How does deterioration of different sources of astaxanthin affect antioxidant activity in rainbow trout?

 In recent years, the culture development of rainbow trout (Oncorhynchus mykiss) in China has been rapid, and the culture output has reached 39.37 million tons by 2019, which is an important cold water economic fish in China. The muscle redness value of rainbow trout is an important criterion for the market to evaluate its quality, and in the production, astaxanthin is often added exogenously to improve the color of rainbow trout muscle. Currently, the main astaxanthin used in production is synthetic astaxanthin (Ast), which accounts for 10%-20% of the total cost of feed [1]. The safety of Ast has been debated due to the presence of different stereoisomers and possible residues of synthetic intermediates. Natural sources of astaxanthin include Haematococcus pluvialis [2], Phafia rhodozyma [3], Spirulina platen-sis [4], Chlorella zofingiensis [5], etc. Natural sources of astaxanthin are more stable, and the safety of astaxanthin has been discussed. Astaxanthin from natural sources is more stable and safe to use, but there are problems such as complicated extraction process and high price.

 

Summer side marigold (Adonis aestivalis L.), also known as Fuchsia, belongs to the buttercup family (Ranunculaceae), side marigold genus (Adon- is L.), its petals are rich in carotenoids, of which the astaxanthin content of the total amount of carotenoids accounted for more than 80%, about 1% of the dry weight of the flower petal, is a high-quality natural astaxanthin source [6]. It is a high quality natural source of astaxanthin [6]. However, studies on summer marigold have mainly focused on cardiac glycosides[7] , and little research has been done on its use as a colorant.Kamata et al.[8] fed rainbow trout with diets containing 5.05% of A. fumigatus petals (AF) and 0.01% of A. fumigatus extracts (AE) (converted to astaxanthin content of 100 mg/kg) for 3 months, and high mortality rates (30%) were observed in the AF group. As a result, the AF group had a high mortality rate (30%), and the AE group increased the muscle redness value, but the amount of astaxanthin deposited in the muscle was very low, only 1.17 mg/kg, which was not up to the market requirement (6 mg/kg).

 

Red algae, belonging to Chlorophyta, Volvocales, Haematococcus, are the organisms with the highest known accumulation of naturally occurring astaxanthin, with astaxanthin accumulation up to 5% of dry weight[9] . Astaxanthin in R. rainieri usually exists in the form of esters, with a pure levulinic structure (3S,3 'S), which is one of the most active configurations in antioxidant activity [10]. However, the thick cell wall of Rhodococcus pyrenoidus prevents fish from absorbing and utilizing the carotenoids [11] and reduces the utilization of the algal meal as bait directly. Therefore, the addition of Rhodococcus aurantium as astaxanthin source to feed, should use appropriate wall breaking method, if the wall breaking method is not appropriate, incomplete wall breaking, will affect the activity and utilization of astaxanthin. The application of Rhodococcus rainbowii algal powder in aquaculture has been reported in rainbow trout [12], Atlantic salmon (Salmo salar) [13], red sea bream (Pagrus pagrus) [14], croaker (Pseudosciaena cro- cea) [15], European catfish (Silurus glanis) [16], etc., and the studies showed that Rhodococcus rainbowii has a good effect on the utilization of astaxanthin. These studies have shown that red algae have good coloring and antioxidant effects, and can improve the growth performance and enhance the immunity of fish. Currently, most of the studies on R. rainbowii are based on algal powder, but there are relatively few studies on R. rainbowii extract (HE)[2,17] , especially in the coloration of salmon and trout.

 

How effective are AF, AE and HE as natural astaxanthin sources in improving meat color and antioxidant properties of rainbow trout? How do they compare with Ast? There is no clear report. Therefore, in this experiment, Ast, AF, AE and HE were added to the feed of rainbow trout to investigate their effects on the growth performance, pigment deposition and antioxidant capacity of rainbow trout, so as to provide a theoretical basis for the rational application of natural astaxanthin sources in aquatic feeds.

 

1 Materials and Methods

1.1 Test materials

Ast, AF, AE and HE (n-butane extraction) were provided by a Guangzhou biotechnology company, and the astaxanthin contents were 10.30%, 1.54%, 2.90% and 2.26%, respectively.

 

1.2 Test feed

Five kinds of nitrogen and energy feeds were formulated, namely, the base feed and the test feed with 1.0 g/kg Ast, 6.5 g/kg AF, 3.4 g/kg AE, 4.4 g/kg HE added to the base feed, which were converted to 100 mg/kg of astaxanthin. Single-screw extruder pelleting into 2.0 mm diameter hard granular sinking feed [pelleting temperature (85 ± 5) ℃], with a blower drying oven at 40 ℃ drying to moisture content of less than 10%, sealed and stored for use. The contents of astaxanthin in these five feeds were 11.00, 95.23, 101.32, 104.25 and 93.52 mg/kg, respectively.

Table 1 Composition and nutrient levels of experimental diets (air-dry basis) g/kg

 

Project Items	control subjects Control group	Synthetic astaxanthin group Ast group	Fuchsia petal group AF group	Forsythia extract AE group	Erythrocystis japonicus extract HE group
Ingredients
Fish meal	250.0	250.0	250.0	250.0	250.0
Soybean meal	200.0	200.0	200.0	200.0	200.0
Soy protein concentrate	110.0	110.0	110.0	110.0	110.0
Flour	260.0	259.0	253.5	256.6	255.6
Pork meat powder	50.0	50.0	50.0	50.0	50.0
Brewers dried yeast	40.0	40.0	40.0	40.0	40.0
Fish oil	30.0	30.0	30.0	30.0	30.0
Soybean meal	30.0	30.0	30.0	30.0	30.0
Vitamin premix1)	5.0	5.0	5.0	5.0	5.0
Mineral premix2)	5.0	5.0	5.0	5.0	5.0
Calcium dihydrogen phosphate Ca(H2  PO )4 2	15.0	15.0	15.0	15.0	15.0

 

Choline chloride	5.0	5.0	5.0	5.0	5.0
Synthesis astaxanthin		1.0
Adonis aestivalis flower			6.5
Adonis aestivalis extract				3.4
Haematococcus pluvialis extract					4.4
Total	1 000.0	1 000.0	1 000.0	1 000.0	1 000.0
Nutrient levels3)
Moisture	39.3	39.0	38.0	41.5	41.7
Crude protein CP	451.3	446.2	445.7	442.9	441.1
Crude fat EE	134.8	134.7	134.5	124.4	125.1
Crude Ash Ash	85.5	84.8	86.8	85.4	85.5

 

 

 

1.3 Test fish and feeding management

The rainbow trout was purchased from Tiangui Aquaculture Farm, Dongpo District, Meishan City, Sichuan Province, China. The rainbow trout were temporarily reared for 2 weeks to acclimatize to the experimental environment. The feeding was stopped for 24 h before the formal experiment, and 375 rainbow trout of healthy and uniform size [average weight (6.28±0.07) g] were randomly assigned to 15 self-inflating recirculating glass tanks (0.60 m×0.60 m×0.50 m) with 25 trout in each tank, and there were 5 groups of 3 replicates in each group. Before the start of the culture experiment, 20 rainbow trout were stored at -20 ℃ for the initial analysis of the whole fish routine composition. During the culture period, the fish were fed twice a day (09:00 and 16:00), and the daily feeding rate was 2%~3% of the body weight, which was adjusted according to the feeding situation of the fish and the weather condition, and the feeding level of each group was basically the same, and no residual bait was preferred in each feeding. The water temperature was 13~18 ℃, the dissolved oxygen content was 6~7 mg/L, the pH was 7.24~7.78, the ammonia nitrogen content was ≤0.2 mg/L, and the nitrite content was ≤0.1 mg/L. The fish were fed for 1~2 h in the morning, and the feces at the bottom of the tank were removed by siphoning, and the water was changed twice a week, and the amount of water was 1/3 of the volume of water in the tank. The culture experiment was conducted in the Fish Nutrition Laboratory of Shanghai Ocean University for 6 weeks.

 

1.4 Sample collection

The samples were collected according to the method of Zhao et al.[18] At the end of the 2nd, 4th and 6th weeks of culture, three fish were randomly taken from each tank after 24 h of starvation and anesthetized with 100 mg/L MS-222. Blood was collected from the tail vein, centrifuged at 8000 r/min for 10 min, and the serum was stored at -80 ℃ for the determination of serum carotenoids. After blood collection, the skin of both dorsal parts was peeled off, the muscle between the lateral line and the dorsal fin was removed, and the color difference was measured, then stored with the skin and the caudal fin at -20 ℃ for determination of the astaxanthin content of the tissues. 6 weeks of the culture test was completed, and the fish were starved for 24 h. All the rainbow trout in the tanks were weighed, and the weights and numbers of tails were recorded, and 3 tails were taken from the tanks, and then anaesthetized and stored at -20 ℃ for determination of the whole fish conventional ingredients and astaxanthin content. The muscle, liver and serum of three fish were collected for the determination of antioxidant capacity, and 1.5 g of each muscle on both sides of the back of the other three fish were collected for the determination of drip loss and freezing loss, respectively.

 

1.5 Measurement indicators

1.5.1 Growth performance

The survival rate, weight gain rate and feed coefficient were calculated based on the first and last weights, number of tails and feeding rate of rainbow trout.

 

1.5.2 Feed and whole fish composition

After crushing the whole fish, the initial whole fish and the feed, the routine composition analysis was carried out with reference to AOAC (2000) as follows: moisture content was determined by drying at 105 ℃; crude protein content was determined by an automatic Kjeldahl nitrogen analyzer (2300-Auto-Analyzer, Fosstecator, Sweden); crude fat content was determined by chloroform-methanol extraction; crude ash content was determined by cauterization at 550 ℃ in a muffle furnace (SXL-1008 muffle furnace, Shanghai Jinghong Experimental Equipment Co., Ltd.). The crude fat content was determined by methanol extraction with chloroform, and the crude ash content was determined by cauterization in a muffle furnace (SXL-1008 muffle furnace, Shanghai Jinghong Experimental Equipment Co., Ltd.) at 550 ℃.

 

1.5.3 Muscle color difference values

After blood sampling and skinning, the muscle between the lateral line and the dorsal fin was taken, and the surface was dried with absorbent paper, then the probe of WSC-S colorimeter (WSC-S colorimeter, o/d light source, with luster, stability ΔY≤0.6, Shanghai Precision Scientific Instruments Co.

 

1.5.4 Astaxanthin content

The astaxanthin content of muscle and whole fish was determined by chloroform-ethanol (1:1) extraction according to the method of Zhang et al. [19], and the astaxanthin content of skin and fins was determined by dichloromethane-methanol (1:3) extraction according to the method of Song et al. [20]. The absorbance (OD) was measured and the astaxanthin content was calculated according to the standard curve of astaxanthin (Astaxanthin standard was purchased from Shanghai Jizhi Biochemical Technology Co.

 

1.5.5 Total serum carotenoids

After 0.2 mL of serum was mixed with 0.4 mL of 95% ethanol, 1 mL of n-hexane was added and centrifuged at 1,000 r/min for 5 min. The OD value of the supernatant was measured at 470 nm, and the total carotenoid content was calculated according to the standard curve of all-trans astaxanthin. The total carotenoid content was calculated from the standard curve of all-trans astaxanthin. The standard curve of all-trans astaxanthin was prepared according to Tolasa et al.

Serum carotenoid content (μg/mL) = OD value × OD value (μg/mL).

10,000/extinction coefficient E.

 

1.5.6 Muscle water holding capacity

The muscle of one side of the back of rainbow trout (1.5 g) was weighed (W1) and suspended by a thin wire in a refrigerator at 4 ℃. The muscle was removed from the refrigerator at 2, 4, and 6 h, and then weighed after gently wiping off the surface water with absorbent paper and the weight was recorded (W2). The other side of the back muscle (1.5 g) was weighed (W3), sealed in a bag and placed in the refrigerator at -20 ℃ for 24 h. After 24 h, the muscle was thawed at room temperature for 10 min, and then weighed after gently removing the surface water with absorbent paper, and the weight was recorded (W4). Drip loss and freezing loss were calculated as follows.

Drip loss (%) = 100 x [ ( W1 -W2 )/W1 ] ; Freezing loss (%) = 100 x [ ( W3 -W4 )/W3 ].

 

1.5.7 Antioxidant capacity of serum, muscle and liver

The liver and back muscle were thawed at 4 ℃, and the supernatant was made into 20% tissue homogenate with 0.9% saline, centrifuged at 2 500 r/min for 10 min. The antioxidant indices of serum, muscle and liver included total protein (TP), malondialdehyde (MDA), total superoxide dismutase (T-SOD) and hydroxyl radical inhibition in serum, muscle and liver. The above indexes were determined according to the instructions of the kit (Nanjing Jianjian Institute of Biological Engineering), in which the total protein was determined by the Coomassie blue method, the MDA content was determined by the thiobarbituric acid (TAB) method, and the ability to inhibit hydroxyl radicals was determined by the Fenton reaction.

 

Definition of Serum and Tissue T-SOD Activity Units (U/mL): The amount of SOD per milliliter of reaction solution and per milligram of tissue protein that results in 50% inhibition of SOD in 1 mL of reaction solution is defined as one SOD activity unit (U).

 

1.6 Statistics and analysis of data

The experimental data were analyzed by one-way ANOVA using SPSS 22.0 and Tukey's method for multiple comparisons.

 

2 Results

2.1 Effects of different sources of astaxanthin on growth performance of rainbow trout (Oncorhynchus mykiss)

As shown in Table 2, after 6 weeks of culture, there were no significant differences in weight gain rate, feed coefficient and survival rate among the Con, Ast, AE and HE groups (P>0.05), while the AF group had a significantly lower weight gain rate (P<0.05) and higher feed coefficient (P<0.05) than the other groups, and the weight gain rate of the AF group was reduced by 13.5% and increased by 0.10 (P<0.05) when compared with the control group. Compared with the control group, the weight gain rate of AF group decreased by 13.5% and the feed coefficient increased by 0.10 (P<0.05).

 

2.2 Effect of different sources of astaxanthin on the conventional composition of whole rainbow trout (Oncorhynchus mykiss)

As shown in Table 3, there was no significant difference (P>0.05) in the composition of whole rainbow trout, including moisture, crude protein, crude ash and crude fat content, among the groups.

 

2.3 Effects of different sources of astaxanthin on the color difference of rainbow trout muscles

As shown in Figure 1, with the increase of culture time, the muscle brightness and redness of rainbow trout decreased and increased in all groups; the muscle brightness of Ast, AF, AE and HE groups was significantly lower than that of the control group (P<0.05), and the redness and yellowness values of Ast, AF, AE and HE groups were significantly higher than those of the control group (P<0.05) at all time points; in the 6th week, there was no significant difference (P>0.05) in the values of muscle brightness and redness of astaxanthin-added groups; the muscle yellowness values of AE and HE groups were significantly higher than those of the Ast and AF groups (P<0.05). At week 6, there was no significant difference between the muscle brightness and redness values of the astaxanthin-added groups (P>0.05), and the muscle yellowness values of the AE and HE groups were significantly higher than those of the Ast and AF groups (P<0.05).

 

Table 2 Effects of different astaxanthin sources on growth performance of rainbow trout

 

sports event	control subjects	Synthetic astaxanthin group	Fuchsia Flower Petal Set	Forsythia extract group	Erythrocystis japonicus Extract Group
Items	Control group	Ast group	AF group	AE group	HE group
Initial weight IBW/g	6.24±0.04	6.34±0.07	6.25±0.05	6.25±0.03	6.24±0.04
Final weight FBW/g	27.62±0.31b	27.16±0.94b	24.10±1.05a	26.19±0.92b	26.27±0.78b
Weight gain rate WGR/%	339.45±1.29b	328.54±12.54b	293.74±16.98a	322.64±10.88b	325.16±6.57b
Survival ratio/%	100	100	100	100	100
Feed coefficient FCR	0.99±0.04a	1.01±0.02a	1.09±0.02b	1.03±0.04a	1.03±0.03a

 

 

Table 3 Effects of different astaxanthin sources on whole body routine composition of rainbow trout g/kg

 

sports event	control subjects	Synthetic astaxanthin group	Fuchsia Petal Set	Forsythia extract group	Erythrocystis japonicus Extract Group
Items	Control group	Ast group	AF group	AE group	HE group
Moisture	715.18±2.45	708.04±9.05	716.56±6.04	710.08±5.82	713.54±7.60
Crude protein CP	161.98±4.46	162.73±6.84	160.03±3.58	162.96±4.12	164.43±6.67
Crude fat EE	75.85±3.05	81.27±9.10	82.28±5.28	76.54±3.13	79.30±4.94
Crude Ash Ash	24.23±0.97	24.81±1.13	24.76±0.51	24.75±0.85	23.95±0.63

 

 

2.4 Effects of different sources of astaxanthin on astaxanthin content and deposition rate in rainbow trout tissues

As shown in Table 4, the astaxanthin content in different tissues of all astaxanthin-added groups increased with the increase of culture time. At weeks 2, 4 and 6, the astaxanthin content in muscle, skin and caudal fin of Ast, AF, AE and HE groups was significantly higher than that of the control group (P<0.05); at week 6, the muscle astaxanthin content was the highest in the HE group, and the skin and caudal fin of the AF group were the highest; there was no significant difference in whole fish astaxanthin content and astaxanthin deposition rate between Ast, AF, AE and HE groups (P>0.05); whole fish astaxanthin content was significantly higher than that of the control group (P>0.05); and whole fish astaxanthin content was significantly higher than that of the control group (P>0.05). In terms of whole fish astaxanthin content and astaxanthin deposition rate, there was no significant difference between the Ast, AF, AE and HE groups (P>0.05), while the whole fish astaxanthin content was significantly higher than that of the control group (P<0.05), and the astaxanthin deposition rate was significantly lower than that of the control group (P<0.05).

 

Table 4 Effects of different astaxanthin sources on astaxanthin content and retention in tissue of rainbow trout

 

Project Items	Farming time Breeding time	control subjects Control group	Synthetic astaxanthin group Ast group	Fuchsia petal group AF group	Forsythia extract AE group	Erythrocystis japonicus extract HE group
Tissue astaxanthin content Astaxanthin		content in tissue/(mg/	kg)
Initial		0.79±0.07A	0.79±0.07A	0.79±0.07A	0.79±0.07A	0.79±0.07A
Muscle Week 2 Week		2 0.81±0.06a,A	1.85±0.30b,B	1.39±0.32b,B	2.97±0.79c,B	3.36±0.17c,B
Flesh Week 4 Week		4 0.87±0.06a,A	4.57±0.33b,C	4.56±0.25b,C	4.55±0.52b,C	4.12±0.44b,B
Week 6 Week		6 1.35±0.50a,B	4.96±0.79b,C	4.81±0.54b,C	5.20±0.91b,C	5.26±0.91b,C
Initial		2.49±0.18A	2.49±0.18A	2.49±0.18A	2.49±0.18A	2.49±0.18A
Skin Week 2 Week		2 1.73±0.48a,A	3.19±0.26b,B	3.04±0.89b,A	2.70±0.84b,A	2.95±0.68b,AB
Skin Week 4 Week		4 2.26±0.37a,A	3.90±0.15b,C	3.78±0.48b,AB	3.87±0.33b,AB	3.23±0.57b,AB
Week 6 Week		6 2.63±0.59a,A	4.02±0.25b,C	4.83±0.52c,B	4.13±0.17bc,B	3.74±0.36b,B
Initial		2.63±0.46A	2.63±0.46A	2.63±0.46A	2.63±0.46A	2.63±0.46A
Caudal Fins Week 2 Week		2 1.77±0.07a,A	10.08±1.13c,B	12.08±1.15c,B	7.11±0.95b,B	9.85±1.94c,B
Caudal fin Week 4 Week		4 1.93±0.37a,A	11.74±0.41c,B	15.35±1.80d,C	10.24±0.96b,C	12.85±0.71c,BC
Week 6 Week		6 2.73±0.52a,A	15.25±0.90b,C	17.84±0.61c,C	15.78±0.41b,D	15.82±0.56b,C
Whole body		5.59±0.27a	7.73±0.39b	7.69±0.35b	7.40±0.16b	7.21±0.06b
Astaxanthin deposition rate Astaxanthin retention/%		63.16±3.49b	10.37±0.57a	8.89±0.36a	9.71±1.67a	9.28±0.08a

 

 

2.5 Effects of different sources of astaxanthin on serum carotenoids in rainbow trout (Oncorhynchus mykiss)

As shown in Table 5, the serum carotenoid content of each group increased with the increase of incubation time, and the serum carotenoid content of Ast, AF (except week 2), AE and HE groups was significantly higher than that of the control group at weeks 2, 4 and 6 (P<0.05), and there was no significant difference between them at weeks 4 and 6 (P>0.05).

 

2.6 Effects of different sources of astaxanthin on the antioxidant capacity of muscle, liver and serum of rainbow trout (Oncorhynchus mykiss)

As shown in Table 6, the T-SOD activity and MDA content of muscle and serum of all astaxanthin-added groups were significantly lower than those of the control group (P<0.05), and the hydroxyl radical inhibition capacity of all tissues of Ast, AF, AE and HE groups was significantly higher than that of the control group (P<0.05). In the AF group, muscle antioxidant indices were not significantly different from those of the AE group (P>0.05), but the ability of liver to inhibit hydroxyl radicals was significantly lower than that of the AE group (P<0.05), and the activity of serum T-SOD was significantly higher than that of the AE group (P<0.05); there were no significant differences in the antioxidant indices of the AE and HE groups in terms of muscle, liver and serum antioxidant indices between these two groups and those of the Ast group (P>0.05).

2.7 Effect of different sources of astaxanthin on water holding capacity of rainbow trout muscle

As shown in Table 7, the drip loss of each group increased with time, and the drip loss (except for the drip loss of Ast group at 2 h) and freezing loss of Ast, AE and HE groups were significantly lower than that of the control group (P<0.05); in addition, the drip loss of AF group at 4 and 6 h was also significantly lower than that of the control group (P<0.05), while the freezing loss was not significantly different from that of the control group (P>0.05). gt;0.05).

 

 

Table 5 Effects of different astaxanthin sources on serum carotenoid contents of rainbow trout μg/mL

 

Farming time	control subjects	Synthetic astaxanthin group	Fuchsia Petal Set	Forsythia extract group	Erythrocystis japonicus Extract Group
Breeding time	Control group	Ast group	AF group	AE group	HE group
Initial	0.14±0.05A	0.14±0.05A	0.14±0.05A	0.14±0.05A	0.14±0.05A
Week 2	0.26±0.04a,AB	0.50±0.07bc,B	0.35±0.06ab,B	0.42±0.07bc,A	0.53±0.09c,B
Week 4	0.26±0.16a,A	1.09±0.18b,C	0.95±0.08b,C	0.90±0.08b,B	1.01±0.14b,C
Week 6	0.32±0.07a,A	1.24±0.07b,C	1.13±0.05b,D	1.18±0.17b,B	1.30±0.12b,D

 

 

Table 6 Effects of different astaxanthin sources on antioxidant ability of flesh , liver and serum of rainbow trout

 

Tissue	Project Items	control subjects Control group	Synthetic astaxanthin group Ast group	AF group	Forsythia extract AE group	Erythrocystis japonicus extract HE group
Muscle Flesh	Total superoxide dismutase T-SOD/(U/mg prot)	8.66±0.41b	6.45±0.66a	6.69±0.74a	6.24±0.53a	6.14±0.51a
	malondialdehyde MDA/(nmol/mg prot)	11.39±1.45b	7.04±0.91a	7.79±0.73a	6.44±0.65a	6.44±0.76a
	Ability to inhibit hydroxyl radicals
	Inhibition ability of -OH/(U/mg prot)	7.25±0.96a	12.84±1.06b	11.26±1.26b	12.23±0.78b	12.99±1.24b
Liver	Total superoxide dismutase T-SOD/(U/mg prot)	5.32±0.16b	4.44±0.17a	4.87±0.19ab	4.57±0.59a	4.58±0.03a
	malondialdehyde MDA/(nmol/mg prot) Ability to inhibit hydroxyl radicals	2.68±0.42c	1.89±0.32ab	2.41±0.50bc	1.63±0.57a	1.85±0.49ab
	Inhibition ability of -OH/(U/mg prot)	12.75±2.34a	19.02±0.98bc	16.84±1.81b	21.74±3.12c	19.70±2.63bc
	Total superoxide dismutase T-SOD/(U/mL)	249.17±8.73c	211.52±5.86ab	216.79±2.43b	204.16±7.00a	208.72±8.27ab
Serum	malondialdehyde MDA/(nmol/mL) Ability to inhibit hydroxyl radicals Inhibition ability of -OH/(U/mL)	13.11±1.05c   89.48±9.88a	4.39±0.71a   193.35±10.78b	7.48±0.91b   190.56±4.19b	5.47±1.55ab   205.60±1.05b	5.77±0.97ab   200.64±9.90b

 

 

3 Discussion

3.1 Effects of different sources of astaxanthin on growth performance of rainbow trout (Oncorhynchus mykiss)

There are different reports on the effects of Ast and HP on the growth of fish, Christiansen et al [21] and Wang Lei et al [22] showed significant improvement in the growth performance of Atlantic salmon (1.75 g) and rainbow trout by adding 50, 70 and 100 mg/kg of Ast to their diets, respectively. However, the addition of 50, 70 and 100 mg/kg Ast by Page et al. [23], Yanar et al. [24] and Amar et al. [25] had no significant effect on the growth performance of rainbow trout (Oncorhynchus mykiss), while the addition of 2.0 g/kg HE (100 mg/kg astaxanthin) by Pham et al. [2] also had a significant effect on the weight gain, specific growth rate and survival of juvenile flounder. There was no significant effect on the weight gain rate, specific growth rate and survival rate of juvenile toothfish. In the present experiment, the additions of Ast and HE also had no significant effect on the growth performance of rainbow trout, although the feed coefficients of the two additions were slightly higher than those of the control group, but there was no significant difference, which might be caused by experimental error. The effects of astaxanthin on the growth performance of fish may be related to fish species, sex, growth stage, feed composition and culture conditions.

 

Table 7 Effects of different astaxanthin sources on flesh water-holding capacity of rainbow trout %

 

Project Items	Drip loss			Thawing loss
	2 h	4 h	6 h
Control group	9.74±1.06b	18.69±1.78c	23.91±1.71c	6.19±0.47b
Synthetic astaxanthin group Ast group	8.73±0.85ab	13.71±1.04b	17.62±2.02b	4.66±0.69a
AF group	8.23±0.63ab	14.18±0.39b	16.96±1.33b	5.57±0.84ab
AE group	7.50±1.11a	10.78±1.51a	14.52±1.50a	4.80±0.55a
Erythrocystis japonicus extract group HE group	7.55±1.27a	12.33±1.61ab	17.47±1.46b	4.86±0.41a

 

 

In this experiment, although the addition of AF did not affect the survival rate of rainbow trout (100%), it reduced the growth performance of the fish, which to a certain extent reflected the negative effects of alkaloids, cardiac glycosides and other toxic and harmful substances contained in the petals on feeding and feed utilization, and indicated that AF should not be added to rainbow trout feed directly. In order to minimize or eliminate the effects of toxic substances in AF, the extraction of astaxanthin is an effective way. Cardiac glycosides are water-soluble substances, and the extraction of astaxanthin is an organic solvent extraction process, so AE is basically free of cardiac glycosides. A study showed that the addition of 0.01% of AE (converted to 100 mg/kg of astaxanthin) to feed had no significant effect on the growth performance of rainbow trout[8] , and the addition of 3.4 g/kg of AE to feed in the present experiment did not have any adverse effect on the growth performance of rainbow trout. In the future, the development and utilization of fuchsia resources should follow the path of active substance extraction.

 

3.2 Effects of different sources of astaxanthin on meat color and astaxanthin deposition in rainbow trout (Oncorhynchus mykiss)

The muscle redness value of rainbow trout is an important quality criterion, which depends on astaxanthin deposition in the body.Rahman et al. [26] significantly increased the muscle redness value of rainbow trout (Oncorhynchus mykiss) weighing 18.5 g fed 100 mg/kg Ast for 10 weeks with muscle astaxanthin content of 6.1 mg/kg.Zhang et al. [19] significantly increased the muscle redness value of rainbow trout (Oncorhynchus mykiss) weighing 101 g fed 100 mg/kg Ast for 60 d, and the astaxanthin content of 8.03 mg/kg was found in the muscle of rainbow trout. Zhang et al. [19] fed 101 g of rainbow trout with 100 mg/kg Ast feed for 60 d, which resulted in a significant increase in muscle redness value and astaxanthin content of 8.03 mg/kg, and De La Mora et al. [27] fed 80 mg/kg Ast feed for 6 weeks to rainbow trout weighing 161 g, which resulted in muscle astaxanthin content of 8.8 mg/kg. After 6 weeks of culture, the muscle redness value and astaxanthin content of both the HE and AE groups increased significantly and reached the same level as that of Ast, but the muscle astaxanthin content was low, ranging from 4.96 to 5.26 mg/kg, which might be related to the short culture period (6 weeks) and the small size of the experimental fish (initial weight of 6.28 g). In aquaculture production, the muscle of rainbow trout is usually colored at a period of time before marketing, and the small size of rainbow trout used in this experiment was mainly because it was easy to culture small-size fish under laboratory conditions, and it could be used to screen astaxanthin sources on a larger scale, thus laying a foundation for the coloration test of rainbow trout adults.

 

In addition, the addition of Ast, AF and AE to the diets significantly increased the muscle redness and yellowness values of rainbow trout in the present study. Among them, there was no significant difference in the muscle redness value and astaxanthin content between the Ast and AE groups at the end of 6 weeks of culture, which was in agreement with the results of the study conducted on rainbow trout by Kamata et al[28] . However, Kamata et al[8] found that the addition of 5.05% AF (100 mg/kg astaxanthin) to the diet did not have a significant effect on the meat color of rainbow trout, which might be due to the slight deterioration in taste of the diets after 5-6 d, which had a greater impact on the intake of rainbow trout, and resulted in poorer deposition of the pigmentation.

 

Most of the astaxanthin in AF and HP is in the form of esters [29], while that in artificial Ast is in the free state [9]. Some studies have shown that esterified astaxanthin is better absorbed by animals, which may be related to the low polarity of astaxanthin esters and their good solubility in the digestive tract[30-31] ; while Henmi et al.[32] suggested that free astaxanthin has better coloring effect than esterified astaxanthin at the same astaxanthin dosage, which may be attributed to the fact that free astaxanthin binds tightly to actin, while mono esterified astaxanthin is weakly bound to actin, while di ester is not bound at all. This may be due to the fact that free astaxanthin binds tightly to actin, monoesterified astaxanthin binds weakly to actin, and diester does not bind at all [32], resulting in poor deposition of esterified astaxanthin. However, in the present study, astaxanthin deposition in the AF, AE and HE groups was not significantly different from that in the Ast group, which is consistent with the findings of Bowen et al.[33] who fed mono-, di- and Ast-enriched feeds to rainbow trout. Schiedt[34] and Zhou[35] concluded that astaxanthin esters need to be hydrolyzed after entering the animal body in order to be absorbed and utilized. In a study by Su Fang[36] , it was found that astaxanthin from red algae Rainbow trout was de-esterified during its delivery in the rainbow trout. These studies showed that the esterification of astaxanthin did not affect its absorption and utilization by rainbow trout.

 

3.3 Effect of different sources of astaxanthin on the antioxidant capacity of rainbow trout (Oncorhynchus mykiss)

Rahman et al.[26] and Zhang et al.[19] significantly reduced serum SOD activity in rainbow trout fed diets supplemented with 50 and 100 mg/kg of astaxanthin, respectively, and muscle MDA levels were significantly reduced in rainbow trout fed diets supplemented with red fife yeast[37] and Ast[18-19] . In addition, the addition of Ast to the diets of horse fat carp (Hyphessobrycon eques Stein- dachner), blue carp (Hyphessobrycon callistus), and spotted prawn (Penaeus monodon) also significantly enhanced the antioxidant capacity of the organisms[38-40] . Meanwhile, the addition of astaxanthin to the diets of rainbow trout (Oncorhynchus mykiss) enhanced the hydroxyl radical inhibition capacity of serum, muscle and liver[18,26] . In the present experiment, the hydroxyl radical inhibition ability of rainbow trout muscle, liver and serum was significantly increased, and the T-SOD activity and MDA content were significantly reduced to the same level as that of Ast when AE and HE were added to the feeds respectively. The antioxidant property of astaxanthin is related to the unsaturated ketone and hydroxyl groups on the violet ketone ring at both ends of the astaxanthin, which have active electronic effects and can attract free radicals or provide electrons to free radicals to ultimately scavenge free radicals and improve antioxidant effects[41] .

 

Hydraulic force refers to the ability to maintain the original water when the muscle is subjected to external forces such as pressure, freezing, etc., is an important indicator of muscle quality, when the muscle is exposed to air, it will be oxidized to a certain extent, resulting in the evaporation of water from the surface of the muscle, so that the drip loss increases; when there are antioxidant substances in the muscle, such as astaxanthin, vitamin E and other antioxidant substances can reduce the degree of oxidation of the cell membrane, enhance the muscle hydraulic force [ 19 ,42]. When antioxidants such as astaxanthin and vitamin E are present in the muscle, it can reduce the oxidation of cell membrane and enhance the muscle system. In this experiment, the drip loss and freezing loss of Ast, AE and HE groups were significantly reduced compared with the control group (except for the 2-h drip loss of Ast group). It can be seen that the addition of Ast, AE and HE to the feed can prolong the shelf life of rainbow trout muscle. It was also found that the muscle drip loss at 6 h in the AE group was lower than that in the Ast and HE groups, which implies that the ability of AE to improve the shelf-life of muscle may be stronger than that of Ast and HE, and whether it is related to other antioxidant substances in AE needs to be further investigated.

 

4 CONCLUSIONS

Addition of AE and HE to the feed can effectively improve the muscle color of rainbow trout and enhance the antioxidant ability of the organism, which can achieve the same effect as that of adding Ast, but AF is not suitable to be used directly as a colorant for rainbow trout.

 

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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.

 

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