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1 Effects of high intensity ultrasound on disaggregation of a macromolecular 2 procyanidin-rich fraction from Vitis vinifera L. seed extract and evaluation of its 3 antioxidant activity 4 5 Ana Muñoz-Labrador, Marin Prodanov and Mar Villamiel* 6 *Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM).C/Nicolás 7 Cabrera 9, Universidad Autónoma de Madrid, 28049 Madrid, España. 8 9 10 11 *Author to whom correspondence should be addressed: 12 Tel: +34 910017951 13 E-mail: [email protected] 14 1 15 Abstract 16 The impact of high intensity ultrasound (US, 45 and 20 kHz) on a purified macromolecular 17 fraction (more than 85% of polymeric procyanidins) from grape seed extract was 18 investigated. Matrix-Assisted Laser Desorption/Ionisation (MALDI TOF), Reverse Phase 19 High Performance Liquid Chromatography (RP-HPLC) and Fourier-transform infrared 20 spectroscopy (FTIR) revealed a modification in the chemical structure of these 21 macromolecules treated by US and, particularly, bath US produced a considerable increase 22 of up to 49, 41 and 35%, respectively, of catechins and oligomeric and polymeric 23 procyanidin contents of the treated purified fraction. Bath US also produced, an important 24 increase in the number of procyanidins with higher molecular mass (up to decamers) and an 25 overall increase in the mass signal intensities in most of the detected B-type procyanidin 26 series, as well as an important increase of the antioxidant activity of the macromolecular 27 fraction of procyanidins. These results could be ascribed to a certain disaggregation of 28 procyanidins linked to other biopolymers, such as proteins and/or polysaccharides, 29 indicating that US is an efficient technology to modify the chemical structure and hence the 30 bioactivity of tannins. 31 32 Key words: ultrasound, procyanidins, grape seeds, dissagregation, antioxidant activity. 2 33 1 Introduction 34 Proanthocyanidins (PCs), also known as polyflavan-3-ols or condensed tannins, are the 35 second most abundant natural phenolics in plants and consist of mixtures of oligomers made 36 of flavan-3-ol monomeric units linked mainly through C-4 and C-8 or C-6 bond [1–4]. 37 Beyond contributing to organoleptic characteristics of foods with astringency, bitterness, 38 sourness, sweetness, salivary viscosity, aroma and color formation, they are responsible for 39 some physiological effects in humans, such as cardiopreventive, anti-inflammatory, 40 antioxidant, antiallergic, antithrombotic, antibacterial and anticarcinogenic activities, among 41 others, supposing benefits to human health [5–9]. 42 Main dietary sources of PCs are beans, cinnamon, nuts, tea tee, apples, cocoa, grapes 43 and a wide number of berries [10]. From industrial point of view, grape seeds are the most 44 interesting because of their high content in procyanidins, low cost and abundance as a by- 45 product of the wine industry. This is one of the main reason for the existence of wide varieties 46 of grape seed extracts (GSE) rich in PCs in the world dietetic and supplementary market 47 [11]. Most valued are those known as oligomer procyanidin (OPC) extracts, because of their 48 high bioavailability and low content of highly polymeric procyanidins (PPC), considered 49 also as potent antinutrients. Nevertheless, purification of OPCs generates an important 50 amount of PPC that should be managed in one way. The most proper possibility to reuse 51 PPCs should be their depolymerisation to catechins or OPCs, using preferably physical 52 treatments [12, 13]. 53 Power or high intensity ultrasound (US) are considered an physical emergent and 54 environmentally friendly process that involves reduced times of treatments and energy, 55 constituting an alternative to conventional processes [14-1612–14]. The US technology has 56 been developed for processing, conservation and extraction, among other techniques, and is 57 based on the application of acoustic waves of intensity beyond the limit of human hearing 3 58 (>16 kHz). Those waves pass through the material in which is spread and the velocity 59 depends on the nature of the wave and the propagation medium. The US propagates by 60 compression and rarefaction series through the medium obtaining the cavitation 61 phenomenon previously mentioned [142]. Due to the concentration of high quantities of 62 energy in different points of the medium treated by US, one of the principal applications is 63 related with the depolymerisation of macromolecules of biological origin. In general, it is 64 considered that whenever there is a decrease in the molecular size, the biological properties 65 can be modified. Hence, there are some reviews [17, 1815,16] on the principal effects of 66 high intensity US on the disaggregation of proteins and polysaccharides, respectively. 67 However, no studies have been previously reported neither on the US effect on the 68 depolymerisation/disaggregation of tannins nor on the modification of its bioactivity. In 69 general, the main factors that can affect these phenomena are the power, frequency, time, 70 and temperature regarding the working conditions; and the type, molecular mass and 71 concentration in what refer to biomolecules. Thereby, the aim of this work was to study the 72 effect of high intensity ultrasound on procyanidins derived from by-products of wine 73 industry and their antioxidant activity. All of this with the purpose of obtaining potential 74 ingredients with improved functionality in order to raise the value of the raw material and to 75 establish a new approach for reusing PPC by-products. 76 2 Material and methods 77 2.1 Samples 78 Grape seed extract (GSE) with 26% of total soluble substances (TSS) was provided 79 by Output Trade S.L., Villafranca del Penedés (Spain). It was obtained from mature fresh 80 grape seeds from Vitis vinifera L. grapes, cv. Airén, cultivated in La Mancha grape vine- 81 growing area (Spain). A high macromolecular fraction was obtained from the above 82 mentioned GSE by cross-flow pressure-driven ultrafiltration (UF) using a membrane of 10 4 83 kDa molecular mass cut-off (0.54 m2 filtration surface, regenerated cellulose spiral-wound) 84 (model Prep-scale 6) from Millipore (Merck, Darmstadt, Germany), as described in Silván 85 et al. [197]. The PPC fraction was diafiltrated to remove minimal quantity of low molecular 86 weight compounds and freeze-dried. An amount of 106.1 g of dry sample was recovered, 87 which represents about 22% of the dry matter of the clarified GSE (data not shown). The 88 process related to the obtainment of procyanidins fraction is shown in Fig. S1. Samples were 89 stored in dark at 4ºC until processing and analysis. 90 2.2 High intensity ultrasound treatments 91 Samples obtained in section 2.1 were diluted with distilled water at 0.01, 0.1 and 1 92 % (w/v) and two different types of assays were performed: i) in ultrasonic bath and ii) in 93 ultrasonic probe. All the treatments were carried out in duplicate. 94 Ultrasonich bath (Brandson Digital Sonifier 450 full power, 12.7 mm Biogen 95 Científica S.L.) with a frequency of 45 kHz was used to sonicate a volume of 10 mL of each 96 sample dilution in continuous and degas mode for 30 min. Temperature was monitored 97 reaching ranges between 25 and 30 ºC. 98 The ultrasonic probe (BrandsonBranson Digital Sonifier 450 full power, 12.7 mm 99 Biogen Científica S.L.) was applied into 50 mL of each sample dilution trough a microtip 100 horn of 12.7 mm diameter at 20 kHz, in pulsed mode (5 s on/5 s off) at 30 and 70% of 101 amplitude whose conditions were digitally set. The temperature was controlled through an 102 ice-water bath to avoid reaching temperatures higher than 60ºC in order to maximize the US 103 effect [186]. Samples were finally freeze-dried before analysis. 5 104 2.3 Characterisation of procyanidin Characterisation of the isolated GSE macromolecular 105 fraction 106 The protein content of samples was determined through a colorimetric assay using 107 Bicinchoninic acid (BCA) and bovine serum albumin as standard protein (0.02-2 mg/mL). 108 The product of the reaction is purple and it is formed by the chelation of two molecules of 109 BCA with a cuprous ion. Samples were incubated with the reagent for 15 min at 60 ºC and 110 aliquots of 300 µL were analyzed at 560 nm. 111 Soluble fraction of carbohydrates was analysed by GC-FID previous derivatisation 112 reaction. Trimethylsilylated oximes (TMSo) of the carbohydrates present in samples were 113 determined following a previous method [2018]. A volume of 100 µL of supernatant of PPCs 114 at 10% (w/v) was added to 400 µL phenyl-B-D-glucoside (0.5 mg/mL, internal standard) 115 and evaporated under vacuum with a rotary evaporator. The sugar oximes formation was 116 carried out by adding 250 µL hydroxylamine chloride (2.5 %) in pyridine and heated at 70 117 ºC for 30 min. Afterwards, the oximes were silylated with hexamethyldisilazane (250 µL) 118 and trifluoroacetic acid (25 µL) at 50 ºC for 30 min. Reaction mixtures were centrifuged at 119 10,000 rpm for 2 min and supernatants were injected in GC with the split mode (1:5). 120 Chromatographic analysis was carried out on an Agilent Technologies gas chromatograph 121 (Mod7890A) (Agilent Technologies, Wilmingon, DE, USA) equipped with a flame 122 ionisation detector (FID). The TMSO were separated using a 15 m x 0.25 mm x 0.10 µm 123 film, capillary column (SGE HT5, North Harrison Road, Bellefont, USA). Nitrogen was 124 used as carrier gas at flow rate of 1 mL/min. Injector and detector temperatures were 280 125 and 385 ºC, respectively. The oven temperature was programmed from 150 to 380 ºC at a 126 heating ratio of 3 ºC/min.

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