How to Extract and Purify Natural Colorants?

1 Natural Colorant Overview

Pigments can be divided into two main categories: natural colorants and synthetic pigments. One type is artificially synthesized chemical pigments, most of which are azo-type compounds. Some of these can be metabolized in the human body to form β-naphthylamine and α-amino-1-naphthol, which have certain toxic side effects on the human body. The other type is pigments derived from natural plants, animals, microorganisms, etc., which are called natural colorants. Since natural colorants are non-toxic and harmless to the human body, high nutritional value, and some have certain biological activities. Therefore, the research and development of non-toxic and harmless Natural Colorant has also become the trend of pigment development.

1.1 A brief history of the development of natural food coloring

Natural food coloring has a long history as a food coloring agent. Ancient Chinese texts such as Shijing and Qimin Yaoju contain records of coloring wine and food with natural plant pigments. In ancient Egypt and India, for example, pigments from sorghum were used to dye items. In the 4th century BC, the people of ancient Britain began to use madder pigment to color wine[2].

In 1856, Professor W.H. Perkins of the United Kingdom invented the world's first synthetic organic pigment, “aniline violet,” and many other organic pigments were synthesized after that [1]. Because these pigments are bright in color, stable in nature, strong in coloring power, easily soluble, uniform in quality, suitable for color mixing, odorless and tasteless, and inexpensive, they quickly replaced most natural colorants.

In the 20th century, with the continuous development of toxicology and analytical chemistry, humans gradually understood the mechanism of transformation of synthetic pigments after entering the human body, and realized that most synthetic pigments entering the human body can cause more serious chronic toxicity and teratogenicity and carcinogenicity. In response, countries around the world have enacted relevant laws and regulations to strictly restrict the use of synthetic pigments. As a result, there are now only a limited number of synthetic food colors available. For example, when synthetic colors were most widely used in various countries around the world, there were more than 100 varieties. However, today there are only seven in the United States, eight in China, and some countries such as Norway have completely banned the use of any synthetic colors [2]. Compared to synthetic colors, although most natural colors are susceptible to various conditions in foods, their colors are less vivid, and they are also more expensive. However, it is safe, has a light color, and gives a subtle feeling of no added coloring, which meets the psychological needs of consumers. With the progress of science and technology in recent years, the stability of Natural Colorant has been improved, and the application of the product is more convenient and safer. Therefore, the food industry's choice of coloring is increasingly trending towards natural coloring.

Natural food coloring has become the mainstream of the coloring market. The current market size of natural food coloring is 250 million U.S. dollars, and if synthetic natural colorants are added, the market value reaches as much as 440 million U.S. dollars. Based on the high food safety and improved stability of natural colorants, it is predicted that the annual growth rate of natural colorants in the future will be 5% to 10% [3]. China's food natural colorant is about 25,000 V years, caramel coloring accounts for 80%, and the rest is plant extracts and microbial fermentation products. There are 65 kinds of food coloring approved for use in China, of which 48 are plant extracts [4]. 1.2 50,000 tons/year, with caramel coloring accounting for 80% and the rest being plant extracts and microbial fermentation products. There are 65 food colorings approved for use in China, of which 48 are plant extracts [4].

1.2 Classification of natural food coloring

Natural food coloring can be classified according to chemical structure, morphological origin and solubility. The most common method is to classify according to chemical structure. Natural colorants in plants can be divided into four categories according to their chemical structures:5 pyrrole derivatives; polyenes (carotenoids); phenolic pigments; and ketones and quinones.

1.3 Characteristics and properties of natural food coloring

Natural food coloring has the following characteristics [6]:

(1) Most natural colorants come from animal and plant tissues, so they are non-toxic and have high safety. Most natural plant pigments are anthocyanins, flavonoids, and carotenoids. Therefore, natural food coloring is not only non-toxic and harmless, but also contains many nutrients essential to the human body or is itself a vitamin or vitamin-like substance, such as riboflavin, lycopene, and β-carotene;

(2) Natural colorants have a more natural hue and can better imitate the colors of natural substances. The hue of the coloring is more natural.

(3) Due to the different chemical structures of natural colorants, the properties of various pigments are also different.

2 Extraction of natural colorants

2.1 Extraction method

This is currently the most commonly used method for extracting natural colorants. The principle is to separate the target components based on their different solubilities in different solvents. The extraction process involves drying and crushing the raw materials, followed by extraction with a solvent, separation, concentration, drying and refinement to obtain the finished product. Take carotenoid pigments as an example. Most carotenoids are highly lipophilic, easily soluble in solvents such as chloroform and acetone, and almost insoluble in water, ethanol and methanol. Hydrocarbon carotenoids are even more lipophilic and can be dissolved in petroleum ether or alkanes. If the structure of a carotenoid contains oxygen-containing groups, the lipophilicity decreases and the hydrophilicity increases with the increase in the number of oxygen-containing groups. The solubility in petroleum ether decreases, while the solubility in ethanol and methanol increases. Different extraction solvents should be selected according to the different properties of the pigment during extraction.

2.2 Microwave extraction method

The process flow of microwave-assisted extraction technology for the extraction of natural colorants is: pretreatment of raw materials → mixing of solvent and raw materials → microwave extraction → cooling → filtration → filtrate → separation of solvent and extracted components → extracted components [”. Yao Zhongming et al. 8 determined the conditions for extracting gardenia yellow pigment by microwave extraction using single-factor experiments: extraction power 210W, extraction agent 500g/L aqueous ethanol solution, extraction time 80s, extraction series 2, and liquid-to-material ratio 1:12. Under these conditions, the pigment extraction rate reached 98.2%, with a color value of 56.94. Experiments have shown that microwave extraction of gardenia yellow pigment has the advantages of high pigment yield, high color value, solvent and time saving, and simple equipment compared to traditional extraction methods. It has broad application prospects in the extraction process of natural colorants. Compared with traditional heating extraction technology, microwave extraction technology has the advantages of short extraction time, low temperature, low energy consumption and high quality. The actual results of using this technology to extract natural food colors show that microwave extraction technology is a new process with good development prospects. Although microwave extraction of natural food colors has achieved some important results in experimental work, its application scope is limited due to its characteristics. It is currently only at the level of small-scale experimentation, and there is still much work to be done before it can be applied industrially.

2.3 Supercritical fluid extraction

Supercritical fluid (SF) refers to a fluid whose thermodynamic state is above the critical point. SF is a fluid in a special state between the gaseous and liquid states when the gas-liquid interface is in a critical state. It has very unique physicochemical properties. Supercritical fluids have a viscosity close to that of a gas, a density close to that of a liquid, and a diffusion coefficient between that of a gas and a liquid. They have the advantages of both gases and liquids, being as easily diffused as gases and having strong solubility like liquids.

Supercritical fluid extraction (SCFE) is a new type of extraction and separation technology. In the past twenty years, SCFE technology has been widely used in the chemical, pharmaceutical, and food industries. In recent years, attempts have been made to apply SCFE technology to the extraction of natural colorants. Shao Wei et al.] conducted a preliminary study on the extraction of red yeast rice pigment by supercritical CO₂, and obtained the optimal operating conditions for the extraction of red yeast rice pigment by supercritical CO₂: extraction temperature 50%, extraction temperature 50%, extraction pressure 20MPa, CO₂ flow rate 10kg/h, extraction time 4h. Li Shoujun [12] used supercritical CO₂ extraction to extract natural red pigments from wolfberries. The experiment confirmed that the sample crushing degree, raw material moisture content, extraction time, temperature, pressure and flow rate are important factors affecting the extraction rate, and the optimal process conditions were determined: The sample is crushed to 40 mesh, the moisture content of the raw material is about 5%, the extraction time is 100 min, the extraction temperature is 35 °C, the extraction pressure is 35 MPa, and the flow rate of the supercritical fluid CO₂ is 25 kg/h. The extraction rate under the optimal conditions is about 88%. However, the main problem affecting its application at present is the high investment cost of supercritical extraction equipment.

2.4 Ultrasonic extraction method

Li Yunyang et al. [13] found in a study on the use of ultrasound to assist in the extraction of pigments from chestnut shells that ultrasonic-enhanced extraction has the advantages of shorter extraction times and higher pigment extraction rates than conventional extraction, which can greatly improve production efficiency. Wang Zhenyu et al. [14] also studied the process of ultrasonic extraction of pigments from the large-flowered kai. The optimal extraction process parameters were determined to be: an ultrasonic frequency of 30 kHs, an extraction agent of dilute H₂SO₄ at 2% by mass, a treatment time of 40 min, and an extraction rate of 50 °C. In the extraction of lycopene, a power of 320 W was used, the extraction time was 6 min, the ultrasonic time was 3 s, the interval time was 4 s, the solid-liquid ratio was 1:2, and the extraction was repeated twice. The lycopene extraction rate was 96.83%. Compared with supercritical CO₂ extraction, it is low in cost, low in investment, and high in extraction efficiency [15]. Wang Qiufen et al. [16 studied the use of ultrasound to enhance the extraction of limonene with organic solvents. The experimental results showed that ultrasonic enhanced extraction not only shortened the extraction time compared with stirring extraction, but also increased the extraction rate. The extraction test of curcumin showed that ultrasonic enhanced extraction had the fastest extraction rate, and its extraction rate was slightly higher than that of the Soxhlet method [17].

3 Pigment purification

The liquid product obtained by concentrating the extract obtained using a conventional extraction process, or the solid product obtained by drying the liquid, is a crude natural colorant. Without purification, the product has a low color value, high impurity content, and some have a special odor from the raw material itself, while others have strong water absorption and cannot be used. These directly affect the stability and coloring properties of natural pigments, limiting their application. Therefore, advanced separation and purification technology should be introduced into the production process of natural colorants for refinement, which can improve the performance of edible natural colorants and expand their scope of application.

3.1 Ultrafiltration refinement method

The purification process for natural colorants can use an ultrafiltration membrane with an appropriate pore size, so that water molecules and even small molecular impurities can pass through the ultrafiltration membrane, while the active ingredients in the solvent are separated, thus purifying the pigment to a certain degree and concentrating it many times. Natural colorants have already been purified using ultrafiltration membranes abroad, and research in this area is also being carried out in China. He Chongyan et al. [18 used a two-step ultrafiltration and nanofiltration method to effectively separate, purify and concentrate beet red pigment, and the product quality met national standards (GB8271–87) and international standards (FAO/WHO).

3.2 Adsorption resin purification method

Adsorption resin is a type of polymer that is characterized by adsorption and has the effect of concentrating and separating organic matter. It is widely used in the separation, preparation and purification of organic matter. The adsorption characteristics of adsorption resin for substances mainly depend on the chemical properties, specific surface area and pore size of the surface of the adsorbent material. According to the surface properties of the resin, adsorption resins are generally divided into three categories: non-polar, medium-polar and polar. Non-polar adsorption resins are suitable for adsorbing non-polar solutes from polar solvents, neutral adsorption resins can be used to adsorb non-polar solutes from polar solvents and also to adsorb solutes with a certain polarity from non-polar solvents. Polar adsorption resins are suitable for adsorbing polar solutes from non-polar solvents. The operation of an adsorption resin generally includes processes such as adsorption, elution or regeneration, and rinsing. The appropriate adsorbent resin material and specific adsorbent and eluent are selected according to the nature of the different pigments. Adsorption and desorption can be used to achieve the purpose of refining pigments [19].

The macroporous resin adsorption method is becoming more and more widely used in pigment refinement due to its low organic solvent consumption, low energy consumption, large adsorption capacity, fast adsorption speed, easy desorption, and reusability. For example, Tianjin Institute of Light Industry used adsorption resin to refine Natural Colorant radish red, and the color value increased by more than 15 times [2]; Bi Hongxia et al. [21] used AB-8 adsorption resin to refine European plum red pigment, and the recovery rate of the pigment crystals was 61.8%, and the color value reached 15.48; Peng Yongfang et al. [22] used macroporous adsorption resin to separate the yellow pigment of Melia azedarach, and the product quality was greatly improved; Ma Yinhai et al. 23 used adsorption resin to adsorb and separate red pigment from kale. 48; Peng Yongfang et al. [22 used macroporous adsorption resin to separate the yellow pigment of the honeysuckle flower, and the quality of the product was greatly improved; Ma Yinhai et al. 23 used adsorption resin to adsorb and separate the red pigment of the kale and also obtained good results.

3.3 Gel chromatography

The principle of gel chromatography for separating pigment solutions is that when a pigment solution passes through a gel column, pigment molecules that are smaller than the gel pores can freely enter the interior of the gel, while molecules that are larger than the gel pores cannot enter the interior of the gel and can only pass through the gaps between the gel particles, so the migration rates are different. Substances with large molecules are not excluded and move ahead with the mobile phase, while substances with small molecules are retained due to diffusion in the pores and move behind the mobile phase, thus separating the two [24]. Gel chromatography has the advantages of simple equipment, convenient operation, no regeneration required for each chromatography, and effective protection of the activity of the separated substances. Gel chromatography has now become an indispensable technique not only for separating and purifying biological macromolecules such as proteins, but also for purifying pigments. Lu Xiaoling et al. [25 used a dextran gel as a support in the study of refining gardenia yellow pigment and achieved a good refining effect with a yield of 48.9%.

4 Conclusion

Natural food coloring ingredients are widely available and come in a wide range of colors. The urgent task facing researchers is to select those that are abundant in resources, low in cost, stable, vibrant in hue, non-toxic and harmless, and in demand in the market. Extensive research is needed to extract new pigments from new resources, develop efficient and economical Natural Colorant extraction and separation techniques, and improve the stability of existing Natural Colorants to light, heat, pH, and metal ions.

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