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The influence of storage conditions on starch and amylose content of South African quality protein maize and normal maize hybrids
Journal of Stored Products Research 56 (2014) 16e20
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
The influence of storage conditions on starch and amylose content of South African quality protein maize and normal maize hybrids Maryke Labuschagne a, *, Lekgolwa Phalafala a, Gernot Osthoff b, Angeline van Biljon a a b
Department of Plant Sciences, University of the Free State, Bloemfontein 9300, South Africa Department of Microbial, Biochemical and Food Technology, University of the Free State, Bloemfontein, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 19 November 2013
The quality of maize grains during storage is affected by unfavourable storage conditions, resulting in physicochemical changes in specifically amylose and starch content that lead to significant product qualitative and quantitative losses. The objective of this study was to evaluate the starch and amylose content of normal maize and quality protein maize (QPM) seed samples at different temperature treatments: in cold storage at 3.6 C, at room temperature (18.5 C) and at 30 C for 0, 6 and 12 month storage periods, respectively. Sixteen genotypes were tested in a single trial at Potchefstroom in South Africa. Due to optimal growing conditions the seed was of excellent quality. Storage at 3.6 C and 18.5 C caused some reduction in amylose and starch content, although for starch it was not significant. On the other hand, storage at 30 C significantly (P 0.05) reduced the starch and amylose content after both 6 and 12 month periods of storage. QPM and non-QPM seed reacted similarly to storage conditions, and there were larger differences between cultivars than between QPM and non-QPM material. Even at relatively high relative humidity, low temperature storage maintained seed quality the best. Therefore high temperature storage is detrimental to starch and amylose content of both normal maize and QPM, especially after 6 months or more of storage. Maize should therefore be stored under low temperatures (around 4 C) or if not possible, at least under 19 C. Ó 2013 Published by Elsevier Ltd.
Keywords: Quality Protein Maize Storage Starch Amylose
1. Introduction Starch, the chief source of carbohydrate in human diets, forms the major energy reserve in cereal grains. Amylose and amylopectin are the two glucopolysaccharide components of starch. Starch granule physical properties are influenced by the distribution of amylose and amylopectin in starch (Corcuera et al., 2007). The starch and amylose contents of seed depend on the quality, physical structure and storage conditions of the seed after harvest (Buckow et al., 2009). Poor storage conditions often prevail and can lead to biochemical changes in chemical compounds of maize kernels including amylose and starch content during storage. The possible causes of these changes are not clear, but high relative humidity (r.h.) and the formation of free radicals during storage have been reported to play a role (MacDonald, 1999, 2006). These changes may affect the starch and amylose content. During storage, part of the starch and amylose has been reported to undergo an
* Corresponding author. Tel.: þ27 514012715. E-mail address: [email protected] (M. Labuschagne). 0022-474X/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jspr.2013.11.004
aggregation process that leads to changes in their content (Liu et al., 2003; Pongsawatmamit et al., 2006). There is a concern in terms of food security, on how to best store seeds, including maize seeds, for a long time without compositional changes, especially in developing countries worldwide. In these countries, efficient maintenance of grain quality is often not possible due to the lack of adequate post-harvest technology, including suitable storage conditions (Doijode, 2001). Quality protein maize (QPM) is nutritionally enhanced and has 70e100% more lysine and tryptophan than normal maize. QPM cultivars are being introduced in a number of African countries to reduce protein deficiencies in areas where maize is the staple food (Sofi et al., 2009). Little is known about how storage conditions affect both normal maize and QPM with special reference to starch and amylose content. It is better to implement preventive management, rather than to solve specific storage problems once they have occurred (Lopes et al., 2008). The aim of this study was to investigate the influence of different storage conditions on starch and amylose content extracted from the South African open pollinated QPM, a QPM hybrid and normal maize samples which were stored for 6 and 12
M. Labuschagne et al. / Journal of Stored Products Research 56 (2014) 16e20
months at 3.6 C, 18.5 C and 30 C compared to an untreated control. 2. Materials and methods 2.1. Planting location The trial was planted in Potchefstroom at the Agricultural Research Council (ARC)-Grain Crops Institute (26 740 S; 27 80 E) in November 2008. The ARC-Grain Crops Institute is located in Potchefstroom in the North West Province and works on a broad diversity of cereal crops grown in South Africa. Potchefstroom is located at an altitude of 1344 m above sea level with an average minimum and maximum temperature of 9.6 C and 25.5 C respectively with an average annual total rainfall of 619 mm (Weather South Africa, 2013). The rainfall during the growing season (November to April) was 409 mm which was well distributed over the season. The temperatures for the last two months of the growing season when grain filling took place ranged between a minimum of 12.6 C and a maximum of 27.9 C. Grain quality and yield was very good for this season. 2.2. Plant material The genotypes used in this study were eight South African open pollinated QPM varieties (SYN2QYQPM, SYN4QYQPM, SYN11QYQPM, SYN13QYQPM, SYN2QWQPM, SYN5QWQPM, SYN12Q WQPM and SYN15QWQPM), one QPM hybrid (QS7608) and six normal maize hybrids (CRW3505, C3505, CB341xI37F2, CB346xI37F2, P6479F2 and CB389xI37F2) obtained from the ARCGrain Crops Institute, Potchefstroom. 2.3. Experimental design and procedures The trial was planted for one season at a density of 50,000 plants per hectare on sandy clay loam. The experimental design was a randomised complete block design with three replications. The genotypes were grown in two-row plots. The rows were 5 m long with 0.3 m spacing apart, and row width was 90 cm. A compound fertilizer was applied at a rate equivalent to 300 kg per hectare of 3:2:1 (N:P:K) and then top-dressed with limestone ammonium nitrate (LAN). Standard cultural practices including ploughing, disking, and application of herbicides were done at the site in order to make nutrients easily accessible to the genotypes and to minimise competition for nutrients between the planted genotypes and weeds, as well as to reduce damage from insect pests. The trial was planted under rain-fed conditions. After harvesting, the seeds were dried to 12.5% moisture content and shelled with a stationary sheller. After shelling, a light table was used to confirm the status of the QPM genotypes. 2.4. Storage conditions Seed sampling was done randomly from each of the three replications after checking for QPM status. All samples were sealed in brown paper bags and stored under controlled conditions in a cold room (3.6 C), laboratory (18.5 C) and oven (30 C) for 6 and 12 months and were compared with an untreated control. The humidity was 76.5% r.h. in the cold room (3.6 C), and 28% r.h. in the laboratory (18.5 C). The laboratory was air conditioned which maintained a constant temperature. In the oven (30 C) moisture was very low or close to zero due to the prolonged exposure to heat. Forty-five bags (15 entries with three replications) were placed in the cold room at 3.6 C for 6 and 12 months. Another 45 bags were placed in the laboratory at 18.5 C for 6 and 12 months. The last 45
17
bags were placed in an oven at 30 C for 6 and 12 months. Each bag contained 90 maize seeds (45 seeds for the 6 months sampling, and 45 for the 12 month sampling). A randomized complete block design was used for the layout with three replications where each replication consisted of 45 seeds. After 6 and 12 month storage periods, 45 seed samples per bag were sampled and bulked and ground to a fine powder using a 1 kA analysis grinder, A10 Yellowline (Merck Chemicals Pty Ltd) with a 1 mm sieve. The processed samples were directly transferred to containers to avoid moisture accumulation. The seeds were then evaluated for starch and amylose content. 2.5. Starch extraction and analysis Starch content was determined according to the Megazyme total starch assay procedure (Megazyme, 2009). A homogenized milled sample (100 mg) of three replications of each genotype was measured into glass test tubes and 0.2 ml of aqueous ethanol (80% v/v) was added to wet the samples to aid dispersion. The tube was stirred on a vortex mixer. Two ml of dimethyl sulphoxide (DMSO) was added immediately and the tube was stirred on a vortex mixer. After that, the tube was placed in a boiling water bath at 95 C for 5 min. Three ml of thermostable a-amylase (300 U) was added in a 3-morpholinopropanesulfonic acid (MOPS) buffer (50 mM, pH 7.0) and the tubes were vigorously stirred on a vortex mixer. The tubes were incubated in a boiling water bath at 95 C for 6 min and stirred vigorously after 2, 4 and 6 min. The tubes were then placed in a water bath at 50 C after which sodium acetate buffer (4 ml, 200 mM, pH 4.5) was added, followed by amyloglucosidase (0.1 ml, 20 U). The volume was adjusted from 7 ml to 10 ml with distilled water and then the tubes were centrifuged at 3000 rpm for 10 min. The duplicate aliquots (0.1 ml) of the diluted solution were transferred to glass test tubes. Three ml of the glucose oxidaseperoxidase (GOPOD) reagent was added to each tube (including the glucose controls and reagent blanks), and the tubes were incubated at 50 C for 20 min. A glucose control was prepared by mixing 0.1 ml of glucose standard solution (1 mg ml1) and 3.0 ml of GOPOD reagent. A reagent blank solution was prepared by mixing 0.1 ml of water and 3.0 ml of GOPOD reagent. The absorbance was read in duplicate at 510 nm for each sample, and the glucose control was read against the reagent blank in duplicate. Total starch was measured as the glucose derived from hydrolyzed starch and was expressed as a percentage of total sample weight on an “as is” basis:
Total starch ¼ DAF FV=0:11=1000100=W 162=180 ¼ DAF=W FV0:9 where DA is the absorbance (reaction) read against the sample blank; F is a factor for the conversion from absorbance values to micrograms of glucose (100 mg of glucose/absorbance for 100 mg of glucose); FV ¼ final volume; 0.1 ¼ volume of sample analysed; 1/ 1000 is a conversion from micrograms to milligrams; 100/ W ¼ factor to express “starch” as a percentage of flour weight; W is the weight in milligrams (“as is” basis) of the flour analysed; and 162/180 is adjustment from free D-glucose to anhydro D-glucose (as occurs in starch). 2.6. Amylose determination Amylose was extracted and estimated by the iodine binding method (Cruz and Khush, 2000). One hundred milligram of homogenized maize flour sample was measured for three replications of each genotype. The samples were wetted with 1 ml of 95%
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M. Labuschagne et al. / Journal of Stored Products Research 56 (2014) 16e20
ethanol followed by 9 ml of 1 M sodium hydroxide (NaOH) to aid dispersion and stirred using a vortex mixer. The samples were placed in a boiling water bath for 15 min and stirred using a vortex mixer every 5 min. The samples were cooled for 1 h at room temperature and centrifuged at 3000 rpm for 5 min. Duplicate 0.1 ml aliquots of the solution were transferred to clean test tubes and 0.1 ml of 1 M acetic acid was added to each test tube followed by the addition of 0.2 ml iodine solution and 9.6 ml distilled water. The contents were vortexed and left to stand for 20 min. The absorbance was read in duplicate against the reagent blank at 620 nm for each sample. The amylose percentage was calculated using the formula:
h Amylose% ¼ Concentration mg ml1
i 1000=mass of the sample ðmgÞ 100
were significant differences between genotypes at all treatments, which were again not related to the type of maize genotype. The starch content of the maize genotypes at 18.5 C ranged between 49.9% and 65.7% after 6 months of storage and from 49.3% to 65.2% after 12 months of storage and at 30 C ranged from 48.5% to 63.5% after 6 months of storage and from 46.3% to 61.3% after 12 months of storage. There was a small reduction in starch content at the 3.6 C and 18.5 C treatments, but it was not significant. The reduction at 30 C was significant at both storage periods and it was 4.7 and 8.2% at 6 and 12 months respectively. The starch content of the non-QPM genotypes was slightly lower than that of the QPM genotypes for all treatments, but the differences were not significant. What was interesting was that SYN4QYQPM ranked in the first three positions for amylose and the last three for starch content while SYN15QWQPM ranked in the last two positions for both amylose and starch content for all the treatments. 4. Discussion
2.7. Statistical analysis Analysis of variance (ANOVA) for the 15 entries, three replications and treatments (control, heat and cold treatments for 6 and 12 months of storage) was used to determine the influence of storage conditions on starch and amylose content compared to an untreated control (Agrobase, 2005). 3. Results Mean squares for genotype and temperature treatments were highly significant (P 0.01), but interaction between cultivar and temperature treatments was not significant, indicating that the ranking of the genotypes remained largely the same at different temperature treatments for both starch and amylose content (Table 1). Amylose content varied significantly amongst tested maize genotypes (Table 2). The open pollinated QPM SYN2QWQPM consistently had the highest amylose content for all treatments. Normal hybrid CB341xI37F2 consistently ranked second or third for amylose content for all treatments. Open pollinated QPM SYN15QWQPM consistently had the lowest amylose content for all treatments. There was an average 10% and 21% reduction in amylose content after 6 and 12 month of storage at 30 C. The reduction was similar for QPM and non-QPM material. The reduction in amylose content at 3.6 C was not significant, irrespective of the time of storage. The amylose reduction at 18.5 C and 30 C was significant for both periods of storage. There were significant differences between genotypes for each treatment, but this was not related to genotypes being QPM or not. Open pollinated genotype SYN2QYQPM consistently had the highest starch content of all the genotypes for all the treatments (Table 3). SYN4QYQPM ranked second or third for all the treatments. Normal hybrid CRW3505 consistently had the lowest starch content for all the treatments, with open pollinated QPM SYN15QWQPM having the second lowest value throughout. There
Table 1 Mean squares for entry, treatment and their interaction for amylose and starch content. Source of variation
Amylose
Starch
Entry Treatment Entry Treatment
14.63** 88.17** 0.184
350.88** 142.62** 0.182
**P 0.01.
Amylose content was more sensitive to storage conditions than starch content, where storage at room temperature and 30 C caused a significant reduction in values, compared to starch where the only significant reduction in values was seen at storage at 30 C. Amylose mainly occurs in the amorphous region of the starch granule, either in an extended shape, or in a left-handed single helix, or in a parallel left-handed double helical form. In the latter case it might also be entwined with amylopectin (Ral et al., 2008). It is possible that the amylose may be partially degraded by amylase during storage, but it might also be that it is transformed from the extended shape to one of the two helix structures. Another reason why the amylose content decreases over storage time may lie in the analytical technique (Zhu et al., 2008; Fitzgerald et al., 2009). Although the iodine test is the most reproducible amylose test, the blue colour is produced due to binding to the single helix. The extended and double helix forms result in a colour of lower intensity. A change to the double helix structure may therefore result in a lower detection although the amylose content was not necessarily decreased. The reduced starch was consistent with other reports (Rehman et al., 2002; Simi c et al., 2007) where starch was reduced when exposed to high temperatures (25 C) for 6 months of storage. This occurrence was reported to be the effect of high temperature and r.h. as the latter showed to have a negative effect on chemical compounds of various seeds including maize starch (Al-Yahya, 2001). In this study the r.h. at 30 C was very low, which indicates that the high temperature by itself caused the reduction in starch content. These findings are in agreement with those reporting that unfavourable storage conditions cause seed quality losses (Al-Yahya, 2001; Rehman et al., 2002). Although the r.h. at the low temperature storage was very high, it did not seem to influence the starch or amylose content negatively. It seemed as though the low temperature was sufficient to preserve the status of the seed in terms of starch and amylose content. It was reported that the storage life of a seed doubles for each 5.6 C decrease in temperature (Desai, 2004). The QPM and normal maize genotypes showed a consistent reduction in both amylose and starch content, producing rankings that were consistent over treatments. The amylose and starch content of open pollinated QPM, the QPM hybrid and normal maize genotypes were equally affected by the different storage conditions over time. Large genotype differences were evident, suggesting that selection for increased starch and/or amylose content is possible within these genotypes. In conclusion, QPM and normal maize reacted the same to storage conditions and periods of storage. Maize should be stored at
M. Labuschagne et al. / Journal of Stored Products Research 56 (2014) 16e20
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Table 2 Effect of different storage conditions on amylose content over time of storage. No.
Genotype
Type
Amylose (%) Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CRW3505 QS7608 C3505 CB341xI37F2 CB346xI37F2 P6479F2 CB389xI37F2 SYN2QYQPM SYN4QYQPM SYN11QYQPM SYN13QYQPM SYN2QWQPM SYN5QWQPM SYN12QWQPM SYN15QWQPM
Normal hybrid QPM hybrid Normal hybrid Normal hybrid Normal hybrid Normal hybrid Normal hybrid Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated
QPM QPM QPM QPM QPM QPM QPM QPM
LSD (0.05) LSD (0.05) across treatments
18.5 C
30 C
0 months
6 months
12 months
6 months
12 months
6 months
12 months
17.62 18.22 17.92 18.73 18.33 17.73 18.18 18.39 16.78 17.27 17.72 19.60 19.12 18.67 16.97
17.47 18.87 18.20 19.35 18.62 17.47 17.97 18.28 16.66 17.13 17.51 19.49 19.04 18.46 16.83
17.28 18.65 18.09 19.09 18.27 17.22 17.78 18.04 16.43 16.88 17.15 19.23 18.96 18.16 16.62
16.35 17. 50 17.74 18.72 17.81 17.12 17.50 17.74 16.24 16.48 16.81 18.86 18.68 17.74 15.90
15.62 16.41 16.77 17.03 16.80 16.12 16.53 16.69 15.22 15.43 15.80 17.81 16.37 16.72 15.35
15.56 17.16 16.29 17.43 16.74 15.58 16.09 16.33 14.72 15.26 15.69 17.54 17.18 16.68 14.93
13.57 15.13 14.28 15.41 14.69 13.63 14.12 14.32 14.14 12.96 13.75 15.56 15.15 14.47 12.64
1.4860 0.4168
Mean Mean for QPM Mean for normal hybrids
3.6 C
18.08 18.08 18.09
1.4899 18.09 18.03 18.18
1.1146 17.86 17.79 17.96
1.3089 17.41 17.33 17.54
1.4964 16.31 16.20 16.48
1.3304 16.21 16.17 16.28
1.1390 14.26 14.24 14.28
Table 3 Effect of different storage conditions on starch content over time of storage. No.
Genotype
Type
Starch (%) Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CRW3505 QS7608 C3505 CB341xI37F2 CB346xI37F2 P6479F2 CB389xI37F2 SYN2QYQPM SYN4QYQPM SYN11QYQPM SYN13QYQPM SYN2QWQPM SYN5QWQPM SYN12QWQPM SYN15QWQPM
Normal hybrid QPM hybrid Normal hybrid Normal hybrid Normal hybrid Normal hybrid Normal hybrid Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated
LSD (0.05) LSD (0.05) across treatments Mean Mean for QPM Mean for normal hybrids
QPM QPM QPM QPM QPM QPM QPM QPM
3.6 C
18.5 C
30 C
0 months
6 months
12 months
6 months
12 months
6 months
12 months
50.06 60.58 56.19 62.65 62.56 58.57 62.56 66.20 63.21 56.60 58.56 62.12 60.80 58.00 53.15
50.01 60.25 56.06 62.20 62.17 58.18 62.17 66.03 63.00 56.20 58.18 62.04 60.40 58.00 53.02
49.88 59.37 55.75 61.86 61.82 57.78 61.84 65.64 62.66 55.93 57.72 61.69 59.65 57.70 52.84
49.92 59.49 55.84 61.93 61.89 57.85 61.91 65.73 62.88 56.07 57.88 61.85 59.83 57.77 52.96
49.28 58.95 55.45 61.70 61.48 57.32 61.41 65.24 62.08 55.95 57.92 61.46 59.30 57.32 52.37
48.45 57.28 53.91 59.28 59.02 55.87 59.63 63.50 59.88 53.71 55.50 59.05 57.95 55.66 50.24
46.30 55.64 51.16 57.44 57.07 53.74 57.62 61.27 57.81 51.39 53.70 57.98 55.89 53.34 48.20
8.1865 1.3093 59.45 59.91 58.77
1.7682 59.19 59.68 58.47
around 4 C if possible, or at least under 19 C to minimize the loss of amylose and starch content. In this study the moisture content did not have a major influence on amylose and starch content. This was a study on only one field trial, and the study needs to be repeated over locations and seasons to confirm the findings. References Agrobase, 2005. AGROBASE Generation II User’s Manual. Version 11, revised ed. Agronomix Software, Winnipeg, MB, Canada www.agronomix.com. Al-Yahya, S.A., 2001. Effect of storage conditions on germination in wheat. J. Agron. Crop Sci. 184, 273e279. Buckow, R., Jankowiak, L., Knorr, D., Versteeg, C., 2009. Pressureetemperature phase diagrams of maize starches with different amylose contents. J. Agric. Food Chem. 57, 11510e11516.
2.1069 58.81 59.24 58.14
2.0295 58.92 59.38 58.22
1.9344 58.48 58.95 57.77
1.4763 56.60 56.97 56.03
1.9190 54.57 55.02 53.89
Corcuera, V., Salmoral, E.M., Salerno, J.C., Krisman, C.R., 2007. Starch molecular fractionation of bread wheat varieties. Agriscientia XXIV, 11e18. Cruz, N.D., Khush, G.S., 2000. Rice grain quality evaluation procedures. In: Singh, R.K., Singh, U.S., Khush, G.S. (Eds.), Aromatic Rice. Science Publishers, Inc., India. Desai, B.B., 2004. Seeds Handbook: Biology, Production, Processing, and Storage, second ed. Marcel Dekker, Inc., New York. Doijode, S.D., 2001. Seed Storage of Horticultural Crops. The Haworth Press, Binghamton, New York. Fitzgerald, M.A., Bergman, C.J., Resurreccion, A.P., Möller, J., Jimenez, R., Reinke, R.F., Martin, M., Blanco, P., Molina, F., Chen, M.-H., Kuri, V., Romero, M.V., Habibi, F., Umemoto, T., Jongdee, S., Graterol, E., Reddy, K.R., Bassinello, P.Z., Sivakami, R., Rani, N.S., Das, S., Wang, Y.J., Indrasari, S.D., Ramli, S., Ahmad, R., Dipti, S.S., Xie, L., Lang, N.T., Singh, P., Toro, D.C., Tavasoli, F., Mestres, C., 2009. Addressing the dilemmas of measuring amylose in rice. Cereal Chem. 86, 492e498. AACC International, Inc. Liu, H., Eskin, N.A., Cui, S.W., 2003. Interaction of wheat and rice starches with yellow mustard mucilage. Food Hydrocoll. 17, 863e869.
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Lopes, D.C., Martins, J.H., Neto, A.J.S., Filho, A.F.L., 2008. Management of stored maize by AERO controller in five Brazilian locations: a simulation study. Biosyst. Eng. 101, 325e330. MacDonald, M.B., 1999. Seed deterioration: physiology, repair and assessment. Seed Sci. Technol. 27, 177e237. MacDonald, M.B., 2006. Physiological Causes of Seed Deterioration. Seed Biology Program, Department of Horticulture and Crop Science, Ohio State University. Seebiology.osu.edu/HCS631_FILES/9B%20Seed%20deterioration.pdf. Megazyme International, 2009. Megazyme Total Starch Assay Procedure K-TSTA 04/ 06. Ireland. Pongsawatmamit, R., Temsiripong, T., Ikeda, S., Nishinari, K., 2006. Influence of tamarind seed xyloglucan on rheological properties and thermal stability of tapioca starch. J. Food Eng. 77, 41e50. Ral, J.P., Cavanagh, C.R., Larroque, O., Regina, A., Morell, M.K., 2008. Structural and molecular basis of starch viscosity in hexaploid wheat. J. Agric. Food Chem. 56, 4188e4197.
Rehman, Z.U., Habib, F., Zafar, S.I., 2002. Nutritional changes in maize (Zea mays) during storage at three temperatures. Food Chem. 77, 197e201. Simi c, B., Popovi c, R., Sudari c, A., Rozman, V., Kalinovi c, I., Cosi c, J., 2007. Effect of storage condition on seed oil content of maize, soybean and sunflower. Agric. Conspec. Sci. 72, 211e213. Sofi, P.A., Wani, S., Rather, A.G., Wani, S.H., 2009. Quality protein maize (QPM): genetic manipulation for nutritional fortification of maize. J. Plant Breed. Crop Sci. 1, 244e253. Weather South Africa, 2013. www.tutiempo.net/en/climate/South_Africa/ZA.html (accessed January 2013). Zhu, T., Jackson, D.S., Wehling, R.L., Geera, B., 2008. Comparison of amylose determination methods and the development of a dual wavelength iodine binding technique. Cereal Chem. 85, 51e58.
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
The influence of storage conditions on starch and amylose content of South African quality protein maize and normal maize hybrids Maryke Labuschagne a, *, Lekgolwa Phalafala a, Gernot Osthoff b, Angeline van Biljon a a b
Department of Plant Sciences, University of the Free State, Bloemfontein 9300, South Africa Department of Microbial, Biochemical and Food Technology, University of the Free State, Bloemfontein, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 19 November 2013
The quality of maize grains during storage is affected by unfavourable storage conditions, resulting in physicochemical changes in specifically amylose and starch content that lead to significant product qualitative and quantitative losses. The objective of this study was to evaluate the starch and amylose content of normal maize and quality protein maize (QPM) seed samples at different temperature treatments: in cold storage at 3.6 C, at room temperature (18.5 C) and at 30 C for 0, 6 and 12 month storage periods, respectively. Sixteen genotypes were tested in a single trial at Potchefstroom in South Africa. Due to optimal growing conditions the seed was of excellent quality. Storage at 3.6 C and 18.5 C caused some reduction in amylose and starch content, although for starch it was not significant. On the other hand, storage at 30 C significantly (P 0.05) reduced the starch and amylose content after both 6 and 12 month periods of storage. QPM and non-QPM seed reacted similarly to storage conditions, and there were larger differences between cultivars than between QPM and non-QPM material. Even at relatively high relative humidity, low temperature storage maintained seed quality the best. Therefore high temperature storage is detrimental to starch and amylose content of both normal maize and QPM, especially after 6 months or more of storage. Maize should therefore be stored under low temperatures (around 4 C) or if not possible, at least under 19 C. Ó 2013 Published by Elsevier Ltd.
Keywords: Quality Protein Maize Storage Starch Amylose
1. Introduction Starch, the chief source of carbohydrate in human diets, forms the major energy reserve in cereal grains. Amylose and amylopectin are the two glucopolysaccharide components of starch. Starch granule physical properties are influenced by the distribution of amylose and amylopectin in starch (Corcuera et al., 2007). The starch and amylose contents of seed depend on the quality, physical structure and storage conditions of the seed after harvest (Buckow et al., 2009). Poor storage conditions often prevail and can lead to biochemical changes in chemical compounds of maize kernels including amylose and starch content during storage. The possible causes of these changes are not clear, but high relative humidity (r.h.) and the formation of free radicals during storage have been reported to play a role (MacDonald, 1999, 2006). These changes may affect the starch and amylose content. During storage, part of the starch and amylose has been reported to undergo an
* Corresponding author. Tel.: þ27 514012715. E-mail address: [email protected] (M. Labuschagne). 0022-474X/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jspr.2013.11.004
aggregation process that leads to changes in their content (Liu et al., 2003; Pongsawatmamit et al., 2006). There is a concern in terms of food security, on how to best store seeds, including maize seeds, for a long time without compositional changes, especially in developing countries worldwide. In these countries, efficient maintenance of grain quality is often not possible due to the lack of adequate post-harvest technology, including suitable storage conditions (Doijode, 2001). Quality protein maize (QPM) is nutritionally enhanced and has 70e100% more lysine and tryptophan than normal maize. QPM cultivars are being introduced in a number of African countries to reduce protein deficiencies in areas where maize is the staple food (Sofi et al., 2009). Little is known about how storage conditions affect both normal maize and QPM with special reference to starch and amylose content. It is better to implement preventive management, rather than to solve specific storage problems once they have occurred (Lopes et al., 2008). The aim of this study was to investigate the influence of different storage conditions on starch and amylose content extracted from the South African open pollinated QPM, a QPM hybrid and normal maize samples which were stored for 6 and 12
M. Labuschagne et al. / Journal of Stored Products Research 56 (2014) 16e20
months at 3.6 C, 18.5 C and 30 C compared to an untreated control. 2. Materials and methods 2.1. Planting location The trial was planted in Potchefstroom at the Agricultural Research Council (ARC)-Grain Crops Institute (26 740 S; 27 80 E) in November 2008. The ARC-Grain Crops Institute is located in Potchefstroom in the North West Province and works on a broad diversity of cereal crops grown in South Africa. Potchefstroom is located at an altitude of 1344 m above sea level with an average minimum and maximum temperature of 9.6 C and 25.5 C respectively with an average annual total rainfall of 619 mm (Weather South Africa, 2013). The rainfall during the growing season (November to April) was 409 mm which was well distributed over the season. The temperatures for the last two months of the growing season when grain filling took place ranged between a minimum of 12.6 C and a maximum of 27.9 C. Grain quality and yield was very good for this season. 2.2. Plant material The genotypes used in this study were eight South African open pollinated QPM varieties (SYN2QYQPM, SYN4QYQPM, SYN11QYQPM, SYN13QYQPM, SYN2QWQPM, SYN5QWQPM, SYN12Q WQPM and SYN15QWQPM), one QPM hybrid (QS7608) and six normal maize hybrids (CRW3505, C3505, CB341xI37F2, CB346xI37F2, P6479F2 and CB389xI37F2) obtained from the ARCGrain Crops Institute, Potchefstroom. 2.3. Experimental design and procedures The trial was planted for one season at a density of 50,000 plants per hectare on sandy clay loam. The experimental design was a randomised complete block design with three replications. The genotypes were grown in two-row plots. The rows were 5 m long with 0.3 m spacing apart, and row width was 90 cm. A compound fertilizer was applied at a rate equivalent to 300 kg per hectare of 3:2:1 (N:P:K) and then top-dressed with limestone ammonium nitrate (LAN). Standard cultural practices including ploughing, disking, and application of herbicides were done at the site in order to make nutrients easily accessible to the genotypes and to minimise competition for nutrients between the planted genotypes and weeds, as well as to reduce damage from insect pests. The trial was planted under rain-fed conditions. After harvesting, the seeds were dried to 12.5% moisture content and shelled with a stationary sheller. After shelling, a light table was used to confirm the status of the QPM genotypes. 2.4. Storage conditions Seed sampling was done randomly from each of the three replications after checking for QPM status. All samples were sealed in brown paper bags and stored under controlled conditions in a cold room (3.6 C), laboratory (18.5 C) and oven (30 C) for 6 and 12 months and were compared with an untreated control. The humidity was 76.5% r.h. in the cold room (3.6 C), and 28% r.h. in the laboratory (18.5 C). The laboratory was air conditioned which maintained a constant temperature. In the oven (30 C) moisture was very low or close to zero due to the prolonged exposure to heat. Forty-five bags (15 entries with three replications) were placed in the cold room at 3.6 C for 6 and 12 months. Another 45 bags were placed in the laboratory at 18.5 C for 6 and 12 months. The last 45
17
bags were placed in an oven at 30 C for 6 and 12 months. Each bag contained 90 maize seeds (45 seeds for the 6 months sampling, and 45 for the 12 month sampling). A randomized complete block design was used for the layout with three replications where each replication consisted of 45 seeds. After 6 and 12 month storage periods, 45 seed samples per bag were sampled and bulked and ground to a fine powder using a 1 kA analysis grinder, A10 Yellowline (Merck Chemicals Pty Ltd) with a 1 mm sieve. The processed samples were directly transferred to containers to avoid moisture accumulation. The seeds were then evaluated for starch and amylose content. 2.5. Starch extraction and analysis Starch content was determined according to the Megazyme total starch assay procedure (Megazyme, 2009). A homogenized milled sample (100 mg) of three replications of each genotype was measured into glass test tubes and 0.2 ml of aqueous ethanol (80% v/v) was added to wet the samples to aid dispersion. The tube was stirred on a vortex mixer. Two ml of dimethyl sulphoxide (DMSO) was added immediately and the tube was stirred on a vortex mixer. After that, the tube was placed in a boiling water bath at 95 C for 5 min. Three ml of thermostable a-amylase (300 U) was added in a 3-morpholinopropanesulfonic acid (MOPS) buffer (50 mM, pH 7.0) and the tubes were vigorously stirred on a vortex mixer. The tubes were incubated in a boiling water bath at 95 C for 6 min and stirred vigorously after 2, 4 and 6 min. The tubes were then placed in a water bath at 50 C after which sodium acetate buffer (4 ml, 200 mM, pH 4.5) was added, followed by amyloglucosidase (0.1 ml, 20 U). The volume was adjusted from 7 ml to 10 ml with distilled water and then the tubes were centrifuged at 3000 rpm for 10 min. The duplicate aliquots (0.1 ml) of the diluted solution were transferred to glass test tubes. Three ml of the glucose oxidaseperoxidase (GOPOD) reagent was added to each tube (including the glucose controls and reagent blanks), and the tubes were incubated at 50 C for 20 min. A glucose control was prepared by mixing 0.1 ml of glucose standard solution (1 mg ml1) and 3.0 ml of GOPOD reagent. A reagent blank solution was prepared by mixing 0.1 ml of water and 3.0 ml of GOPOD reagent. The absorbance was read in duplicate at 510 nm for each sample, and the glucose control was read against the reagent blank in duplicate. Total starch was measured as the glucose derived from hydrolyzed starch and was expressed as a percentage of total sample weight on an “as is” basis:
Total starch ¼ DAF FV=0:11=1000100=W 162=180 ¼ DAF=W FV0:9 where DA is the absorbance (reaction) read against the sample blank; F is a factor for the conversion from absorbance values to micrograms of glucose (100 mg of glucose/absorbance for 100 mg of glucose); FV ¼ final volume; 0.1 ¼ volume of sample analysed; 1/ 1000 is a conversion from micrograms to milligrams; 100/ W ¼ factor to express “starch” as a percentage of flour weight; W is the weight in milligrams (“as is” basis) of the flour analysed; and 162/180 is adjustment from free D-glucose to anhydro D-glucose (as occurs in starch). 2.6. Amylose determination Amylose was extracted and estimated by the iodine binding method (Cruz and Khush, 2000). One hundred milligram of homogenized maize flour sample was measured for three replications of each genotype. The samples were wetted with 1 ml of 95%
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M. Labuschagne et al. / Journal of Stored Products Research 56 (2014) 16e20
ethanol followed by 9 ml of 1 M sodium hydroxide (NaOH) to aid dispersion and stirred using a vortex mixer. The samples were placed in a boiling water bath for 15 min and stirred using a vortex mixer every 5 min. The samples were cooled for 1 h at room temperature and centrifuged at 3000 rpm for 5 min. Duplicate 0.1 ml aliquots of the solution were transferred to clean test tubes and 0.1 ml of 1 M acetic acid was added to each test tube followed by the addition of 0.2 ml iodine solution and 9.6 ml distilled water. The contents were vortexed and left to stand for 20 min. The absorbance was read in duplicate against the reagent blank at 620 nm for each sample. The amylose percentage was calculated using the formula:
h Amylose% ¼ Concentration mg ml1
i 1000=mass of the sample ðmgÞ 100
were significant differences between genotypes at all treatments, which were again not related to the type of maize genotype. The starch content of the maize genotypes at 18.5 C ranged between 49.9% and 65.7% after 6 months of storage and from 49.3% to 65.2% after 12 months of storage and at 30 C ranged from 48.5% to 63.5% after 6 months of storage and from 46.3% to 61.3% after 12 months of storage. There was a small reduction in starch content at the 3.6 C and 18.5 C treatments, but it was not significant. The reduction at 30 C was significant at both storage periods and it was 4.7 and 8.2% at 6 and 12 months respectively. The starch content of the non-QPM genotypes was slightly lower than that of the QPM genotypes for all treatments, but the differences were not significant. What was interesting was that SYN4QYQPM ranked in the first three positions for amylose and the last three for starch content while SYN15QWQPM ranked in the last two positions for both amylose and starch content for all the treatments. 4. Discussion
2.7. Statistical analysis Analysis of variance (ANOVA) for the 15 entries, three replications and treatments (control, heat and cold treatments for 6 and 12 months of storage) was used to determine the influence of storage conditions on starch and amylose content compared to an untreated control (Agrobase, 2005). 3. Results Mean squares for genotype and temperature treatments were highly significant (P 0.01), but interaction between cultivar and temperature treatments was not significant, indicating that the ranking of the genotypes remained largely the same at different temperature treatments for both starch and amylose content (Table 1). Amylose content varied significantly amongst tested maize genotypes (Table 2). The open pollinated QPM SYN2QWQPM consistently had the highest amylose content for all treatments. Normal hybrid CB341xI37F2 consistently ranked second or third for amylose content for all treatments. Open pollinated QPM SYN15QWQPM consistently had the lowest amylose content for all treatments. There was an average 10% and 21% reduction in amylose content after 6 and 12 month of storage at 30 C. The reduction was similar for QPM and non-QPM material. The reduction in amylose content at 3.6 C was not significant, irrespective of the time of storage. The amylose reduction at 18.5 C and 30 C was significant for both periods of storage. There were significant differences between genotypes for each treatment, but this was not related to genotypes being QPM or not. Open pollinated genotype SYN2QYQPM consistently had the highest starch content of all the genotypes for all the treatments (Table 3). SYN4QYQPM ranked second or third for all the treatments. Normal hybrid CRW3505 consistently had the lowest starch content for all the treatments, with open pollinated QPM SYN15QWQPM having the second lowest value throughout. There
Table 1 Mean squares for entry, treatment and their interaction for amylose and starch content. Source of variation
Amylose
Starch
Entry Treatment Entry Treatment
14.63** 88.17** 0.184
350.88** 142.62** 0.182
**P 0.01.
Amylose content was more sensitive to storage conditions than starch content, where storage at room temperature and 30 C caused a significant reduction in values, compared to starch where the only significant reduction in values was seen at storage at 30 C. Amylose mainly occurs in the amorphous region of the starch granule, either in an extended shape, or in a left-handed single helix, or in a parallel left-handed double helical form. In the latter case it might also be entwined with amylopectin (Ral et al., 2008). It is possible that the amylose may be partially degraded by amylase during storage, but it might also be that it is transformed from the extended shape to one of the two helix structures. Another reason why the amylose content decreases over storage time may lie in the analytical technique (Zhu et al., 2008; Fitzgerald et al., 2009). Although the iodine test is the most reproducible amylose test, the blue colour is produced due to binding to the single helix. The extended and double helix forms result in a colour of lower intensity. A change to the double helix structure may therefore result in a lower detection although the amylose content was not necessarily decreased. The reduced starch was consistent with other reports (Rehman et al., 2002; Simi c et al., 2007) where starch was reduced when exposed to high temperatures (25 C) for 6 months of storage. This occurrence was reported to be the effect of high temperature and r.h. as the latter showed to have a negative effect on chemical compounds of various seeds including maize starch (Al-Yahya, 2001). In this study the r.h. at 30 C was very low, which indicates that the high temperature by itself caused the reduction in starch content. These findings are in agreement with those reporting that unfavourable storage conditions cause seed quality losses (Al-Yahya, 2001; Rehman et al., 2002). Although the r.h. at the low temperature storage was very high, it did not seem to influence the starch or amylose content negatively. It seemed as though the low temperature was sufficient to preserve the status of the seed in terms of starch and amylose content. It was reported that the storage life of a seed doubles for each 5.6 C decrease in temperature (Desai, 2004). The QPM and normal maize genotypes showed a consistent reduction in both amylose and starch content, producing rankings that were consistent over treatments. The amylose and starch content of open pollinated QPM, the QPM hybrid and normal maize genotypes were equally affected by the different storage conditions over time. Large genotype differences were evident, suggesting that selection for increased starch and/or amylose content is possible within these genotypes. In conclusion, QPM and normal maize reacted the same to storage conditions and periods of storage. Maize should be stored at
M. Labuschagne et al. / Journal of Stored Products Research 56 (2014) 16e20
19
Table 2 Effect of different storage conditions on amylose content over time of storage. No.
Genotype
Type
Amylose (%) Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CRW3505 QS7608 C3505 CB341xI37F2 CB346xI37F2 P6479F2 CB389xI37F2 SYN2QYQPM SYN4QYQPM SYN11QYQPM SYN13QYQPM SYN2QWQPM SYN5QWQPM SYN12QWQPM SYN15QWQPM
Normal hybrid QPM hybrid Normal hybrid Normal hybrid Normal hybrid Normal hybrid Normal hybrid Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated
QPM QPM QPM QPM QPM QPM QPM QPM
LSD (0.05) LSD (0.05) across treatments
18.5 C
30 C
0 months
6 months
12 months
6 months
12 months
6 months
12 months
17.62 18.22 17.92 18.73 18.33 17.73 18.18 18.39 16.78 17.27 17.72 19.60 19.12 18.67 16.97
17.47 18.87 18.20 19.35 18.62 17.47 17.97 18.28 16.66 17.13 17.51 19.49 19.04 18.46 16.83
17.28 18.65 18.09 19.09 18.27 17.22 17.78 18.04 16.43 16.88 17.15 19.23 18.96 18.16 16.62
16.35 17. 50 17.74 18.72 17.81 17.12 17.50 17.74 16.24 16.48 16.81 18.86 18.68 17.74 15.90
15.62 16.41 16.77 17.03 16.80 16.12 16.53 16.69 15.22 15.43 15.80 17.81 16.37 16.72 15.35
15.56 17.16 16.29 17.43 16.74 15.58 16.09 16.33 14.72 15.26 15.69 17.54 17.18 16.68 14.93
13.57 15.13 14.28 15.41 14.69 13.63 14.12 14.32 14.14 12.96 13.75 15.56 15.15 14.47 12.64
1.4860 0.4168
Mean Mean for QPM Mean for normal hybrids
3.6 C
18.08 18.08 18.09
1.4899 18.09 18.03 18.18
1.1146 17.86 17.79 17.96
1.3089 17.41 17.33 17.54
1.4964 16.31 16.20 16.48
1.3304 16.21 16.17 16.28
1.1390 14.26 14.24 14.28
Table 3 Effect of different storage conditions on starch content over time of storage. No.
Genotype
Type
Starch (%) Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
CRW3505 QS7608 C3505 CB341xI37F2 CB346xI37F2 P6479F2 CB389xI37F2 SYN2QYQPM SYN4QYQPM SYN11QYQPM SYN13QYQPM SYN2QWQPM SYN5QWQPM SYN12QWQPM SYN15QWQPM
Normal hybrid QPM hybrid Normal hybrid Normal hybrid Normal hybrid Normal hybrid Normal hybrid Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated Open pollinated
LSD (0.05) LSD (0.05) across treatments Mean Mean for QPM Mean for normal hybrids
QPM QPM QPM QPM QPM QPM QPM QPM
3.6 C
18.5 C
30 C
0 months
6 months
12 months
6 months
12 months
6 months
12 months
50.06 60.58 56.19 62.65 62.56 58.57 62.56 66.20 63.21 56.60 58.56 62.12 60.80 58.00 53.15
50.01 60.25 56.06 62.20 62.17 58.18 62.17 66.03 63.00 56.20 58.18 62.04 60.40 58.00 53.02
49.88 59.37 55.75 61.86 61.82 57.78 61.84 65.64 62.66 55.93 57.72 61.69 59.65 57.70 52.84
49.92 59.49 55.84 61.93 61.89 57.85 61.91 65.73 62.88 56.07 57.88 61.85 59.83 57.77 52.96
49.28 58.95 55.45 61.70 61.48 57.32 61.41 65.24 62.08 55.95 57.92 61.46 59.30 57.32 52.37
48.45 57.28 53.91 59.28 59.02 55.87 59.63 63.50 59.88 53.71 55.50 59.05 57.95 55.66 50.24
46.30 55.64 51.16 57.44 57.07 53.74 57.62 61.27 57.81 51.39 53.70 57.98 55.89 53.34 48.20
8.1865 1.3093 59.45 59.91 58.77
1.7682 59.19 59.68 58.47
around 4 C if possible, or at least under 19 C to minimize the loss of amylose and starch content. In this study the moisture content did not have a major influence on amylose and starch content. This was a study on only one field trial, and the study needs to be repeated over locations and seasons to confirm the findings. References Agrobase, 2005. AGROBASE Generation II User’s Manual. Version 11, revised ed. Agronomix Software, Winnipeg, MB, Canada www.agronomix.com. Al-Yahya, S.A., 2001. Effect of storage conditions on germination in wheat. J. Agron. Crop Sci. 184, 273e279. Buckow, R., Jankowiak, L., Knorr, D., Versteeg, C., 2009. Pressureetemperature phase diagrams of maize starches with different amylose contents. J. Agric. Food Chem. 57, 11510e11516.
2.1069 58.81 59.24 58.14
2.0295 58.92 59.38 58.22
1.9344 58.48 58.95 57.77
1.4763 56.60 56.97 56.03
1.9190 54.57 55.02 53.89
Corcuera, V., Salmoral, E.M., Salerno, J.C., Krisman, C.R., 2007. Starch molecular fractionation of bread wheat varieties. Agriscientia XXIV, 11e18. Cruz, N.D., Khush, G.S., 2000. Rice grain quality evaluation procedures. In: Singh, R.K., Singh, U.S., Khush, G.S. (Eds.), Aromatic Rice. Science Publishers, Inc., India. Desai, B.B., 2004. Seeds Handbook: Biology, Production, Processing, and Storage, second ed. Marcel Dekker, Inc., New York. Doijode, S.D., 2001. Seed Storage of Horticultural Crops. The Haworth Press, Binghamton, New York. Fitzgerald, M.A., Bergman, C.J., Resurreccion, A.P., Möller, J., Jimenez, R., Reinke, R.F., Martin, M., Blanco, P., Molina, F., Chen, M.-H., Kuri, V., Romero, M.V., Habibi, F., Umemoto, T., Jongdee, S., Graterol, E., Reddy, K.R., Bassinello, P.Z., Sivakami, R., Rani, N.S., Das, S., Wang, Y.J., Indrasari, S.D., Ramli, S., Ahmad, R., Dipti, S.S., Xie, L., Lang, N.T., Singh, P., Toro, D.C., Tavasoli, F., Mestres, C., 2009. Addressing the dilemmas of measuring amylose in rice. Cereal Chem. 86, 492e498. AACC International, Inc. Liu, H., Eskin, N.A., Cui, S.W., 2003. Interaction of wheat and rice starches with yellow mustard mucilage. Food Hydrocoll. 17, 863e869.
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Lopes, D.C., Martins, J.H., Neto, A.J.S., Filho, A.F.L., 2008. Management of stored maize by AERO controller in five Brazilian locations: a simulation study. Biosyst. Eng. 101, 325e330. MacDonald, M.B., 1999. Seed deterioration: physiology, repair and assessment. Seed Sci. Technol. 27, 177e237. MacDonald, M.B., 2006. Physiological Causes of Seed Deterioration. Seed Biology Program, Department of Horticulture and Crop Science, Ohio State University. Seebiology.osu.edu/HCS631_FILES/9B%20Seed%20deterioration.pdf. Megazyme International, 2009. Megazyme Total Starch Assay Procedure K-TSTA 04/ 06. Ireland. Pongsawatmamit, R., Temsiripong, T., Ikeda, S., Nishinari, K., 2006. Influence of tamarind seed xyloglucan on rheological properties and thermal stability of tapioca starch. J. Food Eng. 77, 41e50. Ral, J.P., Cavanagh, C.R., Larroque, O., Regina, A., Morell, M.K., 2008. Structural and molecular basis of starch viscosity in hexaploid wheat. J. Agric. Food Chem. 56, 4188e4197.
Rehman, Z.U., Habib, F., Zafar, S.I., 2002. Nutritional changes in maize (Zea mays) during storage at three temperatures. Food Chem. 77, 197e201. Simi c, B., Popovi c, R., Sudari c, A., Rozman, V., Kalinovi c, I., Cosi c, J., 2007. Effect of storage condition on seed oil content of maize, soybean and sunflower. Agric. Conspec. Sci. 72, 211e213. Sofi, P.A., Wani, S., Rather, A.G., Wani, S.H., 2009. Quality protein maize (QPM): genetic manipulation for nutritional fortification of maize. J. Plant Breed. Crop Sci. 1, 244e253. Weather South Africa, 2013. www.tutiempo.net/en/climate/South_Africa/ZA.html (accessed January 2013). Zhu, T., Jackson, D.S., Wehling, R.L., Geera, B., 2008. Comparison of amylose determination methods and the development of a dual wavelength iodine binding technique. Cereal Chem. 85, 51e58.