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Removal of Glucosinolates from Midas Rapeseed and Mustard Seed by Methanol-Ammonia1
Can. Ins!. Food Sci. Technol. J. Vol. 19, No.2, pp. 75-77, 1986
RESEARCH
Removal of Glucosinolates from Midas Rapeseed and Mustard Seed by Methanol-Ammonia l M. Naczk,z F. Shahidi, L.L. Diosady and L.J. Rubin Department of Chemical Engineering and Applied Chemistry University of Toronto Toronto, Ontario, Canada M5S IA4
3 mg/g by plant breeders in Canada through the development of canola varieties. Nonetheless, these levels are still too high for incorporation into food products. Recently, a novel process for the removal of glucosinolates from canola was developed (Rubin et al., 1984, 1986) in which the crushed seeds are treated with a two-phase extraction solution. The first phase consists of ammonia in methanol (w/v) containing 5070 water (v/v), and hexane which extracts the oil into the non-polar second phase. The glucosinolates in canola were removed in this process below the detection limit (0.2 mg glucosinolates/g meal) of the method of Wetter and Youngs (1976), and a bright coloured meal with a bland taste was obtained (Rubin et al., 1984, 1986; Naczk et al., 1985). In this paper the application of the two-phase solvent system to remove glucosinolates from highglucosinolate rapeseed and mustard seed is reported.
Abstract A two-phase solvent extraction system for the removal of glucosinolates from Midas (high-glucosinolate) rapeseed and mustard seed was studied. The polar phase consisted of different levels of ammonia in methanol containing 5070 water, and hexane was used as the non-polar second phase. The effect of a quiescent contact period, the concentration of ammonia, and the ratio of the volume of the methanol/ammonia phase to the weight of seed (R) on the glucosinolate content of the meal was studied. The optimum conditions for glucosinolate removal at room temperature were a 30-min quiescent period, 10% ammonia concentration, and R = 6.7. Over 98% of all glucosinolate was removed after a second extraction of the resultant meal with the above extraction system under optimum conditions.
Resume L'etude a porte sur un systeme d'extraction au solvant a double phase pour I'extraction des glucosinolates du colza Midas (riche en glucosinolate) et de la graine de moutarde. La phase polaire consista en differents niveaux d'ammoniac dans du methanol contenant 5% d'eau, tandis que l'hexane constitua la phase non polaire. On a etudie l'effet d'un temps de contact au repos, de la concentration en ammoniac, et du rapport (R), phase polaire/graines (VIP) sur la teneur en glucosinolate du tourteau. Les conditions optimales pour I'extraction du glucosinolate a la temperature ambiante furent une periode de repos de 30 min, une concentration en ammoniac de 10%, et R = 6.7. Plus de 98% du glucosinolate fut enleve apres une deuxieme extraction du tourteau avec Ie systeme mentionne et dans les conditions optimales.
Materials and Methods Rapeseed of the Midas variety and mustard seed cultivar L22A were obtained from the POS Pilot Plant Corporation in Saskatoon, Sakatchewan. The seed (30 g) was first ground in a Philips coffee grinder, then treated with the two-phase solvent extraction method developed in our laboratories (Rubin et al., 1984, 1986). The effect of the ammonia concentration in methanol (6-14%), the solvent-to-seed ratio, R (6.7-20), and a quiescent contact period after a 2-min blending time (0-120 min) without any agitation on the glucosinolate content of the meal was studied. The effect of one- and two-stage treatments on the removal of glucosinolates was examined and the effect of agitation after blending, instead of the quiescent period, was also studied. Hexane-extracted meals were prepared by continuous extraction of oil from crushed seed using a Soxhlet apparatus. The defatted meals were dried in a vacuum oven at 40°C. The total glucosinolate content, measured as thioureas, was determined by the method
Introduction Although rapeseed meal has a well balanced amino acid content, its use as a food protein source has been thwarted due to the presence of undesirable components, particularly glucosinolates (thioglucosides), Products of glucosinolate hydrolysis interfere with the function of the thyroid gland and adversely affect growth. Fortunately, the glucosinolate content of rapeseed meal has already been reduced to less than Ipresented at the 28th Annual Meeting of the Canadian Institute of Food Science and Technology, June 23-26, 1985. Toronto, Ontario .. 2Author
Naczk is on leave of absence from the Department of Technical Microbiology, Politechnika Gdanska, Gdansk, Poland.
Copyright
©
1986 Canadian Institute of Food Science and Technology
75
Table I. Chemical Composition of Oilseeds 1 Moisture Crude Protein 2 Oil Glucosinolate Seed (070) ("70) (070) (mg/g meal) Rapeseed 5.6 ± 0.2 21.4 ± 0.4 42.3 ± 0.4 17.0 ± 0.7 Mustard 6.4 ± 0.2 27.6 ± 0.3 33.2 ± 0.6 18.4 + 0.5 I Values reported are averages of four replications and standard deviations given. 2Values are calculated as (N x 6.25).
of Wetter and Youngs (1976) and the enzyme myrosinase was prepared as described by Jones (1979). The crude protein content (N x 6.25) of selected meals was determined using the AACC procedure (1976). Confidence limits were calculated for each mean (P < 0.05). Differences among means were tested by the Tukey's test (Steel and Torries, 1980).
Results and Discussion The chemical composition of Midas rapeseed and mustard seed is summarized in Table 1 along with the glucosinolate content of their corresponding hexaneextracted meals. The effect of the ammonia concentration in methanol containing 5070 water on the removal of glusocinolates from Midas and mustard seed is presented in Figure 1. A 30-min quiescent period was allowed. Approximately 90% of glucosinolates originally present in the Midas and 80% of those present in mustard seed was removed by treatment with 10% ammonia solution. Increasing the ammonia concentration from 10 to 14% did not significantly improve the removal of glucosinolates from rapeseed, but was more effective in the case of mustard seed. The difference in the efficiency of glucosinolate removal from rapeseed and mustard seed may arise from the differ-
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Fig. 1. Effect of ammonia concentration in methanol containing 5"70 water on the glucosinolate content of rapeseed and mustard seed (at R = 6.7 and a quiescent period of 30 minutes). Vertical bars represent confidence limits (P <0.05).
76 / Naczk et al.
ences in their structural composition. Mustard seed is characterized by thicker epidermal and palisade cell sublayers than rapeseed (Young and Seigler, 1981). This may, in turn, bring about coarser particle size distribution of mustard meal and, hence, a lower reduction of the glucosinolates (Figure 1). The influence of the quiescent period on the glucosinolate contents of mustard and rapeseed meals, using 10% ammonia solution and an R of 6.7, is shown in Figure 2. After the initial 2-min blending time (i.e. zero quiescent contact time) some 70% of the glucosinolates present originally in rapeseed and mustard seed were removed. The glucosinolate content of the meal was sharply reduced during the first 30 min of the quiescent period, after which it remained essentially constant (P = 0.025, t-test). This optimum quiescent contact time is somewhat longer than the IS-min period required for canola (Diosady et al., 1985). The effect of the ratio of the volume of ammoniamethanol solution to seed weight (R) on the glucosinolate contents was also studied (Table 2). Increasing R from 6.7 to 13.3 decreased the glucosinolate levels in the Midas and mustard meals by a factor of 2 and 4 respectively. Further increase in R to 20 had the effect of increasing them by a factor of 2 and 4. In this case the glucosinolate content was basically the same as that at R = 6.7. Further dilution may interfere with particle size reduction in the Waring blender bringing about
,....
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3
Table 2. Effect of Polar Phase-to-Seed Ratio on the Removal of Glucosinolates from Oilseeds I R(v/w) Seed 6.7 13.3 20.0 Rapeseed 1.58 ± 0.08 0.87 ± 0.13 1.58 ± 0.Q7 Mustard 3.86 ± 0.03 1.15 + 0.12 3.65 + 0.03 IValues reported are averages of four replications and are expressed in mg glucosinolates/g meal and standard deviations are given. A quiescent period of 30 minutes was applied.
\
2
Rapeseed
t
!-----
S o
40
80
120
Quiescent Time ( min. )
Fig. 2. Effect of a quiescent contact period on removal of glucosinolates from rapeseed and mustard seed by 10"70 ammonia in methanol containing 5"70 water (at R = 6.7). Vertical bars represent confidence limits (P < 0.05). J. Inst. Can. Sci. Technol. Aliment. Vol. 19, No.2, 1986
Table 3. Effect of One- and Two-Stage Treatment on the Crude Protein Content of Oilseed Meals l Crude Protein (N x 6.25, %) Meal One-Stage Two-Stage Hexane-Extracted Treatment Treatment Rapeseed 41.2 ± 0.5 50.2 ± 0.4 53.6 ± 0.5 Mustard 44.2 ± 0.4 49.9 ± 1.1 53.8 ± 0.4 IValues reported are averages of four replications with standard deviations given. An R of 6.7 and a quiescent period of 30 minutes were applied.
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I Mustard
Fig. 3. Effect of one- and two-stage treatment on the glucosinolate content of rapeseed and mustard seed. l) Hexaneextracted; 2) one-stage treatment; 3) one-stage treatment with agitation; 4) two-stage treatment (at R = 6.7 and a quiescent period of 30 minutes).
lower removal of glucosinolates. It is noteworthy that agitation during the quiescent contact time did not improve the removal of the glucosinolates to any appreciable extent (Figure 3). After one treatment with methanol-ammonia, the rapeseed meal still contains about 1.5 mg/g of glucosinolates and the mustard meal about 4. Therefore, in one experiment the effect of a two-stage treatment on the content of glucosinolates was examined. The results shown in Figure 3 indicate that the twostage process removes over 98010 of the glucosinolates originally present in the meals. In Table 3, the crude protein content (N x 6.25) of the hexane extracted meals are compared with those after a one- and two-stage treatment with methanolammonia solution at an R of 6.7 and a quiescent period of 30 min. Results show an increase in the crude protein content of both rapeseed and mustard meals, the increase being more pronounced for meals obtained from the two-stage treatment. This is presumably due to the dissolution of some carbohydrates and other constituents present in the meals in the polar ammonia-methanol phase.
Can. Insi. Food Sci. Technol. J. Vol. \9, No.2. 1986
The optimum conditions for the removal of glucosinolates from high-glucosinolate rapeseeds are: 10% ammonia in methanol, methanol/ammonia-toseed ratio of 6.7, and a quiescent period of 30 min. Under these conditions, the glucosinolate content of the meal drops to the level of untreated canola meal. For mustard seed, 14% ammonia in methanol, in the one-stage process markedly increased the glucosinolate removal, other conditions being the same. A second extraction of the resultant meals with the above extraction system, containing 10% ammonia in methanol under optimum conditions, removed over 98% of the glucosinolates.
Acknowledgement The authors are grateful to M. Lau and R.J. Goncza for technical assistance.
References AACC 1976. Approved Method of the American Association of Cereal Chemists. St. Paul, Minnesota. Diosady, L.L., Rubin, L.J., Phillips, C.R., and Naczk, M. 1984. Effects of Alkanol-Ammonia Treatment on the Glucosinolate Content of Rapeseed Meal. Can. Inst. Food Sci. Technol. J. 18:311. Jones, J.D. 1979. Private communication. Naczk, M., Diosady, L.L. and Rubin, L.J. 1985. Functional Properties of Canola Meals Produced by a Two-Phase Solvent Extraction System. 1. Food Sci. 50: 1685. Rubin, L.J., Diosady, L.L. and Phillips, C.R. 1984. Solvent Extraction of Oil Bearing Seeds. US Patent 4,460,504. Rubin, L.J., Diosady, L.L., Naczk, M. and Halfani, M. 1986. The Alkanol-Ammonia-Water/Hexane Treatment of Canola. Can. Inst. Food Sci. Technol. J. 19:57. Steel, R.G.D. and Torries, J .H. 1980. Principles and Procedures of Statistics. McGraw-Hill Co. Inc., New York. Wetter, L.R. and Youngs, C. 1976. A Thiourea-UV Assay for Total Glucosinolates in Rapeseed Meals. J. Am. Oil Chern. Soc. 53:162. Young, D.A. and Seigler, D.S. 1981. Phytochemistry and Angiosperm Phylogeny. Praeger Publishers, New York. Accepted November 18, 1985
Naczk et al. / 77
RESEARCH
Removal of Glucosinolates from Midas Rapeseed and Mustard Seed by Methanol-Ammonia l M. Naczk,z F. Shahidi, L.L. Diosady and L.J. Rubin Department of Chemical Engineering and Applied Chemistry University of Toronto Toronto, Ontario, Canada M5S IA4
3 mg/g by plant breeders in Canada through the development of canola varieties. Nonetheless, these levels are still too high for incorporation into food products. Recently, a novel process for the removal of glucosinolates from canola was developed (Rubin et al., 1984, 1986) in which the crushed seeds are treated with a two-phase extraction solution. The first phase consists of ammonia in methanol (w/v) containing 5070 water (v/v), and hexane which extracts the oil into the non-polar second phase. The glucosinolates in canola were removed in this process below the detection limit (0.2 mg glucosinolates/g meal) of the method of Wetter and Youngs (1976), and a bright coloured meal with a bland taste was obtained (Rubin et al., 1984, 1986; Naczk et al., 1985). In this paper the application of the two-phase solvent system to remove glucosinolates from highglucosinolate rapeseed and mustard seed is reported.
Abstract A two-phase solvent extraction system for the removal of glucosinolates from Midas (high-glucosinolate) rapeseed and mustard seed was studied. The polar phase consisted of different levels of ammonia in methanol containing 5070 water, and hexane was used as the non-polar second phase. The effect of a quiescent contact period, the concentration of ammonia, and the ratio of the volume of the methanol/ammonia phase to the weight of seed (R) on the glucosinolate content of the meal was studied. The optimum conditions for glucosinolate removal at room temperature were a 30-min quiescent period, 10% ammonia concentration, and R = 6.7. Over 98% of all glucosinolate was removed after a second extraction of the resultant meal with the above extraction system under optimum conditions.
Resume L'etude a porte sur un systeme d'extraction au solvant a double phase pour I'extraction des glucosinolates du colza Midas (riche en glucosinolate) et de la graine de moutarde. La phase polaire consista en differents niveaux d'ammoniac dans du methanol contenant 5% d'eau, tandis que l'hexane constitua la phase non polaire. On a etudie l'effet d'un temps de contact au repos, de la concentration en ammoniac, et du rapport (R), phase polaire/graines (VIP) sur la teneur en glucosinolate du tourteau. Les conditions optimales pour I'extraction du glucosinolate a la temperature ambiante furent une periode de repos de 30 min, une concentration en ammoniac de 10%, et R = 6.7. Plus de 98% du glucosinolate fut enleve apres une deuxieme extraction du tourteau avec Ie systeme mentionne et dans les conditions optimales.
Materials and Methods Rapeseed of the Midas variety and mustard seed cultivar L22A were obtained from the POS Pilot Plant Corporation in Saskatoon, Sakatchewan. The seed (30 g) was first ground in a Philips coffee grinder, then treated with the two-phase solvent extraction method developed in our laboratories (Rubin et al., 1984, 1986). The effect of the ammonia concentration in methanol (6-14%), the solvent-to-seed ratio, R (6.7-20), and a quiescent contact period after a 2-min blending time (0-120 min) without any agitation on the glucosinolate content of the meal was studied. The effect of one- and two-stage treatments on the removal of glucosinolates was examined and the effect of agitation after blending, instead of the quiescent period, was also studied. Hexane-extracted meals were prepared by continuous extraction of oil from crushed seed using a Soxhlet apparatus. The defatted meals were dried in a vacuum oven at 40°C. The total glucosinolate content, measured as thioureas, was determined by the method
Introduction Although rapeseed meal has a well balanced amino acid content, its use as a food protein source has been thwarted due to the presence of undesirable components, particularly glucosinolates (thioglucosides), Products of glucosinolate hydrolysis interfere with the function of the thyroid gland and adversely affect growth. Fortunately, the glucosinolate content of rapeseed meal has already been reduced to less than Ipresented at the 28th Annual Meeting of the Canadian Institute of Food Science and Technology, June 23-26, 1985. Toronto, Ontario .. 2Author
Naczk is on leave of absence from the Department of Technical Microbiology, Politechnika Gdanska, Gdansk, Poland.
Copyright
©
1986 Canadian Institute of Food Science and Technology
75
Table I. Chemical Composition of Oilseeds 1 Moisture Crude Protein 2 Oil Glucosinolate Seed (070) ("70) (070) (mg/g meal) Rapeseed 5.6 ± 0.2 21.4 ± 0.4 42.3 ± 0.4 17.0 ± 0.7 Mustard 6.4 ± 0.2 27.6 ± 0.3 33.2 ± 0.6 18.4 + 0.5 I Values reported are averages of four replications and standard deviations given. 2Values are calculated as (N x 6.25).
of Wetter and Youngs (1976) and the enzyme myrosinase was prepared as described by Jones (1979). The crude protein content (N x 6.25) of selected meals was determined using the AACC procedure (1976). Confidence limits were calculated for each mean (P < 0.05). Differences among means were tested by the Tukey's test (Steel and Torries, 1980).
Results and Discussion The chemical composition of Midas rapeseed and mustard seed is summarized in Table 1 along with the glucosinolate content of their corresponding hexaneextracted meals. The effect of the ammonia concentration in methanol containing 5070 water on the removal of glusocinolates from Midas and mustard seed is presented in Figure 1. A 30-min quiescent period was allowed. Approximately 90% of glucosinolates originally present in the Midas and 80% of those present in mustard seed was removed by treatment with 10% ammonia solution. Increasing the ammonia concentration from 10 to 14% did not significantly improve the removal of glucosinolates from rapeseed, but was more effective in the case of mustard seed. The difference in the efficiency of glucosinolate removal from rapeseed and mustard seed may arise from the differ-
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:= = 3
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NH 3 Concentration ct)
Fig. 1. Effect of ammonia concentration in methanol containing 5"70 water on the glucosinolate content of rapeseed and mustard seed (at R = 6.7 and a quiescent period of 30 minutes). Vertical bars represent confidence limits (P <0.05).
76 / Naczk et al.
ences in their structural composition. Mustard seed is characterized by thicker epidermal and palisade cell sublayers than rapeseed (Young and Seigler, 1981). This may, in turn, bring about coarser particle size distribution of mustard meal and, hence, a lower reduction of the glucosinolates (Figure 1). The influence of the quiescent period on the glucosinolate contents of mustard and rapeseed meals, using 10% ammonia solution and an R of 6.7, is shown in Figure 2. After the initial 2-min blending time (i.e. zero quiescent contact time) some 70% of the glucosinolates present originally in rapeseed and mustard seed were removed. The glucosinolate content of the meal was sharply reduced during the first 30 min of the quiescent period, after which it remained essentially constant (P = 0.025, t-test). This optimum quiescent contact time is somewhat longer than the IS-min period required for canola (Diosady et al., 1985). The effect of the ratio of the volume of ammoniamethanol solution to seed weight (R) on the glucosinolate contents was also studied (Table 2). Increasing R from 6.7 to 13.3 decreased the glucosinolate levels in the Midas and mustard meals by a factor of 2 and 4 respectively. Further increase in R to 20 had the effect of increasing them by a factor of 2 and 4. In this case the glucosinolate content was basically the same as that at R = 6.7. Further dilution may interfere with particle size reduction in the Waring blender bringing about
,....
I
3
Table 2. Effect of Polar Phase-to-Seed Ratio on the Removal of Glucosinolates from Oilseeds I R(v/w) Seed 6.7 13.3 20.0 Rapeseed 1.58 ± 0.08 0.87 ± 0.13 1.58 ± 0.Q7 Mustard 3.86 ± 0.03 1.15 + 0.12 3.65 + 0.03 IValues reported are averages of four replications and are expressed in mg glucosinolates/g meal and standard deviations are given. A quiescent period of 30 minutes was applied.
\
2
Rapeseed
t
!-----
S o
40
80
120
Quiescent Time ( min. )
Fig. 2. Effect of a quiescent contact period on removal of glucosinolates from rapeseed and mustard seed by 10"70 ammonia in methanol containing 5"70 water (at R = 6.7). Vertical bars represent confidence limits (P < 0.05). J. Inst. Can. Sci. Technol. Aliment. Vol. 19, No.2, 1986
Table 3. Effect of One- and Two-Stage Treatment on the Crude Protein Content of Oilseed Meals l Crude Protein (N x 6.25, %) Meal One-Stage Two-Stage Hexane-Extracted Treatment Treatment Rapeseed 41.2 ± 0.5 50.2 ± 0.4 53.6 ± 0.5 Mustard 44.2 ± 0.4 49.9 ± 1.1 53.8 ± 0.4 IValues reported are averages of four replications with standard deviations given. An R of 6.7 and a quiescent period of 30 minutes were applied.
-1 18
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a
till ~ till
a ... ...= 0= 0
-
1
17 c)
I)
'-' Q)
-2
4
Conclusions
...!.
Q)
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3
0
oS
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0 0
....:s 0
2
2
3
-f--
1
4
h Rapeseed
4
I Mustard
Fig. 3. Effect of one- and two-stage treatment on the glucosinolate content of rapeseed and mustard seed. l) Hexaneextracted; 2) one-stage treatment; 3) one-stage treatment with agitation; 4) two-stage treatment (at R = 6.7 and a quiescent period of 30 minutes).
lower removal of glucosinolates. It is noteworthy that agitation during the quiescent contact time did not improve the removal of the glucosinolates to any appreciable extent (Figure 3). After one treatment with methanol-ammonia, the rapeseed meal still contains about 1.5 mg/g of glucosinolates and the mustard meal about 4. Therefore, in one experiment the effect of a two-stage treatment on the content of glucosinolates was examined. The results shown in Figure 3 indicate that the twostage process removes over 98010 of the glucosinolates originally present in the meals. In Table 3, the crude protein content (N x 6.25) of the hexane extracted meals are compared with those after a one- and two-stage treatment with methanolammonia solution at an R of 6.7 and a quiescent period of 30 min. Results show an increase in the crude protein content of both rapeseed and mustard meals, the increase being more pronounced for meals obtained from the two-stage treatment. This is presumably due to the dissolution of some carbohydrates and other constituents present in the meals in the polar ammonia-methanol phase.
Can. Insi. Food Sci. Technol. J. Vol. \9, No.2. 1986
The optimum conditions for the removal of glucosinolates from high-glucosinolate rapeseeds are: 10% ammonia in methanol, methanol/ammonia-toseed ratio of 6.7, and a quiescent period of 30 min. Under these conditions, the glucosinolate content of the meal drops to the level of untreated canola meal. For mustard seed, 14% ammonia in methanol, in the one-stage process markedly increased the glucosinolate removal, other conditions being the same. A second extraction of the resultant meals with the above extraction system, containing 10% ammonia in methanol under optimum conditions, removed over 98% of the glucosinolates.
Acknowledgement The authors are grateful to M. Lau and R.J. Goncza for technical assistance.
References AACC 1976. Approved Method of the American Association of Cereal Chemists. St. Paul, Minnesota. Diosady, L.L., Rubin, L.J., Phillips, C.R., and Naczk, M. 1984. Effects of Alkanol-Ammonia Treatment on the Glucosinolate Content of Rapeseed Meal. Can. Inst. Food Sci. Technol. J. 18:311. Jones, J.D. 1979. Private communication. Naczk, M., Diosady, L.L. and Rubin, L.J. 1985. Functional Properties of Canola Meals Produced by a Two-Phase Solvent Extraction System. 1. Food Sci. 50: 1685. Rubin, L.J., Diosady, L.L. and Phillips, C.R. 1984. Solvent Extraction of Oil Bearing Seeds. US Patent 4,460,504. Rubin, L.J., Diosady, L.L., Naczk, M. and Halfani, M. 1986. The Alkanol-Ammonia-Water/Hexane Treatment of Canola. Can. Inst. Food Sci. Technol. J. 19:57. Steel, R.G.D. and Torries, J .H. 1980. Principles and Procedures of Statistics. McGraw-Hill Co. Inc., New York. Wetter, L.R. and Youngs, C. 1976. A Thiourea-UV Assay for Total Glucosinolates in Rapeseed Meals. J. Am. Oil Chern. Soc. 53:162. Young, D.A. and Seigler, D.S. 1981. Phytochemistry and Angiosperm Phylogeny. Praeger Publishers, New York. Accepted November 18, 1985
Naczk et al. / 77