An enzymatic hydrolysis process and enzymatic hydrolysis tank for scallop umami peptides

Abstract

This invention belongs to the field of food biotechnology, specifically relating to an enzymatic hydrolysis process and enzymatic hydrolysis tank for scallop umami peptides. This invention employs steam flash explosion and ultrasonic treatment combined with hydroxylated nanoparticles to pretreat scallop protein raw materials, followed by sequential enzymatic hydrolysis of the scallop protein solution using alkaline protease and flavor protease. This solves the technical problem of low hydrolysis degree caused by the high aggregation and high helicalization of scallop protein molecules, significantly improving the hydrolysis rate of scallop protein and increasing the utilization of umami substances in scallops.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology, specifically relating to an enzymatic hydrolysis process and enzymatic hydrolysis tank for scallop umami peptides. Background Technology

[0002] Scallops are characterized by their high protein, low fat, and low cholesterol content, and their delicious flavor makes them a favorite among consumers. The umami substances in scallops mainly include organic acids, umami peptides, nucleotides, and certain amino acids. Among these, umami peptides are rich in nutrients, non-antigenic, easily absorbed, and can enhance the rich flavor and aroma of food. As an ideal natural and safe flavoring substance, umami peptides have become a research hotspot in recent years.

[0003] Scallop umami peptides are typically obtained through enzymatic hydrolysis. However, due to the high aggregation and highly helical nature of scallop protein molecules, the degree of hydrolysis during enzymatic treatment is often low. Even with pretreatment methods such as high-temperature treatment of scallop materials, the protein hydrolysis rate can generally only be increased to 35-45%.

[0004] Therefore, the enzymatic hydrolysis process for scallop umami peptides still needs improvement to increase the utilization of umami substances in scallops and avoid waste of raw materials. Summary of the Invention

[0005] To address the above problems, the present invention provides an enzymatic hydrolysis process for scallop umami peptides, which includes the following steps;

[0006] (1) Obtain scallop protein raw material, grind and homogenize it.

[0007] (2) The homogenized scallop protein raw material is subjected to steam flash explosion treatment.

[0008] (3) Water and hydroxylated magnetic nanoparticles were added to the scallop protein raw material that had been treated with steam flash explosion to obtain scallop protein liquid, which was then subjected to ultrasonic treatment.

[0009] (4) The scallop protein solution was enzymatically hydrolyzed by alkaline protease and flavor protease in sequence.

[0010] (5) After inactivating the enzyme in the enzymatic hydrolysate at 90-100℃, the supernatant is collected by centrifugation to obtain the scallop umami peptide solution.

[0011] Further, the homogenization in step (1) includes adding one mass of water to the crushed scallop protein raw material and then homogenizing it in a homogenizer.

[0012] Furthermore, the steam flash explosion treatment conditions are: treatment at 3.0–4.0 MPa for 2–3 minutes, with instantaneous depressurization within 0.1–0.2 seconds.

[0013] Furthermore, in step (3), the amount of water added is three times the mass of the scallop protein raw material treated with steam flash explosion.

[0014] Furthermore, in step (3), the amount of hydroxylated magnetic nanoparticles added is 0.5–1.0 g / L.

[0015] Furthermore, the hydroxylated magnetic nanoparticles are commercially available hydroxylated iron oxide nanoparticles.

[0016] Furthermore, the ultrasonic treatment power is 1000-1500W, and the time is 15-30min.

[0017] Furthermore, the amount of alkaline protease added is 1500-2000 U / g based on scallop protein raw material, the enzymatic hydrolysis temperature is 40-50℃, the pH is 8-10, and the enzymatic hydrolysis time is 5-8h; the amount of flavor protease added is 500-1000 U / g based on scallop protein raw material, the enzymatic hydrolysis temperature is 50-60℃, the pH is 6-7, and the enzymatic hydrolysis time is 5-8h.

[0018] Furthermore, step (4) also includes inactivating the enzyme in the hydrolysate at 90-100°C after alkaline protease hydrolysis and before flavor protease hydrolysis. The inactivation time is further specified as 10-20 minutes.

[0019] Furthermore, step (4) also includes mechanical or ultrasonic stirring during the enzymatic hydrolysis process, wherein the ultrasonic stirring power is 300-500w.

[0020] Furthermore, step (5) also includes removing the hydroxylated magnetic nanoparticles in the enzymatic hydrolysate by magnetic adsorption after enzyme inactivation.

[0021] Furthermore, the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 5-15 min.

[0022] Furthermore, step (5) also includes separating and purifying scallop umami peptides from the scallop umami peptide solution. The separation and purification may include using methods such as column chromatography, membrane filtration, or ultrafiltration to collect scallop peptide components of specific molecular weights from the scallop umami peptide solution to obtain purified scallop umami peptide products. Purification can be performed using any suitable conventional techniques for obtaining purified peptides.

[0023] Furthermore, the scallop umami peptide solution obtained in step (5) can be dried to obtain a dry product of scallop umami peptides. Alternatively, the scallop umami peptides can be separated and purified from the scallop umami peptide solution before drying to obtain a dry product of scallop umami peptides. The drying method includes, but is not limited to, oven drying, freeze drying, spray drying, etc., preferably without impairing the umami effect.

[0024] In other respects, the present invention also provides scallop umami peptides prepared by the enzymatic hydrolysis process described herein.

[0025] The enzymatic hydrolysis process for scallop umami peptides described herein can be implemented using appropriate equipment as needed. For example, homogenization in step (1) can be performed in a homogenizer; steam flash explosion in step (2) can be performed in a steam flash explosion device; ultrasonic treatment in step (3) can be performed using an ultrasonic cell disruptor; and enzymatic hydrolysis in step (4) can be performed using an enzymatic hydrolysis tank. The enzymatic hydrolysis tank can be any enzymatic hydrolysis tank commonly used in the art, such as an enzymatic hydrolysis tank with a built-in mechanical stirring device. When ultrasonic stirring is used in step (4), steps (3) and (4) can both be performed in an ultrasonic reactor, or in an ultrasonic-assisted enzymatic hydrolysis tank known in the art, such as the enzymatic hydrolysis tank containing an ultrasonic generator disclosed in CN214142390U, CN1 02847477A, ​​and CN1 13019214A.

[0026] Therefore, in other respects, the present invention also provides an enzymatic hydrolysis tank for the enzymatic hydrolysis process of the scallop umami peptides described herein, which includes an ultrasonic generator.

[0027] In other respects, the present invention also provides the application of an enzymatic hydrolysis vessel containing an ultrasonic generator in the enzymatic hydrolysis process of scallop umami peptides described herein. The enzymatic hydrolysis vessel containing the ultrasonic generator is used in steps (3) and (4) of the enzymatic hydrolysis process of scallop umami peptides described herein.

[0028] Beneficial effects of the present invention

[0029] To improve the enzymatic hydrolysis process of scallop umami peptides and increase the utilization rate of umami substances in scallops, this invention improves the pretreatment method of scallop protein raw materials. More specifically, this invention uses a method of steam flash explosion and ultrasonic treatment combined with hydroxylated nanoparticles to pretreat scallop protein raw materials, which significantly improves the hydrolysis rate of scallop protein.

[0030] Steam flash explosion technology is an advanced biomass pretreatment technology that has attracted widespread attention from scholars in recent years. It boasts advantages such as simple process, high efficiency, energy saving, no pollution, and low processing cost. The steam flash explosion technology involves placing the material in a pressure vessel and utilizing the micropores on the material's surface to allow high-pressure saturated steam to permeate into the material's structure. Some of the saturated steam forms high-temperature liquid water. Due to the small size of the micropores on the material's surface, when the external pressure instantly drops to atmospheric pressure, only a very small portion of the high-pressure steam inside the material can be released into the atmosphere through these micropores. Most of the saturated steam expands rapidly, while the high-temperature liquid water also quickly boils and flashes. A large amount of steam completes adiabatic expansion in a very short time, converting thermal energy into mechanical energy. The shock wave generated by the steam expansion exerts shear force on the material, thereby destroying its internal structure.

[0031] This invention introduces steam flash explosion technology into the pretreatment of scallop protein, which can loosen the highly aggregated scallop protein tissue and disrupt the helical structure of protein molecules, thereby allowing proteases to enter the scallop protein tissue for protein hydrolysis. This invention also introduces hydroxylated nanoparticles and simultaneously treats the scallop protein solution with ultrasound. On the one hand, ultrasound treatment can disrupt the scallop protein tissue structure and increase the hydrolysis rate; on the other hand, the ultrasonic dispersion of hydroxylated nanoparticles in the scallop protein solution can further disrupt the highly aggregated tissue structure of scallop protein using shear force. Simultaneously, the hydroxyl groups on the nanoparticle surface can form hydrogen bonds with amino acids, thereby disrupting the hydrogen bonds between protein molecules and preventing the formation of hydrogen bonds between protein molecules during hydrolysis, thus contributing to the improvement of the hydrolysis rate. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0033] Example 1

[0034] This embodiment provides an enzymatic hydrolysis process for scallop umami peptides, which includes the following steps;

[0035] (1) Add an equal amount of water to the shelled scallop meat, stir and homogenize.

[0036] (2) The homogenized scallop protein raw material was subjected to steam flash explosion treatment in a steam flash explosion device under the conditions of 4.0 MPa for 2 min, and the pressure was released instantaneously within 0.2 s.

[0037] (3) Add 5 times the mass of water and 1.0 g / L of hydroxylated iron tetroxide nanoparticles (purchased from Ruixi Biotechnology) to the scallop protein raw material that has been treated with steam flash explosion to obtain scallop protein solution, and then treat it with ultrasound at 1000w for 30 min.

[0038] (4) Transfer the scallop protein solution to an enzymatic hydrolysis tank, add alkaline protease to the scallop protein solution at a rate of 2000 U / g based on scallop meat, and hydrolyze at 40℃ and pH 8 for 5 hours. Then, inactivate the enzyme at 100℃ for 10 minutes. Next, add flavor protease at a rate of 1000 U / g based on scallop protein raw material, and hydrolyze at 50℃ and pH 7 for 5 hours. During both stages of enzymatic hydrolysis, maintain mechanical stirring to prevent the protein solution from settling.

[0039] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100°C for 10 min. First, the hydroxylated iron oxide nanoparticles in the enzymatic hydrolysis are removed by a magnet. Then, the supernatant is collected by centrifugation to obtain the scallop umami peptide solution.

[0040] Example 2

[0041] This embodiment provides an enzymatic hydrolysis process for scallop umami peptides, which includes the following steps;

[0042] (1) Add an equal amount of water to the shelled scallop meat, stir and homogenize.

[0043] (2) The homogenized scallop protein raw material was subjected to steam flash explosion treatment in a steam flash explosion device under the conditions of 3.0 MPa for 3 min, and the pressure was released instantaneously within 0.1 s.

[0044] (3) Add 5 times the mass of water and 0.5 g / L of hydroxylated iron tetroxide nanoparticles (purchased from Ruixi Biotechnology) to the scallop protein raw material that has been treated with steam flash explosion to obtain scallop protein solution, and then treat it with ultrasound at 1500w for 20 min.

[0045] (4) Transfer the scallop protein solution to an enzymatic hydrolysis tank, add alkaline protease to the scallop protein solution at a rate of 1500 U / g based on scallop meat, and hydrolyze for 8 hours at 45℃ and pH 10. Then, inactivate the enzyme at 100℃ for 10 minutes. Next, add flavor protease at a rate of 500 U / g based on scallop protein raw material, and hydrolyze for 8 hours at 50℃ and pH 7. During both stages of enzymatic hydrolysis, maintain mechanical stirring to prevent the protein solution from settling.

[0046] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100°C for 10 min. First, the hydroxylated iron oxide nanoparticles in the enzymatic hydrolysis are removed by a magnet. Then, the supernatant is collected by centrifugation to obtain the scallop umami peptide solution.

[0047] Example 3

[0048] This embodiment provides an enzymatic hydrolysis process for scallop umami peptides, which includes the following steps;

[0049] (1) Add an equal amount of water to the shelled scallop meat, stir and homogenize.

[0050] (2) The homogenized scallop protein raw material was subjected to steam flash explosion treatment in a steam flash explosion device under the conditions of 4.0 MPa for 3 min, and the pressure was released instantaneously within 0.2 s.

[0051] (3) Add 5 times the mass of water and 1.0 g / L of hydroxylated iron tetroxide nanoparticles (purchased from Ruixi Biotechnology) to the scallop protein raw material that has been treated with steam flash explosion to obtain scallop protein solution, and then treat it with ultrasonic waves at 1000 W for 30 min in an ultrasonic reactor.

[0052] (4) After the ultrasonic treatment in step (3), alkaline protease was added to the scallop protein solution at a rate of 2000 U / g based on scallop meat in the ultrasonic reactor. The mixture was enzymatically hydrolyzed at 40°C and pH 8 for 5 hours, and then the enzyme was inactivated at 100°C for 10 minutes. Then, flavor protease was added at a rate of 1000 U / g based on the scallop protein raw material. The mixture was enzymatically hydrolyzed at 50°C and pH 7 for 5 hours. During both enzymatic hydrolysis processes, ultrasonic stirring at 500 W was maintained.

[0053] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100°C for 10 min. First, the hydroxylated iron oxide nanoparticles in the enzymatic hydrolysis are removed by a magnet. Then, the supernatant is collected by centrifugation to obtain the scallop umami peptide solution.

[0054] Comparative Example 1

[0055] This comparative example provides an enzymatic hydrolysis process for scallop umami peptides, which includes the following steps;

[0056] (1) Add an equal amount of water to the shelled scallop meat, stir and homogenize.

[0057] (2) Add 5 times the mass of water and 1.0 g / L of hydroxylated iron tetroxide nanoparticles (purchased from Ruixi Biotechnology) to the homogenized scallop protein raw material to obtain scallop protein solution, and then treat it with ultrasound at 1000w for 30 min.

[0058] (3) Transfer the scallop protein solution to an enzymatic hydrolysis tank, add alkaline protease to the scallop protein solution at a rate of 2000 U / g based on scallop meat, and hydrolyze at 40℃ and pH 8 for 5 hours. Then, inactivate the enzyme at 100℃ for 10 minutes. Next, add flavor protease at a rate of 1000 U / g based on scallop protein raw material, and hydrolyze at 50℃ and pH 7 for 5 hours. During both stages of enzymatic hydrolysis, maintain mechanical stirring to prevent the protein solution from settling.

[0059] (4) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100°C for 10 min. First, the hydroxylated iron oxide nanoparticles in the enzymatic hydrolysis are removed by a magnet. Then, the supernatant is collected by centrifugation to obtain the scallop umami peptide solution.

[0060] Comparative Example 2

[0061] This comparative example provides an enzymatic hydrolysis process for scallop umami peptides, which includes the following steps;

[0062] (1) Add an equal amount of water to the shelled scallop meat, stir and homogenize.

[0063] (2) The homogenized scallop protein raw material was subjected to steam flash explosion treatment in a steam flash explosion device under the conditions of 4.0 MPa for 2 min, and the pressure was released instantaneously within 0.2 s.

[0064] (3) Add 5 times the mass of water to the scallop protein raw material that has been treated with steam flash explosion to obtain scallop protein liquid, and then treat it with ultrasound at 1000w for 30min.

[0065] (4) Transfer the scallop protein solution to an enzymatic hydrolysis tank, add alkaline protease to the scallop protein solution at a rate of 2000 U / g based on scallop meat, and hydrolyze at 40℃ and pH 8 for 5 hours. Then, inactivate the enzyme at 100℃ for 10 minutes. Next, add flavor protease at a rate of 1000 U / g based on scallop protein raw material, and hydrolyze at 50℃ and pH 7 for 5 hours. During both stages of enzymatic hydrolysis, maintain mechanical stirring to prevent the protein solution from settling.

[0066] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100℃ for 10 min, and then the supernatant is collected by centrifugation to obtain the scallop umami peptide solution.

[0067] Comparative Example 3

[0068] This comparative example provides an enzymatic hydrolysis process for scallop umami peptides, which includes the following steps;

[0069] (1) Add an equal amount of water to the shelled scallop meat, stir and homogenize.

[0070] (2) The homogenized scallop protein raw material was subjected to steam flash explosion treatment in a steam flash explosion device under the conditions of 4.0 MPa for 2 min, and the pressure was released instantaneously within 0.2 s.

[0071] (3) Add 5 times the mass of water and 1.0 g / L of unmodified iron oxide nanoparticles (purchased from Ruixi Biotechnology) to the scallop protein raw material that has been treated with steam flash explosion to obtain scallop protein solution, and then treat it with ultrasound at 1000w for 30 min.

[0072] (4) Transfer the scallop protein solution to an enzymatic hydrolysis tank, add alkaline protease to the scallop protein solution at a rate of 2000 U / g based on scallop meat, and hydrolyze at 40℃ and pH 8 for 5 hours. Then, inactivate the enzyme at 100℃ for 10 minutes. Next, add flavor protease at a rate of 1000 U / g based on scallop protein raw material, and hydrolyze at 50℃ and pH 7 for 5 hours. During both stages of enzymatic hydrolysis, maintain mechanical stirring to prevent the protein solution from settling.

[0073] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100°C for 10 min. First, the hydroxylated iron oxide nanoparticles in the enzymatic hydrolysis are removed by a magnet. Then, the supernatant is collected by centrifugation to obtain the scallop umami peptide solution.

[0074] Test Example 1: Determination of Protein Hydrolysis Rate

[0075] The total nitrogen content of the scallop umami peptide solutions prepared in Examples 1-3 and Comparative Examples 1-3 was determined by the Kjeldahl method, and the amino nitrogen content was determined by formaldehyde titration. The protein hydrolysis rate was calculated using the following formula:

[0076] Protein hydrolysis rate (%) = (Amino nitrogen content in the hydrolysate / Total nitrogen content in the hydrolysate) × 100.

[0077] Test Example 2: Analysis of Umami Peptide Content

[0078] The peptide distribution in the scallop umami peptide solutions prepared in Examples 1-3 and Comparative Examples 1-3 was tested according to Appendix A of GB / T 22492-2008. Then, the mass percentage of peptides with a molecular weight of 200-1000 Da in the total peptides was calculated to measure the umami peptide content in the scallop umami peptide solutions.

[0079] The results of test examples 1 and 2 are shown in Table 1 below.

[0080] Table 1

[0081]

[0082] As shown in Table 1, the enzymatic hydrolysis processes in Examples 1-3 of this invention achieved a protein hydrolysis rate as high as approximately 50%, and the prepared scallop umami peptide solutions also had a high content of umami peptides. Among these, the enzymatic hydrolysis process involving ultrasonic stirring during enzymatic hydrolysis yielded the best results. In contrast, when the scallop protein raw materials were not subjected to steam flash explosion pretreatment (Comparative Example 1), not subjected to ultrasonic pretreatment with bound nanoparticles (Comparative Example 2), and subjected to ultrasonic pretreatment with bound unhydroxylated nanoparticles (Comparative Example 3), both the protein hydrolysis rate and the umami peptide content decreased significantly, indicating that the technical solution of this invention has a surprising technical effect on improving the utilization of umami substances in scallops.

[0083] It should be noted that while the preferred embodiments of the present invention are provided in this specification, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. An enzymatic process for scallop umami peptides, characterized by, The method comprises the following steps: (1) obtaining scallop protein raw material, stirring and homogenizing, (2) steam flash explosion treatment of the homogenized scallop protein raw material, (3) adding water and hydroxylated magnetic nanoparticles to the steam flash explosion treated scallop protein raw material to obtain a scallop protein liquid, and then performing ultrasonic treatment, wherein the amount of water added is 3 times the mass of the steam flash explosion treated scallop protein raw material; the amount of hydroxylated magnetic nanoparticles added is 0.5-1.0 g / L; the hydroxylated magnetic nanoparticles are hydroxylated ferroferric oxide nanoparticles; (4) sequentially using alkaline protease and flavor protease for enzymolysis of the scallop protein liquid, (5) collecting the supernatant by centrifugation after enzyme inactivation of the enzymolysis liquid at 90-100℃, to obtain a scallop umami peptide solution; The steam flash explosion treatment conditions are 3.0-4.0 MPa for 2-3 min, and instantaneous pressure relief within 0.1-0.2 s; The ultrasonic treatment power is 1000-1500 w, and the time is 15-30 min; The amount of alkaline protease added is 1500-2000 U / g based on the scallop protein raw material, the enzymolysis temperature is 40-50℃, the pH is 8-10, and the enzymolysis time is 5-8 h; the amount of flavor protease added is 500-1000 U / g based on the scallop protein raw material, the enzymolysis temperature is 50-60℃, the pH is 6-7, and the enzymolysis time is 5-8 h.

2. The enzymatic process according to claim 1, characterized in that, Step (4) further comprises enzyme inactivation of the enzymolysis liquid at 90-100℃ after alkaline protease enzymolysis and before flavor protease enzymolysis.

3. The enzymatic process according to claim 1, characterized in that, Step (4) further comprises mechanical or ultrasonic stirring during enzymolysis, and the ultrasonic stirring power is 300-500 w.

4. The enzymatic process according to claim 1, characterized in that, Step (5) further comprises removing the hydroxylated magnetic nanoparticles in the enzymolysis liquid by magnetic adsorption after enzyme inactivation.

5. The enzymatic process according to claim 1, wherein, Step (5) further comprises separation and purification of scallop umami peptides from the scallop umami peptide solution.

6. A scallop umami peptide prepared by the enzymolysis process of any one of claims 1-5.

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