Preparation method of eleocharis tuberosa peel active component, eleocharis tuberosa peel polyphenol and polysaccharide and application thereof

Abstract

The invention relates to the technical field of food processing, in particular to a preparation method of active ingredients of eleocharis tuberosa peel, polyphenols and polysaccharides of the eleocharis tuberosa peel and application of the polyphenols and polysaccharides. The preparation method comprises the following steps: drying and dehydrating the eleocharis tuberosa peel, and then carrying out superfine treatment to obtain eleocharis tuberosa peel powder; mixing the powder with a first DES solution, performing ultrasonic treatment for a certain time, and separating to obtain a target polyphenol active component; and mixing the polyphenol-separated raw material with a second DES solution again, carrying out microwave treatment for a certain time, and separating to obtain the target polysaccharide active component. According to the scheme provided by the invention, a specific component deep-eutectic solvent combination is combined with a specific microwave / ultrasonic process, so that the active components of the eleocharis tuberosa peel polyphenol and the eleocharis tuberosa peel polysaccharide can be efficiently extracted. The polyphenol and the polysaccharide have specific active functions, and the active ingredients can prolong the reproduction rate of putrefying bacteria in the baked food under the condition of reducing the dosage of the preservative. According to the scheme, the variety of active substances is enriched, and the water chestnut resource utilization rate is increased.

Description

Technical Field

[0001] The present application relates to the field of food processing technology, and in particular to a method for preparing active ingredients from water chestnut peel, water chestnut peel polyphenols and polysaccharides, and applications thereof. Background Art

[0002] Water chestnuts, also known as water caltrops, are perennial herbs belonging to the genus Water Chestnut in the Cyperaceae family. They are an important crop in southern China, particularly in Guangdong and Guangxi. Nutritionally, water chestnuts contain flavonoids, polyphenols, polysaccharides, sterols, and terrestrins, as well as vitamin C, calcium, phosphorus, iron, and other beneficial trace elements. Due to their low calorie content and high fiber content, they are often recommended as part of a healthy diet.

[0003] In addition, water chestnuts have certain medicinal value and are believed in traditional Chinese medicine to have diuretic, heat-clearing and detoxifying effects. According to statistics, the annual production of water chestnuts in China exceeds 1 million tons, and water chestnut peels account for approximately 20% of the weight of the water chestnut corm. Water chestnut peels are a byproduct of water chestnut processing and are usually discarded or used as feed. However, water chestnut peels contain brown pigments, flavonoids, polyphenols, polysaccharides, and dietary fiber. Polyphenols are a class of compounds in plant metabolites that are widely found in fruits, vegetables, and other plants. They include flavonoids, phenolic acids, and tannins. Due to their various biological activities, such as antioxidant, antibacterial, anti-inflammatory, and anti-tumor properties, they have been widely used to prevent cardiovascular disease, reduce pituitary blood sugar, oral infections, lower blood pressure, and protect nerves. They are known as the "seventh nutrient" for health. Clearly, current processing methods result in a waste of water chestnut peel resources.

[0004] In summary, water chestnuts are an important edible resource. However, the water chestnut peel, which is rich in polyphenols and polysaccharides, is often treated as waste, resulting in a significant waste of natural byproduct resources. Therefore, seeking a process for efficiently isolating the active ingredients in water chestnut peel is an important means to improve its utilization and increase the added value of the water chestnut industry.

[0005] In recent years, the recycling and high-value utilization of plant resource waste has become a research hotspot. Extracting bioactive components from plant biomass for use in food additives or nutritional supplements is a new resource development direction. Sustainable and environmentally friendly extraction technologies are essential for the development of green chemistry and the realization of sustainable human development. Within this context, green extraction technologies have become a key option for the sustainable utilization of plant raw materials.

[0006] Solvent extraction is a commonly used extraction technique in the field to extract bioactive components from plant biomass. Existing methods often use alcoholic solvents (such as ethanol) to extract substances from plant biomass, or alternatively, use deep eutectic solvents (DES) for optimal extraction, such as the extraction of bioactive compounds such as anthocyanins and carotenoids. However, if traditional alcoholic solvents (such as ethanol) are directly used for maceration extraction of water chestnut peels, the extraction yield of polyphenols and polysaccharides is low, and the extracted polyphenols have poor antibacterial activity, while the polysaccharides have poor enzyme inhibitory activity. Therefore, alternative extraction process routes are worth considering. Because deep eutectic solvents achieve different effects through the synergistic interaction of their components, different deep eutectic solvents are suitable for different raw materials and target products. Therefore, finding an effective combination of solvent components, combined with an optimized extraction process design, to impart superior performance to DES for the efficient extraction of water chestnut polysaccharides and polyphenols is a key area of ​​research and development in this field. However, there are currently no reports on the application of DES in the extraction of active ingredients from water chestnut peels, nor is there any disclosure on what combination of low eutectic solvents and optimized extraction process design can significantly improve the extraction effect. Summary of the Invention

[0007] To solve the problems mentioned in the prior art mentioned in the above background technology, this application provides the following technical solutions: The present application provides a method for preparing an active ingredient from water chestnut peel, which comprises the following steps: Ultrafine treatment of water chestnut peel: drying the water chestnut peel, crushing and sieving, and then ultrafine treatment to obtain water chestnut peel powder; Ultrasonic-assisted DES extraction of water chestnut polyphenols: prepare a first DES solution and dilute it with or without water to reduce its viscosity; add the water chestnut peel powder to the first DES solution and ultrasonically treat it at 40-50°C for 30-60 minutes to obtain a mixture; separate the mixture into solid and liquid, collect the supernatant, place it in an ethanol solution for precipitation, and then separate the solid and liquid and dry the precipitate to obtain water chestnut peel polyphenols and raw material residues; wherein the components of the first DES solution include choline chloride and a first other solvent, and the molar ratio of the choline chloride to the first other solvent is 1:(1-3); the first other solvent is one or more combinations of 1,4-butanediol, 1,2-propylene glycol, malic acid, lactic acid, and citric acid; the ultrasonic treatment conditions are single frequency 20KHz, single frequency 60KHz, single frequency 80KHz, dual frequency 20 / 60KHz, dual frequency 60 / 80KHz or dual frequency 20 / 80 KHz, and its power is 200~600W.

[0008] In some embodiments, the following preparation steps are also included: microwave-assisted DES extraction of water chestnut polysaccharide: mixing the raw material residue after separation of polyphenols with a second DES solution, diluting the second DES solution with or without water to reduce its viscosity; then extracting under microwave conditions for 30 to 60 minutes; then separating the microwave-treated mixture into solid and liquid, collecting the supernatant and placing it in an ethanol solution for precipitation, and then solid-liquid separation and drying the precipitate to obtain water chestnut polysaccharide; wherein the components of the second DES solution include choline chloride and a second other solvent, and the molar ratio of choline chloride to the second other solvent is 1: (1 to 3); the other solvent is one or more combinations of ethylene glycol, glycerol, urea, and 1,6-hexanediol; the conditions of the microwave treatment are: power 250 to 550 W, extraction temperature 70 to 100°C.

[0009] In some embodiments, when the first DES solution is diluted with water, the first DES solution is diluted with water to obtain a first DES dilution, wherein the amount of water used is 10-20% of the mass of the first DES solution; In some embodiments, the first other solvent is 1,4-butanediol and citric acid; in the first DES solution, the molar ratio of choline chloride, 1,4-butanediol and citric acid is: 1:1:(0.5-1); and the ultrasonic treatment condition is dual frequency 20 / 80 KHz.

[0010] In some embodiments, when the second DES solution is diluted with water, the second DES solution is diluted with water to obtain a second DES dilution solution, wherein the amount of water used is 10-20% of the mass of the second DES solution.

[0011] In some embodiments, the second other solvent is 1,6-hexanediol and urea; in the second DES solution, the molar ratio of choline chloride, 1,6-hexanediol and urea is: 1:1:(0.5-1); the microwave treatment conditions are: power 400-550W, extraction temperature 70-100°C.

[0012] In some embodiments, the microwave treatment conditions are: power 550W, extraction temperature 80°C.

[0013] In some embodiments, during the ultrafine treatment of the water chestnut peel, the water chestnut peel is dried and then crushed and sieved; the coarsely crushed water chestnut peel is then ground and ultrafinely processed by heavy-pressure grinding; the ultrafinely processed water chestnut peel powder is then subjected to air flow pulverization and air flow classification to obtain water chestnut peel powder.

[0014] In some embodiments, in the step of ultrasonic-assisted DES extraction of water chestnut polyphenols, choline chloride and a first other solvent are mixed, heated and stirred at 60-80°C until the liquid mixture is uniform and transparent, thereby obtaining a first DES solution; when the first DES solution is diluted with water, the mass ratio of the first DES dilution to the water chestnut peel powder is (15-30):1; when the first DES solution is not diluted with water, the mass ratio of the first DES solution to the water chestnut peel powder is (15-30):1; and the ethanol used in the precipitation process is ethanol at 4-10°C.

[0015] In some embodiments, in the microwave-assisted DES extraction step of water chestnut polysaccharide, when the second DES solution is diluted with water, the mass ratio of the second DES dilution to the raw material residue is (10-20):1; when the second DES solution is not diluted with water, the mass ratio of the second DES solution to the raw material residue is (10-20):1; and the ethanol used in the precipitation process is ethanol at 4-10°C.

[0016] In some embodiments, during the ultrafine treatment of the water chestnut peel, the water chestnut peel is dried and then crushed and sieved, and then the coarsely crushed water chestnut skin is ground at 1000-1500 r / min for 10-30 min to perform heavy pressure grinding ultrafine treatment, and then the ultrafine water chestnut peel powder is passed through an air flow mill at a pressure of 0.1-0.3 MPa and a material processing capacity of 2-5 g / h for air flow classification to finally obtain a water chestnut peel powder sample.

[0017] The present application also provides a water chestnut peel polyphenol, which is prepared using the preparation method described above.

[0018] The present application also provides a water chestnut peel polysaccharide, which is prepared using the preparation method described above.

[0019] The present application also provides the use of the water chestnut peel polyphenols and / or water chestnut peel polysaccharides as described above in food.

[0020] Based on the above, compared with the existing technology, the preparation method of the active ingredient of water chestnut peel provided by this application has the following technical effects: This application utilizes a specific deep eutectic solvent combination combined with a specific microwave / ultrasound process to efficiently extract the active ingredients, water chestnut peel polyphenols and polysaccharides. The water chestnut peel polyphenols and polysaccharides extracted using this application exhibit specific active functions. Water chestnut peel polyphenols inhibit Trichophyton rubrum, while water chestnut peel polysaccharides promote the proliferation of probiotics and exhibit significant inhibitory effects on angiotensin-converting enzyme, α-amylase, and α-glucosidase. Furthermore, these active ingredients can prolong the growth rate of spoilage bacteria in baked goods, particularly bread, while reducing the dosage of preservatives. This application enriches the variety of active substances and improves the resource utilization of water chestnuts.

[0021] Other features and benefits of the present application will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present application. The purpose and other benefits of the present application can be achieved and obtained through the contents shown in the description, claims, and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the polyphenol activity test results of the polyphenol samples prepared in the experimental group; Figure 2 This is a graph showing the test results of the polysaccharide samples prepared in the experimental group on their promoting effect on the proliferation of probiotics.

[0023] Figure 3 This is a graph showing the test results of the polysaccharide samples prepared in the experimental group inhibiting enzyme activity. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments; the technical features designed in different implementation modes of the present application described below can be combined with each other as long as they do not conflict with each other; based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0025] In the description of this application, it should be noted that all terms used in this application (including technical terms and scientific terms) have the same meanings as those commonly understood by ordinary technicians in the field to which this application belongs, and should not be understood as limiting this application; it should be further understood that the terms used in this application should be understood to have meanings consistent with the meanings of these terms in the context of this specification and the relevant field, and should not be understood in an idealized or overly formal sense, unless explicitly defined as such in this application.

[0026] This application provides the following experiments to verify the effectiveness of this application solution: 1. Experimental process: Exploring the effect of DES (Deep Eutectic Solvents, DES) type on polyphenol extraction efficiency (1) Ultrafine treatment of water chestnut peel The water chestnut peel was dried, crushed and sieved, and then the coarsely crushed water chestnut peel was ground at 1250 r / min for 20 min and subjected to heavy pressure grinding ultrafine grinding. The ultrafine water chestnut peel powder was then passed through an air flow mill at a pressure of 0.2 MPa and a material handling capacity of 3 g / h for air flow classification to obtain a water chestnut peel powder sample. (2) Ultrasound-assisted DES extraction of water chestnut polyphenols As shown in Table 1, choline chloride and one or more of 1,4-butanediol, 1,2-propylene glycol, malic acid, lactic acid, and citric acid were mixed in a specific molar ratio (1:1-3). The mixture was heated and stirred at 70°C until the liquid mixture became homogeneous and transparent to obtain DES. The DES was diluted with or without water (15% by weight of the DES solution) to reduce the viscosity. Ultrafinely ground water chestnut peel powder was added to a 25-fold volume of the DES solution and ultrasonicated at 45°C (single frequency 60 kHz, power 400 W) for 45 minutes. After this extraction step, the mixture was centrifuged, and the supernatant was collected, placed in ethanol at 4°C for precipitation, and centrifuged and dried to obtain water chestnut peel polyphenols.

[0027] The extraction rate of the polyphenol samples prepared above was tested, and the results are shown in Table 1: Table 1: Effect of DES type on polyphenol content

[0028] Extraction rate characterization: The results in Table 1 demonstrate significant differences in the extraction efficiency of water chestnut peel polyphenols using DES with different compositions. The best polyphenol extraction efficiency was achieved when the DES composition was choline chloride: 1,4-butanediol: citric acid = 1:1:1 or choline chloride: 1,4-butanediol: citric acid = 1:1:0.5. The optimal ratio was choline chloride: 1,4-butanediol: citric acid = 1:1:1. Introducing an appropriate amount of water into the DES also increased the polyphenol extraction efficiency, as the presence of water promotes the exudation of water-soluble polyphenols. Furthermore, compared to traditional DES extraction methods, the ultrasound-assisted DES extraction employed in this application demonstrated superior extraction efficiency, likely due to the broad polarity of DES, which allows the solvent to interact with phenolic compounds and form intermolecular hydrogen bonds to maintain stability.

[0029] Exploring the effect of ultrasonic treatment on polyphenol extraction rate (1) Ultrafine treatment of water chestnut peel The water chestnut peel was dried, crushed and sieved, and then the coarsely crushed water chestnut peel was ground at 1000 r / min for 30 min, and then ultrafine-mixed by heavy-pressure grinding. The ultrafine-mixed water chestnut peel powder was then passed through an airflow mill at a pressure of 0.3 MPa and a material handling capacity of 3 g / h for airflow classification to obtain the water chestnut peel powder sample. (2) Ultrasound-assisted DES extraction of water chestnut polyphenols Choline chloride, 1,4-butanediol, and citric acid were mixed in a specific molar ratio (1:1:1) and heated and stirred at 60°C until the liquid mixture became homogeneous and transparent to obtain DES. The DES was diluted with 15% water by weight to reduce the viscosity. Ultrafinely ground water chestnut peel powder was then ultrasonicated at 50°C (single frequency 20 kHz, 60 kHz, 80 kHz, dual frequency 20 / 60 kHz, dual frequency 60 / 80 kHz, or dual frequency 20 / 80 kHz; power 400 W) for 30–60 min with 20 times the weight of the DES solution added. Following this extraction step, the mixture was centrifuged, and the supernatant was collected, placed in an ethanol solution at 4°C for precipitation, and then centrifuged and dried to obtain water chestnut peel polyphenols.

[0030] The extraction rate of the polyphenol samples prepared above was tested, and the results are shown in Table 2: Table 2: Effect of ultrasonic treatment on the content of polyphenols

[0031] Extraction rate characterization: At a certain power, different ultrasonic frequencies showed significant differences in the extraction of polyphenols from water chestnut peel. Ultrasonic frequency was proportional to the polyphenol content in the peel, and higher ultrasonic frequencies significantly shortened the extraction time. This may be because, at a certain power, the thermal, mechanical, and cavitation effects generated by high-frequency ultrasonic treatment can help disrupt the cellular structure of the peel and promote the precipitation of polyphenols from the raw water chestnut peel.

[0032] Experimental results indicate that, under the specific polyphenol extraction conditions of this protocol (specific raw materials, specific solvent system, and specific desired extract), dual-frequency ultrasound is more beneficial for promoting polyphenol exudation than single-frequency ultrasound. The mechanism of action may be that, under the specific polyphenol extraction conditions of this protocol, multi-frequency ultrasound with a specific frequency combination significantly enhances cavitation activity and the cavitation effect compared to single-frequency ultrasound. Furthermore, multi-frequency ultrasound exerts a stronger interference effect on the medium, disrupting the continuity of the medium surface and leading to the generation of more cavitation nuclei. This stronger disturbance may significantly enhance bubble-to-bubble interactions in the acoustic field through secondary Bjerknes forces. Furthermore, due to nonlinear interactions, a combined-frequency acoustic field is formed, generating a wider range of cavitation in the sample solution than single-frequency acoustic cavitation.

[0033] Exploring the effect of DES type on polysaccharide extraction rate (1) Ultrafine treatment of water chestnut peel The water chestnut peel was dried, crushed and sieved, and then the coarsely crushed water chestnut peel was ground at 1500 r / min for 10 min and subjected to heavy pressure grinding ultrafine grinding. The ultrafine water chestnut peel powder was then passed through an air flow mill at a pressure of 0.3 MPa and a material handling capacity of 5 g / h for air flow classification to obtain the water chestnut peel powder sample. (2) Ultrasound-assisted DES extraction of water chestnut polyphenols Choline chloride, 1,4-butanediol, and citric acid were mixed in a molar ratio of 1:1:1 and heated at 60°C with stirring until the liquid mixture became homogeneous and transparent to obtain DES. The DES was diluted with 15% water to reduce viscosity. A sample of ultrafine water chestnut peel powder was then ultrasonicated (dual frequency 20 / 80 kHz, power 400 W) at 50°C for 45 minutes with 20 times the weight of the DES solution. Following this extraction step, the mixture was centrifuged, and the supernatant was collected and stored at 4°C for precipitation.

[0034] (3) Microwave-assisted DES extraction of water chestnut polysaccharides The raw material after polyphenol separation was mixed with DES (DES, obtained by mixing choline chloride with one or more of ethylene glycol, glycerol, urea, and 1,6-hexanediol in a molar ratio of 1:1 to 1:3) at a solid-to-liquid mass ratio of 1:15. The DES was diluted with or without water (20% by mass of the DES) to reduce viscosity. The mixture was then extracted at a microwave power of 400 W and a temperature of 80°C for 45 minutes. The resulting mixture was centrifuged, and two volumes of anhydrous chilled ethanol were added to the supernatant. The mixture was incubated at 4°C for 12 hours, then centrifuged, and the resulting precipitate was dried to obtain water chestnut polysaccharide.

[0035] The extraction rate of the polysaccharide sample prepared above was tested, and the results are shown in Table 3: Table 3: Effect of DES type on polysaccharide content

[0036] Different DES components had a significant effect on the extraction rate of water chestnut peel polysaccharides. When choline chloride: 1,6-hexanediol: urea = 1:1:1 or choline chloride: 1,6-hexanediol: urea = 1:1:0.5 was selected, the extraction rate of polysaccharides was significantly improved. Among them, choline chloride: 1,6-hexanediol: urea = 1:1:1 reached the optimal value, indicating that water chestnut peel treatment under these conditions is more conducive to the precipitation of water chestnut peel polysaccharides.

[0037] Ionic liquids (DES) have been recognized as suitable solvents / cosolvents / mediums for microwave-assisted extraction due to their ability to effectively and efficiently absorb microwave radiation. Ionic liquids can accelerate the extraction process due to their excellent solvation and dielectric properties, as well as their surface activity, which allows for enhanced interaction with water molecules. Furthermore, the microenvironment provided by the three DSA components specifically selected in this protocol imparts excellent polysaccharide adsorption. Furthermore, the addition of water further improved the extraction of water chestnut peel polysaccharides. This may be due to the reduced viscosity of the eutectic solvent in the experimental group, which improved fluidity and enhanced mass transfer between the solvent and the polysaccharide.

[0038] Exploring the effect of microwave treatment on polysaccharide extraction rate (1) Ultrafine treatment of water chestnut peel The water chestnut peel was dried, crushed and sieved, and then the coarsely crushed water chestnut peel was ground at 1250 r / min for 20 min and subjected to heavy pressure grinding ultrafine grinding. The ultrafine water chestnut peel powder was then passed through an air flow mill at a pressure of 0.3 MPa and a material handling capacity of 5 g / h for air flow classification to obtain the water chestnut peel powder sample. (2) Ultrasound-assisted DES extraction of water chestnut polyphenols Choline chloride, 1,4-butanediol, and citric acid were mixed in a specific molar ratio (1:1:1) and heated and stirred at 60°C until the liquid mixture became homogeneous and transparent to obtain DES. The DES was diluted with 15% water to reduce viscosity. Ultrafinely processed water chestnut peel powder was then added to a 20-fold volume of DES solution and sonicated at 50°C (dual frequency 20 / 80 kHz; power 400 W) for 45 minutes. Following this extraction step, the mixture was centrifuged, and the supernatant was collected and stored at 4°C for precipitation.

[0039] (3) Microwave-assisted DES extraction of water chestnut polysaccharides The raw material after polyphenol separation was mixed with DES (choline chloride mixed with ethylene glycol, 1,6-hexanediol, and urea in a 1:1:1 molar ratio) at a solid-to-liquid mass ratio of 1:15. The DES was diluted with 20% water to reduce viscosity. The extract was then extracted for various times (30, 45, and 60 min) under microwave power conditions (250 W, 400 W, and 550 W) and temperatures (70, 80, and 100°C). The resulting mixture was centrifuged, and two volumes of chilled anhydrous ethanol were added to the supernatant. The mixture was incubated at 4°C for 24 hours and then centrifuged. The resulting precipitate was dried to obtain water chestnut polysaccharide.

[0040] The extraction rate of the polysaccharide sample prepared above was tested, and the results are shown in Table 4: Table 4 Effect of microwave on polysaccharide extraction rate

[0041] The results in Table 4 show that: Microwave-assisted extraction can effectively improve the extraction efficiency of polysaccharides. This may accelerate the release of the target compound, water chestnut peel polysaccharides, through the following mechanism: Compared with traditional pyrolysis, microwave treatment can generate high temperatures in a shorter time. Microwave pyrolysis is more prone to gas production, which can rupture water chestnut peel cells. Microwave-induced increases in the internal vapor pressure of the peel material effectively disrupt the cell wall structure, leading to better precipitation of water chestnut peel polysaccharides. Specifically, for the specific polysaccharide extraction conditions of this protocol (specific raw materials, specific DES solvent system, and specific desired extract), microwave extraction at a power of 400-550W and an extraction temperature of 70-100°C is the preferred method, with 550W and 80°C being the most optimal.

[0042] The samples prepared above were subjected to the following performance characterizations, and the results are shown below: (1) Activity of polyphenols Different polyphenols prepared in different experimental groups exhibited varying antibacterial abilities. Among them, the polyphenols produced in Experimental Groups 1-1 through 1-14 and Experimental Groups 2-1 through 2-9 all exhibited some antibacterial activity against Trichophyton rubrum, with varying antibacterial activity across different experimental groups. Polyphenols produced in Experimental Group 1-11 (composed of choline chloride: 1,4-butanediol: citric acid = 1:1:1), using ultrasound-assisted optimization of a specific DES, exhibited the best antibacterial activity against Trichophyton rubrum.

[0043] like Figure 1 The antibacterial effects of polyphenols in the extracts against Trichophyton rubrum are shown in Figure 1. A to F represent the inhibition zones obtained by adding polyphenols to experimental groups 1-11 (choline chloride: 1,4-butanediol: citric acid = 1:1:1, diluted with water), experimental groups 1-2 (choline chloride: 1,4-butanediol = 1:3, diluted with water), experimental groups 1-8 (choline chloride: lactic acid = 1:3, diluted with water), comparative experimental group 1-15 (70% ethanol, diluted with water), experimental group 2-1 (ultrasonic extraction at 20 kHz for 60 minutes) in Table 2, and the pure water control group (the same mass of pure water as the polyphenols added in the other antibacterial experiments). This indicates that experimental groups 1-11 exhibit the best antibacterial effect against Trichophyton rubrum.

[0044] (2) Polysaccharides promote the proliferation of probiotics Different doses of water chestnut peel polysaccharides from the experimental groups were added to ice cream containing a certain amount of lactic acid bacteria. After incubation at -25°C for 7 days, the effects of the polysaccharides on the protective ability of lactic acid bacteria during low-temperature storage were observed. The polysaccharides produced in Experimental Groups 3-1 through 3-10 and Experimental Groups 4-1 through 4-9 all improved the freezing tolerance of Streptococcus lactis, with varying degrees of effectiveness across the experimental groups.

[0045] Figure 2 The results showed that adding a certain dose of water chestnut peel polysaccharide can promote the proliferation of Streptococcus lactis. Figure 2 (A) is the control group without adding water chestnut peel polysaccharide. Figure 2 (B) Add 0.2% of the ice cream mass of water chestnut peel polysaccharide (experimental groups 4-7), Figure 2 (C) 0.5% of the weight of ice cream was added with water chestnut peel polysaccharide (experimental groups 4-7). It can be seen that the extracted water chestnut peel polysaccharide has the effect of improving the freezing tolerance of probiotics during the freezing process.

[0046] (3) Polysaccharide inhibition of enzyme activity Polysaccharides extracted using different extraction methods exhibited different functional activities. The polysaccharides produced from Experimental Groups 3-1 to 3-10, and Experimental Groups 4-1 to 4-9, all promoted the proliferation of Streptococcus lactis, but the abilities of the products from different experimental groups varied. This suggests that the polysaccharide structures extracted under different extraction conditions differ.

[0047] like Figure 3 As shown, A, B, and C represent the polysaccharides extracted under the conditions of experimental group 4-3 (250W, 70℃, 30 min), experimental group 4-7 (550W, 80℃, 60 min), and comparative experimental group (0W, 100℃, 60 min) in Table 4, respectively. D represents the polysaccharide extracted from comparative experimental group 3-11 (70% ethanol) in Table 3.

[0048] according to Figure 3 The results showed that sample B had the best ACE inhibition, but its α-amylase and α-glucosidase inhibition rates were lower than those of sample A (i.e., experimental group 4-3). However, the polysaccharide obtained by microwave-assisted DES extraction had a better inhibitory effect on the three enzymes than the polysaccharide obtained without microwave or DES extraction.

[0049] (4) Application of water chestnut peel polyphenols in baked goods As shown in Table 5, high-gluten flour (300g), water (150g), table salt (6g), and yeast (9g), with or without potassium sorbate or water chestnut peel polyphenols, were thoroughly mixed to form a dough. The dough was then proofed for 30 minutes, shaped, and fermented again for 30 minutes before baking. The upper heat temperature was 220°C and the lower heat temperature was 200°C. The bread was sealed and packaged and observed for spoilage.

[0050] Table 5 Microbial growth of bread (stored at 25°C for 30 days)

[0051] Potassium sorbate can effectively extend the shelf life of food and reduce food waste; during storage and transportation, it can prevent food from spoiling due to microbial contamination and ensure food quality; by reducing the risk of microbial contamination, it may have adverse effects on certain groups of people, such as people who are allergic to potassium sorbate may develop skin problems; excessive intake may place a burden on liver and kidney function.

[0052] Therefore, the development of new natural antibacterial raw materials is crucial. Adding 0.3% water chestnut peel polyphenols can reduce the amount of preservatives added and inhibit the proliferation of harmful microorganisms. It can be used as a food additive.

[0053] In summary, the solution provided in this application has at least the following design concepts and beneficial effects: 1. This application utilizes a specific deep eutectic solvent combination with a specific microwave / ultrasound process to efficiently extract the active ingredients water chestnut peel polyphenols and polysaccharides. These polyphenols and polysaccharides possess specific active functions, and the active ingredients can prolong the growth rate of spoilage bacteria in baked goods while reducing the dosage of preservatives. This application enriches the variety of active substances and improves the utilization rate of water chestnut resources. Its design concept is: Water chestnuts are an important edible resource, yet the peels, rich in polyphenols and polysaccharides, are often treated as waste, resulting in a significant waste of natural byproduct resources. Using traditional alcoholic solvents (such as ethanol) for direct immersion extraction of water chestnut peels results in low polyphenol and polysaccharide extraction rates, poor antibacterial activity of the polyphenols, and poor enzyme inhibitory activity of the polysaccharides.

[0054] Deep eutectic solvents (DES) are ionic liquids composed of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) that form a supramolecular network through intermolecular forces. As a more biocompatible and effective alternative to traditional solvents, they have attracted considerable attention due to their environmentally friendly, designable, and low-cost advantages. DES are typically liquids composed of two or three compounds. These compounds self-associate through hydrogen bonds, forming a eutectic mixture with a melting point lower than that of each individual component. For example, in a DES composed of choline chloride and urea, the melting points of choline chloride and urea are 302°C and 133°C, respectively. When these two components are mixed, the mixture melts below 60°C, and the freezing point depression occurs at 12°C, a temperature known as the "eutectic temperature." Generally speaking, the performance of a DES is fundamentally determined by the structure of its hydrogen bond network. Each additional component introduces new hydrogen bonding competition.

[0055] When attempting to optimize multiple performance parameters simultaneously by adding components, it is the synergistic effect between the components, rather than a simple sum, that truly determines the performance of the DES. Although DES is widely used in the extraction of bioactive compounds such as anthocyanins and carotenoids, enhancing drug solubility and its delivery, there is currently a problem. Since deep eutectic solvents achieve different effects through the synergistic coordination of their components, different raw materials and target products are suitable for different deep eutectic solvents. That is, the same deep eutectic solvent is used for different raw materials and target products, and the extraction effect is different. In other words, it is necessary to find an effective combination of specific solvent components to help give DES more superior performance for the efficient extraction of water chestnut polysaccharides and polyphenols.

[0056] In addition, the extraction method is not limited to solvent selection. The type of extraction process and parameter design also affect the extraction effect and the type and activity of the extracted product. Therefore, it is necessary to explore and optimize the use of a combination of low-melting solvents with specific components combined with specific processes to comprehensively improve the extraction effect through coordination of various aspects.

[0057] The core designs of the two steps of polyphenol and polysaccharide extraction in the present application scheme are: a specific component deep eutectic solvent combination, and a specific parameter microwave process or specific parameter ultrasonic process that matches the specific deep eutectic solvent combination. These core designs are indispensable. For the target product polyphenol product, a specific component deep eutectic solvent combination is designed to match the specific parameter ultrasonic process. For the target product polysaccharide product, another specific component deep eutectic solvent combination is designed to match the specific parameter microwave process. All aspects work synergistically to achieve the desired goal, and none of them can be missing. If the specific component deep eutectic solvent combination is not matched with the specific parameter ultrasonic process (no ultrasonic treatment or the ultrasonic process is not consistent), it is difficult to achieve the desired effect of the present application. If the other specific component deep eutectic solvent combination is not matched with the specific parameter microwave process (no microwave treatment or the microwave process is not consistent), it is difficult to achieve the desired effect of the present application.

[0058] In summary, this application utilizes a combination of a specific deep eutectic solvent and a microwave / ultrasonic process designed with specific parameters to efficiently extract the active ingredients, water chestnut peel polyphenols and polysaccharides. These polyphenols and polysaccharides possess specific active functions, and the active ingredients can prolong the growth rate of spoilage bacteria in baked goods while reducing the dosage of preservatives. This application improves the utilization rate of water chestnut resources and enriches the resource base of polysaccharides and polyphenols.

[0059] 2. Based on the above, this application further optimizes the above-mentioned schemes of matching the deep eutectic solvent combination of specific components with the specific parameter ultrasonic process design, and matching the deep eutectic solvent combination of another specific component with the specific parameter microwave process design: (1) Optimization of polyphenol extraction steps: The deep eutectic solvent combination used in this step is diluted with some water to further improve the extraction effect; The optimal deep eutectic solvent combination was a mixture of choline chloride, 1,4-butanediol, and citric acid in a molar ratio of 1:1:(0.5-1), and a dual-frequency ultrasonic treatment of 20 / 80 kHz. This optimized design further improved extraction efficiency and product activity.

[0060] Among them, for the optimization of polysaccharide extraction step: The deep eutectic solvent combination used in this step is diluted with some water to further improve the extraction effect; For the deep eutectic solvent combination, a design combining choline chloride, 1,6-hexanediol, and urea in a molar ratio of 1:1:(0.5-1) was the preferred choice. The microwave treatment conditions of 400-550W power and 70-100°C extraction temperature were also preferred, with 550W power and 80°C extraction temperature being optimal. This optimized design further improved extraction efficiency and product activity.

[0061] In addition, the indicator determination method used in this article is: 1: Content of total phenolic compounds The total phenolic compound content was determined using the Folin-Ciocalteu method modified according to the description of Barbieri et al. The results were based on the gallic acid analysis curve (y = 0.0007x−0.0121, R 2 =0.993) and expressed as mg gallic acid equivalent (GAE) per g sample (wet basis).

[0062] The source of the description by Barbieri et al. is: Barbieri, JB, Goltz, C., Cavalheiro, FB, Toci, AT, Igarashi-Mafra, L., & Mafra, MR (2020). Deep eutecticsolvents applied in the extraction and stabilization of rosemary (Rosmarinusofficinalis L.) phenolic compounds. Industrial Crops and Products , 144 , 112049. 2: Extraction rate of polysaccharides Referring to the method of Zhang et al., the polysaccharide content in the dried water chestnut polysaccharide was measured by the phenol-sulfuric acid method using D-glucose as a standard substance.

[0063] Extraction rate = 100% * polysaccharide content / sample content; The method by Zhang et al. is available from: Zhang, L., & Wang, M. (2017). Optimization of deep eutectic solvent-based ultrasound-assisted extraction of polysaccharides from Dioscorea opposita Thunb. International Journal of Biology Macromolecules , 95 , 675-681. 3: α-amylase and α-glucosidase inhibition rate α-Amylase (AMY) and α-glucosidase inhibition assays (GLU) were performed according to the method described by Zhang et al., with appropriate modifications. α-Amylase or α-glucosidase was dissolved in phosphate-buffered saline (PBS, 0.067 mol / L, pH 6.8). 40 μL of sample solution (4–50 μg / mL) and 40 μL of α-amylase or α-glucosidase solution (0.2 U / mL) were then added to the wells of a 96-well plate. The plate was incubated at 37°C for 15 min, followed by the addition of pNPG (8 mmol / L). After an additional 30 min of incubation at 37°C, the reaction was terminated by the addition of 1 mL of Na2CO3 (1 mol / L). The absorbance (OD) of the mixture was measured at 405 nm. Each sample was tested in triplicate. Inhibition was calculated based on the relationship between inhibition rate and sample concentration as follows: Inhibition rate (%) = (1-(OD1-OD2) / OD3-OD4)*100% Where OD1, OD2, OD3, and OD4 are the absorbances of the sample (enzyme and inhibitor), sample blank (PBS and inhibitor), control (enzyme and PBS), and control blank (PBS), respectively. Plot the inhibition values ​​and half-inhibitory concentration (IC50) values ​​for different concentrations.

[0064] The method described by Zhang et al. is available from: Zhang, XJ, Liu, ZT, Chen, XQ, Zhang, TT, & Zhang, Y. (2023). Deep eutectic solvent combined with ultrasound technology: A promising integrated extraction strategy foranthocyanins and polyphenols from blueberry pomace. Food Chemistry , 422 ,136224. 4: Angiotensin-converting enzyme (ACE) inhibitory activity The ACE inhibition rate of polyphenols was tested with reference to Fan Zhongchao's method. The specific method is as follows: take 20 μL of polyphenols and mix thoroughly with 80 μL of HHL solution, preheat at 37 °C for 5 min, add 10 μL of ACE solution (0.1 U / mL), continue the reaction for 1 h, and add 200 μL of 1 mol / L HCI after the reaction is completed to terminate the reaction. Add 1.2 mL of ethyl acetate to the reaction product for oscillation extraction, centrifuge at 3000 g for 5 min, aspirate 900 μL of the upper ester layer, blow dry the ethyl acetate with nitrogen, add 3.0 mL of ultrapure water to fully dissolve the extract, and measure the ultraviolet absorbance of the reactant at 228 nm. The control group used borate buffer instead of the sample, and the blank group was pre-added with 200 μL of 1 mol / L HCI to terminate the reaction. The calculation formula for ACE inhibition rate (%) is as follows: ACE inhibition rate (%) = 100%*(A1-A) / (A1-A0); Where: A is the absorbance value of the experimental group; A1 is the absorbance value of the control group; A0 is the absorbance value of the blank group.

[0065] The source of Fan Zhongchao's method is: Fan Chaozhong. Preparation, structural analysis and property research of sea cucumber peptide ferrous chelate[D]. Tianjin University of Science and Technology, 2023. 5: Antibacterial activity The fungal inhibitory effect of water chestnut peel polyphenols was determined using the perforation method. Polyphenols were dissolved in a 5 mg / mL solution, and 25 μL was added dropwise to the perforated portion of a culture dish. After 48 hours of incubation, the inhibition zone was observed.

[0066] Source: Zhang, S., Tan, T., Wang, J., Ma, T., & Li, T. (2024). Green extraction of polyphenols from pomegranate seeds by ultrasound-assisted deep eutectic solvent extraction: Optimization and bioactivity. Sustainable Chemistry and Pharmacy , 41 , 101710. 6: Microbiological testing According to GB 4789.15-2016 National Food Safety Standard Food Microbiology Test Mold and Yeast Count, the mold content in bread was tested. According to GB 4789.35-2023 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test, test the lactic acid bacteria content It should be noted that: In this article, “~” is used to indicate a numerical range, and the range indicated by this expression includes two endpoint values.

[0067] In addition, those skilled in the art should understand that, although there are many problems in the prior art, each embodiment or technical solution of the present application may be improved in only one or several aspects, without having to simultaneously solve all the technical problems listed in the prior art or background art. Those skilled in the art should understand that the absence of any content in a claim should not be construed as a limitation on that claim.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing an active ingredient from water chestnut peel, characterized in that: The following steps are involved: Ultrafine treatment of water chestnut peel: drying the water chestnut peel, crushing and sieving, and then ultrafine treatment to obtain water chestnut peel powder; Ultrasonic-assisted DES extraction of water chestnut polyphenols: prepare a first DES solution with or without dilution with water to reduce its viscosity; The water chestnut peel powder is added to the first DES solution and ultrasonically treated at 40-50° C. for 30-60 min to obtain a mixture; the mixture is solid-liquid separated, the supernatant is collected, and the mixture is placed in an ethanol solution for precipitation, followed by solid-liquid separation and drying the precipitate to obtain water chestnut peel polyphenols and a raw material residue; The components of the first DES solution include choline chloride and a first other solvent, and the molar ratio of the choline chloride to the first other solvent is 1:(1-3); the first other solvent is one or a combination of 1,4-butanediol, 1,2-propylene glycol, malic acid, lactic acid, and citric acid; the ultrasonic treatment conditions are single frequency 20 kHz, single frequency 60 kHz, single frequency 80 kHz, dual frequency 20 / 60 kHz, dual frequency 60 / 80 kHz, or dual frequency 20 / 80 kHz, and the power is 200-600 W.

2. The method for preparing the active ingredient from water chestnut peel according to claim 1, wherein: The following preparation steps are also included: Microwave-assisted DES extraction of water chestnut polysaccharides: mixing the raw material residue after separation of polyphenols with a second DES solution, and diluting the second DES solution with or without water to reduce its viscosity; The mixture was then extracted under microwave conditions for 30 to 60 minutes. The microwave-treated mixture was then subjected to solid-liquid separation, the supernatant was collected and placed in an ethanol solution for precipitation, and the solid-liquid separation and drying of the precipitate were performed to obtain water chestnut polysaccharide. The components of the second DES solution include choline chloride and a second other solvent, and the molar ratio of choline chloride to the second other solvent is 1:(1-3); the other solvent is one or a combination of ethylene glycol, glycerol, urea, and 1,6-hexanediol; the conditions of the microwave treatment are: power 250-550W, extraction temperature 70-100°C.

3. The method for preparing the active ingredient from water chestnut peel according to claim 1, wherein: When the first DES solution is diluted with water, the first DES solution is diluted with water to obtain a first DES dilution solution, wherein the amount of water used is 10-20% of the mass of the first DES solution; The first other solvent is 1,4-butanediol and citric acid; in the first DES solution, the molar ratio of choline chloride, 1,4-butanediol and citric acid is: 1:1:(0.5-1); The ultrasonic treatment was performed at a dual frequency of 20 / 80 kHz.

4. The method for preparing the active ingredient from water chestnut peel according to claim 2, wherein: When the second DES solution is diluted with water, the second DES solution is diluted with water to obtain a second DES dilution solution, wherein the amount of water used is 10 to 20% of the mass of the second DES solution; The second other solvent is 1,6-hexanediol and urea; in the second DES solution, the molar ratio of choline chloride, 1,6-hexanediol and urea is: 1:1:(0.5-1); The microwave treatment conditions are: power 400-550W, extraction temperature 70-100°C.

5. The method for preparing the active ingredient from water chestnut peel according to claim 4, characterized in that: The microwave treatment conditions are: power 550W, extraction temperature 80°C.

6. The method for preparing the active ingredient from water chestnut peel according to claim 2, wherein: In the ultrafine treatment process of the water chestnut peel, the water chestnut peel is dried and then crushed and sieved; the coarsely crushed water chestnut peel is then ground and subjected to heavy pressure grinding ultrafine treatment; the ultrafine water chestnut peel powder is then subjected to air flow grinding treatment and air flow classification to obtain water chestnut peel powder; In the ultrasonic-assisted DES extraction step of water chestnut polyphenols, choline chloride and a first other solvent are mixed, heated and stirred at 60-80° C. until the liquid mixture is uniform and transparent, thereby obtaining a first DES solution; when the first DES solution is diluted with water, the mass ratio of the first DES dilution to the water chestnut peel powder is (15-30):1; when the first DES solution is not diluted with water, the mass ratio of the first DES solution to the water chestnut peel powder is (15-30):1; the ethanol used in the precipitation process is 4-10° C. ethanol; In the microwave-assisted DES extraction step of water chestnut polysaccharide, when the second DES solution is diluted with water, the mass ratio of the second DES dilution solution to the raw material residue is (10-20):1; when the second DES solution is not diluted with water, the mass ratio of the second DES solution to the raw material residue is (10-20):1; the ethanol used in the precipitation process is 4-10°C ethanol.

7. The method for preparing the active ingredient from water chestnut peel according to claim 1, wherein: In the ultrafine treatment process of the water chestnut peel, the water chestnut peel is dried and then crushed and sieved, and then the coarsely crushed water chestnut peel is ground at 1000-1500 r / min for 10-30 min to perform heavy pressure grinding ultrafine treatment, and then the ultrafine water chestnut peel powder is passed through an air flow mill at a pressure of 0.1-0.3 MPa and a material processing capacity of 2-5 g / h for air flow classification to finally obtain a water chestnut peel powder sample.

8. A water chestnut peel polyphenol, characterized by: The method is as described in any one of claims 1 to 7.

9. A water chestnut peel polysaccharide, characterized by: The product is prepared by the preparation method according to any one of claims 2 and 4 to 6.

10. Application of water chestnut peel active ingredients in food, characterized by: The water chestnut peel active ingredients include water chestnut peel polyphenols and / or water chestnut peel polysaccharides; The polyphenol is prepared by the preparation method according to any one of claims 1 to 7; the polysaccharide is prepared by the preparation method according to any one of claims 2 and 4 to 6.

Images