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Use of diabetic test kits to assess the recovery of glucosinolates during isolation of canola protein
ARTICLE IN PRESS
LWT 41 (2008) 934–941 www.elsevier.com/locate/lwt
Use of diabetic test kits to assess the recovery of glucosinolates during isolation of canola protein Wan Yuin Sera, Susan D. Arntfielda,, Arnie W. Hydamakaa, Bogdan A. Slominskib a
Department of Food Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Department of Animal Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
b
Received 13 June 2006; received in revised form 14 June 2007; accepted 14 June 2007
Abstract A series of canola fractions representing various stages in the protein isolation process were collected to assess the extent of glucosinolate recovery using two ultrafiltration systems. The total glucosinolate level of all fractions was estimated using a diabetic test kit (DTK), One Touch Blood Glucose Meter, to monitor glucose released on hydrolysis of glucosinolates by endogenous myrosinase and results compared to those obtained using gas chromatography (GC). Of the glucosinolates in the meal, approximately 1.9 g/100 g were recovered in the protein isolated from canola meal, regardless of the type of ultrafiltration system. A major reduction in glucosinolates was evident in the ultrafiltration step where, based on GC data, 63% and 39% of the glucosinolates were recovered in the discarded permeate using an AmiconTM stirred cell and VivaflowTM 200 ultrafiltration, respectively. However, an overestimation of glucose was observed for the DTK results when looking at total glucosinolate recovery, possibly due to the presence of other free glucose in the canola fractions. These findings suggest that low glucosinolate protein isolates can be obtained, in large part due to glucosinolate removal during ultrafiltration of the extracted material. While the DTK method gave good estimates of the glucosinolates in the meal, it cannot be recommended for use with isolation products. r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Diabetic test kit; Glucosinolate; Protein micellar mass; Canola protein; Glucose
1. Introduction As canola meal is used primarily for animal feed, the value of the protein has not been utilized to its fullest. One option to improve this situation is to recover the protein so it can be consumed by humans thus increasing the profitability of this oilseed. The presence of antinutritional factors such as glucosinolates has limited the use of canola meal for human consumption. While glucosinolates in broccoli, which contain 61 mmol glucosinolates per 100 g have been linked to a reduced cancer risk (Song & Thornalley, 2007), the levels in canola meal are considerable higher, at 18–30 mmol/g and have been shown to have antinutritional or toxic effects in animal studies (Sørensen, 1990). As a result, when isolating a protein for human
Corresponding author. Tel.: +1 204 474 9866; fax: +1 204 474 7630.
E-mail address: susan_arntfi[email protected] (S.D. Arntfield).
consumption, it is important to decrease this glucosinolate level. One protein isolation method, which is based on the formation of a protein micellar mass (PMM), has been developed to reduce the problematic antinutritional or toxic factors, including the glucosinolates and their degradation products, associated with canola meal (Ismond & Welsh, 1992). Employing only canola meal, salt and water, this isolation method is claimed to be a mild and selective technique to isolate native protein with low levels of antinutritional factors (Burgess, 1991). The three main stages of the PMM procedure include (i) extraction, (ii) ultrafiltration and (iii) dilution and precipitation (Fig. 1). A sodium chloride (NaCl) solution is the agent most frequently used to extract canola protein with this approach (Burgess, 1991; Ismond & Welsh, 1992; Le´ger & Arntfield, 1993; Murray, 2001). Following the extraction stage, canola protein is concentrated and purified by ultrafiltration. Inclusion of an ultrafiltration step in the
0023-6438/$34.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2007.06.008
ARTICLE IN PRESS W.Y. Ser et al. / LWT 41 (2008) 934–941 I.Ground Defatted Canola Meal EXTRACTION CENTRIFUGATION FILTRATION II.Debris
III. III.Supernatant
ULTRAFILTRATION IV.Filtrate
V.Retentate
COLD WATER DILUTION PRECIPITATION CENTRIFUGATION VI.Supernatant
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ultrafiltration systems. A second objective was to evaluate the use of DTK as a method to monitor the total glucosinolate levels in extracted canola fractions to determine if this technique is viable when lower concentrations of both glucosinolates and myrosinase are present. DTK results will be compared to those obtained with the standard GC method. 2. Materials and methods 2.1. Sample preparation
VII.PMM
Fig. 1. A schematic diagram showing the isolation of canola protein based on the formation of a PMM through (i) extraction, (ii) ultrafiltration and (iii) dilution and precipitation stages. Two series of samples (I, II, III, IV, V, VI and VII) were collected for further analysis.
PMM procedure has proven to be efficient in removing glucosinolates with minimal loss of proteins (Tzeng, Diosady, Chen, & Rubin, 1990). The final step in protein isolation involves reducing the ionic strength of the concentrated protein to promote precipitation. An optimum dilution factor of one to six has been shown to effectively precipitate the purified salt-extracted canola protein through the formation of protein micelles (Burgess, 1991). While glucosinolates can be monitored using a gas chromatography (GC) technique (Slominski, 1995), the diabetic test kit (DTK) technique is claimed to be a simpler and more rapid method for determining glucosinolates based on the measurement of the glucose released from the samples (Vescio, Scott, & Daun, 2001). Water is added to the ground canola seed to initiate and optimize the endogenous myrosinase activities for catalyzing the hydrolysis of glucosinolate and releasing the glucose. The use of endogenous myrosinase within the glucosinolate–myrosinase system contributes to the speed of this technique, which was a motivating factor in its development (Brzenzinski & Mendelewski, 1984; Mawson, Heaney, Piskula, & Kozlowska, 1993). Myrosinases react with the majority of the glucosinolates to produce thiohydroxamate-0-sulfonates and glucose. Exceptions to this are the acyl substituted thioglucose and desulfoglucosinlates which are not catalyzed by myrosinase (Sørensen, 1990). The presence of active endogenous myrosinase in the canola fractions, throughout the isolation process is necessary for this to be an appropriate technique to use. The measurement of glucose has been used successfully as an indication of the glucosinolate levels in canola seeds (Vescio et al., 2001). To extend this technique to other canola fractions, the presence of active endogenous myrosinase is necessary for this to be an appropriate technique to use. The main objective of this study was to determine the mass balance of canola glucosinolates during the PMM protein isolation process, comparing two different
Raw defatted ground canola meal (AL018; low temperature treatment during meal preparation) was obtained from BMW Canada located in Winnipeg, Manitoba and stored at 4 1C until used. Two series of samples representing the various stages of canola protein isolation using PMM methodology procedures (Fig. 1) as adapted from Ismond and Welsh (1992) were collected. The specific samples collected included: (I) canola meal; (II) debris; (III) first supernatant; (IV) filtrate from ultrafiltration; (V) retentate from ultrafiltration; (VI) second supernatant; and (VII) PMM. The ultrafiltration step for the first series was done by using an AmiconTM stirred ultrafiltration cell unit at a pressure of 410–480 kPa with a XM100A Diaflos ultrafiltration membrane (10,000 molecular weight cut-off (MWCO)). A tangential flow module of VivaflowTM 200 ultrafiltration unit (Vivasciences) at a pressure of 250 kPa with a 30,000 MWCO of regenerated cellulose membrane was used in ultrafiltration step of the second series of samples. Due to the difference in sample hold-up volume for the two types of ultrafiltration units, 25 g of meal were mixed for 1 h in 250 mL of 0.5 mol/L NaCl in the first series; whereas 100 g of meal were mixed in 1 L of 0.5 mol/L NaCl for 2 h to prepare the second series of samples. Enough sample was collected at each stage of the isolation process to provide the 600 mg necessary to conduct the glucosinolate analyses. All collected samples were frozen at 40 1C and freeze-dried (Freeze Mobile 6, Unitop 600L; Virtis). These two series of samples were compared in terms of recovery of glucosinolates. For all of the analyses, each series of canola fractions were extracted in duplicate, and each fraction was analyzed in duplicate (or triplicate if enough sample was available). 2.2. GC analysis The type and level of glucosinolates were determined by gas–liquid chromatography as desulpho-trimethylsilyl (TMS) derivatives in accordance with the modified method described by Slominski (1995) which was derived from Slominski and Campbell (1987). As the different chemical structures of the various glucosinolates affect the flame ionization detector response, updated relative response factors (RRF) were calculated as suggested by Slominski (1995) from the ratios of TMS carbon number for benzyl glucosinolate, an internal standard (IS), and the respective
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Table 1 Relative response factor (RRF) on major glucosinolates of canola for GCa Glucosinolate
RRF
Benzyl-b Prop-2-enylBut-3-enylPent-4-enyl2-Hydroxy-but-3-enyl2-Hydroxy-pent-4-enylp-Hydroxy-benzylIndol-3-ylmethyl4-Hydroxyindol-3-ylmethyl-
1.00 1.06 1.02 0.98 0.91 0.88 0.91 0.94 0.90
a
From Slominski (1995). Internal standard used for calculation of relative response factor.
b
glucosinolate TMS carbon number. Glucosinolates levels were adjusted based on these RRFs, as shown in Table 1. 2.2.1. Sample extraction Two milliliters of methanol, 1 mL of each internal standard (0.5 mmol/mL) of benzyl glucosinolate (glucotropaeolin; Raneylab, AG Canada, SK) and allyl glucosinolate (sinigrin monohydrate from horseradish; Sigma S-1647, St. Louis, MO), and 0.1 mL of 0.6 M lead–barium acetate were added to 100 mg of ground samples and shaken for 3 h. The mixture was then centrifuged for 10 min at 3000 rpm. One milliliter of the resulting supernatant was applied to a DEAE-Sephadex A-25 column (pyridine acetate form), which was then washed with 1 mL of 67% methanol, 1 mL of distilled water and 1 mL of 0.02 M pyridine acetate. About 0.05 mL of purified sulphatase solution (from sulphatase EC 3.1.6.1, type H-1; Sigma S-9626, St. Louis, MO) was added to the column and the contents were allowed to stand overnight at ambient temperature. The following day, desulphoglucosinolates were eluted with four washings of 0.6 mL of 60% methanol and combined in a single vial. The contents of the vial were dried using a stream of N2 at 60 1C. 2.2.2. Derivatization of sample extracts and internal standards After drying, 200 mL of premix containing acetone, N,O-bis-trimethyl-silyl-acetamide (BSA; Supleco 33037-U, Bellefonte, PA), trimethyl-cholorosilane (TMCS; Supleco 3-3014, Bellefonte, PA) and 1-methylimidasole (Sigma M-8878, St. Louis, MO) in a ratio of 4:2:0.2:0.1 (v/v) was added to the vial containing sample extracts, which was capped immediately with Teflon-lined caps and let sit for 30 min. These derivatized desulphoglucosinolate samples were then ready to be analyzed by GC. Two of the internal standards (benzyl- and allylglucosinolates) were prepared and derivatized alone, as described for the samples. This was to confirm and compare with the chromatograms obtained by Slominski (1995). Also, internal standard compensated for variations
in conditions during sampling and derivatization, as well as for variation in injection volume and retention times during GC run. 2.2.3. Chromatographic separations In this study, GC analysis of glucosinolates was carried out using a Varian Aerograph Model 3700 gas chromatograph equipped with a flame ionization detector and a Hewlett Packard Integrator model 3390A (Mississauga, ON). A glass column (1.2 m 2 mm i.d.; Supelco, Bellefonte, PA), which was packed tightly with liquid phase of 2% OV-7 on an inert solid support of diatomaceous earth (Chromosorb Ws, AW-DMDCS, 100–200 mesh) coated with a thin film of liquid. Helium was used as the carrier gas flowing at a rate of 20 mL/min. The oven temperature was kept at 185 1C for 4 min, and then increased at 3 1C/ min to 275 1C. Temperatures for the injection port and detector were 280 and 300 1C, respectively. Injection volume was 12 mL of the derivatized desulphoglucosinolate samples. 2.3. Use of diabetic test kits (DTK) The technique used with the DTK was adapted from Vescio et al. (2001) to estimate the total glucosinolate levels in the canola fractions. After mixing a 0.2 g of sample with 1.25 mL of water and letting it stand for 3 min, the sample solution was taken into the One Touchs Blood Glucose Meter (LifeScan Canada Ltd., Burnaby, BC) by capillary action. Then the glucose concentration was read, and used as an indication of the glucosinolate levels where 1 mmol/g of glucose corresponds to 1 mmol/g of total glucosinolates. The glucose meter was calibrated with water. Also, a canola protein isolate obtained from BMW Canola (Winnipeg, MB) was used as a control to check the repeatability of the DTK technique. 2.4. Statistical analysis Statistical analysis was performed with SASs software (SAS Institute, Inc., Cary, NC, USA). Significant differences amongst the canola fractions were determined by analysis of variance (ANOVA). Means were compared using Duncan’s multiple range tests. Statistical significance was declared at po0.05. 3. Results and discussion 3.1. Mass balance of glucosinolates 3.1.1. GC analysis All of the glucosinolates, including internal standards, were well separated and they include allyl- (sinigrin), but-3enyl- (gluconapin), pent-4-enyl- (glucobrassicanapin), 2-hydroxy-but-3-enyl- (progoitrin), 2-hydroxy-pent-4-enyl(gluconapoleiferin), benzyl- (glucotropaeolin), p-hydroxybenzyl- (glucosinalbin), 3-indolyl-methyl- (glucobrassicin,
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Table 2 Total and individual glucosinolates (mmol/g of freezed-dried fraction) present in the different stages of the protein isolation process with the AmiconTM stirred cell unit as the ultrafiltration system Glucosinolate
Extraction I
Aliphatic Sinigrin Gluconapin Glucobrassicanapin Progoitrin Gluconapoleiferin
Ultrafiltration II
Dilution and precipitation
III
IV
V
VI
VII
0.270.0b 11.470.2ab 2.670.5a 13.070.8d 0.770.2a
0.470.0a 5.670.5d 0.770.1d 8.870.5e 0.270.0e
0.170.0c 11.070.4bc 2.170.4b 14.170.9c 0.570.1bc
nd 11.670.3a 1.470.0c 18.770.4a 0.370.0de
0.370.0ab 10.970.2c 3.070.3a 12.570.7d 0.670.1ab
0.170.0c 10.770.4c 1.670.2c 15.670.7b 0.370.0d
nd 1.770.2e 0.270.1e 1.670.2f 0.470.0cd
28.070.4b
15.671.2c
27.671.1b
32.070.5a
27.370.6b
28.470.9b
3.870.6d
0.970.4b 0.670.4b 9.071.2d
0.270.0c 0.170.0c 4.570.9e
1.670.4a 1.370.3a 15.871.1b
0.970.1b 0.670.2b 20.870.5a
1.270.3ab 0.870.3b 10.972.5cd
0.870.2b 0.470.2bc 11.571.5c
0.170.0c 0.170.0c 3.470.9e
Subtotal
10.572.0d
4.871.1e
18.771.2b
22.370.8a
13.072.1c
12.671.9cd
3.670.8e
Overall total
38.471.7d
20.372.2e
46.470.2b
54.370.5a
40.372.1cd
41.071.4c
7.471.0f
Subtotal Aromatic/heterocyclic Glucosinalbin Glucobrassicin 4-OH-glucobrassicin
I, canola meal; II, debris, III, first supernatant; IV, filtrate; V, retentate; VI, second supernatant; VII, protein micellar mass (PMM); and ‘nd’ refers to not detected. For each row, values having different letters are significantly different (po0.05).
Table 3 Total and individual glucosinolates (mmol/g of freezed-dried fraction) present in the different stages of the protein isolation process with the VivaflowTM 200 unit as the ultrafiltration system Glucosinolate
Extraction I
Aliphatic Sinigrin Gluconapin Glucobrassicanapin Progoitrin Gluconapoleiferin Subtotal Aromatic/heterocyclic Glucosinalbin Glucobrassicin 4-OH-glucobrassicin
Ultrafiltration II
0.270.0 11.470.2a 2.670.5a 13.070.8b 0.770.2ab 28.070.4b 0.970.4bc 0.670.4cd 9.071.2b
Dilution and precipitation
III
IV
V
VI
VII
0.270.0 9.170.3b 1.470.2bc 13.771.1b 0.370.1cd
0.270.0 11.670.8a 2.170.3ab 17.170.5a 0.970.0a
nd 8.070.5b 1.470.4bc 11.670.6c 0.570.0bc
0.370.1 10.871.4a 2.870.7a 13.070.4b 0.670.1b
0.370.0 2.470.6d 0.270.2d 1.470.1e nd
24.771.1c
31.871.9a
21.571.5d
27.472.8b
4.370.1f
0.370.1cd 0.170.0d 5.670.8c
1.070.6bc 1.471.2bc 17.070.8a
1.670.0b 1.870.3b 18.471.0a
1.270.0b 1.470.1bc 16.570.8a
2.770.6a 3.170.4a 16.272.1a
0.170.0d 0.270.1d 6.470.3c
0.5701 5.870.2c 0.970.1cd 8.470.1 d 0.270.0d 15.670.4e
Subtotal
10.572.0b
6.070.1c
19.472.5a
21.971.2a
19.170.8a
22.071.1a
6.770.4c
Overall total
38.471.7d
21.671.3e
44.171.64c
53.770.4a
40.670.7d
49.471.4b
10.970.1f
I, canola meal; II, debris, III, first supernatant; IV, filtrate; V, retentate; VI, second supernatant; VII, protein micellar mass (PMM) and ‘nd’ refers to not detected. For each row, values having different letters are significantly different (po0.05).
also known as indole) and 4-hydroxy-3-indolylmethy(4-hydroxyglucobrassicin, also known as 4-hydroxyl indole) glucosinolates (in ascending order in terms of the retention time). The fates of these glucosinolates from two ultrafiltration systems at various stages of the PMM procedure are detailed in Tables 2 and 3. The results show that aliphatic glucosinolates were predominant throughout most of the isolation for both systems. Generally, the total aliphatic
content of glucosinolates did not exceed 30 mmol/g, the value used to define canola meal. The exception was for the filtrate samples in both systems. However, this was not a concern as this product would be discarded. The final PMM protein isolates for the AmiconTM and VivaflowTM systems had only 3.8 or 4.3 mmol of total aliphatic glucosinolate per gram, respectively. Of all these aliphatic glucosinolates, gluconapin and progoitrin together constitute more than half of the total content of glucosinolates in
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the samples. The results also indicate that aromatic/ heterocyclic glucosinolates represent a significant proportion of the total glucosinolate content in the samples with 4-hydroxyglucobrassicin as the predominant glucosinolate. This supports previous work indicating that 4-hydroxyglucobrassicin is the major indole glucosinolate in canola fractions (Daun & McGregor, 1983; Slominski & Campbell, 1987). As in other studies (Campbell & Slominski, 1990; Slominski & Campbell, 1987), it appears that these samples were contaminated by admixtures of weed seeds such as commercial mustard, wild mustard and stinkweed (Campbell & Slominski, 1990) where sinigrin and glucosinalbin were present. The results show that the PMM process successfully reduced the total content of glucosinolates by 81% and 72% in the final protein isolate using the stirred cell and tangential flow ultrafiltration systems, respectively. Maximum removal of the glucosinolates from canola meal took place at the dilution and precipitation stages. In this final stage of the PMM procedure, hydrophobic interactions were promoted upon sudden dilution leading to precipitation of the PMM protein isolates. Under such conditions, most of the glucosinolates remained soluble and were, therefore, excluded from the protein isolates. However, the percent eliminated was lower than what was obtained by Ismond and Welsh (1992) where an average of 92.4% of the glucosinolates in the protein isolate was eliminated using extraction media of 0.01 and 0.1 mol/L of NaCl. The glucosinolate content of the starting canola meal in their study was only 8.1 mmol/g, compared to the 38.4 mmol/g for the low temperature heated canola meal used in this study. The different heat treatments used may have contributed to this initial difference in glucosinolates. When considering the overall effect of the ultrafiltration systems on glucosinolates, lower glucosinolate levels were found in the PMM isolates prepared with the AmiconTM stirred cell system (7.4 mmol/g) compared to the VivaflowTM 200 system (10.9 mmol/g). The reduction in aliphatic glucosinolates for the two systems was similar with 86% and 85% reduction for the AmiconTM stirred cell and VivaflowTM 200 systems, respectively. Therefore, the reason for the difference in overall recovery was related to the behavior of the aromatic glucosinolates where there was a reduction of 66% for the AmiconTM stirred cell system and only 36% for the VivaflowTM 200 system. As a result, the contribution of the aromatic glucosinolates to the total glucosinolate increases due to the isolation procedures and this increase is accentuated with the VivaflowTM 200 ultrafiltration system. 3.1.2. DTK analysis Table 4 shows the estimated total glucosinolate levels from the two ultrafiltration systems for samples representing various stages of the PMM process. The DTK technique estimated a value of approximately 42.5 and 41.9 mmol total glucosinolate per gram of the starting canola meal for the AmiconTM and VivaflowTM series,
Table 4 Total glucosinolate levels in canola samples collected from various stages of canola protein isolation procedure with AmiconTM stirred cell unit (A) and VivaflowTM 200 unit (B) as the ultrafiltration systems using a diabetic test kit expressed as mmol/g in dry weight basis Samples
I Canola meal II Debris III First supernatant IV Filtrate V Retentate VI Second supernatant VII Protein micellar mass (PMM)
Total glucosinolate (mmol/g of freezed-dried fraction) A
B
42.571.8 21.970.9 68.571.4 65.070.0b 69.870.4a 78.071.4 18.071.1
41.970.9 22.570.0 67.871.8 73.370.4a 63.570.0b 73.870.4 16.170.5
One micromol per gram of glucose corresponds to 1 mmol/g of total glucosinolates. Values within the same sample treatment having different letters are significantly different with respect to the effect of the different ultrafiltration systems (po0.05).
respectively. These values are comparable to the results obtained with GC of about 38.4 mmol/g (p values of 0.24 and 0.21 for the AmiconTM and VivaflowTM series, respectively). This demonstrates that this technique has the potential to indirectly determine total glucosinolate content in canola meal based on glucose equivalents. The glucosinolate levels in the debris were estimated to be 21.9 mmol/g (AmiconTM) and 22.5 mmol/g (VivaflowTM) which are not significantly different from the values of 20.3 mmol/g (p ¼ 0.61) and 21.6 mmol/g (p ¼ 0.56) from those obtained by GC for the AmiconTM and VivaflowTM series, respectively. However, the ability of this technique to produce results similar to those from the GC was not achieved for the rest of the samples as the p values were o0.05 for all samples. The total glucosinolate contents estimated from the DTK were consistently higher than those obtained by the GC method. It seems that the DTK technique works best with products that are originally in a dry state, including the canola seeds that were examined by Vescio et al. (2001), and canola meal and debris in the present work. One possible explanation for this result is that the glucose meter detected not only the glucose released from the glucosinolate–myrosinase system, but also other glucose in the samples. According to Shahidi, Naczk, and Myhara (1990), canola meal contains 3.93–5.73 g/100 g sucrose, 0.27–0.62 g/100 g raffinose and 0.83–1.61 g/100 g stachyose as the major soluble sugars, while glucose and fructose were present in trace amounts. It should be noted, however, that the types and amount of these soluble sugars varied, depending on their origin and the chemical analysis method employed (Naczk & Shahidi, 1990). Although the free glucose was present in trace amounts, glucose may also have been released by hydrolysis of other sugars, oligosaccharides and glycosylated materials such as protein in the presence of water.
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72% for the AmiconTM stirred cell and VivaflowTM 200 systems, respectively) (Fig. 2). In addition, the DTK data did not show the difference in the glucosinolates for the two ultrafiltration systems that was noted for the GC data. This may also be attributed to the influence of free glucose on the glucosinolate measurements.
As a result, the glucose levels of the samples would, in part, be related to the conditions present during the three stages of the PMM process. In the extraction stage, these sugars were readily soluble in the first supernatant, and some oligosaccharide hydrolysis may occur releasing more free glucose. This overestimation of glucose was further supported by the fact that about 78% of the glucose was recovered in the first supernatant while an average of 45% was recovered in the debris for both series (Fig. 2), resulting in the first supernatant containing approximately 68 mmol/g of glucose (average for both series; Table 4). As there were only 45 mmol/g (average for both series; Tables 2 and 3) of total glucosinolates in the first supernatant according to the GC results, about 23 mmol/g of glucose existed as non-glucosinolate glucose. This high glucose profile remained in the samples during subsequent stages of the isolation process although minor differences due to the ultrafiltration systems were noted. In the final stage of the isolation process, the soluble glucose remained in the second supernatant leaving lower amounts in the PMM isolates. Comparable results have been reported by Aman and Gilberg (1977) where their canola protein isolates contained 86.0–88.5% less neutral sugars than those originally present in the meal. The presence of this extra glucose clearly influenced the mass balance reported as glucosinolates. While a reduction in total glucosinolates was seen in the isolated protein, the percentage reduction (58% and 62% for the AmiconTM stirred cell and VivaflowTM 200 systems, respectively) were not as high as those noted with the GC analysis (81% and
3.2. Comparison of two ultrafiltration systems Ultrafiltration is an important stage in the PMM process in that it allows the separation of the impurities with low molecular weight, including glucosinolates, from the high molecular weight desirable canola proteins. This is, in fact, an added benefit for ultrafiltration over other concentrating systems. The effect of ultrafiltration and the comparison of two laboratory-scale ultrafiltration systems in terms of the overall recovery of glucosinolates are shown in Fig. 2. For both methods of analyses, the results show that the AmiconTM stirred ultrafiltration cell unit (A), with lower MWCO (10,000 MWCO), allowed significantly more glucosinolates (or glucosinolate and glucose for DTK technique) from first supernatant through the membrane into the filtrate (IV) compared to the VivaflowTM 200 ultrafiltration cell unit (30,000 MWCO) (Fig. 2). This has, in turn, left less glucosinolates in the retentate (V) according to the DTK analysis, although this difference was not significant with the GC analysis. These results are opposite to the statement of Diosady, Tzeng, and Rubin (1984) where the removal of impurities, including glucosinolate and glucose, was best with the higher MWCO
Glucosinolates recoverd (mol / 100 mol in canola meal)
90 a a
80
a 70 b b
60 a a 50
a
b c
a d
40
a 30
b
a c
20
c
b c c
10 a a c b 0 II
III
IV V Sampling step
VI
VII
Fig. 2. Recovery of glucosinolates (mol/100 mol of glucosinolate in the meal) in the fractions obtained during the isolation of canola protein. The sampling steps were as follows: (II) debris from first extraction, (III) supernatant from first extraction, (IV) filtrate from ultrafiltration, (V) retentate from ultrafiltration, (VI) supernatant from the protein precipitation step and (VII) isolated canola protein. Samples using AmiconTM Stirred Cell (A) and VivaflowTM 200 (B) ultrafiltration systems were analyzed with gas chromatography (GC) and a diabetic test kit (DTK). , DTK A; , DTK B; , GC A; , GC B.
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membrane. The reason for this difference is not clear, but it may be related to the different flow behavior of samples for these ultrafiltration systems. The continuous flow for the VivaflowTM 200 system appeared to minimize the selective removal of glucosinolates (or glucose). As a result, while the VivaflowTM 200 unit improved protein recovery, it has also increased the recovery of undesirable glucosinolates compared to the AmiconTM unit. In the final stage of the isolation procedure, a relatively large percentage of the glucosinolates was recovered in the decanted second supernatant (VI) as most remained soluble upon dilution and were separated from the PMM protein isolates (VII) for both systems (Fig. 2). Interestingly, the supernatant from the AmiconTM stirred cell system had significantly higher glucosinolate recovery than the VivaflowTM 200 system when using the DTK analysis but the level of recovery of glucosinolates in the PMM were not significantly different. With the GC analysis, the recoveries in the supernatants were not significantly different but the glucosinolates recovered in the PMM were lower for the AmiconTM stirred cell system. In this case, the increased retention of glucosinolates by the VivaflowTM 200 unit that was noted during ultrafiltration has resulted in increased glucosinolate recovery (Fig. 2) and higher glucosinolate levels in the final product (Tables 2 and 3). 3.3. Comparison of GC and DTK for evaluating glucosinolates By comparing the DTK results in Table 4 to the GC results in Tables 2 and 3, it is clear that the DTK results in an overestimation of glucose in the canola fractions as a consequence of the determination of non-glucosinolate glucose; this is most evident of fractions originally in liquid form (III, IV, V and VI). The fact that this difference varies depending on the stage in the isolation process results in a difference in glucosinolate recovery depending on the method used to evaluate the level of glucosinolates recovered (Fig. 2). While the recoveries in the residue (II) during the first extraction step were unaffected by the method of analysis, the high values for the supernatant with the DTK assay, resulted in unrealistically high overall recoveries (sum of fractions II, IV, VI and VII) of well over 100%. With the CG method, on the other hand the recoveries were much closer to 100%. For the VivaflowTM system, for example, the DTK assay resulted in an overall recovery of 122% whereas the recovery was 101% with the CG system. Overall recoveries were higher with the AmiconTM system for both assays. The effect of the overestimation of glucosinolate recovery in the initial supernatant is reflected in subsequent steps, as the recoveries for the DTK were all significantly higher (po0.05), than the corresponding treatment analyzed using the GC method (Fig. 2). While the DTK method is able to follow the trend in terms of the relative distribution of the glucosinolates during protein
isolation, the absolute values and percent recoveries for the various fractions do not agree with the GC values. Although the DTK technique has been used effectively in the past to evaluate seeds (Vescio et al., 2001), and did a good job for the meal and initial debris, it was unable to accurately measure the glucosinolates in the products of protein isolation. As the values were higher than those obtained with the GC method, it would appear that other sources of glucose were contributing to the DTK results. If the distribution of myrosinase was an issue, than lower values for the DTK test would have been expected. 4. Conclusion The protein isolation procedure used in this study was effective in reducing the glucosinolate level in the isolate to 20 (AmiconTM stirred cell ultrafiltration) to 30% (VivaflowTM 2000 ultrafiltration) of what it was in the original meal. The levels obtained are still more than 10 times higher that those reported for broccoli, with values of 7.4–10.9 mmol/g for the canola protein isolate in this study compared to 0.62 mmol/g in broccoli (Song & Thornalley, 2007). The higher residual glucosinolate level obtained with the VivaflowTM ultrafiltration was due to a higher recovery of aromatic glucosinolates as both systems had similar reductions in aliphatic glucosinolates. Reductions in the glucosinolate levels were associated with the ultrafiltration step where levels of glucosinolates in the retentate were significantly lower than in the filtrate. Reductions in glucosinolates were also observed at the precipitation step where most of the glucosinolates remained in the supernatant. The DTK technique is a simple and rapid that has been used as a non-specific indirect method for measuring glucosinolates (Vescio et al., 2001). However, it is not applicable to those samples in which the endogenous myrosinase enzyme is not active and it measures the glucose regardless of where it comes from. The glucose meter used in this study was capable of determining only the free glucose, whether originally present as the monosaccharide itself or the glucose released from the glucosinolate–myrosinase system, or from the hydrolysis of other glycosylated compounds or oligosaccharides. In the current study, overestimation occurred for most samples as a consequence of the determination of non-glucosinolate glucose by the DTK. While this may be an appropriate method to estimate the total glucosinolate content of canola seeds and mild heat-treated meal, it is not recommended for materials produced during the processing of the meal. Acknowledgment Financial support provided by the Natural Science & Engineering Research Council of Canada (NSERC) is gratefully acknowledged.
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References Aman, P., & Gilberg, L. (1977). Preparation of rapeseed protein isolates: A study of the distribution of carbohydrates in the preparation of rapeseed isolates. Journal of Food Science, 42(1), 114–116. Brzenzinski, W., & Mendelewski, P. (1984). Determination of total glucosinolate content in rapeseed meal with thymol reagent. Zeitschrift fu¨r Pflanzenzu¨chtung, 93(3), 177–183. Burgess, D. A. (1991). Purification, characterization and the micelle response of the 12S canola globulin. M.Sc. thesis. University of Manitoba, Winnipeg, Manitoba. Campbell, L. D., & Slominski, B. A. (1990). Extent of thermal decomposition of indole glucosinolates during the processing of canola seed. Journal of American Oil Chemists Society, 67(2), 73–75. Daun, J. K., & McGregor, D. I. (1983). Glucosinolate analysis of rapeseed (canola) (pp. 1–32). Winnipeg, MB: Agriculture Canada. Diosady, L. L., Tzeng, Y., & Rubin, L. J. (1984). Preparation of rapeseed protein concentrates and isolates using ultrafiltration. Journal of Food Science, 49, 768–777. Ismond, M. A. H., & Welsh, W. D. (1992). Application of new methodology to canola protein isolation. Food Chemistry, 45, 125–127. Le´ger, L. W., & Arntfield, S. D. (1993). Thermal gelation of 12S canola globulin. Journal of American Oil Chemists Society, 70, 853–861. Mawson, R., Heaney, R. K., Piskula, M., & Kozlowska, H. (1993). Rapeseed meal glucosinolates and their antinutritional effects. Part 1. Rapeseed production and chemistry of glucosinolates. Die Nahrung, 37(2), 131–140.
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Murray, E. D. (2001). Isolating protein from canola. Oils and Fats International, 17(3), 19–21. Naczk, M., & Shahidi, E. (1990). Carbohydrate of canola and rapeseed. In F. Shahidi (Ed.), Canola and rapeseed (pp. 211–220). New York: Van Nostrand Reinhold. Shahidi, F., Naczk, M., & Myhara, R. M. (1990). Effect of processing on the soluble sugars of Brassica seeds. Journal of Food Science, 55, 1470–1471. Slominski, B. A. (1995). Relative response factor of canola glucosinolates for GC. Winnipeg, Manitoba: Department of Animal Science, University of Manitoba. Slominski, B. A., & Campbell, L. D. (1987). Gas chromatography determination of indole glucosinolate: A re-examination. Journal of the Science of Food and Agriculture, 40, 131–143. Song, L., & Thornalley, P. J. (2007). Effect of storage, processing and cooking on glucosinolate content of Brassica vegetables. Food and Chemical toxicology, 45, 216–224. Sørensen, H. (1990). Glucosinolates: Structure-properties-function. In F. Shahidi (Ed.), Canola and rapeseed (pp. 149–172). New York: Van Nostrand Reinhold. Tzeng, Y.-M., Diosady, L. L., Chen, B.-K., & Rubin, L. J. (1990). Ultrafiltration rejection coefficient of canola meal components. Canadian Institute of Food Science and Technology Journal, 23(2/3), 131–136. Vescio, K., Scott, C., & Daun, J. (2001). Rapids methods for glucosinolate determination in canola and mustard using diabetic test kits (4pp.). Winnipeg, MB, Canada: Canadian Grain Commission.
LWT 41 (2008) 934–941 www.elsevier.com/locate/lwt
Use of diabetic test kits to assess the recovery of glucosinolates during isolation of canola protein Wan Yuin Sera, Susan D. Arntfielda,, Arnie W. Hydamakaa, Bogdan A. Slominskib a
Department of Food Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Department of Animal Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
b
Received 13 June 2006; received in revised form 14 June 2007; accepted 14 June 2007
Abstract A series of canola fractions representing various stages in the protein isolation process were collected to assess the extent of glucosinolate recovery using two ultrafiltration systems. The total glucosinolate level of all fractions was estimated using a diabetic test kit (DTK), One Touch Blood Glucose Meter, to monitor glucose released on hydrolysis of glucosinolates by endogenous myrosinase and results compared to those obtained using gas chromatography (GC). Of the glucosinolates in the meal, approximately 1.9 g/100 g were recovered in the protein isolated from canola meal, regardless of the type of ultrafiltration system. A major reduction in glucosinolates was evident in the ultrafiltration step where, based on GC data, 63% and 39% of the glucosinolates were recovered in the discarded permeate using an AmiconTM stirred cell and VivaflowTM 200 ultrafiltration, respectively. However, an overestimation of glucose was observed for the DTK results when looking at total glucosinolate recovery, possibly due to the presence of other free glucose in the canola fractions. These findings suggest that low glucosinolate protein isolates can be obtained, in large part due to glucosinolate removal during ultrafiltration of the extracted material. While the DTK method gave good estimates of the glucosinolates in the meal, it cannot be recommended for use with isolation products. r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Diabetic test kit; Glucosinolate; Protein micellar mass; Canola protein; Glucose
1. Introduction As canola meal is used primarily for animal feed, the value of the protein has not been utilized to its fullest. One option to improve this situation is to recover the protein so it can be consumed by humans thus increasing the profitability of this oilseed. The presence of antinutritional factors such as glucosinolates has limited the use of canola meal for human consumption. While glucosinolates in broccoli, which contain 61 mmol glucosinolates per 100 g have been linked to a reduced cancer risk (Song & Thornalley, 2007), the levels in canola meal are considerable higher, at 18–30 mmol/g and have been shown to have antinutritional or toxic effects in animal studies (Sørensen, 1990). As a result, when isolating a protein for human
Corresponding author. Tel.: +1 204 474 9866; fax: +1 204 474 7630.
E-mail address: susan_arntfi[email protected] (S.D. Arntfield).
consumption, it is important to decrease this glucosinolate level. One protein isolation method, which is based on the formation of a protein micellar mass (PMM), has been developed to reduce the problematic antinutritional or toxic factors, including the glucosinolates and their degradation products, associated with canola meal (Ismond & Welsh, 1992). Employing only canola meal, salt and water, this isolation method is claimed to be a mild and selective technique to isolate native protein with low levels of antinutritional factors (Burgess, 1991). The three main stages of the PMM procedure include (i) extraction, (ii) ultrafiltration and (iii) dilution and precipitation (Fig. 1). A sodium chloride (NaCl) solution is the agent most frequently used to extract canola protein with this approach (Burgess, 1991; Ismond & Welsh, 1992; Le´ger & Arntfield, 1993; Murray, 2001). Following the extraction stage, canola protein is concentrated and purified by ultrafiltration. Inclusion of an ultrafiltration step in the
0023-6438/$34.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2007.06.008
ARTICLE IN PRESS W.Y. Ser et al. / LWT 41 (2008) 934–941 I.Ground Defatted Canola Meal EXTRACTION CENTRIFUGATION FILTRATION II.Debris
III. III.Supernatant
ULTRAFILTRATION IV.Filtrate
V.Retentate
COLD WATER DILUTION PRECIPITATION CENTRIFUGATION VI.Supernatant
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ultrafiltration systems. A second objective was to evaluate the use of DTK as a method to monitor the total glucosinolate levels in extracted canola fractions to determine if this technique is viable when lower concentrations of both glucosinolates and myrosinase are present. DTK results will be compared to those obtained with the standard GC method. 2. Materials and methods 2.1. Sample preparation
VII.PMM
Fig. 1. A schematic diagram showing the isolation of canola protein based on the formation of a PMM through (i) extraction, (ii) ultrafiltration and (iii) dilution and precipitation stages. Two series of samples (I, II, III, IV, V, VI and VII) were collected for further analysis.
PMM procedure has proven to be efficient in removing glucosinolates with minimal loss of proteins (Tzeng, Diosady, Chen, & Rubin, 1990). The final step in protein isolation involves reducing the ionic strength of the concentrated protein to promote precipitation. An optimum dilution factor of one to six has been shown to effectively precipitate the purified salt-extracted canola protein through the formation of protein micelles (Burgess, 1991). While glucosinolates can be monitored using a gas chromatography (GC) technique (Slominski, 1995), the diabetic test kit (DTK) technique is claimed to be a simpler and more rapid method for determining glucosinolates based on the measurement of the glucose released from the samples (Vescio, Scott, & Daun, 2001). Water is added to the ground canola seed to initiate and optimize the endogenous myrosinase activities for catalyzing the hydrolysis of glucosinolate and releasing the glucose. The use of endogenous myrosinase within the glucosinolate–myrosinase system contributes to the speed of this technique, which was a motivating factor in its development (Brzenzinski & Mendelewski, 1984; Mawson, Heaney, Piskula, & Kozlowska, 1993). Myrosinases react with the majority of the glucosinolates to produce thiohydroxamate-0-sulfonates and glucose. Exceptions to this are the acyl substituted thioglucose and desulfoglucosinlates which are not catalyzed by myrosinase (Sørensen, 1990). The presence of active endogenous myrosinase in the canola fractions, throughout the isolation process is necessary for this to be an appropriate technique to use. The measurement of glucose has been used successfully as an indication of the glucosinolate levels in canola seeds (Vescio et al., 2001). To extend this technique to other canola fractions, the presence of active endogenous myrosinase is necessary for this to be an appropriate technique to use. The main objective of this study was to determine the mass balance of canola glucosinolates during the PMM protein isolation process, comparing two different
Raw defatted ground canola meal (AL018; low temperature treatment during meal preparation) was obtained from BMW Canada located in Winnipeg, Manitoba and stored at 4 1C until used. Two series of samples representing the various stages of canola protein isolation using PMM methodology procedures (Fig. 1) as adapted from Ismond and Welsh (1992) were collected. The specific samples collected included: (I) canola meal; (II) debris; (III) first supernatant; (IV) filtrate from ultrafiltration; (V) retentate from ultrafiltration; (VI) second supernatant; and (VII) PMM. The ultrafiltration step for the first series was done by using an AmiconTM stirred ultrafiltration cell unit at a pressure of 410–480 kPa with a XM100A Diaflos ultrafiltration membrane (10,000 molecular weight cut-off (MWCO)). A tangential flow module of VivaflowTM 200 ultrafiltration unit (Vivasciences) at a pressure of 250 kPa with a 30,000 MWCO of regenerated cellulose membrane was used in ultrafiltration step of the second series of samples. Due to the difference in sample hold-up volume for the two types of ultrafiltration units, 25 g of meal were mixed for 1 h in 250 mL of 0.5 mol/L NaCl in the first series; whereas 100 g of meal were mixed in 1 L of 0.5 mol/L NaCl for 2 h to prepare the second series of samples. Enough sample was collected at each stage of the isolation process to provide the 600 mg necessary to conduct the glucosinolate analyses. All collected samples were frozen at 40 1C and freeze-dried (Freeze Mobile 6, Unitop 600L; Virtis). These two series of samples were compared in terms of recovery of glucosinolates. For all of the analyses, each series of canola fractions were extracted in duplicate, and each fraction was analyzed in duplicate (or triplicate if enough sample was available). 2.2. GC analysis The type and level of glucosinolates were determined by gas–liquid chromatography as desulpho-trimethylsilyl (TMS) derivatives in accordance with the modified method described by Slominski (1995) which was derived from Slominski and Campbell (1987). As the different chemical structures of the various glucosinolates affect the flame ionization detector response, updated relative response factors (RRF) were calculated as suggested by Slominski (1995) from the ratios of TMS carbon number for benzyl glucosinolate, an internal standard (IS), and the respective
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Table 1 Relative response factor (RRF) on major glucosinolates of canola for GCa Glucosinolate
RRF
Benzyl-b Prop-2-enylBut-3-enylPent-4-enyl2-Hydroxy-but-3-enyl2-Hydroxy-pent-4-enylp-Hydroxy-benzylIndol-3-ylmethyl4-Hydroxyindol-3-ylmethyl-
1.00 1.06 1.02 0.98 0.91 0.88 0.91 0.94 0.90
a
From Slominski (1995). Internal standard used for calculation of relative response factor.
b
glucosinolate TMS carbon number. Glucosinolates levels were adjusted based on these RRFs, as shown in Table 1. 2.2.1. Sample extraction Two milliliters of methanol, 1 mL of each internal standard (0.5 mmol/mL) of benzyl glucosinolate (glucotropaeolin; Raneylab, AG Canada, SK) and allyl glucosinolate (sinigrin monohydrate from horseradish; Sigma S-1647, St. Louis, MO), and 0.1 mL of 0.6 M lead–barium acetate were added to 100 mg of ground samples and shaken for 3 h. The mixture was then centrifuged for 10 min at 3000 rpm. One milliliter of the resulting supernatant was applied to a DEAE-Sephadex A-25 column (pyridine acetate form), which was then washed with 1 mL of 67% methanol, 1 mL of distilled water and 1 mL of 0.02 M pyridine acetate. About 0.05 mL of purified sulphatase solution (from sulphatase EC 3.1.6.1, type H-1; Sigma S-9626, St. Louis, MO) was added to the column and the contents were allowed to stand overnight at ambient temperature. The following day, desulphoglucosinolates were eluted with four washings of 0.6 mL of 60% methanol and combined in a single vial. The contents of the vial were dried using a stream of N2 at 60 1C. 2.2.2. Derivatization of sample extracts and internal standards After drying, 200 mL of premix containing acetone, N,O-bis-trimethyl-silyl-acetamide (BSA; Supleco 33037-U, Bellefonte, PA), trimethyl-cholorosilane (TMCS; Supleco 3-3014, Bellefonte, PA) and 1-methylimidasole (Sigma M-8878, St. Louis, MO) in a ratio of 4:2:0.2:0.1 (v/v) was added to the vial containing sample extracts, which was capped immediately with Teflon-lined caps and let sit for 30 min. These derivatized desulphoglucosinolate samples were then ready to be analyzed by GC. Two of the internal standards (benzyl- and allylglucosinolates) were prepared and derivatized alone, as described for the samples. This was to confirm and compare with the chromatograms obtained by Slominski (1995). Also, internal standard compensated for variations
in conditions during sampling and derivatization, as well as for variation in injection volume and retention times during GC run. 2.2.3. Chromatographic separations In this study, GC analysis of glucosinolates was carried out using a Varian Aerograph Model 3700 gas chromatograph equipped with a flame ionization detector and a Hewlett Packard Integrator model 3390A (Mississauga, ON). A glass column (1.2 m 2 mm i.d.; Supelco, Bellefonte, PA), which was packed tightly with liquid phase of 2% OV-7 on an inert solid support of diatomaceous earth (Chromosorb Ws, AW-DMDCS, 100–200 mesh) coated with a thin film of liquid. Helium was used as the carrier gas flowing at a rate of 20 mL/min. The oven temperature was kept at 185 1C for 4 min, and then increased at 3 1C/ min to 275 1C. Temperatures for the injection port and detector were 280 and 300 1C, respectively. Injection volume was 12 mL of the derivatized desulphoglucosinolate samples. 2.3. Use of diabetic test kits (DTK) The technique used with the DTK was adapted from Vescio et al. (2001) to estimate the total glucosinolate levels in the canola fractions. After mixing a 0.2 g of sample with 1.25 mL of water and letting it stand for 3 min, the sample solution was taken into the One Touchs Blood Glucose Meter (LifeScan Canada Ltd., Burnaby, BC) by capillary action. Then the glucose concentration was read, and used as an indication of the glucosinolate levels where 1 mmol/g of glucose corresponds to 1 mmol/g of total glucosinolates. The glucose meter was calibrated with water. Also, a canola protein isolate obtained from BMW Canola (Winnipeg, MB) was used as a control to check the repeatability of the DTK technique. 2.4. Statistical analysis Statistical analysis was performed with SASs software (SAS Institute, Inc., Cary, NC, USA). Significant differences amongst the canola fractions were determined by analysis of variance (ANOVA). Means were compared using Duncan’s multiple range tests. Statistical significance was declared at po0.05. 3. Results and discussion 3.1. Mass balance of glucosinolates 3.1.1. GC analysis All of the glucosinolates, including internal standards, were well separated and they include allyl- (sinigrin), but-3enyl- (gluconapin), pent-4-enyl- (glucobrassicanapin), 2-hydroxy-but-3-enyl- (progoitrin), 2-hydroxy-pent-4-enyl(gluconapoleiferin), benzyl- (glucotropaeolin), p-hydroxybenzyl- (glucosinalbin), 3-indolyl-methyl- (glucobrassicin,
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Table 2 Total and individual glucosinolates (mmol/g of freezed-dried fraction) present in the different stages of the protein isolation process with the AmiconTM stirred cell unit as the ultrafiltration system Glucosinolate
Extraction I
Aliphatic Sinigrin Gluconapin Glucobrassicanapin Progoitrin Gluconapoleiferin
Ultrafiltration II
Dilution and precipitation
III
IV
V
VI
VII
0.270.0b 11.470.2ab 2.670.5a 13.070.8d 0.770.2a
0.470.0a 5.670.5d 0.770.1d 8.870.5e 0.270.0e
0.170.0c 11.070.4bc 2.170.4b 14.170.9c 0.570.1bc
nd 11.670.3a 1.470.0c 18.770.4a 0.370.0de
0.370.0ab 10.970.2c 3.070.3a 12.570.7d 0.670.1ab
0.170.0c 10.770.4c 1.670.2c 15.670.7b 0.370.0d
nd 1.770.2e 0.270.1e 1.670.2f 0.470.0cd
28.070.4b
15.671.2c
27.671.1b
32.070.5a
27.370.6b
28.470.9b
3.870.6d
0.970.4b 0.670.4b 9.071.2d
0.270.0c 0.170.0c 4.570.9e
1.670.4a 1.370.3a 15.871.1b
0.970.1b 0.670.2b 20.870.5a
1.270.3ab 0.870.3b 10.972.5cd
0.870.2b 0.470.2bc 11.571.5c
0.170.0c 0.170.0c 3.470.9e
Subtotal
10.572.0d
4.871.1e
18.771.2b
22.370.8a
13.072.1c
12.671.9cd
3.670.8e
Overall total
38.471.7d
20.372.2e
46.470.2b
54.370.5a
40.372.1cd
41.071.4c
7.471.0f
Subtotal Aromatic/heterocyclic Glucosinalbin Glucobrassicin 4-OH-glucobrassicin
I, canola meal; II, debris, III, first supernatant; IV, filtrate; V, retentate; VI, second supernatant; VII, protein micellar mass (PMM); and ‘nd’ refers to not detected. For each row, values having different letters are significantly different (po0.05).
Table 3 Total and individual glucosinolates (mmol/g of freezed-dried fraction) present in the different stages of the protein isolation process with the VivaflowTM 200 unit as the ultrafiltration system Glucosinolate
Extraction I
Aliphatic Sinigrin Gluconapin Glucobrassicanapin Progoitrin Gluconapoleiferin Subtotal Aromatic/heterocyclic Glucosinalbin Glucobrassicin 4-OH-glucobrassicin
Ultrafiltration II
0.270.0 11.470.2a 2.670.5a 13.070.8b 0.770.2ab 28.070.4b 0.970.4bc 0.670.4cd 9.071.2b
Dilution and precipitation
III
IV
V
VI
VII
0.270.0 9.170.3b 1.470.2bc 13.771.1b 0.370.1cd
0.270.0 11.670.8a 2.170.3ab 17.170.5a 0.970.0a
nd 8.070.5b 1.470.4bc 11.670.6c 0.570.0bc
0.370.1 10.871.4a 2.870.7a 13.070.4b 0.670.1b
0.370.0 2.470.6d 0.270.2d 1.470.1e nd
24.771.1c
31.871.9a
21.571.5d
27.472.8b
4.370.1f
0.370.1cd 0.170.0d 5.670.8c
1.070.6bc 1.471.2bc 17.070.8a
1.670.0b 1.870.3b 18.471.0a
1.270.0b 1.470.1bc 16.570.8a
2.770.6a 3.170.4a 16.272.1a
0.170.0d 0.270.1d 6.470.3c
0.5701 5.870.2c 0.970.1cd 8.470.1 d 0.270.0d 15.670.4e
Subtotal
10.572.0b
6.070.1c
19.472.5a
21.971.2a
19.170.8a
22.071.1a
6.770.4c
Overall total
38.471.7d
21.671.3e
44.171.64c
53.770.4a
40.670.7d
49.471.4b
10.970.1f
I, canola meal; II, debris, III, first supernatant; IV, filtrate; V, retentate; VI, second supernatant; VII, protein micellar mass (PMM) and ‘nd’ refers to not detected. For each row, values having different letters are significantly different (po0.05).
also known as indole) and 4-hydroxy-3-indolylmethy(4-hydroxyglucobrassicin, also known as 4-hydroxyl indole) glucosinolates (in ascending order in terms of the retention time). The fates of these glucosinolates from two ultrafiltration systems at various stages of the PMM procedure are detailed in Tables 2 and 3. The results show that aliphatic glucosinolates were predominant throughout most of the isolation for both systems. Generally, the total aliphatic
content of glucosinolates did not exceed 30 mmol/g, the value used to define canola meal. The exception was for the filtrate samples in both systems. However, this was not a concern as this product would be discarded. The final PMM protein isolates for the AmiconTM and VivaflowTM systems had only 3.8 or 4.3 mmol of total aliphatic glucosinolate per gram, respectively. Of all these aliphatic glucosinolates, gluconapin and progoitrin together constitute more than half of the total content of glucosinolates in
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the samples. The results also indicate that aromatic/ heterocyclic glucosinolates represent a significant proportion of the total glucosinolate content in the samples with 4-hydroxyglucobrassicin as the predominant glucosinolate. This supports previous work indicating that 4-hydroxyglucobrassicin is the major indole glucosinolate in canola fractions (Daun & McGregor, 1983; Slominski & Campbell, 1987). As in other studies (Campbell & Slominski, 1990; Slominski & Campbell, 1987), it appears that these samples were contaminated by admixtures of weed seeds such as commercial mustard, wild mustard and stinkweed (Campbell & Slominski, 1990) where sinigrin and glucosinalbin were present. The results show that the PMM process successfully reduced the total content of glucosinolates by 81% and 72% in the final protein isolate using the stirred cell and tangential flow ultrafiltration systems, respectively. Maximum removal of the glucosinolates from canola meal took place at the dilution and precipitation stages. In this final stage of the PMM procedure, hydrophobic interactions were promoted upon sudden dilution leading to precipitation of the PMM protein isolates. Under such conditions, most of the glucosinolates remained soluble and were, therefore, excluded from the protein isolates. However, the percent eliminated was lower than what was obtained by Ismond and Welsh (1992) where an average of 92.4% of the glucosinolates in the protein isolate was eliminated using extraction media of 0.01 and 0.1 mol/L of NaCl. The glucosinolate content of the starting canola meal in their study was only 8.1 mmol/g, compared to the 38.4 mmol/g for the low temperature heated canola meal used in this study. The different heat treatments used may have contributed to this initial difference in glucosinolates. When considering the overall effect of the ultrafiltration systems on glucosinolates, lower glucosinolate levels were found in the PMM isolates prepared with the AmiconTM stirred cell system (7.4 mmol/g) compared to the VivaflowTM 200 system (10.9 mmol/g). The reduction in aliphatic glucosinolates for the two systems was similar with 86% and 85% reduction for the AmiconTM stirred cell and VivaflowTM 200 systems, respectively. Therefore, the reason for the difference in overall recovery was related to the behavior of the aromatic glucosinolates where there was a reduction of 66% for the AmiconTM stirred cell system and only 36% for the VivaflowTM 200 system. As a result, the contribution of the aromatic glucosinolates to the total glucosinolate increases due to the isolation procedures and this increase is accentuated with the VivaflowTM 200 ultrafiltration system. 3.1.2. DTK analysis Table 4 shows the estimated total glucosinolate levels from the two ultrafiltration systems for samples representing various stages of the PMM process. The DTK technique estimated a value of approximately 42.5 and 41.9 mmol total glucosinolate per gram of the starting canola meal for the AmiconTM and VivaflowTM series,
Table 4 Total glucosinolate levels in canola samples collected from various stages of canola protein isolation procedure with AmiconTM stirred cell unit (A) and VivaflowTM 200 unit (B) as the ultrafiltration systems using a diabetic test kit expressed as mmol/g in dry weight basis Samples
I Canola meal II Debris III First supernatant IV Filtrate V Retentate VI Second supernatant VII Protein micellar mass (PMM)
Total glucosinolate (mmol/g of freezed-dried fraction) A
B
42.571.8 21.970.9 68.571.4 65.070.0b 69.870.4a 78.071.4 18.071.1
41.970.9 22.570.0 67.871.8 73.370.4a 63.570.0b 73.870.4 16.170.5
One micromol per gram of glucose corresponds to 1 mmol/g of total glucosinolates. Values within the same sample treatment having different letters are significantly different with respect to the effect of the different ultrafiltration systems (po0.05).
respectively. These values are comparable to the results obtained with GC of about 38.4 mmol/g (p values of 0.24 and 0.21 for the AmiconTM and VivaflowTM series, respectively). This demonstrates that this technique has the potential to indirectly determine total glucosinolate content in canola meal based on glucose equivalents. The glucosinolate levels in the debris were estimated to be 21.9 mmol/g (AmiconTM) and 22.5 mmol/g (VivaflowTM) which are not significantly different from the values of 20.3 mmol/g (p ¼ 0.61) and 21.6 mmol/g (p ¼ 0.56) from those obtained by GC for the AmiconTM and VivaflowTM series, respectively. However, the ability of this technique to produce results similar to those from the GC was not achieved for the rest of the samples as the p values were o0.05 for all samples. The total glucosinolate contents estimated from the DTK were consistently higher than those obtained by the GC method. It seems that the DTK technique works best with products that are originally in a dry state, including the canola seeds that were examined by Vescio et al. (2001), and canola meal and debris in the present work. One possible explanation for this result is that the glucose meter detected not only the glucose released from the glucosinolate–myrosinase system, but also other glucose in the samples. According to Shahidi, Naczk, and Myhara (1990), canola meal contains 3.93–5.73 g/100 g sucrose, 0.27–0.62 g/100 g raffinose and 0.83–1.61 g/100 g stachyose as the major soluble sugars, while glucose and fructose were present in trace amounts. It should be noted, however, that the types and amount of these soluble sugars varied, depending on their origin and the chemical analysis method employed (Naczk & Shahidi, 1990). Although the free glucose was present in trace amounts, glucose may also have been released by hydrolysis of other sugars, oligosaccharides and glycosylated materials such as protein in the presence of water.
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72% for the AmiconTM stirred cell and VivaflowTM 200 systems, respectively) (Fig. 2). In addition, the DTK data did not show the difference in the glucosinolates for the two ultrafiltration systems that was noted for the GC data. This may also be attributed to the influence of free glucose on the glucosinolate measurements.
As a result, the glucose levels of the samples would, in part, be related to the conditions present during the three stages of the PMM process. In the extraction stage, these sugars were readily soluble in the first supernatant, and some oligosaccharide hydrolysis may occur releasing more free glucose. This overestimation of glucose was further supported by the fact that about 78% of the glucose was recovered in the first supernatant while an average of 45% was recovered in the debris for both series (Fig. 2), resulting in the first supernatant containing approximately 68 mmol/g of glucose (average for both series; Table 4). As there were only 45 mmol/g (average for both series; Tables 2 and 3) of total glucosinolates in the first supernatant according to the GC results, about 23 mmol/g of glucose existed as non-glucosinolate glucose. This high glucose profile remained in the samples during subsequent stages of the isolation process although minor differences due to the ultrafiltration systems were noted. In the final stage of the isolation process, the soluble glucose remained in the second supernatant leaving lower amounts in the PMM isolates. Comparable results have been reported by Aman and Gilberg (1977) where their canola protein isolates contained 86.0–88.5% less neutral sugars than those originally present in the meal. The presence of this extra glucose clearly influenced the mass balance reported as glucosinolates. While a reduction in total glucosinolates was seen in the isolated protein, the percentage reduction (58% and 62% for the AmiconTM stirred cell and VivaflowTM 200 systems, respectively) were not as high as those noted with the GC analysis (81% and
3.2. Comparison of two ultrafiltration systems Ultrafiltration is an important stage in the PMM process in that it allows the separation of the impurities with low molecular weight, including glucosinolates, from the high molecular weight desirable canola proteins. This is, in fact, an added benefit for ultrafiltration over other concentrating systems. The effect of ultrafiltration and the comparison of two laboratory-scale ultrafiltration systems in terms of the overall recovery of glucosinolates are shown in Fig. 2. For both methods of analyses, the results show that the AmiconTM stirred ultrafiltration cell unit (A), with lower MWCO (10,000 MWCO), allowed significantly more glucosinolates (or glucosinolate and glucose for DTK technique) from first supernatant through the membrane into the filtrate (IV) compared to the VivaflowTM 200 ultrafiltration cell unit (30,000 MWCO) (Fig. 2). This has, in turn, left less glucosinolates in the retentate (V) according to the DTK analysis, although this difference was not significant with the GC analysis. These results are opposite to the statement of Diosady, Tzeng, and Rubin (1984) where the removal of impurities, including glucosinolate and glucose, was best with the higher MWCO
Glucosinolates recoverd (mol / 100 mol in canola meal)
90 a a
80
a 70 b b
60 a a 50
a
b c
a d
40
a 30
b
a c
20
c
b c c
10 a a c b 0 II
III
IV V Sampling step
VI
VII
Fig. 2. Recovery of glucosinolates (mol/100 mol of glucosinolate in the meal) in the fractions obtained during the isolation of canola protein. The sampling steps were as follows: (II) debris from first extraction, (III) supernatant from first extraction, (IV) filtrate from ultrafiltration, (V) retentate from ultrafiltration, (VI) supernatant from the protein precipitation step and (VII) isolated canola protein. Samples using AmiconTM Stirred Cell (A) and VivaflowTM 200 (B) ultrafiltration systems were analyzed with gas chromatography (GC) and a diabetic test kit (DTK). , DTK A; , DTK B; , GC A; , GC B.
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membrane. The reason for this difference is not clear, but it may be related to the different flow behavior of samples for these ultrafiltration systems. The continuous flow for the VivaflowTM 200 system appeared to minimize the selective removal of glucosinolates (or glucose). As a result, while the VivaflowTM 200 unit improved protein recovery, it has also increased the recovery of undesirable glucosinolates compared to the AmiconTM unit. In the final stage of the isolation procedure, a relatively large percentage of the glucosinolates was recovered in the decanted second supernatant (VI) as most remained soluble upon dilution and were separated from the PMM protein isolates (VII) for both systems (Fig. 2). Interestingly, the supernatant from the AmiconTM stirred cell system had significantly higher glucosinolate recovery than the VivaflowTM 200 system when using the DTK analysis but the level of recovery of glucosinolates in the PMM were not significantly different. With the GC analysis, the recoveries in the supernatants were not significantly different but the glucosinolates recovered in the PMM were lower for the AmiconTM stirred cell system. In this case, the increased retention of glucosinolates by the VivaflowTM 200 unit that was noted during ultrafiltration has resulted in increased glucosinolate recovery (Fig. 2) and higher glucosinolate levels in the final product (Tables 2 and 3). 3.3. Comparison of GC and DTK for evaluating glucosinolates By comparing the DTK results in Table 4 to the GC results in Tables 2 and 3, it is clear that the DTK results in an overestimation of glucose in the canola fractions as a consequence of the determination of non-glucosinolate glucose; this is most evident of fractions originally in liquid form (III, IV, V and VI). The fact that this difference varies depending on the stage in the isolation process results in a difference in glucosinolate recovery depending on the method used to evaluate the level of glucosinolates recovered (Fig. 2). While the recoveries in the residue (II) during the first extraction step were unaffected by the method of analysis, the high values for the supernatant with the DTK assay, resulted in unrealistically high overall recoveries (sum of fractions II, IV, VI and VII) of well over 100%. With the CG method, on the other hand the recoveries were much closer to 100%. For the VivaflowTM system, for example, the DTK assay resulted in an overall recovery of 122% whereas the recovery was 101% with the CG system. Overall recoveries were higher with the AmiconTM system for both assays. The effect of the overestimation of glucosinolate recovery in the initial supernatant is reflected in subsequent steps, as the recoveries for the DTK were all significantly higher (po0.05), than the corresponding treatment analyzed using the GC method (Fig. 2). While the DTK method is able to follow the trend in terms of the relative distribution of the glucosinolates during protein
isolation, the absolute values and percent recoveries for the various fractions do not agree with the GC values. Although the DTK technique has been used effectively in the past to evaluate seeds (Vescio et al., 2001), and did a good job for the meal and initial debris, it was unable to accurately measure the glucosinolates in the products of protein isolation. As the values were higher than those obtained with the GC method, it would appear that other sources of glucose were contributing to the DTK results. If the distribution of myrosinase was an issue, than lower values for the DTK test would have been expected. 4. Conclusion The protein isolation procedure used in this study was effective in reducing the glucosinolate level in the isolate to 20 (AmiconTM stirred cell ultrafiltration) to 30% (VivaflowTM 2000 ultrafiltration) of what it was in the original meal. The levels obtained are still more than 10 times higher that those reported for broccoli, with values of 7.4–10.9 mmol/g for the canola protein isolate in this study compared to 0.62 mmol/g in broccoli (Song & Thornalley, 2007). The higher residual glucosinolate level obtained with the VivaflowTM ultrafiltration was due to a higher recovery of aromatic glucosinolates as both systems had similar reductions in aliphatic glucosinolates. Reductions in the glucosinolate levels were associated with the ultrafiltration step where levels of glucosinolates in the retentate were significantly lower than in the filtrate. Reductions in glucosinolates were also observed at the precipitation step where most of the glucosinolates remained in the supernatant. The DTK technique is a simple and rapid that has been used as a non-specific indirect method for measuring glucosinolates (Vescio et al., 2001). However, it is not applicable to those samples in which the endogenous myrosinase enzyme is not active and it measures the glucose regardless of where it comes from. The glucose meter used in this study was capable of determining only the free glucose, whether originally present as the monosaccharide itself or the glucose released from the glucosinolate–myrosinase system, or from the hydrolysis of other glycosylated compounds or oligosaccharides. In the current study, overestimation occurred for most samples as a consequence of the determination of non-glucosinolate glucose by the DTK. While this may be an appropriate method to estimate the total glucosinolate content of canola seeds and mild heat-treated meal, it is not recommended for materials produced during the processing of the meal. Acknowledgment Financial support provided by the Natural Science & Engineering Research Council of Canada (NSERC) is gratefully acknowledged.
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