Cellulase

The black wheat seed was provided by the Cotton Research Institute of the Shanxi Academy of Agricultural Sciences (Taiyuan, China). The xylanase (food grade 50,000 U/g), cellulase (food grade 50,000 U/g), high-temperature α-amylase (food grade 40,000 U/g), and acid protease (food grade 700,000 U/g) were obtained from Solarbio (Beijing Solarbio Technology Co., Ltd., Beijing, China). The α-amylase (15 U/mg) and pepsin (474 U/mg) were purchased from Novozymes (Bagsvaerd, Denmark).

Cuticular membranes from wampee fruit at different stages were prepared following the previous methods with minor modifications (Huang and Jiang, 2019 (link)). The fruits with uniform shape and size at each stage were randomly selected to isolate cuticular membranes. Because of different fruit sizes during development, peel disks from the middle position of wampee fruit at the green, turning, and yellow stages were prepared using a puncher with diameters of 0.5, 0.8, and 1.0 cm, respectively. The cuticular membranes were isolated by immersing the peel disks in 10 mM citric acid buffer containing 1% (w/v) pectinase and 1% (w/v) cellulase (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and incubated under 37°C for 2–3 days. Once most of the tissues were removed from the fruit skin, the cuticular membranes were then washed by 10 mM sodium tetraborate decahydrate (Solarbio Science and Technology Co., Ltd., Beijing, China) to absorb the contamination of free fatty acids. The cuticular membranes were further rinsed with distilled water and air-dried for further analysis.

Pith (0.1 g) pretreated under different conditions was then treated with commercial cellulase® (activity 3,000 IU/g) and β-glucosidase® (activity 100 IU/g) in 50 mL flasks provided by Solarbio (Beijing, China). Pretreated pith samples were added to 0.05M sodium citrate buffer (pH 4.8) to a concentration of 10 g/L, and the resulting mixture was then mixed with 10 mL of deionized water. To these flasks, five different concentrations of enzymes were added (cellulase and β-glucosidase at concentrations of 10 and 20, 15 and 25, 20 and 30, 25 and 35, and 30 and 40 IU/(g dry biomass), respectively). Each concentration was tested in triplicate. Hydrolysis was performed for 72 h (Li et al., 2013 (link)) in a shaking incubator at 50 °C and 150 rpm (Wu et al., 2011 (link); Zheng et al., 2018 (link)). Afterwards, the hydrolysates were centrifuged (10,000 rpm, 10 min) (Singh et al., 2017 (link); Li et al., 2016 (link)), and the liquid fraction was collected for further experiments (Yan et al., 2015 (link)).

Sargassum fusiforme was purchased from Qingdao Fuxingxiang Import & Export Co., Ltd. (Shandong, China). Raw materials were washed with running tap water, then soaked in industrial alcohol for 24 h. The dried powder was acquired and stored at room temperature for further use. The voucher specimen (FSLs-045-2021) was deposited with the Food Science Laboratory of Chaozhou Branch of Chemistry and Chemical Engineering Guangdong Laboratory (Chaozhou, China).
Pectinase, papain and cellulase were purchased from Solarbio Science & Technology Co., Ltd., Beijing, China. Acarbose was produced at Luye Pharmaceutical Co., Ltd., Sichuan, China. Streptozocin (STZ) was obtained from Sigma-Aldrich (Burlington, MA, USA). Other reagents used in this paper were analytical grade.

Pectinase, cellulase, β-galactosidase, dextranase, β-mannase, and standards for maltose (purity ≥98%), maltotriose (purity ≥98%), malttetraose (purity ≥97%), and maltpentaose (purity ≥7%) were products of Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). D-glucose standard (purity ≥98%) was obtained from Sichuan Weikeqi Biotechnology Co., Ltd. (Chengdu, China). Moreover, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) was purchased from Meryer (Shanghai, China) Biochemical Technology Co., Ltd. (Shanghai, China). Aβ_25–35 and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Gibco (Burlington, ON, Canada), Annexin V-APC/PI and Hoechst33342/PI dual staining kits were from Jiangsu KeyGEN BioTECH Co., Ltd. (Nanjing, China), Dulbecco’s modified Eagles medium (DMEM) cell culture medium was purchased from Procell Life Science &Technology Co., Ltd. (Wuhan, China). Trypsin-EDTA from Hyclone Laboratories (Logan, UT, USA). Detection kits for ROS, SOD, LDH and MDA were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent and the RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher (Waltham, MA, USA).