Thymus vulgaris L. Essential Oil: Effect on Fatty Acids and Proteins Oxidation of Little Tuna (Euthynnus alletteratus) Minced during Refrigerated Storage

a Research Unit Valorization and Optimization of Resource Exploitation (UR16ES04), Faculty of Science and Technology of Sidi Bouzid, University of Kairouan, Campus University Agricultural City Sidi Bouzid 9100, Tunisia. b Regional Center of Agricultural Research (CRRA) Sidi Bouzid, Gafsa Road Km 6, PB 357, Sidi Bouzid 9100, Tunisia. c Laboratory of Valorization of Unconventional Waters, INRGREF, University of Carthage, Tunisia. d Department of Biology, Hail University, College of Science, P.O. Box 2440, 81451 Ha’il, Kingdom of Saudi Arabia. e Laboratory of Bioressources: Integrative Biology and Recovery, High Institute of BiotechnologyUniversity of Monastir, Monastir 5000, Tunisia. f Department of Chemistry, College of Science, Qassim University, Buraidah 51452, Saudi Arabia. g Faculty of Sciences of Monastir, University of Monastir, Avenue of the Environment, Monastir 5019, Tunisia. h Laboratory of Genetic, Biodiversity and Valorization of Bioressources, Higher Institute of Biotechnology of Monastir, University of Monastir, Avenue Taher Hadded BP 74, 5000 Monastir, Tunisia. i Faculty of Science and Arts in Baljurashi, Albaha University, P.O. Box (1988), Albaha, Saudi Arabia. j Faculty of Science of Sfax, Department of Chemistry, University of Sfax, B.P. 1171, 3000, Sfax, Tunisia.


INTRODUCTION
Recent advances in discovering new, effective, and non-toxic agents from plant essential oils (EOs) and extracts, have been explored, due to their health promoting properties, and their diverse applications in food technology, pharmaceuticals and phytomedicine [1][2][3][4][5][6]. Their potent antimicrobial and antioxidant properties provide the opportunity to be a good safer alternative to synthetics [7][8][9][10][11][12]. Currently, a major problem in the food industry is to ensure proper preservation of food. In addition, oxidative degradation leads to loss of vitamins, deterioration of lipids and proteins, deterioration of flavour, decrease in nutritional value and even sometimes to the appearance of toxic substances [13]. Nowadays, all over the world, stored food products suffer serious damage, leading to considerable economic losses and especially health risk. 30% of foodborne deaths occur among children under 5 years of age [14]. In the United States, we estimate that foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths each year. 9.4 million cases were caused by several pathogenic microorganisms [15].
Seafood is a major global food commodity and an important part of a healthy diet. It is an important source of proteins. In 2016, 47% of seafood was already supplied by aquaculture and around 17% of animal protein consumed worldwide came from aquatic animals (fish, crustaceans and molluscs) [16]. Several studies on seafood have shown high levels of contamination by several bacteria such as Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas, Salmonella spp [17,18]. Therefore, the aim of this study is to investigate the inhibition effect of TVEO on fatty acids and proteins alteration of little tuna (Euthynnus alletteratus) stored at 4°C for 16 days through evaluating its effect on the addition on the lipids and proteins oxidation as well as on total volatile basic nitrogen (TVB-N).

Thymus vulgaris Essential Oil
T. vulgaris plants were freshly collected (June, 2020) from the mountainous region of Zaghouan (Tunisian locality) (upper semi-arid zone, latitude 36•26'N, longitude 10•46'E, Emberger's pluviothermic coefficient = 55.44, Altitude = 500 m, Rainfall = 400-500 mm/year). In this experiment we used fresh TVEO, the extraction of which was carried out in our research unit. Arial part (stems, leaves and flowers) were dried at room temperature. The 100 g of aerial part were subjected to hydrodistillation for 3 hours with 500 ml distilled water using a Clevengertype apparatus according to the European Pharmacopoeia. The distilled essential oil was dried over anhydrous sodium sulfate, filtered and stored at 4°C. Yield based on dried weight of the sample was calculated. The chemotype of this oil is carvacrol (75%).

Fish Sampling and Processing
Little tuna (Euthynnus alletteratus) was caught in the Northern area of the Tunisian Sea. The fishes were transported on ice to the laboratory where they were eviscerated, headed and filleted. Each piece was immersed in boiling water for 1 min, in order to reduce microorganisms attached to the fillet surface. The pieces of little tuna prepared as above were minced in a sterile grinder. In order to test TVEO effect on limitation of lipid and protein alteration in tuna samples, three lots were prepared: one was left as a control lot, the second was added the BHT and the third was added a 3% of TVEO. All lots were then vacuum packed in polyethylene bags and stored at +4°C for 16 days. Peroxide value (PV), thiobarbituric acid reactive substance (TBARS) and total volatile bases nitrogen (TVB-N) were determined at days 0, 4, 8, 12 and 16.

Determination of Peroxide Value
The peroxide value (PV), expressed as mEq of active O 2 per kg of lipid, was determined using the method described by Guran et al. [19]. The free fatty acid (FFA) content of the lipid was determined volumetrically using aqueous sodium hydroxide (0.1 N) and phenolphthalein indicator (1% ethanol).

Determination of Thiobarbituric Acid
Reactive Substance (TBARS) The TBARS was determined according to the AOCS [20] method and based on the same protocol as done by Hajlaoui et al. [21].

Determination of Total Volatile Bases Nitrogen (TVB-N)
Total volatile basic nitrogen (TVB-N) was determined according to the method described by Varlik et al. [22].

Statistical Analysis
All the experiments were conducted in triplicate and average values were calculated using the SPSS 26.0 statistics package for Windows. The differences in mean were calculated using the Duncan's multiple-range tests for means with 95% confidence limit (P≤0.05).

Determination of Peroxide Value (PV):
Hydroperoxides are among the first products formed during lipid oxidation reactions. These are unstable molecules rapidly broken down into secondary products such as aldehydes, ketones, alcohols, etc. They were assayed directly on methanolic extracts of tuna flesh in order to characterize the degree of primary lipids oxidation. Evolution of hydroperoxides concentrations measured on different samples, stored at 4°C for 16 days without (negative control) and with TVEO and BHT (positive control), are presented in Fig. 1.
These results showed a significant increase (P ≤ 0.05) in PV over storage time of all samples. But this development was dependent on added antioxidant. However, negative control batch PVs were systematically higher than those of stored TVEO and BHT batches for different storage periods, which indicate significant lipids oxidation reaching its maximum after 12 days of storage. Concerning batches treated by TVEO and BHT and for all storage periods, PVs were lower than control ones. This result revealed a significant (P ≤ 0.05) limitation of fatty acids oxidation in flesh   [23] revealed that TBARS value in control was 0.58 mg MDA/kg in vacuum packed sardines while a little fluctuation was mentioned when it was treated with rosemary extract (10g/l) at 3°C reaching 0.84 mg MDA/kg after 20 days of storage. On the other hand, TVEO treatment seems to be more efficient to decrease TBARS value in flesh fish than treatment with powdered thyme. However, the study of Selmi and Sadok [25] revealed that no significant differences between control and fresh tuna (Thunnus thynnus) sprinkled by powdered thyme lots. Several studies showed also that temperatures variation of meat and fish storage were an important factor in lipid oxidation. For example, Rong et al. [26] compared TBARS value of Atlantic mackerel stored in 4 and 0°C which they ranged from 0.23±0.04 mg/kg to 2.60±0.08 mg/kg and to 2.16±0.12 mg/kg after five days of storage at 4°C and 0°C, respectively. This later was confirmed by others studies like Wang et al. [27] which reported that 0° C was more effective than 4°C to inhibiting lipid oxidation of salmon.

Effect on Total Volatile Basic Nitrogen (TVB-N)
TVB-N mainly consists of amines such as methylamine, dimethylamine, and trimethylamine, they were the most widely used index for fish freshness assessment [20]. Fig. 3 [28] suggested that the increase in TVB-N value in fish during cold storage is mainly due to the alkalinity substances produced by the protein degradation by enzymatic reactions and microbial activity.
These results showed that TVEO has a great capacity on limiting flesh tuna proteins deterioration. This ability is explained by the antioxidant and antimicrobial power that this oil has. This limitation of TVB-N is mainly due to the high percentage of carvacrol. Despite this preservation, there is some alteration which is due to several factors initiating the oxidation such as; initiation by activated forms of oxygen, initiation by metals and other environmental factors like; temperature, pH, and oxygen partial pressure. Noting that oxidation is a very complicated phenomenon resulting in the interaction between different compounds in fish flesh. However, after fish death, lipoxygenases would be released by the skin and generate hydroperoxides of lipids within the muscles. These hydroperoxides participate in the initiation and propagation of auto-oxidation [29]. During storage, hydroperoxides and secondary products of lipid oxidation interact with proteins and amino acids. Free radicals produced by the lipid oxidation can promote protein oxidation to form carbonyl groups and change the secondary structure [26,20]. These interactions have a significant impact on degradation of functional, sensory and nutritional properties of foods [27].

CONCLUSION
In summary, TVEO is highly effective in ensuring the preservation of tuna during storage at + 4 ° C for 16 days. However, the addition of 3% of this EO showed a limitation of primary and secondary oxidation of lipids as well as proteins deterioration of tuna flesh. PV, TBARS and TVB-N concentrations measured after 16 days of storage revealed no alteration of little tuna confirming good preservation of its physicochemical parameters. During the different periods of storage, the use of TVEO in preservation is more effective than the synthetic antioxidant (BHT). These results promote the use of TVEO as a preservative for seafood.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.