Gram Research Laboratory. Winnipeg. Manitoba, m ^^^ ^ ^p,^ harvest ripeness

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1 Vol. 79, 1973] 471 CHANGES IN THE FREE SUGAR CONTENT OF BARLEY KERNELS DURING MATURATION By D. E. LaBerge, A. W. MacGregor and W. 0. S. Meredith (Canadian Grain Commission, Winnipeg, Manitoba) Received 22nd June, 1973 The free sugars extracted from kernels of a two-row barley variety, Betzes, were separated and determined quantitatively as negatively-charged borate derivatives by anion-exchange chromatography on substituted polystyrene resin. Sugars were determined at 2 or 3 day intervals immediately following anthesis, until kernels achieved harvest ripeness. Initially, the free sugars accounted for 6*5% of kernel dry matter; reducing sugars comprised 80% of the total free sugars, primarily as fructose (3% of the kernel dry matter) and glucose (2% of kernel dry weight), with lesser amounts of galactose, mannose, and maltose. was the principal non-reducing sugar (1-8% of the kernel dry weight), but traces of raffinose were also detected. As kernel dry matter increased, the relative amount of free sugars decreased rapidly and comprised 2% of the kernel dry matter in mature kernels. At harvest ripeness, non-reducing sugars comprised 90% of the total free sugars. decreased slightly from 1-8 to 1-2% of total kernel dry matter, but actually increased from 90 /tg in immature kernels to 500 /ig in mature kernels; raffinose increased relative to kernel dry matter from trace amounts 20 days after anthesis to 0-7% of kernel dry matter (i.e., 250 pg/kernel) 36 days after anthesis, and declined to 0-4% in mature kernels. Non-reducing, soluble fructosans were present at all stages of kernel maturation, but were not determined quantitatively. Paper No. 327 of the Canadian Grain Commission, sugars of barley kernels immediately follow- Gram Research Laboratory. Winnipeg. Manitoba, m ^^^ ^ ^p,^ harvest ripeness of the kernels. Key words: barley, seed, sugars. Materials and Methods The samples of Betzes two-row malting Introduction barley were harvested during the 1970 crop The present investigation was undertaken year. The complete temperature, sunshine as part of a larger project relating to chemical and precipitation record for Winnipeg during and biochemical changes that occur as barley the 1970 growth period, and the dates of kernels ripen under field conditions. On the seeding, anthesis, cutting, and threshing Canadian Prairies, barley plants normally have been published previously.8 On selected grow vegetatively for 45 to 60 days prior to days after anthesis, 200 to 250 ears were flowering, and kernels require another 35 to harvested randomly from the plot. The 50 days to become fully mature. Malting ears were deep frozen and freeze-dried to barley varieties are screened and selected approximately 4% moisture, for their ability to be fully mature within The dried ears were threshed by placing 95 to 100 days from the date of seeding. them in small cotton sacks and rubbing the Previous studies have dealt with changes sacks by hand until all the kernels had been of protein, ash, fat, fibre, total sugars, and liberated from the ears. The awns and starch,9 and changes of a-amylase and /3- other extraneous materials were removed amylase activities8-8'10 during the process of by hand sieving, and the cleaned kernels at kernel maturation. The present study each sampling date were ground in a Wiley describes changes in the composition of free mill equipped with a 1-mm sieve.

2 472 LABERGE et al.: FREE SUGARS OF MATURING BARLEY [J. Inst. Brew. Sugars were extracted utilizing a Soxhlet extraction apparatus. One gram of ground sample was weighed into a Soxhlet thimble and extracted for 20 hours with 80% ethanol. The extracts were taken to dryness under vacuum on a rotary evaporator, and the residues were dissolved in 10 ml of distilled water. The aqueous extracts were stored frozen until used for chromatography. The sugars were separated as negativelycharged borate derivatives by ion-exchange column chromatography on 8% crosslinked polystyrene resin substituted with quater nary amine groups.2-7 Type S (Technicon, Tarrytown, N.Y.) or Aminex A-25 (BioRad Laboratories, Richmond, Calif.) resins with bead sizes from 15 to 20 microns produced satisfactory chromatograms. Two columns were used for separating sugars. The short column was 6 mm x 75 cm, and the long column was 6 mm x 110 cm. The columns were equilibrated initially with M borate buffer, ph 7. The frozen sugar extracts were thawed, and an aliquot of 0*1 ml was loaded on the columns under pressure, using nitrogen from a highpressure cylinder. Standard sugar solutions were prepared containing 50 /xg of each sugar per 0-1 ml of solution. The sugars were eluted from the ion-exchange resin with a combination of a ph gradient and a linear increase in borate ion concentration. The gradient was produced by a two-vessel gradient device. One vessel contained 220 ml of the limit buffer, 0-6-M borate buffer, ph 10. The buffer in this vessel flowed into a mixing vessel that contained a stirring bar; the latter vessel was charged initially with 220ml of 0075-m borate buffer, ph 7. The columns were operated at a flow rate of 1 ml/min, and the jacketed columns were maintained at 55 C. Column pressures during the chromatography were 150 lb/ina for the short column, and 600 lb/in2 for the long column. The column effluent was monitored con tinuously at 420 nm, utilizing a Technicon AutoAnalyzer, by mixing the column efflu ent with 0-1% orcinol in 70% H2SO4 and heating at 95 C in a bath equipped with a 40 ft coil to develop the colour.2-7 The stream was passed through a jacketed cool ing coil mounted over a fluorescent bulb to increase the sensitivity of the reaction prior to flowing the stream through the colorimeter. The cooling coil and fluorescent lamp were enclosed in a black box. The chart speed of the recorder was 2 in per hour. Sugars were determined quantitatively by calculating the ratio of the area under a particular peak, relative to the area under the curve for a known standard sugar. The peak height absorbance and the width of the peak in mm at one-half the peak height absorbance were used to calculate areas under the curve. Results A typical chromatogram produced by chromatography of a standard sugar solu tion on a short ion-exchange column is shown in the upper portion of Fig. 1. The system will also resolve other sugars. How- Standard sugars Raffinose Mannose Glucose.Fructose ' Elution time (hours) Fig. 1. Separation of borato derivatives of sugars by ion-exchange chromatography. Upper chromatogram standard sample of sugars containing 60 pg of each sugar. Lower chroma togram extract of barley kernels harvested 36 days after anthesis.

3 Vol. 79, 1973] LABERGE tt al.\ FREE SUGARS OF MATURING BARLEY 473 ever, only traces of arabinose and xylose were observed on some chromatograms, and these sugars were omitted from the standard samples. The sugars detected in an extract of a sample that was harvested 36 days after anthesis are shown in the lower chromatogram in Fig. 1. The sugars were resolved by chromatography on the short column, and it was relatively simple to determine accurately the amount of each sugar. The fructosan component was not identified, as no standard fructosan sugars were available. However, it seems likely that the fructosan peak was primarily glucodifructose.11 During the early stages of grain matura tion, fructosans, sucrose and raffinose were not resolved very well by chromatography of extracts on a short ion-exchange column (left portion of Fig. 2). The separation of fructosan, sucrose and raffinose was not sufficient to permit an accurate measure ment of sucrose and raffinose. These sugars were determined on chromatograms obtained by partitioning another aliquot of extract on a longer ion-exchange column (Fig. 2). Fructosans, sucrose, and raffinose were eluted by washing the column for 3 hours with M borate buffer, ph 7, and then terminating the chromatography by washing the column with 6% potassium tetraborate to elute the other sugars that were still absorbed on the ion-exchange resin. The fructosans were separated partially into several components by this procedure. The development of barley kernels at various stages following anthesis is shown in Fig. 3. Initially, the immature kernel consists primarily of husk and pericarp tissue, but the endosperm develops rapidly 10 days after anthesis. As a result, the dry matter of the kernel increases very rapidly in the period between 10 and 30 days after anthesis and levels off during the final stages of grain maturation. It was shown previously that the increase in dry matter was largely due to the production of amylopectin and amylose.9 The changes in the quantities of various sugars during grain ripening are shown in Long column (15 days) Elution time (hours) Fig. 2. Comparison of resolution of sugars on short columns and long columns of anion-exchange resin. Extracts from very immature kernels, e.g.. kernels harvested 15 days after floral anthesis, contain large quantities of fructosan material that can only be separated from sucrose by chromatography on longer columns.

4 474 LABERGE el (U. I FREE SUGARS OF MATURING BARLEY [J. Inst. Brew. "3 30.? I I I 1,1.1 I I Fig. 3. Changes of dry matter during development and maturation of Bctzes barley Galactose 0 F - y yr I, I. I i o Fructose ^.-^_ r * ~«Ov J Raffinose - G^ose* -^^3^ 1 i 1. 1 t 1. 1 i I \ -o- - f\r * Nv Ma tose 1. 1, 1, 1 i Fig. 4. Changes in the content of free sugars during kernel maturation. Note the different scales used on the ordinatc axis of each graph. 50 Fig. 4. was low initially but in creased steadily as ripening progressed until sucrose was the predominant sugar. Fructose and glucose were the predominant sugars in very immature kernels. Following an initial increase, these sugars decreased as the grain matured. Traces of raffinose were detected during initial stages of grain maturation, but this sugar increased rapidly from 20 to 40 days from anthesis, and declined during the final stages of ripening. Mannose and galactose were observed in the very im mature kernels, but only traces of these sugars were detected as ripening progressed. Maltose increased in the period between 10 and 25 days after anthesis, and then de creased to low values in the mature kernel. The increase of maltose closely paralleled the production of free and bound /3-amylase shown previously for these samples of Betzes barley.8 Other interesting relationships can be demonstrated by expressing the changes of the sugar composition at various stages of kernel maturation in terms of percentage of the total dry weight of the kernel. In the very immature kernels, the total soluble sugars accounted for 6 % of the dry weight of the kernel (Fig. 5). The proportion of total sugars relative to the dry matter of the kernel decreased as kernel ripening progressed and accounted for 2% of the weight of the kernel at harvest ripeness. The data in Fig. 5 were based on soluble sugars but omitted the soluble, non-reducing fructosan material, which undoubtedly was also very important. The decrease in sugars was due primarily to a decrease in the proportion of reducing sugars. Initially, reducing sugars accounted for 5% of the dry matter but decreased to 0-25% of the dry matter at harvest ripeness. The nonreducing sugars accounted for approximately 1-8% of the dry matter at all stages of kernel development. The reducing sugars, fructose and glucose, decreased quite rapidly in relation to the rapid increase in the dry matter of the kernel (Fig. 6). Of the non-reducing sugars, sucrose tended to decrease relative to the increase of dry matter, but the values were more variable on the different days that kernels were harvested. This variability was probably the result of diurnal variation of sucrose. Ramnose increased relative to the dry matter of the kernel, starting from

5 Vol. 79, 1973] laberge el al.: free sugars of maturing barley v Fructose.f. Total sugars I 4 i 3 o * I I I J, I, I Fig. 5. Changes in free sugar composition in relation to kernel dry matter. The soluble, non-reducing fructosan sugars were not included in these graphs. Raffinose a low value (0-1%) after 20 days from anthesis to 0-7% of the kernel dry matter after 36 days. The rafnnose content then decreased slightly to 0-4% of the dry matter as the kernels became fully mature. The changes in the amounts of reducing and non-reducing sugars relative to one another are shown in Fig. 7. Initially, the reducing sugars, primarily glucose and fruc tose, were predominant but decreased in proportion to the non-reducing sugars, sucrose and raffinose. Approximately 18 days after anthesis, the non-reducing sugars were major sugar constituents and these sugars continued to increase in proportion to the reducing sugars. In the fully mature kernel, non-reducing sugars comprised 90% of the total soluble sugars. It would be extremely useful to measure the changes in fructosan components in order to determine to what extent these non-reducing sugars would alter the relationships shown in Fig. 7. Discussion There is a considerable amount of informa tion relating to the carbohydrate composition 1.III I I I I I Fig. 6. Changes of fructose, glucose, sucrose and raffinose relative to changes of kernel dry matter. of mature barley * and to changes that occur during malting.*-14-18'19 Except for the earlier work by Harris & MacWUliam*-9 and the recent work by Cerning & Guilbot,3 there is very little information regarding changes in carbo hydrate composition during the development and maturation of barley kernels. Most of the earlier data regarding changes in the composition of free sugars were obtained using paper chromatography to separate the sugars. Recent advances in the use of polystyrene resins to separate borate derivatives of sugars, coupled with automated systems for determining sugars colorimetrically, have resulted in the 50

6 > 60 3 IU 2 I 2 40 o as LABERGE el al.l FREE SUGARS OF MATURING BARLEY [J. Inst. Brew. I I I I Fig. 7. Relative changes of reducing and nonreducing sugars during kernel development. development of carbohydrate analysers with improved accuracy and precision for deter mining sugars. Using such a system, Abou- Guendia & D'Appolonia1 have recently studied the changes in free sugars during maturation of wheat kernels. The present investigation was performed using a Technicon Carbohydrate Analyzer2 to study changes in free sugar composition of barley kernels during the ripening process, and in many respects confirms the results obtained by paper chromatography reported by Harris & MacWilliam5'0 and Cerning & Guilbot.3 In contrast to previous findings,6 fructose and glucose were the principal sugars in very immature kernels. However, as dry matter increased, these sugars decreased and, at the same time, the amount of sucrose increased and was the major sugar during the later stages of kernel maturation. The amount of fructosans appeared to be greatest at very early stages of grain maturation and decreased as the kernel matured, as reported previously." In extracts from very im mature kernels, the large quantities of fructosan material interfered with the separa tion of sucrose. The trisaccharide, glucodifructose, was not determined or identified as purified samples of this sugar were not available. Harris & MacWilliam6 found that this non-reducing sugar was a principal constituent of extracts from early, immature kernels. Glucodifructose was detected origi nally by MacLeod" in extracts from mature barley. The changes in raffinose composition were similar to those reported previously for barley9 and for wheat.1 Initially, traces of this sugar were observed on chromatograms, but between 20 and 35 days after anthesis, the relative amount of raffinose increased and, together with sucrose, it was a major sugar constituent of mature kernels. Galactose, a monosaccharide component of the raffinose molecule, was not detected in kernel extracts following the increase in raffinose content. Raffinose is present primarily in the embryo of the kernel13." and declines slowly during germination with no further accumulation of raffinose in the embryo during germina tion It is likely that raffinose has some special metabolic function in embryo tissue. The kernels of Betzes barley were tested for germination potential following each sampling.8 Initial germination occurred 41 days after anthesis and 50% germination was observed after 48 days. Initial germina tion occurred following the accumulation of raffinose, and it would be interesting to determine if raffinose accumulation occurs before the embryos can germinate for other barley varieties. The maltose content of kernels was low initially, and from 15 days to approximately 30 days from anthesis increased to 50 micrograms per kernel and then fell to low levels in the mature kernels. The increase in maltose content coincides with the appear ance of "free" and "bound" j8-amylase in the same samples of Betzes barley.8 How ever, after reaching maximum activity 32 days after anthesis, the j8-amylase activity remained constant as the kernels became fully ripened. The present study has not accounted for changes in the fructosan component of barley kernels. In future work, attempts will be

7 Vol. 79, 1973] LABERGE d al.\ FREE SUGARS OF MATURING BARLEY 477 made to separate, identify and quantify this major carbohydrate fraction. Acknowledgement. The authors express their gratitude to Miss Joan Morgan for her capable technical assistance. References 1. Abou-Guendia, M., & D'Appolonia, B. L.,, Cereal Chemistry Catravas, G. N., in Automation in Analytical Chemistry, Tcchnicon Symposia, Mcdiad Incorporated, White Plains, N.Y., 1900, 1, Cerning, J., & Guilbot, A., Cereal Chemistry, , Harris, G., & MacWilliam. I. C, Journal of the Institute of Brewing. 1954, 60, Harris, G., & MacWilliam, I. C, Journal of the Institute of Brewing, 1954, 60, Harris, G., & MacWilliam, I. C, Journal of the Institute of Brewing, 1957, 63, Keslcr, R. B., Analytical Chemistry, 1907, 39, LaBerge, D. E., MacGregor, A. W., & Meredith, W. O. S., Canadian Journal of Plant Science, 1971, 51, MacGregor. A. W., LaBerge, D. E., & Meredith, W. O. S., Cereal Chemistry, MacGregor. A. W., Gordon, A. G.. Meredith. W. O. 3., & Lacroix, L.. Journal of the Insti tute of Brewing, 1972, 78, MacLeod, A. M., Journal of the Intsitute of Brewing, 1961, MacLeod, A. M., Journal of the Institute of Brewing, 1952, 58, MacLeod, A. M., Journal of the Institute of Brewing, 1952, 58, MacLeod, A. M., Travis, D. C, & Wreay, D. G. journal of the Institute of Brewing, 1953, 59, MacLeod, A. M., Journal of the Institute of Brewing, 19S3, 59, MacLeod. A. M., & Prcece, I. A.. Journal of the Institute of Brewing, 1954, MacLeod, A. M., New Phytologist, 1957, 56, MacLeod, A. M., Wallerstein Laboratories Com munications. 11)00. 23, MacLeod, A. M., Brewers' Digest, Mar., 1962, Ohms, J. I., Zee, J., Benson, J. V., & Patterson, J. A., Analytical Biochemistry, 1987, 20, Palmer, G. H., Journal of the Institute of Brewing, 1969, 75, 505.