Application of cis-11-methyl-2-dodecenoic acid in regulating intestinal flora imbalance

Abstract

The present invention discloses a kind of application of cis-11-methyl-2-dodecenoic acid in regulating intestinal flora imbalance, relating to the field of intestinal flora regulation technology. The present invention proves through experiments that cis-11-methyl-2-dodecenoic acid can regulate the intestinal flora structure in the zebrafish intestinal flora disorder model, affecting multiple intestinal bacteria to return to normal levels. Further, a probiotic composition prepared by cis-11-methyl-2-dodecenoic acid as an active ingredient and common prebiotics can promote the colonization of probiotics Bifidobacterium and Lactobacillus in the intestine, and can also restore the pathological damage caused by intestinal flora disorder, enhance the tight connection of the intestine, have a good therapeutic effect on intestinal flora disorder, and are beneficial to maintaining intestinal health. And cis-11-methyl-2-dodecenoic acid can play its activity of regulating intestinal flora at extremely low doses, and will not cause secondary damage to the intestine at this dose, which is conducive to low-cost production.

Description

Technical Field

[0001] The present invention relates to the technical field of intestinal flora regulation, and specifically to the use of cis-11-methyl-2-dodecenoic acid in regulating intestinal flora imbalance, and to the use of a probiotic composition containing cis-11-methyl-2-dodecenoic acid as an active ingredient in regulating intestinal flora imbalance. Background Art

[0002] The intestinal microecology is the main component of the human microecology. The intestinal microecology is composed of intestinal microbiota, intestinal epithelial cells and the intestinal mucosal immune system. These components are interdependent and establish a complex interactive network of mutual constraints. According to their impact on the human body, there are three types of commensal bacteria, opportunistic pathogens and pathogenic bacteria. The intestinal microecology participates in digestion and absorption and material metabolism, and inhibits the growth of pathogenic microorganisms. It also acts as the body's natural immune barrier, regulates the innate immunity of the intestine, controls the mucosal barrier function, and participates in the physiological activities of intestinal epithelial cells, such as proliferation or apoptosis. When the homeostatic balance of the intestinal microecology is disturbed, the existing core intestinal microbiota network changes, leading to obesity, diabetes and many other diseases, especially intestinal diseases such as irritable bowel syndrome, inflammatory bowel disease (IBD) and colon malignancies are particularly closely related to the intestinal microecology.

[0003] The dense and intact mucus layer composed of goblet cells and tight junction (TJs) proteins can effectively prevent bacteria from invading the epithelial layer. Current research suggests that the core of the pathogenesis of IBD seems to point to the interaction between the intestinal microbiota and the intestinal epithelium. Increased colonic epithelial permeability is one of the indicators of the severity of IBD. Defects in the regulation of mucus secretion by goblet cells in the gastrointestinal epithelium can lead to changes in the expression of tight junction proteins, thereby causing the destruction of tight junctions in epithelial cells, the imbalance of intestinal homeostasis and the termination of symbiosis. Microbial diversity changes and leads to a decline in immunity. It also affects the differentiation of immune cells, leading to the production of more pro-inflammatory cytokines and aggravated intestinal inflammatory damage.

[0004] Among the many molecules found in the intestinal ecosystem, quorum sensing (QS) molecules are an often overlooked but worthy of attention. Quorum sensing relies on the release of small molecules (autoinducers) by bacteria that accumulate in the environment according to bacterial cell density. These molecules are not only sensed by the microbial community but also interact with host cells to contribute to intestinal homeostasis. Therefore, it seems entirely appropriate to highlight the role of these molecules in the immune system under inflammatory conditions associated with dysbiosis of bacterial populations. In addition, bacterial quorum sensing signals may help to establish symbiotic interactions in some cross-species interaction dynamics.

[0005] Cis-11-methyl-2-dodecenoic acid (diffusible signal factor, quorum sensing signal molecule) was discovered in 1997. It is a small molecule signal substance secreted by wild-type strains of Xanthomonas campestris (Xcc). This small molecule can diffuse in agar culture medium, promote pathogenicity-related gene expression, inhibit the formation of biofilm, promote bacterial colonies to make metabolic adjustments, and adapt to high population density environments. Studies have found that cis-11-methyl-2-dodecenoic acid can induce plant innate immunity, reduce the severity of disease in host plants, and inhibit pathogen growth. At present, cis-11-methyl-2-dodecenoic acid is mainly studied in plant disease prevention and control. Recently, studies have found that it has anti-inflammatory activity in animals. Referring to the record in patent document CN114306307A, it was found that cis-11-methyl-2-dodecenoic acid has the effect of suppressing LPS-induced inflammatory response and oxidative stress response. However, there is no report on cis-11-methyl-2-dodecenoic acid regulating intestinal microorganisms and improving intestinal microecology.

[0006] Colitis is an inflammatory bowel disease characterized by long-term recurrent inflammation accompanied by an influx of neutrophils and macrophages, the production of cytokines, proteases, and free radicals by the intestinal mucosa, which causes inflammatory damage and ulcers in the colon, leading to abdominal pain, diarrhea, bloody stools, and weight loss. Its pathogenesis is complex, involving factors such as genetic susceptibility, intestinal flora imbalance, intestinal barrier function damage, and immune disorders. At present, the treatment drugs for colitis, especially antibiotics and glucocorticoids, will affect the balance of intestinal flora to a certain extent, leading to dysbacteriosis. For example, short-term antibiotic treatment will cause the intestinal microbiota to transform into a long-term alternative imbalance state, which may promote the development and aggravation of the disease. Long-term or broad-spectrum antibiotics will lead to more serious dysbacteriosis and the production of drug-resistant strains (Lange K, Buerger M, Stallmach A, Bruns T. Effects of Antibiotics on Gut Microbiota. Dig Dis. 2016; 34(3): 260-8.). Relevant studies on glucocorticoid treatment have found that it causes a decrease in intestinal animal lactobacilli and leads to femoral head necrosis (Chen CY, Rao SS, Yue T, Tan YJ, Yin H, Chen LJ, Luo MJ, Wang Z, Wang YY, Hong CG, Qian YX, HeZH, Liu JH, Yang F, Huang FY, Tang SY, Xie H. Glucocorticoid-induced loss of beneficial gut bacterial extracellular vesicles is associated with the pathogenesis of osteonecrosis. Sci Adv. 2022 Apr 15; 8(15): eabg8335.). Other immunosuppressants and biological agents also have the risk of organ toxicity and increased bacterial and fungal infections (Pithadia AB, Jain S. Treatment of inflammatory bowel disease (IBD). Pharmacol Rep. 2011; 63(3): 629-42.). Therefore, it is particularly important to find a therapeutic strategy that can restore intestinal inflammation while also optimizing the structure of the intestinal flora. Summary of the Invention

[0007] In response to the shortcomings of the prior art, the present invention aims to provide a method for preparing a product related to regulating intestinal flora imbalance using cis-11-methyl-2-dodecenoic acid as an active ingredient, and a method for preparing a product related to regulating intestinal flora imbalance using a probiotic composition containing the active ingredient. Cis-11-methyl-2-dodecenoic acid, an active ingredient, can regulate and restore the structure of intestinal flora. A probiotic composition containing the active ingredient can promote the colonization and growth of probiotics Bifidobacterium and Lactobacillus in the intestine, repair intestinal mucosal damage caused by intestinal flora imbalance, and enhance intestinal tight junctions, thereby having a good therapeutic effect on intestinal flora disorders.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a use of cis-11-methyl-2-dodecenoic acid as an active ingredient in the preparation of products related to regulating intestinal flora imbalance.

[0010] The chemical structural formula of the active ingredient cis-11-methyl-2-dodecenoic acid of the present invention is:

[0011]

[0012] As a preferred embodiment, the products related to regulating intestinal flora imbalance include products for treating and / or preventing diseases related to intestinal flora imbalance.

[0013] As a preferred embodiment, the product is any one of food, medicine, health care product, cosmetics and skin care product containing cis-11-methyl-2-dodecenoic acid.

[0014] As a preferred embodiment, the products for treating and / or preventing diseases related to intestinal flora imbalance include products for repairing intestinal mucosal damage caused by intestinal flora imbalance, products for restoring intestinal tight junctions and promoting intestinal barrier repair, or products for promoting the colonization of probiotics in the host intestine.

[0015] As a preferred embodiment, the intestinal flora includes at least one of Streptomyces, Bifidobacterium, Bacteroides, Ruminobacterium, Parabacteroides, Archaeobacterium, Lactobacillus, Fusobacterium, Lachnospiraceae, Enterococcus, Streptococcus tigerus, Clostridium, and Flavobacterium.

[0016] In the experimental study of the present invention, it was found that the cis-11-methyl-2-dodecenoic acid can restore the changes in the intestinal flora at the genus level caused by DSS, especially it can regulate the genera Streptomyces, Bifidobacterium, Bacteroides, Ruminobacterium, Parabacteroides, Archaeobacterium, Lactobacillus, Fusobacterium, Lachnospiraceae, Enterococcus, Streptococcus tigerus, Clostridium, Flavobacterium, etc. to return to normal levels.

[0017] As a preferred embodiment, the probiotics include at least one of the genus Lactobacillus and the genus Bifidobacterium. In our experimental studies, we found that cis-11-methyl-2-dodecenoic acid can promote the colonization of exogenous Lactobacillus and Bifidobacterium in the host intestine.

[0018] In a second aspect, the present invention provides an application of a probiotic composition in the preparation of products related to regulating intestinal flora, wherein the probiotic composition contains cis-11-methyl-2-dodecenoic acid as an active ingredient.

[0019] As a preferred embodiment, the product is any one of food, medicine, health care product, cosmetics and skin care product containing the probiotic composition.

[0020] As a preferred embodiment, the prebiotic composition further comprises common prebiotics.

[0021] As a preferred embodiment, the common prebiotics are at least one of polydextrose, xylo-oligosaccharide, xylitol, maltodextrin and seaweed polyphenols.

[0022] As a preferred embodiment, the probiotic composition comprises the following components in parts by weight: 2-5 parts of polydextrose, 5-10 parts of xylo-oligosaccharide, 30-60 parts of xylitol, 10-20 parts of maltodextrin, 1-2 parts of seaweed polyphenols, and 0.01-0.05 parts of cis-11-methyl-2-dodecenoic acid.

[0023] As a further preferred embodiment, the probiotic composition is composed of the following components in parts by weight: 2 parts of polydextrose, 5 parts of xylo-oligosaccharides, 30 parts of xylitol, 10 parts of maltodextrin, 1 part of seaweed polyphenols, and 0.01 part of cis-11-methyl-2-dodecenoic acid.

[0024] As a further preferred embodiment, the probiotic composition is composed of the following components in parts by weight: 3 parts of polydextrose, 7 parts of xylo-oligosaccharides, 40 parts of xylitol, 15 parts of maltodextrin, 2 parts of seaweed polyphenols, and 0.02 parts of cis-11-methyl-2-dodecenoic acid.

[0025] As a further preferred embodiment, the probiotic composition is composed of the following components in parts by weight: 5 parts of polydextrose, 10 parts of xylo-oligosaccharides, 50 parts of xylitol, 20 parts of maltodextrin, 2 parts of seaweed polyphenols, and 0.04 parts of cis-11-methyl-2-dodecenoic acid.

[0026] As a further preferred embodiment, the preparation process of the probiotic complex is as follows: polydextrose, xylo-oligosaccharide, xylitol, maltodextrin, seaweed polyphenols, and cis-11-methyl-2-dodecenoic acid are weighed respectively according to the above weight portions, and then stirred at a speed of 200-400 rpm for 15-25 minutes and mixed evenly to obtain the probiotic complex.

[0027] As a preferred embodiment, the products related to regulating intestinal flora imbalance include products for treating and / or preventing diseases related to intestinal flora imbalance.

[0028] As a preferred embodiment, the products for treating and / or preventing diseases related to intestinal flora imbalance include products for repairing intestinal mucosal damage caused by intestinal flora imbalance, products for restoring intestinal tight junctions and promoting intestinal barrier repair, or products for promoting the colonization of probiotics in the host intestine.

[0029] In the experimental study of the present invention, it was found that the probiotic composition can restore intestinal pathological damage caused by dysbiosis in a zebrafish model. The pathological damage includes: loosening and shedding of intestinal tissue, obvious expansion of the intestine, and a significant increase in mucin granules secreted by goblet cells.

[0030] The experimental study of the present invention also found that the probiotic composition can restore the expression of intestinal tight junction genes in a zebrafish colitis model. The intestinal tight junction genes include claudin1 and occludin1.

[0031] It was further found in the experimental studies of the present invention that the probiotic composition of the present invention can promote the colonization of exogenous probiotics such as bifidobacteria and lactobacilli in the host intestine.

[0032] As a preferred embodiment, the intestinal flora includes at least one of Streptomyces, Bifidobacterium, Bacteroides, Ruminobacterium, Parabacteroides, Archaeobacterium, Lactobacillus, Fusobacterium, Lachnospiraceae, Enterococcus, Streptococcus tigerus, Clostridium, and Flavobacterium.

[0033] As a preferred embodiment, the probiotics include at least one of Lactobacillus and Bifidobacterium.

[0034] The present invention proves through experiments that cis-11-methyl-2-dodecenoic acid can regulate the intestinal flora structure in a zebrafish intestinal flora imbalance model, affecting the recovery of multiple intestinal bacterial genera to normal levels. At the same time, a probiotic composition with cis-11-methyl-2-dodecenoic acid as an active ingredient can promote the colonization and growth of probiotics Bifidobacterium and Lactobacillus in the intestine, enhance the tight connection of the intestine, have a good therapeutic effect on intestinal flora-related diseases, and are beneficial for restoring pathological damage to the intestine. Moreover, cis-11-methyl-2-dodecenoic acid can exert its activity in regulating intestinal flora at an extremely low dose, and will not cause secondary damage to the intestine at this dose, which is conducive to low-cost production.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention first discovered that in a zebrafish intestinal flora imbalance model, cis-11-methyl-2-dodecenoic acid demonstrated its ability to regulate intestinal flora imbalance, including restoring intestinal flora structure. Furthermore, a probiotic composition prepared with cis-11-methyl-2-dodecenoic acid as the active ingredient can promote the colonization and growth of intestinal probiotics and restore intestinal pathological damage caused by intestinal flora imbalance, including restoring the expression of intestinal tight junction genes, and has a good restorative effect on intestinal mucosal damage. Furthermore, the active ingredient cis-11-methyl-2-dodecenoic acid can exert its intestinal flora-regulating effect even at very low doses, and can be used in the production of foods, medicines, or health products that assist in regulating intestinal flora imbalance in a variety of formulations, such as suppositories, tablets, pills, granules, and oral liquids. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0038] Figure 1 This is a genus-level analysis of the effects of cis-11-methyl-2-dodecenoic acid on the composition of intestinal flora in a zebrafish intestinal flora disorder model;

[0039] Figure 2 This is a heat map of the genus level of intestinal flora in the zebrafish intestinal flora disorder model treated with cis-11-methyl-2-dodecenoic acid;

[0040] Figure 3 The present invention is to analyze the effect of cis-11-methyl-2-dodecenoic acid and a probiotic composition on the colonization of probiotic lactobacilli in the zebrafish intestine; Figure 3 A is the fluorescence intensity photograph of each experimental group; Figure 3 B is the relative fluorescence intensity analysis results of each experimental group;

[0041] Figure 4 The present invention is to analyze the effect of cis-11-methyl-2-dodecenoic acid and a prebiotic composition on the colonization of probiotic Bifidobacterium in the intestine of zebrafish; Figure 4 A is the fluorescence intensity photograph of each experimental group; Figure 4 B is the relative fluorescence intensity analysis results of each experimental group;

[0042] Figure 5 Pathological tissue sections showing the effects of cis-11-methyl-2-dodecenoic acid and a prebiotic composition on intestinal structure in a zebrafish intestinal flora disorder model;

[0043] Figure 6 This study analyzed the effects of cis-11-methyl-2-dodecenoic acid and a prebiotic composition on the expression levels of tight junction genes claudin1 and occludin1 in a zebrafish intestinal flora disorder model. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0045] In the following examples, the embryo culture medium was prepared as follows: 0.29 g NaCl (Chinese medicine), 0.012 g KCl (Chinese medicine), 0.036 g CaCl₂ (Chinese medicine), and 0.04 g MgSO₄ (Chinese medicine) were weighed, made up to 1 L with ddH₂O, sterilized at high temperature, and stored at room temperature. DSS was purchased from Meilun Biotechnology, and cis-11-methyl-2-dodecenoic acid was provided by the State Key Laboratory of Microbial Metabolism, International Joint Research Laboratory of Metabolism and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University.

[0046] Example 1: A probiotic composition capable of regulating the structure of intestinal flora

[0047] The probiotic composition described in this embodiment is composed of the following components in parts by weight: 2 parts of polydextrose, 5 parts of xylo-oligosaccharides, 30 parts of xylitol, 10 parts of maltodextrin, 1 part of seaweed polyphenols, and 0.01 part of cis-11-methyl-2-dodecenoic acid.

[0048] The preparation process of the probiotic complex is as follows: polydextrose, xylo-oligosaccharide, xylitol, maltodextrin, seaweed polyphenols, and cis-11-methyl-2-dodecenoic acid are weighed respectively according to the above weight parts, and then stirred at 300 rpm for 20 minutes to mix evenly to obtain the probiotic complex.

[0049] Example 2: A probiotic composition capable of regulating the structure of intestinal flora

[0050] The probiotic composition described in this embodiment is composed of the following components in parts by weight: 3 parts of polydextrose, 7 parts of xylo-oligosaccharides, 40 parts of xylitol, 15 parts of maltodextrin, 2 parts of seaweed polyphenols, and 0.02 parts of cis-11-methyl-2-dodecenoic acid.

[0051] The preparation process of the probiotic complex is as follows: polydextrose, xylo-oligosaccharide, xylitol, maltodextrin, seaweed polyphenols, and cis-11-methyl-2-dodecenoic acid are weighed respectively according to the above weight parts, and then stirred at 300 rpm for 20 minutes to mix evenly to obtain the probiotic complex.

[0052] Example 3: A probiotic composition capable of regulating the structure of intestinal flora

[0053] The probiotic composition described in this embodiment is composed of the following components in parts by weight: 5 parts of polydextrose, 10 parts of xylo-oligosaccharides, 50 parts of xylitol, 20 parts of maltodextrin, 2 parts of seaweed polyphenols, and 0.04 parts of cis-11-methyl-2-dodecenoic acid.

[0054] The preparation process of the probiotic complex is as follows: polydextrose, xylo-oligosaccharide, xylitol, maltodextrin, seaweed polyphenols, and cis-11-methyl-2-dodecenoic acid are weighed respectively according to the above weight parts, and then stirred at 300 rpm for 20 minutes to mix evenly to obtain the probiotic complex.

[0055] Experimental Example 1: Evaluation of the Effect of Cis-11-Methyl-2-Dodecenoic Acid on Regulating Intestinal Microflora

[0056] 1. Collect fish eggs

[0057] The night before the experiment, select adult fish that are in good condition and have not laid eggs within 7 days. Place one male and one female fish in each mating box, separated by a baffle. Remove the baffle the next morning and fertilized eggs can be obtained within 5 minutes. Collect the eggs and transfer them to a culture dish containing embryo culture medium.

[0058] 2. Construction and treatment of zebrafish embryo intestinal flora disorder model

[0059] 3 dpf (3 days post fertilization) eggs were transferred to 6-well plates, with 30 eggs per well, and grouped as follows:

[0060] Control group: 5 ml fresh embryo culture medium;

[0061] Model group: replaced with 5 ml embryo culture medium containing 0.25% (w / v) DSS;

[0062] Experimental group: The medium was replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and cis-11-methyl-2-dodecenoic acid.

[0063] 3. Total DNA Extraction

[0064] After treatment for 7 dpf, zebrafish embryos were collected in EP tubes and total DNA was extracted using a microbial DNA extraction kit. DNA was quantified using Nanodrop, and the quality of the extracted DNA was checked by 1.2% agarose gel electrophoresis.

[0065] 4. PCR amplification of target fragments

[0066] Usually, target sequences such as microbial ribosomal RNA or specific gene fragments that can reflect the composition and diversity of the bacterial community are used as targets. Corresponding primers are designed according to the conserved regions in the sequence, and sample-specific barcode sequences are added to perform PCR amplification on the variable regions (single or multiple consecutive) of the rRNA gene or specific gene fragments.

[0067] PCR amplification uses Quanshijin's Pfu high-fidelity DNA polymerase. The number of amplification cycles is strictly controlled to keep it as low as possible while ensuring consistent amplification conditions across the entire sample batch. Negative controls are also included to detect microbial contamination from environmental and reagent sources. Any sample group showing bands in the negative control amplification should not be used in subsequent experiments.

[0068] 5. Purification and recovery of amplified products using magnetic beads

[0069] (1) Add 0.8 times the volume of magnetic beads (Vazyme VAHTS™ DNA CleanBeads) to 25 μl of PCR amplification product, shake thoroughly to suspend, and adsorb on a magnetic stand for 5 minutes. Carefully aspirate the supernatant with a pipette;

[0070] (2) Add 20 μl of 0.8x magnetic bead washing buffer, shake thoroughly to suspend, place on a magnetic stand for adsorption for 5 minutes, and carefully aspirate the supernatant;

[0071] (3) Add 200 μl of 80% ethanol and place the tube on a magnetic stand. The magnetic beads are adsorbed to the other side of the PCR tube. After sufficient adsorption, aspirate the supernatant.

[0072] (4) Leave it at room temperature for 5 minutes until the alcohol evaporates completely and cracks appear on the magnetic beads.

[0073] (5) Add 25 μl of Elution Buffer for elution;

[0074] (6) Place the PCR tube on the adsorption rack for 5 minutes to allow for sufficient adsorption. Remove the supernatant and store it in a clean 1.5 ml centrifuge tube.

[0075] 6. Fluorescence quantification of amplified products

[0076] The PCR amplification products obtained in step 5 were subjected to fluorescence quantification using the Quant-iT PicoGreen dsDNA Assay Kit and a Microplate reader (BioTek, FLx800). Based on the fluorescence quantification results and the sequencing requirements for each sample, the samples were mixed in the appropriate proportions.

[0077] 7. Sequencing Library Preparation

[0078] The sequencing library was prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit.

[0079] (1) First, the amplified product is subjected to sequence end repair. The protruding base at the 5' end of the DNA sequence is removed using End Repair Mix 2 in the kit, and a phosphate group is added to fill the missing base at the 3' end.

[0080] (2) Adding an A base to the 3' end of the DNA sequence to prevent the DNA fragment from self-ligating and to ensure that the target sequence can be connected to the sequencing adapter (there is a protruding T base at the 3' end of the sequencing adapter);

[0081] (3) Add a sequencing adapter containing a library-specific tag (i.e., index sequence) to the 5' end of the sequence so that the DNA molecule can be fixed on the flow cell;

[0082] (4) Using BECKMAN AMPure XP Beads, the adapter-ligated fragments were removed by magnetic bead screening and the library system after the addition of adapters was purified;

[0083] (5) PCR amplification of the adapter-attached DNA fragments to enrich the sequencing library templates, and the library enrichment products were purified again using BECKMAN AMPure XP Beads;

[0084] (6) The library was subjected to final fragment selection and purification by 2% agarose gel electrophoresis.

[0085] 8. Perform high-throughput sequencing on the machine

[0086] (1) Before sequencing, the library needs to be quality checked on an Agilent Bioanalyzer using the Agilent High Sensitivity DNA Kit. A qualified library has only a single peak and no adapters.

[0087] (2) Afterwards, the library was quantified using the Quant-iT PicoGreen dsDNA Assay Kit on the Promega QuantiFluor fluorescence quantitative system. The qualified library concentration should be above 2 nM.

[0088] (3) After gradient dilution of each qualified sequencing library (index sequence cannot be repeated), mix them in the corresponding proportion according to the required sequencing amount, and denature them into single strands with NaOH for sequencing;

[0089] (4) Double-end sequencing was performed using a MiSeq sequencer, and the corresponding reagent was MiSeq Reagent Kit V3 (600 cycles).

[0090] 9. Analysis process

[0091] (1) First, the original data from high-throughput sequencing is preliminarily screened based on sequence quality; problematic samples are retested and supplemented.

[0092] (2) The original sequences that have passed the initial quality screening are divided into libraries and samples according to the index and barcode information, and the barcode sequence is removed.

[0093] (3) Perform sequence denoising or OTU clustering according to the QIIME2 dada2 analysis process or the Vsearch software analysis process.

[0094] (4) Display the specific composition of each sample (group) at different species taxonomic levels to understand the overall overview.

[0095] Genus-level analysis of the effects of cis-11-methyl-2-dodecenoic acid on the composition of intestinal flora in a zebrafish model of dysbacteriosis Figure 1 As shown in the figure, the heat map of the effect of cis-11-methyl-2-dodecenoic acid on the intestinal flora genus level of the zebrafish model of dysbacteriosis is shown in the figure. Figure 2 The results showed that cis-11-methyl-2-dodecenoic acid could restore the changes in intestinal flora caused by DSS at the genus level, and regulate the genera Streptomyces, Bifidobacterium, Bacteroides, Ruminococcus, Parabacteroides, Runella, Lactobacillus, Fusobacterium, Lachnospiraceae, Enterococcus, Streptococcus tigerus, Clostridium, and Flavobacterium to return to normal levels.

[0096] Experimental Example 2: Experiment on the promotion of colonization of Lactobacillus and Bifidobacterium in the intestine of zebrafish by various probiotic compositions

[0097] 1. Collect zebrafish samples

[0098] The night before the experiment, select adult fish that are in good condition and have not laid eggs within 7 days. Place one male and one female fish in each mating box, separated by a baffle. Remove the baffle the next morning and fertilized eggs can be obtained within 5 minutes. Collect the eggs and transfer them to a culture dish containing embryo culture medium.

[0099] 2. Lactobacillus and Bifidobacterium culture and fluorescent probe incubation

[0100] Lactobacillus and Bifidobacterium were inoculated into MRS medium at a 1% inoculum rate, cultured at 37°C to the logarithmic phase, centrifuged at 5000 rpm for 1 min, and the bacterial pellets were collected. The bacterial pellets were washed three times with sterile PBS solution and then resuspended in PBS solution. CFDA-SE (cell proliferation tracer fluorescent probe) stock solution was added to the lactobacillus suspension and the bifidobacterium suspension, respectively. The cells were incubated at 37°C in the dark for 20 min, centrifuged at 4°C, washed three times with PBS solution, excess fluorescent dye was removed, and the cells were resuspended in PBS solution.

[0101] 3. Zebrafish feeding

[0102] 3 dpf (3 days post fertilization) eggs were transferred to 6-well plates, with 30 eggs per well, and grouped as follows:

[0103] Control group: 5 ml fresh embryo culture medium;

[0104] Experimental group 1: replaced with 5 ml of embryo culture medium containing cis-11-methyl-2-dodecenoic acid;

[0105] Experimental Group 2: The medium was replaced with 5 ml of embryo culture medium containing the probiotic composition prepared in Example 1.

[0106] Experimental Group 3: The medium was replaced with 5 ml of embryo culture medium containing the probiotic composition prepared in Example 2.

[0107] Experimental Group 4: The medium was replaced with 5 ml of embryo culture medium containing the probiotic composition prepared in Example 3.

[0108] After each treatment, zebrafish in each group were immersed in embryo culture medium supplemented with fluorescently labeled Lactobacillus and Bifidobacterium at 7 dpf. After immersion for 4 hours, the zebrafish embryos were washed three times with clean embryo culture medium. The fluorescence intensity of the bacteria within the same field of view was recorded and quantitatively analyzed under a fluorescence microscope.

[0109] Analysis of the effects of cis-11-methyl-2-dodecenoic acid and three groups of probiotic compositions (Examples 1-3) on the colonization of probiotic lactobacilli in the zebrafish intestine Figure 3As shown in Figure 2, the effects of cis-11-methyl-2-dodecenoic acid and three groups of probiotic compositions on the colonization of probiotic Bifidobacterium in the zebrafish intestine were analyzed. Figure 4 The results showed that cis-11-methyl-2-dodecenoic acid and the three groups of probiotic compositions all significantly promoted the colonization of Lactobacillus and Bifidobacterium in the intestines of zebrafish embryos, and the differences were statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001), indicating that cis-11-methyl-2-dodecenoic acid and the probiotic compositions can prevent or treat intestinal flora imbalance and other related diseases by promoting the colonization of probiotics in the intestine.

[0110] The results also show that cis-11-methyl-2-dodecenoic acid and the probiotic composition can significantly promote the growth of probiotics Lactobacillus and Bifidobacterium, and can thus be used to prepare products that promote the growth of probiotics, including any one of medicines, health products, foods, cosmetics, and skin care products.

[0111] Experimental Example 3: H&E staining method to evaluate the histopathological changes of zebrafish intestine and the recovery of intestinal tissue after treatment with each group of probiotic compositions

[0112] 1. Collect fish eggs

[0113] The night before the experiment, select adult fish that are in good condition and have not laid eggs within 7 days. Place one male and one female fish in each mating box, separated by a baffle. Remove the baffle the next morning and fertilized eggs can be obtained within 5 minutes. Collect the eggs and transfer them to a culture dish containing embryo culture medium.

[0114] 2. Construction of a zebrafish embryo intestinal flora disorder model and treatment with a probiotic composition

[0115] 3 dpf (3 days post fertilization) eggs were transferred to 6-well plates, with 30 eggs per well, and grouped as follows:

[0116] Control group: 5 ml fresh embryo culture medium;

[0117] Model group: replaced with 5 ml embryo culture medium containing 0.25% (w / v) DSS;

[0118] Experimental group 1: replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and cis-11-methyl-2-dodecenoic acid;

[0119] Experimental Group 2: The medium was replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and the probiotic composition prepared in Example 1.

[0120] Experimental group 3: The medium was replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and the probiotic composition prepared in Example 2.

[0121] Experimental Group 4: The medium was replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and the probiotic composition prepared in Example 3.

[0122] 3. Intestinal tissue sections and HE staining:

[0123] Each group was treated until 7 dpf (7 days after fertilization), and the whole zebrafish embryos were obtained, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E) to observe the changes in intestinal tissue morphology in each group.

[0124] In order to study the rescue effect of cis-11-methyl-2-dodecenoic acid and prebiotic composition on DSS-induced intestinal damage in zebrafish, H&E staining was used to observe the histopathological changes of zebrafish intestine. Figure 5 As shown in the results, compared with the control group, DSS treatment caused intestinal flora disorder, resulting in loosening and shedding of intestinal tissue in zebrafish embryos, obvious expansion of the intestine, and a significant increase in the number of mucin granules secreted by goblet cells. After adding cis-11-methyl-2-dodecenoic acid and the three groups of probiotic compositions, the above phenomena were restored to a certain extent.

[0125] Experimental Example 4: Real-time fluorescence quantitative PCR detection of the expression levels of tight junction-related factors in zebrafish embryos after DSS-induced intestinal flora disturbance and treatment with each group of probiotic compositions

[0126] 1. Collect fish eggs

[0127] The night before the experiment, select adult fish that are in good condition and have not laid eggs within 7 days. Place one male and one female fish in each mating box, separated by a baffle. Remove the baffle the next morning and fertilized eggs can be obtained within 5 minutes. Collect the eggs and transfer them to a culture dish containing embryo culture medium.

[0128] 2. Construction and treatment of zebrafish embryo intestinal flora disorder model

[0129] 3 dpf (3 days post fertilization) eggs were transferred to 6-well plates, with 30 eggs per well, and grouped as follows:

[0130] Control group: 5 ml fresh embryo culture medium;

[0131] Model group: replaced with 5 ml embryo culture medium containing 0.25% (w / v) DSS;

[0132] Experimental group 1: replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and cis-11-methyl-2-dodecenoic acid;

[0133] Experimental Group 2: The medium was replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and the probiotic composition prepared in Example 1.

[0134] Experimental group 3: The medium was replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and the probiotic composition prepared in Example 2.

[0135] Experimental Group 4: The medium was replaced with 5 ml of embryo culture medium containing 0.25% (w / v) DSS and the probiotic composition prepared in Example 3.

[0136] 3. Extraction of Total RNA from Zebrafish Embryos

[0137] The Trizol method was used, and the specific steps were as follows:

[0138] 1) After each group was treated to 7 dpf, the zebrafish embryos of each group were collected in RNase-free EP tubes. 1 ml of Trizol was added to the EP tubes and the mixture was repeatedly pipetted evenly using a 1 ml syringe (on ice) until the zebrafish embryos were fully lysed.

[0139] 2) Add 200 μl of chloroform (sample to chloroform ratio is 5:1), shake vigorously for 20 seconds, let stand on ice for 3 minutes, and then centrifuge at 4°C, 12,000 rpm for 15 minutes.

[0140] 3) Carefully pipette the top supernatant from the EP tube into a new RNase-free EP tube, add an equal volume of isopropanol to the tube, invert the tube to mix thoroughly, and place at -80°C for 30 minutes.

[0141] 4) Remove the sample from -80°C and centrifuge at 12,000 rpm for 30 min at 4°C.

[0142] 5) Carefully discard the supernatant, then add 600 μl of pre-chilled 75% ethanol, mix well, centrifuge at 4°C, 12,000 rpm for 5 minutes, and carefully remove the supernatant.

[0143] 6) Repeat step 5).

[0144] 7) Discard any remaining 75% ethanol as much as possible, open the lid and let it stand for 5 minutes. When the white precipitate at the bottom of the EP tube becomes transparent, add 20 μl of Nuclease Free Water to fully dissolve it.

[0145] 8) Take 1 μl of the RNA obtained after treatment in step 7) to measure its concentration. Take another 1 μl of RNA and mix it with Loading Buffer. Perform nucleic acid electrophoresis to detect the quality of the total RNA.

[0146] 4. cDNA Synthesis

[0147] Reverse transcription was performed using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit.

[0148] 1) Add to the tube:

[0149]

[0150] 2) Incubate at 65°C for 5 min (performed in a PCR instrument), then immediately remove from the tube and place in ice for 5 min.

[0151] 3) After centrifugation in a microcentrifuge, add:

[0152]

[0153] 4) Mix gently.

[0154] 5) Perform reverse transcription in a PCR instrument. Reaction conditions: 65°C for 5 min, 25°C for 5 min, 42°C for 60 min, 70°C for 5 min, and 4°C for ∞.

[0155] 5. Real-time quantitative PCR (qRT-PCR)

[0156] use qRT-PCR was performed using the FAST Universal Fluorescent Quantitative PCR Kit. The reaction system is as follows:

[0157]

[0158] The qRT-PCR reaction conditions were as follows: 95°C for 3 min, 95°C for 3 s, 60°C for 20 s, 40 cycles, 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.

[0159] Zebrafish-related qRT-PCR primers were designed using qRT-PCR primer design software. The primer sequences are as follows: Tight junction proteins are multi-protein complexes composed of regulatory molecules such as kinases, transmembrane proteins, and peripheral membrane proteins. They play a role in protecting the intestinal epithelial barrier and regulating intestinal permeability. The tight junction protein family mainly includes members such as claudin1, occludins1, and zo1. Figure 6 As shown, compared with the control group, the levels of tight junction-related factors (claudin1 and occludins1) in the model group were significantly reduced, and the differences were statistically significant (#p<0.05, ##p<0.01, ###p<0.001); compared with the model group, the levels of tight junction-related factor indicators in the cis-11-methyl-2-dodecenoic acid group and the three experimental groups in Examples 1-3 were significantly increased, and the differences were statistically significant (*p<0.05, **p<0.01, ***p<0.001), indicating that cis-11-methyl-2-dodecenoic acid and the prebiotic composition have a significant effect on the restoration of the intestinal barrier.

[0160] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, several improvements can be made without departing from the principles of the present invention, and these improvements should also be considered as the scope of protection of the present invention.

Claims

1. Use of cis-11-methyl-2-dodecenoic acid as an active ingredient in the preparation of a product for regulating intestinal flora imbalance; the product is a health product; The intestinal flora includes at least one of Streptomyces, Bifidobacterium, Bacteroides, Ruminobacterium, Parabacteroides, Archaeobacterium, Lactobacillus, Fusobacterium, Lachnospiraceae, Enterococcus, Streptococcus tigerus, Clostridium, and Flavobacterium.

2. Use of cis-11-methyl-2-dodecenoic acid as an active ingredient in the preparation of a drug for repairing intestinal mucosal damage caused by intestinal flora imbalance; The intestinal flora includes at least one of Streptomyces, Bifidobacterium, Bacteroides, Ruminobacterium, Parabacteroides, Archaeobacterium, Lactobacillus, Fusobacterium, Lachnospiraceae, Enterococcus, Streptococcus tigerus, Clostridium, and Flavobacterium.

3. Use of a probiotic composition in the preparation of products related to regulating intestinal flora, characterized in that: The active ingredient of the probiotic composition is cis-11-methyl-2-dodecenoic acid; and the product is a health product.

4. Use of a probiotic composition in the preparation of a medicine for repairing intestinal mucosal damage caused by intestinal flora imbalance, characterized in that: The active ingredient of the probiotic composition is cis-11-methyl-2-dodecenoic acid.

5. The use according to claim 3 or 4, characterized in that The probiotic composition in the application also includes common prebiotics; the common prebiotics are at least one of polydextrose, oligoxylose, xylitol, maltodextrin, and seaweed polyphenols.

6. The use according to claim 5, characterized in that The probiotic composition comprises the following components in parts by weight: 2-5 parts of polydextrose, 5-10 parts of xylo-oligosaccharide, 30-60 parts of xylitol, 10-20 parts of maltodextrin, 1-2 parts of seaweed polyphenols, and 0.01-0.05 parts of cis-11-methyl-2-dodecenoic acid.

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