Composition for promoting proliferation of animal bifidobacterium subsp. Lactis in intestinal tract and application thereof

Abstract

The invention discloses a composition for promoting animal bifidobacterium subsp. Lactis to proliferate in intestinal tracts and application thereof, and belongs to the technical field of probiotics, the composition comprises a prebiotic composition and animal bifidobacterium subsp. Lactis, wherein the prebiotic composition is prepared from at least three of mannose oligomer, cotton seed oligosaccharide, isomaltooligosaccharide and galactooligosaccharide; the number of viable bacteria of the bifidobacterium animalis subsp. Lactis added in each gram of the prebiotic composition is 106-1012. According to the invention, at least three of the mannan-oligosaccharide, the cotton seed oligosaccharide, the isomaltooligosacharide and the galactooligosaccharide are compounded, so that the probiotic effect of the probiotics is coordinated and enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and in particular relates to a prebiotic composition for promoting the proliferation of animal Bifidobacterium lactis subspecies in the intestinal tract and an application thereof. Background Art

[0002] Bifidobacterium animalis subsp. lactis Bifidobacterium animalum subsp. milk ) dates back to 1997, when it was described as a new species Bifidobacterium lactis , which was not identified as a new subspecies until 2004. B. animal ssp. milk They are often isolated from fermented dairy products and are currently one of the most widely used probiotic species. Strains of this subspecies generally have high genetic and physiological similarities and can grow on a variety of carbon sources in an anaerobic environment.

[0003] Bifidobacterium animalis subsp. lactis has the effects of regulating intestinal microbial flora, improving intestinal function, reducing diarrhea, inhibiting pathogens, enhancing intestinal barrier, regulating host immunity, etc. These effects involve adhesion and colonization, antibacterial production, nutritional competition, production of short-chain fatty acids, and communication with the immune system. Obviously, these mechanisms require sufficient number and activity of probiotics to drive. It is worth noting that Bifidobacterium animalis is not an indigenous species in the human intestinal flora. As an oral probiotic of foreign species, its probiotic effect in the colon is limited by competition from the indigenous intestinal flora. Develop prebiotics that are synergistically compounded with Bifidobacterium animalis subsp. lactis to help the target probiotics gain a nutritional competitive advantage over the indigenous flora in the colon, which is more conducive to their health effects.

[0004] Prebiotics benefit the host by improving the survival and colonization of live microbial dietary supplements in the gastrointestinal tract, or by selectively stimulating the growth and / or activating the metabolism of one or a few health-promoting bacteria. Synergistic prebiotics provide nutrients to paired probiotics, thereby enhancing the growth or activity of these microorganisms, and this selective utilization produces specific health benefits that may exceed the effects achieved by using probiotics or prebiotics alone.

[0005] At present, CN118044616A discloses a synbiotic and its use. The prebiotic combination of the invention is mainly screened from the prebiotic spectrum: oligosaccharides of isomaltooligosaccharides, oligoxyloses, soybean oligosaccharides, oligogalactoses, polydextrose, pullulan, lactulose, oligofructose, inulin and resistant starch, and does not cover more matching prebiotic ingredients. In addition, the prebiotic combination is mainly targeted at Bifidobacterium bifidum.

[0006] CN117751982A discloses a nutritional composition for promoting the colonization of bifidobacteria and its application. The prebiotic combination of the invention is mainly screened from the prebiotic spectrum: polydextrose, galacto-oligosaccharide, and fructo-oligosaccharide, and does not cover more matching prebiotic ingredients.

[0007] CN116491655A discloses the use of a probiotic-prebiotic composition in the preparation of a medicine or food for improving intestinal probiotic colonization. The invention mainly examines the health effects of a synbiotic composed of human milk oligosaccharides and bifidobacteria, and does not cover more matching prebiotic ingredients. Summary of the invention

[0008] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0009] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0010] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a composition for promoting the proliferation of animal Bifidobacterium lactis subsp.

[0011] In order to solve the above technical problems, the present invention provides the following technical solutions: a composition for promoting the proliferation of animal Bifidobacterium lactis subspecies in the intestine, comprising a prebiotic composition and animal Bifidobacterium lactis subspecies; wherein the prebiotic composition comprises at least three of oligomannosaccharides, raffinose oligosaccharides, isomaltooligosaccharides and oligogalactoses; The number of live bacteria of animal Bifidobacterium lactis subspecies added per gram of the prebiotic composition is 10 6 -10 12 indivual.

[0012] As a preferred embodiment of the composition of the present invention, the prebiotic composition comprises mannooligosaccharides, raffinose oligosaccharides and isomaltooligosaccharides, wherein, by weight, the mannooligosaccharides are 0.01-50 parts, the raffinose oligosaccharides are 0.01-50 parts, and the isomaltooligosaccharides are 0.01-50 parts. Preferably, the mannooligosaccharides are 16.7-50 parts, the raffinose oligosaccharides are 16.7-50 parts, and the isomaltooligosaccharides are 16.7-50 parts. Further preferably, the mannooligosaccharides are 16.7-33.3 parts, the raffinose oligosaccharides are 16.7-33.3 parts, and the isomaltooligosaccharides are 16.7-33.3 parts.

[0013] As a preferred embodiment of the composition of the present invention, the prebiotic composition comprises mannooligosaccharides, raffinose oligosaccharides and galactooligosaccharides, wherein, by weight, the mannooligosaccharides are 0.01-50 parts, the raffinose oligosaccharides are 0.01-50 parts, and the galactooligosaccharides are 0.01-50 parts; preferably, the mannooligosaccharides are 16.7-50 parts, the raffinose oligosaccharides are 16.7-50 parts, and the galactooligosaccharides are 16.7-50 parts; further preferably, the mannooligosaccharides are 16.7-33.3 parts, the raffinose oligosaccharides are 16.7-33.3 parts, and the galactooligosaccharides are 16.7-33.3 parts.

[0014] As a preferred embodiment of the composition of the present invention, the prebiotic composition comprises cotton oligosaccharides, isomaltooligosaccharides and galacto-oligosaccharides, and in parts by weight, the cotton oligosaccharides are 0.01-50 parts, the isomaltooligosaccharides are 0.01-50 parts, and the galacto-oligosaccharides are 0.01-50 parts; preferably, the cotton oligosaccharides are 16.7-50 parts, the isomaltooligosaccharides are 16.7-50 parts, and the galacto-oligosaccharides are 16.7-50 parts; further preferably, the cotton oligosaccharides are 16.7-33.3 parts, the isomaltooligosaccharides are 16.7-33.3 parts, and the galacto-oligosaccharides are 16.7-33.3 parts.

[0015] As a preferred embodiment of the composition of the present invention, the prebiotic composition comprises oligomannosaccharides, cotton oligosaccharides, isomalto-oligosaccharides and galacto-oligosaccharides, wherein, in parts by weight, the oligomannosaccharides are 0.01-50 parts, the cotton oligosaccharides are 0.01-50 parts, the isomalto-oligosaccharides are 0.01-50 parts, and the galacto-oligosaccharides are 0.01-50 parts; preferably, the oligomannosaccharides are The sugar is 16.7-50 parts, the cottonseed oligosaccharide is 16.7-50 parts, the isomaltooligosaccharide is 16.7-50 parts, and the galacto-oligosaccharide is 16.750 parts; further preferably, the manno-oligosaccharide is 16.7-33.3 parts, the cottonseed oligosaccharide is 16.7-33.3 parts, the isomaltooligosaccharide is 16.7-33.3 parts, and the galacto-oligosaccharide is 16.7-33.3 parts.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide a composition for use in preparing food, health food, and medicine.

[0017] As a preferred embodiment of the application of the present invention, the dosage forms of the food, health food and medicine include soft candy, beverage, powder, tablet, hard capsule and soft capsule.

[0018] Beneficial effects of the present invention: (1) The present invention provides a prebiotic that can efficiently proliferate animal Bifidobacterium lactis subsp. in the intestine, and synergistically enhances the probiotic effect of probiotics by compounding manno-oligosaccharides, raffinose oligosaccharides, isomalto-oligosaccharides and galacto-oligosaccharides.

[0019] (2) The present invention synergistically improves the proliferation of animal Bifidobacterium lactis subsp. lactis in the intestine by compounding mannooligosaccharides, raffinose oligosaccharides, isomaltooligosaccharides and galactooligosaccharides within a suitable ratio; the prebiotic composition can help animal Bifidobacterium lactis subsp. lactis proliferate in the intestine and exert more health effects. DETAILED DESCRIPTION

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0023] Example 1 High-throughput screening of high-efficiency biosimilars for efficient proliferation of BB-12 and HN019: (1) Culture system: MRS carbon-free medium (peptone 10 g / L, beef extract 8 g / L, yeast extract 4 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.2 g / L and Tween 80 0.1% v / v) + prebiotics (0.8%, w / v ), 200 μL volume was placed in a 96-well transparent flat-bottom ELISA plate, and three replicate wells were made for each prebiotic.

[0024] (2) Test strain: Bifidobacterium animalis subsp. lactis BB-12 ( Bifidobacterium animal subsp. milk BB-12, from Novonesis); Bifidobacterium animalis subsp. lactis HN019 ( Bifidobacterium animalum subsp. milkHN019, from Fonterra) (3) Prebiotics to be tested: Cellobiose (Hebei Qiansheng), chitosan oligosaccharides (Shandong Hailongyuan), 2'-fucosyllactose (DSM-Firmenich), fructooligosaccharides (Baolingbao), galactooligosaccharides (Quantum High-tech), isomaltooligosaccharides (Baolingbao), isomerized lactose (Henan Longteng), lactose-N-neotetraose (DSM-Firmenich), mannooligosaccharides (Shandong Zhengsheng), cottonseed oligosaccharides (Inner Mongolia Changhui), stachyose (China Food Fermentation Industry Research Institute), xylo-oligosaccharides (Henan Yichangqing), arabinogalactan (Hebei Hongtao), arabinoxylan (Xi'an Lavia), galactomannan (Beijing Guarrun), glucomannan (Lanzhou Waters), inulin (BENEO), oat β-glucan (Guangzhou Zhongkang), polydextrose (Baolingbao), resistant dextrin (Baolingbao), xylan (Hebei Hongtao), yeast β-glucan (Angel Yeast); Glucose and no carbon source groups were set as controls.

[0025] (4) Culture conditions: Obtain monoclonal colonies of BB-12 and HN019 by streaking on agar plates, and inoculate the single colonies into fresh MRS liquid medium (containing 2% glucose) as seed culture (37°C, anaerobic). After the seed culture reaches the mid-logarithmic phase (approximately 24-32 hours), inoculate 0.5% of the inoculum into a culture medium with prebiotics as the sole carbon source for culture (37°C, anaerobic).

[0026] (5) Detection indicators: Use Tecan high-throughput growth curve tester to measure OD600 from 0 to 24 h. Set up a 9-point detection matrix in a square in each well, and take the average value as the OD reading of the well. The final result is the average of three replicate wells.

[0027] Pure culture experiments were conducted to culture animal Bifidobacterium lactis subsp. lactis strains BB-12 and HN019 using different prebiotics as the sole carbon source, and the results shown in Table 1 were obtained.

[0028] Table 1 Effects of different prebiotics on proliferation of BB-12 and HN019

[0029] Judging from the increase in OD600, BB-12 and HN019 have similar abilities to utilize prebiotics. Among them, the prebiotics with the strongest proliferation ability are galacto-oligosaccharides, isomaltooligosaccharides, mannan-oligosaccharides and cottonseed oligosaccharides. These prebiotics with better proliferation effects can be used as the basis for prebiotic compositions.

[0030] Example 2 In vitro biomimetic intestinal flora fermentation system to screen prebiotic combinations: (1) Bionic system: fecal flora + MRS carbon-free medium + prebiotic combination (0.8%, w / v ) + Live bacteria of Bifidobacterium animalis subsp. lactis (5×10 5 cells per gram of feces) in a 5 mL volume in an anaerobic vial.

[0031] Fecal flora processing method: collect fecal samples from 10 healthy people, weigh 0.8±0.02 g of fecal samples and put them into a centrifuge tube. After vortex mixing, transfer the suspension to a fecal filter to filter out food residues and other particles to obtain fecal flora inoculum, add 5×10 5 Live bacteria of animal Bifidobacterium lactis subsp. lactis (calculated by a hemacytometer) were collected by using a sterile 1 mL syringe with a No. 5 needle to draw 0.5 mL of fecal inoculum through the butyl rubber stopper of the vial and injected into the culture medium for the sample to be fermented.

[0032] The principle of combination is to select at least three prebiotics with the best proliferation effect and combine them, with the maximum content of each ingredient being 50%.

[0033] (2) Fermentation cycle: 0-24 hours, anaerobic culture at 37 °C.

[0034] (3) Detection index: Bifidobacterium animalis subsp. lactis quantitative PCR.

[0035] Quantitative method: Collect the bacteria before and after culture in each group, and extract DNA; The bacterial suspension of Bifidobacterium animalis subsp. bifidum was cultured, the cell number was counted using a hemocytometer, and DNA was extracted as a standard for quantitative PCR.

[0036] Primers (F: 5'-GGC ATC GAT CTG GTT GAA GT-3'; R: 5'-ACT GCA GCA ATCTCC AAG GT-3') were used for quantitative PCR, and the number of animal Bifidobacterium lactis subsp. lactis under different conditions was calculated based on the CT values ​​of the standard and the sample to be tested.

[0037] The intestinal flora bionic culture system contains intestinal flora derived from feces of healthy people, live bacteria of animal Bifidobacterium lactis subsp. as probiotics, and prebiotic compositions of different components and proportions. The system simulates the process of probiotics and prebiotics entering the colon after taking them. The number (cells / g) of animal Bifidobacterium lactis subsp. in the simulated colon contents is obtained by fluorescent quantitative PCR specific to animal Bifidobacterium lactis subsp. The data shown in Table 2 are the average values ​​from bionic cultures of 10 individual flora.

[0038] Table 2 Effect of prebiotic composition on proliferation of BB-12 in intestinal bacteria bionic system

[0039] Before culture, the number of Bifidobacterium animalis subsp. lactis was 6.71E+05 cells / g, which included the number of live bacteria added as probiotics.

[0040] After the prebiotic compositions of the present invention are cultured in a colon bionic system with different combinations, the number of animal Bifidobacterium lactis subspecies of the preferred prebiotic composition can reach 3.46E+07, and its proliferation ability on animal Bifidobacterium lactis subspecies in the colon is 2.28 times that of the commonly used prebiotics galacto-oligosaccharides, 1.45 times that of isomaltooligosaccharides, 3.17 times that of xylo-oligosaccharides, 2.15 times that of fructo-oligosaccharides, 4.11 times that of inulin, 17.74 times that of resistant dextrin, and 95.8 times that of polydextrose.

[0041] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A composition for promoting the proliferation of animal Bifidobacterium lactis subsp. lactis in the intestinal tract, characterized in that: Comprising a prebiotic composition and Bifidobacterium animalis subsp. lactis; wherein, The prebiotic composition comprises at least three of manno-oligosaccharides, raffinose oligosaccharides, isomaltooligosaccharides and galacto-oligosaccharides; The number of live bacteria of animal Bifidobacterium lactis subspecies added per gram of the prebiotic composition is 10 6 -10 12 indivual.

2. The composition according to claim 1, characterized in that: The prebiotic composition comprises mannooligosaccharide, raffinose oligosaccharide and isomaltooligosaccharide, wherein, by weight, the mannooligosaccharide is 0.01-50 parts, the raffinose oligosaccharide is 0.01-50 parts, and the isomaltooligosaccharide is 0.01-50 parts.

3. The composition according to claim 1, characterized in that: The prebiotic composition comprises mannooligosaccharide, raffinose oligosaccharide and galactooligosaccharide, wherein, by weight, the mannooligosaccharide is 0.01-50 parts, the raffinose oligosaccharide is 0.01-50 parts, and the galactooligosaccharide is 0.01-50 parts.

4. The composition according to claim 1, characterized in that: The prebiotic composition comprises cottonseed oligosaccharide, isomaltooligosaccharide and galacto-oligosaccharide, wherein, by weight, the cottonseed oligosaccharide is 0.01-50 parts, the isomaltooligosaccharide is 0.01-50 parts, and the galacto-oligosaccharide is 0.01-50 parts.

5. The composition according to claim 1, characterized in that: The prebiotic composition comprises mannooligosaccharide, raffinose oligosaccharide, isomaltooligosaccharide and galactooligosaccharide, wherein, by weight, the mannooligosaccharide is 0.01-50 parts, the raffinose oligosaccharide is 0.01-50 parts, the isomaltooligosaccharide is 0.01-50 parts, and the galactooligosaccharide is 0.01-50 parts.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of food, health food, and medicine.

7. The use according to claim 6, characterized in that: The dosage forms of the food, health food and medicine include soft candy, beverage, powder, tablet, hard capsule and soft capsule.

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