CN111254124B - Method for recovering and extracting SOD (superoxide dismutase) from acerola cherry residues - Google Patents
Method for recovering and extracting SOD (superoxide dismutase) from acerola cherry residues Download PDFInfo
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- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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Abstract
The invention relates to the field of agricultural and sideline product recycling, in particular to a method for recycling and extracting SOD from acerola cherry residues; the method comprises the following steps: taking acerola residues as a raw material, and obtaining powder after pretreatment and crushing; carrying out ultrasonic crushing and leaching, and filtering to remove filter residues to obtain filtrate; performing graded filtration and interception to obtain a crude extract; and (3) refining the SOD in the crude extract, and then freeze-drying the refined SOD solution to obtain SOD powder. The invention can realize the high-efficiency and rapid recovery and extraction of SOD in acerola residues; the method is simple and efficient, and has low requirements on equipment; the extraction rate of SOD is high, and the extracted SOD has higher purity and higher specific activity.
Description
Technical Field
The invention relates to the field of agricultural and sideline product recycling, in particular to a method for recycling and extracting SOD from acerola cherry residues.
Background
Acerola (Malpighia emarginata D.C.) is the fruit of acerola of the genus acerola of the family acerola, which is rich in various vitamin and mineral components. In particular, it contains a characteristic anti-aging factor (SOD), also known as superoxide dismutase, which is an important component of the antioxidant enzyme system in the organism. Medical research shows that: SOD has certain curative effect on various diseases, especially has obvious curative effect on rheumatoid arthritis, lupus erythematosus, dermatomyositis and the like, and has good performance on radiation protection, anti-aging, anti-tumor and the like.
Most plants and microorganisms contain the substance, which is mainly divided into Cu/Zn-SOD, Fe-SOD and Mn-SOD according to the difference of metal prosthetic groups, wherein the cytoplasm and the chloroplast of most plants contain Cu/Zn-SOD, Mn-SOD exists in mitochondria of prokaryotic cells and eukaryotic cells, and Fe-SOD exists only in prokaryotic cells and few plants.
The acerola cherry contains Fe-SOD, so that the acerola cherry is more beneficial to the absorption of a human body and realizes the effects of maintaining beauty and keeping young, so the acerola cherry is also known as a 'beauty fruit', most of products prepared by the acerola cherry on the market at present are fruit powder, medicinal powder, beverage and the like, and most of the products are prepared by squeezing the acerola cherry fruits and then squeezing the obtained juice.
However, actually, the discarded acerola residue after pressing still contains more SOD components, and the existing acerola residue cannot be effectively utilized, which causes a lot of waste.
For example, according to the patent of China patent office, patent publication No. CN105558256B, discloses acerola cherry vitamin C collagen powder, a preparation method thereof and an application thereof in 9, 13, 2019, month and year, wherein the patent publication No. CN105558256B is granted, fish scales are cleaned, water is added into the fish scales, the fish scales are ground into fish scale slurry, then the pH value is adjusted to be alkaline for enzymolysis, enzyme inactivation is carried out after the enzymolysis, the filtration is carried out, filtrate is decolorized, the pH value is adjusted to be acidic, and finally, the fish scale extracting solution is obtained for later use; cleaning acerola cherry, juicing, filtering, decoloring filtrate, and finally concentrating to obtain acerola cherry juice for later use; and mixing the fish scale extracting solution and the acerola juice, and performing spray drying to obtain the acerola vitamin C collagen powder. In the technical scheme of the invention patent, the utilization of the acerola is only simple juicing utilization, so that the utilization rate of raw materials is low.
Disclosure of Invention
The invention provides a method for recovering acerola residue and extracting SOD (superoxide dismutase) in order to solve the problems that the utilization rate of the existing acerola is low, most of acerola residues are prepared into products by simply squeezing and extracting juice, and the squeezed acerola residues cannot be effectively utilized, so that a large amount of nutrient components are wasted in slag materials in the process of juicing.
The invention aims to: firstly, effectively recycling SOD of acerola residues; secondly, ensuring higher efficiency when extracting SOD from acerola residues; thirdly, ensuring that the extracted SOD has higher specific activity and higher purity; and fourthly, the extraction rate of the SOD is improved, and the vast majority of SOD can be extracted.
In order to achieve the purpose, the invention adopts the following technical scheme.
A method for recovering and extracting SOD from acerola cherry dregs comprises the following steps:
s1, preprocessing and crushing the acerola residues serving as raw materials to obtain powder; the pretreatment comprises screening to remove impurities and cleaning; the removal of impurities through screening, the cleaning and the removal of dust and the like are all conventional operations, and the operations can even comprise the cutting of branches and leaves mixed in during squeezing, which is beneficial to the subsequent operations; the acerola residues are the residues of pressed acerola;
s2, putting the powder obtained in the step S1 and cellulase into water together, and carrying out ultrasonic crushing, wherein the frequency of ultrasonic extraction is 10-25 kHz, the power is 50-150W, and the time duration is 10-15 min; filtering to remove filter residue to obtain filtrate; wherein the ratio of the mass of the powder to the enzymatic activity of the cellulase is 1g: (600-1000) U, wherein the mass ratio of the powder to the water is 1: 2-1: 4;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000-60000 Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using a filtering membrane with the molecular weight cutoff of 10000-30000 Da to obtain a secondary filtrate and a secondary trapped fluid; wherein the filtrate refers to liquid which passes through the filtering membrane, and the trapped liquid refers to liquid which cannot pass through the filtering membrane;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution; adding the mixed solution into an ethanol solution, uniformly mixing, removing precipitates, performing pressure filtration on the obtained liquid at 0.35MPa by using a filtration membrane with the molecular weight cutoff of 50000-60000 Da to obtain a pressure filtrate, filtering the pressure filtrate by using the filtration membrane with the molecular weight of 10000-30000 Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; the ethanol solution is prepared from ethanol and chloroform according to a volume ratio of 1: (0.4-0.6), wherein the volume ratio of the ethanol solution to the mixed solution is 1: (0.5-0.25);
s5, mixing the crude extract obtained in the step S4 and acetone according to the volume ratio of 1: (0.8-1.2), taking the precipitate, dissolving the precipitate in a salt solution, treating at 40-45 ℃ for 10-20 min, standing for 2-4 h, taking the supernatant, and freeze-drying to obtain SOD powder; the concentration of sodium dihydrogen phosphate in the salt solution is 0.4-0.7 g/L, the concentration of disodium hydrogen phosphate is 7-11 g/L, and the balance is water.
In the method, the collection of the SOD is maximized mainly by repeated filtration and interception, a series of impurities such as most of plant protein, cell walls, pectin, cellulose and the like can be removed in the process, the purity of the prepared SOD is improved, the content of the impurities is reduced, in the subsequent process of mixing the crude extract with acetone, the SOD precipitate is separated out by utilizing acetone, then the SOD is dissolved in a salt solution, the heat treatment and the standing are carried out to separate the impurities, the further purification is realized, and finally the supernatant is taken to prepare the SOD powder. The whole method is simple and efficient, the crushing, the ultrasonic extraction, the filtration, the interception, the liquid mixing, the secondary purification and the final drying are carried out, the operation time in the whole process is short, and the adsorption and the elution are carried out without a separation device such as an ion exchange column, so that the loss of SOD caused by the separation device in the extraction process can be reduced.
Preferably, in step S1, the crushing specifically includes: crushing the acerola residues into fragments less than or equal to 2mm by using a beating machine.
Preferably, the filtering of step S2 is performed using a 160 mesh screen.
Preferably, the step S4 of removing the precipitate employs 200 mesh filtration to remove the precipitate.
Preferably, the method for taking out the precipitate in step S5 includes: filtering with 200 mesh sieve, and collecting precipitate; the freeze-drying parameters were: the temperature is-40 ℃, the vacuum degree is 10Pa, and the time is 48 h.
In a preferred mode, the method for recovering and extracting SOD from acerola residues comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder and the cellulase in the step S1 into water with the mass ratio of 1g to 600U, wherein the mass ratio of the powder to the cellulase is 2 times that of the powder, enabling the water to completely cover the powder, carrying out ultrasonic crushing with the frequency of 20kHz and the power of 100W for 10min, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by utilizing a filter membrane with the trapped molecular weight of 50000Da to obtain a filter press solution; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is prepared from absolute ethanol and chloroform according to the volume ratio: 13: 7; the volume ratio of the ethanol solution to the mixed solution is 1: 0.5;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 15min at the temperature of 45 ℃, standing for 3h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4g/L, the concentration of the disodium hydrogen phosphate is 7g/L, and the balance is water.
The invention has the beneficial effects that:
1) SOD in the acerola residue can be efficiently and quickly recovered and extracted;
2) the method is simple and efficient, and has low requirements on equipment;
3) the extraction rate of SOD is high, the yield of SOD powder obtained by extraction is higher, and the contained SOD has higher specific activity.
Drawings
FIG. 1 is a graph comparing the yields of SOD powders prepared in examples of the present invention and comparative examples;
FIG. 2 is a graph comparing the enzymatic activities of SOD powders prepared in examples of the present invention and comparative examples.
Detailed Description
The invention is described in further detail below with reference to specific embodiments and drawings. Those skilled in the art will be able to implement the invention based on these teachings. Moreover, the embodiments of the present invention described in the following description are generally only some embodiments of the present invention, and not all embodiments. Therefore, all other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.
Unless otherwise specified, the raw materials used in the examples of the present invention are all commercially available or available to those skilled in the art; unless otherwise specified, the methods used in the examples of the present invention are all those known to those skilled in the art.
A method for recovering and extracting SOD from acerola cherry dregs,
the method comprises the following steps:
1) taking needle cherry residues as raw materials, and carrying out pretreatment and crushing to obtain powder;
2) putting the powder and the enzyme into water, carrying out ultrasonic crushing, and filtering to remove filter residues to obtain filtrate;
3) filtering the filtrate by using a filtering membrane to obtain primary filtrate and primary trapped fluid, and filtering the primary filtrate by using the filtering membrane to obtain secondary filtrate and secondary trapped fluid;
4) mixing the first-stage trapped fluid and the second-stage filtrate, adding an ethanol solution, uniformly mixing, removing precipitates, performing filter pressing by using a filter membrane to obtain a filter pressing solution, filtering the filter pressing solution by using the filter membrane to obtain trapped fluid, and mixing the second-stage filtrate and the trapped fluid to obtain a crude extract;
5) mixing the crude extractive solution with acetone, filtering to obtain precipitate, dissolving the precipitate in saline solution, heat treating at low temperature, standing, and freeze drying the supernatant to obtain SOD powder.
In the method, the collection of the SOD is maximized mainly by repeated filtration and interception, a series of impurities such as most of plant protein, cell walls, pectin, cellulose and the like can be removed in the process, the purity of the prepared SOD is improved, the content of the impurities is reduced, in the subsequent process of mixing the crude extract with acetone, the SOD precipitate is separated out by utilizing acetone, then the SOD is dissolved in a salt solution, the heat treatment and the standing are carried out to separate the impurities, the further purification is realized, and finally the supernatant is taken to prepare the SOD powder. The whole method is simple and efficient, the crushing, the ultrasonic extraction, the filtration, the interception, the liquid mixing, the secondary purification and the final drying are carried out, the operation time in the whole process is short, and the adsorption and the elution are carried out without a separation device such as an ion exchange column, so that the loss of SOD caused by the separation device in the extraction process can be reduced.
As a preference, the first and second liquid crystal compositions are,
step 1) the pretreatment comprises screening and cleaning;
the particle size of the powder obtained after the crushing in the step 1) is less than or equal to 2 mm.
Removing impurities through screening, cleaning and removing dust and the like are conventional operations, and can even comprise cutting branches and leaves mixed in during squeezing, so that the subsequent operation is facilitated; the acerola cherry residues are crushed into powder with the particle size of less than or equal to 2mm by a conventional crushing method, so that the subsequent ultrasonic leaching process is facilitated, and the leaching efficiency is improved.
As a preference, the first and second liquid crystal compositions are,
the enzyme in the step 2) is cellulase.
The enzyme is added mainly for breaking the walls of plant cells, and the cell walls of a large number of plant cells are still kept intact in the squeezing process, which is one of the main reasons for a large amount of residual SOD in acerola residue, so that the wall breaking is firstly carried out if the high-efficiency extraction of the SOD is to be realized. The common wall breaking methods include a physical method and an enzymatic hydrolysis method, the wall breaking efficiency of the physical method is usually low, the wall breaking effect cannot be guaranteed, the enzymolysis method is easy to introduce small molecular impurities which are difficult to remove, so the invention combines the physical method and the enzymolysis method to carry out simultaneously, the cell wall is thinned to a certain degree by using cellulase, then the wall is further broken by using ultrasonic, the wall breaking method can ensure high wall breaking degree, realize extraction of SOD to the maximum degree, avoid excessive cell wall breakage and introduction of impurities which are difficult to separate, if pectinase is selected for auxiliary enzymolysis wall breaking, the molecular weight of the pectinase is very close to that of Fe-SOD, impurities which are difficult to remove are also easily introduced, however, cellulase is easy to separate, and the rest pectin component is also easy to separate by precipitation.
As a preference, the first and second liquid crystal compositions are,
step 2), the ultrasonic crushing frequency is 10-25 kHz;
step 2), the ultrasonic crushing power is 50-150W;
and 2) carrying out ultrasonic crushing for 10-15 min.
The ultrasonic extraction under the above conditions can ensure the cell wall breaking and SOD extraction and dissolution, and can avoid the decomposition or destruction of SOD caused by too high ultrasonic intensity or too long time.
As a matter of preference,
the molecular weight cutoff of the filtering membrane in the steps 3) and 4) is 50000-60000;
the molecular weight cut-off of the filtering membrane in the steps 3) and 4) is 10000-30000.
The molecular weight of SOD in acerola is about 40000, so the filtering membrane can realize the best filtering and intercepting effects.
As a matter of preference,
and 4) preparing the ethanol solution from ethanol and chloroform according to a volume ratio of 1: (0.4-0.6) by mixing.
Insoluble pectin impurities can be better separated through the mixed solution of ethanol and chloroform, and the product purity is improved.
As a preference, the first and second liquid crystal compositions are,
step 5), mixing the crude extract with acetone according to a volume ratio of 1: (0.8-1.2);
and 5) the salt solution contains 0.4-0.7 g/L of sodium dihydrogen phosphate and 7-11 g/L of disodium hydrogen phosphate.
Mixing the crude extractive solution with acetone to precipitate SOD precipitate, and dissolving with sodium dihydrogen phosphate and disodium hydrogen phosphate solution to improve SOD purity.
As a preference, the first and second liquid crystal compositions are,
and 5) the low-temperature heat treatment is carried out for 10-20 min at the temperature of 40-45 ℃.
The low temperature heat treatment condition can further denature partial impurity protein or enzyme, and precipitate with sodium dihydrogen phosphate and/or disodium hydrogen phosphate to improve SOD purity.
If not specifically stated, the preparation method of the acerola residues comprises the following steps:
example 1
A method for recovering and extracting SOD from acerola cherry residue comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder and the cellulase in the step S1 into water with the mass ratio of 1g to 600U, wherein the mass ratio of the powder to the cellulase is 2 times that of the powder, enabling the water to completely cover the powder, carrying out ultrasonic crushing with the frequency of 20kHz and the power of 100W for 10min, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by utilizing a filter membrane with the trapped molecular weight of 50000Da to obtain a filter press solution; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is prepared from absolute ethanol and chloroform according to the volume ratio: 13: 7; the volume ratio of the ethanol solution to the mixed solution is 1: 0.5;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 15min at the temperature of 45 ℃, standing for 3h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4g/L, the concentration of the disodium hydrogen phosphate is 7g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this example were detected by electrophoresis, and the components were separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected as follows:
in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
The embodiment can also comprise pretreatment steps of solution preparation, cherry residue taking and the like.
Example 2
A method for recovering and extracting SOD from acerola cherry residue comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder and the cellulase in the step S1 into water with the mass ratio of 1g:900U, wherein the mass ratio of the powder to the cellulase is 3 times that of the powder, enabling the water to completely cover the powder, carrying out ultrasonic crushing with the frequency of 20kHz and the power of 125W for 14min, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 55000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using a filtering membrane with the molecular weight cutoff of 20000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by using a filter membrane with the trapped molecular weight of 55000Da to obtain a pressure filtrate; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 20000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is formed by mixing absolute ethanol and chloroform according to the volume ratio of 1: 0.6; the volume ratio of the ethanol solution to the mixed solution is 1: 0.3;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1.1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment at 44 ℃ for 18min, standing for 3h, taking supernatant, and carrying out freeze drying at-40 ℃ and the vacuum degree of 10Pa for 48h to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.6g/L, the concentration of the disodium hydrogen phosphate is 10g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this example were detected by electrophoresis, and the components were separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected as follows: in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
Example 3
A method for recovering and extracting SOD from acerola cherry residue comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder and the cellulase in the step S1 into water with the mass ratio of 1g:700U, wherein the mass ratio of the powder to the cellulase is 2 times that of the powder, enabling the water to completely cover the powder, carrying out ultrasonic crushing with the frequency of 10kHz and the power of 50W for 10min, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by utilizing a filter membrane with the trapped molecular weight of 50000Da to obtain a filter press solution; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is formed by mixing absolute ethanol and chloroform according to the volume ratio of 1: 0.5; the volume ratio of the ethanol solution to the mixed solution is 1: 0.4;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:0.9, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 10min at 42 ℃, standing for 2h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.6g/L, the concentration of the disodium hydrogen phosphate is 9g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this example were detected by electrophoresis, and the components were separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected as follows: in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
Example 4
A method for recovering and extracting SOD from acerola cherry residue comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder and the cellulase in the step S1 into water with the mass ratio of 1g to 800U, wherein the mass ratio of the powder to the cellulase is 3 times that of the powder, enabling the water to completely cover the powder, carrying out ultrasonic crushing with the frequency of 15kHz and the power of 100W for 11min, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 55000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 25000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by using a filter membrane with the trapped molecular weight of 55000Da to obtain a pressure filtrate; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 25000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is formed by mixing absolute ethanol and chloroform according to the volume ratio of 1: 0.5; the volume ratio of the ethanol solution to the mixed solution is 1: 0.3;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:0.9, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 15min at the temperature of 43 ℃, standing for 2.5h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.7g/L, the concentration of the disodium hydrogen phosphate is 9g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this example were detected by electrophoresis, and the components were separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected as follows: in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
Example 5
A method for recovering and extracting SOD from acerola cherry residue comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder and the cellulase in the step S1 into water with the mass ratio of 1g to 1000U, wherein the mass ratio of the powder to the cellulase is 4 times that of the powder, enabling the water to completely cover the powder, carrying out ultrasonic crushing with the frequency of 25kHz and the power of 150W for 15min, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 60000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using a filtering membrane with the molecular weight cutoff of 10000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by utilizing a filter membrane with the trapped molecular weight of 60000Da to obtain a pressure filtrate; filtering the press filtrate by using a filter membrane with the cut-off molecular weight of 10000Da to obtain a third-stage cut-off liquid, and mixing the second-stage cut-off liquid and the third-stage cut-off liquid to obtain a crude extract; wherein the ethanol solution is formed by mixing absolute ethanol and chloroform according to the volume ratio of 1: 0.6; the volume ratio of the ethanol solution to the mixed solution is 1: 0.25;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1.2, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 20min at the temperature of 45 ℃, standing for 4h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.7g/L, the concentration of the disodium hydrogen phosphate is 11g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this example were detected by electrophoresis, and the components were separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected as follows: in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
Comparative example 1
A method for recovering and extracting SOD from acerola cherry residue comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder into enough water to ensure that the water completely does not cover the powder, carrying out ultrasonic crushing with the frequency of 20kHz and the power of 100W for 10min, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using a filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by using a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a pressure filtrate; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is prepared from absolute ethanol and chloroform according to the volume ratio: 13: 7; the volume ratio of the ethanol solution to the mixed solution is 1: 0.5;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 15min at the temperature of 45 ℃, standing for 3h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4g/L, the concentration of the disodium hydrogen phosphate is 7g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this embodiment are detected by electrophoresis, and the components are separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected:
in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
Comparative example 2
A method for recovering and extracting SOD from acerola cherry residue comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder into sufficient water to ensure that the water completely does not cover the powder, carrying out ultrasonic crushing for 5min at the frequency of 30kHz and the power of 300W, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by using a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a pressure filtrate; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is prepared from absolute ethanol and chloroform according to the volume ratio: 13: 7; the volume ratio of the ethanol solution to the mixed solution is 1: 0.5;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 15min at the temperature of 45 ℃, standing for 3h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4g/L, the concentration of the disodium hydrogen phosphate is 7g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this example were detected by electrophoresis, and the components were separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected as follows:
in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
Comparative example 3
A method for recovering and extracting SOD from acerola cherry residues comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, mixing the powder with cellulase according to the mass ratio of 1: adding 0.02 of the weight percentage into enough water, stirring for 1h at constant temperature of 30 ℃, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by utilizing a filter membrane with the trapped molecular weight of 50000Da to obtain a filter press solution; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is prepared from absolute ethanol and chloroform according to the volume ratio: 13: 7; the volume ratio of the ethanol solution to the mixed solution is 1: 0.5;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 15min at the temperature of 45 ℃, standing for 3h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4g/L, the concentration of the disodium hydrogen phosphate is 7g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this example were detected by electrophoresis, and the components were separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected as follows:
in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
Comparative example 4
A method for recovering and extracting SOD from acerola cherry residues comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, mixing the powder with cellulase according to the mass ratio of 1: adding 0.05 into enough water, stirring at constant temperature of 30 deg.C for 1h, filtering with 160 mesh screen to remove filter residue to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by utilizing a filter membrane with the trapped molecular weight of 50000Da to obtain a filter press solution; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is prepared from absolute ethanol and chloroform according to the volume ratio: 13: 7; the volume ratio of the ethanol solution to the mixed solution is 1: 0.5;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 15min at the temperature of 45 ℃, standing for 3h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4g/L, the concentration of the disodium hydrogen phosphate is 7g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this example were detected by electrophoresis, and the components were separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected as follows:
in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
Comparative example 5
A method for recovering and extracting SOD from acerola cherry residues comprises the following steps:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments with the size less than or equal to 2mm by using a beater to obtain powder;
s2, mixing the powder, cellulase and pectinase in a mass ratio of 1: 0.02: adding 0.02 of the weight percentage into enough water, stirring for 1h at constant temperature of 30 ℃, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by utilizing a filter membrane with the trapped molecular weight of 50000Da to obtain a filter press solution; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-stage trapped fluid, and mixing the second-stage trapped fluid and the third-stage trapped fluid to obtain a crude extract; wherein the ethanol solution is prepared from absolute ethanol and chloroform according to the volume ratio: 13: 7; the volume ratio of the ethanol solution to the mixed solution is 1: 0.5;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out low-temperature heat treatment for 15min at the temperature of 45 ℃, standing for 3h, taking supernatant, and carrying out freeze drying for 48h at the temperature of-40 ℃ and the vacuum degree of 10Pa to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4g/L, the concentration of the disodium hydrogen phosphate is 7g/L, and the balance is water.
The filter residue obtained in step S2 and the SOD powder obtained in step S5 of this embodiment are detected by electrophoresis, and the components are separated by polyacrylamide gel electrophoresis, and identified according to the color band formed on the gel strip, and detected:
in this example, almost no SOD component was detected in the residue obtained in step S2, and Fe-SOD was detected in the SOD powder obtained in step S5.
The enzymatic activities of superoxide dismutase and the yields of the SOD powders obtained in examples 1 to 5 and comparative examples 1 to 5 were calculated based on the weight of the SOD powder obtained in example 1 (i.e., 100 wt%).
The results are shown in fig. 1 and fig. 2, respectively. It is evident from fig. 1 and 2 that the stability of the yield of the SOD powder prepared by the technical scheme of the present invention is high, and the stability is substantially maintained high, while the yield of some comparative examples in comparative examples 1 to 5 is higher, but the specific activity of the enzyme in the prepared product is significantly lower than that of the SOD powder obtained by the examples of the present invention, and the impurity content is high.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (7)
1. A method for recovering and extracting SOD from acerola cherry residues is characterized by comprising the following steps:
s1, crushing acerola cherry residues to obtain powder;
s2, putting the powder obtained in the step S1 and cellulase into water together, and carrying out ultrasonic crushing, wherein the frequency of ultrasonic crushing is 10-25 kHz, the power is 50-150W, and the time duration is 10-15 min; filtering to remove filter residue to obtain filtrate; wherein the ratio of the mass of the powder to the enzyme activity of the cellulase is 1g (600-1000) U, and the mass ratio of the powder to the water is 1: 2-1: 4;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000-60000 Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using a filtering membrane with the molecular weight cutoff of 10000-30000 Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution; adding the mixed solution into an ethanol solution, uniformly mixing, removing precipitates, and performing pressure filtration on the obtained liquid at 0.35MPa by using a filter membrane with the molecular weight cutoff of 50000-60000 Da to obtain a pressure filtrate; filtering the press filtrate by using a filter membrane with the molecular weight of 10000-30000 Da to obtain a third-level trapped fluid, and mixing the second-level trapped fluid and the third-level trapped fluid to obtain a crude extract; the ethanol solution is prepared from ethanol and chloroform according to a volume ratio of 1: (0.4-0.6), wherein the volume ratio of the ethanol solution to the mixed solution is 1: (0.5 to 0.25);
s5, mixing the crude extract obtained in the step S4 and acetone according to the volume ratio of 1: (0.8-1.2), taking the precipitate, dissolving the precipitate in a salt solution, carrying out heat treatment at 40-45 ℃ for 10-20 min, standing for 2-4 h, taking the supernatant, and carrying out freeze drying to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4-0.7 g/L, the concentration of the disodium hydrogen phosphate is 7-11 g/L, and the balance is water.
2. The method for recovering and extracting SOD from acerola residue according to claim 1, wherein the crushing in step S1 is specifically: crushing the acerola residues into fragments less than or equal to 2mm by using a beating machine.
3. The method for recovering and extracting SOD from acerola residue as claimed in claim 1, wherein the filtering of step S2 uses a 160-mesh sieve.
4. The method for recovering and extracting SOD from acerola residues as claimed in claim 1, wherein the removing of the precipitate in step S4 is specifically: the precipitate was removed by filtration through a 200 mesh screen.
5. The method for recovering and extracting SOD from acerola residue according to claim 1, wherein the method for collecting the precipitate in step S5 comprises: filtering with 200 mesh sieve, and collecting precipitate.
6. The method for recovering and extracting SOD from acerola residue as claimed in claim 1, wherein the freeze-drying parameters of step S5 are: the temperature is-40 ℃, the vacuum degree is 10Pa, and the time is 48 h.
7. The method for recovering and extracting SOD from acerola residue according to claim 1, comprising the steps of:
s1, taking 1kg of acerola residues as raw materials, screening to remove impurities, cleaning the acerola residues, and crushing the acerola residues into fragments less than or equal to 2mm by using a beater to obtain powder;
s2, adding the powder obtained in the step S1 and cellulase into water with the mass being 2 times that of the powder according to the proportion of 1g to 600U, carrying out ultrasonic crushing with the frequency of 20kHz and the power of 100W for 10min, and filtering by using a 160-mesh screen to remove filter residues to obtain filtrate;
s3, filtering the filtrate obtained in the step S2 by using a filtering membrane with the molecular weight cutoff of 50000Da to obtain a primary filtrate and a primary trapped fluid, and filtering the primary filtrate by using the filtering membrane with the molecular weight cutoff of 30000Da to obtain a secondary filtrate and a secondary trapped fluid;
s4, mixing the primary trapped fluid and the secondary filtrate obtained in the step S3 to obtain a mixed solution, adding the mixed solution into an ethanol solution, uniformly mixing, filtering by adopting a 200-mesh sieve to remove precipitates, and performing pressure filtration on the obtained solution at 0.35MPa by utilizing a filter membrane with the trapped molecular weight of 50000Da to obtain a filter press solution; filtering the press filtrate by using a filter membrane with the molecular weight cutoff of 30000Da to obtain a third-level cutoff liquid; mixing the secondary trapped fluid and the tertiary trapped fluid to obtain a crude extract; wherein the ethanol solution is formed by mixing absolute ethanol and chloroform according to the volume ratio of 13: 7; the volume ratio of the ethanol solution to the mixed solution is 1: 0.5;
s5, mixing the crude extract obtained in the step S3 and acetone according to the volume ratio of 1:1, filtering out precipitates by using a 200-mesh screen, dissolving the precipitates in a salt solution, carrying out heat treatment at a low temperature of 45 ℃ for 15min, standing for 3h, taking supernatant, and carrying out freeze drying at a temperature of-40 ℃ and a vacuum degree of 10Pa for 48h to obtain SOD powder; wherein the concentration of the sodium dihydrogen phosphate in the salt solution is 0.4g/L, the concentration of the disodium hydrogen phosphate is 7g/L, and the balance is water.
Priority Applications (1)
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