CN115166089A - Method for identifying chlorella pyrenoidosa by using methylated sulfated oligosaccharide group - Google Patents

Abstract

The invention provides a methylated sulfated oligosaccharide combination for detecting chlorella pyrenoidosa and a detection method, which are used for identifying chlorella pyrenoidosa by using the detection of the control methylated sulfated oligosaccharide in chlorella powder, and have the advantages of good specificity and accurate and reliable analysis. The detection method has the characteristics of simplicity, convenience, rapidness, high sensitivity and high resolution. By adopting a liquid chromatography-mass spectrometry combined technology, the separation and analysis of the microalgae sulfated oligosaccharide mixture can be completed in one step, the steps of purification, desalination and the like of sulfated oligosaccharides are saved, and the detection time is greatly shortened. The method has high sensitivity, and the required microalgae sample amount is only 10g. The mass spectrum can also detect all trace sulfated oligosaccharides in the oligosaccharide mixture to obtain a full fingerprint spectrogram of sulfated oligosaccharides of the chlorella pyrenoidosa, and the chlorella pyrenoidosa can be accurately identified by analyzing the methylated sulfated oligosaccharides.

Description

A method for identifying Chlorella pyrenoidosa using methylated sulfated oligosaccharide groups

technical field

The invention belongs to the technical field of food detection, in particular to a method for identifying Chlorella pyrenoidosa by using methylated sulfate oligosaccharide group.

Background technique

The annual output of microalgae in my country is about 10,000 tons of dry powder, of which 80% is Spirulina, 10% is Chlorella, and a small amount of Haematococcus pluvialis and Dunaliella salina. Microalgae are rich in nutrients such as protein, lipids, vitamins, natural pigments and minerals, as well as unique functional components such as phycocyanin, astaxanthin, fucoxanthin, growth factors, etc., which have good development and health. potential of food. Especially spirulina and chlorella have high protein content, so they are mainly used as protein source health food and functional food.

Chlorella pyrenoidosa is rich in proteins, lipids, polysaccharides, growth factors, vitamins and other substances. It has been certified as a green and healthy food by the United Nations Food and Agriculture Organization (FAO) and is widely used in aquaculture, cosmetics, environmental protection and medicine. .

However, the prices and active ingredients of Chlorella pyrenoidea, Spirulina and Dunaliella salina are quite different. After crushing and spray drying, it is difficult to distinguish the species when added to food. At present, the accurate detection method of microalgae species mainly adopts PCR method. However, the processing process such as wall breaking and spray drying will destroy the DNA of microalgae, and there is also matrix interference, so PCR method cannot be used for detection. Therefore, it is necessary to establish an efficient and accurate identification method of microalgae species.

There are 10%-25% polysaccharides in microalgae, which have biological activities such as antioxidant, hypolipidemic, anti-asthma, anti-tumor, immune regulation and neuroprotective activities. The polysaccharides of spirulina and chlorella are complex in structure, mainly composed of glucose, galactose, mannose, rhamnose, xylose, arabinose, glucosamine, glucuronic acid and other monosaccharides. Branched chains of lactose, rhamnose, arabinose or uronic acid, some of which have methylation, sulfate and acetylation modifications. Different microalgae species and culture methods are different, so the composition and structure of polysaccharides in microalgae are also different.

The method of analyzing the structure of microalgal polysaccharide mainly adopts NMR analysis, but the microalgal sulfated polysaccharide has a large molecular weight and a complex composition, so the NMR absorption peaks overlap, and the structural differences of sulfated polysaccharides of different varieties of microalgae cannot be identified. At the same time, NMR analysis requires multi-step complex separation and purification steps such as ion exchange chromatography purification, degradation, gel column purification and desalting of polysaccharides, which takes a long time. Therefore, there is an urgent need for a more rapid, simple, and high-resolution analytical method for the detection of microalgal polysaccharides. With the maturity of biological mass spectrometry technology, the analysis technology of using mass spectrometry combined with glycomic technology to analyze the fine structure difference of complex polysaccharides has matured.

SUMMARY OF THE INVENTION

The invention provides a method for using methylated sulfated oligosaccharide group to identify Chlorella pyrenoidosa. The extraction, degradation and oligosaccharide fingerprint analysis of methylated sulfated oligosaccharide of Chlorella pyrenoidosa are established to establish A method for identifying Chlorella pyrenoidosa from the level of methylated sulfated oligosaccharides is presented, thereby making up for the deficiencies of the prior art.

The present invention first provides a combination of methylated sulfated oligosaccharides for detecting Chlorella pyrenoidosa, wherein the oligosaccharide combination is (Hex) _1-4 (Rha) _1-4 (SO ₄ ) _1-3 ( Me) _1-3 ; Wherein Hex is hexose, including galactose, glucose or mannose; Rha is rhamnose; SO ₄ is sulfate; Me is methyl; The number represents the number of monosaccharide or sulfate.

Further, the combination of methylated sulfated oligosaccharides is (Hex) ₄ (Rha) ₄ (SO ₄ ) ₃ (Me) ₂ , (Hex) ₃ (Rha) ₃ (SO ₄ ) ₂ (Me) ₂ and (Hex) ₄ (Rha) ₃ (SO ₄ ) ₃ (Me) ₂ ;

Another aspect of the present invention provides the application of the methylated sulfated oligosaccharide combination in the identification of Chlorella pyrenoidosa;

The present invention also provides a method for detecting Chlorella pyrenoidea, which uses the above-mentioned mass spectrometry detection spectrum of the combination of methylated sulfated oligosaccharides as a standard;

The method includes the following steps:

1) The sample to be tested is subjected to hydrophilic high performance liquid chromatography-mass spectrometry pretreatment to obtain the methylated sulfate oligosaccharide solution to be tested;

2) testing the methylated sulfate oligosaccharide composition of the sample by hydrophilic high performance liquid chromatography-mass spectrometry, analyzing the Chlorella pyrenoidosa composition in the sample, and detecting whether the methylated sulfate oligosaccharide combination occurs;

In the described 1), the sample to be tested is carried out hydrophilic high performance liquid chromatography-mass spectrometry pretreatment, and the steps are as follows:

1) Take the microalgae powder to be detected, and ultrasonically treat it in hot water at 60°C for 5s at 200-400w for 5s, and treat for 20-40min;

2) Add 15mmol/L EDTA-Na ₂ , 5mmol/L cysteine, 0.1mol/L pH 8.0 phosphate buffer solution, neutral protease and papain, and enzymolysis at 40-50°C for 10-24h; After 5 minutes of boiling water bath to inactivate the enzyme, centrifuge, and take the supernatant;

3) Add 95% ethanol, place at 4°C for 24 hours, and after centrifugation, collect the precipitate to complete the extraction of sulfated polysaccharide from Chlorella pyrenoidosa;

4) Dissolving the sulfated polysaccharide of Chlorella pyrenoidosa in the aqueous solution, adding 10% acetic acid solution to adjust the pH value to 3-5, and degrading it at 100-120° C. for 20-40 min; preparation.

Described 2) in the hydrophilic high performance liquid chromatography-mass spectrometry method, its chromatographic condition is as follows:

The chromatographic column was a hydrophilic high performance liquid chromatography column (2.0×150mm, 5μm), mobile phase A was 10% acetonitrile ammonium acetate solution (ammonium acetate 10mmol/L), mobile phase B was 90% acetonitrile-ammonium acetate solution (ammonium acetate) 10mmol/L), the injection volume is 4μL, the elution gradient of mobile phase A is 10%-30%, the elution is 0-40min, and the flow rate is 150μL/min;

The conditions of mass spectrometry are as follows: Thermo Q Exactive ^TM mass spectrometer, negative ion mode, electrospray ion source, Orbitrap-Fourier transform mass analyzer, scanning molecular weight 100-1500 m/z; capillary voltage -35V, tube lens compensation voltage -40V, the capillary temperature was set to 270L/min, the sheath flow rate was 28L/min, the flow rate of the auxiliary gas was 6L/min, and processed in Xcalibur mass spectrometry software.

In described 2), to detect whether the combination of methylated sulfated oligosaccharides occurs, is to compare the mass spectrum results of the sample to be tested with the mass spectrogram of the combination of methylated sulfated oligosaccharides, and the above-mentioned combination of oligosaccharides appears. When the mass spectrometry of each specific methylated sulfated oligosaccharide is detected, it can be judged that the sample contains Chlorella pyrenoidosa.

The method of the invention utilizes the detection of the control methylated sulfated oligosaccharide in the sample to be tested to identify Chlorella pyrenoidosa, with good specificity and accurate and reliable analysis. The detection method has the characteristics of simplicity, rapidity, high sensitivity and high resolution. Using liquid chromatography-mass spectrometry, the separation and analysis of oligosaccharide mixtures in Chlorella pyrenoidosa can be completed in one step, saving the steps of ion exchange column purification, gel chromatography column separation and desalting of oligosaccharides, and greatly shortening the time. detection time. This method has high sensitivity and only needs 10g of microalgae powder. Mass spectrometry can also detect all traces of oligosaccharides in the oligosaccharide mixture, which can accurately identify Chlorella pyrenoidosa by the analysis of methylated sulfated oligosaccharides.

Description of drawings

Figure 1 : Mass spectrum of the disaccharide (Hex) ₁ (Rha) ₁ (Sulph) ₁ (Me) ₁ on a Thermo Q Exactive ^™ mass spectrometer.

Figure 2: Mass spectrum of the tetrasaccharide (Hex) ₃ (Rha) ₁ (SO ₄ ) ₂ (Me) ₁ on a Thermo Q Exactive ^™ mass spectrometer.

Figure 3: Mass spectrum of the pentasaccharide (Hex) ₂ (Rha) ₃ (SO ₄ ) ₂ (Me) ₂ on a Thermo Q Exactive ^™ mass spectrometer.

Figure 4: Mass spectrum of the hexasaccharide (Hex) ₃ (Rha) ₃ (SO ₄ ) _2-3 (Me) ₂ on a Thermo Q Exactive ^™ mass spectrometer.

Figure 5: Mass spectrum of the heptaose (Hex) ₄ (Rha) ₃ (SO ₄ ) ₃ (Me) ₂ on a Thermo Q Exactive ^™ mass spectrometer.

Figure 6: Mass spectrum of octasaccharide (Hex) ₅ (Rha) ₃ (SO ₄ ) ₃ (Me) ₂ on a Thermo Q Exactive ^™ mass spectrometer.

Figure 7: Mass spectrum of octasaccharide (Hex) ₄ (Rha) ₄ (SO ₄ ) _1-2 (Me) _1-2 on a Thermo Q Exactive ^™ mass spectrometer.

Figure 8: Analysis spectrum of (Hex) ₁ (Rha) ₁ (Sulph) ₁ (Me) ₁ by HPLC-MS.

Figure 9: Analysis spectrum of (Hex) ₃ (Rha) ₁ (SO ₄ ) ₂ (Me) ₁ by HPLC-MS.

Figure 10: Analysis spectrum of (Hex) ₂ (Rha) ₃ (SO ₄ ) ₂ (Me) ₂ by HPLC-MS.

Figure 11: Analytical spectrum of (Hex) ₃ (Rha) ₃ (SO ₄ ) _1-2 (Me) ₂ by high performance liquid chromatography-mass spectrometry.

Figure 12: Analytical chromatogram of (Hex) ₄ (Rha) ₃ (SO ₄ ) ₃ (Me) ₂ by high performance liquid chromatography-mass spectrometry.

Figure 13: Analytical chromatogram of (Hex) ₅ (Rha) ₃ (SO ₄ ) ₃ (Me) ₂ by high performance liquid chromatography-mass spectrometry.

Figure 14: Analytical spectrum of (Hex) ₄ (Rha) ₄ (SO ₄ ) _1-2 (Me) _1-2 by high performance liquid chromatography-mass spectrometry.

Figure 15: Total ion chromatogram of oligosaccharides from Chlorella pyrenoidosa by high performance liquid chromatography-mass spectrometry.

Detailed ways

The invention uses liquid chromatography-mass spectrometry to detect the full fingerprint of the oligosaccharides of Chlorella pyrenoidosa, and compares the oligosaccharides from Chlorella vulgaris, Chlorella ellipsoidea, Spirulina and other green algae to identify Methylated sulfated oligosaccharides unique to Chlorella pyrenoidosa.

The present invention will be described in detail below with reference to the embodiments.

Example 1. Structure and comparative information of sulfated oligosaccharides derived from Chlorella pyrenoidea

1. The unique methylated sulfated oligosaccharide of Chlorella pyrenoidosa

Unique methylated sulfated oligosaccharide (Hex) _m (Rha) _n (SO ₄ ) _x (Me) _y (m,n=1-4,x=1-3,y=1 in Chlorella pyrenoidea -2), in the sulfated polysaccharide derived from Chlorella pyrenoidosa, it is composed of rhamnose and hexose (galactose, glucose or mannose) linked, and there are methylation modifications on sulfated hexose and rhamnose . In the sulfated oligosaccharides of Chlorella vulgaris, Chlorella ellipsoidea, Spirulina and other green algae, there are only sulfated oligosaccharides composed of hexose and rhamnose, and there is no methylated sulfated oligosaccharide, so (Hex ) _m (Rha) _n (SO ₄ ) _x (Me) _y (m,n=1-4,x=1-3,y=1-2) is the unique methylated sulfuric acid in Chlorella pyrenoidosa Oligosaccharides.

The mass spectra of the target methylated sulfated oligosaccharides were detected by the Thermo Scientific Q Exactive ^TM mass spectrometer, and the results are shown in Figures 1 to 7. (Hex) _m (Rha) _n (SO ₄ ) _x (Me) _y (m,n=1-4,x=1-3,y=1-2) sugar chain composition, parent ion and charge number are shown in the table 1 shown.

Table 1: Sugar chain composition, ion-to-mass ratio and electric charge of target sulfated oligosaccharides in Chlorella pyrenoidea.

Rha: rhamnose; Hex: hexose, including galactose, glucose or mannose; SO ₄ : sulfate, Me: methyl; the numbers represent the number of monosaccharides or sulfates.

Example 2 Detection steps for Chlorella pyrenoidosa

The steps for analyzing the algal powder sample to be tested are as follows:

1) Sample preprocessing steps:

(1) Solid dry algal powder, weigh 10g of powder, ultrasonically treat it with ultrasonic waves at 200w under 60°C hot water for 5s, stop for 5s, and treat for 60min. 300 mL of 0.1 mol/L pH 8.0 phosphate buffer solution, 15 mmol/L EDTA-Na ₂ , 5 mmol/L cysteine, 0.3 g neutral protease and 0.3 g papain were added, and the hydrolysis was carried out under stirring in a water bath at 50°C for 24 h. After that, the reaction was inactivated by boiling water bath for 5 min, and cooled to room temperature. Centrifuge at 5000 × g for 10 min and take the supernatant.

(2) Concentrate the supernatant obtained in step 1) to 150 mL, add 150 mL of 95% ethanol, place at 4 °C for 5 h, centrifuge (4000 × g, 15 min, 20 °C), and take the precipitate; add 300 mL of 95 % ethanol, placed at 4°C for 5h, centrifuged (4000×g, 15min, 20°C), and the precipitate was collected. The precipitates were combined and washed twice with 50 mL of 95% ethanol to obtain a mixture of sulfated polysaccharides.

(3) Dissolving the crude sulfated polysaccharide obtained in step 2) in 50 mL of aqueous solution, using a 5000 Da dialysis bag to remove salt by dialysis, and then concentrating and lyophilizing.

(4) The lyophilized sample was made into 5 mg/mL, 10% acetic acid solution was added to adjust the pH value to 4.0, and then degraded at 110° C. for 30 min; the preparation of sulfated oligosaccharide of Chlorella pyrenoidosa was completed.

(5) Take 100 μL of the sulfated oligosaccharide solution of Chlorella pyrenoidosa in step (4), add 100 μL of acetonitrile, centrifuge at 10000×g for 10 min, and take the supernatant. Standby check.

2) Liquid chromatography and mass spectrometry conditions

The experimental instruments and reagents are as follows:

Liquid chromatography: Agilent 1260, Mass spectrometry: Thermo Scientific Q Exactive ^™ mass spectrometer. The sulfated polysaccharide degradation product obtained in step (4) was added to an equal volume of acetonitrile, and centrifuged (10000×g, 10 min, 20° C.). Hydrophilic high performance liquid chromatography conditions: Column: Luna HILIC column (2.0×150mm, 5μm), mobile phase A: 10% acetonitrile-ammonium acetate solution (ammonium acetate 10mmol/L), mobile phase B: 90% acetonitrile-acetic acid Ammonium solution (ammonium acetate 10mmol/L), the injection volume is 4μL, the elution gradient of mobile phase A is 10%-30%, 0-40min elution, flow rate 150μL/min;

Conditions for mass spectrometry: A Thermo Q Exactive ^™ mass spectrometer was used. Negative ion mode, electrospray ion source, Orbitrap-Fourier transform mass analyzer, scanning molecular weight 100-1500m/z. Capillary voltage -35V, tube lens compensation voltage -40V, capillary temperature 270L/min, sheath flow 28L/min, auxiliary gas flow 6L/min, processed in Xcalibur mass spectrometry software.

3) Result analysis

1. Identification of methylated sulfated oligosaccharides from Chlorella pyrenoidosa

According to the charge-to-mass ratio of the target oligosaccharide ions, 9 characteristic methylated sulfated oligosaccharides were found in the sulfated oligosaccharide mixture through the extraction, degradation and liquid chromatography-mass spectrometry analysis of crude sugar in Chlorella pyrenoidosa. Based on the extracted ion chromatograms in the liquid chromatography-mass spectrometry analysis of these target oligosaccharides, the ionic strength of each charge was calculated, and the content of each ion was compared. Figures 8 to 14 are the analysis spectra of different combinations in HPLC-MS;

Table 2: Ion-to-mass ratio, number of charges and total ionic strength of sulfated oligosaccharides detected in Chlorella pyrenoidosa in mass spectrometry

The results show that (Hex) ₄ (Rha) ₄ (SO ₄ ) ₃ (Me) ₂ , (Hex) ₃ (Rha) ₃ (SO ₄ ) ₂ (Me) ₂ , (Hex) ₄ (Rha) ₃ (SO ₄ ) ) ₃ (Me) ₂ has the highest content. It indicated that the methylated and sulfated octasaccharide, heptasaccharide and hexasaccharide oligosaccharide fragments were the most abundant in the rhamno-hexose sulfate sugars of Chlorella pyrenoidea, and there were sulfate and methylation modifications on them.

Claims (8)

1. A methylated sulfated oligosaccharide composition for detecting Chlorella pyrenoidosa is characterized in that the oligosaccharide composition is (Hex) _1-4 (Rha) _1-4 (SO ₄ ) _1-3 (Me) _1-3 (ii) a Wherein Hex is hexose, and contains galactose, glucose or mannose; rha is rhamnose; SO (SO) ₄ Is sulfate radical; me is methyl; the numbers represent the number of monosaccharides or sulfates.

2. The methylated sulfated oligosaccharide combination of claim 1, wherein the methylated sulfated oligosaccharide combination is (Hex) ₄ (Rha) ₄ (SO ₄ ) ₃ (Me) ₂ 、(Hex) ₃ (Rha) ₃ (SO ₄ ) ₂ (Me) ₂ And (Hex) ₄ (Rha) ₃ (SO ₄ ) ₃ (Me) ₂ 。

3. Use of a methylated sulfated oligosaccharide combination as claimed in claim 1 or claim 2 for the identification of chlorella pyrenoidosa.

4. A method for detecting chlorella pyrenoidosa, wherein the method comprises using a mass spectrometric detection profile of the methylated sulfated oligosaccharide combination of claim 1 or claim 2 as a standard for detection.

5. The method of claim 4, wherein the method comprises the steps of:

1) Performing hydrophilic high performance liquid chromatography-mass spectrometry pretreatment on a sample to be detected to obtain a methylated sulfated oligosaccharide solution to be detected;

2) Detecting the methylated sulfated oligosaccharide component of the sample by a hydrophilic high performance liquid chromatography-mass spectrometry, analyzing the Chlorella pyrenoidosa component in the sample, and detecting whether a mass spectrometry detection spectrogram of the methylated sulfated oligosaccharide combination of claim 1 or 2 appears.

6. The method as claimed in claim 5, wherein the step of 1) performing hydrophilic high performance liquid chromatography-mass spectrometry pretreatment on the sample to be tested comprises the following steps:

1) Taking microalgae powder to be detected, and performing ultrasonic treatment in hot water by using ultrasonic waves

2) Adding 15mmol/L EDTA-Na ₂ 5mmol/L cysteine, 0.1mol/L phosphoric acid buffer solution with pH of 8.0, neutral protease and papain, and carrying out enzymolysis for 10-24h at 40-50 ℃; deactivating enzyme of the reactant in boiling water bath for 5min, centrifuging, and collecting supernatant;

3) Adding 95% ethanol, standing at 4 deg.C for 24 hr, centrifuging, collecting precipitate, and extracting Chlorella pyrenoidosa sulfated polysaccharide;

4) Dissolving Chlorella pyrenoidosa sulfated polysaccharide in water solution, adding 10% acetic acid solution to adjust pH to 3-5, and degrading at 100-120 deg.C for 20-40min; the preparation of the chlorella pyrenoidosa sulfated oligosaccharide solution is completed.

7. The method as claimed in claim 5, wherein the hydrophilic HPLC-MS in 2) is performed under the following chromatographic conditions:

the chromatographic column adopts a hydrophilic high performance liquid chromatographic column, the mobile phase A is a 10% acetonitrile ammonium acetate solution, the mobile phase B is a 90% acetonitrile-ammonium acetate solution, the sample injection amount is 4 mu L, the elution gradient of the mobile phase A is 10% -30%, the elution is carried out for 0-40min, and the flow rate is 150 mu L/min;

the mass spectrometry conditions were as follows: using Thermo Q active ^TM The mass spectrometer is in a negative ion mode, an electrospray ion source and a mass analyzer are Orbitrap-Fourier transform, and the scanning molecular weight is 100-1500m/z; the capillary voltage is-35V, the compensation voltage of the sleeve lens is-40V, the temperature of the capillary is set to be 270L/min, the flow of the sheath layer is 28L/min, the flow of the auxiliary gas is 6L/min, and the processing is carried out in Xcalibur mass spectrum software.

8. The method of claim 5, wherein the step 2) of detecting whether the methylated sulfated oligosaccharide combination appears or not is to compare the mass spectrum result of the sample to be detected with the mass spectrum of the methylated sulfated oligosaccharide combination, and when the mass spectrum detection spectrum of each specific methylated sulfated oligosaccharide of the oligosaccharide combination appears, the sample can be judged to contain the chlorella pyrenoidosa.

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