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Effect of alkanol-ammonia-water treatment on the glucosinolate content of rapeseed meal
Can. lnsi. Food Sci. Technol. J. Vol. 18. NO.4. pp. 311-315. 1985
RESEARCH
Effect of Alkanol-Ammonia-Water Treatment on the Glucosinolate Content of Rapeseed Meal L.L. Diosady, L.J. Rubin, C.R. Phillips and M. Naczk l Department of Chemical Engineering and Applied Chemistry University of Toronto Toronto, Ontario M5S IA4, Canada
Abstract The effect of alkanol and alkanol-ammonia treatment, with and without water, on the glucosinolate content of laboratory-prepared Altex and commercial rapeseed meals was studied. The meals were treated with methanol, ethanol, isopropanol and t-butanol, and with the same alkanols containing anhydrous ammonia (0-15070 for methanol and 10% for other alkanols), and water (0-35%). The results showed that methanol was the most effective alkanol for the removal of glucosinolates. The treatment of laboratory-prepared meal with methanol removed about 55% of the glucosinolates initially present in the meal. Methanol containing 35% water removed 80% of the glucosinolates. However, treatment of this meal with 10% ammonia in methanol reduced the glucosinolates content to 0.2 mg/g after a quiescent period of 15 minutes. The addition of water to the methanol-ammonia solutions further reduced the glucosinolates to trace levels. Methanol or methanol-ammonia were less effective in reducing the glucosinolate content of commercial meal. This suggests that the treatment of the meal during processing reduces the extractability of the glucosinolates. The effectiveness of glucosinolate removal by aikanols and solutions containing ammonia and water could be ranked as follows: methanol > ethanol > isopropanol > t-butanol The preferred solvent mixture for the removal of glucosinolates is 10% NH 3 in methanol containing 5% H 20.
Resume L'effet de traitement a I'alcanol et a l'alcanol ammoniace, avec et sans eau, a ete etudie sur la teneur en glucosinolates de tourteaux de colza du commerce et prepares en laboratoire (Altex). Les tour· teaux furent traites au methanol, a I'ethanol, al'isopropanol et au t-butanol, et aussi pour chacun des alcanols, en presence d'ammonia anhydre (0-15% pour Ie methanol et 10% pour les autres alcanols) et aussi avec ou sans eau (0-35%). Le methanol s'est revele I'alcanol Ie plus efficace pour I'extraction des glucosinolates. Le traitement du tourteau de laboratoire avec du methanol a extrait environ 35% du glucosinolate initialement present dans Ie tourteau. Du methanol contenant 35% d'eau a permis d'en extraire 80%. L'extraction de ce tourteau avec du methanol contenant 10% d'ammoniac a reduit la teneur en glucosinolates a 0.2 mg/g apres un repos de 15 min. L'addition d'eau aux solutions methanol-ammoniac a reduit davantage les glucosinolates a des niveaux de traces. Le methanol ou Ie I Author
Naczk is on leave of absence from the Department of Food Preservation and Technical Microbiology, Politechnika Gdanska, Gdansk, Poland
Copyright
'C
methanol ammoniace furent moins efficaces pour l'extraction des glucosinolates du tourteau commercial. Ceci indique donc que I'extractabilite des glucosinolates est affectee par certains traitements que subissent les tourteaux lors des operations industrielles. L'efficacite de I'extraction des glucosinolates par des alcanols et par des solutions contenant de I'ammoniac et de l'eau fut dans I'ordre suivant: metanol > etanol > isopropanol > t-butanol. Le melange d'extraction prHere pour les glucosinolates est 10% NH 3 dans du methanol contenant 5% H 20.
Introduction In conventional rapeseed processing the glucosinolates in the seed are preserved and retained in the meal by the thermal destruction of myrosinase, the enzyme responsible for the hydrolysis of glucosinolates. Canadian plant breeders have successfully reduced the glucosinolate content of rapeseed by a factor of 7 to 10, resulting in the "canola" varieties which are low in glucosinolates as well as in erucic acid. Canola meal may be fed to animals without restrictions based on glucosinolate content, although other meal components such as fibre may still limit its use. However, the use of the meal in food cannot be contemplated without a further significant reduction in its glucosinolate content. Many processes have been reported in the literature for the removal of glucosinolates from rapeseed and rapeseed meal. Jones (1979) attributed the nutritional problems associated with using rapeseed meal in animal feed to the high glucosinolate, fibre, and phytate content. He described a technique for producing a rapeseed concentrate low in glucosinolate by water extraction, and counteracted the effect of phyphate by zinc supplementation. Schlingmann and Vertesy (l978a,b) used ammoniamethanol solution for the extraction of lipids in the· production of single-cell protein. The presence of ammonia in the solvent is ·c1aimed to rupture the microbial cell wall, resulting in rapid extraction of the lipids. In a later patents Schlingmann and von Rymon Lipinski (1980, 1982) extended their work with methanol-ammonia to the extraction of oilseeds.It was
1985 Canadian Institute of Food Science and Technology
311
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Concentration of ammonia
30
Quiescent
period (min.)
45
60
ffi (l()
Fig. I. Effect of ammonia concentration in methanol on the glucosinolates content of canola meal.
claimed that the presence of ammonia improved the rate of extraction, and reduced the concentration of "undesirable lipids" such as fatty acid oxidation products, resulting in improved flavour and reduced residual oil content in the meal. The effect of alkanol-ammonia treatment, with or without water, on the glucosinolate content of an Altex meal produced under carefully controlled laboratory conditions is reported here. A rapeseed meal prepared industrially by the conventional pre-press hexaneextraction process was treated similarly for comparison.
Materials and Methods The laboratory-extracted meal was prepared by grinding Altex seed in a Philips coffee mill and blending at low speed (approximately 2,000 rpm) in a 4 L Waring blender for 2 min with hexane at a solvent-toseed ratio (R) of 5. The meal was separated by vacuum filtration using Whatman No. 41 filter paper. The residual oil content was removed by extraction with hexane for 12 h in a Soxhlet apparatus. The defatted meal was dried at 40°C in a vacuum oven. Commercially extracted meal was obtained through the Canola Council of Canada, but the cultivar was not specified. It was produced by the conventional prepress hexane-extraction process. The selected alkanols: methanol, isopropanol, and t-butanol were used as such or with the addition of low levels of water. Anhydrous ammonia was bubbled through these solutions at O°C for 1 h. The ammonia concentration was determined by titration with 1.0 N H 2S04 , and the desired ammonia concentration was achieved by dilution with ammonia-free solvent. The 312 / Diosady et 01.
15
Fig. 2. Effect of the quiescent period on removal of glucosinolates by 10% ammonia in methanol /0-0/, and amount of extracted solids / . - . / (laboratory-prepared meal)
final concentration of ammonia was determined by titration. A 30 g sample of meal was mixed at low speed (approximately 2,000 rpm) in a 2 L Waring blender for 2 min with the selected alkanol-ammonia solution at a solvent-to-meal ratio of 6.7. The solutions were made up to 10% ammonia except for solvents for which the ammonia solubility was below 10010. ThUS, for isopropanol, t-butanol, and t-butanol containing 5% H 20, saturated ammonia solutions with concentrations of 8.9, 6.4 and 7.8% respectively were used. After a quiescent period (without mixing) of 15 min the meal was vacuum filtered using Whatman No. 41 filter paper, rinsed 3 times with 50 mL of alkanol, and dried at 40°C in a vacuum oven. The experimental conditions were varied for methanol as follows: (1) ammonia concentration in methanol: 0, 2,4, 5, 6, 8, 10, 12, 14, 15 and 180/0 (w/w) (2) methanol/ammonia-to-seed ratio: 2.5, 5, 6.7, 10 and 15 (v/w) (3) quiescent period: 0, 15, 30 and 60 minutes.
The water concentration in the various alkanolammonia solutions was 0, 5, 10, and 15% (v/v). Crude protein (Nx6.25) content of the meal was determined using the standard AACC procedure (1976), and the total glucosinolate content was measured by the method of Wetter and Youngs (1976). Myrosinase for this assay was prepared from mustard seeds by the method of Jones (1979).
Results and discussion A significant decrease in the glucosinolates content of the meal was observed with increasing concentrations of ammonia in methanol (Figure 1). However, J. Inst. Can. Sci. Technol. Aliment. Vol. 18, No.4, 1985
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Fig. 3. Effect of methanol-to-meal ratio (v/w) on the glucosinolate content 10-01 and amount of extracted solids I . - . I (laboratory-prepared meal, 10070 NH 3 in methanol)
Fig. 4. Effect of water in methanol and in methanol containing 10070 ammonia on the glucosinolate removal from laboratoryprepared meal
the glucosinolate removal was about 4 times more effective for the laboratory-prepared meal than for commercial meal. After treatment of the meals with 100/0 NH 3 in methanol, the residual glucosinolates in the commercial and laboratory meals were 0.82 ± 0.14 and 0.20 ± 0.03 mg/g respectively. The commercial meal contained 3.5% of hexane-extractable lipid material. Residual lipids in the pores of the meal may hinder the diffusion of methanol-ammonia into the meal and thus interfere with the removal of the glucosinolates. To test this hypothesis a sample of commercial meal was re-extracted with hexane using a Soxhlet apparatus prior to treatment with 10% ammonia in methanol containing 5% water. The treatment removed 78% of the glucosinolates compared with a removal of 72% without the re-extraction with hexane. Statistical analysis of the results indicate that the effect was significant (t-test, P = 0.05). Since the treated defatted commercial meal still contains a higher level of glucosinolates than the laboratory-defatted meals, the lower effectiveness of the treatment of commercial meal may also be influenced by the visually larger particle size of the meal and by the possible alteration of the protein structure by heat during processing. Heat treatment may result in an increase in the number of functional groups available for reaction on the protein surface to bind the glucosinolates. The crude protein content of treated meals was not greatly influenced by the concentration of ammonia in methanol. The average protein content of treated commercial meal was 41.2 ± 0.4% and that of treated laboratory-prepared meals 48.8 ± 0.3%, on a dry basis. The 12% increase in crude protein content of
the treated laboratory-prepared meal was due to the dissolution of up to 16% of the meal solids containing carbohydrates and other constituents. The increase of the crude protein content of treated commercial meals was quite small, and the reason for this is not obvious. The effect of the quiescent period on the glucosinolates in the laboratory-prepared meal is shown in Figure 2. During the initial two minutes of blending with 10% ammonia in methanol, 85% of the glucosinolates originally present in the laboratoryprepared meal were removed. An additional 15 min of quiescent contact reduced the glucosinolate content by 92%, and 60 min by 95%. After a quiescent period of 15 min the glucosinolates content of the meal was close to the detection limit of the method of Wetter and Youngs (1976). Therefore, the difference in the glucosinolate content after 15, 30 and 60 min can be assumed to be negligible. A slight increase in extracted solids with a longer quiescent period was observed. The effect of the ratio of the volume of 10% ammo~ nia in methanol to meal weight (R) on the process is shown in Figure 3. The increase of R from 2.5 to 15 decreased the residual glucosinolates to trace levels. A significant increase in extracted solids from 13.2% for an R of2.5 to 17.5% for an R of 15 was observed. The removal of glucosinolates from laboratoryprepared and commercial meal by methanol, ethanol, isopropanol, and t-butanol is presented in Table 1. Only methanol is effective in reducing the glucosinolates content of the meals in the absence of ammonia. While the methanol removed 54.5% and 38% of glucosinolates from laboratory-prepared and commer-
Can. [nst. Food Sci. Technol. J. Vol. 18. No.4, 1985
Diosady et al. / 313
Table I. Effect of extraction with alkanols on the glucosinolate and crude protein content of meals!.
Alkanol
Water content of solvent (070)
Laboratory-prepared meal untreated
o
Methanol
5 10 IS
Crude protein (N x 6.25) (070)
Water Content (%; v/v)
I
2.55 ± 0.15
43.7 ± 0.3
5 25
1.11 ± 0.12 0.61 ± 0.07
1.16 1.11 1.04 0.92
± ± ± ±
47.6 47.6 48.5 48.8
0.10 0.12 0.07 0.05
± 0.4 ± 0.6 ± 1.0 ± 0.3
o
2.54 ± 0.21
43.8 ± 0.3
Isopropanol
o o
2.53 ± 0.17
43.1 ± 0.4
2.52 ± 0.27
43.8 ± 0.5
Commercial meal untreated
Multiple extraction of laboratory-prepared meal with methanol-water 1
Glucosinolates (mg/g)
Ethanol t-butanol
Table 2.
2.86 ± 0.20
39.7 ± 0.8
Methanol
0
1.78 ± 0.25
41.7 ± 0.1
Ethanol
0
2.71 ± 0.20
40.6 ± 0.1
Isopropanol
0
3.00 ± 0.25
40.9 ± 0.5
t-butanol
0
2.79 ± 0.20
41.1 ± 0.2
1The meals were blended with alkanol or alkanol-water solution for 2 minutes at R = 6.7, and fIltered after a quiescent period of IS minutes.
cial meal, ethanol, isopropanol, and t-butanol had no effect. Van Megen (1983) reported that 30 min of leaching of toasted rapeseed meal with 75lTfo ethanol or 80lTfo methanol removed 78lTfo and 88lTfo of the glucosinolates originally present respectively. The removal of glucosinolates from untoasted, mildly treated meal by 75lTfo ethanol was only 62lTfo. As shown in Figure 4, the leaching of laboratory-prepared meal with methanol containing 35lTfo water resulted in the removal of 79lTfo of the glucosinolates, producing a meal still containing 0.54 mg/g glucosinolates. The quantities of extracted solids increased from 13.9lTfo to 19.0lTfo as the water content in methanol was increased to 35lTfo. These quantities are lower than the solid losses reported by Van Megen (1983) using 80lTfo aqueous methanol.
Glucosinolate content (mg/g) Extraction steps 2 0.96 ± 0.10 0.40 ± 0.03
4
0.54 ± 0.06 0.12 ± 0.04
I At each step the meal was blended for 2 minutes with methanolwater at R = 6.7, and filtered after a quiescent period of IS minutes.
The glucosinolate removal by methanol-water solutions could be improved by multiple extraction of the meal. The results given in Table 2 indicate that about 95lTfo of the glucosinolates can be removed from laboratory-prepared meal after leaching 4 times with methanol containing 25lTfo of water. The loss of protein material was not measured. Table 3 shows the effect of treatment of laboratoryprepared and commercial meals with aliphatic alkanols containing ammonia and water (0-15lTfo) on the level of glucosinolates and crude protein. In all cases where the solubility was high enough, solutions containing lOlTfo ammonia by weight were used. Isopropanol, tbutanol and t-butanol containing 5lTfo water were saturated with ammonia, and used at 8.9, 6.4, and 7.8lTfo ammonia concentration respectively. Methanolammonia solutions were the most effective in reducing the glucosinolate content of the meals. The treatment of laboratory-prepared meal with methanolammonia reduced the glucosinolate content to trace levels. Thus the effect of the presence of water in the methanol-ammonia on the glucosinolates level was not readily measurable with the analytical technique used. Treatment with methanol-ammonia was less effective in the case of commercial meal. While lOlTfo ammonia in methanol removed 59lTfo of the glucosinolates originally present, the methanol-ammonia solution containing 15lTfo H 2 0 removed 86lTfo (Table 3).
Table 3. Effect of water in alkanol-ammonia solutions on glucosinolates and crude protein of meals. Alkanol
Water Content in alkanol (%)
untreated Methanol
o 5 10 IS
Ethanol
Isopropanol
t-Butanol
o 5 10 IS 01 5 10 IS 02 53 10 IS
Glucosinolates (mg/g)
Crude protein (N x 6.25)
laboratory
commercial
laboratory
2.55 ± 0.15
2.86 ± 0.20
43.7 ± 0.3
39.7 ± 0.8
commercial
0.20 0.18 0.11 0.08
± 0.03 ± 0.03 ± 0.01 ± 0.01
1.17 1.12 0.52 0.42
± 0.10 ± 0.14 ± 0.08 ± 0.05
48.8 49.4 49.2 49.8
± 0.6 ± 0.6 ± 1.2
0.6
41.0 ± 0.1 41.2 ± 0.2 40.9 ± 0.4 39.5 ± 0.1
2.36 1.34 0.92 0.77 2.70 2.72 2.10 1.34
± 0.25
2.60 2.19 1.55 0.74 3.13 2.89 2.24 1.57
± 0.20
45.4 47.0 47.2 48.5 44.0 45.2 45.8 46.6
0.7 0.3 0.3 0.6 0.9 0.6 1.0 1.2
40.5 40.3 40.6 40.3 40.2 40.2 40.4 40.7
± 0.14 ± 0.08 ± 0.05 ± 0.17 ± 0.16 ± 0.16 ± 0.15
2.79 2.50 2.16 1.50
± 0.13
± ± ± ± ±
0.05 0.04 0.23 0.22 0.10 ± 0.14
± 0.20 ± 0.20 ± 0.21 ± 0.15
± ± ± ± ± ± ±
± ±
41.0 40.6 39.3 41.0
± 0.2 ± 0.3 ± 0.2 ± 0.2 ± 0.3 ± 0.4 ± 0.4 ± 0.5 ± 0.1 ± 0.3
± 0.1 ± 0.1
18.9% NH 3 26.4% NH 3 37.8% NH3
314 / Diosady et al.
J. InSI. Can. Sci. Technol. Aliment. Vol. 18, No.4, 1985
Table 4. Myrosinase activity of the laboratory-prepared meal treated with methanol or methanol-ammonia solution. Glucosinolate content (mg/g) determined: Solvent with addition of without addition myrosinase of myrosinase untreated methanol methanol containing 5010 ammonia methanol containing 100f0 ammonia
2.55 ± 0.15 1.16 ± 0.10
2.24 ± 0.10 0.05 ± 0.02
0.40 ± 0.02
0.09 ± 0.02
0.20 ± 0.03
0.08 ± 0.03
The glucosinolate content of laboratory-prepared and commercial meals was not significantly affected by the treatment with 10% ammonia in ethanol, 8.9070 ammonia in isopropanol, and 6.4% ammonia in tbutanol (Table 3). An increase in the water content of ammonia solutions in ethanol, isopropanol and tbutanol resulted in lower glucosinolate levels. Similar trends were found with both laboratory-prepared and commercial meals. Ethanol- and isopropanolammonia solutions containing 15% water removed 74% and 48% of the glucosinolates respectively. These results were similar for laboratory-prepared and commercial meals (Table 3), indicating that the efficiency of glucosinolate removal decreases with the increasing number of carbon atoms in the aliphatic alkanols. Apparently, removal increases with increased polarity. The presence of ammonia and water in alkanols increases the effectiveness of the glucosinolate removal (Table 3 and Figure 4). This is not only due to better dissolution of glucosinolates from the meal, as the extraction of laboratory-prepared meal with methanol containing 5% water reduces the glucosinolate content to 1.11 mg/g. However, in the presence of 10% ammonia it drops to 0.18 mg/g. Presumably the more insoluble glucosinolates are first converted to other products which are then dissolved in the polar phase. The treatment of meals with alkanol-ammonia increased the crude protein concentration in the meal as a direct result of the dissolution of non-protein components (Table 3). Other studies showed that about 5% of the crude protein was dissolved in the methanol phase containing 10% ammonia, mainly non-protein nitrogen compounds soluble in a 10% trichloroacetic acid solution. The effect of the treatment of laboratory-prepared meal with methanol or methanol-ammonia solution on the myrosinase activity in the meal was studied. The myrosinase activity was evaluated, somewhat
Can. [nSf. Food Sci. Technol. J. Vol. 18, No.4. 1985
indirectly, using the Wetter and Youngs (1976) procedure, both with and without myrosinase addition. The results given in Table 4 indicate that treatment with methanol or methanol-ammonia solutions reduces the myrosinase activity to trace level.
Conclusions The treatment of rapeseed meal with alkanolammonia solutions effectively removed most of the glucosinolates. The effectiveness of this process depends upon the method of preparation of the meal, the type of alkanol used, and the presence of water in the alkanol-ammonia solution. A methanolammonia solution was found to be the most effective solvent for removal of glucosinolates from rapeseed meal. Treatment of laboratory-prepared meal with 10% ammonia in methanol containing 5% water removed glucosinolates to trace levels, while extraction with ammonia-free methanol containing 5% water removed only 56% of the glucosinolates, These results indicate that the presence of ammonia significantly increases the effectiveness of the detoxification process. The treatment of laboratory-prepared meal with methanol or methanol-ammonia solution reduces the myrosinase activity to trace level.
References AACC. 1976. Approved Methods of the American Association of Cereal Chemists. St. Paul, MN. Jones, J.D. 1979. Private communication. Jones, J.D. 1979. Rapeseed protein concentrate preparation and evaluation. JAOCS 56:716. Van Megen, W.H. 1983. Removal of glucosinolates from defatted rapeseed meal by extraction with aqueous ethanol. Can. Inst. Food Sci. Technol. J. 16:93. Schlingmann, M. and Vertesy, L. 1978a. Single cell proteins with reduced content of nucleic acid and fat, Fette, Seifen, Anstrichsmittel. 80(7):283. Schlingmann, M. and Vertesy, L. 1978b. Reducing the lipid and nucleic acid content in microbial cell masses. German Patent 2,633,666. Schlingmann, M. and von Rymon Lipinski, G.W. 1980. Process for treating meals and flours of oilseeds. U.K. Patent 2,030,441. Schlingmann, M. and von Rymon Lipinski, G.W. 1982. Process for improving the properties of meals and flours of oilseeds. Can. Patent 1,120,779. Wetter, L.R. and Youngs, C. 1976. A thiourea-UV assay for total glucosinolates in rapeseed meals. JAOCS 53: 162.
Accepted April 29, 1985
Diosady et al. / 315
RESEARCH
Effect of Alkanol-Ammonia-Water Treatment on the Glucosinolate Content of Rapeseed Meal L.L. Diosady, L.J. Rubin, C.R. Phillips and M. Naczk l Department of Chemical Engineering and Applied Chemistry University of Toronto Toronto, Ontario M5S IA4, Canada
Abstract The effect of alkanol and alkanol-ammonia treatment, with and without water, on the glucosinolate content of laboratory-prepared Altex and commercial rapeseed meals was studied. The meals were treated with methanol, ethanol, isopropanol and t-butanol, and with the same alkanols containing anhydrous ammonia (0-15070 for methanol and 10% for other alkanols), and water (0-35%). The results showed that methanol was the most effective alkanol for the removal of glucosinolates. The treatment of laboratory-prepared meal with methanol removed about 55% of the glucosinolates initially present in the meal. Methanol containing 35% water removed 80% of the glucosinolates. However, treatment of this meal with 10% ammonia in methanol reduced the glucosinolates content to 0.2 mg/g after a quiescent period of 15 minutes. The addition of water to the methanol-ammonia solutions further reduced the glucosinolates to trace levels. Methanol or methanol-ammonia were less effective in reducing the glucosinolate content of commercial meal. This suggests that the treatment of the meal during processing reduces the extractability of the glucosinolates. The effectiveness of glucosinolate removal by aikanols and solutions containing ammonia and water could be ranked as follows: methanol > ethanol > isopropanol > t-butanol The preferred solvent mixture for the removal of glucosinolates is 10% NH 3 in methanol containing 5% H 20.
Resume L'effet de traitement a I'alcanol et a l'alcanol ammoniace, avec et sans eau, a ete etudie sur la teneur en glucosinolates de tourteaux de colza du commerce et prepares en laboratoire (Altex). Les tour· teaux furent traites au methanol, a I'ethanol, al'isopropanol et au t-butanol, et aussi pour chacun des alcanols, en presence d'ammonia anhydre (0-15% pour Ie methanol et 10% pour les autres alcanols) et aussi avec ou sans eau (0-35%). Le methanol s'est revele I'alcanol Ie plus efficace pour I'extraction des glucosinolates. Le traitement du tourteau de laboratoire avec du methanol a extrait environ 35% du glucosinolate initialement present dans Ie tourteau. Du methanol contenant 35% d'eau a permis d'en extraire 80%. L'extraction de ce tourteau avec du methanol contenant 10% d'ammoniac a reduit la teneur en glucosinolates a 0.2 mg/g apres un repos de 15 min. L'addition d'eau aux solutions methanol-ammoniac a reduit davantage les glucosinolates a des niveaux de traces. Le methanol ou Ie I Author
Naczk is on leave of absence from the Department of Food Preservation and Technical Microbiology, Politechnika Gdanska, Gdansk, Poland
Copyright
'C
methanol ammoniace furent moins efficaces pour l'extraction des glucosinolates du tourteau commercial. Ceci indique donc que I'extractabilite des glucosinolates est affectee par certains traitements que subissent les tourteaux lors des operations industrielles. L'efficacite de I'extraction des glucosinolates par des alcanols et par des solutions contenant de I'ammoniac et de l'eau fut dans I'ordre suivant: metanol > etanol > isopropanol > t-butanol. Le melange d'extraction prHere pour les glucosinolates est 10% NH 3 dans du methanol contenant 5% H 20.
Introduction In conventional rapeseed processing the glucosinolates in the seed are preserved and retained in the meal by the thermal destruction of myrosinase, the enzyme responsible for the hydrolysis of glucosinolates. Canadian plant breeders have successfully reduced the glucosinolate content of rapeseed by a factor of 7 to 10, resulting in the "canola" varieties which are low in glucosinolates as well as in erucic acid. Canola meal may be fed to animals without restrictions based on glucosinolate content, although other meal components such as fibre may still limit its use. However, the use of the meal in food cannot be contemplated without a further significant reduction in its glucosinolate content. Many processes have been reported in the literature for the removal of glucosinolates from rapeseed and rapeseed meal. Jones (1979) attributed the nutritional problems associated with using rapeseed meal in animal feed to the high glucosinolate, fibre, and phytate content. He described a technique for producing a rapeseed concentrate low in glucosinolate by water extraction, and counteracted the effect of phyphate by zinc supplementation. Schlingmann and Vertesy (l978a,b) used ammoniamethanol solution for the extraction of lipids in the· production of single-cell protein. The presence of ammonia in the solvent is ·c1aimed to rupture the microbial cell wall, resulting in rapid extraction of the lipids. In a later patents Schlingmann and von Rymon Lipinski (1980, 1982) extended their work with methanol-ammonia to the extraction of oilseeds.It was
1985 Canadian Institute of Food Science and Technology
311
.4
16
.........
1.5
-
E .3
~ ~
~
.. .. ... .. ....
-!.....to
::!
0
::
15
.. .; II .. .5
i
.2
'0
~
1;
;
:!
'0
... . • .. . !
c;
.1
C;
14
0
o
5
ro
Concentration of ammonia
30
Quiescent
period (min.)
45
60
ffi (l()
Fig. I. Effect of ammonia concentration in methanol on the glucosinolates content of canola meal.
claimed that the presence of ammonia improved the rate of extraction, and reduced the concentration of "undesirable lipids" such as fatty acid oxidation products, resulting in improved flavour and reduced residual oil content in the meal. The effect of alkanol-ammonia treatment, with or without water, on the glucosinolate content of an Altex meal produced under carefully controlled laboratory conditions is reported here. A rapeseed meal prepared industrially by the conventional pre-press hexaneextraction process was treated similarly for comparison.
Materials and Methods The laboratory-extracted meal was prepared by grinding Altex seed in a Philips coffee mill and blending at low speed (approximately 2,000 rpm) in a 4 L Waring blender for 2 min with hexane at a solvent-toseed ratio (R) of 5. The meal was separated by vacuum filtration using Whatman No. 41 filter paper. The residual oil content was removed by extraction with hexane for 12 h in a Soxhlet apparatus. The defatted meal was dried at 40°C in a vacuum oven. Commercially extracted meal was obtained through the Canola Council of Canada, but the cultivar was not specified. It was produced by the conventional prepress hexane-extraction process. The selected alkanols: methanol, isopropanol, and t-butanol were used as such or with the addition of low levels of water. Anhydrous ammonia was bubbled through these solutions at O°C for 1 h. The ammonia concentration was determined by titration with 1.0 N H 2S04 , and the desired ammonia concentration was achieved by dilution with ammonia-free solvent. The 312 / Diosady et 01.
15
Fig. 2. Effect of the quiescent period on removal of glucosinolates by 10% ammonia in methanol /0-0/, and amount of extracted solids / . - . / (laboratory-prepared meal)
final concentration of ammonia was determined by titration. A 30 g sample of meal was mixed at low speed (approximately 2,000 rpm) in a 2 L Waring blender for 2 min with the selected alkanol-ammonia solution at a solvent-to-meal ratio of 6.7. The solutions were made up to 10% ammonia except for solvents for which the ammonia solubility was below 10010. ThUS, for isopropanol, t-butanol, and t-butanol containing 5% H 20, saturated ammonia solutions with concentrations of 8.9, 6.4 and 7.8% respectively were used. After a quiescent period (without mixing) of 15 min the meal was vacuum filtered using Whatman No. 41 filter paper, rinsed 3 times with 50 mL of alkanol, and dried at 40°C in a vacuum oven. The experimental conditions were varied for methanol as follows: (1) ammonia concentration in methanol: 0, 2,4, 5, 6, 8, 10, 12, 14, 15 and 180/0 (w/w) (2) methanol/ammonia-to-seed ratio: 2.5, 5, 6.7, 10 and 15 (v/w) (3) quiescent period: 0, 15, 30 and 60 minutes.
The water concentration in the various alkanolammonia solutions was 0, 5, 10, and 15% (v/v). Crude protein (Nx6.25) content of the meal was determined using the standard AACC procedure (1976), and the total glucosinolate content was measured by the method of Wetter and Youngs (1976). Myrosinase for this assay was prepared from mustard seeds by the method of Jones (1979).
Results and discussion A significant decrease in the glucosinolates content of the meal was observed with increasing concentrations of ammonia in methanol (Figure 1). However, J. Inst. Can. Sci. Technol. Aliment. Vol. 18, No.4, 1985
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Fig. 3. Effect of methanol-to-meal ratio (v/w) on the glucosinolate content 10-01 and amount of extracted solids I . - . I (laboratory-prepared meal, 10070 NH 3 in methanol)
Fig. 4. Effect of water in methanol and in methanol containing 10070 ammonia on the glucosinolate removal from laboratoryprepared meal
the glucosinolate removal was about 4 times more effective for the laboratory-prepared meal than for commercial meal. After treatment of the meals with 100/0 NH 3 in methanol, the residual glucosinolates in the commercial and laboratory meals were 0.82 ± 0.14 and 0.20 ± 0.03 mg/g respectively. The commercial meal contained 3.5% of hexane-extractable lipid material. Residual lipids in the pores of the meal may hinder the diffusion of methanol-ammonia into the meal and thus interfere with the removal of the glucosinolates. To test this hypothesis a sample of commercial meal was re-extracted with hexane using a Soxhlet apparatus prior to treatment with 10% ammonia in methanol containing 5% water. The treatment removed 78% of the glucosinolates compared with a removal of 72% without the re-extraction with hexane. Statistical analysis of the results indicate that the effect was significant (t-test, P = 0.05). Since the treated defatted commercial meal still contains a higher level of glucosinolates than the laboratory-defatted meals, the lower effectiveness of the treatment of commercial meal may also be influenced by the visually larger particle size of the meal and by the possible alteration of the protein structure by heat during processing. Heat treatment may result in an increase in the number of functional groups available for reaction on the protein surface to bind the glucosinolates. The crude protein content of treated meals was not greatly influenced by the concentration of ammonia in methanol. The average protein content of treated commercial meal was 41.2 ± 0.4% and that of treated laboratory-prepared meals 48.8 ± 0.3%, on a dry basis. The 12% increase in crude protein content of
the treated laboratory-prepared meal was due to the dissolution of up to 16% of the meal solids containing carbohydrates and other constituents. The increase of the crude protein content of treated commercial meals was quite small, and the reason for this is not obvious. The effect of the quiescent period on the glucosinolates in the laboratory-prepared meal is shown in Figure 2. During the initial two minutes of blending with 10% ammonia in methanol, 85% of the glucosinolates originally present in the laboratoryprepared meal were removed. An additional 15 min of quiescent contact reduced the glucosinolate content by 92%, and 60 min by 95%. After a quiescent period of 15 min the glucosinolates content of the meal was close to the detection limit of the method of Wetter and Youngs (1976). Therefore, the difference in the glucosinolate content after 15, 30 and 60 min can be assumed to be negligible. A slight increase in extracted solids with a longer quiescent period was observed. The effect of the ratio of the volume of 10% ammo~ nia in methanol to meal weight (R) on the process is shown in Figure 3. The increase of R from 2.5 to 15 decreased the residual glucosinolates to trace levels. A significant increase in extracted solids from 13.2% for an R of2.5 to 17.5% for an R of 15 was observed. The removal of glucosinolates from laboratoryprepared and commercial meal by methanol, ethanol, isopropanol, and t-butanol is presented in Table 1. Only methanol is effective in reducing the glucosinolates content of the meals in the absence of ammonia. While the methanol removed 54.5% and 38% of glucosinolates from laboratory-prepared and commer-
Can. [nst. Food Sci. Technol. J. Vol. 18. No.4, 1985
Diosady et al. / 313
Table I. Effect of extraction with alkanols on the glucosinolate and crude protein content of meals!.
Alkanol
Water content of solvent (070)
Laboratory-prepared meal untreated
o
Methanol
5 10 IS
Crude protein (N x 6.25) (070)
Water Content (%; v/v)
I
2.55 ± 0.15
43.7 ± 0.3
5 25
1.11 ± 0.12 0.61 ± 0.07
1.16 1.11 1.04 0.92
± ± ± ±
47.6 47.6 48.5 48.8
0.10 0.12 0.07 0.05
± 0.4 ± 0.6 ± 1.0 ± 0.3
o
2.54 ± 0.21
43.8 ± 0.3
Isopropanol
o o
2.53 ± 0.17
43.1 ± 0.4
2.52 ± 0.27
43.8 ± 0.5
Commercial meal untreated
Multiple extraction of laboratory-prepared meal with methanol-water 1
Glucosinolates (mg/g)
Ethanol t-butanol
Table 2.
2.86 ± 0.20
39.7 ± 0.8
Methanol
0
1.78 ± 0.25
41.7 ± 0.1
Ethanol
0
2.71 ± 0.20
40.6 ± 0.1
Isopropanol
0
3.00 ± 0.25
40.9 ± 0.5
t-butanol
0
2.79 ± 0.20
41.1 ± 0.2
1The meals were blended with alkanol or alkanol-water solution for 2 minutes at R = 6.7, and fIltered after a quiescent period of IS minutes.
cial meal, ethanol, isopropanol, and t-butanol had no effect. Van Megen (1983) reported that 30 min of leaching of toasted rapeseed meal with 75lTfo ethanol or 80lTfo methanol removed 78lTfo and 88lTfo of the glucosinolates originally present respectively. The removal of glucosinolates from untoasted, mildly treated meal by 75lTfo ethanol was only 62lTfo. As shown in Figure 4, the leaching of laboratory-prepared meal with methanol containing 35lTfo water resulted in the removal of 79lTfo of the glucosinolates, producing a meal still containing 0.54 mg/g glucosinolates. The quantities of extracted solids increased from 13.9lTfo to 19.0lTfo as the water content in methanol was increased to 35lTfo. These quantities are lower than the solid losses reported by Van Megen (1983) using 80lTfo aqueous methanol.
Glucosinolate content (mg/g) Extraction steps 2 0.96 ± 0.10 0.40 ± 0.03
4
0.54 ± 0.06 0.12 ± 0.04
I At each step the meal was blended for 2 minutes with methanolwater at R = 6.7, and filtered after a quiescent period of IS minutes.
The glucosinolate removal by methanol-water solutions could be improved by multiple extraction of the meal. The results given in Table 2 indicate that about 95lTfo of the glucosinolates can be removed from laboratory-prepared meal after leaching 4 times with methanol containing 25lTfo of water. The loss of protein material was not measured. Table 3 shows the effect of treatment of laboratoryprepared and commercial meals with aliphatic alkanols containing ammonia and water (0-15lTfo) on the level of glucosinolates and crude protein. In all cases where the solubility was high enough, solutions containing lOlTfo ammonia by weight were used. Isopropanol, tbutanol and t-butanol containing 5lTfo water were saturated with ammonia, and used at 8.9, 6.4, and 7.8lTfo ammonia concentration respectively. Methanolammonia solutions were the most effective in reducing the glucosinolate content of the meals. The treatment of laboratory-prepared meal with methanolammonia reduced the glucosinolate content to trace levels. Thus the effect of the presence of water in the methanol-ammonia on the glucosinolates level was not readily measurable with the analytical technique used. Treatment with methanol-ammonia was less effective in the case of commercial meal. While lOlTfo ammonia in methanol removed 59lTfo of the glucosinolates originally present, the methanol-ammonia solution containing 15lTfo H 2 0 removed 86lTfo (Table 3).
Table 3. Effect of water in alkanol-ammonia solutions on glucosinolates and crude protein of meals. Alkanol
Water Content in alkanol (%)
untreated Methanol
o 5 10 IS
Ethanol
Isopropanol
t-Butanol
o 5 10 IS 01 5 10 IS 02 53 10 IS
Glucosinolates (mg/g)
Crude protein (N x 6.25)
laboratory
commercial
laboratory
2.55 ± 0.15
2.86 ± 0.20
43.7 ± 0.3
39.7 ± 0.8
commercial
0.20 0.18 0.11 0.08
± 0.03 ± 0.03 ± 0.01 ± 0.01
1.17 1.12 0.52 0.42
± 0.10 ± 0.14 ± 0.08 ± 0.05
48.8 49.4 49.2 49.8
± 0.6 ± 0.6 ± 1.2
0.6
41.0 ± 0.1 41.2 ± 0.2 40.9 ± 0.4 39.5 ± 0.1
2.36 1.34 0.92 0.77 2.70 2.72 2.10 1.34
± 0.25
2.60 2.19 1.55 0.74 3.13 2.89 2.24 1.57
± 0.20
45.4 47.0 47.2 48.5 44.0 45.2 45.8 46.6
0.7 0.3 0.3 0.6 0.9 0.6 1.0 1.2
40.5 40.3 40.6 40.3 40.2 40.2 40.4 40.7
± 0.14 ± 0.08 ± 0.05 ± 0.17 ± 0.16 ± 0.16 ± 0.15
2.79 2.50 2.16 1.50
± 0.13
± ± ± ± ±
0.05 0.04 0.23 0.22 0.10 ± 0.14
± 0.20 ± 0.20 ± 0.21 ± 0.15
± ± ± ± ± ± ±
± ±
41.0 40.6 39.3 41.0
± 0.2 ± 0.3 ± 0.2 ± 0.2 ± 0.3 ± 0.4 ± 0.4 ± 0.5 ± 0.1 ± 0.3
± 0.1 ± 0.1
18.9% NH 3 26.4% NH 3 37.8% NH3
314 / Diosady et al.
J. InSI. Can. Sci. Technol. Aliment. Vol. 18, No.4, 1985
Table 4. Myrosinase activity of the laboratory-prepared meal treated with methanol or methanol-ammonia solution. Glucosinolate content (mg/g) determined: Solvent with addition of without addition myrosinase of myrosinase untreated methanol methanol containing 5010 ammonia methanol containing 100f0 ammonia
2.55 ± 0.15 1.16 ± 0.10
2.24 ± 0.10 0.05 ± 0.02
0.40 ± 0.02
0.09 ± 0.02
0.20 ± 0.03
0.08 ± 0.03
The glucosinolate content of laboratory-prepared and commercial meals was not significantly affected by the treatment with 10% ammonia in ethanol, 8.9070 ammonia in isopropanol, and 6.4% ammonia in tbutanol (Table 3). An increase in the water content of ammonia solutions in ethanol, isopropanol and tbutanol resulted in lower glucosinolate levels. Similar trends were found with both laboratory-prepared and commercial meals. Ethanol- and isopropanolammonia solutions containing 15% water removed 74% and 48% of the glucosinolates respectively. These results were similar for laboratory-prepared and commercial meals (Table 3), indicating that the efficiency of glucosinolate removal decreases with the increasing number of carbon atoms in the aliphatic alkanols. Apparently, removal increases with increased polarity. The presence of ammonia and water in alkanols increases the effectiveness of the glucosinolate removal (Table 3 and Figure 4). This is not only due to better dissolution of glucosinolates from the meal, as the extraction of laboratory-prepared meal with methanol containing 5% water reduces the glucosinolate content to 1.11 mg/g. However, in the presence of 10% ammonia it drops to 0.18 mg/g. Presumably the more insoluble glucosinolates are first converted to other products which are then dissolved in the polar phase. The treatment of meals with alkanol-ammonia increased the crude protein concentration in the meal as a direct result of the dissolution of non-protein components (Table 3). Other studies showed that about 5% of the crude protein was dissolved in the methanol phase containing 10% ammonia, mainly non-protein nitrogen compounds soluble in a 10% trichloroacetic acid solution. The effect of the treatment of laboratory-prepared meal with methanol or methanol-ammonia solution on the myrosinase activity in the meal was studied. The myrosinase activity was evaluated, somewhat
Can. [nSf. Food Sci. Technol. J. Vol. 18, No.4. 1985
indirectly, using the Wetter and Youngs (1976) procedure, both with and without myrosinase addition. The results given in Table 4 indicate that treatment with methanol or methanol-ammonia solutions reduces the myrosinase activity to trace level.
Conclusions The treatment of rapeseed meal with alkanolammonia solutions effectively removed most of the glucosinolates. The effectiveness of this process depends upon the method of preparation of the meal, the type of alkanol used, and the presence of water in the alkanol-ammonia solution. A methanolammonia solution was found to be the most effective solvent for removal of glucosinolates from rapeseed meal. Treatment of laboratory-prepared meal with 10% ammonia in methanol containing 5% water removed glucosinolates to trace levels, while extraction with ammonia-free methanol containing 5% water removed only 56% of the glucosinolates, These results indicate that the presence of ammonia significantly increases the effectiveness of the detoxification process. The treatment of laboratory-prepared meal with methanol or methanol-ammonia solution reduces the myrosinase activity to trace level.
References AACC. 1976. Approved Methods of the American Association of Cereal Chemists. St. Paul, MN. Jones, J.D. 1979. Private communication. Jones, J.D. 1979. Rapeseed protein concentrate preparation and evaluation. JAOCS 56:716. Van Megen, W.H. 1983. Removal of glucosinolates from defatted rapeseed meal by extraction with aqueous ethanol. Can. Inst. Food Sci. Technol. J. 16:93. Schlingmann, M. and Vertesy, L. 1978a. Single cell proteins with reduced content of nucleic acid and fat, Fette, Seifen, Anstrichsmittel. 80(7):283. Schlingmann, M. and Vertesy, L. 1978b. Reducing the lipid and nucleic acid content in microbial cell masses. German Patent 2,633,666. Schlingmann, M. and von Rymon Lipinski, G.W. 1980. Process for treating meals and flours of oilseeds. U.K. Patent 2,030,441. Schlingmann, M. and von Rymon Lipinski, G.W. 1982. Process for improving the properties of meals and flours of oilseeds. Can. Patent 1,120,779. Wetter, L.R. and Youngs, C. 1976. A thiourea-UV assay for total glucosinolates in rapeseed meals. JAOCS 53: 162.
Accepted April 29, 1985
Diosady et al. / 315