An rg-i pectin-based supplement, and methods of making and using the same

Abstract

The application discloses an RG-I pectin-based supplement and a preparation method and application thereof, and belongs to the technical field of food additives.The RG-I pectin rich in galactose side chains is obtained by sequentially performing acid treatment, purification treatment and enzymolysis treatment on a fruit peel; then the RG-I pectin rich in galactose side chains is mixed with a whey protein solution to obtain a probiotic RG-I pectin-based supplement with a double-layer microcapsule structure.The composite probiotic microcapsule has good gastrointestinal anti-digestion property, can significantly improve the utilization rate of the probiotic, and can promote the production of short-chain fatty acids and the proliferation of probiotics in the intestinal tract, better regulate the fermentation balance of the intestinal flora of weaned infants.Therefore, the application has a good application prospect in the preparation of products for regulating the intestinal flora of weaned infants.

Description

Technical Field

[0001] This invention belongs to the field of food additive technology, specifically relating to an RG-I pectin-based supplement, its preparation method, and its application. Background Technology

[0002] Currently, traditional prebiotics such as fructooligosaccharides (FOS), β-galacto-oligosaccharides (GOS), and xylooligosaccharides (XOS) hold a relatively high market share. However, some of these prebiotics exhibit problems with their fermentation properties under specific gut conditions. For example, rapidly fermented prebiotics may cause discomfort and bloating. Pectin has a more comprehensive regulatory effect on the gut microbiota. Traditional prebiotics overemphasize their effects on common probiotics (such as Bifidobacteria and Lactobacilli), while pectin can benefit a wide range of probiotics, including Bacteroides, which constitute a large part of the human gut microbiome. Pectin has been reported to help increase glycolytic activity in the distal colon and enhance cross-feeding interactions between gut bacteria. Therefore, pectin provides better microbiome diversity as a substrate, modulating gut bacteria in a slow but powerful manner, avoiding drastic changes in microbiome composition. In conclusion, the fermentation properties of pectin are desirable and compensate for the shortcomings of traditional prebiotics.

[0003] For young mammals, separation from their mother or weaning is a complex process, often accompanied by physiological, behavioral, and nutritional changes. Infants typically enter the weaning period between 6 and 12 months. The stress response to weaning can easily lead to gastrointestinal imbalances, manifesting as diarrhea or other illnesses, all related to changes in the gut microbiota composition. Reports indicate that Bifidobacterium supplementation can correct gut microbiota and improve intestinal inflammation in weaned infants. Compared to adding a single probiotic to improve the gut microbiota, supplementing with appropriate prebiotics has received more attention. Currently, there are many types of prebiotic products on the market, but their efficacy is unclear and their utilization rate is low. Furthermore, prebiotics usually need to be taken orally, passing through the gastrointestinal tract to reach the colon where they are utilized by gut microbiota to exert their effects. Therefore, the integrity of prebiotics reaching the colon is also a crucial factor affecting their efficacy. Prebiotic microencapsulation helps prebiotics safely reach the colon, but problems remain, such as the ease with which prebiotic microcapsules are digested in the gastrointestinal tract, leading to low prebiotic utilization rates. Summary of the Invention

[0004] The purpose of this invention is to provide an RG-I pectin-based supplement, its preparation method, and its application. The RG-I pectin-based supplement provided by this invention is a double-layered microcapsule obtained by compounding RG-I pectin prebiotics and whey protein prebiotics. This compound prebiotic microcapsule has good gastrointestinal resistance to digestion and can significantly improve the utilization rate of prebiotics.

[0005] In a first aspect, the present invention provides a method for preparing an RG-I pectin-based supplement, comprising the following steps: S1, acid treatment of fruit peel to obtain water-soluble pectin; S2, purification of the water-soluble pectin, followed by enzymatic hydrolysis with a complex enzyme to obtain RG-I pectin rich in galactose side chains; S3, addition of an additive to a whey protein solution to obtain a mixture, mixing the RG-I pectin rich in galactose side chains with the mixture and stirring to obtain a further mixture, centrifuging the mixture and drying the resulting precipitate to obtain the RG-I pectin-based supplement; wherein, in step S2, the complex enzyme includes polygalacturonidase and polygalacturonidase.

[0006] In this invention, the inventors discovered that RG-I pectin rich in galactose side chains can be obtained by using a specific method (first acid treatment, then purification, and finally enzymatic hydrolysis). Then, the RG-I pectin rich in galactose side chains is mixed with whey protein solution to obtain a prebiotic RG-I pectin-based supplement with a double-layer microcapsule structure. This composite prebiotic microcapsule has good gastrointestinal resistance to digestion and can significantly improve the utilization rate of prebiotics.

[0007] In some embodiments, the peel is red dragon fruit peel, and the acid in the acid treatment is citric acid; the preparation of water-soluble pectin includes: adding red dragon fruit peel to a citric acid solution, and then reacting at a temperature of 40-70°C (e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C or other values ​​within this range) for 1-3 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours or other values ​​within this range); after centrifugation, the supernatant is collected, and 1-3 times (e.g., 1 time, 1.5 times, 2 times, 2.5 times, 3 times or other values ​​within this range) of organic solvent is added to the supernatant, the precipitate is filtered, and the precipitate is dried to obtain water-soluble pectin.

[0008] In some embodiments, the citric acid solution has a mass concentration of 0.5% to 1%, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or other values ​​within this range; the red dragon fruit peel has a mass concentration of 1% to 10% (w / v), for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or other values ​​within this range; and the organic solvent is anhydrous ethanol.

[0009] In some implementations, step S2, the purification process includes: sequentially passing the water-soluble pectin through a DEAE cellulose column and a Sephadex G-75 column to obtain a purified solution; the purified solution is then dialyzed and freeze-dried to obtain purified water-soluble pectin; wherein, in the DEAE cellulose column purification, the eluent flow rate is 1 mL / min, and the eluent includes deionized water and 0.1–0.5 M NaCl solution, and the elution method is gradient elution (specifically, the gradient elution program is as follows: 0–30 min deionized water elution, 30–60 min 0.1 M NaCl solution elution, 60–90 min 0.2 M NaCl solution elution, 90–120 min 0.3 M NaCl solution elution, 120–150 min 0.4 M NaCl solution elution, 150–180 min 0.5 M NaCl solution elution), collecting the eluent from 60–90 min, and then loading it onto a Sephadex column. In Sephadex G-75 column separation and purification, the eluent flow rate is 0.15 mL / min, and the eluent includes 0.1–0.5 M (e.g., 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, or other values ​​within this range) NaCl solution, and the elution method is isocratic elution; the dialysis treatment includes: placing the purified solution in a 3.5 kDa dialysis bag and dialyzing for 24–48 h, e.g., 24 h, 30 h, 36 h, 42 h, 48 h, or other values ​​within this range.

[0010] In some embodiments, step S2, the enzymatic hydrolysis includes: adding polygalacturonidase and polygalacturonidase to a purified water-soluble pectin solution with a mass concentration of 1–10 mg / mL (e.g., 1 mg / mL, 3 mg / mL, 5 mg / mL, 7 mg / mL, 9 mg / mL, 10 mg / mL, or other values ​​within this range) to carry out an enzymatic hydrolysate, thereby obtaining an enzymatic hydrolysate; wherein the final concentration of polygalacturonidase and polygalacturonidase in the pectin solution is 1–10 U / mL, for example, 1 U / mL, 3 U / mL, 5 U / mL, 7 U / mL, 9 U / mL, 10 U / mL. L or other values ​​within that range; the mass ratio of polygalacturonidase to polygalacturonidase is (1-3):1, for example, 1:1, 1.5:1, 2:1, 2.5:1, 3:1 or other values ​​within that range; the temperature of the enzymatic hydrolysis reaction is 35-40℃, for example, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃ or other values ​​within that range; the time is 12-24h, for example, 12h, 15h, 18h, 21h, 24h or other values ​​within that range; and after the enzymatic hydrolysis treatment, the step of separating and purifying the hydrolysate to obtain RG-I pectin rich in galactose side chains is also included.

[0011] In some embodiments, the separation and purification process in obtaining RG-I pectin rich in galactose side chains includes: centrifuging the enzymatic hydrolysate, loading the supernatant onto a Sephadex G-75 column, eluting with 0.1M NaCl solution at a flow rate of 0.15 mL / min, and lyophilizing the eluent to obtain RG-I pectin rich in galactose side chains.

[0012] In some embodiments, in step S3, the whey protein solution in the mixture has a mass concentration of 6-12%, for example, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or other values ​​within this range; the additives include glucono-delta-lactone and calcium chloride, and the mass concentrations of glucono-delta-lactone and calcium chloride added are both 0.1-0.4% (w / v), for example, 0.1%, 0.2%, 0.3%, 0.4%, or other values ​​within this range.

[0013] In some embodiments, in step S3, during the process of obtaining the mixture, the volume ratio of RG-I pectin rich in galactose side chains to the mixture is (2-6):1, for example, it can be 2:1, 3:1, 4:1, 5:1, 6:1 or other values ​​within this range; wherein the mass concentration of RG-I pectin rich in galactose side chains is 1-3%, for example, it can be 1%, 1.5%, 2%, 2.5%, 3% or other values ​​within this range; the stirring treatment includes: a stirring speed of 700-900 rpm, for example, it can be 700 rpm, 750 rpm, 800 rpm, 850 rpm, 900 rpm or other values ​​within this range; The interval is 5 to 30 minutes, for example, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min or other values ​​within this range; in the process of obtaining RG-I pectin-based supplement, centrifugation includes: centrifuging at a speed of 7000 to 9000 rpm (for example, 7000 rpm, 7500 rpm, 8000 rpm, 8500 rpm, 9000 rpm or other values ​​within this range) for 5 to 15 minutes, for example, 5 min, 7 min, 9 min, 11 min, 13 min, 15 min or other values ​​within this range.

[0014] In a second aspect, the present invention provides an RG-I pectin-based supplement prepared by any of the above-described preparation methods.

[0015] In this invention, the inventors discovered that the RG-I pectin-based supplement prepared by this invention contains abundant galactose side chains. When applied to the preparation of products that regulate the intestinal flora of weaned infants, it is beneficial to significantly increase the diversity of intestinal microorganisms during the fermentation stage and increase the production of beneficial metabolites.

[0016] In a third aspect, the present invention provides the application of the above-mentioned RG-I pectin-based supplement in the preparation of products that regulate the intestinal flora of weaning infants.

[0017] In this invention, the inventors discovered that when the prebiotic RG-I pectin-based supplement functional food prepared according to this invention is applied to the preparation of products that regulate the intestinal flora of weaning infants, a balance between carbohydrate fermentation and protein fermentation is achieved. After the pectin-based functional food is fermented by the intestinal flora in the colon, it promotes the production of short-chain fatty acids and the proliferation of probiotics, thus better regulating the fermentation balance of the intestinal flora of weaning infants.

[0018] The beneficial effects of this invention are as follows: Unlike the prior art, this invention obtains RG-I pectin rich in galactose side chains by sequentially subjecting the fruit peel to acid treatment, purification treatment, and enzymatic hydrolysis; then, the RG-I pectin rich in galactose side chains is mixed with whey protein solution to obtain a prebiotic RG-I pectin-based supplement with a double-layer microcapsule structure. This composite prebiotic microcapsule has good gastrointestinal resistance to digestion, can significantly improve the utilization rate of prebiotics, and can promote the production of short-chain fatty acids and the proliferation of probiotics in the intestine, thus better regulating the fermentation balance of the intestinal flora in weaning infants. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation method of the RG-I pectin-based supplement of the present invention;

[0020] Figure 2 The above are heatmaps of in vitro fermentation of the RG-I pectin-based supplement prepared in Example 1 of the present invention into the intestinal flora of infants with diarrhea during weaning, fermentation samples of intestinal flora of infants with diarrhea during weaning, and fermentation samples of intestinal flora of normal infants during weaning.

[0021] Figure 3 The total SFCA content is the amount obtained after adding the RG-I pectin-based supplements prepared in Examples 1-6 and Comparative Examples 1-2 of this invention to the intestinal flora of infants with diarrhea during weaning period and fermenting them in vitro for 48 hours. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0024] Please see Figure 1 The flowchart of the preparation method of the RG-I pectin-based supplement of the present invention is shown below, which specifically includes the following steps: S1, acid treatment of fruit peel to obtain water-soluble pectin; S2, purification treatment of water-soluble pectin, and then enzymatic hydrolysis treatment with a compound enzyme to obtain RG-I pectin rich in galactose side chains.

[0025] S3. Add additives to whey protein solution to obtain a mixture. Mix RG-I pectin rich in galactose side chains with the mixture and stir to obtain a mixture. After centrifugation, dry the resulting precipitate to obtain RG-I pectin-based supplement. In step S2, the complex enzyme includes polygalacturonidase and polygalacturonidase.

[0026] Example 1

[0027] A method for preparing an RG-I pectin-based supplement includes the following steps:

[0028] S1. Add 3% (w / v) red dragon fruit peel to a 0.8% citric acid solution, react at 60℃ for 1.5h, centrifuge, take the supernatant, add 2 times the volume of anhydrous ethanol to the supernatant, filter to take the precipitate, and dry to obtain water-soluble pectin.

[0029] S2. The water-soluble pectin obtained in step S1 is loaded onto a DEAE cellulose column and eluted using a gradient of deionized water and 0.2M NaCl solution at a flow rate of 1 mL / min to obtain the principal component. This principal component is then loaded onto a Sephadex G-75 column and eluted using 0.2M NaCl solution. Isocratic elution was performed using NaCl solution at a flow rate of 0.15 mL / min to obtain the purified main fraction. The purified main fraction was dialyzed in a 3.5 kDa dialysis bag for 36 h, then freeze-dried and stored at 4 °C to obtain purified water-soluble pectin. This purified water-soluble pectin was diluted to 5 mg / mL with 50 mM sodium acetate buffer, and then a complex enzyme (polygalacturonidase and polygalacturonidase in a mass ratio of 2:1) was added to a final concentration of 2 U / mL in the pectin solution. The enzymatic hydrolysis reaction was carried out at 37 °C for 20 h, followed by heating at 100 °C for 5 min to terminate the reaction, yielding the enzymatic hydrolysate. After centrifugation, the supernatant was loaded onto a Sephadex G-75 column and treated with 0.1 M... The NaCl solution was eluted at a flow rate of 0.15 mL / min, and the eluent was freeze-dried to obtain RG-I pectin rich in galactose side chains.

[0030] S3. Dissolve whey protein in sterile water to prepare an 8% (w / w) protein solution, and place it in a magnetically stirred water bath. Heat at 8000 rpm and 45°C for 2 hours, then transfer to an 80°C water bath and continue heating for 30 minutes. After heating, cool to room temperature to obtain a whey protein solution. Add 0.3% (w / v) gluconolactone and 0.15% (w / v) calcium chloride to the whey protein solution, and heat and stir at 40°C and 600 rpm for 2 hours to obtain a mixture. Adjust the mass concentration of RG-I pectin rich in galactose side chains obtained in step S2 to 2%, and mix the RG-I pectin rich in galactose side chains with the above mixture at a volume ratio of 4:1. Stir at 800 rpm for 10 minutes. Then centrifuge the mixture at 8000 rpm for 10 minutes, and dry the resulting precipitate to obtain an RG-I pectin-based supplement with a double-layer microcapsule structure.

[0031] Example 2

[0032] The preparation method of RG-I pectin-based supplement in this embodiment is basically the same as that in Example 1. The difference is that in step S1, 5% (w / v) red dragon fruit peel is added to a 0.5% citric acid solution, and then the mixture is reacted at 60°C for 1 hour.

[0033] Example 3

[0034] The preparation method of RG-I pectin-based supplement in this embodiment is basically the same as that in Example 1. The difference is that in step S2, the purified water-soluble pectin is diluted to 10 mg / mL with 50 mM sodium acetate buffer.

[0035] Example 4

[0036] The preparation method of RG-I pectin-based supplement in this embodiment is basically the same as that in Example 1. The difference is that in step S2, the mass ratio of polygalacturonidase to polygalacturonidase is 1:1.

[0037] Example 5

[0038] The preparation method of RG-I pectin-based supplement in this embodiment is basically the same as that in Example 1. The difference is that in step S3, gluconolactone with a mass concentration of 0.2% (w / v) and calcium chloride with a mass concentration of 0.3% (w / v) are added respectively.

[0039] Example 6

[0040] The preparation method of the RG-I pectin-based supplement in this embodiment is basically the same as that in Example 1. The difference is that in step S3, the RG-I pectin rich in galactose side chains is mixed with the above mixture at a volume ratio of 2:1.

[0041] Comparative Example 1

[0042] The preparation method of the RG-I pectin-based supplement in this comparative example is basically the same as that in Example 1. The difference is that in step S3, whey protein is dissolved in sterile water to prepare a protein solution with a mass concentration of 5%.

[0043] Comparative Example 2

[0044] The preparation method of the RG-I pectin-based supplement in this comparative example is basically the same as that in Example 1. The difference is that in step S2, the complex enzyme is replaced with β-galactosidase, and finally RG-I pectin containing arabinose side chains is obtained.

[0045] Performance testing

[0046] The monosaccharide composition, molecular characteristics, and pectin yield of the RG-I pectin rich in galactose side chains prepared in Example 1 were determined, and the results are shown in Table 1 below:

[0047] Table 1. Performance test results of RG-I pectin rich in galactose side chains

[0048]

[0049] As can be seen from the data in Table 1, the galactose content in the RG-I pectin prepared by this invention reaches more than 42%.

[0050] The RG-I pectin-based supplement with a bilayer microcapsule structure prepared in Example 1 was subjected to an in vitro simulated digestion test, including the following steps:

[0051] 1) Prepare separate digestive systems for saliva, gastric juice, and intestinal juice;

[0052] The salivary digestive system is prepared as follows:

[0053] 24 mg NaCl, 30 mg KCl and 200 mg mucin were dissolved in 100 mL distilled water to prepare a stock solution, which was then stored at 4 °C. Subsequently, 20 mL of the stock solution was placed in a 100 mL Erlenmeyer flask, and 3 mg α-amylase was added and mixed well to obtain the salivary digestion system.

[0054] The gastric digestive system is prepared as follows:

[0055] Weigh out 775 mg NaCl, 37.5 mg CaCl2, 275 mg KCl and 150 mg NaHCO3 and dissolve them in 250 mL of deionized water. Adjust the pH to 3 with 0.1 M HCl to prepare a simulated gastric electrolyte solution. Then, take 100 mL of the simulated gastric electrolyte solution and add 23.6 mg pepsin, 25 mg gastric lipase and 1 mL sodium acetate solution (1 M) in sequence. After mixing well, adjust the pH of the resulting solution to 2 with 0.1 M HCl solution to obtain the gastric juice digestion system.

[0056] The intestinal digestive system is prepared as follows:

[0057] Weigh out 5.4g NaCl, 0.65g KCl, 0.33g CaCl2, and 0.3g NaHCO3 respectively, dissolve them in 1L of deionized water, mix thoroughly, and adjust the pH of the solution to 7 with 0.1M NaOH to prepare a simulated small intestinal electrolyte solution. Then, take 200mL of the simulated small intestinal electrolyte solution and add 200mL of pancreatic enzyme solution (7%, w / w), 400mL of bile salt solution (4%, w / w), and 26mg of trypsin. Mix the solution thoroughly and adjust the pH to 7.5 to obtain the intestinal digestion system.

[0058] 2) The RG-I pectin-based supplement prepared in Example 1 was first digested in a salivary digestion system for 1 hour, and samples were taken after 0, 0.25, 0.5, and 1 hour; then it was transferred to a gastric digestion system for 6 hours, and samples were taken after 0, 2, 4, and 6 hours; finally, it was transferred to an intestinal digestion system for 6 hours, and samples were taken after 0, 2, 4, and 6 hours.

[0059] 3) The reducing sugar content and molecular weight in the sample taken in step 2) were detected, and the results are shown in Table 2 below:

[0060] Table 2. Results of reducing sugar content and molecular weight of RG-I pectin-based supplements during in vitro digestion.

[0061]

[0062]

[0063] Typically, digestion by saliva and the gastrointestinal tract can lead to the degradation of pectin and the production of some reducing sugars. Therefore, an increase in reducing sugar content and a decrease in molecular weight can be used to determine the cleavage of glycosidic bonds in pectin. As can be seen from Table 2, the RG-I pectin-based supplement with a double-layer microcapsule structure prepared in this invention showed almost no increase in reducing sugar content and almost no decrease in molecular weight during in vitro digestion. The results indicate that the RG-I pectin-based supplement has good gastrointestinal resistance to digestion.

[0064] Application testing

[0065] Stool samples were collected from weaned infants aged 6-12 months, divided into two groups:

[0066] Normal intestinal control group: Two male infants and two female infants, with no digestive tract diseases and no antibiotic use in the past two months. Equal amounts of feces from each infant were mixed and diluted with sterile physiological saline to prepare a fecal suspension of normal intestinal control group with a mass concentration of 10% (w / v).

[0067] Treatment group: Two male infants and two female infants presented with diarrhea symptoms and had not used antibiotics for nearly two months. Equal amounts of feces from each infant were mixed and diluted with sterile saline to prepare a 10% (w / v) fecal suspension for the treatment group.

[0068] Fermentation medium: Weigh 5g peptone, 4.5g yeast extract, 0.5g bile salts, 0.1g NaCl, 2.0g NaHCO3, 0.5g L-cysteine ​​hydrochloride, 0.04g KH2PO4, 0.04g K2HPO4, 0.01g MgSO4·7H2O, 0.01g CaCl2·6H2O, 0.02g heme chloride, 2mL Tween 80, 10μL vitamin K1, and 1mL resin azure solution (1.0%, w / v) to prepare 1L of fermentation medium. Adjust the pH to 7 with 0.1M HCl and sterilize at 121℃ for 15min before use.

[0069] The steps for determining gut microbiota are as follows:

[0070] 1) The experiment was divided into three groups: blank group: 10 mL of fecal bacterial solution from the treatment group and 90 mL of fermentation medium were added to the Erlenmeyer flask; positive control group: 10 mL of fecal bacterial solution from the normal intestinal control group and 90 mL of fermentation medium were added to the Erlenmeyer flask; experimental group: 10 mL of fecal bacterial solution from the treatment group, 90 mL of fermentation medium and 0.8 g of RG-I pectin-based supplement prepared in Example 1 were added to the Erlenmeyer flask.

[0071] 2) Place all the Erlenmeyer flasks from step 1) in a 37℃ anaerobic incubator, and take samples after 48 hours to determine the intestinal flora. The results are as follows: Figure 2 As shown.

[0072] from Figure 2 As can be seen, after adding the RG-I pectin-based supplement of the present invention to the fecal suspension of the treatment group with diarrhea symptoms, the composition of its flora was almost identical to that of the normal intestinal control group; the results show that the RG-I pectin-based supplement prepared by the present invention can promote the proliferation of probiotics in the intestine and better regulate the fermentation balance of the intestinal flora of weaning infants.

[0073] The procedure for determining short-chain fatty acids (SCFA) is as follows:

[0074] Eight Erlenmeyer flasks were prepared. 10 mL of the treatment group's fecal microbial solution and 90 mL of fermentation medium were added to each flask. Then, 0.8 g of the RG-I pectin-based supplement prepared in Examples 1-6 and Comparative Examples 1-2 was added to each flask. The flasks were placed in an anaerobic incubator at 37°C, and samples were taken after 48 hours to determine the short-chain fatty acids. The results are as follows: Figure 3 As shown.

[0075] from Figure 3 As can be seen from the results, the content of short-chain fatty acids produced by the RG-I pectin-based supplements prepared in Examples 1-6 is significantly higher than that in Comparative Examples 1 and 2. The results further indicate that the RG-I pectin-based supplements prepared in this invention can significantly promote the production of short-chain fatty acids in the intestine.

[0076] The difference between Comparative Example 2 and Example 1 is that the RG-I pectin prepared in Example 1 contains galactose side chains, while the RG-I pectin prepared in Comparative Example 2 contains arabinose side chains. The results show that the RG-I pectin containing specific galactose side chains prepared in this invention can significantly promote the production of short-chain fatty acids in the intestine.

[0077] In summary, the RG-I pectin-based supplement prepared by this invention not only has good gastrointestinal resistance to digestion and can significantly improve the utilization rate of prebiotics, but also promotes the production of short-chain fatty acids and the proliferation of probiotics in the intestine, thus better regulating the fermentation balance of intestinal flora in weaning infants.

[0078] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0079] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing an RG-I pectin-based supplement, characterized in that, Includes the following steps: S1. Acid treatment of the fruit peel yields water-soluble pectin; S2. The water-soluble pectin is purified and then enzymatically hydrolyzed with a compound enzyme to obtain RG-I pectin rich in galactose side chains. S3. Add additives to whey protein solution to obtain a mixture. Mix the RG-I pectin rich in galactose side chains with the mixture and stir to obtain a mixture. After centrifugation, dry the precipitate to obtain RG-I pectin-based supplement. In step S2, the complex enzyme includes polygalacturonidase and polygalacturonidase. In step S1, the peel is red dragon fruit peel, and the acid in the acid treatment is citric acid; the preparation of the water-soluble pectin includes: adding red dragon fruit peel to a citric acid solution, reacting at a temperature of 40~70℃ for 1~3 hours, centrifuging to collect the supernatant, adding 1~3 times the volume of organic solvent to the supernatant, filtering to collect the precipitate, and drying to obtain the water-soluble pectin; The organic solvent is anhydrous ethanol; In step S2, the purification process includes: passing the water-soluble pectin sequentially through a DEAE cellulose column and a Sephadex G-75 column to separate and purify it, obtaining a purified solution, and then dialyzing and freeze-drying the purified solution to obtain purified water-soluble pectin. In the DEAE cellulose column separation and purification, the eluent flow rate is 1 mL / min, and the eluent includes deionized water and 0.1~0.5M NaCl solution, with gradient elution. In the Sephadex G-75 column separation and purification, the eluent flow rate is 0.15 mL / min, and the eluent includes 0.1~0.5M NaCl solution, with isocratic elution. The dialysis treatment includes placing the purified solution in a 3.5 kDa dialysis bag and dialyzing for 24~48 h. In step S3, the whey protein solution in the mixture has a mass concentration of 6-12%, the additives include glucono-delta-lactone and calcium chloride, and the mass concentrations of glucono-delta-lactone and calcium chloride are both 0.1-0.4%.

2. The preparation method of the RG-I pectin-based supplement according to claim 1, characterized in that, The citric acid solution has a mass concentration of 0.5-1%, and the red dragon fruit peel has a mass concentration of 1-10%.

3. The preparation method of the RG-I pectin-based supplement according to claim 1, characterized in that, In step S2, the enzymatic hydrolysis includes: adding the polygalacturonidase and polygalacturonidase to a purified water-soluble pectin solution with a mass concentration of 1~10 mg / mL to carry out the enzymatic hydrolysis reaction, and obtaining the enzymatic hydrolysate; The final concentrations of polygalacturonidase and polygalacturonidase in the pectin solution are 1-10 U / mL, and the mass ratio of polygalacturonidase to polygalacturonidase is (1-3):1; the enzymatic hydrolysis reaction is carried out at a temperature of 35-40℃ for 12-24 hours. Furthermore, after the enzymatic hydrolysis, the process further includes separating and purifying the enzymatic hydrolysate to obtain RG-I pectin rich in galactose side chains.

4. The preparation method of the RG-I pectin-based supplement according to claim 3, characterized in that, In the step of obtaining RG-I pectin rich in galactose side chains, the separation and purification includes: centrifuging the enzymatic hydrolysate, loading the supernatant onto a Sephadex G-75 column, eluting with 0.1M NaCl solution at a flow rate of 0.15 mL / min, and lyophilizing the eluent to obtain RG-I pectin rich in galactose side chains.

5. The method for preparing the RG-I pectin-based supplement according to claim 1, characterized in that, In step S3, during the process of obtaining the mixture, the volume ratio of the RG-I pectin rich in galactose side chains to the mixture is (2~6):1, wherein the mass concentration of the RG-I pectin rich in galactose side chains is 1~3%, and the stirring treatment includes: stirring speed of 700~900 rpm and time of 5~30 min; In the process of obtaining the RG-I pectin-based supplement, the centrifugation includes centrifuging at a speed of 7000~9000 rpm for 5~15 min.

6. The RG-I pectin-based supplement prepared by the preparation method according to any one of claims 1-5.

7. The use of the RG-I pectin-based supplement of claim 6 in the preparation of products for regulating the intestinal flora of weaning infants.

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