A kind of selenium-enriched maltose peptide and production method thereof

Abstract

The invention discloses a kind of selenium-enriched maltose peptide and its production method, wherein, a kind of production method of selenium-enriched maltose peptide, including crushing: selenium-enriched malt crushing;Cooking: adding purified water to malt powder for cooking;Enzyme hydrolysis: adding a-amylase and saccharifying enzyme for enzymolysis, then adding neutral protease and terminal peptidase for enzymolysis;Enzyme inactivation;Glycopeptide synthesis: temperature control to 40-45℃, adding lactic acid bacteria, stirring 1.0-1.5h synthetic glycopeptide under 10-25r / min conditions;Separation and purification, concentration, drying to obtain finished product. The raw material used in the production method of the present invention is selenium-enriched malt, using biological production process, without the addition of any organic solvent, the selenium-enriched black wheat maltose peptide obtained is highly active and has a high yield, and has higher economic benefits.

Description

Technical Field

[0001] The invention relates to the technical field of producing active peptides using plants as raw materials, and in particular to a selenium-enriched maltose peptide and a production method thereof. Background Art

[0002] Selenium-enriched rye malt is a selenium supplement product rich in natural organic selenium. It uses black wheat as an active carrier for selenium conversion. Through the absorption and conversion of the germination process, selenium is enriched in the amino acids, proteins and other molecules contained in the malt. The protein content of selenium-enriched rye malt is about 20%, which is a high-quality protein. It contains a large amount of dietary fiber and 8 essential amino acids for the human body. The organic selenium content reaches 280mg / kg. Selenium-enriched rye malt has the effect of activating the immune system and improving the body's immunity; malt selenium has the effect of preventing cancer and is an essential trace element for the human body.

[0003] In the past, selenium-rich black wheat was only used as a raw material for flour processing, and it had problems with poor water solubility and poor taste. In recent years, with the development of biotechnology, selenium-rich black rye malt can be hydrolyzed to obtain highly active small molecule peptides and oligosaccharides, with a peptide content of about 16% and an oligosaccharide content of about 76%, making it an excellent raw material for the production of glycopeptides.

[0004] Glycopeptides are polypeptides containing a carbohydrate of a glycan. Glycopeptides are produced during peptide synthesis. During peptide synthesis, glycans bind to polypeptides and are linked to other glycan-bound amino acids until a chain is produced. This is a compound formed by connecting short sugar chains and short peptides. There are two main types of glycopeptide bonds: N-glycopeptide bonds and O-glycopeptide bonds. The newly generated glycopeptides are then bound to proteins and lipids through glycosylation. This enzyme-catalyzed process allows glycopeptides to affect the biochemical communication between cells. Therefore, these glycopeptides have important biological effects in the life process of organisms. They also have the functions of resisting diseases and helping the body maintain homeostasis. Since glycans are ubiquitous in all cells of organisms and glycopeptides have the function of maintaining health and preventing diseases, products such as glycopeptide health products and medicines have gradually been developed. In addition, glycopeptide antibiotics have been developed to treat certain types of infections, which has increased their utilization value.

[0005] However, the existing production methods of wheat germ polypeptides for selenium-enriched rye malt have sugars and peptides in the components prepared, making it difficult to produce glycopeptides, and the raw material utilization value and economic benefits are low. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the defect in the prior art that no higher content of glycopeptides can be prepared using selenium-enriched malt by only adopting biological production process without adding any organic solvent, thereby providing a selenium-enriched malt glycopeptide and its production method that solves the above problem.

[0007] A method for producing selenium-enriched maltose peptide, comprising:

[0008] Crushing: crushing of selenium-rich malt;

[0009] Cooking: Add purified water to the malt powder for cooking;

[0010] Enzymatic hydrolysis: add a-amylase and saccharifying enzyme for enzymatic hydrolysis, and then add neutral protease and terminal peptidase for enzymatic hydrolysis;

[0011] Inactivate enzymes;

[0012] Glycopeptide synthesis: Control the temperature to 40-45°C, add lactic acid bacteria, and stir at 10-25r / min for 1.0-1.5h to synthesize glycopeptides;

[0013] The finished product is obtained by separation, purification, concentration and drying.

[0014] The selenium-enriched malt is malt obtained by germinating selenium-enriched black wheat.

[0015] In the cooking step, the amount of purified water added is 8-10 times the weight of the selenium-enriched malt, and the cooking conditions are: 92-96° C., and cooking for 30-35 minutes.

[0016] The amount of α-amylase added is 0.5%-1.0% of the weight of malt powder;

[0017] And / or, the amount of saccharifying enzyme added is 1.0%-1.5% by weight of malt powder;

[0018] and / or, the neutral protease is added in an amount of 1%-1.5% by weight of the malt powder;

[0019] And / or, the amount of the terminal peptidase added is 0.4-0.6% by weight of the malt powder.

[0020] The α-amylase is obtained by fermentation of Bacillus subtilis;

[0021] And / or, the saccharifying enzyme is obtained by fermentation of Monascus purpurogenum;

[0022] And / or, the neutral protease is produced by fermentation of Bacillus subtilis;

[0023] And / or, the terminal peptidase is obtained by fermentation of Aspergillus oryzae.

[0024] The amount of lactic acid bacteria added is 0.05%-0.1% of the weight of the malt powder, and the activity of the lactic acid bacteria is 100 billion cfu / g.

[0025] In the enzymatic hydrolysis step, the enzymatic hydrolysis temperature is 55-58° C.; and / or, the enzymatic hydrolysis time of the a-amylase and the saccharifying enzyme is 1.5-2.0 h, and / or, the enzymatic hydrolysis time of the neutral protease and the terminal peptidase is 2.0-3.0 h;

[0026] And / or, in the enzyme inactivation step, the enzyme inactivation temperature is 90-95° C. and the enzyme inactivation time is 10-15 min.

[0027] The separation and purification process is: first filtering with a plate and frame filter, and then purifying with a tubular high-speed centrifuge.

[0028] The concentration is carried out using a double-effect concentrator.

[0029] The drying is spray drying.

[0030] The technical solution of the present invention has the following advantages:

[0031] 1. The present invention provides a method for producing selenium-enriched maltose peptide, which adopts selenium-enriched malt as raw material, adopts biological production process, and does not add any organic solvent, so that the selenium-enriched black wheat maltose peptide obtained has high activity; the specific implementation mechanism of the biological production process is: firstly, the selenium-enriched malt is steamed to deform the sugar chain and protein, and accelerate the subsequent enzyme catalysis process; then, the sugar chain of the selenium-enriched malt is enzymatically hydrolyzed by a-amylase and saccharifying enzyme, and the macromolecular polysaccharide is decomposed into oligosaccharides, which is beneficial to the next step of synthesis with short peptides; wherein, a-amylase can not only degrade glucose in sugars, but also degrade oligoxylose and oligofructose; saccharifying enzyme can not only saccharify Raffinose in sugars can reduce its side effects and modify sugar chains, which is beneficial to the synthesis of sugars and peptides. Neutral proteases are then used to degrade proteins in malt, and terminal peptidase is used to modify terminal peptide bonds, which is beneficial to the subsequent synthesis of sugars and peptides. Under specific temperature and rotation speed conditions, lactic acid bacteria are added, and polysaccharides bind oligopeptides during biological activities, and are linked to other polysaccharide-bound amino acids until a chain is produced. The newly produced active peptides are then bound to polypeptides and lipids through glycosylation to ultimately form glycopeptides. After synthesis, the finished product is obtained through separation, purification, concentration, and drying. The finished product has the functions of resisting diseases and improving the body's immunity.

[0032] 2. The present invention provides a method for producing selenium-enriched maltoglycopeptide, which has a short preparation time, a high yield of glycopeptide, a simple preparation process and high economic benefits. DETAILED DESCRIPTION

[0033] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0034] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0035] Among them, a-amylase is fermented by Bacillus subtilis; for example, the α-amylase with the product number LM1003 produced by LMAI Bio has an activity of 4000U / g. Glucoamylase is fermented by Monascus; for example, the saccharifying enzyme produced by Henan Xiezhi has an activity of 50,000U / g. Neutral protease is fermented by Bacillus subtilis; for example, the neutral protease produced by Longkote has an activity of 50,000U / g. Terminal peptidase is fermented by Aspergillus oryzae; for example, the peptidase of Kmik has an activity of 500U / g.

[0036] Example 1

[0037] A method for producing selenium-enriched maltose peptide, comprising:

[0038] Weigh 100g of selenium-enriched black wheat malt and crush it; add 800ml of purified water and cook at 96℃ for 30min; then cool to 55℃, add 0.5g of a-amylase and 1g of saccharifying enzyme respectively, and stir for enzymolysis for 1.5h; then add 1g of neutral protease and 0.4g of terminal peptidase, and stir for enzymolysis for 2.0h; heat to 90℃ to inactivate the enzyme for 15min; then lower the temperature to 40℃, add 0.05g of lactic acid bacteria (Lactobacillus bulgaricus-R1), and the activity of the lactic acid bacteria is 100 billion cfu / g; control the speed of the stirrer at 10r / min, and synthesize for 1.0h; first filter with a plate and frame filter (using a filter cloth with a pore size of 1 micron), and then purify with a tubular high-speed centrifuge (17500r / m); concentrate the filtrate with a double-effect concentrator; the concentrate is spray-dried with a spray dryer to obtain a selenium-enriched black maltose peptide product.

[0039] Example 2

[0040] A method for producing selenium-enriched maltose peptide, comprising:

[0041] Weigh 1000g of selenium-enriched black wheat malt and crush it; add 9000ml of purified water and cook at 94°C for 35min; then cool to 58°C, add 7g of a-amylase and 12g of saccharifying enzyme respectively, and stir for enzymolysis for 2.0h; then add 12g of neutral protease and 5g of terminal peptidase, and stir for enzymolysis for 2.5h; heat to 92°C to inactivate the enzyme for 12min; then lower the temperature to 42°C, add 10g of the same lactic acid bacteria in Example 1, and the activity of the lactic acid bacteria is 100 billion cfu / g; control the agitator speed at 15r / min, and synthesize for 1.5h; first filter with a plate and frame filter (using a filter cloth with a pore size of 1 micron), and then purify with a tubular high-speed centrifuge (17500r / m); concentrate the filtrate with a double-effect concentrator; the concentrate is spray-dried with a spray dryer to obtain a selenium-enriched black maltose peptide product.

[0042] Example 3

[0043] A method for producing selenium-enriched maltose peptide, comprising:

[0044] Weigh 10 kg of selenium-enriched black wheat malt and crush it; add 100 L of purified water and cook at 92 ° C for 35 min; then cool to 56 ° C, add 100 g of a-amylase and 150 g of saccharifying enzyme respectively, and stir for enzymolysis for 2.5 h; then add 150 g of neutral protease and 60 g of terminal peptidase, and stir for enzymolysis for 3.0 h; heat to 95 ° C to inactivate the enzyme for 10 min; then lower the temperature to 45 ° C, add 0.8 g of the same lactic acid bacteria in Example 1, and the activity of the lactic acid bacteria is 100 billion cfu / g; control the agitator speed at 25 r / min, and synthesize for 1.5 h; first filter with a plate and frame filter (using a filter cloth with a pore size of 1 micron), and then purify with a tubular high-speed centrifuge (17500 r / m); concentrate the filtrate with a double-effect concentrator; the concentrate is spray-dried with a spray dryer to obtain a selenium-enriched black maltose peptide product.

[0045] Comparative Example 1

[0046] A method for producing selenium-enriched maltose peptide, comprising:

[0047] Weigh 100g of selenium-enriched black wheat malt and crush it; add 800ml of purified water and cook at 96℃ for 30min; then cool to 55℃, add 0.5g a-amylase and 1g saccharifying enzyme respectively, stir and hydrolyze for 1.5h; then add 1g neutral protease and 0.4g terminal peptidase, stir and hydrolyze for 2.0h; heat to 90℃ and react for 45min; first filter with a plate and frame filter (using filter cloth with a pore size of 1 micron), and then purify with a tubular high-speed centrifuge (17500r / m); concentrate the filtrate with a double-effect concentrator; the concentrate is spray-dried with a spray dryer to obtain a selenium-enriched black maltose peptide product.

[0048] Experimental example

[0049] The selenium-enriched black maltose peptide products prepared in Examples 1-3 and Comparative Example 1 were used as samples, and sialic acid glycopeptide (SGP) of 1% by mass of the sample was added to the samples for mass spectrometry analysis, and the peak positions of the O-glycopeptide bond and the N-glycopeptide bond in the samples were obtained respectively, and the peak intensities were recorded to obtain the percentage (%) of the glycopeptide bond content increase in the sample corresponding to the product of Comparative Example 1 compared with the product of the example. The test results are shown in Table 1;

[0050] The percentage increase of glycopeptide bond content (%) = {[(O-glycopeptide bond peak intensity of the sample - O-glycopeptide bond peak intensity of the sample of comparative example 1) / O-glycopeptide bond peak intensity of the sample of comparative example 1] + [(N-glycopeptide bond peak intensity of the sample - N-glycopeptide bond peak intensity of the sample of comparative example 1) / N-glycopeptide bond peak intensity of the sample of comparative example 1)]} / 2.

[0051] In addition, the products of Examples 1-3 and Comparative Example 1 were used to test the antioxidant activity and reducing power. The test results are shown in Table 1. The specific test process is as follows:

[0052] (1) DPPH free radical scavenging ability: Take 1.5 mL of 0.2 mg / mL sample, add 1.5 mL of 99.5% ethanol and 0.675 mL of 0.02% DPPH ethanol solution, mix, oscillate, place in a light-proof water bath at room temperature for 30 min, and then detect the absorbance of the system at 517 nm. The lower the absorbance, the stronger the DPPH free radical scavenging ability of the system. The blank control is to replace 1.5 mL of sample solution with 1.5 mL of deionized water.

[0053] DPPH free radical scavenging ability % = ((blank absorbance value - sample absorbance value) / blank absorbance value) × 100.

[0054] (2) Determination of reducing power: Take 1 mL of 0.5 mg / mL sample, add 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% (mass fraction) potassium ferricyanide solution, mix well, and then heat in a 50°C water bath for 20 min. Take out and cool quickly, add 2.5 mL of 10% (mass fraction) trichloroacetic acid (TCA) solution, mix well, and then centrifuge at 3000g for 10 min. Take 2.5 mL of supernatant, add 2.5 mL of deionized water and 0.5 mL of 1% (mass fraction) ferric chloride solution, mix well, react at room temperature for 10 min, and measure the absorbance at 700 nm. The intensity of the absorbance at 700 nm is used to indicate the reducing power. A reducing power higher than 0.8 Abs is strong, a reducing power lower than 0.6 Abs is weak, and a reducing power between 0.6 and 0.8 Abs is medium.

[0055] Table 1

[0056]

[0057] The above test results show that the method of the present invention can effectively prepare selenium-enriched black maltose peptide products, and the preparation time is short, the yield of the glycopeptide is high, the preparation process is simple, and it has high economic benefits.

[0058] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for producing selenium-enriched maltodextrin, characterized in that: include: Crushing: crushing of selenium-rich malt; Cooking: Add purified water to the malt powder for cooking; Enzymatic hydrolysis: add a-amylase and saccharifying enzyme for enzymatic hydrolysis, and then add neutral protease and terminal peptidase for enzymatic hydrolysis; Inactivate enzymes; Glycopeptide synthesis: Control the temperature to 40-45°C, add lactic acid bacteria, and stir at 10-25r / min for 1.0-1.5h to synthesize glycopeptides; the amount of lactic acid bacteria added is 0.05%-0.1% of the weight of malt powder, and the activity of lactic acid bacteria is 100 billion cfu / g; The finished product is obtained by separation, purification, concentration and drying.

2. The production method according to claim 1, characterized in that The selenium-enriched malt is malt obtained by germinating selenium-enriched black wheat.

3. The production method according to claim 1, characterized in that In the cooking step, the amount of purified water added is 8-10 times the weight of the selenium-enriched malt, and the cooking conditions are: 92-96° C., and cooking for 30-35 minutes.

4. The production method according to any one of claims 1 to 3, characterized in that: The amount of α-amylase added is 0.5%-1.0% of the weight of malt powder; And / or, the amount of saccharifying enzyme added is 1.0%-1.5% of the weight of malt powder; and / or, the neutral protease is added in an amount of 1%-1.5% by weight of the malt powder; And / or, the amount of the terminal peptidase added is 0.4-0.6% by weight of the malt powder.

5. The production method according to any one of claims 1 to 3, characterized in that: The α-amylase is obtained by fermentation of Bacillus subtilis; And / or, the saccharifying enzyme is obtained by fermentation of Monascus purpurogenum; And / or, the neutral protease is produced by fermentation of Bacillus subtilis; And / or, the terminal peptidase is obtained by fermentation of Aspergillus oryzae.

6. The production method according to any one of claims 1 to 3, characterized in that: In the enzymatic hydrolysis step, the enzymatic hydrolysis temperature is 55-58°C; and / or, the enzymatic hydrolysis time of the α-amylase and the saccharifying enzyme is 1.5-2.0h, and / or, the enzymatic hydrolysis time of the neutral protease and the terminal peptidase is 2.0-3.0h; And / or, in the step of inactivating the enzyme, the temperature for inactivating the enzyme is 90-95° C. and the time for inactivating the enzyme is 10-15 min.

7. The production method according to any one of claims 1 to 3, characterized in that: The separation and purification process is: first filtering with a plate and frame filter, and then purifying with a tubular high-speed centrifuge.

8. The production method according to any one of claims 1 to 3, characterized in that: The concentration is carried out using a double-effect concentrator; And / or, the drying is spray drying.

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