How to Extract Astaxanthin from Red Fife Yeast?

 Astaxanthin, also known as astaxanthin, is a ketocarotenoid oxidized from β-carotene, with the molecular formula C40H52O4, and the chemical name 3,3′-dihydroxy-4,4′-diketo-β′-carotene. β′-carotene is fat-soluble [1], and can be soluble in most organic reagents, such as acetone, chloroform, ethanol, and most ethers [2], but not in water. It is soluble in most organic reagents such as acetone, chloroform, ethanol, and most ethers [2], but not in water and has a dark red color. Astaxanthin has strong antioxidant, anti-inflammatory, anticancer, anti-aging, immune-enhancing, and coloring properties, and has a wide range of applications in food, pharmaceuticals, feeds, cosmetics, and other fields [3].

Astaxanthin is mainly produced chemically and biologically. There are two main conformations of chemically produced astaxanthin: 3S,3′R and 3R,3′S, and two conformations of biologically produced astaxanthin: 3R,3′R extracted from the yeast Saccharomyces cerevisiae (Fig. 1) and 3S,3′S extracted from the red alga Rhodococcus pyrenoidosus [5].

 

According to GVR, about 95% of astaxanthin on the market is chemically synthesized [6], but chemical synthesis of astaxanthin has a long lead time, complicated process, and produces a large number of by-products [7], and people prefer to use natural synthesized astaxanthin [8]. In recent years, the market value of natural astaxanthin has been increasing and the global astaxanthin market is estimated to be worth US$2.83 billion by 2024 [6].

 

Currently, biosynthesized astaxanthin is mainly derived from Rhodococcus pyrenoidus [9] and Saccharomyces cerevisiae [10]. Due to the harsh growing conditions, strict light requirements for the growing area and high water quality requirements, only Yunnan and Shandong are suitable for the cultivation of Rhodococcus aureus in China, which leads to the total production of astaxanthin from Rhodococcus aureus is not high, and the pollution is serious [11]. In recent years, red fife yeast has become a popular microorganism for astaxanthin production. Red Fife yeast is a naturally occurring astaxanthin-producing yeast, similar to Saccharomyces cerevisiae, which is capable of a variety of mutation breeding and genetic modification, and is capable of high-density fermentation to obtain a large amount of naturally occurring astaxanthin, making it a promising microorganism for astaxanthin production [12]. The process of astaxanthin production is shown in Fig. 2, but the extraction process of Red Fife yeast astaxanthin has been an important factor limiting its marketability. In view of this, this paper summarizes the progress of wall-breaking and astaxanthin extraction from Saccharomyces cerevisiae, with the aim of providing a reference for the subsequent research on astaxanthin extraction from Saccharomyces cerevisiae.

 

1 Wall-breaking of Red Fife yeasts

The cell wall of Saccharomyces cerevisiae is thicker than that of Saccharomyces cerevisiae, and astaxanthin is located in the cell membrane, so astaxanthin extraction must first break the cell wall. The common ways to break the cell wall are physical, chemical, biological, and a combination of these methods.

 

1.1 Physical breaking

Physical wall breaking is mainly through mechanical vibration or friction to break the red fife yeast cell wall, this method is simple and easy to implement, low cost, but the process of wall breaking may produce a lot of heat, resulting in slow degradation of astaxanthin, so it is necessary to cool down during the process of breaking the wall. gogate et al. [13] used 3 mol/L of lactic acid as the medium, ethanol as the extraction solvent, and the extraction rate reached 90% by breaking the wall for 15 min under the condition of 80W of ultrasonication and 60% duty cycle. GOGATE et al. [13] used 3 mol/L lactic acid as the medium and ethanol as the extraction solvent, and the extracted astaxanthin was broken under ultrasound power of 80 W and duty cycle of 60% for 15 min, and the extraction rate of astaxanthin reached 90%. Low frequency resonance (LFR) is a new green method for physical breakage, and some researchers found that a resonance frequency of 35 Hz and a resonance time of 75 min resulted in a breakage rate as high as 99.9% [15]. In addition, glass bead shearing is also a green and efficient method of wall-breaking. CHEN et al. [16] used glass beads with a particle size of 1 mm for the milling of red Fife yeast, and found that the extraction rate of astaxanthin was 65% when the liquid to material ratio was 20:1 (L:g), the dosage of glass beads was 200 g/L (glass beads:fermentation broth), and the milling time was 6 h. The results of this study were summarized as follows.

 

1.2 Chemical Breakdown

Chemical breakage refers to the chemical degradation of the active ingredients in the cell wall of S. rosenbergii, exposing the cell membrane of S. rosenbergii to facilitate astaxanthin extraction. The main methods of chemical breakage are dimethyl sulfoxide, acid and alkali. Dimethyl sulfoxide is toxic and is generally used as a laboratory extraction method, but not as a downstream extraction process. Acid wall-breaking is usually used to break up the red Fife yeast cells to extract the DNA group, but this method is usually carried out under high temperature, which is prone to degradation of astaxanthin, and the acid in this method will damage the structure of astaxanthin, and the subsequent deacidification process is quite complicated, so it is not suitable for downstream extraction of astaxanthin.

 

SILVA et al. [17] used dimethyl sulfoxide to break the wall of red fife yeast, the dry weight of red fife yeast to dimethyl sulfoxide ratio of 1:40 (g:mL), the heating temperature of 55 ℃, the vortex time of 1 min, the astaxanthin extraction rate was only 28.4%. Lu Baoju et al. [18] extracted astaxanthin using high-temperature wet-heat acid method at a saturated vapor pressure of 0.1 MPa (121 ℃), a hydrochloric acid concentration of 0.5 mol/L, a liquid-to-feed ratio of 30:1 (L:g), and a wall-breaking time of 2 min, with an extraction rate of 84.8% ± 3.2%. JIANG et al. [19] used hydrochloric acid to treat fermented yeast cells to obtain 253.1 mg/L of astaxanthin, with an extraction rate of 3.2%. JIANG et al. [19] used hydrochloric acid to treat the fermented yeast cells and obtained 253.1 mg/L astaxanthin. DA FONSECA et al. [20] used alkaline reagents such as Na2CO3 to break the walls of red fife yeast, but found that the effect was very little.

 

1.3 Biobreaking

Biological wall-breaking is mainly an enzymatic method, which is mild and does not cause harm to human beings and the environment, but there is still the problem of enzyme selection and recycling. Some researchers used lysin produced by Bacillus circulans A1.383-2 to break the wall of red fife yeast, and the extraction rate of astaxanthin could reach 98% in 16.5 h at a hydrolysis temperature of 37 ℃, pH 5, and the addition of 33 mL lysin (1,603.8 U/g of dry yeast)[21] . HARITH et al. [22] used two enzymes, Accellerase 1500 and Glucanex, to extract astaxanthin, and found that the extraction rate of glucanase was higher than that of Accellerase. Then they used a central combination design to optimize the wall-breaking conditions of dextranase, and finally found that dextranase at a temperature of 30.9 ℃, pH 4.5, the extraction rate of astaxanthin reached 94%.

 

In summary, each method for breaking red fife yeast has its own advantages and disadvantages, and there are differences in the places where it can be used. When breaking red fife yeast, it is important to consider the cost and efficiency, and to choose a method that is suitable for the downstream industry. Physical breakage meets the requirements of sustainability and low cost, and can better meet the requirements of industrial large-volume breakage, which is presumed to be the direction of industrial astaxanthin extraction in the future. The various wall-breaking methods and their breakage status are shown in Table 1.

 

2 Extraction of astaxanthin from red fife yeasts

There are two main categories of astaxanthin extraction methods: (1) solid-liquid extraction of astaxanthin using volatile organic reagents; (2) extraction assisted by new green reagents or new methods.

 

2.1 Extraction of conventional volatile organic reagents

A common method of astaxanthin extraction is the organic solvent method, i.e. the extraction of astaxanthin using conventional volatile organic reagents. Conventional volatile organic solvents should be polar reagents, such as methanol, ethanol, acetone, dimethyl sulfoxide, etc., and sometimes some non-polar reagents such as hexane can be used. ZHANG et al. [23] used acetone as the solvent and solid-liquid extraction to extract astaxanthin from red fife yeast, and the amount of astaxanthin extracted was up to 14.7 mg/L after shaking for 30 min at 5 ℃. BATGHARE et al. [24] predicted the amount of astaxanthin extracted from dimethyl sulfoxide:acetone (1:2) by using the UNIFAC method, and verified the extraction amount of astaxanthin under the condition of dimethyl sulfoxide:acetone (1:2) through the experiments. The amount of astaxanthin extracted from red Fife yeast was 1.57 g/L, which was in accordance with the prediction.

 

Some scholars [25] used acetone and methanol composite system for astaxanthin extraction, and the amount of astaxanthin extracted was 4.70 mg/g. ZHANG et al. [26] shook the acetone composite fermentation system (acetone ∶ fermentation broth =1:1) for 1 min, and the amount of astaxanthin extracted under this condition was 19.465 mg/L. However, these polar or nonpolar reagents used in the process of astaxanthin extraction may cause some damage to human health, and improper disposal may also pollute the environment. However, these polar or non-polar reagents used in the astaxanthin extraction process will cause some degree of damage to human health, and improper handling will also pollute the environment. Therefore, laboratory requirements and rules should be strictly adhered to during the process, and further removal should be performed after use to ensure that the astaxanthin product meets the requirements for food or drug use.

 

2.2 Novel green solvent extraction

In recent years, biosolvents and green solvents with low environmental and human impact have been used for astaxanthin extraction. Ionic liquids are emerging green solvents with low or non-toxic components, which are not harmful to the environment and the human body.MUSSAGY et al. [27] used various choline ionic liquids for the extraction of astaxanthin from red fife yeast and found that choline caprylate was the most powerful ionic liquid in extraction. Choline caprylate:water = 1:1 (v/v), extraction temperature of 45 ℃, and extraction time of 60 min resulted in 85% extraction of astaxanthin, and MUSSAGY et al. [28] screened a solvent mixture (ethanol:lauric acid) with high recovery of astaxanthin through the COSMO-AC model, and the antioxidant capacity of astaxanthin extracted from this solvent mixture was higher than that of conventional red fife yeast astaxanthin. The antioxidant capacity of astaxanthin extracted from this solvent mixture was higher than that of traditional red Fife yeast astaxanthin. The use of caprylic/capric triglyceride (GTCC) as an extractant has resulted in the extraction of 539.4 mg/L of astaxanthin, and a finished astaxanthin-caprylic/capric triglyceride product has been successfully prepared [29].

 

In addition to traditional solvents, supercritical fluids offer a new approach to the extraction of astaxanthin from red fife yeast. Supercritical fluids are neither liquids nor gases, but have good fluidity and drivability, and have properties not found in traditional solvents. Harith et al. [22] used a combination of supercritical CO2 and ethanol as a bioassistant solvent to extract astaxanthin, and the extraction rate of astaxanthin was 22% at 76.8 ℃, 360 bar, and 18% v/v of ethanol.

 

In recent years, more and more methods have been developed for the extraction of astaxanthin from red fife yeast. Solvent extraction (polar and non-polar solvents) is more suitable for rapid extraction in the laboratory due to its ease of use. The use of green solvents or their combination with other auxiliary methods is more in line with the concept of sustainable development, and meets the national requirements of harmlessness to human body and the environment, and is suitable for large-scale popularization. Currently, there is no common method for extracting astaxanthin from Red Fife yeast, and different methods are used depending on the needs. The relevant extraction methods and their extraction status are shown in Table 2.

 

3 Outlook

Compared with chemically synthesized astaxanthin, natural astaxanthin is more biocompatible and better able to exert its antioxidant, anti-inflammatory, anti-aging and anticancer effects. The production cost of natural astaxanthin is high, and the downstream cell crushing and astaxanthin extraction process is not yet mature, so the price of the product has remained high.

 

In recent years, the emergence of new wall-breaking methods has promoted the research progress of natural astaxanthin for food and medicinal use, which can achieve high wall-breaking rate without the use of volatile organic reagents, and ensure the green and non-toxicity of the cell wall breaking process. Compared with the other two methods, physical cell wall breaking is a simple, easy and low-cost process, which is the future direction of cell wall breaking. With the emergence of new green solvents, the extraction process of natural products has also made some progress, and promoted the development of the green extraction process of astaxanthin from red Fife yeast, and the use of new green solvents is in line with the requirements of green chemistry, and is also in line with the direction of future development.

 

At present, the downstream crushing and extraction process of natural astaxanthin from red fife yeast is still unstable, and it is necessary to continue efforts to break through the bottlenecks in the downstream process of red fife yeast cell wall breaking and astaxanthin extraction, and to promote the development of the whole astaxanthin industry from red fife yeast.

 

References:

[1] MUSSAGY C U,PEREIRA J F B,DUFOSSÉ L,et al.Advances and trends in biotechnological production of natural astaxanthin by Phaffia rhodozymayeast[J]. Crit Rev Food Sci Nutr,2023,63(13):1862-1876.

[2] MISAWA N,SATOMI Y,KONDO K,et al.Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level[J].J Bacteriol,1995,177(22):6575-6584.

[3] NISHIDA Y,NAWAZ A,HECHT K,et al.Astaxanthin as a novel mitochondrial regulator:a new aspect of carotenoids,beyond antioxidants[J].Nutrients, 2021,14(1):107

[4] Yao Kangfei , Zhang Ruilian , Liu Xiaojuan , et al. Study on anti-lipid peroxidation activity of astaxanthin with different stereoconformations [J]. Chinese Journal of Food ,2018,18(10):86-94.

[5] Meng Aung . Preparation, Stability and Functionality of Astaxanthin Crystals from Rhodococcus rainieri

Research [D]. Jinan : Jinan University ,2020.

[6] MUSSAGY C U,REMONATTO D,PAULA A V,et al.Selective recovery and purification of carotenoids and fatty acids from Rhodotorula glutinis using mixtures of biosolvents[J].Separation and Purification Technology,2021,266:118548.

[7] GONG Z K,WANG H L,TANG J L,et al.Coordinated expression of astaxanthin biosynthesis genes for improved astaxanthin production in Escherichia coli[ J].Journal of Agricultural and Food Chemistry,2020,68(50): 14917-14927.

[8] GVR.Astaxanthin market size,share & trends analysis report by product (oil, softgel, liquid),by source (natural, synthetic), by application (aquaculture & animal feed, nutraceuticals), by region, and segment forecasts,2024-2030[R].San Francisco:Grand View Research,2023.

[9] DEBNATH T,BANDYOPADHYAY T K,VANITHAK,et.al.Astaxanthin from microalgae:a review on structure, biosynthesis,production strategies and application[J].Food Res Int,2024(176):113841.

[10] NAGAL S,RAJA A,ADIN S N,et al. Screening and development of β-carotene enriched Phaffia rhodozyma cell by culture media engineering[J]. Microbiology,2023,92:36-46.

[11] MUHAMMAD G,ALAMA,XIONG WL,et al.Microalgae biomass production:an overview of dynamic operational methods[J].Microalgae Biotechnology for Food,Health and High Value Products,2020,169:415-432.

[12] LIBKIND D,MOLINÉ M,COLABELLA F.Isolation and selection of new astaxanthin-producing strains of Phaffia rhodozyma[J]. 1852:297-310.

[13] GOGATE P R,NADAR S G.Ultrasound-assisted intensification of extraction of astaxanthin from Phaffia rhodozyma[J].Indian Chemical Engineer, 2015,57(3/4):240-255.

[14] HASAN M,AZHAR M,NANGIA H,et al.Influence of high-pressure homogenization, ultrasonication, and supercritical fluid on free astaxanthin extraction from β-glucanase-treated Phaffia rhodozyma cells[J].Prep Biochem Biotechnol,2016,46(2):116-122.

[15] STEFANYSHYN O,HUNCHAK A,STARCHEVSKYY V,et al. Disruption of yeast cells xanthophyllomyces dendrorhous (Phaffia rhodozyma) by vibration resonant low-frequency cavitator[J].Chemistry & Chemical Technology,2023,17(1):188-194.

[16] CHEN L, WANG J L,NI H,et al. Disruption of Phaffia rhodozyma cells and preparation of microencapsulated astaxanthin with high water solubility[J]. Food Sci Biotechnol,2018,28(1):111-120.

[17] SILVA P G P,JÚNIOR D P,DE MEDEIROS BURKERT J F,et al. Carotenoid extraction from Phaffia rhodozyma biomass:downstream strategies and economic evaluation of energy[J].Brazilian Journal of Chemical Engineering,2023,40:93-102.

[18] LU Baoju , XIAO Anfeng , LI Lijun , et al. Extraction of intracellular astaxanthin from Fife yeast by high-temperature, wet-heat and acid breakage [J]. Journal of Bioengineering ,2008(7):1285-1292.

[19] JIANG G L,ZHOU L Y,WANG Y T,et al.Astaxanthin from Jerusalem artichoke:production by fed-batch fermentation using Phaffia rhodozyma and application in cosmetics[J].Process Biochemistry,2017, 63:16-25.

[20] DA FONSECA R A S,RAFAEL R,KALIL S J,et al. Different cell disruption methods for astaxanthin recovery by Phaffia rhodozyma[J].

African Journal of Biotechnology, 2011, 10(7):1165-1171.

[21] JIAN H L,SONG G J.Study on hydrolysis of yeast-wall with lytic enzyme for extraction of astaxanthin from Phaffia rhodozyma[J]. Applied Mechanics and Materials,2014,651/653:297-300.

[22] HARITH Z T, DE ANDRADE LIMA M, CHARALAMPOPOULOS D, et al.

al.Optimized production and extraction of astaxanthin from the Yeast Xanthophyllomyces dendrorhous[J].Microorganisms,2020,8(3):430.

[23] ZHANG J,LI Q R,LIU J H,et al.Astaxanthin overproduction and proteomic analysis of Phaffia rhodozyma under the oxidative stress induced by TiO2[J]. Bioresour Technol,2020,311:123525.

[24] BATGHARE A H,PATI S,ROY K,et al.Mechanistic investigations in ultrasound-assisted extraction of astaxanthin from Phaffia rhodozyma MTCC 7536[J ].Bioresource Technology Reports,2018,4:166-173.

[25] GUAN X Y,ZHANG J,XU N,et al.Optimization of culture medium and scale-up production of astaxanthin using corn steep liquor as substrate by response surface methodology[J].Prep Biochem Biotechnol,2023,53(4):443-453.

[26] ZHANG C,ZHAO X H,YAO M J,et al.High-density cultivation of Phaffia rhodozyma SFAS-TZ08 in sweet potato juice for astaxanthin production[J]. Electronic Journal of Biotechnology,2023,61:1-8.

[27] MUSSAGY C U,FARIAS F O,BILA N M,et al.Recovery of β-carotene and astaxanthin from Phaffia rhodozyma biomass using aqueous solutions of cholinium- based ionic liquids[J].Separation and Purification Technology,2022,290:120852.

[28] MUSSAGY C U,FARIAS F O,SANTOS-EBINUMA V C,et al. Sustainable one-pot platform for the green recovery of carotenoids from Phaffia rhodozyma yeast and their use as natural additives in soap formulation[J].Environmental Technology & Innovation,2023, 29:103029.

[29] CHENG X Y,XIONG Y J,YANG M M,et al.Preparation of astaxanthin mask from Phaffia rhodozyma and its evaluation[J].Separation and Purification Technology,2019,290:195-202.