Eutectic solvent and method for extracting anthocyanin from waxberry residues

Abstract

The invention relates to the technical field of anthocyanin extraction, in particular to a eutectic solvent and a method for extracting anthocyanin from waxberry residues. According to the deep-eutectic solvent, the extraction efficiency and stability of the anthocyanin in the waxberry residues are remarkably improved, the deep-eutectic solvent is acidic, the stability of the anthocyanin in the extraction process is better protected, degradation or inactivation of the anthocyanin caused by acid-base environment changes is reduced, and therefore the purity and quality reliability of the anthocyanin are improved. Besides, the method disclosed by the invention also avoids the use of harmful solvents and extreme conditions, reduces environmental pollution and energy consumption, realizes cyclic utilization of resources, and conforms to the concepts of green chemistry and sustainable development.

Description

Technical Field

[0001] The invention relates to the technical field of anthocyanin extraction, and in particular to a low eutectic solvent and a method for extracting anthocyanin from bayberry residue. Background Art

[0002] Anthocyanins are a class of compounds formed by the glycosidic bond between anthocyanins and sugars. They are widely found in the cell fluid of flowers, fruits, stems, leaves and roots of plants, making them appear red, purple, blue, etc. Anthocyanins are a natural water-soluble pigment. Due to their unique functionality, they are used to remove free radicals in the body, proliferate lutein, fight tumors, fight cancer, fight inflammation, inhibit lipid peroxidation and platelet aggregation, prevent diabetes, lose weight and protect eyesight.

[0003] Bayberry residue, as a byproduct of bayberry processing, is rich in natural pigments such as anthocyanins and has high nutritional value and application potential. However, the traditional process of extracting anthocyanins using organic solvents often has problems such as low extraction efficiency and poor stability. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a deep eutectic solvent and method for extracting anthocyanins from bayberry residue. The deep eutectic solvent of the present invention can efficiently extract anthocyanins from bayberry residue and at the same time can ensure that the anthocyanins have good stability.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a low eutectic solvent for extracting anthocyanins from bayberry residue, comprising a hydrogen bond acceptor and a hydrogen bond donor; or comprising a hydrogen bond acceptor, a hydrogen bond donor and water;

[0007] The pH of the deep eutectic solvent is ≤7;

[0008] When the deep eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is betaine, the hydrogen bond donor is lactic acid, and the molar ratio of betaine to lactic acid is 1:(2-3);

[0009] When the low eutectic solvent comprises a hydrogen bond acceptor, a hydrogen bond donor and water, the water content in the low eutectic solvent is ≤30wt% and is not 0, the hydrogen bond acceptor is betaine, and the hydrogen bond donor is lactic acid, malic acid or citric acid;

[0010] When the water content in the low eutectic solvent is 10 wt %, the hydrogen bond donor is lactic acid, and the molar ratio of betaine to lactic acid is 1:(1-3);

[0011] When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is lactic acid, the molar ratio of betaine to lactic acid is (1-2):(1-3);

[0012] When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is malic acid, the molar ratio of betaine to malic acid is (1-2):(1-3);

[0013] When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is citric acid, the molar ratio of betaine to citric acid is (1-2): (1-2);

[0014] When the water content in the deep eutectic solvent is 30 wt %, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2):(1-3).

[0015] The present invention also provides a method for extracting anthocyanins from bayberry residue, comprising the following steps:

[0016] The bayberry residue is placed in a deep eutectic solvent for extraction to obtain an anthocyanin solution;

[0017] The low eutectic solvent is the low eutectic solvent described in the above technical solution.

[0018] Preferably, before placing the bayberry residue in a low eutectic solvent, the method further comprises freeze-drying, grinding and sieving the bayberry residue in sequence.

[0019] Preferably, the freeze-drying process is to first freeze at -80°C for 24 hours and then freeze-dry under vacuum for 72 hours.

[0020] Preferably, the extraction is carried out under ultrasonic conditions;

[0021] The power of the ultrasound is 100-400W, the temperature is 30-60°C, and the time is 15-60min.

[0022] Preferably, after the extraction is completed, centrifugation and solid-liquid extraction are repeated in sequence.

[0023] Preferably, the centrifugation temperature is 4°C, the rotation speed is 4000 rpm, and the time is 20 min.

[0024] Preferably, the solid-liquid extraction is to use the low eutectic solvent to leaching the precipitate obtained by centrifugation.

[0025] Preferably, the number of repetitions is ≥ 3 times.

[0026] The invention provides a low eutectic solvent for extracting anthocyanins from bayberry residue, comprising a hydrogen bond acceptor and a hydrogen bond donor; or comprising a hydrogen bond acceptor, a hydrogen bond donor and water; the pH of the low eutectic solvent is ≤7; when the low eutectic solvent comprises the hydrogen bond acceptor and the hydrogen bond donor, the hydrogen bond acceptor is betaine, the hydrogen bond donor is lactic acid, and the molar ratio of the betaine to the lactic acid is 1:(2-3); when the low eutectic solvent comprises the hydrogen bond acceptor, the hydrogen bond donor and water, the water content in the low eutectic solvent is ≤30wt% and is not 0, the hydrogen bond acceptor is betaine, the hydrogen bond donor is lactic acid, malic acid or citric acid; when the water content in the low eutectic solvent is 10wt%, the hydrogen bond donor is lactic acid, the betaine ... The molar ratio of betaine to lactic acid is 1:(1-3); when the water content in the low eutectic solvent is 20wt% and the hydrogen bond donor is lactic acid, the molar ratio of betaine to lactic acid is (1-2):(1-3); when the water content in the low eutectic solvent is 20wt% and the hydrogen bond donor is malic acid, the molar ratio of betaine to malic acid is (1-2):(1-3); when the water content in the low eutectic solvent is 20wt% and the hydrogen bond donor is citric acid, the molar ratio of betaine to citric acid is (1-2):(1-2); when the water content in the low eutectic solvent is 30wt%, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2):(1-3). Anthocyanin, as a highly polar compound, has a significant advantage in solubility in water compared to non-polar solvents. What is more complicated is that the chemical form and stability of anthocyanins are deeply affected by changes in pH. Specifically, under acidic conditions with a pH of 1, anthocyanins mainly exist in the form of chalcanthite cations, giving the solution a purple or red color; when the pH range is adjusted to between 2 and 4, the cationic charge on the C-ring lactone of anthocyanins is weakened, prompting it to transform into a blue quinone base form. There is a reversible mutual conversion relationship between these quinone bases and chalcanthite cations in acidic media. However, as the pH value gradually increases, the structure of anthocyanins begins to change significantly, especially in an alkaline environment with a pH value greater than 7. This structural change will directly lead to the degradation of anthocyanins, thereby adversely affecting their extraction efficiency. In view of this, the present invention innovatively introduces a low eutectic solvent, which not only significantly improves the extraction efficiency of anthocyanins in bayberry residue, but also effectively enhances its stability. The acidic nature of the deep eutectic solvent provides a strong guarantee for the stability of anthocyanins during the extraction process, effectively reducing the degradation or inactivation caused by changes in the acid-base environment, thereby ensuring the high purity and quality reliability of anthocyanins. In addition, the method of the present invention also avoids the use of harmful solvents and extreme conditions, reduces environmental pollution and energy consumption, realizes the recycling of resources, and is in line with the concepts of green chemistry and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The physical appearance pictures of the deep eutectic solvents mentioned in Tables 1 to 4 after being placed at room temperature for 0 hours and 24 hours;

[0028] Figure 2 A bar graph of the surface tension of the deep eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 20, 22, 24, 26 and 27;

[0029] Figure 3 A bar graph showing polarity values ​​of the deep eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 20, 22, 24, 26 and 27;

[0030] Figure 4 The physical appearance of the anthocyanin extract after extraction using the deep eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water described in Comparative Example 27, and the acidified ethanol described in Comparative Example 28;

[0031] Figure 5 The content of anthocyanins in the anthocyanin extract extracted with the deep eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water described in Comparative Example 27, and the acidified ethanol described in Comparative Example 28;

[0032] Figure 6 It is a full wavelength scanning diagram of anthocyanin extracts extracted with the deep eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water described in Comparative Example 27, and the acidified ethanol described in Comparative Example 28;

[0033] Figure 7 is the absorbance value at the maximum absorption wavelength of the anthocyanin extract extracted with the deep eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water described in Comparative Example 27, and the acidified ethanol described in Comparative Example 28;

[0034] Figure 8 The L of anthocyanin extract extracted with the deep eutectic solvents of Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water of Comparative Example 27, and the acidified ethanol of Comparative Example 28 * 、a * and b * value;

[0035] Fig. 9are the Hue° and C* values ​​of the anthocyanin extracts extracted with the deep eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water described in Comparative Example 27, and the acidified ethanol described in Comparative Example 28;

[0036] Fig.10 The degradation patterns of the anthocyanin extract extracted with the deep eutectic solvent described in Example 1 after being stored in the dark at different temperatures;

[0037] Fig.11 The degradation patterns of the anthocyanin extract extracted with the deep eutectic solvent described in Example 21 after being stored in the dark at different temperatures;

[0038] Fig.12 The degradation pattern of the anthocyanin extract extracted with the deep eutectic solvent described in Example 1 after being stored under different light conditions;

[0039] Fig.13 This is the degradation pattern of the anthocyanin extract extracted with the low eutectic solvent described in Example 21 after being stored under different lighting conditions. DETAILED DESCRIPTION

[0040] The present invention provides a low eutectic solvent for extracting anthocyanins from bayberry residue, comprising a hydrogen bond acceptor and a hydrogen bond donor; or comprising a hydrogen bond acceptor, a hydrogen bond donor and water;

[0041] The pH of the deep eutectic solvent is ≤7;

[0042] When the deep eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is betaine, the hydrogen bond donor is lactic acid, and the molar ratio of betaine to lactic acid is 1:(2-3);

[0043] When the low eutectic solvent comprises a hydrogen bond acceptor, a hydrogen bond donor and water, the water content in the low eutectic solvent is ≤30wt% and is not 0, the hydrogen bond acceptor is betaine, and the hydrogen bond donor is lactic acid, malic acid or citric acid;

[0044] When the water content in the low eutectic solvent is 10 wt %, the hydrogen bond donor is lactic acid, and the molar ratio of betaine to lactic acid is 1:(1-3);

[0045] When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is lactic acid, the molar ratio of betaine to lactic acid is (1-2):(1-3);

[0046] When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is malic acid, the molar ratio of betaine to malic acid is (1-2):(1-3);

[0047] When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is citric acid, the molar ratio of betaine to citric acid is (1-2): (1-2);

[0048] When the water content in the deep eutectic solvent is 30 wt %, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2):(1-3).

[0049] In the present invention, by regulating the pH value and water content of the low eutectic solvent, the extraction efficiency of the anthocyanins can be further improved, so that the yield of anthocyanins is significantly improved.

[0050] In the present invention, the preparation method of the low eutectic solvent preferably includes: adding water after mixing the hydrogen bond acceptor and the hydrogen bond donor, heating and stirring, cooling and standing in sequence. The present invention does not have any special restrictions on the mixing process, and the process well known to those skilled in the art can be used. In the present invention, the temperature of the heating and stirring is preferably 60°C to 80°C, more preferably 80°C; the rotation speed is preferably 100rpm to 400rpm, more preferably 300rpm; the present invention does not have any special restrictions on the time of the heating and stirring, and it is ensured that a transparent and uniform mixed solution is formed and no precipitation or crystallization is carried out at room temperature. The present invention does not have any special restrictions on the cooling process, and the process well known to those skilled in the art can be used to cool to room temperature. In the present invention, the standing time is preferably 24h.

[0051] In the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0052] The present invention also provides a method for extracting anthocyanins from bayberry residue, comprising the following steps:

[0053] The bayberry residue is placed in a deep eutectic solvent for extraction to obtain an anthocyanin solution;

[0054] The deep eutectic solvent is the deep eutectic solvent described in the above technical solution,

[0055] Before placing the bayberry residue in a low eutectic solvent, the present invention preferably further comprises freeze-drying, grinding and sieving the bayberry residue in sequence.

[0056] In the present invention, the freeze drying process is preferably performed by first freezing at -80°C for 24 hours and then vacuum freeze drying for 72 hours. In the present invention, the above treatment is performed to remove moisture from the material under low temperature and vacuum environment, thereby achieving the purpose of drying.

[0057] The present invention does not have any special limitation on the grinding process, and the grinding process may be performed using a process well known to those skilled in the art.

[0058] In the present invention, the screening is preferably performed through a 100-mesh sieve.

[0059] The present invention preferably further includes vortexing after placing the bayberry residue in the low eutectic solvent. The present invention has no special limitation on the vortexing process, and a process well known to those skilled in the art can be used to ensure that the solvent and the solute are fully mixed.

[0060] In the present invention, the extraction is preferably carried out under ultrasonic conditions, the ultrasonic power is preferably 100W to 400W, more preferably 300W; the temperature is preferably 30°C to 60°C, more preferably 50°C; the time is preferably 15min to 60min, more preferably 30min.

[0061] After the extraction is completed, the present invention also preferably includes centrifugation and solid-liquid extraction repeated in sequence; the centrifugation temperature is preferably 4°C; the centrifugation speed is preferably 4000rpm, and the time is preferably 20min. In the present invention, the solid-liquid extraction is preferably performed by leaching the precipitate obtained by centrifugation with the low eutectic solvent. In the present invention, the number of repetitions is preferably ≥3 times.

[0062] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] Examples 1 to 2 and Comparative Examples 1 to 10

[0064] The hydrogen bond acceptor and the hydrogen bond donor are mixed in a certain molar ratio, and stirred at 80°C at a speed of 300 rpm until a transparent and uniform liquid is obtained, and no precipitation or crystallization occurs at room temperature; cooled to room temperature and allowed to stand for 24 hours to obtain a low eutectic solvent (wherein the types and molar ratios of the hydrogen bond acceptor and the hydrogen bond donor, pH value and other related parameters are shown in Table 1)

[0065] Table 1 Related parameters, physical properties and appearance of the deep eutectic solvents of Examples 1 to 12 and Comparative Examples 1 to 10 after standing for 0 h and 24 h at room temperature

[0066]

[0067]

[0068] Examples 3 to 5 and Comparative Examples 11 to 19

[0069] After mixing the hydrogen bond acceptor and the hydrogen bond donor in a certain molar ratio, water is added, and the mixture is stirred at 80° C. and 300 rpm to obtain a transparent and uniform liquid, which does not precipitate or crystallize at room temperature; the mixture is cooled to room temperature and allowed to stand for 24 hours to obtain a low eutectic solvent (wherein the water content in the low eutectic solvent is 10 wt %, and the types and molar ratios of the hydrogen bond acceptor and the hydrogen bond donor, pH value and other related parameters are shown in Table 2);

[0070] Table 2 Related parameters, physical properties and appearance of the deep eutectic solvents described in Examples 3 to 5 and Comparative Examples 11 to 19 after being placed at room temperature for 0 h and 24 h

[0071]

[0072]

[0073] Examples 6 to 15 and Comparative Examples 20 to 21

[0074] With reference to Examples 3 to 5, a deep eutectic solvent was obtained (wherein the water content in the deep eutectic solvent was 20 wt %, and the types and molar ratios of the hydrogen bond acceptors and hydrogen bond donors, pH value and other related parameters were shown in Table 3);

[0075] Table 3 Related parameters, physical properties and appearance of the deep eutectic solvents of Examples 6 to 15 and Comparative Examples 20 to 21 after standing for 0 h and 24 h at room temperature

[0076]

[0077]

[0078] Examples 16 to 27

[0079] Referring to Examples 3 to 5, a deep eutectic solvent was obtained (wherein the water content in the deep eutectic solvent was 30 wt %, and the types and molar ratios of the hydrogen bond acceptors and hydrogen bond donors, pH value and other related parameters were shown in Table 4)

[0080] Table 4 Parameters, physical properties and appearance of the deep eutectic solvents described in Examples 16 to 27 after standing at room temperature for 0 h and 24 h

[0081]

[0082]

[0083] Note: The test results in Tables 1 to 4 are expressed as mean ± standard deviation. The values ​​of the same index with different letters in different samples are significantly different (p < 0.05).

[0084] Comprehensive Tables 1 to 4 and Figure 1As shown, Examples 1 and 2 are clear and uniform liquids. After cooling to room temperature and standing for 24 hours, they remain stable and do not recrystallize; generally, the mixture formed at a specific eutectic composition establishes a balanced intermolecular force between HBA and HBD through hydrogen bond interactions. These interactions form a stable molecular structure within the eutectic system, thereby preventing the formation of any crystals. When Bet is in excess (2:1) or in an equal molar ratio with LA (1:1), the mixture cannot form a liquid, and solid crystals exist in the mixture. At all four different molar ratios (2:1, 1:1, 1:2 and 1:3), Bet-MA is a white paste, while Bet-CA is always a white solid. When the water content is increased to 10%, it is observed that only Bet-LA forms a liquid at a ratio of 1:1, 1:2 and 1:3, and is solid at a molar ratio of 2:1. Similarly, Bet-MA and Bet-CA are in solid form at all four molar ratios (2:1, 1:1, 1:2 and 1:3). When the water content was further increased to 20%, Bet-LA gradually formed liquids at four molar ratios (2:1, 1:1, 1:2 and 1:3), presenting as clear and uniform liquids. However, within 24 h after synthesis, Bet-LA (2:1) was found to crystallize, and the same phenomenon was observed for Bet-MA (1:3). This may be due to the lack or excess of one of the two components in the system, resulting in weak intermolecular interactions between the two components. Bet-MA and Bet-CA also gradually formed liquids at molar ratios of 2:1, 1:1 and 1:2, but they were highly viscous, with viscosity values ​​ranging from 471.25±0.22cP to 1104.33±0.34cP (Table 2). In fact, compared with other organic solvents, deep eutectic solvents (DESs) exhibit higher viscosities, which is attributed to the presence of hydrogen bond networks inside the compounds. These networks limit the mobility of free radicals in DESs, resulting in reduced extraction efficiency. This is undesirable and difficult to manipulate in subsequent studies, so they were excluded from subsequent experiments. However, the viscosity of DESs is tunable. Usually, during the extraction process in the food industry, an appropriate amount of water is added to reduce the viscosity of DESs and optimize the mass transfer between solid and liquid. Studies have shown that a strategy of adding 30% (v / w) water can be used to reduce the viscosity of the DESs system. When the temperature increases, the DESs molecules gain enough kinetic energy to overcome the intermolecular segmental motion, resulting in weakened van der Waals forces and hydrogen bonding interactions, thereby exhibiting good fluidity. It was found that Bet-LA, Bet-MA, and Bet-CA all formed transparent liquids at four molar ratios (2:1, 1:1, 1:2, and 1:3). However, Bet-MA (1:3) was observed to crystallize within 24 hours after synthesis, and Bet-CA (1:1) had a high viscosity (760.10 ± 0.16 cP). Therefore, they were excluded from subsequent experiments.The pH values ​​of 28 combinations of acidic deep eutectic solvents (ADESs) ranged from 1.53 to 5.08. Studies have shown that pH values ​​greater than 4 are not conducive to the extraction of anthocyanins. The experimental groups including Bet-LA (10% water, 1:1) with a pH value of 4.32, Bet-LA (20% water, 1:1) with a pH value of 4.09, Bet-LA (20% water, 2:1) with a pH value of 5.08, and Bet-LA (30% water, 2:1) with a pH value of 4.86 all had pH values ​​greater than 4. Therefore, they were also excluded from subsequent studies. After excluding ADESs combinations with high viscosity and high pH values, the remaining 15 combinations were used for subsequent anthocyanin extraction. The results were also compared with acidified water and acidified ethanol, the two most suitable and environmentally friendly traditional solvents.

[0085] From Tables 1 to 4, we can see that the density of ADESs is in the range of 1.15 ± 0.007 g / cm 3 To 1.68±0.016g / cm 3It was observed that the density of ADESs was greater than that of water. This was attributed to the presence of different functional groups in LA, MA, and CA, and their different abilities to form hydrogen bonds with Bet. The density of the ADESs mixture was affected by its molecular structure. At the same water content, the order of density of the three ADESs combinations was Bet-CA>Bet-MA>Bet-LA. This may be because CA, a tricarboxylic acid with three carboxyl groups (-COOH) as hydrogen bond donors, exhibited higher flexibility and diversity in forming hydrogen bonds with anions, followed by MA and LA. This suggests that the density of DESs has an important relationship with the structure of HBD. The stronger the interaction between HBD and anions, the lower the mobility of the internal molecules, thereby increasing the density of DESs. For the same type of DESs, under the same molar ratio conditions, the observed density follows a specific rule: ADESs with a water content of 0% have the highest density, followed by ADESs with water contents of 10%, 20%, and 30%. This phenomenon may be due to the fact that the intermolecular interactions within DESs become more intense and complex as the water content in the system gradually decreases when the type and molar ratio of HBD remain unchanged. When the water content is low, the interactions between the solute molecules in the DESs system dominate. These molecules are tightly bound together by hydrogen bonds, van der Waals forces, and other interactions to form a relatively ordered and compact molecular network structure. This tight molecular arrangement increases the viscosity of the entire system because the molecular motion is restricted. Density, as a basic physical property of matter, is closely related to factors such as molecular mass, intermolecular distance, and the tightness of molecular arrangement. Therefore, when the water content is low and the system viscosity is high, the density of ADESs increases accordingly. As the water content gradually increases, water molecules are inserted into the molecular network of ADESs as solvent molecules, destroying the original intermolecular interactions and arrangement. These water molecules not only increase the total volume of the system, but also weaken the intermolecular forces between the solute molecules through solvation, resulting in a decrease in the viscosity of the system. Therefore, at higher water content, the density of ADESs decreases accordingly. When examining the density relationship of ADESs mixtures with the same water content and the same HBD, it was found that the density of ADESs reached a maximum value when the molar ratio was 1:3, and the density of ADESs also increased with the increase in the proportion of organic acid. This may be because as the proportion of organic acid increases, the number of hydrogen bonds in ADESs increases, which strengthens the intermolecular force and makes the molecular arrangement more compact and orderly, thereby further increasing the density. Therefore, during the extraction process, it is necessary to comprehensively consider the effects of HBD structure, water content, and the molar ratio of HBA to HBD on the density of DESs;

[0086] Figure 2Surface tension bar graphs of the ADESs described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 20, 22, 24, 26 and 27; Figure 3 A bar graph showing the polarity values ​​of the ADESs described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 20, 22, 24, 26 and 27; Figure 2 It can be seen that the surface tension of different types of ADESs varies significantly due to the HBD component. Specifically, when Bet-CA (20% water, 1:2) was used, the surface tension reached the highest level (71.63±0.02 mN / m) (p<0.05), followed by Bet-LA (0% water, 1:2), whose surface tension was 70.95±0.02 mN / m. However, Bet-LA (30% water, 1:1) had the lowest surface tension (42.92±0.05 mN / m). For other types of ADESs, their surface tensions ranged from 46.15±0.06 mN / m to 68.51±0.02 mN / m. In summary, the surface tension of ADESs is affected by multiple factors such as HBD components and proportions, water content, etc., which in turn affects its efficiency in the extraction process; Figure 3 It can be seen that the polarity of ADESs has a direct effect on the solubility of anthocyanins. Solvents with appropriate polarity can more effectively penetrate into the plant cell wall and interact with anthocyanin molecules, thereby extracting them from the cells. In addition, these solvents can also provide a stable extraction environment, reduce the degradation and transformation of anthocyanin molecules, thereby retaining their original biological activity and application value. Bet-LA (0% water, 1:2) has the lowest E NR value (47.57±0.06 kcal / mol), corresponding to the highest polarity. It was followed by Bet-CA (30% water, 1:2) and Bet-LA (20% water, 1:2), with E NR The values ​​were 47.62±0.04 kcal / mol and 47.89±0.13 kcal / mol, respectively. In this study, Bet-LA (30% water, 1:1) appears to be the least polar solvent, with its E NR The value is 50.16±0.07 kcal / mol. NR The values ​​ranged from 48.87 ± 0.27 kcal / mol to 49.90 ± 0.14 kcal / mol;

[0087] In summary, there is a certain positive correlation between the surface tension and polarity of DESs, that is, DESs with higher surface tension tend to have relatively higher polarity. However, it is worth noting that this positive correlation is not absolute. In some cases, due to the complexity and diversity of the DESs system, there may be a certain degree of decoupling or nonlinear relationship between surface tension and polarity. For example, some DESs may exhibit relatively low polarity while maintaining a high level of surface tension through special intermolecular interactions or spatial configurations. This complex relationship may be due to multiple factors such as the diversity of hydrogen bond structures within the DESs, the influence of water content, and the interaction between HBD components.

[0088] Comparative Examples 22 to 26

[0089] With reference to Examples 3 to 5, a deep eutectic solvent was obtained (wherein the water content in the deep eutectic solvent was 30 wt %, and the types and molar ratios of the hydrogen bond acceptors and hydrogen bond donors, pH value and other related parameters were shown in Table 5);

[0090] Table 5 Related parameters of the deep eutectic solvents described in Examples 18, 20, 25 and Comparative Examples 22 to 26

[0091]

[0092]

[0093] Comparative Example 27

[0094] The water was adjusted to pH 3 with 1 mol / L hydrochloric acid to obtain acidified water.

[0095] Comparative Example 28

[0096] The pH of ethanol was adjusted to 3 with 1 mol / L hydrochloric acid to obtain acidified ethanol.

[0097] Embodiment 28

[0098] The bayberry residue was placed in a refrigerator at -80°C for 24 hours and then vacuum dried for 72 hours. The dried material was ground in a laboratory mill and sieved through a 100-mesh sieve to obtain the pretreated bayberry residue.

[0099] The pretreated bayberry residue was dispersed into the ADESs described in Examples 1 to 27 and the DESs described in Comparative Examples 1 to 28, vortexed for 1 min, ultrasonicated at 300 W and 50° C. for 30 min, and centrifuged at 4° C. and 400 rpm for 20 min to collect the extract, and the separated precipitate was subjected to solid-liquid extraction using 10 mL of the ADESs described in Examples 1 to 27 and the DESs described in Comparative Examples 1 to 28, the above centrifugation and separation process was repeated, and the obtained supernatants were mixed to obtain anthocyanin extracts;

[0100] The content of total anthocyanins (TAC) in the anthocyanin extract was determined by the pH difference method: 10 μL of the anthocyanin extract was added to the ELISA plate, and then 190 μL of a buffer solution of pH 1.0 (prepared by mixing 0.2 mol / L KCl and 0.2 mol / L HCl in a ratio of 25:67) and pH 4.5 (prepared by mixing 0.2 mol / L sodium acetate and 0.2 mol / L acetic acid in a ratio of 1:1) were added respectively. After mixing, the solution was allowed to stand in the dark for 1 hour. Then, the prepared solution was measured for fluorescence at two different wavelengths of 521 nm and 700 nm using an ELISA instrument. TAC is expressed as milligrams of cyanidin-3-glucoside equivalent per 100 grams of dry weight (mg CGE / g DW). The anthocyanin content was calculated according to formula (1):

[0101]

[0102] Where A represents the absorbance value of anthocyanin, and the calculation formula is A=(A 521pH1.0 -A 700pH1.0 )-(A 521pH4.5 -A 700pH4.5 ), where A 521pH1.0 A is the absorbance value measured at 521 nm after dilution in a buffer solution with a pH of 1.0. 700pH1.0 A is the absorbance value measured at 700 nm after dilution in a buffer solution with a pH of 1.0. 521pH4.5 A is the absorbance value measured at 521 nm after dilution in a buffer solution with a pH of 4.5. 700pH4.5 is the absorbance value measured at 700 nm after dilution in a buffer solution with a pH of 4.5; Mw is the molar mass of cyanidin-3-glucoside, which is 449.2 g / mol; DF is the dilution factor, which is 20; V is the volume of the extract, in milliliters (mL); ε is the molar extinction coefficient of cyanidin-3-glucoside, which is 26900 L / mol·cm; 1 is the optical path length, in centimeters (cm); m is the mass of the sample, in grams (g); 10 3 is the conversion factor from grams to milligrams.

[0103] Figure 4 The physical appearance of the anthocyanin extract after extraction using the deep eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water described in Comparative Example 27, and the acidified ethanol described in Comparative Example 28, Figure 5 The content of anthocyanins in the anthocyanin extract extracted with the deep eutectic solvents of Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water of Comparative Example 27, and the acidified ethanol of Comparative Example 28 is determined by Figures 4-5 It can be seen that most ADESs showed higher extraction efficiency than the traditional solvent water (0.97±0.08mg CGE / g DW). This finding shows that ADESs have significant advantages in improving the extraction efficiency of anthocyanins. Specifically, when acidified ethanol (1.27±0.06mg CGE / g DW) was used as a comparative reference, the extraction efficiency of three ADESs (i.e., Bet-LA, Bet-MA, and Bet-CA) was significantly improved (p<0.05). Among all the ADESs tested, Bet-LA (0% water, 1:2) was particularly outstanding. The TAC extracted from bayberry residue using it was as high as 7.28±0.06mg CGE / g DW. This value is 7.5 times and 5.7 times that of acidified water and acidified ethanol, respectively, fully demonstrating its excellent extraction ability. It was closely followed by Bet-CA (30% water, 1:2), which also performed well with a TAC of 7.05±0.07mg CGE / g DW. Although the extraction efficiencies of the remaining ADESs varied, their TAC values ​​ranged from 1.47±0.03mg CGE / g DW to 6.28±0.46mg CGE / g DW. This significant difference (p<0.05) suggests that the composition and proportion of ADESs have a crucial impact on their ability to extract anthocyanins. It is worth noting that although Bet-LA (0% water, 1:2) and Bet-CA (30% water, 1:2) did not show statistically significant differences in extraction efficiency (p>0.05), the stability of the solvent in practical applications is equally crucial. Therefore, the stability of anthocyanins in these two solvents under different temperature and light conditions was further studied, aiming to achieve more breakthroughs in the efficient extraction and stable preservation of anthocyanins;

[0104] Figure 6 The full wavelength scans of anthocyanin extracts extracted with the deep eutectic solvents of Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water of Comparative Example 27, and the acidified ethanol of Comparative Example 28 are shown in FIG. Figure 6It can be seen that the corresponding extract was obtained and full wavelength scanning analysis was performed. Figure 6 The experimental results shown in the figure show that all extracts exhibited a maximum absorption peak at a specific wavelength of 530nm. The absorption characteristics of anthocyanins at 530nm mainly represent their chrysanthemum salt cationic aglycone structure, which is one of the basic structures of anthocyanins and a key factor in determining their color. The process of extracting anthocyanins using the ADES solvent system did not change the original properties of anthocyanins in bayberry residues, and their molecular structures remained consistent before and after extraction, proving the stability and reliability of the ADES solvent system in extracting anthocyanins;

[0105] Figure 7 is the absorbance value at the maximum absorption wavelength of the anthocyanin extract extracted with the deep eutectic solvents of Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water of Comparative Example 27, and the acidified ethanol of Comparative Example 28, Figure 7 It can be seen that although all anthocyanin extracts showed similar absorption characteristics at 530nm, further comparative analysis showed that there were significant differences in the absorbance values ​​(OD) between different groups (p < 0.05), which indicates that although the structure of anthocyanins remains unchanged, their concentrations in their respective solutions are different;

[0106] Figure 8 The L values ​​of the anthocyanin extracts extracted with the deep eutectic solvents of Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water of Comparative Example 27, and the acidified ethanol of Comparative Example 28 at the maximum absorption wavelength are shown in Table 1. * 、a * and b * Value; L * The value represents brightness, and its value directly reflects the brightness of the extract. * The higher the value, the brighter the extract, which is particularly useful for capturing subtle brightness differences between various extracts. * The a and b values ​​describe the color change between red and green and between yellow and blue, respectively. * A positive value indicates that the extract has a red hue, and a larger value indicates a darker red. * The positive and negative values ​​correspond to yellow and blue respectively; this division of color space helps to grasp the color characteristics of the extracts more comprehensively and accurately. In order to intuitively display these color characteristics, this study used the L values ​​of all ADESs extracts. * 、a * and b *The values ​​are used as the x, y, and z axes, respectively, to construct a three-dimensional color space. In addition, we also use the absorbance value of the extract at 530nm as the color mapping index of each corresponding sphere, thereby realizing a three-dimensional visualization of the color characteristics of the extract; Figure 8 It can be seen that the L * The values ​​range from 23.39 to 60.70, indicating that there is some difference in brightness. In terms of color change between red and green, a * The values ​​vary from 6.41 to 50.83, while b * The values ​​ranged from 2.45 to 34.25. Among them, Bet-LA (0% water, 1:2) obtained the largest a * The value (50.83 ± 0.06) indicates that its red color is the deepest. This result is consistent with the highest absorbance value of the extract at 530nm under this condition, further verifying the accuracy of its color characteristics. In addition, the projection point of Bet-LA (0% water, 1:2) in the three-dimensional color space diagram is relatively far from the projection points of other samples, indicating that there is a significant color difference compared with other samples. The extract closest in color to Bet-LA (0% water, 1:2) is Bet-CA (30% water, 1:2), which has a * The value was 45.39±0.02. Compared with all ADESs anthocyanin extracts, the L * The value increased significantly, while a * and b * The value was significantly decreased (p<0.05);

[0107] Fig. 9 The Hue° and C* values ​​of the anthocyanin extracts extracted with the low eutectic solvents described in Examples 1, 2, 4, 5, 7, 8, 17, 18, 19, 21, 22, 23, 24, 26, and 27, the acidified water described in Comparative Example 27, and the acidified ethanol described in Comparative Example 28 at the maximum absorption wavelength; wherein Hue° represents the angular change in hue, generally between 0 and 180. Specifically, a Hue° of 0 corresponds to purple-red, 90 corresponds to yellow, and 180 corresponds to green. When Hue° is greater than 100, the larger the value, the greener the color; when Hue° is less than 50, the smaller the value, the redder the color. C* represents the vertical distance from the test color to the L axis; the larger the distance, the higher the color saturation; Fig. 9It can be seen that the Hue° values ​​are between 1.25 and 2.55, while the C* values ​​are between 6.86 and 61.30. Among them, the Bet-LA (0% water, 1:2) extract has the smallest Hue° (1.25±0.088) and the largest C* (61.30±0.088), indicating that its color is the darkest red and the hue is the brightest. Compared with the chromaticity values ​​of all anthocyanin extracts extracted using the low eutectic solvent of the present invention, the samples of the acidified water and acidified ethanol control groups show L * There is a trend of increasing ΔH values, decreasing C* values, and increasing Hue° values;

[0108] Fig.10 The degradation patterns of the anthocyanin extract extracted with the deep eutectic solvent described in Example 1 after being stored in the dark at different temperatures are shown in FIG. Fig.11 The degradation patterns of the anthocyanin extract extracted with the deep eutectic solvent described in Example 21 after being stored in the dark at different temperatures; Fig.12 The degradation pattern of the anthocyanin extract extracted with the deep eutectic solvent described in Example 1 after being stored under different light conditions; Fig.13 The degradation patterns of the anthocyanin extract extracted with the deep eutectic solvent described in Example 21 after being stored under different light conditions; Figures 10-11 It can be seen that the anthocyanins extracted from bayberry residue using the low eutectic solvent of the present invention basically follow the first-order reaction kinetics under different temperature and light conditions, and the correlation coefficient is greater than 85%. For Bet-LA (0% water, 1:2) and Bet-CA (30% water, 1:2), the highest degradation rate was observed at 80°C, with k values ​​of 0.1238 and 0.1169, respectively, indicating that anthocyanins are lost rapidly. However, they show the greatest stability at lower temperatures, especially at 4°C, where their k values ​​are the lowest, 0.0015 and 0.0016, respectively, indicating that the degradation rate of anthocyanins is slower. 1 / 2 The value (i.e., the time required for the anthocyanin concentration to be halved) is inversely proportional to the k value. Bet-LA (0% water, 1:2) and Bet-CA (30% water, 1:2) at 4°C 1 / 2The longest, reaching 462.10h and 433.22h respectively, indicating enhanced stability. In contrast, although Bet-CA (30% water, 1:2) had a higher degradation rate at 4°C, its t1 / 2 values ​​were approximately 0.14 days and 0.33 days higher than Bet-LA (0% water, 1:2) at other experimental temperatures, especially 60°C and 80°C. These results indicate that although the initial concentration is important, the degradation rate and the time required for significant degradation are more important for evaluating the effectiveness of the solvent. Bet-CA (30% water, 1:2) has a lower degradation rate and a longer half-life, making it the most effective solvent for preserving anthocyanins under different storage conditions. However, few studies have considered storage time and temperature at the same time, indicating a research gap. It is generally believed that at freezing temperatures (-18°C or below), the degradation rate of anthocyanins slows down due to the inhibition of metabolism and enzyme activity, thereby maintaining stability over time. However, the freezing process may affect the plant cell structure, thereby affecting the stability of anthocyanins. At refrigerator temperature (4-5°C), enzyme activity is not effectively inhibited, which may accelerate degradation, which is consistent with the pattern observed in this study; Figures 12-13 It can be seen that the anthocyanin concentration and degradation trend of Bet-LA (0% water, 1:2) and Bet-CA (30% water, 1:2) under different lighting conditions. It can be seen that after 7 days of storage in the dark, the anthocyanins extracted using different solvent systems are relatively stable, and the preservation rate remains above 90%. Under natural light, the degradation rate of anthocyanin extracts is slow, but the extract obtained using Bet-CA (30% water, 1:2) decreases more than Bet-LA (0% water, 1:2). Direct sunlight has a significant effect on the preservation rate of anthocyanins, and long-term exposure to sunlight causes significant degradation of anthocyanins extracted using different solvents. Studies have shown that long-term exposure to sunlight can cause photodegradation, which may be due to the high content of non-acylated anthocyanins. Sunlight interacts with water molecules and anthocyanin molecules, converting their ground state to an excited state. Fluorescence may have a more destructive effect on anthocyanin content, but short-term exposure to light has little effect on anthocyanins. An important observation was that the degradation rate of anthocyanins was faster under UV exposure compared to samples stored in sunlight, with a loss rate of nearly 95.0%. Therefore, it is recommended to store anthocyanin extracts in amber, sealed containers. Based on the analysis of all observed indicators, including physicochemical properties, extraction efficiency, anthocyanin stability, and solvent cost, Bet-CA (30% water, 1:2) was selected as the optimal solvent for the separation of anthocyanins from bayberry residue.

[0109] In summary, the present invention uses betaine (Bet) as the core component and efficiently extracts anthocyanins from bayberry residue through ultrasonic-assisted extraction (UAE) technology. From the perspective of green chemistry, the combination strategy of ADES and UAE not only takes advantage of DESs as a non-toxic and biodegradable extractant, but also reduces solvent consumption, energy consumption and processing time, thereby enhancing its competitiveness and practical feasibility in industrial applications. Overall, the betaine-based ADES system provides an environmentally friendly and efficient method for extracting anthocyanins from bayberry residue, while ensuring the stability of anthocyanins.

[0110] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A deep eutectic solvent for extracting anthocyanins from bayberry residue, characterized in that: comprising a hydrogen bond acceptor and a hydrogen bond donor; or comprising a hydrogen bond acceptor, a hydrogen bond donor and water; The pH of the deep eutectic solvent is ≤7; When the deep eutectic solvent comprises a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is betaine, the hydrogen bond donor is lactic acid, and the molar ratio of betaine to lactic acid is 1:(2-3); When the low eutectic solvent comprises a hydrogen bond acceptor, a hydrogen bond donor and water, the water content in the low eutectic solvent is ≤30wt% and is not 0, the hydrogen bond acceptor is betaine, and the hydrogen bond donor is lactic acid, malic acid or citric acid; When the water content in the low eutectic solvent is 10 wt %, the hydrogen bond donor is lactic acid, and the molar ratio of betaine to lactic acid is 1:(1-3); When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is lactic acid, the molar ratio of betaine to lactic acid is (1-2):(1-3); When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is malic acid, the molar ratio of betaine to malic acid is (1-2):(1-3); When the water content in the deep eutectic solvent is 20 wt % and the hydrogen bond donor is citric acid, the molar ratio of betaine to citric acid is (1-2): (1-2); When the water content in the deep eutectic solvent is 30 wt %, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2):(1-3).

2. A method for extracting anthocyanins from bayberry residue, characterized in that: The following steps are involved: The bayberry residue is placed in a deep eutectic solvent for extraction to obtain an anthocyanin solution; The deep eutectic solvent is the deep eutectic solvent according to claim 1.

3. The method according to claim 2, characterized in that Before placing the bayberry residue in a low eutectic solvent, the method further includes freeze-drying, grinding and sieving the bayberry residue in sequence.

4. The method according to claim 3, characterized in that The freeze-drying process is to first freeze at -80°C for 24 hours and then freeze-dry in vacuum for 72 hours.

5. The method according to claim 2, characterized in that The extraction is carried out under ultrasonic conditions; The power of the ultrasound is 100-400W, the temperature is 30-60°C, and the time is 15-60min.

6. The method according to claim 5, characterized in that After the extraction is completed, centrifugation and solid-liquid extraction are repeated in sequence.

7. The method according to claim 6, characterized in that The centrifugation temperature is 4°C, the rotation speed is 4000 rpm, and the time is 20 min.

8. The method according to claim 6, characterized in that The solid-liquid extraction is to use the low eutectic solvent to leaching the precipitate obtained by centrifugation.

9. The method according to any one of claims 6 to 8, characterized in that: The number of repetitions is ≥ 3 times.

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