7S protein fermentation liquor and application thereof in reducing blood fat

Abstract

The invention belongs to the technical field of medicines, discloses 7S protein fermentation liquor and application of the 7S protein fermentation liquor in blood fat reduction, and particularly discloses the 7S protein fermentation liquor. The invention provides 7S protein fermentation liquor obtained by fermenting 7S protein through probiotics, and the 7S protein fermentation liquor can effectively inhibit the activity of pancreatic lipase, remarkably reduce the relative fat level in a zebra fish body, effectively reduce the cholesterol and triglyceride level in serum and the content of low-density lipoprotein cholesterol, and improve the stability of the zebra fish. And the 7S protein fermentation broth has no influence on blood routine and liver and kidney functions of human, and has high safety.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a 7S protein fermentation broth and its application in lowering blood lipids. Background Art

[0002] In recent years, with the global prevalence of obesity and metabolic diseases, the health effects of dietary plant protein have received increasing attention. Substituting plant protein for animal protein is associated with lower cardiovascular disease mortality.

[0003] Soy hydrolysates are highly effective in treating hyperlipidemia. The 7S protein in soy protein can effectively lower serum cholesterol and triglyceride levels by regulating serum bile acid metabolism. It also binds to bile acids in the intestine, inhibiting the formation of cholesterol micelles and cholesterol absorption in the intestine. However, because soy protein is a large molecule, the human body's absorption and digestion capacity is limited, requiring large doses to achieve significant results in lowering blood lipids and cholesterol. Furthermore, the finished product of 7S protein has a poor taste and is not widely popular with consumers.

[0004] Fermented dairy products, known for their delicious taste and high nutritional value, have become a mainstream daily food. However, to maintain flavor, most dairy products add large amounts of sugar, which impacts consumers' sugar intake and, consequently, blood lipid levels.

[0005] Therefore, there is an urgent need to develop a fermentation liquid with lipid-lowering effect to assist people with hyperlipidemia in controlling blood lipids, supplementing protein, and reducing dependence on lipid-lowering drugs. Summary of the Invention

[0006] The first aspect of the present invention aims to provide a 7S protein fermentation broth.

[0007] 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.

[0008] The third aspect of the present invention aims to provide an application of the 7S protein fermentation broth of the first aspect of the present invention.

[0009] The fourth aspect of the present invention aims to provide a product.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a 7S protein fermentation broth, which is fermented from the following raw materials: 7S protein, sugar, probiotics, a sweetener and a thickener, wherein the probiotics include at least one of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus helveticus and Lactobacillus paracasei.

[0012] In some embodiments of the present invention, the probiotics include at least two of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus helveticus, and Lactobacillus paracasei.

[0013] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum and Lactobacillus rhamnosus, and the mass ratio between the bacteria is 1:(1-3); preferably 1:1.

[0014] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum and Lactobacillus paracasei, and the mass ratio between the bacteria is 1:(1-3); preferably 1:1.

[0015] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum and Lactobacillus helveticus, and the mass ratio between the bacteria is 1:(1-3); preferably 1:1.

[0016] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei, and the mass ratio between the bacteria is 1:(1-3):(1-3); preferably 1:1:1.

[0017] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus helveticus, and the mass ratio between the bacteria is 1:(1-3):(1-3); preferably 1:1:1.

[0018] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus helveticus, and the mass ratio between the bacteria is 1:(1-3):(1-3); preferably 1:1:1.

[0019] In some embodiments of the present invention, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus helveticus, and the mass ratio between the bacteria is 1:(1-3):(1-3):(1-3); preferably 1:1:1:1.

[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.

[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 helveticus is Lactobacillus helveticus LH05 with a deposit number of CGMCC:23452.

[0026] In some embodiments of the present invention, the sugar includes at least one of glucose, white sugar, and maltose; preferably glucose and white sugar.

[0027] 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.

[0028] In some embodiments of the present invention, the thickener comprises soybean polysaccharide, guar gum and curdlan gum.

[0029] In some embodiments of the present invention, the thickener is composed of guar gum and curdlan gum in a mass ratio of 1:(1-5):(1-5); preferably 1:2:2.

[0030] 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, 10 to 70 parts of probiotics, 20 to 50 parts of sweetener and 2 to 8 parts of thickener.

[0031] 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, 10 to 60 parts of sugar, 20 to 50 parts of probiotics, 20 to 40 parts of sweetener and 2 to 6 parts of thickener.

[0032] 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 40 parts of glucose, 10 to 15 parts of white sugar, 20 to 50 parts of probiotics, 20 to 40 parts of sweetener and 3 to 6 parts of thickener.

[0033] In some embodiments of the present invention, the raw material further comprises water.

[0034] In some embodiments of the present invention, the raw materials further include 400 to 700 parts of water.

[0035] 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:

[0036] Mixing 7S protein and sugar with water and dissolving them to obtain a 7S protein solution;

[0037] inoculating probiotics into the 7S protein solution and fermenting to obtain a fermentation solution;

[0038] The fermentation solution is mixed with a sweetener and a thickener to obtain 7S protein fermentation liquid.

[0039] In some embodiments of the present invention, the fermentation temperature is 30-50° C., and the fermentation time is 4-30 h; preferably, the fermentation temperature is 32-40° C., and the fermentation time is 8-24 h.

[0040] In some embodiments of the present invention, 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.

[0041] In some embodiments of the present invention, the 7S protein solution is cooled to 30-50°C, preferably 35-40°C, before inoculating the probiotics.

[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 the use of 7S protein fermentation broth in any one of (1) to (3):

[0044] (1) Preparation of drugs for lowering blood lipids;

[0045] (2) Inhibit the activity of pancreatic lipase;

[0046] (3) Prepare products that inhibit pancreatic lipase activity.

[0047] In some embodiments of the present invention, the product comprises a reagent or a kit.

[0048] The fourth 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 effect of maintaining healthy blood lipid (cholesterol / triglyceride) levels.

[0051] In some embodiments of the present invention, the product further comprises pharmaceutically acceptable adjuvants and / or excipients.

[0052] The beneficial effects of the present invention are:

[0053] The present invention provides a 7S protein fermentation liquid obtained by fermenting 7S protein with probiotics. The 7S protein fermentation liquid can effectively inhibit the activity of pancreatic lipase, significantly reduce the relative fat level in zebrafish, and effectively reduce the cholesterol, triglyceride levels and low-density lipoprotein cholesterol content in serum. The 7S protein fermentation liquid has no effect on human blood routine and liver and kidney function, and has high safety.

[0054] The present invention provides a method for preparing a 7S protein fermentation broth. This method processes the 7S protein through a unique probiotic fermentation technology, decomposing the macromolecular protein and releasing more small-molecule peptides. This method not only facilitates protein absorption and protein supplementation, but also utilizes the probiotics to metabolize the macromolecular protein into specific small-molecule peptides, thereby better regulating bile acid metabolism and more effectively lowering serum cholesterol and triglyceride levels. Furthermore, this method enhances the lipid-lowering ability of the 7S protein while improving its taste, resulting in a sweet and sour flavor with a bean flavor. This method better meets the public's demand for healthy foods, reduces the public's intake of sugary beverages, and more effectively and rationally controls blood lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The inhibitory effect of the 7S protein fermentation broth of Examples 1 to 7 on pancreatic lipase activity.

[0056] Figure 2 The inhibitory effect of the 7S protein fermentation broth of Examples 8 to 14 on pancreatic lipase activity.

[0057] Figure 3 The inhibitory effect of the 7S protein fermentation broth of Example 11 and Examples 15-16 on the activity of pancreatic lipase.

[0058] Figure 4 The inhibitory effect of the 7S protein fermentation broth of Example 11 and Examples 17 to 19 on the activity of pancreatic lipase.

[0059] Figure 5 The inhibitory effect of the 7S protein fermentation broth of Example 11 and Examples 20 to 22 on the activity of pancreatic lipase.

[0060] Figure 6 These are the sensory evaluation results of the 7S protein fermentation broth of Example 11.

[0061] Figure 7 The results of zebrafish Oil Red O staining in different treatment groups.

[0062] Figure 8 is the relative fat content in zebrafish of different treatment groups.

[0063] Figure 9 This is the in vitro lipid-lowering result of the 7S protein fermentation broth of Example 11.

[0064] Figure 10 These are the results of measuring total cholesterol levels in subjects after taking the 7S protein fermentation broth of Example 11.

[0065] Figure 11 These are the results of determining triglyceride levels in subjects after taking the 7S protein fermentation broth of Example 11.

[0066] Figure 12 These are the results of measuring the low-density lipoprotein cholesterol levels in subjects after taking the 7S protein fermentation broth of Example 11. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below through specific examples.

[0068] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0069] 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.

[0070] The preparation method of the 7S protein raw material is the same as that of Example 3 in Chinese invention patent CN102187934A.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] Lactobacillus helveticus LH05, with a deposit number of CGNCC: 23452, has been disclosed in the Chinese invention patent CN116426426A. The preparation method of Lactobacillus helveticus LH05 powder is referred to the literature "Lei Dan, Han Di, Jiang Deyi, et al. Study on the production process of freeze-dried powder of Lactobacillus helveticus LH-G51 [J]. Chinese Food and Nutrition, 2017, 23(02): 62-65." The viable cell count of the powder is 5×10^9 CFU / g.

[0078] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0079] Example 1

[0080] A 7S protein fermentation liquid is fermented from the following raw materials, calculated by weight: 35 parts of 7S protein, 30 parts of glucose, 15 parts of white sugar, 30 parts of erythritol, 3 parts of a thickener, 20 parts of probiotic powder (Lactobacillus rhamnosus NX-2), and 700 parts of purified water;

[0081] The thickener consists of soybean polysaccharide, guar gum and curdlan gum in a weight ratio of 1:2:2.

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

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

[0084] (2) After the 7S protein solution is cooled to 37°C, probiotic powder is added for fermentation, i.e., Lactobacillus rhamnosus NX-2 is added and fermented at 37°C for 12 hours; after the fermentation stops, erythritol and a thickener are added, mixed, divided and sterilized at 115°C for 5 minutes to obtain the 7S protein fermentation liquid.

[0085] Example 2

[0086] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic powder is Lactobacillus plantarum NX-1.

[0087] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0088] Example 3

[0089] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic powder is Lactobacillus rhamnosus E2.

[0090] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0091] Example 4

[0092] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic powder is Lactobacillus casei LS01.

[0093] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0094] Example 5

[0095] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic powder is Lactobacillus salivarius LF01.

[0096] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0097] Example 6

[0098] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic bacterial powder is Lactobacillus paracasei Lp.R3.

[0099] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0100] Example 7

[0101] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic powder is Lactobacillus helveticus LH05.

[0102] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0103] Example 8

[0104] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic bacterial powder is Lactobacillus plantarum NX-1+Lactobacillus rhamnosus E2 (mass ratio is 1:1).

[0105] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0106] Example 9

[0107] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic bacterial powder comprises Lactobacillus plantarum NX-1+Lactobacillus paracasei Lp.R3 (mass ratio is 1:1).

[0108] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0109] Example 10

[0110] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic bacterial powder is Lactobacillus plantarum NX-1+Lactobacillus helveticus LH05 (mass ratio is 1:1).

[0111] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0112] Example 11

[0113] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic bacterial powder comprises Lactobacillus plantarum NX-1+Lactobacillus rhamnosus E2+Lactobacillus paracasei Lp.R3 (mass ratio is 1:1:1).

[0114] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0115] Example 12

[0116] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic bacterial powder comprises Lactobacillus plantarum NX-1+Lactobacillus rhamnosus E2+Lactobacillus helveticus LH05 (mass ratio is 1:1:1).

[0117] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0118] Example 13

[0119] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic bacterial powder comprises Lactobacillus plantarum NX-1+Lactobacillus paracasei Lp.R3+Lactobacillus helveticus LH05 (mass ratio is 1:1:1).

[0120] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0121] Example 14

[0122] A 7S protein fermentation broth, compared with Example 1, differs in that: the probiotic bacterial powder comprises Lactobacillus plantarum NX-1+Lactobacillus rhamnosus E2+Lactobacillus paracasei Lp.R3+Lactobacillus helveticus LH05 (mass ratio is 1:1:1:1).

[0123] The preparation method of the 7S protein fermentation broth is the same as that in Example 1.

[0124] Example 15

[0125] A 7S protein fermentation broth, compared with Example 11, differs in that: the content of white granulated sugar is 10 parts.

[0126] The preparation method of the above 7S protein fermentation broth is the same as that in Example 11.

[0127] Example 16

[0128] A 7S protein fermentation broth, compared with Example 11, differs in that: the content of white granulated sugar is 20 parts.

[0129] The preparation method of the above 7S protein fermentation broth is the same as that in Example 11.

[0130] Example 17

[0131] A 7S protein fermentation broth, compared with Example 11, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation temperature is 32°C.

[0132] Example 18

[0133] A 7S protein fermentation broth, compared with Example 11, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation temperature is 35°C.

[0134] Example 19

[0135] A 7S protein fermentation broth, compared with Example 11, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation temperature is 40°C.

[0136] Example 20

[0137] A 7S protein fermentation broth, compared with Example 11, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation time is 8 hours.

[0138] Example 21

[0139] A 7S protein fermentation broth, compared with Example 11, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation time is 16 hours.

[0140] Example 22

[0141] A 7S protein fermentation broth, compared with Example 11, differs in that: in the preparation method of the 7S protein fermentation broth, the fermentation time is 24 hours.

[0142] Effect embodiment

[0143] Effect of 1.7S protein fermentation broth on lipase activity

[0144] In the sample group, samples (i.e., the 7S protein fermentation broths of Examples 1 to 22) were added to the reaction substrate 4-nitrophenyl laurate solution, Tris-HCl buffer, and lipase solution, and reacted at 37°C for 2 hours. In the background group, samples were added to the reaction substrate 4-nitrophenyl laurate solution, Tris-HCl buffer, and lipase solution, and reacted at 37°C for 2 hours. In the blank group, ultrapure water was added to the reaction substrate 4-nitrophenyl laurate solution, Tris-HCl buffer, and lipase solution, and reacted at 37°C for 2 hours. After centrifugation, the supernatant was collected and the absorbance at OD410nm was measured. The measured value for the sample group was A1, the measured value for the background group was A2, and the measured value for the blank group was A0. The lipase activity inhibition rate was calculated as follows: the control group was treated with unfermented 7S protein at the same concentration.

[0145]

[0146] The results are as follows Figure 1 The results showed that the pancreatic lipase activity inhibition rate of the 7S protein fermentation broth obtained by fermenting 7S protein using a single strain such as Lactobacillus plantarum NX-1, Lactobacillus rhamnosus E2, Lactobacillus paracasei Lp.R3 and Lactobacillus helveticus LH05 (corresponding to the 7S protein fermentation broths of Examples 2, 6 and 7, respectively) was significantly higher than that of the control group.

[0147] like Figure 2 As shown in the results, the 7S protein fermentation liquid obtained by fermenting 7S protein using a combination of bacteria has a good inhibitory effect on the activity of pancreatic lipase. Among them, the 7S protein fermentation liquid obtained by fermenting the strain combination of Lactobacillus plantarum NX-1 + Lactobacillus rhamnosus E2 + Lactobacillus paracasei Lp.R3 (i.e., the 7S protein fermentation liquid of Example 11) has the highest pancreatic lipase activity inhibition rate, reaching 64.01%, which is significantly higher than that of the non-fermented group (14.8%).

[0148] By optimizing the amount of sugar added to the 7S protein fermentation broth, the results are as follows Figure 3 As shown, when the amount of white sugar added was 15 parts (i.e., the 7S protein fermentation broth of Example 11), the pancreatic lipase activity inhibition rate was the highest (65.3%), so the amount of white sugar added was determined to be 15 parts.

[0149] By optimizing the fermentation temperature and fermentation time in the preparation method of 7S protein fermentation broth, the results are as follows Figures 4 and 5 As shown in Figure 2, when the fermentation temperature was 37°C, the 7S protein fermentation broth had the highest inhibition rate on pancreatic lipase activity (80.2%) ( Figure 4 ); When the fermentation time was 12h, the inhibition rate of 7S protein fermentation broth on pancreatic lipase activity was the highest (80.6%) ( Figure 5 ).

[0150] Sensory evaluation of 2.7S protein fermentation broth

[0151] Twenty-one participants aged 18 to 55 years were selected to conduct a sensory evaluation of the 7S protein fermentation broth of Example 11 based on six aspects: color, texture, aroma, acidity, sweetness, and mouthfeel. The sensory evaluation was conducted using a nine-point scale, with 1 being the worst and 9 being the best. The sensory evaluation criteria for the samples are shown in Table 1.

[0152] Table 1 Sensory evaluation standards

[0153]

[0154]

[0155] Sensory evaluation Figure 6As shown, compared with the negative control group (unfermented 7S protein fermentation broth, compared with Example 11, the difference is that it is not fermented), the texture, aroma, sweetness and taste of the fermented 7S protein fermentation broth are significantly improved. Therefore, the fermentation treatment can significantly improve its taste.

[0156] Effects of 3.7S protein fermentation broth on fat content in zebrafish

[0157] (1) Test system

[0158] The zebrafish used in this test are AB strain zebrafish, which were bred by Guangdong Nanxin Medical Technology Co., Ltd. Laboratory.

[0159] (2) Reagents and main equipment

[0160] Reagents: Oil Red O, zebrafish high-fat diet, zebrafish normal diet, 1,2-propylene glycol, 4% paraformaldehyde, atorvastatin calcium trihydrate, glucose, o-phthalaldehyde, glacial acetic acid, sulfuric acid, lipase, Tris-HCl buffer (pH=8), Triton X-100, sodium acetate, 4-nitrophenyl laurate.

[0161] Equipment: Five-layer single-row independent culture unit; continuous zoom stereo microscope; biochemical incubator; SQP 1 / 10,000 electronic balance; microplate reader; metal bath; centrifuge.

[0162] (3) Model construction

[0163] Normally developed 5 dpf (days-post fertilization) zebrafish larvae were randomly selected and placed in cell culture plates. E3 water was added to the normal group, model group, and test group. The normal group was fed with ordinary feed; the model group, positive control group, and test group were all added with an appropriate amount of 1% glucose solution and fed with a high-fat feed to construct a high-fat model.

[0164] Both ordinary feed and high cholesterol feed were purchased from Nantong Trophy Feed Technology Co., Ltd., ordinary feed item number TP1FM21000, and high fat feed item number TP 1FM21050.

[0165] (4) Intervention

[0166] After the high-fat model was established, E3 water was added to the normal, model, and test group, and atorvastatin calcium solution was added to the positive control group. The normal group continued to be fed a standard diet, while the model and positive control groups were fed a high-fat diet. The test group (7S protein) was added to E3 aquaculture water and fed a 7S protein mixed with a high-fat diet. The test group (7S protein fermentation broth freeze-dried powder) was added to E3 aquaculture water and fed a 7S protein fermentation broth freeze-dried powder mixed with a high-fat diet. The test group (Gynostemma pentaphyllum total glycosides tablets) was added to E3 aquaculture water and fed a gypenoside tablet powder mixed with a high-fat diet. The test group (By-Health fish oil) was added to E3 aquaculture water and fed a By-Health fish oil mixed with a high-fat diet (in the above experiments, the mass ratio of test sample to high-fat diet was 2:1, and the total amount of test sample fed was 1 mg / mL). The intervention period was 48 hours.

[0167] The 7S protein fermentation broth freeze-dried powder was prepared by freeze-drying the fermentation broth of Example 11 and then grinding it. The specific freeze-drying parameters were as follows: freezing stage: -50°C, 240 minutes; main drying stage: -30°C, ramp time 90 minutes, hold time 1200 minutes, vacuum setting 10 Pa; desorption drying stage: 25°C, ramp time 60 minutes, hold time 1100 minutes, vacuum setting 10 Pa. Storage temperature: -20°C.

[0168] The above-mentioned gypenosides tablets were purchased from Ankang Zhengda Pharmaceutical Co., Ltd. with approval number National Medicine Standard Z10900032; By-Health fish oil was purchased from By-Health Co., Ltd. with approval number Food and Health Preparation G202444002683.

[0169] (5) Oil Red O staining

[0170] After the intervention, the zebrafish were washed twice with E3 water and fixed with 4% paraformaldehyde solution at 4°C. They were then rinsed twice with PBS and dehydrated with a gradient of 1,2-propylene glycol. The zebrafish were then placed in a cell culture plate and dyed with Oil Red O. After staining, the cells were decolorized with 1,2-propylene glycol and observed under a stereomicroscope and photographed. Image J software was used to analyze the Oil Red O staining of zebrafish fat and calculate the corresponding grayscale value (S). The formula for calculating the relative fat level of zebrafish is as follows:

[0171]

[0172] (6) Data statistics

[0173] GraphPad Prism 8 software was used to statistically process the data. All experimental data were expressed as mean ± SEM. t-test analysis showed that compared with the blank group, the model group had the following results: #P < 0.05, ##P < 0.01, ###P < 0.001; compared with the 7S protein fermentation broth group and the 7S protein group, the results were: &&&&P < 0.0001. One-way ANOVA showed that compared with the model group, the results were: *P < 0.05, **P < 0.01, ***P < 0.001.

[0174] Depend on Figure 7 and Figure 8 Compared with the normal group, the zebrafish Oil Red O staining in the model group was significantly darker, and the relative fat level in the zebrafish was measured to be 522.50±48.15%, which was significantly higher than the normal group (100.00±15.59%) (P<0.001), indicating that the zebrafish hyperlipidemia model was successfully established. Compared with the model group, the zebrafish Oil Red O staining in the positive control group was lighter, and the relative fat level in the zebrafish was measured to be 354.30±15.54%, which was significantly lower than the model control group (522.50±48.15%) (P<0.01), indicating that this experiment was effective.

[0175] Depend on Figure 7 and Figure 8 It can be seen that compared with the model group, the zebrafish oil red O staining in the gypenosides group became lighter, and the relative fat level in the body was 288.70±33.62%, which was lower than the model group (522.50±48.15%) and had a significant difference (P<0.01). Compared with the model group, the zebrafish oil red O staining in the fish oil group became darker, and the relative fat level in the body was 681.70±64.42%, which was higher than the model group (522.50±48.15%), with no statistical difference (P>0.05). Compared with the model group, the zebrafish oil red O staining in the 7S protein group became lighter, and the relative fat level in the body was 371.50±9.02%, which was lower than the model group (522.50±48.15%) and had a significant difference (P<0.01). The results are as follows Figure 7 and Figure 8 As shown, compared with the model group, the zebrafish oil red O staining in the 7S protein fermentation liquid group became lighter, and the relative fat level in the body was 223.00±18.35%, which was significantly lower than that in the model group (522.50±48.15%) (P<0.001).

[0176] The above results show that 1 mg / mL 7S protein fermentation broth can significantly reduce the relative fat level in zebrafish. At the same concentration, the effect is better than that of unfermented 7S protein, and it has the potential to reduce body fat.

[0177] In vitro lipid-lowering results of 4.7S protein fermentation broth

[0178] In vitro determination of cholesterol binding rate: For the sample group, samples were added to a cholesterol solution and reacted at 37°C for 2 hours. For the background group, samples were added to glacial acetic acid and reacted at 37°C for 2 hours. For the blank group, ultrapure water was added to the cholesterol solution and reacted at 37°C for 2 hours. For each group, o-phthalaldehyde and mixed acid were added and reacted at 37°C for 10 minutes. After centrifugation, the supernatant was collected and the absorbance at OD550nm was measured. The measured value for the sample group was A1, the measured value for the background group was A2, and the measured value for the blank group was A0. The cholesterol binding rate was calculated according to the following formula.

[0179]

[0180] Pancreatic lipase activity inhibition rate: In the sample group, samples were added to the reaction substrate 4-nitrophenyl laurate solution, Tris-HCl buffer, and lipase solution, and reacted at 37°C for 2 hours. In the background group, samples were added to the reaction substrate 4-nitrophenyl laurate solution, Tris-HCl buffer, and lipase solution, and reacted at 37°C for 2 hours. In the blank group, ultrapure water was added to the reaction substrate 4-nitrophenyl laurate solution, Tris-HCl buffer, and lipase solution, and reacted at 37°C for 2 hours. After centrifugation, the supernatant was collected and the absorbance at OD410nm was measured. The measured value for the sample group was A1, the measured value for the background group was A2, and the measured value for the blank group was A0. The pancreatic lipase activity inhibition rate was calculated according to the following formula.

[0181]

[0182] Experimental data were analyzed using GraphPad Prism 8.0 software and expressed as mean ± SEM. T-test analysis showed that the cholesterol binding rate before and after fermentation was ****P < 0.0001; the pancreatic lipase activity inhibition rate before and after fermentation was ####P < 0.0001.

[0183] The results are as follows Figure 9 As shown, compared with the pre-fermentation (i.e., 7S protein solution), the 7S protein fermentation broth of Example 11 significantly improved the cholesterol binding rate and pancreatic lipase inhibition rate after fermentation, with the cholesterol binding rate increasing by 159.4% and the pancreatic lipase inhibition rate increasing by 410.3%.

[0184] Human experimental results of 5.7S protein fermentation broth

[0185] Ten subjects, aged 20 to 50 years, all had elevated cholesterol and triglycerides or elevated cholesterol and low-density lipoprotein cholesterol. Each subject took 200 mL of the 7S protein fermentation broth of Example 11 daily. During the trial, they followed their normal dietary habits. The duration of the trial was 20 days. General information: The subjects' mental state, diet, sleep patterns, urination and bowel movements, etc., were monitored during the trial. A routine blood test, liver and kidney function tests, and a four-component lipid profile were also performed. Tests were performed on Day 0, Day 5, and Day 20.

[0186] Blood routine, liver function, kidney function, and blood lipid tests: Fasting blood is drawn and sent to Foshan Dian Medical Laboratory for testing. For the four blood lipid tests, total cholesterol is measured using the CHOD-PAP method, triglycerides are measured using the GPO-PAP method, and high-density lipoprotein and low-density lipoprotein tests are performed using the direct homogeneous enzymatic method. Testing methods for other items are shown in Table 2.

[0187] The experimental data were analyzed using GraphPad Prism 8.0 software. The experimental data were expressed as mean ± SEM and analyzed using T-test.

[0188] like Figure 10 , Figure 11 and Figure 12 As shown, after 20 days of taking the 7S protein fermentation liquid, the subjects' total cholesterol, triglycerides, and low-density lipoprotein cholesterol all showed significant decreases. Six subjects with abnormally high total cholesterol saw a significant 15.5% decrease after taking the liquid, and three subjects returned to normal levels. Three subjects with abnormally high triglycerides saw a 30.9% decrease after taking the liquid, and three subjects with abnormally high low-density lipoprotein cholesterol saw a 12.5% ​​decrease after taking the liquid. The 7S protein fermentation liquid also had no effect on blood counts or liver and kidney function (Table 2).

[0189] Table 2 Effects of 7S protein fermentation broth on blood routine, liver and kidney function, and renal function

[0190]

[0191]

[0192] The above results show that the 7S protein fermentation broth provided by the present invention has a good lipid-lowering effect both in vivo and in vitro, in animals and humans, and has high safety.

[0193] 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, probiotics, a sweetener and a thickener, wherein the probiotics include at least one of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus helveticus and Lactobacillus paracasei.

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 2, 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, 10 to 70 parts of probiotics, 20 to 50 parts of sweetener and 2 to 8 parts of thickener.

4. The 7S protein fermentation broth according to any one of claims 1 to 3, characterized in that The probiotics include at least two of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus helveticus and Lactobacillus paracasei; Preferably, the probiotics are a combination of Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei.

5. The 7S protein fermentation broth according to claim 4, 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 7S protein and sugar with water and dissolving them to obtain a 7S protein solution; inoculating probiotics into the 7S protein solution and fermenting to obtain a fermentation solution; The fermentation solution is mixed with a sweetener and a thickener to obtain 7S protein fermentation liquid.

7. The preparation method according to claim 6, characterized in that The fermentation temperature is 30-50° C., and the fermentation time is 4-30 hours.

8. The preparation method according to claim 6, characterized in that The dissolution temperature is 100-110° C., and the dissolution time is 20-40 minutes.

9. Use of the 7S protein fermentation broth according to any one of claims 1 to 5 in any one of (1) to (3): (1) Preparation of drugs for lowering blood lipids; (2) Inhibit the activity of pancreatic lipase; (3) Prepare products that inhibit pancreatic lipase activity.

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

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