CN112813125A - Preparation method of walnut peptide - Google Patents
Preparation method of walnut peptide Download PDFInfo
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- CN112813125A CN112813125A CN202110102236.6A CN202110102236A CN112813125A CN 112813125 A CN112813125 A CN 112813125A CN 202110102236 A CN202110102236 A CN 202110102236A CN 112813125 A CN112813125 A CN 112813125A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention belongs to the field of food processing, and discloses a preparation method of walnut peptide, which comprises the following steps: (1) cold pressing; (2) preparing suspension, homogenizing to obtain mixed solution, and soaking in deionized water; adding acid for regulation, standing, taking the lower layer precipitate, washing with water to neutrality, and collecting filtrate; (3) carrying out primary enzymolysis by Alcalase enzyme; enzyme deactivation; (4) secondary enzymolysis with cellulase and moderate-temperature amylase; enzyme deactivation treatment; (5) precipitating and centrifuging to obtain supernatant; adjusting the supernatant to neutral with dilute alkali solution, and ultrafiltering with ultrafiltration membrane; (6) concentrating in vacuum; (7) spray drying or freeze drying to obtain walnut peptide product. The walnut peptide produced by the method is milky white to light yellow powder, is easy to dissolve in water, has fresh taste and no obvious bitter taste, and the solution is clear and transparent, wherein the content of the polypeptide with the relative molecular weight less than 2000Dal is more than 87 percent.
Description
Technical Field
The invention belongs to the field of food processing, and relates to a preparation method of walnut peptide.
Background
The walnut is the unique resource industry of China, is the first of four nuts in the world, has high nutritional value and medical value, is rich in carbohydrate such as protein and the like, also contains calcium, phosphorus, iron, carotene, borrelidin, riboflavin, nicotinic acid and the like, has the functions of tonifying qi and nourishing blood, moistening dryness and reducing phlegm, warming lung and lubricating intestines in medicine, is a good tonic and is always an important material in international trade, so the walnut is favored by people in production areas. The walnut product can enhance brain activity and memory when being eaten, and has good effect on preventing and treating adolescent overstrain and middle-aged and elderly memory decline. Therefore, the walnut essence has the beautiful names of 'health-preserving fruit', 'longevity fruit' and 'brain-strengthening fruit'.
The walnut has vigorous market demand and wide development prospect. The walnut yield in China is 36.5 million tons, in recent years, many excellent new varieties are cultivated in China, multiple economic indexes reach the international excellent variety level, and the yield and the quality of the walnut in China are remarkably improved. If the deep processing of the walnuts is further carried out, export of the walnuts can create more foreign exchanges for the country. Walnut products in developed countries in foreign countries begin to develop towards refined classification, and the added value of various distinctive walnut products is far higher than that of common walnut milk drinks, walnut powder and the like in the market.
Active peptides hidden in food protein can be released by utilizing enzymolysis, the active peptides have wide physiological regulation functions besides the nutrition function, such as blood pressure reduction, opioid activity, antithrombotic property, immunity promotion, nutrient substance digestion and absorption promotion and the like, and the digestion and absorption performance in vivo is obviously superior to that of single amino acid. At present, various bioactive peptides have been isolated from enzymatic hydrolysate or fermentation products of various food proteins such as milk protein, soy protein, zein, fish and shellfish protein, collagen, and the like. They are characterized by wide sources, high edible safety and easy industrial production.
The study of walnut protein polypeptide has been introduced into the attention at home and abroad, and related research reports are continuously reported, but the development of industry is not much. According to market research, a small amount of walnut peptide products on the market fall behind in technology, which is mainly shown in that: the walnut protein obtained by the prior art can only replace other vegetable protein to be used for food processing and is mainly used as a food ingredient.
Disclosure of Invention
According to the technical problems existing in the development and utilization of the walnut peptide at present, the invention provides a preparation method of the walnut peptide. The invention has important significance for the diversification of walnut protein products and the development and utilization of walnut bioactive peptides in functional foods, health-care foods or medicines.
The purpose of the invention is realized by the following technical scheme:
a preparation method of walnut peptide comprises the following steps:
(1) cold pressing the walnuts at the temperature of 30-40 ℃, removing grease, crushing the walnut dregs after the oil pressing, and sieving by a 30-100-mesh sieve to obtain walnut dreg particles;
(2) preparing walnut pulp particles into a suspension by using deionized water, homogenizing the suspension to prepare a mixed solution, wherein the mass of the walnut pulp particles accounts for 5-8% of that of deionized water, adding alkali to adjust the pH value to 8.5-9, soaking for 1-3 h, and centrifuging to remove the lower-layer precipitate; adding acid to adjust the pH of the centrifugate to 4-4.5, standing for 1-3 h, centrifuging, collecting the lower layer precipitate, washing with water to neutrality, and collecting filtrate to obtain walnut protein solution;
(3) carrying out primary enzymolysis on Alcalase enzyme which is 0.1-0.2% of the mass of walnut meal in a walnut protein solution, wherein the primary enzymolysis condition is that the temperature is 45-55 ℃, the pH value is 8.5-9, and the time is 6-8 hours; after the primary enzymolysis is finished, carrying out enzyme deactivation treatment on the primary enzymolysis liquid;
(4) cooling the enzyme-deactivated primary enzymolysis liquid, adding cellulase accounting for 0.1-1.0% of the mass of the walnut protein and medium-temperature amylase accounting for 0.5-2.0%, wherein the secondary enzymolysis conditions are that the temperature is 40-60 ℃, the pH value is 6-7, and the time is 6-8 hours; after the secondary enzymolysis is finished, carrying out enzyme deactivation treatment on the secondary enzymolysis liquid;
(5) adjusting the secondary enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.1-0.5MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(6) performing vacuum concentration on the polypeptide liquid with the molecular weight of more than 500Da obtained by ultrafiltration treatment, and concentrating until the solid content of the polypeptide liquid is 30-50%;
(7) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product.
Further, the walnut pulp suspension is ground into pulp by a colloid mill and then is homogenized by a high-pressure homogenizer to prepare walnut pulp mixed liquor, wherein the homogenization pressure is 25-30 MPa.
Further, the enzyme deactivation treatment process comprises the following steps: heating the primary enzymolysis liquid or the secondary enzymolysis liquid to 80-85 ℃, and keeping the temperature for 10-20 min for enzyme deactivation.
Furthermore, the product is milky white to light yellow powder, is easy to dissolve in water, has fresh taste and no obvious bitter taste, and the content of the polypeptide with the relative molecular weight of less than 2000Dal is more than 87%.
The invention has the beneficial effects that: the invention solves the degradation problem of high-fat walnut protein, can degrade various nutrient components in the degreased walnut meal to generate walnut peptide which can be directly and rapidly absorbed by human bodies and has rich nutrition, has the functions of enhancing the immunity of the human bodies, resisting aging, resisting oxidation, recovering fatigue and the like, greatly improves the conveying capacity and the digestibility of the edible protein of the human bodies, and the obtained walnut peptide product meets the market requirements in the aspects of color, taste, purity and yield; the walnut peptide produced by the method is milky white to light yellow powder, is easy to dissolve in water, has fresh taste and no obvious bitter taste, and the solution is clear and transparent, wherein the content of the polypeptide with the relative molecular weight less than 2000Dal is more than 87%.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1
A preparation method of walnut peptide comprises the following steps:
(1) cold pressing walnut at 30 deg.C to remove oil, pulverizing the pressed walnut cake, and sieving with 30 mesh sieve to obtain walnut cake granule;
(2) preparing walnut pulp particles into suspension by using deionized water, grinding the walnut pulp suspension by using a colloid mill, homogenizing by using a high-pressure homogenizer to prepare walnut pulp mixed solution, wherein the homogenizing pressure is 25MPa, the mass of the walnut pulp particles accounts for 5% of the mass of deionized water, adding alkali to adjust the pH value to 8.5, soaking for 3 hours, and centrifuging to remove lower-layer precipitates; adding acid to adjust the pH of the centrifugate to 4, standing for 3h, performing centrifugal separation, taking the lower layer precipitate, washing with water to neutrality, and collecting filtrate to obtain walnut protein solution;
(3) adding Alcalase enzyme which is 0.1 percent of the mass of the walnut meal into the walnut protein solution for primary enzymolysis, wherein the primary enzymolysis condition is that the temperature is 45 ℃, the pH value is 8.5, and the time is 6 hours; after the primary enzymolysis is finished, heating the primary enzymolysis liquid to 80 ℃, and keeping the temperature for 20min for enzyme deactivation;
(4) cooling the enzyme-deactivated primary enzymolysis liquid, adding cellulase accounting for 0.1 percent of the mass of the walnut protein and medium-temperature amylase accounting for 0.5 percent of the mass of the walnut protein, and performing secondary enzymolysis for 6 hours at the temperature of 40 ℃ and the pH value of 6; after the secondary enzymolysis is finished, heating the secondary enzymolysis liquid to 80 ℃, and keeping the temperature for 20min for enzyme deactivation;
(5) adjusting the secondary enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.1MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(6) concentrating the polypeptide liquid with molecular weight above 500Da obtained by ultrafiltration under vacuum until the solid content of the polypeptide liquid is 30%;
(7) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product, wherein the product is milky-white to light yellow powder, is easy to dissolve in water, has fresh taste and no obvious bitter taste, and the content of the polypeptide with the relative molecular weight of less than 2000Dal is more than 87%.
Example 2
A preparation method of walnut peptide comprises the following steps:
(1) cold pressing walnut at 35 deg.C to remove oil, pulverizing the pressed walnut cake, and sieving with 60 mesh sieve to obtain walnut cake granule;
(2) preparing walnut pulp particles into suspension by using deionized water, grinding the walnut pulp suspension by using a colloid mill, homogenizing by using a high-pressure homogenizer to prepare walnut pulp mixed solution, wherein the homogenizing pressure is 27MPa, the mass of the walnut pulp particles accounts for 5-8% of the mass of deionized water, adding alkali to adjust the pH value to 8.5, soaking for 2 hours, and centrifuging to remove lower-layer precipitates; adding acid to adjust the pH of the centrifugate to 4, standing for 3h, performing centrifugal separation, taking the lower layer precipitate, washing with water to neutrality, and collecting filtrate to obtain walnut protein solution;
(3) adding Alcalase enzyme which is 0.1 percent of the mass of the walnut meal into the walnut protein solution for primary enzymolysis, wherein the primary enzymolysis condition is that the temperature is 50 ℃, the pH value is 8.5, and the time is 7 hours; after the primary enzymolysis is finished, heating the primary enzymolysis liquid to 83 ℃, and keeping the temperature for 15min for enzyme deactivation;
(4) cooling the enzyme-deactivated primary enzymolysis liquid, adding cellulase accounting for 0.1 percent of the mass of the walnut protein and medium-temperature amylase accounting for 0.5 percent of the mass of the walnut protein, and performing secondary enzymolysis for 7 hours at the temperature of 50 ℃ and the pH value of 6; after the secondary enzymolysis is finished, heating the secondary enzymolysis liquid to 83 ℃, and keeping the temperature for 15min for enzyme deactivation;
(5) adjusting the secondary enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.3MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(6) concentrating the polypeptide liquid with molecular weight above 500Da obtained by ultrafiltration under vacuum until the solid content of the polypeptide liquid is 30%;
(7) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product, wherein the product is milky-white to light yellow powder, is easy to dissolve in water, has fresh taste and no obvious bitter taste, and the content of the polypeptide with the relative molecular weight of less than 2000Dal is more than 87%.
Example 3
A preparation method of walnut peptide comprises the following steps:
(1) cold pressing walnut at 40 deg.C to remove oil, pulverizing the pressed walnut cake, and sieving with 100 mesh sieve to obtain walnut cake granule;
(2) preparing walnut pulp particles into suspension by using deionized water, grinding the walnut pulp suspension by using a colloid mill, homogenizing by using a high-pressure homogenizer to prepare walnut pulp mixed solution, wherein the homogenizing pressure is 30MPa, the mass of the walnut pulp particles accounts for 8% of the mass of deionized water, adding alkali to adjust the pH value to 9, soaking for 3 hours, and centrifuging to remove lower-layer precipitates; adding acid to adjust the pH of the centrifugate to 4.5, standing for 3h, performing centrifugal separation, collecting the lower layer precipitate, washing with water to neutrality, and collecting the filtrate to obtain walnut protein solution;
(3) adding Alcalase enzyme which is 0.1 percent of the mass of the walnut meal into the walnut protein solution for primary enzymolysis, wherein the primary enzymolysis condition is that the temperature is 55 ℃, the pH value is 9, and the time is 8 hours; after the primary enzymolysis is finished, heating the primary enzymolysis liquid to 85 ℃, and keeping the temperature for 10min for enzyme deactivation;
(4) cooling the enzyme-deactivated primary enzymolysis liquid, adding cellulase accounting for 0.1 percent of the mass of the walnut protein and medium-temperature amylase accounting for 0.5 percent of the mass of the walnut protein, and performing secondary enzymolysis for 8 hours at the temperature of 60 ℃ and the pH value of 6; after the secondary enzymolysis is finished, heating the secondary enzymolysis liquid to 85 ℃, and keeping the temperature for 10min for enzyme deactivation;
(5) adjusting the secondary enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.5MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(6) concentrating the polypeptide liquid with molecular weight above 500Da obtained by ultrafiltration under vacuum until the solid content of the polypeptide liquid is 30%;
(7) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product, wherein the product is milky-white to light yellow powder, is easy to dissolve in water, has fresh taste and no obvious bitter taste, and the content of the polypeptide with the relative molecular weight of less than 2000Dal is more than 87%.
Comparative example 1
A preparation method of walnut peptide comprises the following steps:
(1) cold pressing walnut at 30 deg.C to remove oil, pulverizing the pressed walnut cake, and sieving with 30 mesh sieve to obtain walnut cake granule;
(2) preparing walnut pulp particles into suspension by using deionized water, grinding the walnut pulp suspension by using a colloid mill, homogenizing by using a high-pressure homogenizer to prepare walnut pulp mixed solution, wherein the homogenizing pressure is 25MPa, the mass of the walnut pulp particles accounts for 5% of the mass of deionized water, adding alkali to adjust the pH value to 8.5, soaking for 3 hours, and centrifuging to remove lower-layer precipitates; adding acid to adjust the pH of the centrifugate to 4, standing for 3h, performing centrifugal separation, taking the lower layer precipitate, washing with water to neutrality, and collecting filtrate to obtain walnut protein solution;
(3) adding Alcalase enzyme which is 0.1 percent of the mass of the walnut meal into the walnut protein solution for primary enzymolysis, wherein the primary enzymolysis condition is that the temperature is 45 ℃, the pH value is 8.5, and the time is 6 hours; after the primary enzymolysis is finished, heating the primary enzymolysis liquid to 80 ℃, and keeping the temperature for 20min for enzyme deactivation;
(4) cooling the enzyme-deactivated primary enzymolysis liquid, adding cellulase which is 0.1 percent of the mass of the walnut protein, and performing secondary enzymolysis for 6 hours at the temperature of 40 ℃ and the pH value of 6; after the secondary enzymolysis is finished, heating the secondary enzymolysis liquid to 80 ℃, and keeping the temperature for 20min for enzyme deactivation;
(5) adjusting the secondary enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.1MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(6) concentrating the polypeptide liquid with molecular weight above 500Da obtained by ultrafiltration under vacuum until the solid content of the polypeptide liquid is 30%;
(7) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product.
Comparative example 2
A preparation method of walnut peptide comprises the following steps:
(1) cold pressing walnut at 30 deg.C to remove oil, pulverizing the pressed walnut cake, and sieving with 30 mesh sieve to obtain walnut cake granule;
(2) preparing walnut pulp particles into suspension by using deionized water, grinding the walnut pulp suspension by using a colloid mill, homogenizing by using a high-pressure homogenizer to prepare walnut pulp mixed solution, wherein the homogenizing pressure is 25MPa, the mass of the walnut pulp particles accounts for 5% of the mass of deionized water, adding alkali to adjust the pH value to 8.5, soaking for 3 hours, and centrifuging to remove lower-layer precipitates; adding acid to adjust the pH of the centrifugate to 4, standing for 3h, performing centrifugal separation, taking the lower layer precipitate, washing with water to neutrality, and collecting filtrate to obtain walnut protein solution;
(3) adding Alcalase enzyme which is 0.1 percent of the mass of the walnut meal into the walnut protein solution for primary enzymolysis, wherein the primary enzymolysis condition is that the temperature is 45 ℃, the pH value is 8.5, and the time is 6 hours; after the primary enzymolysis is finished, heating the primary enzymolysis liquid to 80 ℃, and keeping the temperature for 20min for enzyme deactivation;
(4) cooling the enzyme-deactivated primary enzymolysis liquid, adding medium-temperature amylase which is 0.5 percent of the mass of the walnut protein, and performing secondary enzymolysis for 6 hours at the temperature of 40 ℃ and the pH value of 6; after the secondary enzymolysis is finished, heating the secondary enzymolysis liquid to 80 ℃, and keeping the temperature for 20min for enzyme deactivation;
(5) adjusting the secondary enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.1MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(6) concentrating the polypeptide liquid with molecular weight above 500Da obtained by ultrafiltration under vacuum until the solid content of the polypeptide liquid is 30%;
(7) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product.
Comparative example 3
A preparation method of walnut peptide comprises the following steps:
(1) cold pressing walnut at 30 deg.C to remove oil, pulverizing the pressed walnut cake, and sieving with 30 mesh sieve to obtain walnut cake granule;
(2) preparing walnut pulp particles into suspension by using deionized water, grinding the walnut pulp suspension by using a colloid mill, homogenizing by using a high-pressure homogenizer to prepare walnut pulp mixed solution, wherein the homogenizing pressure is 25MPa, the mass of the walnut pulp particles accounts for 5% of the mass of deionized water, adding alkali to adjust the pH value to 8.5, soaking for 3 hours, and centrifuging to remove lower-layer precipitates; adding acid to adjust the pH of the centrifugate to 4, standing for 3h, performing centrifugal separation, taking the lower layer precipitate, washing with water to neutrality, and collecting filtrate to obtain walnut protein solution;
(3) adding Alcalase enzyme which is 0.1 percent of the mass of the walnut meal into the walnut protein solution for enzymolysis, wherein the enzymolysis condition is that the temperature is 45 ℃, the pH value is 8.5, and the time is 6 hours; after enzymolysis, heating the enzymolysis liquid to 80 ℃, and keeping the temperature for 20min for enzyme deactivation;
(4) adjusting the enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.1MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(5) concentrating the polypeptide liquid with molecular weight above 500Da obtained by ultrafiltration under vacuum until the solid content of the polypeptide liquid is 30%;
(6) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product.
Comparative example 4
A preparation method of walnut peptide comprises the following steps:
(1) cold pressing walnut at 30 deg.C to remove oil, pulverizing the pressed walnut cake, and sieving with 30 mesh sieve to obtain walnut cake granule;
(2) preparing walnut pulp particles into suspension by using deionized water, grinding the walnut pulp suspension by using a colloid mill, homogenizing by using a high-pressure homogenizer to prepare walnut pulp mixed solution, wherein the homogenizing pressure is 25MPa, the mass of the walnut pulp particles accounts for 5% of the mass of deionized water, adding alkali to adjust the pH value to 8.5, soaking for 3 hours, and centrifuging to remove lower-layer precipitates; adding acid to adjust the pH of the centrifugate to 4, standing for 3h, performing centrifugal separation, taking the lower layer precipitate, washing with water to neutrality, and collecting filtrate to obtain walnut protein solution;
(3) adding cellulase and medium temperature amylase which are equivalent to 0.1 percent and 0.5 percent of the mass of the walnut protein into the walnut protein solution, wherein the enzymolysis condition is that the temperature is 40 ℃, the pH value is 6, and the time is 6 hours; after enzymolysis, heating the enzymolysis liquid to 80 ℃, and keeping the temperature for 20min for enzyme deactivation;
(4) adjusting the enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.1MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(5) concentrating the polypeptide liquid with molecular weight above 500Da obtained by ultrafiltration under vacuum until the solid content of the polypeptide liquid is 30%;
(6) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product.
The walnut peptide powder prepared in the examples 1-3 and the comparative examples 1-4 is detected as follows: measuring the color, moisture, ash content and protein content of the walnut peptide powder by methods specified in GB/T5492, GB/T5009.3, GB/T5009.4, GB/T5009.5 and GB/T5009.6 respectively; adding semen Juglandis peptide powder into 50 deg.C pure water to obtain 3% solution, tasting its taste and smell; analyzing the content of the walnut peptide powder peptide by adopting a method specified in GB/T22729-2008 at 6.3; the proportion of the walnut peptide powder with the relative molecular weight of less than 2000Dal and 1000Dal peptides is analyzed by adopting GB/T22729-2008 appendix A high-efficiency gel filtration chromatography, and the result is shown in Table 1.
TABLE 1 analysis and detection results of walnut peptide powder prepared in examples 1-3 and comparative examples 1-4
The walnut peptide prepared by the method of the embodiment 1-3 has the mass percent of protein of 92.67-93.62% (more than or equal to 90%), the peptide content of 86.95-87.69% (more than or equal to 85%), the relative molecular weight of less than 2000Dal peptide is more than or equal to 87%, the relative molecular weight of less than 1000Dal peptide is more than or equal to 50%, and the walnut peptide is a high-quality high-purity protein peptide product.
Compared with the example 1, the medium temperature amylase is not added in the comparative example 1, the cellulase is not added in the comparative example 2, the product is light yellow in color and has no obvious bitter taste, the protein content, the peptide content and the peptide proportion of the relative molecular weight of less than 2000Dal and 1000Dal are all reduced, and the protein content, the peptide content and the small molecular peptide content of the product can be improved by adding the cellulase and the medium temperature amylase.
Compared with the example 1, the comparative example 3 adds Alcalase enzyme for one time of enzymolysis, the comparative example 4 adds cellulase and moderate temperature amylase for one time of enzymolysis, the obtained product has a deepened yellow color and luster, the difference of the flavor and the example 1 is obvious, the special flavor and smell of the product of the example 1 are not generated, the protein content, the peptide content and the relative molecular weight are less than 2000Dal, the 1000Dal peptide accounts for the reduction of the comparative example 1, and the difference of the product and the comparative example 1 and the comparative example 2 is obvious. The effect of singly adopting the cellulase and the medium-temperature amylase on removing the carbohydrate from the walnut protein substrate is not good in the combined use effect of the cellulase and the medium-temperature amylase, and the combined use has obvious synergistic effect; the effect of only carrying out primary enzymolysis and not carrying out secondary enzymolysis is good, and the difference is obvious.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (4)
1. The preparation method of the walnut peptide is characterized by comprising the following steps:
(1) cold pressing the walnuts at the temperature of 30-40 ℃, removing grease, crushing the walnut dregs after the oil pressing, and sieving by a 30-100-mesh sieve to obtain walnut dreg particles;
(2) preparing walnut pulp particles into a suspension by using deionized water, homogenizing the suspension to prepare a mixed solution, wherein the mass of the walnut pulp particles accounts for 5-8% of that of deionized water, adding alkali to adjust the pH value to 8.5-9, soaking for 1-3 h, and centrifuging to remove the lower-layer precipitate; adding acid to adjust the pH of the centrifugate to 4-4.5, standing for 1-3 h, centrifuging, collecting the lower layer precipitate, washing with water to neutrality, and collecting filtrate to obtain walnut protein solution;
(3) carrying out primary enzymolysis on Alcalase enzyme which is 0.1-0.2% of the mass of walnut meal in a walnut protein solution, wherein the primary enzymolysis condition is that the temperature is 45-55 ℃, the pH value is 8.5-9, and the time is 6-8 hours; after the primary enzymolysis is finished, carrying out enzyme deactivation treatment on the primary enzymolysis liquid;
(4) cooling the enzyme-deactivated primary enzymolysis liquid, adding cellulase accounting for 0.1-1.0% of the mass of the walnut protein and medium-temperature amylase accounting for 0.5-2.0%, wherein the secondary enzymolysis conditions are that the temperature is 40-60 ℃, the pH value is 6-7, and the time is 6-8 hours; after the secondary enzymolysis is finished, carrying out enzyme deactivation treatment on the secondary enzymolysis liquid;
(5) adjusting the secondary enzymolysis liquid to weak acidity by using dilute acid, and then precipitating and centrifuging the enzymolysis liquid to obtain supernatant; adjusting the supernatant to be neutral by using dilute alkali liquor, then performing ultrafiltration by using an ultrafiltration membrane, ensuring the operating pressure in the ultrafiltration process to be 0.1-0.5MPa, removing free amino acid components or short peptides with the molecular weight of less than 500Da in the enzymolysis liquid, and separating out the polypeptides with the relative molecular weight of more than 500 Da;
(6) performing vacuum concentration on the polypeptide liquid with the molecular weight of more than 500Da obtained by ultrafiltration treatment, and concentrating until the solid content of the polypeptide liquid is 30-50%;
(7) and drying the polypeptide concentrated solution by adopting a centrifugal spray drying method or a freeze drying method to finally obtain the walnut peptide product.
2. The preparation method of the walnut peptide according to claim 1, wherein the walnut pulp suspension is ground into slurry by a colloid mill and then homogenized by a high-pressure homogenizer at a homogenization pressure of 25-30 MPa to obtain the walnut pulp mixed solution.
3. The method for preparing walnut peptide according to claim 1, wherein the enzyme deactivation treatment process comprises: heating the primary enzymolysis liquid or the secondary enzymolysis liquid to 80-85 ℃, and keeping the temperature for 10-20 min for enzyme deactivation.
4. The method for preparing walnut peptide according to claim 1, wherein the product is milk-white to light yellow powder, easily soluble in water, fresh in taste, and free of significant bitterness, wherein the content of polypeptides with relative molecular weights less than 2000Dal is above 87%.
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| CN114606283B (en) * | 2021-12-17 | 2023-08-15 | 陕西师范大学 | Walnut polypeptide with good emulsifying property and preparation method thereof |
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