黑曲霉发酵制备荔枝果壳原花青素工艺优化及其对HaCaT细胞的增殖和迁移作用

Optimization of the Fermentation Process Using Aspergillus niger to Prepare Proanthocyanidins from Litchi Husks and Their Effects on the Proliferation and Migration of HaCaT Cells

摘要:

采用黑曲霉对荔枝果壳进行发酵，研究发酵时间、接种量、料液比对荔枝果壳原花青素（LPPC）得率的影响，优化荔枝果壳发酵工艺条件，分析发酵前后荔枝果壳中原花青素、总酚和总黄酮含量以及单体酚的组成和含量的变化，评价发酵前后LPPC的抗氧化活性以及对HaCaT增殖和迁移作用。通过单因素结合正交试验优化确定最佳工艺条件为发酵时间7 d，接种量2.0%（V/V），料液比1:10.0（g/mL），在该发酵工艺条件下测得LPPC得率为3.77%（m/m）。与未发酵相比，发酵后荔枝果壳中原花青素、总酚和总黄酮含量分别提高了112.75%、53.86%和73.02%，鉴定出7种主要单体酚含量均显著增加；且发酵后LPPC的ABTS和FRAP抗氧化活性显著增强。发酵前后LPPC在10、20、40 μg/mL质量浓度时对HaCaT细胞无增殖作用，但能显著提高划痕细胞迁移能力，且发酵后LPPC的细胞迁移率较发酵前分别提高了7.89%、40.89%和75.37%。因此黑曲霉发酵能显著增加荔枝果壳中可提取的原花青素含量，提高其抗氧化活性，有效促进角质细胞划痕创伤愈合，研究结果为LPPC应用于表皮损伤修复功能方面提供理论依据。

Abstract:

Aspergillus niger was used to ferment litchi husk, and the effects of fermentation time, inoculum amount and solid-to-solvent ratio on the yield of proanthocyanidins (LPPC) from litchi husk by were investigated, and the process conditions for the fermentation of litchi husk were optimized. The changes in the contents of proanthocyanidins, total polyphenols and total flavonoids and the composition and content of monomeric phenolics in litchi husk before and after fermentation were analyzed. The antioxidant activity of LPPC before and after fermentation and the effect of LPPC on the proliferation and migration of HaCaT cells were evaluated. The optimum process conditions determined through single factor experiments combined with the orthogonal test were: fermentation time, 7 days; inoculum amount, 2% (V/V); solidto-solvent ratio 1:10 (g/mL). The yield of LPPC prepared under such process conditions was 3.77% (m/m). Compared with those of the unfermented sample, the contents of procyanidins, total polyphenols and total flavonoids of the fermented litchi husk increased by 112.75%, 53.86% and 73.02%, respectively, the contents of the seven main monomeric phenolics increased significantly, and the antioxidant activity of LPPC as ABTS scavenging capacity and FRAP significantly increased after fermentation. Before and after fermentation at a mass concentration of 10, 20 or 40 μg/mL, LPPC had no effect on the proliferation of HaCaT cells, but significantly promoted cell migration, with the cell migration rate for the LPPC after fermentation increasing by 7.89%, 40.89% and 75.37%, respectively, compared with that for the unfermented sample. Therefore, the fermentation with Aspergillus niger can significantly increase the content of proanthocyanidins from litchi husk, thereby increasing the antioxidant capacity and promoting wound healing by keratinocytes. The research results will provide a theoretical basis for the application of LPPC in epidermal repair after skin injury.