enzyme-based methods of determination of sucrose,glucose and fructose

An enzyme-based method for the rapiddetermination of sucrose,glucose and fructose in sugar beet roots and the effects of impact damage and postharvest storage in clampsVictoria MT Spackman and Andrew H Cobb *Crop and Environment Research Centre,School of Agriculture,Harper Adams University College,Newport,Shropshire TF108NB,UKAbstract:A high-throughput enzyme-coupled assay is described for the determination of sucrose,glucose and fructose in sugar beet roots.This method is sensitive,rapid and inexpensive and has been used to highlight the increases in sucrose loss following root stresses such as freezing or aggressive harvesting.Sugar beet roots lose 12.5%of their sucrose following an episode of impact damage greater than 2J,rising to 19.7%loss after 8J,with concurrent increases in glucose and fructose.Increases in glucose and fructose are particularly pronounced following a period of clamp storage (up to 2.3and 3.3m gmg À1fresh weight,respectively).#2001Society of Chemical IndustryKeywords:sugar beet;Beta vulgaris ;damage;sucrose;sugar lossINTRODUCTIONThe UK sugar beet crop provides more than half of the UK sugar requirement,the remainder being provided by cane sugar imports.Sucrose losses arising from postharvest damage cost the UK sugar beet industry £12million per annum.Sugar beet bruising or damage at harvest is one of the major problems of crop quality facing the modern processing industry.Impact damage to sugar beet is a major cause of sugar loss in the UK;an estimated 90g kg À1of the sucrose content is lost at harvest and at least 1g kg À1day À1thereafter.1Subsequent reduction in sugar content can be caused by respiration,invasion by bacteria or fungi,and conversion of sucrose to reducing sugars.The losses are much greater in a mild winter when ambient conditions remain warm.2Every stage in the chain of handling of sugar beet from harvesting to processing can cause impact damage to the crop.Strategies are adopted to minimise impacts;for example,taking care when building a clamp to avoid root compression and bruising.Harvester forward speed and the speed of turbine rotation can in¯uence the impact damage received by sugar beet and should be adjusted to suit the individual situation,taking account of soil type and environmental factors.3In order to clean the crop,turbines are required to run at high speed to reduce the quantity of soil adhering to the beet;however,at high speeds it is likely that the sugar beet will receive greater impacts.4A compromise must be struck between theneed to clean the crop and the need to prevent excessive damage.For an average harvest operation,estimates using an electronic beet analogue have predicted sugar losses through impact damage to be 7.4g kg À1of total sugar,representing approximately 63kg sugarha À1from an average crop.2Sugar loss is generally greater when the beet has been stored prior to processing.Storage in a poorly maintained clamp can substantially decrease sucrose content.2Effective management of clamps is based on the need to maintain sugar content at as near harvest levels as possible and to prevent spoilage by frost and microbial infection.The physiological state of the beet at harvest affects its viable time in clamp and its processing potential.5Frost damage affects the internal texture of beet and causes discolouration.Only a small amount of frost-damaged beet is needed to affect processing,particu-larly at the ®ltration stage,when frost-damaged beet can completely halt the extraction operation by causing ®lter blockage.6Many growers adopt effective strategies to minimise the effects of frost on the crop;for example,building clamps between retaining walls of hay bales or using ¯exible sheeting cover.There is currently little information available on the physiological and biochemical changes that occur in sugar beet postharvest,and this is especially true in the area of sugar measurement.It is vital that a quick,accurate and reproducible method of sugar determi-nation can be used to quantify sugar concentrations(Received 8January 2001;revised version received 4July 2001;accepted 11September 2001)*Correspondence to:Andrew H Cobb,Crop and Environment Research Centre,School of Agriculture,Harper Adams University College,Newport,Shropshire TF108NB,UKContract/grant sponsor:British Beet Research OrganisationJournal of the Science of Food and AgricultureJ Sci Food Agric 82:80±86(online:2001)DOI:10.1002/jsfa.1005both in whole beet and in washings from processing. Current methods of sugar analysis comprise HPLC or enzymatic assays for speci®c sugars,which can be time-consuming(5±30min per sample)and relatively expensive.7The method described in this paper is a high-throughput enzyme-based assay.It has been tested with a variety of samples from different harvest situations,including a range of impact energies,the effect of clamp storage and freezing postharvest.MATERIALS AND METHODSHarvesting and sample collectionWhen sampling from the®eld,six sugar beet roots were taken from each treatment.The mature beet were placed in polythene bags and transported from the®eld in protective containers to prevent further impact damage.When hand-harvested beet were impacted in the laboratory under controlled condi-tions,a falling bolt was used to deliver impacts of0±8J onto a marked circular area of1cm radius.The target area was located2cm below the lowest leaf scar of the beet.The impact end of the bolt was semicircular,with a width of15mm and a radius of curvature of15mm. All beet were stored at10°C until sampling or analysis had®nished.The gently and aggressively harvested beet were taken from the same site using a17t beet harvester(Vervaet,Biervliet,The Netherlands).The aggressive harvest was carried out at a forward speed of 6kmhÀ1and with the turbine speed at maximum, whilst the gentle harvest was carried out at a forward speed of4.5kmhÀ1and with the turbine running at 25%of maximum speed.The beet were then stored in a clamp for84days.To investigate the effects of freezing on sugar content,beet were grown outside in large pallets®lled with soil and then frozen toÀ2°C (over3days)and thawed to ambient temperature (over3days).Beet temperature was measured using probes inserted into control roots.The beet were then lifted from the pallets and sampled as before.Analytical methodologyA method for the determination of sugars in potato tubers was adapted for sugar beet roots.8,9Transverse sections(3mm thick)were cut with a razor-blade from cores taken with a10mm cork borer from2cm below the shoulder of the beet,weighed,rapidly frozen in liquid nitrogen and stored atÀ80°C.The samples were freeze-dried using a Modulyo freeze-dryer (Edwards,Crawley,UK),weighed and stored at À80°C.Samples were then®nely ground with a round-ended glass rod,and100mg of each sample was extracted in a sealed container with5ml of20:80(v/v) aqueous ethanol.The containers were immersed in a shaking water bath(model W22,Grant,Royston,UK) at70°C for2h for the extraction of sugars.As the sucrose content in sugar beet is much higher than the reducing sugar content,the ethanolic extract was diluted to within the linear range of the assay,usually1:50(sample/aqueous ethanol).The assay was carried out in Falcon¯at-bottomed,96-well,¯exible microplates(Fahrenheit,Northampton,UK).Undi-luted ethanolic extract(10m l)and distilled water (140m l)were dispensed into two adjacent wells,and the same quantities of diluted ethanolic extract and distilled water were dispensed into the third adjacent well.The initial sample absorbance was determined at 340nm using a Benchmark microplate reader (BioRad,Hemel Hempstead,UK).Enzymic reactions as detailed below were then carried out at room temperature.The plate was shaken automatically within the reader for10s immediately prior to reading,to ensure thorough mixing.Glucose reagent(GR)(Bayer, Newbury,UK)was added undiluted(30m l)to the ®rst well(to provide1.5units hexokinase,1.2units glucose-6-phosphate dehydrogenase,2m mol ATP, 1.2m mol NAD and15m mol Mg2 per well),and the absorbance was determined at340nm after40min. The increase in absorbance obtained was due to the GR-mediated enzymic oxidation of sample glucose to 6-phosphogluconic acid and concomitant reduction of NAD to NADH.The second well was treated with GR containing0.2units phosphoglucose isomerase(PGI) type IV rabbit muscle(Sigma,Poole,UK).In this well the increase in absorbance at340nm after40min was due to the conversion of glucose and fructose into6-phosphogluconic acid.The third well was treated with GR containing0.2units PGI and10units invertase(I) (grade VII from bakers'yeast,Sigma).The absorbance of the third well at340nm was determined after 60min;any increase was due to the conversion of glucose fructose sucrose into6-phosphogluconic acid.Absorbance increases due to individual sugars were obtained by subtracting the initial absorbance from the®nal absorbance in each well.Quanti®cation of sugars was obtained by comparison with absorbance increases obtained with standard sugar solutions(2.8±16.7m g mlÀ1)which were included in each test microplate.Each sample was replicated on at least three occasions.When using this method,several microplates could be processed in parallel,greatly increasing the number of samples that could be analysed.Data analysisAbsorbance data were collected and®tted to a standard curve to obtain sugar concentrations for each ethanolic extract.Extract concentrations were con-verted into m gmgÀ1fresh weight(fwt)and averaged. Statistical analyses(linear regression and t-test)were performed using Genstat5,Release 4.1(Lawes Agricultural Trust,Rothamsted Experimental Station, UK).RESULTSMethod developmentIndividual standard solutions of sucrose,glucose andDetermination of sugars in sugar beet rootsfructose were assayed;the increase in absorbance at 340nm was proportional to the sugar content in each well in the concentration range2.8±16.7m g mlÀ1(Fig 1).Regression analysis was carried out for the individual sugars in the linear range,with a value of R2>0.9981for all three sugars.The time taken for the reaction to proceed to completion varied depending on which sugar was being determined(Fig2).The®nal inverting step in the determination of sucrose took20min longer to complete than the glucose and fructose stages.Studies involving standards(Figs1and2)were replicated on three occasions,and data points presented are means of the replicates.When sucrose was tested,a sig-ni®cant increase in absorbance was noted in the presence of GR PGI at50and60min,probably due to monosaccharide contamination in the 995mggÀ1purity sucrose.The sucrose concentrations in the sugar beet extracts were so high that a1:50dilution of samples was required for the reaction to®t the linear range of the assay.Various ethanol volumes(0±25m l)were added to30m l aliquots of a standard sucrose solution (16.7m g mlÀ1),and the sucrose reaction was moni-tored using GR PGI I.Volumes above10m l proved deleterious to the assay(Fig3).Experimental dataAs impact energy increased from0to8J,the sucrose content of sugar beet roots declined from183to 147m gmgÀ1fwt(p=0.0005).At the same time, increases were noted in glucose and fructose concen-trations(p0.1)(Fig4).The gentle and aggressive harvests were followed by 84days of clamp storage,after which roots were analysed for sugar content.Aggressively harvested beet had a signi®cantly lower sucrose content(170m gmgÀ1 fwt)than gently harvested beet(187m gmgÀ1fwt) (p=0.0005).Once again,as sucrose concentration fell,signi®cant rises in glucose and fructose concen-trations were recorded(p=0.0005)(Fig5).Following a period of storage atÀ2°C,beet were analysed for sugar content.Roots subjected to low-temperature storage contained less total sugar than those stored at ambient temperature.Sucrose con-centration was signi®cantly lower(p=0.0005).The increases in glucose and fructose concentrations previously observed were absent and similar concen-trations of glucose and fructose were recorded despite storage under different temperature regimes(Fig6).DISCUSSIONCurrent methods of sugar analysis in sugar beet are expensive and slow.There is a need for a fast,ef®cient and reliable method of sugar measurement in the sugar beet industry,and the method reported here has been adapted from one used for potatoes.8The linear region of the assay has been optimised and reaction times have been standardised(Figs1and2).As a consequence of the high sucrose concentrations in sugar beet,samples required substantial dilution prior to sucrose determination.In order to determine reducing sugar concentrations,which are much lower, the assay was run with undiluted and1:50diluted samples in parallel.Furthermore,the®nal volume of ethanol in the reaction mixture was kept to<10m l to prevent enzyme denaturation.Since the pH optimum for invertase(pH 4.5) differed from the optima for the coupling enzymes used to metabolise the hexoses,the sucrose reaction took60min compared with40min for the glucose or fructose reaction to be completed.There are advan-tages in using a microplate method rather than a spectrophotometric assay or HPLC.The microplate method is fast and,once samples are extracted,20 fully replicated samples with standards and controls can be dispensed and determined in70min.Substrate costs per sample are low,as small volumes are used. This study has shown that sugar beet roots harvested without impact or freezing damage contain more sucrose and lower levels of reducing sugars(FigsFigure1.Standard curves for glucose (squares),fructose(diamonds)and sucrose(circles)content from ethanolic extracts of lyophilised sugar beet roots in the presence of glucose reagent, phosphoglucose isomerase and invertase.Linear range2.8–16.7m g mlÀ1.Standard errors were smaller than the symbol size(<0.01 ODunit).VMT Spackman,AHCobb4and 6).Sucrose is predominantly located in the vacuoles of sugar beet root cells and is usually compartmentalised away from the cytosolic alkaline invertase and sucrose synthase.Acid invertases are also located in the vacuoles and play an important role in the hydrolysis of sucrose to reducing sugars.10Acid invertases are present in fresh tissue and to a lesser extent in frozen tissue.11In sweet potato studies,vacuolar acid invertase was the most in¯uential invertase in determining the production of reducing sugars.12In sugar beet there is a high level of acid invertase activity in young roots,which declines as the roots become more mature.13At crop maturity and during storage the activities of the cytoplasmic sugar-hydrolysing enzymes (alkaline invertase and sucrose synthases)greatly exceed those of acid invertases.10Berghall et al 10reported that the cytoplasmic inver-tases are required for sucrose hydrolysis and that acid invertase activity increases in response to stress and is responsible for increasing the reducing sugar content.In addition to the balance of hexoses,non-impacted sugar beet tissue contains a high proportion of intact cells and shows no evidence of lignin formation.14Impact damage of 4and 8J to sugar beet roots caused a fall in sucrose concentration and an increase in impurity (glucose and fructose)concentration (Fig 4).A net loss of the three sugars occurred after impact,indicating that impact was not simply causing a movement of sucrose into reducing sugars,but that some sucrose was lost to other processes.Examples of these processes are sucrose leaching,consumption by respiratory processes or microbial activity,and metab-olism in regrowth processes.It is likely that some sucrose is utilised in wound-healing processes;larger falls in sucrose concentration were recorded as impact increased (0.9,12.6and 19.6%reduction in sucrose concentration from 2,4and 8J impact,respectively).Wound healing in sugar beet is vital to prevent the entry of water and micro-organisms and the escape of sucrose,and takes the form of secretion of glycopro-teins,lignin or suberin to seal and waterproof the wound.14In addition to the inversion of sucrose to glucose and fructose,sucrose can be degraded to uridine diphosphate (UDP)glucose by sucrose synthase.This nucleotide sugar is involved in cell wall synthesis as well as other carbohydrate interconver-sions 15and may well play a role in wound healing.The impacts of 0±8J represent realistic forces in terms of a commercial harvest.Numerous studies using electronic beet analogues have determined that average impacts vary from 0.5to 6.5J depending on the position of the beet in the harvester.16Sucrose concentration declined signi®cantly follow-ing a period of freezing to À2°C (Fig 6).In other crops such as sweet potato a drop in temperature to 4.5°C for 7weeks of storage caused a signi®cant increase in invertase activity.11The decline in sucrose content of frozen sugar beet could be due to increases in cytoplasmic invertases following freezing.Sucrose breakdown products may be channelled into wound healing,respiration or regrowth and so may not always contribute to the glucose and fructose concentration in the beet.Increased amounts of microbial invertases in frost-damaged sugar beet add further complexity to this situation.10In raspberry fruit a period offreezingFigure 2.Time-course of enzymic analysis of standards.Glucose,fructose and sucrose with either glucose reagent (GR)alone (squares),GR phosphoglucose isomerase (PGI)(diamonds)orGR PGI invertase (circles).Standard errors were smaller than the symbol size (<0.01ODunit).Figure 3.Negative effect of increasing ethanol volume in the sugar assay.Determination of sugars in sugar beet rootscaused up to 26%reduction in free radical-scavenging capability (measured as antiradical ef®ciency),and signi®cant reductions in vitamin C and ellagic acid concentrations were also reported.17If similar changes affect sugar beet metabolism after freezing,the reduction in antioxidant capability could contribute to the rapid breakdown of sugar beet roots after freezing.In some countries,sugar beet is stored without sugar loss,as the average winter temperatures remain below À9°C;processors in these regions can process the frozen root as long as all of the beet remains frozen.Problems can occur under this type of processing regime if thawed and frozen roots are mixed to-gether.18In the UK it is imperative to avoid freezing roots because of the blockages that frozen beetcanFigure 4.Sugar contents of ethanolic extracts of lyophilised,hand-harvested sugar beet roots given known impacts of 0J (white),2J (light grey),4J (dark grey)and 8J(black).Figure 5.Sugar contents of ethanolic extracts of lyophilised beet cores from roots that were either harvested gently (white)or aggressively (grey)by a commercial harvester and stored in clamp for 84days.VMT Spackman,AHCobbcause in the ®lters during processing.19In addition,when roots thaw,the ruptured cells of the beet start to leak.This encourages rapid growth of bacteria which convert the sucrose to gums and reducing sugars.These reducing sugars are unstable in the factory process and can give rise to acidic compounds whose presence causes still more sucrose to be converted into glucose and fructose,requiring the addition of further alkali during processing.20Lastly,the freezing of roots should be avoided because of the drop in sucrose concentration of the root as demonstrated in this study (Fig 6).Clamp storage is the main method of avoiding frozen beet in store,but clamping can contribute towards sugar losses from the crop.A long clamp storage period can enhance the effects of damage received prior to storage.Immediately after harvest a moderate-sized clamp may contain £20000worth of beet.If the clamp is managed well,the sugar loss after 30days may be valued at only £300.Poor manage-ment can easily increase this loss to £1500.19In this study the decline in sucrose concentration and increase in reducing sugars were far greater following clamp storage (Fig 5)compared with damaged,unstored beet.Although it is often necessary to store sugar beet,the methods for such storage have not been investigated as thoroughly as in the potato industry.21By monitoring the sugar content throughout the various processes of the sugar beet handling chain,areas of sugar loss such as clamping and freezing could be identi®ed and improved upon.ACKNOWLEDGEMENTSThe authors wish to thank the British Beet ResearchOrganisation for funding,British Sugar for the supply of beet and advice,and Victoria Cruxton and Luda Ibrahim for technical assistance.REFERENCES1Jaggard KW,Clark CJA,May MJ,McCullagh S and DraycottAP,Changes in the weight and quality of sugar beet (Beta vulgaris )roots in storage clamps on farms.J Agric Sci Camb 129:287±301(1997).2Hopkinson I and Houghton BJ,Electronic sugar beet measure-ments:assessments of beet handling and harvesting sugar losses.Aspects Appl Biol 52:169±172(1998).3Wyse R,Effect of harvest injury on respiration and sucrose loss insugar beet roots during storage.J Am Soc Sugar Beet Technol 20:193±202(1978).4Brown S and Pilbrow J,Electronic beet and harvestingÐaprogress report.Br Sugar Beet Rev 67:13±17(1999).5Hill P,Temperature is crucial in beet clamp storage.FarmersWeekly (25August):76(2000).6Shore M,Dutton JV and Houghton BJ,Evaluation of deterio-rated beet.Int Sugar J 85:106±110(1983).7Campbell JA,Hansen RW and Wilson 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