阅读说明：本技术 一种提升核桃青皮黄酮提取率及抑菌活性的方法 (Method for improving extraction rate and antibacterial activity of walnut green husk flavone ) 是由 罗安伟 喜梅花 沈荷玉 候妤婕 蔡莹莹 于 2021-09-06 设计创作，主要内容包括：本发明公开了一种提升核桃青皮黄酮提取率及抑菌活性的方法,其中,所述方法包括：对核桃青皮进行清洗、晾晒至水分得率低于10％；对晾晒后的核桃青皮进行研磨,控制核桃青皮粉末到预定目数范围后,进行均匀封装；通过设置不同剂量的电子束对封装后的核桃青皮粉末进行辐照预处理；对预处理后的核桃青皮粉末按照预定条件进行超声提取,获得核桃青皮溶液；对超声提取的核桃青皮溶液进行冷却、离心、浓缩,干燥,获得核桃青皮中的黄酮提取物。解决了现有技术中存在核桃青皮中黄酮类物质的提取缺少强度高、生产成本低的预处理技术导致提取率不高以及提取的黄酮类物质抑菌活性较差等技术问题。(The invention discloses a method for improving extraction rate and bacteriostatic activity of walnut green husk flavone, wherein the method comprises the following steps: cleaning and airing the walnut green husks until the water yield is lower than 10%; grinding the dried walnut green husks, and uniformly packaging after controlling the walnut green husk powder to be in a predetermined mesh range; performing irradiation pretreatment on the packaged walnut green husk powder by setting electron beams with different doses; performing ultrasonic extraction on the pretreated walnut green husk powder according to a preset condition to obtain a walnut green husk solution; and cooling, centrifuging, concentrating and drying the walnut green husk solution extracted by ultrasonic to obtain the flavone extract in the walnut green husk. Solves the technical problems of low extraction rate, poor bacteriostatic activity of extracted flavonoids and the like caused by the lack of a pretreatment technology with high intensity and low production cost in the extraction of flavonoids in walnut green husks in the prior art.)

1. A method for improving extraction rate and bacteriostatic activity of walnut green husk flavone, wherein the method comprises the following steps:

cleaning and airing the walnut green husks until the water yield is lower than 10%;

grinding the aired walnut green husks, controlling walnut green husk powder to reach a preset mesh range, and then uniformly packaging;

performing irradiation pretreatment on the packaged walnut green husk powder by setting electron beams with different doses;

carrying out ultrasonic extraction on the pretreated walnut green husk powder according to a preset condition to obtain a walnut green husk solution;

and cooling, centrifuging, concentrating and drying the walnut green husk solution extracted by ultrasonic to obtain the flavone extract in the walnut green husk.

2. The method as claimed in claim 1, wherein the irradiation pretreatment of the encapsulated walnut green husk powder by setting different doses of electron beams comprises:

and (3) performing electric irradiation pretreatment on the packaged walnut green husk powder by using an electron beam generated by a linear accelerator with an accelerator frequency of 50-60Hz, a tray running speed of 5-10m/min and a 10MeV/20kW electron beam.

3. The method according to claim 1, wherein the irradiation doses are set to 20-60kGy, respectively.

4. The method of claim 1, wherein the walnut green husk powder is uniformly packaged, including packaging in a sealed bag of medicinal PE, 0.3-0.7kg per bag, and 1-2cm thick.

5. The method of claim 1, wherein the ultrasonic extraction of the pretreated walnut green husk powder according to a predetermined condition comprises:

the predetermined conditions are that the ratio of the material to the liquid is 1:45-1:50g/mL, the concentration of the ethanol solution is 75-76%, the ultrasonic power is 480-500W, and the ultrasonic time is 110-130 min.

6. The method as claimed in claim 1, wherein the harvesting time of the walnut green husk is 7-8 months.

7. The method as claimed in claim 1, wherein the predetermined mesh number of the walnut green husk powder is in the range of 60-100 meshes.

Technical Field

The invention relates to the technical field of flavone extraction, and particularly relates to a method for improving extraction rate and antibacterial activity of walnut green husk flavone.

Background

The walnut is considered to have extremely high nutritional value and good health care function all the time, people begin to enjoy quality life and healthy diet, and the walnut market is rapidly developed, but the whole industry is mainly processed by walnut kernels for a long time, the byproducts are numerous but the resource utilization rate is low, and especially in the harvesting season, a large amount of waste walnut green husks accumulated in the market cause the problem of environmental pollution which is difficult to ignore due to the rough treatment mode of people. The flavonoid compounds are widely present in plant raw materials such as fruits, vegetables, grains, tea and the like, and are yellow pigments derived from 2-phenylchromone serving as a mother nucleus, most of the yellow pigments exist in a form of combining glycosides with sugar or in a form of carbon glycosyl, and the number of the determined flavonoid compounds is as much as 4000. The flavonoid compound has polarity, a polar solvent is used for extraction, water, alkali, an organic solvent and the like are usually used for extraction, compared with the traditional extraction method, the ultrasonic wave has four characteristics of thermal property, non-thermal property, high frequency and fluctuation, and the ultrasonic extraction technology has the characteristics of shortened operation time, uniform heating, reduced reagent consumption, easy control and the like, and is a high-efficiency and feasible extraction method, so that the ultrasonic extraction technology is widely applied to the extraction of natural products in the food industry.

However, in the process of implementing the technical solution of the invention in the embodiments of the present application, the inventors of the present application find that the above-mentioned technology has at least the following technical problems:

in the prior art, the extraction of flavonoid substances in walnut green husks is lack of a pretreatment technology with high intensity and low production cost, so that the extraction rate is not high, the extracted flavonoid substances have poor antibacterial activity and the like.

Disclosure of Invention

Aiming at the defects in the prior art, the embodiment of the application aims to solve the technical problems that the extraction rate of flavonoid in walnut green seedcase is low due to the lack of a pretreatment technology with high intensity and low production cost in the extraction of flavonoid in walnut green case, the extracted flavonoid has poor antibacterial activity and the like in the prior art by providing the method for improving the extraction rate and the antibacterial activity of the flavonoid in walnut green case. The technical effects of promoting the effective dissolution of active substances in plant cells, remarkably promoting the extraction of flavone substances in the walnut green husks and improving the bacteriostatic activity of the flavone substances are achieved.

On one hand, the embodiment of the application provides a method for improving the extraction rate and the bacteriostatic activity of walnut green husk flavone, wherein the method comprises the following steps: cleaning and airing the walnut green husks until the water yield is lower than 10%; grinding the aired walnut green husks, controlling walnut green husk powder to reach a preset mesh range, and then uniformly packaging; performing irradiation pretreatment on the packaged walnut green husk powder by setting electron beams with different doses; carrying out ultrasonic extraction on the pretreated walnut green husk powder according to a preset condition to obtain a walnut green husk solution; and cooling, centrifuging, concentrating and drying the walnut green husk solution subjected to ultrasonic extraction to obtain the flavone extract in the walnut green husk.

One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

cleaning and airing the walnut green husks until the water yield is lower than 10%; grinding the aired walnut green husks, controlling walnut green husk powder to reach a preset mesh range, and then uniformly packaging; performing irradiation pretreatment on the packaged walnut green husk powder by setting electron beams with different doses; carrying out ultrasonic extraction on the pretreated walnut green husk powder according to a preset condition to obtain a walnut green husk solution; and cooling, centrifuging, concentrating and drying the walnut green husk solution subjected to ultrasonic extraction to obtain the flavone extract in the walnut green husk. Based on the method, the method for improving the extraction rate and the bacteriostatic activity of the flavone in the walnut green husk can be obtained, and the electron beam irradiation is used as a pretreatment technology to be coupled with an ultrasonic extraction technology, so that the technical effects of promoting the effective dissolution of active substances in plant cells, remarkably promoting the extraction of flavone substances in the walnut green husk and improving the bacteriostatic activity of the flavone substances are achieved.

The foregoing description is only an overview of the technical solutions of the present application, and the present application can be implemented according to the content of the description in order to make the technical means of the present application more clearly understood, and the following detailed description of the present application is given in order to make the above and other objects, features, and advantages of the present application more clearly understandable.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic flow chart of a method for improving extraction rate and bacteriostatic activity of walnut green husk flavone according to an embodiment of the present application;

FIG. 2 is a line graph showing the effect of ethanol concentration on flavone yield;

FIG. 3 is a line graph showing the effect of grind size on flavone yield;

FIG. 4 is a line graph showing the effect of liquid-to-feed ratio on flavone yield;

FIG. 5 is a line graph showing the effect of extraction temperature on flavone yield;

FIG. 6 is a line graph of the effect of ultrasonic power on flavone yield;

FIG. 7 is a line graph of the effect of sonication time on flavone yield;

FIG. 8 is a response surface test interaction diagram of a method for improving extraction rate and bacteriostatic activity of walnut green husk flavone in the embodiment of the application;

FIG. 9 is a bar graph of the effect of metal ions on bacteriostatic stability of a walnut green husk crude extract;

FIG. 10 is a bar graph of the effect of irradiation dose on the bacteriostatic effect of crude extract of exocarpium Juglandis Immaturus.

Detailed Description

The embodiment of the application provides a method for improving the extraction rate and the bacteriostatic activity of the flavone in the walnut green seedcase, and solves the technical problems that the extraction rate is not high due to the fact that the extraction of the flavone in the walnut green seedcase lacks of a pretreatment technology with high intensity and low production cost, the extracted flavone has poor bacteriostatic activity and the like in the prior art. The technical effects of promoting the effective dissolution of active substances in plant cells, remarkably promoting the extraction of flavone substances in the walnut green seedcase and improving the bacteriostatic activity of the flavone substances are achieved.

Hereinafter, technical solutions in example embodiments of the present application will be described in detail with reference to the accompanying drawings. It should be apparent that the described embodiments are merely some embodiments of the present application and not all embodiments of the present application, and it should be understood that the present application is not limited to the example embodiments described herein. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present application without making any creative effort, shall fall within the protection scope of the present application.

Summary of the application

The walnut is considered to have extremely high nutritional value and good health care function all the time, people begin to enjoy quality life and healthy diet, and the walnut market is rapidly developed, but the whole industry is mainly processed by walnut kernels for a long time, the byproducts are numerous but the resource utilization rate is low, and especially in the harvesting season, a large amount of waste walnut green husks accumulated in the market cause the problem of environmental pollution which is difficult to ignore due to the rough treatment mode of people. The flavonoid compounds are widely present in plant raw materials such as fruits, vegetables, grains, tea and the like, and are yellow pigments derived from 2-phenylchromone serving as a mother nucleus, most of the yellow pigments exist in a form of combining glycosides with sugar or in a form of carbon glycosyl, and the number of the determined flavonoid compounds is as much as 4000. The flavonoid compound has polarity, a polar solvent is used for extraction, water, alkali, an organic solvent and the like are usually used for extraction, compared with the traditional extraction method, the ultrasonic wave has four characteristics of thermal property, non-thermal property, high frequency and fluctuation, and the ultrasonic extraction technology has the characteristics of shortened operation time, uniform heating, reduced reagent consumption, easy control and the like, and is a high-efficiency and feasible extraction method, so that the ultrasonic extraction technology is widely applied to the extraction of natural products in the food industry. At present, the technical problems of low extraction rate caused by the lack of a pretreatment technology with high intensity and low production cost in the extraction of flavonoid substances in walnut green husks, poor bacteriostatic activity of the extracted flavonoid substances and the like exist.

In view of the above technical problems, the technical solution provided by the present application has the following general idea:

the embodiment of the application provides a method for improving extraction rate and bacteriostatic activity of walnut green husk flavone, wherein the method comprises the following steps: cleaning and airing the walnut green husks until the water yield is lower than 10%; grinding the aired walnut green husks, controlling walnut green husk powder to reach a preset mesh range, and then uniformly packaging; performing irradiation pretreatment on the packaged walnut green husk powder by setting electron beams with different doses; carrying out ultrasonic extraction on the pretreated walnut green husk powder according to a preset condition to obtain a walnut green husk solution; and cooling, centrifuging, concentrating and drying the walnut green husk solution subjected to ultrasonic extraction to obtain the flavone extract in the walnut green husk.

For better understanding of the above technical solutions, the following detailed descriptions will be provided in conjunction with the drawings and the detailed description of the embodiments.

Example one

As shown in fig. 1, an embodiment of the present application provides a method for improving extraction rate and bacteriostatic activity of walnut green husk flavone, wherein the method includes:

step S100: cleaning and airing the walnut green husks until the water yield is lower than 10%;

specifically, the walnut green seedcase is taken and cleaned, after surface impurities are removed, the walnut green seedcase is aired until the water yield is lower than 10%, the water yield refers to that in the airing process, part of the walnut green seedcase is randomly taken to measure the water content until the water yield is lower than 10%, airing is stopped, the aired walnut green seedcase is collected, after subsequent treatment, the water yield is controlled to be lower than 10%, backwater and rot of the walnut green seedcase can be reduced, long-time storage is facilitated, and the accuracy and reliability of subsequent results can also be guaranteed.

Step S200: grinding the aired walnut green husks, controlling walnut green husk powder to reach a preset mesh range, and then uniformly packaging;

specifically, to extract flavone substances in the walnut green seedcase, firstly, crushing the walnut green seedcase, grinding the walnut green seedcase to a preset mesh range through grinding treatment, wherein the grinding treatment is to crush the walnut green seedcase into powder with different meshes by using a tissue pounder and mainly achieve the purpose by controlling grinding time, and the mesh control method comprises the steps of passing through a screen with the corresponding mesh, collecting undersize, uniformly packaging according to a certain packaging standard, and placing at room temperature for later use, thereby being a practical and tamping basis for the research on the subsequent flavone substance extraction method.

Step S300: performing irradiation pretreatment on the packaged walnut green husk powder by setting electron beams with different doses;

in particular, electron beam irradiation is used as a safe and environment-friendly pretreatment method, and compared with means such as ultraviolet light and X-ray, the method has the advantages of high flexibility, easiness in operation and control, safety and no pollution. The electron beam irradiation pretreatment process is optimized by setting different electron beam irradiation doses, the walnut green husk powder is irradiated by electron beams with different doses, and the plant cell walls can be damaged after the walnut green husk powder is irradiated by the electron beams in an irradiation field, so that the effect of improving the dissolution of active substances is achieved.

Step S400: carrying out ultrasonic extraction on the pretreated walnut green husk powder according to a preset condition to obtain a walnut green husk solution;

specifically, the ultrasonic wave has four characteristics of thermal characteristic, non-thermal characteristic, high frequency and fluctuation, the ultrasonic extraction technology has the characteristics of shortened operation time, uniform heating, reduced reagent consumption, easy control and the like, and the ultrasonic extraction method is a high-efficiency and feasible extraction method.

Step S500: and cooling, centrifuging, concentrating and drying the walnut green husk solution extracted by ultrasonic to obtain the flavone extract in the walnut green husk.

Specifically, after the ultrasonic extraction reaction is finished, cooling the sample liquid to room temperature, centrifuging the sample liquid by a high-speed centrifuge at the rotating speed of 8000r/min for 10min, taking supernatant, discarding precipitates, putting the supernatant on a rotary evaporator, concentrating the supernatant under reduced pressure to recover a solvent to the near-dry state, obtaining dry paste by the rotary evaporator manufacturer of Qingdao poly-wound with the model of JC-ZF-RE5000, obtaining flavone extract in walnut green peel, dissolving the flavone extract by methanol, fixing the volume, and taking the dissolved flavone extract as a sample solution to be tested to perform subsequent index measurement. The electron beam irradiation pretreatment and the ultrasonic extraction method can improve the extraction rate of active flavone and enhance the bacteriostatic activity of the flavone, thereby solving the problem of comprehensive utilization of walnut green husk waste and changing waste into valuable.

Further, the irradiation pretreatment is carried out on the encapsulated walnut green husk powder by setting electron beams with different doses, and the method further comprises the following steps: and (3) performing electric irradiation pretreatment on the packaged walnut green husk powder by using an electron beam generated by a linear accelerator with an accelerator frequency of 50-60Hz, a tray running speed of 5-10m/min and a 10MeV/20kW electron beam.

Specifically, in the process of electron beam irradiation pretreatment, the frequency of an accelerator used is 50-60Hz, and the running speed of an irradiation tray is 5-10 m/min. The type of the high-energy electron beam linear accelerator is 10MeV/20kW ESS-010-03 electron linear accelerator, the rated energy is 10MeV, the power is 10kW, the sweep width is 800cm, the beam current is 2mA, the frequency is 50-60Hz, the high-energy electron beam linear accelerator is classified according to the energy, and can be divided into three energy regions in the category of industrial electron irradiation accelerators, 80keV-0.3MeV is a low-energy accelerator, 0.3MeV-5MeV is an intermediate-energy accelerator, and 5MeV-10MeV is a high-energy accelerator. 10MeV/20kW means that the high-energy electron irradiation accelerator outputs an electron beam with the energy of 10MeV, and the maximum beam power is 20 kW. The walnut green husk powder after irradiation treatment by different electron beam irradiation doses is reserved for standby, and the application of the irradiation pretreatment technology can promote the dissolution of active substances and improve the extraction rate of the active substances.

Further, the embodiment of the present application further includes: the irradiation doses were set to 20-60kGy, respectively.

Specifically, setting the irradiation dose to be 20-60kGy, setting 0kGy as a control group, verifying the electron beam irradiation treatment effect, and when the irradiation dose exceeds 60kGy, the extraction efficiency of flavone substances in the walnut green husks is poor due to overhigh dose. The choice of a dosage range of 20-60kGy is therefore a reliable, reasonable dosage range.

Further, the embodiment of the present application further includes: and uniformly packaging the walnut green husk powder, wherein the packaging comprises sealing bags of medicinal PE, each bag is 0.3-0.7kg, and the thickness is 1-2 cm.

Specifically, the walnut green husk powder is filled into a commercially available medical PE sealing bag, each bag is 0.3-0.7kg, the thickness is 1-2cm, so that the sample can be prevented from being polluted, the electron beam can be ensured to completely penetrate through the sample by the thickness of 1-2cm, the irradiation effect is ensured, the repeatability and the operability of the whole experimental process can be ensured by regulating the weight and the thickness of each bag of sample, the whole process is convenient to standardize, and a foundation is laid for the popularization and the large-scale treatment of the method.

Further, the ultrasonic extraction of the pretreated walnut green husk powder according to a predetermined condition further comprises: the predetermined conditions are that the ratio of the material to the liquid is 1:45-1:50g/mL, the concentration of the ethanol solution is 75-76%, the ultrasonic power is 480-500W, and the ultrasonic time is 110-130 min.

Specifically, the ultrasonic-assisted extraction process comprises liquid-material ratio, ethanol solution concentration, ultrasonic power, time and the like, and the pretreated walnut green husk powder is subjected to ultrasonic extraction according to preset conditions, wherein the preset conditions comprise the liquid-material ratio of 1:45-1:50g/mL, the ethanol solution concentration of 75-76%, the ultrasonic power of 480-500W and the ultrasonic time of 110-130 min. Under the ultrasonic extraction condition, the flavone substances in the walnut green seedcase can be better extracted, and a foundation is laid for a subsequent antibacterial property experiment.

Further, the embodiment of the present application further includes: the harvesting time of the walnut green seedcases is 7-8 months.

Specifically, in 7-8 months, the walnut green husks of five kinds of fresh walnuts including Xinxin No. 2, Xifu No. 1, Xifu No. 2, Xiangling and Dalu kernel are collected in the peak season of walnut harvesting, and a consumer can generate a large number of walnut green husks when purchasing walnuts in the peak season of walnut harvesting, so that the purchased walnut green husk samples can not only ensure the freshness of the walnut green husks, but also can utilize the walnut green husks, and the environmental pollution is reduced. Meanwhile, the accuracy of experimental data can be ensured by using a fresh walnut green husk sample.

Further, the embodiment of the present application further includes: the predetermined mesh range of the walnut green husk powder is 60-100 meshes.

Specifically, in order to explore the influence effect of walnut green husk powder raw materials with different pulverization degrees on the flavone extraction rate, the walnut green husk powder is ground to 60-100 meshes, generally, the higher the pulverization degree of the raw materials is, the finer the powder is, the faster the dissolution, permeation and diffusion in the extraction process is, and the higher the extraction efficiency is. However, the powder is too fine, the surface area of the powder particles is large, and the adsorption action is enhanced to adversely affect the diffusion rate. Moreover, if the raw material contains a large amount of protein and polysaccharide components, the raw material is pulverized too finely, and these components are dissolved out too much, so that the extract is more viscous, and a pepton phenomenon is generated, which affects other operations. Therefore, the study of the walnut green husk powder with different pulverization degrees is crucial to the extraction process by researching the optimal pulverization degree.

Compared with the prior art, the invention has the following beneficial effects:

1. cleaning and airing the walnut green husks until the water yield is lower than 10%; grinding the aired walnut green husks, controlling walnut green husk powder to reach a preset mesh range, and then uniformly packaging; performing irradiation pretreatment on the packaged walnut green husk powder by setting electron beams with different doses; carrying out ultrasonic extraction on the pretreated walnut green husk powder according to a preset condition to obtain a walnut green husk solution; and cooling, centrifuging, concentrating and drying the walnut green husk solution subjected to ultrasonic extraction to obtain the flavone extract in the walnut green husk. Based on the method, the method for improving the extraction rate and the bacteriostatic activity of the flavone in the walnut green husk can be obtained, and the electron beam irradiation is used as a pretreatment technology to be coupled with an ultrasonic extraction technology, so that the technical effects of promoting the effective dissolution of active substances in plant cells, remarkably promoting the extraction of flavone substances in the walnut green husk and improving the bacteriostatic activity of the flavone substances are achieved.

Example two

Accurately weighing 1.0g of walnut green husk powder which is sieved by a 70-mesh sieve, adding 60%, 70%, 80%, 90% and 100% of 5 ethanol solutions with different concentrations into the walnut green husk powder according to a ratio of 1:20g/mL for extraction, carrying out ultrasonic extraction for 120min, setting the ultrasonic temperature at 50 ℃, the extraction power at 420W, centrifuging the walnut green husk powder for 10min after the reaction is finished, setting the rotating speed at 8000r/min, and carrying out constant volume on the supernatant dry extract obtained after rotary evaporation and concentration to 100mL by using methanol to be detected.

The following data were determined for the walnut green husk extract prepared in example two: the method for measuring flavone yield comprises the following steps: using NaNO₂-Al(NO₃)₃Determining total flavone yield by color colorimetric method, taking rutin standard solution mass concentration C (mg/mL) as abscissa, and light absorption value of standard solution with different concentration at 510nm as ordinate, to obtain rutin standard curve regression equation of A ═ 7.1134C-0.0197, wherein A is absorbance, C is rutin mass concentration, and mg/mL is^-1。R²The results are shown in fig. 2, 0.9996.

EXAMPLE III

Accurately weighing 1.0g of dried walnut green husk powder which is sieved by 20, 40, 60, 80 and 100 meshes, respectively, adding 60% ethanol according to a ratio of 1:20g/mL, carrying out ultrasonic extraction for 120min, setting the ultrasonic temperature at 50 ℃, carrying out extraction power at 420W, centrifuging for 10min after reaction is finished, carrying out rotation speed at 8000r/min, carrying out rotary evaporation concentration on the supernatant dry paste to 100mL by using methanol, and carrying out volume fixing to be tested.

The following data were determined for the walnut green husk extract prepared in example three: the method for measuring flavone yield comprises the following steps: using NaNO₂-Al(NO₃)₃Determining total flavone yield by color colorimetric method, taking rutin standard solution mass concentration C (mg/mL) as abscissa, and light absorption value of standard solution with different concentration at 510nm as ordinate, to obtain rutin standard curve regression equation of A ═ 7.1134C-0.0197, wherein A is absorbance, C is rutin mass concentration, and mg/mL is^-1。R²The results are shown in fig. 3, 0.9996.

Example four

Accurately weighing 1.0g of walnut green husk powder which is sieved by a 70-mesh sieve, adding 60% ethanol into the walnut green husk powder according to the ratio of 1:10, 1:20, 1:30, 1:40 and 1:50g/mL, carrying out ultrasonic extraction for 120min at the temperature of 50 ℃, carrying out extraction power of 420W, centrifuging the walnut green husk powder for 10min after the reaction is finished, carrying out rotation speed of 8000r, and carrying out constant volume drying on the supernatant dry paste which is subjected to rotary evaporation concentration to 100mL by using methanol to be detected.

The following data were determined for the walnut green husk extract prepared in example four: the method for measuring flavone yield comprises the following steps: using NaNO₂-Al(NO₃)₃Determining total flavone yield by color colorimetric method, taking rutin standard solution mass concentration C (mg/mL) as abscissa, and light absorption value of standard solution with different concentration at 510nm as ordinate, to obtain rutin standard curve regression equation of A ═ 7.1134C-0.0197, wherein A is absorbance, C is rutin mass concentration, and mg/mL is^-1。R²The results are shown in fig. 4, 0.9996.

EXAMPLE five

Accurately weighing 1.0g of walnut green husk powder which is sieved by a 70-mesh sieve, adding 60% ethanol according to a ratio of 1:20g/mL, ultrasonically extracting for 120min at the temperature of 20, 30, 40, 50 and 60 ℃ at the extraction power of 420W, centrifuging for 10min after the reaction is finished at the rotating speed of 8000r/min, and fixing the volume of the supernatant dry paste after rotary evaporation concentration to 100mL by using methanol to be detected.

The following data were determined for the walnut green husk extract prepared in example five: the method for measuring flavone yield comprises the following steps: using NaNO₂-Al(NO₃)₃Determining total flavone yield by color colorimetric method, taking rutin standard solution mass concentration C (mg/mL) as abscissa, and light absorption value of standard solution with different concentration at 510nm as ordinate, to obtain rutin standard curve regression equation of A ═ 7.1134C-0.0197, wherein A is absorbance, C is rutin mass concentration, and mg/mL is^-1。R²The results are shown in fig. 5, 0.9996.

EXAMPLE six

Accurately weighing 1.0g of walnut green husk powder which is sieved by a 70-mesh sieve, adding 60% ethanol according to a ratio of 1:20g/mL, carrying out ultrasonic extraction for 120min, setting the temperature at 50 ℃, respectively setting the working power of an ultrasonic cleaner at 280W, 350W, 420W, 490W and 560W, centrifuging for 10min after the reaction is finished, setting the rotating speed at 8000r, and carrying out rotary evaporation concentration on the supernatant dry paste to a constant volume of 100mL by using methanol to be measured.

The following data were determined for the walnut green husk extract prepared in example six: the method for measuring flavone yield comprises the following steps: using NaNO₂-Al(NO₃)₃Determining total flavone yield by color colorimetric method, taking rutin standard solution mass concentration C (mg/mL) as abscissa, and light absorption value of standard solution with different concentration at 510nm as ordinate, to obtain rutin standard curve regression equation of A ═ 7.1134C-0.0197, wherein A is absorbance, C is rutin mass concentration, and mg/mL is^-1。R²The results are shown in fig. 6, 0.9996.

EXAMPLE seven

Accurately weighing 1.0g of walnut green husk powder which is sieved by a 70-mesh sieve, adding 60% ethanol according to a ratio of 1:20g/mL, carrying out ultrasonic extraction for 80, 100, 120, 140 and 160min, setting the temperature at 50 ℃, carrying out extraction power at 420W, centrifuging for 10min after the reaction is finished, carrying out rotation speed at 8000r/min, and carrying out constant volume on the supernatant dry paste obtained after rotary evaporation concentration to 100mL by using methanol to be measured.

The following data were determined for the walnut green husk extract prepared in example seven: the method for measuring flavone yield comprises the following steps: using NaNO₂-Al(NO₃)₃Determining total flavone yield by color colorimetric method, taking rutin standard solution mass concentration C (mg/mL) as abscissa, and light absorption value of standard solution with different concentration at 510nm as ordinate, to obtain rutin standard curve regression equation of A ═ 7.1134C-0.0197, wherein A is absorbance, C is rutin mass concentration, and mg/mL is^-1。R²The results are shown in fig. 7, 0.9996.

As shown in fig. 2-7, the extraction process parameters are different, the yield of flavone in the walnut green husk extract is different, and the influence difference of each factor on the flavone is obvious (P is less than 0.05); the general trend is that flavone firstly rises and then falls along with the increase of ethanol concentration, crushing degree, liquid-material ratio, temperature, ultrasonic power and ultrasonic time. Therefore, when the ethanol concentration is 70%, the crushing degree is 60 meshes, the liquid-material ratio is 40:1(mL/g), the temperature is 50 ℃, the ultrasonic power is 490W, and the ultrasonic time is 120min, the extraction effect is most remarkable.

Furthermore, the regression relationship between the flavone content and the crushing degree, the ethanol concentration, the feed-liquid ratio and the ultrasonic time is analyzed by a response surface analysis method to obtain the optimal process parameter conditions.

Designing a Box-Behnken test of 29 experiments of three levels of four factors according to the number of the significant factors, establishing a regression model equation and determining the optimal process parameters of the walnut green husk extract by using a response surface. As can be seen from table 1 and fig. 8, ethanol concentration (B), liquid-to-material ratio (C) and sonication time (D) are the three main variables affecting the extract (P < 0.0001). The flavone yield gradually increases to a peak value along with the increase of the ethanol concentration, the liquid-material ratio and the ultrasonic time, and then starts to decrease along with the increase of B, C, D. AB. AC and BD have significant interaction on flavone yield. The optimal technological parameter conditions obtained by response surface test optimization are that the crushing degree is 70 meshes, the ethanol concentration is 75.479%, the liquid-material ratio is 46:1mL/g, the extraction time is 131.774min, and the flavone yield is 30.29 mg/g.

TABLE 1 response surface test results

Example eight

Taking a new 2-variety walnut green husk powder sample for electron beam irradiation pretreatment, namely packaging walnut green husk powder by a medical PE sealing bag, placing the walnut green husk powder in an irradiation tray, respectively carrying out irradiation treatment of 0 (contrast), 20, 30, 40, 50 and 60kGy, carrying out ultrasonic extraction under optimized optimal ultrasonic technological conditions, filtering and concentrating the extracted feed liquid, and preparing the walnut green husk extract.

The following data were determined for the walnut green husk extract prepared in example eight: the method for measuring flavone yield comprises the following steps: using NaNO₂-Al(NO₃)₃The total flavone yield is determined by color colorimetric method, with rutin standard solution mass concentration C (mg/mL) as abscissa, light absorption value of standard solution with different concentrations at 510nm as ordinate,obtaining rutin standard curve regression equation of A-7.1134C-0.0197, wherein A is absorbance, C is rutin mass concentration, and mg.mL^-1。R²＝0.9996。

As can be seen from Table 2, with the increase of the irradiation dose of the electron beam, the yield of the walnut green husk flavone increases firstly and then decreases, and reaches the maximum value of 38mg/g at 30 kGy. Compared with the non-irradiated walnut green husk, the yield of the irradiated walnut green husk is improved more after ultrasonic-assisted extraction.

TABLE 2 influence of different irradiation dose treatments on the yield of flavone in exocarpium Juglandis Immaturus extract

Further, the yield of the flavone substances extracted from the walnut green seedcases of different varieties by the optimized process is determined. As can be seen from Table 3, after the optimized electron beam irradiation pretreatment and ultrasonic extraction technology are performed on the walnut green husks of five different varieties, the flavone yield of the extract is as follows: west Fu No. 1, Dalu ren, West Fu No. 2, Xiangling and Xinxin No. 2.

TABLE 3 flavone yield in walnut green husk of different varieties

Example nine

And (3) strain culture: the method comprises the steps of respectively carrying out streak culture on staphylococcus aureus, bacillus cereus, escherichia coli and salmonella on an LB solid plate, and standing for 24 hours in an incubator at 37 ℃.

Preparation of bacterial suspension: respectively picking out single colony tested bacteria from an LB plate, putting the single colony tested bacteria into an LB liquid culture medium, culturing for 18h at 37 ℃, sucking 1mL of bacterial liquid into a centrifugal tube at the rotating speed of 8000r/min, centrifuging for 1min, discarding supernatant, cleaning the bacteria for 3 times by using PBS buffer solution, adjusting bacterial suspension to OD by using LB broth_600nmPipette 1mL of each bacterial suspension into a test tube, dilute the suspension with LB broth, and controlThe concentration of the bacterial suspension is 1X 10⁶CFU/mL for standby.

Determination of the inhibition zone: the invention adopts an oxford cup method to measure the diameters of the inhibition zones of four bacteria by using the walnut green seedcase crude extract. Adding 15mL of agar into an aseptic culture dish, cooling and solidifying, respectively and uniformly coating 150 mu L of different bacterial suspensions on a solid culture medium, putting an aseptic oxford cup into the culture dish, sucking 150 mu L of sample liquid 60mg/mL into the oxford cup, culturing for 36h at constant temperature of 37 ℃, and measuring the diameter d (mm) of a bacteriostatic zone by a cross method. Blank control was 70% ethanol and positive control was 100 μ g/mL gentamicin solution, each treatment was repeated three times. Antibacterial effect judgment standard: high sensitivity (d >15mm), moderate sensitivity (10mm < d <15mm), low sensitivity (8mm < d <10 mm).

Influence of metal ions on the bacteriostatic effect of the walnut green seedcase is realized by respectively adding 30 mu L of 0.1mol/L CaCl into each Oxford cup₂、FeCl₂And CuCl₂Solutions, assayed in triplicate.

And (3) adjusting the concentration of flavone in the irradiated walnut green husk extracting solution to 0.06g/mL, respectively measuring the diameters of inhibition zones, and carrying out parallel measurement for three times.

As can be seen from FIG. 9, the metal ions show differences in the improvement of the bacteriostatic effect of the crude extract of exocarpium Juglandis Immaturus. The iron ions and the copper ions have obvious improvement on the bacteriostatic ability of the walnut green seedcase. Fe compared with the control group²⁺The diameter of the inhibition zone of the treatment group is increased by 47-93 percent, and Cu²⁺The treatment group is improved by 49-89%. After the calcium chloride solution is added, the diameter of the inhibition zone of the tested bacteria is not obviously changed. This is probably because copper ions and zinc ions themselves have a certain inhibitory effect on the growth of bacteria, and the inhibitory effect of copper ions and zinc ions is improved by the easy reaction with the hydroxyl structure of the catechol in the active compound. Therefore, when the walnut green husk extract bacteriostatic agent is developed, the iron ions and the copper ions are properly added, so that the bacteriostatic effect can be obviously improved.

The influence of the irradiation dose on the bacteriostasis effect of the walnut green husk crude extract in fig. 10 can be obtained, and the diameters of the bacteriostasis zones of the four tested bacteria show a trend of increasing first and then decreasing as the irradiation dose is gradually increased. When the irradiation dose is 30kGy, compared with the diameter of an inhibition zone of a crude extract of the walnut green seedcase which is not subjected to irradiation treatment, the diameters of the inhibition zones of escherichia coli and staphylococcus aureus reach maximum values, namely 16.79 +/-0.51 mm and 16.32 +/-0.62 mm respectively, high sensitivity is achieved, the diameters of the inhibition zones of bacillus cereus and salmonella are maximum values, namely 13.27 +/-0.86 mm and 11.93 +/-0.89 mm respectively, and the intermediate sensitivity is achieved. The reason is probably that after the walnut green husk powder is irradiated and ultrasonically treated, active substances such as flavone and polyphenol are dissolved out, and the bacteriostatic effect of the walnut green husk crude extract on test bacteria is improved.

Table 4 shows the bacteriostatic effect of the control group, the diameters of the inhibition zones of the 75% ethanol solution on four bacteria are all less than 10mm, no inhibition effect is realized, the diameters of the inhibition zones of the gentamicin solution on escherichia coli, staphylococcus aureus and bacillus cereus are all more than 15mm, high sensitivity is achieved, and the diameter of the inhibition zone of the gentamicin solution on salmonella is 14.48mm, and the gentamicin solution is moderate sensitivity.

TABLE 4 bacteriostatic effect of control group

The diameter of the inhibition zone shows that the crude extract of the walnut green seedcase has an inhibition effect on four food-borne pathogenic bacteria, the inhibition effect of the walnut green seedcase on gram-positive bacteria and gram-negative bacteria is verified to a certain degree, and the walnut green seedcase has a certain potential when being used as a plant-derived natural bacteriostatic agent or a food-grade preservative.

The embodiment of the application provides a method for improving extraction rate and bacteriostatic activity of walnut green husk flavone, wherein the method comprises the following steps: cleaning and airing the walnut green husks until the water yield is lower than 10%; grinding the aired walnut green husks, controlling walnut green husk powder to reach a preset mesh range, and then uniformly packaging; performing irradiation pretreatment on the packaged walnut green husk powder by setting electron beams with different doses; carrying out ultrasonic extraction on the pretreated walnut green husk powder according to a preset condition to obtain a walnut green husk solution; and cooling, centrifuging, concentrating and drying the walnut green husk solution subjected to ultrasonic extraction to obtain the flavone extract in the walnut green husk.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.