Exploring the quality and nutritional profiles of monovarietal oils from millennial olive trees in Tunisia

In Tunisia, the olive tree has been cultivated for more than 3000 years by different civilizations, such as the Phoenicians, Greeks, Carthaginians, Romans, and Arabs. The ancient olive trees, which thrive in harsh environments, are little known and contain a foremost part of the olive germplasm. This study focused on the chemical oil analysis of the Tunisian millennial olive trees dating from the Roman and the Carthaginians periods. Twenty-eight antique olive trees grown in Tunisia's north to south are the subject of this study. Analyzed according to international standards, the tested accessions present oil with high quality, rich in chlorophyll and carotenes, and highly rich in polyphenols. The oil's fatty acid composition is complete with the international norms with a high oleic acid content, low palmitic and linoleic acid concentrations, and a C18:1/C18:2 ratio ≥ 7. Four oils, in particular, contained substantial levels of polyphenols (500–1632 mg/kg) and a C18:1/C18:2 ratio of > 9. Subsequently, it is crucial to prevent the extinction of ancient olive trees to preserve their historical significance and ecological worth, as well as to incorporate the best genotypes into new varieties and boost the competitiveness of Tunisian olive oil on the global market.


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.

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).

Genotyping 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 genotyping [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.

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Fig. 1 Geographical distribution of the analyzed millennial olive trees

Physiochemical quality indices of the oil
Free fatty acids and K 232 and K 270 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 (A 670 *10 6 )/(613*100*d) for chlorophyll and as (A 470 *10 6 )/ (2000*100*d) for carotenoid, where 'A' is the absorbance and 'd' is the thickness of the spectrophotometer cell (1 cm).

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

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.

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 K 232 and K 270 were ≤ 2.5 and ≤ 0.22, respectively, complying with the limits prescribed in [25].

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.
Among the complex phenols, the aldehyde form of elenolic acid combined with hydroxytyrosol (oleocanthal, 3,4-DHPEA-EDA) or tyrosol (oleuropein, p-HPEA-EDA), the ligstroside aglycone (p-HPEA-EA), and an isomer of the oleuropein aglycone (3,4-DHPEA-EA) are the most abundant secoiridoid fractions in virgin olive oil [40][41][42][43][44].The analyzed oils differed significantly in the content of oleocanthal and oleuropein, reaching, in sample "M11", the content of 337.33 mg/kg and 225.33 mg/kg, respectively.This oil was also the richest in oleuropein aglycone (668 mg/kg) and ligustroside aglycone (150.66 mg/kg), two components very important in the metabolism of the human body by reducing the levels of reactive oxygen species [45,46].Other phenols, such as vanillic acid, vanillin, p-coumaric acid, and ferulic acid, were also found, although in low amounts (0.001-5.35 mg/kg).Finally, the lignans pinoresinol and acetoxypinerosol varied from 0.77 to 17.85 mg/kg and from 0.001 to 6.81 mg/kg, respectively, but they were > 12 mg/kg in samples M2, M3, M4, M11, and M28.These compounds belong to the phytoestrogen family and have beneficial health effects [47].In clinical studies, it has been observed that women with a high intake of plant lignans have a lower risk of postmenopausal breast cancer, especially the ER + / PR + subtype, as well as a reduction in the expression of the tyrosine kinase proteins FASN and HER2, which are directly involved in cancer processes [48].

Discussion
Since the twentieth century, efforts to develop the quality of olive oil in Mediterranean countries have greatly increased, extending the study to thousands of trees, along the Aegean Sea.In Crete, the largest Greek island exists a 3000-yearold olive tree in Kavousi.It is an olive tree of exceptional size with a 14.2 m perimeter and nearly 5 m in diameter [51].Italian CNR researchers on the olive genetic resources proved the existence of Millennium olive trees in the Mount of Palestine and Urla, a small Turkish peninsula [52].The Spanish researchers consider that the ancient olives from southern Spain constitute a priceless reservoir of genetic diversity [53].In Tunisia, olive trees were first introduced by Phoenicians following the Second Punic War [54].This study presents the primary analysis of millennium olives in Tunisia.Our preliminary analyses have shown that oils from Tunisian millennial olive trees are of exceptional quality and very rich in polyphenols, carotenes, and monounsaturated fatty acids.The free acidity was below than the upper limit of 0.8% set for the "Extra Virgin Olive Oil" category, and the K 232 and K 270 extensions were ≤ 2.5 and ≤ 0.22, respectively.The small variations observed in these parameters confirm the low influence of location and genotype on the primary and secondary oxidation level [55].In fact, fruit ripeness, careless harvesting, and storage conditions strongly influence oil quality, as triglycerides are gradually hydrolyzed in ripe fruits and poor storage conditions expose the oils to enzymatic activity that increases free acidity and eventually gives the oil an unpleasant taste [56].
Chlorophylls and carotenoids are the main color pigments in olive oil, responsible for the color, green for chlorophylls and yellowish for carotenoids, and taste of the oils [32].They are directly related to the quality of this food and play an essential role in the oxidative activity of the oil acting as antioxidants in the dark and as prooxidants when exposed to light [24,57].Most of the oils had low-to-medium chlorophylls content (average 3.16 ppm), which is consistent with other Tunisian oils [32][33][34], but M6, M19, M20, and M27 had a very high levels of chlorophyll (17.2 to 22.1 ppm) and carotenoids (> 3), which makes them interesting for direct use to improve the color and "fruity" note of olive oil in new blends [58], and makes an important contribution to consumers acceptance [24].
Polyphenols are a very important component of the potential benefits of olive oil, as they have a strong antioxidant and anti-inflammatory effect, making them natural and effective anticancer agents in a balanced diet [38].The phenolic fraction of olive oil consists of a heterogeneous mixture of compounds, all of which influence the chemical properties and quality of the oil [15,59].International standards for the phenolic content of olive oil range from 50 to 1000 mg/kg, depending on several factors: variety, degree of ripeness, edaphic-climatic conditions, and cultivation methods [60][61][62][63].Several oils studied have a total polyphenol content > 500 mg/kg, especially those coming from trees grown in the dry and harsh climatic conditions of the highlands, as environmental stress conditions activate the biosynthetic pathway for the accumulation of polyphenols [64].The composition of phenols was also interesting, with tyrosols in sample M1 reaching 55.7 mg/kg, and hydroxytyrosol in M12 reaching 34.36 mg/kg, making these oils very interesting from a nutritional point of view.The oil "M11" was particularly rich in oleocanthal, oleuropein, oleuropein aglycone, and ligustroside aglycone, phenols that play an important role as chemopreventive compounds [65].
The fatty acid profile of olive oil is strongly influenced by the cultivar, fruit maturity, pedoclimatic growing conditions, and other minor area-related parameters [66].Oleic acid was the most abundant, followed by palmitic acid, linoleic acid, and palmitoleic acid, with levels in agreement with the limits of the international food standards (23,43) and in line with those of other local cultivars [29,47], but significantly better than those of the most commonly grown cultivar "Chemlali", which has a palmitic acid content of > 21% and an oleic acid content of < 60% [67][68][69][70], suggesting that this germplasm contains variants for fatty acid components that could be used to improve commercial Tunisian cultivars.
Among PUFAs, the most abundant was linoleic acid, which cannot be synthesized by humans and must be supplied through the diet [65].Nine oils, derived from ecotypes M5, M6, M8, M9, M10, M11, M14, M15, and M21, had an oleic acid/linoleic acid ratio > 7, the threshold value considered to ensure high quality and long shelf-life of the oil [47,48,71].Previous studies have shown the key role of the enzyme fatty acid desaturase 2 (FAD2) in the oleic acid/ linoleic acid profile, and how SNPs in the gene can influence FAD2 activity [72,73].It will be very interesting to analyze the FAD2 sequence of these nine millennial olive ecotypes to further investigate the role of the FAD2 gene and include them in breeding programs to improve the quality of Tunisian EVOOs.

Conclusion
The compositional profile of the oils obtained from the millennial olive trees surviving in Tunisia makes them very interesting from a nutritional point of view, as beneficial properties are widely attributed to these compounds [43].These trees have survived thousands of years in harsh conditions and are still productive, proving resilient to climate change, disease, and water scarcity, indicating careful selection by ancestors to meet their needs: cooking, lighting, and healing, which explains the high quality of the oil from these historic olive trees.This heritage is a treasure to be preserved and valorized for the development of the Tunisian olive sector, by including them in conservation and breeding programs to protect the environment and promote sustainable production.Future work will include accurate genotyping and authentication of the oil according to the consolidated protocols based on microsatellite markers for effective protection and valorization [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.

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

Table 1
Origin and use of the millennium olive cultivars under investigation Tableusingpure 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.

Table 3
Phenolic compound composition of the studied olive oils

Table 4
Fatty acid composition of the studied millennium olive oils