A plant fermentation composition, its preparation method and applications

Abstract

The present application discloses a plant fermentation composition, a preparation method thereof and an application thereof, belonging to the technical field of food or medicine. In the present application, polygonatum odoratum and smoked plum are used to nourish yin with sweet flavor and specifically supplement the stomach yin; a small amount of tangerine peel is added to promote qi movement and relieve stagnation; licorice is used as an adjuvant to invigorate the middle energizer, relieve spasm and pain, and harmonize various herbs. The whole formula combines dredging and tonifying; and polygonatum odoratum, tangerine peel, licorice, smoked plum and other Chinese herbal medicines with both edible and medicinal properties are fermented by lactic acid bacteria, combining the efficacy components of Chinese herbal medicines with the action of lactic acid bacteria extracellular polysaccharides to comprehensively improve chronic atrophic gastritis from aspects such as antioxidant, immune regulation and gastric mucosa protection.

Description

Technical Field

[0001] This application belongs to the field of food or medicine, and particularly relates to a plant fermentation composition, a preparation method thereof, and an application thereof. Background Art

[0002] Based on the defects existing in the prior art, the purpose of this application is to provide a plant fermentation composition, a preparation method thereof, and an application thereof. Chronic gastritis is a common and frequently-occurring gastric mucosal inflammation, which is divided into chronic non-atrophic gastritis (superficial gastritis) and chronic atrophic gastritis according to the forms and phenotypes at different stages. Among them, the gastric mucosal secretory glands in non-atrophic gastritis are intact, with mild inflammation in the upper layer of the mucosa and normal gastric acid secretion; in atrophic gastritis, due to repeated and continuous damage and stimulation of the gastric mucosal epithelium, the gastric mucosal secretory glands atrophy, there is severe inflammation in the submucosa layer, and gastric acid secretion is damaged.

[0003] Most patients with chronic atrophic gastritis will have abdominal pain, and will also be accompanied by symptoms such as abdominal distension, belching, nausea, and diarrhea after meals. In the later stage, the gastric glands will atrophy and disappear, and intestinal metaplasia will occur in some gastric mucosal parts, that is, the gastric mucosal epithelial cells become intestinal epithelial cells and cannot secrete gastric acid, and may eventually develop into gastric cancer. The main causes of chronic atrophic gastritis are as follows: bad living habits, such as long-term smoking, drinking, irregular diet, and stimulation by spicy and overheated or overcooled foods; intake of non-steroidal anti-inflammatory drugs such as aspirin; Helicobacter pylori infection; iron deficiency anemia, heavy metals, psychological factors, etc.

[0004] The modern medical treatment of chronic atrophic gastritis mainly focuses on acid suppression, gastric mucosal protection, promoting gastric motility, etc., and antibacterial treatment is given to patients with positive Helicobacter pylori. Although the western medicine treatment has a rapid onset, there is a relatively high recurrence rate. Among them, taking proton pump inhibitors for a long time will increase the probability of suffering from hypomagnesemia and cardiovascular and cerebrovascular diseases; traditional gastric mucosal protectants such as sucralfate may cause a lack of phosphorus in the body fluid and lead to hypophosphatemia when taken in large doses for a long time; colloidal bismuth pectin may cause constipation, and the stool is dark brown, and long-term use may cause bismuth encephalopathy, with many side effects; long-term use of antibiotics is likely to produce drug resistance and cause gastrointestinal flora imbalance, leading to secondary infections, making the condition relapse. There are also studies on using probiotics to treat chronic atrophic gastritis, but due to the low pH value of the gastric environment, it has a great impact on the activity of probiotics, making it difficult for probiotics to survive in the stomach and difficult to play a role.

[0005] Therefore, there is an urgent need to develop a product that can be taken for a long time and can effectively improve chronic atrophic gastritis. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a plant fermentation composition, a preparation method thereof, and an application thereof.

[0007] To achieve the above object, in a first aspect, the present application provides a plant fermentation composition mainly fermented from polygonatum odoratum, orange peel, licorice, and smoked plum, wherein the strains used for fermentation include lactic acid bacteria; the mass ratio of polygonatum odoratum, orange peel, licorice, and smoked plum is (1.9 - 6.1) : (0.9 - 2.1) : (0.9 - 2.1) : (0.9 - 2.1).

[0008] The main components of polygonatum odoratum and their functions are as follows:

[0009] Polygonatum odoratum polysaccharide: One of the important active substances of polygonatum odoratum, having various biological activities such as hypoglycemic, antioxidant, anti-tumor, and immune enhancing effects;

[0010] Polygonatum odoratum glycoprotein: It is a plant lectin, having antibacterial, antiviral effects, inhibiting the binding of bacteria to host cells, playing an immunomodulatory role, and can also bind to the surface of immune cells, induce signal transduction, activate immune responses, and also have anti-tumor effects;

[0011] Polygonatum odoratum flavonoids: Having antioxidant, hypoglycemic, anti-tumor and other effects;

[0012] Polygonatum odoratum saponins: Mainly gastric steroid saponins, having antibacterial, hypoglycemic and immunomodulatory effects.

[0013] The main components of orange peel and their functions are as follows:

[0014] Orange peel flavonoids: Polyoxymethylated flavonoids, having anti-inflammatory, anti-cancer and anti-atherosclerotic effects;

[0015] Tangeretin: One of the main components in the polyoxymethylated flavonoid fraction, having anti-inflammatory, antioxidant, antibacterial and anti-cancer activities.

[0016] The main components of licorice and their functions are as follows: The main active components are glycyrrhizin, flavonoid compounds, licorice polysaccharide, etc., having many pharmacological effects such as antioxidant, immunomodulatory, anti-inflammatory and antiviral effects.

[0017] The main components of smoked plum and their functions are as follows: It contains rich organic acids (mainly citric acid), flavonoids, polysaccharides, terpenoids, sterols, esters, alkaloids, etc., having antibacterial, smooth muscle regulating, antioxidant, anti-tumor and other effects.

[0018] Lactic acid bacteria are recognized as microorganisms for food safety. Their extracellular polysaccharides imply the concepts of green and health, and have biocompatibility, non-toxicity, and biodegradability. Lactic acid bacteria extracellular polysaccharides are polysaccharides (EPS) secreted outside the cells. The extracellular polysaccharides adhere to the cell surface in the form of capsular polysaccharides (CPS) or are secreted into the extracellular environment in the form of mucus (mucus polysaccharides SPS). The extracellular polysaccharides produced by different types of lactic acid bacteria are different, with diverse structures and different biological activities. Lactic acid bacteria extracellular polysaccharides have multiple biological activity functions, such as anti-inflammatory, antioxidant, anti-tumor, and immune regulation functions.

[0019] The above-mentioned plant fermentation composition uses Polygonatum odoratum and Prunus mume to nourish yin with sour and sweet flavors, specifically tonifying the stomach yin; adding a small amount of Citrus reticulata peel to promote qi and relieve stagnation; using Glycyrrhiza uralensis Fisch. as an envoy, tonifying the middle qi, relieving spasm and pain, and harmonizing various herbs. The whole formula combines supplementation and dredging. It ferments Polygonatum odoratum, Citrus reticulata peel, Glycyrrhiza uralensis Fisch., and Prunus mume and other Chinese herbal medicines that are both food and medicine with lactic acid bacteria, uses lactic acid bacteria to improve the texture, rheology, and taste of the product, enhances the quality of the plant products that are both food and medicine, and additionally contains various functional components of lactic acid bacteria, combining the efficacy components of Chinese herbal medicines and the effects of lactic acid bacteria extracellular polysaccharides, comprehensively improving chronic atrophic gastritis from aspects such as antioxidant, immune regulation, and protecting the gastric mucosa.

[0020] In some embodiments, the mass ratio of Polygonatum odoratum, Citrus reticulata peel, Glycyrrhiza uralensis Fisch., and Prunus mume is (2.9~6.1) : 1 : 1 : 1 to increase the yield of extracellular polysaccharides obtained by fermentation and better improve chronic atrophic gastritis. Especially when the mass ratio of Polygonatum odoratum, Citrus reticulata peel, Glycyrrhiza uralensis Fisch., and Prunus mume is 5 : 1 : 1 : 1, the yield of extracellular polysaccharides obtained by fermentation is higher and the improvement effect on chronic atrophic gastritis is better.

[0021] In some embodiments, the strains used for fermentation include at least one of Lactobacillus plantarum HCS03-001, Lactobacillus plantarum RHJS001, Lactobacillus fermentum HCS08-005, Lactobacillus paracasei HCS17-040, Lactobacillus rhamnosus HCS01-013, and Lactobacillus reuteri HCS02-001 to increase the yield of extracellular polysaccharides and better improve chronic atrophic gastritis. Especially when the strains used for fermentation include Lactobacillus plantarum RHJS001, the yield of extracellular polysaccharides is the highest and the improvement effect on the yield of extracellular polysaccharides is the best.

[0022] In the second aspect, the present application provides a preparation method of the plant fermentation composition, including the following steps:

[0023] Mix Polygonatum odoratum, Citrus reticulata peel, Glycyrrhiza uralensis Fisch., and Prunus mume, and obtain an extract by water extraction method;

[0024] Inoculate the strains into a culture medium for activation to obtain an activated bacterial solution;

[0025] Inoculate the activated bacterial liquid into the obtained extract, adjust the pH value to 5 - 7, and ferment at 30 - 50 °C for 24 - 96 h to obtain a plant fermentation composition.

[0026] In one embodiment, before inoculating the activated bacterial liquid into the extract, a carbon source is first added to the extract, and the concentration of the carbon source in the extract is 20 - 40 g / L. By adding a carbon source with a concentration of 20 - 40 g / L to the extract, the yield of extracellular polysaccharide and the SOD activity of the plant fermentation composition can be increased, and chronic atrophic gastritis can be better improved. Especially when adding a carbon source with a concentration of 30 g / L to the extract, the yield of extracellular polysaccharide is higher, the SOD activity of the plant fermentation composition is stronger, and the improvement effect on chronic atrophic gastritis is better.

[0027] In one embodiment, the carbon source includes sucrose.

[0028] In one embodiment, during the fermentation process, the pH value is controlled within the range of 5 - 7. Controlling the pH value of the fermentation process within the range of 5 - 7 can increase the yield of extracellular polysaccharide and the SOD activity of the plant fermentation composition, and better improve chronic atrophic gastritis. Especially when controlling the pH value of the fermentation process to be 6, the yield of extracellular polysaccharide is higher, the SOD activity of the plant fermentation composition is stronger, and the improvement effect on chronic atrophic gastritis is better.

[0029] In one embodiment, the volume of the activated bacterial liquid is 2% - 10% of the volume of the extract.

[0030] In one embodiment, the cell concentration in the activated bacterial liquid is 10 8 ~10 9 CFU / mL.

[0031] In one embodiment, the medium includes the following components: peptone 6 - 14 g, beef extract powder 3 - 7 g, yeast extract powder 2 - 6 g, K2HPO4·7H2O 1 - 3 g, ammonium citrate 1 - 3 g, sodium acetate·3H2O 3 - 7 g, glucose 10 - 30 g, Tween 80 0.5 - 1.5 mL, MgSO4·7H2O 0.1 - 0.3 g, MnSO4·4H2O 0.03 - 0.07 g, distilled water 800 - 1200 mL. According to the production scale, the amount of each component can be controlled within the above ratio range to adjust the dosage of the medium.

[0032] In one embodiment, the steps of mixing polygonatum odoratum, tangerine peel, licorice root, and smoked plum and obtaining an extract by water extraction method include:

[0033] After mixing polygonatum odoratum, tangerine peel, licorice root, and smoked plum, add water according to a solid - liquid ratio of 1:4 - 10 (w / w), heat to boiling, maintain for 2 - 4 h, and then filter to obtain a first - stage extract and filter residue;

[0034] Add water to the filter residue at 2 to 4 times the initial weight of polygonatum odoratum, tangerine peel, licorice, and dark plum, heat to boiling, maintain for 2 to 4 hours, and then filter to obtain a secondary extract.

[0035] Mix the first extract and the second extract, centrifuge, and take the supernatant to obtain the target extract.

[0036] Thirdly, the present application provides the use of the plant fermentation composition or the plant fermentation composition prepared by the preparation method in the preparation of foods or drugs for improving chronic atrophic gastritis.

[0037] Fourthly, the present application also provides a plant-based fermented drink, which comprises the plant fermentation composition or the plant fermentation composition prepared by the preparation method.

[0038] Compared with the prior art, the beneficial effects of the present application are as follows: In the present application, polygonatum odoratum and dark plum are used to nourish yin with sour and sweet flavors, specifically tonifying the stomach yin; a small amount of tangerine peel is used to promote qi circulation and relieve stagnation; licorice is used as an adjuvant, tonifying the middle qi, relieving spasm and pain, and harmonizing various herbs. The whole formula combines dredging and tonifying; it ferments traditional Chinese herbs that are both food and medicine, such as polygonatum odoratum, tangerine peel, licorice, and dark plum, with lactic acid bacteria. The lactic acid bacteria are used to improve the texture, rheology, and taste of the product, improve the quality of the product of traditional Chinese herbs that are both food and medicine, and additionally add various functional components of lactic acid bacteria. The combination of the efficacy components of traditional Chinese herbs and the effects of lactic acid bacteria extracellular polysaccharides comprehensively improves chronic atrophic gastritis from aspects such as antioxidant, immune regulation, and gastric mucosa protection. Detailed Embodiments

[0039] In order to better illustrate the purpose, technical solutions, and advantages of the present invention, the present invention will be further described below in conjunction with specific examples and comparative examples. The purpose is to understand the content of the present invention in detail, rather than to limit the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention. The experimental reagents and instruments involved in the implementation of the present invention are all common ordinary reagents and instruments unless otherwise specified.

[0040] 1. Screening of lactic acid bacteria strains producing extracellular polysaccharides

[0041] 1.1 Sample source

[0042] Sour radishes purchased from rural households in Sichuan.

[0043] 1.2 Composition of the culture medium

[0044] Screening medium: 10 g of peptone, 5 g of beef extract powder, 4 g of yeast extract powder, 2 g of K2HPO4·7H2O, 2 g of ammonium citrate, 5 g of sodium acetate·3H2O, 20 g of glucose, 1 mL of Tween 80, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·4H2O, 5 g of calcium carbonate, 15 g of agar, 1000 mL of distilled water.

[0045] Activation medium: 10 g of peptone, 5 g of beef extract powder, 4 g of yeast extract powder, 2 g of K2HPO4·7H2O, 2 g of ammonium citrate, 5 g of sodium acetate·3H2O, 20 g of glucose, 1 mL of Tween 80, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·4H2O, 1000 mL of distilled water.

[0046] 1.3 Screening method

[0047] 1.3.1 Take out the pickled radish samples under aseptic conditions and dilute them by gradient dilution method to 10^(-1), 10^(-2), 10^(-3), 10^(-4), 10^(-5), 10^(-6), 10^(-7), 10^(-8) respectively;

[0048] 1.3.2 Take 0.1 mL of each gradient dilution and spread it on the screening medium plate, and culture it anaerobically at 37°C for 48 h;

[0049] 1.3.3 Among them, the number of colonies grown on the plate cultured with the 10^(-6) gradient dilution was 127, and the colony distribution was uniform. Select the colonies that were round, white, moist, and had a relatively large transparent circle for marking. Then use a sterile bamboo stick to pick the marked colonies and gently pull them outwards to measure the length of the colony drawing. Select the strain with a relatively large drawing length for purification, and obtain 1 strain with a relatively large drawing length. Perform 16S rRNA identification, and it was identified as Lactobacillus plantarum after identification. It was named Lactobacillus plantarum RHJS001 and deposited in the China General Microbiological Culture Collection Center (CGMCC). Its deposit number is: CGMCC No. 23703, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit time was November 1, 2021. At the same time, Lactobacillus plantarum HCS03-001, Lactobacillus fermentum HCS08-005, Bifidobacterium lactis HCS04-002, Lactobacillus paracasei HCS17-040, Lactobacillus rhamnosus HCS01-013, Lactobacillus reuteri HCS02-001 purchased from Jiangxi Renren Health Microecology Technology Co., Ltd. and Lactobacillus casei L.Casei21, Bifidobacterium lactis JYBR-390, Lactobacillus rhamnosus JYLR-005 purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd. were used for the remaining experiments.

[0050] 1.3.4 Inoculate the above strains into the activation medium and culture at 37 °C for 24 h for activation; then inoculate the activated bacterial liquid into the MRS medium at an inoculation amount of 5% (v / v), and anaerobically culture at 37 °C for 48 h. Centrifuge at 4 °C and 10,000 r / min for 10 min to remove the bacteria and impurities in the fermentation broth. After centrifugation, take the supernatant, add 80% (w / w) trichloroacetic acid solution to make the final concentration of trichloroacetic acid in the solution 4% (w / v), place it in a refrigerator at 4 °C for 6 - 8 h, then centrifuge at 4 °C and 10,000 r / min for 15 min to remove the protein precipitate, and take the supernatant; determine the extracellular polysaccharide content by the phenol-sulfuric acid method.

[0051] 1.3.5 Evaluate the ability to produce extracellular polysaccharides. The yield results are shown in Table 1. Select high-yield strains with a yield greater than 50 mg / L for the next step of screening. The strains that meet the requirements are Lactobacillus plantarum HCS03-001, Lactobacillus plantarum RH JS001, Lactobacillus fermentum HCS08-005, Lactobacillus paracasei HCS17-040, Lactobacillus rhamnosus HCS01-013, and Lactobacillus reuteri HCS02-001.

[0052] Table 1 Preliminary determination results of the ability of each strain to produce extracellular polysaccharides

[0053]

[0054] 2. Preparation of the extract from medicinal and edible homologous plants

[0055] 2.1 Mix the four kinds of Chinese herbal pieces of Polygonatum odoratum, Citrus reticulata peel, Glycyrrhiza uralensis, and Prunus mume in a weight ratio of 1:1:1:1, and wash with water to obtain a traditional Chinese medicine composition.

[0056] 2.2 Primary extract: Add water according to the weight ratio of the traditional Chinese medicine composition to water of 1:6, heat to boiling, maintain for 2 h and then filter to obtain the primary extract and the filter residue.

[0057] 2.3 Secondary extract: Add water to the filter residue according to the weight ratio of the initial traditional Chinese medicine composition to water of 1:2, heat to boiling, maintain for 2 h and then filter to obtain the secondary extract.

[0058] 2.4 Mix the primary extract and the secondary extract to obtain a mixed extract.

[0059] 2.5 Centrifuge the mixed extract at 6000 r / min with a tubular centrifuge, take the supernatant to obtain the plant-based extract.

[0060] 3. Screen the strains that ferment medicinal and edible homologous plants to produce extracellular polysaccharides and the optimal formula ratio

[0061] Experimental principle: Since the exopolysaccharide of lactic acid bacteria belongs to crude polysaccharide, the change in the amount of crude polysaccharide before and after fermentation can reflect the ability of producing exopolysaccharide by fermentation.

[0062] 3.1 Strain screening

[0063] 3.1.1 Respectively inoculate Lactobacillus plantarum HCS03-001, Lactobacillus plantarum RH JS001, Lactobacillus fermentum HCS08-005, Lactobacillus paracasei HCS17-040, Lactobacillus rhamnosus HCS01-013 and Lactobacillus reuteri HCS02-001 into the activation medium, culture at 37 °C for 24 h for activation; then take the activated bacterial liquid and inoculate it into the plant-based extract at an inoculation amount of 5% (v / v), adjust the initial pH value to 6.5, culture at 37 °C for 48 h, and detect the content of crude polysaccharide before and after fermentation according to the method in 1.3.4 above (the content of crude polysaccharide before fermentation is the content of blank crude polysaccharide), and compare the change in the amount of crude polysaccharide before and after fermentation.

[0064] 3.1.2 Before and after fermentation, the results of the change in the amount of crude polysaccharide are shown in Table 2. Among them, the increase in the amount of crude polysaccharide of Lactobacillus plantarum RH JS001 is the highest, reaching 0.57 g / L, indicating that it produces the highest exopolysaccharide in the plant-based extract. Therefore, Lactobacillus plantarum RH JS001 is selected as the final fermentation strain, and subsequent fermentation process optimization experiments are carried out with this strain.

[0065] Table 2 Changes in the amount of crude polysaccharide in the fermentation of plant-based extracts of traditional Chinese medicines and foods by various strains

[0066]

[0067] 3.2 Screening of formula ratios

[0068] 3.2.1 Prepare plant-based extracts from four kinds of Chinese herbal pieces of Polygonatum odoratum, Citrus reticulata peel, Glycyrrhiza uralensis, and Prunus mume according to the ratios of the control group, experimental group a, experimental group b, experimental group c, and experimental group d respectively, with the total weight of the traditional Chinese medicine composition remaining unchanged; prepare plant-based extracts according to the method of "2. Preparation of plant-based extracts of traditional Chinese medicines and foods" above. The mass ratios of Polygonatum odoratum, Citrus reticulata peel, Glycyrrhiza uralensis, and Prunus mume in each group are as follows:

[0069] Control group: 1:1:1:1;

[0070] Experimental group a: 2:1:1:1;

[0071] Experimental group b: 1:2:1:1;

[0072] Experimental group c: 1:1:2:1;

[0073] Experimental group d: 1:1:1:2.

[0074] 3.2.2 The strain RH JS001 was inoculated into the activation medium and cultured at 37°C for 24 hours for activation; then the activated bacterial solution was inoculated into the plant-based extract obtained in each group at an inoculum size of 5% (v / v), the initial pH value was adjusted to 6.5, and the culture was carried out at 37°C for 48 hours. The crude polysaccharide content before and after fermentation was detected according to the method in 1.3.4 above (the crude polysaccharide content before fermentation was the blank crude polysaccharide content), and the changes in crude polysaccharides before and after fermentation were compared.

[0075] 3.2.3 The results of crude polysaccharide changes before and after fermentation are shown in Table 3. The increase in crude polysaccharides in experimental group a was higher than that in the control group, reaching 0.75 g / L; the increase in crude polysaccharides in experimental groups b, c, and d were all lower than those in the control group, indicating that increasing the proportion of Polygonatum odoratum in the formula can increase the yield of crude polysaccharides. Therefore, the proportion of Polygonatum odoratum was increased in the subsequent formula ratio optimization experiment.

[0076] Table 3 Changes in crude polysaccharide content of each group of plant-based extracts before and after fermentation

[0077]

[0078] 3.2.4 Prepare plant-based extracts from four kinds of decoction pieces, namely, yuzhu, tangerine peel, licorice and ebony, according to experimental group e, experimental group f, experimental group g, experimental group h and experimental group i, respectively, and the weight of the total Chinese medicine composition remains unchanged; prepare plant-based extracts according to the method of "2. Preparation of plant-based extracts of medicinal and edible homology" above. The mass ratios of yuzhu, tangerine peel, licorice and ebony in each group are as follows:

[0079] Experimental group e: 2:1:1:1;

[0080] Experimental group f: 3:1:1:1;

[0081] Experimental group g: 4:1:1:1;

[0082] Experimental group h: 5:1:1:1;

[0083] Experimental group i: 6:1:1:1.

[0084] 3.2.5 The strain RH JS001 was inoculated into the activation medium and cultured at 37°C for 24 h for activation; then the activated bacterial solution was inoculated into the plant-based extract obtained in each group at an inoculum size of 5% (v / v), the initial pH value was adjusted to 6.5, and the culture was carried out at 37°C for 48 h. The crude polysaccharide content before and after fermentation was detected according to the method in 1.3.4 above (the crude polysaccharide content before fermentation was the blank crude polysaccharide content), and the changes in crude polysaccharides before and after fermentation were compared.

[0085] 3.2.6 The results of the change in crude polysaccharides before and after fermentation are shown in Table 4. Among them, the increase in crude polysaccharides in experimental group h was the highest, reaching 0.90 g / L. Therefore, experimental group h was selected as the formula ratio for preparing plant-based extracts.

[0086] Table 4 Changes in the amount of crude polysaccharide in the plant-based extracts of each experimental group before and after fermentation

[0087]

[0088] 4. Optimization of the fermentation process

[0089] Experimental principle: Research shows that antioxidant therapy can effectively improve the pathological changes of atrophic gastritis. SOD has super antioxidant ability, so the SOD activity is also used as one of the screening indicators.

[0090] 4.1 Optimize the optimal addition amount of sucrose in the fermentation process.

[0091] 4.1.1 Add 20 g / L, 30 g / L, and 40 g / L of sucrose to the plant-based extracts of the experimental groups h respectively as options for process optimization, and use the extracellular polysaccharide yield and SOD activity as selection indicators.

[0092] Experimental group A: Add 20 g / L of sucrose to the plant-based extract;

[0093] Experimental group B: Add 30 g / L of sucrose to the plant-based extract;

[0094] Experimental group C: Add 40 g / L of sucrose to the plant-based extract;

[0095] 4.1.2 Inoculate the strain RH JS001 into the activation medium and culture it at 37 °C for 24 h for activation; then take the activated bacterial liquid and inoculate it into experimental group A, experimental group B, and experimental group C at an inoculation amount of 5% (v / v). Fermentation conditions: Adjust the initial pH value to 6.5, keep the temperature constant at 37 °C, the stirring speed at 50 r / min, and ferment for 48 h.

[0096] 4.1.3 Detect the content of crude polysaccharide according to the method in 1.3.4 above. Use the crude polysaccharide content before fermentation in each experimental group to detect the crude polysaccharide content before and after fermentation according to the method in 1.3.4 above (the crude polysaccharide content before fermentation is the blank crude polysaccharide content), and compare the changes in the amount of crude polysaccharide before and after fermentation.

[0097] Use the total SOD activity detection kit (WST-8 method) (manufacturer: Shanghai Beyotime Biotechnology Co., Ltd.) to detect the SOD activity of each experimental group according to the operation method of the kit. Use the SOD activity before fermentation in each experimental group as a control to compare the changes in SOD activity before and after fermentation.

[0098] 4.1.5 Before and after fermentation, the changes in the content of crude polysaccharides and SOD activity are shown in Table 5. Among them, the increases in the content of crude polysaccharides and SOD activity in experimental group B are the largest, reaching 1.29 g / L and 23.3 u / ml respectively, indicating that its extracellular polysaccharide production is the highest and its SOD activity is the highest. Therefore, 30 g / L of sucrose is added to the production process of this fermented beverage.

[0099] Table 5 Changes in the content of crude polysaccharides and SOD activity in each experimental group

[0100]

[0101] 4.2 Optimize the optimal control conditions of pH in the fermentation process.

[0102] 4.2.1 According to the test results in 4.1, 30 g / L of sucrose is added to each experimental group in the fermentation process. Then, without controlling pH during fermentation (adjust the pH value to 6.5 before natural fermentation), constantly controlling pH at 5.0, constantly controlling pH at 6.0, and constantly controlling pH at 7.0 are set as process optimization options. The extracellular polysaccharide production and SOD activity are used as selection indicators, and the group situations are as follows:

[0103] Experimental group D: Natural fermentation without controlling pH during fermentation;

[0104] Experimental group E: Constantly control pH at 5.0 during fermentation;

[0105] Experimental group F: Constantly control pH at 6.0 during fermentation;

[0106] Experimental group G: Constantly control pH at 7.0 during fermentation;

[0107] 4.2.2 Inoculate the strain RH-FJ01 into the activation medium and culture it at 37 °C for 24 h for activation; then take the activated bacterial liquid and inoculate it into experimental group D, experimental group E, experimental group F, and experimental group G at an inoculation amount of 5% (v / v). Fermentation conditions: Adjust the initial pH to the corresponding value, keep the temperature constant at 37 °C, and the stirring speed at 50 r / min. Control the automatic flow addition of 15% (w / w) sodium hydroxide solution in experimental groups E - G, and ferment for 48 h.

[0108] 4.2.3 Detect the content of crude polysaccharides according to the method in 1.3.4 above. Detect the content of crude polysaccharides before and after fermentation in each experimental group according to the method in 1.3.4 above (the content of crude polysaccharides before fermentation is the blank crude polysaccharide content), and compare the changes in the content of crude polysaccharides before and after fermentation;

[0109] 4.2.4 Use the total SOD activity detection kit (WST-8 method) (manufacturer: Shanghai Beyotime Biotechnology Co., Ltd.) to detect the SOD activity of each experimental group according to the operation method of the kit. Use the SOD activity before fermentation of each experimental group as a control to compare the change in SOD activity before and after fermentation.

[0110] Before and after fermentation, the change amounts of crude polysaccharide and SOD activity are shown in Table 6. Among them, the increase amounts of crude polysaccharide and SOD activity in experimental group F are the largest, reaching 2.86 g / L and 33.2 U / mL respectively, indicating that its extracellular polysaccharide yield is the highest and the SOD activity is the highest. Therefore, the constant pH 6.0 during fermentation is selected as the production process condition for this fermented beverage.

[0111] Table 6 Change amounts of crude polysaccharide and SOD activity in each experimental group

[0112]

[0113] 4.3 Prepare the fermentation broth according to the determined production process conditions above (i.e., experimental group F) to obtain the plant-based fermented beverage for subsequent efficacy example research.

[0114] Efficacy Example 1: Improvement effect of plant-based fermented beverage on gastric mucosa of rats with chronic atrophic gastritis

[0115] 1. Experimental principle: By constructing a disease model of chronic atrophic gastritis in rats, compare the gastric mucosal ulcer index of rats to evaluate the improvement effect of plant-based fermented beverage on chronic atrophic gastritis.

[0116] 2. Scoring criteria for gastric mucosal ulcer index

[0117] Ulcer index: Calculated based on the length (mm) of mucosal ulcers and erosions. For spot erosions ≤ 1 mm, it is counted as 1 point, 1 - 2 mm is counted as 2 points, 2 - 3 mm is counted as 3 points, 3 - 4 mm is counted as 4 points, and so on. For each additional 1 mm, it is counted as 1 point. When the erosion width ≥ 1 mm, the score is doubled. The total score of the whole stomach is the ulcer index of this rat.

[0118] 3. Calculation method:

[0119] Ulcer inhibition rate (100%) = (ulcer index of model group - ulcer index of test group) / ulcer index of model group × 100%.

[0120] 4. Establishment and experiment of rat chronic atrophic gastritis model

[0121] Divide 40 SD rats into three groups, with 10 rats in each group, including 5 male and 5 female rats, divided into groups ①, ②, ③, and ④, where:

[0122] ① Group is the control group: Regularly fed with mixed feed and drinking water for 12 weeks;

[0123] ② The second group was the positive group of chronic atrophic gastritis: The rats were treated with a combination of 20 mM sodium deoxycholate, 60% (v / v) alcohol, and 0.1% (v / v) ammonia water for 12 weeks; 20 mM sodium deoxycholate was administered by gavage daily. Fasting gavage was performed on Wednesdays and Saturdays every week. The feed was removed at 8 pm on Tuesdays and Fridays, and fasting gavage started at 8 am the next morning. The dose was 1 mL / time / rat. 60% (v / v) alcohol was given by fasting gavage on Wednesdays and Saturdays, with the same conditions and gavage dose of 1 mL / time / rat. 1% (v / v) ammonia water was freely available for drinking, and the daily consumption of rats was recorded.

[0124] ③ The third group was the experimental group of plant-based extract: Modeling was performed according to the method of the second group, and at the same time, the rats were given plant-based extract (the plant-based extract obtained from part h of the experimental group in 3.2.4, the same below) by gavage at a dose of 0.5 mL / 100 g once a day for 12 weeks.

[0125] ④ The fourth group was the experimental group of plant-based fermented beverage: Modeling was performed according to the method of the second group, and at the same time, the rats were given plant-based fermented beverage by gavage at a dose of 0.5 mL / 100 g once a day for 12 weeks.

[0126] 5. Sample collection and processing

[0127] After 12 weeks, all rats were anesthetized with 10% (m / v) chloral hydrate and then sacrificed by laparotomy to remove the stomach. The stomach was dissected along the greater curvature, and gross observation of the gastric mucosa specimens was performed. After rinsing with normal saline, it was placed on blue table paper for observation; 1 / 3 of the gastric mucosa was excised. Part of it was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator, and part was used for protein extraction.

[0128] 6. Results and analysis

[0129] Table 7 Results of gastric ulcer index and inhibition rate of rats in each group

[0130]

[0131] The test results are shown in Table 7. It can be seen that compared with the control group ①, the gastric ulcer index of the positive group of chronic atrophic gastritis ② increased significantly; the ulcer inhibition rate of the experimental group of plant-based extract ③ was 27.4%, and the ulcer inhibition rate of the experimental group of plant-based fermented beverage ④ was 57.6%. This shows that the plant-based fermented beverage after fermentation of the plant-based extract has a higher ulcer inhibition rate and better effect in improving chronic atrophic gastritis, indicating that it has a better improvement effect on the gastric mucosa.

[0132] Effect Example 2: Effects of plant-based fermented beverage on the contents of SOD, GSH, and MDA in the gastric mucosa tissue of rats with chronic atrophic gastritis

[0133] 1. Experimental principle: In the development of chronic atrophic gastritis, oxidative stress is one of the important influencing factors, which will further exacerbate the damage of gastric mucosal cells and may even induce the development towards gastric cancer. SOD, GSH, and MDA are important markers in the process of oxidative stress. The increase in MDA levels is related to epithelial cell apoptosis, injury, and death; GSH can act as a non-enzymatic antioxidant to scavenge free radicals; SOD can scavenge oxygen free radicals; low activity of GSH and SOD may exacerbate the damage of gastric mucosa and affect the healing of damaged gastric mucosa, while high activity of GSH and SOD may have a protective effect on gastric mucosa.

[0134] 2. Take the gastric mucosal tissues of rats in each group in Effect Example 1 and detect the contents of SOD, GSH, and MDA respectively.

[0135] 3. The test results are shown in Table 8. It can be seen that compared with the control group ①, the contents of SOD and GSH in the gastric mucosal tissue of the chronic atrophic gastritis positive group ② decreased, while the content of MDA increased; compared with the positive group ②, the contents of SOD and GSH in the plant-based extract test group ③ and the plant-based fermented beverage test group ④ increased, and the content of MDA decreased, indicating that both the plant-based extract test group ③ and the plant-based fermented beverage test group ④ can inhibit oxidative stress, thereby playing a protective role on gastric mucosal cells to improve chronic atrophic gastritis. Among them, the plant-based fermented beverage test group ④ has higher contents of SOD and GSH and lower content of MDA than the plant-based extract test group ③, indicating that the fermented plant-based beverage has a better effect in improving chronic atrophic gastritis.

[0136] Table 8 Contents of SOD, GSH, and MDA in the gastric mucosal tissues of rats in each group

[0137]

[0138] Example 3 Effects of Plant Fermented Beverage on the Contents of IL-6, IL-1β, and TNF-α in the Gastric Mucosal Tissue of Rats with Chronic Atrophic Gastritis

[0139] 1. Experimental principle: Pro-inflammatory cytokines IL-6, IL-1β, and TNF-α play important roles in inflammatory-related diseases. The long-term persistent inflammatory response in chronic atrophic gastritis exacerbates the damage of gastric mucosa and induces the production of a large amount of oxygen free radicals, affecting the repair and healing of the damaged part of gastric mucosa. By detecting the content changes of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, the anti-inflammatory effect of plant fermented beverage on chronic atrophic gastritis is evaluated.

[0140] 2. Take the gastric mucosal tissues of rats in each group in Effect Example 1 and detect the contents of IL-6, IL-1β, and TNF-α respectively.

[0141] 3. The test results are shown in Table 9. It can be seen that compared with the control group ①, the contents of IL-6, IL-1β, and TNF-α in the gastric mucosa tissue of the chronic atrophic gastritis positive group ② increased; compared with the positive group ②, the contents of IL-6, IL-1β, and TNF-α in the plant-based extract test group ③ and the plant-based fermented beverage test group ④ decreased, indicating that both the plant-based extract test group ③ and the plant-based fermented beverage test group ④ have anti-inflammatory effects, thus playing a role in improving chronic atrophic gastritis. Among them, the contents of IL-6, IL-1β, and TNF-α in the plant-based fermented beverage test group ④ were lower than those in the plant-based extract test group ③, indicating that the fermented plant-based beverage has a better effect in improving chronic atrophic gastritis.

[0142] Table 9 Contents of IL-6, IL-1β, and TNF-α in the gastric mucosa tissue of rats in each group

[0143]

[0144] Effect Example 4: Clinical Trial

[0145] 1. 96 patients diagnosed with chronic atrophic gastritis by rapid urease test and gastroscopy were selected, including 48 male patients and 48 female patients; they were divided into a control group and a test group, with 48 cases in each group; Control group: 24 males and 24 females, with an average age of 40.4 years and an average disease course of 8.4 years; Test group: 24 males and 24 females, with an average age of 41.2 years and an average disease course of 8.1 years.

[0146] 2. The control group adopted the PPI triple therapy (omeprazole, clarithromycin, amoxicillin), where omeprazole was 20 mg each time, twice a day; clarithromycin was 500 mg each time, twice a day; amoxicillin was 1.0 g each time, twice a day.

[0147] 3. The test group was treated with a plant-based fermented beverage in addition to the control group, 30 mL each time, twice a day.

[0148] 4. The treatment time was 14 days. Clinical symptoms were compared. The basic clinical symptoms of chronic atrophic gastritis can be summarized as belching, acid reflux, and upper abdominal pain. According to the severity of the clinical symptoms, the symptoms were divided into 0 - 3 points, and then recorded as 0, 1, 2, 3 points. 0 points represented no symptoms, 1 point represented mild symptoms, 2 points represented moderate symptoms, and 3 points represented severe symptoms; among them, mild symptoms were relatively mild and did not affect normal work and life; moderate symptoms were slightly more obvious and had a certain impact on life and work; severe symptoms seriously affected work and life. After treatment, the scores were statistically analyzed, the average score was recorded, and the similarities and differences before and after treatment and between groups were compared.

[0149] 5. The test results are shown in Table 10. Before treatment, the symptom scores of the two groups of patients were basically the same. After 14 days of treatment, compared with before treatment, the symptoms of belching, acid reflux, upper abdominal pain and the total score of the two groups of patients were significantly improved. Among them, the scores of belching, acid reflux, upper abdominal pain and the total score of the experimental group were lower than those of the control group, indicating that it has a better effect on improving the clinical symptoms of chronic atrophic gastritis. Therefore, taking this plant-based fermented drink at a dose of 30 mL once and twice a day has the effect of improving chronic atrophic gastritis.

[0150] Table 10 Clinical trial results

[0151]

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A plant fermentation composition, characterized in that, The plant fermentation composition is fermented from polygonatum odoratum, tangerine peel, licorice and smoked plum, and the strain used for fermentation is lactic acid bacteria; the mass ratio of polygonatum odoratum, tangerine peel, licorice and smoked plum is (2~6.1) : 1 : 1 : 1; The lactic acid bacteria is one of Lactobacillus plantarum HCS03-001, Lactobacillus plantarum RH JS001, Lactobacillus fermentum HCS08-005, Lactobacillus paracasei HCS17-040, Lactobacillus rhamnosus HCS01-013 and Lactobacillus reuteri HCS02-001; The preparation method of the plant fermentation composition comprises the following steps: Mix polygonatum odoratum, tangerine peel, licorice and smoked plum, add water according to the solid-liquid ratio of 1:4~10 (w / w), heat to boiling, maintain for 2~4 h and then filter to obtain a first extract and filter residue; Add water to the filter residue according to 2~4 times the initial weight of polygonatum odoratum, tangerine peel, licorice and smoked plum, heat to boiling, maintain for 2~4 h and then filter to obtain a second extract; Mix the first extract and the second extract, centrifuge, and take the supernatant to obtain the target extract; Inoculate the strain into a culture medium for activation to obtain the activated bacterial liquid; Inoculate the activated bacterial liquid into the obtained target extract, adjust the pH value to 5-7, and ferment at 30-50 °C for 24-96 h to obtain a plant fermentation composition, wherein the volume of the activated bacterial liquid is 2%-10% of the volume of the target extract, and the cell concentration in the activated bacterial liquid is 10 8 ~10 9 CFU / mL.

2. The plant fermentation composition according to claim 1, characterized in that, The mass ratio of polygonatum odoratum, tangerine peel, licorice and smoked plum is (2.9~6.1) : 1 : 1 :

1.

3. The plant fermentation composition according to claim 2, characterized in that, The mass ratio of polygonatum odoratum, tangerine peel, licorice and smoked plum is 5 : 1 : 1 :

1.

4. The plant fermentation composition according to any one of claims 1 to 3, characterized in that, The strain used for fermentation includes Lactobacillus plantarum RH JS001.

5. A method for preparing the plant fermentation composition according to any one of claims 1 to 4, characterized in that, Comprises the following steps: Mix polygonatum odoratum, tangerine peel, licorice and smoked plum, add water according to the solid-liquid ratio of 1:4~10 (w / w), heat to boiling, maintain for 2~4 h and then filter to obtain a first extract and filter residue; Add water to the filter residue according to 2~4 times the initial weight of polygonatum odoratum, tangerine peel, licorice and smoked plum, heat to boiling, maintain for 2~4 h and then filter to obtain a second extract; Mix the first extract and the second extract, centrifuge, and take the supernatant to obtain the target extract; Inoculate the strain into a culture medium for activation to obtain the activated bacterial liquid; Inoculate the activated bacterial liquid into the obtained target extract, adjust the pH value to 5 - 7, and ferment at 30 - 50 °C for 24 - 96 h to obtain a plant fermentation composition, wherein the volume of the activated bacterial liquid is 2% - 10% of the volume of the target extract, and the cell concentration in the activated bacterial liquid is 10 8 ~10 9 CFU / mL.

6. The method for preparing the plant fermentation composition according to claim 5, characterized in that, Before inoculating the activated bacterial liquid into the target extract, first add a carbon source to the extract, and the concentration of the carbon source in the extract is 20~40 g / L.

7. The method for preparing the plant fermentation composition according to claim 6, characterized in that, The concentration of the carbon source in the extract is 30 g / L.

8. The method for preparing the plant fermentation composition according to claim 6, characterized in that, The carbon source includes sucrose.

9. The method for preparing the plant fermentation composition according to claim 5, characterized in that, During the fermentation process, control the pH value within the range of 5~7.

10. The method for preparing the plant fermentation composition according to claim 9, characterized in that, During the fermentation process, control the pH value to be 6.

11. Use of the plant fermentation composition according to any one of claims 1 to 4 or the plant fermentation composition prepared by the preparation method according to any one of claims 5 to 10 in the preparation of a medicament for improving chronic atrophic gastritis.

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