How to extract astaxanthin from Antarctic krill shells by ultrasound-assisted extraction?

 Astaxanthin (3,3′-dihydroxy-4,4′-diketo-β,β,′-carotene) contains conjugated double bonds, β-violet ketone rings and hydroxyl groups in its molecular structure, and is known as the "strongest antioxidant" and the "super-vitamin E"[1] . In addition, astaxanthin has anti-lipid oxidation, anti-inflammatory, anti-diabetic, cardiovascular disease prevention, immunomodulation and anticancer effects, and has demonstrated high economic value and broad application prospects in the fields of feed, food, pharmaceuticals, and cosmetics [2-4]. Currently, astaxanthin is commercially available through chemical synthesis and natural extraction, and more than 90% of astaxanthin is chemically synthesized. However, there is a big gap between chemically synthesized astaxanthin and natural astaxanthin in terms of structure, function, application and safety.

 

In recent years, people's growing awareness of food safety and the government's strict monitoring of astaxanthin safety have made the acquisition of natural astaxanthin from biological resources the focus of extensive attention. However, the alkaline extraction method of serious corrosion of equipment; organic solvent method of reagent residues, the risk of pollution of the environment; vegetable oil viscosity is not conducive to astaxanthin separation and purification. In addition, the traditional extraction process causes partial degradation of astaxanthin due to high temperature and long time, which is not conducive to the maintenance of its stability and biological activity [5]. Due to the weak research base of extraction technology, the production of natural astaxanthin is very limited, so it is far from being able to meet the demand of domestic and foreign markets. Exploring the ideal solvent system for extracting natural astaxanthin has become a technical bottleneck in this field.

 

Ionic liquids have the advantages of low melting point, good thermal stability, strong solvency and designability, and have been successfully used in the extraction of astaxanthin and other natural active substances[6-7] . In addition, Martins et al.[8] showed that ionic liquids do not cause cellular damage at low and medium doses, and they have good potential for use in the food industry. However, ionic liquids alone are not only expensive as extraction agents, but also have high viscosity and are usually used as additives to organic solvents during the extraction process[9-10] . For example, Desai et al [11] used 1-ethyl-3-methylimidazolium dibutyl phosphate aqueous solution (40% by mass) supplemented with ethyl acetate to extract astaxanthin from Rhodococcus pyrenoidus at 45 ℃, but the extraction rate was only 70% due to the unstable nature of the extraction system.

 

Microemulsion is a thermodynamically stable, isotropic and transparent homogeneous dispersion of two immiscible liquids under the action of surfactant interfacial membrane[12] . In recent years, ionic liquid microemulsion systems have been widely used in a variety of fields such as extraction [13-14], catalytic [15], enzymatic processes [16], nanomaterials [17] and biomass pretreatment [18]. For example, Cao et al[13] used 30 μL of sodium alginate sulfate mixed with 0.005 g of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and added 270 μL of 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6) as the aqueous phase to construct ionic liquid microemulsion, and extracted low-density lipoproteins (LDLs) from bovine serum albumin (BSA) with an extraction rate of 83% at room temperature. Similarly, Shu et al. [19] used 50 μL of AOT aqueous solution (0.5 mol/L) and 450 μL of [Bmim]PF6 to construct an ionic liquid reversed-phase microemulsion, and the extraction rate of hemoglobin was as high as 96% after shaking for 10 min at room temperature.

 

Nevertheless, the research of existing microemulsion systems is limited to the use of imidazolium-based ionic liquids as the main component, and the characteristics of microemulsion are more suitable for the extraction of hydrophilic molecules. More importantly, there are still many basic scientific problems that have not been solved for the extraction of natural astaxanthin by microemulsion systems, such as: first, astaxanthin is not soluble in water, and the water content of aqueous microemulsion systems is as high as 50%, so how to provide internal conditions suitable for the dissolution of astaxanthin? Second, the stability of astaxanthin is affected by multiple factors, so how to accurately regulate the external environment conducive to astaxanthin extraction? This is a common problem in basic and applied basic research on microemulsion extraction of hydrophobic molecules.

 

In this paper, in response to the urgent demand for the extraction of natural astaxanthin from biological resources, the technical bottlenecks faced by the traditional extraction methods and the new challenges faced by the modern microemulsion extraction system, a new microemulsion system was constructed by replacing imidazolium ionic liquid with phosphonate ionic liquid (the chemical structure is shown in Fig. 1), and replacing hydrophilic surfactant with lecithin, and ultrasonication-assisted extraction of astaxanthin from the shells of Antarctic krill was used. The effects of three factors on the phase behavior of the microemulsion, namely, the type of ionic liquid, the ratio of surfactant to co-surfactant (Km), and the temperature, were investigated, and the microstructure of the microemulsion system was characterized by the methods of conductivity and dynamic light scattering. Then, the natural astaxanthin was extracted from Antarctic krill shells by using ionic liquid microemulsion as the solvent system to study the extraction rate of astaxanthin under different conditions and the effect of temperature on the stability of astaxanthin. Finally, by analyzing the structural characteristics of shrimp shell residues after astaxanthin extraction, the mechanism of astaxanthin extraction from Antarctic krill shells by ionic liquid microemulsion was revealed.

 

1 Test materials and methods

1.1 Materials

Antarctic krill, Zhanjiang City, Guangdong Province, Guolian Aquatic Food Company Limited; lecithin (from soybeans, purity >98%), n-butanol (purity >99.5%), astaxanthin standard (purity >98%), Aladdin Chemical Reagent Company, Shanghai; tetrabutylphosphonium trifluoride ([P4444]CF3COO), octyltributylphosphonium bromide ([ P4448]Br) (purity >95%), Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences; all organic solvents used for separation were domestic analytical pure. P4448]Br) (purity >95%), Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences; all organic solvents used for the separation were domestically produced in analytical purity.

 

1.2 Instruments and equipment

LD-20AD Gel Permeation Liquid Chromatograph, Shimadzu, Japan; DDSJ-308A Conductivity Meter, Shanghai Yidian Scientific Instrument Company Limited; Zetasizer NANO ZS90 Dynamic Light Scattering Instrument, Malvern Panalytical, UK; KQ-300 Ultrasonic Scrubber, Shanghai JinkoScience Industry Co. Ltd; HH-6 Digital Display Electronic Constant Temperature Bath, Changzhou Guohua Electric Appliance Co., Ltd; AW120 Electronic Balance, Shimadzu, Japan; UV757CRT Ultraviolet Spectrophotometer, Shanghai Jingke Instruments Co.

 

1.3 Test methods

1.3.1 Proposed ternary phase diagrams    

The mass ratio of lecithin to n-butanol (Km = 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8) was fixed, and the ionic liquids and complex surfactants with the mass ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 were weighed accurately, and then vortexed, and then the solutions were added with water step by step at 30 ℃ and 60 ℃ to make them fully shake. When the solution changed from clear and transparent to turbid and stratified, the mass of water was recorded and the mass fraction of each component was calculated, and the ternary phase diagram was plotted by Origin software, and the curves formed by the points corresponding to the mass ratios of each component at the critical point of phase separation were the boundaries of single-phase and multiphase zones in sequence [20].

 

1.3.2 Microstructural characterization    

Fixed Km, prepare solutions with different R values (ratio of ionic liquids to complex surfactants) and place them in a thermostatic water bath to reach thermodynamic equilibrium, then use a pipette gun to add distilled water one by one, and for each addition of water, the vortexer was oscillated to homogeneity, and then after the temperature of the system was stabilized and equilibrated, the conductivity of ionic liquid microemulsion was measured by a conductivity meter, and the amount of water added and the conductivity values were recorded, and the microstructure of microemulsions was classified according to the curve of the change in the mass fraction of water [21]. The microstructure of the microemulsion was classified according to the curve of the change of the conductivity of the system with the mass fraction of water [21].

 

1.3.3 Particle size determination    

Particle size distribution and size change of ionic liquids/lecithin/n-butanol/water at 30 °C and 60 °C were measured by using dynamic light scattering (DLS), i.e., laser particle sizing.

 

1.3.4 Extraction of astaxanthin    

The shells of Antarctic krill were washed, freeze-dried for 48 h, removed and pulverized in a high-speed pulverizer, passed through a 120-mesh sieve and stored at -18 ℃. Subsequently, according to the mass ratio of ionic liquid : lecithin : n-butanol : water = 0.2 : 0.12 : 0.48 : 0.2 to prepare ionic liquid microemulsion. The optimal microemulsion system was determined by extracting astaxanthin with different solvents at a controlled temperature of 30 ℃. The effects of solid-liquid ratio, system components, ultrasonic power and ultrasonic time on astaxanthin extraction were determined under ultrasonication-assisted conditions.

 

1.3.5 Determination of astaxanthin content by high performance liquid chromatography    

The extract was centrifuged, and 1 mL of the supernatant of astaxanthin was taken into a 10 mL brown flask, and then passed through a 0.45 μm organic membrane for use. Chromatographic column: C18; column temperature: 30 ℃; mobile phase: methanol : acetonitrile : dichloromethane : water = 85.0 : 5.5 : 5.0 : 4.5 (v/v) (A), methanol : acetonitrile : dichloromethane : water = 28 : 42.5 : 25 : 4.5 (v/v) (B); flow rate: 1.00 mL/min; detection wavelength: 474 nm; injection volume: 20 μL [22]. .

 

1.3.6 Determination of astaxanthin stability    

The astaxanthin extract obtained from the reverse extraction was dissolved in ethanol and heated at 25-55 ℃, and the absorbance values at 0, 2, 4, 8 and 12 h were determined at 474 nm using an ultraviolet spectrophotometer.

 

1.4 Data processing methods

The tests were performed three times in parallel and analyzed for significance using the software JMP, and statistical differences were found at P<0.05.

 

2 Test results and discussion

2.1 Construction and characterization of ionic liquid microemulsion systems

2.1.1 Phase behavior

(1) Effect of ionic liquid type Ionic liquids consist of asymmetric organic cations and organic or inorganic anions, and their own structure plays a decisive role in the hydrophilic and hydrophobic properties of ionic liquids, which is the primary factor affecting the internal structure of their microemulsions[23] . Figure 2a shows the proposed ternary phase diagram of the microemulsion system formed by [P4444]CF3COO or [P4448]Br with lecithin, n-butanol and water at 60 ℃. The larger area of the single-phase region of the microemulsion indicates that the ability of each component to form microemulsion is stronger[24] . It can be seen that the phase-forming ability of ionic liquids is [P4444] CF3COO>[P4448] Br. According to the literature, although the hydrophilicity index of the anion Br- of ionic liquids is larger than that of CF3COO-, the cation [P4448]+ has a longer side-chain and a larger molar volume than that of [P4444]+, and thus [P4448] Br (HI=6.7) has a greater hydrophilicity than [P4448] Br (HI=6.7), and [P4448] Br (HI=6.7) is a more hydrophilic than [P4448] Br (HI=6.7). 6.7) is more hydrophobic than [P4444]CF3COO (HI=9.0)[25] . It can be seen that the cation molar volume of ionic liquids is the main factor affecting the phase-forming ability of ionic liquids in the ionic liquid/lecithin/n-butanol/water microemulsion system.

 

(2) Effect of Km The ratio of surfactant to co-surfactant, Km, is another important factor affecting the internal structure of microemulsions. For example, Wang et al. [26] constructed a glyceryl trioleate/TX-100/n-butanol/[Bmim]BF4 microemulsion system and showed that Km has an important effect on the phase behavior of glyceryl trioleate-based ionic liquid microemulsion systems. In order to reduce the toxicity of surfactants, lecithin was used as an alternative to traditional hydrophilic surfactants. Lecithin is an important biosurfactant, which is widely used in the fields of medicine and health care, food industry and cosmetics [27-29]. However, lecithin is a solid at room temperature, and its use as a surfactant alone is not conducive to the microemulsification of ionic liquids.Amiri-Rigi et al [30] used lecithin/1-propanol/olive oil/water microemulsion to extract lycopene from industrial tomato pomace with an extraction rate of 88%. The addition of alcohols induced lecithin to dissolve and fill in the surfactant voids, resulting in a reduction of interfacial tension and an increase in fluidity [31-32]. Accordingly, n-butanol was used as a co-surfactant to develop a ternary phase diagram of the ionic liquid/lecithin/n-butanol/water microemulsion system at 35 ℃ at different Km.

 

In the case of [P4444]CF3COO microemulsion, for example, the area of the microemulsion with Km=1 : 8 increased compared to that with Km=1 : 3, but there was no significant change in the range of 1 : 3 to 1: 7, as shown in Fig. 2b . Similar results were obtained when the ionic liquid was [P4448] Br. The effect of the ratio of surfactant and co-surfactant on the phase-forming ability of microemulsions is mainly related to their hydrophilic-lipophilic balance (HLB)[33] . The HLB of lecithin = 7.0 and that of n-butanol = 6.9 are very close to each other, and the differences in the HLB values of the surfactant composites calculated at different Km values are not significant, which explains why the Km value does not have a significant effect on the phase-forming ability of ionic liquid microemulsions. However, when the Km value is less than 1 : 3, it is difficult to form microemulsion with good fluidity due to the large viscosity of the system.

 

3) Effect of temperature  

Alexandridis et al [34] used block copolymers to investigate the effect of temperature change on the transformation behavior between micelles and stretching monomers during micellar extraction, and showed that temperature is an important factor influencing the internal structural changes of microemulsions. Moreover, temperature is a key factor affecting the stability and function of bioactive molecules. For example, Liu et al [35] investigated the temperature-dependent stability of curcumin extracted from a low eutectic solvent synthesized from citric acid and glucose with a 1:1 ratio, methanol, etc. The results showed that curcumin molecules degraded rapidly at too high a temperature, and at too low a temperature, mass transfer in solvent systems was weakened, thus inhibiting curcumin solubilization. Therefore, exploring the effect of temperature on the ionic liquid microemulsion system can provide a favorable external environment for the dissolution of astaxanthin.

 

The phase diagrams of the [P4448]Br and [P4444]CF3COO microemulsion systems are shown in Figs. 2c and 2d, respectively, from 20 to 70 ℃. Overall, the area of the single-phase region increases with increasing temperature, indicating that high temperature is favorable for the formation of microemulsion systems. Similar microemulsion phase behavior is also observed for imidazolium-based ionic liquids[36] . In contrast, in [P4444]CF3COO/ TX-100/n-butanol/water and [P4448]Br/TX-100/n-butanol/water microemulsions, the single-phase area decreased with increasing temperature[23] . This may be due to the fact that lecithin penetrates into the ionic liquid phase and enters more into the barrier layer, and its solubility in ionic liquids increases with elevated temperatures, resulting in an increase in the monophasic area [37]. On the other hand, [P4444] CF3COO microemulsion showed a large variation of single-phase area with temperature, which indicated that the hydrophobicity of the ionic liquid was significantly affected by temperature, and the temperature could be adjusted to achieve the "breaking of the emulsion" and astaxanthin isolation and purification, as well as the recycling of ionic liquid. The phase equilibrium of the [P4448]Br/lecithin/n-butanol/water microemulsion system showed a relatively weak temperature dependence, suggesting that the thermophysical properties of the system are more stable.

 

2.1.2 Microstructure    

Classification of microemulsions into single-phase zones by measuring conductivity is a common method to study the microstructure of microemulsions. As shown in Fig.

As shown in Figure 3, at low water content, the conductivity rises rapidly with increasing water molecule content. When the water content increases to a certain level, the conductive particles are widely spaced and not connected to each other, and can only conduct electricity by the collision of the dispersed water droplets, so the conductivity rises slowly until the mixed system becomes turbid. It was determined that the microstructure of the ionic liquid/lecithin/n-butanol/water microemulsion system mainly consisted of water-in-ionic-liquid (W/IL) and bicontinuous (B) types because lecithin, with its lower HLB value and higher oil content, contributes to the formation of the W/IL type microemulsions. The microstructure of the ionic liquid microemulsion was plotted according to the conductivity relationship (Fig. 4), which provided a basis for selecting the appropriate component ratio for astaxanthin extraction in this solvent system.

 

2.1.3 Particle size distribution    

According to the microregion of the ionic liquid microemulsion, a bicontinuous microemulsion system was prepared according to the mass ratio of ionic liquid: lecithin: n-butanol: water of 0.2 : 0.12 : 0.48 : 0.2, and the particle size distribution and average particle size were measured in the range of 30~60 ℃. As shown in Figure 5, the average particle sizes of [P4448]Br and [P4444]CF3COO at 30 ℃ were 56.74 nm and 60.04 nm, respectively, which were less than 100 nm, proving that [P4448]Br and [P4444]CF3COO could form microemulsions (<100 nm) with lecithin, n-butanol, and water at this temperature. 100 nm) with lecithin, n-butanol and water. This conclusion is consistent with the phase equilibrium results of ionic liquid microemulsions in Figs. 2 and 4.

 

Literature reports that the particle size of 1-butyl-3-methylimidazolium tetrafluoroborate/ TX-100/cyclohexane microemulsion increases with increasing temperature. The main reason for this result is that the increase in temperature leads to an increase in the solubility of the surfactant tailing group in organic solvents, which reduces the interfacial curvature of the surfactant film and the hydrogen bonding or hydration between dispersed water molecules, ultimately leading to an increase in particle size [38]. The enhanced solubility of the activator tail group in organic solvents reduces the interfacial curvature of the surface activator film and hydrogen bonding between dispersed water molecules or hydration, which ultimately leads to an increase in particle size [38]. For [P4448]Br and [P4444]CF3COO microemulsions, the overall increase in particle size with increasing temperature was also observed. When the temperature was increased from 30 ℃ to 60 ℃, the particle size of [P4444]CF3COO microemulsion system increased from 60.04 nm to 1,167 nm, which was more significant than that of [P4448]Br microemulsion (from 56.74 nm to 133.0 nm), and was in line with the pattern of the single-phase region in the phase diagrams (Fig. 2), and this further explained that the particle size of the [P4448]Br and [P4444]CF3COO microemulsions increased with temperature. Br and [P4444]CF3COO microemulsion phase equilibria as a function of temperature. This further explains the difference in the phase equilibria of [P4448]Br and [P4444]CF3COO microemulsions with respect to temperature, i.e., an increase in temperature leads to an increase in the emulsification of the components of the [P4444]CF3COO/lecithin/n-butanol/water system, which results in the formation of microemulsions from the swelling of micelles. However, high temperatures not only lead to rapid volatilization of the system, resulting in a reduced reaction environment, but also to increased particle collision, which destabilizes the microemulsion thermodynamically and ultimately leads to an increase in particle size due to agglomeration.

 

2.2 Ionic liquid microemulsion extraction of astaxanthin

2.2.1 Comparing the effects of different solvents    

Ethanol is a commonly used reagent for astaxanthin extraction in industry[39] . The ability of microemulsions of ionic liquids to extract astaxanthin was first compared with water, organic reagents, and pure ionic liquids, and the results are shown in Figure 6. The ability of different solvents to extract astaxanthin from Antarctic krill shells was as follows: [P4444]CF3COO microemulsion ≈ [P4448]Br microemulsion ≈ ethanol ≈ n-butanol > [P4444]CF3COO > water > [P4448]Br. The results of astaxanthin extraction using [P4444]CF3COO microemulsion were (25.41±0.48) μg/g at 30 ℃, 5% mass fraction and 30 min extraction time.

It is worth noting that pure ionic liquids are often limited in the field of biomolecular extraction due to the fact that the ionic liquid molecules interact with each other in the form of clusters to form a viscous molecular structure that is not conducive to mass transfer [31]. [P4448]Br is viscous at room temperature and difficult to extract astaxanthin. CF3COO [P4444] has some mobility, but the mass transfer rate is still low. The ionic liquid microemulsion system contains a certain mass of water and alcoholic surfactants, resulting in low viscosity, which is favorable for astaxanthin migration. Therefore, the ionic liquid microemulsion not only retains the polar characteristics of pure ionic liquids, but also has low viscosity, which can replace organic solvents as the ideal solvent for astaxanthin.

 

2.2.2 Effect of solid-liquid ratio    

To further promote the solubility of astaxanthin in ionic liquid microemulsion system, ultrasound-assisted microemulsion was used to extract astaxanthin from Antarctic krill shells.Amiri-Rigi et al[40] constructed a microemulsion system using saponin as a surfactant, glycerol as a co-surfactant, and double-distilled water as the aqueous phase, and extracted lycopene from tomato industrial waste, and the solubility of lycopene was significantly enhanced by increasing the dosage of microemulsion in a certain range. When the amount of microemulsion was increased within a certain range, the solubility of lycopene increased significantly. Similarly, the solid-liquid ratio of [P4444]CF3COO microemulsion was increased from 1:10 to 1:90, and the amount of astaxanthin extracted was increased from (9.17 ± 1.5 mm) to (9.5 ± 1.5 mm). 17 ± 1 . (9.17 ± 1.20) μg/g to (55.54 ± 5.22) μg/g, but the further increase in the amount of microemulsion after the solid-liquid ratio of [P4444]CF3COO microemulsions was increased to 1:60 had no significant effect on the amount of astaxanthin extracted (Fig. 6). Therefore, when the ultrasonic power and ultrasonic time were 60 W and 30 min, respectively, and the composition of the microemulsion liquid system was [P4444]CF3COO/lecithin/n-butanol/water (mass ratio=0.2∶0.12∶0.48∶0.2), the astaxanthin extracted reached (45.09 ± 2.67) μg/g by a mass fraction of 1.7%.

 

2.2.3 Effect of composition    

The microstructure of the ionic liquid microemulsions was significantly affected by different component ratios, which in turn determined the solubility of biomolecules. The effect of different mass ratios of components (ionic liquid: lecithin: n-butanol: water) on the amount of astaxanthin extracted from the microemulsions was determined under the conditions of ultrasonic power of 60 W, ultrasonic time of 30 min, and mass fraction of 1.7%.

As can be seen from Fig. 6, the extraction of astaxanthin from ionic liquid microemulsion decreased from (45.09 ± 2.67) μg/g to (38.53 ± 0.08) μg/g when the content of [P4444]CF3COO was increased from 20% to 35%. On the one hand, it is due to the fact that the viscosity of the microemulsion system was increased by the excessive use of [P4444]CF3COO which was not conducive to the mass transfer, and on the other hand, it is consistent with the results of the lower extraction rate of pure ionic liquid of astaxanthin in Fig. 6a. On the one hand, the viscosity of the microemulsion system increased due to the excessive use of [P4444]CF3COO, which was not favorable for mass transfer, and this was consistent with the lower extraction rate of astaxanthin with pure ionic liquid in Figure 6a. On the other hand, the gradual increase of the ionic liquid content changed the microstructure of the microemulsion from a bicontinuous type to a water-in-ionic liquid type (Fig. 4), which made it difficult for astaxanthin to be dissolved in the "pool", and thus the microstructure was not favorable for astaxanthin extraction. When the microemulsion system was composed of [P4444]CF3COO/lecithin/n-butanol/water (mass ratio = 0.2∶0.12∶0.48∶0.2), the microemulsion had a bicontinuous microstructure, which was strong in astaxanthin solubilization.

 

2.2.4 Effect of ultrasound power and time    

Compared with organic reagents such as ethanol, the viscosity of ionic liquid microemulsions is lower than that of pure ionic liquids, but there is still a large internal friction. Therefore, the traditional vegetable oil extraction method is often used to reduce the viscosity of the solvent system temperature extraction. For example, sunflower oil or sunflower oil methyl ester was used to treat shrimp waste at 70 ℃ for 150 min, and the extraction rates of astaxanthin were 60% and 80%, respectively[41] . However, this method not only has a low extraction rate but also is not conducive to the maintenance of biomolecular activity. Ultrasound-assisted extraction is one of the most important aids for the extraction of biomolecules at ambient temperature [42]. Zhao et al. [43] used ultrasound-assisted extraction to investigate the pattern of astaxanthin stability as a function of ultrasound power and time, and the results showed that an increase in the ultrasound time was beneficial for the increase of the effective contact time between the material and the liquid, which facilitated the effective solubilization of astaxanthin, and thus resulted in a higher extraction volume. However, further increase in ultrasonic power and processing time resulted in accelerated degradation of astaxanthin.

 

In order to further improve the extraction efficiency of astaxanthin, ultrasound-assisted extraction of natural astaxanthin from Antarctic krill shells was carried out using [P4444] CF3COO/lecithin/n-butanol/water (mass ratio=0.2∶0.12∶0.48∶0.2) as the medium with a mass fraction of 1.7%, and the results are shown in Figure 7. After 30 min of ultrasound-assisted extraction, the amount of astaxanthin extracted was close to saturation, reaching (45.09±2.67) μg/g. However, 97.98% of the maximum amount of astaxanthin extracted could be reached after 2 min of ultrasound-assisted extraction, which indicated that the [P4444]CF3COO microemulsion system could achieve good extraction results under ultrasound-assisted conditions in a short period of time, and it not only reduced the economic cost but also facilitated the maintenance of astaxanthin extraction. The results showed that the CF3COO microemulsion system could achieve good extraction results in a short period of time under ultrasound-assisted conditions, which not only reduced the economic cost, but also helped maintain the stability of astaxanthin. In addition, the appropriate ultrasonic power can promote the dissolution and dispersion of solutes, but when the ultrasonic power is too high, the mechanical effect generated by ultrasound is too strong, which can damage the microstructure of the extract, leading to a decrease in the amount of astaxanthin extracted. In summary, when the power and time of ultrasound-assisted extraction were 100 W and 2 min, respectively, and the mass fraction was 1.7%, the amount of astaxanthin extracted from [P4444]CF3COO/lecithin/n-butanol/water (mass ratio=0.2∶0.12∶0.48∶0.2) reached (48.21±0.07) μg/g, and the extraction rate was 97.75%.

 

2.3 Stability of astaxanthin

Qiao et al [44], in a study to evaluate the stability of free astaxanthin and astaxanthin esters, clearly pointed out that the degradation of astaxanthin increased when the treatment temperature was increased from 40 ℃ to 80 ℃, which was characterized by thermal sensitivity. The absorbance value is a physical quantity used to measure the degree of light absorption, and the stability of astaxanthin can be evaluated by measuring the absorbance value of astaxanthin at 474 nm. To demonstrate the stability of astaxanthin in the ionic liquid microemulsion system, the absorbance values of astaxanthin in [P4444]CF3COO/lecithin/n-butanol/water microemulsions were determined at 25, 35, 45, and 55 ℃. The results are shown in Fig. 8. The measured absorbance values were 0.653, 0.649, 0.636, 0.624, and the degradation rate increased from 2.54% to 5.88% after 12 h of water bath treatment, respectively. This result indicated that the degradation of astaxanthin increased with the increase of temperature, because the increase of temperature accelerated the molecular movement of the substances in the [P4444]CF3COO/lecithin/n-butanol/water microemulsion, which led to the partial degradation of astaxanthin, the decrease of absorbance value, and the destabilization of astaxanthin.

 

Extraction time is another major factor affecting astaxanthin stability. With the increase of heating time, the hydroxyl groups at both ends of free astaxanthin were easily replaced by fatty acids, which increased the stability of astaxanthin esters, and therefore, astaxanthin was not degraded much in a short period of time[45] . However, as the treatment time was extended to 12 h, the measured absorbance values of astaxanthin at 45 ℃ were 0.654 (2 h), 0.650 (4 h), 0.650 (8 h), 0.644 (10 h), 0.636 (12 h), with the degradation rate increasing from 0% to 2.54%. This is due to the fact that the electronically conjugated double bond structure of astaxanthin is highly sensitive to physical and chemical degradation at high temperatures for a long period of time, leading to increased degradation[46] . Similarly, Parjikolaeib et al [41] treated Arctic sweet shrimp with sunflower oil at 70 ℃ for 2.5 h to extract astaxanthin, and Tan Junxiao et al [47] treated Antarctic krill with alkaline protease, papain and ethanol in a mixed solvent system at 51 ℃ for 2.1 h to extract astaxanthin, which were susceptible to degradation due to high temperature and time-consuming process. In contrast, as shown in Figure 8, the difference in absorbance values of astaxanthin in [P4444] CF3COO/lecithin/n-butanol/water microemulsions incubated in a water bath at 25 ℃ for 12 h and incubated in a water bath at 35, 45, and 55 ℃ for 2 h was statistically insignificant, indicating that astaxanthin stabilization is favored in this range of conditions.

 

2.4 Mechanism of Astaxanthin Extraction by Bicontinuous Ionic Liquid Microemulsions

The hydrogen bonding, π-π bonding, van der Waals forces and inter-ionic forces within the structure of ionic liquids give them good solubilization properties for biomolecules. In order to investigate the solubilizing effect of [P4444]CF3COO and [P4448]Br microemulsions on the shells of Antarctic krill, scanning electron microscopy (SEM) was used to observe the surface morphology of the shells of Antarctic krill before and after extraction. As shown in Fig. 9, the untreated Antarctic krill shells had smooth surfaces and a dense multilayer structure, but the shells treated with [P4444]CF3COO microemulsions showed different degrees of curvature and the multilayer structure was damaged. This may be due to the attraction of astaxanthin molecules with conjugated double bonds and β-violet ketone rings to the crystal space array formed by electrostatic interaction between the ionic liquid cations and the hydrophobic ends of lecithin in the microemulsion system. In addition, it has been shown that the ability of lycopene molecules to interact with the hydrophobic tails of lecithin is important for the good solubility of lycopene in microemulsions[30] .

 

Similarly, Liu et al[48] used cholesterol as the oil phase, lecithin and bile salts as surfactants and co-surfactants, and combined with polyoxyethylene castor oil in the aqueous phase to prepare microemulsions for the extraction of esters such as andrographolide and diterpene lactone from Andrographis paniculata, demonstrating that lipophilic fatty acid structure of lecithin plays an important role in the extraction of hydrophobic molecules. On the other hand, the hydrogen bonding between the ionic liquid anions in the microemulsion system and the conjugated double bonds of n-butanol and astaxanthin molecules is also an important reason for the formation of the porous structure inside the shell of Antarctic krill.

In summary, the mechanism of astaxanthin extraction by bicontinuous ionic liquid microemulsion is as follows (Fig. 10): 1) [P4444]CF3COO/lecithin/n-butanol/water or [P4448]Br/lecithin/n-butanol/water microemulsion facilitates the migration of astaxanthin on the surface of the shell and inside the shell to the solvent by destroying and disintegrating the internal structure of the shells of the krill, and by increasing the contact area between the shells and microemulsion. 2) The conjugated double bonds and β-viniferone ring of astaxanthin have strong electrostatic interactions with the cationic hydrophobic ends of the lecithin and ionic liquid in the microemulsion. (2) The conjugated double bond and β-violet ketone ring of astaxanthin have strong electrostatic interactions with the cationic hydrophobic ends of lecithin and ionic liquid in the microemulsion, and there are strong hydrogen bonding interactions between hydrogen bonding of astaxanthin and the anions of n-butanol and ionic liquid.

 

3 Conclusion

In this paper, lecithin was used as an emulsifier and ionic liquid to construct a bicontinuous microemulsion system, and natural astaxanthin was extracted from Antarctic krill shells. In the range of 20~70 ℃, [P4448]Br and [P4444]CF3COO were able to form a microemulsion system with lecithin, n-butanol and water. The microemulsion system was not only able to disrupt the dense organization of shrimp shells to form a porous and loose structure, but also the hydrophobic and hydrogen-bonding interactions between lecithin and ionic liquids and astaxanthin increased the solubility of astaxanthin. Under ultrasound-assisted conditions (100 W and 2 min), the astaxanthin extracted from the microemulsion of [P4444]CF3COO/lecithin/n-butanol/water (mass ratio=0.2∶0.12∶0.48∶0.2) reached (48.21±0.07) μg/g at a mass fraction of 1.7%. The results provide a new method for the extraction of natural astaxanthin with low temperature, short time, high extraction rate and high stability of astaxanthin. Of course, the ionic liquid microemulsion system has complex components, and it is difficult to break the emulsion, and the separation and purification of biomolecules is a scientific problem that needs to be broken through further.

 

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