Method for extracting konjak flying powder protein by high-voltage discharge assisted eutectic solvent

Abstract

The invention belongs to the technical field of agricultural and sideline food processing, and particularly relates to a method for extracting konjak flying powder protein through a deep-eutectic solvent under the assistance of high-voltage discharge. The preparation method comprises the following steps: mixing konjac flying powder and distilled water, performing high-voltage discharge pretreatment, centrifuging and collecting supernate; mixing and stirring the supernate and a deep eutectic solvent to extract protein of the konjac flying powder, then centrifugally collecting an extracting solution, carrying out alcohol precipitation treatment on the extracting solution, centrifugally collecting precipitate, washing, centrifuging and freeze-drying to obtain konjac flying powder protein; the eutectic solvent is composed of ethylamine hydrochloride and acetamide, and the water content of the eutectic solvent is 20%-40%. According to the method, a high-voltage discharge process and the eutectic solvent are combined, so that mass transfer of protein in the konjac flying powder can be enhanced, the extraction rate of the konjac flying powder protein is effectively increased, reutilization of waste konjac flying powder in konjac production and processing is realized, resource waste is avoided, and the method is environment-friendly and suitable for industrial popularization and application.

Description

Technical Field

[0001] The present application belongs to the technical field of agricultural and sideline food processing, and more specifically, relates to a method for extracting konjac fly starch protein by using a low eutectic solvent assisted by high voltage discharge. Background Art

[0002] Konjac is a traditional Chinese medicinal and edible plant. It belongs to the genus Amorphophallus in the family Araceae. It is rich in glucomannan, protein, starch, alkaloids, trace elements and other nutrients. It is usually processed into konjac flour to improve its economic value. Konjac flour is widely used in food production as a food additive such as thickener, gelling agent and stabilizer due to its low calorie, high dietary fiber, high strength and high viscosity. Konjac fly flour, as the main by-product of konjac flour processing, contains alkaloids and tannin polyphenols, as well as protein and starch polysaccharides. It is a potential plant protein resource. Compared with animal protein foods, foods processed from plant proteins often contain higher levels of fiber and bioactive substances, and have certain lipid-lowering and anti-oxidation effects. However, the edible development and utilization of plant protein in konjac fly flour is low and it is often disposed of as waste.

[0003] Although the alkali dissolution and acid precipitation method is a traditional method for extracting plant protein, it is simple and easy, but it has low targeting and low extraction rate of plant protein, and it also has high solvent consumption and cost, which hinders its widespread promotion and application. Summary of the invention

[0004] In view of the defects of the prior art, the purpose of the present application is to provide a method for extracting konjac fly starch protein with the assistance of a low eutectic solvent using a high-voltage discharge, aiming to solve the technical problems of the prior art such as low extraction rate and high cost of protein from konjac fly starch.

[0005] To achieve the above object, the present application provides a method for extracting konjac fly powder protein by using a high voltage discharge assisted deep eutectic solvent, comprising the following steps:

[0006] S1, konjac powder and distilled water are mixed and pre-treated by high voltage discharge, and the supernatant is collected by centrifugation;

[0007] S2, above-mentioned supernatant and deep eutectic solvent are mixed and stirred to extract the protein of konjak fly powder, then centrifugal collection of extract, above-mentioned extract is centrifugal collection precipitation after alcohol precipitation treatment, then through washing, centrifugation, freeze drying, obtain konjak fly powder protein;

[0008] The above-mentioned low eutectic solvent consists of ethylamine hydrochloride and acetamide, and its water content is 20% to 40%.

[0009] Preferably, in step S1, the energy of the high voltage discharge is 150 kJ / kg to 400 kJ / kg.

[0010] More preferably, the energy of the high voltage discharge is 250 kJ / kg to 350 kJ / kg.

[0011] Preferably, in step S1, the solid-liquid ratio of the konjac fly starch to the distilled water is 1:(5-15).

[0012] Preferably, in step S2, the volume ratio of the supernatant to the low eutectic solvent is 1:(2-8).

[0013] Preferably, in step S2, in the above-mentioned low eutectic solvent, the molar ratio of ethylamine hydrochloride to acetamide is 2:(1-10).

[0014] Preferably, the method for preparing the above-mentioned deep eutectic solvent comprises the following steps:

[0015] The ethylamine hydrochloride and the acetamide are mixed according to a ratio, heated and stirred until they are completely dissolved, thereby obtaining ethylamine hydrochloride-acetamide.

[0016] Preferably, in step S2, the stirring speed is 500 rpm to 800 rpm, and the stirring time is 15 min to 30 min.

[0017] Preferably, in step S2, the extraction temperature is 40°C to 60°C.

[0018] Preferably, in step S2, the specific operation of the alcohol precipitation is: mixing the extract and anhydrous ethanol, and letting it stand for 10 min to 30 min to allow the protein to precipitate; wherein the volume ratio of the extract to anhydrous ethanol is 1:(3-5).

[0019] On the other hand, the method provided in the present application further comprises recovering the low eutectic solvent.

[0020] Preferably, the recovery of the above-mentioned deep eutectic solvent comprises the following steps:

[0021] The extract is subjected to alcohol precipitation treatment, and then centrifuged to collect the supernatant, and then evaporated to remove the anhydrous ethanol, and the low eutectic solvent is recovered.

[0022] In some embodiments, the evaporation temperature is 40°C to 60°C.

[0023] In general, the above technical solutions conceived by this application have the following technical advantages compared with the prior art:

[0024] (1) The method for extracting konjac fly starch protein by high-voltage discharge-assisted low eutectic solvent provided by the present application comprises the following steps: firstly, konjac fly starch and distilled water are mixed and then subjected to high-voltage discharge pretreatment, and the supernatant is collected by centrifugation; the supernatant and the low eutectic solvent are mixed and stirred to extract the protein of konjac fly starch, and then the extract is collected by centrifugation, the extract is subjected to alcohol precipitation treatment and then centrifuged to collect the precipitate, and then washed, centrifuged and freeze-dried to obtain konjac fly starch protein; wherein the low eutectic solvent is composed of ethylamine hydrochloride and acetamide, and its water content is 20% to 40%. The present application combines the high-voltage discharge process with the low eutectic solvent, can enhance the mass transfer of protein in konjac fly starch at ambient temperature, effectively improve the extraction rate of konjac fly starch protein, realize the reuse of konjac fly starch, which is the waste of konjac production and processing, effectively avoid the waste of resources and reduce the pollution to the environment, and is suitable for industrial promotion and application.

[0025] (2) In a preferred embodiment, by regulating the energy of high-voltage discharge and the solid-liquid ratio of konjak fly powder and distilled water, the mass transfer process of protein in konjak fly powder can be accelerated, and the extraction time can be significantly shortened and the protein extraction rate can be improved with minimum energy consumption. On this basis, the supernatant through high-voltage discharge pretreatment is mixed with the deep eutectic solvent composed of ethylamine hydrochloride-acetamide to extract konjak fly powder protein, and the water content of the deep eutectic solvent and the volume ratio of the supernatant and the deep eutectic solvent are limited, the polarity of the system can be increased, the viscosity of the system can be reduced, the interaction of the deep eutectic solvent and the konjak fly powder protein can be strengthened, and the comprehensive effect makes the konjak fly powder protein accelerate dissolution, and the consumption of the solvent can be reduced while the konjak fly powder protein extraction rate is guaranteed, and the cost is effectively saved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow chart of extracting konjac fly flour protein with the assistance of a low eutectic solvent using a high-voltage discharge as provided in the present application;

[0027] Figure 2 is the extraction rate of konjac fly powder protein in Examples 1 and 2 of the present application;

[0028] Figure 3 is the extraction rate of konjac fly powder protein in Examples 1 and 3 of the present application;

[0029] Figure 4 It is the extraction rate of konjac fly powder protein in Example 1 and Comparative Example 3 of the present application. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0031] The present application provides a method for extracting konjac fly powder protein with the aid of a low eutectic solvent using a high voltage discharge, such as Figure 1 As shown, the following steps are included:

[0032] S1, konjac powder and distilled water are mixed and pre-treated by high voltage discharge, and the supernatant is collected by centrifugation;

[0033] S2, above-mentioned supernatant and deep eutectic solvent are mixed and stirred to extract the protein of konjak fly powder, then centrifugal collection of extract, above-mentioned extract is centrifugal collection precipitation after alcohol precipitation treatment, then through washing, centrifugation, freeze drying, obtain konjak fly powder protein;

[0034] The above-mentioned low eutectic solvent consists of ethylamine hydrochloride and acetamide, and its water content is 20% to 40%.

[0035] The inventor found through experiments that after mixing konjac fly powder and distilled water, high-voltage discharge treatment was first performed to promote cell decomposition, thereby enhancing the mass transfer of natural components in konjac fly powder, and then the protein of konjac fly powder was extracted using a low eutectic solvent composed of ethylamine hydrochloride-acetamide, which can effectively improve the extraction rate of protein from konjac fly powder, and the low eutectic solvent used can be recycled, with strong sustainability and environmental friendliness. Further experiments found that the extraction rate of protein from konjac fly powder by the low eutectic solvent ethylamine hydrochloride-acetamide was higher than that of choline chloride-citric acid, choline chloride-lactic acid, choline chloride-glucose and choline chloride-ethylene glycol.

[0036] The water content of the above-mentioned low eutectic solvent in this application is 20% to 40%. Experiments have found that as the water content of the low eutectic solvent system increases, the extraction rate of konjac fly powder protein increases first and then decreases, and the extraction rate is the highest when the water content of the system is 30%. This may be because the increase in water content leads to an increase in the polarity of the mixed system, while Cl - It is easier to be ionized in the low eutectic solvent, the viscosity of the system decreases, and the interaction with konjac fly starch protein is enhanced. The combined effect increases the extraction rate of konjac fly starch protein. But at the same time, with the increase of water content in the low eutectic solvent system, the hydrogen bond interaction between the low eutectic solvent and protein weakens, which may lead to a decrease in the stability and solubility of the protein in the low eutectic solvent, thereby reducing the protein extraction rate.

[0037] In the present application, the term "high-voltage discharge pretreatment" refers to the use of a high-voltage electric field to treat a konjac fly powder sample aqueous solution, destroying its cell tissue through an electrical breakdown phenomenon, thereby promoting the release of intracellular components. In some embodiments, in step S1, the energy of the above-mentioned high-voltage discharge is 150kJ / kg to 400kJ / kg, which can effectively improve the extraction rate of fly powder protein in the konjac fly powder sample. When the energy of the high-voltage discharge is too low, the cell tissue of the konjac fly powder cannot be effectively destroyed, so that the intracellular components cannot be completely released; when the energy of the high-voltage discharge is too high, the cells of the konjac fly powder are completely disintegrated, and other components except protein are also dissolved in large quantities, increasing the difficulty of separating fly powder protein, and free radicals, atomic hydrogen and other oxidizing substances may be produced during the treatment process, thereby reducing the extraction rate of konjac fly powder protein.

[0038] In a preferred embodiment, in step S1, the energy of the high voltage discharge is 250 kJ / kg to 350 kJ / kg.

[0039] In some embodiments, in step S1, the solid-liquid ratio of the konjac fly powder and the distilled water is 1: (5-15), which can maintain low turbulence in the mixed solution during high-voltage discharge pretreatment, and the solvent consumption is small. Experiments have found that as the solid-liquid ratio increases, the effect of high-voltage discharge-assisted extraction of konjac fly powder protein gradually tends to balance; when the solid-liquid ratio is too large, it may cause transitional turbulence, and too much solvent will increase the energy loss of the extraction system and increase the processing cost.

[0040] In some embodiments, in step S2, in the above-mentioned low eutectic solvent, the molar ratio of ethylamine hydrochloride to acetamide is 2:(1-10), and the low eutectic solvent has a suitable viscosity and can promote the mass transfer of various components in konjac fly pollen cells.

[0041] It is to be understood that the present application does not limit the source of the above-mentioned low eutectic solvent, and it can be prepared according to methods known to those skilled in the art. In a specific embodiment of the present application, the preparation method of the above-mentioned low eutectic solvent (ethylamine hydrochloride-acetamide) comprises the following steps:

[0042] The ethylamine hydrochloride and the acetamide are mixed according to a ratio, heated and stirred until they are completely dissolved, thereby obtaining ethylamine hydrochloride-acetamide.

[0043] In some embodiments, in step S2, the volume ratio of the supernatant and the deep eutectic solvent is 1:(2-8), which can accelerate the dissolution of konjac fly starch protein, reduce the amount of solvent while ensuring the extraction rate of konjac fly starch protein, and save costs. Further increasing the amount of the deep eutectic solvent, the extraction rate of konjac fly starch protein is slightly reduced, which may be because, after the solubility of protein in the extract reaches saturation, excessive deep eutectic solvent will further dissolve other compounds in the konjac fly starch, thereby affecting the extraction efficiency of protein.

[0044] In some embodiments, in step S2, the stirring speed is 500 rpm to 800 rpm, and the stirring time is 15 min to 30 min.

[0045] In some embodiments, in step S2, the extraction temperature is 40°C to 60°C, which can reduce the viscosity of the low eutectic solvent system, increase the movement speed of protein molecules, and facilitate the diffusion and dissolution of proteins. At the same time, it will not affect the structural stability of the protein and can ensure the functional characteristics of the protein.

[0046] The present application does not limit the method of controlling the extraction temperature, which may be, but is not limited to, water bath heating.

[0047] In some embodiments, in step S2, the specific operation of the alcohol precipitation is: mixing the above-mentioned extract and anhydrous ethanol, standing for 10 minutes to 30 minutes, allowing the protein to precipitate, and removing excess reagents and interfering compounds, wherein the volume ratio of the extract to anhydrous ethanol can be but is not limited to 1: (3 to 5), as long as the protein can be quickly precipitated, it is within the scope of protection of this application.

[0048] In some embodiments, the washing agent is distilled water, which can remove the remaining anhydrous ethanol and purify the konjac fly starch protein.

[0049] It is understood that the present application does not limit the above centrifugal speed and time, and those skilled in the art can select appropriate centrifugal conditions according to the scale of the konjac fly powder to be processed, which are all within the scope of protection of the present application. In some embodiments, the above centrifugal speed is 8000rpm~10000rpm, and the centrifugal time is 10min~20min.

[0050] In some embodiments, the method further comprises recovering the low eutectic solvent.

[0051] In some embodiments, the recovery of the deep eutectic solvent comprises the following steps:

[0052] The extract is subjected to alcohol precipitation treatment, and then centrifuged to collect the supernatant, and then evaporated to remove the anhydrous ethanol, and the low eutectic solvent is recovered.

[0053] The present application does not limit the specific method of the above evaporation. Those skilled in the art can select a suitable evaporation method according to actual conditions, such as but not limited to rotary evaporation. In some embodiments, the temperature of the above evaporation is 40°C to 60°C. The low eutectic solvent recovered by the above method can be recycled and is sustainable.

[0054] It should be understood that materials with the same or similar types, models, qualities, properties or functions as the reagents and instruments in the following examples can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0055] The following are examples and comparative examples:

[0056] Reagents used in the following examples:

[0057] Konjac fly powder was purchased from Hubei Yizhi Konjac Biotechnology Co., Ltd., ethylamine hydrochloride and acetamide were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., choline chloride was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and citric acid, lactic acid, glucose and ethylene glycol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0058] Example 1

[0059] The method for extracting konjac fly powder protein with the aid of a deep eutectic solvent using a high-voltage discharge provided in this embodiment comprises the following steps:

[0060] S1. High-voltage discharge (HVED) pretreatment: Konjac fly powder and distilled water were mixed at a material-liquid ratio of 1:8 and placed in a generator sample treatment chamber with a high-voltage pulse voltage of 40 kV. The distance between the stainless steel needle and the ground plate electrode was fixed at 1 cm. The generator provided exponential pulses with a pulse frequency of 0.5 Hz and an average energy of 160 J / pulse. The treatment included the application of n consecutive pulses. The energy consumption (W) was calculated as follows:

[0061] W(kJ / kg)=(Wi×n) / m

[0062] Where Wi is the energy consumption per pulse (160 J / pulse), n is the number of pulses applied, and m is the total mass of the sample in the sample processing chamber.

[0063] The initial temperature of the sample mixture was controlled to be 25°C, and then pulses were applied for pretreatment. The energy of the high-voltage discharge in this embodiment was 250 kJ / kg. The pretreatment solution was then centrifuged at 8500 rpm for 10 min, and the supernatant was collected and stored at 4°C for later use.

[0064] S2. Preparation of deep eutectic solvents (DESs): Ethylamine hydrochloride and acetamide were mixed in a molar ratio of 1:1 and placed in a beaker. The mixture was stirred and heated at 80° C. to form a clear homogeneous liquid. The mixture was cooled to room temperature to obtain ethylamine hydrochloride-acetamide with a water content of 30%, i.e., a deep eutectic solvent.

[0065] S3, extracting konjac fly starch protein: the above supernatant and ethylamine hydrochloride-acetamide are mixed in a volume ratio of 1:5, and then stirred at 600 rpm for 20 min at 60° C. to extract the protein of konjac fly starch, and then centrifuged to collect the supernatant, i.e., the extract containing konjac fly starch protein.

[0066] S4. Purification of konjac fly starch protein: The extract was mixed with 4 times the volume of 95% anhydrous ethanol and subjected to alcohol precipitation for 20 min, followed by centrifugation at 10000 rpm for 10 min to obtain a first supernatant and a first precipitate containing konjac fly starch protein, followed by washing the first precipitate with distilled water for 2 times, followed by centrifugation at 10000 rpm for 10 min, and vacuum freeze-drying the second precipitate obtained by centrifugation to obtain konjac fly starch protein.

[0067] S5. Place the first supernatant in a rotary evaporator at a temperature of 50° C. for 20 min to recover the low eutectic solvent.

[0068] Calculate the extraction rate of konjac fly powder protein as follows: the content of protein in the extract containing konjac fly powder protein is standard with BSA, adopts Coomassie brilliant blue method to measure the content of protein in the extract. Configure the BSA standard solution of Xmg / mL (mother liquor), and carry out gradient dilution with distilled water, get 1mL different gradient concentration BSA standard solution and add 5mL Coomassie brilliant blue G-250 dye liquor respectively, shake up and leave standstill 5min, finally measure absorbance under 595nm wavelength, each process repeats 3 times, and the result is averaged. Take BSA standard solution concentration as horizontal coordinate, absorbance is the vertical coordinate drawing standard curve. Get 1mL centrifugal konjac fly powder protein extract, after adopting the same method to handle, measure absorbance at 595nm. Then substitute measured absorbance into the regression equation obtained by standard curve, and calculate the extraction rate of konjac fly powder protein according to the following formula:

[0069] Protein extraction rate (%) = (c × V × N) / (m × 1000) × 100

[0070] Wherein: c is the concentration of protein in the extract containing konjac fly powder protein, mg / mL; V is the volume of the extract, mL; N is the dilution multiple; m is the protein content in the sample, g.

[0071] The extraction rate of konjac fly flour protein is shown in Table 1.

[0072] Example 2

[0073] The method for extracting konjac fly powder protein with the aid of a deep eutectic solvent using a high-voltage discharge provided in this embodiment comprises the following steps:

[0074] S1, after konjac fly powder and distilled water are mixed according to solid-liquid ratio of 1:8, high-voltage discharge pretreatment is carried out according to the method provided in Example 1, wherein the energy of high-voltage discharge is respectively 20kJ / kg, 50kJ / kg, 150kJ / kg, 350kJ / kg, 400kJ / kg, then the pretreatment liquid of each treatment group is centrifuged at 8500rpm for 10min, supernatant is collected, and 4 ℃ are stored for standby use.

[0075] Steps S2, S3 and S4 are the same as those in Example 1.

[0076] The extraction rate of konjac fly powder protein in each treatment group was calculated according to the method provided in Example 1. The results are shown in Figure 2 .

[0077] Example 3

[0078] The method for extracting konjac fly powder protein with the aid of a deep eutectic solvent using a high-voltage discharge provided in this embodiment comprises the following steps:

[0079] S1. Preparation of a low eutectic solvent: ethylamine hydrochloride and acetamide were mixed in a molar ratio of 1:1 and placed in a beaker, heated with stirring at 80° C. to form a clear homogeneous liquid, and cooled to room temperature to obtain ethylamine hydrochloride-acetamide with a water content of 10%, 15%, 20%, 30%, 40%, and 45%, i.e., a low eutectic solvent.

[0080] Steps S2, S3 and S4 are the same as those in Example 1.

[0081] The extraction rate of konjac fly powder protein in each treatment group was calculated according to the method provided in Example 1. The results are shown in Figure 3 .

[0082] Example 4

[0083] The method for extracting konjac fly powder protein with the aid of a deep eutectic solvent using a high-voltage discharge provided in this embodiment comprises the following steps:

[0084] S1. High voltage discharge (HVED) pretreatment: Konjac fly powder and distilled water were mixed at a solid-liquid ratio of 1:5, placed in a high voltage pulse voltage of 40 kV generator for pretreatment, wherein the energy of the high voltage discharge was 350 kJ / kg, and then the pretreated liquid was centrifuged at 8500 rpm for 10 min, the supernatant was collected, and stored at 4°C for later use.

[0085] S2. Preparation of a low eutectic solvent: ethylamine hydrochloride and acetamide were mixed in a molar ratio of 1:5 and placed in a beaker. The mixture was heated at 80° C. with stirring until a clear homogeneous liquid was formed. The mixture was cooled to room temperature to obtain ethylamine hydrochloride-acetamide having a water content of 30% in the system, i.e., a low eutectic solvent.

[0086] S3, extracting konjac fly starch protein: the above supernatant and ethylamine hydrochloride-acetamide are mixed in a volume ratio of 1:2, and then stirred at 500 rpm for 30 min at 60° C. to extract the protein of konjac fly starch, and then centrifuged to collect the supernatant, i.e., the extract containing konjac fly starch protein.

[0087] S4. Purification of konjac fly starch protein: The extract was mixed with 5 volumes of 95% anhydrous ethanol and subjected to alcohol precipitation for 10 min, followed by centrifugation at 8000 rpm for 20 min to obtain a precipitate, followed by washing the precipitate with distilled water for 3 times, followed by centrifugation at 8000 rpm for 20 min, and the precipitate obtained by centrifugation was vacuum freeze-dried to obtain konjac fly starch protein.

[0088] The extraction rate of konjac fly powder protein was calculated according to the method provided in Example 1. The results are shown in Table 1.

[0089] Example 5

[0090] The method for extracting konjac fly powder protein with the aid of a deep eutectic solvent using a high-voltage discharge provided in this embodiment comprises the following steps:

[0091] S1. High voltage discharge (HVED) pretreatment: Konjac fly powder and distilled water were mixed at a solid-liquid ratio of 1:7, placed in a high voltage pulse voltage of 40 kV generator for pretreatment, wherein the energy of the high voltage discharge was 250 kJ / kg, and then the pretreated liquid was centrifuged at 8500 rpm for 10 min, the supernatant was collected, and stored at 4°C for later use.

[0092] S2. Preparation of a low eutectic solvent: ethylamine hydrochloride and acetamide were mixed in a molar ratio of 2:1 and placed in a beaker. The mixture was heated at 80° C. with stirring until a clear homogeneous liquid was formed. The mixture was cooled to room temperature to obtain ethylamine hydrochloride-acetamide having a water content of 20% in the system, i.e., a low eutectic solvent.

[0093] S3. Extracting konjac fly starch protein: The above supernatant and ethylamine hydrochloride-acetamide were mixed in a volume ratio of 1:5, and then stirred at 800 rpm for 15 min at 40° C. to extract the protein from the konjac fly starch, thereby obtaining an extract containing konjac fly starch protein.

[0094] S4. Purification of konjac fly starch protein: The extract was mixed with 4 times the volume of 95% anhydrous ethanol and subjected to alcohol precipitation for 20 min, followed by centrifugation at 8000 rpm for 15 min to obtain a precipitate, followed by washing the precipitate with distilled water for 2 times, followed by centrifugation at 8000 rpm for 15 min, and the precipitate obtained by centrifugation was vacuum freeze-dried to obtain konjac fly starch protein.

[0095] The extraction rate of konjac fly powder protein was calculated according to the method provided in Example 1. The results are shown in Table 1.

[0096] Example 6

[0097] The method for extracting konjac fly powder protein with the aid of a deep eutectic solvent using a high-voltage discharge provided in this embodiment comprises the following steps:

[0098] S1. High voltage discharge (HVED) pretreatment: Konjac fly powder and distilled water were mixed at a solid-liquid ratio of 1:10, placed in a high voltage pulse voltage of 40 kV generator for pretreatment, wherein the energy of the high voltage discharge was 250 kJ / kg, and then the pretreated liquid was centrifuged at 8500 rpm for 10 min, the supernatant was collected, and stored at 4°C for later use.

[0099] S2. Preparation of a low eutectic solvent: ethylamine hydrochloride and acetamide were mixed in a molar ratio of 1:2 and placed in a beaker. The mixture was stirred and heated at 80° C. to form a clear homogeneous liquid. The mixture was cooled to room temperature to obtain ethylamine hydrochloride-acetamide having a water content of 40% in the system, i.e., a low eutectic solvent.

[0100] S3. Extracting konjac fly starch protein: The above supernatant and ethylamine hydrochloride-acetamide were mixed in a volume ratio of 1:8, and then stirred at 800 rpm for 10 min at 50° C. to extract the protein from the konjac fly starch, thereby obtaining an extract containing konjac fly starch protein.

[0101] S4. Purification of konjac fly starch protein: The extract was mixed with 3 volumes of 95% anhydrous ethanol and subjected to alcohol precipitation for 30 min, followed by centrifugation at 10,000 rpm for 10 min to obtain a precipitate, followed by washing the precipitate with distilled water for 3 times, followed by centrifugation at 10,000 rpm for 10 min, and the precipitate obtained by centrifugation was vacuum freeze-dried to obtain konjac fly starch protein.

[0102] The extraction rate of konjac fly powder protein was calculated according to the method provided in Example 1. The results are shown in Table 1.

[0103] Comparative Example 1

[0104] The method for extracting konjac fly starch protein by stirring-assisted deep eutectic solvent provided in this comparative example comprises the following steps:

[0105] S1. Stirring pretreatment: Konjac fly powder and distilled water were mixed at a solid-liquid ratio of 1:8, stirred at 500 rpm for 15 min, and then the pretreated liquid was centrifuged at 8500 rpm for 10 min, the supernatant was collected, and stored at 4° C. for later use.

[0106] Steps S2, S3 and S4 are the same as those in Example 1.

[0107] The extraction rate of konjac fly powder protein was calculated according to the method provided in Example 1. The results are shown in Table 1.

[0108] Comparative Example 2

[0109] The high-voltage discharge-assisted deep eutectic solvent extraction of konjac fly powder protein provided in this comparative example comprises the following steps:

[0110] S1. High voltage discharge (HVED) pretreatment: Konjac fly powder and distilled water were mixed at a solid-liquid ratio of 1:20 and then subjected to high voltage discharge pretreatment according to the method provided in Example 1.

[0111] Steps S2, S3 and S4 are the same as those in Example 1.

[0112] The extraction rate of konjac fly powder protein was calculated according to the method provided in Example 1. The results are shown in Table 1.

[0113] Comparative Example 3

[0114] S1. High voltage discharge (HVED) pretreatment: same as in Example 1.

[0115] S2. Preparation of a low eutectic solvent: choline chloride was mixed with citric acid, lactic acid, glucose and ethylene glycol at a molar ratio of 1:1, respectively, and placed in a beaker, heated with stirring at 80° C. to form a clear homogeneous liquid, and cooled to room temperature to obtain low eutectic solvents of choline chloride-citric acid, choline chloride-lactic acid, choline chloride-glucose and choline chloride-ethylene glycol with a water content of 30%.

[0116] Step S3 and step S4 are the same as in Example 1.

[0117] The extraction rate of konjac fly powder protein in each treatment group was calculated according to the method provided in Example 1. The results are shown in Figure 4 .

[0118] Table 1 Extraction rate of konjac fly powder protein

[0119] Extraction rate of konjac fly powder protein (%) Example 1 80.01±1.81 Example 4 76.17±1.71 Example 5 77.35±0.78 Example 6 76.88±2.03 Comparative Example 1 43.62±2.04 Comparative Example 2 74.43±2.11

[0120] It can be seen from Table 1 that, compared with Comparative Example 1, the embodiment of the present application can effectively improve the extraction rate of konjac fly powder protein by mixing konjac fly powder and distilled water, first subjecting the mixture to high-voltage discharge treatment, and then using a low eutectic solvent composed of ethylamine hydrochloride-acetamide to extract the protein of the konjac fly powder.

[0121] From Table 1 and Figure 2It can be seen that when the energy of high-voltage discharge is too low, when only 20kJ / kg, the extraction rate of konjac fly powder protein is 47.21%, along with the rising of high-voltage discharge energy, the extraction rate of konjac fly powder protein appears the trend of increasing first and then decreasing, this may be because electroporation causes the cell wall and cell membrane of konjac fly powder most to be damaged and destroyed, promotes the dissolving of material composition, thereby improves the extraction rate of fly powder protein.But simultaneously, along with the rising of high-voltage discharge energy, the compound in konjac fly powder excessively decomposes, causes oxidizing substances such as hydroxyl radical, atomic hydrogen and ozone to produce, affects the extraction of konjac fly powder protein, makes the extraction rate of konjac fly powder protein decline.

[0122] From Table 1 and Figure 3 It can be seen that when the water content of the low eutectic solvent ethylamine hydrochloride-acetamide system is 30%, the extraction rate of konjac fly powder protein is the highest. The reason for this may be that when the water content increases from 10% to 30%, the polarity of the mixed system increases, and at the same time, Cl - It is easier to be ionized in the low eutectic solvent, the viscosity of the system decreases, and the interaction with konjac fly starch protein is enhanced. The combined effect increases the extraction rate of konjac fly starch protein. When the water content continues to increase, the hydrogen bond interaction between the low eutectic solvent and the protein is weakened, which may lead to a decrease in the stability and solubility of the protein in the low eutectic solvent, thereby reducing the protein extraction rate.

[0123] Depend on Figure 4 It can be seen that the extraction rate of konjac fly powder protein extracted by using ethylamine hydrochloride-acetamide as the low eutectic solvent in the present application is better than that of choline chloride-citric acid, choline chloride-lactic acid, choline chloride-glucose and choline chloride-ethylene glycol used in Comparative Example 3.

[0124] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A method for extracting konjac fly starch protein using a high voltage discharge assisted deep eutectic solvent, characterized in that: The steps include: S1, konjac powder and distilled water are mixed and pre-treated by high voltage discharge, and the supernatant is collected by centrifugation; S2, described supernatant and deep eutectic solvent are mixed and stirred, to extract the protein of konjac fly powder, then centrifugal collection extract, described extract is centrifugal collection precipitation after alcohol precipitation treatment, then through washing, centrifugation, freeze drying, obtain konjac fly powder protein; The low eutectic solvent consists of ethylamine hydrochloride and acetamide, and its water content is 20% to 40%.

2. The method according to claim 1, characterized in that In step S1, the energy of the high voltage discharge is 150 kJ / kg to 400 kJ / kg.

3. The method according to claim 2, characterized in that The energy of the high-voltage discharge is 250 kJ / kg to 350 kJ / kg.

4. The method according to claim 1, characterized in that: In step S1, the solid-liquid ratio of the konjac fly starch to the distilled water is 1:(5-15).

5. The method according to claim 1, characterized in that In step S2, the volume ratio of the supernatant to the deep eutectic solvent is 1:(2-8).

6. The method according to claim 1, characterized in that In step S2, in the low eutectic solvent, the molar ratio of ethylamine hydrochloride to acetamide is 2:(1-10).

7. The method according to claim 6, characterized in that The preparation method of the deep eutectic solvent comprises the following steps: The ethylamine hydrochloride and the acetamide are mixed according to a ratio, and heated and stirred until they are completely dissolved to obtain ethylamine hydrochloride-acetamide.

8. The method according to claim 1, characterized in that In step S2, the stirring speed is 500 rpm to 800 rpm, and the stirring time is 15 min to 30 min; The extraction temperature is 40°C to 60°C.

9. The method according to claim 1, characterized in that: In step S2, the specific operation of the alcohol precipitation is: mixing the extract and anhydrous ethanol, and standing for 10 minutes to 30 minutes to allow the protein to precipitate; wherein the volume ratio of the extract to anhydrous ethanol is 1:(3-5).

10. The method according to claim 1, characterized in that The method further comprises recovering the deep eutectic solvent, and the recovery of the deep eutectic solvent comprises the following steps: The extract is subjected to alcohol precipitation treatment, and then centrifuged to collect the supernatant, and then evaporated to remove the anhydrous ethanol, and the low eutectic solvent is recovered.

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