Study on Natural Blue Food Coloring in China

Pigments are substances that can color the object being dyed. They are also known as colorants and have a wide range of applications in production, life, and scientific research. The history of pigment development and application around the world is also very long. Since the British W. H. Perkins synthesized the organic pigment aniline violet for the first time in the world in 1856, synthetic pigments have dominated the pigment market due to their excellent performance, low price, and ease of use. However, since countries or institutions such as the United Kingdom (1967), the United States (1973), and the World Health Organization (1984) have successively questioned the safety of synthetic pigments (tar pigments) on the human body, and there have been more and more reports on the research of the hazards of pollutants such as arsenic and lead that may be brought in during the synthesis process, the variety of products using these pigments in various countries is gradually decreasing. The number of synthetic food colors permitted for use in China has also been reduced from more than 30 in the past to 10 at present, and the rapid development of natural pigments will be an inevitable trend [2].

Natural pigments generally refer to pigments made by using substances that exist in nature (such as plant and animal materials) or secondary metabolites produced by cultivation methods and undergoing certain processing. Due to their advantages of being safe and reliable, non-toxic and having no side effects, natural hues and versatility, they have been widely used with the development of the food industry, pharmaceutical industry, daily chemical industry and aquaculture industry. Currently, 43 types of natural pigments are permitted for use in food in China. Natural pigments have already dominated the food coloring market and are growing at a rate of 10% per year [3]. The raw materials for processing natural pigments come from a wide range of sources (from animals and plants, microorganisms, minerals, etc.), and there are many types (as of 2004, there were about 600 recorded types [4]).

However, the natural pigments made from these materials are mainly red and yellow tones, and blue pigments are very rare. They are often mentioned in the literature with words such as “precious”, “very few” and “rare” [5-7]. Among the 56 pigments listed in China's GB2760-2007 “Hygienic Standards for Use of Food Additives”, there are only two blue pigments: gardenia blue pigment and phycocyanin (i.e., algae blue egg white pigment). Blue is one of the three primary colors and can be used to mix a variety of shades. However, natural blue pigments are in short supply in both domestic and international markets due to their rarity. Therefore, active research and development of natural blue pigments is of great practical significance and has an attractive market prospect [8].

1 Natural sources of natural blue pigments

Natural blue pigments are rare among natural pigments, and their natural sources are also very limited. Organic natural blue pigments are mainly derived from plants and microbial materials. The leaves of plants in the genus Indigofera, such as indigo, tea blue, horse blue, Wu blue and woad, can be used to make indigo dye [4]. Gardenia blue is made from gardenia glycoside in the fruits of Gardenia jasminoides [8]. Plant sources such as Ophiopogon japonicus fruit [9–10], Peristrophe baphica [11], Brassica oleracea [12], and purple and blue-grained wheat seeds [13] are used to produce the corresponding blue pigments. Although it has been reported that genetically modified cotton and genetically modified roses can produce blue pigments, these genetically modified plant materials cannot be used to produce natural blue pigments at present due to their rare sources [14–15]. Algae such as spirulina, cyanobacteria and chlamydomonas can be used to produce algin blue pigments [16-18]. The oyster shell algae Haslea ostrearia can produce the blue malachite pigment, which increases the economic value of oysters [19]. Garlic (Allium sativum) can turn green after being pickled in vinegar, and blue pigment can be isolated [20-21]. Some microorganisms such as Streptomyces sp., Pseudo- monas sp., Pseudoalteromonas sp., Duganella sp., Aureobasidium sp., purple non-sulfur bacteria, etc., can produce different kinds of blue pigments during growth using a culture medium. Some genetically engineered strains can produce indigo blue pigments [22-23].

2 Structure and color of natural blue pigments

Although natural blue pigments are similar in hue, their color-producing mechanisms are different. Ultimately, the different color-producing mechanisms are determined by differences in the chemical structure or spatial structure of the pigment molecules. The molecular structures of natural blue pigments prepared from different raw materials or by different methods may be different, and the physical and chemical properties and application scope of the pigments may also differ accordingly. The structures and related properties of common natural blue pigments are shown in Table 1.

The pH range in which rubixanthin turns blue is very narrow, so it cannot technically be considered a natural blue pigment.

3 Microbial natural blue pigments

Although most natural blue pigments are still currently produced from animal and plant materials, the availability of these materials is limited by factors such as season, climate and place of origin, making natural blue pigments very limited in supply and therefore expensive and difficult to use. Microorganisms grow rapidly, and there are a wealth of species in nature that can produce pigments. Using microbial resources to produce natural pigments is basically not limited by resources, the environment, time or space, and therefore has advantages that are unmatched by using materials of plant or animal origin to produce natural pigments. Using microorganisms to produce natural pigments will eventually become the mainstream source of natural pigments [24].

The use of microbial fermentation methods to produce a variety of natural pigments such as blue pigments and red yeast pigments has also become a reality [25]. In fact, the production of the main natural blue pigments on the market currently requires the participation of microorganisms. For example, the preparation of indigo blue pigments and gardenia blue pigments requires the participation of microorganisms in the fermentation process, while the cyanobacteria, spirulina, and chlorella that produce phycocyanin pigments are themselves microorganisms. In addition, there are still many microorganisms in nature that can produce natural blue pigments, but most of the work on using microbial fermentation to produce natural blue pigments is still at the laboratory stage [7]. There is still a long way to go before the industrial production of natural blue pigments using these microorganisms directly fermented culture medium can be realized. The reported blue pigment-producing microorganisms and their pigment-related properties are shown in Table 2.

Research on the use of microorganisms to produce blue pigments mainly includes several aspects: 1. screening and identification of blue pigment-producing microorganisms; 2. determination of the physicochemical properties and some toxicological properties of the blue pigment, such as the effects of temperature, light, pH, metal ions, oxygen, complex compounds, and additives on the stability of the pigment; the spectroscopic properties of the blue pigment, its antioxidant and reducing capacity, antibacterial activity,  (cancer) cytotoxicity, and the ability to scavenge free radicals; 3. Screening and optimization of fermentation blue pigment culture media. The molecular structure of some blue pigments and the metabolic mechanisms of blue pigment-producing microorganisms at the physiological, biochemical, and molecular levels are still unclear. In order to better guide industrial production, a lot of in-depth research is needed to provide a theoretical basis for the industrial development of microbial fermentation blue pigments.

4 Extraction, separation and post-processing of natural blue pigments

Gardenia blue pigment and algae blue pigment are the two dominant blue pigments on the market for natural food colors. Cyanobacteria and red algae are the two main sources of algal blue pigments, with spirulina being the preferred and most cost-effective raw material for the production of algal blue pigments. The processing methods used to release algal blue pigments from algae are mostly the addition of corrosion inhibitors, followed by (ultra) low temperature freezing and thawing, and then homogenisation [17, 40], or the use of a combination of enzyme dissolution and high pressure homogenisation to break up the algae cells [39]. For larger algae such as Nostoc, homogenization is carried out using a high-speed tissue homogenizer, followed by enzymatic lysis (achieved through microbial fermentation and autolysis of the algae) [18]. The phycobiliproteins in the cell homogenate can be separated and purified using silica gel column chromatography [17] or by first extracting the yellow pigments using supercritical CO2 fluid and then using an aqueous extractant to separate and purify the phycobiliproteins [41].

The purified phycocyanin is vacuum-concentrated, and then freeze-dried or spray-dried to obtain a dry pigment powder. Microencapsulation before drying can improve the heat resistance of the pigment [18]. There are currently two processes for preparing gardenia blue pigment from gardenia fruit powder: one is a one-step process in which gardenia blue pigment is produced by fermenting gardenia fruit powder aqueous extract with an enzyme-producing strain; the other is a two-step process in which gardenoside is first separated and concentrated from gardenia fruit powder aqueous extract, and then gardenia blue pigment is produced by enzymatic reaction.

The first process produces gardenia blue with a dull color and low color value. It is difficult to separate and purify the product at a later stage, and the yield is low. The second process better solves these problems [42-43]. Gardenin is found in the waste liquid remaining after the gardenia yellow pigment is extracted from the gardenia fruit powder water infusion using a macroporous adsorption resin (such as HPD100) column. The waste liquid is then concentrated and enriched using techniques such as membrane filtration (microfiltration (0. 1 μm) clarification and nanofiltration (100 Dalton) concentration) [44], dual-phase extraction [45], and high-speed countercurrent chromatography (HSCCC) [46] to prepare high-quality gardenia blue pigment. The prepared pigment is purified and refined by techniques such as ultrafiltration [47], chitosan derivative column chromatography [44], or (D301) macroporous adsorption resin column chromatography [48].

Due to the common disadvantages of poor stability and easy degradation of natural pigments, research aimed at improving the stability of natural pigments is increasing. The water-soluble gardenia blue pigment was esterified with acetic anhydride to obtain a hydrophobic gardenia blue pigment, which expands its application range and also improves its stability to a certain extent [49-50]; the heat and light resistance of the purple cabbage blue pigment was significantly improved after being acylated with ferulic acid and salicylic acid [51], but in general, there are still few studies on the modification of natural blue pigments.

5  Prospects

Among the 43 natural pigments permitted for use in China's current GB2760-2007 edition of Hygienic Standards for Uses of Food Additives, there are only two kinds of blue pigments: gardenia blue pigment and spirulina blue pigment. China's production of gardenia and dried spirulina ranks first in the world [16, 42], but the quality of Chinese gardenia blue pigment is still 20 years behind that of Japan, at the level of the 1980s. Gardenia red has been on the market in Japan for 25 years, but China is still blank [8]. Although 98% of the gardenia blue pigment produced in China is currently exported, overall production of natural blue pigments in China is not high, and the supply of such pigments in China is still in short supply [46].

Faced with the huge natural pigment market that is growing at a rate of 10% annually, the development of natural pigments in China faces problems such as a lack of pigment varieties, a relative lack of sources, and high costs [52]. In view of this, the focus in the future should be on the following areas: 1. improving production processes and technical standards, increasing the production and quality of blue pigments, promoting the transformation of low-end products to high-end products, and increasing the added value of products. 2. Using existing raw materials to develop new natural pigments, such as gardenia red. Third, great importance is attached to the research and utilization of natural microbial resources for blue pigment, and new strains of blue pigment are developed to seize the initiative in international research on natural blue pigments. This is of great and far-reaching significance for changing the situation where natural blue pigments produced from materials of animal and plant origin cannot meet market demand.

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