What Is the Extraction Method of Beta Carotene Powder?

Beta-carotene is a good food additive and nutritional supplement with strong free radical scavenging ability. It also has anti-cancer and anti-aging effects [1-3]. Due to these properties, the research and utilization of β-carotene has increased significantly in recent years. The annual global demand for β-carotene is about 1,000 tons [4-5], and it has been widely used in various fields such as medicine and food. This article reviews the research on β-carotene extraction methods, physiological functions and applications, with the aim of providing a reference for the research and development of β-carotene.

1 β-Carotene extraction methods

There are three main sources of β-carotene powder: chemical synthesis, plant extraction and microbial fermentation. The microbial fermentation method of producing carotene is not limited by environmental conditions and has the advantages of high safety and low cost. The main strains of bacteria used to ferment and produce β-carotene are Cladosporium cladosporioides, Dunaliella salina, Brevibacterium linens and red yeast. Due to the different sources of β-carotene, there are also various extraction methods.

1. 1 Extraction with organic solvents

Chen Yongbin [6] studied the extraction process of β-carotene from sweet potatoes “Hongxin 431” by designing an orthogonal test plan to investigate four factors: water bath temperature, water bath time, extraction solvent ratio and material to liquid ratio. The optimal combination is temperature 60 ° C, extraction time 45 min, liquid ratio 2:50, acetone-petroleum ether ratio 2:3. Azmi Wamik et al. [7] used ultrasonic technology to break red yeast cells, and then extracted with hexane and ethyl acetate (1:1, v/v), obtaining a maximum extraction concentration of 336 μg of β-carotene per gram of stem cell weight.

The advantages of the organic solvent extraction method are low capital investment costs, mature technology and ease of industrialization. However, the extraction rate is low and a variety of solvents are required for extraction. The use of large quantities of organic solvents will cause different degrees of pollution, and they are not easy to recycle. Beta-carotene is unstable to oxygen, heat and light, and this method may cause isomerization and oxidation of beta-carotene.

1. 2 Column chromatography

Liu Huilin et al. [8] used macroporous resin to extract and purify β-carotene produced by the viscous red yeast RM-1, and obtained the optimal adsorption and desorption conditions: X-5 adsorption resin, ether as the eluent, sample loading mass concentration 111.82 μg/mL, adsorption flow rate 1mL/min, and elution flow rate 0.5mL/min. Han Mei et al. [9] used two silica gel columns to extract and purify the main functional components of Saccharomyces cerevisiae oil. The yeast oil was dissolved in petroleum ether at 60–90 °C, and the silica gel column (ID 32 mm × 25 mm, silica gel particle size 200–300 mesh) was loaded. The elution conditions are 200 mL of petroleum ether with a gradient of 20% acetone added to 200 mL of petroleum ether, followed by a gradual gradient of 300 mL of 20% petroleum ether acetone solution, and then 300 mL of ethyl acetate added. The flow rate is 5 mL/min, and each tube is collected for 2 min. Nine components such as β-carotene, γ-carotene and red yeast were extracted and separated from the yeast oil.

Compared with other extraction methods, column chromatography has the advantages of low energy consumption, easy recovery and reuse of the eluent, and high purity of the product. However, it also has the disadvantages of a complex extraction process, long time consumption and a large amount of solvent required. It is difficult to industrialize in actual production.

1. 3 Supercritical CO2 extraction

Sorghum [10] used a response surface method to optimize the supercritical CO2 extraction conditions of β-carotene from watermelon bee pollen. It was found that the optimal extraction conditions were an extraction temperature of 51°C, an extraction pressure of 34 MPa, and an extraction time of 80 min. under these conditions, the extraction rate of β-carotene reached 8. 79 mg /100g. Montero et al. [11] evaluated the temperature, pressure, extraction time, cosolvent and flow rate of five factors required for the extraction of β-carotene from a marine cyanobacterium. At 65 ° C and 30 MPa, with the addition of 5% ethanol, the β-carotene extraction rate was the highest during 2h (flow rate = 4kg/h).

Sabio et al. [12] used tomato peel, a tomato processing by-product, as the raw material and obtained the following extraction conditions: average particle size of the raw material 0.345 mm, extraction temperature 80 °C, extraction pressure 30 MPa, CO2 flow rate 0.792 kg/h, and extraction time 8.2 h. The β-carotene recovery rate was 88%. Compared with the traditional solvent extraction method, supercritical CO2 extraction has no chemical solvent residue, is pollution-free, protects the physiological activity of the active substance, and has a simple process, and has been widely used. Because of the limited solubility of β-carotene in CO2, an entrainer is required to improve the extraction rate.

1. 4 Ultrasonic extraction

Ultrasonic extraction is an energy-saving and efficient extraction method that has emerged in recent years. Chen Li et al. [13] used an orthogonal test to optimize the process conditions for the extraction of β-carotene from Trichoderma reesei by ultrasonic method: 15 times the amount of ethyl acetate (v/w), 35°C, 100W ultrasonic extraction for 40 min, and two-stage extraction. Adding an antioxidant can increase the β-carotene extraction rate to 89.2%. Zhang Haixia [14] used dried carrot powder as the raw material, with operating conditions of an ultrasonic temperature of 55°C, an ultrasonic power of 50W, a frequency of 100KHz, and an ultrasonic time of 90min, and achieved an extraction rate of 85% for β-carotene. Soumen et al. [15] investigated the method of extracting β-carotene from spirulina, and the optimal conditions were at 30°C, 50 mL of n-heptane with the addition of 1.5 g of spirulina (pre-soaked in methanol for 2 min), an ultrasonic intensity of 167 W/cm2 and a duty cycle of 61.5%, an ultrasonic time of 8 min, and a probe tip length of 0.5 cm, with a maximum extraction rate of 47.10%.

Ashwini J. Purohit et al. [16] used two ultrasonic irradiation sources to extract β-carotene from carrot waste using ultrasonic-assisted extraction. The results showed that: ultrasonic irradiation for 50 min, 50 °C temperature, 100 W power, a solid-to-solvent ratio of 0.3:20 (g/mL), and the use of an ultrasonic amplitude rod, the β-carotene extraction rate reached 83.32%; while using an ultrasonic bath, the maximum extraction rate was 64.66%.

Ultrasonic extraction is easy to operate and has a high extraction rate. Ultrasonic extraction is adaptable and can be used to extract Chinese medicinal materials regardless of the nature of the ingredients or molecular weight, and is suitable for extracting most types of Chinese medicinal materials and ingredients. It also has a protective effect on the active ingredients in herbs that are unstable when exposed to heat, such as β-carotene, and are prone to hydrolysis or oxidation. However, ultrasonic extraction is limited by factors such as particle size and water content, and at this stage, ultrasonic extraction is still largely confined to laboratory research. Industrial applications still face problems such as the difficulty of matching ultrasonic power with industrial large-volume extraction tanks.

1.5 Microwave extraction

Chen Lei et al. [17] used microwave-assisted extraction to extract β-carotene from wolfberry, and investigated the effects of microwave extraction power, extraction time, solid-liquid ratio and extraction temperature on β-carotene extraction rate. Based on single factor experiments, the extraction process was optimized by orthogonal experiments. The results showed that the optimal process parameters were: wolfberry in acetone-petroleum ether (3:7) as an extractant, a solid-liquid ratio of 1:15, an extraction temperature of 25°C, an extraction time of 80s, and an extraction power of 400W, with a β-carotene extraction rate of 0.55%. Sun Xiejun et al. [18] established a microwave-assisted extraction method for rapid extraction of β-carotene from saltweed powder: ethyl acetate as solvent, microwave power 500W, liquid-solid ratio 232mL / g, extraction temperature 42℃, extraction time 7. 0min, stirring speed 180r / min, the yield of β-carotene from salt algae was 1. 03%.

Microwave extraction of natural products such as β-carotene reduces the extraction time, reduces solvent consumption, and improves the extraction rate and extraction efficiency. Microwave heating increases the temperature quickly, and the effect of temperature on β-carotene, such as isomerization, needs to be considered.

1. 6 Enzyme-assisted extraction method

Zhang Weiwei et al. [19] studied the key process parameters for enzymatic hydrolysis to extract water-soluble β-carotene from carrots. The results showed that the optimal extraction process conditions were: 50 g of fresh carrots, directly homogenized, then 100 mL of 0.2 g/100 mL Tween-80 solution was added, The extraction conditions obtained from the response surface analysis optimization are 0.49 g of pectinase, enzymatic hydrolysis pH 6.08, ultrasonic time 41.58 min, pH adjusted to 6.0, stirred at 40 °C for 2 h, then 2 g of diatomaceous earth was added and filtered. The β-carotene extraction rate was 12.23 μg/g.

Lv Shuang [20] used enzymatic hydrolysis to assist in the extraction of β-carotene from carrots, and the best process was found to be a mixture of ethyl acetate and absolute ethanol in a volume ratio of 2:1 as the extracting agent. The treatment was carried out using a complex enzyme mixture of cellulase and pectinase in the same buffer system. The amount of cellulase added was 0.7%, the enzymatic hydrolysis temperature was 55°C, time 180min, pH 4. 6; pectinase dosage 12%, enzymatic temperature 55℃, time 60min, pH 4. 6, β-carotene extraction rate 99. 8%. SIMS et al. [21] found that treating carrot puree with pectinase and cellulase can improve the juice yield, The carotene yield was more than twice that of the single solvent extraction method, the stability was improved, and the color of the carrot juice became darker. The enzymatic hydrolysis assisted method for extracting β-carotene has the characteristics of mild reaction, strong specificity and low reaction activation energy, which can greatly improve the extraction rate. This technology has high requirements for the experimental conditions of enzymatic pretreatment, and factors such as enzyme type, optimal enzymatic hydrolysis temperature, pH and time need to be screened to obtain the optimal process conditions.

2 Function of β-carotene

β-carotene is an important source of vitamin A. As early as the 20th century, Steenkbock [22] discovered that β-carotene may have vitamin A activity.

2.1 Antioxidant properties of β-carotene

Beta-carotene is a good free radical quencher. Due to its polyene structure, it has a strong free radical trapping ability, can remove oxygen free radicals, singlet oxygen and reduce the production of lipid oxidation products, and significantly improve the body's antioxidant status. Yuan Lei et al. [23] used the DPPH method, salicylic acid method and ortho-phenylene trioxide self-oxidation method to characterize the ability of β-carotene to scavenge DPPH · free radicals, hydroxyl radicals and superoxide anion radicals, respectively. The results showed that β-carotene has a strong ability to scavenge free radicals, and the scavenging ability follows a dose-effect relationship; at a concentration of 100 μg/mL, the scavenging rates of DPPH radicals, hydroxyl radicals and superoxide anions were 65.50%, 69.22% and 69.50%, respectively. Liu Xiaogeng et al. [24] found that β-carotene has a strong scavenging effect on DPPH radicals. When the β-carotene mass concentration is higher than 80μg/mL, its ability to scavenge DPPH radicals is comparable to that of vitamin E and higher than that of BHT; within a certain range, the higher the β-carotene mass concentration, the stronger its ability to scavenge DPPH radicals. When the β-carotene mass concentration increases to a certain critical value, the DPPH radical scavenging rate remains stable.

Beta-carotene can achieve a good antioxidant effect as an antioxidant within 30 minutes. Liu Mingmei et al. [25] added different levels of beta-carotene to a goat diet. When the added level of beta-carotene in the diet was 82.5 mg/d, the activities of SOD, GSH-Px, and CAT and the level of T-AOC in the goat serum were all extremely significantly higher than those in the control group. The MDA content of the serum from all treated goats was extremely significantly lower than that of the control group, which shows that adding an appropriate amount of β-carotene to the diet can enhance the antioxidant capacity of goats. Daniel et al. [26] evaluated the antioxidant effect of β-carotene in liposome and microsomal membranes: when preparing liposomes, the experimental group added 0.35 mol% β-carotene to the diacylphosphatidylcholine liposomes, which inhibited lipid peroxidation caused by AAPH; no antioxidant effect was observed in the control group of liposome suspensions without added β-carotene.

2.2 Regulation of the immune system by β-carotene

Under certain conditions, β-carotene can enhance the body's cellular immunity, humoral immunity and non-specific immune response, increase resistance to certain diseases and improve health. Qiao Dong et al. [27] used two methods to administer β-carotene to mice: gavage and intraperitoneal injection. The changes in the content of 24 cytokines in mouse serum were measured using a liquid chip detection technique. It was found that β-carotene can promote the production of inflammatory cytokines, and the intensity of the effect on cytokines varies with different pathways.

β-Carotene regulates the body's immune system, and the signal pathways involved in its action involve NF-κB, JAK-STAT and other pathways. Amar et al. [28] used naturally-derived β-carotene to orally treat rainbow trout and fed them for 9 weeks to evaluate its effect on the non-specific defense mechanisms of rainbow trout. The results showed that β-carotene can significantly increase the concentration of serum lysozyme and can regulate some of the innate defense mechanisms of rainbow trout. Cucco et al. [29] showed that adding 27 mg/kg β-carotene to the diet of adult partridges significantly improved the immunity of female birds. High levels of β-carotene were detected in the egg white of these eggs, and the hatchability of these eggs was higher, and the eggs had stronger antibacterial properties. However, there was no significant difference in male birds.

2. 3 Improve reproductive performance

By adding different levels of β-carotene to the diet, reproductive performance and offspring survival rate can be improved. The mechanism by which it improves animal reproductive performance may be that, as a physiological antioxidant, it can protect important cell organelles by protecting highly active follicles and uterine cells from free radical damage. This helps to optimise the function of steroids in ovarian cells and the secretion of important uterine proteins, thereby improving the uterine environment [30]. Ren Yanli et al. [31] added different levels of β-carotene bacterial powder to the basic diet of laying ducks and fed it for 15 days. The results showed that adding 1,000 mg/kg β-carotene bacterial powder can reduce the feed-to-egg ratio by 12.4%, increase the egg production rate by 1.94%, and increase the egg color by 5 degrees.

Ge Jinshan et al. [32] used a non-paired single-factor randomized block design to conduct an experiment on growing sows. The results showed that adding β-carotene to the diet can significantly reduce the number of weak piglets and stillbirths; and increase the average birth weight and individual weight at weaning. Liu Ruxiang et al. [33] used Holstein bulls as the experimental subjects, with a single-factor experimental design and random grouping. The bulls were fed a basal diet and a test diet supplemented with β-carotene (based on the air-dry matter of the diet). The test period was 6 months, the effects of different β-carotene addition levels on the semen quality and serum indicators of the breeding bulls were observed. It was found that the addition of β-carotene can significantly increase the serum β-carotene content of the breeding bulls, while the concentration of testosterone in the serum and seminal plasma first increases and then decreases with the increase of β-carotene addition level; the ejaculate volume and fresh sperm motility of the breeding bulls are significantly higher than those of the control group, and the semen density is also significantly higher than that of the control group.

2. 4 β-Carotene for the prevention and treatment of cancer

β-Carotene has a preventive and therapeutic effect on various cancers through different mechanisms. It can inhibit the proliferation of cancer cells, induce apoptosis in cancer cells and reduce the adverse reactions of chemotherapy. Cui Bokang et al. [34] found that β-carotene treatment can significantly inhibit tumor growth and enhance the levels of blood NK, IL-2, TNF-α, WBC, TP, ALB and A/G levels, and reduced blood ALT, AST and ALP activity. Pathological analysis of liver tissue showed that β-carotene treatment could reduce liver tissue damage in HCC rats. It can be concluded that β-carotene can improve the immune function of HCC rats and inhibit tumor growth.

The gopher experiment demonstrated the effect of β-carotene on oral cancer. using dimethylbenzanthracene to induce a model of oral mucosal cancer in gerbils. Natural carotenoids can significantly reduce the elevated expression of tumor necrosis factor and inhibit the expression of IL-10. Senesse et al. [35] believe that β-carotene has a certain local immunomodulatory effect on the premalignant lesions of the gerbil cheek mucosa, can interfere with the process of tumor induction by carcinogenic factors, and has a chemopreventive effect on oral cancer.

Matos et al. [36] conducted an experiment using an iron-induced oxidative damage model in rat prostates. After intraperitoneal injection of β-carotene (10 mg/kg) for 5 days, the concentration of Fe-NTA residues in the plasma was measured, which was significantly lower than that in the control group, indicating a reduction in lipid damage and proving that β-carotene has a preventive effect on prostate cancer. In addition, Kim et al. [37] found that the incidence of gastric cancer in people who consume more foods rich in β-carotene can be reduced by 33%. Tamimi et al. [38] conducted a plasma test and a nested case-control study on 969 women and found that women with high levels of carotenoids such as β-carotene in their plasma had a 25% to 35% lower risk of breast cancer than women with low levels of carotenoids.

2. 5 Other functions

In order to study the effect of β-carotene on the tight junctions of intestinal epithelial cells, Dong Hongwei et al. [39] pretreated porcine jejunal epithelial cells with β-carotene and stimulated them with lipopolysaccharide. Cell viability and transmembrane electrical resistance were measured. The results showed that compared with the control group and the lipopolysaccharide stimulation group, the β-carotene group had the highest IPEC-J2 cell viability and transmembrane resistance, indicating that β-carotene has a protective effect on the tight junctions of intestinal epithelial cells.

Rauscher et al. [40] added natural β-carotene extracted from vegetables and fruits to a histidine-deficient strain of Salmonella typhimurium and found that the bacterial mutagenicity of AFB1, BaP, CP and IQ was reduced by 72%, 67%, 53% and 27% respectively, demonstrating the antimutagenic effect of β-carotene in vitro. Bechor et al. [41] found that β-carotene can inhibit atherosclerosis by improving the outflow of cholesterol from macrophages. β-Carotene is a relatively safe and stable natural food coloring agent that has been widely used in the food and natural coloring industries. In addition, β-carotene also has the effect of protecting against light, preventing eye diseases, cataracts, and preventing multiple degenerative diseases caused by aging and senility [42-43]. Oral β-carotene can reduce the risk of Alzheimer's disease [44].

3 Application

Beta-carotene is a permitted coloring agent in GB 2760-2011 “National Food Safety Standard - Food Additive Use Standards”, and is recognized as an excellent nutrient pigment in Class A. It is also permitted for use in 52 countries and regions around the world. It is reported that the annual global demand for β-carotene is about 1,000 tons, and its annual sales in China are about 4 to 5 tons [4]. The color of β-carotene itself can cover all color systems from red to yellow, which is very suitable for the development of oily products and protein products. After being processed with microcapsules, β-carotene is soluble in water and can be used in almost all foods and feeds. Due to its coloring, antioxidant, and immunity-enhancing properties, β-carotene has been widely used in the production of natural colorants, food additives, nutritional foods, health products, pharmaceuticals, cosmetics, and bioengineering products.

4 Outlook

The combination of modern technologies such as ultrasonic extraction, microwave extraction, and enzymatic hydrolysis with traditional methods of organic solvent extraction can effectively increase the extraction rate of β-carotene and shorten the extraction time. In addition, methods suitable for large-scale industrial production need to be found to meet the demand for β-carotene. At the same time, the sources of β-carotene need to be expanded, such as the use of fruit peels and fruit and vegetable waste residues for extraction.

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