A method for preparing protein peptides with improved flavor and function and stability

Abstract

The present application belongs to the technical field of polypeptide processing, and particularly relates to a preparation method of protein peptide capable of improving flavor and function; the method uses silver carp as raw material, uses food-grade protease, and obtains full fish protein peptide through moderate enzymolysis; under mild conditions, nitrogen is used to replace air to perform glycosylation reaction, which can effectively improve the flavor and antioxidant function of the full fish protein peptide; compared with the original full fish protein peptide and the full fish protein glycopeptide prepared by using air for glycosylation reaction, the full fish protein glycopeptide prepared by the method has lighter fishy smell and bitterness, can effectively improve the acceptance of the public, has strong antioxidant function and good storage stability, and can be widely applied in special food and nutritional food.

Description

Technical Field

[0001] This invention belongs to the field of peptide processing technology, and more specifically, relates to a method for preparing protein peptides that can improve flavor, function and stability. Background Technology

[0002] Fish such as silver carp are rich in protein and have a balanced amino acid composition, making them a high-quality raw material for producing protein peptides. Fish protein peptides have functions such as anti-oxidation, regulating blood lipids, and improving atherosclerosis, making them a good dietary supplement. However, the presence of fishy and bitter tastes seriously affects consumers' acceptance of silver carp protein peptides, limiting their application. Existing technologies for improving the unpleasant flavor of protein peptides mainly include (1) physical mixing, which involves simple physical mixing with sweeteners, flavor enhancers, or taste improvers to mask the unpleasant flavor of protein peptides. (2) physical adsorption, which involves treating protein peptides with activated carbon, resin, or other adsorbents to remove bitterness and fishy taste. (3) microencapsulation, which involves encapsulating protein peptides in tiny capsules to mask unpleasant flavors. (4) membrane separation technology, which utilizes membrane technologies such as ultrafiltration, nanofiltration, or reverse osmosis to selectively separate small molecule peptides that cause fishy and bitter tastes based on molecular size or charge.

[0003] Regarding the technologies mentioned above, the following problems may exist: (1) Physical mixing does not truly change its flavor characteristics. Undesirable flavors may gradually appear during product storage or taste experience. (2) Physical adsorption may not only remove bitter and fishy substances, but may also remove beneficial components in protein peptides, such as nutrients and bioactive molecules, thereby reducing the nutritional value and physiological efficacy of the product. (3) Microencapsulation equipment, materials, and processes are often more expensive. This will increase the production cost of the product and may lead to an increase in the price of the final product. (4) Membrane separation equipment and special membrane materials are usually expensive, which will also increase the production cost of the product. In recent years, oligosaccharides have been widely used in the food and health fields due to their low calories and good prebiotic effects. However, there are still insufficient invention patents and research on using oligosaccharides to improve the flavor characteristics of protein peptides and enhance their antioxidant capacity. Summary of the Invention

[0004] In view of the above-mentioned problems in the prior art, the purpose of the present invention is to provide a method for preparing protein peptides that can improve flavor, function and stability, which can solve the problems of high cost and poor effect in the prior art, and improve the flavor characteristics, antioxidant capacity and storage stability of whole fish protein peptides.

[0005] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing protein peptides that can improve flavor, function, and stability, the method comprising the following steps:

[0007] S1: Use silver carp as raw material, remove the gills, internal organs and black membrane, wash and mince into fish paste;

[0008] S2: Take the minced fish meat and add deionized water at a ratio of 1:2 (w / v) to 1:3 (w / v). After stirring thoroughly, place the whole mixture in a high-pressure steam sterilizer and cook at 121°C for 60 to 120 minutes to obtain a fish meat solution.

[0009] S3: The fish paste solution obtained in step S2 is heated to 50°C by water bath heating, and then food-grade protease is added. The amount of food-grade protease added is 0.5%~1.0%, and the enzymatic hydrolysis time is 1~3h.

[0010] S4: After enzymatic hydrolysis, the whole fish was placed in a water bath at 95°C for 15 minutes to inactivate the enzyme. Then it was cooled to room temperature and centrifuged at 4000 r / min for 20 minutes. The supernatant was then freeze-dried under vacuum to obtain silver carp whole fish protein peptides.

[0011] S5: The xylooligosaccharide and the silver carp whole fish protein peptide obtained in step S4 are fully dissolved in deionized water at a ratio of 0.25:1 (w / w) to 1:1 (w / w). Then, the mixture is transferred to a sealed screw-cap glass tube containing not less than 92% nitrogen and heated at 70℃ to 90℃ for 1 to 2 hours. After heating, the mixture is quickly placed in an ice-water bath to terminate the reaction. After vacuum freeze-drying, the silver carp whole fish protein glycopeptide is obtained.

[0012] Furthermore, in step S5, 95% to 100% nitrogen is used to replace air.

[0013] Furthermore, in step S5, the oligosaccharide is xylooligosaccharide.

[0014] Furthermore, the concentration of the whole silver carp protein peptide is 5% to 10%.

[0015] Furthermore, the food-grade protease is a complex protease.

[0016] Furthermore, the composition ratio of the complex protease includes alkaline protease: papain: flavor protease = 1~2: 1~2: 1.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) The whole fish protein glycopeptides prepared by this method have a lighter fishy and bitter taste compared with the original whole fish protein peptides, which can effectively improve the public's acceptance. At the same time, they have a high antioxidant function and can be widely used in special food and nutritional food.

[0019] (2) In this method, the use of xylooligosaccharides can effectively improve the flavor and antioxidant function of whole fish protein peptides;

[0020] (3) In this method, nitrogen was used to replace air for glycosylation reaction, thereby significantly improving the flavor and antioxidant function of whole fish protein peptides;

[0021] (4) In this method, the whole fish protein glycopeptide developed using whole fish protein peptides and oligosaccharides does not require the addition of acid or alkali to adjust pH during the entire processing process, which can effectively maintain the good functional properties and safety of the product.

[0022] (5) The preparation process of this method is safe and efficient. It uses food-grade protease, and the whole fish protein peptides obtained by moderate enzymatic hydrolysis react with xylooligosaccharides under mild conditions without any additives. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments.

[0024] Example 1:

[0025] In this embodiment, the preparation method includes the following steps:

[0026] S1: Use silver carp as raw material, remove the gills, internal organs and black membrane, wash with tap water and mince into fish meat paste;

[0027] S2: Take the minced fish meat and add deionized water at a ratio of 1:2 (w / v). After stirring thoroughly, place the whole mixture in a high-pressure steam sterilizer and cook at 121°C for 120 minutes to inactivate endogenous enzymes and obtain a fish meat solution.

[0028] S3: Heat the fish paste solution obtained in step S2 to 50°C using a water bath, then add a complex protease (alkaline protease: papain: flavor protease = 1:1:1) at a rate of 0.5%, and hydrolyze for 3 hours.

[0029] S4: After enzymatic hydrolysis, the whole fish was placed in a water bath at 95°C for 15 minutes to inactivate the enzyme. Then it was cooled to room temperature and centrifuged at 4000 r / min for 20 minutes. The supernatant was then freeze-dried under vacuum to obtain silver carp whole fish protein peptides.

[0030] S5: The xylooligosaccharide and the silver carp whole fish protein peptide obtained in step S4 were fully dissolved in deionized water at a ratio of 1:1 (w / w) (the concentration of silver carp whole fish protein peptide was 10%). Then, the solution was transferred to a sealed screw-cap glass tube containing nitrogen and heated at 90°C for 2 hours. After heating, the solution was quickly placed in an ice-water bath to terminate the reaction. After vacuum freeze-drying, the silver carp whole fish protein glycopeptide was obtained. Example 2:

[0031] In this embodiment, the preparation method includes the following steps:

[0032] S1: Use silver carp as raw material, remove the gills, internal organs and black membrane, wash with tap water and mince into fish meat paste;

[0033] S2: Take the minced fish meat and add deionized water at a ratio of 1:2 (w / v). After stirring thoroughly, place the whole mixture in a high-pressure steam sterilizer and cook at 121°C for 120 minutes to inactivate endogenous enzymes and obtain a fish meat solution.

[0034] S3: Heat the fish paste solution obtained in step S2 to 50°C using a water bath, then add a complex protease (alkaline protease: papain: flavor protease = 1:1:1) at a rate of 0.5%, and hydrolyze for 3 hours.

[0035] S4: After enzymatic hydrolysis, the whole fish was placed in a water bath at 95°C for 15 minutes to inactivate the enzyme. Then it was cooled to room temperature and centrifuged at 4000 r / min for 20 minutes. The supernatant was then freeze-dried under vacuum to obtain silver carp whole fish protein peptides.

[0036] S5: The oligo-milk hemi-saccharide and the silver carp whole fish protein peptide obtained in step S4 were fully dissolved in deionized water at a ratio of 1:1 (w / w) (the concentration of silver carp whole fish protein peptide was 10%). Then, the solution was transferred to a sealed screw-cap glass tube containing 100% nitrogen and heated at 90°C for 2 hours. After heating, the solution was quickly placed in an ice-water bath to terminate the reaction. After vacuum freeze-drying, the silver carp whole fish protein glycopeptide was obtained. Example 3:

[0037] In this embodiment, the preparation method includes the following steps:

[0038] S1: Use silver carp as raw material, remove the gills, internal organs and black membrane, wash with tap water and mince into fish meat paste;

[0039] S2: Take the minced fish meat and add deionized water at a ratio of 1:2 (w / v). After stirring thoroughly, place the whole mixture in a high-pressure steam sterilizer and cook at 121°C for 120 minutes to inactivate endogenous enzymes and obtain a fish meat solution.

[0040] S3: Heat the fish paste solution obtained in step S2 to 50°C using a water bath, then add a complex protease (alkaline protease: papain: flavor protease = 1:1:1) at a rate of 0.5%, and hydrolyze for 3 hours.

[0041] S4: After enzymatic hydrolysis, the whole fish was placed in a water bath at 95°C for 15 minutes to inactivate the enzyme. Then it was cooled to room temperature and centrifuged at 4000 r / min for 20 minutes. The supernatant was then freeze-dried under vacuum to obtain silver carp whole fish protein peptides.

[0042] S5: Dissolve the oligofructose and the silver carp whole fish protein peptide obtained in step S4 in deionized water at a ratio of 1:1 (w / w) (the concentration of silver carp whole fish protein peptide is 10%). Then transfer it to a sealed screw-cap glass tube containing 100% nitrogen and heat it at 90°C for 2 hours. After heating, quickly place it in an ice-water bath to terminate the reaction. After vacuum freeze-drying, silver carp whole fish protein glycopeptide is obtained.

[0043] Comparative Example 1:

[0044] In this comparative example, the preparation method includes the following steps:

[0045] S1: Use silver carp as raw material, remove the gills, internal organs and black membrane, wash with tap water and mince into fish meat paste;

[0046] S2: Take the minced fish meat and add deionized water at a ratio of 1:3 (w / v). After stirring thoroughly, place the whole mixture in a high-pressure steam sterilizer and cook at 121°C for 60 minutes to inactivate endogenous enzymes and obtain a fish meat solution.

[0047] S3: Heat the fish paste solution obtained in step S2 to 50°C using a water bath, then add a complex protease (alkaline protease: papain: flavor protease = 2:1:1) at a rate of 0.5%, and hydrolyze for 3 hours.

[0048] S4: After enzymatic hydrolysis, the whole fish was placed in a water bath at 95°C for 15 minutes to inactivate the enzyme. Then it was cooled to room temperature and centrifuged at 4000 r / min for 20 minutes. The supernatant was then freeze-dried under vacuum to obtain silver carp whole fish protein peptides.

[0049] S5: The xylooligosaccharide and the silver carp whole fish protein peptide obtained in step S4 were fully dissolved in deionized water at a ratio of 0.5:1 (w / w) (the concentration of silver carp whole fish protein peptide was 7%). Then, the solution was transferred to a sealed screw-cap glass tube containing 95% nitrogen and heated at 70°C for 2 hours. After heating, the solution was quickly placed in an ice-water bath to terminate the reaction. After vacuum freeze-drying, the silver carp whole fish protein glycopeptide was obtained.

[0050] Comparative Example 2:

[0051] In this comparative example, the preparation method includes the following steps:

[0052] S1: Use silver carp as raw material, remove the gills, internal organs and black membrane, wash with tap water and mince into fish meat paste;

[0053] S2: Take the minced fish meat and add deionized water at a ratio of 1:3 (w / v). After stirring thoroughly, place the whole mixture in a high-pressure steam sterilizer and cook at 121°C for 90 minutes to inactivate endogenous enzymes and obtain a fish meat solution.

[0054] S3: Heat the fish paste solution obtained in step S2 to 50°C using a water bath, then add a complex protease (alkaline protease: papain: flavor protease = 2:2:1) at a rate of 0.6%, and hydrolyze for 1 hour.

[0055] S4: After enzymatic hydrolysis, the whole fish was placed in a water bath at 95°C for 15 minutes to inactivate the enzyme. Then it was cooled to room temperature and centrifuged at 4000 r / min for 20 minutes. The supernatant was then freeze-dried under vacuum to obtain silver carp whole fish protein peptides.

[0056] S5: Dissolve the galacto-oligosaccharide and the silver carp whole fish protein peptide obtained in step S4 in deionized water at a ratio of 1:1 (w / w) (the concentration of silver carp whole fish protein peptide is 8%). Then transfer it to a sealed screw-cap glass tube containing 95% nitrogen and heat it at 70°C for 1 hour. After heating, quickly place it in an ice-water bath to terminate the reaction. After vacuum freeze-drying, silver carp whole fish protein glycopeptide is obtained.

[0057] Comparative Example 3:

[0058] In this comparative example, the preparation method includes the following steps:

[0059] S1: Use silver carp as raw material, remove the gills, internal organs and black membrane, wash with tap water and mince into fish meat paste;

[0060] S2: Take the minced fish meat and add deionized water at a ratio of 1:2 (w / v). After stirring thoroughly, place the whole mixture in a high-pressure steam sterilizer and cook at 121°C for 120 minutes to inactivate endogenous enzymes and obtain a fish meat solution.

[0061] S3: Heat the fish paste solution obtained in step S2 to 50°C using a water bath, then add a complex protease (alkaline protease: papain: flavor protease = 1:2:1) at a concentration of 0.75%, and hydrolyze for 1 hour.

[0062] S4: After enzymatic hydrolysis, the whole fish was placed in a water bath at 95°C for 15 minutes to inactivate the enzyme. Then it was cooled to room temperature and centrifuged at 4000 r / min for 20 minutes. The supernatant was then freeze-dried under vacuum to obtain silver carp whole fish protein peptides.

[0063] S5: Dissolve the oligofructose and the silver carp whole fish protein peptide obtained in step S4 in deionized water at a ratio of 0.5:1 (w / w) (the concentration of silver carp whole fish protein peptide is 8%). Then transfer it to a sealed screw-cap glass tube containing 95% nitrogen and heat it at 70°C for 1 hour. After heating, quickly place it in an ice-water bath to terminate the reaction. After vacuum freeze-drying, silver carp whole fish protein glycopeptide is obtained.

[0064] Comparative Example 4:

[0065] In this comparative example, the other steps are the same as in Example 1, except that in step S5, the contents are transferred to a sealed screw-cap glass tube containing only ordinary air.

[0066] Comparative Example 5:

[0067] In this comparative example, the other steps are the same as in Example 1, except that in step S5, the silver carp whole fish protein peptide obtained in step S4 is fully dissolved in deionized water (the concentration of the silver carp whole fish protein peptide is 10%).

[0068] Comparative Example 6:

[0069] In this comparative example, only the xylooligosaccharide from Example 1 was fully dissolved in deionized water (the concentration of xylooligosaccharide was 10%).

[0070] Comparative Example 7:

[0071] In this comparative example, only the galactooligosaccharide from Example 2 was fully dissolved in deionized water (the concentration of galactooligosaccharide was 10%).

[0072] Comparative Example 8:

[0073] In this comparative example, only the fructooligosaccharides from Example 3 were fully dissolved in deionized water (the concentration of fructooligosaccharides was 10%).

[0074] Experimental Example 1:

[0075] Sensory evaluation test of whole fish protein glycopeptides based on this method.

[0076] To demonstrate the effect of this method on improving the flavor of whole fish protein peptides, sensory evaluation tests were conducted on Examples 1 to 3 and Comparative Examples 1 to 8.

[0077] The sensory evaluation test was conducted as follows:

[0078] The sensory evaluation panel consisted of 30 members (12 men and 18 women), aged 20-30. The experiment employed a descriptive analysis of seven sensory qualities: caramel, bitterness, saltiness, sourness, umami, and fishiness. Prior to the formal experiment, all panel members were informed of the definitions of these tastes and received training on using relevant reference materials to perceive these qualities. Before testing, the sample solutions were incubated at 45°C to minimize the potential impact of temperature on the evaluation process. Each sample was then assigned a three-digit code, which was randomly distributed to each member participating in the sensory evaluation. After tasting each sample, participants rated the intensity of each sensory quality on a scale of 0 to 5 (0 = none / very weak, 5 = very strong). The final sensory evaluation scores are shown in Table 1 below.

[0079] Table 1 Sensory Evaluation Scores

[0080] ;

[0081] Experimental Example 2:

[0082] To demonstrate the effect of this method on enhancing the antioxidant capacity of whole fish protein peptides, the antioxidant capacity of Examples 1 to 3 and Comparative Examples 1 to 8 was measured.

[0083] The specific measurement method includes the following steps:

[0084] (1) Total reducing power determination: Take 1 mL of 0.5 mg / mL sample, add 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% (mass fraction) potassium ferricyanide solution, mix well, and then heat in a water bath at 50℃ for 20 min. Remove and cool rapidly, add 2.5 mL of 10% (mass fraction) trichloroacetic acid (TCA) solution, mix well, and then centrifuge at 3000 g for 10 min. Take 2.5 mL of the supernatant, add 2.5 mL of deionized water and 0.5 mL of 1% (mass fraction) ferric chloride solution, mix thoroughly, react at room temperature for 10 min, and measure the absorbance at a wavelength of 700 nm. The reducing power can be expressed as the absorbance value at a wavelength of 700 nm.

[0085] (2) Ferric ion reducing power: The assay reagent consisted of 2,4,6-tripyridyltriazine (10 mM, dissolved in 40 mM hydrochloric acid), ferric chloride (20 mM), and acetate buffer (0.3 M, pH 3.6) in a ratio of 1:1:10. The diluted sample was mixed with the assay reagent and incubated in the dark at 37°C for 30 min. The absorbance of the mixture was then measured at 593 nm using a spectrophotometer. A standard curve was constructed using 0.1–1 mM ferric sulfate heptahydrate (FeSO4·H2O). Ferric ion reducing power was calculated as mM FeSO4 / mg sample.

[0086] (3) DPPH free radical scavenging ability: Take 1.5 mL of 0.5 mg / mL sample, add 1.5 mL of 99.5% ethanol and 0.675 mL of 0.02% DPPH ethanol solution, mix well by shaking, and incubate in a light-protected water bath at room temperature for 30 min. Then, detect the absorbance of the system at 517 nm. The lower the absorbance, the stronger the DPPH free radical scavenging ability of the system. The blank control is to replace 1.5 mL of sample solution with 1.5 mL of deionized water. Trolox (water-soluble vitamin E) at concentrations of 50-300 μmol / L was used to construct a standard curve. The activity of the sample is expressed as μmol Trolox equivalent (TE) / mg protein.

[0087] The specific data for the three antioxidant capabilities mentioned above are shown in Table 2 below:

[0088] Table 2 Antioxidant Capacity

[0089] ;

[0090] Experimental Example 3:

[0091] To demonstrate the storage stability of the whole fish protein glycopeptides prepared by this method, the sensory evaluation scores and antioxidant capacity of Examples 1 to 3 and Comparative Example 5 at different storage time points were measured.

[0092] Storage stability tests were conducted according to the following method:

[0093] The whole fish protein glycopeptides obtained in Examples 1, 2, 3, and Comparative Example 5, as well as the whole fish protein peptides obtained only after heating, were separately packaged into composite film vacuum packaging bags, vacuum-sealed, and then stored at room temperature (25°C) away from light for 3 months. Sensory evaluation and antioxidant capacity determination were performed on samples stored for 0 months, 1 month, 2 months, and 3 months. The results are shown in Tables 3 and 4 below.

[0094] Table 3 Sensory evaluation scores at different storage times

[0095] ;

[0096] ;

[0097] Table 4 Antioxidant capacity at different storage times

[0098] ;

[0099] ;

[0100] As shown in Table 1, all the oligosaccharides mentioned in this method can reduce the fishy and bitter taste of whole fish protein peptides and enhance their caramel flavor after modification, which is beneficial to improving the acceptability of whole fish protein peptides. In all examples, Example 1 showed the most significant improvement in the fishy and bitter taste of silver carp protein peptides, indicating that xylooligosaccharides have a significantly better flavor-improving effect on silver carp protein peptides than other oligosaccharides. Furthermore, compared to Example 1, Comparative Example 4 showed a certain degree of decrease in the caramel flavor and acceptability of the whole fish protein glycopeptides, indicating that replacing air with nitrogen during glycosylation has a significant effect on improving the flavor of whole fish protein glycopeptides.

[0101] As shown in Table 2, compared with the comparative examples, the embodiments described in this method all enhanced the total reducing power, iron ion reducing power, and DPPH free radical scavenging ability of silver carp protein peptides to varying degrees. This indicates that oligosaccharide modification not only improves the flavor of whole fish protein peptides but also significantly enhances their antioxidant capacity. Among all oligosaccharides, xylooligosaccharides showed the most significant enhancement of the antioxidant capacity of silver carp protein peptides. In the storage stability test of whole fish protein glycopeptides (Tables 3 and 4), it was found that with the extension of storage period, xylooligosaccharides could better maintain the caramel flavor, acceptability, and antioxidant capacity of whole fish protein glycopeptides. Furthermore, replacing air with nitrogen had a significant effect on improving the storage stability of whole fish protein glycopeptides.

[0102] The above experimental results demonstrate that using oligosaccharides for glycosylation modification of whole fish protein peptides with nitrogen instead of air can effectively improve their flavor, specifically reducing fishy and bitter tastes, exhibiting more caramel flavors, and improving overall acceptability. Simultaneously, the antioxidant capacity of whole fish protein glycopeptides modified with nitrogen instead of air is significantly enhanced compared to those modified with air. Replacing air with nitrogen during the glycosylation reaction effectively improves the storage stability of whole fish protein glycopeptides. Therefore, using oligosaccharides through glycosylation combined with nitrogen to replace air is an effective method to improve the flavor, antioxidant function, and storage stability of whole fish protein peptides.

Claims

1. A method for preparing protein peptides that can improve flavor, function, and stability, characterized in that, The method includes the following steps: S1: Use silver carp as raw material, remove the gills, internal organs and black membrane, wash and mince into fish paste; S2: Take the minced fish meat and add deionized water at a ratio of 1:2w / v to 1:3w / v. After stirring thoroughly, place the whole mixture in a high-pressure steam sterilizer and cook at 121°C for 60 to 120 minutes to obtain a fish meat solution. S3: The fish paste solution obtained in step S2 is heated to 50°C by water bath heating, and then food-grade protease is added at a dosage of 0.5%~1.0% for 1~3 hours. The food-grade protease is a complex protease with a component ratio of alkaline protease: papain: flavor protease = 1~2:1~2:

1. S4: After enzymatic hydrolysis, the whole fish was placed in a water bath at 95°C for 15 minutes to inactivate the enzyme. Then it was cooled to room temperature and centrifuged at 4000 r / min for 20 minutes. The supernatant was then freeze-dried under vacuum to obtain silver carp whole fish protein peptides. S5: The oligosaccharide and the silver carp whole fish protein peptide obtained in step S4 are fully dissolved in deionized water at a ratio of 0.25:1w / w to 1:1w / w. Then, the solution is transferred to a sealed screw-cap glass tube containing not less than 92% nitrogen and heated at 70℃ to 90℃ for 1 to 2 hours. After heating, the solution is quickly placed in an ice-water bath to terminate the reaction. After vacuum freeze-drying, the silver carp whole fish protein glycopeptide is obtained. The oligosaccharide is xylooligosaccharide.

2. The method for preparing protein peptides with improved flavor, function, and stability according to claim 1, characterized in that, In step S5, the amount of nitrogen in the sealed capped glass tube is 95%~100%.

3. The method for preparing protein peptides with improved flavor, function, and stability according to claim 1, characterized in that, In step S5, the concentration of the silver carp whole fish protein peptide is 5%~10%.