A prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats and a preparation method thereof

Abstract

The invention provides a prebiotic composition for promoting the proliferation of intestinal probiotics of dogs and cats and a preparation method thereof. The prebiotic composition comprises a prebiotic combination, a prebiotic reinforcing factor combination, and probiotics lactobacillus and bifidobacterium; the prebiotic combination comprises one or two or more of oligofructose, oligogalactose or oligomannose; the prebiotic reinforcing factor combination comprises one or two of docosahexaenoic acid (DHA) and phytosterols. The prebiotic composition for promoting the proliferation of intestinal probiotics of dogs and cats and a preparation method thereof can significantly enhance the proliferation and metabolism of Lactobacillus acidophilus and Bifidobacterium animalis, and the prebiotics and probiotics are embedded by embedding technology to prevent the probiotics from losing activity in gastric juice.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of prebiotic compositions, and in particular relates to a prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats and a preparation method thereof. Background Art

[0002] Dogs and cats are the most common animal companions in human life. With the formation of scientific pet-raising concepts, pet nutrition and health have attracted more and more attention. There are a huge number of microorganisms in the intestines of dogs and cats, including bacteria, fungi, viruses and protozoa, which constitute a unique microbial ecosystem in the intestines of dogs and cats. Intestinal flora plays an important role in both the physiological and mental health of animals. Changes in flora will lead to changes in the content of metabolites such as bile acid and short-chain fatty acids (SCFA) in the intestine, which in turn affects the health of the body.

[0003] Prebiotics are a new type of feed additive that regulates the composition or vitality of intestinal microorganisms through the metabolism of intestinal microorganisms. Currently, substances that can be used as prebiotics include: functional oligosaccharides, polysaccharides, polyols, protein hydrolysates, plant extracts, etc. Among them, functional oligosaccharides are the most important and most studied type of prebiotics. Functional oligosaccharides include fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), mannose oligosaccharides (MOS), xylooligosaccharides (XOS), isomaltooligosaccharides (IMO) and stachyose. Adding prebiotics to pet food can improve intestinal microecology, promote the absorption of minerals, regulate ester metabolism, and enhance immunity. The ingested prebiotics are used by beneficial intestinal bacteria and converted into their monomer form or generate short-chain fatty acids through degradation or fermentation. Prebiotic monomers can effectively form competitive inhibition on intestinal microorganisms and reduce the adhesion of harmful pathogens; at the same time, they can be degraded and utilized by probiotics, thereby promoting their growth and reproduction, forming a microecological competitive advantage. Short-chain fatty acids can lower the pH of the intestinal environment and inhibit the reproduction of harmful bacteria; provide energy for the intestinal epithelium, promote the proliferation of intestinal epithelial cells, the proliferation of crypts, and improve the absorption of nutrients; finally, interact with immune cytokines, regulate the differentiation of immune cells, and ensure the normal exercise of the body's immune function.

[0004] Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid that can be metabolized by α-linolenic acid in animals, but the amount produced is low and is mainly supplemented through food. DHA can regulate the abundance of bifidobacteria in the intestines, inhibit the growth of pathogenic microorganisms, and has antioxidant and immunomodulatory properties. In addition, phytosterols have been shown to have a good anti-tumor effect. They can promote the direct excretion of cholesterol from the body, reduce the decomposition of cholesterol by microorganisms and produce metabolites that are easy to induce colon cancer and inflammation. Phytosterols can also promote the growth of intestinal probiotics and inhibit the reproduction of bacteria and fungi. DHA is docosahexaenoic acid, and its olefinic bond, i.e., carbon-carbon double bond chemical structure, is very unstable and easily oxidized, resulting in low storability of fish oil. Air, light, and metal ions in the processing process may all lead to its oxidative decomposition, reducing its efficacy.

[0005] As three highly effective prebiotics, the application research of FOS, GOS and MOS in animal feed mainly focuses on pigs, chickens and fish, and there are few reports on their application in other animals such as dogs and cats. In addition, the promotion effect of a single prebiotic on probiotics is not broad-spectrum. Therefore, scientifically combining prebiotics with probiotics in a synergistic form can promote the growth of multiple bacterial communities and drive the overall improvement of the intestinal flora structure. This is also one of the key research and development directions for the application of prebiotics in pet food in the future. Prebiotics provide nutrition for probiotics, selectively stimulate their growth, activate metabolism, and give probiotics a competitive advantage in the intestine, thereby beneficially affecting the host. Summary of the invention

[0006] In view of this, the present invention aims to propose a prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats and a preparation method thereof. The composition can significantly enhance the proliferation and metabolism of Lactobacillus acidophilus and Bifidobacterium animalis, and utilizes encapsulation technology to encapsulate prebiotics and probiotics to prevent the probiotics from losing their activity in gastric juice.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] A prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats, comprising a prebiotic combination, a prebiotic strengthening factor combination, and probiotics lactobacillus and bifidobacterium;

[0009] Preferably, the prebiotic combination includes one or two or more of fructo-oligosaccharides, galacto-oligosaccharides or manno-oligosaccharides;

[0010] Preferably, the prebiotic strengthening factor combination includes one or both of docosahexaenoic acid (DHA) and phytosterols.

[0011] The mass ratio of fructooligosaccharide, galacto-oligosaccharide and manno-oligosaccharide is (1-3):(3-1):(0.1-0.3).

[0012] Preferably, the mass ratio is 1:3:0.1, 1:3:0.2, 1:3:0.3, 2:2:0.1, 2:2:0.2, 2:2:0.3, 3:1:0.1, 3:1:0.2, 3:1:0.3. In a culture medium with a mass ratio of fructo-oligosaccharide, galacto-oligosaccharide and manno-oligosaccharide of (1:3:0.1) to (3:1:0.3), the OD of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 is 600 The values ​​were higher than the OD values ​​in the medium containing oligofructose, oligogalactose and oligomannose alone. 600 At the same time, the pH values ​​of the fermentation broths of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 were lower than the pH values ​​of the fermentation broths in the culture medium containing oligofructose, oligogalactose or oligomannose alone.

[0013] Preferably, in the above prebiotic composition, the mass ratio of fructo-oligosaccharide, galacto-oligosaccharide and oligomannosaccharide is 1:3:0.3, 2:2:0.1, 2:2:0.2, 2:2:0.3, 3:1:0.1, 3:1:0.2. In the culture medium with the mass ratio of fructo-oligosaccharide, galacto-oligosaccharide and oligomannosaccharide being 1:3:0.3, 2:2:0.1, 2:2:0.2, 2:2:0.3, 3:1:0.1, 3:1:0.2, the OD of animal Bifidobacterium BNCC185341 growth is 600 The values ​​were significantly higher than those in the medium containing FOS, GOS or MOS alone. 600 Meanwhile, the pH value of the fermentation broth of Bifidobacterium animalis BNCC185341 was significantly lower than that of the fermentation broth in the medium containing FOS, GOS or MOS alone.

[0014] Furthermore, the above prebiotic composition also contains DHA and phytosterols. The addition of DHA promotes the antioxidant activity of lactobacillus and bifidobacterium. Phytosterols can promote the proliferation of probiotics in the intestines of animals and have antibacterial effects.

[0015] Furthermore, the prebiotic composition also contains Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341.

[0016] Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 can adjust the balance of intestinal flora, inhibit the proliferation of harmful intestinal microorganisms by secreting bacteriocins such as lactic acid and acetic acid, and can also release substances that are beneficial to the growth of probiotics to increase the number of probiotics and enhance their activity. The combination of the two can quickly restore the normal balance of intestinal flora, inhibit the proliferation of pathogenic bacteria, improve the utilization rate of calcium, phosphorus and iron, promote the absorption of iron and vitamin D, produce vitamin K and vitamin B, and reduce the absorption of cholesterol, which has a good nutritional and health care effect.

[0017] The present invention microencapsulates prebiotics and probiotics, and comprises the following steps:

[0018] (1) weighing γ-cyclodextrin and dissolving it in water at a certain temperature to prepare a saturated aqueous solution of γ-cyclodextrin;

[0019] (2) dissolving a certain amount of DHA and phytosterols in anhydrous ethanol to prepare a DHA-phytosterol anhydrous ethanol solution;

[0020] (3) adding DHA and phytosterol anhydrous ethanol solution dropwise into a saturated aqueous solution of γ-cyclodextrin at 35-45° C., stirring with a magnetic stirrer at 400-600 rpm for 30-40 min, to obtain an inclusion complex of γ-cyclodextrin-encapsulated DHA and phytosterol;

[0021] (4) Weigh FOS, GOS and MOS, and dissolve them in distilled water to obtain a prebiotic solution;

[0022] (5) dissolving sodium carboxymethylcellulose and lactoferrin solids in distilled water;

[0023] (6) adding the inclusion compound prepared in step (3) and the prebiotic solution prepared in step (4) to the wall material solution prepared in step (5);

[0024] (7) adding bacterial solutions of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 to the mixture obtained in step (6), and stirring at 20-25° C. and 300-500 rpm for 20-30 min;

[0025] (8) mixing the CaCl2 aqueous solution and the gellan gum aqueous solution to obtain a curing agent, mixing the mixture obtained in step (7) with the curing agent, and allowing to stand to obtain a precipitate;

[0026] (9) freeze-drying the precipitate obtained in step (8) to obtain a microcapsule product that double-encapsulates the prebiotic strengthening factor, prebiotics, and probiotics.

[0027] Preferably, the temperature of the water in step (1) is 35-45°C.

[0028] Preferably, the mass ratio of DHA, phytosterols and anhydrous ethanol in step (2) is (1-3):(2-5):(30-200).

[0029] Preferably, the mass ratio of FOS, GOS, MOS and distilled water in step (4) is (1-3):(3-1):(0.1-0.3):(50-300).

[0030] Preferably, the mass ratio of sodium carboxymethyl cellulose, lactoferrin and distilled water in step (5) is (1-2):(2-4):(30-100).

[0031] Preferably, the mass ratio of the inclusion compound, the prebiotic solution and the wall material solution mixed in step (6) is 1:(2-5):(40-100).

[0032] Preferably, the final concentrations of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 in the mixed solution in step (7) are 1×10 7 ~10 9 CFU / mL, 2×10 7 ~10 11 CFU / mL.

[0033] Preferably, the mass concentration of CaCl2 in step (8) is 1-3%, the mass ratio of CaCl2 aqueous solution to gellan gum aqueous solution is (1-6):1, the mass ratio of the mixture to the curing agent is 1:(2-5), and the mixture is allowed to stand for 30-60 minutes.

[0034] Preferably, the conditions in step (9) are freeze drying at -60 to -80°C for 12 to 24 hours.

[0035] The present invention innovatively proposes: (1) embedding hydrophobic DHA and phytosterols as prebiotic strengthening factors into the nano-scale hydrophobic inner cavity cage of γ-cyclodextrin, which reduces the contact of DHA with the external environment and causes oxidative degradation, and is conducive to the slow release of DHA and phytosterols. The hydrophilic hydroxyl groups on the outside of the γ-cyclodextrin molecule are used to hang the hydrophilic prebiotic composition FOS, GOS, and MOS on the outside of the γ-cyclodextrin molecule. At the same time, the DHA carboxyl group in the inner cavity of γ-cyclodextrin can form an intermolecular force with the hydroxyl group in the prebiotic composition, forming a "hand-in-hand" chain structure distributed on the inside and outside of γ-cyclodextrin. (2) The strong gel properties of sodium carboxymethyl cellulose and lactoferrin are used to coat the outside of the prebiotic composition, forming a double composite microcapsule with a double-layer structure coating the prebiotic strengthening factor and a single-layer structure coating the prebiotic composition. Prebiotics fortifying factors need to cross two layers of packaging wall to act on probiotics, and the sustained release effect is better, while prebiotics combination needs to cross one layer of packaging wall, but due to the "hand-in-hand" effect with prebiotics fortifying factors, it still has a good sustained release effect on probiotics. It can achieve slow release of prebiotics and their fortifying factors, continuously act on probiotics, effectively improve the gastrointestinal resistance of probiotics, and efficiently increase the proliferation of probiotics in the intestine.

[0036] Compared with the prior art, the prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats and the preparation method thereof described in the present invention have the following beneficial effects:

[0037] 1. A method for preparing a prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats. Microbial polymers and animal protein substances can be used as wall materials for gastrointestinal probiotic microcapsules. Gellan gum is a high-molecular linear anionic polysaccharide obtained by fermentation of Pseudomonas eloidi. It is non-toxic, biodegradable, has good film-forming properties, and has excellent gelling properties, heat stability and acid resistance.

[0038] 2. Sodium carboxymethyl cellulose is an anionic water-soluble polysaccharide obtained by carboxylmethylation of cellulose, the most abundant cellulose in nature. It has the advantages of good biocompatibility, biodegradability, water solubility and low price. Adding sodium carboxymethyl cellulose to hydrogel can stabilize insoluble components. Sodium carboxymethyl cellulose contains a large number of active groups such as carboxyl and hydroxyl groups, which have high reactivity and can complex with metal ions to improve the mechanical strength of hydrogel.

[0039] 3. Lactoferrin as a probiotic embedding wall material can alleviate the effect of gastric acid on the activity of probiotics. Through the cross-linking of enzymes, acids and calcium ions, lactoferrin can form a dense gel at room temperature, and the reaction conditions are mild, which is conducive to the embedding of bioactive ingredients that are easily inactivated. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0041] Figure 1 : These are the growth curves of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 in different carbon source culture media (A is Lactobacillus acidophilus BNCC185342, and B is Bifidobacterium animalis BNCC185341);

[0042] Figure 2 is the pH value of the fermentation broth of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 in different carbon source culture media in the present invention (A is Lactobacillus acidophilus BNCC185342, and B is Bifidobacterium animalis BNCC185341);

[0043] Figure 3 is the OD value of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 grown in the composite prebiotic medium. 600 value;

[0044] Figure 4 It is the pH value of the fermentation liquid of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 in the composite prebiotic culture medium of the present invention. DETAILED DESCRIPTION

[0045] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0046] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0047] Fructooligosaccharide was purchased from Shanghai Juming Biotechnology Co., Ltd., galacto-oligosaccharide was purchased from Henan Jimei Chemical Products Co., Ltd., and manno-oligosaccharide was purchased from Hangzhou Qiancheng Biotechnology Co., Ltd. Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 were purchased from Beina Biotechnology Research Center. Docosahexaenoic acid was purchased from Zhengzhou Yuhe Food Additive Co., Ltd., and phytosterols were purchased from Shaanxi Fuyubang Biotechnology Co., Ltd.

[0048] Example 1

[0049] Sugar-free MRS medium was selected to study the growth of bacteria under different sugars as the sole carbon source. Different carbon sources were added to the medium at a final concentration of 2% (w / v), and the medium was sterilized at 121°C for 20 min, with glucose added as a positive control. Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 were activated in MRS broth medium, anaerobically cultured at 37°C for 24 h, and seed liquid was obtained. The seed liquid was inoculated in MRS broth medium containing different carbon sources at an inoculum size of 5%, and cultured in an anaerobic incubator at 37°C for 48 h. At 0, 2, 4, 8, 12, 16, 24, 36, and 48 h of fermentation, 200 μL of the fermentation medium was taken out and transferred to a 96-well plate, and the fermentation medium of each strain was recorded at 600nm (OD 600 ) to construct a growth curve and monitor the pH value of the fermentation broth at the same time.

[0050] Figure 1 The results show that compared with the sugar-free group, the addition of FOS, GOS, and MOS to the culture medium can significantly increase the OD of Lactobacillus acidophilus and Bifidobacterium animalis. 600 The final OD value of Lactobacillus acidophilus in Glu, FOS, GOS and MOS medium was 1.347 μg / mL, which promoted the proliferation of strains. However, different strains had different utilization efficiencies of oligosaccharides. 600 The values ​​were 0.9752, 0.9888, 0.9640 and 0.8746, respectively. The influence effect was FOS>Glu>GOS>MOS. The final OD of animal Bifidobacterium 600 The values ​​are 0.97053, 0.79747, 0.98727 and 1.18003 respectively, and the impact effect is MOS>GOS>Glu>FOS.

[0051] Figure 2It is the pH value of the fermentation liquid of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 in the culture medium of different carbon sources in the present invention. The results show that for Lactobacillus acidophilus, the pH value of the fermentation liquid of the FOS group decreases the most, the lowest pH value is 4.20, and the pH value changes to the extent of FOS>Glu>GOS>MOS; Since Bifidobacterium animalis can make good use of these oligosaccharides, the reduction in the pH value of the fermentation liquid is significantly higher than that of the glucose group, and the pH value of the fermentation liquid of the MOS group decreases the most, the lowest pH value is 4.25, and the pH value changes to the extent of MOS>GOS>FOS>Glu. After adding FOS, GOS, and MOS to the culture medium, the pH value of the fermentation liquid of Lactobacillus acidophilus and Bifidobacterium animalis can be significantly reduced, indicating that FOS, GOS, and MOS can promote the acid production capacity of Lactobacillus acidophilus and Bifidobacterium animalis.

[0052] Example 2

[0053] Lactobacillus acidophilus, Bifidobacterium animalis, Bacteroides fragilis, Escherichia coli, and Salmonella were activated in MRS broth medium, cultured anaerobically at 37°C for 24 hours, and seed solution was obtained. The seed solution was inoculated in MRS broth medium containing different carbon sources at an inoculum volume of 2% and co-cultured, and cultured in an anaerobic incubator at 37°C for 48 hours. At the end of the fermentation, a drop of bacterial solution was taken out and Gram-stained and counted on a glass slide. Lactobacillus acidophilus and Bifidobacterium animalis are Gram-positive bacteria, which are purple after staining, while Bacteroides fragilis, Escherichia coli, and Salmonella are Gram-negative bacteria, which are red after staining.

[0054] After co-culture of Lactobacillus acidophilus, Bifidobacterium animalis, Bacteroides fragilis, Escherichia coli and Salmonella in different carbon source culture media, Lactobacillus acidophilus and Bifidobacterium animalis were dominant bacteria, indicating that FOS, GOS and MOS had no proliferation-promoting effect on Bacteroides fragilis, Escherichia coli and Salmonella, but could promote the growth of Lactobacillus acidophilus and Bifidobacterium animalis and produce metabolites that inhibit Escherichia coli and Salmonella.

[0055] Example 3

[0056] FOS, GOS, and MOS with a mass ratio of 1:3:0.1, 1:3:0.2, 1:3:0.3, 2:2:0.1, 2:2:0.2, 2:2:0.3, 3:1:0.1, 3:1:0.2, and 3:1:0.3 were added to MRS broth medium, inoculated with Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341, and cultured anaerobically at 37°C for 48 hours. At the end of fermentation, 200 μL of the fermentation medium was taken out and transferred to a 96-well plate, and the OD of each strain was recorded using an ELISA reader. 600 The pH value of the fermentation broth was also measured.

[0057] The experimental results are as follows Figure 3 , Figure 4 As shown in the table, * indicates the difference compared with single use of FOS, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, # indicates the difference compared with single use of GOS, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, + indicates the difference compared with single use of MOS, + indicates P < 0.05, ++ indicates P < 0.01, +++ indicates P < 0.001.

[0058] Figure 3 is the OD value of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 grown in the composite prebiotic medium. 600 Compared with the culture medium containing FOS, GOS or MOS alone, when the mass ratio of FOS, GOS and MOS was in the range of (1-3):(3-1):(0.1-0.3), the OD of Lactobacillus acidophilus and Bifidobacterium animalis increased significantly. 600 The value will increase, indicating that the proliferation ability of the prebiotic combination on the two probiotics is better than that of a single prebiotic.

[0059] The OD of animal Bifidobacterium BNCC185341 grown in the culture medium with the mass ratio of FOS, GOS and MOS of 1:3:0.3, 2:2:0.1, 2:2:0.2, 2:2:0.3, 3:1:0.1 and 3:1:0.2 600 The values ​​were significantly higher than those in the medium containing FOS, GOS or MOS alone. 600 value.

[0060] In the medium with a mass ratio of FOS, GOS and MOS of 1:3:0.3, the OD of Lactobacillus acidophilus BNCC185342 was 600 The values ​​were significantly higher than those in the medium containing FOS, GOS or MOS alone. 600 value.

[0061] Figure 4 It is the pH value of the fermentation broth of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 in the composite prebiotic medium of the present invention. When the mass ratio of FOS, GOS and MOS is in the range of (1-3):(3-1):(0.1-0.3), the pH value of the fermentation broth is lower than the pH value of the fermentation broth in the medium containing FOS, GOS and MOS alone, indicating that the prebiotic composition has a better ability to proliferate and produce acid for the two probiotics than a single prebiotic.

[0062] In the culture medium with a mass ratio of FOS, GOS to MOS of 1:3:0.3, 2:2:0.1, 2:2:0.2, 2:2:0.3, 3:1:0.1, and 3:1:0.2, the pH value of the fermentation broth of Bifidobacterium animalis BNCC185341 was significantly lower than the pH value of the fermentation broth in the culture medium containing FOS, GOS or MOS alone.

[0063] Compared with the culture medium containing FOS, GOS and MOS separately, the pH value of the fermentation broth of Lactobacillus acidophilus BNCC185342 was significantly reduced when the mass ratio of FOS, GOS and MOS was 1:3:0.3.

[0064] The above results indicate that the combination of FOS, GOS and MOS has a certain enhancing effect on the proliferation and acid production of Lactobacillus acidophilus and Bifidobacterium animalis, and FOS:GOS:MOS=(1-3):(3-1):(0.1-0.3) can be used as the optimal ratio range of compound prebiotics.

[0065] Example 4

[0066] This embodiment provides a method for preparing an inclusion compound, comprising the following steps:

[0067] (1) Weigh γ-cyclodextrin and dissolve it in 35° C. water to prepare a saturated aqueous solution of γ-cyclodextrin;

[0068] (2) using 30 times the mass of anhydrous ethanol to dissolve DHA and phytosterols in a mass ratio of 1:2 to prepare a DHA-phytosterol anhydrous ethanol solution;

[0069] (3) DHA and phytosterol anhydrous ethanol solution were added dropwise into a saturated aqueous solution of γ-cyclodextrin, and stirred at 35°C and 800 rpm for 40 min using a magnetic stirrer to obtain an inclusion complex of γ-cyclodextrin-encapsulated DHA and phytosterol.

[0070] Example 5

[0071] This embodiment provides a method for preparing a group of prebiotic compositions, which contains the inclusion compound provided in Example 4 and FOS, GOS, MOS Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341. The method comprises the following steps:

[0072] (1) Weighing FOS, GOS and MOS in a mass ratio of 1:3:0.1, dissolving them in 50 times the mass of distilled water to obtain a prebiotic solution;

[0073] (2) dissolving sodium carboxymethylcellulose and lactoferrin solids in a mass ratio of 1:1 in 15 times the mass of distilled water;

[0074] (3) mixing the inclusion compound provided in Example 4, the prebiotic solution prepared in step (1), and the wall material solution prepared in step (2) in a mass ratio of 1:2:40;

[0075] (4) Adding bacterial solutions of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 to the mixture obtained in step (3) to a final concentration of 1×10 7 CFU / mL, 2×10 7 CFU / mL, stirred at 20°C, 500 rpm for 30 min;

[0076] (5) mixing a 3% CaCl2 aqueous solution and a gellan gum aqueous solution at a mass ratio of 1:1 to obtain a curing agent, and mixing the mixture obtained in step (4) and the curing agent at a mass ratio of 1:2, and letting it stand for 30 minutes to obtain a precipitate;

[0077] (6) freeze-drying the precipitate obtained in step (5) at -60°C for 24 hours to obtain a microcapsule product that double-encapsulates prebiotic strengthening factors, prebiotics and probiotics.

[0078] Determination of microcapsule particle size: dilute the microcapsules a certain multiple, randomly select 10 fields of view under an optical microscope, and measure the particle size of the microcapsules in each field of view.

[0079] Determination of probiotic embedding rate: Take 0.1g microcapsule product and break the wall in 10mL phosphate buffer, take 1mL from it for gradient dilution, then spread on the plate, and count the live bacteria after anaerobic culture at 37℃ for 24h. The embedding rate is calculated according to the following formula (1):

[0080]

[0081] Determination of prebiotic embedding rate: Prepare a mixed solution of 0.1-0.2 mmol / L FOS, GOS, and MOS, and use a UV spectrophotometer to measure the absorbance at 245 nm. Use different concentrations of prebiotics as the horizontal axis and the corresponding absorbance as the vertical axis to draw a standard curve. In an 80°C water bath, add 80% ethanol to 0.1 g of the microcapsule product while stirring, let it stand for 1 hour, centrifuge at 5000 r / min for 10 minutes, dissolve the precipitate in distilled water, measure its absorbance at a wavelength of 245 nm, and substitute it into the standard curve equation to obtain the concentration of prebiotics in the microcapsule. The embedding rate is calculated according to the following formula (2):

[0082]

[0083] The encapsulation rate of microcapsule probiotics is 75%, the encapsulation rate of prebiotics is 80%, and the average particle size is 700μm.

[0084] Example 6

[0085] Compared with Example 5, the difference is that in step (1), the mass ratio of FOS, GOS, MOS and distilled water is 3:1:0.3:300, and the other conditions are the same as those in Example 5.

[0086] The encapsulation rate of microcapsule probiotics is 70%, the encapsulation rate of prebiotics is 75%, and the average particle size is 600μm.

[0087] Example 7

[0088] Compared with Example 5, the difference is that in step (2), the mass ratio of sodium carboxymethyl cellulose, lactoferrin and distilled water is 1:4:100, and other conditions are the same as those in Example 5.

[0089] The encapsulation rate of microcapsule probiotics is 70%, the encapsulation rate of prebiotics is 75%, and the average particle size is 500μm.

[0090] Example 8

[0091] Compared with Example 5, the difference is that in step (3), the mass ratio of the inclusion compound, the prebiotic solution and the wall material solution of Example 4 is 1:5:100, and the other conditions are the same as those of Example 5.

[0092] The encapsulation rate of microcapsule probiotics is 75%, the encapsulation rate of prebiotics is 80%, and the average particle size is 600μm.

[0093] Example 9

[0094] Compared with Example 5, the difference is that in step (5), the mass concentration of CaCl2 is 1%, the mass ratio of CaCl2 aqueous solution to gellan gum aqueous solution is 6:1, the mass ratio of the mixture to the curing agent is 1:5, and the mixture is allowed to stand for 60 minutes. Other conditions are the same as those in Example 5.

[0095] The encapsulation rate of microcapsule probiotics is 75%, the encapsulation rate of prebiotics is 75%, and the average particle size is 700μm.

[0096] Comparative Example 1

[0097] The difference from Example 5 is that γ-cyclodextrin is not added.

[0098] Comparative Example 2

[0099] The difference from Example 5 is that the wall material solution (sodium carboxymethylcellulose and lactoferrin) is not added.

[0100] Comparative Example 3

[0101] The difference from Example 5 is that no curing agent is added.

[0102] Comparative Example 4

[0103] The difference from Example 5 is that the inclusion compound provided in Example 4 is not used.

[0104] Effect experiment:

[0105] 60 mice were randomly divided into 10 groups, and fed with common mouse food and gavaged once a day for 35 days, while the control group was only fed with common mouse food. The mice's condition, body weight, food intake, and feces were observed every week.

[0106] The fecal flora test is performed by aseptically collecting mouse feces and then diluting them in a gradient manner with sterile saline. The supernatant is taken after centrifugation and the different gradients of bacterial liquid are spread on the culture medium. After cultivation, the lactobacilli and bifidobacteria are identified and counted by colony morphology, Gram staining, biochemical reactions, etc., and the number of probiotics in each gram of wet feces is calculated.

[0107] Table 1 Effect of Examples 5-9 and Comparative Examples 1-4 on the number of fecal flora in mice CFU / g

[0108]

[0109] Table 2 Effects of Examples 5-9 and Comparative Examples 1-4 on Mouse Condition

[0110]

[0111]

[0112] Table 3 Effect of Examples 5-9 and Comparative Examples 1-4 on Mouse Weight g

[0113]

[0114] Table 4 Effects of Examples 5-9 and Comparative Examples 1-4 on the Food Intake of Mice g

[0115]

[0116] Table 5 Effects of Examples 5-9 and Comparative Examples 1-4 on the Hardness of Mouse Feces

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[0118]

[0119] The results in Tables 1 to 5 show that the mice were active, had normal weight, normal feces, and good appetite, and the prebiotic compositions of Examples 5 to 9 could effectively increase the content of lactobacillus and bifidobacterium in the intestine, and the effects were better than those of Control Examples 1 to 4. It can be seen that the use of sodium carboxymethylcellulose, lactoferrin, and gellan gum for embedding can slowly release prebiotics and strengthening factors, continuously act on probiotics, and effectively promote the proliferation of lactobacillus and bifidobacteria.

[0120] The products obtained in Examples 5-9 and Comparative Examples 1-4 were mixed with canine simulated gastrointestinal fluid, and the time required for complete release was measured at 38° C., and the release rate was also measured.

[0121]

[0122]

Claims

1. A prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats, characterized in that: It includes a prebiotic combination, a prebiotic strengthening factor combination and probiotics; the probiotics are lactobacillus and bifidobacterium; the lactobacillus is Lactobacillus acidophilus BNCC185342, and the bifidobacterium is Bifidobacterium animalis BNCC185341; The prebiotic combination is fructooligosaccharide, galacto-oligosaccharide and manno-oligosaccharide; The mass ratio of fructooligosaccharide, galacto-oligosaccharide and manno-oligosaccharide is (1-3):(3-1):(0.1-0.3); the prebiotic strengthening factor combination is docosahexaenoic acid and phytosterol; The preparation method of the prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats comprises the following steps: S1: adding DHA and phytosterols to a solvent to prepare a mixed solution; at a certain temperature, dripping the mixed solution dropwise into a saturated aqueous solution of γ-cyclodextrin, and stirring to obtain an inclusion complex of DHA and phytosterols embedded in γ-cyclodextrin; S2: Dissolve fructooligosaccharides, galacto-oligosaccharides and manno-oligosaccharides in distilled water to obtain a prebiotic solution; S3: dissolving sodium carboxymethylcellulose and lactoferrin solids in distilled water to obtain a wall material solution; S4: adding the inclusion compound prepared in step S1 and the prebiotic solution prepared in step S2 to the wall material solution prepared in step S3, then adding bacterial liquid of Lactobacillus acidophilus and Bifidobacterium animalis, stirring at a certain temperature to obtain a mixture; mixing the mixture with a curing agent, standing to obtain a precipitate, and drying the precipitate to obtain a microcapsule product of double-embedded prebiotic reinforcing factors, prebiotics and probiotics; the mass ratio of DHA, phytosterols and anhydrous ethanol in step S1 is (1-3):(2-5):(30-200); the mass ratio of oligofructose, oligogalactose, oligomannosaccharide and distilled water in step S2 is (1-3):(3-1):(0.1-0.3):(50-300), the mass ratio of sodium carboxymethyl cellulose, lactoferrin and distilled water in step S3 is (1-2):(2-4):(30-100), the inclusion compound, prebiotic solution and wall material solution in step S4 are as follows: The mass ratio of the material solution is 1:(2-5):(40-100); The preparation of the curing agent in step S4 includes the following steps: mixing the CaCl2 aqueous solution and the gellan gum aqueous solution to obtain the curing agent.

2. A prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats according to claim 1, characterized in that: The preparation of the saturated aqueous solution of γ-cyclodextrin in step S1 comprises the following steps: weighing γ-cyclodextrin and dissolving it in water at a certain temperature to prepare a saturated aqueous solution of γ-cyclodextrin.

3. A prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats according to claim 2, characterized in that: The preparation of the saturated aqueous solution of γ-cyclodextrin in step S1 comprises the following steps: weighing γ-cyclodextrin and dissolving it in water at a certain temperature, the temperature being 20-30°C.

4. A prebiotic composition for promoting the proliferation of intestinal probiotics in dogs and cats according to claim 1, characterized in that: The step S1 comprises the steps of dripping DHA and phytosterol anhydrous ethanol solution into a saturated aqueous solution of γ-cyclodextrin at 35-45° C., stirring with a magnetic stirrer at 400-600 rpm for 30-40 min, to obtain an inclusion complex of γ-cyclodextrin-embedded DHA and phytosterol; in the step S4, bacterial liquids of Lactobacillus acidophilus BNCC185342 and Bifidobacterium animalis BNCC185341 are added, stirring at 20-25° C. and 300-500 rpm for 20-40 min; the preparation of the curing agent in the step S4 comprises the steps of mixing a CaCl2 aqueous solution and a gellan gum aqueous solution to obtain a curing agent.

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