Lutein

Lutein (purity > 90%), alcalase (enzyme activity > 200 U/mg), bile salts, and molecular weight standards including bacitracin, aprotinin, myohemoglobin, bovine serum albumin, and thyroglobulin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Soybeans were bought from Fengyuan Zhongye Co., Ltd. (Lianyungang, China). Pepsin (enzyme activity > 2500 U/mg), trypsin (enzyme activity > 2500 U/mg), 8-aniline-1-naphthalensulfonic acid (ANS), and trinitrobenzenesulfonic acid (TNBS) were purchased from Sigma-Aldrich Co., (St. Louis, MO, USA). All other reagents and solvents were of at least analytical grade.

Carotenoids were extracted from the hepatopancreases and ovaries according to the method of Li et al. [20 (link)]. Briefly, we homogenized samples in acetone in an ultrasonic cleaner for dissociation of the pigment. Subsequently, we removed the homogenates after centrifugation. We re-extracted the samples in the dark using acetone until the samples were colorless. Total carotenoids were analyzed spectrophotometrically at 478 nm against acetone as the blank. The concentration of total carotenoids was estimated based on commercially available standards of β-carotene in acetone. A portion of the carotenoids extracted from each sample was used to analyze free carotenoids. Samples were passed through a 0.45 μm syringe filter for high-performance liquid chromatography (HPLC) analysis. Carotenoids were identified based on five commercially available standards of known concentration (astaxanthin, lutein, zeaxanthin, and β-carotene from Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China; canthaxanthin from Dr. Ehrenstorfer GmbH., Augsburg, Germany). We performed quantitative determination using an external standard method. A detailed protocol is provided in the Supplementary Materials Text S3.

Capsanthin (purity 99%) was purchased from Sigma Aldrich (St. Louis, MO, USA). Zeaxanthin, lutein, β-cryptoxanthin and β-carotene (purity 99%) were purchased from Yuanye Bio-Technology (Shanghai, China). HPLC-grade tetrahydrofuran, acetonitrile, methanol (MeOH), acetone, methyl tert-butyl ether (MTBE) and isopropanol were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Analytical-grade N-hexane, MeOH, ethanol, acetone, ethyl acetate, petroleum ether and anhydrous ether were purchased from the Sinopharm Group (Beijing, China). Potassium hydroxide (KOH) was purchased from Lanyi Chemical Company (Beijing, China). Ultrapure water was generated by a Milli-Q integrated water purification system (Millipore, Billerica, MA, USA).
Nine varieties of fresh and red chili peppers, which were selected to be investigated because of the higher and richer carotenoids, were supplied from Comprehensive Experiment Stations of China Agriculture Research System, which were located in Henan, Yunnan, Gansu, Guizhou, Neimen, Shandong (six provinces in China). Dried chili peppers, chili oil, fried chili sauce and fermented chili sauce were all purchased in a market (China).