Salty and fresh peptide RRRRR with salty and fresh increasing function and screening method

Abstract

The invention relates to the technical field of small molecular weight salty peptides, and particularly discloses a salty and fresh peptide RRRRR with a salty and fresh increasing function and a screening method, the salty and fresh peptide is RRRRR, and the amino acid sequence of the salty and fresh peptide is Arg-Arg-Arg-Arg-Arg; the molecular formula of the salty fresh peptide is C30H62N20O6; mixing bovine bone powder with water; adding trypsin, compound protease and flavourzyme to carry out enzymolysis reaction; enzyme deactivation; centrifuging the solution through a centrifugal assembly, and taking supernate by adopting a suction assembly; enabling the supernate to sequentially pass through ultrafiltration membranes of 3kDa and 1kDa to obtain a molecular weight gt; 3 kDa, 1 kDa to 3 kDa, and lt; a component of 1 kDa; the molecular weight 1t is taken; carrying out gel permeation chromatography separation and purification on the components of 1 kDa and 1 kDa to obtain a peak group; performing amino acid sequence identification on components corresponding to the peak group, and performing preliminary screening through C-terminal amino acid composition of peptide; and carrying out molecular docking on the primarily screened peptide and a salty receptor, and obtaining the salty and fresh peptide RRRRR according to binding energy. The invention has the characteristics of no reduction of sensory quality, easy and rapid absorption and simple and convenient preparation steps.

Description

Technical Field

[0001] The present invention relates to the technical field of small molecular weight salty peptides, and in particular to a salty and fresh peptide RRRRR with saltiness and freshness enhancing functions and a screening method thereof. Background Art

[0002] Salt, as the most basic factor affecting human appetite, has an important impact on the physiological functions of the human body. However, high salt can change the body's fluid balance and cause many cardiovascular diseases. People have been studying how to increase the taste of salt without increasing the amount of salt intake. At present, the most commonly used salt reduction strategy in the food industry is to use non-sodium salt substitutes in food formulations, such as potassium chloride and calcium chloride as substitutes for sodium chloride, because they have similar physical, chemical and salty properties to sodium chloride; however, excessive use of these salt substitutes will produce metallic taste and unpleasant bitterness, resulting in a decrease in the sensory quality of food. Another strategy to reduce salt intake is to add flavor enhancers to processed foods, which may or may not have salty taste, but when used with sodium chloride, they can enhance their salty taste perception. Studies have shown that some food-derived peptides have salt-enhancing properties, and many small peptides in various food proteins, such as soy sauce, yeast extract and protamine, have a certain salt taste enhancement effect. In addition, compared with free amino acids, small molecule peptides are easier to absorb. Studies have shown that they have the characteristics of rapid absorption, easy utilization by the human body and not easy to saturate.

[0003] China is a major producer of livestock and poultry meat in the world, and also a major consumer of meat products. The weight of livestock and poultry bones is generally about one-fifth of the total weight of the animal. As a by-product of slaughter, livestock and poultry bones are rich in high-quality protein resources that have not been better and more efficiently utilized, especially in the deep processing of food, which is limited to the primary processing of bone powder and bone glue. It is very necessary to develop high-value utilization and deep processing of livestock and poultry bones. Therefore, it is of great significance to study the salty or salty-enhancing peptides in livestock and poultry bones that make important contributions to saltiness using livestock and poultry bones as the basic raw materials, using protease-controlled enzymatic hydrolysis technology and modern peptide separation and purification technology, and electronic tongue evaluation technology. On the other hand, the existing methods for preparing or predicting salty peptides have the defects of complex operation steps and long operation time, making it difficult to effectively screen salty peptides.

[0004] The patent document with the publication number "CN101755996A" discloses a method for continuous production of casein phosphopeptides by hydrolyzing raw cheese with immobilized enzymes and separating with ultrafiltration membranes, and the steps include: a. extraction of trypsin; b. preparation of a trypsin immobilized reaction column; c. hydrolysis of raw cheese; d. separation and purification of casein phosphopeptides (CPP) with ultrafiltration membranes; e. column chromatography of the ultrafiltrate of CPP; the process is reasonably designed, highly operable, has a high concentration of intermediate products, and has low production energy consumption. However, the scheme adopts a continuous hydrolysis method, and the operation steps are relatively complicated.

[0005] Therefore, existing products for reducing salt intake have the problems of reduced sensory quality, difficulty in rapid absorption and relatively complicated preparation steps. Summary of the invention

[0006] In order to solve the above-mentioned technical problems existing in existing products for reducing salt intake, the present invention provides a salty and fresh peptide RRRRR with the function of increasing saltiness and freshness and a screening method, which has the characteristics of not reducing sensory quality, being easy and quickly absorbed and having relatively simple preparation steps.

[0007] The first technical solution of the present invention: a salty and fresh peptide having the function of increasing saltiness and freshness, wherein the salty and fresh peptide is RRRRR, and the amino acid sequence of the salty and fresh peptide is Arg-Arg-Arg-Arg-Arg; the molecular formula of the salty and fresh peptide is C 30 H 62 N 20 O6; the molecular structure of the salty peptide is The invention is composed of 5 amino acids and is a short peptide with a molecular weight of less than 500Da, which is easier to be quickly absorbed and utilized by the human body; the invention has good saltiness and umami taste, low bitterness, and can be used to partially replace sodium chloride in food without affecting the sensory quality of the food.

[0008] The second technical solution of the present invention is a method for screening salty and fresh peptides with saltiness and freshness enhancing functions, comprising the following steps:

[0009] (S01) taking an appropriate amount of cattle bone powder and mixing it with water, and performing pretreatment;

[0010] (S02) adding trypsin, compound protease and flavor protease to the solution pretreated in step (S01) to perform ultrasonic enzymatic hydrolysis reaction;

[0011] (S03) after the ultrasonic enzymatic hydrolysis reaction in step (S02) is completed, heating up and inactivating the enzyme;

[0012] (S04) centrifuging the solution after the enzyme inactivation in step (S03) through a centrifugal assembly, and then taking out the supernatant using a suction assembly to obtain a bovine bone protein hydrolysate;

[0013] (S05) passing the bovine bone protein hydrolysate from step (S04) through 3 kDa and 1 kDa ultrafiltration membranes in sequence to obtain three components with molecular weights >3 kDa, 1 kDa to 3 kDa, and <1 kDa;

[0014] (S06) taking an appropriate amount of the component with a molecular weight less than 1 kDa in step (S05) and performing gel permeation chromatography separation and purification, and obtaining a peak group of the purified product at a characteristic wavelength of 280 nm;

[0015] (S07) performing amino acid sequence identification on the corresponding components corresponding to the peak group in step (S06), and performing preliminary screening based on the C-terminal amino acid composition of the peptides;

[0016] (S08) Molecular docking of the peptides initially screened out in step (S07) with the salty taste receptor is performed to obtain the salty and fresh peptide RRRRR based on the binding energy index. In the present invention, the bovine bone powder is mixed with water and pretreated, so that the bovine bone powder can be fully dissolved in water, which is convenient for subsequent good enzymatic hydrolysis treatment; trypsin, composite protease and flavor protease are added to the pretreated solution for ultrasonic enzymatic hydrolysis reaction, so that the protein in the bovine bone powder can be fully enzymatically hydrolyzed into peptide molecules, which is convenient for screening out the required small molecular weight, good saltiness and umami taste, low bitterness, and can be used for partial replacement of sodium chloride in food; the solution after the ultrasonic enzymatic hydrolysis reaction is heated to kill the enzyme, so as to avoid the enzyme affecting the purity and quality of the peptides obtained by subsequent separation; the present invention centrifuges the enzyme-killed solution, and uses a physical method to quickly separate components of different densities, so as to quickly obtain the required supernatant components; the present invention centrifuges the bovine bone protein hydrolysate through ultrafiltration, passes through 3kDa and 1kDa ultrafiltration membranes in sequence, and relatively quickly obtains three components with molecular weights greater than 3kDa, 1kDa to 3kDa and less than 1kDa, thereby narrowing the screening range and quickly and accurately obtaining the required small molecule peptides. Peptide; the present invention takes components with a molecular weight of <1kDa for gel permeation chromatography separation and purification, further improves the purity of the components, and further avoids interference from impurities; obtains a peak group of the purified product at a characteristic wavelength of 280nm, identifies the corresponding components corresponding to the peak group by amino acid sequence, and performs preliminary screening through the C-terminal amino acid composition of the peptide, and then performs molecular docking of the peptide preliminarily screened with the salty taste receptor, and obtains the salty and fresh peptide RRRRR according to the binding energy index, and combines computer simulation screening technology to save time and effort while quickly and accurately obtaining the required salty and fresh peptide; the present invention adopts a simultaneous enzymatic hydrolysis method of three proteases as a whole, which is different from traditional continuous hydrolysis, has the advantages of rapidity, high efficiency, convenience and economy, and uses cattle by-products as raw materials, has low production cost, high economic benefits, and is suitable for industrial production; the present invention can be directed prepared from cattle bone enzymatic hydrolysis products, and can also be obtained in large quantities by solid phase synthesis, which is relatively easy to obtain; the entire preparation and screening process of the present invention has simple operating steps, combined with computer simulation screening, does not rely on complex technology, and can save a lot of manpower and material resources.

[0017] Preferably, the cattle bone powder in step (S01) is yak bone powder. Yak bone powder has higher protein nutritional value.

[0018] Preferably, the method for obtaining the cattle bone powder in step (S01) is:

[0019] Take the cattle bones, wash and roughly chop, high-temperature steam, dry, crush and sieve in sequence to obtain cattle bone powder. Washing and roughly chopping cattle bones and high-temperature steaming can make the cattle bones quickly cracked into a breakable form, which is convenient for subsequent good drying and crushing into powder; drying and crushing can well crush the cattle bones into fine pieces, preparing for subsequent sufficient enzymatic hydrolysis; sieving makes the bone powder particles of subsequent enzymatic hydrolysis more delicate and uniform, which will have a better enzymatic hydrolysis effect.

[0020] Preferably, the sieving is through a 150-250 mesh sieve. More preferably, the sieving is through a 180-230 mesh sieve. More preferably, the sieving is through a 190-220 mesh sieve. More preferably, the sieving is through a 200-210 mesh sieve. The limited sieving mesh number allows only particles that meet the particle size requirements to pass through, thereby ensuring a better subsequent enzymolysis effect.

[0021] Preferably, the solid-liquid ratio of the bovine bone powder and water in the step (S01) is 1:15-25. More preferably, the solid-liquid ratio of the bovine bone powder and water in the step (S01) is 1:17-23. More preferably, the solid-liquid ratio of the bovine bone powder and water in the step (S01) is 1:19-21. More preferably, the solid-liquid ratio of the bovine bone powder and water in the step (S01) is 1:20. The limited solid-liquid ratio will be able to more fully dissolve the bovine bone powder, facilitating subsequent more sufficient enzymatic hydrolysis.

[0022] Preferably, the pretreatment in step (S01) is cooling to room temperature after boiling water bath. Boiling water bath pretreatment can denature the protein so that the protease can achieve enzymatic cleavage more efficiently.

[0023] Preferably, the boiling water bath time is 20 min to 60 min. More preferably, the boiling water bath time is 25 min to 55 min. More preferably, the boiling water bath time is 30 min to 50 min. More preferably, the boiling water bath time is 35 min to 45 min. More preferably, the boiling water bath time is 40 min. The limited boiling water bath time ensures that the protein can be denatured so that the protease can achieve enzymatic cleavage more efficiently while taking into account timeliness.

[0024] Preferably, the amount of trypsin added in the step (S02) is 8000U / g substrate mass to 14000U / g substrate mass. More preferably, the amount of trypsin added in the step (S02) is 9000U / g substrate mass to 13000U / g substrate mass. More preferably, the amount of trypsin added in the step (S02) is 10000U / g substrate mass to 12000U / g substrate mass. More preferably, the amount of trypsin added in the step (S02) is 11000U / g substrate mass. Limiting the amount of trypsin added can better selectively hydrolyze arginine and lysine peptide chains, and better hydrolyze the proteins therein into polypeptides or amino acids.

[0025] Preferably, the amount of the compound protease added in step (S02) is 15% to 18% relative to the solution. More preferably, the amount of the compound protease added in step (S02) is 16% to 17% relative to the solution. The compound protease with a limited amount of addition can cooperate with the other two enzymes to better hydrolyze the protein in the solution into polypeptides or amino acids.

[0026] Preferably, the amount of flavor protease added in step (S02) relative to the solution is 20% to 25%. More preferably, the amount of flavor protease added in step (S02) relative to the solution is 21% to 24%. More preferably, the amount of flavor protease added in step (S02) relative to the solution is 22% to 23%. The limited amount of flavor protease added can cooperate with the other two enzymes to better hydrolyze the protein in the solution into polypeptides or amino acids, and can hydrolyze the flavor precursors in the solution to release flavor substances, thereby enhancing and improving the flavor of the food; it can also control the bitterness of the peptide, and its principle is to cut the peptide bonds inside the polypeptide by endoprotease to form short-chain peptides, some of which contain hydrophobic amino acids and thus become bitter peptides, and use exoenzymes to cut and release one amino acid from the end of the polypeptide chain each time, thereby completely degrading the bitter peptides into amino acids.

[0027] Preferably, the enzymolysis in step (S02) is a heated water bath enzymolysis, which can make the entire ultrasonic enzymolysis reaction proceed quickly and stably.

[0028] Preferably, the enzymolysis temperature in step (S02) is 40° C. to 45° C. More preferably, the enzymolysis temperature in step (S02) is 41° C. to 44° C. More preferably, the enzymolysis temperature in step (S02) is 42° C. to 43° C. The limited enzymolysis temperature can ensure the stability and rapid progress of the entire enzymolysis process.

[0029] Preferably, the enzymolysis time in step (S02) is 4 hours to 6 hours. More preferably, the enzymolysis time in step (S02) is 4.5 hours to 5.5 hours. More preferably, the enzymolysis time in step (S02) is 5 hours. The limited enzymolysis time ensures that the entire enzymolysis process is complete while taking into account timeliness.

[0030] Preferably, the step (S03) of inactivating the enzyme by heating is a boiling water bath, which can inactivate the protease simply and quickly.

[0031] Preferably, the enzyme inactivation time in step (S03) is 5 min to 15 min. More preferably, the enzyme inactivation time in step (S03) is 7 min to 13 min. More preferably, the enzyme inactivation time in step (S03) is 9 min to 11 min. More preferably, the enzyme inactivation time in step (S03) is 10 min. The limited enzyme inactivation time is to ensure complete enzyme inactivation while taking into account timeliness.

[0032] Preferably, the centrifugal speed in step (S04) is 4000 r / min to 4500 r / min. More preferably, the centrifugal speed in step (S04) is 4100 r / min to 4400 r / min. More preferably, the centrifugal speed in step (S04) is 4200 r / min to 4300 r / min. The limited centrifugal speed can quickly achieve the stratification of the components in the solution.

[0033] Preferably, the centrifugation time in step (S04) is 25 min to 35 min. More preferably, the centrifugation time in step (S04) is 28 min to 32 min. More preferably, the centrifugation time in step (S04) is 30 min. The limited centrifugation time ensures that the entire centrifugation process is complete while taking into account timeliness.

[0034] Preferably, the gel permeation chromatography separation and purification conditions in step (S06) are that the protein purifier is equipped with a Sephadex G-15 column and the mobile phase is ultrapure water. The defined gel permeation chromatography separation and purification conditions can better improve the purity of the components.

[0035] Preferably, the concentration of the component in the step (S06) is 10 mg / mL to 100 mg / mL. More preferably, the concentration of the component in the step (S06) is 20 mg / mL to 90 mg / mL. More preferably, the concentration of the component in the step (S06) is 30 mg / mL to 80 mg / mL. More preferably, the concentration of the component in the step (S06) is 40 mg / mL to 70 mg / mL. More preferably, the concentration of the component in the step (S06) is 50 mg / mL to 60 mg / mL. Defining the concentration of the component enables better separation and purification on gel permeation chromatography, thereby ensuring a more accurate peak group of the purified product at a characteristic wavelength of 280 nm.

[0036] Preferably, the loading amount of the component in step (S06) is 5 mL to 8 mL. More preferably, the loading amount of the component in step (S06) is 6 mL to 7 mL. The limited loading amount of the component can ensure the good progress of the entire separation and purification.

[0037] Preferably, the elution rate of the components in step (S06) is 1 mL / min to 4 mL / min. More preferably, the elution rate of the components in step (S06) is 2 mL / min to 3 mL / min. The limited elution rate can better elute and purify the sample.

[0038] Preferably, there are three peak groups in the step (S06).

[0039] Preferably, the amino acid sequence identification in step (S07) adopts UPLC-Q-TOF MS system. UPLC-Q-TOF MS system can more accurately and effectively identify the amino acid sequence corresponding to the corresponding component.

[0040] Preferably, the parameters of the UPLC-Q-TOF MS system are:

[0041] HILIC column, column temperature 25°C; flow rate 0.3 mL / min; injection volume 2 μL;

[0042] Mobile phase A, water, 25 mM ammonium acetate, and 25 mM ammonia;

[0043] Mobile phase B, acetonitrile;

[0044] The gradient elution program was as follows: 0-1 min, 95% mobile phase B; 1 min-14 min, mobile phase B linearly changed from 95% to 65%; 14 min-16 min, mobile phase B linearly changed from 65% to 40%; 16 min-18 min, mobile phase B maintained at 40%; 18 min-18.1 min, mobile phase B linearly changed from 40% to 95%; 18.1 min-23 min, mobile phase B maintained at 95%;

[0045] After separation on a HILIC column, mass spectrometry analysis was performed using electrospray ionization positive ion and electrospray ionization negative ion modes;

[0046] The scanning range of TOF MS is 60Da to 1000Da; the scanning accumulation time of TOF MS is 0.20s / spectra;

[0047] The product ion scanning range of TOF MS is 25Da~1000Da; the product ion scanning accumulation time of TOF MS is 0.05s / spectra. The specific UPLC-Q-TOF MS system parameters defined will enable more accurate and effective identification of the amino acid sequence corresponding to the corresponding component.

[0048] Preferably, in step (S07), the C-terminal amino acid composition of the peptide is initially screened for peptides containing glutamic acid, aspartic acid, arginine and lysine. Defining the C-terminal amino acid composition of the peptide will enable more accurate and effective screening of the amino acid sequence corresponding to the corresponding component.

[0049] Preferably, the salty taste receptor in step (S08) is constructed by AlphaFold. AlphaFold can construct salty taste receptors quickly and accurately.

[0050] Preferably, the salty taste receptor in step (S08) is derived from the UniProtKB database, and the salty taste receptor number is Q7Z404-TMC4-HUMAN. The selection of a specific salty taste receptor can better perform molecular docking with the initially screened peptides, thereby more accurately screening out the desired salty and fresh peptides.

[0051] Preferably, the salty taste receptor in step (S08) is transmembrane channel-like protein 4. The selection of a specific salty taste receptor can better perform molecular docking with the initially screened peptides, thereby more accurately screening out the desired salty and fresh peptides.

[0052] Preferably, the molecular docking method in the step (S08) is the CDOCKER program in the Discovery Studio software, with -CDOCKER_ENERGY as the index, the docking ligand is a dipeptide, the docking receptor is TMC4, and the docking force field is CHARMm;

[0053] The docking pocket is x: 17.7065, y: 11.7845, z: -14.9743, and the radius is 19.7. The limited docking method can more accurately and quickly achieve rapid and accurate molecular docking of salty taste receptors with the initially screened peptides, thereby more accurately screening out the desired salty and fresh peptides.

[0054] The third technical solution of the present invention: a salty and fresh peptide screening system with salty and freshness enhancing functions, comprising a reactor, a heating reactor and a filter cartridge; a stirring assembly is arranged inside the reactor, a centrifugal inner barrel is rotatably arranged inside the reactor, the stirring assembly is located inside the centrifugal inner barrel, a water bath is coated on the outside of the reactor, and a boiling water circulation assembly is connected between the heating reactor and the water bath;

[0055] A centrifugal ring is rotatably arranged in the filter cartridge, and two sets of ultrafiltration membranes are arranged on the centrifugal ring, and the pore size of the upper ultrafiltration membrane is larger than that of the lower ultrafiltration membrane. A liquid guide tube is connected between the bottom of the reactor and the top of the filter cartridge, and a partition assembly is arranged on the liquid guide tube;

[0056] A suction tube is connected to the circumferential surface on the back of the filter cartridge, the bottom end of the suction tube is located below the ultrafiltration membrane on the lower side, and the other end of the suction tube is connected to the top of the reactor. The reactor in the present invention is used for the mixed dissolution of bovine bone powder and water, and the ultrasonic enzymatic hydrolysis reaction after adding trypsin, compound protease and flavor protease, as well as the heating and enzyme inactivation and centrifugation to obtain the bovine bone protein hydrolysate are all carried out in the reactor; the heating kettle is used to provide a heat source, and provide heat to the reactor when heating is required; the filter cartridge is used to separate the obtained bovine bone protein hydrolysate into the required three components with molecular weights >3kDa, 1kDa~3kDa and <1kDa; the three major components of the whole system have a simple structure and can well achieve the preparation and screening effect of bovine bone protein hydrolysate with the required molecular weight; the stirring component in the present invention is used to promote the full mixing and dissolution of bovine bone powder and water and the subsequent efficient ultrasonic enzymatic hydrolysis reaction; the centrifugal inner barrel is used for the rapid stratification of the solution after the enzymatic hydrolysis is completed, forming the upper layer of bovine bone protein hydrolysate clear liquid and the impurities at the bottom; the water bath is connected to the heating kettle to provide the required The invention discloses a stable heat source of temperature; the boiling water circulation component is used to circulate the hot water generated in the heating kettle to the water bath, so as to provide a stable heat source of required temperature for the reaction in the heating kettle; the centrifugal ring in the invention provides centrifugal force for the ultrafiltration centrifugal process, and obtains three components with molecular weights>3kDa, 1kDa~3kDa and <1kDa as soon as possible under the ultrafiltration action of two sets of ultrafiltration membranes; the liquid guide tube is used to smoothly transport the bovine bone protein hydrolysate in the centrifugal inner barrel to the filter cylinder for centrifugal ultrafiltration in the next process; the separation component separates the upper bovine bone protein hydrolysate clear liquid and the bottom impurities obtained in the centrifugal inner barrel, first separates and removes the centrifugal impurities at the bottom, so that only the upper bovine bone protein hydrolysate clear liquid that meets the quality requirements can enter the filter cylinder; the suction tube provides negative pressure in the filter cylinder, assists the ultrafiltration centrifugation of the centrifugal ring, and obtains the three components with molecular weights>3kDa, 1kDa~3kDa and <1kDa more quickly.

[0057] Preferably, a feeding port is provided at the top of the reactor, a discharge pipe is connected to the bottom of the reactor, the discharge pipe is rotatably connected to the inside of the centrifugal inner barrel, an air duct is connected to the top of the reactor, the top of the suction pipe is connected to the air duct, a valve 1 is provided on the discharge pipe, and the bottom end of the liquid duct is connected to the discharge pipe; a first centrifugal motor is provided at the bottom of the reactor, and the driving end of the first centrifugal motor passes through the reactor and is connected to the centrifugal inner barrel; a water inlet is provided at the top of the heating kettle, a drain outlet is provided at the bottom of the heating kettle, a heater is fixedly installed inside the heating kettle, and sealing covers are provided at the top of the feeding port, the top of the water inlet and the bottom of the drain outlet. The feeding port is used to add cattle bone powder, water and protease materials into the centrifugal inner barrel; the discharge pipe is used to slowly discharge the layered solution formed after reaction and centrifugation to the separation component, the inlet of the discharge pipe is connected with the inside of the centrifugal inner barrel, the discharge pipe and the bottom of the centrifugal inner barrel are sealed and rotatably connected, during centrifugation, the centrifugal inner barrel is sealed and rotatably connected to the discharge pipe through a sealed bearing, and the centrifugal inner barrel rotates under the driving force of the first centrifugal motor, and the housing of the first centrifugal motor is installed at the bottom of the heating kettle; an ultrasonic generator is installed inside the reactor, and during enzymatic hydrolysis, ultrasound can improve the enzymatic hydrolysis rate to a certain extent, so that the protease can achieve the enzymatic hydrolysis and cutting effect more efficiently; the air guide tube and the suction tube together send the gas in the filter cartridge into the reactor, so that negative pressure is formed in the filter cartridge, which promotes ultrafiltration and forms positive pressure in the reactor. The solution is preferably discharged from the discharge pipe; the discharge of the solution from the discharge pipe can be controlled by controlling the opening and closing of valve one; the liquid guide pipe is used to guide the liquid discharged from the discharge pipe into the separation component; the first centrifugal motor provides centrifugal power for the centrifugal rotation of the centrifugal inner barrel; the water inlet is used to inject water into the heating kettle; the drain outlet is used to discharge the waste water after the final heating is completed from the heating kettle; the heater provides a heat source for the addition of water to the heating kettle; the sealing cover forms a closed environment in the reactor, which is convenient for good control of the pressure change in the reactor; the sealing cover forms a relatively closed environment in the heating kettle, and the top of the heating kettle is connected to an exhaust port to avoid the danger of excessive pressure in the heating kettle during the heating process; pressure gauges and thermometers are connected to the reactor and the heating kettle, which is convenient for real-time understanding and regulation of the temperature and pressure values ​​at the reactor and the heating kettle.

[0058] Preferably, the filter cartridge includes a filter cartridge body, the centrifugal ring is rotatably arranged on the inner wall surface of the filter cartridge body, a filter cartridge cover is arranged on the top of the filter cartridge body, a feed pipe is connected to the bottom of the filter cartridge body, a valve 2 is arranged on the feed pipe, and a connecting pipe 1 is connected to the top of the filter cartridge cover. The filter cartridge body provides a stable nanofiltration environment for the entire nanofiltration process; the centrifugal ring is rotatably arranged on the inner wall of the filter cartridge body through a rotating bearing, and the centrifugal ring can well drive the two groups of nanofiltration membranes to rotate, thereby realizing fast and efficient centrifugation; the filter cartridge cover forms a closed environment inside the entire filter cartridge body, ensuring a stable negative pressure inside the filter cartridge body; the feed pipe is used to export the polypeptide components with a molecular weight <1kDa obtained in the end, for subsequent peptide group identification; by controlling the opening and closing of valve 2, the polypeptide components with a molecular weight <1kDa can be controlled to be discharged from the feed pipe; through the connecting pipe 1, the upper bovine bone protein hydrolysate clear liquid separated by the partition component can smoothly enter the filter cartridge body.

[0059] Preferably, a support ring is fixedly installed at the bottom of the inner wall of the centrifugal ring, a sleeve 1 is inserted in the centrifugal ring, an ultrafiltration membrane 1 is fixedly installed at the bottom of the sleeve 1, the membrane pore size of the ultrafiltration membrane 1 is 1kDa, and the sleeve 1 abuts against the support ring;

[0060] A second sleeve is inserted into the top of the centrifugal ring, and the second sleeve is located in the upper half of the filter cartridge body. An ultrafiltration membrane second is fixedly installed at the bottom of the second sleeve, and the membrane pore size of the second ultrafiltration membrane is 3kDa;

[0061] A second centrifugal motor is provided at the bottom of the filter cartridge body, a rotating shaft is provided at the driving end of the second centrifugal motor, the rotating shaft is rotatably sealed at the bottom of the filter cartridge body, a rotating frame is provided on the rotating shaft, and the rotating frame is fixedly connected to the bottom of the centrifugal ring;

[0062] An air pump is provided on the suction tube, and the bottom end of the suction tube is located below the support ring. The support ring supports the sleeve 1 placed in the centrifugal ring to prevent the sleeve 1 from falling out of the centrifugal ring under a large centrifugal force; the sleeve 1 provides a centrifugal environment for the bovine bone protein hydrolysate with a molecular weight less than 3kDa passing through the ultrafiltration membrane 2, thereby intercepting the bovine bone protein hydrolysate with a molecular weight of 1kDa to 3kDa; the ultrafiltration membrane 1 allows the bovine bone protein hydrolysate with a molecular weight less than 1kDa to pass through, and intercepts the bovine bone protein hydrolysate with a molecular weight of 1kDa to 3kDa; the sleeve 2 provides a centrifugal environment for the bovine bone protein hydrolysate supernatant initially fed in, thereby intercepting the bovine bone protein hydrolysate with a molecular weight greater than 3kDa; the sleeve 2 It is plugged into the top of the centrifugal ring and can rotate centrifugally with the centrifugal ring; the second ultrafiltration membrane allows bovine bone protein hydrolysate with a molecular weight less than 3kDa to pass through, and intercepts bovine bone protein hydrolysate with a molecular weight greater than 3kDa; the second centrifugal motor provides centrifugal power for the centrifugal rotation of the centrifugal ring; the housing of the second centrifugal motor is installed at the bottom of the filter cartridge body, and the rotating shaft is rotatably arranged at the bottom of the filter cartridge body through a rotating sealed bearing, and the rotating frame stably applies the rotating force of the rotating shaft to the centrifugal ring to achieve stable centrifugal rotation of the centrifugal ring; the air pump extracts the gas in the filter cartridge body, so that the filter cartridge body is under negative pressure, which better promotes centrifugal ultrafiltration.

[0063] Preferably, the stirring assembly comprises a motor fixedly mounted at the top center of the reactor, a threaded rod is fixedly mounted at the bottom output end of the motor, the threaded rod is arranged along the axis of the reactor, a sleeve is sleeved on the outer side of the threaded rod, a rotating ring is fixedly mounted on the top outer wall of the sleeve, two groups of guide grooves are opened through the surface of the sleeve, and the guide grooves are arranged along the length direction of the sleeve;

[0064] The threaded rod is threadedly connected with an internal threaded tube, the internal threaded tube is slidably connected in the first sleeve, two groups of connecting pieces are fixedly installed on the outer wall of the internal threaded tube, the two groups of connecting pieces are respectively slidably connected in the guide groove, the outer side of the first sleeve is sleeved with the second sleeve, the connecting piece is fixedly installed on the inner wall of the top end of the second sleeve, and a U-shaped rod is fixedly installed on the outer wall of the bottom end of the second sleeve;

[0065] The outer side of the rotating ring is sleeved with a rotation limiter. The motor provides power for the stirring of the stirring assembly; the threaded rod is used to transmit the stirring force of the motor; the sleeve one can guide the sleeve two, thereby realizing the function of adjusting the U-shaped rod up and down, and the stirring effect of the solution in the reactor can be adjusted by adjusting the U-shaped rod up and down; the rotating ring is used to receive the limiting force of the rotation limiter, thereby realizing the function of adjusting the sleeve two up and down and rotating with the sleeve two at the same time; the two groups of guide grooves are used to realize the stable guidance of the sleeve two, thereby realizing the function of stable adjustment of the U-shaped rod up and down; the internal threaded tube cooperates with the threaded rod to receive the thread rotation force of the threaded rod, thereby realizing stable adjustment of the sleeve two up and down; the two groups of connecting pieces are respectively slidably connected in the guide grooves, realizing the function of stable adjustment and rotation of the sleeve two up and down; the sleeve two moves up and down and rotates with the internal threaded tube, thereby realizing stable adjustment and rotation of the U-shaped rod up and down; the U-shaped rod realizes efficient stirring of the solution in the reactor.

[0066] Preferably, the rotation limiter comprises an annular cover sleeved on the outer side of the swivel, an array of hollow beams are fixed on the outer wall of the annular cover, the other end of the hollow beam is fixedly mounted on the inner wall of the reactor, the hollow beam is connected to the annular cover, a micro cylinder is fixedly mounted on the inner wall of the hollow beam, a friction plate is fixedly mounted on the output end of the micro cylinder, the other side of the friction plate is an arc surface, and the friction plate is attached to the circumferential surface of the swivel. The annular cover limits the swivel so that it can only rotate in the annular cover and will not move downward; the hollow beam supports the sleeve one so that the sleeve one can only rotate and will not fall downward; the hollow beam also provides an installation position for the micro cylinder so that the telescopic end of the micro cylinder can be stably telescoped as needed to achieve locking and unlocking of the swivel; the micro cylinder controls the locking and unlocking of the swivel; the friction plate cooperates with the surface of the swivel to achieve stable locking and unlocking of the swivel.

[0067] Preferably, the boiling water circulation assembly comprises a water supply pipe, a reflux pipe and a water pump, one end of the water supply pipe is connected to the top of the water bath, the other end of the water supply pipe is connected to the bottom of the heating kettle, and the water pump is arranged on the water supply pipe;

[0068] One end of the reflux pipe is connected to the bottom of the water bath, and the other end of the reflux pipe is connected to the bottom of the side of the heating kettle. Under the action of the water pump, the water supply pipe delivers the heated water in the heating kettle into the water bath; the reflux pipe returns the waste water heated in the water bath to the heating kettle; the water pump provides power to deliver the heated water in the heating kettle into the water bath.

[0069] Preferably, the reflux pipe comprises a second connecting pipe, a cladding pipe and a third connecting pipe, wherein the cladding pipe is L-shaped, the cladding pipe is clad on the outside of the water supply pipe, one end of the second connecting pipe is connected to the bottom of the water bath, and the other end of the second connecting pipe is connected to the right side of the horizontal part of the cladding pipe;

[0070] One end of the connecting pipe 3 is connected to the side of the top of the cladding pipe, and the other end of the connecting pipe 3 is connected to the upper part of one side of the heating kettle. The cladding pipe covers the outside of the water supply pipe to prevent the water supply pipe from directly contacting the outside air and excessive heat loss. Under the action of the reflux water, the water temperature in the water supply pipe is maintained constant; the connecting pipe 2 re-introduces the waste water in the water bath into the cladding pipe; the connecting pipe 3 guides the waste water in the cladding pipe into the heating kettle.

[0071] Preferably, the partition assembly comprises a transverse tube connected to the top end of the liquid guide tube, the liquid guide tube and the transverse tube are perpendicular to each other, a slag discharge pipe is connected to the front end of the transverse tube, and a connecting pipe is connected to the rear end of the transverse tube;

[0072] The transverse cylinder is slidably connected with a sealing column, and arc plates are fixedly installed at the bottom of both ends of the sealing column, and a pusher for driving the sealing column to move forward and backward is arranged on the transverse cylinder. The transverse cylinder provides a separation environment for the solution sent from the reactor; the slag discharge pipe is used to discharge the residual solution at the bottom of the reactor after centrifugal separation; the connecting pipe 1 introduces the upper layer of bovine bone protein hydrolysate clear liquid remaining after the residual solution at the bottom of the reactor is discharged into the filter cylinder; the sealing column slides left and right in the transverse cylinder to form a slag discharge channel and a clear liquid channel, so that the residual solution at the bottom of the reactor after centrifugal separation and the upper layer of bovine bone protein hydrolysate clear liquid enter the corresponding channels respectively; the arc plates fixedly installed at the bottom of both ends of the sealing column slide and match with the transverse cylinder, and the chute at the bottom of the transverse cylinder is closed in real time to prevent the solution from leaking out; the pusher can drive the sealing column to slide in the transverse cylinder as needed, thereby forming a slag discharge channel and a clear liquid channel respectively.

[0073] Preferably, the pusher comprises a sliding column fixedly mounted at the bottom of the sealing column, a sliding groove is formed through the bottom of the transverse cylinder, the sliding groove is arranged along the length direction of the transverse cylinder, the sliding column is slidably connected in the sliding groove, and the arc plate is attached to the inner wall of the transverse cylinder and seals the sliding groove;

[0074] A slider is fixedly installed at the bottom of the slide column, and support seats are fixedly installed at the bottom of both ends of the transverse tube. A threaded rod is rotatably connected between the two support seats, and a threaded hole is opened on the slider, and the slider is threadedly connected to the threaded rod. The slide column matches the slide groove and slides back and forth in the slide groove, thereby driving the sealing column to slide left and right in the transverse tube to form a slag discharge channel and a clear liquid channel; the slider matches the threaded rod, and under the forward and reverse rotation of the threaded rod, the slider drives the slider to slide left and right, thereby driving the sealing column to slide left and right in the transverse tube to form a slag discharge channel and a clear liquid channel; a motor can be set separately to automatically control the forward and reverse rotation of the threaded rod, or the staff can manually adjust and rotate the threaded rod according to needs.

[0075] The present invention has the following beneficial effects:

[0076] (1) It is composed of 5 amino acids and is a short peptide with a molecular weight of less than 500 Da, which is more easily absorbed and utilized by the human body;

[0077] (2) It has good saltiness and umami taste, low bitterness, and can be used to partially replace sodium chloride in food without affecting the sensory quality of the food;

[0078] (3) mixing the bovine bone meal with water and pre-treating the mixture so that the bovine bone meal can be fully dissolved in water, which is convenient for subsequent enzymatic hydrolysis; adding trypsin, compound protease and flavor protease to the pre-treated solution for ultrasonic enzymatic hydrolysis, so that the protein in the bovine bone meal can be fully enzymatically hydrolyzed into peptide molecules, which is convenient for screening out small molecule peptides that meet the requirements, have a small molecular weight, good saltiness and umami taste, low bitterness, and can be used to partially replace sodium chloride in food; heating the solution after the ultrasonic enzymatic hydrolysis reaction to inactivate the enzyme, so as to prevent the enzyme from affecting the purity and quality of the peptides obtained by subsequent separation;

[0079] (4) Centrifuging the enzyme-killed solution, and using a physical method to quickly separate the components of different densities, so that the required supernatant components can be obtained more quickly; the present invention centrifuges and ultrafilters the bovine bone protein hydrolysate, and passes it through 3kDa and 1kDa ultrafiltration membranes in turn, and relatively quickly obtains three components with molecular weights >3kDa, 1kDa-3kDa and <1kDa, respectively, thereby narrowing the screening range and quickly and accurately obtaining the required small molecule dipeptide; taking the component with a molecular weight <1kDa for gel permeation chromatography separation and purification, further improving the purity of the component, and further avoiding interference from impurities; obtaining a peak group of the purified product at a characteristic wavelength of 280nm, identifying the amino acid sequence of the corresponding components corresponding to the peak group, and performing a preliminary screening based on the C-terminal amino acid composition of the peptide, and then molecularly docking the peptides screened out with the salty taste receptor, and obtaining the salty and fresh peptide RRRRR according to the binding energy index, and combining computer simulation screening technology, while saving time and effort, quickly and accurately obtaining the required salty and fresh peptide;

[0080] (5) The overall method of using three proteases for simultaneous enzymatic hydrolysis is different from the traditional continuous hydrolysis method, which has the advantages of being fast, efficient, convenient and economical. In addition, the method uses cattle by-products as raw materials, which has low production costs and high economic benefits, and is suitable for industrial production;

[0081] (6) It can be prepared from the enzymatic hydrolysate of bovine bones, or obtained in large quantities by solid phase synthesis, which is relatively easy to obtain; the entire preparation and screening process has simple operation steps, combined with computer simulation screening, does not rely on complex technology, and can save a lot of manpower and material resources;

[0082] (7) The reaction kettle is used for mixed dissolution of bovine bone powder and water, ultrasonic enzymatic hydrolysis after adding trypsin, compound protease and flavor protease, and heating enzyme inactivation and centrifugation to obtain bovine bone protein hydrolysate are all carried out in the reaction kettle; the heating kettle is used to provide a heat source, and heat is provided to the reaction kettle when heating is required; the filter cartridge is used to separate the obtained bovine bone protein hydrolysate into three components with required molecular weights >3kDa, 1kDa to 3kDa and <1kDa; the whole system has three major components, with a simple structure, and can achieve good preparation and screening effects of bovine bone protein hydrolysate with required molecular weights;

[0083] (8) A stirring assembly is used to promote the full mixing and dissolution of the bovine bone powder and water and the subsequent efficient ultrasonic enzymatic hydrolysis reaction; a centrifugal inner barrel is used to quickly stratify the solution after the enzymatic hydrolysis is completed, forming an upper layer of bovine bone protein hydrolysate clear liquid and impurities at the bottom; a water bath is connected to the heating kettle to provide a stable heat source at a required temperature for the heating kettle; a boiling water circulation assembly is used to circulate the hot water generated in the heating kettle to the water bath to provide a stable heat source at a required temperature for the reaction in the heating kettle;

[0084] (9) The centrifugal ring provides centrifugal force for the ultrafiltration centrifugal process, and the three components with molecular weights >3kDa, 1kDa~3kDa and <1kDa are obtained as quickly as possible under the ultrafiltration action of two sets of ultrafiltration membranes; the liquid guide tube is used to smoothly transport the bovine bone protein hydrolysate in the centrifugal inner barrel to the filter cartridge for the next step of centrifugal ultrafiltration; the separation component separates the upper bovine bone protein hydrolysate clear liquid and the bottom impurities obtained in the centrifugal inner barrel, first separating and removing the bottom centrifugal impurities, so that only the upper bovine bone protein hydrolysate clear liquid that meets the quality requirements can enter the filter cartridge; the suction tube provides negative pressure in the filter cartridge to assist the ultrafiltration centrifugation of the centrifugal ring, and more quickly obtains the three components with molecular weights >3kDa, 1kDa~3kDa and <1kDa. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 The 3D structure diagram of the salty peptide Arg-Arg-Arg-Arg-Arg (RRRRR) of the present invention;

[0086] Figure 2 This is the mass spectrum of the salty and fresh peptide Arg-Arg-Arg-Arg-Arg (RRRRR) in Example 4 of the present invention;

[0087] Figure 3 This is the electronic tongue taste characteristic diagram of the salty and fresh peptide Arg-Arg-Arg-Arg-Arg (RRRRR) in the experimental example of the present invention;

[0088] Figure 4 This is the electronic tongue taste characteristic diagram of the standard product (Reference) in the comparative example of the present invention;

[0089] Figure 5 It is a first structural schematic diagram of the system in the present invention;

[0090] Figure 6 It is a second structural schematic diagram of the system in the present invention;

[0091] Figure 7 It is a structural schematic diagram of the water bath of the present invention;

[0092] Figure 8 It is a structural schematic diagram of the U-shaped rod of the present invention;

[0093] Fig. 9 It is a third structural schematic diagram of the system in the present invention;

[0094] Fig.10 It is a fourth structural schematic diagram of the system in the present invention;

[0095] Fig.11 It is a structural schematic diagram of the separation component of the present invention;

[0096] Fig.12 It is a schematic diagram of the structure of the embedded ring of the present invention;

[0097] Fig.13 It is a structural schematic diagram of the sealing column of the present invention;

[0098] Fig.14 It is a structural schematic diagram of the threaded rod of the present invention;

[0099] Fig.15 It is a structural schematic diagram of the hollow beam of the present invention;

[0100] Fig.16 It is a structural schematic diagram of the internal threaded pipe of the present invention.

[0101] The markings in the attached drawings are: 100-reaction kettle; 200-heating kettle; 300-filter cartridge; 400-stirring assembly; 500-water bath; 600-boiling water circulation assembly; 700-liquid guide tube; 800-separation assembly; 900-suction tube; 101-feeding port; 102-discharge pipe; 103-air guide pipe; 104-valve 1; 201-water inlet; 202-drainage port; 203-heater; 301-filter cartridge body; 302-filter cartridge cover; 303-discharge pipe; 304-valve 2; 305-connecting pipe 1; 306-embedded ring; 307-support ring; 308-sleeve 1; 309-ultrafiltration membrane 1; 310-sleeve 2; 311-ultrafiltration membrane 2; 401-motor; 402-threaded rod; 403-sleeve one; 404-swivel; 405-guide groove; 406-internal threaded tube; 407-connecting plate; 408-sleeve two; 409-U-shaped rod; 410-annular cover; 411-hollow beam; 412-micro cylinder; 413-friction plate; 601-water supply pipe; 602-return pipe; 603-water pump; 6021-connecting pipe two; 6022-cladding pipe; 6023-connecting pipe three; 801-transverse cylinder; 802-slag discharge pipe; 803-chute; 804-sealing column; 805-arc plate; 806-sliding column; 807-sliding block; 808-support seat; 809-threaded rod; 901-air pump. DETAILED DESCRIPTION

[0102] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0103] like Figure 1 The salty and fresh peptide with the function of increasing saltiness and freshness shown is RRRRR, and the amino acid sequence of the salty and fresh peptide is

[0104] Arg-Arg-Arg-Arg-Arg; the molecular formula of salty peptide is C 30 H 62 N 20 O6; the molecular structure of salty peptide is

[0105]

[0106] The method for screening salty and fresh peptides having the function of increasing saltiness and freshness comprises the following steps:

[0107] (S01) taking an appropriate amount of cattle bone powder and mixing with water, and pre-treating; the cattle bone powder in step (S01) is yak bone powder; the method for obtaining the cattle bone powder in step (S01) is to take cattle bones, wash and roughly chop, high-temperature steam, dry, crush and sieve in sequence to obtain cattle bone powder; the solid-liquid ratio of cattle bone powder to water in step (S01) is 1:15-25; sieving is sieving through a 150-mesh sieve; the pre-treatment in step (S01) is cooling to room temperature after boiling water bath; the boiling water bath time is 20min-60min;

[0108] (S02) adding trypsin, composite protease and flavor protease to the solution pretreated in step (S01) to carry out ultrasonic enzymatic hydrolysis reaction; the amount of trypsin added in step (S02) is 8000U / g substrate mass to 14000U / g substrate mass; the amount of composite protease added in step (S02) relative to the solution is 15% to 18%; the amount of flavor protease added in step (S02) relative to the solution is 20% to 25%; the enzymatic hydrolysis in step (S02) is heated water bath enzymatic hydrolysis; the enzymatic hydrolysis temperature in step (S02) is 40°C to 45°C; the enzymatic hydrolysis time in step (S02) is 4h to 6h;

[0109] (S03) after the ultrasonic enzymatic hydrolysis reaction in step (S02) is completed, the temperature is raised to inactivate the enzyme; the temperature-raising enzyme inactivation in step (S03) is inactivation in a boiling water bath; the enzyme inactivation time in step (S03) is 5 min to 15 min;

[0110] (S04) centrifuging the solution after the enzyme inactivation in step (S03) to obtain the supernatant to obtain a bovine bone protein hydrolysate; the centrifugal speed in step (S04) is 4000 r / min to 4500 r / min; the centrifugal temperature in step (S04) is room temperature; the centrifugal time in step (S04) is 25 min to 35 min;

[0111] (S05) centrifuging and ultrafiltration the bovine bone protein hydrolysate from step (S04), and passing through 3 kDa and 1 kDa ultrafiltration membranes in sequence to obtain three components with molecular weights >3 kDa, 1 kDa to 3 kDa, and <1 kDa;

[0112] (S06) taking an appropriate amount of the component with a molecular weight of less than 1 kDa in step (S05) for gel permeation chromatography separation and purification, and obtaining a peak group of the purified product at a characteristic wavelength of 280 nm; the conditions for gel permeation chromatography separation and purification in step (S06) are as follows: the protein purifier is equipped with a Sephadex G-15 dextran gel column, and the mobile phase is ultrapure water; the concentration of the component in step (S06) is 10 mg / mL to 100 mg / mL; the loading amount of the component in step (S06) is 5 mL to 8 mL; the elution rate of the component in step (S06) is 1 mL / min to 4 mL / min; there are three peak groups in step (S06);

[0113] (S07) performing amino acid sequence identification on the corresponding components corresponding to the peak group in step (S06), and performing preliminary screening by the C-terminal amino acid composition of the peptide; the amino acid sequence identification in step (S07) adopts UPLC-Q-TOF MS system;

[0114] The parameters of the UPLC-Q-TOF MS system are:

[0115] HILIC column, column temperature 25°C; flow rate 0.3 mL / min; injection volume 2 μL;

[0116] Mobile phase A, water, 25 mM ammonium acetate, and 25 mM ammonia;

[0117] Mobile phase B, acetonitrile;

[0118] The gradient elution program was as follows: 0-1 min, 95% mobile phase B; 1 min-14 min, mobile phase B linearly changed from 95% to 65%; 14 min-16 min, mobile phase B linearly changed from 65% to 40%; 16 min-18 min, mobile phase B maintained at 40%; 18 min-18.1 min, mobile phase B linearly changed from 40% to 95%; 18.1 min-23 min, mobile phase B maintained at 95%;

[0119] After separation on a HILIC column, mass spectrometry analysis was performed using electrospray ionization positive ion and electrospray ionization negative ion modes;

[0120] The scanning range of TOF MS is 60Da to 1000Da; the scanning accumulation time of TOF MS is 0.20s / spectra;

[0121] The product ion scanning range of TOF MS is 25Da to 1000Da; the product ion scanning accumulation time of TOF MS is 0.05s / spectra;

[0122] In step (S07), the C-terminal amino acid composition of the peptide is initially screened to contain four amino acids: glutamic acid Glu (E), aspartic acid Asp (D), arginine Pro (R) and lysine Lys (K);

[0123] (S08) Molecular docking is performed between the peptides initially screened out in step (S07) and the salty taste receptor, and a salty and fresh peptide RRRRR is obtained according to the binding energy index; the salty taste receptor in step (S08) is constructed by AlphaFold; the salty taste receptor in step (S08) is derived from the UniProtKB database, and the salty taste receptor is numbered as Q7Z404-TMC4-HUMAN; the salty taste receptor in step (S08) is transmembrane channel-like protein 4; the molecular docking method in step (S08) is the CDOCKER program in the Discovery Studio software, with -CDOCKER_ENERGY as the index, the docking ligand is a dipeptide, the docking receptor is TMC4, and the docking force field is CHARMm;

[0124] The docking pocket is x: 17.7065, y: 11.7845, z: -14.9743, and the radius is 19.7; its binding energy for docking with TMC4 is -89.7093 kcal / mol.

[0125] like Figure 5 and Figure 6 The salty and fresh peptide screening system with saltiness and freshness enhancing function shown in the figure comprises: Figure 7 The reactor 100, the heating reactor 200 and the filter cartridge 300 are shown; a stirring assembly 400 is arranged inside the reactor 100, a centrifugal inner barrel is arranged inside the reactor 100, and the stirring assembly 400 is located inside the centrifugal inner barrel. Fig. 9 The water bath 500 shown in the figure is connected to the heating kettle 200 and the water bath 500. Fig.10 The boiling water circulation assembly 600 is shown;

[0126] The filter cartridge 300 is provided with a rotating Fig.12 The centrifugal ring 306 shown in the figure has two sets of ultrafiltration membranes, the pore size of the upper ultrafiltration membrane is larger than the pore size of the lower ultrafiltration membrane, and a liquid guide tube 700 is connected between the bottom of the reactor 100 and the top of the filter cartridge 300, and the liquid guide tube 700 is provided with Fig.11 The partition assembly 800 shown;

[0127] A suction pipe 900 is connected to the circumferential surface of the back of the filter cartridge 300 , the bottom end of the suction pipe 900 is located below the ultrafiltration membrane on the lower side, and the other end of the suction pipe 900 is connected to the top of the reaction kettle 100 .

[0128] A feeding port 101 is provided at the top of the reactor 100, a discharge pipe 102 is connected to the bottom of the reactor 100, the discharge pipe 102 is connected to the inside of the centrifugal inner barrel for rotation, an air guide pipe 103 is connected to the top of the reactor 100, the top of the suction pipe 900 is connected to the air guide pipe 103, a valve 104 is provided on the discharge pipe 102, and the bottom end of the liquid guide pipe 700 is connected to the discharge pipe 102; a first centrifugal motor is provided at the bottom of the reactor 100, and the driving end of the first centrifugal motor passes through the reactor 100 and is connected to the centrifugal inner barrel; a water inlet 201 is provided at the top of the heating kettle 200, a drain port 202 is provided at the bottom of the heating kettle 200, a heater 203 is fixedly installed inside the heating kettle 200, and sealing covers are provided at the top of the feeding port 101, the top of the water inlet 201 and the bottom of the drain port 202.

[0129] The filter cartridge 300 includes a filter cartridge body 301, a centrifugal ring 306 rotatably arranged on the inner wall surface of the filter cartridge body 301, a filter cartridge cover 302 is arranged on the top of the filter cartridge body 301, a feed pipe 303 is connected to the bottom of the filter cartridge body 301, a valve 2 304 is arranged on the feed pipe 303, and a connecting pipe 1 305 is connected to the top of the filter cartridge cover 302. A support ring 307 is fixedly installed at the bottom of the inner wall of the centrifugal ring 306, a sleeve 308 is inserted in the centrifugal ring 306, an ultrafiltration membrane 309 is fixedly installed at the bottom of the sleeve 308, the membrane pore size of the ultrafiltration membrane 309 is 1kDa, and the sleeve 308 abuts on the support ring 307; a sleeve 2 310 is inserted at the top of the centrifugal ring 306, the sleeve 2 310 is located in the upper half of the filter cartridge body 301, and an ultrafiltration membrane 2 311 is fixedly installed at the bottom of the sleeve 2 310, and the membrane pore size of the ultrafiltration membrane 2 311 is 3kDa; a second centrifugal motor is provided at the bottom of the filter cartridge body 301, and a rotating shaft is provided at the driving end of the second centrifugal motor, and the rotating shaft is rotatably sealed at the bottom of the filter cartridge body 301, and a rotating frame is provided on the rotating shaft, and the rotating frame is fixedly connected to the bottom of the centrifugal ring 306; an air pump 901 is provided on the suction pipe 900, and the bottom end of the suction pipe 900 is located below the support ring 307.

[0130] The stirring assembly 400 includes a motor 401 fixedly mounted at the top center of the reactor 100, and a Fig.14 The threaded rod 402 is arranged along the axis of the reactor 100, and the outer side of the threaded rod 402 is sleeved with a Fig.15 The sleeve 403 shown in the figure has a swivel 404 fixedly mounted on the outer wall of the top of the sleeve 403. Two sets of guide grooves 405 are formed on the surface of the sleeve 403. The guide grooves 405 are arranged along the length direction of the sleeve 403. The threaded connection on the threaded rod 402 is as shown in the figure. Fig.16The internal threaded tube 406 is slidably connected in the sleeve 1 403, and two sets of connecting pieces 407 are fixedly installed on the outer wall of the internal threaded tube 406. The two sets of connecting pieces 407 are respectively slidably connected in the guide groove 405. The outer sleeve 1 403 is sleeved with a sleeve 2 408, and the connecting piece 407 is fixedly installed on the inner wall of the top end of the sleeve 2 408. The outer wall of the bottom end of the sleeve 2 408 is fixedly installed as shown in FIG. Figure 8 The U-shaped rod 409 shown in the figure is provided with a rotation limiter on the outer side of the rotating ring 404.

[0131] The rotation limiter includes an annular cover 410 sleeved on the outside of the rotating ring 404, and hollow beams 411 are fixed in an array on the outer wall of the annular cover 410. The other end of the hollow beams 411 is fixedly installed on the inner wall of the reactor 100. The hollow beams 411 are connected to the annular cover 410, and a micro cylinder 412 is fixedly installed on the inner wall of the hollow beam 411. A friction plate 413 is fixedly installed on the output end of the micro cylinder 412, and the other side of the friction plate 413 is an arc surface, and the friction plate 413 is attached to the circumferential surface of the rotating ring 404.

[0132] The boiling water circulation component 600 includes a water supply pipe 601, a return pipe 602 and a water pump 603. One end of the water supply pipe 601 is connected to the top of the water bath 500, and the other end of the water supply pipe 601 is connected to the bottom of the heating kettle 200. The water pump 603 is arranged on the water supply pipe 601; one end of the return pipe 602 is connected to the bottom of the water bath 500, and the other end of the return pipe 602 is connected to the upper side of the heating kettle 200. The reflux pipe 602 includes a connecting pipe 2 6021, a wrapping pipe 6022 and a connecting pipe 3 6023. The wrapping pipe 6022 is L-shaped and wraps around the outside of the water supply pipe 601. One end of the connecting pipe 2 6021 is connected to the bottom of the water bath 500, and the other end of the connecting pipe 2 6021 is connected to the right side of the horizontal part of the wrapping pipe 6022; one end of the connecting pipe 3 6023 is connected to the top side of the wrapping pipe 6022, and the other end of the connecting pipe 3 6023 is connected to the upper part of one side of the heating kettle 200.

[0133] The partition assembly 800 includes a Fig.13The transverse cylinder 801 shown in the figure, the liquid guiding tube 700 and the transverse cylinder 801 are perpendicular to each other, the front end of the transverse cylinder 801 is connected to the slag discharge pipe 802, and the rear end of the transverse cylinder 801 is connected to the connecting pipe 305; the interior of the transverse cylinder 801 is slidably connected with a sealing column 804, and the bottom of the front and rear ends of the sealing column 804 are fixedly installed with an arc plate 805, and the transverse cylinder 801 is provided with a pushing member for driving the sealing column 804 to move forward and backward. The pusher includes a sliding column 806 fixedly installed at the bottom of the sealing column 804, a sliding groove 803 is opened through the bottom of the transverse cylinder 801, and the sliding groove 803 is arranged along the length direction of the transverse cylinder 801. The sliding column 806 is slidably connected in the sliding groove 803, and the arc plate 805 fits on the inner wall of the transverse cylinder 801 and seals the sliding groove 803; a sliding block 807 is fixedly installed at the bottom of the sliding column 806, and support seats 808 are fixedly installed at the bottom of both ends of the transverse cylinder 801. A threaded rod 809 is rotatably connected between the two support seats 808, and a threaded hole is opened on the sliding block 807, and the sliding block 807 is threadedly connected to the threaded rod 809.

[0134] Embodiment 1:

[0135] The method for screening salty and fresh peptides having the function of increasing saltiness and freshness comprises the following steps:

[0136] (S01) taking an appropriate amount of cattle bone powder and mixing with water, and pre-treating; the cattle bone powder in step (S01) is yak bone powder; the method for obtaining the cattle bone powder in step (S01) is to take cattle bones, wash and roughly chop, high-temperature steam, dry, crush and sieve in sequence to obtain cattle bone powder; the solid-liquid ratio of cattle bone powder to water in step (S01) is 1:15; sieving is sieving through a 150-mesh sieve; the pre-treatment in step (S01) is cooling to room temperature after boiling water bath; the boiling water bath time is 20 minutes;

[0137] (S02) adding trypsin, composite protease and flavor protease to the solution pretreated in step (S01) to carry out ultrasonic enzymatic hydrolysis reaction; the amount of trypsin added in step (S02) is 8000U / g substrate mass; the amount of composite protease added in step (S02) relative to the solution is 15%; the amount of flavor protease added in step (S02) relative to the solution is 20%; the enzymatic hydrolysis in step (S02) is heated water bath enzymatic hydrolysis; the enzymatic hydrolysis temperature in step (S02) is 40°C; the enzymatic hydrolysis time in step (S02) is 4h;

[0138] (S03) after the ultrasonic enzymatic hydrolysis reaction in step (S02) is completed, the temperature is raised to inactivate the enzyme; the temperature-raising enzyme inactivation in step (S03) is inactivation in a boiling water bath; the enzyme inactivation time in step (S03) is 5 minutes;

[0139] (S04) centrifuging the solution after the enzyme inactivation in step (S03) to obtain the supernatant to obtain a bovine bone protein hydrolysate; the centrifugal speed in step (S04) is 4000 r / min; the centrifugal temperature in step (S04) is room temperature; the centrifugal time in step (S04) is 25 min;

[0140] (S05) centrifuging and ultrafiltration the bovine bone protein hydrolysate from step (S04), and passing through 3 kDa and 1 kDa ultrafiltration membranes in sequence to obtain three components with molecular weights >3 kDa, 1 kDa to 3 kDa, and <1 kDa;

[0141] (S06) taking an appropriate amount of the component with a molecular weight <1 kDa in step (S05) for gel permeation chromatography separation and purification, and obtaining a peak group of the purified product at a characteristic wavelength of 280 nm; the conditions for gel permeation chromatography separation and purification in step (S06) are as follows: the protein purifier is equipped with a Sephadex G-15 dextran gel column, and the mobile phase is ultrapure water; the concentration of the component in step (S06) is 10 mg / mL; the loading amount of the component in step (S06) is 5 mL; the elution rate of the component in step (S06) is 1 mL / min; there are three peak groups in step (S06);

[0142] (S07) performing amino acid sequence identification on the corresponding components corresponding to the peak group in step (S06), and performing preliminary screening by the C-terminal amino acid composition of the peptide; the amino acid sequence identification in step (S07) adopts UPLC-Q-TOF MS system;

[0143] The parameters of the UPLC-Q-TOF MS system are:

[0144] HILIC column, column temperature 25°C; flow rate 0.3 mL / min; injection volume 2 μL;

[0145] Mobile phase A, water, 25 mM ammonium acetate, and 25 mM ammonia;

[0146] Mobile phase B, acetonitrile;

[0147] The gradient elution program was as follows: 0-1 min, 95% mobile phase B; 1 min-14 min, mobile phase B linearly changed from 95% to 65%; 14 min-16 min, mobile phase B linearly changed from 65% to 40%; 16 min-18 min, mobile phase B maintained at 40%; 18 min-18.1 min, mobile phase B linearly changed from 40% to 95%; 18.1 min-23 min, mobile phase B maintained at 95%;

[0148] After separation on a HILIC column, mass spectrometry analysis was performed using electrospray ionization positive ion and electrospray ionization negative ion modes;

[0149] The scanning range of TOF MS is 60Da to 1000Da; the scanning accumulation time of TOF MS is 0.20s / spectra;

[0150] The product ion scanning range of TOF MS is 25Da to 1000Da; the product ion scanning accumulation time of TOF MS is 0.05s / spectra;

[0151] In step (S07), the C-terminal amino acid composition of the peptide is initially screened for peptides containing glutamic acid, aspartic acid, arginine and lysine;

[0152] (S08) Molecular docking is performed between the peptides initially screened out in step (S07) and the salty taste receptor, and a salty and fresh peptide RRRRR is obtained according to the binding energy index; the salty taste receptor in step (S08) is constructed by AlphaFold; the salty taste receptor in step (S08) is derived from the UniProtKB database, and the salty taste receptor is numbered as Q7Z404-TMC4-HUMAN; the salty taste receptor in step (S08) is transmembrane channel-like protein 4; the molecular docking method in step (S08) is the CDOCKER program in the Discovery Studio software, with -CDOCKER_ENERGY as the index, the docking ligand is a dipeptide, the docking receptor is TMC4, and the docking force field is CHARMm;

[0153] The docking pocket is, x:17.7065, y:11.7845, z:-14.9743, with a radius of 19.7.

[0154] Embodiment 2:

[0155] The method for screening salty and fresh peptides having the function of increasing saltiness and freshness comprises the following steps:

[0156] (S01) taking an appropriate amount of cattle bone powder and mixing with water, and pre-treating; the cattle bone powder in step (S01) is yak bone powder; the method for obtaining the cattle bone powder in step (S01) is to take cattle bones, wash and roughly chop, high-temperature cook, dry, crush and sieve in sequence to obtain cattle bone powder; the solid-liquid ratio of cattle bone powder to water in step (S01) is 1:25; sieving is sieving through a 250-mesh sieve; the pre-treatment in step (S01) is cooling to room temperature after boiling water bath; the boiling water bath time is 60 minutes;

[0157] (S02) adding trypsin, composite protease and flavor protease to the solution pretreated in step (S01) to carry out ultrasonic enzymatic hydrolysis reaction; the amount of trypsin added in step (S02) is 14000U / g substrate mass; the amount of composite protease added in step (S02) relative to the solution is 18%; the amount of flavor protease added in step (S02) relative to the solution is 25%; the enzymatic hydrolysis in step (S02) is heated water bath enzymatic hydrolysis; the enzymatic hydrolysis temperature in step (S02) is 45°C; the enzymatic hydrolysis time in step (S02) is 6h;

[0158] (S03) after the ultrasonic enzymatic hydrolysis reaction in step (S02) is completed, the temperature is raised to inactivate the enzyme; the temperature-raising enzyme inactivation in step (S03) is inactivation in a boiling water bath; the enzyme inactivation time in step (S03) is 15 minutes;

[0159] (S04) centrifuging the solution after the enzyme inactivation in step (S03) to obtain the supernatant to obtain a bovine bone protein hydrolysate; the centrifugal speed in step (S04) is 4500 r / min; the centrifugal temperature in step (S04) is room temperature; the centrifugal time in step (S04) is 35 min;

[0160] (S05) centrifuging and ultrafiltration the bovine bone protein hydrolysate from step (S04), and passing through 3 kDa and 1 kDa ultrafiltration membranes in sequence to obtain three components with molecular weights >3 kDa, 1 kDa to 3 kDa, and <1 kDa;

[0161] (S06) taking an appropriate amount of the component with a molecular weight <1 kDa in step (S05) for gel permeation chromatography separation and purification, and obtaining a peak group of the purified product at a characteristic wavelength of 280 nm; the conditions for gel permeation chromatography separation and purification in step (S06) are as follows: the protein purifier is equipped with a Sephadex G-15 dextran gel column, and the mobile phase is ultrapure water; the concentration of the component in step (S06) is 100 mg / mL; the loading amount of the component in step (S06) is 8 mL; the elution rate of the component in step (S06) is 4 mL / min; there are three peak groups in step (S06);

[0162] (S07) performing amino acid sequence identification on the corresponding components corresponding to the peak group in step (S06), and performing preliminary screening by the C-terminal amino acid composition of the peptide; the amino acid sequence identification in step (S07) adopts UPLC-Q-TOF MS system;

[0163] The parameters of the UPLC-Q-TOF MS system are:

[0164] HILIC column, column temperature 25°C; flow rate 0.3 mL / min; injection volume 2 μL;

[0165] Mobile phase A, water, 25 mM ammonium acetate, and 25 mM ammonia;

[0166] Mobile phase B, acetonitrile;

[0167] The gradient elution program was as follows: 0-1 min, 95% mobile phase B; 1 min-14 min, mobile phase B linearly changed from 95% to 65%; 14 min-16 min, mobile phase B linearly changed from 65% to 40%; 16 min-18 min, mobile phase B maintained at 40%; 18 min-18.1 min, mobile phase B linearly changed from 40% to 95%; 18.1 min-23 min, mobile phase B maintained at 95%;

[0168] After separation on a HILIC column, mass spectrometry analysis was performed using electrospray ionization positive ion and electrospray ionization negative ion modes;

[0169] The scanning range of TOF MS is 60Da to 1000Da; the scanning accumulation time of TOF MS is 0.20s / spectra;

[0170] The product ion scanning range of TOF MS is 25Da to 1000Da; the product ion scanning accumulation time of TOF MS is 0.05s / spectra;

[0171] In step (S07), the C-terminal amino acid composition of the peptide is initially screened for peptides containing glutamic acid, aspartic acid, arginine and lysine;

[0172] (S08) Molecular docking is performed between the peptides initially screened out in step (S07) and the salty taste receptor, and a salty and fresh peptide RRRRR is obtained according to the binding energy index; the salty taste receptor in step (S08) is constructed by AlphaFold; the salty taste receptor in step (S08) is derived from the UniProtKB database, and the salty taste receptor is numbered as Q7Z404-TMC4-HUMAN; the salty taste receptor in step (S08) is transmembrane channel-like protein 4; the molecular docking method in step (S08) is the CDOCKER program in the Discovery Studio software, with -CDOCKER_ENERGY as the index, the docking ligand is a dipeptide, the docking receptor is TMC4, and the docking force field is CHARMm;

[0173] The docking pocket is, x:17.7065, y:11.7845, z:-14.9743, with a radius of 19.7.

[0174] Embodiment 3:

[0175] The method for screening salty and fresh peptides having the function of increasing saltiness and freshness comprises the following steps:

[0176] (S01) taking an appropriate amount of cattle bone powder and mixing with water, and pre-treating; the cattle bone powder in step (S01) is yak bone powder; the method for obtaining the cattle bone powder in step (S01) is to take cattle bones, wash and roughly chop, high-temperature cook, dry, crush and sieve in sequence to obtain cattle bone powder; the solid-liquid ratio of cattle bone powder to water in step (S01) is 1:20; sieving is sieving through a 200-mesh sieve; the pre-treatment in step (S01) is cooling to room temperature after boiling water bath; the boiling water bath time is 40 minutes;

[0177] (S02) adding trypsin, composite protease and flavor protease to the solution pretreated in step (S01) to carry out ultrasonic enzymatic hydrolysis reaction; the amount of trypsin added in step (S02) is 10000U / g substrate mass; the amount of composite protease added in step (S02) relative to the solution is 16%; the amount of flavor protease added in step (S02) relative to the solution is 22%; the enzymatic hydrolysis in step (S02) is heated water bath enzymatic hydrolysis; the enzymatic hydrolysis temperature in step (S02) is 42°C; the enzymatic hydrolysis time in step (S02) is 5h;

[0178] (S03) after the ultrasonic enzymatic hydrolysis reaction in step (S02) is completed, the temperature is raised to inactivate the enzyme; the temperature-raising enzyme inactivation in step (S03) is inactivation in a boiling water bath; the enzyme inactivation time in step (S03) is 10 minutes;

[0179] (S04) centrifuging the solution after the enzyme inactivation in step (S03) to obtain the supernatant to obtain a bovine bone protein hydrolysate; the centrifugal speed in step (S04) is 4200 r / min; the centrifugal temperature in step (S04) is room temperature; the centrifugal time in step (S04) is 30 min;

[0180] (S05) centrifuging and ultrafiltration the bovine bone protein hydrolysate from step (S04), and passing through 3 kDa and 1 kDa ultrafiltration membranes in sequence to obtain three components with molecular weights >3 kDa, 1 kDa to 3 kDa, and <1 kDa;

[0181] (S06) taking an appropriate amount of the component with a molecular weight <1 kDa in step (S05) for gel permeation chromatography separation and purification, and obtaining a peak group of the purified product at a characteristic wavelength of 280 nm; the conditions for gel permeation chromatography separation and purification in step (S06) are as follows: the protein purifier is equipped with a Sephadex G-15 dextran gel column, and the mobile phase is ultrapure water; the concentration of the component in step (S06) is 50 mg / mL; the loading amount of the component in step (S06) is 7 mL; the elution rate of the component in step (S06) is 3 mL / min; there are three peak groups in step (S06);

[0182] (S07) performing amino acid sequence identification on the corresponding components corresponding to the peak group in step (S06), and performing preliminary screening by the C-terminal amino acid composition of the peptide; the amino acid sequence identification in step (S07) adopts UPLC-Q-TOF MS system;

[0183] The parameters of the UPLC-Q-TOF MS system are:

[0184] HILIC column, column temperature 25°C; flow rate 0.3 mL / min; injection volume 2 μL;

[0185] Mobile phase A, water, 25 mM ammonium acetate, and 25 mM ammonia;

[0186] Mobile phase B, acetonitrile;

[0187] The gradient elution program was as follows: 0-1 min, 95% mobile phase B; 1 min-14 min, mobile phase B linearly changed from 95% to 65%; 14 min-16 min, mobile phase B linearly changed from 65% to 40%; 16 min-18 min, mobile phase B maintained at 40%; 18 min-18.1 min, mobile phase B linearly changed from 40% to 95%; 18.1 min-23 min, mobile phase B maintained at 95%;

[0188] After separation on a HILIC column, mass spectrometry analysis was performed using electrospray ionization positive ion and electrospray ionization negative ion modes;

[0189] The scanning range of TOF MS is 60Da to 1000Da; the scanning accumulation time of TOF MS is 0.20s / spectra;

[0190] The product ion scanning range of TOF MS is 25Da to 1000Da; the product ion scanning accumulation time of TOF MS is 0.05s / spectra;

[0191] In step (S07), the C-terminal amino acid composition of the peptide is initially screened for peptides containing glutamic acid, aspartic acid, arginine and lysine;

[0192] (S08) Molecular docking is performed between the peptides initially screened out in step (S07) and the salty taste receptor, and a salty and fresh peptide RRRRR is obtained according to the binding energy index; the salty taste receptor in step (S08) is constructed by AlphaFold; the salty taste receptor in step (S08) is derived from the UniProtKB database, and the salty taste receptor is numbered as Q7Z404-TMC4-HUMAN; the salty taste receptor in step (S08) is transmembrane channel-like protein 4; the molecular docking method in step (S08) is the CDOCKER program in the Discovery Studio software, with -CDOCKER_ENERGY as the index, the docking ligand is a dipeptide, the docking receptor is TMC4, and the docking force field is CHARMm;

[0193] The docking pocket is, x:17.7065, y:11.7845, z:-14.9743, with a radius of 19.7.

[0194] Embodiment 4:

[0195] like Figure 2 The method for screening salty and fresh peptides having the function of increasing saltiness and freshness comprises the following steps:

[0196] (S01) taking an appropriate amount of cattle bone powder and mixing with water, and pre-treating; the cattle bone powder in step (S01) is yak bone powder; the method for obtaining the cattle bone powder in step (S01) is to take cattle bones, wash and roughly chop, high-temperature steam, dry, crush and sieve in sequence to obtain cattle bone powder; the solid-liquid ratio of cattle bone powder to water in step (S01) is 1:20; sieving is to sieve through a 150-250 mesh sieve; the pre-treatment in step (S01) is to cool to room temperature after boiling water bath; the boiling water bath time is 30 minutes;

[0197] (S02) adding trypsin, composite protease and flavor protease to the solution pretreated in step (S01) to carry out ultrasonic enzymatic hydrolysis reaction; the amount of trypsin added in step (S02) is 12000U / g substrate mass; the amount of composite protease added in step (S02) relative to the solution is 16%; the amount of flavor protease added in step (S02) relative to the solution is 24%; the enzymatic hydrolysis in step (S02) is heated water bath enzymatic hydrolysis; the enzymatic hydrolysis temperature in step (S02) is 43°C; the enzymatic hydrolysis time in step (S02) is 5h;

[0198] (S03) after the ultrasonic enzymatic hydrolysis reaction in step (S02) is completed, the temperature is raised to inactivate the enzyme; the temperature-raising enzyme inactivation in step (S03) is inactivation in a boiling water bath; the enzyme inactivation time in step (S03) is 5 min to 15 min;

[0199] (S04) centrifuging the solution after the enzyme inactivation in step (S03) to obtain the supernatant to obtain a bovine bone protein hydrolysate; the centrifugal speed in step (S04) is 4000 r / min to 4500 r / min; the centrifugal temperature in step (S04) is room temperature; the centrifugal time in step (S04) is 25 min to 35 min;

[0200] (S05) centrifuging and ultrafiltration the bovine bone protein hydrolysate from step (S04), and passing through 3 kDa and 1 kDa ultrafiltration membranes in sequence to obtain three components with molecular weights >3 kDa, 1 kDa to 3 kDa, and <1 kDa;

[0201] (S06) taking an appropriate amount of the component with a molecular weight <1 kDa in step (S05) for gel permeation chromatography separation and purification, and obtaining a peak group of the purified product at a characteristic wavelength of 280 nm; the conditions for gel permeation chromatography separation and purification in step (S06) are as follows: the protein purifier is equipped with a Sephadex G-15 dextran gel column, and the mobile phase is ultrapure water; the concentration of the component in step (S06) is 10 mg / mL to 100 mg / mL; the loading amount of the component in step (S06) is 5 mL to 8 mL; the elution rate of the component in step (S06) is 1 mL / min to 4 mL / min; there are three peak groups in step (S06), namely G1, G2, and G3;

[0202] (S07) performing amino acid sequence identification on the corresponding components corresponding to the G3 peak group in step (S06), and performing preliminary screening by the C-terminal amino acid composition of the peptide; the amino acid sequence identification in step (S07) adopts the UPLC-Q-TOF MS system;

[0203] The parameters of the UPLC-Q-TOF MS system are:

[0204] HILIC column, column temperature 25°C; flow rate 0.2 mL / min; injection volume 2 μL;

[0205] Mobile phase A, 50% water, 25 mM ammonium acetate, and 25 mM ammonia;

[0206] Mobile phase B, 50% acetonitrile;

[0207] The gradient elution program was as follows: 0-1 min, 95% mobile phase B; 1 min-14 min, mobile phase B linearly changed from 95% to 65%; 14 min-16 min, mobile phase B linearly changed from 65% to 40%; 16 min-18 min, mobile phase B maintained at 40%; 18 min-18.1 min, mobile phase B linearly changed from 40% to 95%; 18.1 min-23 min, mobile phase B maintained at 95%;

[0208] After separation on a HILIC column, mass spectrometry analysis was performed using electrospray ionization positive ion and electrospray ionization negative ion modes;

[0209] The scanning range of TOF MS is 60Da to 1000Da; the scanning accumulation time of TOF MS is 0.20s / spectra;

[0210] The product ion scanning range of TOF MS is 25Da to 1000Da; the product ion scanning accumulation time of TOF MS is 0.05s / spectra;

[0211] In step (S07), the C-terminal amino acid composition of the peptide is initially screened for peptides containing glutamic acid, aspartic acid, arginine and lysine;

[0212] (S08) Molecular docking is performed between the peptides initially screened out in step (S07) and the salty taste receptor, and a salty and fresh peptide RRRRR is obtained according to the binding energy index; the salty taste receptor in step (S08) is constructed by AlphaFold; the salty taste receptor in step (S08) is derived from the UniProtKB database, and the salty taste receptor is numbered as Q7Z404-TMC4-HUMAN; the salty taste receptor in step (S08) is transmembrane channel-like protein 4; the molecular docking method in step (S08) is the CDOCKER program in the Discovery Studio software, with -CDOCKER_ENERGY as the index, the docking ligand is a dipeptide, the docking receptor is TMC4, and the docking force field is CHARMm;

[0213] The docking pocket is, x:17.7065, y:11.7845, z:-14.9743, with a radius of 19.7.

[0214] Experimental example:

[0215] The salty peptide RRRRR was dissolved in water to prepare a 1 mg / mL solution. Before data collection, the electronic tongue was activated and the sensor check was performed to ensure the stability of the sensor and the reliability and stability of the test data. 100 mL of filtrate was placed in a special container for the electronic tongue, and the appropriate sensor and test mode were selected. Sample collection and cleaning were performed alternately. Each sample was measured 4 times and 3 stable electronic tongue response values ​​were selected for subsequent analysis.

[0216] The test setup mainly includes the following aspects:

[0217] 1) Number of test cycles: generally set to 4 times;

[0218] 2) Enter the file name where the experimental results are saved;

[0219] 3) Sample quantity;

[0220] 4) Sample name;

[0221] 5) Test sensor set selection: Select "Foodstuff_sensors" for conventional 5-flavor detection;

[0222] Test Mode:

[0223] Before the sample is tested, the cleaning (reference solution) is performed three times for a total of 330 seconds. After cleaning, the sensor is self-checked, each time for 30 seconds. When the output results of the two self-checks are less than 0.5, the self-check is considered to have passed. After the self-check, the sample is measured, and the sample measurement time is 30 seconds. After the sample is tested, the cleaning (reference solution) is performed twice for a total of 6 seconds, the cut-off time is 30 seconds, the collection cycle is 1 second, the collection delay is 0 seconds, and the detection environment temperature is room temperature.

[0224] like Figure 3 The figure shows the electronic tongue taste characteristic diagram of the salty and fresh peptide RRRRR. Its salty value in aqueous solution is 3.19, and its salty value in 0.35% sodium chloride solution is 6.45; its umami value in aqueous solution is 5.68, and its umami value in 0.35% sodium chloride solution is 7.34. Therefore, the salty and fresh peptide RRRRR has certain salty and umami taste characteristics and good saltiness and umami enhancement effects.

[0225] Experimental comparative example:

[0226] like Figure 4 As shown, reference evaluation standards are set, and their concentrations are: anhydrous citric acid (0.08%, w / v) as a standard solution for sourness, caffeine (0.08%, w / v) as a standard solution for bitterness, tea polyphenols (0.08%, w / v) as a standard solution for astringency, sodium chloride NaCl (0.35%, w / v) as a standard solution for saltiness, and monosodium glutamate MSG (0.35%, w / v) as a standard solution for umami.

[0227] Other operations are the same as those in the experimental example.

[0228] like Figure 4 The figure shows the electronic tongue taste characteristic diagram of the standard (Reference), in which the saltiness value of 0.35% sodium chloride solution is 4.28, and the umami value of 0.35% sodium glutamate solution is 7.94.

[0229] The salty and fresh peptide screening system with the function of increasing saltiness and freshness includes a reactor 100, a heating reactor 200 and a filter cartridge 300. A stirring assembly 400 is arranged inside the reactor 100. A centrifugal inner barrel is rotatably arranged inside the reactor 100. The stirring assembly 400 is located in the centrifugal inner barrel. A water bath cartridge 500 is coated on the outside of the reactor 100. A boiling water circulation assembly 600 is connected between the heating reactor 200 and the water bath cartridge 500. The boiling water circulation assembly 600 forms an annular flow channel between the water bath cartridge 500 and the heating reactor 200, so that boiling water circulates in the annular flow channel to ensure the water bath effect on the reactor 100. A centrifugal ring 306 is rotatably provided in the filter cartridge 300, and two sets of ultrafiltration membranes are provided on the centrifugal ring 306, and the pore size of the upper ultrafiltration membrane is larger than that of the lower ultrafiltration membrane. A liquid guide tube 700 is connected between the bottom of the reactor 100 and the top of the filter cartridge 300, so that when the supernatant is added to the inside of the filter cartridge 300 through the liquid guide tube 700, it passes through two ultrafiltration membranes in sequence and is filtered twice. A partition assembly 800 is provided on the liquid guide tube 700, and through the provision of the partition assembly 800, the bottom layer solution can be discharged from the partition assembly 800, and the upper layer solution can pass through the partition assembly 800 and the liquid guide tube 700 and enter the filter cartridge 300.

[0230] A suction pipe 900 is connected to the circumferential surface of the back of the filter cartridge 300, the bottom end of the suction pipe 900 is located below the ultrafiltration membrane on the lower side, and the other end of the suction pipe 900 is connected to the top of the reactor 100. The suction pipe 900 is used to suck the air inside the filter cartridge 300 and inject the air into the reactor 100, so that the inside of the filter cartridge 300 is in a negative pressure state, which promotes the filtering effect. The inside of the reactor 100 is in a positive pressure state, and the pressure on the surface of the supernatant is uniform, ensuring that the bottom solution is discharged smoothly.

[0231] A feeding port 101 is provided at the top of the reactor 100, a discharge pipe 102 is connected to the lowest point of the bottom of the reactor 100, the discharge pipe 102 is connected to the inside of the centrifugal inner barrel, an air guide pipe 103 is connected to the top of the reactor 100, and the top of the suction pipe 900 is connected to the air guide pipe 103. A valve 104 is provided on the discharge pipe 102, and the bottom end of the liquid guide pipe 700 is connected to the discharge pipe 102; a first centrifugal motor is provided at the bottom of the reactor 100, and the driving end of the first centrifugal motor passes through the reactor 100 and is connected to the centrifugal inner barrel;.

[0232] The stirring assembly 400 includes a motor 401 fixedly mounted at the top center of the reactor 100, a threaded rod 402 fixedly mounted at the bottom output end of the motor 401, and the threaded rod 402 is arranged along the axis of the reactor 100. A sleeve 403 is sleeved on the outer side of the threaded rod 402, a rotating ring 404 is fixedly mounted on the outer wall of the top end of the sleeve 403, and two groups of guide grooves 405 are opened through the surface of the sleeve 403, and the guide grooves 405 are arranged along the length direction of the sleeve 403.

[0233] An internal threaded tube 406 is threadedly connected to the threaded rod 402, and the internal threaded tube 406 is slidably connected in the sleeve 1 403. Two groups of connecting plates 407 are fixedly installed on the outer wall of the internal threaded tube 406. The two groups of connecting plates 407 are respectively slidably connected in the guide groove 405. A sleeve 2 408 is sleeved on the outside of the sleeve 1 403. The connecting plate 407 is fixedly installed on the inner wall of the top end of the sleeve 2 408, and a U-shaped rod 409 is fixedly installed on the outer wall of the bottom end of the sleeve 2 408; a rotation limiter is sleeved on the outer side of the swivel 404. The rotating ring 404 is limited by rotating the limiting piece to prevent the sleeve 1 403 from rotating, and then the threaded rod 402 is driven to rotate by the motor 401, thereby driving the internal threaded tube 406, the connecting piece 407, the sleeve 2 408 and the U-shaped rod 409 to move up and down along the guide groove 405, and then the limiting effect on the rotating ring 404 is released by rotating the limiting piece, and then the threaded rod 402 is driven to rotate by the motor 401, which can drive the internal threaded tube 406, the connecting piece 407, the sleeve 2 408, the U-shaped rod 409, the sleeve 1 403 and the rotating ring 404 to rotate synchronously, and the U-shaped rod 409 stirs the solution.

[0234] The rotation limiter includes an annular cover 410 sleeved on the outside of the rotating ring 404, and hollow beams 411 are fixed in an array on the outer wall of the annular cover 410. The other end of the hollow beams 411 is fixedly installed on the inner wall of the reactor 100. The hollow beams 411 are connected to the annular cover 410. A micro cylinder 412 is fixedly installed on the inner wall of the hollow beam 411. A friction plate 413 is fixedly installed on the output end of the micro cylinder 412. The other side of the friction plate 413 is an arc surface, and the friction plate 413 is attached to the circumferential surface of the rotating ring 404. The output end of the micro cylinder 412 is extended, so that the friction plate 413 is closely attached to the surface of the rotating ring 404, and the rotation of the sleeve 1 403 is suppressed. The output end of the micro cylinder 412 is contracted, and the friction plate 413 is separated from the rotating ring 404, so that the sleeve 1 403 can rotate freely.

[0235] The top of the heating kettle 200 is connected to a water inlet 201, and the lowest point of the bottom of the heating kettle 200 is connected to a drain outlet 202. Water is injected into the heating kettle 200 through the water inlet 201, and all waste water in the heating kettle 200 can be discharged through the drain outlet 202. A heater 203 is fixedly installed inside the heating kettle 200, and sealing covers are provided at the top of the feed inlet 101, the top of the water inlet 201, and the bottom of the drain outlet 202.

[0236] The filter cartridge 300 includes a filter cartridge body 301, a centrifugal ring 306 rotatably disposed on the inner wall surface of the filter cartridge body 301, a filter cartridge cover 302 disposed on the top of the filter cartridge body 301, and the filter cartridge cover 302 is used to seal the top opening of the filter cartridge body 301 to ensure the air pressure inside the filter cartridge 300. The top of the filter cartridge body 301 is higher than the liquid level inside the reactor 100. A feed pipe 303 is connected to the lowest point at the bottom of the filter cartridge body 301, and a valve 2 304 is disposed on the feed pipe 303. A connecting pipe 1 305 is connected to the top of the filter cartridge cover 302, and the top of the liquid guide tube 700 is connected to the top of the connecting pipe 1 305.

[0237] A centrifugal ring 306 is rotatably provided in the middle of the inner wall of the filter cartridge body 301, a supporting ring 307 is fixedly installed at the bottom of the inner wall of the centrifugal ring 306, a sleeve 1 308 is inserted in the centrifugal ring 306, the outer diameter of the sleeve 1 308 is equal to the inner diameter of the centrifugal ring 306, an ultrafiltration membrane 1 309 is fixedly installed at the bottom of the sleeve 1 308, the membrane pore size of the ultrafiltration membrane 1 309 is 1kDa, and the sleeve 1 308 abuts against the top of the supporting ring 307. A sleeve 2 310 is inserted at the top of the centrifugal ring 306, the sleeve 2 310 is located in the upper half of the filter cartridge body 301, and an ultrafiltration membrane 2 311 is fixedly installed at the bottom of the sleeve 2 310, the pore size of the ultrafiltration membrane 2 311 is 3kDa, and the pore size of the ultrafiltration membrane 1 309 is 1kDa. A second centrifugal motor is provided at the bottom of the filter cartridge body 301, and a rotating shaft is provided at the driving end of the second centrifugal motor. The rotating shaft is rotatably sealed at the bottom of the filter cartridge body 301, and a rotating frame is provided on the rotating shaft. The rotating frame is fixedly connected to the bottom of the centrifugal ring 306; an air pump 901 is provided on the suction pipe 900, and the bottom end of the suction pipe 900 is located below the support ring 307.

[0238] The boiling water circulation assembly 600 includes a water supply pipe 601, a return pipe 602 and a water pump 603. One end of the water supply pipe 601 is connected to the top of the water bath 500, and the other end of the water supply pipe 601 is connected to the bottom of the heating kettle 200. The water pump 603 is arranged on the water supply pipe 601; one end of the return pipe 602 is connected to the bottom of the water bath 500, and the other end of the return pipe 602 is connected to the upper side of the heating kettle 200. Thus, an annular flow channel is formed between the heating kettle 200, the water supply pipe 601, the return pipe 602 and the water bath 500, so that the boiling water circulates along the annular flow channel. The water pump 603 cooperates with the water supply pipe 601 to suck boiling water from the inside of the heating kettle 200 and transport it to the top of the water bath 500. Afterwards, under the action of gravity and hydraulic pressure, the waste water in the bottom of the water bath 500 flows back to the inside of the heating kettle 200 through the return pipe 602.

[0239] The reflux pipe 602 includes a second connection pipe 6021, a wrapping pipe 6022 and a third connection pipe 6023. The wrapping pipe 6022 is L-shaped and wraps around the outside of the water supply pipe 601. One end of the second connection pipe 6021 is connected to the bottom of the water bath 500, and the other end of the second connection pipe 6021 is connected to the right side of the horizontal part of the wrapping pipe 6022. One end of the third connection pipe 6023 is connected to the top side of the wrapping pipe 6022, and the other end of the third connection pipe 6023 is connected to the upper part of one side of the heating kettle 200. The wrapping pipe 6022 wraps around the outside of the water supply pipe 601, so that the outer shell of the water supply pipe 601 is in contact with the refluxed boiling water, preventing the outer shell of the water supply pipe 601 from directly contacting the air, thereby ensuring the temperature of the boiling water transported by the water supply pipe 601 to the water bath 500.

[0240] The partition assembly 800 includes a transverse tube 801 connected to the top of the liquid guiding tube 700. The liquid guiding tube 700 and the transverse tube 801 are perpendicular to each other. The front end of the transverse tube 801 is connected to a slag discharge pipe 802, and the rear end of the transverse tube 801 is connected to a connecting pipe 305.

[0241] A sealing column 804 is slidably connected inside the transverse cylinder 801, and the diameter of the sealing column 804 is equal to the inner diameter of the transverse cylinder 801. Arc plates 805 are fixedly installed at the bottom of the front and rear ends of the sealing column 804, and a pusher for driving the sealing column 804 to move forward and backward is provided on the transverse cylinder 801. When the sealing column 804 moves to the rear half of the transverse cylinder 801, the bottom solution of the reactor 100 can pass through the discharge pipe 102, the liquid guide pipe 700, the front half of the transverse cylinder 801, and the slag discharge pipe 802 to be discharged; when the sealing column 804 moves to the front half of the transverse cylinder 801, the supernatant of the reactor 100 can pass through the discharge pipe 102, the liquid guide pipe 700 and the rear half of the transverse cylinder 801 to enter the filter cylinder 300.

[0242] The pusher includes a slide post 806 fixedly mounted at the bottom of the sealing post 804. A slide groove 803 is provided through the bottom of the transverse cylinder 801. The slide groove 803 is arranged along the length direction of the transverse cylinder 801. The slide post 806 is slidably connected in the slide groove 803. The arc plate 805 is attached to the inner wall of the transverse cylinder 801 and seals the slide groove 803. A slider 807 is fixedly mounted at the bottom of the slide post 806. Support seats 808 are fixedly mounted at the bottom of both ends of the transverse cylinder 801. A threaded rod 809 is rotatably connected between the two support seats 808. A threaded hole is provided on the slider 807. The slider 807 is threadedly connected to the threaded rod 809. By rotating the threaded rod 809, the slider 807 and the slide post 806 are driven to move along the slide groove 803, and the sealing post 804 is driven to move inside the transverse cylinder 801. In addition, during the movement of the sealing post 804, the arc plate 805 and the bottom of the sealing post 804 always maintain the sealing effect on the slide groove 803.

[0243] When using:

[0244] Raw material processing: by closing valve 104, the discharge pipe 102 is sealed, and the bovine bone meal and water are added into the centrifugal inner barrel through the feeding port 101, and the output end of the micro cylinder 412 is extended, and the friction plate 413 is closely attached to the surface of the rotating ring 404, so as to inhibit the rotation of the sleeve 1 403, and then the motor 401 drives the threaded rod 402 to rotate forward, so that the internal threaded tube 406 moves downward along the guide groove 405, so that the U-shaped rod 409 is inserted into the solution, and then the output end of the micro cylinder 412 is contracted, and the friction plate 413 is separated from the rotating ring 404, and the motor 401 continues to drive the threaded rod 402 to rotate forward, driving the internal threaded tube 406, the connecting piece 407, the sleeve 2 408, the U-shaped rod 409, the sleeve 1 403, and the rotating ring 404 to rotate synchronously, and the U-shaped rod 409 stirs and mixes the dissolved bovine bone meal and water, and then protease is added for enzymatic hydrolysis, and the ultrasonic generator works at the same time to promote enzymatic hydrolysis;

[0245] Enzyme inactivation treatment: After the enzymolysis time is reached, water is injected into the heating kettle 200 through the water inlet 201, and the heater 203 heats the water to boiling. The boiling water is then transported to the upper part of the water bath 500 through the cooperation of the water pump 603 and the water delivery pipe 601. The boiling water in the bottom of the water bath 500 then flows back to the heating kettle 200 through the reflux pipe 602, and the enzyme is inactivated in the reaction kettle 100 by boiling water bath.

[0246] Solution stratification: Motor 401 continues to drive threaded rod 402 to rotate forward, driving internal threaded tube 406, connecting piece 407, sleeve 2 408, U-shaped rod 409, sleeve 1 403, and rotating ring 404 to rotate synchronously, U-shaped rod 409 stirs the solution again, and then extends through the output end of micro motor 412, friction plate 413 fits with rotating ring 404, and motor 401 drives threaded rod 402 to rotate in the opposite direction, so that sleeve 2 408 and U-shaped rod 409 can move upward along guide groove 405; the first centrifugal motor drives the centrifugal inner barrel to rotate to complete solution stratification;

[0247] Discharge of bottom solution: The air inside the filter cartridge 300 is sucked by the air pump 901 in cooperation with the suction pipe 900, and transported to the inside of the reactor 100, the air pressure inside the reactor 100 is increased, the valve 104 is opened, and the threaded rod 809 is screwed to move the sealing column 804 backward, so that the bottom solution inside the reactor 100 is discharged from the discharge pipe 102, the front half of the horizontal cylinder 801 and the slag discharge pipe 802 under the action of air pressure;

[0248] Supernatant filtration: After the bottom solution is discharged, the threaded rod 809 is screwed to move the sealing column 804 to the front half of the transverse cylinder 801, so that the supernatant passes through the discharge pipe 102, the liquid guide tube 700 and the rear half of the transverse cylinder 801 into the interior of the filter cylinder 300. Since the air pump 901 sucks air from the inside of the filter cylinder 300, the bottom of the filter cylinder 300 is in a negative pressure state; the support ring supports the sleeve 1 placed in the centrifugal ring to prevent the sleeve 1 from escaping from the centrifugal ring under a large centrifugal force; the sleeve 1 provides a centrifugal environment for the bovine bone protein hydrolysate with a molecular weight less than 3kDa passing through the ultrafiltration membrane 2, thereby intercepting the bovine bone protein hydrolysate with a molecular weight of 1kDa to 3kDa; the ultrafiltration membrane 1 allows the bovine bone protein hydrolysate with a molecular weight less than 1kDa to pass through, and intercepts the bovine bone protein hydrolysate with a molecular weight of 1kDa to 3kDa a bovine bone protein hydrolysate; sleeve two provides a centrifugal environment for the bovine bone protein hydrolysate supernatant initially fed in, thereby intercepting the bovine bone protein hydrolysate with a molecular weight greater than 3kDa; sleeve two is plugged into the top of the centrifugal ring and can be centrifugally rotated together with the centrifugal ring; ultrafiltration membrane two allows bovine bone protein hydrolysate with a molecular weight less than 3kDa to pass through, and intercepts the bovine bone protein hydrolysate with a molecular weight greater than 3kDa; the second centrifugal motor provides centrifugal power for the centrifugal rotation of the centrifugal ring; the housing of the second centrifugal motor is installed at the bottom of the filter cartridge body, the rotating shaft is rotatably arranged at the bottom of the filter cartridge body through a rotating sealing bearing, and the rotating frame stably acts the rotating force of the rotating shaft on the centrifugal ring to achieve stable centrifugal rotation of the centrifugal ring; the air pump extracts the gas in the filter cartridge body, so that the filter cartridge body is in a negative pressure, which better promotes centrifugal ultrafiltration;

[0249] By opening valve two 304, the clear liquid in the bottom layer of the filter cartridge 300 is discharged through the discharge pipe 303 to obtain a polypeptide component with a particle size less than 1 kDa; then the filter cartridge cover 302 is opened, and the sleeve two 310 is taken out to obtain a polypeptide component with a particle size greater than 3 kDa; then the sleeve one 308 is taken out to obtain a polypeptide component with a particle size greater than 1 kDa and less than 3 kDa.

[0250] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A salty and fresh peptide with the function of increasing saltiness and freshness, characterized by: The salty and fresh peptide is RRRRR, and the amino acid sequence of the salty and fresh peptide is Arg-Arg-Arg-Arg-Arg; the molecular formula of the salty and fresh peptide is C 30 H 62 N 20 O6; the molecular structure of the salty peptide is 2. A method for screening salty and fresh peptides with saltiness and freshness enhancing functions, characterized by: Includes the following step, (S01) taking an appropriate amount of cattle bone powder and mixing it with water, and performing pretreatment; (S02) adding trypsin, compound protease and flavor protease to the solution pretreated in step (S01) to perform ultrasonic enzymatic hydrolysis reaction; (S03) after the enzymatic hydrolysis reaction in step (S02) is completed, heating and inactivating the enzyme; (S04) centrifuging the solution after the enzyme inactivation in step (S03) to obtain the supernatant to obtain bovine bone protein hydrolysate; (S05) subjecting the bovine bone protein hydrolysate from step (S04) to centrifugal ultrafiltration through 3 kDa and 1 kDa ultrafiltration membranes in sequence to obtain three components with molecular weights >3 kDa, 1 kDa to 3 kDa, and <1 kDa; (S06) taking an appropriate amount of the component with a molecular weight less than 1 kDa in step (S05) and performing gel permeation chromatography separation and purification, and obtaining a peak group of the purified product at a characteristic wavelength of 280 nm; (S07) performing amino acid sequence identification on the corresponding components corresponding to the peak group in step (S06), and performing preliminary screening based on the C-terminal amino acid composition of the peptides; (S08) Molecular docking of the peptides initially screened out in step (S07) with the salty taste receptor is performed to obtain the salty and fresh peptide RRRRR based on the binding energy index.

3. The method for screening salty and fresh peptides having the function of increasing saltiness and freshness according to claim 2, characterized in that: The method for obtaining the bovine bone powder in step (S01) is: Take cattle bones, wash and roughly chop, steam at high temperature, dry, crush and sieve in sequence to obtain cattle bone powder; The sieving is through a 150-250 mesh sieve; In the step (S01), the solid-liquid ratio of the cattle bone powder to water is 1:15-25; The pretreatment in step (S01) is cooling to room temperature after boiling water bath; The boiling water bath time is 20 min to 60 min.

4. The method for screening salty and fresh peptides having the function of increasing saltiness and freshness according to claim 2, characterized in that: In the step (S02), the amount of trypsin added is 8000U / g substrate mass to 14000U / g substrate mass; In the step (S02), the amount of the composite protease added relative to the solution is 15% to 18%; In the step (S02), the amount of flavor protease added to the solution is 20% to 25%; The enzymatic hydrolysis in the step (S02) is enzymatic hydrolysis in a heated water bath; The enzymatic hydrolysis temperature in the step (S02) is 40°C to 45°C; The enzymatic hydrolysis time in the step (S02) is 4 h to 6 h.

5. The method for screening salty and fresh peptides having the function of increasing saltiness and freshness according to claim 2, characterized in that: The step (S03) of inactivating the enzyme by heating is inactivating the enzyme by boiling water bath; The enzyme inactivation time in the step (S03) is 5 min to 15 min.

6. The method for screening salty and fresh peptides having the function of increasing saltiness and freshness according to claim 2, characterized in that: The centrifugal speed in the step (S04) is 4000 r / min to 4500 r / min; The centrifugation time in the step (S04) is 25 min to 35 min.

7. The method for screening salty and fresh peptides having the function of increasing saltiness and freshness according to claim 2, characterized in that: The conditions for gel permeation chromatography separation and purification in the step (S06) are as follows: the protein purifier is equipped with a Sephadex G-15 dextran gel column and the mobile phase is ultrapure water; The concentration of the components in step (S06) is 10 mg / mL to 100 mg / mL; The loading amount of the components in the step (S06) is 5 mL to 8 mL; The elution rate of the components in step (S06) is 1 mL / min to 4 mL / min; There are three peak groups in the step (S06).

8. The method for screening salty and fresh peptides having the function of increasing saltiness and freshness according to claim 2, characterized in that: The amino acid sequence identification in the step (S07) adopts UPLC-Q-TOF MS system; The parameters of the UPLC-Q-TOF MS system are: HILIC column, column temperature 25°C; flow rate 0.3 mL / min; injection volume 2 μL; Mobile phase A, water, 25 mM ammonium acetate, and 25 mM ammonia; Mobile phase B, acetonitrile; The gradient elution program was as follows: 0-1 min, 95% mobile phase B; 1 min-14 min, mobile phase B linearly changed from 95% to 65%; 14 min-16 min, mobile phase B linearly changed from 65% to 40%; 16 min-18 min, mobile phase B maintained at 40%; 18 min-18.1 min, mobile phase B linearly changed from 40% to 95%; 18.1min-23min, mobile phase B was maintained at 95%; After separation on a HILIC column, mass spectrometry analysis was performed using electrospray ionization positive ion and electrospray ionization negative ion modes; The scanning range of TOF MS is 60Da to 1000Da; the scanning accumulation time of TOF MS is 0.20s / spectra; The product ion scanning range of TOF MS is 25Da to 1000Da; the product ion scanning accumulation time of TOF MS is 0.05s / spectra.

9. The method for screening salty and fresh peptides having the function of increasing saltiness and freshness according to claim 2, characterized in that: In the step (S07), the C-terminal amino acid composition of the peptide is initially screened to contain peptides containing glutamic acid, aspartic acid, arginine and lysine.

10. The method for screening salty and fresh peptides having the function of increasing saltiness and freshness according to claim 2, characterized in that: The salty taste receptor in the step (S08) is constructed by AlphaFold; The salty taste receptor in the step (S08) is derived from the UniProtKB database, and the number of the salty taste receptor is Q7Z404-TMC4-HUMAN; The salty taste receptor in the step (S08) is transmembrane channel-like protein 4; The molecular docking method in the step (S08) is the CDOCKER program in the Discovery Studio software, with -CDOCKER_ENERGY as the index, the docking ligand is a dipeptide, the docking receptor is TMC4, and the docking force field is CHARMm; The docking pocket is, x:17.7065, y:11.7845, z:-14.9743, with a radius of 19.7.

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