WO2023050716A1 - 一种融合魏斯氏菌、培养方法及其应用 - Google Patents

一种融合魏斯氏菌、培养方法及其应用 Download PDF

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WO2023050716A1
WO2023050716A1 PCT/CN2022/078752 CN2022078752W WO2023050716A1 WO 2023050716 A1 WO2023050716 A1 WO 2023050716A1 CN 2022078752 W CN2022078752 W CN 2022078752W WO 2023050716 A1 WO2023050716 A1 WO 2023050716A1
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weissella
fusion
wsg1
fused
cultivation
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PCT/CN2022/078752
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French (fr)
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施柔安
施军辉
施并辉
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上海信元宠物食品有限公司
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Priority to JP2023552588A priority Critical patent/JP2024518221A/ja
Publication of WO2023050716A1 publication Critical patent/WO2023050716A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 technical field of microbes, in particular to a fusion Weissella, a culture method and an application thereof.
  • the purpose of the present invention is to provide a probiotic strain that can improve the digestive ability of dogs and inhibit common pathogenic bacteria that cause diarrhea, so as to provide a new optional strain for the development of probiotics that can maintain the intestinal health of dogs.
  • the invention provides a fusion Weissella bacterium, which is Weissella confusa WSG1 fusion, which was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on June 11, 2021. The number is CGMCC NO.22697.
  • nucleotide sequence of the 16S rRNA of the fusion Weissella is shown in SEQ: NO.1.
  • the present invention also provides a culture method for fusion Weissella confusa WSG1, wherein the fusion Weissella confusa WSG1 is inoculated in the medium for cultivation, the initial pH of the cultivation is 5.0-6.5, and the cultivation The temperature is 35-38°C, and the cultivation time is 15-17 hours;
  • the culture medium includes a carbon source and a nitrogen source, and the contents of the carbon source and the nitrogen source in the culture medium are independently 1.0-3.0%.
  • the carbon source is one or more of glucose, sucrose, and starch.
  • the nitrogen source is one or more of peptone, beef extract, and yeast powder.
  • the invention also provides an application of the fused Weissella in the preparation of a drug for aiding digestion.
  • the drug is a veterinary drug.
  • the veterinary drug is a dog-specific drug.
  • the invention also provides an application of fusion Weissella in feed preparation.
  • the invention also provides an application of the fused Weissella in the preparation of medicine for treating diarrhea.
  • the invention provides a fused Weissella confusa WSG1 with the ability to assist in digestion.
  • the safety of the strain has been confirmed through hemolytic evaluation and drug resistance evaluation, and it can survive in the gastrointestinal tract environment and is resistant to antibiotics. It has good medicinal properties, has a significant inhibitory effect on common pathogenic bacteria that cause diarrhea, has the ability to assist digestion, and is non-toxic when taken orally.
  • a new alternative strain is provided for the development of probiotics for maintaining the intestinal health of dogs.
  • Figure 1 is the cover of the identification report of the Institute of Microbiology, Chinese Academy of Sciences;
  • Figure 2 is the identification report content 1 of the Institute of Microbiology, Chinese Academy of Sciences;
  • Figure 3 is the identification report content 2 of the Institute of Microbiology, Chinese Academy of Sciences;
  • Figure 4 shows the production of proteolytic circles by strain WSG1.
  • CGMCC General Microorganism Center
  • the invention provides a fusion Weissella bacterium, which is Weissella confusa WSG1 fusion, which was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on June 11, 2021.
  • the number is CGMCC NO.22697.
  • the nucleotide sequence of the 16S rRNA fused to Weissella in the present invention is preferably shown in SEQ: NO.1.
  • the present invention also provides a method for cultivating Weissella confusa fusion, inoculating the Weissella confusa WSG1 fusion into a culture medium for cultivation.
  • the initial pH of the culture is preferably 5.0-6.5, more preferably 5.5-6.2, and also preferably 5.8-6.0;
  • the temperature of the culture in the present invention is preferably 35-38°C, more preferably 36-6.0 37°C;
  • the cultivation time of the present invention is preferably 15-17 hours, more preferably 15.5-16.5 hours, and still more preferably 15.8-16.2 hours;
  • the medium preferably includes a carbon source and a nitrogen source.
  • the carbon source in the present invention is preferably one or more of glucose, sucrose, and starch, and the content of the carbon source is preferably 1.0-3.0%. , more preferably 1.5 to 2.5%, still more preferably 1.7 to 2.0%.
  • the nitrogen source of the present invention is preferably one or more of peptone, beef extract, and yeast powder, the peptone is preferably casein peptone, and the content of the nitrogen source is preferably 1.0-3.0%, more preferably 1.4-2.8% %, also preferably 1.9 to 2.5%;.
  • the present invention also provides an application of fused Weissella in the preparation of a drug for aiding digestion.
  • the drug is preferably a veterinary drug, and more preferably a dog-specific drug.
  • the live bacteria concentration of Weissella fused to the drug is 1 ⁇ 10 8 to 6 ⁇ 10 8 CFU/kg, more preferably 2 ⁇ 10 8 to 5 ⁇ 10 8 CFU/kg, and more preferably 3 ⁇ 10 8 ⁇ 4 ⁇ 10 8 CFU/kg;
  • the drug preferably uses Weissella fusus as the only active ingredient; more preferably, the drug also includes other active ingredients that aid digestion;
  • the drug includes Adjuvant, the present invention has no special limitation on the type of the adjuvant, and the conventional pharmaceutical adjuvant in the field can be used.
  • the present invention also provides an application of fused Weissella in the preparation of feed, the feed is preferably dog feed, more preferably dog special feed.
  • the live bacteria concentration of Fused Weissella in the feed is 1 ⁇ 10 8 to 6 ⁇ 10 8 CFU/kg, more preferably 2 ⁇ 10 8 to 5 ⁇ 10 8 CFU/kg, and more preferably 3 ⁇ 10 8 ⁇ 4 ⁇ 10 8 CFU/kg;
  • the feed preferably uses Weissella fusion as the only active ingredient to assist digestion; further preferably, the feed also includes other active ingredients to assist digestion;
  • the feed includes nutritional components, and the present invention has no special limitation on the types of the nutritional components, and conventional nutritional components in the field can be used.
  • the present invention also provides an application of fusion Weissella in the preparation of a medicine for treating diarrhea.
  • the medicine is preferably a veterinary medicine, more preferably a dog-specific medicine.
  • the live bacteria concentration of Weissella fused to the drug is 1 ⁇ 10 8 to 6 ⁇ 10 8 CFU/kg, more preferably 2 ⁇ 10 8 to 5 ⁇ 10 8 CFU/kg, and more preferably 3 ⁇ 10 8 ⁇ 4 ⁇ 10 8 CFU/kg;
  • the drug preferably uses Weissella fusus as the only active ingredient; more preferably, the drug also includes other active ingredients for treating diarrhea;
  • the drug includes Adjuvant, the present invention has no special limitation on the type of the adjuvant, and the conventional pharmaceutical adjuvant in the field can be used.
  • the fusion Weissella confusa WSG1 was isolated from the feces of puppies, and was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on June 11, 2021, with the preservation number CGMCC NO.22697; the fusion Weissella The nucleic acid sequence of the bacterium is shown in SEQ: NO.1.
  • the activated strain WSG1 (preservation number CGMCC NO.22697) was inoculated into the culture medium with a 2% inoculation amount, and cultured at 37°C for 16h.
  • the initial pH of the medium is 6.0;
  • the specific formula of the culture medium is as follows:
  • Viable bacteria were counted by the plate method, and the measured concentration was 5.52 ⁇ 10 11 CFU/mL.
  • strain WSG1 The resistance of strain WSG1 to common antibiotics was studied by K-B method and MIC method, including: vancomycin, chloramphenicol, cephalothin, amoxicillin, clindamycin, enrofloxacin, penicillin, cephalosporin Ampicillin, cephradine, azithromycin, oxacillin, cefazolin, tetracycline, ciprofloxacin, marbofloxacin (MIC method).
  • WSG1 has good resistance to antibiotics.
  • Experimental process simulate the gastrointestinal tract environment, inoculate the strain WSG1 in the medium containing different pH and bile salt concentration, and study the change of bacterial content to determine the tolerance of the strain to acid and alkali (pH2.0, 3.0, 4.0, 5.0 and bile salt concentration 0.1%, 0.3%, 0.5%).
  • Table 3 shows the survival rate of strain WSG1 under different conditions.
  • WSG1 has the ability to survive in the environment of gastrointestinal tract.
  • Indicator bacteria Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Clostridium perfringens ATCC 13124.
  • the strain WSG1 was cultured for 24 hours, and the supernatant was obtained by centrifugation and filtered with a 0.22 ⁇ m filter membrane. The inhibitory effect of the supernatant on the three indicator bacteria was studied by punching method.
  • the supernatant of table 4 bacterial strain WSG1 is to the inhibition zone of 3 kinds of indicator bacteria
  • Bacterial liquid preparation Inoculate strain WSG1 into MRS medium, culture for 24 hours, plate count, and the bacterial content reaches 4.75 ⁇ 10 11 CFU/mL.
  • mice Twenty 7-week-old ICR mice were randomly divided into 2 groups after a week of adaptation to feeding.
  • the mice in the experimental group were given oral bacterial suspension diluted with sterile normal saline at an amount of 2.5 ⁇ 10 9 CFU/Kg body weight/day.
  • the mice in the control group were treated with normal saline orally, with a dose of 1 mL, once orally.
  • the activity status, signs of toxicity and death of the mice were continuously observed within 10 days.
  • mice in the experimental group Compared with the control group, the weight gain of mice in the experimental group decreased, but there was no significant difference.
  • Bacterial solution preparation operate next to the alcohol lamp, use a 100 ⁇ L pipette gun to draw 90 ⁇ L of WSG1 bacterial solution, pour it into a 50mL centrifuge tube, then add 30mL of 10% glucose solution to the centrifuge tube, and mix well.
  • the experimental dogs were equally divided into two groups (control group and experimental group).
  • the dogs in the control group were fed normally every day without restriction of drinking water.
  • the dogs in the experimental group were fed normally every day without restriction of drinking water, and each dog was fed the WSG1 bacterial solution configured in step 1 at a dose of 0.5 mL/kg every day (finally equivalent to the daily feeding of each dog 1.5 ⁇ L probiotic stock solution: 7 ⁇ 10 8 CFU/Kg body weight/day), fed in 2 times.
  • the feeding continued for 10 days, and the feces of each dog were observed every day, and the feces of each dog were scored according to the Weihao feces scoring system. Each dog was weighed before and after feeding.
  • test group
  • Juvenile Beagle One of them had no feces formed on the first day after being fed probiotics; the feces were sticky on the 3rd and 4th days; the feces of the other was viscous and not formed on the 2nd and 3rd days; the rest of the time the feces were good.
  • 4Adult Teddy One of them had viscous and unshaped feces on the 6th day; the rest of the time the feces were good.
  • 5Adult Fadou one of them had soft feces on the third day, but it was basically formed; the rest of the time the feces were good.
  • Juvenile Beagle The feces are sticky and not formed on the first and fourth days; the feces are soft but basically formed on the third day; the feces are good the rest of the time.
  • the other animal had viscous and unshaped feces on the first and third days, and the feces were good the rest of the time.
  • the body weight of the experimental group and the control group increased during the experimental period, but there was no difference between the groups, as shown in Table 6.
  • WSG1 is not toxic to dogs.
  • the present invention provides a fusion Weissella confusa WSG1 that can promote digestion and inhibit common diarrhea-causing pathogenic bacteria. It has good resistance to antibiotics, has a significant inhibitory effect on common diarrhea-causing pathogens, has the ability to assist digestion, and is non-toxic when taken orally.
  • a new alternative strain is provided for the development of probiotics for maintaining the intestinal health of dogs.

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Abstract

提供了一种具有辅助消化能力的融合魏斯氏菌Weissella confusa WSG1，该菌株的保藏编号为CGMCC NO.22697，16S rRNA的核苷酸序列如SEQ ID NO.1所示。该菌株能够在胃肠道环境中生存，对抗生素的耐药性良好，对常见引起腹泻病原菌具有显著抑制效果，具有辅助消化的能力，且口服无毒性，可用作维持犬类机体肠道健康能力益生菌。

Description

一种融合魏斯氏菌、培养方法及其应用 技术领域
本发明涉及微生物技术领域，尤其涉及一种融合魏斯氏菌、培养方法及其应用。
背景技术
在犬类未被家养驯化前，他们在自然环境中摄取各类的生食，通过生食来补充自己体内的益生菌。但是宠物犬早就脱离了自然环境，在家中豢养，长期吃狗粮、罐头之类的加工食品，可能没法从这些食品中获得适量益生菌的补充，甚至这些食品中额外的添加剂会破坏宠物犬体内的菌落平衡，使宠物犬发生腹泻、呕吐等问题。某些犬类的消化系统未发育完全，消化能力较弱，腹泻、呕吐等现象更为常见。现有技术中常用抗生素等药物进行治疗，使得病原菌产生抗药性，甚至出现病情反复的情况。
发明内容
本发明的目的在于提供一株能提高犬类消化能力、抑制常见引起腹泻的病原菌的益生菌菌株，为维持犬类机体肠道健康能力益生菌的开发提供新的可选菌株。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种融合魏斯氏菌，所述融合魏斯氏菌为融合魏斯氏菌Weissella confusa WSG1，于2021年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.22697。
优选的，所述融合魏斯氏菌的16S rRNA的核苷酸序列如SEQ:NO.1所示。
本发明还提供了一种融合魏斯氏菌的培养方法，将所述融合魏斯氏菌Weissella confusa WSG1接种于培养基中进行培养，所述培养的初始pH为5.0～6.5、所述培养的温度为35～38℃，所述培养的时间15～17h；
所述培养基包括碳源和氮源，所述培养基中碳源和氮源的含量独立地为1.0～3.0％。
优选的，所述碳源为葡萄糖、蔗糖、淀粉中的一种或几种。
优选的，所述氮源为蛋白胨、牛肉膏、酵母粉中的一种或几种。
本发明还提供了一种融合魏斯氏菌在制备辅助消化的药物中的应用。
优选的，所述药物为兽药。
优选的，所述兽药为犬类专用药。
本发明还提供了一种融合魏斯氏菌在制备饲料中的应用。
本发明还提供了一种融合魏斯氏菌在制备治疗腹泻的药物中的应用。
本发明的技术效果和优点：
本发明提供了一种具有辅助消化能力的融合魏斯氏菌Weissella confusa WSG1，通过溶血性评价和耐药性评价证实了菌株的安全性，能够在胃肠道环境中生存，且对抗生素的耐药性良好，对常见引起腹泻的病原菌具有显著抑制效果，具有辅助消化的能力，且口服无毒性。为维持犬类机体肠道健康能力益生菌开发提供了一种新的可选菌株。
附图说明
图1为中国科学院微生物研究所鉴定报告封面；
图2为中国科学院微生物研究所鉴定报告内容1；
图3为中国科学院微生物研究所鉴定报告内容2；
图4为菌株WSG1产生溶蛋白圈。
保藏说明
融合魏斯氏菌Weissella confusa WSG1，保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号，保藏日期为2021年6月11日，保藏编号为CGMCC NO.22697。
具体实施方式
本发明提供了一种融合魏斯氏菌，所述融合魏斯氏菌为融合魏斯氏菌Weissella confusa WSG1，于2021年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.22697。本发明中融合魏斯氏菌的16S rRNA的核苷酸序列优选如SEQ:NO.1所示。
本发明还提供了一种融合魏斯氏菌的培养方法，将所述融合魏斯氏菌Weissella confusa WSG1接种于培养基中进行培养。
在本发明中，所述培养的初始pH优选为5.0～6.5，进一步优选为5.5～6.2，还优选为5.8～6.0；本发明所述培养的温度优选为35～38℃，进一步优选为 36～37℃；本发明所述培养的时间优选为15～17h，进一步优选为15.5～16.5h，还优选为15.8～16.2h；
在本发明中，所述培养基优选包括碳源和氮源，本发明所述碳源优选为葡萄糖、蔗糖、淀粉中的一种或几种，所述碳源的含量优选为1.0～3.0％，进一步优选为1.5～2.5％，还优选为1.7～2.0％。本发明所述氮源优选为蛋白胨、牛肉膏、酵母粉中的一种或几种，所述蛋白胨优选为酪蛋白胨，所述氮源的含量优选为1.0～3.0％，进一步优选为1.4～2.8％，还优选为1.9～2.5％；。
本发明还提供了一种融合魏斯氏菌在制备辅助消化的药物中的应用，所述药物优选为兽药，进一步优选为犬类专用药。在本发明中，所述药物中融合魏斯氏菌的活菌浓度为1×10 8～6×10 8CFU/kg，进一步优选为2×10 8～5×10 8CFU/kg，还优选为3×10 8～4×10 8CFU/kg；所述药物优选的以融合魏斯氏菌为唯一活性成分；进一步优选的，所述药物还包括其它辅助消化的活性成分；所述药物包括辅料，本发明对所述辅料的种类没有特殊限定，采用本领域常规的药物辅料即可。
本发明还提供了一种融合魏斯氏菌在制备饲料中的应用，所述饲料优选为犬类饲料，进一步优选为犬类专用饲料。在本发明中，所述饲料中融合魏斯氏菌的活菌浓度为1×10 8～6×10 8CFU/kg，进一步优选为2×10 8～5×10 8CFU/kg，还优选为3×10 8～4×10 8CFU/kg；所述饲料优选的以融合魏斯氏菌为唯一辅助消化的活性成分；进一步优选的，所述饲料还包括其它辅助消化的活性成分；所述饲料包括营养组分，本发明对所述营养组分的种类没有特殊限定，采用本领域常规的营养组分即可。
本发明还提供了一种融合魏斯氏菌在制备治疗腹泻的药物中的应用，所述药物优选为兽药，进一步优选为犬类专用药。在本发明中，所述药物中融合魏斯氏菌的活菌浓度为1×10 8～6×10 8CFU/kg，进一步优选为2×10 8～5×10 8CFU/kg，还优选为3×10 8～4×10 8CFU/kg；所述药物优选的以融合魏斯氏菌为唯一活性成分；进一步优选的，所述药物还包括其它治疗腹泻的活性成分；所述药物包括辅料，本发明对所述辅料的种类没有特殊限定，采用本领域常规的药物辅料即可。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把 它们理解为对本发明保护范围的限定。
实施例1
从幼犬粪便中分离得到融合魏斯氏菌Weissella confusa WSG1，于2021年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.22697；所述融合魏斯氏菌的核酸序列如SEQ:NO.1所示。
使用MRS培养基进行培养，进行相关性能测定。
实施例2
将活化好的菌株WSG1(保藏编号CGMCC NO.22697)以2％接种量接种至培养基中，37℃培养16h。
培养基初始pH为6.0；
培养基具体配方如下：
MRS培养基：
Figure PCTCN2022078752-appb-000001
用平板法进行活菌计数，测得浓度为5.52×10 11CFU/mL。
实验例1安全性评价-溶血性评价
实验过程:将菌株WSG1的新鲜培养物划线接种到含5％绵羊血的固体培养基上，观察是否有溶血现象产生，不产生溶血现象的判断为安全菌株。
实验结果：并未产生溶血现象。
实验例2安全性评价-耐药性评价
实验过程:通过K-B法和MIC法研究菌株WSG1对常见抗生素的耐药性，包括：万古霉素、氯霉素、头孢噻吩、阿莫西林、克林霉素、恩诺沙星、青霉素、头孢氨苄、头孢拉定、阿奇霉素、苯唑西林、头孢唑林、四环素、环丙沙星、马波沙星(MIC法)。
实验结果：抑菌圈直径(mm)及MIC值如表1、表2所示。
表1菌株WSG1对抗生素的抑菌圈直径(mm)
Figure PCTCN2022078752-appb-000002
表2菌株WSG1的MIC(马波沙星)
  MIC
WSG1 1.024mg/ml
可见，WSG1对抗生素的耐药性良好。
实验例3菌株鉴定
样品送至中国科学院微生物研究所进行鉴定，鉴定报告如图1～3所示。
实验例4耐酸耐胆盐能力评价
实验过程：模拟胃肠道环境，将菌株WSG1接种于含不同pH和胆盐浓度的培养基中，研究菌含量变化以确定菌株对酸碱的耐受性(pH2.0，3.0，4.0，5.0和胆盐浓度0.1％，0.3％，0.5％)。
实验结果：菌株WSG1在不同条件下的生存率如表3所示。
表3菌株WSG1在不同条件下的生存率
Figure PCTCN2022078752-appb-000003
Figure PCTCN2022078752-appb-000004
可见，WSG1具有在胃肠道环境中生存的能力。
实验例5对常见引起腹泻病原菌的抑制作用
实验过程：指示菌：大肠杆菌ATCC 25922，鼠伤寒沙门菌ATCC 14028，产气荚膜梭菌ATCC 13124。将菌株WSG1培养24小时，离心获得上清液并用0.22μm滤膜过滤，通过打孔法研究上清液对3种指示菌的抑制作用。
实验结果:菌株WSG1的上清液对3种指示菌的抑菌圈(mm)如表4所示，具有显著抑菌效果。
表4菌株WSG1的上清液对3种指示菌的抑菌圈
大肠杆菌 鼠伤寒沙门菌 产气荚膜梭菌
9.5 7.5 10.5
实验例6辅助消化能力-产蛋白酶能力研究
实验过程：将菌株WSG1过夜培养后离心并过滤，得到无细胞上清液，打孔法接入到1％脱脂奶平板中，通过观察其是否产生溶蛋白圈，判断其产蛋白酶能力，有蛋白溶解现象即为有降解蛋白能力。
实验结果：能够产生溶蛋白圈，如图4所示。
实验例7对小鼠的口服毒性试验
菌液制备：将菌株WSG1接种到MRS培养基中，培养24小时，平板计数，菌含量达4.75×10 11CFU/mL。
7周龄ICR小鼠20只，经一周适应饲养后，随机分成2组，实验组小鼠按2.5×10 9CFU/Kg体重/天的量给予口服用无菌生理盐水稀释的菌悬液，对照组小鼠给予生理盐水口服处理，剂量为1mL，口服1次。10d内连续观察小鼠的活动状态，毒性迹象以及死亡情况。
实验结果：菌株WSG1口服急性毒性实验期间，未出现小鼠死亡情况，小鼠皮毛、饮水、躯体活动和行为模式均表现正常，未发现急性中毒体征和相关的毒性反应。
实验组小鼠体重增加与对照组相比有所下降，但无明显差异。
实验例8对犬的口服毒性试验
菌液制备：在酒精灯旁进行操作，用100μL移液枪吸取90μLWSG1菌 液，注入至50mL离心管中，之后向离心管中加入30mL的10％葡萄糖溶液，混匀。
20只实验犬(4只幼年比格，4只成年比格，4只成年法斗，4只成年泰迪，4只成年中华田园犬)
将实验犬只平均分为两组(对照组与实验组)。对照组犬只每日正常饲喂，不限制饮水。实验组犬只每日正常饲喂，不限制饮水，并每只犬只每日按0.5mL/kg的剂量饲喂步骤1中配置的WSG1菌液(最终等同于每只犬只每日饲喂1.5μL的益生菌原液：按7×10 8CFU/Kg体重/天)，分2次饲喂。持续饲喂10日，每日观察每只犬只的粪便情况，并按照威豪粪便评分系统对每只犬的粪便进行评分。饲喂前后对每只犬只进行称重。
实验结果：
实验组：
①幼年比格：其中1只喂食益生菌后第1天粪便不成型；第3、4天粪便粘稠；另一只在2、3天粪便粘稠不成型；其余时间粪便良好。
②成年比格：其中一只第7天粪便较软，但基本成型；其余时间粪便良好。
③成年中华田园犬：全程未见明显粪便异常。
④成年泰迪：其中一只第6天粪便粘稠不成型；其余时间粪便良好。
⑤成年法斗：其中一只第3天粪便较软，但基本成型；其余时间粪便良好。
对照组：
①幼年比格：其中第1、4天粪便粘稠不成型；第3天粪便较软，但基本成型；其余时间粪便良好。另一只第1、3天粪便粘稠不成型，其余时间粪便良好。
②成年比格：其中一只第1天粪便粘稠，基本不成型；其余时间粪便良好。
③成年中华田园犬：全程未见明显粪便异常。
④成年泰迪：全程未见明显粪便异常。
⑤成年法斗：全程未见明显粪便异常。
采用曼-惠特宁检验再次对第九日数据进行分析，差异不显著，如表5所示，两组的粪便评分在十天内并无显著性差异。
表5
Figure PCTCN2022078752-appb-000005
体重在实验期内实验组与对照组有所增加，但无组间差异性，如表6所示。
表6
Figure PCTCN2022078752-appb-000006
因此，WSG1对犬并无毒性。
由以上实施例可知，本发明提供了一株能促进消化、抑制常见引起腹泻病原菌的融合魏斯氏菌Weissella confusa WSG1，溶血性评价和耐药性评价证实了菌株的安全性，能够在胃肠道环境中生存，且对抗生素的耐药性良好，对常见引起腹泻病原菌具有显著抑制效果，具有辅助消化的能力，且口服无毒性。为维持犬类机体肠道健康能力益生菌开发提供了一种新型可选菌株。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种融合魏斯氏菌，其特征在于，所述融合魏斯氏菌为融合魏斯氏菌Weissella confusa WSG1，于2021年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.22697。
  2. 根据权利要求1所述的融合魏斯氏菌，其特征在于，所述融合魏斯氏菌的16S rRNA的核苷酸序列如SEQ:NO.1所示。
  3. 权利要求1或2所述的融合魏斯氏菌的培养方法，其特征在于，将所述融合魏斯氏菌Weissella confusa WSG1接种于培养基中进行培养，所述培养的初始pH为5.0～6.5、所述培养的温度为35～38℃，所述培养的时间15～17h；
    所述培养基包括碳源和氮源，所述培养基中碳源和氮源的含量独立地为1.0～3.0％。
  4. 根据权利要求3所述的融合魏斯氏菌的培养方法，其特征在于，所述碳源为葡萄糖、蔗糖、淀粉中的一种或几种。
  5. 根据权利要求4所述的融合魏斯氏菌的培养方法，其特征在于，所述氮源为蛋白胨、牛肉膏、酵母粉中的一种或几种。
  6. 权利要求1或2所述的融合魏斯氏菌在制备辅助消化的药物中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述药物为兽药。
  8. 根据权利要求7所述的应用，其特征在于，所述兽药为犬类专用药。
  9. 权利要求1或2所述的融合魏斯氏菌在制备饲料中的应用。
  10. 权利要求1或2所述的融合魏斯氏菌在制备治疗腹泻的药物中的应用。
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