Methods for detecting the degree of protein hydrolysis

Abstract

This invention relates to a method for detecting the degree of protein hydrolysis. The invention is based on a standard curve of trinitrobenzenesulfonic acid concentration, and uses the difference in the amount of trinitrobenzenesulfonic acid consumed before and after protein hydrolysis to determine the hydrolyzed amino groups based on the standard curve, thereby calculating the degree of protein hydrolysis.

Description

Technical Field

[0001] This invention belongs to the field of detection, specifically relating to the detection of biological macromolecular components, and more specifically, relating to a method for detecting the degree of protein hydrolysis in animal milk. Background Technology

[0002] Protein is an essential nutrient for human growth. At different stages of life, protein plays a vital role in human development, health promotion and maintenance.

[0003] For protein sources, various proteins have been widely obtained from animal and plant sources, such as various plant proteins (soy protein, etc.) and various milk proteins from mammalian milk.

[0004] Proteins from these sources may require further processing, such as purification, separation, and hydrolysis, in certain situations.

[0005] For example, milk, an important source of protein, is one of the most common foods that cause allergies in children. Reports indicate that approximately 2-3% of children worldwide suffer from milk allergy.

[0006] Therefore, researchers have attempted to moderately degrade / enzymatically hydrolyze milk proteins to inhibit or alleviate allergic reactions. Enzymatic hydrolysis of milk proteins is an effective way to alter protein properties, improve digestibility, reduce allergenicity, and produce peptides with better functions. However, excessive hydrolysis can produce bitter peptides, altering the protein's flavor and affecting its application in food. Therefore, controlling the degree of protein hydrolysis is crucial during the enzymatic process.

[0007] The degree of hydrolysis of milk protein is called the degree of hydrolysis (DH). The polypeptides generated after milk protein hydrolysis, along with their amino acid sequence and molecular weight distribution (MWD), are three important parameters for monitoring the degree of protein hydrolysis.

[0008] In the existing technology, there are many methods for determining DH, which in principle mainly measure the protons released during hydrolysis (pH-stat method) or free amino groups (TNBS, OPA or ninhydrin method).

[0009] pH-stat techniques are performed in proton titrations of peptide release, where matching equipment with a pH-stat is typically necessary to maintain a constant pH in the reaction system. However, the DH obtained using this method often requires correction using other methods, such as the TNBS or OPA methods.

[0010] In free amino group methods, considering that the number of free amino groups in protein hydrolysates increases with hydrolysis, the content of free amino nitrogen in the hydrolysates can also characterize the degree of protein hydrolysis. Therefore, commonly used methods include the trinitrobenzenesulfonic acid (TNBS) method, formaldehyde titration, ninhydrin method, o-phthalaldehyde (OPA) method, and fluorescein method. The free amino groups released during hydrolysis can react with TNBS, OPA, or ninhydrin to form intermediates, which can be quantified using spectroscopic methods. However, it should be noted that these methods always use specific types of amino acids (such as leucine or serine) as standards to construct working curves for quantification. However, protein hydrolysates are much more complex, and quantification using a single standard inevitably leads to systematic errors. Furthermore, these methods have poor spectral resolution for changes in analyte concentration, resulting in low resolution when measuring DH after protein hydrolysis in some experiments.

[0011] For example, Reference 1 describes a method for determining the degree of protein hydrolysis: the molecular weight distribution of peptides in the protein hydrolysate is determined by high-performance gel filtration chromatography, the mass percentage of peptides in different molecular weight ranges is calculated, and then the peptide content, free amino acid content and degree of protein hydrolysis are calculated; it can also accurately determine the free amino acid content, peptide content and degree of protein hydrolysis in a series of protein standard solutions with different degrees of hydrolysis, and generate a standard curve for more accurate quantification.

[0012] Reference 2 describes a method for detecting the degree of protein hydrolysis and a reagent kit for the detection. First, a standard curve and a standard comparison card are prepared, and then the degree of protein hydrolysis in the sample is detected. This technical approach does not rely on measuring changes in the amino acid content after hydrolysis; instead, it indirectly measures the degree of protein hydrolysis by precipitating the protein from the sample. Therefore, it can resist interference from various organic substances such as polysaccharides and lipids.

[0013] Reference 3 describes how alkaline protease hydrolyzes large-molecule collagen to generate small-molecule peptides, while the content of free amino acids in the hydrolysate gradually increases. The amount of increase is positively correlated with the degree of hydrolysis. Therefore, the degree of hydrolysis can be calculated by detecting the total amount of amino acids or the content of a certain amino acid (such as glutamic acid) in the hydrolysate.

[0014] In fact, although the methods mentioned above have been used or studied, they are all indirect methods for measuring protein hydrolysis, and their accuracy remains questionable. Therefore, there is still an urgent need for more direct, accurate, and convenient methods to determine protein hydrolysis.

[0015] In addition, liquid chromatography-mass spectrometry (LC-MS) is a powerful analytical tool that has been developed in recent years. It can directly detect the molecular weight of each component in a complex system and perform qualitative and quantitative analysis of these components. However, there are currently no reports on using this method to detect the degree of hydrolysis in proteins.

[0016] References:

[0017] Reference 1: CN110836936A

[0018] Reference 2: CN102590107A

[0019] Reference 3: CN101246140A Summary of the Invention

[0020] Problems to be solved by the invention

[0021] As mentioned earlier, current technologies for detecting proton release-based titration methods require additional auxiliary calibration steps. Furthermore, in methods involving free amino acids, there are systematic biases in the comparison standards of standard curves based on specific amino acids. For example, the degree of hydrolysis calculation in reference 1 relies on the average molecular weight of the hydrolyzed amino acids, reference 2 primarily relies on spectrophotometric analysis, and reference 3 uses the content of one amino acid in glutamic acid to calculate the degree of hydrolysis.

[0022] Therefore, the reliability of the above methods remains a concern, especially since the reference material used for detection is often a data fitting or simplification benchmark, leading to unavoidable systematic biases. For example, traditionally, trinitrobenzenesulfonic acid-based spectroscopic methods are commonly used for DH analysis. In this method, free amino groups released during hydrolysis react with trinitrobenzenesulfonic acid to generate products that can be quantified using spectroscopic methods. DH is calculated by analyzing the differences in the UV-Vis (UV-VIS) absorption spectra of proteins and peptides after the reaction with trinitrobenzenesulfonic acid. L-Leucine is often used to generate standard curves. However, protein hydrolysates in real samples are more complex than single amino acids; different sequence lengths and structures of peptides can affect the reaction efficiency between trinitrobenzenesulfonic acid and free amino groups, as well as the UV-VIS absorption intensity detection during the process, potentially leading to measurement uncertainties.

[0023] Therefore, the present invention primarily provides a novel method for detecting the degree of hydrolysis of proteins, especially animal-derived proteins. This invention creatively combines the detection method of free TNBS with liquid chromatography-mass spectrometry (LC-MS) technology. By measuring the difference in TNBS consumed before and after hydrolysis, the amount of free amino groups in the hydrolysate can be calculated, thus establishing a new method for DH analysis by detecting free TNBS. This technique is simple, accurate, and reproducible, providing a new approach for monitoring protein hydrolysis.

[0024] Solutions for solving problems

[0025] Through long-term research, the inventors discovered that the above-mentioned technical problems can be solved by implementing the following technical solution:

[0026] [1]. This invention provides a method for detecting the degree of protein hydrolysis, the method firstly comprising providing a target protein, and providing a hydrolysate obtained by hydrolyzing the target protein, wherein,

[0027] The method further includes:

[0028] The standard curve for trinitrobenzenesulfonic acid is established using a liquid chromatography-mass spectrometry detection system, based on the area of ​​the mass spectral peaks of trinitrobenzenesulfonic acid at different concentrations.

[0029] The step of detecting the trinitrobenzenesulfonic acid consumed by the target protein involves reacting the target protein with a known amount of trinitrobenzenesulfonic acid, then using the liquid chromatography-mass spectrometry detection system to detect the mass spectrum peak area of ​​the remaining trinitrobenzenesulfonic acid, and using the standard curve to determine the amount of trinitrobenzenesulfonic acid consumed by the target protein, A0.

[0030] The step of detecting the trinitrobenzenesulfonic acid consumed by the hydrolysate involves reacting the hydrolysate with a known amount of trinitrobenzenesulfonic acid, then using the liquid chromatography-mass spectrometry detection system to detect the mass spectrum peak area of ​​the remaining trinitrobenzenesulfonic acid, and determining the amount of trinitrobenzenesulfonic acid A1 consumed by the hydrolysate using the standard curve.

[0031] The degree of hydrolysis of the target protein is calculated using the relationship between A0 and A1.

[0032] In the step of detecting trinitrobenzenesulfonic acid consumed by the target protein, the mass of the target protein in the test sample during liquid chromatography-mass spectrometry (LC-MS) detection is B0 (dry weight). In the step of detecting trinitrobenzenesulfonic acid consumed by the hydrolysate, the mass of the hydrolysate in the test sample during LC-MS detection is B1 (dry weight). Therefore, the mass of target protein required to generate B1 during the hydrolysis is B1' (dry weight). Thus, B0 and B1' are convertible.

[0033] The detection conditions of the liquid chromatography-mass spectrometry detection system are essentially the same in each step of the method.

[0034] [2]. According to the method of [1], wherein the target protein is selected from one or more proteins derived from animal milk.

[0035] [3]. The method according to [1] or [2], wherein the hydrolysis is carried out in the presence of an enzyme.

[0036] [4]. The method according to any one of [1] to [3], wherein the liquid chromatography-mass spectrometry detection system is ultra-high performance liquid chromatography (UPLC).

[0037] [5]. The method according to any one of [1] to [4], wherein, in the liquid chromatography-mass spectrometry detection system, a reversed-phase column is used in the liquid chromatography.

[0038] [6]. The method according to any one of [1] to [5], wherein the mass spectrometer in the liquid chromatography-mass spectrometry detection system has an electrochemical ion source.

[0039] [7]. The method according to any one of [1] to [6], wherein the mass spectrometer in the liquid chromatography-mass spectrometry detection system is a quadrupole mass spectrometer (Q-MS).

[0040] [8]. According to the method described in [7], when the quadrupole mass spectrometer (Q-MS) is used for detection, the ion pair 291.9(Q1) / 227.9(Q3) is selected as the quantitative monitoring channel.

[0041] [9]. The method according to any one of [1] to [8], wherein each test sample in the step of establishing the standard curve of the trinitrobenzenesulfonic acid, the step of detecting the trinitrobenzenesulfonic acid consumed by the target protein, and the step of detecting the trinitrobenzenesulfonic acid consumed by the hydrolysate contains or uses the same concentration of terminator, the terminator being used to terminate the reaction of trinitrobenzenesulfonic acid with free amino groups.

[0042]

[10] . According to the method of [9], wherein each of the test samples contains or uses the same concentration of buffering components.

[0043]

[11] . The method according to any one of [1] to

[10] , wherein B0 and B1' have equal mass.

[0044] The effects of the invention

[0045] By implementing the above technical solution, the present invention can achieve the following technical effects:

[0046] 1) This invention first provides a new method for testing the degree of protein hydrolysis, especially the degree of animal protein hydrolysis. This method is based on the reaction of trinitrobenzenesulfonic acid with amino groups in the protein and can provide a relatively convenient method for detecting the degree of protein hydrolysis by liquid chromatography-mass spectrometry.

[0047] 2) Unlike existing technologies that rely on fitting or simulating quantitative standards based on specific amino acid (average) molecular weights or standard curves, the standard curve used for quantification in this invention does not depend on proteins, peptides, or amino acids, but only on the content of trinitrobenzenesulfonic acid. Therefore, it can avoid the systematic bias that cannot be avoided in existing hydrolysis degree tests.

[0048] 3) The detection method of the present invention has been verified in practice and can efficiently and accurately detect the degree of protein hydrolysis. Attached Figure Description

[0049] Figure 1 Example: TNBS extraction ion chromatogram (a) and TNBS standard curve (b) in liquid chromatography-mass spectrometry (LC-MS) analysis.

[0050] Figure 2 Compare the standard curve of leucine in the spectroscopic analysis in Example 1. Detailed Implementation

[0051] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0052] <Definition>

[0053] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0054] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0055] In this specification, the term "systematic deviation" is used, which has a different meaning from the "systematic error" commonly referred to in this field. The former refers to the deviation in theoretical accuracy caused by theoretical deficiencies in the test method itself, while the latter refers to the possibility that, although there is no theoretical deviation, limitations of test instruments or real-world conditions may lead to a certain acceptable error between the test results and the theory.

[0056] In this specification, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0057] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0058] In this specification, the terms "optional" or "optional / optionally" are used to indicate the use or non-use of certain substances, components, procedures, application conditions, etc.

[0059] Unless otherwise specified, “room temperature” or “room temperature” as used in this instruction manual usually refers to a temperature of 23±3℃.

[0060] In this specification, when discussing reaction processes, test sample preparation, or LC-MS detection conditions, the term "substantially identical" is used to indicate that they are considered identical except for uncontrollable systematic errors.

[0061] All unit names used in this manual are international standard unit names, and unless otherwise stated, the "%" used refers to weight or mass percentage content.

[0062] In this manual, "mass spectrum intensity" or "mass spectrum signal intensity" can be calculated by integrating the peak area of ​​the mass spectrum peak.

[0063] In this manual, the term "quantity" refers to "mass" unless otherwise specified.

[0064] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0065] This invention provides a novel method for detecting the degree of protein hydrolysis. Based on a standard curve of trinitrobenzenesulfonic acid (TNBS) concentration, this invention determines the difference in the amount of TNBS consumed before and after protein hydrolysis (using liquid chromatography-mass spectrometry detection), thereby determining the amount of primary amino groups / amino groups hydrolyzed based on the standard curve, and then calculating the degree of protein hydrolysis.

[0066] Unlike existing methods, the quantitative detection of peptides, amino acids, and other components obtained after hydrolysis in this invention is not based on a standard curve for a specific amino acid, nor does it require a manually fitted average molecular weight of the amino acid. Therefore, it avoids the unavoidable systematic biases in existing hydrolysis degree testing methods. More specifically, the standard curve for quantitative analysis in this invention relies solely on trinitrobenzenesulfonic acid, thus achieving improved testing accuracy both theoretically and in terms of practical operability.

[0067] (Target protein)

[0068] This invention provides a method for detecting the degree of hydrolysis of a target protein. In principle, there are no special requirements regarding the source or type of the target protein.

[0069] In some specific implementations, these proteins can be of plant or animal origin.

[0070] Plant-derived proteins can typically be legume proteins, wheat proteins, and rice proteins, such as soy protein powder (SF), soy protein concentrate (SPC), soy protein isolate (SPI), and textured soy protein (TSP).

[0071] As animal-derived proteins, they can typically be proteins found in the milk secreted by various mammals, such as cow's milk, sheep's milk, horse's milk, and camel's milk.

[0072] In some preferred embodiments of the present invention, the target protein is derived from animal milk, more preferably from cow's milk, sheep's milk, or horse's milk. Examples of proteins from these sources include casein and whey protein.

[0073] In some specific embodiments of the present invention, the target protein may be a protein from one source or a mixture of proteins from multiple sources, such as a mixture of proteins from multiple animal sources, a mixture of proteins from multiple plant sources, or a mixture of proteins from animal and plant sources.

[0074] In some other specific embodiments of the invention, the target protein may be a single protein or a mixture of multiple proteins. For example, it may consist only of whey protein or be a mixture of whey protein and other proteins (e.g., casein).

[0075] Furthermore, in some more preferred embodiments of the present invention, the target protein of the present invention is whey protein.

[0076] Furthermore, the target protein of this invention can be a whole protein that has not undergone significant hydrolysis, or a protein that has undergone hydrolysis to any degree, as long as these proteins still possess the ability to be further hydrolyzed in the hydrolysis process described below. In some preferred embodiments, the target protein of this invention can be a (whole) protein that has not undergone hydrolysis.

[0077] Furthermore, there are no particular limitations in principle regarding the state of the target protein of the present invention, since various pretreatment methods are allowed. In some preferred embodiments, the target protein of the present invention can be a powdered solid protein.

[0078] The target protein of this invention can be prepared by conventional separation methods in the art or can be obtained directly from commercial purchases.

[0079] (hydrolysis)

[0080] The hydrolysis described in this invention refers to the process of breaking the peptide chain of the target protein. There are no particular limitations on the method of hydrolysis, as long as it is a suitable method known in the art.

[0081] In some specific embodiments of the present invention, from the perspective of hydrolysis efficiency, the hydrolysis is carried out in the presence of enzymes.

[0082] There are no particular restrictions on the enzymes that can be used. In some preferred embodiments, microbial proteases can be used. Such proteases may include one or more of the following: protease A, protease S, protease U, protease N, protease P3, protease SEB, neutral protease, etc.

[0083] Furthermore, protein hydrolysis can be carried out under suitable temperature and pH conditions, which can be performed with reference to methods known in the art. In some specific embodiments, the hydrolysis temperature can be below 65°C, preferably 35–60°C, more preferably 40–55°C; in other specific embodiments, the pH range can be between 6 and 8 (e.g., adjusted by a buffering agent). Additionally, the hydrolysis reaction time is typically between 0.5 and 6 hours.

[0084] (Liquid Chromatography-Mass Spectrometry Detection System)

[0085] The detection system of this invention is primarily used to determine the difference in the amount of trinitrobenzenesulfonic acid (TNBS) consumed by a protein before and after hydrolysis. This difference represents the amount of primary amino groups / amino groups hydrolyzed during the hydrolysis process. These "amino groups" can originate from short-chain peptides or be single amino acids.

[0086] Specifically, before hydrolysis, the protein has terminal amino groups that can consume a certain amount of TNBS. Furthermore, after hydrolysis, the protein produces additional short peptide chains and / or amino acids, which can also consume a certain amount of TNBS. The difference in the amount of TNBS consumed by the two processes can represent the amount of short peptide chains and / or amino acids produced by hydrolysis.

[0087] For TNBS, the reaction process with free amino groups (the ends of peptide chains or groups in amino acids) is as follows:

[0088]

[0089] The product on the right side of the reaction can be stabilized by adding a terminator. Such terminators can typically include inorganic acidic substances, such as hydrochloric acid or bisulfite.

[0090] Liquid chromatography (LC)

[0091] The liquid chromatography in the detection system of the present invention can be liquid chromatography with a reversed-phase column, preferably ultra-high performance liquid chromatography (UPLC) with an autosampler.

[0092] The reversed-phase chromatography column can typically be a long-chain alkyl-modified silica gel column as the stationary phase. In some preferred embodiments of the present invention, the reversed-phase chromatography column can be a C18 column.

[0093] In addition, the mobile phase for liquid chromatography can typically be water and a polar solvent, typically a nitrile solvent, such as acetonitrile.

[0094] Furthermore, there are no particular restrictions on the operating conditions of liquid chromatography; the gradient operating conditions of the mobile phase can be determined according to the specific specifications and conditions of the instrument.

[0095] Mass spectrometry (MS)

[0096] For mass spectrometry applicable to this invention, there are no particular limitations in principle as long as it can detect free trinitrobenzenesulfonic acid.

[0097] In some specific embodiments of the present invention, a suitable mass spectrometer may be equipped with an electrochemical ion source, especially a mass spectrometer equipped with an electrospray ionization (ESI) source.

[0098] Furthermore, the mass spectrometer suitable for this invention can be a quadrupole mass spectrometer (Q-MS) or a time-of-flight mass spectrometer (TOF-MS). From the perspective of convenience and efficiency in the quantitative detection of trinitrobenzenesulfonic acid, a quadrupole mass spectrometer (Q-MS) is suitable for this invention.

[0099] Furthermore, for quadrupole mass spectrometers, in some specific implementation schemes, a multi-reaction monitoring mode can be used, selecting the ion pair 291.9(Q1) / 227.9(Q3) as the quantitative monitoring channel.

[0100] In addition, other detection conditions for the mass spectrometer, such as gas, temperature, and voltage, can be selected based on operating methods known in the art.

[0101] The specific instruments for the liquid chromatography and mass spectrometry described above in this invention can be purchased from manufacturers such as Thermo Fisher Scientific, Waters, Shimadzu, and Agilent.

[0102] (Detection of trinitrobenzenesulfonic acid consumed by the target protein)

[0103] The amount of TNBS consumed by the target protein can be determined by reacting the target protein with a known amount of TNBS and then detecting the remaining, free TNBS by LC-MS analysis of the product. Furthermore, for any desired purpose, surfactants, buffer solutions, and reaction terminators commonly used in methods known in the art, as well as those described above, can be used in the reaction process between the target protein and TNBS.

[0104] Specifically, the target protein can be reacted by mixing it with a known amount of TNBS in solution.

[0105] In some specific implementations, the target protein can be dissolved in a solution system containing a buffer solution, and the solid content can be adjusted as needed. Preferably, a surfactant, such as an SDS solution, can be used at any point in the above process to ensure the complete unfolding of the protein's secondary structure, which is beneficial for improving test accuracy.

[0106] Furthermore, the solution containing the target protein is mixed with a TNBS solution of known concentration to allow the protein to react with the TNBS. Optionally, if necessary, the reaction can also be carried out with the addition of an additional buffer solution. The reaction conditions can be carried out at a temperature of 40–65°C, preferably 50–60°C. There are no particular limitations on the reaction time, which is typically between 0.5 and 3 hours.

[0107] Subsequently, a terminator can be used to stabilize or fix the reaction results described above. Furthermore, the detection sample (S0) for liquid chromatography-mass spectrometry can be prepared under diluted or undiluted conditions. For the detection sample of this invention, it refers to the sample directly detected on the instrument, the same applies below.

[0108] (Detection of trinitrobenzenesulfonic acid consumed by hydrolysate)

[0109] The amount of TNBS consumed by the hydrolysate can be determined by reacting the hydrolysate with a known amount of TNBS and then analyzing the product by LC-MS to detect the remaining, free TNBS. Similarly, for any desired purpose, surfactants, buffer solutions, and reaction terminators commonly used in methods known in the art, as well as those described above, can be used in the reaction process between the hydrolysate and TNBS.

[0110] In some preferred embodiments, from the perspective of convenience and accuracy of quantitative detection, the hydrolysate reaction process with TNBS and the preparation process of the detection sample (S1) of the present invention are substantially the same as the target protein reaction process with TNBS and the preparation process of the detection sample (S0) described above.

[0111] Alternatively, for the test sample (S1), it has or satisfies one or all of the following conditions:

[0112] i) The hydrolyzed protein component contained in S1 is B1 on a dry weight basis, and its dry weight before hydrolysis is B1'. Meanwhile, the target protein contained in S0 is B0 on ​​a dry weight basis. Then B0 and B1' are convertible, or B0 and B1' are convertible multiples. Preferably, B0 and B1' are equal.

[0113] ii) If surfactants, buffering components derived from buffer solutions, terminators, etc., are used in S1 and S0, then their contents in their respective samples are equal on a dry weight basis.

[0114] Furthermore, preferably, the temperature, time, and timing of the reaction between the amino group and TNBS during the preparation of S1 are substantially the same as those during the preparation of S0.

[0115] (Establishment of the standard curve for trinitrobenzenesulfonic acid)

[0116] In this invention, the basis for quantification is the TNBS concentration-mass spectrometry signal intensity standard curve. The principle is to use TNBS-containing solutions of different concentrations for liquid chromatography-mass spectrometry detection, and the liquid chromatography-mass spectrometry detection conditions are substantially the same as those used in the detection of trinitrobenzenesulfonic acid consumed by the target protein / hydrolysate described above.

[0117] In some preferred embodiments, to further improve the accuracy of the standard curve, it can be prepared in the manner described above for the detection of trinitrobenzenesulfonic acid consumed by the target protein / hydrolysate, using sample S0 or S1.

[0118] In some specific implementations, TNBS solutions of different concentrations can be mixed with buffer solutions, surfactants, and other components, and then stored at a temperature of 40–65°C, preferably 50–60°C, for 0.5–3 hours before adding a terminator.

[0119] Let C (C1, C2, C3…) represent TNBS standard test samples of different concentrations. For each sample C, if a buffer solution, surfactant, or terminator was used in S0 and S1, the content of the buffer solution (based on the buffering component), surfactant, or terminator in each C is substantially the same as in S0 and S1.

[0120] Furthermore, by detecting samples C with different TNBS concentrations, a standard curve of TNBS concentration versus mass spectrometry signal intensity can be obtained. Preferably, the R-value of this curve is... 2 Above 0.999.

[0121] (Calculation of degree of hydrolysis)

[0122] Using the method described above, the amount of TNBS consumed by the target protein (as background), A0, and the amount of TNBS consumed by the hydrolysate of the target protein, A1, can be accurately determined. The values ​​corresponding to A1-A0 then reflect the peptides and / or amino acids released by the target protein during hydrolysis. Based on this data, the degree of hydrolysis of the target protein can be calculated according to any standard for the degree of hydrolysis.

[0123] Example

[0124] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0125] Example 1

[0126] (Equipment and conditions)

[0127] In this embodiment, DH analysis is performed by detecting free TNBS.

[0128] TNBS was detected using a Waters UPLC (model I-class Acquity) liquid chromatograph and a Sciex QTrap 4500 mass spectrometer.

[0129] Chromatographic column: Waters CSH C18 reversed-phase column (1.7μm, 2.1mm ID×100mm), temperature setting: 40℃.

[0130] Mobile phase A was ultrapure water, mobile phase B was pure acetonitrile, the flow rate was 400 μL / min, the separation time was 4 min per sample, and the gradient elution parameters were as follows:

[0131] (1)0.0-1.0min, 10%B-10%B;

[0132] (2)1-2.8min, 10%B-98%B;

[0133] (3)2.8-3.2min, 98%B-98%B;

[0134] (4)3.2-3.3min,98%B-10%B;

[0135] (5)3.3-4.0min,10%B-10%B.

[0136] The mass spectrometry electrospray ionization source is set as follows:

[0137] Curtain gas (CUR) 25, collision gas (CAD) medium, temperature 550℃, ion source gas 1 (GS1) 45, ion source gas 2 (GS2) 50, electrospray voltage - 4500V.

[0138] Multiple reaction monitoring (MRM) mode was used for quantitative analysis of TNBS, with the following ion pair settings: Q1: 291.9, Q3: 227.9, collision energy: 21V. The peak area of ​​TNBS was used for standard curve construction and DH calculation.

[0139] (Construction of TNBS standard curve using liquid chromatography-mass spectrometry)

[0140] Dilute a commercially available 5% (w / v) TNBS aqueous solution 100 times with water to obtain a 500 μg / mL TNBS working solution. Add 1, 5, 10, 20, 30, 40, and 50 μL of TNBS working solution to a 96-well plate, respectively, and add 49, 45, 40, 30, 20, 10, and 0 μL of water to a total volume of 50 μL. Then add 15 μL of 1% SDS solution and 45 μL of PBS buffer (pH 8.5).

[0141] The prepared samples were incubated in a 55°C oven in the dark for 1 hour. Then, 90 μL of 0.1 M HCl solution was added to each well to stop the reaction. 5 μL of the solution was then diluted with water to 1000 μL. After vortexing, 1 μL of the sample was injected for mass spectrometry analysis. A standard curve was established based on the TNBS peak area and concentration.

[0142] Standard curve data can be found Figure 1 .

[0143] (Liquid chromatography-mass spectrometry for protein hydrolysis (DH))

[0144] Five protein hydrolysates were obtained through commercial purchase. DI-3072, IF-3070 IF-3080, IF-3085, IF-3090) and α-lactalbumin. The hydrolysate described above is a hydrolysate of the α-lactalbumin.

[0145] The five protein hydrolysates and α-lactalbumin were dissolved separately in Tris-HCl (10 mg / mL) and diluted 20-fold with 1% SDS solution (500 μg / mL). In a 96-well plate, 50 μL of TNBS (500 μg / mL) working solution, 15 μL of sample solution, and 45 μL of PBS buffer were added sequentially, and the plate was incubated at 55°C in the dark for 1 h. The reaction was stopped by adding 90 μL of HCl (0.1 M) to each well, and then 5 μL of the solution was diluted with water to 1000 μL. After vortexing, 1 μL of sample was injected for liquid chromatography-mass spectrometry analysis.

[0146] The degree of protein hydrolysis (DH) was calculated based on the TNBS peak area and the TNBS standard curve.

[0147] The data is shown in Table 1:

[0148]

[0149] The data shows that the coefficient of variation (CV) of the DH data obtained by the new scheme constructed in this invention is <12.5%, and the DH data is basically within the range of DH given in the product description of hydrolyzed protein. This indicates that the method has good reproducibility, the obtained data is reliable, and the method is feasible.

[0150] The calculation method for the above DH test is explained as follows:

[0151] For DH determination, firstly, the amount of free TNBS after reaction with α-lactalbumin and five protein hydrolysates is obtained based on the TNBS standard curve and mass spectrometry signal; secondly, the amount of TNBS reacting with α-lactalbumin (n1, μg) and five protein hydrolysates (n2, μg) is obtained by subtracting the free TNBS from the total TNBS. Then, the formula for calculating the degree of hydrolysis of the DH sample is as follows:

[0152]

[0153] The peak area of ​​TNBS in the reaction system was determined by mass spectrometry, and the degree of hydrolysis of five protein hydrolysis samples was calculated according to the above formula, as shown in Table 1 above. Each sample was measured in three parallel replicates, and the test deviations are also listed in the table.

[0154] Comparative Example 1

[0155] DH% was determined using the TNBS method.

[0156] A commercially available 5% (w / v) TNBS aqueous solution was diluted 100-fold with water to obtain 0.5 mg / mL TNBS. 15 μL of 0, 0.6, 0.9, 1.2, 1.5, 2.4, 3.0, 3.6, 4.5, and 6.0 mM leucine standard solutions (1% SDS) were prepared in a Costar 96-well plate (Corning, Kennebunker, USA). 45 μL of TNBS (0.5 mg / mL) solution, 15 μL of sample solution, and 45 μL of PBS buffer (pH 8.5) were added sequentially to each well of the 96-well plate. The prepared samples were incubated in a 55°C oven in the dark for 1 h. 90 μL of 0.1 M HCl solution was added to each well to stop the reaction. The absorbance was then measured at 340 nm using a microplate reader (Molecular Devices SpectraMax i3BioTek, USA) to construct a standard curve.

[0157] Five protein hydrolysates (same as in the previous example) and α-lactalbumin (same as in the previous example) were dissolved in Tris-HCl (10 mg / mL) and further diluted 20-fold with 1% SDS as the sample solution (0.5 mg / mL). 45 μL of TNBS (0.5 mg / mL) solution, 15 μL of sample solution, and 45 μL of PBS buffer (pH 8.5) were added sequentially to each well of a 96-well plate. The prepared samples were incubated in a 55°C oven in the dark for 1 hour. 90 μL of HCl solution (0.1 M) was added to each well to stop the reaction, and the absorbance was measured at 340 nm using a microplate reader (Molecular Devices SpectraMax i3 BioTek, USA).

[0158] See data Figure 2 And Table 2.

[0159] Table 2:

[0160]

[0161] The DH measured by spectrophotometry is significantly higher than that specified in the product manual. This indicates that traditional methods need further improvement.

[0162] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0163] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for detecting the degree of protein hydrolysis, the method comprising providing a target protein and providing a hydrolysate obtained by hydrolyzing the target protein, characterized in that, The method further includes: The standard curve for trinitrobenzenesulfonic acid is established using a liquid chromatography-mass spectrometry detection system, based on the area of ​​the mass spectral peaks of trinitrobenzenesulfonic acid at different concentrations. The step of detecting the trinitrobenzenesulfonic acid consumed by the target protein involves reacting the target protein with a known amount of trinitrobenzenesulfonic acid, then using the liquid chromatography-mass spectrometry detection system to detect the mass spectrum peak area of ​​the remaining trinitrobenzenesulfonic acid, and using the standard curve to determine the amount of trinitrobenzenesulfonic acid consumed by the target protein, A0. The step of detecting the trinitrobenzenesulfonic acid consumed by the hydrolysate involves reacting the hydrolysate with a known amount of trinitrobenzenesulfonic acid, then using the liquid chromatography-mass spectrometry detection system to detect the mass spectrum peak area of ​​the remaining trinitrobenzenesulfonic acid, and determining the amount of trinitrobenzenesulfonic acid A1 consumed by the hydrolysate using the standard curve. The degree of hydrolysis of the target protein is calculated using the relationship between A0 and A1. In the step of detecting trinitrobenzenesulfonic acid consumed by the target protein, the mass of the target protein in the test sample during liquid chromatography-mass spectrometry (LC-MS) detection is B0 (dry weight). In the step of detecting trinitrobenzenesulfonic acid consumed by the hydrolysate, the mass of the hydrolysate in the test sample during LC-MS detection is B1 (dry weight). Therefore, the mass of target protein required to generate B1 during the hydrolysis is B1' (dry weight). Thus, B0 and B1' are convertible. The detection conditions of the liquid chromatography-mass spectrometry detection system are essentially the same in each step of the method. The operating conditions for liquid chromatography in the liquid chromatography-mass spectrometry detection system are as follows: Chromatographic column: Waters CSH C18 reversed-phase column, 1.7 μm, 2.1 mm ID × 100 mm, temperature set at 40 ℃. Mobile phase A is ultrapure water, mobile phase B is pure acetonitrile, the flow rate is 400 μL / min, the separation time is 4 min per sample, and the gradient elution parameters are as follows: (1) 0.0 - 1.0 min, 10%B - 10%B; (2) 1 - 2.8 min, 10%B - 98%B; (3) 2.8 - 3.2 min, 98%B - 98%B; (4) 3.2 - 3.3 min, 98%B - 10%B; (5)3.3 -4.0 min, 10%B - 10%B.

2. The method according to claim 1, characterized in that, The target protein is selected from one or more proteins derived from animal milk; the liquid chromatography-mass spectrometry detection system uses a Waters I-class Acquity liquid chromatograph and a Sciex QTrap 4500 mass spectrometer.

3. The method according to claim 1 or 2, characterized in that, The hydrolysis is carried out in the presence of enzymes.

4. The method according to claim 1, characterized in that, The mass spectrometer in the liquid chromatography-mass spectrometry detection system has an electrochemical ion source.

5. The method according to claim 1, wherein The mass spectrometer in the liquid chromatography-mass spectrometry detection system is a quadrupole mass spectrometer (Q-MS).

6. The method according to claim 5, characterized in that, When using the quadrupole mass spectrometer (Q-MS) for detection, the ion pair 291.9 (Q1) / 227.9 (Q3) was selected as the quantitative monitoring channel.

7. The method according to claim 1 or 2, characterized in that, In the steps of establishing the standard curve of trinitrobenzenesulfonic acid, detecting the trinitrobenzenesulfonic acid consumed by the target protein, and detecting the trinitrobenzenesulfonic acid consumed by the hydrolysate, each test sample in the liquid chromatography-mass spectrometry detection contains or uses the same concentration of terminator, which is used to terminate the reaction between trinitrobenzenesulfonic acid and free amino groups.

8. The method according to claim 7, characterized in that, Each of the test samples contained or used the same concentration of buffering components.

9. The method according to claim 1 or 2, characterized in that, The masses of B0 and B1' are equal.

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