic solution for several minutes, take it out and rinse it several times. Cut the intestinal wall into small pieces, add the enzyme digestion solution composed of 0.2g/L collagenase I, 0.02% EDTA and 0.2g/L collagenase dissolved in D-Hank's solution, shake and digest in a 28℃ water bath for 20 minutes, and pipette repeatedly until A large number of cells and cell clusters appear under the light microscope, add DMEM (containing 5% fetal bovine serum) to terminate the digestion, centrifuge at 1000 r/min for 5 minutes, discard the supernatant, add antibiotic-containing to prepare the cell suspension, and minimize the processing process time.
(3)培养：将细胞悬液接种于孔培养板，培养板底壁涂有I型胶原，台盼蓝染色计数，每孔接种浓度为104，高糖培养液总体积1mL，含5％FBS、0.01mg/L表皮生长因子(EGF)、200U/L胰岛素、100mg/L阿莫西林、100mg/L链霉素和10万U/L庆大霉素。在28℃，5％CO2培养箱培养。分别培养30、90、150min后，把培养液连同未贴壁的细胞转入新的培养板培养，观察肠道上皮细胞是否能到纯化。每48h更换培养液一次，待基本铺满底壁时传代。传代时移去培养液，加入少许D-Hank’s液，浸润所有细胞后移去，加入0.25％的胰蛋白酶溶液消化，接种新的培养板培养。(3) Cultivation: Inoculate the cell suspension on a well culture plate, the bottom wall of the culture plate is coated with type I collagen, and counted by trypan blue staining. The inoculation concentration per well is 104. The total volume of high-sugar culture medium is 1 mL, containing 5% FBS. , 0.01mg/L epidermal growth factor (EGF), 200U/L insulin, 100mg/L amoxicillin, 100mg/L streptomycin and 100,000 U/L gentamicin. Incubate at 28°C, 5% CO2 incubator. After culturing for 30, 90, and 150 minutes, transfer the culture medium and the non-adherent cells to a new culture plate to observe whether the intestinal epithelial cells can be purified. Change the culture medium every 48h, and pass it down when the bottom wall is basically covered. Remove the culture medium during subculture, add a little D-Hank’s solution, infiltrate all the cells and remove them, add 0.25% trypsin solution for digestion, and inoculate a new culture plate for culture.
最优培养时间在90min后，转移原培养液效果较好，这时大量的肠道上皮未粘附，悬浮于原培养液中After the optimal culture time is 90 minutes, the transfer of the original culture medium is better. At this time, a large amount of intestinal epithelium is not adhered and suspended in the original culture medium.
(4)测定丁酸梭菌对鳗鲡肠道上皮细胞的粘附率：采用活菌计数法测定丁酸梭菌对鳗鲡肠道上皮细胞的粘附率。(4) Determination of the adhesion rate of Clostridium butyricum to eel intestinal epithelial cells: The viable bacteria count method was used to determine the adhesion rate of Clostridium butyricum to eel intestinal epithelial cells.
A、粘附率计算方式：A. Calculation method of adhesion rate:
细菌粘附率(％)＝(Trion处理后释放出细菌浓度/所加入细菌的浓度)×100Bacterial adhesion rate (%) = (Concentration of bacteria released after Trion treatment/Concentration of bacteria added)×100
B、步骤：B. Steps:
将原代培养的细胞，用0.25％胰蛋白酶消化，制成细胞悬液，吸取20μL悬液和等量的2％台盼蓝液混合，滴入细胞计数板，计数未着色的活细胞，按照活细胞量进行相应的稀释，使最终浓度为1x105细胞/mL，细胞稀释液采用DMEM，细胞接种于24孔培养板，28℃，5％CO2培养箱培养待细胞铺满底壁后(约3-5d)，除去培养液，加入新培养液0.5ml。分三组，第一组加入500μL丁酸梭菌(1x108CFU/mL)，第二组加入500μL丁酸梭菌(1x108CFU/mL)和500μL胶质芽孢杆菌(1x108CFU/mL)，第三组加入500μL丁酸梭菌(1x108CFU/mL)和5ml胶质 芽孢杆菌(1x109CFU/mL)，每组重复12个孔，继续培养90min。用Hank’s液轻轻冲洗3次，并将冲洗液去除干净，除掉未粘附的细菌，之后加入含1％TrionX-100的PBS液0.2ml于各孔，裂解细胞膜、释放胞内及胞膜上的菌，用移液枪轻轻混匀，使粘附的细菌分离充分，置室温下10min，一般认为TrionX-100处理在30min以内不影响细菌存活率，再加入PBS0.8ml终止反应，吸出各孔悬液，用无菌生理盐水100倍稀释，平板活菌计数法测定细菌数。The primary cultured cells were digested with 0.25% trypsin to prepare a cell suspension. Mix 20 μL of the suspension with an equal amount of 2% trypan blue solution, drop it into a cell counting plate, and count the unstained living cells. Dilute the amount of viable cells to a final concentration of 1x105 cells/mL. Use DMEM as the cell diluent. The cells are seeded in a 24-well culture plate at 28°C and 5% CO2 in an incubator. After the cells are covered with the bottom wall (about 3 -5d), remove the culture solution and add 0.5ml of new culture solution. Divided into three groups, the first group was added with 500μL of Clostridium butyricum (1x108CFU/mL), the second group was added with 500μL of Clostridium butyricum (1x108CFU/mL) and 500μL of Bacillus glia (1x108CFU/mL), and the third group was added with 500μL Clostridium butyricum (1x108CFU/mL) and 5ml of Bacillus glaci (1x109CFU/mL), each group repeated 12 holes, and continued to culture for 90min. Rinse gently with Hank's solution 3 times, and remove the rinse solution to remove unadhered bacteria. Then add 0.2ml of PBS containing 1% TrionX-100 to each well to lyse the cell membrane and release the intracellular and cell membranes. Use a pipette to mix gently to separate the adhered bacteria. Leave it at room temperature for 10 minutes. It is generally believed that TrionX-100 treatment will not affect the survival rate of bacteria within 30 minutes. Add 0.8ml of PBS to stop the reaction and aspirate. The suspension in each well was diluted 100 times with sterile normal saline, and the number of bacteria was determined by the plate viable bacteria counting method.
实验结果：第一组的平均粘附率为8.1％，第二组的平均粘附率13.1％，第三组的平均粘附率为12.3％。Experimental results: The average adhesion rate of the first group was 8.1%, the average adhesion rate of the second group was 13.1%, and the average adhesion rate of the third group was 12.3%.
本发明利用胶质芽孢杆菌分泌一种于细胞壁外具有一定厚度的稳定粘液状物质(即荚膜)，大量的荚膜附着在丁酸梭菌表面，提高了丁酸梭菌粘附于肠道上皮细胞的能力；即提高了丁酸梭菌在肠道定植的能力，有利于益生菌在宿主肠道内长时间逗留、繁殖，以及发挥持久益生作用。The invention utilizes Bacillus glacius to secrete a stable mucus-like substance (ie capsule) with a certain thickness outside the cell wall. A large number of capsules are attached to the surface of Clostridium butyricum, which improves the adhesion of Clostridium butyricum to the intestinal tract. The ability of epithelial cells; that is, the ability of clostridium butyricum to colonize in the intestine is improved, which is conducive to probiotics staying and multiplying in the host's intestine for a long time, and exerting a lasting probiotic effect.
虽然以上描述了本发明的具体实施方式，但是熟悉本技术领域的技术人员应当理解，我们所描述的具体的实施例只是说明性的，而不是用于对本发明的范围的限定，熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化，都应当涵盖在本发明的权利要求所保护的范围内。Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described by us are only illustrative, and are not used to limit the scope of the present invention. The equivalent modifications and changes made by the skilled person in accordance with the spirit of the present invention should all fall within the protection scope of the claims of the present invention.

Claims (3)

  1. 一种提高丁酸梭菌在鳗鲡肠道的粘附性的复合菌剂，其特征在于：所述复合菌剂包括丁酸梭菌和胶质芽孢杆菌，所述丁酸梭菌和胶质芽孢杆菌的活菌数比为1∶1-100。A composite bacterial agent for improving the adhesion of Clostridium butyricum in the intestinal tract of an eel, characterized in that: the composite bacterial agent includes Clostridium butyricum and Bacillus glaciens, and the Clostridium butyricum and glial spores The ratio of viable bacteria number of bacilli is 1:1-100.
  2. 根据权利要求1所述的一种提高丁酸梭菌在鳗鲡肠道的粘附性的复合菌剂，其特征在于：所述丁酸梭菌和胶质芽孢杆菌的活菌数比为1∶1。The compound bacterial agent for improving the adhesion of Clostridium butyricum in the intestine of eel according to claim 1, wherein the ratio of the number of viable bacteria of the Clostridium butyricum and Bacillus mucillis is 1: 1.
  3. 根据权利要求1所述的一种提高丁酸梭菌在鳗鲡肠道的粘附性的复合菌剂，其特征在于：所述丁酸梭菌和胶质芽孢杆菌的活菌浓度独立的为(1-10)x10 8CFU/mL。 The compound bacterial agent for improving the adhesion of Clostridium butyricum in the intestinal tract of an eel according to claim 1, wherein the viable bacteria concentration of the Clostridium butyricum and Bacillus mucillis is independently ( 1-10)x10 8 CFU/mL.
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