Fatty acid profile and safety aspects of the edible oil prepared by artisans' at small‐scale agricultural companies

Abstract The aim of this study was to analyze the fatty acid (FA) profiles and mycotoxin and polycyclic aromatic hydrocarbon (PAH) concentrations in sea buckthorn (SB1, SB2), flaxseed (FL3, FL4, FL5), hempseed (HE6, HE7, HE8), camelina (CA9, CA10), and mustard (MU11) edible oils, prepared by artisans’ by artisanal at small‐scale agricultural companies in Lithuania. The dominant FAs were palmitic and oleic acids in SB; palmitic, stearic, oleic, linoleic, and α‐linolenic acids in FL; palmitic, stearic, oleic, linoleic, and α‐linolenic acids in HE; palmitic, oleic, linoleic, α‐linolenic, eicosenoic, and erucic acids in CA; and oleic, linoleic, α‐linolenic, eicosenoic, and erucic acids in MU. In SB2 oil samples, T‐2 toxin and zearalenone concentrations higher than 1.0 µg/kg were found (1.7 and 3.0 µg/kg, respectively). In sample FL4, an ochratoxin A concentration higher than 1.0 µg/kg was established (1.2 µg/kg); also, in HE8 samples, 2.0 µg/kg of zearalenone was found. None of the tested edible oils exceeded the limits for PAH concentration. Finally, because of the special place of edible oils in the human diet, not only should their contamination with mycotoxins and PAHs be controlled but also their FA profile, as an important safety characteristic, must be taken into consideration to ensure higher safety standards.

genic effects (Sun et al., 2014;Thompson & Raizada, 2018). It is very important to point out that the synergistic activity of mycotoxins leads to multiple, sometimes cumulative, toxic effects; for this reason, the presence of mycotoxins in foodstuffs raises the risk of associated public health concerns (Alassane-Kpembi et al., 2017). Mycotoxicosis is characterized by an accumulation of the above-mentioned toxins in body organs, tissues, and the central nervous system (Gherbawy et al., 2012). Low concentrations of aflatoxin can lead to long-term effects; the most common effect of the majority of mycotoxins is cancerogenic, as DNA replication is influenced by some mycotoxins, and incompatible effects appear. Aflatoxin is involved in immunosuppression and mutagenic, teratogenic, and carcinogenic actions (Fan et al., 2013). The International Agency for Research on Cancer (IARC) indicates that aflatoxin B1 is a Group 1 agent (carcinogen), and ochratoxin belongs to Group 2B (probable carcinogen) (Fashandi et al., 2018;Ostry et al., 2017). The technological steps applied to the refining and extraction of edible oils vary according to the type of edible oil and refining technology. Some have an influence on the mycotoxin concentrations in edible oils and others do not; however, reports of a high occurrence of mycotoxin contamination in edible oils worldwide have been published (Bordin et al., 2014;Cavaliere et al., 2007;Karunarathna et al., 2019;Shephard, 2018). It should be mentioned that nowadays, many consumers select edible oils from nontraditional plants; moreover, products prepared at smallscale agricultural companies are associated with the characteristics "natural," "ecological", and "'healthier" (sometimes proven, sometimes not). However, in such types of edible oil, as well as the stock from which they are prepared, mycotoxin contamination is not controlled. For this reason, it is very important to know about the challenges in the small-scale edible oil industry, especially because most of the technological steps included in high-capacity edible oil technology are not used on a small scale.
According to the European Food Safety Authority (EFSA), TFAs may originate from various sources, including the bacterial conversion of unsaturated FAs in the rumen of ruminants, industrial hydrogenation (used to produce semi-liquid and solid fats, can be used to produce margarine, shortening, biscuits, etc.), deodorization of unsaturated vegetable oils (or occasionally fish oils) with a high PUFA content (a necessary step of refining), and heating and frying oil at excessively high temperatures (>220°C). TFAs do not play a positive role in any vital functions. On the contrary, the intake of TFAs may harm human health. Evidence suggests that ruminant-derived TFAs have similar adverse effects on blood lipids and lipoproteins to TFAs from industrial sources. Sufficient evidence is still needed to reveal whether a difference exists between equivalent amounts of ruminant and industrially produced TFAs in terms of the risk of coronary heart disease .
Another challenge related to the safety of edible oils is contamination with polycyclic aromatic hydrocarbons (PAHs). Although the safety of foodis strictly controlled throughout the world (Ji et al., 2020), edible oil is one of the major sources of PAH contamination, due to the hydrophobic characteristics of PAHs (Barranco et al., 2004;Sannino, 2016). PAHs are organic contaminants released through incomplete combustion  or pyrolysis of organic materials (Drabova et al., 2013). They contain more than one fused aromatic ring (Tfouni et al., 2014), and their toxicity depends on the number of rings: the higher the number of rings, the more toxic and stable the PAH (Li et al., 2003). The 16 most toxic PAHs are indicated as environmental priority pollutants (Zelinkova & Wenzl, 2015), and benzo[a]pyrene (BaP) is indicated as one of the most toxic PAHs (IARC, 2021 well as to give recommendations on edible oil consumption and improve public health.
The aim of this study was to analyze the FA profile, and mycotoxin and PAH concentrations in sea buckthorn, mustard, flaxseed, hempseed, and camelina seed edible oils, prepared at small-scale companies in Lithuania.

| Samples of edible oils used for analysis
In total, 11 samples of edible oil were analyzed (

| Fatty acid profile analysis
The FA composition of edible oils was determined using gas

| Analysis of mycotoxins in edible oil samples
Deoxynivalenol ( Then, acetonitrile (10 ml) was gradually added to the tubes, and extraction was started by mixing for 5 min on a mechanical shaker.
The obtained mixtures were centrifuged (1,313 × g, 5 min), and the supernatants were transferred to 15-ml centrifuge tubes and stored for 15 min at −80°C in a Heto PowerDry ® freeze dryer (Thermo Fisher Scientific). After removal, the extracts were immediately centrifuged (2,626 × g, 5 min) at 10°C. For each sample, replicate volumes (500 µl) were transferred to 10-mL glass tubes, whereas the remaining extracts (5 ml) were transferred to QuEChERS dSPE centrifuge tubes for clean-up. The tubes were shaken for 5 min and centrifuged (2,626 × g, 5 min) at room temperature to obtain purified extracts. The initial fractions (500 µl) and the purified extracts Chromatographic separation was performed on a reversed-phase analytical column (Kinetex C18, 1.7 µm, 100 Å, 50 × 3.00 mm; Phenomenex) at a 0.35 ml/min flow rate. A ternary gradient elution was carried out using 0.1% formic acid in water (eluent A), 0.1% formic acid in methanol (eluent B), and 0.1% formic acid in acetonitrile (eluent C) according to the following gradient program: 0-1.5 min: 0% B and 30% C; 2.0-2.7 min: 15% B and 35% C; 5.5-6.5 min: 40% B and 58% C; 8.0 min: 5% B and 93% C; 8.5-9.5 min: 0% B and 10% C; 10.0 min: 0% B and 30% C. The autosampler was maintained at 4°C, and the column temperature was 40°C. The sample injection volume was 15 µl. Ion monitoring was conducted in both positive and negative ion modes, and the mass analysis was performed in selective reaction monitoring (SRM) mode. The following instrumental settings were used: spray voltage 3.5 kV (positive ion mode); 2.5 kV (negative ion mode); vaporizer temperature 350°C; ion transfer temperature 300°C; sheath gas 55 arbitrary units (arb); auxiliary gas 25 arb; and sweep gas 5 arb. Data processing was performed with Xcalibur™ software (Thermo Fisher Scientific).

| Determination of polycyclic aromatic hydrocarbons in edible oil samples
The solvents employed were cyclohexane, hexane, dichloromethane,

| Statistical analysis
In order to evaluate the influence of the type of edible oil on the FA profile, and mycotoxin and PAH concentrations, data were analyzed by one-way ANOVA (statistical program R 3.2.1). The results were recognized as statistically significant at p ≤ .05.

| Fatty acid profiles of sea buckthorn, flaxseed, hempseed, and camelina seed oils
The FA profiles of sea buckthorn (SB1 and SB2), flaxseed (FL3, FL4, and FL5), hempseed (HE6, HE7, and HE8), camelina seed (CA9 and CA10), and mustard (MU11) edible oils are shown in In a comparison of samples HE6, HE7, and HE8, the highest content of palmitic, stearic, oleic, linoleic, and arachidic acids was found in sample HE7. However, the lowest ALA content was established in HE7. The highest oleic acid content was found in HE7. Sample HE7 showed the highest SFA, MUFA, omega-6, and omega-9 concentrations; however, the highest content of PUFAs and omega-3 was found in sample HE8. The dominant FAs in the hemp seed oil sample group were palmitic, stearic, oleic, linoleic, and ALA, and their con-   (Popa, et al., 2021). The The dominant FAs s mustard seed oil (MU11) were oleic, linoleic, ALA, gondoic, and erucic acids (14.58%, 11.00%, 13.66%, 9.49%, and 42.66% of total fat content, respectively). The SFA content in MU11 was, on average, 5.32% of the total fat content, and the predominant FAs in MU11 were MUFAs (69.65% of the total fat content). The PUFA content in MU11 was, on average, 2.9 times lower than the MUFA content. Comparing FA series, the most abundant in MU11 was omega-9 (69.40% of total fat content), and the omega-3 and 6 content in MU11 was, on average, 5.1 and 6.1 times lower,

respectively.
Our results are in agreement with Stamenković et al. (Stamenković et al., 2018) and Mitrović et al. (Mitrović et al., 2020), who reported that the main FAs in mustard seed oil are UFAs (oleic, eicosenoic, erucic, linoleic, and linolenic). The main specific characteristic of mustard seed oil is its high content of erucic acid, which can range from 32.81% to 60.29% (Mitrović et al., 2020). Erucic acid is a longchain FA, classified as a natural toxin due to its detrimental effects on heart muscle function (Vetter et al., 2020) and lipid degeneration of the heart (Krist, 2020). Oxidation of mitochondrial FAs plays a key role in liver lipid metabolism; therefore, it is possible that hepatic metabolism of erucic acid might decrease mitochondrial FA oxidation . One of the main sources of erucic acid in the human diet is oil prepared from Brassicaceae plants, for example, mustard (Vetter et al., 2020). According to EFSA recommendations, the tolerable daily intake of erucic acid is 7 mg/kg of body weight (EFSA, 2016).
Finally, it can be stated that the FA profile of an edible oil is a very important characteristic, which shows not only a functional aspect but also a safety aspect. The data on FAs in edible oils should be disseminated to a wide audience and, if some of the oils are not recommended for daily consumption as food ingredients, perhaps they could be used in other industries, for example, for cosmetology, etc.

| Mycotoxin contamination in tested edible oil samples
The major mycotoxins in food are aflatoxins, OTA, ZEN, fumonisins, and trichothecenes (Vasseghian et al., 2020). Mycotoxin contamination of the tested edible oil samples is shown in Table 3. In SB2, T-2 and ZEN concentrations higher than 1.0 µg/kg were found (1.7 and 3.0 µg/kg, respectively). As the awareness and understanding of ZEN exposure-associated risks have increased, the European Commission (EC) has established and enforced a maximum 400 µg/ kg ZEN level in refined corn oil, and the tolerable daily intake (TDI) of ZEN has been set at 0.25 µg/kg b.w. based on collected toxicity assessment and exposure data (EC, 2021). ZEN shows distinct lipophilic properties, in contrast to the high water solubility of trichothecenes (Lacko- Bartošová et al., 2017), and this characteristic can facilitate absorption through the gut. One of the most toxic mycotoxins is T-2, which is a metabolite of F. acuminatum and F. equiseti, mainly found in cold climate regions (Kang et al., 2020;Ling et al., 2020). T-2 is harmful to mammals, and its lipophilic characteristics imply that it is easily absorbed through the gut, skin, and pulmonary mucosa . Based on these toxic effects, the Note: Data are represented as means (n = 5) ± SE. set the TDI for the sum of T-2 and HT-2 at 100 ng/kg body weight (EU, 2013). In sample FL4, an OTA concentration higher than 1.0 µg/ kg was established (1.2 µg/kg). The limits for OTA range from 0 to 50 µg/kg in food (Mazumder & Sasmal, 2001). Ochratoxin is associated with immunotoxic, teratogenic, ascertained nephrotoxic, and carcinogenic effects (Meucci et al., 2021). In oil sample HE8, 2.0 µg/ kg of ZEN was determined. ZEN is a metabolite of Fusarium species; its estrogenic activity, hepatotoxicity, teratogenicity, genotoxicity, carcinogenicity, hematotoxicity, and immunotoxicity to mammals are well known (Alshannaq & Yu, 2017;Gallo et al., 2015;Häggblom & Nordkvist, 2015;Kowalska et al., 2016).
Mycotoxins are thermostable toxins, resistant to high pressure, transportation conditions, etc. (Amirahmadi et al., 2017;Heshmati et al., 2019). The formation of mycotoxins depends not only on the fungal strain but also on environmental conditions, and the reasons for the low concentration of these fungal metabolites in the tested edible oils may be associated with low fungal contamination of the raw material. There is a set maximum concentration for mycotoxin contamination in some foods (Li et al., 2016;Nabizadeh et al., 2018). do not use many technological steps in the process of oil purification. Usually, local producers offer consumers nonrefined products, which are considered healthier and safer options. However, there have been publications about ZEN and trichothecene contamination in both nonrefined and refined oils from soybean, sunflower, and corn germ (Schollenberger et al., 2008). Also, refining cannot protect against Fusarium mycotoxin contamination of edible oils (Kamimura et al., 1986).
Assessment of mycotoxin contamination usually focuses on the main food products and the main mycotoxins for which regulatory limits have been set to protect human health (Fontaine et al., 2015).
Therefore, further research is needed, as the results of the present study suggest minor contamination of some of the tested edible oils with ZEN, T-2, and OTA. However, contamination of raw material with fungi is usually due to climatic conditions and many other agricultural factors. For this reason, not only oils but also seeds, as the raw material for oil preparation, must be controlled. Finally, considering that the popularity of edible oils prepared from various nontraditional raw materials is growing, and that such types of product are associated with a healthy lifestyle, it is very important to ensure their safety in terms of mycotoxin contamination.

| Polycyclic aromatic hydrocarbon contamination of tested oil samples
The PAH contamination in the tested edible oils is shown in In a comparison of ∑PAHs in the tested edible oil samples, the highest ∑PAH concentration was found in samples HE7 and CA10 (on average, 9.34 µg/kg). The lowest ∑PAHs was shown in samples CA9 and MU11 (0.56 and 0.74 µg/kg, respectively). The results of ANOVA indicated that the separate PAH concentrations and ∑PAHs were significantly influenced by the type of oil (p ≤.05) (Table 5).
Finally, not one of the tested edible oil samples exceeded the PAH concentration limits, which are for BaP in oil samples ˂2 µg/kg and for ∑PAHs ˂ 10 µg/kg.
European regulation sets limits for some PAHs for the category of oils and fats, that is, 2.0 mg/kg for BaP and 10.0 mg/kg for PAH4 (EC, 2011). PAHs may be generated during stock pretreatment (usually drying), and stock that is already contaminated with PAHs may further spread the contamination to the final product, edible oils (Lee et al., 2020). Edible oils are consumed directly, to improve the organoleptic properties of food or for thermal treatment of food (roasting). PAHs consumed in the diet are easily absorbed through the intestinal tract (Stavric & Klassen, 1994). Usually, contamination of edible oils with PAHs is a consequence of environmental pollution of the raw oilseeds (Drabova et al., 2013;Menichini et al., 1990), technological processes (contact with direct smoke during the drying process and solvent extraction), or the introduction of nonfood grade mineral oils (Hollosi & Wenzl, 2011). PAHs can be generated during high-temperature and long-duration frying . In any case, because of the special aspects of edible oils in the human diet, analysis of PAHs in edible oils is necessary (Mohammadi et al., 2020).

| CON CLUS IONS
The dominant FAs were palmitic, oleic, and linoleic acids in SB oil samples; palmitic, stearic, oleic, linoleic, and ALA in FL oil; palmitic, stearic, oleic, linoleic, and ALA in HE; palmitic, oleic, linoleic, ALA, gondoic, and erucic acids in CA; and oleic, linoleic, ALA, gondoic, and erucic acids in MU. According to the results obtained, the FA profile is a very important safety characteristic of an edible oil, and if some of the oils are not recommended for daily consumption as food ingredients, perhaps they could be used in other industries, for example, cosmetology, taking into account their other desirable bioactive compounds. Concentrations of 1.7 µg/kg T-2 and 3.0 µg/kg ZEN in SB2 oil samples, and 1.2 µg/ kg OTA in FL4 and 2.0 µg/kg ZEN in HE8 oil were found. The type of edible oil was a significant factor (p ≤.05) for separate PAH concentrations, as well as ∑PAHs; however, none of the tested edible oils exceeded the upper limits for PAH concentrations (for BaP content in oil samples ˂ 2 µg/kg and for ∑PAHs ˂ 10 µg/kg).
Finally, because of the special place of edible oils, in the human diet, not only should their contamination with mycotoxins and PAHs be controlled but also their FA profile must be taken into consideration to avoid adulteration of these products.