WO2023284848A1 - Fructooligosaccharide having high kestose content and application thereof - Google Patents
Fructooligosaccharide having high kestose content and application thereof Download PDFInfo
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- WO2023284848A1 WO2023284848A1 PCT/CN2022/105867 CN2022105867W WO2023284848A1 WO 2023284848 A1 WO2023284848 A1 WO 2023284848A1 CN 2022105867 W CN2022105867 W CN 2022105867W WO 2023284848 A1 WO2023284848 A1 WO 2023284848A1
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- kestose
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- fructooligosaccharide
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- ODEHMIGXGLNAKK-OESPXIITSA-N 6-kestotriose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 ODEHMIGXGLNAKK-OESPXIITSA-N 0.000 title claims abstract description 45
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 title claims abstract description 35
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
Definitions
- the invention relates to the field of biotechnology, in particular to a kind of fructooligosaccharide with high content of kestose and its application.
- Prebiotics are a class of substances that are selectively utilized by microorganisms to confer health benefits on the host, including fructo-oligosaccharides, galacto-oligosaccharides, and human milk oligosaccharides.
- fructo-oligosaccharides include fructo-oligosaccharides, galacto-oligosaccharides, and human milk oligosaccharides.
- human milk oligosaccharides include fructo-oligosaccharides, galacto-oligosaccharides, and human milk oligosaccharides.
- Precise prebiotics are based on the research and deep understanding of the physical and chemical properties, structural components, and physiological mechanism of prebiotics, guided by the laws and requirements of modern precision nutrition, aiming at the flora, metabolism, nutritional characteristics and needs of different groups of people or individuals , in an independent or synergistic manner, realize the precise customization and scientific formulation of prebiotics in micro-ecological health products, biomedical research
- Fructooligosaccharides widely exist in natural plants that people often eat, such as bananas, garlic, wheat, onions, yacon, Jerusalem artichoke, etc. contain fructooligosaccharides, which are a type of natural oligosaccharides. Its molecular structure is generally composed of sucrose molecules with ⁇ -(1 ⁇ 2) glycosidic bonds and 1-6 fructose molecules. The degree of polymerization is 2-7, and the average degree of polymerization is about 2.7. Fructose-oligosaccharide has been proven to be an all-natural ingredient with dual physiological properties of super bifidus factor and water-soluble dietary fiber, and has been recognized by more than 200 countries and regions around the world. In the Chinese market, fructooligosaccharides are approved as "food ingredients", “cosmetic raw materials” and “nutritional enhancers” and are widely used in food, health care, medicine, cosmetics and feed fields.
- fructo-oligosaccharides are a mixture whose main components are kestose (GF2), nystose (GF3) and fructopentose (1- ⁇ -fructofuranosyl-nystose, GF4). There are some differences in the components of fructo-oligosaccharides produced by the process. Kestose can effectively proliferate Bifidobacterium spp. and Faecalibacterium prausnitzii and relieve atopic dermatitis in infants.
- GF2 kestose
- GF3 nystose
- fructopentose (1- ⁇ -fructofuranosyl-nystose
- the purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a high kestose triose content fructooligosaccharide that can regulate intestinal flora and immune function, and overcome the confusion and ambiguity in the application level of existing prebiotics , arbitrariness and other limitations, providing a scientific basis and typical demonstration for the application of prebiotics in biomedical fields such as precision nutrition and microecological medicine.
- the present invention provides a fructooligosaccharide with high kestose content, wherein the mass percentage of kestose in dry matter is greater than 80%, and the mass percentage of kestose in fructooligosaccharide is greater than 90%.
- the daily dosage of the high-kestose-content fructo-oligosaccharide is 4.5 g/kg-28.6 g/kg.
- the daily dose of fructooligosaccharide with high kestose content is 4.5 g/kg, 14.3 g/kg or 28.6 g/kg.
- the present invention provides the application of the above-mentioned fructooligosaccharide with high kestose content in regulating intestinal flora.
- the intestinal flora includes one of Akkermansia muciniphila, Bifidobacterium spp. and Lactobacillus spp. one or more species.
- the regulation of intestinal flora is to increase rodent feces and cecal contents of Akkermansia muciniphila (Akkermansia muciniphila), Bifidobacterium (Bifidobacterium spp.) , Lactobacillus spp. and its metabolites short-chain fatty acids.
- the adjustment of the intestinal flora is by increasing the water content of the feces of rats so as to laxative the intestines.
- the present invention provides the application of the above-mentioned fructooligosaccharide with high kestose content in regulating immune function.
- the regulation of immune function includes enhancing the secretion of ⁇ -interferon from human peripheral blood lymphocytes and their passage cells and inhibiting the inflammatory response of intestinal epithelial cells induced by EHEC.
- the present invention provides the application of the above-mentioned fructooligosaccharide with high kestose content in the preparation of medicines for treating and/or preventing obesity, type 2 diabetes, Crohn's disease, atopic dermatitis or respiratory system infection.
- the present invention provides a compound feed, comprising the following raw materials in weight percentage: 70% low fiber feed AIN-93M and 30% high kestose content fructooligosaccharides.
- high kestose triose content fructooligosaccharide of the present invention can increase the mucinophilic Akkermansia (Akkermansia muciniphila), bifidobacterium (Bifidobacterium spp.), Lactobacillus (Lactobacillus spp.) and their metabolites short-chain fatty acids, and can increase the water content of rat feces to achieve the effect of laxative, and also enhance the human peripheral blood lymphocytes and their passage cells ⁇ -Interferon secretion, inhibits EHEC-induced inflammatory response of intestinal epithelial cells.
- the present invention has the following beneficial effects:
- the present invention accurately evaluates the physiological functions and effects of fructooligosaccharides with high kestose content, overcomes the limitations of non-repeatability, ambiguity, and randomness in the application level of existing prebiotics, and provides prebiotics in precision nutrition and microecological medical care.
- the application in the field of biomedicine provides scientific basis and typical demonstration.
- Fig. 1 is the result schematic diagram of Akkermansia muciniphila (Akkermansia muciniphila) mucinophila (Akkermansia muciniphila) result that high kestose triose content fructooligosaccharide increases rat cecal contents;
- Figure 2 is a schematic diagram of the results of short-chain fatty acids in cecum contents of mice increased by high kestose-oligosaccharide content fructooligosaccharides;
- Fig. 3 is the result schematic diagram that high kestose triose content fructooligosaccharide increases the water content of rat feces;
- Fig. 4 is a schematic diagram of the results of enhancing the secretion of gamma-interferon by human peripheral blood lymphocytes and passaged cells thereof with high fructooligosaccharide content;
- Fig. 5 is a schematic diagram of the results of inhibition of EHEC-induced intestinal epithelial cell inflammatory response by fructooligosaccharides with high kestose content.
- the mass percentage of kestose in dry matter is greater than 80%, and the mass percentage of kestose in fructooligosaccharide is greater than 90%.
- the fructooligosaccharide with high kestose content of the present invention can be obtained.
- -2014 National Food Safety Standard Food Additive Use Standard
- Q/QHB 0003S-2021 Fibertose Oligosaccharides
- Embodiment 2 high kestose triose content fructooligosaccharides increase the mucinophilic Akkermansia (Akkermansia muciniphila) of rat cecum contents and the water content of rat feces
- the rats were male SD rats aged 7-8 weeks, and they were adaptively fed for at least 7 days; 40 rats were randomly divided into 5 groups, 8 rats in each group, fed in a single cage, and fed per kilogram of rat body weight every day 10ml, the intervention of each group was (1) pure water twice a day; (2) loperamide hydrochloride twice a day (4 mg per kilogram of rat body weight per day); (3) pure water once a day; (4) Loperamide hydrochloride (8 mg per kilogram of rat body weight per day) in the morning, pure water in the afternoon; (5) Loperamide hydrochloride (8 mg per kilogram of rat body weight per day in the morning) high in the afternoon and low in kestose Fructose (4.5g per kilogram of rat body weight per day); after the rats were fasted for 16 hours (17:00-9:00) in the afternoon of the 10th day, 10ml of 10% activated carbon was administered per kilogram of rat body weight With 5% gum arabic suspension, the animal was euthanized after
- fructooligosaccharides with high kestose content can increase Akkermansia muciniphila in rat cecal contents, and high fructooligosaccharides content can also increase fecal and cecal contents. Bifidobacterium spp. and Lactobacillus spp. in the contents.
- FIG. 3 compare the effects of different concentrations of loperamide hydrochloride and different administration methods on rat constipation, and try to use fructooligosaccharides with high kestose content to improve the symptoms of constipation in rats.
- FOS with high kestose content can increase the water content of rat feces, thereby making the rats laxative and improving the symptoms of constipation in rats.
- Embodiment 3 high kestose triose content fructooligosaccharide increases the short-chain fatty acid of mouse cecum content
- mice were 4-week-old female BALB/c mice from the same littermate, and they were adaptively fed with low-fiber diet AIN-93M for 2 weeks; 40 mice were randomly divided into 5 groups, 8 in each group, and fed separately (1) Low fiber feed AIN-93M+30% cellulose; (2) low fiber feed AIN-93M+30% fructooligosaccharides; (3) low fiber feed AIN-93M+15% cellulose+15% fructooligosaccharides; ( 4) Low fiber feed AIN-93M+30% high kestose content fructooligosaccharide; (5) Low fiber feed AIN-93M+15% cellulose+15% high kestose content fructooligosaccharide; For 6 weeks, feces samples were collected every two weeks and cecal contents were collected after the sixth week for intestinal flora and their metabolites short-chain fatty acids (Total-SCFA, acetic-acid, butyric-acid, isobutyric-acid, lactic
- fructooligosaccharides with high kestose content can increase the short-chain fatty acids in the cecal contents of mice.
- low-fiber feed is set as the basic background to better highlight the high fructooligosaccharides content and set up two concentration gradients to clarify the dose effect of high kestose triose content fructooligosaccharides.
- the immune cells are derived from human peripheral blood mononuclear cells (PBMCs) T cells; 5 ⁇ 10 6 human peripheral blood mononuclear cells (PBMCs) per well are seeded in a 24-well plate and incubated for 2 hours, and the non-adherent cells are lymphocytes ; 2-5 ⁇ 10 5 lymphocytes per well were inoculated on a 96-well plate coated with 5ug/mL anti-CD3 and anti-CD28 one day in advance, and 10ng/mL IL-2 was added and cultured for 72 hours.
- PBMCs peripheral blood mononuclear cells
- PBMCs human peripheral blood mononuclear cells
- a human peripheral blood lymphocyte differentiation model was constructed, and fructooligosaccharides with high kestose content were used to regulate T cell differentiation and the ability to secrete cytokines.
- fructooligosaccharides with high kestose content can enhance the secretion of ⁇ -interferon from human peripheral blood lymphocytes and their subcultured cells.
- the relative proportion of IFN- ⁇ +T cells significantly increased by 50%-90%, and the mass concentration of IFN- ⁇ in the supernatant of IFN- ⁇ +T cell culture fluid significantly increased by 80%-120%.
- Example 5 Fructo-oligosaccharides with high kestose content inhibit the inflammatory response of intestinal epithelial cells induced by EHEC
- the intestinal epithelial cells HCT-8, HCT-116 and HT-29 were inoculated on a 24-well plate with a healing degree of 25%-30%, and 25 mg/mL high kestose triose-content fructooligosaccharides were added at the same time.
- Set a blank control without addition cultivate for 48 hours, and then add E. coli EHEC with a multiplicity of infection (MOI) of 100:1 for treatment. Starting from the addition of E. coli, collect the samples at 0, 15, 30, 45, 60, and 90 minutes respectively.
- the cell lysates were detected by Western blot, and the detection indicators were inflammation-related cell signaling molecules NF- ⁇ B/p65, phospho-NF- ⁇ B/p65 and I ⁇ B- ⁇ .
- an intestinal epithelial cell inflammation model was constructed, and fructooligosaccharide with high kestose content was tried to inhibit the inflammatory response.
- fructooligosaccharide (GF2) with high kestose content can inhibit the inflammatory response of EHEC-induced intestinal epithelial cells HCT-8, HCT-116 and HT-29.
- the gray level of the inflammatory indicator molecule phospho-NF- ⁇ B/p65 in the Western blot gel electrophoresis graph was significantly reduced by 45%-65%.
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Abstract
The present invention relates to the field of biotechnology, and specifically discloses fructooligosaccharide having high kestose content and an application thereof. In the fructooligosaccharide having high kestose content in the present invention, the mass percentage of kestose among dry matter is greater than 80%, and the mass percentage of kestose in the fructooligosaccharide is more than 90%. The fructooligosaccharide having high kestose content in the present invention can increase Akkermansia muciniphila, Bifidobacterium, Lactobacillus and metabolite short-chain fatty acids thereof in rodent fecal and cecal contents, can increase the water content in rat feces to achieve a laxative effect, and can also enhance the secretion of γ-interferon from human peripheral blood lymphocytes and passage cells thereof, and inhibit inflammatory responses of intestinal epithelial cells induced by EHEC. In addition, the fructooligosaccharide having high kestose content in the present invention is helpful for preventing, improving or treating obesity, type 2 diabetes, Crohn's disease, atopic dermatitis, respiratory system infection and other diseases.
Description
本发明涉及生物技术领域，尤其是涉及一种高蔗果三糖含量低聚果糖及其应用。The invention relates to the field of biotechnology, in particular to a kind of fructooligosaccharide with high content of kestose and its application.
益生元是被微生物选择性利用从而赋予宿主健康效益的一类物质，包括低聚果糖、低聚半乳糖、母乳低聚糖等。随着技术进步，益生元精准化逐渐成为重要发展方向。精准益生元是通过对益生元的理化性质、结构成分、生理作用机制的研究和深刻理解，以现代精准营养的规律和要求为指导，针对不同人群或个体的菌群、代谢、营养特征和需求，以独立或协同作用的方式，实现益生元在微生态健康产品、生物医药研发、医疗服务上的精准化定制和科学配方。要实现上述精准益生元的科学应用，至少必须在以下几个层面上对各种特定的益生元进行深入研究：(1)在分子生物学、细胞生物学的层面上，研究、阐明特定益生元的直接生理功能机制，为进一步指导和应用该益生元提供坚实的科学基础；(2)分离、纯化单一化学组分的益生元，在此基础上进行单一组分益生元的功效研究，建立该组分益生元“结构-菌群-功能”方面的对应关系，为单组分益生元向药物研究和临床应用提供精准的依据。Prebiotics are a class of substances that are selectively utilized by microorganisms to confer health benefits on the host, including fructo-oligosaccharides, galacto-oligosaccharides, and human milk oligosaccharides. With the advancement of technology, the precision of prebiotics has gradually become an important development direction. Precise prebiotics are based on the research and deep understanding of the physical and chemical properties, structural components, and physiological mechanism of prebiotics, guided by the laws and requirements of modern precision nutrition, aiming at the flora, metabolism, nutritional characteristics and needs of different groups of people or individuals , in an independent or synergistic manner, realize the precise customization and scientific formulation of prebiotics in micro-ecological health products, biomedical research and development, and medical services. In order to realize the scientific application of the above-mentioned precise prebiotics, it is necessary to conduct in-depth research on various specific prebiotics at least at the following levels: (1) at the level of molecular biology and cell biology, research and elucidate specific prebiotics The direct physiological function mechanism of the prebiotic provides a solid scientific basis for further guidance and application of the prebiotic; (2) separates and purifies the prebiotic of a single chemical component, and conducts research on the efficacy of the single component prebiotic on this basis, and establishes the prebiotic The corresponding relationship of component prebiotics in terms of "structure-flora-function" provides an accurate basis for single-component prebiotics in drug research and clinical application.
低聚果糖(Fructooligosaccharides，FOS)广泛存在于人们经常食用的天然植物中，如香蕉、大蒜、小麦、洋葱、雪莲果、菊芋等都含有低聚果糖，是一类天然的低聚糖。其分子结构一般是蔗糖分子以β-(1→2)糖苷键与1-6个果糖分子组成，聚合度在2-7，平均聚合度约2.7。低聚果糖已被证实是同时具有超强双歧因子和水溶性膳食纤维的双生理学特性的全天然配料，在世界范围内被200多个国家和地区政府所认可。在中国市场，低聚果糖被批准为“食品配料”、“化妆品原料”以及“营养强化剂”并被广泛应用于食品、保健、医药、化妆品以及饲料领域。Fructooligosaccharides (FOS) widely exist in natural plants that people often eat, such as bananas, garlic, wheat, onions, yacon, Jerusalem artichoke, etc. contain fructooligosaccharides, which are a type of natural oligosaccharides. Its molecular structure is generally composed of sucrose molecules with β-(1→2) glycosidic bonds and 1-6 fructose molecules. The degree of polymerization is 2-7, and the average degree of polymerization is about 2.7. Fructose-oligosaccharide has been proven to be an all-natural ingredient with dual physiological properties of super bifidus factor and water-soluble dietary fiber, and has been recognized by more than 200 countries and regions around the world. In the Chinese market, fructooligosaccharides are approved as "food ingredients", "cosmetic raw materials" and "nutritional enhancers" and are widely used in food, health care, medicine, cosmetics and feed fields.
常规低聚果糖为一种混合物，主要组分为蔗果三糖(kestose,GF2)，蔗果四糖 (nystose,GF3)和蔗果五糖(1-β-fructofuranosyl-nystose,GF4)，不同工艺生产的低聚果糖组分存在一定的差异。蔗果三糖可以有效地增殖双歧杆菌(Bifidobacterium spp.)与普拉梭菌(Faecalibacterium prausnitzii)并缓解婴幼儿特应性皮炎。Conventional fructo-oligosaccharides are a mixture whose main components are kestose (GF2), nystose (GF3) and fructopentose (1-β-fructofuranosyl-nystose, GF4). There are some differences in the components of fructo-oligosaccharides produced by the process. Kestose can effectively proliferate Bifidobacterium spp. and Faecalibacterium prausnitzii and relieve atopic dermatitis in infants.
目前，高蔗果三糖含量低聚果糖在肠道菌群与免疫功能的应用上还未有报道。At present, there is no report on the application of fructooligosaccharides with high kestose content in intestinal flora and immune function.
发明内容Contents of the invention
本发明的目的在于克服上述现有技术的不足之处而提供一种具有调节肠道菌群与免疫功能的高蔗果三糖含量低聚果糖，克服了现有益生元应用层面的混杂模糊性、随意性等局限，为益生元在精准营养与微生态医疗等生物医药领域的应用提供科学依据与典型示范。The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a high kestose triose content fructooligosaccharide that can regulate intestinal flora and immune function, and overcome the confusion and ambiguity in the application level of existing prebiotics , arbitrariness and other limitations, providing a scientific basis and typical demonstration for the application of prebiotics in biomedical fields such as precision nutrition and microecological medicine.
为实现上述目的，本发明采取的技术方案为：In order to achieve the above object, the technical scheme that the present invention takes is:
第一方面，本发明提供了一种高蔗果三糖含量低聚果糖，蔗果三糖占干物质的质量百分比大于80％，蔗果三糖占低聚果糖的质量百分比大于90％。In a first aspect, the present invention provides a fructooligosaccharide with high kestose content, wherein the mass percentage of kestose in dry matter is greater than 80%, and the mass percentage of kestose in fructooligosaccharide is greater than 90%.
作为本发明所述高蔗果三糖含量低聚果糖的优选实施方式，当动物给药时，高蔗果三糖含量低聚果糖每天给药量为4.5g/kg-28.6g/kg。As a preferred embodiment of the high-kestose-content fructo-oligosaccharide of the present invention, when animals are administered, the daily dosage of the high-kestose-content fructo-oligosaccharide is 4.5 g/kg-28.6 g/kg.
更为优选地，当动物给药时，高蔗果三糖含量低聚果糖每天给药量为4.5g/kg、14.3g/kg或28.6g/kg。More preferably, when animals are administered, the daily dose of fructooligosaccharide with high kestose content is 4.5 g/kg, 14.3 g/kg or 28.6 g/kg.
第二方面，本发明提供了上述高蔗果三糖含量低聚果糖在调节肠道菌群中的应用。In the second aspect, the present invention provides the application of the above-mentioned fructooligosaccharide with high kestose content in regulating intestinal flora.
作为本发明所述应用的优选实施方式，所述肠道菌群包括嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)、双歧杆菌(Bifidobacterium spp.)和乳酸杆菌(Lactobacillus spp.)中的一种或多种。As a preferred embodiment of the application of the present invention, the intestinal flora includes one of Akkermansia muciniphila, Bifidobacterium spp. and Lactobacillus spp. one or more species.
作为本发明所述应用的优选实施方式，所述调节肠道菌群为增加啮齿类动物粪便及盲肠内容物的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)、双歧杆菌(Bifidobacterium spp.)、乳酸杆菌(Lactobacillus spp.)及其代谢产物短链脂肪酸。As a preferred embodiment of the application of the present invention, the regulation of intestinal flora is to increase rodent feces and cecal contents of Akkermansia muciniphila (Akkermansia muciniphila), Bifidobacterium (Bifidobacterium spp.) , Lactobacillus spp. and its metabolites short-chain fatty acids.
作为本发明所述应用的优选实施方式，所述调节肠道菌群为通过增加大鼠粪便含水量从而润肠通便。As a preferred embodiment of the application of the present invention, the adjustment of the intestinal flora is by increasing the water content of the feces of rats so as to laxative the intestines.
第三方面，本发明提供了上述高蔗果三糖含量低聚果糖在调节免疫功能中的应用。In the third aspect, the present invention provides the application of the above-mentioned fructooligosaccharide with high kestose content in regulating immune function.
作为本发明所述应用的优选实施方式，所述调节免疫功能包括增强人外周血淋巴细胞及其传代细胞γ-干扰素分泌和抑制EHEC诱导的肠上皮细胞的炎症响应。As a preferred embodiment of the application of the present invention, the regulation of immune function includes enhancing the secretion of γ-interferon from human peripheral blood lymphocytes and their passage cells and inhibiting the inflammatory response of intestinal epithelial cells induced by EHEC.
第四方面，本发明提供了上述高蔗果三糖含量低聚果糖在制备治疗和/或预防肥胖症、2型糖尿病、克罗恩病、特应性皮炎或呼吸系统感染药物中的应用。In a fourth aspect, the present invention provides the application of the above-mentioned fructooligosaccharide with high kestose content in the preparation of medicines for treating and/or preventing obesity, type 2 diabetes, Crohn's disease, atopic dermatitis or respiratory system infection.
第五方面，本发明提供了一种复合饲料，包括以下重量百分比的原料：70％低纤维饲料AIN-93M和30％高蔗果三糖含量低聚果糖。In the fifth aspect, the present invention provides a compound feed, comprising the following raw materials in weight percentage: 70% low fiber feed AIN-93M and 30% high kestose content fructooligosaccharides.
根据实施例2-5的实验数据证明了本发明的高蔗果三糖含量低聚果糖可以增加啮齿类动物粪便及盲肠内容物的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)、双歧杆菌(Bifidobacterium spp.)、乳酸杆菌(Lactobacillus spp.)及其代谢产物短链脂肪酸，并且可以增加大鼠粪便含水量从而达到润肠通便的效果，还增强了人外周血淋巴细胞及其传代细胞γ-干扰素分泌，抑制EHEC诱导的肠上皮细胞的炎症响应。According to the experimental data of embodiment 2-5, it has been proved that high kestose triose content fructooligosaccharide of the present invention can increase the mucinophilic Akkermansia (Akkermansia muciniphila), bifidobacterium (Bifidobacterium spp.), Lactobacillus (Lactobacillus spp.) and their metabolites short-chain fatty acids, and can increase the water content of rat feces to achieve the effect of laxative, and also enhance the human peripheral blood lymphocytes and their passage cells γ-Interferon secretion, inhibits EHEC-induced inflammatory response of intestinal epithelial cells.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明精准评价高蔗果三糖含量低聚果糖的生理功能和作用，克服现有益生元应用层面的不可重复性、模糊性、随意性等局限，为益生元在精准营养与微生态医疗等生物医药领域的应用提供科学依据与典型示范。The present invention accurately evaluates the physiological functions and effects of fructooligosaccharides with high kestose content, overcomes the limitations of non-repeatability, ambiguity, and randomness in the application level of existing prebiotics, and provides prebiotics in precision nutrition and microecological medical care. The application in the field of biomedicine provides scientific basis and typical demonstration.
图1是高蔗果三糖含量低聚果糖增加大鼠盲肠内容物的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)结果示意图；Fig. 1 is the result schematic diagram of Akkermansia muciniphila (Akkermansia muciniphila) mucinophila (Akkermansia muciniphila) result that high kestose triose content fructooligosaccharide increases rat cecal contents;
图2是高蔗果三糖含量低聚果糖增加小鼠盲肠内容物的短链脂肪酸结果示意图；Figure 2 is a schematic diagram of the results of short-chain fatty acids in cecum contents of mice increased by high kestose-oligosaccharide content fructooligosaccharides;
图3是高蔗果三糖含量低聚果糖增加大鼠粪便含水量的结果示意图；Fig. 3 is the result schematic diagram that high kestose triose content fructooligosaccharide increases the water content of rat feces;
图4是高蔗果三糖含量低聚果糖增强人外周血淋巴细胞及其传代细胞γ-干扰素分泌的结果示意图；Fig. 4 is a schematic diagram of the results of enhancing the secretion of gamma-interferon by human peripheral blood lymphocytes and passaged cells thereof with high fructooligosaccharide content;
图5是高蔗果三糖含量低聚果糖抑制EHEC诱导肠上皮细胞炎症响应的结果示意图。Fig. 5 is a schematic diagram of the results of inhibition of EHEC-induced intestinal epithelial cell inflammatory response by fructooligosaccharides with high kestose content.
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.
在以下实施例中，所使用的实验方法如无特殊说明，均为常规方法，所用的材料、试剂等，如无特殊说明，均可从商业途径得到。In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified.
实施例1Example 1
本发明提供的高蔗果三糖含量低聚果糖(GF2)，蔗果三糖占干物质的质量百分比大于80％，蔗果三糖占低聚果糖的质量百分比大于90％。In the fructooligosaccharide (GF2) with high kestose content provided by the invention, the mass percentage of kestose in dry matter is greater than 80%, and the mass percentage of kestose in fructooligosaccharide is greater than 90%.
按照专利申请CN202011039079.0的技术方法制备可以得到本发明的高蔗果三糖含量低聚果糖，将高蔗果三糖含量低聚果糖根据GB/T 23528-2009《低聚果糖》、GB 2760-2014《食品安全国家标准食品添加剂使用标准》、Q/QHB 0003S-2021《低聚果糖》等依据进行测试。According to the technical method of patent application CN202011039079.0, the fructooligosaccharide with high kestose content of the present invention can be obtained. -2014 "National Food Safety Standard Food Additive Use Standard", Q/QHB 0003S-2021 "Fructose Oligosaccharides" and other basis for testing.
表1测试结果Table 1 Test results
实施例2、高蔗果三糖含量低聚果糖增加大鼠盲肠内容物的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)及大鼠粪便含水量 Embodiment 2, high kestose triose content fructooligosaccharides increase the mucinophilic Akkermansia (Akkermansia muciniphila) of rat cecum contents and the water content of rat feces
实验：大鼠为7-8周龄的雄性SD大鼠，适应性饲养至少7天；40只大鼠随机分为5组，每组8只，单笼饲养，每天每千克大鼠体重灌胃10ml，每组的干预为(1)每天两次纯水；(2)每天两次盐酸洛哌丁胺(每天每千克大鼠体重灌胃4mg)；(3)每天一次纯水；(4)上午盐酸洛哌丁胺(每天每千克大鼠体重灌胃8mg)，下午纯水；(5)上午盐酸洛哌丁胺(每天每千克大鼠体重灌胃8mg)下午高蔗果三糖含量低聚果糖(每天每千克大鼠体重灌胃4.5g)；大鼠于第10天下午禁食16小时(17:00-9:00)后，每千克大鼠体重灌胃10ml的10％活性碳与5％阿拉伯胶混悬液，30分钟后将动物安乐死，剖腹剥离胃肠道；每天记录粪便数量、粪便湿重、粪便干重、粪便含水量，第0天、第10天粪便取样以及第11天盲肠取样用于肠道菌群的检测。Experiment: The rats were male SD rats aged 7-8 weeks, and they were adaptively fed for at least 7 days; 40 rats were randomly divided into 5 groups, 8 rats in each group, fed in a single cage, and fed per kilogram of rat body weight every day 10ml, the intervention of each group was (1) pure water twice a day; (2) loperamide hydrochloride twice a day (4 mg per kilogram of rat body weight per day); (3) pure water once a day; (4) Loperamide hydrochloride (8 mg per kilogram of rat body weight per day) in the morning, pure water in the afternoon; (5) Loperamide hydrochloride (8 mg per kilogram of rat body weight per day in the morning) high in the afternoon and low in kestose Fructose (4.5g per kilogram of rat body weight per day); after the rats were fasted for 16 hours (17:00-9:00) in the afternoon of the 10th day, 10ml of 10% activated carbon was administered per kilogram of rat body weight With 5% gum arabic suspension, the animal was euthanized after 30 minutes, and the gastrointestinal tract was removed by laparotomy; the number of feces, wet weight of feces, dry weight of feces, water content of feces were recorded every day, feces were sampled on the 0th day, the 10th day and Cecum samples were taken on day 11 for the detection of intestinal flora.
参考图1，高蔗果三糖含量低聚果糖可以增加大鼠盲肠内容物的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)，同样高蔗果三糖含量低聚果糖也可以增加粪便及盲肠内容物的双歧杆菌(Bifidobacterium spp.)及乳酸杆菌(Lactobacillus spp.)。Referring to Figure 1, fructooligosaccharides with high kestose content can increase Akkermansia muciniphila in rat cecal contents, and high fructooligosaccharides content can also increase fecal and cecal contents. Bifidobacterium spp. and Lactobacillus spp. in the contents.
参考图3，比较不同浓度的盐酸洛哌丁胺及不同给药方式对大鼠便秘的诱导效果，尝试使用高蔗果三糖含量低聚果糖改善大鼠便秘症状。高蔗果三糖含量低聚果糖可以增加大鼠粪便含水量，从而使得大鼠润肠通便，改善了大鼠便秘症状。Referring to Figure 3, compare the effects of different concentrations of loperamide hydrochloride and different administration methods on rat constipation, and try to use fructooligosaccharides with high kestose content to improve the symptoms of constipation in rats. FOS with high kestose content can increase the water content of rat feces, thereby making the rats laxative and improving the symptoms of constipation in rats.
实施例3、高蔗果三糖含量低聚果糖增加小鼠盲肠内容物的短链脂肪酸 Embodiment 3, high kestose triose content fructooligosaccharide increases the short-chain fatty acid of mouse cecum content
实验：小鼠为同窝4周龄雌性BALB/c小鼠，低纤维饲料AIN-93M适应性喂养2周；40只小鼠随机分为5组，每组8只，分别饲喂(1)低纤维饲料AIN-93M+30％纤维素；(2)低纤维饲料AIN-93M+30％低聚果糖；(3)低纤维饲料AIN-93M+15％纤维素+15％低聚果糖；(4)低纤维饲料AIN-93M+30％高蔗果三糖含量低聚果糖；(5)低纤维饲料AIN-93M+15％纤维素+15％高蔗果三糖含量低聚果糖；饲喂6周，每两周收集粪便样本以及第6周结束后取盲肠内容物用于肠道菌群及其代谢产物短链脂肪酸(Total-SCFA、acetic-acid、butyric-acid、isobutyric-acid、lactic-acid、propionate-acid、valerate-acid)的检测。Experiment: The mice were 4-week-old female BALB/c mice from the same littermate, and they were adaptively fed with low-fiber diet AIN-93M for 2 weeks; 40 mice were randomly divided into 5 groups, 8 in each group, and fed separately (1) Low fiber feed AIN-93M+30% cellulose; (2) low fiber feed AIN-93M+30% fructooligosaccharides; (3) low fiber feed AIN-93M+15% cellulose+15% fructooligosaccharides; ( 4) Low fiber feed AIN-93M+30% high kestose content fructooligosaccharide; (5) Low fiber feed AIN-93M+15% cellulose+15% high kestose content fructooligosaccharide; For 6 weeks, feces samples were collected every two weeks and cecal contents were collected after the sixth week for intestinal flora and their metabolites short-chain fatty acids (Total-SCFA, acetic-acid, butyric-acid, isobutyric-acid, lactic -acid, propionate-acid, valerate-acid) detection.
参考图2，高蔗果三糖含量低聚果糖可以增加小鼠盲肠内容物的短链脂肪酸，本实施例通过设置低纤维饲料作为基础背景从而更好地凸显高蔗果三糖含 量低聚果糖的作用，并且设置两个浓度梯度从而明确高蔗果三糖含量低聚果糖的剂量效应。Referring to Figure 2, fructooligosaccharides with high kestose content can increase the short-chain fatty acids in the cecal contents of mice. In this example, low-fiber feed is set as the basic background to better highlight the high fructooligosaccharides content and set up two concentration gradients to clarify the dose effect of high kestose triose content fructooligosaccharides.
实施例4、高蔗果三糖含量低聚果糖增强人外周血淋巴细胞及其传代细胞γ-干扰素分泌Example 4. High-kestose-content fructooligosaccharides enhance the secretion of gamma-interferon from human peripheral blood lymphocytes and their subcultured cells
本实施例中，免疫细胞来源于人外周血单个核细胞PBMC的T细胞；每孔5×10
6个人外周血单个核细胞PBMC接种在24孔板并孵育2小时，非贴壁细胞为淋巴细胞；每孔2-5×10
5个淋巴细胞接种于提前一天以5ug/mL的anti-CD3和anti-CD28包被的96孔板，加入10ng/mL的IL-2并培养72小时即为T细胞；在T细胞培养基中加入5mg/mL的低聚果糖、高蔗果三糖含量低聚果糖并设置无添加的空白对照，通过流式细胞术与酶联免疫吸附测定法检测IFN-γ+T细胞的相对比例以及细胞培养液上清中IFN-γ的质量浓度。
In this example, the immune cells are derived from human peripheral blood mononuclear cells (PBMCs) T cells; 5×10 6 human peripheral blood mononuclear cells (PBMCs) per well are seeded in a 24-well plate and incubated for 2 hours, and the non-adherent cells are lymphocytes ; 2-5×10 5 lymphocytes per well were inoculated on a 96-well plate coated with 5ug/mL anti-CD3 and anti-CD28 one day in advance, and 10ng/mL IL-2 was added and cultured for 72 hours. Cells; add 5 mg/mL fructo-oligosaccharides and high-kestose-triose-content fructo-oligosaccharides to the T cell culture medium and set up no added blank control, and detect IFN-γ by flow cytometry and enzyme-linked immunosorbent assay The relative proportion of +T cells and the mass concentration of IFN-γ in the cell culture supernatant.
本实施例中，构建人外周血淋巴细胞分化模型，尝试使用高蔗果三糖含量低聚果糖调节T细胞分化及其分泌细胞因子的能力。In this example, a human peripheral blood lymphocyte differentiation model was constructed, and fructooligosaccharides with high kestose content were used to regulate T cell differentiation and the ability to secrete cytokines.
参考图4，高蔗果三糖含量低聚果糖可以增强人外周血淋巴细胞及其传代细胞γ-干扰素分泌。其中，IFN-γ+T细胞相对比例显著增加50％-90％，IFN-γ+T细胞培养液上清中IFN-γ的质量浓度显著增加80％-120％。Referring to FIG. 4 , fructooligosaccharides with high kestose content can enhance the secretion of γ-interferon from human peripheral blood lymphocytes and their subcultured cells. Among them, the relative proportion of IFN-γ+T cells significantly increased by 50%-90%, and the mass concentration of IFN-γ in the supernatant of IFN-γ+T cell culture fluid significantly increased by 80%-120%.
实施例5、高蔗果三糖含量低聚果糖抑制EHEC诱导的肠上皮细胞的炎症响应Example 5. Fructo-oligosaccharides with high kestose content inhibit the inflammatory response of intestinal epithelial cells induced by EHEC
本实施例中，将肠上皮细胞HCT-8、HCT-116及HT-29以25％-30％的愈合度接种于24孔板，同时加入25mg/mL高蔗果三糖含量低聚果糖并设置无添加的空白对照，培养48小时，再加入感染复数(MOI)为100:1的大肠杆菌EHEC处理，以加入大肠杆菌为起点，分别收集第0、15、30、45、60、90分钟的细胞裂解液进行Westernblot检测，检测指标为炎症相关细胞信号分子NF-κB/p65、phospho-NF-κB/p65与IκB-α。In this example, the intestinal epithelial cells HCT-8, HCT-116 and HT-29 were inoculated on a 24-well plate with a healing degree of 25%-30%, and 25 mg/mL high kestose triose-content fructooligosaccharides were added at the same time. Set a blank control without addition, cultivate for 48 hours, and then add E. coli EHEC with a multiplicity of infection (MOI) of 100:1 for treatment. Starting from the addition of E. coli, collect the samples at 0, 15, 30, 45, 60, and 90 minutes respectively. The cell lysates were detected by Western blot, and the detection indicators were inflammation-related cell signaling molecules NF-κB/p65, phospho-NF-κB/p65 and IκB-α.
本实施例中构建肠上皮细胞炎症模型，尝试使用高蔗果三糖含量低聚果糖抑制炎症反应。In this example, an intestinal epithelial cell inflammation model was constructed, and fructooligosaccharide with high kestose content was tried to inhibit the inflammatory response.
参考图5，与对照组相比，加入高蔗果三糖含量低聚果糖(GF2)可以抑制EHEC诱导的肠上皮细胞HCT-8、HCT-116及HT-29的炎症响应。其中，炎症指 标分子phospho-NF-κB/p65在Western blot凝胶电泳图中的灰度显著下降45％-65％。Referring to FIG. 5 , compared with the control group, the addition of fructooligosaccharide (GF2) with high kestose content can inhibit the inflammatory response of EHEC-induced intestinal epithelial cells HCT-8, HCT-116 and HT-29. Among them, the gray level of the inflammatory indicator molecule phospho-NF-κB/p65 in the Western blot gel electrophoresis graph was significantly reduced by 45%-65%.
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
- 一种高蔗果三糖含量低聚果糖，其特征在于，蔗果三糖占干物质的质量百分比大于80％，蔗果三糖占低聚果糖的质量百分比大于90％。A fructooligosaccharide with high kestose content is characterized in that the mass percentage of kestose in dry matter is greater than 80%, and the mass percentage of kestose in fructooligosaccharide is greater than 90%.
- 如权利要求1所述的高蔗果三糖含量低聚果糖，其特征在于，当动物给药时，高蔗果三糖含量低聚果糖每天给药量为4.5-28.6g/kg。The high-kestose-content fructo-oligosaccharide according to claim 1, characterized in that, when the animal is administered, the daily dosage of the high-kestose-content fructo-oligosaccharide is 4.5-28.6 g/kg.
- 如权利要求1所述的高蔗果三糖含量低聚果糖在调节肠道菌群中的应用。The application of the fructooligosaccharide with high kestose content as claimed in claim 1 in regulating intestinal flora.
- 如权利要求3所述的应用，其特征在于，所述肠道菌群包括嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)、双歧杆菌(Bifidobacterium spp.)和乳酸杆菌(Lactobacillus spp.)中的一种或多种。application as claimed in claim 3, is characterized in that, described intestinal flora comprises mucinophilic Akkermansia (Akkermansia muciniphila), Bifidobacterium (Bifidobacterium spp.) and Lactobacillus (Lactobacillus spp.) one or more of.
- 如权利要求3所述的应用，其特征在于，所述调节肠道菌群为增加啮齿类动物粪便及盲肠内容物的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)、双歧杆菌(Bifidobacterium spp.)、乳酸杆菌(Lactobacillus spp.)及其代谢产物短链脂肪酸。The application according to claim 3, wherein the regulation of intestinal flora is Akkermansia muciniphila (Akkermansia muciniphila), Bifidobacterium spp. .), Lactobacillus (Lactobacillus spp.) and its metabolites short-chain fatty acids.
- 如权利要求3所述的应用，其特征在于，所述调节肠道菌群为通过增加大鼠粪便含水量从而润肠通便。The application according to claim 3, characterized in that the regulation of intestinal flora is by increasing the water content of rat feces so as to laxative the intestines.
- 如权利要求1所述的高蔗果三糖含量低聚果糖在调节免疫功能中的应用。The application of the fructooligosaccharide with high kestose content as claimed in claim 1 in regulating immune function.
- 如权利要求7所述的应用，其特征在于，所述调节免疫功能包括增强人外周血淋巴细胞及其传代细胞γ-干扰素分泌和抑制EHEC诱导的肠上皮细胞的炎症响应。The application according to claim 7, characterized in that the regulation of immune function includes enhancing the secretion of γ-interferon from human peripheral blood lymphocytes and their passage cells and inhibiting the inflammatory response of intestinal epithelial cells induced by EHEC.
- 如权利要求1所述的高蔗果三糖含量低聚果糖在制备治疗和/或预防肥胖症、2型糖尿病、克罗恩病、特应性皮炎或呼吸系统感染药物中的应用。Application of the fructooligosaccharide with high kestose content as claimed in claim 1 in the preparation of medicines for treating and/or preventing obesity, type 2 diabetes, Crohn's disease, atopic dermatitis or respiratory system infection.
- 一种复合饲料，其特征在于，包括以下重量百分比的原料：70％低纤维饲料AIN-93M和30％如权利要求1所述的高蔗果三糖含量低聚果糖。A compound feed is characterized in that it comprises the following raw materials in percentage by weight: 70% low-fiber feed AIN-93M and 30% fructooligosaccharides with high kestose content as claimed in claim 1.
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