WO2022110727A1 - 副干酪乳杆菌et-22提升肠道细菌感染抗性和肠道免疫力的应用 - Google Patents

副干酪乳杆菌et-22提升肠道细菌感染抗性和肠道免疫力的应用 Download PDF

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WO2022110727A1
WO2022110727A1 PCT/CN2021/096959 CN2021096959W WO2022110727A1 WO 2022110727 A1 WO2022110727 A1 WO 2022110727A1 CN 2021096959 W CN2021096959 W CN 2021096959W WO 2022110727 A1 WO2022110727 A1 WO 2022110727A1
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lactobacillus paracasei
intestinal
cfu
cells
pathogenic bacteria
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French (fr)
Inventor
刘伟贤
怀斯·吉塞拉·阿德里安娜
凡·鲁-鲍曼·卡洛琳·安妮卡
施密特·盖瑞特
赵雯
洪维鍊
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Inner Mongolia Yili Industrial Group Co Ltd
Inner Mongolia Dairy Technology Research Institute Co Ltd
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Inner Mongolia Yili Industrial Group Co Ltd
Inner Mongolia Dairy Technology Research Institute Co Ltd
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Priority to AU2021388477A priority Critical patent/AU2021388477B2/en
Priority to US18/254,989 priority patent/US20240091283A1/en
Priority to EP21896237.1A priority patent/EP4252554A4/en
Publication of WO2022110727A1 publication Critical patent/WO2022110727A1/zh
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/165—Paracasei
    • 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
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of microorganisms, in particular to a new application of Lactobacillus paracasei ET-22 (deposit number CGMCC No. 15077) in improving intestinal bacterial infection resistance and intestinal immunity.
  • Intestinal epithelial cells are the body's first barrier against pathogenic bacteria.
  • the adhesion or invasion of pathogenic bacteria on epithelial cells will stimulate intestinal epithelial cells to produce inflammatory factors or chemokines.
  • Some soluble mediators can modulate the gut immune system or induce cellular innate immune responses. Appropriate immune responses and good epithelial cell barrier function will help protect the host from pathogenic infection.
  • Probiotics are a class of live microorganisms that, when ingested in sufficient amounts, can produce benefits to human health. As an important component of the gut microbiome, probiotics play an important role in human health, such as nutrient supply, vitamin synthesis, digestion, promotion of angiogenesis and enteric nerve function.
  • An object of the present invention is to provide a new use of Lactobacillus paracasei ET-22.
  • the invention provides a new application of a strain of Lactobacillus paracasei, which is named ET-22 in the present invention.
  • the strain has been preserved in the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee (CGMCC) on December 18, 2017 (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), classified and named : Lactobacillus paracasei; deposit number CGMCC No.15077.
  • CGMCC General Microbiology Center
  • CGMCC General Microbiology Center
  • CGMCC China Microbial Culture Collection Management Committee
  • Lactobacillus paracasei ET-22 was promoted by using Enterotoxigenic Escherichia coli ETEC H10407 and Enteropathogenic Escherichia coli EPEC O119 as pathogenic bacteria strains for inducing intestinal infection in vitro A functional study of intestinal immunity. Both types of E. coli are common pathogens, including infantile diarrhea, traveler's diarrhea, and in developing countries or areas with poor sanitation. ETEC H10407 is a relatively mature and commonly used model strain in experiments, and is often used to evaluate the adhesion ability and inflammatory signaling pathway of probiotics to pathogenic bacteria.
  • Lactobacillus paracasei ET-22 ie Lactobacillus paracasei with preservation number CGMCC No. 15077
  • Lactobacillus paracasei ET-22 ie Lactobacillus paracasei with preservation number CGMCC No. 15077
  • the adhesion of cells improves the integrity of the intestinal barrier, and has the effect of inhibiting the production of intestinal inflammatory factors, which can alleviate the inflammatory response caused by ETEC H10407.
  • the present invention provides the application of Lactobacillus paracasei in the preparation of a composition for improving intestinal immunity, the deposit number of said Lactobacillus paracasei is CGMCC No.15077.
  • the Lactobacillus paracasei is used to prepare the composition in the form of a solid or liquid bacterial preparation of living bacteria and/or dead bacteria.
  • the improving intestinal immunity includes: improving the resistance of intestinal bacterial infection, resisting the invasion of pathogenic bacteria in the intestinal system, maintaining the intestinal barrier function, and/ Or prevent diarrhea caused by pathogenic bacteria.
  • the improving intestinal immunity includes: reducing the adhesion ability of pathogenic bacteria to intestinal epithelial cells, and/or reducing the release of inflammation from intestinal cells caused by pathogenic bacteria Factors IL-8 and /IP-10.
  • the application amount of the Lactobacillus paracasei is 1.0 ⁇ 10 3 CFU to 1.0 ⁇ 10 12 CFU/day, or 0.001 ⁇ g to 1000 mg based on the weight of the cells /sky.
  • the application amount of the Lactobacillus paracasei is 10 7 CFU to 10 11 CFU/day, or 10 ⁇ g to 10 mg/day based on the weight of the cells.
  • the composition may comprise a food composition, a feed composition or a pharmaceutical composition.
  • the composition may be used in animals or humans.
  • the composition may also include ingredients conventional in the art.
  • an appropriate amount of adjuvants may be included, and the adjuvants may be excipients, diluents, fillers, absorption enhancers, and the like.
  • the Lactobacillus paracasei of the present invention can be produced according to the food containing Lactobacillus paracasei in the prior art, and the composition can adopt different forms according to the needs of the recipient. For example, powders, lozenges, granules, microcapsules, liquid preparations, and the like.
  • the composition is a food composition
  • the food can be a liquid beverage, a solid beverage, an oral liquid, a dairy product, a tablet or a capsule, such as a fermented dairy product (such as a fermented dairy product).
  • a fermented dairy product such as a fermented dairy product.
  • milk, flavored fermented milk, fermented milk beverages, etc. cheese, milk-containing beverages, probiotic solid beverages or milk powder, etc.
  • the composition is a feed composition.
  • Other components in the feed composition can be selected with reference to conventional techniques in the field of probiotic feed.
  • the composition is a pharmaceutical composition.
  • Other components in the pharmaceutical composition can be selected with reference to conventional techniques in the field of probiotic medicine.
  • the present invention also provides a method for enhancing intestinal immunity, the method comprising:
  • Lactobacillus paracasei is administered to the subject to enhance intestinal immunity; wherein, the deposit number of the Lactobacillus paracasei is CGMCC No.15077.
  • the present invention provides Lactobacillus paracasei ET-22 for enhancing intestinal immunity.
  • the improving intestinal immunity includes: improving the resistance of intestinal bacteria to infection, resisting the invasion of pathogenic bacteria in the intestinal system, maintaining the intestinal barrier function, preventing pathogenic bacteria Diarrhea caused by pathogens, reduce the adhesion of pathogenic bacteria to intestinal epithelial cells, and/or reduce the release of inflammatory factors IL-8 and/or IP-10 from intestinal cells caused by pathogens.
  • the application amount of the Lactobacillus paracasei is 1.0 ⁇ 10 3 CFU to 1.0 ⁇ 10 12 CFU/day, or 0.001 ⁇ g to 1000 mg based on the weight of the cells /sky;
  • the application amount of the Lactobacillus paracasei is 10 7 CFU to 10 11 CFU/day, or 10 ⁇ g to 100 mg/day based on the weight of the cells.
  • the present invention provides a new use of Lactobacillus paracasei ET-22, which has the functions of significantly reducing the adhesion of pathogenic bacteria to intestinal epithelial cells, improving intestinal barrier function, and activating intestinal immunity. , can be used to prepare food, medicine and feed with anti-infection, regulating intestinal health and immunity, and has a wide application prospect.
  • Fig. 1 shows the determination curve of the viable count of Lactobacillus paracasei ET-22.
  • Figure 2 shows the effect of Lactobacillus paracasei ET-22 on inhibiting the adhesion of pathogenic E. coli ETECH10407 to Caco-2 (p ⁇ 0.01).
  • Figure 3 shows the effect of Lactobacillus paracasei K56 on inhibiting the adhesion of pathogenic E. coli EPEC O119 to Caco-2 (p ⁇ 0.01).
  • Figures 4A and 4B show the effect of the combination of Lactobacillus paracasei ET-22 with a mixture of breast milk oligosaccharides on the intestinal barrier.
  • Figure 5 shows the changes of TEER ( ⁇ .cm 2 ) after co-culture of Lactobacillus paracasei ET-22 and ETEC H10407 for 1 h, 2 h, 3 h and 4 h.
  • Figure 6 shows the effect of Lactobacillus paracasei ET-22 on the expression of proinflammatory factor IL-8 in Caco-2 cells.
  • Figure 7 shows the effect of Lactobacillus paracasei ET-22 on the inflammatory factor IP10 of Caco-2 cells.
  • Lactobacillus paracasei ET-22 of the present invention Lactobacillus paracasei ET-22 of the present invention:
  • CGMCC General Microbiology Center of China Microorganism Culture Collection Management Committee
  • Classification name Lactobacillus paracasei.
  • Lactobacillus paracasei ET-22 used in the embodiment:
  • Lactobacillus paracasei ET-22 of the present invention has been stored in the General Microbiology Center of China Microorganism Culture Collection Management Committee CGMCC on December 18, 2017 (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Research Institute of Microbiology, Chinese Academy of Sciences Institute), classification name: Lactobacillus paracasei; deposit number CGMCC No.15077.
  • Morphological characteristics 1. When cultured in MRS medium, the cells are short-medium rods with rounded ends, usually in chains, and occasionally in pairs. 2. Gram-positive bacilli, do not produce spores, do not have catalase, oxidase and motility, can grow in both aerobic and anaerobic environments, the most suitable growth temperature is 37 ⁇ 1 °C, belonging to facultative heterogeneity Fermentative strain, no gas is produced when glucose is metabolized.
  • the fermentation conditions of the ET-22 strain were: MRS liquid medium: peptone, 10.0g; beef extract, 10.0g; yeast extract, 5.0g; glucose, 20.0g; dipotassium hydrogen phosphate, 5.0g; diammonium hydrogen citrate , 2.0g; sodium acetate, 5.0g; magnesium sulfate heptahydrate, 0.5g; manganese sulfate tetrahydrate, 0.2g; Tween 80, 1.0g; agar 15.0g; distilled water 1000mL. Adjust pH to between 6.2 and 6.4, and sterilize at 121°C for 15 minutes.
  • Lactobacillus paracasei ET-22 is a micro-aerobic bacterium, which grows better in facultative anaerobic environment, produces lactic acid, has acid resistance, can tolerate pH 2.5 acid environment and 0.4% bile salt environment for 4 hours.
  • Mesophilic bacteria the growth temperature ranges from 15 to 45 °C, and the optimum growth temperature is around 37 °C.
  • Lactobacillus paracasei ET-22 (Deposit No. CGMCC No. 15077), a single colony was picked out by the plate coating method, 16S identification, and frozen at -80°C in a glycerol tube.
  • the strains were cultured in MRS medium at 37°C. Grow to log phase centrifugation medium to enrich bacteria. Fluorescence activated cell sorting (FACS) was used to determine the number of viable bacteria and bacterial activity, and the results are shown in Figure 1. The strain entered the growth plateau at 8h, the number of viable bacteria tended to be stable, and the number of viable bacteria was stable in the range of 10 8 cfu/mL at 8h. The number of viable bacteria of the strains used in each example of the present invention was divided into 1 ⁇ 10 8 CFU/mL.
  • the co-culture method was used to explore whether Lactobacillus paracasei ET-22 had Caco-2 adhesion resistance to EPEC O119.
  • Caco-2 cells were seeded in 24-well plates. Caco-2 cells were washed with PBS buffer, and the strain and Caco-2 cells were co-cultured for 1 h.
  • EPEC O119 was added to Caco-2 cells at a multiplicity of infection (MOI) of 50:1 for co-culture for 1 h (37°C), and the final viable count was 1 ⁇ 10 7 CFU/mL.
  • MOI multiplicity of infection
  • the Caco-2 group without the addition of Lactobacillus paracasei ET-22 was used as a negative control.
  • E. coli As a positive control group, 1 mM zinc oxide (Zinc Oxide, ZnO) was used instead of Lactobacillus paracasei ET-22. After incubation, Caco-2 cells were washed and lysed, and then pathogenic bacteria were plated on agar. After overnight incubation on agar plates at 37°C, the number of bacterial CFU colonies was counted to measure pathogen adsorption. The number of growing E. coli colonies was counted and recorded as CFU/mL. In parallel with the anti-adherence test, E. coli (final concentration) 10 7 CFU/mL was added to 1 mL of the test substance (ET-22 bacterial broth) and co-cultured at 37° C. for 1 hour to measure the activity.
  • Zinc Oxide ZnO
  • E. coli was collected from each sample by centrifugation, resuspended in PBS, and plated on agar plates. After overnight incubation on agar plates at 37°C, the number of bacterial CFU colonies was counted to measure pathogen adsorption. The number of growing E. coli colonies was counted and recorded as CFU/mL.
  • the present invention confirms that Lactobacillus paracasei ET-22 has an inhibitory effect on enteropathogenic Escherichia coli EPEC O119.
  • the inhibitory ability of Lactobacillus paracasei ET-22 at a concentration of 10 8 CFU/mL on ETEC H10407 is shown in Figure 2.
  • the adhesion number of enterotoxic E. coli ETEC H10407 to Caco-2 cells was as high as 3.75 ⁇ 10 6 CFU/mL (6.57 log CFU/mL).
  • enterotoxic E. coli ETEC H10407 and Lactobacillus paracasei ET-22 act together, the number of adherent enteropathogenic E. coli ETEC H10407 is 7.67 ⁇ 10 5 CFU/mL (5.88log CFU/mL), and the inhibition rate is 7.67 ⁇ 10 5 CFU/mL (5.88log CFU/mL). 79.54%.
  • the experimental results showed that Lactobacillus paracasei ET-22 had a significant inhibitory effect on enteropathogenic E. coli ETEC H1040 (p ⁇ 0.01).
  • Lactobacillus paracasei ET-22 has inhibitory effects on both enterotoxic E. coli ETEC H10407 and enteropathogenic E. coli EPEC O119.
  • TEER transepidermal electrical resistance
  • Transepithelial electrical resistance was measured with EVOM2 epidermal voltmeter purchased from World Precision Instruments to measure barrier integrity.
  • Lactobacillus paracasei ET-22 was based on whether it could reduce the production of IL-8 and IP-10 induced by ETCT H10407Caco-2 cells.
  • Caco-2 cells were grown to appropriate abundance in 96-well plates. At the beginning of the experiment, cells were washed once with antibiotic-free medium. Monolayers of cells were co-cultured with test substances for 1 hour at 37°C in medium without antibiotics, repeated three times. Add E. coli to stimulate cells (MOI 200:1). After 1 hour incubation, monolayers were co-cultured with pathogenic bacteria, washed and incubated overnight with a medium containing the test substance and 50 ⁇ g/mL gentamicin.
  • FIG. 7 The experimental results of the effect of Lactobacillus paracasei ET-22 at a concentration of 10 8 CFU/mL on the inflammatory factor IL-8 in Caco-2 cells are shown in Figure 6 .
  • the experimental results of the influence of 2-cell inflammatory factor IP10 are shown in FIG. 7 .
  • the IL-8 content in the cell culture medium increased from 2.62 ⁇ 0.44 pg/mL to 473.84 ⁇ 21.9 pg/mL, and after adding ET-22, the IL-8 expression returned to 17.09 ⁇ 1.89pg/mL, indicating that Lactobacillus paracasei ET-22 has a significant inhibitory effect on the increase of IL-8 content induced by ETEC H10407 (p ⁇ 0.001).
  • Lactobacillus paracasei ET-22 could significantly enhance the adhesion of ETEC H1407 and EPEC O119 to Caco-2 cells, and relieve the inflammatory response caused by ETEC H10407. It indicated that Lactobacillus paracasei ET-22 has good potential for preventing infant diarrhea, traveler's diarrhea, intestinal inflammation, etc.

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Abstract

副干酪乳杆菌（Lactobacillus paracasei）在制备用于提升肠道免疫力的组合物中的应用，所述副干酪乳杆菌的保藏编号为CGMCC No. 15077。所述组合物包括食品组合物、饲料组合物或药品组合物。

Description

副干酪乳杆菌ET-22提升肠道细菌感染抗性和肠道免疫力的应用 技术领域
本发明涉及微生物技术领域，尤其是涉及一种副干酪乳杆菌(Lactobacillus paracasei)ET-22(保藏编号CGMCC No.15077)在提升肠道细菌感染抗性和肠道免疫能力的新应用。
背景技术
肠道上皮细胞是机体抵抗病原菌的第一道屏障。病原菌在上皮细胞上的黏附或侵入，将会刺激肠道上皮细胞产生炎症因子或趋化因子。一些可溶性的介质可以调节肠道免疫系统或者诱发细胞的先天免疫反应。适当的免疫反应及良好的上皮细胞屏障功能将有助于保护宿主免受病原菌的感染。
益生菌是指在摄入足量的数量时，可以对人体健康产生益处的一类活的微生物。益生菌作为肠道微生物组重要的组成成分，对人体健康发挥着不可忽视的重要作用，如营养供给，合成维生素，有助于消化、促进血管新生和肠神经功能。
发明内容
本发明的一个目的在于提供一种副干酪乳杆菌ET-22的新用途。
本发明提供了一株副干酪乳杆菌(Lactobacillus paracasei)的新应用，该菌株本发明中命名为ET-22。该菌株已于2017年12月18日保存于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)(地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，分类命名：副干酪乳杆菌(Lactobacillus paracasei)；保藏编号CGMCC No.15077。
在本发明中，以肠毒性大肠杆菌(Enterotoxigenic Escherichia coli)ETEC H10407和致肠病性大肠杆菌(Enteropathogenic Escherichia coli)EPEC O119作为诱发体外肠道感染的病原菌菌株进行了副干酪乳杆菌ET-22提升肠道免疫力的功能研究。两种类型的E.coli都是常见的致病菌，包含婴儿腹泻、旅行者腹泻、以及在发展中国家或者卫生较差的地区。ETEC H10407是较为成熟、实验常用的模式菌株，也经常用于评价益生菌对于致病菌的黏附能力和炎症信号通路。
本发明的研究发现，副干酪乳杆菌ET-22(即保藏编号为CGMCC No.15077的副干酪乳杆菌)菌株具有抑制肠道病原菌的黏附的作用，能够显著ETEC H1407和EPEC O119对Caco-2细胞的黏附作用，提高肠道屏障完整性，并且具有抑制肠道炎症因子产生的作用，能够缓解由于ETEC H10407引起的炎症反应。
从而，一方面，本发明提供了副干酪乳杆菌(Lactobacillus paracasei)在制备用于提升肠道免疫力的组合物中的应用，所述副干酪乳杆菌的保藏编号为CGMCC No.15077。
根据本发明的具体实施方案，本发明的应用中，所述副干酪乳杆菌以活菌和/或死菌的固态或液态菌制剂的形式用于制备所述组合物。
根据本发明的具体实施方案，本发明的应用中，所述提升肠道免疫力包括：提升肠道细菌感染抗性，在肠道系统中抵御致病菌侵入，维持肠道屏蔽功能，和/或预防致病菌引起的腹泻。
根据本发明的具体实施方案，本发明的应用中，所述提升肠道免疫力包括：降低致病菌对肠道上皮细胞的黏附能力，和/或降低致病菌引起的肠道细胞释放炎症因子IL-8和/IP-10。
根据本发明的具体实施方案，本发明的应用中，所述副干酪乳杆菌的应用量为1.0×10 3CFU～1.0×10 12CFU/天，或者以菌体的重量计为0.001μg～1000mg/天。优选地，所述副干酪乳杆菌的应用量为10 7CFU～10 11CFU/天，或者以菌体的重量计为10μg～10mg/天。
根据本发明的具体实施方案，所述组合物可以包括食品组合物、饲料组合物或药品组合物。
根据本发明的具体实施方案，所述组合物可使用于动物或是人类。所述组合物还可包括所属领域中的常规用料组分。例如，对于药物组合物，可包括适量的辅料，所述辅料可以为赋型剂、稀释剂、填充剂、吸收促进剂等。对于食品组合物，本发明的副干酪乳杆菌可以按照现有技术中含副干酪乳杆菌的食品进行生产，所述组合物可根据受施予者的需要，而采用不同形态。例如粉剂、锭剂、造粒、微胶囊、液体制剂等。
在本发明的一具体实施方案中，所述组合物为食品组合物，所述食品可以为液体饮料、固体饮料、口服液、奶制品、片剂或胶囊，例如可以为发酵乳制品(例如发酵乳、风味发酵乳、发酵乳饮料等)、乳酪、含乳饮料、益生菌固体饮料或乳粉等。
在本发明的另一具体实施方案中，所述组合物为饲料组合物。所述饲料组合物中的其他组分可以参照益生菌饲料领域的常规技术进行选择。
在本发明的另一具体实施方案中，所述组合物为药物组合物。所述药物组合物中的其他组分可以参照益生菌药物领域的常规技术进行选择。
另一方面，本发明还提供了一种提升肠道免疫力的方法，该方法包括：
给予受试者有效量的副干酪乳杆菌(Lactobacillus paracasei)，以提升肠道免疫力；其中，所述副干酪乳杆菌的保藏编号为CGMCC No.15077。
换而言之，本发明提供了用于提升肠道免疫力的副干酪乳杆菌(Lactobacillus paracasei)ET-22。
根据本发明的具体实施方案，本发明的方法中，所述提升肠道免疫力包括：提升肠道细菌感染抗性，在肠道系统中抵御致病菌侵入，维持肠道屏蔽功能，预防致病菌引起的腹泻，降低致病菌对肠道上皮细胞的黏附能力，和/或降低致病菌引起的肠道细胞释放炎症因子IL-8和/或IP-10。
根据本发明的具体实施方案，本发明的方法中，所述副干酪乳杆菌的应用量为1.0×10 3CFU～1.0×10 12CFU/天，或者以菌体的重量计为0.001μg～1000mg/天；
优选地，所述副干酪乳杆菌的应用量为10 7CFU～10 11CFU/天，或者以菌体的重量计为10μg～100mg/天。
综上所述，本发明提供了副干酪乳杆菌ET-22的新用途，该菌具有显著降低致病菌对肠道上皮细胞的黏附能力、提高肠道屏障功能、激活肠道免疫能力的作用，可以用于制备具有抗感染、调节肠道健康和免疫能力的食品、药物及饲料等，具有广泛的应用前景。
附图说明
图1显示了副干酪乳杆菌ET-22的活菌数测定曲线。
图2显示了副干酪乳杆菌ET-22抑制致病性大肠杆菌ETEC H10407对Caco-2的黏附效果(p<0.01)。
图3显示了副干酪乳杆菌K56抑制致病性大肠杆菌EPEC O119对Caco-2的黏附效果(p<0.01)。
图4A和图4B显示了副干酪乳杆菌ET-22与母乳低聚糖混合物的组合对肠道屏 障的影响。
图5显示了副干酪乳杆菌ET-22与ETEC H10407共培养1h、2h、3h、4h后TEER(Ω.cm 2)变化。
图6显示了副干酪乳杆菌ET-22对Caco-2细胞促炎症因子IL-8表达的影响。
图7显示了副干酪乳杆菌ET-22对Caco-2细胞炎症因子IP10的影响。
专利程序的微生物保存：
本发明的副干酪乳杆菌ET-22：
保藏日期：2017年12月18日；
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)；
保藏单位地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所
保藏编号：CGMCC No.15077；
分类命名：副干酪乳杆菌(Lactobacillus paracasei)。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例及对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例中所用副干酪乳杆菌ET-22：
本发明的副干酪乳杆菌ET-22已2017年12月18日保存于中国微生物菌种保藏管理委员会普通微生物中心CGMCC(地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，分类命名：副干酪乳杆菌(Lactobacillus paracasei)；保藏编号CGMCC No.15077。
根据16S rDNA序列分析以及API细菌鉴定系统分析结果来确认ET-22菌株在分类学上的特征。具体如下：
形态学特征：1.于MRS培养液培养时，菌体呈中短杆状，二端呈圆形，通常成链状，偶尔成对出现。2.革兰氏染色阳性杆菌，不生成孢子，不具触酶、氧化酶及运动性，在好氧及厌氧环境均能生长，最适宜的生长温度为37±1℃，属于兼性异质发酵性菌株，葡萄糖代谢时不产生气体。
ET-22菌株的发酵条件为：MRS液体培养基：蛋白胨，10.0g；牛肉膏，10.0g；酵母浸粉，5.0g；葡萄糖，20.0g；磷酸氢二钾，5.0g；柠檬酸氢二铵，2.0g；乙酸钠，5.0g；七水合硫酸镁，0.5g；四水合硫酸锰，0.2g；吐温80，1.0g；琼脂15.0g；蒸馏水1000mL。调整pH至6.2～6.4之间，121℃灭菌15分钟。
副干酪乳杆菌ET-22为微好氧菌，兼性厌氧环境生长较佳，产乳酸，具有耐酸性，可耐受pH值2.5的酸性环境及耐0.4％的胆盐环境4小时，嗜中温菌，生长温度范围在15～45℃，最适生长温度在37℃左右。
实施例中所用副干酪乳杆菌ET-22的活菌数测定：
副干酪乳杆菌ET-22(保藏编号CGMCC No.15077)，平板涂布法挑出单菌落，16S鉴定后于甘油管-80℃冷冻保藏。实验前菌株于MRS培养基，37℃培养。生长至对数期离心培养基富集菌体。使用荧光细胞分选法(Flourescence activated cell sorting，FACS)测定活菌数和菌活性，测定结果见图1。菌株在8h进入生长平台期，活菌数变化趋于稳定，在8h活菌数稳定在10 8cfu/mL范围。本发明各实施例用菌株活菌数分为1×10 8CFU/mL。
实施例中数据分析：
实验中的所有数据至少重复3次，实验结果表示为平均值±标准差；采用SPSS17.0软件进行数据分析(SPSS，Chicago，IL，USA)，显著性分析采用LSD的方法进行单因素方差分析，P-value<0.05被认为有统计学上的显著差异，显著性差异按照：*p<0.05、**p<0.01、***p<0.001、****p<0.0001区分。图的绘制采用GraphPad Prism 5.0。
实施例1.抗黏附能力测定
采用共培养的方法探究副干酪乳杆菌ET-22对EPEC O119是否具有Caco-2黏附抗性。Caco-2细胞接种于24孔板中培养。Caco-2细胞使用PBS缓冲液清洗，菌株和Caco-2细胞共同培养1h。EPEC O119以感染复数(multiplicity of infection,MOI)50：1添加至Caco-2细胞中共同培养1h(37℃)，最终活菌数为1×10 7CFU/mL。以无副干酪乳杆菌ET-22添加的Caco-2组作为阴性对照。使用1mM氧化锌(Zinc Oxide，ZnO)替代副干酪乳杆菌ET-22作为阳性对照组。孵育后，清洗并裂解Caco-2细胞，然后致病菌被接种在琼脂上。在37℃琼脂平板上培养过夜后，数细菌的CFU菌落数以衡量致病菌吸附。数生长中的大肠杆菌菌落数并记录为CFU/mL。与抗粘附测试平 行的，将大肠杆菌(终浓度为)10 7CFU/mL添加到1mL受试物(ET-22菌液)中，并在37℃共培养1小时以测量活性。培养后，从每个样品离心收集大肠杆菌，再悬浮于PBS中，并在琼脂平板上接种。在37℃琼脂平板上培养过夜后，数细菌的CFU菌落数以衡量致病菌吸附。数生长中的大肠杆菌菌落数并记录为CFU/mL。
通过上述以人体肠道细胞Caco-2为模型，本发明证实了副干酪乳杆菌ET-22对肠致病性大肠杆菌EPEC O119具有抑制作用。
在本发明的一些批次的实验中，10 8CFU/mL浓度的副干酪乳杆菌ET-22对ETEC H10407的抑制能力如图2所示，在无副干酪乳杆菌ET-22干预条件下，肠毒性大肠杆菌ETEC H10407对Caco-2细胞的黏附数高达3.75×10 6CFU/mL(6.57log CFU/mL)。而当肠毒性大肠杆菌ETEC H10407和副干酪乳杆菌ET-22共同作用时，黏附的肠致病性大肠杆菌ETEC H10407数目为7.67×10 5CFU/mL(5.88log CFU/mL)，抑制率为79.54％。实验结果表明，副干酪乳杆菌ET-22对肠致病性大肠杆菌ETEC H1040具有显著抑制作用(p<0.01)。
在本发明的一些批次的实验中，10 8CFU/mL浓度的副干酪乳杆菌ET-22对EPEC O119抑制能力的实验结果如图3所示。在没有副干酪乳杆菌ET-22的干预条件下，肠致病性大肠杆菌EPEC O119对Caco-2细胞的黏附数高达1.73×10 6CFU/mL(6.23log CFU/mL)。而当肠致病性大肠杆菌EPEC O119和副干酪乳杆菌ET-22共同作用时，黏附的肠致病性大肠杆菌EPEC O119数目为6.00×10 5CFU/mL(5.78log CFU/mL)，抑制率为65.32％。实验结果表明，副干酪乳杆菌ET-22对肠致病性大肠杆菌EPEC O119具有显著抑制作用(p<0.01)。
上述实验结果表明，副干酪乳杆菌ET-22对肠毒性大肠杆菌ETEC H10407和肠致病性大肠杆菌EPEC O119均具有抑制作用。
实施例2.肠道屏障完整性测试
理想的小肠上皮屏障功能是保护宿主免受致病菌侵入和/或致病菌来源毒素的先决条件。在此研究中，体外的屏障完整性通过测定肠道细胞层的跨表皮的电阻(TEER)体现。为了研究益生菌对于感染的作用，测定了TEER在大肠杆菌感染前和感染后随时间变化的情况。
Caco-2细胞以2×10 4cell/cm 2的浓度被接种到Transwell聚碳酸酯细胞培养插入物中，平均孔径为0.4um，面积为0.33cm 2直至完全分化(±1000Ω)。用世界精准仪 器购入的EVOM2表皮伏特测量器测量跨上皮细胞电阻(TEER)以衡量屏障完整性。
在测试当天，细胞被冲洗，并在37℃下用不含抗生素和血清、但含有受试物质的培养基中培养1小时。紧接着加入大肠杆菌到受试物质之上(感染倍数MOI为200:1)，并培养6小时。在实验开始前(t＝-1)，受试物质暴露1小时后及添加致病菌前(t＝0)，并在致病菌接触后1小时、2小时、3小时、4小时和6小时分别测定TEER。在与致病菌接触后单独条件下的TEER值与其各自在t＝0时的TEER值相关，且被表述成ΔTEER(Ω.Cm2)。阴性对照(只加入大肠杆菌)与未接触致病菌或受试物质的阳性对照也在实验组别中。所有条件重复测定三次，一些对照组被重复测定6次。
ETEC H10407(MOI＝50)攻毒时间达到6h时，空白对照组表现为不理想的TEER数值，表明当攻毒时间达到6小时后，细胞的细胞存活率显著下降，因此，初步确定ETECH10407攻毒时间应控制在4h内。
如图4A和图4B所示，在不同的时间点测试了受试物质对于跨膜电阻的影响，1×10 8CFU/mL的ET-22可在多个时间点，于一定程度上有提升跨膜电阻TEER值或防止其下降的趋势。图中MixA、MixB、MixC、MixD为母乳低聚糖混合物，当加入ET-22与这些母乳低聚糖的组合物后，跨膜电阻TEER值提升或防止下降的趋势更显著，比空白组效果有提升的趋势。
图5显示了本发明另一批次的实验中10 8CFU/mL浓度的副干酪乳杆菌ET-22与ETEC H10407共培养1h、2h、3h、4h后TEER(Ω.cm 2)变化。由图5可知，在添加ET-22后，跨膜电阻增加。处理4h后，ET-22组别跨膜电阻以时间依赖方式降低，在t＝2h、t＝3h时，与ETEC H10407组别相比，具有显著差异(p<0.01)。表明副干酪乳杆菌ET-22的干预缓解了由于ETEC H10407引起的肠道细胞屏障的完整性破坏。
实施例3.免疫调节能力测定
副干酪乳杆菌ET-22的免疫调节能力是基于是否能降低ETCT H10407Caco-2细胞诱发的IL-8、IP-10的产生。将Caco-2细胞在96孔板养到适合的丰度。在实验最初，用不含抗生素的培养基冲洗细胞一次。将单层细胞与受试物质在37℃下用不含抗生素的培养基中共培养1小时，重复三次。加入大肠杆菌刺激细胞(MOI 200:1)。在1小时培养后，单层细胞与致病菌共培养，并用含有受试物质和50μg/mL庆大霉素的培养液冲洗和培养过夜。作为空白对照(Blank)，只用了培养液而没用大肠杆 菌刺激。用大肠杆菌刺激但没用受试物质的培养液被用作大肠杆菌应答的对照。此外，作为Caco-2细胞应答的对照，用含有Rec TNFα(10ng/mL)以及Rec IFNγ(5ng/mL)(均从英国阿宾顿的R&D systems购入)的混合物培养液刺激细胞。刺激24小时候收集上清液并储存于-20℃。按照生产商的使用指导，用Bio-Plex试剂盒(美国加州BioRad公司)测试IL-8、IP-10。
10 8CFU/mL浓度的副干酪乳杆菌ET-22对Caco-2细胞炎症因子IL-8的影响的实验结果参见图6。10 8CFU/mL浓度的副干酪乳杆菌ET-22对Caco-2细胞炎症因子IP10的影响的实验结果参见图7。由图6可知，ETEC H10407胁迫后，细胞培养液中的IL-8含量从2.62±0.44pg/mL提升至473.84±21.9pg/mL,加入ET-22处理后，IL-8表达回复至17.09±1.89pg/mL，表明副干酪乳杆菌ET-22对ETEC H10407诱导的IL-8含量增加具有显著抑制作用(p<0.001)。由图7可知，ETEC H10407胁迫后后，细胞培养液中的IP10含量从5±0.00pg/mL提升至1454.08±193.31pg/mL,加入ET-22处理后，IP10表达回复至10.94±8.41pg/mL，表明副干酪乳杆菌ET-22对ETEC H10407诱导的IP10含量增加具有显著抑制作用(p<0.001)。实验结果表明副干酪乳杆菌ET-22具有抑制炎症因IL-8、IP10产生，显著抑制ETEC 10407引起的炎症反应(p<0.001),进一步说明ET-22具有一定程度降低ETEC 10407引起炎症反应的潜力。
以上研究结果证实：副干酪乳杆菌ET-22能够显著ETEC H1407和EPEC O119对Caco-2细胞的黏附作用，缓解由于ETEC H10407引起的炎症反应。表明副干酪乳杆菌ET-22对于预防婴儿腹泻、旅行者腹泻、肠道炎症等具有良好的潜力。

Claims (13)

  1. 一种副干酪乳杆菌(Lactobacillus paracasei)在制备用于提升肠道免疫力的组合物中的应用，所述副干酪乳杆菌的保藏编号为CGMCC No.15077。
  2. 根据权利要求1所述的应用，其中，所述副干酪乳杆菌以活菌和/或死菌的固态或液态菌制剂的形式用于制备所述组合物。
  3. 根据权利要求1所述的应用，其中，所述提升肠道免疫力包括：提升肠道细菌感染抗性，在肠道系统中抵御致病菌侵入，维持肠道屏蔽功能，和/或预防致病菌引起的腹泻。
  4. 根据权利要求1所述的应用，其中，所述提升肠道免疫力包括：降低致病菌对肠道上皮细胞的黏附能力，和/或降低致病菌引起的肠道细胞释放炎症因子IL-8和/或IP-10。
  5. 根据权利要求3或4所述的应用，其中，所述致病菌为大肠杆菌。
  6. 根据权利要求5所述的应用，其中，所述大肠杆菌包括肠毒性大肠杆菌ETEC H10407和/或致病性大肠杆菌EPEC O119。
  7. 根据权利要求3或4所述的应用，其中，所述副干酪乳杆菌的应用量为1.0×10 3CFU～1.0×10 12CFU/天，或者以菌体的重量计为0.001μg～1000mg/天。
  8. 根据权利要求7所述的应用，其中，所述副干酪乳杆菌的应用量为10 7CFU～10 11CFU/天，或者以菌体的重量计为10μg～100mg/天。
  9. 根据权利要求1所述的应用，其中，所述组合物包括食品组合物、饲料组合物或药品组合物。
  10. 根据权利要求9所述的应用，其中，所述食品为液体饮料、固体饮料、口服液、奶制品、片剂或胶囊。
  11. 一种提升肠道免疫力的方法，该方法包括：
    给予受试者有效量的副干酪乳杆菌(Lactobacillus paracasei)，以提升肠道免疫力；其中，所述副干酪乳杆菌的保藏编号为CGMCC No.15077。
  12. 根据权利要求11所述的方法，其中，所述提升肠道免疫力包括：提升肠道细菌感染抗性，在肠道系统中抵御致病菌侵入，维持肠道屏蔽功能，预防致病菌引起的腹泻，降低致病菌对肠道上皮细胞的黏附能力，和/或降低致病菌引起的肠道细胞释放炎症因子IL-8和/或IP-10。
  13. 根据权利要求11或12所述的方法，其中，所述副干酪乳杆菌的应用量为1.0×10 3CFU～1.0×10 12CFU/天，或者以菌体的重量计为0.001μg～1000mg/天；
    优选地，所述副干酪乳杆菌的应用量为10 7CFU～10 11CFU/天，或者以菌体的重量计为10μg～100mg/天。
PCT/CN2021/096959 2019-11-29 2021-05-28 副干酪乳杆菌et-22提升肠道细菌感染抗性和肠道免疫力的应用 Ceased WO2022110727A1 (zh)

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