What Are Astaxanthin Uses in Aquaculture?

Astaxanthin is a type of carotenoid that not only has a good coloring effect on aquatic animals, but also has the effect of preventing discoloration and deterioration and keeping the food fresh[1]. Astaxanthin has functions such as high-efficiency anti-oxidation, anti-cancer, immune enhancement, eye protection and central nervous system protection, and is currently widely used in the production of medicine, feed, food and cosmetics[2]. Astaxanthin sources include artificial synthesis and natural extraction. At present, the common natural astaxanthin products at home and abroad are mainly derived from aquatic product waste, microorganisms and genetically modified plants. In the aquaculture industry, astaxanthin is mainly used as a new and highly effective feed additive and is widely used.

1 Introduction to astaxanthin

Astaxanthin has the molecular formula C40H52O4. It is a fat-soluble and water-soluble pigment. Crystalline astaxanthin has a melting point of 224°C. It is a dark purple-brown powder with a pink color. It is insoluble in water, but soluble in organic solvents such as acetone, benzene and chloroform. Astaxanthin is widely found in living organisms, especially in fish, shrimp, crab, and the feathers of birds such as flamingos and ibises, as well as in the flesh of salmon and trout, and the shells of shrimp and crab. Animals cannot synthesize astaxanthin on their own, and although some crustaceans can convert other carotenoids into astaxanthin, they cannot meet their body's needs, so they must be ingested from food. Most marine fish and crustaceans contain astaxanthin, which is generally obtained from phytoplankton and zooplankton through the food chain [3-4]. Astaxanthin not only gives aquatic animals a good appearance, it is also an essential nutrient for animal growth and development.

Astaxanthin molecules have one hydroxyl group (-OH) at each end of the cyclic structure, which can form monoesters and diesters with fatty acids. The esterified groups in organisms act as a bridge for astaxanthin to bind to proteins. The free or esterified state affects the stability of astaxanthin in the body, the degree of binding to proteins and the rate of metabolism. For example, the surface of live healthy shrimp and crabs is green, and when cooked it turns orange-red, which is caused by the separation of esterified astaxanthin from the protein.

2 Forms and sources of astaxanthin

2.1 Forms of astaxanthin

Astaxanthin is distributed differently in different animals and tissues, and is more stable when stored in living organisms. Unoxidized astaxanthin is esterified astaxanthin. The skin, scales and roe of fish mainly contain esterified astaxanthin, while muscles, blood plasma and internal organs mainly contain free astaxanthin. In crustaceans such as crabs and shrimp, the esterified astaxanthin is mainly deposited on the shell, gonads and hepatopancreas.

2.2 Astaxanthin sources

Currently, the mature astaxanthin production processes include biological extraction and chemical synthesis. Common natural astaxanthin is mainly derived from aquatic product waste, microorganisms and genetically modified plants. Molecular structure of astaxanthin (Figure 1): four isoprene units are linked in the form of a conjugated unsaturated double bond. The long conjugated unsaturated double bond structure is very sensitive to light, heat, acids, alkalis, oxides and enzymes [5]. Therefore, how to optimize the extraction process to extract natural astaxanthin with maximum efficiency has become an international research hotspot.

2.2.1 Chemical synthesis method

There are two methods for synthesizing astaxanthin: direct synthesis and indirect synthesis. The direct synthesis method commonly uses synthetic carotenoid monomers for direct synthesis, while the indirect synthesis method obtains astaxanthin by oxidizing other carotenoids. The synthesis processes for both methods are very complex, and the astaxanthin obtained is 100% free, and mostly in the cis configuration (natural astaxanthin is mostly in the trans configuration).

2.2.2 Extraction from aquatic processing waste

In 2022, China's annual output of aquatic products will reach 68.69 million tons. Aquatic products waste is a rich resource, and the extraction of astaxanthin from aquatic products waste can bring huge economic benefits and promote the sustainable development of China's aquaculture industry. Traditional astaxanthin extraction methods include alkaline extraction, oil solubilization, Soxhlet extraction, and organic solvent extraction. In recent years, new methods such as enzymatic extraction, negative pressure cavitation, high-pressure homogenization, ionic liquids, pulsed electric fields, and supercritical fluid extraction have been used to extract astaxanthin with low consumption and high efficiency.

Zu Yuangang et al. [6] conducted a preliminary study on the various process conditions for the extraction of astaxanthin by the negative pressure cavitation method, and obtained the optimal extraction process parameters: an extraction solvent of 80% ethanol by mass, an extraction time of 35 min, and an aeration volume of 0.2 m3/h. Martínez et al. [7] compared the extraction rates of astaxanthin from Haematococcus pluvialis by pulsed electric field treatment, grinding, freeze-thawing, heat treatment, and ultrasonic treatment. The results showed that the extraction rate of astaxanthin after pulsed electric field treatment was 96%, and the highest extraction rate of other extraction methods was 80%.

Zhang Ye et al. [8] studied the effect of cellulase, pectinase and complex enzyme on the wall-breaking of Haematococcus pluvialis, and optimized the enzymatic wall-breaking assisted extraction of astaxanthin by response surface. It was found that when the ratio of cellulase and pectinase enzyme activity was 1:1 (U/ U), enzyme amount 7 000 U/mL, pH 4.9, temperature 49 ℃, time 6 h, astaxanthin extraction rate 71.08%, and the composite enzyme method is simple, mild, green, safe and efficient. In addition, purification methods for astaxanthin such as column chromatography, high-performance liquid chromatography, recrystallization, and high-speed countercurrent chromatography are also constantly being developed. The astaxanthin content in aquatic product waste is relatively low, and the extraction process is complex and expensive. Therefore, the cost-effective extraction of astaxanthin has become a pressing problem for the production industry.

2.2.3 Microbial production

Many types of naturally occurring microorganisms (algae, fungi, bacteria, etc.) can synthesize natural astaxanthin. At present, Xanthophyllomyces dendrorhous and Haematococcus Pluvialis are the most widely studied and used for production [9-10]. A good Xanthophyllomyces dendrorhous strain can accumulate astaxanthin accounting for about 0.5% of the dry weight, and the fermentation process is mature, so the product can be obtained in a short time [11]. However, it is greatly affected by fermentation conditions such as carbon source, nitrogen source, temperature, pH, and dissolved oxygen, and the fermentation cost is high. The astaxanthin produced is the dextrorse isomer with low antioxidant activity, so Xanthophyllomyces dendrorhous cannot be used as the best natural astaxanthin production tool.

The main production tool at present is Haematococcus pluvialis, which can accumulate astaxanthin accounting for 4% to 5% of the dry weight of the strain. However, the growth conditions of Haematococcus pluvialis are extremely harsh, with high requirements for water quality, light and culture environment are demanding, the culture cycle is long, the technical requirements are strict, and the accumulation of astaxanthin in its body occurs under stress conditions that are not suitable for the accumulation of cell biomass. Therefore, large-scale production is difficult [12-13].

2.2.4 Production of genetically modified plants

The precursors β-carotene and β-carotene hydroxylase required for astaxanthin synthesis are ubiquitous in higher plants, but they do not contain β-carotene ketolase and therefore cannot synthesize astaxanthin. Current research has successfully introduced β-carotene ketolase into plants to produce astaxanthin in tobacco [14-15], potatoes [16], Arabidopsis [17], lotus [18], corn [19], and other plants. However, the astaxanthin content produced in transgenic plants is unstable, and there are problems such as the accumulation of intermediate metabolites. Therefore, the discovery and utilization of the plant's own astaxanthin synthesis-related genes (for example, the petals of the marigold plant contain astaxanthin, with a content of about 1% of the dry weight of the petals [20]) will become an important research direction for the genetic engineering of astaxanthin production.

3 Application of astaxanthin in aquaculture

Astaxanthin has been used in the food, pharmaceutical and feed industries, but is currently mainly used in aquaculture as a new and highly effective feed additive.

3.1 Coloring effect

Astaxanthin can combine with different types of protein to produce red, orange, yellow, green, blue, purple and other colors.

3.1.1 Promoting the coloring of farmed fish

Adding astaxanthin to the feed can make the skin and muscles of farmed fish such as salmon and sturgeon appear bright red, and the meat tastes more delicious [21]. Nickell et al. [22] found that the coloring degree and efficiency of astaxanthin increased with the increase of the content of lipid substances in the feed by feeding rainbow trout with feed of different fat levels. Zhang Chunyan et al. [23] found that the redness and yellowness values of the muscle of Oncorhynchus mykiss were significantly higher in the group fed 1.0 g/kg synthetic astaxanthin and the group fed 0.1 g/kg astaxanthin-containing Haematococcus pluvialis extract than in the control group.

Nogueira et al. [24] found that dietary supplementation with astaxanthin (50 or 80 mg/kg for 6 months; or 50 mg/kg for 3 months, followed by 80 mg/kg for 3 months) had a positive effect on the skin tone and chroma of the dorsal fin and tail of red snapper, and the tone and chroma values were close to those of wild individuals. Li Yao-peng et al. [25] selected more than 170,000 triploid rainbow trout (Oncorhynchus mykiss) with an average weight of about 1 kg to conduct a pilot test on the effects of dietary astaxanthin levels on the growth performance , yield and muscle coloration.

It was found that adding 40 mg/L and 30 mg/L astaxanthin to the feed, respectively, and feeding it 7 months and 9 months before the rainbow trout is marketed, the meat color can meet the standards. Wang Hongyu et al. [26] fed hexagramma otakii (Hexagrammos otakii) with astaxanthin-added feed. The results after 60 days showed that when the additive amount was 0.10%-0.20%, the brightness, redness, and yellowness of the dorsal surface of the fish, the redness and yellowness of the abdomen, and the brightness and yellowness of the tail were significantly higher than those of the control group. When the additive amount was 0.05%-0.20%, the astaxanthin deposition on the dorsal, abdominal, and caudal skin of the fish was significantly higher than that of the control group. were significantly higher than those of the control group. When the additive content was 0.05% to 0.20%, the astaxanthin deposition on the skin of the back, abdomen and tail of the fish was significantly higher than that of the control group.

3.1.2 Promoting the coloring of ornamental fish

The body color of ornamental fish is caused by the accumulation of the pigments astaxanthin and canthaxanthin in the body, which produce the panchromatic color. Ornamental fish cannot synthesize these two pigments and must obtain them from the feed. Ornamental fish feed must meet the needs of both the growth and development of the fish and the need to maintain their bright body color. Astaxanthin, as the best coloring agent available, can help ornamental fish maintain their bright body color. Chen Xiaoming et al. [27] found after a 60-day experiment that adding 60 mg/kg astaxanthin to the feed could make the coloring of goldfish more natural and vibrant. Wang Rui et al. [28] found that adding 30 mg/kg astaxanthin to the feed could significantly improve the effect of pigment deposition in guppies, red swordtails and goldfish.

Sun Xueliang et al. [29] studied the combination of astaxanthin with different carriers (phospholipids, vitamin E) and found that the combination of astaxanthin with the two carriers vitamin E and phospholipids significantly reddens the body color of parrot fish. Wang Junhui et al. [30] studied the effect of astaxanthin on the body color of koi carp (Cyprinus carpio L.) and found that the redness and yellowness values in the body color reached a maximum when the astaxanthin addition was 400 mg/kg.

3.1.3 Coloring effect on shrimp and crab

The body color of shrimp and crab determines their market value. Astaxanthin combines with chitin in shrimp and crab to appear greenish-blue. After high-temperature heating, the protein is separated from the original astaxanthin, and the color changes to orange-red. Jin Zhengyu et al. [31] fed Macrobrachium rosenbergii with 60 mg/kg astaxanthin for 35 days. The results showed that the total carotenoid content in the shrimp was the highest (119.38 g/kg), which was 40% higher than that of the control group. Chien et al. [32] added 50 and 100 mg/kg astaxanthin in the feed of Japanese tiger prawns, and after 63 days, it was found that the deposition rate of astaxanthin in the shell and muscle of the prawns had significantly increased.

Long et al. [33] added Haematococcus pluvialis powder, which is rich in natural astaxanthin, to the feed of adult Chinese mitten crabs (Eriocheir sinensis), and found that the redness of the crab ovaries and carapaces increased significantly with the increase in the amount of Haematococcus pluvialis powder added. This was also confirmed by Su Fang's [34] experiments, which found that feeding Chinese mitten crabs with Haematococcus pluvialis feed can significantly improve the color and quality of crab products, with a significant increase in the astaxanthin content of the ovaries, hepatopancreas, carapace and epidermis of the crabs. There is a significant dose-effect relationship, and the higher the addition of Haematococcus pluvialis, the higher the accumulation of astaxanthin in the body. Ma Nan et al. [35] proposed that the addition of synthetic astaxanthin to fattening feed can significantly increase the total carotenoid content, color and antioxidant capacity in the head and thorax, liver and pancreas, and ovaries of the Chinese mitten crab, and suggested that the synthetic astaxanthin content added to the fattening feed of female crabs should be about 90 mg/kg.

3.2 Strong antioxidant effect

Shimidzu et al. [36] found in an in vitro study that astaxanthin has a higher ability to quench singlet oxygen and scavenge free radicals than lutein and zeaxanthin, and this was also confirmed by Lee et al. [37]. Wang Jiqiao et al. [38] fed juvenile Apostichopus japonicus with feed containing 30, 60, and 90 mg/kg of β-carotene and astaxanthin, respectively, under laboratory conditions of water temperature 11.0–20.0 °C, salinity 35, and pH 7.5. After feeding for 80 d, it was found that the average total antioxidant capacity of the body cavity fluid of the groups supplemented with astaxanthin (12.77 U/mL) was higher than that of the groups supplemented with β-carotene (8.7).  After 80 days, it was found that the mean total antioxidant capacity value (12.77 U/mL) of the body cavity fluid of each group of Apostichopus japonicus fed with astaxanthin was 45.61% higher than the mean value (8.77 U/mL) of each group fed with β-carotene, indicating that the antioxidant capacity of astaxanthin is higher than that of β-carotene.

Feng Minglei et al. [39] added 31.50 mg/kg red yeast rice astaxanthin (P-AST) and 32.96 mg/kg synthetic astaxanthin (S-AST) to the basal diet, respectively, and fed rainbow trout for 112 days. They found that both S-AST and P-AST could regulate the function of the antioxidant system and lipid metabolism-related genes in the red muscle of rainbow trout. . After feeding rainbow trout with 31.50 mg/kg P-AST and 32.96 mg/kg S-AST for 112 days, it was found that both S-AST and P-AST could regulate the function of the antioxidant system in rainbow trout red muscle and the expression of genes related to lipid metabolism.

Wang Zhaoxin et al. [40] designed three kinds of feed with different concentrations of astaxanthin by adding Astaxanthin Plus (containing 10% astaxanthin) to an isonitrogenous and isoleucine-rich feed, and fed it to Litopenaeus vannamei prawns. After 112 days, the study showed that adding an appropriate amount of astaxanthin to the feed can improve the antioxidant capacity and immune function of the prawns. In terms of food preservation, Han Qingyou [41] found that astaxanthin can not only be used in the preservation of fruits and foods, but also provide a scientific basis for extending the shelf life of fruits. Li Nian et al. [42] showed that a shrimp-astaxanthin-carboxymethylchitosan composite coating of 60 and 90 mg/L is a safe, effective, and feasible method for preserving Litopenaeus rossensis. It can inhibit the decline in sensory quality of Litopenaeus rossensis during refrigeration, delay lipid oxidation, and extend the shelf life of Litopenaeus rossensis by 3–4 days.

3.3 Anti-stress effect

Jyonouchi et al. [43] showed that astaxanthin can enhance the activity of Th1 (T helper cell 1) and Th2 (T helper cell 2) in humoral immune response, and also increase the production of immunoglobulins IgA, IgM, and IgG, so that animals have higher immune regulatory activity. Zhang et al. [44] found that The addition of 125–150 mg/kg astaxanthin to the diet of vanamei shrimp can enhance the antioxidant capacity of the shrimp body and its tolerance to hypoxic stress. Jiang et al. [45] found that the addition of Haematococcus pluvialis powder to the diet of juvenile Chinese mitten crabs can reduce the mortality of juvenile crabs during ammonia stress. Xie et al. [46] found that the addition of Haematococcus pluvialis reduced the inflammatory response in golden pomfret (Trachinotus ovatus) after 80 days of feeding and an acute hypoxic stress test (1.2 mg/L). Tizkar et al. [47] showed that after 70 days of feeding a diet containing astaxanthin (50–150 mg/kg), Japanese pond shrimp (Macrobrachium nipponense) could tolerate various physical and chemical stresses such as hypoxia, ammonia stress and cold stress.

3.4 Promotes growth, reproduction and development

Adding astaxanthin to the feed can significantly improve the growth and reproductive performance of rainbow trout, increase the survival rate of young shrimp, the buoyancy and survival rate of fish eggs, and increase the fertilization rate, survival rate and growth rate of salmon eggs. Jin Zhengyu et al. [31] showed that astaxanthin can significantly increase the weight gain rate of Litopenaeus vannamei. Li Chenlu [48] showed that astaxanthin has a significant alleviating effect on the oxidative stress response and oxidative damage caused by microcystin in zebrafish (Barchydanio rerio var.), and the higher the astaxanthin concentration, the better the effect on improving oxidative stress in the body. Wang Zhaoxin et al. [40] showed that adding the right amount of astaxanthin to the feed can increase the ovary yolk protein content, fertilized egg hatch rate, larval metamorphosis rate, and the number of amoeboid larvae and copepod larvae, thereby improving the reproductive performance of the parent shrimp.

4 Safety and application prospects of astaxanthin

Astaxanthin is found in large quantities in everyday foods. Shrimp and crab foods contain 80–100 mg/kg astaxanthin, wild sockeye salmon contain 30–58 mg/kg, fish average astaxanthin concentrations of about 40 mg/kg, and shellfish average astaxanthin concentrations of about 10 mg/kg. In recent years, the results of many toxicological studies on animals and humans have also shown that astaxanthin is safe and non-toxic [49-50].

Due to its important physiological functions and economic value, astaxanthin has great application potential in aquaculture, food additives, cosmetics and pharmaceutical products. With the development of various industries at home and abroad, the demand for astaxanthin will continue to increase. At present, the synthesis and extraction of astaxanthin internationally generally have the disadvantages of complex synthesis and extraction methods, low yields, and high costs, and cannot meet the needs of large-scale commercial production. The use of modern biotechnology to carry out research on the breeding of high-yield astaxanthin strains has broad development and application prospects and needs to be included in key research plans.

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