Sei sulla pagina 1di 29

Accepted Manuscript

Optimization of microwave-assisted extraction of natural antioxidants from spent espresso coffee grounds by response surface methodology

Marija Ranic , Marina Nikolic , MSc Marija Pavlovic , MSc Aneta Buntic , MSc Slavica Siler-Marinkovic , PhD Suzana Dimitrijevic-Brankovic , PhD

PII:

S0959-6526(14)00537-X

DOI:

Reference:

JCLP 4353

To appear in:

Journal of Cleaner Production

Received Date: 6 July 2013

Revised Date:

Accepted Date: 20 May 2014

5 May 2014

2013 Revised Date: Accepted Date: 20 May 2014 5 May 2014 Please cite this article as:

Please cite this article as: Ranic M, Nikolic M, Pavlovic M, Buntic A, Siler-Marinkovic S, Dimitrijevic- Brankovic S, Optimization of microwave-assisted extraction of natural antioxidants from spent espresso coffee grounds by response surface methodology, Journal of Cleaner Production (2014), doi: 10.1016/

j.jclepro.2014.05.060.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Reference: JCLP 4353

Editorial reference: JCLP_JCLEPRO-D-13-00940

To be published in: Journal of Cleaner Production

Abstract: Optimization of microwave-assisted extraction of natural antioxidants from spent espresso

coffee grounds by response surface methodology

Espresso spent coffee grounds (SCG) that is a waste material abundantly produced by restaurants,

cafeterias and in domestic environment could be used as a low-cost and rich source of valuable

polyphenol compounds. The benefit would be twofold: extraction of health beneficial natural

polyphenol antioxidants and reducing the cost to facilitate SCG waste management. The overall

objective of this study was to examine an optimal range of extraction conditions for extraction of

antioxidants from spent espresso coffee. Optimization of the extraction process from SCG was

carried out using response surface methodology (RSM). Microwave-assisted extraction (MAE) has

been used as a potential alternative to conventional solvent extraction for the isolation of

polyphenol compounds from SCG. A complete central composite 2³ factorial experimental design

has been used to monitor the extraction characteristics, as affected by different variables, extraction

time (ET), liquid-to-solid ratio (LSR), and microwave power (MWP). Low concentration ethanol in

aqueous solutions was employed as non-toxic extracting media. With the reduced time of

extraction, low power and medium liquid to solid ratio while using minimal concentration of

ethanol, the polyphenols extract with high antioxidant activity can be achieved. The obtained

experimental values were in solid agreement with predicted values. The FRAP and the DPPH

antioxidative activity showed good correlation with the total polyphenol content (TPC), with high

correlation factors. The presented data could be a reliable guidelines for establishing full-scale,

sustainable cost-effective and resource-effective industrial process.

ACCEPTED MANUSCRIPT

A CCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

7328 words in total

Optimization of microwave-assisted extraction of natural antioxidants from spent espresso coffee grounds by response surface methodology

Marija Ranic a* , Marina Nikolic a , MarijaPavlovic b , AnetaBuntic , Slavica Siler-Marinkovic , Suzana

Dimitrijevic-Brankovic b

b

b

a Institute for Medical Research, Centre of Research Excellence in Nutrition and Metabolism, University of

Belgrade, TadeusaKoscuska 1, PAK 104 201, 11 158 Belgrade, Serbia

b Faculty of Technology and Metallurgy, Division: Biochemical Engineering and Biotechnology, University

of Belgrade, Karnegijeva 4, 11120 Belgrade, Serbia

Marina Nikolic, MSc, marina.nikolic12@yahoo.com

Marija Pavlovic, MSc, marija.pavlovic87.tmf@hotmail.com

Aneta Buntic, MSc, anetabuntic@gmail.com

Slavica Siler-Marinkovic, PhD, siler@tmf.bg.ac.rs

Suzana Dimitrijevic-Brankovic, PhD, suzana@tmf.bg.ac.rs

Keywords: Spent espresso coffee, Microwave-assisted extraction, Response surface methodology, Total

polyphenol content, DPPH, FRAP

Abbreviations: SCG, Spent coffee grounds; SCGE Spent coffee grounds extract; MAE, Microwave-assisted

extraction; RSM, Response surface methodology; TEY, Total extraction yield; TPC, Total polyphenol

content; ET, Extraction time; LSR, Liquid-to-solid ratio; MWP, Microwave power; d.w., dry weight; DPPH,

2,2-diphenyl-1-picrylhydrazyl; FRAP, ferric reducing/antioxidant power.

* Corresponding author: Tel: +381 113281564; fax: +381 112030169.

E-mail address: marija.ranic@gmail.com (Marija Ranic).

ACCEPTED MANUSCRIPT

1. Introduction

Antioxidant activity in foods and beverages is one of the properties that have generated much

interest within the scientific community (Gulcin, 2012). It has to be mentioned that food commodities are usually fortified with additional quantities of antioxidants, either natural, or synthetic due to depletion of antioxidants present in raw food when subjected to processing, preservation and storage.

The synthetic antioxidants have proven to be cheaper and more effective in the retardation process of

oxidative degradation, comparing to the natural ones. The synthetic antioxidants, introduced a few

decades ago, are mostly butylated phenols and polyphenols, such as butylated hydroxyl toluene (BHT),

an effective, widely used and low cost antioxidant (Mukhopadhay, 2006). However, due to safety

concerns, interest in natural antioxidants has intensified (Shahidi, 2000) and synthetic antioxidants are

subjected to evaluating the safety. Many studies were investigated whether BHT possess

mutagenic/genotoxic risk to man (Bomhard et al., 1992). Recently, the EFSA Panel (European Food

Safety Authority) gave the Scientific opinion on the re-evaluation of BHT as a food additive (EFSA, 2012).

Despite contradictory recommendations, BHT it is still in use for food products preservation specially in

developing countries, thanks to cost advantages and availability and it is still very popular antioxidant in

many different fields; 80.000 – 100.000 tons per year is been producing in USA, Western Europe and

Japan (Mukhopadhay, 2006). The superiority of natural antioxidants has to be emphasized vs. synthetic

in terms of tolerance, safety and non-toxicity. In addition to their safety advantages, food phenolics

may augment body’s source of natural antioxidants.

Polyphenol compounds have received substantial attention due to their beneficial effects on

human health, such as a protective action against chronic degenerative diseases (cataracts, macular

degeneration, neurodegenerative diseases, and diabetes mellitus), cancer and cardiovascular diseases,

which is attributed to their antioxidant activity (Mussatto et al., 2011a). Recent studies report

considerable evidence of health benefits for healthy adults as a result of moderate coffee consumption

(Butt and Sultan, 2011). The beneficial effect of coffee consumption is partially attributed to the

antioxidant activity of the polyphenols present in coffee (Vignoli et al., 2011; Cammerer and Kroh, 2006).

Tannins are consider to be the main phenolic compounds in coffee pulp, while in the seed, polyphenol

compounds are present predominantly as a family of esters formed between certain hydroxycinnamic

acids and quinic acid, known as chlorogenic acids (CGA) (Farah and Donangelo, 2006).

Coffee, as the one of the most popular beverages, is consumed by millions of people all over the

world, several times a day. In the coffee industry, during the manufacture of soluble and concentrate

coffee, huge amount of solid residue known as spent coffee grounds (SCG) is produced. It was estimated

that only from the Province of Rome over 10,000t of SCG are available per year for the production of

polyphenols rich extracts and bioenergy, which means that around 1400t/year of polyphenol extract

could be produced (Zuorro at al., 2012). With regard to spent coffee, two types should be

differentiated. The soluble coffee industry bringing together almost 50% of the world coffee production,

with a proportional amount of spent coffee residues. Another 50% is used for preparation of beverages.

Depending on the intended reuse, espresso spent coffee bring out a similar or even greater reuse

potential than the one expected from spent grounds obtained from the soluble coffee industry,

exhausted of most of its soluble components (Cruz at al., 2012). Regardless of the brewing process applied, during the preparation of coffee beverages, extraction efficiency is clearly lower comparing to industrial level. Therefore, the residues, being richer in unextracted coffee components comparing to coffee brew, opening the series of potential applications. That fact leads us to the assumption that spent coffee produced in tons by restaurants, cafeterias and in domestic environment, could be important source of natural antioxidants, also from an economical point of view (Bravo et al., 2012). Adams and

ACCEPTED MANUSCRIPT

Ghaly, 2006 suggested an alternative use of coffee industry by-products, among others, for maximizing the sustainability of the coffee industry, through identification of new opportunities and cost and risk reductions. Waste minimization can also be achieved by turning wastes into value added products of economic value. Several previous studies have dealt with residues generated during the entire coffee processing (Yen et al., 2005; Adams and Ghaly, 2006, Kondamudi et al., 2008; Murthy et al., 2010; Bravo et al., 2013; Mussatto et al., 2011a; Esquivel et al., 2012; Zuorro et al., 2012; Cruz et al., 2012). In

addition, the more recent studies continue to deal with different aspects of possible SCG utilization: oil

extracted from SCG as a substrate for the production of poly (3-hydroxybutyrate) (Obruca et al., 2014),

production of polyhydroxyalkanoates from SCG oil obtained by supercritical fluid extraction technology

(Cruz et al., 2014),) potential feedstock for mannooligosaccharides (Chiyanzu et al., 2014) and mannans

from SCG (Passos et al., 2014), etc.

Extraction is the first step in the isolation of polyphenol compounds from SCG. Different

techniques have been applied to recover antioxidant polyphenol compounds from natural sources

including solid–liquid extraction with organic solvents, ultrasound-assisted extraction (UAE), microwave-

assisted extraction (MAE), supercritical fluid extraction, and high pressure processes (Mussato et al.,

2011). Different extraction techniques have certain limitations in terms of extraction time, energy,

solvent consumption and cost and also influenced by several factors such as the type and concentration

of solvent, the solvent/solid ratio, pH, time, temperature, etc. Comparing to the SCG extracts (SCGE)

obtained with continuous (Soxlet, 1h and 3h) and discontinuous methods (solid-liquid extraction) (Bravo

et al., 2011), MAE has been considered as a potential alternative to conventional solvent extraction for

the isolation of polyphenol compounds from SCG (Upadhyay et al., 2012). So far, it has been used for

extracting a number of components from different sample matrices (Chen and Spiro, 1994; Young, 1995;

Lopez-Avila et al., 1996; Bureau et al. 1996; Kwon al., 2003; Rostagno et al., 2007; Zhanga et al., 2008;

Ballard et al., 2009; Hyun-Ku et al., 2012; Li et al., 2012; Singh et al., 2011). Optimization of the

extraction procedure of plant constituents through the traditional method, i.e., “one variable at-a-time”,

is time consuming and not cost effective, while statistical optimization methods provide insight about

the interactions of the variables when generating the response. In the context of optimization of the

whole system, response surface methodology (RSM) is the most widely used (Bezerra et al., 2008). It

derives a model equation that fits the experimental data and it calculates the optimal response of the

system with a limited number of experiments. The RSM was used so far for optimization of antioxidant

activity of different extracts (Karacabey et al., 2010; Saha et al., 2011; Gupta et al., 2011; Ghafoor et al.,

2011; Singh et al., 2011). In the work of Zuorro et al. 2012, a two-level factorial design was used to

study the effects of temperature, extraction time and liquid-to-solid ratio, and ethanol concentration on

the recovery of phenolic compounds from SCG. They suggest future improvement by subsequent

optimization based, like on RSM.

Over the last few decades, an espresso coffee started to be dominant in the restaurants and

cafeterias. Increase consumption of espresso coffee generates a considerable amount of SCG, as a

waste, that could be reused for industrial scale-up. The overall objective of this study was to identifying

optimal range of extraction conditions and to develop an effective and eco-friendly MAE strategy to

optimize the extraction of polyphenol compounds from samples of spent espresso coffee, using RSM.

The correlation between the antioxidant activities and polyphenol content (Dudonné et al., 2009) was

studied by employing two well-established assays for antioxidant activity, 2, 2-diphenyl-1-picrylhydrazyl

(DPPH . ) decolorization assay and ferric reducing antioxidant power (FRAP) assay. To the best of our knowledge, such an approach has not been reported elsewhere, so far.

ACCEPTED MANUSCRIPT

2.1 Chemicals and reagents

We used „Doncafe - Espresso Aromatico-cialde“ corresponding to Arabica/Robusta blends in

undisclosed exact proportion. All reagents that were used in this work were of analytical grade and were purchased from Sigma Chemical Co. (St. Luis, USA), Aldrich Chemical Co. (Steineheim, Germany), Merck (Darmstadt, Germany) and Sineks laboratory (Serbia). The ethanol (20%) and distilled water were used

as solvents. Total polyphenol content was measured using Folin-Ciocalteau reagent and then calculated

using Gallic Acid (GA) as a standard. The antioxidant capacity of spent coffee extracts was measured

using 2, 2-diphenyl-1-picrylhydrazyl (DPPH . ) decolorization assay. The FRAP method, was performed

using complex ferric ion-TPTZ (2, 4, 6-tri (2-pyridyl) - 1, 3, 5-triazine).

2.2 Spent coffee preparation

A total number of samples „Doncafe - Espresso Aromatico - cialde“ were donated by a local

company Strauss-Adriatic d.o.o. Doncafe Espresso Aromatico is mix obtained from Arabica (below 50%)

and Robusta (above 50%) (supplied from Santos, Guatemala, Columbia, India, Honduras and Vietnam

depending on the current offer on the market, while taking into account similarity in composition).

Espresso coffee was prepared on Italian Frog Espresso Coffee Pod Machine (Power: 450W; Voltage:

230/120V) that use cialda – pre-packaged espresso ground coffee in coffee pods. 30 ml of espresso

coffee brew was obtained from 7g of ground coffee in cialda, within 25s of extraction process at about

90 o C. After brewing, spent coffee grounds from coffee pods, was widespread in a very thin layer, with a

large contact area, thus shortening the duration of the next phase (drying stage). Samples of collected

spent coffee were preserved by oven-drying at 40

during the experiment.

o C (Memmert). The samples were stored at 3-4°C

2.3 Experimental design

The RSM was employed to optimize the MAE of phenolic compounds from SCG samples, and to

determine the optimum extraction characteristic by employing the central composite design (CCD)

(Cochran et al., 1992). In this case, the RSM was used to study the effects of extraction time (ET), liquid -

to-solid ratio, (LSR) and microwave power (MWP) to total polyphenol content (TPC) in the extract. Our

previous work showed that 20% ethanol solution in water is optimal for polyphenol extraction from

spent coffe so this parameter was not considered in this work (Pavlović et al., 2013).

The experimental domain was defined taking into account the results obtained in preliminary

tests, as well as the operational limits of the instrument and all significant parameters in a typical MAE

process were chosen as independent variables: ET (A=11.0-209.0s), LSR (B=4.76-13.24ml/g) and MWP

(C= 240, 400 and 560W). The range and levels of 3 independent variables and code values are

presented in Table 1. The variables A and B have been coded at 5 levels -α, -1, 0, 1, +α while variable C

was coded at 3 levels (-1, 0, 1). The response variable were: total extraction yield (TEY; in mg/g d.w. SCG), total polyphenol content (TPC; % (w/w) in extract), FRAP (mmolFe 2+ /l) and DPPH (% of DPPH inhibition). The complete design consists of 30 experiments that were conducted in triplicate (Table 2). Experimental runs were randomised in order to minimize the effects of unexpected variability in the observed responses. The data from CCD were analysed by multiple regression to fit to a second-order polynomial regression model containing the coefficient of linear, quadratic, and two factor interaction

ACCEPTED MANUSCRIPT

effects. The model equation of response (Y) of three independent variables (A, B and C) is given in the following equation:

Y=β

3

i=1

β

3

i=1

(1)

where Y is the dependent variable, β 0 is the constant coefficient, β

is the quadratic coefficient, and is the two factor interaction coefficients. The model adequacy

was determined by evaluating the lack of fit, coefficient of determination (R

is the linear coefficient (main effect),

2 ), and the Fisher test value

(F-value) obtained by analysis of variance (ANOVA) that was generated by the Design Expert 8 software

(Stat-Ease, Inc., Minneapolis, MN, USA). Differences were considered significant if p<0.05. Optimal

independent variables (extraction conditions) for maximised responses were pre-established by

superimposing the corresponding contour plots, by which two subsequent confirmatory experiments

were carried out to validate their equations.

2.4 Analysis of responses

Total extraction yield (TEY): MAE was performed using a domestic microwave oven (SAMSUNG

CE104VD,1250W). In brief, 3g d.w. SCG was macerated with appropriate volume of 20% (v/v) ethanol in

250ml Erlenmeyer flasks. Extraction with different ET, LSR, and MWP was performed according to the

experimental design (Table 2) in a total number of 30 samples. After extraction, solid phase was

separated using vacuum-pump (BÜCHI Vacuum Pump V-700), and extracts are amended to the initial

volume for further analysis. The TEY of each sample was assessed by plotting the dry matter calibration

curve made by optical density measurement (green filter, 570nm) of different concentration of dry

samples, obtained using the Büchi Mini Spray Dryer B-290 (BüchiLabortechnik AG, Flawil, Switzerland)

after dissolving in 20% (v/v) ethanol concentration, which is the working condition of this unit, using

20% (v/v) ethanol concentration as a blind test.

Total polyphenol content (TPC): TPC in spent espresso coffee extracts was measured using the

Folin-Ciocalteau reagent according to the modified method of Dordjevic et al. 2009. Briefly, water

solution (100µl) of the investigated extracts (adjusted to 1mg d.w. /ml) was shaken for 60s on the Vortex

mixer with 500 L of Folin–Ciocalteu reagent and 6 mL of methanol. After that, 2 mL of 15% Na 2 CO 3 was

added and the mixturewas shaken once again for 30s. Finally, the solution was brought up to 10 mL by

adding distilled water. After 2 h, the absorbance was read on the UV/visible spectrophotometer

(Ultrospec 3300 pro, Amersham Bioscience, Uppsala, Sweden) at 750 nm (25 o C) using glass cuvettes

against a blank (100µL of distilled water instead test samples). The TPC was assessed by plotting the

gallic acid calibration curve (from 1 to 1500 µg/ mL) and the results expressed as gallic acid equivalent

are recalculated as the percent of dry extract.

The antioxidant capacity of DPPH assay: The ability to neutralize DPPH• (2, 2-diphenyl-1-

picrylhydrazyl), stable free radical is a method commonly used to determine the antioxidant activity

(Pisoschi et al., 2011). The free radical scavenging capacities of spent espresso coffee extracts were

determined according to the previously described method (Cuendet et al., 1997). Briefly, the aqueous solutions of the extracts (50μl) with concentrations 1mg d.w. /mL were further dissolved in methanol up to 4ml. After that 1 mL of 0.2mmol DPPH in methanol was added. The obtained solutions were vigorously shaken and left at room temperature in the dark. After 30 min of incubation the absorbance was measured against a blank (methanol) at 517 nm using the UV/visible spectrophotometer (Ultrospec 3300 pro, Amersham Bioscience, Uppsala, Sweden). Inhibition of DPPH radical was calculated as a

percentage (%) using the equation (2):

ACCEPTED MANUSCRIPT

Scavenging activity (%) = [(A control-A sample) /A control] *100

(2)

where, A control is the absorbance of the control reaction (containing all reagents except tested extract), and A sample is the absorbance of the tested extract. Ascorbic acid was used as a positive control and all tests were carried out in triplicates.

Ferric reducing antioxidant power (FRAP): FRAP method is based on the ability of phenolic

substances, soluble in water, to reduce Fe 3+ to Fe 2+ . FRAP assay uses antioxidants as reductants in a

redox-linked colorimetric method. The FRAP method relies on the reduction by the antioxidants, of the

complex ferric ion-TPTZ (2,4,6-tri(2-pyridyl)- 1,3,5-triazine) (Pisoschi et al., 2011). FRAP method

described previously (Szöllösi and Szöllösi, 2002) was used to assess overall antioxidant activity. Water

solution (150 µL) of espresso SCGE was mixed with 4.5 mL of FRAP reagent. In the FRAP method, the

yellow Fe 3+ –TPTZ complex is reduced to the blue Fe

under acidic conditions. Any electron donating substance with a half reaction of lower redox potential

than Fe 3+ /Fe 2+ –TPTZ will drive the reaction and the formation of the blue complex forward. The

absorbance readings started after 5 min at 593 nm using a spectrophotometer (Ultrospec 3300 pro,

Amersham Bioscience, Sweden). The blank probe consisted of FRAP reagent. The final absorbance of

each sample was compared with those obtained from the standard curve made from ferric sulphate

(FeSO 4 · 7H2O) (200, 400, 600, 800, and 1000 µmol/L). The results were expressed in mmol Fe

extract.

2+

–TPTZ complex by electron- donating substances

2+

/g of

2.

Results and discussion

3.1

Selection of the solvent

A constant solvent concentration of 20% (v/v) ethanol was used based on our preliminary

results. The influence of solvent to polyphenols extraction was investigated extensively (Bilek S. E., 2010;

Zhang et al., 2011; Wu et al., 2011). Ethanol as a solvent was chosen based on comparing the phenolic

content obtained from spent espresso using different solvent systems (water < methanol < ethanol

extract) (Milutinovic et al., 2013), confirmed by DPPH and FRAP assays. Further analyses included

phenolics extraction from espresso SCG under MAE conditions, optimised using one factor design

under RSM. The investigated factors, ET and percent of ethanol in water solution ranged from 40s

to 360s and 20% to 80% (v/v), respectively (unpublished results).The yield of TPC reached a

maximum using

20% (v/v) ethanol concentration as it is shown on Fig. 2 (I). Our results are in

agreement with results of Jokić et al., 2010 that are also found that lower percent of ethanol in water

positively affected polyphenols recovery from soybean. Aqueous ethanol is a safe and efficient solvent,

and also bio-solvent that can be generated through fermentation of different raw materials, with

possibilities for recycling, as a basic element for sustainable industrial process (Kiassos et al., 2009).

3.2 Optimization of the total extract yield

The yield of total dry extracts was optimized by using RSM approach. In Table 2 actual and predicted values for total extract yield (TEY) were given. The yield ranged from 7,694 to 31.216 mg/g d.w SCG. The maximum yield was recorded under following experimental conditions: 180s ET, 12 ml/g LSR, and 550W MWP (run 24). The lowest result is recorded at following conditions: 40s extraction

ACCEPTED MANUSCRIPT

time, 6 ml/g LSR, and 400W MWP (run 1). According to analysis from present study an increase of ET (A), LSR (B) and MWP (C) result in an increase of TEY.

By applying a multiple regression analysis of the experimental data, the response variable

related to the second-order polynomial equation is given in Table 3. In Table 4 analysis of variance (ANOVA) for the regression equation is shown. The linear term and quadratic term were very significant

(p<0.01). The most significant interaction between independent variables is between A and C. The value

of lack of fit (0,0790) verifies the adequacy of the proposed model indicating that the model fits the

experimental data adequately (p>0.05). The relationship between the experimental and predicted

values showed that the plotted points cluster around the diagonal line in Fig. 1 (A), indicating good

fitness with a low dispersion, because the value of predicted R-squared of 0.9586 is in satisfactory

agreement with adjusted R-squared of 0.9402. Rather low value of coefficient of variance C.V.% of 7,304

indicated a high degree of precision and reliability of the experimental values. Fig. 2 (A) shows the three-

dimensional response surface plots of the TEY as affected by A (ET) and C (MWP), indicated as significant

with a p value of 0.0002. An increase of ET (160-180s) with higher values of MWP (480-550W) results in

an increase of TEY to a maximum at a certain level. Verification experiments (A and B in Table 2), carried

out in the predicted conditions, showed values reasonably close to predicted ones and further

confirmed the adequacy of predicted models.

3.3 Optimization of the total polyphenol content

Present experimental and predicted data for TPC are given in Table 2 expressed as percent (%,

w/w) in dry SCGE. The yield ranged from 18.83 up to 79.83%w/w. The maximum yield was recorded in

sample run 16 under following experimental conditions: 40s extraction time, 240 W MWP and 6 fold

solvent to SCG ratio. The response variable related to the second-order polynomial equation is given in

Table 3. In Table 4, analysis of variance (ANOVA) for the regression equation is shown. The linear and

quadratic term were very significant (p<0.01). The most significant interaction between independent

variables is between A and C, and B and C. The value of lack of fit (0,0679) indicating that the model fits

the experimental data adequately (p>0.05). The relationship between the experimental and predicted

values showed that the plotted points group around the diagonal line as it is shown at Fig. 1 (B),

indicating good fitness with a low dispersion, the value of predicted R-squared of 0.845 and adjusted R-

squared of 0.892. Coefficient of variance C.V.% of 13.667 indicated a satisfactory degree of precision and

reliability of the experimental values. Fig. 2 (B) shows the three-dimensional response surface plots of

the TPC as affected by A and C indicated as significant with a p value of 0.0007, and B and C, Fig. 2 (C),

with a p value of 0.0013. The results suggest that shorter ET (40-110s) and lower range of MWP lead to

higher TPC (plot B). The LSR lower than 6mg/g with the use of 240-400W MWP also results in higher TPC

(plot C).

Similar results regarding to ET and MWP can be found in a work of Zhang et al., 2012, where 35s

of ET were enough to reach the maximal yield of polyphenols from Lotus seeds with MWP of 400W.

Further increasing of these two factors leads to decreasing of polyphenols yields in Lotus seeds extracts.

Somporn et al., 2011, showed that temperature applied to roasting process can influence the TPC in coffee extracts and that higher temperature and longer time of roasting decrease the TPC in extracts. The influence of LSR is a little more difficult to compare with other authors due to the fact that we investigated the lower level of LSR in a narrow range, from 4.76 to 13.24 ml/g SCG. Some investigations showed that the TPC total yield is higher with the higher LSR. For example, Mussatto et al., 2011a showed that 25 LSR of methanol solution (in range of 10-50) was optimal for polyphenols recovery from spent coffee grounds. Zuorro and Lavecchia, 2012 are concluded that the increasing of LSR (in range of

ACCEPTED MANUSCRIPT

10-50, ml/g) lead to higher TPC in spent coffee grounds extracts. However, Franco et al, 2008 showed that phenolic recovery from grape pomace was higher with the lowest LSR (in range of 1-5, ml/g of ethanol solution). In addition, Mukhopadhyay et al., 2006 showed that a higher portion of solids in the black cohosh extraction process (ranged from 0-200 S/L, mg/ml) positively influence the TPC in extract.

Comparing our results of TPC with the results of total extraction yield - TEY (total solids of

extracts) it is clear that the TPC is in inverse proportion to TEY, as it is shown in Fig. 3 (A). The value of

correlation factor is -0.747, negative slope. Experimental conditions that are optimal for the maximum

of total extraction yield (longer ET, higher LSR and higher MWP) are not optimal for the maximum

content of TPC (shorter ET, lower LSR and lower MWP). A possible explanation for this outcome could be

that the extracts with higher values of dry matter also content an increase level of other (ballast)

substances such as polysaccharides, proteins and lipids, thus reducing the proportion of TPC (Mussatto

et al., 2011b).The similar results can be noticed at work of Wu et al., 2011 when recalculations of TPC

contribution in total yield were carried out.

3.4 Optimum extraction conditions based on the DPPH method

Experimental and predicted data for antioxidative activity of SCGE determined by the DPPH

method are given in Table 2. The antioxidative activity ranged from 36,560%, in run 6 up to 98,240% in

run 28, under experimental conditions of 11s ET, 9 fold solvent to SCG ratio and 240 W, MWP. The

response variable related to the second-order polynomial equation is given in Table 3. In Table 4,

analysis of variance (ANOVA) for the regression equation is shown. The used model is significant

(p<0.01). The most significant interaction between independent variables is between A and C. The value

of lack of fit (0,1377) indicating that the model fits the experimental data adequately (p>0.05). The

relationship between the experimental and predicted values showed that the plotted points cluster

around the diagonal line (Fig. 1 (C), indicating good fitness with a low dispersion, with the value of

predicted R-squared of 0.9734 and adjusted R-squared of 0.9805, R 2 is 0.9845. Coefficient of variance

C.V.% of 3.711 indicated a very high degree of precision and reliability of the experimental values.

Fig. 2 (D) shows the three-dimensional response surface plots of the DPPH as affected by A and

C

indicated as significant with a p value <0.001. Shorter MAE time (40-120s) with the use of lower range

of

MWP (240-400W) results in higher % in neutralization of DPPH radicals.These results are in direct

correlation with TPC (correlation factor 0,841) and in inverse correlation to TEY (correlation factor -

0.786), as it is shown in Fig. 3 (A) and (B),resp. It should be noticed that the samples in our investigations

were adjusted to a concentration of 1 mg/ml of extracts, so these findings also confirm the influence of

investigated factors on TPC proportions in total solids of SCGE. The other researcher also noticed

positive correlation of TPC and DPPH inhibition activity (Franco et al., 2008, Wang et al., 2011).

3.5 Optimum extraction conditions based on the FRAP activity

Experimental and predicted data for antioxidative capacity of SCGE determined by FRAP method

are given in Table 2. The antioxidative activity ranged from 2.620mmol Fe 2+ /l , run 12, up to 6.660mmol

Fe 2+ /l in run 28, under experimental conditions of 11s ET, 9 fold solvent to SCG ratio and 240W MWP.

The response variable related to the second-order polynomial equation is given in Table 3. In Table 4, analysis of variance (ANOVA) for the regression equation is shown. The used model is significant (p<0.01), with all significant parameters. The most significant interaction between independent variables is between A and C. The value of lack of fit (0,4664) indicating that the model fits the

ACCEPTED MANUSCRIPT

experimental data adequately (p>0.05). The relationship between the experimental and predicted

values showed that the plotted points cluster around the diagonal line (Fig. 1 (D), indicating good fitness with a low dispersion. The squared correlation coefficient R 2 of the model was 0.9433, which confirms that the model can adequately represent the true relationship between the parameter chosen. Coefficient of variance C.V.% of 8.324 indicated a high degree of precision and reliability of the experimental values. FRAP value as affected by relation of A and C indicated as significant with a p value

<0.0001. As evident in Fig. 2 (E) shorter MAE time (40-110s) with the use of the lower range of MWP

(240-300W) are more effective in extracting antioxidative polyphenols from espresso SCG using MAE.

This result is favorable since using of higher MWP with extended ET may lead to thermal degradation of

the polyphenol compounds (Chen et al., 2007).

It can be noticed that FRAP values are also in direct correlation with TCP (correlation factor

0,859), Fig. 3 (D) and in inverse proportion to TEY (correlation factor -0.770), as it is shown in Fig. 3 (E).In

general, the relationship of FRAP was the similar to what was observed in the DPPH assay. Correlation

between DPPH and FRAP values (Fig. 3 (F) shows that samples with high antioxidative activity

determined by DPPH method, also have high FRAP values. The highest FRAP value in the range from 6.0

to 7.0 mmol Fe 2+ /l, and the highest antioxidant capacity determined by DPPH (70% to 100%) correspond

to TPC range from 65% and 80%of dry SCGE.9 fold solvent to SCG ratio, shorter MAE time (40-110s), and

a lower range of (240-400W) gives the optimal DPPH and FRAP values. The minor deviations could be

the result of different solubility of polyphenols showing antioxidative properties in different solvents

(methanol for DPPH and water for FRAP method).

The results presented in this study show that used models are efficient to predict the outcome

variables (TEY, TPC, DPPH and FRAP) of espresso SCG using the independent variables (ET, LSR, MWP).

The results obtained from all models are highly correlated, which means that each model can be used to

predict the outcome based on listed independent variables. Validation of the models showed no

statistically significant differences between the results obtained by the experiment and those predicted

by the models.

Overall, espresso SCG could be an inexpensive source of natural antioxidants. Extracts obtain

form espresso SCG in this study had high TPC contents (up to 79.83%) of dry matter obtained under

optimal extraction conditions. In this regards the SCG can be compared to other sources that were used

for the same purpose, such as kinema – fermented soybean food with 135 mg GAE/g d.w. (Saha et al.,

2011); potato peels, 3.94 mg/g d.w. (Singh et al., 2011); grape (Vitis vinifera) seeds, 5.44mg GAE/100mL

(Ghafoor et al., 2009); tomato, 4.89-9.97mg GAE/g d.w. (Li et al., 2012); or onion (Allium cepa) solid

waste, which is theoretically predicted to have 93.42±14.35mg GAE/g d.w., under optimal conditions

(Kiassos et al., 2009).

Many companies from food and cosmetic industry used MAE for plant nutrient extraction. It is

worth to mention CRODAROM and CODIF companies that use microwaves for extraction of antioxidant

and colours of plants and marine algae, respectively (Ying Li et al., 2013). The industrial scale microwave

equipment is still developing especially for continuous processes (Radoiu, 2013 ). Mandal et al., 2007.,

gives a detailed review of

process optimisation for the maximum yield. Kumar Das and coworker, 2013, compared various method for extraction of lupeol from Ficus racemosa leaves. They concluded that the MAE is more rapid, convenient and appropriate extraction method, with a higher yield and lower solvent consumption when compared with conventional extraction techniques. They also concluded that the response surface methodology can be applied to all natural products and if explored properly, can prove to be efficient for large-scale industrial application.

MAE for medicinal plant extraction and point out the significance of MAE

ACCEPTED MANUSCRIPT

Our results also suggested that MAE can be an efficient technique for waste coffee polyphenolic

extraction. With the short time of extraction, low power and medium liquid to solid ratio as well as with minimal concentration of ethanol the polyphenol extract with high antioxidant activity can be obtained. The SCG is widely available from restaurants with no-costs. It gives great importance to the economic feasibility of its use. In addition, an environmental standard promotes proper, responsible and safe management of waste and successful waste prevention that not only benefits the environment, but also

has the potential to eliminate certain amount in annual disposal costs. (Nelson and Pound, 2009).

Starbucks Corporation, an American global coffee company and coffee house chain, possess 20,891

Starbucks stores, as of March 2013 (Loxcel, 2013) in 61 countries and territories. Starbucks used to make

spent espresso/coffee grounds available for free. Costa Coffee, a British multinational coffee house

company, internationally operates to over 1,700 stores across 29 countries (Costa Coffee, 2013). In

2009, 1.6 billion K-cups were sold, mostly fulfilled with different kind of espresso coffee (Keurig, 2013).

It is worth to mention an ecological device for quick removal of the spent espresso coffee grounds (EP

1803380 A2). This device allows the user to remove, with little effort, in just one quick stage the used

coffee grounds from the metallic filter, housed in the portafilter of an espresso machine, after the

preparation of a coffee beverage (Campos, 2012). The foregoing facts contribute to Waste-to-Product

(WtP) novel concept (Bosmans, 2013), in a sustainable context. Turning waste into value added

products, ie. proposed cyclic reuse of coffee waste is shown in Fig. 4.

4. Conclusions

The sustainability of the coffee processing system can be substantially improved through the use

of by-products, by adoption of new technologies that maximize process profitability. The espresso

coffee grounds are non-cost by product of coffee consumption. It contains a great amount of valuable

phenolic and other components with high antioxidative properties. In our study, MAE along with RSM

proved to be effective in estimating the effects of three independent variables (ET, LSR and MWP) on

the phenolics extraction from espresso SCG. We were able to extract high level of phenolics from SCG,

using less solvent with considerably shorter extraction time than previously reported. Although a

detailed cost-benefit analysis is needed to assess the economic practicability of the proposed approach,

the analysis and the results reported in this study could be of great significance for future exploitation of

espresso SCG as a valuable source of natural antioxidant in industrial scale-up. All presented facts could

be a reliable guidelines for development of a full-scale project and good business opportunities for

SMEs, producing fortified foods or dietary supplements with pharmacological doses and safety

assessments of the applied doses performed (Mennen et al., 2005).

Acknowledgments

This work is funded by the Ministry of Education, Science and Technological Development, Republic of

Serbia, Projects TR31035 and III 41030. Language help and proof reading the article provided by Romana Novakovic, PhD is greatly appreciated. The authors would also like to thank the Strauss Adriatic d.o.o,

Šimanovci, Serbia for providing materials for investigation.

ACCEPTED MANUSCRIPT

References

Adams, M., Ghaly, , A.E., 2007. Maximizing sustainability of the Costa Rican coffee industry. Journal of Cleaner Production, 15, 1716-1729.

Ballard, T.S.,Mallikarjunan, P., Zhou, K.,O’Keefe, S.F., 2009. Optimizing the extraction of phenolic

antioxidants from peanut skins using response surface methodology. Journal of Agriculture and

Food Science, 57, 3064-3072.

Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., Escaleira, L. A., 2008. Response surface

methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 76 (5), 965-977.

Bilek, S.E., 2010. The effects of time, temperature, solvent: solid ratio and solvent composition on

extraction of total phenolic compound from dried olive (Oleaeuropaea L.) leaves. GIDA, 35, 6,

411-416.

Bomhard, E.M,, Bremmer, J.N., Herbold, B.A., 1992. Review of the mutagenicity/genotoxicity of

butylated hydroxytoluene. Mutation Research, 277, 187-200.

Bosmans , A., Vanderreydt , I., Geysen, D., Helsen, L. , 2013. The crucial role of Waste-to-Energy

technologies in enhanced landfill mining: a technology review. Journal of Cleaner Production,

55, 10-23.

Bravo, J., Juániz, I., Monente, C., Caemmerer, B., Kroh, L. W., De Peña, M. P., Cid, C. , 2012. Evaluation of

Spent Coffee Obtained from the Most Common Coffeemakers as a Source of Hydrophilic

Bioactive Compounds. Journal of Agricultural and Food Chemistry, 60 (51), 12565-12573.

Bravo, J., Monente, C., Juániz, I., De Peña, M. P., Cid, C., 2013. Influence of extraction process on

antioxidant capacity of spent coffee. Food Research International, 50 (2), 610-616.

Bureau, S., Razungles, A., Baumes, R., Bayonove, C. , 1996. Glycosylated flavour precursor extraction by

microwave from grape juice and grapes. Journal of Food Science, 61, 557–561.

Butt, M.S., Sultan, M.T., 2011. Coffee and its consumption: benefits and risks. Crit Rev Food Sci Nutr, 51,

4,363-73.

Cammerer, B., Kroh, L.W. , 2006. Antioxidant activity of coffee brews. European Food Research and

Technology, 223, 469–474.

Campos, D.A., 2012. Ecological device for quick removal and without knocking impact of the spent

espresso coffee grounds, EP 1803380 A3

(http://www.google.com/patents/EP1803380A2?hl=sr&cl=en).

Chen, S.S., Spiro, M., 1994.Study of microwave process of essential oil constituents from plant materials. The Journal of Microwave Power and Electromagnetic Energy, 29, 231–241.

Chen, Y., Xie, M.-Y., Gong, X.-F., 2007. Microwave-assisted extraction used for the isolation of total triterpenoid saponins from Ganoderma atrum. Journal of Food Engineering, 81 (1), 162–170.

ACCEPTED MANUSCRIPT

Chiyanzu, I., Brienzo, M., García-Aparicio, M.P., Görgens, J.F., 2014. Application of Endo-β-1,4,D- mannanase and Cellulase for the Release of Mannooligosaccharides from Steam-Pretreated Spent Coffee Ground. Appl Biochem Biotechnol, 172, 7, 3538-57.

Cochran, W. G., Cox, G. M., 1992. Some Methods for the Study of Response Surfaces. In Experimental Designs; John Wiley and Sons, Inc., the United States of America, 335-375.

Costa Coffee, 2013. (http://www.costacoffee.com/)

Cruz, M.V., Paiva, A., Lisboa, P., Freitas, F., Alves, V.D., Simões, P., Barreiros, S., Reis, M.A., 2014.

Production of polyhydroxyalkanoates from spent coffee grounds oil obtained by supercritical

fluid extraction technology. Bioresour Technol, 157, 360-3.

Cuendet, M., Hostettmann, K., Potterat, O., 1997. Iridoid glucosides with free radical scavenging

properties from Fagraea blume. Helvetica Chimica Acta, 80, 1144–1152.

Das, A.K., Mandal, V., Mandal,S.C., 2013. Design of Experiment Approach for the Process Optimisation of

Microwave Assisted Extraction of Lupeol from Ficus racemosa Leaves Using Response Surface

Methodology. Phytochem. Anal., 24, 230–247.

Dordevic,T.M., Siler-Marinkovic, S.S., Dimitrijevic-Brankovic, S.I. , 2009. Effect of fermentation on

antioxidant properties of some cereals and pseudo cereals, Food Chemistry, 119, 957–963.

Dudonné, S., Vitrac, X., Coutière, P., Woillez, M., Mérillon, JM., 2009. Comparative Study of Antioxidant

Properties and Total Phenolic Content of 30 Plant Extracts of Industrial Interest Using DPPH,

ABTS, FRAP, SOD, and ORAC Assays. Journal of Agricultural and Food Chemistry, 57 (5), 1768-

1774.

EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), 2012. Scientific Opinion on the

re evaluation of butylated hydroxytoluene BHT (E 321) as a food additive. EFSA Journal, 10, 3,

2588.

Esquivel, P., Jiménez, V.M. , 2012. Functional properties of coffee and coffee by-products. Food

Research International, 46, 488-495.

Farah, A., Donangelo, C.M., 2006. Phenolic compounds in coffee. Braz. J. Plant Physiol. 18, 1.

Franco, D., Sineiro, J., Rubilar, M., Sánchez, M., Jerez, M., Pinelo, M., Costoya, N., Núñez,M.J.,

2008.Polyphenols from plant materials: extraction and antioxidant power. EJEAFChe, 7, 8, 3210-

3216.

Ghafoor, K., Choi, Y. H., Jeon, J. Y., Jo, I. H., 2009. Optimization of Ultrasound-Assisted Extraction of

Phenolic Compounds, Antioxidants, and Anthocyanins from Grape (Vitisvinifera) Seeds. Journal

of Agricultural and Food Chemistry, 57 (11), 4988-4994.

Gulcin, I., 2012. Antioxidant activity of food constituents: an overview. Archives of Toxicology, 86,345–

391.

Gupta, S., Banerjee, R., 2011. Radical scavenging potential of phenolics from Bryophyllumpinnatum (LAM.) OKEN. Prep BiochemBiotechnol, 41 (3), 305-19.

ACCEPTED MANUSCRIPT

Jokić, S., Velić, D., Bilić, M., Bucić-Kojić,A., Planinić, M.,Tomas, S., 2010. Modelling of the Process of Solid-Liquid Extraction of Total Polyphenols from Soybeans, Czech J. Food Sci. 28, 3, 206–212.

Keurig, 2013. (http://www.keurig.com/)

Kiassos, E., Mylonaki, S., Makris, D. P., Kefalas, P., 2009. Implementation of response surface

methodology to optimise extraction of onion (Allium cepa) solid waste phenolics. Innovative

Food Science and Emerging Technologies, 10 (2), 246-252.

Kwon, J.H., Belanger, J.M.R. Pare, J.R.J. , 2003. Optimization of microwave assisted extraction (MAP) for

ginseng components by response surface methodology. Journal of Agriculture and Food Science,

51, 1807-1810.

Li, H., Deng, Z., Wu, T., Liu, R., Loewen, S., Tsao, R., 2012. Microwave-assisted extraction of phenolics

with maximal antioxidant activities in tomatoes. Food Chemistry, 130 (4), 928-936.

Li, Y., Fabiano-Tixier, AS., Abert-Vian, M., Chemat, F., 2013. Microwave-assisted Extraction of

Antioxidants and Food Colors, in Microwave-assisted Extraction for Bioactive Compounds: Theory

and Practice, edited by Farid Chémat, Giancarlo Cravotto, Springer, pp. 103-126.

Lopez-Avila, V., Young ,R., Teplitsky, N.Y.R. , 1996. Microwave-assisted process as an alternative to

Soxhlet, sonication and supercritical fluid process. J AOAC Int, 79, 142–156.

Loxcel, 2013. (http://www.loxcel.com/sbux-faq.html)

Lu,J., Zhou, C., Rong, O., Xu,Y., Zhou, B., and Zhonghai, L., 2013.Optimization of Microwave-assisted

Extraction of Flavonoids from Cryptotaenia japonica Hassk using Response Surface

Methodology. Advance Journal of Food Science and Technology 5, 3, 310-317.

Mandal, V., Mohan, Y., Hemalatha, S., 2007.Microwave Assisted Extraction – An Innovative and

Promising Extraction Tool for Medicinal Plant Research. Pharmacognosy Reviews, 1, 1, 7-18.

Mennen, L.I., Walker, R., Bennetau-Pelissero, C., Scalbert, A., 2005. Risks and safety of polyphenol

consumption. Am J Clin Nutr, 81(suppl), 326S–9S.

Milutinović,M.D., Šiler-Marinković, S.S., Antonović, D.G., Mihajlovska, K.R., Pavlović, M.D., Dimitrijević-

Branković, S.I., 2013. The antioxidant properties of dried extracts from the spent espresso

coffee. Chemical Industry, 67, 2, 261-267.

Mukhopadhyay, A.K., 2006. Antioxidants – Natural and Synhtetics, Armani International Publishers, Kiel.

Mukhopadhyay, S.,Luthri, D.L., Robbins, R.J., 2006. Optimization of extraction process for phenolic acids

from black cohosh (Cimicifugaracemosa) by pressurized liquid extraction. J Sci Food Agric, 86,

156–162.

Mussatto, S. I., Ballesteros, L.F., Silvia, M., Teixeira, J. A ., 2011a. Extraction of antioxidant phenolic compounds from spent coffee grounds. Separation and Purification Technology, 83, 173-179.

ACCEPTED MANUSCRIPT

Mussatto, S., Carneiro, L.M., Silva, J.P.A., Roberto, I.C., Teixeira, J.A., 2011b. A study on chemical constituents and sugars extraction from spent coffee grounds. Carbohydrate Polymers, 83,2,

368-374.

Nelson, V. Pound,B., 2009. The Last Ten Years: A Comprehensive Review of the Literature on the Impact of Fairtrade Natural Resources Institute (NRI), University of Greenwich

(http://www.fairtrade.org.uk/resources/natural_resources_institute.aspx)

Obruca, S., Petrik, S., Benesova, P., Svoboda, Z., Eremka, L., Marova, I., 2014. Utilization of oil extracted

from spent coffee grounds for sustainable production of polyhydroxyalkanoates. Appl Microbiol

Biotechnol, [Epub ahead of print].

Passos, C.P., Moreira, A.S., Domingues, M.R., Evtuguin, D.V., Coimbra, M.A., 2014. Sequential microwave

superheated water extraction of mannans from spent coffee grounds. Carbohydr Polym, 103,

333-8.

Pavlović MD, Buntić AV, Šiler-Marinković SS, Dimitrijević-Branković SI, 2013. Ethanol influenced fast

microwave-assisted extraction for natural antioxidants obtaining from spent filter coffee.

Separation and Purification Technology 118, 503-510.

Pisoschi, A.M.,and Negulescu, G.P., 2011. Methods for Total Antioxidant Activity Determination: A

Review. Biochemistry and Analytical Biochemistry, 1,106.

Radoiu, M. The scale-up of microwave-assisted extraction, International Congress on Green Extraction of

Natural Products - GENP Avignon, France, April 16 – 17, 2013, book of abstract, pp. 119.

Rostagno, M. A., Palma, M., Barroso, C. G., 2007. Microwave assisted extraction of soyisoflavones.

AnalyticaChimicaActa, 588, 274–282.

Saha, J., Biswas, A., Chhetri, A., Sarkar, P.K., 2011. Response surface optimization of antioxidant

extraction from kinema a Bacillus-fermented soybean food. Food Chemistry, 129, 507-513.

Shahidi,F., 2000. Antioxidants in food and food antioxidants. Nahrung, 44, 3, 158-63.

Singh, A., Sabally, K., Kubow, S., Donnelly, D. J., Gariepy, Y., Orsat, V., Raghavan, G. S. V., 2011.

Microwave-Assisted Extraction of Phenolic Antioxidants from Potato Peels. Molecules, 16 (3),

2218-2232.

Somporn, C., Kamtuo, A., Theerakulpisut, P., Siriamornpun,S., 2011. Effects of roasting degree on radical

scavenging activity, phenolics and volatile compounds of Arabica coffee beans (Coffeaarabica L.

cv. Catimor), International Journal of Food Science and Technology, 46, 2287–2296.

Szöllösi, R., Szölloösi Varga, I., 2002. Total antioxidant power in some species of Labiate (adaptation of

FRAP method). Acta Biologica Szegediensis, 46, 125–127.

Upadhyay, R., K. R., Mohan Rao, L.J., 2012. Microwave-assisted extraction of chlorogenic acids from green coffee beans.Food Chemistry, 130, 188.

Vignoli, J. A., Bassoli, D. G., Benassi, M. T., 2011. Antioxidant activity, polyphenols, caffeine and melanoidins in soluble coffee: The influence of processing conditions and raw material. Food Chemistry, 124, 863–868.

ACCEPTED MANUSCRIPT

Wang, Z., Pan, Z., Ma, H., Atungulu, G.G., 2011. Extract of Phenolics From Pomegranate Peels. The Open Food Science Journal, 5, 17-25.

Wu,X., Yu, X., Jing, H., 2011. Optimization of Phenolic Antioxidant Extraction from Wuweizi (Schisandrachinensis) Pulp Using Random-Centroid Optimazation Methodology.Int. J. Mol. Sci. 12, 6255-6266.

Young, J.C., 1995. Microwave-assisted process of the fungal metabolite ergosterol and total fatty acids.

Journal of Agricultural Food Chemistry, 43, 2904–2910.

Zhang, Y., Zheng, B., Tian, Y., Huang, S., 2012. Microwave-assisted Extraction and Anti-oxidation Activity

of Polyphenols from Lotus (NelumbonuciferaGaertn.)Seeds.Food Sci. Biotechnol. 21,6, 1577-

1584.

Zhang,L., Wang, Y., Wu, D., Xu, M., Chen, J., 2011. Microwave-Assisted Extraction of Polyphenols from

Camellia oleifera Fruit Hull. Molecules, 16, 4428-4437.

Zhanga, B., Yang, R., Liua, C. Z., 2008. Microwave-assisted extraction of chlorogenic acid from flower

buds of Lonicera japonica Thunb. Separation and Purification Technology, 62, 480–483.

Zuorro, A., Lavecchia, R., 2012. Spent coffee grounds as a valuable source of phenolic compounds and

bioenergy. Journal of Cleaner Production, 34, 49-56.

ACCEPTED MANUSCRIPT

Figure captions

Fig. 1.

(C) and FRAP (D)

The relationship between the experimental and predicted values for TEY (A), TPC (B), DPPH

Fig. 2.

and LSR and MWP (C) to TPC;

Response surface plots showing the combined effects of ethanol and ET (I), ET and MWP (B)

ET and MWP to TEY (A); ET and MWP to DPPH (D); ET and MWP to

FRAP (E)

Fig. 3.

Different bivariate plots: TEY vs. TPC (A), DPPH vs. TPC (B), DPPH vs. TEY (C), FRAP vs. TPC

(D), FRAP vs. TEY (E) and FRAP vs. DPPH (F)

Fig. 4. Proposed model for cyclic reuse of coffee waste

ACCEPTED MANUSCRIPT

Table 1.

optimization of SCG extraction

Independent variables and their levels employed in a Central Composite Rotatable Design for

Independent variable

Unit

Label

 

Coded levels

 

-1

0

1

Time of extraction

s

A

11.0

40.0

110

180.0

209.0

Liquid-to-solid ratio

ml/g

B

4.76

6.00

9

12.0

13.24

Microwave power

W

C

240

400

560

ACCEPTED MANUSCRIPT

Table 2.

conditions of extraction time (ET), liquid-to-solid-ratio (LSR), and microwave power (MWP) based on central composite design for response surface

Experimental data on total extract yield (TEY), total polyphenol content, (TPC), DPPH, and FRAP of spent espresso coffee extracts under different

analysis

 

Independent variables

 

Response variables

 

Exp.

ET (s)

LSR (ml/g)

MWP (W)

TEY (mg/g d.w. SCG)

TPC (% in dry extract)

DPPH (%)

FRAP (mmol Fe 2+/ l)

No.

 

Actual

Predicted

Actual

Predicted

Actual

Predicted

Actual

Predicted

1

40,00 (-1) a

6,00 (-1)

400 (0)

7,694 b

9,357

55.918

49.631

81,520

77,860

4,440

4,723

2

110,00 (0)

4,76 (- )

550 (1)

17,245

16,970

29.423

29.763

51,870

51,784

3,940

4,282

3

40,00 (-1)

12,00 (1)

240 (-1)

13,840

15,396

66.100

64.563

91,350

92,584

6,260

6,142

4

180,00 (1)

6,00 (-1)

400 (0)

22,064

20,344

19.055

24.547

45,250

47,786

3,540

3,173

5

110,00 (0)

9,00 (0)

240 (-1)

15,269

15,429

69.372

59.380

80,600

78,097

5,910

5,498

6

208,99 (+ )

9,00 (0)

550 (1)

27,387

29,221

31.100

30.082

36,560

35,088

2,750

2,751

7

40,00 (-1)

12,00 (1)

400 (0)

16,031

17,917

46.286

42.368

77,870

77,899

3,860

3,947

8

11,01 (- )

9,00 (0)

550 (1)

17,541

18,115

54.782

50.051

70,760

69,322

3,670

3,370

9

110,00 (0)

4,76 (- )

400 (0)

12,713

12,755

30.464

34.522

64,050

62,618

4,870

4,445

10

110,00 (0)

9,00 (0)

400 (0)

15,870

16,279

36.327

37.528

64,520

66,541

3,560

3,896

11

110,00 (0)

9,00 (0)

550 (1)

22,195

20,494

37.555

40.067

54,240

55,707

4,170

3,733

12

208,99 (+ )

9,00 (0)

240(-1)

28,053

28,736

29.100

33.373

51,380

48,904

2,620

2,890

13

11,01 (- )

9,00 (0)

400 (0)

12,433

11,684

48.372

55.266

85,860

84,305

4,240

4,320

14

208,99 (+ )

9,00 (0)

400 (0)

26,768

27,222

19.055

19.791

39,070

41,773

2,630

2,127

15

180,00 (1)

12,00 (1)

400 (0)

30,620

28,904

18.827

17.283

45,320

47,824

2,680

2,396

16

40,00 (-1)

6,00 (-1)

240 (-1)

8,408

6,836

79.827

82.833

91,630

92,545

6,660

6,919

17

40,00 (-1)

12,00 (1)

550 (1)

25,741

23,699

41.100

44.584

61,290

64,131

3,430

3,227

18

180,00 (1)

6,00 (-1)

240 (-1)

21,534

21,165

39.010

46.054

54,320

56,213

3,850

4,181

19

110,00 (0)

13,24 (+ )

400 (0)

26,678

24,860

26.464

24.250

59,140

62,673

2,990

3,347

20

180,00 (1)

6,00 (-1)

550 (1)

23,109

22,992

33.691

27.407

42,110

39,885

3,440

3,566

21

110,00 (0)

9,00 (0)

400 (0)

14,729

16,279

41.945

37.528

67,390

66,541

3,460

3,896

22

110,00 (0)

13,24 (+ )

550 (1)

28,270

29,076

36.918

34.085

54,090

51,839

3,130

3,184

23

40,00 (-1)

6,00 (-1)

550 (1)

13,823

15,139

39.827

41.528

63,470

64,093

4,040

4,004

24

180,00 (1)

12,00 (1)

550 (1)

31,216

31,552

27.055

30.463

41,770

39,924

2,630

2,790

ACCEPTED MANUSCRIPT

25 110,00 (0)

9,00 (0)

550 (1)

21,228

20,494

36.645

40.067

54,330

55,707

3,440

3,733

26 110,00 (0)

13,24 (+ )

240 (-1)

22,578

24,011

31.736

38.318

78,100

74,229

5,290

4,949

27 110,00 (0)

9,00 (0)

240 (-1)

15,112

15,429

68.645

59.380

79,610

78,097

5,860

5,498

28 11,01 (- )

9,00 (0)

240 (-1)

10,451

8,471

76.464

85.386

98,240

100,286

6,660

6,761

29 180,00 (1)

12,00 (1)

240 (-1)

30,581

29,725

29.418

27.784

56,710

56,251

2,940

3,405

30 110,00 (0)

4,76 (- )

240 (-1)

11,277

11,905

71.555

64.156

72,260

74,174

6,240

6,047

 

Data for validation

 

A 40

6,78

240

7,876*

7,694

76.941*

79.641

92,963*

92,593

6,647*

6,775

B 40

8

240

8.259*

8.565

82.453*

80.362

93.912*

94.289

6,782*

6.660

a Numbers in parentheses are coded symbols for levels of independent parameters as per text

b Min and max values are given in bold

*Not significant

ACCEPTED MANUSCRIPT

Table 3.

Second-order polynomial equations calculated for extraction conditions of spent espresso

coffee

Response variable

Second-order polynomial equation 1

Total extract yield

Y TEY = 16,195 + 5,546*A + 4,280*B + 2,533*C – 1,619*A*C + 1,587*A 2 +

 

1,264*B 2 + 1,766*C 2

TPC

Y TPC =37.83 – 12.72*A – 3.80*B – 9.66*C + 5.66*A*C + 5.33*B*C – 4.07*B 2

11.90*C 2

DPPH

Y DPPH =66,902 – 15,135*A + 0,019*B – 11,195*C + 3,031*A*C - 1,751A

1,948*B 2

2

FRAP

Y FRAP =66,902 – 15,135*A + 0,019*B – 11,195*C + 3,031*A*C - 1,751A

2

1,948*B 2

1 A, extraction time (s), B, Liquid-to-solid-ratio (ml/g), C, microwave power (W)

ACCEPTED MANUSCRIPT

Table 4.

Regression coefficients and ANOVA results

Source

Total extract

TPC

DPPH

FRAP

yield

 

P value Prob>F

Model

< 0.0001

<0.0001

< 0.0001

< 0.0001

A

< 0.0001

< .0001

< 0.0001

< 0.0001

B

< 0.0001

0.0040

0.9684

< 0.0001

C

< 0.0001

<0.0001

< 0.0001

< 0.0001

AC

0.0002

0.0007

< 0.0001

< 0.0001

BC

0.0013

A

2

0.0005

0.0117

0.0005

B

2

0.0036

0.0087

0.0057

C

2

0.0044

<0.0001

< 0.0001

Lack of Fit

0.0790

0.0679

0.1377

0.4664

R-Squared

0.968599

0.918165

0.984501

0.943311

Adj R-Squared

0.958607

0.892127

0.980458

0.928522

Pred R-Squared

0.940209

0.844539

0.97341

0.90661

C.V. %

7.304383

13.667171

3.710808

8.324233

ACCEPTED MANUSCRIPT

A CCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

A CCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

A CCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

A CCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

HIGHLIGHTS

RSM was used to monitor the extraction characteristics of espresso SCG

The effect of extraction time, liquid-to-solid ratio, and microwave power were investigated

The results obtained from all models were highly correlated

The FRAP and the DPPH antioxidative activity showed substantial correlation with TPC

The model can be useful tool for optimization at industrial scale