Biorefining of Bilberry (Vaccinium myrtillus L.) Pomace Using

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Biorefining of bilberry (Vaccinium myrtillus L.) pomace using microwave hydro-diffusion and gravity, ultrasound assisted and bead milling extraction Harish Karthikeyan Ravi, Cassandra Breil, Maryline Abert Vian, Farid Chemat, and Petras Rimantas Venskutonis ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04592 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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ACS Sustainable Chemistry & Engineering

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Biorefining of Bilberry (Vaccinium myrtillus L.) Pomace Using Microwave

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Hydro-Diffusion and Gravity, Ultrasound Assisted and Bead Milling

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extraction

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Harish Karthikeyan Ravi 1-2, Cassandra Breil 2, Maryline Abert Vian 2, Farid Chemat 2, Petras

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Rimantas Venskutonis 1

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1

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Lithuania

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2

10

*

Department of Food Science and Technology, Kaunas University of Technology, Radvilėnų pl. 19, LT-50254,

Université d’Avignon et des Pays de Vaucluse, INRA, UMR408, GREEN Team Extraction, F-84000 Avignon,

France

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*Corresponding author: [email protected]

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ABSTRACT: Bio-refining of bilberry pomace using innovative technologies such as

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microwave hydro diffusion and gravity extraction (MHG) and ultrasound assisted extraction

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(UAE) with different concentrations of ethanol/water as the solvent was established. Bead

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milling was also utilized in this sequential extraction scheme to remove the lipophilic

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fraction. Solubility index of target polyphenols was predicted using a computational tool

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(COSMO-RS) and compared to experimental results obtained by in vitro antioxidant activity

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assessments. MHG extracts obtained using microwave power of 2 W/g had the highest Folin-

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Ciocalteu reducing capacity (43.46 ± 0.48 mg GAE/g of extract), total flavonoid (4.17 ± 0.04

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mg QE/g of extract), total monomeric anthocyanin content (12.19 ± 0.13 mg D3GE/g of

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extract) and radical scavenging capacity (22.64 ± 2.23 mg TE/g of extract). In UA

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ethanol/water extracts the highest flavonoid (10.41 ± 0.08 mg QE/g of extract) and

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anthocyanin content (12.19 ± 0.51 D3GE mg/g of extract) was present in ethanol (100%),

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these results were in good correlation with computational prediction. The lipid fraction

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recovered from pomace using bead milling extraction was mainly composed of oil rich in

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polyunsaturated linoleic and linolenic acids.

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KEYWORDS: bilberry pomace, microwave, ultrasound, polyphenol, lipid, COSMO-RS

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INTRODUCTION

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Bilberry or European wild blueberry belongs to Vaccinium genus and is predominantly found

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in North America and European countries. It contains a wide range of bioactive compounds

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and nutrients such as anthocyanins, flavonols, flavan-3-ols, stilbenes, procyanidins, tannins,

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vitamins, phenolic and hydroxycinnamic acids.1 High concentration of polyphenols and other

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secondary metabolites in wild bilberries can be attributed to the elevated environmental stress

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exposure, which significantly modulates their phytochemical profile thereby enabling them to

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accumulate larger amounts of defensive phytochemicals than their cultivated relatives.2,3

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Bilberry is considered to have a protective role in human health against cardiovascular

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disorders, advanced age-induced oxidative stress, inflammatory responses, and diverse

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degenerative diseases. Bilberry extracts have demonstrated a protective effect against

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restraint stress-induced liver damage in mice4, cytoprotective effect against oxidative damage

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of intoxicated rat hepatocytes5 and all these effects were attributed to the antioxidant potential

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of its constituents. During juice processing, a considerable amount of polyphenol-rich seeds and skins of

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berries are discarded resulting in a relatively lower concentration of polyphenols in juice. 49

Enzyme-assistant pressing enables manufacturers to enhance polyphenol content in juice, yet 50

there is a significant amount of polyphenols trapped in pomace.6-8 Consequently there is a

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great interest in recovery of valuable phytochemicals from berry pomace for their 52

applications in foods and other products. Moreover, there is a preference of using for such 53

purposes green chemistry based extraction technologies instead of conventional methods, 54

which often apply hazardous organic solvents. Therefore, development of complex multistep 55

processing methods, which include conventional and novel techniques for the recovery of 56

high added value constituents, may be considered as a very promising trend in biorefining the 57 58

by-products of agro-food industry.9 Emerging technologies were reported as particularly promising for the production of nutraceuticals from agricultural by-products.10

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The benefits of green methodology involving simultaneous ultrasound (UAE) and 60

microwave assisted (MAE) extraction were recently demonstrated for the recovery of EOs 61 62

and pectin from citrus waste.11 MAE process was tested for producing nutrient-rich antioxidant ingredients from tomato fruit processing residues.12 A solvent-free MAE process

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was shown as an eco-friendly and effective method for obtaining high-quality essential oils 64 65

from citrus fruits.13 Water, as dispersing medium, and microwaves, as energy source, were applied for extracting pectin and d-limonene from waste orange and lemon peel.14 ACS Paragon Plus Environment

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Microwave, ultrasound, pulsed electric field, instant controlled pressure drop, supercritical 67

fluid processing techniques as well application of deep eutectic solvents were recently 68

reviewed in terms of the strategies and the tools available to make preservation, 69

transformation and extraction greener.15-17 Microwave Hydro-diffusion and Gravity (MHG)

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technique was shown as much better than conventional methods for dehydration of onion 71

slices in terms of end product quality and process efficiency.18

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MHG is a novel technology with enormous potential for a variety of extractive 73

applications including production of aromatic juices and extracts. Extraction of different 74

compounds with respect to microwave time from various fruits was studied by Cendres et 75

al.19; the authors concluded that at different steps of extraction certain classes of compounds

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are recovered with MHG. For instance, in ‘dry biorefinery’ of ginger MHG was applied to 77

recover essential oil, whereas the residues were further used for recovery of gingerols and 678

shogaol by UAE.20 A MAE of essential oil from leaves of lemongrass was shown as a better

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alternative to hydrodistillation in terms of yield, extraction time, biological activity of the 80 81

extract, energy efficiency and environmental friendliness.21 Similarly, the merits of UAE for food and natural products in general15 and for the recovery of polyphenols in particular22

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were comprehensively reviewed and its application includes intensification of extraction 83

among others. For instance, acoustic cavitation as a novel approach for extraction of oil from 84 85

waste date seeds reduced the environmental impact23 and considerably improved energy cost for extracting pectin from mango peels compared to the conventional process.24

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The application of pressurized and ionic liquids comprises another important group of 87

green extraction. A combination of supercritical carbon dioxide, pressurized water/ethanol 88

and enzyme assisted extractions were successfully applied for the recovery of valuable 89 90 91

substances from the pomace of raspberries25; guelder-rose berries26, chokeberries27-29; black currants30 and sea-buckthorn.31 Pseudosubcritical water extraction as a green extraction methodology was employed to extract potential platform molecules from pea vine waste.32

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Ionic liquids have been extensively used in many research and industry fields as green 93 94

solvents.33 A green alternative procedure employing ultrasound-assisted supercritical carbon dioxide extraction was applied to isolate clove oil and its major bioactive constituents.34

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This study aimed to develop a new schematic approach (Fig. 1) for biorefining of

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bilberry pomace by incorporating innovative and green extraction technologies such as MHG

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and UAE. Those innovative techniques were employed sequentially to extract target

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polyphenols from bilberry pomace. The scope of the study also includes the utilization of

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computational prediction software (COSMO-RS) to obtain theoretical values of solubility ACS Paragon Plus Environment


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index of target polyphenols at different ethanol:water concentrations and its comparison to

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the experimental results from various in vitro antioxidant assays, namely Folin-Ciocalteu

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reducing capacity, total flavonoid content, total monomeric anthocyanin content and radical

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scavenging capacity of extracts. The anthocyanin composition in all extracts was quantified

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with HPTLC and compared to the results obtained by UV-Vis spectrophotometry. As a final

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valorization step, all waste residues were subjected to bead milling in order to remove the

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lipophilic fraction. The fatty acid profile was determined by gas chromatography-flame

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ionization detection (GC-FID) and distribution of lipids by high-performance thin layer

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chromatography (HPTLC).

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MATERIALS AND METHODS

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Standards and reagents

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Folin-Ciocalteu phenol reagent (Panreac Quimica S.L. U, Spain), sodium carbonate, 2,2-

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diphenyl-2-picryl-hydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic

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acid (Trolox) and gallic acid were purchased from Sigma-Aldrich Chemie (Steinheim,

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Germany). Aluminium chloride (Fluka Analytical), potassium acetate, quercetin, primuline

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and sodium acetate were from Sigma-Aldrich (USA); potassium chloride and sodium

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chloride from VWR international (Leuven, Belgium); delphinidin-3-O-glucoside and

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cyanidin-3-O-glucoside from Extrasynthese S.A (Genay, France). All solvents were of

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analytical grade and were purchased from Merck KGaA (Darmstadt, Germany). For the

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purpose of extractions in this study 95% ethanol is referred as 100% ethanol, just to indicate

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that it was not additionally diluted by the water.

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Plant material

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Frozen bilberries (Vaccinium myrtillus L.) were procured from the local supermarket

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(Auchan) in Avignon, France. The berries at room temperature were subjected to pressing at

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3000 psi (10 cycles) using a lab scale hydraulic press R.E.U.S. (Contes, France) to obtain

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juice and pomace. The pomace collected was stored at -18°C until further extraction.

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Extraction procedure

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Extraction of polyphenol fraction by MHG

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A patented MHG apparatus35, Milestone ETHOS-X microwave laboratory oven, was

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used for MHG extraction.36 Bilberry pomace (300 g) were placed in an extraction vessel and ACS Paragon Plus Environment

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three levels of microwave power, 300 W (1 W/g), 450 W (1.5 W/g), and 600 W (2 W/g) were

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employed for MHG extraction. From preliminary analysis, it was found that bilberries, when

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subjected to MHG produce two extract fractions: (1) colored fraction (in situ water with

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polyphenols) and (2) colorless fraction (only in situ water). Therefore, only the colored

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fraction was collected and used for all analyses. Post MHG, the solid residues were labeled as

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MHG residues and stored at -18 °C until further extraction. The temperature, time and weight

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of extracts collected were recorded. All extractions were performed in triplicate. The best

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extraction conditions (Table. 1) were selected based on two parameters, specific energy (E)

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and productivity (Pr). All extracts obtained from MHG were frozen and lyophilized to

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identify the global yield after microwave treatment. The freeze-dried extracts were

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reconstituted in methanol prior to all analysis.

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Extraction of polyphenol fraction by UAE

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An ultrasonic extraction reactor PEX 1 (R.E.U.S., Contes, France) with 24 kHz input

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power was used for UAE37 with different ethanol/water concentrations as the solvent system.

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The MHG residue obtained at optimal extraction conditions was subjected to UAE. The time

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for sonication was selected based on initial analysis, in which 30 g of MHG residue was

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extracted with 300 mL (100% ethanol) for 10, 20, 30, 40, 50 and 60 min respectively (data

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not included). The highest yield was obtained at 30 min of sonication; therefore this time was

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employed for all subsequent extractions with different ethanol/water concentrations. All

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extracts obtained from UAE were frozen and lyophilized to identify the global yield after

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ultrasound treatment. The freeze-dried extracts were reconstituted in methanol prior to all

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analysis.

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Extraction of lipophilic fraction by bead milling

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All biomass, including bilberries, its pomace, MHG residues (1;1.5;2 W/g) and all UAE

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ethanol/water (100:0; 80:20; 60:40; 40:60; 20:80; 0:100) residues were dried in an air oven at

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30 °C for 2 days and used for extraction by bead milling to investigate the distribution of

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lipophilic fraction. For this purpose ULTRA-TURRAX® Tube Drive (UTTD, Ika, Germany)

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operating in a 20 mL tube with 20 g of ceramic beads, 0.3 g of dried biomass and 15 mL of

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hexane was used. The drive tube operated at 4000 rpm for 60 min. The hexane phase was

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recovered and stored at -4 °C until analysis.

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Analysis of hydrophilic and lipophilic fractions ACS Paragon Plus Environment


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Anthocyanin quantitation by high-performance thin layer chromatography (HPTLC)

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Silica gel 60 F254 20 × 10 cm HPTLC plates with layer thickness of 150 - 200 µm and

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particle size of 5 - 7 µm (Merck, Darmstadt, Germany) were pre-developed with a mixture of

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chloroform/methanol (2/1, v/v) and dried at 110 °C for 60 min on the TLC plate heater

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(CAMAG, Muttenz, Switzerland). About 10 mg of anthocyanin standards (delphinidin-3-O-

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glucoside chloride and cyanidin-3-O-glucoside chloride) were dissolved in 10 mL acidified

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methanol (0.5% HCl) to obtain reference solutions. For MHG and UAE extracts, 50 mg of

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each were dissolved in 10 mL acidified methanol as well. All sample and stock solutions

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were stored in the dark at -20 °C until analysis. The analysis was performed with an ATS 5

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automatic TLC sampler (CAMAG). Development was performed in an ADC 2 automatic

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developing chamber (CAMAG) with a mixture of ethyl acetate-methyl ethyl ketone-formic

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acid-water (7:3:1.2:0.8; v/v/v/v) as a solvent.38 Anthocyanins were quantified by a CAMAG

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3 TLC scanning densitometer at a measurement wavelength of 555 nm. Anthocyanin

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quantitation was performed in duplicate and all data recorded were processed with winCATS

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software (CAMAG). CAMAG TLC visualizer was used to capture the image of plates

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analyzed.

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Analysis of fatty acid methyl esters (FAME) by GC-FID

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FAME were prepared from the lipophilic fraction using acid-catalyzed transmethylation

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as described by Morrison et al.39 Triheptadecanoin (C17:0 TAG) was used as an internal

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standard. One mL of acidified methanol (5%) solution was added to the known volume of

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lipid extracted. The mixture was then heated for 90 min at 85 °C. Later, the mixture was

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cooled down to room temperature, to which 1.5 mL of sodium chloride (0.9%) solution and 1

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mL of n-hexane were added. The mixture was transferred to a vial and vigorously shaken for

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1 min. Briefly, 800 µL of the organic layer was recovered and transferred to small vials

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before being injected into GC-FID for analysis. FAME were separated, identified and

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quantified by GC-FID in an Agilent (Kyoto, Japan) gas chromatograph. The instrument was

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equipped with a BD-EN14103 capillary column 30 m × 320 µm × 0.25 µm (Agilent

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Technologies), and the velocity of the carrier gas (He) was 33 cm/s. Two µL of sample were

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injected in split mode (split ratio 1:20), and the injector temperature was set at 250 °C. The

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oven temperature was initially 50 °C for 1 min and then progressed at a rate of 20 °C/min

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from 50 °C to 180 °C and then increased from 180 °C to 220 °C at a rate of 2 °C/min. The

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temperature was then held at 230 °C for 10 min. FAME in each extract was identified by


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retention time and comparison with purified FAME standards (Sigma Co., St. Louis, MO,

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USA).

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Lipid composition by HPTLC

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Pre-development of the plate was done similarly to the protocol followed in the

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anthocyanin quantitation. Chloroform was used as a solvent to prepare stock solutions; about

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10 mg of each lipid was dissolved in 50 mL solvent (0.2 mg/mL). A known quantity of

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lipophilic fraction was dissolved in 20 mL chloroform. Extracts were loaded as a spot onto 20

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× 10 cm Silica gel 60 F254 HPTLC plates using an ATS 5 automatic TLC sampler. The

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HPTLC silica gel plates were developed with a mixture of solvents in an ADC 2 automatic

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developing chamber. The eluent to separate neutral lipids was a mixture of n-hexane/diethyl

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ether/glacial acetic acid in a ratio of 70:30:2 v/v/v to a height of 7 cm from the origin. After

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drying the plate was dipped for 2 s in a reagent (10 mg of primuline, 160 mL of acetone, 40

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mL of distilled water), then scanned using a TLC Scanner. The lipid classes present in the

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lipophilic fraction were quantified by densitometer with identification against known neutral

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lipid standards.40

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In vitro antioxidant assays

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In vitro antioxidant assays of polyphenol fraction by colorimetry

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The Folin-Ciocalteu reagent reducing capacity (FCRC) method41 was used to determine

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the reducing capacity of the MHG and UAE extracts. Briefly, 20 µL of extract/gallic acid

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standard was allowed to react with 80 µL of 7.5% Na2CO3 in a 96-well microplate and placed

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in SPECTROstar omega microplate reader with UV-Vis spectrophotometer. 100 µL of FC

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reagent were added to all wells previously equilibrated with extract and Na2CO3. The

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absorbance of the mixture was recorded at 750 nm for every 5 min over a period of 60 min

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with distilled water as blank at 25 °C. Results were expressed in mg of gallic acid equivalent

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(GAE) per gram of extract.

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The Total Flavonoid Content (TFC) was determined by AlCl3 assay with quercetin as a

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standard.42 The standard solution or extract (500 µL) was mixed with 1.5 mL of 95% ethanol,

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100 µL of 10% AlCl3, 100 µL of 1M potassium acetate and 2.8 mL of distilled water. The

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mixture was allowed to equilibrate at room temperature for 45 min after which absorbance

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was measured at 415 nm in a spectrophotometer (Biochrom, Libra S22, UK). Results were

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expressed in mg of quercetin equivalents (QE) per gram of extract.



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Total monomeric anthocyanin content (TMAC) was determined using pH differential

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method with delphinidin-3-O-glucoside as a standard.43 200 µL of standards or extracts were

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added to 2 mL of potassium chloride buffer and similarly, 200 µL of extracts to 2 mL of

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sodium acetate buffer. The absorbance was measured spectrophotometrically at 520 and 700

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nm. Results were expressed in mg of delphinidin-3-O-glucoside equivalents (D3GE) per

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gram of extract.

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DPPH• scavenging capacity (RSC) of the extracts was measured using trolox as a

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standard44. 50 µL of 0.5 mM methanolic DPPH• solution were added to 50 µL of extracts or

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trolox in a microplate and the absorbance was read at 520 nm for every 5 min over a period

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of 60 min. All experiments were carried out in triplicate and the final results expressed in mg

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of Trolox equivalents (TE) per gram of extract.

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Computational prediction: COSMO-RS

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The Conductor-like Screening Model for Real Solvents (COSMO-RS) is a computational

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prediction model based on the electrostatic interaction between the solutes and a solvent,

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which calculates the thermodynamic properties for solvation. Developed by Klamt and co-

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workers45 it is a known powerful tool for molecular description and solvent screening based

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on quantum-chemical approach.

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COSMO-RS prediction is a two-step procedure. First, the microscopic step when

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simulation is performed in a virtual conductor environment for the molecules. In the given

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environment, molecule induced polarization charge density is achieved on the surface (σ-

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surface). Therefore, the solute molecule is converted to its energetically optimal state in the

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conductor with respect to its geometry and electron density, via the quantum calculation self-

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consistency algorithm. Secondly, a macroscopic step, which is an integrated sequential

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approach established to determine the σ-profile, which is a 3D distribution of the polarization

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charges on the surface of each molecule converted into a surface composition function, thus,

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enabling a wide array of data on the molecular polarity distribution of the molecule. The

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thermodynamics of the molecular interactions that were based on the obtained σ-profile were

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used to calculate the chemical potential of the surface segment (σ-potential) using

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COSMOthermX program (version C30 release 13.01). The standard quantum chemical

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methods, triple zeta valence polarized basis set (TZVP) was used in this study. The σ-

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potential can be associated with the affinity of the solvent to the solute.


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In this work, the model is based on the prediction of the chemical potential of individual

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solute in the ethanol/water solvent system. The solutes were selected after exhaustive

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literature review and each solute corresponds to an individual class of polyphenol.

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Calculation of the relative solubility of target polyphenols delphinidin-3-O-glucoside

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(anthocyanin), quercetin-3-O-galactoside (flavonol), epicatechin (flavan-3-ol), chlorogenic

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acid (phenolic acid), trans-resveratrol (stilbene) and ascorbic acid (vitamin C) in different

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ethanol/water ratios were elucidated by implementing this COSMO-RS model in

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COSMOtherm software (C30 1401, CosmothermX14, COSMOlogic GmbH &Co. KG). The

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relative solubility is calculated from the following equation:

275 ()

μ'

log ( ) = log

, ! " #$

% (Equation 1)

: chemical potential of pure compound j (Joule/mol)

μ? @A : chemical potential of j at infinite dilution (Joule/mol) '

ΔGj, fusion: free energy of fusion of j (Joule/mol) x' ∶ solubility of j (g/g solvent). 276

Relative solubility is always calculated in infinite dilution. The logarithm of the best

277

solubility is set to 0 and all other solvents are ranked relatively to the best or reference

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solvent (Table. 2).

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RESULTS AND DISCUSSION

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Microwave extraction

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Extraction of biomolecules from plant material using MHG technique is a patented

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process.35 The concentration of microwave power on the plant matrix results in a rapid

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increase in temperature, nearing the boiling point of water as it is the major constituent. This

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accelerated increase initiates evaporation of the in situ water, which in turn leads to the

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rupture of cells thereby facilitating the release of in situ water, which acts as a carrier of free

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polyphenols present in the berries.

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Fig. 2a depicts the temperature profile in the microwave system at different powers: 1,

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1.5 and 2 W/g. As microwave power increased the sample temperature with respect to its

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corresponding time increased. The time required to reach 90 °C decreased with increase in

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microwave power, thus reducing the total extraction time. The total extraction time refers to



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the time required to collect only the colored (polyphenol-rich) fraction. Therefore, the yield

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of MHG represents the recovery of Polyphenol-Rich Fraction (PRF).

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The yield of individual microwave power is shown in Fig. 2b; it is evident that the time

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required obtaining the desired polyphenol fraction reduced substantially with an increase in

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power. Microwave power 300 W (1 W/g) required nearly 16 min to produce the PRF

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whereas, 450 W (1.5 W/g) took 9 min and 600 W (2 W/g) required only 6.5 min to yield the

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PRF. It is interesting to note that the distinction to obtain the PRF was better at higher

300

powers, as the transition between the PRF and in situ water fraction was precise. Based on the

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specific power and productivity of MHG extraction the appropriate power of 2 W/g was

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chosen (Table 1) for further extraction. MHG extracts possessed a pleasant characteristic

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aroma regardless of the processing conditions. Lyophilisation of PRF resulted in pasty

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extracts owing to the high sugar content in pomace.

305 306

Effect of microwaves on antioxidant activity of extracts

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The highest phenolic, flavonoid, anthocyanin content and RSC were found for the

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extracts obtained by MHG at 600 W (2 W/g). From Fig. 3 it is evident that no particular trait

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corresponding to the microwave power was observed as 1 and 1.5 W/g extracts had relatively

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lower values. The concentration of anthocyanin was higher than that of flavonoids in all

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MHG extracts with an average of 10.38 mg D3GE and 3.55 mg QE per gram of extract. It

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should be noted that power input may modify the activity of other constituents as it was

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recently shown in case of using pulsed electric fields to modify the thermostability of

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ascorbic acid oxidase in carrots.46

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Ultrasound Assisted Extraction (UAE)

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MHG residue (2 W/g) was subjected to UAE at maximum amplitude wherein different

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ethanol/water concentrations were employed to extract trapped polyphenols. The yield of

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extracts decreased with a decrease in ethanol concentration in the solvent mixture. Pure

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ethanol (100:0) gave the highest yield of 7.45 ± 0.18% whereas the lowest was found for pure

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water (0:100) with 5.11 ± 0.17%. The general trend in lower yields can be attributed to

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decreasing ethanol concentration in the solvent system resulting in the decrease of its

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dielectric constant.

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Effect of solvent on reducing and radical scavenging capacity of extracts and on flavonoid and anthocyanin content ACS Paragon Plus Environment

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Ethanol/water extracts at different concentrations had varying reducing and radical

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scavenging capacity (Fig. 4). FCRC of the extracts was in the following decreasing order of

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EtOH/H2O: 80:20 > 60:40 > 40:60 > 100:0. Sanchez-Rangel et al.41 proposed that the FCRC

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of any extract is not limited to the presence of polyphenols but also depends on the reducing

331

sugar and ascorbic acid content of the fruit extracts, as both reducing sugars and ascorbic acid

332

have the highest impact on hampering the accuracy of the FC reducing assay. The steep

333

increase in the reducing power of EtOH/H2O (80:20) extract when compared to 100% ethanol

334

extract values could be attributed to the theory stated above.

335

The highest RSC was exhibited by extracts obtained with EtOH/H2O (60:40) followed by

336

EtOH/H2O (80:20) with 30.21 ± 1.98 and 29.65 ± 1.21 mg TE per gram of extract,

337

respectively. EtOH/H2O (80:20), EtOH/H2O (0:100) had the lower and lowest reducing and

338

scavenging capacity, respectively. Though low concentration of ethanol in the solvent system

339

enhances the polarity, the bioactivity of their respective extract tends to be significantly

340

lower.

341

EtOH/H2O (100:0) extracts had the highest flavonoid and anthocyanin content with 10.14

342

± 0.08 mg QE and 12.19 ± 0.51 mg D3GE per gram of extract which was three-fold higher

343

than that of EtOH/H2O (0:100) extracts. Ethanol concentration in the solvent system had a

344

direct correlation to the flavonoid and anthocyanin concentration (Fig. 4).

345 346

Analysis of anthocyanins

347

The anthocyanins are the principal pigments determining the colour as well as many of

348

the beneficial effects attributed to berries and their products.47,48 Total anthocyanin content

349

was quantified by HPTLC in all MHG and UAE extracts. In total, 5 anthocyanins were

350

identified and individual concentration of delphinidin-3-O-glucoside and cyanidin-3-O-

351

glucoside were quantified with respective standards. Other anthocyanins (anthocyanins 3, 4

352

and 5) were quantified as cyanidin-3-O-glucoside equivalents (Fig. 5). The total anthocyanin

353

concentration in all extracts followed a trait similar to that of total monomeric anthocyanin

354

content elucidated by spectrophotometric method. The concentration of individual

355

anthocyanin followed a specific order anthocyanin 4 > cyanidin-3-O-glucoside > anthocyanin

356

5 > anthocyanin 3 > delphinidin-3-O-glucoside. Surprisingly, distribution of individual

357

anthocyanins in both MHG and UAE ethanol/water extracts were similar. It can be speculated

358

that the low concentration of delphinidin-3-O-glucoside in both MHG and UAE extracts

359

might be because the extraction was performed from pomace and not from the whole berry.



360

The results (Fig. 6) obtained by spectrophotometric and chromatographic method are in a

361

good correlation. Relatively, the total anthocyanin concentration in UAE ethanol/water

362

extracts determined by UV-Vis spectrophotometry was in good agreement with the data

363

obtained by HPTLC. This correlation validates the accuracy of total monomeric anthocyanin

364 365

content quantitation by a pH differential method with delphinidin-3-O-glucoside as standard. The total anthocyanin content in extracts from bilberry press cake using supercritical

366

carbon dioxide with ethanol as co-solvent varied in the range of 13.67 ± 0.25 mg/g dry 367

weight to 43.66 +/- 0.79 mg/g dry weight, dependent on the choice of drying technique, 368

temperature, and moisture content.49 The maximum anthocyanin yield (98.46 ± 4.92 mg/100

369

g) was found after applying PEF treatment and supplementary extraction with hot water at 50 370

°C.50

371 372

Computational prediction and correlation: COSMO-RS

373

The simulation predicts the solubility index of different solutes in the solvent system. The

374

solutes (target polyphenols) were selected after exhaustive literature review and each solute

375

represents a polyphenol class. The relative solubility log10(x_RS) values are given in Table 2

376

showing that the best solvent for extraction of all solutes was found to be ethanol (100%),

377

thus justifying it as the reference solvent. The values highlighted in green indicates that these

378

solvents possess higher solubility index (0 to -1) when compared to the other proportions of

379

ethanol/water in the solvent system. The values highlighted in yellow and brown cells

380

stipulates that these particular values have relatively medium (-1 to -2) and lower solubility

381

index (> -2) than that of the reference solvent. For anthocyanins (delphinidin-3-O-glucoside)

382

ethanol:water (100:0; 80:20) had better solubility index. Similarly, flavonoids (flavonol

383

hyperoside, flavanol epicatechin) and stilbenes (trans-resveratrol) had good solubility index

384

in ethanol:water (100:0; 80:20 and 60:40) as the solvent system. Ironically, ascorbic acid

385

being a water soluble vitamin had good solubility index in almost all ethanol/water

386

concentrations with the best in pure ethanol (100%) and the lowest in pure water (100%).

387

Though only selected colorimetry assays were performed to quantify the polyphenol,

388

flavonoid and anthocyanin content, these results can be used for correlation and comparison

389

with the theoretical prediction since each solute represents a particular class of polyphenol.

390 391

Lipophilic fraction

392

The lipid fraction was extracted using bead milling, which is an innovative method to

393

extract lipids from a limited quantity of raw material. Meullemiestre et al.51 have already ACS Paragon Plus Environment

Page 12 of 30

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394

demonstrated that bead milling is more efficient than the conventional method for extraction

395

of lipids. All samples (bilberries, pomace, MHG and UAE residues) had similar fatty acid

396

profiles. As shown in Fig. 7a lipid extracts from all samples comprised of 6 individual fatty

397

acids primarily palmitic (C16), stearic (C18), vaccenic (C18:1n7), linoleic (C18:2n6), α-

398

linolenic (C18:3n3) and cis-11-eicosenoic (C20:1). Linoleic acid had the highest relative

399

percentage, approx. 40%, followed by α-linolenic acid with 30% cumulatively taking the total

400

of polyunsaturated fatty acids (PUFA) to 70%. These results were in good agreement with

401

Bunea et al.52, who used a modified Folch method to extract lipids from two varieties of

402

bilberries from Romania. Yet, the variation in fatty acid profile can be attributed to the

403

geographical location, genotype, harvesting period, extraction condition etc. PUFA,

404

particularly linoleic and linolenic acids are widely known for their preventive action against

405

cardiovascular disorders and diabetes.

406

The HPTLC analysis was carried to determine the lipid composition of all samples as

407

stated above. Only neutral lipids monoacylglycerol (MAG), diacylglycerol (DAG),

408

triacylglycerol (TAG) and free fatty acids (FFA) were present in the samples. A standard

409

mixture containing all (MAG, DAG, TAG and FFA) individual lipid classes was used as a

410

reference for quantification. TAGs were the predominant lipid class (Fig. 7b) present in the

411

lipophilic fraction of bilberries. The lipid class distribution was TAG (87%), DAG (8.7%),

412

FFA (2%) and MAG (1%). So, it is safe to assume that neither microwave nor ultrasound had

413

a degradative effect on the lipophilic fraction of bilberry.

414 415

CONCLUSION

416 417

This study sheds light on the utilization of new innovative extraction techniques such as

418

microwave hydro diffusion gravity and ultrasound assisted extraction with different

419

concentrations of ethanol/water, as efficient means to extract free and trapped polyphenols

420

from bilberry pomace thereby, establishing a new biorefining scheme for the valorization of

421

an industrial by-product like bilberry pomace. The highest concentrations of polyphenols,

422

flavonoids, monomeric anthocyanin content and the best radical scavenging capacity were

423

observed in the extracts obtained via MHG using a 2 W/g microwave power density. The

424

ultrasound-assisted extraction of the MHG residue affords highest concentration of flavonoid

425

and anthocyanin using 100% ethanol as extraction solvent. On the other hand, the highest

426

polyphenol content was found using ethanol/water (80:20) extract and the highest radical

427

scavenging capacity with ethanol/water (60:40). Therefore, solvents ethanol/water (100:0; ACS Paragon Plus Environment


428

80:20) could be considered efficient for the extraction of target polyphenols, as their

429

respective extracts possessed the highest bioactivity in the studied in vitro assays. A

430

conductor-like screening model for real solvents (COSMO-RS) was used for prediction of the

431

solubility index of solute representing each class of polyphenols in the solvent system

432

employed. COSMO-RS prediction was in good correlation with experimental results and

433

supported the argument that ethanol/water (100:0; 80:20) was the suitable solvent for

434

extraction. This work elaborates the constituents of the lipophilic fraction obtained by bead

435

milling. Fatty acid profile, and lipid composition distribution after each treatment in residues

436

were identified and quantified. Linoleic acid and α-linolenic acid were the major fatty acids

437

(70%) and triacylglycerols were the major lipid class (87%) found in all bilberry residues.

438 439

AUTHOR INFORMATION

440

Corresponding Author

441

*Tel: +370-37-456647, Fax: +370-37-300155.

442

E-mail: [email protected]

443

Notes

444

The authors declare no competing financial interest.

445 446

ACKNOWLEDGEMENTS

447

The study was supported by Research Council of Lithuania, grant no. S-MIP-17-100

448 449

REFERENCES

450 451

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615

FIGURE LEGENDS

616 617

Figure 1. Graphical representation of experimental scheme

618

Figure 2a. MHG extract yield profile under different microwave power

619

Figure 2b. Temperature index during MHG extraction at different microwave power

620

Figure 3. Effect of microwave power on antioxidant activity of extracts; different lowercase

621

letters above the bars for the same assay indicate significant differences at p

Biorefining of Bilberry (Vaccinium myrtillus L.) Pomace Using

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