Oxidative Stability of Long-Chain Fatty Acids with Different Unsaturation Degrees Into Layered Double Hydroxides

applied sciences

Article Oxidative Stability of Long-Chain Fatty Acids with Different Unsaturation Degrees into Layered Double Hydroxides

Francesca Blasi 1,† , Claudia Chiesi 1,†, Roberto Spogli 2, Lina Cossignani 1,3,* and Morena Nocchetti 1

1 Department of Pharmaceutical Sciences, University of Perugia, 06100 Perugia, Italy; [email protected] (F.B.); [email protected] (C.C.); [email protected] (M.N.) 2 Prolabin & Tefarm Srl, Via dell’Acciaio 9, 06134 Perugia, Italy; [email protected] 3 Center for Perinatal and Reproductive Medicine, Santa Maria della Misericordia University Hospital, University of Perugia, Sant’Andrea delle Fratte, 06132 Perugia, Italy * Correspondence: [email protected]; Tel.: +39-075-5857959 † Both authors contributed equally to this work.

Abstract: Nowadays, there is increasing evidence that the intake of essential fatty acids (FAs) and oleic acid has high nutritional importance. However, the vulnerability of these FAs to oxidation deserves special attention. FA oxidation may be avoided or delayed by intercalation of its anionic form in inorganic matrices as layered double hydroxides (LDH). Thus, the aim of the study was to evaluate the protective effects of MgAl LDH towards oleate (O), linoleate (L) and α-linolenate (Ln) degradation. The incorporation and the loading of different FAs in anionic form in LDH was determined by X-ray diffraction and thermogravimetric analysis (TGA), respectively. In order to study the selectivity of LDH towards the FA, the inorganic solid was equilibrated with a mixture of O, L and Ln (1:1:1). TGA and gas chromatography showed that Ln was preferentially intercalated. Citation: Blasi, F.; Chiesi, C.; Free FA (FFA) and intercalated FA (IFA) were heated at 40 ◦C in the dark and then analyzed weekly Spogli, R.; Cossignani, L.; Nocchetti, for a maximum of 42 days. Their oxidative stability was evaluated by monitoring the primary M. Oxidative Stability of Long-Chain Fatty Acids with Different and secondary oxidative compounds. The volatile compounds were determined by solid-phase Unsaturation Degrees into Layered microextraction, coupled with gas chromatography–mass spectrometry. Peroxide values were higher Double Hydroxides. Appl. Sci. 2021, in FFA samples than in IFA samples, such as hexanal and trans,trans-2,4-heptadienal % contents. The 11, 7035. https://doi.org/10.3390/ results showed the potential of LDH intercalation for FA preservation from oxidative modiﬁcation. app11157035 Keywords: oleic acid; linoleic acid; α-linolenic acid; intercalation; layered double hydroxides; hydro- Academic Editor: Natalia talcites; oxidative stability; SPME-HRGC-MS Casado Navas

Received: 2 July 2021 Accepted: 28 July 2021 1. Introduction Published: 30 July 2021 Epidemiologic studies provide important insight into the relationship between dietary fatty acid (FA) intake and the risk of disease development. The scientific community Publisher’s Note: MDPI stays neutral agrees on the importance of essential fatty acids (EFAs), linoleic and α-linolenic acids, and with regard to jurisdictional claims in published maps and institutional affil- oleic acid in human nutrition and disease prevention [1]. In recent decades, researchers iations. have focused their attention on n-3 polyunsaturated fatty acids (PUFAs), because of their health, therapeutic, and prophylactic properties [2]. It is known that for the maintenance of optimal health conditions, n-3 and n-6 PUFA must be consumed in a balanced proportion; the optimal n-6:n-3 PUFA ratio is 4:1 [1]. The minimum intake values to prevent deficiency symptoms are estimated to be 2.5% of total energy from linoleic acid (C18:2 n-6) plus 0.5% Copyright: © 2021 by the authors. from α-linolenic acid (C18:3 n-3) [1]. Moreover, the scientific literature has attributed many Licensee MDPI, Basel, Switzerland. interesting properties for human health to monounsaturated FAs (MUFAs), in particular, This article is an open access article distributed under the terms and oleic acid (C18:1 n-9), [3]. For example, oleic acid content is responsible for a reduction in conditions of the Creative Commons blood pressure, and it has been shown that a diet rich in high-oleic sunflower oil favorably Attribution (CC BY) license (https:// alters some blood parameters [4]. In the daily diet, natural sources of these FAs are creativecommons.org/licenses/by/ vegetable and animal foods [5,6]. However, sometimes, recourse to the use of supplements 4.0/). or functional foods enriched with particular FAs is necessary [7].

Appl. Sci. 2021, 11, 7035. https://doi.org/10.3390/app11157035 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 7035 2 of 12

It should not be overlooked that unsaturated FAs are particularly prone to undergo oxidation reactions. The primary products of autoxidation are hydroperoxides and peroxy radicals, unstable compounds that decompose into secondary oxidation products (e.g., unsaturated aldehydes, ketones and other reactive substances) [8]. The greater suscepti- bility of PUFA in respect to MUFA to oxidative and rancidity processes further decreases organoleptic properties, nutrition value and the shelf life of food. This condition makes the development of strategies and techniques to protect n-3 and n-6 PUFA from oxidation necessary; in fact, this topic is of great importance in the food field, and is still an open challenge for researchers [9]. In order to prevent/avoid the oxidative degradation of the fats during storage, technological, and household treatments, the most popular method is the addition of antioxidants, which are often molecules obtained by chemical synthesis [10]. Nowadays, the replacement of synthetic antioxidants with natural antioxidants in the food and pharmaceutical fields is of great interest, for greater safety and acceptance by the consumer [11]. Another strategy for preventing oxidative phenomena is the complexation of organic molecules (guest), such as FAs, in organic or inorganic structures with cavities (host), a process based on host–guest chemistry (e.g., molecular encapsulation, or nanoencapsulation). For example, the cyclodextrins (CD), natural cyclic oligosaccharides, containing six (αCD), seven (βCD), eight (γCD), or more glucopyranose moieties, are widely used as host molecules [12]. The capacity of nanoencapsulation of the oleic acid and different volatile oils in CD and the thermal stability of linoleic acid encapsulated in α- and β-CD was investigated elsewhere [13]. Among inorganic structures, layered double hydroxides (LDH) are versatile layered hosts of molecular anions with biological activity. LDH have the following general formula, n− [M(II)1−xM(III)x(OH)2](A )x/n nH2O, where M(II) is Mg, Zn, Co, Cu, etc., M(III) is Al, Ga, or Fe, and An− is the anion, located into the interlayer region, which counterbalances the positive charge of the lamellae. In the last two decades, LDH have found wide advanced applications in the biomedical field because of their biocompatibility. For example, they are nanovehicles for drug delivery (i.e., anti-inflammatory, antimicrobial, antacid, anti- cancer), DNA segments, cosmeceuticals, as well as nanoparticles for diagnoses through fluorescent or magnetic analyses [14]. Some authors have studied the intercalation of FA: lauric, myristic and palmitic acids [15], caprylic, lauric, stearic, and behenic acids [16], oleic and stearic acids [17], elaidate, oleate, linoleate, and linolenate [18]. It was also reported that LDH could be used in water treatment, as adsorbents, as ion exchangers, and in other interesting agricultural and nutraceutical applications [19,20]. Recently, novel technological approaches for the protection of $–3 PUFA against oxidation processes have been comprehensively reviewed [9]. The purpose of this work was to study the ability of LDH, constituted of Mg and Al, to intercalate anions deriving from oleic, linoleic, and linolenic acids. The selectivity of LDH towards the three different anions was investigated by means of thermogravimetric analysis and gas chromatography. Moreover, the thermal stability of mixtures of intercalated FA (IFA) was investigated and compared with that of free FA (FFA).

2. Materials and Methods 2.1. Chemicals

Oleic acid (≥99%), sodium chloride [NaCl (≥99.5%)], aluminum nitrate [Al(NO3)3· 9H2O (98%)] and xylenol orange disodium salt were obtained from Fluka (Chemika, Buchs, Switzerland). Linoleic acid (≥99%), α-linolenic acid (≥99%), sodium sulfate (Na2SO4), and ethyl acetate (99.8%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol (MeOH), ethanol absolute (EtOH) and n-hexane were purchased from Panreac (Barcelona, Spain). Sodium carbonate (Na2CO3) and hydrochloric acid 37-38% (HCl) were purchased from J.T. Baker, Mallinckrodt Baker B.V. (Denventer, Holland). Magnesium nitrate [Mg(NO3)2·6H2O] (98%) and nitric acid (HNO3) solution was from Carlo Erba reagenti Srl (Milan, Italy). Urea (98%) was purchased from Alfa Aesar (Ward Hill, MA, USA). Appl. Sci. 2021, 11, 7035 3 of 12

2.2. Preparation of LDH

MgAl LDH in carbonate form with the formula [Mg0.63Al0.37(OH)2](CO3)0.185·0.5H2O (LDH-CO3) was prepared according to the urea method [21]. The solid was ﬁrst con- verted into the corresponding chloride form as follows: 1 g of LDH-CO3 was dispersed in a 0.1 mol/dm3 NaCl solution and then titrated, at room temperature (r.t.), with a 0.1 mol/dm3 HCl solution by means of a radiometer automatic titrator operating in pH stat mode, and with a pH value of 5. The recovered solid of formula [Mg0.63Al0.37(OH)2](Cl)0.37·0.6H2O 3 3 was suspended in a CO2-free aqueous solution 0.5 mol/dm of NaNO3 (1g/50 cm of − − solution; molar ratio NO3 /Cl = 10) and gently stirred for 24 h, at r.t. [22]. The recovered solid was washed three times with CO2-free deionized water and ﬁnally dried over, at r.t., with a saturated NaCl solution (75% of relative humidity). It had a formula of [Mg0.63Al0.37(OH)2](NO3)0.37·0.6H2O (LDH-NO3).

2.3. Intercalation of O, L and Ln The intercalation compounds were obtained by ion exchange reactions achieved by equilibrating LDH-NO3 with solutions containing the species of interest in anionic form. The compound containing oleate (O) was prepared by equilibrating the LDH- 3 NO3 with an aqueous solution of 0.1 M sodium oleate (0.2 g/6.4 cm of solution; molar ratio O/NO3 = 1), for 24 h at r.t. in the dark. The same experimental conditions were used for linoleate (L), linolenate (Ln) and for the mixture of the three anions (molar ratio O:L:Ln = 1:1:1). Intercalation compounds with O, L and Ln were also prepared by using a mixture of H2O/EtOH (1:1, v/v) as solvent. Subsequently, the intercalation compounds will be referred to as MgAl-O, MgAl-L, MgAl-Ln, and MgAl-OLLn (IFA).

2.4. Sample Characterization The samples were characterized by chemical analysis, X-ray powder diffraction (XRPD), and thermal analysis. XRPD patterns were taken with a Philips X’PERT PRO MPD diffractometer operating at 40 kV and 40 mA, with a step size 0.0170 2θ degrees, and step scan 20 s, using Cu Kα radiation and an X’Celerator detector. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were performed with Netzsch STA 449C apparatus, in air ﬂow and with a heating rate of 10 ◦C/min. Metal analyses were performed with Varian 700-ES series inductively coupled plasma-optical emission spectrometers (ICP-OES), using solutions prepared by dissolving the samples in concentrated HNO3 and properly diluted.

2.5. Heating of FFA and IFA Seven 2 mL amber glass vials, capped with a PTFE (polytetraﬂuoroethylene) septum, containing a mixture of O, L, Ln in equimolar ratios (FFA), were placed in an oven maintained at 40 ◦C for 42 days. Another seven vials, containing IFA, were subjected to the same treatment. Every seven days, a vial containing FFA and a vial containing IFA were taken out and subjected to analyses reported in the following paragraphs (IFA samples were extracted as reported in Section 2.6. Then all samples were subjected to the analyses described in the following Sections 2.7–2.9.

2.6. Procedure of Extraction of FA from IFA The LDH functionalized with anions deriving from FA were suspended in water and maintained under magnetic stirring at r.t. for 10 min; then, Na2CO3 was added. The samples were stirred for another 40 min. HCl was added until pH 3–4. Ethyl acetate was used for the extraction. The collected organic phases were washed with saturated NaCl solution, dried over anhydrous Na2SO4, and totally dried under nitrogen ﬂow.

2.7. Peroxide Value Determination The peroxide value (PV) was determined by FOX (ferrous oxidation–xylenol orange) assay and expressed as meq peroxide/kg sample [23]. Appl. Sci. 2021, 11, 7035 4 of 12

2.8. Analysis of Volatiles Volatile compounds were analyzed by solid-phase microextraction (SPME), coupled with high-resolution gas chromatography–mass spectrometry (HRGC-MS), as reported in a previous paper [6]. SPME was performed using a 50/30 µm Divinylben- zene/Carboxen/Polydimethylsiloxane StableFlex (DVB/Carboxen/PDMS) ﬁber mounted in an SPME manual holder gray assembly (Supelco, Bellefonte, PA, USA). Compounds were identiﬁed by comparing their mass spectra with those reported in the Wiley138 mass spectral library and using the linear retention indexes from the literature [24].

2.9. Analysis of FA Percentage Composition by HRGC FA percentage composition was determined after the derivatization of FA to methyl es- ters with methanol, as reported by Maurelli et al. (2009) [25], and successive HRGC analysis with a ﬂame ionization detector was carried out, as reported in a previous paper [26].

2.10. Statistical Analysis The reported results were expressed as the mean and standard deviation of duplicate samples. Microsoft Excel 2007 (Microsoft Corporation, Redmond, WA, USA) was used for data analysis.

3. Results 3.1. Characterization of IFA and FA The compounds obtained by O, L and Ln intercalation were characterized by XRPD patterns and TGA. The solvent used in the synthetic procedure, water or water/ethanol mixture, did not dramatically affect the X-ray diffraction patterns and the thermal profile of the intercalated compounds; thus, the data related to the samples obtained from water/ethanol will be discussed. The powder diffraction data provide information about the interlayer distance and thereby indirectly gives information about the arrangement of chains in the interlayer region. Figure1 reports the XRPD of the LDH intercalated with O, L and Ln compared with the pristine LDH-NO3. The interlayer distance of the LDH increases markedly upon intercalation of the organic anions with respect to that of LDH-NO3, which is 8.8 Å. Note that the pattern of LDH-NO3 shows two reflections at low 2theta angles ascribable to the diffraction of the reticular planes (003) and (006); the first, related to LDH metal atomic planes, gives the interlayer distance between the layers. Conversely, the first and the second peaks of the XRPD patterns of the intercalated LDH correspond to the (006) and (009) reticular planes, respectively. The interlayer distance in these compounds may be obtained by multiplying the distance of (006) planes by two; thus, interlayer distances of 30.8 Å, 30.0 Å, 30.0 Å for MgAl-O, MgAl-L and MgAl-Ln, respectively, were obtained. The patterns of MgAl-O and MgAl-L showed a small reflection at 9.9 2theta, corresponding to the nitrate phase, suggesting that nitrate anions are not completely exchanged by the organic anions. This reflection is lacking in the MgAl-Ln X-ray pattern, attesting that in this compound, a more efficient exchange occurred. However, the presence of residual nitrate anions cannot be excluded due to the formation of solid solution in which the nitrate phase is solubilized in the Ln phase [27]. The interlayer distances of the LDH intercalated with anions deriving from FA are close to that of the LDH intercalated with stearate (30.6 Å) [17,28], the corresponding saturate anion. The simulation of the arrangement of the stearate anions between the LDH sheets suggested the formation of a partially interdigitated monolayer. It can be supposed that this arrangement is also adopted from O, L and Ln. Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 13 Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 13

Appl. Sci. 2021, 11, 7035 tion of the arrangement of the stearate anions between the LDH sheets suggested the5 of for- 12 tion of the arrangement of the stearate anions between the LDH sheets suggested the formation of a partially interdigitated monolayer. It can be supposed that this arrangement mation of a partially interdigitated monolayer. It can be supposed that this arrangement is also adopted from O, L and Ln. is also adopted from O, L and Ln.

Figure 1. X-ray diffraction patterns of LDH-NO3 (a) and of MgAl-O (b), MgAl-L (c) and MgAl-Ln FigureFigure 1. 1.X-ray X-ray diffraction diffraction patterns patterns of LDH-NOof LDH-NO3 (a3) ( anda) and of MgAl-O of MgAl-O (b), ( MgAl-Lb), MgAl-L (c) and (c) MgAl-Lnand MgAl-Ln (d) (d) prepared in H2O/EtOH as solvent. prepared(d) prepared in H2 inO/EtOH H2O/EtOH as solvent. as solvent. In order to study the thermal behavior of the intercalation compounds, TGA of the InIn order order to to study study the the thermal thermal behavior behavior of of the the intercalation intercalation compounds, compounds, TGA TGA of of the the samplessamples prepared prepared in Hin O/EtOHH2O/EtOH were were performed performed and and reported reported in Figure in Figure2. The 2. results The wereresults samples prepared in2 H2O/EtOH were performed and reported in Figure 2. The results were similar for all the considered samples (MgAl-O; MgAl-L; MgAl-Ln). The first weight similarwere similar for all thefor consideredall the considered samples samples (MgAl-O; (MgAl-O; MgAl-L; MgAl-L; MgAl-Ln). MgAl-Ln). The ﬁrst The weight first weight loss, loss, ranging from◦ 20 °C◦ to 150 °C, was due to the loss of the hydration water. After this, rangingloss, ranging from 20 fromC to 20 150 °C toC, 150 was °C, due was tothe due loss to the of the loss hydration of the hydration water. After water. this, After a series this, a series of processes occurred: exothermic degradation of the organic moieties prevailed ofa processesseries of processes occurred: occurred: exothermic exothermic degradation degradation of the organic of the moieties organic prevailed moieties andprevailed took placeand concomitantlytook place concomitantly with the de-hydroxylation with the de-hydroxylation of the brucite of layers the brucite and the layers decomposition and the de- and took place concomitantly with the de-hydroxylation of the brucite layers and the de- ofcomposition residual nitrate of residual anions, leadingnitrate toanions, the formation leading to of mixedthe formation metal oxides of mixed [29–32 metal]. Notably, oxides composition of residual nitrate anions, leading to the formation of mixed metal oxides the[29–32]. position Notably, of the TGAthe position curve, andof the the TGA curves curve, of MgAl-Land the andcurves MgAl-Ln of MgAl-L shifted and towardsMgAl-Ln [29–32]. Notably, the position of the TGA curve, and the curves of MgAl-L and MgAl-Ln highshifted temperature, towards suggestinghigh temperature, that the combustionsuggesting that of organic the combustion anions is delayed of organic with anions respect is shifted towards high temperature, suggesting that the combustion of organic anions is todelayed that of Owith in MgAl-O. respect to The that DTA of curvesO in MgAl-O. are also The different; DTA thecurves exothermic are also eventsdifferent; in MgAl-L the exo- delayed with respect to that of O in MgAl-O. The DTA curves are also different; the exo- andthermic MgAl-Ln events occurred in MgAl-L in abroader and MgAl-Ln temperature occurred range in a because broader the temperature LDH lamellae range produced because thermic events in MgAl-L and MgAl-Ln occurred in a broader temperature range because athe barrier LDH effect lamellae to oxygen produced diffusion, a barrier protecting effect to the oxygen organic diffusion, speciesfrom protecting the degradation. the organic the LDH lamellae produced a barrier effect to oxygen diffusion, protecting the organic Moreover,species from it can the be degradation. assumed that Moreover, the packing it ofcan L be and assumed Ln into thethat interlayer the packing region of L is and more Ln species from the degradation. Moreover, it can be assumed that the packing of L and Ln efﬁcientinto the to interlayer hinder the region oxygen is more diffusion. efficient to hinder the oxygen diffusion. into the interlayer region is more efficient to hinder the oxygen diffusion.

Figure 2. TGA and DTA curves of the indicated samples prepared in H2O/EtOH as a solvent com- Figure 2. TGA and DTA curves of the indicated samples prepared in H2O/EtOH as a solvent com- Figurepared 2.withTGA LDH-NO and DTA3. Operative curvesof conditions: the indicated air, 10 samples °C/min. prepared in H2O/EtOH as a solvent pared with LDH-NO3. Operative conditions: air, 10 °C/min.◦ compared with LDH-NO3. Operative conditions: air, 10 C/min.

The water contents obtained in MgAl-O, MgAl-L and MgAl-Ln were 5.7%, 4.0% and 4.6%, respectively. Manzi et al. (2009) prepared MgAl-LDH containing oleate, and the water content of this compound, calculated by TGA, was 7.1% (air, 20 ◦C/min, 50–800 ◦C). Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 13

Appl. Sci. 2021, 11, 7035 6 of 12 The water contents obtained in MgAl-O, MgAl-L and MgAl-Ln were 5.7%, 4.0% and 4.6%, respectively. Manzi et al. (2009) prepared MgAl-LDH containing oleate, and the water content of this compound, calculated by TGA, was 7.1% (air, 20 °C/min, 50–800 °C). TheThe amount amount of of anion anion intercalated intercalated was was obtained obtained by TGAby TGA considering considering the presencethe presence of nitrate of ni- totrate balance to balance the positive the positive charge charge of the of lamellae, the lamellae, and theand obtainedthe obtained values values are reportedare reported in Tablein Table1. In 1. MgAl-O In MgAl-O and MgAl-L,and MgAl-L, the percentage the percentage of exchanged of exchanged nitrate nitrate ranged ranged between between 62% and62% 68%, and whereas68%, whereas in MgAl-Ln, in MgAl-Ln, it was it about was about 78%, and78%, it and seemed it seemed that H that2O/EtOH H2O/EtOH solvent sol- slightlyvent slightly favors favors the intercalation the intercalation process. process. The higherThe higher amount amount of nitrate of nitrate found found by TGA by TGA in MgAl-Oin MgAl-O and and MgAl-L MgAl-L than than in MgAl-Ln in MgAl-Ln is in is agreement in agreement with with the XRPDthe XRPD data. data.

TableTable 1. 1.Molar Molar fractions fractions of of O, O, L L and and Ln Ln intercalated intercalated in in LDH-NO LDH-NO3 using3 using different different solvents. solvents.

Intercalation [Mg[Mg0.630.63AlAl0.370.37(OH)(OH)22](A)](A)x(NOx(NO3)3y)·ynH·nH2O2O mmolmmol A/g A/g A Intercalation Solvent Solvent xx yy n n LDHLDH HH2O 0.230.23 0.140.14 0.440.44 1.641.64 OleateOleate 2 HH22O:EtOHO:EtOH (1:1,(1:1,v/v v/v)) 0.240.24 0.13 0.13 0.450.45 1.68 1.68 HH2O 0.250.25 0.120.12 0.320.32 1.761.76 LinoleateLinoleate 2 HH22O:EtOHO:EtOH (1:1,(1:1,v/v v/v)) 0.250.25 0.12 0.12 0.300.30 1.76 1.76 HH2O 0.280.28 0.090.09 0.380.38 1.881.88 αα-Linolenate-Linolenate 2 HH22O:EtOHO:EtOH (1:1,(1:1,v/v v/v)) 0.290.29 0.08 0.08 0.380.38 1.92 1.92

3 TheThe selectivity selectivity of of LDH-NO LDH-NO3 towards towards the the different different anions anions was was studied studied by equilibratingby equilibrat- theing solid the solid with with an equimolar an equimolar solution solution of O, of LO, and L and Ln. Ln. The The XRPD, XRPD, the the TGA TGA and and DTA DTA of of MgAl-OLLnMgAl-OLLn are are shown shown in in Figure Figure3. The3. The interlayer interlayer distance distance of 30.6 of 30.6 Å is Å very is very close close to those to those of theof the intercalated intercalated single single anions, anions, and and does does not givenot give information information on the on typethe type of anions of anions present pre- insent the in interlayer the interlayer region. region. The DTA The proﬁle DTA profile is different is different from the from previous the previous results, results, suggesting sug- thegesting contribution the contribution of more thanof more one than anion. one anion.

Figure 3. XRPD (a); TGA and DTA, operative conditions: air, 10 °C/min (b) of MgAl-OLLn. Figure 3. XRPD (a); TGA and DTA, operative conditions: air, 10 ◦C/min (b) of MgAl-OLLn.

TGATGA and and HRGC-FID HRGC-FID data data were were combined combined to to determine determine the the content content of of each each anion anion in in MgAl-OLLnMgAl-OLLn (IFA), (IFA), obtaining obtaining the following empiricalthe following formula: [Mgempirical0.63Al0.37(OH) formula:2](O)0.1 (L)[Mg0.110.63(Ln)Al0.370.14(OH)(NO23](O))0.020.1·0.33H(L)0.11(Ln)2O.0.14 The(NO LDH3)0.02·0.33H showed2O. a The higher LDH selectivity showed a towards higher selectiv- Ln, as expectedity towards from Ln, the as expected data of the from intercalation the data of the of singleintercalation anions. of Thissingle slightly anions. preferential This slightly intercalationpreferential couldintercalation be ascribed could to be higher ascribed interaction to higher among interaction the Ln anionsamong into the theLn anions interlayer into regionthe interlayer than the region solution. than the solution.

3.2.3.2. Oxidative Oxidative Stability Stability FFAFFA and and IFA IFA mixtures mixtures were were heated heated to evaluateto evaluate their their thermal thermal stability stability and and to investigate to investi- thegate eventual the eventual protective protective role ofrole LDH of LDH on theon the oxidative oxidative processes. processes. Then, Then, aliquots aliquots of of the the thermallythermally stressed stressed mixtures mixtures (FFA (FFA and and IFA IFA samples) samples) were were withdrawn withdrawn periodically periodically after after seven days for analysis. The primary and secondary oxidative products, in addition to the FA composition, were evaluated. Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 13

Appl. Sci. 2021, 11, 7035 7 of 12 seven days for analysis. The primary and secondary oxidative products, in addition to the FA composition, were evaluated. TheThe evaluationevaluation ofof thethe primaryprimary oxidationoxidation productsproducts waswas carriedcarried outout usingusing thethe FOXFOX spectrophotometricspectrophotometric assay.assay. FigureFigure4 4 shows shows the the evolution evolution of of peroxide peroxide values values (PV, (PV, meq/kg) meq/kg) duringduring thethe heatingheating ofof FFAFFA andand IFAIFA samples.samples. TheThe determinationdetermination ofof PVPV isis thethe typicaltypical andand ofﬁcialofficial methodmethod toto evaluateevaluate thethe primaryprimary oxidativeoxidative lipidlipid process,process, includingincluding inin vegetablevegetable foodsfoods [[8].8]. FattyFatty acidacid oxidationoxidation hashas beenbeen recognizedrecognized asas aa freefree radicalradical chainchain reaction,reaction, withwith thethe abstractionabstraction ofof hydrogenshydrogens atat thethe allylicallylic positionposition ofof thethe doubledouble bondbond ofof unsaturatedunsaturated fattyfatty acidsacids toto formform hydroperoxideshydroperoxides andand freefree radicalsradicals onon aa newnew fattyfatty acid.acid. HydroperoxidesHydroperoxides areare consideredconsidered the primary primary oxidation oxidation products; products; therefore, therefore, the the PV PV parameter parameter represents represents an anin- indicatordicator of of initial initial fat fat autoxidation autoxidation [8,9]. [8,9 ].

20 PV (meq/kg) IFA FFA 0 7 1421283542 Time (days) FigureFigure 4.4. PeroxidePeroxide valuesvalues (PV,(PV, mean mean value value± ±SD) SD) ofof FFA FFA (free (free fatty fatty acid; acid; OLLn) OLLn) and and IFA IFA (intercalated (interca- fattylated acid; fatty MgAl-OLLn)acid; MgAl-OLLn) series series during during heating. heating.

TheThe resultsresults showedshowed thatthat thethe hydroperoxidehydroperoxide contentcontent waswas alwaysalways lowerlower forfor thethe IFAIFA seriesseries thanthan forfor thethe correspondingcorresponding FFA,FFA, atat differentdifferent heatingheating times.times. TheseThese resultsresults areare veryvery signiﬁcantsignificant becausebecause theythey demonstratedemonstrate thethe protectiveprotective effecteffect ofof LDHLDH onon thethe oxidation.oxidation. InIn thisthis work,work, the the secondary secondary oxidative oxidative compounds compounds of of the the thermally thermally stressed stressed FA mixtureFA mix- wereture were evaluated evaluated using using SPME-HRGC-MS SPME-HRGC-MS analysis, analysis, a procedure a procedure used inused a previous in a previous paper pa- to evaluateper to evaluate the volatile the volatile proﬁle profile of vegetable of vege foodstable and foods FA and oxidative FA oxidative status [ 6status,33]. [6,33]. TableTable2 2shows shows the the main main volatile volatile categories categories detected detected for FFAfor FFA and IFAand series. IFA series. Aldehydes Alde- werehydes the were most the represented most represented compounds compounds in FFA and in IFAFFA series, and IFA among series, the among many categoriesthe many ofcategories volatiles. of Alcoholsvolatiles. andAlcohols ketones and were ketone thes were second the most second represented most represented category category in FFA andin FFA IFA and series, IFA respectively. series, respectively. Moreover, Moreover, alkylfurans alkylfurans and hydrocarbons and hydrocarbons were also were detected, also togetherdetected, with together carboxylic with carboxylic acids in FFA acids series. in FFA series.

Table 2. Main categories ofTable volatile 2. Main compounds categories in FFAof volatile and IFA compounds during heating in FFA (area and percentage, IFA during mean heating value (area± SD). percentage, mean value ± SD). Time (days) 7 14 21 28 Time (days) 35 42 FFA 71.3 ± 3.2 60.2 ± 2.0 7 56.6 ± 1.8 14 52.621± 1.328 53.8 ± 1.535 50.6 ±421.6 Aldehydes IFA 80.1 ± 2.8 58.7FFA± 1.8 71.3 ± 3.2 49.1 60.2± 1.1 ± 2.0 56.649.7 ±± 1.81.4 52.6 49.7± 1.3± 1.3 53.8 ± 1.5 45.8 50.6± 1.4 ± 1.6 Aldehydes FFA 2.4 ± 0.5 6.6IFA± 0.8 80.1 ± 2.8 4.1 ± 58.70.5 ± 1.8 49.1 3.3 ± ± 0.61.1 49.7 ± 3.4 1.4± 0.6 49.7 ± 1.3 3.6 45.8± 0.5 ± 1.4 Ketones IFA 11.9 ± 1.0 16.2FFA± 1.0 2.4 ± 0.5 24.0 ± 6.61.0 ± 0.8 23.6 4.1 ±± 0.50.9 3.3 ± 23.8 0.6± 0.9 3.4 ± 0.6 26.3 3.6± 1.0± 0.5 Ketones FFA 12.6 ± 1.1 11.8IFA± 0.9 11.9 ± 1.0 12.7 16.2± 0.9 ± 1.0 24.012.0 ±± 1.0.80 23.6 11.8± 0.9± 0.7 23.8 ± 0.9 10.3 26.3± 0.1 ± 1.0 Alcohols IFA 6.0 ± 0.5 4.0FFA± 0.4 12.6 ± 1.1 5.3 ± 11.80.1 ± 0.9 12.7 5.4 ± ± 0.20.9 12.0 ± 4.9 0.8± 0.4 11.8 ± 0.7 4.5 10.3± 0.1 ± 0.1 Alcohols FFA 2.9 ± 0.3 5.8IFA± 0.5 6.0 ± 0.5 6.0 ± 4.00.2 ± 0.4 5.35.6 ±± 0.10.1 5.4 ± 4.9 0.2± 0.5 4.9 ± 0.4 5.2 4.5± 0.2 ± 0.1 Alkylfurans IFA 0.8 ± 0.0 8.7FFA± 0.3 2.9 ± 0.3 9.7 ± 5.80.3 ± 0.5 10.2 6.0 ±± 0.20.3 5.6 ± 9.8 0.1± 0.3 4.9 ± 0.5 13.1 5.2± 0.5± 0.2 FFAAlkylfurans 0.5 ± 0.0 1.6 ± 0.5 2.5 ± 0.5 1.8 ± 0.5 1.7 ± 0.2 3.3 ± 0.3 Hydrocarbons IFA 0.8 ± 0.0 8.7 ± 0.3 9.7 ± 0.3 10.2 ± 0.3 9.8 ± 0.3 13.1 ± 0.5 IFA 1.2 ± 0.1 7.2 ± 0.7 5.2 ± 0.2 4.6 ± 0.5 5.7 ± 0.3 6.1 ± 0.4 FFA 0.5 ± 0.0 1.6 ± 0.5 2.5 ± 0.5 1.8 ± 0.5 1.7 ± 0.2 3.3 ± 0.3 FFAHydrocarbons 0.7 ± 0.0 1.5 ± 0.4 2.7 ± 0.1 7.2 ± 0.3 6.7 ± 0.4 9.2 ± 0.6 Carboxylic acids IFA 1.2 ± 0.1 7.2 ± 0.7 5.2 ± 0.2 4.6 ± 0.5 5.7 ± 0.3 6.1 ± 0.4 IFA ------FFA 0.7 ± 0.0 1.5 ± 0.4 2.7 ± 0.1 7.2 ± 0.3 6.7 ± 0.4 9.2 ± 0.6 ± ± ± ± ± ± ˆ FFA 9.7 0.9 12.5 1.0 15.6 1.1 17.3 1.2 17.5 1.2 17.8 1.2 Others IFA - 5.2 ± 0.4 6.7 ± 0.5 6.4 ± 0.5 6.2 ± 0.4 4.2 ± 0.3 FFA, free fatty acid; IFA, intercalated fatty acid; -, not detected; ˆ, unidentiﬁed compounds. Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 13

Carboxylic ac- IFA ------ids FFA 9.7 ± 0.9 12.5 ± 1.0 15.6 ± 1.1 17.3 ± 1.2 17.5 ± 1.2 17.8 ± 1.2 Appl. Sci. 2021, 11, 7035 Others ^ 8 of 12 IFA - 5.2 ± 0.4 6.7 ± 0.5 6.4 ± 0.5 6.2 ± 0.4 4.2 ± 0.3 FFA, free fatty acid; IFA, intercalated fatty acid; -, not detected; ^, unidentified compounds.

AmongAmong alcohols, 1-penten-3-ol 1-penten-3-ol and and 1-pentanol, 1-pentanol, typical typical autoxidation autoxidation products products of lin- of linoleicoleic and and α-linolenicα-linolenic acids acids [8], [8 ],were were produced produced during during the the heat heat treatments treatments of of FFA FFA and IFA,IFA, whereaswhereas 3-exen-2-one3-exen-2-one andand 3,5-octadien-2-one3,5-octadien-2-one werewere detecteddetected amongamong ketonesketones inin bothboth series.series. TheThe resultsresults ofof TableTable2 2show show that that the the maximum maximum value value for for the the aldehyde aldehyde category category was was observedobserved after seven days days of of treatment, treatment, both both for for FFA FFA and and IFA IFA series; series; then, then, this this value value de- decreasedcreased with with the the increase increase inin the the heating heating time time (42 (42 days). days). FigureFigure5 5shows shows the the aldehydes aldehydes grouped grouped as as saturated saturated (S), (S), monounsaturated monounsaturated (M), (M), and and polyunsaturatedpolyunsaturated (P) for FFA and IFA IFA series. series. It It can can be be observed observed that that monounsaturated monounsaturated al- aldehydesdehydes are are the the most most represented represented in in the the FFA FFA category, whereaswhereas saturatedsaturated aldehydesaldehydes areare thethe mostmost representedrepresented inin thethe IFAIFA category. category.

FFA 60

40 Area Area (%) 20

0 SMPSMPSMPSMPSMPSMP 7 1421283542 Time (days)

IFA 60

40 Area Area (%)

0 SMPSMPSMPSMPSMPSMP 7 1421283542 Time (days) FigureFigure 5.5. AldehydeAldehyde content (area %, mean value value ±± SD)SD) in in FFA FFA and and IFA IFA series series during during the the thermal thermal treatment.treatment. S,S, saturated;saturated; M,M, monounsaturated;monounsaturated; P,P, polyunsaturated. polyunsaturated.

Aldehydes,Aldehydes, amongamong all all the the compounds compounds derived derived from from the the oxidation oxidation process, process, are are consid- con- eredsidered the mostthe most important important breakdown breakdown products produc andts the and largest the largest contributors contributors to ﬂavor to [ 33flavor,34]. It[33,34]. has been It has reported been reported that saturated that saturated aldehydes alde arehydes more are stable more than stable unsaturated than unsaturated aldehydesaldehydes [35], and [35], that and unsaturated that unsaturated aldehydes aldehydes can be further can be oxidizedfurther oxidized and additional and additional volatile productsvolatile products may be formed may be [ 34formed]. [34]. RegardingRegarding thethe aldehydesaldehydes inin bothboth FFAFFA andand IFAIFA series, series, hexanal, hexanal, octanal, octanal, nonanal,nonanal, andand decanaldecanal werewere detecteddetected amongamong saturatedsaturated aldehydes,aldehydes, whereaswhereas 2-butenal,2-butenal,trans trans-2-pentenal,-2-pentenal, transtrans-2-hexenal,-2-hexenal,trans trans-2-eptenal,-2-eptenal,trans trans-2-octenal,-2-octenal,trans trans-2-nonenal,-2-nonenal,trans -2-decenaltrans-2-decenal and trans and, trans-2,4-esadienal, trans, trans-2,4-eptadienal and trans, trans-2,4-nonadienal were the main unsaturated aldehydes. Propanal, pentanal, and heptanal were detected among the compounds identiﬁed in saturated aldehydes from IFA. Propanal is a typical volatile compound from α-linolenic acid oxidation, whereas pentanal and heptanal originated from the decomposition of linoleate 13- and 11-hydroperoxides. Hexanal, octanal, and nonanal are the typical volatile compounds of oleic acid oxidation, in particular, from 8-O•, 10-O•, and 11-O• [34–36]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 9 of 13

Appl. Sci. 2021, 11, 7035 9 of 12

trans, trans-2,4-esadienal, trans, trans-2,4-eptadienal and trans, trans-2,4-nonadienal were the main unsaturated aldehydes. Propanal,Among pentanal, saturated and compounds, heptanal were hexanal detected was particularly among the representedcompounds in identified the IFA series, in saturatedbut also aldehydes in the FFA from series. IFA. MoralesPropanal et is al. a typical (1997) [volatile37] reported compound that the from hexanal:nonanal α-linolenic acidratio oxidation, could be whereas an appropriate pentanal way and toheptanal detect theoriginated beginning from of oxidationthe decomposition and its evolution; of li- noleatethey 13- found and that 11-hydroperoxides. the ratio changed Hexanal, quickly oc fromtanal, the and highest nonanal values are forthe high-quality typical volatile extra- compoundsvirgin olive of oleic oil to acid the oxidation, lowest for in oxidized particular, oils. from In this 8-O paper,•, 10-O as•, and an interesting 11-O• [34–36]. result, the hexanal:nonanalAmong saturated ratio compounds, decreased forhexanal the FFA was series, particularly whereas represented it increased in for the the IFA IFA se- series ries,(data but also not shown),in the FFA indicating series. Morales a better et protection al. (1997) of[37] IFA reported with respect that the to hexanal:nonanal FFA. ratio couldAs anbe an example, appropriate Figure way6a–c to shows detect thethe trendbeginning of octanal, of oxidationtrans-2-octenal, and its evolution; and trans, theytrans found-2,4-heptadienal that the ratio duringchanged heating, quickly showing from the lower highest percentage values for contents high-quality in IFA extra- samples trans virgincompared olive oil to to the the FFA lowest series. for oxidized-2-Octenal oils. was In this formed paper, from as thean decompositioninteresting result, of linoleic the acid 11-hydroperoxide. Xu et al. (2017) [36] reported, for example, that trans-2-octenal hexanal:nonanal ratio decreased for the FFA series, whereas it increased for the IFA series percentage content increased with oxidation in soybean and peanut oils, although it was (data not shown), indicating a better protection of IFA with respect to FFA. not found in the rapeseed and linseed oils. This difference was linked to the different As an example, Figure 6a–c shows the trend of octanal, trans-2-octenal, and trans, FA composition; in fact, linoleic acid was more abundant in soybean and peanut oils, trans-2,4-heptadienal during heating, showing lower percentage contents in IFA samples with respect to the other two edible oils. trans, trans-2,4-heptadienal was formed by the compared to the FFA series. trans-2-Octenal was formed from the decomposition of lino- decomposition of α-linolenic acid 12-hydroperoxide. It has been reported that its content leic acid 11-hydroperoxide. Xu et al. (2017) [36] reported, for example, that trans-2-octenal increased with oxidation in linseed and rapeseed oils, in particular, for linseed oil where percentage content increased with oxidation in soybean and peanut oils, although it was α-linolenic acid is the main FA [36]. not found in the rapeseed and linseed oils. This difference was linked to the different FA Regarding alkylfurans, 2-pentylfuran was the main alkylfuran detected in FFA, composition; in fact, linoleic acid was more abundant in soybean and peanut oils, with whereas 2-ethylfuran was the main for IFA. High amounts of 2-pentylfuran were detected respectin edible to the oils other with two relatively edible oils. high trans, linoleic trans acid-2,4-heptadienal [35]. was formed by the decom- positionFor of furtherα-linolenic investigation, acid 12-hydroperoxide. HRGC-FID analysis It has was been performed reported inthat order its tocontent evaluate in- the creasedFA percentage with oxidation composition in linseed during and rapeseed the thermal oils, treatment in particular, of IFA for and linseed FFA oil series, where and α- the linolenicresults acid are reportedis the main in FA Figure [36].7.

16 IFA FFA 12

8 Area Area (%)

0 7 1421283542 Time (days)

(a)

16 IFA FFA 12

8 Area Area (%) 4

0 7 1421283542 Time (day)

(b)

Figure 6. Cont. Appl. Sci. 2021, 11, x FOR PEER REVIEW 10 of 13

16 IFA FFA 12 Appl.Appl. Sci. 2021 Sci. 2021, 11, ,x11 FOR, 7035 PEER REVIEW 10 of 1013 of 12

8 Area Area (%) 4 16 IFA 0 FFA 12 7 1421283542 Time (days)

8 (c) Area Area (%) Figure 46. Content (area percentage, mean value ± SD) of octanal (a), trans-2-octenal (b), and trans, trans-2,4-heptadienal (c) in FFA and IFA series during heating. 0 Regarding7 alkylfurans, 1421283542 2-pentylfuran was the main alkylfuran detected in FFA, Time (days) whereas 2-ethylfuran was the main for IFA. High amounts of 2-pentylfuran were detected in edible oils with relatively( chigh) linoleic acid [35]. For further investigation, HRGC-FID analysis was performed in order to evaluate the FigureFAFigure percentage 6. Content 6. Content composition (area (area percentage, percentage, during mean meanthe value thermal value ± SD)± treatment SD)of octanal of octanal of(a), IFA trans (a), andtrans-2-octenal FFA-2-octenal series, (b), and (b ),and andtrans thetrans, , trans-2,4-heptadienal (c) in FFA and IFA series during heating. resultstrans are-2,4-heptadienal reported in (Figurec) in FFA 7. and IFA series during heating.

Regarding alkylfurans, 2-pentylfuran was the main alkylfuran detected in FFA, whereas100 2-ethylfuran was the main for IFA. High amounts of 2-pentylfuran were detected O L Ln FFA in edible80 oils with relatively high linoleic acid [35]. For further investigation, HRGC-FID analysis was performed in order to evaluate the

FA percentage60 composition during the thermal treatment of IFA and FFA series, and the

results Area % are reported in Figure 7. 40

10020 O L Ln FFA 800 7 1421283542

60 Time (days) 100 Area % 40 O L Ln IFA 80

20 60 0

Area % 40 7 1421283542 Time (days)

10020 O L Ln IFA 800 7 1421283542 Time (days) 60

Area % FigureFigure 7.40 Fatty 7. Fatty acid acid composition composition (area (area percentage, percentage, mean mean value value ± SD)± SD)of FFA of FFAand andIFA IFAseries series during during the thethermal thermal treatment. treatment. 20 Regarding the FFA series, the FA percentage contents of oleic, linoleic and α-linolenic Regarding0 the FFA series, the FA percentage contents of oleic, linoleic and α-linolenic acidsacids were were fairly7 fairly constant 1421283542 constant for the for first the 21 ﬁrst days; 21 days;after that, after linoleic that, linoleic and α-linolenic and α-linolenic acid per- acid percentage contents decreased,Time (days) reaching values of 21.5% and 7.5%, respectively, after centage contents decreased, reaching values of 21.5% and 7.5%, respectively, after 42 days. Clearly,42 days. the increase Clearly, in the oleic increase acid percentage in oleic acid content percentage is a consequence content is a of consequence the percentage of the Figuredecreasepercentage 7. Fatty in linoleic acid decrease composition and inα-linolenic linoleic (area andpercentage, acids.α-linolenic mean acids. value ± SD) of FFA and IFA series during the thermalThe treatment. trend observed for the FFA mixture was expected because it is known that the unsaturation degree is inversely related to the oxidative stability of FA. Very interesting resultsRegarding were the obtained FFA series, for the the IFA FA mixturepercentage during contents the heating; of oleic, in linoleic fact, minimal and α-linolenic changes in acidsthe were FA percentagefairly constant content for the were first observed. 21 days; In after particular, that, linoleic after 42and days α-linolenic of thermal acid treatment, per- centagethe linoleic contents acid decreased, percentage reaching content values was almostof 21.5% similar and 7.5%, to the respectively, initial value. after Regarding 42 days. the Clearly,percentage the increase content in of oleicα-linolenic acid percentage acid, only content a slight is decrease a consequence was observed, of the andpercentage it explains decreasethe slight in linoleic increase and in α oleic-linolenic acid percentage acids. content. These results indicate that intercalation could represent a valid approach to protect unsaturated FAs from oxidative processes. Appl. Sci. 2021, 11, 7035 11 of 12

4. Conclusions Layered compounds, belonging to the family of biocompatible layered double hydroxides, are versatile hosts of a wide variety of molecular anions with biological activity. The obtained results showed a high percentage of incorporation of all three FA with the preference of α-linolenate in respect to oleate and linoleate. The data indicated that inorganic sheets offer protection to labile biomolecules such as long-chain FAs with different unsaturation degrees. The results of oxidation products and the FA percentage composition showed a higher stability of IFA with respect to FFA samples. This study has provided scientiﬁc evidence for the practical applications of improvements in the oxidative stability of oleic, linoleic, and α-linolenic acid during thermal treatment. LDH offer interesting perspectives for developing new innovative inorganic–organic materials to be applied in food, nutraceutical, pharmaceutical, and cosmeceutical ﬁelds.

Author Contributions: Conceptualization, L.C. and R.S.; methodology, F.B. and M.N.; validation, L.C., M.N. and R.S.; formal analysis, C.C. and F.B.; investigation, L.C., M.N. and R.S.; data curation, F.B., C.C. and M.N.; writing—original draft preparation, C.C. and F.B.; writing—review and editing, L.C. and M.N.; visualization, L.C. and M.N.; supervision, L.C. and M.N. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank Giuseppa Verducci for her support in the chemical analysis. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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