CN116406733A - Protein glutaminase modified casein and preparation method and application thereof - Google Patents
Protein glutaminase modified casein and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
- A23J3/08—Dairy proteins
- A23J3/10—Casein
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
- A23J1/202—Casein or caseinates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
- A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
- A23J3/344—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins of casein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
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- 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
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- 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
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Abstract
The invention discloses protein glutaminase modified casein, and a preparation method and application thereof. The method comprises the following steps: dispersing casein in water to obtain casein dispersion; adding protein glutaminase to perform deamination reaction and termination reaction; and freeze-drying to obtain deamidated modified casein. Compared with unmodified casein, the modified casein has similar protein molecular main structure, but compared with the unmodified casein, the modified casein has obviously reduced glutamine residue proportion, increased glutamic acid residue proportion and increased water solubility, has better emulsifying property and emulsifying stability, and can effectively solve the problems of protein precipitation, layering and the like in the high-content protein milk product when being particularly used as a protein content supplement or an emulsifying stabilizer of the milk product. Meanwhile, the modified casein is used as a food-grade natural emulsifier and an emulsion stabilizer, has high safety and good effect, and is a preferable protein for food production and processing and other applications.
Description
Technical Field
The invention belongs to the technical field of dairy products, and particularly relates to protein glutaminase modified casein, a preparation method thereof and application thereof in improving application performance of casein.
Background
Milk has been widely consumed as a basic nutrient for human life for thousands of years, and with the development of civilization and advances in production technology, the variety of milk products, such as cheese, milkshake, yogurt, milk powder, etc., has also increased greatly, and these milk products have been applied to various aspects of the demands of human production and life. Casein is the main protein component in milk, and this protein can form one unique nanometer structure component, casein micelle, with average size of 150-200nm, and through non-covalent bond, several protein molecules are polymerized together to form sponge colloid particle with high hydration. Casein micelles comprise αs1-, αs2-, β -, kappa-casein, which is a carrier for minerals in milk, and mineral calcium and phosphate components, and are a major source of nutrition for calcium, phosphate and amino acids, which are capable of meeting the growth and energy demands of the human body, and which have gradually become an integral part of the daily diet of humans.
Because of its viscosity, water-holding capacity, emulsifying properties, solubility and other application properties and high nutritional value, casein and its products are widely used in the food industry and other industrial sectors, for example in meat products, baked goods, margarine, coffee partners, cheese, desserts, beverages, yoghurt, grease and the like. Although casein has good application performance and prospect, the application performance such as solubility, emulsifying property, stability and the like still cannot meet higher requirements in certain fields. In addition, the improvement of the performance of casein is beneficial to the application of casein in the existing industry, for example, casein and products thereof are added into meat products, and the water retention and the emulsifying property of casein can be utilized to maintain the water-moist degree and enhance the meat quality and flavor, and the protein content is enhanced, but the heat resistance of casein is poor; in the preparation process of milk beverage and yoghourt, the homogenization can cause the destruction of a curd stabilizing system to a certain extent, so that protein precipitation, precipitation and flocculation are caused; in addition, the problems of unstable system or exceeding the saturation degree of the solution, aggregation and precipitation of casein and the like can also occur when a large amount of casein is added. Therefore, strategies such as increasing casein solubility, reducing casein addition to achieve the same application performance as the original addition, reducing adverse effects caused by casein addition, enhancing functional properties of casein, saving economic costs, and improving flavor and mouthfeel have been further explored and improved by modifying casein.
The current method for modifying proteins mainly comprises a physical method, a chemical method and a biological enzyme method. The physical method usually uses heat treatment, ultrasonic treatment, ultrafiltration treatment and other methods to modify proteins, but due to the extreme physical means, the method may cause hydrolysis, denaturation, bad flavor generation and high energy consumption of proteins, and has disadvantages to a certain extent. The chemical method usually adopts acid, alkali and salt treatment, but often has difficult purification or residual chemical reagent after the treatment, and a certain risk can still exist in the aspect of eating. The biological enzyme method mainly comprises protease, glutamine transaminase, peptide glutaminase and the like, but has certain defects in application due to the problems of proteolysis, protein crosslinking and substrate restriction respectively.
Disclosure of Invention
The invention overcomes the defects and the shortcomings of the prior art and provides a method for improving the application performance of casein by modifying casein with protein glutaminase.
The Protein Glutaminase (PG) has been newly discovered in recent yearsAn enzyme mainly produced by fermentation of Flavobacterium prion (Chryseobacterium proteolyticum), mature PG enzyme consisting of 185 amino acids with a relative molecular mass of 19.8kDa, which is capable of specifically catalyzing the conversion of glutamine residues to glutamic acid residues and releasing NH in proteins 4 + Thus exhibiting deamidation activity on the substrate protein. Because PG enzymes only have deamidation activity on side chains of proteins, have no protease and glutamine transaminase activity, and do not show deamidation activity on free glutamine, the proteins treated by PG enzymes do not cause cleavage of peptide chains, proteolysis and crosslinking, thereby generating adverse factors such as bad flavor. In addition, since many glutamine residues and asparagine residues are contained in a protein, a structure such as hydrogen bond is easily formed between these amide groups, so that the solubility of the protein is lowered, and thus, the flocculation, precipitation and other phenomena of the protein occur, and further, the characteristic functions such as the emulsifying property of the protein are lowered, whereas in general, the emulsifying property of the protein is related to the structural characteristics of the protein, and the protein can form a thin adsorption layer on the oil-water interface, thereby forming an emulsified stable state, and preventing the aggregation and flocculation of the protein. After deamidation of protein by glutaminase, the solubility, net charge and surface hydrophobicity of the protein are increased, so that the hydrophilic-lipophilic balance is changed and emulsification enhancement is realized; the improvement of the emulsification stability of the protein after deamidation is mainly caused by the enhancement of electrostatic repulsion of carboxyl groups after deamidation and the interaction of protein-protein at a water-oil interface, so that the emulsification stability is enhanced.
The technical scheme provided by the invention is as follows: a method for protein glutaminase modification of casein to improve the performance of casein application, comprising the steps of:
the first step: dispersing casein in water to obtain casein dispersion;
and a second step of: adding food-grade protein glutaminase into the casein dispersion liquid to perform deamidation reaction, heating the solution in water bath after the reaction is finished, stopping the enzymatic reaction of the protein glutaminase, and cooling to obtain casein solution;
and a third step of: and (3) carrying out vacuum freeze drying on the casein solution after the deamidation reaction is terminated to obtain the deamidated modified casein.
In one embodiment, the method comprises the steps of:
the first step: preparation of Casein Dispersion
Dispersing casein in water to obtain casein suspension with mass-volume ratio (W/V) of 1-10%;
and a second step of: protein glutaminase deamidation casein reaction and termination reaction
Adding food-grade protein glutaminase into the casein suspension to perform deamidation reaction, heating the solution to 80 ℃ in water bath after the reaction is finished to terminate the enzymatic reaction of the protein glutaminase, and cooling to obtain casein solution;
and a third step of: freeze-drying
And (3) placing the casein solution sample after the deamidation reaction is terminated in a freeze dryer for vacuum freeze drying to obtain the deamidation modified casein.
In a specific embodiment, the Enzyme activity to Substrate ratio (E/S), i.e. the mass of protein glutaminase to casein is 1-5U/g protein, the enzymatic reaction temperature is 30-50 ℃ and the enzymatic reaction time is 1-4 h. The modified casein of the invention is food grade and is added in proportion during application.
Preferably, in the second step, the food-grade protein glutaminase is added to perform a deamidation reaction, the protein glutaminase is added in an amount of 1-5U/g protein.
Preferably, in the second step, the protein glutaminase is subjected to a deamidation reaction at a temperature of 30-50 ℃.
Preferably, in the second step, the protein glutaminase is subjected to a deamidation reaction for a period of 1 to 5 hours.
Preferably, in the second step, the heating time is 10min.
Preferably, in the second step, the cooling is an ice bath for 30min.
Preferably, in the third step, the vacuum freeze-drying condition is-50-15 ℃, the vacuum degree is 15Pa, and the drying time is 35 hours.
Preferably, the deamidated casein obtained in the third step is subjected to a solubility test, i.e. a sample of the casein solution after termination of the deamidation reaction is taken for the solubility test.
Preferably, the solubility test is performed by subjecting the resulting sample to centrifugation at 12000rpm for 10min, taking the soluble fraction of the supernatant and measuring the protein concentration in the solution by BCA method, solubility (%) = concentration of the supernatant/(initial concentration x 100%).
Preferably, the deamidated casein obtained in the third step is tested for its emulsifying property and stability, i.e., a sample of the casein solution after termination of the deamidation reaction is taken for testing for its emulsifying property and emulsion stability.
Preferably, the condition is to dilute the modified casein solution to 1%, add soybean oil with the volume ratio of the modified casein solution to the soybean oil being 3:1, homogenize the oil-protein mixed solution for 2min by a homogenizer at 20000rpm, take the emulsion at the bottom of the container after homogenization, add into 0.1% SDS solution, mix uniformly, OD 500nm The absorbance value (A0) was measured and the Emulsifiability (EAI) of the protein solution was calculated, EAI (m 2 /g)＝2×2.303×A0÷0.25。
Preferably, the conditions are that the homogenized emulsion is allowed to stand for 10min, the absorbance value at OD500 (a 10) is again detected, and the Emulsion Stability (ESI) is calculated, ESI (min) =a0+ (A0-a 10) ×10.
The invention also provides protein glutaminase modified casein prepared by the method.
The protein glutaminase modified casein obtained by the method has increased net charge and surface hydrophobicity; the solubility and the emulsibility of the water-soluble polyurethane are greatly increased; the electrostatic repulsive force of the modified casein and the interaction of protein-protein at the water-oil interface are enhanced.
Specifically, the protein glutaminase modified casein has deamidation degree of 0-87.88%, hydrolysis degree of 2.66-7.80%, precipitation rate of 4.94-100%, and emulsifying property of 20.79-55.06 m 2 The emulsion stability per gram is 17.02-307.47 min, and the foamability is 37.67-165.00%.
The protein glutaminase modified casein obtained by the method disclosed by the invention has the advantages that the application performance of the casein is enhanced, the application scene of the casein is widened, and the problems of bitter taste, peculiar smell, flocculation, precipitation and the like generated by other modified casein are effectively solved.
The invention also provides the preparation method and application of the protein glutaminase modified casein in improving the performance of casein by enzymatically modifying casein.
The beneficial effects of the invention include: the method for improving the application performance of the casein by using the protein glutaminase modified casein provided by the invention takes the casein obtained by degreasing, concentrating and drying milk as a raw material, does not need any chemical reagent treatment, has wide and safe sources and low cost, and is a protein source which is eaten by people daily and has rich nutrition. The food-grade protein glutaminase adopted in the method disclosed by the invention has the advantages that on the premise of deamidation, the reaction is mild, adverse side reactions and limiting conditions such as hydrolysis, crosslinking and substrate limitation on protein during other enzymatic reactions are avoided, adverse reactions and unsafe addition such as hydrolysis, high energy consumption and chemical residues caused by modifying the protein by a physical or chemical method are avoided, and the industrialization cost is reduced. Compared with the protein before modification, the modified casein obtained by deamidation modification of the protein glutaminase has the advantages that the main structure of protein molecules is not changed significantly, the net charge and the surface hydrophobicity of the modified casein are increased, the solubility and the emulsifying property are greatly increased, the electrostatic repulsive force of the modified casein and the interaction of protein-protein at a water-oil interface are enhanced, the emulsifying property and the emulsifying stability are obviously improved, the application performance of the casein is enhanced, the application scene of the casein is widened, and the problems of bitter taste, peculiar smell, flocculation, precipitation and the like generated by the modified casein in other modes are effectively solved. The whole process has low cost, mild and safe reaction, simple and quick operation, low equipment requirement and easy industrialized implementation.
Compared with unmodified casein, the food-grade modified casein has similar protein molecular main structure, but the glutamine residue proportion of the modified casein is obviously reduced compared with the unmodified casein, the glutamic acid residue proportion is increased, the water solubility is increased, the food-grade modified casein has better emulsifying property and emulsifying stability, and particularly when the food-grade modified casein is used as a protein content supplement or emulsifying stabilizer of a milk product, the problems of protein precipitation, layering and the like in the milk product with high protein content can be effectively solved. Meanwhile, the modified casein is used as a food-grade natural emulsifier and an emulsion stabilizer, has high safety and good effect, and is a preferable protein for food production and processing and other applications.
Drawings
FIG. 1 shows the precipitation after various time modifications of casein.
FIG. 2 shows the precipitation of modified casein at different casein concentrations with different enzyme to substrate ratios.
FIG. 3 shows the precipitation after modification of casein at different temperatures.
FIG. 4 shows the sedimentation after modification of casein under different pH conditions.
Detailed Description
The invention will be described in further detail with reference to the following specific examples and drawings. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited.
In the invention, each detection method comprises the following steps:
(1) Determination of degree of deamidation:
ammonia content resulting from complete deamidation of casein: preparing casein suspension with the same concentration as protein glutaminase deamidating casein sample, taking 500ul casein sample in 1.5ml EP tube, adding equal volume of 2N sulfuric acid solution, heating in 100deg.C water bath for 4h, centrifuging at 12000rpm for 10min, taking appropriate amount of supernatant, and measuring NH in the supernatant by ammonia detection kit (Sigma-Aldrich) 4 + 。
Protein glutaminase catalyzes the ammonia content produced by casein deamidation: preparing casein suspension with proper concentration, adding protein glutaminase for catalytic deamidation, and then carrying out 12000rpm is centrifuged for 10min, a proper amount of supernatant is taken and NH in the supernatant is measured by using an ammonia detection kit 4 + . The degree of deamidation is the percentage of ammonia content produced by the deamidation of casein catalyzed by the protein glutaminase over that produced by the complete deamidation of casein (cf. Inthawoot Suppavorasatit, elvira Gonzalez De Mejia, keith R Cadwallader, optimization of the enzymatic deamidation of soy protein by protein-glutaminase and its effect on the functional properties of the protein, J Agric Food chem.2011 Nov 9;59 (21): 11621-8.Doi:10.1021/jf2028973.Epub 2011 Oct 11.).
(2) Determination of the degree of hydrolysis:
measurement of the degree of complete hydrolysis of casein: preparing casein suspension with the same concentration as that of a protein glutaminase deamidating casein sample, taking 500ul of casein sample in a 2ml EP tube, adding an equal volume of 2N sulfuric acid solution, heating in a water bath at 100 ℃ for 4 hours, adding 1ml of 0.2N trichloroacetic acid solution, centrifuging at 12000rpm for 10 minutes, taking 10ul of supernatant in a 96-well microplate, and measuring the total soluble protein content in the supernatant by using a BCA protein concentration measuring method, namely the total hydrolysis degree of casein.
Measurement of degree of hydrolysis after casein deamidation: preparing casein suspension with proper concentration, adding protein glutaminase to catalyze deamination reaction, stopping reaction in water bath at 80 ℃ for 10min after the reaction is completed, ice-bathing for 30min, taking 1ml of modified casein sample, adding 1ml of 0.2N trichloroacetic acid solution into 2ml of EP tube, centrifuging at 12000rpm for 10min, taking 10ul of supernatant into 96-hole micro-pore plates, and measuring the content of soluble protein after the deamination reaction of casein in the supernatant by using BCA (broadcast control channel) protein concentration method, namely the hydrolysis degree after the deamination reaction of casein. The degree of hydrolysis is the percentage of the degree of hydrolysis after deamidation of casein compared to the degree of complete hydrolysis of casein
(3) Measurement of solubility:
measurement of casein solubility: preparing casein suspension with the same concentration as that of protein glutaminase deamidating casein sample, and measuring protein content in unmodified casein solution by BCA protein concentration measurement method. The protein content in the modified casein solution is measured by BCA protein concentration measurement method by taking the modified casein solution with the same concentration. Solubility is the percentage of protein content in the modified casein solution compared to the protein content in the unmodified casein solution.
(4) Determination of protein concentration:
protein concentration was determined using BCA method: preparing a developing solution A (pH=11.25) containing 1%Bicinchonininc acid (BCA) disodium salt solution, 2% anhydrous sodium carbonate, 0.16% sodium tartrate, 0.4% sodium hydroxide and 0.95% sodium bicarbonate, preparing 4% copper sulfate solution as developing solution B, adding a proper amount of sample to be tested and 200ul of working solution into a 96-hole micro-plate, reacting at 37 ℃ for 30min with an OD (optical density) by taking 1.5mg/ml standard crystalline bovine serum albumin solution as a protein concentration standard solution, adding 200ul of developing solution A+4ul of developing solution B as working solution, adding a proper amount of sample to be tested and 200ul of working solution into the 96-hole micro-plate, and reacting at 37 ℃ for 30min with an OD (optical density) of the protein 562nm Absorbance values were measured and protein concentrations were scaled.
(5) Determination of the emulsibility:
soybean oil volume ratio of 1% modified casein solution equal to 3:1, placing the mixture in a tissue homogenizer at 20000rpm for 2min, immediately sucking 50ul of the emulsion from the bottom of the solution in 5ml of 0.1% SDS solution, mixing, OD 500nm Detecting absorbance, designated as A0, and calculating the Emulsibility (EAI) of the protein solution, EAI (m 2 /g)＝2×2.303×A0÷0.25。
(6) Determination of emulsion stability:
soybean oil volume ratio of 1% modified casein solution equal to 3:1, placing the mixture in a tissue homogenizer at 20000rpm for 2min, immediately sucking 50ul of the emulsion from the bottom of the emulsion in 5ml of 0.1% SDS solution, mixing, and OD 500nm After the absorbance value is recorded as A0 and standing at room temperature for 10min, 50ul of the emulsion is again sucked from the bottom of the emulsion and mixed in 5ml of 0.1% SDS solution, the absorbance value is recorded as A10 by OD500 detection, and the Emulsion Stability (ESI) is calculated, ESI (min) =A0/(A0-A10). Times.10.
Example 1: protein glutaminase modifies casein solutions at different times
Weighing a proper amount of casein, adding the casein into a beaker containing a proper amount of distilled water while stirring, continuously stirring for 10min, preparing a casein suspension with a final concentration of 5%, adding a proper amount of food-grade protein glutaminase, stirring to ensure that the mass ratio E/S of the protein glutaminase to the casein is 1U/g of protein, placing the reaction system into a constant-temperature shaking table for culture, placing the reaction system into a constant-temperature water bath pot with the temperature of 30 ℃ and the pH value of 7 at the speed of 200rpm for reaction, placing the reaction system into a water bath with the constant temperature of 80 ℃ for 10min for stopping the reaction after the reaction is finished, then placing the reaction system into ice for ice bath for cooling for 30min, taking a small amount of samples for detection of deamidation degree, solubility, emulsifying property and emulsion stability after cooling, and placing the rest samples into a freeze dryer for freeze drying. The vacuum freeze-drying conditions were: the temperature is minus 50 ℃, the vacuum degree is 15Pa, and the time is 35 hours.
The experimental results are shown in table 1 and fig. 1 below.
TABLE 1
In this example, the experimental results show that: in the range of 0 to 4 hours, the deamidation degree of casein gradually increases with the increase of the reaction time, but the hydrolysis degree does not change in a trend; furthermore, the precipitation rate of casein decreases with increasing deamidation degree, and the emulsifying property, emulsifying stability and foamability characteristics increase with increasing deamidation degree. Therefore, deamidation reaction is carried out on casein for 1-4 hours to obtain deamidation modified casein with different degrees, and the deamidation reaction effect of casein for 4 hours is optimal by combining various characteristics of modified casein.
Example 2: protein glutaminase modified casein solution with different concentrations
Weighing a proper amount of casein, adding the casein into a beaker containing proper amount of distilled water while stirring, continuously stirring for 10min, preparing casein suspension with the final concentration of 1%, 5% and 10% respectively, adding proper amount of food-grade protein glutaminase, stirring to ensure that the mass ratio E/S of the protein glutaminase to the casein is 0-5U/g of protein, placing the reaction system into a constant-temperature shaking table for culturing at the temperature of 50 ℃, the pH value of the reaction system is=7, the rotating speed of the reaction system is 200rpm, reacting for 2h, placing the reaction system into a constant-temperature water bath kettle at the temperature of 80 ℃ for water bath for 10min to terminate the reaction after the reaction, then placing the reaction system into an ice bath for cooling for 30min, taking a small amount of samples for detection of deamidation degree, solubility, emulsifying property and emulsion stability after cooling, and placing other samples into a freeze dryer for freeze drying.
The vacuum freeze-drying conditions were the same as in example 1.
The detection results are shown in the following table 2 and fig. 2.
TABLE 2
In this example, experiments show that: the concentration of casein of 1%, 5% and 10% can be effectively deamidated, the deamidation degree is continuously increased along with the increase of the ratio of enzyme to substrate, the hydrolysis degree is not changed in a trend along with the increase of time, and the hydrolysis degree is kept to fluctuate within the range of 2.66-7.36%; the precipitation rate decreases with increasing deamidation, indicating that the solubility increases with decreasing amount of glutamine in the protein; the emulsifying property, the emulsifying stability and the foamability are all increased along with the increase of the deamidation degree, which shows that the function of the casein after deamidation is improved, and the application scene and the characteristics of the casein are increased.
As can be seen in table 2, the preferred conditions are: when the enzyme to substrate ratio E/s=5, the modified casein at different concentrations has a higher deamidation degree, a lower precipitation rate, and better emulsifying property, emulsion stability, and foamability than E/s=1 or E/s=3.
Example 3: protein glutaminase modifies casein solutions at different temperatures
Weighing a proper amount of casein, adding the casein into a beaker containing proper amount of distilled water while stirring, continuously stirring for 10min, preparing a casein suspension with a final concentration of 5%, adding a proper amount of food-grade protein glutaminase, stirring to ensure that the mass ratio E/S of the protein glutaminase to the casein is=3U/g of protein, placing a reaction system into a constant-temperature shaking table for culture, reacting at different temperatures at 200rpm, reacting for 2h or not, pH=7, placing the reaction system into a constant-temperature water bath kettle at 80 ℃ for water bath for 10min to terminate the reaction after the reaction is finished, then placing the reaction system into ice bath on ice for cooling for 30min, taking a small amount of samples for detection of deamidation degree, solubility, emulsifying property and emulsion stability after cooling, and placing other samples into a freeze dryer for freeze drying.
The vacuum freeze-drying conditions were the same as in inventive example 1.
The experimental results are shown in table 3 below and fig. 3.
TABLE 3 Table 3
In this example, the experimental results show that: in the range of 30-60 ℃, the deamidation degree of casein increases with the increase of the reaction temperature, the hydrolysis degree does not change in a trend, the increase of the deamidation degree promotes the reduction of the precipitation rate of casein, and the emulsifying property, the emulsifying stability and the foaming property of casein are gradually enhanced. Therefore, the deamidation of casein can be realized at different degrees at 30-60 ℃, and the deamidation of casein at 50-60 ℃ is more than 50% by combining various characteristics of modified casein, so that the effect is ideal.
Example 4: protein glutaminase modified casein solutions at different pH conditions
Weighing a proper amount of casein, adding the casein into a beaker containing proper amount of distilled water while stirring, continuously stirring for 10min to prepare casein suspension with a final concentration of 5%, adding proper amount of food-grade protein glutaminase, stirring to ensure that the mass ratio E/S of the protein glutaminase to the casein is=3U/g of protein, placing the reaction system into a constant-temperature shaking table for culture, placing the reaction system into a constant-temperature water bath pot with the temperature of 50 ℃ and the pH value of 5-9 at the rotating speed of 200rpm for 2h or no reaction, placing the reaction system into a constant-temperature water bath with the temperature of 80 ℃ for water bath for 10min to terminate the reaction after the reaction is finished, then placing the reaction system into ice bath for cooling for 30min, taking a small amount of samples for detection of deamidation degree, solubility, emulsifying property and emulsion stability after cooling, and placing other samples into a freeze dryer for freeze drying. The vacuum freeze-drying conditions were the same as in inventive example 1.
The experimental results are shown in table 4 below and fig. 4 below.
TABLE 4 Table 4
In this example, the experimental results show that: in the pH range of 5-9, the deamidation degree increases and then decreases with the increase of the pH, and when the pH=7-8, the deamidation degree is higher; the change in the degree of hydrolysis does not show a tendency; the changes in the precipitation rate, the emulsifying property, the emulsion stability and the foamability are consistent with the changes in the deamidation degree, and the pH is preferably 7 to 8, which indicates that the deamidation reaction of the present invention is best performed under the condition of pH of 7 to 8.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
Claims (13)
1. A method for preparing protein glutaminase modified casein, said method comprising the steps of:
the first step: dispersing casein in water to obtain casein dispersion;
and a second step of: adding protein glutaminase to perform deamidation reaction, heating and stopping the reaction;
and a third step of: freeze drying to obtain deamidated modified casein.
2. The method according to claim 1, wherein in the first step casein is dispersed in water to obtain a casein suspension having a mass-to-volume ratio of 1-10%.
3. The method according to claim 1, wherein in the second step, food-grade protein glutaminase is added to the casein dispersion to perform deamidation, and after the reaction is completed, the solution is heated in a water bath to 80 ℃ to terminate the enzymatic reaction of the protein glutaminase, and the casein solution is obtained by cooling; and/or, the heating time is 10min; and/or the temperature of the deamidation reaction of the protein glutaminase is 30-50 ℃; and/or the time for deamidation reaction of the protein glutaminase is 1-5h.
4. A method according to claim 3, wherein the protein glutaminase is added in an amount of E/S of 1-5U/g protein; and/or, the cooling is an ice bath for 30min.
5. The method according to claim 1, wherein in the third step, the casein solution sample after the deamidation reaction is terminated is put in a freeze dryer to be freeze-dried in vacuum; wherein, the vacuum freeze drying condition is: the temperature is-50 ℃ to 15 ℃, the vacuum degree is 15Pa, and the drying time is 35 hours.
6. The method according to claim 1, wherein after the second step, a sample of the casein solution after the deamidation reaction has been terminated is taken for solubility detection.
7. The method according to claim 6, wherein the solubility test is carried out by subjecting the obtained sample to centrifugation at 12000rpm for 10 minutes, collecting the supernatant soluble fraction, detecting the protein concentration in the solution by BCA method,
the calculation formula is as follows:
solubility (%) = supernatant concentration +.initial concentration x 100%.
8. The method according to claim 1, wherein after the second step, a sample of the casein solution after the deamidation reaction has been terminated is taken for the detection of the emulsifying property and the emulsion stability.
9. The method according to claim 8, characterized in that theThe emulsifying property detection comprises the steps of diluting modified casein solution to 1%, adding soybean oil with the volume ratio of modified casein solution to soybean oil being 3:1, homogenizing the oil-protein mixed solution for 2min at 20000rpm by a homogenizer, taking an emulsion at the bottom of a container after homogenizing, adding into 0.1% SDS solution, uniformly mixing, and OD 500nm Detecting absorbance value A0, calculating the emulsifying EAI of the protein solution,
the calculation formula is as follows:
EAI(m 2 /g)＝2×2.303×A0÷0.25。
10. the method according to claim 8, wherein the homogenized emulsion is allowed to stand for 10min and the OD is detected again 500nm Absorbance value at (a 10), and Emulsion Stability (ESI) was calculated,
the calculation formula is as follows:
ESI(min)＝A0÷(A0-A10)×10。
11. a protein glutaminase-modified casein produced by the method of any one of claims 1 to 10.
12. The protein glutaminase-modified casein according to claim 11, wherein the degree of deamidation thereof is from 0 to 87.88%; the degree of hydrolysis is 2.66-7.80%; the precipitation rate is 4.94-100%; the emulsifying property is 20.79-55.06 m 2 /g; the emulsion stability is 17.02-307.47 min; the foamability was 37.67 to 165.00%.
13. Use of the protein glutaminase-modified casein of claim 11 or 12 for enzymatically modifying casein to improve casein performance.
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| CN102480980A (en) * | 2009-08-31 | 2012-05-30 | 味之素株式会社 | Low-fat or fat-free yoghurt, and process for production thereof |
| US20170044513A1 (en) * | 2014-03-07 | 2017-02-16 | Ajinomoto Co., Inc. | Protein deamidase |
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