Slick Extra Virgin

 

Polyphenol concentration in extra virgin olive oil can be measured in three ways.

Method 1: Lets start with the classic, basic foundation  method used for total polyphenols everywhere.

The total polyphenol concentrations in extra virgin olive oil (EVOO/AOVE) and many other foods and beverages have routinely been made either directly or indirectly using the Folin-Ciocalteau (F-C) method developed in the 1950’s.

This is how the F-C test works. 1) total polyphenols are extracted from the EVOO/AOVE, 2) the extract is mixed with the reagent developed by Drs F and C, and 3) the oxidising F–C reagent reacts with the polyphenols (which are reducing agents to various degrees) to form a complex which is vividly blue in colour. The deeper the blue colour the greater the concentration of polyphenols in the EVOO.

For fun, I did the test on three Australian EVOO’s. Placed against a window (Figure 1), the differences in blueness from the three EVOO’s are shown (Cropped images from the test tubes are shown in Figure 2).

Figure 1: An example of the reaction between EVOO polyphenols and the F-C reagent.

Figure 2: The reaction of three EVOO’s with different polyphenol concentrations with the F-C reagent.

 

But how do you take that ‘blueness’ to a number such as milligrams of polyphenols in a kilogram of oil? The blue colour is caused by the F-C/polyphenol complex absorbing light at 765 nm. The absorbance of light at that wavelength is measured by a machine called a spectrophotometer, but the spectro just spits out arbitrary absorbance units. The concentration of polyphenols is proportional to the amount of absorbance. More absorbance, means deeper blue, and therefore proportionally more polyphenols.

Ok, now that we have moved on from subjective by sight ‘blueness’ to a semi objective measure called absorbance units, how can an absorbance unit be turned into an actual practical value of polyphenols in mg/kg that producers of EVOO can refer to, or compare with other producers?

This how it is done.

A phenolic compound which may or may not be found in EVOO (THE STANDARD) is put through the F-C test at different concentrations spanning a typical range found in EVOO, i.e 100 to 1000 mg/kg. The absorbance at the different concentrations is measured and plotted against the concentration of the phenolic compound chosen (the plot is called a standard curve). This plot is used to reverse track the absorbance of the EVOO sample to an actual concentration number (Figure 3).

Here is a real example of why it is important. Again, I did this myself. I produced two standard curves using two phenolic standards – the commonly used caffeic acid, and coumaric acid. (despite the acid at the ends of their names they are phenolic compounds). The absorbance of the ‘middle blue’ EVOO in Figure 3 was determined using both standard curves. A massive 85mg/kg difference in polyphenol reading. The same oil!

Figure 3: The effect of using different phenolic standards on quantifying total polyphenols in EVOO/AOVE.

 

So which polyphenol value is correct? Both. Just think of it as one is using cms and the other is using inches (not to scale 😊).

The fundamental message is that any total polyphenol concentration statement must specify the phenolic compound used for the standard curve to be meaningful. Aka 355 mg/kg of total polyphenols as caffeic acid equivalents or tyrosol equivalents or whatever equivalents.

 

Method 2: NIR (Near Infrared Spectroscopy)

For context. NIR results are by far and away the most quoted polyphenol results anywhere because they are rapid and affordable. Olive mills use them, as do large commercial laboratories reporting results.

Having said that, the reported results for total polyphenols have almost certainly been done by the F-C method using a particular phenolic standard, but indirectly.

The NIR sends ‘light’ across a range in the near infrared through a droplet of the oil. The bonds that connect the atoms together are stretched & bent leading to a unique characteristic spectrum. This spectrum is compared to the spectrum of hundreds of other EVOO’s, of which their total polyphenol are known using an early form of AI called chemometrics.

The known total polyphenols of the oils were almost certainly previously determined using the F-C method, and the NIR was ‘trained’ on these inputted values to predict the total polyphenol concentrations of new EVOO’s presented to it.

Therefore, (as an example), if the NIR was trained on polyphenol concentrations determined using a curve (Fig 3) using caffeic acid as a standard, then the NIR will estimate polyphenol concentrations of new samples presented to it in caffeic acid equivalents.

 

 

Method 3: High Performance Liquid Chromatography (HPLC)

Many years ago the International Olive Council method (since pulled) was effectively using the F-C method except they didn’t specify which phenolic should be used as the reference. They should have kept the method with a standard phenolic reference. But no, that would be far too easy.

Then a few years ago, someone conned the EU into thinking that only a few phenolic compounds in EVOO are responsible for their health benefits, and these needed to be quantified before any health benefit claim could be made on the producers’ label. So, the search was on for a method to quantify these specific compounds.

Other than not quantifying  total polyphenols which was the only thing most producers, and consumers really cared about, it was totally successful!

That method used high performance liquid chromatography (HPLC) whereby the total phenolic pool from an EVOO is loaded into a thin pipe (column) filled with stuff that phenolics stick to (C18 resin). Then the machine pumps through water and solvents and mixtures thereof in a defined way. The different phenolics in the initial mixture get washed out of the column at different times and as they are washed through, a detector records the time and the relative amount of polyphenols by measuring the absorbance of light typically associated with polyphenols (280nm). The result is a time-line called a chromatogram (Figure 4) which comprise peaks. Each peak has a time stamp indicating the identity of the polyphenol and an area which corresponds in a relative way to its concentration.

 

Figure 4: A typical HPLC chromatogram for polyphenols in EVOO / AOVE (Source: International Olive Council)

Great if you are quantifying a few compounds for a health claim, as the area of a few clearly defined early peaks can be calibrated to actual concentrations by running those simple cheap compounds at known concentrations effectively producing a standard curve for each of them.

But then…..

The IOC mandated a full HPLC test to measure total polyphenols where peak areas are added up to obtain a total polyphenol measure. While this seems ok, it has significant issues. Two related reasons. Firstly, not all phenolics absorb light maximally at 280nm. Those that don’t will produce smaller peak areas than those that do, making their contribution to the total polyphenol count smaller. In the total vote count, the vote of some phenolics are worth less than others!

Importantly, pure standards for most phenolics in EVOO shown on the chromatogram are unavailable. So just like the simple F-C test, the absorbance of a standard phenolic compound needs to be used as a yardstick to be able to determine the relative concentrations of the others, The IOC stipulated tyrosol.

Yes, It is what is called in HPLC as an internal standard. You include tyrosol at one concentration, measure its peak area, and then compute every other polyphenol concentration according to that standard.

In short, the F-C reaction does it all at once. The official IOC method effectively does the same thing using tyrosol as what is effectively a single point standard curve to every HPLC peak. Then the areas of all the peaks are added up to get a total polyphenol measure.

It was not surprising that a comparative study of polyphenol results using the F-C method with the official IOC HPLC method (Olmo-García et al., 2018),  showed highly correlated polyphenol results (r>0.9). Additionally, the IOC method, which uses tyrosol as the phenolic standard, produced polyphenol values that were on average 67% higher than those obtained by the F-C method using caffeic acid as the standard.

Olmo-Garcia et al. also concluded that “This (the IOC) protocol requires relatively expensive instrumentation, operational skills, and time of analysis incompatible with the necessity of categorizing EVOO rapidly during bottling”. My comment is that they are putting it politely.

So what is the solution to the confusion?

Someone needs to discover and publish in a reputable journal, a caffeic to tyrosol equivalence conversion factor by creating standard curves for both caffeic acid and tyrosol under 1) IOC specified HPLC conditions and 2) under F-C conditions. The latter will enable the the widely accepted new world industry caffeic acid equivalent measure based on tens of thousands of New World EVOO’s  to be converted to IOC official tyrosol equivalents, and of course vice versa.

Reference:

Lucía Olmo-García et al. (2019).  Evaluating the reliability of specific and global methods to assess the phenolic content of virgin olive oil: Do they drive to equivalent results? J Chromatogr A. 1585:56-69 DOI: 10.1016/j.chroma.2018.11.031,

 

Important Geeky Questions on this topic

So why do the different phenolic standards produce standard curves with different slopes?

I’ll try to keep this simple without being ridiculous. The phenolic family of compounds all have one thing in common. Their chemical structure consists of at least one ring of six carbon atoms bonded together (like chicken wire with a carbon on each corner), AND at least one hydroxyl group (an oxygen-hydrogen, OH) attached to the ring off one of the carbons. But like human families, there are lots of differences (like your older uncle who shouts at plane contrails at Christmas lunch, your lovely unmarried auntie who loves dogs, and a throwback strange scientist 😊), phenolic compounds can have more than one OH group hanging off the carbon ring, and they hang off the carbons in different positions. They typically have some other chemical group like an alcohol, an acid, a methoxy, a sugar, or in case of the unique EVOO phenolic oleocanthal an aldehyde hanging off the ring.

Now remember the F-C reagent. It reacts with the polyphenols (which are reducing agents) to form the important blue complex. The ability of the polyphenol to reduce the reagent depends primarily on the number of hydroxyl (OH) groups and their position(s) on the ring.  To a lesser extent reducing power also depends on the type of hanger-offerers chemical groups. Therefore, if the phenolic standard used is a weak reducing agent it will produce a shallow sloped standard curve and therefore will produce higher polyphenol readings. Tyrosol, frequently referred to by Italian researchers looks suspiciously like such a candidate (but at 20K a gram I’d have to take out a mortgage on my house to test this).

So why do many labs use phenolic compounds not found in EVOO to determine equivalents?

Cost, availability and solubility. Firstly cost. Standard good scientific practice demands that for every analysis run of samples, a new standard curve is produced at the same time. Currently caffeic acid (in a high purity HPLC grade) sells for $US6.20 per gram. Tyrosol, a phenolic found in EVOO sells for $US 20,700 per gram! Secondly, important phenolic compounds in EVOO like oleocanthal just can’t be purchased not for love nor money. Finally, many polyphenols take a lot of time and effort to completely dissolve, and unless they do, accurate standard curve cannot be produced. Hence the popularity of the very soluble and cheap phenolic, caffeic acid for the F-C test and by default most NIR results, and tyrosol for HPLC needs tiny amounts for a single point reference standard.

 

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I often get asked whether extra virgin olive oil is more fattening than other edible oils. Given the frequency that the question is posed, I’ll assume that there is some ‘information’ out there somewhere saying that it must be so. Or perhaps it is just a case of people thinking that if extra virgin olive oil is so healthy for you then it must have some downside. Can’t have your cake and eat it too right?

So I searched the peer-reviewed scientific literature to find out the energy (or calorific) values of different edible oils. Two papers provided some relevant data.

Fassinou et al. (2010) measured the energy values for a wide range of edible oils (Figure 1), but unfortunately olive oil was not one of them. However I’ve provided a logical estimate of the energy value of extra virgin olive oil based on the predictive model that they derived (1).

Figure 1: Energy values of various edible oils. Value for extra virgin olive oil is an estimate. See footnote 1.

Sadrameli et al. (2008) determined the energy values of the free fatty acids themselves (actual edible oils comprise 3 fatty acids of various sorts joined together to a glycerol molecule, not as individual free fatty acids). However, these values can presumably be good proxies of the energy values of the edible oils that are rich in a particular fatty acid (oleic acid makes up on average ¾ of the fatty acids in olive oil for example) (Figure 2). However as all edible fats comprise a mixture of fatty acids combined in various combinations in groups of three, this data is at best an estimate of the energy values of real edible oils.

Figure 2: Energy values of different fatty acids. Examples of edible fats that have high proportions of that fatty acid are given in parenthesis.

So what’s the conclusion? There is no practical difference in the energy values of the different edible oils.

OK, these results do not consider that our bodies may absorb one type of fat differently from another. Some have suggested that we absorb short chain fatty acids such as those found in coconut oil differently. You can read more about the good, the bad and the ugly of this possibility elsewhere in this blog (search for coconut oil in the search box to the right), but based purely on the maximum potential of each fat to contribute to our daily energy intake, there is little evidence of any practical differences between edible oils when it comes to their energy quotient (2).

References

Fassinou, W.F., Sako, A., Fofana, A., Koua, K.B. and Toure, S. (2010) Fatty acids composition as a means to estimate the high heating value (HHV) of vegetable oils and biodiesel fuels. Energy, 35, 4949-4954. doi:10.1016/j.energy.2010.08.030

Sadrameli, S.M., Seames, W. and Mann, M. (2008) Prediction of higher heating values for saturated fatty acids from their physical properties. Fuel, 87, 1776-1780. doi:10.1016/j.fuel.2007.10.020

Footnotes:

(1) The energy value can be predicted from the ‘carbon number’ of the oil. The carbon number is the average carbon chain length of the fatty acids that comprise the oil. In olive oil the carbon number averages 17.5 (based on the fatty acid profiles of 500+ extra virgin olive oils reported by Mailer and Ayrton 2008). The energy value of the ‘typical’ extra virgin olive was then estimated using the predictive model derived by Fassinou et al. (2010)..

(2) To save you scrambling for your abacuses paleo people, coconut oil has on average 3% lower energy value compared with the other oils, which is due to the lower average carbon number of the predominant fatty acids in coconut oil. 3% less energy but 90% less antioxidants. Take your pick.

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What is Extra Virgin Olive Oil?

The ‘oily’ olive fruit juice extracted from fresh olives using a mechanical process which does not involve the application of excessive heat, additives or solvents.

Should I only buy first cold pressed oil?

Most extra virgin olive oil is produced by spinning the lighter oil away from the other heavier components of the olive such as water and olive matter using a centrifuge. So the term ‘cold pressed’ is redundant.

What’s the difference between extra virgin olive oil and other types of olive oil?

Extra virgin olive oil is a whole, unprocessed food and therefore contains higher amounts of healthy antioxidants. Conversely, ‘light’, ‘pure’, ‘olive oil’ and pomace oils are highly processed products.

Is ‘Pure’ olive oil better than Extra Virgin Olive Oil?

No. The word ‘Pure’ (together with ‘Light’) are purely marketing terms applied to refined olive oil. Pure sounds better than refined.

Do ‘light’ olive oils contain fewer calories than Extra Virgin Olive Oil?

No. All olive oils (and indeed all edible oils) have the same energy values.

This oil is bitter and peppery. Is it ok?

The bitterness and pepperyness indicates that the oil contains healthful antioxidants.. These characters complement the taste of strongly flavoured foods.

Is eating Extra Virgin Olive Oil good for my health?

EVOO’s contain significantly higher levels of monounsaturated fats and antioxidants than any other oil. These attributes are sought after by the health conscious.

Does the colour of olive oil indicate quality?

No. High quality extra virgin olive oils can appear emerald green through to golden yellow.

Where should I store Extra Virgin Olive Oil?

They should be stored in a cool dark place. For longer term storage where oils are used infrequently, refrigeration is a good option.

How long does Extra Virgin Olive Oil last?

They are best consumed young as fresh flavours and healthful polyphenols decline over time.  So when the new season oils are released, buy them.

I’ve heard that you can’t cook with Extra Virgin Olive Oil as its smoke point is too low. Is this true?

The temperature at which good quality (low acidity) extra virgin olive oil begins to smoke is significantly higher than what is needed to fry food (180C). So you can fry in EVOO.

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