WO2021147162A1 - 一株母乳来源罗伊乳杆菌调节母婴免疫功能的应用 - Google Patents

一株母乳来源罗伊乳杆菌调节母婴免疫功能的应用 Download PDF

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WO2021147162A1
WO2021147162A1 PCT/CN2020/080353 CN2020080353W WO2021147162A1 WO 2021147162 A1 WO2021147162 A1 WO 2021147162A1 CN 2020080353 W CN2020080353 W CN 2020080353W WO 2021147162 A1 WO2021147162 A1 WO 2021147162A1
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intestinal
infants
lactobacillus reuteri
application according
women
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WO2021147162A9 (zh
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孙进
李双祁
齐策
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湖南菲勒生物技术有限公司
孙进
广州菲勒生物科技有限公司
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Publication of WO2021147162A1 publication Critical patent/WO2021147162A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • 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
    • 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
    • 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
    • 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
    • A23V2400/173—Reuteri

Definitions

  • the present invention relates to the application of a strain of Lactobacillus reuteri from breast milk to regulate the immune function of mothers and infants, belonging to the field of microbial technology and food science, and in particular to a Lactobacillus reuteri Fn041 combined with breast milk sIgA in promoting the gastrointestinal function of infants and young children.
  • the initiation and maturation of the human immune system are closely related to intestinal microbes.
  • the bacteria colonized by the intestinal tract of infants and young children play an important role in this process.
  • Breast milk is the main source of the intestinal flora of healthy newborn full-term infants.
  • bifidobacteria and lactobacilli are the sub-dominant microorganisms.
  • These bacteria can be continuously inoculated into the intestines of infants and young children through breastfeeding to promote the formation of normal intestinal flora.
  • the normal intestinal flora of infants and young children affects the intestinal immunity and protection of infants and young children through a variety of mechanisms.
  • pathogenic microorganisms which can produce broad-spectrum antibacterial substances, compete with pathogenic bacteria for fermentation substrates, or compete for adhesion to intestinal wall mucin and other binding sites, thereby inhibiting pathogenic microorganisms in the intestines of infants and young children. Colonization and growth in the tract protect infants and young children from infection and promote the maturity of the immune system. Second, the intestinal flora of infants and young children stimulates the neonatal gastrointestinal tract to initiate an immune response, promote the development of the immune system, and reduce inflammation. Due to the effects of microbes from breast milk, the incidence of infectious diseases in breastfed infants is significantly lower than that of infants and young children fed with formula milk.
  • infants and young children After infants and young children ingest these bacteria, they can bind to the sIgA in the intestinal tract, mediate their entry into the small intestine PP of infants and young children, and penetrate the development of immune cells in the PP, especially the maturation of IgA-producing plasma cells.
  • the combination of sIgA and bacteria can promote the colonization of these bacteria in the mucosa, act on epithelial cells at a closer distance, and regulate the expression of tight junction protein and antibacterial peptides in intestinal epithelial cells.
  • the sIgA-bound Lactobacillus or Bifidobacterium can be separated from breast milk, and the breast-feeding mother or infant can induce the development of the infant's immune system after ingesting such bacteria.
  • IgA in breast milk affects the composition and maturity of the intestinal flora of breast-fed infants. Infants fed mainly with formula milk lack the effect of IgA, and the growth of intestinal flora is too fast.
  • IgA in breast milk mainly encapsulates specific breast milk bacteria, thereby affecting the development of intestinal bacteria in breast-fed infants.
  • the IgA-bound lactic acid bacteria in breast milk is a typical member of healthy term infants, a candidate strain of probiotics suitable for infants and young children, and can be used to promote the intestinal immune development of infants and young children.
  • pregnancy diseases such as eclampsia and gestational diabetes also cause breast milk flora to be different from healthy mothers, and migration to infants and young children may affect the health of offspring.
  • probiotic supplements to infants and young children to promote the healthy development of intestinal flora.
  • the mother's intake of sIgA-binding beneficial bacteria isolated from breast milk can also enhance immunity.
  • these bacteria can migrate to breast milk and be ingested by infants and young children through breastfeeding.
  • the technical problem to be solved by the present invention is to promote the development of intestinal immunity in the form of probiotic supplements for infants and young children who cannot take breast milk or whose breast milk flora is disordered, and women who need to enhance immunity during pregnancy and lactation. , To ensure the intestinal barrier and protective function.
  • the present invention provides an application of Lactobacillus reuteri Fn041 in the preparation of products for enhancing the immunity of the target population and establishing or strengthening the intestinal barrier;
  • the target population is: pregnancy Women, lactating women and infants;
  • the Lactobacillus reuteri Fn041 is Lactobacillus reuteri with the deposit number GDMCC No. 60546, which has been disclosed in the patent application document with the publication number CN110205261A.
  • the product includes, but is not limited to, food, medicine or health care products.
  • the application is to prepare a product with any function (1) to (5):
  • the establishment or strengthening of the intestinal barrier includes, but is not limited to: resisting the invasion of pathogenic bacteria or pathogenic bacteria; the pathogenic bacteria include Salmonella.
  • the age of the infant is 0-36 months.
  • Lactobacillus reuteri Fn041 is provided in food or medicine at a daily dose of 1 ⁇ 10 4 to 1 ⁇ 10 11 , preferably 1 ⁇ 10 7 to 1 ⁇ 10 11 cfu for pregnancy. Or breastfeeding women, or infants.
  • the Lactobacillus reuteri is added to the composition at 1 ⁇ 10 4 to 1 ⁇ 10 12 cfu/g dry matter for administration to pregnant or lactating women, or infants.
  • the Lactobacillus reuteri in the product is live cells, inactivated cells, or in the form of fermentation products or metabolites, or a mixture of any of these states.
  • the product is a food or medicine
  • its dosage form includes, but is not limited to: tablets, capsules, solid powders or oral liquids.
  • the product also contains prebiotics, including but not limited to: inulin, fructooligosaccharides, short-chain fructooligosaccharides, galacto-oligosaccharides, human milk oligosaccharides or cow milk oligosaccharides.
  • prebiotics including but not limited to: inulin, fructooligosaccharides, short-chain fructooligosaccharides, galacto-oligosaccharides, human milk oligosaccharides or cow milk oligosaccharides.
  • Lactobacillus reuteri Fn041 in the added food or medicine contains but is not limited to one or more of the following ingredients: prebiotics, euglena powder or euglena extract, Tangerine peel powder or tangerine peel extract, bilberry powder or bilberry extract, wolfberry powder or wolfberry extract, hemp seed powder, hemp seed protein, milk protein, animal and plant hydrolyzed protein or peptide.
  • the prebiotics include, but are not limited to: inulin, fructooligosaccharides, short-chain fructooligosaccharides, galactooligosaccharides, human milk oligosaccharides or cow milk oligosaccharides, or a combination of the foregoing multiple prebiotics.
  • the present invention provides a new use of Lactobacillus reuteri Fn041 in enhancing the immunity of infants and mothers, that is, enhancing the mucosal barrier, promoting the production of intestinal antibacterial peptides and IgA, and promoting the development of the immune system of infants and young children.
  • Lactobacillus reuteri Fn041 can promote the intestinal immune development of newborn mice, and achieve the following effects:
  • TLR4 gene promotes the expression of antimicrobial peptides in intestinal epithelial cells, the expression of TLR4 gene is inhibited by about 0.5 times, and the expression of TLR-9 gene is activated by 0.5 to 1 times respectively;
  • IgA intestinal immunoglobulin A pathway
  • Lactobacillus reuteri Fn041 can enhance the immunity of mother mice during pregnancy or lactation.
  • the specific manifestations are as follows:
  • Figure 1 shows the effect of Lactobacillus reuteri Fn041 treatment on intestinal villus height (A), crypt depth (B), chorionic gland ratio (C) and mucosal morphology (D) of young mice; from left to right are control, Inf, Lacm, CoT, among them, Inf, baby mice gavage Lactobacillus; Lacm, lactating mother mice gavage Lactobacillus; CoT lactating mothers and pups both gavage Lactobacillus; **, compared with the control group is significantly different (p ⁇ 0.01); *, the difference is significant compared with the control (p ⁇ 0.05);
  • Figure 2 shows the effect of Lactobacillus reuteri Fn041 treatment on the number of IgA plasma cells in the intestinal mucosa of young mice; A, the average number of IgA + cells per villi; B, the optical density per unit area; C, the typical immunohistochemical staining image; where, Figure A From left to right in Figure B and B are the control, Inf, Lacm, CoT; Inf, gavage Lactobacillus in young mice; Lacm, gavage Lactobacillus in lactating mothers; CoT, lactating mothers and young mice, both gavage Lactobacillus; *, the difference is significant compared with the control (p ⁇ 0.05);
  • Figure 3 shows the effect of Lactobacillus reuteri Fn041 treatment on the expression of genes in the intestinal antimicrobial peptide pathway of young mice; Inf, young mice gavage Lactobacillus; Lacm, lactating mothers gavage Lactobacillus; CoT lactating mothers and pups both gavage milk Bacillus; **, significant difference compared with control (p ⁇ 0.01); *, significant difference compared with control (p ⁇ 0.05);
  • Figure 4 shows the effect of Lactobacillus reuteri Fn041 treatment on the bactericidal activity of small intestinal crypts of young mice; Inf, young mice gavage Lactobacillus; Lacm, lactating mothers gavage Lactobacillus; CoT lactating mothers and young mice both gavage Lactobacillus; **, the difference is significant compared with the control (p ⁇ 0.01); *, the difference is significant compared with the control (p ⁇ 0.05);
  • Figure 5 shows the effect of Lactobacillus reuteri Fn041 treatment on the gene expression of IgA + cell production pathway in the intestine of young mice; Inf, young mice gavage Lactobacillus; Lacm, lactating mothers gavage Lactobacillus; CoT lactating mothers and The young mice are all gavaged with Lactobacillus; **, the difference is significant compared with the control (p ⁇ 0.01); *, the difference is significant compared with the control (p ⁇ 0.05);
  • Figure 6 The effect of Lactobacillus reuteri Fn041 treatment on the expression of genes related to the intestinal mucosal barrier of young mice; Inf, young mice gavage Lactobacillus; Lacm, lactating mothers gavage Lactobacillus; CoT lactating mothers and pups both gavage Lactobacillus ; **, the difference is significant compared with the control (p ⁇ 0.01); *, the difference is significant compared with the control (p ⁇ 0.05);
  • Figure 7 shows the effect of Lactobacillus reuteri Fn041 treatment on the serum IgA, IgE, IgG2a and intestinal sIgA antibody concentrations of adult female mice; control: control group; LR: Lactobacillus reuteri Fn041 treatment group; *the two groups have significant differences after T test, p ⁇ 0.05;
  • Figure 8 shows the effect of Lactobacillus reuteri Fn041 treatment on the content of IgA plasma cells in the intestinal villi intestinal of adult female mice; CON: control group; LR: Lactobacillus reuteri Fn041 treatment group. *The two groups were significantly different after T test, p ⁇ 0.05;
  • Figure 9 shows the effect of Lactobacillus reuteri Fn041 treatment on the morphology of the small intestinal villi of adult female mice; CON: control group; LR: Lactobacillus reuteri Fn041 treatment group; the two groups are significantly different after T test, **p ⁇ 0.01; ***p ⁇ 0.001 ;
  • Figure 10 shows the effect of Lactobacillus reuteri Fn041 treatment on the gene expression of Peyer’s junction IgA+ cell production pathway in adult female mice; control: control group; LR: Lactobacillus reuteri Fn041 treatment group; significant difference between the two groups after T test, *p ⁇ 0.05; ***p ⁇ 0.01;
  • Figure 11 shows the effect of Lactobacillus reuteri Fn041 treatment on the expression of genes related to the intestinal mucosal barrier of adult female mice; control: control group; LR: Lactobacillus reuteri Fn041 treatment group; significant difference after T test, **p ⁇ 0.01;
  • Figure 12 shows the effect of Lactobacillus reuteri Fn041 treatment on the expression of antibacterial peptide pathway genes in adult female mice; control: control group; LR: Lactobacillus reuteri Fn041 treatment group; antibacterial peptides have significant differences after T test, *p ⁇ 0.05;*** p ⁇ 0.01.
  • the data analysis method used in the following examples are all expressed by (mean value ⁇ standard deviation).
  • ANOVA one-way analysis of variance
  • Tukey test for comparison between groups.
  • the homogeneity of variance is less than 0.05
  • Use Kruskal-Wallis test to analyze the data. It is significant The level is P ⁇ 0.05, the extremely significant level is P ⁇ 0.01, and the very significant level is P ⁇ 0.001.
  • CON another group of Lactobacillus reuteri FN041 (1 ⁇ 10 10 CFU/mouse ⁇ d) was given to the rats.
  • mice were grouped into: 1) Con group, both mother and pups were given normal saline; 2) Inf group, mothers were given normal saline, and pups were given Lactobacillus reuteri FN041 (1 ⁇ 10 9 CFU/mouse ⁇ d); 3) Lacm group, mother rats were given Lactobacillus reuteri FN041 (1 ⁇ 10 10 CFU/mouse ⁇ d), pups were given normal saline; 4) Co-T group, mother rats and The pups were gavaged with Lactobacillus reuteri FN041, the gavage dose of Lactobacillus reuteri FN041 in mother mice was 10 10 cfu/mouse ⁇ d, and the gavage dose of young rats was 10 9 cfu/mouse ⁇ d.
  • Sectioning Use a Leica microtome to slice 5 ⁇ m thick jejunum tissue slices, spread the slices in a water bath at 42°C, and bake slices at 70°C for 30 minutes.
  • Antigen retrieval Take 200 mL of EDTA buffer at pH 9.0, add it to the staining box, place the deparaffinized and hydrated tissue section in the staining box on the plastic slice rack, heat it in the pressure cooker to full pressure, and then continue heating 5min, turn off the power, take out the staining box after 10min, cool at room temperature for 30min, soak in PBS buffer for 3min ⁇ 3 times.
  • Dehydration and transparency Treat each with 75% ethanol, 85% ethanol, 95% ethanol for 3 minutes, then 100% ethanol for 5 minutes, repeat three times, then soak in xylene for 5 minutes, and then soak in xylene for 3 minutes .
  • Inverted microscope eyepiece 10 times, objective lens 20 times, observe and take pictures, use Image-Pro Plus software for imaging analysis, calculate the average optical density density (mean), density (mean) cumulative optical density IOD/area and microscope statistical average of each villi The number of plasma cells contained on it.
  • the ratio of villi height to crypt depth can comprehensively reflect the digestion and absorption function of the small intestine.
  • the depth of intestinal crypts represents the rate of cell formation. The shallower the crypts, the more mature the cell maturity and the more mature the secretory function.
  • Figure 1 shows that ingestion of Lactobacillus reuteri significantly increased the ratio of villus height and crypt depth (p ⁇ 0.01), and the ratio of villus height and crypt depth (p ⁇ 0.05) can also be increased by the administration of lactating female mice. Lactobacillus ileus promotes the intestinal absorption function and the maturation of crypt cells, and gavage combined with infant mice with breast milk can significantly increase the content of villus IgA plasma cells (Figure 2) (p ⁇ 0.05).
  • Example 3 Gene expression and antibacterial activity of antimicrobial peptides in small intestinal epithelial cells of young mice:
  • the jejunum and small intestine Peyer's knot RNA extraction and cDNA preparation Take out the liver and spleen of the mouse in the ultra-clean table, cut out 0.1g jejunum and quickly put it into Trizol. The Trizol method was used to extract RNA from the jejunum and Peyer's knot. NanoDrop measures OD260/280 and concentration, adjusts RNA concentration to 1,000ng/ ⁇ L, and OD 260/280 is in the range of 1.8-2.0. Perform the reverse transcription of RNA according to the reaction system in Table 1.
  • Jejunal Reverse Transcription Fluorescence Quantitative PCR Reaction Quantitative PCR analysis is performed with jejunal reverse transcription cDNA as the amplification template.
  • the reaction system is: 2 ⁇ SYBR Rreen Master Mix 5 ⁇ L; upstream and upstream primers (10 ⁇ M, sequence See Table 2) 0.4 ⁇ L each; 0.3 ⁇ L cDNA; 3.7 ⁇ L DEPC water.
  • the amplification conditions were 95°C, 5min; 95°C, 20s; 60°C, 30s; 72°C, 1min, 40 cycles, 72°C, 2min, using ⁇ -actin as the internal control, and using the 2- ⁇ Ct method to analyze the data analyze.
  • Preparation of small intestinal crypts and evaluation of antibacterial activity of antibacterial peptides Wash a section of mouse small intestinal lumen with pre-cooled sterile water. The intestinal segment was turned over and shaken in Ca++ and Mg ++- free PBS buffer containing 30 mM EDTA to elute the crypts. The villi and crypts were eluted several times at 5 min intervals, centrifuged to pellet (700 g), and resuspended in PBS buffer. Use a capillary pipette to transfer individual crypts to siliconized microcentrifuge tubes.
  • the antimicrobial peptides expressed by Paneth cells at the base of the small intestinal crypts are important defensive effector molecules in the intestinal tract, and are secreted into the intestinal mucus layer to resist the invasion of pathogenic bacteria and potentially pathogenic commensal bacteria.
  • the peptidoglycan, endotoxin, bacterial DNA released by bacterial cells bind to Paneth cell toll-like receptors (TLR2, TLR4, TLR9, etc.), and the signal transduction mediated by the transprotein MyD88 can make the cytoplasmic Nf- ⁇ B subunit
  • TLR2, TLR4, TLR9, etc. Paneth cell toll-like receptors
  • the protein is released, enters the cell and induces a variety of antimicrobial expression.
  • FIG 3 shows that the expression of TLR4 gene in the small intestine of the three groups of mice was significantly inhibited by about 0.5 times, and the expression of TLR-9 gene was activated by 0.5 to 1 times (p ⁇ 0.05).
  • Direct gavage of pups also induced the expression of TLR2 Significantly increased, increased by 1 times compared with the control (p ⁇ 0.05).
  • the three treatments all inhibited the expression of Myd88 (p ⁇ 0.05), and significantly induced the expression of the three antimicrobial peptides ⁇ -defense peptide, CRS1C and CRS4C.
  • Direct gavage of the pups also significantly induced the expression of reg3g and CRS1C.
  • the expression of Reg3g and a-defensin even increased 2 times and 1.5 times respectively (p ⁇ 0.05).
  • Figure 4 shows that the three methods of treatment of small intestinal crypt cells of mice also significantly inhibited Salmonella. Among them, only treating 1000 crypt secretions of young mice can reach a 50% lethality rate in 5 minutes. Treating only the female mice or simultaneously The killing rate of Salmonella can be reached 50% in 10 minutes in female rats and mice, and the antibacterial effect is significantly higher than that of the control.
  • IgA is the most important active molecule of the small intestinal immune system. It is mainly released into the mucus layer in the form of secreted IgA (sIgA) to limit the excessive proliferation and migration of pathogenic bacteria.
  • the small intestine Peyer's knot is the initial induction site of IgA plasma cells.
  • the bacteria are taken up by the M cells of Peyer’s junction and passed to the dendritic cells (DC), which activates the DC, makes it through TLR and follicular dendritic cells (FDC), induces the FDC to produce TGF ⁇ 1 and CLCL13, and promotes Foxp3-T
  • DC dendritic cells
  • FDC follicular dendritic cells
  • Tfh follicular helper T
  • CXCR5 follicular helper T
  • This cell migrates to the lamina intestinal immune function and can also be converted into IgA + cells under the action of APPIL.
  • RNA extraction and the real-time PCR reaction (RT-PCR) reaction were carried out.
  • the results are shown in Figure 5.
  • Both treatment of pups or mothers alone and treatment of pups and mothers at the same time can significantly induce the expression of Peyer’s knot CXCR5, CXCL13, APPIL, TGF- ⁇ and Foxp3 (P ⁇ 0.01).
  • the amount of increase has reached about 1 times. It indicated that the treatment of Lactobacillus reuteri caused Foxp3 + T to chemoattract the intestinal tract and participate in the production of IgA + plasma cells.
  • Example 5 Expression of genes related to the small intestinal mucosal barrier of young rats corresponding to different treatments
  • mice were gavaged with 4kDa fluorescein isothiocyanate-dextran (FD4, 125mg/mL) (600mg/kg body weight). After 4 hours, the eyeballs were removed and blood was collected. The treatment needs to be protected from light. The blood was incubated at room temperature (RT) for at least 1 hour to clot, and centrifuged at high speed for 10 minutes to separate the serum. The serum aliquot was diluted 1:1 with PBS in duplicate, and the fluorescence readings at 488/530nm were analyzed. The concentration of FD4 in the serum sample was determined according to the value of the standard curve of serial dilution of FD4 in PBS. The control mouse sera of all serum samples FD4 were normalized.
  • FD4 fluorescein isothiocyanate-dextran
  • the three treatments all reduced the FD4 concentration entering the blood, indicating that the mucosal permeability of the pups increased.
  • the FD4 concentration can be reduced by at least 20 when only the pups are treated and the pups/mothers are treated at the same time. %.
  • the first line of defense for infants’ intestines against commensal bacteria and pathogenic bacteria is the intestinal mucosal barrier, which includes biochemical and immune components; the physical barrier is mainly composed of intestinal epithelial cells through tight junctions, chemical barriers It is mainly composed of mucus that covers the surface of the intestine. Tight junction is a complex composed of intracellular occluding egg (ZO1), Claudin, and intercellular occludin (Occludin). When the mucosal barrier becomes weak, the expression of Claudin-2 is down-regulated, while the expression of ZO1 and Occludin genes is up-regulated. Mucus is mainly composed of mucin 2 expressed by epithelial cells, and its enhanced expression is conducive to the maintenance of the mucus barrier.
  • ZO1 intracellular occluding egg
  • Occludin intercellular occludin
  • Figure 6 shows that the intake of Lactobacillus reuteri Fn041 by young rats can significantly up-regulate the expression of ZO-2 and ZO-1 mRNA and mucin-2, and the increase can reach 0.5 to 3 times. Ingestion of female mice can significantly inhibit Claudin-2 and induce mucin 2mRNA, indicating that different ways of intake of Lactobacillus reuteri Fn041 can improve the mucosal barrier.
  • Example 6 The effect of different treatments on the immune function, intestinal morphology and mucosal barrier of the small intestine of mother mice
  • the small intestine tissue was processed according to the method of Example 1, and the content of sIgA in the small intestine content of the mother mouse was determined: strictly in accordance with the sIgA ELISA kit instructions.
  • Figure 9 shows that the small intestinal villus height and chorionic gland ratio are increased by more than 60% after breast milk is treated with Lactobacillus reuteri, and the expression of PIgR, mucin-2 and Occludin genes are increased by 0.5 times, 4 times and 1.4 times, respectively.
  • Lactobacillus treatment The FITC-F000 entering the blood was reduced from 3.2 ⁇ g/mL to 2.1 ⁇ g/mL ( Figure 11), indicating that the structure of the small intestine and the integrity of the mucosa were improved.
  • the mouse crypts were isolated and cultured according to the method of Example 4. It was found that the ability of Lactobacillus to treat the mouse crypts to kill Salmonella was significantly higher than that of the control (p ⁇ 0.01, Figure 12).
  • Lactobacillus reuteri Fn041 also significantly enhanced the mother
  • the expression of multiple genes related to the antimicrobial peptide pathway in mucosal mucosa increased the expression intensity of a-defesin, MyD88, RegIII-gamma, CRS1C and TLR2 by 3.5 times, 0.25 times, 0.3 times, 3.1 times and 2.2 times, respectively.
  • TLR2, TLR4, and TLR9 recognize bacterial peptidoglycan/lipoteichoic acid, lipopolysaccharide, and CpG unmethylated DNA, respectively.
  • TRL2 expression is significantly up-regulated, indicating that Lactobacillus reuteri is mainly through cellular peptidoglycan /Lipoteichoic acid stimulates the expression of antimicrobial peptides.
  • Lactobacillus reuteri Fn041 was inoculated into MRS medium, cultivated at 35 ⁇ 37°C until the bacteria concentration was ⁇ 1 ⁇ 10 3 CFU/mL, and the bacteria were collected.
  • a protective agent is added to the bacterial cells to prepare bacterial powder.
  • the protective agent may be selected from: monosaccharides, oligosaccharides, polysaccharides, polyols or mixtures thereof, such as trehalose, sorbitol, mannitol and the like.
  • the Lactobacillus reuteri Fn041 prepared by any of the foregoing methods is calculated based on the unit mass (g) or monomer volume (mL), according to 1 ⁇ 10 4 to 1 ⁇ 10 11 , preferably 1 ⁇ 10 7 to 1 ⁇ 10 11 cfu / The amount per unit mass or unit volume is added to the food.
  • the food also contains prebiotics, euglena powder or euglena extract, tangerine peel powder or tangerine peel extract, bilberry powder or bilberry extract, wolfberry powder or wolfberry extract, hemp seed powder, fire Hemp kernel protein, milk protein, animal and plant hydrolyzed protein or peptide.
  • prebiotics euglena powder or euglena extract, tangerine peel powder or tangerine peel extract, bilberry powder or bilberry extract, wolfberry powder or wolfberry extract, hemp seed powder, fire Hemp kernel protein, milk protein, animal and plant hydrolyzed protein or peptide.
  • prebiotics include, but are not limited to: inulin, fructooligosaccharides, short-chain fructooligosaccharides, galactooligosaccharides, human milk oligosaccharides or milk oligosaccharides, or a combination of the foregoing multiple prebiotics.

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Abstract

一株母乳来源罗伊乳杆菌调节母婴免疫功能的应用，罗伊乳杆菌(Lactobacillus reuteri)Fn041增强妊娠期或哺乳期女性以及婴幼儿的免疫力，即增强粘膜屏障、促进肠道抗菌肽产生和IgA产生，具有促进婴幼儿免疫系统的发育，预防病原菌感染，降低过敏疾病发生率等效果。

Description

一株母乳来源罗伊乳杆菌调节母婴免疫功能的应用 技术领域
本发明涉及一株母乳来源罗伊乳杆菌调节母婴免疫功能的应用，属于微生物技术和食品科学领域，具体涉及一种母乳sIgA结合的罗伊乳杆菌(Lactobacillus reuteri)Fn041在促进婴幼儿胃肠道免疫发育和增强成人免疫力中的应用。
背景技术
人体免疫系统的启动和发育成熟与肠道微生物密切相关，婴幼儿早期肠道定植的细菌在此过程发挥重要作用。母乳是健康初生足月儿肠道菌群的主要来源，其中双歧杆菌和乳杆菌是次优势微生物，这些细菌可通过哺乳不断接种到婴幼儿肠道，促进正常肠道菌群形成。正常婴幼儿肠道菌群通过多种机制影响婴幼儿肠道免疫和保护。首先，他们构成了抵御病原微生物的第一道防线，可产生广谱抗菌物质、与病原菌竞争性发酵底物，或竞争粘附肠壁粘蛋白等结合位点，从而抑制病原微生物在婴幼儿肠道内定植生长，保护婴幼儿抗感染和促进免疫系统功能的成熟。第二，婴幼儿肠道菌群刺激新生儿胃肠道启动免疫反应、促进免疫系统发育，降低炎症反应。由于来自母乳微生物的作用，母乳喂养婴幼儿感染性疾病的发生率明显低于配方奶粉喂养的婴幼儿。
母乳中的部分活性细菌是由母亲肠道的特定细菌通过生理异位迁移到乳腺的。母亲小肠存在丰富的sIgA，可以选择性地结合特定细菌。这些和细菌结合的sIgA可被小肠派伊尔结(PP)表面的M细胞识别和摄取，并被转移到PP内的树突细胞(DC)而由其携带进入乳腺。婴幼儿摄入这些细菌后，又可和肠道的sIgA结合，介导进入婴幼儿小肠PP，刺进PP内免疫细胞的发育，尤其是产IgA浆细胞的成熟。sIgA和细菌结合后可促进这些细菌在黏膜定植，以更近的距离作用于上皮细胞，调节肠道上皮细胞紧密连接蛋白和抗菌肽的表达。从母乳可分离sIgA结合的乳杆菌或双歧杆菌，哺乳母亲或婴幼儿摄取这类细菌后可诱导婴幼儿免疫系统发育。有研究已经证明，母乳中母体来源的IgA影响着母乳喂养婴幼儿肠道菌群组成以及成熟度，以配方奶粉喂养为主的婴幼儿缺乏IgA的作用，肠道菌群的发育速度过快。母乳中IgA主要包裹特定的母乳细菌，从而影响母乳喂养婴幼儿肠道细菌的发育。母乳中IgA结合的乳酸菌是典型的健康足月儿有益菌成员，是适用于婴幼儿的益生菌候选菌株，可用于促进婴幼儿肠道免疫发育。
世界卫生组织、美国儿科学会、中国营养学会等都鼓励母亲延长母乳喂养的时间，至少坚持到1周岁或更久，而且6月龄内最好坚持纯母乳喂养。有些母亲母乳不足或者由于某些 原因造成母乳不适合哺乳，不得已选择婴幼儿配方奶粉作为母乳的补充品。造成婴幼儿无法获得来自母乳的细菌。我国不同地区母乳细菌构成存在显著地域差异，有些地区母乳的有益菌含量明显低于其他地区。同时，子痫和妊娠糖尿病等孕期疾病也造成母乳菌群异于健康母亲，迁移给婴幼儿可能影响子代健康。在这些情况有必要给婴幼儿给予益生菌补充剂，以促进肠道菌群健康发育。母亲摄取从母乳中分离的sIgA结合性有益菌也可增强免疫力，同时这些细菌可迁移到母乳，通过哺乳被婴幼儿摄取。
发明内容
本发明要解决的技术问题是对于无法摄取母乳或母乳菌群紊乱的婴幼儿，以及需增强免疫力的妊娠期与哺乳期的女性，通过益生菌补充剂的形式，以促进肠道免疫的发育，保障肠道屏障和保护功能。
为解决上述问题，本发明提供了一种罗伊乳杆菌(Lactobacillus reuteri)Fn041在制备用于增强目标人群免疫力、建立或强化肠道屏障的产品中的应用；所述目标人群为：妊娠期女性、哺乳期女性以及婴幼儿；所述罗伊乳杆菌(Lactobacillus reuteri)Fn041为保藏号GDMCC No.60546的罗伊氏乳杆菌，已公开于公开号为CN110205261A的专利申请文件中。
在一种实施方式中，所述产品包括但不限于食品、药品或保健品。
在一种实施方式中，所述应用是制备具有(1)～(5)任一功能的产品：
(1)提高妊娠期或哺乳期女性，以及婴幼儿肠道抗菌肽的表达和分泌量；
(2)促进妊娠期或哺乳期女性，以及婴幼儿肠道产免疫球蛋白A以及肠道IgA浆细胞数量的提高；
(3)增强妊娠期或哺乳期女性，以及婴幼儿肠道粘膜的屏障功能；
(4)促进婴幼儿免疫系统的发育；
(5)增强妊娠期或哺乳期女性的免疫力；
(6)增加哺乳期女性母乳中Lactobacillus reuteri的数量。
在一种实施方式中，所述建立或强化肠道屏障包括但不限于：抵御病原菌或致病菌的入侵；所述致病菌包括沙门氏菌。
在一种实施方式中，所述婴幼儿年龄为0-36个月。
在一种实施方式中，罗伊乳杆菌(Lactobacillus reuteri)Fn041在食品或药品中以1×10 4～1×10 11，优选1×10 7～1×10 11cfu的日剂量提供给妊娠期或哺乳期女性，或婴幼儿。
在一种实施方式中，所述罗伊氏乳杆菌以1×10 4至1×10 12cfu/g干物质添加到组合物施用给妊娠期或哺乳期女性，或婴幼儿。
在一种实施方式中，所述产品中的罗伊氏乳杆菌是活细胞、失活的细胞，或以发酵产物或代谢物的形式，或任何这些状态的混合物。
在一种实施方式中，所述产品为食品或药品，其剂型包括但不限于：片剂、胶囊剂、固体粉剂或口服液。
在一种实施方式中，所述产品中还含有益生元，包括但不限于：菊粉、低聚果糖、短链低聚果糖、低聚半乳糖、人乳低聚糖或牛乳低聚糖中的一种或多种。
在一种实施方式中，罗伊乳杆菌(Lactobacillus reuteri)Fn041在添加的食品或药品中，含有但不限于以下成分中的一种或多种：益生元、裸藻粉或裸藻提取物、陈皮粉或陈皮提取物、越橘粉或越橘提取物、枸杞粉或枸杞提取物、火麻仁粉、火麻仁蛋白、牛奶蛋白、动植物水解蛋白或肽。所述益生元包括但不限于：菊粉、低聚果糖、短链低聚果糖、低聚半乳糖、人乳低聚糖或牛乳低聚糖或上述多种益生元的组合物。
有益效果：本发明提供了Lactobacillus reuteri Fn041在增强婴幼儿及母体的免疫力，即增强粘膜屏障、促进肠道抗菌肽产生和IgA产生，以及促进婴幼儿免疫系统发育方面的新用途。
针对幼体，经动物实验表明，Lactobacillus reuteri Fn041可促进初生幼鼠肠道免疫发育，达到如下效果：
1)促进肠道上皮细胞表达抗菌肽，TLR4基因的表达受到约0.5倍的抑制，TLR-9基因表达分别被激活了0.5～1倍；
2)促进肠道产免疫球蛋白A(IgA)通路的基因表达和IgA浆细胞形成，使CXCR5、CXCL13、APPIL、TGF-β和Foxp3的表达量提高的幅度达到1倍左右；
3)增强肠道粘膜屏障功能相关基因表达，可使ZO-2和ZO-1mRNA和粘液素-2的表达提高0.5～3倍；
4)提高小肠绒毛/隐窝深度比例，增强致病菌抵抗能力，在5min就可以达到50％的沙门氏菌致死率。
针对母体，经动物实验表明，Lactobacillus reuteri Fn041可增强妊娠期或哺乳期母鼠的免疫力，具体体现为：
1)增加血清和肠道抗体浓度，血清IgA由11.97ng/mL提高至12.10ng/mL，IgE由11.55ng/mL提高至11.73ng/mL；
2)促进粘膜抗体产生细胞数量，使IgA浆细胞数量增加约1倍；
3)促进肠道粘膜屏障和抗菌肽相关基因表达，分别使PIgR、mucin-2和Occludin基因表 达强度分别提高0.5倍、4倍和1.4倍；
4)改善小肠绒毛形态，增强致病菌抵抗能力，在10min既可达到沙门氏菌50％的杀死率。
附图说明
图1为Lactobacillus reuteri Fn041处理对幼鼠肠道绒毛高度(A)、隐窝深度(B)、绒腺比(C)和粘膜形态(D)的影响；自左向右依次为对照、Inf、Lacm、CoT，其中，Inf，幼鼠灌胃乳杆菌；Lacm，哺乳母鼠灌胃乳杆菌；CoT哺乳母鼠和幼鼠都灌胃乳杆菌；**,与对照组比较差异显著(p<0.01)；*,与对照比较差异显著(p<0.05)；
图2为Lactobacillus reuteri Fn041处理对幼鼠肠道粘膜IgA浆细胞数量的影响；A,单位绒毛平均IgA +细胞数量；B，单位面积光密度；C，典型免疫组化染色图；其中，A图和B图中从左向右依次为对照、Inf、Lacm、CoT；Inf，幼鼠灌胃乳杆菌；Lacm，哺乳母鼠灌胃乳杆菌；CoT哺乳母鼠和幼鼠都灌胃乳杆菌；*,与对照比较差异显著(p<0.05)；
图3为Lactobacillus reuteri Fn041处理对幼鼠肠道抗菌肽通路基因表达的影响；Inf，幼鼠灌胃乳杆菌；Lacm，哺乳母鼠灌胃乳杆菌；CoT哺乳母鼠和幼鼠都灌胃乳杆菌；**,与对照比较差异显著(p<0.01)；*,与对照比较差异显著(p<0.05)；
图4为Lactobacillus reuteri Fn041处理对幼鼠小肠隐窝杀菌活力的影响；Inf，幼鼠灌胃乳杆菌；Lacm，哺乳母鼠灌胃乳杆菌；CoT哺乳母鼠和幼鼠都灌胃乳杆菌；**,与对照比较差异显著(p<0.01)；*,与对照比较差异显著(p<0.05)；
图5为Lactobacillus reuteri Fn041处理对幼鼠肠道派伊尔结IgA +细胞产生途径基因表达的影响；Inf，幼鼠灌胃乳杆菌；Lacm，哺乳母鼠灌胃乳杆菌；CoT哺乳母鼠和幼鼠都灌胃乳杆菌；**,与对照比较差异显著(p<0.01)；*,与对照比较差异显著(p<0.05)；
图6 Lactobacillus reuteri Fn041处理对幼鼠肠道粘膜屏障相关基因表达的影响；Inf，幼鼠灌胃乳杆菌；Lacm，哺乳母鼠灌胃乳杆菌；CoT哺乳母鼠和幼鼠都灌胃乳杆菌；**,与对照比较差异显著(p<0.01)；*,与对照比较差异显著(p<0.05)；
图7为Lactobacillus reuteri Fn041处理对成年母鼠血清IgA、IgE、IgG2a和肠道sIgA抗体浓度的影响；对照：对照组；LR：Lactobacillus reuteri Fn041处理组；*两组经过T检验差异显著，p<0.05；
图8为Lactobacillus reuteri Fn041处理对成年母鼠小肠绒毛固有层IgA浆细胞含量的影响；CON：对照组；LR：Lactobacillus reuteri Fn041处理组。*两组经过T检验差异显著，p<0.05；
图9为Lactobacillus reuteri Fn041处理对成年母鼠小肠绒毛形态的影响；CON：对照组； LR：Lactobacillus reuteri Fn041处理组；两组经过T检验差异显著，**p<0.01；***p<0.001；
图10为Lactobacillus reuteri Fn041处理对成年母鼠肠道派伊尔结IgA+细胞产生途径基因表达的影响；对照：对照组；LR：Lactobacillus reuteri Fn041处理组；两组经过T检验差异显著，*p<0.05；***p<0.01；
图11为Lactobacillus reuteriFn041处理对成年母鼠肠道粘膜屏障相关基因表达的影响；对照：对照组；LR：Lactobacillus reuteri Fn041处理组；经过T检验差异显著，**p<0.01；
图12为Lactobacillus reuteri Fn041处理对成年母鼠肠道抗菌肽通路基因表达的影响；对照：对照组；LR：Lactobacillus reuteri Fn041处理组；抗菌肽经过T检验差异显著，*p<0.05；***p<0.01。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
下述实施例中采用的数据分析方法：实验结果均以(平均值±标准偏差)表示。采用SPSS25.0对数据进行统计分析，单因素方差分析(ANOVA)，Tukey检验进行组间比较，当方差齐性小于0.05时采用非参数独立双样本t检验，用Kruskal-Wallis检验分析数据，显著水平为P<0.05，极显著水平为P<0.01，十分显著水平为P<0.001。
实施例1：动物实验
1)实验方法
动物分组：将40只BALB/C雌鼠，14只BALB/C雄鼠，昼夜周期保持在8:00-20:00，室温22-24℃，湿度40-70％，喂食普通饲料。雌雄分开单独适应一周，一周后按照雌:雄＝3:1合笼，待雌鼠怀孕后，将雄鼠拿出，雌鼠分娩后，将哺乳雌鼠分为两组，一组灌胃生理盐水(CON)，另外一组灌胃罗伊乳杆菌FN041(1×10 10CFU/只·d)，至幼鼠达到3周龄时，自然断奶，将每组母乳哺乳过得雄性幼鼠分别分为两组，分别灌胃生理盐水或罗伊乳杆菌。14天后处死所处理的幼鼠。以上所述小鼠分组为：1)Con组，母鼠和幼鼠都灌胃生理盐水；2)Inf组，母鼠灌胃生理盐水，幼鼠灌胃罗伊乳杆菌FN041(1×10 9CFU/只·d)；3)Lacm组，母鼠灌胃罗伊乳杆菌FN041(1×10 10CFU/只·d)，幼鼠灌胃生理盐水；4)Co-T组，母鼠和幼鼠都灌胃罗伊乳杆菌FN041，母鼠的罗伊乳杆菌灌胃剂量为10 10cfu/只·d，幼鼠的灌胃剂量为10 9cfu/只·d。
实施例2 小肠IgA +浆细胞免疫组化分析
(1)脱水：将小肠组织分别放入在50％乙醇；75％乙醇；85％乙醇；90％乙醇震荡脱水，每次15min，然后放入100％乙醇处理30min，每15min时更换一次乙醇。
(2)透明：将乙醇和正丁醇按1:1配制混合液，将小肠组织放入混合液中震荡20min； 倒掉液体，转移至正丁醇溶液中处理40min，每20min更换一次正丁醇。
(3)浸蜡：在70℃石蜡Ⅰ中浸没3h；再转移至70℃石蜡Ⅱ中浸没3h。
(4)包埋：将步骤(3)处理后的小肠组织包埋在石蜡中。
(5)切片：使用徕卡切片机切5μm厚的空肠组织切片，42℃水浴摊片，70℃烤片30min。
(6)脱蜡、水化：在二甲苯中浸泡三次，每次5min；在100％乙醇浸泡两次，每次5min；分别依次在95％乙醇、85％乙醇、75％乙醇各浸泡3min，然后用去离子水浸洗5min。
(7)抗原修复：取200mL pH 9.0的EDTA缓冲液，加入染色盒中，将脱蜡水化后的组织切片置于染色盒内塑料切片架，置于高压锅内加热至饱压，然后继续加热5min，关闭电源，10min后取出染色盒，室温冷却30min，PBS缓冲液浸3min×3次。
(8)每张切片加100μL 3％H 2O 2，室温下孵育10min，PBS浸洗3min×3次。
(9)除去PBS缓冲液，加100μL 2.5％山羊血清，室温孵育30min，除去封闭液。
(10)每张切片加100μL稀释的兔抗鼠IgA单克隆抗体(一抗)，25℃孵育1h，PBS浸洗3min×3次。
(11)除去PBS，加100μL羊抗兔IgG单克隆抗体(二抗)，25℃孵育30min，PBS浸洗3min×3次。
(12)除去PBS，加100μL现配DAB显色液，镜下显色5min，自来水终止反应。
(13)苏木素复染12s，自来水冲洗。
(14)1％盐酸酒精分化1s，自来水稍洗，自来水返蓝5min。
(15)脱水、透明：分别用75％乙醇、85％乙醇、95％乙醇处理各3min，再用100％乙醇处理5min，重复三次，再于二甲苯中浸泡5min，再于二甲苯中浸泡3min。
(16)中性树胶封片，60℃烘箱内烘20min。
倒置显微镜目镜10倍，物镜20倍，观察拍照，用Image-Pro Plus软件进行成像分析，计算平均光密度density(mean)，density(mean)＝累计光密度IOD/area及显微镜统计平均每条绒毛上含有的浆细胞个数。
绒毛高度与隐窝深度的比值可综合反映小肠消化吸收功能状况，绒毛越高则肠道表面积越大，肠道的吸收功能越强。肠隐窝深度代表细胞的生成率，隐窝越浅细胞成熟度越完善，分泌功能越成熟。图1显示，幼鼠摄取罗伊乳杆菌显著提高了绒毛高度和隐窝深度比(p<0.01)，通过哺乳母鼠给予也可提高绒毛高度和隐窝深度比(p<0.05)，说明罗伊乳杆菌促进了肠道吸收功能和隐窝细胞的成熟，通过母乳给予结合幼鼠灌胃可显著提高绒毛IgA浆细胞含量(图2)(p<0.05)。
实施例3 幼鼠小肠上皮细胞抗微生物肽基因表达和抑菌活性：
空肠及小肠派伊尔结RNA提取及cDNA制备：在超净台中取出小鼠的肝脏、脾脏，剪取0.1g空肠迅速放入Trizol中。采用Trizol法提取空肠和派伊尔结RNA。NanoDrop测定OD260/280和浓度，调节RNA浓度至1,000ng/μL，OD 260/280在1.8-2.0范围内。按表1的反应体系进行RNA的反转录，第一步反应体系混匀后70℃，5min，冰浴；随后加入第二步体系混匀后，37℃水浴1h，95℃，3min，冰浴，反转录后的cDNA于-80℃保存。
空肠逆转录荧光定量PCR反应(RT-PCR)：以空肠反转录后的cDNA作为扩增模板进行定量PCR分析，反应体系为：2×SYBR Rreen Master Mix 5μL；上、游引物(10μM，序列见表2)各0.4μL；cDNA0.3μL；DEPC水3.7μL。扩增条件为95℃，5min；95℃，20s；60℃，30s；72℃，1min，40个循环，72℃，2min，以β-actin作为内参，采用2-△△Ct法对数据进行分析。
表1反转录反应体系
Figure PCTCN2020080353-appb-000001
表2实时荧光定量RT-PCR引物
Figure PCTCN2020080353-appb-000002
Figure PCTCN2020080353-appb-000003
小肠隐窝制备和抗菌肽抑菌活性评价：用预冷无菌水冲洗一段小鼠小肠肠腔。将肠段外翻，并在含有30mM EDTA的不含Ca ++和Mg ++的PBS缓冲液中振摇，以洗脱隐窝。以5min间隔多次洗脱绒毛和隐窝，离心沉淀(700g)，并重悬于PBS缓冲液中。用毛细管移液器将单个隐窝转移到硅化微量离心管中。用血细胞计数法估计约1000个隐窝，重悬于2ml iPIPES缓冲液中(10mM PIPES+137mM NaCl，pH 7.4)，按1000CFU/隐窝加入沙门氏菌，在37℃孵育30分钟。离心沉淀隐窝，取10μl上清液，涂布于营养琼脂平板上，并过夜生长后确定菌落数。分别测定1、2.5、5、10、15、30min的杀菌率。
小肠隐窝基底部潘氏细胞表达的抗微生物肽是肠道重要的防御效应分子，被分泌到肠道黏液层中作为抵御病原菌和潜在致病的共生菌的入侵。细菌细胞释放的肽聚糖、内毒素、细菌DNA和潘氏细胞的toll样受体(TLR2、TLR4、TLR9等)结合，通过接转蛋白MyD88介导的信号传导可使细胞浆Nf-κB亚蛋白释放，进入细胞和诱导多种抗微生物表达。图3显示，三组小鼠小肠TLR4基因的表达被显著抑制了约0.5倍，TLR-9基因表达分别得到了0.5～1倍激活(p<0.05)，直接灌胃幼鼠也诱导TLR2表达的显著提高，与对照相比提高1倍(p<0.05)。同时，三种处理都抑制了Myd88表达(p<0.05)，并显著诱导了α-防御肽、CRS1C和CRS4C三种抗微生物肽的表达，直接灌胃幼鼠也显著诱导了reg3g和CRS1C的表达，使Reg3g和a-defensin的表达甚至分别提高了2倍和1.5倍(p<0.05)。
图4显示，三种方式处理小鼠的小肠隐窝细胞也显著抑制了沙门氏菌，其中仅处理幼鼠的1000个隐窝分泌物在5min就可以达到50％致死率，仅处理母鼠或同时处理母鼠和小鼠时在10min既可达到沙门氏菌50％的杀死率，且抑菌作用都显著高于对照。
实施例4 幼鼠小肠派伊尔结IgA基因表达：
IgA是小肠免疫系统最重要的活性分子，主要以分泌型IgA(sIgA)形式被释放到粘液层， 限制病原菌过度增殖和迁移。小肠派伊尔结是IgA浆细胞初始诱导部位。细菌被派伊尔结的M细胞摄取后传递给树突细胞(DC)，从而激活DC，使其通过TLR和滤泡树突细胞(FDC)作用，诱导FDC产生TGFβ1和CLCL13，促使Foxp3-T细胞转化为滤泡辅助T(Tfh)细胞，后者表达CXCR5在TGFβ参与下使初始型IgM +B细胞表达的抗体发生类别转换，分化为IgA +B细胞，这种细胞迁移到固有层后转变为IgA浆细胞，可产生并分泌IgA。固有层的IgM +细胞在实现肠道免疫功能成熟，在APPIL作用下也可转换为IgA +细胞。
按照实施例3的方法进行RNA的提取及荧光定量PCR反应(RT-PCR)反应。结果如图5所示，单独处理幼鼠或母鼠和同时处理幼鼠及母鼠，都可显著诱导派伊尔结CXCR5、CXCL13、APPIL、TGF-β和Foxp3表达(P<0.01)，表达量提高的幅度都达到了1倍左右。说明罗伊乳杆菌处理引起Foxp3 +T下表向肠道趋化，并参与IgA +浆细胞的产生。
实施例5 不同处理方式对应的幼鼠小肠粘膜屏障相关基因表达情况
按照实施例1的分组进行动物实验，给小鼠灌胃4kDa异硫氰酸荧光素-右旋糖酐(FD4，125mg/mL)(600mg/kg体重)，4小时后，摘眼球采血，采血后的所有处理需要避光。将血液在室温(RT)下孵育至少1小时以凝血，高速离心10分钟分离血清。将血清等分试样用PBS 1:1稀释，一式两份，分析488/530nm处的荧光读数。血清样品中FD4的浓度根据PBS连续稀释FD4标准曲线值确定。将所有血清样品FD4的对照小鼠血清标准化。
如图6显示，三种处理方式都使进入血液的FD4浓度减小，说明幼鼠粘膜通透性提高，其中，仅处理幼鼠和同时处理幼鼠/母鼠时可将FD4浓度降低至少20％。
婴幼儿肠道抵抗共生细菌和致病菌的第一道防线就是肠粘膜屏障，该物理屏障包括生物化学和免疫成分；其中物理屏障主要由肠上皮细胞通过紧密连接(tight junctions)构成，化学屏障主要由覆盖于肠道表面的粘液构成。紧密连接是细胞内闭合带蛋(ZO1)、Claudin，和细胞间的闭合蛋白(Occludin)构成的复合体。粘膜屏障变弱时Claudin-2表达下调，而ZO1和Occludin基因表达上调。粘液主要有上皮细胞表达的粘液素2构成，其表达增强有利于粘液屏障的维持。
图6显示，幼鼠摄入罗伊乳杆菌Fn041可显著上调ZO-2和ZO-1mRNA和粘液素-2的表达，提高量可达0.5～3倍。通过母鼠摄入可显著抑制Claudin-2并诱导粘液素2mRNA，说明不同途径摄入罗伊乳杆菌Fn041都可以提高粘膜屏障。
实施例6 不同处理方式对母鼠小肠免疫功能、肠道形态和粘膜屏障的影响
按照实施例1的方法处理小肠组织，并对母鼠小肠内容物中sIgA含量测定：严格按照sIgA ELISA试剂盒说明书进行测定。
用罗伊乳杆菌处理后处理后，母鼠血清IgA、IgE和肠道sIgA显著提高(p<0.05)(图7)，血清IgA由11.97ng/mL提高至12.10ng/mL，IgE由11.55ng/mL提高至11.73ng/mL，说明粘膜免疫和系统免疫能力都有所增强。同时，母鼠小肠粘膜固有层IgA浆细胞数量增加约1倍(p<0.05，图8)，小肠派伊尔结中与IgA浆细胞转换相关的CXCR5(p<0.05)和CXCL13(p<0.01)两个基因的表达显著提高(图10)，进一步说明粘膜免疫得到增强，从而可有助于预防细菌感染和腹泻的发生、以及预防湿疹等过敏性疾病的发生。图9显示，母乳经罗伊乳杆菌处理后小肠绒毛高度和绒腺比均提高60％以上，PIgR、mucin-2和Occludin基因表达强度分别提高了0.5倍、4倍和1.4倍，乳杆菌处理使进入血液的FITC-F000由3.2μg/mL降低至2.1μg/mL(图11)，说明小肠组织结构和粘膜完整性得到改善。按照实施例4的方法分离并培养小鼠隐窝，发现乳杆菌处理小鼠隐窝杀死沙门氏菌的能力显著高于对照(p<0.01，图12)，罗伊乳杆菌Fn041也显著增强了母鼠小肠粘膜多个与抗菌肽通路相关基因的表达，分别使a-defesin、MyD88、RegIII-gamma、CRS1C和TLR2的表达强度提高3.5倍、0.25倍、0.3倍、3.1倍和2.2倍。在受体中，TLR2、TLR4和TLR9分别识别细菌肽聚糖/脂磷壁酸、脂多糖和CpG未甲基DNA，其中仅TRL2表达被显著上调，说明罗伊乳杆菌主要通过细胞肽聚糖/脂磷壁酸刺激抗菌肽表达。
实施例7 应用罗伊氏乳杆菌Fn041制备配方食品
将罗伊氏乳杆菌Fn041接种于MRS培养基中，35～37℃培养至菌浓≥1×10 3CFU/mL，收集菌体。
可选地，向菌体中加入保护剂，制备菌粉。保护剂可选自：单糖、低聚糖、多糖、多元醇或其混合物，例如海藻糖、山梨醇、甘露醇等。
将前述任一方法制备的罗伊氏乳杆菌Fn041以单位质量(g)或单体体积(mL)计，按照1×10 4至1×10 11，优选1×10 7至1×10 11cfu/单位质量或单位体积的量加入至食品中。
可选地，所述食品还含有益生元、裸藻粉或裸藻提取物、陈皮粉或陈皮提取物、越橘粉或越橘提取物、枸杞粉或枸杞提取物、火麻仁粉、火麻仁蛋白、牛奶蛋白、动植物水解蛋白或肽。
其中，益生元包括但不限于：菊粉、低聚果糖、短链低聚果糖、低聚半乳糖、人乳低聚糖或牛乳低聚糖或上述多种益生元的组合物。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (14)

  1. 罗伊乳杆菌(Lactobacillus reuteri)Fn041在制备用于增强目标人群免疫力、提高肠道抗菌肽的表达和分泌量、促进免疫球蛋白A以及肠道IgA浆细胞数量的提高、建立或强化肠道屏障的产品中的应用；所述目标人群为：妊娠期女性、哺乳期女性和/或婴幼儿。
  2. 罗伊乳杆菌(Lactobacillus reuteri)Fn041在制备用于增强目标人群免疫力、建立或强化肠道屏障的产品中的应用；所述目标人群为：妊娠期女性、哺乳期女性和/或婴幼儿；所述产品包括但不限于食品、药品。
  3. 根据权利要求2所述的应用，其特征在于，所述增强目标人群免疫力、建立或强化肠道屏障具体为(1)～(5)任一所示功能：
    (1)提高妊娠期或哺乳期女性，或婴幼儿肠道抗菌肽的表达和分泌量；
    (2)促进妊娠期或哺乳期女性，或婴幼儿肠道产免疫球蛋白A以及肠道IgA浆细胞数量的提高；
    (3)增强妊娠期或哺乳期女性，以及婴幼儿肠道粘膜的屏障功能；
    (4)增强妊娠期或哺乳期女性的免疫力；
    (5)促进婴幼儿免疫系统的发育；
    (6)增加哺乳期女性母乳中Lactobacillus reuteri的数量。
  4. 根据权利要求3所述的应用，其特征在于，所述婴幼儿年龄为0-36个月。
  5. 根据权利要求3所述的应用，其特征在于，所述建立或强化肠道屏障包括但不限于：抵御病原菌或致病菌的入侵；所述致病菌包括沙门氏菌。
  6. 根据权利要求3所述的应用，其特征在于，所述产品以1×10 4至1×10 11，优选1×10 7至1×10 11cfu罗伊氏乳杆菌Fn041的日剂量提供给妊娠期或哺乳期女性，或婴幼儿。
  7. 根据权利要求3所述的应用，其特征在于，所述罗伊氏乳杆菌Fn041以1×10 4至1×10 12cfu/g干物质添加到组合物施用给妊娠期或哺乳期女性，或婴幼儿。
  8. 根据权利要求6～7任一所述的应用，其特征在于，所述罗伊氏乳杆菌Fn041的形态是活细胞、失活的细胞，或含有发酵产物或代谢物的形式，或上述任一状态的混合物。
  9. 根据权利要求8所述的应用，其特征在于，所述产品为食品。
  10. 根据权利要求9所述的应用，其特征在于，所述食品为发酵食品，包括但不限于发酵乳制品、发酵豆制品、发酵果蔬制品。
  11. 根据权利要求10所述的应用，其特征在于，所述产品为药品，其剂型包括但不限于：片剂、胶囊剂、固体粉剂或口服液。
  12. 根据权利要求11所述的应用，其特征在于，所述药品还含有药学上可接受的载体。
  13. 根据权利要求10～12任一所述的应用，其特征在于，所述产品中还含有但不限于以 下成分中的一种或多种：益生元、裸藻粉或裸藻提取物、陈皮粉或陈皮提取物、越橘粉或越橘提取物、枸杞粉或枸杞提取物、火麻仁粉、火麻仁蛋白、牛奶蛋白、动植物水解蛋白或肽。
  14. 根据权利要求13所述的应用，其特征在于，所述益生元包括但不限于：菊粉、低聚果糖、短链低聚果糖、低聚半乳糖、人乳低聚糖或牛乳低聚糖或上述多种益生元的组合物。
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