Anthocyanin extraction method and application thereof

Abstract

The invention provides an anthocyanin extraction method and application thereof.The method comprises the steps that purple corn processing by-product spike stalks serve as raw materials, a deep eutectic solvent prepared from choline chloride and D-lactic acid according to the molar ratio of 1: 2 serves as the basis, and extraction is conducted through ultrasonic assistance; the optimal extraction process parameters are as follows: the DES water content is 29%, the material-liquid ratio is 1: 7, the ultrasonic temperature is 50 DEG C, and the ultrasonic time is 39 min. The method is high in extraction efficiency, and the anthocyanin extraction amount can reach 56.23 mg / g DW and is 1.33 times that of a traditional 70% ethanol extraction method. Meanwhile, the DES extraction system can significantly enhance the thermal stability and storage stability of the anthocyanin, the thermal degradation half-life period at 80 DEG C is prolonged by 51.8%, and the retention rate is increased to 38.57% after the anthocyanin is stored in a dark place for 15 days. The method is green, environment-friendly, efficient and stable, and a new way is provided for comprehensive utilization of purple corn resources and green preparation of high-value natural anthocyanin.

Description

Technical Field

[0001] This application relates to the field of natural product extraction technology, and in particular to a method for extracting anthocyanins from purple corn cobs using ultrasound-assisted eutectic solvents and its application. Background Technology

[0002] Anthocyanins, composed of glycosidones (also known as anthocyanins) and carbohydrate residues, are flavonoid compounds and pigments found in various grains, vegetables, fruits, and other plant tissues. They are non-toxic, harmless, and environmentally friendly; their structure and color can change with pH, ​​leading to their use in designing smart packaging and real-time monitoring of food spoilage; they possess antioxidant and antibacterial properties, enabling packaging films to protect nutrients and extend shelf life; and they have potential therapeutic properties, such as preventing cardiovascular disease and diabetes, fighting cancer, and improving vision and brain function, offering numerous health benefits to the human body.

[0003] Purple corn cobs are a byproduct of purple corn processing and are often discarded or used as low-value fuel. Studies have shown that the anthocyanin content in purple corn cobs is even higher than in the kernels, representing a valuable but underutilized resource. Currently, anthocyanin extraction from plants typically employs traditional solvent extraction methods, using conventional organic solutions or ionic liquids such as anhydrous ethanol or ethanol-citric acid mixtures. These methods suffer from drawbacks including high solvent toxicity, low extraction efficiency, high energy consumption, and easy degradation of heat-sensitive anthocyanins, and are also environmentally unfriendly.

[0004] Solvent extraction is the most common method for obtaining anthocyanins from plant materials. It involves extraction with polar solvents or mixtures thereof. These solvents increase the diffusion rate of anthocyanins, and the addition of citric acid or acetic acid can protect the anthocyanins from degradation. Netravati et al. found that extracting anthocyanins from butterfly pea flowers using an acidified solvent of 50% ethanol and 1% citric acid significantly improved the total anthocyanin content and recovery rate compared to using 50% ethanol alone. Heinonen et al. used acetic acid-acidified ethanol to extract anthocyanins from purple sweet potatoes, determining the optimal extraction time and ethanol concentration. While solvent extraction is simple and inexpensive, it suffers from low extraction efficiency, prolonged heating leads to anthocyanin degradation and reduced activity, and some organic solvents are toxic, difficult to degrade, and harmful to the environment and human health.

[0005] Deep eutectic solvents (DESs) consist of two components: a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD). HBD and HBA form a eutectic phase through hydrogen bonding, and its melting point is lower than that of either component. The advantages of DESs are that they are non-toxic, non-flammable, easy to produce, and inexpensive, overcoming the disadvantages of traditional organic solvents, such as toxicity, difficulty in synthesis, and low biodegradability. However, the main drawback of DESs as extraction solvents is their high viscosity, which is difficult to adjust by increasing the temperature for heat-sensitive bioactive compounds. Furthermore, the anthocyanins prepared from DESs exhibit poor stability to environmental factors (pH, ultraviolet radiation, oxygen, etc.), increasing costs for manufacturers and limiting their application in the food industry. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a highly efficient, green, and stable method for extracting anthocyanins from purple corn cobs using ultrasound-assisted eutectic solvents, along with its applications. This method utilizes a specific composition of DES for efficient extraction of anthocyanins under ultrasound assistance, and the resulting anthocyanin extract exhibits excellent thermal and storage stability.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of this application provides a method for extracting anthocyanins from purple corn cobs using an ultrasound-assisted eutectic solvent, comprising the following steps:

[0009] S1: Raw material pretreatment: Cut the purple corn cobs into small pieces, dry them in a 40℃ forced-air drying oven to constant weight, pulverize them in a high-speed multi-functional pulverizer, pass them through a 60-mesh sieve to obtain purple corn cob powder, seal them in a brown bottle and store them at 4℃ for later use.

[0010] S2: Preparation of eutectic solvent: Using choline chloride as hydrogen bond acceptor (HBA) and D-lactic acid as hydrogen bond donor (HBD), the raw materials were weighed at a molar ratio of HBA to HBD of 1:2 and stirred at 80°C until a homogeneous and transparent liquid was formed to obtain the DES base solution; distilled water was added to the DES base solution to adjust its water content to 20%-40% (w / w), and stirred evenly to obtain the DES extract, which was then sealed and stored away from light;

[0011] S3: Ultrasonic-assisted extraction: Accurately weigh the purple corn cob powder obtained in S1, add it to the DES extraction solution prepared in S2 at a material-liquid ratio of 1:5-1:15 (g / mL), mix well, and extract for 30-50 min under ultrasonic temperature of 40-60℃ and ultrasonic power of 240W.

[0012] S4: Subsequent processing: The mixture obtained in S3 was centrifuged at 4℃ and 8000rpm for 15min, and then the supernatant was filtered through a 0.45μm nylon filter membrane to obtain anthocyanin extract from purple corn cob.

[0013] Preferably, in step S2, the water content of the DES extract is 29% (w / w).

[0014] Preferably, in step S3, the material-to-liquid ratio is 1:7 (g / mL).

[0015] Preferably, in step S3, the ultrasonic temperature is 50°C.

[0016] Preferably, in step S3, the ultrasound time is 39 minutes.

[0017] A second aspect of this application provides an application of ultrasound-assisted eutectic solvent extraction of anthocyanins from purple corn cobs, and the anthocyanins obtained by the above method are used in food, cosmetics, and agricultural and livestock additives.

[0018] The beneficial effects of this application are:

[0019] This solution aims to address the issues of low anthocyanin extraction efficiency, environmentally unfriendly traditional solvents, and poor anthocyanin stability in purple corn cobs (agricultural waste). It establishes an efficient and green extraction method while simultaneously improving the thermal and storage stability of anthocyanins.

[0020] 1. High extraction efficiency: This invention is the first to use a choline chloride / D-lactic acid (1:2) eutectic solvent system to extract anthocyanins from purple corn cobs. This DES has low viscosity, suitable polarity, and is weakly acidic, enabling it to form a hydrogen bond network with anthocyanins, greatly improving extraction efficiency. After optimization using response surface methodology, the anthocyanin extraction yield reached 56.23 mg / g DW, which is 1.33 times that of the traditional 70% ethanol extraction method (42.29 mg / g DW).

[0021] 2. Green and environmentally friendly: The eutectic solvent used is mainly composed of biodegradable and low-toxicity components, replacing traditional toxic and volatile organic solvents, which meets the requirements of green chemistry development.

[0022] 3. Significantly Enhanced Stability: The extraction method of this invention not only achieves efficient extraction but also significantly enhances the stability of anthocyanins. Experiments show that, compared with the 70% ethanol extract, the thermal degradation half-life (t~1 / 2~) of anthocyanins in the DES extract of this invention is prolonged by 51.8% (at 80℃), and the retention rate after 15 days of light-protected storage increases from 13.19% to 38.57%, significantly improving photostability and storage stability. Attached Figure Description

[0023] Figure 1 This is a comparison chart of the anthocyanin extraction yields of DESs and 70% ethanol from purple corn cobs in this application.

[0024] Figure 2 This is a graph showing the effect of the water content of the eutectic solvent on the anthocyanin extraction yield of purple corn cobs in this application.

[0025] Figure 3 This is a graph showing the effect of the material-to-liquid ratio on the anthocyanin extraction yield of purple corn cobs in this application.

[0026] Figure 4 This is a graph showing the effect of ultrasonic temperature on the anthocyanin extraction yield of purple corn cob in this application.

[0027] Figure 5 This is a graph showing the effect of ultrasound time on the anthocyanin extraction yield of purple corn cob in this application.

[0028] Figure 6 This is a graph showing the effect of the molar ratio on the anthocyanin extraction yield of purple corn cob in this application;

[0029] Figure 7 This is a comparative graph showing the thermal degradation kinetics of anthocyanins from purple corn cobs in this application at different temperatures in choline chloride / lactic acid (1:2)-water (71:29, w / w).

[0030] Figure 8 This is a comparative diagram showing the thermal degradation kinetics of anthocyanins from purple corn cobs at different temperatures in 70% ethanol, as presented in this application.

[0031] Figure 9 This is a comparison of the storage stability of anthocyanins from purple corn cobs in this application in choline chloride / lactic acid (1:2)-water (71:29, w / w) and 70% ethanol. Detailed Implementation

[0032] The following detailed description, with appropriate reference to the accompanying drawings, discloses an anthocyanin extraction method and its application embodiments according to this application. However, some unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of essentially identical structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided to enable those skilled in the art to fully understand this application and are not intended to limit the subject matter of the claims.

[0033] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0034] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0035] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0036] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0037] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0038] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0039] Purple corn cobs are a byproduct of purple corn processing, primarily used as fuel and chemical feedstock. However, the significant anthocyanin content within the cobs is often overlooked. Literature on anthocyanin extraction from purple corn cobs is limited, and existing studies typically employ conventional organic solutions or ionic liquids as extraction solvents, which are often detrimental to the environment. DESs, on the other hand, demonstrate excellent extraction and biodegradability when used to extract bioactive compounds, significantly improving extraction rates compared to classic methods (impregnation and heat-assisted extraction). To date, no research literature reports the use of DESs for anthocyanin extraction from purple corn cobs.

[0040] Anthocyanins are flavonoid compounds containing glycosidic ketones and carbohydrate residues, possessing both natural pigment functions and health benefits (antioxidant, antibacterial, and potential for cardiovascular disease prevention), but they are sensitive to environmental factors such as pH and temperature and are easily degraded. This invention, through extensive research, selects a eutectic solvent (DES) prepared from choline chloride (HBA) and D-lactic acid (HBD) in a 1:2 molar ratio. This DES is weakly acidic (pH≈1.10), protecting the acylation structure of anthocyanins from damage; it can form a strong hydrogen bond network with anthocyanin molecules, enhancing the solubility of anthocyanins and reducing their degradation during subsequent storage and heating through intermolecular interactions; furthermore, this DES has a low viscosity (223 mPa·s), and the viscosity can be further reduced by adjusting the water content (optimal 29%), thus improving mass transfer efficiency. Ultrasonic treatment generates a cavitation effect through high-frequency mechanical vibration, which disrupts the cell wall structure of purple corn cob, forming micro-cracks, reducing the binding force between anthocyanins and the cell wall, and accelerating the penetration and mass transfer of DES in the cell, greatly promoting the release of anthocyanins into the solvent.

[0041] Experiments verified that the optimal process conditions, optimized using response surface methodology, were: DES water content 29%, solid-liquid ratio 1:7 (g / mL), ultrasonic temperature 50℃, and ultrasonic time 39 min. Under these conditions, the anthocyanin extraction yield reached 56.23 mg / g DW, a 33% increase compared to the traditional 70% ethanol extraction method (42.29 mg / g DW), with good process reproducibility. The anthocyanins in the obtained DES extract exhibited excellent stability: the thermal degradation half-life (t1 / 2) at 80℃ was 58.3 min, a 51.8% increase compared to the ethanol extract (38.4 min); after 15 days of storage at room temperature in the dark, the anthocyanin retention rate was 38.57%, significantly higher than the 13.19% of the ethanol extract.

[0042] This application employs ultrasound-assisted DES (Discharge-Assisted Extraction) to extract anthocyanins from purple corn cobs, while simultaneously investigating the stability of the anthocyanins within the cobs. The aim is to overcome the shortcomings of traditional solvents, such as their lack of environmental friendliness and low extraction efficiency, and to provide a green and efficient method for extracting anthocyanins from purple corn cobs. This extraction method combines a specific eutectic solvent system with ultrasound-assisted technology, specifically designed for extracting anthocyanins from purple corn cobs while significantly improving the stability of the extracted anthocyanins—a method not previously reported. Furthermore, this provides a direction for fully utilizing purple corn processing byproducts and providing an inexpensive and readily available source of anthocyanins, promoting the development of the purple corn industry and the application of anthocyanins in the food industry.

[0043] A method for extracting anthocyanins from purple corn cobs using an ultrasound-assisted eutectic solvent, characterized by comprising the following steps:

[0044] S1: Raw material pretreatment: Cut the purple corn cob into pieces, dry, crush and sieve to obtain purple corn cob powder, and store it in a sealed container;

[0045] S2: Preparation of eutectic solvent: Choline chloride is used as a hydrogen bond acceptor and D-lactic acid is used as a hydrogen bond donor. They are mixed in a molar ratio of 1:2 and heated and stirred until a uniform and transparent liquid is formed to obtain the DES base solution; distilled water is added to the DES base solution to prepare a DES extract with a water content of 20%-40%.

[0046] S3: Ultrasonic-assisted extraction: Add the DES extract obtained in S2 to the purple corn cob powder obtained in S1 at a material-liquid ratio of 1:5-1:15 (g / mL), and extract for 30-50 min at an ultrasonic temperature of 40-60℃ and an ultrasonic power of 240W.

[0047] S4: Subsequent processing: Centrifuge and filter the mixture obtained in S3 to obtain anthocyanin extract from purple corn cob.

[0048] In some embodiments, in step S2, the water content of the DES extract is 29%.

[0049] In some embodiments, in step S3, the material-to-liquid ratio is 1:7 (g / mL).

[0050] In some embodiments, the ultrasonic temperature in step S3 is 50°C.

[0051] In some embodiments, the ultrasound time in step S3 is 39 minutes.

[0052] In some embodiments, in step S1, the sieving is through a 60-mesh sieve; in step S4, the filtration is performed using a 0.45μm nylon filter membrane.

[0053] An application of ultrasound-assisted eutectic solvent extraction of anthocyanins from purple corn cobs; the anthocyanins obtained by the above method are used in food, cosmetics, and agricultural and livestock additives.

[0054] As natural food colorings, anthocyanins are favored for their natural and safe properties compared to synthetic pigments (such as carmine and amaranth). Applications include: beverages, confectionery and baked goods, dairy products, other jams, canned goods, and meat substitutes.

[0055] Its application in health foods and dietary supplements has antioxidant and anti-inflammatory effects, protects eyesight and relieves eye fatigue, prevents cardiovascular diseases, combats obesity and improves metabolic syndrome, protects against neurological damage and improves cognitive function, and has anti-cancer potential.

[0056] Applications in cosmetics and skincare products (“edible cosmetics”): anti-aging, sun protection and repair, soothing and anti-allergy, such as serums, creams, masks, lotions, etc.

[0057] Applications in agriculture and animal husbandry: as a plant resistance inducer, to improve the quality of agricultural products, and as an animal feed additive.

[0058] Example

[0059] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0060] Example 1

[0061] A method for extracting anthocyanins from purple corn cobs using an ultrasound-assisted eutectic solvent includes the following steps:

[0062] S1: Raw material pretreatment: Cut the purple corn cobs into small pieces, dry them in a 40℃ forced-air drying oven to constant weight, pulverize them in a high-speed multi-functional pulverizer, pass them through a 60-mesh sieve to obtain purple corn cob powder, seal them in a brown bottle and store them at 4℃ for later use.

[0063] S2: Preparation of eutectic solvent: Using choline chloride as hydrogen bond acceptor (HBA) and D-lactic acid as hydrogen bond donor (HBD), the raw materials were weighed at a molar ratio of HBA to HBD of 1:2 and stirred at 80°C until a homogeneous and transparent liquid was formed to obtain the DES base solution; distilled water was added to the DES base solution to adjust its water content to 29% (w / w), and stirred evenly to obtain the DES extract, which was then sealed and stored in the dark.

[0064] S3: Ultrasonic-assisted extraction: Accurately weigh the purple corn cob powder obtained in S1, add it to the DES extraction solution prepared in S2 at a material-to-liquid ratio of 1:7 (g / mL), mix well, and extract for 39 min under ultrasonic temperature of 50℃ and ultrasonic power of 240W.

[0065] S4: Subsequent processing: The mixture obtained in S3 was centrifuged at 4℃ and 8000rpm for 15min, and then the supernatant was filtered through a 0.45μm nylon filter membrane to obtain anthocyanin extract from purple corn cob.

[0066] Example 2

[0067] A method for extracting anthocyanins from purple corn cobs using an ultrasound-assisted eutectic solvent includes the following steps:

[0068] S1: Raw material pretreatment: Cut the purple corn cobs into small pieces, dry them in a 40℃ forced-air drying oven to constant weight, pulverize them in a high-speed multi-functional pulverizer, pass them through a 60-mesh sieve to obtain purple corn cob powder, seal them in a brown bottle and store them at 4℃ for later use.

[0069] S2: Preparation of eutectic solvent: Using choline chloride as hydrogen bond acceptor (HBA) and D-lactic acid as hydrogen bond donor (HBD), the raw materials were weighed at a molar ratio of HBA to HBD of 1:2 and stirred at 80°C until a homogeneous and transparent liquid was formed to obtain the DES base solution; distilled water was added to the DES base solution to adjust its water content to 40% (w / w), and stirred evenly to obtain the DES extract, which was then sealed and stored in the dark.

[0070] S3: Ultrasonic-assisted extraction: Accurately weigh the purple corn cob powder obtained in S1, add it to the DES extraction solution prepared in S2 at a material-to-liquid ratio of 1:5 (g / mL), mix well, and extract for 45 min under ultrasonic temperature of 45℃ and ultrasonic power of 240W.

[0071] S4: Subsequent processing: The mixture obtained in S3 was centrifuged at 4℃ and 8000rpm for 15min, and then the supernatant was filtered through a 0.45μm nylon filter membrane to obtain anthocyanin extract from purple corn cob.

[0072] Example 3

[0073] A method for extracting anthocyanins from purple corn cobs using an ultrasound-assisted eutectic solvent includes the following steps:

[0074] S1: Raw material pretreatment: Cut the purple corn cobs into small pieces, dry them in a 40℃ forced-air drying oven to constant weight, pulverize them in a high-speed multi-functional pulverizer, pass them through a 60-mesh sieve to obtain purple corn cob powder, seal them in a brown bottle and store them at 4℃ for later use.

[0075] S2: Preparation of eutectic solvent: Using choline chloride as hydrogen bond acceptor (HBA) and D-lactic acid as hydrogen bond donor (HBD), the raw materials were weighed at a molar ratio of HBA to HBD of 1:2 and stirred at 80°C until a homogeneous and transparent liquid was formed to obtain the DES base solution; distilled water was added to the DES base solution to adjust its water content to 35% (w / w), and stirred evenly to obtain the DES extract, which was then sealed and stored in the dark.

[0076] S3: Ultrasonic-assisted extraction: Accurately weigh the purple corn cob powder obtained in S1, add it to the DES extraction solution prepared in S2 at a material-to-liquid ratio of 1:10 (g / mL), mix well, and extract for 40 min under ultrasonic temperature of 50℃ and ultrasonic power of 240W.

[0077] S4: Subsequent processing: The mixture obtained in S3 was centrifuged at 4℃ and 8000rpm for 15min, and then the supernatant was filtered through a 0.45μm nylon filter membrane to obtain anthocyanin extract from purple corn cob.

[0078] Example 4

[0079] A method for extracting anthocyanins from purple corn cobs using an ultrasound-assisted eutectic solvent includes the following steps:

[0080] S1: Raw material pretreatment: Cut the purple corn cobs into small pieces, dry them in a 40℃ forced-air drying oven to constant weight, pulverize them in a high-speed multi-functional pulverizer, pass them through a 60-mesh sieve to obtain purple corn cob powder, seal them in a brown bottle and store them at 4℃ for later use.

[0081] S2: Preparation of eutectic solvent: Using choline chloride as hydrogen bond acceptor (HBA) and D-lactic acid as hydrogen bond donor (HBD), the raw materials were weighed at a molar ratio of HBA to HBD of 1:2 and stirred at 80°C until a homogeneous and transparent liquid was formed to obtain the DES base solution; distilled water was added to the DES base solution to adjust its water content to 30% (w / w), and stirred evenly to obtain the DES extract, which was then sealed and stored away from light;

[0082] S3: Ultrasonic-assisted extraction: Accurately weigh the purple corn cob powder obtained in S1, add it to the DES extraction solution prepared in S2 at a material-to-liquid ratio of 1:15 (g / mL), mix well, and extract for 50 min at an ultrasonic temperature of 60℃ and an ultrasonic power of 240W.

[0083] S4: Subsequent processing: The mixture obtained in S3 was centrifuged at 4℃ and 8000rpm for 15min, and then the supernatant was filtered through a 0.45μm nylon filter membrane to obtain anthocyanin extract from purple corn cob.

[0084] To verify the research results of this application, the specific experimental process is as follows.

[0085] 1. Experimental raw materials and reagents

[0086] Purple corn was purchased from the market. The reagents used in the experiment included tartaric acid, citric acid, fructose, D-lactic acid, glycerol, choline chloride, anhydrous ethanol, 4 mol / L hydrochloric acid, potassium chloride, and anhydrous sodium acetate, all of analytical grade.

[0087] 2. Instruments and Equipment

[0088] Table 1. Main Instruments and Equipment Used in the Experiment

[0089]

[0090] 3. Pretreatment of raw materials

[0091] Cut the purple corn cobs into small pieces and dry them in a 40℃ forced-air drying oven until constant weight. Then, grind them into powder using a high-speed multi-functional pulverizer and pass them through a 60-mesh sieve. Finally, seal the cob powder in a brown bottle and store it in a 4℃ refrigerator.

[0092] 4. Preparation and determination of physicochemical properties of eutectic solvents

[0093] Choline chloride and different HBDs were mixed at a specified molar ratio (see Table 2) and stirred at 80-90°C for a period of time. When the mixture became a homogeneous liquid without any precipitated crystals, the DESs were successfully prepared. A certain amount of distilled water was added to the DESs to dilute it to a 30% water content, and then it was stored in a sealed glass flask protected from light. The prepared eutectic solvent remained clear and transparent after being stored at room temperature for one week and could be used for subsequent experiments.

[0094] The viscosity and pH of the five prepared DESs were determined using an NDJ-8S rotational viscometer (digital display) and an ST3100 laboratory pH meter. Density was calculated based on the mass and volume measurements of the DES mixtures. All measurements were performed in triplicate, and the average value was taken.

[0095] Table 2 Composition and preparation conditions of different DESs

[0096]

[0097] 5. DES Filtering Methods

[0098] Weigh 0.1g of purple corn cob powder into a centrifuge tube and add 2ml of DESs with a water content of 30%. Sonicate at 40℃ and 240W for 30 minutes, then remove the tube. Subsequently, centrifuge the mixture at 4℃ and 8000rpm for 15 minutes, and then filter the supernatant through a filter membrane. As a control, repeat the above method to extract anthocyanins from purple corn cobs using 70% ethanol as the extraction solvent. Compare the anthocyanin extraction yields of the five DESs to screen for the optimal DES system.

[0099] Experiment 1: Single-factor experiment on the extraction of anthocyanins from purple corn cobs using the DES system.

[0100] Using the anthocyanin extraction yield of purple corn cobs as an indicator, the effects of moisture content, material-to-liquid ratio, extraction time, extraction temperature, and molar ratio of DES on the anthocyanin extraction yield of purple corn cobs were investigated to determine the optimal parameter range for the process.

[0101] (1) Selection of DES water content. Under the conditions of molar ratio of 1:2, material-liquid ratio of 1:20, ultrasonic temperature of 40℃ and ultrasonic time of 30min, the changes in anthocyanin extraction amount when the water content of DES is 10%, 20%, 30%, 40% and 50% were investigated.

[0102] (2) Selection of material-liquid ratio. Under the conditions of DES water content of 30%, ultrasonic time of 30 min, molar ratio of 1:2 and ultrasonic temperature of 40℃, the changes in anthocyanin extraction amount were investigated when the material-liquid ratio was 1:5, 1:10, 1:15, 1:20 and 1:25 g / mL.

[0103] (3) Selection of ultrasonic temperature. Under the conditions of DES water content of 30%, molar ratio of 1:2, material-liquid ratio of 1:10 and ultrasonic time of 30 min, the changes in anthocyanin extraction amount were investigated at extraction temperatures of 30, 40, 50, 60 and 70℃.

[0104] (4) Selection of ultrasonic time. Under the conditions of DES water content of 30%, molar ratio of 1:2, material-liquid ratio of 1:10 and temperature of 50℃, the changes in anthocyanin extraction amount were investigated when the extraction time was 20, 30, 40, 50 and 60 min respectively.

[0105] (5) Selection of molar ratio. Under the conditions of DES water content of 30%, material-liquid ratio of 1:10, temperature of 50℃ and ultrasonic time of 40min, the effect of DES prepared with HBA to HBD molar ratio of 1:2, 1:3, 1:4, 1:5 and 1:6 on anthocyanin extraction was investigated.

[0106] After centrifuging each experimental mixture in a high-speed refrigerated centrifuge, the supernatant was taken and filtered through a 0.45μm nylon membrane. The crude content of anthocyanins extracted from purple corn cobs was calculated. Three parallel experiments were performed for each sample.

[0107] For DES-1 and DES-2, after heating at 90°C for several hours, solutions with molar ratios of 1:1 and 1:2 still contained solids, while the solution with a molar ratio of 2:1 was successfully prepared after 4 hours. For DES-5, a solid precipitated upon cooling to room temperature at a molar ratio of 1:1, while a clear solution could not be formed at a molar ratio of 2:1. DES-3 and DES-4 formed homogeneous and transparent solutions at all three molar ratios. Based on practical preparation experience and literature, the appropriate molar ratio for each DES was finally determined. During the preparation process, it was found that solutions of DES-2, DES-3, and DES-5 exhibited a pale yellow color when the preparation temperature exceeded 80°C, consistent with the findings reported by Qamar et al. Yellowing or browning is a drawback of heating methods for preparing DES; appropriate temperatures can be used to avoid discoloration. Furthermore, DES with all solid components were more difficult to prepare than those with one liquid component, requiring longer preparation times and higher temperatures.

[0108] The physicochemical properties of DESs affect their ability to extract bioactive substances. Table 3 shows that the densities of all five DESs with a 30% water content are greater than water. The pH value of the eutectic solvent (HBD) for organic acids is lower than that for alcohols and sugars. Viscosity is one of the most important properties of DESs; high viscosity is a characteristic of DESs, but it affects the extraction rate. DES-5 has the lowest viscosity among the five eutectic solvents. Compared to DES-5, the acidic DESs DES-1 and DES-2 have higher viscosities due to their different hydrogen bond donors. Lactic acid contains only one carboxyl group, while tartaric acid and citric acid contain multiple carboxyl groups, resulting in stronger intermolecular interactions and higher viscosity in the resulting DESs. The viscosity of small-molecule polyol groups is lower than that of eutectic solvents containing sugars or organic acids; therefore, the viscosity of DES-4 is lower than that of DES-3. Experimental studies have shown that increasing the temperature and adding water decreases the viscosity of DESs. Therefore, this application compensates for the shortcomings of DESs by reducing viscosity when extracting bioactive substances by increasing temperature or water content.

[0109] Table 3 Properties of 5 DESs

[0110]

[0111] Anthocyanins were extracted from the cob of purple corn using five prepared DESs, and compared with 70% ethanol (by mass fraction). The results are as follows: Figure 1 As shown.

[0112] The extraction yields of DES-2 and DES-3 were lower than those of 70% ethanol, while the anthocyanin extraction yields of DES-1, DES-4, and DES-5 were significantly higher than those of 70% ethanol. The extraction efficiency of DESs is related to properties such as polarity, pH, and viscosity. DES-5 exhibited the highest anthocyanin extraction yield because of its lowest viscosity, which facilitates mass transfer. Furthermore, its hydrogen bond donors are organic acids, resulting in a lower pH value for the DES, which improves anthocyanin stability compared to DESs with hydrogen bond donors such as alcohols and sugars, thus benefiting anthocyanin extraction. Although DES-1 and DES-2 are also acidic DESs with high polarity, citric acid and tartaric acid, with multiple carboxyl and hydroxyl groups, limited extraction efficiency through steric hindrance, hindering intermolecular interactions. In contrast, lactic acid, with only one carboxyl group, is more conducive to intermolecular interactions, thus leading to the higher extraction rate of DES-5. Although the hydrogen bond donors of DES-4 are alcohols, the anthocyanin extraction yield is relatively high. This may be related to the multiple hydrogen bond interactions. The hydrogen bond network formed between the components results in a polarity close to that of anthocyanins, leading to high extraction efficiency.

[0113] In summary, DES-5, which has the highest extraction yield and is beneficial to the stability of purple corn cobs, was selected for subsequent experiments.

[0114] Experiment 2: Selection of water content in the preparation method

[0115] Moisture content is one of the important factors affecting the extraction efficiency of eutectic solvents. Adding water can not only reduce the viscosity of eutectic solvents, but also affect their polarity. For example... Figure 2 As shown, when the moisture content of DES increased from 10% to 50%, the extraction yield of anthocyanins from purple corn cobs first increased significantly and then decreased. This is because increasing the moisture content reduces the viscosity of DES, decreasing mass transfer resistance, and a certain amount of water makes the polarity of acidic DES closer to that of anthocyanins, thus facilitating the extraction of anthocyanins from the cob powder. However, when the moisture content exceeded 30%, the anthocyanin extraction yield decreased. This may be because excessive water disrupts the molecular network structure formed between HBA and HBD, weakening the interaction between DES and anthocyanin compounds, resulting in a solvent property closer to water and reducing the extraction rate. Adding a certain amount of water can not only affect the extraction capacity of DES but also reduce the amount of DES used, saving costs. Therefore, a moisture content of 30% for choline chloride / lactic acid was chosen for subsequent optimization.

[0116] Experiment 3: Selection of the material-liquid ratio in the preparation method

[0117] The solid-liquid ratio is another important factor affecting anthocyanin extraction. When the solid-liquid ratio increased from 1:5 (g / mL) to 1:25 (g / mL), the anthocyanin extraction yield from purple corn cobs showed a trend of first gradually increasing and then significantly decreasing. This is because the amount of extraction solvent was too small to dissolve all the solids, resulting in incomplete extraction. As the solid-liquid ratio increases, the contact area between DES and cob powder increases, and the higher concentration gradient between the cob powder and DES allows anthocyanins to diffuse more easily into the DES, thus increasing the extraction yield. However, when the solid-liquid ratio continued to increase, the anthocyanin extraction yield decreased significantly. This may be because excessive DES inhibits mass transfer of the solids and weakens the penetration ability of ultrasound, thus reducing the anthocyanin extraction yield. Therefore, a ratio of 1:10 (g / mL) was selected for further optimization. Figure 3 As shown.

[0118] Experiment 4: Selection of Ultrasonic Temperature in the Preparation Method

[0119] Depend on Figure 4As shown, the extraction yield of anthocyanins from purple corn cobs gradually increased as the temperature rose from 30℃ to 50℃. This is because higher temperatures accelerate mass transfer and reduce the viscosity and surface tension of DES, thereby enhancing the effect of ultrasound and increasing the anthocyanin extraction yield. However, when the temperature increased from 50℃ to 70℃, the extraction yield of anthocyanins from purple corn cobs decreased significantly. This is because anthocyanins decompose thermally at temperatures exceeding 50℃, and excessively high ultrasonic temperatures weaken the cavitation effect. Therefore, excessively high temperatures are detrimental to anthocyanin extraction, and 50℃ was selected as the optimal ultrasonic temperature for subsequent optimization.

[0120] Experiment 5: Selection of Ultrasonic Time in the Preparation Method

[0121] When the ultrasonic time increased from 20 min to 40 min, the extraction yield of anthocyanins from purple corn cobs significantly increased to a maximum value, and then showed a decreasing trend with further extension of ultrasonic time. Figure 5 As shown, sound waves can rupture plant cell walls, allowing anthocyanins to flow out of the cells more easily and disperse better in DES, thus improving extraction efficiency. However, excessively long ultrasonic times can cause complete cell wall rupture and release other intracellular components, which in turn reduces the amount of anthocyanins extracted. Furthermore, prolonged ultrasonic time can also cause localized overheating of anthocyanins and induce the generation of free radicals, leading to anthocyanin degradation. Therefore, 40 minutes was selected as the optimal ultrasonic time for extracting anthocyanins from purple corn cobs.

[0122] Experiment 6: Selection of Molar Ratio in the Preparation Method

[0123] The ratio of hydrogen bond donors to hydrogen bond acceptors affects the formation of hydrogen bonds between anthocyanins and components, thus influencing the extraction yield of anthocyanins. Figure 6 It was found that the extraction yield of anthocyanins from purple corn cobs decreased with increasing proportion of lactic acid in the fraction. This may be because anthocyanins more readily form hydrogen bonds with choline quaternary cations in hydrogen bond acceptors, and excessive lactic acid hinders the formation of hydrogen bonds between anthocyanins and choline, weakening the molecular interaction between them and leading to a reduced extraction yield. Therefore, a molar ratio of 1:2 was chosen as the optimal molar ratio for extracting anthocyanins from purple corn cobs.

[0124] Experiment 7: Thermokinetic Study of Anthocyanins in Purple Maize Cob in DES

[0125] The DES extract used in the study of anthocyanin thermal degradation kinetics and storage stability of purple corn cobs was DES-5 (29% water content, w / w) optimized by response surface methodology.

[0126] In addition to ensuring high anthocyanin extraction efficiency, the extraction solvent also needs to maintain the stability of anthocyanins under the heating conditions during the extraction process. For example... Figure 7As shown, the thermal degradation process of anthocyanins in purple corn cobs at 60℃, 70℃, and 80℃ follows a simple first-order kinetic reaction. The anthocyanin degradation rate constant k and Rc at different temperatures are also shown. 2 Value and half-life t 1 / 2 Value. Whether in DES-5 or in 70% ethanol, the k value gradually increases with increasing temperature, t 1 / 2 The value gradually decreases, indicating that high temperature promotes the degradation of anthocyanins in purple corn cobs, thus reducing their stability. Compared with 70% ethanol ( Figure 8 At the same temperature, the anthocyanin k-value in DES-5 extract was lower, and t 1 / 2 The higher value indicates that anthocyanins from purple corn cobs have better thermal stability in DES-5. Especially at 80℃, the t-value of anthocyanins in the DES-5 extract is higher. 1 / 2 The value is 1.5 times that of 70% ethanol. This is likely because DES-5 has a pH < 2, which helps protect anthocyanins. Furthermore, the hydrogen bonds formed between DES and anthocyanins help prevent the structure of anthocyanins from being destroyed during heating, thus enhancing the thermal stability of anthocyanins.

[0127] Experiment 8: Storage stability of anthocyanins in purple corn cobs in DES

[0128] At room temperature, the DES-5 extract and the 70% ethanol extract were placed in light and dark environments for 15 days. The retention rate of anthocyanins in the extracts was measured every 3 days. The results are as follows: Figure 9 As shown in the figure, anthocyanins in both extracts degraded over time under different light conditions. Compared to the darkened conditions, the retention rate of anthocyanins under indoor light was significantly lower, indicating that light causes anthocyanin decomposition and is detrimental to anthocyanin storage. After 15 days of storage under this environment, the anthocyanin content in the 70% ethanol extract was only 13.19% of the initial anthocyanin content, while the retention rate of anthocyanins in the DES-5 extract was 38.57%, indicating that anthocyanins from purple corn cobs have higher photostability and storage stability in DES-5. This may be because oxygen has lower solubility in DES than in ethanol and water, and the interaction between DES and anthocyanin molecules can reduce the contact time between anthocyanins and oxygen, preventing their oxidative degradation. The low pH environment provided by DES-5 also helps protect anthocyanins from degradation. In addition, the strong hydrogen bonds formed in the aqueous DES system can maintain the spatial structure of bioactive compounds and protect them from environmental factors.

[0129] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for extracting anthocyanins, characterized by, The method comprises the following steps: S1: raw material pretreatment: cutting, drying, crushing, and sieving purple corn cob to obtain purple corn cob powder, which is sealed and stored; S2: preparation of a deep eutectic solvent: mixing choline chloride as a hydrogen bond acceptor and D-lactic acid as a hydrogen bond donor at a molar ratio of 1:2, heating and stirring until a uniform transparent liquid is formed to obtain a DES base liquid; adding distilled water to the DES base liquid to prepare a DES extraction liquid with a water content of 20%-40%; S3: ultrasonic-assisted extraction: adding the DES extraction liquid obtained in S2 to the purple corn cob powder obtained in S1 at a solid-liquid ratio of 1:5-1:15, and extracting at an ultrasonic temperature of 40-60℃ and an ultrasonic power of 240W for 30-50min; S4: subsequent treatment: centrifuging and filtering the mixture obtained in S3 to obtain a purple corn cob anthocyanin extraction liquid.

2. The anthocyanin extraction method according to claim 1, characterized by, In step S2, the water content of the DES extraction liquid is 25-35%.

3. The anthocyanin extraction method according to claim 1 or 2, characterized by, In step S2, the water content of the DES extraction liquid is 29%.

4. The anthocyanin extraction method according to claim 1, characterized by, In step S3, the solid-liquid ratio is 1:6-1:

9.

5. The anthocyanin extraction method according to claim 1 or 4, characterized by, In step S3, the solid-liquid ratio is 1:

7.

6. The anthocyanin extraction method according to claim 1, characterized by, In step S3, the ultrasonic temperature is 45-50℃.

7. The anthocyanin extraction method according to claim 1 or 6, characterized by, In step S3, the ultrasonic temperature is 50℃.

8. The anthocyanin extraction method of claim 1, wherein In step S3, the ultrasonic time is 35-40min.

9. The anthocyanin extraction method according to claim 1 or 8, characterized by, In step S3, the ultrasonic time is 39min.

10. Use of anthocyanins, characterized in that The anthocyanin obtained by the method of claims 1-9 is used in food, cosmetics, skin care products, and livestock and poultry additives.

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