CN115011660A

CN115011660A - Metabolism promoting and oxidation resisting oat protein peptide and preparation method and application thereof

Info

Publication number: CN115011660A
Application number: CN202210946529.7A
Authority: CN
Inventors: 张光明; 董洪波; 赵杰; 王颖; 张琦峰; 万宏颖; 洪伟雄
Original assignee: Beijing Happy Energy Health Technology Co ltd
Current assignee: Beijing Happy Energy Health Technology Co ltd
Priority date: 2022-08-09
Filing date: 2022-08-09
Publication date: 2022-09-06
Anticipated expiration: 2042-08-09
Also published as: CN115011660B

Abstract

The invention provides an oat protein peptide with the functions of promoting metabolism and resisting oxidation, and a preparation method and application thereof, and belongs to the technical field of protein peptides. The method comprises the steps of crushing oat, extracting by using an organic solvent to obtain oat protein, carrying out primary enzymolysis by using pepsin and acid protease, carrying out secondary enzymolysis by using neutral protease and papain, carrying out tertiary enzymolysis by using alkaline protease and flavourzyme to obtain a tertiary oat protein enzymolysis liquid, fermenting by using lactobacillus plantarum and bacillus coagulans to obtain a primary oat protein fermentation product, and fermenting by using selenium-enriched yeast to obtain the metabolism promoting and oxidation resisting oat protein peptide. The product prepared by the invention contains rich short peptides, oligopeptides, polypeptides and oat protein peptide-selenium chelate, has excellent effects of promoting metabolism, resisting oxidation, diminishing inflammation, resisting bacteria, enhancing immunity, reducing blood pressure, blood fat and blood sugar, losing weight and fat and controlling body weight, is simple in preparation method, has good effect, and can be applied to preparation of products for losing weight and controlling body weight.

Description

Metabolism promoting and oxidation resisting oat protein peptide and preparation method and application thereof

Technical Field

The invention relates to the technical field of protein peptides, in particular to oat protein peptide capable of promoting metabolism and resisting oxidation, and a preparation method and application thereof.

Background

The peptide is formed by dehydration condensation of 2 or more than 2 amino acids, is a fragment of a protein mechanism and function, enables the protein to have various physiological functions, and has strong special physiological functions. The peptide is formed by dehydration condensation of 2 or 3 amino acids, which are respectively dipeptide and tripeptide, and so on, tetrapeptide, pentapeptide and the like. Generally, the number of amino acids in a peptide chain is 10 or less, 10 to 50 are polypeptides, and 50 or more are proteins. The polypeptide is an active substance and a nutrient substance which exist in the human body and are necessary, is almost widely distributed in the whole body of animals and human beings, particularly the brain, and has a regulating effect on used cells, such as deletion, and disorder of an immune system and various functional systems can occur.

The traditional protein extraction method adopts an alkali dissolution and acid precipitation method, obtains separated protein, and then prepares hydrolyzed protein peptide through hydrolysis of an enzyme preparation, and the production period is long; the environment is not friendly: a large amount of acid and alkali are used in the industrial production process, so that environmental pollution to a certain degree can be caused; large amount of waste water: the traditional industrial production method has the disadvantages of large wastewater discharge amount, high salt content, large wastewater treatment difficulty and high operation cost. Meanwhile, plant polypeptide products in the market have large average molecular weight, the ratio of molecular weight in a specific interval is not controllable, and the action mechanisms and the action effects of polypeptides with different molecular weights are different, so that the controllability of the functions of the polypeptides is poor.

Chinese patent CN103243144B discloses collagen powder rich in collagen tripeptide and a preparation process thereof. The invention aims to provide collagen powder rich in collagen tripeptide and a preparation process of the collagen powder. The fish skin with low fat content is selected as the raw material, and the method omits the soaking in dilute alkali solution to remove fat in the preparation process, thereby reducing harmful substances generated by acid-base treatment; in addition, after the fish skin is subjected to enzymolysis by adopting protease and flavor enzyme sold in the market, part of protein peptide is further degraded into collagen tripeptide by adopting self-made collagenase, so that not only can the extraction rate of collagen in the raw material be improved, but also the functionality of the collagen powder is obviously improved

Japanese patent laid-open publication No. 2005-80668 discloses: provided is a soybean protein hydrolysis method which hydrolyzes soybean protein using an enzyme having both endo-and exo-peptidase activities, thereby increasing the soluble fraction, avoiding the bitter fraction, and enhancing or improving the antioxidant ability. However, the soluble soy protein fraction described in this patent contains about 3kDa to 30kDa peptides and is substantially free of low molecular weight soy proteins. As is not described in this patent, in soy proteins, dipeptides and tripeptides are best absorbed in the intestinal tract, and larger peptides are often difficult to absorb directly. Therefore, the peptide described in this patent is too large to be absorbed and is not considered to be practical because it does not exhibit a significant action in vivo. Furthermore, the peptide described in Japanese patent application laid-open No. 10-203994 also has a size of 500-5000A, and is considered to be impractical because it is too large as compared with the dipeptide and tripeptide.

Disclosure of Invention

The oat protein peptide contains rich short peptides, oligopeptides, polypeptides and oat protein peptide-selenium chelate, has excellent effects of promoting metabolism, resisting oxidation, diminishing inflammation, resisting bacteria, enhancing immunity, reducing blood pressure, blood fat, blood sugar, weight and fat and controlling body weight, is simple in preparation method, has a good effect, and can be applied to preparation of products for weight reduction and weight control.

The technical scheme of the invention is realized as follows:

the invention provides a preparation method of oat protein peptide for promoting metabolism and resisting oxidation, which comprises the steps of crushing oat, extracting by using an organic solvent to obtain oat protein, carrying out primary enzymolysis by using pepsin and acid protease, carrying out secondary enzymolysis by using neutral protease and papain, carrying out tertiary enzymolysis by using alkaline protease and flavourzyme to obtain a tertiary enzymolysis liquid of the oat protein, fermenting by using lactobacillus plantarum and bacillus coagulans to obtain a primary fermentation product of the oat protein, and fermenting by using selenium-enriched yeast to obtain the oat protein peptide for promoting metabolism and resisting oxidation.

As a further improvement of the invention, the method comprises the following steps:

s1, extracting oat protein: cleaning oat, drying, crushing, sieving to obtain oat powder, adding the oat powder into trichloroacetic acid-acetone-acetonitrile solution, uniformly mixing, precipitating for a first time period, centrifuging, adding insoluble substances into the acetone-acetonitrile solution, uniformly mixing, precipitating for a second time period, centrifuging, washing the insoluble substances, drying, adding the insoluble substances into a lysate for cracking, taking supernatant, and concentrating by using an ultrafiltration membrane to obtain an oat protein extracting solution;

s2, primary enzymolysis: adjusting the pH value of the oat protein extract prepared in the step S1 to 2.5-3.5, heating to the temperature of 35-40 ℃, adding pepsin and acid protease, performing enzymolysis, inactivating enzymes, and filtering to obtain a filtrate, namely an oat protein first-level enzymolysis liquid;

s3, secondary enzymolysis: adjusting the pH value of the first-stage oat protein enzymolysis liquid prepared in the step S2 to 6.5-7, heating to 50-55 ℃, adding neutral protease and papain, performing enzymolysis, inactivating enzymes, and filtering to obtain a filtrate, namely a second-stage oat protein enzymolysis liquid;

s4, three-stage enzymolysis: adjusting the pH value of the filtrate obtained in the step S3 to be the second-level enzymatic hydrolysate of the oat protein to be 8-9, heating to the temperature of 45-55 ℃, adding alkaline protease and flavourzyme, carrying out enzymolysis, enzyme deactivation and filtration, wherein the filtrate is the third-level enzymatic hydrolysate of the oat protein;

s5, activating strains: respectively inoculating lactobacillus plantarum, bacillus coagulans and selenium-enriched yeast to a Gauss culture medium, culturing under a micro-anoxic condition, and respectively culturing into strain seed solutions;

s6, primary fermentation: inoculating strain seed solutions of lactobacillus plantarum and bacillus coagulans to the oat protein three-level enzymatic hydrolysate prepared in the step S4, and performing fermentation culture under a micro-anoxic condition to obtain an oat protein first-level fermentation product;

s7, secondary fermentation: inoculating the selenium-enriched yeast strain seed liquid to the oat protein primary fermentation product prepared in the step S6, adding a solution containing calcium ions, carrying out fermentation culture under the condition of hypoxia for a third time period, then adding a vitamin solution, continuing fermentation culture for a fourth time period, filtering by a microfiltration membrane, separating and removing non-hydrolyzed insoluble components which cannot penetrate through the membrane, then concentrating by a nanofiltration membrane, and carrying out freeze drying to prepare the metabolism promoting and oxidation resisting oat protein peptide.

In step S7, the stress resistance of the selenium-enriched yeast is improved and the fermentation time of the selenium-enriched yeast is prolonged by adding the calcium ion solution, and after the selenium-enriched yeast is fermented for a period of time, the vitamin solution is added to prolong the stabilization period of the selenium-enriched yeast, so that a large amount of beneficial products including oat protein peptide-selenium chelate can be produced, and the yield of the product is improved.

As a further improvement of the invention, the solid-to-liquid ratio of the oat flour and the trichloroacetic acid-acetone-acetonitrile solution in the step S1 is 1: 3-5 g/mL; trichloroacetic acid in the trichloroacetic acid-acetone-acetonitrile solution: acetone: the volume ratio of the acetonitrile is 0.5-1: 7-12: 3-5; the solid-liquid ratio of the insoluble substances to the acetone-acetonitrile solution is 1:2-4g/mL, and the ratio of acetone in the acetone-acetonitrile solution is as follows: the volume ratio of the acetonitrile is 3-5: 4-7; the first time period is 0.5-1.5 h; the second time period is 1-2 h; the lysis solution is an aqueous solution containing 3-5mol/L urea, 1-2mol/L thiourea and 0.5-1wt% dithiothreitol; the cracking time is 2-4 h; the solid-liquid ratio of the insoluble substance to the lysate is 1:2-5 g/mL; the pore diameter of the ultrafiltration membrane is 3000-5000D.

As a further improvement of the invention, the mass ratio of the oat protein extract, the pepsin and the acid protease in the step S2 is 100:1-5: 3-7; the enzymolysis time is 1-3 h; in the step S3, the mass ratio of the oat protein primary enzymolysis liquid to the neutral protease to the papain is 100:2-5:3-6, and the enzymolysis time is 2-4 h; in the step S4, the mass ratio of the oat protein secondary enzymolysis liquid to the neutral protease to the papain is 100:3-5:1-3, and the enzymolysis time is 0.5-1.5 h.

As a further improvement of the invention, the micro-anoxic condition comprises 5-12% of oxygen, 3-5% of carbon dioxide and the balance of nitrogen, wherein the percentage is volume percentage; the enzyme deactivation method comprises 1000-1500W ultrasonic treatment for 15-20 min.

As a further improvement of the invention, the culture temperature in the step S5 is 35-40 ℃, and the culture time is 18-24h, soThe strain seed liquid has a bacteria content of 10 ⁷ -10 ⁹ cfu/mL; in the step S6, the inoculation amounts of the lactobacillus plantarum and the bacillus coagulans are respectively 2-5% and 1-3%; the fermentation culture condition is that the temperature is 36-38 deg.C, and the time is 24-36 h.

As a further improvement of the invention, the inoculation amount of the selenium-enriched yeast in the step S7 is 3-5%, and the volume ratio of the oat protein primary fermentation product, the solution containing calcium ions and the vitamin solution is 10: 1-2: 0.5 to 1; the calcium ion concentration in the solution containing calcium ions is 5-12wt%, and the vitamin solution is an aqueous solution containing 2-5wt% of vitamin B1 and 1-3wt% of vitamin B12; the fermentation culture condition is 36-38 ℃, and the third time period is 12-18 h; the fourth time period is 18-24 h; the aperture range of the micro-filtration membrane is 0.1-0.5 mu m, and the aperture range of the nano-filtration membrane is not more than 2 nm.

As a further improvement of the invention, the method specifically comprises the following steps:

s1, extracting oat protein: washing oat, drying, crushing and sieving to obtain oat powder, adding the oat powder into trichloroacetic acid-acetone-acetonitrile solution, wherein the solid-to-liquid ratio of the oat powder to the trichloroacetic acid-acetone-acetonitrile solution is 1: 3-5g/mL, wherein the ratio of trichloroacetic acid in the trichloroacetic acid-acetone-acetonitrile solution is as follows: acetone: the volume ratio of acetonitrile is 0.5-1: 7-12:3-5, uniformly mixing, precipitating for 0.5-1.5h, centrifuging, adding an insoluble substance into an acetone-acetonitrile solution, wherein the solid-to-liquid ratio of the insoluble substance to the acetone-acetonitrile solution is 1:2-4g/mL, and the mass ratio of acetone in the acetone-acetonitrile solution is as follows: the volume ratio of acetonitrile is 3-5:4-7, uniformly mixing, precipitating for 1-2h, centrifuging, washing insoluble substances, drying, adding the dried insoluble substances into a lysate for cracking for 2-4h, wherein the solid-to-liquid ratio of the insoluble substances to the lysate is 1:2-5g/mL, the lysate is an aqueous solution containing 3-5mol/L urea, 1-2mol/L thiourea and 0.5-1wt% dithiothreitol, taking supernatant, and concentrating through an ultrafiltration membrane with the pore diameter of 3000-;

s2, primary enzymolysis: adjusting the pH value of 100 parts by weight of the oat protein extract prepared in the step S1 to 2.5-3.5, heating to the temperature of 35-40 ℃, adding 1-5 parts by weight of pepsin and 3-7 parts by weight of acid protease, performing enzymolysis for 1-3h, inactivating enzymes, and filtering to obtain a filtrate, namely an oat protein first-stage enzymolysis liquid;

s3, secondary enzymolysis: adjusting the pH value of 100 parts by weight of the first-stage enzymatic hydrolysate of the oat protein prepared in the step S2 to 6.5-7, heating to 50-55 ℃, adding 2-5 parts by weight of neutral protease and 3-6 parts by weight of papain, carrying out enzymolysis for 2-4h, inactivating enzyme, and filtering to obtain filtrate which is a second-stage enzymatic hydrolysate of the oat protein;

s4, three-stage enzymolysis: adjusting the pH value of 100 parts by weight of the filtrate obtained in the step S3 to 8-9 of the oat protein secondary enzymolysis liquid, heating to 45-55 ℃, adding 3-5 parts by weight of alkaline protease and 1-3 parts by weight of flavor protease, carrying out enzymolysis for 0.5-1.5h, inactivating enzymes, filtering, and obtaining the filtrate which is the oat protein tertiary enzymolysis liquid;

s5, activating strains: inoculating Lactobacillus plantarum, Bacillus coagulans and selenium-rich yeast to Gao's medium, culturing at 35-40 deg.C under anoxic condition for 18-24 hr, respectively, to obtain strain seed solution with a bacteria content of 10 ⁷ -10 ⁹ cfu/mL；

S6, primary fermentation: inoculating strain seed solutions of lactobacillus plantarum and bacillus coagulans to the oat protein three-level enzymolysis solution prepared in the step S4, wherein the inoculation amounts of the lactobacillus plantarum and the bacillus coagulans are respectively 2-5% and 1-3%, and performing fermentation culture for 24-36h at the temperature of 36-38 ℃ under the condition of micro-hypoxia to obtain an oat protein first-level fermentation product;

s7, secondary fermentation: inoculating selenium-enriched yeast strain seed liquid to 10 volume parts of the oat protein primary fermentation product prepared in the step S6, wherein the inoculation amount of the selenium-enriched yeast is 3-5%, adding 1-2 volume parts of a solution containing 5-12wt% of calcium ions, performing fermentation culture at 36-38 ℃ for 12-18h under the condition of micro-hypoxia, then adding 0.5-1 volume part of a vitamin solution, wherein the vitamin solution is an aqueous solution containing 2-5wt% of vitamin B1 and 1-3wt% of vitamin B12, continuing fermentation culture at 36-38 ℃ for 18-24h, filtering through a microfiltration membrane with the pore size of 0.1-0.5 mu m, separating and removing non-hydrolyzed insoluble components which cannot permeate through the membrane, then concentrating through a nanofiltration membrane with the pore size of not more than 2nm, freeze-drying, preparing oat protein peptide with metabolism promoting and antioxidant effects;

the micro-anoxic condition comprises 5-12% of oxygen, 3-5% of carbon dioxide and the balance of nitrogen, wherein the volume percentage is;

the enzyme deactivation method comprises 1000-1500W ultrasonic treatment for 15-20 min.

The invention further protects the oat protein peptide with the functions of promoting metabolism and resisting oxidation, which is prepared by the preparation method.

The invention further protects the application of the oat protein peptide for promoting metabolism and resisting oxidation in preparing products for losing weight and controlling body weight.

The invention has the following beneficial effects: the oat flour is directly extracted by trichloroacetic acid-acetone-acetonitrile solution/acetone-acetonitrile solution for the second time, the trichloroacetic acid-acetone-acetonitrile solution is adopted to replace the traditional trichloroacetic acid-acetone solution, the protein extraction rate is higher, nitrogen elements on cyano groups contained in the adopted acetonitrile are easy to form hydrogen bond with carboxylic acid structures of protein amino acids, so that the protein in the oat flour is promoted to be extracted and precipitated in the presence of acetone, the extraction time is short, the repetition rate is high, the influence of metabolic substances in a sample can be reduced, and the protein loss can be reduced;

further, the prepared oat protein extract is respectively subjected to enzymolysis under acidic, neutral and alkaline conditions, and is subjected to enzymolysis by pepsin and acidic protease which are suitable for acidity under the acidic condition, the proportion of small-molecule peptides in hydrolysate is very high, and the produced polypeptides are almost all small-molecule peptides; under neutral conditions, the enzymolysis is carried out by neutral protease suitable for neutral and papain, the neutral protease is a metal protease, the peptide bond between leucine and phenylalanine is preferentially cut, the papain can decompose the peptide bond in a hydrophobic region, including threonine, tryptophan, phenylalanine and the like, the hydrolysis degree of the enzymolysis product is high, the antioxidant activity is high, under alkaline conditions, the enzymolysis is carried out by alkaline protease suitable for alkaline and flavourzyme, the alkaline protease can decompose the peptide bond, including phenylalanine, tyrosine, tryptophan and lysine carboxyl, and the flavourzyme can preferentially break the peptide bond between leucine and proline or between proline and proline; the enzymolysis speed is high, the enzymolysis effect is good, the yield of hydrolysate small peptides is high, the polypeptide after enzymolysis has higher emulsification stability, after three-stage enzymolysis, most of oat protein in the oat protein extracting solution is enzymolyzed into small molecular short peptides and oligopeptide substances, and the oat protein extracting solution has excellent antioxidant activity;

the prepared third-level enzymolysis liquid of the oat protein also comprises a plurality of long-chain protein molecules and unhydrolyzed protein, lactobacillus plantarum and bacillus coagulans are further added for fermentation, probiotics further ferment and hydrolyze the protein molecules, and most of the unhydrolyzed long-chain protein is hydrolyzed into amino acid, short peptide, oligopeptide or polypeptide substances, so that a first-level fermentation product of the oat protein is prepared;

then, adding selenium-enriched yeast into the oat protein primary fermentation product, further performing enzymolysis, and simultaneously chelating the product polypeptide with selenium mineral elements in organic selenium substances to form oat protein peptide-selenium chelate, wherein the oat protein peptide-selenium chelate is formed by mutual coordination between the selenium element and N-terminal amino, amino acid side chain, C-terminal carboxyl and carbonyl and imino in a peptide chain in the oat protein peptide. Compared with a chelate formed by the interaction of simple amino acid and selenium element, the chelate of the oat protein peptide and the selenium element has better stability and matching rate, has the advantages of high biological benefit, excellent absorption effect, excellent nutrition and the like, and can combine the complementary functional activities of the oat protein peptide and the selenium element, such as metabolism promotion, oxidation resistance, inflammation diminishing, bacteria resistance, immunity enhancement, blood pressure reduction, blood fat reduction, blood sugar reduction, weight losing, fat reduction, weight control and the like.

The oat protein peptide with the functions of promoting metabolism and resisting oxidation, which is prepared by the method disclosed by the invention, comprises an oat protein peptide-selenium chelate, and the oat protein peptide has an excellent anti-oxidation effect after detection, wherein most of the oat protein peptide is short peptide and oligopeptide substances with low molecular weight, and the short peptide and oligopeptide substances with low molecular weight are more easily close to free radicals than bulky parent proteins of the oat protein peptide and oligopeptide substances, and can inhibit peroxidation mediated by the free radicals. The short peptide and oligopeptide substances provide hydrogen atoms or electrons through peptide bonds and hydroxyl groups of the substances, and free radicals lacking hydrogen ions or electrons are eliminated, so that the destructive effect on biological molecules is eliminated; at the same time, the formation of ROS and free radicals can be inhibited by inhibiting certain pro-oxidases and chelating transition metal ions involved in catalyzing the generation of free radicals. The short peptide and oligopeptide substances can also inhibit the conversion of free radicals into hydrogen peroxide, and can be decomposed into harmless metabolites such as water and oxygen by endogenous antioxidants (including superoxide dismutase, catalase and glutathione), thereby having high antioxidant effect.

The oat protein peptide for promoting metabolism and resisting oxidation contains rich short peptide, oligopeptide, polypeptide and oat protein peptide-selenium chelate, has excellent effects of promoting metabolism, resisting oxidation, diminishing inflammation, resisting bacteria, enhancing immunity, reducing blood pressure, reducing blood fat, reducing blood sugar, losing weight and fat and controlling body weight, has a simple preparation method, has a good effect, and can be applied to preparation of products for losing weight and controlling body weight.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a graph showing the change of body weight with time in each group of mice in test example 4 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The pepsin is provided by Cangzhou summer enzyme biotechnology limited, 10 ten thousand U/g, the optimum pH value is 2.0-3.0, and the optimum temperature is 30-50 ℃. The acidic protease is provided by bioscience and technology Limited liability company of Henghuadao, Dongning, 3 ten thousand U/g, the optimum pH value is 2.5-3.5, and the optimum temperature is 30-40 ℃. The neutral protease is provided by Nanning Dong Henghuadao biological science and technology Limited liability company, and has an optimum pH value of 20 ten thousand U/g, an optimum pH value of 6.5-7.5, and an optimum temperature of 40-50 deg.C. The papain is provided by Nanning Dong Henghuadao biological science and technology Limited liability company, 10 ten thousand U/g, 99.5% of enzyme activity preservation rate, 7.0-8.0 of optimum pH value and 50-55 ℃ of optimum temperature. The alkaline protease is provided by bioscience and technology Limited liability company of Henghuadao, Dongning, 20 ten thousand U/g, the optimum pH value is 9-12, and the optimum temperature is 40-55 ℃. The flavourzyme is provided by Nanning Dong Henghuadao biological science and technology Limited liability company, 5 ten thousand U/g, the optimum pH value is 5-9, and the optimum temperature is 50-55 ℃.

Lactobacillus plantarum was provided by Weifang Rui Biotechnology Co., Ltd, 50 hundred million cfu/g; the bacillus coagulans is provided by the biological technology company Limited in the exhibition of Xian, 100 hundred million cfu/g, the selenium-rich yeast is provided by the biological technology company Limited in the Jiaodong Jiaoyang, and the selenium content is more than 300mg/kg, wherein the content of organic selenium is more than 95 percent.

In the examples, each step is independent of the other, and the above steps may be repeated several times before the next step is performed to obtain a suitable amount of the product.

Unit D is short for dalton.

Example 1

The embodiment provides a metabolism promoting and antioxidant oat protein peptide, which specifically comprises the following steps:

s1, extracting oat protein: cleaning 100g of oat, drying, crushing, sieving with a 100-mesh sieve to obtain oat powder, adding into 300mL of trichloroacetic acid-acetone-acetonitrile solution, uniformly mixing, precipitating for 0.5h, centrifuging at 10000r/min for 10min, and adding 70g of insoluble substances into 140mL of acetone-acetonitrile solution; mixing, precipitating for 1h, centrifuging at 10000r/min for 10min, washing 50g of insoluble substance, drying at 60 deg.C for 2h, adding 100mL of lysate for cracking for 2h, collecting supernatant, and concentrating with ultrafiltration membrane with pore diameter of 3000D to obtain oat protein extract;

the lysis solution is an aqueous solution containing 3mol/L urea, 1mol/L thiourea and 0.5wt% dithiothreitol;

trichloroacetic acid in the trichloroacetic acid-acetone-acetonitrile solution: acetone: the volume ratio of acetonitrile is 0.5: 7: 3;

acetone in the acetone-acetonitrile solution: the volume ratio of the acetonitrile is 3: 4;

s2, primary enzymolysis: adjusting the pH value of 100g of the oat protein extract prepared in the step S1 to 2.5, heating to the temperature of 35 ℃, adding 1g of pepsin and 3g of acid protease, performing enzymolysis for 1 hour, inactivating enzymes, and filtering to obtain a filtrate, namely a first-stage enzymolysis liquid of the oat protein;

s3, secondary enzymolysis: adjusting the pH value of 100g of the first-stage enzymatic hydrolysate of the oat protein prepared in the step S2 to 6.5, heating to 50 ℃, adding 2g of neutral protease and 3g of papain, carrying out enzymolysis for 2h, inactivating enzymes, and filtering to obtain a filtrate, namely a second-stage enzymatic hydrolysate of the oat protein;

s4, three-stage enzymolysis: adjusting the pH value of 100g of the filtrate prepared in the step S3 to be the oat protein secondary enzymolysis liquid to be 8, heating to the temperature of 45 ℃, adding 3g of alkaline protease and 1g of flavourzyme, carrying out enzymolysis for 0.5h, inactivating enzyme, and filtering to obtain a filtrate which is oat protein tertiary enzymolysis liquid;

s5, activating strains: inoculating Lactobacillus plantarum, Bacillus coagulans and selenium-rich yeast to Gao's medium, culturing under anoxic condition at 35 deg.C for 18 hr to obtain strain seed solution with a strain content of 10 ⁷ cfu/mL；

S6, primary fermentation: inoculating strain seed solutions of lactobacillus plantarum and bacillus coagulans to 100mL of the oat protein three-level enzymolysis solution prepared in the step S4, wherein the inoculation amounts of the lactobacillus plantarum and the bacillus coagulans are respectively 2% and 1%, and performing fermentation culture at the temperature of 36 ℃ for 24h under the condition of micro-hypoxia to obtain an oat protein first-level fermentation product;

s7, secondary fermentation: inoculating selenium-enriched yeast strain seed liquid to 100mL of oat protein primary fermentation product prepared in the step S6, wherein the inoculation amount of the selenium-enriched yeast is 3%, adding 10mL of calcium chloride solution containing 5wt% of calcium ions, performing fermentation culture at 36 ℃ for 12h under the condition of micro-hypoxia, then adding 5mL of vitamin solution, continuing fermentation culture at 36 ℃ for 18h, filtering through a microfiltration membrane with the pore size range of 0.1 mu m, separating and removing unhydrolyzed insoluble components which cannot permeate the membrane, concentrating through a nanofiltration membrane with the pore size range of not more than 2nm, and performing freeze drying to prepare metabolism promoting and oxidation resisting oat protein peptide;

the vitamin solution is an aqueous solution containing 2wt% of vitamin B1 and 1wt% of vitamin B12;

the micro-anoxic condition comprises 5% of oxygen, 3% of carbon dioxide and the balance of nitrogen, wherein the volume percentage is;

the enzyme deactivation method is 1000W ultrasonic treatment for 15 min.

Example 2

s1, extracting oat protein: cleaning 100g of oat, drying, crushing, sieving with a 100-mesh sieve to obtain oat powder, adding into 500mL of trichloroacetic acid-acetone-acetonitrile solution, uniformly mixing, precipitating for 1.5h, centrifuging at 10000r/min for 10min, and adding 75g of insoluble substances into 300mL of acetone-acetonitrile solution; mixing uniformly, precipitating for 2h, centrifuging at 10000r/min for 10min, washing 60g of insoluble substances, drying at 60 ℃ for 2h, adding 300mL of lysate for cracking for 4h, taking supernatant, and concentrating by an ultrafiltration membrane with the pore diameter of 5000D to obtain an oat protein extract;

the lysis solution is an aqueous solution containing 5mol/L urea, 2mol/L thiourea and 1wt% dithiothreitol;

trichloroacetic acid in the trichloroacetic acid-acetone-acetonitrile solution: acetone: the volume ratio of acetonitrile is 1: 12: 5;

acetone in the acetone-acetonitrile solution: the volume ratio of the acetonitrile is 5: 7;

s2, primary enzymolysis: adjusting the pH value of 100g of the oat protein extract prepared in the step S1 to 3.5, heating to 40 ℃, adding 5g of pepsin and 7g of acid protease, performing enzymolysis for 3 hours, inactivating enzymes, and filtering to obtain a filtrate, namely a first-stage enzymolysis solution of the oat protein;

s3, secondary enzymolysis: adjusting the pH value of 100g of the first-stage enzymatic hydrolysate of the oat protein prepared in the step S2 to 7, heating to 55 ℃, adding 5g of neutral protease and 6g of papain, carrying out enzymolysis for 4h, inactivating enzymes, and filtering to obtain a filtrate, namely a second-stage enzymatic hydrolysate of the oat protein;

s4, three-stage enzymolysis: adjusting the pH value of 100g of the filtrate prepared in the step S3 to 9, heating to 55 ℃, adding 5g of alkaline protease and 3g of flavourzyme, performing enzymolysis for 1.5h, inactivating enzyme, and filtering to obtain a filtrate, namely a third-level enzymolysis solution of the oat protein;

s5, strain activation: inoculating Lactobacillus plantarum, Bacillus coagulans and selenium-rich yeast to Gao's medium, culturing under anoxic condition at 40 deg.C for 24 hr to obtain strain seed solution with a strain content of 10 ⁹ cfu/mL；

S6, primary fermentation: inoculating strain seed solutions of lactobacillus plantarum and bacillus coagulans into 100mL of the oat protein three-level enzymolysis solution prepared in the step S4, wherein the inoculation amounts of the lactobacillus plantarum and the bacillus coagulans are respectively 5% and 3%, and performing fermentation culture at 38 ℃ for 36h under the condition of micro-hypoxia to obtain an oat protein first-level fermentation product;

s7, secondary fermentation: inoculating a selenium-enriched yeast strain seed solution to 100mL of the oat protein primary fermentation product prepared in the step S6, wherein the inoculation amount of the selenium-enriched yeast is 5%, adding 20mL of a calcium chloride solution containing 12wt% of calcium ions, performing fermentation culture at 38 ℃ for 18h under the condition of micro-hypoxia, then adding 10mL of a vitamin solution, continuing fermentation culture at 38 ℃ for 24h, filtering through a microfiltration membrane with the pore size range of 0.5 mu m, separating and removing non-hydrolyzed insoluble components which cannot permeate the membrane, concentrating through a nanofiltration membrane with the pore size range of not more than 2nm, and performing freeze drying to prepare the metabolism promoting and oxidation resisting oat protein peptide;

the vitamin solution is an aqueous solution containing 5wt% of vitamin B1 and 3wt% of vitamin B12;

the micro-anoxic condition comprises 12% of oxygen, 5% of carbon dioxide and the balance of nitrogen, wherein the volume percentage is;

the enzyme deactivation method comprises 1500W ultrasonic treatment for 20 min.

Example 3

s1, extracting oat protein: cleaning 100g of oat, drying, crushing, sieving with a 100-mesh sieve to obtain oat powder, adding the oat powder into 400mL of trichloroacetic acid-acetone-acetonitrile solution, uniformly mixing, precipitating for 1h, centrifuging at 10000r/min for 10min, and adding 72g of insoluble substances into 220mL of acetone-acetonitrile solution; mixing uniformly, precipitating for 1.5h, centrifuging at 10000r/min for 10min, washing 53g of insoluble substances, drying at 60 ℃ for 2h, adding 190mL of lysate for cracking for 3h, taking supernatant, and concentrating by an ultrafiltration membrane with the pore diameter of 4000D to obtain an oat protein extract;

the lysis solution is an aqueous solution containing 4mol/L urea, 1.5mol/L thiourea and 0.7wt% dithiothreitol;

trichloroacetic acid in the trichloroacetic acid-acetone-acetonitrile solution: acetone: the volume ratio of acetonitrile is 0.7: 10: 4;

acetone in the acetone-acetonitrile solution: the volume ratio of the acetonitrile is 4: 5;

s2, primary enzymolysis: adjusting the pH value of 100g of the oat protein extract prepared in the step S1 to 3, heating to the temperature of 37 ℃, adding 3g of pepsin and 5g of acid protease, carrying out enzymolysis for 2h, inactivating enzymes, and filtering to obtain a filtrate, namely an oat protein first-level enzymolysis liquid;

s3, secondary enzymolysis: adjusting the pH value of 100g of the first-stage enzymatic hydrolysate of the oat protein prepared in the step S2 to 6.7, heating to 52 ℃, adding 3.5g of neutral protease and 5g of papain, carrying out enzymolysis for 3h, inactivating enzyme, and filtering to obtain a filtrate, namely a second-stage enzymatic hydrolysate of the oat protein;

s4, three-stage enzymolysis: adjusting the pH value of 100g of the filtrate prepared in the step S3 to be the oat protein secondary enzymolysis liquid to be 8.5, heating to 50 ℃, adding 4g of alkaline protease and 2g of flavourzyme, performing enzymolysis for 1 hour, inactivating enzyme, and filtering to obtain a filtrate which is the oat protein tertiary enzymolysis liquid;

s5, activating strains: inoculating Lactobacillus plantarum, Bacillus coagulans and selenium-rich yeast to Gao's medium, culturing under anoxic condition at 37 deg.C for 21 hr, and culturing to obtain strain seed solution with a strain content of 10 ⁸ cfu/mL；

S6, primary fermentation: inoculating strain seed solutions of lactobacillus plantarum and bacillus coagulans into 100mL of the oat protein three-level enzymolysis solution prepared in the step S4, wherein the inoculation amounts of the lactobacillus plantarum and the bacillus coagulans are respectively 3.5% and 2%, and performing fermentation culture at the temperature of 37 ℃ for 30h under the condition of micro-hypoxia to obtain an oat protein first-level fermentation product;

s7, secondary fermentation: inoculating selenium-enriched yeast strain seed liquid to 100mL of oat protein primary fermentation product prepared in the step S6, wherein the inoculation amount of the selenium-enriched yeast is 4%, adding 15mL of calcium chloride solution containing 10wt% of calcium ions, performing fermentation culture at 37 ℃ for 16h under the condition of micro-hypoxia, then adding 7mL of vitamin solution, continuing fermentation culture at 37 ℃ for 22h, filtering through a microfiltration membrane with the pore size range of 0.3 mu m, separating and removing non-hydrolyzed insoluble components which cannot permeate the membrane, concentrating through a nanofiltration membrane with the pore size range of not more than 2nm, and performing freeze drying to prepare metabolism promoting and oxidation resisting oat protein peptide;

the vitamin solution is an aqueous solution containing 3.5wt% of vitamin B1 and 2wt% of vitamin B12;

the micro-anoxic condition comprises 9% of oxygen, 4% of carbon dioxide and the balance of nitrogen, wherein the volume percentage is;

the enzyme deactivation method is 1250W ultrasonic treatment for 17 min.

Example 4

Compared to example 3, the trichloroacetic acid-acetone-acetonitrile solution in step S1 was replaced by a trichloroacetic acid-acetone solution, trichloroacetic acid: the volume ratio of acetone is 0.7: 14, all other conditions were unchanged.

Example 5

In step S7, 15mL of a calcium chloride solution containing 10wt% of calcium ions was not added, and the other conditions were not changed, as compared with example 3.

The method comprises the following specific steps:

s7, secondary fermentation: inoculating selenium-enriched yeast strain seed liquid to 100mL of the oat protein primary fermentation product prepared in the step S6, wherein the inoculation amount of the selenium-enriched yeast is 4%, performing fermentation culture at 37 ℃ for 16h under the condition of micro-hypoxia, adding 7mL of vitamin solution, continuing performing fermentation culture at 37 ℃ for 22h, filtering through a microfiltration membrane with the pore size range of 0.3 mu m, separating and removing non-hydrolyzed insoluble components which cannot permeate the membrane, concentrating through a nanofiltration membrane with the pore size range of not more than 2nm, and performing freeze drying to obtain the metabolism promoting and oxidation resisting oat protein peptide.

Example 6

In step S7, 7mL of the vitamin solution was not added, compared to example 3, and the other conditions were not changed.

The method comprises the following specific steps:

s7, secondary fermentation: inoculating selenium-enriched yeast strain seed liquid to 100mL of oat protein primary fermentation product prepared in the step S6, wherein the inoculation amount of the selenium-enriched yeast is 4%, adding 15mL of calcium chloride solution containing 10wt% of calcium ions, performing fermentation culture at 37 ℃ for 38h under the condition of micro-hypoxia, filtering through a microfiltration membrane with the aperture range of 0.3 mu m, separating and removing non-hydrolyzed insoluble components which cannot permeate through the membrane, concentrating through a nanofiltration membrane with the aperture range of not more than 2nm, and freeze-drying to obtain the metabolism promoting and oxidation resisting oat protein peptide.

Example 7

In step S7, 15mL of calcium chloride solution containing 10wt% calcium ions and 7mL of vitamin solution were not added, as compared with example 3, and other conditions were not changed.

The method comprises the following specific steps:

s7, secondary fermentation: inoculating the selenium-enriched yeast strain seed liquid to 100mL of the oat protein primary fermentation product prepared in the step S6, wherein the inoculation amount of the selenium-enriched yeast is 4%, performing fermentation culture at 37 ℃ for 38h under the condition of micro-hypoxia, filtering through a microfiltration membrane with the pore size range of 0.3 mu m, separating and removing non-hydrolyzed insoluble components which cannot permeate through the membrane, then concentrating through a nanofiltration membrane with the pore size range of not more than 2nm, and performing freeze drying to prepare the metabolism promoting and oxidation resisting oat protein peptide.

Comparative example 1

Compared with the embodiment 3, the first-stage enzymolysis of the step S1 is not carried out, and other conditions are not changed.

Comparative example 2

Compared with the example 3, the secondary enzymolysis of the step S2 is not carried out, and other conditions are not changed.

Comparative example 3

Compared with the example 3, the tertiary enzymolysis of the step S3 is not carried out, and other conditions are not changed.

Comparative example 4

Compared with the example 3, the steps S1-S3 are not carried out, and other conditions are not changed.

Comparative example 5

Compared with example 3, no lactobacillus plantarum was used in step S6, and other conditions were unchanged.

Comparative example 6

In step S6, Bacillus coagulans was not inoculated, and other conditions were not changed, as compared with example 3.

Comparative example 7

Step S6 was not performed, and other conditions were not changed, as compared with example 3.

Comparative example 8

Step S7 was not performed, and other conditions were not changed, as compared with example 3.

Comparative example 9

Compared with example 3, common baker' S yeast (available from Angel Yeast Co., Ltd.) was used in step S7 in place of the selenium-enriched yeast, and other conditions were not changed.

Comparative example 10

As compared with example 3, the fermentation steps S6-S7 were not performed, and other conditions were not changed.

Test example 1

The protein content of the oat protein extract obtained in step S1 of examples 1-4 was measured according to GB 5009.5-2016, "determination of proteins in national food safety standards".

The extraction rate R of the oat protein is calculated according to the following formula:

R=m/M×100%

in the formula: r is protein extraction rate,%; m is the total mass of protein in the extracting solution, g; m is the protein mass in oat, g.

The results are shown in Table 1.

TABLE 1

As can be seen from the above table, the method described in step S1 in examples 1-3 of the present invention provides a higher extraction rate of oat protein.

Test example 2 determination of molecular weight distribution

Reference is made to the following documents: li Y H, et al, analytical and free radial-scanning activities of Chippea Proteins Hydrosate (CPH) [ J ]. Food Chemistry, 2008, 106 (2): 444-450.

The pro-metabolic and antioxidant avenin peptides prepared in examples 1 to 7 and comparative examples 1 to 10 were prepared into a sample solution of 2mg/mL to be analyzed, and filtered through a microfiltration membrane. A10. mu.L sample of the sample to be analyzed was taken with a hand-operated sampler, injected into a TSK gel G2000 SWXL column, and eluted with a 42% acetonitrile solution containing 0.15% trifluoroacetic acid at a flow rate of 0.2mL/min and a detection wavelength of 214 nm. The standard curve was obtained by plotting elution time against log molecular weight using standard aprotinin (6512D), bactetracin (1423D), WPWW (tetrapeptide, 674D), NCS (tripeptide, 322D), Gly-Ser (dipeptide, 146D): y = -0.1892x +6.5172 (y: logMW, x: elution time), the results are shown in Table 2.

TABLE 2

As can be seen from the above table, the molecular weight of the pro-metabolic and antioxidant avenin peptides prepared in examples 1-3 of the present invention is mainly below 1000D, and studies have shown that small molecular peptides are more easily absorbed, which indicates that the polypeptide obtained from the pro-metabolic and antioxidant avenin peptides is smaller in molecular weight and mainly exists in a form of less than pentapeptide.

Test example 3 Oxidation resistance test

1. Determination of DPPH radical scavenging Capacity

Preparing the metabolism promoting and oxidation resisting avenin peptides prepared in the examples 1-7 and the comparative examples 1-10 into 10mg/mL sample solution, uniformly mixing 1mL sample solution and 4mL of 0.12mmol/L DPPH solution, sealing and standing at room temperature for 30min, detecting the absorbance at the wavelength of 517nm, and using a comparison group to be 10mg/mL vitamin C solution, wherein the clearance rate calculation formula is as follows:

clearance (%) = (1-a) ₁ -A ₂ ）/A ₃ ×100%

In the formula: a. the ₁ Is the absorbance of the solution after the sample is added; a. the ₂ Is the absorbance of the sample; a. the ₃ Absorbance of the solution without sample.

The results are shown in Table 3.

TABLE 3

As can be seen from the above table, the oat protein peptides of examples 1-3 of the present invention have excellent DPPH radical scavenging effect.

2. Determination of OH free radical scavenging Capacity

0.1mL of 10mmol/LFeSO was added to the centrifuge tube in sequence ₄ ·7H ₂ O, 0.1mL of 10mmol/L EDTA, 0.1mL of 10 mmol/L2-deoxyribose, 0.1mL of 10mg/mL of the pro-metabolic, antioxidant avenin peptide solutions prepared in examples 1 to 7 and comparative examples 1 to 10, 0.5mL of 0.1mol/L PBS (pH = 7.4) and 0.1mL of 10mmol/L H ₂ O ₂ After being mixed uniformly, the mixture is reacted in a constant temperature water bath kettle at 37 ℃ for 60min, then 0.5mL of 2.8 percent trichloroacetic acid and 0.5mL of 1 percent thiobarbituric acid are added, boiled in boiling water for 15min, cooled in cold water, and the absorbance is measured under the condition of 532 nm. An equal amount of distilled water was used as a blank, and a 10mg/mL vitamin C solution was used as a control.

The clearance calculation formula is as follows:

clearance (%) = (a) ₀ -A _s ）/A ₀ ×100%

In the formula: a. the ₀ Absorbance of blank group, A _s And the absorbance of the sample group to be detected.

The results are shown in Table 4.

TABLE 4

As can be seen from the above table, the oat protein peptides with metabolism promoting and antioxidant effects prepared in examples 1-3 of the present invention have excellent OH free radical scavenging effects.

3. Determination of superoxide anion radical scavenging ability

The pro-metabolic and anti-oxidative avenin peptides prepared in examples 1-7 and comparative examples 1-10 were prepared as 10mg/mL sample solutions, 1mL sample solution and 1.85mL 50mmol/L Tris-HCl (pH = 8.2) were placed in a water bath at 25 ℃ for reaction for 10min, then 0.15mL 3mmol/L pyrogallol (prepared in 10mmol/L HCl) was added, and the absorbance of the solution was measured at 325nm, and the absorbance was read every 30s until 4 min. Determining the oxidation rate of pyrogallol (A) from the rate of change of absorbance _{Sample (I)} ) The oxidation rate of pyrogallol (A) was measured by replacing the sample with an equal volume of distilled water _{Blank space} ) As a blank group, a 10mg/mL vitamin C solution was used as a control group. The clearance calculation formula is as follows:

clearance (%) = (1-a) _{Sample (I)} /A _{Blank space} ）×100%

In the formula: a. the _{Blank space} Absorbance for blank group; a. the _{Sample (I)} Is the absorbance of the sample to be measured.

The results are shown in Table 5.

TABLE 5

As can be seen from the above table, the oat protein peptides with metabolism promoting and oxidation resisting effects prepared in the embodiments 1-3 of the present invention have a very good superoxide anion free radical scavenging effect.

In conclusion, the oat protein peptides with metabolism promoting and oxidation resisting effects prepared in examples 1-3 of the present invention have excellent oxidation resisting effects.

Test example 4 weight loss mouse test

After 1 week acclimation of C57 mice, the mice were randomly divided into 19 groups: normal control group, high fat diet group, examples 1 to 7 groups, comparative examples 1 to 10 groups, 10 mice per group. Normal control group was given normal mouse diet (purchased from multi-meter agriculture technology ltd, linqing) and other groups were given high fat diet (purchased from cooperative medical bioengineering ltd, jiangsu province, 60% fat content, free diet). Examples 1-5 and comparative examples 1-10 groups were additionally administered the prepared metabolism-promoting, antioxidant avenin peptide 10g/kg body weight daily, and the normal control group, the high fat diet group, and the same amount of water. All mice were continuously fed for 8 weeks, during which the animal body weight was measured 1 time per week at a fixed time and recorded, and at the end of 8 weeks, all animals were fasted overnight, anesthetized with 10% chloral hydrate intraperitoneal injection (100 μ L/10g body weight), blood was collected from the heart, left at room temperature for 30min, centrifuged, and serum TG, TC, HDL, LDL were measured by an enzymatic method.

The results are shown in FIG. 1 and Table 6.

As can be seen from FIG. 1, the mice in the groups of examples 1-3 had their body weights significantly controlled after eating the prepared metabolism-promoting, antioxidant avenin peptides, and the body weights were significantly lower than those of the high-fat diet group.

TABLE 6

As can be seen from the above table, the TG content of the mice fed with the high-fat feed is obviously lower than that of the mice in the normal control group, and the TC, HDL and LDL contents are obviously higher than that of the mice in the normal control group, and the content of TG in the metabolism-promoting and oxidation-resisting oat protein peptides prepared by the examples 1-3 of the invention is obviously improved, and the content of TC, HDL and LDL is obviously reduced, so that the metabolism-promoting and oxidation-resisting oat protein peptides prepared by the examples 1-3 of the invention have better effects of controlling blood fat, promoting in vivo metabolism and controlling body weight.

Example 4 compared to example 3, the trichloroacetic acid-acetone-acetonitrile solution in step S1 was replaced by a trichloroacetic acid-acetone solution, trichloroacetic acid: the volume ratio of acetone is 0.7: 14. the extracted protein content is obviously reduced, the antioxidation effect is reduced, the weight loss effect is reduced, and the blood fat reducing effect is reduced. The oat flour is directly extracted by trichloroacetic acid-acetone-acetonitrile solution/acetone-acetonitrile solution for the second time, the trichloroacetic acid-acetone-acetonitrile solution is adopted to replace the traditional trichloroacetic acid-acetone solution, the protein extraction rate is higher, the nitrogen element on the cyano group contained in the adopted acetonitrile is easy to form hydrogen bond with the carboxylic acid structure of protein amino acid, so that the protein extraction in the oat flour is promoted and is precipitated in the presence of acetone, the extraction time is short, the repetition rate is high, and the influence of metabolic substances in a sample can be reduced and the protein loss can be reduced.

Example 5 compared to example 3, no 15mL of calcium chloride solution containing 10wt% calcium ions was added in step S7. Example 6 compared to example 3, no 7mL vitamin solution was added in step S7. Example 7 compared to example 3, 15mL of calcium chloride solution containing 10wt% calcium ions and 7mL of vitamin solution were not added in step S7. The content of the prepared protein peptide less than 1000D is reduced, and the effects of resisting oxidation, reducing weight and reducing blood fat are reduced. In comparative example 8, compared with example 3, the content of the produced protein peptide less than 500D was significantly decreased without performing step S7, and the antioxidant effect, weight loss effect, and blood lipid lowering effect were significantly decreased. Comparative example 9 compared with example 3, the antioxidant effect and the weight loss effect of the protein peptide prepared by using common baker' S yeast (purchased from Angel Yeast Co., Ltd.) instead of the selenium-enriched yeast in step S7 were reduced. Adding selenium-enriched yeast into the oat protein primary fermentation product, further performing enzymolysis, and simultaneously chelating product polypeptide with selenium mineral elements in organic selenium substances to form oat protein peptide-selenium chelate, wherein the chelate of the oat protein peptide and the selenium element is formed by mutual coordination between the selenium element and N-terminal amino, amino acid side chain, C-terminal carboxyl and carbonyl and imino in a peptide chain in the oat protein peptide. Compared with a chelate formed by the interaction of simple amino acid and selenium element, the chelate of the oat protein peptide and the selenium element has better stability and matching rate, has the advantages of high biological benefit, excellent absorption effect, excellent nutrition and the like, and can combine the complementary functional activities of the oat protein peptide and the selenium element, such as metabolism promotion, oxidation resistance, inflammation diminishing, bacteria resistance, immunity enhancement, blood pressure reduction, blood fat reduction, blood sugar reduction, weight losing, fat reduction, weight control and the like.

Compared with the example 3, the comparative examples 1, 2 and 3 do not carry out the first-stage enzymolysis of the step S1, the second-stage enzymolysis of the step S2 and the third-stage enzymolysis of the step S3 respectively, so that the relative content of the small molecular weight of the prepared protein peptide is reduced, and the antioxidant effect is obviously reduced. Compared with the example 3, the comparative example 4 has no steps of S1-S3 enzymolysis, the relative content of the small molecular weight of the prepared protein peptide is obviously reduced, the antioxidation effect is obviously reduced, and the weight-losing effect and the blood fat reducing effect are obviously reduced. The prepared oat protein extract is respectively subjected to enzymolysis under acidic, neutral and alkaline conditions, the acidic pepsin and the acidic protease which are suitable for the acidity are used for enzymolysis under the acidic condition, the proportion of small-molecule peptides in hydrolysate is very high, and the produced polypeptides are almost all small-molecule peptides; under neutral conditions, the enzymolysis is carried out by neutral protease suitable for neutral and papain, the neutral protease is a metal protease, the peptide bond between leucine and phenylalanine is preferentially cut, the papain can decompose the peptide bond in a hydrophobic region, including threonine, tryptophan, phenylalanine and the like, the hydrolysis degree of the enzymolysis product is high, the antioxidant activity is high, under alkaline conditions, the enzymolysis is carried out by alkaline protease suitable for alkaline and flavourzyme, the alkaline protease can decompose the peptide bond, including phenylalanine, tyrosine, tryptophan and lysine carboxyl, and the flavourzyme can preferentially break the peptide bond between leucine and proline or between proline and proline; the enzymolysis speed is fast, the enzymolysis effect is good, the yield of hydrolysate small peptides is high, the polypeptide after enzymolysis has higher emulsification stability, after three-stage enzymolysis, most of oat protein in the oat protein extracting solution is enzymolyzed into micromolecular short peptides and oligopeptide substances, and the antioxidant activity is very good.

Comparative examples 5 and 6 compared with example 3, no Lactobacillus plantarum or Bacillus coagulans was used in step S6. Compared with example 3, the comparative example 7 has no step S6, the relative content of the prepared protein peptide less than 1000D is obviously reduced, and the weight-reducing effect and the blood fat-reducing effect are reduced. The prepared third-level enzymolysis liquid of the oat protein also comprises a plurality of long-chain protein molecules and unhydrolyzed protein, lactobacillus plantarum and bacillus coagulans are further added for fermentation, probiotics ferment and hydrolyze the protein molecules further, and most of the unhydrolyzed long-chain protein is hydrolyzed into amino acid, short peptide, oligopeptide or polypeptide substances, so that a first-level fermentation product of the oat protein is prepared.

Comparative example 10 compared to example 3, the fermentation steps S6-S7 were not performed. The relative content of the prepared protein peptide less than 1000D is obviously reduced, and the effects of oxidation resistance, weight loss and blood fat reduction are obviously reduced. The oat protein peptide with the functions of promoting metabolism and resisting oxidation, which is prepared by the method disclosed by the invention, comprises an oat protein peptide-selenium chelate, and the oat protein peptide has an excellent anti-oxidation effect after detection, wherein most of the oat protein peptide is short peptide and oligopeptide substances with low molecular weight, and the short peptide and oligopeptide substances with low molecular weight are more easily close to free radicals than bulky parent proteins of the oat protein peptide and oligopeptide substances, and can inhibit peroxidation mediated by the free radicals. The short peptide and oligopeptide substances provide hydrogen atoms or electrons through peptide bonds and hydroxyl groups of the substances, and free radicals lacking hydrogen ions or electrons are eliminated, so that the destructive effect on biological molecules is eliminated; at the same time, the formation of ROS and free radicals can be inhibited by inhibiting certain pro-oxidases and chelating transition metal ions involved in catalyzing the generation of free radicals. The short peptide and oligopeptide substances can also inhibit the conversion of free radicals into hydrogen peroxide, and can be decomposed into harmless metabolites such as water and oxygen by endogenous antioxidants (including superoxide dismutase, catalase and glutathione), thereby having high antioxidant effect.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A preparation method of oat protein peptide with metabolism promotion and oxidation resistance is characterized by comprising the steps of crushing oat, extracting by using an organic solvent to obtain oat protein, performing primary enzymolysis by using pepsin and acid protease, performing secondary enzymolysis by using neutral protease and papain, performing tertiary enzymolysis by using alkaline protease and flavourzyme to obtain tertiary enzymolysis liquid of the oat protein, fermenting by using lactobacillus plantarum and bacillus coagulans to obtain a primary fermentation product of the oat protein, and fermenting by using selenium-enriched yeast to obtain the oat protein peptide with metabolism promotion and oxidation resistance.

2. The method of claim 1, comprising the steps of:

s1, extracting oat protein: cleaning oat, drying, crushing, sieving to obtain oat powder, adding the oat powder into a trichloroacetic acid-acetone-acetonitrile solution, uniformly mixing, precipitating for a first time period, centrifuging, adding an insoluble substance into the acetone-acetonitrile solution, uniformly mixing, precipitating for a second time period, centrifuging, washing the insoluble substance, drying, adding the insoluble substance into a lysate for cracking, taking a supernatant, and concentrating by using an ultrafiltration membrane to obtain an oat protein extracting solution;

3. The preparation method of claim 2, wherein the solid-to-liquid ratio of the oat flour and the trichloroacetic acid-acetone-acetonitrile solution in the step S1 is 1: 3-5 g/mL; trichloroacetic acid in the trichloroacetic acid-acetone-acetonitrile solution: acetone: the volume ratio of the acetonitrile is 0.5-1: 7-12: 3-5; the solid-liquid ratio of the insoluble substances to the acetone-acetonitrile solution is 1:2-4g/mL, and the ratio of acetone in the acetone-acetonitrile solution is as follows: the volume ratio of the acetonitrile is 3-5: 4-7; the first time period is 0.5-1.5 h; the second time period is 1-2 h; the lysis solution is an aqueous solution containing 3-5mol/L urea, 1-2mol/L thiourea and 0.5-1wt% dithiothreitol; the cracking time is 2-4 h; the solid-liquid ratio of the insoluble substance to the lysate is 1:2-5 g/mL; the pore diameter of the ultrafiltration membrane is 3000-5000D.

4. The preparation method according to claim 2, wherein the mass ratio of the oat protein extract, the pepsin and the acid protease in the step S2 is 100:1-5: 3-7; the enzymolysis time is 1-3 h; in the step S3, the mass ratio of the oat protein primary enzymolysis liquid to the neutral protease to the papain is 100:2-5:3-6, and the enzymolysis time is 2-4 h; in the step S4, the mass ratio of the oat protein secondary enzymolysis liquid to the neutral protease to the papain is 100:3-5:1-3, and the enzymolysis time is 0.5-1.5 h.

5. The method of claim 2, wherein the micro-anoxic conditions are an oxygen content of 5-12%, a carbon dioxide content of 3-5%, and the balance nitrogen, wherein% is volume percent; the method for killing the enzyme comprises 1000-1500W ultrasonic treatment for 15-20 min.

6. The method according to claim 2, wherein the cultivation temperature in step S5 is 35-40 ℃, the cultivation time is 18-24 hours, and the bacteria content of the seed solution of the strain is 10 ⁷ -10 ⁹ cfu/mL; in the step S6, the inoculation amounts of the lactobacillus plantarum and the bacillus coagulans are respectively 2-5% and 1-3%; the fermentation culture condition is that the temperature is 36-38 deg.C, and the time is 24-36 h.

7. The method according to claim 2, wherein the inoculation amount of the selenium-enriched yeast in step S7 is 3-5%, and the volume ratio of the oat protein primary fermentation product, the solution containing calcium ions and the vitamin solution is 10: 1-2: 0.5 to 1; the calcium ion concentration in the solution containing calcium ions is 5-12wt%, and the vitamin solution is an aqueous solution containing 2-5wt% of vitamin B1 and 1-3wt% of vitamin B12; the fermentation culture condition is 36-38 ℃, and the third time period is 12-18 h; the fourth time period is 18-24 h; the aperture range of the micro-filtration membrane is 0.1-0.5 mu m, and the aperture range of the nano-filtration membrane is not more than 2 nm.

8. The preparation method according to claim 2, characterized by comprising the following steps:

s1, extracting oat protein: washing oat, drying, crushing and sieving to obtain oat powder, adding the oat powder into trichloroacetic acid-acetone-acetonitrile solution, wherein the solid-to-liquid ratio of the oat powder to the trichloroacetic acid-acetone-acetonitrile solution is 1: 3-5g/mL, wherein the ratio of trichloroacetic acid in the trichloroacetic acid-acetone-acetonitrile solution is as follows: acetone: the volume ratio of the acetonitrile is 0.5-1: 7-12:3-5, uniformly mixing, precipitating for 0.5-1.5h, centrifuging, adding an insoluble substance into an acetone-acetonitrile solution, wherein the solid-to-liquid ratio of the insoluble substance to the acetone-acetonitrile solution is 1:2-4g/mL, and the mass ratio of acetone in the acetone-acetonitrile solution is as follows: the volume ratio of acetonitrile is 3-5:4-7, uniformly mixing, precipitating for 1-2h, centrifuging, washing insoluble substances, drying, adding the dried insoluble substances into a lysate for cracking for 2-4h, wherein the solid-to-liquid ratio of the insoluble substances to the lysate is 1:2-5g/mL, the lysate is an aqueous solution containing 3-5mol/L urea, 1-2mol/L thiourea and 0.5-1wt% dithiothreitol, taking supernatant, and concentrating through an ultrafiltration membrane with the pore diameter of 3000-;

s4, three-stage enzymolysis: adjusting the pH value of 100 parts by weight of the filtrate prepared in the step S3 to be the oat protein secondary enzymolysis liquid to 8-9, heating to 45-55 ℃, adding 3-5 parts by weight of alkaline protease and 1-3 parts by weight of flavor protease, carrying out enzymolysis for 0.5-1.5h, inactivating enzymes, and filtering to obtain a filtrate which is the oat protein tertiary enzymolysis liquid;

s5, strain activation: respectively inoculating Lactobacillus plantarum, Bacillus coagulans and selenium-rich yeast to Gao's medium, culturing under anoxia condition at 35-40 deg.C for 18-24 hr to obtain strain seed solutions with a bacteria content of 10 ⁷ -10 ⁹ cfu/mL；

S6, primary fermentation: inoculating strain seed solutions of lactobacillus plantarum and bacillus coagulans to the oat protein tertiary enzymolysis solution prepared in the step S4, wherein the inoculation amounts of lactobacillus plantarum and bacillus coagulans are respectively 2-5% and 1-3%, and the fermentation culture is carried out for 24-36h at the temperature of 36-38 ℃ under the condition of micro-hypoxia, so as to obtain an oat protein primary fermentation product;

9. A metabolically-enhanced, antioxidative avenin peptide produced by the method of any one of claims 1-8.

10. Use of the pro-metabolic, anti-oxidant avenin peptide of claim 9 in the manufacture of a weight loss, weight control product.