Low-molecular-weight oyster oligopeptide and preparation method thereof

Abstract

The invention discloses low-molecular-weight oyster oligopeptide and a preparation method thereof, and belongs to the technical field of food processing. Fresh oyster meat is used as a raw material, an enzymolysis method and a membrane filtration method are combined to treat oyster meat liquid and oyster cooking normal juice for multiple times, glutamic acid decarboxylase (GAD) is added into an oyster enzymolysis product for enzymolysis and filtration, and finally an oyster enzymolysis filtrate with the molecular weight smaller than 3 kDa and rich in gamma-aminobutyric acid is obtained. Concentrating and freeze-drying to obtain the low-molecular-weight oyster oligopeptide. On the basis of ensuring freshness and nutritional value, the added value of the oysters is remarkably improved, the oyster meat liquid and the oyster cooking juice are subjected to multi-stage multi-time enzymolysis through a combined enzymolysis method and a membrane filtration method, oyster protein is subjected to enzymolysis more thoroughly, and then oyster oligopeptides which are low in molecular weight (3kDa) and can be fully absorbed by organisms are obtained; glutamic acid decarboxylase (GAD) is added to catalyze glutamic acid to be converted into gamma-aminobutyric acid (GABA), and the oyster oligopeptide is endowed with a nutritional supplement function and a health-care function.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a low-molecular-weight oyster oligopeptide and a preparation method thereof. Background Art

[0002] Oysters are a type of molluscs, belonging to the order Ostreidae, class Bivalvia, and are commonly known as oysters, raw oysters, or sea oysters. Oysters are the world's largest cultured shellfish and an important economic cultured shellfish in my country. Oysters are known as "undersea milk" for their freshness, deliciousness, and rich nutrition. Their soft layer is rich in protein, animal glycogen, unsaturated fatty acids, essential amino acids, trace elements, and other nutrients, making them the most ideal food source. Oysters are the world's largest cultured shellfish and an important economic cultured shellfish in my country. my country's offshore areas are rich in oyster resources, and with the rapid development of artificial aquaculture technology, my country's total oyster production has reached 6.1995 million tons by 2022.

[0003] With the deepening of modern medical research, it is gradually discovered that oysters also have a variety of biological activities, such as improving the body's immunity, anti-tumor, and protecting the liver. Oyster peptides, as an extracted product of oysters, have attracted the attention of researchers for their health effects such as anti-oxidation, lowering blood pressure, and lowering blood sugar. There are various technologies for obtaining oyster peptides from oysters. For example, a Chinese patent with publication number CN110074378A and application date May 23, 2019 discloses a flavor peptide isolated from oyster hydrolysate, its preparation method and application. This invention isolates and identifies a new flavor peptide from oyster hydrolysate for the first time, and uses composite enzymatic hydrolysis technology, ethanol fractionation and UPLC-MS / MS technology for separation and identification. The flavor peptide has rich taste and can provide a theoretical basis for industrial synthesis; however, its technology is complex, and cost control and large-scale production are difficult. The stability of the flavor peptide and its application effect in different food systems need to be studied; a Chinese patent with publication number CN113718006A and application date August 9, 2021 discloses a method for extracting oyster peptides. This invention innovatively uses disodium EDTA treatment to remove heavy metals and extract The invention has high food safety, and uses calcium bicarbonate and microcrystalline cellulose to reduce the chelation of heavy metals and polypeptides, improves the protein recovery rate, and destroys the interaction between protein and fat through lipase treatment to improve the separation effect; however, the disodium EDTA treatment is likely to have a potential impact on the flavor and nutritional value of oyster peptides, increasing the cost during large-scale production; the Chinese patent with publication number CN106359840A and application date of September 12, 2016 provides a method for preparing low-sugar and low-salt oyster peptides by low-temperature enzymatic hydrolysis of oyster meat, using a shrimp head composite enzyme preparation for low-temperature enzymatic hydrolysis, reducing energy consumption and maintaining biological activity, and improving the product taste through soaking in a decolorant solution, repeated soaking in clean water to remove salt, hot water extraction to remove sugar and other steps, and the prepared oyster peptide has antioxidant and antihypertensive effects; however, the low-temperature enzymatic hydrolysis in the preparation process affects the stability and activity of the enzyme preparation, which invisibly increases the cost investment.

[0004] Oyster oligopeptides usually refer to oyster peptides with a smaller molecular weight. Since oligopeptides are structural functional fragments between proteins and amino acids, oyster oligopeptides can be quickly absorbed without digestion, and the absorption rate is higher than that of free amino acids. Compared with oyster peptides, oyster oligopeptides have the characteristics of being 100% quickly and efficiently absorbed by the human body. However, there are still many gaps in the current research on the preparation method of oyster oligopeptides, especially low-molecular-weight oyster oligopeptides. The Chinese patent with publication number CN114634961A and application date of March 30, 2022 uses an accelerated solvent extraction system to improve the extraction rate of oyster protein, uses deep eutectic solvents to assist enzymatic hydrolysis, accelerates the enzymatic reaction process, and improves the yield of oyster peptides. The molecular weight of oyster peptides is further reduced by high-pressure homogenization treatment to improve the yield of oligomeric peptides; however, the invention has problems including high extraction cost, recovery and reuse of solvents, and difficulty in large-scale production, and high-pressure homogenization treatment is easy to affect the biological activity of oyster peptides.

[0005] In combination with my country's abundant oyster seafood resources and the high nutritional value, health care functions and medicinal value of oysters themselves, the research and development of methods for efficiently preparing low molecular weight, high bioactive oyster oligopeptides and at the same time giving them functionality or added value will help promote the development of high-quality marine bioactive peptide materials, which is of great significance for improving the marketization level of oyster peptide products and improving the economic benefits. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a low molecular weight oyster oligopeptide and a preparation method thereof, which gives the oyster oligopeptide nutritional supplement function and health care function on the basis of ensuring biological activity and nutritional value, thereby significantly improving the added value of oysters.

[0007] The technical solution of the present invention is as follows:

[0008] One of the purposes of the present invention is to provide a method for preparing a low molecular weight oyster oligopeptide. The preparation method uses fresh oyster meat as a raw material, combines enzymatic hydrolysis and membrane filtration to treat oyster meat liquid and oyster cooking juice for multiple times, then adds glutamate decarboxylase (GAD) to the oyster enzymatic hydrolysis product for enzymatic hydrolysis and filtration, obtains an oyster enzymatic hydrolysis filtrate with a molecular weight of less than 3kDa and rich in γ-aminobutyric acid, and concentrates and freeze-dries to obtain the low molecular weight oyster oligopeptide.

[0009] Furthermore, the preparation method comprises the following steps:

[0010] S1, steaming fresh oysters as raw materials, and then immediately cooling the fresh oyster meat with running water;

[0011] S2, placing the cooled oysters in an IQF machine, and performing spiral IQF in batches until the oysters are completely frozen;

[0012] S3, subjecting the frozen oysters to a drum-type meat extraction process, discarding the viscera of the oysters, retaining the fresh oyster meat, crushing the fresh oyster meat in an ice bath and uniformly mixing it with purified water, discarding the upper suspended matter after centrifugation, and subjecting the lower oyster meat sediment to sequential processing by a colloid mill and a high-pressure homogenizer;

[0013] S4, mixing the composite protease with the homogenized oyster meat sediment for enzymatic hydrolysis, filtering the enzymatic hydrolysis reaction product with a three-stage membrane, retaining the filtrate with a Da content less than 3 kDa after the filtration, and retaining the retentate with a Da content greater than 3 kDa for secondary enzymatic hydrolysis;

[0014] S5, adding a composite protease to the retained product greater than 3 kDa in S4 for secondary enzymolysis, controlling the enzymolysis time to obtain a secondary enzymolysis reaction product, filtering the second enzymolysis reaction product with a three-stage membrane, retaining a secondary filtrate less than 3 kDa after filtration, and retaining the retained product greater than 3 kDa for the third enzymolysis;

[0015] S6, using a composite protease to perform a third enzymatic hydrolysis on the retained product greater than 3 kDa in S5, and after sufficient enzymatic hydrolysis, using a three-stage membrane to filter the third enzymatic hydrolysis reaction product, and retaining the tertiary filtrate less than 3 kDa after the filtration;

[0016] S7, combining the filtrates below 3 kDa obtained by three enzymatic hydrolysis, adding glutamate decarboxylase (GAD) thereto for enzymatic hydrolysis, filtering using a 3 kDa filter membrane, concentrating the obtained enzymatic hydrolysis filtrate by RO and drying, to obtain the low molecular weight oyster oligopeptide.

[0017] Furthermore, the oyster cooking time is 5 to 10 minutes, and the running water temperature is 0 to 4°C.

[0018] Furthermore, the freezing speed of the single freezing in S2 is 1.1 to 18.3 mm / min, the freezing temperature is -40 to -35°C, and the complete freezing of the oysters means that the central temperature of the oysters reaches -18 to -7°C.

[0019] Furthermore, in S3, a drum-type method is used to extract fresh oyster meat. The diameter of the circular holes for extracting meat on the inner wall of the drum is 2-5 mm, and two adjacent circles of circular holes are circumferentially staggered with a staggered distance of 3-5 mm. The drum speed is 400-1400 r / min, and the process temperature during the meat extraction process is controlled below 4°C, and it is ensured to be completed under spraying conditions.

[0020] Furthermore, the temperature of the pure water in S3 is below 10° C., and the mass ratio of the mixed liquid of the crushed oyster meat and the pure water is 1:5-10.

[0021] Furthermore, the composite protease in S4 is composed of one or both of pepsin and trypsin, the usage of the composite protease is 2-5% of the mass of fresh oyster meat, and the enzymatic hydrolysis time is 3-5h; the filtration specifications of the tertiary membrane are 3kDa-10kDa, 10kDa-30kDa, and greater than 30kDa, the filtration temperature is 25°C, and the equipment operating power is 50Hz.

[0022] Furthermore, the composite protease in S5 is composed of one or both of pepsin and trypsin, the usage of the composite protease is 2-5% of the mass of fresh oyster meat, the enzymatic hydrolysis time is 1-4 hours, the filtration specifications of the tertiary membrane are 3kDa-10kDa, 10kDa-30kDa, and greater than 30kDa, and the equipment operating power is 50Hz.

[0023] Furthermore, the composite protease in S6 is composed of one or both of pepsin and trypsin, the usage of the composite protease is 1-2% of the mass of the oyster, the filtration specifications of the tertiary membrane are 3kDa-10kDa, 10kDa-30kDa, and greater than 30kDa, and the equipment operating power is 50Hz.

[0024] Furthermore, the added amount of glutamate decarboxylase GAD in S7 is 0.2-0.5% of the mass of fresh oyster meat, the enzymolysis time is 0.5-4h, the enzymolysis temperature is 30-70°C, and the pH is 3.5-5.5.

[0025] Furthermore, the concentration equipment operates at 50 Hz, with a reflux rate of 12 LPM and a membrane area of ​​2.5 m 2 , concentrated to 50 times.

[0026] Furthermore, the membrane flux of the concentration equipment is 19.7-37.6 LMH, the dialysis volume is 0-6.0 L, the concentration multiple is 1.0-9.0 times, the inlet pressure is 8.0-20.0 bar, the outlet pressure is 6.0-19 bar, and the operation time is 60-600 min.

[0027] Furthermore, the concentrated filtrate is freeze-dried or spray-dried to obtain the low molecular weight oyster oligopeptide.

[0028] A second object of the present invention is to provide a low molecular weight oyster oligopeptide.

[0029] Furthermore, the low molecular weight oyster oligopeptide is milky white or white (Lab value: L*=80.36-94.33, a*=1.91--1.34, b*=21.84-4.82).

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention uses fresh oyster meat as raw material, combines enzymatic hydrolysis and membrane filtration to process oyster meat liquid in multiple stages and multiple times, while fully retaining oyster active peptides, accurately and thoroughly enzymatically hydrolyzes proteins or macromolecular peptides, improves the separation effect and purity of oyster oligopeptides, and obtains low molecular weight (3kDa) oyster oligopeptides that can be fully absorbed by the body. The oyster oligopeptides retain the original vitamins, trace elements, taurine and other nutrients of the oyster, and because of their small molecular weight, they are more easily absorbed and utilized by the human body, and combine with human cells to exert higher biological activity, and the multi-stage and multiple preparation process of oyster oligopeptides removes harmful substances and allergens that may exist in themselves to the maximum extent, so they are safer.

[0032] 2. The present invention proposes for the first time to add glutamate decarboxylase (GAD) during the enzymatic hydrolysis of oyster oligopeptides. GAD, as a pyridoxal phosphate (PLP)-dependent enzyme, irreversibly catalyzes the α-decarboxylation of L-glutamate or glutamate to generate γ-aminobutyric acid (GABA), thereby releasing more amino acids and peptides, reducing the difficulty of enzymatic hydrolysis of protein in oyster meat, and promoting the decomposition of protein in oyster meat and the generation of oyster oligopeptides. At the same time, since the reaction of GAD catalyzing the conversion of glutamate into GABA is specific, it avoids the generation of other unnecessary by-products that affect the purity and quality of oyster oligopeptides, and finally obtains higher quality oyster oligopeptides by working together with enzymatic hydrolysis and membrane filtration.

[0033] 3. In the present invention, the addition of glutamate decarboxylase (GAD) when preparing oyster oligopeptides not only promotes the purity and quality of oyster oligopeptides, but also the increase of GABA can enhance the nutritional value of oyster oligopeptides. The addition of GAD catalyzes the conversion of glutamate in oyster meat into GABA with multiple physiological functions. As an important neurotransmitter, GABA in oyster oligopeptides can inhibit the excitability of neurons by binding to GABA receptors on neurons, thereby assisting in regulating the nervous system. It has important application value in improving sleep, relieving anxiety, and lowering blood pressure, further enhancing the functionality and added value of oyster oligopeptides. In addition, since the present invention can obtain high-quality oyster oligopeptide powder, the prepared oyster oligopeptides can be directly used as a nutritional supplement.

[0034] 4. The present invention discloses a method for preparing functional low-molecular-weight oyster oligopeptides, which does not require enzyme inactivation after multi-stage multiple enzymatic hydrolysis, has a simple processing technology, has low equipment requirements, and is suitable for industrial production. The oyster oligopeptides produced meet consumers' demand for healthy food, realize the high-value utilization of oyster resources, provide a theoretical basis for the application of oysters in the fields of health food, special medical formula food, etc., and promote the high-value utilization of marine biological resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is a comparison chart of oyster oligopeptides below 3 kDa prepared without and with the preparation method of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with preferred embodiments. The endpoints of the ranges disclosed in the present invention and any values ​​are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be deemed to be specifically disclosed herein.

[0037] The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1

[0040] This embodiment provides a method for preparing a low molecular weight oyster oligopeptide, comprising the following steps:

[0041] (1) 20 kg of fresh oysters were shelled, washed and steamed for 5 min. After steaming, they were immediately cooled with running water at 1°C until the oysters entered the single freezing machine;

[0042] (2) spirally freezing the cooled oyster meat at a freezing speed of 15 mm / min and a freezing temperature of -35°C until the core temperature of the oyster reaches -7°C and is completely frozen;

[0043] (3) The frozen oysters were subjected to a drum-type meat extraction process. The diameter of the meat extraction circular holes on the inner wall of the drum was 2 mm. Two adjacent circles of circular holes were arranged in a staggered manner in the circumferential direction with a staggered distance of 5 mm. The drum speed was 400 r / min. The process temperature during the meat extraction process was controlled below 4°C and was completed under spraying conditions.

[0044] (4) crushing the completely frozen fresh oyster meat in an ice bath, uniformly mixing the crushed oyster meat with purified water below 10°C at a material-liquid ratio of 1:5, and subjecting the lower layer of oyster meat sediment to sequential processing by a colloid mill and a high-pressure homogenizer;

[0045] (5) 0.4 kg of a composite protease composed of pepsin and trypsin was mixed with the homogenized oyster meat sediment for enzymolysis for 3 hours without inactivating the enzyme during the enzymolysis reaction. After the enzymolysis reaction, the enzymolysis reaction product was filtered using a three-stage membrane with filtration specifications of 3 kDa to 10 kDa, 10 kDa to 30 kDa, and greater than 30 kDa. The filtration temperature was 25 ° C, the equipment operating power was 50 Hz, and the filtrate less than 3 kDa was retained, and the retentate of the oyster meat sediment greater than 3 kDa was retained for the second enzymolysis;

[0046] (6) adding 0.4 kg of composite protease to the retentate greater than 3 kDa retained in S4 for a second enzymatic hydrolysis for 1 h, and filtering the obtained enzymatic hydrolyzate for a second time, using the same filtration membrane and conditions as in step (5);

[0047] (7) using 0.2 kg of composite protease to perform a third enzymatic hydrolysis on the retentate greater than 3 kDa retained in step (6), and after sufficient enzymatic hydrolysis, perform a third filtration, using the same filtration membrane and conditions as step (5);

[0048] (8) The filtrates below 3 kDa obtained from the three enzymatic hydrolysis were combined, 0.04 kg of glutamate decarboxylase (GAD) was added, and the mixture was enzymatically hydrolyzed at 30 °C and pH 3.5 for 0.5 h. The mixture was filtered using a 3 kDa filter membrane and then concentrated by RO. The concentration equipment was operated at 50 Hz, with a reflux rate of 12 LPM and a membrane area of ​​2.5 m 2 , concentrated to 50 times, the membrane flux of the concentration equipment is 20MH, the dialysis volume is 2.0L, the concentration multiple is 5.0 times, the inlet pressure is 8.0bar, the outlet pressure is 6.0bar, the running time is 60min, and the concentrated enzymatic filtrate is freeze-dried to obtain the low molecular weight oyster oligopeptide.

[0049] Example 2

[0050] This embodiment provides a method for preparing a low molecular weight oyster oligopeptide, comprising the following steps:

[0051] (1) 30 kg of fresh oysters were shelled, washed and steamed for 7 min. After steaming, they were immediately cooled with running water at 2°C until the oysters entered the single freezing machine;

[0052] (2) spirally freezing the cooled oyster meat at a freezing speed of 17 mm / min and a freezing temperature of -37°C until the core temperature of the oyster reaches -9°C and is completely frozen;

[0053] (3) The frozen oysters were subjected to a drum-type meat extraction process. The diameter of the meat extraction circular holes on the inner wall of the drum was 3 mm. Two adjacent circles of circular holes were arranged in a staggered manner in the circumferential direction with a staggered distance of 4 mm. The drum speed was 800 r / min. The process temperature during the meat extraction process was controlled below 4°C and was completed under spraying conditions.

[0054] (4) crushing the completely frozen fresh oyster meat in an ice bath, uniformly mixing the crushed oyster meat with purified water below 10°C at a material-liquid ratio of 1:7, and subjecting the lower layer of oyster meat sediment to sequential processing by a colloid mill and a high-pressure homogenizer;

[0055] (5) 0.6 kg of pepsin was mixed with the homogenized oyster meat sediment and enzymolyzed for 4 hours without inactivating the enzyme. After the enzymolysis reaction, the enzymolysis reaction product was filtered using a three-stage membrane with filtration specifications of 3 kDa to 10 kDa, 10 kDa to 30 kDa, and greater than 30 kDa. The filtration temperature was 25 ° C. The equipment operating power was 50 Hz. The filtrate less than 3 kDa was retained, and the retentate greater than 3 kDa of the oyster meat sediment was retained for secondary enzymolysis;

[0056] (6) Add 0.6 kg of pepsin to the retentate greater than 3 kDa retained in S4 for a second enzymatic hydrolysis for 2 h, and filter the obtained enzymatic hydrolyzate for a second time using the same filtration membrane and conditions as in step (5);

[0057] (7) using 0.3 kg of pepsin to perform a third enzymatic hydrolysis on the retentate greater than 3 kDa retained in step (6), and after sufficient enzymatic hydrolysis, perform a third filtration, using the same filtration membrane and conditions as step (5);

[0058] (8) The filtrates below 3 kDa obtained from the three enzymatic hydrolysis were combined, 0.06 kg of glutamate decarboxylase (GAD) was added, and the enzymatic hydrolysis was carried out at 70°C and pH 5.5 for 2 h. The filtrates were filtered using a 3 kDa filter membrane and then concentrated by RO. The operating power of the concentration equipment was 50 Hz, the reflux rate was 12 LPM, and the membrane area was 2.5 m 2 , concentrated to 50 times, the membrane flux of the concentration equipment is 25LMH, the dialysis volume is 4.0L, the concentration multiple is 7.0 times, the inlet pressure is 10.0bar, the outlet pressure is 8.0bar, the operation time is 120min, and the concentrated enzymatic filtrate is freeze-dried to obtain the low molecular weight oyster oligopeptide.

[0059] Example 3

[0060] This embodiment provides a low molecular weight oyster oligopeptide, and the preparation method thereof comprises the following steps:

[0061] (1) 40 kg of fresh oysters were shelled, washed and steamed for 10 min. After steaming, they were immediately cooled with running water at 4°C until the oysters entered the single freezing machine;

[0062] (2) spirally freezing the cooled oyster meat at a freezing rate of 1.1 mm / min and a freezing temperature of -39°C until the core temperature of the oyster reaches -18°C and is completely frozen;

[0063] (3) The frozen oysters were subjected to a drum-type meat extraction process. The diameter of the meat extraction circular holes on the inner wall of the drum was 5 mm. Two adjacent circles of circular holes were arranged circumferentially in a staggered manner with a staggered distance of 3 mm. The drum speed was 1400 r / min. The process temperature during the meat extraction process was controlled below 4°C and was completed under spraying conditions.

[0064] (4) crushing the completely frozen fresh oyster meat in an ice bath, uniformly mixing the crushed oyster meat with purified water below 10° C. at a material-liquid ratio of 1:10, and subjecting the lower layer of oyster meat sediment to sequential processing by a colloid mill and a high-pressure homogenizer;

[0065] (5) Mix the composite protease composed of 0.8 kg of trypsin and the homogenized oyster meat sediment for enzymolysis for 5 hours without inactivating the enzyme during the enzymolysis reaction. After the enzymolysis reaction, the enzymolysis reaction product is filtered using a three-stage membrane with filtration specifications of 3 kDa to 10 kDa, 10 kDa to 30 kDa, and greater than 30 kDa. The filtration temperature is 25 ° C, the equipment operating power is 50 Hz, and the filtrate less than 3 kDa is retained, and the retentate of the oyster meat sediment greater than 3 kDa is retained for secondary enzymolysis;

[0066] (6) Add 0.8 kg of trypsin to the retentate greater than 3 kDa retained in S4 for a second enzymatic hydrolysis for 4 h, and filter the obtained enzymatic hydrolyzate for a second time, using the same filtration membrane and conditions as in step (5);

[0067] (7) using 0.4 kg of trypsin to perform a third enzymatic hydrolysis on the retentate greater than 3 kDa retained in step (6), and after sufficient enzymatic hydrolysis, perform a third filtration, using the same filtration membrane and conditions as step (5);

[0068] (8) The filtrates below 3 kDa obtained from the three enzymatic hydrolysis were combined, 0.08 kg of glutamate decarboxylase (GAD) was added, and the mixture was enzymatically hydrolyzed at 35 °C and pH 4.5 for 4 h. The mixture was filtered using a 3 kDa filter membrane and then concentrated by RO. The concentration equipment was operated at 50 Hz, with a reflux rate of 12 LPM and a membrane area of ​​2.5 m 2 , concentrated to 50 times, the membrane flux of the concentration equipment is 30LMH, the dialysis volume is 6.0L, the concentration multiple is 9.0 times, the inlet pressure is 12.0bar, the outlet pressure is 10.0bar, the operation time is 180min, and the concentrated enzymatic filtrate is spray-dried to obtain the low molecular weight oyster oligopeptide.

[0069] Performance Testing

[0070] 1. Freeze-drying yield test

[0071] First, prepare two clean centrifuge tubes, weigh and record their masses respectively, and weigh and record the masses of oyster hydrolysate solutions of different molecular weights that need to be freeze-dried; then, load the oyster hydrolysate solutions of different molecular weights into centrifuge tubes respectively and put them into a freeze dryer for freeze drying; finally, after freeze drying, take out the centrifuge tubes, weigh and record the total mass of the freeze-dried centrifuge tubes and the solution.

[0072] The freeze-drying yield was calculated using the formula:

[0073] Freeze-drying yield = (mass of sample after freeze-drying / mass of liquid before freeze-drying) * 100%.

[0074] Table 1 Freeze-dried yield of oyster hydrolysate with different molecular weights

[0075]

[0076] As shown in Table 1, the data in the table show the freeze-drying yields of oyster hydrolysate solutions with different molecular weights, and the following conclusions can be drawn: the freeze-drying yield of oyster hydrolysate solutions with a molecular weight of less than 3 kDa is approximately 32.97% to 33.64%; the freeze-drying yield of oyster hydrolysate solutions with a molecular weight of 3-10 kDa is approximately 59.08% to 60.68%; the freeze-drying yield of oyster hydrolysate solutions with a molecular weight of 10-30 kDa is approximately 41.08% to 47.08%.

[0077] The results show that the yields of oyster hydrolyzate solutions with different molecular weights during freeze-drying are different, among which the freeze-drying yield of the solution with a molecular weight of 10-30 kDa is the highest, while the freeze-drying yield of the solution with a molecular weight of less than 3 kDa is the lowest. However, since the smaller the molecular weight, the higher the requirements for the process, the method of the present invention can obtain low-molecular-weight oyster oligopeptides with a good yield.

[0078] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. The terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprises a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0079] The above are only preferred embodiments of the present application and do not constitute any form of limitation to the present application. Any technician familiar with the profession can make some changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present application. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present application without departing from the content of the technical solution of the present application still fall within the scope of the technical solution of the present application.

Claims

1. A method for preparing low molecular weight oyster oligopeptides, characterized in that: The preparation method uses fresh oyster meat as raw material, combines enzymatic hydrolysis and membrane filtration to treat oyster meat liquid and oyster cooking juice for multiple times, then adds glutamate decarboxylase (GAD) to the oyster enzymatic hydrolysis product for enzymatic hydrolysis and filtration, obtains oyster enzymatic hydrolysis filtrate with a molecular weight of less than 3kDa and rich in γ-aminobutyric acid, and obtains the low molecular weight oyster oligopeptide after concentration and freeze-drying.

2. The method for preparing a low molecular weight oyster oligopeptide according to claim 1, characterized in that: The preparation method comprises the following steps: S1, steaming fresh oysters as raw materials, and then immediately cooling the fresh oyster meat with running water; S2, placing the cooled oysters in an IQF machine, and performing spiral IQF in batches until the oysters are completely frozen; S3, subjecting the frozen oysters to a drum-type meat extraction process, discarding the viscera of the oysters, retaining the fresh oyster meat, crushing the fresh oyster meat in an ice bath and uniformly mixing it with purified water, discarding the upper suspended matter after centrifugation, and subjecting the lower oyster meat sediment to sequential processing by a colloid mill and a high-pressure homogenizer; S4, mixing the composite protease with the homogenized oyster meat sediment for enzymatic hydrolysis, filtering the enzymatic hydrolysis reaction product with a three-stage membrane, retaining the filtrate with a Da content less than 3 kDa after the filtration, and retaining the retentate with a Da content greater than 3 kDa for secondary enzymatic hydrolysis; S5, adding a composite protease to the retained product greater than 3 kDa in S4 for secondary enzymolysis, controlling the enzymolysis time to obtain a secondary enzymolysis reaction product, filtering the second enzymolysis reaction product with a three-stage membrane, retaining a secondary filtrate less than 3 kDa after the filtration, and retaining the retained product greater than 3 kDa for the third enzymolysis; S6, using a composite protease to perform a third enzymatic hydrolysis on the retained product greater than 3 kDa in S5, and after sufficient enzymatic hydrolysis, using a three-stage membrane to filter the third enzymatic hydrolysis reaction product, and retaining the tertiary filtrate less than 3 kDa after the filtration; S7, combining the filtrates below 3 kDa obtained by three enzymatic hydrolysis, adding glutamate decarboxylase (GAD) thereto for enzymatic hydrolysis, filtering using a 3 kDa filter membrane, concentrating the obtained enzymatic hydrolysis filtrate by RO and drying, to obtain the low molecular weight oyster oligopeptide.

3. The method for preparing a low molecular weight oyster oligopeptide according to claim 1, characterized in that: The oyster cooking time in S1 is 5 to 10 minutes, and the running water temperature is 0 to 4°C.

4. The method for preparing a low molecular weight oyster oligopeptide according to claim 1, characterized in that: The freezing speed of the single freezer in S2 is 1.1 to 18.3 mm / min, the freezing temperature is -40 to -35°C, and the complete freezing of oysters means that the central temperature of the oysters reaches -18 to -7°C.

5. The method for preparing a low molecular weight oyster oligopeptide according to claim 1, characterized in that: In S3, a drum-type method is used to extract fresh oyster meat. The diameter of the circular holes for extracting meat on the inner wall of the drum is 2-5 mm. Two adjacent circles of circular holes are arranged circumferentially in an alternating manner with a staggered distance of 3-5 mm. The drum speed is 400-1400 r / min. The temperature during the meat extraction process is controlled below 4° C. and is ensured to be completed under spraying conditions. The temperature of the purified water is below 10°C, and the mass ratio of the mixed liquid of the crushed oyster meat and purified water is 1:5-10.

6. The method for preparing a low molecular weight oyster oligopeptide according to claim 1, characterized in that: The compound protease in S4 is one or both of pepsin and trypsin, the amount of compound protease used is 2-5% of the weight of fresh oyster meat, and the enzymolysis time is 3-5h; The filtration specifications of the three-stage membrane are 3kDa-10kDa, 10kDa-30kDa, and greater than 30kDa, the filtration temperature is 25°C, and the equipment operating power is 50Hz.

7. The method for preparing a low molecular weight oyster oligopeptide according to claim 1, characterized in that: The composite protease in S5 is composed of one or both of pepsin and trypsin, the usage of the composite protease is 2-5% of the mass of fresh oyster meat, the enzymolysis time is 1-4 hours, the filtration specifications of the tertiary membrane are 3kDa-10kDa, 10kDa-30kDa, and greater than 30kDa, and the equipment operating power is 50Hz.

8. The method for preparing a low molecular weight oyster oligopeptide according to claim 1, characterized in that: The composite protease in S6 is composed of one or both of pepsin and trypsin, and the usage of the composite protease is 1-2% of the mass of the oyster. The filtration specifications of the tertiary membrane are 3kDa-10kDa, 10kDa-30kDa, and greater than 30kDa, and the equipment operating power is 50Hz.

9. The method for preparing a low molecular weight oyster oligopeptide according to claim 1, characterized in that: The amount of glutamate decarboxylase (GAD) added in S7 is 0.2-0.5% of the mass of fresh oyster meat, the enzymolysis time is 0.5-4h, the enzymolysis temperature is 30-70°C, the pH is 3.5-5.5, the concentration equipment operating power is 50Hz, the reflux rate is 12LPM, and the membrane area is 2.5m 2 , concentrated to 50 times.

10. A low molecular weight oyster oligopeptide obtained according to the method of any one of claims 1 to 9.

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