WO2020098396A1 - 一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用 - Google Patents

一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用 Download PDF

Info

Publication number
WO2020098396A1
WO2020098396A1 PCT/CN2019/108390 CN2019108390W WO2020098396A1 WO 2020098396 A1 WO2020098396 A1 WO 2020098396A1 CN 2019108390 W CN2019108390 W CN 2019108390W WO 2020098396 A1 WO2020098396 A1 WO 2020098396A1
Authority
WO
WIPO (PCT)
Prior art keywords
fermentation broth
stevia
ethyl acetate
mastitis
bacillus
Prior art date
Application number
PCT/CN2019/108390
Other languages
English (en)
French (fr)
Inventor
朴美子
Original Assignee
青岛海思达生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 青岛海思达生物科技有限公司 filed Critical 青岛海思达生物科技有限公司
Publication of WO2020098396A1 publication Critical patent/WO2020098396A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/287—Chrysanthemum, e.g. daisy
    • 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
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • 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
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

Definitions

  • the invention belongs to the technical field of extraction and application of plant active substances, and particularly relates to an extract of stevia fermentation broth and its polar components and application in the preparation of bacteriostatic agents and additives.
  • Stevia is rich in nutrients and has good application prospects in many industries. Adding stevia leaf residues to poultry feed can play a good role in preventing poultry dysentery and other diseases. Adding stevia leaf residues to beverages as a drink for livestock such as cows and sheep can effectively increase its milk production. Increase the nutrients in milk.
  • Qiu Guangzhong and others studied the effect of "stevia extract” on the performance of laying hens, and found that adding 1% "stevia extract” premix to the basic diet of laying hens can improve the performance of laying hens. Studies have reported the antioxidant, antibacterial and antihistamine activities of stevia fermentation broth, as well as the separation of new compounds in stevia fermentation broth. After fermentation, stevia can obtain active products and produce antibacterial active substances, which is beneficial to the comprehensive development and utilization of stevia.
  • Mastitis is a frequently-occurring disease in dairy cows and one of the most serious diseases in the dairy industry. Dairy cow mastitis can reduce milk production, reduce fresh milk quality and increase herd replacement costs. Mastitis is an inflammation of the breast, and its occurrence is often closely related to microbial infections.
  • the treatment methods for cow mastitis are: antibacterial treatment, traditional Chinese medicine treatment and vaccine treatment. The antibacterial mechanism of stevia fermentation broth against dairy cow mastitis bacteria has not been reported. Studying the antibacterial mechanism of antibacterial agents will help their application, develop safe and efficient natural antibacterial agents, and conduct safety evaluation on stevia fermentation broth.
  • Antibacterial drugs such as penicillins, cephalosporins, fludioxolone, etc., are an effective means of preventing and treating dairy cow mastitis, especially in the treatment of acute, multiple and subacute mastitis, quality control of dairy products or as a An important part of prevention and control, has important application value. Therefore, antibiotics were selected as a positive control for this experiment.
  • Hou Xiaoshen used compound amoxicillin breast injection to treat clinical dairy cow mastitis, the effect was significant, and the clinical efficacy of dairy cows was 92.00%.
  • Feng Yanyan et al. Used cefquinoxime sulfate breast injection to treat dairy cow mastitis. The injection has a good therapeutic effect on lactating dairy cow mastitis. The effective rate is higher than 80% and the safety is high. It does not cause adverse reactions in lactating dairy cows.
  • the object of the present invention is to provide an extract of stevia fermentation broth and its polar components and application in the preparation of bacteriostatic agents and additives.
  • Natural antibacterial agents of plant origin have broad application prospects. Studying the antibacterial mechanism of additives will help their application, as well as the development of safe and efficient natural antibacterial agents.
  • the invention provides an extract of stevia fermentation broth.
  • the preparation method of the extract of stevia fermentation broth includes the following steps:
  • the mass volume ratio of stevia powder mixed with water in the step (1) is 1: 15-1: 25.
  • the volume ratio of chloroform and methanol in the mixed solution in step (3) is 100: 0, 100: 1, 100: 4, 100: 8 and 100: 16 respectively.
  • the ratio of chloroform to methanol in the mixed solution is 100: 8, and the polar component obtained has the highest activity.
  • the invention provides the application of the stevia fermentation broth in the preparation of bacteriostatic agent, the bacteriostatic agent is a bacteriostatic agent for inhibiting Bacillus mastitis, and the use concentration of the stevia fermentation broth is 50mg / mL ⁇ 100mg / mL.
  • the invention provides the application of the ethyl acetate phase of the fermentation broth in the preparation of bacteriostatic agents
  • the bacteriostatic agent is a bacteriostatic agent for inhibiting Bacillus mastitis
  • the concentration of the ethyl acetate phase of the fermentation broth is 25 mg / mL ⁇ 100mg / mL.
  • the invention provides the application of the polar component FSE4 in the preparation of bacteriostatic agents
  • the bacteriostatic agent is a bacteriostatic agent for inhibiting Bacillus mastitis
  • the use concentration of the polar component FSE4 is 5 mg / mL ⁇ 100mg / mL.
  • the invention provides the application of the stevia fermentation broth in the preparation of feed additives for dairy cows.
  • the invention provides the application of the ethyl acetate phase of the fermentation broth in the preparation of feed additives for dairy cows.
  • the invention provides the application of the polar component FSE4 in the preparation of feed additives for dairy cows.
  • the present invention provides Stevia fermented broth extract and its polar components and application in the preparation of bacteriostatic agents and additives, from cell membrane permeability and
  • experiments on the antibacterial mechanism were carried out in order to promote the application of stevia, because the stevia extract of the present invention, the ethyl acetate phase of the fermentation broth and the polar component FSE4 are all on the breast Bacillus pneumoniae has a good bacteriostatic effect, so it can be used as a feed additive for dairy cows to prevent mastitis in dairy cows.
  • the raw material utilization rate of stevia ferment is improved, the application range of stevia is further expanded, and the market application prospect is good.
  • the dried stevia is crushed with a pulverizer, stevia powder and distilled water are mixed at 1:20, 40% of the liquid volume is added to the cone-shaped bottle, and the pH is adjusted to 7.0 with NaOH.
  • the tampons are sealed and sealed with plastic wrap.
  • the filled Erlenmeyer flask was heated in a 60 ° C water bath for 30 minutes. After cooling, Lactobacillus plantarum was inserted at 7% inoculation amount.
  • the Erlenmeyer flask containing the inoculum was incubated in a 37 ° C incubator at a speed of 160 r / m for 3 days.
  • the obtained stevia fermentation broth (FS) is filtered with gauze.
  • the filtrate is centrifuged at 3000r / m for 10min to remove the stevia powder as much as possible.
  • the phase (FSE) the remaining phase was extracted with ethyl acetate and then extracted three times with n-butanol 1: 1 to obtain the n-butanol phase (FSB) and the remaining aqueous phase (FSW) of the fermentation broth.
  • FS, FSE, FSB and FSW were solid samples prepared by freeze-drying, which were used in the next bacteriostasis experiment.
  • Example 2 Determination of antibacterial activity of the extract of stevia fermentation broth
  • the inhibition zone was measured by agar diffusion-punching method.
  • the test bacteria were inoculated into sterilized LB broth, and cultured in a 37 ° C incubator at a speed of 160r / m for 24h, and the concentration of the bacterial suspension was adjusted to 1.5 ⁇ 10 8 CFU / mL.
  • FS, FSE, FSB and FSW were tested at concentrations of 25, 50 and 100 mg / mL. Except FS and FSW were dissolved in sterile water, the rest of the samples were dissolved in dimethyl sulfoxide. Take 100 ⁇ g / mL chloramphenicol (Chloramphenicol, Chl) as a positive control. FS and FSW use water as the solvent control, and FSE and FSB use dimethyl sulfoxide as the solvent control.
  • Variance can accurately reflect the degree of dispersion between the parallel test results of each group, showing the accuracy of the test results.
  • the standard deviation is used as a benchmark to make an error line, so that the true value can be accurately reflected within the range of the error line.
  • SPSS was used to analyze single factor variance and difference significance.
  • the bacteriostatic zone experiment can compare the antibacterial activity of antibacterial agents from different sources in vitro, and facilitate the screening of natural antibacterial agents.
  • Table 1 shows that FS has an inhibitory effect on strains at 100 mg / mL.
  • Bacillus.sp.1 and 2 are common bacillus mastitis commercially available, and have the strongest inhibitory effect on Bacillus.sp.1. 15mm. At a concentration of 50 mg / mL, it has an inhibitory effect on Bacillus.sp.2. However, there was no inhibitory effect on the strain at 25 mg / mL, and the effect was concentration-dependent.
  • the dosage of crude plant extracts with bacteriostatic activity is often large, but the preparation of crude extracts is relatively easy and does not incorporate organic solvents, which can reduce costs and expand the scope of use.
  • the fermentation broth was extracted with ethyl acetate.
  • FSE exhibits bacteriostatic activity from 25 mg / mL to 100 mg / mL, as shown in Table 2.
  • the antibacterial effect of FSB on Bacillus.sp.1 and Bacillus.sp.2 is similar to FS, but only when the concentration reaches 100mg / mL.
  • the inhibitory effect on Bacillus.sp.1 is the most obvious, the inhibition zone reaches 10.50mm, and FSB has no inhibitory effect at the other two concentrations.
  • the experimental results show that the product dissolved in n-butanol failed to produce strong antibacterial activity.
  • FSW showed no antibacterial activity at three concentrations for Bacillus.sp.1 and Bacillus.sp.2.
  • Example 3 Antibacterial activity of FSE chromatographically separated components against Bacillus mastitis
  • Example 2 To study the antibacterial activity of the components separated by FSE chromatography on Bacillus mastitis. The method of determination of the inhibition zone is the same as in Example 2. The five polar components obtained by FSE silica gel column chromatography were tested at concentrations of 5, 10, and 25 mg / mL. The sample was dissolved in dimethyl sulfoxide. Take 100 ⁇ g / mL chloramphenicol as a positive control and dimethyl sulfoxide as a solvent control.
  • the silica gel column was used to further separate the FSE, and the five polar component products were selected to carry out bacteriostasis tests at three concentrations of 5, 10 and 25 mg / mL.
  • the experimental results are shown in Table 5.
  • the inhibitory effect of FSE1 on Bacillus mastitis strain Bacillus.sp.1 and Bacillus.sp.2 is weak, and the inhibitory effect is only produced at 25 mg / mL.
  • the inhibition of FSE1 is less than that of FSE. Stronger than FSB and FSW.
  • FSE2 The antibacterial activity of FSE2 is shown in Table 6. FSE2 only inhibited Bacillus.sp.1 and Bacillus.sp.2 at 10 and 25 mg / mL. FSE2 has a lower concentration than FSE1 in producing inhibitory effects. As the polarity of the eluent increases, the content of antibacterial components in the eluent increases, gradually increasing the antibacterial activity.
  • FSE3 The antibacterial activity of FSE3 is close to that of FSE2, as shown in Table 7.
  • FSE3 has no inhibitory effect on Bacillus.sp.1 and Bacillus.sp.2 at 5 mg / mL, but only at 10 and 25 mg / mL. Among them, the inhibition zone of Bacillus.sp.1 reached 12.75mm at 25mg / mL.
  • FSE4 has an inhibitory effect on Bacillus.sp.1 and Bacillus.sp.2 at three concentrations, and the inhibitory effect increases with increasing concentration.
  • the inhibition effect on Bacillus.sp.1 The strongest, at 5 and 25mg / mL, the inhibition zone was 10.5 and 15mm, respectively.
  • FSE5 has inhibitory effects on Bacillus.sp.1 and Bacillus.sp.2 at three concentrations. The inhibitory effect increases with increasing concentration, and the inhibitory effect is close to FSE4. FSE5 had the strongest inhibitory effect on Bacillus.sp.1, and the bacteriostatic circles at the concentrations of 5 and 25 mg / mL were 10 and 14.25 mm, respectively.
  • Example 4 MIC of Stevia fermented broth extract on Bacillus mastitis
  • the antimicrobial activity of the stevia fermented liquid extract against Bacillus mastitis was determined by MIC.
  • the MIC measurement method is the same as in Example 3.
  • FS selection concentration is 62.50-8000.00 ⁇ g / mL for experiment
  • FSB selection concentration is 15.63-2000.00 ⁇ g / mL for experiment
  • FSW concentration is 125.00-16000.00 ⁇ g / mL for experiment
  • the MIC of the antibacterial agent is a quantitative description of the antibacterial activity.
  • the antimicrobial agent with a smaller MIC is more sensitive to microorganisms, the concentration of the inhibitory effect is low, and the bacteriostatic effect is stronger.
  • the combination of bacteriostatic circle and MIC value can better reflect the inhibitory effect of antibacterial agents on microorganisms.
  • the MIC of stevia rebaudiana extract against Bacillus mastitis is shown in Table 10.
  • the MIC of FSE and FSE silica gel column chromatography separation components is the lowest, between 31.25-250 ⁇ g / mL, although FS and FSB have antibacterial activity, However, the MIC is much larger than that of FSE and its silica gel column chromatography.
  • Example 5 The stevia fermented extract is used as an alternative to antibiotics to prepare additives for dairy cows
  • Stevia fermented material is used to prepare additives for dairy cattle feeding
  • the antibacterial activity (inhibition circle, mm) of fermented stevia against Bacillus mastitis was 10.80 ⁇ 0.71 and 9.60 ⁇ 0.50, 60.8% and 54.1% of the chloramphenicol positive control inhibition zone (17.75 ⁇ 1.06), respectively. .
  • the antibacterial activity of stevia fermentate against Bacillus mastitis (bacteriostatic zone, mm) was measured at a concentration of 100 mg / mL and chloramphenicol was 100 ug / mL.
  • the ethyl acetate phase of stevia fermentation broth is used to prepare additives for dairy cattle feeding
  • the filtrate was centrifuged at 3000 rpm / min for 10 min. Ethyl acetate was extracted. After repeated three times, the extracts were combined, concentrated by rotation to dryness, and the residual extractant was removed by freeze-drying to obtain the ethyl acetate phase of stevia fermentation broth.
  • the antibacterial activity of the stevia fermentation broth ethyl acetate against Bacillus mastitis was 14.50 ⁇ 0.71 and 14.00 ⁇ 0.00, respectively, and the chlortetracycline positive control inhibition zone (17.75 ⁇ 1.06) was 81.7 % And 78.9%.
  • the antibacterial activity of the stevia fermentation broth ethyl acetate against Bacillus mastitis was measured at a concentration of 100 mg / mL and chloramphenicol was 100 ug / mL.
  • the fermented polar component FSE4 is used to prepare feed additives for dairy cows.
  • the antibacterial activity of the polar component FSE4 against Bacillus mastitis (bacteriostatic zone, mm) was 15.00 ⁇ 0.11 and 14.50 ⁇ 0.00, respectively, 87.0% and 84.1 of the chloramphenicol positive control inhibitory zone (17.25 ⁇ 0.35) %.
  • the concentration of the polar component FSE4 against Bacillus mastitis (bacteriostatic zone, mm) was measured at a concentration of 25 mg / mL and chloramphenicol was 100 ug / mL.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Polymers & Plastics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Husbandry (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Sustainable Development (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physiology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Pregnancy & Childbirth (AREA)
  • Epidemiology (AREA)
  • Endocrinology (AREA)
  • Reproductive Health (AREA)
  • Mycology (AREA)
  • Medical Informatics (AREA)
  • Botany (AREA)
  • Birds (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

一种甜叶菊发酵液提取物及其极性组分，其制备方法如下：将甜叶菊粉末与蒸馏水混合，调节pH，水浴加热，冷却后接入植物乳杆菌，在培养箱培养，得到的甜叶菊发酵液，过滤，离心，用乙酸乙酯萃取发酵液，得到乙酸乙酯相；将乙酸乙酯相用硅胶柱进一步的分离，用氯仿和甲醇混合液进行洗脱得到五个极性组分。其中，氯仿和甲醇的比例为100:8得到的极性组分FSE4活性最高。所述发酵液、乙酸乙酯相和极性组分FSE4能抑制乳房炎芽孢杆菌，可以在奶牛饲料添加剂中应用。

Description

一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用 技术领域
本发明属于植物活性物质提取及应用技术领域，具体涉及一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用。
背景技术
甜叶菊营养丰富，在多个行业中有很好的应用前景。将甜菊叶残渣按添加到禽类饲料中，能对禽类的痢疾等疾病发挥很好的预防作用，将甜菊叶残渣添加至饮料中作为奶牛、奶羊等牲畜的饮品，可有效增加其产奶量，增加了奶中的营养成分。邱光忠等研究了“甜叶菊提取物”对蛋鸡生产性能的影响，发现在蛋鸡基础日粮中添加1％“甜叶菊提取物”预混剂，能提高蛋鸡的生产性能。已有研究报道了甜叶菊发酵液的抗氧化、抗菌和抗组胺等活性，以及甜叶菊发酵液中新化合物的分离等。甜叶菊经发酵后，可获取活性产物，产生抑菌活性物质，从而有利于甜叶菊的综合开发利用。
乳房炎是奶牛的一种多发病，是造成奶牛业损失最严重的疾病之一。奶牛乳房炎能造成产奶量减少，降低鲜奶质量和增加牛群更替成本。乳房炎作为乳腺的一种炎症，其发生常与微生物感染有密切关系。目前奶牛乳房炎的治疗方法有：抗菌药物的治疗、中药治疗和疫苗治疗等。甜叶菊发酵液对奶牛乳房炎细菌的抑菌机理尚未有报导。研究抗菌剂的抑菌机理有助于其应用，开发安全、高效的天然抗菌剂，以及对甜菊发酵液进行安全性评价。
抗菌药物如青霉素类、头抱菌素、氟喳诺酮等，是防治奶牛乳房炎的一种有效手段，特别是在急性、多发性和亚急性乳房炎治疗、控制奶产品质量上或作为综合防治中的一个重要组成部分，具有重要应用价值。因此选择抗生素作为本实验阳性对照。侯晓申用复方阿莫西林乳房注入剂治疗临床型奶牛乳房炎，效果显著，奶牛临床疗效为92.00％。冯言言等用硫酸头孢喹肟乳房注入剂治疗奶牛乳房炎，注入剂对泌乳期奶牛乳房炎有良好的治疗作用，有效率高于80％，且安全性高，未引起泌乳期奶牛不良反应。
发明内容
本发明的目的是提供了一种甜叶菊发酵液提取物及其极性组分和在制备抑 菌剂和添加剂中的应用。植物源天然抗菌剂具有广泛的应用前景，研究添加剂的抑菌机理有助于其应用，也有利于开发安全、高效的天然抗菌剂。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一种甜叶菊发酵液提取物，所述甜叶菊发酵液提取物的制备方法包括如下步骤：
(1)将甜叶菊干燥并粉碎后，将甜叶菊粉末与水混合，调节pH为6.5-7.5，水浴加热；冷却后，接入植物乳杆菌，培养2-5d得到甜叶菊发酵液；
(2)将所述甜叶菊发酵液过滤，离心，用乙酸乙酯以体积比1∶1萃取发酵液得到发酵液乙酸乙酯相；
(3)将所述发酵液乙酸乙酯相固体样品用硅胶柱进行进一步的分离，用氯仿和甲醇的混合液进行洗脱分别得到五个极性组分FSE1、FSE2、FSE3、FSE4和FSE5。
进一步的：所述步骤(1)中甜叶菊粉末与水混合的质量体积比为1∶15-1∶25。
进一步的：所述步骤(3)中混合液中氯仿和甲醇的体积比例分别为100∶0、100∶1、100∶4、100∶8和100∶16。
进一步的：所述步骤(3)中混合液中氯仿和甲醇的比例为100∶8得到的极性组分活性最高。
本发明提供了所述甜叶菊发酵液在制备抑菌剂中的应用，所述抑菌剂为抑制乳房炎芽孢杆菌的抑菌剂，所述甜叶菊发酵液的使用浓度为50mg/mL～100mg/mL。
本发明提供了所述发酵液乙酸乙酯相在制备抑菌剂中的应用，所述抑菌剂为抑制乳房炎芽孢杆菌的抑菌剂，所述发酵液乙酸乙酯相的使用浓度为25mg/mL～100mg/mL。
本发明提供了所述极性组分FSE4在制备抑菌剂中的应用，所述抑菌剂为抑制乳房炎芽孢杆菌的抑菌剂，所述极性组分FSE4的使用浓度为5mg/mL～100mg/mL。
本发明提供了所述甜叶菊发酵液在制备奶牛饲用添加剂中的应用。
本发明提供了所述发酵液乙酸乙酯相在制备奶牛饲用添加剂中的应用。
本发明提供了所述极性组分FSE4在制备奶牛饲用添加剂中的应用。
与现有技术相比，本发明的优点和技术效果是：本发明提供了甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用，从细胞膜通透性和结构方面对抑菌机理进行了实验，以便于促进甜叶菊的应用，因为本发明所述的甜叶菊发酵液提取物、所述发酵液乙酸乙酯相和所述极性组分FSE4均对乳房炎芽孢杆菌具有良好的抑菌效果，所以可以用于添加到奶牛饲料添加剂中，用于防治奶牛的乳房炎。通过本发明的技术方案，提高了甜叶菊发酵物的原料利用率，进一步扩大甜叶菊的应用范围，具有良好的市场应用前景。
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步详细的说明。
实施例1：甜叶菊发酵液提取物的制备
1、将MRS斜面保存的植物乳杆菌接种到灭菌的MRS液体培养基，在37℃培养箱以转速160r/m培养24h。
2、干燥甜叶菊用粉碎机粉碎，甜叶菊粉末与蒸馏水按1∶20混合，以40％装液量加入锥型瓶中，用NaOH调节pH为7.0。棉塞封口，并用保鲜膜密封。装液后的锥形瓶在60℃水浴锅中加热30min。冷却后，以7％接种量接入植物乳杆菌。装有接种液的锥形瓶在37℃培养箱以转速160r/m培养3d。
3、得到的甜叶菊发酵液(FS)用纱布过滤，过滤液以转速3000r/m离心10min以尽可能除去甜叶菊粉末，用乙酸乙酯以1∶1萃取发酵液三次得到发酵液乙酸乙酯相(FSE)，乙酸乙酯萃取剩余相再用正丁醇以1∶1萃取3次，得到发酵液正丁醇相(FSB)和剩余水相(FSW)。FS、FSE、FSB和FSW经冷冻干燥制得固体样品，用于下一步抑菌实验。
4、FSE用硅胶柱进行进一步的分离，分别用氯仿∶甲醇＝100∶0、100∶1、100∶4、100∶8和100∶16进行洗脱得到FSE硅胶柱层析五个极性组分(分别记为FSE1、FSE2、FSE3、FSE4和FSE5)，不同极性组分经冷冻干燥制得固体样品，用于下一步抑菌实验。
实施例2：甜叶菊发酵液提取物抑菌活性测定
抑菌圈通过琼脂扩散-打孔法进行测定。将供试菌接种到灭菌的LB肉汤，在37℃培养箱以转速160r/m培养24h，调节菌悬液浓度为1.5×10 8CFU/mL。在 无菌平板中加入0.1mL上述菌液，再加入20mL灭菌融化的LB营养琼脂并与菌液混匀，室温静置至培养基凝固。凝固后用6mm打孔器打孔，每孔加样50μL，37℃培养24h，采用十字交叉法测量抑菌圈直径。
1：甜叶菊发酵液及有机溶剂萃取相对乳房炎芽孢杆菌的抑菌活性
研究发酵液及有机溶剂萃取相对乳房炎芽孢杆菌的抑菌活性。抑菌圈测定方法同上。FS、FSE、FSB和FSW用25、50和100mg/mL的浓度进行实验。除FS和FSW用无菌水溶解，其余样品均用二甲基亚砜溶解。以100μg/mL氯霉素(Chloramphenicol，Chl)为阳性对照。FS和FSW以水为溶剂对照，FSE和FSB以二甲基亚砜为溶剂对照。
方差能准确反映各组平行试验结果之间的离散程度，显示出试验结果的精确性。本试验以标准差为衡量基准，做出误差线，使得真实值能准确的反映在误差线范围之内。利用SPSS进行单因素方差和差异显著性分析。
通过实验发现抗菌剂对微生物的抑制作用可用抑菌圈反映，相同实验条件下，抑菌圈越大，抑菌作用越强。抑菌圈实验能在体外对不同来源的抗菌剂的抑菌活性进行比较，方便的对天然抗菌剂进行筛选。
表1.FS对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000001
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)
表1表明，FS在100mg/mL时对菌株都有抑制效果，Bacillus.sp.1和2为市面销售的常见乳房炎芽孢杆菌，对Bacillus.sp.1的抑制作用最强，抑菌圈达到15mm。在浓度为50mg/mL时，对Bacillus.sp.2有抑制效果。而在25mg/mL时对菌株均无抑制作用，作用效果呈现浓度依赖性。具有抑菌活性的植物粗提物使用剂量往往较大，但粗提物的制备相对容易，且不混入有机溶剂，能够降低成本和扩大使用范围。
2、FSE对乳房炎芽孢杆菌的抑菌活性
为了充分提取和利用甜叶菊发酵液中的活性组分，用乙酸乙酯对发酵液进 行萃取。与FS产生抑菌作用的浓度范围不同，FSE在25mg/mL～100mg/mL均显示出了抑菌活性，如表2所示。
表2.FSE对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000002
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)。
由表2可以看出，随着使用剂量增大，对Bacillus.sp.1和Bacillus.sp.2的抑制作用均增强。FSE较好的抑菌活性说明有较多的具有抗菌活性的天然物质溶解在乙酸乙酯中，因此，选择FSE进行进一步的分离。
3、FSB对乳房炎芽孢杆菌的抑菌活性
如表3所示，FSB对Bacillus.sp.1和Bacillus.sp.2的抑菌效果与FS类似，但只有浓度达到100mg/mL时才产生抑制作用。在产生抑制作用的所有菌株中，对Bacillus.sp.1的抑制作用最明显，抑菌圈达到10.50mm，FSB在其它两个浓度无抑制效果。实验结果说明溶解在正丁醇中的产物未能产生很强的抑菌活性。
表3.FSB对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000003
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)。
4、FSW对乳房炎芽孢杆菌的抑菌活性
如表4所示，FSW对Bacillus.sp.1和Bacillus.sp.2在三个浓度均未显示出抑菌活性。甜叶菊发酵液中的抗菌组分，经乙酸乙酯和正丁醇萃取后，在相应溶剂中得到进一步浓缩，使FSW中具有抑制作用的产物浓度低于FS，FSW抗菌组分含量较低而未能显示出抑制作用。
表4.FSW对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000004
Figure PCTCN2019108390-appb-000005
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)。
实施例3：FSE层析分离组分对乳房炎芽孢杆菌的抑菌活性
研究FSE层析分离组分对乳房炎芽孢杆菌的抑菌活性。抑菌圈测定方法同实施例2。FSE硅胶柱层析得到的五个极性组分用5、10和25mg/mL的浓度进行实验。样品用二甲基亚砜溶解。以100μg/mL氯霉素为阳性对照，二甲基亚砜为溶剂对照。
1、FSE1对乳房炎芽孢杆菌的抑菌活性
选用硅胶柱对FSE进一步分离，对五个极性组分产物选择5、10和25mg/mL三个浓度进行抑菌试验。实验结果如表5所示。
表5.FSE1对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000006
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)。
如表5所示，FSE1对乳房炎芽孢杆菌株Bacillus.sp.1和Bacillus.sp.2的抑制作用较弱，只在25mg/mL时才产生抑制效果。与未经硅胶柱层析处理的同浓度FSE相比，FSE1的抑制小于FSE，说明FSE中主要的抗菌成分未洗脱到FSE1中，产物中含有较多的色素等杂质，但FSE1的抑制作用强于FSB和FSW。
2、FSE2对乳房炎芽孢杆菌的抑菌活性
FSE2的抑菌活性如表6所示，FSE2对Bacillus.sp.1和Bacillus.sp.2只在10和25mg/mL有抑制作用。FSE2比FSE1产生抑制作用的浓度更低。随着洗脱剂极性的增大，洗脱液中抗菌组分含量增加，使抗菌活性逐渐增强。
表6.FSE2对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000007
Figure PCTCN2019108390-appb-000008
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)。
3、FSE3对乳房炎芽孢杆菌的抑菌活性
FSE3的抗菌活性与FSE2接近，如表7所示。FSE3对Bacillus.sp.1和Bacillus.sp.2在5mg/mL时不产生抑制作用，只在10和25mg/mL浓度下产生抑制效果。其中，在25mg/mL时对Bacillus.sp.1的抑菌圈达到12.75mm。
表7.FSE3对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000009
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)。
4、FSE4对乳房炎芽孢杆菌的抑菌活性
FSE4对乳房炎芽孢杆菌的抑菌活性实验结果如表8所示。
表8.FSE4对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000010
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)。
由表8可以看出，FSE4对Bacillus.sp.1和Bacillus.sp.2在三个浓度均产生了抑制作用，抑制效果随浓度增大而增强，其中，对Bacillus.sp.1的抑制作用最强，在5和25mg/mL时抑菌圈分别为10.5和15mm。
5、FSE5对乳房炎芽孢杆菌的抑菌活性
表9.FSE5对乳房炎芽孢杆菌的抑菌活性
Figure PCTCN2019108390-appb-000011
注：相同上标代表数值差异不显著，不同上标代表数值差异显著(P＜0.05)。
如表9所示，FSE5对Bacillus.sp.1和Bacillus.sp.2在三个浓度均有抑制效果，抑制作用随浓度增大而增强，抑制效果与FSE4接近。FSE5对Bacillus.sp.1的抑制作用最强，在5和25mg/mL浓度下的抑菌圈分别为10和14.25mm。
实施例4：甜叶菊发酵液提取物对乳房炎芽孢杆菌的MIC
以MIC判断甜叶菊发酵液提取物对乳房炎芽孢杆菌的抗菌活性。MIC测定方法同实施例3。FS选择浓度为62.50-8000.00μg/mL进行实验，FSB选择浓度为15.63-2000.00μg/mL进行实验，FSW用浓度为125.00-16000.00μg/mL进行实验，FSE及其硅胶柱层析分离组分用浓度为7.81-1000.00μg/mL进行实验。除FS和FSW用无菌水溶解，其余样品均用二甲基亚砜溶解。以未加菌液的LB肉汤作空白对照，加入氨苄青霉素(Ampicillin，Amp)的肉汤为阳性对照，FS和FSW选择加入无菌水的肉汤为溶剂对照，其余样品以加入二甲基亚砜的肉汤为溶剂对照。
表10.甜叶菊发酵液提取物对乳房炎芽孢杆菌的MIC
Figure PCTCN2019108390-appb-000012
Figure PCTCN2019108390-appb-000013
不同于抑菌圈的定性测量，抗菌剂的MIC是对抗菌活性的定量描述。MIC较小的抗菌剂，对微生物更加敏感，发挥抑制作用的浓度低，抑菌作用更强。结合抑菌圈和MIC值，能更好反映抗菌剂对微生物的抑制作用。
甜叶菊发酵液提取物对乳房炎芽孢杆菌的MIC如表10所示，FSE和FSE硅胶柱层析分离组分的MIC最低，在31.25-250μg/mL之间，FS和FSB虽然具有抗菌活性，但MIC远大于FSE及其硅胶柱层析分离组分。研究发现中药蒲公英对奶牛隐性乳房炎的杀菌作用最强，对金黄色葡萄球菌MIC和MBC分别为8mg/mL。胡少辉等发现中草药黄连和鱼腥草对奶牛隐形乳房炎致病菌金黄色葡萄球菌的最小抑菌浓度达到1.91mg/mL。与中草药相比，甜叶菊发酵液提取物中的FSE和FSE分离组分有更低的最小抑菌浓度。
实施例5、所述甜叶菊发酵提取物用于替代抗生素制备奶牛饲用添加剂
1、甜叶菊发酵物用于制备奶牛饲用添加剂
将实施例1步骤(2)制得的甜叶菊发酵液先用纱布过滤后，滤液在3000rpm/min转速下离心10min，澄清液进行冷冻干燥。甜叶菊发酵物对乳房炎芽孢杆菌的抑菌活性(抑菌圈，mm)分别为10.80±0.71和9.60±0.50，达氯霉素阳性对照抑菌圈(17.75±1.06)的60.8％和54.1％。甜叶菊发酵物对乳房炎芽孢杆菌的抑菌活性(抑菌圈，mm)测定浓度为100mg/mL，氯霉素为100ug/mL。
2、甜叶菊发酵液乙酸乙酯相用于制备奶牛饲用添加剂
将实施例1步骤(2)制得的甜叶菊发酵液先用纱布过滤后，滤液在3000rpm/min转速下离心10min，澄清液浓缩至发酵液的1/3，按体积1∶2比例添加乙酸乙酯进行萃取，重复三次后，将萃取液进行合并，旋转浓缩至干燥，残余的萃取剂通过冷冻干燥除掉，得到甜叶菊发酵液乙酸乙酯相。甜叶菊发酵液乙酸乙酯相对对乳房炎芽孢杆菌的抑菌活性(抑菌圈，mm)分别为14.50±0.71和14.00±0.00，达氯霉素阳性对照抑菌圈(17.75±1.06)的81.7％和78.9％。甜叶菊发酵液乙酸乙酯相对乳房炎芽孢杆菌的抑菌活性(抑菌圈，mm)测定浓度为100mg/mL，氯霉素为100ug/mL。
3、甜叶菊发酵物分离组分奶牛饲用添加剂的制备
将实施例1步骤(2)中得到的甜叶菊发酵乙酸乙酯萃取物，通过硅胶柱进一步分离，以氯仿∶甲醇＝100∶8比例进行洗脱后的洗脱液进行冷冻干燥，获得甜叶菊发酵物极性组分FSE4用于制备奶牛饲用添加剂。极性组分FSE4对乳房炎芽孢杆菌的抑菌活性(抑菌圈，mm)分别为15.00±0.11和14.50±0.00，达氯霉素阳性对照抑菌圈(17.25±0.35)的87.0％和84.1％。极性组分FSE4对乳房炎芽孢杆菌的抑菌活性(抑菌圈，mm)测定浓度为25mg/mL，氯霉素为100ug/mL。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。

Claims (10)

  1. 一种甜叶菊发酵液提取物，其特征在于所述甜叶菊发酵液提取物的制备方法包括如下步骤：
    (1)将甜叶菊干燥并粉碎后，将甜叶菊粉末与水混合，调节pH为6.5-7.5，水浴加热；冷却后，接入植物乳杆菌，培养2-5d得到甜叶菊发酵液；
    (2)将所述甜叶菊发酵液过滤，离心，用乙酸乙酯以体积比1∶1萃取发酵液得到发酵液乙酸乙酯相；
    (3)将所述发酵液乙酸乙酯相固体样品用硅胶柱进行进一步的分离，用氯仿和甲醇的混合液进行洗脱分别得到五个极性组分FSE1、FSE2、FSE3、FSE4和FSE5。
  2. 根据权利要求1所述的甜叶菊发酵液提取物，其特征在于：所述步骤(1)中甜叶菊粉末与水混合的质量体积比为1∶15-1∶25。
  3. 根据权利要求1所述的甜叶菊发酵液提取物，其特征在于：所述步骤(3)中混合液中氯仿和甲醇的体积比例分别为100∶0、100∶1、100∶4、100∶8和100∶16。
  4. 根据权利要求3所述的甜叶菊发酵液提取物，其特征在于：所述步骤(3)中混合液中氯仿和甲醇的比例为100∶8得到的极性组分活性最高。
  5. 权利要求1步骤(1)制得的所述甜叶菊发酵液在制备抑菌剂中的应用，其特征在于所述抑菌剂为抑制乳房炎芽孢杆菌的抑菌剂，所述甜叶菊发酵液的使用浓度为50mg/mL～100mg/mL。
  6. 权利要求1步骤(2)制得的所述发酵液乙酸乙酯相在制备抑菌剂中的应用，其特征在于所述抑菌剂为抑制乳房炎芽孢杆菌的抑菌剂，所述发酵液乙酸乙酯相的使用浓度为25mg/mL～100mg/mL。
  7. 权利要求1步骤(3)制得的所述极性组分FSE4在制备抑菌剂中的应用，其特征在于所述抑菌剂为抑制乳房炎芽孢杆菌的抑菌剂，所述极性组分FSE4的使用浓度为5mg/mL～100mg/mL。
  8. 权利要求1步骤(1)制得的所述甜叶菊发酵液在制备奶牛饲用添加剂中的应用。
  9. 权利要求1步骤(2)制得的所述发酵液乙酸乙酯相在制备奶牛饲用添加剂中的应用。
  10. 权利要求1步骤(3)制得的所述极性组分FSE4在制备奶牛饲用添加剂中的应用。
PCT/CN2019/108390 2018-11-16 2019-09-27 一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用 WO2020098396A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811372090.1 2018-11-16
CN201811372090.1A CN109498668A (zh) 2018-11-16 2018-11-16 一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用

Publications (1)

Publication Number Publication Date
WO2020098396A1 true WO2020098396A1 (zh) 2020-05-22

Family

ID=65748931

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/108390 WO2020098396A1 (zh) 2018-11-16 2019-09-27 一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用

Country Status (2)

Country Link
CN (1) CN109498668A (zh)
WO (1) WO2020098396A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109498668A (zh) * 2018-11-16 2019-03-22 青岛海思达生物科技有限公司 一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109349333A (zh) * 2018-11-16 2019-02-19 青岛海思达生物科技有限公司 一种甜叶菊发酵产物及其极性组分和在制备香肠防腐剂中的应用
CN109498668A (zh) * 2018-11-16 2019-03-22 青岛海思达生物科技有限公司 一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102283315A (zh) * 2011-08-18 2011-12-21 南昌大学 复合菌发酵高纤维农副产品生产动物益生菌饲料的方法
CN106265810B (zh) * 2016-08-29 2017-08-04 晨光生物科技集团股份有限公司 一种甜叶菊酚类提取物及其在制备抑菌制品中的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109349333A (zh) * 2018-11-16 2019-02-19 青岛海思达生物科技有限公司 一种甜叶菊发酵产物及其极性组分和在制备香肠防腐剂中的应用
CN109498668A (zh) * 2018-11-16 2019-03-22 青岛海思达生物科技有限公司 一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHAO LEI ET AL: "Antibacterial activity and stability of stevia rebaudiana waste extract", SCIENCE AND TECHNOLOGY OF FOOD INDUSTRY, vol. 37, no. 24, 31 December 2016 (2016-12-31), pages 168 - 172, XP009521123, ISSN: 1002-0306 *

Also Published As

Publication number Publication date
CN109498668A (zh) 2019-03-22

Similar Documents

Publication Publication Date Title
Saisithi et al. Microbiology and chemistry of fermented fish
CN112011481B (zh) 一株防治畜禽细菌性腹泻的罗伊氏乳杆菌及其应用
CN109259146B (zh) 鼠李糖乳杆菌在制备具有抑制脂肪肝病变功效的组合物中的应用
CN108186784B (zh) 蒲公英复合酵素及其制备方法和用途
CN106222158B (zh) 一种包埋乳酸杆菌的微胶囊制备方法
CN113881591B (zh) 一种产多糖的副干酪乳酸杆菌slp16及其应用以及利用该菌株制备得到的饲料添加剂
CN113755409B (zh) 一种缓解胰岛素抵抗的长双歧杆菌及其应用
CN115927045A (zh) 具有降胆固醇、缓解由高脂血症引起肝损伤功能的唾液乳杆菌069及其应用
CN113797232B (zh) 具有缓解胰岛素抵抗功能的组合物及其应用
WO2020098396A1 (zh) 一种甜叶菊发酵液提取物及其极性组分和在制备抑菌剂和添加剂中的应用
CN106674331B (zh) 抗菌脂肽bacaucin及其制备方法与应用
CN116747245A (zh) 动物双歧杆菌乳亚种bx-245在抑菌和/或生产功能活性物质中的应用
CN115505543B (zh) 一株乳双歧杆菌br001及其发酵方法与改善过敏的应用
RU2415604C2 (ru) Способ приготовления силоса
KR101862132B1 (ko) 홍국 추출물을 포함하는 장 건강 개선용 조성물
Odekina et al. Antimicrobial and antioxidant activities of novel marine bacteria (Bacillus 2011SOCCUF3) isolated from marine sponge (Spongia officinalis)
CN103239497B (zh) 一种恩诺沙星包合物及其制备方法
Oraibi et al. Investigation the antibacterial activity of Portulaca oleracea L. tissue cultures in vitro
KR20080078460A (ko) 신규한 락토바실러스 브츠네리 및 이의 용도
CN114621886A (zh) 一种枯草芽孢杆菌发酵产抗菌肽工艺及制备方法
CN113604391A (zh) 荔枝果肉黄酮类提取物在制备促乳杆菌增殖和抗致病菌黏附制剂方面的应用
KR20110049576A (ko) 미역 정유 추출물을 유효성분으로 함유하는 항균 조성물
CN117089505B (zh) 一种副干酪乳杆菌vb306及其应用
CN111567807A (zh) 抑制发炎反应和抗阴道炎的含鼠李糖乳杆菌组合物及应用
Krema et al. Evaluation of novel culture media prepared from plant substrates for isolation and identifi cation of Cryptococcus neoformans species complex

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19884502

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19884502

Country of ref document: EP

Kind code of ref document: A1