CN115152848A - Enzymolysis method for improving antioxidant activity of oat milk and prepared enzymolysis oat milk - Google Patents
Enzymolysis method for improving antioxidant activity of oat milk and prepared enzymolysis oat milk Download PDFInfo
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C11/00—Milk substitutes, e.g. coffee whitener compositions
- A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
- A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
- A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
- A23C11/106—Addition of, or treatment with, microorganisms
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Cereal-Derived Products (AREA)
Abstract
The invention discloses an enzymolysis method for improving antioxidant activity of oat milk and prepared enzymolysis oat milk. Comprises soaking herba Avenae Fatuae in hot water, taking out, rinsing with clear water, and cooling to room temperature; soaking in clear water; pulping; adding 80U/mL-160U/mL cellulase, 30U/mL-40U/mL pectinase, 2U/mL-10U/mL alpha-amylase and 2U/mL-10U/mL glucoamylase into the oat milk with dregs, carrying out enzymolysis, deactivating enzyme, and sieving to obtain enzymolysis oat milk 1; adding papain with the mass fraction of 0.05-0.1% into the enzymatic oat milk 1, carrying out enzymolysis, and inactivating enzyme to obtain enzymatic oat milk 2; adding alkaline protease with the mass fraction of 0.75% -1% into the enzymatic oat milk 2, carrying out enzymolysis, inactivating enzyme, blending, homogenizing, and sterilizing to obtain the enzymatic oat milk with antioxidant activity. The invention establishes an enzymolysis method for improving the antioxidant activity of oat milk through the synergistic effect of protease and cellulase, and provides technical reference for large-scale production of antioxidant oat milk.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to an enzymolysis method for improving antioxidant activity of oat milk and the prepared enzymolysis oat milk.
Background
The oat is a gramineous annual herbaceous plant, belongs to small coarse cereals, contains lipid, protein, antioxidant and other nutritional ingredients, is a high-quality cereal, has balanced proportion of nutrients in the oat, is easy to absorb by a human body, and has good health care functions, such as functions of preventing and treating cardiovascular and cerebrovascular diseases, delaying human body aging and the like.
When dried oats swell with water, dormant enzymes are activated to increase metabolic activity, produce primary and secondary metabolites, and cause complex physical, chemical and structural changes in the kernels, thereby improving the nutritional and functional properties of the oats. The soaking process is not only the process of seed imbibition, but also can cause the content change of some anti-nutritional ingredients and mineral substances in the seeds, and simultaneously, the soaking process can soften the oat grains and improve the dissolution rate of protein and glucan in the oat. Heat shock can break physical dormancy by breaking the outer seed coat, allowing moisture-impermeable seeds to absorb water, promoting seed germination, which Baskin JM believes can also accelerate the maturation of seeds with water-permeable seed coats, with the highest germination rate occurring at 120 ℃ for 5min, and higher temperatures or longer durations that are lethal to many species. The germination response of seeds to heat shock varies according to species regeneration strategies and distribution ranges, most species of seeds are tolerant to high temperature, only a small part of the seeds can be stimulated by heat, and about 20 percent of the seeds have germination inhibition responses; suitable heat shock treatments activate the endogenous enzymatic activity of the seeds at the same time as the activation of seed germination, so that the seed extract is clearly distinguished from the non-heat shock treatment solution.
The protein contained in the oat is high-quality cereal protein, and the content of the oat protein in the oat is far higher than that in other grain crops. The oat polypeptide has strong inoxidizability, free radical scavenging function and anti-aging function, and the small molecular polypeptide is hydrolyzed into oat bran protein by utilizing the protease catalysis, so that the inoxidizability of the oat milk can be improved.
Dextran is a generic name for polysaccharides composed of glucose. There are many kinds of beta-glucans in nature, and the beta-glucans from different sources have different functions due to differences of glucose linkage type, polymerization degree, branching and the like. The oat glucan is beta-1, 3/1, 4-glucan, and the beta-glucan in the oat is used as a main functional component of the oat, has certain scavenging capacity on free radicals, hydroxyl free radicals and hydrogen peroxide, and has certain inhibiting capacity on lipid peroxidation. At present, there are some researches on the change of the edible quality after the enzymolysis of glucan, but no researches on the biological activity after the enzymolysis of beta-glucanase are reported.
Disclosure of Invention
The invention aims to provide an enzymolysis method for improving the antioxidant activity of oat milk.
The enzymolysis method for improving the antioxidant activity of the oat milk provided by the invention comprises the following steps:
(1) Heat shock: heating oat in hot water, taking out, and rinsing with clear water to room temperature;
(2) Soaking: soaking the heat-shocked oat in clear water;
(3) Pulping: adding water into the soaked oat, pulping, and sieving the residues with a 60-100 mesh sieve to obtain oat milk with residues;
(4) Starch total enzymatic hydrolysis and glucan restriction: adding 80U/mL-160U/mL (namely adding 80U-160U of cellulase in 1mL of oat milk with dregs, the same is applied below) of cellulase, 30U/mL-40U/mL of pectinase, 2U/mL-10U/mL of alpha-amylase and 2U/mL-10U/mL of glucoamylase into the oat milk with dregs, carrying out enzymolysis, inactivating the enzymes, and sieving to obtain an enzymolysis oat milk 1;
(5) And (3) protease enzymolysis: adding papain with the mass fraction of 0.05-0.1% (referring to adding 0.05-0.1 g papain to 100g of the enzymolyzed oat milk 1, the same below) into the enzymolyzed oat milk 1, and carrying out enzymolysis and enzyme inactivation to obtain an enzymolyzed oat milk 2;
(6) Performing compound enzymolysis by alkaline protease: adding alkaline protease with the mass fraction of 0.75% -1% into the enzymolysis oat milk 2, and carrying out enzymolysis and enzyme deactivation to obtain enzymolysis oat milk;
(7) Adding or not adding sweetener into the obtained enzymolysis oat milk, adjusting the temperature to 70 ℃, homogenizing under 30Mpa, and sterilizing at 121 ℃ for 15min to obtain the enzymolysis oat milk with antioxidant activity.
In the step (1), the heat shock is 60-150 s in water with the temperature of 90-100 ℃;
in the step (2) of the method, the soaking is to make the thermally excited oat absorb water and expand to 1.5-2 times of the original mass;
in the step (3), the weight ratio of oat to water may be 1:8-10 (calculated by dry weight of oat) (1 g dry weight of oat with 8-10g water);
the water temperature of pulping water can be 70-90 ℃;
in the step (4) of the method, the cellulase may specifically be 80U/mL or 160U/mL; the pectinase can be 30U/mL or 40U/mL; the alpha-amylase can be 5U/mL or 10U/mL specifically; the saccharifying enzyme can be 5U/mL or 10U/mL;
the temperature of the enzymolysis can be 50-55 ℃, and the time can be 0.5-1.0 h;
the enzyme inactivation is carried out by keeping the temperature at 100 ℃ for 8min-10 min;
the sieving is to sieve through 100 meshes to 200 meshes;
in the step (5), the mass fraction of the papain can be specifically 0.075% and 0.1%;
the temperature of the enzymolysis can be 50-55 ℃, and the time can be 1.0-3.0 h;
the enzyme inactivation is carried out by keeping the temperature at 100 ℃ for 8min-10 min;
in the step (6), the alkaline protease may be 0.75% or 1% by mass;
the temperature of the enzymolysis can be 50-55 ℃, and the time can be 1.0-3.0 h;
the enzyme inactivation is carried out by keeping the temperature at 100 ℃ for 8min-10 min.
The method may further include the following operations: adding sweetener into the obtained enzymolysis oat milk or not, adjusting temperature to 70 deg.C, homogenizing under 30Mpa, and sterilizing at 121 deg.C for 15min.
The enzymes used above apply only to the following sources and combinations of enzymes:
the enzymes used in said steps (4) to (6) are derived from:
cellulase: enzyme activity 500000U/g from Beijing Soluna science and technology Limited (Solarbio);
papain: the enzyme activity of Nanning Pompe bioengineering GmbH is 100000U/g;
alkaline protease: cangzhou Xiusheng enzyme biotechnology limited company, the enzyme activity is 200000U/g;
and (3) pectinase: the enzyme activity of Shandonglong Kort enzyme preparation, inc. is 30000U/g;
alpha-amylase: the enzyme activity of the Beijing Oobozoxin biotechnology Limited liability company is greater than 3700U/g;
saccharifying enzyme: the enzyme activity of Beijing Boototta science and technology Limited (Biotopped) is 100000U/g.
The enzymatic oat milk prepared by the enzymatic hydrolysis method also belongs to the protection scope of the invention.
The invention also protects the application of the enzymatic oat milk in preparing an antioxidant product and/or an anti-aging product.
The invention has the advantages and positive effects that:
endogenous enzyme is activated by heat shock treatment, seed germination is promoted, the water absorption rate is increased, the type and content of the oat milk extract substances are finally changed, and the whole substance composition of the oat milk is changed; the extraction rate of glucan and protein is increased by the alpha-amylase, the saccharifying enzyme and the pectinase; by the synergistic effect of the specific protease and the cellulase restriction enzyme, the oat polypeptide and the oat beta-glucan with appropriate molecular weight are obtained, so that the antioxidant activity of the oat milk is effectively improved, the process is simple, and the method is suitable for industrial production.
Drawings
FIG. 1 is an HPLC chromatogram of example 1;
FIG. 2 is an HPLC chromatogram of example 2;
FIG. 3 is an HPLC chromatogram of comparative example 1;
FIG. 4 is an HPLC chromatogram of comparative example 2;
FIG. 5 is a comparative example 3HPLC chromatogram;
FIG. 6 is a HPLC chromatogram of comparative example 4;
FIG. 7 is a comparative example 5HPLC chromatogram;
FIG. 8 is a comparative example 6HPLC chromatogram;
FIG. 9 is a glucose content standard curve;
FIG. 10 is a standard curve for protein content;
FIG. 11 is a dextran relative molecular weight standard curve.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The enzymes in the following examples and comparative examples 1-3 and 5 were from:
cellulase: enzyme activity 500000U/g from Beijing Soluna science and technology Limited (Solarbio);
and (3) papain: the enzyme activity of Nanning Pompe bioengineering GmbH is 100000U/g;
alkaline protease: cangzhou Xiusheng enzyme biotechnology limited company, the enzyme activity is 200000U/g;
and (3) pectinase: the enzyme activity of Shandonglong Kort enzyme preparation, inc. is 30000U/g;
alpha-amylase: the enzyme activity of the Beijing Olympic Star biotechnology Limited liability company is more than 3700U/g;
saccharifying enzyme: the enzyme activity of Beijing Boototta science and technology Limited (Biotoped) is 100000U/g.
Example 1
(1) Heat shock: removing impurities in oat, thermally exciting oat in boiling water (100 ℃) for 120s, immediately taking out the oat, placing the oat in clear water, and quickly rinsing the oat until the temperature is reduced to room temperature;
(2) Soaking: soaking the heat-excited oat in clear water at room temperature to ensure that the oat absorbs water and expands to 1.8 times of the original mass;
(3) Pulping: mixing the soaked oat according to the proportion of 1:8 (calculated by the dry weight of the oat) adding water (water temperature is 90 ℃) to pulp, and sieving the slag by 100 meshes to obtain oat milk with slag;
(4) Starch total enzymatic hydrolysis and glucan restriction: adding 160U/mL cellulase, 40U/mL pectinase, 5U/mL alpha-amylase and 10U/mL glucoamylase into the oat milk with residues, performing enzymolysis for 1.0h at 50 ℃, then preserving heat at 100 ℃ for 10min to inactivate enzyme activity, and filtering by a 200-mesh sieve to obtain enzymolysis oat milk 1;
(5) And (3) protease enzymolysis: adding papain with the mass fraction of 0.1% into the enzymatic oat milk 1, performing enzymolysis at 50 deg.C for 1h, and keeping the temperature at 100 deg.C for 8min to inactivate enzyme to obtain enzymatic oat milk 2;
(6) Performing compound enzymolysis by alkaline protease: adding 1% of alkaline protease into the enzymolysis oat milk 2, carrying out enzymolysis at 50 ℃ for 1h, and then preserving heat at 100 ℃ for 10min to inactivate enzyme to obtain enzymolysis oat milk 3;
(7) Adjusting the temperature of the enzymolysis oat milk 3 to 70 ℃, homogenizing under 30Mpa, and sterilizing at 121 ℃ for 15min to obtain the enzymolysis oat milk.
Example 2
(1) Heat shock: removing impurities in oat, thermally exciting oat in water of 90 deg.C for 60s, immediately taking out, placing in clear water, and rapidly rinsing to room temperature;
(2) Soaking: soaking the thermally excited oat in clear water at room temperature to ensure that the oat absorbs water and expands to 1.5 times of the original mass;
(3) Pulping: mixing the soaked oat according to the proportion of 1:10 Adding water (water temperature is 90 ℃) into the dry weight of the oat for pulping, and enabling the residue to pass through 100 meshes to obtain oat milk with the residue;
(4) Starch total enzymolysis and glucan restriction: adding 80U/mL cellulase, 30U/mL pectinase, 10U/mL alpha-amylase and 5U/mL glucoamylase into the oat milk with residues, carrying out enzymolysis for 0.5h at 55 ℃, then preserving heat at 100 ℃ for 8min to inactivate enzyme activity, and filtering by a 200-mesh sieve to obtain enzymolysis oat milk 1;
(5) And (3) protease enzymolysis: adding papain with the mass fraction of 0.075% into the enzymatic oat milk 1, carrying out enzymolysis for 1h at 55 ℃, then keeping the temperature at 100 ℃ for 10min, and inactivating the enzyme to obtain enzymatic oat milk 2;
(6) Performing compound enzymolysis by alkaline protease: adding alkaline protease with the mass fraction of 0.75% into the enzymolysis oat milk 2, carrying out enzymolysis at 55 ℃ for 1h, and then preserving the heat at 100 ℃ for 8min to inactivate the enzyme, thus obtaining enzymolysis oat milk 3;
(7) Adjusting the temperature of the enzymolysis oat milk 3 to 70 ℃, homogenizing under 30Mpa, and sterilizing at 121 ℃ for 15min to obtain the enzymolysis oat milk.
Comparative example 1
(1) Soaking: removing impurities in the oat, and soaking the oat in clear water at room temperature to ensure that the oat absorbs water and expands to 1.8 times of the original mass;
(2) Pulping: mixing the soaked oat according to the proportion of 1:8 (calculated by the dry weight of the oat) adding water (water temperature is 90 ℃) to pulp, and sieving the slag by 100 meshes to obtain oat milk with slag;
(3) And (3) starch complete enzymolysis: adding 40U/mL pectinase, 5U/mL alpha-amylase and 10U/mL glucoamylase into the oat milk with residues, performing enzymolysis for 0.5h at 55 ℃, then preserving heat for 8min at 100 ℃ to inactivate enzyme activity, and filtering by a 200-mesh sieve to obtain an enzymolysis oat milk 1;
(4) Adjusting the temperature of the enzymolysis oat milk 1 to 70 ℃, homogenizing under 30Mpa, and sterilizing at 121 ℃ for 15min to obtain the enzymolysis oat milk.
Comparative example 2
(1) Heat shock: removing impurities in oat, thermally exciting oat in boiling water for 120s, immediately taking out the oat, placing the oat in clear water, and quickly rinsing the oat until the temperature is reduced to room temperature;
(2) Soaking: soaking the thermally excited oat in clear water at room temperature to ensure that the oat absorbs water and expands to 1.8 times of the original mass;
(3) Pulping: mixing the soaked oat according to the weight ratio of 1:8 (calculated by the dry weight of the oat) adding water (water temperature is 90 ℃) to pulp, and sieving the slag by 100 meshes to obtain oat milk with slag;
(4) And (3) starch complete enzymolysis: adding 40U/mL pectinase, 5U/mL alpha-amylase and 10U/mL glucoamylase into the oat milk with residues, carrying out enzymolysis for 1.0h at 55 ℃, then preserving heat for 8min at 100 ℃ to inactivate enzyme activity, and filtering by a 200-mesh sieve to obtain enzymolysis oat milk 1;
(5) Adjusting the temperature of the enzymolysis oat milk 1 to 70 ℃, homogenizing under 30Mpa, and sterilizing at 121 ℃ for 15min to obtain the enzymolysis oat milk.
Comparative example 3
(1) Heat shock: removing impurities in oat, thermally exciting oat in boiling water for 120s, immediately taking out, placing in clear water, and rapidly rinsing to room temperature;
(2) Soaking: soaking the thermally excited oat in clear water at room temperature to ensure that the oat absorbs water and expands to 1.8 times of the original mass;
(3) Pulping: mixing the soaked oat according to the weight ratio of 1:8 (calculated by the dry weight of the oat) adding water (water temperature is 90 ℃) to pulp, and sieving the slag by 100 meshes to obtain oat milk with slag;
(4) Starch total enzymatic hydrolysis and glucan restriction: adding 640U/mL cellulase, 40U/mL pectinase, 5U/mL alpha-amylase and 10U/mL glucoamylase into the oat milk with dregs, carrying out enzymolysis for 1.0h at 55 ℃, then preserving heat for 10min at 100 ℃ to inactivate enzyme activity, and filtering by a 200-mesh sieve to obtain an enzymolysis oat milk 1;
(5) And (3) protease enzymolysis: adding papain with the mass fraction of 0.2% into the enzymatic oat milk 1, carrying out enzymolysis for 1h at 55 ℃, and then keeping the temperature at 100 ℃ for 8min to inactivate the enzyme, thereby obtaining enzymatic oat milk 2;
(6) Performing compound enzymolysis by alkaline protease: adding alkaline protease with the mass fraction of 1.2% into the enzymolysis oat milk 2, carrying out enzymolysis at 55 ℃ for 1h, and then keeping the temperature at 100 ℃ for 10min to inactivate the enzyme, thereby obtaining enzymolysis oat milk 3;
(7) Adjusting the temperature of the enzymolysis oat milk 3 to 70 ℃, homogenizing under 30Mpa, and sterilizing at 121 ℃ for 15min to obtain the enzymolysis oat milk.
Comparative example 4
(1) Preparing a sample: oat beta-glucan (neat) solution was prepared according to the polysaccharide content in comparative example 2. Oat beta-glucan was purchased from shenyou biotechnology limited, zhejiang.
Comparative example 5
(1) Preparing a sample: preparing a solution of oat beta-glucan (pure) according to the polysaccharide content in comparative example 2;
(2) Restriction of glucan: referring to example 1, after adding 160U/mL cellulase for enzymolysis at 55 ℃ for 1h, keeping the temperature at 100 ℃ for 10min to inactivate the enzyme, thus obtaining the oat beta-glucan enzymolysis liquid.
Comparative example 6
(1) Heat shock: removing impurities in oat, thermally exciting oat in boiling water (100 ℃) for 120s, immediately taking out, placing in clear water, and quickly rinsing to reduce the temperature to room temperature;
(2) Soaking: soaking the thermally excited oat in clear water at room temperature to ensure that the oat absorbs water and expands to 1.8 times of the original mass;
(3) Pulping: mixing the soaked oat according to the proportion of 1:8 (calculated by the dry weight of the oat), adding water (the water temperature is 90 ℃) and pulping to ensure that the residues pass through 100 meshes to obtain oat milk with residues;
(4) Starch total enzymatic hydrolysis and glucan restriction: adding 160U/mL cellulase, 40U/mL pectinase, 5U/mL alpha-amylase and 10U/mL glucoamylase into the oat milk with residues, performing enzymolysis for 1.0h at 50 ℃, then preserving heat at 100 ℃ for 10min to inactivate enzyme activity, and filtering by a 200-mesh sieve to obtain enzymolysis oat milk 1;
(5) And (3) protease enzymolysis: adding papain with the mass fraction of 0.1% into the enzymolytic oat milk 1, carrying out enzymolysis at 50 ℃ for 1.0h, and then preserving the heat at 100 ℃ for 8min to inactivate the enzyme, thereby obtaining an enzymolytic oat milk 2;
(6) Performing compound enzymolysis by alkaline protease: adding 1% of alkaline protease into the enzymolysis oat milk 2, carrying out enzymolysis at 50 ℃ for 1h, and then preserving heat at 100 ℃ for 10min to inactivate enzyme to obtain enzymolysis oat milk 3;
the cellulase is obtained from CEL-01 type cellulase of Cangzhou summer enzyme biotechnology limited, and the enzyme activity is 11000U/g; papain is purchased from Nanning Hengdonghua David Biotechnology Limited liability company, and has enzyme activity of 100000U/g; the alkaline protease is obtained from 2.4L Novoxil food-grade alkaline protease Alcalase, and the enzyme activity is 200000U/g; the pectinase is purchased from Shandong Kete enzyme preparation Co., ltd, and the enzyme activity is 30000U/g; the alpha-amylase is purchased from Oboxing biotechnology Limited liability company of Beijing, and the enzyme activity is more than 3700U/g; saccharifying enzyme is purchased from Beijing Bootouda science and technology Limited (Biotopped), and the enzyme activity is 100000U/g;
(7) Adjusting the temperature of the enzymolysis oat milk 3 to 70 ℃, homogenizing under 30Mpa, and sterilizing at 121 ℃ for 15min to obtain the enzymolysis oat milk.
The relevant detection methods and results of the present invention are as follows:
the products obtained in the examples and comparative examples were subjected to polysaccharide and polypeptide content analysis, antioxidant property analysis and polysaccharide molecular weight analysis.
1. Determination of polysaccharide content
4mL of the products prepared in the examples and the comparative examples are respectively taken, added with 4 times of volume of absolute ethyl alcohol for precipitation, kept overnight, dried and re-dissolved in 2mL of deionized water to obtain the coarse polysaccharide solution of the oat milk. And measuring the polysaccharide content by adopting a phenol-sulfuric acid method.
(1) Drawing a standard curve: accurately sucking 0.1mg/mL glucose standard solution 0.2, 0.4, 0.6, 0.8 and 1.0mL into a test tube, supplementing water to 1.0mL, adding 0.5mL phenol solution with mass fraction of 6%, slowly adding 5mL concentrated sulfuric acid solution along the wall of the test tube, uniformly mixing, carrying out boiling water bath for 5min, and cooling to room temperature. Using 1.0mL of distilled water as a blank control, measuring the absorbance value at 490nm by an ultraviolet spectrophotometer, repeatedly taking the average value for three times, taking the glucose content as the abscissa and the OD 490nm On the ordinate, a standard curve is plotted as shown in FIG. 9. The standard curve linear regression equation is known as y =8.2243x-0.0087 2 =0.999, wherein y is the absorbance and x is the glucose content (mg/mL).
(2) Determination of polysaccharide in sample solution: accurately sucking 1mL of sample crude polysaccharide solution diluted by a certain time, adding 0.5mL of phenol solution with the mass fraction of 6%, slowly adding 5mL of concentrated sulfuric acid solution along the wall of the test tube, uniformly mixing, carrying out boiling water bath for 5min, and cooling to room temperature. And measuring an absorbance value at 490nm by using an ultraviolet spectrophotometer by taking the reagent blank as a reference. And calculating the polysaccharide content in the solution according to a standard curve.
2. Determination of polypeptide content
5mL of the product prepared in the example and the comparative example is taken, 5mL of trichloroacetic acid solution with the mass fraction of 10% is added, and after centrifugation at 8000rpm for 3min, the supernatant is taken for testing.
(1) Drawing a standard curve: accurately sucking 0.16, 0.32, 0.48, 0.64 and 0.8mL of 10mg/mL bovine serum albumin standard solution into a test tube, replenishing water to 0.8mL, adding 3.2mL of biuret reagent, uniformly mixing, and reacting for 30min at 25 ℃. Using 1.0mL of distilled water as blank control, measuring absorbance value at 540nm wavelength by an ultraviolet spectrophotometer, repeatedly taking an average value for three times, taking protein content as abscissa and OD 540nm As an ordinate, a standard curve was plotted as shown in FIG. 10. It can be seen that the standard curve linear regression equation is y =0.0537x-0.002 2 =0.9999, wherein y is absorbance and x is protein content (mg/mL).
(2) Determination of the polypeptides of the sample solution: accurately sucking 0.8mL of sample solution to be detected, adding 3.2mL of biuret reagent, uniformly mixing, and reacting at 25 ℃ for 30min. And measuring an absorbance value at the wavelength of 540nm by using a reagent blank as a reference through an ultraviolet spectrophotometer. Calculating the polypeptide content of the solution according to a standard curve.
3. Determination of hydroxyl radical scavenging Rate
Diluting the products prepared in the examples and the comparative examples by 5 times, adding 0.375mL ferrous sulfate solution (2 mg/mL), 1mL 1% hydrogen peroxide solution and 1mL salicylic acid ethanol solution (1.5 mg/mL) into 1mL diluent, mixing uniformly, shaking, performing water bath at 37 ℃ for 1h, and measuring absorbance C at 526nm 1 (ii) a Distilled water is used for replacing hydrogen peroxide solution for testing, and the background absorbance C of the sample is measured 2 (ii) a The test is carried out by replacing salicylic acid ethanol with distilled water, and the absorbance C of the blank control solution is measured 0 . Calculating the formula:
hydroxyl radical clearance (%) = [1- (C) 1 -C 2 )/C 0 ]×100％
4. Determination of DPPH radical scavenging Rate
Diluting the products obtained in examples and comparative examples by 5 times, collecting 2mL of the diluted solution, adding 2mL of a DPPH absolute ethanol solution (0.2 mmol/L), vortexing, standing in the dark for 30min, and measuring B at 517nm using distilled water as a blank solution (adjusted to 0) 1 The absolute ethyl alcohol is used for replacing DPPH ethanol solution for carrying out the experiment, and the background absorbance B of the sample is measured 2 (ii) a Using distilled water to replace the sample solution to measure the absorbance B of the blank control solution 0 . Calculating the formula:
DPPH radical clearance (%) = [1- (B) 1 -B 2) /B 0 ]×100％
5. Measurement of reducing Power
Diluting the products prepared in the examples and the comparative examples by 5 times, taking 1mL of diluent, adding 2.5mL of phosphate buffer solution (0.2 mol/L, pH 6.6), adding 2.5mL of 1% potassium ferricyanide solution, carrying out vortex mixing, keeping the temperature in a water bath at 50 ℃ for 20min, adding 2.5mL of 10% trichloroacetic acid, centrifuging for 10min, taking 3mL of supernatant, sequentially adding 3mL of distilled water and 0.6mL of 0.1% ferric trichloride solution, mixing uniformly, standing for 10min, and measuring the absorbance A at 700nm 1 The absorbance A is measured by using distilled water as a blank instead of a ferric trichloride solution 0 With a reducing power of A 1 -A 0 。
The measurement results of the polysaccharide content, the polypeptide content and the in vitro antioxidant activity of the examples and the comparative examples of the invention are as follows:
TABLE 1 polysaccharide content, polypeptide content and antioxidant Activity in the examples of the invention and comparative examples
As can be seen from Table 1, the polysaccharide elution with heat-shocked oats (comparative example 2) was significantly increased by a factor of 2.86 over that of non-heat-shocked oats (comparative example 1), while there was no significant difference in the elution of protein, indicating the importance of heat shock in the process of the invention; the polysaccharide content of soaked oat after heat shock is not increased by adopting proper amount of cellulase hydrolysis (80U/mL-160U/mL), however, compared with comparative example 2, the peptide yield of example 1 and example 2 is improved by 1.93-2.07 times, which shows the importance of the combination and proportion of protease and cellulase used in the patent; higher addition levels of protease and cellulase did not significantly increase polysaccharide and polypeptide content, indicating that the protease and cellulase combination used in this patent needs to be controlled within a certain ratio range to be effective (comparative example 3 compared to examples 1 and 2).
The hydroxyl radical clearance rate, DPPH radical clearance rate and reducing power of the soaked oat milk (comparative example 1) only added with amylase, saccharifying enzyme and pectinase are the lowest among various treatments (except comparative examples 4 and 5), the antioxidant activity of the soaked oat milk (comparative example 2) after heat shock is improved, and the hydroxyl radical clearance rate, DPPH radical clearance rate and reducing power are improved by 1.30 times, 73.3 times and 11.4 times compared with comparative example 1, so that the heat shock of the patent can activate the endogenous enzyme activity of the oat and release antioxidant factors in the subsequent soaking process. The specific protease and cellulase combinations and ratios used in examples 1 and 2 further increased antioxidant activity over heat shock, 37.8% -41.0%, 32.9% -35.8%, and 21.3% -55.2% higher hydroxyl radical scavenging, DPPH radical scavenging, and reducing power, respectively, than those of the heat shock group (comparative example 2).
The enzymatic oat milk treated by protease and cellulase with higher addition amount (comparative example 3) has higher total polysaccharide content than that of example 1 and example 2, but the antioxidant activity (hydroxyl radical clearance, DPPH radical clearance and reducing power) is lower than that of the example and lower than that of the heat shock oat milk (comparative example 2), which shows that the molecular weight of peptide and glucan in the oat milk is further reduced and the activity is lost by the treatment of protease and cellulase with higher addition amount, thus further proving the importance of the combination and proportion of the characteristic protease and cellulase used in the patent.
The solution (comparative example 4) is directly prepared by adopting the oat glucan extract with similar glucan content in heat shock oat, the result shows that the antioxidant activity of the glucan alone is lower, and the antioxidant activity (hydroxyl radical clearance rate, DPPH radical clearance rate and reducing power) of the oat beta-glucan (comparative example 5) which is subjected to enzymolysis by cellulase with the same enzyme activity as that in example 1 is improved by more than 1 time than that of the oat beta-glucan which is not subjected to enzymolysis (comparative example 4), thereby showing that the antioxidant activity can be improved by the enzymolysis of the glucan by restriction enzyme.
The enzyme preparation sources are different and the activities of protease and cellulase are the same, the enzymolysis conditions are the same as those of the example 1, the obtained enzymolysis oat milk (comparative example 6) has the same total polysaccharide content as those of the example 1 and the example 2, but the antioxidant activity (hydroxyl radical clearance, DPPH free radical clearance and reducing power) is lower than that of the example and than that of the high-enzyme-activity treatment group (comparative example 3), and the results show that even if the protease with the same enzyme activity is adopted, the types and the contents of the oat peptides in the enzymolysis oat milk are different due to different enzyme-cutting amino acid connection sites in the proteases of different manufacturers; the same cellulase activity enables the molecular weight of glucan in oat milk to be similar, but because cellulase of different manufacturers contains cellobiase, endonuclease and exonuclease in different proportions and the polysaccharide branch enzyme cutting capacity is different, the antioxidant activity of the obtained oat milk is weaker than that of the enzyme combination used in the patent, and the importance of the combination and proportion of the characteristic protease and the cellulase used in the patent is further proved.
Therefore, the patent improves the antioxidant activity of the oat milk by adding the characteristic protease and the cellulase on the basis of reducing the viscosity and the starch content of the oat milk by combining heat shock and soaking and by traditionally applying the amylase, the saccharifying enzyme and the pectinase.
6. Determination of polysaccharide molecular weight:
and 4mL of the product prepared in the example and the comparative example is taken, added with 4 times of volume of absolute ethyl alcohol for alcohol precipitation, kept overnight, dried and re-dissolved in 2mL of deionized water to obtain the coarse polysaccharide solution of the oat milk.
The polysaccharide molecular weight was determined using High Performance Liquid Chromatography (HPLC). The conditions were as follows:
chromatograph: agilent 1260; a chromatographic column: agiLent PL aquageL L-OH MIXED-M (300 mm. Times.8 mm); a detector: a differential refractive detector; mobile phase: 0.1mol/LNaNO 3 A solution; flow rate: 1.0mL/min; sample introduction amount: 20 mu L of the solution; column temperature: at 30 ℃.
With 0.1mol/L NaNO 3 The solutions were separately dissolved in dextran standards of molecular weight 2700, 5250, 9750, 13050, 36800, 64650, 135350g/mol, which were sequentially injected after passing through a 0.22 μm microporous filter. The results are shown in FIG. 11, where LgMw is plotted on the ordinate and retention time is plotted on the abscissa as a standard curve. The formula for calculating the relative molecular weight and retention time of the polysaccharide is: y = -0.5281x +8.9252, R2=0.9911, where x is the retention time.
The crude polysaccharide solution is added with 0.1mol/L NaNO 3 The solution was diluted, passed through a 0.22 μm millipore filter, injected and the retention time recorded. The relative molecular mass of the polysaccharide was calculated.
Table 2 shows the results of the molecular weight measurement of the polysaccharides in the examples of the present invention and the comparative examples.
TABLE 2 molecular weight determination of polysaccharides in inventive examples and comparative examples
- - - -: no peak was detected
As can be seen from Table 2, the ratio of 20KDa polysaccharide in the oat milk soaked after heat shock (comparative example 2) is obviously higher than that in the oat milk soaked directly (comparative example 1), which shows that heat shock can activate the endogenous enzyme activity in oat seeds and increase the polysaccharide extraction rate, and proves that the heat shock treatment of the invention is effective for dissolving out effective substances in oat.
The ratio of glucan with molecular weight of about 20KDa in example 1 and example 2 is obviously higher than that in comparative example 1, and the glucan with molecular weight of about 13KDa has higher ratio, while the comparative example 1 and comparative example 2 without adding cellulase have no component, which shows that the glucan is subjected to the restrictive hydrolysis by the proper characteristic cellulase, and the glucan with certain molecular weight and lower polymerization degree is obtained.
Comparative example 5 further demonstrates that a proportion of the characteristic cellulase can hydrolyze 11-12kDa glucans to 4.5kDa glycans.
The addition of the cellulase with a certain characteristic (comparative example 3) improves the dissolution of the oat macromolecular polysaccharide, but the low molecular weight glucan with about 7KDa is completely decomposed compared with 2 examples, still shows that the oligosaccharide with a certain molecular weight has a certain antioxidation effect, and simultaneously, the excessive cellulase further reduces the ratio of the oligosaccharide with the endogenous molecular weight of about 700Da in the oat compared with the examples, which shows that more endogenous oligosaccharide is decomposed into monosaccharide, so the addition amount of the cellulase with a certain characteristic is necessary for maintaining the oxidation activity of the glucan, and the excessive cellulase and the insufficient cellulase influence the content and the molecular weight of the glucan with the antioxidation activity.
The enzyme activities of the same cellulase of different manufacturers for enzymolysis of oat milk (comparative example 6) enable the oat macromolecular polysaccharide to be similar to the examples, but the proportion of glucan with 14KDa molecular weight is increased compared with 2 examples, which shows that the cellulase of the same enzyme activity source and different manufacturers can hydrolyze the oat milk to obtain the oat glucan with certain molecular weight, but the cellulase of different manufacturers is a mixture of different enzymes, comprising endonuclease, exonuclease and cellobiase with different proportions, and simultaneously containing a trace amount of non-glucan hydrolase, so that the addition amount of the cellulase of a specific manufacturer is necessary for keeping the antioxidant activity of the glucan.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (4)
1. An enzymolysis method for improving the antioxidant activity of oat milk comprises the following steps:
(1) Heat shock: heating oat in hot water, taking out, rinsing with clear water, and cooling to room temperature;
(2) Soaking: soaking the heat-shocked oat in clear water;
(3) Pulping: adding water into the soaked oat, pulping, and sieving the residue with a 60-100 mesh sieve to obtain oat milk with residue;
(4) Starch total enzymolysis and glucan restriction: adding 80U/mL-160U/mL cellulase, 30U/mL-40U/mL pectinase, 2U/mL-10U/mL alpha-amylase and 2U/mL-10U/mL diastase into the oat milk with dregs, carrying out enzymolysis, inactivating enzymes, and sieving to obtain enzymolysis oat milk 1;
(5) And (3) protease enzymolysis: adding papain with the mass fraction of 0.05-0.1% into the enzymatic oat milk 1, carrying out enzymolysis, and inactivating enzyme to obtain enzymatic oat milk 2;
(6) Performing compound enzymolysis by alkaline protease: adding alkaline protease with the mass fraction of 0.75% -1% into the enzymolysis oat milk 2, and carrying out enzymolysis and enzyme deactivation to obtain enzymolysis oat milk;
wherein, the cellulase: beijing Soilebao Tech Co., ltd (Solarbio), enzyme activity 500000U/g;
and (3) papain: the enzyme activity of Nanning Pompe bioengineering GmbH is 100000U/g;
alkaline protease: cangzhou Xiusheng enzyme biotechnology limited company, the enzyme activity is 200000U/g;
and (3) pectinase: shandonglongket enzyme preparation Co., ltd., enzyme activity 30000U/g;
alpha-amylase: the enzyme activity of the Beijing Olympic Star biotechnology Limited liability company is more than 3700U/g;
saccharifying enzyme: the enzyme activity of the Beijing Boototta science and technology Limited (Biotoped) is 100000U/g;
(7) Adding or not adding sweetener into the enzymolysis oat milk, adjusting the temperature to 70 ℃, homogenizing under 30Mpa, and sterilizing at 121 ℃ for 15min to obtain the enzymolysis oat milk with antioxidant activity.
2. The method of claim 1, wherein: in the step (1), the heat shock is 60-150 s in water with the temperature of 90-100 ℃;
in the step (2), the soaking is to make the thermally excited oat absorb water and expand to 1.5-2 times of the original mass;
in the step (3), the weight ratio of the oat to the water is 1:8-10;
the water temperature of pulping water is 70-90 ℃;
in the step (4), the enzymolysis temperature is 50-55 ℃, and the time is 0.5-1.0 h;
the enzyme deactivation operation is to deactivate enzyme by keeping the temperature at 100 ℃ for 8min-10 min;
the sieving is to sieve through a 100-200 mesh sieve;
in the step (5), the enzymolysis temperature is 50-55 ℃, and the time is 1.0-3.0 h;
the enzyme deactivation operation is to deactivate enzyme by keeping the temperature at 100 ℃ for 8min-10 min;
in the step (6), the enzymolysis temperature is 50-55 ℃, and the time is 1.0-3.0 h;
the enzyme inactivation is carried out by keeping the temperature at 100 ℃ for 8min-10 min.
3. An enzymatically hydrolyzed oat milk produced by the enzymatic hydrolysis method of claim 1 or 2.
4. Use of the enzymatically hydrolyzed oat milk of claim 3 in the preparation of an antioxidant product and/or an anti-aging product.
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| CN115887341A (en) * | 2022-12-22 | 2023-04-04 | 广州雷诺生物科技有限公司 | Red rice enzymolysis extract and preparation method thereof |
| CN117797057A (en) * | 2024-02-29 | 2024-04-02 | 广州诗妃生物科技有限公司 | Composition containing hyaluronic acid and application of composition in preparation of moisturizing products |
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| CN111771963A (en) * | 2020-08-06 | 2020-10-16 | 吴璇 | Heat shock and biological corrosion prevention combined fresh-keeping method |
| CN113875916A (en) * | 2021-10-08 | 2022-01-04 | 内蒙古工业大学 | Oat beverage and preparation method thereof |
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| CN111771963A (en) * | 2020-08-06 | 2020-10-16 | 吴璇 | Heat shock and biological corrosion prevention combined fresh-keeping method |
| CN113875916A (en) * | 2021-10-08 | 2022-01-04 | 内蒙古工业大学 | Oat beverage and preparation method thereof |
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| 陈东方;师俊玲;胡新中;: "纤维素酶水解处理提高燕麦全粉中总多酚含量与抗氧化活性", 食品科学, vol. 37, no. 01 * |
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| CN115633718A (en) * | 2022-10-19 | 2023-01-24 | 中国农业科学院油料作物研究所 | Method for improving plant milk enzymolysis efficiency through low-consumption physical double-field cooperation |
| CN115633718B (en) * | 2022-10-19 | 2024-03-15 | 中国农业科学院油料作物研究所 | Method for improving plant lactosis enzymolysis efficiency through low-consumption physical double-field cooperation |
| CN115887341A (en) * | 2022-12-22 | 2023-04-04 | 广州雷诺生物科技有限公司 | Red rice enzymolysis extract and preparation method thereof |
| CN115887341B (en) * | 2022-12-22 | 2023-12-08 | 广州雷诺生物科技有限公司 | Red rice enzymolysis extract and preparation method thereof |
| CN117797057A (en) * | 2024-02-29 | 2024-04-02 | 广州诗妃生物科技有限公司 | Composition containing hyaluronic acid and application of composition in preparation of moisturizing products |
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