Application of lactobacillus rhamnosus KF7 in improving intestinal barrier injury and regulating intestinal flora and functional food containing lactobacillus rhamnosus KF7

Abstract

The invention relates to the technical field of microorganisms, in particular to application of lactobacillus rhamnosus KF7 in improving intestinal barrier injury and regulating intestinal flora and functional food containing the lactobacillus rhamnosus KF7. The invention provides application of lactobacillus rhamnosus KF7 in preparation of a product for improving intestinal barrier injury and / or regulating intestinal flora. The preservation number of the lactobacillus rhamnosus KF7 is CGMCC (China General Microbiological Culture Collection Center) NO.6430. The invention further provides application of the lactobacillus rhamnosus KF7 in preparation of a product for improving intestinal barrier injury and / or regulating intestinal flora. The invention discloses the novel application of the caseus rhamnosus KF7 in the aspects of improving the normal intestinal epithelial barrier function, protecting the normal intestinal epithelial barrier from lipopolysaccharide induced barrier injury, recovering animal intestinal barrier injury caused by rhythm disorder, regulating intestinal flora disorder and improving growth, development and metabolic states for the first time. The method can be used for producing functional food products such as fermented milk, lactic acid bacteria beverages, probiotic powder, viable bacteria water, milk powder and cheese.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to the use of a strain of Lactobacillus rhamnosus in improving intestinal barrier damage and regulating intestinal flora, and a functional food containing the strain. Background Art

[0002] The intestine is the main digestive organ and an important immune organ of the human body. It not only participates in the absorption and transport of nutrients, but also can resist the invasion of foreign pathogens and harmful substances. The intestinal barrier is called "the first line of defense for physical health" and is mainly composed of a mucus layer, symbiotic bacteria, intestinal epithelial cells and immune cells. Among them, the physical and mechanical barrier mainly composed of the mucus layer, intestinal epithelial cells and the tight junction structure between cells is the main component of the intestinal barrier. Mucin-2 (MUC-2) is the main component of the intestinal mucus layer, covering the top of intestinal epithelial cells and playing an important role in resisting the invasion of intestinal pathogenic bacteria and harmful substances. The tight junction (TJ) between intestinal epithelial cells is the main determinant of paracellular permeability and is composed of a transmembrane protein family (occludin and claudin), a perimembrane protein family (zonula occludens, ZO), etc., making adjacent cells closely adhere to each other and playing roles such as selective permeability and maintaining the homeostasis of the intracellular environment. The impairment of intestinal barrier function is related to many diseases, such as irritable bowel syndrome, inflammatory bowel disease (Crohn's disease and ulcerative colitis), diabetes, etc. Therefore, maintaining the integrity of the intestinal barrier is of great importance to human health.

[0003] A variety of internal and external factors can cause damage to the human intestinal barrier. Lipopolysaccharides (LPS) are the main components of the cell wall of Gram-negative bacteria, mainly composed of lipids and polysaccharides. LPS is a powerful stimulator of the immune system, which can lead to the production of pleiotropic cytokines / chemokines, trigger an inflammatory signaling cascade, and then regulate inflammatory, innate immune, and adaptive immune responses. There are a large number of Gram-negative commensal bacteria in the human colon, and they maintain a balance with Gram-positive bacteria in the healthy human intestine. However, when the intestinal flora is imbalanced and the abundance of Gram-negative bacteria increases, the elevated LPS level in the intestinal lumen can lead to intestinal inflammation and damage the intestinal epithelial barrier function. The circadian rhythm is the variation of life activities with a cycle of about 24 hours generated by organisms to synchronize with the solar day. The rhythm system is crucial for maintaining the synchronization between internal physiology, behavior, and external environmental cues (such as sunlight, temperature, humidity). When this synchronization is lost, circadian rhythm disorders occur. The disruption of the rhythm can change the composition of the gut microbiota and the integrity of the intestinal barrier. Studies have found that compared with the normal control group, the relative abundance of Odoribacter bacteria, which can produce short-chain fatty acids, in the intestines of subjects with circadian rhythm disorders is significantly reduced. Animal experiments have also found that the disruption of the circadian rhythm in mice leads to a significant increase in the abundance of Clostridium in the intestine. Some circadian rhythm-related diseases (such as diabetes, hypertension, sleep disorders, and chronic inflammation) all show clinical symptoms of damage to the intestinal barrier integrity.

[0004] Probiotics are considered to be live microorganisms that confer health benefits on the host when ingested in sufficient amounts. Lacticaseibacillus rhamnosus (synonym: Lactobacillus rhamnosus) belongs to the genus Lactobacillus and is an important probiotic with a variety of beneficial effects on the health of the body. It is widely used in fermented dairy products. Lacticaseibacillus rhamnosus KF7 is a probiotic strain isolated from Chinese Yunnan kefir. There is currently no report on the improvement of damaged intestinal barrier and intestinal flora disorder by Lacticaseibacillus rhamnosus KF7, nor is there a report on the preparation of related functional products using this strain, including but not limited to fermented milk, lactic acid bacteria beverages, probiotic powders, live bacteria water, milk powder, and cheese. Summary of the Invention

[0005] Based on the above technical problems, the present invention provides the use of a strain of Lacticaseibacillus rhamnosus (synonym: Lactobacillus rhamnosus) KF7 in improving the intestinal barrier, regulating the intestinal flora, and producing related functional foods.

[0006] The first aspect of the present application provides the use of Lactobacillus casei rhamnosus KF7 in the preparation of a product for improving intestinal barrier damage and / or regulating the intestinal flora, and the preservation number of Lactobacillus casei rhamnosus KF7 is CGMCC NO. 6430.

[0007] The second aspect of the present application provides a fermented milk product, the raw materials of which include Lactobacillus casei rhamnosus KF7; optionally, it further includes one or a combination of more than one of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum.

[0008] The third aspect of the present application provides a lactic acid bacteria beverage product, the raw materials of which include Lactobacillus casei rhamnosus KF7; optionally, it further includes one or a combination of more than one of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum.

[0009] The fourth aspect of the present application provides a probiotic agent, which is obtained by freeze-drying a fermented culture strain after adding a freeze-drying protectant, and the strain includes Lactobacillus casei rhamnosus KF7; optionally, it further includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum, Kluyveromyces.

[0010] The fifth aspect of the present application provides a probiotic powder product, which is obtained by adding a dietary supplement, vitamins, fructooligosaccharide, maltodextrin and pectin to the aforementioned probiotic agent, mixing evenly and then freeze-drying.

[0011] The sixth aspect of the present application provides a live bacteria aquatic product, which is obtained by adding vitamin C and a stabilizer to the aforementioned probiotic agent. Based on the quality of the live bacteria aquatic product, the concentration of the probiotic agent is 10 6 ~10 9 CFU / g.

[0012] The seventh aspect of the present application provides a milk powder product, the raw materials of which include Lactobacillus casei rhamnosus KF7, and optionally, it further includes one or a combination of more than one of whole milk powder, skim milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin and compound nutrients.

[0013] The eighth aspect of the present application provides a cheese product, the raw materials of which include Lactobacillus casei rhamnosus KF7, and optionally, it further includes one or a combination of more than one of raw cow milk, commercial starter and rennet.

[0014] Compared with the prior art, the present invention uses Lactobacillus rhamnosus (synonym: Lactobacillus rhamnosus) KF7 (CGMCC NO. 6430), and for the first time discloses that Lactobacillus rhamnosus KF7 can not only improve the intestinal epithelial barrier function, protect it from lipopolysaccharide-induced barrier damage, but also restore the intestinal barrier damage caused by circadian rhythm disorder, regulate the intestinal flora, and improve the growth and development and metabolic status of animals. Lactobacillus rhamnosus KF7 can strengthen the intestinal barrier function by upregulating the expression level of mucin-related genes, and its fermented milk supernatant can improve the integrity of the intestinal epithelial barrier structure by increasing the expression of tight junction protein genes, and improve the inflammatory response and the permeability between intestinal epithelial cells by inhibiting the gene expression of the TLR / MyD88 / NF-κB signaling pathway and downstream MLCK. Consuming Lactobacillus rhamnosus KF7 can reduce the increased intestinal permeability caused by circadian rhythm disorder, increase the diversity of the intestinal flora, regulate the balance of the intestinal microbiota, and improve the growth and development and metabolic level. It can be used for the production of functional food products such as fermented milk, lactic acid bacteria beverages, probiotic powders, live bacteria water, milk powder, and cheese. Brief Description of the Drawings

[0015] Figure 1 . Schematic diagram of the dual-environment co-culture system.

[0016] Figure 2 . Effects of co-culture of Lactobacillus rhamnosus KF7 and Caco-2 cell monolayers on intestinal epithelial barrier TEER and mucin-related gene expression. (A) Changes in TEER of Caco-2 cell monolayers; (B) Expression level of MUC-2 gene. *P<0.05. Significant differences are indicated by different lowercase letters.

[0017] Figure 3 . Effects of fermented milk supernatant of Lactobacillus rhamnosus KF7 on TEER of Caco-2 cell monolayers. Significant differences are indicated by different lowercase letters.

[0018] Figure 4 . Effects of fermented milk supernatant of Lactobacillus rhamnosus KF7 on the expression of tight junction protein-related genes. Significant differences are indicated by different lowercase letters.

[0019] Figure 5 . Effects of fermented milk supernatant of Lactobacillus rhamnosus KF7 on the inflammatory response in Caco-2 cells. (A) Expression level of pro-inflammatory cytokine TNF-α; (B) Expression level of pro-inflammatory cytokine IL-1β; (C) Expression level of pro-inflammatory cytokine IL-6; (D) Expression level of pro-inflammatory cytokine IL-8; (E) Expression level of anti-inflammatory cytokine TGF-β; (F) Expression level of anti-inflammatory cytokine IL-10. Significant differences are indicated by different lowercase letters.

[0020] Figure 6 . Effects of the supernatant of Lactobacillus rhamnosus KF7 fermented milk on the inflammatory signaling pathway and the expression of downstream MLCK gene in Caco-2 cells. (A) Expression level of MyD88; (B) Expression level of NF-κB p65; (C) Expression level of MLCK. Significant differences are indicated by different lowercase letters.

[0021] Figure 7 . Effects of Lactobacillus rhamnosus KF7 on the intestinal permeability of rhythm-disordered nematodes. (A) Intestinal leakage of nematodes after staining with food coloring brilliant blue; (B) Total fluorescence intensity of nematodes after staining with fluorescein isothiocyanate-dextran (FD4). *P<0.05; **P<0.01; ns indicates no statistical significance.

[0022] Figure 8 . Effects of Lactobacillus rhamnosus KF7 on the intestinal flora composition of rhythm-disordered nematodes. (A) α-diversity; (B) β-diversity (PCoA plot based on Unweighted Unifrac distance); (C) Relative abundances of intestinal microorganisms at the phylum level; (D) Relative abundances of intestinal microorganisms at the genus level. *P<0.05.

[0023] Figure 9 . Effects of Lactobacillus rhamnosus KF7 on the body shape of rhythm-disordered nematodes. (A) Body length; (B) Body width. ***P<0.001; ns indicates no statistical significance.

[0024] Figure 10 . Effects of Lactobacillus rhamnosus KF7 on the metabolism (pharyngeal pumping frequency) of rhythm-disordered nematodes. ***P<0.001; ns indicates no statistical significance. Detailed implementation manners

[0025] In order to make the invention purpose, technical solutions and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. Those familiar with this technology can easily understand other advantages and effects of the invention of this application from the content disclosed in this specification.

[0026] The present application provides the use of Lactobacillus rhamnosus KF7 in the preparation of products for improving intestinal barrier damage and / or regulating intestinal flora, and the preservation number of the Lactobacillus rhamnosus KF7 is CGMCC NO.6430.

[0027] Lactobacillus rhamnosus KF7 is a probiotic strain isolated from Chinese Yunnan kefir. Lactobacillus rhamnosus (synonym: Lactobacillus rhamnosus) belongs to the genus Lactobacillus.

[0028] In the use provided by this application, the product has any one or more of the following functions:

[0029] 1) Improve intestinal barrier integrity;

[0030] 2) Promote the expression of genes related to intestinal cell tight junction proteins and mucin-related genes;

[0031] 3) Up-regulate the expression level of inflammatory factors;

[0032] 4) Reduce the expression level of key molecules in the inflammatory signaling pathway;

[0033] 5) Improve intestinal flora diversity;

[0034] 6) Increase the relative abundance of beneficial bacteria;

[0035] 7) Reduce the relative abundance of harmful bacteria;

[0036] 8) Regulate the balance of the intestinal microbiota;

[0037] 9) Improve growth and development indicators;

[0038] 10) Improve the reduced metabolic level.

[0039] In some embodiments, the subjects of the above research are human colorectal adenocarcinoma cells (Caco-2) and Caenorhabditis elegans. The structure and function of Caco-2 cells are similar to those of differentiated small intestinal epithelial cells, with the same cell polarity, tight junctions, and microvilli and other structures. Under cell culture conditions, cells grown on porous permeable polycarbonate membranes can fuse and differentiate into intestinal epithelial cells, forming a continuous monolayer, which is widely used to study the integrity of the intestinal epithelial barrier. Caenorhabditis elegans is a simple multicellular organism that lives in soil and feeds on microorganisms. 40% of its genome is homologous to human genes, and the homology with human disease-related genes is as high as 65%. Therefore, Caenorhabditis elegans is widely used to simulate complex human diseases. In previous studies, it has been found that the movement, metabolism, protein activity, and transcriptional regulation of C. elegans all exhibit circadian rhythms. Whole-genome analysis found that a large number of genes in C. elegans exhibit rhythmic characteristics under light or temperature entrainment, and compared with light, temperature has a greater impact on the circadian rhythm of C. elegans. This may be due to the fact that C. elegans usually lives in soil, and the direct impact of the sun on it is reflected in the change of temperature.

[0040] In the use provided by this application, improving intestinal barrier integrity includes increasing the trans-epithelial electrical resistance value of the intestinal epithelial cell monolayer. The trans-epithelial electrical resistance value provides information on the ionic current resistance across the cell monolayer and is related to the integrity of the tight junctions between cells. The change in the trans-epithelial electrical resistance value (TEER value) of each cell monolayer is calculated using the following formula: TEER value change (%) = TEER (Ω.cm2 ) / Baseline TEER (Ω·cm 2 ) × 100%.

[0041] In the use provided by the present application, the intestinal cell tight junction proteins are selected from Occludin, Claudin, or ZO; the genes related to the intestinal cell tight junction proteins are selected from Claudin-1, Occludin, or ZO-1. The tight junctions located at the apical side membrane of intestinal epithelial cells are the main factors determining paracellular permeability, including tight junction proteins such as Occludin, Claudin, and ZO. The integrity of tight junction proteins in the epithelial monolayer can be represented by a quantitative index, such as trans-epithelial electrical resistance (TEER), so as to determine the degree of intestinal barrier damage.

[0042] In the use provided by the present application, the intestinal cell mucin is selected from MUC-2; the gene related to the intestinal cell mucin is selected from MUC-2. The intestinal mucus layer is a natural barrier against the invasion of intestinal pathogenic bacteria and harmful substances, and MUC is its main component. As a secreted mucin, MUC-2 is widely expressed in the intestine. Its monomer is a homologous oligomer containing an N-terminal oxidized glycosylation domain and a C-terminal cysteine-rich domain, which constitutes the backbone of the intestinal mucus layer, can provide biological and adhesion sites for bacteria and immunoglobulins, and plays an important role in the intestinal mucosal barrier.

[0043] In the use provided by the present application, the inflammatory factors are selected from TNF-α, IL-1β, IL-6, IL-8, TGF-β or IL-10. The key molecules of the inflammatory signaling pathway are selected from MyD88, NF-κB p65 or MLCK. The TLR / MyD88 / NF-κB signaling pathway is an important pathway in the body's inflammatory system, widely present in various tissue cells, and is involved in the occurrence and regulation of various diseases. MyD88 (Myeloid Differentiation factor 88) is an important transduction protein in this pathway and is an important node for signal transduction downstream. NF-κB is the main transcription factor responsible for regulating innate and adaptive immune responses. In the body, NF-κB usually functions as a heterodimer formed by the binding of p50 and p65. In the resting state, the heterodimer binds to the inhibitor IκB (inhibitory κB) and exists in an inactive state; when stimulated, IκB is phosphorylated and then ubiquitinated and degraded, releasing the p50 / p65 complex, which is transported into the nucleus and binds to the promoter of specific genes, thereby regulating the expression of inflammatory factors (such as TNF-α, IL-1β, IL-6 and IL-8). TGF-β and IL-10 are anti-inflammatory cytokines and play a key role in controlling intestinal homeostasis and immunity. MLCK is widely expressed in various tissues and organs, and its main function is to catalyze the phosphorylation of myosin light chain (MLC) to regulate cell contraction. There is a structure called the perijunctional actomyosin ring (PAMR) near the tight junctions between intestinal epithelial cells. MLCK causes the contraction of PAMR by phosphorylating MLC, pulling the tight junction proteins connected to it, thereby increasing the adjacent cell gap and increasing the permeability between intestinal epithelial cells. TNF-α and IL-1β have been found to cause high expression of MLCK by activating the enzyme activities of ERK1 / 2 and NF-κB p65, respectively.

[0044] The present application study found that after co-culture with Caco-2 cell monolayer, Lactobacillus rhamnosus KF7 can strengthen the intestinal barrier function by upregulating the expression level of mucin-related genes. When lipopolysaccharide is used to induce Caco-2 cell monolayer barrier damage, the fermented milk supernatant of Lactobacillus rhamnosus KF7 can restore the integrity of the intestinal epithelial barrier structure by increasing the expression of tight junction protein genes, and improve the inflammatory response and intercellular permeability of intestinal epithelial cells by inhibiting the TLR / MyD88 / NF-κB signaling pathway and the gene expression of downstream MLCK. The present application study found that when the rhythm disorder of Caenorhabditis elegans is induced by temperature cycle changes, the intervention of Lactobacillus rhamnosus KF7 helps to restore the integrity of the intestinal barrier of the rhythm-disordered nematodes, improve the diversity of the intestinal flora of the nematodes, regulate the balance of the intestinal microbiota, and improve the growth, development and metabolic state of the nematodes. The present invention broadens the scope of application of Lactobacillus rhamnosus as a probiotic, and provides a new method for improving intestinal barrier damage and intestinal flora disorder.

[0045] In the application provided in this application, the diversity of intestinal flora is α-diversity. α-diversity analysis can reflect the abundance and diversity of microbial communities, where ACE and Chao indexes are used to calculate flora abundance, and the larger the index value, the more species there are; Shannon and Simpson indexes are used to calculate flora diversity, and the larger the Shannon index value, the higher the community diversity, and conversely, the larger the Simpson index value, the lower the community diversity; Coverage represents the sequencing depth, and the higher the value, the higher the probability that the sequence in the sample is detected.

[0046] In the use provided in the present application, the beneficial bacteria include Firmicutes and / or Planctomyces; further, the beneficial bacteria include a combination of one or more of the genera Cystomyces, Salinosporium, and Lactobacillus; further, the beneficial bacteria include a combination of one or more of Clostridium, Lactobacillus, Enterobacter, and Enterococcus. Firmicutes mainly include some beneficial bacteria that produce short-chain fatty acids, such as Clostridium, Lactobacillus, Enterobacter, and Enterococcus, and a reduction in their number can lead to colon inflammation. Lactobacillus is a Gram-positive, non-spore-forming bacillus. It has a strong ability to decompose sugars, and also has the effects of regulating intestinal flora, enhancing immunity, protecting gastric mucosa, improving intestinal function, and anti-oxidation.

[0047] In the use provided in the present application, harmful bacteria include Proteobacteria and Cyanobacteria; further, harmful bacteria include one or more combinations of Corynebacterium, Achromobacter, and Escherichia. Increased Proteobacteria and decreased Firmicutes are the main manifestations of intestinal inflammation. An increased proportion of Proteobacteria is a potential marker for diagnosing microbial community imbalance and disease risk. Proteobacteria are closely related to inflammation caused by host nutritional and metabolic disorders and immune disorders.

[0048] In the uses provided by this application, the growth and development indicators include body size. In some embodiments, the body size can be measured by body length and body width. Body length and body width can reflect the development speed and growth of nematodes from the side, and are common indicators for evaluating the growth and development of nematodes.

[0049] In the uses provided by this application, the metabolic level is reflected by the pharyngeal pumping frequency. The pharyngeal pumping frequency can reflect the feeding ability and metabolic level of nematodes.

[0050] In the uses provided by this application, intestinal barrier damage is caused by rhythm disorder or lipopolysaccharide; further, the rhythm disorder is caused by cyclic temperature changes; the cyclic temperature changes are cycling between culturing at 20-25 °C for 4-20 h and culturing at 15-18.5 °C for 4-20 h. The disorder of rhythm will change the composition of intestinal microbiota and the integrity of the intestinal barrier. Studies have found that compared with the normal control group, the relative abundance of Odoribacter bacteria that can produce short-chain fatty acids in the intestines of subjects with circadian rhythm disorder is significantly reduced. Animal experiments have also found that the disruption of the circadian rhythm in mice leads to a significant increase in the abundance of Clostridium in the intestines. Some diseases related to circadian rhythm (such as diabetes, hypertension, sleep disorders, and chronic inflammation) all show clinical symptoms of damaged intestinal barrier integrity. Lipopolysaccharides (LPS) are the main components of the cell walls of Gram-negative bacteria, mainly composed of lipids and polysaccharides. LPS is a powerful stimulator of the immune system, which will cause the production of pleiotropic cytokines / chemokines, trigger an inflammatory signal cascade reaction, and then regulate inflammatory, innate immune, and adaptive immune responses. There are a large number of Gram-negative symbiotic bacteria in the human colon, which maintain a balance with Gram-positive bacteria in the healthy human intestine. However, when the intestinal flora is imbalanced and the abundance of Gram-negative bacteria increases, the increase in the level of LPS in the intestinal lumen will lead to intestinal inflammation and damage the function of the intestinal epithelial barrier.

[0051] In the uses provided by this application, the product dosage form is solid, liquid, gel, semi-liquid, aerosol or powder. In the uses provided by this application, the products include but are not limited to foods, health products, drugs, etc.

[0052] In some embodiments, the products are selected from fermented milk products, lactic acid bacteria beverage products, probiotic agents, probiotic powder products, live aquatic product products, milk powder products or cheese products.

[0053] In some embodiments, the stabilizer is pectin.

[0054] In some embodiments, the thickener is a conventional thickener well-known to those skilled in the art, for example, it can be pectin, agar, carrageenan, locust bean gum, gelatin, sodium caseinate, xanthan gum, modified starch, etc.

[0055] On the other hand, the present application provides a fermented milk product, the raw materials of which include Lactobacillus rhamnosus KF7, animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum, or a combination of one or more of them.

[0056] In some embodiments, the preparation method of the fermented milk product is as follows:

[0057] 1. Inoculate the activated Lactobacillus rhamnosus KF7 into whole milk at 3% - 5% (mass percentage), and culture it under suitable conditions to prepare a starter culture.

[0058] 2. Heat the whole milk to about 55 - 60 °C, add 4 - 7% sucrose (mass percentage) and an appropriate amount of thickener, stir to fully dissolve and mix evenly, then heat up to about 60 - 70 °C and perform homogenization under a pressure of 18 - 22 MPa, where the primary pressure is 16 - 18 MPa and the secondary pressure is 2 - 4 MPa. After cooling to 35 - 42 °C, add the starter culture prepared in step 1 at an inoculation amount of 3 - 5% (volume percentage), ferment at 35 - 42 °C until coagulation, and the acidity at the end of fermentation is 70 - 90 °T. After cooling, store at 4 - 6 °C to obtain the fermented milk product with the effects of improving intestinal barrier damage and regulating the intestinal flora.

[0059] In the above preparation method of the fermented milk product, the activation in step 1 is a strain activation step well-known to those skilled in the art. For example, it can be to pick a single colony of Lactobacillus rhamnosus KF7 with an inoculation loop and streak it on an MRS solid plate, culture it in a 37 °C constant temperature anaerobic incubator for two days, and repeat three times. Finally, pick a single colony into an MRS liquid medium and culture it overnight at 37 °C to prepare an activated Lactobacillus rhamnosus KF7 bacterial solution.

[0060] In the above preparation method of the fermented milk product, in step 1, a suitable culture environment can be, for example: inoculate the activated bacterial solution into whole milk (heat-treated at 95 °C for 5 min) at 3% - 5% (mass percentage), culture at 37 °C for 24 h, and cool to 4 °C to be used as a starter culture (the viable count is about 10 8 CFU / mL) for standby.

[0061] In the above preparation method of the fermented milk product, after adding the starter culture in step 2, optionally, Lactobacillus bulgaricus powder and Streptococcus thermophilus powder can be added according to experimental needs. Based on the mass of the mixed fermented milk product, the concentration of Lactobacillus bulgaricus powder is 0.01 - 0.02 g / kg; the concentration of Streptococcus thermophilus powder is 0.015 - 0.03 g / kg. The functions of Lactobacillus bulgaricus powder and Streptococcus thermophilus powder here are to act as a starter culture, shorten the fermentation time, and improve the flavor of the fermented milk.

[0062] On the other hand, the present application provides a lactic acid bacteria beverage product, the raw materials of which include Lactobacillus rhamnosus KF7; optionally, it further includes one or a combination of more than one of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum.

[0063] In some embodiments, the product obtained according to the preparation method of the aforementioned fermented milk product is used as a fermented milk base material.

[0064] In some embodiments, the preparation method of the lactic acid bacteria beverage product is as follows:

[0065] 1. Mix white granulated sugar, stabilizer and water evenly by stirring, cool after high-pressure homogenization and sterilization;

[0066] 2. Add the aforementioned fermented milk base material to step 1, stir evenly, adjust the acidity, preheat to 55-60 °C, then carry out aseptic homogenization under a pressure of 18-22 MPa, and aseptically fill after cooling to about 10-15 °C, thus obtaining a lactic acid bacteria beverage product with the functions of improving intestinal barrier damage and regulating intestinal flora.

[0067] On the other hand, the present application provides a probiotic agent, which is obtained by freeze-drying a fermented and cultured strain after adding a freeze-drying protectant. The strain includes Lactobacillus rhamnosus KF7; optionally, it further includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum, Kluyveromyces.

[0068] In some embodiments, the strain is Lactobacillus rhamnosus KF7. The fermentation and culture of the strain is a method well-known to those skilled in the art. For example, the strain can be first activated, then inoculated into MRS liquid medium, expanded cultured under suitable temperature conditions, and then inoculated into a fermenter of the fermentation medium for further expanded fermentation to obtain the product.

[0069] In some embodiments, the freeze-drying protectant can be an acid regulator, protein protectant, saccharide protectant, surfactant, vitamin C, and sodium glutamate, etc. Further, the acid regulator can be phosphate buffer solution, cysteine hydrochloride, etc. The protein protectant can be skim milk powder, whey protein powder, etc. The saccharide protectant can be sucrose, maltodextrin, lactose, trehalose, etc. The surfactant can be glycerol, polyethylene glycol, etc. Those skilled in the art can add appropriate freeze-drying protectants according to experimental needs.

[0070] On the other hand, the present application provides a probiotic powder product, which is obtained by adding a dietary supplement, vitamin, fructooligosaccharide, maltodextrin and pectin to the probiotic agent in the aforementioned use, mixing evenly and then freeze-drying.

[0071] In some embodiments, the preparation method of the probiotic powder product is as follows:

[0072] Dissolve 15 - 30% wt of the aforementioned probiotic agent in distilled water, heat it to 20 - 30 °C, and add 10 - 12% wt of dietary supplement, 8 - 10% wt of vitamins, 10 - 15% wt of fructooligosaccharide, 18 - 47% wt of maltodextrin, and 10 - 15% wt of pectin. After mixing evenly, perform freeze-drying to obtain a probiotic powder product with the functions of improving intestinal barrier damage and regulating the intestinal flora.

[0073] In some embodiments, the viable count of the probiotic powder product is 10 8 - 10 11 CFU / g; specifically, it can be 10 8 - 10 9 CFU / g, 10 9 - 10 10 CFU / g, or 10 10 - 10 11 CFU / g, etc.

[0074] In some embodiments, the probiotic powder product is a mixed probiotic powder product, and the fermented milk raw material therein is Lactobacillus rhamnosus KF7 and other strains. The other strains include one or more strains selected from Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum, Kluyveromyces. The fermentation and culture of the other strains are methods well-known to those skilled in the art. In a specific embodiment of the present application, the other strains are subjected to activation treatment and enlarged culture. The culture conditions are a temperature of 37 °C and a culture time of 15 h. The enlarged culture solution of the strain is inoculated into the fermenter of the fermentation medium and stirred and cultured at 37 °C for 12 h, and the pH value is controlled at about 5.5.

[0075] On the other hand, the present application provides a live bacteria aquatic product, which is obtained by adding vitamin C and a stabilizer to the probiotic agent in the aforementioned use. Based on the mass of the live bacteria aquatic product, the concentration of the probiotic agent is 10 6 - 10 9 CFU / g.

[0076] In some embodiments, based on the mass of the live bacteria aquatic product, the concentration of the probiotic agent is 10 6 - 10 9 CFU / g; specifically, it can be 10 6 - 10 7 CFU / g, 10 7 - 10 8 CFU / g, or 10 8 - 10 9 CFU / g, etc.

[0077] In some embodiments, the preparation method of the live bacteria aquatic product is as follows:

[0078] Dissolve the aforementioned probiotic agent in water, which also contains 100 - 300 mg / 100 ml of vitamin C and 0.1 - 0.2% wt of pectin stabilizer. After mixing evenly, a live bacteria aquatic product with the functions of improving intestinal barrier damage and regulating intestinal flora is prepared.

[0079] On the other hand, the present application provides a milk powder product, the raw materials of which include Lactobacillus rhamnosus KF7, and also include one or more combinations of whole milk powder, skim milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin and compound nutrients.

[0080] In some embodiments, the compound nutrients can be one or more combinations of ultramicro calcium carbonate, vitamin B6, vitamin C, vitamin E, vitamin A, vitamin D, ferric pyrophosphate, zinc oxide, sodium selenite, taurine, folic acid, arachidonic acid, docosahexaenoic acid, niacin.

[0081] In some embodiments, optionally, the milk powder product further includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum.

[0082] In some embodiments, the preparation method of the milk powder product is as follows:

[0083] 1. Mix whole milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin and compound nutrients evenly, add water and then perform homogenization treatment, high-temperature sterilization, stir evenly, and spray dry to obtain milk powder;

[0084] 2. Add 0.04 - 0.08% wt of Lactobacillus rhamnosus KF7 to the milk powder before packaging to obtain a milk powder product with the functions of improving intestinal barrier damage and regulating intestinal flora.

[0085] On the other hand, the present application provides a cheese product, the raw materials of which include Lactobacillus rhamnosus KF7, and also include one or more combinations of raw milk, commercial starter culture and rennet.

[0086] In some embodiments, the preparation method of the cheese product is as follows:

[0087] 1. Standardize the raw milk so that the weight ratio of fat to protein in it is 1.5 - 1.8:1 to obtain raw material milk;

[0088] 2. Pasteurize the raw material milk and cool the raw material milk to 30 - 32 °C; inoculate commercial starter culture and Lactobacillus rhamnosus KF7, add 0.002 - 0.006 g / L of rennet, stir and ferment to obtain curd.

[0089] 3. Cut the fermented curd into curds with a volume of 1.2 - 1.5 cm 3 and stir slowly for 10 - 30 min. Wash the curds with water and keep stirring for 20 - 30 min. Drain the whey, add square modules, and press for 60 - 100 min;

[0090] 4. Cure with 1 - 3% wt of salt and ripen at 10 - 15 °C for 5 - 6 months to obtain a cheese product with the function of improving intestinal barrier damage and regulating the intestinal flora.

[0091] In some embodiments, in step 2 of the preparation method of the cheese product, the pasteurization is carried out at 73 °C for 15 s.

[0092] Compared with the prior art, the beneficial effects of the present application are as follows:

[0093] The present application first discloses that Lactobacillus rhamnosus KF7 can not only improve the intestinal epithelial barrier function, protect it from lipopolysaccharide-induced barrier damage, but also restore the intestinal barrier damage caused by disrupted rhythm in Caenorhabditis elegans, regulate the intestinal flora, and improve the growth, development and metabolic status of C. elegans. It can be used in the production of functional food products such as fermented milk, lactic acid bacteria beverages, probiotic powders, live bacteria water, milk powders and cheeses.

[0094] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0095] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments, rather than limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include the plural forms.

[0096] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any value therebetween can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0097] Statistical analysis was performed using SPSS 20.0 software, and various statistical charts were made using GraphPad Prism 7. All data were expressed as mean ± standard deviation. Unpaired t-tests (Students test) were used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for comparisons among multiple groups (three groups and above). P < 0.05 was considered statistically significant. The QIIME (Quantitative Insights Into Microbial Ecology) and R software were used to analyze the microbiota sequencing data. The RDP classifer Bayesian algorithm was used to annotate the species of the representative sequences of OTUs (Operational Taxonomic Units) at the 97% similarity level, and the community composition of each sample was statistically analyzed at each taxonomic level. Significant differences were indicated by *P < 0.05, **P < 0.01, ***P < 0.001, and different lowercase letters.

[0098] The present application will be further illustrated by the following examples, but the scope of the present application is not limited thereby.

[0099] Example 1

[0100] Co-culture of Lactobacillus rhamnosus KF7 with Caco-2 cell monolayers

[0101] Caco-2 cells (ATCC, HTB37) were cultured in Medium 199 culture medium (ThermoFisher Scientific, 11150059) containing 10% fetal bovine serum (HyClone, SV30208.02) and 1% non-essential amino acids (ThermoFisher Scientific, 11140050) in a cell culture incubator at 37 °C and 5% CO2. Before the co-culture experiment, Caco-2 cells were seeded at 8 × 10 4Cells were inoculated at a concentration of and cultured on a Transwell polycarbonate membrane (6.5 mm, 0.4 μm pores) (Greiner bio-one, 665641) for 17 days. The Caco-2 cell monolayer was transferred to a dual-environment co-culture system in an anaerobic workstation (refer to the experimental method of Maier et al., doi: 10.3390 / nu9121349). The specific method is as follows: Each well of the co-culture chamber contains 3 mL (aerobic) Medium 199 culture medium (containing 1% non-essential amino acids and 10% fetal bovine serum), and a Transwell insert containing the Caco-2 monolayer is placed at the top of each well. The Transwell insert contains 260 μL (anaerobic) Medium 199 culture medium (containing 1% non-essential amino acids, and fetal bovine serum is not added to prevent excessive bacterial growth). The Transwell insert separates the aerobic environment from the anaerobic environment of the anaerobic workstation, so that the Caco-2 cell monolayer is exposed to the anaerobic environment at the top, and oxygen diffuses through the semi-permeable membrane from the bottom of the chamber, ensuring the survival of Caco-2 cells ( Figure 1 ). On the day of the co-culture experiment, the Caco-2 cell monolayer was incubated under anaerobic conditions at the top for about 1 h, and then the cells of Lactobacillus rhamnosus KF7 (the number is about 1000 times the number of Caco-2 cells) were added to the (anaerobic) Medium 199 culture medium for co-culture with the cells.

[0102] Example 2

[0103] Enhanced intestinal epithelial barrier function after co-culture of Lactobacillus rhamnosus KF7 with Caco-2 cell monolayer

[0104] The co-culture system in Example 1 was connected to a CellZscope trans-epithelial electrical resistance automatic monitoring system (NanoAnalytics, Germany, software version 2.2.3) to measure the TEER (trans-epithelial electrical resistance) value of the Caco-2 cell monolayer. The change in the TEER value of each cell monolayer was calculated using the following formula: Change in TEER value (%) = TEER (Ω.cm 2 ) / Baseline TEER (Ω.cm 2 ) × 100%.

[0105] As Figure 2 shown in A, the barrier integrity of the Caco-2 cell monolayer remained stable in the dual-environment co-culture system. Within 0 - 12 h, the TEER values of the two groups of cell monolayers showed a downward trend in the first hour, which may be the adaptation stage after the cells were transferred from the aerobic environment to the top anaerobic environment. The TEER value of the control group (No treatment) continued to decline within 1 - 12 h, but the TEER value was > 500 Ω.cm throughout the process 2, it was considered that the barrier integrity was not damaged during the entire experimental stage. The TEER value of the KF7 group was significantly higher than that of the control group within the range of 6 - 12 h (P < 0.05), indicating that the co - culture of Lactobacillus rhamnosus KF7 could strengthen the intestinal epithelial barrier function.

[0106] According to the TEER experimental results, we collected Caco - 2 cells at the 4 - h and 8 - h time points. According to the RNA extraction kit (TaKaRa, 9767) and the FastKing one - step genomic DNA removal and cDNA first - strand synthesis premix kit ( KR118 - 02), RNA extraction and cDNA synthesis were carried out. Then, PCR amplification was performed according to the instructions of the QuantiNova SYBR Green PCR Kit (QIAGEN, 208054). The reaction system was 20 μL and the number of cycles was 40. The primer sequences used are shown in Table 1, where β - actin is the internal reference gene.

[0107] As Figure 2 shown in B, there was no significant difference in the expression level of MUC - 2 between the KF7 group and the control group at 4 h, but the expression level of MUC - 2 in the KF7 group was significantly higher than that of the control group at 8 h (P < 0.05). The intestinal mucus layer is a natural barrier against the invasion of intestinal pathogenic bacteria and harmful substances, and MUC is its main component. As a secreted mucin, MUC - 2 is widely expressed in the intestine. Its monomer is a homologous oligomer containing an N - terminal oxidized glycosylation domain and a C - terminal cysteine - rich domain, which constitutes the skeleton of the intestinal mucus layer and can provide biological and adhesion sites for bacteria and immunoglobulins, playing an important role in the intestinal mucosal barrier. The above results indicate that the co - culture of Lactobacillus rhamnosus KF7 and Caco - 2 cell monolayers may strengthen the intestinal barrier function by up - regulating the expression level of the MUC - 2 gene.

[0108] Example 3

[0109] Preparation of the supernatant of Lactobacillus rhamnosus KF7 fermented milk

[0110] Using an inoculation loop, pick a single colony of Lactobacillus rhamnosus KF7 and streak it on an MRS ( Merck, Germany, product number: 1106610500) solid plate, and culture it in a 37 °C constant - temperature anaerobic incubator for two days, repeating three times. Inoculate a single colony into 3 mL of sterilized 10% (mass percentage) skim milk and ferment the curd at 37 °C. Inoculate the above - mentioned fermentation broth into sterilized 10% (mass percentage) skim milk at an inoculation amount of 3% (volume percentage) and continue to culture for 20 h to obtain Lactobacillus rhamnosus KF7 fermented milk (1.73×10 8CFU / mL). Centrifuge at 9000 r / min at 4 °C for 10 min, discard the precipitate. After adjusting the pH of the supernatant to 7.3, centrifuge again at 9000 r / min at 4 °C for 10 min, discard the precipitate, take the supernatant, and filter it through a 0.22 μm filter membrane as the subsequent intervention sample, and store it at -20 °C for later use.

[0111] Example 4

[0112] The supernatant of Lactobacillus rhamnosus KF7 fermented milk protects the Caco-2 cell monolayer from LPS-induced barrier damage

[0113] Caco-2 cells were cultured in MEM medium (Corning, 10-009-CV) containing 20% fetal bovine serum (HyClone, SV30208.02) and 1% antibiotics (ThermoFisher Scientific, 15140122) in a cell culture incubator at 37 °C and 5% CO2. After growing to the logarithmic phase, Caco-2 cells were seeded on Transwell polycarbonate membranes (0.4 μm pores) at a concentration of 1×10 5 cells / well, and the culture medium was changed every two days, and the TEER value was detected using an epithelial volt-ohmmeter (Merck, ERS-2). After the monolayer resistance value was relatively stable, 1%, 3%, and 5% of the KF7 fermented milk supernatant were added for intervention for 4 h respectively, and then 10 μg / mL of LPS (Sigma-Aldrich, L2880) was added and incubated for 24 h to cause monolayer barrier damage of the cells. The groups were as follows: normal control group (NC), LPS model group (LPS), low-dose KF7 group (1% KF7 + LPS), medium-dose KF7 group (3% KF7 + LPS), and high-dose KF7 group (5% KF7 + LPS). The TEER values were measured before and after the intervention. The change in TEER value (%) = (TEER after intervention / TEER before intervention) × 100%.

[0114] As Figure 3 shown, compared with the NC group, LPS significantly induced a decrease in the transepithelial electrical resistance of the Caco-2 cell monolayer (P = 0.0042). Compared with the LPS group, the intervention with the supernatant of Lactobacillus rhamnosus KF7 fermented milk could significantly increase the transepithelial electrical resistance of the cell monolayer (P = 0.0267, P = 0.0024, P = 0.0141). The above results indicate that the supernatant of Lactobacillus rhamnosus KF7 fermented milk can protect the intestinal epithelial cell monolayer from LPS-induced barrier function damage.

[0115] Example 5

[0116] Effect of the supernatant of Lactobacillus rhamnosus KF7 fermented milk on the expression of tight junction protein-related genes in Caco-2 cells

[0117] After culturing Caco-2 cells to the logarithmic phase according to the method in Example 4, they were inoculated onto a 6-well plate at a concentration of 2×10 5 cells per well. After overnight culture, the old culture medium was removed, and MEM culture medium containing 1%, 3%, and 5% fermented milk supernatant of Lactobacillus rhamnosus KF7 was added respectively, and cultured for 4 h, then 10 μg / mL of LPS was added to intervene for 24 h. RNA extraction and cDNA synthesis were carried out according to the instructions of the RNA extraction kit (TaKaRa, 9767) and the FastKing one-step genomic DNA removal and cDNA first-strand synthesis premix kit ( KR118-02). Then, PCR amplification was carried out according to the instructions of the QuantiNova SYBR Green PCR Kit (QIAGEN, 208054). The reaction system was 20 μL and the number of cycles was 40 times. The primer sequences used are shown in Table 1, where β-actin is the internal reference gene.

[0118] Table 1 Primer sequences

[0119]

[0120] As Figure 4 shown, compared with the NC group, the expressions of Claudin-1 and Occludin genes in the LPS group were significantly decreased (P = 0.0127, P = 0.0039), and the expression of the ZO-1 gene showed a downward trend, but there was no significant difference. The intervention of the fermented milk supernatant of Lactobacillus rhamnosus KF7 could up-regulate the decrease in the expression of tight junction protein-related genes caused by LPS to varying degrees. Among them, the fermented milk supernatant at a concentration of 5% had the most significant improvement effect on the expressions of Claudin-1 and Occludin genes (P = 0.0333, P = 0.0162). Tight junctions located at the apical side membrane of intestinal epithelial cells are the main factors determining paracellular permeability, including tight junction proteins Occludin, Claudin, ZO, etc. The integrity of tight junction proteins in the epithelial monolayer can be represented by a quantitative index, such as trans-epithelial electrical resistance (TEER), so as to determine the degree of intestinal barrier damage. The above results indicate that the fermented milk supernatant of Lactobacillus rhamnosus KF7 can improve the expression of tight junction protein-related genes among Caco-2 cells induced by LPS.

[0121] Example 6

[0122] Effect of fermented milk supernatant of Lactobacillus rhamnosus KF7 on inflammatory signaling pathways and downstream signaling molecules in Caco-2 cells

[0123] Detect the expression levels of genes related to the inflammatory signaling pathway and downstream signaling molecules in Caco-2 cells according to the method in Example 5. The primer sequences used are shown in Table 2, where β-actin is the reference gene.

[0124] Table 2 Primer Sequences

[0125]

[0126]

[0127] As Figure 5 shown in A-D, compared with the NC group, the LPS group significantly up-regulated the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 (P<0.05). The high-dose KF7 group significantly decreased the expression level of TNF-α (P = 0.0205), and the low-, medium-, and high-dose KF7 groups all significantly decreased the expression levels of IL-1β and IL-6 (P<0.05), but had little effect on the expression level of IL-8. As Figure 5 shown in E and F, compared with the NC group, neither the LPS group nor the KF7 group had a significant effect on the expression level of TGF-β, but LPS could significantly down-regulate the expression level of the anti-inflammatory cytokine IL-10 (P = 0.0200), while the supernatant of Lactobacillus casei rhamnosus KF7 fermented milk could significantly up-regulate its expression (P<0.001).

[0128] As Figure 6 shown, compared with the NC group, the expression levels of MyD88 and NF-κB p65 genes in the LPS group were both significantly increased (P<0.001), while the low-, medium-, and high-dose KF7 groups could all significantly decrease the expression levels of both (P<0.001). In addition, we also detected the gene expression of myosin light chain kinase (MLCK) and found that LPS significantly increased the expression of the MLCK gene (P = 0.0009), while its expression level was significantly decreased in the KF7 group (P<0.01).

[0129] The TLR / MyD88 / NF-κB signaling pathway is an important pathway in the body's inflammatory system, widely present in various tissue cells, and is involved in the occurrence and regulation of various diseases. MyD88 (Myeloid Differentiation factor 88) is an important transduction protein in this pathway and a key node for signal transduction downstream. NF-κB is the main transcription factor responsible for regulating innate and adaptive immune responses. In the body, NF-κB usually functions as a heterodimer formed by the binding of p50 and p65. In the resting state, the heterodimer binds to the inhibitor IκB (inhibitory κB) and exists in an inactive state; when stimulated, IκB is phosphorylated and then ubiquitinated and degraded, releasing the p50 / p65 complex, which is transported into the nucleus and binds to the promoter of specific genes, thereby regulating the expression of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8, etc.). TGF-β and IL-10 are anti-inflammatory cytokines and play a key role in controlling intestinal homeostasis and immunity. MLCK is widely expressed in various tissues and organs, and its main function is to catalyze the phosphorylation of myosin light chain (MLC) to regulate cell contraction. There is a structure called the perijunctional actomyosin ring (PAMR) near the tight junctions between intestinal epithelial cells. MLCK causes the contraction of PAMR by phosphorylating MLC, pulling the tight junction proteins connected to it, thereby increasing the adjacent cell gap and increasing the permeability between intestinal epithelial cells. TNF-α and IL-1β have been found to cause high expression of MLCK by activating the enzyme activities of ERK1 / 2 and NF-κB p65, respectively. The above results indicate that the fermented milk supernatant of Lactobacillus casei rhamnosus KF7 may improve the LPS-induced cellular inflammatory response and the increased intercellular permeability caused by inflammatory factors by inhibiting key molecules in the inflammatory signaling pathway, thereby restoring intestinal barrier function.

[0130] Example 7

[0131] Culture of Escherichia coli OP50:

[0132] Pick a single colony of Escherichia coli OP50 with an inoculation loop into LB liquid medium (tryptone 1 g, yeast extract 0.5 g, NaCl 1 g, add distilled water to a volume of 100 mL, autoclave at 121 °C for 15 min), and incubate at 37 °C with constant shaking at 100 - 200 rpm for 18 - 20 h. The viable cell count is measured to be 1.43×10 9 CFU / mL. Store at 4 °C in the refrigerator for later use.

[0133] Example 8

[0134] Culture of Lactobacillus rhamnosus KF7 and Preparation of Probiotic Bacterial Solution for Intervention

[0135] Use an inoculation loop to pick a single colony of Lactobacillus rhamnosus KF7 and streak it on an MRS ( Merck, Germany, product number: 1106610500) solid plate, and culture it in a 37°C constant temperature anaerobic incubator for two days, repeating three times. Finally, pick a single colony and culture it overnight in MRS liquid medium to prepare a Lactobacillus rhamnosus KF7 bacterial solution. Take the above bacterial solution and add it to a new MRS liquid medium at a concentration of 3% (volume percentage), and culture it in a 37°C incubator for 20 - 24 h. The viable count is measured to be 1.73×10 8 CFU / mL. Store it in a 4°C refrigerator for later use.

[0136] Concentrate the above Lactobacillus rhamnosus KF7 bacterial solution by 10 times, resuspend it in LB liquid medium, and mix it with the Escherichia coli OP50 bacterial solution prepared in Example 7 at a volume ratio of 1:1 to obtain the OP50 + KF7 bacterial solution for feeding the nematodes in the probiotic intervention group.

[0137] Example 9

[0138] Preparation of Nematode Growth Medium (NGM)

[0139] Weigh 0.3 g of NaCl, 2 g of agar powder, and 0.25 g of bacteriological peptone, add deionized water and make up the volume to 100 mL. After shaking well, autoclave at 121°C for 15 min. After autoclaving, cool to 55°C, and add 0.1 mL of CaCl2 (1 mol / L) ion solution, 0.1 mL of MgSO4 (1 mol / L), 2.5 mL of K3PO4 (1 mol / L), and 0.32 mL of cholesterol solution (5 mg / mL absolute ethanol) that have been sterilized by microporous filtration. Mix well. Pour the above solution into a 6 cm diameter petri dish, and after solidification, place it at room temperature for 2 days to evaporate water and check for contamination by miscellaneous bacteria. Store it in a 4°C refrigerator for later use.

[0140] Aspirate 200 μL of the Escherichia coli OP50 bacterial solution prepared in Example 7, drop it onto the center of the NGM plate and spread it evenly, and place it in a 37°C incubator for 8 h. Prepare the NGM plate coated with Escherichia coli OP50 and store it in a 4°C refrigerator for later use.

[0141] Aspirate 200 μL of the OP50 + KF7 bacterial solution prepared in Example 8, drop it onto the center of the NGM plate and spread it evenly, and place it in a 37°C incubator for 8 h. Prepare the NGM plate coated with OP50 + KF7 and store it in a 4°C refrigerator for later use.

[0142] Example 10

[0143] Culture and synchronization of Caenorhabditis elegans:

[0144] Use a dissecting needle to transfer a single nematode in the egg-laying period to a new NGM plate, or use a sterile scalpel to cut an appropriate-sized agar block with nematodes on the old NGM plate, invert it face down on the surface of the new NGM plate, make a mark, and culture it in a 20°C biochemical incubator. Subculture every 3-4 days to maintain the activity of the nematodes.

[0145] Select a plate with more adult nematodes in the reproductive period for nematode synchronization. Add 2 mL of M9 buffer (0.5 g of NaCl, 0.6 g of Na2HPO4, 0.3 g of KH2PO4, add distilled water to a volume of 100 mL, after sterilization, add 0.1 mL of 1 mol / L MgSO4 filtered and sterilized through a 0.45 μM microporous filter) to the plate, pipette with a sterile pipette to wash the egg-laying nematodes off the NGM plate, transfer them to a 1.5 mL centrifuge tube, centrifuge at 800×g for 1 min, and discard the supernatant. Add 1 mL of M9 buffer, centrifuge at 800×g for 1 min again to wash the worms, and discard the supernatant. Add 1 ml of fresh nematode lysis solution (1 mL of 8 mol / L NaOH, 0.6 mL of 9% sodium hypochlorite, 3.4 mL of distilled water), immediately vortex for 10 s, centrifuge at 800×g for 1 min, and discard the supernatant. Immediately add 1 mL of M9 buffer to wash the eggs, centrifuge at 800×g for 1 min, and discard the supernatant. Repeat the washing 2 times. Finally, there is about 50 μL of suspension left in the tube. Use a sterile pipette to drop the eggs onto the NGM plate coated with OP50 in Example 9, culture at 20°C, and after about 48 h, a large number of L1-stage larvae can be seen under the microscope, confirming successful synchronization.

[0146] Example 11

[0147] Experimental grouping of Caenorhabditis elegans:

[0148] Control group (Control): Transfer the synchronized L1-stage nematodes to the NGM plate coated with Escherichia coli OP50 in Example 9, and culture them for 7 days under the temperature cycle conditions of darkness, 20°C for 12 h; 18.5°C for 12 h.

[0149] Rhythm disorder model group (Model): Transfer the synchronized L1-stage nematodes to the NGM plate coated with Escherichia coli OP50 in Example 9, culture them for 4 days under the temperature cycle conditions of darkness, 20°C for 12 h; 18.5°C for 12 h, and then transfer them to a constant temperature of 20°C for 3 days.

[0150] Probiotic intervention group (KF7): Synchronized L1-stage nematodes were transferred onto NGM plates coated with Escherichia coli OP50 in Example 9 and cultured for 4 days under the temperature cycling conditions of darkness, 20°C for 12 h; 18.5°C for 12 h. Then the nematodes were transferred onto NGM plates coated with OP50+KF7 in Example 9 and cultured for 3 days in a constant temperature environment of 20°C.

[0151] Example 12

[0152] Effect of Lactobacillus rhamnosus KF7 on intestinal permeability of rhythm-disordered nematodes:

[0153] The intestinal permeability of the nematodes in Example 11 was detected using food coloring brilliant blue (Sigma, USA, product number: 861146) and fluorescein isothiocyanate-dextran (Sigma, USA, product number: FD4).

[0154] Food coloring brilliant blue method: Prepare 2.5% (wt / vol) food coloring brilliant blue (2.5 g food coloring brilliant blue + 100 mL LB solution containing OP50). After culturing the three groups of nematodes under different conditions for 7 days, they were collected in centrifuge tubes containing food coloring brilliant blue and placed in the dark at 20°C for 3 h. After staining, the nematodes were washed 3 times with M9 buffer and transferred to NGM plates to recover for 45 min. The distribution of the dye in the intestinal lumen and body cavity was observed under a 40-fold microscope. If there is damage to the nematode intestine, the brilliant blue dye will penetrate from the intestine into the body cavity and cannot be excreted from the body.

[0155] Fluorescein isothiocyanate-dextran method: Prepare 0.5% wt / vol fluorescein isothiocyanate-dextran solution (0.025 g fluorescein isothiocyanate-dextran + 5 mL distilled water). After culturing the three groups of nematodes in Example 11 under different conditions for 7 days, they were collected in centrifuge tubes containing fluorescein isothiocyanate-dextran solution and placed in the dark at 20°C for 3 h. After staining, the nematodes were washed 3 times with M9 buffer and transferred to NGM plates to recover for 45 min. The nematode density was adjusted to 100 - 200 nematodes / 50 μL and added dropwise to a 96-well black plate with a white bottom. The total fluorescence intensity was detected using a microplate reader (excitation wavelength 480 nm, emission wavelength 520 nm).

[0156] The results of the food coloring brilliant blue method are as Figure 7 shown in A. Leakage of the blue dye was observed in the nematodes of the rhythm-disorder model group (indicated by the black arrow), and no leakage was observed in the nematodes of the control group and the probiotic intervention group.

[0157] The results of the fluorescein isothiocyanate-dextran method are as Figure 7As shown in Figure B. The fluorescence intensity of the rhythm disorder model group was significantly higher than that of the control group (P<0.05), indicating that intestinal leakage occurred in the nematodes of the model group. The fluorescence intensity of the probiotic intervention group was significantly reduced (P<0.01), and there was no statistical difference from the control group.

[0158] The above results indicate that rhythm disorder increases the intestinal permeability of nematodes, while consuming Lactobacillus casei rhamnosus KF7 can restore the intestinal barrier of rhythm-disordered nematodes to a certain extent.

[0159] Example 13

[0160] Effect of Lactobacillus casei rhamnosus KF7 on the intestinal microbiota of rhythm-disordered nematodes:

[0161] After culturing for 7 days, nematodes in the control group, rhythm disorder model group, and probiotic intervention group (about 400 in each group) were collected, washed 3 times with M9 buffer to clean the OP50 adhered to the nematode surface. Four samples were prepared for each group of nematodes and stored at -80°C. The samples were sent to Shanghai Jingzhou Gene Technology Co., Ltd. by dry ice for 16S sequencing.

[0162] α-diversity analysis can reflect the abundance and diversity of microbial communities. Among them, ACE and Chao indices are used to calculate the abundance of bacterial communities. The larger the index value, the more species there are in total. Shannon and Simpson indices are used to calculate the diversity of bacterial communities. The larger the Shannon index value, the higher the community diversity. On the contrary, the larger the Simpson index value, the lower the community diversity. Coverage represents the sequencing depth, and the higher the value, the higher the probability that the sequences in the sample are detected. The results of α-diversity analysis are as Figure 8 shown in Figure A and Table 3. Compared with the control group, the ACE, Chao, and Shannon indices in the rhythm disorder model group all showed a downward trend (P>0.05); the Simpson index showed an upward trend (P>0.05). Compared with the rhythm disorder model group, the ACE, Chao, and Shannon indices in the probiotic intervention group all showed an upward trend, and the ACE index increased significantly (P<0.05); the Simpson index showed a downward trend (P>0.05). These results indicate that Lactobacillus casei rhamnosus KF7 can improve the intestinal microbiota diversity of rhythm-disordered nematodes to a certain extent.

[0163] Table 3 α-diversity indices of intestinal microbiota of nematodes in each group (n = 4)

[0164]

[0165] β-diversity analysis is to compare and analyze the microbial community compositions of samples between different groups. Principal coordinates analysis (PCoA) is a classical multi-dimensional scaling analysis method, which can consider the phylogenetic relationship between two community members when comparing the differences between different community samples. By calculating the UniFrac distance between microbial community samples, the similarity between two community samples can be more objectively reflected. In this experiment, PCoA analysis was performed based on the unweighted UniFrac distance, and non-parametric multi-factor variance analysis (Adonis) was combined to study the differences in the compositions of the intestinal microbial communities of each group of nematodes. The results of β-diversity analysis are as Figure 8 shown in Figure B and Table 4. There were no significant differences in the β-diversity of the intestinal microbiota among the control group, the circadian rhythm disorder model group, and the probiotic intervention group (P>0.05).

[0166] Table 4 Results of Adonis analysis based on Unweighted Unifrac distance

[0167]

[0168] Note: Df represents degrees of freedom; Sums Of Sqs represents total variance (sum of squared deviations); MeanSqs represents mean variance; F.Model represents the F-test value; R2 represents the explanatory power of different groups for sample differences, and the larger the value, the higher the explanatory power of the grouping for the differences; Pr(>F) represents the P-value obtained through permutation test, and the smaller the P-value, the stronger the significance of differences between groups.

[0169] At the phylum level classification, the comparison results of the relative abundances of the intestinal microbiota of each group of nematodes are as Figure 8As shown in Table 5. Actinobacteria, Proteobacteria, and Firmicutes are the main components of the nematode gut microbiota, and the combined relative abundance of these three bacteria reaches over 90% in all three groups of nematodes. Among them, the relative abundance of Proteobacteria in the rhythm disorder model group increased from 18.31 ± 4.99% in the control group to 38.66 ± 23.88% (P = 0.08), and returned to 19.85 ± 18.92% (P = 0.25) after probiotic intervention; the relative abundance of Firmicutes in the rhythm disorder model group decreased from 16.26 ± 18.04% in the control group to 2.83 ± 4.56% (P = 0.39), and increased to 32.13 ± 11.62% (P = 0.02) after probiotic intervention. In addition to the high-abundance microorganisms, two low-abundance microorganisms were also found to be significantly different among the three groups of nematodes. Cyanobacteria were found to be unique to the rhythm disorder model group and were not detected in the other two groups. In contrast, Planctomycetes were detected in the control group and the probiotic intervention group, but not in the rhythm disorder model group. Some studies have found that an increase in Proteobacteria and a decrease in Firmicutes are the main manifestations of intestinal inflammation. Firmicutes mainly include some beneficial bacteria that produce short-chain fatty acids, such as Clostridium, Lactobacillus, Enterobacter, and Enterococcus, and a decrease in their numbers can lead to colonic inflammation. An increase in the proportion of Proteobacteria is a potential marker for diagnosing microbial community dysbiosis and disease risk, and Proteobacteria are closely related to host nutritional and metabolic disorders and immune disorder inflammation.

[0170] Relative abundances of gut microbiota of nematodes in each group at the phylum level (n = 4)

[0171]

[0172] Note: P1 represents the P value of the comparison between the rhythm disorder model group (Model) and the control group (Control); P2 represents the P value of the comparison between the probiotic intervention group (KF7) and the rhythm disorder model group (Model).

[0173] At the genus level classification, the comparison results of the relative abundances of gut microbiota of nematodes in each group are as Figure 8As shown in Table 6. Compared with the control group, the relative abundances of Corynebacterium_1, Achromobacter, and Escherichia-Shigella all showed an upward trend in the rhythm disorder model group (51.69±36.63%, 28.20±26.00%, and 6.86±6.72%, respectively), and the relative abundances all showed a downward trend after probiotic intervention (30.79±37.34%, 10.83±19.83%, and 0.95±1.68%, respectively). On the contrary, compared with the control group, Nannocystis and Salinispora were not detected in the rhythm disorder model group, but their relative abundances were found to be 0.25±0.48% and 0.17±0.32% in the probiotic intervention group (P = 0.04). It should be noted that the relative abundance of Lactobacillus was very low in both the control group and the rhythm disorder model group (0.01±0.01% and 0.03±0.05%, respectively), but its relative abundance increased significantly to 15.08±18.15% in the probiotic intervention group (P = 0.02). Lactobacillus is a Gram-positive, sporeless bacillus. It has strong sugar decomposition ability and also has functions such as regulating the intestinal flora, enhancing immunity, protecting the gastric mucosa, improving intestinal function, and antioxidant effects.

[0174] Relative abundances of nematode intestinal microorganisms at the genus level in each group (n = 4)

[0175]

[0176] Note: P1 represents the P value of the comparison between the rhythm disorder model group (Model) and the control group (Control); P2 represents the P value of the comparison between the probiotic intervention group (KF7) and the rhythm disorder model group (Model).

[0177] The above results indicate that rhythm disorder reduces the α-diversity of nematode intestinal microorganisms and changes the intestinal flora composition, while consuming Lactobacillus rhamnosus KF7 can, to a certain extent, increase the diversity of the intestinal flora of rhythm-disordered nematodes, increase the relative abundance of beneficial bacteria, reduce the relative abundance of harmful bacteria, and regulate the balance of the nematode intestinal microbiota.

[0178] Example 14

[0179] Effect of Lactobacillus rhamnosus KF7 on the body length and body width of rhythm-disordered nematodes:

[0180] At the end of the 7-day culture, nematodes in the control group, the circadian rhythm disorder model group, and the probiotic intervention group were collected. 5-6 nematodes were randomly selected from each group and transferred to NGM plates coated with OP50 bacterial solution, and nematode photos were taken under a 40× microscope. The body length and body width of the nematodes were calculated using Image Plus 3.0 image processing software.

[0181] The body length and body width can reflect the development speed and growth of nematodes from the side, and are common indicators for evaluating the growth and development of nematodes. The results are as Figure 9 shown. Compared with the control group, there was no statistical difference in the body length of the circadian rhythm disorder model group (P>0.05), while the body width was significantly reduced (P<0.001). After intervention with Lactobacillus rhamnosus KF7, the body length of the nematodes was significantly higher than that of the control group and the circadian rhythm disorder model group (P<0.001), and the body width was significantly higher than that of the circadian rhythm disorder model group (P<0.001). The above results indicate that consuming Lactobacillus rhamnosus KF7 can significantly increase the body size of nematodes and improve the growth and development indicators.

[0182] Example 15

[0183] Effect of Lactobacillus rhamnosus KF7 on the metabolism (pharyngeal pumping frequency) of circadian rhythm disorder nematodes:

[0184] At the end of the 7-day culture, nematodes in the control group, the circadian rhythm disorder model group, and the probiotic intervention group were collected. 6-7 nematodes were randomly selected from each group and transferred to NGM plates coated with OP50 bacterial solution (control group, circadian rhythm disorder model group) and OP50+KF7 bacterial solution (probiotic intervention group) respectively. After allowing the nematodes to adapt to the plate for 1 min, the pharyngeal pumping frequency of the nematodes was observed under a 40× microscope. Each nematode was video-recorded for 10 s, and the video was played back slowly until the number of pharyngeal pumping vibrations of the nematodes could be observed.

[0185] The pharyngeal pumping frequency can reflect the feeding ability and metabolic level of nematodes. The results are as Figure 10 shown. Compared with the control group, the pharyngeal pumping frequency of the nematodes in the circadian rhythm disorder model group was significantly reduced (P<0.001). After intervention with Lactobacillus rhamnosus KF7, the pharyngeal pumping frequency of the nematodes increased significantly (P<0.001) and returned to the control group level (no statistical difference from the control group). The above results indicate that consuming Lactobacillus rhamnosus KF7 can significantly improve the reduction in the metabolic level of nematodes caused by circadian rhythm disorder.

[0186] Example 16

[0187] Fermented milk product containing Lactobacillus rhamnosus KF7 and having the effects of improving intestinal barrier damage and regulating intestinal flora:

[0188] Use an inoculation loop to pick a single colony of Lactobacillus rhamnosus KF7 and streak it on an MRS solid plate. Incubate it in a 37°C constant temperature anaerobic incubator for two days, and repeat three times. Finally, pick a single colony and inoculate it into MRS liquid medium, and incubate it overnight at 37°C to prepare an activated Lactobacillus rhamnosus KF7 bacterial solution. Take the above-activated bacterial solution and inoculate it into whole milk (heat-treated at 95°C for 5 min) at 3% - 5% (volume percentage), and incubate it at 37°C for 24 h. After cooling to 4°C, it is used as a starter culture (the viable count is about 10 8 CFU / mL) for standby. Heat the whole milk to about 55°C, add 4% - 7% (mass percentage) sucrose and an appropriate amount of thickener, and stir for 15 min to fully dissolve and mix evenly. After heating to about 65°C, homogenize it under a pressure of 20 MPa. After cooling to 38°C, add the above starter culture at an inoculation amount of 3% - 5% (volume percentage), and ferment it at 37°C until curdled (the viable count is 10 6 ~10 9 CFU / mL), and the acidity is 70°T. After cooling, store it at 4°C - 6°C, and a fermented milk product with the functions of improving intestinal barrier damage and regulating intestinal flora is obtained.

[0189] Example 17

[0190] A lactic acid bacteria beverage product containing Lactobacillus rhamnosus KF7 and having the functions of improving intestinal barrier damage and regulating intestinal flora:

[0191] Use the fermented milk product in Example 16 as the fermented milk base material. Mix 10% wt white granulated sugar, 45% wt water, and 0.5% wt evenly, cool it after high-pressure homogenization and sterilization. Add 40% of the fermented milk base material, stir and mix evenly, and adjust the acidity to pH 4.4. After preheating to 60°C, perform aseptic homogenization under a pressure of 20 MPa. After cooling to about 15°C, perform aseptic filling, and a lactic acid bacteria beverage product with the functions of improving intestinal barrier damage and regulating intestinal flora is obtained (the viable count is about 10 6 ~10 9 CFU / g).

[0192] Example 18

[0193] A probiotic powder product containing Lactobacillus rhamnosus KF7 and having the functions of improving intestinal barrier damage and regulating intestinal flora:

[0194] After activating Lactobacillus rhamnosus KF7 according to the method in Example 16, it was inoculated into MRS liquid medium and expanded culture was carried out at 37 °C for 24 h. The expanded culture solution of the strain was inoculated into a fermenter of the fermentation medium and stirred and cultured at 37 °C for 12 h (the pH value was controlled at about 5.5). A freeze-drying protectant (10% wt soluble starch, 3% wt sodium salt, 12% wt skim milk powder, 14% wt fructooligosaccharide) was added and mixed evenly, and then obtained a probiotic agent after freeze-drying treatment. Take 18% wt of the probiotic agent and add it to distilled water, heat it to 25 °C, and add 12% wt of dietary supplement, 8% wt of vitamins, 12% wt of fructooligosaccharide, 38% wt of maltodextrin and 12% wt of pectin, mix evenly and then freeze-dry to obtain a probiotic powder product with the functions of improving intestinal barrier damage and regulating intestinal flora (the viable count is about 10 8 ~10 11 CFU / g).

[0195] Example 19

[0196] A mixed probiotic powder product containing Lactobacillus rhamnosus KF7 and having the functions of improving intestinal barrier damage and regulating intestinal flora:

[0197] After activating Lactobacillus rhamnosus KF7 according to the method in Example 16, it was inoculated into MRS liquid medium and expanded culture was carried out at 37 °C for 24 h. Lactobacillus animalis subsp. lactis BB-12, Lactobacillus casei BDII, Lactobacillus rhamnosus B6, Lactobacillus paracasei BD5115, Lactiplantibacillus plantarum ST-III, Kluyveromyces marxianus KM3 were respectively activated and expanded in culture. The expanded culture solution of the strain was inoculated into a fermenter of the fermentation medium and stirred and cultured at 37 °C for 12 h (the pH value was controlled at about 5.5). A freeze-drying protectant was added and mixed evenly, and then obtained a probiotic agent after freeze-drying treatment.

[0198] Take 18% wt of the probiotic agent and add it to distilled water, heat it to 25 °C, and add 12% wt of dietary supplement, 8% wt of vitamins, 12% wt of fructooligosaccharide, 38% wt of maltodextrin and 12% wt of pectin, mix evenly and then freeze-dry to obtain a mixed probiotic powder product with the functions of improving intestinal barrier damage and regulating intestinal flora (the viable count is about 10 8 ~10 11 CFU / g).

[0199] Example 20

[0200] A live probiotic aquatic product containing Lactobacillus rhamnosus KF7 and having the functions of improving intestinal barrier damage and regulating intestinal flora:

[0201] After the Lactobacillus rhamnosus KF7 was expanded and cultured according to the method in Example 18 and then freeze-dried, a probiotic powder was obtained. It was diluted and added to pure water containing 200 mg / 100 ml of vitamin C and 0.2% wt of pectin stabilizer to prepare a live bacteria aquatic product with a final concentration of 10 6 ~10 9 CFU / g, which has the effects of improving intestinal barrier damage and regulating the intestinal flora.

[0202] Example 21

[0203] A milk powder product containing Lactobacillus rhamnosus KF7, which has the effects of improving intestinal barrier damage and regulating the intestinal flora:

[0204] The milk powder formula includes: 74.96% wt of whole milk powder, 10% wt of demineralized whey powder, 8% wt of isomaltooligosaccharide, 3.5% wt of concentrated whey protein powder, 2.5% wt of inulin, 1% wt of compound nutrients (1000 parts of ultramicro calcium carbonate, 150 g of vitamin B6, 8.5 g of vitamin C, 14.2 g of vitamin E, 10 g of vitamin A, 1 g of vitamin D, 18 g of ferric pyrophosphate, 10 g of zinc oxide, 0.3 g of sodium selenite, 6.6 g of taurine, 0.5 g of folic acid, 0.5 g of arachidonic acid, 0.5 g of docosahexaenoic acid, 2 g of nicotinic acid), 0.04% wt of Lactobacillus rhamnosus KF7, and added with Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus casei BDII, Lactobacillus rhamnosus B6, Lactobacillus paracasei BD5115, Lactiplantibacillus plantarum ST-III.

[0205] Mix the whole milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrients evenly, add water and then homogenize, sterilize at high temperature, stir evenly, and spray-dry to obtain milk powder. Before packaging, add 0.04% wt of Lactobacillus rhamnosus KF7 to the milk powder to obtain a milk powder product with the effects of improving intestinal barrier damage and regulating the intestinal flora.

[0206] Example 22

[0207] A cheese product containing Lactobacillus rhamnosus KF7, which has the effects of improving intestinal barrier damage and regulating the intestinal flora:

[0208] Standardize the raw milk so that the weight ratio of fat to protein in it is 1.8:1 to obtain raw milk; after pasteurizing the raw milk (15 s at 73 °C), cool it to 30 °C; inoculate the raw milk with the commercial starter culture CHOOZITTM TM(Danisco) and Lactobacillus rhamnosus KF7. Add 0.006 g / L rennet (Chr. Hansen, Fromase 750XLG), stir for 3 min, and ferment the curd at a constant temperature of 32 °C; cut the fermented curd into curds with a volume of 1.4 cm 3 blocks, and stir slowly for 20 min. Wash the curd blocks with water at 35 °C and keep stirring for 25 min. Then drain the whey, add square modules, and press for 75 min. Cure with 2% wt salt for 4 days. Age at 12 °C for 6 months to obtain a cheese product with the effects of improving intestinal barrier damage and regulating the intestinal flora.

[0209] In summary, this application for the first time discloses that Lactobacillus rhamnosus KF7 can not only improve the human intestinal epithelial barrier function, protect it from lipopolysaccharide-induced barrier damage, but also restore the intestinal barrier damage of Caenorhabditis elegans caused by disrupted rhythm, regulate the intestinal flora, and improve the growth, development and metabolic status of C. elegans. Compared with the monolayer of Caco-2 cells cultured normally, co-culture with Lactobacillus rhamnosus KF7 can strengthen the intestinal barrier function by up-regulating the expression levels of mucin-related genes. Compared with the Caco-2 cell monolayer barrier damage model induced by lipopolysaccharide, the intervention of the fermented milk supernatant of Lactobacillus rhamnosus KF7 can restore the integrity of the intestinal epithelial barrier structure by increasing the expression of tight junction protein genes, and improve the inflammatory response and the permeability between intestinal epithelial cells by inhibiting the gene expression of the TLR / MyD88 / NF-κB signaling pathway and downstream MLCK. Compared with the C. elegans with disrupted rhythm, feeding on Lactobacillus rhamnosus KF7 can reduce the intestinal permeability of C. elegans, increase the diversity of the intestinal flora of C. elegans, regulate the balance of the intestinal microbiota, and increase the body size and metabolic level of C. elegans. It can be used for the production of functional food products such as fermented milk, lactic acid bacteria beverages, probiotic powders, live bacteria water, milk powder and cheese.

[0210] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. Use of Lactobacillus rhamnosus KF7 in the preparation of a product for improving intestinal barrier damage and / or regulating intestinal flora, wherein the deposit number of Lactobacillus rhamnosus KF7 is CGMCC NO.6430.

2. The use according to claim 1, characterized in that, The product has any one or more of the following functions: 1) Improve intestinal barrier integrity; 2) Promote the expression of genes related to intestinal cell tight junction proteins and mucin-related genes; 3) Up-regulate the expression level of inflammatory factors; 4) Reduce the expression level of key molecules in the inflammatory signaling pathway; 5) Increase intestinal flora diversity; 6) Increase the relative abundance of beneficial bacteria; 7) Reduce the relative abundance of harmful bacteria; 8) Regulate the balance of the intestinal microbiota; 9) Improve growth and development indicators; 10) Improve the reduced metabolic level.

3. The use according to claim 2, characterized in that, The improvement of intestinal barrier integrity includes increasing the trans-epithelial resistance value of the intestinal epithelial cell monolayer; and / or, the intestinal cell tight junction proteins are selected from Occludin, Claudin, or ZO; the genes related to the intestinal cell tight junction proteins are selected from Claudin-1, Occludin, or ZO-1; and / or, the intestinal cell mucins are selected from MUC-2; the genes related to the intestinal cell mucins are selected from MUC-2; and / or, the inflammatory factors are selected from TNF-α, IL-1β, IL-6, IL-8, TGF-β, or IL-10; and / or, the key molecules in the inflammatory signaling pathway are selected from MyD88, NF-κB p65, or MLCK; and / or, the intestinal flora diversity is α-diversity. and / or, the beneficial bacteria include Firmicutes and / or Planctomycetes; further, the beneficial bacteria include one or more combinations of the genera Blautia, Halomonas, and Lactobacillus; still further, the beneficial bacteria include one or more combinations of Clostridium, Lactobacillus, Enterobacter, and Enterococcus; and / or, the harmful bacteria include Proteobacteria, Cyanobacteria; further, the harmful bacteria include one or more combinations of the genera Corynebacterium, Achromobacter, and Escherichia; and / or, the growth and development indicators include body size; and / or, the metabolic level is reflected by the pharyngeal pump frequency.

4. The use according to claim 1, characterized in that, The intestinal barrier damage is caused by circadian rhythm disorder or lipopolysaccharide; further, the circadian rhythm disorder is caused by cyclic temperature changes; the cyclic temperature changes are cycling between culturing at 20 - 25°C for 4 - 20 h and culturing at 15 - 18.5°C for 4 - 20 h.

5. The use according to claim 1, characterized in that, The product includes food, health products, or drugs; preferably, the product is selected from fermented milk products, lactic acid bacteria beverage products, probiotic agents, probiotic powder products, live aquatic products, milk powder products, or cheese products.

6. A fermented milk product, the raw materials of which comprise Lactobacillus rhamnosus KF7; optionally, it further comprises one or a combination of more of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum.

7. A lactic acid bacteria beverage product, the raw materials of which comprise Lactobacillus rhamnosus KF7; optionally, it further comprises one or a combination of more of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum.

8. A probiotic agent, which is obtained by freeze-drying a fermentation-cultured strain after adding a freeze-drying protectant. The strain includes Lactobacillus rhamnosus KF7; optionally, it further includes one or more strains selected from Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum, and Kluyveromyces.

9. When it comes to a probiotic powder product, the probiotic powder product is obtained by adding a dietary supplement, vitamins, fructooligosaccharide, maltodextrin, and pectin to the probiotic agent as described in claim 8, mixing them evenly, and then freeze-drying.

10. When it comes to a live probiotic aquatic product, the live probiotic aquatic product is obtained by adding vitamin C and a stabilizer to the probiotic agent as described in claim 8. Based on the mass of the live probiotic aquatic product, the concentration of the probiotic agent is 10 6 ~10 9 CFU / g.

11. A milk powder product, whose raw materials include Lactobacillus rhamnosus KF7. Optionally, it further includes a combination of one or more of whole milk powder, skim milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrients.

12. A cheese product, whose raw materials include Lactobacillus rhamnosus KF7. Optionally, it further includes a combination of one or more of raw cow milk, commercial fermenting agent, and rennet.

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