Patent Grant
12133874
The disclosure relates to application of breast milk-derived Lactobacillus reuteri in regulating the maternal and infant immune function, belonging to the fields of microbiological technology and food science, and particularly relates to application of secretory immunoglobulin A coated Lactobacillus reuteri Fn041 from breast milk in promoting the gastrointestinal immune development of infants and enhancing the immunity of adults.
The initiation and maturation of the human immune system are closely related to intestinal microbes, and bacteria colonized in the intestinal tract in early infancy play an important role in this process. Breast milk is the main source of intestinal microbiota of healthy full-term infants. Bifidobacterium and Lactobacillus in breast milk can be continuously inoculated into intestinal tract of infants by breastfeeding to promote the development of normal intestinal microbiota. The normal intestinal microbiota of infants affects the intestinal immunity and protection of infants through various mechanisms. Firstly, the intestinal microbiota constitutes the first line of defense against pathogenic microorganisms, and can produce broad-spectrum antibacterial substances, and fermentation substrates competing with pathogenic bacteria, or competing for adhesion to intestinal mucin and other binding sites, thus inhibiting the colonization and growth of pathogenic microorganisms in infants' intestines, protecting infants against infection and promoting the maturation of immune system functions. Secondly, the intestinal microbiota of infants stimulates the gastrointestinal tract of newborns to initiate an immune response, promote the development of the immune system and reduce an inflammatory response. Due to the action of microorganisms from breast milk, the incidence of infectious diseases of infants fed breast milk is significantly lower than that of infants fed formula milk powder.
Some active bacteria in breast milk refer to that specific bacterium in mothers' intestinal tract migrate to the mammary gland through physiological ectopia. Mothers' intestine is rich in secretory immunoglobulin A (sIgA), which can selectively bind to specific bacteria. The sIgA coated bacteria can be recognized and ingested by M cells on the surfaces of intestinal Peyer's patches (PP), and transferred to dendritic cells (DC) in the PP to be carried into the mammary gland. After being ingested by infants, these bacteria can be combined with sIgA in the intestinal tract and be mediated into the intestinal PP to promote the development of immune cells in the PP, especially the maturation of IgA-producing plasma cells. The coating of bacteria with sIgA can promote the colonization of these bacteria in the mucosa, so that the bacteria can act on epithelial cells at a closer distance, and regulate the expression of tight junction proteins and antibacterial peptides in intestinal epithelial cells. Lactobacillus or Bifidobacterium coated with sIgA can be isolated from breast milk, which can induce the development of the immune system of infants after being ingested by lactating mothers or infants. Some studies have proved that sIgA derived from mothers' milk affects the composition and maturity of the intestinal microbiota of breast-fed infants, while infants mainly fed with formula milk lack sIgA, leading to excessively fast development of the intestinal microbiota. SlgA in breast milk mainly coates specific breast milk bacteria, affecting the development of intestinal bacteria in breast-fed infants. sIgA-coated lactic acid bacteria in breast milk are typical beneficial bacteria of healthy full-term infants, and are probiotic candidate strains suitable for infants, which can be used to promote the intestinal immune development of infants.
The World Health Organization, American Academy of Pediatrics, Chinese Nutrition Society, etc. all encourage mothers to extend breastfeeding time to at least one year or longer, and exclusive breastfeeding is strongly recommended for the first 6 months after birth. Some mothers have insufficient breast milk or are unsuitable for breastfeeding due to some reasons, so they have to choose formula milk powder as a supplement to breast milk, which hinders infants from getting bacteria from breast milk. There are significant regional differences in the bacterial composition of breast milk in China, and the content of beneficial bacteria in breast milk in some regions is obviously lower than that in other regions. Meanwhile, diseases during pregnancy such as eclampsia and gestational diabetes also cause breast milk microbiota to be different from healthy mothers, the transmission of which to infants may affect their health. In these cases, it is necessary to give probiotics supplements to infants to promote the healthy development of intestinal microbiota. Mothers ingesting sIgA-coacted beneficial bacteria isolated from breast milk can also enhance immunity, and these bacteria can transfer to breast milk and be ingested by infants through breastfeeding.
The technical problem to be solved by the disclosure is to promote the development of intestinal immunity and ensure the intestinal barrier and protection function by means of probiotic supplements for infants who cannot be breastfed or infants who consume breast milk with disturbed microbiota and lactating women who need to enhance immunity.
To solve the above problem, the disclosure provides application of Lactobacillus reuteri Fn041 in preparing products for enhancing the immunity of a target population, increasing the expression and secretion of an intestinal antibacterial peptide, promoting the increase of numbers of immunoglobulin A (IgA) and intestinal IgA plasma cells, establishing or strengthening an intestinal barrier, and ability to killing pathogens such as Salmonella. The target population is pregnant women, lactating women and infants. The Lactobacillus reuteri Fn041 is Lactobacillus reuteri with the preservation number GDMCC No. 60546, which has been disclosed in the patent application document with the publication number CN110205261A.
In one embodiment, the products include, but are not limited to, food, medicine or health care products.
In one embodiment, the application is to prepare products with any one of the functions shown in (1) to (5):
(1) increasing the expression and secretion of the intestinal antibacterial peptide for pregnant or lactating women and infants;
(2) promoting the increase of numbers of intestinal IgA and intestinal IgA plasma cells for pregnant or lactating women and infants;
(3) enhancing the barrier function of intestinal mucosae of pregnant or lactating women and infants;
(4) promoting the development of the immune system of infants;
(5) enhancing the immunity of pregnant or lactating women; and
(6) increasing the abundance of Lactobacillus reuteri in breast milk of lactating women.
In one embodiment, establishing or strengthening the intestinal barrier includes, but is not limited to: resisting the invasion of pathogenic bacteria, the pathogenic bacteria including Salmonella.
In one embodiment, the age of the infants is 0-36 months.
In one embodiment, food or medicine is provided to pregnant or lactating women or infants at a daily dose of 1×104 to 1×1011, preferably 1×107 to 1×1011 cfu of Lactobacillus reuteri Fn041.
In one embodiment, the Lactobacillus reuteri is added to a composition at a dose of 1×104 to 1×1012 cfu/g dry matter to be provided to pregnant or lactating women or infants.
In one embodiment, Lactobacillus reuteri in the products is a living cell, an inactivated cell, a form of fermentation products or metabolites, or a mixture of any of the above forms.
In one embodiment, the product is food or medicine, whose dosage forms include, but are not limited to, tablets, capsules, solid powder or oral liquid.
In one embodiment, the product also contains prebiotics, and the prebiotics include, but are not limited to, one or more of inulin, fructooligosaccharides, short-chain fructooligosaccharides, galactooligosaccharides, human milk oligosaccharides or cow milk oligosaccharides.
In one embodiment, the food or medicine to which Lactobacillus reuteri Fn041 is added also contains, but is not limited to, one or more of the following components: prebiotics, Euglena powder or Euglena extract, pericarpium citri reticulatae powder or pericarpium citri reticulatae extract, huckleberry fruit powder or huckleberry fruit extract, wolfberry fruit powder or wolfberry fruit extract, fructus cannabis powder, fructus cannabis protein, milk protein, and 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 above prebiotics.
Beneficial effects: The disclosure provides new application of Lactobacillus reuteri Fn041 in enhancing the immunity of infants and mothers, namely enhancing the mucosal barrier, promoting the production of the intestinal antibacterial peptide and IgA, and promoting the development of the immune system of infants.
For the young, animal experiments show that Lactobacillus reuteri Fn041 can promote the intestinal immune development of newborn mice and achieve the following effects:
(1) promoting the expression of genes of the antimicrobial peptide pathway in intestinal epithelial cells. The expression of the TLR4 gene is inhibited by about 0.5 times, and the expression of the TLR-9 gene is activated by 0.5-1 time;
(2) promoting the gene expression of the intestinal immunoglobulin A (IgA) pathway and the formation of IgA plasma cells, wherein the expression of CXCR5, CXCL13, APPIL, TGF-β and Foxp3 is increased by about one time;
(3) enhancing the expression of genes related to the intestinal mucosal barrier function, wherein the expression of ZO-2 and ZO-1 mRNA and mucin-2 can be increased by 0.5-3 times; and
(4) increasing the intestinal villi to crypt depth ratio and enhancing the antimicrobial activity of crypt secretions, wherein a Salmonella inhibition rate of 50% can be achieved within 5 min.
For the mother, animal experiments show that Lactobacillus reuteri Fn041 can enhance the immunity of pregnant or dams, which is embodied as follows:
(1) increasing serum and intestinal antibody concentration, wherein serum IgA is increased from 11.97 ng/mL to 12.10 ng/mL, and IgE from 11.55 ng/mL to 11.73 ng/mL;
(2) promoting the number of cells producing mucosal antibodies, wherein the number of IgA plasma cells is increased by about one time;
(3) promoting the intestinal mucosal barrier and the expression of antimicrobial peptide related genes, wherein the expression intensity of PIgR, mucin-2 and occludin genes is increased by 0.5 times, 4 times and 1.4 times respectively; and
(4) improving the morphology of intestinal villi and enhancing the resistance to pathogenic bacteria, wherein a Salmonella inhibition rate of 50% can be achieved within 10 min.
The disclosure will be further explained with specific examples below.
In the data analysis methods adopted in the following examples, experimental results are all expressed in (mean±standard deviation). SPSS 25.0 was used for statistical analysis of data, and one-way analysis of variance (ANOVA) and a Tukey test were used for comparison between groups. When the homogeneity of variance was less than 0.05, a nonparametric independent two-sample T test was conducted, and a Kruskal-Wallis test was used to analyze the data. Significant level: P<0.05, very significant level: P<0.01, and extremely significant level: P<0.001.
(1) Experimental Method
Animal grouping: 40 BALB/C female mice and 14 BALB/C male mice were fed with common feed at room temperature 22-24° C. and humidity 40-70% with the day and night cycle maintained at 8:00-20:00. The male mice and the female mice were separated for one week, and then put together according to a female to male ratio of 3:1 after one week. After the female mice were pregnant, the male mice were taken out. After the female mice gave birth, the dams were divided into two groups, one group was given normal saline (CON) through intragastric administration, the other group was given Lactobacillus reuteri Fn041 (1×101° CFU per mouse per day) through intragastric administration, and when pups reached 3 weeks old, they were weaned naturally. Each group of breast-fed male pups were divided into two groups, which were given normal saline or Lactobacillus reuteri respectively through intragastric administration. After 14 days, the treated pups were killed. The above pups were divided into: (1) Con group, in which both damsdams and pups were given normal saline through intragastric administration; (2) Inf group, in which the damsdams were given normal saline through intragastric administration, and the pups were given Lactobacillus reuteri Fn041 (1×109 CFU per mouse per day); (3) Lacm group, in which the damsdams were given Lactobacillus reuteri Fn041 (1×1010 CFU per mouse per day) through intragastric administration, and the pups were given normal saline through intragastric administration; and (4) CO-T group, in which both damsdams and pups were given Lactobacillus reuteri Fn041 through intragastric administration, and the dose for the damsdams was 1010 CFU per mouse per day and 109 CFU per mouse per day for pups.
(1) Dehydration: Intestine tissue was put into 50% ethanol, 75% ethanol, 85% ethanol and 90% ethanol respectively for shaking dehydration for 15 min, and then was put in 100% ethanol for treatment for 30 min, wherein the ethanol was replaced every 15 min.
(2) Transparentizing: Ethanol and n-butanol was mixed according to the ratio of 1:1 to obtain a mixed solution, the intestine tissue was put into the mixed solution, shaking was performed for 20 min, the liquid was poured out, and the intestine tissue was transferred to an n-butanol solution for treatment for 40 min, wherein n-butanol was replaced every 20 min.
(3) Wax immersion: Immersing was performed in 70° C. paraffin I for 3 h, and then transferring to 70° C. paraffin II was performed for immersion for 3 h.
(4) Embedding: The intestine tissue treated in step (3) was embedded in paraffin.
(5) Slicing: Jejunum tissue was sliced into pieces with a thickness of 5 μm with a Leica microtome, the pieces were spread in a water bath at 42° C., and the pieces were baked at 70° C. for 30 min.
(6) Dewaxing and hydration: Soaking was performed in xylene three times, each time lasting 5 min; soaking was performed in 100% ethanol twice, each time lasting 5 min; soaking was performed in 95% ethanol, 85% ethanol and 75% ethanol sequentially for 3 min respectively; and then washing with deionized water was performed for 5 min.
(7) Antigen repair: 200 mL of EDTA buffer solution with pH 9.0 was added into a dyeing box, the tissue slices after dewaxing and hydration were placed on a plastic slice rack of the dyeing box, heating was performed in an autoclave till saturation, then continuing heating was performed for 5 min, a power supply was turned off, the dyeing box was taken out after 10 min, the dyeing box was cooled at room temperature for 30 min, and the dyeing box was soaked in a PBS buffer solution for 3 min×3 times.
(8) 100 μL of 3% H2O2 was added to each slice, incubating at room temperature was performed for 10 min, and soaking was performed in PBS for 3 min×3 times.
(9) The PBS buffer solution was removed, 100 μL of 2.5% goat serum was added, incubating at room temperature was performed for 30 min, and a blocking solution was removed.
(10) 100 μL of diluted rabbit anti-mouse IgA monoclonal antibody (primary antibody) was added to each slice, incubating was performed at 25° C. for 1 h, and soaking was performed in PBS for 3 min×3 times.
(11) PBS was removed, 100 μL of goat anti-rabbit IgG monoclonal antibody (secondary antibody) was added, incubating was performed at 25° C. for 30 min, and soaking was performed in PBS for 3 min×3 times.
(12) PBS was removed, 100 μL of DAB color developing solution prepared in real time was added, color development was performed under a microscope for 5 min, and the reaction was stopped with tap water.
(13) Redyeing was performed with hematoxylin for 12 sec and tap water was used for rinsing.
(14) Differentiating was performed with 1% hydrochloric acid alcohol for 1 sec, tap water was used for washing, and returning to blue with tap water was performed for 5 min.
(15) Dehydration and transparentizing: treating was performed with 75% ethanol, 85% ethanol and 95% ethanol respectively for 3 min, then treating was performed with 100% ethanol for 5 min, the operation was repeated three times, and then soaking was performed in xylene for 5 min and then 3 min.
(16) Sealing with neutral gum and baking in an oven at 60° C. for 20 min were performed.
An eyepiece of a microscope was inverted by 10 times and an objective lens 20 times, and after observation and photo shooting, and Image-Pro Plus software was used for imaging analysis. The mean optical density (density (mean)) was calculated, density (mean)=integrated optical density (IOD)/area. The mean number of plasma cells contained in each villus was counted with the microscope.
The villi height to crypt depth ratio can comprehensively reflect the digestion and absorption function of intestines. The higher the villi, the larger the intestinal surface area and the stronger the intestinal absorption function. The intestinal crypt depth represents the cell production rate. The shallower the crypt, the better the cell maturity and the more mature the secretion function.
RNA extraction and cDNA preparation of jejunum and intestine Peyer's patch, comprising the following steps: taking out the liver and spleen of mice in an ultra-clean table, cutting off 0.1 g of jejunum and putting it into Trizol quickly, extracting RNA from the jejunum and Peyer's patch by a Trizol method, determining OD260/280 and concentration by NanoDrop, adjusting the RNA concentration to 1,000 ng/μL, making OD260/280 within the range of 1.8-2.0, conducting reverse transcription of RNA according to reaction systems in Table 1, mixing with the reaction system in step 1 evenly and then bathing in ice at 70° C. for 5 min, then mixing with the second system evenly and bathing in water at 37° C. for 1 h and in ice at 95° C. for 3 min, and storing the cDNA after reverse transcription at −80° C.
Reverse transcription fluorescence quantitative PCR in jejunum (RT-PCR), comprising the following steps: conducting quantitative PCR analysis by using cDNA after reverse transcription in jejunum as an amplification template, wherein the reaction system: 2×SYBR Rreen Master Mix 5 μL, 0.4 μL for upstream primer and 0.4 μL for downstream primer (10 μM, see Table 2 for sequence), cDNA 0.3 μL, and DEPC water 3.7 μL; amplification conditions: 95° C., 5 min; 95° C., 20 sec; 60° C., 30 sec; 72° C., 1 min, 40 cycles, 72° C., 2 min; and using β-actin as internal reference, and analyzing the data by a 2-ΔΔCt method.
Preparation of intestine crypt and evaluation of antibacterial activity of antibacterial peptide, comprising the following steps: washing a segment of intestine cavity of mice with precooled sterile water, everting the intestinal segment and shaking in a PBS buffer containing 30 mM EDTA without Ca++ and Mg++ to elute the crypts, eluting the villi and crypts several times at 5 min intervals, conducting centrifugation (700 g), resuspending in the PBS buffer, transferring a single crypt into a silicified microcentrifuge tube with a capillary pipette, estimating about 1000 crypts by a blood cell counting method, resuspending in 2 ml of iPIPES buffer (10 mM PIPES+137 mM NaCl, pH 7.4), adding Salmonella at 1000 CFU/crypt, incubating at 37° C. for 30 min, conducting centrifugation on the crypts, take 10 μl of supernatant, spreading it on a nutrient agar plate, determining the colony number after growing overnight, and measuring the germicidal rates for 1, 2.5, 5, 10, 15 and 30 min respectively.
The antimicrobial peptide expressed by Paneth cells at the base of the intestinal crypt is an important defensive molecule in the intestinal tract, which is secreted into the intestinal mucus layer to resist the invasion of pathogenic bacteria and potentially pathogenic symbiotic bacteria. Peptidoglycan, endotoxin, bacterial DNA released by bacterial cells are combined with toll-like receptors (TLR2, TLR4, TLR9, etc.) of Paneth cells, and through signal transduction mediated by transducing protein MyD88, Nf-κB sub-protein in cytoplasm can be released, enter cells and induce a variety of antimicrobial expressions.
IgA is the most important active molecule of the intestinal immune system, which is mainly released into the mucous layer in the form of secretory IgA (sIgA) to restrain the excessive proliferation and migration of pathogenic bacteria. Intestinal Peyer's patch is the initial induction site of IgA plasma cells. Bacteria are absorbed by M cells of the Peyer's patch and then transmitted to dendritic cells (DC), so as to activate DC, induce follicular dendritic cells (FDC) to produce TGFβ1 and CLCL13 through TLR and FDC, and promote Foxp3-T cells to transform into follicular helper T(Tfh) cells, which express CXCR5. Under the participation of TGFβ, the antibodies expressed by initial IgM+ B cells undergo class conversion and are differentiated into IgA+ B cells. After migrating to the lamina propria, these cells are transformed into IgA plasma cells, which can produce and secrete IgA. IgM+ cells in the lamina propria can be transformed into IgA+ cells under the action of APPIL on the premise that the intestinal immune function is mature.
RNA extraction and fluorescent quantitative PCR (RT-PCR) were carried out according to the method of Example 3. Results are shown in
According to the grouping of Example 1, an animal experiment was carried out. The mice were given 4 kDa fluorescein isothiocyanate-dextran (FD4, 125 mg/mL) (600 mg/kg body weight) through intragastric administration. After 4 hours, the eyeballs were taken for blood collection, and all treatments after blood collection needed to be protected from light. Blood was incubated at room temperature (RT) for at least 1 h to coagulate blood, and high-speed centrifugation was conducted for 10 min to separate serum. Serum aliquot was diluted with PBS 1:1 in duplicate, and fluorescence readings at 488/530 nm were analyzed. The concentration of FD4 in serum samples was determined according to the FD4 standard curve value of PBS continuous dilution. The control mouse serum of FD4 in all the serum samples was standardized.
As shown in
The first line of defense against symbiotic bacteria and pathogenic bacteria in infants' intestines is the intestinal mucosal barrier, and this physical barrier includes biochemical and immune components. The physical barrier is mainly composed of intestinal epithelial cells through tight junctions, and the chemical barrier is mainly composed of mucus covering the intestinal surface. The tight junction is a complex composed of intracellular zona occludens (ZO1), Claudin, and intercellular Occludin. The expression of Claudin-2 was down-regulated and the expression of ZO1 and Occludin genes was up-regulated when the mucosal barrier weakened. Mucus is mainly composed of mucin 2 expressed by epithelial cells, and its enhanced expression is beneficial to the maintenance of the mucus barrier.
Intestine tissue was treated according to the method of Example 1, and the content of sIgA in the intestine of the damsdams was determined strictly according to the instructions of sIgA ELISA kit.
After treatment with Lactobacillus reuteri, serum IgA, IgE and intestinal sIgA of the damsdams increased significantly (p<0.05) (
Six-week-old male C57BL/6J mice without specific pathogen were divided into three groups (30 mice in each group): (1) control group: treated with 0.1 mL of phosphate buffer through intragastric administration; (2) Salmonella infection group (SI): treated with 0.1 mL of phosphate buffer through intragastric administration for 10 days, and then infected with 1.0×106 Salmonella typhimurium SL1344 for 10 days every day; (3) Lactobacillus reuteri Fn041 intervention group (Fn041+SI): firstly treated with Lactobacillus reuteri Fn041 for 20 days (1×109 CFU per mouse per day, suspended in 100 μL of phosphate buffer), and treated with Salmonella infection every day from the 11th day to the 20th day. The experiment was ended on the 21st day.
As shown in
An de Man, Rogosa and Sharpe medium was inoculated with Lactobacillus reuteri Fn041, culturing was performed at 35-37° C. until the concentration of bacteria was ≥1×103 CFU/mL, and the bacterial cells were collected.
Optionally, a protective agent was added to the bacterial cells to prepare bacterial powder. The protective agent can be selected from monosaccharide, oligosaccharide, polysaccharide, polyol or a mixture thereof, such as trehalose, sorbitol, mannitol, etc.
The Lactobacillus reuteri Fn041 prepared by any one of the above methods was added to food in an amount of 1×104 to 1×1011, preferably 1×107 to 1×1011 cfu/unit mass (g) or unit volume (mL).
Optionally, the food also contains prebiotics, Euglena powder or Euglena extract, pericarpium citri reticulatae powder or pericarpium citri reticulatae extract, huckleberry fruit powder or huckleberry fruit extract, wolfberry fruit powder or wolfberry fruit extract, fructus cannabis powder, fructus cannabis protein, milk protein, and 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 above prebiotics.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010076261.7 | Jan 2020 | CN | national |
| Number | Date | Country |
|---|---|---|
| 110205261 | Sep 2019 | CN |
| Entry |
|---|
| Li et al (Food & Function (Aug. 1, 2019 and online published Jun. 28, 2019), 10(8), 4705-4715). |
| Lara-Villoslada et al (British Journal of Nutrition (2007), 98, Suppl. 1, S96-S100). |
| Weizman et al (Pediatrics. 2005. Jan. 115(1): 5-9). |
| Ding, Mengfan et al. “A study on human milk microbiota and their dominant lactic acid bacteria influening gut mucosal immune development in neonatal mice”, Thesis, China Academic Journal electric publishing house, Dec. 31, 2019, english translation for abstract only. only abstract considered. |
| Number | Date | Country | |
|---|---|---|---|
| 20220133820 A1 | May 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2020/080353 | Mar 2020 | WO |
| Child | 17578537 | US |
