Effects of processing and storage on chlorophyll derivatives in commercially extracted canola oil

This study characterizes the chlorophyll pigments present in canola oil immediately after commercial extraction and following oil storage to determine the best storage conditions for analytical samples and to examine the changes that chlorophyll derivatives undergo during oil processing and storage. Samples of pressed, solvent-extracted, crude and degummed canola oils, obtained from a commercial crushing plant, were stored for one month under four different conditions—in the freezer, in a refrigerator and at room temperature both in the light and in the dark. Chlorophyll derivatives (chlorophylls, pheophytins, pyropheophytins) were measured by high-performance liquid chromatography immediately after sampling and then on a weekly basis. The main pigments present in commercially extracted canola oil were pheophytin a, pyropheophytin a, chlorophyll a and chlorophyll b. The “a” derivatives comprised 81 to 100% of total chlorophyll pigments in the fresh oil samples. During degumming, the remaining chlorophylls were converted to pheophytins and pyropheophytins. During oil storage, exposure to light at room temperature affected the composition of chlorophyll derivatives as chlorophyll b was converted to pheophytin b and chlorophyll a was converted first to pheophytin a, then to pyropheophytin a.     

Determination of chlorophyll pigments in crude and degummed canola oils by HPLC and spectrophotometry

Chlorophyll pigments in crude and degummed canola oils were analyzed by spectrophotometry using a modified AOCS Method and by reversed phase HPLC. HPLC showed that crude canola oils contained very littlechlorophyll a orb, these pigments having been converted to pheophytins and other pigments with similar spectral properties. The ratio ofchlorophyll a∶b in the seed was found to be about 3∶1 while the ratio ofpheophytin a∶b in the oil was about 9∶1. As the AOCS Method for determining oil chlorophyll was calibrated for pure chlorophyll, the use of this method on crude canola oil results in a significant error. Recalibration of the spectrophotometric procedure with pheophytin gave better agreement with the HPLC method. Paper No. 635 of the Canadian Grain Commission, Grain Research Laboratory, 1404-303 Main Street, Winnipeg., Manitoba, Canada R3C 3G8. Presented at the A.O.C.S. 79th Annual Meeting, Phoenix.     

Behavior of chlorophyll derivatives in canola oil processing

Chlorophyll derivatives in canola oil were analyzed quantitatively by reversed-phase high-performance liquid chromatography without any pretreatment. The main components were pheophytin (pheo) a and b and pyropheophytin (pyro) a and b. The factors affecting the types and concentration of chlorophyll derivatives in oil have been investigated during seed preparation, expelling, extraction, degumming and alkali-refining processes. Bleaching tests of alkali-refined canola oil with activated earth indicated the adsorption of each derivative to decrease in the following order: pheo a > pyro a >> pheo b > pyro b. In bleaching with activated carbon, however, the following order was observed: pyro b > pheo b > pheo a > pyro a.     

Characterization of chlorophyll pigments in ripening canola seed (Brassica napus)

This study characterizes the chlorophyll pigments in ripeningBrassica napus seed. Seed samples, collected weekly as the crop ripened, were analyzed by high-performance liquid chromatography to characterize chlorophyll pigment composition. Chlorophyll A, chlorophyll B, pheophytin A and pheophytin B were the predominant pigments, while pheophorbide A, methylpheophorbide A and pyropheophytin A were minor components. No differences in pigment composition were observed between the three cultivars tested or between early and late seeding dates. There were differences in pigment composition between the two years of the study, which may result either from seed aging during storage or from environmental influences. Pigment composition was dependent on seed maturity, with physiologically mature green seeds containing both chlorophylls and pheophytins, but fully mature seeds containing only chlorophylls. Pheophytins and the minor components appeared transiently, presumably formed from the chlorophylls and subsequently degraded. The ratio of chlorophyll A/B increased during seed ripening, with fully mature canola seed having a chlorophyll A/B ratio twice that of physiologically mature green seed. The “B” derivatives degraded faster than the “A” derivatives, suggesting enzymatic reactions. The initial steps in the chlorophyll breakdown pathway in canola seed appear to be:      

Detection of chlorophyll derivatives in soybean oil by HPLC

Chlorophyll derivatives have been isolated from a degummed soybean oil by cellulose column chromatography and resolved by reversed phase HPLC. The HPLC separation was performed on a Zorbax ODS column using acetone-methanol (75:25) as the mobile phase. Seven major components were detected by visible (650 nm) light absorption. Pheophytin A is the predominant component of the mixture (40–45% of the total). Pheophytin A’, pyropheophytin A, and three unidentified pigments having spectral features resembling pheophytin A comprise the other major pigments. No evidence was found for the presence of chlorophylls A and B in this oil.     

Effect of storage on the original pigment profile of spanish virgin olive oil

The present study was carried out on 12 virgin olive oils to determine whether one year's storage under mild conditions of 15°C and darkness affected the initial pigment composition of recently extracted virgin olive oil. Although the total pigment content remained constant, the individual contribution of each pigment changed. The acid compounds liberated from the fruits during the oil extraction process promote the beginning of chlorophyll pheophytinization and the isomerization of the 5,6-epoxide groups of the minor xanthophylls. During the first 3 mon of storage, there was a generalized increase in pheophytinization that was different for each oil (P<0.01, Duncan test) but was not correlated with the free acidity measured in them. At the same time, isomerized xanthophylls and allomerized pheophytins increased slightly. Following this stage, pyropheophytin a (a pigment not present in the initial oils), was detected; its concentration increased during storage. There were no significant differences in the final percentages of pyropheophytin a among the 12 oils, and the concentration of this new compound represented around 3% of the chlorophyll fraction. The pheophytin a/pyropheophytin a ratio always exceeded 20. All these small pigment transformations were signs that the oil had been stored. The content and class of pigments present in virgin olive oil are authentic indicators of its history prior to marketing.     

Studies on the pigments of some citrus,prune and cucurbit seed oils when processed with or without cottonseed oil

Pigments of citrus, prune and cucurbit fruit seed oils were studied spectrophotometrically. The citrus fruits used were: orange (O), mandarin (M), bitter orange (BO) and lemon (L). The prunes used were apricot (A), peach (P) and plum (PL); while melon (M), watermelon (WM) and Winter squash (S) were the cucurbits. Absorption spectra and Lovibond color were studied for crude, refined and bleached oils. Cottonseed oil (CSO) was mixed with some of the previous oils in the crude state, then refined and bleached. Absorption spectra of the crude fruit seed oils revealed carotenoid pigments at 400, 425, 455 and 480 nm, chlorophyll at 610 and 670 nm and unknown pigments at 525, 570 and 595 nm. Refining did not remove these pigments, whereas bleaching eliminated them completely. In oil mixtures of CSO+A, CSO+M and CSO+S, interference occurred between gossypol ‘360 nm’ from CSO and the pigments of A, M and S seed oils. Refining the oil mixtures removed gossypol, but its effect on carotenoids, chlorophyll and unknown pigments was limited. Bleaching completely removed all these residual pigments. Lovibond color for all bleached oils was very low (0.2–2 yellow). The refined oils, except those containing Winter squash seed oil, were found to have an acceptable color (0.8–15 yellow). Results of the proposed process reveals the possibility of mixing crude edible oil with crude fruit seed oils, then processing the oil mixture by the conventional methods of refining and bleaching.     

Possible causes for decreased stability of canola oil processed from green seed

Canola oil extracted from seeds with a high-chlorophyll content can contain chlorophyll derivatives in excess of 30 ppm. When processed, this oil has been observed to be less stable than oil (typically containing 5 to 25 ppm chlorophyll) processed from high-grade seed. Possible causes for this phenomenon were investigated in this study. The effect of initial pheophytin content was examined by mixing fully saturated oil (tricapryloylglycerol) with increasing amounts of pheophytin and then by subjecting the mixtures to processing conditions. When the processed oils were combined with an unsaturated oil (canola oil), the oxidative stabilities decreased as the pre-processing content of pheophytin increased. Examination of the effect of increased bleaching to remove excessive levels of pheophytin showed that oil stability decreased with increasing exposure to bleaching clay. Additionally, processing treatments did not remove secondary autoxidation products from oil that was abused prior to processing. Such a finding revealed the importance of initial oil quality on processed oil stability, i.e., the greater abuse of the crude oil (resulting in greater contents of secondary oxidation products), the lower the stability of the processed oil. Finally, previous reports by other researchers of pheophytin's pro-oxidative effect in oil stored in light were confirmed.     

Spectrophotometric analysis of chlorophyll pigments in canola and rapeseed oils

Of all the vegetable oils commonly in production, rapeseed and canola oil have the highest level of undesirable chlorophyll pigments. Chlorophyll is desirable as a flavour and colour agent in olive oil, the other species with significant levels. In canola and rapeseed oils, chlorophyll is not desired because it imparts an undesirable colour, especially in North America where consumers are accustomed to colourless oils. It also makes the processed oils more susceptible to oxidation, even after the chlorophyll components have been removed. The content and role of chlorophyll in canola seed and oil have been well reviewed recently [1].     

Content of chlorophylls and carotenes in rapeseed oil

The content of the green components in crude rapeseed oil, namely chlorophylls A and B, as well as the products of their decomposition pheophytins A and B, were determined by means of the spectrophotometric method of analysis. No chlorophyll A was found in any of the analyzed samples but the content of pheophytin A was quite high and amounted to from 17.99 to 25.65 ppm. Because of the fact that the oils were investigated 5 to 6 months after their extraction, the results obtained are not unexpected, bearing in mind that chlorophyll contained in oil easily changes into pheophytin. No chlorophyll B was found in some of the samples, investigated and in the remaining samples from 0.14 to 1.79 ppm was found. This can be explained by the hypothesis of slower change of chlorophyll B into pheophytin B. The content of pheophytin B in the investigated samples was between 0.52 and 6.15 ppm. This confirms the results obtained by Mingot (3). With old oils, the results of determining such components, whose content approaches nil in the sample, are less precise because of the influence of the oil which was used as a blank. The content of carotenes in crude rapeseed oils was also determined by the spectrophotometric method after isolating them by means of the column chromatography on Al₂O₃. In spite of the small amount of carotenes determined in rapeseed oils, a correlation can be observed between the quantity of chlorophylls and carotenes. It has been found that the higher the chlorophyll content, the higher also the carotene content, even if very slight in some cases, in all the investigated oils either extracted or pressed.     

Canola check sample series

Four separate check series for canola seed, canola meal, crude and crude degummed canola oil and refined, bleached and deodorized canola oil were started in July 1980. They are devised to help laboratories calibrate analytical procedures to be used on canola products. This paper describes the organization of the program and special features of canola analysis. It indicates and discusses less than reliable analyses and describes the active participation of the program organizers to improve reliability of analyses. Deceased, 1983. POS Pilot Plant Corporation (the initials POS denote protein, oil and starch) is a private company set up by the federal and two provincial Canadian governments and a number of food corporations to provide pilot plant services on a fee-for-use basis to clients.     

Sulfur content of rapessed oils

Sulfur contents in rapeseed oils were determined by reduction with Raney nickel, acidification, and titration of released H₂S with mercuric acetate. The sulfur contents decreased with successive steps of industrial processing, i.e., crude oil, 17–31 ppm S; degummed, 16 ppm; alkali refined, 4–9 ppm; bleached, 3–5 ppm; and deodorized, <1 ppm. Laboratory-extracted oil from sound seed contained no detectable sulfur, regardless of the glucosinolate content of the seed. Heating of the seed or addition of water to the seed prior to extraction increased the sulfur in the oil-less, however, for low-glucosinolate seed than for high-glucosinolate seed. Laboratory-extracted oils from green, frost-damaged, and bin-heated seed contained appreciable amounts of sulfur. Contribution No. 403, Department of Plant Science, University of Manitoba.     

Chlorophyllase biocatalysis in an aqueous/miscible organic solvent medium containing canola oil

Chlorophyllase catalyzes the bioconversion of chlorophyll into chlorophyllide by replacing the phytol group with a hydrogen atom. There is an increased interest in the biotechnological application of chlorophyllase for the removal of green pigments from edible oil and its potential as an alternative to the use of the conventional bleaching technique. Partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity in an aqueous/miscible organic solvent system containing refined-bleached-deodorized (RBD) canola oil, using chlorophyll and pheophytin as substrate models. The results indicated that chlorophyllase biocatalysis could be successfully carried out in an aqueous/miscible organic system containing RBD canola oil. The presence of 20% RBD canola oil decreased the hydrolytic activity of chlorophyllase by 2.2 and 6.7 times, using chlorophyll and pheophytin as substrates, respectively. In addition, acetone acted as an activator of chlorophyllase activity at low concentrations and an inhibitor at higher ones. The optimal reaction conditions for chlorophyllase biocatalysis in the aqueous/miscible organic system were determined to consist of 20% RBD oil and 10% acetone at a 200 rpm agitation speed and at a temperature and substrate concentration of 35°C and 12.6 μM for chlorophyll, and 30°C and 9.3 μM for pheophytin.     

Oxidative stability of high-fatty acid rice bran oil at different stages of refining

The contents of natural antioxidants and the oxidative stability of rice bran oils at different refining steps were determined. Tocopherols and oryzanols were constant in crude and degummed oils but decreased in alkali-refined, bleached and deodorized oils. The process of degumming, alkali-refining, bleaching and deodorization removed 34% of the tocopherols and 51% of the oryzanols. During storage of deodorized oil for 7 wk, 34% of the tocopherols and 19% of the oryzanols were lost. The maximum weight gain, peroxide value and anisidine value were obtained from alkali-refined oil during storage. The order of oxidation stability was crude ≥ degummed > bleached = deodorized > alkali-refined oil.     

Effects of processing on the quality and stability of three unconventional Sudanese oils

In a refining experiment, on a laboratory scale, crude oils from Sclerocarya birrea (SCO), sorghum bugs (SBO), water‐extracted melon bugs (MBO H₂O) and solvent‐extracted melon bugs (MBO SOL) were processed by alkali refining. Quality changes were characterized by the determination of free fatty acids (FFA), peroxide value, tocopherols, sterols, phosphatides and stability against oxidation (Rancimat test). In addition, the fatty acid composition was determined. It is clear that the contents of phosphatides, peroxides, tocopherols, sterols as well as oxidative stability were reduced during processing, while FFA were nearly totally removed. The content of phosphorus was reduced in SCO, SBO, MBO H₂O and MBO SOL by 26, 19, 12, and 78%, respectively, while complete oil processing removed 95, 99, 96 and 99% of the FFA in crude oils, respectively. The level of total tocopherols decreased during processing by 38.7, 83.8, 100, and 33.3%, respectively. The color decreased through the processing steps up to bleaching; then, in the deodorization step, it darkened sharply in all samples. No change in the fatty acid composition was observed. The order of oxidation stability was crude > degummed > deodorized > neutralized > bleached, in SCO; and crude > degummed > neutralized > bleached = deodorized, in MBO H₂O; and crude > degummed > deodorized > neutralized > bleached in MBO SOL; while in SBO, the order of oxidative stability was deodorized > crude > degummed > neutralized = bleached. Total sterols decreased by 42–92% in the processed oils, compared with crude oils.     

Processing of cottonseed

1. The pigmentation of cooked cottonseed has been shown to depend principally upon the moisture content and period of heating of the seed.
2. Several samples of crude hydraulic-pressed and screw-pressed oils produced under known processing conditions were found to differ markedly from each other with respect to their original colors and refining characteristics.
3. The screw-pressed crude oils were more deeply colored and contained one principal pigment, whereas the hydraulic-pressed oils contained two principal pigments.
4. The absence of significant amounts of gossypol in the crude oils has been demonstrated by means of a new technic for the quantitative isolation of gossypol.
5. The crude oil pigments differed from gossypol, but like gossypol, they were removed during alkali refining.
6. The pigmentation of the crude oils has been shown to depend principally upon the pigmentation of the original seed and the moisture content of the seed during cooking.
7. On the basis of their absorption spectra it has been deduced that the alkali-refined hydraulic-pressed oils contain two to three pigments originally present in the crude oils whereas the alkali-refined serewpressed oils contain these same pigments as well as a large number of decomposition products of the principal crude oil pigment.

     

Canola oil processing in Canada

Canola is the registered trademark of the Canola Council of Canada for the seed, oil and meal derived from rapeseed cultivars low in erucic acid and low in glucosinolates. Conversion to canola cultivars on a commercial scale started in 1976; in 1981, ca. 87% of the brassica-based oil crop in Canada was of canola quality. Canola oil is the most important oil in Canada. Processing of the oil is, in its essentials, conventional. A few problems not usually encountered with other oils are its chlorophyll content which requires extra processing and analytical effort, and certain limitations in crystallization behavior when highly hydrogenated. Advantages are that stable oils can be produced at moderate degree of hydrogenation, and without hydrogenation in the case of salad oil. New developments in processing of the oil have led to the production of acid-degummed, crude oil on a commercial scale. This opens the possibility to apply physical refining to the oil.     

Effects of processing steps on the contents of minor compounds and oxidation of soybean oil

The processes of degumming, alkali refining, bleaching and deodorization removed 99.8% phospholipids, 90.7% iron, 100% chlorophyll, 97.3% free fatty acids and 31.8% tocopherols from crude soybean oil. The correlation coefficient between the removals of phosphorus and iron in soybean oil during processing was r = 0.99. The relative ratios of α-, β -, γ- and δ-tocopherols in crude oil, degummed oil, refined oil, bleached oil and deodorized soybean oil were almost constant, γ- and δ -tocopherols represented more than 94% of tocopherols in soybean oil. The order of oxidation stability of oil is crude > deodorized > degummed > refined > bleached oil.     

Effect of sprouting on the quality and composition of canola seed and oil

Sprouting has been considered as a damage factor in grading canola. This project deals with the evaluation of the effect of sprouting on the quality and composition of canola seed and oil. Sprouted seeds had lower oil content than nonsprouted seeds as determined by exhaustive petroleum ether extraction. The difference, although statistically significant, was small, less than 0.1% oil at the maximum level of sprouting allowed in topgrade canola. There were no differences in chlorophyll contents or moisture contents between sound and sprouted seeds. Sprouted seeds had significantly higher levels of FFA and crude protein than sound seeds. Oxidation parameters (diene and aldehyde) were higher in oils from sound seeds than oils from sprouted seeds, but there was no statistically significant difference in PV. Sprouted seeds had higher levels of tocopherols and sucrose, but lower levels of raffinose, stachyose, and total sugars than sound seeds. There was no difference in overall FA composition of the oil between sound and sprouted seeds. The second extraction of the Federation of Oils Seeds and Fat Associations (FOSFA) extraction method, which allowed the extraction of more polar lipids, contained significantly more saturated FA. However, this was not significant in the overall FA composition of the oils because this fraction counted for about 2% of the total lipid content. The presence of sprouted seed had an effect on results for oil and crude protein determined by NIR as compared with results by FOSFA extraction, or pulsed NMR for oil and Dumas combustion for crude protein. Addition of sprouted seed samples to the NIR, calibration set overcame this problem. These results suggested that sprouting did not have a highly damaging effect on the quality and composition of canola seed and oil when less than 10% of the seeds in a sample were sprouting.     

Chemical aspects of chlorophyll breakdown products and their relevance to canola oil stability

The production of prooxidant compounds brought about through subjecting chlorophyll a or pheophytin a to laboratory-scale processing in the presence of canola oil or tricapryloylglycerol was investigated. The addition of chlorophyll a (60 ppm) to canola oil prior to processing resulted in an oil of lowered stability. No large contribution to the produced instability by any one processing step was found when pheophytin a was added (60 ppm) to canola oil prior to processing. To isolate the effect of processing on the pigment, tricapryloylglycerol was used in the place of unsaturated canola oil as a carrier for pheophytin a (60 ppm). A control consisted of processed tricapryloylglycerol that had no added pheophytin prior to processing. The subsequent addition of pigment-treated processed tricapryloylglycerol to linseed oil (1:1, w/w) caused a decrease in the stability of the latter, when compared with the control. No differences were observed between the prooxidant tricapryloylglycerol and the control tricapryloylglycerol by methods involving ultraviolet spectroscopy and thin-layer or gas chromatography.