7S protein fermentation liquor and application thereof in regulating intestinal flora

Abstract

The invention belongs to the technical field of food processing, discloses 7S protein fermentation liquor and application of the 7S protein fermentation liquor to regulation of intestinal flora, and particularly discloses the 7S protein fermentation liquor which is prepared from the following raw materials through fermentation: 7S protein, sugar, cow milk, probiotics, a sweetening agent and a thickening agent. Wherein the probiotics comprise at least one of lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus casei, lactobacillus salivarius, lactobacillus paracasei and lactobacillus gasseri. The invention provides 7S protein fermentation liquor obtained by fermenting 7S protein through probiotics. The 7S protein fermentation liquor can regulate the composition of intestinal flora, improve the relative abundance of beneficial bacteria in intestinal microorganisms, improve the intestinal microenvironment and relieve intestinal inflammation. Meanwhile, the 7S protein fermentation liquor has a relatively high inhibition effect on escherichia coli (the inhibition rate is as high as 95.9%).

Description

Technical Field

[0001] The present invention belongs to the field of food processing technology, and specifically relates to a 7S protein fermentation broth and its application in regulating intestinal flora. Background Art

[0002] The intestinal microbiota refers to the community of microorganisms present in the human intestine, including bacteria, archaea, viruses, fungi, and parasites. These microorganisms form a complex ecosystem in the intestine that is closely related to the health and disease of the host. The composition of the intestinal microbiota can be changed by many factors such as aging, physiological state, medication, various diseases, diet, and stress. An imbalance in the intestinal microbiota, or "microbiome dysbiosis," can lead to the occurrence and development of many diseases, such as obesity, diabetes, inflammatory bowel disease, and colorectal cancer. Therefore, maintaining a balance of the intestinal microbiota is crucial to maintaining the health of the host.

[0003] The bioactive peptides from soy hydrolysates have a strong regulatory effect on intestinal flora. The 7S protein in soy protein can reshape the composition of the intestinal microbiota by selectively inhibiting Gram-negative bacteria involved in lipopolysaccharide (LPS)-peptide interactions. This process is accompanied by a reduction in the LPS load in the intestine. However, soy protein is a large molecular protein with limited absorption and digestion capacity in the human body. Large amounts of it are required to achieve a good effect on regulating the flora. Furthermore, the finished product of 7S protein has a poor taste, which limits its product development. There is an urgent need to develop a 7S protein product that is easily absorbed and better meets the public's demand for healthy food. Summary of the Invention

[0004] The present invention aims to develop a 7S protein fermentation liquid that has the effect of regulating intestinal flora and can help people with intestinal inflammation or intestinal flora disorder to relieve symptoms by taking a small amount.

[0005] The first aspect of the present invention aims to provide a 7S protein fermentation broth.

[0006] The purpose of the second aspect of the present invention is to provide a method for preparing the 7S protein fermentation broth of the first aspect of the present invention.

[0007] The third aspect of the present invention aims to provide a use of the 7S protein fermentation broth of the first aspect of the present invention in the preparation of a product that helps regulate intestinal flora.

[0008] The fourth aspect of the present invention aims to provide a product.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a 7S protein fermentation broth, which is fermented from the following raw materials: 7S protein, sugar, cow's milk, probiotics, a sweetener and a thickener; wherein the probiotics include at least one of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus paracasei and Lactobacillus gasseri.

[0011] In some embodiments of the present invention, the sugar includes at least one of glucose, white sugar, and maltose; preferably, it is a combination of glucose and white sugar.

[0012] In some embodiments of the present invention, the sweetener includes at least one of erythritol, xylitol, maltitol, sorbitol, steviol glycosides, sucralose, aspartame, acesulfame potassium and neotame; preferably erythritol.

[0013] In some embodiments of the present invention, the thickener comprises soybean polysaccharide, guar gum and curdlan gum.

[0014] In some embodiments of the present invention, the thickener comprises, by weight, 4 to 6 parts of soybean polysaccharide, 4 to 8 parts of guar gum, and 4 to 8 parts of curdlan gum.

[0015] In some embodiments of the present invention, the probiotics include at least three of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus gasseri.

[0016] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus casei and Lactobacillus gasseri, and the mass ratio of the probiotics is 1:(1-3):(1-3).

[0017] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus gasseri, and the mass ratio of the probiotics is 1:(1-3):(1-3).

[0018] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus gasseri, and the mass ratio of the probiotics is 1:(1-3):(1-3).

[0019] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus gasseri, and the mass ratio of the probiotics is 1:(1-3):(1-3):(1-3).

[0020] In some embodiments of the present invention, the Lactobacillus rhamnosus includes Lactobacillus rhamnosus NX-2 with a preservation number of CGMCC: 20110 or Lactobacillus rhamnosus E2 with a preservation number of CGMCC: 21770; preferably, Lactobacillus rhamnosus NX-2.

[0021] In some embodiments of the present invention, the Lactobacillus plantarum is Lactobacillus plantarum NX-1 with a deposit number of CGMCC:20109.

[0022] In some embodiments of the present invention, the Lactobacillus casei is Lactobacillus casei LS01 with a deposit number of CGMCC:22006.

[0023] In some embodiments of the present invention, the Lactobacillus salivarius includes Lactobacillus salivarius LF01 with a deposit number of CGMCC:23313.

[0024] In some embodiments of the present invention, the Lactobacillus paracasei is Lactobacillus paracasei Lp.R3 with a deposit number of CGMCC:22008.

[0025] In some embodiments of the present invention, the Lactobacillus gasseri is Lactobacillus gasseri LS03 with a deposit number of CGMCC:22009.

[0026] In some embodiments of the present invention, the 7S protein fermentation broth is fermented from the following raw materials, calculated by weight: 20 to 70 parts of 7S protein, 10 to 70 parts of sugar, 100 to 500 parts of milk, 10 to 70 parts of probiotics, 20 to 60 parts of sweetener and 2 to 8 parts of thickener.

[0027] In some embodiments of the present invention, the 7S protein fermentation broth is fermented from the following raw materials, calculated by weight: 20 to 60 parts of 7S protein, 20 to 70 parts of sugar, 150 to 500 parts of milk, 20 to 60 parts of probiotics, 20 to 50 parts of sweetener and 2 to 6 parts of thickener.

[0028] In some embodiments of the present invention, the 7S protein fermentation broth is fermented from the following raw materials, calculated by weight: 20 to 60 parts of 7S protein, 20 to 60 parts of sugar, 150 to 450 parts of milk, 20 to 50 parts of probiotics, 20 to 40 parts of sweetener and 3 to 6 parts of thickener.

[0029] In some embodiments of the present invention, the 7S protein fermentation broth is fermented from the following raw materials, measured by mass: 20 to 60 parts of 7S protein, 20 to 40 parts of glucose, 10 to 15 parts of white sugar, 150 to 450 parts of milk, 20 to 50 parts of probiotics, 20 to 40 parts of sweetener and 3 to 6 parts of thickener.

[0030] In some embodiments of the present invention, the raw material further comprises water.

[0031] In some embodiments of the present invention, the raw materials further include 400 to 700 parts of water.

[0032] The second aspect of the present invention provides a method for preparing the 7S protein fermentation broth of the first aspect of the present invention, comprising the following steps:

[0033] Mixing the 7S protein with water and dissolving it to obtain a 7S protein solution;

[0034] Mixing sugar and milk and dissolving them to obtain a sugar-milk mixture;

[0035] The 7S protein solution and the sugar-milk mixture are mixed, cooled, inoculated with probiotics, and fermented to obtain a fermentation solution;

[0036] The fermentation solution is mixed with a sweetener and a thickener to obtain 7S protein fermentation liquid.

[0037] In some embodiments of the present invention, the fermentation temperature is 30-50° C., and the fermentation time is 5-30 h; preferably, the fermentation temperature is 32-40° C., and the fermentation time is 8-24 h.

[0038] In some embodiments of the present invention, the cooling is to 30-50°C; preferably, the cooling is to 32-40°C.

[0039] In some embodiments of the present invention, during the preparation of the 7S protein solution, the dissolution temperature is 100-110° C., and the dissolution time is 20-40 min; preferably, the dissolution temperature is 100-105° C., and the dissolution time is 20-30 min.

[0040] In some embodiments of the present invention, during the preparation of the sugar-milk mixture, the dissolution temperature is 90-98° C., and the dissolution time is 20-40 min; preferably, the dissolution temperature is 90-95° C., and the dissolution time is 20-30 min.

[0041] In some embodiments of the present invention, before the S protein solution and the sugar-milk mixture are mixed, the temperature of the S protein solution is lowered to 88-95°C, preferably to 90-95°C.

[0042] In some embodiments of the present invention, the preparation method further comprises a sterilization step, and the sterilization conditions are 110-130° C. for 5-20 min.

[0043] The third aspect of the present invention provides use of the 7S protein fermentation broth of the first aspect of the present invention in preparing a product that helps regulate intestinal flora.

[0044] In some embodiments of the present invention, the product includes a medicine and / or a health food.

[0045] In some embodiments of the present invention, the product further comprises pharmaceutically acceptable adjuvants and / or excipients.

[0046] The fourth aspect of the present invention provides the use of the 7S protein fermentation broth of the first aspect of the present invention in antibacterial treatment or in the preparation of antibacterial products.

[0047] In some embodiments of the present invention, the bacteria are Gram-negative bacteria; preferably Escherichia coli.

[0048] The fifth aspect of the present invention provides a product comprising the 7S protein fermentation broth of the first aspect of the present invention.

[0049] In some embodiments of the present invention, the products include reagents, foods, health foods and medicines.

[0050] In some embodiments of the present invention, when the product is a health food or medicine, the product has the function of regulating intestinal flora.

[0051] In some embodiments of the present invention, when the product is a reagent or a drug, the product has a bacteriostatic (eg, Escherichia coli) function.

[0052] In some embodiments of the present invention, the product further comprises pharmaceutically acceptable adjuvants and / or excipients.

[0053] The beneficial effects of the present invention are:

[0054] The present invention provides a 7S protein fermentation broth produced by fermenting 7S protein with probiotics. This broth can regulate the composition of intestinal flora, increase the relative abundance of beneficial bacteria in the intestinal microorganisms, improve the intestinal microenvironment, and alleviate intestinal inflammation. Furthermore, this broth has a high inhibitory effect on Escherichia coli (with an inhibition rate of up to 95.9%).

[0055] The present invention provides a method for preparing a 7S protein fermentation liquid, in which probiotics are used to ferment the 7S protein, decompose the macromolecular protein, and release more small-molecule peptides, which not only helps the absorption of protein but also supplements protein. The specific small-molecule peptides metabolized by probiotics can better regulate the composition of the intestinal flora. At the same time, probiotics can directly increase the relative abundance of beneficial bacteria in intestinal microorganisms, improve the intestinal microenvironment, and relieve intestinal inflammation. In addition, this preparation method improves the taste of the 7S protein while enhancing the ability of the 7S protein to regulate the intestinal flora. The protein is sweet and sour, with a bean flavor, which is more in line with the public's demand for healthy food. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The antibacterial effect of the 7S protein fermentation broth of Examples 1 to 7 on Escherichia coli.

[0057] Figure 2 The antibacterial effect of the 7S protein fermentation broth of Examples 8 to 14 on Escherichia coli.

[0058] Figure 3 The antibacterial effect of the 7S protein fermentation broth of Examples 15 to 16 on Escherichia coli.

[0059] Figure 4 The antibacterial effect of the 7S protein fermentation broth of Examples 17 to 19 on Escherichia coli.

[0060] Figure 5 The antibacterial effect of the 7S protein fermentation broth of Example 14 and Examples 20 to 22 on Escherichia coli.

[0061] Figure 6 The antibacterial effect of the 7S protein fermentation broth of Example 14 and Examples 23 to 25 on Escherichia coli.

[0062] Figure 7 These are the results of the in vitro antibacterial test of the 7S protein fermentation liquid of Example 14. **** represents p<0.0001.

[0063] Figure 8 This is a graph showing the changes in the total quantitative score of intestinal inflammation symptoms of volunteers before and after the trial.

[0064] Figure 9 This is a graph showing the quantitative integral changes in specific symptoms of intestinal inflammation in volunteers before and after the trial.

[0065] Figure 10 Figure 2 is an analysis of the results of 16S rRNA sequencing in human feces; (a) shows the distribution of bacterial species identified by 16S rRNA sequencing, (b) shows the number of bacterial species in feces before and after 7S protein fermentation liquid intervention, (c) shows the relative abundance of Lactobacillus before and after 7S protein fermentation liquid intervention, (d) shows the relative abundance of Firmicutes before and after 7S protein fermentation liquid intervention, (e) shows the relative abundance of Monoglobus, Colidextribacter, and Intestinibacter before and after 7S protein fermentation liquid intervention, (f) shows the relative abundance of Bacteroidetes before and after 7S protein fermentation liquid intervention, (g) shows the relative abundance of Alistipes and Parabacteroides before and after 7S protein fermentation liquid intervention, (h) shows the diversity analysis results of the α-intestinal microbial community, and (i) shows the relative abundance of Oscillospirales, Lachnospirales, Bacteroidales, and Fusobacteriales before and after 7S protein fermentation liquid intervention. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below through specific examples.

[0067] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0068] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0069] The preparation method of the 7S protein raw material is the same as Example 3 in Chinese invention patent CN102187934A.

[0070] Lactobacillus rhamnosus NX-2, with a deposit number of CGMCC: 20110, has been disclosed in the Chinese invention patent CN111826315A. The preparation method of Lactobacillus rhamnosus NX-2 powder refers to the literature "Du Hanxiao, Ran Junjian, Sun Junliang, et al. Preparation and process optimization of freeze-dried powder of Lactobacillus rhamnosus zrx01 [J]. Journal of Henan Institute of Science and Technology: Natural Science Edition, 2023, 51(6): 14-25.", and the number of viable bacteria in the powder is 5×10^9 CFU / g.

[0071] Lactobacillus plantarum NX-1, with a preservation number of CGMCC: 20109, has been disclosed in the Chinese invention patent CN111733111A. The preparation method of Lactobacillus plantarum NX-1 powder refers to the literature "Liu Xiaocui, Zhao Jingshu. Research on the drying process of Lactobacillus plantarum probiotic powder [J]. Grain and Feed Industry, 2024(5): 29-33." The number of viable bacteria in the powder is 5×10^9 CFU / g.

[0072] Lactobacillus rhamnosus E2, with a deposit number of CGMCC: 21770, has been disclosed in the Chinese invention patent CN114790430A. The preparation method of Lactobacillus rhamnosus E2 powder refers to the literature "Du Hanxiao, Ran Junjian, Sun Junliang, et al. Preparation and process optimization of freeze-dried powder of Lactobacillus rhamnosus zrx01 [J]. Journal of Henan Institute of Science and Technology: Natural Science Edition, 2023, 51(6): 14-25." The number of viable bacteria in the powder is 5×10^9 CFU / g.

[0073] Lactobacillus casei LS01, with a deposit number of CGMCC: 22006, has been disclosed in Chinese invention patent CN113197256A. The preparation method of Lactobacillus casei LS01 powder is referenced in the literature "Ouyang Biyan, Cui Shumao, Mao Bingyong, Tang Xin, Ma Fangli, Zhao Jianxin, Zhang Hao, Chen Wei. Preparation of acid-resistant freeze-dried powder of probiotic Lactobacillus casei CCFM711 [J]. Food and Fermentation Industries: 62-68." The viable cell count of the powder is 5×10^9 CFU / g.

[0074] Lactobacillus salivarius LF01, with a deposit number of CGMCC: 23313, has been disclosed in Chinese invention patent CN115786207A. The preparation method of Lactobacillus salivarius LF01 powder is referred to in the literature "Ouyang Biyan, Cui Shumao, Mao Bingyong, Tang Xin, Ma Fangli, Zhao Jianxin, Zhang Hao, Chen Wei. Preparation of acid-resistant freeze-dried powder of probiotic Lactobacillus casei CCFM711 [J]. Food and Fermentation Industries: 62-68." The viable cell count of the powder is 5×10^9 CFU / g.

[0075] Lactobacillus paracasei Lp.R3, with a deposit number of CGMCC:22008, has been disclosed in Chinese invention patent CN114350555A. The preparation method of Lactobacillus paracasei Lp.R3 powder refers to the reference "Xu Xiaofang. Research on high-density fermentation and freeze-drying process of Lactobacillus paracasei [D]. Huazhong Agricultural University, 2024." The number of viable bacteria in the powder is 5×10^9 CFU / g.

[0076] Lactobacillus gasseri LS03, with a deposit number of CGMCC:22009, has been disclosed in Chinese invention patent CN114767615A. The preparation method of Lactobacillus gasseri LS03 powder refers to the reference "Chen Xiaocen. Research on high-density culture and freeze-drying protection of Lactobacillus bulgaricus NQ2508 [D]. Jiangnan University, 2023." The viable cell count of the powder is 5×10^9 CFU / g.

[0077] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0078] Example 1

[0079] A 7S protein fermentation liquid is fermented from the following raw materials, calculated by weight: 35 parts of 7S protein raw material, 30 parts of glucose, 15 parts of white sugar, 30 parts of skim milk powder, 20 parts of Lactobacillus rhamnosus NX-2 bacterial powder, 30 parts of sweetener, 3 parts of thickener, and 700 parts of pure water;

[0080] The food additive consists of soybean polysaccharide, guar gum and curdlan gum in a weight ratio of 1:2:2;

[0081] The sweetener includes 30 parts of erythritol.

[0082] The method for preparing the 7S protein fermentation broth comprises the following steps:

[0083] (1) Weigh the 7S protein raw material, add pure water, and preliminarily homogenize and dissolve it in a mixing tank. Then, pass it into a fermentation tank and dissolve it at 105°C for 30 minutes to obtain a 7S protein solution;

[0084] (2) Weigh glucose and white sugar, add skim milk powder, and homogenize and dissolve in a mixing tank at 95°C for 30 minutes;

[0085] (3) When the 7S protein solution is cooled to 90° C., the material dissolved in step (2) is introduced into the fermentation tank of step (1), cooled to 37° C., and inoculated and fermented, i.e., Lactobacillus rhamnosus NX-2 powder is inoculated and fermented at 37° C. for about 12 h; after the fermentation is stopped, sweeteners and food additives are added, mixed, packaged, and sterilized at 115° C. for 5 min to obtain a 7S protein fermentation liquid.

[0086] Example 2

[0087] A 7S protein fermentation broth, compared with Example 1, differs in that the probiotic is Lactobacillus plantarum NX-1.

[0088] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0089] Example 3

[0090] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic is Lactobacillus rhamnosus E2.

[0091] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0092] Example 4

[0093] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic is Lactobacillus casei LS01.

[0094] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0095] Example 5

[0096] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic is Lactobacillus salivarius LF01.

[0097] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0098] Example 6

[0099] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic is Lactobacillus paracasei Lp.R3.

[0100] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0101] Example 7

[0102] A 7S protein fermentation broth, compared with Example 1, differs in that the probiotic is Lactobacillus gasseri LS03.

[0103] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0104] Example 8

[0105] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotics are Lactobacillus plantarum NX-1+Lactobacillus gasseri LS03 (mass ratio is 1:1).

[0106] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0107] Example 9

[0108] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotics are Lactobacillus casei LS01+Lactobacillus gasseri LS03 (mass ratio is 1:1).

[0109] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0110] Example 10

[0111] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotics are Lactobacillus paracasei Lp.R3+Lactobacillus gasseri LS03 (mass ratio is 1:1).

[0112] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0113] Example 11

[0114] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotics are Lactobacillus plantarum NX-1+Lactobacillus casei LS01+Lactobacillus gasseri LS03 (mass ratio is 1:1:1).

[0115] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0116] Example 12

[0117] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotics are Lactobacillus plantarum NX-1+Lactobacillus paracasei Lp.R3+Lactobacillus gasseri LS03 (mass ratio is 1:1:1).

[0118] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0119] Example 13

[0120] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotics are Lactobacillus paracasei Lp.R3+Lactobacillus casei LS01+Lactobacillus gasseri LS03 (mass ratio is 1:1:1).

[0121] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0122] Example 14

[0123] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotics are Lactobacillus plantarum NX-1+Lactobacillus paracasei Lp.R3+Lactobacillus casei LS01+Lactobacillus gasseri LS03 (mass ratio is 1:1:1:1).

[0124] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0125] Example 15

[0126] A 7S protein fermentation broth, compared with Example 14, differs in that: the mass fraction of white granulated sugar is 10 parts.

[0127] The preparation method of the above 7S protein fermentation broth is the same as that in Example 14.

[0128] Example 16

[0129] A 7S protein fermentation broth, compared with Example 15, differs in that: the mass parts of white granulated sugar are 20 parts.

[0130] The preparation method of the above 7S protein fermentation broth is the same as that in Example 15.

[0131] Example 17

[0132] A 7S protein fermentation broth, compared with Example 14, differs in that: the mass fraction of glucose is 10 parts.

[0133] The preparation method of the above 7S protein fermentation broth is the same as that in Example 16.

[0134] Example 18

[0135] A 7S protein fermentation broth, compared with Example 17, differs in that: the mass parts of glucose are 20 parts.

[0136] The preparation method of the above 7S protein fermentation broth is the same as that in Example 16.

[0137] Example 19

[0138] A 7S protein fermentation broth, compared with Example 17, differs in that: the mass parts of glucose are 40 parts.

[0139] The preparation method of the above 7S protein fermentation broth is the same as that in Example 16.

[0140] Example 20

[0141] A 7S protein fermentation broth, compared with Example 14, the difference is that: in the preparation method of the 7S protein fermentation broth, the fermentation temperature in step (3) is 32°C.

[0142] Example 21

[0143] A 7S protein fermentation broth, compared with Example 20, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation temperature in step (3) is 35°C.

[0144] Example 22

[0145] A 7S protein fermentation broth, compared with Example 20, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation temperature in step (3) is 40°C.

[0146] Example 23

[0147] A 7S protein fermentation broth, compared with Example 14, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation time of step (3) is about 8 hours.

[0148] Example 24

[0149] A 7S protein fermentation broth, compared with Example 23, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation time of step (3) is about 16 hours.

[0150] Example 25

[0151] A 7S protein fermentation broth, compared with Example 23, the difference is that: in the preparation method of the 7S protein fermentation broth, the fermentation time of step (3) is about 24 hours.

[0152] Effect embodiment

[0153] In vitro antibacterial test of 1.7S protein fermentation broth

[0154] 2 mL of 7S protein fermentation broth from Examples 1 to 25 was added to 8 mL of liquid LB, and the OD 600 For A1, 10 μL of E. coli seed solution was inoculated and cultured for 12 h, and the OD was measured. 600 The antibacterial activity of the 7S protein fermentation broths of Examples 1 to 27 against Gram-negative bacteria was evaluated by calculating ΔA = A2 - A1. The uninoculated 7S protein solution (i.e., the 7S protein solution obtained in step (1)) was used as a negative control, and LB culture medium was used as a blank control.

[0155] The results are as follows Figures 1 to 6 shown.

[0156] Compared with the negative control, the 7S protein fermentation liquid obtained by fermenting the 7S protein solution using 7 strains of bacteria alone (i.e., the 7S protein fermentation liquid of Examples 1 to 7) can significantly inhibit the growth of Escherichia coli. Among them, the inhibition rate of the 7S protein fermentation liquid obtained by using Lactobacillus rhamnosus NX-2, Lactobacillus plantarum NX-1, Lactobacillus casei LS01, Lactobacillus paracasei Lp.R3 and Lactobacillus gasseri LS03 as probiotics reached more than 90% ( Figure 1 ).

[0157] By combining different probiotics to prepare 7S protein fermentation broth (i.e., the 7S protein fermentation broth of Examples 8 to 14), the results showed that the 7S protein fermentation broth (i.e., the 7S protein fermentation broth of Example 14) fermented by the combined fermentation of four strains of Lactobacillus rhamnosus NX-2, Lactobacillus plantarum NX-1, Lactobacillus paracasei Lp.R3, and Lactobacillus gasseri LS03 had the highest inhibition rate on Escherichia coli, reaching 95.9% ( Figure 2 ), so the subsequent experiments selected the strain combination of Lactobacillus rhamnosus NX-2, Lactobacillus plantarum NX-1, Lactobacillus paracasei Lp.R3 and Lactobacillus gasseri LS03.

[0158] The amount of added white sugar in the 7S protein fermentation broth was optimized (i.e., the 7S protein fermentation broths of Examples 14 to 16). The results showed that when the amount of added white sugar was 1.5% (i.e., Example 14), the inhibition rate of the 7S protein fermentation broth on Escherichia coli was the highest ( Figure 3 ), so the amount of white sugar added is 15 parts.

[0159] The amount of glucose added to the 7S protein fermentation broth was optimized (i.e., the 7S protein fermentation broths of Examples 14 and 17-19). The results showed that when the amount of glucose added was 3% (i.e., Example 14), the 7S protein fermentation broth had the highest inhibition rate on Escherichia coli ( Figure 4 ), so the amount of glucose added was selected as 30 parts.

[0160] The fermentation temperature during the preparation of the 7S protein fermentation broth was optimized (i.e., the 7S protein fermentation broths of Examples 14 and 20-22). The results showed that the 7S protein fermentation broth obtained by fermentation at 37°C (i.e., Example 14) had the lowest inhibition rate on Escherichia coli ( Figure 5 ), so the fermentation temperature was selected as 37℃.

[0161] The fermentation time during the preparation of the 7S protein fermentation broth was optimized (i.e., the 7S protein fermentation broths of Examples 14 and 23-25). The results showed that the 7S protein fermentation broth obtained by fermentation for 12 h (i.e., Example 14) had the lowest inhibition rate on Escherichia coli ( Figure 6 ), so the fermentation time was selected as 12h.

[0162] The antibacterial effect of the optimal 7S protein fermentation broth (i.e., the 7S protein fermentation broth of Example 14) was further tested. At the same time, the 7S protein solution that was not inoculated and fermented (i.e., the 7S protein solution obtained in step (1)) was used as a negative control, and the LB culture medium was used as a blank control. The detection process was the same as above. The experimental data were analyzed and processed using GraphPad Prism 8.0 software, and the experimental data were expressed as mean ± SEM. The results are shown in Figure 2. Figure 7 As shown, compared to the blank control, the 7S protein fermentation broth significantly inhibited the growth and reproduction of E. coli, with an inhibition rate of up to 95.9%. However, the same concentration of 7S protein (negative control) promoted the growth of E. coli in vitro. T-test analysis showed a significant difference in the inhibition rate of E. coli in vitro between the blank control and the fermented broth (**** indicates p < 0.0001).

[0163] 2. Population Experiment

[0164] A total of 10 subjects, aged 20 to 50 years old, all had symptoms of gastrointestinal discomfort such as irritable bowel syndrome or chronic gastritis. Each subject took 200 mL of 7S protein fermentation liquid (Example 14) daily. During the experiment, they ate according to their normal eating habits. The duration of administration was 20 days. The subjects' mental state, diet, sleep, urination, etc. during the trial period were recorded, and a symptom quantitative score table (Table 1) was completed; routine blood tests; liver and kidney function tests; feces were collected before and after the experiment for 16S-rRNA sequencing. The experimental data were analyzed and processed using GraphPad Prism 8.0 software, and the experimental data were expressed as mean ± SEM. Using T-test analysis, the quantitative scores of intestinal inflammation symptoms before and after taking 7S protein fermentation liquid were compared: *p ​​< 0.05, **p < 0.01.

[0165] Table 1 Clinical symptom score quantification table

[0166]

[0167] Fecal 16S-rRNA sequencing:

[0168] Genomic DNA extraction and PCR amplification: The genomic DNA of the sample was extracted using the CTAB method, and then the purity and concentration of the DNA were tested by agarose gel electrophoresis. An appropriate amount of sample DNA was placed in a centrifuge tube and diluted to 1 ng / μL with sterile water. The diluted genomic DNA was used as a template, and specific primers with barcodes were used according to the selection of the sequencing region. High-Fidelity PCR Master Mix with GCBuffer and high-efficiency, high-fidelity enzymes were used for PCR to ensure amplification efficiency and accuracy. Primers for the 16S V4 region (515F and 806R) were used to identify bacterial diversity; primers for the 18S V4 region (528F and 706R) were used to identify eukaryotic microbial diversity; and primers for the ITS1 region (ITS5-1F-F and ITS1-1F-R) were used to identify fungal diversity. In addition, amplification regions included: 16S V3-V4 / 16S V4-V5 / 16SV5-V7; Archaeal 16S V4-V5 / Archaeal 16S V8; and 18SV9 and ITS2 regions. Primer sequences are as follows:

[0169] 515F: 5'-GTGYCAGCMGCCGCGGTAA-3' (SEQ ID NO: 1);

[0170] 806R: 5'-GGACTACNVGGGTWCTTAAT-3' (SEQ ID NO: 2);

[0171] 528F: 5'-GCGGTAATTCCAGCTCCAA-3' (SEQ ID NO: 3);

[0172] 706R: 5'-AATCCRAGAATTTCACCTCT-3' (SEQ ID NO: 4);

[0173] ITS5-1F-F:5'-GGAAGTAAAAGTCGTAACAAGG-3' (SEQ ID NO:5);

[0174] ITS1-1F-R:5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO:6);

[0175] Primers for amplifying bacterial 16S V3-V4 region:

[0176] 341F: 5'-CCTACGGGNGGCWGCAG-3' (SEQ ID NO: 7);

[0177] 806R: 5'-GGACTACNVGGGTWCTTAAT-3' (SEQ ID NO: 8);

[0178] Bacterial 16S V4-V5 region amplification primers:

[0179] F: 5'-TATGGTAATTGTGCCAGCMGCCGCGGTAA-3' (SEQ ID NO: 9);

[0180] R: 5'-AGTCAGTCAGGCCCTGTAATTCMTTRAGT-3' (SEQ ID NO: 10);

[0181] Primers for amplifying bacterial 16S V5-V7 region:

[0182] 799F: 5'-AACMGGATTAGATACCCKG-3' (SEQ ID NO: 11);

[0183] 1193R: 5'-ACGTCATCCCCACCTTCC-3' (SEQ ID NO: 12);

[0184] Primers for amplifying the archaeal 16S V4-V5 region:

[0185] Arch519F:5'-CAGCCGCCGCGGTAA-3'(SEQ ID NO:13);

[0186] Arch915R:5'-GTGCTCCCCCGCACATTCCT-3' (SEQ ID NO:14);

[0187] Primers for amplifying the archaeal 16S V8 region:

[0188] F:5'-GGATTAGATACCC-3'(SEQ ID NO:15);

[0189] R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 16);

[0190] 18S V9 region amplification primers:

[0191] F: 5'-CCCTGCCHTTTGTACACAC-3' (SEQ ID NO: 17);

[0192] R: 5'-CCTTCYGCAGGTTCACCTAC-3' (SEQ ID NO: 18);

[0193] 18S ITS2 region amplification primers:

[0194] ITS3:5'-GCATCGATGAAGAACGCAGC-3' (SEQ ID NO: 19);

[0195] ITS4:5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO:20);

[0196] PCR amplification and library construction: PCR products were detected by electrophoresis on 2% agarose gel; equal amounts of samples were mixed according to the concentration of PCR products, and after thorough mixing, PCR products were detected by electrophoresis on 2% agarose gel. The target bands were recovered using the gel recovery kit provided by Qiagen. The library was constructed using the DNA PCR-Free Sample Preparation Kit. The constructed library was quantified by Qubit and Q-PCR. Once qualified, it was sequenced using the NovaSeq6000.

[0197] Bioinformatics analysis: The original data is in FASTQ format. After the data is downloaded, cutadapt software is first used to cut out the primer sequence from the rawdata sequence. Then, DADA2 is used to perform quality control analysis such as quality filtering, noise reduction, splicing, and chimera removal on the qualified double-end raw data in the previous step according to the default parameters of QIIME 2 to obtain representative sequences and ASV abundance tables. After using the QIIME 2 software package to select the representative sequences of each ASV, all representative sequences are aligned and annotated with the database. 16S is aligned using the Silva (version 138) database. Species alignment annotations are analyzed using the default parameters of the q2-feature-classifier software. The experimental data were analyzed and processed using GraphPad Prism 8.0 software. The experimental data are expressed as mean ± SEM and analyzed using T-test.

[0198] The results showed that after continuous consumption of 7S protein fermentation liquid, the volunteers' scores on the symptom quantitative score questionnaire were significantly reduced ( Figure 8 ), indicating that taking 7S protein fermentation liquid significantly alleviated the overall physical symptoms of the volunteers. Taking 7S protein fermentation liquid also has a certain effect on alleviating the symptoms of intestinal inflammation, among which belching, loss of appetite, diarrhea or constipation, chronic enteritis / gastritis and abnormal defecation are alleviated by more than 50% ( Figure 9 ).

[0199] 16S rRNA sequencing was performed on the feces of volunteers before and after the experiment. The results showed that 274 bacterial operational taxonomic units (OTUs) were identified by 16S rRNA sequencing, covering 12 phyla, 17 classes, 32 orders, 53 families, 151 genera, and 274 species. The identified bacteria mainly belonged to the Firmicutes and Bacteroidetes phyla, and also involved the Fusobacteriota, Actinobacteriota, Verrucomicrobia, and Archaea. Figure 10 It can be observed that the relative abundance of Firmicutes increased significantly before and after the intervention. Many members of the Firmicutes are beneficial bacteria, including Lactobacillus ( Figure 10 c), indicating that 7S protein fermentation broth provides a better intestinal environment for probiotics. In this phylum, three bacteria, Monoglobus, Colidextribacter and Intestinibacter, showed a significant increase in abundance overall ( Figure 10 Among them, Monoglobus is a pectin-degrading specialist bacterium in the human colon, Colidextribacter is associated with hyperlipidemia, and Intestinibacter is associated with a reduced risk of type 1 diabetes.

[0200] Bacteroidetes are very successful competitors in the intestinal ecosystem, showing considerable nutritional flexibility and the ability to respond to stresses imposed by the host and the intestinal environment. It is difficult to say whether intestinal Bacteroidetes have negative or positive effects on the host. Bacteroidetes are able to benefit the host by preventing infection by potential pathogens that may colonize and infect the intestine. As members of the polysaccharide degradation alliance, they help release energy from dietary fiber and starch, and they are likely to be the main source of propionate. However, they are also involved in the release of toxic products during protein breakdown, and some members of this group have activities that may help suppress inflammation, but they may also promote inflammation, and some are known opportunistic pathogens. It can be seen from the experiment that the relative abundance of Bacteroidetes decreased significantly before and after intervention with 7S protein fermentation broth ( Figure 10 In the Bacteroidetes phylum, two bacteria, Alistipes and Parabacteroides, showed a significant decrease in abundance overall ( Figure 10 (g) Excessive amounts of Alistipes have been implicated as causative agents of disease in clinical and preclinical studies. Studies have shown that their presence is associated with a healthy phenotype, such as protective effects in conditions such as colitis, autism spectrum disorder, and various liver and cardiovascular fibrotic diseases. In stark contrast, Alistipes has a pathogenic role in conditions such as anxiety, myalgic encephalomyelitis / chronic fatigue syndrome, depression, PDD-NOS, and CRC. Parabacteroides exhibit dual potential between pathogenicity and probiotic ability, and our current understanding of the bacteria's potential to regulate health or cause disease is suboptimal and incomplete. Of 14 studies of Parabacteroides distasonis, five strains were presumed to be pathogenic, one was a probiotic strain, and seven strains were neither presumed to be probiotic nor pathogenic.

[0201] At the family level, Bacteroidales was the most abundant bacterial family, followed by Lachnospirales. Others included Oscillospirales, Fusobacteriales, Verrucomicrobiales, Bifidobacteriales, Peptostreptococcales-Tissierellales, Erysipelotrichales, Clostridia, and Coriobacteriales. The inventors observed that the abundance of Oscillospirales and Lachnospirales increased after intervention, while the abundance of Bacteroidales and Fusobacteriales decreased ( Figure 10 i) Decrease. Oscillospirales bacteria are associated with obesity, emaciation, gallstones, and chronic constipation at the population level, and have been shown to correlate positively or negatively with changes in the course of their illness. Bacteria that produce short-chain fatty acids (SCFAs) such as butyrate are also listed as candidates for next-generation probiotics, as butyrate is an important indicator for screening "next-generation probiotics." Lachnospirales bacteria are found in the intestines of most healthy people and may be potential beneficial bacteria. They are particularly active in the metabolism of various carbohydrates, especially pectin (a complex dietary fiber and prebiotic) found in fruits and vegetables. Fermentation leads to the production of acetic acid and butyric acid, which provide the host with a major source of energy. Bacteroidales are important cornerstone bacteria that reside in the human intestine and have a symbiotic relationship with humans. They help break down food and produce the nutrients and energy needed by the body. However, when Bacteroides enter the body outside the gastrointestinal area, they can cause or exacerbate infections such as abscesses. Fusobacteriaceae and Fusobacterium necroticum are among the formidable anaerobic pathogens in the genus Fusobacteriales, most commonly found in the oral cavity and dental plaque, and are associated with periodontal disease, acute necrotizing gingivitis, oral cancer, ulcerative colitis, Crohn's disease, and colorectal cancer.

[0202] α-Diversity analysis of intestinal microbial communities showed that the diversity of the microbial community after intervention was greater than that before intervention, and the intestinal microbial diversity of patients with inflammatory bowel disease was reduced ( Figure 10 h), thus indicating that 7S protein fermentation broth can alleviate inflammatory bowel disease by increasing the diversity of intestinal flora.

[0203] From the above results, it can be seen that 7S protein fermentation broth has the effect of regulating intestinal flora both in vivo and in vitro.

[0204] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A 7S protein fermentation broth, fermented from the following raw materials: 7S protein, sugar, milk, probiotics, sweetener and thickener; wherein, The probiotics include at least one of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus paracasei and Lactobacillus gasseri.

2. The 7S protein fermentation broth according to claim 1, characterized in that The sugar includes at least one of glucose, white sugar, and maltose; and / or the sweetener includes at least one of erythritol, xylitol, maltitol, sorbitol, steviol glycosides, sucralose, aspartame, acesulfame potassium, and neotame; and / or the thickener includes soybean polysaccharide, guar gum, and curdlan gum.

3. The 7S protein fermentation broth according to claim 1, characterized in that The probiotics include at least three of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus gasseri; Preferably, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus casei and Lactobacillus gasseri; Preferably, the probiotic is a combination of Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus gasseri; Preferably, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus gasseri.

4. The 7S protein fermentation broth according to any one of claims 1 to 3, characterized in that The 7S protein fermentation broth is fermented from the following raw materials, calculated by weight: 20 to 70 parts of 7S protein, 10 to 70 parts of sugar, 100 to 500 parts of milk, 10 to 70 parts of probiotics, 20 to 60 parts of sweetener and 2 to 8 parts of thickener.

5. The 7S protein fermentation broth according to any one of claims 1 to 3, characterized in that The raw materials also include water.

6. The method for preparing the 7S protein fermentation broth according to claim 5, comprising the following steps: Mixing the 7S protein with water and dissolving it to obtain a 7S protein solution; Mixing sugar and milk and dissolving them to obtain a sugar-milk mixture; The 7S protein solution and the sugar-milk mixture are mixed, cooled, inoculated with probiotics, and fermented to obtain a fermentation solution; The fermentation solution is mixed with a sweetener and a thickener to obtain a 7S protein fermentation liquid; Preferably, the fermentation temperature is 30-50°C and the fermentation time is 5-30h; Preferably, the temperature is lowered to 30-50°C.

7. The preparation method according to claim 6, characterized in that During the preparation of the 7S protein solution, the dissolution temperature is 100-110° C. and the dissolution time is 20-40 min; and / or, during the preparation of the sugar-milk mixture, the dissolution temperature is 90-98° C. and the dissolution time is 20-40 min; and / or, before the S protein solution and the sugar-milk mixture are mixed, the temperature of the S protein solution is lowered to 88-95° C.

8. Use of the 7S protein fermentation broth according to any one of claims 1 to 5 in the preparation of a product that helps regulate intestinal flora; Preferably, the product comprises a medicine and / or a health food.

9. Use of the 7S protein fermentation broth according to any one of claims 1 to 5 in antibacterial treatment or in the preparation of antibacterial products; Preferably, the bacteria are Gram-negative bacteria.

10. A product comprising the 7S protein fermentation broth according to any one of claims 1 to 5.

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