How to prepare emulsion-embedded astaxanthin?

 Astaxanthin is a terpenoid unsaturated compound with a conjugated double bond and a six-membered isoprene ring at both ends of its molecular structure, which gives it a strong electronic effect and enables it to effectively scavenge free radicals, mono-linear oxygen, and hydroxyl radicals [1]. In addition, astaxanthin can improve skin condition[2] , protect the eyes[3] , maintain the function of the cardiovascular system and nervous system[4-5] , and regulate the immune response[6] . As a functional active factor, astaxanthin has great potential for application in the food, pharmaceutical, and cosmetic industries[7]. It has been widely used in soft gels, microcapsules, creams, energy drinks, and other fields[8] . However, the low molecular polarity and poor water solubility of astaxanthin limit its application in water-mediated food products; astaxanthin is also chemically unstable and is susceptible to oxidative degradation and loss of biological activity during processing and storage, resulting in low bioaccessibility[9] . These problems have become a key factor limiting the astaxanthin industry's development.

 

Emulsification is a common method to protect lipophilic nutrients, and it can effectively protect astaxanthin [10 ]. The emulsion is formed by dissolving the fat-soluble components in the oil phase, and then dispersing the oil phase in the aqueous system containing emulsifier under external force, and the oil droplets are surrounded by an interfacial layer composed of emulsifier molecules. Boonlao et al. dissolved the astaxanthin oleoresin in cod liver oil, and then prepared an astaxanthin emulsion by emulsification, which had a better stability at low temperature. The emulsion has good storage stability at low temperature, but the solubility of astaxanthin oleoresin in cod liver oil is only about 1%, and the amount of astaxanthin embedded in the emulsion is relatively low[11] .

 

Dai et al. dissolved astaxanthin in dichloromethane and prepared astaxanthin microparticles by emulsification-solvent evaporation method, and obtained astaxanthin microcapsules by spray drying; the surface of the resulting astaxanthin microcapsules was smooth, with a low content of surface oils and good liquidity; however, the method used dichloromethane to dissolve astaxanthin, which posed certain safety and health hazards, and the dissolution of astaxanthin was only about 1%, and the embedded amount was low [12]. However, this method uses dichloromethane to dissolve astaxanthin, which has some safety and health risks, and the solubility of astaxanthin is only about 1%, which makes the embedded amount low [12]. Considering that astaxanthin  Considering that astaxanthin has limited solubility in organic solvents such as chloroform and acetone and that there are safety concerns, exploring effective emulsion embedding systems that can safely and healthily dissolve astaxanthin and increase its solubility is the key to the successful preparation of astaxanthin emulsions.

 

Deep eutectic solvent (DES) is a eutectic formed by mixing two or more components in a specific molar ratio and interacting with each other through hydrogen bonding, where one component is a hydrogen bond acceptor (HBA) and the other is a hydrogen bond donor (HBD) [13 ]. The melting point of DES is lower than the melting point of any single component it contains, and it has the advantages of stable chemical properties, simple components, low toxicity, low price, biodegradable, environmentally friendly, etc. It has a strong solubility and a high solubility of astaxanthin [14].

 

In this study, the hydrophobic DES consisting of muscimol and lauric acid was used as the solvent for the oil phase, and astaxanthin was dissolved in DES to form the oil phase of astaxanthin-DES, while gelatin and gum arabic were used as the emulsifiers for the aqueous phase, and the astaxanthin emulsions were prepared by high-speed shearing. On this basis, the morphology, stability, rheological properties, bioaccessibility and resistance to environmental factors of the obtained astaxanthin emulsions were further investigated to explore safe, healthy and efficient astaxanthin emulsion embedding systems.

 

1 Materials and Methods

1.1 Materials and reagents

Astaxanthin was purchased from Xinhecheng Co., Ltd; gelatin (type B, 240 g Bloom) and lauric acid (analytically pure, 98%) were purchased from Aladdin Reagents Co.

 

1.2 Instruments and equipment

MICCRA-D9 Shear, Miccra GmbH, Germany; EX31 Microscope, Sunny Group Instrument Co. KQ-300DB Ultrasonic Bath Cleaner, Kunshan Ultrasonic Instrument Co. Discovery DHR-2 Rheometer, TA Instruments, USA; TURBISCAN TOWER Multiple Light Scattering Analyzer, Formulaction Ltd.

1.3 Astaxanthin Emulsion Preparation

DES was prepared by mixing muscimol and lauric acid in a molar ratio of 3:1 with magnetic stirring in a water bath at 70 ℃ until clarified and transparent, and then left to cool to room temperature. Add astaxanthin into DES (5% astaxanthin by mass), and stir the mixture magnetically at 80 ℃ for 5.3 h until astaxanthin was dissolved. The oil phase of the emulsion was gelatin, and the aqueous phase of the emulsion was gelatin. The aqueous phase of the emulsion was a mixture of gelatin and gum arabic. The aqueous phase of the emulsion was a mixture of gelatin and gum arabic at different gelatin/gum arabic mass ratios (1, 2, 5, 10). 5, 5, 10) and different emulsifier (gelatin + gum arabic) mass fractions (1%, 1.5%, 2%, 3%). The water phase with different gelatin/gum arabic mass ratios (1, 2.5, 5, 10) and emulsifier (gelatin + gum arabic) mass fractions (1%, 1.5%, 2%, 3%) was added to the oil phase drop by drop and emulsified by high-speed shear in a shear machine at a rotational speed of 11 000 r/min. The mass ratio of aqueous phase to oil phase was maintained at 9:1.

 

1.4 Microscopic observation

One drop of the emulsion was placed on a smooth, clean slide, covered with a coverslip without air bubbles, and placed on a carrier stage to observe the microscopic morphology of the emulsion under a 40x lens. The particle size of the emulsion was obtained by Image J counting software, and the counting area of the optical micrograph was randomly selected, and the number of droplets counted was not less than 300.

 

1.5 Stability Measurements

The stability of emulsions with different emulsifier contents was determined by using a multiple light scattering analyzer. The stability of emulsions with different emulsifier contents was determined by using the Gravimetric Light Scattering Analyzer (GLSA). 20 mL of emulsions were placed in cylindrical glass tubes and measured at 25 ℃ for 12 h. The samples were scanned every 10 min.

 

1.6 Determination of rheological properties

The rheological properties of the emulsions with different gelatin/gum arabic mass ratios and different emulsifier mass fractions were tested using a rotational rheometer equipped with a 60 mm aluminum cone plate. All the tests were carried out at 25 ℃ under the following conditions: full strain scanning with a frequency of 1 Hz and a strain range of 0.01%~100%; frequency scanning with an angular frequency in the range of 1~100 rad/s for appropriate strain values; and steady state scanning with a shear rate in the range of 0.1~100 s-1 for appropriate strain values. 1 ~ 100 s-1 , for steady state scanning.

 

1.7 Measurement of the effect of environmental factors on emulsions

A gelatin/gum arabic mass ratio of 5 and an emulsifier mass fraction of 1.5% were used to study the effect of environmental factors on the emulsions. Samples with gelatin/gum arabic mass ratio of 5 and emulsifier mass fraction of 1.5% were used to study the effect of environmental factors on the emulsions. Firstly, to study the effect of temperature on the emulsions, the emulsion samples were heated in a water bath at 25 ℃, 80 ℃, 90 ℃ and 100 ℃ for 2 h, and then the samples were taken out to observe the microscopic morphology of the samples and to determine the astaxanthin content; secondly, the samples were irradiated by UV irradiation of the astaxanthin emulsion and the astaxanthin solution (in contrast to the oil) for 41 h. The astaxanthin content was then determined at different time intervals (0 h, 18 h, 29 h, 41 h). Finally, the astaxanthin emulsion and astaxanthin solution (oil phase control) were stored at 27 ℃ and protected from light, and the astaxanthin content of the samples was determined at different times (0 d, 5 d, 10 d, 20 d).

 

1.8 Bioaccessibility determination

The bioaccessibility study was based on the method of Zhang et al. Simulation of gastric fluid digestion: dissolve HCl and NaCl in ultrapure water, take 15 mL of the above solution and add it to the astaxanthin sample to obtain the sample mixed salt solution. Pepsin was mixed with 15 mL of phosphate buffer (10 mmol/L, pH = 7) and added into the mixed salt solution, and the pH value was adjusted to 2.5°C and stirred in a 37 ℃ water bath. The final mass concentration of pepsin in the system was controlled to be 3.2 g/L, the final concentration of HCl was 84 mmol/L, and the final mass concentration of NaCl was 2 g/L. The final mass concentration of astaxanthin emulsion was taken from 0.5 mL of the sample. The final mass concentration of NaCl was 2 g/L. 0.1 g of astaxanthin emulsion was taken. 0.1 g of astaxanthin was taken for astaxanthin lotion, and the same amount of astaxanthin was taken for astaxanthin solution (oil phase control) as for astaxanthin lotion.

 

Simulated enteric digestion: CaCl2-NaCl mixed salt solution was obtained by dissolving CaCl2 and NaCl in ultrapure water. For simulated enteric digestion, the bile salt solution and trypsin suspension were freshly prepared in phosphate buffer (10 mmol/L, pH = 7). The pH was adjusted to 7 by adding 1.5 mL of CaCl2-NaCl2 -NaCl2 -NaCl2 solution. 5 mL of CaCl2 -NaCl mixed salt solution and 2.5 mL of bile salt solution. 5 mL of bile salt solution, adjust pH to 7 again, and continue to add 3.5 mL of trypsin suspension. 5 mL of trypsin suspension was added to obtain the simulated intestinal fluid. The concentrations of CaCl2 and NaCl in the control system were 5 mmol/L and 15 mmol/L, and the mass concentrations of bile salt solution and trypsin suspension were 5 mg/mL and 1.6 mg/mL, respectively. The mass concentrations of bile salt solution and trypsin suspension were 5 mg/mL and 1.6 mg/mL, respectively.

The digested samples of simulated gastric and intestinal fluids were centrifuged at 9 500 r/min for 30 min at 25 ℃, and the supernatant (micelles) was extracted.

 

1.9 Data analysis

All experimental data were expressed as mean ± standard deviation, and the data were processed by Origin software, and analyzed for significance by SPSS software, with P < 0.05 considered significant.

 

2 Results and analysis

2.1 Micromorphology and Stability of Emulsions

Figure 1 shows optical micrographs of emulsions with different gelatin/gum Arabic mass ratios. The emulsions embedded with astaxanthin were reddish-brown in color with no delamination, and the micrographs showed bright red spherical droplets with no astaxanthin crystals precipitated from the external continuous phase, indicating that the emulsions loaded with astaxanthin were stable and the astaxanthin was embedded in the internal dispersed phase. From the results of the emulsion particle size in Table 1, it can be seen that the average particle size of the emulsion decreased with the increase of the gelatin/gum arabic mass ratio, from 3.84 μm to 2.69 μm. The average particle size decreased from 3.84 μm to 2.69 μm. When the gelatin/gum arabic mass ratio was 5 and 10, the particle size was more uniform and the emulsion system was more stable. Therefore, the sample with gelatin/gum Arabic mass ratio of 5 was selected to further investigate the effect of gelatin-gum Arabic emulsifier on the particle size and stability of the emulsion.

 

Figure 2 shows the optical micrographs of the emulsions with different emulsifier mass fractions. With the increase of emulsifier mass fraction, the particle size of the emulsions decreased gradually, and the average particle size decreased from 4.11 μm to 1.71 μm (Table 2). This is due to the better emulsifying property of gelatin, which is more conducive to the stabilization of the oil-water interface. With the increase of the emulsifier mass fraction, the protein coverage of the droplet interface increases, which can stabilize more small droplets, and the probability of the emulsion droplets agglomerating during high-speed shear is reduced, resulting in smaller and more homogeneous emulsions[16] .

 

The results of emulsion stability test in Fig. 3 show that the stability indices of emulsions with different emulsifier mass fractions are all less than 1, which means that the emulsions are all stable. The stability indices of the emulsion samples showed an increasing and then flattening trend with time. The stability index of the emulsion samples showed an increasing and then decreasing trend with the increase of the mass fraction of emulsifier from 1% to 3% within 12 h. The stability index of the emulsion samples was less than 1, indicating that the emulsions were stable. The emulsion stability index was smallest at 2% emulsifier mass fraction, indicating that the emulsions were most stable after 12 h of storage. Therefore, when the gelatin/arabinose mass ratio was 5 and the emulsifier mass fraction was 2%, the emulsion had a smaller and more homogeneous particle size and was the most stable.

 

2.2 Rheological Properties of Emulsions

Figures 4a and 4b show the results of viscosity determination for emulsions with different gelatin/gum arabic mass ratios and different emulsifier mass fractions, respectively. In the range of 0 . The viscosities of all emulsion samples decreased with the increase of shear rate in the range of 0.1~100 s-1 , showing the typical shear-thinning characteristics of pseudoplastic fluids. This is due to the fact that the increase in shear rate disrupts the structure of the entangled polymer network, the recovery rate of intermolecular entanglement is slower than the destruction rate, and the intermolecular resistance of the emulsions to flow at higher shear rates is lower [17 ]. In addition, the emulsion viscosity decreased with the increase of gelatin/arabinose mass ratio because the concentration of gum arabic in the emulsifier decreased with the increase of gelatin/arabinose mass ratio, and gum arabic, as a macromolecular thickening agent, had a significantly higher ability to maintain the viscosity of the system than gelatin. The significant increase in emulsion viscosity was also accompanied by an increase in the mass fraction of emulsifier.

 

The compound viscosities of emulsions with different gelatin/gum arabic mass ratios and different emulsifier mass fractions are shown in Figs. 4c and 4d, respectively. The compound viscosities of all emulsions decreased with increasing angular frequency in the range of 1-100 rad/s. The initial compound viscosities of the emulsions decreased with the content of gelatin/gum arabic and emulsifier. The initial composite viscosity of the emulsions increased with the increase of gum arabic and emulsifier content in the system. The results are consistent with the viscosity changes.

 

The results of the Herschel-Bulkley model fit in Table 3 show that the yield stress (τ0 ) and K value both increase with increasing emulsifier mass fraction, indicating that the emulsion viscosity increases with increasing emulsifier mass fraction. The flow factor (n) of all the emulsion samples is less than 0.5 and increases with emulsifier mass fraction. The flow factor (n) of all emulsion samples was less than 0.5 and decreased with the increase of emulsifier mass fraction. n value reflects the degree of pseudoplasticity of emulsions, the smaller the value of n, the worse the fluidity of emulsions, n = 1 emulsions are Newtonian fluids, n < 1 emulsions are pseudoplastic fluids. Therefore, emulsions with different emulsifier mass fractions are pseudoplastic fluids with shear-thinning behavior; as the emulsifier mass fraction increases, the viscosity of emulsions increases, and the fluidity deteriorates, showing a gel-like structure.

 

2.3 Effects of environmental factors on emulsions

The emulsions were heated at 25, 80, 90 and 100 ℃ for 2 h. The optical micrographs of the emulsions are shown in Fig. 5. As the heating temperature increased, the emulsion was emulsified, thermal aggregation phenomenon appeared, and the emulsion particle size increased obviously. When the heating temperature was increased from 25 ℃ to 100 ℃, the average particle size of the emulsion increased from 2.50 μm to 3.50 μm. When the heating temperature was increased from 25 ℃ to 100 ℃, the average particle size of emulsion increased from 2.50 μm to 3.99 μm. When the heating temperature was increased from 25 ℃ to 100 ℃, the average particle size of emulsion increased from 2.50 μm to 3.99 μm, and the particle size distribution was not uniform (Table 4). This is due to the thermal denaturation of adsorbed proteins, which weakens the emulsification of the emulsion [18]. The thermal denaturation of adsorbed proteins on the droplet surface exposes their nonpolar sulfhydryl groups, which increases the interactions between protein molecules and leads to droplet aggregation [19]. After the aggregation of droplets, the phenomenon of agglomeration is also more likely to occur, so the emulsion particle size increases significantly after the temperature increases. The degradation rates of astaxanthin in emulsions at 80 ℃ and 90 ℃ were 2.2% and 5.5%, respectively (Table 4). The degradation rates of astaxanthin emulsion at 80 ℃ and 90 ℃ were 2.2% and 5.5%, respectively (Table 4), indicating that the embedded astaxanthin emulsion had excellent thermal stability.

 

Unembedded astaxanthin solution (oil phase control) and embedded astaxanthin emulsion were exposed to UV light and the astaxanthin content of the samples at different irradiation times was determined, and the results are shown in Figure 6a. The retention of astaxanthin in the emulsified astaxanthin emulsion was higher than that in the unembedded astaxanthin solution during the UV irradiation time, which may be attributed to the ability of emulsified droplets to scatter light waves, making it difficult for harmful light waves to enter the interior of the emulsion, and resulting in better photostability of the emulsified astaxanthin emulsion [20 ].

 

The special structure of astaxanthin makes it sensitive to environmental factors and is susceptible to isomerization during storage [21 ]. The samples were stored in a light-proof environment for 20 d, and the astaxanthin contents of the samples at different storage times were determined, and the results are shown in Figure 6b. The retention of astaxanthin decreased in all samples during storage; after 20 d of storage, the retention of astaxanthin in the embedded emulsion was 83%, which was 5% higher than that of the unembedded astaxanthin solution. This was attributed to the formation of a protein-polysaccharide interfacial membrane layer on the surface of the embedded astaxanthin, which was able to reduce the influence of the external environment on astaxanthin. In addition, the retention rate of astaxanthin in these emulsions was better than that of the reported astaxanthin-embedding systems. Zhang et al. embedded astaxanthin in the form of microspheres, and the retention rate of the resulting astaxanthin microspheres was only 50.4% after 14 d of storage at 25 ℃ [22 ]. Zhao et al. embedded astaxanthin oleoresin in the form of emulsified-spray-drying resin, and the retention rate of the sample was 79% after 10 d of storage [23]. The retention rate of astaxanthin was about 79% after 10 d of storage [23].

 

2.4 Bioaccessibility of Astaxanthin

The bioaccessibility of hydrophobic nutrients depends on physicochemical and biochemical processes, including release from food, solubilization in gastrointestinal fluids, chemical or biochemical transformations, and uptake by intestinal epithelial cells [24-26]. Astaxanthin is highly hydrophobic, and the apical surface of intestinal epithelial cells is less permeable, making astaxanthin less bioaccessible [27]. In this study, the effect of emulsion embedding on the bioaccessibility of astaxanthin was investigated, and the results are shown in Figure 7. The bioaccessibility of astaxanthin was 81.5%, 84.7%, 83.4%, and 86.0% for 1%, 1.5%, 2%, and 3% emulsions, respectively, which were not significantly different from each other, whereas the bioaccessibility of astaxanthin in the unemulsified astaxanthin solution (the control) was only 43%, which was much lower than that of the emulsified astaxanthin solution. This is due to the fact that the emulsion embedding improved the solubility of astaxanthin in aqueous solution, and the embedded astaxanthin was able to dissolve effectively in the mixed micelles after digestion, thus increasing its bioaccessibility compared with the control.

 

3 Conclusion

In this study, we used DES solubilized astaxanthin as the oil phase and a protein-polysaccharide mixture as the emulsifier to prepare astaxanthin emulsions, and investigated the effects of gelatin/gum arabic mass ratio in the aqueous phase and the mass fraction of emulsifier on astaxanthin emulsions, as well as the effects of emulsification on the properties of astaxanthin. Increasing the gelatin/gum arabic mass ratio and emulsifier mass fraction reduced the emulsion size, and the prepared astaxanthin emulsions were stable, exhibited shear-thinning properties, and had gel-like behavior. The astaxanthin retention rate of the embedded emulsion increased by 5% to 83% after 20 d of storage compared with that of the unembedded astaxanthin solution. The bioaccessibility of astaxanthin in the embedded emulsion was 83%, which was about 37% higher than that of the unembedded astaxanthin solution. The constructed embedding system is made of food-grade raw materials, which is safe, easy to prepare, low cost, and the solubility of astaxanthin in DES is higher than that of traditional solvents. The construction of the embedding system using DES as a new green solvent provides a new strategy for the dissolution and embedding of astaxanthin, and broadens the application of astaxanthin in aqueous matrices.

 

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