Method for extracting and modifying papaya polysaccharide through multi-stage low-temperature plasma

Abstract

The invention relates to the technical field of extraction and modification of papaya polysaccharide, and discloses a method for extracting and modifying papaya polysaccharide by multistage low-temperature plasma, which comprises the following steps: S1, taking fresh papaya pulp, and freeze-drying to obtain papaya powder; s2, performing low-temperature plasma treatment on the pawpaw powder; s3, removing impurities to obtain papaya polysaccharide powder; and S4, performing low-temperature plasma treatment on the papaya polysaccharide powder. As a brand-new extraction and modification process, the extraction yield of the papaya polysaccharide is remarkably improved, the combination of all the steps can achieve the effect of modifying the papaya polysaccharide, the oxidation resistance of the papaya polysaccharide is remarkably improved, and the application prospect is wide; based on the basic steps of the method for extracting and modifying the papaya polysaccharide through the multi-stage low-temperature plasma, papaya polysaccharide powder is added into a glucose solution for modification in the final modification step, the emulsibility and antibacterial activity of the papaya polysaccharide can be accidentally improved, and the specific application of the papaya polysaccharide with high oxidation resistance is facilitated.

Description

[0001] This application claims priority to the prior Chinese application, application number: 202411985461.9, filing date December 31, 2024, the specification, claims, and abstract of which are incorporated herein by reference in their entirety as part of this application. Technical Field

[0002] The invention relates to the technical field of extraction and modification of papaya polysaccharide, in particular to a method for extracting and modifying papaya polysaccharide by multi-stage low-temperature plasma. Background Art

[0003] Papaya (Carica Papaya L.), belonging to the genus Carica of the family Caricapae, is widely planted in Guangdong, Guangxi, Fujian, Yunnan, and Taiwan in my country. It has multiple biological activities such as anti-cancer and antibacterial, immune regulation, anti-diabetes, and antioxidant. Papaya polysaccharide is one of its important active ingredients. Polysaccharides have a strong ability to scavenge free radicals and can be used as a raw material for antioxidants, so they have great development potential. Polysaccharide antioxidants are currently widely used in the fields of chemical industry, medicine, food, and beauty. As a natural polymer compound, it has superior biocompatibility compared to chemical antioxidants and composite antioxidants, and has a wide range of sources and is green and safe. However, most natural polysaccharides have poor antioxidant activity and weak stability, making it difficult to use them directly as antioxidants.

[0004] According to the SAR theory of compound structure-activity relationship, the biological function of polysaccharides is affected by their physical and chemical structure (conformation, side chain distribution, etc.) and determines their application in the field of industrial production. At present, the structure of polysaccharides is often modified by physical, chemical, and enzymatic methods to enhance their applicability in the fields of biomedicine and food. Physical methods (radiation, heat treatment) can avoid the use of chemical reagents to a certain extent, but the essence is still heat treatment, which is not conducive to the nutritional components and sensory quality of polysaccharides. Chemical methods (acetylation, sulfation) have chemical toxicity and pollute the environment, and it is difficult to ensure the integrity of the packaging material. Biological methods (hydrolases, oxidases) are specific, highly selective, and have mild conditions, but the yield is low, and most by-products need to be purified.

[0005] Regarding the extraction of papaya polysaccharides and the maintenance of their antioxidant activity, existing literature usually adopts the water extraction and alcohol precipitation method, and some further adopt ultrasound assistance, but the best they can do is not to destroy the antioxidant activity of papaya polysaccharides. As a natural polysaccharide, the antioxidant activity of papaya polysaccharides is still relatively low.

[0006] Therefore, it is urgent to improve the extraction process of papaya polysaccharides and introduce modification processes to increase the yield of papaya polysaccharide extraction and improve its antioxidant activity, so as to expand the application of papaya polysaccharides. Summary of the invention

[0007] In order to solve the above existing problems, the present invention provides a method for extracting and modifying papaya polysaccharides by multi-stage low-temperature plasma, comprising the steps of: S1, taking fresh papaya pulp and freeze-drying to obtain papaya powder; S2, subjecting the papaya powder to low-temperature plasma treatment; S3, removing impurities such as protein to obtain papaya polysaccharide powder; S4, subjecting the papaya polysaccharide powder to low-temperature plasma treatment. The above treatment can achieve: first, improving the extraction yield and purity of papaya polysaccharides; second, improving the antioxidant activity of papaya polysaccharides; third, shortening the process flow of papaya polysaccharide extraction and modification.

[0008] The research and development team of the present invention has conducted in-depth research in the field of natural product extraction from papaya. For example, a method for extracting papain with a low-temperature plasma-assisted low eutectic solvent has been proposed before (CN202310374820.6). The natural products in papaya are complex and diverse and difficult to extract, especially the extraction of papaya polysaccharides is more difficult. The existing methods either have extremely low yields and a lot of impurities, or seriously damage the antioxidant activity of papaya polysaccharides. In addition, the existing methods also have the addition of chemical substances, which are not green and natural. The research and development team of the present invention has obtained the method of low-temperature plasma extraction and modification of papaya polysaccharides of the present invention after a lot of theoretical research and experiments.

[0009] Although the electron temperature is very high during the discharge process of low temperature plasma, the temperature of heavy particles is very low, and the entire system is in a low temperature state, so it is also called cold plasma or non-equilibrium plasma.

[0010] In one aspect, the present invention provides a method for extracting and modifying papaya polysaccharides using multi-stage low-temperature plasma, comprising the steps of:

[0011] S1, taking fresh papaya pulp and freeze-drying it to obtain papaya powder;

[0012] S2, subjecting the papaya powder to low-temperature plasma treatment;

[0013] S3, removing impurities to obtain papaya polysaccharide powder;

[0014] S4, subjecting the papaya polysaccharide powder to low-temperature plasma treatment.

[0015] First, step S1 freeze-dries the papaya pulp. Compared with traditional crushing or other pre-treatment methods, the present invention finds that direct freeze-drying is beneficial to improving the yield. The principle may be that freeze-drying can minimize sample loss, dehydrate more thoroughly, and the sample can maintain biological activity to a large extent after freeze-drying. In addition, freeze-drying may make various natural active substances in papaya more fully exposed to low-temperature plasma during the next low-temperature plasma treatment. Low-temperature plasma is a new non-thermal extraction technology that contains a variety of active ingredients, such as active oxygen, active nitrogen, charged particles, electrons and photons, etc. It has strong biological activity and oxidizability, can quickly promote the separation of impurities and papaya polysaccharides, and promote the improvement of yield.

[0016] In step S2, the papaya powder may be directly treated with low-temperature plasma, or the papaya powder may be first dissolved and then the dissolved solution may be treated with low-temperature plasma. This step promotes the separation of impurities and papaya polysaccharides, thereby achieving the extraction function.

[0017] The substrate of step S3 is the product treated with low-temperature plasma in step S2, so the separation degree between its internal impurities and papaya polysaccharide is significantly improved, the protein can be easily removed by sevage method or other methods, and the impurities above 10000Da can be removed by dialysis bag. The main contribution of this purification step still comes from the treatment with low-temperature plasma.

[0018] Step S4 is a modification step. Generally speaking, low-temperature plasma is considered to be oxidizing and may have a destructive effect on the antioxidant properties of certain natural products. However, the present invention finds that after the above steps S1-S3, low-temperature plasma treatment of papaya polysaccharide powder can improve its antioxidant properties. The reasons may be: First, in the treatment of low-purity papaya polysaccharides such as those extracted by bioenzymatic method, low-temperature plasma cannot significantly increase its antioxidant properties, and even reduces its antioxidant properties. This is because impurities seize the effect of low-temperature plasma, and charged particles react with impurities first, and the oxidized impurities are oxidized by papaya polysaccharides. Reduction leads to oxidation of papaya polysaccharide. Therefore, it can be inferred that after steps S1-S3 of the present invention, papaya polysaccharide has high purity and directly becomes a substrate for low-temperature plasma. Previous studies could not extract such high-purity papaya polysaccharide, so such effect was not understood. Secondly, ROS / RNS free radicals generated by low-temperature plasma and their degradation of macromolecules affect the conformation of papaya polysaccharide. As a macromolecule, papaya polysaccharide has a variety of sites with free radical scavenging effects at the molecular level. The conformation of papaya polysaccharide affects the exposure of its antioxidant reaction sites, thereby affecting the antioxidant activity.

[0019] Therefore, the difference between the method of extracting and modifying papaya polysaccharides by multi-stage low-temperature plasma of the present invention and the conventional method of extracting polysaccharides by low-temperature plasma is that: 1. A freeze-drying step is introduced; 2. Two-step low-temperature plasma treatment, one step is extraction and the other step is modification; 3. An impurity removal step is added between the two steps of low-temperature plasma treatment; based on the current research of the present invention, when the above four steps are replaced, the extraction of papaya polysaccharides can still be carried out, but the antioxidant modification effect cannot be achieved; in addition, the present invention is only for papaya polysaccharides, and the extraction of other polysaccharides may be carried out according to the present invention, but once modification is involved, it must be highly related to the structure and spatial configuration of the corresponding polysaccharide, and therefore cannot be transplanted to other polysaccharides. Similarly, the modification of other polysaccharides cannot be transplanted to papaya polysaccharides.

[0020] The "multi-stage" in multi-stage low-temperature plasma extraction and modification means multiple uses of low-temperature plasma for treatment. Low-temperature plasma has a strong disinfection and sterilization effect. Therefore, after two treatments of extraction and modification, the final papaya polysaccharide product does not have bacterial and other contamination, and is extremely safe when used in the food, pharmaceutical and cosmetics industries.

[0021] In some embodiments, the discharge form of the low-temperature plasma is selected from one of glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge, and jet discharge.

[0022] Preferably, the discharge form of the low-temperature plasma is dielectric barrier discharge (DBD).

[0023] Dielectric barrier discharge is usually driven by a sinusoidal AC high voltage power supply. As the supply voltage increases, the state of the reaction gas in the system will undergo three stages of change, that is, it will gradually change from an insulating state to a discharge and finally breakdown. When the supply voltage is relatively low, although some gases will have some ionization and free diffusion, the current is too small because the content is too small to cause a plasma reaction in the gas in the reaction zone. At this time, the current is zero. As the supply voltage gradually increases, the number of electrons in the reaction area also increases. However, when the breakdown voltage of the reaction gas is not reached, the electric field between the two electrodes is relatively low and cannot provide enough energy for the electrons to cause inelastic collisions of the gas molecules. The lack of inelastic collisions results in the number of electrons not being able to increase significantly. Therefore, the reaction gas is still in an insulating state and cannot produce discharge. At this time, the current increases slightly with the increase in the voltage applied by the electrodes, but is almost zero. If the supply voltage continues to increase, when the electric field between the two electrodes is large enough to cause the gas molecules to collide inelastically, the gas will increase significantly due to the inelastic collision of ionization. When the electron density in the space is higher than a critical value and the Paschen breakdown voltage, many microdischarge filaments will be generated and conducted between the two electrodes. At the same time, luminescence can be clearly observed in the system. At this time, the current will increase rapidly with the increase of the applied voltage.

[0024] According to the principle of the above-mentioned dielectric barrier discharge, further analysis shows that the reason why it has a better effect on the modification of papaya polysaccharide may be that the dielectric barrier discharge is uniform, diffuse and stable, and can regularly adjust the conformation of papaya polysaccharide, so that most of the treated papaya polysaccharide molecules have similar conformations. As macromolecules, polysaccharide molecules have a certain complexity. There may be antagonistic effects on chemical reactions between polysaccharide molecules with different conformations. The conformation of papaya polysaccharide molecules treated with DBD is more uniform, reducing the probability of antagonism. At the same time, most of the papaya polysaccharide molecules have been modified to enhance antioxidant properties, and the overall antioxidant activity has been significantly improved.

[0025] In some embodiments, step S2 includes: dissolving the papaya powder in distilled water at a mass-to-volume ratio of 1:15-1:35, treating with low-temperature plasma, taking the supernatant and concentrating it to 1 / 3-1 / 10 of the original volume.

[0026] Furthermore, in step S2, the discharge form of the low-temperature plasma includes dielectric barrier discharge, the operating voltage of the low-temperature plasma is 10-50V, the processing time is 5-20min, and the processing frequency is 50-150Hz.

[0027] In some embodiments, step S3 includes: removing proteins using the Sevage method, removing impurities using a dialysis bag, centrifuging, concentrating, and freeze-drying after graded alcohol precipitation to obtain papaya polysaccharide powder.

[0028] Furthermore, in step S3, the Sevage method uses chloroform and n-butanol, the volume ratio of chloroform to n-butanol is 1:1-5:1, the molecular weight of the dialysis bag is 10000Da, and the dialysis time is 48h.

[0029] Preferably, the volume ratio of chloroform to n-butanol is 3:1, at which time the sugar content of papaya crude polysaccharide is the highest and the impurity removal effect is the best.

[0030] In some embodiments, in step S4, the discharge form of the low-temperature plasma includes dielectric barrier discharge, the low-temperature plasma operating voltage is 10-50V, the processing time is 5-20min, and the processing frequency is 50-150Hz.

[0031] Preferably, the low-temperature plasma operating voltage is 40 kV, the frequency is 150 Hz, and the treatment time is 10 min.

[0032] In some embodiments, the step S4 is: adding the papaya polysaccharide powder to a glucose solution, and subjecting the obtained mixed solution to low-temperature plasma treatment.

[0033] Based on the technical solution of low-temperature plasma extraction and modification of papaya polysaccharides discovered by the present invention, papaya polysaccharides with good antioxidant properties were obtained. The research and development team of the present invention considered increasing the efficiency of the last step of modification to maximize the antioxidant enhancement effect of low-temperature plasma on papaya polysaccharide modification. In a large number of experiments, the present invention unexpectedly found that adding papaya polysaccharide powder to glucose solution and then subjecting the mixed solution to low-temperature plasma modification, although there was no significant effect on the antioxidant properties of the product papaya polysaccharide, the emulsification of the papaya polysaccharide was significantly enhanced in subsequent tests, which is very beneficial for its application in the fields of food, medicine and cosmetics, and glucose can be separated from papaya polysaccharide through a semipermeable membrane, and even if there is a certain amount of residue, it is not polluting; the working principle of glucose may be that certain glycoside fragments of papaya polysaccharide tend to The formation of tight hydrogen bonds with glucose molecules is determined by the glycoside arrangement structure of papaya polysaccharide and the molecular structure of glucose. When impacted by charged particles of low-temperature plasma, glucose molecules have a protective effect on certain fragments of papaya polysaccharide. Low-temperature plasma will change the conformation of papaya polysaccharide, and further addition of glucose will affect the change of the conformation of papaya polysaccharide; the change of conformation may affect the spatial arrangement of hydrophilic and lipophilic groups on the surface of papaya polysaccharide molecules, thereby changing the emulsification of papaya polysaccharide products. This change may enhance the emulsification or weaken the emulsification; the present invention only provides a possible principle, and does not exclude other possible principles at the same time; and the present invention happens to find that low-temperature plasma + glucose solution has a significant improvement in the emulsification of papaya polysaccharide products. At the same time, the combined treatment of low-temperature plasma and glucose does not involve chemical additives, and glucose is completely safe for the human body.

[0034] In addition, when testing papaya polysaccharides, it was found that the antibacterial properties of the modified papaya polysaccharides were significantly improved. This may be based on a principle similar to the above. Through the conformational change of papaya polysaccharides, its tendency to bind to bacteria and substances within bacteria is enhanced, similar to how polyphenols can have antibacterial properties on bacteria, acting on structures such as bacterial cell membranes, causing damage to the bacterial structure, thereby achieving an antibacterial effect.

[0035] In some embodiments, the glucose solution is a 5% glucose solution.

[0036] 5% is only the concentration of the glucose solution when low-temperature plasma and glucose solution work synergistically in the embodiment. It can be understood that, based on the above-mentioned principle of glucose action, any concentration of glucose solution has the effect of improving the emulsification of papaya polysaccharide modified by low-temperature plasma.

[0037] Papaya polysaccharides with improved antioxidant, emulsifying and antibacterial properties can be used in the following applications: in the food field, to improve the stability, emulsifying and water holding capacity of food; in the pharmaceutical field, as an auxiliary material to improve the stability of drugs, improve the efficacy of drugs, etc., and papaya polysaccharides with improved antibacterial properties can inhibit common pathogenic microorganisms; in the cosmetics field, they can be used to prepare antioxidant products with better emulsifying properties, such as lotions, creams, etc., and have an inhibitory effect on common pathogenic microorganisms on the skin surface; there can also be corresponding applications in other fields. The papaya polysaccharide obtained by the method provided by the present invention has broad application prospects.

[0038] On the other hand, the present invention provides a papaya polysaccharide with enhanced antioxidant properties, and the preparation method of the papaya polysaccharide includes the method of multi-stage low-temperature plasma extraction and modification of papaya polysaccharide.

[0039] On the other hand, the present invention provides a papaya polysaccharide with enhanced antioxidant and emulsifying properties, and the preparation method of the papaya polysaccharide includes the method of multi-stage low-temperature plasma extraction and modification of papaya polysaccharide.

[0040] On the other hand, the present invention provides a papaya polysaccharide with enhanced antioxidant, emulsifying and antibacterial properties, and the preparation method of the papaya polysaccharide includes the multi-stage low-temperature plasma extraction and modification method of the papaya polysaccharide.

[0041] On the other hand, the present invention provides a use of glucose for preparing an agent for improving the emulsification of low-temperature plasma-modified papaya polysaccharides, and the preparation method of the low-temperature plasma-modified papaya polysaccharides includes the multi-stage low-temperature plasma extraction and modification method of papaya polysaccharides.

[0042] On the other hand, the present invention provides a use of glucose for preparing an agent for improving the antibacterial properties of low-temperature plasma-modified papaya polysaccharides, and the preparation method of the low-temperature plasma-modified papaya polysaccharides includes the multi-stage low-temperature plasma extraction and modification method of papaya polysaccharides.

[0043] It is worth noting that the above-mentioned use of glucose is based on papaya polysaccharide modified by low-temperature plasma, that is, the papaya polysaccharide obtained by the above-mentioned multi-stage low-temperature plasma extraction and modification method of papaya polysaccharide of the present invention, which is further discovered and optimized on the basis of improving antioxidant properties.

[0044] In summary, the present invention has the following beneficial technical effects:

[0045] 1. The present invention provides a method for extracting and modifying papaya polysaccharides using multi-stage low-temperature plasma. As a new extraction and modification process, the extraction yield of papaya polysaccharides is significantly improved. The combination of each step can modify the papaya polysaccharides and significantly improve the antioxidant activity of papaya polysaccharides.

[0046] 2. Multi-stage low-temperature plasma extraction and modification enhance the disinfection and sterilization effect of low-temperature plasma on papaya polysaccharides. The final papaya polysaccharide product is free of bacterial contamination and has good safety.

[0047] 3. The present invention discovered that the basic steps of the method for extracting and modifying papaya polysaccharides based on multi-stage low-temperature plasma, in which papaya polysaccharide powder is added to a glucose solution for modification in the final modification step, can unexpectedly improve the emulsification of papaya polysaccharides. The improvement in emulsification is beneficial to the specific application of its high antioxidant properties. Based on a similar principle, the antibacterial property of papaya polysaccharides is significantly improved, and it has broad application prospects. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific examples, which are only used to explain the present invention and are not used to limit the scope of the present invention. The materials, reagents, etc. used in the following examples are reagents and materials available from commercial sources unless otherwise specified.

[0049] In the invention, methods such as the Sevage method, DPPH, OH, and ABTS free radical scavenging rate tests are all commonly used experimental methods and are performed according to the methods recorded in relevant textbooks or literature; the low-temperature plasma treatment scheme can be performed according to the product manual of the purchased low-temperature plasma reaction chamber, and any routine adjustments will not affect the final experimental results.

[0050] Example 1: Method for extracting and modifying papaya polysaccharides using multi-stage low-temperature plasma

[0051] The preferred multi-stage low-temperature plasma extraction and modification method of papaya polysaccharide of the present invention is as follows:

[0052] 1) Papaya raw material processing: the papaya raw material is peeled and the pulp is cut into thin slices; the papaya pulp is freeze-dried and crushed to obtain papaya powder, which is passed through a 60-mesh sieve, and the sample powder is collected and stored in a room temperature dryer.

[0053] 2) Low-temperature plasma extraction: Dissolve the sample in step 1 with distilled water and place it in a dielectric barrier discharge low-temperature plasma reaction chamber, and treat it at a frequency of 150 Hz and a high voltage of 40 kV for 10 min; centrifuge the sample after low-temperature plasma treatment (5000 r / min, 15 min) to obtain the supernatant, and concentrate it to 4 mL; add 12 mL of anhydrous ethanol to the supernatant, place it at 4°C for 12 h, obtain the precipitate, and dry and collect it.

[0054] 3) Impurity removal: The powder was subjected to the Sevage method to remove protein, wherein the ratio of chloroform to n-butanol was 3:1 (v / v), and the impurities were removed by dialysis for 48 h using a 10,000 Da dialysis bag. After graded alcohol precipitation, the powder was centrifuged (5,000 r / min, 15 min), concentrated, and freeze-dried.

[0055] 4) Low-temperature plasma modification: The papaya polysaccharide powder obtained by freeze-drying was placed in a dielectric barrier discharge low-temperature plasma reaction chamber and treated at a frequency of 150 Hz and a high voltage of 40 kV for 10 min respectively.

[0056] According to the formula papaya polysaccharide yield = papaya polysaccharide mass / total papaya powder weight, the papaya polysaccharide yield was calculated to be 24.01%.

[0057] The papaya polysaccharide powder obtained in step 3 and step 4 was tested for antioxidant activity (DPPH, OH, ABTS free radical scavenging rate), and the results are shown in Table 1.

[0058] Table 1: Antioxidant test results of papaya polysaccharides before and after low-temperature plasma modification

[0059] Antioxidant Before low temperature plasma modification After low temperature plasma modification DPPH free radical scavenging rate 39.36％ 86.95％ OH radical scavenging rate 42.40％ 81.83％ ABTS free radical scavenging rate 50.77％ 94.27％

[0060] The results showed that the antioxidant activity of papaya polysaccharide was significantly improved after modification by low-temperature plasma.

[0061] Further experimental results show that the above steps can be: dissolving the papaya powder in distilled water at a mass volume ratio of 1:15-1:35, treating with low-temperature plasma, taking the supernatant and concentrating it to 1 / 3-1 / 10 of the original volume; the low-temperature plasma working voltage is 10-50V, the treatment time is 5-20min, and the treatment frequency is 50-150Hz. The above treatment has similar effects, but the preferred scheme at the beginning of this embodiment is the best.

[0062] Example 2: Comparative test of raw material processing steps

[0063] According to Example 1, the preferred method of the present invention selects the principle processing step of freeze-drying papaya pulp. This example compares the effect of other principle processing methods on the final papaya polysaccharide yield and antioxidant activity, and introduces the following raw material processing steps with reference to some existing extraction processes:

[0064] Sun-drying method: peel the papaya raw material and cut the pulp into thin slices; crush the papaya pulp after drying it in the sun and sieve it through a 60-mesh sieve to obtain the powder;

[0065] High temperature drying method: peel the papaya raw material and cut the pulp into thin slices; place the papaya pulp in a 50°C oven to dry, crush it, and sieve it through a 60-mesh sieve to obtain the powder;

[0066] Pulping and filtering method: Peel the papaya raw material and cut the pulp into thin slices; Mix the papaya pulp with distilled water and pulp it into a paste, use a gauze to filter the solids, bake the filtrate at 50°C to dry, and sieve through a 60-mesh sieve to obtain the powder;

[0067] Three groups of experiments were set up respectively, combining the above three raw material processing steps with steps 2-4 in Example 1 to measure the final yield and antioxidant activity of papaya polysaccharide. The results are shown in Table 2.

[0068] Table 2: Yield and antioxidant test results of papaya polysaccharides in different raw material processing steps

[0069] Lyophilization (Example 1) Sun drying method High temperature drying method Beating Filtration Yield 24.01％ 12.84％ 13.69％ 8.22％ DPPH free radical scavenging rate 86.95％ 32.48％ 32.81％ 31.66％ OH radical scavenging rate 81.83％ 36.55％ 34.59％ 37.14％ ABTS free radical scavenging rate 94.27％ 41.07％ 42.32％ 44.31％

[0070] According to the above results, compared with the freeze-drying method, the yields of other raw material processing methods are lower. The reason is that when the papaya powder obtained by other methods is treated with low-temperature plasma, papaya polysaccharides are more difficult to separate from other substances, and therefore it is difficult to extract them from biological tissues. Based on the above experimental results, the combination of freeze-drying and low-temperature plasma extraction significantly improved the extraction yield of papaya polysaccharides.

[0071] Regarding antioxidant properties, the antioxidant properties of papaya polysaccharides obtained by other methods are still very low even after a secondary low-temperature plasma treatment modification step. The reason is that the papaya powder treated by other methods is difficult to separate from other substances under low-temperature plasma treatment, so the purity of the final papaya polysaccharide is not high. Other substances in the papaya polysaccharide powder will affect the effect of low-temperature plasma modification, so the final antioxidant properties are not much different from those of the unmodified ones.

[0072] Example 3: Comparative test of extraction steps

[0073] According to Example 1, the preferred method of the present invention selects dielectric barrier discharge low-temperature plasma for extraction. This example compares whether other extraction steps have an effect on the yield and antioxidant activity of the final papaya polysaccharide, and refers to some existing extraction processes, and introduces the following extraction steps:

[0074] Heat treatment: Add papaya powder to an appropriate amount of hot water, adjust the pH value and perform water bath extraction. After extraction, centrifuge at 4000r / min for 10 minutes, discard the precipitate, and concentrate the supernatant at 60°C to 1 / 5 of the original volume;

[0075] Hydrolytic enzyme: Add papaya powder to an appropriate amount of distilled water, adjust the pH value and add cellulase, pectinase and protease, incubate with shaking for 30 minutes, discard the precipitate, and concentrate the supernatant to 1 / 5 of the original volume at 60°C.

[0076] Two groups of experiments were set up respectively, combining the above three extraction steps with other steps in Example 1 to determine the final yield and antioxidant activity of papaya polysaccharide. The results are shown in Table 3.

[0077] Table 3: Yield and antioxidant test results of papaya polysaccharides in different extraction steps

[0078] DBD (Example 1) Heat Treatment Hydrolases Yield 24.01％ 19.45％ 17.34％ DPPH free radical scavenging rate 86.95％ 34.77％ 36.58％ OH radical scavenging rate 81.83％ 34.29％ 34.21％ ABTS free radical scavenging rate 94.27％ 43.65％ 44.66％

[0079] According to the above results, compared with DBD, the yield of other extraction steps is only slightly lower. The reason is that there are a lot of impurities after extraction, and the actual extraction yield calculated according to the purity will be very low. In the antioxidant test, the antioxidant properties of papaya polysaccharides obtained by other extraction steps are far inferior to the papaya polysaccharides obtained in Example 1. The reason may be that the papaya polysaccharides obtained by other extraction methods are still poor in the end even after being modified by low-temperature plasma due to their low biological activity and low purity.

[0080] Further considering the effects of different low-temperature plasma extractions (including a modification step, with the same type of low-temperature plasma used in both steps) on the yield and antioxidant properties of papaya polysaccharides, different types of low-temperature plasma reaction chambers developed by the research and development team of the present invention were used, including glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge, and jet discharge. The results are shown in Table 4.

[0081] Table 4: Yield and antioxidant test results of papaya polysaccharides extracted by different low-temperature plasmas

[0082]

[0083] The final results show that the extraction results of papaya polysaccharides by the above different types of low-temperature plasmas are not much different. The reason is that they all belong to low-temperature plasmas and have similar working principles. Among them, the yield of papaya polysaccharides extracted by dielectric barrier discharge is the highest, mainly because of the discharge principle of dielectric barrier discharge itself.

[0084] Example 4: Comparative test of impurity removal step

[0085] According to Example 1, the preferred method of the present invention selects the sevage method and dialysis bag for impurity removal. This embodiment compares the effect of specific impurity removal process on the yield and antioxidant property of papaya polysaccharide. The papaya powder is deproteinized by the Sevage method, wherein the ratio of chloroform to n-butanol is 1:1, 2:1, 3:1, 4:1, 5:1 (v / v), and the molecular weight is 10000Da dialysis bag for dialysis for 48h to remove impurities, centrifuged (5000r / min, 15min) after graded alcohol precipitation, concentrated, and freeze-dried. The sugar content in papaya crude polysaccharide is detected, and it is found that the sugar content of papaya crude polysaccharide is the highest when the ratio of chloroform to n-butanol is 3:1, which is 76%.

[0086] It is worth noting that although the core step is a two-step low-temperature plasma treatment, the impurity removal effect is still correlated with the effect of the next step of low-temperature plasma modification. The higher the purity of papaya polysaccharide during modification, the better. Therefore, the impurity removal provided in this embodiment is only an implementation method, and does not exclude that other impurity removal methods can have an equivalent or better impurity removal effect on papaya polysaccharide.

[0087] Example 5: Comparative test of modification steps

[0088] According to Example 1, the preferred method of the present invention selects dielectric barrier discharge low-temperature plasma for modification. This example compares whether other modification steps have an impact on the yield and antioxidant properties of the final papaya polysaccharide, and refers to some existing modification processes, and introduces the following chemical modification steps:

[0089] Sulfation: Take 40 mL of a 3:1 (v / v) mixture of concentrated sulfuric acid and n-butanol in a dry conical flask, add 15 mL of concentrated sulfuric acid, cool to 0°C with ice water, slowly add 0.5 g of polysaccharide sample, stir thoroughly to dissolve, react at 0°C for 1.5 h, add 100 g / L NaOH solution pre-cooled to 0°C to adjust the pH to neutral, centrifuge to obtain the precipitate, add to a dialysis bag for dialyze for 48 h, concentrate to 1 / 5 of the original volume, and freeze-dry.

[0090] Acetylation: Weigh 500 mg of papaya polysaccharide and prepare a saturated solution with deionized water. Use 100 g / L NaOH solution to adjust the pH of the polysaccharide solution to 9.0. After heating to 50°C, slowly add a certain amount of acetic anhydride (the liquid-to-solid ratio of acetic anhydride to papaya polysaccharide is 40:1). After the addition is complete, keep the constant temperature for 3 hours. After the reaction is completed, use 1 mol / L hydrochloric acid solution to adjust the pH of the reaction solution to neutral, add a dialysis bag for 48 hours, concentrate to 1 / 5 of the original volume, and freeze-dry.

[0091] Two groups of experiments were set up respectively, combining the above three modification steps with other steps in Example 1 to measure the antioxidant activity of the final papaya polysaccharide. The results are shown in Table 5.

[0092] Table 5: Yield and antioxidant test results of papaya polysaccharides at different modification steps

[0093] DBD (Example 1) Sulfation Acetylation DPPH free radical scavenging rate 86.95％ 40.29％ 42.60％ OH radical scavenging rate 81.83％ 38.67％ 38.24％ ABTS free radical scavenging rate 94.27％ 47.55％ 46.11％

[0094] According to the results, chemical modification did not significantly improve the antioxidant properties of papaya polysaccharide. The papaya polysaccharide extracted and modified by low-temperature plasma in the present invention has a significant high antioxidant property. Sulfation modification mainly improves the hydrophilicity of papaya polysaccharide, which increases its solubility in water. Acetylation modification can improve the emulsification property to a certain extent.

[0095] Example 6: Emulsification improvement test

[0096] Based on the above embodiments, the present invention obtains papaya polysaccharides with high antioxidant properties through the steps of freeze-drying-low-temperature plasma extraction-impurity removal-low-temperature plasma modification. The research and development team of the present invention also conducted some experiments during the research, trying to further play the role of low-temperature plasma modification to further improve the antioxidant properties of papaya polysaccharides. The present invention tested the relevant properties of papaya polysaccharides after the test results and found that one of the papaya polysaccharides had outstanding emulsifying properties, which is very beneficial for the practical application of papaya polysaccharides. Therefore, the process scheme was traced back and several process schemes that may have similar effects were expanded, as follows:

[0097] Process scheme: The method of Example 1 is followed, but in step 4, the papaya polysaccharide powder is added to a 5% glucose solution and mixed, and then DBD low-temperature plasma modification is performed according to the same parameters, and the papaya polysaccharide and glucose are separated by a semipermeable membrane, and the papaya polysaccharide product is obtained by low-temperature drying;

[0098] Extension 1: Follow the process plan, but replace 5% glucose solution with 5% fructose solution;

[0099] Extension 2: Follow the process plan, but replace 5% glucose solution with 5% maltose solution;

[0100] Reference substance 1: papaya polysaccharide product obtained in the manner of Example 1;

[0101] Reference substance 2: the papaya polysaccharide product obtained by acetylation modification in Example 5.

[0102] The above five products were mixed with glycerol at a mass ratio of 1:5, dispersed under an ultrasonic probe for 30 minutes, diluted 10 times with distilled water and magnetically stirred for 30 minutes (100r / min), and the obtained emulsion was measured for Zeta potential and particle size by Malvern nanoparticle size analyzer. The preparation and test were repeated 5 times, and the average of the 5 tests was calculated; the smaller the particle size and the higher the absolute value of Zeta potential, the higher and more uniform the emulsification stability of the emulsion is, indicating that the corresponding papaya polysaccharide has better emulsification. The results are shown in Table 6.

[0103] Table 6: Comparison of emulsification properties of papaya polysaccharides in different process schemes (n=5)

[0104] Process solution Expansion Plan 1 Expansion Plan 2 Reference 1 Reference 2 Zeta Potential -48±0.23 -33±0.30 -33±0.26 -33±0.27 -39±0.25 Particle size (nm) 257.88 362.75 367.15 364.93 311.40

[0105] The results showed that the papaya polysaccharide modified by low-temperature plasma combined with glucose solution had the best emulsifying property because its Zeta potential absolute value was the highest and its particle size was the smallest; the second was the papaya polysaccharide modified by acetylation, whose emulsifying property was not as good as the above papaya polysaccharide; the emulsifying property of the papaya polysaccharide obtained in Example 1 and the papaya polysaccharide modified by low-temperature plasma combined with maltose solution or fructose was not much different, indicating that maltose solution or fructose solution had no obvious effect on the modification of emulsifying property.

[0106] The principle of low-temperature plasma combined with glucose solution for modification may be that there is a hydrogen bond combination arrangement between glucose molecules and papaya polysaccharide macromolecules. Under the action of low-temperature plasma, glucose molecules may have a certain influence on the conformational transformation of papaya polysaccharide macromolecules, and this influence is just reflected in the improvement of emulsification. At the same time, glucose is a substance that can be directly absorbed and utilized by the human body, and its use is completely safe.

[0107] Based on the above possible principles, the relevant properties were further studied and it was found that the antibacterial property was also significantly improved. Specifically, in the antibacterial test, the colony count of the low-temperature plasma + glucose modified group was significantly lower than that of the papaya polysaccharide in Example 1 and other related groups; the principle may be that the transformation of the conformation changes the spatial position of the group that binds to the bacteria, and the spatial conformation of the papaya polysaccharide modified by low-temperature plasma + glucose is more conducive to binding with bacteria and substances in bacteria, especially with structures such as bacterial cell membranes, thereby destroying the bacterial structure and achieving an antibacterial effect.

[0108] The improvement of the emulsifying and antibacterial properties of papaya polysaccharide is very beneficial to its application, especially the papaya polysaccharide with high antioxidant properties prepared in this application. For example, it can be prepared into a cream with antioxidant effect, which can improve the water retention of food in the food industry, increase the stability of drugs in the pharmaceutical industry, etc., and the safety is also improved.

[0109] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto, and any equivalent modifications or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting and modifying papaya polysaccharides using multi-stage low-temperature plasma, characterized in that: Includes steps: S1, taking fresh papaya pulp and freeze-drying it to obtain papaya powder; S2, subjecting the papaya powder to low-temperature plasma treatment; S3, removing impurities to obtain papaya polysaccharide powder; S4, subjecting the papaya polysaccharide powder to low-temperature plasma treatment.

2. The method for extracting and modifying papaya polysaccharides by multi-stage low-temperature plasma according to claim 1, characterized in that: The discharge form of the low-temperature plasma is selected from one of glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge and jet discharge.

3. The method for extracting and modifying papaya polysaccharides by multi-stage low-temperature plasma according to claim 1, characterized in that: The step S2 comprises: dissolving the papaya powder in distilled water at a mass volume ratio of 1:15-1:35, treating with low temperature plasma, taking the supernatant and concentrating it to 1 / 3-1 / 10 of the original volume.

4. The method for extracting and modifying papaya polysaccharides by multi-stage low-temperature plasma as claimed in claim 3, characterized in that: In the step S2, the discharge form of the low-temperature plasma includes dielectric barrier discharge, the working voltage of the low-temperature plasma is 10-50V, the processing time is 5-20min, and the processing frequency is 50-150Hz.

5. The method for extracting and modifying papaya polysaccharides by multi-stage low-temperature plasma according to claim 1, characterized in that: The step S3 comprises: removing protein by using the Sevage method, removing impurities by using a dialysis bag, centrifuging, concentrating and freeze-drying after graded alcohol precipitation to obtain papaya polysaccharide powder.

6. The method for extracting and modifying papaya polysaccharides by multi-stage low-temperature plasma according to claim 5, characterized in that: In step S3, the Sevage method uses chloroform and n-butanol, the volume ratio of chloroform to n-butanol is 1:1-5:1, the molecular weight of the dialysis bag is 10000Da, and the dialysis time is 48h.

7. The method for extracting and modifying papaya polysaccharides using low-temperature plasma as claimed in claim 1, characterized in that: In the step S4, the discharge form of the low-temperature plasma includes dielectric barrier discharge, the working voltage of the low-temperature plasma is 10-50V, the processing time is 5-20min, and the processing frequency is 50-150Hz.

8. The method for extracting and modifying papaya polysaccharides by multi-stage low-temperature plasma according to claim 1, characterized in that: The step S4 is: adding the papaya polysaccharide powder into the glucose solution, and subjecting the obtained mixed solution to low-temperature plasma treatment.

9. A papaya polysaccharide with enhanced antioxidant properties, characterized in that: The method for preparing papaya polysaccharide comprises the method for extracting and modifying papaya polysaccharide by multi-stage low-temperature plasma as described in any one of claims 1 to 8.

10. A use of glucose for preparing a reagent for improving the emulsification of papaya polysaccharide modified by low-temperature plasma, characterized in that: The method for preparing the low-temperature plasma modified papaya polysaccharide comprises the method for extracting and modifying papaya polysaccharide by multi-stage low-temperature plasma as described in any one of claims 1 to 8.

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