A method for preparing walnut peptide from walnut meal through microbial fermentation and walnut peptide

Abstract

The application discloses a method for preparing walnut peptide by fermenting walnut meal through microorganisms, and comprises the following steps: walnut meal is soaked in distilled water, and then high-pressure cooking is carried out; after the cooking is completed, the walnut meal is cooled; flour, aspergillus niger and aspergillus oryzae are added, and then uniform stirring and culture are carried out; uniform stirring is carried out again, and then the walnut meal is loaded into a solid fermentation tank; after sugar water is added, the walnut meal is continuously cultured; then bacillus subtilis is added for culture; saccharomyces cerevisiae is added for fermentation; then the material is distilled; the remaining material is dried; and finally, walnut peptide-containing raw materials are obtained. The four kinds of bacteria are used for fermentation in different stages, and the amount of the produced peptide is higher than that of the peptide produced by the currently reported fermentation methods, so that the method has the advantage of high efficiency. The method changes the traditional plant protein fermentation process in which salt water is added, and instead uses sugar water with the same osmotic pressure, so that the peptide is subsequently fermented by the yeast to produce alcohol from sugar, and other substrates in the fermentation process are effectively removed, which is beneficial to the production of high-purity peptide raw materials. The walnut peptide after fermentation has the function of regulating blood sugar.

Description

Technical Field

[0001] This invention belongs to the field of walnut peptide preparation technology, and more specifically relates to a method for preparing walnut peptides by microbial fermentation of walnut meal and the walnut peptides themselves. Background Technology

[0002] Walnut kernels are mainly composed of fat (60%–70%) and protein (15%–22%). As the second largest woody oilseed tree species, walnuts are distributed across 28 provinces in my country, producing 33,000 tons of walnut oil annually, serving as a new engine for addressing the current oilseed crisis in the country. In the process of walnut oil production, the high-value utilization of walnut meal has become another way to increase walnut income. Currently, there are two types of walnut meal: one containing the outer pericarp (hard shell) and the other without. Walnut meal containing the hard shell is currently discarded as waste, resulting in resource waste and environmental pollution. Walnut meal without the outer pericarp, containing the inner pericarp, has poor taste and color and is often used as animal feed, with very low added value. Walnut meal is mainly composed of walnut protein, which primarily consists of four proteins: glutenin, prolyl glutenin, albumin, and globulin, accounting for 70.11%, 5.33%, 17.57%, and 6.81% of the total, respectively. It is rich in essential amino acids needed by the human body, and its protein amino acid score is between 67.02 and 87.98. Walnut peptides obtained by enzymatic hydrolysis using walnut protein resources have antioxidant, ACE inhibitory, anti-hyperuric acid, anti-proliferative, neuroprotective and hypoglycemic activities.

[0003] There are several main methods for preparing bioactive peptides, including enzymatic hydrolysis, fermentation, and directed synthesis. Enzymatic hydrolysis offers mild, rapid, safe, and easily controllable conditions. Protein pretreatment can improve enzyme sensitivity, and enzymatic hydrolysis technology allows control over enzyme cleavage sites, ensuring a certain molecular weight of the product. Its limitations lie in the need to select suitable enzymes and determine the required time, temperature, pH, and other conditions, and its relatively high production cost. Directed synthesis includes liquid-phase methods, solid-phase methods, enzymatic synthesis, and DNA recombination. Liquid-phase methods are suitable for small peptide synthesis; DNA recombination improves the yield and purity of bioactive peptides. However, liquid-phase methods are not suitable for situations where the solubility of reaction intermediates is low; solid-phase synthesis is limited to 40–200 amino acids, and auxiliary agents used in the synthesis process often remain in the peptide product, affecting product quality.

[0004] Therefore, how to develop an economical and effective method for preparing walnut peptides from walnut meal through microbial fermentation that is beneficial to the healthy development of the walnut industry is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing walnut peptides by microbial fermentation of walnut meal and the walnut peptides themselves.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing walnut peptides by microbial fermentation of walnut meal includes the following steps:

[0008] After soaking walnut meal in distilled water for 30-35 hours, it was pressure-cooked at a pressure of 103.4-104.5 kPa, a temperature of 121±6℃, and a cooking time of 15±3 min. After cooking, it was cooled until the temperature dropped below 30℃. Then, flour, Aspergillus niger, and Aspergillus oryzae were added and stirred evenly. The mixture was then cultured at 30±2℃ for 50±3 h. After stirring evenly, it was placed in a solid fermentation tank. Sugar water was added and the mixture was cultured at 40±2℃ for 72±3 h. Then, Bacillus subtilis was added and the mixture was cultured at 36±1℃ for 96±3 h. Saccharomyces cerevisiae was added and the mixture was fermented at 40±3℃ for 96±3 h. The material was then distilled at 100±5℃. Distillation was stopped when the volume-to-mass ratio of the distillate to the walnut meal was (100±5):(100±5) mL / g. The remaining material was dried to obtain the raw material containing walnut peptides.

[0009] The mass ratio of the above-mentioned walnut meal, distilled water, flour, Aspergillus niger, Aspergillus oryzae, Bacillus subtilis and Saccharomyces cerevisiae is (100±5):(20±5):(0.5±0.3):(0.002±0.0008):(0.001±0.0008):(0.003±0.0006):(0.5±0.3);

[0010] The volume-to-mass ratio of the sugar water to the walnut meal was (100±5):(100±5)mL / g.

[0011] Furthermore, the concentration of sucrose in the above-mentioned sugar solution is 0.15-0.17 g / ml.

[0012] Furthermore, the remaining material is dried at a temperature of 105-110℃ for 24-36 hours.

[0013] Furthermore, the method also includes extracting the raw material containing walnut peptides with water at a material-to-liquid ratio of 1:1.2-1.5, an extraction temperature of 20-30℃, and an extraction time of 10-15 minutes. The extract is then freeze-dried under vacuum at a temperature of -45 to -50℃, a pressure of 100-110 Pa, and a time of 20-24 hours to obtain a raw material with a walnut peptide content of more than 50%.

[0014] The present invention also provides a method for preparing walnut peptides by microbial fermentation of walnut meal.

[0015] The beneficial effects of this invention are as follows: This invention utilizes four types of bacteria for fermentation at different stages, producing a higher amount of peptides than currently reported fermentation methods, demonstrating high efficiency. It changes the traditional technique of adding brine during plant protein fermentation, replacing it with sugar water of the same osmotic pressure. This allows the peptides to subsequently be fermented into alcohol by yeast, effectively removing other substrates during fermentation and facilitating the production of higher purity peptide raw materials. Animal experiments on the fermented walnut peptides showed a blood sugar regulating effect. Attached Figure Description

[0016] Figure 1 The graph shows the effect of walnut peptides on food intake in mice.

[0017] Figure 2 The graph shows the effect of walnut peptides on blood glucose levels in mice.

[0018] Figure 3 The graph shows the effect of walnut peptides on glucose tolerance in mice.

[0019] Figure 4 This is a graph showing the effect of walnut peptides on the area under the blood glucose curve in mice.

[0020] Figure 5 The graph shows the effect of walnut peptides on insulin tolerance in mice.

[0021] Figure 6 The figure shows the effect of walnut peptide on insulin tolerance in mice. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] In the examples, Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, and Saccharomyces cerevisiae were all purchased from the China Industrial Microbial Culture Collection Center: Aspergillus nyzae CICC2085; Aspergillus niger CICC2395; Bacillus subtilis CICC20519; and Saccharomyces cerevisiae CICC31294.

[0024] Example 1

[0025] A method for preparing walnut peptides by microbial fermentation of walnut meal includes the following steps:

[0026] Soak 100g of walnut meal in 20g of distilled water for 33 hours, then pressure cook at 104kPa, 121℃, and 15min. After cooking, cool the mixture until it reaches 28℃, then add 0.5g of flour, 0.002g of Aspergillus niger, and 0.001g of Aspergillus oryzae. Stir well and incubate at 30℃ for 50h. Stir well again, then transfer the mixture to a solid-state fermentation tank, add 100mL of sugar water, and continue incubating at 40℃ for 72 hours. The concentration of sucrose in the sugar water was 0.16 g / ml. Then, 0.003 g of Bacillus subtilis was added and cultured at 36℃ for 96 hours. Then, 0.5 g of Saccharomyces cerevisiae was added and fermented at 40℃ for 96 hours. Then, the material was distilled at 100℃. Distillation was stopped when the volume-to-mass ratio of the distillate to walnut meal was 100:100 mL / g. The remaining material was dried at 108℃ for 30 hours to obtain raw material containing walnut peptides.

[0027] The raw material containing walnut peptides was extracted with water at a material-to-liquid ratio of 1:1.3, an extraction temperature of 25℃, and an extraction time of 13 minutes. The extract was then freeze-dried under vacuum at a temperature of -48℃, a pressure of 105Pa, and a time of 22 hours to obtain a raw material with a walnut peptide content of 63%.

[0028] Example 2

[0029] A method for preparing walnut peptides by microbial fermentation of walnut meal includes the following steps:

[0030] Soak 95g of walnut meal in 15g of distilled water for 30 hours, then pressure cook at 103.4kPa, 115℃, and 12min. After cooking, cool the mixture until it reaches 27℃, then add 0.2g of flour, 0.0012g of Aspergillus niger, and 0.0002g of Aspergillus oryzae. Stir well and incubate at 28℃ for 47h. Stir well again, transfer to a solid-state fermentation tank, add 95mL of sugar water, and continue incubating at 38℃ for 6 hours. After 9 hours, the sucrose concentration in the sugar water was 0.15 g / ml. Then, 0.0024 g of Bacillus subtilis was added and cultured at 35℃ for 93 hours. 0.2 g of Saccharomyces cerevisiae was added and fermented at 37℃ for 93 hours. The material was then distilled at 95℃. Distillation was stopped when the volume-to-mass ratio of the distillate to walnut meal was 95:105 mL / g. The remaining material was dried at 105℃ for 24 hours to obtain raw material containing walnut peptides.

[0031] The raw material containing walnut peptides was extracted with water at a material-to-liquid ratio of 1:1.2, an extraction temperature of 20℃, and an extraction time of 10 minutes. The extract was then freeze-dried under vacuum at a temperature of -45℃, a pressure of 100Pa, and a time of 20 hours to obtain a raw material with a walnut peptide content of 59%.

[0032] Example 3

[0033] A method for preparing walnut peptides by microbial fermentation of walnut meal includes the following steps:

[0034] 105g of walnut meal was soaked in 25g of distilled water for 35 hours, then pressure-cooked at 104.5kPa, 127℃, and for 18 minutes. After cooking, the mixture was cooled to 29℃, and then 0.8g of flour, 0.0028g of Aspergillus niger, and 0.0018g of Aspergillus oryzae were added. The mixture was stirred evenly and then incubated at 32℃ for 53 hours. After stirring evenly, the mixture was transferred to a solid-state fermentation tank, 105mL of sugar water was added, and the fermentation continued at 42℃ for 7 hours. After 5 hours, the sucrose concentration in the sugar water was 0.17 g / ml. Then, 0.0036 g of Bacillus subtilis was added and cultured at 37°C for 99 hours. 0.8 g of Saccharomyces cerevisiae was added and fermented at 43°C for 99 hours. The material was then distilled at 105°C. Distillation was stopped when the volume-to-mass ratio of the distillate to walnut meal was 105:95 mL / g. The remaining material was dried at 110°C for 36 hours to obtain the raw material containing walnut peptides.

[0035] The raw material containing walnut peptides was extracted with water at a material-to-liquid ratio of 1:1.5, an extraction temperature of 30℃, and an extraction time of 15 minutes. The extract was then freeze-dried under vacuum at a temperature of -50℃, a pressure of 110Pa, and a time of 24 hours to obtain a raw material with a walnut peptide content of 70%.

[0036] Animal experiments

[0037] The raw material with a walnut peptide content of 63% in Example 1 was divided into low, medium and high dose groups.

[0038] 1. Effects of walnut peptides on food intake and body weight changes in mice

[0039] Table 1. Effects of walnut peptides on the body weight of experimental animals.

[0040]

[0041] Note: For statistically significant differences, P < 0.05.

[0042] Table 1 shows that there was no significant difference in mouse body weight among the groups during the gavage period, and the weight gain was stable. However, over the five weeks of gavage, significant differences emerged within each group as the gavage duration increased. Figure 1 As shown, the food intake of mice in each group was similar before modeling. During the 5-week experimental period after modeling, the food intake of mice in each group showed an upward trend. The food intake of mice that were given gavage with walnut peptides gradually stabilized.

[0043] 2. Effects of walnut peptides on fasting blood glucose (FBG) changes in mice.

[0044] After successful model establishment, the mice underwent a 5-week experiment.

[0045] like Figure 2 As shown, after successful modeling, the blood glucose level of diabetic mice was >11.1 mol / L. The blood glucose level of the blank group was lower than that of the model group, but its blood glucose level was >8, which is considered to be a stress response in the mice. In the first week, the blood glucose levels of mice in all groups decreased to varying degrees. Among them, the blood glucose level of mice in the low-dose group tended to normal levels, while the blood glucose level of the dimethyl biguanide hydrochloride group increased. Considering that glimepiride promotes insulin secretion, it can be determined that the mice have type II diabetes caused by insufficient insulin secretion. With continued gavage, except for the model group, the blood glucose levels of mice in other groups showed a trend of first rising and then falling. This indicates that walnut peptides help control blood glucose.

[0046] 3. Effects of walnut peptides on glucose tolerance in mice

[0047] Mice underwent an oral glucose tolerance test after 5 weeks of feeding. Blood glucose levels were measured after 12 hours of fasting and at 0, 0.5, 1, and 2 hours after gavage administration of glucose. Figure 3It was found that before glucose administration, i.e., after fasting for 12 hours, the blood glucose levels in the model group mice were significantly higher than those in other groups. The differences between the blank control group and the metformin group and the other three groups were significant (P<0.05). Thirty minutes after glucose administration, blood glucose levels in all groups significantly increased, reaching peak values. The model group had the highest blood glucose level (33.3 mg / mL), the metformin group showed a slower rate of increase, and the low-dose group had the lowest (13.1 mg / mL). Considering that metformin's main function is to improve insulin resistance, this result indicates that metformin has good blood glucose regulation. There was no significant difference between the walnut peptide group and the metformin group (P<0.05), indicating that walnut peptide also has a good effect on blood glucose regulation. The negative control group and the metformin group showed significant differences (P<0.05). One hour after glucose administration, the blood glucose levels in the negative control group were significantly different (P<0.05), while the walnut peptide group and the metformin group were significantly lower than those in the model group (P<0.05), and blood glucose remained relatively stable. Two hours later, the blood glucose levels in the negative control group, dimethyl biguanide group, low-dose group, medium-dose group, and high-dose group were significantly lower than those in the model group (P<0.05).

[0048] Depend on Figure 4 The results showed that the areas under the curve (AUC) of the blank walnut peptide group, model walnut peptide group, model metformin group, and model glimepiride group were significantly lower than those of the model group (P<0.05), indicating that all groups improved glucose tolerance in mice. Among them, the walnut peptide group and the metformin group showed significantly higher effects than the other two groups (P<0.05). This indicates that walnut peptide can effectively inhibit the rapid rise in postprandial blood glucose.

[0049] 4. Effects of walnut peptides on insulin tolerance in mice

[0050] The insulin tolerance test can determine the body's sensitivity to insulin based on changes in blood glucose levels. Figure 5-6 The changes in blood glucose levels after subcutaneous injection of insulin in each group of mice were recorded.

[0051] like Figure 5 As shown in the insulin tolerance curves, after 4 hours of fasting, the blood glucose level in the model group was significantly higher than that in the other groups (P<0.05), and the medium-dose group was higher than the other four groups. Thirty minutes after subcutaneous insulin injection, blood glucose levels decreased in all groups. The medium-dose group showed a faster rate of decrease, while the model group showed a slower rate of decrease and significantly higher blood glucose levels than the other groups (P<0.05). After 1 hour, the model group was significantly higher than the other groups (P<0.05). After 2 hours, blood glucose levels in all groups essentially returned to their original levels. Figure 6The percentage decrease curve provides a more intuitive observation of the rate of blood glucose decline. After insulin injection, the blood glucose levels of mice in each group first decreased rapidly and then rose slowly. The rate of blood glucose decline reflects the mice's sensitivity to insulin. The blood glucose levels of the mice in the walnut peptide group remained within the normal range throughout the experiment, indicating that the mice fed with walnut peptides had enhanced insulin regulation capabilities and were less affected by sudden changes in insulin dosage.

[0052] The residual protein in walnut meal was broken down into peptides through microbial fermentation. This method leverages the rapid reproduction of microorganisms to obtain more peptides. The extracted crude walnut peptides were purified by dextran G-50 gel electrophoresis and high-performance liquid chromatography, yielding six different components. In vitro antioxidant and hypoglycemic activities of these components were measured, yielding good results. In the antioxidant activity assay, component 5 showed better performance than the other components, exhibiting higher scavenging rates of 84%, 93%, and 83% for DPPH, ABTS, and hydroxyl radicals, respectively. In the hypoglycemic activity assay, component 4 showed the best activity, inhibiting DPP-IV, α-amylase, and glucosidase at 92%, 29%, and 76%, respectively. The molecular weight of component 4 ranged from 3360 to 6160, while that of component 5 ranged from 1400 to 3360. Finally, animal experiments were conducted to further verify the hypoglycemic activity of the walnut active peptides, with different groups used to demonstrate the activity. By measuring food intake, body weight, blood glucose level, glucose tolerance, and insulin tolerance, it was found that walnut peptides have a certain effect on lowering blood sugar.

[0053] The description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing walnut peptide from walnut meal by microbial fermentation, characterized in that, The method comprises the following steps: After the walnut meal is soaked in distilled water for 30-35 hours, high-pressure cooking is performed at a pressure of 103.4-104.5 kPa and a temperature of 121±6℃ for 15±3 min. After the cooking is completed and the temperature is reduced to below 30℃, flour, Aspergillus niger and Aspergillus oryzae are added, and the mixture is stirred and incubated at a temperature of 30±2℃ for 50±3 h. Then, the mixture is stirred and loaded into a solid fermentation tank. After the addition of sugar water, the mixture is further incubated at a temperature of 40±2℃ for 72±3 h. Then, Bacillus subtilis is added, and the mixture is incubated at a temperature of 36±1℃ for 96±3 h. Then, Saccharomyces cerevisiae is added, and the mixture is fermented at a temperature of 40±3℃ for 96±3 h. Then, the mixture is distilled at a temperature of 100±5℃ until the volume-to-mass ratio of the distilled liquid to the walnut meal is (100±5):(100±5) mL / g. The remaining mixture is dried to obtain a raw material containing walnut peptides. The mass ratio of the walnut meal, the distilled water, the flour, the Aspergillus niger, the Aspergillus oryzae, the Bacillus subtilis and the Saccharomyces cerevisiae is (100±5):(20±5):(0.5±0.3):(0.002±0.0008):(0.001±0.0008):(0.003±0.0006):(0.5±0.3). The volume-to-mass ratio of the sugar water to the walnut meal is (100±5):(100±5) mL / g.

2. The method for preparing walnut peptides by microbial fermentation of walnut meal according to claim 1, characterized in that, The concentration of sucrose in the sugar water is 0.15-0.17 g / ml.

3. The method for preparing walnut peptides by microbial fermentation of walnut meal according to claim 1, characterized in that, The remaining mixture is dried at a temperature of 105-110℃ for 24-36 h. 4.The method of claim 1, wherein the walnut meal is fermented by the microorganism to prepare the walnut peptide. The raw material containing walnut peptides is extracted with water at a solid-to-liquid ratio of 1:1.2-1.5, an extraction temperature of 20-30℃ and an extraction time of 10-15 min. The extraction liquid is vacuum freeze-dried at a temperature of -45 to -50℃, a pressure of 100-110 Pa and for a time of 20-24 h to obtain a raw material containing walnut peptides at a content of 50% or more.

5. A walnut peptide prepared by the method of any one of claims 1-4.

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