Improvement of stability and lipophilicity of pelargonidin-3-glucoside by enzymatic acylation with aliphatic dicarboxylic acid.

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Improvement of stability and lipophilicity of pelargonidin-3-glucoside by enzymatic acylation with aliphatic dicarboxylic acid

Anthocyanins are common natural pigments, and some anthocyanin-based compounds have been approved by legislation in the EU , the United States and Asian countries as natural pigment additives. Pelargonidin-3-glucoside (Pg3G) is one of the most representative anthocyanins and is found in strawberries and raspberries. The biological activity of Pg3G has attracted much attention in recent years. Substantial evidence demonstrates that Pg3G has potential positive health effects, including antioxidant, anti-diabetic, anti-obesity, and anti-inflammatory effects. However, common anthocyanins are sensitive to pH, light, and heat treatment, and the fat solubility of and is relatively low, which limits their application in the food industry.

Acylated anthocyanins are the products of the acylation reaction between anthocyanins and organic acids. There are many types of acyl involved in natural acylated anthocyanins, including fatty acids (such as acetic acid , malic acid and lactic acid) and phenolic acids (such as gallic acid , p-coumaric acid , caffeic acid and ferulic acid ). A large number of studies have proven that acylated anthocyanins are superior to non-acylated anthocyanins in terms of stability and lipophilicity. Therefore, enzymatic acylation reactions have been developed in recent years to obtain acylated anthocyanins. Most of these studies use Candida antarctica lipase B (CALB) as an efficient catalyst and use fatty acids as substrates to obtain higher acylation rates.

Aliphatic dicarboxylic acids (such as oxalic acid, malonic acid and succinic acid) are also involved in the acylation of anthocyanins in strawberries, cereals and other plants. Anthocyanins acylated with aliphatic dicarboxylic acids have a unique free carboxyl group to form zwitterions, which can improve structural stability or biological activity. However, the linkage between the fatty acyl moiety and the sugar is sensitive to the inorganic acid , which is common during the extraction and isolation of anthocyanins. Furthermore, the detection and identification of aliphatic acyl moieties is complicated by their lack of UV/visible light absorption. Therefore, it is difficult to isolate sufficient amounts of acylated anthocyanins using dicarboxylic acids for further studies. Therefore, few studies have focused on the properties of acylated anthocyanins from aliphatic dicarboxylic acid derivatives.

In this work, the authors synthesized and characterized Pg3G-dicarboxylic acid derivatives and studied the lipophilicity and thermal stability of Pg3G and its acylated derivatives, and subsequently explored the thermal stability mechanism of acylated Pg3G derivatives. .

Effects of acyl donors and solvents on enzymatic acylation reactions . The acylation reaction of anthocyanins is a reversible reaction . During the acylation reaction, the influence of the acyl donor or solvent on the acylation reaction is relatively complex. In this study, four aliphatic dibasic acids with different carbon lengths (oxalic acid, malonic acid, succinic acid and glutaric acid ) were used as acyl donors. The conversion rate of Pg3G after 24 h is shown in Figure 1A Show. The results show that the conversion rate is positively correlated with the carbon chain length of the dibasic acid. When oxalic acid was used as the reactant, no product was found. The conversion rates of malonic acid, succinic acid and glutaric acid are 6%~36%, 60%~65% and 70%~78% respectively. As shown in Figure 1B, the initial reaction rate is related to the carbon chain length of the dibasic acid, and the reaction reaches equilibrium in approximately 24 hours. A similar phenomenon was found in previous studies using fatty acids. The effect of diacids on acylation efficiency may be attributed to their solubility, hydrophobicity, and active center affinity.

Figure 1

Solvent not only has a critical impact on the solubility of the reactants (acyl donors and anthocyanins), but also affects the interaction with the active center, thereby affecting the catalytic activity. In this study, three common solvents (acetonitrile, tert-butanol and tert-amyl alcohol) were used as reaction media. When using succinic acid and glutaric acid as raw materials, there is no significant difference in the conversion rates of the three solvents. However, when malonic acid was used as the reactant, the conversion rates of tert-butanol (28%) and tert-amyl alcohol (35%) were significantly higher than that of acetonitrile (6%) (Figure 1A). Therefore, tert-butanol and tert-amyl alcohol are recommended as reaction media for the acylation reaction of Pg3G with aliphatic dibasic acids.Identification of

Pg3G-dibasic acid derivatives. uses a semi-preparative high-performance liquid chromatography system to separate the required Pg3G-dibasic acid derivatives and analyze them with LC-MS respectively. The analysis shows that the acylation reaction occurs in the part of the glycoside. The structure of Pg3G-dibasic acid derivatives was further analyzed by NMR studies. Analysis shows that the acylation reaction between dibasic acid and Pg3G occurs at the 6’’-hydroxyl group. Therefore, the preliminary acylation products were identified as pelargonidin-3-(6''-malonyl)glucoside, pelargonidin-3-(6''-succinoyl)glucoside and pelargonidin-3-(6 ''-Glutaryl)glucoside. The results also indicate that the enzymatic acylation is regioselective, consistent with previous studies in which fatty acids were used as acyl donors.

Table 1

Figure 2

Dibasic acid acylation enhances lipophilicity. In order to determine the lipophilicity of Pg3G (1) and its acylated derivatives (2-4), the octanol-water partition coefficient method was used, and the results are shown in Figure 3. Pelargonidin-3-(6''-malonyl)glucoside, Pelargonidin-3-(6''-succinoyl)glucoside, and Pelargonidin-3-(6''-glutaryl)glucoside The log P values ​​of are −1.18, −0.93, −0.74 and −0.54 respectively. Due to the presence of free carboxyl in the product, dicarboxylic acid acylation slightly increases the lipophilicity of anthocyanins compared with fatty acid acylation. Therefore, the lipophilicity of anthocyanins can be improved to a certain extent through the enzymatic acylation method by adding dibasic acids or fatty acids.

diacid acylation improves thermal stability. anthocyanins are stable under acidic conditions but relatively unstable under neutral conditions. According to Table 1, the degradation rate constant (k) increases with increasing temperature, and the half-life of (t_1/2) decreases with increasing temperature. The half-lives of Pg3G at 37℃, 50℃ and 60℃ are 7.45h, 3.96h and 1.83h respectively. In contrast, the t_1/2 values ​​for all acylated derivatives are significantly larger at each temperature. During the thermal degradation process, the E_a value of Pg3G (51.3kJ⋅mol⁻¹) was smaller than the E_a value of the acylated product (53.0, 69.3 and 69.7kJ⋅mol⁻¹). This result confirms that the dibasic acid- acylation reaction can improve the thermal stability of Pg3G. In addition, the kinetic parameters in Table 1 show that the thermal stability of acyl groups is positively correlated with the carbon chain length. This phenomenon may be attributed to the steric hindrance of the acyl group, which affects the hydration of anthocyanin molecules and prevents degradation. Notably, Pg3G and its dicarboxylic acid acylated derivatives exhibit similar color characteristics under strongly acidic conditions or neutral conditions (Figure 3), although there are significant differences in their thermal stability. This phenomenon indicates that the aliphatic dibasic acid-acylation reaction has little effect on the color characteristics, and therefore, the dibasic acid-mediated acylated anthocyanins have potential applications as pH color indicators.

Figure 3

Polymorphism of anthocyanins. anthocyanins produce a variety of pH-dependent states in aqueous solutions, usually consisting of flavylium cation (AH⁺), quinoidal base (A), hemiketal (B), cis-chalcone (C_c) and trans-chalcone ( C_t) composition. These species share an interconnected equilibrium network through several reversible reactions. Generally, under strongly acidic conditions, AH⁺ is the dominant species. But as the pH value of increases and increases, AH⁺ participates in different reactions and transforms into other species.

The first step is the transfer of protons to form A, which reaches equilibrium within microseconds . When pH≫pKa, AH⁺ is immediately and completely converted into A, and the absorption spectrum is red-shifted, with the maximum absorption peak located at 522nm. The second step of

involves two reactions: hydration to form B, and then tautomerization to form C_c. Hydration is a rate-limiting step because it is much slower than tautomerization. The last step of

refers to the isomerization reaction of C_c to generate C_t, and reaches the final equilibrium within a few hours.

mole fraction distribution of Pg3G and Pg3G-diacid derivatives. The mole fraction distribution is only related to the hydrogen ion concentration. At pH3, AH⁺ was dominant, and at pH3, B and C_c were dominant (Figure 4). Furthermore, when the pH rises above 7, A will lose a proton and transform into the anion A−.However, under strongly alkaline conditions, anthocyanins auto-oxidize and degrade rapidly. It is worth noting that under mild acidic or neutral conditions, the acylation reaction of dicarboxylic acids changes the equilibrium distribution of AH⁺, A, B, C_c and C_t. The mole fraction of C_t from the acylated derivative is much lower than that of Pg3G (Fig. 4). Since the formation of C_t triggers the degradation of anthocyanins, the smaller fraction of C_t indicates a higher stability of anthocyanins. This may be the main reason why acylated Pg3G derivatives exhibit better thermal stability under neutral conditions. The results of this study indicate that acylation of dibasic acid reduces the formation of unstable C_t, which provides a new perspective on the mechanism by which acylates to improve the stability of anthocyanins.

Figure 4

In summary, pelargonidin-3-glucoside is acylated on the 6''-hydroxyl group by different dibasic acids, and tert-butanol and tert-amyl alcohol are recommended as the acylation reaction of Pg3G with aliphatic dibasic acids. Medium, conversion rate is positively related to the carbon chain length of the dibasic acid. Three primary acylation products were purified and characterized, demonstrating the regioselectivity of the enzymatic reaction. Acylated Pg3G derivatives have better lipophilicity and thermal stability. Based on the analysis of the mole fraction distribution of Pg3G and its acylated Pg3G derivatives, it is speculated that under weakly acidic or neutral conditions, the dicarboxylic acid acylation reaction changes flavylium cation (AH⁺), quinoidal base (A), hemiketal ( B), the balanced distribution of cis-chalcone (C_c) and trans-chalcone (C_t), so that the acylated Pg3G derivative shows better thermal stability than Pg3G. Overall, this study provides insights into the improvement of stability and lipophilicity following enzymatic acylation of pelargonidin-3-glucoside with aliphatic dibasic acids.

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Article information:

PMID: 35500410

DOI: 10.1016/j.foodchem.2022.133077