CN114703247B - High-absorptivity composite protein composition and preparation method and application thereof - Google Patents

High-absorptivity composite protein composition and preparation method and application thereof Download PDF

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CN114703247B
CN114703247B CN202210631568.8A CN202210631568A CN114703247B CN 114703247 B CN114703247 B CN 114703247B CN 202210631568 A CN202210631568 A CN 202210631568A CN 114703247 B CN114703247 B CN 114703247B
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张光明
董洪波
赵杰
王颖
张琦峰
万宏颖
洪伟雄
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Beijing Happy Energy Health Technology Co ltd
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Abstract

The invention provides a high-absorptivity composite protein composition, a preparation method and application thereof, and belongs to the technical field of proteins. Adding the whey protein powder into a disodium hydrogen phosphate-citric acid buffer solution, inoculating lactobacillus plantarum, bacillus coagulans and bifidobacterium adolescentis for fermentation, adding compound protease into fermentation liquor for enzymolysis, further performing synchronous ultrasonic and ozone treatment on the obtained enzyme hydrolysate, performing enzymolysis by using cross-linked compound enzyme, adding a metal ion solution, heating and stirring for reaction, and thus obtaining the high-absorptivity compound protein composition. The high-absorptivity composite protein composition prepared by the invention has moderate molecular weight, does not cause liver first-pass effect, has high absorption and utilization rate, has rich protein functions, and has obvious functions of resisting aging and oxidation, promoting fat combustion and promoting metabolism.

Description

High-absorptivity composite protein composition and preparation method and application thereof
Technical Field
The invention relates to the technical field of protein, in particular to a high-absorptivity composite protein composition and a preparation method and application thereof.
Background
Aerobic organisms including humans take in oxygen through respiration for survival, and a part of the oxygen taken in the body is converted into active oxygen species such as peroxide radicals, hydrogen peroxide, hydroxyl radicals and the like during energy metabolism. Such active oxygen species are originally involved in organism defense and the like represented by a mechanism of removing a pathogen from macrophages when infected with bacteria or viruses, and play an important role in maintaining health. However, environmental factors such as air pollution and ultraviolet rays, living habits such as smoking, stress, etc. break down the balance in the living body, and when active oxygen species become excessive, they react with proteins, lipids, DNA, etc. in the living body, and cause protein denaturation, lipid peroxide production, genetic damage, etc., resulting in occurrence of living habit diseases and aging promotion. Therefore, it is gradually recognized that the intake of antioxidant active ingredients from the diet is very important for maintaining health in addition to the mechanism possessed by the organism, and it is recommended to actively take antioxidant substances as the 7 th nutrient.
The synthetic antioxidant has the advantages of low price, high yield and the like, and is widely applied to the industries of food, skin care products and cosmetics. However, with the economic development, people pay more and more attention to health, so that people prefer safe, purely natural and non-toxic and side-effect natural antioxidants, such as tea polyphenol, ascorbic acid, protein peptide and the like. There are many kinds of food-derived antioxidant peptides, and they are classified into antioxidant peptides of plant origin, animal origin, milk origin, egg origin, and the like according to their source. The plant source mainly comprises corn, soybean, wheat grain, rice bran, rapeseed, peanut, bean olecranon, rice residue, pricklyash seed, walnut, black bean, wheat germ, etc.
The method for preparing the antioxidant peptide by adopting the enzyme hydrolyzed protein is a mainstream method, but because a plurality of proteases have specific enzyme cutting sites and uniqueness of protein amino acid sequences, different proteins and enzyme combinations are selected to have important significance for generating the peptide compound with unique functionality. In the case of the existing enzymatic hydrolysis methods, some proteases without specificity or with wide specificity are used, such methods have low production repeatability and can generate free amino acids, or the peptide chain can be hydrolyzed too small to cause reduced functionality, the yield of the final product is also low, the absorption and utilization rate of the antioxidant peptide by human body is reduced, and the method for preparing the antioxidant peptide with high absorption rate stably and efficiently is also lacked.
Whey is a cheese by-product with a high annual yield. Whey protein is the main component of whey, and contains a plurality of components, mainly including beta-lactoglobulin, alpha-lactalbumin, immunoglobulin, bovine serum albumin and the like. Whey protein contains abundant essential amino acids, is easy to digest and absorb by organisms, has the reputation of 'the king of protein', is used as an important raw material and ingredient of functional food, nutritional products and the like, and is widely applied to food production. At present, whey protein products are mainly prepared by ultrafiltration technology or diafiltration technology, concentrated whey protein can be classified into 4 types, namely WPC34, WPC50, WPC75 and WPC80 according to the difference of protein content, and the separated whey protein refers to the whey protein products with the protein relative content higher than 90%.
Some components in whey protein easily cause allergic reaction, and beta-lactoglobulin and alpha-lactalbumin are main allergens and can enable an organism to generate immune response, so that strong sensitization is shown. The property of the whey protein is unstable, and the high-level structure is easily damaged in the processing and production process, so that the physical, chemical and biological functions of the whey protein are changed, and the application of the whey protein is limited. The distribution of hydrophobic groups, spatial arrangement conformation, amino acid composition and the like of the whey protein can be changed by modifying the whey protein, so that the functional property of the whey protein is improved or new functional characteristics are developed.
Disclosure of Invention
The invention aims to provide a high-absorptivity composite protein composition, a preparation method and application thereof, the preparation method is simple, the prepared high-absorptivity composite protein composition has moderate molecular weight, can be well absorbed and utilized by a human body, does not cause the first-pass effect of the liver, has high absorptivity, and simultaneously has rich protein functions, and has obvious functions of resisting aging and oxidation, promoting fat burning and promoting metabolism.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a high-absorptivity composite protein composition, which comprises the steps of adding whey protein powder into disodium hydrogen phosphate-citric acid buffer solution, inoculating lactobacillus plantarum, bacillus coagulans and bifidobacterium adolescentis for fermentation, adding composite protease into fermentation liquor for enzymolysis, further carrying out synchronous ultrasonic and ozone treatment on obtained enzyme hydrolysate, carrying out enzymolysis by using cross-linked composite enzyme, adding metal ion solution, heating and stirring for reaction, and preparing the high-absorptivity composite protein composition;
the compound protease is a compound of alkaline protease and papain;
the cross-linked complex enzyme is a complex of glutamine transaminase and laccase.
As a further improvement of the invention, the method comprises the following steps:
s1, preparation of a protein solution: adding whey protein powder into a disodium hydrogen phosphate-citric acid buffer solution to obtain a protein solution;
s2, activating strains: respectively inoculating Lactobacillus plantarum, Bacillus coagulans and Bifidobacterium adolescentis into sterile MRS liquid culture medium, activating, culturing, centrifuging, discarding supernatant, washing thallus, centrifuging, discarding supernatant, and using sterile CaCl 2 The normal saline suspends the somatic cells to prepare a resting cell suspension;
s3, fermentation: sequentially inoculating the resting cell suspension of the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis prepared in the step S2 into the protein solution prepared in the step S1, fermenting, culturing, centrifuging, and taking supernatant to obtain fermentation liquor;
s4, enzyme hydrolysis: adding compound protease into the fermentation liquor prepared in the step S3, performing enzymolysis reaction, inactivating enzyme, and centrifuging to obtain enzyme hydrolysate; the compound protease is a compound of alkaline protease and papain;
s5, ultrasonic wave and ozone synergistic treatment: carrying out ultrasonic treatment on the enzymatic hydrolysate prepared in the step S4, and simultaneously introducing ozone for treatment to obtain a treatment solution;
s6, enzyme weak crosslinking: adding malic acid into the treatment liquid prepared in the step S5, uniformly mixing, adding a cross-linked complex enzyme, carrying out enzymolysis reaction, inactivating the enzyme, and centrifuging to obtain a weak cross-linked protein peptide solution; the cross-linked complex enzyme is a compound of glutamine transaminase and laccase;
s7, metal ion complexing: and (4) adding a metal ion solution into the weak cross-linked protein peptide solution prepared in the step S6, heating, stirring, reacting, and freeze-drying to obtain the high-absorptivity composite protein composition.
As a further improvement of the invention, in the step S1, the whey protein powder is at least one selected from the group consisting of separated whey protein powder, concentrated milk protein powder and hydrolyzed whey protein powder, wherein one selected from the group consisting of a compound mixture of separated whey protein powder, concentrated milk protein powder and hydrolyzed whey protein powder in a mass ratio of 5-10:3-5: 2-5; the pH value of the disodium hydrogen phosphate-citric acid buffer solution is 7.2-8; the solid-liquid ratio of the protein powder to the disodium hydrogen phosphate-citric acid buffer solution is 1: 50-100 g/mL.
As a further improvement of the invention, the condition of the activation culture in the step S2 is 36-40 ℃, under the micro-aerobic condition, 20-50r/min, the culture lasts 18-24 h; the micro-aerobic condition is that the oxygen concentration is 5-10 percent and CO 2 The concentration is 3-7%, the balance is nitrogen, wherein the volume percentage is; the centrifugal rotating speed is 3000-5000r/min, and the time is 10-15 min; the resting cell suspension is measured to have an optical density value OD of 600nm 600 Is 18-25 OD/mL.
As a further improvement of the invention, the inoculation amounts of the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis in the step S3 are respectively 2-4%, 1-3% and 3-5%; the fermentation culture condition is 36-38 ℃, and the culture is carried out for 24-48h at 20-50r/min under the micro-aerobic condition; the micro-aerobic condition is that the oxygen concentration is 5-10 percent and CO 2 The concentration is 3-7%, and the balance is nitrogen, wherein the% is volume percentage; the centrifugal rotating speed is 3000-5000r/min, and the time is 10-15 min.
As a further improvement of the invention, in the step S4, the mass ratio of the alkaline protease to the papain is 3-7: 2-5; the solid-liquid ratio of the compound protease to the fermentation liquor is 1: (100-200) g/mL; the temperature of the enzymolysis reaction is 40-50 ℃, and the time is 3-7 h; the enzyme killing method comprises 1000-1500W ultrasonic treatment for 10-15 min; in the step S5, the ultrasonic treatment condition is 100-200W ultrasonic treatment, the flow rate of the ozone treatment gas is 15-25L/h, the concentration of ozone is 30-70mg/L, and the treatment time is 2-5S.
As a further improvement of the invention, the mass ratio of the malic acid to the cross-linked complex enzyme in the step S6 is 1-2: 5-12; the mass ratio of the cross-linked complex enzyme to the glutamine transaminase is 2-5: 3-7; the enzymolysis reaction is carried out under the conditions of 40-50 ℃ for 1-2h, the enzyme deactivation method is 1000-1500W ultrasonic treatment for 10-15min, the centrifugal rotation speed is 3000-5000r/min, and the time is 10-15 min; in step S7, the metal ion solution contains copper ions, zinc ions and iron ions, and the contents are 1-3wt%, 2-4wt% and 3-5wt%, respectively; the mass ratio of the metal ion solution to the weak cross-linked protein peptide solution is 2-5: 10; heating at 55-60 deg.C for 20-40 min.
As a further improvement of the invention, the method specifically comprises the following steps:
s1, preparation of a protein solution: uniformly mixing 5-10 parts by weight of separated whey protein powder, 3-5 parts by weight of concentrated milk protein powder and 2-5 parts by weight of hydrolyzed whey protein powder to obtain protein powder, and adding the protein powder into a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 7.2-8 to obtain a protein solution; the solid-liquid ratio of the protein powder to the disodium hydrogen phosphate-citric acid buffer solution is 1: (50-100) g/mL;
s2, activating strains: respectively inoculating Lactobacillus plantarum, Bacillus coagulans and Bifidobacterium adolescentis into sterile MRS liquid culture medium, activating and culturing at 36-40 deg.C under micro-aerobic condition for 18-24 hr for 20-50r/min, centrifuging, discarding supernatant, washing thallus, centrifuging, discarding supernatant, and adding sterile CaCl 2 The physiological saline of (1) suspending the somatic cells, preparing a resting cell suspension, which is measured for its optical density value OD at 600nm 600 Is 18-25 OD/mL;
s3, fermentation: sequentially inoculating the resting cell suspension of the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis prepared in the step S2 into the protein solution prepared in the step S1, wherein the inoculation amounts are 2-4%, 1-3% and 3-5%, respectively, the inoculation amounts are 36-38 ℃, the fermentation culture time is 24-48h under the micro-aerobic condition, and the fermentation broth is obtained after centrifugation and supernatant fluid taking;
s4, enzyme hydrolysis: adding compound protease into the fermentation liquor prepared in the step S3, wherein the solid-to-liquid ratio of the compound protease to the fermentation liquor is 1: (100) g/mL, carrying out enzymolysis reaction at 40-50 ℃ for 3-7h, inactivating enzyme, and centrifuging to obtain enzyme hydrolysate; the compound protease is a compound of alkaline protease and papain; the mass ratio of the alkaline protease to the papain is (3-7): 2-5;
s5, ultrasonic wave and ozone synergistic treatment: synchronously performing 100-200W ultrasonic treatment and ozone treatment on the enzyme hydrolysate prepared in the step S4, wherein the gas flow is 15-25L/h, the ozone concentration is 30-70mg/L, and the treatment time is 2-5S to obtain a treatment solution;
s6, enzyme weak crosslinking: adding 1-2 parts by weight of malic acid into the treatment fluid prepared in the step S5, uniformly mixing, adding 5-12 parts by weight of cross-linked complex enzyme, carrying out enzymolysis reaction at 40-50 ℃ for 1-2h, inactivating the enzyme, and centrifuging to obtain a weakly cross-linked protein peptide solution; the cross-linked complex enzyme is a compound of glutamine transaminase and laccase; the mass ratio of the cross-linked complex enzyme to the glutamine transaminase is 2-5: 3-7;
s7, metal ion complexing: adding 2-5 parts by weight of metal ion solution into 10 parts by weight of the weakly cross-linked protein peptide solution prepared in the step S6, wherein the metal ion solution contains 1-3wt%, 2-4wt% and 3-5wt% of copper ions, zinc ions and iron ions respectively, heating to 55-60 ℃, stirring and reacting for 20-40min, and freeze-drying to obtain a high-absorptivity composite protein composition;
the micro-aerobic condition is that the oxygen concentration is 5-10 percent and CO is 2 The concentration is 3-7%, the balance is nitrogen, wherein the volume percentage is; the centrifugal rotating speed is 3000-5000r/min, and the time is 10-15 min; the enzyme deactivation method is ultrasonic treatment of 1000-1500W for 10-15 min.
The invention further protects the high-absorptivity composite protein composition prepared by the preparation method.
The invention further protects the application of the high-absorptivity composite protein composition in preparing anti-aging products and/or anti-oxidation products.
The invention has the following beneficial effects:
according to the invention, the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis are fermented by the composite probiotics, the composite protein powder containing the isolated whey protein powder, the concentrated milk protein powder and the hydrolyzed whey protein powder can be primarily fermented to generate a large amount of micromolecular protein peptides, micromolecular short peptides and a large amount of amino acid substances, and meanwhile, the bacillus coagulans has good alkali resistance, and the three can cooperate with each other to achieve the effect of efficiently fermenting the protein powder;
the alkaline protease acts on amide bonds at carboxyl terminals of all hydrophobic and aromatic amino acids on carboxyl side chains to catalyze peptide bonds in protein molecules to hydrolyze to generate peptides with smaller molecular mass, so that antigen binding sites in whey protein molecules are damaged, and the allergenicity is obviously reduced. The alkaline protease has good hydrolysis effect, and the beta-lactoglobulin can only be completely hydrolyzed by the alkaline protease, so that the antigenicity of the alpha-lactalbumin and beta-lactoglobulin antibodies can be obviously reduced, and the allergenicity of the whey protein can be greatly reduced. The papain is a single-chain endonuclease, can hydrolyze peptide bonds at carboxyl terminals of lysine, arginine and glycine in polypeptide, amide and ester, has strong hydrolysis capacity, can strongly destroy epitope of whey protein, and has good antigenicity.
The protein is hydrolyzed by enzyme method, and then polypeptide chain is degraded, thereby realizing the reconnection of whey protein molecules and improving functional characteristics. The invention utilizes alkaline protease and papain to carry out double enzymolysis, wherein the alkaline protease is nonspecific protein endopeptidase, has wide action substrates, can hydrolyze all amino acids with hydrophobicity or aromatic at carboxyl terminals, and the papain and the peptide bond catalyzed by the alkaline protease have complementarity, thereby hydrolyzing protein to the maximum extent.
Generally, peptide fragments of hydrolyzed protein are shorter than intact protein, free amino acids enter blood circulation faster after being digested and absorbed by gastrointestinal tract, once the concentration of the free amino acids is higher than the net synthetic capacity of the protein, the first-pass effect of liver is increased, the amino acids are oxidized and metabolized, so that more amino acids and protein cannot be utilized by human body, therefore, the utilization rate and the absorption rate of the protein are not higher, the protein is not properly crosslinked, and the protein has secondary and tertiary structures, so that the protein is more beneficial to the absorption and utilization.
Therefore, after the protein is subjected to enzymolysis and fermentation to obtain small molecular structures such as small molecular short peptides, amino acids and the like, the small molecular structures are further subjected to proper crosslinking to obtain a particle structure, so that the protein composition disclosed by the invention is further promoted to be absorbed and utilized in a human body, and thus, the protein composition has better physiological activity.
In the low-power ultrasonic treatment process, the structure of the protein allergen can be changed under the action of ultrasonic waves, and the repeated combination reaction of protein peptide substances in the enzyme hydrolysate and ozone can be promoted, so that the effect of promoting the modification of ozone is achieved; the concentration of alpha-lactalbumin and beta-lactoglobulin monomers of protein peptides after the short-time oxidation treatment by ozone is reduced, the allergenicity is reduced, and simultaneously, the content of disulfide bonds is increased, because sulfydryl on side chains of two molecules of cysteine are aggregated to form new disulfide bonds, so that simple dimeric peptides are obtained, and the molecular weight of the short peptides is preliminarily improved; because the time is short, other reducing amino acids on the protein peptide have not yet come to have oxidation reaction, so the property of the protein peptide is not greatly influenced;
the cross-linked complex enzyme comprises glutamine transaminase and laccase; wherein, the glutamine transaminase is a transferase which can be used for cross-linking polymerization, deamidation and primary amine introduction, the enzyme takes amino groups of lysine residues on protein chains, epsilon-amino groups of free amino acids and the like as acceptors, acyl donors are gamma-hydroxyamide groups on the glutamine residues, and epsilon- (gamma-glutamine) lysine cross-linked chains are formed in molecules and among molecules, so as to obtain a weakly cross-linked protein peptide product; in addition, the laccase is polyphenol oxidase, contains copper elements, can form a compound with malic acid, and catalyzes and oxidizes phenol, the protein peptide substance prepared in the steps forms substrate free radicals after reacting with the laccase-malic acid compound, and simultaneously, the laccase and copper ions in the laccase act synergistically to oxidize-SH in the protein peptide into S-S, tyrosine residues are oxidized into quinone, and the quinone can perform Schiff base addition or Michael addition with active amino groups such as primary amine of free amino acids, so that the weak crosslinking effect of the protein peptide is realized. After the compound cross-linking action of glutamine transaminase and laccase, small molecular short peptide, short polypeptide and amino acid can be combined with each other, so that a protein peptide compound with moderate molecular weight is prepared, the protein peptide compound is beneficial to absorption and utilization of a human body, the digestion and absorption rate of gastrointestinal tracts is moderate, the first pass effect of liver cannot be caused, the absorption and utilization rate of the prepared protein peptide is greatly improved, and the quality of protein is improved.
Furthermore, the invention adds metal ion solution containing copper ion, zinc ion and iron ion into the weak cross-linking protein peptide, these ions are easy to complex with the group on the protein peptide to form protein-metal complex, after these complexes enter into human body, they can release metal ion under proper condition, and participate in the enzyme reaction of human body, so as to play a good role in resisting senility, resisting oxidation, promoting fat combustion and promoting metabolism.
The high-absorptivity composite protein composition prepared by the invention has a simple preparation method, has moderate molecular weight, can be well absorbed and utilized by a human body, does not cause the first-pass effect of the liver, has high absorptivity, has rich protein peptide functions, and has obvious effects of resisting aging and oxidation, promoting fat combustion and promoting metabolism.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Whey protein isolate (92%, food grade) purchased from Henan Natural, New Zealand; concentrated milk protein powder (effective component >80%, food grade) purchased from Qingdao Hevesen Biotech limited; hydrolyzed whey protein powder (99%, food grade), brand Hilmar, available from australian biotechnology limited, south river;
alkaline protease Alkaline (200000U/g) and Papain Papain (800000U/g) are available from Beijing Soilebao Tech technologies, Inc. Glutamine transaminase (2000000U/g), available from Hirschhorn China Biotechnology, Inc., Dongning; laccase (50000U/g) from Qiansheng Biotechnology, Inc., Hebei.
MRS (DeManRogosa-sharp) liquid medium: 10g of beef extract powder, 10g of soybean peptone, 20g of glucose, 5g of yeast powder, 801mL of Tween, 2g of dipotassium phosphate, 5g of anhydrous sodium acetate, 2g of triammonium citrate, 200mg of magnesium sulfate and 50mg of manganese sulfate, and if the beef extract powder is a solid culture medium, 15g of agar powder is added. Adding water to 1L, pH6.2, sterilizing at 121 deg.C for 15 min.
Lactobacillus plantarum (50 hundred million cfu/g) purchased from Weifang Rui Biotechnology Limited; bacillus coagulans (100 hundred million cfu/g) available from Luoyang Ohioc Biotech GmbH; bifidobacterium adolescentis (100 hundred million cfu/g) was purchased from Jiayi bioengineering technology, Inc., of Shandong, Zhongzhou.
Example 1
A preparation method of a high-absorptivity composite protein composition specifically comprises the following steps:
s1, preparation of a protein solution: uniformly mixing 5 parts by weight of separated whey protein powder, 3 parts by weight of concentrated milk protein powder and 2 parts by weight of hydrolyzed whey protein powder to obtain protein powder, and adding the protein powder into a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 7.2 to obtain a protein solution; the solid-liquid ratio of the protein powder to the disodium hydrogen phosphate-citric acid buffer solution is 1: 50 g/mL;
s2, activating strains: inoculating Lactobacillus plantarum, Bacillus coagulans and Bifidobacterium adolescentis into sterile MRS liquid culture medium respectively, and culturing at 36 deg.C under microaerophilic condition (oxygen concentration of 5% and CO content of microaerophilic condition) 2 The concentration is 3 percent,the balance being nitrogen, wherein the volume percent) is 20r/min, activated and cultured for 18h, centrifuged for 10min at 3000r/min, the supernatant fluid is discarded, and sterile 5wt percent CaCl is used 2 Washing thallus with physiological saline, centrifuging at 3000r/min for 10min, discarding supernatant, and using sterile 5wt% CaCl 2 The physiological saline of (1) suspending the somatic cells, preparing a resting cell suspension, which is measured for its optical density value OD at 600nm 600 Is 18 OD/mL;
s3, fermentation: sequentially inoculating the resting cell suspension of the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis prepared in the step S2 into the protein solution prepared in the step S1, wherein the inoculation amounts are respectively 2%, 1% and 3%, and 36 ℃, and the micro-aerobic conditions are micro-aerobic conditions (the micro-aerobic conditions are that the oxygen concentration is 5%, and the CO concentration is 5%) 2 3 percent of concentration and the balance of nitrogen, wherein the percent is volume percent), fermenting and culturing for 24 hours at 20r/min, centrifuging at 3000r/min for 10min, and taking supernatant to obtain fermentation liquor;
s4, enzyme hydrolysis: adding compound protease into the fermentation liquor prepared in the step S3, wherein the solid-to-liquid ratio of the compound protease to the fermentation liquor is 1: performing enzymolysis reaction at 40 ℃ for 3h at 100g/mL, performing ultrasonic treatment at 1000W for 10min, and centrifuging at 3000r/min for 10min to obtain enzymatic hydrolysate; the compound protease is a compound of alkaline protease and papain, and the mass ratio of the compound protease to the papain is 3: 2;
s5, ultrasonic wave and ozone synergistic treatment: synchronously carrying out 100 parts by weight of ultrasonic treatment and ozone treatment by 100W on the enzyme hydrolysate prepared in the step S4, wherein the gas flow is 15L/h, the ozone concentration is 30mg/L, and the treatment time is 2S, so as to obtain a treatment solution;
s6, enzymatic weak crosslinking: adding 1 part by weight of malic acid into 100 parts by weight of the treatment solution prepared in the step S5, uniformly mixing, adding 5 parts by weight of cross-linked complex enzyme, carrying out enzymolysis reaction at 40 ℃ for 1h, carrying out ultrasonic treatment at 1000W for 10min, and centrifuging at 3000r/min for 10min to obtain a weakly cross-linked protein peptide solution; the cross-linked complex enzyme is a compound of glutamine transaminase and laccase with the mass ratio of 2: 3;
s7, metal ion complexing: and (2) adding 2 parts by weight of metal ion solution into 10 parts by weight of the weakly cross-linked protein peptide solution prepared in the step S6, heating the metal ion solution to 55 ℃, stirring and reacting for 20min, and freeze-drying to obtain the high-absorptivity composite protein composition, wherein the metal ion solution contains 1wt% of copper ions, 2wt% of zinc ions, 3wt% of iron ions and the balance of deionized water.
Example 2
A preparation method of a high-absorptivity composite protein composition specifically comprises the following steps:
s1, preparation of a protein solution: uniformly mixing 10 parts by weight of separated whey protein powder, 5 parts by weight of concentrated milk protein powder and 5 parts by weight of hydrolyzed whey protein powder to obtain protein powder, and adding the protein powder into a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 8 to obtain a protein solution; the solid-liquid ratio of the protein powder to the disodium hydrogen phosphate-citric acid buffer solution is 1: 100 g/mL;
s2, activating strains: inoculating Lactobacillus plantarum, Bacillus coagulans and Bifidobacterium adolescentis into sterile MRS liquid culture medium respectively, and culturing at 40 deg.C under microaerophilic condition (oxygen concentration is 10%, CO is 2 Concentration of 7 percent and the balance of nitrogen gas, wherein the percent is volume percent), at 50r/min, activating and culturing for 24h, centrifuging for 15min at 5000r/min, discarding supernatant, and using sterile 5wt percent CaCl 2 Washing thallus with physiological saline, centrifuging at 5000r/min for 15min, discarding supernatant, and using sterile 5wt% CaCl 2 The physiological saline of (1) suspending the somatic cells, preparing a resting cell suspension, which is measured for its optical density value OD at 600nm 600 Is 25 OD/mL;
s3, fermentation: sequentially inoculating the lactobacillus plantarum, bacillus coagulans and bifidobacterium adolescentis resting cell suspension prepared in the step S2 into the protein solution prepared in the step S1, wherein the inoculation amounts are 4%, 3% and 5%, 38 ℃, and the micro-aerobic conditions are micro-aerobic conditions (the micro-aerobic conditions are that the oxygen concentration is 10%, and CO is used for oxygen concentration) 2 Concentration of 7% and the balance of nitrogen gas, wherein the% is volume percentage), fermenting and culturing for 48h at 50r/min, centrifuging at 5000r/min for 15min, and taking supernatant to obtain fermentation liquor;
s4, enzyme hydrolysis: adding compound protease into the fermentation liquor prepared in the step S3, wherein the solid-to-liquid ratio of the compound protease to the fermentation liquor is 1: performing enzymolysis reaction at 50 ℃ for 7h at 200g/mL, performing 1500W ultrasonic treatment for 15min, and centrifuging at 5000r/min for 15min to obtain enzymatic hydrolysate; the compound protease is a compound of alkaline protease and papain, and the mass ratio of the compound protease to the papain is 7: 5;
s5, ultrasonic wave and ozone cooperation treatment: synchronously performing ultrasonic treatment and ozone treatment on 100 parts by weight of the enzyme hydrolysate prepared in the step S4 at 200W, wherein the gas flow is 25L/h, the ozone concentration is 70mg/L, and the treatment time is 5S to obtain a treatment solution;
s6, enzyme weak crosslinking: adding 2 parts by weight of malic acid into 100 parts by weight of the treatment solution prepared in the step S5, uniformly mixing, adding 12 parts by weight of cross-linked complex enzyme, carrying out enzymolysis reaction at 50 ℃ for 2 hours, carrying out 1500W ultrasonic treatment for 15min, and centrifuging at 5000r/min for 15min to obtain a weakly cross-linked protein peptide solution; the cross-linked complex enzyme is a compound of glutamine transaminase and laccase with the mass ratio of 5: 7;
s7, metal ion complexing: and (2) adding 5 parts by weight of metal ion solution into 10 parts by weight of the weakly cross-linked protein peptide solution prepared in the step S6, heating the metal ion solution to 60 ℃, stirring and reacting for 40min, and freeze-drying to obtain the high-absorptivity composite protein composition, wherein the metal ion solution contains 3wt% of copper ions, 4wt% of zinc ions, 5wt% of iron ions and the balance of deionized water.
Example 3
A preparation method of a high-absorptivity composite protein composition specifically comprises the following steps:
s1, preparation of a protein solution: uniformly mixing 7 parts by weight of separated whey protein powder, 4 parts by weight of concentrated milk protein powder and 3.5 parts by weight of hydrolyzed whey protein powder to obtain protein powder, and adding the protein powder into a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 7.7 to obtain a protein solution; the solid-liquid ratio of the protein powder to the disodium hydrogen phosphate-citric acid buffer solution is 1: 70 g/mL;
s2, activating strains: inoculating Lactobacillus plantarum, Bacillus coagulans and Bifidobacterium adolescentis into sterile MRS liquid culture medium respectively at 37 deg.C under microaerophilic condition (microaerophilic condition is oxygen concentration of 7%, CO) 2 5 percent of concentration and the balance of nitrogen gas, wherein the percent is volume percentage), 35r/min, 21h of activation culture, 4000r/min of centrifugation for 12min, discarding the supernatant, and using 5 percent of sterile CaCl 2 Washing thallus with physiological saline, centrifuging at 4000r/min for 12min, discarding supernatant, and using sterile 5wt% CaCl 2 The physiological saline of (1) suspending the somatic cells, preparing a resting cell suspension, which is measured for its optical density value OD at 600nm 600 Is 20 OD/mL;
s3, fermentation: sequentially inoculating the lactobacillus plantarum, bacillus coagulans and bifidobacterium adolescentis resting cell suspension prepared in the step S2 into the protein solution prepared in the step S1, wherein the inoculation amounts are 3%, 2% and 4%, respectively, and the micro-aerobic conditions are 37 ℃ and are micro-aerobic conditions (the micro-aerobic conditions are that the oxygen concentration is 7%, and CO is used for preparing the protein solution) 2 5 percent of concentration and the balance of nitrogen, wherein the percent is volume percentage), 35r/min, 36h of fermentation culture, 4000r/min of centrifugation for 12min, and taking supernatant fluid to obtain fermentation liquor;
s4, enzyme hydrolysis: adding compound protease into the fermentation liquor prepared in the step S3, wherein the solid-to-liquid ratio of the compound protease to the fermentation liquor is 1: performing enzymolysis reaction at 45 ℃ for 5h at 150g/mL, performing ultrasonic treatment at 1250W for 12min, and centrifuging at 45000r/min for 12min to obtain enzyme hydrolysate; the compound protease is a compound of alkaline protease and papain, and the mass ratio of the compound protease to the papain is 5: 3;
s5, ultrasonic wave and ozone synergistic treatment: synchronously performing 150W ultrasonic treatment and ozone treatment on 100 parts by weight of the enzyme hydrolysate prepared in the step S4, wherein the gas flow is 20L/h, the ozone concentration is 50mg/L, and the treatment time is 3S to obtain a treatment solution;
s6, enzymatic weak crosslinking: adding 1.5 parts by weight of malic acid into 100 parts by weight of the treatment solution prepared in the step S5, uniformly mixing, adding 9 parts by weight of cross-linked complex enzyme, carrying out enzymolysis reaction at 45 ℃ for 1.5h, carrying out ultrasonic treatment at 1250W for 12min, and centrifuging at 4000r/min for 12min to obtain a weakly cross-linked protein peptide solution; the cross-linked complex enzyme is a compound of glutamine transaminase and laccase with the mass ratio of 3.5: 5;
s7, metal ion complexing: adding 3.5 parts by weight of metal ion solution into 10 parts by weight of the weakly cross-linked protein peptide solution prepared in the step S6, heating the metal ion solution to 57 ℃, stirring and reacting for 30min, and freeze-drying to obtain the high-absorptivity composite protein composition, wherein the metal ion solution contains 2wt% of copper ions, 3wt% of zinc ions, 4wt% of iron ions and the balance of deionized water.
Example 4
Compared with the example 3, the metal ion solution in the step S7 does not contain copper ions, and other conditions are not changed.
Example 5
Compared with the example 3, the metal ion solution in the step S7 does not contain zinc ions, and other conditions are not changed.
Example 6
Compared with the example 3, the metal ion solution in the step S7 does not contain iron ions, and other conditions are not changed.
Comparative example 1
In step S3, lactobacillus plantarum was not added, as compared to example 3, and other conditions were not changed.
Comparative example 2
In step S3, Bacillus coagulans was not added, and the other conditions were not changed, as compared with example 3.
Comparative example 3
Compared with example 3, bifidobacterium adolescentis was not added in step S3, and other conditions were not changed.
Comparative example 4
Compared with example 3, the complex protease in step S4 was replaced by a single alkaline protease, and other conditions were not changed.
Comparative example 5
Compared with the example 3, the compound protease in the step S4 is replaced by the single papain, and other conditions are not changed.
Comparative example 6
In step S5, ultrasonic treatment was not performed, and other conditions were not changed, as compared with example 3.
Comparative example 7
Step S5 was not performed, and other conditions were not changed, as compared with example 3.
Comparative example 8
Compared with the example 3, the crosslinking compound enzyme in the step S6 is replaced by single glutamine transaminase, and other conditions are not changed.
Comparative example 9
Compared with the embodiment 3, the cross-linked compound enzyme in the step S6 is replaced by single laccase, and other conditions are not changed.
Comparative example 10
Step S7 was not performed, and other conditions were not changed, as compared with example 3.
Test example 1
The high absorption rate complex protein compositions prepared in examples 1 to 6 of the present invention and comparative examples 1 to 10 were tested, and the results are shown in Table 1.
The method for measuring the crude protein adopts a Kjeldahl method according to GB5009.5-2016 (determination of protein in food safety national standard food).
Crude protein content (%) = crude protein mass/sample mass × 100%
The determination method of amino acid refers to GB 5009.124-2016 determination of amino acid in food safety national standard food.
Amino acid content (%) = total mass of amino acid/sample mass × 100%
TABLE 1
Group of Crude protein content (%) Amino acid content (%)
Example 1 95.2 5.8
Example 2 95.7 5.9
Example 3 96.9 6.1
Example 4 94.4 5.6
Example 5 94.9 5.4
Example 6 94.2 5.5
Comparative example 1 93.5 4.0
Comparative example 2 93.7 3.8
Comparative example 3 94.0 3.6
Comparative example 4 94.2 2.8
Comparative example 5 94.4 1.6
Comparative example 6 92.2 6.7
Comparative example 7 91.7 7.4
Comparative example 8 90.2 8.0
Comparative example 9 91.8 8.2
Comparative example 10 91.0 5.2
As can be seen from the above table, the high absorption rate composite protein compositions prepared in examples 1 to 3 of the present invention are rich in protein and amino acids, wherein the protein content is the crude protein content minus the amino acid content.
Test example 2
Grouping and feeding: 170 male broilers of 20 days old were selected and randomly divided into 17 groups, including the test groups of examples 1 to 6 and comparative examples 1 to 10, and the commercial group, respectively. After the male broilers ate for 32 hours, food formulas containing the high absorption rate composite protein compositions prepared in examples 1-6 and comparative examples 1-10 were respectively fed to the nitrogen-free treated group, and the formulas of each group are shown in table 2. During the test period, the patients had free access to food and water.
TABLE 2
Components and contents Test group Commercial group
High absorption rate composite protein composition (wt%) 20 -
Commercial whey protein isolate (wt%) - 20
Starch (wt%) 30 30
Glucose (wt%) 40 20
Vitamin (wt%) 0.5 0.5
Microelement (wt%) 1.2 1.2
Choline chloride (wt%) 0.4 0.4
Inositol (wt%) 0.01 0.01
Calcium powder (wt%) 1.5 1.5
Sodium hydrogen phosphate (wt%) 1.39 1.39
Peanut oil (wt%) 3 3
Salt (wt%) 0.5 0.5
Sucrose (wt%) 1 1
Chromium oxide (wt%) 0.5 0.5
Wherein chromium oxide is used as an indicator.
The test method comprises the following steps: after each group of male broiler freely eats the daily ration for 36 hours, all male broiler are anesthetized and then dissected, ileal chyme is collected, mixed, frozen and dried to constant weight, and the mixture is poured into a sample bag to be tested after being porphyrized.
Determining the content of chromium sesquioxide in the test daily ration and the chyme by a colorimetric method;
the content of protein in the test daily ration and the chyme is determined by adopting a Kjeldahl method according to GB5009.5-2016 (determination of protein in national food safety standard).
Calculating the protein uptake rate:
protein absorption (%) =100 × (chromium oxide content in ration × protein content in chyme)/(chromium oxide content in chyme × protein content in ration)
The results of each group are shown in Table 3.
TABLE 3
Group of Protein absorption Rate (%)
EXAMPLE 1 group 96.53
EXAMPLE 2 group 97.23
EXAMPLE 3 group 97.75
EXAMPLE 4 group 95.22
EXAMPLE 5 group 94.56
EXAMPLE 6 group 94.12
Comparative example 1 group 90.21
Comparative example 2Group(s) 89.57
Comparative example 3 group 86.77
Comparative example 4 group 84.52
Comparative example 5 group 83.72
Comparative example 6 group 91.25
Comparative example 7 group 87.72
Comparative example 8 group 82.02
Comparative example 9 group 81.75
Comparative example 10 group 92.52
Market group 75.66
As can be seen from the above table, the high absorption rate complex protein compositions prepared in examples 1 to 3 of the present invention have excellent absorption rates.
Test example 3
The high absorption rate complex protein compositions prepared in examples 1 to 6 of the present invention and comparative examples 1 to 10 were subjected to an antioxidant capacity test, and their antioxidant activities were compared by measuring relative ORAC values (μmol TE/mmol), and the results are shown in Table 4.
ORAC method references: evaluation of antioxidant Activity of whey protein isolate hydrolysate of Lactobacillus plantarum A-2 [ J ] Chinese food additive, 2021,6,11-14.
TABLE 4
Group of Relative ORAC value (mmol TE/mmol)
Example 1 0.622
Example 2 0.635
Example 3 0.647
Example 4 0.425
Example 5 0.417
Example 6 0.431
Comparative example 1 0.511
Comparative example 2 0.504
Comparative example 3 0.498
Comparative example 4 0.442
Comparative example 5 0.437
Comparative example 6 0.552
Comparative example 7 0.525
Comparative example 8 0.409
Comparative example 9 0.392
Comparative example 10 0.402
As can be seen from the above table, the high absorption rate composite protein compositions prepared in examples 1 to 3 of the present invention have good antioxidant activity.
Test example 4 anti-aging test
Molding: dividing 134 healthy SPF mice of 40 + -10 g into 18 groups, adaptively feeding for 1w, performing intraperitoneal injection molding with D-galactose of 100mg/kg BW, wherein the injection amount is 0.1mL/10g, and continuously molding for 6w 1 time per day. Another 14 mice were blank groups and were injected i.p. with an equal amount of physiological saline.
Grouping and administration: after the model is established, the model is randomly divided into a model group, 8 groups in each group, examples 1-6 groups and comparative examples 1-10 groups based on the MDA level of each mouse serum, and the MDA levels of the model group and the mice in each test group have no obvious difference. Blank group 14 only. Each test group was administered 1.5g/kg BW per day by gavage with the high absorption rate complex protein composition prepared in the corresponding example or comparative example, and the model group and the blank group were administered with the same amount of physiological saline. At the same time of administration, the model group and each test group mice continued to be injected with D-galactose. After 30 days, the mice were sacrificed by cervical dislocation, serum, liver and brain tissue were separated, and MDA content in the serum, liver and brain tissue was measured using a kit, and the results are shown in table 4.
TABLE 4
Group of Serum MDA content (nmol/mL) Liver MDA content (nmol/mL) Brain MDA content (nmol/mL)
Blank group 28.23±19.21 1.47±0.27 3.62±0.45
Model set 55.26±24.56* 2.32±1.17* 6.98±1.72*
Example 1 31.02±19.52# 1.57±0.32# 4.26±0.47#
Example 2 30.27±20.11# 1.54±0.28# 4.17±0.42#
Example 3 29.89±18.57# 1.52±0.34# 4.05±0.38#
Example 4 35.72±19.24 1.89±0.31 4.72±0.32
Example 5 36.59±18.51 1.87±0.28 4.70±0.42
Example 6 35.89±17.29 1.92±0.35 4.76±0.38
Comparative example 1 35.25±20.22 1.80±0.26 4.85±0.41
Comparative example 2 34.73±19.57 1.78±0.30 4.90±0.43
Comparative example 3 36.25±18.46 1.85±0.29 4.82±0.34
Comparative example 4 38.27±18.59 1.98±0.25 4.99±0.36
Comparative example 5 39.77±19.45 2.02±0.34 5.04±0.44
Comparative example 6 34.42±20.82 1.77±0.39 4.56±0.39
Comparative example 7 35.17±21.11 1.85±0.27 4.78±0.40
Comparative example 8 40.24±20.48 2.10±0.21 5.07±0.47
Comparative example 9 39.98±19.26 2.07±0.22 5.17±0.39
Comparative example 10 39.22±18.49 2.10±0.33 5.03±0.38
And (3) annotation: p <0.05 compared to blank group and P <0.05 compared to model group.
The biochemical and physiological decline related to aging is caused by the accumulation of oxidative damage to cells by the formation of reactive oxygen radicals during cellular metabolism, and the imbalance of the cellular generation of free radicals and the self-antioxidant defense/damage repair mechanism occurs with the aging, which is one of the reasons behind aging. As can be seen from the above table, the high absorption rate complex protein compositions prepared in examples 1 to 3 of the present invention have excellent anti-aging activity by reducing neurotoxic hyperoxidation products such as MDA of serum, liver and brain to achieve anti-aging effect.
Examples 4, 5 and 6 compared with example 3, the metal ion solution in step S7 does not contain copper ion, zinc ion or iron ion, so that the relative ORAC value is decreased, MDA value of serum, liver and brain is decreased, antioxidant and anti-aging activities are significantly decreased, comparative example 10 compared with example 3, without performing step S7, the ORAC value was significantly decreased, and MDA values of serum, liver and brain were significantly increased, the present invention added metal ion solution containing copper ion, zinc ion and iron ion to the weakly cross-linked protein peptide prepared, which has synergistic effect, these ions tend to complex with groups on the protein peptide to form protein-metal complexes which, after entry into the human body, under proper conditions, the metal ions can be released to participate in the enzyme reaction of human bodies, and the effects of resisting aging, resisting oxidation, promoting fat combustion and promoting metabolism are achieved.
Comparative examples 1, 2 and 3 compared with example 3, in step S3, without lactobacillus plantarum, bacillus coagulans or bifidobacterium adolescentis, the protein absorption rate was decreased, the relative ORAC value was decreased, the MDA value of serum, liver and brain was decreased, and the antioxidant and anti-aging activities were decreased. According to the invention, the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis are fermented by the composite probiotics, and the composite protein powder containing the isolated whey protein powder, the concentrated milk protein powder and the hydrolyzed whey protein powder can be primarily fermented to produce a large amount of small-molecule protein peptides, small-molecule short peptides and a large amount of amino acid substances, so that the antioxidant and anti-aging effects of the composition are improved, and the three can cooperate with each other to achieve the effect of efficiently fermenting the protein powder.
In comparison with example 3, in step S4, the complex protease is replaced by single alkaline protease or papain, the protein content is increased, the amino acid content is decreased, however, the protein absorption rate is decreased, the relative ORAC value is decreased, the MDA value of serum, liver and brain is decreased, and the antioxidant and anti-aging activities are decreased. The alkaline protease acts on amide bonds of all hydrophobic and aromatic amino acid carboxyl terminals on a carboxyl side chain to catalyze peptide bonds in protein molecules to hydrolyze to generate peptides with smaller molecular mass, so that antigen binding sites in whey protein molecules are damaged, and the allergenicity is obviously reduced. The alkaline protease has good hydrolysis effect, and the beta-lactoglobulin can only be completely hydrolyzed by the alkaline protease, so that the antigenicity of the alpha-lactalbumin and beta-lactoglobulin antibodies can be obviously reduced, and the allergenicity of the whey protein can be greatly reduced. The papain is a single-chain endonuclease, can hydrolyze peptide bonds at carboxyl terminals of lysine, arginine and glycine in polypeptide, amide and ester, has strong hydrolysis capacity, can strongly destroy epitope of whey protein, and has good antigenicity. The protein is hydrolyzed by enzyme method, and then polypeptide chain is degraded, thereby realizing the reconnection of whey protein molecules and improving functional characteristics. The invention utilizes alkaline protease and papain to carry out double enzymolysis, wherein the alkaline protease is nonspecific protein endopeptidase, has wide action substrates, can hydrolyze all amino acids with hydrophobicity or aromatic at carboxyl terminals, and the papain and the peptide bond catalyzed by the alkaline protease have complementarity, thereby hydrolyzing protein to the maximum extent.
Comparative example 6 compared with example 3, the ultrasonic treatment was not performed in step S5, and comparative example 7 compared with example 3, the ultrasonic treatment was not performed in step S5, the protein content was decreased, the amino acid content was increased, the protein polymer was decreased, and at the same time, the protein absorption rate was decreased, the relative ORAC value was decreased, the MDA value of serum, liver and brain was decreased, and the antioxidant and anti-aging activities were decreased. In the low-power ultrasonic treatment process, the structure of the protein allergen can be changed under the action of ultrasonic waves, and the repeated combination reaction of protein peptide substances in the enzyme hydrolysate and ozone can be promoted, so that the effect of promoting the modification of ozone is achieved; the concentration of the protein peptide alpha-lactalbumin and beta-lactoglobulin monomers after the short-time oxidation treatment by ozone is reduced, the allergenicity is reduced, and simultaneously, the content of disulfide bonds is increased, because sulfydryl on two molecular cysteine side chains is aggregated to form a new disulfide bond, so that simple dimeric peptide is obtained, and the molecular weight of the short peptide is preliminarily increased; due to the short time, other reducing amino acids on the protein peptide have not yet been subjected to oxidation reaction, so that the property of the protein peptide is not greatly influenced.
Compared with the example 3, the cross-linked compound enzyme in the step S6 is replaced by the single glutamine transaminase or laccase, so that the protein content is reduced, the amino acid content is improved, the protein polymer is reduced, the protein absorption rate is reduced, the relative ORAC value is reduced, and the MDA value of serum, liver and brain is reduced. The cross-linked complex enzyme comprises glutamine transaminase and laccase; wherein, the glutamine transaminase is a transferase which can be used for cross-linking polymerization, deamidation and primary amine introduction, the enzyme takes amino groups of lysine residues on protein chains, epsilon-amino groups of free amino acids and the like as acceptors, acyl donors are gamma-hydroxyamide groups on the glutamine residues, and epsilon- (gamma-glutamine) lysine cross-linked chains are formed in molecules and among molecules, so as to obtain a weakly cross-linked protein peptide product; in addition, the laccase is polyphenol oxidase, contains copper elements, can form a compound with malic acid, and catalyzes and oxidizes phenol, the protein peptide substance prepared in the steps forms substrate free radicals after reacting with the laccase-malic acid compound, and simultaneously, the laccase and copper ions in the laccase act synergistically to oxidize-SH in the protein peptide into S-S, tyrosine residues are oxidized into quinone, and the quinone can perform Schiff base addition or Michael addition with active amino groups such as primary amine of free amino acids, so that the weak crosslinking effect of the protein peptide is realized. After the compound cross-linking action of glutamine transaminase and laccase, small molecular short peptide, short polypeptide and amino acid can be combined with each other, so that a protein peptide compound with moderate molecular weight is prepared, the protein peptide compound is beneficial to absorption and utilization of a human body, the digestion and absorption rate of gastrointestinal tracts is moderate, the first pass effect of liver cannot be caused, the absorption and utilization rate of the prepared protein peptide is greatly improved, and the quality of protein is improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims (9)

1. A method for preparing a high absorption rate complex protein composition, comprising the steps of:
s1, preparation of a protein solution: adding whey protein powder into a disodium hydrogen phosphate-citric acid buffer solution to obtain a protein solution;
s2, activating strains: respectively inoculating Lactobacillus plantarum, Bacillus coagulans and Bifidobacterium adolescentis into sterile MRS liquid culture medium, activating, culturing, centrifuging, discarding supernatant, washing thallus, centrifuging, discarding supernatant, and using sterile CaCl 2 The normal saline suspends the somatic cells to prepare a resting cell suspension;
s3, fermentation: sequentially inoculating the resting cell suspension of the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis prepared in the step S2 into the protein solution prepared in the step S1, fermenting, culturing, centrifuging, and taking supernatant to obtain fermentation liquor;
s4, enzyme hydrolysis: adding compound protease into the fermentation liquor prepared in the step S3, performing enzymolysis reaction, inactivating enzyme, and centrifuging to obtain enzyme hydrolysate; the compound protease is a compound of alkaline protease and papain;
s5, ultrasonic wave and ozone cooperation treatment: performing ultrasonic treatment on the enzyme hydrolysate prepared in the step S4, and introducing ozone for treatment to obtain a treatment solution;
s6, enzymatic weak crosslinking: adding malic acid into the treatment liquid prepared in the step S5, uniformly mixing, adding a cross-linked complex enzyme, carrying out enzymolysis reaction, inactivating the enzyme, and centrifuging to obtain a weak cross-linked protein peptide solution; the cross-linked complex enzyme is a compound of glutamine transaminase and laccase;
s7, metal ion complexing: and (4) adding a metal ion solution into the weak cross-linked protein peptide solution prepared in the step S6, heating, stirring, reacting, and freeze-drying to obtain the high-absorptivity composite protein composition.
2. The preparation method according to claim 1, wherein the whey protein powder in step S1 is at least one selected from the group consisting of a whey protein isolate powder, a milk protein concentrate powder and a whey protein hydrolysate powder, wherein one selected from the group consisting of a mixture of a whey protein isolate powder, a milk protein concentrate powder and a whey protein hydrolysate powder in a mass ratio of 5-10:3-5: 2-5; the pH value of the disodium hydrogen phosphate-citric acid buffer solution is 7.2-8; the solid-liquid ratio of the protein powder to the disodium hydrogen phosphate-citric acid buffer solution is 1: 50-100 g/mL.
3. The method according to claim 1, wherein the activation culture in step S2 is carried out at 36-40 ℃ under the micro-aerobic condition at 20-50r/min for 18-24 h; the micro-aerobic condition is that the oxygen concentration is 5-10 percent and CO is 2 The concentration is 3-7%, the balance is nitrogen, wherein the volume percentage is; the centrifugal rotating speed is 3000-5000r/min, and the time is 10-15 min; the resting cell suspension is measured to have an optical density value OD under 600nm 600 Is 18-25 OD/mL.
4. The method of claim 1, wherein the step ofIn the step S3, the inoculation amounts of the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis are respectively 2-4%, 1-3% and 3-5%; the fermentation culture condition is 36-38 ℃, and the culture is carried out for 24-48h at 20-50r/min under the micro-aerobic condition; the micro-aerobic condition is that the oxygen concentration is 5-10 percent and CO is 2 The concentration is 3-7%, and the balance is nitrogen, wherein the% is volume percentage; the centrifugal rotating speed is 3000-5000r/min, and the time is 10-15 min.
5. The preparation method according to claim 1, wherein the mass ratio of the compound protease in step S4 to the alkaline protease to the papain is 3-7: 2-5; the solid-liquid ratio of the compound protease to the fermentation liquor is 1: 100-200 g/mL; the temperature of the enzymolysis reaction is 40-50 ℃, and the time is 3-7 h; the enzyme killing method comprises 1000-1500W ultrasonic treatment for 10-15 min; in the step S5, the ultrasonic treatment condition is 100-200W ultrasonic treatment, the ozone treatment gas flow is 15-25L/h, the ozone concentration is 30-70mg/L, and the treatment time is 2-5S.
6. The preparation method according to claim 1, wherein the mass ratio of the malic acid to the cross-linked complex enzyme in the step S6 is 1-2: 5-12; the mass ratio of the cross-linked complex enzyme to the glutamine transaminase is 2-5: 3-7; the enzymolysis reaction is carried out under the conditions of 40-50 ℃ for 1-2h, the enzyme deactivation method is 1000-plus 1500W ultrasonic treatment for 10-15min, the centrifugal rotating speed is 3000-plus 5000r/min, and the time is 10-15 min; in step S7, the metal ion solution contains copper ions, zinc ions and iron ions, and the contents are 1-3wt%, 2-4wt% and 3-5wt%, respectively; the mass ratio of the metal ion solution to the weak cross-linked protein peptide solution is 2-5: 10; the heating temperature is 55-60 deg.C, and the heating time is 20-40 min.
7. The preparation method according to claim 1, comprising the following steps:
s1, preparation of a protein solution: uniformly mixing 5-10 parts by weight of separated whey protein powder, 3-5 parts by weight of concentrated milk protein powder and 2-5 parts by weight of hydrolyzed whey protein powder to obtain protein powder, and adding the protein powder into a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 7.2-8 to obtain a protein solution; the solid-liquid ratio of the protein powder to the disodium hydrogen phosphate-citric acid buffer solution is 1: 50-100 g/mL;
s2, activating strains: respectively inoculating Lactobacillus plantarum, Bacillus coagulans and Bifidobacterium adolescentis into sterile MRS liquid culture medium, activating and culturing at 36-40 deg.C under micro-aerobic condition for 18-24 hr, centrifuging, discarding supernatant, washing thallus, centrifuging, discarding supernatant, and adding sterile CaCl 2 The physiological saline of (1) suspending the somatic cells, preparing a resting cell suspension, which is measured for its optical density value OD at 600nm 600 18-25 OD/mL;
s3, fermentation: sequentially inoculating the resting cell suspension of the lactobacillus plantarum, the bacillus coagulans and the bifidobacterium adolescentis prepared in the step S2 into the protein solution prepared in the step S1, wherein the inoculation amounts are 2-4%, 1-3% and 3-5%, respectively, the inoculation amounts are 36-38 ℃, the fermentation culture time is 24-48h under the micro-aerobic condition, and the fermentation broth is obtained after centrifugation and supernatant fluid taking;
s4, enzyme hydrolysis: adding compound protease into the fermentation liquor prepared in the step S3, wherein the solid-to-liquid ratio of the compound protease to the fermentation liquor is 1: performing enzymolysis reaction at 40-50 ℃ for 3-7h at the concentration of 100-200g/mL, inactivating enzyme, and centrifuging to obtain enzyme hydrolysate; the compound protease is a compound of alkaline protease and papain; the mass ratio of the alkaline protease to the papain is 3-7: 2-5;
s5, ultrasonic wave and ozone synergistic treatment: synchronously performing 100-200W ultrasonic treatment and ozone treatment on the enzyme hydrolysate prepared in the step S4, wherein the gas flow is 15-25L/h, the ozone concentration is 30-70mg/L, and the treatment time is 2-5S to obtain a treatment solution;
s6, enzymatic weak crosslinking: adding 1-2 parts by weight of malic acid into the treatment solution prepared in the step S5, uniformly mixing, adding 5-12 parts by weight of cross-linked complex enzyme, carrying out enzymolysis reaction at 40-50 ℃ for 1-2h, inactivating the enzyme, and centrifuging to obtain a weakly cross-linked protein peptide solution; the cross-linked complex enzyme is a compound of glutamine transaminase and laccase; the mass ratio of the glutamine transaminase to the laccase is 2-5: 3-7;
s7, metal ion complexing: adding 2-5 parts by weight of metal ion solution into 10 parts by weight of the weakly cross-linked protein peptide solution prepared in the step S6, wherein the metal ion solution contains 1-3wt%, 2-4wt% and 3-5wt% of copper ions, zinc ions and iron ions respectively, heating to 55-60 ℃, stirring and reacting for 20-40min, and freeze-drying to obtain a high-absorptivity composite protein composition;
the micro-aerobic condition is that the oxygen concentration is 5-10 percent and CO is 2 The concentration is 3-7%, the balance is nitrogen, wherein the volume percentage is; the centrifugal rotating speed is 3000-5000r/min, and the time is 10-15 min; the enzyme deactivation method is ultrasonic treatment of 1000-1500W for 10-15 min.
8. A high absorption rate complex protein composition prepared by the preparation method according to any one of claims 1 to 7.
9. Use of the high absorption rate complex protein composition according to claim 8 for the preparation of an anti-ageing product and/or an anti-oxidant product.
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