AU2020256314B1 - Superoxide dismutase solid preparation and method for preparing the same - Google Patents

Abstract

Provided are a superoxide dismutase, a superoxide dismutase solid preparation, methods for preparing the same and use thereof. The superoxide dismutase solid preparation comprises a superoxide dismutase derived fromRosa roxburghii. The superoxide dismutase derived from Rosa roxburghii has the amino acid sequence of SEQ ID NO: 1.

Description

SUPEROXIDE DISMUTASE SOLID PREPARATION AND METHOD FOR PREPARING THE SAME CROSS-REFERENCE TO RELATED APPLICATION

This application is based upon and claims a priority to Chinese Patent Application No. 202010121491.0 filed on February 26 2020, Chinese Patent Application No. 202010360991.X

filed on April 30 2020 and Chinese Patent Application No. 202010362023.2 filed on April 30

2020, the entire contents of which are incorporated herein by reference.

FIELD The present disclosure relates to the field of biology. Specifically, the present disclosure relates to a superoxide dismutase, a method for preparing a superoxide dismutase, a superoxide

dismutase solid preparation, a method for preparing a superoxide dismutase solid preparation and

use thereof.

BACKGROUND Superoxide dismutase (SOD) is a class of redox metal enzyme that can remove superoxide free radical anions and effectively prevent its damage to the body. The superoxide dismutase is

the top killer of oxygen free radical in the body and is the foundation of life and health. At

present, the SOD enzyme is mainly produced by extracting from animal blood or liver, which is difficult for popular application due to pollution and limited resources. Therefore, many

researchers have actively explored the microbial fermentation to produce the SOD enzyme

because of advantages of large-scale culture and easy access. Therefore, a superoxide dismutase, a superoxide dismutase solid preparation and methods

for preparing the same still need to be studied.

SUMMARY Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent.

In an aspect, the present disclosure in embodiments provides a superoxide dismutase solid

preparation, comprising a superoxide dismutase derived from Rosa roxburghii, wherein the

superoxide dismutase derived from Rosa roxburghii has the amino acid sequence of SEQ ID NO: 1, the superoxide dismutase derived from Rosa roxburghiiis encoded by the nucleotide sequence of SEQ ID NO: 2, and the superoxide dismutase solid preparation is prepared by steps: expressing a genetically engineered bacterium containing an expression vector connected with the nucleotide sequence of SEQ ID NO: 2 to obtain a bacterial medium comprising the superoxide dismutase, concentrating the bacterial medium via centrifugation and resuspending the concentrated bacterial cells, followed by ultrasound treatment, centrifuged to collect a supernate and

SDS-PAGE electrophoresis of total protein of the bacterial cells to obtain the superoxide

dismutase, subjecting the superoxide dismutase to high pressure activation under a pressure of 550 MPa

and at a temperature of 16.5°C for 5 minutes, thereby obtaining an activated product, and

mixing the activated product with p-cyclodextrin (at a mass ratio of 10% based on a mass of the activated product) followed by frozen-drying at a cold trap temperature of -67°C under a

vacuum degree of 26 Pa for 48 hours, thereby obtaining the superoxide dismutase solid

preparation.

In an embodiment, the superoxide dismutase solid preparation in the aspect further comprises at least one of prebiotics and probiotics.

In another aspect, the present disclosure in embodiments provides a method for preparing a superoxide dismutase, comprising steps:

cultivating a recombinant cell expressing the superoxide dismutase to obtain a culture

medium,

isolating the superoxide dismutase from the culture medium followed by purification, and

subjecting the purified superoxide dismutase to high pressure activation under a pressure of

550 MPa and at a temperature of 16.5°C for 5 minutes, wherein the superoxide dismutase comprises the amino acid sequence of SEQ ID NO: 1,

the superoxide dismutase is derived from Rosa roxburghii, the superoxide dismutase is encoded by the nucleotide sequence of SEQ ID NO: 2, and the recombinant cell is obtained by transforming a receptor cell with an expression vector

connected with the nucleotide sequence of SEQ ID NO: 2.

In an embodiment, the method for preparing a superoxide dismutase further comprises subjecting an activated product obtained by high pressure activation to a drying treatment to

obtain a superoxide dismutase solid preparation,

wherein the drying treatment is frozen-drying treatment, the activated product is mixed with a drying aid agent before the drying treatment and the drying aid agent is p-cyclodextrin at a mass ratio of 10% based on a mass of the activated product, and the frozen-drying treatment is performed at a cold trap temperature of -67C under a vacuum degree of 26 Pa for 48 hours.

BRIEF DESCRIPTION OF THE DRAWINGS The above and/or additional aspects and advantages of the present disclosure will become apparent and easily understood from the description of embodiments in combination with the accompanying drawings, in which: Figure 1 shows a schematic flow chart of a method for preparing a superoxide dismutase solid preparation according to an embodiment of the present disclosure. Figure 2 shows a schematic diagram for thermal stability analysis of a superoxide dismutase solid preparation according to an embodiment of the present disclosure.

DETAILED DESCRIPTION Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions. Terms used herein in the description of the present disclosure are only for the purpose of describing specific embodiments, but they should not be construed to limit the present disclosure. As used in the description of the present disclosure and the appended claims, "a", "an" and "the" in singular forms mean including plural forms, unless clearly indicated in the context otherwise. It should also be understood that, as used herein, the term "and/or" represents and contains any one and all possible combinations of one or more associated listed items. It should be further understood that, when used in the specification, terms "comprising" and/or "containing" specify the presence of stated features, operations, elements and/or components, but do not exclude the presence or addition of one or more other features, operations, elements, components and/or groups thereof. In addition, terms such as "first" and "second" are used herein for purposes of description and are not intended to indicate or imply relative importance or significance or impliedly indicate quantity of the technical feature referred to. Thus, the feature defined with "first" and

"second" may comprise one or more this feature. In the description of the present disclosure, "a

plurality of' means two or more than two this features, unless specified otherwise.

High Pressure Processing (HPP), Ultra-High Pressure (UHP) or High Hydrostatic Pressure

(HHP) refer to the treatment of food under a pressure of 100 to 1,000 MPa for sterilization, enzyme inactivation and food processing. In general, high pressure can make the secondary

structure or tertiary structure of protein destroyed and the activity center of enzyme changed

orbroken, thereby inactivating the enzyme. However, an appropriate pressure may induce the change of conformation of some enzymes, the exposure of active center of enzyme and

increased contact of enzyme with corresponding substrate, thereby activating enzyme.

Present inventors found that high pressure treatment cannot improve the enzyme activity of all SOD enzymes, such as SOD enzyme of Rosa chinensis. Moreover, the existing SOD

enzymes may exhibit significantly decreased activity at a relatively high temperature due to low

thermal stability. Therefore, SOD enzymes have strict requirements on ambient temperature,

which cannot be stored and applied conveniently. In view of this, the present inventors have found that Rosa roxburghii which already has a

high SOD enzyme activity can exhibit significantly improved SOD enzyme activity after the high pressure treatment. Moreover, the high-pressure treated SOD enzyme of Rosa roxburghii

has high thermal stability, which can maintain the high enzyme activity even at about 80°C,

showing low requirement for ambient temperature, thus can be stored and applied conveniently. Further, the present inventors cloned the SOD gene of Rosa roxburghii which had not been

disclosed, thus obtained the amino acid sequence of the SOD enzyme as well as the

corresponding nucleotide sequence. By analyzing the amino acid sequence of the Rosa roxburghii SOD, the present inventors concluded that the amino acids at positions 15, 67, 87

and 143 may be the functional sites affecting enzyme activity, behaviors after high pressure

treatment and thermal stability. Furthermore, the present inventors performed high pressure activation on the SOD enzyme

of Rosa roxburghii to improve the enzyme activity, and prepared an oral liquid or solid powder

of the SOD enzyme of Rosa roxburghii with significantly improved activity and thermal stability, which can be useful in the fields of food, medicine and cosmetics, with a broad

application prospect.

Superoxide dismutase In a first aspect of the present disclosure, provided in embodiments is a superoxide dismutase. According to an embodiment of the present disclosure, the superoxide dismutase has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having a homology of at least 80%, for example, at least 85%, 90%, 95% or 99% to the amino acid sequence of SEQ ID

NO: 1, in which the amino acid at position 15 is Threonline (T), the amino acid at position 67 is

Glycine (G), the amino acid at position 87 is Isoleucine (I), and the amino acid at position 143 is Valine (V). MAKGVAVLCSSEGVTGTILFTQEGDGPTTVTGNVSGLKPGLHGFHVHALGDTTNGC

MSTGPHFNPAGKEHGAPEDENRHAGDLGNIIVGDDGTATFTIVDKQIPLTGPHSIIGRAV VVHGDPDDLGKGGHELSKSTGNAGGRVACGIIGLQG (SEQ ID NO: 1)

According to embodiments of the present disclosure, the superoxide dismutase further has the following additional technical features. According to an embodiment of the present disclosure, the superoxide dismutase is derived

from Rosa roxburghii.

Nucleic acid encoding a superoxide dismutase In a second aspect of the present disclosure, provided in embodiments is a nucleic acid for encoding the superoxide dismutase as described in the above aspect. As mentioned above, the

superoxide dismutase encoded by the nucleic acid in this aspect has high enzyme activity and thermal stability, which also exhibits significantly improved enzyme activity after high pressure

treatment, showing a broad application prospect.

According to an embodiment of the present disclosure, the nucleic acid for encoding the superoxide dismutase comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide

sequence having a homology of at least 80%, for example, at least 85%, 90%, 95% or 99% to

the nucleotide sequence of SEQ ID NO: 2,in which the nucleotides at positions 43 to 45 are ACG, the nucleotides at positions 199 to 201 are GGC, the nucleotides at positions 259 to 261

are ATT and the nucleotides at positions 427 to 429 are GTA.

The presents inventors have found that the superoxide dismutase encoded by the nucleic acid in this aspect has high enzyme activity and thermal stability and exhibits significantly

improved enzyme activity after high pressure treatment.

ATGGCAAAGGGTGTTGCTGTACTTTGCTCCAGTGAGGGTGTTACGGGAACTATCCT CTTCACCCAAGAGGGAGATGGCCCAACTACTGTGACTGGAAACGTTTCTGGCCTCA AGCCTGGGCTTCATGGTTTCCATGTTCATGCTCTTGGTGACACAACAAACGGTTGCA TGTCAACTGGACCACACTTCAATCCTGCTGGCAAAGAGCATGGTGCTCCTGAAGAT GAGAATCGTCATGCTGGTGATCTTGGAAATATCATTGTTGGGGATGATGGAACTGCTA

CCTTCACAATTGTTGACAAGCAGATTCCTCTCACTGGACCACATTCTATCATTGGTAG GGCGGTTGTTGTCCATGGAGACCCTGATGACCTTGGCAAGGGTGGACATGAGCTTA GCAAATCCACTGGAAATGCTGGAGGCAGGGTAGCTTGTGGTATTATTGGTCTCCAAG GATGA (SEQ ID NO: 2) Expression vector In a third aspect of the present disclosure, provided in embodiments is an expression vector. According to an embodiment of the present disclosure, the expression vector is connected with the nucleic acid for encoding the superoxide dismutase as described in the above aspect. Thus, after introduced into a receptor cell, the expression vector connected with the nucleic acid for encoding the superoxide dismutase is capable of expressing the superoxide dismutase which has high enzyme activity and thermal stability and exhibits significantly improved enzyme activity after high pressure treatment. Recombinant cell In a fourth aspect of the present disclosure, provided in embodiments is a recombinant cell. According to an embodiment of the present disclosure, the recombinant cell is obtained by transforming the receptor cell with the expression vector as described in the above aspect. Thus, the nucleic acid for encoding the superoxide dismutase in the recombinant cell is capable of expressing the superoxide dismutase which has high enzyme activity and thermal stability and exhibits significantly improved enzyme activity after high pressure treatment. Method for preparing a superoxide dismutase In a fifth aspect of the present disclosure, provided in embodiments is a method for preparing the superoxide dismutase as described in the above aspect. According to an embodiment of the present disclosure, the method for preparing a superoxide dismutase comprises cultivating a recombinant cell expressing the superoxide dismutase to obtain a culture medium, isolating the superoxide dismutase from the culture medium followed by purification, optionally subjecting the purified superoxide dismutase to high pressure activation under a pressure of 400 to 600 MPa and at a temperature of 6 to 18°C for I to 20 minutes. According to the method in the embodiment of the present disclosure, the superoxide dismutase prepared has high enzyme activity and thermal stability. The superoxide dismutase when subjected to high pressure activation can improve its enzyme stability significantly. The present inventors have developed the optimized high pressure activation conditions as described above through a large number of experiments, and the prepared superoxide dismutase exhibits significantly improved enzyme activity. Thus, the superoxide dismutase obtained by the method according to the embodiment has high enzyme activity and thermal stability, and exhibits significantly improved enzyme activity after high pressure treatment, showing low requirement for ambient temperature, thus can be stored and applied conveniently. Moreover, the method is simple to operate, easy to implement and suitable for large-scale production.

According to an embodiment of the present disclosure, the method further comprises subjecting an activated product obtained by high pressure activation to a drying treatment to

obtain a superoxide dismutase solid preparation.

The activated product can be useful in preparation of an oral liquid of superoxide dismutase. The present inventors have found that the conformation of the superoxide dismutase

is changed after high pressure activation, leading to increase of enzyme activity. However, the

conformation of the superoxide dismutase can be easily restored after long-time storage, resulting in decrease of enzyme activity. It is found that the superoxide dismutase of the present

disclosure after high pressure activation is not easily subjected to conformation restoration

when is in a solid form, thus its solid preparation prepared by a drying treatment is capable of

preventing the decrease of enzyme activity and retaining the high enzyme activity for a long time.

According to an embodiment of the present disclosure, the drying treatment comprises frozen-drying treatment and/or spray-drying treatment. Thus, a superoxide dismutase solid

preparation is prepared through the drying treatment.

According to an embodiment of the present disclosure, the activated product is mixed with a drying aid agent before the drying treatment. The drying aid agent is selected from of

p-cyclodextrin, maltodextrin and soluble starch at a mass ratio of 0 to 50% based on a mass of the activated product. Thus, the solid preparation obtained can have an effectively improved dryness, which can prevent moisture absorption.

According to an embodiment of the present disclosure, the frozen-drying treatment is

performed at a cold trap temperature is -45 to -80°C under a vacuum degree of 15 to 30 Pa for 12 to 48 hours. Thus, the frozen-drying conditions can accelerate the drying of the activated

product, preventing the decrease of superoxide dismutase activity.

According to an embodiment of the present disclosure, the spray-drying treatment is performed at an air inlet temperature of 140 to 180°C and an air outlet temperature of 55 to 70°C

under a feeding flow of 3 to 12 mL/min. Thus, the spray-drying conditions can accelerate the

drying of the activated product, preventing the decrease of superoxide dismutase activity. According to a specific embodiment of the present disclosure, the air inlet temperature is 160°C, the air outlet temperature is 60°C, and the feeding flow is 3 ml/min. According to the method in embodiments of the present disclosure, the solid preparation of superoxide dismutase prepared comprises the superoxide dismutase as described in above aspects. Thus, the superoxide dismutase solid preparation prepared by the method shows low requirement for ambient temperature, which can be stored and applied conveniently. Moreover, the superoxide dismutase in the solid preparation has high enzyme activity, a long shelf life, and a good efficacy. According to an embodiment of the present disclosure, the superoxide dismutase solid preparation is prepared by the method for preparing a superoxide dismutase as described in above aspect. Thus, the obtained superoxide dismutase solid preparation exhibits a high and stable enzyme activity, a long shelf life and a good efficacy. According to an embodiment of the present disclosure, the enzyme activity of the superoxide dismutase in the solid preparation is decreased by 20% or below, for example, 15% or below, 10% or below or 6% or below after storage at 4°C for 60 days. Thus, preparation of the high pressure activated superoxide dismutase into a solid preparation is capable of avoiding the conformation restoration of the superoxide dismutase, thus preventing the decrease of enzyme activity, retaining the high enzyme activity in a long time and prolonging the shelf life. Superoxide dismutase oral liquid In a sixth aspect of the present disclosure, provided in embodiments is an oral liquid of superoxide dismutase. According to an embodiment of the present disclosure, the superoxide dismutase oral liquid comprises the superoxide dismutase as described in above aspect. Thus, the superoxide dismutase oral liquid prepared by the method for preparing a superoxide dismutase according to the embodiments shows low requirement for ambient temperature, which can be stored and applied conveniently, with a broad application prospect. According to an embodiment of the present disclosure, the superoxide dismutase oral liquid is prepared by the method for preparing a superoxide dismutase as described in above aspect. Thus, the superoxide dismutase in the superoxide dismutase oral liquid obtained has high enzyme activity. Superoxide dismutase solid preparation The present inventors have also found that the increased SOD enzyme activity obtained by high pressure activation can be maintained in a short time, but is easily decreased with the prolonged storage time due to the conformation restoration. In view of this, the present inventors conducted an in-depth study on high pressure activated SOD enzymes and found the existence form of SOD enzymes may significantly affect the enzyme activity. The superoxide dismutase when presented in a solid form can effectively prevent the conformation restoration compared to when presented in a liquid form, so as to avoid the decrease of enzyme activity and maintain the high enzyme activity for a long time, with a prolonged shelf life. In a seventh aspect of the present disclosure, provided in embodiments is a superoxide dismutase solid preparation. According to an embodiment of the present disclosure, the superoxide dismutase solid preparation comprises the superoxide dismutase as described in above aspect, in which the superoxide dismutase is subjected to high pressure activation in advance. As mentioned above, the high pressure activated superoxide dismutase when presented in a form of solid preparation can effectively prevent the restoration of conformation formed via the high pressure activation, so as to avoid the decrease of enzyme activity and maintain the high enzyme activity for a long time, with a prolonged shelf life. Thus, according to the superoxide dismutase solid preparation in the embodiment of the present disclosure, the superoxide dismutase has high enzyme activity and a long shelf life, which can be useful in the fields of food, medicine and cosmetics, with a broad application prospect. It should be noted that the present disclosure does not make strict limitations on the form of the solid preparation, for example, the solid preparation may be in a block form, a powder form and the like, which can be flexibly selected according to the actual situation. Preferably, the solid preparation is in the form of a powder, thereby generating convenience for carry and eating, as well as absorption. According to an embodiment of the present disclosure, the superoxide dismutase in the superoxide dismutase solid preparation is derived from edible plants or microorganisms. Compared to the animal-derived superoxide dismutase, the plant or microorganism-derived superoxide dismutase is more easily accessible. According to an embodiment of the present disclosure, the edible plants can be selected from Actinidia Chinensis, Chaenomeles sinensis Koehne, Citrus limon (L.) Burm. f, fruit of Morus alba Linn., Hippophae rhamnoides, Vaccinium Spp or Rosa roxburghii. Such edible plants contain superoxide dismutase with high enzyme activity and thermal stability. The present inventors have found that high pressure treatment cannot improve the enzyme activity of all SOD enzymes, such as SOD enzyme of Rosa chinensis. However, all of the edible plants as mentioned above can exhibit significantly improved SOD enzyme activity after high pressure treatment, preferably, Rosa roxburghii and Hippophae rhamnoides, more preferably Rosa roxburghii. Rosa roxburghii which already has a good SOD enzyme activity can exhibit significantly improved SOD enzyme activity after the high pressure treatment and retain its high thermal stability, with unchanged enzyme activity even at 90°C or below, showing low requirement for ambient temperature, thus can be stored and applied conveniently.

According to an embodiment of the present disclosure, the microorganism is selected from

Escherichiacoli or yeast. The microorganism which can express the superoxide dismutase can be either a wild-type microorganism such as wild-type Escherichia coli or yeast, or a

genetically engineered bacterium such as Escherichia coli or yeast carrying an expression

vector connected with a gene encoding superoxide dismutase, so as to achieve high expression of the superoxide dismutase.

According to an embodiment of the present disclosure, the superoxide dismutase solid preparation further contains at least one of prebiotics and probiotics, thereby further improving the beneficial efficacy of the superoxide dismutase and increasing the nutritional value. The

present disclosure does not make strict limitations on the types of probiotics and prebiotics,

which can be chosen from those known in the art according to actual needs. For example,

prebiotics are selected from dietary fiber, inulin, microalgae, oligosaccharides, and some natural plant extracts such as fresh vegetables, Chinese herbal medicine, wild plants and the like,

protein hydrolysates, etc. The probiotics are selected from Bifidobacterium, Lactobacillus

paracasei,Streptococcus thermophilus, Lactobacillus acidophilus and the like. According to an embodiment of the present disclosure, the superoxide dismutase in the

superoxide dismutase solid preparation has the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having a homology of at least 80%, for example, at least 85%, 90%, 95%

or 99% to the amino acid sequence of SEQ ID NO: 1, in which the amino acid at position 15 is

Threonline (T), the amino acid at position 67 is Glycine (G), the amino acid at position 87 is Isoleucine (I) and the amino acid at position 143 is Valine (V). Such a superoxide dismutase

having the amino acid sequence of SEQ ID NO: 1 is the superoxide dismutase derived from

Rosa roxburghii, which already has a high SOD enzyme activity and a high thermal stability and can exhibit significantly improved enzyme activity after the high pressure treatment.

Further, the present inventors also have found that the superoxide dismutase having an amino

acid sequence with a homology of at least 80%, for example, at least 85%, 90%, 95% or 99% to the amino acid sequence of SEQ ID NO: 1 exhibits similar high enzyme activity and thermal

stability, as well as similar high enzyme activity after the high pressure treatment, compared to

the superoxide dismutase having the amino acid sequence of SEQ ID NO: 1. Furthermore, the present inventors concluded that the amino acids at positions 15, 67, 87 and 143 are the functional sites affecting enzyme activity, behaviors after high pressure treatment and thermal stability. MAKGVAVLCSSEGVTGTILFTQEGDGPTTVTGNVSGLKPGLHGFHVHALGDTTNG

CMSTGPHFNPAGKEHGAPEDENRHAGDLGNIIVGDDGTATFTIVDKQIPLTGPHSIIGRA

VVVHGDPDDLGKGGHELSKSTGNAGGRVACGIIGLQG (SEQ ID NO: 1) According to an embodiment of the present disclosure, the nucleic acid for encoding the

superoxide dismutase comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide

sequence having a homology of at least 80%, for example, at least 85%, 90%, 95% or 99% to the nucleotide sequence of SEQ ID NO: 2, in which the nucleotides at positions 43 to 45 are

ACG, the nucleotides at positions 199 to 201 are GGC, the nucleotides at positions 259 to 261

are ATT and the nucleotides at positions 427 to 429 are GTA. Thus, the superoxide dismutase encoded by the nucleic acid has high enzyme activity and thermal stability and exhibits

significantly improved enzyme activity after high pressure treatment.

ATGGCAAAGGGTGTTGCTGTACTTTGCTCCAGTGAGGGTGTTACGGGAACTATC CTCTTCACCCAAGAGGGAGATGGCCCAACTACTGTGACTGGAAACGTTTCTGGCCT CAAGCCTGGGCTTCATGGTTTCCATGTTCATGCTCTTGGTGACACAACAAACGGTTG CATGTCAACTGGACCACACTTCAATCCTGCTGGCAAAGAGCATGGTGCTCCTGAAGA TGAGAATCGTCATGCTGGTGATCTTGGAAATATCATTGTTGGGGATGATGGAACTGCT ACCTTCACAATTGTTGACAAGCAGATTCCTCTCACTGGACCACATTCTATCATTGGTA GGGCGGTTGTTGTCCATGGAGACCCTGATGACCTTGGCAAGGGTGGACATGAGCTT AGCAAATCCACTGGAAATGCTGGAGGCAGGGTAGCTTGTGGTATTATTGGTCTCCAA

GGATGA (SEQ ID NO: 2)

According to an embodiment of the present disclosure, the microorganism is a genetically engineered bacterium, which contains an expression vector connected with the nucleotide

sequence of SEQ ID NO: 2 or a nucleotide sequence having a homology to SEQ ID NO: 2.

Thus, the superoxide dismutase expressed by the genetically engineered bacterium has high enzyme activity and thermal stability, showing low requirement for ambient temperature, thus

can be stored and applied conveniently. Specifically, the genetically engineered bacterium can

be selected from Escherichiacoli or yeast, and the expression vector can be a plasmid. According to an embodiment of the present disclosure, the enzyme activity of the

superoxide dismutase is 2x105 U/g or above. Thus, the superoxide dismutase has high enzyme

activity. According to an embodiment of the present disclosure, the enzyme activity of the superoxide dismutase in the solid preparation is decreased by 20% or below, for example, 15% or below, 10% or below or 6% or below after storage at 4°C for 60 days. Thus, preparation of the high pressure activated superoxide dismutase into a solid preparation is capable of avoiding the conformation restoration of the superoxide dismutase, thus preventing the decrease of enzyme activity, retaining the high enzyme activity in a long time and prolonging the shelf life.

Method for preparing a superoxide dismutase solid preparation In an eighth aspect of the present disclosure, provided in embodiments is a method for

preparing a superoxide dismutase solid preparation. According to an embodiment of the present disclosure, referring to Figure 1, the method for preparing a superoxide dismutase solid

preparation comprises the following steps.

S100 Extraction treatment In this step, a raw material is subjected to extraction treatment to obtain an extract

containing the superoxide dismutase. Thus, the superoxide dismutase is extracted from the raw

material.

According to an embodiment of the present disclosure, the extraction treatment includes crushing edible plant cells or microorganism cells in the fermentation medium to a slurry to

obtain an extract of superoxide dismutase, thus facilitating the release of superoxide dismutase. According to an embodiment of the present disclosure, the microorganism is a genetically

engineered bacterium, which contains an expression vector connected with the nucleotide

sequence of SEQ ID NO: 2 or a nucleotide sequence having a homology to SEQ ID NO: 2. Thus, the superoxide dismutase of Rosa roxburghii can be expressed by the genetically

engineered bacterium, which has high enzyme activity and thermal stability, showing low

requirement for ambient temperature and can be stored and applied conveniently. According to an embodiment of the present disclosure, crushing edible plant cells or

microorganism cells to a slurry is performed at a temperature of 0 to 30°C, preferably 4 to10°C, thus preventing the decrease of enzyme activity. According to an embodiment of the present disclosure, the extraction treatment further

includes at least one of the following steps: filtering and centrifuging the slurry to collect a

supernatant; and subjecting the supernatant to double-channel ultrafiltration separation to collect a retentate fluid, thereby obtaining the extract of superoxide dismutase. The filtering and

centrifuging is performed for removing fiber and other substances in the slurry. The

double-channel ultrafiltration separation is performed for obtaining the superoxide dismutase with a high purity.

According to an embodiment of the present disclosure, centrifuging is performed at a speed of 1,000 to 4,000 rpm and at a temperature of 0 to10°C for 0 to 30 minutes (excluding the point of 0 minute), thereby removing the impurity and avoiding the precipitation of superoxide dismutase. According to an embodiment of the present disclosure, the double-channel ultrafiltration separation includes a first separation performed by a tubular membrane with an aperture of 60 to 120 kD and a second separation performed by a coiled membrane with an aperture of 8 to 30 kD, thereby obtaining the superoxide dismutase with high yield and purity. S200 High pressure activation treatment In this step, the extract of superoxide dismutase obtained in S100 is subjected to high pressure activation to obtain an activated product. According to an embodiment of the present disclosure, high pressure activation treatment is performed under a pressure of 200 to 600 MPa and at a temperature of 6 to 18°C for 1 to 20 minutes. The present inventors obtained the optimized conditions for high pressure activation as described above through a large number of experiments, thus obtaining superoxide dismutase with high enzyme activity. S300 Drying treatment In this step, the activated product obtained in S200 is subjected to drying to obtain a superoxide dismutase solid preparation. According to an embodiment of the present disclosure, the drying treatment includes frozen-drying and/or spray-drying. Thus, the drying treatment can prevent the decrease of superoxide dismutase activity. In some embodiments, the superoxide dismutase powder obtained by the drying treatment can be compressed into a formulation in a block or sheet form. The particular manufacture process can be flexibly selected according to actual situations, which is not strictly limited in the present disclosure. According to an embodiment of the present disclosure, the activated product is mixed with a drying aid agent before the drying treatment. The drying aid agent is selected from of p-cyclodextrin, maltodextrin and soluble starch at a mass ratio of 0 to 50% based on a mass of the activated product. Thus, the solid preparation obtained can have an effectively improved dryness, which can prevent moisture absorption. According to an embodiment of the present disclosure, the frozen-drying treatment is performed at a cold trap temperature of -45 to -80°C under a vacuum degree of 15 to 30 Pa for

12 to 48 hours. Thus, the frozen-drying conditions can accelerate the drying of the activated

product, preventing the decrease of superoxide dismutase activity.

According to an embodiment of the present disclosure, the spray-drying treatment is performed at an air inlet temperature of 140 to 180°C and an air outlet temperature of 55 to 700 C

under a feeding flow of 3 to 12 mL/min. Thus, the spray-drying conditions can accelerate the drying of the activated product, preventing the decrease of superoxide dismutase activity.

According to an embodiment of the present disclosure, the method for preparing a superoxide dismutase solid preparation further includes allowing the superoxide dismutase solid preparation to be in a vacuum packaging container, followed by vacuumizing , purging with a

gas and heat sealing. The purged gas is selected from at least one of CO 2 and N 2 . The

weight-to-volume ratio in g/mL of the superoxide dismutase to the gas is 1:0.2 to 1:2. Thus, the superoxide dismutase solid preparation sealed and stored under the above conditions can have

an extended shelf life, which can be stored at 0 to 10°C for a long time.

It would be appreciated by those skilled in the art that the features and advantages

described above for the superoxide dismutase solid preparation are also suitable to the method for preparing a superoxide dismutase solid preparation, which will not be repeated here.

Reference will be made in detail to examples of the present disclosure. It would be appreciated by those skilled in the art that the following examples are explanatory and cannot

be construed to limit the scope of the present disclosure. If the specific technology or conditions

'0 are not specified in the examples, a step will be performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. If

the manufacturers of reagents or instruments are not specified, the reagents or instruments may

be commercially available.

EXAMPLES

Example 1 In this example, a superoxide dismutase (SOD) solid powder was prepared according to

the following steps.

1. Extraction of total RNA from Rosa roxburghii The total RNA of Rosa roxburghiiwas extracted by using the Aidlab RNA Extraction Kit.

The specific steps were as follows:

(1) 200 mg of fruit tissue of Rosa roxburghii was ground in the presence of liquid nitrogen, followed by transferred to 1 ml cell lysis buffer (CLB) containing 10% mercaptoethanol preheated at 65°C, pipetted up and down to mix with a pipette tip and vortexed for 30 to 60 seconds immediately, incubated at 65°C for 5 minutes, and centrifuged at 13,000 rpm for 10 minutes.

(2) The mixture obtained in (1) was transferred to a new centrifuge tube, which was added

with 0.5 times the volume of absolute ethanol, followed by blown and mixed with a pipette tip. (3) The mixture obtained in (2) was transferred to a genome removal column and

centrifuged at 13,000 rpm for 2 minutes, followed by discarding the waste liquid.

(4) The genome removal column in (3) was transferred to a new centrifuge tube. 500 1 RLT Plus lysis buffer was added into the tube and centrifuged at 13,000 rpm for 30 seconds,

followed by adding 0.5 times the volume of absolute ethanol, blown and mixed with a pipette

tip. (5) The mixture obtained in (4) was transferred to an adsorption column RA and

centrifuged at 13,000 rpm for 2 minutes to discard the waste liquid.

(6) 700 1 of protein removal buffer RW1 was added, incubated for 1 minute at room temperature, centrifuged at 13,000 rpm for 30 seconds, and the waste liquid was discarded.

(7) 500 1 of washing buffer RW (added with absolute ethanol before use) was added, centrifuged at 13,000 rpm for 30 seconds, and the waste liquid was discarded. (8) The step (7) was repeated once.

(9) The adsorption column RA transferred to an empty collection tube was centrifuged at

13,000 rpm for 2 minutes to remove the washing buffer completely. (10) The adsorption column RA in (9) was transferred to a RNase-free centrifuge tube, 40

1 RNase-free water was added to the middle part of adsorption column RA and incubated for 1

minute. (11) The RNase-free centrifuge tube in (10) was centrifuged at 12,000 rpm for 1 minute,

and the liquid collected was the total RNA of Rosa roxburghii.

(12) Detection of RNA concentration and quality: 5 1 of total RNA was subjected to ordinary agarose gel electrophoresis (1.2%), and 1 1 of total RNA was detected for

concentration and OD 260/OD 280 ratio by the Nanodrop.

2. Synthesis of the first strand of cDNA The total RNA was reversely transcribed into cDNA by using the following reaction

system and procedure.

Reaction system is as below: Reagents Volume (pl) 5x FastKing-RT SuperMix 4 Total RNA 2 Rnase-Free ddH20 14

Reaction procedure: 42°C for 15 minutes and 95°C for 3 minutes. 3. PCR amplification of SOD gene of Rosa roxburghii A conserved region of superoxide dismutase (SOD) gene was subjected to PCR amplification in the presence of the cDNA first strand obtained in step 2 as a template and a pair of primers of SOD conserved region (SOD-F of SEQ ID NO: 3 and SOD-R of SEQ ID NO: 4), followed by 3'RACE and 5'RACE, thus obtaining the full length cDNA sequence. The nucleotide sequence of superoxide dismutase gene of Rosa roxburghii is shown as SEQ ID NO: 2 and the amino acid sequence encoded by the nucleotide sequence is shown as SEQ ID NO: 1. SOD-F: TCTCCTGGCCTTCATGGTTTCCATAT (SEQ ID NO: 3) SOD-R: GTAGTCTTGCTAAGTTCATGTCCACC (SEQ ID NO: 4) Reaction system is as below: Reagents Volume (pl) template 1 supermix 10 ddH20 7 SOD-R 1 SOD-F 1

Reaction procedure is as below: denaturation at 98°C 2 minutes denaturation at 98°C 10 seconds annealing at 57°C 20 seconds 30 cycles extension at 72°C 10 seconds extension at 72°C 5 minutes

4. Obtaining SOD of Rosa roxburghii

(1) The nucleotide sequence encoding SOD of Rosa roxburghii was subjected to PCR amplification by using the full length cDNA sequence obtained in step 3 as a template. The

amplified fragment was inserted into pET-30a followed by introduced into E.coli BL21(DE3).

BL21(DE3) with pET-30a-SOD was selected and inoculated in 20 ml Luria Broth (LB) medium of 50 g/ml, followed by shaken at 220 rpm and 37°C for 12 hours.

(2) 10 ml of bacterial cells in (1) were further inoculated in 1 L LB medium of 50 pg/ml,

and cultured with shaking at 220 rpm and 37C. (3) Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1

mM when OD6 0 0 reached 0.9, followed by culturing with shaking at 220 rpm and 37°C for 5

hours. (4) The bacterial medium was transferred to a 200 ml centrifuge flask and centrifuged at

7,000g and 4°C for 5 minutes, after which the concentrated bacterial cells were resuspended

with 20 ml cell lysis buffer. The resuspended bacterial cells were broken via ultrasound treatment, centrifuged to collect a supernate, and then subjected to SDS-PAGE electrophoresis

of total protein of the bacterial cells to obtain the superoxide dismutase.

5. High pressure activation The obtained SOD was subjected to high pressure activation under a pressure of 550 MPa

and at a temperature of 16.5°C for 5 minutes, thereby obtaining an activated product, which can

be used as a superoxide dismutase oral liquid. 6. Preparation of a superoxide dismutase solid powder

The activated product was mixed with p-cyclodextrin (at a mass ratio of 10% based on a

mass of the activated product), followed by frozen-drying at a cold trap temperature of -67°C under a vacuum degree of 26 Pa for 48 hours, thereby obtaining the superoxide dismutase solid

preparation.

Example 2 Analysis of enzyme activity 1. SOD of Rosa roxburghii, SOD of Rosa chinensis and SOD of Hippophae rhamnoides

were obtained according to the following steps. 0.2 g of Rosa roxburghii, 0.2 g of Rosa chinensis or 0.2 g of Hippophae rhamnoides were

weighed accurately, which was respectively added with 1.8 ml of phosphate buffer saline (PBS).

The samples were crushed and then ground in a grinding bowl on ice bath to prepare a slurry with 10% sample. Then 8 ml of PBS solution was added to wash the bowl and the collected washing mixture was transferred to the slurry, forming a 5-fold dilution of slurry. The slurry was centrifuged at 10,000 g and 4°C for 10 minutes, after which the supernatant was collected, which was a crude SOD extract of Rosa roxburghii, a crude SOD extract of Rosa chinensis and a crude SOD extract of Hippophae rhamnoides. The amino acid sequence of Rosa chinensis SOD is shown as SEQ ID NO: 5 and the amino acid sequence of Rosa roxburghii SOD is shown as SEQ ID NO: 1. makgvavlcs segvkgtilf tqegdgpttv tgnvsglkpg lhgfhvhalg dttngcmstg phfnpaakeh gapedenrha gdlgnitvgd dgtatftivd kqipltgphs iigravvvhg dpddlgkggh elskstgnag griacgiigl qg (SEQ ID NO: 5)

2. The crude SOD extract ofRosa roxburghii, the crude SOD extract of Rosa chinensis and the crude SOD extract of Hippophae rhamnoides were respectively subjected to high pressure activation under a pressure of 550 MPa and at a temperature of 16.5°C for 5 minutes. 3. The crude SOD extract and high pressure activated SOD extract of Rosa roxburghii, the crude SOD extract and high pressure activated SOD extract of Rosa chinensis and the crude SOD extract and high pressure activated SOD extract of Hippophae rhamnoides were respectively detected with the SOD Enzyme Activity Detection Kit (Jiancheng, Nanjing) for enzyme activity. Results are shown in Table 1, indicating that Rosa roxburghii SOD has a higher enzyme activity than that of Rosa chinensis SOD. Moreover, enzyme activity of Rosa roxburghiiSOD is significantly increased after high pressure treatment, while enzyme activity of Rosa chinensis SOD is not increased, which suggests that high pressure treatment cannot improve enzyme activity of all SOD enzymes. The Hippophae rhamnoides SOD which has an enzyme activity lower than that of Rosa roxburghii, exhibited increased enzyme activity after high pressure treatment, which is superior over that of Rosa chinensis. Moreover, it is concluded that the amino acids at positions 15, 67, 87 and 143 may be the functional sites affecting enzyme activity and behaviors after high pressure treatment through comparison of the amino acid sequences of Rosa roxburghii SOD and Rosa chinensis SOD.

Table 1 Enzyme activity of SODs Enzyme activity before Enzyme activity after

high pressure treatment high pressure treatment

(U/g) (U/g)

Rosa roxburghii SOD (duplication 1) 3625.9 4713.67

Rosa roxburghii SOD (duplication 2) 3894.5 4868.13

Hippophae rhamnoides SOD (duplication 1) 1423.74 1667.66

Hippophae rhamnoides SOD (duplication 2) 1649.23 1929.33

Rosa chinensis SOD (duplication 1) 993.14 983.12

Rosa chinensis SOD (duplication 2) 1076.92 1036.58

Example 3 Thermal stability analysis The SOD obtained in step 4 of Example 1 was subjected to thermal stability analysis by incubating the SOD at fixed temperatures of 50°C, 60°C, 70°C, 80°C and 90°C for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes respectively, followed by transferred to ice water immediately and measuring the enzyme activity within 2 hours. The results show that the SOD can maintain the high enzyme activity for a long time at a temperature ranging from 50°C to 80°C. When the SOD is incubated at a high temperature of 90°C for a short time such as 0 to 20 minutes, the enzyme activity of SOD almost maintains unchanged; however, when the SOD is incubated at a high temperature of 90°C for a long time such as more than 20 minutes, the enzyme activity of SOD starts to decrease significantly, thus suggesting a high thermal stability for the SOD of the present disclosure. Meanwhile, the crude SOD extract of Rosa chinensis and the crude SOD extract of Rosa roxburghii obtained in step 1 of Example 2 were incubated at fixed temperatures 50°C and 90°C for 20 minutes and 60 minutes respectively, followed by transferred to ice water immediately and measuring the enzyme activity within 2 hours. The results are shown in Table 2 and Table 3, indicating that the Rosa roxburghii SOD exhibited a significantly slower decreased enzyme activity compared to Rosa chinensis. Thus, the Rosa roxburghii SOD has an excellent thermal stability.

Table 2 enzyme activity (U/g) change rate of Rosa chinensis SOD under different heating

treatments 0 min 20 mins Change rate (%) 60 mins Change rate (%)

50 0 C 993.13 935.92 -5.76 1039.50 4.67 90 0 C 1076.92 868.63 -19.34 589.79 -45.23

Table 3 enzyme activity (U/g) change rate of Rosa roxburghii SOD under different heating

treatments 0 min 20 mins Change rate (%) 60 mins Change rate (%) 500 C 3625.90 3654.55 0.79 3627.04 0.03 900 C 3679.51 3646.99 -0.88 3057.79 -16.89

Example 4 In this example, the SOD solid powder was prepared according to the following steps.

(1) Fresh fruit of Rosa roxburghii without rotting was selected as a raw material.

(2) The fresh fruit of Rosa roxburghiiwas cleaned.

(3) The cleaned fresh fruit of Rosa roxburghii was mixed with ice water and crushed to a slurry at 4 to 10°C.

(4) The slurry was filtrated with 60-mesh filter to remove peel, pulp residue and seed, thereby obtaining juice of Rosa roxburghii.

(5) The juice was centrifuged at a speed of 3,000 rpm for 10 minutes to collect a

supernatant. (6) The supernatant was subjected to double-channel ultrafiltration separation to collect a

retentate fluid, in which the double-channel ultrafiltration separation includes a first separation

performed by a tubular membrane with an aperture of 100 kD and a second separation performed by a coiled membrane with an aperture of 10 kD.

(7) The retentate fluid was subjected to high pressure activation under a pressure of 550

MPa and at a temperature of 16.50 C for 5 minutes, thereby obtaining an activated product. (8) The activated product was mixed with p-cyclodextrin (at a mass ratio of 10% based on

a mass of the activated product).

(9) The mixture obtained in (8) was frozen-dried at a cold trap temperature of -67C under a vacuum degree of 26 Pa for 48 hours, thereby obtaining the SOD solid powder.

(10) The SOD solid powder transferred to a vacuum packaging container was vacuumizd,

purged with a gas (1:1 of CO 2 : N 2 and 1:1 of total gas volume to SOD solid powder mass) and heat sealed.

Example 5 In this example, the SOD solid powder was prepared according to the following steps.

(1) Fresh fruit of Rosa roxburghii without rotting was selected as a raw material. (2) The fresh fruit of Rosa roxburghiiwas cleaned.

(3) The cleaned fresh fruit of Rosa roxburghii was mixed with ice water and crushed to a

slurry at 4 to 10°C. (4) The slurry was filtrated with 60-mesh filter to remove peel, pulp residue and seed,

thereby obtaining juice of Rosa roxburghii.

(5) The juice was centrifuged at a speed of 2,000 rpm for 15 minutes to collect a supernatant.

(6) The supernatant was subjected to double-channel ultrafiltration separation to collect a

retentate fluid, in which the double-channel ultrafiltration separation includes a first separation

performed by a tubular membrane with an aperture of 80 kD and a second separation performed by a coiled membrane with an aperture of 20 kD.

(7) The retentate fluid was subjected to high pressure activation under a pressure of 450 MPa and at a temperature of 25°C for 4 minutes, thereby obtaining an activated product.

(8) The activated product was mixed with p-cyclodextrin (at a mass ratio of 2.5% based on

a mass of activated product). (9) The activated product in (8) was spray-dried at an air inlet temperature of 160°C and an

air outlet temperature of 60°C under a feeding flow of 5 ml/min, thereby obtaining the SOD

solid powder. (10) The SOD solid powder transferred to a vacuum packaging container was vacuumizd,

purged with a gas (1:1 of CO 2 : N 2 and 1:1 of total gas volume to SOD solid powder mass) and

heat sealed.

Comparative Example 1 The SOD solid powder in this example was prepared according to the procedure of Example 4, except that the supernatant obtained in step (6) was directly subjected to step (8)

without step (7).

Example 6 Stability analysis 1. The high pressure activated SOD solid powder prepared in Example 4 and Example 5, the inactivated SOD powder prepared in Comparative Example 1 and the high pressure

activated SOD liquid obtained in step (7) of Example 4 were respectively stored at 4°C for 60

days, and the enzyme activity before and after the storage was detected by the SOD Enzyme Activity Detection Kit (Jiancheng, Nanjing).

The results are shown in Table 4. It can be seen from the results of Example 4 and

Comparative Example 1 that the high pressure activated SOD powder exhibits an enzyme activity being 247.9% higher than that of inactivated SOD powder. Further, the activated SOD

in a power form (Example 4 and Example 5) is capable of maintaining the enzyme activity for a

longer storage time than the activated SOD in a liquid form. It is concluded that the high pressure activated SOD when presented in a liquid can be easily restored to the conformation

before high pressure activation, resulting in the decrease of enzyme activity. Moreover, the

inactivated SOD power (Comparative Example 1) despite a high retention rate of SOD activity,

exhibits a low enzyme activity; however, such a SOD in a power form cannot effectively improve its enzyme activity through the high pressure activation since the pressure transmission

depends on a liquid. Table 4 Stability analysis

SOD activity U/g SOD activity U/g retention rate of Samples (before storage) (after storage) SOD activity

Example 4 (activated, power) 245567.84 215469.22 87.74%

Example 5 (activated, power) 322847.13 304124.33 94.20%

Comparative example 1 99055.84 97817.10 98.750% (inactivated, power)

Example 4 (activated, liquid) 27748.44 18091.98 65.2%

2. The high pressure activated SOD powder prepared in Example 4 was incubated at fixed

temperatures of 50°C, 60°C, 70°C, 80°C and 90°C for 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes respectively, followed by transferred to ice water

immediately and measuring the enzyme activity within 2 hours.

As the results shown in Figure 2, the SOD can maintain the high enzyme activity for a long time at a temperature ranging from 50°C to 80°C. When the SOD is incubated at a high

temperature of 90°C for a short time such as 0 to 20 minutes, the enzyme activity of SOD almost maintains unchanged; however, when the SOD is incubated at a high temperature of

90°C for a long time such as more than 20 minutes, the enzyme activity of SOD starts to decrease significantly, thus suggesting a high thermal stability for the SOD solid powder of the

present disclosure.

Reference throughout this specification to "an embodiment", "some embodiments", "one

embodiment", "another example", "an example", "a specific example" or "some examples"

means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the

present disclosure. Thus, the appearances of the phrases such as "in some embodiments", "in

one embodiment", "in an embodiment", "in another example", "in an example", "in a specific example" or "in some examples" in various places throughout this specification are not

necessarily referring to the same embodiment or example of the present disclosure. Furthermore,

the particular features, structures, materials, or characteristics may be combined in any suitable

manner in one or more embodiments or examples. Although explanatory embodiments have been shown and described, it would be

appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the

embodiments without departing from spirit, principles and scope of the present disclosure.

Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and

"including" will be understood to imply the inclusion of a stated integer or group of integers,

but not the exclusion of any other integer or group of integers. The reference to any prior art in this specification is not, and should not be taken as, an

acknowledgement or any form of suggestion that such prior art forms part of the common

general knowledge. It will be appreciated by those skilled in the art that the invention is not restricted in its use

to the particular application described. Neither is the present invention restricted in its preferred

embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments

disclosed, but is capable of numerous rearrangements, modifications and substitutions without

departing from the scope of the invention as set forth and defined by the following claims.

Claims (4)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A superoxide dismutase solid preparation, comprising a superoxide dismutase derived from Rosa roxburghii,

wherein the superoxide dismutase derived from Rosa roxburghii has the amino acid

sequence of SEQ ID NO: 1, the superoxide dismutase derived from Rosa roxburghii is encoded by the nucleotide

sequence of SEQ ID NO: 2, and

the superoxide dismutase solid preparation is prepared by steps: expressing a genetically engineered bacterium containing an expression vector connected

with the nucleotide sequence of SEQ ID NO: 2 to obtain a bacterial medium comprising the

superoxide dismutase, concentrating the bacterial medium via centrifugation and resuspending the concentrated

bacterial cells, followed by SDS-PAGE of total protein of the bacterial cells to obtain the

superoxide dismutase, subjecting the superoxide dismutase to high pressure activation under a pressure of 550

MPa and at a temperature of 16.5°C for 5 minutes, thereby obtaining an activated product, and

mixing the activated product with p-cyclodextrin which is at a mass ratio of 10% based on a mass of the activated product, followed by frozen-drying at a cold trap temperature of -67°C

under a vacuum degree of 26 Pa for 48 hours, thereby obtaining the superoxide dismutase solid

preparation.

2. The superoxide dismutase solid preparation according to claim 1, further comprising at

least one of prebiotics and probiotics.

3. A method for preparing a superoxide dismutase, comprising steps: cultivating a recombinant cell expressing the superoxide dismutase to obtain a culture

medium,

isolating the superoxide dismutase from the culture medium followed by purification, and subjecting the purified superoxide dismutase to high pressure activation under a pressure

of 550 MPa and at a temperature of 16.5°C for 5 minutes,

wherein the superoxide dismutase comprises the amino acid sequence of SEQ ID NO: 1,

the superoxide dismutase is derived from Rosa roxburghii,

the superoxide dismutase is encoded by the nucleotide sequence of SEQ ID NO: 2, and the recombinant cell is obtained by transforming a receptor cell with an expression vector connected with the nucleotide sequence of SEQ ID NO: 2.

4. The method according to claim 3, further comprising subjecting an activated product obtained by high pressure activation to a drying treatment to obtain a superoxide dismutase

solid preparation,

wherein the drying treatment is frozen-drying treatment, the activated product is mixed with a drying aid agent before the drying treatment and the

drying aid agent is p-cyclodextrin at a mass ratio of 10% based on a mass of the activated

product, and the frozen-drying treatment is performed at a cold trap temperature of -67°C under a

vacuum degree of 26 Pa for 48 hours.