CN116615213A

CN116615213A - Composition for prevention, treatment or risk control of colorectal tumours

Info

Publication number: CN116615213A
Application number: CN202280007454.4A
Authority: CN
Inventors: 黄秀娟; 陈家亮; 梁巧仪
Original assignee: Chinese University of Hong Kong CUHK
Current assignee: Chinese University of Hong Kong CUHK
Priority date: 2021-09-10
Filing date: 2022-09-08
Publication date: 2023-08-18
Also published as: WO2023036214A1; CN115786156A; TW202313078A

Abstract

Probiotic compositions comprising bifidobacterium adolescentis (Bifidobacterium adolescentis), bifidobacterium bifidum (Bifidobacterium bifidum) and bifidobacterium longum (Bifidobacterium longum) are provided. Prebiotic compositions comprising inulin, galacto-oligosaccharides and resistant starch/polydextrose are also provided. Also provided are dietary compositions comprising the above probiotic compositions and prebiotic compositions. Further provided is the use of the above composition, in particular in the prevention, alleviation, adjuvant treatment, risk control or reduction of colorectal tumours.

Description

Composition for prevention, treatment or risk control of colorectal tumours

Citation of related application

The present application claims priority from chinese patent application No. 202111059596.9 filed on 10, 9, 2021, which is incorporated by reference in its entirety for all purposes.

Technical Field

The present application relates generally to the fields of pharmaceuticals, foods, and health products. In particular, the present application provides compositions and uses thereof for preventing, alleviating and/or aiding in the treatment of colorectal tumors (e.g. colorectal cancer or colorectal adenoma), or controlling or reducing the risk of colorectal tumors (e.g. colorectal cancer or colorectal adenoma).

Background

Colorectal tumors are common tumor types in the gastrointestinal tract, and malignant tumors are colorectal cancers @Colo Rectal Can, herein referred to as CRC), mostly from colorectal adenoma. The early symptoms of colorectal cancer are not obvious, the symptoms such as bowel movement habit change, hematochezia, diarrhea, constipation, local abdominal pain and the like are shown along with the increase of tumors, the symptoms such as anemia, weight loss and the like are shown in the late stage, the incidence rate is higher in malignant tumors of the digestive system than gastric cancer, esophageal cancer and primary liver cancer, and the death rate is inferior to lung cancer and liver cancer.

Intestinal microecological imbalance has been found to be associated with the occurrence of colorectal tumours or colorectal cancers. For example, some of the risk bacteria, such as fusobacterium nucleatum (Fusobacterium nucleatum), have been shown to play an important role in the development of CRC, while some probiotics maintain intestinal microecological balance through different mechanisms, beneficial to intestinal microecology. For example, some probiotics can directly affect colonisation of intestinal microorganisms by producing inhibitory compounds (e.g. bacteriocins, short Chain Fatty Acids (SCFA), etc.) and matrices (e.g. secreted extracellular polysaccharides, vitamins, etc.) that nourish other microorganisms. Some probiotics may also indirectly regulate intestinal micro-ecology by affecting the host immune system and the integrity of the intestinal barrier. For CRC, the antitumor effect of probiotics has been reported. In vitro studies have shown that co-culture with certain probiotic strains can inhibit proliferation and induce apoptosis of colon cancer cells. In vivo studies have shown that certain probiotics are effective in reducing the incidence of cancer or inhibiting tumor growth in animal models treated with carcinogens. Clinically, probiotics have been used as an adjunct treatment during anti-cancer chemotherapy, reducing the risk of postoperative complications. In addition, a prospective study conducted in italy over ten years ago showed that individuals who had increased yogurt intake for a long period of 12 years had a lower incidence of CRC, which may be that some probiotics in yogurt were exerting a prophylactic effect on CRC. However, it is not yet disclosed how probiotics act as a prophylactic for CRC by modulating the intestinal microbiota, nor is it clear whether probiotics inhibit the development of CRC by ameliorating a CRC-related microbiota imbalance.

Summary of The Invention

In a first aspect, the present application provides a probiotic (probiotics) composition comprising bifidobacterium adolescentis (Bifidobacterium adolescentis), bifidobacterium bifidum (Bifidobacterium bifidum) and bifidobacterium longum (Bifidobacterium longum).

In some embodiments, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in colony forming units is (1-2): 1 (0.5-1.5). In some embodiments, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in colony forming units is (1-2): 1 (0.5-1).

In some embodiments, the probiotic composition further comprises streptococcus thermophilus (Streptococcus thermophilus). In some embodiments, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, and streptococcus thermophilus in colony forming units is (1-2): 1 (0.5-1.5): 0-2. In some embodiments, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, and streptococcus thermophilus in colony forming units is (1-2): 1 (0.5-1): 0.5-1.

In some embodiments, the probiotic composition is in unit dosage form and the amount of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus is independently 10 in colony forming units ⁴ To 10 ¹² Magnitude of CFU. In some embodiments, the total amount of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus is 10 ⁶ To 10 ¹² Magnitude of CFU.

In some embodiments, the amount of bifidobacterium adolescentis is 3.08X10 ⁵ -4.00×10 ¹¹ CFU. In some embodiments, the amount of bifidobacterium bifidum is 1.54×10 ⁵ -4.00×10 ¹¹ CFU. In some embodiments, the amount of bifidobacterium longum is 2.00 x 10 ⁵ -3.01×10 ¹¹ CFU or 2.00×10 ⁵ -2.31×10 ¹¹ CFU. In some embodiments, the Streptococcus thermophilus is in an amount of 0 to 3.08X10 ¹¹ CFU。

In some embodiments, the probiotic composition is free of other probiotics than those described in the various embodiments of the application. In some embodiments, the probiotic composition is free of probiotics other than bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus. In some embodiments, the probiotic composition is free of bifidobacteria other than bifidobacteria adolescentis, bifidobacteria bifidus, and bifidobacteria longum. In some embodiments, each of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum or streptococcus thermophilus may have two or more strains (strains) within the species (species) thereof contained within the probiotic composition.

In a second aspect, the present application provides a prebiotic (pre-conjugates) composition comprising (a) inulin, (b) galactooligosaccharides and (c) resistant starch and/or polydextrose.

In some embodiments, the prebiotic composition comprises resistant starch and the ratio of the amounts of inulin, galactooligosaccharide and resistant starch by weight is (2-5): 1 (2-4).

In some embodiments, the prebiotic composition comprises polydextrose, and the ratio of the amounts of inulin, galactooligosaccharide and polydextrose by weight is (2.5-5): 1 (2-3).

In some embodiments, the prebiotic composition is in unit dosage form and the total amount of (a) inulin, (b) galactooligosaccharides, and (c) resistant starch and/or polydextrose is from 0.1 to 12g by weight. In some embodiments, the prebiotic composition is in unit dosage form and the total amount of (a) inulin, (b) galactooligosaccharides, and (c) resistant starch and/or polydextrose is from 0.1 to 4g by weight.

In some embodiments, the amount of inulin is 0.04g to 3g or 0.04g to 2g. In some embodiments, the amount of galacto-oligosaccharides is 0.01g to 0.8g. In some embodiments, the amount of resistant starch is 0.04g to 1.6g. In some embodiments, the amount of polydextrose is from 0.01 to 1.6g.

In some embodiments, the prebiotic composition does not comprise a prebiotic component other than (a) inulin, (b) galacto-oligosaccharides, and (c) resistant starch and/or polydextrose.

In a third aspect, the present application provides a dietary composition (also referred to in some cases as a "synbiotics" composition) comprising a probiotic composition of the first aspect and a prebiotic composition of the second aspect.

In some embodiments, the probiotic composition and the prebiotic composition are formulated as a single composition.

In some embodiments, the probiotic composition and the prebiotic composition are physically separate.

In some embodiments, the dietary composition comprises bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum as probiotics and (a) inulin, (b) galactooligosaccharide and (c) resistant starch and/or polydextrose as prebiotics, wherein the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in Colony Forming Units (CFU) is (1-2): 1 (0.5-1.5), preferably (1-2): 1 (0.5-1),and the total of the three bacteria is 1×10 ¹⁰ -2×10 ¹¹ CFU and when the prebiotic composition comprises resistant starch, the ratio of inulin, galactooligosaccharide and resistant starch is (2-5) 1 (2-4) by weight, when the prebiotic composition comprises polydextrose, the ratio of inulin, galactooligosaccharide and polydextrose amounts is (2.5-5) 1 (2-3) by weight, and the total amount of (a) inulin, (b) galactooligosaccharide and (c) resistant starch and/or polydextrose is 1.2-2g.

In some embodiments of the first to third aspects, the probiotic composition or prebiotic composition or dietary composition is formulated for oral administration. In some embodiments, oral administration includes oral, mixed with oral-like products, tube feeding, and the like.

In some embodiments of the first to third aspects, the probiotic composition or prebiotic composition or dietary composition is a food supplement, food additive or food.

In some embodiments of the first to third aspects, the probiotic composition or prebiotic composition or dietary composition is formulated as a powder, granule, tablet or capsule.

In some embodiments of the first to third aspects, the probiotic composition or prebiotic composition or dietary composition is administered to an individual for preventing, alleviating and/or aiding in the treatment of colorectal neoplasms (e.g. colorectal cancer or colorectal adenoma), controlling or reducing the risk of colorectal neoplasms (e.g. colorectal cancer or colorectal adenoma), or inhibiting the growth of clostridium nucleatum (Fusobacterium nucleatum) or clostridium harveyi (Clostridium hathewayi). In some embodiments of the above use, the level of clostridium nucleatum, clostridium harveyi (Clostridium hathewayi) or lachnoclostrichum sp.m3 in the fecal sample of the individual is higher than the corresponding level in the fecal sample of a healthy individual. In some embodiments of the above uses, the combined score (e.g., 4Bac score) of clostridium nucleatum, clostridium clarkii (Bacteroides clarus), clostridium hardtii, and lachnoclostricium sp.m3 in the fecal sample of the individual is higher than the corresponding score in the fecal sample of a healthy individual.

In a fourth aspect, the present application provides the use of a probiotic composition of the first aspect or a prebiotic composition of the second aspect or a dietary composition of the third aspect for the manufacture of a dietary product or medicament for the prevention, alleviation and/or adjuvant treatment of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), for controlling or reducing the risk of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), or for inhibiting the growth of clostridium nucleatum (Fusobacterium nucleatum) or clostridium harveyi (Clostridium hathewayi).

In a fifth aspect, the application provides a method for preventing, alleviating and/or aiding in the treatment of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), controlling or reducing the risk of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), or inhibiting the growth of clostridium nucleatum (Fusobacterium nucleatum) or clostridium hardtii (Clostridium hathewayi), comprising administering to a subject in need thereof the probiotic composition of the first aspect, or the prebiotic composition of the second aspect, or the dietary composition of the third aspect. In some embodiments, the method comprises determining the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium harveyi (Clostridium hathewayi), or lachnoclostrichum sp.m3 in the fecal sample of the individual, and administering the probiotic composition or prebiotic composition or dietary composition to the individual if the determined level is higher than the corresponding level in the fecal sample of a healthy individual. In some embodiments, the method comprises determining the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium clarkii (Bacteroides clarus), clostridium harveyi (Clostridium hathewayi), and lachnoclostrichum sp.m3 in the individual's fecal sample and deriving a composite score (e.g., a 4Bac score), and administering the probiotic composition or prebiotic composition or dietary composition to the individual if the measured score is higher than the corresponding score in the fecal sample of a healthy individual.

Brief description of the drawings

FIGS. 1A-1C show the correlation of bacterial species with colorectal tumor progression, wherein FIG. 1A shows the Spearman correlation of detected probiotic species and CRC-related species with colorectal tumor progression; FIG. 1B shows specific correlation parameters of Bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum in normal individuals (N), adenoma patients (A) and CRC patients; FIG. 1C compares the relative abundance of four target species in stool samples from normal individuals (N), adenoma patients (A), and CRC patients.

Fig. 2A to 2D show the correlation of probiotic species with CRC-related bacterial gene markers. FIG. 2A shows 20 bacterial gene markers previously determined by the inventors, which have differential levels between healthy individuals and CRC patients; FIG. 2B shows that Bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum are inversely or positively correlated with CRC-related gene markers; figures 2C and 2D show fold changes in CRC gene markers due to the presence of 3 bifidobacteria.

Figures 3A-3E show that exemplary tests of compositions comprising bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum intervene in reducing the microbial risk of CRC. FIG. 3A shows a clinical intervention study flow chart; figure 3B shows that baseline levels of CRC-related biomarkers are similar in the intervention group and the non-intervention group. Figure 3C shows that there was no significant change in 3 bifidobacteria and CRC-related microbial markers in the non-intervening group; figures 3D and 3E show significant changes in 3 bifidobacteria and CRC-related biomarkers in the intervention group.

Figure 4 shows that co-culture in vitro with bifidobacteria alone or in combination with bifidobacteria significantly inhibited the growth of clostridium nucleatum (Fn) compared to control treatment with clostridium aldenensis (e.aldensis, ea). Treatment dose for all groups: fn=1:1.

Figures 5A to 5B show that the risk of CRC microorganisms is significantly reduced in individuals taking synbiotic formulas comprising bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus, inulin, galactooligosaccharides and polydextrose for one month, wherein figure 5A shows that the 4Bac composite score is significantly reduced after one month compared to baseline; fig. 5B shows that individual CRC marker levels varied over a month with significantly reduced Fn and Ch levels compared to baseline. The p-value was analyzed by wilcoxon symbol rank test.

Figure 6 shows that probiotic compositions comprising bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum and streptococcus thermophilus significantly inhibited the growth of fusobacterium nucleatum (Fn). * : p <0.05.

Detailed Description

The inventors of the present application have conducted intensive studies on the effect of intestinal flora on the occurrence and development of colorectal tumors, in particular colorectal carcinoma and colorectal adenoma. In one previous study by the inventors, 20 bacterial gene markers associated with CRC were determined, and the levels of these 20 bacterial gene markers were significantly different between healthy individuals and CRC patients. Another previous study by the inventors found that a novel pathogenic species Lachnoclotridium sp.m3 (abbreviated herein as "m 3") associated with the development of colorectal adenoma and colorectal carcinoma and revealed that a microorganism marker panel consisting of Clostridium nucleatum (Fusobacterium nucleatum, abbreviated herein as "Fn"), clostridium harveyi (Clostridium hathewayi, abbreviated herein as "Ch"), m3 and Clark-like bacillus (Bacteroides clarus, abbreviated herein as "Bc") could well assess the risk of CRC and colorectal adenoma. The decrease in the combined score (4 Bac) of Fn, ch, m3 and/or the four microbial markers (Fn, ch, m3, bc) reflects a decrease in the microbial risk (environmental risk factor in the gut) of CRC/colorectal adenoma. Since most colorectal cancers are transformed by adenomas, adenomas are also known as precancerous lesions. Thus, decreasing the levels of Fn, ch, m3 and/or the combined score of the four microbial markers (4 Bac) may decrease environmental risk factors in the gut and prevent colorectal cancer or decrease the risk of the application.

Using the previously determined 20 bacterial gene markers associated with CRC, the inventors performed a systematic correlation analysis of the metagenomic sequencing data of the study cohort to find probiotic species that are negatively correlated with these identified bacterial gene markers at risk for CRC, and designed probiotic, prebiotic, and synbiotics compositions using these probiotics, which can be used to prevent, alleviate, and/or assist in the treatment of colorectal tumors, controlling or reducing the risk of colorectal tumors. The synbiotic compositions of the application comprise identified probiotic species that combat CRC and prebiotics that nourish the growth of these probiotics. The inventors have further determined the effect of the synbiotic composition on the reduction of microbial risk of colorectal neoplasms (e.g., colorectal cancer or colorectal adenoma) by studying the effect of the synbiotic composition on Fn, ch, m3 levels and 4Bac composite scores in individuals via clinical intervention trials.

In general, the inventors identified a group of probiotics, including bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum, whose levels in the gut were not only significantly reduced with the development of the tumor, but also positively correlated with the increased bacteria in the CRC patient, by correlation analysis with tumor development and CRC-related bacterial markers. The inventors of the present application combine the identified probiotics and prebiotics beneficial to their growth into a synbiotic composition and further demonstrate, through clinical intervention trials, that the synbiotic composition of the present application has significant utility in reducing the risk of microorganisms associated with the development of CRC, conforming to the CRC risk assessment test previously devised by the inventors. The application demonstrates for the first time that probiotic compositions comprising bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum play an important role in combating colorectal tumours. Antitumor studies of bifidobacteria including these three bifidobacteria and other probiotics have also been reported in the art, but the disclosure of the relevance of colorectal neoplasms (e.g. colorectal cancer or colorectal adenoma) to bifidobacteria adolescentis, bifidobacteria bifidus and bifidobacterium longum, especially in the prevention and risk control of illness, was first suggested in the art.

Hereinafter, the present application will be further described according to some specific embodiments. However, the specific embodiments are set forth for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art will recognize that a particular feature of one embodiment described below may be used in any other embodiment without departing from the spirit of the application.

Unless otherwise defined, terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. All patent documents, academic papers, and other publications cited herein are incorporated by reference in their entirety.

It is to be understood that the specific values given herein are not only to be understood as individual values, but also to be considered as providing endpoints of a certain range, and that other ranges may be provided in combination with each other. For example, when a composition is disclosed as having a content of 1, 2 or 3g, it is also disclosed that the content of the component may be 1 to 2g, 1 to 3g or 2 to 3g.

In this context, when the context clearly states that a certain component is present, and when the lower limit of its content range is described as "0", it means more than 0. For example, when the present application describes that the amount of Streptococcus thermophilus is 0-3.08X10 ¹¹ CFU indicates that the amount of streptococcus thermophilus is greater than 0.

As used herein, a "probiotic composition" refers to a composition having probiotics as active ingredient, without excluding the presence of auxiliary ingredients required for the culture, isolation and purification of the probiotics and/or auxiliary ingredients for formulating the composition according to the desired purpose.

In some embodiments, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in colony forming units is (1-2): 1 (0.5-1.5). In some embodiments, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in colony forming units is (1-2): 1 (0.5-1). For example, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in terms of colony forming units may be (1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2) 1 (0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4 or 1.5).

Colony Forming Units (CFU) are a form of characterizing the amount of microorganisms common in the art. Unless otherwise specified, the amounts of microorganisms described herein are calculated in colony forming units.

In some embodiments, the probiotic composition further comprises streptococcus thermophilus (Streptococcus thermophilus). Streptococcus thermophilus has been shown to have an inhibitory effect on intestinal cancer in vitro and in vivo studies, which inhibits the growth of intestinal cancer cells by secretion of β -galactosidase, relying on galactose produced by β -galactosidase to initiate oxidative phosphorylation in intestinal cells and inhibit the Hippo signaling pathway, thereby mediating cancer-inhibiting effects. The inventors of the present application expected and initially demonstrated that the addition of Streptococcus thermophilus further provided the efficacy of the probiotic composition of the present application.

In some embodiments, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, and streptococcus thermophilus in colony forming units is (1-2): 1 (0.5-1.5): 0-2. In some embodiments, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, and streptococcus thermophilus in colony forming units is (1-2): 1 (0.5-1): 0.5-1. For example, the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum and streptococcus thermophilus may be (1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2) 1 (0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4 or 1.5) in terms of colony forming units (0.0001, 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2).

Those skilled in the art will appreciate that in the field of microbiology, a variety of strains are included within a single species, then for the purposes of the present application, when the probiotic composition comprises bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum or streptococcus thermophilus, a single strain may be included in each species within the probiotic composition, or each species may independently include two or more strains within the probiotic composition.

As described in detail in the examples below, the inventors of the present application studied the average relative abundance of bifidobacteria adolescentis, bifidobacteria bifidus and bifidobacterium longum in harbour people, their abundance changes in healthy people, colorectal adenoma patients and colorectal cancer group of people, their relevance to 20 CRC-related bacterial gene markers, and their ability to regulate the composite score (4 Bac) with Fn, ch, m3 and/or four microbial markers (Fn, ch, m3, bc) in clinical intervention trials. Through scientific design based on the above-mentioned research results, the inventors set the ratio of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in the probiotic composition of the present application to (1-2): 1 (0.5-1.5). In some embodiments, the probiotic composition further comprises Streptococcus thermophilus, bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, and Streptococcus thermophilus in a ratio of (1-2) 1 (0.5-1.5) to (0-2). The classification number (NCBI: txid) of 4 probiotics in the national center for Biotechnology information is set forth below.

TABLE 1

Probiotic species	NCBI:txid
Bifidobacterium adolescentis (Ba)	1680
Bifidobacterium bifidum (Bb)	1681
Bifidobacterium longum (Bl)	216816
Streptococcus thermophilus (St)	1308

In some embodiments, the probiotic composition is in unit dosage form and the amount of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus is independently 10 in colony forming units ⁴ To 10 ¹² Magnitude of CFU. In some embodiments, the total amount of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus is 10 ⁶ To 10 ¹² Magnitude of CFU. It will be appreciated that the probiotic composition of the present application does not necessarily all comprise bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus and that, as such, "total amount" herein refers to the total amount of these four probiotics present in the probiotic composition.

As used herein, "unit dosage form" refers to a composition of individual or separately packaged single administration doses, which may typically be presented in individual tablets, capsules or powder/granule pouches and the like. In some embodiments, for ease of administration, unit dosage forms are prepared containing daily doses of the composition.

In some embodiments, the amount of bifidobacterium adolescentis is 3.08X10 ⁵ -4.00×10 ¹¹ CFU. In some embodiments, the amount of bifidobacterium bifidum is 1.54×10 ⁵ -4.00×10 ¹¹ CFU. In some embodiments, the amount of bifidobacterium longum is 2.00 x 10 ⁵ -2.31×10 ¹¹ CFU. In some embodiments, the amount of bifidobacterium longum is 2.00 x 10 ⁵ -3.01×10 ¹¹ CFU. In some embodiments, the Streptococcus thermophilus is in an amount of 0 to 3.08X10 ¹¹ CFU。

In some embodiments, the probiotic composition is free of other probiotics than those described in the various embodiments of the application.

In the technical context of such embodiments, "free" should be understood as "substantially free" and does not exclude the presence of trace or trace amounts of other probiotics due to factors such as culture, isolation and purification of the strain. In some embodiments, the amount of other probiotics is no more than 5%, preferably no more than 1% of the total probiotic of the composition.

In some embodiments, the probiotic composition is free of probiotics other than bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus. In some embodiments, the probiotic composition is free of bifidobacteria other than bifidobacteria adolescentis, bifidobacteria bifidus, and bifidobacteria longum.

In a second aspect, the present application provides a prebiotic (pre-conjugates) composition comprising (a) inulin, (b) galactooligosaccharides and (c) resistant starch and/or polydextrose. The composition comprises inulin and galactooligosaccharides, and further comprises one or both of resistant starch and polydextrose.

As used herein, a "prebiotic composition" refers to a composition having a prebiotic as the active ingredient, without excluding the presence of auxiliary ingredients introduced by synthesis, isolation, purification, etc. of the prebiotic and/or auxiliary ingredients for formulating the composition according to the intended purpose.

The prebiotics and probiotics are generally complementary, and it is therefore advantageous to add the prebiotics specifically based on the identification and selection of the probiotics of the first aspect. The prebiotics selected by the inventors for the purpose of identifying the species of probiotics identified in the above studies, and for which it has been shown that they have a role in the prevention of intestinal diseases, in particular tumours, include inulin, galactooligosaccharides and resistant starches.

Inulin consists of 2-60 fructose units and one terminal glucose molecule. Inulin is a common name covering all beta- (2, 1) fructopolysaccharides of different degrees of polymerization. Sources of inulin include chicory root, dahlia, artichoke. Digestive enzymes in the small intestine cannot digest inulin, and after the inulin reaches the large intestine intact, it is fermented into short chain fatty acids and gases by the bacteria of the large intestine. Studies have shown that inulin has a proliferation promoting effect on bifidobacteria.

Galactooligosaccharides and resistant starches can promote the growth of a variety of bifidobacteria. The molecular structure of galactooligosaccharides is generally that 1-7 galactosyl groups are connected on galactose or glucose molecules, and the galactooligosaccharides are functional oligosaccharides with natural properties. The galacto-oligosaccharide has good palatability, water solubility and stability, and can proliferate probiotics, especially bifidobacteria in human intestinal canal after entering human body, and inhibit the growth of putrefying bacteria. Probiotics in the intestinal tract can produce a large amount of extracellular polysaccharide while utilizing galacto-oligosaccharides to proliferate. The extracellular polysaccharide not only has anti-tumor activity and immune activity, but also can promote the long-term colonization of probiotics in intestinal tracts. Resistant starch is a dietary fiber that is used for digestive health benefits. Resistant starch (e.g., corn dietary fiber) absorbs some of the water and promotes increased intestinal peristalsis, and accelerates fecal discharge, thereby reducing rectal stress and preventing and reducing intestinal disease. At the same time, resistant starch can be fermented by microorganisms such as bifidobacteria to produce short chain fatty acids.

Polydextrose is a soluble fiber having a molecular weight of about 80,000Da. Polydextrose has been shown to have an effect on promoting immune function by producing short chain fatty acids and a protective effect on carcinogenesis in several ways, for example increasing the number of bifidobacteria that can produce protective fermentation metabolites for cancer.

Based on the proportions of the probiotics of the first aspect determined in the study and the nature of the prebiotics described above, the inventors have further scientifically designed the proportions of the prebiotics.

In some embodiments, the prebiotic composition comprises resistant starch and the ratio of the amounts of inulin, galactooligosaccharide and resistant starch by weight is (2-5): 1 (2-4). For example, the ratio of the amounts of inulin, galacto-oligosaccharides and resistant starch may be (2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0) 1 (2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0) by weight.

In some embodiments, the prebiotic composition comprises polydextrose, and the ratio of the amounts of inulin, galactooligosaccharide and polydextrose by weight is (2.5-5): 1 (2-3). For example, the ratio of the amounts of inulin, galacto-oligosaccharides and polydextrose may be (2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0) 1 (2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0).

In some embodiments, the prebiotic composition is in unit dosage form and the total amount of (a) inulin, (b) galacto-oligosaccharides and (c) resistant starch and/or polydextrose is 0.1 to 12g, e.g. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12g by weight. In some embodiments, the prebiotic composition is in unit dosage form and the total amount of (a) inulin, (b) galacto-oligosaccharides and (c) resistant starch and/or polydextrose is 0.1-4g by weight, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4g.

In some embodiments, the amount of inulin is between 0.04g and 3g. In some embodiments, the amount of inulin is between 0.04g and 2g. In some embodiments, the amount of galacto-oligosaccharides is 0.01g to 0.8g. In some embodiments, the amount of resistant starch is 0.04g to 1.6g. In some embodiments, the amount of polydextrose is from 0.01 to 1.6g.

In the technical context of such embodiments, "free" should be understood as "substantially free" of other prebiotics in trace or trace amounts due to factors such as synthesis, extraction, isolation and purification of the prebiotics. In some embodiments, the amount of other prebiotics is no more than 5%, preferably no more than 1% of the total amount of all prebiotics in the composition.

In a third aspect, the present application provides a dietary composition (sometimes also referred to as a "synbiotics" composition) comprising a probiotic composition of the first aspect and a prebiotic composition of the second aspect.

In some embodiments, the probiotic composition and the prebiotic composition are formulated as a single composition, i.e., the probiotic composition and the prebiotic composition are present in the form of a mixture or a compounded formulation.

In some embodiments, the probiotic composition and the prebiotic composition are physically separate. By "physically separated" is meant that the probiotic composition and the prebiotic composition are each present in separate spaces (e.g. separate packaging treatments, containers, capsules), the substances themselves not touching each other. Furthermore, while the probiotic composition and the prebiotic composition may be physically separate in the dietary composition product, they may be mixed together for co-administration prior to administration or may be administered sequentially.

It will also be appreciated by those skilled in the art that the above-described dietary composition may be, but is not limited to, a probiotic composition of the first aspect and a probiotic composition of the second aspect separately formulated and then mixed or compounded. As long as one dietary composition covers all features of one embodiment of the probiotic composition of the first aspect and all features of one embodiment of the prebiotic composition of the second aspect, i.e. belongs to the dietary composition of the third aspect of the application.

In some embodiments, the dietary composition comprises bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum as probiotics and inulin, galactooligosaccharide and resistant starch as prebiotics, wherein the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in Colony Forming Units (CFU) is (1-2): 1 (0.5-1.5), and the total of three bacteria is about 2 x 10 ¹¹ CFU and the ratio of inulin, galacto-oligosaccharides and resistant starch by weight is (2-5): 1 (2-4), and the total amount of inulin, galacto-oligosaccharides and resistant starch is 1.2-2g.

As a non-limiting example, the dietary composition of the present application may have the following formulation (daily amounts, which may be provided in unit dosage form):

TABLE 2

The main mode of application of the probiotic composition or prebiotic composition or dietary composition of the application is to the gastrointestinal tract of an individual. Direct oral administration is a convenient way, but for some specific individuals (e.g. bedridden patients) it may also be assisted by gavage or the like.

The probiotic composition or prebiotic composition or dietary composition of the application may be in a wide variety of product forms, for example, may be prepared as a separate dietary supplement (e.g. capsule, tablet, powder, granule) for administration with or without meal; the preparation method can also be used for preparing various solid/semisolid foods, blended powder/granule foods, beverages and other additive products which are added or blended before being ingested by an individual; can also be used as direct constituent of various solid/semisolid foods, powder/granule foods, and beverages. Examples of food products include, but are not limited to, beverage mixes, dairy products, yogurt, ice cream, dry cereal mixes, porridge, edible oils, and the like.

In some embodiments of the first to third aspects, the probiotic composition or prebiotic composition or dietary composition is administered to an individual for the prevention, alleviation and/or adjuvant treatment of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), or for controlling or reducing the risk of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), or for inhibiting the growth of clostridium nucleatum (Fusobacterium nucleatum) or clostridium harveyi (Clostridium hathewayi). For controlling or reducing the risk of colorectal neoplasms (e.g., colorectal cancer or colorectal adenoma), more particularly may be manifested in reducing the risk of microorganisms of colorectal neoplasms (e.g., colorectal cancer or colorectal adenoma), the risk microorganisms include clostridium nucleatum (Fusobacterium nucleatum), clostridium hardtii (Clostridium hathewayi), or lachnoclostricium sp.m3, as discussed in more detail in the examples below. Thus in some embodiments, the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium haljensis (Clostridium hathewayi) or lachnoclotridium sp.m3 in the fecal sample of the individual is higher than the corresponding level in the fecal sample of a healthy individual, thus having an increased risk of developing colorectal neoplasms (e.g., colorectal cancer or colorectal adenoma). In some embodiments, the combined score (e.g., 4Bac score, e.g., the calculation method shown in the examples of the application) of clostridium nucleatum, clostridium clarkii (Bacteroides clarus), clostridium harveyi, and lachnocladium sp.m3 in the fecal sample of the individual is higher than the corresponding score in the fecal sample of a healthy individual, and thus has an increased risk of developing colorectal neoplasms (e.g., colorectal cancer or colorectal adenoma).

In a fourth aspect, the present application provides the use of a probiotic composition of the first aspect or a prebiotic composition of the second aspect or a dietary composition of the third aspect for the manufacture of a dietary product or medicament for the prevention, alleviation and/or adjuvant treatment of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), or for controlling or reducing the risk of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), or for inhibiting the growth of clostridium nucleatum (Fusobacterium nucleatum) or clostridium harveyi (Clostridium hathewayi).

In the absence of conflict, the preparation of the probiotic composition or prebiotic composition or dietary composition of the present application may be referred to in the art as conventional processing of probiotic or prebiotic-like products. For example, the various probiotics or prebiotic ingredients may be mixed into the product sequentially or simultaneously or as a lyophilized premix by conventional processing techniques.

In a fifth aspect, the application provides a method for preventing, alleviating and/or assisting in the treatment of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), or controlling or reducing the risk of colorectal tumours (e.g. colorectal cancer or colorectal adenoma), or inhibiting the growth of clostridium nucleatum (Fusobacterium nucleatum) or clostridium hardtii (Clostridium hathewayi), comprising administering to a subject a probiotic composition of the first aspect, or a probiotic composition of the second aspect, or a dietary composition of the third aspect. As described above and shown in the examples below, when the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium hardtii (Clostridium hathewayi) or lachnoclostidium sp.m3 in a fecal sample of an individual is higher than the corresponding level in a fecal sample of a healthy individual, the individual has an elevated risk of developing a colorectal tumor (e.g., colorectal cancer or colorectal adenoma), and thus in some embodiments the method comprises determining (e.g., by qPCR method) the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium hardtii (Clostridium hathewayi) or lachnoclostidium sp.m3 in the fecal sample of the individual, and administering the probiotic composition or prebiotic composition or dietary composition to the individual if the measured level is higher than the corresponding level in the fecal sample of a healthy individual. Furthermore, the dosage, frequency and/or period of administration may also be adjusted based on the magnitude of the increase in the risk microorganisms. The combined score (e.g., a 4Bac score, such as the calculation method shown in the examples of the present application) of clostridium nucleatum, clostridium clavatum (Bacteroides clarus), clostridium harbouring and lachnoclotridium sp.m3 in the fecal sample of the individual is higher than the corresponding score in the fecal sample of a healthy individual, and the individual has an increased risk of developing colorectal neoplasms (e.g., colorectal cancer or colorectal adenoma), so in some embodiments the method comprises determining the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium clarkii (Bacteroides clarus), clostridium harbouring (Clostridium hathewayi) and lachnoclotridium sp.m3 in the fecal sample of the individual and deriving the combined score (e.g., a 4Bac score), and administering the probiotic composition or prebiotic composition or dietary composition to the individual if the determined score is higher than the corresponding score in the fecal sample of a healthy individual.

The following examples are given for the purpose of illustration only and are not intended to limit the scope of the application.

Example 1

Method

Metagenomic dataset

The inventors analyzed metagenomic sequencing data of 589 harbour individuals (184 CRC patients, 185 colorectal adenoma patients, and 220 normal control individuals) collected in previous studies, including a discovery cohort of 74 CRC patients and 54 normal control individuals, for identifying 20 bacterial genes associated with colorectal cancer. The present study has been approved by The Internet of clinical research ethical Committee of The New world Dong Hospital, university of hong Kong (The Joint CUHK-NTEC CREC, CREC Ref.No. 2017.369). All individuals participating in the study signed written informed consent. The levels of 20 bacterial gene markers were analyzed using previous studies. The relative abundance of the species was generated as MetaPhlAn 3.

Clinical trial

The inventors performed an interventional clinical trial in the university of hong Kong, weiership, king Hospital (clinical trial registration number: NCT04581018; https:// clinicaltrias gov). The Wilski king hospital is a third-class transfer center. The 48 subjects were randomly assigned to the intervention and non-intervention groups. The present study has been approved by The Internet of clinical research ethics Committee of The New world Hospital, university of hong Kong (The Joint CUHK-NTEC CREC, CREC Ref. No. 2020.407). All individuals participating in the study signed written informed consent. In the intervention group, 40 subjects took the synbiotic composition (formula shown in table 2 of the specification) daily for 4 weeks. At baseline (week 0), week 2, week 4, week 5, week 8 and week 12, the inventors collected stool samples from all subjects, including the intervention group and the non-intervention group (n=8). Faeces were stored in Norgen faeces preservative (Norgen Biotek Corp, ontario, canada) and stored in-80℃refrigerator over 24 hours until the next analysis was performed.

Fecal DNA extraction and use of double-stranded quantitative PCR (qPCR) for microbial markers Quantization

UsingRSC PureFood GMO and Authentication Kit (Promega) after extracting fecal DNA, the abundance of four colorectal cancer (CRC) -related microbial markers (Fn, m3, bc and Ch) and three bifidobacteria (bifidobacterium adolescentis (Ba), bifidobacterium bifidum (Bb), bifidobacterium longum (Bl)) in the feces was quantified by quantitative PCR (qPCR). Table 3 shows the primer sequences and probe sequences for the respective markers and 16s rDNA internal controls. Each probe carries the 5 'reporter dye FAM (6-carboxyfluorescein) or VIC (4, 7,2' -trichloro-7 '-phenyl-6-carboxyfluorescein) and the 3' quencher dye TAMRA (6-carboxytetramethyl rhodamine). Primers and hydrolysis probes were synthesized by Invitrogen (Carlsbad, calif.). qPCR amplification was performed on an ABI Quantum studio sequence detection system with thermal cycler parameters of 95℃for 10 minutes and (95℃for 15 seconds, 60℃for 1 minute) X45 cycles of 10. Each experiment contained positive and negative controls for the markers (H ₂ O-templates). Measurements were performed in triplicate for each sample. By using the Δcq method, the relative abundance of each marker was calculated as compared to the internal control (Power (2, - (Cq target-Cq control))andshown as a logarithmic value of "×10e6+1".

TABLE 3 nucleotide sequences of primers and probes used in this study

* C:16S internal control for calculating relative quantification

Scoring algorithm

Of the 20 microorganism gene markers associated with CRC, 8 microorganism gene markers were increased and 12 were decreased in CRC patients compared to control individuals. The "8Up" score was calculated as Log10[ Sum (8 increased markers) +1e-20]. The "12Down" score was calculated as Log10[ Sum (12 reduced markers) +1e-20]. The composite scores (4 Bac) for the four microbial markers were calculated using the logistic regression model (4 Bac score=0.23162×fn+0.13451×m3-0.10075×bc+0.32841×ch-2.73836) determined by previous studies.

Statistical analysis

All values are expressed as mean ± standard deviation or median (quarter-bit distance (IQR)). The difference in bacterial abundance between the two groups was determined by the Mann-Whitney U test or paired t test. The correlation between bacterial species level/gene marker level/score was analyzed by pearson correlation. The correlation between bacterial levels and colorectal tumor stage was analyzed by spearman correlation. Marker changes at different time points were analyzed by one-way anova multiplex comparison, where appropriate, to derive linear trends. All tests were performed by Graphpad Prism 5.0 (Graphpad Software inc., san Diego, CA) or Statistical Software 19.6 version 19.6 (MedCalc Software Ltd, ostend, belgium; https:// www.medcalc.org; 2020). P is p<0.05 is considered to represent statistical significance.

Results

Correlation analysis determined three bifidobacteria (bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium bifidum) Bifidobacterium longum) in the course of colorectal tumor progression

To determine the presence of probiotic species associated with colorectal tumour development, the inventors analyzed the spearman correlation between all detected levels of probiotic species and the normal control, colorectal adenoma and CRC groups, and the results showed that three bifidobacteria were inversely correlated with disease progression from normal to CRC, including bifidobacterium adolescentis Ba (rho= -0.144; p < 0.001), bifidobacterium longum Bl (rho= -0.092; p < 0.05) and bifidobacterium bifidum Bb (rho= -0.081; p < 0.05) (fig. 1A and 1B). The three bifidobacteria of the CRC group were significantly reduced compared to the normal control group, as were the Ba and Bl of the colorectal adenoma group (all p <0.005; fig. 1C). Unexpectedly, the inventors found that some lactic acid bacteria species significantly increased from the normal control group to the colorectal adenoma group and the CRC group, such as lactobacillus salivarius (rho=0.188; p < 0.0001), compared to the normal control group (p < 0.0001) (fig. 1A and 1C). For reference, the inventors also analyzed four risk bacterial species associated with CRC. As expected, the CRC-risk bacteria, fusobacterium nucleatum (Fusobacterium nucleatum, fn) and clostridium harveyi (Clostridium hathewayi, ch) increased significantly from the normal control group to the colorectal adenoma group and to the CRC group, while both beneficial species entero-ross-bayesian (Roseburia intestinalis, rn) and clarithromycin (Bacteroides clarus, bc) were in a decreasing trend, although Bc was not significant (fig. 1A). Thus, these findings indicate that bifidobacterium adolescentis, bifidobacterium longum and bifidobacterium bifidum are important probiotics against CRC.

Correlation analysis determined three bifidobacteria (bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium bifidum) Bifidobacterium longum) is inversely related to the risk bacterial gene marker of CRC and is related to beneficial bacteria Gene markers are positively correlated

To determine the class of probiotic species that can modulate the markers associated with CRC, the inventors further analyzed the correlation between individual probiotic species levels and 20 CRC-related bacterial gene marker levels determined by previous studies by the inventors. Of the 20 bacterial gene markers, the inventors combined 8 markers Up-regulated in CRC and 12 markers Down-regulated in CRC, respectively, resulting in combined scores of 8Up and 12Down (fig. 2A), and performed correlation analysis with probiotic species levels with these two combined scores. In addition, the inventors have also performed correlation analysis of four individual gene markers with probiotic species levels. These four markers are four qPCR targets in the non-invasive CRC and colorectal adenoma diagnostic test previously established by the inventors, including Fn, ch, bc and lachnocrostrinium sp.m3. Interestingly, these analyses also determined that the same three bifidobacteria (bifidobacterium adolescentis, bifidobacterium longum and bifidobacterium bifidum) were significantly correlated with one or more CRC markers, including bifidobacterium adolescentis and bifidobacterium longum being inversely correlated with Fn (rho values of-0.1 and-0.096, respectively, with p < 0.05), bifidobacterium longum being positively correlated with Bc (rho=0.106, p=0.01), bifidobacterium adolescentis, bifidobacterium longum and bifidobacterium bifidum being inversely correlated with 12Down (rho values of 0.135, 0.134 and 0.126, respectively, with all p < = 0.002) (fig. 2B).

Furthermore, the inventors split the stool samples into two groups for each probiotic species, containing (right hand side of each figure in fig. 2D) and not containing (left hand side of each figure in fig. 2D) the probiotic species, and compared the fold change in CRC gene markers between the two groups. The results show that the presence of each of the three bifidobacteria described above is significantly correlated with the decrease of at least one risk marker (Fn, ch, m3 and 8 Up) and the increase of at least one beneficial marker (Bc and 12 Down) (fig. 2C and 2D). These findings further indicate that bifidobacterium adolescentis, bifidobacterium longum and bifidobacterium bifidum are important probiotic species against CRC.

Comprises Bifidobacterium adolescentis, bifidobacterium longum and Bifidobacterium bifidumSynbiotic composition of bifidobacteria Intervention significantly reduces the microbial risk of CRC

To evaluate the role of the identified probiotics in regulating the risk of microorganisms associated with the development of CRC, the inventors performed a clinical intervention trial. The synbiotic composition used in the intervention test contained bifidobacterium adolescentis, bifidobacterium longum and bifidobacterium bifidum (probiotic formulations see table 2 above), and prebiotics capable of nourishing the growth of these probiotics (prebiotic formulations optimized according to the results of the present experiment are shown in table 2). In the intervention group, 40 individuals took the synbiotic composition daily for 4 weeks. The inventors collected stool samples of all individuals at baseline (week 0), week 2, week 4, week 5, week 8 and week 12, including the intervention group (n=40) and the non-intervention group (n=8) (fig. 3A), and performed qPCR on the samples to quantify three bifidobacteria and 4 CRC risk markers (Fn, m3, ch and Bc) and calculated their composite score of 4Bac. At baseline, the level of CRC risk marker was not significantly different between the intervention group and the non-intervention group (fig. 3B), so the change difference between the two groups thereafter could reflect the effect of the intervention.

In the non-intervention group, the levels of the three bifidobacteria (Ba, bb, bl) and CRC markers (Fn, m3, 4 Bac) were not significantly changed at each time point compared to baseline (fig. 3C). In the intervention group, there was a significant increase in three bifidobacteria at week 2, while some bifidobacteria were also increased at weeks 4 and 5, but no significant change at weeks 8 and 12, by paired comparison with baseline levels (fig. 3D). This indicates that bifidobacteria cannot colonize the colon of the individual and return to baseline levels within 1 month after withdrawal of the synbiotic composition. On the other hand, the levels of Fn and m3 and the 4Bac composite score decreased from week 2; their levels were significantly reduced at almost all time points compared to baseline, including at week 12 (two months after cessation of synbiotic composition administration) (fig. 3D). Furthermore, the inventors compared the levels of the three bifidobacteria and CRC markers to baseline by the mann-whitney U test and multiple comparison trend analysis. Three bifidobacteria all tended to increase from baseline to week 4, but only the increase in bifidobacteria had statistical significance (linear trend p < 0.05), probably due to the relatively high levels of bifidobacteria adolescentis and bifidobacterium longum at baseline, resulting in insignificant elevation after ingestion. After the synbiotic composition was stopped, the levels of all three bifidobacteria decreased, and the decrease in bifidobacteria was significant from week 4 to week 12 (p < 0.05) (fig. 3E). Interestingly, fn, m3 levels, and 4Bac composite scores were significantly decreased from baseline to week 4 or from baseline to week 12 (fig. 3E). Fn, m3 levels and 4Bac composite scores reflect that a synbiotic composition containing bifidobacterium adolescentis, bifidobacterium longum and bifidobacterium bifidum can reduce the microbial risk of CRC. The reduction in Fn, m3 levels and 4Bac composite scores well demonstrates that a synbiotic composition containing bifidobacterium adolescentis, bifidobacterium longum and bifidobacterium bifidum can reduce the risk of CRC microorganisms. Furthermore, the effect of reducing the risk of microorganisms may last for at least 12 weeks with daily intake of the synbiotic composition for 4 consecutive weeks.

Example 2

Method

Bacterial culture conditions

Bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, fn (ATCC 25586) and clostridium aldensonii (Enterocloster aldenensis) (ATCC BAA 1318; an anaerobic bacterium which has no correlation with Fn as a control) were all cultured in RCM medium (Sigma-Aldrich, UK) under anaerobic conditions at 37 ℃.

Bacterial co-culture assay

To investigate the inhibition of Fn growth by bifidobacteria, a co-cultivation experiment was performed in which a single bifidobacterium species (bifidobacterium adolescentis, bifidobacterium longum and bifidobacterium bifidum), a combination of three bifidobacterium species, clostridium aldensonii (control) were co-cultivated with Fn for 12 hours in an anaerobic environment at 37 ℃ respectively. Specifically, 5 co-culture groups were provided in total: (1) Bifidobacterium adolescentis (10) ⁹ CFU)+Fn(10 ⁹ CFU), (2) bifidobacterium longum (10) ⁹ CFU)+Fn(10 ⁹ CFU), (3) bifidobacterium bifidum(10 ⁹ CFU)+Fn(10 ⁹ CFU), (4) three bifidobacteria combinations (total 10 ⁹ CFU)+Fn(10 ⁹ CFU), and (5) clostridium aldencia (10) ⁹ CFU)+Fn(10 ⁹ CFU). For the three bifidobacterium combinations (co-culture group (4)), the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum was in the range of (1-2): 1 (0.5-1.5). After 12 hours of co-culture, the bacteria were thoroughly mixed with the medium, 10. Mu.L of each was diluted with 90. Mu.L of ultrapure water, and boiled at 100℃for 30 minutes to obtain a bacterial DNA solution. qPCR was performed on the bacterial DNA samples in the same amount (equivalent to the equivalent volume of the culture medium bacterial mixture) to obtain the copy number of Fn, to evaluate the growth of Fn. The effect of each bifidobacterium and bifidobacterium combination on Fn growth was calculated relative to the effect of clostridium aldendorsum on Fn growth. Each co-culture group was repeated three times. The nucleotide sequences of the Fn primers and probes used in this study are shown in table 3.

Results

As shown in FIG. 4, the co-culture experiments showed that Bifidobacterium adolescentis, bifidobacterium longum and Bifidobacterium bifidum have an effect of inhibiting the growth of Fusobacterium nucleatum (Fn) in vitro (26-70% inhibition; all P < 0.0001). The inhibition of Fn growth (70% inhibition) by the combination of three bifidobacteria was significantly higher than that of the individual bifidobacteria species (bifidobacterium adolescentis 65%, bifidobacterium longum 26% and bifidobacterium bifidum 62%; P < 0.05). These data support that the three bifidobacteria combinations have a synergistic effect on inhibiting Fn growth.

Example 3

Synbiotic formulation pair reduces the composite score of four microbial markers (Fn, ch, m3, bc) (4 Bac) Effect

Study design

This example is a preliminary study aimed at determining the effectiveness of a synbiotic formulation (containing Ba, bb, bl, st) to alter the microbiota associated with colorectal cancer risk in feces.

Study object

Subjects who have completed testing for fecal 4Bac composite scores and have provided colonoscopy and/or pathology reports and met the following qualification criteria.

Inclusion criteria

1.18 years old or older;

2. the 4Bac comprehensive score in the latest fecal test within one year belongs to middle and high; and

3. informed consent was given consent.

Exclusion criteria

1. Unwilling to follow the study procedure;

2. have been diagnosed with colorectal cancer;

3. any complications that may impede study assessment or study formula compliance;

4. the probiotics, prebiotics and antibiotics are taken orally 30 days before the administration. However, if they agree to discontinue the administration, the same subject may be re-invited to receive an assessment of the addition to the study after 30 days.

Subject recruitment and assessment

At baseline, demographic and clinical data including age, gender, date of birth, medical history, concomitant medication and eating habits were collected, and the latest 4Bac composite score over the year, colonoscopy and pathology results were collected as the baseline for the study.

Research intervention

All eligible subjects will be assigned to take an inclusion meta-formulation daily for 3 months. The synbiotic formulation comprises bifidobacterium adolescentis (Ba), bifidobacterium bifidum (Bb), bifidobacterium longum (Bl), streptococcus thermophilus (St), inulin, galacto-oligosaccharides and polydextrose. Wherein St comprises two strains of Streptococcus thermophilus. Research products are delivered by express delivery or by person. The ratio of probiotics among them, including bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum and streptococcus thermophilus, in Colony Forming Units (CFU), is (1-2): 1: (0.5-1): (0.5-1) probiotics per kit of formula The total amount is 5x10 ¹⁰ . The ratio of inulin, galacto-oligosaccharides and polydextrose is in the range of (2.5-5) by weight: 1: (2-3).

Biological sample collection

All subjects obtained a study package in which a fecal sample collection bottle with a preservative was provided. Subjects were sent back to the laboratory using designated double-bag sealed waterproof packages after collection of stool samples or scheduled for rapid delivery for collection by researchers.

4Bac composite score

Calculation methods for fecal DNA extraction and quantification of microbial markers using double-stranded quantitative PCR (qPCR), and the 4Bac comprehensive scoring algorithm were performed as described in example 1.

Statistical analysis

The differences in 4Bac composite score and individual CRC marker levels at different time points were analyzed by the wilcoxon symbol rank test. All tests were performed by R (version 4.2.1) and RStudio (version 2022.07.1+554). p <0.05 is considered to represent statistical significance.

Preliminary results

A total of 12 have been included in the study and taken a synbiotic formulation for 1 month. The 9 out of 12 had a reduced 4Bac composite score compared to baseline. The wilcoxon symbol rank test analysis showed that after taking the synbiotic formulation for one month, the 4Bac composite score (p=0.009, fig. 5A), fn level (p=0.0015) and Ch level (p=0.036) decreased significantly compared to baseline (fig. 5B).

Example 4

Method

Bacterial culture

Fusobacterium nucleatum (Fn) standard strain DSM20482 was purchased from the Lebunidz institute-German collection of microorganisms and cell cultures (German Collection of Microorganisms and Cell Cultures). 5 probiotics (Bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, streptococcus thermophilus strain 1 and Streptococcus thermophilus strain 2) and Fn were cultured overnight. The culture conditions were 37 degrees celsius, 100% carbon dioxide.

Bacterial co-culture assay

Bacterial co-culture experiments were used to investigate the effect of bifidobacteria and streptococcus thermophilus combinations on Fn growth. The combination of each bifidobacterium species (bifidobacterium adolescentis, bifidobacterium longum, bifidobacterium bifidum) and two strains of streptococcus thermophilus (strain 1 and strain 2) was co-cultivated with Fn for 12 hours in an anaerobic environment at 37 ℃. The experiment comprises 1 control group and 1 treatment group, and the concrete steps are as follows: 1) Fn (10) ⁹ CFU) control group, 2) three bifidobacteria in combination with two streptococcus thermophilus strains (total 10) ⁹ CFU) and Fn (10) ⁹ CFU) co-culture. The specific composition ratios of the 5 strain compositions in group 2 are as follows: bifidobacterium bifidum: bifidobacterium longum: two streptococcus thermophilus (in Colony Forming Units (CFU)) were (1-2): 1: (0.5-1): (0.5-1). All experiments were repeated three times.

Bacterial DNA extraction

The bacterial solutions after overnight culture were thoroughly mixed, and 200. Mu.l of each group was extracted. ZYMO Quick-DNA was used according to the instructions ^TM Bacterial DNA extraction was performed by Fecal/oil Microbe Kit (ZYMO, USA). The concentration and quality of bacterial DNA was detected using Nanodrop One (sameimer, usa).

Real-time quantitative PCR (polymerase chain reaction) detection of bacterial DNA (deoxyribonucleic acid) content

2. Mu.l of bacterial DNA was extracted for detection. Using TB according to instructionsPremix Ex Taq (TAKARA, japan) was subjected to real-time quantitative PCR detection in a 10. Mu.l total system. The fusobacterium nucleatum primers used in this experiment are shown in table 3. All experiments were repeated three times. Fn DNA copy number in each experimental group was analyzed by One-Way analysis of variance (One Way ANOVA).

Results

After overnight incubation, the content of F.nucleatum DNA in the culture system was detected using real-time quantitative PCR (FIG. 6). The DNA content of clostridium nucleatum was significantly lower than that of the control group (p=0.018) after treatment with the probiotic composition comprising 5 strains, suggesting that the probiotic composition was able to significantly inhibit the growth of clostridium nucleatum.

While exemplary embodiments of the application have been described, modifications and improvements to the described exemplary embodiments of the application can be made by those skilled in the art without departing from the spirit and scope of the application, and the resulting variations or equivalents thereof fall within the scope of the application.

Claims

A probiotic composition comprises Bifidobacterium adolescentis (Bifidobacterium adolescentis), bifidobacterium bifidum (Bifidobacterium bifidum) and Bifidobacterium longum (Bifidobacterium longum).
A probiotic composition according to claim 1, wherein the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in colony forming units is (1-2): 1 (0.5-1.5), preferably (1-2): 1 (0.5-1).
The probiotic composition of claim 1 or 2, further comprising streptococcus thermophilus (Streptococcus thermophilus).
A probiotic composition according to claim 3, wherein the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum and streptococcus thermophilus in colony forming units is (1-2): 1 (0.5-1.5): 0-2, preferably (1-2): 1 (0.5-1): 0.5-1.
The probiotic composition of any one of claims 1-4, wherein the probiotic composition is in unit dosage form and is in colony forming units, bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longumThe amount of bacteria and streptococcus thermophilus is independently 10 ⁴ To 10 ¹² Magnitude of CFU; optionally, the total amount of Bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium longum, and Streptococcus thermophilus is 10 ⁶ To 10 ¹² Magnitude of CFU.
The probiotic composition of claim 5, wherein

The amount of Bifidobacterium adolescentis is 3.08X10 ⁵ -4.00×10 ¹¹ CFU; and/or

The amount of Bifidobacterium bifidum is 1.54X10 ⁵ -4.00×10 ¹¹ CFU; and/or

The amount of Bifidobacterium longum was 2.00X 10 ⁵ -3.01×10 ¹¹ CFU or 2.00×10 ⁵ -2.31×10 ¹¹ CFU; and/or

The Streptococcus thermophilus has an amount of 0-3.08X10 ¹¹ CFU。
The probiotic composition of any one of claims 1-6, wherein the probiotic composition is free of bifidobacteria adolescentis, bifidobacteria bifidus, bifidobacterium longum, probiotics other than streptococcus thermophilus, such as free of bifidobacteria adolescentis, bifidobacterium bifidum, bifidobacterium other than bifidobacterium longum.
A prebiotic composition comprising (a) inulin, (b) galacto-oligosaccharides and (c) resistant starch and/or polydextrose.
The prebiotic composition of claim 8 wherein the prebiotic composition comprises resistant starch and the ratio of the amounts of inulin, galactooligosaccharide and resistant starch by weight is (2-5): 1 (2-4).
The prebiotic composition of claim 8 wherein the prebiotic composition comprises polydextrose and the ratio of the amounts of inulin, galactooligosaccharide and polydextrose by weight is (2.5-5): 1 (2-3).
A prebiotic composition according to any of claims 8 to 10, wherein the prebiotic composition is in unit dosage form and the total amount of (a) inulin, (b) galacto-oligosaccharides and (c) resistant starch and/or polydextrose is 0.1 to 12g, such as 0.1 to 4g, by weight.
The prebiotic composition of claim 11, wherein

The inulin amount is 0.04g-3g or 0.04g-2g; and/or

The amount of galacto-oligosaccharide is 0.01g-0.8g; and/or

The amount of resistant starch is 0.04g-1.6g; and/or

The amount of polydextrose is 0.01-1.6g.
A prebiotic composition according to any of claims 8 to 12, which does not comprise a prebiotic component other than (a) inulin, (b) galacto-oligosaccharides and (c) resistant starch and/or polydextrose.
A dietary composition comprising the probiotic composition of any one of claims 1-7 and the prebiotic composition of any one of claims 8-13; preferably, the dietary composition comprises as probiotics bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum and as probiotics (a) inulin, (b) galactooligosaccharide and (c) resistant starch and/or polydextrose, wherein the ratio of the amounts of bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in terms of Colony Forming Units (CFU) is (1-2): 1 (0.5-1.5), preferably (1-2): 1 (0.5-1), and the total of the three bacteria is 1 x 10 ¹⁰ -2×10 ¹¹ CFU and when the prebiotic composition comprises resistant starch, the ratio of inulin, galacto-oligosaccharide and resistant starch is (2-5) 1 (2-4) by weight, when the prebiotic composition comprises polydextrose, the ratio of inulin, galacto-oligosaccharide and polydextrose amounts is (2.5-5) 1 (2-3) by weight, and (a) inulin, (b)The total amount of galactooligosaccharides and (c) resistant starch and/or polydextrose is 1.2-2g.
The dietary composition of claim 14, wherein the probiotic composition and prebiotic composition are formulated as a single composition.
The dietary composition of claim 14, wherein the probiotic composition and the prebiotic composition are physically separate.
The probiotic composition of any one of claims 1-7, or the prebiotic composition of any one of claims 8-13, or the dietary composition of any one of claims 14-16, formulated for oral administration, such as oral administration, mixing with an oral product, or tube feeding.
The probiotic composition of any one of claims 1-7, 17, or the prebiotic composition of any one of claims 8-13, 17, or the dietary composition of any one of claims 14-17, which is a food supplement, food additive or food.
The probiotic composition of any one of claims 1-7, 17-18, or the prebiotic composition of any one of claims 8-13, 17-18, or the dietary composition of any one of claims 14-18, formulated as a powder, granule, tablet, or capsule.
The probiotic composition of any one of claims 1-7, 17-19, or the prebiotic composition of any one of claims 8-13, 17-19, or the dietary composition of any one of claims 14-19, for administration to a subject for preventing, alleviating and/or aiding in the treatment of colorectal neoplasms (e.g., colorectal cancer or colorectal adenoma), controlling or reducing the risk of colorectal neoplasms (e.g., colorectal cancer or colorectal adenoma), or inhibiting the growth of fusobacterium nucleatum (Fusobacterium nucleatum) or clostridium harveyi (Clostridium hathewayi).
The probiotic composition or prebiotic composition or dietary composition for use according to claim 20, wherein the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium hardtii (Clostridium hathewayi) or lachnoclostricium sp.m3 in the faecal sample of the individual is higher than the corresponding level in the faecal sample of a healthy individual, or the combined score (e.g. 4Bac score) of clostridium nucleatum, clostridium clarkii (Bacteroides clarus), clostridium hardtii and lachnoclostricium sp.m3 is higher than the corresponding score in the faecal sample of a healthy individual.
Use of the probiotic composition of any one of claims 1-7, 17-19, or the prebiotic composition of any one of claims 8-13, 17-19, or the dietary composition of any one of claims 14-19, for the manufacture of a dietary product or medicament for preventing, alleviating and/or aiding in the treatment of colorectal neoplasms (e.g. colorectal cancer or colorectal adenoma), controlling or reducing the risk of colorectal neoplasms (e.g. colorectal cancer or colorectal adenoma), or inhibiting the growth of clostridium nucleatum (Fusobacterium nucleatum) or clostridium harveyi (Clostridium hathewayi).
A method for preventing, alleviating and/or assisting in the treatment of colorectal tumors (e.g. colorectal cancer or colorectal adenoma), controlling or reducing the risk of colorectal tumors (e.g. colorectal cancer or colorectal adenoma), or inhibiting the growth of clostridium nucleatum (Fusobacterium nucleatum) or clostridium hardtii (Clostridium hathewayi), comprising administering to a subject in need thereof the probiotic composition of any one of claims 1-7, 17-19, or the prebiotic composition of any one of claims 8-13, 17-19, or the dietary composition of any one of claims 14-19.
The method of claim 23, comprising determining the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium harveyi (Clostridium hathewayi), or lachnoclostrichum sp.m3 in a fecal sample of the individual, and administering the probiotic composition or prebiotic composition or dietary composition to the individual if the determined level is higher than the corresponding level in a fecal sample of a healthy individual.
The method of claim 23, comprising determining the level of clostridium nucleatum (Fusobacterium nucleatum), clostridium clarkii (Bacteroides clarus), clostridium harveyi (Clostridium hathewayi) and lachnoclostrichum sp.m3 in the individual's fecal sample and deriving a composite score (e.g., a 4Bac score), and administering the probiotic composition or prebiotic composition or dietary composition to the individual if the determined score is higher than the corresponding score in the fecal sample of a healthy individual.