基于生物酶法的酰基化桑椹花青素的制备与特性

Preparation and characterization of acylated mulberry anthocyanins using biological enzyme method

为尽可能减少花青素的降解,提高其稳定性,采用生物酶法,对桑椹花青素进行酰基化修饰,并与非酰基化花青素进行对比研究。运用单因素试验筛选脂肪酶、反应溶剂和酰基供体,分析对花青素酰基转化率的影响。确定酰基化反应的优化条件为南极假丝酵母脂肪酶为酰基化催化酶、吡啶为催化反应溶剂、苯甲酸甲酯为酰基供体,酰基化效果最好,转化率最大为13.5%。采用傅里叶红外、高效液相色谱对产物进行分析,经鉴定,酰基化产物为单酰基或多酰基花青素。此外,研究了酰基化对花青素稳定性和抗氧化性的影响。酰基化可提高花青素的热稳定性、光稳定性和耐酸碱稳定性。相同温度下,酰基化花青素保留率提高约5.0%,光照6d后,酰基化花青素的保留率仍高达96.1%。酰基化可以显著增加花青素体外抗氧化性,增强DPPH自由基清除能力,总还原能力比非酰基化花青素提高30%,金属离子螯合能力高达90%。酰基化花青素肿瘤细胞活性抑制率可达81%,而非酰基化花青素仅为50%。该研究为花青素稳定性提高和性能改善提供参考的理论依据。

Mulberry is rich in anthocyanins. The main component is cyanidin-3-O-glucoside. Anthocyanins are hydrophilic and water-soluble polyphenolic plant pigments and metabolites, which are one of the important antioxidants. They possess a wide range of pharmacological properties, such as anti-oxidant, anti-aging, anti-inflammatory, antimicrobial, and anti-cancer. Therefore, anthocyanins have great potential in various fields, such as the pharmaceutical and food industries. However, the relatively low stability of anthocyanin limits its bioavailability and effective applications, particularly on the degradation of anthocyanin. Taking the mature mulberry fruit as research objects, this study aims to prepare and characterize acylated mulberry anthocyanins using biological enzymes. The anthocyanin was extracted with acidified ethanol solvent and then purified by D101 macroporous resin. In order to reduce the degradation of anthocyanin while improving its stability, the mulberry anthocyanin was acylated by biological enzymatic and compared with that of non-acylated anthocyanin. Lipase, reaction solvent, and acyl donor were screened by single factor experiment to explore the anthocyanin acyl conversion rate. Taking the conversion rate as the evaluation index, the optimum conditions for acylation reaction were as follows: Candida Antarctica lipase as an acylating catalytic enzyme, pyridine as a catalytic reaction solvent, methyl benzoate as acyl donor, and the acylation effect was the best. The maximum conversion rate of mulberry anthocyanin was 13.5%. The products were analyzed by Fourier Transform Infrared Spectroscopy(FTIR), Ultraviolet-visible spectrophotometer(UV-Vis), and High-Performance Liquid Chromatography-Mass Spectrometry(HPLC-MS). Acylated anthocyanin was composed of C=O absorption peak at 1 650-1 870 cm-1,-OH bending vibration absorption, and C-O-C stretching vibration absorption of phenolic molecules at 1 000-1 300 cm-1. There was also a skeleton vibration peak of the benzene ring(1 420-1 600 cm-1). The non-acylated anthocyanin sample had obvious UV absorption at 280 nm, and the absorption at this wavelength was the most stable. It showed that anthocyanin contained a benzene ring and a phenolic hydroxyl group on the benzene ring. The absorption peak of acylated anthocyanins moved forward from 280 nm to 271 nm, indicating that the structure of anthocyanins had changed. In addition, an attempt was made to explore the effects of acylation on the stability and antioxidant properties of anthocyanin. The acylation could improve the thermal stability, photostability, and acid-base resistance of anthocyanin. At the same temperature, the preservation rate of acylated anthocyanin increased by 5.0%, and it could be better preserved at 40, 50, and 60 ℃. Light had a great influence on the stability of anthocyanin. The preservation rate of non-acylated anthocyanin decreased linearly to 77.3% within 10 days. After 6 days of light exposure, the preservation rate of acylated anthocyanin was still as high as 96.1%. At the p H values of 2, 3 and 8, the stability of acylated anthocyanin was improved. The acylation significantly increased the antioxidant activity of anthocyanin in vitro. DPPH radical scavenging activity was enhanced. The total reducing ability was 30% higher than that of the non-acylated anthocyanin. The chelating ability of metal ions was 90% higher. The inhibition rate of acylated anthocyanin on tumor cell activity was up to 81%, while that of non-acylated anthocyanin was only 50%. Therefore, acylated anthocyanin can effectively inhibit the proliferation of tumor cells. This finding can provide a theoretical basis and technical support for the stable application and performance improvement of anthocyanin in the production fields of functional food, biomedicine, botanical pesticides, and daily cosmetics fields.