A low-bitter casein hydrolysate and preparation method thereof

Abstract

This invention relates to the field of food processing technology, specifically to a low-bitterness casein hydrolysate and its preparation method. The preparation method provided by this invention includes: performing three enzymatic hydrolysis treatments on casein to obtain the casein hydrolysate; the enzyme used in the first enzymatic hydrolysis is trypsin, the enzyme used in the second enzymatic hydrolysis is flavor protease, and the enzyme used in the third enzymatic hydrolysis is TG enzyme; by mass, the amount of trypsin is 0.4-2.2% of the casein; the amount of flavor protease is 0.08-0.5% of the casein; and the amount of TG enzyme is 0.4-2.2% of the casein. This invention significantly reduces the bitterness of the obtained hydrolysate by optimizing the enzymatic hydrolysis conditions for casein, obtaining a casein hydrolysate with virtually no bitterness without adding exogenous non-enzymatic substances.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, in particular to a low-bitter casein enzymatic hydrolysate and a preparation method thereof. Background Art

[0002] Cow's milk is a food with high nutritional value. Its proteins have multiple physiological functions, including α-lactalbumin, β-lactoglobulin, lactoferrin, etc., which play an important biological role in the human body. However, cow's milk protein, especially cow's milk casein, will produce bitterness after enzymatic hydrolysis. This is mainly because the released bitter peptides contain hydrophobic amino acids. These hydrophobic amino acids are exposed in the polypeptide chain and can stimulate the taste buds and produce bitterness. Bitterness is a taste with a very low threshold, which will lead to a decrease in sensory quality. It can be detected when the content is low, affecting the acceptance of food and further affecting the application of casein enzymatic hydrolysates.

[0003] The bitter taste can be masked by using masking agents such as malic acid, citric acid, cyclodextrin, maltodextrin, starch and pectin, but this method will introduce other ingredients and limit the industrial application of the enzymatic hydrolysate. Microorganisms that can produce peptidases, such as Aspergillus oryzae, Aspergillus niger, yeast, lactic acid bacteria, etc., can be used to further hydrolyze bitter peptides and reduce bitterness, but this method will also introduce other ingredients and limit the industrial application of the enzymatic hydrolysate.

[0004] Utilizing the high adhesion of hydrophobic peptides to activated carbon, resins or solvents for adsorption, or separating bitter peptides with smaller molecular weight through ultrafiltration technology can significantly reduce their bitterness, but at the same time it will also remove a large number of active peptides and reduce their biological activity.

[0005] In summary, the current methods for debittering protein hydrolysates have certain limitations in application. Therefore, it is necessary to develop a method for preparing low-bitter casein hydrolysates without introducing other non-enzyme components and without losing active peptides during the enzymatic preparation process. Summary of the invention

[0006] The invention provides a casein hydrolysate with low bitterness and a preparation method thereof, so as to solve the defect that the casein hydrolysate in the prior art has a relatively large bitterness.

[0007] In a first aspect, the present invention provides a method for preparing a casein hydrolysate, comprising: performing three enzymatic hydrolysis treatments on casein to obtain a casein hydrolysate; the enzyme used in the first enzymatic hydrolysis is trypsin, the enzyme used in the second enzymatic hydrolysis is flavor protease, and the enzyme used in the third enzymatic hydrolysis is TG enzyme;

[0008] The amount of trypsin used is 0.4-2.2% of casein by mass;

[0009] The amount of the flavor protease used is 0.08-0.5% of the casein;

[0010] The dosage of the TG enzyme is 0.4-2.2% of the casein.

[0011] The present invention finds that after three enzymatic hydrolysis treatments are carried out in sequence using three specific enzymes, the degree of casein hydrolysis is high and the types and contents of bitter peptides are significantly reduced, and the bitterness of the hydrolysate is significantly alleviated. The coordinated action of the above three enzymes is critical for improving the bitterness of the hydrolysate. Replacing these three enzymes with other commonly used casein hydrolysis enzymes will destroy this coordinated action, resulting in an increase in the bitterness of the hydrolysate; and the order of the three enzymatic hydrolysis treatments and the amount of enzymes used will affect the bitterness intensity of the final enzymatic hydrolysis product. For example, increasing the amount of the enzymes used will increase the bitterness of the enzymatic hydrolysis product. Similarly, the bitterness of the enzymatic hydrolysis product obtained by adjusting the enzymatic hydrolysis order will also increase significantly.

[0012] In the preparation method provided by the present invention, before the first enzymatic hydrolysis, the temperature of the casein solution is 37-45°C.

[0013] In the preparation method provided by the present invention, the temperature of the enzymatic hydrolysis reaction is 40-60° C., the pH is 6-8, and the time of the enzymatic hydrolysis reaction is 20 min-120 min.

[0014] Preferably, the temperature of the first enzymatic hydrolysis is 40-45° C., the pH is 7.0-7.5, and the enzymatic hydrolysis time is 45-120 min.

[0015] The temperature of the second enzymatic hydrolysis is 50-60°C, the pH is 6.5-7.5 (preferably 6.8-7.2), and the enzymatic hydrolysis time is 20-70 minutes.

[0016] The temperature of the third enzymatic hydrolysis is 50-60°C, the pH is 7-8 (preferably 7.8-8.0), and the enzymatic hydrolysis time is 20-120 minutes.

[0017] In the preparation method provided by the present invention, after each enzymatic hydrolysis treatment, an enzyme inactivation treatment is performed.

[0018] Preferably, the enzyme inactivation treatment is carried out at 90-95°C for 10-15 min.

[0019] As a specific embodiment of the present invention, the preparation method of casein enzymatic hydrolysate comprises:

[0020] (1) Add trypsin accounting for 0.4-2.2% of the casein mass to the casein solution, the enzymatic hydrolysis temperature is 40-45°C, the pH is 7.0-7.5, after enzymatic hydrolysis for 45 min-120 min, inactivate the enzyme at 90-95°C for 10-15 min, and obtain an enzymatic hydrolyzate;

[0021] (2) adding flavor protease accounting for 0.08-0.5% of the casein mass to the enzymatic hydrolysate obtained in step (1), the enzymatic hydrolysis temperature is 50-60° C., the pH is 6.5-7.5, after enzymatic hydrolysis for 20-70 min, the enzyme is inactivated at 90-95° C. for 10-15 min, to obtain an enzymatic hydrolysate;

[0022] (3) Add TG enzyme accounting for 0.4-2.2% of the casein mass to the enzymatic hydrolysate obtained in step (2), the enzymatic hydrolysis temperature is 50-60°C, the pH is 7-8, after enzymatic hydrolysis for 20-120 minutes, inactivate the enzyme at 90-95°C for 10-15 minutes, and obtain a casein enzymatic hydrolysate.

[0023] In the preparation method provided by the present invention, NaOH solution is used to adjust the pH of the enzymatic reaction system.

[0024] In the preparation method provided by the present invention, the concentration of the casein solution is 4-8%.

[0025] In the preparation method provided by the present invention, the casein enzymatic hydrolysate obtained in step (3) is centrifuged, and the supernatant is collected to obtain a casein enzymatic hydrolysate; at this time, the casein enzymatic hydrolysate is in a liquid state; or, the casein enzymatic hydrolysate obtained in step (3) is centrifuged, and the supernatant is collected and then dried to obtain a casein enzymatic hydrolysate, and at this time, the casein enzymatic hydrolysate is in a solid state, and can also be ground after drying to obtain a casein enzymatic hydrolysate powder.

[0026] In a second aspect, the present invention provides application of the above preparation method in reducing the bitterness of casein hydrolysate.

[0027] In a third aspect, the present invention provides a casein enzymatic hydrolysate prepared by the above-mentioned preparation method.

[0028] The beneficial effects of the present invention are:

[0029] The present invention optimizes the enzymatic hydrolysis conditions for casein, significantly reduces the bitterness of the obtained enzymatic hydrolysate, and obtains a casein hydrolysate with substantially no bitterness without adding exogenous non-enzymatic substances. Compared with the same type of bitterness reduction technology provided by the prior art, the bitterness in the casein hydrolysate can be reduced by 30-40%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the bitterness value result obtained by using the electronic tongue in Experimental Example 1 of the present invention.

[0031] Figure 2 This is the hydrolysis degree result of the casein enzymatic hydrolysis product in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The method for preparing the milk casein solution used in the following examples is as follows: 36 g of micellar casein powder is weighed at room temperature for use. About 500 mL of deionized water is then measured and placed in an enzyme reactor, the temperature is raised to 50-60° C., the micellar casein powder is added to the enzyme reactor, stirred evenly, and the volume is fixed after hydration for 30-60 minutes to prepare a 5% (w / w) casein solution.

[0034] The various enzymes used in the following examples and comparative examples are all commercially available products, among which the enzyme activity of trypsin is 1000-2000 BAEE U / mg, the enzyme activity of flavor protease is 500 LAPU / g, the enzyme activity of TG enzyme is 130.5 U / g, the enzyme activity of alkaline protease is 2.4 AU / g, and the enzyme activity of papain is 6000 U / mg.

[0035] Example 1

[0036] This embodiment provides a method for preparing a casein hydrolysate with low bitterness, and the steps are as follows:

[0037] (1) Cool the casein solution to 37°C, adjust the solution pH to 7 with 1 mol / L NaOH solution, and add trypsin accounting for 0.42% of the casein substrate mass. During the enzymatic hydrolysis process, the pH of the reaction system is maintained at 7-7.5 with 1 mol / L NaOH standard solution, the enzymatic hydrolysis temperature is 45°C, and the enzymatic hydrolysis time is 45 minutes. Inactivate the enzyme in a 90-95°C water bath for 10-15 minutes to terminate the enzymatic hydrolysis reaction and obtain an enzymatic hydrolyzate.

[0038] (2) After the enzymatic hydrolysate obtained in step (1) is cooled to 55-60°C, flavor protease accounting for 0.084% of the mass of the casein substrate is added. The enzymatic hydrolysis reaction temperature is 50°C, the pH is 7, and the enzymatic hydrolysis time is 20 minutes. The enzyme is inactivated in a water bath at 90-95°C for 10-15 minutes to obtain an enzymatic hydrolysate.

[0039] (3) After the enzymatic hydrolysate obtained in step (2) is cooled to an appropriate temperature, TG enzyme accounting for 0.42% of the mass of the casein substrate is added. The enzymatic hydrolysis reaction temperature is 50°C, the pH is 8, and the enzymatic hydrolysis time is 20 minutes. The enzyme is inactivated in a 90-95°C water bath for 10-15 minutes, the obtained casein enzymatic hydrolysate is centrifuged, and the supernatant is collected to obtain a casein enzymatic hydrolysate.

[0040] Example 2

[0041] This embodiment provides a method for preparing a casein hydrolysate with low bitterness, and the steps are as follows:

[0042] (1) Cool the casein solution to 45°C, adjust the solution pH to 7 with 1 mol / L NaOH solution, and add trypsin accounting for 2.1% of the casein substrate mass. During the enzymatic hydrolysis process, the pH of the reaction system is maintained at 7-7.5 with 1 mol / L NaOH standard solution, the enzymatic hydrolysis temperature is 40°C, and the enzymatic hydrolysis time is 120 minutes. Inactivate the enzyme in a 90-95°C water bath for 10-15 minutes to terminate the enzymatic hydrolysis reaction and obtain an enzymatic hydrolyzate.

[0043] (2) After the enzymatic hydrolysate obtained in step (1) is cooled to 55-60°C, flavor protease accounting for 0.42% of the mass of the casein substrate is added. The enzymatic hydrolysis reaction temperature is 60°C, the pH is 7, and the enzymatic hydrolysis time is 60 min. The enzyme is inactivated in a water bath at 90-95°C for 10-15 min to obtain an enzymatic hydrolysate.

[0044] (3) After the enzymatic hydrolysate obtained in step (2) is cooled to an appropriate temperature, TG enzyme accounting for 2.1% of the mass of the casein substrate is added. The enzymatic hydrolysis reaction temperature is 60°C, the pH is 8, and the enzymatic hydrolysis time is 120 min. The enzyme is inactivated in a 90-95°C water bath for 10-15 min, the obtained casein enzymatic hydrolysate is centrifuged, and the supernatant is collected to obtain a casein enzymatic hydrolysate.

[0045] Example 3

[0046] This embodiment provides a method for preparing a casein hydrolysate with low bitterness, and the steps are as follows:

[0047] (1) Cool the casein solution to 40°C, adjust the solution pH to 7 with 1 mol / L NaOH solution, and add trypsin accounting for 1.26% of the casein substrate mass. During the enzymatic hydrolysis process, the pH of the reaction system is maintained at 7-7.5 with 1 mol / L NaOH standard solution, the enzymatic hydrolysis temperature is 45°C, and the enzymatic hydrolysis time is 75 minutes. Inactivate the enzyme in a 90-95°C water bath for 10-15 minutes to terminate the enzymatic hydrolysis reaction and obtain an enzymatic hydrolyzate.

[0048] (2) After the enzymatic hydrolysate obtained in step (1) is cooled to 55-60°C, flavor protease accounting for 0.25% of the mass of the casein substrate is added. The enzymatic hydrolysis reaction temperature is 55°C, the pH is 7, and the enzymatic hydrolysis time is 70 minutes. The enzyme is inactivated in a water bath at 90-95°C for 10-15 minutes to obtain an enzymatic hydrolysate.

[0049] (3) After the enzymatic hydrolysate obtained in step (2) is cooled to an appropriate temperature, TG enzyme accounting for 1.26% of the mass of the casein substrate is added. The enzymatic hydrolysis reaction temperature is 55°C, the pH is 8, and the enzymatic hydrolysis time is 70 minutes. The enzyme is inactivated in a 90-95°C water bath for 10-15 minutes, the obtained casein enzymatic hydrolysate is centrifuged, and the supernatant is collected to obtain a casein enzymatic hydrolysate.

[0050] Comparative Example 1

[0051] This comparative example provides a method for preparing casein hydrolysate, which differs from the method of Example 2 in that the enzymatic hydrolysis steps of steps (2) and (3) are swapped in order, i.e., TG enzyme hydrolysis is performed in step (2), and flavor protease hydrolysis is performed in step (3).

[0052] Comparative Example 2

[0053] This comparative example provides a method for preparing casein hydrolysate, which differs from the method in Example 2 in that the trypsin in step (1) is replaced by alkaline protease accounting for 0.21% of the mass of the casein substrate.

[0054] Comparative Example 3

[0055] This comparative example provides a method for preparing casein hydrolysate, which differs from the method of Example 2 in that: in step (2), papain accounting for 0.63% of the mass of the casein substrate is additionally added, that is, step (2) uses flavor protease and papain for enzymatic hydrolysis.

[0056] Comparative Example 4

[0057] This comparative example provides a method for preparing casein hydrolysate, which differs from the method of Example 2 in that: in step (1), trypsin is added in an amount of 2.5% by weight of the casein substrate.

[0058] Comparative Example 5

[0059] This comparative example provides a method for preparing casein hydrolysate, which differs from the method of Example 2 in that: in step (2), flavor protease is added in an amount accounting for 0.84% ​​of the mass of the casein substrate.

[0060] Comparative Example 6

[0061] This comparative example provides a method for preparing casein hydrolysate, which differs from the method of Example 2 in that: in step (3), TG enzyme is added in an amount of 2.5% of the mass of the casein substrate.

[0062] Experimental Example 1 Determination of bitterness value

[0063] The present invention uses an electronic tongue to measure the bitterness value. The Smartongue electronic tongue system consists of four parts: a sensor array, a signal conditioning system, a test platform, and application software. The sensor array consists of six working electrodes (platinum, gold, palladium, tungsten, titanium, and silver) and an auxiliary electrode to form an independent unit mechanism and a reference electrode to form a complete sensor array; the signal conditioning system consists of a signal excitation unit, a signal conditioning unit, and a data acquisition unit; the test platform provides a strict working environment for sample placement and detection. The application software is an application program running on a computer for controlling smart tongue detection, analyzing data, and outputting results.

[0064] The method for measuring bitterness using electronic tongue is as follows:

[0065] (1) Sample preparation: Thaw the frozen sample at 4°C overnight until the sample is completely dissolved, then place it at room temperature for 2 hours to ensure that the sample reaches room temperature. Mix the solution before weighing the sample to ensure the uniformity of the solution. Accurately measure 40mL of solution into a 250mL beaker, dilute with 80mL of deionized water, and stir with a glass rod. Then dispense into special beakers for electronic tongues, pour 15mL of solution into each beaker, and each sample is six parallel.

[0066] (2) How to operate the electronic tongue: Turn on the electronic tongue host, automatic sampler and electronic tongue software in sequence. After the instrument is turned on, set the instrument parameters. The instrument parameters are as follows:

[0067] The initial voltage and end voltage are 1V and -1V respectively, and the voltage step is 0.2; the sensitivity of the six sensors is set to 1E-5; sensor types: Pt, Au, Pd, W, Ti, Ag.

[0068] Then, place the sensor in the electronic tongue test beaker containing 15mL of 0.01 mol / L potassium chloride solution, click the "preheat" button to activate the sensor, take out the sensor after the preheating, place it in deionized water, and click the "clean" button to clean the sensor. Then edit the sample information in the sample setting interface, and place the samples to be tested in sequence on the sample tray. Before the test starts, perform a "pre-inspection" on the samples to be tested. After the pre-inspection, click "start" to perform a sample sequence test. The bitterness value results obtained from the test are shown in Figure 1 The results showed that the bitterness values ​​of the casein hydrolysates of Examples 1-3 were significantly lower than those of Comparative Examples 1-6.

[0069] Experimental Example 2 Determination of the degree of hydrolysis of enzymatic hydrolysis products

[0070] The degree of hydrolysis of the casein enzymatic hydrolysis products obtained in each embodiment and comparative example was determined with reference to the pH-Stat method (Adler-Nissen J., 1986). The specific calculation formula is as follows:

[0071]

[0072] In the above calculation formula, B is the volume of alkali solution consumed in the enzymatic reaction (mL); N b is the molar concentration of alkali solution (NaOH) (mol / L); Mp is the mass of protein in the substrate (g); h tot is the total number of peptide bonds in the protein (mmol / g). Relative to micellar casein, h tot =8.2 mmol / g; α is the dissociation degree of α-NH2, which is a function of the reaction temperature. The hydrolysis degree test results are shown in Figure 2 The results showed that the casein hydrolysates of Examples 1-3 had a more appropriate degree of hydrolysis.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a casein hydrolysate, characterized in that: include: The casein is subjected to three enzymatic hydrolysis treatments to obtain a casein enzymatic hydrolysate; the enzyme used in the first enzymatic hydrolysis is trypsin, the enzyme used in the second enzymatic hydrolysis is flavor protease, and the enzyme used in the third enzymatic hydrolysis is TG enzyme; The amount of trypsin used is 0.4-2.2% of casein by mass; The amount of the flavor protease used is 0.08-0.5% of the casein; The dosage of the TG enzyme is 0.4-2.2% of casein; The temperature of enzymatic hydrolysis is 40~60℃, the pH is 6~8, and the time of enzymatic hydrolysis is 20min~120min.

2. The preparation method according to claim 1, characterized in that: The temperature of the first enzymatic hydrolysis is 40-45°C, the pH is 7.0-7.5, and the enzymatic hydrolysis time is 45-120 min; and / or, the temperature of the second enzymatic hydrolysis is 50-60° C., the pH is 6.5-7.5, and the enzymatic hydrolysis time is 20-70 min; And / or, the temperature of the third enzymolysis is 50-60° C., the pH is 7-8, and the time of the enzymolysis is 20-120 min.

3. The preparation method according to claim 1 or 2, characterized in that: After each enzymatic hydrolysis treatment, the enzyme is inactivated.

4. The preparation method according to claim 3, characterized in that: The enzyme inactivation treatment is carried out at 90-95° C. for 10-15 min.

5. The preparation method according to any one of claims 1, 2 and 4, characterized in that: include: (1) Add trypsin accounting for 0.4-2.2% of the casein mass to the casein solution, the enzymatic hydrolysis temperature is 40-45°C, the pH is 7.0-7.5, and after enzymatic hydrolysis for 45-120 minutes, inactivate the enzyme at 90-95°C for 10-15 minutes to obtain an enzymatic hydrolyzate; (2) adding flavor protease accounting for 0.08-0.5% of the casein mass to the enzymatic hydrolysate obtained in step (1), the enzymatic hydrolysis temperature is 50-60° C., the pH is 6.5-7.5, after enzymatic hydrolysis for 20-70 min, the enzyme is inactivated at 90-95° C. for 10-15 min, to obtain an enzymatic hydrolysate; (3) adding TG enzyme accounting for 0.4-2.2% of the casein mass to the enzymatic hydrolysate obtained in step (2), the enzymatic hydrolysis temperature is 50-60°C, the pH is 7-8, after enzymatic hydrolysis for 20-120 minutes, inactivating the enzyme at 90-95°C for 10-15 minutes, to obtain a casein enzymatic hydrolysate; The concentration of the casein solution is 4-8%.

6. The preparation method according to claim 5, characterized in that: The casein enzymatic hydrolysate obtained in step (3) is centrifuged, and the supernatant is collected to obtain a casein enzymatic hydrolysate; or, the casein enzymatic hydrolysate obtained in step (3) is centrifuged, and the supernatant is collected and then dried to obtain a casein enzymatic hydrolysate.

7. Use of the preparation method according to any one of claims 1 to 6 in reducing the bitterness of casein enzymatic hydrolysates.

8. A casein enzymatic hydrolysate, characterized in that The method is prepared according to any one of claims 1 to 6.

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