Hawthorn pectin as well as preparation method and application thereof

Abstract

The invention discloses hawthorn pectin as well as a preparation method and application thereof, and belongs to the field of medicines. The preparation method comprises the following steps: adding compound enzyme into fresh hawthorn fruit homogenate for enzymolysis, adding probiotics (lactobacillus and bifidobacterium) for fermentation, carrying out alcohol precipitation, separating and purifying, and drying to obtain the hawthorn pectin. The hawthorn pectin can play a role in preventing and / or treating enteritis, remarkably increase the length of colon villus and the thickness of muscular layers of mice with colitis, promote the protein expression of perimembrane protein family protein 1 (ZO-1) and occludin (Occludin), increase the species number of cecum content flora and increase the activity of colitis. According to the present invention, with the application of the cecum extract to the preparation of the cecum extract, the abundance of the Mucispiril bacterial genus, the desulfo vibrio genus, the prevotella genus and the Bilophila genus in the cecum content can be inhibited, and the related metabolic level in the KEGG pathway and the MetaCyc pathway of the cecum content flora can be adjusted;

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to hawthorn pectin and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Inflammatory bowel disease (IBD) is a group of chronic intestinal diseases with unknown etiology, including ulcerative colitis (UC) and Crohn's disease (CD). With the changes in people's lifestyles and the improvement of living standards in recent years, its incidence has shown an increasing trend year by year, and has become the main cause of chronic diarrhea and bloody stools in my country. At present, studies speculate that the internal cause of IBD is the damage to the intestinal barrier and the disorder of the intestinal immune system. After the barrier function is damaged, symbiotic bacteria and microbial products are induced to enter the intestinal wall from the intestinal cavity, leading to the activation of immune cells and the production of cytokines. At the same time, chronic inflammation can also cause damage to intestinal tissues and barriers. In addition, external factors such as genetic factors, environmental factors, and the interaction between hosts and microorganisms play an important role in the occurrence and development of IBD. There are many drugs for the treatment of IBD in clinical practice, which can be roughly divided into aminosalicylic acid, antibiotics, glucocorticoids, immunomodulators, cytokine blockers, growth factors, recombinant human cytokines, etc. However, the above-mentioned therapeutic drugs and methods have the disadvantages of large side effects in long-term use, increased complications, unstable efficacy, strict requirements on drug dosage, and high prices. Diet is an important factor in the development of IBD. Reasonable diet and nutritional management can reduce the incidence of related diseases such as enteritis. Therefore, a healthy diet is a safe and effective choice for alleviating inflammatory bowel disease. At present, more and more food-borne functional ingredients have been discovered to inhibit intestinal inflammation. For example, polysaccharides extracted from Chinese medicinal materials, fruits and vegetables, mushrooms, etc. have received widespread attention. These sugars have good anti-enteritis effects and fewer side effects, and have potential clinical application value. The common anti-enteritis mechanisms of the above polysaccharides include direct or indirect effects, such as improving intestinal barrier function, reducing inflammation levels, and remodeling intestinal flora. It is of great significance to human health to explore more food-borne active ingredients for inhibiting enteritis and to study the mechanism of action in depth.

[0004] Hawthorn is a plant of the genus Crataegus in the Rosaceae family. It is both a medicine and food, rich in nutrients and contains functional factors such as hawthorn pectin. The currently known functions of hawthorn pectin include antioxidant, anti-inflammatory, anti-cancer and anti-fatigue; regulating cholesterol, blood sugar and lipid metabolism; improving constipation and enhancing immunity. At present, hawthorn products on the domestic market are still mainly food. For example, traditional hawthorn candied foods represented by moutan bark and hawthorn cake have not been further processed with hawthorn as a raw material to prepare active ingredients and used in the preparation of anti-enteritis products.

[0005] The research on the extraction technology of bioactive components in hawthorn is the focus of hawthorn application research. For the extraction of hawthorn pectin, the methods reported in literature and patents are hot water extraction and ultrasonic extraction. In addition, enzyme extraction, acid extraction, microwave extraction and other methods have also been reported, and there are also several extraction methods that are coordinated, such as acid and high pressure, enzyme and ultrasound. At present, the preparation of hawthorn pectin has problems such as high equipment requirements or excessive energy and reagent consumption, low extraction rate, and poor product quality. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a hawthorn pectin and a preparation method and application thereof. The present invention utilizes a composite enzyme method in coordination with a probiotic fermentation method to achieve efficient preparation and effective separation of hawthorn pectin, and the hawthorn pectin prepared by the coordinated method has a better effect when used.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] In a first aspect, the present invention provides a method for preparing hawthorn pectin, wherein the hawthorn pectin is prepared by an enzyme-assisted fermentation method, comprising the following steps:

[0009] The method comprises adding compound enzyme to the fresh hawthorn fruit homogenate for enzymolysis, adding probiotics for fermentation, precipitating with alcohol, separating and purifying, and drying to obtain the product.

[0010] Preferably, the probiotics are lactobacillus and bifidobacterium. The lactobacillus may be one or more of Lactobacillus reuteri CICC-6132, Lactobacillus casei NRRL B-1922, Lactobacillus rhamnosus AS 1.2466, Lactobacillus acidophilus NRRL B-4495, Lactobacillus brevis NRRL B-4527, Lactobacillus mansoni ATCC 7830 and Lactobacillus bulgaricus ATCC 11842; the bifidobacterium may be one or more of Bifidobacterium longum NRRL B-41409, Bifidobacterium breve NRRL B-41408, Bifidobacterium adolescentis ATCC 15703, Bifidobacterium bifidum ATCC29521 and Bifidobacterium lactis HN019.

[0011] In one or more embodiments, the fresh hawthorn fruit is prepared according to the following steps: selecting hawthorns that are free of mold and insects, washing with clean water to remove surface dirt, draining the water and then drying naturally.

[0012] Furthermore, the hawthorn fresh fruit homogenate is obtained by mixing the hawthorn fresh fruit with water and breaking the fruit wall.

[0013] Furthermore, the solid-liquid ratio of the fresh hawthorn fruit to water is 1:(2-10), measured in g / mL; preferably 1:3.

[0014] In one or more embodiments, the pH is adjusted before adding the complex enzyme, specifically, citric acid is used to adjust the pH to 5.6-6.0.

[0015] In one or more embodiments, the complex enzyme is selected from a combination of at least two of pectinase, protease, xylanase, cellulase, β-glucanase, α-amylase and glucose oxidase; preferably, the complex enzyme may be a combination of pectinase and cellulase, and the enzyme activity ratio of pectinase and cellulase may be (1:10) to (10:1), preferably 1:1.

[0016] Furthermore, the amount of the complex enzyme added is 50U / g to 1000U / g of fresh hawthorn fruit, preferably 300U / g of fresh hawthorn fruit.

[0017] Furthermore, the enzyme extraction pH and temperature are determined according to the optimal pH and temperature of the enzyme used. The enzyme extraction temperature and time can be 40-60°C and 0.5-5h, respectively; preferably, the enzyme extraction is performed at 50°C for 2h.

[0018] Furthermore, after the enzyme extraction is completed, the temperature is rapidly raised to 90-110° C., and the enzyme is inactivated for 5-15 minutes; preferably, after the enzyme extraction is completed, the temperature is rapidly raised to 100° C., and the enzyme is inactivated for 10 minutes.

[0019] Furthermore, the total inoculation amount of the lactobacillus and bifidobacterium is 0.2% to 5.0% of the weight of the fresh hawthorn fruit, preferably 1% to 2%.

[0020] Furthermore, the lactobacillus and bifidobacterium cooperate with each other, and the mass ratio of lactobacillus to bifidobacterium is (1:10) to (10:1), preferably (2:3) to (3:2).

[0021] In one or more embodiments, the fermentation temperature and time are 30-45° C. for 12-60 h, respectively, preferably 35-40° C. for 24-36 h.

[0022] In one or more embodiments, after the fermentation is completed, the supernatant is obtained by centrifugation to remove the precipitate. The centrifugation to remove the precipitate is performed at 5000-7000 rpm for 10-20 min.

[0023] In one or more embodiments, the alcohol precipitation is to concentrate the supernatant obtained after fermentation to remove water, add alcohol to the concentrate, perform alcohol precipitation at low temperature, centrifuge, and obtain a precipitate.

[0024] Furthermore, the alcohol is ethanol; the mass ratio of the concentrated solution to the alcohol is 1:(2-6); preferably 1:(2-3).

[0025] Furthermore, the alcohol precipitation is carried out at a temperature of 4 to 25°C for 6 to 24 hours, preferably at a temperature of 4 to 10°C for 10 to 14 hours.

[0026] Furthermore, the centrifugation is performed at 4000-6000 rpm / min for 5-15 min.

[0027] In one or more embodiments, the purification is to separate, elute and concentrate using a chromatographic column, and then freeze-dry the supernatant to prepare sugar powder to obtain hawthorn pectin.

[0028] Furthermore, the chromatographic column is a DEAE-Sephadex A-25 dextran gel chromatography column.

[0029] Furthermore, the elution is first eluted with distilled water and then eluted with 0.2-0.5 mol / L NH4HCO3.

[0030] In a second aspect, the present invention provides a hawthorn pectin prepared by the above method.

[0031] In a third aspect, the present invention provides the use of the hawthorn pectin in at least one of the following aspects:

[0032] 1) Application in the preparation of products for preventing and / or treating enteritis;

[0033] 2) Application in the preparation of products for improving intestinal tissue morphology;

[0034] 3) Application in the preparation of products for improving intestinal barrier damage;

[0035] 4) Application in the preparation of products for increasing the species composition of bacterial flora;

[0036] 5) Application in the preparation of products for inhibiting the proliferation of pathogenic bacteria;

[0037] 6) Application in the preparation of products for regulating bacterial metabolic pathways.

[0038] In one or more embodiments, the enteritis is colitis or proctitis.

[0039] In one or more embodiments, the intestinal tissue is colon, cecum or small intestine, specifically mouse colon tissue.

[0040] In one or more embodiments, the improvement of intestinal barrier damage is to increase the expression of tight junction proteins in enteritis or enteritis. The tight junction proteins can specifically be pericytin family proteins (ZO), occludin, and the like.

[0041] In one or more embodiments, the increasing of the flora species composition is increasing the number of enteritis or enteritis intestinal flora species.

[0042] In one or more embodiments, the inhibition of pathogenic bacteria proliferation is inhibition of enteritis or enteritis pathogenic bacteria proliferation, or inhibition of pathogenic bacteria proliferation in vitro.

[0043] Preferably, the pathogenic bacteria include Mucispirillum, Desulfovibrio, Prevotella and Bilophila.

[0044] In one or more embodiments, the metabolic pathway of the microbiota is a metabolic pathway for regulating enteritis or enteritis intestinal microbiota. The metabolic pathway can specifically be a metabolic pathway of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and a metabolic pathway database (MetaCyc).

[0045] In some embodiments, the product is a medicine, a health product, a food (such as a functional food) or a preparation. When the hawthorn pectin is used as a functional food or a health product, it can be used alone or mixed with other ingredients.

[0046] In clinical application, it is used as a food component in special medical foods or health products to prevent and / or treat enteritis, regulate bacterial metabolic pathways, etc. It can also be used alone or in combination with other drugs to prepare drugs of different dosage forms that can be used clinically by conventional preparation processes, such as adding excipients or flavoring agents and preservatives to prepare capsules for preventing and / or treating enteritis or oral liquids for preventing and / or treating enteritis.

[0047] In some embodiments, the dosage form of the drug includes capsules, oral solutions, injection solutions, tablets, powders or granules.

[0048] In a fourth aspect, the present invention provides a method of treatment using the hawthorn pectin in at least one of the following aspects.

[0049] 1) Method for preventing and / or treating enteritis using hawthorn pectin;

[0050] 2) Methods of improving intestinal tissue morphology using hawthorn pectin;

[0051] 3) A method for improving intestinal barrier damage by using hawthorn pectin;

[0052] 4) A method for increasing the species composition of bacterial flora by using hawthorn pectin;

[0053] 5) A method for inhibiting the proliferation of pathogenic bacteria by using hawthorn pectin;

[0054] 6) A method for preparing a method for regulating bacterial metabolic pathways using hawthorn pectin.

[0055] The beneficial effects of the present invention are:

[0056] (1) The present invention adopts compound enzyme enzymolysis and probiotic (Lactobacillus and Bifidobacterium) fermentation to prepare hawthorn pectin, realizing efficient preparation and effective separation of hawthorn pectin, with a hawthorn pectin yield of 6.8%. The process is simple, the product purity is high, the production cycle is short, the energy consumption is low, and it is suitable for industrial production.

[0057] (2) The hawthorn pectin prepared by the present invention using compound enzyme hydrolysis and probiotic fermentation has a synergistic effect in preventing and / or treating enteritis. The compound enzyme and probiotics play a significant role in preventing and / or treating enteritis, significantly increasing the length of colon villi and the thickness of the muscular layer of colitis mice, promoting the protein expression of pericyin family protein 1 (ZO-1) and occludin, increasing the number of species in the cecal content flora, inhibiting the abundance of Mucispirillum, Desulfovibrio, Prevotella and Bilophila in the cecal content, and regulating the relevant metabolic levels in the KEGG and MetaCyc pathways of the cecal content flora.

[0058] (3) The hawthorn pectin prepared by the present invention using complex enzymes and probiotics significantly alleviated the colonic physiological indicators and intestinal barrier damage of dextran sulfate sodium (DSS)-induced colitis model mice, increased the number of bacterial species, inhibited the proliferation of pathogenic bacteria, and regulated the metabolic pathways of the bacterial flora. The present invention provides a new option for the treatment or prevention of enteritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0060] Figure 1Effects of hawthorn pectin prepared in Example 1 and Comparative Examples 1 and 2 of the present invention on the colon of DSS-induced colitis mice; wherein, (A) is the experimental design idea, (B) is a representative image of the colon, (C) is the colon length, (D) is the villus length, and (E) is the muscle layer thickness; data are expressed as mean ± SEM, compared with the control group, *p < 0.05 is significant, **p < 0.01 is extremely significant; compared with the model group, #p < 0.05 is significant, ##p < 0.01 is extremely significant;

[0061] Figure 2 Effects of hawthorn pectin prepared in Example 1 of the present invention and Comparative Examples 1 and 2 on the expression levels of pericytin family protein 1 (ZO-1) and occludens in the colon of DSS-induced colitis mice; wherein, (A) is the expression level of ZO-1 protein, (B) is the expression level of Occludin protein, and the data are expressed as mean ± SEM. Compared with the control group, *p<0.05 is significant, **p<0.01 is extremely significant; compared with the model group, #p<0.05 is significant, and ##p<0.01 is extremely significant;

[0062] Figure 3 Effects of hawthorn pectin prepared in Example 1 and Comparative Examples 1 and 2 of the present invention on the species composition of the cecum contents of mice with DSS-induced colitis; wherein the number of OTUs refers to the number of species they represent;

[0063] Figure 4 Effects of hawthorn pectin prepared in Example 1 of the present invention and Comparative Examples 1 and 2 on the abundance of pathogenic bacteria in the cecal contents of DSS-induced colitis mice; wherein, (A) is the abundance of the genus Mucispirillum, (B) is the abundance of the genus Desulfovibrio, (C) is the abundance of the genus Prevotella, and (D) is the abundance of the genus Bilophila; data are expressed as mean ± SEM, and compared with the control group, *p < 0.05 is significant, and **p < 0.01 is extremely significant; compared with the model group, #p < 0.05 is significant, and ##p < 0.01 is extremely significant;

[0064] Figure 5 Effects of hawthorn pectin prepared in Example 1 of the present invention and Comparative Examples 1 and 2 on metabolic pathways of flora in the cecal contents of DSS-induced colitis mice; wherein (A) is a KEGG metabolic pathway, and (B) is a MetaCyc metabolic pathway. DETAILED DESCRIPTION

[0065] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0066] Example 1 - Preparation of hawthorn pectin by fermentation with complex enzymes in cooperation with lactobacillus and bifidobacterium

[0067] Hawthorn pectin is prepared by fermentation of compound enzymes in cooperation with lactobacillus and bifidobacteria. The preparation method is briefly described as follows: compound enzymes, lactobacillus and bifidobacteria are sequentially added to the fresh hawthorn fruit homogenate, and fermentation, alcohol precipitation, separation and purification are performed, and drying is performed to obtain the pectin. Specifically:

[0068] Take 10 kg of fresh hawthorn fruit and add it into a water reaction container, add 30 L of distilled water, and use a 500 W wall-breaking machine to homogenize for 5 minutes.

[0069] The pH was adjusted to 5.6-6.0 with citric acid, and pectinase and cellulase (enzyme activity ratio of 1:1) were added at 300 U / g fresh hawthorn, mixed well, and then extracted at 50°C for 2 h. After the enzyme extraction was completed, the temperature was quickly raised to 100°C and the enzyme was inactivated for 10 min.

[0070] Then, 1% (calculated by the weight of fresh hawthorn fruit) of Lactobacillus casei NRRL B-1922 and Bifidobacterium adolescentis ATCC 15703 were inoculated, and fermented at 37°C for 36 hours. After the fermentation was completed, the mixture was centrifuged at 6000 rpm for 15 minutes, the precipitate was removed, and the supernatant was taken. The supernatant obtained after the fermentation was concentrated and water was removed by a rotary evaporator, and 3 times the volume of ethanol was slowly added to the concentrated solution (while stirring), and the mixture was precipitated at 4°C for 12 hours, and then centrifuged at 5000 rpm / min for 10 minutes, the supernatant was removed, and the precipitate was taken.

[0071] Add purified water and stir to dissolve the precipitate. Separate using DEAE-Sephadex A-25 dextran gel chromatography column, first eluting with distilled water and then eluting with 0.3 mol / L NH4HCO3.

[0072]

[0073] After appropriate concentration, the supernatant was freeze-dried to make sugar powder (yield 6.8%), thus obtaining hawthorn pectin with a purity of 93.6%. The monosaccharide composition is shown in Table 1.

[0074] Table 1

[0075]

[0076] Comparative Example 1- Preparation of Hawthorn Pectin by Composite Enzyme

[0077] The preparation method of hawthorn pectin using compound enzyme is briefly described as follows: adding compound enzyme to fresh hawthorn fruit homogenate for enzymolysis, alcohol precipitation, separation and purification, and drying to obtain the pectin. Specifically:

[0078] Take 10 kg of fresh hawthorn fruit and add it into a water reaction container, add 30 L of distilled water, and use a 500 W wall-breaking machine to homogenize for 5 minutes.

[0079] The pH was adjusted to 5.6-6.0 with citric acid, and pectinase and cellulase were added at 300 U / g fresh hawthorn (enzyme activity ratio of 1:1), mixed well, and then enzyme extraction was performed at 50°C for 2 h.

[0080] After the enzyme extraction is completed, the temperature is quickly raised to 100°C to inactivate the enzyme for 10 minutes. Centrifuge at 6000 rpm for 15 minutes, remove the precipitate, and take the supernatant.

[0081] The supernatant was concentrated by rotary evaporator to remove water, 3 volumes of ethanol were slowly added to the concentrate (stirring while adding), and the mixture was precipitated at 4°C for 12 hours, followed by centrifugation at 5000 rpm / min for 10 minutes, the supernatant was removed, and the precipitate was taken.

[0082] Add purified water and stir to dissolve the precipitate. Separate using a DEAE-cellulose chromatography column, first eluting the column with distilled water and then with 0.3 mol / L NH4HCO3.

[0083] After appropriate concentration, the mixture was freeze-dried to produce sugar powder (yield: 3.6%), thus obtaining hawthorn pectin with a purity of 86.8%. The monosaccharide composition of the hawthorn pectin is shown in Table 2.

[0084] Table 2

[0085]

[0086] Comparative Example 2 - Preparation of Hawthorn Pectin by Fermentation with Lactobacillus and Bifidobacterium

[0087] Hawthorn pectin is prepared by synergistic fermentation of Lactobacillus and Bifidobacterium. The preparation method is briefly described as follows: Lactobacillus and Bifidobacterium are added to the fresh hawthorn fruit homogenate, precipitated with alcohol, separated and purified, and dried to obtain the pectin. The specific preparation method is as follows:

[0088] Take 10 kg of fresh hawthorn fruit and add it into a water reaction container, add 30 L of distilled water, and use a 500 W wall-breaking machine to homogenize for 5 minutes.

[0089] 1% (calculated by the weight of fresh hawthorn fruit) of Lactobacillus reuteri CICC-6132 and Bifidobacterium longum NRRL B-41409 were inoculated and fermented at 37°C for 36 hours. After the fermentation, the mixture was centrifuged at 6000 rpm for 15 minutes to remove the precipitate and take the supernatant.

[0090] The supernatant obtained after fermentation was concentrated by rotary evaporator to remove water, and 3 volumes of ethanol were slowly added to the concentrate (stirring while adding), and the mixture was precipitated at 4°C for 12 hours, followed by centrifugation at 5000 rpm / min for 10 minutes, the supernatant was removed, and the precipitate was taken.

[0091] Add purified water and stir to dissolve the precipitate. Separate using a DEAE-cellulose chromatography column, first eluting the column with distilled water and then with 0.3 mol / L NH4HCO3.

[0092] After appropriate concentration, the supernatant was freeze-dried to prepare sugar powder (yield 4.2%), thus obtaining hawthorn pectin with a purity of 87.2%. The monosaccharide composition is shown in Table 3.

[0093] Table 3

[0094]

[0095] Application examples:

[0096] 1. Effect of hawthorn pectin on the colon of mice with DSS (dextran sulfate sodium)-induced colitis

[0097] Eight-week-old male C57BL / 6J mice were adaptively fed for one week in an SPF animal room (temperature: 22±2℃; humidity: 50±10%; lighting: 12h light-dark alternation, 8:00-20:00 as daytime hours). The mice were divided into 5 groups (8 mice in each group), namely normal group, model group, hawthorn pectin group of comparative example 1, hawthorn pectin group of comparative example 2 and hawthorn pectin group of Example 1. On days 1 to 15, the mice in each group were gavaged as follows: the normal group and the DSS group were gavaged with normal saline; the hawthorn pectin groups of comparative example 1, comparative example 2 and example 1 were all given 900mg / kg / day of hawthorn pectin of comparative example 1, comparative example 2 and example 1. On days 8 to 15, the drinking water of all groups except the normal group was replaced with drinking water containing 2.5% DSS ( Figure 1 A) 24 hours after the last treatment, the mice were killed by cervical dislocation, and the colon was removed and the length of the colon was measured.

[0098] like Figure 1 As shown in B-1C, compared with the normal group, the colon length of the DSS-induced colitis mice was significantly reduced. Compared with the model group, the colon length of the mice in the hawthorn pectin intervention group of Comparative Example 1, Comparative Example 2 and Example 1 increased by 5.6%, 13.9% and 54.2% respectively (p<0.01). Figure 1 As shown in D-1E, compared with the normal group, the length of colon villi and the thickness of the muscular layer of the DSS-induced colitis mice were significantly reduced. Compared with the model group, the length of villi and the thickness of the muscular layer of the mice in the hawthorn pectin intervention group of Example 1 were significantly increased, with an increase of 53.9% and 120.6% (both p<0.01), respectively, while the increase in comparative example 1 was only 6.4% and 10.6% (both p>0.05) and the increase in comparative example 2 was only 13.3% and 21.1% (both p>0.05).

[0099] 2. Effect of hawthorn pectin on the expression of ZO-1 and occludin in the colon of mice with DSS-induced colitis

[0100] Animal grouping and intervention were the same as above. The effect of hawthorn pectin on the expression levels of ZO-1 and Occludin proteins in the colon of mice with DSS-induced colitis was determined by immunofluorescence. Figure 2 As shown in A-2B, compared with the control group, the expression levels of ZO-1 and Occludin proteins in the colon of DSS-induced colitis mice were significantly reduced. Compared with the DSS group, the expression levels of ZO-1 and Occludin proteins in the hawthorn pectin group of Example 1 were significantly increased by 511.5% and 432.0%, respectively (both p<0.01), while the expression level of ZO-1 in the hawthorn pectin group of Comparative Example 1 was reduced by 11.0%, and the expression level of Occludin protein was only increased by 4.8%; the expression levels of ZO-1 and Occludin proteins in the hawthorn pectin group of Comparative Example 2 were only increased by 34.4% and 47.4%, respectively (both p>0.05).

[0101] 3. Effect of hawthorn pectin on species composition of cecal contents in mice with DSS-induced colitis

[0102] Animal grouping and intervention were the same as above. The number of OTUs, i.e. the number of species they represent. Figure 3 As shown, compared with the control group, the number of bacterial species in the model group decreased by 27.3%. Compared with the model group, the number of bacterial species in the colitis mice treated with hawthorn pectin in Comparative Example 1 decreased by 3.6%, while the number of bacterial species in the colitis mice treated with hawthorn pectin in Comparative Example 2 and Example 1 increased by 5.3% and 37.0%, respectively.

[0103] 4. Effect of hawthorn pectin on the abundance of pathogenic bacteria in the cecum of mice with DSS-induced colitis

[0104] Animal grouping and intervention were the same as above. Figure 4As shown in A-4D, compared with the control group, the relative abundance of Mucispirillum, Desulfovibrio, Prevotella and Bilophila in the model group was higher than that in the control group. Compared with the model group, the relative abundance of Mucispirillum, Desulfovibrio, Prevotella and Bilophila in the hawthorn pectin group of Example 1 was significantly reduced by 74.1%, 90.9%, 91.1% and 93.1% respectively (all p<0.01), while in the hawthorn pectin group of Comparative Example 1, Mucispirillum, Desulfovibrio and Bilophila were only reduced by 6.7%, 9.2% and 26.0% respectively, and Prevotella increased by 5.2%; in the hawthorn pectin group of Comparative Example 2, Mucispirillum, Desulfovibrio and Prevotella were only reduced by 12.0%, 18.0% and 7.0% respectively, and Bilophila increased by 2.1%.

[0105] 5. Effect of hawthorn pectin on metabolic pathways of cecal contents in mice with DSS-induced colitis

[0106] Animal grouping and intervention were the same as above. Functional prediction of intestinal flora in cecal contents was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaCyc database. Figure 5 As shown in A, based on the KEGG pathway, compared with the control group, the energy metabolism, cofactor and vitamin metabolism, transmembrane transport and nucleotide metabolism levels in the cecal content flora of the model group mice were significantly reduced, and the hawthorn pectin intervention in comparative example 1 did not change the metabolic levels of the above pathways in mice, while the hawthorn pectin intervention in comparative example 2 and example 1 increased the metabolic levels of the above pathways in mice, especially the hawthorn pectin in example 1. Figure 5 As shown in Figure B, based on the MetaCyc pathway, compared with the control group, the metabolic levels of photosynthetic carbon cycle (CALVIN-PWY), gondoate biosynthesis (anaerobic) (PWY-7663), cis-vaccenate biosynthesis (PWY-5973) and superpathway of L-threonine biosynthesis (THRESYN-PWY) in the cecal contents of the model group mice were significantly reduced, and the hawthorn pectin intervention in Comparative Example 1 did not change the metabolic levels of the above pathways in mice, while the hawthorn pectin intervention in Comparative Example 2 and Example 1 increased the metabolic levels of the above pathways in mice, especially the hawthorn pectin in Example 1.

[0107] In summary, the hawthorn pectin prepared by the composite enzyme and probiotics in Example 1 has a more significant effect when used, which is much higher than the effects of Comparative Examples 1 and 2, indicating that the composite enzyme and probiotics have a synergistic effect.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing hawthorn pectin, characterized in that: The steps include: The method comprises adding compound enzyme to the fresh hawthorn fruit homogenate for enzymolysis, adding probiotics for fermentation, precipitating with alcohol, separating and purifying, and drying to obtain the product.

2. The method according to claim 1, characterized in that: The probiotics are lactobacillus and bifidobacterium; The complex enzyme is selected from a combination of at least two of pectinase, protease, xylanase, cellulase, β-glucanase, α-amylase and glucose oxidase; The added amount of the complex enzyme is 50U / g to 1000U / g of fresh hawthorn fruit; The total amount of lactobacillus and bifidobacterium inoculated is 0.2% to 5.0% of the weight of fresh hawthorn fruit; The mass ratio of Lactobacillus to Bifidobacterium is (1:10) to (10:1).

3. The method according to claim 1 or 2, characterized in that: The lactobacillus is one or more of Lactobacillus reuteri CICC-6132, Lactobacillus casei NRRL B-1922, Lactobacillus rhamnosus AS1.2466, Lactobacillus acidophilus NRRL B-4495, Lactobacillus brevis NRRL B-4527, Lactobacillus mansoni ATCC 7830 and Lactobacillus bulgaricus ATCC 11842; The bifidobacterium is one or more of Bifidobacterium longum NRRL B-41409, Bifidobacterium breve NRRL B-41408, Bifidobacterium adolescentis ATCC 15703, Bifidobacterium bifidum ATCC 29521 and Bifidobacterium lactis HN 019; The compound enzyme is a compound of pectinase and cellulase; the enzyme activity ratio of pectinase to cellulase is (1:10) to (10:1); Before adding the complex enzyme, adjust the pH to 5.6-6.0; During the enzymolysis, the temperature of enzyme extraction is 40-60° C., and the time is 0.5-5 h. After the enzyme extraction is completed, the temperature is rapidly raised to 90-110° C., and the enzyme is inactivated for 5-15 min.

4. The method according to claim 1, characterized in that: The hawthorn fresh fruit homogenate is obtained by mixing the hawthorn fresh fruit with water and breaking the wall; The solid-liquid ratio of the fresh hawthorn fruit to water is 1:(2-10), measured in g / mL.

5. The method according to claim 1, characterized in that The fermentation temperature and time are 30-45°C and 12-60h respectively; After the fermentation is finished, the supernatant is obtained by centrifugation to remove the precipitate; the centrifugation to remove the precipitate is performed at 5000-7000 rpm for 10-20 min.

6. The method according to claim 1, characterized in that The alcohol precipitation is to concentrate the supernatant obtained after fermentation, add alcohol to the concentrate, perform alcohol precipitation at low temperature, centrifuge, and take the precipitate; Or, the alcohol is ethanol; The mass ratio of the concentrated solution to the alcohol is 1:(2-6); The temperature of alcohol precipitation is 4-25℃ and the time is 6-24h; The centrifugation is performed at 4000-6000 rpm / min for 5-15 min.

7. The method according to claim 1, characterized in that The purification comprises the following steps: using a chromatographic column to separate, elute and concentrate, and then taking the supernatant and freeze-drying it to make sugar powder, thereby obtaining hawthorn pectin; The elution is firstly eluted with distilled water and then eluted with 0.2-0.5 mol / L NH4HCO3.

8. A hawthorn pectin prepared by the method according to any one of claims 1 to 7.

9. Use of the hawthorn pectin according to claim 8 in at least one of the following aspects: 1) Application in the preparation of products for preventing and / or treating enteritis; 2) Application in the preparation of products for improving intestinal tissue morphology; 3) Application in the preparation of products for improving intestinal barrier damage; 4) Application in the preparation of products for increasing the species composition of bacterial flora; 5) Application in the preparation of products for inhibiting the proliferation of pathogenic bacteria; 6) Application in the preparation of products for regulating bacterial metabolic pathways.

10. The use according to claim 9, characterized in that The enteritis is colitis or proctitis; Or, the intestinal tissue is colon, cecum or small intestine; Or, the improvement of intestinal barrier damage is to increase the expression of tight junction proteins in enteritis or enteritis intestine; the tight junction proteins are perimelin family proteins and occludens; Or, the increasing of the flora species composition is to increase the number of enteritis or enteritis intestinal flora species; Or, the inhibition of pathogenic bacteria proliferation is to inhibit the proliferation of enteritis or enteritis pathogenic bacteria, or inhibit the proliferation of pathogenic bacteria in vitro; the pathogenic bacteria include Mucispirillum, Desulfovibrio, Prevotella and Bilophila; Or, the metabolic pathway for regulating the flora is a metabolic pathway for regulating enteritis or enteritis intestinal flora; the metabolic pathway is a KEGG and MetaCyc metabolic pathway; Or, the product is a medicine, a health product, a food or a preparation; the dosage form of the medicine includes capsules, oral liquids, injections, tablets, powders or granules.

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