Method for activating, hydrolyzing and polymerizing papaya procyanidine by using plasma

Abstract

The invention discloses a method for activating, hydrolyzing and polymerizing papaya procyanidine by adopting plasma. The method mainly comprises the following steps: extracting and purifying procyanidine from pawpaw; a plasma device is adopted to prepare plasma activated water, the output power is 50-70 W, and the treatment time is 2-6 min; stirring the papaya procyanidine, the nucleophilic reagent and the plasma activated water according to the mass ratio of 1: 1: 98 at the temperature of 15-25 DEG C for 8-18 minutes; and carrying out centrifugal separation on the fully and uniformly mixed liquid, and carrying out freeze-drying treatment on the supernate at the temperature of 60 DEG C below zero for 36 hours to finally obtain the depolymerized papaya procyanidine. The papaya proanthocyanidins are subjected to plasma activation hydrolytic polymerization treatment, the polymerization degree of the papaya proanthocyanidins can be reduced, the biological activity of the papaya proanthocyanidins can be improved, the papaya proanthocyanidins can be absorbed by the human body more easily, and the method is safer to operate and can be applied to industrialized production; and meanwhile, no extra chemical reagent is added in the depolymerization treatment process, so that the method conforms to the green, environment-friendly and healthy development trend.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a method for hydrolyzing polypapain proanthocyanidins by plasma activation. Background Art

[0002] Papaya (Chaenomeles sinensis) is abundant in Asia. The ripe papaya has a rich aroma and golden color. The fruit contains a large number of bioactive ingredients, such as organic acids, flavonoids, phenolic acids, etc. Papaya is rich in proanthocyanidins, which can be developed and utilized as functional active ingredients. Proanthocyanidins are a class of polyphenolic compounds widely found in plants, with many important biological activities and health benefits. Proanthocyanidins are polymers formed by the condensation of different numbers of catechins or epicatechins through CC bonds, and their structures are complex and diverse. It has many benefits, such as anti-oxidation, anti-inflammatory, vision protection, immunity enhancement, and cardiovascular health.

[0003] When the proanthocyanidins in papaya are often present in the form of high polymers, they are difficult to be directly absorbed and utilized by the human body. Through depolymerization, high polymer proanthocyanidins can be converted into oligomers, thereby improving their bioavailability in the body. In addition, the antioxidant activity of oligomeric papaya proanthocyanidins is usually higher than that of high polymer papaya proanthocyanidins. Therefore, the depolymerized papaya proanthocyanidins have stronger antioxidant capacity and can better exert their physiological effects. In the fields of food, health care products, etc., the stability of papaya proanthocyanidins is affected by their degree of polymerization. In addition, the depolymerized papaya proanthocyanidins have a wider range of applications. For example, in the field of cosmetics, oligomeric papaya proanthocyanidins have better permeability and stability, and can more effectively exert antioxidant, anti-aging and other effects. Therefore, it is necessary to use depolymerization treatment to reduce the degree of polymerization of papaya proanthocyanidins.

[0004] At present, the common methods for depolymerizing proanthocyanidins on the market include acid hydrolysis, alkaline hydrolysis, chromatographic adsorption separation, etc. Among them, the strong acid in the acid hydrolysis method may over-degrade the proanthocyanidins, resulting in the formation of substances with weak antioxidant capacity, thereby reducing the biological activity of the product, and the large-scale use of strong acids may cause environmental pollution, requiring strict wastewater treatment measures. The high concentration of alkali solution in the alkaline hydrolysis method may increase production costs, and waste liquid treatment also requires additional costs. It is also necessary to accurately control the reaction conditions (such as temperature, time, alkali solution concentration, etc.) to ensure the depolymerization effect, and the operation is relatively complicated. The chromatographic adsorption separation method is relatively cumbersome to operate, requiring multiple steps and a long processing time, and its processing capacity is relatively small and the efficiency is limited. Therefore, exploring a new, environmentally friendly and efficient method for depolymerizing papaya proanthocyanidins is of great significance to improving its biological activity and protecting the ecological environment. Summary of the invention

[0005] The present invention aims to solve the above problems and provides a method for hydrolyzing polypapain proanthocyanidins by plasma activation.

[0006] According to the technical solution of the present invention, the method of hydrolyzing polypapain proanthocyanidins by plasma activation comprises the following steps:

[0007] S1: Wash the papaya with distilled water, cut it into 3 mm slices using a slicer, and then put it into a dryer for drying at 50 °C for 2 h;

[0008] S2: The dried papaya slices were crushed into powder, passed through an 80-mesh sieve, and 70% methanol aqueous solution was used as a solvent, mixed at a solid-liquid ratio of 1:30, and extracted at 60°C by magnetic stirring for 1 h. The supernatant was collected and concentrated by a rotary evaporator to obtain a crude proanthocyanidin extract;

[0009] S3: The crude proanthocyanidin extract and AB-8 macroporous resin were dynamically adsorbed at a mass ratio of 1:14. Impurities were removed with distilled water, and then the proanthocyanidin was eluted with 70% ethanol. The eluate was collected, concentrated and freeze-dried at a freeze dryer temperature of -60°C for 36 hours to obtain purified papaya proanthocyanidin;

[0010] S4: using a dielectric barrier discharge cold plasma with an output power of 50-70W to treat the distilled water for 2-6 minutes to obtain plasma activated water;

[0011] S5: mixing papaya proanthocyanidins, nucleophilic reagents (catechins, epicatechins, phloroglucinol, etc.) and plasma-activated water in a mass ratio of 1:1:98 at a temperature of 15-25° C. for a mixing time of 8-18 min to obtain a mixed solution;

[0012] S6: The mixed solution is centrifuged in a centrifuge at a centrifugal speed of 4000 r / min. The supernatant is freeze-dried at a temperature of -60°C for 36 hours.

[0013] Furthermore, in step S4, the reactor for plasma-activated water is a dielectric barrier discharge, the pH of plasma-activated water is 2.38-3.74, the redox potential is 399.00-557.65 mV, and the conductivity is 212.00-581.15 μS / cm.

[0014] The beneficial technical effects of the present invention are embodied in the following aspects:

[0015] The present invention interacts with water molecules through the active substances such as ions and free radicals in the ion body, thereby generating an aqueous solution rich in active ingredients such as active oxygen and active nitrogen, these active ingredients have extremely high chemical reactivity, resulting in changes in the structure of the proanthocyanidin molecule, thereby reducing its degree of polymerization, and at the same time, in the acidic plasma-activated water environment and the joint action of nucleophilic reagents, the proanthocyanidin molecule may undergo a hydrolysis reaction, and the oligomers or monomers produced by hydrolysis have a lower degree of polymerization, which effectively reduces the degree of polymerization of papaya proanthocyanidins, and achieves a more accurate depolymerization effect. Plasma-activated water does not need to add additional chemicals during the treatment process, and the treated wastewater usually has a lower chemical oxygen demand and biological oxygen demand, which is environmentally friendly. In addition, plasma-activated water does not produce harmful by-products during the treatment process, which meets the requirements of green chemistry and sustainable development. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0017] Example 1

[0018] Wash the papaya with distilled water, cut it into 3mm slices using a slicer, and then put it into a dryer for drying at 50°C for 2h. Grind the dried papaya slices into powder, pass through an 80-mesh sieve, use 70% methanol aqueous solution as a solvent, mix at a solid-liquid ratio of 1:30, and extract by magnetic stirring at 60°C for 1h. Collect the supernatant and concentrate it with a rotary evaporator to obtain a crude extract of proanthocyanidins. Then dynamically adsorb the above crude extract of proanthocyanidins and AB-8 macroporous resin at a mass ratio of 1:14. Remove impurities with distilled water, then elute the proanthocyanidins with 70% ethanol, collect the eluate, concentrate and freeze-dry, the temperature of the freeze dryer is -60°C, the time is 36h, and purified papaya proanthocyanidins are obtained. Plasma-activated water is obtained by treating water in a dielectric barrier discharge plasma reactor, wherein the cold plasma output power is 50 W, the cold plasma activation time is 6 min, and the pH value of the plasma-activated water is 3.74, the redox potential is 399.00 mV, and the conductivity is 212.00 μS / cm; papaya proanthocyanidins, a nucleophilic agent (catechin) and plasma-activated water are mixed in a mass ratio of 1:1:98, the temperature is 20°C, and the mixing time is 18 min to obtain a mixed solution; the solution is centrifuged at a rate of 4000 r / min using a centrifuge, and the supernatant is placed in a freeze dryer at a temperature of -60°C for 36 hours.

[0019] In this embodiment, the average polymerization degree of the obtained papaya proanthocyanidins is 7.53±0.37, which is significantly lower than that of papaya proanthocyanidins (17.93±0.90).

[0020] Example 2

[0021] Wash the papaya with distilled water, cut it into 3mm slices using a slicer, and then put it into a dryer for drying at 50°C for 2h. Grind the dried papaya slices into powder, pass through an 80-mesh sieve, use 70% methanol aqueous solution as a solvent, mix at a solid-liquid ratio of 1:30, and extract by magnetic stirring at 60°C for 1h. Collect the supernatant and concentrate it with a rotary evaporator to obtain a crude extract of proanthocyanidins. Then dynamically adsorb the above crude extract of proanthocyanidins and AB-8 macroporous resin at a mass ratio of 1:14. Remove impurities with distilled water, then elute the proanthocyanidins with 70% ethanol, collect the eluate, concentrate and freeze-dry, the temperature of the freeze dryer is -60°C, the time is 36h, and purified papaya proanthocyanidins are obtained. Plasma-activated water is obtained by treating water in a dielectric barrier discharge plasma reactor, wherein the cold plasma output power is 60 W, the cold plasma activation time is 2 min, and the pH value of the plasma-activated water is 2.82, the redox potential is 439.74 mV, and the conductivity is 298.36 μS / cm; papaya proanthocyanidins, a nucleophilic agent (epicatechin) and plasma-activated water are mixed in a mass ratio of 1:1:98, the temperature is 25°C, and the mixing time is 8 min to obtain a mixed solution; the solution is centrifuged at a rate of 4000 r / min using a centrifuge, and the supernatant is placed in a freeze dryer at a temperature of -60°C for 36 hours.

[0022] In this embodiment, the average polymerization degree of the obtained papaya proanthocyanidins is 8.26±0.49, which is significantly lower than that of papaya proanthocyanidins (17.93±0.90).

[0023] Example 3

[0024] Wash the papaya with distilled water, cut it into 3mm slices using a slicer, and then put it into a dryer for drying at 50°C for 2h. Grind the dried papaya slices into powder, pass through an 80-mesh sieve, use 70% methanol aqueous solution as a solvent, mix at a solid-liquid ratio of 1:30, and extract by magnetic stirring at 60°C for 1h. Collect the supernatant and concentrate it with a rotary evaporator to obtain a crude extract of proanthocyanidins. Then dynamically adsorb the above crude extract of proanthocyanidins and AB-8 macroporous resin at a mass ratio of 1:14. Remove impurities with distilled water, then elute the proanthocyanidins with 70% ethanol, collect the eluate, concentrate and freeze-dry, the temperature of the freeze dryer is -60°C, the time is 36h, and purified papaya proanthocyanidins are obtained. Plasma-activated water is obtained by treating water in a dielectric barrier discharge plasma reactor, the cold plasma output power is 50 W, the cold plasma activation time is 2 min, the pH of the plasma-activated water is 3.22, the redox potential is 495.28 mV, and the conductivity is 341.23 μS / cm; papaya proanthocyanidins, a nucleophilic agent (phloroglucinol) and plasma-activated water are mixed in a mass ratio of 1:1:98, the temperature is 15°C, the mixing time is 14 min, and a mixed solution is obtained; the solution is centrifuged at a rate of 4000 r / min using a centrifuge, and the supernatant is placed in a freeze dryer at a temperature of -60°C for 36 hours.

[0025] In this embodiment, the average polymerization degree of the obtained papaya proanthocyanidins is 9.17±0.53, which is significantly lower than that of papaya proanthocyanidins (17.93±0.90).

[0026] Example 4

[0027] Wash the papaya with distilled water, cut it into 3mm slices using a slicer, and then put it into a dryer for drying at 50°C for 2h. Grind the dried papaya slices into powder, pass through an 80-mesh sieve, use 70% methanol aqueous solution as a solvent, mix at a solid-liquid ratio of 1:30, and extract by magnetic stirring at 60°C for 1h. Collect the supernatant and concentrate it with a rotary evaporator to obtain a crude extract of proanthocyanidins. Then dynamically adsorb the above crude extract of proanthocyanidins and AB-8 macroporous resin at a mass ratio of 1:14. Remove impurities with distilled water, then elute the proanthocyanidins with 70% ethanol, collect the eluate, concentrate and freeze-dry, the temperature of the freeze dryer is -60°C, the time is 36h, and purified papaya proanthocyanidins are obtained. Plasma-activated water is obtained by treating water in a dielectric barrier discharge plasma reactor, wherein the cold plasma output power is 70 W, the cold plasma activation time is 6 min, and the pH value of the plasma-activated water is 2.38, the redox potential is 557.65 mV, and the conductivity is 581.15 μS / cm; papaya proanthocyanidins, a nucleophilic agent (catechin) and plasma-activated water are mixed in a mass ratio of 1:1:98, the temperature is 22° C., and the mixing time is 16 min to obtain a mixed solution; the solution is centrifuged at a rate of 4000 r / min using a centrifuge, and the supernatant is placed in a freeze dryer at a temperature of -60° C. for 36 hours.

[0028] In this embodiment, the average polymerization degree of the obtained papaya proanthocyanidins is 6.83±0.36, which is significantly lower than that of papaya proanthocyanidins (17.93±0.90).

[0029] Comparative Example 1

[0030] Wash the papaya with distilled water, cut the papaya into 3mm slices using a slicer, and then put it into a dryer for drying at a temperature of 50°C for 2h. The dried papaya slices were crushed into powder, passed through an 80-mesh sieve, and 70% methanol aqueous solution was used as a solvent, mixed at a solid-liquid ratio of 1:30, and extracted by magnetic stirring at 60°C for 1h. The supernatant was collected and concentrated using a rotary evaporator to obtain a crude extract of proanthocyanidins. The above-mentioned crude extract of proanthocyanidins and AB-8 macroporous resin were then dynamically adsorbed at a mass ratio of 1:14. Impurities were removed with distilled water, and then the proanthocyanidins were eluted with 70% ethanol, the eluate was collected, concentrated and freeze-dried, the temperature of the freeze dryer was -60°C, and the time was 36h to obtain purified papaya proanthocyanidins. Hydrochloric acid was used instead of the plasma-activated water in Examples 1 to 4, the nucleophilic reagent was catechins, and the other implementation methods were the same. The pH of the hydrochloric acid used is 1.89, the redox potential is 412.13 mV, and the conductivity is 224.57 μS / cm. The hydrochloric acid and papaya proanthocyanidins are mixed and soaked at a soaking temperature of 20° C. for 16 minutes to obtain a hydrochloric acid depolymerized papaya proanthocyanidin solution; the solution is centrifuged at a rate of 4000 r / min using a centrifuge, and the supernatant is placed in a freeze dryer at a temperature of -60° C. for 36 hours.

[0031] In this comparative example, the average degree of polymerization of the obtained papaya proanthocyanidins is 13.18±0.66. Compared with comparative example 1, the average degree of polymerization in Examples 1 to 4 is significantly reduced. Compared with the traditional acid hydrolysis method, the plasma activated hydrolysis of polypapain proanthocyanidins is a method with unique physical and chemical properties, which can reduce the average degree of polymerization in papaya proanthocyanidins.

[0032] Comparative Example 2

[0033] Wash the papaya with distilled water, cut it into 3mm slices using a slicer, and then put it into a dryer for drying at 50°C for 2h. Grind the dried papaya slices into powder, pass through an 80-mesh sieve, use 70% methanol aqueous solution as a solvent, mix at a solid-liquid ratio of 1:30, and extract by magnetic stirring at 60°C for 1h. Collect the supernatant and concentrate it with a rotary evaporator to obtain a crude extract of proanthocyanidins. Then dynamically adsorb the above crude extract of proanthocyanidins and AB-8 macroporous resin at a mass ratio of 1:14. Remove impurities with distilled water, then elute the proanthocyanidins with 70% ethanol, collect the eluate, concentrate and freeze-dry, the temperature of the freeze dryer is -60°C, the time is 36h, and purified papaya proanthocyanidins are obtained. Plasma-activated water is obtained by treating water in a dielectric barrier discharge plasma reactor, wherein the cold plasma output power is 40 W, the cold plasma activation time is 6 min, and the pH value of the plasma-activated water is 4.88, the redox potential is 281.74 mV, and the conductivity is 136.53 μS / cm; papaya proanthocyanidins, a nucleophilic agent (catechin) and plasma-activated water are mixed in a mass ratio of 1:1:98, the temperature is 15° C., and the mixing time is 14 min to obtain a mixed solution; the solution is centrifuged at a rate of 4000 r / min using a centrifuge, and the supernatant is placed in a freeze dryer at a temperature of -60° C. for 36 hours.

[0034] In this comparative example, the average polymerization degree of papaya proanthocyanidins obtained was 14.02±0.53, and the average polymerization degrees in Examples 1 to 4 were significantly reduced compared with Comparative Example 2. When the output power of the cold plasma device for preparing plasma-activated water was 40W, the depolymerization effect of plasma-activated water on papaya proanthocyanidins was not ideal.

[0035] Plasma activated water depolymerization treatment can generate active ingredients such as active oxygen and active nitrogen, which can promote the depolymerization of macromolecular substances and improve the depolymerization efficiency. In contrast, conventional acid hydrolysis treatment mainly works through its ionized hydrogen ions, and the depolymerization effect may be relatively simple. Moreover, since plasma activated water does not require the addition of additional chemicals during the treatment process, it is environmentally friendly and does not produce harmful by-products. This makes it advantageous in some application scenarios with high environmental protection requirements. Conventional acid hydrolysis treatment may need to consider its impact on the environment and subsequent treatment issues during use.

[0036] Example 5

[0037] The measurement results of the above-mentioned comparative example 1, comparative example 2 and embodiments 1-4 are analyzed and sorted out, and the specific results are shown in the following Table 1.

[0038] Table 1 Comparison of the average polymerization degree of papaya proanthocyanidins in comparative examples and examples

[0039] Blank papaya proanthocyanidins Comparative Example 1 Comparative Example 2 Example 1 Example 2 Example 3 Example 4 Average degree of polymerization 17.93±0.90 13.18±0.66 14.02±0.53 7.53±0.37 8.26±0.49 9.17±0.53 6.83±0.36

[0040] Note: The average polymerization degree of each condition was obtained by three replicates, and the results are expressed as mean ± standard deviation.

[0041] It can be seen from the above results that the average degree of polymerization of Examples 1-4 of the present invention using the plasma activated water depolymerization treatment method is significantly reduced compared with Comparative Example 1, Comparative Example 2 and blank papaya proanthocyanidins.

[0042] Example 6

[0043] Before the determination, methanol was used to prepare proanthocyanidin sample solutions with different concentration gradients (10-400 μg / mL). 0.5 mL of proanthocyanidin sample solution was mixed with 19.5 mL of 0.1 mmol / L DPPH ethanol solution. Methanol and ascorbic acid (i.e., vitamin C) were used as blank control and positive control groups, respectively. During the experiment, the mixed solution was placed at room temperature and reacted in a light-proof environment for 20 minutes. Subsequently, its absorbance value was measured at a wavelength of 517 nm. To ensure the accuracy of the data, each sample was measured three times. The DPPH free radical scavenging activity percentage (%) was calculated using the formula provided below:

[0044] Clearance rate (%) = (A0-A1) / A0×100%

[0045] Where A0 is the absorbance value of the blank solution, and A1 is the absorbance of the sample solution after the reaction.

[0046] Results using IC 50 (the concentration required to achieve 50% clearance), IC 50 The value is inversely proportional to the DPPH free radical scavenging ability.

[0047] Table 2 DPPH free radical scavenging ability IC of papaya proanthocyanidins in comparative examples and examples 50 contrast

[0048] Blank papaya proanthocyanidins Comparative Example 1 Comparative Example 2 Example 1 Example 2 Example 3 Example 4 <![CDATA[IC 50 (mg / mL)]]> 0.31±0.03 0.24±0.06 0.26±0.05 0.14±0.03 0.14±0.04 0.12±0.03 0.11±0.05

[0049] Note: The DPPH free radical scavenging capacity IC of each condition 50 The results were expressed as mean ± standard deviation, and were repeated three times.

[0050] As shown in Table 2, the DPPH free radical scavenging ability of Examples 1-4 of the present invention using the plasma activated water decomposition treatment method is significantly enhanced compared with Comparative Example 1, Comparative Example 2 and blank papaya proanthocyanidins.

[0051] It can be seen that the method of using plasma activated hydrolysis of papaya proanthocyanidins provided by the present invention can effectively and significantly reduce the degree of polymerization of papaya proanthocyanidins, making papaya proanthocyanidins more easily absorbed and utilized by the human body, and can effectively improve the DPPH free radical scavenging ability and enhance the antioxidant capacity of papaya proanthocyanidins. In addition, unlike the traditional acid hydrolysis of papaya proanthocyanidins, the plasma activated water decomposition treatment method has a more efficient depolymerization effect and better application flexibility. Since no additional chemical reagents are required to be added during the treatment of plasma activated hydrolysis of papaya proanthocyanidins, and no harmful by-products are produced, the risk of secondary pollution is reduced, making it advantageous in some application scenarios with high environmental protection requirements, and achieving the goals of environmental protection and sustainability. By optimizing the process flow and improving the processing efficiency, the plasma activated water decomposition technology helps to achieve higher processing effects and lower overall energy consumption to reduce energy consumption. The method of using plasma activated hydrolysis of papaya proanthocyanidins is fully in line with the development trend of natural, green and healthy, and has a wide range of application prospects.

Claims

1. A method for hydrolyzing polypapain proanthocyanidins using plasma activation, characterized in that: The following steps are involved: S1: Wash the papaya, cut it into thin slices, and then put it into a dryer for drying at 50°C for 2 h; S2: The dried papaya slices were crushed into powder, sieved, and mixed with 70% methanol aqueous solution as solvent at a solid-liquid ratio of 1:

30. The mixture was extracted by magnetic stirring at 60 °C for 1 h, and the supernatant was collected and concentrated to obtain a crude proanthocyanidin extract; S3: The obtained crude proanthocyanidin extract was subjected to dynamic adsorption and decontamination, and then the proanthocyanidin was eluted with 70% ethanol, and the eluate was collected, concentrated and freeze-dried at a freeze dryer temperature of -60°C for 36 h to obtain purified papaya proanthocyanidin; S4: The distilled water was treated with a dielectric barrier discharge cold plasma with an output power of 50-70 W for 2-6 min to obtain plasma-activated water with a pH of 2.38-3.74, an oxidation-reduction potential of 399.00-557.65 mV, and a conductivity of 212.00-581.15 μS / cm; S5: mixing papaya proanthocyanidins, nucleophilic reagent and plasma activated water in a mass ratio of 1:1:98, at a temperature of 15-25° C., for a mixing time of 8-18 min, to obtain a mixed solution; S6: The mixed solution is centrifuged and then the obtained supernatant is freeze-dried at a freeze dryer temperature of -60°C for 36 h.

2. The method for hydrolyzing polypapain proanthocyanidins by plasma activation according to claim 1, characterized in that: In step S1, the papaya is cut into 3 mm thin slices.

3. The method for hydrolyzing polypapain proanthocyanidins by plasma activation according to claim 1, characterized in that: In the step S3, the crude proanthocyanidin extract is dynamically adsorbed on AB-8 macroporous resin at a mass ratio of 1:

14.

4. The method for hydrolyzing polypapain proanthocyanidins by plasma activation according to claim 1, characterized in that: The centrifugal speed of the centrifuge in step S6 is 4000 r / min.

5. The method for hydrolyzing polypapain proanthocyanidins by plasma activation according to claim 1, characterized in that: The nucleophilic reagent in step S5 is one or more of catechin, epicatechin, and phloroglucinol.