Compound for improving tolerance and multiplication capacity of probiotics as well as preparation method and application of compound

Abstract

The invention provides a compound for improving tolerance and multiplication capacity of probiotics as well as a preparation method and application of the compound, and the method comprises the following steps: extracting rosa laevigata polysaccharide, mixing the rosa laevigata polysaccharide with cyanidin-3-O-glucoside (C3G) and silybum marianum extract to form the compound, and culturing the probiotics, so that the tolerance and multiplication capacity of the probiotics are improved. Experimental results show that the compound can significantly improve the survival rate and multiplication capacity of lactobacillus acidophilus and bacillus coagulans, and has application potential in the fields of functional food, health care products and the like.

Description

Technical Field

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

[0002] Probiotics are defined as living microorganisms that can have a beneficial effect on the host's health when the host ingests a certain amount. Currently, 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 structure of intestinal microbial communities, inhibiting the growth of pathogens, and enhancing the intestinal barrier function. However, probiotics will encounter various stress factors such as low pH, digestive enzymes and bile during their passage through the gastrointestinal tract. 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 a variety of methods, including the development of new prebiotics. The so-called "prebiotics" are a type of substance that can selectively promote the growth of probiotics and provide probiotic activity, including natural active products extracted from plants or trace elements and other nutrients existing in nature.

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

[0005] According to the article “Isolation and Purification of Old Xianghuang Polysaccharide and Its In Vitro Gastric Mucosal Protective Activity” published by Yang Dan et al., Old Xianghuang Polysaccharide, as a natural plant polysaccharide, has a certain protective effect on gastric mucosa in vitro, but the article did not study its role as a prebiotic in promoting the tolerance and proliferation of beneficial bacteria. Based on the characteristics of Old Xianghuang Polysaccharide, it should also have the characteristics of a prebiotic, but there are relatively few studies on its prebiotic activity. The combined use of anthocyanins and Old Xianghuang Polysaccharide may produce a synergistic effect, more effectively improving the tolerance and proliferation of probiotics, but this synergistic effect has not yet been fully utilized. Summary of the invention

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

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] Firstly, the present invention provides a method for extracting and purifying old fragrant yellow polysaccharide, which comprises mechanically beating 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 old fragrant yellow crude polysaccharide through heating water extraction and alcohol precipitation steps; after defatting, decolorizing and deproteinizing the old fragrant yellow crude polysaccharide, eluting through a DEAE-52 cellulose ion exchange column and a Sephadex G-100 glucan gel column, and freeze-drying to obtain purified old fragrant yellow polysaccharide.

[0009] Furthermore, the present invention provides a method for preparing a composite, which comprises mixing the purified old fragrant yellow polysaccharide with anthocyanins and milk thistle extract in a certain mass ratio to form a composite with a synergistic effect.

[0010] Furthermore, the mass ratio of the old fragrant yellow polysaccharide, anthocyanin and milk thistle extract is 1:0.5-5:1.

[0011] Furthermore, the anthocyanin is cyanidin-3-O-glucoside (ie, C3G).

[0012] Secondly, the present invention utilizes the complex comprising old fragrant yellow polysaccharide, milk thistle extract and anthocyanin to culture Lactobacillus acidophilus and Bacillus coagulans respectively, and it is found that the complex of the present invention can effectively promote the proliferation of probiotics.

[0013] Therefore, the present invention claims to protect the use of the complex in promoting the proliferation of probiotics.

[0014] The present invention also claims to protect the use of the complex in preparing a product for promoting the proliferation of probiotics.

[0015] The present invention also claims to protect the use of the complex in regulating the balance of intestinal flora or in preparing a product 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 a compound containing old fragrant yellow polysaccharide, anthocyanin and milk thistle extract to culture Lactobacillus acidophilus and Bacillus coagulans respectively, and finds that the compound can promote the proliferation of the above-mentioned probiotics, and can be used to prepare a product that promotes the proliferation of probiotics, thereby helping to regulate the balance of intestinal flora and maintain the stability of the intestinal environment. The present invention not only enriches the source of prebiotics that can promote the proliferation of probiotics, but also enriches the application field of old fragrant yellow polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 shows the growth curves of two probiotic strains, wherein A: Lactobacillus acidophilus (LA), and B: Bacillus coagulans (BC).

[0019] Figure 2 This is the DEAE-52 cellulose column chromatogram of old fragrant yellow polysaccharide.

[0020] Figure 3 This is the Sephadex G-100 gel column chromatogram of old fragrant yellow polysaccharide.

[0021] Figure 4A This is the high-performance gel permeation chromatogram of old fragrant yellow polysaccharide, and the upper right corner is the standard curve of dextran.

[0022] Figure 4B for Figure 4A Dextran standard curve graph in the upper right corner.

[0023] Figure 5 is the ion chromatogram of old fragrant yellow polysaccharide (A is the mixed standard, B is old fragrant yellow polysaccharide). DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

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

[0027] The old fragrant yellow was purchased from Wenxiang Food Factory, Wenci Town, Chaoan District, Chaozhou City, and is one year old fragrant yellow.

[0028] Example 1 Growth curve of probiotics

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

[0030] The activated secondary bacteria were inoculated into MRS broth at a rate of 5 v / v%, mixed thoroughly and placed in a 37°C incubator for static culture. An appropriate amount of culture medium was taken every 2 hours, and the uninoculated MRS was used as a blank control. The optical density (OD) at 600 nm was measured using a microplate reader, referred to as OD600 nm.

[0031] The growth curve of probiotics was drawn 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 structural identification of old fragrant yellow polysaccharide

[0033] The sarcodactylis polysaccharide of the present invention is extracted from sarcodactylis medica (Citrus medicavar.sarcodactylis), and the extraction and purification process thereof comprises the following steps:

[0034] S1. Mechanically beat the old fragrant yellow and freeze-dry it in vacuum to obtain the old fragrant yellow freeze-dried powder;

[0035] S2. sequentially performing heating water extraction and alcohol precipitation, collecting the alcohol precipitate, and obtaining Laoxianghuang crude polysaccharide;

[0036] S3. Defatting the crude polysaccharide of Laoxianghuang obtained in step S2 to obtain defatted crude polysaccharide of Laoxianghuang;

[0037] S4. Decolorizing the defatted Laoxianghuang crude polysaccharide obtained in step S3 to obtain decolorized Laoxianghuang crude polysaccharide;

[0038] S5. Deproteinizing the decolorized old fragrant yellow crude polysaccharide obtained in step S4 to obtain deproteinized old fragrant yellow crude polysaccharide;

[0039] S6. Take the deproteinized old fragrant yellow crude polysaccharide obtained in step S5, separate it using a DEAE-52 cellulose ion exchange column, elute it with water or a sodium chloride solution, and obtain old fragrant yellow graded polysaccharides. Use Sephadex G-100 polyacrylamide gel column to separate the eluted old fragrant yellow graded polysaccharides, elute it with water or a sodium chloride solution, and obtain old fragrant yellow polysaccharides.

[0040] Among them, step S1 specifically includes: mechanically beating the old fragrant yellow, vacuum freeze-drying for 48 hours, and obtaining the old fragrant yellow freeze-dried powder.

[0041] Step S2 specifically includes: mixing the freeze-dried powder of Laoxianghuang with deionized water at a material-liquid ratio of 1g:20mL, extracting at 80°C for 3h, filtering, and combining the filtrate; concentrating under reduced pressure at 60°C to obtain a concentrated solution; adding 95v / v% ethanol in an amount of 4 times the volume of the concentrated solution to the concentrated solution, and standing in a refrigerator at 4°C for 24h; centrifuging at 10000rpm for 10min, discarding the supernatant, washing the obtained precipitate three times with 95v / v% ethanol, concentrating under reduced pressure at 55°C, and freeze-drying for 48h to obtain Laoxianghuang crude polysaccharide powder.

[0042] Step S3 specifically includes: weighing the crude polysaccharide powder of old fragrant yellow, wrapping it in filter paper and placing it in a Soxhlet extractor, adding petroleum ether 4 times the volume of the crude polysaccharide of old fragrant yellow, and refluxing at 90°C for 2h, evaporating the solvent to remove the lipid components in the crude polysaccharide of old fragrant yellow.

[0043] Step S4 specifically includes: dissolving the defatted old fragrant yellow crude polysaccharide in deionized water, adding pretreated AB-8 macroporous resin (refer to the instructions for use of AB-8 resin for the pretreatment method), adsorbing and oscillating at 37°C and 160r / min for 6h for decolorization, standing overnight, filtering to remove the resin, repeating until the color of the polysaccharide becomes lighter, and removing part of the pigment in the old fragrant yellow polysaccharide.

[0044] Step S5 specifically comprises: adding Sevag reagent (chloroform: n-butanol=4:1, v / v) to the decolorized old fragrant yellow crude polysaccharide solution, wherein the volume ratio of the Sevag reagent to the decolorized old fragrant yellow crude polysaccharide solution is 1:4, oscillating for 20 minutes, centrifuging at 10000rpm for 10 minutes, absorbing the upper polysaccharide solution, discarding the lower organic phase and protein precipitation, repeating the extraction 4-5 times, and removing the protein in the solution; using a rotary evaporator to spin-dry the old fragrant yellow polysaccharide layer solution from which the protein is removed, removing the organic reagent contained therein, re-dissolving it with a small amount of deionized water, and dialyzing it with a dialysis bag with a molecular cutoff of 8000-14000Da for 48 hours, and changing the water every 4 hours; and vacuum freeze-drying to obtain deproteinized old fragrant yellow crude polysaccharide powder.

[0045] Step S6 specifically comprises: taking the obtained deproteinized Laoxianghuang crude polysaccharide powder and re-dissolving it in deionized water to make its final concentration 20 mg / mL; separating it with a DEAE-52 cellulose ion exchange column (2.5×40 cm, filler purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) with a sample volume of 10 mL, and eluting it 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 in sequence, and obtaining the elution curve of the DEAE-52 cellulose column as shown in the following figure: Figure 2As shown in FIG. 1 , the elution curve reaches peak values ​​at 0.1 mol / L, 0.2 mol / L and 0.3 mol / L, among which the component eluted with 0.2 mol / L NaCl solution has the highest sugar content and absorbance, so this component is retained for purification. The old fragrant yellow polysaccharide obtained by separation of DEAE-52 cellulose ion exchange column (i.e., the component corresponding to the elution of 0.2 mol / L NaCl solution described in S6) is prepared into an old fragrant yellow polysaccharide solution with a final concentration of 10 mg / mL by adding deionized water; with a sample volume of 1 mL, it is separated by Sephadex G-100 dextran gel column (2.5×40 cm, filler purchased from Shanghai MacLean Biochemical Technology Co., Ltd.), eluted with deionized water at 0.2 mL / min, and collected at 5 mL / tube. The elution curve is shown in FIG. Figure 3 As shown, the components obtained from tubes 5 to 9 were retained and freeze-dried to obtain the old fragrant yellow polysaccharide.

[0046] 1. Experimental methods

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

[0048] The molecular weight of polysaccharide from S6 was determined by HPGPC. The gel column of HPGPC was BRT105103-101 series gel column (8×300mm) and RI-20A differential detector.

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

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

[0051] Chromatographic column: BRT105-103-101 gel column in series (8×300mm); detector: differential detector RID-20A; mobile phase: 0.05mol / LNaCl solution; mobile phase flow rate: 0.8mL / min; injection volume: 25μL; column temperature: 40℃; analysis time: 60min.

[0052] In logM w And t to make a standard curve.

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

[0054] Sixteen kinds of monosaccharide standards were prepared into standard solutions (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid), and each monosaccharide standard solution was taken as a mixed standard. The mass of different monosaccharides was determined according to the absolute quantitative method, and then the molar ratio was calculated according to the molar mass of the monosaccharide.

[0055] Sample preparation: Weigh 5.0 mg of S6 polysaccharide sample into an ampoule, add 2 mL of 3 mol / L TFA (i.e. trifluoroacetic acid), and hydrolyze at 120°C for 3 h. Blow dry with nitrogen, add 5 mL of deionized water, vortex mix, then draw 50 μL and add 950 μL of deionized water, centrifuge at 12000 rpm for 5 min. Take the supernatant for ion chromatography analysis.

[0056] Chromatographic method:

[0057] Column: Dionex TM Carbopac TM PA20 (3*150mm); mobile phase: A: H2O; B: 15mmol / LNaOH; C: 15mmol / LNaOH and 100mmol / LNaAc; injection volume: 25μL; flow rate: 0.3mL / min; column temperature: 30℃; elution gradient: 0minA phase / B phase / C phase (98.8:1.2:0, v / v), 18minA phase / B phase / C phase (98.8:1.2:0, v / v), 20minA phase / B phase / C phase (50:50:0, v / v), 30minA phase / B phase / C phase phase (50:50:0, v / v), 30.1minA phase / B phase / C phase (0:0:100, v / v), 46minA phase / B phase / C phase (0:0:100, v / v), 46.1minA phase / B phase / C phase (0:100:0, v / v), 50minA phase / B phase / C phase (0:100:0, v / v), 50.1minA phase / B phase / C phase (98.8:1.2:0, v / v), 80minA 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 old fragrant yellow polysaccharide

[0060] As can be seen from Figure 4, the purified S6 old fragrant yellow polysaccharide sample is a single symmetrical peak, and no other molecular weight polysaccharide peaks appear, indicating that the obtained old fragrant yellow polysaccharide is a uniform component. At the same time, according to the retention time of polysaccharides 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, M w is the average molecular weight, and the relative average molecular weight of Laoxianghuang polysaccharide is calculated to be 470086Da, the retention time is 25.963min, and the weight average molecular weight M w The number average molecular weight is 485528Da. n The molecular weight distribution width (M w / M n ) is 1.02.

[0061] 2.2 Analysis of monosaccharide composition of old fragrant yellow polysaccharide

[0062] The results are shown in Figure 5. Compared with the retention time of monosaccharide standards ( Figure 5A ) can be known that Figure 5B The monosaccharide components in the middle and old fragrant yellow polysaccharide are fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, with a molar ratio of 0.4:1.9:10:22.7:27.7:0.4:0.6:35.7:0.6, and the molar ratio of total neutral monosaccharide residues to uronic acid residues is 63.7:36.3. The results show that the monosaccharide composition of the old fragrant yellow polysaccharide is mainly glucose and galactose, and also contains some fucose, rhamnose, xylose and mannose.

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

[0064] 1. Experimental methods

[0065] 1.1-1.3 Test samples

[0066] Test samples:

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

[0068] B: 0.6 mg / mL old fragrant yellow polysaccharide + 0.9 mg / mL deionized water;

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

[0070] D: 1.5 mg / mL complex of old fragrant yellow polysaccharide, G3C, and milk thistle extract in a mass ratio of 1:0.5:1;

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

[0072] F: 0.6 mg / mL milk thistle 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 milk thistle extract + 0.6 mg / mL deionized water.

[0074] 1.1 Acid resistance test.

[0075] With MRS liquid culture medium as blank control, the activated strains were inoculated with 2% (v / v) in the sample MRS liquid culture medium with pH 3.0 and blank (con) MRS control culture medium with sterile film added, of which there were 7 groups of sample MRS liquid culture medium. After adding the test samples, they were cultured at 37°C for 4h. 10-fold serial dilution was performed with sterile physiological saline, and 3 groups were repeated for each dilution. 1mL of the appropriate dilution was transferred to a sterile empty plate, and about 15mL of MRS solid culture medium was poured into the culture plate, mixed evenly, and counted after static culture at 37°C for 48h. The survival rate calculation formula is as follows: survival rate (%) = N' / N0×100%. In the formula: N' is the number of viable bacteria under pH 3.0; N0 is the number of viable bacteria in the blank control.

[0076] 1.2 Simulated gastric fluid test

[0077] The activated bacteria were inoculated into MRS liquid medium at a 2% (v / v) inoculation rate for 24 h, centrifuged at 5000 g for 10 min, and the bacterial sludge was collected and resuspended in PBS buffer solution to prepare a resuspension. The concentration was adjusted to 1.0 × 10 8 CFU / mL, 10 mL were taken and inoculated into 100 mL of artificial gastric juice with a pH of 3.0 and added with different test samples (the artificial gastric juice contained 0.20% NaCl and 0.30% pepsin by mass fraction, and the pH was adjusted with HCl / sodium hydroxide), and artificial gastric juice without adding samples was used as blank (con) control. The samples were allowed to stand at 37°C for 0 h and 4 h, and then diluted 10 times in a gradient manner into MRS solid culture medium for counting, and the survival rate was calculated, survival rate (%) = N1 / N0×100%, wherein N1: the number of viable bacteria after 4 h of artificial gastric juice treatment; N0: the number of viable bacteria after 0 h of artificial gastric juice treatment.

[0078] 1.3 Simulated intestinal fluid test

[0079] The activated bacteria were inoculated into MRS liquid medium at a 2% (v / v) inoculation rate for 24 h, centrifuged at 5000 g for 10 min, and the bacterial sludge was collected and resuspended in PBS buffer solution to prepare a resuspension. The concentration was adjusted to 1.0 × 10 8 CFU / mL, 10mL was inoculated into 100mL artificial intestinal fluid with pH 3.0 and added with different test samples (different MRS liquid culture media were prepared with 0.1mol / L phosphate buffered saline, the pH of the liquid culture medium was adjusted to 7 with hydrochloric acid / sodium hydroxide, sterilized by high temperature and high pressure treatment at 121°C for 15min, 1g of trypsin was added to 100mL of the sterilized liquid culture medium to obtain in vitro simulated intestinal fluid), artificial intestinal fluid without adding samples was used as blank (con) control, and the cells were allowed to stand at 37°C for 0h and 4h, and then diluted 10-fold into MRS solid culture medium for counting, and the survival rate was calculated, survival rate (%) = N1 / N0×100%, wherein N1: the number of viable bacteria after 4h of artificial intestinal fluid treatment; N0: the number of viable bacteria after 0h of artificial intestinal fluid treatment.

[0080] 2. Experimental results

[0081] Table 1 Effects of C3G, old fragrant yellow polysaccharide, milk thistle extract and their complexes on the survival rate of Lactobacillus acidophilus and Bacillus coagulans under different environments

[0082]

[0083] "*" indicates significant difference from "con (blank)" control, p < 0.01

[0084] 2.1 Effects of different additives on the survival rate of probiotics in an acidic environment

[0085] It can be seen from Table 1 that, whether in an acidic environment or in gastric juice simulation or intestinal juice simulation, the effect of the complex on the survival rate of Lactobacillus acidophilus and Bacillus coagulans is stronger than that of C3G or old fragrant yellow polysaccharide or milk thistle extract used alone or in combination of the two. Therefore, it can be concluded that there is a certain synergistic effect between C3G, old fragrant yellow polysaccharide and milk thistle extract. In view of this survival characteristic, the following proliferation experiment only explores the proliferation effect of the complex of C3G, old fragrant yellow polysaccharide and milk thistle extract on Lactobacillus acidophilus probiotics at different mass ratios.

[0086] Example 4 Effect of the composite of C3G, old fragrant yellow polysaccharide and milk thistle extract on the growth and proliferation of probiotics

[0087] 1. Experimental methods

[0088] The activated Lactobacillus acidophilus strain was inoculated into MRS liquid medium at a 2% (v / v) inoculation rate for 24 h, and centrifuged at 5000 g for 10 min. The bacterial sludge was collected and resuspended in PBS buffer solution to prepare a resuspension. The concentration was adjusted to 1.0×10 8 CFU / mL, 10 mL was inoculated into 100 mL of MRS medium with a pH of 7.0 and added with 1.5 mg / mL of different samples (AG), and 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 different mass ratios of the complex of old fragrant yellow polysaccharide, C3G and milk thistle extract on the proliferation rate of Lactobacillus acidophilus.

[0092]

[0093] P value is the significance analysis between af ​​group and blank group.

[0094] It can be seen from Table 2 that when the mass ratio of old fragrant yellow polysaccharide, C3G and milk thistle extract falls within the numerical range specified by the present invention (i.e., groups a, b, and c), the composite has little difference in the growth and proliferation effect of Lactobacillus acidophilus, and has a certain proliferation effect compared to the blank. When the mass ratio of the three exceeds the numerical range specified by the present invention (i.e., groups d and f), compared with group a, the results show that its proliferation rate is decreasing, and when the mass ratio of old fragrant yellow polysaccharide increases (group e), it will lead to an increase in the proliferation rate of Lactobacillus acidophilus, but compared with the amount of old fragrant yellow polysaccharide added, the increase in its proliferation rate is lower, and the cost performance is lower.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A composite, characterized in that The mass ratio of old fragrant yellow polysaccharide, anthocyanin and milk thistle extract in the complex is 1:0.5-5:1, and the anthocyanin is cyanidin-3-O-glucoside.

2. A method for preparing the composite according to claim 1, characterized in that: The preparation method comprises the following steps: S1. Mechanically beat the old fragrant yellow and freeze-dry it in vacuum to obtain the old fragrant yellow freeze-dried powder; S2. The freeze-dried powder of Laoxianghuang was mixed with deionized water at a material-liquid ratio of 1g:20mL, extracted with water at 80℃ for 3h, filtered to obtain a filtrate, concentrated under reduced pressure at 60℃ to obtain a concentrate, 95v / v% ethanol in an amount of 4 times the volume of the concentrate was added to the concentrate, allowed to stand at 4℃ for 24h, and then centrifuged at 10000rpm for 10min, the supernatant was discarded, the precipitate was washed with 90-98v / v% ethanol, concentrated under reduced pressure at 55℃, and freeze-dried to obtain Laoxianghuang crude polysaccharide powder; S3. Weigh the crude polysaccharide powder of Laoxianghuang and perform Soxhlet extraction with petroleum ether to defat it; S4. The defatted crude polysaccharide of Laoxianghuang was dissolved in deionized water and decolorized by passing through AB-8 macroporous resin; S5. The decolorized crude polysaccharide solution of old fragrant yellow was deproteinized with Sevag reagent and then freeze-dried to obtain crude polysaccharide powder of old fragrant yellow; S6. The crude polysaccharide powder of old fragrant yellow treated by S5 was separated by DEAE-52 cellulose ion exchange column and further purified by Sephadex G-100 polysaccharide gel chromatography column to obtain old fragrant yellow polysaccharide; S7. The obtained old fragrant yellow polysaccharide is mixed with anthocyanins and milk thistle extract according to a mass ratio to obtain a composite.

3. The preparation method according to claim 2, characterized in that: The S5 freeze-drying step also includes a dialysis step using an 8000-14000Da dialysis bag before the step.

4. A method for improving the tolerance and proliferation ability of probiotics using the compound according to claim 1, characterized in that: The following steps are involved: T1: inoculating the probiotics into an MRS medium containing 1-5 mg / mL of the complex of claim 1; T2: Cultivate probiotics at 37±0.5℃ for 12-48 hours; The probiotics are Lactobacillus acidophilus or Bacillus coagulans.

5. A functional food, characterized in that: Comprising the complex according to claim 1.

6. A health product, characterized in that: Comprising the complex according to claim 1.

7. An application of a composite, characterized in that: The complex according to claim 1 is used to promote the proliferation of probiotics.

8. An application of a composite, characterized in that: The complex according to claim 1 is used for regulating the balance of intestinal flora.

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