Preparation method and detection method of corn oligopeptide rich in tripeptide

Abstract

The invention provides a preparation method and a detection method of corn oligopeptide rich in tripeptide, and particularly relates to the technical field of oligopeptide preparation. The preparation method comprises the following steps: A, preparing a corn protein raw material into homogenate, adding protease into the homogenate for primary enzymolysis, and centrifuging the material subjected to primary enzymolysis to obtain supernate; b, carrying out secondary enzymolysis on the supernate, and then carrying out enzyme deactivation, cooling and centrifugation to obtain corn protein zymolyte; and C, concentrating and drying the corn protein zymolyte to obtain the corn oligopeptide rich in tripeptide. According to the preparation method provided by the invention, the preparation efficiency of the tripeptide is remarkably improved, and the whole preparation process is simplified. According to the method, efficient release of the tripeptide is successfully realized, the corn oligopeptide rich in the tripeptide is prepared, the problems of low efficiency and complex preparation process in a traditional enzymolysis technology are solved, and a new effective way is provided for high-value utilization of corn protein.

Description

Technical Field

[0001] The invention relates to the technical field of oligopeptide preparation, in particular to a preparation method of corn oligopeptide rich in tripeptides and a detection method thereof. Background Art

[0002] As people's interest in healthy diet and functional foods increases, bioactive peptides show great application potential in the field of functional foods and biomedicine due to their important value in nutrition and health care, especially corn oligopeptides. This low molecular weight polypeptide extracted from natural corn protein has many health benefits such as lowering blood pressure, sobering up, protecting the liver and anti-oxidation, and has attracted great attention from researchers around the world.

[0003] When producing these active peptides, researchers usually pursue high hydrolysis degree and high content of small molecule (molecular weight less than 1000 Dalton) components to ensure the excellent digestion and absorption characteristics of corn oligopeptides. In the process of raw material or product development, excessive emphasis on improving the degree of hydrolysis and the proportion of low molecular weight components may ignore the combination specificity of the enzymatic treatment process and the impact of these processes on the release characteristics of active peptides at the molecular level. This emphasis has led to problems such as unclear characteristics of existing technologies, unclear composition characteristics of enzymatic hydrolysis products, low content of active ingredients, and weak technical barriers. With the development of science and technology and industry, this product development strategy guided by high hydrolysis degree and low molecular weight component proportion has gradually become popular, resulting in intensified competition for homogeneous oligopeptide products among different manufacturers, a mixed bag of products in the market, and vicious competition from time to time.

[0004] In addition, the existing quality control of active peptide raw materials mainly relies on indicators such as degree of hydrolysis, molecular weight distribution and amino acids, and these methods have limitations in raw material quality control. Due to the complex composition of peptides in protein hydrolysates and the presence of many isomers, this has caused great interference to the peptide detection and quality control methods based on liquid chromatography. At present, there is no reported method for preparing corn oligopeptides with the goal of releasing specific bioactive peptides, nor is there a method for detecting corn characteristic peptides based on mass spectrometry technology.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a method for preparing a corn oligopeptide rich in tripeptides, aiming to solve at least one of the above-mentioned technical problems in the prior art.

[0007] The second object of the present invention is to provide a corn oligopeptide rich in tripeptides.

[0008] The third object of the present invention is to provide a method for detecting corn oligopeptides rich in tripeptides.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] The first aspect of the present invention provides a method for preparing a tripeptide-rich corn oligopeptide comprising the following steps:

[0011] A. preparing a zein raw material into a homogenate, adding a protease into the homogenate for primary enzymolysis, and centrifuging the primary enzymolysis material to obtain a supernatant;

[0012] B. performing secondary enzymolysis on the supernatant, and after completion, performing enzyme inactivation, cooling and centrifugation to obtain corn protein hydrolysate;

[0013] C. Concentrating and drying the corn protein hydrolysate to obtain the tripeptide-rich corn oligopeptide.

[0014] Furthermore, in the corn protein raw material, the corn protein content is ≥ 60%.

[0015] Preferably, the corn gluten raw material comprises corn gluten meal and / or corn yellow meal.

[0016] Preferably, in step A, the pH of the homogenate is 8.0-8.5.

[0017] Preferably, the temperature of the primary enzymatic hydrolysis is 50-60°C and the time is 8-16 hours.

[0018] Preferably, in step B, the pH of the secondary enzymatic hydrolysis is 7.0-7.5.

[0019] Preferably, the temperature of the secondary enzymatic hydrolysis is 50-60°C and the time is 8-16 hours.

[0020] Furthermore, the protease is capable of hydrolyzing Leu amino acid residues from the N-terminus and Phe amino acid residues from the C-terminus.

[0021] Furthermore, the protease includes papain, neutral protease and alkaline protease.

[0022] Preferably, the protease is alkaline protease and papain in a mass ratio of 1:1.

[0023] Preferably, the added amount of the protease is 1.0-1.5% of the mass of the corn protein raw material.

[0024] Furthermore, the preparation method also includes a filtration process after obtaining the corn protein hydrolysate and before concentration.

[0025] Preferably, the filtration is performed using a microfiltration membrane;

[0026] Preferably, the pore size of the microfiltration membrane is 0.45-1 μm.

[0027] Furthermore, the preparation method of the homogenate is as follows:

[0028] The corn protein raw material is dispersed in water, then homogenized and homogenized, and then heat-treated and cooled to obtain the homogenate.

[0029] Preferably, the heat treatment is carried out at a temperature of 110 to 130° C. and for a time of 30 to 60 minutes.

[0030] The second aspect of the present invention provides a corn oligopeptide rich in tripeptides, which is prepared by the preparation method described in the first aspect.

[0031] The third aspect of the present invention provides a method for detecting tripeptides in corn oligopeptides, comprising the following steps:

[0032] Standard solution: Leucine-proline-phenylalanine was used as a tripeptide standard to prepare a standard solution;

[0033] Test solution: First, corn oligopeptide was made into an aqueous solution, then an equal volume of methanol was added to the aqueous solution, mixed evenly, and then centrifuged, and the supernatant was used as the test solution;

[0034] Detection: The standard solution and the test solution are loaded onto a liquid chromatography-mass spectrometer for quantitative analysis of the tripeptide.

[0035] Furthermore, the liquid chromatography-mass spectrometer is an ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer;

[0036] Preferably, the chromatographic column of the ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer is a RPLCC18 column or a HSS T3 chromatographic column, the flow rate is 0.1-0.5 mL / min, the column temperature is 35-40° C., and the injection volume is 1-3 μL.

[0037] Preferably, a mixed solution of mobile phase A and mobile phase B is used as the mobile phase for gradient elution.

[0038] The mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile.

[0039] Preferably, the volume concentration of the formic acid aqueous solution is 0.05-0.15%.

[0040] Preferably, the gradient elution comprises the following procedure:

[0041] From 0 min to 1.5 min, the volume fraction of mobile phase A is maintained at 88% to 92%, and the volume fraction of mobile phase B is maintained at 8% to 12%.

[0042] From 1.5 min to 7.5 min, the volume fraction of mobile phase A decreased from 88% to 92% to 58% to 62%, and the volume fraction of mobile phase B increased from 8% to 12% to 38% to 42%.

[0043] From 7.5min to 8.5min, the volume fraction of mobile phase A decreased from 58% to 62% to 8% to 12%, and the volume fraction of mobile phase B increased from 38% to 42% to 88% to 92%.

[0044] 8.5min~10min, maintain the volume fraction of mobile phase A at 8%~12%, and the volume fraction of mobile phase B at 88%~92%.

[0045] From 10min to 10.5min, the volume fraction of mobile phase A increased from 8% to 12% to 93% to 97%, and the volume fraction of mobile phase B decreased from 88% to 92% to 3% to 7%.

[0046] 10.5min~15min, maintain the volume fraction of mobile phase A at 93%~97%, and the volume fraction of mobile phase B at 3%~7%.

[0047] Preferably, the detection mode of the ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer is MRM multiple reaction ion monitoring mode.

[0048] Preferably, in mass spectrometry detection, the primary precursor ion monitoring is set to 376.2 m / z, and the secondary fragmentation ion monitoring is set to 263.1 m / z and 211.1 m / z, respectively.

[0049] Preferably, in mass spectrometry detection, the ion declustering voltage is 100-110 V, and the fragmentation cell collision energy is 20-30 eV.

[0050] Furthermore, the liquid chromatography-mass spectrometry instrument is an ultra-high pressure liquid chromatography-electrospray ionization-quadrupole-high resolution mass spectrometry instrument.

[0051] Preferably, the chromatographic column of the ultra-high pressure liquid chromatography-electrospray ionization-quadrupole-high resolution mass spectrometer is a RPLC C18 column or a HSS T3 chromatographic column, the flow rate is 0.1-0.5 mL / min, the column temperature is 35-40° C., and the injection volume is 1-3 μL.

[0052] Preferably, a mixed solution of mobile phase A and mobile phase B is used as the mobile phase for gradient elution.

[0053] The mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile.

[0054] Preferably, the volume concentration of the formic acid aqueous solution is 0.05-0.15%.

[0055] Preferably, the gradient elution comprises the following procedure:

[0056] From 0 min to 6 min, the volume fraction of mobile phase A was maintained at 88% to 92%, and the volume fraction of mobile phase B was maintained at 8% to 12%.

[0057] From 6 to 30 minutes, the volume fraction of mobile phase A decreased from 88% to 92% to 58% to 62%, and the volume fraction of mobile phase B increased from 8% to 12% to 38% to 42%.

[0058] From 30 to 35 minutes, the volume fraction of mobile phase A decreased from 58% to 62% to 8% to 12%, and the volume fraction of mobile phase B increased from 38% to 42% to 88% to 92%.

[0059] From 35 to 40 min, the volume fraction of mobile phase A is maintained at 8% to 12%, and the volume fraction of mobile phase B is maintained at 88% to 92%.

[0060] From 40 to 42 minutes, the volume fraction of mobile phase A increased from 8% to 12% to 93% to 97%, and the volume fraction of mobile phase B decreased from 88% to 92% to 3% to 7%.

[0061] From 42 min to 60 min, the volume fraction of mobile phase A is maintained at 93% to 97%, and the volume fraction of mobile phase B is maintained at 3% to 7%.

[0062] Preferably, the mass spectrometry data is identified using PepOSX-Search library search software, with a primary ion mass-to-charge ratio deviation of 20 ppm, a secondary ion mass-to-charge ratio deviation of 0.02 Da, a residue coverage threshold of 50%, an ion extraction chromatogram mass-to-charge ratio deviation of 20 ppm, and a chromatographic peak recognition method of Gaussian recognition to analyze the tripeptide composition characteristics of the enzymatic hydrolysate.

[0063] Compared with the prior art, the present invention has at least the following beneficial effects:

[0064] The preparation method provided by the present invention adopts a two-step multi-enzyme synergistic treatment technology to efficiently treat corn protein to prepare corn oligopeptides rich in tripeptides. In the first step, the macromolecular protein in the corn protein is degraded into small molecular polypeptides. Subsequently, soluble corn small molecular polypeptides and enzyme preparations are extracted by centrifugation. In the second step, the enzyme preparation is further used to perform deep hydrolysis treatment to fully release the corn tripeptides. This continuous process not only significantly improves the preparation efficiency of the tripeptides, but also simplifies the entire preparation process. The present invention successfully achieves the efficient release of the tripeptides and prepares corn oligopeptides rich in tripeptides. This method overcomes the problems of low efficiency and complex preparation process in traditional enzymatic hydrolysis technology, and provides a new and effective way for the high-value utilization of corn protein.

[0065] The tripeptide-rich corn oligopeptide provided by the present invention has an LPF content significantly increased to more than 0.35%. Through peptidomics analysis, the relative composition of the tripeptide accounts for more than 30%, and its composition characteristics are obvious. It is mainly composed of key tripeptides such as LPF, NQL (asparagine-glutamic acid-leucine), SQL (serine-glutamic acid-leucine), and LGA (leucine-glycine-alanine), which improves the quality and functionality of the corn oligopeptide and opens up a new way for the high-value utilization of corn protein.

[0066] The detection method provided by the present invention is a mass spectrometry detection method based on monitoring of target peptide precursor ions and characteristic fragment ions, and is used for accurate detection and quality control of LPF polypeptides in enzymatic hydrolysates. This method has significant advantages such as strong analytical specificity, good reproducibility, and high accuracy, and is also easy to operate and has a fast analysis speed. Compared with traditional hydrolysis degree and molecular weight monitoring methods, the method of the present invention has achieved significant improvements in quality control specificity and analytical performance. In addition, compared with traditional liquid chromatography methods, the present invention further enhances the specificity and stability of detection by reducing the interference of co-eluting products, thereby providing a more efficient and reliable technical means for the detection of LPF polypeptides in enzymatic hydrolysates. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0068] Figure 1 This is the MRM detection spectrum of the corn oligopeptide obtained in Example 3;

[0069] Figure 2The material spectrum provided in Result Example 1 with the peak emitting at the same time as the LPF polypeptide;

[0070] Figure 3 This is a histogram of the length distribution of polypeptide composition of corn oligopeptide hydrolysate in Example 3;

[0071] Figure 4 It is the spectrum identification diagram of LPF;

[0072] Figure 5 It is the spectrum identification diagram of NQL;

[0073] Figure 6 It is the spectrum identification graph of SQL;

[0074] Figure 7 This is the spectrum identification diagram of LGA. DETAILED DESCRIPTION

[0075] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0076] Hereinafter, the terms "including", "having" and their cognates, which may be used in various embodiments of the present invention, are intended only to indicate specific features, numbers, steps, operations, elements, components or combinations of the foregoing items, and should not be understood as first excluding the existence of one or more other features, numbers, steps, operations, elements, components or combinations of the foregoing items or adding the possibility of one or more features, numbers, steps, operations, elements, components or combinations of the foregoing items.

[0077] The first aspect of the present invention provides a method for preparing a tripeptide-rich corn oligopeptide comprising the following steps:

[0078] A. preparing a zein raw material into a homogenate, adding a protease into the homogenate for primary enzymolysis, and centrifuging the primary enzymolysis material to obtain a supernatant;

[0079] B. performing secondary enzymolysis on the supernatant, and after completion, performing enzyme inactivation, cooling and centrifugation to obtain corn protein hydrolysate;

[0080] C. Concentrating and drying the corn protein hydrolysate to obtain the tripeptide-rich corn oligopeptide.

[0081] The preparation method provided by the present invention adopts a two-step multi-enzyme synergistic treatment technology to efficiently treat corn protein to prepare corn oligopeptides rich in tripeptides. In the first step, the macromolecular protein in the corn protein is degraded into small molecular polypeptides. Subsequently, soluble corn small molecular polypeptides and enzyme preparations are extracted by centrifugation. In the second step, the enzyme preparation is further used to perform deep hydrolysis treatment to fully release the corn tripeptides. This continuous process not only significantly improves the preparation efficiency of the tripeptides, but also simplifies the entire preparation process. The present invention successfully achieves the efficient release of the tripeptides and prepares corn oligopeptides rich in tripeptides. This method overcomes the problems of low efficiency and complex preparation process in traditional enzymatic hydrolysis technology, and provides a new and effective way for the high-value utilization of corn protein.

[0082] Furthermore, in the corn protein raw material, the corn protein content is ≥ 60%.

[0083] Preferably, the corn gluten raw material comprises corn gluten meal and / or corn yellow meal.

[0084] Preferably, in step A, the pH of the homogenate is 8.0-8.5.

[0085] Typically but not limiting, the pH of the homogenate may be, for example, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5, or any value within the range of 8.0 to 8.5.

[0086] Preferably, the temperature of the primary enzymatic hydrolysis is 50-60°C and the time is 8-16 hours.

[0087] Typically but not restrictively, the temperature of the primary enzymolysis can be, for example, 50°C, 52°C, 54°C, 56°C, 58°C or 60°C, or any value within the range of 50-60°C; the time of the primary enzymolysis can be, for example, 8h, 10h, 12h, 14h or 16h, or any value within the range of 8-16h.

[0088] Preferably, in step B, the pH of the secondary enzymatic hydrolysis is 7.0-7.5.

[0089] Typically but not limiting, the pH of the secondary enzymatic hydrolysis may be, for example, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5, or any value within the range of 7.0 to 7.5.

[0090] Preferably, the temperature of the secondary enzymatic hydrolysis is 50-60°C and the time is 8-16 hours.

[0091] Typically but not limiting, the temperature of the secondary enzymolysis can be, for example, 50°C, 52°C, 54°C, 56°C, 58°C or 60°C, or any value within the range of 50-60°C; the time of the secondary enzymolysis can be, for example, 8h, 10h, 12h, 14h or 16h, or any value within the range of 8-16h.

[0092] Furthermore, the protease is capable of hydrolyzing Leu amino acid residues from the N-terminus and Phe amino acid residues from the C-terminus.

[0093] Furthermore, the protease includes papain, neutral protease and alkaline protease.

[0094] Preferably, the protease is alkaline protease and papain in a mass ratio of 1:1.

[0095] Preferably, the added amount of the protease is 1.0-1.5% of the mass of the corn protein raw material.

[0096] Typically but not limiting, the added amount of the protease may be, for example, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5% of the mass of the corn protein raw material, or any value within the range of 1.0-1.5%.

[0097] Furthermore, the preparation method also includes a filtration process after obtaining the corn protein hydrolysate and before concentration.

[0098] Preferably, the filtration is performed using a microfiltration membrane;

[0099] Preferably, the pore size of the microfiltration membrane is 0.45-1 μm.

[0100] Typically but not limiting, the pore size of the microfiltration membrane can be, for example, 0.45 μm, 0.50 μm, 0.55 μm, 0.60 μm, 0.65 μm, 0.70 μm, 0.75 μm, 0.80 μm, 0.85 μm, 0.90 μm, 0.95 μm or 1.00 μm, or any value within the range of 0.45 to 1 μm.

[0101] Furthermore, the preparation method of the homogenate is as follows:

[0102] The corn protein raw material is dispersed in water, then homogenized and homogenized, and then heat-treated and cooled to obtain the homogenate.

[0103] Preferably, the heat treatment is carried out at a temperature of 110 to 130° C. and for a time of 30 to 60 minutes.

[0104] Typically but not restrictively, the temperature of the heat treatment may be, for example, 110°C, 115°C, 120°C, 125°C or 130°C, or any value within the range of 110-130°C; the time of the heat treatment may be, for example, 30 min, 35 min, 40 min, 45 min, 50 min or 60 min, or any value within the range of 30-60 min.

[0105] The second aspect of the present invention provides a corn oligopeptide rich in tripeptides, which is prepared by the preparation method described in the first aspect.

[0106] The tripeptide-rich corn oligopeptide provided by the present invention has an LPF content significantly increased to more than 0.35%. Through peptidomics analysis, the relative composition of the tripeptide accounts for more than 30%, and its composition characteristics are obvious. It is mainly composed of key tripeptides such as LPF, NQL, SQL, and LGA, which improves the quality and functionality of the corn oligopeptide and opens up a new way for the high-value utilization of corn protein.

[0107] The third aspect of the present invention provides a method for detecting tripeptides in corn oligopeptides, comprising the following steps:

[0108] Standard solution: Leucine-proline-phenylalanine was used as a tripeptide standard to prepare a standard solution;

[0109] Test solution: First, corn oligopeptide was made into an aqueous solution, then an equal volume of methanol was added to the aqueous solution, mixed evenly, and then centrifuged, and the supernatant was used as the test solution;

[0110] Detection: The standard solution and the test solution are loaded onto a liquid chromatography-mass spectrometer for quantitative analysis of the tripeptide.

[0111] The detection method provided by the present invention is a mass spectrometry detection method based on monitoring of target peptide precursor ions and characteristic fragment ions, and is used for accurate detection and quality control of LPF polypeptides in enzymatic hydrolysates. This method has significant advantages such as strong analytical specificity, good reproducibility, and high accuracy, and is also easy to operate and has a fast analysis speed. Compared with traditional hydrolysis degree and molecular weight monitoring methods, the method of the present invention has achieved significant improvements in quality control specificity and analytical performance. In addition, compared with traditional liquid chromatography methods, the present invention further enhances the specificity and stability of detection by reducing the interference of co-eluting products, thereby providing a more efficient and reliable technical means for the detection of LPF polypeptides in enzymatic hydrolysates.

[0112] The tripeptide standard used is artificially chemically synthesized, with a purity of more than 95%, and is desalted leucine-proline-phenylalanine.

[0113] When preparing the test solution, the corn oligopeptide is first subjected to a constant temperature treatment at 50-55°C in order to allow the tripeptide in the sample to be fully dissolved in the sample solution.

[0114] Preferably, when preparing the test solution, the centrifugation temperature is 0-5°C; after centrifugation, the supernatant is filtered using an organic filter membrane.

[0115] Furthermore, the liquid chromatography-mass spectrometer is an ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer;

[0116] Preferably, the chromatographic column of the ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer is a RPLCC18 column or a HSS T3 chromatographic column, the flow rate is 0.1-0.5 mL / min, the column temperature is 35-40° C., and the injection volume is 1-3 μL.

[0117] Typically but not limiting, the heat flow rate can be, for example, 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min, or any value in the range of 0.1 to 0.5 mL / min; the column temperature can be, for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, or any value in the range of 35 to 40°C; the injection volume can be, for example, 1 μL, 1.5 μL, 2 μL or 3 μL, or any value in the range of 1 to 3 μL.

[0118] Preferably, a mixed solution of mobile phase A and mobile phase B is used as the mobile phase for gradient elution.

[0119] The mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile.

[0120] Preferably, the volume concentration of the formic acid aqueous solution is 0.05-0.15%.

[0121] Typically but not limiting, the volume concentration of the formic acid aqueous solution may be, for example, 0.05%, 0.075%, 0.1%, 0.125% or 0.15%, or any value within the range of 0.05-0.15%.

[0122] Preferably, the gradient elution comprises the following procedure:

[0123] From 0 min to 1.5 min, the volume fraction of mobile phase A is maintained at 88% to 92%, and the volume fraction of mobile phase B is maintained at 8% to 12%.

[0124] From 1.5 min to 7.5 min, the volume fraction of mobile phase A decreased from 88% to 92% to 58% to 62%, and the volume fraction of mobile phase B increased from 8% to 12% to 38% to 42%.

[0125] From 7.5min to 8.5min, the volume fraction of mobile phase A decreased from 58% to 62% to 8% to 12%, and the volume fraction of mobile phase B increased from 38% to 42% to 88% to 92%.

[0126] 8.5min~10min, maintain the volume fraction of mobile phase A at 8%~12%, and the volume fraction of mobile phase B at 88%~92%.

[0127] From 10min to 10.5min, the volume fraction of mobile phase A increased from 8% to 12% to 93% to 97%, and the volume fraction of mobile phase B decreased from 88% to 92% to 3% to 7%.

[0128] 10.5min~15min, maintain the volume fraction of mobile phase A at 93%~97%, and the volume fraction of mobile phase B at 3%~7%.

[0129] Preferably, the detection mode of the ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer is MRM multiple reaction ion monitoring mode.

[0130] Preferably, in mass spectrometry detection, the primary precursor ion monitoring is set to 376.2 m / z, and the secondary fragmentation ion monitoring is set to 263.1 m / z and 211.1 m / z, respectively.

[0131] Preferably, in mass spectrometry detection, the ion declustering voltage is 100-110 V, and the fragmentation cell collision capacity is 20-30 eV.

[0132] Furthermore, the liquid chromatography-mass spectrometry instrument is an ultra-high pressure liquid chromatography-electrospray ionization-quadrupole-high resolution mass spectrometry instrument.

[0133] Preferably, the chromatographic column of the ultra-high pressure liquid chromatography-electrospray ionization-quadrupole-high resolution mass spectrometer is a RPLC C18 column or a HSS T3 chromatographic column, the flow rate is 0.1-0.5 mL / min, the column temperature is 35-40° C., and the injection volume is 1-3 μL.

[0134] Preferably, a mixed solution of mobile phase A and mobile phase B is used as the mobile phase for gradient elution.

[0135] The mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile.

[0136] Preferably, the volume concentration of the formic acid aqueous solution is 0.05-0.15%.

[0137] Preferably, the gradient elution comprises the following procedure:

[0138] From 0 min to 6 min, the volume fraction of mobile phase A was maintained at 88% to 92%, and the volume fraction of mobile phase B was maintained at 8% to 12%.

[0139] From 6 to 30 minutes, the volume fraction of mobile phase A decreased from 88% to 92% to 58% to 62%, and the volume fraction of mobile phase B increased from 8% to 12% to 38% to 42%.

[0140] From 30 to 35 minutes, the volume fraction of mobile phase A decreased from 58% to 62% to 8% to 12%, and the volume fraction of mobile phase B increased from 38% to 42% to 88% to 92%.

[0141] From 35 to 40 min, the volume fraction of mobile phase A is maintained at 8% to 12%, and the volume fraction of mobile phase B is maintained at 88% to 92%.

[0142] From 40 to 42 minutes, the volume fraction of mobile phase A increased from 8% to 12% to 93% to 97%, and the volume fraction of mobile phase B decreased from 88% to 92% to 3% to 7%.

[0143] From 42 min to 60 min, the volume fraction of mobile phase A is maintained at 93% to 97%, and the volume fraction of mobile phase B is maintained at 3% to 7%.

[0144] Preferably, the mass spectrometry data is identified using PepOSX-Search library search software, with a primary ion mass-to-charge ratio deviation of 20 ppm, a secondary ion mass-to-charge ratio deviation of 0.02 Da, a residue coverage threshold of 50%, an ion extraction chromatogram mass-to-charge ratio deviation of 20 ppm, and a chromatographic peak recognition method of Gaussian recognition to analyze the tripeptide composition characteristics of the enzymatic hydrolysate.

[0145] The present invention is further described below by specific examples and comparative examples, but it should be understood that these examples are only used for more detailed description and should not be understood as limiting the present invention in any form. The raw materials used in the examples and comparative examples of the present invention, if no specific conditions are specified, are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0146] Example 1

[0147] This embodiment provides a method for preparing a corn oligopeptide rich in tripeptides, and the preparation steps are as follows:

[0148] (1) Pretreatment of corn protein raw materials: corn protein powder (protein content 75%) was fully dispersed in warm water (material-water ratio was 1:9), homogenized and slurried, and then heat-treated at 121° C. for 30 min. After cooling to room temperature, a corn protein homogenate was obtained.

[0149] (2) Enzymatic hydrolysis of corn protein: The pH of the homogenate described in (1) was adjusted to 8.5, and alkaline protease and papain (compound ratio of 1:1, total addition of 1.0% by weight of corn protein powder) were added and stirred at 55°C for 12 h, the supernatant was collected by centrifugation, the pH was adjusted to 7.0, and the enzymatic hydrolysis was continued at 55°C for 12 h, then the enzyme was inactivated at 95°C for 15 min, cooled, and centrifuged at 8000 rpm for 20 min to obtain the supernatant to obtain corn protein hydrolysate.

[0150] (3) Refining and drying of corn oligopeptides: The corn protein hydrolysate described in (2) was passed through a 1 μm microfiltration membrane, low-temperature concentrated at 55° C., and then spray-dried to obtain corn oligopeptides rich in corn tripeptides.

[0151] The obtained corn oligopeptides are tested, and the testing steps are as follows:

[0152] 1. Accurately weigh LPF (chemically synthesized, with a purity greater than 95% and desalted) peptide standard, and use ultrapure water to prepare a series of standard solutions with gradient concentrations of 0, 2, 4, 6, 8, and 10 μg / mL. Pass through a 0.22 μm organic filter membrane to obtain an LPF standard solution.

[0153] 2. Disperse corn oligopeptide fully in ultrapure water (concentration 4000 μg / mL), stir at constant temperature for 10 minutes, aspirate the sample solution and add an equal volume of methanol, mix well, let stand for 5 minutes, then centrifuge to obtain the supernatant, filter through a 0.22 μm organic filter membrane, and obtain the test solution.

[0154] 3. Load the above LPF standard solution and the test solution onto an ultra-high pressure liquid chromatography-triple quadrupole mass spectrometer (SCIEX Triple QuadTM 5500, AB Company, USA) for analysis.

[0155] ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 100 mm, The mobile phase A was a 0.1% volume concentration of formic acid in water, and the mobile phase B was acetonitrile.

[0156] Perform gradient elution according to the following program:

[0157] From 0 min to 1.5 min, the volume fraction of mobile phase A was maintained at 90%, and the volume fraction of mobile phase B was maintained at 10%.

[0158] From 1.5 min to 7.5 min, the volume fraction of mobile phase A decreased from 90% to 60%, and the volume fraction of mobile phase B increased from 10% to 40%.

[0159] From 7.5 min to 8.5 min, the volume fraction of mobile phase A decreased from 60% to 10%, and the volume fraction of mobile phase B increased from 40% to 90%.

[0160] 8.5min~10min, maintain the volume fraction of mobile phase A at 10%, and the volume fraction of mobile phase B at 90%.

[0161] From 10 min to 10.5 min, the volume fraction of mobile phase A increased from 10% to 95%, and the volume fraction of mobile phase B decreased from 90% to 5%.

[0162] From 10.5 min to 15 min, the volume fraction of mobile phase A was maintained at 95%, and the volume fraction of mobile phase B was maintained at 5%.

[0163] The flow rate was 0.40 mL / min, the column temperature was 40 °C, and the injection volume of the standard and sample was 1 μL.

[0164] The mass spectrometer ion source worked in positive ion mode, the detection mode was MRM multiple reaction ion monitoring mode, the primary precursor ion monitoring was set to 376.2 m / z, the secondary fragmentation ion monitoring was set to 263.1 and 211.1 m / z, the ion declustering voltage was 100 v, and the fragmentation pool collision capacity was 25 eV. After the two fragment ions were detected simultaneously to achieve the qualitative analysis of LPF, the external standard method was used to perform quantitative analysis of LPF based on the peak area.

[0165] Example 2

[0166] In this example, the corn oligopeptides obtained in Example 1 were detected, and the detection method was as follows:

[0167] 1. The detection steps are the same as those in Example 1.

[0168] 2. The detection step is the same as in Example 1.

[0169] 3. Load the test solution onto an ultra-high pressure liquid chromatography-high resolution mass spectrometer (SCIEX X500R LC-QTOF, AB Company, USA) for peptide analysis.

[0170] ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 100 mm, The mobile phase A was a 0.1% volume concentration of formic acid in water, and the mobile phase B was acetonitrile.

[0171] Perform gradient elution according to the following program:

[0172] From 0 min to 6 min, the volume fraction of mobile phase A was maintained at 90%, and the volume fraction of mobile phase B was maintained at 10%.

[0173] From 6 to 30 minutes, the volume fraction of mobile phase A decreased from 90% to 60%, and the volume fraction of mobile phase B increased from 10% to 40%.

[0174] From 30 to 35 minutes, the volume fraction of mobile phase A decreased from 60% to 10%, and the volume fraction of mobile phase B increased from 40% to 90%.

[0175] From 35 to 40 min, the volume fraction of mobile phase A was maintained at 10%, and the volume fraction of mobile phase B was maintained at 90%.

[0176] From 40 to 42 minutes, the volume fraction of mobile phase A increased from 10% to 95%, and the volume fraction of mobile phase B decreased from 90% to 5%.

[0177] From 42 min to 60 min, the volume fraction of mobile phase A was maintained at 95%, and the volume fraction of mobile phase B was maintained at 5%.

[0178] The flow rate was 0.20 mL / min, the column temperature was 40 °C, and the injection volume of the standard and sample was 2 μL.

[0179] The mass spectrometry data were identified using PepOSX-Search library search software, with a primary ion mass-to-charge ratio deviation of 20 ppm, a secondary ion mass-to-charge ratio deviation of 0.02 Da, a residue coverage threshold of 50%, and a sequence library downloaded from Uniprot (keyword: "maize-reviewed"). The mass-to-charge ratio deviation of the ion extraction chromatogram was 20 ppm, and the chromatographic peak identification method was Gaussian recognition to analyze the composition characteristics of the tripeptide hydrolysate.

[0180] Example 3

[0181] This embodiment provides a method for preparing a corn oligopeptide rich in tripeptides, and the preparation steps are as follows:

[0182] (1) Pretreatment of corn protein raw material: the same as the step in Example 1.

[0183] (2) Enzymatic hydrolysis of corn protein: The pH of the homogenate described in (1) was adjusted to 8.5, and alkaline protease and papain (compound ratio of 1:1, total addition of 1.5% by weight of corn protein powder) were added and stirred at 55°C for 12 h. The supernatant was collected by centrifugation, and the pH was adjusted to 7.5. The enzymatic hydrolysis was continued at 55°C for 12 h. The enzyme was then inactivated, cooled, and the supernatant was collected by centrifugation to obtain corn protein hydrolysate.

[0184] (3) Refining and drying of corn oligopeptides: The corn protein hydrolysate described in (2) is passed through a 0.45 μm microfiltration membrane, low-temperature concentrated at 45° C., and then spray-dried to obtain corn oligopeptides rich in corn tripeptides.

[0185] The obtained corn oligopeptides are tested, and the testing steps are as follows:

[0186] 1. Accurately weigh LPF (chemically synthesized, with a purity greater than 95% and desalted) peptide standard, use ultrapure water to prepare a series of standard solutions with gradient concentrations of 0, 2.5, 5, 7.5, and 10 μg / mL, and filter through a 0.22 μm organic filter membrane to obtain an LPF standard solution.

[0187] 2. Disperse corn oligopeptide fully in ultrapure water (concentration 400 μg / mL), stir at constant temperature for 10 minutes, aspirate the sample solution and add an equal volume of methanol, mix well, let stand for 5 minutes, then centrifuge to obtain the supernatant, pass through a 0.22 μm organic filter membrane, and obtain the test solution.

[0188] 3. The difference from this step in Example 1 is that the column temperature of the chromatographic column is 35° C., and the other parameters are the same as those in this step in Example 1.

[0189] Example 4

[0190] In this example, the corn oligopeptides obtained in Example 2 were detected using the detection method in Example 2.

[0191] Example 5

[0192] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that alkaline protease is used for enzymatic hydrolysis, and the remaining raw materials and steps are the same as those in Example 1, which will not be repeated here.

[0193] Example 6

[0194] This embodiment provides a method for preparing a corn oligopeptide rich in tripeptides and a method for detecting the same. The difference from Embodiment 1 is that papain is used for enzymatic hydrolysis, and the remaining raw materials and steps are the same as those in Embodiment 1, which will not be described again.

[0195] Example 7

[0196] This embodiment provides a method for preparing a corn oligopeptide rich in tripeptides and a method for detecting the same. The difference from Example 1 is that acidic protease is used for enzymatic hydrolysis, and the remaining raw materials and steps are the same as those in Example 1, which will not be repeated here.

[0197] Example 8

[0198] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Embodiment 1 is that bromelain is used for enzymatic hydrolysis, and the remaining raw materials and steps are the same as those in Embodiment 1, which will not be repeated here.

[0199] Example 9

[0200] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that neutral protease is used for enzymatic hydrolysis, and the remaining raw materials and steps are the same as those in Example 1, which will not be repeated here.

[0201] Example 10

[0202] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that alkaline protease and neutral protease are compounded in a 1:1 ratio in enzymatic hydrolysis. The remaining raw materials and steps are the same as those in Example 1 and are not described again.

[0203] Embodiment 11

[0204] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that alkaline protease and bromelain are compounded in a ratio of 1:1 in enzymatic hydrolysis. The remaining raw materials and steps are the same as those in Example 1 and are not described again.

[0205] Example 12

[0206] This embodiment provides a method for preparing a tripeptide-rich corn oligopeptide and a detection method thereof. The difference from Example 1 is that alkaline protease and acidic protease are compounded in a 1:1 ratio in enzymatic hydrolysis. The remaining raw materials and steps are the same as those in Example 1 and will not be repeated here.

[0207] Embodiment 13

[0208] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that the temperature of the heat treatment process is 100°C and the time is 30 minutes. The remaining raw materials and steps are the same as those in Example 1 and will not be repeated here.

[0209] Embodiment 14

[0210] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that the temperature of the heat treatment process is 100° C. and the time is 60 min. The remaining raw materials and steps are the same as those in Example 1 and will not be repeated here.

[0211] Embodiment 15

[0212] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that the temperature of the heat treatment process is 110° C. and the time is 30 min. The remaining raw materials and steps are the same as those in Example 1 and will not be repeated here.

[0213] Example 16

[0214] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that the temperature of the heat treatment process is 110° C. and the time is 60 min. The remaining raw materials and steps are the same as those in Example 1 and will not be repeated here.

[0215] Embodiment 17

[0216] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that the temperature of the heat treatment process is 121° C. and the time is 60 min. The remaining raw materials and steps are the same as those in Example 1 and will not be repeated here.

[0217] Embodiment 18

[0218] This example provides a method for preparing a tripeptide-rich corn oligopeptide and a method for detecting the same. Different from Example 1, the enzymatic hydrolysis of corn protein is performed according to the following steps:

[0219] The pH of the homogenate in (1) was adjusted to 8.5, alkaline protease (0.5% by weight of corn protein powder) was added and the mixture was stirred at a constant temperature of 55°C for 12 hours, and then the enzyme was inactivated at 95°C for 15 minutes, papain (0.5% by weight of corn protein powder) was added and the mixture was stirred at a constant temperature of 55°C for 12 hours, and then the enzyme was inactivated at 95°C for 15 minutes, cooled, and centrifuged at 8000 rpm for 20 minutes to obtain the supernatant to obtain corn protein hydrolysate.

[0220] The remaining materials and steps are the same as those in Example 1 and will not be described again.

[0221] Embodiment 19

[0222] This example provides a method for preparing a tripeptide-rich corn oligopeptide and a method for detecting the same. Different from Example 1, the enzymatic hydrolysis of corn protein is performed according to the following steps:

[0223] The pH of the homogenate in (1) is adjusted to 8.5, alkaline protease (0.5% by weight of corn protein powder) is added and the mixture is stirred and enzymolyzed at 55°C for 12 hours, centrifuged at 8000 rpm for 20 minutes to obtain the supernatant, papain (0.5% by weight of corn protein powder) is added and the mixture is stirred and enzymolyzed at 55°C for 12 hours, then the enzyme is inactivated at 95°C for 15 minutes, cooled, and centrifuged at 8000 rpm for 20 minutes to obtain the supernatant to obtain corn protein hydrolysate.

[0224] The remaining materials and steps are the same as those in Example 1 and will not be described again.

[0225] Embodiment 20

[0226] This example provides a method for preparing a tripeptide-rich corn oligopeptide and a method for detecting the same. Different from Example 1, the enzymatic hydrolysis of corn protein is performed according to the following steps:

[0227] The pH of the homogenate in (1) is adjusted to 8.5, alkaline protease (0.5% by weight of corn protein powder) is added and the mixture is stirred and enzymolyzed at 55°C for 12 hours, and the mixture is centrifuged at 8000 rpm for 20 minutes to obtain a supernatant. After the supernatant is inactivated at 95°C for 15 minutes, papain (0.5% by weight of corn protein powder) is added and the mixture is stirred and enzymolyzed at 55°C for 12 hours, and then the enzyme is inactivated at 95°C for 15 minutes, cooled, and centrifuged at 8000 rpm for 20 minutes to obtain a supernatant to obtain a corn protein hydrolysate.

[0228] The remaining materials and steps are the same as those in Example 1 and will not be described again.

[0229] Embodiment 21

[0230] This embodiment provides a preparation method of a corn oligopeptide rich in tripeptides and a detection method thereof. The difference from Example 1 is that the compounding ratio of alkaline protease to papain is 5:1, and the remaining steps are the same as those in Example 1 and will not be repeated here.

[0231] Embodiment 22

[0232] This embodiment provides a preparation method of a corn oligopeptide rich in tripeptides and a detection method thereof. The difference from Example 1 is that the compounding ratio of alkaline protease to papain is 4:2, and the remaining steps are the same as those in Example 1 and will not be repeated here.

[0233] Embodiment 23

[0234] This embodiment provides a preparation method of a corn oligopeptide rich in tripeptides and a detection method thereof. The difference from Example 1 is that the compounding ratio of alkaline protease to papain is 2:4, and the remaining steps are the same as those in Example 1 and will not be repeated here.

[0235] Embodiment 24

[0236] This embodiment provides a method for preparing a tripeptide-rich corn oligopeptide and a detection method thereof. The difference from Example 1 is that the compounding ratio of alkaline protease to papain is 1:5, and the remaining steps are the same as those in Example 1 and will not be repeated here.

[0237] Embodiment 25

[0238] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that the total addition amount of alkaline protease and papain is 0.5% of the mass of corn protein powder. The remaining steps are the same as those in Example 1 and will not be repeated here.

[0239] Embodiment 26

[0240] This embodiment provides a preparation method of a corn oligopeptide rich in tripeptides and a detection method thereof. The difference from Example 1 is that the total addition amount of alkaline protease and papain is 0.75% of the mass of corn protein powder. The remaining steps are the same as those in Example 1 and will not be repeated here.

[0241] Embodiment 27

[0242] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that the total addition amount of alkaline protease and papain is 1.25% of the mass of corn protein powder. The remaining steps are the same as those in Example 1 and will not be repeated here.

[0243] Embodiment 28

[0244] This embodiment provides a preparation method of corn oligopeptides rich in tripeptides and a detection method thereof. The difference from Example 1 is that the total addition amount of alkaline protease and papain is 1.5% of the mass of corn protein powder. The remaining steps are the same as those in Example 1 and will not be repeated here.

[0245] Embodiment 29

[0246] This embodiment provides a method for preparing a tripeptide-rich corn oligopeptide and a method for detecting the same. The difference from Embodiment 1 is that the temperature of the two enzymatic hydrolysis steps is 45° C. The remaining steps are the same as those in Embodiment 1 and will not be described in detail herein.

[0247] Embodiment 30

[0248] This embodiment provides a method for preparing a corn oligopeptide rich in tripeptides and a method for detecting the same. The difference from Example 1 is that the temperature of the two enzymatic hydrolysis steps is 50° C. The remaining steps are the same as those in Example 1 and will not be repeated here.

[0249] Embodiment 31

[0250] This embodiment provides a method for preparing a tripeptide-rich corn oligopeptide and a detection method thereof. The difference from Example 1 is that the temperature of the two enzymatic hydrolysis steps is 60° C., and the remaining steps are the same as those in Example 1, which will not be repeated here.

[0251] Embodiment 32

[0252] This embodiment provides a method for preparing a tripeptide-rich corn oligopeptide and a method for detecting the same. The difference from Embodiment 1 is that the temperature of the two enzymatic hydrolysis steps is 65° C. The remaining steps are the same as those in Embodiment 1 and will not be described in detail herein.

[0253] Comparative Example 1

[0254] This comparative example provides a preparation method of corn oligopeptides and a detection method thereof. The difference from Example 1 is that in the enzymatic hydrolysis step of corn protein, only one enzymatic hydrolysis is performed for 22 hours. The remaining raw materials and steps are the same as those in Example 1 and will not be repeated here.

[0255] Test Example 1

[0256] The dry basis recovery rate and soluble nitrogen recovery rate of the corn oligopeptides obtained in Examples 5-9 were measured.

[0257] The dry basis content of corn oligopeptides was determined by direct drying method according to GB 5009.3-2016 to calculate the dry basis recovery rate, and the soluble nitrogen content of corn oligopeptides was determined according to national standard GB5009.5-2016 to calculate the nitrogen recovery rate.

[0258] The obtained data are shown in Table 1 below.

[0259] Table 1

[0260]

[0261] As can be seen from Table 1, the effects of different proteases on the hydrolysis of corn protein are quite different. Among them, alkaline protease has the most obvious effect on improving nitrogen recovery rate and dry basis recovery rate, and its nitrogen recovery rate is close to 60%, followed by bromelain, acid protease and papain. In addition, the overall trend of nitrogen recovery rate is consistent with the dry basis recovery rate, indicating that there is a good consistency between the two, which is closely related to the higher protein content in corn protein meal.

[0262] Test Example 2

[0263] The LPF content obtained from the corn oligopeptides obtained in Examples 1 and 10-12 was statistically analyzed and listed in Table 2, and the soluble nitrogen content of the corn oligopeptides was measured according to the reference national standard GB5009.5-2016 to calculate the nitrogen recovery rate.

[0264] Table 2

[0265]

[0266] As can be seen from Table 2, different compounding methods have a significant effect on the nitrogen recovery rate and LPF content of the enzymatic hydrolysate. Among them, after the alkaline protease is compounded with the neutral protease, the nitrogen recovery rate is relatively high, while the nitrogen recovery rate of the enzymatic hydrolysate obtained by other compounding methods is relatively low. However, it is worth noting that after the alkaline protease is compounded with papain in Example 1, the LPF content of the enzymatic hydrolysate is relatively high, exceeding 0.2%, which is 19 times the lowest (Example 12). It can be seen that the compounding of alkaline protease and papain is conducive to the release of tripeptides.

[0267] Taking into full consideration the objectives of this study and the relatively small variation in nitrogen recovery rate under other non-enzyme type variation factors, the nitrogen recovery rate was no longer tested in the subsequent examples and comparative examples performance test experiments, and was only retested in the final experimental results.

[0268] Test Example 3

[0269] The LPF contents measured in Example 1 and Examples 13-32 were statistically analyzed and listed in Table 3.

[0270] Table 3

[0271]

[0272] It can be seen from Table 3 that high temperature treatment has a positive effect on the release of LPF polypeptide in corn protein. With the increase of treatment temperature or the extension of treatment time, the LPF content in the hydrolysate increases to a certain extent, especially when the treatment temperature is 121 ° C and the treatment time is 30 or 60 min, the LPF content of the hydrolysate is close to 0.3%.

[0273] For the enzymatic release of the target polypeptide, an appropriate segmented enzymatic treatment method is crucial. As shown in Table 3, different segmentation methods have a significant effect on the content of target polypeptide LPF. The LPF content of the enzymatic hydrolysate obtained by the segmentation method of Example 1 is significantly higher than that of other samples, while the segmented enzymatic release effects of Examples 15 and 17 are relatively poor, reflecting that the dual enzyme synergy in the enzymatic hydrolysis process is of great significance for the efficient release of the target polypeptide, and the dual enzyme synergy is divided into two-step mixed synergistic enzymatic hydrolysis without inactivating the enzyme, which has a significant effect on improving the target peptide content of the enzymatic hydrolysate.

[0274] The influence of other different enzymatic hydrolysis conditions on the LPF in the hydrolysate varies, and there are better values ​​that can promote the improvement of the enzymatic hydrolysis effect. Among them, in Example 1, when the ratio of the dual enzyme compound is 1:1, the enzymatic hydrolysis time is 24h, the enzyme addition amount is 1.0%, and the enzymatic hydrolysis temperature is 55°C, a better enzymatic hydrolysis effect is achieved. It is worth noting that the extension or increase of the enzymatic hydrolysis time and the enzyme addition amount does not show the improvement of the enzymatic hydrolysis effect as in the traditional enzymatic hydrolysis experiment, but instead causes the LPF content in the enzymatic hydrolysate to decrease. The possible reason is that the excessive enzymatic hydrolysis time or the excessive enzyme addition amount leads to further degradation or breakage of the LPF polypeptide. This also shows that for the target polypeptide in the released protein, the technical characteristics of the enzymatic hydrolysis process are more obvious than the original process, and a small change in the process parameters can also lead to a significant change in the content of the target polypeptide in the enzymatic hydrolysate.

[0275] Result Example 1

[0276] The LPF contents in the corn oligopeptides obtained in Example 1 and Example 3 were 0.38% and 0.40%, respectively. The corn oligopeptide obtained in Example 1 was measured in parallel three times on the same day, and the RSD value was 1.13%. The corn oligopeptide obtained in Example 1 was measured at intervals of 0, 6, 18, 24, and 48 h, and the RSD value was 2.58%, reflecting that the mass spectrometry detection method of the LPF polypeptide has good reproducibility and stability.

[0277] The MRM detection spectrum of corn oligopeptides obtained in Example 3 is as follows Figure 1 As shown. Figure 1 It can be seen that the chromatographic peak of the target substance LPF in the method described in the present invention is bilaterally symmetrical and presents a strong response peak, without interferences such as shoulder peaks and miscellaneous peaks, and the separation and detection effects are good.

[0278] The substances analyzed by peptideomics that peaked at the same time as the LPF peptide are as follows Figure 2 .Depend on Figure 2 It can be seen that there are many strong ions or substances co-eluting with LPF during the chromatographic elution process, and the use of the mass spectrometry detection method described in the present invention can better avoid the interference of these non-target substance ions on the detection results.

[0279] Result Example 2

[0280] The length distribution characteristics of the corn oligopeptide polypeptide composition in Example 3 were analyzed using peptidomics as follows Figure 3 , Figure 3 The number of tripeptides identified reached about 38%, and the relative proportion of their ion peak areas was about 32%, and they were mainly LPF, NQL, SQL, and LGA, with the relative proportion of their peak areas being about 8:8:4:3. The secondary spectrum identification diagrams of each peptide are shown in Figure 4 , Figure 5 , Figure 6 and Figure 7 .

[0281] Finally, it should be noted that the above-described embodiments are only specific implementations of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above-described embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify the technical solutions recorded in the above-described embodiments within the technical scope disclosed by the present invention, or can easily think of changes, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing a corn oligopeptide rich in tripeptides, characterized in that: The following steps are involved: A. preparing a zein raw material into a homogenate, adding a protease into the homogenate for primary enzymolysis, and centrifuging the primary enzymolysis material to obtain a supernatant; B. performing secondary enzymolysis on the supernatant, and after completion, performing enzyme inactivation, cooling and centrifugation to obtain corn protein hydrolysate; C. Concentrating and drying the corn protein hydrolysate to obtain the tripeptide-rich corn oligopeptide.

2. The preparation method according to claim 1, characterized in that: The corn protein raw material has a corn protein content of ≥60%; Preferably, the corn protein raw material comprises corn protein meal and / or corn yellow meal; Preferably, in step A, the pH of the homogenate is 8.0 to 8.5; Preferably, the temperature of the primary enzymatic hydrolysis is 50-60°C and the time is 8-16h; Preferably, in step B, the pH of the secondary enzymatic hydrolysis is 7.0 to 7.5; Preferably, the temperature of the secondary enzymatic hydrolysis is 50-60°C and the time is 8-16 hours.

3. The preparation method according to claim 1, characterized in that: The protease is capable of hydrolyzing a Leu amino acid residue from the N-terminus and a Phe amino acid residue from the C-terminus.

4. The preparation method according to claim 3, characterized in that: The proteases include papain, neutral protease and alkaline protease; Preferably, the protease is alkaline protease and papain in a mass ratio of 1:1; Preferably, the added amount of the protease is 1.0-1.5% of the mass of the corn protein raw material.

5. The preparation method according to any one of claims 1 to 4, characterized in that: It also includes a filtering process after obtaining the corn protein hydrolysate and before concentrating; Preferably, the filtration is performed using a microfiltration membrane; Preferably, the pore size of the microfiltration membrane is 0.45-1 μm.

6. The preparation method according to any one of claims 1 to 4, characterized in that: The preparation method of the homogenate is as follows: Dispersing the corn protein raw material in water, then homogenizing and homogenizing, then heat treating, and cooling to obtain the homogenate; Preferably, the heat treatment is carried out at a temperature of 110 to 130° C. and for a time of 30 to 60 minutes.

7. A corn oligopeptide rich in tripeptides, characterized in that: The preparation method is described in any one of claims 1 to 6.

8. A method for detecting tripeptides in corn oligopeptides according to claim 7, characterized in that: The following steps are involved: Standard solution: Leucine-proline-phenylalanine was used as a tripeptide standard to prepare a standard solution; Test solution: First, corn oligopeptide was made into an aqueous solution, then an equal volume of methanol was added to the aqueous solution, mixed evenly, and then centrifuged, and the supernatant was used as the test solution; Detection: The standard solution and the test solution are loaded onto a liquid chromatography-mass spectrometer for quantitative analysis of the tripeptide.

9. The detection method according to claim 8, characterized in that: The liquid chromatography-mass spectrometer is an ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer; Preferably, the chromatographic column of the ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer is a RPLC C18 column or a HSS T3 chromatographic column, the flow rate is 0.1-0.5 mL / min, the column temperature is 35-40° C., and the injection volume is 1-3 μL; Preferably, a mixed solution of mobile phase A and mobile phase B is used as the mobile phase for gradient elution; The mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile; Preferably, the volume concentration of the formic acid aqueous solution is 0.05-0.15%; Preferably, the gradient elution comprises the following procedure: 0min~1.5min, maintain the volume fraction of mobile phase A at 88%~92%, and the volume fraction of mobile phase B at 8%~12%; From 1.5 to 7.5 minutes, the volume fraction of mobile phase A decreased from 88% to 92% to 58% to 62%, and the volume fraction of mobile phase B increased from 8% to 12% to 38% to 42%; From 7.5 to 8.5 minutes, the volume fraction of mobile phase A decreased from 58% to 62% to 8% to 12%, and the volume fraction of mobile phase B increased from 38% to 42% to 88% to 92%; 8.5min~10min, maintain the volume fraction of mobile phase A at 8%~12%, and the volume fraction of mobile phase B at 88%~92%; From 10 to 10.5 minutes, the volume fraction of mobile phase A increased from 8% to 12% to 93% to 97%, and the volume fraction of mobile phase B decreased from 88% to 92% to 3% to 7%; 10.5min~15min, keep the volume fraction of mobile phase A at 93%~97%, and the volume fraction of mobile phase B at 3%~7%; Preferably, the detection mode of the ultra-high pressure liquid chromatography-electrospray triple quadrupole mass spectrometer is MRM multiple reaction ion monitoring mode; Preferably, in mass spectrometry detection, the primary precursor ion monitoring is set to 376.2 m / z, and the secondary fragmentation ion monitoring is set to 263.1 m / z and 211.1 m / z respectively; Preferably, in mass spectrometry detection, the ion declustering voltage is 100-110 V, and the fragmentation cell collision energy is 20-30 eV.

10. The detection method according to claim 8, characterized in that: The liquid chromatography-mass spectrometer is an ultra-high pressure liquid chromatography-electrospray-quadrupole-high resolution mass spectrometer; Preferably, the chromatographic column of the ultra-high pressure liquid chromatography-electrospray-quadrupole-high resolution mass spectrometer is a RPLCC18 column or a HSS T3 chromatographic column, the flow rate is 0.1-0.5 mL / min, the column temperature is 35-40° C., and the injection volume is 1-3 μL; Preferably, a mixed solution of mobile phase A and mobile phase B is used as the mobile phase for gradient elution; The mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile; Preferably, the volume concentration of the formic acid aqueous solution is 0.05-0.15%; Preferably, the gradient elution comprises the following procedure: 0min~6min, maintain the volume fraction of mobile phase A at 88%~92%, and the volume fraction of mobile phase B at 8%~12%; From 6 to 30 minutes, the volume fraction of mobile phase A decreased from 88% to 92% to 58% to 62%, and the volume fraction of mobile phase B increased from 8% to 12% to 38% to 42%; From 30 to 35 minutes, the volume fraction of mobile phase A decreased from 58% to 62% to 8% to 12%, and the volume fraction of mobile phase B increased from 38% to 42% to 88% to 92%; 35min-40min, maintain the volume fraction of mobile phase A at 8%-12%, and the volume fraction of mobile phase B at 88%-92%; From 40 to 42 minutes, the volume fraction of mobile phase A increased from 8% to 12% to 93% to 97%, and the volume fraction of mobile phase B decreased from 88% to 92% to 3% to 7%; 42min-60min, maintain the volume fraction of mobile phase A at 93%-97%, and the volume fraction of mobile phase B at 3%-7%; Preferably, the mass spectrometry data is identified using PepOSX-Search library search software, with a primary ion mass-to-charge ratio deviation of 20 ppm, a secondary ion mass-to-charge ratio deviation of 0.02 Da, a residue coverage threshold of 50%, an ion extraction chromatogram mass-to-charge ratio deviation of 20 ppm, and a chromatographic peak recognition method of Gaussian recognition to analyze the tripeptide composition characteristics of the enzymatic hydrolysate.

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