该试验对二步发酵法制备雪花梨酵素的工艺进行了优化。以雪花梨为原料，采用Plackett-Burman试验从影响酵母菌发酵的6因素中筛选出料液比、发酵温度、接种量、发酵时间4个显著影响因素，在此基础上以总酚含量和蛋白酶活力为评价指标通过Box-Behnken试验对酵母菌的发酵条件进行优化，以超氧化物歧化酶（Superoxide Dismutase，SOD）活力和可滴定酸含量为评价指标，采用单因素试验对植物乳杆菌的发酵条件进行优化，并以总酚含量、蛋白酶活力、SOD活力、可滴定酸含量、pH值及可溶性固形物含量（Soluble Solids Content，SSC）确定最终植物乳杆菌接种时间，最终确定酵母菌和植物乳杆菌二步发酵制备雪花梨酵素最佳工艺参数为：发酵液起始pH值4.5、料液比1:1、糖添加量13%，一步发酵接种酵母菌0.14%、在26 ℃下发酵24 h后，二步发酵接种植物乳杆菌1.50%、在39 ℃下发酵36 h。此条件制得雪花梨酵素酸甜适中，色泽均匀，体态澄清，无杂质，具有发酵梨果特有香气，总酚含量32.32 μg/mL，蛋白酶活力45.15 U/mL，SOD活力1 140 U/mL，可滴定酸4.43 g/L，pH值3.49，SSC 11.3 Brix%，符合食用液态植物酵素对理化指标的要求。该结果可为雪花梨酵素的综合开发提供理论依据。

Fermentation experiments were performed on Xuehua pears using the Plackett–Burman experimental design to optimize the two-step fermentation process for the preparation of Xuehua pear ferment. Four out of six factors indicating yeast fermentation were identified as significant; the solid-to-liquid ratio, fermentation temperature, inoculum size, and fermentation time. On this basis, the Box–Behnken design was used to optimize the yeast fermentation conditions based on total phenolic content and protease activity, and a single-factor design was used to optimize the conditions for Lactobacillus plantarum fermentation based on superoxide dismutase (SOD) activity and titratable acidity. The final timing for L. plantarum inoculation was determined based on the total phenolic content, protease activity, SOD activity, titratable acidity, pH, and soluble solids content (SSC). The results suggest that the optimal parameters for the two-step fermentation of Xuehua pears by yeast and L. plantarum are an initial broth pH of 4.5, solid-to-liquid ratio of 1:1, sugar content of 13%, yeast inoculum size of 0.14% for the first fermentation step, fermentation period and temperature of 24 hours and 26 ℃ for the first fermentation step, a L. plantarum inoculum size of 1.50% for the second fermentation step, and a fermentation period and temperature of 36 hours and 39 ℃ for the second fermentation step. Xuehua pear ferment obtained under these conditions shows an appropriate level of sourness and sweetness, uniform color, and an aroma unique to fermented pears, whilst being clear and free of impurities. The physicochemical indices of the obtained ferment are a total phenolic content of 32.32 μg/mL, protease activity of 45.15 U/mL, SOD activity of 1 140 U/mL, titratable acidity of 4.43 g/L, pH value of 3.49, and SSC of 11.3 Brix%, which are in line with the requirements of edible liquid plant-based ferments. The results obtained can be used as a theoretical reference for the comprehensive development of Xuehua pear ferments.

财政部和农业农村部：现代农业产业（梨）技术体系资助项目（CARS-28-23）