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

Method for identifying chlorella pyrenoidosa by using methylated sulfated oligosaccharide group Download PDF

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CN115166089A
CN115166089A CN202210804632.8A CN202210804632A CN115166089A CN 115166089 A CN115166089 A CN 115166089A CN 202210804632 A CN202210804632 A CN 202210804632A CN 115166089 A CN115166089 A CN 115166089A
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methylated
sulfated
chlorella pyrenoidosa
sulfated oligosaccharide
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赵雪
何启煜
张鸿伟
安子哲
张晓梅
卢海燕
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Ocean University of China
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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

Method for identifying chlorella pyrenoidosa by using methylated sulfated oligosaccharide group
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a method for identifying chlorella pyrenoidosa by utilizing a methylated sulfated oligosaccharide group.
Background
The annual output of microalgae in China is about ten thousand tons of dry powder, wherein 80 percent of the dry powder is spirulina, 10 percent of the dry powder is chlorella, and a small amount of haematococcus pluvialis and dunaliella salina exist. The microalgae is rich in nutritional ingredients such as protein, lipid, vitamins, natural pigments and minerals, and also has unique functional ingredients such as phycocyanin, astaxanthin, fucoxanthin, growth factors and the like, and has good potential for developing health foods. Especially spirulina and chlorella have high protein content, so that the spirulina and chlorella are mainly used as health food and functional food of protein sources.
Chlorella pyrenoidosa is rich in proteins, lipids, polysaccharides, growth factors, vitamins and other substances, has been certified by FAO of the food and agriculture organization of the United nations as a green health food, and is widely applied to the fields of cultivation, cosmetics, environmental protection, medicines and the like.
However, the price and the active ingredients of the chlorella pyrenoidosa, the spirulina and the dunaliella salina are greatly different, and the chlorella pyrenoidosa, the spirulina and the dunaliella salina are difficult to distinguish types when being added into food after being crushed and processed by spray drying. At present, the accurate method for detecting the microalgae species mainly adopts a PCR method, but the processing processes of wall breaking, spray drying and the like can damage the DNA of the microalgae, and simultaneously matrix interference exists, so that the PCR method cannot be adopted for detection. Therefore, an efficient and accurate identification method of microalgae varieties is needed.
The microalgae contains 10% -25% of polysaccharide, and has biological activities of resisting oxidation, reducing blood lipid, resisting asthma, resisting tumor, regulating immunity, protecting nerve, etc. The spirulina and chlorella polysaccharide has complex structure, mainly consists of glucose, galactose, mannose, rhamnose, xylose, arabinose, glucosamine, glucuronic acid and other monosaccharides, the sugar chain also has galactose, rhamnose, arabinose or uronic acid branched chains, and methylation, sulfate radical and acetylation modification exist in part of the sugar chain. Different microalgae species and different culture modes are different, so the composition and the structure of polysaccharide in microalgae are different.
The method for analyzing the microalgae polysaccharide structure mainly adopts NMR analysis, but the microalgae sulfated polysaccharide has large molecular weight and complex composition, so that NMR absorption peaks are overlapped, and the structural difference of sulfated polysaccharide of different varieties of microalgae cannot be identified. Meanwhile, NMR analysis requires multiple complicated separation and purification steps such as ion exchange chromatography purification, degradation, gel column purification, desalination and the like on polysaccharides, and the time consumption is long. Therefore, an analysis method for detecting microalgae polysaccharide with rapidness, simplicity and high resolution is urgently needed. With the maturation of the biological mass spectrometry technology, the analysis technology for analyzing the fine structure difference of complex polysaccharides by using mass spectrometry combined with glycomics technology has been matured.
Disclosure of Invention
The invention provides a method for identifying chlorella pyrenoidosa by utilizing a methylated sulfated oligosaccharide group, which establishes a method for identifying chlorella pyrenoidosa from the level of methylated sulfated oligosaccharides by extracting and degrading the methylated sulfated oligosaccharides of the chlorella pyrenoidosa and analyzing an oligosaccharide fingerprint spectrogram, thereby making up the defects of the prior art.
The invention firstly provides a methylated sulfated oligosaccharide combination for detecting chlorella pyrenoidosa, wherein the oligosaccharide combination 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.
Further, 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 ；
In still another aspect, the invention provides the use of the methylated sulfated oligosaccharide combination in the identification of Chlorella pyrenoidosa;
the invention also provides a method for detecting the chlorella pyrenoidosa, which uses the mass spectrum detection spectrogram of the methylated sulfated oligosaccharide combination as a standard;
the method comprises the following steps:
1) Carrying out 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 methylated sulfated oligosaccharide components of the sample by a hydrophilic high performance liquid chromatography-mass spectrometry combined method, analyzing the Chlorella pyrenoidosa components in the sample, and detecting whether the methylated sulfated oligosaccharide combination appears;
the method comprises the following steps of 1) carrying out hydrophilic high performance liquid chromatography-mass spectrometry pretreatment on a sample to be detected, wherein the steps are as follows:
1) Taking microalgal powder to be detected, and carrying out ultrasonic treatment for 5s at 200-400w for 5s in hot water at 60 ℃ for 20-40min;
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; and finishing the preparation of the chlorella pyrenoidosa sulfated oligosaccharide solution.
The hydrophilic high performance liquid chromatography-mass spectrometry combination method in the step 2) has the following chromatographic conditions:
the chromatographic column adopts a hydrophilic high performance liquid chromatographic column (2.0 multiplied by 150mm,5 mu m), the mobile phase A is a 10% acetonitrile ammonium acetate solution (ammonium acetate 10 mmol/L), the mobile phase B is a 90% acetonitrile-ammonium acetate solution (ammonium acetate 10 mmol/L), 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, the negative ion mode, the electrospray ion source and the mass analyzer areOrbitrap-Fourier transform, scanning molecular weight 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.
The step 2) of detecting whether the methylated sulfuric acid oligosaccharide combination appears or not is to compare the mass spectrum result of the sample to be detected with the mass spectrum spectrogram of the methylated sulfuric acid oligosaccharide combination, and when the mass spectrum detection spectrogram of each specific methylated sulfuric acid oligosaccharide of the oligosaccharide combination appears, the sample can be judged to contain the chlorella pyrenoidosa.
The method of the invention utilizes the detection of the control methylated sulfated oligosaccharide in the sample to be detected to identify the chlorella pyrenoidosa, and has 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 oligosaccharide mixture in the chlorella pyrenoidosa can be completed in one step, the steps of oligosaccharide ion exchange column purification, gel chromatographic column separation, desalination and the like are saved, and the detection time is greatly shortened. The method has high sensitivity and only needs 10g of microalgae powder. The mass spectrum can also detect all trace oligosaccharides in the oligosaccharide mixture, and the chlorella pyrenoidosa can be accurately identified by analyzing the control methylated sulfated oligosaccharides.
Drawings
FIG. 1: disaccharide (Hex) 1 (Rha) 1 (Sulph) 1 (Me) 1 In Thermo Q active TM Mass spectrum on mass spectrum.
FIG. 2: tetrasaccharide (Hex) 3 (Rha) 1 (SO 4 ) 2 (Me) 1 In Thermo Q active TM Mass spectrum on mass spectrum.
FIG. 3: pentasaccharide (Hex) 2 (Rha) 3 (SO 4 ) 2 (Me) 2 In Thermo Q active TM Mass spectrum on mass spectrum.
FIG. 4: six candies (Hex) 3 (Rha) 3 (SO 4 ) 2-3 (Me) 2 In Thermo Q active TM Mass spectrum on mass spectrum.
FIG. 5: seven candies (Hex) 4 (Rha) 3 (SO 4 ) 3 (Me) 2 In Thermo Q active TM Mass spectrum on mass spectrum.
FIG. 6: eight candies (Hex) 5 (Rha) 3 (SO 4 ) 3 (Me) 2 In Thermo Q active TM Mass spectrum on mass spectrum.
FIG. 7: eight candies (Hex) 4 (Rha) 4 (SO 4 ) 1-2 (Me) 1-2 In Thermo Q active TM Mass spectrum on mass spectrum.
FIG. 8: (Hex) 1 (Rha) 1 (Sulph) 1 (Me) 1 And (3) analyzing the spectrogram by high performance liquid chromatography-mass spectrometry.
FIG. 9: (Hex) 3 (Rha) 1 (SO 4 ) 2 (Me) 1 And (3) analyzing the spectrogram by high performance liquid chromatography-mass spectrometry.
FIG. 10: (Hex) 2 (Rha) 3 (SO 4 ) 2 (Me) 2 And analyzing the spectrogram by high performance liquid chromatography-mass spectrometry.
FIG. 11: (Hex) 3 (Rha) 3 (SO 4 ) 1-2 (Me) 2 And (3) analyzing the spectrogram by high performance liquid chromatography-mass spectrometry.
FIG. 12: (Hex) 4 (Rha) 3 (SO 4 ) 3 (Me) 2 And (3) analyzing the spectrogram by high performance liquid chromatography-mass spectrometry.
FIG. 13: (Hex) 5 (Rha) 3 (SO 4 ) 3 (Me) 2 And (3) analyzing the spectrogram by high performance liquid chromatography-mass spectrometry.
FIG. 14: (Hex) 4 (Rha) 4 (SO 4 ) 1-2 (Me) 1-2 And (3) analyzing the spectrogram by high performance liquid chromatography-mass spectrometry.
FIG. 15: the total ion flow diagram of the oligosaccharide of the chlorella pyrenoidosa in the high performance liquid chromatography-mass spectrometry combination.
Detailed Description
The invention utilizes a liquid chromatography-mass spectrometry combined technology to detect the full fingerprint spectrum of the oligosaccharide of the chlorella pyrenoidosa, and identifies the specific methylated sulfated oligosaccharide of the chlorella pyrenoidosa by comparing the oligosaccharide from the chlorella vulgaris, the ellipsoidal chlorella, the spirulina and other green algae.
The present invention will be described in detail with reference to examples.
Example 1 Structure and comparative information on Chlorella pyrenoidosa-derived specific sulfated oligosaccharides
1. Methylated sulfated oligosaccharides unique to Chlorella pyrenoidosa
Methylated sulfated oligosaccharides (Hex) unique to Chlorella pyrenoidosa m (Rha) n (SO 4 ) x (Me) y (m, n =1-4, x =1-3, y = 1-2), is derived from Chlorella pyrenoidosa sulfated polysaccharide, is composed of rhamnose and hexose (galactose, glucose or mannose) linkage, and has methylation modification on sulfated hexose and rhamnose. While the sulfated oligosaccharides of Chlorella vulgaris, chlorella ellipsoidea, spirulina and other green algae are only sulfated oligosaccharides composed of hexose and rhamnose, and sulfated oligosaccharides without methylation modification, therefore (Hex) m (Rha) n (SO 4 ) x (Me) y (m, n =1-4, x =1-3, y = 1-2) is a methylated sulfated oligosaccharide unique to Chlorella pyrenoidosa.
Adopts Thermo Scientific Q active TM Mass spectrograms of the target methylated sulfated oligosaccharides were detected by mass spectrometry, and the results are shown in FIGS. 1 to 7. (Hex) m (Rha) n (SO 4 ) x (Me) y The sugar chain composition, parent ion and charge number of (m, n =1-4, x =1-3, y = 1-2) are shown in table 1.
Table 1: sugar chain composition, ion-to-mass ratio and charge of the target sulfated oligosaccharide in the chlorella pyrenoidosa.
Figure BDA0003736373990000051
Figure BDA0003736373990000061
Wherein R isha: rhamnose; hex: hexoses, including galactose, glucose or mannose; SO (SO) 4 : sulfate, me: a methyl group; the numbers represent the number of monosaccharides or sulfates.
Example 2 detection procedure for Chlorella pyrenoidosa
The steps for analyzing the algae powder sample to be detected are as follows:
one) sample pretreatment step:
(1) Weighing 10g of powder, and ultrasonically treating with ultrasonic wave at 60 deg.C in hot water at 200w for 5s and 5s for 60min. 300mL of 0.1mol/L phosphoric acid buffer solution having a pH of 8.0 and 15mmol/L EDTA-Na were added 2 5mmol/L cysteine, 0.3g neutral protease and 0.3g papain, and stirring in a water bath at 50 ℃ for enzymolysis for 24 hours. Then the reactant is inactivated in boiling water bath for 5min and cooled to room temperature. Centrifuging at 5000 Xg for 10min, and collecting supernatant.
(2) Concentrating the supernatant obtained in the step 1) to 150mL, adding 150mL of 95% ethanol, standing at 4 ℃ for 5h, centrifuging (4000 Xg, 15min,20 ℃), and taking the precipitate; the supernatant was added with 300mL of 95% ethanol, left at 4 ℃ for 5 hours, centrifuged (4000 Xg, 15min,20 ℃) and the precipitate was collected. The precipitates were combined and washed 2 times with 50mL 95% ethanol to obtain a mixture of sulfated polysaccharides.
(3) Dissolving the crude sulfated polysaccharide obtained in the step 2) in 50mL of aqueous solution, dialyzing by using a 5000Da dialysis bag to remove salt, and then concentrating and freeze-drying.
(4) Preparing the freeze-dried sample into 5mg/mL, adding 10% acetic acid solution to adjust the pH value to 4.0, and degrading at 110 ℃ for 30min; the preparation of the chlorella pyrenoidosa sulfated oligosaccharide is completed.
(5) And (4) taking 100 mu L of the chlorella pyrenoidosa sulfated oligosaccharide solution in the step (4), adding 100 mu L of acetonitrile, centrifuging at 10000 Xg for 10min, and taking supernatant. And (5) waiting for computer detection.
II) liquid chromatography and mass spectrometry conditions
The experimental instruments and reagents were as follows:
liquid chromatography: agilent 1260, ms: thermo Scientific Q active TM A mass spectrometer. Adding the sulfated polysaccharide degradation product obtained in the step (4) into acetonitrile with the same volume, and centrifuging (10000 Xg)10min,20 deg.C. Hydrophilic high performance liquid chromatography conditions: a chromatographic column: luna HILIC column (2.0X 150mm,5 μm), mobile phase A:10% acetonitrile-ammonium acetate solution (ammonium acetate 10 mmol/L), mobile phase B:90% acetonitrile-ammonium acetate solution (ammonium acetate 10 mmol/L), the sample amount is 4 μ L, the elution gradient of mobile phase A is 10% -30%, elution is carried out for 0-40min, and the flow rate is 150 μ L/min;
conditions of mass spectrum: using Thermo Q active TM A mass spectrometer. Negative ion mode, electrospray ion source, mass analyzer for Orbitrap-Fourier transform, scanning molecular weight 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.
Three) analysis of results
1. Identification of methylated sulfated oligosaccharides of Chlorella pyrenoidosa
Through crude sugar extraction, degradation and liquid chromatography-mass spectrometry combined analysis of chlorella pyrenoidosa, 9 characteristic methylated sulfated oligosaccharides are found in a sulfated oligosaccharide mixture according to the charge-to-mass ratio of target oligosaccharide ions. According to the extracted ion chromatogram in the liquid chromatography-mass spectrometry analysis of the target oligosaccharides, the ion intensity of each charge is calculated, and the contents of the ions are compared. FIGS. 8 to 14 show different combinations of analysis spectra in HPLC-MS; the sugar chain composition, charge-to-mass ratio, number of charges and total ionic strength of the ions are shown in Table 2.
Table 2: ionic charge-mass ratio, charge number and total ionic strength table of sulfated oligosaccharide detected in chlorella pyrenoidosa in mass spectrum
Figure BDA0003736373990000081
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 The highest content of (A). Say thatThe sulfuric acid rhamnose hexose of the chlorella pyrenoidosa contains the most methylated and sulfated octasaccharide, heptasaccharide and hexasaccharide oligosaccharide fragments, and has sulfate modification and methylation modification.

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.
CN202210804632.8A 2022-07-08 2022-07-08 Method for identifying chlorella pyrenoidosa by using methylated sulfated oligosaccharide group Pending CN115166089A (en)

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Publication number Priority date Publication date Assignee Title
CN116535532A (en) * 2023-02-07 2023-08-04 广西中医药大学 Chlorella mannogalactan or sulfate compound and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116535532A (en) * 2023-02-07 2023-08-04 广西中医药大学 Chlorella mannogalactan or sulfate compound and application thereof
CN116535532B (en) * 2023-02-07 2024-03-29 广西中医药大学 Chlorella mannogalactan or sulfate compound and application thereof

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