Introduction

Olive oil is a foodstuff steeped in myths and cultures, with many important properties related to its organoleptic characteristics, nutritional value, and cultural influences. The development of olive cultivation in the Mediterranean is closely linked to human nutrition and, together with wheat and grapes, forms the “Mediterranean trilogy” on which the diet of the same name is based, widely recognized for its health benefits [1].

Extra virgin olive oil (EVOO) is defined as oil obtained directly from fresh fruits through a mechanical process without refining steps, which play an important role in the final quality of EVOO [2]. It is a high-quality nutrient due to its content of fatty acids, essentially unsaturated, and its minor components, such as polyphenols, tocopherols, and carotenoids [3]. These give it a high antioxidant activity that can help prevent or delay the onset of certain degenerative and cardiovascular diseases [4, 5].

One of the olive-growing countries that attract more and more attention for its high-quality oil is Tunisia, a North African country covered with olive groves and where olive oil presents an essential part of gastronomy. In this country, olive cultivation dates back to the Berbers, long before the arrival of the Carthaginians. Since then, the dietary habits of Tunisia’s indigenous population have been based on olive fruit and oil, used by rural women to prepare bread, soup, porridge, and many other dishes [6]. Throughout history, olive oil has also been used to treat diseases, such as rheumatic and cardiovascular diseases, stomach ailments, and burns [7], which is why olive oil can be considered an integral part of Tunisia’s history and the culture of its inhabitants. The centuries-old tradition of olive cultivation has given rise to today’s olive groves which cover an area of 1.89 million hectares, including 75,000 ha of certified organic cultivation [8]. Today, the olive sector employs 269,000 farmers, or 57% of all farmers, and contributes to 45% of agricultural exports, averaging 120,000 tons per year [9, 10].

Research on Tunisian olive germplasm began in the twentieth century. Initially, studies focused on the morphological and biochemical traits of a limited number of olive varieties, such as the most widely cultivated “Chetoui” and “Chemlali” and some minor cultivars, such as “Oueslati”, “Besbessi”, and “Gerboui” [11,12,13,14,15]. Over time, studies moved to using molecular markers, which allowed the identification of more than 83 varieties distributed throughout Tunisia [16, 17] and the registration of 56 cultivars in the national catalogue [18]. However, given that Tunisia harbours a great genetic diversity of Olea europaea L., this number falls far short of reality. Many monumental trees whose age exceeds thousand years have never been studied for their oil composition and quality. Mnasri et al. [12, 19] studied their geographical distribution across the country and their thriving in desert and arid areas and explained their survival by people’s belief in their sacredness. Using morphological and molecular data, the authors pointed out a great genetic diversity that could be used in olive breeding programs. Thus, there is an urgent need to protect and study these thousand-year-old olive trees to prevent their extinction in the face of constantly intensified agriculture. For decades, Tunisian olive oil was sold in large quantities to other countries to be blended with other oils under European brand names. Today, Tunisia is making considerable efforts to produce and sell oils under Tunisian brands that are competitive on the world market, such as Teboursouk olive oil, which is registered as a protected designation of origin (PDO) in the international register of designations of origin [20].

The study aimed to examine the quality of 28 oils from millennial olive trees from different regions of Tunisia to obtain information on their oil quality and composition. The results will help us understand the flavor and quality of the oils consumed by our ancestors and select those whose quality complies with international standards by a composition rich in oleic acid, carotenes, chlorophyll, and polyphenols, to integrate them into the national breeding programs to increase the nutritional value of Tunisian oil and the competitiveness of the Tunisian olive industry on an international scale.

Materials and methods

Olive samples

The National GeneBank of Tunisia carried out extensive surveys throughout Tunisia and found 28,000-year-old olive trees of impressive size and appearance in the mountainous and arid inland areas known for their difficult climatic conditions (Figs. 1, 2). Trees at each sampling site were selected on the basis of the growth habit, structure, trunk thickness, using information from local growers and trunk dimensions as indicators of tree age according to [21], selecting trees with a girth of 6–8 m measured at 1.3 or 1.4 m above the ground (Table 1).

Fig. 1
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Geographical distribution of the analyzed millennial olive trees

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Fig. 2
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Some of the monumental olive trees of Tunisia located in A Beja (North), B Kairouan (Center), and C Gafsa (South)

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Table 1 Origin and use of the millennium olive cultivars under investigation
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Genoty** of olives

Fresh, healthy leaves were collected from each plant in July 2021, genomic DNA was extracted according to [22], and DNA quality and concentration were checked on 0.8% agarose gel and a Nano Drop TM ND2000c (Thermo Scientific, MA, USA) spectrophotometer. DNA concentrations were normalized to 50 ng/μL using 0.1 X TE buffer (10 mM Tris–HCl pH 8.0 and 1 mM EDTA) and polymerase chain reactions were carried out using a set of 10 highly informative SSR markers consolidated for olive genoty** [23]. PCR reactions were performed in a final volume of 12.5 µL and contained 1X Dream Taq buffer, 0.15 mM dNTP, 0.25 μM primer mix, 0.3 U Dream Taq, and 50 ng genomic DNA. Amplicons were separated using the automatic capillary sequencer ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with the internal size standard GeneScan 600 LIZ (Applied Biosystems). Allele size was estimated using GeneMapper v.5.0 software (Applied Biosystems, Foster City, CA, USA).

For three consecutive harvest years (2018–2021), 1 kg of fresh olives was hand-picked from different parts of each tree at full and equal ripeness index (RI = 3), based on the assessment of olive skin and pulp color [24]. All fruits were visually inspected and damaged fruits or those affected by pests and diseases were discarded. The olives were washed and processed within 24 h of harvest in a laboratory mill of the National GeneBank, Tunisia (MC2 Ingenieria Y Sistemas, Seville, Spain) equipped with a metal crusher, a mixer, and a basket centrifuge to obtain extra virgin olive oils (EVOO) through a cold extraction process. The mill was washed between each batch of olive fruits. The oil samples obtained were stored in dark glass bottles at 4 °C until they were used for the analyses, which were carried out in triplicate.

Physiochemical quality indices of the oil

Free fatty acids and K232 and K270 extinction coefficients spectrophotometric were determined according to the official methods described in the European Union standard methods Regulations 2568/91 and the subsequent amendments by the Commission of the European Union.20 [25]. All parameters were determined in triplicate for each sample.

Chlorophyll and carotenoid pigments

The content of chlorophylls and carotenoids pigments was expressed as mg of pheophytin a or lutein per kg of oil. Carotenoids are conjugated terpene compounds in various forms (α, β and γ) of which the β-carotene, the biochemical precursor of vitamin A, is the most abundant. Pigments were determined according to the method described in [26] by measuring the absorbance at 670 and 470 nm of a mixture obtained by dissolving 7.5 g of oil in cyclohexane to a final volume of 25 ml. The absorbance was determined as (A670*106)/(613*100*d) for chlorophyll and as (A470*106)/ (2000*100*d) for carotenoid, where ‘A’ is the absorbance and ‘d’ is the thickness of the spectrophotometer cell (1 cm).

Phenolic profile

Phenolic compounds’ extraction and purification

The phenolic fraction was extracted according to [27] by mixing 2.5 g of fresh oil with 250 ml of the internal standard solution (15 ppm syringic acid in methanol). The emulsion was vortexed for 10 min and evaporated in a rotary evaporator at 35 °C under vacuum. The aliquot was dissolved in 6 ml of hexane. The phenols were purified according to [28]. Diol SPE cartridges (Supelco Co., Bellefonte, PA, USA) were placed in a vacuum elution apparatus to refine the phenolic phase. The cartridge was conditioned by the sequential addition of 6 ml methanol and 6 ml hexane. The oil solution was injected into the column after releasing the vacuum. The solvent was drawn through the column leaving the sample and standard on the solid phase. The sample container was washed with 6 ml hexane followed by 4 ml hexane/ethyl acetate (85:15, v/v), and the solvents were removed from the cartridge and discarded. The phenolic phase was eluted from the column with 15 ml methanol, vortexes for 5 min, and the solvent removed on a low-speed rotary evaporator under vacuum at room temperature. Finally, the residue was dissolved in 250 ml of methanol/water (1:1, vol/vol).

Phenolic composition

The total phenolic content, expressed as mg gallic acid equivalent (GAE) per kg EVOO, was determined by the spectrophotometric Folin–Ciocalteu method of Singleton and Rossi [29]. Gallic acid was used in the external calibration at different concentrations, including 0.00, 0.25, 0.50, 0.75, and 1 mM. After 30 min, the solution was tested to determine its absorbance at 750 nm using a UV–Vis spectrophotometer. The total phenolic content was calculated as mM gallic acid equivalent (mM GAE) using the calibration curve. The phenolic compounds were identified using the Agilent 1100 liquid chromatography system equipped with an automatic injector, a column oven (Waters Co., Milford, MA, USA), and a diode array UV detector. A Spherisorb S3 ODS2 column (250*4.6 mm i.e., 5 µm particle size) was maintained at 30 °C, with a flow rate of 1.0 ml/min and an injection volume of 20 µl. The mobile phases consisted of solvent A (water/acetic acid 95:5, vol/vol), methanol (B), and acetonitrile (C). The following multi-step linear gradient was applied over a period of 50 min: it started at 95% A, 2.5% B, and 2.5% C and changed to 34% A, 33% B and 33% C. The phenols were quantified at 280 nm using syringic acid as an internal standard, and the response factors were determined by comparing their retention times with maximum absorbance according to [29]. Simple phenols were identified using pure standards injected under the same conditions as the olive oil extracts. Complex phenols were tentatively identified with two replicate analyses based on their retention times, and phenolic compound concentrations expressed as mg/kg syringic acid.

Fatty acid composition

The fatty acid composition was determined by gas chromatography according to the International Olive Council [30]. Fatty acid methyl esters (FAME) were dissolved by mixing 0.2 g of oil in 3 ml of hexane. The solution was mixed with 0.4 ml of 2N methanolic potassium hydroxide before being analyzed by gas phase chromatography. The carrier gas was nitrogen (1 ml/min), the injector and detector (FID) temperatures were 240 °C and 260 °C, respectively, and the oven temperature was 210 °C. The spillage ratio was 1/5 and the injection volume was 1 µl. Fatty acids were identified by comparing each sample with fatty acid methyl ester standards.

Statistical analysis

Statistical analysis was performed using XLSTAT v. 6.1 software (Addinsoft, Paris, France) and the statistical package SPSS software (IBM version 17). Results were reported as tables of mean values and standard deviations. One-way analysis of variance (ANOVA) was used to evaluate the effects of the genotype and the growing location on the composition of the oil and its quality parameters. The significance of differences at 1% (p < 0.01) between mean values was determined using Turkey’s test.

Results

Quality parameters

The results for quality parameters of the analyzed EVOO oils are presented in Table 2. At the fruit ripening stage RI = 3, all the oils except M24 and M27 had a free acidity content lower than the upper limit of 0.8% which is the threshold limit established for the “Extra Virgin Olive Oil” category. The specific extensions’ values of K232 and K270 were ≤ 2.5 and ≤ 0.22, respectively, complying with the limits prescribed in [25].

Table 2 Mean values and standard deviations (SD) of quality parameters of olive oils obtained from fruits at Maturity index = 3. Free acidity, UV spectrophotometer indices, and total chlorophylls and carotenes’ pigments’ content are listed. In the same column, the means marked by different lowercase letters differ significantly (Tukey’s test, p 0.05).
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Chlorophyll and carotenoids

The chlorophyll content of the analyzed EVOOs ranged from 0.28 (sample M1) to 22.1 ppm (sample M27) with significant differences among the oils (p value < 0.01) (Table 2). Most of oils had low-to-medium content (average 3.16 ppm), in line with other Tunisian oils [31,32,33], but oils M6, M19, M20, and M27 had a very high chlorophyll content (17.2 to 22.1 ppm (Table 2). Carotenoids also varied significantly (0.21–6.64 ppm), and were > 1 ppm, which is the threshold requested for an effective inhibition of photo-oxidation, in most of oils. Although many factors influence the pigment profile of olive fruits, such as ripening degree and growing conditions [34,35,36], four oils, M9, M19, M20, and M27, had a carotene content > 3 ppm.

Polyphenols

In the studied oils, the polyphenol content, expressed as mg gallic acid equivalent (GAE) per kg EVOO, varied significantly (p < 0.01) ranging from 91.2 to 1632 mg/kg (Table 3). Several oils had content > 500 mg/kg, in particular those originating from the arid regions of Kesra (M10, M11, M12), El Alaa (M21, M22), and Gafsa and Medenine (M3, M5, M7, M24, and M25) (Table 3).

Table 3 Phenolic compound composition of the studied olive oils
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As for the phenolic composition, chromatography revealed 14 compounds (Table 3). Among the monomeric phenols, tyrosol (p-hydroxyphenyl ethanol: p-HPEA), hydroxytyrosol (3,4-dihydroxy phenyl ethanol: 3,4-DHEA), and their derivatives were the most abundant. These phenols have important biological activities as free radical scavengers and metal-chelators [74, 75]. We propose to recognize and protect these trees as living monuments and elements of the “biocultural heritage” and to preserve them in the arboretum of the Tunisian national Gene bank.