Efficient fresh polypeptide preparation method based on proteolysis

Abstract

The invention discloses an efficient fresh polypeptide preparation method based on proteolysis, and belongs to the field of food. The invention relates to an efficient fresh polypeptide preparation method based on proteolysis, which comprises the following steps: sample pretreatment: cleaning fresh chicken skeleton and chicken breast, mixing the chicken skeleton and the chicken breast according to a certain proportion, sterilizing, and crushing to obtain minced chicken; ultrasonic treatment: uniformly mixing the minced chicken obtained in the step 1 with distilled water in proportion, and performing ultrasonic treatment; and performing enzymolysis: adding compound protease into the minced chicken solution subjected to ultrasonic treatment in the step 2 for enzymolysis, performing enzyme deactivation after enzymolysis is completed, performing cooling, performing centrifugation, taking supernate, adjusting the pH value to be neutral, and performing freeze drying.

Description

Technical Field

[0001] The invention relates to the field of food, and in particular to a method for preparing a highly efficient fresh polypeptide based on proteolysis. Background Art

[0002] Food flavoring agents are a type of product that can enrich and improve the flavor of food or dishes by using a small amount, thus giving them a fresh, fragrant and delicious taste. Among them, basic flavoring agents such as flavor enhancers, sweeteners, and saltiness enhancers are not only directly used in food and dish making, but are also widely used in various compound seasonings, greatly enriching the application and development of food compound seasonings.

[0003] Flavor peptides are a new type of peptide-based seasoning that has gradually developed in recent years. Because of their safety, nutrition, and health, they have broad application prospects in the production of processed foods, catering, and compound seasonings. The flavor peptides that have been discovered so far are usually obtained from food through enzymatic extraction, and a small number of short peptides are obtained through chemical synthesis and identification, with a molecular weight between 1 and 5 kDa. Flavor peptides mainly include six categories: sweet peptides, sour peptides, umami peptides, salty peptides, bitter peptides, and kokumi peptides. In addition, they also include some more special flavor peptides that can have multiple flavor effects at the same time, such as Gly-Asp, which has both umami and sour tastes. Flavor peptides are widely present in protein-rich animal and plant foods such as meat, beans, and fish. So far, a variety of flavor peptides have been isolated and identified from a variety of natural foods such as soybeans, meat and poultry, soy sauce, and pufferfish.

[0004] Umami peptides are a class of peptide components with umami flavor that can give food a delicious taste and have great application potential in the development of new umami seasoning bases. Currently available umami peptides are mainly obtained by enzymatic extraction and identification from protein-rich animal and plant foods such as soybeans, fish, livestock and poultry meat, and ham. While bringing a pleasant taste experience, they can also provide the human body with peptide / amino acid nutrients. In addition, they are easy to absorb, have good processing characteristics, are non-antigenic, and have certain physiological activities. They are in line with the "green and safe" development trend and are the most promising new generation of umami agents.

[0005] The preparation methods of umami peptides mainly include acid-base hydrolysis and biological enzymatic hydrolysis. The degree of hydrolysis in the acid-base method is difficult to control, the content of active peptides is low, and the amino acids are destroyed and even carcinogenic substances are generated, which greatly damages the nutritional value of the protein. Protein enzymatic hydrolysis mainly involves the action of proteases on the peptide bonds of proteins, which gradually hydrolyzes the proteins into polypeptides, small molecule peptides, free amino acids, etc. This method has few side reactions, mild conditions, easy to control the degree of hydrolysis, and the amino acids will not be destroyed, while retaining the nutrients to the maximum extent.

[0006] Although single enzyme hydrolysis is simple to operate and has low cost, the degree of protein hydrolysis is not high due to the single enzyme cleavage site, the enzymatic hydrolysis time is long, and the aroma of the thermal reaction product after single enzyme hydrolysis is not prominent. When it is difficult to achieve the expected effect with only one enzyme, it is very necessary to provide an efficient fresh polypeptide preparation method based on proteolysis. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the defects of the prior art and provide a method for preparing fresh polypeptides based on proteolysis. In order to shorten the reaction time and increase the umami taste, the embodiment of the present invention adopts the following technical solution: a method for preparing fresh polypeptides based on proteolysis, comprising the following steps:

[0008] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts are cleaned, the chicken skeletons and chicken breasts are mixed in a certain proportion, sterilized, and then crushed to obtain chicken mince;

[0009] (2) Ultrasonic treatment: the chicken paste prepared in step (1) is mixed with distilled water in a certain proportion and then subjected to ultrasonic treatment;

[0010] (3) Enzymatic hydrolysis: adding a composite protease to the chicken mince solution treated with ultrasound in step (2) for enzymatic hydrolysis for 2-5 hours, inactivating the enzyme after enzymatic hydrolysis, cooling and centrifuging at 3500-4200 r / min for 15-25 minutes, collecting the supernatant, adjusting the pH value to neutral, and freeze-drying to obtain the fresh polypeptide.

[0011] Furthermore, in step (1), the mass ratio of the chicken skeleton to the chicken breast is (6-9):2.

[0012] Furthermore, in step (1), the sterilization temperature is 120-140° C. and the time is 50-70 min.

[0013] Furthermore, the ultrasonic conditions in step (2) are as follows: power 600-900W, and ultrasonic time 20-40min.

[0014] Furthermore, the mass volume ratio of the chicken paste to distilled water in step (2) is 1:2 (g / ml).

[0015] Furthermore, the compound protease in step (3) is a composite protease and a flavor protease.

[0016] Furthermore, the mass ratio of the composite protease to the flavor protease is 1:1.

[0017] Furthermore, the dosage of the composite protease in step (3) is 0.1-0.5% of the mass fraction of the chicken paste.

[0018] Furthermore, the pH value of step (3) is 7.5 and the temperature is 50°C.

[0019] Furthermore, the enzyme inactivation temperature in step (3) is 90° C. and the time is 10-20 min.

[0020] Compared with existing technologies:

[0021] (1) The present invention helps to promote the hydrolysis of chicken skeletons and chicken breasts through ultrasonic treatment, and the ultrasonic treatment increases the content of umami amino acids in the enzymatic hydrolysate and enhances the umami taste.

[0022] (2) The present invention uses a compound protease for simultaneous hydrolysis, which greatly shortens the reaction time and significantly enhances the umami taste.

[0023] (3) The present invention adopts the process of ultrasound and compound protease, which greatly improves the hydrolysis degree and shortens the reaction time, and obtains umami peptides with prominent sweet and umami taste, weak fishy smell and bitterness, rich nutrients and good seasoning effect. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Example 1

[0026] The embodiment of the present invention provides a method for preparing an efficient fresh polypeptide based on proteolysis, comprising the following steps:

[0027] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts were cleaned, mixed with chicken skeletons and chicken breasts in a mass ratio of 8:2, sterilized at 130°C for 60 min, and then crushed to obtain chicken mince.

[0028] (2) Ultrasonic treatment: The chicken paste prepared in step 1 was evenly mixed with distilled water in a ratio of 1:2 (g / ml), and then ultrasonically treated at 800 W for 30 min.

[0029] (3) Enzymatic hydrolysis: add commercially available composite protease and flavor protease in a mass ratio of 1:1 to the chicken paste solution ultrasonically treated in step 2, with the amount of protease being 0.3% of the mass fraction of the chicken paste, the enzymatic hydrolysis time being 3 h, and the pH value being 7.5. After completion, the enzyme is inactivated at 90°C for 15 min. After cooling, centrifuge at 4000 r / min for 20 min, collect the supernatant, adjust the pH to neutral, and freeze-dry at -80°C for 10 h.

[0030] Example 2

[0031] The embodiment of the present invention provides a method for preparing an efficient fresh polypeptide based on proteolysis, comprising the following steps:

[0032] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts were cleaned, mixed with chicken skeletons and chicken breasts in a mass ratio of 6:2, sterilized at 120°C for 70 min, and then crushed to obtain chicken mince.

[0033] (2) Ultrasonic treatment: The chicken paste prepared in step 1 was evenly mixed with distilled water in a ratio of 1:2 (g / ml), and then ultrasonically treated at 600 W for 40 min.

[0034] (3) Enzymatic hydrolysis: add commercially available composite protease and flavor protease in a mass ratio of 1:1 to the chicken paste solution ultrasonically treated in step 2, with the amount of protease being 0.1% of the mass fraction of the chicken paste, the enzymatic hydrolysis time being 2 h, and the pH value being 7.5. After completion, the enzyme is inactivated at 90°C for 10 min. After cooling, the solution is centrifuged at 3500 r / min for 25 min, the supernatant is collected, the pH is adjusted to neutral, and freeze-dried at -80°C for 10 h.

[0035] Example 3

[0036] The embodiment of the present invention provides a method for preparing an efficient fresh polypeptide based on proteolysis, comprising the following steps:

[0037] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts were cleaned, mixed with chicken skeletons and chicken breasts in a mass ratio of 9:2, sterilized at 140°C for 50 min, and then crushed to obtain chicken mince.

[0038] (2) Ultrasonic treatment: The chicken paste prepared in step 1 was evenly mixed with distilled water in a ratio of 1:2 (g / ml), and then ultrasonically treated at 900 W for 20 min.

[0039] (3) Enzymatic hydrolysis: add commercially available composite protease and flavor protease in a mass ratio of 1:1 to the chicken paste solution ultrasonically treated in step 2, with the amount of protease being 0.5% of the mass fraction of the chicken paste, the enzymatic hydrolysis time being 5 h, and the pH value being 7.5. After completion, the enzyme is inactivated at 90°C for 20 min. After cooling, centrifuge at 4200 r / min for 15 min, collect the supernatant, adjust the pH to neutral, and freeze-dry at -80°C for 10 h.

[0040] Comparative Example 1

[0041] The embodiment of the present invention provides a method for preparing an efficient fresh polypeptide based on proteolysis, comprising the following steps:

[0042] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts were cleaned, mixed with chicken skeletons and chicken breasts in a mass ratio of 8:2, sterilized at 130°C for 60 min, and then crushed to obtain chicken mince.

[0043] (2) Enzymatic hydrolysis: The chicken paste prepared in step 1 was uniformly mixed with distilled water in a ratio of 1:2 (g / ml), and commercially available composite protease and flavor protease were added in a mass ratio of 1:1. The amount of protease was 0.3% of the mass fraction of the chicken paste, the enzymatic hydrolysis time was 3 h, and the pH value was 7.5. After completion, the enzyme was inactivated at 90°C for 15 min. After cooling, the mixture was centrifuged at 4000 r / min for 20 min, the supernatant was collected, the pH was adjusted to neutral, and freeze-dried at -80°C for 10 h.

[0044] Comparative Example 2

[0045] The embodiment of the present invention provides a method for preparing an efficient fresh polypeptide based on proteolysis, comprising the following steps:

[0046] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts were cleaned, mixed with chicken skeletons and chicken breasts in a mass ratio of 8:2, sterilized at 130°C for 60 min, and then crushed to obtain chicken mince.

[0047] (2) Ultrasonic treatment: The chicken paste prepared in step 1 was evenly mixed with distilled water in a ratio of 1:2 (g / ml), and then ultrasonically treated at 800 W for 30 min.

[0048] (3) Enzymatic hydrolysis: add 0.3% by mass of commercially available composite protease (based on the mass of chicken paste) to the chicken paste solution ultrasonically treated in step 2, the enzymatic hydrolysis time is 3 h, the pH value is 7.5, and the enzyme is inactivated at 90°C for 15 min. After cooling, centrifuge at 4000 r / min for 20 min, collect the supernatant, adjust the pH to neutral, and freeze-dry at -80°C for 10 h.

[0049] Comparative Example 3

[0050] The embodiment of the present invention provides a method for preparing an efficient fresh polypeptide based on proteolysis, comprising the following steps:

[0051] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts were cleaned, mixed with chicken skeletons and chicken breasts in a mass ratio of 8:2, sterilized at 130°C for 60 min, and then crushed to obtain chicken mince.

[0052] (2) Ultrasonic treatment: The chicken paste prepared in step 1 was evenly mixed with distilled water in a ratio of 1:2 (g / ml), and then ultrasonically treated at 800 W for 30 min.

[0053] (3) Enzymatic hydrolysis: add 0.3% by mass of commercially available flavor protease (based on the mass of chicken paste) to the chicken paste solution ultrasonically treated in step 2, the enzymatic hydrolysis time is 3 h, the pH value is 7.5, and the enzyme is inactivated at 90°C for 15 min. After cooling, centrifuge at 4000 r / min for 20 min, collect the supernatant, adjust the pH to neutral, and freeze-dry at -80°C for 10 h.

[0054] Comparative Example 4

[0055] The embodiment of the present invention provides a method for preparing an efficient fresh polypeptide based on proteolysis, comprising the following steps:

[0056] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts were cleaned, mixed with chicken skeletons and chicken breasts in a mass ratio of 8:2, sterilized at 130°C for 60 min, and then crushed to obtain chicken mince.

[0057] (2) The chicken paste of step 1 was uniformly mixed with distilled water in a ratio of 1:2 (g / ml), and a commercially available composite protease (based on the mass of the chicken paste) was added at a mass fraction of 0.3% (based on the mass of the chicken paste). The enzymatic hydrolysis time was 3 h and the pH value was 7.5. After completion, the enzyme was inactivated at 90°C for 15 min. After cooling, the mixture was centrifuged at 4000 r / min for 20 min, the supernatant was collected, the pH was adjusted to neutral, and freeze-dried at -80°C for 10 h.

[0058] Comparative Example 5

[0059] The embodiment of the present invention provides a method for preparing an efficient fresh polypeptide based on proteolysis, comprising the following steps:

[0060] (1) Sample pretreatment: Fresh chicken skeletons and chicken breasts were cleaned, mixed with chicken skeletons and chicken breasts in a mass ratio of 8:2, sterilized at 130°C for 60 min, and then crushed to obtain chicken mince.

[0061] (2) The chicken paste of step 1 was uniformly mixed with distilled water in a ratio of 1:2 (g / ml), and 0.3% mass fraction of commercially available flavor protease (based on the mass of the chicken paste) was added, the enzymatic hydrolysis time was 3 h, and the pH value was 7.5. After completion, the enzyme was inactivated at 90°C for 15 min. After cooling, it was centrifuged at 4000 r / min for 20 min, the supernatant was collected, the pH was adjusted to neutral, and freeze-dried at -80°C for 10 h.

[0062] Performance Testing

[0063] 1. Determination method of hydrolysis degree

[0064] The enzymatic hydrolysate was centrifuged at a speed of 4000r / min for 20min, the supernatant was collected, and the degree of hydrolysis was determined by the ninhydrin colorimetric method. After the enzymatic hydrolysate was diluted a certain multiple, 1mL of 2% ninhydrin solution and phosphate buffer (pH 8.0) were added, mixed and heated at 90℃ for 15min. After cooling to room temperature, the volume was adjusted to 25mL, the absorbance value was measured at 570nm, and the degree of hydrolysis was calculated according to the following formula:

[0065]

[0066] 2. Determination of peptide mass concentration

[0067] The biuret method was used to determine the polypeptide content. Bovine albumin (BSA) was used as the standard sample. Solutions of 0.5, 1, 2, 3, and 4 mg / mL of polypeptide mass concentration were prepared. 2 mL of BSA solution was mixed with 3 mL of biuret reagent. After standing for 30 minutes, the absorbance of the standard sample was measured at 540 nm, and a standard curve was drawn. When the polypeptide mass concentration in the sample was determined, 2 mL of polypeptide enzymatic hydrolyzate was taken, an equal volume of 10% trichloroacetic acid was added, and after uniform mixing, the supernatant was obtained by high-speed refrigerated centrifugation at 4°C and 4,000 r / min for 20 minutes. The supernatant was taken and the corresponding absorbance value was determined according to the standard curve determination method. The polypeptide mass concentration was calculated according to the following formula:

[0068]

[0069] Where: A is the absorbance value, n is the dilution multiple.

[0070] 3. Sensory evaluation method

[0071] The sensory evaluation adopted a 5-point scoring method, and 7 food professionals who had been trained in sensory evaluation evaluated the umami taste of chicken protein hydrolysate (Table 1). The standard substance was 0.1% monosodium glutamate by mass concentration, and the standard substance was scored as 5 points. Take 1g of freeze-dried enzymatic hydrolysate, add 100mL of pure water and mix evenly, and compare and score with the standard substance. The average of the scores was taken as the umami evaluation result.

[0072] Table 1 Umami rating criteria for enzymatic hydrolysates

[0073]

[0074] Table 2 Performance test results of Examples 1-3 and Comparative Examples 1-5

[0075]

[0076]

[0077] As can be seen from Table 2, under ultrasonic conditions, the composite protease and the flavor protease simultaneously enzymatically hydrolyzed chicken breast and chicken skeleton, the hydrolysis degree reached 20.87%, the polypeptide concentration reached 6.16 mg / mL, and the flavor substances in the enzymatic hydrolysis products were mainly small molecule peptides that contribute to the flavor, flavor amino acids and volatile flavor substances, etc., so the enzymatic hydrolysate prepared by the present invention has a strong aroma.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing highly efficient fresh polypeptides based on proteolysis, characterized in that: The following steps are involved: (1) Sample pretreatment: fresh chicken skeletons and chicken breasts are cleaned, the chicken skeletons and chicken breasts are mixed in a certain proportion, sterilized, and then crushed to obtain chicken mince; (2) Ultrasonic treatment: the chicken paste prepared in step (1) is mixed with distilled water in a certain proportion and then subjected to ultrasonic treatment; (3) Enzymatic hydrolysis: adding a composite protease to the chicken mince solution treated with ultrasound in step (2) for enzymatic hydrolysis for 2-5 hours, inactivating the enzyme after enzymatic hydrolysis, cooling and centrifuging at 3500-4200 r / min for 15-25 minutes, collecting the supernatant, adjusting the pH value to neutral, and freeze-drying to obtain the fresh polypeptide.

2. The method for preparing an efficient fresh polypeptide by proteolysis according to claim 1, characterized in that: In the step (1), the mass ratio of the chicken skeleton to the chicken breast is (6-9):

2.

3. The method for preparing highly efficient fresh polypeptide by proteolysis according to claim 1, characterized in that: In the step (1), the sterilization temperature is 120-140° C. and the time is 50-70 min.

4. The method for preparing highly efficient fresh polypeptide by proteolysis according to claim 1, characterized in that: The ultrasonic conditions in step (2) are: power 600-900W, and ultrasonic time 20-40min.

5. The method for preparing highly efficient fresh polypeptide by proteolysis according to claim 1, characterized in that: The mass volume ratio of the chicken paste to distilled water in the step (2) is 1:2 (g / ml).

6. The method for preparing highly efficient fresh polypeptide by proteolysis according to claim 1, characterized in that: The compound protease in step (3) is a composite protease and a flavor protease.

7. The method for preparing highly efficient fresh polypeptide by proteolysis according to claim 6, characterized in that: The mass ratio of the composite protease to the flavor protease is 1:

1.

8. The method for preparing a highly efficient fresh polypeptide by proteolysis according to claim 1, 6 or 7, characterized in that: The dosage of the composite protease in step (3) is 0.1-0.5% of the mass fraction of the chicken paste.

9. The method for preparing a highly efficient fresh polypeptide by proteolysis according to claim 2, 6 or 7, characterized in that: The pH value of step (3) is 7.5 and the temperature is 50°C.

10. The method for preparing highly efficient fresh polypeptide by proteolysis according to claim 2, characterized in that: The enzyme inactivation temperature in step (3) is 90° C. and the time is 10-20 min.

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