Preparation method of cereal fermentation broth metaplasm and application of cereal fermentation broth metaplasm in improvement of systemic chronic inflammation and reduction of cholesterol and low-density lipoprotein

Abstract

The invention provides a preparation method of cereal fermentation broth postbiotics and application of the cereal fermentation broth postbiotics in improvement of systemic chronic inflammation and reduction of cholesterol and low-density lipoprotein, and belongs to the technical field of fermentation engineering. The invention provides a preparation method of cereal fermentation broth metabiotics, which is characterized in that mixed cereals are used as raw materials, anaerobic fermentation is carried out by using compound enzymes and various bacteria, and polysaccharides and proteins in the cereal raw materials can be quickly converted into functional small molecular substances, such as polypeptides, total phenols and total flavonoids. The cereal fermentation broth metagen can inhibit secretion of a macrophage proinflammatory medium, inhibit generation of inflammatory factors of liver, heart and intestinal tissues, and improve activation of adipose tissue macrophages related to body inflammation. After the cereal fermentation liquor is subjected to post-biogen treatment, glucose and lipid metabolism disorder can be effectively improved, liver function damage can be relieved, and the intestinal barrier function can be effectively improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermentation engineering, and in particular relates to a method for preparing postbiotics from cereal fermentation liquid and an application thereof in improving systemic chronic inflammation and lowering cholesterol and low-density lipoprotein. Background Art

[0002] Obesity associated with systemic chronic inflammation is a major factor in inducing chronic metabolic syndromes such as hypertension, cardiovascular disease, and type 2 diabetes. On the one hand, the endogenous release of free fatty acids in adipocytes activates macrophages and increases the level of systemic proinflammatory factors. On the other hand, the imbalance of intestinal microecology mediated by a high-fat diet has a negative impact on the diversity of intestinal microbiota and intestinal integrity. Endotoxins such as lipopolysaccharide (LPS) from Gram-negative bacteria enter the submucosal layer, causing a subclinical increase in the level of proinflammatory mediators and inducing systemic inflammation. Currently, chronic inflammatory-related diseases are a major challenge facing the global public health system and have imposed a serious economic burden on the sick population.

[0003] At present, many bioactive compounds in food have anti-inflammatory activity and are used as potential methods to prevent and improve chronic inflammation. Plant-based foods are low in cholesterol and almost free of saturated fat, which can effectively limit calorie intake and reduce the incidence of chronic metabolic syndrome. Cereals such as millet, sorghum, glutinous rice, fragrant rice, and rice are rich in dietary fiber and phenolic plant compounds and have strong anti-inflammatory potential. The latest "Expert Consensus on Anti-Inflammatory Diet for Cancer Prevention" also emphasizes that whole grain carbohydrates are an important component of anti-inflammatory diet. Cereal fermentation can degrade indigestible polysaccharides and oligosaccharides, dissociate antinutrients from polysaccharides and proteins, improve the digestibility and absorbability of products, and improve the bioavailability of functional ingredients in cereals. Therefore, fermented products with cereals as the main raw materials have broad application prospects in the consumer market for gastrointestinal diseases, but the use of fermentation to improve the bioavailability of functional ingredients in cereals, especially the clinical effects on chronic inflammatory diseases, is not thoroughly studied at present. Summary of the invention

[0004] The present invention provides a method for preparing postbiotics from cereal fermentation broth and its application in improving systemic chronic inflammation and lowering cholesterol and low-density lipoprotein. The method can inhibit the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells, and improve the body's chronic inflammation-related glucose and lipid metabolism disorders and impaired intestinal barrier function.

[0005] The present invention provides a method for preparing postbiotics from a cereal fermentation liquid, which mainly comprises the following steps: mixing mature mixed cereals with a composite enzyme and fermentation bacteria and then performing anaerobic fermentation to obtain the postbiotics from the cereal fermentation liquid; The complex enzyme comprises saccharifying enzyme, amylase, cellulase and pectinase; The fermentation bacteria include a composite bacteria of yeast and lactic acid bacteria.

[0006] In a preferred embodiment of the present invention, the types of mixed cereals include black glutinous rice, fragrant rice, rice, millet and corn.

[0007] In a preferred embodiment of the present invention, the mixed cereals include the following raw materials in parts by mass: 2-5 parts of black glutinous rice, 3-6 parts of fragrant rice, 4-7 parts of rice, 4-6 parts of millet and 5-8 parts of corn.

[0008] In a preferred embodiment of the present invention, the mass of the complex enzyme is 0.4% of the mass of the mixed grains; Among them, the mass ratio of saccharifying enzyme, amylase, cellulase and pectinase is (1.5~2.8):(2~3.5):(0.4~0.8):(0.6~1.0).

[0009] In a preferred embodiment of the present invention, the amount of live bacteria added to the fermentation bacteria is (0.3-3)×10 7 CFU / g mixed grains; The yeast in the fermentation bacteria includes at least one of baker's yeast and sweet wine yeast; The lactic acid bacteria in the fermentation bacteria include at least one of Lactobacillus delbrueckii subspecies and Streptococcus salivarius subspecies.

[0010] In a preferred embodiment of the present invention, the temperature of the anaerobic fermentation is 30° C. and the time is 7 to 8 days.

[0011] The present invention also provides postbiotics from cereal fermentation liquid prepared by the above preparation method.

[0012] The present invention also provides the use of the postbiotics of the cereal fermentation liquid in preparing food.

[0013] The present invention also provides the use of the above-mentioned cereal fermentation broth postbiotics in the preparation of medicines, which have at least one of the following effects: improving chronic inflammation of the body, reducing serum cholesterol content, reducing low-density lipoprotein content, reducing the level of serum pro-inflammatory cytokines in the body, inhibiting the activation of adipose tissue macrophages, maintaining blood sugar and insulin levels and maintaining intestinal barrier function.

[0014] The present invention also provides a medicine comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the above-mentioned cereal fermentation liquid postbiotics.

[0015] Beneficial effects: The present invention provides a method for preparing postbiotics from grain fermentation liquid, which uses mixed grains as raw materials and utilizes complex enzymes and multiple bacteria for anaerobic fermentation. After anaerobic fermentation, polysaccharides and proteins in the grain raw materials can be rapidly converted into functional small molecules, which is manifested in a significant increase in the content of polypeptides, total phenols and total flavonoids after the anaerobic fermentation.

[0016] The postbiotics of the cereal fermentation broth of the present invention can inhibit the secretion of pro-inflammatory mediators of macrophages, such as reducing the release of TNF-α, NO and IL-6 pro-inflammatory cytokines in macrophages, while inhibiting the generation of inflammatory factors in liver, heart and intestinal tissues, and can also improve the activation of adipose tissue macrophages related to inflammation in the body. The occurrence of chronic inflammation in the body is accompanied by impaired liver function, disorder of glycolipid metabolism and impaired intestinal barrier function. After being treated with the postbiotics of the cereal fermentation broth of the present invention, it can effectively improve the disorder of glycolipid metabolism, alleviate liver function damage, and effectively improve the intestinal barrier function. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the results of the determination of the basic components of postbiotics in cereal fermentation broth. In the graph, A: pH value of each fermentation broth; B: polypeptide content in each fermentation broth; C: total phenol content in each fermentation broth; D: total flavonoid content in each fermentation broth; Figure 2 This is a statistical chart of inflammatory factor release in the macrophage model; Figure 3 This is a graph showing weight changes in a chronic inflammation mouse model; Figure 4 This is a graph showing changes in the secretion of inflammatory factors in a chronic inflammation mouse model; Figure 5 These are H&E stained pathological sections of inflammatory organs in a chronic inflammation mouse model, where AE is liver tissue and FJ is heart tissue; Figure 6 This is a graph showing changes in glucose and lipid metabolism-related indicators in a chronic inflammation mouse model; Figure 7 This is the result of H&E section of intestinal tissue in the chronic inflammation mouse model; Figure 8 This is a graph showing changes in colon barrier function in mice with chronic inflammation; Fig. 9 This is the immunohistochemistry result of macrophages in adipose tissue of mice with chronic inflammation. DETAILED DESCRIPTION

[0018] The present invention provides a method for preparing postbiotics from a cereal fermentation liquid, which mainly comprises the following steps: mixing mature mixed cereals with a composite enzyme and fermentation bacteria and then performing anaerobic fermentation to obtain the postbiotics from the cereal fermentation liquid; The complex enzyme comprises saccharifying enzyme, amylase, cellulase and pectinase; The fermentation bacteria are selected from a composite bacteria of yeast and lactic acid bacteria.

[0019] The mixed grains of the present invention are a mixture of grains, and the so-called grains refer to plant seeds that can obtain starchy seeds and are suitable for food, such as wheat, corn, rice, buckwheat, oats, millet, and potatoes. In one embodiment of the present invention, the mixed grains include black glutinous rice, fragrant rice, rice, millet, and corn. In terms of mass, black glutinous rice can be 2 to 5 parts, such as 2 parts, 3 parts, 4 parts, or 5 parts; fragrant honey can be 3 to 6 parts, such as 3 parts, 4 parts, 5 parts, or 6 parts; rice can be 4 to 7 parts, such as 4 parts, 5 parts, 6 parts, or 7 parts; millet can be 4 to 6 parts, such as 4 parts, 5 parts, or 6 parts; corn can be 5 to 8 parts, such as 5 parts, 6 parts, 7 parts, or 8 parts. In the present invention, compared with other types of grain mixing, the active peptide release is best after fermenting black glutinous rice, fragrant rice, rice, millet, and corn.

[0020] In one embodiment of the present invention, black glutinous rice, fragrant rice, rice, millet and corn are mixed, soaked in sterile distilled water, and then steam sterilized to obtain cooked grains. The soaking in the present invention can be carried out in a ratio of 1:2 between the volume of sterile distilled water and the total mass of grains, and the soaking time can be 2 hours. The soaking can allow the grains to fully absorb water, which is convenient for subsequent steaming and maturation. Sufficient soaking is conducive to sufficient steaming of the grains within the specified time (1 hour) to achieve the required maturity and softness for fermentation. The present invention does not specifically limit the method of steam sterilization, and the conventional steam sterilization scheme in the art can be used, such as the conventional parameters for steam sterilization for 1 hour in the embodiment.

[0021] The present invention places the cooked mixed grains in a fermentation basin, and adds a composite enzyme and fermentation bacteria. In one embodiment, the composite enzyme can be added first and then the fermentation bacteria, wherein the composite enzyme includes saccharifying enzyme, amylase, cellulase and pectinase, and the mass of the composite enzyme is 0.4% of the mass of the mixed grains; wherein the mass ratio of saccharifying enzyme, amylase, cellulase and pectinase is (1.5-2.8): (2-3.5): (0.4-0.8): (0.6-1.0). The addition of the composite enzyme of the present invention can achieve effective degradation of macromolecular starch and protein in grains, thereby facilitating microbial fermentation and utilization. The composite enzyme of the present invention can fully play a role in grain fermentation and will not be used as a nitrogen source by microorganisms: when the grains are mixed and fermented, the composite enzyme is first added for enzymolysis, and then the mixed bacteria are added for fermentation after 1 hour. The time difference between the composite enzyme and the mixed bacteria in the mixed grain system can ensure that the composite enzyme fully plays a degradation role and will not be metabolized and utilized by microorganisms; in addition, the mixed grains contain a large amount of carbon source and nitrogen source, and the amount of composite enzyme added is relatively small, and will not be metabolized and utilized by the strain as a nitrogen source.

[0022] In the present invention, fermentation bacteria are added to the fermentation basin, and the amount of live bacteria added to the fermentation bacteria is (0.3~3)×10 7 CFU / g mixed grains. The fermentation bacteria of the present invention are selected from yeast and lactic acid bacteria, wherein the yeast can be at least one of baker's yeast and sweet wine yeast. In one embodiment, the effective live bacteria ratio of the baker's yeast, sweet wine yeast and lactic acid bacteria is 2:1:1. The lactic acid bacteria of the present invention can be at least one of Lactobacillus delbrueckii subspecies and Streptococcus salivarius subspecies, and the effective live bacteria ratio of the Lactobacillus delbrueckii subspecies and Streptococcus salivarius subspecies is 2:1~10:1. For example, Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius thermophilus subspecies are selected in the embodiment. There is no limitation on the strains used in the fermentation process of the present invention, and the mixed strains are all commercial products. The strains are inoculated into the mixed grain fermentation system in the form of bacterial powder.

[0023] The present invention also provides postbiotics from cereal fermentation liquid prepared by the above preparation method.

[0024] In an embodiment of the present invention, the anaerobic fermentation is carried out in a fermentation basin, wherein the temperature of the anaerobic fermentation is 30°C and the time is 7-8 days. After the mixed grains are fermented as described in the present invention, the fermentation supernatant is extracted to obtain the postbiotics of the grain fermentation liquid, which can significantly reduce the level of serum proinflammatory cytokines, inhibit the activation of adipose tissue macrophages, maintain blood sugar and insulin levels, and maintain intestinal barrier function through diet therapy. It can be used to inhibit the secretion of proinflammatory mediators of macrophages and intestinal epithelial cells, improve the body's chronic inflammation-related glucose and lipid metabolism disorders and impaired intestinal barrier function.

[0025] The present invention also provides the use of the postbiotics of the cereal fermentation liquid in preparing food.

[0026] The fermented liquid obtained by the invention can be eaten after being sterilized by boiling or other methods.

[0027] The present invention also provides the use of the above-mentioned cereal fermentation broth postbiotics in the preparation of medicines, which have at least one of the following effects: improving chronic inflammation of the body, reducing serum cholesterol content, reducing low-density lipoprotein content, reducing the level of serum pro-inflammatory cytokines in the body, inhibiting the activation of adipose tissue macrophages, maintaining blood sugar and insulin levels and maintaining intestinal barrier function.

[0028] In the present invention, yeast and lactic acid bacteria co-ferment to produce a synergistic effect, which can significantly increase the accumulation of polypeptides, total phenols and total flavonoids in the fermentation product, and significantly alleviate macrophage inflammatory response. The use of the cereal fermentation broth of the present invention to feed a chronic inflammation model showed that the fermentation broth has a significant improvement effect on the weight loss caused by inflammation in the body, inhibits the generation of inflammatory factors and endotoxins in the liver, heart, intestinal tissue and plasma, alleviates the inflammation level of the main inflammatory organs, and can also reduce serum cholesterol and low-density lipoprotein.

[0029] The present invention also provides a medicine comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the above-mentioned cereal fermentation liquid postbiotics.

[0030] The present invention has no special limitation on the dosage form of the drug, and the drug can be prepared using conventional dosage forms and auxiliary materials in the art.

[0031] To further illustrate the present invention, the method for preparing postbiotics from cereal fermentation broth provided by the present invention and its application in improving systemic chronic inflammation, lowering cholesterol and low-density lipoprotein are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0032] In the embodiments of the present invention, unless otherwise specified, the materials used are conventional commercially available products in the art.

[0033] Example 1 300 g of black glutinous rice, 300 g of fragrant rice, 300 g of rice, 300 g of millet and 300 g of corn were mixed, and then 600 mL of sterilized distilled water was added to soak for 2 h, followed by steam sterilization for 1 h to obtain cooked grains; the cooked grains were transferred to a fermentation basin, and 6 g of complex enzymes were added, including 1.846 g of saccharifying enzyme (product number: 60883074703, trade name Jianshi food grade saccharifying enzyme), 1.846 g of amylase (product number: 60883074703, trade name Jianshi food grade saccharifying enzyme), and 1.846 g of amylase (product number: 60883074703, trade name Jianshi food grade saccharifying enzyme). 59985, trade name a-starch medium temperature enzyme food grade) 2.769g, cellulase (product number: 60883065873, trade name Jianshi food grade cellulase) 0.462g, pectinase (product number: 60883082113608830882113, pectinase food grade edible enzyme) 0.923g, the product numbers of the above enzymes are all the product numbers of JD.com, and the enzymes are from Henan Wanbang Industrial Co., Ltd. The above-mentioned cooked grains and compound enzyme mixed raw materials were enzymatically hydrolyzed for 1 hour and marked as Cere; Inoculate (0.3~3)×10 7CFU / g of fermentation bacteria of grain raw materials, including baker's yeast, sweet wine yeast and mixed lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius thermophilus subsp., with an effective live bacteria count ratio of 2:1-10:1), of which baker's yeast, sweet wine yeast and mixed lactic acid bacteria were purchased from Angel Yeast Co., Ltd., with an effective live bacteria ratio of 2:1:1. They were fermented at 30°C in a closed environment for 7-8 days, and the fermented juice was filtered through gauze to obtain postbiotics from grain fermentation liquid, recorded as HFC.

[0034] Inoculate (0.3~3)×10 6 CFU / g of fermentation bacteria of grain raw materials, including baker's yeast, sweet wine yeast and mixed lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius thermophilus subsp., with an effective live bacteria count ratio of 2:1-10:1), of which baker's yeast, sweet wine yeast and mixed lactic acid bacteria were purchased from Angel Yeast Co., Ltd., with an effective live bacteria ratio of 2:1:1. They were fermented at 30°C in a closed environment for 7-8 days, and the fermented juice was filtered through gauze to obtain postbiotics from grain fermentation liquid, recorded as HFC1.

[0035] Inoculate (0.3~3)×10 5 CFU / g of fermentation bacteria of grain raw materials, including baker's yeast, sweet wine yeast and mixed lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius thermophilus subsp., with an effective live bacteria count ratio of 2:1-10:1), among which baker's yeast, sweet wine yeast and mixed lactic acid bacteria were purchased from Angel Yeast Co., Ltd., with an effective live bacteria ratio of 2:1:1. They were fermented at 30°C in a closed environment for 7-8 days, and the fermented juice was filtered through gauze to obtain postbiotics from grain fermentation liquid, recorded as HFC2.

[0036] Inoculate (0.3~3)×10 8 CFU / g of fermentation bacteria of grain raw materials, including baker's yeast, sweet wine yeast and mixed lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius thermophilus subsp., with an effective live bacteria count ratio of 2:1-10:1), among which baker's yeast, sweet wine yeast and mixed lactic acid bacteria were purchased from Angel Yeast Co., Ltd., with an effective live bacteria ratio of 2:1:1. They were fermented at 30°C in a closed environment for 7-8 days, and the fermented juice was filtered through gauze to obtain postbiotics from grain fermentation liquid, recorded as HFC3.

[0037] Inoculate (0.3~3)×10 7 The effective live bacteria ratio of baker's yeast and sweet wine yeast CFU / g grain raw materials is 2:1. The baker's yeast and sweet wine yeast were purchased from Angel Yeast Co., Ltd. and fermented at 30°C for 7-8 days in a closed environment. The fermented juice was filtered through gauze to obtain postbiotics from grain fermentation liquid, which was recorded as SFC.

[0038] Inoculate (0.3~3)×10 7 CFU / g of lactic acid bacteria from cereal raw materials (the lactic acid bacteria were purchased from Angel Yeast Co., Ltd.) were fermented in a closed environment at 30°C for 7-8 days, and the fermented juice was filtered through gauze to obtain postbiotics from cereal fermentation liquid, which was recorded as LFC.

[0039] Example 2 Determination of the content of postbiotic components in cereal fermentation broth The changes in the acidity of samples in Cere and fermentation broths HFC, HFC1, HFC2, HFC3, SFC and LFC in Example 1 were measured using a fully automatic pH measuring instrument. The measuring method is as follows: 2.1 Take 1 mL of the test solution and add it to 5 mL of distilled water, mix well and then measure the pH value.

[0040] The results are as follows Figure 1 As shown in A, after inoculation of fermentation bacteria, the pH values ​​of all groups decreased as the fermentation progressed. The pH values ​​of HFC3 and LFC groups decreased most significantly, from the initial 6.175±0.17 to 3.935±0.05 and 3.707±0.06, respectively; the pH values ​​of HFC2 and SFC groups changed least significantly, decreasing to 4.645±0.02 and 5.195±0.12, respectively, after 24 hours of fermentation; the pH values ​​of HFC and HFC1 groups changed moderately, decreasing to 4.26±0.08 and 4.34±0.03, respectively.

[0041] 2.2 The changes in the sample polypeptide content in Cere and fermentation broths HFC, HFC1, HFC2, HFC3, SFC and LFC in Example 1 were determined using the biuret method. The determination method is as follows: The test solution was mixed with an equal volume of 10% trichloroacetic acid, allowed to stand for 10 min, centrifuged at 10000 g for 10 min, the supernatant was mixed with biuret reagent at a volume ratio of 1:4, reacted at room temperature for 30 min, and then the OD value was measured at 310 nm. Glutathione tripeptide was used as the standard to calculate the polypeptide content.

[0042] The results are as follows Figure 1 As shown in B, the polypeptide release of all groups increased after inoculation of fermentation bacteria, among which the polypeptide content of HFC and HFC1 groups increased most significantly, from the initial 1.65±0.10 mg / mL to 2.77±0.15 mg / mL and 2.75±0.10 mg / mL, respectively.

[0043] 2.3 Determination of the contents of polyphenols and flavonoids in Cere and fermentation broths HFC, HFC1, HFC2, HFC3, SFC and LFC prepared in Example 1.

[0044] 2.3.1 Determination of total phenol content by Folin-phenol method: Take 200 μL of the test solution diluted 5 times into a 10 mL volumetric flask, add 2.5 mL of Folin reagent, vortex, add 2 mL of saturated sodium carbonate, add distilled water to make up to 10 mL, and measure the absorbance at 760 nm. Total polyphenols are expressed as gallic acid equivalent per mL of test solution.

[0045] 2.3.2 Determination of total flavonoids content by aluminum chloride-potassium acetate colorimetric method: take 100 μL of the test solution diluted 5 times into a 10 mL volumetric flask, add anhydrous ethanol to 5 mL, add 300 μL of 5% sodium nitrite solution, vortex and let stand for 6 min, add 2 mL of 1 M NaOH, make up to 10 mL with anhydrous ethanol, measure the absorbance at 510 nm, and the total flavonoids are expressed as rutin equivalent per mL of fermentation liquid.

[0046] The results of polyphenol and flavonoid content determination are as follows Figure 1 As shown in C and D, the release of total phenols and total flavonoids in all groups increased after inoculation of fermentation bacteria, among which the content of HFC and HFC1 groups increased most significantly. The total phenols increased from the initial 0.116±0.01 mg / mL to 0.266±0.00 mg / mL and 0.247±0.00 mg / mL, respectively; the total flavonoids increased from the initial 0.171±0.03 mg / mL to 0.333±0.03 mg / mL and 0.318±0.03 mg / mL, respectively.

[0047] Therefore, too little inoculation amount (HFC2) is not enough to complete the conversion of polysaccharides and proteins in cereal raw materials into functional small molecules within 24 hours, which is manifested as a higher acidity value and a lower polypeptide content; while too much inoculation amount (HFC3) accelerates the fermentation process, produces more organic acids, and thus inhibits the polysaccharide and protein metabolic activities of lactic acid bacteria, which is manifested as a lower acidity value and a lower polypeptide content. Therefore, (0.3~3)×10 7 CFU / g of cereal raw material was the optimal inoculum.

[0048] In addition, compared with lactic acid bacteria fermentation alone (LFC) or yeast fermentation alone (SFC), at the same inoculation amount, there is metabolic synergy in the mixed fermentation of yeast and lactic acid bacteria (HFC, HFC1), which is manifested in the accumulation of higher amounts of peptides, total phenolics and total flavonoids in the mixed fermentation products.

[0049] Example 3 Cereal fermentation broth postbiotics inhibit the secretion of pro-inflammatory mediators by macrophages The inflammatory macrophage model was established using the RAW264.7 cell line. The cells were cultured at 3×10 5The cells were inoculated into 24-well cell culture plates and cultured for 24 hours. Then, 5-fold diluted Cere, HFC, LFC and SFC were added to treat the cells for 2 hours. Finally, 100 ng / mL LPS was used to treat the cells for 24 hours to stimulate cellular inflammatory response. The cell culture supernatant was collected and the secretion of macrophage proinflammatory mediators (NO, TNF-α, IL-6) was determined using a kit. The NO kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. with a product number of A013-2-1; the TNF-α and IL-6 kits were purchased from Jiangsu Enzyme Biotechnology Co., Ltd. with product numbers of MB6426A and MB50054A, respectively.

[0050] Inflammatory macrophage model results Figure 2 As shown in the figure, the release of TNF-α, NO and IL-6 proinflammatory cytokines in macrophages increased significantly after LPS treatment, which was about 2 times, 7.4 times and 2.3 times higher than that of the control group (Control). Cere, HFC, SFC and LFC can inhibit LPS-induced macrophage inflammatory response to varying degrees. Among them, the effect of HFC intervention was the most significant, reducing TNF-α secretion by about 26%, NO secretion by about 29% and IL-6 secretion by about 25%; followed by the LFC group, which reduced TNF-α secretion by about 10%, NO secretion by about 18% and IL-6 secretion by about 18%; and the SFC group, which reduced TNF-α secretion by about 10% and NO secretion by about 15%; Cere intervention only reduced TNF-α secretion by about 6%. It can be seen that compared with the unfermented treatment (Cere), microbial fermentation of grains can significantly alleviate the inflammatory response of macrophages, among which the mixed fermentation group of yeast and lactic acid bacteria (HFC) had the most significant effect.

[0051] Example 4 Animal Experiment The experimental animals were specific pathogen-free C57BL / 6J mice (8 weeks old, female) with a body weight of 15-17 g. The mice were placed in a polycarbonate box under the controlled conditions of temperature 22±2°C, humidity 65%±5%, and light / dark photoperiod of 12 h. After one week of adaptive feeding, all mice were randomly divided into 5 groups, namely, postbiotic intervention group with fermented cereal (HFC), intervention group with unfermented cereal (Cere), chronic inflammation model group (HFL), positive control group with fermented chestnut protein (FCP) and blank control group (NC). All groups were provided with normal diet from 0 to 4 weeks. From 4 to 16 weeks, except for the blank control group, the intervention group and model group were provided with 400 µg / Kg LPS drinking water and high-fat diet, and the weight changes were recorded. At the same time, the intervention group was continuously gavaged with postbiotics from fermented cereal (HFC), unfermented cereal (Cere) (0.3 mL / mouse / day) and positive control group with fermented chestnut protein (FCP), and the model group (HFL) was gavaged with an equal amount of sterile water. At the 16th week, the mice were killed by cervical dislocation, and serum, adipose tissue and intestinal tissue were collected for further analysis.

[0052] The changes in mouse body weight Figure 3 As shown, the body weight of mice in the chronic inflammation model group (HFL) was significantly reduced compared with that of mice in the blank control group (NC). The Cere and HFC groups were gavaged with unfermented grains and postbiotics from grain fermented broth, which effectively improved the weight loss of mice. During the intervention period of 4 to 11 days, the effect of the HFC group was significantly better than that of the Cere and positive control (FCP) groups. This shows that postbiotics from unfermented grains and postbiotics from grain fermented broth have an improvement effect on weight loss caused by inflammation in the body, and postbiotics from grain fermented broth are more effective.

[0053] Example 5 4.1 After the 16th week of intervention in Example 4, serum was collected and the levels of inflammatory factors and endotoxin LPS in the mouse serum were measured using Elisa kits. TNF-α, IL-1β, IL-6 and LPS kits were purchased from Jiangsu Enzyme Biotechnology Co., Ltd. with product numbers MB-2868A, MB-2776A, MB-2899A and MB-3418A, respectively.

[0054] The results of the test are as follows Figure 4As shown, the plasma TNF-α content (119.2±9.828 pg / mL), IL-1β content (88.77±2.006 pg / mL) and IL-6 content (137.2±7.490 pg / mL) of mice in the HFL group were significantly higher than those in the NC group (the corresponding inflammatory factor contents were 104.8±8.106 pg / mL, 82.57±2.828 pg / mL and 123.3±4.536 pg / mL, respectively). In addition, the serum endotoxin LPS level of inflammatory mice was 163.2±9.790 EU / L, which was significantly increased compared with 132.3±11.88 EU / L in the NC group (P<0.001). Oral administration of unfermented cereals (Cere) and composite fermented broth (HFC) could inhibit the production of plasma inflammatory factors and endotoxins. The HFC group could most significantly reduce the levels of plasma IL-1β, IL-6 and endotoxin LPS (P<0.05), and there was no significant difference compared with the positive control group.

[0055] 4.2 Elisa kits were used to measure the secretion of inflammatory factors (TNF-α and IL-1β) in the heart, liver and intestinal tissues of mice. The TNF-α and IL-1β kits were purchased from ELISA reagent company with product numbers MM-0132M1 and MM-0040M1, respectively.

[0056] The results are as follows Figure 4 As shown in the data, the secretion levels of TNF-α in the heart and liver of mice in the HFL group were 236.8±6.554 pg / mL and 250±13.79 pg / mL, and the secretion levels of IL-1β were 34.04±1.629 pg / mL and 51.26±3.715 pg / mL, respectively; the secretion level of IL-6 in the intestinal tissue was 7.23±0.279 pg / mL. The secretion levels of the above three inflammatory factors were significantly higher than those in the NC group (heart TNF-α: 206.8.8±9.741 pg / mL, IL-1β: 28.13±1.779 pg / mL; liver TNF-α: 209.9±9.467 pg / mL, IL-1β: 46.41±2.681 pg / mL; intestinal IL-6: 6.89±0.165 pg / mL). Intragastric administration of postbiotics (HFC) from fermented grains can significantly inhibit the production of inflammatory factors in liver, heart and intestinal tissues, and the effect is significantly better than that of unfermented grains (P<0.05).

[0057] 4.3 Heart and liver were harvested for histological analysis. The tissues were placed in 10% neutral buffered formaldehyde and embedded in paraffin after 24 h. The sections (4 μm) were placed on glass slides and stained with H&E. The histopathological changes of the heart and liver of each group of mice in the H&E sections were observed under an optical microscope.

[0058] The results of H&E staining pathological sections are as follows Figure 5 As shown, compared with the control group (NC), the vacuoles around the blue nuclei in the liver tissue of the chronic inflammation mouse model group (HFL) were enlarged, and the nucleus and cytoplasm were separated, which was consistent with the pathological condition of fatty liver. In addition, there was inflammatory infiltration. After gavage with unfermented grains (Cere) and postbiotics (HFC) of grain fermentation broth, the inflammatory infiltrating cells decreased (outlined by red boxes), and the area of ​​vacuoles around the nuclei also decreased. Among them, the effect of the HFC group was the most significant, and the section results were closest to those of the NC and positive control groups (FCP). It can be seen from the heart tissue sections that the cytoplasm of the model group was obviously separated, and there were fat vacuoles, congestion (indicated by blue arrows) and inflammatory infiltration (outlined by blue boxes). After gavage with unfermented grains (Cere) and postbiotics (HFC) of grain fermentation broth, fat vacuoles decreased, interstitial congestion decreased, and inflammatory infiltrating cells decreased. Among them, the effect of the HFC group was the most significant, and the section results were closest to those of the NC and positive control groups (FCP).

[0059] In summary, postbiotics from grains and grain fermentation broth can improve the inflammatory condition of mice, reduce serum proinflammatory factors and endotoxin levels, and alleviate the inflammatory levels of major inflammatory organs. The effect of postbiotics from grain fermentation broth is significantly better than that of unfermented grains.

[0060] Example 6 Animal experiments refer to the relevant operations of Example 4, and a glucose tolerance test (GTT) is performed at week 15 to determine the occurrence of insulin resistance. Before the test, the mice were fasted for 12 to 14 hours (food was removed but water was retained). After fasting, the tail tip of the mouse was cut off. A drop of blood was immediately squeezed out from the incision and placed in a blood glucose meter to measure the initial glucose level. Then, 200 μL of 2 g / kg BW oral glucose solution was immediately given to each mouse by gavage. The same protocol as above was then used to measure and record the mouse glucose levels at 15, 30, 60 and 120 minutes; the total cholesterol, triglyceride, low-density / high-density lipoprotein and transaminase (alanine and aspartate transaminase) levels in the mouse serum were determined using the Nanjing Jiancheng kit. Among them, the catalog numbers of the lipid index TC, TG, LDL-C and HDL-C test kits are A111-1-1, A110-1-1, A113-1-1 and A112-1-1 respectively; the catalog numbers of the transaminase ALT and AST test kits are C009-2-1 and C010-2-1 respectively.

[0061] Glucose tolerance test results Figure 6As shown in the figure, the GTT results evaluated by the area under the curve (AUC) showed that the AUC value of the HFL group (1335±70.41 mmol / L×120 min) was higher than that of the NC group (1047±64.12 mmol / L×120 min). Compared with the HFL group, the glucose AUC of Cere and HFC supplementation decreased, and the effect of HFC was significantly better than that of Cere and positive control (P<0.05). Cereals and postbiotics of cereal fermentation broth can play a positive regulatory role in glucose metabolism in mice with chronic inflammation, and the effect of postbiotics of cereal fermentation broth is significantly better than that of unfermented cereals.

[0062] The levels of total cholesterol, triglyceride and low-density lipoprotein in the serum of mice in the chronic inflammation model group (HFL) were 2.58±0.362 mmol / L, 1.74±0.167 mmol / L and 0.72±0.167 mmol / L, respectively, which were significantly higher than those in the NC group (P<0.05); while the level of high-density lipoprotein in the model group (4.45±0.328 mmol / L) was lower than that in the NC group (4.93±0.568mmol / L). Oral administration of unfermented cereals (Cere) could not significantly improve the lipid metabolism imbalance induced by chronic inflammation, while postbiotics from cereal fermentation liquid (HFC) could significantly inhibit the abnormal increase of serum total cholesterol and total triglyceride levels, and significantly reduce the content of low-density lipoprotein and increase the content of high-density lipoprotein, thereby maintaining the balance of glucose and lipid metabolism and playing a positive role in regulating blood lipid levels, and the effect was not significantly different from that of the positive control.

[0063] In addition, the serum transaminase activity (alanine aminotransferase ALT: 8.233±2.106 U / L, aspartate aminotransferase AST: 24.60±2.337 U / L) of the chronic inflammation model group (HFL) was significantly higher than that of the NC group (ALT: 5.572±0.7804 U / L, AST: 16.70±5.050 U / L), and the liver index (liver / body weight) increased, indicating that chronic inflammation caused liver damage and increased the permeability of liver cell membranes. Oral administration of unfermented grains (Cere) can reduce ALT activity and liver index, but has no effect on AST activity. Oral administration of postbiotics (HFC) of fermented grain broth can significantly inhibit the abnormal changes in serum transaminase levels, reduce the activity of ALT and AST, and significantly reduce the liver index (P<0.05), and the effect is not significantly different from that of the positive control.

[0064] In summary, the occurrence of chronic inflammation in the body is accompanied by liver damage and lipid metabolism disorders. The postbiotic products of grain fermentation broth can effectively improve lipid metabolism disorders and alleviate liver damage, and the effect is significantly better than that of unfermented grains.

[0065] Example 7 After the 16-week intervention in Example 4, the colon tissue of mice was collected, the colon morphology was observed, and the length of the cecum was measured. 2-3 cm of the colon was taken for histological analysis. The tissue was placed in 10% neutral buffered formaldehyde and embedded in paraffin after 24 h. The sections (4 μm) were placed on glass slides and stained with H&E. The histopathological changes of the colon of each group of mice in the H&E sections were observed under an optical microscope.

[0066] Total RNA was extracted from colon tissue using TRIzol reagent. After determining RNA concentration and purity, it was reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit. qRT-PCR was used to detect the mRNA expression levels of genes related to intestinal barrier damage. β-actin was used as an internal reference and 2 -ΔΔCt The relative quantitative analysis was performed by the method, and the primer information of the relevant genes is shown in Table 1.

[0067] Table 1 Primer information of related genes

[0068] Depend on Figure 7 It can be seen that compared with the NC group, the colon tissue section of the HFL group showed obvious inflammatory cell infiltration (outlined by the yellow box in the figure). Supplementation of unfermented cereals (Cere) and postbiotics from cereal fermentation broth (HFC) can significantly improve the inflammatory infiltration of colon tissue, among which the colon tissue morphology of the HFC group is closest to that of the control group (NC).

[0069] The results of mRNA expression level determination of genes related to intestinal barrier damage in colon tissue are as follows Figure 8 As shown, compared with the NC group, the contents of the main proinflammatory factors IL-1β, TNF-α and IL-6 in the colon tissue of mice in the HFL chronic inflammation model group increased by about 5.07 times, 3.92 times and 3.02 times, respectively. Compared with the HFL group, supplementation with unfermented grain Cere reduced the IL-1β level by 2.59 times, and the TNF-α and IL-6 levels increased by about 1.36 and 1.3 times, respectively; supplementation with grain fermentation broth postbiotics HFC reduced the IL-1β, TNF-α and IL-6 levels by 3.58 times, 3.27 times and 1.88 times, respectively. From the above results, it can be seen that supplementation with grain fermentation broth postbiotics can significantly inhibit the expression of colon proinflammatory factors, and there is no significant difference with the positive control FCP. This result is consistent with Figure 6 The results of colon tissue sections were consistent with those of

[0070] In addition, in order to further evaluate the changes in colon barrier function, the expression of intestinal tight junction proteins in colon tissue was measured. Figure 8As shown in the figure, compared with the NC group, the expression levels of the main tight junction proteins ZO-1, Claudin-1 and Occludin in the colon tissue of mice in the HFL chronic inflammation model group were reduced by about 2.02 times, 1.53 times and 1.89 times, respectively. When supplemented with unfermented cereal Cere, the expression of Claudin-1 and ZO-1 did not change significantly, while the expression of ZO-1 and Occludin increased by about 1.51 and 2.41 times, respectively; when supplemented with cereal fermentation liquid postbiotic HFC, the expression of ZO-1, Claudin-1 and Occludin increased by 3.01, 1.55 and 10.50 times, respectively. The above results show that supplementation with cereal fermentation liquid postbiotics can significantly promote the expression of colon tight junction proteins, and the effect is better than that of unfermented cereals and the positive control FCP.

[0071] In summary, the occurrence of chronic inflammation in the body is accompanied by impaired intestinal barrier function. Postbiotic products from grain fermentation broth can effectively improve intestinal barrier function, and the effect is significantly better than that of unfermented grains.

[0072] Example 8 After the 16-week intervention in Example 4, the abdominal white adipose tissue of mice was collected, and the distribution of macrophages was observed by immunohistochemistry. The tissue was placed in 10% neutral buffered formaldehyde, embedded in paraffin after 24 hours, and then sliced. The tissue sections were placed in a microwave oven filled with citric acid antigen retrieval buffer (pH6.0) or EDTA antigen retrieval buffer (pH9.0) for antigen retrieval, and the sections were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark to block endogenous peroxidase. After drying the sections, serum was used for sealing and mouse F4 / 80 primary antibody (purchased from Santa Cruz Biotechnology, item number sc-52664) and HRP-labeled goat anti-mouse secondary antibody (purchased from sino biological, item number SSA006) were added in sequence. The sections were stained with DAB colorimetric solution, and the cell nuclei were counterstained with hematoxylin staining solution. After dehydration and sealing, the sections were observed under a microscope.

[0073] The results are as follows Fig. 9As shown, hematoxylin stains the nucleus in blue, and DAB shows the positive expression of macrophages in brown-yellow. In the NC group, adipose tissue cells were closely arranged in a honeycomb shape, and the immature macrophages (blue particles) in the interstitial space were few in number and unevenly distributed. Compared with the NC group, the number of macrophages in the adipose tissue of mice in the HFL chronic inflammation model group increased, and significant activation and aggregation occurred (indicated by the blue arrow). After gavage with unfermented grains (Cere) and postbiotics from grain fermentation broth (HFC), the area of ​​yellow positive expression (activated macrophages) in the interstitial space of adipose tissue was significantly reduced, among which the effect of the HFC group was the most significant, and the section results were closest to those of the NC and positive control groups (FCP). In summary, grains and postbiotics from grain fermentation broth can improve the activation of adipose tissue macrophages associated with inflammation in mice, and the effect of postbiotics from grain fermentation broth is significantly better than that of unfermented grains.

[0074] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing postbiotics from cereal fermentation broth, characterized in that: The main steps include: The cooked mixed grains are mixed with a complex enzyme and fermentation bacteria and then subjected to anaerobic fermentation to obtain the grain fermentation liquid postbiotics; The complex enzyme comprises saccharifying enzyme, amylase, cellulase and pectinase; The fermentation bacteria include a composite bacteria of yeast and lactic acid bacteria.

2. The preparation method according to claim 1, characterized in that: The types of the mixed grains include black glutinous rice, fragrant rice, rice, millet and corn.

3. The preparation method according to claim 2, characterized in that: In parts by mass, the mixed cereals include the following raw materials in parts by mass: 2-5 parts of black glutinous rice, 3-6 parts of fragrant rice, 4-7 parts of rice, 4-6 parts of millet and 5-8 parts of corn.

4. The preparation method according to claim 1, characterized in that: The mass of the complex enzyme is 0.4% of the mass of the mixed grains; Among them, the mass ratio of saccharifying enzyme, amylase, cellulase and pectinase is (1.5~2.8):(2~3.5):(0.4~0.8):(0.6~1.0).

5. The preparation method according to claim 1, characterized in that: The amount of live bacteria added to the fermentation bacteria is (0.3~3)×10 7 CFU / g mixed grains; The yeast in the fermentation bacteria includes at least one of baker's yeast and sweet wine yeast; The lactic acid bacteria in the fermentation bacteria include at least one of Lactobacillus delbrueckii subspecies and Streptococcus salivarius subspecies.

6. The preparation method according to claim 1, characterized in that: The temperature of the anaerobic fermentation is 30°C and the time is 7 to 8 days.

7. Cereal fermentation liquid postbiotics prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the postbiotics of the cereal fermentation liquid according to claim 7 in preparing food.

9. The use of the cereal fermentation liquid postbiotics in the preparation of medicines according to claim 7, characterized in that: The drug has at least one of the following effects: improving chronic inflammation of the body, reducing serum cholesterol content, reducing low-density lipoprotein content, reducing serum pro-inflammatory cytokine levels, inhibiting activation of adipose tissue macrophages, maintaining blood sugar and insulin levels, and maintaining intestinal barrier function.

10. A drug, characterized in that It comprises active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the postbiotics of the cereal fermentation liquid according to claim 7.

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