Method for recovering and extracting SOD from conifer cherry residues

A technology of acerola and crude extract, applied in biochemical equipment and methods, enzymes, oxidoreductases, etc., can solve the problem of low utilization rate of acerola, ineffective utilization of acerola slag, waste of SOD, etc. problem, to achieve the effect of high specific activity, low equipment requirements, and high extraction rate

Active Publication Date: 2020-06-09

DALIAN POLYTECHNIC UNIVERSITY

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AI-Extracted Technical Summary

Problems solved by technology

[0007] In order to solve the low utilization rate of the existing acerola cherries, most of them only extract the juice after simple pressing and use the juice to make products, and the pressed acerola cherry pomace cannot be effectively uti...

Method used

Adopt the ultrasound of above-mentioned condition to carry out leaching, can guarantee the fragmentation of cell wall, the extraction and dissolving of SOD, can avoid the decomposition or the destruction of SOD that ultrasound intensity is excessive or the time is too long again.

After the thick extract is mixed with acetone, the SOD precipitation can be separated out, and further utilize sodium dihydrogen phosphate and disodium hydrogen phosphate solution to cooperate to dissolve it, and improve the purity of SOD.

In aforesaid method, mainly by repeated filtration and retention, realize the collection of maximum SOD, and can remove a series of impurities such as most vegetable protein, cell wall, pectin and cellulose in this process, improve all The purity of SOD is obtained to reduce the content of impurities. In the subsequent process of mixing the crude extract with acetone, use acetone to precipitate SOD, then dissolve SOD in a salt solution, heat-treat and stand to separate impurities to achieve further purification. , and finally take the supernatant to obtain SOD powder. The overall method is simple and efficient, including crushing, ultrasonic extraction, filtration, interception, liquid mixing and secondary purification, and finally drying. The operation time in the overall process is short, and there is no need to use separation devices such as ion exchange columns for adsorption and elution, which can Reduce the loss of SOD due to the separation device during the extraction process.

Removing impurities by sub-sieving, and cleaning and removing dust etc. are routine operations, and it can even include cutting off the branches and leaves mixed in when pressing, which is conducive to the carrying out of follow-up operations; the coniferous cherry residue is broken by conventional crushing methods It is a powder with a particle size of ≤2mm, which is beneficial to the subsequent ultrasonic extraction process and improves the extrac...

Abstract

The invention relates to the field of recycle of agricultural and sideline products, and in particular to a method for recovering and extracting SOD from conifer cherry residues. The method comprisesthe following steps: pre-treating and crushing conifer cherry residues serving as raw materials to obtain crushed powder; carrying out ultrasonic crushing and leaching, carrying out filtering, and removing filter residues to obtain a filtrate; carrying out grading filtration and interception in cooperation to obtain a crude extract; and refining SOD in the crude extract, and freeze-drying the refined SOD solution to obtain SOD powder. The method disclosed by the invention can be used for efficiently and rapidly recovering and extracting SOD in conifer cherry residues; the method is simple andefficient, and has low requirements on equipment; and the extraction rate of SOD is high, and the extracted SOD has a higher purity and high specific activity.

Application Domain

Oxidoreductases

Technology Topic

EngineeringBiotechnology +3

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Examples

Experimental program(5)
Comparison scheme(5)

Example Embodiment

[0070] Example 1

[0071] A kind of coniferous cherry pomace reclaims and extracts the method for SOD, and the steps are as follows:

[0072] S1. Take 1 kg of coniferous cherry residue as a raw material, carry out sub-sieving to remove impurities, clean the coniferous cherry residue and crush it with a beater into fragments of ≤ 2mm to obtain crushed powder;

[0073] S2. Add the crushed powder and cellulase described in step S1 to water twice the mass of the crushed powder at a mass ratio of 1g:600U, so that the water is completely submerged in the crushed powder, and the frequency is 20kHz, and the power is 100W. Ultrasonic crushing for 10 minutes, using a 160-mesh sieve to filter and remove the filter residue to obtain the filtrate;

[0074] S3. Use a filter membrane with a molecular weight cut-off of 50000Da to filter the filtrate described in step S2 to obtain a primary filtrate and a primary retentate, and then use a filter membrane with a molecular weight cut-off of 30000Da to filter the primary filtrate to obtain a secondary filter. filtrate and secondary retentate;

[0075] S4. Mix the primary retentate and the secondary filtrate described in step S3 to obtain a mixed solution, add the mixed solution to the ethanol solution, and after mixing uniformly, filter with a 200-mesh sieve to remove the precipitate, and then filter the obtained solution with a molecular weight cut-off of 50000Da The membrane is subjected to 0.35MPa pressure filtration to obtain a press filtrate; the filter membrane with a molecular weight cut-off of 30000Da is used to filter the press filtrate to obtain a tertiary retentate, and the secondary retentate and the tertiary retentate are mixed to obtain a crude extract Liquid; wherein, the ethanol solution is mixed by absolute ethanol and chloroform in a volume ratio: 13:7; the volume ratio of the ethanol solution and the mixed solution is 1:0.5;

[0076] S5. Mix the crude extract described in step S3 with acetone at a volume ratio of 1:1, filter out the precipitate using a 200-mesh screen, then dissolve the precipitate in a salt solution, and heat treat at a low temperature at 45°C 15min, let it stand for 3h, take the supernatant at a temperature of -40°C, a vacuum of 10Pa, and freeze-dry for 48h to obtain SOD powder; wherein, the concentration of sodium dihydrogen phosphate in the saline solution is 0.4g/L, phosphoric acid The concentration of disodium hydrogen is 7g/L, and the balance is water.

[0077] The filter residue described in step S2 of this embodiment and the SOD powder obtained in step S5 are detected respectively by electrophoresis, and the components are separated by polyacrylamide gel electrophoresis, and are identified according to the color bands on the gel strip. After detection:

[0078] Almost no SOD component was detected in the filter residue described in step S2 of this embodiment, and Fe-SOD was detected in the SOD powder obtained in step S5.

[0079] This embodiment may also include pretreatment steps such as solution preparation and cherry pomace extraction.

Example Embodiment

[0080] Example 2

[0081] A kind of coniferous cherry pomace reclaims and extracts the method for SOD, and the steps are as follows:

[0082] S1. Take 1 kg of coniferous cherry residue as a raw material, carry out sub-sieving to remove impurities, clean the coniferous cherry residue and crush it with a beater into fragments of ≤ 2mm to obtain crushed powder;

[0083] S2. Add the crushed powder and cellulase described in step S1 to water with a mass 3 times the mass of the crushed powder at a mass ratio of 1g:900U, so that the water is completely submerged in the crushed powder, and the frequency is 20kHz, and the power is 125W. Ultrasonic crushing for 14 minutes, using a 160-mesh sieve to filter and remove the filter residue to obtain the filtrate;

[0084] S3. Use a filter membrane with a molecular weight cut-off of 55000Da to filter the filtrate described in step S2 to obtain a primary filtrate and a primary retentate, and then use a filter membrane with a molecular weight cut-off of 20000Da to filter the primary filtrate to obtain a secondary filter. filtrate and secondary retentate;

[0085] S4. Mix the primary retentate and the secondary filtrate described in step S3 to obtain a mixed solution, add the mixed solution to the ethanol solution, mix evenly and filter with a 200-mesh sieve to remove the precipitate, and then filter the obtained solution with a molecular weight cut-off of 55000Da The membrane is subjected to 0.35MPa pressure filtration to obtain a press filtrate; the filter membrane with a molecular weight cut-off of 20000Da is used to filter the press filtrate to obtain a tertiary retentate, and the secondary retentate and the tertiary retentate are mixed to obtain a crude extract liquid; wherein, the ethanol solution is mixed by absolute ethanol and chloroform in a volume ratio of 1:0.6; the volume ratio of the ethanol solution and the mixed solution is 1:0.3;

[0086] S5. Mix the crude extract described in step S3 with acetone at a volume ratio of 1:1.1, filter out the precipitate using a 200-mesh screen, then dissolve the precipitate in a salt solution, and heat treat at a low temperature at 44°C 18min, let it stand for 3h, take the supernatant at a temperature of -40°C, a vacuum of 10Pa, and freeze-dry for 48h to obtain SOD powder; wherein, the concentration of sodium dihydrogen phosphate in the saline solution is 0.6g/L, phosphoric acid The concentration of disodium hydrogen is 10g/L, and the balance is water.

[0087] The filter residue described in step S2 of this embodiment and the SOD powder obtained in step S5 are detected respectively by electrophoresis, and the components are separated by polyacrylamide gel electrophoresis, and are identified according to the color bands on the gel strip. After detection: Almost no SOD component was detected in the filter residue described in step S2 of this embodiment, and Fe-SOD was detected in the SOD powder obtained in step S5.

Example Embodiment

[0088] Example 3

[0089] A kind of coniferous cherry pomace reclaims and extracts the method for SOD, and the steps are as follows:

[0090] S1. Take 1 kg of coniferous cherry residue as a raw material, carry out sub-sieving to remove impurities, clean the coniferous cherry residue and crush it with a beater into fragments of ≤ 2mm to obtain crushed powder;

[0091] S2. Add the crushed powder and cellulase described in step S1 to water twice the mass of the crushed powder at a mass ratio of 1g:700U, so that the water is completely submerged in the crushed powder, and the frequency is 10kHz, and the power is 50W. Ultrasonic crushing for 10 minutes, using a 160-mesh sieve to filter and remove the filter residue to obtain the filtrate;

[0092] S3. Use a filter membrane with a molecular weight cut-off of 50000Da to filter the filtrate described in step S2 to obtain a primary filtrate and a primary retentate, and then use a filter membrane with a molecular weight cut-off of 30000Da to filter the primary filtrate to obtain a secondary filter. filtrate and secondary retentate;

[0093] S4. Mix the primary retentate and the secondary filtrate described in step S3 to obtain a mixed solution, add the mixed solution to the ethanol solution, and after mixing uniformly, filter with a 200-mesh sieve to remove the precipitate, and then filter the obtained solution with a molecular weight cut-off of 50000Da The membrane is subjected to 0.35MPa pressure filtration to obtain a press filtrate; the filter membrane with a molecular weight cut-off of 30000Da is used to filter the press filtrate to obtain a tertiary retentate, and the secondary retentate and the tertiary retentate are mixed to obtain a crude extract Liquid; wherein, the ethanol solution is mixed by absolute ethanol and chloroform in a volume ratio of 1:0.5; the volume ratio of the ethanol solution and the mixed solution is 1:0.4;

[0094] S5. Mix the crude extract described in step S3 with acetone at a volume ratio of 1:0.9, filter out the precipitate using a 200-mesh screen, then dissolve the precipitate in a salt solution, and heat treat at a low temperature at 42°C 10min, let it stand for 2h, take the supernatant at a temperature of -40°C, a vacuum of 10Pa, and freeze-dry for 48h to obtain SOD powder; wherein, the concentration of sodium dihydrogen phosphate in the saline solution is 0.6g/L, phosphoric acid The concentration of disodium hydrogen is 9g/L, and the balance is water.

[0095] The filter residue described in step S2 of this embodiment and the SOD powder obtained in step S5 are detected respectively by electrophoresis, and the components are separated by polyacrylamide gel electrophoresis, and are identified according to the color bands on the gel strip. After detection: Almost no SOD component was detected in the filter residue described in step S2 of this embodiment, and Fe-SOD was detected in the SOD powder obtained in step S5.

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Classification and recommendation of technical efficacy words

The method is simple and efficient
low equipment requirements

Method for recovering and extracting SOD from conifer cherry residues

AI-Extracted Technical Summary

Problems solved by technology

Method used

Abstract

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Examples

Example Embodiment

Example Embodiment

Example Embodiment

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Description & Claims & Application Information

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