Discussion of Protein purification

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Both PFJ and re-suspended powder were used for protein purification and both start materials could be separated into relatively pure patatin and PI fractions. At an industrial scale PFJ needs to be processed immediately and fractionated during the potato harvest. If a spray dried isolate is made during the harvest, it can be further fractionation in the remaining months of the year, preferably by using some of the same equipment. Compared to PFJ, more concentrated re-suspended solutions can be made, thus decreasing the size of expensive chromatographic equipment. Re-suspended powder however needs to be concentrated and spray dried one additional time compared to PFJ which could increase cost. Purification of either PFJ or re-suspended powder led to differences in colour of the final fractions. For PFJ the patatin fraction had an acceptable light colour and many of the dark coloured compounds could be separated by the stepwise elution on IEX. The PI fraction achieved an unacceptable dark colour, but this was mostly due to a longer processing time and due to freezing and thawing of this fraction giving PPO more time to act. If the PI fraction had been processed immediately a light colour would also be achievable. For the re-suspended powder did the PI fraction have a white hue while the patatin fraction was dark. A higher salt concentration was needed to elute bound patatin, compared to patatin in PFJ, and therefore did the coloured compounds also elute with patatin. A solution could be screening of additional chromatographic materials to assess if better separation could be found or change the stepwise gradient to two steps resulting in two patatin fractions with the first having improved colour and the second being brown. The relative proportion of the two glycoalkaloids α-solanine and α-chaconine changed during purification by IEX and HIC. The PI fraction had a higher content of α-solanine and the patatin fraction a higher content of α-chaconine. HIC purification led to an enrichment of α- solanine in the least hydrophobic PI fraction while α-chaconine was enriched in the most hydrophobic patatin fraction, suggesting different ionic and hydrophobic properties of the two glycoalkaloid types. Both PI HIC fractions had a total glycoalkaloid content below 150 ppm, while only one patatin HIC fraction was below this threshold. The PI fractions were concentrated by ultrafiltration at pH 3 which could lead to lower glycoalkloid content according to an earlier published patent, so the low values might not only be a result of IEX and HIC purification but also to some degree the final concentration and diafiltration.

8.2 Protein determination methods To determine the protein concentration is of fundamental importance when working with protein purification or functionality. The presence of interfering substances makes this task far from trivial. In PFJ and re-suspended powder are numerous polyphenols and oxidations products hereof present which interacts with the most common protein determination techniques. Protein concentration based on UV signal at 280 nm cannot be used due to interference with polyphenols and an increased signal if the polyphenols are bound to the proteins. The most common protein determination techniques like BCA, Bradford and Lowry are all affected resulting in either under-or overestimation. Pre-treatments like acid precipitation or binding of phenols with hydrophobic materials have been used to correct for interference and a modified Lowry method developed that also quantifies covalently bound proteins. Measurements were in this study conducted by the BCA method. For solutions based on spray dried powder this assay seemed to perform satisfactory and solubility data was agreeing to analysis performed at KMC (Brande, Denmark) based on the Kjeldahl method. Overestimations was however seen when PFJ and PI from PFJ was measured. The Bradford method was tested but this method let to underestimation. For future studies it is needed to perform a thorough test of different samples and protein determination methods. A modified Lowry method able to correct for bound phenolic substances was tested by a colleague but with no success.

8.3 Determination of total phenol content The Folin-Ciocalteu is the most common method to estimate phenol content in foods and crops. Unfortunately can the Folin-Ciocalteu reagent react with a long list of substances e.g. proteins, vitamins, thiols, nucleotides and inorganic ions also react causing overestimation. A way to assess interfering substances is measurements of colour development before addition of alkali. No colour development was appearing in the potato protein fraction tested. A surprising observation was however made with the PI proteins that dissolved and gelled in methanol and ethanol solutions used for phenol extraction. By using more dilute suspension samples could be mixed with the Folin-Ciocalteu reagent but it is not clear to which degree this affected the assay. The Fast Blue BB assay which is claimed to only react with phenols (Medina 2011) was also tested earlier in this study on spray dried powder. The results were however not convincing, sometimes producing results with negative values and precipitates making measurement impossible. Other authors have also noted problems with this assay (Granato et al. 2016). Preliminary studies with the spectrophotometric method of (Dao and Friedman 1992) were made, and future studies of this method and the Prussian Blue assay is needed to get a trustworthy result.