Article pubs.acs.org/JAFC
Effect of Microwave Treatment on the Efficacy of Expeller Pressing of Brassica napus Rapeseed and Brassica juncea Mustard Seeds Yanxing Niu,†,‡ Anna Rogiewicz,‡ Chuyun Wan,† Mian Guo,† Fenghong Huang,† and Bogdan A. Slominski*,‡ †
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, Hubei Province 430062, People’s Republic of China ‡ Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 ABSTRACT: A study was conducted to evaluate the effect of microwave heating on the efficacy of expeller pressing of rapeseed and mustard seed and the composition of expeller meals in two types of Brassica napus rapeseed (intermediate- and lowglucosinolate) and in Brassica juncea mustard (high-glucosinolate). Following microwave treatment, the microstructure of rapeseed using transmission electron microscopy showed a significant disappearance of oil bodies and myrosin cells. After 6 min of microwave heating (400 g, 800 W), the oil content of rapeseed expeller meal decreased from 44.9 to 13.5% for intermediateglucosinolate B. napus rapeseed, from 42.6 to 11.3% for low-glucosinolate B. napus rapeseed, and from 44.4 to 14.1% for B. juncea mustard. The latter values were much lower than the oil contents of the corresponding expeller meals derived from the unheated seeds (i.e., 26.6, 22.6, and 29.8%, respectively). Neutral detergent fiber (NDF) contents showed no differences except for the expeller meal from the intermediate-glucosinolate B. napus rapeseed, which increased from 22.7 to 29.2% after 6 min of microwave heating. Microwave treatment for 4 and 5 min effectively inactivated myrosinase enzyme of intermediate-glucosinolate B. napus rapeseed and B. juncea mustard seed, respectively. In low-glucosinolate B. napus rapeseed the enzyme appeared to be more heat stable, with some activity being present after 6 min of microwave heating. Myrosinase enzyme inactivation had a profound effect on the glucosinolate content of expeller meals and prevented their hydrolysis to toxic breakdown products during the expelling process. It appeared evident from this study that microwave heating for 6 min was an effective method of producing expeller meal without toxic glucosinolate breakdown products while at the same time facilitating high yield of oil during the expelling process. KEYWORDS: microwave heating, expeller pressing, rapeseed, mustard, chemical composition
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INTRODUCTION Rapeseed is a good source of edible oil and meal as well as a good source of oil for biodiesel production. It ranks second among oilseed crops and is widely grown in China, Canada, Europe, and India. New technologies that could improve the quality of rapeseed oil and meal such as cold pressing,1 microwave heating,2 or aqueous enzymatic extraction3 have been proposed. Microwave heating is a dielectric treatment, which is fundamentally different from the conventional cooking in the prepress solvent extraction process.4 It has been reported that following microwave treatment rapeseed oil contains more phytosterols and tocopherols, and hence its oxidative stability could be improved.5 The flavor of rapeseed oil could also be significantly improved by microwave treatment. According to Zhou et al.6 microwave heating of rapeseed for 6 min before oil extraction by pressing gave a pleasant roasted flavor to the oil when compared to the crude oil. Microwave treatment can also significantly decrease the seed lipase activity.7 The effects of microwave treatment on rapeseed cake and meal quality parameters have been investigated. Sadeghi et al.8 evaluated the effects of microwave treatment on ruminal degradability of canola (low-glucosinolate rapeseed) meal protein and concluded that 4 min of microwave heating of canola meal effectively protected proteins from their degradation in the rumen. Ebrahimi et al.9 reported that phytic acid and © 2015 American Chemical Society
glucosinolate contents of canola seed linearly decreased with increased time of microwave heating. The conventional heat treatment of the seed in the prepress solvent extraction process involves cooking or conditioning throughout the steam-heated drum or stack-type cookers. This promotes coalescing of oil droplets, reduces oil viscosity, and denatures (inactivates) myrosinase enzymes.10 Till now, the effect of microwave treatment on the efficacy of oil extraction by expeller-pressing has not been adequately studied. The objective of this study was to investigate the effect of the duration of microwave heating on the efficacy of oil extraction and the chemical composition of expeller meals from rapeseed and mustard seeds.
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MATERIALS AND METHODS
Plant Materials. Intermediate-glucosinolate (∼60 μmol/g, fat-free basis) Brassica napus rapeseed var. Mianyou 309 (moisture content = 10.8%) was provided by the Mianyang Academy of Agricultural Sciences, Szechwan Province, People’s Republic of China. Lowglucosinolate (∼20 μmol/g, fat-free basis) B. napus rapeseed var. Zhongshuang 11 (moisture content = 11.0%) was provided by the Oil Received: Revised: Accepted: Published: 3078
October 8, 2014 March 12, 2015 March 13, 2015 March 13, 2015 DOI: 10.1021/jf504872x J. Agric. Food Chem. 2015, 63, 3078−3084
Article
Journal of Agricultural and Food Chemistry
Figure 1. Transmission electron microscopy of two types of Brassica napus rapeseed and Brassica juncea mustard without or with microwave treatment: (A) B. napus rapeseed (intermediate-glucosinolate; as-is); (B) B. napus rapeseed (intermediate-glucosinolate) microwaved for 5 min; (C) B. napus rapeseed (low-glucosinolate; as-is); (D) B. napus rapeseed (low-glucosinolate) microwaved for 5 min; (E) B. juncea mustard (highglucosinolate; as-is); (F) B. juncea mustard (high glucosinolate) microwaved for 5 min. CW, cell wall; PB, protein bodies; MG, myrosin cells; OB, oil bodies. Magnification: ×4000. temperature of no more than 90 °C (KOMET CA59G, IBG Monforts Oekotec GmbH & Co. KG, Germany). Microscopy Analysis. The seed samples were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) for 4 h, washed three times with phosphate buffer, postfixed with 1% of OsO4 in phosphate buffer (pH 7.0) for 1 h, and washed three times in the phosphate buffer. The samples were then dehydrated in the progressive ethanol solutions of 50, 70, 80, 90, 95, and 100% for about 15−20 min at each solution, embedded in acetone and Spurr resin. Sections 70−90 nm in size were cut from the embedded tissue using a microtome (Ultracut E, Reichert-Jung, Vienna, Austria), double stained with uranyl acetate and lead citrate, and then were examined under a transmission electron microscope (H-7650, Hitachi Instrument, Tokyo, Japan).
Crops Research Institute, Chinese Academy of Agricultural Sciences, Hubei Province, People’s Republic of China. Brassica juncea mustard (high-glucosinolate; ∼130 μmol/g, fat-free basis; moisture content = 11.0%) was provided by the Institute of Economic Crops, Xinjiang Academy of Agricultural Sciences, Xinjiang Province, People’s Republic of China. All seeds were produced in 2012. Sample Preparation and Treatments. The seed samples (400 g each) were subjected to microwave irradiation (MarsX, CEM, Matthews, NC, USA) at a frequency of 2450 MHz at a medium power of 800 W for 1, 2, 3, 4, 5, and 6 min. All microwave-treated samples were allowed to cool to room temperature for 60 min. The samples without or with microwave heating were expeller-pressed using an oil expeller at a feeding rate of 3 kg h−1 and barrel 3079
DOI: 10.1021/jf504872x J. Agric. Food Chem. 2015, 63, 3078−3084
Article
Journal of Agricultural and Food Chemistry Chemical Analyses. The samples were ground and were adjusted to the same moisture content by exposure to room humidity for 7 days at 25 °C.11 The samples were then defatted with hexane for 6 h and air-dried. Oil content and neutral detergent fiber (NDF) were analyzed according to AOAC (1990) method 920.39 and Van Soest et al.,12 respectively. Glucosinolate analysis was performed as described by Slominski and Campbell.13 Briefly, 100 mg of high-glucosinolate or 300 mg of lowglucosinolate samples was weighed into 15 mL centrifuge tubes. Two milliliters of methanol, 1.0 mL of benzyl glucosinolate (internal standards, 0.5 mM), and 0.1 mL of lead−barium acetate were added to the tubes, extracted for 3 h at room temperature, and then centrifuged. Two milliliters of supernatant was transferred to a DEAE-Sephadex column, which was washed successively with 1 mL each of 67% methanol, water, and pyridine acetate. Purified sulfatase solution was then added to the column, and the contents were incubated at room temperature overnight. The resulting desulfated glucosinolates were eluted with 2 mL of 60% methanol and evaporated under nitrogen. The dry residue was trimethylsilylated by adding 0.2 mL of a mixture of anhydrous acetone/N,O-bis(trimethylsilyl)acetamide (BSA)/trimethylchlorosilane (TMCS)/1-methylimidazole (2:1:0.1:0.05 v/v) and incubated for 30 min at room temperature. The trimethylsilyl derivatives of desulfoglucosinolates were separated by gas−liquid chromatography using a glass column (1.2 m × 2 mm i.d.) packed with 2% OV-7 on Chromosob W (HP) (100−120 mesh) with helium gas at a flow rate of 40 mL/min. The oven temperature was kept at 200 °C for 4 min and then increased at 5 °C min−1 to 275 °C. Injection port and detector temperatures were 280 and 300 °C, respectively. Myrosinase activity was determined by difference between total sample glucosinolate content and the glucosinolates remaining following incubation of the sample with distilled water (autolysis) at 40 °C for a defined period of time. Glucosinolate analysis was conducted as described above. One unit of myrosinase activity was defined as the amount of enzyme that catalyzes the hydrolysis of 1 μmol of glucosinolate per 1 min. The analyses of moisture content, oil content, NDF, and glucosinolate content were run in triplicate, wheereas those for myrosinase activity were run in duplicate. Statistical Analysis. All of the statistical analysis was conducted by the SAS program (version 9.1, SAS Institute Inc., Cary, NC, USA). Means were separated by Tukey’s honestly significant difference. All statements of significance were based on P ≤ 0.05.
amine, 14% of phosphatidylinositol, and 20% of phosphatidylserine. According to Valentov et al.,15 microwave treatment could improve the content of phospholipids in the crude oil by more than twice, which would be a consequence of the membrane rupture. A strong relationship between oil bodies’ stability and oil extractability has been reported,16 indicating that the oil from the microwave-treated rapeseed should be easier to extract. Myrosin cells are the subcellular compartments associated with enzyme myrosinase. Normally, glucosinolates are located in vacuoles and are separated from the myrosin cells.17 The presence of the glucosinolate−myrosinase system is characteristic of the Brassicaceae family, and it serves as a defense system against biotic and abiotic stressors.18 Disruption of the tissue induced by mechanical damage, infection, or pest attack brings glucosinolates into contact with myrosinase,19 resulting in their hydrolysis to biologically active and often toxic breakdown products, including isothiocyanates, goitrin, and nitriles. After microwave treatment, it appeared evident that the structure of myrosin cells was effectively damaged, indicating the potential for myrosinase to come into contact with glucosinolates. In Figure 1 images A, C, and E, the cells of rapeseed were well coherent, and the shape was smooth, whereas in the microwave-treated samples (i.e., images B, D, and F) the cell walls became folded. This phenomenon may be ascribed to the water loss of the cells, as documented by Niu et al.20 As illustrated in Figure 2, there was a significant decline in moisture content of B. napus rapeseed and B. juncea mustard.
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RESULTS AND DISCUSSION Effect of Microwave Treatment on Microstructure of the Seed. The effect of microwave heating (400 g, 800 W) on the microstructure of the seed samples is shown in Figure 1. The microstructure of the intact (unheated) seed revealed welldefined pattern of protein bodies and intact myrosin cells amid oil bodies with smooth cell walls. In high-glucosinolate B. juncea (E) and intermediate-glucosinolate B. napus (A) myrosin cells were quite visible, whereas there were almost no visible myrosin cells in the seed of low-glucosinolate rapeseed (C). The microstructure of all samples changed significantly after 5 min of microwave heating (see images F, B, and D) with the oil bodies and myrosin cells almost disappearing. The microscopy images clearly demonstrate that microwave treatment disrupts the membranes of oil bodies as well as other intracellular organelles. Most of the biological membranes of rapeseed are composed of phospholipids and proteins (i.e., oleosins) with the oil bodies covered by a single-layer membrane. Eighty percent of this layer is filled with phospholipids, and the other 20% is occupied by oleosins. Phospholipids constitute a polar headgroup and a nonpolar fatty acid region.14 It has been reported that the composition of phospholipids in the oil bodies would account for 60% of phosphatidylcholine, 6% of phosphatidylethanol-
Figure 2. Effect of time of microwave heating on mean moisture content of B. napus rapeseed and B. juncea mustard.
Effect of Microwave Treatment on Oil Content of Expeller Meal. The effect of time of microwave heating on the oil content of seed and expeller meal of two types of B. napus rapeseed and B. juncea mustard is shown in Table 1. The oil contents of B. napus rapeseed (intermediate- and lowglucosinolate) and B. juncea mustard were similar. After expeller-pressing, however, the oil content of expeller meal decreased significantly with increased time of microwave heating. After 6 min of microwave heating, the oil content of intermediate- and low-glucosinolate B. napus rapeseed and of B. juncea mustard expeller meal decreased from 26.6, 22.6, and 29.8 to 13.5, 11.3, and 14.1%, respectively. Overall, the microwave treatment decreased the oil content of expeller meal by 49−53% with an oil extraction rate of 79−83%. The oil contents of expeller meals are in agreement with the changes in microstructure of microwave-treated seed showing 3080
DOI: 10.1021/jf504872x J. Agric. Food Chem. 2015, 63, 3078−3084
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Table 1. Effect of Time of Microwave Heating on Oil Content of Seed and Expeller Meal of Two Types of Brassica napus Rapeseed and Brassica juncea Mustard (Percent, As-Is Basis) time of microwave heating sample
untreated
B. napus rapeseed (intermediate-glucosinolate) seed 44.9 ± 0.3aa expeller meal 26.6 ± 0.8a B. napus rapeseed (low-glucosinolate) seed 42.6 ± 0.3a expeller meal 22.6 ± 0.5a B. juncea mustard (high-glucosinolate) seed 44.4 ± 0.4a expeller meal 29.8 ± 0.4a a
1 min
2 min
3 min
4 min
5 min
6 min
45.0 ± 0.4a 23.6 ± 0.9b
44.2 ± 0.5a 20.6 ± 0.2c
44.4 ± 0.7a 18.0 ± 0.3d
44.9 ± 0.9a 16.7 ± 1.0e
44.0 ± 0.2a 15.3 ± 0.6f
43.7 ± 0.6a 13.5 ± 0.3g
42.0 ± 0.8a 22.9 ± 0.3a
42.5 ± 0.1a 19.4 ± 1.0b
41.3 ± 0.2a 17.4 ± 0.7c
41.5 ± 0.2a 15.2 ± 0.9d
42.5 ± 0.6a 14.2 ± 0.5d
42.4 ± 0.4a 11.3 ± 0.9e
44.1 ± 0.2a 27.3 ± 0.5b
44.1 ± 0.2a 22.4 ± 0.1c
44.0 ± 0.2a 18.8 ± 0.3d
43.4 ± 0.1a 15.9 ± 0.7e
43.2 ± 0.2a 14.4 ± 0.6f
43.1 ± 0.2a 14.1 ± 0.3g
Mean ± SD. Values within rows with the same letter (a−g) are not significantly different (P ≤ 0.05).
Table 2. Effect of Time of Microwave Heating on Neutral Detergent Fiber (NDF) Content of Seed and Expeller Meal of Two Types of Brassica napus Rapeseed and Brassica juncea Mustard (Percent, As-Is, Fat-Free Basis) time of microwave heating sample
untreated
B. napus rapeseed (intermediate-glucosinolate) seed 23.7 ± 0.1aa expeller meal 22.7 ± 0.4b B. napus rapeseed (low-glucosinolate) seed 22.0 ± 0.5a expeller meal 23.6 ± 0.3a B. juncea mustard (high-glucosinolate) seed 17.5 ± 1.3a expeller meal 17.4 ± 1.0a a
1 min
2 min
3 min
4 min
5 min
6 min
23.8 ± 0.5a 24.1 ± 0.7b
24.1 ± 0.4a 23.7 ± 0.1b
23.8 ± 0.7a 23.1 ± 0.4b
24.3 ± 0.5a 23.2 ± 0.2b
23.9 ± 0.5a 24.7 ± 0.2b
24.6 ± 0.7a 29.2 ± 2.2a
22.4 ± 1.0a 24.8 ± 0.4a
23.0 ± 0.2a 23.0 ± 0.1a
22.4 ± 0.4a 24.1 ± 1.7a
22.4 ± 0.4a 25.1 ± 1.7a
22.2 ± 0.9a 25.9 ± 1.3a
22.1 ± 0.7a 25.6 ± 1.4a
18.1 ± 0.5a 16.8 ± 0.2a
17.5 ± 1.3a 18.9 ± 2.2a
18.6 ± 1.5a 17.3 ± 1.3a
18.3 ± 0.5a 16.8 ± 0.3a
18.0 ± 0.8a 18.8 ± 2.8a
19.6 ± 1.0a 17.6 ± 0.8a
Mean ± SD. Values within rows with the same letter (a, b) are not significantly different (P ≤ 0.05).
Table 3. Effect of Time of Microwave Heating on Myrosinase Activity of Seed of Two Types of Brassica napus Rapeseed and Brassica juncea Mustard (Units per Minute, As-Is, Fat-Free Basis) time of microwave heating
a
sample
untreated
1 min
2 min
3 min
4 min
5 min
6 min
B. napus rapeseed (intermediate-glucosinolate) B. napus rapeseed (low-glucosinolate) B. juncea mustard (high-glucosinolate)
32.7 ± 0.9aa 11.6 ± 0.4b 49.2 ± 2.5a
27.6 ± 1.5b 14.9 ± 0.1a 26.2 ± 4.2b
22.8 ± 1.5c 13.6 ± 0.2a 26.9 ± 1.7b
16.8 ± 0.6d 8.0 ± 0.5c 11.2 ± 1.7c
5.6 ± 0.6e 5.5 ± 0.4d nd
3.6 ± 0.8f 5.2 ± 0.2d nd
ndb 4.2 ± 0.4d nd
Mean ± SD. Values within rows with the same letter (a−f) are not significantly different (P ≤ 0.05). bNot detected.
The contents of NDF in the seed and expeller meal of two types of B. napus rapeseed and B. juncea mustard were different, with B. juncea showing significantly less fiber than B. napus. This is due to the fact that B. juncea mustard is yellow-seeded, whereas B. napus rapeseed is black-seeded. The yellow seed coat color is a visual marker of lower polyphenol (i.e., proanthocyanidin) content and a thinner seed hull. As a result, black-seeded rapeseed contains more fiber than its yellowseeded B. napus counterparts or B. juncea mustard.23 After microwave heating for 6 min, the NDF contents of all seed samples were similar, so there was no significant effect of heat treatment on fiber content. In the expeller meals, however, the NDF content of intermediate-glucosinolate B. napus rapeseed increased from 24.1 to 29.2% after 6 min of microwave heating, whereas in low-glucosinolate B. napus rapeseed and B. juncea mustard no significant changes were observed. It is well-known that heat treatment of oilseeds during processing may lead to losses of digestible amino acids due to the formation of Maillard reaction products. This highly indigestible fraction, often referred to as a neutral detergent insoluble crude protein
the broken membranes of oil bodies, which, in turn, facilitated higher efficacy of oil extraction during expeller-pressing. The oil extraction rate by expeller-pressing after 6 min of microwave heating used in the current study was similar to the conditioning of the seed at 80 °C for 30 min, flaking of the seed, and then expeller-pressing.21 In another study, similar results were achieved when the seed was subjected to 40 min of heat treatment with the temperature increased to 80 °C and then heat treatment at 80 °C for 30 min followed by 40 min of flaking to 0.3 mm thickness and expeller pressing under a barrel temperature of 105−110 °C.1 According to Grageola et al.22 expeller-pressing recovers around 75% of oil from canola seed. This indicates that the microwave treatment could be an effective method of increasing the efficacy of oil extraction by expeller pressing and would facilitate oil extraction down to the industry standards of 8−10% of residual oil. which, in turn, would prevent the need for double pressing. Effect of Microwave Treatment on NDF Content of Expeller Meal. The effect of time of microwave heating on NDF content of seed and expeller meal is shown in Table 2. 3081
DOI: 10.1021/jf504872x J. Agric. Food Chem. 2015, 63, 3078−3084
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Table 4. Effect of Time of Microwave Heating on Total Glucosinolate Content of Seed and Expeller Meal of Two Types of Brassica napus Rapeseed and Brassica juncea Mustard (Micromoles per Gram, As-Is, Fat-Free Basis) time of microwave heating sample
untreated
B. napus rapeseed (intermediate-glucosinolate) seed 58.4 ± 6.2aa,b expeller meal 12.2 ± 0.1ef B. napus rapeseed (low-glucosinolate) seed 18.3 ± 2.7abb expeller meal 7.7 ± 0.1e B. juncea mustard (high-glucosinolate) seed 134.9 ± 2.1ac expeller meal 20.8 ± 1.5e
1 min
2 min
3 min
4 min
5 min
6 min
53.9 ± 3.0ab 11.6 ± 0.5f
54.4 ± 1.7ab 16.4 ± 2.7e
54.4 ± 3.1ab 28.9 ± 2.1d
51.2 ± 3.7abc 44.9 ± 2.9b
46.8 ± 2.8bc 53.5 ± 3.7a
43.0 ± 3.4c 38.7 ± 2.0c
18.3 ± 1.4ab 8.0 ± 0.3de
20.8 ± 2.3a 9.3 ± 0.3cd
17.4 ± 0.9ab 11.2 ± 0.3ab
15.1 ± 0.5bc 11.9 ± 0.6a
13.9 ± 1.0bc 10.2 ± 1.5bc
11.6 ± 1.7c 7.7 ± 0.1e
137.3 ± 17.6a 29.8 ± 3.0e
135.7 ± 1.9a 67.1 ± 5.4d
146.4 ± 16.2a 106.5 ± 9.1c
135.4 ± 4.8a 150.5 ± 9.9a
131.0 ± 3.9a 134.4 ± 2.9b
135.9 ± 5.1a 132.1 ± 9.5b
Mean ± SD. Values within rows with the same letter (a−f) are not significantly different (P ≤ 0.05). bIncludes gluconapin (3-butenyl), glucobrassicanapin (4-pentenyl), progoitrin (2-hydroxy-3-butenyl), gluconapoleiferin (2-hydroxy-4-pentenyl), glucobrassicin (3-indolylmethyl), and 4-hydroxyglucobrassicin (4-hydroxy-3-indolylmethyl). cIncludes sinigrin (2-propenyl), gluconapin (3-butenyl), progoitrin (2-hydroxy-3-butenyl), gluconapoleiferin (2-hydroxy-4-pentenyl), and 4-hydroxyglucobrassicin (4-hydroxy-3-indolylmethyl). a
(NDICP), would contribute to the increase in the NDF content of heat-treated products and would significantly affect the nutritive value of rapeseed/canola meal.24 In the current study, expeller pressing of intermediate-glucosinolate B. napus rapeseed microwave heated for 6 min resulted in some protein damage as documented by the increase in NDF content which, in turn, could lead to reduced protein and amino acid digestibilities. Therefore, extending the time of microwave treatment could further contribute to protein damage and the increase in NDICP content of expeller meal. Effect of Microwave Treatment on Myrosinase Activity of the Seed. As illustrated in Table 3, there was a significant decline in myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) activity with increased time of microwave heating for two types of B. napus and B. juncea seed. This is in agreement with a study by Verkerk and Dekker,25 who demonstrated that myrosinase in microwave-treated cabbage almost completely lost its hydrolytic capacity, despite the fact that the stability of myrosinase is strongly dependent on the water content,26 which was most likely much higher in cabbage than in the seed samples used in the current study (i.e., 10%). When compared with B. napus intermediate-glucosinolate rapeseed and B. juncea mustard, the myrosinase activity in B. napus low-glucosinolate rapeseed was relatively low, although the enzyme per se appeared to be more heat-stable with some activity still being present after 6 min of microwave treatment. Among other reasons, application of heat treatment in any crushing operation of rapeseed is essential for an effective inactivation of myrosinase enzyme. In the presence of moisture and following rupture of the seed, this enzyme would hydrolyze glucosinolates to unstable aglucones, which would then break down to yield a range of products, including isothiocyanates, goitrin, nitriles, and thiocyanates, that interfere with the function of the thyroid gland and would adversely affect growth performance of monogastric animals.24 Therefore, it is critical that myrosinase is effectively inactivated by heat treatment prior to seed expelling. Effect of Microwave Treatment on Glucosinolate Content of Seed and Expeller Meal. The effect of time of microwave heating on total glucosinolate content in seed and expeller meal is shown in Table 4. The total glucosinolate content of B. napus seed samples decreased after 4 min of microwave heating. It is of interest to note that in the seed of B. juncea mustard the total glucosinolate content did not change
during microwave heating. The glucosinolate content of the corresponding expeller meals was much lower and reflected the hydrolysis of glucosinolates by myrosinase during the expelling process. However, their contents increased with increased time of microwave heating and, thus, myrosinase inactivation and then decreased due to excessive time of microwave heating, which coincided with the decrease in glucosinolate content in the seed samples for 5−6 min. More specifically, the glucosinolate content of expeller meal of B. juncea mustard and intermediate-glucosinolate B. napus rapeseed increased significantly during microwave heating for 4 and 5 min, respectively, and then decreased. Somewhat similar changes, although not significant, were observed for low-glucosinolate B. napus rapeseed. According to their structure, glucosinolates can be divided into aliphatic glucosinolates, including sinigrin, gluconapin, glucobrassicanapin, and progoitrin, and indole glucosinolates, including glucobrassicin and 4-hydroxyglucobrassicin. In different Brassica species the compositions of glucosinolates are different. The major glucosinolates of B. napus rapeseed are gluconapin, progoitrin, and 4-hydroxyglucobrassicin, whereas sinigrin and gluconapin are the predominant glucosinolates of B. juncea mustard. It is well-known that heat treatment and myrosinase activity are the factors that affect the glucosinolate content during processing.27 It has also been shown that indole glucosinolates are more susceptible to thermal degradation than aliphatic glucosinolates. 27−30 As a result, the decrease in total glucosinolate content due to microwave heating observed in the current study was more pronounced for two types of B. napus rapeseed than for B. juncea mustard. The effect of time of microwave heating on the major glucosinolate contents in the seed and expeller meal samples of two types of B. napus rapeseed and B. juncea mustard are shown in Figures 3, 4, and 5. When compared to the seed samples, the aliphatic glucosinolates gluconapin and progoitrin contents of the corresponding expeller meal samples of intermediateglucosinolate B. napus rapeseed (Figure 3) increased significantly after 3 min, which was directly related to myrosinase inactivation and thus lack of glucosinolate autolysis. The decrease after 6 min of microwave treatment was a consequence of extended microwave heating. The indole glucosinolate hydroxyglucobrassicin content of expeller meal demonstrated a less clear pattern, probably due to its 3082
DOI: 10.1021/jf504872x J. Agric. Food Chem. 2015, 63, 3078−3084
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Journal of Agricultural and Food Chemistry
glucosinolate hydroxyglucobrassicin showing a significant thermal decomposition after 4, 5, and 6 min. Overall, the content of hydroxyglucobrassicin in the seed of intermediateand low-glucosinolate B. napus rapeseed decreased by 50 and 53%, respectively, after 6 min of microwave heating. As aliphatic glucosinolates are less sensitive to heat treatment, sinigrin and gluconapin contents of B. juncea mustard seed showed no significant differences with increased time of microwave heating (Figure 5). However, their content after 4, 5, and 6 min of microwave treatment reached the level of that of the seed, which was a consequence of complete myrosinase inactivation. There was a strong negative relationship between myrosinase activity in the seed (Table 3) and the total glucosinalate content in the corresponding expeller meals (Table 4) with intermediate-glucosinolate B. napus rapeseed and B. juncea mustard showing R2 values of −0.92 and −0.92, respectively. In a study on the effect of microwave treatment on the glucosinolate content of canola seed, Ebrahimi et al.9 reported that the glucosinolate content after 2, 4, and 6 min of microwave heating decreased by 42, 55, and 59%, respectively, which was significantly higher than that observed in the current study. The main reason for the difference could be related to the processing method used. In their study, canola seed was dried at 55 °C for 48 h, after which time water was added to increase the moisture content to 25%. It is very probable that drying of the seed inactivated the myrosinase activity, whereas the heat produced during microwave treatment under high moisture conditions facilitated glucosinalate degradation. In conclusion, it would appear evident that microwave heating of 2 W kg−1 for 6 min could be an effective method of producing expeller meal without toxic glucosinolate breakdown products while at the same time increasing the yield of oil during the expelling process.
Figure 3. Effect of time of microwave heating on major glucosinolate of B. napus intermediate-glucosinolate rapeseed and expeller meal.
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AUTHOR INFORMATION
Corresponding Author
Figure 4. Effect of time of microwave heating on major glucosinolate of B. napus low-glucosinolate rapeseed and expeller meal.
*(B.A.S.) Phone: (204) 474-8291. Fax: (204) 474-7628. Email: [email protected]. Funding
Funding of this study was provided by the National Natural Science Foundation of China (Grant 31201461) and the Canola Council of Canada. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge Ruibao Zhou, He’nan University of Technology, and Junying Li, Zhejiang University, for their assistance in microstructure analysis and Zirong Guo, Mianyang Academy of Agricultural Sciences, Sichuan, for providing the samples.
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ABBREVIATIONS USED NDF, neutral detergent fiber; BSA, N,O-bis(trimethylsilyl)acetamide; TMCS, trimethylchlorosilane; NDICP, neutral detergent insoluble crude protein
Figure 5. Effect of time of microwave heating on major glucosinolate of B. juncea high-glucosinolate mustard seed and expeller meal.
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pronounced thermal decomposition in the seed during 5 or 6 min of microwave heating. The glucosinolate content of the seed and expeller meal samples of low-glucosinolate B. napus rapeseed (Figure 4) showed a similar pattern with the indole
REFERENCES
(1) Prior, E. M.; Vadke, V. S.; Sosulski, F. W. Effect of heat treatments on canola press oils. I. Non-triglyceride components. J. Am. Oil Chem. Soc. 1991, 68, 401−406.
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Journal of Agricultural and Food Chemistry (2) Azadmard-Damirchi, S.; Habibi-Nodeh, F.; Hesari, J.; Nemati, M.; Achachlouei, B. F. Effect of pretreatment with microwaves on oxidative stability and nutraceuticals content of oil from rapeseed. Food Chem. 2010, 121, 1211−1215. (3) Lanzani, A.; Petrini, M. C.; Cozzoli, O.; Gallavresi, P.; Carola, C.; Jacini, G. On the use of enzymes for vegetable-oil extraction. A preliminary report. Riv. Ital. Sostanze Grasse 1975, L11, 226−229. (4) Oberndorfer, C.; Pawelzik, E.; Lückea, W. Prospects for the application of dielectric heating processes in the pre-treatment of oilseeds. Eur. J. Lipid Sci. Technol. 2000, 102, 487−493. (5) Szydlowska-Czerniak, A.; Trokowski, K.; Karlovits, G.; Szlyk, E. Determination of antioxidant capacity, phenolic acids, and fatty acid composition of rapeseed varieties. J. Agric. Food Chem. 2010, 58, 7502−7509. (6) Zhou, Q.; Yang, M.; Huang, F.; Zheng, C.; Deng, Q. Effect of pretreatment with dehulling and microwaving on the flavor characteristics of cold-pressed rapeseed oil by GC-MS-PCA and electronic nose discrimination. J. Food Sci. 2013, 78, C961−C970. (7) Ponne, C. T.; Moller, A. C.; Tijskens, M. M.; Bartels, P. V.; Meijer, M. M. T. Influence of microwave and steam heating on lipase activity and microstucture of rapeseed (Barassica napus). J. Agric. Food Chem. 1996, 44, 2818−2824. (8) Sadeghi, A. A.; Shawrang, P. Effects of microwave irradiation on ruminal degradability and in vitro digestibility of canola meal. Anim. Feed Sci. Technol. 2006, 127, 45−54. (9) Ebrahimi, S. R.; Nikkhah, A.; Sadeghi, A. A. Changes in nutritive value and digestion kinetics of canola seed due to microwave irradiation. Asian−Aust. J. Anim. Sci. 2010, 23, 347−354. (10) Wiesenborn, D.; Doddapaneni, R.; Tostenson, K.; Kangas, N. Cooking indices to predict screw-press performance for crambe seed. J. Am. Oil Chem. Soc. 2001, 78, 467−471. (11) Tao, K. J. Factors causing variations in the conductivity test for soybean seeds. J. Seed Technol. 1978, 3, 10−18. (12) Van Soest, P. J.; Robertson, J. B.; Lewis, B. A. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 1991, 74, 3583−3597. (13) Slominski, B. A.; Campbell, L. D. Gas chromatographic determination of indole glucosinolates − a re-examination. J. Sci. Food Agric. 1987, 40, 131−143. (14) Deleu, M.; Vaca-Medina, G.; Fabre, J.-F.; Roı̈z, J.; Valentin, R.; Mouloungui, Z. Interfacial properties of oleosins and phospholipids from rapeseed for the stability of oil bodies in aqueous medium. Colloid Surf. B 2010, 80, 125−132. (15) Valentová, O.; Novotná, Z.; Svoboda, Z.; Schwarz, W.; Kás,̌ J. Microwave heating and γ-irradiation treatment of rapeseed (Brassica napus). J. Food Lipids 2000, 7, 237−245. (16) Jolivet, P.; Deruyffelaere, C.; Boulard, C.; Quinsac, A.; Savoire, R.; Nesi, N.; Chardot, T. Deciphering the structural organization of the oil bodies in the Brassica napus seed as a means to improve the oil extraction yield. Ind. Crops Prod. 2013, 44, 549−557. (17) Bones, A. M.; Rossiter, J. T. The myrosinase-glucosinolate system, its organization and biochemistry. Physiol. Plant. 1996, 97, 194−208. (18) Ahuja, I.; Borgen, B. H.; Hansen, M.; Honne, B. I.; Muller, C.; Rohloff, J.; Rossiter, J. T.; Bones, A. M. Oilseed rape seeds with ablated defence cells of the glucosinolate-myrosinase system. Production and characteristics of double haploid MINELESS plants of Brassica napus L. J. Exp. Bot. 2011, 62, 4975−4993. (19) Chen, S.; Andreasson, E. Update on glucosinolate metabolism and transport. Plant Physiol. Biochem. 2001, 39, 743−758. (20) Niu, Y.; Jiang, M.; Wan, C.; Yang, M.; Hu, S. Effect of microwave treatment on sinapic acid derivatives in rapeseed and rapeseed meal. J. Am. Oil Chem. Soc. 2013, 90, 307−313. (21) Kraljic, K.; Skevin, D.; Pospisil, M.; Obranovic, M.; Nederal, S.; Bosolt, T. Quality of rapeseed oil produced by conditioning seeds at modest temperatures. J. Am. Oil Chem. Soc. 2013, 90, 589−599. (22) Grageola, F.; Landero, J. L.; Beltranena, E.; Cervantes, M.; Araiza, A.; Zijlstra, R. T. Energy and amino acid digestibility of
expeller-pressed canola meal and cold-pressed canola cake in ilealcannulated finishing pigs. Anim. Feed Sci. Technol. 2013, 186, 169−176. (23) Slominski, B. A.; Jia, W.; Rogiewicz, A.; Nyachoti, C. M.; Hickling, D. Low-fiber canola. Part 1. Chemical and nutritive composition of the meal. J. Agric. Food Chem. 2012, 60, 12225−12230. (24) Khajali, F.; Slominski, B. A. Factors that affect the nutritive value of canola meal for poultry. Poult. Sci. 2012, 91, 2564−2575. (25) Verkerk, R.; Dekker, M. Glucosinolates and myrosinase activity in red cabbage (Brassica oleracea L. var. capitata f. rubra DC.) after various microwave treatments. J. Agric. Food Chem. 2004, 52, 7318− 7323. (26) Oliviero, T.; Verkerk, R.; Van Boekel, M. A. J. S.; Dekker, M. Effect of water content and temperature on inactivation kinetics of myrosinase in broccoli (Brassica oleracea var. italica). Food Chem. 2014, 163, 197−201. (27) Campbell, L. D.; Slominski, B. A. Extent of thermal decomposition of indole glucosinolates during the processing of canola seed. J. Am. Oil Chem. Soc. 1990, 67, 73−75. (28) Slominski, B. A.; Campbell, L. D. Formation of indole glucosinolate breakdown products in autolyzed, steamed and cooked Brassica vegetables. J. Agric. Food Chem. 1989, 37, 1297−1299. (29) Jensen, S. K.; Liu, Y.G.; Eggum, B. O. The effect of heat treatment on glucosinolates and nutritive value of rapeseed meal in rat. Anim. Feed Sci. Technol. 1995, 53, 17−28. (30) Oliviero, T.; Verkerk, R.; Van Boekel, M. A. J. S.; Dekker, M. Effect of water content and temperature on glucosinolate degradation kinetics in broccoli (Brassica oleracea var. italica). Food Chem. 2012, 132, 2037−2045.
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Effect of Microwave Treatment on the Efficacy of Expeller Pressing of Brassica napus Rapeseed and Brassica juncea Mustard Seeds Yanxing Niu,†,‡ Anna Rogiewicz,‡ Chuyun Wan,† Mian Guo,† Fenghong Huang,† and Bogdan A. Slominski*,‡ †
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, Hubei Province 430062, People’s Republic of China ‡ Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 ABSTRACT: A study was conducted to evaluate the effect of microwave heating on the efficacy of expeller pressing of rapeseed and mustard seed and the composition of expeller meals in two types of Brassica napus rapeseed (intermediate- and lowglucosinolate) and in Brassica juncea mustard (high-glucosinolate). Following microwave treatment, the microstructure of rapeseed using transmission electron microscopy showed a significant disappearance of oil bodies and myrosin cells. After 6 min of microwave heating (400 g, 800 W), the oil content of rapeseed expeller meal decreased from 44.9 to 13.5% for intermediateglucosinolate B. napus rapeseed, from 42.6 to 11.3% for low-glucosinolate B. napus rapeseed, and from 44.4 to 14.1% for B. juncea mustard. The latter values were much lower than the oil contents of the corresponding expeller meals derived from the unheated seeds (i.e., 26.6, 22.6, and 29.8%, respectively). Neutral detergent fiber (NDF) contents showed no differences except for the expeller meal from the intermediate-glucosinolate B. napus rapeseed, which increased from 22.7 to 29.2% after 6 min of microwave heating. Microwave treatment for 4 and 5 min effectively inactivated myrosinase enzyme of intermediate-glucosinolate B. napus rapeseed and B. juncea mustard seed, respectively. In low-glucosinolate B. napus rapeseed the enzyme appeared to be more heat stable, with some activity being present after 6 min of microwave heating. Myrosinase enzyme inactivation had a profound effect on the glucosinolate content of expeller meals and prevented their hydrolysis to toxic breakdown products during the expelling process. It appeared evident from this study that microwave heating for 6 min was an effective method of producing expeller meal without toxic glucosinolate breakdown products while at the same time facilitating high yield of oil during the expelling process. KEYWORDS: microwave heating, expeller pressing, rapeseed, mustard, chemical composition
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INTRODUCTION Rapeseed is a good source of edible oil and meal as well as a good source of oil for biodiesel production. It ranks second among oilseed crops and is widely grown in China, Canada, Europe, and India. New technologies that could improve the quality of rapeseed oil and meal such as cold pressing,1 microwave heating,2 or aqueous enzymatic extraction3 have been proposed. Microwave heating is a dielectric treatment, which is fundamentally different from the conventional cooking in the prepress solvent extraction process.4 It has been reported that following microwave treatment rapeseed oil contains more phytosterols and tocopherols, and hence its oxidative stability could be improved.5 The flavor of rapeseed oil could also be significantly improved by microwave treatment. According to Zhou et al.6 microwave heating of rapeseed for 6 min before oil extraction by pressing gave a pleasant roasted flavor to the oil when compared to the crude oil. Microwave treatment can also significantly decrease the seed lipase activity.7 The effects of microwave treatment on rapeseed cake and meal quality parameters have been investigated. Sadeghi et al.8 evaluated the effects of microwave treatment on ruminal degradability of canola (low-glucosinolate rapeseed) meal protein and concluded that 4 min of microwave heating of canola meal effectively protected proteins from their degradation in the rumen. Ebrahimi et al.9 reported that phytic acid and © 2015 American Chemical Society
glucosinolate contents of canola seed linearly decreased with increased time of microwave heating. The conventional heat treatment of the seed in the prepress solvent extraction process involves cooking or conditioning throughout the steam-heated drum or stack-type cookers. This promotes coalescing of oil droplets, reduces oil viscosity, and denatures (inactivates) myrosinase enzymes.10 Till now, the effect of microwave treatment on the efficacy of oil extraction by expeller-pressing has not been adequately studied. The objective of this study was to investigate the effect of the duration of microwave heating on the efficacy of oil extraction and the chemical composition of expeller meals from rapeseed and mustard seeds.
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MATERIALS AND METHODS
Plant Materials. Intermediate-glucosinolate (∼60 μmol/g, fat-free basis) Brassica napus rapeseed var. Mianyou 309 (moisture content = 10.8%) was provided by the Mianyang Academy of Agricultural Sciences, Szechwan Province, People’s Republic of China. Lowglucosinolate (∼20 μmol/g, fat-free basis) B. napus rapeseed var. Zhongshuang 11 (moisture content = 11.0%) was provided by the Oil Received: Revised: Accepted: Published: 3078
October 8, 2014 March 12, 2015 March 13, 2015 March 13, 2015 DOI: 10.1021/jf504872x J. Agric. Food Chem. 2015, 63, 3078−3084
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Journal of Agricultural and Food Chemistry
Figure 1. Transmission electron microscopy of two types of Brassica napus rapeseed and Brassica juncea mustard without or with microwave treatment: (A) B. napus rapeseed (intermediate-glucosinolate; as-is); (B) B. napus rapeseed (intermediate-glucosinolate) microwaved for 5 min; (C) B. napus rapeseed (low-glucosinolate; as-is); (D) B. napus rapeseed (low-glucosinolate) microwaved for 5 min; (E) B. juncea mustard (highglucosinolate; as-is); (F) B. juncea mustard (high glucosinolate) microwaved for 5 min. CW, cell wall; PB, protein bodies; MG, myrosin cells; OB, oil bodies. Magnification: ×4000. temperature of no more than 90 °C (KOMET CA59G, IBG Monforts Oekotec GmbH & Co. KG, Germany). Microscopy Analysis. The seed samples were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) for 4 h, washed three times with phosphate buffer, postfixed with 1% of OsO4 in phosphate buffer (pH 7.0) for 1 h, and washed three times in the phosphate buffer. The samples were then dehydrated in the progressive ethanol solutions of 50, 70, 80, 90, 95, and 100% for about 15−20 min at each solution, embedded in acetone and Spurr resin. Sections 70−90 nm in size were cut from the embedded tissue using a microtome (Ultracut E, Reichert-Jung, Vienna, Austria), double stained with uranyl acetate and lead citrate, and then were examined under a transmission electron microscope (H-7650, Hitachi Instrument, Tokyo, Japan).
Crops Research Institute, Chinese Academy of Agricultural Sciences, Hubei Province, People’s Republic of China. Brassica juncea mustard (high-glucosinolate; ∼130 μmol/g, fat-free basis; moisture content = 11.0%) was provided by the Institute of Economic Crops, Xinjiang Academy of Agricultural Sciences, Xinjiang Province, People’s Republic of China. All seeds were produced in 2012. Sample Preparation and Treatments. The seed samples (400 g each) were subjected to microwave irradiation (MarsX, CEM, Matthews, NC, USA) at a frequency of 2450 MHz at a medium power of 800 W for 1, 2, 3, 4, 5, and 6 min. All microwave-treated samples were allowed to cool to room temperature for 60 min. The samples without or with microwave heating were expeller-pressed using an oil expeller at a feeding rate of 3 kg h−1 and barrel 3079
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Journal of Agricultural and Food Chemistry Chemical Analyses. The samples were ground and were adjusted to the same moisture content by exposure to room humidity for 7 days at 25 °C.11 The samples were then defatted with hexane for 6 h and air-dried. Oil content and neutral detergent fiber (NDF) were analyzed according to AOAC (1990) method 920.39 and Van Soest et al.,12 respectively. Glucosinolate analysis was performed as described by Slominski and Campbell.13 Briefly, 100 mg of high-glucosinolate or 300 mg of lowglucosinolate samples was weighed into 15 mL centrifuge tubes. Two milliliters of methanol, 1.0 mL of benzyl glucosinolate (internal standards, 0.5 mM), and 0.1 mL of lead−barium acetate were added to the tubes, extracted for 3 h at room temperature, and then centrifuged. Two milliliters of supernatant was transferred to a DEAE-Sephadex column, which was washed successively with 1 mL each of 67% methanol, water, and pyridine acetate. Purified sulfatase solution was then added to the column, and the contents were incubated at room temperature overnight. The resulting desulfated glucosinolates were eluted with 2 mL of 60% methanol and evaporated under nitrogen. The dry residue was trimethylsilylated by adding 0.2 mL of a mixture of anhydrous acetone/N,O-bis(trimethylsilyl)acetamide (BSA)/trimethylchlorosilane (TMCS)/1-methylimidazole (2:1:0.1:0.05 v/v) and incubated for 30 min at room temperature. The trimethylsilyl derivatives of desulfoglucosinolates were separated by gas−liquid chromatography using a glass column (1.2 m × 2 mm i.d.) packed with 2% OV-7 on Chromosob W (HP) (100−120 mesh) with helium gas at a flow rate of 40 mL/min. The oven temperature was kept at 200 °C for 4 min and then increased at 5 °C min−1 to 275 °C. Injection port and detector temperatures were 280 and 300 °C, respectively. Myrosinase activity was determined by difference between total sample glucosinolate content and the glucosinolates remaining following incubation of the sample with distilled water (autolysis) at 40 °C for a defined period of time. Glucosinolate analysis was conducted as described above. One unit of myrosinase activity was defined as the amount of enzyme that catalyzes the hydrolysis of 1 μmol of glucosinolate per 1 min. The analyses of moisture content, oil content, NDF, and glucosinolate content were run in triplicate, wheereas those for myrosinase activity were run in duplicate. Statistical Analysis. All of the statistical analysis was conducted by the SAS program (version 9.1, SAS Institute Inc., Cary, NC, USA). Means were separated by Tukey’s honestly significant difference. All statements of significance were based on P ≤ 0.05.
amine, 14% of phosphatidylinositol, and 20% of phosphatidylserine. According to Valentov et al.,15 microwave treatment could improve the content of phospholipids in the crude oil by more than twice, which would be a consequence of the membrane rupture. A strong relationship between oil bodies’ stability and oil extractability has been reported,16 indicating that the oil from the microwave-treated rapeseed should be easier to extract. Myrosin cells are the subcellular compartments associated with enzyme myrosinase. Normally, glucosinolates are located in vacuoles and are separated from the myrosin cells.17 The presence of the glucosinolate−myrosinase system is characteristic of the Brassicaceae family, and it serves as a defense system against biotic and abiotic stressors.18 Disruption of the tissue induced by mechanical damage, infection, or pest attack brings glucosinolates into contact with myrosinase,19 resulting in their hydrolysis to biologically active and often toxic breakdown products, including isothiocyanates, goitrin, and nitriles. After microwave treatment, it appeared evident that the structure of myrosin cells was effectively damaged, indicating the potential for myrosinase to come into contact with glucosinolates. In Figure 1 images A, C, and E, the cells of rapeseed were well coherent, and the shape was smooth, whereas in the microwave-treated samples (i.e., images B, D, and F) the cell walls became folded. This phenomenon may be ascribed to the water loss of the cells, as documented by Niu et al.20 As illustrated in Figure 2, there was a significant decline in moisture content of B. napus rapeseed and B. juncea mustard.
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RESULTS AND DISCUSSION Effect of Microwave Treatment on Microstructure of the Seed. The effect of microwave heating (400 g, 800 W) on the microstructure of the seed samples is shown in Figure 1. The microstructure of the intact (unheated) seed revealed welldefined pattern of protein bodies and intact myrosin cells amid oil bodies with smooth cell walls. In high-glucosinolate B. juncea (E) and intermediate-glucosinolate B. napus (A) myrosin cells were quite visible, whereas there were almost no visible myrosin cells in the seed of low-glucosinolate rapeseed (C). The microstructure of all samples changed significantly after 5 min of microwave heating (see images F, B, and D) with the oil bodies and myrosin cells almost disappearing. The microscopy images clearly demonstrate that microwave treatment disrupts the membranes of oil bodies as well as other intracellular organelles. Most of the biological membranes of rapeseed are composed of phospholipids and proteins (i.e., oleosins) with the oil bodies covered by a single-layer membrane. Eighty percent of this layer is filled with phospholipids, and the other 20% is occupied by oleosins. Phospholipids constitute a polar headgroup and a nonpolar fatty acid region.14 It has been reported that the composition of phospholipids in the oil bodies would account for 60% of phosphatidylcholine, 6% of phosphatidylethanol-
Figure 2. Effect of time of microwave heating on mean moisture content of B. napus rapeseed and B. juncea mustard.
Effect of Microwave Treatment on Oil Content of Expeller Meal. The effect of time of microwave heating on the oil content of seed and expeller meal of two types of B. napus rapeseed and B. juncea mustard is shown in Table 1. The oil contents of B. napus rapeseed (intermediate- and lowglucosinolate) and B. juncea mustard were similar. After expeller-pressing, however, the oil content of expeller meal decreased significantly with increased time of microwave heating. After 6 min of microwave heating, the oil content of intermediate- and low-glucosinolate B. napus rapeseed and of B. juncea mustard expeller meal decreased from 26.6, 22.6, and 29.8 to 13.5, 11.3, and 14.1%, respectively. Overall, the microwave treatment decreased the oil content of expeller meal by 49−53% with an oil extraction rate of 79−83%. The oil contents of expeller meals are in agreement with the changes in microstructure of microwave-treated seed showing 3080
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Table 1. Effect of Time of Microwave Heating on Oil Content of Seed and Expeller Meal of Two Types of Brassica napus Rapeseed and Brassica juncea Mustard (Percent, As-Is Basis) time of microwave heating sample
untreated
B. napus rapeseed (intermediate-glucosinolate) seed 44.9 ± 0.3aa expeller meal 26.6 ± 0.8a B. napus rapeseed (low-glucosinolate) seed 42.6 ± 0.3a expeller meal 22.6 ± 0.5a B. juncea mustard (high-glucosinolate) seed 44.4 ± 0.4a expeller meal 29.8 ± 0.4a a
1 min
2 min
3 min
4 min
5 min
6 min
45.0 ± 0.4a 23.6 ± 0.9b
44.2 ± 0.5a 20.6 ± 0.2c
44.4 ± 0.7a 18.0 ± 0.3d
44.9 ± 0.9a 16.7 ± 1.0e
44.0 ± 0.2a 15.3 ± 0.6f
43.7 ± 0.6a 13.5 ± 0.3g
42.0 ± 0.8a 22.9 ± 0.3a
42.5 ± 0.1a 19.4 ± 1.0b
41.3 ± 0.2a 17.4 ± 0.7c
41.5 ± 0.2a 15.2 ± 0.9d
42.5 ± 0.6a 14.2 ± 0.5d
42.4 ± 0.4a 11.3 ± 0.9e
44.1 ± 0.2a 27.3 ± 0.5b
44.1 ± 0.2a 22.4 ± 0.1c
44.0 ± 0.2a 18.8 ± 0.3d
43.4 ± 0.1a 15.9 ± 0.7e
43.2 ± 0.2a 14.4 ± 0.6f
43.1 ± 0.2a 14.1 ± 0.3g
Mean ± SD. Values within rows with the same letter (a−g) are not significantly different (P ≤ 0.05).
Table 2. Effect of Time of Microwave Heating on Neutral Detergent Fiber (NDF) Content of Seed and Expeller Meal of Two Types of Brassica napus Rapeseed and Brassica juncea Mustard (Percent, As-Is, Fat-Free Basis) time of microwave heating sample
untreated
B. napus rapeseed (intermediate-glucosinolate) seed 23.7 ± 0.1aa expeller meal 22.7 ± 0.4b B. napus rapeseed (low-glucosinolate) seed 22.0 ± 0.5a expeller meal 23.6 ± 0.3a B. juncea mustard (high-glucosinolate) seed 17.5 ± 1.3a expeller meal 17.4 ± 1.0a a
1 min
2 min
3 min
4 min
5 min
6 min
23.8 ± 0.5a 24.1 ± 0.7b
24.1 ± 0.4a 23.7 ± 0.1b
23.8 ± 0.7a 23.1 ± 0.4b
24.3 ± 0.5a 23.2 ± 0.2b
23.9 ± 0.5a 24.7 ± 0.2b
24.6 ± 0.7a 29.2 ± 2.2a
22.4 ± 1.0a 24.8 ± 0.4a
23.0 ± 0.2a 23.0 ± 0.1a
22.4 ± 0.4a 24.1 ± 1.7a
22.4 ± 0.4a 25.1 ± 1.7a
22.2 ± 0.9a 25.9 ± 1.3a
22.1 ± 0.7a 25.6 ± 1.4a
18.1 ± 0.5a 16.8 ± 0.2a
17.5 ± 1.3a 18.9 ± 2.2a
18.6 ± 1.5a 17.3 ± 1.3a
18.3 ± 0.5a 16.8 ± 0.3a
18.0 ± 0.8a 18.8 ± 2.8a
19.6 ± 1.0a 17.6 ± 0.8a
Mean ± SD. Values within rows with the same letter (a, b) are not significantly different (P ≤ 0.05).
Table 3. Effect of Time of Microwave Heating on Myrosinase Activity of Seed of Two Types of Brassica napus Rapeseed and Brassica juncea Mustard (Units per Minute, As-Is, Fat-Free Basis) time of microwave heating
a
sample
untreated
1 min
2 min
3 min
4 min
5 min
6 min
B. napus rapeseed (intermediate-glucosinolate) B. napus rapeseed (low-glucosinolate) B. juncea mustard (high-glucosinolate)
32.7 ± 0.9aa 11.6 ± 0.4b 49.2 ± 2.5a
27.6 ± 1.5b 14.9 ± 0.1a 26.2 ± 4.2b
22.8 ± 1.5c 13.6 ± 0.2a 26.9 ± 1.7b
16.8 ± 0.6d 8.0 ± 0.5c 11.2 ± 1.7c
5.6 ± 0.6e 5.5 ± 0.4d nd
3.6 ± 0.8f 5.2 ± 0.2d nd
ndb 4.2 ± 0.4d nd
Mean ± SD. Values within rows with the same letter (a−f) are not significantly different (P ≤ 0.05). bNot detected.
The contents of NDF in the seed and expeller meal of two types of B. napus rapeseed and B. juncea mustard were different, with B. juncea showing significantly less fiber than B. napus. This is due to the fact that B. juncea mustard is yellow-seeded, whereas B. napus rapeseed is black-seeded. The yellow seed coat color is a visual marker of lower polyphenol (i.e., proanthocyanidin) content and a thinner seed hull. As a result, black-seeded rapeseed contains more fiber than its yellowseeded B. napus counterparts or B. juncea mustard.23 After microwave heating for 6 min, the NDF contents of all seed samples were similar, so there was no significant effect of heat treatment on fiber content. In the expeller meals, however, the NDF content of intermediate-glucosinolate B. napus rapeseed increased from 24.1 to 29.2% after 6 min of microwave heating, whereas in low-glucosinolate B. napus rapeseed and B. juncea mustard no significant changes were observed. It is well-known that heat treatment of oilseeds during processing may lead to losses of digestible amino acids due to the formation of Maillard reaction products. This highly indigestible fraction, often referred to as a neutral detergent insoluble crude protein
the broken membranes of oil bodies, which, in turn, facilitated higher efficacy of oil extraction during expeller-pressing. The oil extraction rate by expeller-pressing after 6 min of microwave heating used in the current study was similar to the conditioning of the seed at 80 °C for 30 min, flaking of the seed, and then expeller-pressing.21 In another study, similar results were achieved when the seed was subjected to 40 min of heat treatment with the temperature increased to 80 °C and then heat treatment at 80 °C for 30 min followed by 40 min of flaking to 0.3 mm thickness and expeller pressing under a barrel temperature of 105−110 °C.1 According to Grageola et al.22 expeller-pressing recovers around 75% of oil from canola seed. This indicates that the microwave treatment could be an effective method of increasing the efficacy of oil extraction by expeller pressing and would facilitate oil extraction down to the industry standards of 8−10% of residual oil. which, in turn, would prevent the need for double pressing. Effect of Microwave Treatment on NDF Content of Expeller Meal. The effect of time of microwave heating on NDF content of seed and expeller meal is shown in Table 2. 3081
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Table 4. Effect of Time of Microwave Heating on Total Glucosinolate Content of Seed and Expeller Meal of Two Types of Brassica napus Rapeseed and Brassica juncea Mustard (Micromoles per Gram, As-Is, Fat-Free Basis) time of microwave heating sample
untreated
B. napus rapeseed (intermediate-glucosinolate) seed 58.4 ± 6.2aa,b expeller meal 12.2 ± 0.1ef B. napus rapeseed (low-glucosinolate) seed 18.3 ± 2.7abb expeller meal 7.7 ± 0.1e B. juncea mustard (high-glucosinolate) seed 134.9 ± 2.1ac expeller meal 20.8 ± 1.5e
1 min
2 min
3 min
4 min
5 min
6 min
53.9 ± 3.0ab 11.6 ± 0.5f
54.4 ± 1.7ab 16.4 ± 2.7e
54.4 ± 3.1ab 28.9 ± 2.1d
51.2 ± 3.7abc 44.9 ± 2.9b
46.8 ± 2.8bc 53.5 ± 3.7a
43.0 ± 3.4c 38.7 ± 2.0c
18.3 ± 1.4ab 8.0 ± 0.3de
20.8 ± 2.3a 9.3 ± 0.3cd
17.4 ± 0.9ab 11.2 ± 0.3ab
15.1 ± 0.5bc 11.9 ± 0.6a
13.9 ± 1.0bc 10.2 ± 1.5bc
11.6 ± 1.7c 7.7 ± 0.1e
137.3 ± 17.6a 29.8 ± 3.0e
135.7 ± 1.9a 67.1 ± 5.4d
146.4 ± 16.2a 106.5 ± 9.1c
135.4 ± 4.8a 150.5 ± 9.9a
131.0 ± 3.9a 134.4 ± 2.9b
135.9 ± 5.1a 132.1 ± 9.5b
Mean ± SD. Values within rows with the same letter (a−f) are not significantly different (P ≤ 0.05). bIncludes gluconapin (3-butenyl), glucobrassicanapin (4-pentenyl), progoitrin (2-hydroxy-3-butenyl), gluconapoleiferin (2-hydroxy-4-pentenyl), glucobrassicin (3-indolylmethyl), and 4-hydroxyglucobrassicin (4-hydroxy-3-indolylmethyl). cIncludes sinigrin (2-propenyl), gluconapin (3-butenyl), progoitrin (2-hydroxy-3-butenyl), gluconapoleiferin (2-hydroxy-4-pentenyl), and 4-hydroxyglucobrassicin (4-hydroxy-3-indolylmethyl). a
(NDICP), would contribute to the increase in the NDF content of heat-treated products and would significantly affect the nutritive value of rapeseed/canola meal.24 In the current study, expeller pressing of intermediate-glucosinolate B. napus rapeseed microwave heated for 6 min resulted in some protein damage as documented by the increase in NDF content which, in turn, could lead to reduced protein and amino acid digestibilities. Therefore, extending the time of microwave treatment could further contribute to protein damage and the increase in NDICP content of expeller meal. Effect of Microwave Treatment on Myrosinase Activity of the Seed. As illustrated in Table 3, there was a significant decline in myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) activity with increased time of microwave heating for two types of B. napus and B. juncea seed. This is in agreement with a study by Verkerk and Dekker,25 who demonstrated that myrosinase in microwave-treated cabbage almost completely lost its hydrolytic capacity, despite the fact that the stability of myrosinase is strongly dependent on the water content,26 which was most likely much higher in cabbage than in the seed samples used in the current study (i.e., 10%). When compared with B. napus intermediate-glucosinolate rapeseed and B. juncea mustard, the myrosinase activity in B. napus low-glucosinolate rapeseed was relatively low, although the enzyme per se appeared to be more heat-stable with some activity still being present after 6 min of microwave treatment. Among other reasons, application of heat treatment in any crushing operation of rapeseed is essential for an effective inactivation of myrosinase enzyme. In the presence of moisture and following rupture of the seed, this enzyme would hydrolyze glucosinolates to unstable aglucones, which would then break down to yield a range of products, including isothiocyanates, goitrin, nitriles, and thiocyanates, that interfere with the function of the thyroid gland and would adversely affect growth performance of monogastric animals.24 Therefore, it is critical that myrosinase is effectively inactivated by heat treatment prior to seed expelling. Effect of Microwave Treatment on Glucosinolate Content of Seed and Expeller Meal. The effect of time of microwave heating on total glucosinolate content in seed and expeller meal is shown in Table 4. The total glucosinolate content of B. napus seed samples decreased after 4 min of microwave heating. It is of interest to note that in the seed of B. juncea mustard the total glucosinolate content did not change
during microwave heating. The glucosinolate content of the corresponding expeller meals was much lower and reflected the hydrolysis of glucosinolates by myrosinase during the expelling process. However, their contents increased with increased time of microwave heating and, thus, myrosinase inactivation and then decreased due to excessive time of microwave heating, which coincided with the decrease in glucosinolate content in the seed samples for 5−6 min. More specifically, the glucosinolate content of expeller meal of B. juncea mustard and intermediate-glucosinolate B. napus rapeseed increased significantly during microwave heating for 4 and 5 min, respectively, and then decreased. Somewhat similar changes, although not significant, were observed for low-glucosinolate B. napus rapeseed. According to their structure, glucosinolates can be divided into aliphatic glucosinolates, including sinigrin, gluconapin, glucobrassicanapin, and progoitrin, and indole glucosinolates, including glucobrassicin and 4-hydroxyglucobrassicin. In different Brassica species the compositions of glucosinolates are different. The major glucosinolates of B. napus rapeseed are gluconapin, progoitrin, and 4-hydroxyglucobrassicin, whereas sinigrin and gluconapin are the predominant glucosinolates of B. juncea mustard. It is well-known that heat treatment and myrosinase activity are the factors that affect the glucosinolate content during processing.27 It has also been shown that indole glucosinolates are more susceptible to thermal degradation than aliphatic glucosinolates. 27−30 As a result, the decrease in total glucosinolate content due to microwave heating observed in the current study was more pronounced for two types of B. napus rapeseed than for B. juncea mustard. The effect of time of microwave heating on the major glucosinolate contents in the seed and expeller meal samples of two types of B. napus rapeseed and B. juncea mustard are shown in Figures 3, 4, and 5. When compared to the seed samples, the aliphatic glucosinolates gluconapin and progoitrin contents of the corresponding expeller meal samples of intermediateglucosinolate B. napus rapeseed (Figure 3) increased significantly after 3 min, which was directly related to myrosinase inactivation and thus lack of glucosinolate autolysis. The decrease after 6 min of microwave treatment was a consequence of extended microwave heating. The indole glucosinolate hydroxyglucobrassicin content of expeller meal demonstrated a less clear pattern, probably due to its 3082
DOI: 10.1021/jf504872x J. Agric. Food Chem. 2015, 63, 3078−3084
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Journal of Agricultural and Food Chemistry
glucosinolate hydroxyglucobrassicin showing a significant thermal decomposition after 4, 5, and 6 min. Overall, the content of hydroxyglucobrassicin in the seed of intermediateand low-glucosinolate B. napus rapeseed decreased by 50 and 53%, respectively, after 6 min of microwave heating. As aliphatic glucosinolates are less sensitive to heat treatment, sinigrin and gluconapin contents of B. juncea mustard seed showed no significant differences with increased time of microwave heating (Figure 5). However, their content after 4, 5, and 6 min of microwave treatment reached the level of that of the seed, which was a consequence of complete myrosinase inactivation. There was a strong negative relationship between myrosinase activity in the seed (Table 3) and the total glucosinalate content in the corresponding expeller meals (Table 4) with intermediate-glucosinolate B. napus rapeseed and B. juncea mustard showing R2 values of −0.92 and −0.92, respectively. In a study on the effect of microwave treatment on the glucosinolate content of canola seed, Ebrahimi et al.9 reported that the glucosinolate content after 2, 4, and 6 min of microwave heating decreased by 42, 55, and 59%, respectively, which was significantly higher than that observed in the current study. The main reason for the difference could be related to the processing method used. In their study, canola seed was dried at 55 °C for 48 h, after which time water was added to increase the moisture content to 25%. It is very probable that drying of the seed inactivated the myrosinase activity, whereas the heat produced during microwave treatment under high moisture conditions facilitated glucosinalate degradation. In conclusion, it would appear evident that microwave heating of 2 W kg−1 for 6 min could be an effective method of producing expeller meal without toxic glucosinolate breakdown products while at the same time increasing the yield of oil during the expelling process.
Figure 3. Effect of time of microwave heating on major glucosinolate of B. napus intermediate-glucosinolate rapeseed and expeller meal.
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AUTHOR INFORMATION
Corresponding Author
Figure 4. Effect of time of microwave heating on major glucosinolate of B. napus low-glucosinolate rapeseed and expeller meal.
*(B.A.S.) Phone: (204) 474-8291. Fax: (204) 474-7628. Email: [email protected]. Funding
Funding of this study was provided by the National Natural Science Foundation of China (Grant 31201461) and the Canola Council of Canada. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge Ruibao Zhou, He’nan University of Technology, and Junying Li, Zhejiang University, for their assistance in microstructure analysis and Zirong Guo, Mianyang Academy of Agricultural Sciences, Sichuan, for providing the samples.
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ABBREVIATIONS USED NDF, neutral detergent fiber; BSA, N,O-bis(trimethylsilyl)acetamide; TMCS, trimethylchlorosilane; NDICP, neutral detergent insoluble crude protein
Figure 5. Effect of time of microwave heating on major glucosinolate of B. juncea high-glucosinolate mustard seed and expeller meal.
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pronounced thermal decomposition in the seed during 5 or 6 min of microwave heating. The glucosinolate content of the seed and expeller meal samples of low-glucosinolate B. napus rapeseed (Figure 4) showed a similar pattern with the indole
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