Method for detecting protein amino acid in microalgae

Abstract

The invention discloses a method for detecting protein amino acids in microalgae. The method comprises the following steps: dividing a microalgae sample into a first sample and a second sample; carrying out acid hydrolysis treatment on the first sample to obtain an acid hydrolysis sample solution, carrying out deacidification treatment on the acid hydrolysis sample solution, and then carrying out first dilution treatment to obtain a first sample solution to be detected; performing alkaline hydrolysis treatment on the second sample to obtain an alkaline hydrolysis sample solution, adjusting the alkaline hydrolysis sample solution to be neutral, and performing second dilution treatment to obtain a second sample solution to be detected; and performing liquid chromatography-tandem mass spectrometry detection on the first to-be-detected sample solution and the second to-be-detected sample solution. Derivation operation is not needed in sample pretreatment, interference of derivative by-products is avoided, conditions in an instrument method are simple, reagents are low in cost, the operation time is greatly shortened, the difficulty of detection steps is reduced, the consumable and time cost of the whole analysis process is reduced, and the detection efficiency is improved. The protein amino acid in the microalgae can be quickly, simply and conveniently detected and analyzed with high sensitivity.

Description

Technical Field

[0001] The present application belongs to the field of biochemical detection technology, and in particular relates to a method for detecting protein amino acids in microalgae. Background Art

[0002] Microalgae is not a taxonomic term, but refers to a group of tiny algae that can only be identified under a microscope. Microalgae generally refers to microorganisms that contain chlorophyll and can perform photosynthesis, and are a type of protists. Microalgae that are cultivated or produced in large quantities using biotechnology generally belong to four algae phyla: Cyanobacteria, Chlorophyta, Chrysophyta, and Rhodophyta.

[0003] Protein, as the primary metabolite of microalgae, is one of the most important components, accounting for 23% to 63% of the dry matter of microalgae. Microalgae as a protein supplement is mainly used to increase the nutritional value and functional properties of food. Microalgae protein is rich in a variety of essential amino acids, so the study of microalgae protein must analyze amino acids.

[0004] At present, there are many methods for amino acid detection and analysis, including amino acid automatic analyzer, high performance liquid chromatography, gas chromatography, capillary electrophoresis, ion chromatography-integrated pulse amperometry, spectrophotometry, near infrared spectroscopy, supercritical fluid chromatography, etc. Among them, capillary electrophoresis is less sensitive than other methods; ion chromatography-integrated pulse amperometry is expensive and highly specific, and requires high operator requirements; spectrophotometry can only measure the content of a single or a class of amino acids; near infrared spectroscopy and supercritical fluid chromatography require the establishment of mathematical models and supercritical CO2 elution. Since most amino acids do not have ultraviolet and fluorescent chromophores, the commonly used amino acid automatic analyzer method and high performance liquid chromatography both require derivatization. The former is separated by cation exchange column and then derivatized after column, and then detected by ninhydrin color development. This method has high instrument cost, cumbersome mobile phase preparation, and long operation time; the latter requires pre-column derivatization, so the by-products of the derivatization are easy to interfere with the separation of the target. Summary of the invention

[0005] The purpose of the present application is to provide a method for detecting amino acids in microalgae proteins, aiming to solve the technical problem of how to better detect and analyze the amino acids in microalgae proteins.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] The present application provides a method for detecting protein amino acids in microalgae, comprising:

[0008] providing a microalgae sample, and dividing the microalgae sample into a first sample and a second sample;

[0009] The first sample is subjected to an acid hydrolysis treatment to obtain an acid hydrolysis sample solution, and the acid hydrolysis sample solution is subjected to a first dilution treatment after being subjected to a deacidification treatment to obtain a first sample solution to be tested;

[0010] The second sample is subjected to alkaline hydrolysis treatment to obtain an alkaline hydrolysis sample solution, and the alkaline hydrolysis sample solution is adjusted to neutrality and then subjected to a second dilution treatment to obtain a second sample solution to be tested;

[0011] The first sample solution to be tested and the second sample solution to be tested are subjected to liquid chromatography tandem mass spectrometry detection.

[0012] The present application provides a method for detecting protein amino acids in microalgae, first subjecting the microalgae samples to a series of processes such as acid hydrolysis and alkaline hydrolysis, respectively, to obtain a first test sample solution and a second test sample solution, and then subjecting the obtained test sample solution to liquid chromatography tandem mass spectrometry detection. The entire pretreatment process of the detection does not require a derivatization operation, there is no interference from derivative byproducts, the conditions in the instrument method are simple, the reagents are low-cost and the running time is greatly shortened, which not only reduces the difficulty of the detection step, but also reduces the cost and time of consumables for the detection, and can detect and analyze protein amino acids in microalgae at low cost, quickly, simply and with high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 is a multiple reaction monitoring (MRM) spectrum of the amino acid mixed standard solution in the embodiment of the present application;

[0015] Figure 2 This is a diagram showing the separation effects of His, Asp, Ile and Leu under different chromatographic column conditions in the examples of the present application. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0017] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0018] In the present application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0019] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0020] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0021] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0022] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0023] Microalgae cells contain: proteins, lipids, algae polysaccharides, β-carotene, various inorganic elements (such as Cu, Fe, Se, Mn, Zn, etc.) and other high-value nutrients and chemical raw materials. Its rich protein content makes microalgae a high-quality non-animal protein source, with great application potential in the fields of food and feed. The amino acid composition of protein is an important parameter for evaluating its nutritional value. Due to the complex matrix in microalgae cells and the characteristics of the amino acid structure itself, if the amino acid of protein in microalgae is analyzed based on the existing amino acid detection technology, there will be many problems: either the detection steps are cumbersome and costly, or the sensitivity is low / repeatability is poor and there is a certain interference.

[0024] In order to achieve rapid and sensitive detection of the content and type of protein amino acid components in microalgae and reduce the cost of consumables, sample processing and instrument operation time for detection, the embodiment of the present application aims to introduce a non-derivatization method for determining protein amino acids in microalgae based on liquid chromatography-tandem mass spectrometry. The specific scheme is as follows.

[0025] The present application embodiment provides a method for detecting protein amino acids in microalgae. The detection method provided in the present application embodiment comprises the following steps:

[0026] S01: providing a microalgae sample, and dividing the microalgae sample into a first sample and a second sample;

[0027] S02: subjecting the first sample to an acid hydrolysis treatment to obtain an acid hydrolysis sample solution, subjecting the acid hydrolysis sample solution to a deacidification treatment and then to a first dilution treatment to obtain a first sample solution to be tested;

[0028] S03: performing alkaline hydrolysis treatment on the second sample to obtain an alkaline hydrolysis sample solution, adjusting the alkaline hydrolysis sample solution to neutrality and then performing a second dilution treatment to obtain a second sample solution to be tested;

[0029] S04: Performing liquid chromatography tandem mass spectrometry detection on the first sample solution and the second sample solution.

[0030] The detection method based on liquid chromatography-mass spectrometry in the embodiment of the present application does not require any pre-treatment or instrument derivatization, and has simple processing, high sensitivity, short analysis time, and high throughput, and can quickly detect protein amino acids in microalgae.

[0031] In some embodiments, a standard solution may be prepared before the detection of the embodiments of the present application. The step of liquid chromatography tandem mass spectrometry detection also includes the detection of the standard solution. Wherein, the standard solution includes an acidic standard solution and an alkaline standard solution; wherein, the standard substances in the acidic standard solution include aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, alanine, proline, cystine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine, and the standard substances in the alkaline standard solution include tryptophan. Specifically, the amino acid standards in the acidic standard solution may be gradiently diluted in sequence with a formic acid aqueous solution having a volume percentage of 0.1% to obtain an acidic standard solution: the amino acid concentration may be 10 μmol / L, 5 μmol / L, 2.5 μmol / L, 1 μmol / L, 0.5 μmol / L, 0.25 μmol / L, 0.1 μmol / L, 0.050 μmol / L, 0.025 μmol / L, 0.010 μmol / L; and then a standard linear equation is made. The amino acid standard in the alkaline standard solution can be diluted with water in sequence to obtain an alkaline standard solution: the amino acid concentration can be 10μmol / L, 5μmol / L, 2.5μmol / L, 1μmol / L, 0.5μmol / L, 0.25μmol / L, 0.1μmol / L, 0.050μmol / L, 0.025μmol / L, 0.010μmol / L; then a standard linear equation is made. The linear correlation coefficients of the 18 amino acids are all above 0.99, and they show a good linear relationship in the range of 10nmol / L to 10μmol / L.

[0032] In some embodiments, the microalgae sample is a freeze-dried microalgae powder (also referred to as freeze-dried algae powder), and the microalgae sample is divided into two equal parts: a first sample and a second sample, and a first test sample solution and a second test sample solution are prepared respectively. Specifically, the freeze-dried algae powder can be pre-treated, for example, the freeze-dried algae powder is mixed with ceramic beads and then frozen, and then oscillated and crushed to better break the wall.

[0033] In some embodiments, for the preparation of the first sample solution to be tested, the acid hydrolysis treatment step includes: dissolving the first sample in an acid solution, and then hydrolyzing it at 100-115° C. for 20-24 hours.

[0034] The acid solution used includes a hydrochloric acid solution with a concentration of 4 to 6 mol / L, and the mass volume ratio of the first sample to the hydrochloric acid solution is 1 mg: 0.05 to 0.50 mL.

[0035] In some embodiments, for the preparation of the first sample solution to be tested, the deacidification step includes: blowing the acid-hydrolyzed sample solution dry with nitrogen in a water bath at 55-60° C.

[0036] The subsequent first dilution treatment step includes: redissolving the sample obtained after deacidification with formic acid aqueous solution. In the first sample solution obtained by dilution after redissolution, the amino acid concentration can be within the linear range of the sample analyte response, which is 10nmol / L to 10μmol / L.

[0037] After the acid hydrolysis in the embodiment of the present application is completed, the deacidification and dilution steps are adopted, and then the machine is directly tested. The whole pre-treatment process reduces the derivatization operation steps, avoids the poor repeatability problem caused by the derivatization efficiency and the interference of the derivatization byproducts with the detection of the target product, and reduces the difficulty of experimental operation; nitrogen blowing deacidification and redissolution can be carried out without adjusting the pH to neutral, which not only greatly saves the pre-treatment time cost, but also can batch process samples at the same time. The deacidified sample can be further diluted with the initial mobile phase at a low multiple multiple in the future, which not only saves the filtration time, but also further reduces the interference of the sample matrix.

[0038] In some embodiments, for the preparation of the second sample solution to be tested, the alkaline hydrolysis treatment step includes: dissolving the second sample in an alkaline solution, and then hydrolyzing it at 100-115° C. for 20-24 hours.

[0039] Wherein, in the step of alkaline hydrolysis treatment: the alkaline solution includes a lithium hydroxide solution with a concentration of 4 to 6 mol / L, and the mass volume ratio of the second sample to the lithium hydroxide solution is 1 mg: 0.05 to 0.50 mL.

[0040] In some embodiments, for the preparation of the second sample solution to be tested, the step of adjusting the alkaline hydrolysis sample solution to neutrality comprises: adding hydrochloric acid to the alkaline hydrolysis sample solution to pH=7.

[0041] The subsequent second dilution treatment step includes: diluting with water. In the diluted second test sample solution, the amino acid concentration can be within the linear range of the sample test object response, which is 10 nmol / L to 10 μmol / L.

[0042] Because the microalgae culture cycle is long, the biomass obtained under different experimental conditions is very precious. In the embodiment of the present application, the sample pretreatment process optimizes the sample weighing amount and tests it. 10 mg, 20 mg and 50 mg of microalgae freeze-dried powder are weighed for acid hydrolysis and alkaline hydrolysis respectively. Each weighing amount is parallel. The test results on the machine are converted to the mass dry weight ratio. It can be seen that the weighing amount of 10-50 mg does not affect the results in the 5 mL hydrolysis system, and it needs to be diluted thousands of times before the machine. Therefore, the 10 mg weighing amount is sufficient to meet the detection limit and quantitative limit of the method, and the linear range of the method is from 10 nmol / L to 10 μmol / L, and the linear range is wide enough to meet the amino acid quantification of samples with different protein contents, and it is also more suitable for the analysis of amino acids in microalgae with low protein content.

[0043] In some embodiments, in the step of liquid chromatography tandem mass spectrometry detection, the liquid chromatography conditions include: mobile phase A: formic acid-water solution; mobile phase B: formic acid-acetonitrile solution; flow rate: 0.15-0.25 mL / min; column temperature: 34-36°C; sample chamber temperature: 9-11°C.

[0044] Specifically, the volume concentration of formic acid in the mobile phase A is 0.05-0.15%, and the volume concentration of formic acid in the mobile phase B is 0.05-0.15%. Further, the mobile phase A and the mobile phase B are used for gradient elution, and the gradient elution program is: 0-3 minutes, keep 100% of the mobile phase A; 3-6 minutes, linearly change to 90% of the mobile phase A and 10% of the mobile phase B; 6-10 minutes, linearly change to 65% of the mobile phase A and 35% of the mobile phase B; 10-11 minutes, linearly change to 5% of the mobile phase A and 95% of the mobile phase B; 11-13.5 minutes, continue to keep 5% of the mobile phase A and 95% of the mobile phase B; 13.5-18 minutes, keep 100% of the mobile phase A for 4.5 minutes.

[0045] If ammonium formate buffer salt of a certain concentration and pH value is used, the experimental operation level is required to be high, and the pH value preparation is prone to errors. The effect of pH on amino acids may cause chromatographic peak overlap, tailing, and poor separation. However, in the embodiments of the present application, a simple 0.1% formic acid-water solution and 0.1% formic acid-acetonitrile solution mobile phase is used. The mobile phase does not need to add buffer salts to reduce the risk of clogging the chromatographic column and pipelines, and also reduces the time and reagent costs for preparing complex mobile phases. The mobile phase gradient starts with 100% aqueous phase, and the entire elution gradient consumes very little organic phase. The entire detection process not only reduces the frequency of maintaining instruments and chromatographic columns, but also saves corresponding reagent and consumable costs.

[0046] In some embodiments, in the step of liquid chromatography tandem mass spectrometry detection, the mass spectrometry conditions include: ion source: electrospray ion source; scanning mode: positive ion mode; capillary voltage: 2.5-3.3 kV; desolvation temperature: 540-560°C; desolvation gas flow rate: 800-1200 L / Hr; collision gas flow rate: 0.14-0.18 mL / min; ion source temperature: 140-160°C.

[0047] Some embodiments of the present application further use an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer. The triple quadrupole mass spectrometer can directly use the positive ion scanning mode of the ESI ion source to lock the mass-to-charge ratio of the target analyte, generate characteristic fragments according to the different cone voltages applied in the source and the collision voltage in the quadrupole, and use the multiple reaction monitoring mode to achieve targeted qualitative and quantitative analysis, and the samples do not need to be derivatized; ultra-high performance liquid chromatography-tandem mass spectrometry can greatly shorten the analysis time and achieve the purpose of batch sampling in a short time.

[0048] In summary, the method for detecting protein amino acids in microalgae provided in the embodiments of the present application has a simple pretreatment technology, does not require derivatization, reduces the influence of derivatization byproducts on the analyte and the problem of poor repeatability caused by derivatization efficiency. At the same time, the mobile phase in the liquid chromatography tandem mass spectrometry detection is simple to prepare, low in cost, high in analysis efficiency and sensitivity, and the detection limit can meet the requirements of protein amino acids in microalgae. It is a reliable method for rapid, comprehensive and accurate determination of protein amino acids in microalgae.

[0049] The following describes the invention in conjunction with specific embodiments.

[0050] Example

[0051] 1. Instrumental method

[0052] 1.1 Liquid phase condition setting

[0053] Chromatographic column: Waters ACQUITY HSS T3 column (150×2.1 mm, 1.7 μm);

[0054] Mobile phase: A: 0.1% formic acid-water solution; B: 0.1% formic acid-acetonitrile solution;

[0055] Flow rate: 0.2 mL / min;

[0056] Column temperature: 35°C;

[0057] Sample chamber temperature: 10°C;

[0058] Injection volume: 2 μL;

[0059] The elution gradient is shown in Table 1:

[0060] Table 1 Liquid phase elution gradient

[0061]

[0062] 1.2 Mass spectrometry condition settings

[0063] Ion source: electrospray ion source (ESI), scanning mode: positive ion mode (ES+);

[0064] Capillary voltage: 3kV;

[0065] Desolventization temperature: 550°C;

[0066] Desolvation gas flow rate: 1000L / Hr;

[0067] Collision gas flow rate: 0.16 mL / min;

[0068] Ion source temperature: 150°C.

[0069] 2. Standard curve drawing

[0070] 2.1 Preparation of acid standard solution

[0071] (I) Preparation of standard mixed solution and stock solution (stored at -80°C).

[0072] ① A mixed solution of 17 amino acid standards (purchased from Waters Technology Shanghai Co., Ltd.) with a concentration of 1 nmol / μL, i.e. 1 mmol / L, of which cystine is 0.5 mmol / L.

[0073] ②100 μmol / L 17 kinds of amino acid stock solution: Take 100 μL of the above standard mixed solution into a 1.5 mL centrifuge tube, add 0.1% formic acid water to 1 mL, mix well as the stock solution, and then dilute it step by step.

[0074] (ii) Concentration range and gradient dilution (the standard working solution was stored at 4°C).

[0075] ① A 0.1% formic acid aqueous solution was diluted in sequence (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.050, 0.025, 0.010 μmol / L).

[0076] ② Target compounds: aspartic acid (Asp), serine (Ser), glutamic acid (Glu), glycine (Gly), histidine (His), arginine (Arg), threonine (Thr), alanine (Ala), proline (Pro), cystine (2Cys), tyrosine (Tyr), valine (Val), methionine (Met), lysine (Lys), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe).

[0077] 2.2 Preparation of alkaline standard solution

[0078] Tryptophan (Trp) standard (purchased from Sigma, USA, stored at 4°C).

[0079] ①1mmol / L stock solution: weigh 2.04mg of tryptophan powder and dissolve it in 10mL of pure water.

[0080] ② Use pure water to perform gradient dilution in sequence (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.050, 0.025, 0.010 μmol / L).

[0081] According to the liquid phase and mass spectrometry conditions in the above instrumental method, a series of linear equations were obtained as shown in Table 2: the linear correlation coefficients of the 18 amino acids were all above 0.99, and they showed a good linear relationship in the range of 10 nmol / L to 10 μmol / L.

[0082] Table 2 Amino acid regression equation, linear coefficient, detection limit and quantification limit

[0083]

[0084] 3. Sample pretreatment and testing

[0085] 3.1 Equipment and consumables

[0086] Equipment: Ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer ACQUITY H-Class Plus-XevoTQ-XS, high-throughput sample disruptor TissueLyserIl (QIAGEN, Japan), water bath nitrogen blowdown apparatus WT-12A (Hangzhou Miou Instrument Co., Ltd.), and air drying oven DHG-9015A (Shanghai Yiheng Scientific Instrument Co., Ltd.).

[0087] Consumables: chromatographic grade acetonitrile (Merk), mass spectrometry grade formic acid (Fisher), 15 mL explosion-proof glass bottle (transparent bottle + gasket + cap), polytetrafluoroethylene tube (white bottle + cap), 2 mL centrifuge tube, 0.22 μm PTFE filter (acid-resistant), 1 mL syringe, pH test paper.

[0088] Solvent: 6mol / L hydrochloric acid: Take 100mL of concentrated hydrochloric acid, add 100mL of deionized water, and mix well. 5mol / L LiOH solution: Add 41.96g of LiOH·H2O into 200mL of deionized water and dissolve by ultrasonic.

[0089] 3.2 Determination of 17 amino acids in microalgae by acid hydrolysis

[0090] ① Sample hydrolysis

[0091] Take 50.0 mg of freeze-dried algae powder in a 2 mL centrifuge tube (3 parallels), add 4 ceramic beads, freeze in a -80 ° C refrigerator overnight, oscillate at 30 Hz for 10 min, repeat the oscillation three times, and finally add 1 mL of 6 mol / L HCl, oscillate again for 10 min to break the sample and facilitate transfer, use 4 mL of 6 mol / L HCl to transfer the algae solution in batches into 15 mL threaded transparent explosion-proof bottles, blow nitrogen for 1 min to remove the upper air in the bottle, quickly tighten the bottle cap, and place it in an oven at 110 ° C for constant temperature drying and hydrolysis for 24 h.

[0092] ②Sample filtration, deacidification, and dilution

[0093] Sample filtration: After the hydrolysis is completed, the sample is cooled to room temperature, then mixed, the tube is opened, and the hydrolyzate is transferred to a centrifuge tube by filtering with a 0.22 μm PTFE filter membrane.

[0094] Sample deacidification: Take 100 μL of hydrolyzate and evaporate to dryness in a centrifugal concentrator at 55°C / blow dry in a 55°C water bath with nitrogen for deacidification. Wash with water 1 to 2 times and evaporate to dryness if necessary.

[0095] Sample dilution: Dissolve the evaporated sample in 1 mL of 0.1% formic acid aqueous solution (i.e., dilute 10 times and keep as a backup), mix well, and obtain the total protein result according to the Dumas nitrogen analyzer. Continue to dilute to an appropriate multiple so that the sample analyte response is within the linear range (for example, for microalgae sample hydrolyzate with a protein content of more than 25%, 50 μL can be diluted to 1 mL (diluted 20 times) and mixed well, and then 100 μL can be diluted to 1 mL (diluted 10 times) in a sampling vial, mixed well, and tested, that is, the hydrolyzate is diluted 2000 times after drying), and stored at 4°C for testing.

[0096] 3.3 Determination of tryptophan in microalgae by alkaline hydrolysis

[0097] ① Sample hydrolysis

[0098] Take 50.0 mg of freeze-dried algae powder in a 2 mL centrifuge tube (3 parallels), add 4 ceramic beads, freeze in a -80 ° C refrigerator overnight, oscillate the crusher at 30 Hz for 10 min, repeat the oscillation three times, then place it at the bottom of a polytetrafluoroethylene tube, add 5 mL of 5 mol / L lithium hydroxide, blow nitrogen for 1 min to deoxygenate, quickly tighten the bottle cap, hydrolyze at 110 ° C for 24 h, and cool to room temperature.

[0099] ②Neutralization, filtration, volume determination and dilution of hydrolyzate

[0100] Cool, mix, open the tube, add appropriate amount of 6 mol / L hydrochloric acid, take 1 μL and measure with pH (1-14) test paper until it is neutral.

[0101] Filter with qualitative filter paper into a 25 mL volumetric flask, make up to volume with pure water, and mix again.

[0102] Dilution: According to the total protein result obtained by the Dumas nitrogen analyzer, continue to dilute the above-mentioned solution to an appropriate multiple so that the sample analyte response is within the linear range (for example, for microalgae samples with a protein content of more than 50%, dilute them 200 times in stages after the volume is fixed, and dilute as appropriate if the sample amount is small), and store at 4°C for testing.

[0103] 3.4 Sample testing

[0104] According to the above “1.1 Liquid Phase Condition Setting” and “1.2 Mass Spectrometry Condition Setting”, prepare the mobile phase, balance the chromatographic column, and prepare to analyze the mixed standard and actual samples in the MRM positive mode with the optimized mass spectrometry parameters (see Table 3). Figure 1 This is the MRM diagram of 18 amino acid mixed standards under this condition. The total running time is 18 minutes, and all 18 amino acids are eluted within 11 minutes, which greatly improves the analysis efficiency. To verify the stability of the method, 6 samples were taken as parallel samples and hydrolyzed together for detection on the machine.

[0105] Due to the polar functional groups in the amino acid structure, the common C18 column cannot achieve the effect of strong retention and better separation of multiple amino acids. Most of the existing LC-MS method literature uses HILIC and Amide columns with hydrophilic effects. The present application example compares the BEH Amide column and HSS T3 column of Waters. Due to the different column usage methods, the gradient elution ratio is different. By testing the same concentration mixed standard solution, such as Figure 2 The results show that the former column has severe Asp tailing and His tailing at low concentrations. The T3 column can not only achieve better separation effect, but also has symmetrical peaks without tailing, higher separation of isomers Ile and Leu, and higher response to standards of the same concentration. In order to avoid interference from miscellaneous peaks, the separation effects of 100mm and 150mm T3 columns were compared. It is obvious that the 150mm column shows better separation effect, which effectively avoids analyte peak overlap and interference from miscellaneous peaks in the sample. In addition, the T3 column is a common chromatographic column in the laboratory and is the preferred choice for analyzing polar compounds. It has a higher utilization rate and is more durable.

[0106] Table 4 shows the results of protein amino acids measured in actual sample microalgae (green algae from heterotrophic culture under different conditions) using the same detection method mentioned above. Among them, the three parallelisms of 18 amino acid contents in four microalgae samples (i.e., sample 1, sample 2, sample 3, and sample 4) were good, and the relative errors of the final mass ratio results were all below 0.3; the relative standard deviation RSD of the peak area responses of 6 parallel samples of the same sample of another microalgae sample (i.e., sample 5) was all below 0.04, indicating that the method has good stability.

[0107] Table 3 Mass spectrometry acquisition parameters for 18 amino acids in MRM mode

[0108]

[0109] Note: * indicates quantitative ion.

[0110] Table 4 Types, contents (unit: g / g) and stability of amino acids in microalgae samples

[0111]

[0112]

[0113] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for detecting protein amino acids in microalgae, characterized in that: include: providing a microalgae sample, and dividing the microalgae sample into a first sample and a second sample; The first sample is subjected to an acid hydrolysis treatment to obtain an acid hydrolysis sample solution, and the acid hydrolysis sample solution is subjected to a first dilution treatment after being subjected to a deacidification treatment to obtain a first sample solution to be tested; The second sample is subjected to alkaline hydrolysis treatment to obtain an alkaline hydrolysis sample solution, and the alkaline hydrolysis sample solution is adjusted to neutrality and then subjected to a second dilution treatment to obtain a second sample solution to be tested; The first sample solution to be tested and the second sample solution to be tested are subjected to liquid chromatography tandem mass spectrometry detection.

2. The detection method according to claim 1, characterized in that The acid hydrolysis treatment step includes: dissolving the first sample in an acid solution, and then hydrolyzing it at 100 to 115° C. for 20 to 24 hours; Alternatively, the alkaline hydrolysis treatment step includes: dissolving the second sample in an alkaline solution, and then hydrolyzing the second sample at 100 to 115° C. for 20 to 24 hours.

3. The detection method according to claim 2, characterized in that In the step of acid hydrolysis treatment: the acid solution includes a hydrochloric acid solution with a concentration of 4 to 6 mol / L, and the mass volume ratio of the first sample to the hydrochloric acid solution is 1 mg: 0.05 to 0.50 mL; Alternatively, in the alkaline hydrolysis treatment step: the alkaline solution includes a lithium hydroxide solution with a concentration of 4 to 6 mol / L, and the mass volume ratio of the second sample to the lithium hydroxide solution is 1 mg: 0.05 to 0.50 mL.

4. The detection method according to claim 1, characterized in that The deacidification step includes: blowing the acid hydrolyzed sample solution dry with nitrogen in a water bath at 55-60° C.; And / or, the first dilution treatment step includes: redissolving the sample obtained after the deacidification treatment with an aqueous formic acid solution.

5. The detection method according to claim 1, characterized in that The step of adjusting the alkaline hydrolysis sample solution to neutrality comprises: adding hydrochloric acid to the alkaline hydrolysis sample solution until the pH value is 7; And / or, the second dilution treatment step includes: diluting with water.

6. The detection method according to any one of claims 1 to 5, characterized in that: In the first sample solution obtained after the first dilution treatment, the amino acid concentration is 10 nmol / L to 10 μmol / L; And / or, in the first test sample solution obtained after the second dilution treatment, the amino acid concentration is 10 nmol / L to 10 μmol / L.

7. The detection method according to any one of claims 1 to 5, characterized in that: The step of liquid chromatography tandem mass spectrometry detection also includes detecting a standard solution, and the standard solution includes an acidic standard solution and an alkaline standard solution; wherein the standard substances in the acidic standard solution include aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, alanine, proline, cystine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine, and the standard substances in the alkaline standard solution include tryptophan.

8. The detection method according to any one of claims 1 to 5, characterized in that: In the step of liquid chromatography tandem mass spectrometry detection, the liquid chromatography conditions include: mobile phase A: formic acid-water solution; mobile phase B: formic acid-acetonitrile solution; flow rate: 0.15-0.25 mL / min; column temperature: 34-36° C.; sample chamber temperature: 9-11° C.

9. The detection method according to claim 8, characterized in that: The formic acid concentration in the mobile phase A is 0.05-0.15%, and the formic acid concentration in the mobile phase B is 0.05-0.15%; And / or, the mobile phase A and the mobile phase B are subjected to gradient elution, and the program of the gradient elution is: 0-3 min, maintaining 100% of the mobile phase A; 3-6 min, linearly changing to 90% of the mobile phase A and 10% of the mobile phase B; 6-10 min, linearly changing to 65% of the mobile phase A and 35% of the mobile phase B; 10-11 min, linearly changing to 5% of the mobile phase A and 95% of the mobile phase B; 11-13.5 min, continuing to maintain 5% of the mobile phase A and 95% of the mobile phase B; 13.5-18 min, maintaining 100% of the mobile phase A for 4.5 min.

10. The detection method according to any one of claims 1 to 5, characterized in that: In the step of liquid chromatography tandem mass spectrometry detection, the mass spectrometry conditions include: ion source: electrospray ion source; scanning mode: positive ion mode; capillary voltage: 2.5-3.3 kV; desolvation temperature: 540-560°C; desolvation gas flow rate: 800-1200 L / Hr; collision gas flow rate: 0.14-0.18 mL / min; ion source temperature: 140-160°C.

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