*3.1. Concentration and pH*

Ascorbic acid is unstable in aqueous solutions, and its degradation has been considered the main cause of quality and color changes during food storage and processing. Stability analysis of ascorbic acid is a key point in its application. In addition to the interference of external factors, the concentration of ascorbic acid in the solution will also affect its stability. As reported, after storage at room temperature in the presence of light for 27 days, an aqueous solution of 1% concentration of ascorbic acid lost around 21% of its initial concentration, while the 10% ascorbic acid system only degraded about 8% [56]. In order to ensure sufficient content of ascorbic acid in the product, reinforcing the ascorbic acid content is a method commonly used in the food industry. The content of ascorbic acid in fortified milk decreased from 36.4 mg/L to 26.1 mg/L after sterilization, while the ascorbic acid content of normal milk dropped from 12.2 mg/L to 8.3 mg/L. Although the loss content of ascorbic acid increased with the addition of ascorbic acid, the loss efficiency decreased compared with the initial concentration of ascorbic acid. According to the stability and the degradation kinetics of ascorbic acid, a higher concentration of ascorbic acid has a lower degradation rate constant [57]. However, some studies have shown that excessive ascorbic acid (AH2) is prone to auto-oxidation to produce dehydroascorbic acid anions, according to the following reaction [58]:

$$\begin{aligned} \mathrm{AH\_2} &\rightarrow \mathrm{AH^-} + \mathrm{H} \\ \mathrm{AH^- + O\_2} &\rightarrow \mathrm{A^- + O\_2}^- + \mathrm{H^+} \end{aligned}$$

Furthermore, the autoxidation of ascorbic acid depends on pH. Without transition metal catalysis, its spontaneous oxidation under neutral conditions is quite slow. At pH 7, 99.9% of ascorbic acid is in the form of ascorbate (AH−), as well as a small amount of ascorbic acid (AH2, ~0.1%) and ascorbate dianion (A2<sup>−</sup>, 0.005%) [59].

$$\mathrm{AH\_2} \xrightarrow{\mathrm{pK\_4} \mathrm{4.2}} \mathrm{AH^-} \xrightarrow{\mathrm{pK\_4} \mathrm{11.6}} \mathrm{A^{2-}}$$

$$\mathrm{A^{2-} + \mathrm{O\_2} \to \mathrm{A^- + \mathrm{O\_2}^-}}$$

The amount of A2<sup>−</sup> increases 10-fold for every unit increase in pH. Auto-oxidation occurs through A2<sup>−</sup>, thus accelerating the oxidation rate of ascorbic acid and hydrolyzing to 2,3-diketogulonic acid in alkaline solution [60]. It is worthy to note that the pH value of the ascorbic acid solution changes with its degradation process. Ascorbic acid with an initial pH of alkaline would degrade to about 7.3, which was affected by degradation products generated from ascorbic acid once the reaction began [61]. In acidic solutions, the degradation products of ascorbic acid are also related to oxygen. Under aerobic conditions, dehydroascorbic acid is further degraded to form 2-furoic acid and 3-hydroxypyrone. The main degradation product in anaerobic environment is furfural, and the intermediate product does not involve dehydroascorbic acid [62]. However, in alkaline solutions, the main products of ascorbic acid are 2-methylfuran, 2,4-dimethylfuran, 2 acetyl-5-methylfuran and 2-methyl-2-cyclopentanone [61]. Compared with the hydrolytic pathway that directly cleaves the lactone ring of ascorbic acid under anaerobic conditions, the oxidoreductive pathway in which ascorbic acid forms dehydroascorbic acid in the

presence of oxygen is more common in food systems [62]. Although the headspace can be filled with nitrogen, the dissolved oxygen in the solution is difficult to remove. Previous researchers found that there is a linear relationship between the first-order kinetic constant of ascorbic acid degradation in juice and the initial headspace oxygen concentration during storage at 22 ◦C [63].
