A method for extracting freeze-dried puree enriched in polypeptide species from cactus fruit pomace

Abstract

This invention discloses a freeze-dried fruit pulp extraction method for enriching polypeptides from cactus pomace, comprising the following steps: Step 1: Raw material pretreatment: Select fresh cactus pomace, remove residual pulp and impurities, dry in a vacuum freeze-drying device, pulverize and pass through a sieve to prepare freeze-dried fruit pulp for later use; Step 2: Water extraction sample preparation: Weigh the freeze-dried fruit pulp, add distilled water for the first reflux extraction; filter and collect the supernatant, reflux extraction again; combine the two supernatants, concentrate in a vacuum concentrator until no obvious water remains, and then dry under vacuum to obtain the water-extracted sample; Step 3: 60% ethanol extraction sample preparation: Weigh the freeze-dried fruit pulp, add 60% ethanol solution as the extraction solvent to obtain the 60% ethanol extraction sample; Step 4: Pretreatment for LC-MS detection; Step 5: LC-MS / MS detection. This process has clear parameters and significant polypeptide enrichment effect, and is suitable for the efficient extraction and research application of cactus polypeptides.

Description

Technical Field

[0001] This invention relates to the field of natural plant polypeptide extraction and enrichment technology, and in particular to a freeze-dried fruit pulp extraction method for enriching polypeptide types in cactus pomace. Background Technology

[0002] Cactus pomace is rich in polypeptides with potential bioactivity, but it is often discarded as waste during processing, resulting in a waste of polypeptide resources.

[0003] Current research on the extraction of cactus peptides has significant shortcomings:

[0004] First, the pretreatment method was improper, directly using fruit juice or unfrozen fruit pomace as raw materials, resulting in extremely low detection levels of polypeptide types (only 54 types in fruit juice), which failed to fully exploit polypeptide resources.

[0005] Secondly, the extraction process parameters are vague, and there is a lack of standardized solvent dosage and extraction time settings, resulting in large differences in the types of peptides in different batches of samples.

[0006] Third, the desalting step before detection is missing or not standardized, and impurities interfere with the accuracy of LC-MS detection, further reducing the detection rate of peptides.

[0007] Therefore, in order to solve the problems of low detection rate, non-standard process and inaccurate detection in the existing cactus polypeptide extraction, it is an urgent technical problem to be solved by those skilled in the art to provide a method with "efficient pretreatment - standardized extraction - accurate detection" as the core, which can significantly improve the detection rate of polypeptide types, establish a standardized extraction and detection process, and provide technical support for the development of cactus polypeptide resources. Summary of the Invention

[0008] In view of this, the present invention provides a method for extracting freeze-dried fruit pulp that enriches polypeptides from cactus pomace.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] A method for extracting polypeptides from freeze-dried fruit pulp of cactus fruit pomace, comprising the following steps:

[0011] Step 1: Raw material pretreatment

[0012] Select fresh cactus pomace, remove residual pulp and impurities, dry it in a vacuum freeze-drying device, crush it and pass it through a sieve to make freeze-dried fruit puree for later use.

[0013] Step 2: Preparation of water-extracted samples

[0014] Weigh the freeze-dried fruit puree, add distilled water for the first reflux extraction; filter and collect the supernatant, then reflux extract again; combine the two supernatants, place them in a vacuum concentrator to concentrate until there is no obvious water, and then dry under vacuum to obtain the water extract sample;

[0015] Step 3: Preparation of 60% alcohol extract sample

[0016] Weigh the freeze-dried fruit pulp and add 60% ethanol solution as the extraction solvent to obtain a 60% ethanol-extracted sample; Step 4: Pretreatment for LC-MS detection

[0017] Take the above-mentioned water-extracted / alcohol-extracted samples or freeze-dried fruit pulp directly, dissolve them thoroughly in trifluoroacetic acid (TFA) solution, and transfer them into EP tubes;

[0018] Take the ZipTip C18 solid phase extraction head, rinse it with acetonitrile ACN / TFA solution, and then wash it with TFA solution;

[0019] The dissolved sample is drawn into and discharged from the extraction head to allow for full adsorption of peptides, and the waste liquid is discharged.

[0020] Clean the extraction head with TFA solution to remove impurities;

[0021] Elute the peptides into a new EP tube with ACN / TFA solution, and then vacuum dry the eluent to complete the desalting process.

[0022] Step 5: LC-MS / MS detection

[0023] The desalted peptides were dissolved in formic acid solution and then thoroughly vortexed.

[0024] Place in a centrifuge, centrifuge, and transfer the supernatant to a sample tube;

[0025] 3 μL of the supernatant was extracted and analyzed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RPLC-MS / MS). The peptide type and sequence were determined by searching the UniProt protein database.

[0026] Preferably, in step 1, the vacuum freeze-drying conditions are: temperature -40 to -35°C, vacuum degree 0.08-0.10 MPa, and drying time 8-10 hours.

[0027] Preferably, in step 1, the material is pulverized and then passed through a 60-mesh sieve.

[0028] Preferably, in step 2, the first reflux extraction conditions are as follows: distilled water is added at a ratio of 12 times the mass of the freeze-dried fruit puree, and reflux extraction is performed for 1 hour.

[0029] Preferably, in step 2, the conditions for refluxing and extracting are as follows: add 10 times the mass of the freeze-dried fruit pulp to the filter residue with distilled water and reflux for 1 hour.

[0030] Preferably, in step 2, the vacuum concentration conditions are: temperature 50-55℃, vacuum degree 0.07-0.09MPa, concentrated until there is no obvious moisture; the vacuum drying conditions are: temperature 50℃, vacuum degree 0.09MPa.

[0031] Preferably, in step 4, the solution is fully dissolved in 0.1% trifluoroacetic acid (TFA) solution, rinsed 10 times with 50 μL of 60% acetonitrile (ACN) / 0.1% TFA solution, and then washed 10 times with 10 μL of 0.1% TFA solution.

[0032] The dissolved sample was aspirated and expelled from the extraction head 20 times; the extraction head was washed 5 times with 10 μL of 0.1% TFA solution, and the peptides were eluted into a new EP tube with 10 μL of 60% ACN / 0.1% TFA solution.

[0033] Preferably, step 5: LC-MS / MS detection

[0034] The desalted peptide fragments were dissolved in 20 μL of 0.1% formic acid solution;

[0035] Preferably, in step 5, the centrifugation is performed at 17000g speed and 4℃ for 20 minutes.

[0036] Preferably, in step 5, the chromatographic conditions are as follows: a C18 column with a diameter of 2.1 × 150 mm and a diameter of 1.8 μm; mobile phase A is 0.1% formic acid aqueous solution and mobile phase B is 0.1% formic acid acetonitrile solution; gradient elution is performed for 0-30 min; phase B is 5%-35%; the flow rate is 0.3 mL / min; and the column temperature is 35 °C.

[0037] The mass spectrometry conditions were: electrospray ionization (ESI), positive ion mode, primary mass spectrometry scan range m / z 300-1800, and secondary mass spectrometry using data-dependent acquisition (DDA).

[0038] Database search: Match peptide sequences using the UniProt database to count the types of peptides.

[0039] The present invention achieves the following technical effects compared to the prior art:

[0040] (1) The present invention significantly enriches the types of polypeptides: after pretreatment of freeze-dried fruit pulp, the types of polypeptides reach 903, which is 16.7 times that of fruit juice, 4.3 times that of water extract sample and 4.6 times that of alcohol extract sample, thus fully tapping the polypeptide resources in cactus pomace.

[0041] (2) Standardization of process parameters in this invention: The preparation conditions of freeze-dried fruit puree, the amount of extraction solvent (12 times / 10 times), the extraction time (1h / time), the amount of desalting reagent (50μL for rinsing, 10μL for elution), and the centrifugation parameters (17000g / 4℃ / 20min) are clearly defined to ensure good repeatability of different batches of samples and stable detection of polypeptide types;

[0042] (3) The present invention has high detection accuracy: TFA, salts and other impurities are removed by ZipTip C18 desalting to avoid interference with LC-MS detection. Combined with UniProt database retrieval, the peptide sequence identification is accurate.

[0043] (4) The invention has strong operational compatibility: the equipment used (freeze dryer, reflux extractor, centrifuge, LC-MS / MS instrument) are all conventional instruments, which do not require special customization and are convenient for laboratory research and industrial scale-up;

[0044] (5) The invention utilizes resources in a high-value manner: waste cactus pomace is converted into samples with high polypeptide content, reducing resource waste and achieving both economic and environmental benefits. Attached Figure Description

[0045] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] This invention discloses a method for extracting polypeptides from freeze-dried pectin pomace, comprising the following steps:

[0048] Step 1: Raw material pretreatment

[0049] Select fresh cactus pomace, remove residual pulp and impurities, dry it in a vacuum freeze-drying device, crush it and pass it through a sieve to make freeze-dried fruit puree for later use.

[0050] Step 2: Preparation of water-extracted samples

[0051] Weigh the freeze-dried fruit puree, add distilled water for the first reflux extraction; filter and collect the supernatant, then reflux extract again; combine the two supernatants, place them in a vacuum concentrator to concentrate until there is no obvious water, and then dry under vacuum to obtain the water extract sample;

[0052] Step 3: Preparation of 60% alcohol extract sample

[0053] Weigh the freeze-dried fruit pulp and add 60% ethanol solution as the extraction solvent to obtain a 60% ethanol-extracted sample; Step 4: Pretreatment for LC-MS detection

[0054] Take the above-mentioned water-extracted / alcohol-extracted samples or freeze-dried fruit pulp directly, dissolve them thoroughly in trifluoroacetic acid (TFA) solution, and transfer them into EP tubes;

[0055] Take the ZipTip C18 solid phase extraction head, rinse it with acetonitrile ACN / TFA solution, and then wash it with TFA solution;

[0056] The dissolved sample is drawn into and discharged from the extraction head to allow for full adsorption of peptides, and the waste liquid is discharged.

[0057] Clean the extraction head with TFA solution to remove impurities;

[0058] Elute the peptides into a new EP tube with ACN / TFA solution, and then vacuum dry the eluent to complete the desalting process.

[0059] Step 5: LC-MS / MS detection

[0060] The desalted peptides were dissolved in formic acid solution and then thoroughly vortexed.

[0061] Place in a centrifuge, centrifuge, and transfer the supernatant to a sample tube;

[0062] 3 μL of the supernatant was extracted and analyzed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RPLC-MS / MS). The peptide type and sequence were determined by searching the UniProt protein database.

[0063] In step 1, the vacuum freeze-drying conditions are: temperature -40 to -35℃, vacuum degree 0.08-0.10MPa, and drying time 8-10 hours.

[0064] In step 1, the powder is pulverized and then passed through a 60-mesh sieve.

[0065] In step 2, the conditions for the first reflux extraction are as follows: add distilled water at a ratio of 12 times the mass of the freeze-dried fruit puree, and reflux for 1 hour.

[0066] In step 2, the conditions for refluxing and extraction are as follows: add 10 times the weight of the freeze-dried fruit puree to the filter residue with distilled water and reflux for 1 hour.

[0067] In step 2, the vacuum concentration conditions are: temperature 50-55℃, vacuum degree 0.07-0.09MPa, concentrated until there is no obvious moisture; the vacuum drying conditions are: temperature 50℃, vacuum degree 0.09MPa.

[0068] In step 4, dissolve thoroughly with 0.1% trifluoroacetic acid (TFA) solution, rinse 10 times with 50 μL of 60% acetonitrile (ACN) / 0.1% TFA solution, and then wash 10 times with 10 μL of 0.1% TFA solution.

[0069] The dissolved sample was aspirated and expelled from the extraction head 20 times; the extraction head was washed 5 times with 10 μL of 0.1% TFA solution, and the peptides were eluted into a new EP tube with 10 μL of 60% ACN / 0.1% TFA solution.

[0070] Step 5: LC-MS / MS detection

[0071] The desalted peptide fragments were dissolved in 20 μL of 0.1% formic acid solution;

[0072] In step 5, centrifuge at 17000g speed and 4℃ for 20 minutes.

[0073] In step 5, the chromatographic conditions are as follows: C18 column with a diameter of 2.1 × 150 mm and a diameter of 1.8 μm; mobile phase A is 0.1% formic acid aqueous solution and mobile phase B is 0.1% formic acid acetonitrile solution; gradient elution is performed for 0-30 min; phase B is 5%-35%; flow rate is 0.3 mL / min; and column temperature is 35℃.

[0074] The mass spectrometry conditions were: electrospray ionization (ESI), positive ion mode, primary mass spectrometry scan range m / z 300-1800, and secondary mass spectrometry using data-dependent acquisition (DDA).

[0075] Database search: Match peptide sequences using the UniProt database to count the types of peptides.

[0076] Example 1:

[0077] 1. Experimental Materials

[0078] Raw materials: cactus pomace (processing byproduct), cactus juice (control sample);

[0079] Reagents: distilled water, 60% ethanol solution, 0.1% trifluoroacetic acid (TFA), 60% acetonitrile (ACN) / 0.1% TFA mixture, 0.1% formic acid solution, ZipTip C18 solid-phase extraction head;

[0080] Instruments and equipment: vacuum freeze dryer, reflux extraction device, vacuum concentrator, vacuum drying oven, high-speed refrigerated centrifuge (supporting 17000g speed), reversed-phase high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), UniProt protein database retrieval system, analytical balance.

[0081] 2. Experimental Procedure

[0082] Preparation of freeze-dried fruit puree:

[0083] Take 1000g of fresh cactus pomace, rinse it three times with water to remove impurities, freeze dry it under vacuum (-38℃, 0.09MPa) for 9 hours, pulverize it and pass it through a 60-mesh sieve to obtain 190g of freeze-dried pomace.

[0084] Sample extraction (parallel experiments):

[0085] Preparation of water-extracted samples: Weigh 50g of freeze-dried fruit pulp, add 600mL of distilled water (12 times the solvent), and reflux for 1h; filter and collect the first supernatant; add 500mL of distilled water (10 times the solvent) to the filter residue and reflux for 1h again; combine the supernatants, concentrate under reduced pressure (52℃, 0.08MPa) to 10mL, and dry under reduced pressure (50℃, 0.09MPa) to obtain 3.2g of water-extracted sample.

[0086] Preparation of 60% ethanol extract sample: Weigh 50g of freeze-dried fruit pulp, add 600mL of 60% ethanol solution (12 times the solvent), reflux extract, concentrate and dry as described above, to obtain 2.8g of 60% ethanol extract sample.

[0087] Control sample preparation: Take 200mL of cactus juice directly as the juice control sample; take 50g of freeze-dried fruit puree directly as the freeze-dried fruit puree control sample (without extraction).

[0088] Desalination treatment:

[0089] Take 10 mg each of water extract, 60% alcohol extract, freeze-dried fruit puree, and fruit juice, and dissolve them in 1 mL of 0.1% TFA solution.

[0090] Take a ZipTip C18 extraction head, rinse 10 times with 50 μL of 60% ACN / 0.1% TFA, and wash 10 times with 10 μL of 0.1% TFA;

[0091] Sample solution was aspirated / expelled from the extraction head 20 times, washed 5 times with 10 μL 0.1% TFA, eluted into an EP tube with 10 μL 60% ACN / 0.1% TFA, and then vacuum dried.

[0092] Dissolve the eluent in 20 μL of 0.1% formic acid, centrifuge at 17000g / 4℃ for 20 min, and transfer the supernatant to the sample tube.

[0093] LC-MS / MS detection:

[0094] Chromatographic conditions: C18 column (2.1×150mm, 1.8μm), mobile phase A (0.1% formic acid aqueous solution), mobile phase B (0.1% formic acid acetonitrile solution), gradient elution (0-30min, phase B 5%-35%), flow rate 0.3mL / min, column temperature 35℃;

[0095] Mass spectrometry conditions: electrospray ionization (ESI), positive ion mode, primary mass spectrometry scan range m / z 300-1800, secondary mass spectrometry using data-dependent acquisition (DDA).

[0096] Database search: Match peptide sequences using the UniProt database to count the types of peptides.

[0097] 3. Test Results and Analysis

[0098] The test results are shown in Table 1;

[0099] Table 1:

[0100] .

[0101] As shown in Table 1, freeze-dried fruit pulp pretreatment is the core step in enriching the types of peptides in cactus pomace, with the number of peptides (903) being significantly higher than other samples. Although water extraction and 60% alcohol extraction can extract peptides, some peptides are lost during the extraction process, resulting in a lower number of peptides compared to freeze-dried fruit pulp that can be directly detected. Therefore, freeze-dried fruit pulp pretreatment is the optimal solution and can be directly used for the preparation and detection of samples with high peptide types.

[0102] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for extracting polypeptides from freeze-dried fruit pulp of cactus pomace, characterized in that, Includes the following steps: Step 1: Raw material pretreatment Select fresh cactus pomace, remove residual pulp and impurities, dry it in a vacuum freeze-drying device, crush it and pass it through a sieve to make freeze-dried fruit puree for later use. Step 2: Preparation of water-extracted samples Weigh the freeze-dried fruit puree, add distilled water and perform a first reflux extraction; filter and collect the supernatant, then reflux extract again. The supernatants from both experiments were combined and concentrated in a vacuum concentrator until no obvious water was found. The samples were then dried under reduced pressure to obtain the water-extracted sample. Step 3: Preparation of 60% alcohol extract sample Weigh the freeze-dried fruit pulp and add 60% ethanol solution as the extraction solvent to obtain a 60% ethanol-extracted sample; Step 4: Pretreatment for LC-MS detection Take the above-mentioned water-extracted / alcohol-extracted samples or freeze-dried fruit pulp directly, dissolve them thoroughly in trifluoroacetic acid (TFA) solution, and transfer them into EP tubes; Take the ZipTip C18 solid phase extraction head, rinse it with acetonitrile ACN / TFA solution, and then wash it with TFA solution; The dissolved sample is drawn into and discharged from the extraction head to allow for full adsorption of peptides, and the waste liquid is discharged. Clean the extraction head with TFA solution to remove impurities; Elute the peptides into a new EP tube with ACN / TFA solution, and then vacuum dry the eluent to complete the desalting process. Step 5: LC-MS / MS detection The desalted peptides were dissolved in formic acid solution and then thoroughly vortexed. Place in a centrifuge, centrifuge, and transfer the supernatant to a sample tube; 3 μL of the supernatant was extracted and analyzed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RPLC-MS / MS). The peptide type and sequence were determined by searching the UniProt protein database.

2. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, In step 1, the vacuum freeze-drying conditions are: temperature -40 to -35℃, vacuum degree 0.08-0.10MPa, and drying time 8-10 hours.

3. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, In step 1, the powder is pulverized and then passed through a 60-mesh sieve.

4. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, In step 2, the first reflux extraction conditions are as follows: add distilled water at a ratio of 12 times the mass of freeze-dried fruit puree, and reflux extract for 1 hour.

5. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, In step 2, the conditions for refluxing and extraction are as follows: add 10 times the mass of the freeze-dried fruit pulp to the filter residue with distilled water and reflux for 1 hour.

6. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, In step 2, the vacuum concentration conditions are: temperature 50-55℃, vacuum degree 0.07-0.09MPa, concentrated until there is no obvious moisture; the vacuum drying conditions are: temperature 50℃, vacuum degree 0.09MPa.

7. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, In step 4, the solution is fully dissolved in 0.1% trifluoroacetic acid (TFA) solution, rinsed 10 times with 50 μL of 60% acetonitrile (ACN) / 0.1% TFA solution, and then washed 10 times with 10 μL of 0.1% TFA solution. The dissolved sample was drawn into and expelled from the extraction head 20 times; Wash the extraction head 5 times with 10 μL of 0.1% TFA solution, and elute the peptides into a new EP tube with 10 μL of 60% ACN / 0.1% TFA solution.

8. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, Step 5: LC-MS / MS detection The desalted peptide fragments were dissolved in 20 μL of 0.1% formic acid solution.

9. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, In step 5, centrifuge at 17000g speed and 4℃ for 20 minutes.

10. The method for extracting polypeptides from cactus pomace using freeze-dried fruit pulp according to claim 1, characterized in that, In step 5, the chromatographic conditions are as follows: the chromatographic column is a C18 column with a diameter of 2.1 × 150 mm and a thickness of 1.8 μm; the mobile phase A is 0.1% formic acid aqueous solution and the mobile phase B is 0.1% formic acid acetonitrile solution; the gradient elution is 0-30 min; the B phase is 5%-35%; the flow rate is 0.3 mL / min; and the column temperature is 35 °C. The mass spectrometry conditions were: electrospray ionization (ESI), positive ion mode, primary mass spectrometry scan range m / z 300-1800, and secondary mass spectrometry using data-dependent acquisition (DDA). Database search: Match peptide sequences using the UniProt database to count the types of peptides.

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