A complex for improving the tolerance and proliferation ability of probiotics, and its preparation method and application

Abstract

The present invention provides a complex for improving the tolerance and proliferation ability of probiotics, and its preparation method and application. By extracting polysaccharides from aged Xanthii Fructus and mixing them with cyanidin-3-O-glucoside (C3G) and silymarin extract to form a complex, it is used for the cultivation of probiotics, thereby enhancing the tolerance and proliferation ability of probiotics. The experimental results show that this complex can significantly improve the survival rate and proliferation ability of Lactobacillus acidophilus and Bacillus coagulans, and has application potential in the fields of functional foods, health products, etc.

Description

Technical Field

[0001] The present invention belongs to the field of food biotechnology, and particularly relates to a complex for improving the tolerance and proliferation ability of probiotics, and a preparation method and application thereof. Background Art

[0002] Probiotics are defined as live microorganisms that can have beneficial effects on the health of the host when ingested in a certain amount by the host. At present, probiotics have become a research hotspot in the fields of food technology and nutrition and health. They enhance the host's immunity by regulating the intestinal microbial community structure, inhibiting the growth of pathogenic bacteria, and strengthening the intestinal barrier function. However, probiotics will encounter various stress factors such as low pH value, digestive enzymes, and bile during the process through the gastrointestinal tract, and these stress factors will seriously affect the survival rate and efficacy of probiotics in the gastrointestinal tract.

[0003] In order to improve the survival rate of probiotics in the gastrointestinal tract, researchers have explored various methods, including the development of new prebiotics. The so-called "prebiotics" are substances that can selectively promote the growth of probiotics and provide the activity of probiotics, and they include natural active products extracted from plants, trace elements existing in nature, and other nutrients.

[0004] Anthocyanins are a class of natural pigments widely present in plants, with biological activities such as antioxidant and anti-inflammatory effects, and show their potential as prebiotics in many research reports. For example, anthocyanins can promote the proliferation of probiotics and inhibit the growth of harmful bacteria. In addition, natural plant polysaccharides have attracted attention due to their role in promoting the growth of probiotics and regulating intestinal health.

[0005] According to the research "Isolation, purification and in vitro gastric mucosal protective activity of polysaccharides from Garcinia oblongata" published by Yang Dan et al., polysaccharides from Garcinia oblongata, as a kind of natural plant polysaccharides, have a certain protective effect on the in vitro gastric mucosa, but this article did not study its promotion of the tolerance and proliferation ability of beneficial bacteria as a prebiotic. Based on the characteristics of polysaccharides from Garcinia oblongata, it should also have the characteristics of prebiotics, but the current research on its prebiotic activity is relatively few. The combined use of anthocyanins and polysaccharides from Garcinia oblongata may produce a synergistic effect and more effectively improve the tolerance and proliferation ability of probiotics, but this synergistic effect has not been fully utilized. Summary of the Invention

[0006] To solve the related problems, the purpose of the present invention is to provide a complex for improving the tolerance and proliferation ability of probiotics, and a preparation method and application thereof. This complex will also have good application prospects in promoting the proliferation of probiotics and enhancing their use in functional foods, health products and other fields.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] First, the present invention provides a method for extracting and purifying polysaccharides from old fragrant yellow, which includes mechanically pulping old fragrant yellow and freeze-drying to obtain old fragrant yellow freeze-dried powder; mixing the old fragrant yellow freeze-dried powder with deionized water, and obtaining crude polysaccharides from old fragrant yellow through hot water extraction and alcohol precipitation steps; after the crude polysaccharides from old fragrant yellow are subjected to defatting, decolorization and protein removal treatments, they are eluted through a DEAE-52 cellulose ion exchange column and a SephadexG-100 dextran gel column, and freeze-dried to obtain purified polysaccharides from old fragrant yellow.

[0009] Further, the present invention provides a method for preparing a complex, which includes mixing the purified polysaccharides from old fragrant yellow, anthocyanins and silymarin extract in a certain mass ratio to form a complex with a synergistic effect.

[0010] Further, the mass ratio of the polysaccharides from old fragrant yellow, anthocyanins and silymarin extract is 1:0.5 - 5:1.

[0011] Further, the anthocyanin is cyanidin-3-O-glucoside (i.e., C3G).

[0012] Secondly, the present invention uses the complex containing polysaccharides from old fragrant yellow, silymarin extract and anthocyanins to culture Lactobacillus acidophilus and Bacillus coagulans respectively, and finds that the complex in the present invention can effectively promote the proliferation of probiotics.

[0013] Therefore, the present invention requests protection for the application of the complex in promoting the proliferation of probiotics.

[0014] The present invention also requests protection for the application of the complex in the preparation of products for promoting the proliferation of probiotics.

[0015] The present invention also requests protection for the application of the complex in regulating the balance of intestinal flora or in the preparation of products for regulating the balance of intestinal flora; the regulation of the balance of intestinal flora is achieved by promoting the proliferation of probiotics.

[0016] The present invention has the following beneficial effects:

[0017] The present invention uses the complex containing polysaccharides from old fragrant yellow, anthocyanins and silymarin extract to culture Lactobacillus acidophilus and Bacillus coagulans respectively and finds that the complex can promote the proliferation of the above probiotics, and can be used to prepare products for promoting the proliferation of probiotics, thus contributing to regulating the balance of intestinal flora and maintaining the stability of the intestinal environment. The present invention not only enriches the sources of prebiotics that can promote the proliferation of probiotics, but also enriches the application fields of polysaccharides from old fragrant yellow. Description of the Drawings

[0018] Figure 1 shows the growth curves of two probiotic strains, where A: Lactobacillus acidophilus (LA), B: Bacillus coagulans (BC).

[0019] Figure 2 It is the DEAE-52 cellulose column chromatography diagram of the polysaccharide from Laoxianghuang.

[0020] Figure 3 It is the Sephadex G-100 gel column chromatography diagram of the polysaccharide from Laoxianghuang.

[0021] Figure 4A It is the high performance gel permeation chromatography diagram of the polysaccharide from Laoxianghuang. The dextran standard curve diagram is in the upper right corner.

[0022] Figure 4B It is Figure 4A The dextran standard curve diagram in the upper right corner.

[0023] Figure 5 is the ion chromatography diagram of the polysaccharide from Laoxianghuang (A is the mixed standard, B is the polysaccharide from Laoxianghuang). Specific implementation manners

[0024] The following further illustrates the present invention in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0025] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0026] To detect the influence effect of the complex described in the present invention on the proliferation of probiotics, the following strains were selected as experimental bacteria in the present invention, mainly including: Lactobacillus acidophilus (LA05) strain with the preservation number of CGMCC NO.23546 and Bacillus coagulans (BC99) strain with the preservation number of CGMCC NO.21801. The above strains were purchased from Microkang Probiotics Co., Ltd.

[0027] The Laoxianghuang was purchased from Wenxiang Food Factory, Wenci Town, Chao'an District, Chaozhou City, and it is one-year-old Laoxianghuang.

[0028] Example 1 Growth curve of probiotics

[0029] The Lactobacillus acidophilus and Bacillus coagulans were respectively inoculated into MRS broth medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) at an inoculation amount of 2 v / v%, cultured at 37 °C for 12 h, and the activated secondary strains were obtained by culturing for two generations according to the above method.

[0030] The activated secondary strain was inoculated into MRS broth medium at an inoculum size of 5 v / v%, and after thorough mixing, it was placed in a 37 °C incubator for static culture. An appropriate amount of the culture solution was taken every 2 h, and the MRS without inoculated bacteria was used as a blank control. The optical density (OD) at 600 nm was measured using a microplate reader, abbreviated as OD600nm.

[0031] The growth curve of the probiotic was plotted with the culture time and absorbance value as the horizontal and vertical coordinates, respectively. The results are shown in Figure 1.

[0032] Example 2 Extraction, Purification and Structure Identification of Preserved Aged Citrus polysaccharide

[0033] The preserved aged citrus polysaccharide described in the present invention is extracted from Preserved Aged Citrus (Citrus medicavar. sarcodactylis), and its extraction and purification process includes the following steps:

[0034] S1. The Preserved Aged Citrus was mechanically pulped and vacuum freeze-dried to obtain Preserved Aged Citrus freeze-dried powder;

[0035] S2. Hot water extraction and alcohol precipitation were carried out in sequence, and the alcohol precipitation precipitate was collected to obtain the crude Preserved Aged Citrus polysaccharide;

[0036] S3. The crude Preserved Aged Citrus polysaccharide obtained in step S2 was defatted to obtain defatted crude Preserved Aged Citrus polysaccharide;

[0037] S4. The defatted crude Preserved Aged Citrus polysaccharide obtained in step S3 was decolorized to obtain decolorized crude Preserved Aged Citrus polysaccharide;

[0038] S5. The decolorized crude Preserved Aged Citrus polysaccharide obtained in step S4 was deproteinized to obtain deproteinized crude Preserved Aged Citrus polysaccharide;

[0039] S6. The deproteinized crude Preserved Aged Citrus polysaccharide obtained in step S5 was separated using a DEAE-52 cellulose ion exchange column and eluted with water or sodium chloride solution to obtain the Preserved Aged Citrus fractionated polysaccharide. The Preserved Aged Citrus fractionated polysaccharide obtained by elution was separated using a Sephadex G-100 dextran gel column and eluted with water or sodium chloride solution to obtain the Preserved Aged Citrus polysaccharide.

[0040] Among them, step S1 specifically includes: the Preserved Aged Citrus was mechanically pulped and vacuum freeze-dried for 48 h to obtain the Preserved Aged Citrus freeze-dried powder.

[0041] Step S2 specifically includes: the Preserved Aged Citrus freeze-dried powder was mixed with deionized water at a solid-liquid ratio of 1 g:20 mL, extracted at 80 °C for 3 h, filtered, and the filtrates were combined; the combined filtrates were concentrated under reduced pressure at 60 °C to obtain a concentrated solution; 95 v / v% ethanol with a volume 4 times that of the concentrated solution was added to the concentrated solution, and it was left standing in a 4 °C refrigerator for 24 h; centrifuged at 10000 rpm for 10 min, the supernatant was discarded, the obtained precipitate was washed three times with 95 v / v% ethanol, concentrated under reduced pressure at 55 °C, and freeze-dried for 48 h to obtain the crude Preserved Aged Citrus polysaccharide powder.

[0042] Step S3 specifically includes: Weigh the crude polysaccharide powder of old fragrant yellow, wrap it in filter paper and place it in a Soxhlet extractor. Add petroleum ether with a volume 4 times that of the crude polysaccharide of old fragrant yellow, and reflux at 90 °C for 2 h. Evaporate the solvent to remove lipid components in the crude polysaccharide of old fragrant yellow.

[0043] Step S4 specifically includes: Dissolve the defatted crude polysaccharide of old fragrant yellow in deionized water, add pretreated AB-8 macroporous resin (the pretreatment method refers to the AB-8 resin instruction manual), and perform adsorption oscillation at 37 °C and 160 r / min for 6 h for decolorization. Let it stand overnight, filter to remove the resin, repeat until the color of the polysaccharide becomes lighter, and remove some pigments in the old fragrant yellow polysaccharide.

[0044] Step S5 specifically includes: Add Sevag reagent (chloroform: n-butanol = 4:1, v / v) to the decolorized crude polysaccharide solution of old fragrant yellow. The volume ratio of the Sevag reagent to the decolorized crude polysaccharide solution of old fragrant yellow is 1:4. Oscillate for 20 min, centrifuge at 10000 rpm for 10 min, aspirate the upper polysaccharide solution, discard the lower organic phase and protein precipitate, and repeat the extraction 4 - 5 times to remove all proteins in the solution; Spin-dry the old fragrant yellow polysaccharide layer solution with a rotary evaporator to remove the organic reagents contained therein, redissolve it with a small amount of deionized water, dialyze it with a dialysis bag with a molecular cut-off of 8000 - 14000 Da for 48 h, and change the water every 4 h; Vacuum freeze-dry to obtain the defatted and deproteinized crude polysaccharide powder of old fragrant yellow.

[0045] Step S6 specifically includes: Take the obtained defatted and deproteinized crude polysaccharide powder of old fragrant yellow and redissolve it with deionized water to make its final concentration 20 mg / mL; With a sample loading volume of 10 mL, separate it using a DEAE-52 cellulose ion exchange column (2.5×40 cm, the packing is purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.), and elute it successively with NaCl solutions with concentrations of 0 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L to obtain the DEAE-52 cellulose column elution curve as Figure 2As shown, the elution curve reaches peaks at 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L. Among them, the sugar content and absorbance of the components eluted with 0.2 mol / L NaCl solution are the highest. Therefore, this component is retained for purification. Take the polysaccharide from old fragrant yellow (i.e., the component corresponding to the elution with 0.2 mol / L NaCl solution described in S6) separated by DEAE-52 cellulose ion exchange column, and add deionized water to prepare an old fragrant yellow polysaccharide solution with a final concentration of 10 mg / mL; with a sample loading volume of 1 mL, separate it using a Sephadex G-100 dextran gel column (2.5×40 cm, the packing material is purchased from Shanghai Macklin Biochemical Co., Ltd.), elute with deionized water at a flow rate of 0.2 mL / min, collect in 5 mL tubes, and the elution curve is as Figure 3 shown. Retain the components obtained from the 5th to 9th tubes, and freeze-dry to obtain the polysaccharide from old fragrant yellow.

[0046] 1. Experimental methods

[0047] 1.1 Determination of the molecular weight of the polysaccharide from old fragrant yellow

[0048] The molecular weight of the polysaccharide from old fragrant yellow in S6 was determined by HPGPC. The gel column equipped for HPGPC is a BRT105103-101 tandem gel column (8×300 mm), and it is also equipped with a RI-20A differential refractometer detector.

[0049] Preparation of the standard solution: Weigh accurately 5 mg each of dextran standards Dextran standards 1153, P5, P10, P20, P50, P100, P200, P400, P800, and P2000, dissolve them in 1 mL of the mobile phase solution, prepare a 5 mg / mL solution, and transfer the sample to a 1.8 mL injection vial.

[0050] Preparation of the sample solution: Weigh accurately 5 mg of the polysaccharide sample from old fragrant yellow, dissolve it in 1 mL of the mobile phase solution, sonicate for 10 min, centrifuge at 12000 rpm for 10 min, aspirate the supernatant and filter it through a 0.22 μm aqueous microfiltration membrane, and transfer the sample to an injection vial.

[0051] Chromatographic column: BRT105-103-101 tandem gel column (8×300 mm); Detector: Differential refractometer detector RID-20A; Mobile phase: 0.05 mol / L NaCl solution; Mobile phase flow rate: 0.8 mL / min; Sample injection volume: 25 μL; Column temperature: 40 °C; Analysis time: 60 min.

[0052] Use logM w and t to prepare the standard curve.

[0053] 1.2 Analysis of the monosaccharide composition of the polysaccharide from old fragrant yellow

[0054] Sixteen monosaccharide standards were used to prepare a standard solution (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, D-galactosamine hydrochloride, D-glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid). Each monosaccharide standard solution was used as a mixed standard, and the mass of different monosaccharides was determined according to the absolute quantification method, and then the molar ratio was calculated based on the molar mass of the monosaccharides.

[0055] Sample preparation: Weighed 5.0 mg of the Laoxianghuang polysaccharide sample in S6 and placed it in an ampoule bottle. Added 2 mL of TFA (trifluoroacetic acid) with a concentration of 3 mol / L and hydrolyzed it at 120 °C for 3 h. Dried it with nitrogen blowing, added 5 mL of deionized water, vortexed and mixed evenly, then pipetted 50 μL and added 950 μL of deionized water, and centrifuged at 12000 rpm for 5 min. Took the supernatant for ion chromatography analysis.

[0056] Chromatographic method:

[0057] Chromatographic column: Dionex TM Carbopac TM PA20 (3 * 150 mm); Mobile phase: A: H2O; B: 15 mmol / L NaOH; C: 15 mmol / L NaOH and 100 mmol / L NaAc; Injection volume: 25 μL; Flow rate: 0.3 mL / min; Column temperature: 30 °C; Elution gradient: 0 min A phase / B phase / C phase (98.8:1.2:0, v / v), 18 min A phase / B phase / C phase (98.8:1.2:0, v / v), 20 min A phase / B phase / C phase (50:50:0, v / v), 30 min A phase / B phase / C phase (50:50:0, v / v), 30.1 min A phase / B phase / C phase (0:0:100, v / v), 46 min A phase / B phase / C phase (0:0:100, v / v), 46.1 min A phase / B phase / C phase (0:100:0, v / v), 50 min A phase / B phase / C phase (0:100:0, v / v), 50.1 min A phase / B phase / C phase (98.8:1.2:0, v / v), 80 min A phase / B phase / C phase (98.8:1.2:0, v / v); Detector: Electrochemical detector.

[0058] 2. Experimental results

[0059] 2.1 Molecular weight of Laoxianghuang polysaccharide

[0060] As can be seen from Figure 4, the purified polysaccharide sample of Laosianghuang in S6 shows a single symmetric peak, and no polysaccharide impurity peaks of other molecular weights appear, indicating that the obtained polysaccharide of Laosianghuang is a homogeneous component. At the same time, according to the retention time of the polysaccharide and the standard curve of dextran with different known molecular weights, that is, LogM w =-0.2225t + 11.457, R 2 = 0.9945, where t is the elution time and M w is the average molecular weight. The relative average molecular weight of the polysaccharide of Laosianghuang can be calculated to be 470086 Da, the retention time is 25.963 min, the weight-average molecular weight M w is 485528 Da, the number-average molecular weight M n is 474476 Da, and the molecular weight distribution width (M w / M n ) is 1.02.

[0061] 2.2 Analysis of monosaccharide composition of polysaccharide of Laosianghuang

[0062] The results are shown in Figure 5. By comparing the retention times of the monosaccharide standards ( Figure 5A ), it can be known that Figure 5B the monosaccharide components in the polysaccharide of Laosianghuang are fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid in sequence, and the molar ratio is 0.4:1.9:10:22.7:27.7:0.4:0.6:35.7:0.6. Among them, the molar ratio of the total neutral monosaccharide residues to the uronic acid residues is 63.7:36.3. The results show that the monosaccharide composition of the polysaccharide of Laosianghuang is mainly glucose and galactose, and at the same time contains some fucose, rhamnose, xylose, and mannose.

[0063] Example 3 Effects of different additives on the survival rates of probiotics in acid, stomach, and intestine

[0064] 1. Experimental method

[0065] 1.1 - 1.3 Test samples

[0066] Test samples:

[0067] A: 0.3 mg / mL C3G + 1.2 mg / mL deionized water;

[0068] B: 0.6 mg / mL polysaccharide of Laosianghuang + 0.9 mg / mL deionized water;

[0069] C: 0.6 mg / mL silymarin extract + 0.9 mg / mL deionized water;

[0070] D: 1.5 mg / mL complex of polysaccharide of Laosianghuang, C3G, and silymarin extract with a mass ratio of 1:0.5:1;

[0071] E: 0.3 mg / mL C3G + 0.6 mg / mL old fragrant yellow polysaccharide + 0.6 mg / mL deionized water;

[0072] F: 0.6 mg / mL silymarin extract + 0.6 mg / mL old fragrant yellow polysaccharide + 0.3 mg / mL deionized water;

[0073] G: 0.3 mg / mL C3G + 0.6 mg / mL silymarin extract + 0.6 mg / mL deionized water.

[0074] 1.1 Acid resistance test.

[0075] Using MRS liquid medium as the blank control, the twice-activated strains were inoculated into the sample MRS liquid medium with a pH of 3.0 and the blank (con) MRS control medium with a sterile membrane added at an inoculation amount of 2% (v / v). Among them, there were 7 groups of sample MRS liquid medium. After adding the test samples respectively, they were cultured at 37 °C for 4 h. Serial 10-fold dilutions were made with sterile normal saline. For each dilution, 3 replicates were made. 1 mL of the appropriate dilution of the bacterial liquid was transferred into a sterile empty petri dish, and about 15 mL of MRS solid medium was poured into the petri dish, mixed evenly, and cultured statically at 37 °C for 48 h and then counted. The survival rate calculation formula is as follows: Survival rate (%) = N' / N0 × 100%. In the formula: N' is the number of viable bacteria under the condition of pH 3.0; N0 is the number of viable bacteria in the blank control.

[0076] 1.2 Simulated gastric juice test

[0077] The twice-activated strains were inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) and cultured for 24 h, centrifuged at 5000 g for 10 min, the bacterial sludge was collected, and the bacterial sludge was resuspended in PBS buffer solution to prepare a resuspension. The concentration was adjusted to 1.0×10 8 CFU / mL with a McFarland turbidimetric tube. 10 mL was taken and inoculated into 100 mL of artificial gastric juice with different test samples added and a pH of 3.0 (by mass fraction, the artificial gastric juice contains 0.20% NaCl and 0.30% pepsin, and the pH was adjusted with HCl / sodium hydroxide). Using the artificial gastric juice without adding samples as the blank (con) control, it was statically placed at 37 °C for 0 h and 4 h, serially 10-fold diluted into MRS solid medium for counting, and its survival rate was calculated. Survival rate (%) = N1 / N0 × 100%, where N1: the number of viable bacteria after 4 h of treatment with artificial gastric juice; N0: the number of viable bacteria after 0 h of treatment with artificial gastric juice.

[0078] 1.3 Simulated intestinal juice test

[0079] The strain that has been activated twice is inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) and cultured for 24 h. Then, it is centrifuged at 5000 g for 10 min, and the bacterial sludge is collected. The bacterial sludge is resuspended in PBS buffer solution to prepare a resuspended solution. The concentration is adjusted to 1.0×10 8 CFU / mL with a McFarland turbidimeter. Take 10 mL and inoculate it into 100 mL of artificial intestinal fluid with a pH of 3.0 and different test samples added (different MRS liquid media are prepared using 0.1 mol / L phosphate buffer solution, and the pH of the liquid medium is adjusted to 7 with hydrochloric acid / sodium hydroxide, sterilized by autoclaving at 121 °C for 15 min, and 1 g of trypsin is added to 100 mL of the sterilized liquid medium to obtain in vitro simulated intestinal fluid). Use the artificial intestinal fluid without adding samples as the blank (con) control. Let it stand at 37 °C for 0 h and 4 h, dilute it by 10-fold gradient and count it on MRS solid medium, and calculate its survival rate. The survival rate (%) = N1 / N0×100%, where N1: the number of viable bacteria after 4 h of treatment with artificial intestinal fluid; N0: the number of viable bacteria at 0 h of treatment with artificial intestinal fluid.

[0080] 2. Experimental results

[0081] Table 1 Effects of C3G, Laoshuanghuang polysaccharide, silymarin extract and their complexes on the survival rates of Lactobacillus acidophilus and Bacillus coagulans in different environments

[0082]

[0083] "*" indicates a significant difference compared with the "con (blank)" control, p < 0.01

[0084] 2.1 Effects of different additives on the survival rates of probiotics in acidic environments

[0085] It can be seen from Table 1 that whether in an acidic environment, gastric juice simulation or intestinal fluid simulation, the effects of the complex on the survival rates of Lactobacillus acidophilus and Bacillus coagulans are stronger than those of C3G or Laoshuanghuang polysaccharide or silymarin extract used alone or in combination of two of them. Therefore, it can be concluded that there is a certain synergistic effect among C3G, Laoshuanghuang polysaccharide and silymarin extract. In view of this survival characteristic, the following proliferation experiment only explores the proliferation effects of the complexes of C3G, Laoshuanghuang polysaccharide and silymarin extract on Lactobacillus acidophilus probiotics at different mass ratios.

[0086] Example 4 Proliferation effects of the complex of C3G, Laoshuanghuang polysaccharide and silymarin extract on the growth of probiotics

[0087] 1. Experimental method

[0088] The Lactobacillus acidophilus strain activated twice was inoculated into MRS liquid medium at an inoculum size of 2% (v / v) and cultured for 24 h. Then, it was centrifuged at 5000 g for 10 min, and the bacterial sludge was collected. The bacterial sludge was resuspended in PBS buffer solution to prepare a resuspension. The concentration was adjusted to 1.0×10 8 CFU / mL with a McFarland turbidimeter. 10 mL was taken and inoculated into 100 mL of MRS medium with different samples (A - G) added at 1.5 mg / mL and a pH of 7.0. The MRS medium without added samples was used as the blank (con) control. The bacterial solution was cultured at 37 °C for 24 h, and the OD value of the bacterial solution was measured at 600 nm, with the medium without addition as the blank control.

[0089] Proliferation rate = (OD 样品 - OD 空白 ) / OD 空白

[0090] 2. Experimental results

[0091] Table 2 Effects of complexes of different mass ratios of laoxianghuang polysaccharide, C3G, and silymarin extract on the proliferation rate of Lactobacillus acidophilus.

[0092]

[0093] The P value is the significance analysis of groups a - f compared with the blank group respectively.

[0094] As can be seen from Table 2, when the mass ratio of laoxianghuang polysaccharide, C3G, and silymarin extract falls within the numerical range defined in the present invention (i.e., groups a, b, c), the effects of the complexes on the growth and proliferation of Lactobacillus acidophilus are not very different, and there is a certain proliferation effect compared with the blank. When the mass ratio of the three exceeds the numerical range defined in the present invention (i.e., groups d, f), the results compared with group a show that the proliferation rate decreases. When the mass ratio of laoxianghuang polysaccharide increases (group e), it will lead to an increase in the proliferation rate of Lactobacillus acidophilus, but compared with the added amount of laoxianghuang polysaccharide, the increase in the proliferation rate is relatively low, and the cost performance is relatively low.

[0095] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A composite, characterized in that, In the complex, the mass ratio of old sweet basil polysaccharide, anthocyanin, and silymarin extract is 1:0.5 - 5:1, and the anthocyanin is cyanidin-3-O-glucoside; The preparation method of the complex includes the following steps: S1. Mechanically beat old sweet basil and freeze-dry it under vacuum to obtain old sweet basil freeze-dried powder; S2. Mix the old sweet basil freeze-dried powder with deionized water at a solid-liquid ratio of 1 g:20 mL, extract with water at 80°C for 3 h, filter to obtain a filtrate, concentrate under reduced pressure at 60°C to obtain a concentrated solution, add 95 v / v% ethanol with a volume 4 times that of the concentrated solution, let it stand at 4°C for 24 h, then centrifuge at 10000 rpm for 10 min, discard the supernatant, wash the precipitate with 90 - 98 v / v% ethanol, concentrate under reduced pressure at 55°C, and freeze-dry to obtain old sweet basil crude polysaccharide powder; S3. Weigh the old sweet basil crude polysaccharide powder and perform Soxhlet extraction with petroleum ether to defat; S4. Dissolve the defatted old sweet basil crude polysaccharide in deionized water and decolorize it through AB-8 macroporous resin; S5. Treat the decolorized old sweet basil crude polysaccharide solution with Sevag reagent to remove proteins, and then freeze-dry to obtain old sweet basil crude polysaccharide powder; S6. The old sweet basil crude polysaccharide powder treated in S5 is separated by a DEAE-52 cellulose ion exchange column and further purified by a SephadexG-100 dextran gel chromatography column to obtain old sweet basil polysaccharide; S7. Mix the obtained old sweet basil polysaccharide with anthocyanin and silymarin extract according to the mass ratio to obtain the complex.

2. The composite according to claim 1, characterized in that, Before the freeze-drying step in S5, there is also a step of dialysis using a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da.

3. A method for enhancing the tolerance and proliferation ability of probiotics by using the complex described in claim 1, characterized in that, Including the following steps: T1: Inoculate probiotics into an MRS medium containing 1 - 5 mg / mL of the complex described in claim 1; T2: Culture the probiotics at 37 ± 0.5°C for 12 - 48 hours; The probiotics are Lactobacillus acidophilus or Bacillus coagulans.

4. A functional food, characterized in that, Containing the complex described in claim 1.

5. A health product, characterized in that, Containing the complex described in claim 1.

6. Use of a complex, characterized in that, The complex described in claim 1 is used for promoting the proliferation of probiotics.

7. Use of a complex, characterized in that, The complex described in claim 1 is used for regulating the balance of the intestinal flora.

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