Microbial compound preparation containing chitosan oligosaccharide and application of microbial compound preparation in oxidation resistance

Abstract

The invention belongs to the technical field of microbial compound preparations, and particularly discloses a microbial compound preparation containing chitosan oligosaccharide and application of the microbial compound preparation in oxidation resistance. The microbial compound preparation containing chitosan oligosaccharide comprises the following raw materials in parts by mass: 1-2 parts of a compound microbial inoculant, 1-2 parts of mesoporous silica and 1-2 parts of chitosan oligosaccharide or active chitosan oligosaccharide, the complex microbial inoculants comprise lactic acid bacteria and bifidobacteria; in the complex microbial inoculant, the effective viable count of lactic acid bacteria is 2-6 * 10 < 9 > / g, and the viable count of bifidobacterium is 1-3 * 10 < 9 > / g. The microbial compound preparation containing chitosan oligosaccharide is applied to the following (1) or (2): (1) preparing a weight-losing product; and (2) preparing an antioxidant product. The weight of naturally aged mice can be effectively reduced, the antioxidant activity of the mice can be improved, the content of short-chain fatty acid in intestinal tracts can be improved, the diversity of intestinal flora is improved, and the content of probiotics is increased.

Description

Technical Field

[0001] The invention relates to the technical field of microbial composite preparations, in particular to a microbial composite preparation containing chitosan oligosaccharide and its application in anti-oxidation. Background Art

[0002] Aging is an inevitable physiological process, an inevitable law of life, and a high-risk factor for some major diseases (such as tumors, diabetes, cardiovascular diseases, and neurodegenerative diseases). Therefore, aging has always been a long-standing research field for scientists.

[0003] As the problem of global population aging becomes increasingly serious and life expectancy increases, various aging diseases have gradually become major diseases that threaten people's lives and health. Therefore, the study of the mechanism of aging and delaying aging has attracted attention from many scholars.

[0004] Chitosan oligosaccharides have attracted wide attention in the field of human health care, such as the treatment and prevention of obesity and age-related dysfunction. Chitosan oligosaccharides are produced by the degradation of macromolecular chitosan by specific tool enzymes, and have the advantages of good stability, high uniformity, and easy absorption and utilization. Previous studies have found that chitosan oligosaccharides can change the composition and function of the intestinal flora of mice and increase the content of short-chain fatty acids. However, chitosan oligosaccharides contain only a single ingredient and have a relatively simple effect, which also has certain limitations in practical applications.

[0005] Based on previous research work, this application uses in vitro fermentation models and mouse models to study the effects of chitosan oligosaccharides and microbial preparations on intestinal health homeostasis and metabolic pathways in the elderly, revealing its mechanism of action for anti-glycosylation, which has important scientific significance and research value for the intervention of obesity and senile metabolic dysfunction. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a microbial composite preparation containing chitosan oligosaccharide and its application in anti-oxidation.

[0007] A microbial composite preparation containing chitosan oligosaccharide, wherein the raw materials include, by mass, 1-2 parts of a composite bacterial agent, 1-2 parts of mesoporous silica, and 1-2 parts of chitosan oligosaccharide or active chitosan oligosaccharide; the composite bacterial agent includes lactic acid bacteria (Lactobacillus) and Bifidobacterium (Bifidobacterium); in the composite bacterial agent, the effective live bacterial count of lactic acid bacteria is 2-6×10 9 / g, and the number of viable bifidobacteria is 1-3×10 9 Pieces / g.

[0008] Preferably, the raw materials also include: polyvinyl alcohol, sodium alginate, and sodium dihydrogen phosphate saturated boric acid solution.

[0009] More preferably, the concentration of the saturated boric acid solution of sodium dihydrogen phosphate is 0.5-1.2 mol / L.

[0010] More preferably, the mass ratio of the composite bacterial agent, polyvinyl alcohol, sodium alginate, and sodium dihydrogen phosphate saturated boric acid solution is 1-2:5-10:1-2:100-200.

[0011] Preferably, the active chitosan oligosaccharide is prepared by the following specific operation: adding chitosan oligosaccharide to phosphate buffer solution and stirring for 1-2 hours, adding wheat oligopeptide thereto and continuing stirring for 10-20 minutes, adding transglutaminase, stirring at 5-20°C for 1-2 hours, heating to 70-80°C, continuing stirring for 5-10 minutes, centrifuging, dialysis purification, and freeze-drying.

[0012] More preferably, the mass ratio of chitosan oligosaccharide, wheat oligopeptide and transglutaminase is 1-2:0.1-0.2:0.01-0.02.

[0013] More preferably, the molecular weight of the wheat oligopeptide is 600-800 Da.

[0014] More preferably, the pH value of the phosphate buffer solution is 4.5-5.5.

[0015] Preferably, the average relative molecular weight of chitosan oligosaccharide is 500-700, and the degree of polymerization is 2-8.

[0016] Preferably, the particle size of the mesoporous silica is 80-100 nm, and the Zeta potential is -20.2 to -23.6 mV.

[0017] The preparation method of the microbial composite preparation containing chitosan oligosaccharide comprises the following steps: adding mesoporous silica and composite bacterial agent into physiological saline for ultrasonic treatment for 10-20 minutes at an ultrasonic frequency of 5-15kHz, adding chitosan oligosaccharide thereto and stirring for 10-30 minutes, filtering, washing and vacuum drying.

[0018] The method for preparing the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps:

[0019] S1. Add chitosan oligosaccharide or active chitosan oligosaccharide, polyvinyl alcohol and sodium alginate into water, stir at 60-80°C for 1-2h, sterilize at 100-110°C for 1-3min, and cool to 30-40°C to obtain a prefabricated material;

[0020] S2. Add mesoporous silica and composite bacterial agent to physiological saline and ultrasonically treat for 10-20 minutes, add prefabricated material and stir for 10-30 minutes, add dropwise to a stirred sodium dihydrogen phosphate saturated boric acid solution, continue stirring for 10-30 minutes after complete addition, filter, wash and vacuum dry.

[0021] Preferably, in S2, the ultrasonic frequency is 5-15 kHz.

[0022] The use of the above-mentioned microbial composite preparation containing chitosan oligosaccharide in the following (1) or (2):

[0023] (1) preparing weight loss products;

[0024] (2) Preparation of antioxidant products.

[0025] Preferably, the product is a medicine or a health food.

[0026] The present invention uses a composite bacterial agent loaded on mesoporous silica, and then combined with chitosan oligosaccharide or active chitosan oligosaccharide to ensure that the composite bacterial agent is highly dense and maintains biological activity, while resisting miscellaneous bacteria and having good toxicity resistance. After cross-linking and curing, the product has good impact load resistance, significantly improves the ability to withstand harsh environments, and has high stability.

[0027] The invention combines wheat oligopeptides of specific molecular weight to the surface of chitosan oligosaccharide, which can not only enhance the stability and activity of chitosan oligosaccharide, but also compound the active chitosan oligosaccharide with polyvinyl alcohol and sodium alginate, and coat the mesoporous silica loaded with the composite bacterial agent. The mouse test confirms that the invention can effectively reduce the weight of naturally aged mice, improve their antioxidant activity, and improve the content of short-chain fatty acids in the intestine, and increase the diversity of intestinal flora and the content of probiotics. At the same time, the preparation method of the invention is simple, easy to control, and suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a comparison chart of the body weights of mice in the YCK group, ECK group, ES3 group, ES6 group and ES9 group.

[0029] Figure 2 This is a comparison chart of intestinal acetic acid, propionic acid and butyric acid in mice in YCK group, ECK group, ES3 group, ES6 group and ES9 group.

[0030] Figure 3 This is a comparison chart of intestinal isobutyric acid, valeric acid and isovaleric acid in mice in YCK group, ECK group, ES3 group, ES6 group and ES9 group.

[0031] Figure 4 This is a comparison chart of serum SOD activity and serum T-AOC capacity of mice in YCK group, ECK group, ES3 group, ES6 group and ES9 group.

[0032] Figure 5 This is a comparison chart of serum CAT activity and serum GSH-Px activity of mice in YCK group, ECK group, ES3 group, ES6 group and ES9 group.

[0033] Figure 6This is a comparison chart of the ACE index and Xiangnong diversity index of the intestinal flora of mice in the YCK group, ECK group, ES3 group, ES6 group and ES9 group.

[0034] Figure 7 This is a comparison of the relative abundance of intestinal flora at the genus level in mice in the YCK group, ECK group, ES3 group, ES6 group and ES9 group. DETAILED DESCRIPTION

[0035] The present invention will be further explained below in conjunction with specific embodiments.

[0036] The chitosan oligosaccharides used below were sourced from Zhejiang Golden Shell Pharmaceutical Co., Ltd.

[0037] The Lactobacillus johnsonii used in the following is from China General Microbiological Culture Collection Center, and its collection number is CGMCC 1.3221.

[0038] The Bifidobacterium breve used in the following is from China General Microbiological Culture Collection Center, and its collection number is CGMCC 1.3001.

[0039] Example 1

[0040] A chitosan oligosaccharide-containing microbial composite preparation, the raw materials of which include: 10g of composite bacterial agent, 10g of mesoporous silica and 10g of chitosan oligosaccharide.

[0041] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 2×10 9 The number of viable bacteria of Bifidobacterium breve is 1×10 9 Pieces / g.

[0042] The preparation method of the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps: adding mesoporous silica and composite bacterial agent to 100g of physiological saline for ultrasonic treatment for 10 minutes at an ultrasonic frequency of 5kHz, adding chitosan oligosaccharide thereto, stirring at a speed of 500r / min for 10 minutes, filtering, washing, and vacuum drying.

[0043] Example 2

[0044] A chitosan oligosaccharide-containing microbial composite preparation, the raw materials of which include: 20g of composite bacterial agent, 20g of mesoporous silica and 20g of chitosan oligosaccharide.

[0045] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 6×10 9 / g, and the number of viable bacteria of Bifidobacterium breve was 3×10 9 Pieces / g.

[0046] The preparation method of the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps: adding mesoporous silica and composite bacterial agent to 200g of physiological saline for ultrasonic treatment for 20 minutes at an ultrasonic frequency of 15kHz, adding chitosan oligosaccharide thereto, stirring at a speed of 1000r / min for 30 minutes, filtering, washing, and vacuum drying.

[0047] Example 3

[0048] A chitosan oligosaccharide-containing microbial composite preparation, the raw materials of which include: 15g of composite bacterial agent, 15g of mesoporous silica and 15g of chitosan oligosaccharide.

[0049] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 4×10 9 / g, and the number of viable bacteria of Bifidobacterium breve was 2×10 9 Pieces / g.

[0050] The preparation method of the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps: adding mesoporous silica and composite bacterial agent to 150g of physiological saline for ultrasonic treatment for 12 minutes at an ultrasonic frequency of 10kHz, adding chitosan oligosaccharide thereto, stirring at a speed of 800r / min for 20 minutes, filtering, washing, and vacuum drying.

[0051] Example 4

[0052] A chitosan oligosaccharide-containing microbial composite preparation comprises raw materials of: 10 g of composite bacterial agent, 20 g of mesoporous silica, 10 g of chitosan oligosaccharide, 100 g of polyvinyl alcohol, 10 g of sodium alginate, and 1000 g of 1.2 mol / L sodium dihydrogen phosphate saturated boric acid solution.

[0053] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 6×10 9 The number of viable bacteria of Bifidobacterium breve is 1×10 9 Pieces / g.

[0054] The method for preparing the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps:

[0055] S1. Add chitosan oligosaccharide, polyvinyl alcohol and sodium alginate to 800 g of deionized water, stir at 60° C. for 2 h at a stirring speed of 500 r / min, sterilize at 110° C. for 1 min, and cool to 40° C. to obtain a prefabricated material.

[0056] S2. Add mesoporous silica and composite bacterial agent to 100g of normal saline and ultrasonically treat for 20 minutes at an ultrasonic frequency of 5kHz. Add the prefabricated material thereto, stir at a speed of 1000r / min for 10 minutes, and add dropwise to a stirred saturated boric acid solution of sodium dihydrogen phosphate. After the addition is complete, continue stirring for 30 minutes at a stirring speed of 1000r / min, filter, wash, and vacuum dry.

[0057] Example 5

[0058] A chitosan oligosaccharide-containing microbial composite preparation comprises the following raw materials: 20 g of composite bacterial agent, 10 g of mesoporous silica, 20 g of chitosan oligosaccharide, 50 g of polyvinyl alcohol, 20 g of sodium alginate, and 2000 g of a 0.5 mol / L sodium dihydrogen phosphate saturated boric acid solution.

[0059] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 2×10 9 / g, and the number of viable bacteria of Bifidobacterium breve was 3×10 9 Pieces / g.

[0060] The method for preparing the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps:

[0061] S1. Add chitosan oligosaccharide, polyvinyl alcohol and sodium alginate to 400 g of deionized water, stir at 80° C. for 1 h at a stirring speed of 1000 r / min, sterilize at 100° C. for 3 min, and cool to 30° C. to obtain a prefabricated material.

[0062] S2. Add mesoporous silica and composite bacterial agent to 200g of normal saline and ultrasonically treat for 10 minutes at an ultrasonic frequency of 15kHz. Add the prefabricated material thereto, stir at a speed of 500r / min for 30 minutes, and add dropwise to a stirred saturated boric acid solution of sodium dihydrogen phosphate. After the addition is complete, continue stirring for 10 minutes at a stirring speed of 2000r / min, filter, wash, and vacuum dry.

[0063] Example 6

[0064] A chitosan oligosaccharide-containing microbial composite preparation comprises the following raw materials: 15 g of composite bacterial agent, 15 g of mesoporous silica, 15 g of chitosan oligosaccharide, 80 g of polyvinyl alcohol, 15 g of sodium alginate, and 1500 g of a 0.8 mol / L sodium dihydrogen phosphate saturated boric acid solution.

[0065] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 4×10 9 / g, and the number of viable bacteria of Bifidobacterium breve was 2×10 9 Pieces / g.

[0066] The method for preparing the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps:

[0067] S1. Add chitosan oligosaccharide, polyvinyl alcohol and sodium alginate to 600 g of deionized water, stir at 70° C. for 90 min at a stirring speed of 800 r / min, sterilize at 105° C. for 2 min, and cool to 35° C. to obtain a prefabricated material.

[0068] S2. Add mesoporous silica and composite bacterial agent to 150g of normal saline and ultrasonically treat for 15 minutes at an ultrasonic frequency of 9kHz. Add the prefabricated material thereto, stir at a speed of 800r / min for 15 minutes, and add dropwise to a stirred saturated boric acid solution of sodium dihydrogen phosphate. After the addition is complete, continue stirring for 15 minutes at a stirring speed of 1500r / min. Filter, wash, and vacuum dry.

[0069] Example 7

[0070] A microbial composite preparation containing chitosan oligosaccharide, the raw materials of which include: 13g of composite bacterial agent, 18g of mesoporous silica, 15g of active chitosan oligosaccharide, 90g of polyvinyl alcohol, 13g of sodium alginate, and 1200g of saturated boric acid solution of sodium dihydrogen phosphate with a concentration of 1 mol / L.

[0071] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 5×10 9 The number of viable bacteria of Bifidobacterium breve was 1.5×10 9 Pieces / g.

[0072] The method for preparing the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps:

[0073] S1. Add active chitosan oligosaccharide, polyvinyl alcohol and sodium alginate to 700 g of deionized water, stir at 65° C. for 100 min at a stirring speed of 600 r / min, sterilize at 108° C. for 2 min, and cool to 33° C. to obtain a prefabricated material.

[0074] The above-mentioned active chitosan oligosaccharide is prepared by the following specific operation: 20g of chitosan oligosaccharide is added to 100g of phosphate buffer solution with a pH value of 4.5-5.5, stirred at a speed of 2000r / min for 80min, 2g of wheat oligopeptide with a molecular weight of 600Da is added thereto, stirring is continued for 20min, 0.1g of transglutaminase is added, stirring is continued at a temperature of 20°C for 80min, the temperature is raised to 80°C, stirring is continued for 5min, centrifuged, dialyzed for purification, and freeze-dried.

[0075] S2. Add mesoporous silica and composite bacterial agent to 180g of normal saline and ultrasonically treat for 13 minutes at an ultrasonic frequency of 12kHz. Add the prefabricated material thereto, stir at a speed of 700r / min for 25 minutes, and add dropwise to a stirred saturated boric acid solution of sodium dihydrogen phosphate. After the addition is complete, continue stirring for 15 minutes at a stirring speed of 1800r / min. Filter, wash, and vacuum dry.

[0076] Example 8

[0077] A microbial composite preparation containing chitosan oligosaccharide, the raw materials of which include: 17g of composite bacterial agent, 12g of mesoporous silica, 19g of active chitosan oligosaccharide, 70g of polyvinyl alcohol, 17g of sodium alginate, and 1800g of 0.6mol / L sodium dihydrogen phosphate saturated boric acid solution.

[0078] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 3×10 9 / g, and the number of viable bacteria of Bifidobacterium breve was 2.5×10 9 Pieces / g.

[0079] The method for preparing the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps:

[0080] S1. Add active chitosan oligosaccharide, polyvinyl alcohol and sodium alginate to 500 g of deionized water, stir at 75° C. for 80 min at a stirring speed of 900 r / min, sterilize at 102° C. for 2 min, and cool to 37° C. to obtain a prefabricated material.

[0081] The above-mentioned active chitosan oligosaccharide is prepared by the following specific operation: 10g of chitosan oligosaccharide is added to 200g of phosphate buffer solution with a pH value of 4.5-5.5, stirred at a speed of 1000r / min for 100min, 1g of wheat oligopeptide with a molecular weight of 800Da is added thereto, stirring is continued for 10min, 0.2g of transglutaminase is added, stirring is carried out at a temperature of 5°C for 100min, the temperature is raised to 70°C, stirring is continued for 10min, centrifugation, purification by dialysis, and freeze-drying.

[0082] S2. Add mesoporous silica and composite bacterial agent to 120g of normal saline and ultrasonically treat for 17 minutes at an ultrasonic frequency of 6kHz. Add the prefabricated material thereto, stir at a speed of 900r / min for 15 minutes, and add dropwise to a stirred saturated boric acid solution of sodium dihydrogen phosphate. After the addition is complete, continue stirring for 25 minutes at a stirring speed of 1200r / min, filter, wash, and vacuum dry.

[0083] Example 9

[0084] A microbial composite preparation containing chitosan oligosaccharide, the raw materials of which include: 15g of composite bacterial agent, 15g of mesoporous silica, 17g of active chitosan oligosaccharide, 80g of polyvinyl alcohol, 15g of sodium alginate, and 1500g of 0.8mol / L sodium dihydrogen phosphate saturated boric acid solution.

[0085] The effective viable count of Lactobacillus johnsonii in the composite bacterial agent is 4×10 9 / g, and the number of viable bacteria of Bifidobacterium breve was 2×10 9 Pieces / g.

[0086] The method for preparing the above-mentioned microbial composite preparation containing chitosan oligosaccharide comprises the following steps:

[0087] S1. Add active chitosan oligosaccharide, polyvinyl alcohol and sodium alginate to 600 g of deionized water, stir at 70° C. for 90 min at a stirring speed of 800 r / min, sterilize at 105° C. for 2 min, and cool to 35° C. to obtain a prefabricated material.

[0088] The above-mentioned active chitosan oligosaccharide is prepared by the following specific operation: 15g of chitosan oligosaccharide is added to 150g of phosphate buffer solution with a pH value of 4.5-5.5, stirred at a speed of 1500r / min for 90min, 1.5g of wheat oligopeptide with a molecular weight of 700Da is added thereto, stirring is continued for 15min, 0.15g of transglutaminase is added, stirring is continued at a temperature of 10°C for 90min, the temperature is raised to 75°C, stirring is continued for 8min, centrifuged, dialyzed for purification, and freeze-dried.

[0089] S2. Add mesoporous silica and composite bacterial agent to 150g of normal saline and ultrasonically treat for 15 minutes at an ultrasonic frequency of 9kHz. Add the prefabricated material thereto, stir at a speed of 800r / min for 20 minutes, and add dropwise to a stirred saturated boric acid solution of sodium dihydrogen phosphate. After the addition is complete, continue stirring for 20 minutes at a stirring speed of 1500r / min. Filter, wash, and vacuum dry.

[0090] The natural aging mouse model was used to study the chitosan oligosaccharide-containing microbial composite preparation. The chitosan oligosaccharide-containing microbial composite preparations obtained in Examples 3, 6 and 9 were intragastrically administered to C57 / BL6 male mice, as follows:

[0091] C57 / BL6 male mice weighing 25.5±0.5g were housed in a 12-hour light-dark cycle environment with room temperature (25±2°C) and humidity of 50±5%. After a 7-day adaptation period, the mice were randomly divided into 5 groups:

[0092] (1) Young blank group (YCK): 3-month-old mice were gavaged with sterile saline;

[0093] (2) Elderly blank group (ECK): 13-month-old mice were gavaged with sterile saline;

[0094] (3) Elderly Example 3 group (ES3): Oral administration of the chitosan oligosaccharide-containing microbial composite preparation obtained in Example 3 (1.5 g / kg / day);

[0095] (4) Elderly Example 6 group (ES6): Oral administration of the chitosan oligosaccharide-containing microbial composite preparation obtained in Example 6 (1.5 g / kg / day);

[0096] (5) Elderly Example 9 group (ES9): Oral administration of the chitosan oligosaccharide-containing microbial composite preparation obtained in Example 9 (1.5 g / kg / day);

[0097] During the experiment, mice in each group were fed with standard feed. After 8 months of oral gavage, blood was collected from the eyeballs and the mice were killed by spinal dislocation. The centrifuge tubes were placed at room temperature for 2 hours, refrigerated at 4°C for 3 hours, and centrifuged at 3000g for 10 minutes. The serum was collected and frozen at -80°C for later use.

[0098] The abdominal cavity of the mouse was opened, and the cecum and small segment of colon were collected and placed in EP tubes and stored at -80°C for analysis.

[0099] Determination of short-chain fatty acids:

[0100] The concentration of short-chain fatty acids was determined by gas chromatography (GC). The culture medium filtrate was passed through a DB-FFAP gas chromatography column (0.32 mm × 30 m × 0.5 um) using a H2 flame ionization detector and crotonic acid (trans-2-butenoic acid) as an internal standard. The gas chromatography conditions were as follows:

[0101] Inlet parameters: temperature 250℃, injection volume: 1μL, carrier gas type: N2, purge flow 3.0mL / L, split ratio: 8:1, pressure: 54.2kPa, control mode: linear speed, linear velocity: 28.1cm / s, total flow: 16.1mL / min, column flow: 1.46mL / min.

[0102] Column box parameters: programmed heating, the heating program is as follows: initial temperature: 80℃, heating at a rate of 10℃ / min, maintaining for 0.50min after rising to 190℃; then reaching the final temperature at a rate of 40℃ / min: 230℃, maintaining for 4min, the complete program takes 16.5min, and detection is stopped at 17min. FID detector parameters: temperature 250℃, tail gas type N2, tail gas flow rate: 30mL / min, hydrogen flow rate: 40mL / min, air flow rate: 400mL / min

[0103] Sample pretreatment process: Dilute the feces 10 times with 0.1mM PBS to make a 10% feces suspension stock solution, add 0.06M crotonic acid-metaphosphoric acid mixed solution, shake well and mix, and place in a -30℃ low-temperature refrigerator for acidification reaction for more than 24 hours. Before loading, thaw the reaction solution on ice, centrifuge at 13000rpm and 4℃ for 3 minutes, use a disposable syringe to draw the supernatant through a 0.22μm aqueous filter membrane, place it in a 1.5mL centrifuge tube, take 100μL of the filtrate and add it to the rubber foot insert of the gas phase sample bottle, cover it with a lid to remove bubbles and then load the sample.

[0104] Determination of intestinal flora: Collect the contents of mouse cecum, extract DNA from feces according to the method and steps in the DNA rapid extraction kit manual, perform PCR amplification on the V3-V4 region of the sample 16S rDNA, purify and recover the amplified fragment using a gel recovery kit, and transfer it to a sequencing company for sequencing and bioinformatics analysis.

[0105] like Figure 1 As shown in the figure, after the experiment, there was no significant difference in the body weight of mice in the YCK group and the ECK group (P>0.05), and the body weight of mice in the ES3 group, ES6 group and ES9 group was lower than that in the YCK group and the ECK group (P<0.05). The body weight of mice in the ES9 group was the lowest, which was better than that in the ES3 group and the ES6 group (P<0.05).

[0106] like Figure 2 and Figure 3 As shown, the contents of short-chain fatty acids in ES3, ES6 and ES9 groups were higher than those in YCK and ECK groups, and the content of short-chain fatty acids in ES9 group was the highest, higher than that in the other groups (P < 0.05).

[0107] like Figure 4 and Figure 5 As shown in the figure, the antioxidant indexes of the YCK group were better than those of the ECK group (P < 0.05), and the antioxidant indexes of the ES3, ES6 and ES9 groups were also better than those of the ECK group (P < 0.05). The serum CAT activity of the ES9 group was slightly lower than that of the YCK group, but there was no significant difference (P > 0.05), and the other three antioxidant indexes were better than those of the YCK group (P < 0.05).

[0108] The α diversity of intestinal flora of each group of mice was analyzed. Figure 6 As shown in the figure, the ACE index and Xiangnong diversity index of the YCK group were higher than those of the ECK group (P<0.05), indicating that the richness of the microbiota of the ECK mice in the old control group was reduced, and the diversity was significantly reduced; while the ACE index and Xiangnong diversity index of the ES3 group, ES6 group and ES9 group were higher than those of the ECK group, indicating that the chitosan oligosaccharide-containing microbial composite preparation obtained by the present invention can effectively improve the richness of the microbiota of old mice and increase the diversity of the microbiota.

[0109] The intestinal flora of each group of mice was analyzed at the genus level. Figure 7 As shown, the contents of probiotic genera such as Bifidobacterium in the intestines of mice in groups ES3, ES6 and ES9 were significantly higher than those in groups YCK and ECK, while the contents of lipopolysaccharide (LSP)-producing genera such as Mycobacterium were significantly lower than those in groups YCK and ECK, thereby enhancing the intestinal immunity and antioxidant capacity of mice.

[0110] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A microbial composite preparation containing chitosan oligosaccharide, characterized in that: The raw materials include, by mass: 1-2 parts of composite bacterial agent, 1-2 parts of mesoporous silica, and 1-2 parts of chitosan oligosaccharide or active chitosan oligosaccharide; The compound bacterial agent includes: Lactobacillus, Bifidobacterium; In the compound bacterial agent, the effective viable count of lactic acid bacteria is 2-6×10 9 / g, and the number of viable bifidobacteria is 1-3×10 9 Pieces / g.

2. The microbial composite preparation containing chitosan oligosaccharides according to claim 1, characterized in that: Its raw materials also include: polyvinyl alcohol, sodium alginate, sodium dihydrogen phosphate saturated boric acid solution; The concentration of the saturated boric acid solution of sodium dihydrogen phosphate is 0.5-1.2 mol / L; The mass ratio of the composite bacterial agent, polyvinyl alcohol, sodium alginate and sodium dihydrogen phosphate saturated boric acid solution is 1-2:5-10:1-2:100-200.

3. The microbial composite preparation containing chitosan oligosaccharide according to claim 1, characterized in that: Active chitosan oligosaccharide is prepared by the following specific operation: chitosan oligosaccharide is added to phosphate buffer solution and stirred for 1-2 hours, wheat oligopeptide is added thereto and stirring is continued for 10-20 minutes, transglutaminase is added, stirring is continued at 5-20° C. for 1-2 hours, heating to 70-80° C., stirring is continued for 5-10 minutes, centrifugation is performed, purification is performed by dialysis, and freeze-drying is performed.

4. The microbial composite preparation containing chitosan oligosaccharide according to claim 3, characterized in that: The mass ratio of chitosan oligosaccharide, wheat oligopeptide and transglutaminase is 1-2:0.1-0.2:0.01-0.

02.

5. The microbial composite preparation containing chitosan oligosaccharide according to claim 3, characterized in that: The molecular weight of wheat oligopeptides is 600-800Da.

6. The microbial composite preparation containing chitosan oligosaccharide according to claim 3, characterized in that: The pH value of phosphate buffer solution is 4.5-5.

5.

7. The microbial composite preparation containing chitosan oligosaccharide according to any one of claims 1 to 6, characterized in that: The average relative molecular weight of chitosan oligosaccharide is 500-700, and the degree of polymerization is 2-8.

8. The microbial composite preparation containing chitosan oligosaccharide according to claim 1, characterized in that: The particle size of mesoporous silica is 80-100 nm, and the Zeta potential is -20.2 to -23.6 mV.

9. A method for preparing a microbial composite preparation containing chitosan oligosaccharide according to any one of claims 2 to 8, characterized in that: The steps include: S1. Add chitosan oligosaccharide or active chitosan oligosaccharide, polyvinyl alcohol and sodium alginate into water, stir at 60-80°C for 1-2h, sterilize at 100-110°C for 1-3min, and cool to 30-40°C to obtain a prefabricated material; S2. Add mesoporous silica and composite bacterial agent to physiological saline and ultrasonically treat for 10-20 minutes, add prefabricated material and stir for 10-30 minutes, add dropwise to a stirred sodium dihydrogen phosphate saturated boric acid solution, continue stirring for 10-30 minutes after complete addition, filter, wash and vacuum dry.

10. Use of a microbial composite preparation containing chitosan oligosaccharides as claimed in any one of claims 1 to 8 in the following (1) or (2): (1) preparing weight loss products; (2) Preparation of antioxidant products.

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