1 Enzyme-Assisted Extraction Enhancing the Phenolic Release from 2 Cauliflower (Brassica oleracea L. var. botrytis) Outer Leaves 3 Huynh Thai Nguyen, †,‡,§ Guy Smagghe, ‡ Gerard Bryan Gonzales, †,‡,§ John Van Camp, † 4 and Katleen Raes* ,§ 5 † Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, and ‡ Department of Crop Protection, Faculty of 6 Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium 7 § Laboratory of Food Microbiology and Biotechnology, Department of Industrial Biological Sciences, Faculty of Bioscience 8 Engineering, Ghent University - Campus Kortrijk, Graaf Karel de Goedelaan 5, 8500 Kortrijk, Belgium 9 ABSTRACT: Phenolic compounds are highly present in byproducts from the cauliflower (Brassica oleracea L. var. botrytis) 10 harvest and are thus a valuable source for valorization toward phenolic-rich extracts. In this study, we aimed to optimize and 11 characterize the release of individual phenolic compounds from outer leaves of cauliflower, using two commercially available 12 polysaccharide-degrading enzymes, Viscozyme L and Rapidase. As major results, the optimal conditions for the enzyme 13 treatment were: enzyme/substrate ratio of 0.2% for Viscozyme L and 0.5% for Rapidase, temperature 35 °C, and pH 4.0. Using a 14 UPLC-HD-TOF-MS setup, the main phenolic compounds in the extracts were identified as kaempferol glycosides and their 15 combinations with different hydroxycinnamic acids. The most abundant components were kaempferol-3-feruloyldiglucoside and 16 kaempferol-3-glucoside (respectively, 37.8 and 58.4 mg rutin equiv/100 g dry weight). Incubation of the cauliflower outer leaves 17 with the enzyme mixtures resulted in a significantly higher extraction yield of kaempferol-glucosides as compared to the control 18 treatment. 19 KEYWORDS: Brassica oleracea, cauliflower outer leaves, enzyme-assisted extraction, release of phenolic compounds, kaempferol, 20 flavonoids 1. INTRODUCTION 21 Cauliflower (Brassica oleracea L. var. botrytis) is one of the 22 cruciferous vegetables belonging to the Brassicaceae family that 23 are widely consumed all over the world. These products contain 24 considerable amounts of health beneficial compounds, such as 25 phenolic compounds, glucosinolates, and vitamins. 1-4 These 26 vegetables are also characterized by their high amount of 27 nonedible parts, such as outer leaves, stems, and pods. These 28 nonedible parts are now valorized only as raw materials for 29 industrial fertilizer, animal feed, 5 and fiber production, 6,7 or 30 they are left on the fields. However, as they contain high 31 amounts of bioactive compounds, their valorization potential 32 can be much higher. 33 In the past, a number of techniques have been applied to 34 obtain phenolic compounds from plant materials, such as cold 35 pressing, supercritical fluid extraction, and organic solvent 36 extraction. 8,9 Nevertheless, the drawback of these methods is 37 the low extraction yield as the phenolic compounds are bound 38 to plant cell wall material. In cauliflower leaves, just as in other 39 vegetables, phenolic compounds may be classified as bound 40 phenolics found in cell walls in which they are linked to 41 polysaccharides by ester bonds, hydrophobic interactions, and 42 hydrogen bonds, and as free phenolic compounds found in the 43 vacuoles of plant cells. 10-12 As a consequence, preprocessing 44 techniques prior to extraction may be used to reduce the loss of 45 bioactive components and to improve the yields of the 46 extraction process. Degradation and disruption of the cell-wall 47 matrix have been considered as an appropriate step to improve 48 the release of phenolic compounds, keeping their stability and 49 antioxidant activity. 13,14 The mechanism for this treatment is 50 based on the use of cell-wall degrading enzymes to 51 depolymerize cell-wall polysaccharides, 15 and to hydrolyze the 52 glycosidic linkages between phenolic compounds and cell-wall 53 polymers. 12 In addition, enzyme systems originating from 54 microorganisms can transglycosylate the target compounds. 16,17 55 As a result, not only the structure of cell walls can be weakened 56 and broken down, whereby intracellular materials are more 57 exposed for extraction, 13,14 but also the solubility of the target 58 compounds in the extractant can be improved. 16 59 The successful application of carbohydrate-cleaving enzymes 60 for the extraction of phenolic compounds has been reported in 61 several studies, mainly focusing on other plant sources, such as 62 apple peel, citrus peel, grape pomace, Thymus vulgaris, Ginkgo 63 biloba leaves, berries, and oat bran. 10,13-16,19-21 However, the 64 investigation field was restricted to the factors influencing 65 enzyme-assisted extraction of phenolic components, 14,22 and 66 information on the impact of enzymatic treatment on the 67 release of individual components from plant waste material is 68 lacking. The aim of this study was to investigate the potential of 69 using enzyme-assisted extraction and to evaluate its effect on 70 the yield and the profile of extracted phenolic compounds from 71 Brassica cauliflower outer leaves. Received: March 21, 2014 Revised: July 1, 2014 Accepted: July 3, 2014 Article pubs.acs.org/JAFC © XXXX American Chemical Society A dx.doi.org/10.1021/jf502543c | J. Agric. Food Chem. XXXX, XXX, XXX-XXX jwp00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.6.i5 HF01:4227 | 2.0 alpha 39) 2014/03/19 08:04:00 | PROD-JCAVA | rq_3711677 | 7/10/2014 09:20:35 | 9 | JCA-DEFAULT