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J. of Supercritical Fluids 84 (2013) 113– 120

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

jou rn al hom epage: www.elsev ier .com/ locate /supf lu

upercritical fluid extraction of �- and �-acids from hops comparedo cyclically pressurized solid–liquid extraction

ndrea Formatoa, Monica Gallob, Domenico Iannielloa,omenico Montesanoc, Daniele Navigliod,∗

Department of Agriculture, University of Naples Federico II, via Università, 100, Portici, 80055 Naples, ItalyDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, via Pansini, 5, 80131 Naples, ItalyDepartment of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Naples, ItalyDepartment of Chemical Sciences, University of Naples Federico II, via Cintia, 80126 Naples, Italy

r t i c l e i n f o

rticle history:eceived 5 June 2013eceived in revised form6 September 2013ccepted 17 September 2013

eywords:

a b s t r a c t

In this paper, two solid–liquid extraction techniques, supercritical fluid extraction (SFE) with and withoutmodifiers and cyclically pressurized solid–liquid extraction with a Naviglio Extractor, were compared onthe basis of extraction of acidic compounds contained in hops flowers. The hops extracts were analyzedby electro-kinetic capillary chromatography (MECK). The results showed that the technique using super-critical carbon dioxide was more effective for the isolation of � acids; the use of ethanol as a co-solvent,as reported in the literature, produced a heterogeneous extract, while cyclically pressurized solid–liquid

op extractionBD modelO2

FE modelingaviglio Extractor

extraction showed a greater extraction capacity for � acids. Consequently, both techniques are valid forthe extraction of � and � acids from hops. By suitably varying the parameters of the two extractive pro-cedures, it will be possible to obtain extracts for use in the production of beer and dietary supplementsand drugs. Furthermore, based on the SFE CO2 extraction process, a mathematical model was appliedto the examined process, and a numerical simulation was performed, leading to a model that providesdirection for the optimization of further experiments.

. Introduction

.1. Technological properties

Hops are the bitter and spicy components of malts and are essen-ial in beer. Historically, before the use of hops, various bitteringpices were used to balance the sweetness of the malt. The hopssed for brewing are the cone flowers of Humulus lupulus. Theubstances responsible for this activity are mainly the hops acids,hich are classified as � acids (humulones), � acids (lupulones) and

heir oxidation products. These substances consist of a mixture ofomologous compounds [1].

.2. Biological importance

Hops have been recognized as a medicinal plant for centuries,nd different medicinal activities of hops are currently being

iscovered and investigated. In addition to being used in the pro-uction of beer, hops are also used in other sectors and in differentays. They are antibacterial, promote the coagulation of protein

∗ Corresponding author. Tel.: +39 81 674063; fax: +39 81 674063.E-mail addresses: naviglio@unina.it, daniele.naviglio@yahoo.it (D. Naviglio).

896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.supflu.2013.09.021

© 2013 Elsevier B.V. All rights reserved.

and provide good stable beer foam [2]. Due to their numerousactive ingredients, hops have many interesting properties and havebeen used in herbal applications. Modern herbal medicine practi-tioners use hops as a sedative and mild hypnotic, as well as fortheir endocrine, free-radical scavenging and anti-tumoral activ-ities [3]. In a recent study, the antiproliferative mechanisms oflupulones in hops in human colon carcinoma metastasis wereevaluated [4]. Hops bitter acids have been shown to have differ-ent biological activities, including inhibition of angiogenesis [5];inhibition of tumor promotion by phorbol esters [6]; inductionof apoptosis [7]; suppression of cyclooxygenase-2 gene transcrip-tion [8]; and antioxidant [9], antibacterial [10] and antifungal[11] actions. Today, these substances are recognized as potentialchemotherapeutic or chemopreventive agents with antioxidativeand antitumoral properties. The iso-� acids also show interestingantiseptic properties against Gram-positive bacteria in particu-lar and contribute to the stabilization of beer foam. The � acids(or lupulones) contribute to the constitution of beer flavor but ifsubjected to oxidative phenomena, they are capable of producingunwanted substances [12]. In addition to the main components

mentioned above, hops also contain secondary components suchas essential oils and polyphenols. Polyphenols with low molecu-lar weight are natural antioxidants that contribute to the reducingpower of the must and improve the aromatic stability of beer.

114 A. Formato et al. / J. of Supercritica

Nomenclature

� density [kg m−3]�1 dynamic viscosity [Pa s]De effective diffusion coefficient in a solid substrate

(m2 s−1)Kf film mass transfer coefficient (m s−1)r radial position within the particle (m)� one of the coordinatesϕ one of the coordinates�t adaptive time step (s)R radius of spherical solid (m)t extraction time (s)mt total cumulative amount of the solute extracted (g)m0 total weight of the solute in the fixed bed (maximum

extractable solute) (g)(r, �, ϕ) spherical coordinates (m)D12 external diffusion coefficient (m2 s–1) of the solute

Pt

1

aau[lbmabctsfpipwrttfldsltoFtstiraSamp

of the supercritical fluid or the solute–solvent binarydiffusivity coefficient

olyphenols with a high molecular weight tend to precipitateogether with the protein fraction during the mashing phase [13].

.3. Extraction systems

Because of their poor shelf life due to the oxidation of � and �cids, the inflorescences of hops are subjected to various chemicalnd/or physical processes to obtain a more homogeneous prod-ct that maintains the native sensory characteristics over time14]. For this reason, hops are sold mainly in the form of pel-ets or extracts on the market. The former are mainly obtainedy physical treatment but also, in some cases, by chemical treat-ents (iso-pellets); the latter are obtained by the extraction of the

ctive principles by means of CO2 or ethanol. The extracts obtainedy the two techniques result in products with very similar resinontents [15]. However, supercritical CO2 is a non-polar solventhat is selective for apolar substances [16], making it particularlyuitable to extract the “soft resin” and the chemicals responsibleor the aromas of hops, almost completely excluding polar com-onents. Conversely, ethanol, because of its polar characteristics,

s less selective, producing a heterogeneous extract that is rich inolyphenols. Such extracts can also be found in isomerized forms,hich means that boiling is not required; if they are treated with

educing agents, then they are transformed into rho-hydro-�-acids,etrahydro-�-acids and hexahydro-�-acids, which are also resis-ant to post-fermentation oxidation processes [12]. Supercriticaluid extraction (SFE) technology is a reliable alternative to tra-itional solvent extraction. In fact, it has numerous advantages,uch as the absence of solvent residues in the food extract and aow environmental impact. In addition, carbon dioxide, which ishe most frequently used supercritical fluid, does not contain freexygen; thus, the extracts undergo limited oxidation damage [17].inally, the parameters of SFE-based equipment can be modulatedo obtain different extracts after the addition of modifiers (polarolvents). Cyclically pressurized extraction is a process in whichhe pressure applied to the extracting liquid in which samples aremmersed for hydration periodically varies during an establishedange of time. The process was performed at room temperature,nd the energy cost was primarily due to liquid pressurization.

everal methods are used for the determination of hops bittercids [18–20]. However, capillary electrophoresis methods, such asicellar electro-kinetic chromatography (MEKC), have emerged as

romising separation techniques for the analysis of hops acids and

l Fluids 84 (2013) 113– 120

have been successfully used for the separation of humulones andlupulones, including their isomers [20–24]. Further, in addition toextensive experimental research, many efforts have also been madeto mathematically simulate the SFE processes in terms of a time-dependent extraction curve. Mathematical modeling studies areof great importance, as they may enable researchers to generalizethe experimental observations derived from the studied systemsto optimize process conditions or to extend the observations tonew systems. Subsequently, these models may be qualitativelyand quantitatively used in promoting the development of scaling-up procedures for subsequent extraction applications. Moreover,knowledge of the mass-transfer mechanism, kinetic parametersand thermodynamics restrictions could be used to economicallyevaluate the extraction process from the laboratory scale to thepilot and industrial scales. In the meantime, an understanding ofvarious process variables and how they can be connected to atheoretical model to describe the extraction kinetics may also beobtained. For these reasons, many mathematical models for SFEhave been proposed and developed, as summarized in two recentreview papers [24–26].

1.4. Objective

The objective of this work was to separate � and � acids by MECKand to compare hops extracts obtained by different extraction tech-niques to assess the extractive efficiency of supercritical CO2, withor without a coeluent, and an innovative method of cyclically pres-surized solid liquid extraction using the Naviglio Extractor [27–30].Finally, a mathematical model was considered, and a numericalsimulation of the considered process was performed.

2. Materials and methods

2.1. Raw material characterization and preparation of samples

The isomerized extract and hops flowers (cones) were pur-chased from an Italian market. Hops flowers were collected in June2012. The flowers were dried in oven at 60 ◦C. The flowers werecomminuted in a knife mill (Marconi, model MA 340, Naples, Italy).The ground raw material was classified according to particle sizeusing a vibratory system (Bertel, model 1868, Naples, Italy) with8–80 mesh sieves (Tyler series, Wheeling, IL, USA) and then storedin a domestic freezer at −20 ◦C prior to extraction. The mean parti-cle diameter, 3 mm, was determined according to ASAE Standards[31]. The moisture content of the raw material, 9.3 ± 0.7%, wasdetermined in triplicate by the xylene distillation method [32]. Trueparticle density (�s) was determined by pycnometry with heliumgas (Micrometrics, Multivolume Pycnometer 1305, Norcross, GA) tobe 1417 ± 74 kg m−3. The apparent bed density (�a) was calculatedby dividing the feed mass by the vessel volume (266 ± 3 kg m−3).The bed porosity was calculated as 1 − �a/�s (0.8297).

2.2. MECK analysis

MECK analysis was used to separate the ionizable � and � acidscontained in the extracts. The electrophoretic analysis was per-formed using the BioFocus 3000 system (Biorad, CA, USA) followingthe protocol of Royle et al. [18]. The separation was obtained withan uncoated capillary (60 cm inlet to outlet; 56 cm to the detec-tor, i.d. 50 �m) using 50 mM borate buffer containing 40 mM SDSat pH 9.3. The electrophoretic separation was obtained by applying

a 25 kV differential with detection at 200 nm. The capillary tube atthe end of each run was washed with methanol for 200 s. After fil-tering on a Millex-GS (MCE, MF 22 �M), samples were injected ata pressure of 350 mbar for 0.2 s.

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A. Formato et al. / J. of Super

.3. Extraction procedures

Supercritical extractions were performed using the Model020/690 Spe-SFE system from Applied Separations (Allentown,A, USA), with supercritical carbon dioxide as the extraction fluid.ops material was loaded into a 21 mL stainless steel extractionessel, and the remaining volume was filled with glass wool. Threeifferent extracts, SFE I (CO2 supercritical), SFE II (CO2 supercriti-al and ethanol) and one obtained by extraction with the Naviglioxtractor (NE) 500 mL model (Atlas Filtri Engineering, Padua, Italy),ere obtained with the following conditions:

SFE I: Sample weight: 7 g; Tvessel = 35 ◦C; Tvalv = 50 ◦C; = 350 bar; stationary phase: 10 min; dynamic phase: 260 min; col-ected sample: 21 mL.

SFE II: Sample weight: 7 g; Tvessel = 35 ◦C; Tvalv = 50 ◦C; = 350 bar; stationary phase: 10 min; dynamic phase: 260 min; col-ected sample: 21 mL.

NE: Sample weight: 21 g; static phase: 2 min; dynamic phase: cycles with 12 sec of stop piston; total cycles: 360 (24 h); sol-ent: ethyl alcohol; collected sample: 500 mL. To achieve the sameolid/liquid ratio (1:3) adopted for the extraction in supercriticalO2, the ethanol extract (500 mL) was concentrated by rotary evap-rator to 63 mL before MECK analysis.

The Naviglio Extractor is formed by two extraction chambers,ach consisting of a steel cylinder with a piston. Two porous septahat allow only the liquid phase to pass through are on the bot-om of the chambers. The two extraction chambers are connectedy a pipe with an electric valve that is closed during the extrac-ion process and opened to evacuate the liquid from the systemt the end of the cycle. When the maximum value of the pro-rammed pressure is reached, it is maintained for a predeterminedime (generally 2 min); in this manner, equilibrium between theolid and liquid matrices (static phase) is established. After this timestatic phase), the pressurized air that acts on the pistons is quicklyvacuated with a consequent pressure reduction, resulting in a neg-tive pressure gradient between the inside and outside of the solidatrix (dynamic phase). During this phase, compounds that are not

hemically bound to the solid matrix are physically extracted andransferred to the liquid phase, and the active ingredients are thusxtracted [33].

.4. Overall extraction curve determination

Laboratory-scale equipment (Applied Separations, model 7071,llentown, PA, USA) equipped with a 290 mL extraction vessel wassed to determine the overall extraction curve (OEC). The solventsed was carbon dioxide (99.9% purity, SOL, Caserta, Italy). Thexperimental conditions were T = 313 K and P = 35 MPa. The rawaterial was inserted in the vessel, and after pressurization, the

ystem was held for 10 min in the static phase. Then, the CO2 wasntroduced at a constant flow rate of 1.155 ± 0.001 × 10−4 kg s−1.he total extraction time was set at 260 min. The separatoronsisted of a 50 mL glass vial immersed in an ice bath at envi-onmental pressure. Sample extracts were periodically collected atntervals from 15 to 60 min. These parameters were chosen accord-ng to the instructions for use and maintenance of the equipmentsed and the procedures outlined by Rahimi et al. [34].

The data were determined in triplicate. Each variable wasubjected to statistical analysis using a multifactorial analysis ofariance (ANOVA). The statistical significance of each factor underonsideration was calculated using the LSD test. The data weretatistically analyzed with Statgraphic Plus 5.1 software.

.5. Numerical simulation

A numerical simulation for the considered process was gener-ted by ANSYS CFX 14.5 program code. This approach enabled us

l Fluids 84 (2013) 113– 120 115

to simulate two-component fluid dynamics in an SFE extractionsystem, namely, modeling of the diffusion of a hops essential oil(dispersed phase) in a stream of SCCO2 (continuous component),considering the mass and motion quantity balance of both the masstypes considered, which were the solute (hops mass extract) andthe solvent (SCCO2). Because the pressure gradients in this type ofsystem are usually low and do not cause significant compressibilityeffects in the supercritical phase, both components were consid-ered incompressible. Calculations were performed for a transientflow with an adaptive time step, varying from �t = 0.1 to 5.0 s.Moreover, the flow was assumed to be laminar due to the lowvelocity of the continuous phase. In this model, the SCCO2 velocitywas equal to 0.01 m s−1. All calculations were carried out for theoperative conditions considered. The SCCO2 density and dynamicviscosity were � = 628.59 kg m−3 and �1 = 4.022 × 10−5 Pa s, respec-tively.

Further, the coefficients De (effective diffusion coefficient(m2 s−1)) and Kf (external mass transfer coefficient (m s−1)) wereevaluated with the “Hot Ball Diffusion” theory. Extracted particlesare assumed to be spherical with initially uniformly distributedsolute diffusing through the matrix in a manner analogous to heatdiffusion, and the solute concentration in the solvent is assumed tobe close to zero (Bartle et al. [34], Reverchon et al. [37] and Esquívelet al. [35–48]).

Therefore, the material balance across an internal particle sur-face of radius r (m) for a constant density � and diffusivity De canbe obtained in solid and spherical coordinates (r, �, ϕ) accordingto Fick’s first law. In the technical literature, the most recognizedanalytical solution for this model is that of Incropera et al. [49] andCarillo et al. [50].

Applying Fourier transforms and the heat–mass transfer anal-ogy, with mt denoting the total cumulative amount of the soluteextracted and m0 denoting the total weight of the solute in thefixed bed (maximum extractable solute), it is possible to obtain ageneral solution for the concentration value based on the extrac-tion time and the intra-particle position, as given below [38,44,47]:

mt

m012

∞∑k

(sin ˇk − ˇk cos ˇk

)2

ˇ3k

(2ˇk − sin 2ˇk

) .

{1 − exp

[−(

ˇk

R

)2

Det

]}(1)

where the term ˇk allows tailoring for a given system, and eachterm of the series is defined by:

ˇk cot ˇk = 1 − KfR

De(2)

where Kf is the external mass transfer coefficient in m s−1.The extraction calculations obtained from Eq. (1) can thus be

compared to the experimental data by applying a least-squaresmethod in which the solute solid diffusivity De is used as the modelparameter to obtain the best fit of the experimental data. To modelthe extraction curve, ˇk must be calculated from Eq. (2), so theexternal mass transfer coefficient Kf has to be evaluated. Usually, Kfcan be estimated by using a number of correlations that relate thiscoefficient to dimensionless numbers [25,26]. Such dimensionlessnumbers (according to Sherwood, Reynolds, and Schmidt), include� the viscosity of the supercritical solvent phase (g/m*s); dp, theparticle diameter (m); �f, the density of the SCCO2 fluid (g/m−3); u,the superficial velocity of the SCCO2 solvent (m s−1); and D12, theexternal diffusion coefficient (m2 s−1) of the solute into the super-critical fluid, also known as the solute–solvent binary diffusivitycoefficient. The values of the parameter D12 may be similarly eval-

uated using existing correlations and available data. For example,Reverchon et al. [37] assumed a D12 value of 1 × l0−6 m2 s−1 whendescribing the SCCO2 extraction of essential oils and waxes frombasil, marjoram and rosemary leaves [38–45].

116 A. Formato et al. / J. of Supercritical Fluids 84 (2013) 113– 120

e hot

miStBtBtpi

F(

Fig. 1. Particle scheme for th

When the value of Kf/De is very low, a large solubility limitationay occur during the extraction process, making the HBD model

nappropriate. For this reason, some researchers such as Macías-ánchez et al. [44] have suggested that the most appropriate massransfer model can be determined by evaluating the values of theiot number obtained from the internal diffusion coefficient De,he external mass transfer coefficient kf and the particle dimensioni = 2kfR/De. If the Biot number is very high, perhaps greater than 10,he internal diffusion may be the controlling factor in the extraction

rocess. In this case, the HBD model could be appropriate. However,

f the Biot number is less than 10, the controlling factor is the mass

ig. 2. Inflow boundary condition (a) and mesh detail of the sphere boundary layerb).

ball diffusion (HBD) model.

transfer in the interstitial fluid. The HBD model may fail in such acase, and other models should be evaluated (Fig. 1).

The binary diffusion coefficient of the dispersed phase in thecontinuous phase (hops essential oil diffusivity) was assumed tobe equal to D12 = 1 × 10−6 m2 s−1 [37]. The CFD simulations werecarried out for the geometry reported in Fig. 2. The hops essentialoil flux mass enters the main stream through the surface of theextracted particle, which is assumed to be spherical and embeddedin a uniform surrounding. Due to symmetry of the system, a quarterof the extractor was solved.

The mesh consists of 102,955 tetrahedral elements (with anelement characteristic length varying from 0.1 to 0.3 mm). All calcu-lations were carried out using ANSYS Workbench 14.5. Further, theexperimental extraction curve for the considered process was con-sidered, and the hop mass flux curve was evaluated by means of thenumerical derivative of the mass extraction divided by the equiv-alent surface of the sphere with a diameter of 3 mm. This methodwas also applied in other numerical simulations [51].

The experimental extraction curve was derived from the hopsoverall extraction curve, performed in this study for a 7 g sample.This model, calibrated by the analytical solution, was the startingpoint for a more complex model consisting of several spheres filledwith CO2 that were fixed in space and enclosed in an adiabatic vol-ume. In this model, the areas surrounding each sphere are not alloccupied by the gas due to the presence of other spheres and theadiabatic plane on which the spheres lie. Fig. 2 shows the meshdetail near the sphere–CO2 interface.

The characteristic diameter of the spheres was assumed to beequal to 3 mm. The physical properties of the spheres consideredin the calculations are reported in Table 1.

3. Results and discussion

In the present report, the extractive capacities of acid com-ponents of hops were evaluated by two methods, supercriticalphase extraction (SFE), which allows the extraction of analytes

Table 1Thermodynamic properties of the spheres (hop) and liquid CO2.

Hop spheres Liquid CO2

density (�) [Kg m−3] 520 628.59Specific thermal capacity Cp [J (Kg K)−1] 1600 2050Thermal conductivity [W (m K)−1] 0.042 0.132

A. Formato et al. / J. of Supercritical Fluids 84 (2013) 113– 120 117

Fc

ac(�mesawhnIuf

3

3mc1((as

wdnHpmt

3

owt[

tnswwai

Fig. 4. MECK electropherograms of the following hops extracts: commercial sam-ples (a), supercritical CO2 extracts (b), supercritical CO2 with coeluent extracts (c)

ig. 3. Experimental kinetics data for SFE at 313 K and 35 MPa (mass extractionurve).

nd active ingredients through the use of CO2 in the supercriti-al phase, with and without co-solvent, and solid–liquid extractionNaviglio Extractor). The obtained extracts containing hops � and

acids were analyzed by capillary electrophoresis. In the experi-ental conditions used, extraction by supercritical CO2 was more

ffective for the extraction of � acids, while the Naviglio Extractorhowed a greater extractive capacity for � acids. Ethanol produces

more heterogeneous extract because of its polarity comparedith apolar solvents, as reported in the literature. Many studiesave contributed to an increased understanding of the medici-al properties of hops beyond its use in the production of beer.

n fact, clinical and scientific results indicate that hops may besed more extensively in both medicine and nutrition in theuture.

.1. Kinetic curve of the SFE process

In Fig. 3, the experimental kinetics data for SFE I at 323 K and5 MPa are shown as a yield (in terms of the mass of extract (mt) perass of raw material (MT)) versus time diagram. By means of this

urve, the parameter De can be evaluated at different times. After00 min, the total extract in SFE I (supercritical CO2) was 2.3 ± 0.1%w/w); for SFE II (supercritical CO2 and ethanol), it was 2.4 ± 0.1%w/w); and for NE, it was 3.0 ± 0.1% (w/w). The results obtainedre in accordance with the increasing polarity of the extractionolvent.

Using Eq. (2), the diffusion coefficient De was calculated toithin a range of 0.75–0.77 × 10−13 m2 s−1 in the operative con-ition, and Kf was calculated to be 3.02–3.12 × 10−10. The Biotumber obtained was 12.08. Based on the previous discussion, theBD model is valid here. The obtained coefficients were used toerform a numerical simulation of the considered process. The hopass flux curve was derived by calculating the first derivatives of

he mass extraction curve (Fig. 3).

.2. Experimental test results

Fig. 4 shows MECK electropherograms obtained from an analysisf a commercial isomerized extract (Fig. 4a) and extracts obtainedith the two techniques used in this study (Fig. 4b–d). The charac-

erization of the profiles was performed as reported by Royle et al.18].

The first compounds eluted are the iso-� acids, followed byhe � acids and finally the � acids. These last compounds didot interact efficiently with the column. A qualitative compari-on of electropherogram profiles shows that the extracts obtained

ith supercritical CO2 were rich in � acids, the extracts obtainedith supercritical CO2 and coeluent showed intermediate values,

nd the extracts obtained with the Naviglio Extractor were richn � acids; these peaks are indicated by the letters F, G and I,

and solid–liquid extracts with Naviglio Extractor (d).

respectively (See Fig. 4). This result confirms that extractive tech-niques that involve the use of ethanol are less selective than thoseusing only supercritical CO2. The integration of the peak areasallowed a quantitative analysis. Table 2 shows the distribution of� acids, � acids and iso-� acids of hops extracts obtained with thetwo techniques used, expressed as the percentage of the total foreach class of compounds extracted, for a sum of 100% of the totalarea of the peaks obtained by electrophoretic separation.

The results obtained for � acids show an extractive capacity of21.5% for supercritical CO2, 28.3% for supercritical CO2 with cosol-vent and 50.2% with the Naviglio Extractor, with respect to the total� acid content. In contrast, for the � acids, we found that the situ-ation was reversed. In fact, the results show an extractive capacityof 46.2% for extracts obtained with supercritical CO2, 37.5% withthe supercritical CO2 with cosolvent and 16.3% with the NaviglioExtractor, with respect to the total �-acid content. Extraction withCO2 and co-solvent was less selective in respect of other techniquesthat use more polar solvents, in agreement with the literature

[16]. Undoubtedly, the use of solid–liquid pressure in the NaviglioExtractor increases the extractive capacity of ethanol compared tothe classic technique of maceration [28].

118 A. Formato et al. / J. of Supercritical Fluids 84 (2013) 113– 120

Table 2Distribution of soft resin hops extracts obtained with the two techniques used.

Extractive techniques �-acids �-acids Iso-�-acids Total soft resin

CO2 supercritical Total area % on total 3115 ± 40 25.6 ± 0.3 8469 ± 75 69.6 ± 0.6 584 ± 20 4.8 ± 0.2 12169 ± 65 100 ± 0.5CO2 coeluent Total area % on total 5760 ± 50 35.4 ± 0.3 9551 ± 80 58.7 ± 0.5 960 ± 35 5.9 ± 0.2 16271 ± 70 100 ± 0.4

85 69.2 ± 0.4 5159 ± 45 21.5 ± 0.2 2231 ± 45 9.3 ± 0.2 23996 ± 90 100 ± 0.4

count for the dilution. All reported results are the average of three determinations.

t(s(tfa�unicawsctttaPogacCf�(st(Feafiwect(pwAofis

TD

Solid–liquid extraction (Naviglio Extractor) Total area % on totala 16606 ±a Value obtained from the experimentally calculated and multiplied by two to ac

The iso-� acids showed similar behavior in terms of the yield ofhe extraction with supercritical CO2 with and without a co-solvent5.8 and 4.9%), while the Naviglio extractor, using ethanol as theolvent, led to the extraction of a greater amount of iso-� acids9.3%) (Table 3). In the modern brewing process, it is more commono use products that have been extracted from hops, especially inorms that can be added post-fermentation, because this approachllows better control of the bitter taste. These hops products include

acids that have been pre-isomerized (iso-� acids) and prod-cts that have been reduced using hydrogen in the presence of aoble metal catalyst. Therefore, our preliminary results may be of

nterest to the beverage industry; in fact, they indicate that theomposition of the alcohols obtained by the Naviglio Extractor has

greater concentration of � acids than that of extracts obtainedith supercritical CO2. Thus, reliable methods for the separation of

uch mixtures, especially iso-� acids and their derivatives, are ofonsiderable interest. Therefore, it would be interesting to evaluatehe contribution that a sensory extract could give to beer relativeo commercial extracts and those obtained by supercritical extrac-ion. Several studies have described the various potent biologicalctivities of � acids, iso-� acids or a mixture of hops acids [32].reviously, there were few data regarding the biological propertiesf lupulones. In a recent study, it was shown that lupulones trig-er apoptosis by several different death-related signaling pathwaysnd inhibit the formation of tumors in an experimental model ofarcinogenesis of the colon. According to our results, supercriticalO2 is selective for this class of compounds, which is interesting

rom a medical point of view. Moreover, analysis of the profiles of acids, limiting the quantitative comparison to the peaks E and H

common to all extracts), shows that supercritical CO2 is the mostuitable solvent for extracting these components. In fact, underhese conditions, they are present in significantly higher amounts65 and 56%) than the extracts from the Naviglio Extractor (Table 2).ig. 2 shows the profiles of a commercial isomerized extract (a), anxtract with supercritical CO2 (b) and a 50% mixture (c); the profilesre limited to the first 10 min of analysis to facilitate the identi-cation of the peaks corresponding to iso-� acids, in accordanceith the technique of Royle et al. [18]. From this comparison, the

xistence of quali-quantitative differences is evident; in particular,omponents A and D are absent in all extracts obtained with twoechniques, likely due to differences related to the cultivar of originFig. 4b). The extraction of iso-� acids from the commercial sam-le presents a more complete distribution of isomers of � acids,hich, however, are present in all the extracts obtained (Fig. 5).

s a result of increased understanding of the medicinal propertiesf hops, their use beyond beer is increasing. Scientific and clinicalndings indicate the possibility that hops will be used more exten-ively in both medicine and nutrition in the future. Their use as a

able 3istribution of �-acids of hops extracts obtained with the two techniques used.

Extractive techniques �

CO2 supercritical Total area % on total 1CO2 coeluent Total area % on total 1Solid–liquid extraction (Naviglio Extractor) Total area % on totala

a Value obtained from the experimentally calculated and multiplied by two to account

Fig. 5. MECK electropherograms of the following hops extracts: commercial sample(a) supercritical CO2 sample (b), 50% mixture of (a) and (b) in the first 10 min ofanalysis (c).

health food is just as conceivable as the processing of individualhops components in a pure form for use as dietary supplements orin medicines.

3.3. Numerical simulation results

By means of numerical simulations, the hop fraction mass forthe process was obtained at the beginning of, during and at the

-acids peak E �-acids peak H Total �-acids

564 ± 30 52.2 ± 1.0 1123 ± 25 37.5 ± 0.8 2997 ± 30 100 ± 1.0801 ± 45 38.3 ± 1.0 776 ± 25 16.3 ± 0.5 4703 ± 35 100 ± 0.7247 ± 10 11.5 ± 0.5 109 ± 10 5.9 ± 0.5 2154 ± 20 100 ± 0.9

for the dilution. All reported results are the average of three determinations.

A. Formato et al. / J. of Supercritical Fluids 84 (2013) 113– 120 119

eagio

(

(

tgoiv

Fig. 6. HOP mass fraction fields at various times.

nd of the process. The HOP mass fraction fields at various timesre depicted in Fig. 6. Details of the relative mass fraction andradient mass fraction around the extracted particle are reportedn Fig. 7. From the obtained data results, the following can bebserved:

1) The hypothesis that the transversal diffusivity is negligible(because the laminar flow and the diffusive phenomena aredeveloped only in the longitudinal direction, based on numer-ous mathematical models) is validated in this case.

2) Because of the presence of a recirculating zone around thespheres, there are zones with increased solute mass, with a con-sequent reduction of the concentration gradient between thesphere surface and the surrounding fluid, with the reduction ofthe diffusivity. Such a condition due to the laminar flow couldbe resolved by introducing a turbulent, which would avoid thisproblem.

Further, numerical simulation of the considered model allowedhe evaluation of temperature and pressure distributions with a

iven CO2 flow rate; the De and the whole process strongly dependn this rate. Indeed, the considered model is able to predict changesn the parameters at specified nodes within the selected contrololume at every time examined.

Fig. 7. Detail of the mass fraction (a) and gradient mass fraction (b) around theextracted particle at t = 500 s.

4. Conclusions

The preliminary results obtained in this study allow us to affirmthat both methods may be used to produce high yields of hopsextracts. Furthermore, selection of the method to be used dependson the component to be extracted. In fact, the possibility of mod-ulating the experimental parameters of the two methods makeseach method selective for a certain class of compounds, or even forindividual components extracted from hops in pure form (for usein brewing), in accordance with the current trend, which is mainlyoriented toward hops with high bittering potential (high levels of� acids) or the use of hops as dietary supplements and medicines.Further, it is possible to simulate the SFE process and thereforeto evaluate the extraction process in different time periods and toexamine the examined diffusion process. The finite element modelis able to predict the diffusion distribution at any given point withina cylindrical chamber as a function of time.

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