How to prepare astaxanthin/bovine serum albumin nanocomplexes

 Astaxanthin (AST), chemical name 3,3′-dihydroxy-4,4′-diketo-β,β,′-carotene, molecular formula C40 H52 O4, molecular weight 596.86, melting point 224 ℃, insoluble in water, soluble in some organic solvents, such as methylene dichloride, chloroform and so on [1]. The molecular structure of astaxanthin is a six-membered ring consisting of two isopentadiene units connected by a series of conjugated double bonds [2]; its molecular linear part has multiple double bonds, which can neutralize free radicals and scavenge reactive oxygen species (ROS) [3], and it is the strongest natural antioxidant discovered so far [4]. In addition, astaxanthin has physiological activities such as anti-inflammation [5], inhibition of cell apoptosis [6], protection of the central nervous system [7], and modulation of the mitochondrial membrane potential [8], and it is currently used as a nutritional dietary supplement for human beings in the food industry, and as an antioxidant, photoprotectant, and anti-inflammatory ingredient in pharmaceuticals and cosmetics industries [4].

 

Astaxanthin exists in two types of aggregates in hydrophilic mixtures of organic solvents and water, namely, the H-aggregate with a stuck-bun structure and a blue-shifted spectrum, which is dominated by conjugated chains and hydrogen bonding [1], and the J-aggregate, which is head-to-tail, red-shifted in the spectrum, and does not contain hydrogen bonds [9]. Astaxanthin exists mostly in free or esterified form in nature. Its aggregates can be obtained by precisely regulating the volume ratio of aqueous to organic solutions (e.g., dimethylsulfoxide [1], ethanol [10], and acetone [11]). The formation of H aggregates is favored by the increase in the number of hydrogen bonds in the solution [9]. Dai et al. [12] showed that the π-π conjugation structure and intermolecular hydrogen bonding in the structure of H-aggregate astaxanthin resulted in higher electron transfer efficiency, and thus H-aggregates showed higher scavenging efficiency than J-aggregates and astaxanthin monomers in the 1,1-diphenyl-2-trinitrohydrazine and hydroxyl radical scavenging assays in vitro; furthermore, the combination of H-aggregate and J-aggregate astaxanthin nano-complexes was also found to be more efficient than J-aggregates and astaxanthin monomers in the in vitro 1,1-diphenyl-2-trinitrohydrazine and hydroxyl radical scavenging assays. In addition, when H-aggregate and J-aggregate astaxanthin nanocomplexes were applied to H2 O2 pretreated Caco-2 cells, the results showed that H-aggregate astaxanthin had a stronger ability to promote the scavenging of intracellular reactive oxygen species (ROS), and thus exhibited better cytoprotective effects.

 

However, astaxanthin aggregates are poorly stabilized and are prone to metamorphosis among themselves, which makes it difficult to be stably stored in the aqueous phase for a long period of time [1,9]. Currently, the strategy of using nanotechnology to improve the stability and bioavailability of astaxanthin has been popularized, but no systematic study has been reported on the improvement of water dispersibility and application of astaxanthin aggregates; therefore, in this study, we attempted to use protein molecules as nanomaterials to encapsulate astaxanthin aggregates to improve the water dispersibility and stability, and to expand its application in food and pharmaceuticals. Whey protein is a high-quality globular protein that accounts for about 20% of the total protein content of cow's milk and contains β-lactoglobulin (β-lg), α-lactalbumin (α-la), bovine serum albumin, immunoglobulins, lactoferrin, and other minor proteins [13-14]. Pan et al. [15] investigated whey isolate proteins and astaxanthin liposomes, and found that whey isolate proteins could protect liposomes from gastric juice damage, which made astaxanthin more effective in the treatment of astaxanthin. Gastric juice can protect the liposomes from being damaged by gastric juice and increase the release of astaxanthin into the intestinal fluid, thus improving the bioavailability and stability of astaxanthin.

 

Bovine serum albumin (BSA), with a molecular mass of 69 kDa [ 16], can encapsulate nonpolar groups inside the molecule through hydrophobic interactions, form a hydrophobic core after folding, and further form a stable and compact internal structure under the effects of hydrogen bonding, electrostatic interactions and Vander Waals forces, which gives it gel-forming and emulsifying properties, It has the properties of gelation, emulsification, coating and microencapsulation [13,17], and is widely used as a carrier to assist the targeted delivery of drugs in the field of food and pharmaceuticals [18-19]. An et al. [20] combined bovine serum albumin and indocyanine green (ICG) to form ICG-BSA-NC nanocomplexes by molecular self-assembly, and showed that their combination could enhance the hydrolysis of ICG and improve the hydrolysis of ICG. It was shown that the combination of ICG-BSA-NC could improve the hydrolysis stability and photoluminescence quantum yield (PLQY) of ICG, which resulted in its efficient passive targeting ability and significantly improved the tumor accumulation and near-infrared fluorescence imaging in vivo.

 

In the present study, H-aggregate astaxanthin/bovine serum albumin nanocomplexes (H-ABNs) and J-aggregate astaxanthin/bovine serum albumin nanocomplexes (J-ABNs) could be prepared by using astaxanthin and bovine serum albumin as the main materials, and the binding mechanism of the two aggregates could be preliminarily investigated by characterization. By adjusting the ratio of ethanol to water, we investigated the effect of volume fraction of ethanol on the formation of H-aggregate astaxanthin and J-aggregate astaxanthin and screened the optimal conditions for the formation of astaxanthin aggregates. Under the optimal conditions, stable and well dispersed H-ABNs and J-ABNs were prepared. Dynamic light scattering, transmission electron microscopy, ultraviolet visible absorption spectroscopy, Fourier infrared spectroscopy and fluorescence spectroscopy were utilized to investigate the binding mechanism of the nanocomplexes, and to provide data support and theoretical basis for expanding the application of whey proteins and the development of stable astaxanthin and its aggregates.

 

1 Materials and Methods

1.1 Test materials

Astaxanthin (98% purity): Shanghai Aladdin Biochemical Technology Co., Ltd; bovine serum albumin (97% purity): Beijing Soleilbao Science and Technology Co., Ltd; anhydrous ethanol, potassium bromide, phosphotungstic acid, dichloromethane, methanol, etc. were analytically pure: Sinopharm Chemical Reagent Co.

 

1.2 Instruments and equipment

AMM-6T Magnetic Stirrer :Beijing Otesaense Instrument Co., Ltd; TECAN SPARK 10M Multi-functional Enzyme Labeler :Swiss Tecan; T6 Ultraviolet Spectrophotometer :Beijing PU Analytical Instrument Co. Nano-S90 Malvern Dynamic Light Scattering Instrument, Nano-Z Malvern Dynamic Light Scattering Instrument: Malvern, UK; HT-7000 Transmission Microscope: Hitachi, Japan; VERTEX70 Fourier Infrared Spectrometer: Bruker, Sweden; RF-6000 Fluorescence Spectrophotometer: Shimadzu, Japan.

1.3 Test methods

 

1. 3. 1 Preparation of solutions

The ethanol solution of 0.03 mg/mL astaxanthin was prepared according to the method of Zhao Yingyuan et al [21] and stored at 4 ℃.

100 mL of distilled water was accurately measured and placed in a sample bottle, 10 mg of BSA was added, and the solution was stirred at 300 r/min for 1 h at room temperature to obtain a mass concentration of 0.1 mg/mL BSA. Store at 4 ℃.

 

1. 3. 2 Effect of volume fraction of ethanol and storage time on astaxanthin aggregates

In order to investigate the effects of different ethanol v/v and storage time on astaxanthin monomers or their aggregation, the solution dilution method of Lu et al. [22] was used to prepare astaxanthin-ethanol/water solution with 10%~100% ethanol v/v. The solution was stirred at 25 ℃ for 10 min at 200 r/min and then scanned at 200~800 nm in UV region immediately with TECAN enzyme marker. Immediately after stirring for 10 min at 25 ℃ and 200 r/min, the solution was scanned at 200-800 nm in the UV region with a TECAN enzyme marker. According to the results of UV-vis full-wavelength scanning, 20% ethanol-ethanol/water solution of astaxanthin was used for the preparation of H-astaxanthin aggregates, and 35% ethanol-ethanol/water solution of astaxanthin was used for the preparation of J-astaxanthin aggregates, and the state of astaxanthin aggregates was recorded at room temperature after 0, 2, 4, 6, 8, and 24 h of storage, and the full-wavelength scanning was carried out by UV-vis.

 

1. 3. 3 Preparation of H-ABNs and J-ABNs

According to the results of 1.3.2 and the method used by Zhao Yingyuan et al [ 14], 20% ethanol was used for the preparation of H-ABNs, and the final mass concentration of AST was 0.003 mg/mL, and the final mass concentration of BSA was 0.01, 0.02, and 0.05 mg/mL for the preparation of H-ABNs solution. Use 35% alcohol to prepare J-ABNs, and prepare J-ABNs solution with final mass concentration of 0.003 mg/mL for AST and 0.01, 0.02, 0.05 mg/mL for BSA respectively, and set aside.

 

1. 3. 4 Particle size potential measurements of H-ABNs and J-ABNs

Following the method used by Zhao et al. [10], 1 mL of freshly prepared sample was taken and tested, with a test angle of 90 °, a test temperature of 25 ℃, an equilibrium time of 120 s, 90 cycles, and a dispersant R Ⅰ of 1.300 for water and 1.45 for Material R Ⅰ to determine the hydrated particle size. The Zeta potential was determined by adding 1 mL of freshly prepared sample into a potentiometric cuvette at a test angle of 90 ° and a test temperature of 25 ℃ with an equilibration time of 120 s and a cycle time of 20 times.

 

1. 3. 5 Transmission electron microscopy (TEM) observation of H-ABNs and J-ABNs

The morphology and microstructure of H-ABNs and J-ABNs were observed by TEM using the method of Zhu et al [23] with slight modification. A copper mesh was placed on top of the filter paper, and 1 drop of the sample dispersion was placed on the carbon carrier membrane of the copper mesh grid and air-dried naturally for 10 min. Afterwards, 0.01 g/mL phosphotungstic acid was added onto the grid dropwise. The sample was dried at room temperature and observed by TEM at 100 kV.

 

1. 3. 6 Calculation of bonding and loading rates of H-ABNs and J-ABNs

Preparation of standard curve: 3 mg of astaxanthin was accurately weighed and dissolved in 100 mL of dichloromethane-methanol (1:1) solution, and stirred at 200 r/min for 1 h at 25 ℃ to obtain the mass concentration of 0.03 mg/mL of astaxanthin-dichloromethane-methanol solution. The absorbance at 478 nm of the astaxanthin methanol solution with the mass concentration of 0.002~0.007 mg/mL was determined by ultraviolet spectrophotometer, and the linear equation of astaxanthin-dichloromethane methanol solution was obtained according to the Lambert-Beer law [24], with the linear equation of y = 265. 321 43x-0. 081 57, R2 = 0. 997 72.

Calculation of binding efficiency and loading efficiency: Freshly prepared H-ABNs nano-complex solution was collected in a tomato bottle, and the unloaded astaxanthin was extracted by adding 2 mL of methylene dichloride and 2 mL of methanol, and then shaken for 2 min. The organic phase was filtered through an organic membrane with a pore size of 0.22 μm, and the absorbance at 478 nm was measured by the UV-vis method, and the binding efficiency (BE) [25] and loading efficiency (LE) [26] were calculated based on the standardized curve of astaxanthin. The binding efficiency (BE) [25] and loading efficiency (LE) [26] were calculated from the standard curve of astaxanthin; the same was done for J-ABNs. The formula is as follows.

r1 = mAST/mAST total; r2 = mAST/m(BSA+AST) total where: r1 is BE ,%; r2 is LE ,%; mAST is the amount of astaxanthin encapsulated in the complex, mg; mAST total is the total amount of astaxanthin in the test, mg; m(BSA+AST) total is the total amount of BSA and astaxanthin, mg.

 

1. 3. 7 Measurement of ultraviolet-visible absorption spectra (UV-vis) of H-ABNs and J-ABNs

The full-wavelength scanning of H/J-ABNs was performed with a TECAN enzyme labeling instrument. Freshly prepared H-ABNs and J-ABNs were scanned at 200~800 nm in the ultraviolet region in 200 μL of each sample, and the spectral effects of the samples were investigated.

 

1. 3. 8 Scanning of fluorescence spectra of H-ABNs and J-ABNs

The fluorescence spectra of H-ABNs and J-ABNs were measured by an RF-6000 fluorescence spectrophotometer with the emission wavelength of 280 nm, the scanning range of 280~400 nm, the scanning speed of 6000 nm/min, and the width of the excitation and emission slit of 10 nm, with a slight modification of the method used by Ling Huang [27].

 

1. 3. 9 Fourier Transform Infrared Spectroscopy (FTIR) Measurements of H-ABNs and J-ABNs

The H-ABNs and J-ABNs solutions were added dropwise onto a KBr slide, dried and examined under the conditions of 25 ℃, 4 cm-1 resolution, 400-4000 cm-1 scanning range, and 16 scanning times, and the same method was applied to the powdered mixtures of BSA, AST, and BSA-AST. The infrared spectra were analyzed by OMNIC 8.2 software.

 

1.4 Data processing

Each group of tests was repeated three times and the results were presented as mean ± standard deviation. The experimental data were processed by Microsoft Excel 2019, OM-NIC 8.2, Origin 8.0, and IBM SPSS 22.0.

 

2 Results and analysis

2.1 Effect of volume fraction of ethanol and storage time on astaxanthin morphology

Astaxanthin can form aggregates in different ratios of organic and aqueous mixtures, and the increase of hydrogen bonding in the system is favorable to the formation of yellowish H aggregates [28]; when the organic phase is the main dispersing medium, the hydrogen bonding content in the system decreases, and the astaxanthin tends to exist in the solution as an orange-colored M monomer [11]. When 0.03 mg/mL of astaxanthin was added into different ethanol/distilled water ratios, the properties of astaxanthin in the solution were shown in Fig. 1(a), and the solution was yellowish when the volume fraction of ethanol was 10%-20%, which was presumed to be because of the large proportion of the water phase in the system, and the more hydrogen bonding, which led to the formation of card-packed astaxanthin with a yellowish color, The maximum absorption peak was around 387 nm (Fig. 1(b)); the solution became pinkish-purple when the volume fraction of ethanol was 25%~50%, and it was assumed that as the proportion of the organic phase increased, the amount of hydrogen bonding decreased, which led to the formation of head-to-tail, pinkish-purple, and spectrally red-shifted J-aggregates, and the UV peaks shifted to the right to form a side by side peak, respectively, at 510~510 nm. The UV absorption peaks were gradually shifted to the right to form side-by-side peaks between 510~530 nm and 550~580 nm, respectively (Figure 1(c)). When the volume fraction of ethanol was more than 60%, the color of the solution gradually became darker and orange, and the proportion of the water phase was greatly reduced, and there were not enough water molecules to participate in the formation of astaxanthin aggregates, which led to the gradual increase in the formation of M-monomers, and the absorption peak was a single peak at 480 nm (Figure 1(d)). The optical properties of astaxanthin aggregates formed under different ethanol volume fractions in this experiment are consistent with the results of Lu et al [22].

 

Based on the UV-vis spectra of astaxanthin in different volume fractions of the organic phase, H-AST was prepared with 20% of the organic phase and J-AST with 35% of the organic phase, and the samples were stored at 25 ℃, and the color and ultraviolet absorbance of the samples were observed to change at different time intervals, as shown in Fig. 2. The H-aggregate astaxanthin solution was light yellow at 0 h, and the solution gradually changed to a purplish-red color after 6 h, and the ultraviolet spectrum showed a significant decrease in the characteristic absorption peak of H-aggregate astaxanthin after 6 h, and the maximum absorption peak gradually shifted from 388 nm to a peak at 520 nm and 560 nm, which is a significant decrease in the absorption peak at 520 nm and 560 nm respectively. The spectrum showed that the characteristic absorption peaks of H-aggregate astaxanthin began to decrease greatly after 6 h. The maximum absorption peaks gradually shifted from 388 nm to the shoulder absorption peaks with peaks around 520 nm and 560 nm, which indicated that the single H-aggregate was unstable, and gradually shifted to the J-aggregate during the storage process.The J-aggregate was purplish-red in the beginning, and there was no obvious change in the color of the J-aggregate in the process of 24 h. This indicated that the stability of J-aggregate astaxanthin was not stable. The stability of astaxanthin in J aggregates was better than that of astaxanthin in H aggregates.

 

2. 2 Preparation and particle size potential analysis of H-ABNs and J-ABNs

H-ABNs and J-ABNs (AST final mass concentration of 0.003 mg/mL) were prepared by using BSA solution with mass concentrations of 0.01, 0.02 and 0.05 mg/mL as the carriers, and the results were shown in Fig. 3.The H-ABNs were pale yellow and the J-ABNs were pinkish purple. Both of them were clear and transparent, with uniform color, no flocculation and no precipitation. The Tyndall phenomena of both of them were uniform and the optical paths were homogeneous, indicating that the dispersion of both of them was good.

The particle size potentials of H-ABNs and J-ABNs samples are shown in Table 1. With the increase of BSA mass concentration, the PDI of both samples decreased gradually, indicating that the dispersion degree and stability of the nanosome system improved gradually. The particle size of H-ABNs was the smallest when the BSA concentration was 0.05 mg/mL, which was ( 146 ± 24) nm, and the PDI was 0.43. At the same time, the particle size of J-ABNs was also the best, which was ( 266 ± 8) nm, and the PDI was 0.19, which indicated that the solution was monodisperse, with homogeneous particle size and good degree of dispersion (PDI<0.3). The potentials of H-ABNs and J-ABNs were (-9.69 ± 0.89) mV and (-6.16 ± 0.45) mV, respectively, at a mass concentration of 0.05 mg/mL. Compared with the single aggregates, the nanosystems enabled the astaxanthin aggregates to exist in a more stable manner.

 

2. 3 TEM analysis of H-ABNs and J-ABNs

H-ABNs and J-ABNs were prepared by using AST at a final mass concentration of 0.003 mg/mL and BSA at a final mass concentration of 0.05 mg/mL, and their transmission electron microscope images are shown in Figure 4. The H-ABNs were nearly spherical, with compact particles, the peripheral protein molecules were more tightly arranged than those of J-ABNs, and a small amount of gray flocculent existed in the periphery of the particles, which was presumed to be the exposure of the hydrophobic sites of the proteins; the particles were uniform in size, non-adhesive, and well dispersed, and their average TEM particle sizes were close to that of DLS hydrated particles and both were less than 200 nm; the J-ABNs were spherical in shape, and the sphere was composed of the black solid region and the loose protein peripheral ring, which was the most important part of the protein. J-ABNs were spherical, consisting of a black solid region and a loose protein periphery, uniform in size, non-adhesive and well dispersed, with an average DLS hydrated particle size of 200~300 nm and an average TEM particle size of about 200 nm. The TEM particle sizes were slightly smaller than the DLS particle sizes due to the fact that the nano-complexes in the hydrated particles were in the solution state and the molecular state was relatively loose. The size and morphology of H-ABNs and J-ABNs particles were consistent with the results of the study by Zhao, Yingyuan et al [14]. It is suggested that astaxanthin is successfully encapsulated in the hydrophobic core formed by bovine serum albumin.

 

2. 4 UV-vis analysis of H-ABNs and J-ABNs

The UV-vis of H-ABNs and J-ABNs with BSA concentrations of 0.01, 0.02 and 0.05 mg/mL are shown in Figure 5. The maximum absorption peak of H-ABNs was around 388 nm, and there was a weak shoulder peak in the range of 460~500 nm. With the increase of BSA content, the absorbance of H-ABNs increased and then decreased, while the absorbance of the shoulder peak decreased and then increased, so it was assumed that with the increase of BSA mass concentration, the H aggregates encapsulated in H-ABNs were gradually transformed. The J-ABNs had two absorption peaks at about 530 nm and 580 nm. As shown in Fig. 5, the peak absorbance of J-ABNs gradually increased with the increase of BSA content, indicating that increasing the mass concentration of BSA within a certain range is favorable to the formation of J-ABNs. Compared with the astaxanthin nanocomplexes prepared by Zhao et al [ 10], the UV absorption spectra of H-ABNs were basically similar; the maximum absorption peaks of J-ABNs were basically the same as those of J-ABNs, but there was a difference in the size of the shoulder peaks, which was presumed to be a change of the specificity of the interaction between astaxanthin and BSA with the increase of the concentration of BSA.

 

2. 5 Binding and loading analysis of H-ABNs with J-ABNs

The binding and loading rates of BSA at a mass concentration of 0.05 mg/mL for H aggregate astaxanthin were 61.10% and 3.46%, respectively; and for J aggregate astaxanthin, the binding and loading rates were 66.47% and 9.72%, respectively. Comparing with the astaxanthin-casein binary nano-complexes in the previous study of the team [21], the encapsulation and loading of casein to H-aggregate astaxanthin were 44.8% and 10.3%, respectively; and to J-aggregate astaxanthin, the encapsulation and loading rates were 22.7% and 4.54%, respectively, which indicated that the bovine serum albumin used in the present study had a better binding effect on astaxanthin and improved loading rate to J-aggregate astaxanthin. The loading rate of astaxanthin in J aggregates was improved.

 

2. 6 Fluorescence spectral analysis of H-ABNs and J-ABNs

Yao Huifang et al. [29] found that the fat-soluble active molecules combined with BSA through hydrogen bonding, electrostatic interaction, van der Waals force and hydrophobic interaction, etc., and changed the internal structure of BSA in the process of forming new complexes, thus causing the fluorescence burst, and meanwhile, it was proved that anthocyanins could bind with the tryptophan residues on the surface of BSA molecules, so as to change their spatial positions and form new complexes and then cause the static burst. The fluorescence spectra of H-ABNs and J-ABNs are shown in Figure 6, the AST monomer is fluorescence-free, and the BSA has the maximum emission wavelength at 342 nm, which is broadband fluorescence, and it can be identified as tryptophan residue [30]. As shown in Figure 6, the fluorescence intensity of H-ABNs and J-ABNs decreased dramatically due to the fluorescence burst of BSA, and the maximum emission wavelengths of H-ABNs and J-ABNs shifted by 10 nm with respect to that of BSA. It is assumed that the combination of the aggregated astaxanthin with BSA led to an increase in hydrophobicity in the microcosm of the amino acid residues, which suggests that the hydrophobicity around the liposoluble small molecule astaxanthin was increased by the combination of the aggregated astaxanthin and BSA. The hydrophobic microregion constructed by BSA was successfully encapsulated with the lipid-soluble active small molecule astaxanthin.

 

2.7 FTIR analysis of H-ABNs and J-ABNs

The characteristic absorption peaks of BSA molecules at 1,600-1,700 cm- 1 are mainly the -OH stretching peaks of BSA at 1,500-1,600 cm- 1, which are mainly caused by the stretching vibration of the C-N bond and the bending vibration of the N-H bond [32], and the -OH stretching peaks of BSA at 3,5003,400 cm- 1 [33]. -BSA, AST, AST, AST, AST, AST, AST, AST, AST, AST, AST, AST and AST. The infrared spectra of BSA, AST, AST-BSA powder physical mixtures, H-ABNs and J-ABNs solutions are shown in Figure 7. As shown in Fig. 7, the amide Ⅰ band, amide Ⅱ band, and -OH stretching peaks of H/J-ABNs were significantly weakened, indicating that the AST interacted with BSA and changed the internal structure of BSA; and the multiple peaks of the AST molecule in the range of 900~1,700 cm-1 almost disappeared, which suggests that the AST was wrapped up in the internal core of BSA successfully. The infrared peaks of the physical mixture of AST-BSA combined the peaks of AST and BSA, which did not interact with each other and their peaks remained unchanged.

 

3 Conclusion

In this study, the complexes of astaxanthin and bovine serum albumin were constructed by molecular self-assembly method, and the H-aggregated astaxanthin/bovine serum albumin nanocomplexes (H-ABNs) and J-aggregated astaxanthin/bovine serum albumin nanocomplexes (J-ABNs) could be prepared. After dissolving astaxanthin in different volume fractions of ethanol, the color of the solution changed, and the H-aggregated astaxanthin appeared in a light yellow color when the volume fraction of ethanol was 10%~20%; J-aggregated astaxanthin appeared in a purplish-red color when the volume fraction of ethanol was 25%~50%; and it appeared in an orange color when the volume fraction of ethanol was more than 60%, and it was presumed to be the M-aggregate of astaxanthin. The optimal conditions for the preparation of H-ABNs were 20% v/v astaxanthin ethanol solution and 35% v/v astaxanthin ethanol solution for J-ABNs. Dynamic light scattering particle size analysis (DLS) showed that the hydrated particle sizes of H-ABNs and J-ABNs were at the smallest nanoscale when the concentration of BSA was 0.05 mg/mL, and the dispersion of J-ABNs was better, and the size of J-ABNs was at the smallest nanoscale when the concentration was 0.05 mg/mL. The degree of dispersion of J-ABNs was better, and the stability of astaxanthin was improved compared with that of single aggregates. Combined with the changes of the characteristic peaks in the infrared spectra and the decrease in fluorescence intensity and the blue shift of the maximum emission spectrum due to the fluorescence burst of BSA, it was hypothesized that the hydrophobicity of the microenvironment of the amino acid residues of BSA was increased by the combination of astaxanthin and BSA through hydrogen bonding, electrostatic interactions, van der Waals forces and hydrophobic interactions, which proved that astaxanthin was encapsulated in the hydrophobic microregion constructed by BSA successfully. In this study, the preparation of H/J-ABNs was successfully obtained, and the binding mechanism of H/J-ABNs complexes was preliminarily investigated through various characterizations, which provided theoretical basis for improving the stability and bioavailability of astaxanthin aggregates, and is expected to expand the application of astaxanthin and its aggregates in the fields of food and medicine.

 

References .

 

[ 1] BROTOSUDARMO T H P , LIMANTARA L ,SETIYONO E , et al. Structures of astaxanthin and their consequences for therapeutic application [J] . International journal of food science , 2020 , 2020 : 2156582.

[2] STACHOWIAK B , SZULC P. Astaxanthin for the food industry [J] . Molecules , 2021 , 26(9) : 2666.

[3] JANNEL S , CARO Y , BERMUDES M ,et al. Novel insights into the biotechnological production of Haematococcus pluvialis-derived astaxanthin : advances Novel insights into the biotechnological production of Haematococcus pluvialis-derived astaxanthin : advances and key challenges to allow its industrial use as novel food ingredi- ent [J] . Journal of marine science and engi- neering , 2020 , 8( 10) : 789.

[4] NISHIDA Y , NAWAZ A , HECHT K , et al. Astaxanthin as a novel mitochondrial regula- tor : a new aspect of carotenoids , beyond an- tioxidants [ J ] .  Nutrients , 2021 , 14 ( 1 ) : 107.

[5] WU L W , MO W H , FENG J , et al. Astaxanthin attenuates hepatic damage and mito- chondrial dysfunction in non-alcoholic fatty liver disease by up- regulating the FGF21/ PGC-1 α pathway [ J ] .  British journal of pharmacology , 2020 , 177 ( 16 ): 3760 - 3777.

[6] CHUNG B Y , PARK S H , YUN S Y , et al. Astaxanthin protects ultraviolet B-induced ox- idative stress and apoptosis in human kerati- nocytes via intrinsic apoptotic pathway [J]. Annals of dermatology , 2022 , 34 (2) : 125- 131.

[7] RAHMAN S O , PANDA B P , PARVEZ S ,et al. Neuroprotective role of astaxanthin in hippocampal insulin resistance induced by Aβ peptides in animal model of Alzheimer's disease [J]. Biomedicine & pharmacothera- py , 2019 , 110 : 47-58.

[8] SUN L Y , KIM S , MORI R , et al. Astaxanthin exerts immunomodulatory effect by regu- lating SDH-HIF- 1α axis and reprogramming mitochondrial metabolism in LPS - stimulated RAW264.7 cells [J] . Marine drugs , 2022 , 20( 11) : 660.

[9] Y.Y. Zhao , Z.X. Wang , W.J. Xue , et al. Progress of Astaxanthin Aggregates [J]. Modern Food Science and Technology , 2021 ,37(7) : 327-334.

[ 10] ZHAO Y Y , LI J , DAI M Q , et al. Discrimi- native preparation of stable H - or J - aggre- gates of astaxanthin in waterborne chitosan/ DNA nanoparticles [ J ]. .  Chemistry letters , 2019 , 48(4) : 345-348.

[ 11] SUBRAMANIAN B , TCHOUKANOVA N , DJAOUED Y , et al. Raman spectroscopic in- vestigations on intermolecular interactions in aggregates and crystalline forms of trans- astaxanthin [J] . Journal of Raman spectros- copy , 2013 , 44(2) : 219-226.

[ 12] DAI M Q , LI C J , YANG Z , et al. The astaxanthin aggregation pattern greatly influ- ences its antioxidant activity : a comparative study in Caco - 2 cells [ J ]. J ] .  Antioxidants , 2020 , 9(2) : 126.

[ 13] Zhang Jianqiang , Diao Jingjing , Li Hao , et al. Research on functional properties of whey protein and its peptide products [J]. Packaging and Food Machinery , 2013 , 31(2) : 34-36 , 66.

[ 14] Zhao Yingyuan , Zhang Shengmeng , Li Yifan , et al. Study on the interaction between astaxanthin and whey protein based on fluorescence and ultraviolet spectroscopy [ J ]. Food Industry Technology , 2022 , 43 (2) : 126- 134.

[ 15] PAN L , LI H , HOU L F , et al. Gastrointes- tinal digestive fate of whey protein isolate coa- ted liposomes loading astaxanthin : lipolysis , release , and bioaccessibility [J] .  Food bio- science , 2022 , 45 : 101464.

[ 16] KARAMI E , BEHDANI M , KAZEMI-LOME- DASHT F. Albumin nanoparticles as nano- carriers for drug delivery : focusing on anti- body and nanobody delivery and albumin- based drugs [ J ] . and albumin- based drugs [ J ].  Journal of drug delivery science and technology , 2020 , 55 : 101471.

[ 17] WAGNER J , BILIADERIS C G , MOS- CHAKIS T. Whey proteins : musings on de- naturation , aggregate formation and gelation [ J ] .  Critical reviews in food science and nutrition , 2020 , 60(22) : 3793-3806.

[ 18] BABA W N , MCCLEMENTS D J , MAQ- SOOD S. Whey protein-polyphenol conju- gates and complexes : production , character- ization , and applications [J] . Food chemis- try , 2021 , 365 : 130455.

[ 19] BOURBON A I , PEREIRA R N , PASTRA- NA L M , et al. Protein-based structures for food applications : from macro to nanoscale [J ] . Frontiers in sustainable food systems , 2018 , 2 : 77.

[20] AN F F , YANG Z , ZHENG M C , et al. Ra- tionally assembled albumin/indocyanine green nano complex for enhanced tumor ima- ging to guide photothermal therapy [ J ]. Journal of nanobiotechnology , 2020 , 18( 1) : 49.

[21] ZHAO Yingyuan , DANG Yucheng , TANG Zhongyi , et al. Preparation, characterization and stability of astaxanthin-casein nanocomposite[J]. Food Science and Technology , 2021 , 46( 11) : 236-243.

[22] LU L P , HU T P , XU Z G. Structural char- acterization of astaxanthin aggregates as re- vealed by analysis and simulation of optical spectra [ J ] .  Spectrochimica acta part A : molecular and biomolecular spectroscopy , 2017 , 185 : 85-92.

[23] ZHU J X , SUN X W , WANG S H , et al. Formation of nano complexes comprising whey proteins and fucoxanthin : characterization , spectroscopic analysis , and molecular doc- king [ J ] . and molecular doc- king [ J ].  Food hydrocolloids , 2017 , 63 : 391-403.

[24] Hou Yahui . Design and realization of food safety rapid detector based on Lambert-Beer law [ D ] . Beijing : China University of Geosciences , 2017.

[25] DU Chen , YIN Huanhuan , ZHAO Chengbin , et al. Effects of ultrasonic treatment on the structure and functional properties of red bean protein-lutein complex [J]. Food Science , 2020 , 41( 11) : 104- 112.

[26] Wang Xiaoyi , Deng Xiaoyu , Han Lishu , et al. Effect of gastrointestinal environment on the stabilization of Pickering high internal phase emulsion and its astaxanthin loading by silkworm pupa protein [J] . Food Science , 2023 , 44(6) : 41-48.

[27] Huang Ling . Fatty acid-mediated self-assembly of astaxanthin-protein and absorption properties of the complex[D] . Fujian : Jimei University ,2021.

[28] Zhao Yingyuan , Jia Huihui , Li Ziwei , et al. Progress of carotenoid aggregates [J]. JOURNAL OF HENAN INDUSTRIAL UNIVERSITY (NATURAL SCIENCE EDITION) , 2021 , 42(6) : 134- 140.

[29] Yao Huifang , Jing Hao . Interaction between anthocyanins from Vaccinium myrtillus and bovine serum albumin [J] . Food Science , 2013 , 34(23) : 6- 10.

[30] ZHANG Qingqing . Study on the structural and functional effects of glutamine aminotransferase on whey protein [ D ]. Hohhot : Inner Mongolia University of Technology , 2021.

[31] SHI Yan , LIU Fan , GE Hui , et al. Infrared spectroscopy of protein secondary structure changes during microencapsulation [J]. Spectroscopy and Spectral Analysis , 2012 , 32 (7 ) : 1815-1819 .

[32] JIN C G , PATEL A , PETERS J , et al. Quantum cascade laser based infrared spec- troscopy : a new paradigm for protein second- ary structure measurement [J ] . Pharmaceuti- cal research , 2023 , 40(6) : 1507- 1517.

[33] Chen Sha . Preparation of BCC natural composite gum and encapsulation of Rhodiola rosea glycosides and Vc [ D ] .  Nanchang University , 2016.