Sugarbeet Biochemical Quality Changes During Pile Storage. Part I

January-March 2001 Biochemical Quality Changes During Pile Storage 35

Sugarbeet Biochemical Quality Changes During Pile Storage. Part I. Sugars.

2 Susan S. Martini, Judy A. Narum , and Ken H. Chambers3

lUSDA, ARS, Crops Research LaboratOl)" 1701 Center Avenue, Fort Collins Co. 80526; 2(jormerl}) Beet Sugar Development Foundation, 800 Grant St., Suite 300, Denver Co. 80203; and 3Holly Sugar COIporation, P.o. Box 9, Sugar Land, Texas 77487

ABSTRACT

Sucrose and quality losses in pile-stored sugarbeets have financial implications for growers and processers alike. Groups oflocally adapted varieties were grown and piJe stored at Sidney MT, Worland WY, and Hereford TX, to investigate varietal effect on losses during storage. Paired root samples were prepared at harvest. One of each pair immediately was analyzed for sucrose by polarimetry (pol), and a portion of each sucrose filtrate was frozen for later HPLC analysis for "true" sucrose, glucose, fructose, and raffinose. The second sample of each pair, in an air-permeable bag, was placed into the factory storage pile for 110 d at Sidney, 90 d at Worland, or 56 d at Hereford, then recovered and analyzed similarly to the unstored samples. Data were analyzed separately for each location. Both at harvest and after storage, pol sucrose overestimated true sucrose concentration as determined by HPLC. At the three locations, the average pol error, the percentage by which pol sucrose minus HPLC sucrose differed from the HPLC sucrose value, was +2% to +]9% at harvest,and +9% to +14% after storage. Glucose and fructose concentrations were low at harvest and increased significantly with storage at each location. Significant varietal difference after storage occurred for glucose at Worland and for fructose at Sidney and Worland. Raffinose concentra tions at harvest were low at Sidney and Worland, 36 Journal of Sugar Beet Research Vol 38 No 1

increasing significantly with storage. Varietal difference for raffinose after pile storage occurred at Worland. The raffinose concentration was unexpectedly high at harvest at Hereford, but did not increase significantly during piJe storage. Additional Key Words: Beta vulgaris, sucrose, glucose, fructose, raffmose, pol sucrose, HPLC sucrose, true sucrose, quality.

Most of the U.S. sugarbeet (Beta vulgaris L.) crop is planted in the spring and harvested in the fall. Such beets are grown mainJy in temperate areas with relatively short growing seasons. Because photosynthesis and sugar accumulation continue until below-freezing temperatures kill the tops, it is desirable to allow as long a growth period as possible. Nevertheless, sugarbeet roots must be removed from the ground before it freezes, and sugar factories must be run for several months to process the crop economically. Because of these factors, the harvest period usually is compressed iuto a short period of time, and large quantities of sugarbeet roots are stored for lengthy periods prior to processing. For many years sugarbeet agriculturists have been aware that significant amounts of recoverable sucrose were lost during such storage. McGinnis (1982) summarized U.S. industry storage practice and the factors leading to loss of sucrose during storage, and reviewed the literature on these topics prior to 1982. A more recent review was provided by Bugbee (1993), who summarized sucrose loss in storage, its causes, and methods for reducing losses. Vukov and Hangyal (1985) and Harvey and Dutton (1993) reviewed factors affecting root quality and its relationship to storage and processing quality. Sugarbeet cultivars differ in the rate and degree to which they lose sucrose or recoverable sugar during storage (e.g., Wyse and Dexter, 1971 b; Akeson and Widner, 1981). Wyse and his co-workers pointed out the importance of respiration in storage losses and found variation in respiration among cultivars (Wyse, 1973; Wyse, 1978; Wyse and Peterson, 1979; Wyse et aI., 1979). In addition, pre-harvest and harvest factors affect sugarbeet quality (e.g., Cole, 1977), which in tum may affect storage and processing quality. However, such studies often were limited in the number or types ofcultivars included, frequently including breeding lines and employing special, limited or controlled storage conditions. In addition, sucrose loss in such experiments was detelmined by polarimetry ofsamples pre- and post-storage. Polarimetric (pol) sucrose determination as practiced in sugarbeet tare labs depends on an assumption that the optical rotatory property of a given sample is due only to sucrose-i.e., January-March 2001 Biochemical Quality Changes During Pile Storage 37 that optically active extract components such as other sugars and amino acids are present only in very low concentration or have rotatory properties that tend to cancel one another. Neither assumption is necessarily true, particularly for stored or degraded samples. With more sensitive and specific techniques now available for analysis of sucrose, other sugarbeet sugars, and major quality components, we decided to re-explore the question of varietal effect on losses in storage. Our objective was to compare the biochemical changes that occurred during factory pile storage oflocally adapted, commercial cultivars that were harvested by standard conunercial practice and stored in sugar factory storage piles. The experiment included three locations, in each case with a group ofcultivars accepted for cultivation there. Only the sugars data are reported here; the nonsugar components also determined in this study will be reported separately.

MATERIALS AND METHODS

This experiment was done during the 1989 growing season at Holly Sugar Corporation factories in Sidney, Montana; Worland, Wyoming; and Hereford, Texas. At each location a different set oflocally adapted varieties was grown (Table 1); thus, each location was analyzed

Table 1. Experimental locations (1989), sugarbeet cultivars studied at each location, and length offactory pile storage.

Pile Location Cultivars Storage (days)

Sidney, Montana Beta 1996 (Betaseed), K W 1745, 11 0 M865 (Maribo/American Crystal), Monohikari (Seedex), MonoHy 110 I [Hilleshog (Novartis)], MonoHy 1105

Worland, Wyoming HH44 (Holly), HH50, MonoHy R2 90 (Hilleshog), ACH 164 (American Crystal), ACH 191

Hereford, Texas HH42 (Holly), TX 18 (Hilleshog) 56 separately for this report. Holly Sugar Corporation 's agricultural personnel at each location grew strip trials and harvested the varieties chosen, following local commercial practice for planting, fertilization, irrigation, 38 Journal of Sugar Beet Research Vol 38 No 1

and harvest. In each case, sugarbeet root samples were collected by random selection from mUltiple rows. First, several rows of the variety to be sampled were selected randomly from the strip trial; next, from the selected rows, two samples often beets each were randomly selected and placed into paired tare bags. Five replicate sample pairs were collected for each cultivar and labelled by variety and replicate. Samples were transported immediately to the factory where one of each sample pair was weighed, placed into an air-permeable sack, and placed into a factory pile. Sample placement was chosen so that a moderately long period of pile storage would result before that portion ofthe pile was to be processed. On the same day one sample of the pair was placed in the pile, the other sample of each pair was weighed, washed, reweighed for tare determination, and processed through the factory tare lab for polarimetric sucrose analysis after the standard clarification by aluminum sulfate. A portion of each aluminum-clarified sample was placed in a vial and immediately frozen. These "laboratory samples" were transported on dry ice to the USDA-ARS sugarbeet laboratory in Fort Collins, Colorado, where they were held at -20 C until further processing as described below. When the portion of each factory pile containing the captive samples was reached for normal pile processing, all samples were recovered by hand and immediately washed, weighed, and processed through the factory tare lab as described above. Sucrose filtrate samples were similarly collected, frozen, transported, and held at Fort Collins until analyzed. Samples were placed in the pile and removed from the pile, respectively, on October Il1January 29 at Sidney; October 12/January 10 at Worland; and November 21/January 16 at Hereford. Thus, pile storage time was 110 d at Sidney, 90 d at Worland, and 56 d at Hereford. For analysis of sugar components and betaine, sucrose filtrate samples were thawed at room temperature (approximately 2 hr at 21 C), diluted I + 2 v/v (sample + HPLC-grade water), vortex mixed, then passed through Waters (Waters Corporation, Milford MA) carboxymethyl (CM) Sep-Paks to remove aluminum III, discarding the fust 2 ml then collecting the next ml eluted. Each sample was then filtered (0.45 t-tm) and transferred to an autoinjector vial. Twenty microliter samples were autoinjected (Waters WISP 712) for separation by HPLC on a Waters SugarPak I cation exchange column in the calcium form, held at 85 C by a FIAtron CH-30 column heater (Rainin Instrument Co., Wobul1l MA). The column was preceded by a 0.22 t-tm prefilter. Sugars were eluted with 0.2 roM calcium EDTA solution prepared with freshly boiled HPLC-grade water and degassed with helium. Eluant flow of 0.5 ml min· t was maintained by a Spectra-Physics SP8800 ternary solvent pump; detection was by refractive index (Spectra-Physics SP6040; Spectra-Physics Corp., San Jose CA) January-March 2001 Biochemical Quality Changes During Pile Storage 39

and data were recorded by a Perkin Elmer LC-l 00 computing integrator (Perkin-Elmer Corp., Norwalk CT). Three working standards containing varied concentrations ofsucrose, glucose, fructose, raffinose, and betaine were prepared daily from frozen stock solutions. The analytical procedure was standardized each day with three standards analyzed before and after each group of ten experimental samples. To provide greatest accuracy, calculations for each sample group were made from regressions established from the standards run before and after the group. If the regression coefficient (r) for standards was less than 0.995, that group of samples was rerun with new standards. The HPLC procedure gave excellent separation of glucose, fructose, sucrose and raffinose in standards; galactose also was separated but was not tabulated because detectable quantities occurred only sporadically. We note specifically that our procedure was not designed to separate other trisaccharide sugars that could be present in small amounts, such as the kestoses (fructosyl-sucroses: l-kestose, 6-kestose, and neokestose; Kand ler and Hopf, 1982). Ifpresent, these sugars probably would have co-eluted with raffinose and been tabulated as that compound. No commercial kestose standards were available to examine this possibility. From measured pol sucrose and HPLC ("true") sucrose values, pol error as a percent of "true" sucrose was calculated as [(pol sucrose HPLC sucrose )IHPLC sucrose] X 100. Data summary statistics and analyses ofvariance for each component at each location were calculated with SAS (SAS Institute, Inc., Cary NC). Where the anova F-test was significant, means were separated by Duncan's multiple range test.

RESULTS AND DISCUSSION

Sucrose Across the varieties tested at each location, pol sucrose at harvest averaged 16.1% of root fresh weight at Sidney, 18.5% at Worland, and 15.7% at Hereford. After pile storage for 110 d, 90 d, and 56d, respectively, those values decreased to 14.6%, 18.1 %, and 14.6%. Storage conditions at Sidney were considered "the worst in years," according to local factory personnel, and the stored beet bags at the time of removal were observed to be a few feet away from a rotted spot with aLmost totally decayed, black beets. However, the pile stored beet samples for this experiment were not appreciably diseased or abnormal in appearance. Beets removed from the Worland and Hereford piles also were sound. In every case, HPLC-detetmined "true" sucrose concentrations (% of root fresh weight) were lower than the corresponding pol sucrose values. At harvest, average HPLC sucrose contents were 15.9% at Sidney, 40 Journal of Sugar Beet Research Vol 38 No 1

16.6% at Worland, and 12.7% at Hereford, declining after storage to 13.0%, 16.5%, and 12.6%, respectively (Figure 1). Thus, in every case pol sucrose

20 Sucrose (% of root fresh weight)

5 o Sidney MT Worland WY Hereford TX

~ Pol, at harvest o HPLC, at harvest IlIIlll Pol, post-storage !88! HPLC, post-storage

Figure 1. Pol sucrose vs. true (HPLC-determined) sucrose, at harvest and after factory pile storage, at tlu"ee locations. Data cannot be compared among locations, as different sets of locally adapted varieties were used. Storage period was 110 d at Sidney, 90 d at Worland, and 56 d at Hereford. determination overestimated the true sucrose content of the roots. At harvest the pol error (error expressed as a percentage of the HPLC "tme" sucrose value) was minimal at Sidney, but considerable at Worland and Hereford (about 10% and 19%, respectively; Figure 2). After storage, pol error ranged from 9% to 14% at the three locations (Figure 2). Polarimetric sucrose determination is recognized as an approximation that depends on the assumption that sucrose is the only component in the prepared extract that rotates the plane of polarized light, or that other rotating compounds such as non-sucrose sugars and some amino acids are present in low concentrations or have optical properties that tend to cancel one another. HPLC sucrose determination is a specific analytical method; assuming the equipment is properly standardized and operated, more accurate, "true" sucrose data are obtained. Pol sucrose values often are thought to represent reasonable approximations at harvest, but after storage the accumulation of monosaccharides, trisaccharides such as raffinose, and perhaps other optically active compounds can be expected January-March 2001 Biochemical Quality Changes During Pile Storage 41

Pol error, % 20 .------~

5 o Sidney MT Worland WY Hereford TX I~At harvest DAfter storage

Figure 2. Summary of pol error (% by which pol-detennined sucrose exceeds HPLC-detennined sucrose) at three locations. Data cannot be compared among locations, as different sets of locally adapted varieties were used. Storage period was 110 d at Sidney, 90 d at Worland, and 56 d at Hereford.

to result in pol values that may be poor estimates ofactual sucrose content of the sampled beets. From this experiment, however, we conclude that even at harvest the pol sucrose values were poor estimates ofactual sucrose content at two of the three locations (Figure 2, Worland and Hereford). Correlation coefficients between pol sucrose and HPLC sucrose ofsamples at harvest were 0.89 (Sidney), 0.92 (Worland), and 0.55 (Hereford). The poor correlation between methods for the Texas samples could be caused by an unusual quantity ofoptically active non-sucrose components in the pol sucrose extract. The amino acid contents of Hereford samples at harvest averaged approximately twice that of Sidney samples, and four times that of Worland samples (data not shown), and many amino acids are optically active; thus, this could have contributed to the pol error at Hereford. Post-storage correlation coefficients between methods were 0.75 (Sidney), 0.90 (Worland), and 0.84 (Hereford). The post-storage drop in correlation between methods at Sidney probably is explained by the poor storage conditions at Sidney and the potential for pol sucrose 42 Journal of Sugar Beet Research Vol 38 No 1

values to be in error when appreciable amounts of non-sucrose sugars or other optically active compounds accumulate during storage (see data below). The analysis ofvmiance for sucrose content expressed as percent of root fresh weight showed a highly significant difference between analytical methods at all three locations, but a highly significant difference with storage time only at Sidney. The variance among samples at Worland and Hereford and the relatively good conditions during the 90 d and 56 d storage periods, respectively, masked the small sucrose loss that undoubtedly occurred.

Glucose and Fructose Glucose content in roots at harvest was low, from 0.24 to 0.39 g/ 100 g sucrose [subsequently abbreviated gIlOOS] at the three locations. Glucose content at harvest and after storage differed significantly, with means increasing from the low levels at harvest to a post-storage range of 1.6 to 2.3 gil OOS (Table 2). A significant difference among cultivars

Table 2. Glucose, fructose, and raffinose content, across varieties, of sugarbeet roots at harvest and after pile storage at three locations. Statistical comparisons are made only within a location because a different . set of locally adapted varieties was used at each location.

Sampling Location Time Glucose Fructose Raffinose

------gil 00 g sucrose ------

SidneyMT At harvest 0.38 0.14 0.45 Stored 110 d 2.26 ** 1.62 ** 1.90 **

Worland WY At harvest 0.24 0.03 0.36 Stored 90 d 1.59 ** 1.03 ** 1.70 **

Hereford TX At harvest 0.39 0.14 0.75 Stored 56 d 2.23 ** 1.74 ** 1.05 ns

•• For each chemical compound at each location, data pairs followed by two asterisks are highly significantly different (P < 0.01). ns = not significantly different. January-March 2001 Biochemical Quality Changes During Pile Storage 43

occurred only after pile storage at Worland, when ACHI64 (2.7 gIlOOS) and HH50 (2.2 g/ IOOS) were significantly higher in glucose content than ACH191 (1.3), HH44 (0.89), and HM-R2 (0.84) (Table 3). Table 3. Glucose and fructose content of sugarbeet cultivars at harvest and after factory pile storage for 90 d at Sidney MT or 110 d at Worland WY. No significant cultivar differences occurred for glucose and fructose contents at harvest or after 56 d storage at Hereford TX.

--- Glucose -- --- Fructose -- At Post At Post Location Cultivar Harvest Storage Harvest Storage

gil 00 g Sucrose Sidney MT Beta1996 0.29 b 1.54 ct 0.13 1.16 b KWI745 0.45 a 2.24 b 0.21 1.38 b M865 0.49 a 4.01 a 0.14 3.24 a NIH 1101 0.29 b 2.01 b 0.08 1.29 b NlHI105 0.43 a 2.01 b 0.14 1.30 b Monohikari 0.31 b 1.77 bc 0.13 1.38 b

Worland WY ACH164 0.22 2.73 a 0.02 1.69 a ACH191 0.24 1.30 b 0.03 1.04 b HH44 0.26 0.89 c 0.04 0.58 c HH50 0.22 2.21 a 0.00 1.32 b HM-R2 0.26 0.84 c 0.07 0.52 c tWithin columns and locations, means followed by a different letter are significantly different at the 0.05 level of probability (significant F-test; mean separation by Duncan's mUltiple range test). A highly significant difference occurred at all three locations in root fructose content at harvest vs. after storage. Very low levels at harvest (0.03-0.14 g/lOOS range of means for the three locations) increased at least lO-fold after harvest (Table 2). In addition, significant cultivar differences and significant interaction between time and cultivar occurred at Sidney and Worland (Table 3). At Sidney, cultivar M865 contained significantly greater amounts of fructose after storage (3 .2 gil OOS) than all others, although it did not contain the largest amount at harvest. After storage at Worland, ACH 164 had a significantly greater fructose level 44 JournaJ of Sugar Beet Research Vol 38 No 1

(1.7 gil OOS) than other tested cultivars. A significant interaction of time and cultivar in the analysis ofvariance occurred because cultivars ACHI64 and HH50 had the least fructose at harvest, but the greatest amount after storage. Glucose to fructose ratios (G:F) varied considerably from individual sample to sample, even within cultivars (data not shown). At Worland, considered to have the best storage conditions of the three locations studied, many samples contained no detectable fructose at harvest (below a minimum detectable level of0.002 gIlOOS), so a glucose:fructose ratio could not be determined. Where fructose was detected, the glucose:fructose ratio ranged from 2.7 to 4.7 (data not shown). After storage, glucose:fructose ratios ranged from l.1 to 1.9, averaging about l.5. These findings agree well with the reports of Burba and Nitzschke (1973) for sugarbeet at harvest, and of Schiweck and Biisching (1974) for sugarbeet during the campaign. These ratios are of interest because they sometimes provide insight into the biochemical processes affecting the monosaccharide quantities observed. For example, one would expect equimolar amounts, (i.e., approximately a 1:1 ratio), of glucose and fructose if the amounts found in stored sugarbeets arose from sucrose decomposition without further biochemical use of the "invert sugar" produced. The G:F ratio can be affected by such factors as respiratory use and the ability of the sucrose-degrading enzyme invertase to add fructose to sucrose to fonn one of the three kestoses. Glucose and fructose are the only "reducing sugars" reported in significant amounts in sugarbeet roots, although traces of galactose and arabinose sometimes have been found (Silin, 1964; Burba and Nitzschke, 1973). Prior to the advent of rapid, specific analytical methods for individual determination of sugars, "reducing sugar" or "reducing substance" was reported as the result ofanalyses ofthis property. Reducing sugars are undesirable because they break down during processing to yield organic acids, which in turn affect juice pH and subsequent processing requirements, with melassigenic consequences. For example, Dexter et al. (1970) calculated that ifsodium carbonate were added during processing to neutralize the acid produced from one pound of reducing sugar, 4 pounds of sucrose would be lost (lib by direct loss to glucose and fructose and 3 Ib loss to molasses from sodium addition). Reducing sugars also contribute to color formation (Harvey and Dutton, 1993). For comparison of our data with earlier data, the sum of the individual values for glucose and fructose (Table 2) is approximately equivalent to "reducing sugar." Reducing sugar concentrations (glucose January-March 2001 Biochemical Quality Changes During Pile Storage 45

+ fructose) at harvest at Sidney and Hereford were about 0.5 g/lOOS (Table 2), increasing about eight-fold in storage. Concentrations of reducing sugars for the Worland cultivars and storage conditions were lower, both at harvest and after storage, but the same significant increase occurred during storage. Kamik et al. (1970) reported similar amounts ofreducing sugars at harvest, and found approximately a four-fold increase after 90 d storage under controlled conditions (35 F, 0.03% CO2and 21.0% 0 2)' McCready and Goodwin (1966) reported that sugarbeet roots increased in reducing sugar content with time in controlled storage at 2C, approximately tripling their at-harvest levels after about 90 days. Other work in our laboratory has shown that sugarbeet roots stored under pile storage conditions accumulate more reducing sugar than roots from the same varieties stored under controlled laboratory storage conditions (4C without appreciable fluctuation, high humidity) (Martin and Narum, unpublished data). The larger increases in reducing sugars under pile storage could be attributed in part to the effects of external fungi or other rotting organisms, although the samples in this study were observed to be nearly free of visible decay. Other sources ofreducing sugar accumulation in storage include respiration, the action of the enzyme invertase, and the action of endogenous bacteria. Respiration is a requirement for all living organisms, and the sugarbeet in a storage pile is no exception. Through the action of a catabolic enzyme, sucrose is broken down into its constituent monosaccharides, glucose and fructose. These sugars, in tum, serve as substrates for the multitude of reactions that are required to maintain structural integrity and life functions. Ifstorage conditions were "perfect," with beets suffering no harvest injury, having no disease or mold, no loss of sucrose to raffinose synthesis or endogenous bacterial action, and no other factor causing sucrose loss, only respiration would remain and would account for 100% of sucrose loss. Under the best controlled storage conditions (i.e., controlled temperature, minimal injury or disease, no desiccation) respiration is estimated to account for about 50 to 75% of direct sucrose losses (Wyse and Dexter, 1971 b). Reducing sugars can double or triple during storage, even under ideal controlled conditions (Cole, 1977). Uncontrolled factory storage piles cannot be expected to produce such ideal conditions, with stressful conditions such as temperature variation, desiccation, and injury and disease being inevitable. In such circumstances respiration increases (e.g., Dilley, Wood and Brimhall, 1970; Mumford and Wyse, 1976; Cole, 1977; Wyse, 1978). A varietal difference for respiration rate has been reported (Cole, 1977; Wyse

------_ ._-- 46 Jownal of Sugar Beet Research Vol 38 No 1

et aI., 1979), which could have contributed to the varietal difference in reducing sugar concentrations found in our study. Another sugarbeet root enzyme, sucrose synthase, also can catalyze the breakdown ofsucrose to monosaccharides. Sucrose synthase activity is retained even through prolonged high quality storage, and may be an important source of reducing sugar accumulation during storage (Wyse and Dexter, 1971b; Wyse, 1973). Even healthy sugarbeet roots at harvest contain endogenous bacteria (Tervet and Hollis, 1948; MacDonald et aI., 1963). Bugbee et al. (1975) found that bacterial populations increased several-fold during storage at 5C, with variation among cultivars. In the latter study, reducing sugar amounts increased with storage time in one cultivar, but remained unchanged from at-harvest amounts in another. Bugbee et a1. (1975) pointed out that 20 of the 36 bacterial genera isolated from sugarbeet showed the ability to hydrolyze sucrose in vitro, and suggested that these microbes might be involved in sucrose losses during storage. Cole and Bugbee (1976) further showed that bacterial populations increase rapidly when oxygen is depleted, and that those bacteria were capable of and appeared to be responsible for rapid sucrose hydrolysis. Thus, differing numbers, types, and activities of endogenous bacteria also could be involved in producing the reducing sugar concentrations observed in sugarbeet roots that were pile stored at the three locations studied. Although in this study we did not measure respiration rates, enzyme activities, or numbers ofendogenous bacteria, each ofthose factors may have contributed in some degree to the large changes and varietal differences we recorded in reducing sugars after pile storage. In particular, further studies ofthe dynamics ofthe enzymes ofsucrose degradation are needed to understand their relative contributions to the monosaccharide pool from which sugar nucleotides and other substrates for synthesis of detrimental and melassigenic compounds are derived.

Raffinose At harvest, raffinose levels at Sidney and Worland were low (about 0.4 g/IOOS), as expected for healthy sugarbeet roots (Table 2). Storage led to significant increases in raffinose content at these locations. In contrast, the raffinose concentration of Hereford roots at harvest was significantly higher than at the other locations, averaging 0.75 g/lOOS. The raffinose concentrations of the Hereford samples at harvest were consistently high (range 0.66-0.86, standard deviation = 0.07). After storage at Hereford, raffinose content increased above at-harvest levels in some samples, but not in others (mean = 1.05; range 0.51-2.36, standard

-----_._- - January-March 2001 Biochemical Quality Changes During Pile Storage 47

deviation = 0.53). The increase in raffinose in some Hereford sample pairs but not in others occurred in both genotypes. Cultivars differed significantly for raffmose content after storage at Worland, with ACH164 and HH50 accumulating significantly more rafftnose than the other three cultivars studied. Varietal difference for raffmose accumulation has been reported (e.g., Finkner et aI., 1959; Wyse and Dexter, 1971 a), and the raffmose content found in our study at harvest and after pile storage were similar to amounts previously reported by others (e.g., Walker et aI., 1960; McCready and Goodwin, 1966). The accumulation ofrafflOose is related to frost-tolerance in plants (Kandler and Hopf, 1982), with decreasing photoperiod and temperature in the fall stimulating the plant's preparations for winter. Raffinose can be synthesized in leaves of frost-tolerant plants and transported to roots (Wiemken and Ineichen, 1993), but in fall harvested sugarbeet this mechanism seldom leads to much raffmose accumulation by harvest. Once harvested, the conditions considered ideal for unfrozen storage (ca. 4 C with air circulation) also are ideal for the cold induction of galactinol synthetase. This enzyme, which is responsible for the synthesis of galactinol (galactosyl-myo-inositol), usually the galactosyl donor in the formation ofraffmose by raffmose synthase (Bourne et al. 1965; Bachmann et ai., 1994), is cold-inducible in leaves and seeds ofsoybeans and French beans (Castillo et ai., 1990) and must also be inducible in sugarbeet judging by its known propensity to accumulate raffinose in cold storage (e.g., McCready and Goodwin, 1966; Wyse and Dexter, 1971a). In our study, the raffinose concentration in sugarbeet roots at harvest was greater at the Hereford TX location than at the two more northerly study sites, a fmding that seems anomalous. In part, this can be explained by the fact that raffmose is expressed relative to the amount of sucrose present, and the Hereford sugarbeet roots contained considerably less sucrose at harvest than roots from the varieties grown in Wyoming and Montana (Fig. 1). Because different sets of locally adapted varieties were grown at each location, one must be very cautious in making comparisons between locations. Nevertheless, the pre-harvest environmental conditions at Hereford appeared to induce more raffinose formation, or the cultivars grown at Hereford were more prone to raffmose accumulation. The former seems less likely, but cannot be ruled out. Also, the data interpreted as raffinose in the Hereford samples might have included kestoses or other compounds that were not separated from raffmose by our HPLC procedure. The previously noted poor correlation between pol sucrose and HPLC sucrose for Hereford samples at harvest may support the latter explanation. After harvest, the Hereford pile-stored sugarbeet roots on average had not accumulated significantly more raffinose than at harvest, Journal of Sugar Beet Research Vol 38 No 1

although several individual samples had greater raffmose contents than any individual sample at harvest. The shorter storage period and wanner temperatures ofTexas vs. Wyoming and Montana presumably led to less raffinose accumulation in Texas samples. The statistically significant difference among cultivars for raffinose accumulation after pile storage at Worland suggests that variation exists among cultivars for galactinol induction. Finkner et al. (1958) found significant difference in at-harvest raffinose contents ofthree cultivars grown in the field atEast Grand Forks, Mirmesota. They also determined galactinol by paper chromatography, but their statistical summary and text statements are contradictory as to whether galactinol did or did not differ significantly among the three varieties (Finkner et aI., 1958). McCready and Goodwin, also using paper chromatography, did not find detectable amounts of galactinol in one sugarbeet cultivar (US-75) grown in Califomia. Harvey and Dutton (1993) list galactinol as typically OCCUlTing at a level of about 0.1 gil OOS. Additional studies with new methods now available are needed to clarify the biochemistry and genetic control ofgalactinol and raffmose synthesis and accmTIulation in sugarbeets. Wood et al. (1956) reported that raffinose production is a multigenic character involving additive gene action. If one critical step within the overall biosynthetic pathway for raffmose could be inhibited, an important improvement in sugarbeet storability could result. Because galactinol does not accumulate appreciably in cold stored sugarbeet roots, galactinol:sucrose-6-galactosyltransferase, the enzyme presumably responsible for catalyzing the reaction of galactinol with sucrose to form raffinose (Kandler and Hopf, 1982), apparently is not limiting. The induction ofgalactinol may be a point at which genetic modification to block or diminish the induction of one enzyme could be practical.

Sucrose Losses in Storage McCready and Goodwin ( 1966) summarized the ways sucrose can be lost in stored sugarbeet roots as: (1) losses resulting from the action of microorgan.isms, (2) respiration of the stored sugarbeet roots, and (3) biochemical transfomlations of sucrose into other sugars. In addition to these direct causes ofsucrose loss during storage, other changes can occur in storage that are detrimental to recovery of the sucrose that remains. Melassigenic extractable impurities, both sugar and non-sugar (CalTUthers et a!. , 1962; Dexter et a!., 1967; McGirmis, 1982), have the effect of reducing recoverable sucrose. Wyse and Dexter (1971 b) reported that in sugarbeets stored for 130d at 3C, losses in recoverable sugar were about equally divided between direct loss in storage and losses due to impurity accumulation. Bugbee (1993) also noted that physiological factors January-March 2001 Biochemical Quality Changes During Pile Storage 49

affecting sugarbeet quality at harvest can influence their subsequent storability. Any of these sources of sucrose loss, or any combination of them, can assume paramount importance depending on the combined effect offactors such as variety; quality at harvest; injury during harvest, piling, and storage; the presence and nature of storage rot inducing organisms; and the conditions and length ofstorage. From our data we conclude that even under relatively good outdoor pile storage conditions, accumulation ofreducing sugars and raffinose remains a significant factor contributing to sucrose loss in processing. Potential exists for varietal improvement to reduce accumulation of these impurities, particularly of raffinose.

ACKNOWLEDGMENTS

We thank agricultmal and tare lab personnel ofHolly Sugar Corporation at Sidney MT, Worland WY, and Hereford TX for their cooperation and assistance with this research, and Christina G. Andre for careful technical assistance with the analyses performed at Fort Collins.

LITERATURE CITED

Akeson, W. R. , and J. N. Widner. 1981. Differences among sugarbeet cultivars in sucrose loss during storage. J. Am. Soc. Sugar Beet Techno!. 21: 80-91.

Bachmann, M., P. Matile, and F. Keller. 1994. Metabolism ofthe raffinose family oligosaccharides in leaves of Ajuga replans L. Plant Physio!. 105: 1335-1345.

Bourne, E. J. , M . W. Walter, and J. B. Pridham. 1965. The biosynthesis of raffinose. Biochem. J. 97: 802-806.

Bugbee, W. M . 1993. Storage. Pages 551-570. In D. A. Cooke and R. K. Scott (eds.). The Sugar Beet Crop. Chapman and HaIl, London. 675 pp.

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