Nanoencapsulation systems to improve solubility and antioxidant efficiency of a grape marc extract into hazelnut paste

Journal of Food Engineering 114 (2013) 207–214

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Nanoencapsulation systems to improve solubility and antioxidant efficiency of a grape marc extract into hazelnut paste Giorgia Spigno a,⇑, Francesco Donsì b, Danila Amendola a, Mariarenata Sessa b, Giovanna Ferrari b,c, D. Marco De Faveri a a b c

Università Cattolica del Sacro Cuore, Institute of Oenology and Agro-Food Engineering, via Emilia Parmense, 84-29122 Piacenza, Italy University of Salerno, Department of Industrial Engineering, Salerno, Italy ProdAl Scarl, University of Salerno, Italy

a r t i c l e

i n f o

Article history: Received 23 April 2012 Received in revised form 10 July 2012 Accepted 15 August 2012 Available online 25 August 2012 Keywords: Antioxidants Hazelnut paste Nanoemulsions Phenolic extract Shelf-life

a b s t r a c t Encapsulation of a phenolic grape marc extract to enhance its lipid solubility and antioxidant efficiency for application as a natural preserving agent of hazelnut paste was investigated. Three nanoemulsion based encapsulation systems were tested: an oil/water nanoemulsion; a powder obtained by maltodextrin-assisted spraydrying of the previous and an ethanol/solid–lipid nanoemulsion (the third also applied to resveratrol for comparison). All the samples were mixed with hazelnut paste at different phenolic concentrations (from 1200 to less than 10 ppm of gallic acid equivalents for the original and encapsulated extracts, respectively). An accelerated shelf-life test at 60 °C was carried out until 59 and 98 days (for the original and encapsulated extracts, respectively) with peroxides value monitoring. Paste oxidation followed a first-order kinetics model and was significantly inhibited by extract addition. Encapsulation improved phenolic efficiency against lipid oxidation, by increasing extract dispersability in the paste and preserving the antioxidant activity, with the oil/water nanoemulsion resulting as the best system. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction In previous studies (Amendola et al., 2010) the authors obtained a powdery phenolic extract from red-grape marc, which is a typical wine-making by-product. The extract was obtained by hydroalcoholic extraction and subsequent freeze-drying and it showed stable total phenolic content and antioxidant activity when stored at 25 °C in a closed container and in the dark for up to 300 days. This extract offers potential applications as a natural low-cost additive to improve quality and extend shelf-life of foods without the use of synthetic additives, such as BHA and BHT, which have restricted use because of their toxicity. This phenolic extract could be used as an innovative and natural active additive, which can improve preservation of food products, such as hazelnut paste. Hazelnut paste is a food ingredient used in different processed foodstuff such as ice-creams, confectionery and bakery products. After processing into paste, hazelnuts become more prone to oxidation because of increased surface area exposed to oxygen and damaged structure that cause deterioration reactions to proceed faster (Cam and Kilic, 2009). The few studies reported on hazelnut paste mainly deal with rheological aspects, oil adulteration problems or ⇑ Corresponding author. Tel.: +39 0523599181; fax: +39 0523599232. E-mail addresses: [email protected] (G. Spigno), [email protected] (F. Donsì), [email protected] (D. Amendola), [email protected] (M. Sessa), [email protected] (G. Ferrari), [email protected] (D. M. De Faveri). 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.08.014

its use for bread enrichment (Bonvehi and Coll, 2009; D’Addio et al., 2012; Ercan and Dervisoglu, 1998; Oliete et al., 2008), but not with its oxidative stability. The previously obtained freeze-dried extract (Amendola et al., 2010) exhibited a very low water and lipid solubility, making its application into most food systems extremely difficult. While the antioxidant, antimicrobial and pharmacological activity of phenolic extracts were thoroughly investigated, only a few studies focused on the technological implications that their addition into real foods might have, especially concerning the important aspects related to miscibility, stability and interaction with the other food components (Nanditha and Prabhasankar, 2009). Recently, nanotechnology significantly contributed to the development of nanometric delivery systems, capable of encapsulating the antioxidant compounds, of protecting them from undesired reactions, of minimising the impact on the organoleptic properties of the product, as well as of enhancing their activity by promoting the mass transfer rates to sites of action (Donsì et al., 2010a, 2011; Sessa et al., 2011). Different types of delivery systems exist, whose preparation requires an adequate formulation and opportune processing conditions. Among the nanoencapsulation systems, nanoemulsions and lipid nanoparticles appear particularly suitable for food applications. Nanoemulsions are very fine, kinetically stable emulsions, made of lipid droplets of nanometric size (50– 200 nm), dispersed in aqueous phase, prepared by means of high pressure homogenisation and the use of an adequate emulsifier at

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water/oil interface (Schultz et al., 2004). Depending on the emulsifier employed and the relative ratio of lipid and aqueous phase, delivery systems can be prepared to be specifically suitable for the incorporation in foods with high lipid content. In our case, grape marc phenolics can be solubilised in an ethanol aqueous solution, which is subsequently nanodispersed in a lipid phase. Moreover, physical processing methods used for nanoemulsion preparation, such as high-pressure homogenization, also contribute to reduce particle size of polyphenolic powder particles and generate an amorphous state, with the consequence of further improving their dispersability (Donsì et al., 2010b). Based on these premises, the aims of this study were to:  Investigate the exploitation of the protective effect of a grape marc phenolic freeze-dried extract against the oxidation of commercial hazelnut pastes.  Investigate the encapsulation of the marc extract into different nanoemulsion based delivery systems to improve the dispersion of the phenolic compounds into the hazelnut paste and enhance their antioxidant efficiency. As a reference compound, due to its well-known antioxidant properties, pure resveratrol was also tested for preparation of a nanoemulsion (Chachay et al., 2011).  Find out a kinetic model to describe hazelnut paste oxidation and evaluate the inhibition efficiency of the different extract formulations (as freeze-dried powder or in encapsulated form).

2. Materials & methods 2.1. Materials Barbera red-grape marc extract (crude extract, CE) was obtained by solvent extraction with ethanol:water/60:40, at 60 °C, followed by freeze-drying, according to the procedure described in (Amendola et al., 2010). The total phenols content was evaluated according to the Folin–Ciocalteau assay. Dark natural hazelnut paste (NP) and dark fluid hazelnut paste (FP) (added with monoglycerides as emulsifiers) were kindly provided by an Italian company. The two pastes were obtained from Turkish hazelnuts through a process consisting in a roasting step, followed by peels removal and milling. NP and FP differed only for the monoglycerides addition and their average composition, kindly provided by the company, is reported in Table 1. All the used solvents were of analytical grade. The nanoemulsions were prepared using peanut and sunflower oil (Sagra, Lucca, Italy) and stearic acid (Sigma–Aldrich s.r.l., Milan, Italy) as lipid phase and soy lecithin Solec IP (a kind gift from Solae Italia s.r.l., Milan, Italy) as emulsifier. Resveratrol, extracted from grape skin (purity > 98%), was a kind gift from Organic Herb Inc., China. Maltodextrins with dextrose equivalent 16.5–19.5 (Sigma–Aldrich s.r.l., Milan, Italy) were used as spray-drying carriers. The used reagents and standards were: DPPH (Sigma–Aldrich s.r.l., Milan, Italy), Folin–Ciocalteau reagent (Fluka, Darmstadt, Germany),

Table 1 Natural and fluid hazelnut paste characterization. Values are reported as means ± s.d. Component

% (w/w)

Proteins Fat Sugars Moisture Ash Fibre

15.09 ± 2.23 65.61 ± 2.01 5.56 ± 0.14 0.90 ± 0.07 2.41 ± 0.07 10.43 ± 4.52

starch paste 1% in water (Carlo Erba, Rodano (M) Italy), gallic acid and catechin (Fluka, Buchs, Switzerland). 2.2. Experimental work The efficiency of the grape marc extract as natural preservative agent into hazelnut paste against lipid oxidation was investigated through accelerated shelf-life tests. The first trials were carried out on the CE, adding it into two different hazelnut commercial pastes: a NP and a FP. In the so called ‘‘fluid’’ paste, monoglycerides were added to stabilise the paste and prevent oil separation, which instead occurs at significant extent in the ‘‘natural’’ paste after a few days. In the experiments, CE was handily mixed with the paste and added at a concentration of 5000 ppm (w/w) (chosen based on common maximum allowed additives incorporation into foods). Samples were kept into open 10 mL amber glass vials in oven at 60 °C. Each vial contained 5 g of paste, with the entire content being used for the analyses at each sampling time (approximately every week for 5 and 8 weeks for the NP and FP, respectively), including also blank samples (paste without extract addition). For the analysis, the lipid phase was separated and analysed for the peroxides value (PV), while the phenolic compounds were extracted (only from the FP samples) with methanol from the degreased paste and analysed for the total phenolics content (as reducing power by the Folin–Ciocalteau assay). The next trials were carried out with grape extract encapsulated into three different systems and with resveratrol encapsulated into one system. In this case only FP was used. Each formulation was added at a concentration of 5000 ppm (w/w). The accelerated shelf-life tests were carried out as described for the CE, but sampling occurred weekly for 8 weeks and, then, after 12 and 14 weeks. Commercial hazelnut pastes usually exhibit a quite long shelflife, from 12 up to 36 months if properly stored (generally declared in the darkness and away from both light and heat sources, therefore a reference room temperature of 21 °C is assumed). According to literature data (Allen and Hamilton, 1994), a 10–25 kcal/mol activation energy can be assumed for reactions of oxidative rancidity: therefore, applying the Arrhenius law, a week storage at 60 °C can be considered equivalent to a minimum of 7 weeks at 21 °C. The minimum duration of 8 weeks for the accelerated shelf-life tests of this study was, then, calculated in order to simulate the minimum 1-year shelf-life at 21 °C. The experimental data of PV obtained during accelerated shelf-life studies were used for kinetics interpretation of hazelnut paste oxidation. Influence of the nanoencapsulation process on the antioxidant activity of both marc extract and resveratrol, was assessed by measuring the antioxidant activity (DPPH assay) before and after encapsulation. 2.3. Nanoencapsulation Table 2 reports the final composition of the different encapsulated formulations investigated in this study. Formulation 1 (F1) consisted of an O/W nanoemulsion, where the CE, firstly dissolved in ethanol (5% w/w) was dispersed in the lipid phase, constituted by sunflower oil and soy lecithin, on a hot plate at a temperature of 50 °C in order to evaporate the ethanol. Following complete ethanol evaporation, monitored by weight reduction until the target value, the resulting lipid phase was dispersed in water by High Shear Homogenization (HSH), using an Ultra Turrax T25 (IKA Labortechnik, Germany) at 24000 rpm for 5 min to form the primary emulsion, and subsequently reduced to nanometric dimensions by high pressure homogenization (HPH) in a Nano DeBEE Electric Bench-top Laboratory homogeniser (BEE International, USA) five times at 150 MPa. Formulation 2 (F2) consisted of a powder obtained by spraydrying F1 in a Mini Spray Dryer B-290 Basic (Büchi Italia s.r.l., Mi-

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G. Spigno et al. / Journal of Food Engineering 114 (2013) 207–214 Table 2 Composition of the different nanoemulsion formulations used in this study. F: formulation; O: oil; W: water; EtOH: ethanol.

a

Component (wt%)/size (nm)

F1 O/W nanoemulsion

F2 Powder from F1

F3 EtOH/O nanoemulsion

Grape extract (wt.%) Resveratrol (wt.%) Sunflower oil (wt.%) Peanut oil (wt.%) Stearic acid (wt.%) Soy lecithin (wt.%) Ethanol (wt.%) Water (wt.%) Maltodextrins (wt.%) Average size ± s.d (nm)

0.25

0.7

0.5

9

25

F4 EtOH/O nanoemulsion 0.05

1

2.8

89.75

2 69.5 290 ± 5a

210 ± 5

83.1 4.4 2 10

83.55 4.4 2 10

325 ± 50

90 ± 20

Droplet size measured upon rehydration of the powder.

lan, Italy). The emulsion was mixed with maltodextrins and passed at a spray rate of 4 mL/min, with drying and outlet temperatures fixed at 180 and 120 °C, respectively. Dry air flow was set at 800 L/h, and the atomization pressure was set at 1.5 bar. Formulation 3 (F3) was an ethanol–oil nanoemulsion. The CE was dissolved in ethanol (10%) and homogenised by HSH with a hot melt of stearic acid and peanuts oil using soy lecithin as emulsifier. The emulsion was further comminuted by HPH (5 passes at 150 MPa). Formulation 4 (F4) was identical to F3, using instead of grape extract a lower amount of resveratrol in consideration of its higher antioxidant activity. A photon correlation spectrometer (HPPS, Malvern Instruments, Malvern, UK) was used for the particle size measurement of the emulsion droplets. The droplet size distribution was characterised in terms of the mean droplet size (z-diameter) by measuring the backscattered (173°) light through samples diluted 1:100 with bidistilled water to avoid multiple scattering effects within polystyrene cuvettes. Measurements were carried out at 25 °C for O/ W emulsions (F1–F2), and at 50 °C for ethanol-in-oil emulsions (F3–F4) in order to maintain the lipid phase in liquid state. Each measurement was replicated twice, with the means and the standard deviations being calculated. In the case of ethanol-in-oil emulsions, photon correlation spectrometry analysis required the values of viscosity and refractive index of the disperdant medium (the lipid phase consisting of peanut oil and stearic acid), which were preliminary characterised at 50 °C by means of a dynamic shear rheometer (TA Instruments, New Castle, DE) with a shear rate profile from 1 to 100 s1, and of an ABBE refractometer (ATAGO Italia s.r.l., Milan, Italy) with jacketed cell. The values of viscosity and refractive index were 35 cP and 1.466, respectively. Antioxidant activity of final formulations was assessed by the DPPH radical assay.

2.4. Analyses 2.4.1. Peroxides value (PV) One of the most common objective methods to assess oxidation degrees of oils is the peroxide value, which measures hydroperoxide formation. Samples were defatted twice with 15 ml of hexane mixing for 2 min with a vortex (Velp scientific s.r.l., Milano Italy) and centrifuging at 10000 g for 10 min. Hexane was removed under vacuum (Rotavapor Büchi R-114) and the recovered oil was used to evaluate PV. Briefly, 1 g of oil was weighed into a 250 ml flask, 25 ml of mixture of acetic acid/chloroform (3/2 v/v) and 0.5 ml of saturated solution of potassium iodide were added. The sample was left in the dark for 2 min. After this time, 25 ml of water were added to dilute the sample and 0.5% starch solution

was added as indicator. Sample was then titrated with Na2S2O3 0.01 N and PV calculated as:

PV

  meqO2 mlNa2 S2 O3  NNa2 S2 O3 ¼  1000 kgoil gsample

ð1Þ

The absence of any effect of the extraction procedure on the PV of the sample was preliminary verified by analysing the PV of a sunflower oil sample before and after extraction. Oxidation inhibition was calculated as percent inhibition of peroxides formation (PVI):

PVI ¼

PVblank  PVextract  100 PVblank

ð2Þ

Considering the peroxides reduction given by extract addition at a certain time t (DPVt) , the fact that the PV is referred to 1 g of lipid phase, the lipid content of the paste (Table 1) and the added amount of polyphenols with the extract, the specific peroxides inhibition power of total phenols (PIP0 ) was calculated as:

PIP0 ¼

DPVt ¼ mgGAE

ðPVblank  PVextract Þt glipid 0:6561 glipid =gpaste



5 mgextract=formulation gpaste

 X mg

mgGAE

ð3Þ

extract=formulation

Finally, peroxides formation rate (PFR) in a certain time interval was calculated as:

PFR ¼

PVt2  PVt1 t2  t1

ð4Þ

where (t2–t1) is the considered time interval, PVt1 and PVt2 is the peroxides value at the beginning and end of the time interval, respectively. 2.4.2. Reducing power (Folin–Ciocalteau assay) The Folin–Ciocalteau assay is commonly used to evaluate the total phenolics content and known as the Total Phenolics Assay but, being the basic mechanism an oxidation/reduction reaction, it can be considered also a method to measure the antioxidant capacity of a sample (Prior et al., 2005). The assay was used to evaluate both the total phenols content of the CE and the reducing power of the hazelnut paste. In the first case, the extract was dissolved in ethanol:water/60:40, while in the second phase a phenolic extract was obtained from the paste. In this work the hazelnut phenolics were extracted with methanol according to a procedure adapted from Yurttas et al. (2000) through preliminary trials where a known amount of catechin was added to the paste and a 100% recovery was verified. Briefly, the defatted hazelnut paste was extracted twice with 25 ml of aqueous methanol (70%) for 1 h in an orbital shaker (HT INFORS AG CH-4103, Bottmingen, Switzerland, rotation speed set at 150 rpm) and centrifuged at 3000 g for 10 min. The supernatant was collected and used for the analysis (Amendola et al., 2010). An aliquot of

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the sample (0.5 ml) was mixed with 2.5 ml of Folin–Ciocalteau reagent; then 5 ml of Na2CO3 20% were added and the mixture was brought to 50 ml with distilled water. A blank sample was included (with 0.5 ml of methanol 70%). The samples were kept in a thermostatic bath at 40 °C for 30 min, then the absorbance of the samples at 750 nm was measured against the blank. Results were expressed as equivalents of gallic acid (GAE) by means of a calibration curve prepared for a concentration range of 100–750 mg/L. Preliminary tests with addition of the crude extract to the paste showed a recovery of about 80% of the extract phenolics. 2.4.3. Antioxidant activity (DPPH assay) Antioxidant activity of CE, resveratrol and final formulations, was assessed by the DPPH radical assay according to a previously reported procedure (Lachman et al., 2007; Lucas-Abellán et al., 2011) with minor modifications (after mixing the sample and the DPPH solution, the absorbance readings at 515 nm were taken after 5 min at 25 °C). The stock solution was prepared by dissolving 24 mg of DPPH with 100 ml of methanol, and then stored at -20 °C in the dark until needed. The working solution was obtained by diluting 10 ml of stock solution with 45 ml of methanol. For the analysis, the CE was dissolved in ethanol at a concentration of 0.125 wt.%; resveratrol was dissolved in ethanol at a concentration of 0.05 wt.%; F2 was rehydrated in water at a concentration of 28.2 wt.%; F1, F3 and F4 were used directly. For each sample, density was measured. A volume of 100 ll of the sample was added to 3.9 ml of DPPH working solution. After 5 min, allowing to the reaction to reach the steady state, the absorbance readings were taken using a V-650 UV–Vis spectrophotometer (Jasco Instruments, Easton, MD, USA) set at 515 nm at 25 °C. The antioxidant activity was expressed as mg of ascorbic acid equivalents (AAE)/ml by using an ascorbic acid calibration curve prepared for a concentration range of 0–5 lM. Each sample was analysed in triplicate. Based on sample density, the result was expressed as mgAAE/g and then, based on extract or resveratrol concentration, specific antioxidant activity was also calculated as mgAAE/gmarc extract and mgAAE/gresveratrol, respectively. Finally, combining the dosing level in the paste, the extract concentration in the formulation and the calculated specific antioxidant activity, data were elaborated to give the antioxidant activity expressed as mgAAE/ghazelnut paste. 2.4.4. SEM observations A scanning electron microscope (FEI Quanta Feg 250 Esem, FEI, Hillsboro, OR, USA) was used to examine the morphology and surface appearance of crude marc extract and nanoemulsions by environmental SEM technique. The samples were analysed immediately after their production.

2.5. Statistical analysis Shelf-life experiments were carried out on duplicate and chemical analyses on triplicate; mean values and standard deviations were calculated for each case. Analysis of variance (ANOVA) followed by Tukey’s post-hoc test was performed at the p 6 0.02 level using IBM SPSS Statistics 19 (Inc, Chicago, IL, USA) to assess the influence of: storage time on the reducing power and PV; extract addition on PV and encapsulation process on antioxidant activity.

3. Results & discussion 3.1. Addition of crude extract to different types of hazelnut paste The hazelnut pastes tested in the experiments exhibited a very different physical stability, with the fluid paste, containing monoglycerides, showing during the accelerated shelf-life test only a very limited separation of the oil on the surface. In fact, while in the NP samples almost 40% of the sample volume separated as a lipid layer already after 1 week, in the FP samples this percentage was <5% at the end of the storage. The effects of extract addition into NP and FP in terms of peroxides formation during storage at 60 °C, are reported in Table 3. Addition of CE to the NP brought to a significant inhibition of lipid oxidation, even though the antioxidant effect was limited by the separation of the paste over time in two phases (with oil on the surface) and the extract rapidly precipitated to the bottom of the samples. For this reason the trial was stopped after 5 weeks. The paste presented a certain peroxides content (PV ¼ 2:5 meqO2 =kgoil ) at time zero, while the final values resulted to be in agreement with literature data. In fact, Özcan and Arslan (2011) reported a PV of 85 after 14 days for hazelnut oil stored at 50 °C, while Yalcin (2011) found a PV of 530 for a sample of refined hazelnut oil stored at 40 °C in an incubator exposed to light and air for 120 days. Similarly to NP, in the FP the CE did not solubilise completely and over time it tended to precipitate, but still showed a significant antioxidant effect. Oxidation was observed only after an induction period (IP) of 14 days, after which it followed a similar trend as observed with the NP, even though FP oxidation was about 50% slower compared to the NP, both with and without extract (compare PV after 35 days in Table 3). The addition of the emulsifier to FP likely prevented the separation of the oil from the paste and the formation of an upper layer in direct contact with air during the test conditions, consequently slowing oxidation. In order to possibly distinguish the effect of added antioxidants from that of endogen compounds, the total phenolics content was assessed in both the blank and the extract-enriched-sample. In

Table 3 Peroxides values (meqO2 =kgoil ) of all the samples. Values are reported as means ± sd. Superscripts: Same letters in the same column, or same numbers in the same raw inside a trial, refer to statistically not significant different values according to ANOVA and Tukey’s post-hoc test. NP: natural paste. FP: fluid paste. CE: crude extract. F: formulation. Trial NP and CE

FP and CE

FP and encapsulated extract

Time (days)

Blank

CE

Blank

CE

Blank

F1

F2

F3

F4

0 7 14 21 28 35 42 49 59 83 98

2.5 ± 0.0a,1 4.4 ± 0.1b,2 11.0 ± 0.0c,2 22.0 ± 0.0d,2 43.0 ± 0.0e,2 80.8 ± 0.3f,2

2.5 ± 0.0a 2.8 ± 0.3a,1 9.0 ± 0.0b,1 14.5 ± 0.5c,1 28.0 ± 0.0d,1 48.0 ± 0.0e,1

0.0 ± 0.0a 1.1 ± 0.1b,1 9.2 ± 0.5c,1 18.2 ± 0.5d,1 30.1 ± 0.5e,1 36.4 ± 0.2f,1 60.5 ± 0.1g,1 76.5 ± 0.3h,1 243.7 ± 0.2i,1

0.0 ± 0.0a 0.0 ± 0.0a,2 6.5 ± 0.5ab,2 14.0 ± 1.0bc,12 21.8 ± 0.8cd,12 29.5 ± 0.5d,2 41.5 ± 0.5e,2 78.0 ± 0.5f,2 79.5 ± 7.0g,2

0.0 ± 0.0a 2.0 ± 0.0a,1,2 9.3 ± 0.8ab,1,2 18.8 ± 1.2b,2 36.3 ± 4.8c,1,2 59.1 ± 3.9d,2,3 63.8 ± 4.8de,2 77.0 ± 2.0e,3 151.0 ± 9.0f,2 230.6 ± 3.5g,1 491.5 ± 8.5h,3

0.0 ± 0.0a 1.3 ± 0.0a,1 4.5 ± 0.5ab,1 7.8 ± 0.3ab,1 22.5 ± 0.5bc,1 36.5 ± 1.0cd,1 41.0 ± 5.0cd,1 45.5 ± 2.0de,1 63.5 ± 8.5e,1 235.5 ± 3.5f,1 465.0 ± 15.0g,2,3

0.0 ± 0.0a 2.0 ± 0.0a,1,2 7.8 ± 1.3a,1,2 15.3 ± 0.3ab,2 26.8 ± 1.3b,1 50.8 ± 7.2c,1,2 69.3 ± 2.3cd,2 77.0 ± 7.0d,3 85.5 ± 1.5d,1 230.0 ± 12.0e,1 432.5 ± 7.5f,1

0.0 ± 0.0a 2.0 ± 0.0a,1,2 9.5 ± 2.0b,1,2 14.3 ± 0.8b,2 31.8 ± 3.3c,1,2 40.5 ± 3.0d,1 63.5 ± 0.5e,2 62.5 ± 2.5e,2 71.5 ± 2.5f,1 223.8 ± 2.5g,1 478.5 ± 2.5h,3

0.0 ± 0.0a 3.8 ± 0.0a,2 13.0 ± 2.5a,2 20.4 ± 2.6ab,3 43.5 ± 7.5bc,2 75.0 ± 5.0d,3 66.5 ± 10.0cd,2 63.4 ± 1.6cd,2 127.5 ± 12.5e,2 223.0 ± 12.0f,1 445.0 ± 7.0g,1,2

G. Spigno et al. / Journal of Food Engineering 114 (2013) 207–214

mgGAE /gpaste

2.5

a

a,b

a,b a,b,c a,b,c a,b

b,c

2.0 a,b

1.5

a

a b,c,

1.0

c,d

the extract-enriched paste, the reducing power of only the added polyphenols was calculated as the difference between that of the extract-enriched paste and that of the blank. The resulting trend showed a strong reduction after 21 days, which would confirm the active role of the extract in the inhibition of paste oxidation.

c

d b,c,d

211

a,b,c

3.2. Addition of nanoencapsulated extract

0.5 0.0 0

15

FP

30

FP with CE

45

Days 60

FP-(FP with CE)

Fig. 1. Total phenolic content (or reducing power) of fluid paste (FP) samples in the accelerated shelf-life test carried out with crude extract (CE). GAE: gallic acid equivalents. Error bars indicate ±s.d. For each series, same letters indicate means not statistically different according to ANOVA and Tukey’s post-hoc test.

Table 4 Composition of the different nanoemulsion formulations with reference to antioxidant activity and final grape extract concentration in the hazelnut paste. AAE: acid ascorbic equivalents. F: formulation. HP: hazelnut paste. ME: marc extract. Values of antioxidant activity referred to formulation or marc extract weight are reported as mean ± s.d. Antioxidant activity

Formulation ME in HP ppm (w/ w)

Phenolic compounds in HP ppm (w/w)

Crude extract F1

5000

1200

0.11 ± 0.01#

91.72 ± 5.14a

12.5

3

0.19 ± 0.01

76.00 ± 4.41b

mgAAE/gF

mgAAE/gME

mgAAE/ gHP

0.46 0.95103

F2

35

8.4

0.57 ± 0.03

80.80 ± 4.44b

2.82103

F3

25

6

0.19 ± 0.01

38.20 ± 1.87c

0.96103

F4

2.5*

2.5*

0.079 ± 0.00

157.30 ± 8.49*

0.39103

Resveratrol

0.13 ± 0.01## 269.38 ± 14.01*

(a,b,c)

Same letter refers to statistically not significant different values according to ANOVA and Tukey’s post-hoc test. * Values referred to resveratrol. # Value referred to a solution 0.125 wt.% of crude extract in ethanol. ## Value referred to a solution 0.05 wt.% of resveratrol in ethanol.

fact, the hazelnut paste was made starting from the whole fruit, and it has been reported that hazelnuts contain antioxidant compounds other than tocopherols (Yurttas et al., 2000; Shahidi et al., 2007; Ghirardello et al., 2010; Jakopic et al., 2011; Solar and Stampar, 2011). The total phenolics content of the blank was 1.25 ± 0.13 mgGAE/ g, in agreement with literature, where values ranging from 0.07 to 7 mgGAE/g have been reported (Jakopic et al., 2011; Cristofori et al., 2008). The found differences can be due to the different solvents used for extraction, but also to differences in cultivars and years, since phenolic compounds are secondary metabolites whose content varies in response to several external factors, including environmental constraints. Addition of the CE obviously increased the total phenolics content of the paste (Fig. 1) which remained apparently constant until after 45 days, while that of the blank increased after 21 days. This increase, followed by a decrease, might be ascribed to the ability of polyphenols to oxidise and further polymerise to form oligomers with a higher antioxidant activity (Di Mattia et al., 2009) and, actually, the Folin assay measures the reducing power of samples. Assuming the same increase in the hazelnut phenolics occurred in

Tables 2 and 4 compare the different types of used nanosized delivery systems in terms of particle size, antioxidant activity and grape extract content. F4, similar to F3 in formulation and fabrication, but containing resveratrol instead of marc extract, showed the lowest average size and a multimodal distribution, having a first peak around 200 nm and a second peak around 2.8 lm. Also F3 showed a multimodal distribution with a first peak around 240 nm and a second peak around 3 lm. F2, the powdery version of F1, retained the original mean droplet size (210 ± 5 nm) after mixing with maltodextrins and the final powder, upon rehydration, retained a nanometric size, exhibiting only a moderate increase in the z-diameter . As it concerns the effect of encapsulation on the antioxidant activity of the extract, Table 4 shows that the nanoencapsulation process caused a 17% activity loss for F1 and a 58% for F3. The spray-drying of F1 did not cause any additional activity loss. Encapsulation of resveratrol (F4) caused an activity loss of almost the 40%. The highest antioxidant activity was that of F2, however the highest specific antioxidant activity was exhibited by F4 due to the well-known antioxidant properties of resveratrol, followed by F1 and F2. F3, the ethanol solution-in-solid lipid emulsion, exhibited the lowest antioxidant activity probably due to the lower availability of the marc extract (related to the higher mean particle size), which was completely embedded in the stearic acid/peanut oil/lecithin lipid phase. Considering the content of extract in the formulations (Table 2) and the 5000 ppm dosing level in the hazelnut paste, the final extract concentrations ranged from 12.5 to 35 ppm (a 400–140-fold lower amount than in the trials with CE addition), while the consequent concentration of extract phenols in the paste was always lower than 10 ppm (Table 4). None of the nanoemulsion formulations gave any precipitate over the tested storage time, showing an improved lipid solubility of the extract. The results of accelerated shelf-life tests (Table 3) showed again that paste oxidation started after an IP of 14 days. F1 proved to be the most efficient encapsulation solution since it was the only one with PV significantly lower than the blank until 59 days. Similar results were obtained with F3, in agreement with the fact that the specific antioxidant activity referred to the hazelnut paste was the same as for F1. Even though the addition of F2 corresponded to addition into the paste of the highest dose of antioxidant activity, PV of the resulting samples were not significantly different from the blank. Incorporation of resveratrol through F4 showed an initial prooxidant effect (higher, although not statistically significantly, PV than the blank) and appeared to start exerting an antioxidant effect against lipid oxidation after 49 days. Since all the tested formulations were not active anymore after 83 days, it will be required to increase the initial concentration of the phenolics into the delivery systems, in order to prolong the inhibition over longer times. Furthermore, the choice of the appropriate antioxidant molecules is a challenge since the efficiency of the compounds in emulsions and complex systems is influenced by molecular polarity (Di Mattia et al., 2009). Application of the analytical procedure to nanoemulsion samples for paste degreasing and phenolic recovery showed that the added formulations remained in the degreased phase but the

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fine powder, composed of spherical particles of average size <10 lm, where the nanoemulsion is embedded and released upon rehydration. SEM observation of F1 was not possible because of the technical limitations of the instruments. SEM images of F3 instead showed a uniform phase, consisting of lipid components, such as peanut oil, stearic acid and lecithin, where the phenolic crystals are annealed. Preparation procedure of F3 (at 55 °C) induced, in fact, a significant evaporation of the solvent (ethanol), which caused the precipitation of the phenolic crystals in smaller size because crystal growth and agglomeration was prevented by the lipid phase. Measurement of residual ethanol content in F3 and F4 resulted in 6.4 wt.% and 3.5 wt.%, respectively, which is slightly lower than the theoretical value of 10 wt.%. As a consequence, the resulting phenolic crystals had a smaller average size than in freeze-dried samples and were completely covered by the lipid phase. The crystals embedment in the lipid phase also caused the significant reduction in the antioxidant activity shown in Table 4.

3.3. Kinetics interpretation Experimental data of PV over storage time were tentatively elaborated according to different kinetics model in order to find out the best model to predict hazelnut paste oxidation and evaluate the inhibition efficiency of the different extract formulations. The results are summarised in Table 5. Kinetics of fats oxidation has been widely investigated in literature since Labuza (1971). In particular, oxidation of vegetable oils has been found to follow half-order kinetics according to Eq. (5) (Erkan et al., 2009).

PVt1=2  PV1=2 ¼ ð1=2ÞkM ½RH0   t 0

ð5Þ

where PVt is the peroxides value at time t, PV0 the peroxides value at time zero, kM is the mixed rate constant which takes into account an initiation, a propagation, a termination rate constant, corresponding to the three steps of the oxidation of lipids, and [RH0] is the initial concentration of lipid substrate. Table 5 Results of kinetics elaboration of experimental data. CE: crude extract. NP: natural paste. FP: fluid paste. F: formulation. Same letters refer to statistically not significant different values according to ANOVA and Tukey’s post-hoc test for each trial and kinetics model.

Fig. 2. SEM observation of different grape marc extract formulations. From the top: freeze-dried raw extract, Formulation 2 and Formulation 3.

emulsions could not be broken. Therefore, only the reducing power of the hazelnut compounds was actually measured and for all the samples the same trend as that reported in Fig. 1 was observed. This also confirms that endogen reducing power of hazelnut paste is not involved neither diminished by lipid oxidation. 3.2.1. Morphological characterization of crude and encapsulated extracts Observation of the grape marc extract elaborated under different formulation types, clearly showed substantial differences in the physical structure (Fig. 2). The crude extract was obtained through a freeze-drying process in which the initial liquid extract had been slowly frozen in a refrigerator at 20 °C, leading to the formation of large crystals, which partly explains the limited solubility into both water and lipid media. Fig. 2 shows an average crystal size >100 lm in the CE. In contrast, spray-drying, especially when applied to a nanoemulsion, leads to a more homogenous and

*

Trial

Kinetics model

Reaction rate*

r2

Time range (days)

PFR

Blank NP CE NP Blank FP CE FP Blank FP CE FP Blank FP F1 FP F2 FP F3 FP F4 FP Blank FP F1 FP F2 FP F3 FP F4 FP Blank FP F1 FP F2 FP F3 FP F4 FP

First-order

0.100a 0.084b 0.067a 0.057b 0.210a 0.161b 0.092ab 0.110b 0.092ab 0.077a 0.090ab 0.033b 0.043d 0.033ab 0.038c 0.028a 0.205c 0.182a 0.192b 0.191b 0.200c

0.99 0.97 0.97 0.94 0.91 0.97 0.98 0.98 0.99 0.95 0.99 0.95 0.98 0.99 0.98 0.98 0.97 0.93 0.97 0.95 0.96

0–35

2.24b 1.30a 5.32b 1.66a 4.12b 1.35a 2.49bc 1.60a 2.15ab 1.55a 3.10c 6.86b 6.80b 6.06a 6.95b 5.87a 5.02c 4.74bc 4.41a 4.88c 4.54ab

First-order Half-order First-order

First-order

Half-order

14–59 0–59 14–35

35–98

0–98

[k] according to Eq. (6) for the first-order model; [1/2 kMRH0] according to Eq. (5) for the half-order model.

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1000

(A) PV

PVI

100

10

1

0

20

Blank-NP

40

CE-NP

Blank-FP

70 60 50 40 30 20 10 0

(A)

0

Day 60

20

40

FP

CE-FP

Day

NP

60

35

1000

(B)

(B)

25 15

PV

PVI

100

5 -5

10

-15 -25

1

0

20

Blank-FP

40 FP-F1

60 FP-F2

0

80 Day 100 FP-F3

40 F2

60 F3

80 F4

100 Day

FP-F4

Fig. 3. Interpretation of peroxides value (PV) trend according to a first-order kinetics model (continuous lines). PV (meqO2 =kgoil ) are reported on a logarithmic axis. (A) Results from the trials with crude extract (CE); (B) results from the trials with nanoemulsion formulations (F1, F2, F3, F4). NP: natural paste. FP: fluid paste.

It was reported that the addition of prooxidants and antioxidants can change the half-order to first-order, while in complex food systems the data sometimes fit zero-order kinetics (Colakoglu, 2007; Shim and Lee, 2011). Furthermore, oxidation rate varies with the partial pressure of oxygen and when this is high the oxidation rate is independent of oxygen concentration but directly dependent on substrate concentration and can be studied by the first-order reaction kinetics, according to Eq. (6):

lnðPVt Þ ¼ k  t þ lnðPV0 Þ

20 F1

ð6Þ

where k is the apparent first-order kinetics constant. In our case, hazelnut paste is a complex system consisting in a thick semi-solid product with hazelnut particles ranging in size from 7 to 13 lm, being a fine paste dispersed in hazelnut oil (Ercan and Dervisoglu, 1998). In the NP the oil phase separated at the top resulting in a single phase, which, in the open vials used in the experiment was in direct contact with atmospheric air, with constant oxygen concentration, therefore explaining the observed first-order kinetics for oxidation of NP, with and without addition of CE (Fig. 3A). Extract addition slowed down oxidation by a 19%, as calculated comparing the reaction rates. According to the calculated kinetics equations, the percent inhibition of peroxides formation given by the extract was also assessed (Fig. 4A). Results indicate an increase of PVI with time, even though it should be expected that at a certain time a maximum PV will be reached or that the antioxidant activity of the extract polyphenols will end. Similarly to what previously observed, also in the FP an apparent first-order kinetics for PV formation was observed (Fig. 3A), with a reaction rate in the blank a 18% faster than in the sample with the extract. However, the first-order model accurately fitted all the data only after the induction period. When considering the PV evolution over the entire storage period, the half-order kinetics model (Eq. (5)) resulted to fit accurately only the experimental data for the extract-enriched paste but not for the blank (Table 5). The calculated percent inhibition of peroxides formation was slightly lower than the one observed for the natural paste (Fig. 4A), probably

Fig. 4. Trend of calculated percent inhibition of peroxides formation (PVI) after crude extract addition into both natural paste (NP) and fluid hazelnut paste (FP) (A) and after addition of the different investigated nanoformulations (F1, F2, F3, F4) into FP (B).

due to the combination of different effects. First of all, because of its higher stability, the fluid paste was less prone to oxidation, and consequently the effect of the extract was less intense. Secondly, the presence of the emulsifier at the fat/water interfaces might have limited the interaction between fat and antioxidants. The obtained PIP0 values are reported in Table 6. Since the maximum formation of PV was not reached, the added polyphenols resulted still active in inhibiting oxidation at the end of the trials, therefore the calculated PIP0 underestimate the real ones. Globally, the effect of crude extract addition was an inhibition of lipid oxidation with reduced formation of peroxides, but not a delay in the induction period of oxidation reactions. In the case of extract nanoformulations added into FP, experimental data could be accurately fitted both by a first-order kinetics model (after the IP and with a reaction rate decreasing after 35 days) and by a half-order kinetics model through all the storage time. Since the PV at the beginning and the end of the considered time intervals might be different for the different samples, the performance of the formulations should be evaluated considering the reaction rates together with the PFR, the PVI and the PIP0 . If the first-order model is assumed, samples enriched with nanoformulations, were not statistically different from the blank in the first period (14–35 days) based on the reaction rates, while F1 and F3 allowed for the highest lipid inhibition based on the PFR. In the second period (35–98 days) it was exactly the opposite, since F1 and F3 showed reaction rates and PFR comparable to the blank, while F2 and F3 showed both significantly lower oxidation rate and significantly lower PFR. The percent inhibition of peroxides formation for the different formulations, calculated according to the first-order kinetics rates of Table 5, showed a decreasing trend for F1 and F2, and an increasing trend for F3 and F4 (Fig. 4B). Considering the PIP0 (Table 6), the specific peroxides inhibition power of the grape marc extract after 59 days at 60 °C was the same independently of the used form, as crude freeze-dried extract or as nanoncapsulated extract. However F1 and F3 were already active in the first 35 days, while F2 and F4 started exerting their activity only after 35 days. All these results can be interpreted

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Table 6 Specific peroxides inhibition power of total phenols in the accelerated shelf-life trials. PIP0 : specific peroxides inhibition of total phenols. CE: crude extract. NP: natural paste. FP: fluid paste. PV: peroxides value. 0*

Storage time (day)

PIP

35

19.4 7.4 113.7 0 34.4 0 54.6 57.9 43.4 52.6 0 0 11.72 0 26.24

59

98

Sample CE into NP CE into FP F1 into FP F2 into FP F3 into FP F4 into FP CE into FP F1 into FP F2 into FP F3 into FP F4 into FP F1 into FP F2 into FP F3 into FP F4 into FP

*

PIP0 was considered zero for PV not significantly different from the blank according to Table 3.

in terms of release kinetics of the encapsulated compounds, with resveratrol characterised by a slower release than marc extract from the EtOH/O systems (F3 and F4), and F2 system characterised by a slower release of marc extract than F1 and F3. When a half-order kinetics model is assumed to fit the lipid oxidation data over the entire 98 days storage period, F1 resulted the best formulation based on the reaction rate, followed by F2 and F3, while F2 and F4 appeared to be the best ones based on PFR and PIP0 (Tables 5 and 6), which is in contradiction with data analysis reported in Table 3. Furthermore, calculation of the PVI gave a constant protection effect of 20.99%, 12.17%, 13.07% and 0.96% for Formulation 1–4, respectively, which is again in contradiction with experimental data. Therefore, based on qualitative and quantitative considerations on the fitting of the different sets of experimental data with the two kinetic models, the first-order kinetics model results as the more appropriate to describe the oxidation behaviour of hazelnut paste, both with crude and encapsulated extract, and is quite accurate after the induction period. 4. Conclusions This study showed the efficiency of a phenolic grape marc extract in improving the shelf-life of hazelnut paste by inhibiting its oxidation, in spite of the limited solubility of the extract in such a high lipid content matrix. The oil-in-water nanoemulsion resulted the best encapsulation solution for the potential production of a natural preservative agent, while the slower release of phenolic compounds shown by the ethanol-in-oil nanoemulsion could be potentially exploited for the production of an healthy-functional ingredient. Oxidation reaction can be accurately described by first-order kinetics models, whose related mathematical parameters can be used to compare the efficiency of the different formulations. Further research is needed to better understand the stability and action mechanism of the nanoemulsions and to optimise formulation (since the encapsulation process caused a reduction in the antioxidant activity of the extract) and dosing levels, from different points of view, such as costs (particularly if the nanoemulsions are meant to be used as preserving additive for low-cost products) and sensorial quality of the final product. Acknowledgments This research was partly supported by the Doctoral School on the Agro-Food System (Agrisystem) of the Università Cattolica

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