Method for preparing postbiotics from cereal fermentation broth and its application in improving systemic chronic inflammation, reducing cholesterol and low-density lipoprotein

Abstract

The present invention provides a method for preparing postbiotics from cereal fermentation broth and its application in improving systemic chronic inflammation, reducing cholesterol and low-density lipoprotein, belonging to the technical field of fermentation engineering. The present invention provides a method for preparing postbiotics from cereal fermentation broth. Using mixed cereals as raw materials, anaerobic fermentation is carried out with composite enzymes and various bacteria. Polysaccharides and proteins in the cereal raw materials can be rapidly converted into functional small-molecule substances, such as polypeptides, total phenols and total flavonoid substances. The postbiotics from cereal fermentation broth of the present invention can inhibit the secretion of pro-inflammatory mediators by macrophages, and at the same time inhibit the generation of inflammatory factors in liver, heart and intestinal tissues, and can also improve the activation of adipose tissue macrophages related to body inflammation. After treatment with the postbiotics from cereal fermentation broth of the present invention, it can effectively improve glucose and lipid metabolism disorders, relieve liver function damage, and effectively improve intestinal barrier function.

Description

Technical Field

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

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

[0003] Currently, many food bioactive compounds have anti-inflammatory activities and are used as potential methods for preventing and improving 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 syndromes. Cereals such as millet, broomcorn millet, glutinous rice, fragrant rice, rice, etc. are rich in dietary fiber and phenolic phytochemicals 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 diets. Cereal fermentation can degrade indigestible polysaccharides and oligosaccharides, dissociate the binding of antinutrients to polysaccharides and proteins, improve the digestibility and absorption of products, and increase the bioavailability of functional components in cereals. Therefore, fermentation products mainly based on cereals have broad application prospects in the consumer market for gastrointestinal diseases. However, the use of fermentation means to increase the bioavailability of functional components in cereals, especially to have a clinical effect on chronic inflammation-related diseases, is not well 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, reducing cholesterol and low-density lipoprotein, which can inhibit the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells, and improve the glycolipid metabolism disorder and impaired intestinal barrier function related to chronic inflammation of the body.

[0005] The present invention provides a method for preparing postbiotics from cereal fermentation broth, which mainly includes the following steps: mixing cooked mixed cereals with a complex enzyme and fermenting bacteria, and then performing anaerobic fermentation to obtain the postbiotics from the cereal fermentation broth;

[0006] The complex enzyme includes glucoamylase, amylase, cellulase and pectinase;

[0007] The fermenting bacteria include a complex bacteria of yeast and lactic acid bacteria.

[0008] In a preferred embodiment of the present invention, the types of the mixed grains include black glutinous rice, fragrant rice, rice, millet and foxtail millet.

[0009] In a preferred embodiment of the present invention, by mass, the mixed grains 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 foxtail millet.

[0010] In a preferred embodiment of the present invention, the mass of the complex enzyme is 0.4% of the mass of the mixed grains;

[0011] Among them, the mass ratio of glucoamylase, amylase, cellulase and pectinase is (1.5 - 2.8):(2 - 3.5):(0.4 - 0.8):(0.6 - 1.0).

[0012] In a preferred embodiment of the present invention, the viable bacteria addition amount of the fermenting bacteria is (0.3 - 3)×10 7 CFU / g of mixed grains;

[0013] The yeast in the fermenting bacteria includes at least one of baker's yeast and sweet wine yeast;

[0014] The lactic acid bacteria in the fermenting bacteria includes at least one of Lactobacillus delbrueckii subsp. and Streptococcus salivarius subsp.

[0015] In a preferred embodiment of the present invention, the temperature of the anaerobic fermentation is 30°C and the time is 7 - 8 d.

[0016] The present invention also provides a postbiotic of the cereal fermentation broth prepared by the above preparation method.

[0017] The present invention also provides the application of the above postbiotic of the cereal fermentation broth in the preparation of foods.

[0018] The present invention also provides the application of the above postbiotic of the cereal fermentation broth in the preparation of drugs, and the drugs have at least one of the following effects: improving chronic inflammation of the body, reducing the serum cholesterol content, reducing the low - density lipoprotein content, reducing the level of serum pro - inflammatory cytokines in the body, inhibiting the activation of adipose tissue macrophages, maintaining blood glucose and insulin levels, and maintaining intestinal barrier function.

[0019] The present invention also provides a drug, which includes an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient includes the above postbiotic of the cereal fermentation broth.

[0020] Beneficial effects: The present invention provides a method for preparing postbiotics from a cereal fermentation broth. Using mixed cereals as raw materials, anaerobic fermentation is carried out with a composite enzyme and various bacteria. Through anaerobic fermentation, polysaccharides and proteins in the cereal raw materials can be rapidly converted into functional small-molecule substances, manifested by a significant increase in the contents of polypeptides, total phenols, and total flavonoids after the anaerobic fermentation.

[0021] The postbiotics from the cereal fermentation broth of the present invention can inhibit the secretion of pro-inflammatory mediators by macrophages, such as reducing the release amounts of pro-inflammatory cytokines TNF-α, NO, and IL-6 in macrophages. At the same time, it inhibits the generation of inflammatory factors in liver, heart, and intestinal tissues, and can also improve the activation of adipose tissue macrophages related to body inflammation. The occurrence of chronic inflammation in the body is accompanied by impaired liver function, disorders of glucose and lipid metabolism, and impaired intestinal barrier function. After treatment with the postbiotics from the cereal fermentation broth of the present invention, it can effectively improve disorders of glucose and lipid metabolism, relieve liver function damage, and effectively improve intestinal barrier function. Brief description of the drawings

[0022] Figure 1 It is a graph showing the determination results of the basic components of the postbiotics from the cereal fermentation broth. In the figure, A: pH values of each fermentation broth; B: polypeptide contents in each fermentation broth; C: total phenol contents in each fermentation broth; D: total flavonoid contents in each fermentation broth;

[0023] Figure 2 It is a statistical graph of the release of inflammatory factors in the macrophage model;

[0024] Figure 3 It is a graph showing the change in body weight of the chronic inflammation mouse model;

[0025] Figure 4 It is a graph showing the change in the secretion amount of inflammatory factors in the chronic inflammation mouse model;

[0026] Figure 5 It is a pathological section diagram of H&E staining of inflammatory organs in the chronic inflammation mouse model. In the figure, A-E are liver tissues, and F-J are heart tissues;

[0027] Figure 6 It is a graph showing the change in related indicators of glucose and lipid metabolism in the chronic inflammation mouse model;

[0028] Figure 7 It is a result diagram of H&E section of intestinal tissues in the chronic inflammation mouse model;

[0029] Figure 8 It is a result diagram of the change in the colon barrier function of the chronic inflammation mouse;

[0030] Figure 9 It is a result diagram of immunohistochemistry of adipose tissue macrophages in the chronic inflammation mouse. Detailed implementation manners

[0031] The present invention provides a method for preparing postbiotics from a cereal fermentation broth, which mainly comprises the following steps: mixing the cooked mixed cereals with a composite enzyme and a fermentation bacterium, and then carrying out anaerobic fermentation to obtain the postbiotics from the cereal fermentation broth;

[0032] The composite enzyme includes glucoamylase, amylase, cellulase and pectinase;

[0033] The fermentation bacterium is selected from a composite bacterium of yeast and lactic acid bacteria.

[0034] The mixed cereals in the present invention are a mixture of cereals. The cereals referred to herein mean the seeds of plants that can obtain starch-containing seeds and are suitable for making food, such as wheat, corn, rice, buckwheat, oats, millet, and tubers. In an embodiment of the present invention, the mixed cereals include black glutinous rice, fragrant rice, rice, millet, and foxtail millet. In terms of mass parts, the black glutinous rice can be 2-5 parts, such as 2 parts, 3 parts, 4 parts, or 5 parts; the fragrant honey can be 3-6 parts, such as 3 parts, 4 parts, 5 parts, or 6 parts; the rice can be 4-7 parts, such as 4 parts, 5 parts, 6 parts, or 7 parts; the millet can be 4-6 parts, such as 4 parts, 5 parts, or 6 parts; the foxtail millet can be 5-8 parts, such as 5 parts, 6 parts, 7 parts, or 8 parts. In the present invention, compared with other types of cereal mixtures, the release of bioactive peptides is optimal after fermenting black glutinous rice, fragrant rice, rice, millet, and foxtail millet.

[0035] In an embodiment of the present invention, after mixing black glutinous rice, fragrant rice, rice, millet, and foxtail millet, they are added to sterilized distilled water and soaked, and then steam sterilized to obtain cooked cereals. In the present invention, the ratio of the volume of sterilized distilled water to the total mass of the cereals for soaking can be 1:2, and the soaking time can be 2 h. Soaking can make the cereals fully absorb water, facilitating subsequent steaming and cooking. Sufficient soaking is beneficial for the cereals to be steamed thoroughly within a specified time (1 h) to reach the required ripeness and softness for fermentation. The present invention does not have any special limitation on the method of steam sterilization, and a conventional steam sterilization scheme in the art can be adopted. For example, in the embodiment, steam sterilization is carried out for 1 h with conventional parameters.

[0036] The present invention places the matured mixed grains in a fermentation basin, and adds a compound enzyme and fermentation bacteria. In one embodiment, the compound enzyme can be added first and then the fermentation bacteria. The compound enzyme includes glucoamylase, amylase, cellulase, and pectinase, and the mass of the compound enzyme is 0.4% of the mass of the mixed grains. Among them, the mass ratio of glucoamylase, amylase, cellulase, and pectinase is (1.5 - 2.8):(2 - 3.5):(0.4 - 0.8):(0.6 - 1.0). The addition of the compound enzyme of the present invention can effectively degrade macromolecular starch and protein in the grains, thereby facilitating microbial fermentation and utilization. The compound enzyme of the present invention can fully play its role during grain fermentation and will not be utilized by microorganisms as a nitrogen source: when the grains are mixed and fermented, the compound enzyme is added first for enzymatic hydrolysis, and after 1 h, the mixed bacteria are then inoculated for fermentation. The time difference between the addition of the compound enzyme and the mixed bacteria to the mixed grain system can ensure that the compound enzyme fully plays its 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 addition amount of the compound enzyme is relatively small and will not be metabolized and utilized by the strains as a nitrogen source.

[0037] The present invention adds fermentation bacteria to the fermentation basin, and the viable bacteria addition amount of the fermentation bacteria is (0.3 - 3)×10 7 CFU / g of mixed grains. The fermentation bacteria of the present invention are selected from yeast and lactic acid bacteria. Among them, the yeast can be at least one of baker's yeast and sweet wine yeast. In one embodiment, the effective viable 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 subsp. and Streptococcus salivarius subsp., and the effective viable bacteria number ratio of the Lactobacillus delbrueckii subsp. and Streptococcus salivarius subsp. is 2:1 - 10:1. For example, in the embodiment, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus are selected. There is no limitation on each strain used in the fermentation process of the present invention, and the mixed strains are all commercial products, and the strains are inoculated into the mixed grain fermentation system in the form of bacterial powder.

[0038] The present invention also provides a postbiotic of a grain fermentation broth prepared by using the above preparation method.

[0039] In the embodiment of the present invention, the anaerobic fermentation is carried out in a fermentation basin, where the temperature of the anaerobic fermentation is 30°C and the time is 7 - 8 d. After the mixed grains are fermented according to the present invention, the fermentation supernatant is extracted to obtain the postbiotic of the grain fermentation broth. The postbiotic of the grain fermentation broth can significantly reduce the level of serum pro-inflammatory cytokines in the body, inhibit the activation of adipose tissue macrophages, maintain blood glucose and insulin levels, and maintain intestinal barrier function through diet therapy, and can be applied to inhibiting the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells, improving the disorders of glucose and lipid metabolism related to chronic inflammation in the body and the impairment of intestinal barrier function, etc.

[0040] The present invention also provides the application of the postbiotics in the above-mentioned cereal fermentation broth in the preparation of food.

[0041] The fermentation broth obtained in the present invention can be eaten after being sterilized by boiling or other means.

[0042] The present invention also provides the application of the postbiotics in the above-mentioned cereal fermentation broth in the preparation of drugs, and the drugs have at least one of the following effects: improving chronic inflammation of the body, reducing the content of serum cholesterol, reducing the content of low-density lipoprotein, reducing the level of serum pro-inflammatory cytokines in the body, inhibiting the activation of adipose tissue macrophages, maintaining blood glucose and insulin levels, and maintaining intestinal barrier function.

[0043] In the present invention, yeast and lactic acid bacteria jointly 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 the macrophage inflammatory response. Feeding the chronic inflammation model with the postbiotics in the cereal fermentation broth of the present invention shows that the fermentation broth has a significant improvement effect on the weight loss caused by body inflammation, 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.

[0044] The present invention also provides a drug, which includes an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient includes the postbiotics in the above-mentioned cereal fermentation broth.

[0045] The present invention does not particularly limit the dosage form of the drug, and it can be prepared by using conventional dosage forms and excipients in the art.

[0046] In order to further illustrate the present invention, the following examples are used to describe in detail the preparation method of the postbiotics in the cereal fermentation broth provided by the present invention and its application in improving systemic chronic inflammation, reducing cholesterol and low-density lipoprotein, but they should not be construed as limiting the protection scope of the present invention.

[0047] In the examples of the present invention, unless otherwise specified, the materials used are all conventional commercially available products in the art.

[0048] Example 1

[0049] After mixing 300 g of black glutinous rice, 300 g of fragrant rice, 300 g of rice, 300 g of broomcorn millet and 300 g of foxtail millet, add 600 mL of sterilized distilled water and soak for 2 h, then steam sterilize for 1 h to obtain cooked grains; transfer the above cooked grains to a fermentation basin, add 6 g of a complex enzyme, including 1.846 g of glucoamylase (product number: 60883074703, product name: Jianshi food-grade glucoamylase), 2.769 g of amylase (product number: 60883059985, product name: a-amylase medium-temperature enzyme food-grade), 0.462 g of cellulase (product number: 60883065873, product name: Jianshi food-grade cellulase), and 0.923 g of pectinase (product number: 60883082113608830882113, pectinase food-grade edible enzyme). The product numbers of the above enzymes are all product numbers on JD.com, and the enzymes are sourced from Henan Wanbang Industrial Co., Ltd. After enzymolysis of the above cooked grains and complex enzyme mixture for 1 h, it is labeled as Cere;

[0050] Inoculate the above Cere with (0.3 - 3)×10 7 CFU / g of grain raw material fermentation bacteria. The fermentation bacteria include baker's yeast, sweet wine yeast and mixed lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, with an effective viable count ratio of 2:1 - 10:1). Among them, baker's yeast, sweet wine yeast and mixed lactic acid bacteria are purchased from Angel Yeast Co., Ltd., with an effective viable count ratio of 2:1:1. Ferment at 30 °C for 7 - 8 days in a closed environment, filter with gauze to obtain the juice, and obtain the postbiotic of the grain fermentation broth, denoted as HFC.

[0051] Inoculate the above Cere with (0.3 - 3)×10 6 CFU / g of grain raw material fermentation bacteria. The fermentation bacteria include baker's yeast, sweet wine yeast and mixed lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, with an effective viable count ratio of 2:1 - 10:1). Among them, baker's yeast, sweet wine yeast and mixed lactic acid bacteria are purchased from Angel Yeast Co., Ltd., with an effective viable count ratio of 2:1:1. Ferment at 30 °C for 7 - 8 days in a closed environment, filter with gauze to obtain the juice, and obtain the postbiotic of the grain fermentation broth, denoted as HFC1.

[0052] Inoculate the above Cere with (0.3 - 3)×10 5Fermentation bacteria per gram of cereal raw material: The fermentation bacteria include baker's yeast, sweet wine yeast, and mixed lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, with the ratio of viable bacteria being 2:1 - 10:1). Among them, baker's yeast, sweet wine yeast, and mixed lactic acid bacteria are purchased from Angel Yeast Co., Ltd., and the ratio of viable bacteria is 2:1:1. In a sealed environment, ferment at 30 °C for 7 - 8 days, filter with gauze to obtain the fermented juice, and obtain the postbiotic of the cereal fermentation broth, denoted as HFC2.

[0053] Inoculate (0.3 - 3) × 10 8 Fermentation bacteria per gram of cereal raw material: The fermentation bacteria include baker's yeast, sweet wine yeast, and mixed lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, with the ratio of viable bacteria being 2:1 - 10:1). Among them, baker's yeast, sweet wine yeast, and mixed lactic acid bacteria are purchased from Angel Yeast Co., Ltd., and the ratio of viable bacteria is 2:1:1. In a sealed environment, ferment at 30 °C for 7 - 8 days, filter with gauze to obtain the fermented juice, and obtain the postbiotic of the cereal fermentation broth, denoted as HFC3.

[0054] Inoculate (0.3 - 3) × 10 7 Baker's yeast and sweet wine yeast per gram of cereal raw material, with the ratio of viable bacteria being 2:1. Among them, baker's yeast and sweet wine yeast are purchased from Angel Yeast Co., Ltd. In a sealed environment, ferment at 30 °C for 7 - 8 days, filter with gauze to obtain the fermented juice, and obtain the postbiotic of the cereal fermentation broth, denoted as SFC.

[0055] Inoculate (0.3 - 3) × 10 7 Lactic acid bacteria per gram of cereal raw material (where the lactic acid bacteria are purchased from Angel Yeast Co., Ltd.). In a sealed environment, ferment at 30 °C for 7 - 8 days, filter with gauze to obtain the fermented juice, and obtain the postbiotic of the cereal fermentation broth, denoted as LFC.

[0056] Example 2 Determination of component content in the postbiotic of cereal fermentation broth

[0057] Use an automatic pH meter to measure the change in sample acidity in Cere and the fermentation broths HFC, HFC1, HFC2, HFC3, SFC, and LFC in Example 1. The measurement method is as follows:

[0058] 2.1 Take 1 mL of the test solution and mix it with 5 mL of distilled water, and then measure the pH value.

[0059] The results are as Figure 1As shown in A, after inoculating the fermenting bacteria, the pH values of all groups decreased with the progress of fermentation. The pH values of the 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 value changes of the HFC2 and SFC groups were the least significant, and decreased to 4.645 ± 0.02 and 5.195 ± 0.12 respectively after 24 h of fermentation; the pH value changes of the HFC and HFC1 groups were moderate, and decreased to 4.26 ± 0.08 and 4.34 ± 0.03 respectively.

[0060] 2.2 Using the biuret method to determine the changes in the polypeptide content of samples in Cere and the fermentation broths HFC, HFC1, HFC2, HFC3, SFC, and LFC in Example 1. The determination method is as follows:

[0061] Mix the test solution with 10% trichloroacetic acid in equal volume, let it stand for 10 min, centrifuge at 10000 g for 10 min, take the supernatant and mix it with the biuret reagent in a volume ratio of 1:4, react at room temperature for 30 min, and then measure its OD value at 310 nm. Using glutathione tripeptide as the standard product, calculate the polypeptide content.

[0062] The results are as Figure 1 shown in B. After inoculating the fermenting bacteria, the polypeptide release of all groups increased. Among them, the polypeptide contents of the 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.

[0063] 2.3 Determine the polyphenol and flavonoid contents in Cere and the fermentation broths HFC, HFC1, HFC2, HFC3, SFC, and LFC prepared in Example 1.

[0064] 2.3.1 Determination of the total phenol content by the Folin-Ciocalteu 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 and then add 2 mL of saturated sodium carbonate, add distilled water to make up the volume to 10 mL, and measure the absorbance at 760 nm. The total polyphenols are expressed as the gallic acid equivalent per mL of the test solution.

[0065] 2.3.2 Determination of the total flavonoid content by the aluminum trichloride 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 it stand for 6 min, add 2 mL of 1 M NaOH, and make up the volume to 10 mL with anhydrous ethanol, measure the absorbance at 510 nm. The total flavonoids are expressed as the rutin equivalent per mL of the fermentation broth.

[0066] The determination results of the polyphenol and flavonoid contents are as Figure 1As shown in C and D, the release of total phenols and total flavonoids in all groups increased after inoculation with fermenting bacteria. The increase was most significant in the HFC and HFC1 groups. The content of 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 content of 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.

[0067] Therefore, too little inoculum (HFC2) was not sufficient to complete the conversion of polysaccharides and proteins in cereal raw materials into functional small molecule substances within 24 h, resulting in a higher acidity value and a lower polypeptide content; while too much inoculum (HFC3) accelerated the fermentation process, producing more organic acids, which inhibited the polysaccharide and protein metabolic activities of lactic acid bacteria, resulting in a lower acidity value and a lower polypeptide content. Therefore, (0.3 - 3)×10 7 CFU / g of cereal raw material is the optimal inoculum.

[0068] In addition, compared with the single fermentation of lactic acid bacteria (LFC) or the single fermentation of yeast (SFC), at the same inoculum level, the mixed fermentation of yeast and lactic acid bacteria (HFC, HFC1) had a metabolic synergistic effect, manifested as a higher accumulation of polypeptides, total phenols and total flavonoids in the mixed bacteria fermentation products.

[0069] Example 3: Postbiotics in cereal fermentation broth inhibit the secretion of pro-inflammatory mediators by macrophages

[0070] An inflammatory macrophage model was established using the RAW264.7 cell line. The cells were inoculated into a 24-well cell culture plate at 3×10 5 cells / well and cultured for 24 h. Subsequently, 5-fold diluted Cere, HFC, LFC and SFC were added to treat the cells for 2 h. Finally, 100 ng / mL of LPS was used to treat the cells for 24 h to stimulate the cell inflammatory response. The cell culture supernatant was collected, and a kit was used to measure the secretion of macrophage pro-inflammatory mediators (NO, TNF-α, IL-6). Among them, the NO kit was purchased from Nanjing Jiancheng Bioengineering Institute, product number A013-2-1; the TNF-α and IL-6 kits were purchased from Jiangsu Enzyme-linked Biotechnology Co., Ltd., product numbers MB6426A and MB50054A respectively.

[0071] The results of the inflammatory macrophage model are as Figure 2As shown, after LPS treatment, the release of pro-inflammatory cytokines such as TNF-α, NO, and IL-6 in macrophages increased significantly, which were approximately 2-fold, 7.4-fold, and 2.3-fold higher than those in the control group (Control), respectively. Cere, HFC, SFC, and LFC could all inhibit the inflammatory response of macrophages induced by LPS to varying degrees. Among them, the effect of HFC intervention was the most significant, reducing the secretion of TNF-α by approximately 26%, NO by approximately 29%, and IL-6 by approximately 25%; followed by the LFC group, reducing the secretion of TNF-α by approximately 10%, NO by approximately 18%, and IL-6 by approximately 18%; then the SFC group, reducing the secretion of TNF-α by approximately 10% and NO by approximately 15%; Cere intervention only reduced the secretion of TNF-α by approximately 6%. It can be seen that compared with unfermented cereals (Cere), fermented cereal by microorganisms can significantly alleviate the inflammatory response of macrophages, and the group with mixed fermentation of yeast and lactic acid bacteria (HFC) has the most significant effect.

[0072] Example 4 Animal Experiment

[0073] 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, and the controlled conditions were a temperature of 22 ± 2 °C, a humidity of 65% ± 5%, and a light / dark cycle of 12 h. After one week of adaptive feeding, all mice were randomly divided into 5 groups: the postbiotic intervention group of fermented cereal liquid (HFC), the unfermented cereal intervention group (Cere), the chronic inflammation model group (HFL), the positive control group of fermented chestnut protein (FCP), and the blank control group (NC); from 0 to 4 weeks, all groups were provided with normal diet. From 4 to 16 weeks, except for the blank control group, the intervention groups and the model group were provided with LPS drinking water at 400 μg / Kg and a high-fat diet, and the body weight changes were recorded. At the same time, the intervention groups were continuously gavaged with the postbiotic of fermented cereal liquid (HFC), unfermented cereal (Cere) (0.3 mL / only / day), and the positive control group of fermented chestnut protein (FCP), and the model group (HFL) was gavaged with an equal amount of sterile water; at the 1st 16th week, the mice were sacrificed by cervical dislocation, and serum, adipose tissue, and intestinal tissue were collected for further analysis.

[0074] The changes in the body weight of the mice were as Figure 3 shown. The body weight of the mice in the chronic inflammation model group (HFL) was significantly lower than that of the mice in the blank control group (NC). The groups gavaged with unfermented cereals and the postbiotic of fermented cereal liquid in the Cere and HFC groups effectively improved the weight loss of the mice, and during the intervention period from 4 to 11 days, the effect of the HFC group was significantly better than that of the Cere and positive control (FCP) groups. It shows that unfermented cereals and the postbiotic of fermented cereal liquid have an improving effect on the weight loss caused by body inflammation, and the postbiotic of fermented cereal liquid has a better effect.

[0075] Example 5

[0076] 4.1 After the intervention at the 16th week in Example 4 ended, sera were collected, and the levels of inflammatory factors and endotoxin LPS in the sera of mice were measured using an Elisa kit. The TNF-α, IL-1β, IL-6, and LPS kits were all purchased from Jiangsu Enzyme-Linked Immunosorbent Assay Biotechnology Co., Ltd., with product numbers MB-2868A, MB-2776A, MB-2899A, and MB-3418A respectively.

[0077] The measurement results are as Figure 4 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 the mice in the HFL group were significantly higher than those of the mice 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 endotoxin LPS level in the sera of the 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). However, intragastric administration of unfermented grains (Cere) and the complex fermented broth (HFC) could both inhibit the production of plasma inflammatory factors and endotoxin. Among them, the HFC group could most significantly reduce the plasma IL-1β, IL-6, and endotoxin LPS levels (P < 0.05), and there was no significant difference from the positive control group.

[0078] 4.2 The Elisa kit was used to measure the secretion amounts 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 an enzyme immunoassay reagent company, with product numbers MM-0132M1 and MM-0040M1 respectively.

[0079] The results are as Figure 4As shown, the TNF-α secretion levels in the hearts and livers of the mice in the HFL group were 236.8 ± 6.554 pg / mL and 250 ± 13.79 pg / mL, respectively, and the IL-1β secretion levels were 34.04 ± 1.629 pg / mL and 51.26 ± 3.715 pg / mL, respectively; the IL-6 secretion level 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 of the mice in the NC group (heart TNF-α: 206.8 ± 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). However, intragastric administration of postbiotics from fermented cereal broth (HFC) could significantly inhibit the generation of inflammatory factors in the liver, heart, and intestinal tissues, and the effect was significantly better than that of unfermented cereals (P < 0.05).

[0080] 4.3 Take the hearts and livers for histological analysis. Place the tissues in 10% neutral buffered formaldehyde and embed them in paraffin after 24 h. Cut sections (4 μm) and place them on glass slides, and stain them with H&E. Observe the histopathological changes of the hearts and livers of the mice in each group in the H&E sections under an optical microscope.

[0081] The results of the H&E-stained pathological sections are as Figure 5 shown. Compared with the control group (NC), in the liver tissue of the chronic inflammation mouse model group (HFL), the vacuoles around the blue cell nuclei enlarged, the nuclear-cytoplasmic separation occurred, which was consistent with the pathological conditions of fatty liver. In addition, there was inflammatory infiltration. After intragastric administration of unfermented cereals (Cere) and postbiotics from fermented cereal broth (HFC), the inflammatory infiltrating cells decreased (framed by the red box), and the vacuole area around the cell nuclei also decreased. Among them, the effect of the HFC group was the most significant, and the section results were the closest to those of the NC and positive control groups (FCP). It can be seen from the heart tissue sections that the cytoplasmic separation was obvious in the model group, there were fat vacuoles, congestion (indicated by the blue arrow), and inflammatory infiltration (framed by the blue box). After intragastric administration of unfermented cereals (Cere) and postbiotics from fermented cereal broth (HFC), the fat vacuoles decreased, the interstitial congestion decreased, and the inflammatory infiltrating cells decreased. Among them, the effect of the HFC group was the most significant, and the section results were the closest to those of the NC and positive control groups (FCP).

[0082] In summary, it can be seen that cereals and postbiotics from fermented cereal broth can improve the body's inflammatory conditions in mice, reduce the levels of serum pro-inflammatory factors and endotoxins, relieve the inflammatory levels of the main inflammatory organs, and the effect of postbiotics from fermented cereal broth is significantly better than that of unfermented cereals.

[0083] Example 6

[0084] Animal experiments were conducted with reference to the relevant operations in Example 4. A glucose tolerance test (GTT) was performed at the 15th week to determine the occurrence of insulin resistance. Before the test, the mice were fasted for 12 - 14 hours (food was removed, but water was retained). After fasting, the tip of the mouse's tail was cut off. Immediately, a drop of blood was squeezed out from the incision and placed on a blood glucose meter to measure the initial glucose level. Then, each mouse was immediately given 200 μL of 2 g / kg BW oral glucose solution by gavage. Then, the mouse glucose levels were measured and recorded at 15, 30, 60, and 120 minutes using the same protocol as above; the levels of total cholesterol, triglycerides, low-density / high-density lipoproteins, and transaminases (alanine and aspartate transaminases) in the mouse serum were determined using Nanjing Jiancheng kits. Among them, the kit numbers for lipid indices TC, TG, LDL-C, and HDL-C were A111-1-1, A110-1-1, A113-1-1, and A112-1-1 respectively; the kit numbers for transaminases ALT and AST were C009-2-1 and C010-2-1 respectively.

[0085] The results of glucose tolerance measurement are as Figure 6 shown. 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 AUC of both Cere and HFC glucose supplementation decreased, and the effect of HFC was significantly better than that of Cere and the positive control (P < 0.05). Postbiotics from grains and fermented grain broths can play a positive regulatory role in glucose metabolism in mice with chronic inflammation, and the effect of postbiotics from fermented grain broths is significantly better than that of unfermented grains.

[0086] The contents of total cholesterol, triglycerides, and low-density lipoproteins 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 high-density lipoprotein level in the model group (4.45 ± 0.328 mmol / L) was lower than that in the NC group (4.93 ± 0.568 mmol / L). Gavage with unfermented grains (Cere) could not significantly improve the lipid metabolism imbalance induced by chronic inflammation, while postbiotics from fermented grain broths (HFC) could significantly inhibit the abnormal increase in the contents of serum total cholesterol and total triglycerides, and significantly reduce the content of low-density lipoproteins and increase the content of high-density lipoproteins, thereby maintaining glucose and lipid metabolism balance and playing a positive role in regulating blood lipid levels, and there was no significant difference in the effect compared with the positive control.

[0087] In addition, the serum transaminase activities in the mice of the chronic inflammation model group (HFL) (alanine aminotransferase ALT: 8.233 ± 2.106 U / L, aspartate aminotransferase AST: 24.60 ± 2.337 U / L) were significantly higher than those in the NC group (ALT: 5.572 ± 0.7804 U / L, AST: 16.70 ± 5.050 U / L). At the same time, the liver index (liver / body weight) increased, indicating that chronic inflammation caused liver injury and increased the permeability of the liver cell membrane. Gavage with unfermented grains (Cere) could reduce the ALT activity and the liver index, but had no effect on the AST activity. Gavage with postbiotics from fermented grain liquid (HFC) could significantly inhibit the abnormal changes in serum transaminase levels, reduce the activities of ALT and AST, and significantly reduce the liver index (P<0.05), and the effect was not significantly different from that of the positive control.

[0088] In summary, the occurrence of chronic inflammation in the body is accompanied by impaired liver function and disorders of glucose and lipid metabolism. The postbiotic products of fermented grain liquid can effectively improve glucose and lipid metabolism disorders, relieve liver function injury, and the effect is significantly better than that of unfermented grains.

[0089] Example 7

[0090] After the 16-week intervention in Example 4, the mouse colon tissues were collected, the colon morphology was observed, and the cecum colon length was measured. Take 2 - 3 cm of the colon for histological analysis. The tissues were placed in 10% neutral buffered formalin and embedded in paraffin after 24 h. Sections (4 μm) were placed on slides and stained with H&E. The histopathological changes of the colon of mice in each group were observed under an optical microscope in the H&E sections.

[0091] Total RNA was extracted from the colon tissues using TRIzol reagent. After measuring the RNA concentration and purity, it was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit. The mRNA expression levels of genes related to intestinal barrier injury were detected by qRT-PCR, with β-actin as the internal reference, and relative quantitative analysis was performed using the 2 -ΔΔCt -ΔΔCt method. The primer information of the related genes is shown in Table 1.

[0092] Table 1 Primer information of related genes

[0093]

[0094] From Figure 7It can be seen that compared with the NC group, obvious inflammatory cell infiltration occurred in the cross-section of the colon tissue in the HFL group (framed by the yellow box in the figure). Supplementing unfermented grains (Cere) and postbiotics from fermented grain liquid (HFC) can significantly improve the inflammatory infiltration of the colon tissue, and the colon tissue morphology in the HFC group is closest to that of the control group (NC).

[0095] The results of measuring the mRNA expression levels of genes related to intestinal barrier damage in the colon tissue are as Figure 8 shown. Compared with the NC group, the contents of the main pro-inflammatory 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-fold, 3.92-fold, and 3.02-fold, respectively. Compared with the HFL group, after supplementing unfermented grains Cere, the level of IL-1β decreased by 2.59-fold, and the levels of TNF-α and IL-6 increased by about 1.36 and 1.3-fold, respectively; after supplementing postbiotics HFC from fermented grain liquid, the levels of IL-1β, TNF-α, and IL-6 decreased by 3.58-fold, 3.27-fold, and 1.88-fold, respectively. From the above results, it can be known that supplementing postbiotics from fermented grain liquid can significantly inhibit the expression of colon pro-inflammatory factors, and there is no significant difference from the positive control FCP. This result is consistent with the Figure 6 results of the colon tissue section

[0096] In addition, in order to further evaluate the changes in colon barrier function, the expression of intestinal tight junction proteins in the colon tissue was measured. As Figure 8 shown, 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 decreased by about 2.02-fold, 1.53-fold, and 1.89-fold, respectively. Compared with the HFL group,

[0097] after supplementing unfermented grains Cere, the expression levels of Claudin-1 and ZO-1 did not change significantly, and the expression levels of ZO-1 and Occludin increased by about 1.51 and 2.41-fold, respectively; after supplementing postbiotics HFC from fermented grain liquid, the expression levels of ZO-1, Claudin-1, and Occludin increased by 3.01-fold, 1.55-fold, and 10.50-fold, respectively. From the above results, it can be known that supplementing postbiotics from fermented grain liquid can significantly promote the expression of colon tight junction proteins, and the effect is better than that of unfermented grains and the positive control FCP.

[0098] In summary, the occurrence of chronic inflammation in the body is accompanied by impaired intestinal barrier function. Postbiotics from fermented grain liquid can effectively improve intestinal barrier function, and the effect is significantly better than that of unfermented grains.

[0099] Example 8

[0100] 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 h, and then sectioned. The tissue sections were placed in a microwave oven filled with citrate antigen retrieval buffer (pH 6.0) or EDTA antigen retrieval buffer (pH 9.0) for antigen retrieval. The sections were placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark to block endogenous peroxidase. After draining the sections, the slides were sealed with serum, and mouse-derived F4 / 80 primary antibody (purchased from Santa Cruz Biotechnology, catalog number sc-52664) and HRP-labeled goat anti-mouse secondary antibody (purchased from sino biological, catalog number SSA006) were added in sequence. The sections were stained with DAB chromogenic solution, and the cell nuclei were counterstained with hematoxylin solution. After dehydration and sealing, the sections were observed under a microscope.

[0101] The results are as Figure 9 shown. The cell nuclei stained with hematoxylin are blue, and the positive expression of macrophages shown by DAB is brownish yellow. In the NC group, the adipose tissue cells were closely arranged in a honeycomb shape, and the immature macrophages (blue granules) in the tissue 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 blue arrows). After gavage with unfermented cereals (Cere) and postbiotics from fermented cereal broth (HFC), the area of yellow positive expression (activated macrophages) in the adipose tissue space was significantly reduced, and the effect was most significant in the HFC group, and the section results were closest to those of the NC and positive control groups (FCP). In summary, cereals and postbiotics from fermented cereal broth can improve the activation of adipose tissue macrophages related to body inflammation in mice, and the effect of postbiotics from fermented cereal broth is significantly better than that of unfermented cereals.

[0102] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of postbiotics from cereal fermentation broth in the preparation of a drug for relieving liver function injury caused by chronic inflammation, characterized in that, The preparation method of the postbiotic of the cereal fermentation broth comprises the following steps: anaerobically fermenting the ripened mixed cereals after mixing them with a complex enzyme and a fermenting bacterium to obtain the postbiotic of the cereal fermentation broth; By mass, the mixed cereals are composed of 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 broomcorn millet, and 5-8 parts of foxtail millet; The complex enzyme is composed of glucoamylase, amylase, cellulase, and pectinase; the mass of the complex enzyme is 0.4% of the mass of the mixed cereals; the mass ratio of glucoamylase, amylase, cellulase, and pectinase is (1.5-2.8):(2-3.5):(0.4-0.8):(0.6-1.0); The fermenting bacteria are a composite of yeast and lactic acid bacteria; the viable bacteria addition amount of the fermenting bacteria is (0.3 - 3) × 10 7 CFU / g of mixed grains; The temperature of the anaerobic fermentation is 30 °C, and the time is 7-8 d.

2. The application according to claim 1, wherein At least one of baker's yeast and sweet wine koji yeast is included in the yeast in the fermenting bacterium; at least one of Lactobacillus delbrueckii subsp. and Streptococcus salivarius subsp. is included in the lactic acid bacteria in the fermenting bacterium.

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