CN114431485A - Microecological preparation for preventing colorectal cancer and application thereof - Google Patents

Abstract

The invention specially prepares a preparation containing excellent strains for preventing colorectal cancer, which is prepared by mixing fructo-oligosaccharide, galacto-oligosaccharide, inulin, apple powder, blueberry powder, lactobacillus acidophilus, lactobacillus rhamnosus, bifidobacterium adolescentis, bifidobacterium longum, bifidobacterium animalis, lactobacillus reuteri, lactobacillus bulgaricus and streptococcus thermophilus. The microecological preparation can effectively relieve the colonic inflammation and intestinal mucosa barrier damage conditions of CAC mice, improve intestinal flora disorder and metabolic disorder, regulate the expression of Wnt/beta-catenin signal pathway protein, promote apoptosis and inhibit proliferation, thereby preventing the formation of colorectal cancer and playing the role of preventing and treating the colorectal cancer in an auxiliary way.

Description

Microecological preparation for preventing colorectal cancer and application thereof

Technical Field

The invention belongs to the field of medicines and functional health products, and particularly relates to a microecological preparation for inhibiting inflammation and preventing or treating colorectal cancer.

Background

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide, especially in individuals under the age of 50 with a gradual rise in the prevalence [1,2 ]]. Heterogeneous disease as intestinal epithelium, characterized by accumulation of mutations and dysregulation of immune response [3]. Although the actual pathogenesis of CRC has not been addressed, there is considerable evidence that Inflammatory Bowel Disease (IBD) -related inflammation is one of its risk factors [ 4]]. It has been reported that IBD patients exhibit a 2-8 fold higher risk of CRC carcinogenesis and that anti-inflammatory drugs can effectively improve the incidence of CRC [5,6]However, its side effects may be life threatening and should not be used for a long time [7]. Therefore, it is important to develop a more effective and safer natural drug as an alternative to prevent colon cancer and explore its mechanism of action. The synbiotics mainly comprise prebiotics and probiotics, and are a dietary intervention method aiming at intestinal microbiota. The probiotic is a live microorganismThe product is beneficial for health when administered in sufficient amount, and typical probiotic bacteria include Bifidobacterium, Lactobacillus, and Streptococcus [8, 9 ]]. Prebiotics are non-digestible food ingredients that exert a beneficial effect on the host by selectively stimulating the growth and/or activity of one or a limited number of probiotic species in the colon [10]Including but not limited to inulin, FOS and GOS [11]. The colon of mammals has long evolved with a diverse microbial ecosystem, promoting mutual benefits and win-win between hosts and microbial communities to a certain extent by forming a powerful immune system in the host [12 ]]. Research has found that lactobacillus reuteri can maintain the number of intestinal stem cells and promote the proliferation of intestinal epithelium by activating Wnt/beta-catenin signal channel, thereby promoting the repair of the intestinal epithelium after being damaged and enhancing the barrier function of the intestinal epithelium to inhibit pathogenic bacteria infection [13 ]]. The exopolysaccharide EPS116 of the lactobacillus plantarum NCU116 can improve Disease Activity Index (DAI) of a DSS-induced colitis mouse model, reduce immune cell infiltration, promote regeneration of colon crypts of mice and proliferation and differentiation of Intestinal Stem Cells (ISCs) to repair Intestinal barriers, change the flora structure of DSS mice, increase the abundance of microorganisms related to Intestinal regeneration and glycan metabolism, and promote Intestinal homeostasis by regulating proliferation and differentiation of ISCs [14 ]]. It has also been found that the synbiotic preparation group (Lactobacillus rhamnosus GG or GG-PB12 combined with soluble corn fiber SCF) can increase NK cell activity, increase Oscillatoria abundance, and improve intestinal flora and immune system of the elderly [15]. The effect of lactobacillus paracasei 01 on the aspect of treating colitis is researched by utilizing a mouse animal model, and the probiotics reaches 10⁸CFU/mL can alleviate symptoms of colitis in mice [16]. In addition, administration of Bifidobacterium breve Bif195 reduced intestinal injury caused by long-term low-dose aspirin [17]And can restore the ratio of firmicutes/bacteroidetes and increase actinomycetes [18 ]]. Further studies have shown that Lactobacillus and Bifidobacterium, Bifidobacterium animalis subsp lactis, Lactobacillus reuteri, Lactobacillus rhamnosus, respectively, can reduce the risk of severe necrotizing enterocolitis by 65%, 69%, 45%, 56% [19 ]]"Rushuang" for treating mammary gland hyperplasiaTriple combination of bifidobacteria CECT 8145, Bifidobacterium longum CECT 7347 and Lactobacillus casei CECT 9104 is effective in helping young atopic dermatitis patients [20]. In addition, it has been shown that S.thermophilus is also a new strain for preventing mouse CRC, and that S.thermophilus can reduce mouse tumor formation significantly by gavage at a certain dosage [21 ]]. The supplement of fructo-oligosaccharide can obviously increase the bifidobacteria in intestinal flora [22 ]]The recovery effect of the lactobacillus and the fructo-oligosaccharide on the disturbance of the intestinal flora of the mice caused by cefixime is better than that of the lactobacillus or the fructo-oligosaccharide which is used alone; the application of fructo-oligosaccharide alone can increase the amount of beneficial bacteria such as Akk bacteria, and enhance immunity [23 ]]. Therefore in this study we tried to study the effect of synbiotic microecological intervention formulations made from the above mentioned prebiotic components etc. in combination with these specifically functioning probiotics (lactobacillus acidophilus, lactobacillus rhamnosus, bifidobacterium adolescentis, bifidobacterium longum, bifidobacterium animalis, lactobacillus reuteri, lactobacillus bulgaricus and streptococcus thermophilus) in AOM/DSS (Azoxymethane/Dextran Sulfate Sodium Salt, DSS) induced CAC mouse model. We assessed their regulatory role in CAC development from colonic barrier function, level of inflammation, flora structure, metabolic regulation, and possibly involved signaling pathways, respectively, to understand their possible mechanisms of action.

Reference to the literature

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[2]GBD 2017 Colorectal Cancer Collaborators. The global, regional, and national burden of colorectal cancer and its attributable risk factors in 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017 [published correction appears in Lancet Gastroenterol Hepatol. 2020 Mar;5(3):e2]. Lancet Gastroenterol Hepatol. 2019;4(12):913-933. doi:10.1016/S2468-1253(19)30345-0

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[11]Gibson GR, Hutkins R, Sanders ME, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol. 2017;14(8):491-502. doi:10.1038/nrgastro.2017.75

[12]Lloyd-Price J, Abu-Ali G, Huttenhower C. The healthy human microbiome. Genome Med. 2016;8(1):51. Published 2016 Apr 27. doi:10.1186/s13073-016-0307-y

[13]Wu H, Xie S, Miao J, et al. Lactobacillus reuteri maintains intestinal epithelial regeneration and repairs damaged intestinal mucosa. Gut Microbes. 2020;11(4):997-1014. doi:10.1080/19490976.2020.1734423

[14]Zhou X, Zhang D, Qi W, et al. Exopolysaccharides from Lactobacillus plantarum NCU116 Facilitate Intestinal Homeostasis by Modulating Intestinal Epithelial Regeneration and Microbiota. J Agric Food Chem. 2021;69(28):7863-7873. doi:10.1021/acs.jafc.1c01898

[15]Costabile A, Bergillos-Meca T, Rasinkangas P, Korpela K, de Vos WM, Gibson GR. Effects of Soluble Corn Fiber Alone or in Synbiotic Combination with Lactobacillus rhamnosus GG and the Pilus-Deficient Derivative GG-PB12 on Fecal Microbiota, Metabolism, and Markers of Immune Function: A Randomized, Double-Blind, Placebo-Controlled, Crossover Study in Healthy Elderly (Saimes Study). Front Immunol. 2017;8:1443. Published 2017 Dec 12. doi:10.3389/fimmu.2017.01443

[16]Pan T, Guo HY, Zhang H, Liu AP, Wang XX, Ren FZ. Oral administration of Lactobacillus paracasei alleviates clinical symptoms of colitis induced by dextran sulphate sodium salt in BALB/c mice. Benef Microbes. 2014;5(3):315-322. doi:10.3920/BM2013.0041

[17]Mortensen B, Murphy C, O'Grady J, et al. Bifidobacteriumbreve Bif195 Protects Against Small-Intestinal Damage Caused by Acetylsalicylic Acid in Healthy Volunteers. Gastroenterology. 2019;157(3):637-646.e4. doi:10.1053/j.gastro.2019.05.008

[18]Quagliariello A, Aloisio I, Bozzi Cionci N, et al. Effect of Bifidobacterium breve on the Intestinal Microbiota of Coeliac Children on a Gluten Free Diet: A Pilot Study. Nutrients. 2016;8(10):660. Published 2016 Oct 22. doi:10.3390/nu8100660

[19]Morgan RL, Preidis GA, Kashyap PC, Weizman AV, Sadeghirad B; McMaster Probiotic, Prebiotic, and Synbiotic Work Group. Probiotics Reduce Mortality and Morbidity in Preterm, Low-Birth-Weight Infants: A Systematic Review and Network Meta-analysis of Randomized Trials. Gastroenterology. 2020;159(2):467-480. doi:10.1053/j.gastro.2020.05.096

[20]Navarro-López V, Ramírez-Boscá A, Ramón-Vidal D, et al. Effect of Oral Administration of a Mixture of Probiotic Strains on SCORAD Index and Use of Topical Steroids in Young Patients With Moderate Atopic Dermatitis: A Randomized Clinical Trial. JAMA Dermatol. 2018;154(1):37-43. doi:10.1001/jamadermatol.2017.3647

[21]Bomhof MR, Parnell JA, Ramay HR, et al. Histological improvement of non-alcoholic steatohepatitis with a prebiotic: a pilot clinical trial. Eur J Nutr. 2019;58(4):1735-1745. doi:10.1007/s00394-018-1721-2

[22]Shi Y, Zhai Q, Li D, et al. Restoration of cefixime-induced gut microbiota changes by Lactobacillus cocktails and fructooligosaccharides in a mouse model. Microbiol Res. 2017;200:14-24. doi:10.1016/j.micres.2017.04.001

[23]Parang B, Barrett CW, Williams CS. AOM/DSS Model of Colitis-Associated Cancer. Methods Mol Biol. 2016;1422:297-307. doi:10.1007/978-1-4939-3603-8_26。

Disclosure of Invention

According to the invention, by reasonably selecting and combining the functional components of the micro powder and the functional probiotics, the obtained microecological preparation has the effect of inhibiting inflammation, and can be used for preventing or treating colorectal cancer.

The results of the invention show that, compared with the AOM/DSS model group, the synbiotics after the intervention treatment has the protection effect on the body weight and the colon length of the mice, can reduce the number of tumor cells and the tumor volume, and suggest that the synbiotics can recover the damaged colon cells, and the histopathology also supports the recovery effect. The present invention detects and compares the expression of various pro-inflammatory (IL-1 beta, TNF-alpha, IL-17, cox-2, IL-12, and IL-23) and anti-inflammatory cytokines (IL-4 and IL-10) from colon tissue, and finds that AOM/DSS mice have a significant increase in pro-inflammatory cytokines and a significant decrease in anti-inflammatory cytokines, however, administration of synbiotic intervention can significantly reverse this trend. The synbiotic intervention formulation was suggested to have anti-inflammatory effects in the CAC mouse model. The invention uses AOM/DSS to treat normal mice to cause epithelial cell inflammation and release proinflammatory cytokines, such as TNF-alpha, IL-17 and IL-1 beta. These cytokines can cause a decrease in the intestinal claudin and ZO-1, resulting in a loss of the colonic barrier. Whereas the mRNA and protein levels showed significant increases following synbiotic intervention, suggesting that synbiotics could overcome intestinal barrier dysfunction by restoring intestinal tight junctions, thereby inhibiting AOM/DSS-induced CAC (A, B in fig. 3).

The invention discovers that synbiotic intervention obviously increases colonLactobacillus、Bifidobacterium、AkkThe relative abundance of beneficial bacteria such as bacteria, etc., and reduces the number of pathogenic bacteria such as Escherichia/Shigella,Bacteroides_fragilisrelative abundance of (a). This result suggests that synbiotic intervention is given during CAC development, or that gut flora architecture can be modulated. In the research, the content of SCFAs in the excrement of the model group mice is obviously reduced, and is possibly related to the reduction of the relative abundance of short-chain fatty acid-producing bacteria in intestinal tracts, and the level of butyric acid-producing bacteria can be obviously increased by providing synbiotics intervention, so that the generation of SCFAs is promoted, and the generation of inflammatory reaction, the damage of intestinal tracts and the formation of CAC are inhibited to a certain extent. Data indicate that tryptophan and 5-HT are obviously accumulated in excrement of mice in a CAC model group, compared with normal mice, the tryptophan metabolism of microorganisms with imbalance caused by the remarkably reduced levels of tryptophan metabolites IAA, IPA and IA can be remarkably recovered through synbiotic intervention, which is probably due to the regulating effect of the tryptophan metabolism on intestinal microorganisms, and research shows that the tryptophan metabolites can regulate the composition of the intestinal microorganisms, so that the intestinal barrier function is improved, and an immune system is activated.

The results of the discovery of the invention that the levels of each metabolite gradually return to normal levels after synbiotic intervention indicate that the tailor-made synbiotic agent used in this experiment can regulate bile acid metabolism by altering the flora intestinal flora composition, thereby inhibiting colonic inflammation and tumor development. The invention finds that synbiotics intervention can obviously reduce the level of related protein, inhibit the activation of a Wnt/beta-catenin signal channel to inhibit the canceration and proliferation of cells so as to inhibit the occurrence and development of colorectal cancer; synbiotic intervention treatment can obviously up-regulate Bcl-2 expression and down-regulate Bax and ki67 expression. It was further demonstrated that supplementation of synbiotics during CAC formation could promote apoptosis and inhibit cell proliferation, thereby inhibiting the development and progression of colorectal cancer.

Based on this, the technical scheme provided by the invention is as follows:

the invention provides a microecological preparation which mainly comprises the following functional components: FOS, GOS, inulin, apple powder, blueberry powder and functional probiotics, wherein the functional probiotics are formed by mixing lactobacillus acidophilus, lactobacillus rhamnosus, bifidobacterium adolescentis, bifidobacterium longum, bifidobacterium animalis, lactobacillus reuteri, lactobacillus bulgaricus and streptococcus thermophilus, preferably the functional probiotics are formed by mixing lactobacillus acidophilus TYCA06, lactobacillus rhamnosus F-1, bifidobacterium adolescentis BH-20, bifidobacterium longum BETA 536, bifidobacterium animalis bb12, lactobacillus reuteri GL-104, lactobacillus bulgaricus 11842 and streptococcus thermophilus grx02, preferably in the form of embedded bacteria powder.

Preferably, FOS, GOS, inulin, blueberry powder, apple powder and the like are mixed in equal mass ratio to form a micro powder mixture, embedding bacterium powder of each strain is mixed in equal mass ratio to form a bag, and then the bag is mixed with the bacterium powder mixture, wherein the ratio of the FOS to the embedding bacterium powder is 1-2: 1-4, preferably 2: 1.

The invention provides the microecological preparation which is characterized by comprising the following steps:

(1) freeze-drying powder preparation: the inulin, the blueberry powder and the apple powder are fresh fruits and are respectively smashed and juiced, filtered and then respectively freeze-dried to prepare dry powder; weighing FOS, GOS, inulin, blueberry powder and apple powder in equal proportion according to the mass ratio, and uniformly mixing to obtain a micro powder mixture;

(2) preparing embedded bacterium powder, namely performing fermentation culture on each strain respectively, then performing centrifugal concentration, fixing the strain by adopting protein polypeptide and calcium alginate gel for embedding, then performing freeze drying and crushing to obtain the embedded bacterium powder, and uniformly mixing various embedded bacterium powders according to equal proportion to obtain an embedded bacterium powder mixture;

(3) and (3) weighing and uniformly mixing the mixture obtained in the step (1) and the mixture obtained in the step (2) according to the mass ratio of the mixture to the mixture.

In particular, each step is operated under sterile conditions and finally sterile packaging is employed.

The invention also provides application of the microecological preparation in prevention and adjuvant treatment of inflammation and colorectal cancer.

Further provides application of the microecological preparation in preparation of products for relieving and preventing intractable constipation, constipation caused by radiotherapy/chemotherapy, or enhancing intestinal immunity, or improving dysbacteriosis caused by antibiotics.

Meanwhile, the invention also provides a preparation method of the microecological preparation, which is characterized by comprising the following steps:

(1) freeze-drying powder preparation: the inulin, the blueberry powder and the apple powder are fresh fruits and are respectively smashed and juiced, filtered and then respectively freeze-dried to prepare dry powder; weighing FOS, GOS, inulin, blueberry powder and apple powder according to a proportion, and uniformly mixing to obtain a micro powder mixture;

(2) preparing embedded bacterium powder, namely performing fermentation culture on each strain respectively, then performing centrifugal concentration, fixing the strain by adopting protein polypeptide and calcium alginate gel for embedding, then performing freeze drying and crushing to obtain the embedded bacterium powder, and uniformly mixing various embedded bacterium powders according to a proportion to obtain an embedded bacterium powder mixture;

(3) and (3) weighing and uniformly mixing the mixture obtained in the step (1) and the mixture obtained in the step (2) according to the mass ratio of the mixture to the mixture.

Drawings

FIG. 1 development of AOM/DSS mouse tumors and recovery of synbiotics dried prognosis pathological model of colorectal cancer (CAC).

Wherein, (A) an AOM/DSS-induced CAC model and an experimental method of synbiotics intervention treatment are established. The model group mice were first intraperitoneally injected with AOM (10mg/kg), then allowed to freely drink 2.5% DSS drinking water for one week, and then changed to sterilized water for 2 weeks for recovery; this was repeated 3 times for one cycle. The mice in the intervention group are irrigated with stomach synbiotics intervention preparation every day. (B) Percent change in mean body weight for each group of mice; control group mice (control), AOM-DSS-induced CAC mice (AOM/DSS), and synbiotics intervention group (AOM/DSS _ synbiotics). (C) Measuring and comparing the colon length of each group after the molding is finished and (D) a colon length statistical analysis chart. (E) Statistical plots of colon tumor number and size distribution for each group of mice; (F) colon H & E staining schematic (scale, 50 μm). (ii) a Data are expressed as mean ± SEM;. p <0.05,. p <0.01,. p <0.001,. p <0.0001, (n =5-10), Student's t-test.).

FIG. 2. Synbiotics inhibited AOM/DSS-induced intestinal barrier disruption and colonic inflammation in tumor mice.

Wherein (A) the mRNA levels of ZO-1 and Occludin in colon tissues of each group are detected by qRT-PCR, and GAPDH is used as an internal reference gene; (B) detecting the expression conditions of the TJ proteins ZO-1 and Ocplus of each group by using western blotting, performing statistical analysis, and using beta-actin as an internal reference protein; (C) qRT-PCR was used to detect the mRNA level of cytokines in each group of colon tissues and to perform statistical analysis, using GAPDH as reference gene; (D) detecting the expression conditions of the prebiotics, synbiotics TNF-alpha, IL-1 beta and IL-23 by using western blotting, and performing statistical analysis; data are expressed as mean ± SEM;. p <0.05,. p <0.01,. p <0.001,. p <0.0001, (n =5-10), Student's t-test.).

FIG. 3.16 SrDNA sequencing analysis of gut microbial changes in AOM/DSS-induced mouse colitis-associated tumors following synbiotic intervention.

Wherein, (A) the number of OTUs in each group of samples is counted. (B) The alpha diversity of the microorganisms is Simpson index, Chao1 index and Shannon index; (C) and (4) beta diversity analysis, namely evaluating the difference significance of the sample community distribution among groups based on the PCoA analysis of the Bray Curtis distance matrix algorithm. (D) Statistical analysis was performed at the gate (L2) level for each group, and the relative abundance of the different species was compared between groups. (E) Relative abundance of Firmicutes, Bacteroides and ratio of Firmicutes to Bacteroides for each group. (H) Comparison of key species differences at the genus level. (I) Statistical analysis of species Akk that differ at the species level. Data are expressed as mean ± SEM;. p <0.05,. p <0.01,. p <0.001,. p <0.0001, (n =5-10), Student's t-test.).

Figure 4 screening for key differential metabolites using non-targeted metabolomics approach.

Wherein (a) the analysis of the similarity of intestinal metabolites between groups by the OPLS-DA method and (B) a statistical heat map of the differential metabolites between the comparison synbiotic intervention group and the model group; (C) a map of metabolic pathway enrichment bubbles plotted against the significant TOP-20 differential metabolite between the CAC model group and the synbiotic intervention group. (D statistical graphs comparing the content of intestinal SCFA (acetic acid, propionic butyric acid, valeric acid) between different groups (E) statistical analysis of the intestinal content of Tryptophan levels (L-Tryptophan, Asparaginyl-Tryptophan, 5-Hydroxy-L-Tryptophan) and Tryptophan metabolite levels (IPA, IAA and IA), (F) statistical graphs of bile acid levels for each group [ data are mean + -SEM ], [ p <0.05 ], p <0.01, [ p <0.001 ], p <0.0001, (n =5-10), Student's t-test ].

FIG. 5. synbiotics intervene or inhibit the Wnt/beta-catenin signaling pathway.

Wherein (a) KEGG enriches top20 bubble map. Colon tumor tissue transcriptome sequencing and Pathway analysis of the differential protein encoding gene was performed using the KEGG database. (B) The IL-23 and the Wnt/beta-catenin signal channel are subjected to correlation analysis and are shown in the drawing. (C) WB verifies the expression condition (D) of key protein molecules in the Wnt/beta-catenin signal channel and carries out statistical analysis. (E) IHC staining detected Ki67 expression. Data are expressed as mean ± SEM;. p <0.05,. p <0.01,. p <0.001,. p <0.0001, (n =5-10), Student's t-test.).

Detailed Description

All data in the following examples are expressed as mean ± standard deviation. All data were analyzed using GraphPad Prism software. The differences between the two groups in the colony analysis were statistically analyzed by the rank-sum test, and the differences between the other two groups were statistically analyzed by the t-test. P <0.05 is a statistical difference between the two groups.

Example 1 preparation of Synbiotic Microecological preparation

The synbiotic microecologics used in the examples were mainly composed of the following functional components: fructosooligosaccharide (FOS) + galactooligosaccharide (Galactooligosaccharides, GOS) + inulin + apple powder + blueberry powder) 2 g/kg b.w. + functional probiotic (10)⁹CFU/kg b.w.), the functional probiotics are formed by mixing lactobacillus acidophilus, lactobacillus rhamnosus, bifidobacterium adolescentis, bifidobacterium longum, bifidobacterium animalis, lactobacillus reuteri, lactobacillus bulgaricus and streptococcus thermophilus, and specifically are formed by mixing lactobacillus acidophilus TYCA06, lactobacillus rhamnosus F-1, bifidobacterium adolescentis BH-20, bifidobacterium longum BETA 536, bifidobacterium animalis bb12, lactobacillus reuteri GL-104, lactobacillus bulgaricus 11842 and streptococcus thermophilus grx02 according to a certain proportion. The specific preparation method and the dosage of each component need to provide instructions.

The preparation method comprises the following steps:

(1) freeze-drying powder preparation: the inulin, the blueberry powder and the apple powder are prepared by respectively mashing fresh fruits, juicing, filtering, and respectively freeze-drying to prepare dry powder. The specific operation is as follows: respectively selecting fresh unrotten Jerusalem artichoke, blueberry and apple, adding drinking water according to the mass ratio of fresh fruits to water of 1:4, adding 0.04% of food-grade citric acid according to the mass percentage, blending uniformly, repeatedly grinding by using a colloid mill for 2 times to squeeze juice, then adding 0.4% of food-grade pectase, carrying out enzymolysis for 2 hours at 42-46 ℃, and carrying out water bath at 80 ℃ for 15 min after enzymolysis; adding food-grade sodium carboxymethylcellulose and cyclodextrin, pre-freezing for 3h at-40 deg.C under vacuum degree of 8-10Pa and sublimation heating temperature of 35 deg.C);

(2) and (3) culturing microorganisms: the strains are firstly fermented and cultured respectively. The specific operation is as follows: liquid bacteria preserved at-80 deg.CRapidly thawing the seeds at 37 ℃, then inoculating the seeds into a TPY liquid culture medium according to the inoculation amount of 3-5% of the volume ratio, and carrying out anaerobic culture at 37 ℃ for 36 h; and (3) continuously activating the above culture bacterial liquid for 2 times under the above conditions, inoculating the activated bacterial liquid into an anaerobic fermentation tank, continuously fermenting for 50-72 hours in batches at 37 ℃, properly feeding 3 XTPY liquid culture medium for supplementing nutrients, and measuring the number of viable bacteria according to a conventional method after the fermentation is finished. TPY liquid culture medium (casein peptone 10.0g, yeast extract powder 2.5 g, glucose 5.0 g, Tween 801.0 ml, L-cysteine 0.5 g. mixed salt solution 5.0 ml, distilled water 995 ml; mixed salt solution: potassium dihydrogen phosphate 20.0g, water and magnesium chloride 5.0 g, water and zinc sulfate 2.5 g, calcium chloride 1.5 g, ferric chloride 0.5 g, distilled water 100 ml, pH 7.2 adjusted, 0.1MPa sterilized 20 min. then centrifuged 30min at 4000 g at 4 ℃ to concentrate the strain, and the strain was embedded by immobilizing protein polypeptide and calcium alginate gel by placing sodium alginate of 20g/L mass concentration and protein polypeptide solution of 20g/L (mass ratio 1: 2), sterilizing at 121 ℃ for 20 min, naturally cooling, and storing at 4 ℃ about 1-2 × 10⁷Mixing cfu/mL bacterial suspension and mixed solution according to a ratio (3: 20), stirring evenly at 4 ℃, extruding the mixed solution into 0.1 mol/L calcium chloride solution by using an injector, standing for 30min, centrifuging for 10 min at 4 ℃ 2000 g, washing for 3 times by using sterile water, centrifuging for 10 min at 4 ℃ 2000 g to obtain embedded bacterial strains, then freezing, drying and crushing to obtain embedded bacterial powder (pre-freezing for 3h at (-40 ℃), and crushing to obtain 50-200 mu m probiotic powder at 0 ℃ by using a low-temperature crusher, and evenly mixing various embedded bacterial powders in the formula according to an equal ratio, wherein the temperature in the sublimation drying stage is-30 ℃.

(3) Weighing FOS and GOS, the prepared inulin, blueberry powder and apple powder according to the mass ratio in equal proportion, and uniformly mixing. As the raw materials for FOS and GOS, products of Shaoshan Changbutong biological Co., Ltd can be used, for example.

(4) And (4) weighing and uniformly mixing the mixture obtained in the step (2) and the mixture obtained in the step (3) according to the mass ratio of 1:2, and then carrying out sterile packaging to obtain the synbiotic microecological preparation. All of the above steps are required to be performed in a sterile environment.

Example 2 validation of the effectiveness of Synbiotic Microecological Agents

1. AOM/DSS-induced CAC animal model

Healthy male 6-week-old SPF-grade C57BL/6J mice weighing 18.0-20.0 g were purchased from Schlekshirta laboratory animals, Inc. in Hunan. All experiments were performed under approval of the local ethics committee and according to the laboratory animal care and use guidelines of the animal testing center of the medical college of the university of hannam, the breeding conditions of the mice were: the temperature is 20-25 ℃, the humidity is 40-70%, the daily and night rhythm is 12h, and the water and the diet are freely drunk.

After an adaptation period of 7 days, mice were randomly assigned to 3 groups (n = 10/group), including a blank Control group (Control), a CAC model group (AOM/DSS), a synbiotics intervention group (AOM/DSS _ synthetic, using the synbiotics prepared in example 1). Two weeks before AOM/DSS modeling and before sampling is finished, the mice of the blank control group and the model group are respectively administered with sterilized water by intragastric administration every day, and the synbiotics intervention group is respectively administered with synbiotics by intragastric administration every day.

Blank control group mice: drinking sterilized water freely every day; CAC model group mice: the AOM/DSS and AOM/DSS _ Synbiotic groups were administered by intraperitoneal injection of 10mg/kg AOM (Sigma-Oldrich) once at week 1, and after one week, the drinking water in both groups was changed to 2.5% DSS (Sigma-Oldrich) solution prepared with sterilized water, and allowed to drink freely for 1 week, and then changed to sterilized water again for 2 weeks to recover. DSS was repeated 3 times throughout the trial period ( weeks 2, 5, 8) (a in figure 1), although AOM was administered only once on week 1. During the study we measured body weight once daily. Mice were euthanized at week 10 and their colons were excised. After measurement of colon length, longitudinal dissection was performed and the intestinal contents were collected for metabonomic analysis and washed with PBS to count tumor numbers. Thereafter, 2cm below the cecum was cut out for colony and transcriptome sequencing specimens, the distal 1cm colon was left for paraffin section specimens, and the intestine was fixed with 10% neutralizing protein Fulmalin (Sigma-Aldrich). The remaining intestinal sections were stored in liquid nitrogen for later extraction and real-time PCR, and Westernblot analysis. All experiments were performed under approval of the local ethics committee and according to the laboratory animal care and use guidelines of the animal testing center of the medical college of the university of hanceau, hunan.

3. Histopathological analysis

Fixed colon sections were embedded in paraffin and sectioned in a semi-automatic paraffin microtome at 3 μm thickness for H & E staining and immunohistochemistry using standard procedures. Observation and image acquisition were performed using an OlympusCKX41 microscope (tokyo, japan). Each group of 5 mice, each mouse randomly selected 20 times the objective lens under 3 visual field analysis.

4. Detection of cytokine levels

Western Blot (WB) tissue lysate (200. mu.L tissue lysate/10 mg tissue) was added to the mouse colon in proportion, ground thoroughly on ice with a tissue homogenizer, allowed to stand for 30min, fully lysed, centrifuged at 5000 g at 4 ℃ for 5 min, and the supernatant was collected. Then, determining the concentration of the protein of the sample by using a BCA kit to adjust the proper sample loading amount, adding a protein loading buffer solution in proportion, boiling for 10 min, and separating the protein by using SDS-PAGE electrophoresis with proper concentration, wherein the electrophoresis condition of the upper layer of concentrated gel is 80V 0.5 h, and the electrophoresis condition of the lower layer of separation gel is 120V 1h, so as to fully electrophoretically separate the protein sample; transferring the separated protein sample to a PVDF membrane in a wet-transfer mode; sealing with 5% skimmed milk powder on a shaking table at room temperature for 1.5 h, and washing with 1xTBST for 3 times and 5 min/time; immersing the PVDF membrane into a corresponding protein antibody diluted by an antibody diluent, incubating overnight at 4 ℃, and washing the membrane for 3 times and 10 min/time by using 1 xTBST; then immersing the PVDF membrane into a secondary antibody diluted by 1xTBST, incubating for 1h on a shaking bed at room temperature, and washing the membrane for 3 times and 10 min/time by using 1 xTBST; uniformly dripping ECL chemiluminescence liquid on a PVDF membrane, scanning the PVDF membrane by using a Tannon full-automatic chemiluminescence imaging analysis system, and displaying a specific protein band; finally, the grey value of the protein band is analyzed by using software Image J for quantification

5. Total RNA extraction and real-time fluorescent quantitative PCR

Total RNA extraction was performed using TRIzol reagent (Beijing Dingguoshang organism) according to the instructions of the reagent manufacturer. RNA was then quantified using a SynergyHTX microplate reader (Burton, USA), and cDNA synthesis was performed using a reverse transcription kit (Escirey). Real-time fluorescence quantificationUse of PCRPro Taq The HS Premix Probe qPCR Kit (Ecori organism), (Bio-Rad) CFX96 ™ quantitative PCR instrument was used for qPCR. Primer sequences are shown in table S2. Real-time fluorescent quantitative PCR data were analyzed by the 2- Δ Δ Ct method, and normalized with GAPDH.

6. Microbial community analysis

Extracting the mouse colon mucosa tissue microorganism, extracting the genome DNA of a sample by using a DNA extraction kit of Shanghai Europe and Yi biomedicine science and technology Limited company, and performing PCR amplification by using a specific primer with Barcode to establish a 16S amplicon library. And (3) removing impurities of the original double-ended sequence by using Trimmomatic software, and performing the double-ended sequence after removing the impurities by using FLASH software. After the sequencing data are preprocessed to generate a high-quality sequence, the sequence is classified into a plurality of OTUs according to the similarity of the sequence by adopting Vsearch software. Selecting representative sequences of each OTU by using QIIME software, comparing all the representative sequences with a database Greenenenes, counting species compositions on phylum, class, order, family, genus and species level, annotating by using RDP classifier software, and simultaneously carrying out Alpha diversity and Beta diversity on samples.

7. Ginseng transcriptome analysis

After extracting total RNA of a sample and digesting DNA by using DNase, enriching eukaryotic mRNA by using magnetic beads with oligo (dT); adding an interrupting reagent to break mRNA into short segments, synthesizing first-strand cDNA by using a six-base random primer by using the broken mRNA as a template, preparing a second-strand synthesis reaction system to synthesize second-strand cDNA, and purifying the double-strand cDNA by using a kit; carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, then carrying out fragment size selection, and finally carrying out PCR amplification; the constructed library was qualified by Agilent 2100 Bioanalyzer quality testing and sequenced using Illumina HiSeqTM 2500 or Illumina HiSeq X Ten other sequencers to generate 125bp or 150bp paired end data. Raw data (raw reads) generated in high throughput sequencing are fastq format sequences. To obtain high quality reads that can be used for subsequent analysis, the raw reads are further quality filtered. Firstly, using Trimmomatic software to carry out quality control and remove joints, and filtering out low-quality bases and N bases on the basis to finally obtain high-quality clean reads. Clean reads were aligned to the reference genome of the species using hisat2, with software parameters as default parameters, and the condition of the samples was assessed by genome alignment. The results of the clean reads alignment with the reference genome are stored in a binary file, i.e., a bam file. Gene FPKM expression values were quantified using cufflinks software. In calculating the difference in expression amount of genes, the number of reads of genes that fell into each sample was obtained by htseqcount software, data was normalized using the estimazefactors function of DESeq (2012) R package, and pvalue and foldchange values for difference comparison were calculated using the nbinomTest function. And (3) selecting the differential genes with the p value less than 0.05 and the difference multiple more than 2, and carrying out GO and KEGG enrichment analysis on the differential genes to judge the biological functions or pathways mainly influenced by the differential genes. And meanwhile, carrying out unsupervised hierarchical clustering on the differential genes, and displaying the expression modes of the differential genes among different samples in a heat map mode.

8. LC/MS non-targeted metabolome analysis

The method comprises the steps of carrying out metabolome analysis on the intestinal contents of a mouse by a liquid chromatography-mass spectrometry system consisting of a Dionex U3000 UHPLC ultra-high performance liquid phase tandem QE plus high-resolution mass spectrometer by Shanghai Europe biomedical science and technology Limited company, carrying out baseline filtration, peak identification, integration, retention time correction, peak alignment and normalization on original data by metabonomics processing software Progenetics QI v2.3 software (Nonlinear Dynamics, New castle, UK), deleting ion peaks with deletion values (0 value) >50% in a group from the data, replacing the 0 value by half of the minimum value, classifying (Score) according to the qualitative result of the compound, screening the qualitatively obtained compound with the screening standard of 40 points (60 points), and considering that the qualitative result below 40 points is inaccurate and deleted. And finally combining the positive and negative ion data into a data matrix table, wherein the matrix contains all information which can be used for analysis and is extracted from the original data, and the subsequent analysis is based on the information and comprises multivariate statistical analysis, univariate statistical analysis, differential metabolite screening, correlation analysis and metabolic pathway enrichment analysis.

Results of the experiment

1. Improving pathological indexes

Prevention of colorectal carcinogenesis by synbiotics was observed using the AOM/DSS-induced CAC model (A in FIG. 1). After induction of DSS, the mice lost weight significantly, but with discontinuation of the DSS solution, they gradually recovered, and with synbiotic intervention, the mice lost weight (fig. 1B) was alleviated. In addition, compared with the normal control group, the colon of the mouse in the model group is obviously shortened, obvious tumor occurrence is realized, synbiotic intervention can restore the length of the colon, the number of tumors is reduced, the volume of the tumors is reduced, and the ratio of the tumors with the diameter of more than 2mm is obviously reduced (C-F in figure 1). In addition, we performed HE staining of colon disease sections to observe colon inflammation and mucosal damage in mice. The colon of the mice in the normal control group has intact colon epithelium, intestinal gland, mesenchyme and submucosa, while the mice in the AOM/DSS group have shown surface epithelial erosion, crypt destruction, mucosal muscularis destruction, submucosal edema and inflammatory cell infiltration, low-level or high-level intraepithelial neoplasia of the colon mucosa, large proliferation of cancer cells, nuclear enlargement and deep staining and nuclear-to-cytoplasmic ratio increase. The crypt structure of the intestinal mucosa remained relatively well and its histological features were significantly improved or restored compared to after synbiotic intervention (G in fig. 1).

2. Restoration of colonic mucosal barrier

It is known that tight junctions play a crucial role in maintaining cell-cell integrity and their loss can accelerate the pathological onset and progression of various gastrointestinal disorders (e.g., IBD, irritable bowel syndrome and even colorectal cancer). Therefore, we examined the mRNA and protein levels of the tight junction proteins ZO-1, Occludin. Compared with the normal control group, the mRNA expression of the AOM/DSS control group is obviously inhibited. However, after administration of synbiotic intervention, the level of claudin mRNA expression and the corresponding protein expression was significantly restored (fig. 2A, fig. 2B). This result indicates that the preventive supplementation of synbiotic formulations at AOM/DSS-induced CAC can protect the intestinal epithelial barrier by restoring expression of tight junction proteins.

3. Reduction of inflammatory response

Inflammatory molecules are involved in various processes in colon carcinogenesis associated with colitis. To elucidate the AOM/DSS-induced inflammatory response in CAC mice, we measured various proinflammatory and anti-inflammatory cytokines at mRNA and protein levels in colon tissue. In contrast to the results obtained with AOM/DSS induction, mRNA expression of various proinflammatory factors (IL-1. beta., IL-12b, IL-17A, TNF-alpha., IL-23, COX-2) was significantly increased in colon tissues of mice, but gradually decreased after intervention with synbiotic agents, and in CAC model group mice showed significantly decreased mRNA expression of anti-inflammatory cytokines such as IL-4 and IL-10, whereas synbiotic intervention significantly increased the expression of anti-inflammatory cytokines IL-4, IL-10 (FIG. 2C). Meanwhile, the expression of TNF-. alpha.IL-1. beta., IL-23 protein levels was verified by Western Blotting (FIG. 2D). The results indicate that synbiotics therapy used in this experiment can effectively protect the intestinal barrier by reducing AOM/DSS-induced colonic inflammatory responses.

4. Modulating microbial community architecture

We predict that synbiotic intervention can alter the gut microbiota composition of AOM/DSS-induced CAC mice. Thus, at the end of the experiment, we collected stool samples from each group and analyzed colonic mucosa microbial composition using 16sr DNA flora sequencing. We found that the number of AOM/DSS-induced CAC mice OTU was only 1861, which was significantly reduced compared to Control group (2304). Given the synbiotic stem prognosis, the number of OTUs (2104) was significantly increased. Alpha diversity analysis is a comprehensive indicator of species abundance (Chao1) and diversity (Shannon and Simpson indices). As shown in fig. 3, the bacterial population in colon was significantly reduced in the mice of the model group compared to the control group, while the relative abundance of intestinal microbes was significantly increased after synbiotic intervention and the diversity tended to increase, but there was no significant difference (fig. 3B). Principal Component Analysis (PCA) showed significant differences in the similarity between the control group and the AOM/DSS model group and the synbiotic intervention group (fig. 3C), with significant changes in the interclass flora structure. Therefore, we further investigated the bacterial classification profile at the intergate level (fig. 3D). 19 gates were detected in all samples, of whichBacteroidetes(Bacteroides) of,Firmicutes(thickness ofMuramida) is the most abundant door. Of mice in CAC group compared with normal control groupFirmicutesThe relative abundance is remarkably reduced, and the content of the active ingredients is reduced,Bacteroidetesthe relative abundance of the active peptide is increased,FirmicutesandBacteroidetesthe ratio is significantly reduced. Synbiotic intervention can be initiated by reversing this trend (fig. 3E)). At the scientific level, synbiotic intervention may lead toBurkholderiaceae(Burkholderia family)Peptococcaceae(of the family of the digestive coccaceae),Deferribacteraceae(the family of deferribacteriaceae) shows an increasing trend,Pasteurellaceae(Pasteuria family),Streptococcaceae(of the family of Streptococcus),Sphingomonadaceae(of the family of the sphingolipid monadaceae),Pseudomonadaceae(Pseudomonas family),Enterococcaceae(enterococcaceae) was significantly reduced (fig. 3F). In addition, we screened genera with significant differences between groups and found several opportunistic pathogens such as pathogenic Escherichia coli, Shigella, Escherichia coli,Bacteroides_fragilis、Acinetobacter、Anaerotruncus、Fusobacterium nucleatumEtc. of specific microorganisms are significantly elevated, andLactobacillus、Bifidobacterium、 RuminococcusRoseburia、Butyricicoccus、Faecalibacterium、Roseburia、 Subdoligranulu、Clostridium leptumsignificant reductions in relative abundance of beneficial bacteria occurred, and synbiotic intervention was given to restore them to normal levels (fig. 3G-I). Subsequently, we further compared the species level of the differential bacteria to find the probiotic bacteriaAkkermansiaSignificantly increased in synbiotic intervention group (fig. 3J). The results show that the synbiotics intervention is given in the CAC forming process, the disorder of the intestinal flora structure can be effectively relieved, and the relative abundance of pathogenic bacteria can be reduced by increasing the relative abundance of intestinal probiotics, so that the occurrence of inflammation is reduced, and the formation and development of CAC are inhibited.

5. Promote intestinal flora to produce SCFA, and regulate metabolism of tryptophan and bile acid

The effects of gut microbes on the host involve a complex series of host-microbe axis interactions. While small molecule metabolites are key to coordinating this interaction, observing gut microbiologically regulated metabolite changes may allow us a better understanding of the biochemical mechanisms by which organisms target gut microbes to improve AOM/DSS-induced CAC. Therefore, we used non-targeted metabolomics methods to analyze metabolites in different cohort stool specimens in an attempt to screen out differential metabolites that are associated with AOM/DSS-induced CAC development. Consistent with the expectations, there were significant metabolic differences between the different groups (fig. 4A). Synbiotic intervention was effective in regulating the levels of certain related metabolites and restoring them to normal group levels (fig. 4B).

SCFA are produced by the intestinal flora, in particular lactobacilli and bifidobacteria, and play an important role in maintaining the balance of the intestinal flora and preventing intestinal diseases. In this study, the use of tailor-made synbiotic formulations on AOM/DSS-induced CAC mice resulted in a significant increase in lactobacilli, bifidobacteria and Ackermansia, these bacteria being termed SCFA producers [24 ]. The results show that acetic acid is the most abundant SCFA in the stool. The contents of acetic acid, propionic acid, butyric acid and valeric acid in the feces of the model group are all obviously reduced, and the content of 4 short-chain fatty acids in the feces is obviously increased by synbiotic treatment (figure 4C). And it was reported that SCFA production inhibits the growth of certain pathogens in the gut [25], we hypothesize that synbiotic agents might inhibit the growth of specific pathogens in the CAC mouse model by increasing SCFA production. The results show that synbiotic preparation used in the experiment can reduce the proliferation of pathogenic bacteria by stimulating the growth of AOM/DSS-induced specific beneficial bacteria in the intestinal tract of CAC mice and the generation of SCFA, thereby inhibiting the generation of inflammation.

At the same time, we found that metabolic differences between groups also involved tryptophan metabolism and bile acid metabolism. The analysis result shows that the levels of L-Tryptophan (Tryptophan), 5-Hydroxy-L-Tryptophan (5-Hydroxy-L-Tryptophan) and Asparaginyl-Tryptophan (Asparaginyl-Tryptophan) in the model group are obviously increased, and the levels of indoleacetic acid (IAA), indolepropionic acid (IPA) and Indoleacrylic Acid (IA) are obviously reduced; compared with the normal group, the primary bile acid (CA) and the chenodeoxycholic acid (CDCA) are remarkably accumulated in the model group, and the levels of the secondary bile acid (DCA) and the lithocholic acid (LCA) are obviously reduced. Administration of synbiotic intervention clearly reversed the trend of these metabolites and gradually returned them to normal (fig. 4E-F).

6. Inhibiting Wnt/beta-catenin signal channel, promoting apoptosis, and inhibiting proliferation

In addition, to further explore the specific molecular mechanisms of the binding organisms' intervention in CAC, we also performed transcriptome sequencing of colon tissues. Pathway analysis of the differential protein encoding gene using the KEGG database showed that the Wnt/β -catenin signaling Pathway plays a key role (fig. 5A). Furthermore, previous results showed that the reduction of all inflammatory factor IL-23 levels after synbiotic intervention was most significant. Therefore, the IL-23 and the Wnt/beta-catenin signal pathway are subjected to correlation analysis. Based on the cor value, cor >0 is positively correlated, 23 genes which are positively correlated with IL-23 in the Wnt/beta-catenin pathway are found, namely Dkk2, Notum, Axin2, Nlk, Wif1, Tcf7, Ccnd1, Mmp7, Wnt6, Wnt10a, Senp2, Vangl2, Lef1, Vangl1, Nkd1, Fzd10, Wnt16, Wnt3, Bambi, Nfatc3, Wisp1, Gsk3b and Plcb2, and the cor <0 is negatively correlated, and 6 genes which are negatively correlated are found: fzd1, Sfrp2, Wnt5a, Tbl1x, pp3cb, dam 1 (fig. 5B). Therefore, we further verified the significantly related molecules using WB and qPCR, and the results showed that Wnt3 and Wnt10a signals were significantly enhanced, the Frizzled 9 expression was significantly increased, and β -catenin expression was also increased in model group mice, but synbiotic intervention could significantly reverse this expression trend (fig. 5C, E). In addition, compared with a control group, the expression of a downstream transcription factor LEF/TCF combined with beta-catenin in the model group is obviously increased, the expression of a downstream target gene MMP8 is obviously increased, the level of a pro-apoptotic protein Bax is obviously reduced, and the level of an anti-apoptotic protein Bcl-2 is obviously increased. The expression levels were clearly reversed after treatment with synbiotic intervention (FIG. 5D, E). Meanwhile, IHC staining is used for detecting the expression condition of a cell proliferation index Ki67 among groups, and the positive rate of a model group is found to be remarkably higher than that of a blank control group, while the positive rate of a synbiotic intervention group is obviously reduced compared with that of the model group (FIG. 5F). The results indicate that in AOM/DSS-induced CAC, a Wnt/beta-catenin signal path is abnormally activated, cancer cell proliferation is promoted, apoptosis is inhibited, and IL-23 mediated inflammatory reaction is enhanced. Whereas synbiotic intervention may inhibit the development of CAC by inhibiting the activation of this pathway.

Claims (9)

1. A microecological preparation mainly comprises the following functional components: FOS, GOS, inulin, apple powder, blueberry powder and functional probiotics, wherein the functional probiotics are formed by mixing bacterial powder of lactobacillus acidophilus, lactobacillus rhamnosus, bifidobacterium adolescentis, bifidobacterium longum, bifidobacterium animalis, lactobacillus reuteri, lactobacillus bulgaricus and streptococcus thermophilus.

2. The microecological formulation according to claim 1, wherein the functional probiotic bacteria are a mixture of powders of Lactobacillus acidophilus TYCA06, Lactobacillus rhamnosus F-1, Bifidobacterium adolescentis BH-20, Bifidobacterium longum BETA 536, Bifidobacterium animalis bb12, Lactobacillus reuteri GL-104, Lactobacillus bulgaricus 11842 and Streptococcus thermophilus grx 02.

3. The microecological formulation of claim 2, wherein the bacterial powder is in the form of embedded bacterial powder.

4. The microecological preparation according to claim 1, wherein FOS, GOS, inulin, blueberry powder and apple powder are mixed in equal mass ratio to form a micro powder mixture, the embedded bacteria powder of each strain is mixed in equal mass ratio to form a packaged bacteria powder mixture, and the ratio of the former to the latter is 1-2: 1-4, preferably 2: 1.

5. The microecological formulation according to claim 4, wherein the preparation process comprises:

(1) freeze-drying powder preparation: the inulin, the blueberry powder and the apple powder are fresh fruits and are respectively smashed and juiced, filtered and then respectively freeze-dried to prepare dry powder; weighing FOS, GOS, inulin, blueberry powder and apple powder in equal proportion according to the mass ratio, and uniformly mixing to obtain a micro powder mixture;

(2) preparing embedded bacterium powder, namely performing fermentation culture on each strain respectively, then performing centrifugal concentration, fixing the strain by adopting protein polypeptide and calcium alginate gel for embedding, then performing freeze drying and crushing to obtain the embedded bacterium powder, and uniformly mixing various embedded bacterium powders according to equal proportion to obtain an embedded bacterium powder mixture;

(3) and (3) weighing and uniformly mixing the mixture obtained in the step (1) and the mixture obtained in the step (2) according to the mass ratio of the mixture to the mixture.

6. The microecological formulation of claim 3, wherein each step is performed under sterile conditions and is finally packaged aseptically.

7. Use of the probiotic according to any one of claims 1 to 5 for the prevention, co-treatment of inflammation, colorectal cancer.

8. Use of a probiotic according to any one of claims 1 to 5 for the preparation of a preparation for alleviating, preventing, or enhancing the immunity of the intestine, intractable constipation, constipation induced by radiotherapy/chemotherapy, or for ameliorating dysbacteriosis induced by antibiotics.

9. A method for preparing a microecological formulation according to any one of claims 1 to 5, comprising the steps of:

(1) freeze-drying powder preparation: the inulin, the blueberry powder and the apple powder are fresh fruits and are respectively smashed and juiced, filtered and then respectively freeze-dried to prepare dry powder; weighing FOS, GOS, inulin, blueberry powder and apple powder according to a proportion, and uniformly mixing to obtain a micro powder mixture;

(2) preparing embedded bacterium powder, namely performing fermentation culture on each strain respectively, then performing centrifugal concentration, fixing the strain by adopting protein polypeptide and calcium alginate gel for embedding, then performing freeze drying and crushing to obtain the embedded bacterium powder, and uniformly mixing various embedded bacterium powders according to a proportion to obtain an embedded bacterium powder mixture;

(3) and (3) weighing and uniformly mixing the mixture obtained in the step (1) and the mixture obtained in the step (2) according to the mass ratio of the mixture to the mixture.

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