Study on Synthesis of Beta Carotene

Beta-carotene is a carotenoid, an orange-yellow fat-soluble compound. It is one of the most common and most stable natural pigments in nature. It is widely found in plants and is a polyene compound. It is an antioxidant with detoxification effects and an essential nutrient for maintaining the health of humans and animals. It strengthens the immune system, thereby enhancing the immunity of humans or animals and promoting animal growth. Beta-carotene is a precursor of vitamin A and can be converted into vitamin A in the body after entering the body of animals.

Beta-carotene is a fat-soluble pigment that can have a yellow to red color depending on its concentration. It can be used as a food additive or animal feed additive. Beta-carotene has been recommended by the Joint FAO/WHO Expert Committee on Food Additives as a food additive and nutritional supplement and is classified as a Class A nutritional food fortifier. Beta-carotene can be used as an animal feed additive to enhance the animal's immune system, improve the survival rate of animals raised, enhance animal reproductive capacity, improve production performance, increase the color of livestock, poultry and aquatic animal products, and improve animal growth rate and meat quality. It is reported that the domestic and international demand for β-carotene is increasing year by year, and it has good economic benefits in the international market. Therefore, this article reviews the extraction, synthesis and separation methods of β-carotene and its application in the feed industry.

1. The chemical structure and properties of β-carotene

β-carotene, with the molecular formula C40 H56 and a relative molecular mass of 536.88, has a melting point of 176–180 °C, a slight peculiar or foreign smell, and a purplish red or dark red crystalline powder. A dilute solution is orange-yellow to yellow. It is insoluble in water, propylene glycol and glycerin, slightly soluble in ethanol and ether, soluble in chloroform, hexane, carbon disulfide, acetone, benzene and oil. It is unstable in the presence of light and heat and is easily oxidized. 

2 Production of β-carotene

2. 1 Extraction process of natural β-carotene

2.1.1 Extraction with organic solvents

The extraction method with organic solvents is the most traditional method for extracting natural pigments. The principle is to select a suitable solvent based on the principle of like dissolves like, and then heat the mixture for a period of time to dissolve the β-carotene in the solvent. Commonly used solvents include petroleum ether, acetone, ethyl acetate and chloroform. Specific steps: sample pretreatment → weighing the sample → adding liquid at a certain ratio → heating and extracting at a suitable temperature and time → filtering → vacuum concentration of the filtrate under reduced pressure → obtaining crude β-carotene. This method is simple to operate and requires few instruments and equipment. It is widely used for extracting β-carotene from some plants. However, the extraction with organic solvents has the disadvantages of using a large amount of solvent, taking a long time, causing greater environmental pollution, having a low recovery rate of the organic solvent, and damaging the pigment to a certain extent.

Zhang Yan et al. used single factor and orthogonal experiments to investigate the effects of extraction solvent, extraction time, extraction temperature, liquid-to-material ratio and extraction times on the extraction of β-carotene from white lotus powder. The results showed that the optimal extraction conditions for β-carotene from white lotus powder were ethyl acetate as the organic extraction solvent, extraction time 180 min, temperature 45 °C, liquid-to-material ratio 1:6, extracted twice, and the extraction rate can reach 679. 864 μg / g.

Wu Yanmiao et al. used low-grade tea as raw material and determined the optimal process through single-factor testing. The optimal process is as follows: extraction temperature 50 ℃, liquid-to-material ratio 1∶3, extraction time 60 min, extracted twice, The extraction rate can reach 80%. The β-carotene in the extract was analyzed by high performance liquid chromatography (HPLC), and the content of β-carotene was found to be about 0.2%. Saponification and column chromatography were used to obtain a β-carotene product with a purity of 22%.

2. 1. 2 Supercritical fluid extraction

Supercritical CO2 is the most common type of supercritical fluid. This method makes use of the effects of temperature and pressure on the solubility of supercritical fluids. Supercritical fluids above the critical temperature and pressure have the fluidity of a gas and the solubility of a liquid. After contacting with the sample, the extractant can selectively dissolve components with different polarities, boiling points and relative molecular masses from the sample in sequence. Then, at another temperature and pressure, the supercritical fluid is turned into a normal gas, reducing the solubility of the target product and causing it to precipitate completely or almost completely, thereby achieving the goal of separation and purification. This method has the advantages of being cheap and easy to obtain, safe and reliable, simple in process, no solvent residue, non-toxic, harmless and non-polluting, and is widely used in the extraction of some natural products.

Yao Ping et al. used spinach as raw material and supercritical CO2 extraction technology to extract β-carotene. The main factors affecting the extraction rate were studied and analyzed by orthogonal experiments, and the optimal process conditions for the extraction of β-carotene from spinach were determined to be an entrainment agent dosage of 10%, an extraction temperature of 40°C, an extraction pressure of 20 MPa, and an extraction time of 120 min. Under these conditions, the extraction rate of β-carotene reached 5.04 mg/100 g.

Su Haijian et al. used the supercritical CO2 method to extract carotenoids from waste tobacco leaves. Using the carotenoid extraction rate as an indicator, and based on single factor experiments, the Box-Behnken response surface method was used to optimize extraction pressure, extraction temperature, extraction time and CO2 flow rate. The results show that the interaction of extraction temperature, extraction pressure and CO2 flow rate has a very significant effect on the extraction rate of carotenoids in tobacco leaves; the effect of extraction pressure is significant. The optimal process conditions obtained through optimization were an extraction pressure of 23.53 MPa, an extraction time of 1.72 h, an extraction temperature of 50.00 °C, and a CO2 flow rate of 8.05 L/h. Under these conditions, the extraction rate of carotenoids in tobacco leaves was 285.1 μg/100 g.

2. 1. 3 Ultrasonic-assisted extraction method

Ultrasonic extraction makes use of the strong oscillation, cavitation effect and thermal effect of ultrasound. Cavitation bubbles are generated by ultrasonic oscillation, and the cavitation bubbles continue to move in the oscillating environment. During the movement, they continue to grow and then burst. When they burst, they absorb energy in the sound field and release it in a very short time and a very small space, creating a high-temperature and high-pressure environment accompanied by a shock wave. This causes the cells to burst, releasing their contents and dissolving the target product in the solvent. Ultrasonic-assisted extraction technology greatly shortens the extraction time compared with traditional organic solvent extraction, has a higher extraction rate, is simple to operate, has low extraction process costs, is widely adaptable, and has relatively few impurities in the extract. In recent years, this method has been widely used in the extraction of natural products and the extraction of active ingredients from medicinal herbs.

Zhou Mingqian et al. studied the process of ultrasonic-enhanced extraction of β-carotene from Dunaliella salina. The yield of β-carotene was used as the evaluation index. Based on single-factor testing, orthogonal experiments were used to determine the optimal process conditions for extracting β-carotene from Dunaliella salina under the combined effects of extraction temperature, extraction time, ultrasonic enhancement time and liquid-to-material ratio. that is, a liquid-to-solid ratio of 1:6 (g:mL), an ultrasonic enhancement time of 70 s, an extraction temperature of 20 °C, and an extraction time of 9 min. Under these conditions, the yield of β-carotene can reach 4.418%.

Ma Shaojun et al. used sweet orange peel as a raw material to extract carotenoids using ultrasonic technology. A Box-Behnken orthogonal test design was used to analyze the effect on the composition of carotenoids by high performance liquid chromatography. The results showed that the drying method of the peel was freeze-drying, the particle size was 100 to 120 mesh, the liquid-to-solid ratio was 1:50 (g:mL), the ultrasonic power was 270 W, the ultrasonic time was 7 to 10 min, the ultrasonic temperature was 30 to 50 °C, and the extraction was performed 4 to 5 times. optimized, the carotenoid content ranged from 0. 130 to 0. 150 mg / g, and the verified value was 0. 152 mg / g. High performance liquid chromatography analysis showed that ultrasonic extraction had no significant effect on the main components of carotenoids under the test conditions.

2. 1.4 Microwave-assisted extraction method

Microwaves are high-frequency electromagnetic waves with a frequency of between 300 MHz and 300 GHz. They have powerful penetrating power and can penetrate the extraction medium directly to the microtubular bundles and glandular cell systems of the sample material, generating high temperatures inside the cells and causing the internal pressure to exceed the cells' ability to withstand it, thereby rupturing the cells and releasing the active ingredients inside. Moreover, the electromagnetic field generated by microwaves can accelerate the rate at which the extracted molecules diffuse from the interior of the sample to the interface between the sample cells and the solvent, thereby accelerating the release rate of the target product. Microwave-assisted extraction uses a small amount of sample, saves energy, causes less pollution, has a simple process, is highly efficient, shortens the extraction time, and is simple to follow up. As a new extraction technology, it has obvious advantages in the extraction of natural products and has been widely used in the extraction of various natural products in recent years.

Wang Ying et al. used a mixture of ethyl acetate and absolute ethanol to extract β-carotene from carrots by microwave. The effects of the liquid-to-material ratio, microwave time and microwave power on the extraction rate were investigated. The results showed that the optimal process conditions for β-carotene extraction were a liquid-to-material ratio of 1:5 (g:mL), a microwave time of 40 s and a microwave power of 400 W. Under the optimal conditions, the extraction rate can reach 47.8%.

Chen Lei et al. extracted β-carotene from wolfberry using microwave-assisted extraction, screened the solvent for extracting β-carotene, and investigated the effects of microwave power, extraction time, liquid-to-solid ratio and extraction temperature on the extraction rate of β-carotene. On the basis of single factor, the extraction process was optimized by orthogonal test. The results showed that the optimal process parameters are microwave power 400 W, time 80 s, temperature 25 ℃, liquid-to-solid ratio 1:15. Under these conditions, the extraction rate of β-carotene is 0.55%.

2. 1. 5 Enzyme reaction method

The enzyme reaction method uses the specificity of enzymes to produce β-carotene. Commonly used enzymes include cellulase and pectinase. Plant cells are composed of a large amount of cellulose and pectin. Cellulase can lyse the cell walls of plants, and when used in combination with pectinase, it can effectively reduce the resistance of mass transfer barriers such as cell walls and cytoplasm to the outward diffusion of effective ingredients in the cells, so that more effective ingredients are released. Compared with traditional extraction methods, the enzymatic method has a higher extraction rate and milder extraction conditions.

Yuan Xuhong et al. used sea buckthorn fruit as a raw material to study the optimal conditions for the extraction and purification of carotenoids by the composite enzyme method. The results showed that the optimal conditions for the extraction of sea buckthorn carotenoids by the composite enzyme method were cellulase: pectinase 2:1 (g:g), enzymatic temperature 30 °C, enzymatic time 25 min, enzyme addition amount 0.20%, enzymatic pH 7, and the extraction rate under these conditions was 89.88%. The carotenoid content was increased from 23.08% to 69.53% by purifying the crude carotenoid extract by saponification, and the saponified carotenoids were further purified by silica gel column chromatography, increasing the content from 69.52% to 84.36%.

Comparing the above extraction methods, the organic solvent extraction method is more suitable when the experimental conditions are relatively simple and a small amount of crude β-carotene is required. Supercritical fluid extraction is more suitable when purer β-carotene is required. Ultrasonic-assisted extraction and microwave-assisted extraction have higher extraction rates, shorter times and less pollution. These two methods are more suitable for large-scale industrial production because their advantages are in line with the current production concepts of most companies – high efficiency and environmental protection. Because the conditions for extracting β-carotene by enzymatic reaction are milder, β-carotene with higher activity can be obtained. When studying the properties of extracted β-carotene, the enzymatic reaction method is more suitable.

2. 2 Synthesis of β-carotene

2. 2. 1 Microbial fermentation

The use of microorganisms to produce β-carotene is mainly concentrated in the use of Trichoderma reesei and red yeast.

Microbial fermentation is a method that uses microbial culture technology to allow microorganisms to synthesize β-carotene in vivo, and then isolate β-carotene from the microorganisms. This method has the advantages of fast microbial growth, strong ability to produce β-carotene, relatively good quality of the obtained β-carotene, easy control under safe and non-toxic conditions, and quick and convenient measurement.

Wang Aijun et al. optimized the seed medium and fermentation medium in the method of producing natural β-carotene by fermentation of Trichoderma reesei. The results showed that: starch was selected as the medium, and ethyl acetate was used as the solvent for fermentation. The starch content was 2.3%, the pH was 6.6, and the fermentation unit was increased by 94.21%.

It has been reported that using red yeast treated with high hydrostatic pressure, the fermentation medium can be optimized by response surface analysis to produce β-carotene up to 13.43 mg/L.

2.2.2 Genetic engineering

With the development of genetic technology, the use of genetic methods to produce carotenoids has received a lot of attention from many scholars in recent years. The application of genetic engineering technology has greatly increased the amount of β-carotene synthesized in organisms, thereby increasing the amount of β-carotene extracted. Dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) are the two common precursors for carotenoid production. There are currently two known synthetic pathways: the 2-C-methyl-D-erythritol (MEP) pathway, which is mainly found in bacteria and plant organisms; and the methyl-D-erythritol-4-phosphate (MVA) pathway, which is mainly found in the cytoplasm or endoplasmic reticulum of archaea, fungi and plants.

Zhao Jing et al. used six artificially regulated elements with greatly different strengths to study the regulation of eight genes in the terpenoid synthesis pathway. The results showed that the optimal strength of the regulatory element varied for different genes. The regulation of the eight genes increased β-carotene production by 1.2 to 3.5 times. It was also found that regulatory elements of suitable strength could also increase β-carotene production after regulating the dxr, ispG and ispH genes can also increase β-carotene production. The combined regulation of dxr and idi genes can increase β-carotene production by 8 times, and ultimately β-carotene production can reach 17.59 mg/g dry mass of cells.

2. 2. 3 Chemical synthesis

Chemical synthesis is a method of artificially synthesizing β-carotene using organic chemical raw materials and chemical synthesis reactions. At present, the main routes are: using vitamin A as raw material, converting vitamin A into xanthaldehyde and methyl viashin reagent, and then condensing to form β-carotene; β-ionone as a raw material, the Roche company route characterized by the Grignard reaction; β-ionone as a raw material, synthesized by vinyl-β-ionol, C15+C2+C15 Wittig reaction.

Jin Xiao et al. used furan as a raw material, the key intermediate 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde for β-carotene synthesis was synthesized in four steps: hydrolysis, in situ hydrolysis-Wittig reaction, reduction, and oxidation. The intermediate was reacted with a quaternary phosphonium salt in the Wittig reaction to synthesize β-carotene, with a total yield of 43%.

Fan Guixiang used vitamin A aldehyde as a raw material. By combining a transition metal with vitamin A aldehyde, the metal was oxidized, the carbonyl group of vitamin A aldehyde was reduced and coupled to form a double bond, and a β-carotene product with a content of more than 98% was obtained, with a recovery rate of more than 80%.

3 Separation and purification of β-carotene

The main methods for the separation and purification of β-carotene include macroporous resin adsorption and separation, silica gel column chromatography, ion exchange resin separation, enzyme purification, membrane separation and purification, and recrystallization separation and purification.

Liu Huilin et al. used X-5 macroporous adsorption resin and ether as the eluent to separate and purify β-carotene produced by the adhesive red yeast RM-1, and obtained β-carotene with a purity of 33.29%, which is 6.87 times higher than that of unpurified. Jiaoyuzhi et al. used a magnesium oxide exchange column to isolate and purify β-carotene from selenium-rich wheat flour, and the yield of β-carotene could reach 93.37%. Xia Wei et al. used column chromatography and recrystallization to separate and purify β-carotene extracted from moldy tobacco or crushed tobacco extract, obtaining β-carotene with a purity of 98% and a recovery rate of up to 80%. Tang Dandan used β-ionone as a raw material to synthesize β-carotene via the Darzens + Wittig-Horner reaction route, and then separated and purified it by recrystallization. The purity of β-carotene was 96% as determined by HPLC, and the recovery rate was 81.58%.

In comparison, macroporous adsorption resins have a large specific surface area, good selectivity, fast adsorption, mild desorption conditions, convenient regeneration, a long service life and energy savings. Ion exchange resins are renewable and relatively low cost, but have poor selectivity. Silica gel chromatography and membrane separation methods have higher purity and shorter production cycles. The recrystallization separation method is simple to operate and energy-saving, but for some β-carotene extracted from plants, it does not achieve the desired results.

4. Application in the feed industry

Beta-carotene is a natural pigment that is non-toxic and harmless. It has good coloring properties and a stable and uniform color. It is a precursor of vitamin A, and its efficiency in converting to vitamin A varies greatly depending on the animal species. Beta-carotene can enhance the transmission of information between cells. Its molecule has 11 conjugated double bonds. This special structure allows it to scavenge toxic oxygen radicals and quench singlet oxygen in animals, acting as an antioxidant to cut off chain reactions. It also enhances the animal's own immunity against attacks by bacteria and viruses, thereby improving the body's immune capacity and increasing the survival rate of animal breeding; in animals, carotenoids can hinder lipid peroxidation and protect germ cells from the damage caused by oxidative reactions, thereby enhancing animal fertility and and improve production performance.

Because β-carotene is naturally yellow or orange, it is also an effective coloring agent. Adding coloring agents to feed can increase the color of livestock, poultry and aquatic animal products, such as the buttery color of milk, the color of the yolk and outer skin of poultry eggs, and the color of poultry feathers. It can also change the color of feed to stimulate the appetite of livestock and poultry. With the development of the feed industry, various feed additives are increasingly being used in compound feeds. β-carotene is one of them. As a feed additive, β-carotene can improve the growth rate of animals and the quality of meat, enhance the reproductive capacity of cows, horses and pigs, and also enhance the color and quality of fish and shrimp, and deepen the color of poultry eggs. Studies have shown that adding 50, 150 and 200 mg/kg of β-carotene to the feed of breeder chickens can increase their egg production rates by 2.15%, 2.73% and 5.97% respectively compared to the control group, and improve fertilisation and hatchability rates. The egg yolks are also darker in colour and of better quality.

It has been reported that cows fed a diet without β-carotene often show “fever without heat” and delayed ovulation, follicular cysts, reduced and delayed corpus luteum formation, which in severe cases can lead to reproductive disorders and placenta stasis. However, adding β-carotene to the diet can correct these symptoms. During the production process, it was found that the milk of the group with a high level of carotene supplementation was slightly yellow in color compared to the control group and the low-supplementation group. This is because carotene itself is also a pigment that is easily stored in fat. The group with a high level of carotene supplementation also had a higher mass concentration of β-carotene in the milk, which caused a change in the color of the milk fat and resulted in a yellowish milk color. Supplementing bulls with a certain amount of β-carotene can increase the number of sperm produced and improve sperm motility. Conversely, an insufficient supply will result in an abnormally high ratio of abnormal chromosomes compared to normal semen.

Beta-carotene can also promote the growth of Chinese mitten crabs. Yuan Chunyang et al. fed beta-carotene-enriched experimental feed to Chinese mitten crabs, which increased the quality of the Chinese mitten crabs, improved the survival rate of the cultured crabs, and increased the percentage of blood cell phagocytosis in Chinese mitten crabs, reduced the activity of serum superoxide dismutase, significantly increasing the ovary index and oocyte diameter of the Chinese mitten crab. Carotenoids play an important role in the maturation of fish gonads, embryonic development and larval development. Studies have shown that feeding carotenoid-enriched feed during the breeding period can significantly affect the quality of salmon and trout eggs and the health and survival of early larvae. When the carotenoid content of salmon and trout eggs is 1 to 3 mg/kg, the hatching rate of the eggs is about 60%; if the carotenoid content of the eggs is below this level, the hatching rate of the eggs is below 50%.

5 Prospects

Beta-carotene has a variety of physiological functions, in particular it can increase the body's immune capacity, enhance the anti-cancer ability of the human immune system, promote animal growth, and improve reproductive capacity. It has been widely used in the development of animal feed, human health products and pharmaceuticals. Due to its effect of beautifying and nourishing the skin, it has also been well used in cosmetics. International demand for beta-carotene is also growing year by year, and the market demand for beta-carotene will be even greater in the future. It has a very promising market with good economic benefits. Researchers are studying the mechanism of action of beta-carotene, finding more natural sources of beta-carotene extraction and synthetic pathways, improving the yield and purity of beta-carotene, and finding more extensive uses for it. We hope that scholars will conduct further research.

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