Characterization of chlorophyll pigments present in canola seed,meal and oil

Chlorophyll pigments present in canola seed, meal and crude and degummed oils were analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector. Chlorophylls a and b, low levels of pheophytin a, and occasionally traces of pheophorbide and its methyl ester were present in canola seed. Meals and oils contained magnesium-deficient chlorophyll pigments such as pheophorbide a, methylpheophorbide a, pheophytins a and b, and pyropheophytins a and b but not chlorophyll a or b. The amounts of chlorophyll pigments were oil > seed >> meal. Both crude and degummed oils contained pheophytin a and pyropheophytin a as main components, but the ratio of pyropheophytin a to pheophytin a was markedly higher in degummed oils. No pheophorbides were detected in degummed oils. These results suggest that oil processing steps such as extraction and degumming affect the composition of chlorophyll pigments. Publication No. 678 Canadian Grain Commission.     

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.     

Distribution of chlorophylls and carotenoids in ripening olives and between oil and alperujo when processed using a two-phase extraction system

The present work has studied the content and type of pigments present in olive fruits and the respective oils and alperujos. The concentration of isochromic pigment fractions—chlorophylls and carotenoids—decreased with fruit ripening, more markedly in the former than in the latter. This implied that the ratio between the two pigment fractions also decreased in parallel in the three products studied. However, the value in the oil was lower than in the fruit, and that in the alperujo was much higher. During the extraction process, the release of acids may have caused pheophytinization reactions in the chlorophyll fraction, increasing pheophytin content in alperujos and oils, whereas the carotenoid fraction was affected only in the alperujo. Chlorophyll b derivatives were destroyed in greater proportion than chlorophyll a derivatives during transfer to the oil. During processing the destruction of lutein was greater than that of β-carotene. The balance of matter between fruit, alperujo, and oil indicated that not all the fruit pigmentation was released from the structures, and most remained occluded in the alperujo. The rest of the pigmentation, and particularly the chlorophyll fraction, was partly destroyed during its transfer to the oil.     

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.     

Evaluation of the CP-Sil 88 and SP-2560 GC columns used in the recently approved AOCS official method Ce 1h-05: Determination of cis-, trans-, saturated, monounsaturated, and polyunsaturated fatty acids in vegetable or non-ruminant animal oils and fats by capillary GLC method

The AOCS Official Method Ce 1h-05 was recently approved at the 96th AOCS Annual Meeting (2005) by the Uniform Methods Committee as the official method for determining cis and trans FA in vegetable or non-ruminant fats and oils. A series of experiments was undertaken using a margarine (hydrogenated soybean oil) sample containing approximately 34% total trans FA (28% 18∶1 trans, 6% 18∶2 trans, and 0.2% 18∶3 trans), a low-trans oil (ca. 7% total trans FA), and a proposed system suitability mixture (12∶0, 9c−18∶1, 11c−18;1, 9c,12c,15c−18∶3, 11c−20∶1, and 21∶0) in an effort to evaluate and optimize the separation on the 100-m SP-2560 and CP-Sil 88 flexible fused-silica capillary GC columns recommended for the analysis. Different carrier gases and flow rates were used during the evaluation, which eventually lead to the final conditions to be used for AOCS Official Method Ce 1h-05.     

Occurrence of mixtures of geometrical isomers of conjugated octadecatrienoic acids in some seed oils: Analysis by open-tubular gas liquid chromatography and high performance liquid chromatography

Analytical methods to obtain the detailed compositions of the fatty acids in oils containing more than one conjugated octadecatrienoic acid by open-tubular gas liquid chromatography (GLC) and by reversed-phase high performance liquid chromatography (HPLC) were established. Effective GLC separations ofcis,trans,trans-9,11,13-octadecatrienoic acid (ctt-9,11,13–18∶3),ctc-9,11,13–18∶3,ttc-9,11,13–18∶3,ttt-9,11,13–18∶3,ttc-8,10,12–18∶3, andttt-8,10,12–18∶3 were obtained with an opentubular column coated with the nonpolar liquid phase OV-1 using an instrument having all-glass carrier gas pathways. The HPLC method also gave satisfactory separations for the isomeric conjugated octadecatrienoates on the basis of number of thecis andtrans double bonds. Two or three minor conjugated trienoic acids were found along with the principal conjugated trienoic acid in tung oil, and seed oils of cherry,Prunus sp., Momordica charantia, Trichosanthes anguina, Punica granatum, Catalpa ovata, andCalendula officinalis. The mechanism for the formation of the conjugated trienoic acid mixtures in the seed oils is discussed. TheC. ovata seed oil also containedct andtt-9,12-octadecadienoic acids. Thett isomer is presumed to be a precursor ofttc-9,11,13–18∶3, the main conjugated trienoic acid in this oil.     

Low erucic acid canola oil does not induce heart triglyceride accumulation in neonatal pigs fed formula

Canola oil is not approved for use in infant formula largely because of concerns over possible accumulation of triglyceride in heart as a result of the small amounts of erucic acid (22∶1n−9) in the oil. Therefore, the concentration and composition of heart triglyceride were determined in piglets fed from birth for 10 (n=4–6) or 18 (n=6) d with formula containing about 50% energy fat as 100% canola oil (0.5% 22∶1n−9) or 100% soybean oil, or 26% canola oil or soy oil (blend) with palm, high-oleic sunflower and coconut oil, providing amounts of 16∶0 and 18∶1 closer to milk, or a mix of soy, high-oleic sunflower and flaxseed oils with C₁₆ and C₁₈ fatty acids similar to canola oil but without 22∶1. Biochemical analysis found no differences in heart triglyceride concentrations among the groups at 10 or 18 d. Assessment of heart triglycerides using Oil Red O staining in select treatments confirmed no differences between 10-d-old piglets fed formula with 100% canola oil (n=4), 100% soy oil (n=4), or the soy oil blend (n=2). Levels of 22∶1n−9 in heart triglyceride and phospholipid, however, were higher (P<0.01) in piglets fed 100% canola oil or the canola oil blend, with higher levels found in triglycerides compared with phospholipids. The modest accumulation of 22∶1n−9 associated with feeding canola oil was not associated with biochemical evidence of heart triglyceride accumulation at 10 and 18 d.     

Characterization and oxidative stability of enzymatically produced fish and canola oil-based structured lipids

Two-kilogram quantities of structured lipids (SL) of menhaden fish and canola oils containing caprylic acids (8∶0) were produced in a laboratory-scale packed-bed bioreactor by acidolysis catalyzed by an immobilized lipase, Lipozyme IM, from Rhizomucor miehei. SL were characterized and their oxidative stabilities investigated. The SL contained 29.5% 8∶0 for fish oil and 40.15 for canola oil. Polyunsaturated fatty acids (PUFA) of fish oil remained unchanged after the modification while PUFA of canola oil were reduced from 29.6 to 21.2%. Monoenes, especially 18∶1n−9, were completely replaced by 8∶0 in fish oil and reduced from 61.9 to 34.7% in canola oil. Downstream processing of enzymatically produced SL led to loss in natural total tocopherol contents of the fish and canola oils. The effects of antioxidants such as α-tocopherol (TOC), tert-butylhydroxyquinone (TBHQ), and combinations thereof on the oxidative stability of SL were investigated. SL were analyzed for oxidative stability index, peroxide value, conjugated diene content, free fatty acid content, iodine value, saponification number, and thiobarbituric acid value. Iodine value of unmodified fish oil (154.71) was reduced to 144.10 and that of canola oil (114.49) to 97.27 after modification. The SN increased from 183.72 to 242.63 for fish oil and from 172.50 to 227.90 for canola oil. TBHQ exhibited better antioxidant effects than TOC. A combination of TBHQ/TOC also proved to be an effective antioxidant for SL. We suggest the addition of antioxidants to enzymatically produced and purified SL.     

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:      

Hematological and lipid changes in newborn piglets fed milk replacer diets containing vegetable oils with different levels of n−3 fatty acids

To test if linolenic acid (18∶3n−3) from vegetable oils would affect bleeding times and platelet counts in new-borns, piglets were used as a model fed milk replacer diets containing 25% (by wt) vegetable oils or oil mixtures for 28 d and compared to sow-reared piglets. The oils tested included soybean, canola, olive, high oleic sunflower (HOAS), a canola/coconut mixture and a mixture of oils mimicking canola in fatty acid composition. All piglets fed the milk replacer diets showed normal growth. Bleeding times increased after birth from 4–6 min to 7–10 min by week 4 (P<0.001), and were higher in pigs fed diets containing 18∶3n−3, as well as in sowreared piglets receiving n−3 polyunsaturated fatty acids (PUFA) in the milk, as compared to diets low in 18∶3n−3. Platelet numbers increased within the first week in newborn piglets from 300 to 550×10⁹/L, and remained high thereafter. Milk replacer diets, containing vegetable oils, generally showed a transient delay in the rise of platelet numbers, which was partially associated with an increased platelet volume. The oils showed differences in the length of delay, but by the third week of age, all platelet counts were >500×10⁹/L. The delay in rise in platelet counts appeared to be related to the fatty acid composition of the oil, as the effect was reproduced by a mixture of oils with a certain fatty acid profile, and disappeared upon the addition of saturated fatty acids to the vegetable oil. There were no alterations in the coagulation factors due to the dietary oils. Blood plasma, platelets and red blood cell membranes showed increased levels of 18∶3n−3 and long-chain n−3 PUFA in response to dietary 18∶3n−3. The level of saturated fatty acids in blood lipids was generally lower in canola and HOAS oil-fed piglets as compared to piglets fed soybean oil or reared with the sow. The results suggest that consumption of milk replacer diets containing vegetable oils rich in 18∶3n−3 does not represent a bleeding risk, and that the transient lower platelet count can be counterbalanced by the addition of saturated fatty acids to the vegetable oils.     

Supported gold catalysis in the hydrogenation of canola oil

The catalytic activity of gold supported on silica orγ-alumina has been studied in the hydrogenation of canola oil. In the hydrogenation of butadiene and pentene using these catalysts, high stability, low yield oftrans-isomers and high monoene selectivity have been reported in the literature. Catalysts containing 1% and 5% Au w/w on porous silica andγ-alumina were active in hydrogenating canola oil in the range of 150 to 250 C and 3550 to 5620 kPa. The activity level of these catalysts was about 30 times lower than that shown by the standard AOCS Ni catalyst based on the concentration of metal (g Au/L oil). Up to 91% monoene content was obtained using these catalysts in comparison with a maximum of 73% for the AOCS standard Ni catalysts. Gold catalysts can be recovered easily by filtration and reused several times without a decrease in activity. The hydrogenated oil was nearly colorless. No gold was detectable in the oil. Contrary to claims in the patent literature, the gold catalyst produces higher concentrations oftrans-isomers than does nickel. However, using gold catalysts the complete reduction of linolenic acid in canola oil can be achieved at a lowertrans-isomer content in the products than that obtained by using the AOCS standard nickel catalyst.     

Tocol-derived minor constituents in selected plant seed oils

Various crude and processed seed oils were analyzed for tocopherols (T) and tocotrienols (T₃) by reversed-phase HPLC with fluorescence detection (FL). The oils included processed canola oil, crude corn oil, crude milkweed oil, crude palm oil, crude/processed rice bran oils, crude/processed soybean oil, crude/processed sunflower oil, and related modified oil, crude/processed sunflower oil, and related modified oil varieties. The HPLC system consisted of a pentafluorophenylsilica (PFPS) column and a mobile phase of methanol and water. The results of comparative methodological studies with rice bran oils and milkweed oils indicated that the reversed-phase PEPS-HPLC method in conjunction with the use of less hazardous solvents proved to be superior and a viable alternative to the conventional normal-phase HPLC method. Unlike the traditional nonpolar octadecylsilica phase, which fails to resolve β-γ pairs of T and T₃, HPLC with the unique polar PFPS column enables separations of all compounds of interest. Except for palm oil, βT and γT were detected in all other crude oils. Although most milkweed oils contained moderale levels of βT and γT, the βT species was present in relatively low abundance in edible oils despite the observation of fairly high concentrations of γT in the latter oils. βT₃ and γT₃ were detected along with αT₃ and σT₃ only in palm and rice bran oils. Tocolderived antioxidant distribution data for zero-time processed oils provided potential utility in correlation studies of frying quality and stability. The variable distribution data for crude oils shed some light on market profitability of oilseeds with rich sources of vitamin E-related minor constituents.     

The relationship between rapeseed chlorophyll,rapeseed oil chlorophyll and percentage green seeds

Oils with high levels of chrlorphyll have become a major problem in the Canadian crushing industry. It was not possible to compare visually the color of samples of rapeseed oil from various crushing plants in Western Canada with the nickel sulfate standard used as a trade standard. Comparison was easy using samples of oil prepared from seed in the laboratory. The difficulty in comparison was probably caused by conversion of green-colored chlorophyll to russet-colored pheophytin in the crushing process. An “apparent chlorophyll” standard with a maximum of 20 ppm (measured by AOCS Cc 13d-55) is recommended. The “percentage green seed” count used in the Canadian grading system was found to correlate poorly (r²<05) with the chlorophyll level in the seed or oil. A maximal chlorophyll level of 12 ppm was found to be allowable in the top grade of seed. It is recommended that a rapid, accurate and inexpensive procedure for chlorophyll measurement be developed to supplement the grading system. Contribution No. 478 from the Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main St., Winnipeg, Manitoba, Canada R3C 3G9.     

Chlorophyll and β-carotene pigments in moroccan virgin olive oils measured by high-performance liquid chromatography

Chlorophyll and β-carotene concentrations were determined by high-performance liquid chromatography (HPLC) in virgin olive oils, which were press-extracted from green and semi-black olives. Pheophytin A was found to be the major chlorophyll isomer in all oil samples. The occurrence of this pigment at higher concentrations in oil extracted from green olives is a possible indication of its time-related destruction during olive ripening. Some evidence for thein vivo existence of pheophytin A is also presented. Beta-carotene concentration in oils was found to decrease during olive ripening.     

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.     

Preparative chromatographic isolation of hydroxy acids fromLesquerella fendleri andL. gordonii seed oils

To conduct product development research onLesquerella seed oils, we explored methods to obtain >100 g quantities of lesquerolic (14-hydroxy-cis-11-eicosenoic) acid. Preliminary experiments with open-column silica gel chromatography showed thatL. fendleri oil could be separated into 3 triglyceride (TG) fractions. The first (10%) contained nonhydroxy 16-(13%) and 18-carbon acids (65% 18∶1,2,3). The second fraction (15%) contained monolesquerolins (39% lesquerolic acid). The major TG fraction (73%) was mainly dilesquerolins (66% lesquerolic acid) showing that a hydroxy acid-enriched TG oil was obtainable by this procedure. Silica gel chromatography easily separatedL. fendleri fatty acid methyl esters (FAME) into a hydroxy-free ester fraction (40–44%) consisting largely of 18∶1 (39%), 18∶2 (19%) and 18∶3 (31%), and a hydroxy ester fraction (56–60%) that was largely methyl lesquerolate (94%) with small amounts of auricolate (5%) (14-hydroxy-cis-11,cis-17-eicosadienoate) and traces of 18-carbon hydroxy esters. This process for isolating the hydroxy FAME ofLesquerella oil was scaled up 15-to 100-fold with a preparative high performance liquid chromatograph. Thirty-gram samples ofL. gordonii FAME were dissolved in eluting solvent, pumped onto the high performance liquid chromatography (HPLC) silica column and eluted with 97∶3 hexane/ethyl acetate. In an 8-hr period, up to 200 g of methyl lesquerolate could be obtained with a purity >98%, the only contaminants being methyl auricolate and methyl ricinoleate. Presented at the AOCS meeting in Phoenix, AZ, May 1988. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

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].     

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.     

Seed oils ofAcacia holosericea A. Cunn. ex G. Don.: Characteristics and composition

Like the fruits ofElaeis guineensis, the seeds ofAcacia holosericea have two types of oils. One is present in the yellow aril (56%), which is attached to the black seed, and the other is in the kernel of the seed (12%). The proximate composition of seed and the physicochemical characteristics of the solvent-extracted oils are reported. The aril fat is quite different from the seed oil in all respects. In descending order, the major fatty acids in aril fat are 18∶1 (54.35%), 16∶0 (29.3%), and 18∶2 (8.0%), whereas in seed (−aril) oil, the order is 18∶2 (59.45%) 18∶1 (20.2%), and 16∶0 (10.0%). In whole seed (+aril) oil, the order is 18∶2 (53.3%), 18∶1 (25%), 16∶0 (12.6).     

Chlorophyllase-Catalyzed Chlorophyll Removal from Vegetable Oils Using Recombinant Eukaryotic and Prokaryotic Enzymes

Chlorophyllase converts chlorophyll and pheophytin into their colorless derivatives (chlorophyllide/pheophorbide and phytol). This activity can be used in chlorophyll removal from vegetable oils. Chlorophyllase genes from Oscillatoria acuminata (OscChlase) and Citrus aurantium (CitChlase) were isolated, cloned, and expressed in E. coli. Bioinformatics analysis showed that both chlorophyllases shared a conserved GHSXG lipase motif responsible for their catalytic activity. SDS-PAGE and immunoblot assays revealed that both enzymes had a molecular weight of 35 kDa. The purified chlorophyllases were stable at a broad range of temperatures and showed the highest activity at 40 °C. OscChlase and CitChlase exhibited the highest activity at pH 6.0 and 7.0, respectively. Enzyme kinetics analysis revealed that OscChlase was able to hydrolyze bacteriochlorophyll-a more efficiently than the recombinant CitChlase (Vmax/Km of 0.38 for OscChlase vs. 0.01 min⁻¹ mg protein for CitChlase). Instead, CitChlase hydrolyzed chlorophyll-b more efficiently than OscChlase. Both enzymes were able to reduce the chlorophyll content of olive (from 623.1 to as low as 87.2 mg per kg oil) and canola oil (from 537.2 to as low as 101.1 mg per kg oil). The ratio of oil to the aqueous reaction media affected chlorophyll hydrolysis (P < 0.05). The lower the oil ratio was (10%), the higher the chlorophyll removal was (75–86%). The efficiency of CitChlase in chlorophyll removal was higher than that of OscChlase at oil ratios of 10 and 20, but lower at 30% ratio (P < 0.05). This is the first report on the application of recombinant OscChlase and CitChlase in chlorophyll removal (up to 86%) from vegetable oils.