Fish in Vitro Digestion: Influence of Fish Salting on the Extent of

Article pubs.acs.org/JAFC

Fish in Vitro Digestion: Influence of Fish Salting on the Extent of Lipolysis, Oxidation, and Other Reactions Bárbara Nieva-Echevarría, Encarnación Goicoechea, María J. Manzanos, and María D. Guillén* Food Technology, Faculty of Pharmacy, Lascaray Research Center, University of the Basque Country (UPV/EHU), Paseo de la Universidad n° 7, 01006 Vitoria, Spain S Supporting Information *

ABSTRACT: A study of the various chemical reactions which take place during fish in vitro digestion and the potential effect of fish salting on their extent is addressed for the first time. Farmed European sea bass fillets, raw, brine-salted or dry-salted, were digested using a gastrointestinal in vitro model. Fish lipid extracts before and after digestion were analyzed by 1H NMR, and the headspace composition of the digestates was investigated by SPME-GC/MS. During digestion, not only lipolysis, but also fish lipid oxidation took place. This latter was evidenced by the generation of conjugated dienes supported on chains having also hydroperoxy- and hydroxy-groups (primary oxidation compounds), by the increase of volatile secondary oxidation products, and by the decrease of the antioxidant 2,6-di-tert-butyl-hydroxytoluene (BHT). Likewise, esterification and Maillard-type reactions also occurred. Salting, and especially dry-salting, enhanced all these reactions, except for lipolysis, during digestion. KEYWORDS: fish, 1H NMR, in vitro digestion, lipid oxidation, salting, SPME-GC/MS, volatiles

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INTRODUCTION Research into the influence of the digestion process on food in order to better understand its effect on human health is a current trend in food technology and nutrition. In spite of its great relevance, apart from hydrolysis, knowledge is still lacking concerning the potential chemical transformation of food components during digestion. Recently, it has been suggested that lipid oxidation may take place as a result of pro-oxidant conditions of the gastrointestinal tract.1,2 In this sense, fish lipids may be especially prone to oxidation during digestion due to their high content in polyunsaturated acyl groups, as reported by the few in vitro studies on fish oils carried out to date.3−6 In these studies, lipid oxidation was determined either by investigating the occurrence of very few oxidation markers (malondialdehyde, 4-hydroxy-2-nonenal, and/or 4-hydroxy-2hexenal) or by using classical methodologies, such as the determination of lipid hydroperoxides by iodometric titration (Peroxide Value) or by ferrous ion oxidation in the presence of xylenol orange or thiocyanate or the TBARS test. However, study of the extent of oxidation reactions undergone during digestion requires additional approaches by means of other techniques. In fact, the drawbacks of classical methodologies have been widely commented on in the last few years.7,8 Based on the results obtained in these previous works on marine oils, some oxidation could be expected to occur during in vitro digestion of fish meat. Nevertheless, the latter is a much more complex matrix than fish oil: the presence of other components together with fish lipids in the food bolus, such as proteins or endogenous fish antioxidants, among others, might greatly influence the advance of oxidation reactions occurring under digestive conditions. In a recent study, the formation of malondialdehyde (measured as TBARS value) and 4-hydroxy2-hexenal was reported during in vitro digestion of salmon.9 However, further knowledge concerning the nature of lipid oxidation products that may be generated during fish digestion © 2017 American Chemical Society

would be useful. Furthermore, other chemical reactions in addition to oxidation might take place and deserve further attention.10,11 Thus, a detailed study of the occurrence of lipid oxidation and other reactions during fish meat digestion has not been undertaken to date, nor has the potential effect, if any, on the extent of these reactions of common technological processes applied to fish, such as salting, been studied. The salting process has been traditionally carried out on fish in order to extend their shelf life. Its preservative effect relies on the decreased water activity that this process provokes, which prevents microbiological growth. However, certain studies reported that salting can reduce the oxidative stability of fish lipids under frozen, refrigerated, or thermo-oxidative conditions.12−15 Using spectroscopic techniques, such as FTIR and 1 H NMR, it was observed that salting did not provoke any immediate oxidation, but when submitted to pro-oxidative conditions, fish lipid oxidation evolved at a greater rate in salted than in unsalted fish fillets. Nevertheless, when farmed and wild specimens of European sea bass were salted and studied by means of SPME-GC/MS, very slight lipid oxidation could be observed in wild samples immediately after the most intense salting process.16 This pro-oxidant effect could be attributed to the loss of water-soluble antioxidants and to the increase of prooxidant agent concentration in contact with the lipid phase.17 Nevertheless, the degree of salting might also be determinant.12 In this context, the aim of this work is to investigate fish meat in vitro digestion, paying special attention to the hydrolysis reaction and to the occurrence of lipid oxidation, without forgetting that other chemical reactions are also possible. Furthermore, potential differences occurring during digestion Received: Revised: Accepted: Published: 879

September 28, 2016 December 15, 2016 January 4, 2017 January 4, 2017 DOI: 10.1021/acs.jafc.6b04334 J. Agric. Food Chem. 2017, 65, 879−891

Article

Journal of Agricultural and Food Chemistry of unsalted and salted fillets will be addressed, and the influence of the degree of salting evaluated. For this purpose, unsalted, brine-salted, or dry-salted farmed European sea bass samples were in vitro digested following a static model simulating the digestion processes occurring in the mouth, stomach, and small intestine. Fish lipids were extracted before and after digestion and studied by 1H NMR, which allows analysis of both their lipolytic and oxidative status. Moreover, the headspace composition of nondigested and digested samples was investigated by SPME-GC/MS, which is a much more sensitive technique that can provide a great deal of information not only about volatile products coming from lipid oxidation, but also about those from other reactions. It must be noted that SPMEGC/MS was not employed with the aim of quantifying concentrations of volatile compounds, but with that of obtaining valuable data for comparative purposes among samples. Therefore, both tools were employed simultaneously in order to get a holistic view of the reactions occurring in each sample during digestion.

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methodology and the acquisition parameters were the same as in previous studies.13,21−23 In order to select the most appropriate values to obtain accurate quantitative results in the shortest possible period of time, a very broad range of recycling times and relaxation delays were tested in the acquisition of the 1H NMR spectra. The lipid composition of unsalted and salted fish fillets before digestion, expressed as molar percentage of the different kind of acyl groups, and the content of the several kinds of lipolytic products present in digested lipid extracts were determined from 1H NMR spectral data, as in previous studies.20,23 Study by SPME-GC/MS of the Headspace Composition. Solid Phase Microextraction. The extraction of the volatile components of the headspace of the several samples (0.5 g in 10 mL screw-cap vial) was accomplished automatically using a CombiPAL autosampler (Agilent Technologies, Santa Clara, CA, USA). These were sea bass samples before digestion (U, BS, DS), digestive juices employed and submitted to digestion conditions in the absence of fish (J), mixtures made up of juices submitted to digestive conditions and of unsalted, brine-salted, or dry-salted sea bass before digestion mixed in the same proportions as in the digestates (U+J, BS+J, and DS+J), and fish digestates (DU, DBS, DDS). The fiber used was coated with DVB/ CAR/PDMS (50/30 μm film thickness, 1 cm long), acquired from Supelco (Sigma-Aldrich), which was inserted into the headspace of the sample and maintained for 60 min (50 °C). The selection of the fiber type (polarity and thickness of the coating) and of the extraction operating conditions (sample and headspace volumes, extraction time, and temperature) was previously studied in our laboratory in order to ensure reproducible and reliable results. The effectiveness of the SPME fiber used was periodically verified by testing its extraction efficiency with a reference sample of known composition. Gas Chromatography/Mass Spectrometry. The headspace components extracted and retained on the SPME fiber were desorbed, separated, identified, and semiquantified by means of GC/MS. To this aim, the fiber with the adsorbed compounds was injected into an Agilent Technologies gas chromatograph model 7890A equipped with a mass selective detector 5975C inert MSD with a Triple Axis Detector (Agilent Technologies) and a HP Compaq LE2202x computer operating with the ChemStation program. The column used was a fused-silica capillary column (60 m long × 0.25 mm inner diameter × 0.25 μm film thickness) coated with a nonpolar stationary phase (HP-5MS, 5% phenyl methyl siloxane) from Agilent Technologies. The operating conditions were as follows: the oven temperature was set initially at 50 °C (5 min hold) and increased to 290 °C at 4 °C/min (2 min hold), the temperatures of the ion source and the quadrupole mass analyzer were kept at 230 and 150 °C, respectively. Helium was used as carrier gas at a pressure of 18.611 psi, the injector temperature was held at 250 °C, and the splitless mode was used for injection with a purge time of 5 min. Mass spectra were recorded at an ionization energy of 70 eV, and the data acquisition mode employed was Scan. In order to avoid carry-over problems, after each run, the fiber was submitted to a 20 min bake out at 250 °C in the Fiber Cleaning and Conditioning Station of the CombiPAL autosampler. A reference sample of known composition was periodically analyzed in order to verify the reproducibility of the chromatographic runs. Most of the components were identified by using commercial standards (asterisked compounds in the tables) acquired from SigmaAldrich. When standards were not available, matching of the mass spectra with those obtained from a commercial library higher than 85% (W9N08, combined ninth edition of the Wiley Registry of Mass Spectral Data with National Institute of Standards and Technology (NIST) 2008 library) was taken as identification criteria. The semiquantification of the components was based on the area counts of the base peak (Bp) of the mass spectrum of each compound divided by 106. When the Bp of a compound overlapped with the same ion peak of the mass spectrum of another compound, an alternative ion peak was selected for the quantification of the former. Although the chromatographic response factor of each compound is different, the area counts are valid for comparative purposes because they reflect differences among the samples. Compounds detected having lower

MATERIALS AND METHODS

Fish Specimens. Four specimens of farmed European sea bass (Dicentrarchus labrax) were acquired from a local supplier within 48 h of harvest. The average body weight of the specimens was 1337.3 ± 51.1 g. On the same day of purchase, specimens were gutted, cleaned, filleted, and skinned. From each specimen, one fillet was maintained unsalted (U; n = 4) and the other fillet was submitted to salting. The average weight of sea bass fillet (n = 8) was 261.2 ± 12.1 g. Fish Salting. Two salting methods were carried out in this study: dry-salting and brine-salting. The dry-salted fillet (DS; n = 2) was obtained by covering it completely with coarse sea salt for 8 h at 4 °C, whereas the brine-salted one (BS; n = 2) was immersed for 15 min in a 15% brine-solution of salt at room temperature with a brine-to-fish proportion of 1:6 (w/v). Afterward, both fillets were rinsed with water to remove the remaining surface salt or brine. Salted fish fillets were vacuum-packed, frozen, and stored at −80 °C for up to 24 h until in vitro digestion experiments. In Vitro Digestion. Minced fish samples (4.5 g), either previously salted or not, were digested in duplicate, following an in vitro gastrointestinal model,18 slightly modified as described in detail before.19 This model involves a three-step procedure which simulates digestive processes in the mouth, stomach, and small intestine by adding the corresponding digestive juices in sequence. The transit times employed for oral, gastric, and duodenal in vitro digestion were 5 min, 2 h, and 4 h, respectively. Digestive juices (saliva, gastric juice, duodenal juice, and bile juice) were prepared artificially in accordance with the original model, although some modifications were performed in order to increase the lipolysis degree reached. These were the addition of lipase from Aspergillus niger to the gastric juice (100 U/ mL) and the use of a lower bile concentration in the bile juice (18.75 g/L). All the reagents for the preparation of the digestive juices were acquired from Sigma-Aldrich (St. Louis, MO, USA). Thus, the digestates obtained from the in vitro digestion of unprocessed sea bass were named DU (n = 8); those obtained from brine-salted sea bass samples were named DBS (n = 4), and those from dry-salted ones were named DDS (n = 4). In addition, blank samples, corresponding to digestive juices submitted to in vitro digestion conditions in the absence of fish (J) were also undertaken (n = 4) for further analysis. Fish Lipid Extraction and Study by 1H NMR. Lipid Extraction. Lipids from sea bass fillets before digestion (U, BS, DS), from the digested fish samples (DU, DBS, DDS), and from the juices submitted to digestion conditions in the absence of food (J) were extracted with dichloromethane (CH2Cl2, Sigma-Aldrich), as in previous studies.20 The average lipid content per fillet was 7.5 ± 1.2% (ww). 1 H NMR Spectra Acquisition and Derived Data. 1H NMR spectra of lipid extracts were recorded in duplicate on a Bruker Avance 400 spectrometer operating at 400 MHz. The sample preparation 880

DOI: 10.1021/acs.jafc.6b04334 J. Agric. Food Chem. 2017, 65, 879−891

Article

Journal of Agricultural and Food Chemistry values than 50000 area counts were not considered and are indicated in the tables as traces (tr). Statistical Analysis. Statistical analysis was performed using the statistical package SPSS v.22 (IBM, NY, USA). The significance of the differences in the molar percentages of lipolytic products among samples was determined by one-way variance analysis followed by post hoc Tukey b test at 0.05 threshold.

observed in this region in any of the spectra (see U, Bs, Ds), evidencing that none of the salting processes performed provoked a lipid oxidation detectable by this technique. Neither were any signals detected in the spectral region of aldehydic protons (see spectral region at 9.2−9.9 ppm in Figure 1). This is in total agreement with previous studies carried out in our laboratory concerning the salting of European sea bass and Atlantic salmon.13,15 1.2. Extent of Lipolysis Reaction during Fish in Vitro Digestion. Sea bass lipids before digestion consisted mainly of triglycerides, but during digestion some of them were hydrolyzed, yielding diglycerides, monoglycerides, fatty acids, and glycerol. As is well-known, this hydrolytic process is catalyzed by lipases present in gastric, and especially in duodenal digestive juices. To evaluate its extent, the molar percentages of fatty acids and acyl groups bounded to the different kinds of glycerides in relation to the total number of moles of fatty acids plus acyl groups present in the lipid extracts of nondigested and digested fish samples were determined by 1 H NMR. These are shown in Table 1. As can be observed, a very high degree of lipolysis was reached during in vitro digestion of unsalted sea bass (DU). Approximately 95% of triglycerides underwent a hydrolysis reaction, and the average value of fatty acids released was 62.2%, in line with a previous study.19 These values are very similar to those obtained in the lipid extracts of brine-salted (DBs) and dry-salted sea bass digestates (DDs), which indicates that under the conditions of this study neither the salting process nor the intensity of this latter affected the advance of fish lipid hydrolysis. 1.3. Generation of Lipid Oxidation Products during Fish in Vitro Digestion. The oxidation status of the several digested fish samples was also studied by 1H NMR, and the potential occurrence of lipid oxidation during digestion was evaluated by comparing the spectra of fish lipids extracted before (U, Bs, Ds) and after digestion (DU, DBs, DDs). As Figure 1 shows, new signals due to primary oxidation compounds appear in the spectra of lipid extracts (see U and DU) after in vitro digestion of unsalted sea bass. The main signals are centered at 6.00 and 6.58 ppm, named “a”, due to two protons of the (Z,E)-conjugated dienic sytem supported on chains also having hydroperoxy groups.24 In addition, also in very low intensity, signals “b” appeared at 6.48 and 5.98 ppm. These are due to the same two protons of the (Z,E)-conjugated dienic sytem but supported on chains having hydroxy groups.24 It must be noted that these conjugated double bonds supported on chains also having hydroperoxy- or hydroxy-groups can be located in either dienes or trienes, which are generated in the oxidation of polyunsaturated ω-6 or ω-3 acyl groups, respectively. None of these signals were observed in the spectra of the lipids extracted from the juices (J) submitted to the same digestion process. Thus, it is proved that an oxidation process took place during in vitro digestion of fish. In the general scheme of lipid oxidation, these oxidation products (conjugated dienic systems associated with hydroperoxy and hydroxy groups) are considered to form at the initial stages of the process. In this sense, it must be noted that oxidation reactions took place to a very low extent during sea bass in vitro digestion, considering the low intensity of signals a and b related to the above-mentioned primary oxidation compounds and the absence of aldehydic proton signals in the spectral region at 9.2−9.9 ppm. This latter fact evidences that if typical secondary oxidation products were formed, these are in

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RESULTS AND DISCUSSION Apart from hydrolysis, the occurrence of other chemical reactions undergone by food components during digestion has been little studied. In recent years, attention has been given to oxidation reactions which take place under gastrointestinal conditions,1,2 although other reactions cannot be discarded. As previously commented, fish samples before and after digestion were studied by means of two different techniques: one able to provide information about the hydrolysis degree and the nature of primary and secondary lipid oxidation products, namely 1H NMR; and the other able to detect volatile secondary oxidation products and other markers coming from different processes, which is to say SPME-GC/MS. 1. Information Obtained from 1H NMR Study. 1.1. Lipid Composition and Oxidation Status of Fish Samples before Digestion. The lipid extracts of farmed sea bass fillets, either unsalted (U), brine-salted (Bs), or dry-salted (Ds), were studied by 1H NMR, and their lipid composition was estimated. The molar percentages of the several kinds of acyl groups in U were 21.6 ± 0.4% of saturated and 78.4 ± 0.4% of total unsaturated acyl groups; these latter consisted of 41.5 ± 0.4% of oleic plus other minor unsaturated, 22.7 ± 0.4% of diunsaturated ω-6 (mainly linoleic), 0.1 ± 0.0% of ω-1, and 14.1 ± 0.2% of polyunsaturated ω-3 acyl groups; the main ω-3 lipids were DHA (5.6 ± 0.1%) and EPA acyl groups (5.1 ± 0.1%). Immediately after salting (Bs, Ds), no changes in the lipid composition of sea bass were noticed (data shown in Table S1 in the Supporting Information), which proves that if oxidation of fish lipids occurred as a result of brine- or drysalting, it was to a very little extent. This is also corroborated by the analysis of the 1H NMR spectral regions where oxidation products give signals. Figure 1 shows the enlargements from 5.8 to 6.7 ppm of these 1H NMR spectra where proton signals related to primary oxidation compounds could be visible, if present. No signals were

Figure 1. Enlargement of the region between 5.9 and 6.9 ppm and between 9.2 and 9.8 ppm of the 1H NMR spectra of the lipid extracts of unsalted and salted sea bass before digestion (U, BS, DS) and of digested sea bass samples, either unsalted (DU), brine-salted (DBS), or dry-salted (DDS): multiplets due to protons of (Z,E)-conjugated dienes supported in chains having hydroperoxy (a) or hydroxy (b) groups. 881


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Table 1. Molar Percentages of Acyl Groups (AG) Supported on the Different Kinds of Glycerides, Together with That of Fatty Acids (FA) Found in the Lipid Extracts of Unsalted (U), Brine-Salted (Bs), and Dry-Salted (Ds) Fish Samples before and after in Vitro Digestion (DU, DBs, DDs)a AGTG% AG1,2‑DG% AG2‑MG% AG1‑MG% FA%

U

Bs

Ds

DU

DBs

DDs

99.1 ± 0.2a − − − 0.9 ± 0.2a

99.2 ± 0.3a − − − 0.8 ± 0.3a

99.8 ± 0.3a − − − 0.2 ± 0.3a

5.7 ± 2.5b 18.4 ± 2.4a 10.0 ± 0.7a 3.8 ± 0.4a 62.2 ± 4.4b

5.8 ± 2.6b 16.6 ± 0.6a 10.1 ± 0.1a 3.5 ± 0.3a 63.9 ± 2.8b

6.1 ± 2.1b 18.3 ± 0.4a 9.9 ± 0.4a 4.3 ± 0.4a 61.3 ± 1.7b

a Different letters within each row indicate a statistically significant difference (p < 0.05). Abbreviations: TG: triglycerides; DG: diglycerides; MG: monoglycerides.

concentrations which are below the limit of detection of 1H NMR spectroscopy. The formation of lipid-derived hydroxy-dienes has been previously observed during in vivo digestion in rats.25 Certain authors consider these compounds to be biomarkers of in vivo oxidative stress of high physiological relevance.26 The fact that lipid-derived hydroxy-dienes are also formed during in vitro digestion would indicate that similar oxidation pathways and derived oxidation compounds are generated in both in vivo and in vitro systems, corroborating the usefulness of the results obtained using in vitro methodologies. In the lipid extracts of salted fish digestates (DBS, DDS), the same signals a and b were newly detected (see Figure 1). Therefore, the same kind of lipid oxidation products were generated under simulated gastrointestinal conditions from unsalted and salted sea bass. In order to evaluate to what extent fish salting influences the lipid oxidation level reached during its digestion, integration of the corresponding areas was carried out. Quantitative data obtained did not show any significant difference among the three kinds of digestates regarding the intensity of signals a and b. Thus, considering the evidence provided by 1H NMR, fish salting does not seem to favor oxidation reactions occurring under in vitro gastrointestinal conditions. 2. Information Obtained from SPME-GC/MS Study: Changes Provoked by in Vitro Digestion. Due to its high sensitivity, the information extracted by SPME-GC/MS can reinforce the above results, which were obtained by 1H NMR, and even provide further information not only about the lipid oxidation process undergone during digestion, but also about other reactions that may occur. First, the headspace composition of minced fish samples before digestion (U, Bs, Ds) was also studied by SPME-GC/ MS, and it showed that before digestion they had similarly low oxidation levels. This is in total accordance both with two recently published studies carried out in our laboratory where the subject of the study was the effect of dry- and brine-salting on the oxidative stability of farmed and wild European sea bass by means of SPME-GC/MS,16,27 as well as with the information extracted from the 1H NMR study regarding the composition and oxidation status of U, Bs, and Ds lipid extracts. However, 0.5 g of minced sea bass samples and 0.5 g of digestates not only have totally different matrices, but also contain a different amount of lipids. For this reason, mixtures made of fish samples before digestion and juices submitted to digestive conditions in the absence of food, in the same proportions as in the digestates, were prepared and their headspace studied. This was performed in order to obtain nondigested and digested samples showing a similar matrix and

with the same lipid content, whose headspace composition could thus be compared. Tables 2 and 3 and S2 show the volatile compounds identified in digestive juices (J) employed, in the mixtures prepared (U+J, BS+J, DS+J), and in the three kinds of digestates (DU, DBS, DDS). It must be noted that saliva, gastric, duodenal, and bile juices employed for in vitro digestion are mainly composed of water and inorganic salts, although they also contain the following components in negligible amounts: lipids (mainly oleic and saturated acyl groups, and lower proportions of linoleic and phospholipids, among others); proteins (enzymes and bovine serum albumin); and sugars. As a consequence, the formation of volatile compounds coming from these juice components cannot be ruled out. In fact, the submission of juices to the digestion conditions in the absence of fish meat proved this fact. For this reason, in order to differentiate between volatile compounds arising from fish components and from juices components, their origin is indicated in the above-mentioned tables. Figure 2 shows the total ion chromatograms of the headspace of digested samples (DU, DBS, DDS) and of the mixture made of unsalted sea bass before digestion with juices submitted to digestive conditions in the absence of fish (U+J). 2.1. Occurrence of Oxidation during in Vitro Digestion of Unsalted Sea Bass. Volatile Markers of Fish Lipid Oxidation. The main volatile components coming from lipid oxidation identified in the above-mentioned samples are summarized in Table 2. As can be observed, the headspace composition of the mixture U+J and that of J were very similar. This fact indicates again the low content of volatile compounds in unsalted sea bass fillets before digestion (U), in agreement with previous studies,16,27 especially regarding typical fish lipid oxidation markers, such as 1-penten-3-ol, (Z)-octa-1,5-dien-3-ol, hexanal, 2,4-heptadienals, 1-penten-3-one, or 2,3-pentanedione.27,28 Nevertheless, when comparing the headspace composition of U+J and DU, the increased abundance of a great number of volatile compounds arising from lipid oxidation is evident. This can be attributed to oxidation reactions occurring during digestion. Indeed, during lipid oxidation, volatile secondary oxidation products such as alcohols, acids, aldehydes, ketones, and furan derivatives are generated, and compounds belonging to all these kinds of families were detected in much higher (p < 0.05) abundances in DU than in the U+J and J headspace. This indicates that the main origin of the compounds generated during in vitro digestion of U samples was fish lipids instead of those lipids present in negligible amounts in juices. With reference to alcohols, it is worth noting the presence of 1-penten-3-ol (peak 1 in Figure 2), which is a well-known compound arising from the oxidation of polyunsaturated ω-3 groups; likewise, other oxidation markers mostly derived from 882


58 43 43 44 70 41 57 56 70 55 41 41 41 70 55 70 70 81 81 81 41 81

F F/J F F F F/J F/J F F F F/J F F F F

45 60 60 60 60 60

59 57 42 56 57 57

Bp

F/J F F/J F/J F/J F F/J

F F F F F F/J

Acetic acid (60)* Butanoic acid (88)* Butanoic acid, 3-methyl- (102) or is Pentanoic acid (102)* Hexanoic acid (116)* Octanoic acid (144)*

Alkanals Propanal (58) Butanal (72)* Pentanal (86)* Hexanal (100)* Heptanal (114)* Octanal (128)* Nonanal (142)* Alkenals (E)-2-Propenal (56) (E)-2-Butenal (70) (E)-2-Pentenal (84)* (E)-2-Hexenal (98)* (Z)-4-Heptenal (112)* (E)-2-Heptenal (112)* (E)-2-Octenal (126)* (E)-2-Nonenal (140)* (E)-2-Decenal (154)* (E)-2-Undecenal (168) Alkadienals 2,4-Hexadienal (96) (Z,E)-2,4-Heptadienal (110) (E,E)-2,4-Heptadienal (110)* (E,E)-2,6-Nonadienal (138) (E,E)-2,4-Nonadienal (138)*

F F/J F F/J F/J F

Origin

1-Propanol (60) 1-Penten-3-ol (86)* 1-Pentanol (88)* 1-Hexanol (102)* 1-Octen-3-ol (128)* (Z)-Octa-1,5-dien-3-ol (126)

Compound (molecular weight)

b

33.3 ± 1.7ab − 8.9 ± 3.8a 26.3 ± 12.6a 2.0 ± 0.5a − 4.5 ± 2.0a − 1.8 ± 0.7b − − − − 2.1 ± 1.1a − − − − − − − −

− 114.3 ± 13.7a − − − 1.6 ± 0.3a 2.3 ± 0.3a − − −

883

1.1 ± 0.4a − − − −

− − − − −

− 3.5 ± 0.0b − − − − 1.7 ± 0.2a − − −

29.2 ± 1.2ab − 7.0 ± 1.7a 26.5 ± 5.1a 1.8 ± 0.5a − 4.9 ± 0.3a

− − − − −

− 3.9 ± 0.6b − −− − 1.2 ± 0.7a − − −

29.1 ± 1.1ab − 7.6 ± 1.8a 29.5 ± 2.7a 1.5 ± 0.5a − 4.0 ± 0.4a

5.8 ± 1.1a − − − 0.7 ± 0.1a 2.8 ± 0.1a

Acids 2.8 ± 1.3a 6.9 ± 0.8a − − − − − − 0.9 ± 0.4a 0.7 ± 0.2a 2.1 ± 1.4a 2.0 ± 0.1a Aldehydes

− − − − − 0.9 ± 0.4a

27.0 ± 1.0a − 7.8 ± 0.5a 33.3 ± 0.2a 2.5 ± 0.6a − 3.3 ± 0.9a

− 14.8 ± 1.7a 7.0 ± 0.7a 5.4 ± 1.2a 8.9 ± 0.5a −-

± ± ± ± 2.1a 0.9a 0.0a 0.2a

− 8.1 5.8 3.9 5.8 −

− 11.5 ± 2.3a 10.3 ± 3.2a 5.6 ± 2.0a 8.5 ± 2.0a −

DS+J

− 3.2 ± 1.0a − 5.4 ± 2.2a 10.8 ± 1.1a −

BS+J

U+J Alcohols

Nondigested fish samples

J

Juices

5.0 ± 0.4b 13.1 ± 0.2a 34.3 ± 2.0a 6.8 ± 0.3a 2.3 ± 0.2a

1.2 ± 0.3a 33.9 ± 9.3c 30.7 ± 0.3a 22.8 ± 1.6a 4.4 ± 1.3a 21.9 ± 3.3b 18.2 ± 1.1b 2.4 ± 0.3a 4.7 ± 0.4a 0.9 ± 0.0a

41.0 ± 8.7bc 4.4 ± 0.9a 162.3 ± 10.9b 470.4 ± 6.8b 34.0 ± 0.1b 34.1 ± 4.8a 42.4 ± 3.8b

23.1 ± 4.1b 0.9 ± 0.3a 0.5 ± 0.1ab 1.5 ± 0.2a 7.6 ± 0.5b 9.4 ± 2.8b

6.0 ± 1.5a 463.0 ± 35.3b 73.5 ± 1.4c 58.8 ± 4.8b 183.6 ± 11.5b 18.4 ± 1.6a

DU

7.0 ± 1.2c 17.2 ± 0.7b 38.7 ± 9.4b 12.9 ± 3.0b 3.4 ± 0.1b

1.6 ± 0.6a 47.0 ± 0.4d 51.4 ± 0.0b 31.8 ± 6.4b 10.6 ± 2.6b 41.6 ± 13.5c 27.1 ± 5.7c 3.9 ± 1.0b 11.0 ± 2.8b 1.5 ± 0.2b

48.9 ± 2.2c 9.5 ± 2.6b 247.8 ± 43.4c 450.8 ± 131.6b 41.7 ± 9.3b 49.3 ± 10.6b 67.1 ± 3.0c

26.2 ± 4.5b 0.7 ± 0.4a 1.1 ± 0.3bc 0.7 ± 0.1b 5.5 ± 0.6c 17.6 ± 4.9bc

15.6 ± 8.2b 436.9 ± 12.4b 56.3 ± 2.6b 125.4 ± 14.6c 283.5 ± 57.4c 27.2 ± 8.5ab

DBS

Fish digestates

9.0 ± 0.8d 16.8 ± 0.8b 54.9 ± 1.6c 14.0 ± 2.6b 2.6 ± 0.2c

1.3 ± 0.3a 50.1 ± 0.0d 59.5 ± 0.4c 44.5 ± 0.0c 15.1 ± 0.9c 56.8 ± 0.8d 35.5 ± 2.3d 4.7 ± 0.8b 10.1 ± 0.4b 1.4 ± 0.2b

41.5 ± 6.8bc 10.2 ± 3.4b 288.3 ± 15.2c 652.5 ± 2.5c 55.6 ± 1.8c 65.2 ± 5.3c 67.2 ± 9.8c

28.8 ± 6.8b 1.5 ± 0.2a 1.6 ± 0.8c 1.3 ± 0.2a 8.5 ± 0.6b 23.8 ± 10.4c

13.8 ± 7.8b 480.2 ± 22.0b 68.0 ± 5.0c 159.4 ± 4.8d 417.8 ± 23.5d 29.4 ± 4.1b

DDS

Table 2. Main Lipid Oxidation-Related Volatile Compounds Detected by SPME-GC/MS in the Headspace of Juices (J) Submitted to Digestion Conditions in the Absence of Food, Mixtures Made of Juices Submitted to Digestive Conditions and Unsalted (U+J), Brine-Salted (BS+J), or Dry-Salted (DS+J) Sea Bass before Digestion, And Digested Sea Bass Samples, Either Unsalted (DU), Brine-Salted (DBS) or Dry-Salted (DDS)a

Journal of Agricultural and Food Chemistry Article


884

68 82 81 81 81 81 107 81

55 43 43 43 43 55 43 43 43 55 95 58 95 58

81 81

Bp

0.5ab

0.7a

0.2a

0.9a 0.1a 0.5a 2.7a

− − − − − 1.9 ± 0.6a − −

− 9.7 ± 6.1 ± 1.0 ± 8.8 ± − Tr a Tr a 3.8 ± Tr a − 2.9 ± − 1.5 ±

− −

J

Juices Aldehydes − −

BS+J

Nondigested fish samples

Ketones − − 4.8 ± 3.0a 4.5 ± 0.7a 6.5 ± 2.6a 5.2 ± 1.8a 4.8 ± 1.6a 3.9 ± 0.3a 7.3 ± 2.8a 5.4 ± 0.3a − − Tr a Tr a Tr a Tr a 57.2 ± 12.9b 68.2 ± 1.8b − − − − 1.4 ± 0.7a 1.0 ± 0.1a − − 0.5 ± 0.3b 0.5 ± 0.1b Furan derivatives − − − − 0.9 ± 0.6a 1.1 ± 0.3a − − − − 4.6 ± 1.3a 4.3 ± 0.3a − − − −

− −

U+J

− − 1.0 ± 0.0a − − 3.5 ± 0.4a − −

− 4.8 ± 0.2a 5.9 ± 0.7a 3.2 ± 0.0a 5.2 ± 0.1a − Tr a Tr a 40.5 ± 6.1b − − 0.8 ± 0.2a − 0.4 ± 0.2b

− −

DS+J

0.6 ± 0.1a 2.2 ± 0.6a 91.9 ± 21.4b 2.1 ± 0.0a 2.1 ± 0.1a 43.1 ± 10.5b 7.7 ± 1.4a 0.6 ± 0.2a

57.5 ± 4.4a 14.4 ± 0.6b 45.0 ± 1.3b 16.9 ± 2.8b 20.8 ± 0.4b 2.2 ± 0.1a 2.3 ± 0.1b 2.1 ± 0.8b 108.7 ± 18.5c − 12.3 ± 1.4a 9.4 ± 0.2b 8.7 ± 0.3a 1.7 ± 0.0a

3.8 ± 0.3a 7.4 ± 0.7ab

DU

0.7 ± 0.1a 3.4 ± 0.2b 132.6 ± 0.2c 5.0 ± 0.6b 3.4 ± 0.4b 68.7 ± 13.2c 14.7 ± 2.4b 1.4 ± 0.0b

64.5 ± 0.0b 19.6 ± 1.0bc 59.1 ± 4.0c 21.5 ± 2.2c 34.8 ± 5.8c 3.7 ± 0.8b 4.2 ± 0.9c 3.7 ± 1.3bc 140.0 ± 8.3d 13.6 ± 5.2b 18.6 ± 0.3b 23.5 ± 3.9c 20.7 ± 4.7b 3.2 ± 0.7c

4.1 ± 0.1a 10.2 ± 4.4a

DBS

Fish digestates

0.7 ± 0.2a 3.1 ± 0.0ab 113.7 ± 2.8c 6.0 ± 0.4b 3.7 ± 0.9b 54.5 ± 1.7bc 12.6 ± 0.1b 2.6 ± 0.0c

66.5 ± 1.2b 23.0 ± 0.9c 62.7 ± 2.1c 28.0 ± 0.3d 51.8 ± 0.1d 6.9 ± 0.4c 4.1 ± 1.4c 4.0 ± 1.0c 226.5 ± 3.5e 14.7 ± 0.3b 21.5 ± 1.5c 24.6 ± 1.6c 18.5 ± 2.1b 3.8 ± 0.0c

4.0 ± 0.1a 5.3 ± 0.4b

DDS

a Data are expressed as average area counts of their mass spectral base peak (Bp) divided by 106, together with their standard deviation. Abbreviations: −: not detected: tr: traces; is: isomer. Asterisked compounds (*) were acquired commercially and used as standards for identification purposes. bF: fish; J: juices submitted to digestive conditions.

F F F F F F/J F F

F F/J F/J F/J F/J F F/J F/J F/J F/J F F/J F F/J

1-Penten-3-one (84) 2-Pentanone (86)* 2,3-Pentanedione (100)* 2-Hexanone (100)* 2-Heptanone (114)* Cyclohexanone (98) 2-Heptanone, 4-methyl- (128) 2-Heptanone, 6-methyl- (128) 2,5-Octanedione (142) 3-Octen-2-one (126) 3,5-Octadien-2-one (124) 2-Nonanone (142)* 3,5-Octadien-2-one (124) is 2-Decanone (156)*

Furan (68) Furan, 2-methyl- (82) Furan, 2-ethyl- (96) Furan, 2-propyl- (110) Furan, 2-butyl- (124) Furan, 2-pentyl- (138)* Furan, 2-(2-pentenyl)- (136) Furan, 2-hexyl- (152)

F F

Originb

(Z,E)-2,4-Decadienal (152)* (E,E)-2,4-Decadienal (152)*


Table 2. continued



885

F

Octanoic acid, ethyl ester (172)

88

0.5 ± 0.1a Tr a − 24.3 ± 10.4ab − 221.1 ± 94.0b 0.5 ± 0.1b Esters 0.3 ± 0.5a

33.6 ± 8.3ab − 538.3 ± 56.3a 1.1 ± 0.3ab

4.4 ± 2.4b 4.4 ± 1.8b

19.9 ± 1.6a 11.1 ± 2.2a − Tr a −

2.4 ± 0.4a 9.8 ± 3.6b 4.4 ± 2.7b

−

BS+J

Nondigested fish samples

−

21.0 ± 1.4a − 190.1 ± 14.2b 0.4 ± 0.1b

0.4 ± 0.0a Tr a −

3.7 ± 0.5b 4.8 ± 0.9b

2.6 ± 0.6a 10.3 ± 1.0b 5.0 ± 0.4b

BHT and derived metabolites 219.3 ± 61.4b 172.0 ± 2.4b 3.3 ± 0.9a 3.0 ± 0.0a 0.3 ± 0.1b 0.2 ± 0.0b 0.1 ± 0.0a 0.1 ± 0.0a − − 0.8 ± 0.1b 0.4 ± 0.1b Amino acid derivatives

U+J

2.9 ± 0.3a 19.2 ± 3.2a 14.2 ± 0.7a

0.2 ± 0.0a − 1.0 ± 0.3a − − 59.7 ± 13.5a

J

Juices

−

21.9 ± 1.2a − 235.4 ± 0.3b 0.5 ± 0.1b

0.4 ± 0.0a Tr a −

3.8 ± 0.6b 5.5 ± 0.4b

2.5 ± 0.3a 11.6 ± 1.1b 5.8 ± 0.8b

153.6 ± 8.1b 2.5 ± 0.2a 0.2 ± 0.0b 0.1 ± 0.0a − 0.7 ± 0.2b

DS+J ± ± ± ± ± ± 4.2a 0.0b 0.2a 0.3b 0.0a 1.4c

± ± ± ±

1.2abc 0.4a 1.9b 0.2a 0.8 ± 0.0a

42.9 2.0 228.1 1.2

0.5 ± 0.1a 0.4 ± 0.0a 0.3 ± 0.3a

60.3 ± 2.2c 11.6 ± 0.7a

4.4 ± 1.7b 18.5 ± 1.1a 7.2 ± 0.7ab

8.2 0.3 1.4 1.4 0.1 3.7

DU ± ± ± ± ± ± 1.4a 0.1b 0.1a 0.3b 0.0a 0.9c

± ± ± ±

11.5bc 0.4b 70.7ab 0.2a 2.5 ± 0.6b

49.7 2.7 348.1 1.2

1.0 ± 0.0b 1.1 ± 0.9ab 1.0 ± 0.3b

81.6 ± 23.1 cd 18.0 ± 4.0c

8.0 ± 2.7c 24.0 ± 0.7a 8.7 ± 0.8ab

16.6 0.4 1.6 1.8 0.1 3.9

DBS

Fish digestates

± ± ± ± ± ±

0.3a 0.1b 0.2a 0.2b 0.0a 0.8bc

± ± ± ±

0.5c 0.1b 26.4ab 0.2a 2.3 ± 0.3b

61.5 3.2 382.2 1.5

1.2 ± 0.2c 1.6 ± 0.4b 1.6 ± 0.0c

85.1 ± 0.4d 21.1 ± 0.7c

9.2 ± 1.0c 23.6 ± 0.6a 9.1 ± 0.7ab

21.8 0.5 1.5 1.5 0.1 2.5

DDS

Data are expressed as average area counts of their mass spectral base peak (Bp) divided by 106, together with their standard deviation. Abbreviations: −: not detected; Tr: traces; is: isomer. Asterisked compounds (*) were acquired commercially and used as standards for identification purposes. bF: fish; J: juices submitted to digestive conditions.

a

93 106 106 135

106 91

F/J F/J

F/J F J J

41 43 57

F/J J J

47 94 48

205 161 177 165 219 191

Bp

F F/J F

F/J F F/J F F F/J

Originb

Alkylated alkanals Propanal, 2-methyl- (72) Butanal, 3-methyl- (86) Butanal, 2-methyl- (86) Aromatic aldehydes Benzaldehyde (106)* Benzeneacetaldehyde (120)* Sulfur derivatives Methanethiol (48) Disulfide, dimethyl- (94) Methional (104) Nitrogenated compounds Pyridine, 2-methyl- (93)* Pyridine, 2-ethyl- (107)* Pyridine, 5-ethyl-2-methyl- (121) or is Pyrazine, 3-ethyl-2,5-dimethyl- (136) or is

BHT (220)* BHT-QM (218)* BHT-Q (220)* BHT-OH (236)* BHT-CHO (234)* DBP (206)*


Table 3. Other Volatile Compounds of Interest Detected by SPME-GC/MS in the Headspace of Juices (J) Submitted to Digestion Conditions in the Absence of Food, Mixtures Made of Juices Submitted to Digestive Conditions and Unsalted (U+J), Brine-Salted (BS+J), or Dry-Salted (DS+J) Sea Bass before Digestion, and Digested Sea Bass Samples, Either Unsalted (DU), Brine-Salted (DBS), or Dry-Salted (DDS)a



Article


Figure 2. Total ion chromatograms obtained by SPME-GC/MS of the headspace components of farmed sea bass digestates, either unsalted (DU), brine-salted (DBS), or dry-salted (DDS), together with that of the mixture of unsalted sea bass before digestion with juices submitted to digestive conditions (U+J). Peaks identified as 1: 1-penten-3-ol; 2: pentanal; 3: 2-ethylfuran; 4: (E)-2-pentenal; 5: 4-methylheptane; 6: hexanal; 7: 2,4dimethyl-1-heptene; 8: (E)-2-hexenal; 9: 1-hexanol; 10: heptanal; 11: (E)-2-heptenal; 12: 1-octen-3-ol; 13: octanal; 14: 5-ethyl-2-methylpyridine or isomer; 15: (E)-2-octenal; 16: nonanal; 17: pentadecane; 18: BHT; *: fiber coating-derived compounds.

linoleic acyl groups, such as 1-octen-3-ol (peak 12) and 1hexanol (peak 9), were found.29−31 Most acids detected in the headspace of DU were generated during sea bass in vitro digestion, because they were absent or showed significantly lower abundance (p < 0.05) in the juices submitted to digestion conditions in the absence of food (J) and in the mixtures of juices and nondigested unsalted sea bass (U+J), as can be observed in Table 2. Acetic acid and octanoic

acid were the main ones. This latter has been previously identified as a volatile decomposition product of triolein and trilinolein under frying conditions.32 However, the low abundances generally found could be expected due to the mild conditions of the digestion process (6 h at 37 °C). A large number of aldehydes, considered the main volatile secondary compounds formed in any lipid oxidation process, was also generated during fish in vitro digestion. Among them, 886


Article


Figure 3. (a) Main metabolites arising from the oxidation of 2,6-di-tert-butylhydroxytoluene (BHT): 2,6-di-tert-butyl-4-methyl-4-hydroperoxy-2,5cyclohexadien-1-one (BHT-OOH); 2,6-di-tert-butyl-2,5-cyclohexadien-1,4-dione (BHT-Q); 2,6-di-tert-butyl-4-hydroxy-4-methyl-2,5-cyclohexadien1-one (BHT-OH); 3,5-ditert-butyl-4-hydroxybenzaldehyde (BHT-CHO), according to previous studies.39,40 (b) Valine, leucine, isoleucine, phenylalanine, and methionine amino acids and some of their possible derived volatile metabolites.42,43,45

agreement with previous studies carried out by SPME-GC/MS on the in vitro digestion of nonoxidized and thermo-oxidized edible oils,10,11 in which oxygenated α,β-unsaturated aldehydes were not detected in the digestates of nonoxidized oils, and the abundance of those initially present in the thermo-oxidized oils was notably reduced after digestion. Another important family of volatile compounds generated in lipid oxidation processes is ketones. As shown in Table 2, compounds considered fish lipid oxidation markers, such as 2,5octanedione, 1-penten-3-one, and 2,3-pentanedione,27,28 were mainly generated. Note the occurrence of methyl-2-heptanones in the headspace of DU, when these are almost absent in J and U+J. 6-Methyl-2-heptanone has been previously identified in the headspace of oxidized Atlantic horse mackerel muscle,28 and recently, some authors have considered it a cholesterol oxidation marker.34 Furan and its alkyl derivatives were also identified in the headspace of DU, whereas they were almost absent in the corresponding mixture. Among others, they may come from the transformation of the corresponding lipid-derived α,β-unsaturated aldehyde, like 2,4-alkadienals or 2-alkenals.35 As shown in Table 2, the main alkylfurans generated during digestion of minced sea bass were 2-ethylfuran (see peak 3 in Figure 2) and 2-pentylfuran, derived from ω-3 and ω-6 acyl groups,

hexanal and pentanal, derived mainly from linoleic acyl groups,32,33 show the highest abundances in digested sea bass samples (DU), as can be observed in peaks 6 and 2, respectively, of DU chromatogram in Figure 2. Concerning unsaturated aldehydes, the DU headspace contained alkenals from 3 to 11 carbon atoms and alkadienals from 6 to 10 carbon atoms. Among them, those mainly derived from ω-3 acyl groups oxidation, that is (E)-2-butenal, (E)-2-pentenal (see peak 4 in Figure 2), and 2,4-heptadienals, were in higher abundance than those related to ω-6 acyl groups degradation, such as (E)-2-hexenal, (E)-2-heptenal, and 2,4-decadienals.29,31−33 This is explained by the higher susceptibility to oxidation of ω-3 than of ω-6 lipids. The fact that most of the above-mentioned unsaturated aldehydes were absent in U+J samples (with the exception of (E)-2-butenal and (E)-2octenal), confirms the increase of the sea bass lipid oxidation level during digestion. Although a great number of α,β-unsaturated aldehydes were detected in the headspace of fish digestates, it must be pointed out that no toxic oxygenated α,β-unsaturated aldehydes, like 4hydroxy-2-hexenal or 4-hydroxy-2-nonenal, were found. If generated, they would probably have reacted through the oxygenated groups or the double bond with phospholipids, fish proteins, or digestive enzymes, yielding Schiff bases, Michael adducts, or other derived compounds. These results are in 887


Article


(dry-salting) undergo oxidation to a highest extent during in vitro digestion. 2.4. Evolution during in Vitro Digestion of the Abundance of Antioxidants and Derivatives Present in Sea Bass. Table 3 shows the abundance of BHT (peak 18 in Figure 2) and some of its metabolites found in the headspace of unsalted and salted sea bass samples before and after digestion. The formation pathways of some of the latter from BHT are indicated in Figure 3a. The presence of this synthetic antioxidant and some of its metabolites in the headspace of farmed sea bass has been described before27 and could be due to its incorporation via fish feed.38 BHT was also present in the juices (J), but in very low concentrations compared with fish meat. However, one of its metabolites (BHT-Q) and another antioxidant (DBP) were found in J in higher abundances than BHT was. The presence of DBP can be explained by its wide industrial use as antioxidant and as an ultraviolet stabilizer for hydrocarbonbased products, ranging from petrochemicals to plastics. In fact, it was evidenced that DBP was present in high abundances in certain organic components employed for the preparation of the juices, such as bovine serum albumin (≈300 × 106 area counts); α-amylase (≈30 × 106 area counts); pepsin (≈26 × 106 area counts); mucine (≈21 × 106 area counts); pancreatic lipase (≈18 × 106 area counts); pancreatin (≈16 × 106 area counts); and bovine bile extract (≈16 × 106 area counts). As can be observed in Table 3, slightly higher (p > 0.05) abundances of BHT were detected in the headspace of U+J than in those of BS+J and DS+J. This could be due mainly to the variability existing among fish fillets rather than to the occurrence of certain oxidation reactions during fish salting or during the preparation of mixtures, which would lead to decreased BHT abundance. In fact, the analysis of the headspace of the sea bass fillets before and after salting did not show clear differences in BHT abundance, and all the BHTmetabolites detected in the three kinds of mixtures were found in very similar abundances. It must be noted that they were already present, together with BHT, in the headspace of sea bass fillets before digestion. In spite of the above comment, a very noteworthy decrease in BHT abundance occurs during in vitro digestion of unsalted fish (compare U+J and DU in Table 3). This can be explained by its performance as antioxidant during the in vitro digestion process. Anyway, the results obtained evidenced that the amount of this phenolic compound initially present in farmed sea bass fillets was not able to completely avoid fish lipid oxidation during digestion. Moreover, four derived-metabolites were also identified in the headspace of DU: BHT-Q, BHT−OH, BHT-QM, and BHT-CHO. They can be formed during BHT oxidation,38 and bearing in mind the abundance of BHT in J and U+J, it could be considered that most of the BHT derivatives found in the digestates are due to the BHT contained in fish samples. As can be observed in Table 3, during in vitro digestion, the abundance of BHT-QM decreases, whereas that of BHT-OH, BHT-Q, and BHT-CHO increases. In fact, previous studies reported that the major oxidation product of BHT is BHT-OOH,39 which in turn is degraded under thermolysis conditions into BHT-OH, BHTQ, and BHT-CHO.40 These results suggest that similar BHT decomposition pathways could be followed under in vitro digestion conditions, as Figure 3a shows. Nonetheless, the formation of other nonvolatile BHT metabolites cannot be discarded.

respectively; 2-(2-pentenyl)-furan was also detected, which also may be formed during ω-3 acyl groups oxidation.29 Although the origin of hydrocarbons is uncertain, the abundances detected generally increased after fish in vitro digestion (see U+J and DU in Table S2 in the Supporting Information). Their generation could be related to the slight oxidation process27 taking place during digestion and/or to other reactions, such as decarboxylation. In fact, it must be noted that a great number of hydrocarbons were already detected in the headspace of minced sea bass before digestion. The aliphatic hydrocarbons that show the highest abundances in the digestates are 2,4-dimethyl-1-heptene (see peak 7 in Figure 2), 4-methylheptane (see peak 5), 2,2,4,6,6-pentamethylheptane, and 2,4-dimethylheptane. 2.2. Differences in the Oxidation Extent Reached during in Vitro Digestion of Unsalted and Salted Fish. The results obtained by SPME-GC/MS confirm those above-mentioned in the 1H NMR study: lipid oxidation takes place during in vitro digestion of unsalted sea bass. In order to analyze the potential differences occurring during digestion of unsalted and salted fish, the headspace composition of the digestates obtained from brine-salted (DBs) and dry-salted sea bass (DDs) was also analyzed. A greater degree of oxidation might take place under gastrointestinal conditions in the case of salted fish than in that of unsalted ones, bearing in mind previous results showing that under pro-oxidative conditions the oxidative stability of salted fish lipids is lower than that of unsalted fish lipids.13,15 As can be observed in Table 2, the headspace composition of the mixtures made of salted fish before digestion and juices submitted to digestive conditions (Bs+J, Ds+J) is very similar to that of the unsalted sea bass mixture (U+J). However, most of the compounds related to the lipid oxidation process presented significantly higher (p < 0.05) abundances in the headspace of salted fish digestates (DBs and DDs) than in the unsalted ones (DU). These include alcohols, such as 1-hexanol, 1-octen-3-ol, and (Z)-octa-1,5-dien-3-ol, which could be derived from ω-3 acyl groups oxidation;36 octanoic acid; almost all the alkanals, (E)-2-alkenals, and 2,4-alkadienals, being worth noting those above-mentioned coming from ω-3 acyl groups and (Z)-4-heptenal, which has been reported to come from 2,6-nonadienal;37 ketones like 3-octen-2-one, which was only detected in digested salted samples, and 3,5-octadien-2one, a well-known fish lipid oxidation product;28,31 and 2-(2pentenyl)-furan, that can be generated from ω-3 acyl groups.29 These results would clearly indicate that salted fish undergoes higher oxidation during digestion than the unsalted one. 2.3. Influence of Fish Salting Degree on the Advance of Oxidation Reactions during Digestion. When comparing the abundances found in the headspace of digested dry-salted (DDs) and brine-salted (DBs) sea bass, it can be noticed that, although very similar, almost all the alkanals, (E)-2-alkenals, and ketones identified showed higher values in DDS than in DBS. Moreover, oxidation pathways occurring during in vitro digestion of Ds may significantly (p < 0.05) favor the generation of 1-octen-3-ol, hexanal, previously considered as a fish lipid oxidation marker,27,28 and 2,5-octanedione. Thus, it seems that carrying out salting could provoke an enhancement of fish lipid oxidation during human digestion, regardless of the intensity of the method employed, leading inevitably to a loss of nutritive value of fish lipids and to increased generation of potentially reactive aldehydes. However, samples submitted to the most intense salting process 888


Article

Journal of Agricultural and Food Chemistry Regarding in vitro digestion of salted fish, the same trend was observed (see Bs+J and DBs, and Ds+J and DDs). 2.5. Occurrence of Amino Acid Degradation. Evidence of Maillard Type Reactions Markers. Volatile compounds potentially related to amino acid degradation, such as alkylated alkanals, aromatic aldehydes, sulfur-derivatives, and nitrogenated compounds were also detected in the headspace of nondigested and digested samples (Table 3). Their occurrence and/or their higher concentration in digested samples suggest that the in vitro digestion process promotes their formation by means of various reactions involving the loss of essential amino acids. Figure 3b shows some volatile compounds detected in the headspace of the digestates, together with their possible amino acid precursor. Three branched alkanals and two aromatic aldehydes were identified in the headspace of unsalted sea bass after in vitro digestion (DU). 2-Methylpropanal, 2-methylbutanal, 3-methylbutanal, and benzeneacetaldehyde are commonly known as Strecker aldehydes and can be generated from the deamination and decarboxylation of valine, isoleucine, leucine, and phenylalanine, either by enzymatic action or by reaction with carbonyl compounds produced in carbohydrate degradation or lipid oxidation reactions.41,42 Benzaldehyde may be formed by further oxidation of benzeneacetaldehyde,43 although its generation from linoleic or linolenic groups oxidation processes cannot be discarded.29,31 In spite of the occurrence of these five aldehydes in digestive juices, higher abundances are observed in DU than in U+J, suggesting that, in addition to digestive juices components, these compounds may also be generated from sea bass proteins. In fact, the nature of the proteolytic products generated during in vitro digestion of sea bass has been recently addressed, and among the amino acids (or residues) detected by 1H NMR, there were all the above commented.44 Furthermore, the authors reported a selective release of aromatic amino acids (or residues) during the intestinal step, which is in agreement with the significantly higher (p < 0.05) abundances of aromatic aldehydes reported in Table 3. Sulf ur-containing compounds such as methional, methanethiol, and dimethylsulfide were also identified in the headspace of digested sea bass. These compounds can also be produced from chemical or enzymatic degradation of methionine or cysteine.42,45 The low abundances detected can be explained by the low content of this kind of amino acids in sea bass (up to 2.7 g/100 g of protein).46 In addition, four nitrogen derivatives typically associated with Maillard reactions were detected in the headspace of DU. However, only one of them can be exclusively attributed to fish origin and not to digestive juice components. This is 2ethylpyridine, which is known to be formed due to the reaction between 2,4-heptadienal and amino acids.47 This compound was the main pyridine identified in the headspace of fish oil heated with cysteine and trimethylamine oxide.48 Thus, its presence in DU and its absence in U+J evidences that Maillardtype reactions take place during the digestion process, in agreement with previous in vitro studies on edible oils.10,11 Likewise, all these amino acid-derived compounds were also found in the headspaces of digested salted samples. Nonetheless, significantly higher (p < 0.05) abundances of 2methylpropanal, benzeneacetaldehyde, methanetiol, methional,and 2-ethylpyridine were found in DBS and DDS than in DU headspace. This would suggest that degradation reactions of amino acids occurring during in vitro digestion of sea bass may be enhanced by the salting process, although no clear effect of

the degree of salting was observed. Indeed, almost no significant (p < 0.05) differences were found between DDs and DBs headspaces. 2.6. Evidence of Esterification Reactions during in Vitro Digestion. Three esters were detected in the headspace of the digested sea bass, but only one of them, ethyl octanoate, was considered to have come from fish lipids due to its absence in juices submitted to digestive conditions (J in Tables 3 and S2). This compound might be derived from esterification reactions taking place among ethanol and octanoic acid, which are both present in the digestates, with the involvement of enzymes or not. The latter hypothesis is in accordance with previous studies in which the occurrence of this kind of reaction was described during the in vitro digestion of a commercial vegetable oil which contained small proportions of proteins (1%) and carbohydrates (

Fish in Vitro Digestion: Influence of Fish Salting on the Extent of

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