A method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharide

Abstract

The present application relates to the technical field of extraction and modification of papaya polysaccharide, and discloses a method for extracting and modifying papaya polysaccharide by using 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 papaya 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 method can significantly improve the extraction yield of papaya polysaccharide, and the combination between steps can play a modification effect on the papaya polysaccharide, thereby significantly improving the antioxidant property of the papaya polysaccharide and having a wide application prospect. Based on the basic steps of the method for extracting and modifying papaya polysaccharide by using multistage low-temperature plasma, the papaya polysaccharide powder is added into a glucose solution for modification in the last modification step, and the emulsifying property and bacteriostatic property of the papaya polysaccharide can be unexpectedly improved, which is beneficial to the specific application of the high antioxidant property of the papaya polysaccharide.

Description

[0001] This application claims priority to a prior Chinese application, application number 202411985461.9, filed on December 31, 2024, the description, claims and abstract of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to the field of papaya polysaccharide extraction and modification technology, and in particular to a method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides. Background Technology

[0003] Papaya (Carica Papaya L.), belonging to the Caricaceae family and the Carica genus, is widely cultivated in Guangdong, Guangxi, Fujian, Yunnan, and Taiwan in my country. It possesses various biological activities, including anti-cancer, antibacterial, immunomodulatory, anti-diabetic, and antioxidant effects. Papaya polysaccharides are one of its important active components. Polysaccharides have a strong ability to scavenge free radicals and can be used as raw materials for antioxidants, thus showing great development potential. Polysaccharide antioxidants are currently widely used in the chemical, pharmaceutical, food, and cosmetic industries. As a natural polymer compound, it has superior biocompatibility compared to chemical and compound antioxidants, and is widely available, green, and safe. However, most natural polysaccharides suffer from poor antioxidant activity and weak stability, making them difficult to use directly as antioxidants.

[0004] According to the SAR theory of structure-activity relationships, the biological functions of polysaccharides are influenced by their physicochemical structure (conformation, side chain distribution, etc.) and determine their applications in industrial production. Currently, polysaccharide structures are often modified using physical, chemical, and enzymatic methods to enhance their applicability in the biopharmaceutical and food fields. Physical methods (radiation, heat treatment) can avoid the use of chemical reagents to some extent, but they are still essentially heat treatments, which are detrimental to the nutritional components and sensory quality of polysaccharides. Chemical methods (acetylation, sulfation) involve chemical toxicity and environmental pollution, and it is difficult to ensure the integrity of the encapsulated material. Biological methods (hydrolases, oxidases) are specific and selective, with mild conditions, but the yield is low, and many byproducts require purification.

[0005] For the extraction of papaya polysaccharides and the preservation of their antioxidant activity, existing literature usually adopts water extraction and alcohol precipitation methods, and some further use ultrasonic assistance. However, these methods can only achieve the goal of not destroying the antioxidant activity of papaya polysaccharides. As a natural polysaccharide, papaya polysaccharides still have low antioxidant activity.

[0006] Therefore, there is an urgent need to improve the extraction process of papaya polysaccharides and introduce modification processes to increase the yield of papaya polysaccharides and enhance their antioxidant activity, so as to expand the application of papaya polysaccharides. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides, comprising the following 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 such as protein to obtain papaya polysaccharide powder; S4, subjecting the papaya polysaccharide powder to low-temperature plasma treatment. This treatment achieves the following: firstly, it improves the extraction yield and purity of papaya polysaccharides; secondly, it enhances the antioxidant activity of papaya polysaccharides; and thirdly, it shortens the extraction and modification process of papaya polysaccharides.

[0008] The research team of this invention has conducted in-depth research in the field of natural product extraction from papaya. For example, a method for extracting papain using low-temperature plasma-assisted eutectic solvent has been proposed before (CN202310374820.6). Natural products in papaya are complex and diverse, and difficult to extract, especially papaya polysaccharides. Existing methods either have extremely low yields and many impurities, or severely damage the antioxidant activity of papaya polysaccharides. Moreover, existing methods also involve the addition of chemical substances, which are not green and natural. The research team of this invention has obtained the method for low-temperature plasma extraction and modification of papaya polysaccharides of this invention through a large number of theoretical studies 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 exhibits a low-temperature state, so it is also called cold plasma or non-equilibrium plasma.

[0010] On one hand, the present invention provides a method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides, comprising the following steps:

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

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

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

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

[0015] First, in step S1, the papaya pulp was freeze-dried. Compared with traditional crushing or other pretreatment methods, this invention found that direct freeze-drying is beneficial to improving the yield. The principle may be that freeze-drying can minimize sample loss, achieve more thorough dehydration, and retain biological activity to a greater extent after freeze-drying. Furthermore, freeze-drying may allow various natural active substances in papaya to be more fully exposed to low-temperature plasma in the next step of low-temperature plasma treatment. Low-temperature plasma is a novel non-thermal extraction technology that contains a variety of active ingredients, such as reactive oxygen species, reactive nitrogen species, charged particles, electrons, and photons. It has strong biological activity and oxidizing properties, which can quickly promote the separation of impurities from papaya polysaccharides and improve the yield.

[0016] Step S2 can be performed by directly treating the papaya powder with low-temperature plasma, or by first dissolving the papaya powder and then treating the dissolved solution with low-temperature plasma. This step promotes the separation of impurities from papaya polysaccharides, thereby achieving the extraction function.

[0017] The substrate for step S3 is the product treated with low-temperature plasma in step S2. Therefore, the separation degree between the internal impurities and papaya polysaccharides is significantly improved. Proteins can be easily removed using the sevage method or other methods, and impurities above 10,000 Da can be removed using dialysis bags. The main contribution of this purification step comes from the treatment with low-temperature plasma.

[0018] Step S4 is the modification step. Generally speaking, low-temperature plasma is considered to have oxidizing properties and may damage the antioxidant properties of some natural products. However, this invention found that after the above steps S1-S3, low-temperature plasma treatment of papaya polysaccharide powder can actually improve its antioxidant properties. The reason may be: Firstly, in the treatment of low-purity papaya polysaccharide extracted by methods such as bio-enzyme extraction, low-temperature plasma cannot significantly increase its antioxidant properties, and may even reduce them. This is because impurities preempt the effect of low-temperature plasma; charged particles preferentially react with impurities, and the oxidized impurities are absorbed by the papaya polysaccharide. The reduction process oxidizes papaya polysaccharides, suggesting that the high purity of the papaya polysaccharides obtained through steps S1-S3 of this invention allows them to directly become substrates for low-temperature plasma treatment. Previous studies have not been able to extract such high-purity papaya polysaccharides, thus their role remains unknown. Secondly, the ROS / RNS free radicals generated by the low-temperature plasma and their degradation effect on macromolecules influence the conformation of papaya polysaccharides. As a macromolecule, papaya polysaccharides possess multiple sites at the molecular level that scavenge free radicals. The conformation of papaya polysaccharides affects the exposure of its antioxidant reaction sites, thereby influencing its antioxidant activity.

[0019] Therefore, the method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides in this invention differs from conventional methods for low-temperature plasma extraction of polysaccharides in the following ways: 1. A freeze-drying step is introduced; 2. Two-step low-temperature plasma treatment, one for extraction and the other for modification; 3. A purification step is added between the two low-temperature plasma treatments. Based on current research, when the above four steps are replaced, papaya polysaccharide extraction can still be performed, but the antioxidant modification effect cannot be achieved. Furthermore, this invention only applies to papaya polysaccharides. The extraction of other polysaccharides may be carried out according to this 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 transferred to other polysaccharides. Similarly, the modification of other polysaccharides cannot be transferred to papaya polysaccharides.

[0020] The "multi-stage" in multi-stage low-temperature plasma extraction and modification means that the product is processed multiple times using low-temperature plasma. Low-temperature plasma has a strong disinfection and sterilization effect. Therefore, after two treatments of extraction and modification, the final papaya polysaccharide product is free from bacterial contamination and has extremely high safety when used in the food, pharmaceutical and cosmetic industries.

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

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

[0023] Dielectric barrier discharge is typically driven by a sinusoidal AC high-voltage power supply. As the supplied voltage increases, the state of the reactant gas in the system undergoes three stages of change: from an insulating state to discharge and finally breakdown. When the supplied voltage is relatively low, although some gas undergoes ionization and diffusion, the amount is too small and the current is too low to induce a plasma reaction in the reaction zone; at this point, the current is zero. As the supplied voltage gradually increases, the number of electrons in the reaction zone also increases. However, before reaching the breakdown voltage of the reactant gas, the electric field between the two electrodes is too low to provide sufficient energy for the electrons to undergo inelastic collisions with the gas molecules. The lack of inelastic collisions results in a limited increase in the number of electrons; therefore, the reactant gas remains in an insulating state and cannot produce a discharge. At this point, the current increases slightly with the voltage applied to the electrodes, but remains almost zero. If the supply voltage is further increased, when the electric field between the two electrodes becomes large enough to cause gas molecules to undergo inelastic collisions, the gas will increase significantly due to ionization from these inelastic collisions. When the electron density in the space exceeds a critical value and the Paschen breakdown voltage is reached, many microdischarges will be generated between the two electrodes, and the phenomenon of light emission can be clearly observed in the system. At this point, the current will increase rapidly as the applied voltage increases.

[0024] Based on the principle of dielectric barrier discharge (DBD), further analysis suggests that its superior effect on papaya polysaccharide modification may be due to the fact that DBD is uniform, diffuse, and stable, allowing for regular adjustment of the conformation of papaya polysaccharides. This results in most treated papaya polysaccharide molecules having similar conformations. As macromolecules, polysaccharides possess a certain degree of complexity, and there may be antagonistic effects between polysaccharide molecules with different conformations on chemical reactions. However, the conformations of papaya polysaccharide molecules treated with DBD are more uniform, reducing the probability of antagonism. Furthermore, most papaya polysaccharide molecules undergo modification to enhance their antioxidant properties, resulting in a significant improvement in overall antioxidant activity.

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

[0026] Furthermore, in step S2, 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.

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

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

[0029] Preferably, the volume ratio of chloroform to n-butanol is 3:1, at which point 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 operates at a voltage of 40kV, a frequency of 150Hz, and a processing time of 10min.

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

[0033] Based on the low-temperature plasma extraction and modification technique for papaya polysaccharides discovered in this invention, papaya polysaccharides with good antioxidant properties were obtained. The research team considered increasing the efficiency of the final modification step to maximize the antioxidant enhancement effect of low-temperature plasma on the modified papaya polysaccharides. In numerous experiments, this invention unexpectedly discovered that adding papaya polysaccharide powder to a glucose solution followed by low-temperature plasma modification of the mixture did not significantly affect the antioxidant properties of the resulting papaya polysaccharide. However, subsequent tests showed a significant enhancement in the emulsifying properties of the papaya polysaccharide, which is highly beneficial for its application in the food, pharmaceutical, and cosmetic fields. Furthermore, glucose can be separated from papaya polysaccharides through a semi-permeable membrane, and any residue is not contaminating. The mechanism of action of glucose may be that certain glycoside fragments of papaya polysaccharides tend to… The tendency of papaya polysaccharide to form tight hydrogen bonds with glucose molecules is determined by the glycosidic arrangement of papaya polysaccharide and the molecular structure of glucose. When bombarded by charged particles from low-temperature plasma, glucose molecules protect certain segments of papaya polysaccharide. Low-temperature plasma alters the conformation of papaya polysaccharide, and the further addition of glucose affects this conformational change. This conformational change may affect the spatial arrangement of hydrophilic and lipophilic groups on the surface of papaya polysaccharide molecules, thereby altering the emulsifying properties of the papaya polysaccharide product. This change may enhance or weaken the emulsifying properties. This invention only provides one possible principle and does not exclude the possibility of other possible principles. This invention has discovered that low-temperature plasma combined with glucose solution significantly improves the emulsifying properties of papaya polysaccharide products. Furthermore, the combined treatment with low-temperature plasma and glucose does not involve the addition of chemical additives, and glucose is completely safe for human use.

[0034] Furthermore, during the testing of papaya polysaccharides, it was found that the antibacterial properties of the modified papaya polysaccharides were significantly enhanced. This may be based on a similar principle to the above, where the conformational change of papaya polysaccharides enhances their tendency to bind with bacteria and substances within bacteria. This is similar to how polyphenols can exert antibacterial effects on bacteria by acting on bacterial cell membranes and other structures, causing damage to bacterial structures, thereby achieving an antibacterial effect.

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

[0036] The 5% figure is only the concentration of glucose solution in the example where the low-temperature plasma and glucose solution work synergistically. It can be understood that, based on the above-mentioned glucose action principle, any concentration of glucose solution can improve the emulsifying properties of papaya polysaccharide modified by low-temperature plasma.

[0037] Papaya polysaccharides with enhanced antioxidant, emulsifying, and antibacterial properties can be applied in the following ways: In the food industry, they can improve food stability, emulsifying properties, and water-holding capacity; in the pharmaceutical industry, they can be used as excipients to improve drug stability and efficacy, as well as exhibiting antibacterial properties by inhibiting common pathogenic microorganisms; in the cosmetics industry, they can be used to prepare antioxidant products with better emulsifying properties, such as lotions and creams, and to inhibit common pathogenic microorganisms on the skin surface; they can also have corresponding applications in other fields. The papaya polysaccharides obtained by the method provided in this invention have broad application prospects.

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

[0041] In another aspect, the present invention provides the use of glucose as a reagent for preparing a low-temperature plasma modified papaya polysaccharide, wherein the preparation method of the low-temperature plasma modified papaya polysaccharide includes the method of multi-stage low-temperature plasma extraction and modification of papaya polysaccharide.

[0042] In another aspect, the present invention provides the use of glucose in preparing a reagent to improve the antibacterial properties of low-temperature plasma modified papaya polysaccharide, wherein the preparation method of the low-temperature plasma modified papaya polysaccharide includes the method of multi-stage low-temperature plasma extraction and modification of papaya polysaccharide.

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

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

[0045] 1. This invention provides a method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides. As a novel extraction and modification process, it significantly improves the extraction yield of papaya polysaccharides. The combination of each step can modify papaya polysaccharides and significantly improve their antioxidant properties.

[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 from bacterial contamination and has good safety.

[0047] 3. This invention discovers the basic steps of a method for extracting and modifying papaya polysaccharides based on multi-stage low-temperature plasma. In the final modification step, papaya polysaccharide powder is added to a glucose solution for modification, which unexpectedly improves the emulsification properties of papaya polysaccharides. The improved emulsification properties are beneficial for its specific application of high antioxidant properties. Furthermore, based on a similar principle, the antibacterial properties of papaya polysaccharides are significantly enhanced, showing broad application prospects. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only for explaining the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

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

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

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

[0052] 1) Papaya raw material processing: Peel the papaya raw material and cut the pulp into thin slices; freeze-dry the papaya pulp, pulverize it to obtain papaya powder, pass it through a 60-mesh sieve, collect the sample powder and store it in a desiccator at room temperature.

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

[0054] 3) Removal of impurities: The above powder was subjected to the Sevage method to remove proteins, wherein the ratio of chloroform to n-butanol was 3:1 (v / v), and impurities were removed by dialyzing with a dialysis bag with a molecular weight of 10000 Da for 48 hours. After fractionation and alcohol precipitation, the powder was centrifuged (5000 r / min, 15 min), concentrated, and lyophilized.

[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] Based on the formula: papaya polysaccharide yield = papaya polysaccharide mass / total weight of papaya powder, the papaya polysaccharide yield was calculated to be 24.01%.

[0057] The papaya polysaccharide powders obtained in steps 3 and 4 were tested for their antioxidant properties (DPPH, OH, and ABTS free radical scavenging rates), and the results are shown in Table 1.

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

[0059] Antioxidant properties 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 properties of papaya polysaccharide were significantly improved after modification with low-temperature plasma.

[0061] Further experimental results show that the above steps can be as follows: dissolve the papaya powder in distilled water at a mass-to-volume ratio of 1:15-1:35, treat with low-temperature plasma, take the supernatant and concentrate 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 optimal.

[0062] Example 2: Comparative Experiment of Raw Material Processing Steps

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

[0064] Sun-drying method: Peel the papaya raw material and cut the pulp into thin slices; after sun-drying the papaya pulp, grind it into powder and pass it through a 60-mesh sieve.

[0065] High-temperature drying method: Peel the papaya raw material and cut the pulp into thin slices; place the papaya pulp in a 50℃ oven to dry, pulverize, and pass through a 60-mesh sieve to obtain powder;

[0066] Pulping and Filtration 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; filter the solids through a mesh screen; bake the filtrate at 50°C to dry it; and pass it through a 60-mesh sieve to obtain powder.

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

[0068] Table 2: Yield and antioxidant properties of papaya polysaccharides from different raw material processing steps

[0069] Freeze-drying method (Example 1) Sun-drying method High-temperature drying method Pulping and Filtration Method 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] Based on the above results, compared with the freeze-drying method, the yields of other raw material processing methods are all lower. This is because the papaya powder obtained by other methods is more difficult to separate from other substances during low-temperature plasma treatment, making it difficult to extract from biological tissues. Based on the above experimental results, combining freeze-drying and low-temperature plasma extraction significantly improves the extraction yield of papaya polysaccharides.

[0071] Regarding antioxidant properties, papaya polysaccharides obtained by other methods still exhibit low antioxidant properties even after undergoing a two-stage low-temperature plasma treatment modification step. This is because papaya powder processed by other methods is difficult to separate from other substances under low-temperature plasma treatment, resulting in low purity of the final papaya polysaccharide. Furthermore, other substances in the papaya polysaccharide powder can affect the effect of low-temperature plasma modification, thus the final antioxidant properties are not significantly different from those without modification.

[0072] Example 3: Comparative Experiment of Extraction Steps

[0073] According to Example 1, the preferred method of this invention uses dielectric barrier discharge low-temperature plasma for extraction. This example compares the effects of other extraction steps on the final yield and antioxidant properties of papaya polysaccharides. Referring to some existing extraction processes, the following extraction steps are introduced:

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

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

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

[0077] Table 3: Yield and antioxidant properties of papaya polysaccharides obtained from different extraction steps

[0078] DBD (Example 1) Heat treatment hydrolytic enzymes 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] Based on the above results, compared with DBD, the yield of other extraction steps is only slightly lower. This is because there are a large number of impurities after extraction, and the actual extraction yield will be very low when calculated according to purity. In the antioxidant test, the antioxidant properties of papaya polysaccharides obtained by other extraction steps are far inferior to those of papaya polysaccharides obtained in Example 1. This may be because even after low-temperature plasma modification, the final antioxidant properties of papaya polysaccharides obtained by other extraction methods are still poor due to their low biological activity and low purity.

[0080] To further consider the effects of different low-temperature plasma extraction methods (including modification steps, 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 team of this 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 properties of papaya polysaccharides extracted by different low-temperature plasma methods

[0082]

[0083] The final results showed that the extraction results of papaya polysaccharides by the different types of low-temperature plasmas were not significantly different. This is because they are all low-temperature plasmas with similar working principles. Among them, the yield of papaya polysaccharides was the highest when extracted by dielectric barrier discharge, mainly due to the discharge principle of dielectric barrier discharge itself.

[0084] Example 4: Comparative Experiment of Impurity Removal Steps

[0085] According to Example 1, the preferred method of this invention uses the Sevage method and dialysis bags for impurity removal. This example compares the effects of specific impurity removal processes on the yield and antioxidant properties of papaya polysaccharides. Papaya powder was treated with the Sevage method to remove proteins, with chloroform to n-butanol ratios of 1:1, 2:1, 3:1, 4:1, and 5:1 (v / v). Impurities were removed by dialysis using a 10000 Da dialysis bag for 48 hours. After fractionation and alcohol precipitation, the mixture was centrifuged (5000 r / min, 15 min), concentrated, and lyophilized. The sugar content in the crude papaya polysaccharide was measured, and the results showed that the highest sugar content (76%) was achieved when the chloroform to n-butanol ratio was 3:1.

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

[0087] Example 5: Comparative Experiment of Modification Steps

[0088] According to Example 1, the preferred method of this invention uses dielectric barrier discharge low-temperature plasma for modification. This example compares the effects of other modification steps on the final yield and antioxidant properties of papaya polysaccharide. Referring to some existing modification processes, the following chemical modification steps are introduced:

[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 collect the precipitate, add to a dialysis bag and 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. Adjust the pH of the polysaccharide solution to 9.0 with 100 g / L NaOH solution. 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, maintain a constant temperature for 3 hours. After the reaction is complete, adjust the pH of the reaction solution to neutral with 1 mol / L hydrochloric acid solution, add a dialysis bag and dialyze for 48 hours. Concentrate to 1 / 5 of the original volume and freeze-dry.

[0091] Two sets of experiments were set up to combine the above three modification steps with other steps in Example 1, and the antioxidant properties of the final papaya polysaccharide were determined. The results are shown in Table 5.

[0092] Table 5: Yield and antioxidant properties of papaya polysaccharides after 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] The results show that chemical modification did not significantly improve the antioxidant properties of papaya polysaccharides. The papaya polysaccharides extracted and modified using low-temperature plasma in this invention exhibit significantly high antioxidant properties. Sulfation modification primarily affects the hydrophilicity of papaya polysaccharides, increasing their solubility in water, while acetylation modification can improve emulsifying properties to some extent.

[0095] Example 6: Emulsification Improvement Test

[0096] Based on the above embodiments, this invention obtains papaya polysaccharides with high antioxidant properties through freeze-drying-low-temperature plasma extraction-impurity removal-low-temperature plasma modification. During the research, the research team also conducted experiments to further enhance the effect of low-temperature plasma modification and improve the antioxidant properties of the papaya polysaccharides. In testing the relevant properties of the papaya polysaccharides after the experiments, this invention discovered that one type of papaya polysaccharide exhibits outstanding emulsifying properties, which is highly beneficial for the practical application of papaya polysaccharides. Therefore, this process scheme was traced back to, and several other process schemes with potentially similar effects were explored, as detailed below:

[0097] Process scheme: The process is carried out in accordance with the method of Example 1, 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. The papaya polysaccharide and glucose are separated through a semi-permeable membrane and dried at low temperature to obtain the papaya polysaccharide product.

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

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

[0100] Reference standard 1: The papaya polysaccharide product obtained according to the method of Example 1;

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

[0102] The five products were mixed with glycerin at a mass ratio of 1:5, dispersed under ultrasonic probe for 30 min, diluted 10 times with distilled water, and magnetically stirred for 30 min (100 r / min). The resulting emulsions were analyzed using a Malvern nanoparticle size analyzer to determine the zeta potential and particle size. The preparation and testing were repeated five times, and the average value of the five tests was calculated. Smaller particle size and higher absolute value of zeta potential generally indicate higher emulsification stability and more uniformity of the emulsion, indicating better emulsification properties of the corresponding papaya polysaccharide. The results are shown in Table 6.

[0103] Table 6: Comparison of emulsifying properties of papaya polysaccharides processed using different methods (n=5)

[0104] Process scheme Expansion Plan 1 Extension Plan 2 Reference Standard 1 Reference Standard 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 papaya polysaccharide modified by low-temperature plasma combined with glucose solution had the best emulsifying properties because it had the highest absolute value of Zeta potential and the smallest particle size; followed by papaya polysaccharide modified by acetylation, whose emulsifying properties were not as good as the above-mentioned papaya polysaccharide; the papaya polysaccharide obtained in Example 1 and the papaya polysaccharide modified by low-temperature plasma combined with maltose solution or fructose had little difference in emulsifying properties, suggesting that maltose solution or fructose solution did not have a significant effect on the modification of emulsifying properties.

[0106] The principle behind modifying glucose solution using low-temperature plasma may be that there is a hydrogen bond 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 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, further research was conducted on its related properties, and it was found that the antibacterial activity was also significantly improved. Specifically, in the antibacterial test, the number of colonies in the low-temperature plasma + glucose modified group was significantly lower than that in the papaya polysaccharide of Example 1 and other related groups. The principle may be that the conformational transformation changes the spatial position of the groups that bind to bacteria. The spatial conformation of the papaya polysaccharide modified by low-temperature plasma + glucose is more conducive to binding with bacteria and substances within bacteria, especially with structures such as the bacterial cell membrane, thereby destroying the bacterial structure and achieving the antibacterial effect.

[0108] The enhanced emulsifying and antibacterial properties of papaya polysaccharides are highly beneficial for their application, especially when combined with the highly antioxidant papaya polysaccharides prepared in this application. For example, they can be prepared into creams with antioxidant effects, improve the water-holding capacity of food in the food industry, increase the stability of drugs in the pharmaceutical industry, and enhance safety.

[0109] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent modifications or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides, characterized in that, Including the following steps: S1, Take fresh papaya pulp and freeze-dry it to obtain papaya powder; S2, first dissolve the papaya powder, then treat the dissolved solution with low-temperature plasma; S3, remove impurities to obtain papaya polysaccharide powder; S4, the papaya polysaccharide powder is subjected to low-temperature plasma treatment.

2. The method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides as described in claim 1, characterized in that, The discharge mode of the low-temperature plasma is selected from one of the following: glow discharge, corona discharge, dielectric barrier discharge, radio frequency discharge, sliding arc discharge, and jet discharge.

3. The method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides as described in claim 1, characterized in that, Step S2 includes: dissolving the papaya powder in distilled water at a mass-to-volume ratio of 1:15-1:35, treating it with low-temperature plasma, taking the supernatant and concentrating it to 1 / 3-1 / 10 of the original volume.

4. The method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides as described in claim 3, characterized in that, In step S2, the discharge form of the low-temperature plasma includes dielectric barrier discharge, the operating voltage of the low-temperature plasma is 10-50kV, the processing time is 5-20min, and the processing frequency is 50-150 Hz.

5. The method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides as described in claim 1, characterized in that, Step S3 includes: removing proteins using the Sevage method, removing impurities using a dialysis bag, centrifuging, concentrating, and freeze-drying after fractionation and alcohol precipitation to obtain papaya polysaccharide powder.

6. The method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides as described in claim 5, characterized in that, In step S3, the Sevage method uses chloroform and n-butanol, with a volume ratio of chloroform to n-butanol of 1:1 to 5:

1. The molecular weight of the dialysis bag is 10,000 Da, and the dialysis time is 48 h.

7. The method for low-temperature plasma extraction and modification of papaya polysaccharides as described in claim 1, characterized in that, In step S4, the discharge form of the low-temperature plasma includes dielectric barrier discharge, the operating voltage of the low-temperature plasma is 10-50kV, the processing time is 5-20min, and the processing frequency is 50-150 Hz.

8. The method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharides as described in claim 1, characterized in that, Step S4 involves adding the papaya polysaccharide powder to a glucose solution and subjecting the resulting mixed solution to low-temperature plasma treatment.

9. A papaya polysaccharide with enhanced antioxidant properties, characterized in that, The method for preparing papaya polysaccharide includes the method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharide as described in any one of claims 1-8.

10. The use of glucose as a reagent for preparing emulsifying agents that improve the emulsifying properties of low-temperature plasma-modified papaya polysaccharides, characterized in that, The method for preparing the low-temperature plasma modified papaya polysaccharide includes the method for multi-stage low-temperature plasma extraction and modification of papaya polysaccharide as described in any one of claims 1-8.

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