CN103923964A - Method for preparing casein hydrolysate with stable functionality by using different hydrolysis conditions - Google Patents

Abstract

The invention discloses a method for preparing stable functional casein hydrolyzate by using different hydrolysis conditions, which belongs to the field of casein hydrolysis technology and processing. It first selects a substrate (casein)-enzyme (alkaline protease) as the reaction system, and controls the pH of the hydrolysis process to be constant through the pH-stat method. At a certain temperature (T), enzyme-to-substrate ratio (E/S) and Change the three hydrolysis conditions within the range of substrate concentration (S). When the hydrolyzate reaches a predetermined degree of hydrolysis (DH), the enzyme is deactivated and dried, and the properties of the hydrolyzate obtained with the same degree of hydrolysis (DH) have no significant difference. is stable. The invention can be adjusted according to different production conditions, and the hydrolyzate of the same DH can be obtained by hydrolysis, so that it has equivalent properties. It can be used as a method to guide industrial production. It can flexibly change production conditions and still ensure product stability.

Description

A method for preparing stable functional casein hydrolyzate by using different hydrolysis conditions

technical field

The invention relates to a method for preparing a hydrolyzate with specific functional properties by combining different hydrolysis temperatures and enzyme-to-substrate ratios, and belongs to the field of casein hydrolysis technology and processing.

Background technique

Protease is a kind of hydrolase, which mainly hydrolyzes the peptide bonds of proteins and polypeptides. According to the source of the enzyme, proteases can be divided into animal-derived proteases, plant-derived proteases, and microbial-derived proteases. According to the different catalytic positions, proteases can be divided into endopeptidases and exopeptidases. Endopeptidase is an enzyme that is widely used today, and endopeptidase can be divided into four types according to the active site of action: serine protease, cysteine protease, metalloprotease and asparagine protease. Humans have used protease as early as hundreds of years ago, and the typical example is fermented soybean products in China. In recent years, protease has been increasingly used in industrial production and experimental research due to its many advantages. People are increasingly using protease to hydrolyze various proteins in order to improve their structure and function, such as improving the emulsification, foaming, solubility, etc. of soybean protein; Peptide substances with physiological functions. These studies are often based on studying the effects of different enzymes on a certain substrate, or a certain enzyme on multiple substrates, hydrolysis for different times or different degrees of hydrolysis, in order to obtain optimized hydrolysis conditions or optimized degree of hydrolysis.

As for casein, it is well known that it is a protein aggregate formed when milk encounters acid. Casein accounts for about 80% of the total protein in milk, about 3% of its mass, and is also the main component of cheese. Casein is an amphoteric protein with an isoelectric point of pH4.6. It is a mixture of various (about 20) phosphoproteins secreted by bovine mammary glands. It is compounded with dicalcium phosphate, tricalcium phosphate or both in milk Existing in the form of objects, the structure is extremely complex. As a mixed protein with high content and flexible conformation in milk, casein is easily hydrolyzed by protease, and its hydrolyzed products mainly include peptides, a small amount of amino acids, sugars, vitamins, etc. These products have physiological functions such as antibacterial and disease resistance and regulation of gastrointestinal absorption. Under certain conditions, they can also promote the proliferation of animal cells in vitro and enhance the expression of products. Therefore, more and more researchers use casein as a substrate for enzymatic hydrolysis. To prepare polypeptides with functional properties. Such as the preparation of casein phosphopeptides with antioxidant properties, polypeptides with blood pressure lowering function, polypeptides easily absorbed by the human body, and polypeptides that promote the growth and reproduction of beneficial intestinal flora.

In addition, Zheng Lina and Zhang Lanwei from the School of Food Science and Technology of Northeast Agricultural University found that the optimal hydrolysis conditions for alkaline protease to hydrolyze casein to prepare casein phosphopeptides are: substrate concentration 15%, enzyme concentration 800μ/g, pH 9.0, temperature 55°C, time 2.5h. The optimal process conditions for Cai Weirong and Xue Zhenglian to hydrolyze casein with alkaline protease to prepare casein phosphopeptide: substrate concentration 10%, enzyme amount 1500μ/g, reaction temperature 45℃, pH 10.5, reaction time 150min. These researchers all used alkaline protease and casein to prepare casein phosphopeptides, but the optimal preparation conditions obtained were quite different. How to obtain stable hydrolyzate under different enzymolysis conditions is quite important.

In industrial production, product stability is more important. For a given enzymatic hydrolysis system, there are five main factors affecting the process, namely: reaction temperature (T), substrate concentration (S), pH, enzyme-to-substrate ratio (E/S), degree of hydrolysis (DH) or Hydrolysis time (t). Alder-Nissen used the pH-stat method to control the pH of the reaction process of alkaline protease to hydrolyze soybean protein isolate, to measure the properties of its hydrolyzate at a lower degree of hydrolysis, and to study different hydrolysis conditions such as temperature, pH, and enzyme-to-substrate ratio and the effect of substrate concentration on the properties of the hydrolyzate. However, in industrial production, it is difficult to completely control these five hydrolysis conditions, and due to the limitation of production conditions, different production batches often cannot guarantee that the hydrolysis conditions are the same every time. Therefore, it is very important to obtain hydrolysates with stable functions and properties under flexible production conditions.

At present, there are not many studies on this aspect, and the present invention hopes to provide guidance for industrial production, so as to obtain enzymolysis products with stable functional properties under different temperature-enzyme-to-substrate ratio combinations.

Contents of the invention

The purpose of the present invention is to provide a method for preparing casein hydrolyzate with stable functions and properties under different hydrolysis conditions.

In the technical scheme of the present invention, casein is used as a raw material, selected to be hydrolyzed under different hydrolysis conditions, and its hydrolysis is controlled to obtain hydrolyzates with a degree of hydrolysis of 5%, 10% and 15% under each hydrolysis condition. Under the same degree of hydrolysis, the trichloroacetic acid (TCA) soluble nitrogen content and DPPH clearance rate of the hydrolyzate obtained under different conditions did not show significant differences (P<0.05). Specifically, the obtained casein hydrolyzate powder The nature is stable. The steps are:

(1) Preparation of enzymatic hydrolysis substrate: take casein, swell with 10-15 times the mass of distilled water, use 0.5M NaOH solution to adjust the pH value to 8.5±0.1, until the pH value is constant, and then quantify it with distilled water to make the casein The final mass concentration is 4%-8%, and the enzymatic hydrolysis substrate is obtained;

(2) Enzymolysis: Take the enzymolysis substrate prepared in step (1) and place it in an enzyme reactor, control the temperature at 40-60°C, and conduct a circulating water bath for 5 minutes; add alkaline protease to the enzymolysis substrate to make the enzyme and The mass ratio of the substrate protein is 1%-3%; during the enzymolysis process, the pH of the reaction is maintained at 8.5±0.1 by adding alkaline solution;

(3) Enzyme inactivation: When the degree of hydrolysis reaches a fixed value between 5% and 15%, heat at 95oC for 10 minutes to inactivate the enzyme;

(4) Cooling: the hydrolyzed solution after deactivation in step (3) is cooled and dried to obtain light yellow or milky white casein hydrolyzate powder.

The alkaline solution in step (2) is a 0.1M alkaline solution.

The obtained casein hydrolyzate powder is stable in nature, and the specific test results are as follows:

(1) Comparing the difference of TCA soluble nitrogen content of hydrolyzate under the same degree of hydrolysis, when the significance level is p>0.05, the difference is not significant.

(2) Comparing the difference of DPPH clearance rate of hydrolyzate under the same degree of hydrolysis, the difference is not significant when the significance level is p>0.05.

(3) Comparing the molecular weight distribution (SEC-HPLC) of the hydrolyzate under the same degree of hydrolysis, the molecular weight distribution percentage of the hydrolyzate in the four intervals of molecular weight greater than 10kD, 1-10kD, 0.5-1kD and less than 0.5kD and the reference group conditions Compared with the value below, the deviation is within 5%, E/S=1/100, T=50°C as the reference group.

Analytical method:

1. The pH-stat method controls the hydrolysis reaction, and the method for calculating the degree of hydrolysis is as follows:

In the formula: B-consumed volume of standard alkali (mL);

N _b -concentration of standard base (mol/L);

Degree of dissociation of α-α amino groups

M _p - content of protein in the substrate (g);

h _tot - total number of peptide bonds per unit mass of protein (mmol/g), for casein h _tot = 8.2 mmol/g.

2. Determination of nitrogen content: semi-micro Kjeldahl method (GB 5009.5—1985 ).

3. Soluble nitrogen content of trichloroacetic acid (TCA): The solubility of the hydrolyzate was determined by the trichloroacetic acid method. The specific operation steps are: weigh 2.0 g of freeze-dried sample, add 10 mL of 15% trichloroacetic acid (TCA) solution, and mix well. After standing for 5 minutes, transfer the solution quantitatively, centrifuge at 4000r/min for 10 minutes, and take all the supernatant. The nitrogen content was determined by the Kjeldahl method (casein protein conversion factor is 6.38). Calculated as follows:

4. DPPH clearance rate: Take 4mL of sample solution with a certain concentration, add 1mL of DPPH solution prepared with methanol, and make the final concentration of DPPH 0.2mmol/L. Vigorously shake and mix well, then place it in a dark room for 30min, and measure the absorbance at 517nm. Calculate the DPPH clearance rate according to the formula.

In the formula: _Ax is the absorbance after adding the sample solution, _Ax0 is the absorbance of the sample solution background, and _A0 is the absorbance of the blank control solution.

5. The molecular weight distribution of the hydrolyzate was determined. The specific operation steps are as follows: prepare sample solution 10 mg/mL with mobile phase, filter through a cellulose acetate membrane with a pore size of 0.22 μm, and detect with a high-performance liquid chromatograph and an ultraviolet detector. Prepare the peptide standard solution at the same time, inject the sample after passing through the membrane, and make a relative molecular mass calibration curve. Amino acid-arginine (451Da), bacillus enzyme (1450Da), cytochrome C (12500Da). The chromatographic conditions are: chromatographic column: TSKgel G2000SWXL, 300mm*7.8mm; mobile phase: acetonitrile: water: trifluoroacetic acid = 45:55:0.1; flow rate: 0.5mL/min; injection volume: 20μL; detection wavelength: 220nm; Column temperature: 30°C, signal acquisition time 30min.

Beneficial effects of the present invention: the present invention can be adjusted according to different production conditions, casein hydrolyzed to obtain the casein hydrolyzate of the same DH, so that it has equivalent properties. The invention can guide industrial production, and can still ensure the stability of the product by elastically changing the production conditions.

Description of drawings

Figure 1 Molecular weight distribution diagram of 5% casein hydrolysis degree.

Figure 2 Molecular weight distribution of casein with 10% degree of hydrolysis.

Figure 3 Molecular weight distribution of casein with a degree of hydrolysis of 15%.

Detailed ways

The present invention will be further described below in conjunction with specific examples, and its specific examples should be understood as illustrations only, not limiting, and the protection scope of the present invention cannot be limited by the following examples.

Example 1

The mass concentration of casein substrate is 5%, and the hydrolysis temperature is controlled by circulating water bath to 50±0.2°C. According to the ratio of enzyme to substrate protein (E/S) 1/100, alkaline protease is added, and by adding 0.1M Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. Under these conditions, hydrolysates with degrees of hydrolysis of 5%, 10% and 15% were prepared. The soluble nitrogen content of DH5%, 10% and 15% TCA was determined to be 54.36%, 64.31% and 75.34%, respectively, and the DPPH clearance rate was 34.77%, 53.62% and 69.78%, respectively.

Example 2

The mass concentration of casein substrate is 5%, and the hydrolysis temperature is controlled by circulating water bath to 50±0.2°C. According to the ratio of enzyme to substrate protein (E/S) 2/100, alkaline protease is added, and by adding 0.1M Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. Under these conditions, hydrolysates with degrees of hydrolysis of 5%, 10% and 15% were prepared. The hydrolyzate was cooled and freeze-dried to obtain the product. The soluble nitrogen contents of DH5, 10 and 15% TCA were determined to be 54.43%, 64.70% and 76.40%, respectively, and the DPPH clearance rates were 33.88%, 54.49% and 69.48%, respectively.

Example 3

The mass concentration of casein substrate is 5%, and the hydrolysis temperature is controlled by circulating water bath to 50±0.2°C. According to the ratio of enzyme to substrate protein (E/S) 3/100, alkaline protease is added, and by adding 0.1M Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. Under these conditions, hydrolysates with degrees of hydrolysis of 5%, 10% and 15% were prepared. The hydrolyzate was cooled and freeze-dried to obtain the product. The soluble nitrogen content of TCA of DH5%, 10% and 15% was determined to be 53.77%, 64.48% and 76.1%, respectively, and the DPPH clearance rate was 33.89%, 54.42% and 69.15%, respectively.

Example 4

The mass concentration of casein substrate is 5%, the hydrolysis temperature is controlled by circulating water bath to 60±0.2°C, the ratio of enzyme to substrate protein (E/S) is 1/100, alkaline protease is added, and the Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. Under these conditions, hydrolysates with degrees of hydrolysis of 5%, 10% and 15% were prepared. The hydrolyzate was cooled and freeze-dried to obtain the product. The soluble nitrogen contents of TCA with DH5%, 10% and 15% were determined to be 54.12%, 63.96% and 75.54%, respectively, and the DPPH clearance rates were 34.60%, 54.14% and 69.67%, respectively.

Example 5

The mass concentration of casein substrate is 5%, and the hydrolysis temperature is controlled by circulating water bath to 60±0.2°C. According to the ratio of enzyme to substrate protein (E/S) 2/100, alkaline protease is added, and by adding 0.1M Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. The soluble nitrogen contents of TCA with DH5%, 10% and 15% were determined to be 55.06%, 64.46% and 77.53%, respectively, and the DPPH clearance rates were 35.01%, 54.08% and 70.10%, respectively.

Example 6

The mass concentration of casein substrate is 5%, and the hydrolysis temperature is controlled by circulating water bath to 60±0.2°C. According to the ratio of enzyme to substrate protein (E/S) 3/100, alkaline protease is added, and by adding 0.1M Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. The soluble nitrogen contents of TCA with DH5%, 10% and 15% were determined to be 54.12%, 64.23% and 78.92%, respectively, and the DPPH clearance rates were 34.57%, 54.23% and 70.47%, respectively.

Comparative Example 1

The mass concentration of casein substrate is 5%, and the hydrolysis temperature is controlled by a circulating water bath to 70±0.2°C. According to the ratio of enzyme to substrate protein (E/S) 1/100, alkaline protease is added, and by adding 0.1M Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. The soluble nitrogen contents of TCA with DH5%, 10% and 15% were determined to be 44.97%, 63.80% and 63.13%, respectively, and the DPPH clearance rates were 30.16%, 53.52% and 56.67%, respectively.

Comparative example 2

The mass concentration of casein substrate is 5%, and the hydrolysis temperature is controlled by circulating water bath to 70±0.2°C. According to the ratio of enzyme to substrate protein (E/S) 2/100, alkaline protease is added, and by adding 0.1M Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. The soluble nitrogen content of TCA of DH5%, 10% and 15% was determined to be 49.33%, 64.40% and 72.13%, respectively, and the DPPH clearance rate was 37.93%, 54.03% and 64.62%, respectively.

Comparative Example 3

The mass concentration of the casein substrate is 5%, and the hydrolysis temperature is controlled by a circulating water bath to 70±0.2°C. According to the ratio of enzyme to substrate protein (E/S) 3/100, alkaline protease is added. Control the pH to 8.5±0.1 with standard alkali to reach the corresponding degree of hydrolysis (DH), heat at 95oC for 10 minutes to inactivate the enzyme, and dry to obtain the enzymatic hydrolyzate product. The soluble nitrogen contents of TCA with DH5%, 10% and 15% were determined to be 50.02%, 63.39% and 71.65%, respectively, and the DPPH clearance rates were 37.62%, 54.00% and 63.34%, respectively.

From Comparative Examples 1-3, it can be seen that the content of trichloroacetic acid (TCA) soluble nitrogen and the DPPH clearance rate of the hydrolyzed products obtained when the hydrolysis temperature is controlled at 70±0.2°C are significantly different (P≥0.05) , the properties of the obtained casein hydrolyzate powder are relatively unstable.

Example 7 Stability evaluation of hydrolyzate prepared under different hydrolysis conditions.

The stability of the hydrolyzate is determined by examining whether there are significant differences in the properties of the hydrolyzate with the same degree of hydrolysis obtained under different temperature-enzyme-to-substrate ratio combinations. TCA soluble nitrogen content and DPPH clearance rate were analyzed by variance analysis (p<0.05) by SPSS software, and the results are shown in Table 1. The hydrolyzate obtained under the conditions of T=50°C and E/S=1/100 was used as the reference group.

Under the same degree of hydrolysis, the results of the analysis of variance (F-test) of the properties of the hydrolyzate The results with the bold * are significant differences (p<0.05). E/S=1/100, T=50℃ as the control group, F _0.05 (1,2)=18.5, if F>18.5, it means that there is a significant difference at the significance level of a=0.05.

Table 1

The molecular weight distribution of the hydrolyzate prepared under different enzymolysis conditions was compared with the reference group, the molecular weight distribution (SEC-HPLC) of the hydrolyzate with the same degree of hydrolysis, in the four categories of molecular weight greater than 10kD, 1-10kD, 0.5-1kD and less than 0.5kD Compared with the value under the conditions of the reference group, the molecular weight distribution percentage of the hydrolyzate in each interval deviates within 5%. The molecular weight distributions of different DH are shown in Figures 1 to 3, among which the molecular weight distribution of 5% casein hydrolysis degree is shown in Figure 1, and the molecular weight distribution of 10% casein hydrolysis degree is shown in Figure 2, and the casein hydrolysis degree of 15% is shown in Figure 2 The molecular weight distribution is shown in Figure 3.

Claims (2)

1. utilize different hydrolysising condition preparations to there is a method for stable functional Hydrolysates of Casein, it is characterized in that step is:

(1) preparation enzymolysis substrate: get casein, swelling with the distilled water of 10-15 times of quality, adopt the NaOH solution of 0.5M to regulate pH value to 8.5 ± 0.1, until pH value is constant, quantitative with distilled water again, making caseic final quality concentration is 4%-8%, obtains enzymolysis substrate;

(2) enzymolysis: get enzymolysis substrate prepared by step (1) and be placed in enzyme reactor, controlling temperature is 40-60 DEG C, carries out circulator bath 5min; Sumizyme MP is added in enzymolysis substrate, make enzyme-to-substrate protein mass than being 1%-3%; In enzymolysis process, be 8.5 ± 0.1 by the pH that adds alkaline solution to maintain reaction;

(3) enzyme that goes out: in the time that degree of hydrolysis reaches a fixed value in 5%-15%, the 95oC heating 10min enzyme that goes out;

(4) cooling: the hydrolyzed solution that step (3) is gone out after enzyme, through cooling, be dried and obtain yellowish or milky Hydrolysates of Casein powder.

2. utilize according to claim 1 different hydrolysising condition preparations to there is the method for stable functional Hydrolysates of Casein, it is characterized in that: the alkaline solution that the described alkaline solution of step (2) is 0.1M.