Study on the Stability of Natural Anthocyanidin

Food products all have certain color characteristics, and the quality of the color directly affects consumers' acceptability of the food and their evaluation of its quality. The two main types of food coloring used in the food processing industry are synthetic and natural pigments. Synthetic pigments are highly stable, have strong coloring power and are inexpensive, but as research has progressed, it has been discovered that many of the synthetic food coloring that was once allowed to be used have more or less harmful effects on the human body, so the market for natural pigments is expanding [1]. Anthocyanins are a type of natural pigment that is widely used. They belong to a class of polyphenolic compounds. Most flowers, fruits and vegetables in nature have bright colors because they are rich in anthocyanins [2]. Anthocyanins not only have a bright color, but also have strong antioxidant activity. They are an excellent natural antioxidant and free radical scavenger [3] and can reduce the incidence of coronary heart disease, cancer and cerebrovascular disease [4].

However, due to the high activity of anthocyanins, factors such as temperature, pH, oxygen, ascorbic acid, and metal particles can all affect the stability of anthocyanins [2]. Anthocyanins are degraded under external influence into brown or colorless degradation products [4], which affect their color and clarity, placing certain restrictions on the use of anthocyanin pigments. Therefore, how to improve the stability of anthocyanins is the key to the current promotion and use of anthocyanin pigments [5].

1 Factors affecting the stability of anthocyanins

1.1 Effect of pH on anthocyanins

pH can change the structure or composition of anthocyanins, thereby changing their color. Studies have shown that simple sugar anthocyanins exist as 2-phenylbenzopyran cations (AH+) at pH<2, and as quinone-type pseudobases (B) or chalcone (C) at pH4 to 5. At this time, they are colorless, and in the form of the alcohol-type A at pH>6 [6]. It is precisely these structural changes that cause anthocyanins to appear in different colors at different pH values. At pH<2, they appear bright bright red; at neutral pH, they are purple; at alkaline pH, they appear blue; and at pH>11, they can appear dark green [7].

Yang et al. found that the absorbance of rose anthocyanin does not change much at pH 2 to 7. The smaller the pH, the brighter the color. Above pH 7, the absorbance changes greatly and the color changes [8]. Ayeg-ul K et al. studied the stability of anthocyanin by heating anthocyanin solutions of different pH. They found that after heating in a water bath at 70 °C for 8 h, the half-life of the pigment sample with a pH below 4.0 was significantly higher than that of the pigment sample with a pH above 5.0 [4]. This shows that anthocyanin is relatively stable under acidic conditions, and that anthocyanin is generally suitable as an additive for acidic foods.

1.2 Effect of light on anthocyanins

Light (especially ultraviolet light) can induce the decomposition or oxidation of natural pigments, causing them to lose their color. Natural pigments are generally more stable at low temperatures or in a dry state. Heating or high temperatures can accelerate the discoloration reaction, and they are especially prone to oxidation and fading when heated to the boiling point [5]. Cao et al. placed an aqueous solution of mulberry pigment under direct sunlight for 6 hours and found that the pigment degraded by 30%. When placed indoors in the dark for 2 months, the absorbance degraded by 0.03% [9]. Zhu et al. used different monochromatic light to irradiate rose hip pigment and studied the trend of pigment content changes. They found that the pigment was destroyed most quickly under blue light, while it was destroyed most slowly under red light. Under the same light intensity, the order of the destruction rates of pigments by each monochromatic light, from largest to smallest, was: blue light, white light, green light, purple light, yellow light, orange light, and red light [10].

1.3 Effect of heat on anthocyanin pigments

Heating can promote the degradation of anthocyanin pigments, which will lose their bright color. A large number of studies have shown that the degradation rate of anthocyanin increases and the half-life decreases after heating. Cuipeppe, Garzon K, Aysegul K[4, 11, 12] and other researchers have found that the thermal degradation of anthocyanins follows the first thermodynamic formula, and the degradation of anthocyanins accelerates with the increase of heating temperature and time. Heating a rose anthocyanin solution for 2 hours resulted in a greater rate of change in the absorbance of the pigment above 60°C [8].

The half-life of a carrot anthocyanin solution at 70°C is 16.7 hours, while at 80°C it is reduced to 10.1 hours and at 90°C it is only 5.0 hours. Low temperatures are conducive to preventing the degradation of anthocyanins.   The half-life of a carrot anthocyanin solution at 37°C is 4.1 weeks, while at 20°C it is 18.7 weeks. If stored at 4°C, its half-life is 71.8 weeks, and the anthocyanin degrades by less than 36% within one year [4].

1.4 Effect of metal ions

Natural pigments generally do not react with common main group metal ions such as K+, Ca2+, and Na+. Only some metal ions with a slightly higher molecular weight, a high valence state, and metal activity, such as A13+, Zn2+, Cu2+, and Fe3+, react with the pigments, affecting the stability of the pigments and causing the pigments to fade or precipitate [5]. Yang et al. found that N, K+, A3l+, Ba2+, Cd2+, Ca2+, Zn2+, Cu2+, Mg2+, and Pb2+ ions have no adverse effect on the stability of the pigment, while Fe3+ causes the pigment solution to darken and Sn4+ and B3i+ cause the pigment to precipitate [8]. Peng et al. found that Fe3+ and Sn2+ had a significant effect on the absorbance of the pigment, while salt and sucrose had little effect on the pigment [13]. Du et al. found that Fe3+, Zn2+, and Cu2+ ions had a certain effect on the stability of the pigment. As the storage time increased, the absorbance value decreased, with Zn2+ having the greatest effect. Ca2+ ions have a certain protective effect on color [14].

1.5 Effect of other additives

Chen et al. found that ascorbic acid can significantly reduce the color stability of myrtle anthocyanins, accelerate the fading of the pigment solution, and the greater the concentration of ascorbic acid, the worse the stability of anthocyanins. It is analyzed that ascorbic acid should not be used to protect the color or to enhance the content of ascorbic acid in the processing of fruits and vegetables rich in anthocyanins [15]. Yang et al. added sucrose and vitamin C to the aqueous solution of mulberry anthocyanin separately and found that the absorbance of the pigment was not affected when the sucrose concentration was 0–7 mg/mL. When the vitamin C content was 0 to 4 mg/mL, its presence caused a certain degree of increase in the maximum absorbance of the pigment, and the higher the content, the higher the absorbance [8].

Li et al. studied the stability of anthocyanins in pomegranate juice, and found that the three sweeteners sucrose, protein sugar and aspartame had no effect on its stability. The addition of vitamin C caused the absorbance of the pomegranate juice anthocyanins to decrease, and as the concentration of vitamin C increased, the absorbance of the pigment decreased, and the color became lighter. It was concluded that ascorbic acid caused the degradation of the anthocyanins in the juice, thereby causing the color of the pomegranate juice to fade [16]. Xu et al. found that glucose, sucrose, sodium benzoate and potassium sorbate, which are commonly used in foods, had no significant adverse effects on mulberry anthocyanins; vitamin C had a dual effect on mulberry anthocyanins, while H2O2 and NSO3 had a serious damaging effect [17]. At the same time, the presence of oxygen also has an adverse effect on the stability of anthocyanins.

In summary, natural anthocyanins are relatively unstable and are prone to fading, discoloration and precipitation due to various factors during storage or food processing. The stability of pigments varies under different pH conditions due to differences in pigment structure. Heating and the presence of some metal ions are not conducive to the preservation of anthocyanins. Vitamin C has a dual effect on anthocyanins. When present in small amounts, it has a stabilizing effect on anthocyanins. It is precisely these properties of anthocyanins that limit their use in food. To enhance the use of anthocyanins in food, it is necessary to improve the stability of the pigment and prevent discoloration of natural pigments during food processing and distribution.

2 Protective measures

2.1 Change the storage environment of natural pigments

Studies have shown that anthocyanins are more stable at low temperatures and in the dark. Therefore, anthocyanins should be stored, processed and transported at low temperatures and in the dark. Anthocyanins are sensitive to some metal ions, so metal containers should be avoided as much as possible during the extraction, storage and processing of anthocyanins. Some metal chelating agents, such as EDTA, can be added to block metal ions, eliminate the influence of metal ions, and improve the stability of natural pigments. To prevent oxygen from oxidizing anthocyanins, products with added anthocyanin pigments are sealed to prevent oxygen from entering. Given these storage conditions for anthocyanins, as well as the bright color and excellent physiological functions of anthocyanins themselves, I believe that the use of anthocyanin pigments in yogurt, ice cream, fruit juice drinks, fruit vinegar, and other applications will have extremely bright prospects.

2.2 Refining and purifying natural pigments

Natural pigment products generally contain a variety of impurities. There is no clear conclusion as to whether the presence of these impurities has a negative impact on the stability of the pigment. However, the presence of impurities in the pigment can affect the color intensity and color value of the pigment. At the same time, unrefined pigments are not easy to make into powder and are prone to absorb moisture. The main methods of refining and purifying pigments are enzymatic methods, ion exchange methods, membrane separation methods, and comprehensive technical methods. Among these methods, adsorption by macroporous resin is one of the most commonly used methods for purifying pigments in recent years.

Peng et al. used macroporous resin adsorption and separation to purify mulberry red pigment, and compared the adsorption of five resins on mulberry red pigment. The results showed that the use of AB-8 resin as an adsorbent was the most effective. Compared with the traditional method, the color value of the product is higher, reaching a maximum of 38.50. In contrast, the color value of the unpurified pigment is only 5.35 to 5.65. At the same time, AB-8 resin is very stable. After 18 uses, its adsorption rate only decreases by 2.3% [18]. Liu et al. studied the adsorption and separation of mulberry anthocyanins by D101A macroporous adsorption resin. The results showed that the resin had good adsorption capacity for mulberry anthocyanins, the color value of the purified mulberry anthocyanins was greatly improved, it was easy to make into a powder, and it was not easy to absorb moisture [19].    The adsorption rates of five resins, D3520, D4020, X-5, NKA-9, and AB-8, were also studied, and it was found that X-5 had the highest adsorption rate [20].

2.3 Adding auxiliary pigments

Recent studies have found that when anthocyanin molecules bind to certain compounds, it can change the stability of anthocyanin [21, 22]. These compounds that can bind to anthocyanin molecules are usually colorless, but when they are added to the pigment solution and bind to anthocyanin molecules, it will change the color of the solution to some extent. These compounds include substances such as some amino acids, organic acids, nucleotides, flavonoids, polyphenols or anthocyanins themselves. They are generally referred to as copigments. Copigments are rich in electron cloud systems and can form molecular complexes with anthocyanins through hydrophobic and hydrogen bonding,   thus to a certain extent excluding the hydration of the pigment molecule by water molecules and nucleophilic attacks, thereby increasing the stability of anthocyanins [22]. When a compound binds to an anthocyanin molecule, it usually causes a red shift in the maximum absorption wavelength of the pigment and an increase in the maximum absorbance. This chemical reaction exists under conditions of pH 1 to pH 7 [23].

Anna et al. selected substances such as rutin, astragaloside, chlorogenic acid, tannic acid and polyphenols derived from the root of Scutellaria baicalensis Georgi, a Chinese medicinal herb, as co-pigments, and studied the stability of anthocyanins. Experiments were carried out by heating the mixed pigment solution and exposing it to ultraviolet light. It was found that the Scutellaria baicalensis Georgi polyphenols had the greatest effect on improving pigment stability, and that the co-pigment effect was strongest at a pH of about 3.5 [6]. Plamen et al. added polyphenols extracted from rose petals to strawberry drink. The stability of standard pigment solution (PSA), drink, drink and supplementary pigment (RPP), pigment solution (PSA) and supplementary pigment (RPP) under heating conditions was studied. The results showed that the thermal degradation of anthocyanins still conformed to the first thermodynamic formula after the addition of supplementary pigments.   This result is consistent with other researchers, [24, 25]. The half-life of PSA at 85°C is 131min, while the half-life of the PSA+RPP sample under the same conditions is 173min, an increase of about 0.3 times. The stability of the beverage +RP is greater than that of PSA+RPP. It is analyzed that the strawberry beverage itself contains some polyphenols, which have a certain effect on the stability of the beverage's pigments [26].

Some scholars have pointed out that the reaction of polyphenol anthocyanins is a complex reaction of molecular recognition. The molecular configuration of polyphenols can be deformed, the molecular weight is large, and those with a p-coumaroyl group usually have a strong binding ability to anthocyanins. When gelatin is added to the system, it can be observed that the secondary color reaction immediately disappears, which indicates that the polyphenols are involved in the binding of proteins. When salts are present in the system, the polyphenol-anthocyanin complex reaction can be promoted. The bright red color of wine is due to the presence of catechins, condensed tannins and various other flavonoids [27]. In the reaction mode of polyphenols and anthocyanins, the combination of the two is achieved through the combined action of hydrogen bonds and hydrophobic bonds [27].

2.4 Summary

As can be seen from the above, the main determinant of the stability of anthocyanins is the structure of the anthocyanins. To change their stability, there are generally two methods: one is to change the storage environment, for example, by changing the storage temperature, storing it in airtight containers protected from light, and removing substances that have a greater effect on stability, such as oxygen and metal ions. The second is to change its structure, for example, by using anthocyanins in combination with flavonoids and polyphenols, which also have extremely strong physiological functions.

In order to improve the stability of natural food colours, further research is required, especially in terms of the molecular structure of the pigments, to improve the stability of anthocyanins and meet the needs of the rapidly developing food industry.

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