What Is Astaxanthin Made From?

Abstract: This paper introduces that astaxanthin is not only a pink antioxidant pigment, but also has significant biological functions and can be widely used in the feed, food, pharmaceutical and chemical industries. The sources of astaxanthin, especially the breeding of astaxanthin-producing Schizochytrium, the process and the extraction of the pigment, and other research progress at home and abroad in recent years are discussed.

1 Introduction

Astaxanthin, 3,3'-dihydroxy-4,4'-dione-beta,beta'-carotene, is a keto-carotene with a pink color, fat-soluble, insoluble in water, and soluble in organic solvents such as chloroform, acetone, benzene and carbon disulfide. It is widely found in the living world, especially in aquatic animals such as shrimp, crab, fish, and in the feathers of birds, where it plays a role in coloration. It can regulate the deposition of pigments and is different from progesterone.

When added to feed, astaxanthin is deposited in the egg yolk after consumption by poultry, which deepens the color. Astaxanthin is a non-vitamin A carotenoid that cannot be converted into vitamin A in animals. However, astaxanthin is a chain-breaking antioxidant with extremely strong antioxidant properties. Animal experiments have shown that astaxanthin can remove NO2, sulfides, and disulfides, and can also reduce lipid peroxidation and effectively inhibit lipid peroxidation caused by free radicals. In addition, astaxanthin also has strong physiological effects such as inhibiting tumorigenesis and enhancing immune function. Therefore, it has broad application prospects in food additives, aquaculture, cosmetics, health products and the pharmaceutical industry. With the rapid development of high-end aquaculture, there has been a huge market demand for astaxanthin since the mid-1980s, and it has been increasing rapidly in recent years.

2 Sources of astaxanthin

2.1 Chemical synthesis

Astaxanthin is a carotenoid synthesis end point, and the transformation of β-carotene to astaxanthin requires the addition of two ketone groups and a hydroxyl group. Artificial chemical synthesis is relatively difficult, and most of it is cis-structured. The US FDA (Food and Drug Administration) only approves trans-astaxanthin as an additive for aquaculture. Therefore, artificially synthesized trans-astaxanthin is expensive (currently about 2,000 USD/kg on the international market) [1], which limits its widespread use.

At present, since the content of astaxanthin from biological sources is not high enough, chemically synthesized astaxanthin still has a certain competitive advantage. F. Hoffmann-La Roche of Switzerland has completed the synthesis of all-trans astaxanthin and has been approved for use as a feed additive for salmon [2].

However, some astaxanthin-containing microorganisms have the advantages of fast growth, short fermentation cycles, and the fact that the astaxanthin-extracted single-cell protein can be used as bait and feed additives. With the rise of all-natural foods worldwide, it will gradually become the focus of current research.

2.2 Biological sources

In contrast, astaxanthin extracted from living organisms is mostly of the trans configuration, safe to use and environmentally friendly, and has broad development prospects. The current biological sources of astaxanthin are mainly: extraction from the waste of the aquatic product processing industry and production by microbial fermentation.

2.2.1 Extraction of astaxanthin from the waste of the aquatic product processing industry

At present, the foreign crayfish processing industry produces 10 million tons of crustacean aquatic product waste each year. The extraction system using polymerization agents can be used to extract astaxanthin, astaxanthin esters and shrimp red pigment from this waste, with a yield of up to 153 μg/(g waste). According to analysis, astaxanthin accounts for more than 90% of the extracted carotenoids. Recently, the Norwegian marine fisheries industry has adopted the technology of ensiling waste. After ensiling, the recovery rate has increased by 10%, and the purity of astaxanthin has also been greatly improved.

Due to the low astaxanthin content in aquatic product waste, the extraction cost is high, and due to resource constraints, this method is not suitable as a large-scale source of astaxanthin and has little development potential. However, since no better methods have been found yet, this method still exists abroad.

2.2.2 Microbial fermentation production

The distribution of astaxanthin in the microbial world is somewhat similar to that of canthaxanthin. Studies have found that microorganisms that produce astaxanthin include a genus of fungi in the Basidiomycota phylum (the genus Phaffia), two species of bacteria that assimilate hydrocarbons, and many green algae that grow in nitrogen-deficient environments [3].

(1) Cultivating algae to produce astaxanthin

Among the many astaxanthin-producing algae, Haematococcus pluvialis is an important astaxanthin-producing bacterium and was once considered a microalga with great prospects for commercial astaxanthin production. This algae can both carry out autotrophy and heterotrophy. During cultivation, if there is a lack of nitrogen sources, astaxanthin will accumulate in the algae.

At present, the astaxanthin content in the body of foreign high-quality Haematococcus pluvialis is as high as 0.2% to 2%, generally accounting for more than 90% of the total carotenoids. In addition, Chlorococcum sp. has the advantages of high temperature resistance, extreme pH, fast growth rate and easy outdoor cultivation, and is considered to be an algae with great potential for large-scale astaxanthin production [3]. However, the autotrophic cycle of algae is long, the production site is limited to a certain extent due to the need for light, and it is difficult to break the cell wall of algae to release astaxanthin. Therefore, it is also more difficult to carry out large-scale production.

(2) Using bacteria to produce astaxanthin

Two strains of bacteria are known to produce astaxanthin: Mycobacterium lacticola, which produces astaxanthin only on hydrocarbon media and does not produce astaxanthin on nutrient agar; and another strain, Bevibacterium brevis 103, which grows in petroleum and has a biomass of 3 g/L at the end of fermentation, with only 0.03 mg/g of pigment. Considering the disadvantages of hydrocarbon fermentation and its low yield, as well as the availability of Pichia pastoris, the future biotechnological application of the above two bacteria seems unlikely.

(3) Using Pichia pastoris to produce astaxanthin

In 1976, Andrewes and Phaff discovered astaxanthin in Pichia pastoris, which attracted a great deal of attention. Since then, many biotechnology companies have made considerable efforts in the research of Phaffia yeast and have made some progress [4].

3 Research progress in the production of astaxanthin using Phaffia yeast

Phaffia yeast was isolated in 1970 from the exudates of deciduous trees in the mountains of Alaska and Hokkaido, Japan [4]. It was later identified as a new genus of the Basidiomycetes and named as the Phaffia genus [3]. Phaffia yeast appears to be quite special among the yeasts of the Basidiomycetes, mainly because it can ferment sugars and contains astaxanthin, which is different from the strict aerobiosis of other red yeasts, and the pigment is mainly β-carotene or monocyclic carotene. Astaxanthin was discovered in the yeast Haematococcus shortly after it was discovered, and research began on the feasibility of using it as a feed additive in fish and poultry feed and its effect on the pigment formation of organisms, with good results. In the subsequent 20 years of research, research efforts have focused on the following three areas: (1) strain improvement; (2) fermentation process optimization; and (3) astaxanthin extraction from cells.

3.1 Breeding of high-yield astaxanthin strains

Now people have focused on breeding mutant strains with excessive astaxanthin synthesis. In recent years, scholars at home and abroad have made some progress in this area. For example, the astaxanthin content of the obtained mutant strain of Rhodotorula glutinis was increased by 232%, reaching 1500 mg/(kg stem cells) [5]. A mutant strain of Haematococcus pluvialis NRRLY-17269, JB2, was screened using an alcohol waste liquid medium, and the yield of (2,010 + 170) mg of carotenoids per 1 kg of dry cells was obtained in a 5 L fermenter test [1]. In addition, research on the construction of high-yield astaxanthin gene engineering bacteria using DNA recombination technology has been carried out, and progress has been made in the transformation system of Pichia pastoris, the key enzymes in the biosynthesis of astaxanthin pathway and the genes encoding these enzymes.

3.2 Research progress in production process

3.2.1 Control of optimal fermentation conditions

The yield of astaxanthin is related to the culture conditions in addition to the strain. Using the yeast UCD67-210 as the experimental strain, several important parameters affecting fermentation were studied, such as pH, temperature, type and concentration of carbon source, dissolved oxygen and light. The optimal parameters for fermentation were obtained: pH 5. 0; temperature 20 ~ 22 ℃; optimal carbon source, cellobiose; sugar mass concentration exceeding 1.5% will reduce the astaxanthin content per unit weight of cells; however, due to the increase in biomass, the astaxanthin content per unit volume will still increase; dissolved oxygen 3.6 ~ 108 mmoL/(L · h); light has little effect on astaxanthin [3].

When studying the online control of pH during continuous culture of Pichia pastoris, it was found that the pH of the added glucose solution (5.02) was higher than that of the culture medium (5.00), and the growth of Pichia pastoris was relatively slow (0.055 h-1). However, when the pH of the added sugar was controlled at 4.98, the growth rate reached 0. 095 h-1. It was also found that the interval between sugar additions has a significant effect on yeast growth [7].

When the effect of glucose mass concentration on astaxanthin production was studied using the yeast NCHU-FS501, it was found that when the mass concentration of glucose reached 35 g/L, the astaxanthin production reached 16. 33 mg/L; when the mass concentration of glucose reaches or exceeds 45 g/L, astaxanthin formation is inhibited [2]. Recently, French scholars used glycerol as a carbon source to cultivate the yeast PR190, increasing astaxanthin production from 0.78 mg/(g stem cells) to 0.97 mg/(g stem cells). It was also found that the highest astaxanthin yield was achieved when the growth rate of the yeast was 0.075 h-1; after 168 h of fermentation, the astaxanthin yield could reach 33.7 mg/L (1800 μg/(g dry cell)) [8].

Mexican scholars used the juice of yucca as the sole carbon source, and when the mass concentration of reducing sugar was 22.5 g/L, the astaxanthin production reached 6.170 mg/L, which was 2.5 times higher than that using YM medium [9]. It is worth mentioning that when tomato juice is added, the precursor substances that may contain astaxanthin will increase the pigment content. Domestic scholars have optimized the shaking bottle conditions for astaxanthin production by Haematococcus pluvialis, and the highest astaxanthin yield obtained was 11.63 mg/L (1770 μg/(g dry cell)) [10]. Overall, there has been no breakthrough in simply optimizing the fermentation medium to increase the astaxanthin content.

3.2. 2 Reduce fermentation costs

In addition to the low yield of astaxanthin, another factor that adversely affects the commercial application of the yeast is the relatively high cost of the medium required for the growth of the yeast (yeast nitrogen base medium with added sugar). Some cheap food processing waste, such as alfalfa residue, can effectively promote yeast proliferation, but at the same time inhibit astaxanthin formation. This inhibition is due to the presence of saponins.

A mutant strain JB2 of the yeast Pichia pastoris NRRLY-17269 was screened using starch and alcohol waste liquid, and cultivated in distiller's grains to produce 1,330–1,750 mg/kg dry matter of carotenoids, which greatly reduced the cost of the culture medium [1]. It has also been reported that the use of molasses as a cheap fermentation raw material instead of glucose as a carbon source to cultivate Phaffia can increase the production of astaxanthin by about 3 times to 15. 3 mg/L [12]. In addition, xylose can be obtained in large quantities by hydrolyzing wood or industrial and agricultural solid waste, and is also an inexpensive carbon source. Some scholars used xylose as a carbon source, and after process optimization, the astaxanthin yield was 5.2 mg/L [13].

3.3 Extraction of astaxanthin

At present, astaxanthin is mainly extracted by first breaking the cell wall using various methods and then extracting with an organic solvent. Studies have shown that the extraction rate is lower when ethanol is used than when dimethyl sulfoxide (DMso) is used [5]. Domestic scholars have also achieved good results by treating the cells with acid heat and then extracting with acetone. Recently, Japanese scholars have selected a strain of Streptomyces rochei DB-34 that produces a highly active constitutive lyase. This enzyme exhibits activity in the hydrolysis of β-1,6-glucan, and it has also been found that adding this enzyme at the later stages of the culture of Pichia pastoris can effectively extract astaxanthin [14].

When used as a feed additive, the yeast must be broken down so that astaxanthin can be deposited in the fish or egg yolk. To make the pigment more readily available, pre-autolysis in distilled water or citric acid buffer is a promising method, or the tough cell walls can be broken down using an enzyme secreted by Bacillus circulans. Before adding Bacillus circulans, the yeast needs to be heat-killed and the pH adjusted. It is therefore more convenient to cultivate the two microorganisms together. Another advantage of this is that the cell-free culture broth can be reused. Because it still supports the growth of the yeast after some nutrients have been removed for fermentation, and it contains certain lytic enzymes that modify the cell wall. A process scheme for filtering and recycling the mixed fermentation broth has been proposed, with the aim of meeting environmental requirements in large-scale production. Unfortunately, mixed fermentation to some extent inhibits astaxanthin production [3].

4 Development and application prospects

Astaxanthin is currently being widely developed and applied in the production of foods, medicines, cosmetics and animal feed. Although astaxanthin is a carotenoid, some of its biological effects are much stronger than those of other carotenoids. Astaxanthin is fat-soluble, has a bright color and strong antioxidant properties. In foods, it not only colors, but also effectively preserves, preventing discoloration, off-flavors and spoilage.

Astaxanthin-containing red oil can be used to marinate vegetables, seaweed and fruit, as well as to color drinks, noodles and condiments. Patents have also been reported. Astaxanthin has stronger photoprotective effects than β-carotene, and there are patents for cosmetics containing astaxanthin abroad. The pharmaceutical and food industries use the antioxidant, anti-inflammatory and immune-promoting effects of astaxanthin to prevent oxidative tissue damage and formulate health foods. At the same time, because astaxanthin has a bright color and can non-specifically bind to actin, adding it to aquaculture feed can improve the skin and muscle color of farmed fish and increase their disease resistance. In addition, astaxanthin plays an important role in the growth and reproduction of fish. It can be used as a hormone to promote the fertilization of fish eggs, reduce the mortality rate of developing embryos, promote individual growth, increase maturity and fertility. Astaxanthin can also be used as a nutrient to promote the growth of poultry and increase egg production.

There is no doubt that astaxanthin has powerful physiological functions and is widely used. In recent years, the demand for astaxanthin has been increasing both domestically and abroad. In addition to extracting astaxanthin from the waste of the aquatic product processing industry, astaxanthin is also produced by industrial fermentation using microorganisms such as yeast and algae. However, compared with other mature fermented products, the scale of industrial production of astaxanthin using microorganisms is still far behind. The main problems are still low yield and high fermentation cost. Therefore, the further development and application of astaxanthin will benefit from the screening of high-yielding strains, the improvement of fermentation processes, and the timely introduction of genetic modification techniques to increase yields and reduce costs.

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