Ergosterol as a marker for the use of degraded olives in the production of olive oil
Introduction
The quality of olive oils is largely determined by the sensory properties, and a first requirement for the quality is the absence of what is considered as an off-flavor. Off-flavors may be generated by substances from degraded olives, from degradation of the extracted oil (e.g. oxidation) or from varieties of olives naturally containing unpleasant components.
Numerous chemical parameters are used for determining olive oil quality. In fact, since oil quality is affected by different factors (as e.g. olives health state, oil production process, oil shelf life, etc.), different types of defects can originate from the different factors and different analytical parameters are needed for their assessment. The best ones are those which are most closely related to the cause of the defects.
High acidity stands for hydrolysis by lipase that is activated by degraded cell compartmentation. The degradation of cells also causes the release of methanol from pectin, which can be determined indirectly by fatty acid methyl esters formed by transesterification with glycerides in the olives (Aparicio and Harwood, 2013, Biedermann et al., 2008, Sadkaoui et al., 2016).
Oxidation may occur in the olives as well as during storage of the oil. It is determined by the peroxide value, expressed as [meq O2/kg], or by the UV absorbance at 232 and 270 nm (K270 and K232) detecting conjugated dienes and trienes (Giuffrè, Caracciolo, Zappia, Capocasale, & Poiana, 2018). Limits are set out for these parameters by the European Union legislation for an olive oil to be sold as extra virgin olive oil. The degradation products of chlorophylls a and a’ can also be determined to assess the quality of the olive oils. The distribution and the content of chlorophylls in olive oil are highly dependent on the agroclimatic conditions, the treatment of the fruit and the time and type of food processing (Inanc, 2016).
Thermal treatment or aging of olive oil have been determined using the isomerization of 1,2-diacylglycerols to 1,3-diacylglycerols as indicator: fresh olive oils almost exclusively contain 1,2- diacylglycerols (Okparanta, Daminabo, & Solomon, 2018). Sterenes (dehydroxylated sterols), are used for assessing refining or fraud by admixture of other refined oils (Crews et al., 2014, Grob et al., 1992, Grob et al., 1994, Grob et al., 1994, Jee, 2009).
Linear wax esters from the skin enable the detection of extraction procedures that are unacceptable for extra virgin olive oils, such as solvent extraction or second pressing/centrifugation of the pomace (Biedermann et al., 2008, Giuffrè, 2013), but are also indicative for overripe olives with soft skins (Biedermann et al., 2008, Mariani and Bellan, 2008, Mariani and Bellan, 2011).
Use of olives attacked by insects or degraded by microorganisms is commonly detected by fatty acid ethyl esters, as such damage tends to be accompanied by activity of yeast forming ethanol. Due to high solubility in water, ethanol is largely removed during extraction of the oil, but transesterification with the triglycerides in the fruit forms fatty acid ethyl esters that are transferred to the oil (same as mentioned above for methanol). However, this determination is indirect and hampered by the degree of transesterification depending on time, temperature and acidity during storage of the olives, i.e. the amount of ethanol formed cannot accurately be derived (Biedermann, Bongartz, et al., 2008). Furthermore, fatty acid ethyl esters are rather easily removed through deodorization.
Here the use of ergosterol (ergosta-5,7,22-trien-3β-ol) is proposed for detecting the use of degraded olives in producing oil. Ergosterol is produced by mold and yeast (Hashim et al., 2016), but not by olives; ergosterol should be absent in high quality extra virgin olive oils.
Advantages are that ergosterol is virtually completely transferred to the oil owing to its lipophilicity, it is produced by yeast as well as mold and it cannot easily be removed by deodorization.
The ergosterol content in olive oils was determined by on-line HPLC-GC-MS. This method was derived from the on-line HPLC-GC method for total sterol concentrations (Biedermann, Grob, & Mariani, 1993). Transesterification of the triglycerides to methyl esters reduces the retention of the bulk constituents in normal phase HPLC, improving their separation from the sterols, but also cleaving the sterol esters, including the ergosterol esters, thus yielding data on the total ergosterol content. The reaction is performed under conditions optimized to result in virtually complete transesterification of the sterol-type esters, but little saponification. The efficiency of HPLC had been used for the pre-separation of the sterols for the determination of isomerized Δ7-sterols, markers for refined sunflower oil (fraudulently) added to, e.g., olive oil (Biedermann, Grob, Mariani, & Schmid, 1996). This efficiency is also needed for the separation of ergosterol from the major sterols that would severely overload GC otherwise.
As a proof of concept, concentrations of ergosterol in various olive oils and degraded olives are presented. Furthermore, ergosterol concentrations are compared to ethyl ester contents and sensory quality.
Section snippets
Reagents and standards
The analytical standards of 7-dehydrocholesterol (CAS Number 434-16-2), ergosterol (CAS Nr. 57-87-4) and vitamin D2 (Vit D2; CAS Nr. 1217448-46-8) were purchased from Sigma-Aldrich (St. Louis, MO), cholesterol (CAS Nr. 57-88-5) from Fluka (Buchs, Switzerland). Sodium methylate (CAS Nr. 124-41-4) and citric acid (CAS Nr. 77-92-9) were purchased from Sigma-Aldrich (St. Louis, MO) and VWR-Merck (Darmstadt, Germany) respectively. Stock solutions of each compound were prepared by dissolving 10 mg in
Analytical method
A critical point in the method for determining ergosterol was the separation from the dominant and structurally similar sterols present at roughly 1000 times higher concentrations in order to avoid overloading of GC. This was achieved by HPLC. Another one was the stability of the HPLC retention time affected by the matrix. Transesterification was used to liberate ergosterol from esters, but also to convert triglycerides to fatty acid methyl esters that are eluted earlier than triglycerides and
Conclusions
As the defects in olive oils may have numerous reasons, several chemical parameters are needed for their assessment. They should be specific in indicating the cause of the defect. The ergosterol concentration could be a promising one, as it is clearly and directly links to degraded olives.
Presently microbial attack is mainly determined by fatty acid ethyl esters. These esters are indicators of yeast. As the concentration of these esters not only depends on the amount of ethanol formed, but also
CRediT authorship contribution statement
Maria Chiara Boarelli: Formal analysis, Data curation, Validation, Visualization, Investigation. Maurus Biedermann: Supervision, Methodology. Martin Peier: Supervision, Data curation, Validation. Dennis Fiorini: Writing - original draft, Writing - review & editing, Funding acquisition, Resources. Koni Grob: Conceptualization, Writing - original draft, Writing - review & editing, Funding acquisition, Resources.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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