Study on the Extraction and Stability of Natural Food Coloring

Natural food coloring refers to organic colorants obtained by processing natural substances (including metabolites from plants, animals or microorganisms). Most plant-based natural colorings are relatively safe and are considered green food additives. Currently, there are more than 40 kinds of natural food colorings permitted for use in China, and corresponding national standards have been formulated for each of them. In recent years, it has been discovered that most synthetic pigments have chronic toxicity and carcinogenic effects. It has been realized that synthetic pigments pose an increasingly serious threat to health, and restrictions on their use have been imposed. The development of natural coloring has become the general trend in the development of practical pigments in the world. Compared with synthetic pigments, natural coloring is a food coloring derived from natural resources, and has the following outstanding advantages:

1) Most natural colorings have no toxic side effects and are highly safe.

2) Generally, natural food coloring retains many natural substances (such as vitamins, amino acids, nucleotides, small molecule active peptides, aromatic substances and some of their essential elements, etc.), or is itself a nutrient (such as riboflavin, β-carotene, etc.), which has certain nutritional value and health functions.

3) Some natural coloring also has certain pharmacological effects and can prevent and treat certain diseases.

4) Natural Coloring has a more natural coloring, closer to the color of natural substances, etc. [1].

However, in the food processing process, Natural Coloring is easily affected by external conditions, such as light and heat, pH value and many other factors, and the stability of the color and the extraction rate are also affected by the extraction process. Due to the above reasons, the application of Natural Coloring is subject to certain restrictions. Therefore, how to improve the stability of Natural Coloring has become the key to promoting the use of Natural Coloring.

At present, although China is still in a state of coexistence and simultaneous development of synthetic pigments and natural coloring, natural food coloring is bound to be the main direction of development of food coloring in China. China is rich in natural coloring resources and has a wide variety of them. The species that can be developed and utilized are also diverse. Therefore, more natural colorings that are beneficial to human health will continue to be developed and applied.

1. Purple sweet potato red pigment

Sweet potatoes, also known as yams, red potatoes, white potatoes, potatoes, or sweet potatoes, are a kind of grain crop widely cultivated in tropical and subtropical regions around the world. The root tubers of sweet potatoes come in various colors, including white, yellow, orange, and purple. There are about 500 varieties, all of which can be used as food or as raw materials for making starch and alcohol. Orange varieties are rich in β-carotene, yellow varieties are rich in flavonoid pigments, and purple varieties are rich in bright red anthocyanins.

However, because the content is very low (generally only about 1 color value), there has been very little production of sweet potato red pigments in the past [2]. Japan has obtained a new variety after hybridization and named it “Aya Purple”. This sweet potato contains 8 times the anthocyanin content of the original variety, and due to improvements in cultivation characteristics, the yield per mu has increased significantly, so it can be used as a raw material for the production of natural food coloring, which makes the industrialization of PSPC possible. In China, the yield of purple sweet potatoes has always been very low, making it difficult to achieve commercial production of PSPC. In 1980, varieties such as “Purple Rose” were introduced from abroad. After crossbreeding and improvement, a strain with both high yield and pigment content was obtained, which can meet the requirements of industrial pigment extraction [3].

1.1 Extraction methods

The extraction of purple sweet potato red pigment mainly includes the solvent method and the fermentation method. At present, the solvent method is mostly used, and the commonly used extraction agents include acetic acid, hydrochloric acid, sulfuric acid, formic acid, citric acid, ethanol, etc. [4]. For the extraction of sweet potato red pigment, acidified methanol was used abroad, while 0.5% citric acid, ethanol or acidified ethanol (volume ratio 85:15) was used domestically [5]. Lu Guoquan et al. found that the extraction effect of acidified methanol on PSPC was the best, followed by dilute hydrochloric acid and dilute citric acid [6]. However, methanol is not suitable as an extraction agent for natural coloring because it is highly volatile and toxic. The pigments used in food require a relatively high level of safety, and citric acid is a commonly used acidic medium in the food industry, so it is more suitable to use citric acid as an extractant.

Basic process of the fermentation method: a certain amount of purple sweet potato is washed, steamed, cooled, crushed, mixed with a primary mash prepared with a certain amount of rice, fermented for a few days, a secondary mash is prepared, the secondary mash is filtered, and the pigment liquid is obtained by vacuum concentration. The pigment liquid obtained by this method contains very little starch and is a clear pigment liquid.

1. 2 Stability study

1. 2. 1 The effect of heating on the stability of the pigment

Purple sweet potato red pigment was prepared as a pigment solution, placed in a 90°C water bath and heated for 3 hours. After removal, it was quickly cooled to room temperature, and the absorbance was measured at its characteristic absorption peak. It can be seen that purple sweet potato red pigment has strong thermal stability. However, if the original absorbance is compared after being heated at 100 °C for 5 hours, it is found to have decreased by about 50%. Therefore, the higher the heating temperature and the longer the heating time, the more detrimental it is to the stability of the pigment. In addition, the effect of temperature on light density seems to be related to pH. When the pH is 3, the pigment shows relative stability to heat; when the pH is 5 and the temperature is high for a long time, the stability of the pigment decreases, but the decrease is not significant, indicating that it is relatively heat-resistant. However, in actual production, in order to reduce damage to the pigment, high temperatures and long heating times should be avoided as much as possible.

1. 2. 2 Effect of light on the stability of the pigment

When purple sweet potato red pigment was stored for several weeks at pH = 3 in a dark room, in a room with natural light, in natural light outdoors and under ultraviolet light, the pigment still showed considerable stability and the change in optical density was minimal. Lu Guoquan et al. compared purple sweet potato pigment, grape skin pigment, perilla pigment and black rice pigment under the same light conditions and found that purple sweet potato pigment had the best stability [7].

1. 2. 3 The effect of metal ions on pigment stability

Common metal ions such as Fe2+, Al3+, Fe3+, K+, Cu2+, Mg2+, Ca2+, etc. have no effect on purple sweet potato red pigment, so the material of the container used in actual production can be ignored. However, when Fe3+ is added, the solution turns purple-brown, but it cannot be concluded that Fe3+ enhances the color, because Fe3+ itself also has a color, so its mechanism needs to be further studied.

2 Raspberry red pigment

Raspberries are perennial shrubs in the genus Rubus of the Rosaceae family. Studies have shown that raspberry fruits are rich in vitamins, amino acids, sugars, organic acids and trace amounts of potassium, zinc, iron, copper, manganese and other nutrients such as enzyme cofactors [8]. In addition, raspberries are also rich in secondary metabolites such as ellagic acid, flavonoids, salicylic acid, caffeic acid, pigments, etc. [9-10], so they can be widely used in food, pharmaceutical and other industries [11].

2.1 Extraction method

Liu Huqi, Chen Tieshan and others studied the extraction solvent, method and removal of impurities of raspberry pigment, and selected the best extraction method. Raspberry pigment is a water-soluble pigment. The researchers used five different extraction solvents, namely water, petroleum ether, chloroform, ether and ethanol, to carry out comparative tests on pigment extraction, and selected the best extraction solvent. The colorimetric results showed that the water extract had the darkest color, the ethanol extract had a lighter color, and the other extracts contained very little colored components. Therefore, water was the best solvent for extracting raspberry pigment. The researcher also used water as the extracting agent to further investigate the impact of different extraction methods on the extraction effect, in order to determine the best extraction method. The results showed that the percolation method, the heating reflux method and the water vapor distillation method were more effective.

Based on the above experiments, it can be concluded that using water as the extraction solvent and using the percolation method, the heating reflux method and the water vapor distillation method can effectively extract the pigment components. However, considering that raspberries are rich in volatile oil components, so the water vapor distillation method should be the preferred and economical method. While extracting the pigment components (distributed in the aqueous solution), the volatile oil components (distributed in the distillate) are also extracted, which can be said to kill two birds with one stone. In addition, experiments were carried out to remove the main impurities in the aqueous solution of raspberry pigment extraction, such as lipids, proteins, sugars, fats, waxes and other components. The removal of these impurities is mainly based on their properties. 95% ethanol is added to the concentrated aqueous extract. Lipids, proteins, sugars, fats, waxes, etc. that do not dissolve in ethanol will gradually precipitate and be separated out. For impurities such as fats and waxes that are not easily separated, petroleum ether can be added to the concentrated solution to remove them. After impurity removal, the color of the pigment remains unchanged and the purity is increased.

Yang Wanzheng, Liu Haiqing and others also carried out experiments to extract raspberry pigments. By collecting ripe raspberry fruits, removing the stalks, washing, drying, and then crushing them, and using water as a solvent, the fruits were extracted at room temperature. The extract was then filtered to remove solid insoluble matter, and the filtrate was concentrated to obtain a purplish red viscous concentrate. The concentrate was then washed with petroleum ether to remove fat-soluble substances, vacuum drying to obtain a brown extract-like solid, mainly anthocyanins, with a yield of 32.5%. Here, it is reminded that all personnel in contact with raspberry raw materials must use stainless steel, acid- and alkali-resistant ceramic or glass products, and the solvent water must also be deionized water to prevent metal ions from contaminating the product.

2. 2 Stability study

2. 2. 1 Effect of acidity on pigment stability

Liu Huqi, Chen Tieshan and others studied the acid and alkali stability of raspberry pigments by measuring the color of raspberry pigment aqueous solutions at different pH values. The results showed that at pH 2 to 5, the pigment solution was red; at pH 5 to 8, the pigment solution was light red to orange red; and at pH 9 to 11, the pigment solution was purple. Researchers believe that pH = 4 is the optimal pH value for raspberry pigment, when the pigment is a bright red. Yang Wanzheng, Liu Haiqing and others have also confirmed this conclusion in their research, and have analysed and explained the reason for the change in the colour of raspberry pigment with pH value, i.e. the change in the molecular structure of anthocyanin at different pH values. Since the oxygen atom in the pyran ring of the anthocyanin molecule is four valence, it is basic and can accept protons; and the phenolic hydroxyl group in the molecule can dissociate, giving a proton and being acidic, which gives this type of substance the characteristic of changing structure with changes in the pH of the medium.

2. 2. 2 The effect of temperature on pigment stability

Liu Huqi, Chen Tieshan and others studied the thermal stability of raspberry pigment by observing the change in the extinction value (540 nm) of raspberry pigment solution after treatment at different temperatures and pH values. The results of the experiment show that within 100 °C and a pH range of 2 to 5, the extinction value decreases slightly, indicating that raspberry pigment is basically stable. When the temperature exceeds 100 °C, the stability of raspberry pigment decreases, but overall it still has a certain degree of high temperature resistance. Yang Wanzheng, Liu Haiqing and others also investigated the thermal stability of raspberry pigment at pH = 4. The pigment extract was placed in a constant temperature hot water bath at different temperatures and heated for 1 h. After cooling, the absorbance was measured at 510 nm. The results showed that when the heating temperature was changed from room temperature to 100°C, the absorbance value decreased from 0.169 to 0.162, a change of only 0.007, indicating that raspberry pigment has good thermal stability.

2.2.3 Effect of light on pigment stability

Zhang Cunli, Zhang Hongchang and others studied the light stability of raspberry pigments by observing the change in extinction value after treating the pigments with different light times. The experiment selected light exposure times from 0 to 70 days, and measured the corresponding extinction values, which changed from 0.54 to 0.41, a decrease of only 0.13. It can be seen that raspberry pigments are quite stable under light exposure conditions and have better light stability than other pigments.

3 Strawberry red pigment

Strawberries are in the Rosaceae family. They are perennial berries and anthocyanin pigments. The main component is pelargonidin 232 glucoside. In addition, four other pelargonidin glycosides and two derivatives of cyanidin glycosides have been found. Pelargonium 232 glucoside is more stable than other anthocyanins because it contains a phenolic hydroxyl group at the 4' position of the β-ring. Strawberries are sweet, slightly acidic, and cool in nature. They quench thirst, invigorate the stomach and promote digestion, and are used to treat thirst, loss of appetite, and indigestion [12]. The natural strawberry red pigment extracted from strawberries is a safe and non-toxic food coloring, with certain nutritional value and health benefits. It is a natural food coloring with great development potential [13].

3.1 Extraction method

Luo Kai, Hu Tingzhang and others conducted experimental research on the extraction of strawberry pigments, including the screening of extraction solvents and the determination of the optimal extraction conditions, such as the concentration and pH value of the extraction agent, the extraction temperature and time. The extraction solvent screening experiment selected commonly used solvents, including petroleum ether, ether, water, acetone, 95% ethanol and 50% ethanol, and carried out the immersion extraction of pigments. The results showed that the pigment was almost insoluble in petroleum ether, slightly soluble in ether, and more soluble in water, acetone, 95% ethanol and 50% ethanol. However, the acetone extract was slightly turbid, so it was not suitable for research.

Considering the price and edibility, water and ethanol were selected as the more suitable extraction solvents. The optimal extraction conditions experiment shows that the extraction agent ethanol has the best effect at a concentration of 50%; the pH of the extraction agent has a greater effect on the extraction rate of the pigment, and the extraction rate of the pigment increases with a decrease in pH. The greater the pH, the more unstable the red pigment, so it is appropriate to extract under acidic conditions; the extraction temperature experiment shows that the red pigment is more stable when extracted in the range of 20-80°C, However, considering industrial production and energy conservation, it is more practical to extract at 20-40°C. The extraction time was found to be optimal at 1.5-2.0 h. In summary, the optimal extraction conditions are: 50% ethanol at pH = 4, extracted at 20-40°C for 1.5-2.0 h.

Yang Peirong, Kang Jianbiao and others also studied the optimal conditions for the extraction of strawberry pigments. They selected anhydrous ether, acetone, trichloromethane, water, ethanol and hydrochloric acid as the extraction agents. The results showed that strawberry pigments are easily soluble in water, ethanol and hydrochloric acid, insoluble in anhydrous ether, acetone, and trichloromethane, slightly soluble in 0.4% NaOH solution, so hydrochloric acid (1.5 mol/L) - ethanol (97.5%) solution and water were selected as the extraction agent to determine the best process. Extraction temperature experiments showed that the optimum extraction temperature was 50°C. The lower the temperature, the less complete the extraction of the pigment, but if the temperature is too high, the pigment is easily decomposed and the absorbance decreases. Experiments on extraction time show that the extraction rate of the pigment increases with the extension of the extraction time, but the increase is slow after 2 hours, so it is more appropriate to choose an extraction time of 1.5 to 2.0 hours.

3.2 Stability study

3.2.1 Effect of pH on pigment stability

Yang Peirong, Kang Jianbiao and others dissolved strawberry pigment in aqueous solutions of different pH values, left them for 0.5 h and then observed the color change to examine its stability to pH. The results showed that as the pH increased, the red color of the strawberry pigment gradually weakened and the yellow color gradually intensified. When the pH is 1, 2, or 3, the solution is reddish-orange; when the pH is 4, the solution is pink; when the pH is 7, 8, or 9, the solution is purple-brown; and when the pH is 12, the solution is yellow. This indicates that the molecular structure of the pigment has changed [14–15]. The absorbance of each pigment solution with a pH of 1 to 6 was measured at a wavelength of 501 nm after being left at room temperature for 1, 2, or 3 days. The results showed that under acidic conditions with a pH ≤ 2, the absorbance of the strawberry pigment changed from 0.581 to 0.535 after being left for 3 days. The pigment basically did not degrade, and the color basically remained stable. However, when the pH is ≥4, the pigment solution becomes cloudy or precipitates after standing.

3. 2. 2 Effect of temperature on pigment stability

Yang Peirong and Kang Jianbiao dissolved strawberry pigment in a pH = 2 solvent and placed it in a water bath at room temperature and different temperatures for 1 h. The absorbance was measured at a wavelength of 501 nm to investigate thermal stability. The experimental results show that the absorbance of strawberry pigment decreases with increasing temperature. It is relatively stable below 60°C, but if the temperature is higher than 70°C, the color gradually becomes lighter, which shows that high temperature has a certain degrading effect on the pigment.

3. 2. 3 The effect of metal ions on the stability of pigments

Researchers studied the stability of strawberry pigment solutions by adding solutions containing different metal ions. The concentrations of the solutions containing various metal ions were 0.005, 0.05 and 0.1 mol/L, respectively. 5 mL of the pigment stock solution was taken and 5 mL of each metal ion solution was added, and the solutions were placed under the same conditions for 1, 2 and 3 hours, respectively. The absorbance was measured at a wavelength of 501 nm. It can be seen from the experiment that when Cu2+, Fe2+, Fe3+, and Al3+ are added, the higher ion concentration and longer contact time will cause the pigment to degrade, resulting in a decrease in the absorbance of the pigment and even turbidity. It is clear that the addition of these four metal ions affects the stability of the pigment. However, in the presence of Zn2+, Mg2+, K+, Na+, and Ca2+ ions, the pigments are very stable and retain their bright color, indicating that these last five metal ions have no effect on the stability of the strawberry pigments.

3. 2. 4 The effect of oxidants and reductants on pigment stability

Yang Peirong, Kang Jianbiao and others investigated the effect of oxidants and reductants on pigment stability by observing the change in absorbance after adding oxidants or reductants to the strawberry pigment stock solution. Specific method: Take 5 mL of each pigment solution and add 5% hydrogen peroxide (oxidant), 5% sodium sulfite (reductant), and 5% VC (reductant) respectively. Add 5 mL of distilled water to the control group and measure the absorbance at 501 nm. The results show that hydrogen peroxide, sodium sulfite and VC can all change the color of the pigment from red to colorless, and the absorbance decreases significantly compared to the control group. Since strawberry pigment has a polyphenol structure and is highly oxidizable, it can cause the degradation of strawberry pigment [16]. It can be seen that oxidants or reducing agents can have a significant effect on the stability of the strawberry pigment, causing it to lose its color.

References

[1] Xiang Wenbin, Gao Jianrong. Natural Coloring (Practical Handbook of Natural Products) [M]. Beijing: Chemical Industry Press, 2004: 7-12. [2] Ling Guanting. Purple Sweet Potato Pigment and Its Physiological Function [J].  Grain and Oil, 2002 (11): 47-50.

[3] Lu Guoquan, Qiu Yongjun, Lou Xiaobo. Research on the extraction technology of purple pigment from purple sweet potato. Journal of Zhejiang Agricultural University, 1997, 23 (1): 105-107. [4] Wu Qiaoling. Research progress on the extraction process of purple pigment from purple sweet potato.  Science and Technology Today, 2003 (6): 43-46.

[5] Yin Qinghong, Liu Youzhou, Xie Yizhi, et al. Extraction conditions of anthocyanins from purple sweet potatoes. Jiangsu Agricultural Journal, 2002, 18 (4): 236-240.

[6 ] Lu Guoquan, Qiu Yongjun, Lou Xiaobo. Research on the extraction technology of purple pigment from purple sweet potato. Journal of Zhejiang Agricultural University, 1997, 23 (1): 105-107.

[7] Lu Guoquan, Li Xiuling. Stability comparison of purple sweet potato pigment with other similar pigments [J]. Journal of Zhejiang University, 2001, 27 (6): 635-638.

[8] Wang Wenzhi. Preliminary report on the nutritional composition of raspberry fruits [J]. Northwest Horticulture, 2001 (2): 13-14.

[9 ] Li Weilin . Research on the volatile oil composition of blackberry fruit[J]. Chinese Journal of Pharmacy, 1998, 33 (6), 335 -336 .

[10] Shen Zuijun . Physicochemical properties of blackberry juice and changes in nutrient composition during storage[J]. Plant Resources and Environment, 1997, 6 (1) :20 -24 .

[11] Ma Z. Natural food coloring chemistry and production technology [M]. Beijing: China Forestry Publishing House, 1994.

[12] Liu C.R., Hu X.L., Jiang F.S., et al. Study on the physical and chemical properties of strawberry pigments [J]. Food Science and Technology, 2002 (2): 38-39.

[13] Liu Cheng, Zhou Ruzhong. Encyclopedia of Food Additive Use [M]. Beijing: Beijing University of Technology Press, 1995.

[14] Xu Yaqin, Yu Zeyuan, Shao Tiehua. Study on the stability of strawberry pigment [J]. Food and Fermentation Industry, 2000 (4): 13-16.

[15] Gao Fuxing, Luo Wei, Li Genqiang, et al. Extraction stability of strawberry pigment [J]. Journal of Xinyang Normal University, 2001 (10): 448-449.

[16] Finema. Food Chemistry [M]. Wang Zhang, trans. Beijing: China Light Industry Press, 1991.