Determination of phenolic antioxidants in tuna fillets canned in hydrosols with HPLC-DAD Free

Abstract

Canned tuna was fortified with a mixture of brine and hydrosols of aromatic plants (i.e. oregano, laurel, sage and lemon balm). An HPLC-DAD method was developed and validated for the simultaneous determination of thirteen antioxidants in tuna fillets, including phenolic acids (gallic acid, vanillic acid, syringic acid and rosmarinic acid), flavonoids (catechin, epicatechin, vanillin, myricetin, rutin, quercetin, luteolin and apigenin) and one hydroxybenzaldehyde (syringaldehyde). The analytes showed satisfying recovery efficiency (82.1–92.1%), and the method presented excellent linearity (r² > 0.99). The precision limit was ≤5.6% RSD_r for intra-day and ≤7.2% RSD_R for inter-day experiments. The determined analytes ranged between 8.86 mg (quercetin) and 512 mg (rosmarinic acid) per 100g tuna flesh (n = 10), verifying that the hydrosols fortified the tuna fillets.

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Introduction

The demand for high-quality fish products is continuously growing. Fish and other seafood products are healthy food sources representing the third major source of dietary protein after cereals and milk (FAO, 2018). Τhe frequent consumption of fishery products has been associated with several health benefits, such as the reduced risk of coronary heart disease, lowering blood pressure and triglyceride concentration, protecting against breast and colorectal cancer as well as sustaining proper brain function and enhancing the immune system, as it has already been reviewed by Tacon et al. (2020). In this context, fish food could be characterised as nutraceutical, combining the words ‘nutrition’ and ‘pharmaceutical’.

Industries have begun to consider the use of plant antioxidants as potential food additives in foods because of their natural origin and good acceptance among consumers (Maqsood et al.,  2013). The most popular antioxidants presently used in the food industry are ascorbic acid, tocopherols and polyphenols (Kulawik et al.,  2013). The health-related properties of these naturally occurring phenolic bioactive constituents in various functional foods have already been reported (Kalogiouri & Samanidou, 2019; Kalogiouri et al.,  2020a; Kritikou et al.,  2020). As a consequence of these known health benefits, food scientists have focussed on the fortification of seafood with phenolic antioxidants. According to the literature, scientists have used rosemary extracts to fortify sardine (Ozogul et al.,  2011), gilthead seabream (Özyurt et al.,  2011) and surimi fillets (Pérez-Mateos et al.,  2006). Green tea extracts have been used for the fortification of surimi, rainbow trout (Saito et al.,  2002), mince horse mackerel (He & Shahidi, 1997) and tench fillets (Gai et al.,  2014). Furthermore, lemon, thyme and sage essential oils have been used to enrich swordfish fillets (Kykkidou et al.,  2009) and minced chub mackerel (Erkan & Bilen, 2010). Phenolic extracts from witch hazel were used as efficient antioxidants against lipid peroxidation of minced Atlantic mackerel (González et al.,  2010). However, these studies focus mainly on the use of phenolic antioxidants to extend the shelf life of seafood, without measuring the phenolic profile of the proposed product.

The challenge is to produce seafood fortified with bioactive phenolic microconstituents following the food industry's current trends towards the simple, low-cost and effective production of functional foods, according to consumers’ demands. An innovative idea would be to use alternative ingredients such as the by-products from the distillation of the essential oils for food fortification (Stübler et al.,  2020). The hydrosols (also known as flower or floral waters) are a mixture of a variable quantity of essential oil and water-soluble, secondary metabolites (Labadie et al.,  2015), exhibiting antioxidant activity due to the presence of phenolic microconstituents that characterise them with their functional groups (D’Amato et al.,  2018).

The objective of this work was to use for the first-time hydrosols as packaging constituents in canned tuna fillets and examine with chromatographic analysis if the tuna flesh was enhanced with phenolic antioxidants. Considering that tuna is an important food source an important food source with high economic value and extensive international trade, yellowfin tuna (Thunnus albacares) was selected as a case study and was canned in a mixture of brine and hydrosols of oregano (Origanum vulgare), laurel (Laurus nobilis), sage (Salvia officinalis) and lemon balm (Melissa officinalis). The fortified tuna fillets' phenolic profile was assessed six months after canning with the development of a novel high-pressure liquid chromatographic method coupled to diode array detector (HPLC-DAD). To the best of our knowledge, this is the first reported attempt of using hydrosols as natural additives for the production and further analysis of high-quality fortified tuna fillets, rich in phenolic antioxidants, that efficiently meet the increasing demand of modern consumers for novel functional seafood products.

Materials and Methods

The sections 2.1. Reagents and Standards; 2.2. Instrumentation; 2.3 Hydrosols preparation; and 2.4 Tuna fillets processing of Materials and Methods are described in the Supporting Information.

Sample preparation

Τhe tuna fillets were removed from the cans and were drained using a vacuum filtration device to remove the hydrosols based brine broth. Then, the drained fillets were homogenised in a porcelain mortar. Methanol and water are widely used for the extraction of phenolic compounds at different ratios, according to Kalogiouri et al. (2020d). The samples were extracted using a mixture of MeOH: H₂O (80:20, v/v) for the extraction of the phenolics from the matrix, according to Kritikou et al. (2020). Small pieces of 2 g from each homogenised sample were weighted and extracted twice with 5 mL of a mixture of MeOH:H₂O (80:20, v/v) in 15-mL centrifuge tubes. The centrifuge tubes with the homogenised tuna fillets and extractor were vortexed for 2 min, and the phenolic extraction was carried out in an ultrasonic bath for 15 min in 25 °C. Then, the samples were centrifuged for 5 min at 8000 rpm, and the upper methanolic phase was collected. The extraction procedure was repeated, and the methanolic extracts were combined and filtered through 0.22 μm nylon membrane syringe filters (QMax RR, Frisenette ApS). Finally, 20 μL of the extract was injected into the chromatographic system for analysis. The same extraction protocol was followed for ten samples of canned tuna fillets preserved in the same mixtures of brine and hydrosols (85:15, v/v), as well as for three samples of canned tuna fillets preserved solely in brine prior to analysis.

HPLC-DAD analysis

A LiChroCART-LiChrospher RP-C18 analytical column (250 × 4 mm, 5 μm particle size) from Merck Darmstadt was used to determine the phenolic compounds. The system was operated at 270 nm in gradient mode at 30 °C, using (i) 2% acetic acid in water, and (ii) MeOH: ACN (50:50, v/v), as mobile phases. The flow rate was set at 1 mL min⁻¹. The adopted elution gradient started with 15% of organic phase B and gradually increased to 35% B in the next 5 min, increasing to 50% up to 20 min, and finally increasing to 70% B until 35 min, and remaining constant for the next 13 min. Initial conditions (15% B) were restored within 48.01 min for 5 min to re-equilibrate the column for the next injection.

HPLC-DAD method development and validation

This section is described in the Supporting Information.

Results

Method validation results

All the analytical parameters of the HPLC-DAD method including linearity (calibration curves and regression coefficient (r²)), LODs and LOQs, precision (expressed as inter-day (RSD_r) and intra-day precision (expressed as intra-day precision RSD_R)) and recoveries (RE%) were calculated and are presented in the Supporting Information (Table S1). The calibration curves were linear with r² > 0.999 in all cases. The precision limit was ≤5.6% RSD_r for intra-day experiments and ≤7.2% RSD_R for inter-day experiments, indicating the good precision of the developed method. LODs and LOQs were adequate and ranged between 0.02 (gallic acid)–0.65 (vanillin) mg kg⁻¹ and 0.05 (gallic acid)–1.98 (vanillin) mg kg⁻¹, respectively. The analytes showed satisfying recovery efficiency over the range 82.1–92.1%.

Determination of phenolic compounds in tuna fillets with HPLC-DAD

HPLC-DAD analysis was carried out to scan the presence of all the analytes in ten fortified tuna fillets. Gallic acid, syringic acid, vanillic acid and rosmarinic acid were determined from the class of phenolic acids. Catechin, epicatechin, apigenin, quercetin, rutin, luteolin and myricetin were determined from the class of flavonoids. From the class of hydroxybenzaldehydes, syringaldehyde was determined. Table S2 presents the identified phenolic compounds providing information about the experimental retention time; and Fig. 1 illustrates the chromatographic separation of the phenolic compounds in a real sample spiked at 3 mg kg⁻¹ concentration level. The chromatogram was monitored at 270 nm. Three tuna fillets preserved solely in brine were analysed and used as reference, and no peaks were detected.

Quantification results

The detected compounds were quantified, taking into consideration that quantitative results are crucial to offering a comprehensive overview of the antioxidant fortification of the tuna fillets. The identified phenolic compounds were quantified based on their reference standards using the calibration curves presented in Table S1. Before analysis, the ten extracts were spiked with the mixture of standards of all the analytes at a final concentration of 3 mg kg⁻¹. The concentration of each compound was calculated after subtracting the absorbance of the neat extract from the absorbance of the spiked extract. Four phenolic acids, 8 flavonoids and 1 hydroxybenzaldehyde were determined with HPLC-DAD and quantified in the tuna fillets, suggesting that the tuna fillets were fortified with the phenolic constituents of the hydrosols. The determination of the phenolic compounds over the concentration ranged from 8.86 to 512 mg per 100 g (Table S3) attesting that the tuna fillets were fortified with antioxidants.

Discussion

The determination and further quantification of the phenolic compounds in the tuna fillets suggests that the fillets were enhanced with the phenolic constituents of the hydrosols. According to the results, the tuna fillets preserved in oregano, laurel, sage and lemon balm’s hydrosols mixed with brine at the 15:85 (v/v) were fortified with thirteen phenolic compounds, specifically four phenolic acids, seven flavonoids and one syringaldehyde. Rosmarinic acid from phenolic acids (512 mg/100 g) and myricetin (176 mg/100 g) from flavonoids were the most abundant.

The four phenolic acids (rosmarinic, gallic, syringic and vanillic acid) have many health benefits which are summarised in Table S4. The pharmaceutical and therapeutic potential but also the pharmaceutical applications of gallic acid has been well emphasised (Al Zahrani et al.,  2020; Kalogiouri et al.,  2020b). Syringic acid functions as strong pharmaceutical and therapeutic agent (Zheng et al.,  2021). Rosmarinic acid exerts powerful therapeutic effects and deliberates its therapeutic potential against a wide variety of diseases (Nadeem et al., 2019), and also, vanillic acid has been reported to confer among others, several health benefits (Mathew et al.,  2018). The main pharmaceutical potentials of the four determined phenolic acids include anti-cancer, antioxidant and anti-inflammatory properties. In addition, they also act as efficient anti-bacterial and neuroprotective agents and even as efficient preventives of Alzheimer’s disease (syringic and vanillic acids, Table S4). Aside from their health benefits, the gallic acid exhibits extensive applications in food (Kalogiouri & Samanidou, 2020c) and vanillic acid is widely used as flavouring and scent agent due to its pleasant and creamy odour (Kaur & Chakraborty, 2013).

The seven flavonoids (catechin, epicatechin, rutin, apigenin, quercetin, luteolin, myricetin) demonstrate significant biological properties which are summarised in Table S5. Catechin and epicatechin have been shown to protect against modern diseases (i.e. arterial hypertension, obesity, diabetes, metabolic syndrome and ischaemic stroke, Bahadori et al.,  2020). They also demonstrate activities against Alzheimer’s and Parkinson’s diseases, but also, oral and breast cancers (Bahadori et al.,  2020). The most common properties these seven flavonoids may yield are the promising anti-cancer effects since they all intervene in carcinogenesis through different ways, including in tumour cell proliferation, apoptosis, metastasis and inflammation (Table S5). Catechins demonstrate activities against oral and breast cancer (Bahadori et al.,  2020). Apigenin may yield anti-proliferative and anti-metastatic effects, suppressing formation of malignant tumour cells (Zheng et al.,  2021) or/and prevent skin or colon cancer (Zheng et al.,  2021). Myricetin acts as anti-neoplastic agent in humans, and it has demonstrated strong suppressive effects on cellular activities of cancer cells (Martínez-Poveda et al.,  2019). Vanillin and luteolin they also act as they yield anti-mutagenic and anti-neoplastic activities (Jegal et al.,  2020). The identified flavonoids also have other significant biological properties like promising antioxidant potential, protecting cells from ROS induced damage (i.e. rutin, Babou et al.,  2016, apigenin, Zheng et al.,  2021, quercetin, Nour et al.,  2017, vanillin, luteolin, Jegal et al.,  2020). They also act as anti-inflammatory compounds (i.e. rutin, Rakshit et al., 2021, apigenin Zheng et al.,  2021, quercetin and luteolin, Jegal et al.,  2020), or they have anti-allergic and anti-bacterial activities (rutin, Ganeshpurkar & Saluja, 2017, apigenin, Zheng et al.,  2021, vanillin, luteolin, Jegal et al.,  2020).

Furthermore, these flavonoids are widely used in food industry as promising food additives for inhibiting the oxidation of meat and maintain quality while increasing shelf life (i.e. catechin, Tian & Huang, 2019), or to protect meat products against bacteria (i.e. quercetin in synergy with myricetin, Tamkutė et al.,  2019) or for improving the quality of vegetable oils (sunflower, corn, peanut and hazelnut oils) based on their antioxidant stability (Şahin et al.,  2020), or to improve the nutritional value of foods (i.e. vanillin, Falagán et al.,  2016) or as flavouring agents (i.e. vanillin, Huang et al.,  2012, apigenin, Nour et al.,  2017), adjuvant agents (apigenin, Nour et al.,  2017) or dietary supplement in beverages and foods (i.e. quercetin, Tamkutė et al.,  2019).

Syringaldehyde was the only identified phenolic compound from the benzaldehydes. Clinical results suggest that it may produce anti-hyperglycaemic effects, increasing plasma glucose utilisation and insulin sensitivity (Huang et al.,  2012). In the food industry, it is used as an additive, specifically, as a flavouring agent (Dufour & Sauvaitre, 2000).

It becomes evident that the phenolic compounds of the hydrosols diffused into tuna fillets, thus added nutrients resulting to fortified tuna fillets with increased functional properties. These tuna fillets meant to improve nutrition and add health benefits combining tuna benefits and phenolic based benefits from aromatic herbs. This study highlights the promising potential of hydrosols as packaging constituents in functional seafood, as a cost-effective alternative processing procedure, meeting consumers’ needs for quality products with increased nutritional value and health benefits.

Concluding remarks

Fortified tuna fillets canned in a mixture of brine and hydrosols of oregano, laurel, sage and lemon balm (85:15, v/v) were analysed with HPLC-DAD method developed and validated to determine thirteen phenolic compounds over the range 8.86 (quercetin) to 512 mg/100 g (rosmarinic acid). Gallic, syringic, vanillic and rosmarinic acids were determined from phenolic acids. Myricetin, vanillin, catechin, epicatechin, apigenin, quercetin, rutin and luteolin were determined from flavonoids, and syringaldehyde from the class of hydroxybenzaldehydes.

The potential use of hydrosols derived from aromatic herbs was evaluated in an attempt to enhance the nutritional profile of canned tuna fillets. Tuna fillets preserved in highly antioxidant hydrosols based brine, comprise nutritional benefits from combined consumption of both fish and herbs, fortifying positive health effects. The use of hydrosols to preserve fillets rich in phenolic content is undoubtedly an innovative and low-cost idea that will enhance researchers and industry to meet the needs and expectations of modern consumers for health-promoting seafood products. The present work suggests a simple, low-cost and effective way to fortify seafood (or/and different food products) using by-products of the essential oil industry. The suggested methodology could be expanded to different cases of food fortification, as well.

Acknowledgments

The authors would like to thank the Thinkgreen Natural Goods SA company (Thessaloniki, Greece) for providing the tuna fillets canned in hydrosols based brine.

Funding

This research received funding from the Research Project: ‘BLUE_BOOST- Boosting the innovation potential of the triple helix of Adriatic-Ionian traditional and emerging BLUE growth sectors clusters through an open source/knowledge sharing and community based approach’, Interreg V-B Adriatic-Ionian Cooperation Program and was co-financed by the European Union (European Regional Development Fund-Instrument for Pre-Accession II Fund).

Conflict of interest

All authors declare no conflict of interest.

Author contributions

Natasa P. Kalogiouri: Data curation (lead); Formal analysis (lead); Investigation (equal); Methodology (lead); Validation (equal); Writing-original draft (lead); Writing-review & editing (lead). Lambros E. Kokokiris: Data curation (lead); Funding acquisition (lead); Investigation (equal); Methodology (supporting); Project administration (lead); Supervision (equal); Validation (equal); Writing-review & editing (equal). Stephania Doulgeraki: Investigation (equal); Visualization (equal). Athanassios Papadopoulos: Conceptualization (equal); Resources (equal). Victoria F. Samanidou: Resources (equal); Supervision (equal); Visualization (equal).

Ethical approval

Ethics approval was not required for this research.

Peer review

The peer review history for this article is available at https://publons.com/publon/10.1111/ijfs.15034.

Data availability statement

Research data are not shared.

References

Annotated References