Study on Annatto Colorant

A food coloring is a food additive that colors or improves the hue and color of food. It is present in very small quantities in food, but has a significant effect on the quality and taste of the food. In recent years, natural pigments have been highly regarded because their hue is close to the natural hue of the food, they are safer than synthetic pigments, and they have certain physiological activities. The proportion of natural pigments used in food in some developed countries in the West has reached 85%, and there is a trend to completely replace synthetic pigments. At present, more than 40 kinds of natural food colors are allowed to be used in China. However, the main variety produced and used is caramel color. Other natural pigments are rarely used domestically due to high cost and instability, and are mainly used for export. Therefore, it is urgent to develop commercially valuable pigment resources and natural food colors with good stability. The stability, safety, physiological function and distribution, and preparation of pigments will be discussed from several aspects.

1 Purple sweet potato pigment

Purple sweet potato is a special variety of sweet potato. The sweet potato is an annual plant in the Convolvulaceae family and is one of China's main food crops. According to the Compendium of Materia Medica, sweet potatoes have the effect of “replenishing weakness, invigorating the qi, strengthening the spleen and stomach, and strengthening the kidney yin”. In addition to the various nutrients contained in the common sweet potato, the purple sweet potato root is also rich in natural red pigments such as anthocyanins (known as anthocyanins when combined with sugar) that have significant natural antioxidant and free radical scavenging physiological activities, thus exhibiting some unique physiological activities.

1.1 Physiological functions

1.1.1 Antioxidant

The main chemical components of purple sweet potato anthocyanins are two types of glycosides with acyl groups: cyanidin 3-O-glucoside and peonidin 3-O-glucoside. These have multiple phenolic hydroxyl groups in their structures, which act as hydroxyl donors and free radical scavengers. Therefore, purple sweet potato pigments can not only bind to proteins to prevent oxidation, but also provide protons to effectively scavenge lipid free radicals, interrupt the chain reaction of lipid oxidation, and prevent lipid peroxidation [1-3]. Choong C. Teow [4] and others used ORAC (oxygen radical absorbance capacity), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] and DPPH (1,1-diphenyl-2-picrylhydrazyl), etc., to determine the antioxidant properties of purple sweet potatoes. The research shows that the darker the purple colour of the sweet potatoes, the more pigments they contain, and the stronger their antioxidant properties.

1.1.2 Antitumor

Anthocyanins can promote collagen synthesis, increase collagen resistance, and inhibit the activity of collagenase. Therefore, it is speculated that purple sweet potato anthocyanins have anti-tumor properties [1]. According to Nie Linghong [5], American scientists have discovered that sweet potatoes contain a chemical called dehydroepiandrosterone, which can prevent colon cancer and breast cancer.

1.1.3 Anti-mutagenicity

A study of the mutagenicity of Salmonella using refined purple sweet potato anthocyanins found that the purified purple sweet potato anthocyanins had strong anti-mutagenic properties [6-7].

1.1.4 Anti-hyperglycemia

Japanese scholars have isolated a diacyl anthocyanin from purple sweet potato tubers that can lower blood sugar by inhibiting α-glucosidase. 10 mg/kg body weight of diacyl anthocyanin can lower blood sugar levels in the human body [1].

1.2 Stability [8]

1.2.1 Stability to light and metal ions

Purple sweet potato pigment is highly resistant to heat and light. It shows no significant changes under natural outdoor light, indoor reflected light, or direct ultraviolet light. After heating at 100 °C for 0.5 h, the absorbance decreased, but the decrease was very slow, and the absorbance only decreased by 5% for 5.5 h. It also has good stability to metal ions. In a pigment solution with added Fe2+, Fe3+, Cu2+, Zn2+, Mg2+, Al3+ and K+, the characteristic absorption peak of anthocyanin was not changed after 72 h of treatment, so it is relatively stable. Treatment with ascorbic acid, sodium sulfite and hydrogen peroxide showed that purple sweet potato red pigment has good resistance to reduction and oxidation.

1.2.2 Stability to pH

pH changes have a significant effect on this pigment. At pH 3.0 or below, it is a bright dark red. As the pH increases, the color of the solution gradually transitions from light red to purplish red, purple, violet blue, and blue. However, as long as the conditions remain acidic, a more ideal bright red color can be obtained.

1.2.3 Stability to temperature

The effect of temperature on the pigment is related to pH. At a pH of 3, the pigment is quite stable to heat. When the pH is 5 and after a long period of high temperature treatment, the color value of the pigment decreases slightly, indicating that it is more heat-resistant.

1.3 Distribution and preparation

Sweet potatoes are a type of coarse grain crop that is widely cultivated in tropical and subtropical regions around the world. Sweet potatoes are widely cultivated in China, and are grown in all parts of the country except the Qinghai-Tibet Plateau, especially in the Huanghuai Plain, Sichuan, and the hilly areas of the southeastern coastal provinces. The extraction of purple sweet potato pigments mainly involves the solvent method and the fermentation method. The solvent method mainly uses 0.5% citric acid, which is relatively safe. The fermentation method involves washing a certain amount of purple sweet potatoes, steaming them, cooling and crushing them, mixing them with a primary mash made with a certain amount of rice, fermenting them for a few days, making a secondary mash, filtering the secondary mash, concentrating under reduced pressure, and then you can get a concentrated pigment solution. The concentrated pigment solution is treated with cation exchange resin to remove impurities, and the pure pigment is obtained by acidified ethanol exchange and concentration, with a yield of up to 89.4%.

2 Annatto [9]

Annatto is a pigment extracted from the aril of the mature seeds of the annatto tree. It contains four types of carotenoids: bixin (bixin), norbixin (norbixin), β-carotene (β-carotene) and crocin (crocin). It can provide three shades of yellow, orange and red.

2.1 The physiological functions of bixin

Bixin has antioxidant and free radical scavenging effects, and has anti-cancer and anti-aging health benefits [10-12]. Zhao Wen'en et al. of Zhengzhou University [13] confirmed through pulsed radiation research that the efficacy of bixin in scavenging active free radicals is comparable to that of β-carotene, providing strong evidence for the functional pigments of the annatto genus. Sotirios Kiokias et al. [14] showed that, compared with several other carotenoids, the water-soluble annatto can more effectively delay the oxidative degradation of oil and oil-in-water emulsions, and is more effective than ascorbic acid, VC palmitate and vitamin E.

Cecilia Rodrigues Silva et al. [15] used Wistar mice in their study. After an intraperitoneal injection of 5.0 mg/kg body weight of (Z)-diaminedichloroplatinum 24 h, chromosomal deletions and abnormal metaphase cells were observed in the bone marrow cells of the mice. The most frequent event was the breakage of chromatids. In contrast, treatment with annatto at even the lowest dose (2.5 mg/kg body weight) reduced chromosomal aberrations by 21%. Mice that were injected with annatto alone showed no significant chromosomal aberrations or division abnormalities. TBARS (TBARS is a substance formed by the reaction of thiobarbituric acid (TBA) and malondialdehyde, a lipid peroxide product. The amount of TBARS can be used to measure the degree of lipid peroxidation) produced in kidney tissue can be used to measure lipid peroxides. (Z)-Dichlorodiammineplatinum can increase lipid peroxide production, while the amount of lipid peroxide in mice pretreated with annatto did not increase. This may be because annatto can react with oxygenated compounds, scavenge free radicals and react with oxygen molecules produced by (Z)-dichlorodiammineplatinum, thereby providing protection. Di Mascio et al. [16] found that annatto is a good biological singlet oxygen molecule quencher.

2.2 Stability

Bixin is an oxygenated derivative of conjugated polyenes, i.e. an ester of a carotenoid dicarboxylic acid. Carotenoid compounds with a carboxylic acid group are stable in air, unlike other carotenoids, which can absorb oxygen from the air and become resinified and heavier. Therefore, they are very stable to oxygen.

S. V. Najar et al. [17] investigated the effects of light, air, antioxidants and pro-oxidants on carmine. The results showed that the pigment is most sensitive to light, and the damage to the pigment by light increases with the light intensity. This is followed by the damaging effect of free radical promoters. Adding any free radical promoter (such as benzoyl peroxide) to the pigment will accelerate the loss of the pigment. In the absence of light, mahogany pigments are not easily oxidized by air; when antioxidants (such as ascorbyl palmitate) are added to mahogany pigments, the stability of the pigments can be improved. Prentice Hernandez et al. [18] showed that the use of microencapsulation technology to embed mahogany pigments can significantly improve their light stability. K. Balaswamy et al. [19] showed that during the storage of mahogany pigments, under the same temperature and light conditions, the pigment stored in an oily resin is more stable than in powder form, significantly reducing the decomposition and fading of the pigment.

2.3 Distribution and preparation

Large areas of the red-lip tree are cultivated in Yunnan and Fujian, where it exhibits fast growth (seed propagation, first flowers and fruit in three years, production in four years, and entering the peak production period in six years); strong stress resistance (rarely affected by pests and diseases, strong cold resistance); and a long economic lifespan of several decades, with low management costs after reaching adulthood. It can be cultivated in pure stands, mixed stands, or scattered around the front and back of houses. The seeds of the annatto tree are conical or subtriangular in shape, with a groove on one side and a bent fold in the cotyledon. The outer seed coat is fleshy and red, while the inner seed coat is lignified and the wood layer is hard and dense. Soaking the seeds in distilled water at room temperature for 15 h only caused an increase in weight of 2 %.

The inner seed coat can effectively prevent water from entering the seed. Therefore, when the pigment contained in the outer seed coat is extracted, due to the barrier effect of the inner seed coat, many ingredients in the seed are difficult to mix into the pigment extraction solution with the soaking and stirring, which makes the extraction solution contain less non-pigment impurities, greatly simplifies the purification and purification process of the pigment, reduces pollution, and protects the environment. After the pigment is extracted, the seeds are basically intact and can be further processed and utilized. At present, some scholars [20] have used biotechnology to obtain high levels of annatto orange pigment through genetic modification. The extraction method is also simple and easy to carry out. The optimal extraction conditions are: temperature 60 °C, solid-liquid ratio 1/10, alkali (NaOH or KOH) concentration 10%, extraction time 40 min, acid concentration 5%, then purified with acetone as the extracting agent, solid-liquid ratio 1/20, and after 4 extractions at 55 °C, the best redwood pigment is obtained [21-22]. Thus, from the functional stability of the annatto tree to the cultivation and extraction, it has unique advantages and great development potential.

3 Purple corn and purple corn cob pigment

Purple corn is native to central America. In 1976, seeds were introduced from the United States for improved breeding. In 1979, it was officially promoted for planting. It is one of the crops that has been promoted extremely rapidly in recent years. The harvest season is from October to May of the following year. The inhabitants of the Inca Empire used it as food. It is the same size as ordinary corn, but the seeds, husks and kernels are all a dark purple. Purple corn is grown in large quantities in China, mainly in Shandong and Jilin provinces, and the raw material source is very rich. However, many purple corn kernels are not fully utilized, resulting in a waste of resources.

3.1 Purple corn kernel pigments

Purple corncobs contain cornflower pigments, geranium pigments and peony pigments, which have good physiological activity [23] and have good market prospects in the food and health industries.

3.1.1 Physiological functions of purple corncob pigments

So far, there has been relatively little research on the functions of purple corncob pigments. Purple corn core pigment belongs to the anthocyanin pigment class. Anthocyanins have antioxidant and free radical scavenging effects, and are known for their high efficiency, low toxicity and high bioavailability. Zhang Zhong et al. [24] showed that purple corn core pigment has the ability to scavenge superoxide anion (O2- ·) free radicals and hydroxyl free radicals (·OH). This provides a theoretical basis for the application of this pigment in food. Purple corn pigment at different concentrations has a certain scavenging effect on superoxide anion radicals and hydroxyl radicals. Within the range of 0.02 g/L to 0.10 g/L, the higher the concentration, the better the scavenging effect on O2- · and ·OH.

3.1.2 Stability of purple corn core pigment

1) Purple corncore pigment is soluble in polar solvents and is a water-soluble pigment. It is sensitive to hydrogen peroxide (H2O2) and reducing agents have a certain damaging effect on purple corncore. The higher the concentration, the greater the damaging effect. The damaging effect of VC on the pigment increases with increasing concentration and duration. Sodium sulfite has a color-enhancing effect on the pigment within the first 80 minutes, with a tendency for the absorbance to increase. As time goes on, the color-enhancing effect disappears and the absorbance slowly decreases. Fe3+, Al3+, Cu2+, Na+, etc. can change the color of the pigment, and the absorbance also increases significantly, while Zn2+ and Ca2+ have no effect on the stability of the pigment.

(2) Purple corn core pigment can maintain a good color effect under high temperature conditions and has good thermal stability. After being kept at a constant temperature of 100 °C for 3 h, the pigment retains about 95% of its original color.

3) Stability to food additives and carbohydrates: Citric acid, malic acid, soluble starch, lactic acid and tartaric acid have a color-enhancing effect on this pigment, and the effect increases significantly with increasing concentration. Glucose, maltose, sucrose, etc. have no significant effect on the stability of the pigment. In addition, sodium benzoate has a certain degrading effect on the pigment. When applying it, add a small amount of preservative according to national standards.

(4) It is relatively stable to light, and even when exposed to direct sunlight, the pigment will not fade significantly. However, purple corn core pigment is a kind of anthocyanin compound, which is sensitive to changes in pH. Under acidic conditions, it appears in its original red color, and as the pH decreases, the red color gradually deepens and is not easy to fade. Under alkaline conditions, the color is very unstable and changes irregularly.

3.1.3 Preparation of pigment

Purple corn core pigment extraction is relatively simple. The optimal extraction process conditions are an extraction solution pH of 1, an extraction temperature of 80 °C, a liquid-to-solid ratio of 1:50, and an immersion time of 1.5 h. The extraction efficiency and overall effect are best when the extraction level is 2 [25]. Zhang Zhong et al. [26] used ultrasonic waves to assist in the extraction of purple corn core pigments. The study showed that the extraction rate of pigments using ultrasonic waves was higher than that of the conventional extraction method, and the stability was basically the same as that of the pigments extracted by the conventional method.

3.2 Purple corn pigments

Purple corn pigment is composed of cornflower, geranium and peony pigments and their linked glucosides, which are six types of anthocyanins.

3.2.1 Physiological activity of purple corn pigment

3.2.1.1 Antioxidant properties [27]

Polyphenols such as various anthocyanins and anthocyanidins can effectively inhibit the chain oxidation reaction of free radicals. The main anthocyanin in purple corn pigment is cornflower anthocyanin, which accounts for 44%, and it has two -OH groups that can capture free radicals, so it has a very good antioxidant effect.

3.2.1.2 Anticancer effect

Japanese researchers Akihiro Hagiwara et al. [28] have shown that purple maize pigment has an inhibitory effect on colon cancer in rats. The rats were given subcutaneous injections of DMH (1,2-dimethylhydrazine) as an initial treatment, and then fed a diet containing PhlP (2-amino-1-butyl-6-phenyl-imidazo[4,5-c]pyridine), which can induce colorectal cancer, for 32 weeks. It was found that the tumor incidence of the group of rats fed a diet containing purple corn pigment powder was significantly lower than that of the group not added with purple corn pigment. This indicates that purple corn pigment has a significant inhibitory effect on PhlP-induced colorectal cancer.

3.2.1.3 Mutation inhibition

The Ames mutagenicity test found that there is a clear quantitative relationship between the mutagenicity of purple corn pigment and PhlP. When the amount of purple corn pigment is 5,000 times the amount of PhlP, the mutagenicity of PhlP can be completely inhibited.

3.2.2 Stability of purple corn pigment

(1) Purple corn pigment is a dark red liquid or powder. At pH 4–2, the color of purple corn pigment changes from pink to red, and at neutral pH it is red to blue-violet, while at alkaline pH it is dark green. It is soluble in water, propylene glycol, acetic acid, etc., but insoluble in oil, ether, and anhydrous propyl alcohol.

(2) Heat resistance: Purple corn pigment has poor heat resistance and is very unstable at 98 °C. In a heat preservation test at 50 °C, the retention rates of the color values of the pigment at different concentrations after 3 days were different. The retention rate of the pigment with a concentration of 0.04% was 70%, and the retention rate of the pigment with a concentration of 0.7% was 75%. The retention rates after 6 days were 58% and 68%, respectively. This shows that the higher the concentration of purple corn pigment, the stronger its heat resistance [29].

3) Light resistance: Purple corn pigment is not light-resistant. After 5 h of exposure to light in a light tester, the residual color value of 0.05% purple corn pigment and 0.2% citric acid aqueous solution (color value 60) decreased to 50%, and after 10 h of exposure, the residual color value decreased to 20%. If used in combination with enzyme-treated isocholestanol (0.05%), the heat and light resistance is greatly improved.

3.2.3 Preparation of purple corn pigment

This pigment has a simple extraction process, is easy to operate, and has a high pigment content. The pigment was extracted with a 0.05 mol/L HCl solution at a sample-to-extractant ratio of 1:20 for 24 h. After two extractions at 60 °C, the temperature was reduced to 60 °C for concentration under reduced pressure and the vacuum was set to 0.9 MPa. Finally, the pigment was dried under reduced pressure (temperature 60 °C, vacuum of 0.9 MPa) to obtain the pigment [26].

In general, purple corn core pigment has good stability, bright color and large extraction amount. It is a kind of natural food coloring, which can be widely used as food additives in beverages, fruit juices, candies, jams, health care drinks or tablets, as well as in the fields of medicine and cosmetics. Purple corn pigment is used to color beverages, candies, fruit wines, etc., and it appears bright red. When used to color dairy products, ham, sausages and other protein products, it appears dark purple to dark brown. Purple corn pigment can be developed into functional foods because it has the function of inhibiting and preventing colorectal cancer.

4 Conclusion and outlook

Although natural pigments have many advantages that synthetic pigments cannot match, they also have some problems. An important issue facing natural pigments is to determine the chemical structure of the main components, the properties, functions and safety of the pigment components. In addition, the safety of natural pigments also deserves attention. Safety issues related to the raw materials, processing and use of natural pigments, such as the disposal of harmful substances contained in the raw materials, the safety of the materials and reagents used in the extraction process and the control of the amount added, need to be resolved to pave the way for the development of more natural pigments of higher quality. With the continuous improvement of the quality of natural pigments in China, the development of natural food colors will be one of the important development directions.

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