Method for preparing small-molecule active peptide through enzymolysis

Abstract

The invention provides a method for preparing small-molecule active peptide through enzymolysis, and belongs to the technical field of biology. The method comprises the following steps: mixing shiny-leaved yellowhorn meal with brown sugar water, sterilizing, and inoculating a complex microbial inoculant for fermentation; after the fermentation is finished, grinding the fermentation product to obtain ground pulp; adding a biological compound enzyme I into the ground slurry, and carrying out enzymolysis to obtain an enzymatic hydrolysate I; and adding a biological compound enzyme II into the enzymatic hydrolysate I for enzymolysis to obtain enzymatic hydrolysate II, and performing ultrafiltration on the enzymatic hydrolysate II by adopting a membrane with the molecular weight cutoff of 1000 Daltons to obtain the small-molecule active peptide. According to the method, the xanthoceras sorbifolia bunge meal can be fully decomposed into the small-molecule active peptide, the utilization rate of the xanthoceras sorbifolia bunge meal is increased, and meanwhile the xanthoceras sorbifolia bunge meal small-molecule active peptide with antioxidant activity is obtained.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing small molecule active peptides by enzymolysis. Background Art

[0002] Xanthoceras sorbifolia is a plant of the genus Xanthoceras in the family Sapindaceae. The kernel of Xanthoceras sorbifolia is an excellent protein resource. At present, the utilization of Xanthoceras sorbifolia kernels is mainly used as raw materials for oil extraction, and the cake produced by oil extraction is often directly discarded. However, the cake of Xanthoceras sorbifolia contains rich protein, and directly discarding it is a serious waste of resources.

[0003] Peptides have a variety of physiological regulatory functions, such as relieving physical fatigue, anti-oxidation, and enhancing hypoxia tolerance. Peptides have unique physiological activities and medical and health care effects that original proteins and monomeric amino acids do not have, and have the triple functions of nutrition, health care, and treatment. The molecular weight range between 50 and 2000 Daltons is called small peptides, oligopeptides, and oligopeptides, also known as small molecule active peptides. Small molecule active peptides are easy to absorb and have become a hot spot for research and development. Therefore, providing a method for obtaining small molecule active peptides using Xanthoceras sorbifolia meal is beneficial to improving the utilization rate of Xanthoceras sorbifolia. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preparing small molecule active peptides by enzymatic hydrolysis, which effectively avoids the waste of Xanthoceras sorbifolia resources and at the same time obtains small molecule active peptides with high antioxidant activity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A method for preparing small molecule active peptides by enzymatic hydrolysis comprises the following steps: sterilizing after mixing Xanthoceras sorbifolia meal with brown sugar water, inoculating a composite bacterial agent for fermentation; after the fermentation is completed, grinding the fermentation product to obtain grinding liquid; adding biological composite enzyme I to the grinding liquid for enzymatic hydrolysis to obtain enzymatic hydrolysis liquid I; adding biological composite enzyme II to the enzymatic hydrolysis liquid I for enzymatic hydrolysis to obtain enzymatic hydrolysis liquid II, and ultrafiltering the enzymatic hydrolysis liquid II using a membrane with a molecular weight cutoff of 1000 Daltons to obtain small molecule active peptides.

[0007] Preferably, the composite bacterial agent is composed of Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis in a weight ratio of 1-2:1-2:3-5.

[0008] Preferably, the inoculation amount of the composite bacterial agent is 8% to 12% of the mass of the mixture of Xanthoceras sorbifolia meal and brown sugar water.

[0009] Preferably, the fermentation temperature is 26-32°C.

[0010] Preferably, the fermentation time is 12 to 24 hours.

[0011] Preferably, the biological composite enzyme I is composed of lipase and cellulase in a weight ratio of 1:3-5.

[0012] Preferably, the added amount of the biological complex enzyme I is 1% to 3% of the mass of the grinding liquid.

[0013] Preferably, the biological composite enzyme II is composed of papain and bromelain in a weight ratio of 1:2 to 6.

[0014] Preferably, the added amount of the biological composite enzyme II is 0.5% to 1.5% of the mass of the enzymatic solution I.

[0015] The present invention also provides a small molecule active peptide, which is prepared by adopting the above-mentioned method for preparing the small molecule active peptide by enzymatic hydrolysis.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a method for preparing small-molecule active peptides by enzymatic hydrolysis. The method comprises the following steps: fermenting Xanthoceras sorbifolia meal by using a composite bacterial agent, and then enzymolyzing the fermentation product in stages by using an enzyme preparation, so that the Xanthoceras sorbifolia meal can be fully decomposed into small-molecule active peptides, thereby improving the utilization rate of the Xanthoceras sorbifolia meal and obtaining small-molecule active peptides from the Xanthoceras sorbifolia meal with antioxidant activity. DETAILED DESCRIPTION

[0018] The invention provides a method for preparing small-molecule active peptides by enzymolysis, comprising the following steps: sterilizing after mixing Xanthoceras sorbifolia meal with brown sugar water, inoculating a composite bacterial agent for fermentation; after the fermentation is completed, grinding the fermentation product to obtain grinding liquid; adding biological composite enzyme I to the grinding liquid for enzymolysis to obtain enzymolysis liquid I; adding biological composite enzyme II to the enzymolysis liquid I for enzymolysis to obtain enzymolysis liquid II, and ultrafiltering the enzymolysis liquid II by using a membrane with a molecular weight cutoff of 1000 Daltons to obtain small-molecule active peptides.

[0019] In the present invention, preferably, the Xanthoceras sorbifolia seed meal and brown sugar water are mixed at a material-liquid ratio of 1g:2-3mL, and the brown sugar water concentration is 5%-15%, and preferably the brown sugar water concentration is 8%-12%. The brown sugar water concentration in the present invention refers to the mass (g) of brown sugar in 100mL of brown sugar water, such as brown sugar water with a concentration of 5%-15%, that is, 100mL of brown sugar water contains 5-15g of brown sugar.

[0020] In the present invention, the composite bacterial agent is composed of plant lactobacillus, saccharomyces cerevisiae and bacillus subtilis in a weight ratio of 1-2:1-2:3-5, and the number of viable bacteria in the composite bacterial agent is ≥6.0×10 8 The microorganisms in the composite bacterial agent of the present invention can be obtained through common commercial channels.

[0021] In the present invention, the inoculation amount of the composite bacterial agent is 8% to 12% of the mass of the mixture of Xanthoceras sorbifolia meal and brown sugar water, preferably 9% to 11%, and more preferably 10%.

[0022] In the present invention, the fermentation temperature is 26-32° C., preferably 30° C.; the fermentation time is 12-24 h, preferably 15-20 h.

[0023] The invention mixes Xanthoceras sorbifolia soybean meal with brown sugar water and then ferments the mixture, which helps to produce a rich variety of small molecule active peptides, and can also improve the yield of small molecule active peptides and shorten the subsequent enzymolysis time.

[0024] After the fermentation of the present invention is completed, the fermentation product is ground using a colloid mill to make the fineness reach less than 200 μm to obtain a grinding liquid. A biological composite enzyme I is added to the grinding liquid, wherein the biological composite enzyme I is composed of lipase and cellulase in a weight ratio of 1:3 to 5, and preferably the lipase and cellulase are composed of a weight ratio of 1:4; the addition amount of the biological composite enzyme I is 1% to 3% of the mass of the grinding liquid, preferably 1.5% to 2.5% of the mass of the grinding liquid.

[0025] The present invention uses biocomplex enzyme I for enzymatic hydrolysis followed by centrifugation, wherein the centrifugal parameters are: rotation speed 4000-6000 rpm, centrifugal time 15-30 min, the rotation speed is preferably 4500-5500 rpm, and the centrifugal time is preferably 18-24 min; after the centrifugation, the supernatant is collected as enzymatic hydrolysate I.

[0026] The present invention adds a biological composite enzyme II to an enzymolysis solution I for enzymolysis, wherein the biological composite enzyme II is composed of papain and bromelain in a weight ratio of 1:2-6, and preferably papain and bromelain in a weight ratio of 1:3-5; the addition amount of the biological composite enzyme II is 0.5%-1.5% of the mass of the enzymolysis solution I, and preferably 0.8%-1.2% of the mass of the enzymolysis solution I. After the enzymolysis is completed, the enzymolysis solution II is obtained, and the enzymolysis solution II is ultrafiltered using a membrane with a molecular weight cutoff of 1000 Daltons to obtain small molecular active peptides.

[0027] The present invention uses biological composite enzyme II to further enzymatically hydrolyze the peptides in the enzymatic solution I into small molecular active peptides.

[0028] The present invention also provides a small molecule active peptide, which is prepared by adopting the above-mentioned method for preparing small molecule active peptide by enzymatic hydrolysis. The small molecule active peptide provided by the present invention has high antioxidant activity.

[0029] The present invention has no particular limitation on the sources of the raw materials, and commercially available products in the art may be used.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1

[0032] A method for preparing small molecule active peptides by enzymatic hydrolysis, comprising the following steps:

[0033] (1) Xanthoceras sorbifolia seed meal and brown sugar water were mixed at a material-liquid ratio of 1 g:2 mL, and the mixture was sterilized at 121° C. for 15 min. After sterilization, the mixture was cooled to 25° C. and inoculated with a composite bacterial agent, the inoculation amount of the composite bacterial agent being 10% of the mass of the mixture, and fermented at 30° C. for 15 h;

[0034] (2) After the fermentation is completed, the fermentation product is ground using a colloid mill to obtain a grinding liquid with a fineness of less than 200 μm; biological compound enzyme I is added to the grinding liquid for enzymatic hydrolysis, and the amount of biological compound enzyme I added is 2% of the mass of the grinding liquid. The enzymatic hydrolysis is performed for 40 minutes, and the temperature is raised to 90° C. after the enzymatic hydrolysis, and the enzyme is inactivated for 1 minute. The mixture is then centrifuged at 5000 rpm for 20 minutes, and the supernatant is collected to obtain enzymatic hydrolyzate I;

[0035] (3) Adding biocomplex enzyme II to the hydrolyzate I for enzymolysis, the amount of biocomplex enzyme II added is 1% of the mass of the hydrolyzate I, the enzymolysis is carried out for 1 hour, the temperature is raised to 100°C after the enzymolysis, the enzyme is inactivated for 1 minute, and the hydrolyzate II is obtained. The hydrolyzate II is ultrafiltered using a membrane with a molecular weight cutoff of 1000 Daltons to obtain small molecule active peptides.

[0036] In this embodiment:

[0037] Brown sugar water concentration: 10%;

[0038] Composite bacterial agent: Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis in a weight ratio of 1:1:3, with a viable cell count of ≥6.0×10 8 cfu / g;

[0039] Biocomplex enzyme I: lipase and cellulase in a weight ratio of 1:4, lipase activity is 100u / mg, cellulase activity is 10u / mg;

[0040] Biocomplex enzyme II: papain and bromelain are composed in a weight ratio of 1:5, with papain activity of 10u / mg and bromelain activity of 300u / mg.

[0041] Example 2

[0042] A method for preparing small molecule active peptides by enzymatic hydrolysis. The difference between this embodiment and embodiment 1 is that the composite bacterial agent is composed of Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis in a weight ratio of 2:2:5.

[0043] Example 3

[0044] A method for preparing small molecule active peptides by enzymatic hydrolysis, the difference between this embodiment and embodiment 1 is that: biological compound enzyme I is composed of lipase and cellulase in a weight ratio of 1:3;

[0045] Example 4

[0046] A method for preparing small molecule active peptides by enzymatic hydrolysis. The difference between this embodiment and embodiment 1 is that the biological compound enzyme I is composed of lipase and cellulase in a weight ratio of 1:5.

[0047] Example 5

[0048] A method for preparing small molecule active peptides by enzymatic hydrolysis. The difference between this embodiment and embodiment 1 is that the biological compound enzyme II is composed of papain and bromelain in a weight ratio of 1:3.

[0049] Example 6

[0050] A method for preparing small molecule active peptides by enzymatic hydrolysis. The difference between this embodiment and embodiment 1 is that the biological compound enzyme II is composed of papain and bromelain in a weight ratio of 1:6.

[0051] Comparative Example 1

[0052] The difference from Example 1 is that after the Xanthoceras sorbifolia meal and brown sugar water are mixed and sterilized, no fermentation treatment is performed, and steps (2) and (3) are directly performed.

[0053] Comparative Example 2

[0054] The difference from Example 1 is that step (2) is not performed.

[0055] Comparative Example 3

[0056] The difference from Example 1 is that after step (2), the enzymatic hydrolyzate I is ultrafiltered using a membrane with a molecular weight cutoff of 1000 Daltons to obtain small molecule active peptides, and step (3) is not performed.

[0057] Comparative Example 4

[0058] The difference from Example 1 is that the biological compound enzyme II is bromelain.

[0059] Comparative Example 5

[0060] The difference from Example 1 is that the biological composite enzyme I is composed of pectinase and cellulase in a weight ratio of 1:4, wherein the activity of pectinase is 30u / mg.

[0061] Experimental Example 1

[0062] Compare the DPPH free radical inhibition rates of different small molecule active peptides.

[0063] 0.5 mL of each of the small molecule active peptides prepared in Examples 1-6 and Comparative Examples 1-5 was taken into a colorimetric tube, and 1.5 mL of 1 mM DPPH solution was added respectively. The mixture was allowed to stand at room temperature in the dark for 30 min, and the absorbance value A was measured at 517 nm. 肽 , add 1.5 mL of 1 mM DPPH solution to 0.5 mL of distilled water, let stand at room temperature in the dark for 30 min, and the absorbance value A0 at 517 nm is the negative control value. The inhibition rate of DPPH is calculated as follows: Inhibition rate (%) = (A0-A 肽 ) / A0*100%. The results are shown in Table 1.

[0064] Table 1 DPPH free radical inhibition rate of different small molecule active peptides

[0065]

[0066] It can be seen from the results in Table 1 that the small molecule active peptide prepared by the present invention has antioxidant activity and can effectively inhibit DPPH free radicals.

[0067] The method provided by the present invention can decompose Xanthoceras sorbifolia meal into small molecule active peptides with a molecular weight lower than 1000 Daltons, thereby increasing the yield of small molecule active peptides and further increasing the utilization rate of Xanthoceras sorbifolia meal; the obtained small molecule active peptides have high antioxidant activity.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing small molecule active peptides by enzymatic hydrolysis, characterized in that: The method comprises the following steps: sterilizing the mixture of Xanthoceras sorbifolia meal and brown sugar water, inoculating a composite bacterial agent for fermentation; after the fermentation is completed, grinding the fermentation product to obtain grinding liquid; adding biological composite enzyme I to the grinding liquid for enzymolysis to obtain enzymolysis liquid I; adding biological composite enzyme II to the enzymolysis liquid I for enzymolysis to obtain enzymolysis liquid II, and ultrafiltering the enzymolysis liquid II with a membrane with a molecular weight cutoff of 1000 Daltons to obtain small molecule active peptides.

2. The method according to claim 1, characterized in that The composite bacterial agent is composed of plant lactobacillus, brewer's yeast and bacillus subtilis in a weight ratio of 1-2:1-2:3-5.

3. The method according to claim 1, characterized in that The inoculation amount of the composite bacterial agent is 8% to 12% of the mass of the mixture of Xanthoceras sorbifolia meal and brown sugar water.

4. The method according to claim 1, characterized in that The fermentation temperature is 26-32°C.

5. The method according to claim 1, characterized in that The fermentation time is 12 to 24 hours.

6. The method according to claim 1, characterized in that The biological composite enzyme I is composed of lipase and cellulase in a weight ratio of 1:3-5.

7. The method according to claim 1, characterized in that The added amount of the biological complex enzyme I is 1% to 3% of the mass of the grinding liquid.

8. The method according to claim 1, characterized in that: The biological compound enzyme II is composed of papain and bromelain in a weight ratio of 1:2-6.

9. The method according to claim 1, characterized in that: The added amount of the biological composite enzyme II is 0.5% to 1.5% of the mass of the enzymolysis solution I.

10. A small molecule active peptide, characterized in that: The method is prepared by any one of claims 1 to 9.

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