stAGE OF sum-2513: AGRONOMIC, PHYSIOLOGECAL AND QUALITY ASPECTS Thesis for the Degree of Ph. D. MICWGAN STATE UNWERSETY ROGER E. WYSE 1969 THEStS This is to certify that the thesis entitled Storage of Sugarbeets: Agronomic Physiological and Quality Aspects presented by Roger E. Wyse has been accepted towards fulfillment of the requirements for Ph.D. degree in C1702 SCience Date 0C t Ob er 0-169 y “‘M.\ ? amomc av ‘5 "MB & 50W 5 9995mm; ;m§2.sua§;gwls Wyh ‘J size - ABSTRACT STORAGE OF SUGARBEETS: AGRONOMIC, PHYSIOLOGICAL AND QUALITY ASPECTS BY Roger B. Wyse A study was made of changes in the composition and integrity of beets during storage and to develop methods for evaluating beets for desirable storage and processing char- acteristics. ExPeriments were conducted in 1967 and 1968 to determine the influence of nitrogen fertilization, harvest date, tOpping and preharvest sprays on recoverable sucrose losses in storage. The factors affecting sucrose losses included those related to the direct loss of sucrose (respi- ration and sugar conversions) and those affecting sucrose losses in the factory (clear juice purity, acid-base balance, melassigenic impurities). .Samples were analyzed for percent sucrose, clear juice purity, raffinose, reducing sugar, amino acid, sodium, potassium and chloride. A study was also made of marc stability in storage. Included in the storage practices were temperatures, desiccation and modi- fied atmOSpheres including carbon monoxide inhibition of resPiration. Roger E. Wyse Methods are presented for the rapid determination of raffinose and reducing sugars and a technique for producing uniform storage samples by specific gravity sorting. This technique for sample preparation increased sample uniformity 400 percent. Raffinose, reducing sugars, amino acids, sodium, potassium and chloride accounted for approximately 65 per- cent of the total impurities at harvest and in beets stored below 4 C. Changes in raffinose, reducing sugars and amino acids accounted for a large prOportion of the changes in total impurities in storage below 4 C. Above 4 C other impurities not determined in this study accumulated. A formula was derived to correct the polarimetric sucrose determination for the Optical activity of raffinose and invert. The effect of major impurities accumulating during storage on sucrose losses to molasses are discussed. A relationship is develOped whereby the acid-base balance of the clear juice can be used to evaluate stored beets on the basis of loss of recoverable sucrose due to sodium carbonate addition. The recoverable sugar per ton estimate, as modi- fied for stored beets, was used to evaluate the influence of agronomic and storage practices on the loss of sucrose in several sugarbeet varieties during storage. Slightly over 50 percent of the RSPT losses in storage were due to respiration and sugar conversion. The balance of the losses were due to increased factory losses Roger B. Wyse resulting from the accumulation of melassigenic substances in the clear juice of stored beets. Other carbohydrates besides sucrose contributed substantial amounts of substrate for respiration. This was evident since the dry matter loss in storage was consider- ably greater than the loss of sucrose. Means of reducing respirational losses were studied as were the enzyme systems reSponsible for sucrose degradation. Respiration in the beet showed extreme sensitivity to regulation by high levels of carbon dioxide and inhibition by carbon monoxide. Three percent carbon monoxide inhibited respiration 20 percent in the intact beets. The enzymatic hydrolysis of sucrose was found to involve two enzymes, one with a pH Optimum at 5, the other at pH 7. The significance of these two enzymes on the accumulation of reducing sugars in storage is discussed. The marc content remained relatively constant during storage at 3 C,if molding and desiccation were prevented. The marc remaining after 20 C extraction declined during storage but the residue after 70 or 80 C extraction was very stable. The 70 C residue actually increased in some cases under ideal storage conditions. STORAGE OF SUGARBEETS: AGRONOMIC, PHYSIOLOGICAL AND QUALITY ASPECTS BY Roger B: Wyse A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of CrOp and Soil Sciences 1969 €29/77J-2 //~z 7—~ 70 ACKNOWLEDGMENTS I wish to eXpress my deepest gratitude to Dr. S. T. Dexter for the unique insight and subtle prodding with which he guided my growth as a scientist. The Opportunities which he provided and his confidence in my abilities were greatly appreciated. Appreciation is eXpressed for the OOOperation and work of the following men and others in my behalf. Mr. Richard Zielke, a fellow graduate student, OOOperated in the field experiments. Mr. M. G. Frakes ran the routine sugar analyses and made the facilities of the Michigan Sugar Com- pany, Agricultural Research Laboratory readily available. Dr. F. W. Snyder assisted in running some of the routine analyses and comprehensively reviewed this manuscript. Dr. D. R, Dilley made his Specialized equipment readily available. Dr. C. M. Harrison reviewed the manuscript. Finally I would like to thank the USDA-ARS for providing the financial assistance (Contract No. 12-14—100- 8467-34) which made this study possible. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . 3 Loss of Sucrose During Storage . . . . . . . . . . 3 Sugar Losses Due to Respiration . . . . . . . 3 Indirect—-Factory Losses . . . . . . . . . . 5 Loss of Physical Integrity During Storage . . 6 Breakdown of Marc . . . . . . . . . . . . . 6 Pectic Substances as Sources of Impurities . . 7 Changes in the Pectic Substances During Storage . . . . . . . . . . . . . . . . . . 8 Effect of Pectic Substances on Processing . . 9 Non-Sucrose Components of the Beet Root . . . . . 9 Reducing Sugars . . . . . . . . . . . . . . . 9 Raffinose . . . . . . . . . . . . . . . . . . 11 Effect of Agronomic Practices on Beet Root Composition and Storage Characteristics . . . . 13 Nitrogen Fertilization . . . . . . . . . . . . 13 PhOSphorus and Potassium Fertilization . . . . 15 Harvest Date . . . . . . . . . . . . . . . . . 15 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 16 1966 Preliminary EXperiments . . . . . . . . . 16 1967 EXperiments . . . . . . . . . . . . . . . 16 1968 EXperiments . . . . . . . . . . . . . 17 Specific Gravity Sorting and Sample Preparation . . . . . . . . . . . . . . . . 18 Outline of Major Storage Experiments . . . . . . . 19 1967 Date of Harvest . . . . . . . . . . . . . 19 1967 TOpping and Row Width . . . . . . . . . . 21 Desiccation EXperiment . . . . . . . . . . . . 21 1967 Variety . . . . . . . . . . . . . . . . . 22 1968 Date of Harvest . . . . . . . . . . . . . 22 1968 Variety and Temperature . . . . . . . . . 22 1968 Field Sprays . . . . . . . . . . . . . . 23 1968 Vernalization . . . . . . . . . . . . . 23 1968 Controlled AtmOSphere . . . . . . . . . . 24 Laboratory Analyses . . . . . . . . . . . . . 24 ReSpiratory Analyses . . . . . . . . . . . . . 26 iii Page RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 30 Preliminary Results . . . . . . . . . . . . . . . 30 1966 Desiccation Experiment . . . . . . . . . 30 FACTORS CONTROLLING THE MAJOR NON-SUCROSE COMPONENTS OF THE BEET AT HARVEST AND DURING STORAGE . . . . . 37 Factors Controlling the Raffinose Content . . . . 37 Storage Temperature . . . . . . . . . . . . . 37 Variety-Temperature Interaction . . . . . . . 37 Variety . . . . . . . . . . . . . . . . . . . 40 Effect of Harvest Date . . . . . . . . . . . . 42 Effect of Nitrogen Fertilization . . . . . . . 44 Effect of TOpping . . . . . . . . . . . . . . 48 Preharvest Sprays . . . . . . . . . . . . . . 49 Storage in Modified Atmospheres . . . . . . . 50 Factors Controlling the Reducing Sugar Content . . 52 Storage Temperature. . . . . . . . . . . . . . 52 Variety—Temperature Interaction . . . . . . . 52 Variety . . . . . . . . . . . . . . . . . . . 54 Effect of Harvest Date . . . . . . . . . . . . 55 Variety-Nitrogen Interaction . . . . . . . . . 58 Effect of TOpping . . . . . . . . . . . . . . 62 Preharvest Sprays . . . . . . . . . . . . . . 62 Storage in Modified Atmospheres . . . . . . . 64 Factors Controlling the Amino Acid Content . . . . 66 Effect of Nitrogen Fertilizer . . . . . . . . 66 Variety-Nitrogen Interaction . . . . . . . . . 68 Variety-Temperature Interaction . . . . . . . 71 Effect of Topping . . . . . . . . . . . . . . 72 Preharvest Sprays . . . . . . . . . . . . . . 72 Storage of Modified Atmospheres . . . . . . . 74 Factors Controlling the Sodium and Potassium Content of the Root . . . . . . . . . . . . . . 76 Variety-Nitrogen Interaction . . . . . . . . . 76 Harvest Date . . . . . . . . . . . . . . . . . 78 Preharvest Sprays . . . . . . . . . . . . . . 78 Factors Controlling the Chloride Content . . . . . 81 Variety-NitrogenéHarvest Date Interaction . . 81 Effect of Thermal Induction on Chemical Composition of Beets Stored at 5 C . . . . . . . 84 Effect of Prestorage Heating on Raffinose, Reducing Sugar, and Amino Acid Content After Storage . . . . . . . . . . . . . . . . . 88 iv Comparison of Analyzed vs Total Non—Sucrose Components of the Clear Juice . . . . . . Summary of the Factors Controlling Several Of the Non-Sucrose Components in the Clear Juice . . . . . . . . . . . . . . . . . . . CHANGE IN MARC CONTENT DURING HARVEST AND IN STOMGE O O O O C O O O O O O O O O O O O O O C At Harvest . . . . . . . . . . . . . . . . . . Variety-Harvest Interaction . . . . . . . Nitrogen . . . . . . . . . . . . . . . . . During Storage . . . . . . . . . . . . . . . . Changes in Marc During Storage . . . . . . Variety-Temperature Interaction . . . . . Effect of Harvest Date . . . . . . . . . . Summary of Marc Content . . . . . . . . . . . FACTORS INVOLVED IN THE DIRECT LOSS OF SUCROSE IN ' STOMGE . O O O O O O O O O O O O O O O O O O . ReSpiration . . . . . . . . . . . . . . . . . Temperature, Injury and Wilting . . . . . Internal Atmosphere . . . . . . . . . Carbon Monoxide Inhibition Of ReSpiration Effect of Preharvest Sprays on Thermal Induction and Respiration . . . . . . . PrOportion of Sucrose Losses Accounted for by Respiration . . . . . . . . . . . . . . . . Carbon Dioxide Evolution . . . . . . . . . Sucrose vs Dry Matter Losses . . . . . . Enzymatic Degradation of Sucrose in Stored Beets . . . . . . . . . . . . . . . . . . . Enzyme Analysis . . . . . . . . . . . . pH Profile of Sucrase Activity in Root Homogenates . . . . . . . . . . Distribution of Hydrolytic Activity in the Beet Root . . . . . . Possible Importance of Hydrolytic Activity at pH 5 and pH 7 . . . . . . . . . . . . Effect of Several Preharvest Sprays on Hydrolytic Activity at pH 5 and 7.0 . . Summary of Factors Influencing the Direct Loss of Sucrose in Storage . . . . . . . . . Page 90 98 101 101 101 103 103 103 108 111 113 114 114 114 114 116 119 122 122 125 127 127 128 129 131 133 134 ASSESSMENT OF QUALITY IN STORED BEETS Use of the Impurity Index in Evaluating Stored Beets Formula for Correcting Sucrose Determinations for Raffinose and Invert Base-Acid Balance in the Clear Juice Utilizing the Residual Alkalinity in the Calculation of the RSPT Quality Evaluation of Fresh and Stored Beets the Basis of Recoverable Sugar . Recoverable Sugar Yields at Harvest Effect of Harvest Date on Storage Characteristics Comparison of Varieties in Storage on the Basis of RSPT Variety-Nitrogen Interaction . Preharvest Sprays Modified Atmosphere PrOportion of Recoverable Sugar per Ton Loss in Storage Accounted for by the Direct Loss of Sucrose Summary of Recoverable Sugar Yields at Harvest and After Storage SUMMARY AND CONCLUSIONS APPENDIX A. B. C. BIBLIOGRAPHY ABBREVIATIONS USED INVERT DEGRADATION vi DEVELOPMENT OF ENZYME METHOD FOR RAFFINOSE DETERMINATION Page 136 137 137 140 144 148 148 149 152 154 156 158 158 162 164 170 171 174 176 Table 10. 11. LIST OF TABLES Influence of temperature in the production of raffinose by several varieties during storage for 100 days at 3 and 10 C (1968) Interaction between harvest date, variety and length of storage on the raffinose content at harvest and after storage at 3 C in 1967 . . . . . . . . . . . . . . . Effect of nitrogen fertilization on the average raffinose content of three vari— eties stored at 3 C in 1967 . . . . . . . Nitrogen x removal interaction for raffi- nose accumulation in storage (1967) . . . Effect of tOpping on raffinose content of beets stored at 3 and 7 C . . . . . . . . Effect of giberellic acid and maleic hydrazide on the raffinose content of fresh and stored beets (1968) . . . . . . . . . Effect of modified atmospheres on the accumulation of raffinose after 40 days of storage at 5 C . . . . . . . . . . . . Interaction of variety with temperature in the accumulation of reducing sugars after 100 days of storage at 3 and 10 C . . . . Variety x removal interaction in reducing sugar accumulation (1967) for beets stored at 3 C O O O I O I O O O O O O O O O O 0 Harvest x removal interaction for reducing sugar accumulation (1967) ix: beets stored at 3 C O O O O O O O C O O C O C O O O 0 Harvest x variety x removal interaction for reducing sugar accumulation of beets stored at 3 C (1967) . . . . . . . . . . . . . . vii Page 39 46 47 47 48 49 50 54 55 56 57 Table 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. Variety x nitrogen x removal interaction for reducing sugar accumulation in five varieties grown on 24 and 150# nitrogen per acre and stored at 3 C for 65 and 130 days . . . . . . Effect of tOpping on the accumulation of reducing sugars after 50 and 100 days of storage (average of 3 and 7 C) (1967) . . . . Effect of various preharvest foliar Sprays on the accumulation of reducing sugars during storage at 4 C (1968) . . . . . . . . . . . . Effect of modified storage atmospheres on the accumulation of reducing sugars after 40 days of storage at 5 C . . . . . . . . . . . . . . Effect of nitrogen fertilization on the free amino acid content of three varieties at harvest (1967) O O O O O O O O O O O O O O 0 Variety x nitrogen interaction in amino acid content of five varieties . . . . . . . . . . The effect of storage temperature on the amino acid content of beets stored for 100 days 0 O O O O O O O O O O O . O O O O 0 Effect of several preharvest Sprays on the amino acid content of fresh and stored beets O O O O O O O O O O I O O O I I O 0 O 0 Effect of modified atmOSpheres on the amino acid content of stored beets . . . . . . . . Effect of nitrogen fertilization on the sodium and potassium content of five varieties harvested on October 26, 1967 . . . Effect of nitrogen fertilization on the average sodium and potassium content of three varieties harvested on three dates in 1967 . . . . . . . . . . . . . . . . . . . Effect of harvest date on the sodium and potassium content of fresh and stored beets in 1968 . . . . . . . . . . . .-. . . . viii Page 60 62 64 65 68 7O 71 74 75 77 77 79 Table 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. Effect of several preharvest foliar sprays on the sodium and potassium content of fresh and stored beets . . . . . . . . . . . Interaction between variety, nitrogen, and harvest date on the chloride content in 1967 O O O O O O O O O O O O O O O O O O O . Variety x nitrogen interaction for chloride content of five varieties grown on high (150#) and low (24#) nitrogen . . . . . . . . Effect of thermal induction on the raffinose, amino acid and reducing sugar content of beets stored at 5 C (1968) . . . . . . . . . Effect Of postharvest heating on raffinose, invert, and amino acid production in subse- quent cold storage (1968) . . . . . . . . . . Comparison of the sum of analyzed impurities to the total calculated impurities in five varieties stored for 65 and 130 days at 3 C (1967) . . . . . . . . . . . . . . . . . . . Increase in total impurities accounted for by raffinose, reducing sugars and amino acids in five varieties stored 65 and 130 days at 3 C (1967) . . . . . . . . . . . . . . . . . . . Comparison Of the total analyzed impurities (TAI) to the total impurities in three vari- eties stored for 100 days at 3 and 10 C' (1968) . . . . . . . . . . . . . . . . . . . Increase in total impurities accounted for by raffinose, reducing sugars and amino acids in three varieties stored 100 days at 3 and 10 C (1968) . . . . . . . . . . . . . . . . . . . Effect of variety on the percent marc at harvest and after storage at 3 C (70 C extraction) (1967) . . . . . . . . . . . . . Effect of harvest date on the percent marc at harvest and after storage at 3 C . . . . . Effect of extraction temperature on the percent marc of three varieties at harvest (1968) o o o o o o o o O o o o o o o o o o 0 ix Page 80 82 82 87 89 91 94 95 97 106 107 108 Table Page 36. The average percent marc of three varieties at harvest and after 100 days of storage at 3 and 10 C O C O O O O O O O O O O O O O O O O 109 37. The loss in marc of three varieties stored 100 days at 3 and 10 C . . . . . . . . . . . . 110 38. Percent marc at three harvest dates and the loss in marc during 120 days Of storage at 3 C O O O O O O O O O O O O O O O O O O O O O O 111 39. RSSpiration rate of beets subjected to several postharvest treatments (1968) . . . . . 115 40. PrOportion of sucrose losses accounted for by reSpiration and interconversions in 112 day storage at 5 C . . . . . . . . . . . . . . 124 41. Average loss of dry matter and sucrose in 1967 date of harvest study after 65 and 130 days of storage at 3 C . . . . . . . . . . . . 125 42. Loss of dry matter and sucrose in five vari- eties during storage at 3 C for 130 days . . . 126 43. Distribution of inversion activity at pH 5 and 7.2 between supernatant and cell wall material I O C O O O O O O O O O O O O O O O 0 131 44. Effect of storage temperature on the enzymatic inversion of sucrose at pH 5 and 7 O C . . O . O O . . O O O O C O I O O . O 132 45. Effect of preharvest sprays on the degrada- tion of sucrose by two enzymes assayed at pH 5 and pH 7 O O O O O O O O O O O O O O O C O 134 46. Comparison of total impurities with the total analyzed impurities and the impurity index on the evaluation of three varieties stored 65 and 130 days at 3 C (1967) . . . . . 138 47. Magnitude of corrections required due to errors in polarimeter reading caused by raffinose and invert . . . . . . . . . . . . . 141 48. The residual alkalinity of five varieties stored at 3 C for 130 days . . . . . . . . . . 143 Table 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. Page Calculation of sucrose losses due to sodium carbonate addition . . . . . . . . . . . . . . 145 Effect of base-acid balance corrections on the RSPT of five varieties at harvest and after storage at 3 C (1967) . . . . . . . . . . 146 Effect of base-acid balance corrections on the RSPT of three varieties at harvest and after storage at 3 and 10 C (1968) . . . . . . 147 Increase in RSPA yields of thee varieties at harvest grown on high (150#/A) and low (24#/A) levels of nitrogen during 1967 . . . . . . . . 149 Effect of harvest date on the percent sucrose, clear juice purity, and RSPT at harvest and after storage at 3 C (1967) . . . . . . . . . . 151 Effect Of harvest date on the percent sucrose, CJP and RSPT in storage (1968) at 3 C . . . . . 153 The decline in RSPT of three varieties harvested October 26, November 6 and stored for 65 and 130 days at 3 C (1967) . . . . . . . 154 Recoverable sugar per acre yield at harvest and after 130 days of storage for five vari— eties grown on 24 and 150 pounds nitrogen per acre and stored at 3 C . . . . . . . . . . 155 The effect of nitrogen fertilization on the loss of RSPT of three varieties in storage at 3 C (1967) O O O O O O O C C O C O O O C O O 157 Effect of modified atmosphere on clear juice purity, sucrose and RSPT after 40 days at 5 C (1968) . O O O O O O O O O O O C O O O O O 159 PrOportion of RSPT losses due to loss of sucrose in five varieties stored 130 days at 3 C (1967) o o o o o o o o o o o o o o o o o 160 PrOportion of RSPT losses due to loss of sucrose in three varieties stored 100 days at 3 and 10 C (1968) . . . . . . . . . . . . . 161 xi Figure 1. 10. 11. 12. 13. 14. LIST OF FIGURES Internal and external views of a beet stored for 140 days at 3 C . . . . . . . . . . . . Diagram of carbon dioxide analyzing system Method used to determine time required for sample to evolve 20 mg Of C02 . . . . . . . . Effect of temperature on raffinose formation Effect of wilting on raffinose formation . Effect of temperature on invert formation . Effect of wilting on reducing sugar formation 0 O O O O O O O O O O O I O O O 0 Effect of storage temperature on the raffinose content of beets stored for 35, 70, 105 and 145 days (1967) . . . . . . . . Variety x storage interaction for raffinose accumulation at 3 C . . . . . . . . . . . . The effect of harvest date on the accumula- tion of raffinose during storage at 3 C in 1967 I O O O O I O O O O O O O O O O O O 0 Effect of harvest date on raffinose accumu- lation in storage at 3 C . . . . . . . . . Effect of temperature on the accumulation of reducing sugars in beets stored for up to 145 days 0 O O C O O C O C O C C O O C 0 Effect of harvest date on the accumulation of reducing sugars in storage at 3 C . . . Effect Of nitrogen fertilization on the accumulation of reducing sugars in three varieties during storage at 3 C . . . . . . xii Page 20 27 28 31 32 34 35 38 41 43 45 53 59 61 Figure 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Page Effect of preharvest applications of GA and MH-30 on the reducing sugar content in stor— age at 5 C . . . . . . . . . . . . . . . . . . 63 Harvest x nitrogen x removal interaction for amino acid content in beets stored at 3 C for 65 and 130 days . . . . . . . . . . . . . . 67 Amino acid content in storage at 3 C . . . . . 69 Effect of preharvest applications of MH—30 and gibberellic acid On the amino acid content of stored beets . . . . . . . . . . . . 73 Effect of thermal induction on the raffinose, reducing sugar and amino acid content of beets stored at 5 C . . . . . . . . . . . . . . 85 Change in non-TAI content of five varieties in storage 65 and 130 days . . . . . . . . . . 92 Percent marc of three varieties on three harvest dates in 1967 . . . . . . . . . . . . . 102 Yield of marc in tons per acre at harvest for three varieties in 1967 . . . . . . . . . . 104 Marc yield of beets grown on high (150#/A) and low (24#/A) nitrogen on three harvest dates in 1967 O O I O O O O _. O O O O O O O O O 105 The effect of incubation temperatures on the percent marc before and after storage . . . 112 Carbon monoxide inhibition of carbon dioxide eVOlution O O 0 O O O O O O O O O O C O C O O O 118 Carbon dioxide evolution of samples treated with MH-30, gibberellic acid and stored for 102 days at 5 C . . . . . . . . . . . . . . . . 120 Carbon dioxide evolution during 102 days of storage at 5 C . . . . . . . . . . . . . . . 123 Effect of pH on the hydrolysis of sucrose by a beet root homogenate . . . . . . . . . . . . 130 xiii INTRODUCTION Sugarbeet acreage and yields, in the past 20 years, have increased greatly without a corresponding increase in daily factory processing capacity. As a result an in— creasing percentage Of each year's crOp must be stored for considerable time prior to processing. In a factory in Michigan no beets were piled between 1946 and 1959 and the processing campaign averaged 45 days. During the next five years enough beets were piled annually to Operate the fac- tory an extra 73 days. In 1969 over 75 percent of all beets processed were stored. Storage losses of extractable sucrose can be divided into two general categories. One is the direct loss of sucrose due to reSpiration or sugar transformations; the other is the loss incurred during processing. These factory losses are dependent on the physical integrity and chemical composition of the root. Since the majority of beets processed are stored, the storage characteristics of vari- eties and the effect of storage practices on changes in the chemical composition of the beet have become Of utmost importance. This study investigated factors affecting changes in the composition and integrity of the beet during storage and developed methods for evaluating beets for desirable storage and processing characteristics. REVIEW OF LITERATURE In 1924 A. D. Pack wrote a series of articles on the storage of sugarbeets in which he referred to storage work conducted in 1876 to determine the losses incurred. Loss of Sucrosegguringigtorage Sgggr Losses Que to Respiration Loss of sucrose by respiration can be substantial particularly at higher storage temperatures (Silin, 1964; McG innis, 1951) . Barr 2; 31. (1940) determined the loss of sugar by respiration at various temperatures over a 47-day storage period and found that the amount of carbon dioxide evolved accounted for approximately 60 percent of the total apparent sucrose losses. The percentage of carbon dioxide evolved varied with temperature. Stout and Smith (1950) stored beets in large drums at 20 C for 45 days and found approximately 80 percent of the sucrose loss was due to respiration and 20 percent to inversion. The loss of sucrose by respiration has been esti— mated at between 0.3 and 0.5 pounds per ton per day at 21 C (Stout and Spikes, 1957). The early work of Barr t 31. (1940) indicated a very strong temperature effect where losses varied from 0.1 pound per ton per day at 3 C to 1.8 pound per ton per day at 35 C. Dilley e£_§1, (1969) found the respiration Q10 to be approximately 2 between 10 and 20 C. In the first few days after harvest the respiration rate was high but declined steadily after a few days in storage to a much lower and essentially constant rate. Bruising or other injury dras- tically increased the reSpiratory losses in the period immediately following harvest (Dilley e£_§1,, 1969; Stout and Smith, 1950) and also increased the susceptibility to mold invasion. The rate of respiration of the sugarbeet is closely correlated to its surface area. This phenomenon was first Observed by Stout (1954) and more recently a mathematical relationship has been developed to relate surface area to respiration rate (Vajna, 1960). 14C labeled sugars introduced into harvested beet roots have shown that sucrose, in short term storage, is almost exclusively the substrate for respiration in the sugarbeet (Barbour and‘Wang, 1961; Wang and Barbour, 1961). Several studies have been made to reduce respiration losses by modifying storage atmospheres. Reductions in stOr- age losses of up to 65 percent have been found (Stout, 1954; Vajna, 1960). Oxygen levels below 5 percent and carbon diox- ide above 15 percent were found detrimental (Vajna, 1960). Indirect--Factory Losses The decrease in recoverable sucrose during storage is not solely a result of sucrose lost as CO but is also 2. due to an increase in impurities in the factory thin juice (McGinnis, 1951; Silin, 1964; Carruthers _£__1,, 1962; Dexter eE_§1,, 1965). Factory losses incurred in processing the sugarbeet can be predicted by using the purity of the clear juice in conjunction with a formula derived by the Great Western Sugar Company. From this formula a 1 percent loss in clear juice purity will result in approximately a 6 pound or 2 percent loss in recoverable sugar per ton of beets. In an extensive examination Of compositional changes in diffusion juice from stored beets, Walker _E_31. (1960) found a decrease in purity from 92.2 percent to 87.5 percent in 90 days at 10 C. The apparent impurities calculated from thin juice purity increased from 8,500 to 14,700 mg per 100 gms sugar. The only significant compositional change found was an increase in invert at the expense of sucrose with no loss in total sugars. The 6042 mg per 100 gm sugar increase in total impurities was approximately equal to the 6200 mg per 100 gms sugar increase in invert. Carruthers 45 _1. (1962) derived the relationship of (3.5 x Na) + (2.5 x K) + (10 x NH2) which accounted for a high percentage of the total impurities in fresh beets. Dexter et_ 1. (1966) using Carruther's factors for Na, K, and ONH in the clear juice of stored beets found that 2 as the apparent impurities increased particularly at warm temperatures the percent of impurities unaccounted for by Na, K, and oNH -N increased sharply. If this relationship 2 is applied to the data presented by Walker the same trend is noted. Dexter prOposed that these unaccounted-for impu- rities may be weak acids with soluble calcium salts formed in the decomposition of sugars and/or cell wall constituents. _I£@s of Physicalggpteqrity During Storage One of the major problems in processing stored beets is the decrease in the filtration rate of limed diffusion juice which drastically reduces factory efficiency. The problem is caused by the formation of calcium pectate gels as a result of the solubilizing of cell wall pectin due to poor storage conditions (McGinnis, 1951; Silin, 1964). Breakdown of Marc Sugarbeet marc is the insoluble residue which remains after extraction of the beet with water (McGinnis, 1951). The marc is composed primarily of cellulose (~25%), hemicellulose (~25%), and pectic substances (~25%) (McCready, 1966). The pectic substances are stable only in cold water and swell and become Soluble in hot water (Silin, 1964). The marc averages approximately 5 percent of the fresh weight of the beet (Silin, 1964). In a test Of 15 varieties, Owens t 1. (1954) found the marc content to vary from 3 to 6 percent. Pectic Substances as Sources of Impurities The pectic substances are high molecular weight poly- mers, derivatives of pectic acid, and contain galacturonic acid residues. In pectic acid, the carboxyls are free and can readily react with calcium to form insoluble calcium pectate. In pectin and protOpectin the carboxyls are ester— ified with methanol (Joslyn, 1962). In the sugarbeet the free carboxyls may be as high as 50 percent methylated (Goodban and McCready, 1965). From 6 percent (Kertesz, 1951) to 30 percent (Goodban and McCready, 1965) of the hydroxyls in the two and three positions may be acetylated. The pectic substances exist in two forms: water soluble and water insoluble. The water insoluble form is protOpectin, the structure of which is unknown, but which is thought to exist in a matrix complex of pectin, cellulose, and hemicellulose. ProtOpectin upon hydrolysis yields the water soluble pectic acid (Kertesz, 1951 and Joslyn, 1962). The breakdown of protOpectin by water is highly temperature dependent. ProtOpectin when treated with hot water swells and gradually dissolves as pectin (Silin, 1964 and Owen et_ .21., 1955). Silin (1931) extracted dried beet pulp and found only a slight increase in solubility up to 80 C., how- ever, the amount of pectin extracted at 90 C was 30 times greater than at 80 C. The soluble forms of the pectic substances are pectin, pectic and pectinic acids. Pectic and pectinic acids have insoluble calcium salts, but are highly esteri- fied and yield methanol and soluble acetic acid salts when heated in alkaline solution (Kertesz, 1951). Changes in the Pectic Substances During Storage The soluble pectins after an initial increase in the early stages of growth, decrease steadily until harvest. The amount of total pectic substances decreased under des- iccation. Fertilizer has no significant effect (Gaponenkov, 1943). Silin (1964) reports that microbial activity and sprouting increase the quantity of soluble pectins during storage. Walker _§__1, (1960) found no significant change in the pectin content over a 48-day storage period when beets remained in good condition, but when stored for 161 days at 34 F, the soluble pectin content decreased by one- third. If freezing occurs during storage the pectin, which in a normal healthy beet is a large macromolecule, will be degraded by enzyme action to a smaller and more soluble colloidal size (Claassen, 1943). Effect of Pectic Substances on Processing The first noticeable effect Of pectic substances in the factory is in the diffuser. Beets which have been exposed to conditions which weaken the protOpectin stability must be diffused at lower temperatures to prevent the cos— settes from losing their resilience. Loss of resilience will prevent the free flow of water through the diffuser. For frozen or spoiled beets the diffuser temperature should not be above 70 to 75 C. Silin (1964) found a 110 percent increase in pectic substances in diffusion juices by increas— ing the temperature from 75.5 to 85.1 C. During defecation the pectinic acid present in the diffusion juice is de-esterified, splitting off methanol and acetic acid. The acetic acid forms soluble calcium acetate and the polygalacturonic acid residue forms an insoluble calcium pectate (Silin, 1964; Goodban and McCready, 1965). Non-Sucrose Components of the Beet Root Reducingigugars The predominant reducing sugars occurring in the beet are glucose and fructose. Free galactose and arabinose are found only in trace amounts (Silin, 1964). Reducing sugars are presumably destroyed during lime defecation and occur in very low amounts in beet molasses (McGinnis, 1951; Silin, 1964). In the process of alkali decomposition the reducing sugars from stable clear juices are degraded to 10 acids (lactic, formic, acetic, saccharinic) (Carruthers et_ 21,, 1959) which must be neutralized by the addition of sodium carbonate before evaporation to prevent sucrose inversion (Silin, 1964; McGinnis, 1951). As a result of sodium addition molasses quantity and purity are increased resulting in increased sucrose losses (Silin, 1963; McCready, 1969). The reducing sugar content of beets at harvest is very low and sodium carbonate addition is not required. ”However during storage the level of invert sugar may in- crease drastically, particularly if mold or rotting occurs (Stout, 1954; Walker _£__1,, 1960). Even under storage conditions which prevent molding, Sprouting, and wilting, reducing sugars may accumulate, particularly at storage temperatures above 10 C, because of changes in the metabolic balance in the beet root (Walker _E.§1., 1960). This accumu- lation is accelerated by storage conditions which allow des- iccation of the beet root (Atterson e£_§1,, 1963). In a beet devoid of microbial activity the degrada- tion of sucrose to reducing sugars can occur via the action of two enzymes: invertase and the reversal of sucrose syn- thetase (Milner and Aulgad, 1964). The invertase activity of the beet root is very low and has been considered absent by many wOrkers (Vaughn and MacDonald, 1967b). Invertase activity has been reported to increase during storage (Khelemskii, 1963), but no correlation between invertase 11 activity, respiration and reducing sugar accumulation was made. The existence Of a sucrose synthesizing enzyme in the beet root was first discovered by Dutton e£_§1, (1961). In the recent work of Avigad (1966) the possible role of sucrose synthetase in the breakdown of sucrose in the root was studied. The Keq for sucrose synthetase is 1.3 i 0.2 at pH 7.2 and estimated to be 0.2 - 0.5 at pH 6.0 and is therefore easily reversible. The sucrose cleavage activity in root homogenates by sucrose synthetase was found to be much greater than that by invertase. Therefore sucrose Synthetase is capable of playing an important role in the formation of reducing sugars in storage (Avigad, 1966). Raffinose The major trisaccharide occurring in the sugarbeet is raffinose which usually Occurs as 0.3 to 0.5 percent of the sucrose present (McGinnis, 1951). The amount in beets increases with prolonged cool periods during growth or stor- age. Raffinose is as chemically resistant to lime defeca- tion as sucrose and therefore accumulates in the molasses. Since raffinose forms insoluble calcium saccharates as does sucrose, it causes problems by greatly increasing the raf- finose content of Steffen molasses. A high raffinose con— tent in the thick juice produces distorted and elongated sucrose crystals (McGinnis, 1951). 12 The level of raffinose, occurring naturally in the beet and the degree to which it accumulates during growth and storage, is a varietal characteristic. Several pOpu- 1ations of mass selected beets were shown by Wood 35 31. (1956) to vary over a 10 fold range in raffinose content. Five varieties stored for 29 weeks at 4 C showed a signif- icant variety x storage interaction. The effect of temperature on raffinose accumulation was illustrated by storing beets at l C and 12 C (Walker g£_§1,, 1960). At 1 C the raffinose content increased four times after 60 days and then declined with prolonged stor- age. IAt 12 C the raffinose content almost doubled but then remained essentially constant. The effect of temperature was found to be reversible since beets allowed to accumulate raffinose at 2 C for 120 days when shifted to 25 C storage declined almost to their harvest level after 25 days (Mc- Cready and Goodwin, 1966). Atterson e£_§1, (1963) found that not only tempera- ture but also wilting affected the raffinose content of the beet root. Raffinose decreased markedly in beets stored under conditions permitting weight loss. The biochemical pathway of raffinose synthesis and degradation in the sugarbeet has not been elucidated. Pridham and Hassid (1965) found that raffinose is synthe- sized in the broad bean (Vicia faba) by the transfer of UDP-galactose onto sucrose. Other workers found trans- galactosidase activity between raffinose and sucrose in 13 wheat gernlwhich did not require nucleotides as coenzymes. More recently Avigad (1968) found UDP-glucose-4-epimerase activity in sugarbeet root. Since sucrose degradation may occur via sucrose synthetase to yield UDP-glucose this enzyme may have particular significance in the elucidation of the synthetic pathway in the beet. Effect of Agronomic Practices on Beet Root Composition and Storage Characteristics Nitrogen Fertilization The early work of Ulrich in the 1950's (Ulrich, 19547 Ulrich, 1955) on factors controlling the accumulation of sucrose in the beet root pointed out the very dominant effect Of nitrogen fertilization. .Sucrose accumulation in the root is inhibited by high levels of nitrogen (Haddock t 1., 1959; Odgen t 1., 1958; Haddock et 1., 19597 Rounds 2; 31, 1958; Haddock e5_ 1., 1956; Finkner t 1, 1958; Finkner _EI_1., 1964). This effect appears to be partially due to the decreased transport of sucrose from the leaf to the root. This is no doubt a result of continued vegetative growth late into the fall harvest season under high nitrogen regimes (Snyder and Tolbert, 1966). Excessive nitrogen fertilization also causes an increased accumulation of non—sucrose substances resulting in a decreased purity (Henry e£_§1,, 1961: Haddock et a1., 1956). Glutamic acid, the dominant amino acid found in the 14 beet root, appears to be a very sensitive indicator Of the nitrogen fertility level and consequently has shown a high negative correlation with the sucrose content of the beet root (Woolley and Bennet, 1959; Walker t 1., 1950; Hac _§ _1_. , 1950) . Nitrogen not only increases the glutamic acid con- tent Of the beet but all of the other nitrogen containing components. Although a significant variety x nitrogen interaction is commonly found the nitrogen effect is by far the most dominant (Finkner _£_§1,, 1958; Henry _£H§l., 1961). Excessive nitrogen fertilization also increases the ash content primarily sodium, calcium, and to a lesser extent potassium (Henry e£_§l,, 1961; Finkner 93.21,, 1958). Nitrogen fertilization has little effect on the raffinose or galactinol content (Rounds §£_§l,, 1958; Finkner _§_§l,, 1958). Due to the strong melassignic prOperties of sodium, potassium, and the amino acids excessive nitrogen fertiliza- tion is highly detrimental in terms of beet quality for processing (Rounds _E_§1,, 1958). Because of the increased prOportion of stored beets to the total beets processed, the effect of nitrogen fertil- ization on storage losses was studied by Dexter e£_§1, (1966). In general their results indicated that the higher quality, low-nitrogen beets had superior storing character- istics since the loss of extractable sugar per ton was 30 percent less than for high nitrogen beets. 15 PhOSphorus and Potassium Fertilization Studies to determine the effect of potassium and phOSphoruS have indicated little or no effect on beet quality when rates are increased above adequate levels (Finkner _t_§l,, 1964; Finkner _§.§1., 1958; Henry _E.31., 1961). Larmer (1937) noted that increased levels of phos- phorus improved storage characteristics. Harvest Date Due to the rapid accumulation of sucrose in the root during the fall, delaying harvest will normally increase the RSPA* yield. However the cool temperatures during this period tend to promote the accumulation of raffinose. The amount of raffinose accumulated is related to the average daily temperature, incidence of frost and the genetic make- up of the variety (Finkner _t_§l,, 1959; Wood et_§l,, 1956). Beets harvested early lose less sucrose in storage than late harvested beets (Vajna, 1960). However, the soluble pectin content is higher (Silin, 1964). *See Appendix A for explanation Of abbreviations. MATERIALS AND METHODS 1966 Preliminary EXperiments In 1966, samples containing 10 beets each and weigh— ing 16-20 pounds were stored either for 70 or 140 days at either 3 C or 11 C. Various degrees of desiccation were allowed to develOp by storing the beets in Single and double canvas bags and in polyethylene bags containing small perfor- ated bags of wet wood shavings. These treatments were then designated as severe, moderate, and slight wilt, reSpec- tively. The clear juice from the beets in this eXperiment was used to develOp rapid methods of raffinose and invert determinations to be used in subsequent studies. 1967.Experiments The beets for the storage eXperiments in 1967 were grown near Sebewaing, Michigan. In the fall of 1966, 375 pounds of 0-0-60 was broadcast and plowed down. Just prior to planting, 900 pounds of 0—20-0 was applied broadcast and incorporated with a field cultivator. On May 3, 1967 the beets were planted in 28-inch rows with 200 pounds of 12-6-6 applied as starter fertilizer. .After thinning to 120 beets per 100 feet of row, the beets in plots designated as high nitrogen were Side-dressed with 125 pounds of nitrogen per 16 17 acre for a total of 150 pounds of applied nitrogen per acre. The beets with low nitrogen received only the 24 pounds per acre of nitrogen applied as a row fertilizer at the time of planting. The five varieties and their quality ratings based on sucrose, CJP, and yield are given below: Variety .Quality Number Variety Type Rating 1 SP5481-0 Multigerm Average 2 SP63194-0 Monogerm Poor 3 02 Clone Multigerm Good 4 SP6322—0 Multigerm Excellent 5 (SL (129 x 133) ms x SP6322-0) Hybrid Monogerm Excellent Harvests were made on three dates: October 6, October 26, and November 6. All five varieties were har- vested on October 26 but only varieties 2, 3 and 5 on the other two dates. Beets from six field replications were pooled at harvest and lightly tOpped to remove the terminal crown bud. The samples were then transported to the Michigan Sugar Company Agricultural Research LaboratOries in Saginaw for analysis and storage. 1968 EXperiments Beets for the 1968 eXperiments were grown near St. Charles, Michigan and received 500 pounds of 6—24-12 at planting. The beets were thinned to a 120 beets per 100 feet of row four weeks after planting. The variety used was the current commercial (SL (129 x 133) ms x SP6322-0). At harvest, all field replications were pooled for analysis. 18 Specific Gravity Sortinqiand Sample Preparation After washing with high pressure cold water, the pooled samples were sorted by Specific gravity (Dexter, Frakes, Wyse, 1969). The average Specific gravity of beets from each field treatment (i.e., variety 1—-high nitrogen) was determined by weighing a representative portion (40 pounds) in air and in water. Two salt solutions were then prepared for each field treatment one of which.was 0.003 specific gravity units above the average and another 0.003 units below. This range correSponded to approximately 1 percent sucrose on beets. Thirty beets were then added to the tank containing the highest Specific gravity. Those beets which floated were transferred to the second tank containing the lower specific gravity. The concentration of the two tanks was then adjusted so that twenty beets floated in the first tank and ten in the second. The beets which sank in the second tank were used for storage eXperiments. This method eliminated the very high and very low quality beets and increased sample uniformity 400 percent. After sorting, the beets were rinsed to remove any adhering Sodium chloride and after surface drying were made into samples of uniform weight for storage or immediate analysis. All storage samples except those in the desiccation eXperiments were stored in 10" x 14" x 28" polyethylene bags. Each sample bag contained a one pint perforated polyethylene 19 bag containing wet wood chips to saturate the atmosphere and prevent wilting. In 1967 the wood chips were too wet and as a result free water formed on the beet surfaces causing some mold with prolonged storage. This problem was alleviated in 1968 by draining the chips before adding to the sample bags. Using this storage technique the loss in weight was less than 2 percent in 140-day storage (Figure l). The concen- tration of oxygen and carbon dioxide inside the bags was measured by extracting a sample with a syringe and analyzing on a Perkin-Elmer Vapor-fractionator. The average oxygen content was 19.6 percent and the carbon dioxide ranged between 0.8 and 1 percent. Outline of Major Storage Experiments 1967 Date of Harvest Objectives: 1. To determine the effect of harvest date on storing ability with primary emphasis on marc breakdown during storage. 2. To determine the changes in the non-sucrose components during the harvest period and their subsequent effect later in storage. 3. To determine the differences between varieties in both their physical and chemical response to storage. Procedure: Three varieties of beets were harvested on OctOber 6, OctOber 26, and November 6. Ten beet samples, weighing 20 to 28 pounds, of three varieties (No's. 2, 3 and 5) grown at 20 Figure 1. Internal and external views of a beet stored for 140 days at 3 C. 21 two nitrogen levels were washed, sorted and stored for 65 and 130 days at 3 C. All storage treatments were replicated three times. 1967 Topping and Row Width Objectives: To determine the effect of harvest quality on losses in recoverable sugar in storage. To determine if the crown affected the accumulation of non-sucrose constituents in storage. Beets grown in blocks Of 14 and 28 inch rows were tOpped either at the lowest leaf scar or by simply trimming off the terminal crown bud. Samples were stored for 100 days at 3 and 7 C. Desiccation Experiment Objectives: To separate the effects of desiccation and temperature on losses of RSPT. To determine the temperature at which Specific sugar transformations occur. Three levels of shrink were maintained by storing beets in a single canvas bag, a polyethylene bag and a polyethylene bag with wet chips added. All treatments were replicated three times and stored at 2, 4.5, 7.2, and 12.8 C for 35, 70, 105 and 140 days. 22 1967 Variety, Objective: To study the storage response of a wide Spectrum of varieties. Five varieties (see page 17) grown with 24 and 150 pounds of applied nitrogen were harvested on October 26. All samples were treated as in the 1967 date Of harvest eXperiment. Storage was for either 65 or 130 days at 3 C. 1968 Date of Harvest Objective: To confirm the results of the 1967 date of harvest experiment. The commercial variety (SL (129 x 133) ms x SP6322-0) was harvested on September 1, October 1, and November 1 from a uniform stand in a field near Saginaw, Michigan. Four replications were stored for either 50 or 100 days at 3 C for each harvest date. 1968 Variety and Temperature Objective: TO determine the variety x temperature interaction with particular emphasis On sugar transformation. Three varieties were harvested on November 10 from the sugarbeet breeding nursery at the Michigan State Univer- sity ExPeriment Station. Number Variety 5 (SL (129 x 133) ms x SP6322-0) 6 SP6721-01 ms 7 129 x 4661 x 4661 23 Ten beet samples of each variety were stored for 100 days at either 3 C or 10 C. All treatments except temperature were replicated three times. 1968 Fielgggprayg Objective: To determine the effect of several pre- harvest foliar sprays on the loss of RSPT in storage. Preharvest foliar applications of vanadium sulfate (3#/acre), pyrocatechol (3#/acre) and CCC (2-chloroethyl- trimethylammonium—chloride at 3000 ppm) were applied to run off with a hand sprayer ten days prior to harvest. The rates of application of vanadium sulfate and pyrocatechol were those recommended by Dr. D. J.‘Wort (1968). No rain occurred between the time of application and harvest but the weather was cool (40-50 F) for most of the ten day period. Some leaf burn.was noted on the plants treated with vanadium sulfate. Other treatments produced no visible effects. Samples were stored for either 50 or 100 days at 3 C with three replications. 1968 Vernalization Objective: To determine if cold induction has an effect on invert accumulation and on the rate Of reSpiration in storage. MH-30 and GA were applied ten days prior to harvest 3 at concentrations of 3000 ppm. Chemicals were applied with a hand sprayer to "run—off." The crowns were trimmed at harvest to remove all petioles and the beets were stored at 5 C. Respiration rates were determined at weekly intervals 24 on three replications of each treatment. The chemical composition of a second lot of beets from each treatment was analyzed after five, eight and fifteen weeks of storage. These beets were cut on a slight diagonal to prevent crown bud injury and were then planted in sterilized soil in a greenhouse maintained at 18 to 21 C. A sixteen hour day was maintained with florescent lights. The incidence of bolting was recorded after six weeks. 1968 Controlled AtmOSphere Objective: To determine the effect of modified atmospheres on the accumulation of non- sucrose constituents in storage. Gas mixtures of 5 percent oxygen, 5 percent carbon dioxide, and 90 percent nitrogen plus or minus 1000 ppm ethylene were purchased from Matheson. Each storage treat- ment consisted of three ten beet samples in separate con— tainers. The gas mixtures were allowed to flow in series through three chambers (the replicate samples of each treat- ment) at a rate of approximately 1 ft3/hr. The storage period was 8 weeks at 5 C. Laboratory Analyses The percent sucrose was determined by the Dexter, Frakes, and Snyder Method (1967) and the clear juice purity by a modification of the method of Carruthers (1962). The resulting clear juice samples were analyzed for potassium and sodium using a Coleman flame photometer. Total amino 25 acids were determined with ninhydrin by the method of Moore and Stein (1954). Reducing sugars were determined with 3, Sn-dinitrosalicylic acid(Bernfe1d, 1951). A coupled enzyme system was develOped for the determination of raffinose (Appendix B). Dry matter determinations were made by weighing 25 grams of well mixed brei directly into tared weighing bottles and drying at 105 C for 48 hours. Marc determina- tions were made using 50 grams of well mixed brei. The brei was placed in a 300 ml beaker and approximately 200-250 ml of water at the desired temperature was added. The beaker was then placed in a water bath at the apprOpriate temper— ature for 20 minutes. After incubation the samples were quantitatively transferred to tared 9 cm plastic Buchner funnels containing a powdered cellulose pad. The residue was then extracted with 25 C distilled water by repeated washing with ten 200 ml aliquots. The filters were dried at 105 C for 48 hours and the percent marc calculated as percent fresh weight. The weight loss during storage was determined on all samples. All analyses expressed on beet weight were corrected to the original weight at harvest. All sucrose analyses were corrected for raffinose and invert sugar (see page 139 for correction equation). 26 ReSpiration Analyses The carbon dioxide evolved was monitored with a Beckman Model IR—2 Infrared Analyzer and a Sargent SR recorder. ReSpiration rates were determined by measuring the time required for the evolution of 20 mg Of carbon dioxide. The samples consisted of 10 beets in a five gallon pail with a tight fitting lid. The lids were sealed with plastic tape only during the actual period of analysis. The air stream was pumped into the analyzer under a positive pressure of 60 cm of water with a diaphram pump (Figure 2). Pressure and flow rate were regulated with an adjustable flow meter. The air stream was passed through an ice bath to condense excess moisture before passing into the analyzer. .At the beginning of an analysis the sample container was sealed and coupled into the system to produce a closed circuit. Carbon dioxide was allowed to accumulate until a uniform rate of increase was obtained (1 to 2 minutes). At this point 10 cc or 20 mg of carbon dioxide was injected in- to the system and the recorder deflection noted. Since the samples were releasing carbon dioxide during the time required to reach a new equilibrium the deflection was measured by extrapolation back to the time of injection (Figure 3). The system was Opened to the air to flush out the accumulated carbon dioxide. After flushing to atmo- spheric carbon dioxide concentration the system was again 27 1 i3 4 C: 5 ~ 48 1. Analyzer 2. Recorder 3. Flowmeter 4. Moisture trap 5. Sample 6. Diaphram pump Figure 2. Diagram of carbon dioxide analyzing system. 28 .moo_mo me on m>Ho>o ou mHmEmm How OOHHSUOH MEAD mcflEprmp ou pom: tonne: .m musmflm commm Ummoau mammamcd Hmcwmucoo \\\\\ omuommcH Noo me on m>Ho>m -41. on omuwsomn made .-I couuomammp me om 'lllull'll‘ ll'llllllll'll OHOSQmOEum on omcwmo Emummm me o -..-- 1---; 29 closed and the time required fOr the sample to produce 20 mg of carbon dioxide was measured. This method gave very reproducible results and elim- inated errors due to changes in atmospheric pressure and instrument sensitivity over the extended period of the eXperiment. RESULTS AND DISCUSSION Preliminary Results 1966 Desiccation EXperiment The effect of desiccation on the loss of RSPT was studied at two temperatures. The results were used as a basis for planning subsequent studies. The clear juice was later used to develOp rapid methods of analysis for raffi- nose and reducing sugar. Raffinose The raffinose content at harvest was very low (300 mg/lOO RDS) but the average of all treatments increased almost three fold after 70 days Of storage at 3 C (Figure 4). Between 70 and 140 days the average raffinose content decreased sharply. At 11 C the raffinose level decreased in a linear fashion to less than 100 mg/100 RDS after 140 days of storage. The accumulation of raffinose was closely associated with the degree of desiccation during storage. Since the trends were the same, the wilt treatments for the two tem— peratures were pooled and the results given in Figure 5. 3O 31 1000 o 800 H l i 600 8 3 C :1 m o E: a? m 8 -A 400 m- In S m 200 0 70 140 Storage Period, Days Figure 4. Effect of temperature on raffinose formation. 32 700 600 500 400 300 Raffinose,Mg(100 RDS)-l 200 100 0 70 140 Storage Period, Days Figure 5. Effect of wilting on raffinose formation. NO‘wilt 0'; slight‘wilt ' ; heavy wilt ° ; wilt factor ( ). 33 Raffinose in samples which did not wilt in storage increased approximately 200 mg while in those allowed to wilt substan- tially it decreased to zero. After prolonged storage, the raffinose concentration in the Slightly wilted samples was equal to that at harvest. Reducing Sugars Increasing the storage temperature 8 C, from 3 to 11 C, increased the reducing sugar accumulation by 100 per- cent (Figure 6). At 3 C the level decreased over the first 70 days but then increased at approximately the same rate as the 11 C storage. The rapid rise in the last 70 days was caused in part by small amounts of mold, particularly in the root tip area. Beets which were not allowed to wilt accumulated very little reducing sugar at either 3 or 11 C, however, those wilted to 74 and 46 percent of their original weight more than tripled in reducing sugars (Figure 7). The highly wilted beets were in very poor physical condition and showed evidence of slight surface mold. ChromatOgraphic analysis* indicated the presence of kestose in amounts inversely prOportional to the raffinose content. Treatments which increased raffinose decreased kestose. *Paper-AWhatman #1; solvent--Butanol:Acetic Acid: Water, 4:1:5. 1400 1200 H l G a 1000 O O 2', U) 2 ~ 800 m H to 0‘ :3 U) U) G -H 600 U :3 '8 04 400 200 Figure 6. 34 70 140 Storage Period, Days Effect of temperature on invert formation. -1 35 1600 1400 1200 1000 800 600 Reducing Sugar, Mg(100 RDS) 400 200 0 70 140 Storage Period, Days Figure 7. Effect of wilting on reducing sugar formation. No wilt 0 ; slight wilt u ; heavy wilt ° ; wilt factor ( ). 36 The results confirm the work of Atterson _g.gl. (1963) who found the same relationship between wilting and trisaccharide formation. The formation of kestose indicates high invertase activity. Accumulation of reducing sugars in desiccated beets apparently was caused by this enzyme (Allen and Bacon, 1956). These preliminary studies indicated the importance of controlling all storage variables particularly desicca~ tion and surface molds when studying raffinose and invert formation. All subsequent samples were stored in poly— ethylene bags with a small amount of wet wood shavings added to prevent wilting. FACTORS CONTROLLING THE MAJOR NON-SUCROSE COMPONENTS OF THE BEET AT HARVEST AND DURING STORAGE Factors Controlling the Raffinose Content Storage Temperature The preliminary results indicated that the equilib- rium temperature for raffinose production was between 3 and 7 C. In 1967 beets were stored at four temperatures, 2, 4.5, 7.2 and 12.8 C to better define the threshold temperature for raffinose production. However due to refrigeration failure the samples at 2 C were lost. Based on results of other eXperiments, raffinose content may double or triple in 65 days of storage (same variety) at 3.C, therefore 2 C was well below the threshold temperature. At 4.5 C (Figure 8) the raffinose content remained essentially constant and declined at all higher temperatures. In terms of raffinose accumulation, the ideal storage temperature would be slightly above 4 C . Variety-Temperature Interaction Three varieties were stored in 1968 at temperatures well above and below the threshold level for raffinose pro- duction. This was an attempt to develOp a method for more 37 2000 1800 1600 1400 1200 1000 Raffinose, Mg Kg-l 800 600 400 200 35 70 105 145 Storage Period, Days Figure 8. Effect of storage temperature on the raffinose content of beets stored for 35, 70, 105 and 145 days (1967). 39 critically detecting varieties prone to raffinose production. Raffinose increased substantially in all varieties tested previously at 3 C, while storage temperatures above 6 C resulted in a general decline in raffinose content. Variety 5 had an intermediate raffinose content at harvest and variety 7 was very low considering that a week of freezing temperatures preceded harvest (Table 1). Variety 6 was extremely high at harvest, indicating sensi- tivity to temperature. In storage, variety 5 increased slightly (400 mg) in raffinose over the harvest level when stored warm and increased approximately 1000 mg when stored cold. Variety 7 reacted in the same manner but the raffi- nose doubled at 10 C and tripled at 3 C. Variety 6 nearly doubled at both storage temperatures. Variety 6 would be Table 1. Influence of temperature in the production of raffinose by several varieties during storage for 100 days at 3 and 10 C (1968) After Storage At At Variety Harvest 3 C 10 C Mg Kg-1 5 1288 2212 1693 6 2966 5015 4864 7 890 2602 1790 Average 1715 3310 2782 4o undesirable for processing after storage at either tempera- ture because of its extremely high raffinose content. The metabolic processes involved in raffinose pro- duction and degradation are very temperature sensitive and genetically controlled. Apparently two separate systems exist for raffinose production and degradation. At temper— atures above 5 C the degradative system Operates at a rate equal to or greater than the synthesizing system in most varieties. The Opposite is true below 5 C. All the vari- eties tested had the mechanism for synthesizing considerable quantities of raffinose as evidenced by its general occur- rence. The turnover rate of the raffinose pool has not been studied and therefore no estimate of the amount of sucrose lost via this system can be made. The rate of accumulation and degradation (McCready and Goodwin, 1966) (see page 88) is rather Slow but this merely indicates the differential rates of degradation and synthesis and not turnover rate. Ideally a superior variety would be one which did not accumulate raffinose. Variety Five varieties were stored in 1967 to study further the variety effect on raffinose content, reported to be substantial by Wood e£_gl, (1956). At harvest there was a small but significant difference between varieties (Figure 9). 3600 3400 3200 3000 T :3: 2800 U1 2: 5 2600 U) o c: «4 33 m 2400 m 2200 2000 ’ 1800 I 1600 0 Figure 9. 41 65 Storage Period, Days 130 Variety x storage interaction for raffinose accumulation at 3 C. Variety: 3,15; 4,1D; 5,I . 1.“ : 2.0 : 42 Varieties 2 and 4 had higher levels in the first 65 days of storage causing their overall means to be significantly higher. Variety 3 reacted very differently than the other varieties by increasing in an almost linear fashion over the entire storage period. Effect of Harvest Date The raffinose content was four to six times higher at harvest in 1967 than in 1966. This was related to the very cool, damp weather during October in 1967. Prior to the November 6 harvest a severe frost caused visible freez- ing injury to some of the beet crowns. (No frost damaged beets were stored.) The raffinose content increased approximately 35 percent from 1300 to 1850 mg/kg between the early and late harvests. There was little difference between harvest dates in the amount Of raffinose accumulated after 65 days of storage at 3 C (Figure 10). The harvest date x removal interaction was significant due to the greater increase in raffinose for the early harvested beets. The very high raffinose levels at harvest and during storage caused a considerable error in the percent sucrose and clear juice purity determinations resulting in an overestimation of the RSPT. These results indicated the need for correcting the percent sucrose and CJP determination in stdred beets and in some years, at harvest. 43 3100 O o I/ October 6 Nmmmmrlo 2500 ° 0 / . October 26 ow S d 2100 (D o I: -a tH w- m Of- 1700 1300 0 65 130 Storage Period, Days Figure 10. The effect of harvest date on the accumulation of raffinose during storage at 3 C in 1967. 44 In 1968 the effect of harvest date was again studied. The weather during the fall of 1968 was very warm and dry and the raffinose content at harvest was much lower than in 1967. However as in the 1967 the less mature beets showed the greatest increase in raffinose content in storage (Figure 11). The general decline with prolonged storage Observed in 1967 occurred only in the case of the late harvested beets in 1968. In the 1967 eXperiment there was a significant three- way interaction between harvest date, variety and length of storage (Table 2). Although the difference between vari- eties was statistically highly significant from a practical standpoint there was little difference. All three varieties increased in raffinose content during harvest at a very uniform rate. However there was a considerable difference between varieties in the amount of raffinose accumulated in the first 65 days of storage. Variety 5 was considerably lower for all harvests. Raffinose decreased in all vari- eties during the final 65 days of storage. Taking an aver- age of each variety over the entire storage period variety 5 was significantly lower than varieties 2 and 3. Effect of Nitrogen Fertilization The raffinose content of varieties 2 and 3 Showed little reSponse to nitrogen fertilization (Table 3). How- ever the 150 pounds of applied nitrogen per acre resulted in an increase in the raffinose content of 400 mg in variety 5. 45 October 1 O 3200 2800 September 1 O 2400 a November 1 O a 2200 2000 1000 600 0 50 100 Storage Period, Days Raffinose, Mg Kg-l Figure 11. Effect of harvest date on raffinose accumula- tion in storage at 3 C. 46 mvem mmsm emma hmmm comm «mpg mama Hmom coma mmmum>¢ HNHN Hmam mmmm mama meow mama swam mama HNON «mam mamm wmma m hhmm wmmm ogam moon thm Namm OHNN mmna Oemm Hemm maem OONH m mmmm emmm ommm ewna omen mmhm hmem hmha Hmmm mmwm Oewm HOMH m m m m m m 0 HI M 2 HI K 2 an M S m>< OMH mo umm>umm m>¢ Oma mm umm>umm m>< oma mo umw>umm humanm> DC pd u< mmmuoum mmmuoum wmmuoum SA mama SH mama SH mama m HOQEO>OZ om Honouoo m HwQODOO hwma Ga 0 m an mmmnoum Hmumm pom umm>umn um ucoucoo mmocammmu OED co wmmuoum mo numcma cam humflnm> .mumo umm>nm£ cmm3uon SOADOSHODGH .N manna 47 Table 3. Effect of nitrogen fertilization on the average raffinose content of three varieties stored at 3 C in 1967 Nitrogen, #/A Variety 24#/A ‘ 150#/A Mg Kg-1 2 2428 2507 3 2524 2402 5 1872 2255 Average 2274 2388 There was no variety x nitrogen x removal interac- tion. The nitrogen x removal interaction (Table 4) combines three varieties and three harvest dates and was highly sig— nificant. »Again high nitrogen fertilization increased the Table 4. Nitrogen x removal interaction for raffinose accumulation in storage (1967) Days in Storage At Nitrogen Harvest 65 130 Average -1 #/A Mg Kg 24 1749 3017 2127 2298 150 1778 2954 2675 2469 48 raffinose content, but only 170 mg. Nitrogen had no effect on raffinose build—up during storage but did retard its decline between 65 and 130 days of storage. Effect of Topping Beets tOpped at the lowest leaf scar were higher in raffinose than beets with only the terminal crown bud removed (Table 5), suggesting that the crown area was appar- ently lower in raffinose than the tap root. Leaving the crown on caused a slightly greater accumulation of raffinose in storage at 3 C. However the tOpping x temperature inter- action was not significant. Table 5. Effect of tOpping on raffinose content of beets stored at 3 and 7 C Stored 100 Days At At Harvest 3 C 7 C Mg Kg.-l T0pped 1519 1876 596 UntOpped 1330 2161 599 49 Preharvest Sprays GA and MH-30 applied prior to harvest significantly increased the level of raffinose at harvest (Table 6). Beets from these treatments tended to accumulate more raffi- nose in storage than the control. However the spray x removal interaction was not significant. The raffinose content doubled during the first 5 weeks of storage (Table 6). Although the content continued to increase, the treatment differences remained constant. Therefore in relatively short-term storage eXperiments it may be possible to screen varieties and treatments for their effect on raffinose accumulation. Several other Sprays (vanadium sulfate, pyrocatechol and CCC) did not affect raffinose content at harvest or dur— ing storage. Table 6. Effect of giberellic acid and maleic hydrazide on the raffinose content of fresh and stored beets (1968) Weeks at 5 C At Spray Harvest 5 8 12 Average Mg Kg'1 MH-30 1144 2673 3174 3341 2583 GA 1185 2943 3719 3430 2819 Control 904 2202 2714 3076 2224 Average 1078 2606 3202 3282 50 Storage in Modified Atmospheres Beets stored in modified atmOSpheres containing 5 percent carbon dioxide and 5 percent oxygen accumulated 2.5 times more raffinose than those stored at normal atmospheric concentrations (Table 7). The eXperimental design did not permit determining if the effect was caused by high carbon dioxide or by low oxygen. However the control containers accumulated approximately 1-2 percent carbon dioxide (due to insufficient air movement) and since these treatments did not result in accumulated raffinose the cause was apparently related to the low oxygen concentration. Ethylene, applied continuously throughout the storage period, had no effect. Table 7. .Effect of modified atmOSpheres on the accumulation of raffinose after 40 days of storage at 5.C Raffinose Content Ethylene AtmOSphere 0 1000 ppm Average Mg Kg-l Control 691 872 782 5%'02, 5% C02, 90% N2 1958 1899 1929 Average 1325 1386 At harvest 890 51 At normal levels of oxygen the raffinose content decreased slightly at 5 C. Decreasing the level of oxygen allowed raffinose to accumulate. Therefore raffinose accu— mulation was not only temperature dependent but also depended on oxygen concentration. Since oxygen directly affects the rate of reSpiration, the metabolism of raffinose may also be closely associated with respiration. Assuming oxygen affects the general metabolic level of the root, raffinose and invert would both be eXpected to change in relatively the same manner. However, oxygen had no effect on invert sugars. Therefore the metabolism of raffinose must be more directly associated to reSpiration than that due merely to the general metabolic level of the beet root. Whether the effect is due to the rate of synthesis or degra— dation could not be determined in this eXperiment. The brei from beets stored under low oxygen did not turn black upon prolonged eXposure to air at room tempera— ture while the brei from beets stored under normal oxygen turned black immediately. Apparently under low oxygen either the phenol oxidase activity decreased or the accumu- lation of phenolic substrates did not occur. Controlled atmosphere storage of beets may not be evaluated merely from the aspect of reduced respirational losses, but the changes in non-sucrose compounds must also be studied before it can be recommended for use. 52 Factorstontrolling the Reducing Sugar Content Preliminary results indicated that storage tempera- tures above 5 C accelerated accumulation of reducing sugars. In 1967 beets were stored at temperatures ranging from 2 to 12.8 C to determine if a threshold temperature similar to that found for raffinose existed for the accumulation of reducing sugars. Storage Temperature There was no significant difference between temper- atures in the accumulation of reducing sugars in the first 70 days of storage (Figure 12). Beets stored at 12.8 C began to sprout after 70 days and this may account for the rapid increase in reducing sugars. Beets stored at 4.5 C had some small Spots of mold and this could account for the rapid rise in invert after 105 days of storage. At 7.2 C the beets remained in perfect condition and the increase in reducing sugars was small. If molds and Sprouting are pre- vented, beet roots can apparently tolerate prolonged storage in temperatures up to 7 C without appreciable accumulation of reducing sugars. Variety-Temperature Interaction A highly significant variety-temperature interaction for reducing sugar accumulation occurred in 1968 when sam- ples of three varieties were stored at 3 and 10 C (Table 8). 53 .mhmo mga on a: Mom omuoum muomn CH gunman osmosomu mo coaumassouum on» so muoumummEmu mo vacuum mama .ooHumm mmmuoum .NH ousmam oom H m. oom m m. .b S n .b D. I cema m w 5 .U 0 0 oosa m “[— OOHN 54 Table 8. Interaction of variety with temperature in the accumulation of reducing sugars after 100 days of storage at 3 and 10,C Storage Temperature,C At Variety Harvest 3 C 10 C Mg Kg-1 5 656 658 1297 6 916 1095 1530 7 831 965 1115 Average 801 906 1314 The accumulation of reducing sugars was very small for all varieties stored at 3 C but all increased substantially at 10 C. The sensitivity of the varieties to 10 C storage was very different. Variety 5 doubled in concentration while variety 7 increased only 35 percent. Since no molds occurred in.the 1968 storage it appears that the plant breeder may be able to select varieties which can be stored above 5 C with- out the production of reducing sugars. Variety The significant variety x removal interaction (Table 9) indicated that for the first 65 days of storage very little reducing sugar accumulated but in the last 65 days the reducing sugar content approximately doubled. 55 Table 9. Variety x removal interaction in reducing sugar accumulation (1967) for beets stored at 3 C Stored (days) Variety Hagtest 65 130 Average Mg Kg-1 2 848 1241 1953 1347 3 638 856 2833 1442 5 745 1135 1931 1270 Average 743 1077 2239 In this experiment the initial increase was a varietal char- acteristic typical of normal healthy beets in storage and the later rise was due to mold invasion. Although no vari- etal differences in susceptibility to mold invasion were noted, varietal differences have been shown by other workers (Gaskill, 1950). Effect of Harvest Date The reducing sugar content of the beet decreased slightly during the 1967 harvest period. The harvest x removal interaction was highly significant (Table 10). The early harvested beets remained lower throughout the 130 day storage period. The occurrence of very high levels of reducing sugar after 130 days of storage was due to small amounts of rot occurring in the crown region of the beets. 56 Table 10. Harvest x removal interaction for reducing sugar accumulation (1967) for beets stored at 3.C Stored (days) Harvest At Date Harvest 65 130 Mg Kg-l October 6 771 944 1175 October 26 765 1203 2549 November 6 695 1084 2994 These regions were high in reducing sugars and since no attempt was made to remove them, the result was a high aver- age reducing sugar content for the entire beet. Treatments did not differ in the amount of rotting which was due to moisture condensation between the polyethylene bag and the beet surface. Mold also occurred where free water condensed at the points of beet to beet contact. Mold formation is an important consideration if the practice of washing beets prior to storage is used commercially. The harvest date x variety x removal date was highly significant (Table 11). Variety 3 remained low in reducing sugars for all harvest and removal dates, except for the 130-day storage of the late harvest. However this large increase was due to molding. In general, the early harvested beets did not increase as much in reducing sugars as the later harvested beets. S7 Table 11. Harvest x variety x removal interaction for reducing sugar accumulation of beets stored at 3 C (1967) Stored (days) At Variety Harvest 65 130 Average Mg Kg-1 October 6: 2 864 997 1172 1011 3 663 798 987 816 5 787 1036 1366 1063 Average 772 944 1175 October 26: 2 869 1514 2650 1677 3 642 799 2636 1392 5 783 1295 2260 1446 Average 764 1202 2549 November 6: 2 810 1212 2038 1353 3 609 968 4775 2117 5 666 1072 2167 1301 Average 694 1084 2993 58 In the 1968 date of harvest eXperiment the harvest x removal interaction was again significant (Figure 13). The problem of mold in the previous year was eliminated and the increase in reducing sugars with storage was not as great. The early harvested beets remained almost constant while the amount of reducing sugars in the late harvested beets doubled after 100 days of storage. Therefore two years data indicated that late harvested beets tended to accumulate more reducing sugars under prolonged ideal low temperature storage conditions than beets harvested prior to November 1. The September 1 harvest was extremely high in reducing sugars for fresh beets, the reducing sugars decreased substantially at the later harvests. Variety-Nitrogen7Igteraction There was a significant variety x nitrogen x removal interaction when five varieties were stored in 1967 (Table 12). The significant interaction occurred in the 65-130 day period of storage. Again this variation was apparently due to the incidence of molds. During the first 65 days of storage all of the varieties except variety 3 approximately doubled in reducing sugars. Variety 3 increased by only 25 percent. Nitrogen fertilization had no effect on the amount of reducing sugars accumulated in the first 65 days of storage. 59 1400 November 1 0 October 1 O 1200 'm x m September 1 2 o m‘ H '3, 1000 s m 8‘ -H o o o m m 800 600 0 50 100 Storage Period, Days Figure 13. Effect of harvest date on the accumulation of reducing sugars in storage at 3 C. 60 Table 12. Variety x nitrogen x removal interaction for reducing sugar accumulation in five varieties grown on 24 and 150# nitrogen per acre and stored at 3 C for 65 and 130 days Stored 130 Days At Harvest Stored 65 Days Variety 24# 150# Avg. 24# 150# Avg. 24# 150# Avg. Mg Kg-1 1 640 679 660 1021 1059 1040 4724 2697 3711 2 961 777 869 1626 1401 1513 1906 3393 2649 3 613 670 641 781 818 799 2314 3159 2736 4 628 685 656 1190 1099 1144 2653 2561 2607 5 794 772 783 1246 1342 2394 2579 1941 2260 Average 727 716 1173 1144 2835 2751 The variety x nitrogen x removal interaction was also highly significant (Figure 14). All treatments in- creased essentially parallel in the first 65 days of storage. However in the final 65 days varieties 2 and 3 grown on high nitrogen increased by approximately 100 mg over the beets with 103 nitrogen. For variety 5 the reverse trend was true. The threedway interactions were apparently due to the inci- dence of molds and are therefore not indicative of normal beet reSponses. However these results indicate the drastic effect of only small amounts of mold on the composition and subsequent processing characteristics of stored beets (see page 147). 61 O o ~m uu~m M.\N omH vN ummmo oma umum< "huwaum> mummsm moansomu mo Godumaoeouum on» no coflumuaaauumm oomouuac mo uommmm coo coca oowa oomH CONN OOON ooom oo¢m .0 m um wmmuoum magnum mmaumfinm> wounu CH 4 mna cmmouufiz omflu so I - n n.1|||||||||||||a o olll'llllln|lll O/O whom no Hound OOOH oovH oomH CONN oooN ooom oo¢m .cH mucous cm cmH . coo u.|I|II|III|IIIIu ° ‘ o o coca coca coma coma coco ooom coco uww>umm ufl I_6x 6w ’sxefins Buronpea 62 Effect of TOpping Although the increase in reducing sugars during storage was great, topping of beets prior to storage did not significantly affect the accumulation (Table 13). Table 13. Effect of tOpping on the accumulation of reducing sugars after 50 and 100 days of storage (average of 3 and 7 C) (1967) Stored (days) At Harvest 50 100 Mg Kg-l TOpped 863 1891 3089 UntOpped 905 1484 3305 Preharvest Sprays The application of GA and MH-30 prior to harvest significantly increased the accumulation of reducing sugars after eight weeks of storage. Prior to eight weeks no sig- nificant difference existed (Figure 15). Vanadium sulfate, pyrocatechaol and CCC were applied preharvest in an experiment separate from the GA and MH-30 application. Although no statistically significant spray x removal interaction was found all treatments were lower in 63 1600 My-BO GA 0 1400 Control a 1200 H 'm M 1000 a; S U, 800 s m 8‘ -H 8 600 to 3 400 200 0 Harvest 5 8 12 Storage Period, Weeks Figure 15. Effect of preharvest applications of GA and MH-30 on the reducing sugar content in stor- age at 5 C. 64 reducing sugars than the control at harvest and during stor- age (Table 14). The much reduced level with CCC treatment warrants further study. Table 14. Effect of various preharvest foliar sprays on the accumulation of reducing sugars during storage at 4 C (1968) Stored (days) At Spray Harvest 50 100 Average Mg Kg-l Control 762 742 1274 926 Vanadium Sulfate 712 710 1116 846 Catechol 716 715 1133 855 CCC 698 647 930 759 Storage in Modified Atmospheres Altering the carbon dioxide and oxygen concentration in the storage atmosphere had no effect on the accumulation of reducing sugars at 5 C (Table 15). Ethylene also had no effect. This is in contrast to the work reported previously where oxygen levels below 5-7 percent were reported to cause invert accumulation due to the onset of anaerobic respira- tion (Vajna, 1960). 65 Table 15. .Effect of modified storage atmospheres on the accumulation of reducing sugars after 40 days of storage at 5 C Ethylene Atmosphere 0 1000 ppm Average Mg K9"l Control 768 862 815 5%.02, 5% C02, 90% N2 906 909 910 Average 837 886 At Harvest 831 The ideal storage conditions under which these experiments were conducted produced atypical results com- pared to reducing sugar accumulation in commercial piles. Factories in Michigan have Observed that the reducing sugar levels in the early stages of the campaign are approximately 1000 mg/kg and may increase to 9,500 to 12,500 mg/kg after prolonged storage. Therefore, the losses due to reducing sugar production in commercial Operations are much greater than those observed in these eXperiments. 66 Factors Controlling the Amino Acid Content Effect of Nitrogen Fertilizer Beets grown with low nitrogen (24#/A) became nitro- gen deficient several weeks prior to harvest, in 1967, as evidenced by leaf yellowing. The high nitrogen (150#/A) plants were still very succulent and green at the first harvest date (October 6). The free amino acid content of the root decreased almost linearly during the harvest period at the low nitrogen level (Figure 16, dotted lines). The beets were apparently nitrogen deficient and the free amino acid pool in the root was being depleted. The beets with high nitrogen remained high in free amino acids until October 25 after which time the amino acid content* declined rapidly. On November 6 the beets grown on high nitrogen were equal in amino acid content to the low nitrogen beets harvested on October 6. The amino acid content declined during storage at 3 C. This was found previously by Dexter _£_§1, (1966) and is apparentlydue to synthesis of new enzymes and other pro- teins. The beets grown on low nitrogen declined rapidly in amino acid content during the first 65 days of storage and then remained essentially constant at 1200 to 1500 mg/kg. *a—amino nitrogen times 10. 67 0 “085502 OMH .mmmo oma can no now u m on cmuoum mummn om ucmucoo owom Canm How ooHuomumuca Hm>OEmH x ammonuflc x umm>umm .oa mama .oOAHmm mmmHODm mm om umnouoo OMH mm o ¢\¢wma .Illllllllln O c umnouuo whomflm OONH Coma OOON oovm oomN OONm Doom ’sprov’ourmv 6x 6w 68 The high nitrogen beets declined almost linearly (Figure 16). The rate of decline in amino acids of both the high and low nitrogen beets appeared to be related to the amino acid con- tent at harvest (Figure 17). The higher the harvest level the greater the decline during storage. Variety-Nitrogen Interaction The variety x nitrogen interaction at harvest (Table 16) was significant indicating a substantial differ- ence between varieties in their sensitivity to excessive nitrogen fertilization. Variety 5 was extremely sensitive having about twice the amino acid content at the 150 pound compared to the 24 pound per acre level of fertilization. Table 16. .Effect of nitrogen fertilization on the free amino acid content of three varieties at harvest (1967) Nitrogen Applied, Lbs/A Variety 24 150 M9 Kg"1 2 1540 2462 3 1663 2832 5 1636 3360 Average 1613 2884 69 3300 3000 2700 2400 2100 1800 1500 Amino Acids, Mg Kg-1 1200 900 600 300 65 130 Storage Period, Days Figure 17. Amino acid content in storage at 3 C. 70 This sensitivity was also evident in the RSPA yield of vari- ety 5 (p. 155). At the low level of nitrogen fertilization all varieties had essentially identical concentrations of amino acids. There was no significant variety x nitrogen x removal interaction but the harvest x removal x nitrogen interaction was significant (Figure 16). When five varieties were stored for 65 and 130 days at 3 C no variety x removal or nitrogen x removal interac- tions were found. However the variety x nitrogen interac— tion was highly significant (Table 17). Varieties 3 and 5 appeared to be highly sensitive to nitrogen fertilization since their amino acid content was almost doubled under high nitrogen fertilization. Table 17. Variety x nitrogen interaction in amino acid content of five varieties Nitrogen Applied, Lbs/A Variety 24 150 Mg K94 1‘ 1643 2153 2 1454 2493 3 1609 3036 4 1322 2443 5 1541 3564 Average ‘ 1514 2738 71 Variety-Temperature Interaction Cold storage resulted in no accumulation of amino acids in three varieties stored at 3 C for 100 days in 1968 (Table 18). However at 11 C the amino acid content in- creased 50 percent. Variety 7 was relatively insensitive in its response to storage temperature. The extremely high amino acid content of variety 6 at harvest increased sub- stantially at both temperatures. Variety 5 was sensitive t0'warm storage temperatures, accumulating 464 mg/kg. Table 18. The effect of storage temperature on the amino acid content of beets stored for 100 days Storage Temperature Variety Harvest 3 C 11 C Mg Kg"1 5 1241 1128 (-113)a 1705 (+464)a 6 2267 2567 (+300) 2749 (+482) 7 808 864 ( +56) 969 (+161) Average 1439 1520 2117 aParentheses indicate change during storage. 72 In storing beets at various temperatures, varieties differed considerably with respect to the level of raffinose, invert, and amino acids. These compounds make up the major- ity of the non-sucrose components in the clear juice. Through breeding and prOper agronomic and storage practices the level of these important impurities can be controlled. The selection of a beet which would tolerate storage at 11 C without the accumulation of reducing sugars or amino acids appears to be possible and would greatly reduce the cost of refrigerated pile cooling. Effect of TOpping TOpping of beets prior to harvest had no effect on the changes in the amino acid content at either 3 or 7 C storage. TOpped beets were slightly lower in amino acids than untOpped beets (1776 vs 2070) indicating that the crown was somewhat higher in amino acids than the tap root. Preharvest_§prays MH-30 and GA applied prior to harvest decreased the free amino acid content of the root at harvest. The amino acids of beets pretreated with GA also remained lower throughout storage, but with MH-30 they increased for the first 8 weeks of storage and then fell rapidly to the same level as the plants treated with GA (see Figure 18). MH-30 has been shown to inhibit pyrimidine synthesis which may have reduced new protein synthesis (Pavlinova, 1967). 3300 3000 2700 2400 2100 1800 1500 Amino Acid, Mg Kg-l 1200 900 600 300 Figure 18. 73 MH-30 Control 5 8 12 Storage Period, Weeks Effect of preharvest applications of MH-30 and gibberellic acid on the amino acid content of stored beets. 74 This could result in the accumulation of amino acids due to normal protein degradation. Vanadium sulfate, pyrocatechol and CCC applied prior to harvest had no effect on the amino acid content either at harvest or after storage (Table 19). Table 19. Effect of several preharvest sprays on the amino acid content of fresh and stored beets Days in Storage Spray Harvest 50 100 Average Mg Kg-1 Control 2471 2072 2243 .2262 VaSO4 2251 2051 1668 1990 Pyrocatechol 2557 2204 1873 2211 CCC 2285 1753 1878 1972 ’Average 2391 2019 1916 Storage in Modified Atmospheres The storage of beets at 5.C in modified atmospheres for 40 days resulted in a 56 percent increase in amino acids over the control (Table 20). ,Ethylene (1000 ppm) increased the amino acid content 500 mg in normal atmosphere but caused a decline in the modified atmosphere. However the 75 increase in amino acids over the harvest level was much greater than in other experiments at the same storage temperature. Table 20. .Effect of modified atmospheres on the amino acid content of stored beets Ethylene Atmosphere 0 ppm 1000 ppm Average Mg Kg"l Control 1660 2114 1887 Modified Atmosphere 3126 2767 2946 Average 2393 2441 At Harvest 808 76 Factors Controlling the Sodium and Potassium Content of the Root Sodium and potassium are two very important melassigenic constituents of the beet root (Stark, 1968; Khualkouskii, 1964; Silina, 1964). They have been pro— posed for use as a quality index in selecting breeding lines (Wood, 1958). Carruthers prOposed their use in estimating the purity of the clear juice (1963). Variety—Nitrogenygnteraction In 1967 the potassium and sodium content varied between varieties although the differences were not great (Table 21). Nitrogen fertilization had a major effect on both minerals and sodium in particular (Table 22). The nitrogen effect on sodium content was greater than the variety effect. This increase in sodium and potassium caused by high nitrogen fertilization was apparently due in part to the need for more base to neutralize the increased amino acid content of the beets grown on high nitrogen. Nitrggen Meg_Amino Acids Meq K and Na 150#/A 13 24 + 3 = 27 24#/A 7 21 + 2 = 23 Difference 6 4 77 Table 21. Effect of nitrogen fertilization on the sodium and potassium content of five varieties harvested on October 26, 1967 Sodium Potassium Nitrogen Nitrogen Variety 24#/A 150#/A Average 24#/A 150#/A Average Mg Kg_l Mg Kg-1 1 81 87 84 1531 1465 1498 2 78 96 87 1376 1571 1474 3 74 112 93 1159 1335 1247 4 67 107 87 1456 1566 1511 5 72 108 90 1325 1412 1369 Average 74 102 1369 1470 Table 22. Effect of nitrogen fertilization on the average sodium and potassium content of three varieties harvested on three dates in 1967 Sodium Potassium Nitrogen Nitrogen Variety 24#/A 150#/A Average 24#/A 150#/A Average Mg Kg'l Mg Kg"1 2 79 97 88 1436 1681 1506 3 81 114 97 1287 1440 1310 5 77 110 93 1301 1321 1417 Average 79 107 1341 1481 * 78 However the increase in free amino acids was greater than the combined increase in sodium and potassium. Harvest Date The harvest x removal interaction for potassium in 1968 was significant indicating that more potassiumwwas available in stored than in fresh beets (Table 23). This same trend was found by Dexter (1966). The total potassium content in the root cannot change during storage, therefore the potassium in the beet apparently became associated with more soluble anions and was more readily squeezed out of the brei in the process of taking the sugar sample. In many of the other experiments the increased potassium in the clear juice after storage was nearly significant. In fact the increase was large enough to prevent using potassium as a basis for eXpressing changes in the chemical composition of the beets during storage. This method has been prOposed by Vajna (1960), but its effectiveness would require an analysis of total potassium in the ash fraction. ~Preharvest Sprays Foliar Sprays applied 10 days prior to harvest significantly affected the sodium content of the root at harvest and caused a significant increase in both the sodium and potassium content during storage (Table 24). Vanadium sulfate and CCC significantly reduced the level of sodium at harvest. Although the Spray x removal interaction 79 moga mafia moma mm ooa om mmwam>¢ hooa mama Sega mama nma aoa mwa mma a amaam>oz gmma ocaa amga hoaa no mm am am a Honouoo coca mmca mama coma co ooa me eoa a nonsmuomm ox m: m m an an x 2 0mmHm>< ooa om um0>amm 0mwam>< ooa om umm>umm um0>umm Ammmov u< Ammmov pd omaoum omaoum Edammmuom ESaUOm moma Ga mummn cohoum com nmmam mo acoucou Ecammmuom cam EnaoOM map so 0066 umm>um£ mo uomumm .mm manna 80 coca coma coca asammmuoo 65am anacom mama amoa mama Nma mNa maa wmmam>¢ mmga mmma mmma omma moa moa maa mm 000 mmma mmwa mama mmma Nga mma oma moa aonomumooamm moma moma omwa Nmoa aaa mma maa mm mummasm Egaomom> mmoa hmoa Saga nmma ooa moa hma mNa aouucoo m m m m HIM z HIM 2 mmmum>< ooa om umm>amm wmmnm>4 ooa om umm>amm mmamm anamov um ammmov um concum omnoum Edammmuom Edaoom mumwn ooa0um cam nwmam mo uomucoo Edammmuom pom ESaUOm on» so mamamm HmaaOM umm>am£mam amam>mm mo uommmm .wm magma 81 was non-significant, the increase in sodium and potassium during 100 days of storage was highly significant. GA and MH-30 had no effect on the Sodium and potassium content either at harvest or in storage. Factors Controlling the Chloride Content The chloride content of the clear juice is thought to have a high positive correlation to molasses purity (Stark §£_gl,, 1968). Increasing the chloride content increases both the quantity and purity of molasses, thus reducing RSPT. Variety-Nitrogen-Harvest Date Interaction The variety x nitrogen x harvest date interaction for chlorides was significant (Table 25). The average chloride content declined slightly with delayed harvest. The beets grown on low nitrogen contained a higher chloride content than the high nitrogen beets in all varieties except variety 3 harvested late. This is the only non-sucrose com— ponent which showed this reSponse to nitrogen, all others increased with increased nitrogen applications. Variety 5 was significantly higher in chlorides on the average for all Ilarvest dates. The variety x nitrogen interaction has highly signif— icant for the five varieties harvested on October 26 (Table 26). The beets grown on low nitrogen were again higher on 82 Table 25. Interaction between variety, nitrOgen, and har- vest date on the chloride content in 1967 October 6 October 26 November 6 Nitrogen Nitrogen Nitrogen Variety Variety 24# 150# 24# 150# 24# 150# Average M9 K9-1 2 217 137 196 133 210 157 175 3 206 164 133 171 168 171 167 5 255 220 252 182 189 166 212 Average 226 173 194 162 189 171 Harvest Average 200 178 178 Table 26. Variety x nitrogen interaction for chloride con- tent of five varieties grown on high (150#) and low (24#) nitrogen Nitrogen Variety 24#/A 150#/A Mg Kg-1 1 252 189 2 196 133 3 133 172 4 203 224 5 252 182 Average 208 180 83 the average but varieties 3 and 4 were slightly higher in chloride content at high nitrogen fertilization. There was no significant treatment x storage interaction in chloride content. If the millequivalent decrease in chloride content with high levels of nitrogen is considered in the hypothesis that the increased base content is due to the higher concen- tration of amino acids, an interesting relationship develOps. The chloride content was 1 millequivalent lower (lgg-vs Egg) under low nitrogen fertilization. Therefore the balance is as follows: Nitrggen 22:3; Chlorides nggl, Meg K and Na Mg Kg-l 24#/A 7 5.8 12.8 21 + 2 = 23 l50#/A 13 4.8 17.8 24 + 3 = 27 Difference 5.0 4 In the field the acid-base balance of the beet is essentially balanced and any adjustments needed in process- ing arise either from reducing sugar and breakdown products or differential ion elimination in clarification. 84 Effect of Thermal Induction on Chemical Composition of Beets Stored at 5 C Beets from the same origin as those used in the res- piration study (p. 117) and stored at the same temperature (5 C) were analyzed after 5, 8 and 12 weeks of storage. Besides the usual chemical analyses beets from each treat- ment were planted in the greenhouse to determine the degree of thermal induction. After 5 weeks of storage at 5 C, 87 percent of the GA treated and 56 percent of the control plants bolted. The plants treated with MH—30 produced only very stunted and deformed leaves. After 8 weeks of storage all plants from the GA and control treatments bolted. MH-30 inhibited normal Sprout formation for the entire 15 week storage period. Figure 19 is an average of all preharvest treatments and shows the effect of thermal induction on the raffinose, invert and amino acid content in storage. The non-induced beets were 35 percent higher than the induced beets in raffinose after 5 weeks in storage. However only the rate of accumulation was reduced because the induced samples increased to a high level after continued storage. The results from previous years showed that after 60 days of storage at 5 C, the reducing sugars began to increase. Since approximately 60-70 days at 5 C fulfills the thermal requirement for bolting, induced samples might 85 .Il.0mooooa 1:. 0aom oanm 0cm Hmmsm mcaosoou .omoochlcoz .0 m an omuoum mummn mo ucmucoo .mmocammmu on» no GOauoocca amEHmnu mo uommmm mxmmz..00anmm mmmuoum .ma whomam Na m m Na m m 0 0 0 00¢ 00¢ 00¢ ./. ooa-co. 00m 000 00m coma coma coma 000a 000a 000a 000m 000m 000m m0au< OCaE¢ mummsm mcaoawmm omocammmm 6x 5w 86 accumulate reducing sugars at a more rapid rate than the non-induced samples. However, the average of the three treatments shows that the non-induced plants were not sig- nificantly higher in reducing sugars. The reducing sugar content did increase rapidly later in the storage period but this could not be attributed to the induction process since all treatments were fully induced. The non-induced plants were also lower in amino acid content than the induced plants, but again the differences were very small. Table 27 gives the amino acid, raffinose, and reduc- ing sugar content of the induced vs non-induced for all pre- harvest treatments. The difference between the induced and non-induced roots was greatest for the control. Induction decreased raffinose and reducing sugars 600 and 125 mg/lOO RDS reSpectively. The GA treatments showed the same trend but the differences were much smaller. The plants treated with MH did not Sprout normally so no analysis of induction could be made. However the trend for the MH treatment was the same as for the non-bolters in the control and GA treat- ments. From this tenuous information MH may have inhibited induction completely as Opposed to the stimulation by GA (Stout, 1959; Stout, 1959). MH is considered a general growth retardant and has been shown to inhibit pyrimidine synthesis in the sugarbeet (Pavlinova, 1967: Kursanov, 1967). 87 000 0cm 0m¢ 0a¢ mmoa m¢0 mamuaomlcoz 000 mmo awn amm 00¢ mmm 0mma mmma mmma mamuaom mm0mu0>¢ ¢a0 0mm 000 mun 0N0 mom 00¢ m0m 0¢ma ¢mma a0ma a00 mumuaomlcoz cmummua cmumz mob 00h ¢m¢ 00m mmma >00 mamuaomlcoz 50m 00m nob 0¢0 mam 0m¢ mm0a mNaN 0¢¢a mamuaom CmummHB £0 0¢0 0c0 00¢ ~¢¢ 000a 00m mumuaomlcoz 000 ¢0c 000 can aa¢ mam 000a ~00a 000 mumuaom ammmmmw mom coa\0z ma 0 m 0 Na 0 m 0 ma 0 m 0 omuoum mxmoz moaod oaae< A Hmmsm moaooomm mmocammmm 0Ga05©ma 0cm caom ocaEm a000av 0.0 pm cmaoum mummn mo ucmucoo Hmmsm .mmocaummu may no ceauosooa amEHmnu mo uummmm .hN wanB 88 Effect of Prestorage Heating on Raffinose, Reducing Sugar! and Amino Acid Content After Storage Due to warm fall temperatures, interior pile temper- atures may increase to 20-32 C for several days. Since this is a very common occurrence in commercial piles it seemed pertinent to study the effect of excessive heating early in the storage period on subsequent storage. Samples were held at 27 C for 5 days in polyethylene bags. After the heating period the samples were placed in storage at 3 C. The effect on the raffinose, reducing sugar and amino acid content after storage is given in Table 28. The raffinose content declined 30 percent during the five days at 27 C while the control at 3 C remained constant. However the heating treatment had no effect on the subsequent accumu- lation of raffinose in storage. The amino acid and reducing sugar levels were not affected. Apparently beets can tolerate warm temperatures for short periods. The only loss would be due to the increased respiration. In the commercial pile these warm temperatures also facilitate mold growth and Sprouting which are major factors contributing to sugar loss in commercial piles. {Table 28. Effect of postharvest heating on raffinose, and amino acid production in subsequent cold storage (1968) invert, Days Stored At Harvest 5 50 100 M9 K9-l Raffinose 27 C--5 days then at 3 C 1071 734 2152 1985 Control (3 C) 1071 1076 2352 2218 Invert 27 C--5 days 'then at 3 C 762 897 981 1290 Control (3 C) 762 939 744 1272 Amino Acids 27 C--5 days then at 3 C 2471 2635 2637 2303 Control (3 C) 2471 2700 2155 2243 90 Comparison of Analyzed vs Total Non-Sucrose Components of the Clear Juice The preceding sections have discussed the content of several major impurities found in the clear juice at harvest and during storage. These non-sucrose components of the clear juice were composited as total analyzed impurities (TAI) and compared to the total impurities* in the clear juice as calculated from the clear juice purity (Table 29). The TAI comprised approximately 65 percent of the total impurities in fresh and stored beets. However the changes in the TAI and non-TAI during storage varied widely between varieties. The total impurities increased in the first 65 days of storage in all varieties. However varieties 2, 3 and 4 decreased slightly in total impurities during the second 65- day period. This indicates an apparent shift in the meta- bolic pattern of these varieties after prolonged storage. The change in the unknown impurities gives a good illustra- tion of this shift (Table 29, Figure 20). In variety 1 the unknown impurities increased steadily during the entire stor- age period. In varieties 2, 3, 4 and 5 a decline in unknown impurities occurred in the last 65 days. This decline indi— cates a metabolic shift towards the catabolism of these unknown compounds possibly via reSpiration (see p. 122). *Impurities include all soluble non—sucrose constituents found in the thin juice. 91. .mwauaudmfia amuOu mo ucmouwm n mmmmnusmumm ca ngaon . Ammo Am.aao , Acao Ammo .cao 00m: 0cm 0a0 0ma¢ ha0ma 0o¢0 a¢0a ¢¢ha m0hm a0mm 00mu0>< 00: 0¢a| ohm ~00¢ 00¢aa 00¢h m0¢a m0am 00mm ¢00a 0 00¢: ¢nmu m0a 000¢ ¢amma 0mm0 000a a00a 000m m0¢m ¢ 00a: 0m0a ¢m0m b0¢¢ 0nmma aa00 m0ma 0>0a 0mhm ma0m m n0o| 00c: m¢ h0mm hooma 0a00 m00a m00a 0¢0m 00pm m 00m ¢mmm 0>0a ¢mn¢ m00ma 00m0 000a 00¢a aahm aa¢m a 0ma on 00 . (mumn oma Umuoum mam0v mA0.mav mabav macaw ma¢mv mmmaa mmmc coma cmom. mmaa 040m momum>¢ 000a+ m00a 00: omm¢ mmmaa 0aao ¢a¢a m0am ¢0ma hmmm 0 m¢o+ 0mmm 000a ¢¢0¢ 000ma ¢¢00 000a omna ¢¢aa 000m , ¢ 000+ 00ma . 00¢ m00¢ 0¢¢aa. >0>0 00ma ha¢m 00> 0amm m cmm+ ¢0¢m hmam ¢0a¢ m0hma 0000 mm0a n00a mama hm¢m m mmm+ a0ma 0a0a 0c¢¢ 00>aa 00mm mama ¢00a 0¢0a mhhm a 00 00 0 wwwo 00 omuoum «acme 6.0.cao mAmNV 6.80 mamao 0¢0m m0a0a 0¢00 m0¢a comm mmh m0ha 000u0>< 0¢0m m000 ¢0m> 0h¢a 0mmm m0h mm0a 0 a00m 0¢m0a 0¢¢0 m00a 00mm 000 m00a ¢ boom 00a0a m0m0 mama 000m a¢0 00ha m h00m 00m0a am¢0 50¢a 0¢mm 000 hmha m 0¢a¢ >0¢0a a0m0 m00a m0mm 000 00ba a umm>umm 00 0x 0: HI mczochD amuoe H48 czocxco amuoa Has Edaoom + mcao¢ mummam mmocammmm muwaum> . Edammmuom oan¢ moauswwmm Ca 00Cmnu mwauaHDQEH ammoao o m 06 ammo cma can mm mom 000000 mmaumaam> 0>am Ca mmauausmEa cwumasoamo amuOu mnu ou mmauausmEa cmuhamcm no 550 0:» mo cowaummeoo .0m manna 4800 4400 a 4000 0 z: . 3600 U) G) a 4) "-4 3' 3200 H S g 2800 o 2400 2000 Figure 20. 92 01 a O o '3 / 05 I ‘\\\.4 '2 W 65 130 Storage Period, Days Change in non-TAI content of five varieties in storage 65 and 130 days. 93 In Table 30 the changes in the metabolicly active components of the TAI (raffinose, reducing sugars, and amino acids) are compared to the changes in total impurities. The increase in total impurities in the first 65 days of storage was due to the accumulation of raffinose and invert in varieties 1, 2 and 4. Varieties 3 and 5 did not accumulate large amounts of raffinose and invert but the increase in the total impurities was still considerable. After pro- longed storage the total impurities decreased presumably because raffinose, amino acids, and some unknown impurities were catabolized. The beet accumulates impurities, primarily raffinose and invert, during the early part of the storage period. With prolonged cold storage amino acids and some unknown impurities are catabolized presumably via reSpiration. This catabolism may occur at a rate greater than the accumulation of raffinose and invert and actually cause a decline in unknown as well as total impurities. Comparing the very different chemical composition of the three varieties stored at 3 and 10 C illustrates this metabolic shift further (Table 31). The TAI were only 53 percent of the total non-sucrose compounds at harvest but increased to 64 percent in storage at 3 C. »At 10 C the percent TAI remained constant. In varieties 6 and 7 the unknown impurities decreased approximately 400 mg/kg at 3 C. 94 m0m am: 000 mm0l 0¢al 0 aam 00: m0¢a m0aal ¢hml ¢ 000a h¢0l bm0a 000 0m0a m 00 ¢0¢I 0maa m¢0l 00ml m ac0a 0mal ah0m m00| ¢mmm a mama 0ma vmaoum aml bmaal aa0 000 m00a 0 ¢00a 0m0l 00¢ m0>a 0mmm ¢ mmm m0m| 00a hm¢ 00ma m m00a a0ml ¢¢0 000a ¢0¢m m 000 000i a0m, coma a0ma a 0009 m0 umuoum almx 02 m©ao< oca8< m0ao¢ mammsm mmOCammmm mmauaHSmEH mumaam> .mammom 0ca05000 ocaam moaoscmm amuoe ca .mmocammmm Ga 0mmmHUGH mmmcmno m0 850 "ca 00cmno anomao U m um mmmo oma com 00 vmaoum mmaumaam> o>am Ga mwaom oanm com mammsm moaoscmu .omoaammma ma now cmuosooom mmauaasmfia amuou Ca mmmmHUGH .0m manna 95 .mmauaudmea amuou mo unmouom u ammunucmumm ca uwnaozm mammo maaao 61a. mamao maaao mamao nmm+ 000a m¢ma 0m0¢ 0a0aa a000 0hma 00a naam ¢ama ¢0ha mmmu0>< c0c+ a00a ¢m0 0mc¢ m000a 00m0 500a 00 0mma 0aaa 005a n ma+ mmvm 466m chem mamma mccm mmma mom aacm cmma mcom m mmm+ 0maa cmm aomm cmmca 04mm mama mma moma moma coo m anu¢ macmo maoao mxao mamao mace macmo 0mm: 0mmm 00cm ¢m0¢ 000ma 0h00 0mma bma 0m0a 500 a0m¢ 000u0>¢ 00¢: ¢m0m 00mm am0m mmmaa mach 000 00 ¢00 000 ¢00¢ h ¢0m| moam 0000 a0m0 0000a 00¢0a 000a ¢mm 500m 000a 0a00 0 ¢¢a+ m00a 0a0a m00¢ 00n0a ¢a00 m¢aa 0aa 0maa 000 000m 0 000.0 mammo mamao maao mamao name mamao 00c¢ 0000a 0000 ¢mma maa 0m¢a m00 amba 000u0>< ho0m 00¢0 mm¢¢ 0¢0a 00 000 mm0 m00a n 00c0 mmhma 0000 m¢¢a 00a h0mm 0a0 mamm 0 0¢0¢ ¢¢00 00¢¢ 00aa 0ma a¢ma >00 00ma 0 um0>umm ad 0 0 an x z mczocch amuoe ace czocxco amuoa H49 Enammmuom Enavom 00a04 080050 mmocammmm aumaum> I) ooasd moaoacmm ca 00cmno mmauausmEH momma. 0 ca can m 06 ammo ooa now mquum mmaumaum> mmunu Ga mmauauomEa amuOu 020 Cu aadav mwauauamsa cmuxamCM amuou 0:» mo cemaummfioo .am manna 96 Again these varieties were apparently metabolizing the non-TAI components. At 10 C the unknown fraction increased greatly in variety 5 and 7 but in variety 6 the increase in TAI accounted for the total increase in impurities. These results further illustrate the variation between varieties in the interaction of storage temperature and the basic metabolic systems of the beet root. Table 32 compares the change in total impurities to the change in raffinose, reducing sugars and amino acids in the variety-temperature experiment. The increase in total impurities was lower at 10 C. This was due partially to the lack of raffinose develOpment. The increased recoverable sugar losses at warmer storage temperatures were primarily caused by increased reSpiration and not to increased impu- rities (assuming no rot, mold, or Sprouting). However the impurities which increase under warm storage (amino acids, invert) are more melassigenic than raffinose and may result in a greater loss of sucrose to molasses (Carruthers, 1959). The increase in raffinose and invert compared to the increase in total impurities varies greatly between varieties. How- ever these two components account for the majority of the impurities which accumulate in storage below 5 C. Above 5 C other impurities not analyzed in these eXperiments were accumulating as evidenced by the low prOportion (63%) of the increase in total impurities accounted for by raffinose, amino acids and reducing sugars. 97 m0 ¢0ma 000 mam m0 0¢0a mmmum>4 am 000 hm¢ 00m 00 a00a a a0 0¢mm ¢¢aa ¢a0 00¢ m0¢m 0 ®¢ 00> ¢0¢ 0¢0 00ml 00¢a 0 wlolalfla. moa aamm am. om cmmm mmmm mmmum>m 0aa ¢0mm 0m. 00 aham ¢m0m 5 00a 00mm 00m 00a m00m m0am 0 ¢0 000a maal m 000a m00a 0 fl unmoumm almx 0: H emacsoood .mammom moaoscmm ooafid moaosomm amuoa Ga amuos .mmocammmm Ca mmmmuocH ca 00cwso 000cmnu no 850 ca 00cmso. a000av 0 0a 0cm m um mmmo 00a omHODm mmaumaum> mmnnu ca moaom oanm 0cm .mmocammma an Mom wmucsooom mmauauomaa amuou ca mmmmnoca mammsm moaoscma .mm magma 98 Summary of the Factors Controlling Several of the Non-Sucrose Components in the Clear Juice The variation in the chemical composition of the beet root was predominantly a varietal characteristic. In fact variety was the dominant factor controlling the non— sucrose content of the beet at harvest. Delayed harvest increased raffinose due to cool temperatures but the magni- tude of this increase was strongly variety dependent. Nitrogen fertilization had little effect on the analyzed non-sucrose components except the amino acids. However susceptibility to excessive nitrogen fertilization was strongly influenced by variety. Under storage conditions which prevented excessive wilting, storage temperature played a dominant role in deter- mining the impurity content of stored beets. Temperatures below 5 C caused an accumulation of raffinose while temper— atures above 51C caused a reduction in the raffinose content. However the threshold temperature and the amount of raffinose which accumulated were variety dependent. Storage temperature also affected the reducing sugar content of stored beets but this was primarily a result of secondary effects on mold and Sprout formation. These factors caused a rapid increase in reducing sugars. The interaction between temperature and variety for reducing sugar accumulation was not as great as that for raffinose but was significant. 99 Storage temperature also affected the change in amino acid content during storage. The amino acid content declined in storage below 8 C at a rate inversely prOpor- tional to the amino acid content at harvest. Again the response to temperature was variety dependent. TOpping beets prior to storage had little effect on the non-sucrose components in storage. Delaying harvest reduced the accumulation of raf- finose but increased the reducing sugar content in storage at 3 C. Preharvest applications of maleic hydrazide and gibberellic acid increased the raffinose and reducing sugar content at harvest and after storage. GA decreased the amino acid content in storage while MH—30 caused an increase. The low oxygen content of controlled atmosphere stor- age caused a 200 percent accumulation of raffinose and amino acids over the control. Reducing sugars were not affected. Excessive nitrogen fertilization increased the sodium and potassium content of the beets but decreased the chloride content. The variety-nitrogen interaction was highly significant in all cases. Fulfilling the thermal requirement for bolting caused an increase in the raffinose content in storage. The amino acid and reducing sugar contents were not signifi- cantly affected. 100 Short term high temperature treatments prior to storage reduced the raffinose content 35 percent in five days but had no effect on its accumulation later in storage. Reducing sugars and amino acids were not affected. The TAI accounted for approximately 65 percent of the total impurities in beets stored at 3 C and a slightly lower percentage of the total in beets stored warm. ‘With prolonged storage a metabolic shift occurred from the accumulation of raffinose and unknown impurities to the catabolism of these compounds. A decline in total impuri- ties resulted in some instances. High sucroSe losses in storage above 4 C are probably due to higher rates of respiration and not to the accumulation of impurities (if rot, mold and Sprouting are prevented). CHANGE IN MARC CONTENT DURING HARVEST AND IN STORAGE In 1967 the percent marc was determined on all samples in the date of harvest and variety exPerimentS. Fifty-gram brei samples were mixed with 200 ml of 70 C distilled water and incubated for 20 minutes at 70 C in a controlled temperature water bath. The mixture was then filtered through a powdered cellulose filter and washed with ten 200 ml aliquots of 25 C tap water. The residue was dried at 105 C for 48 hours and the marc content calculated as percent fresh weight. At Harvest Variety-HarvestIpteraction In 1967 varieties 2 and 3 were higher than variety 5 in percent marc for all harvest dates (Figure 21). The decline in percent marc for the October 6 harvest was caused by a heavy rain three to four days before harvest. As a result the beets took up water and all determinations based on percent fresh weight declined. Dry weather has been pre- viously shown to increase the percent marc (Dubourg, 1960). However the total yield of marc per acre increased in a 101 102 4.4 o 2 4.3 0 ° 3 4.2 a _ 4.1 O 4.) c 8 u 4.0 0 0+ ‘5‘; 3.9 m 4.) I: 8 o 5 o 3.8 H m z 3.7 O 3.6 \\ O 3.5 L_I___|__| October 6 October 26 November 6 Harvest Date ' Figure 21. Percent marc of three varieties on three harvest dates in 1967. 103 linear fashion during the harvest period except for variety 3 (Figure 22). Nitrggen The percent marc was higher in the beets grown on low nitrogen at all harvest dates than in the beets grown on high nitrogen (average 4.2 vs 4.0). The beets grown on low nitrogen showed severe nitrogen deficiency and had stOpped growing while the high nitrogen beets were still dark green and succulent. The higher percent marc in the low nitrogen beets may have been due to a maturation effect in the non— growing plants resulting in a shift in the pectin content to a more insoluble form. Although the percentage marc was lower for the high nitrogen beets the total marc yield per acre was greater (Figure 23) since there were more tons of high N than of low N beets. These differences were small but the increased pulp yield per factory would be considerable. Duringg§torage Changes in Marc Duringggtorage In 1967 after 65 and 130 days of storage at 3 C the average percent marc for three varieties increased 0.2 per- cent (Table 33). These results were very different from those found in the preliminary experiments of the previous year. The previous studies had shown a considerable decline in the percent marc during storage. The determinations were 104 O/7ii z>er. 117 Due to the bulky nature of the beet root, the use of many reSpiration inhibitors has been largely unsuccessful (Vajna, 1960). Carbon monoxide should be of practical importance, if effective, due to its ease of application. To test the effectiveness of carbon monoxide as a respiration inhibitor in intact beet roots the following eXperiment was performed. Six samples, consisting of ten uniform beets each, were placed in sample containers as described previously. Three samples were connected in series with a potassium hydroxide CO trap and the air 2 circulated with a diaphram pump. Carbon monoxide was then added to the system using a gas burette to the desired con— centration. A six hour equilibriumnwas allowed after each carbon monoxide addition before the rate of reSpiration was determined. The three other samples were used as controls. The samples were held at 5 C. Very low levels (l-Z%) of carbon monoxide (Figure 25) inhibited the carbon dioxide output by 20 percent. Increas- ing the concentration to 10 percent did not increase the degree of inhibition. Between 10 and 20 percent carbon monoxide the rate of respiration declined steadily to 65 percent of the control. Gassing beets in covered piles with carbon monoxide may reduce the sugar losses occurring in the first few days of storage. A 20 percent reduction in these losses would be of substantial economic interest. 118 ON .GOausao>0 mOaanc conumo mo ceauananca mnaxocoa conamu unmouwm .moaxocoz conumo 0a 0a 0. .0m musmam e. m z a A o T. n a... To on o .u d a 1 a .m cm u .. .4 .. o u I. a O . m a cm a . 1 .. o o. T. OOa 119 Effect of Preharvest Sprays on Thermal Induction and ReSpiration The respiration rate of samples treated with GA and MH-30 prior to harvest were monitored periodically for 102 days at 5 C. Samples of GA and MH treated roots were removed after 5, 8 and 12 weeks of storage in the same cooler as the reSpiration eXperiment and the degree of thermal induc- tion was determined. Beet halves were planted in sterilized soil in the greenhouse at 18 to 21 C in 16 hour days and the percent bolters recorded after six weeks. Due to the rapid fluctuations in the respiration rate immediately after harvest (Dilley, 1969; Stout, 1957) the first respiration analyses were delayed until seven days after the beets were placed in storage. After 30 days in storage the rate of respiration for the plants treated with GA was 15 percent higher than the control (Figure 26). At this time 87 percent of the plants treated with GA were induced to flower as compared to only 57 percent of the control plants. Sprouting was inhibited to such an extent in the plants treated with MH that no induction observations could be made. Although no definite conclusion can be drawn, the degree of induction appeared to affect the respiration rate. The increase in respiration between 70 and 110 days was probably due to surface desic- cation. 120 . 0 491.300 m a £0 1 o.omnm= .o m 06 ammo moa How 600608 can can. oaaamumnnao .0mlmz nua3_00ummuu mma08mm mo coau5a0>0 00ax0a0 conumu mama .OOaumm mmmuoum cm g cm cm . om ca .0m 0H50am l‘ O) H ,4 ma Jq I_6x 6w 'paAtoaa apxxorq uoqxea I— 121 Stout (1949) found a high correlation between reSpiration rate and the degree of flower induction. The reSpiration rate increased with prolonged cold temperatures. When the beets were subjected to temperatures of 23 C to cause a reversal of induction the rate of respiration declined. However he did not report the temperature at which the respiration analyses were made which would be a prime factor in explaining his results. Moving beets from the cold to a warmer temperature caused a burst in the rate of reSpiration lasting for 2-3 days (Dilley, 1969). This increase in reSpiration was apparently due to substrate availability and not to a change in carbon dioxide solubil- ity (Stout, 1954). Lang (1956) reported that GA would fulfill the ther— mal induction requirements of some biennials. Gibberellic acid has been shown to partially fulfill the thermal require- ments for induction in sugarbeets (Stout, 1959; Snyder and Wittwer, 1959; Stout and Owen, 1959). The very prominent increase in respiration for the first 20 days of storage has not been shown previously. Previous workers found the reSpiration rate declined to a low level after a high rate in the first few hours (Dilley, 1969; Stout, 1957). However the present results correlate very well with observations of commercially stored beets. In the first few weeks of storage the beets go through a "sweat" period when pile temperatures are hard to control. 122 Again in late January the pile temperatures increase causing substantial sugar losses. These increased pile temperatures may be due to the heat of reSpiration (Figure 27). PrOportion of Sucrose Losses Accounted for by Respiration Carbon Dioxide Evolution Loss of sucrose through respiration is generally considered to account for most of the total sucrose loss in storage. Previous workers have shown that the decrease in sucrose and the total carbon dioxide evolved were not sig- nificantly different (Vajna, 1960). The prOportion of the sucrose lost by carbon dioxide evolution varied between varieties (Stout, 1950). However the incidence of mold in his eXperiments caused an unusually high accumulation of invert sugars which might have caused considerable over- estimation of sucrose losses by polarimetry. Normally the sucrose losses might be expected to be greater than the loss accounted for by carbon dioxide evolution since reducing sugars and trisaccharides are formed from sucrose. The total carbon dioxide evolved during the 102 day 'storage period described in the previous section was 18.8 gm/kg. Table 40 gives a balance Sheet for the losses due to reSpiration and sugar conversions. The 4.7 gm/kg excess carbon dioxide evolved apparently represented the reSpira- tion of non-sucrose compounds derived from carbohydrates other than sucrose. This excess was approximately equal to 123 oaa OOa 0a 00 Oh O0 00 O¢ Om ON 0a Oma .O 0 um m0mnoum mo m>m0 m0a 0caH50 GOausao>0 00ax0a0 conumo mama .OOanmm mmmuoum .hm masmam O In H O (x H O0a (0 H N Keg I_6x 6w 'UOanIOAH Z0:) 0 m N I- OmN OhN 124 Table 40. Proportion of sucrose losses accounted for by reSpiration and interconversions in 112 day storage at 5 C 9M9 CO2 lost . . . . . . . . . . . . . . . . . . . . 18.85 Sucrose lost . . . . . . . . . . . . . . . . . . 8.00 Raffinose lost . . . . . . . . . . . . . . . . . 0.90 Invert gained . . . . . . . . . . . . . . . . . 1.40 Total sugar loss . . . . . . . . . . . 8 + 0.9 = 8.90 Total sugar gained . . . . . . . . . . . . . . . 1.40 Net loss . . . . . . . . . . . . . . . . . . 7.50 Since 1 gm of CO is derived from 0.648 gms of sucrose, 2 18.85 gms CO is equivalent to 12.21 gms of sucrose reSpired. 2 12.2 —7.5 4.7 gms/kg excess CO2 evolved 125 the amount of insoluble cell wall material which became soluble in this type and length of storage (see marc sec- tion). It was therefore possible that part of the cell wall polysaccharides were hydrolyzed and subsequently reSpired. Sucrose vs Dry Matter Losses In 1967 the amount of respiration in storage was estimated by measuring the loss of dry matter. Although this method was not as precise as measuring actual carbon dioxide evolved it was a good estimate. No significant differences were found between agro- nomic practices in the amount of dry matter lost. The prOportion of sucrose lost by respiration and transformation decreased in the last 65 days of the 130 day storage period (Table 41). Table 41. Average loss of dry matter and sucrose in 1967 date of harvest study after 65 and 130 days of storage at 3 C Days Stored Sucrose Loss Dry Matter Loss Pounds/Ton 65 —8 -12 130 -12 -22 126 Dry matter losses, which can occur only by gaseous evolution, were considerably greater than the sucrose losses in all eXperiments. These results substantiate the loss of total impurities in the clear juice which may occur in the final 65 days (page 90). The difference between dry matter and sucrose losses varied among varieties. In variety 1 and 4 the losses were equivalent, while in the other three varieties the dry mat- ter loss was much greater (Table 42). Table 42. Loss of dry matter and sucrose in five varieties during storage at 3 C for 130 days Non-Sucrose Sucrose Dry Matter Compounds Variety Lost Lost ReSpired Pounds/Ton 1 21.6 24.0 2.4 2 4.6 28.6 24.0 3 14.8 16.4 1.6 4 13.0 15.6 2.6 5 13.4 23.6 10.2 Average 13.5 21.6 127 The non-sucrose constituents in the beet root normally increased during storage resulting in a decreased clear juice purity. If the difference between sucrose loss and dry matter losses came from only the soluble fraction of the beet the purity would increase considerably. The slight decline in total impurities in the clear juice was several magnitudes less than the dry matter minus sucrose losses. Therefore the cell wall or insoluble fraction must contrib- ute to any increased impurities in the clear juice and also to the carbon dioxide evolved. However it was possible that the source of non-sucrose compounds reSpired, is partly precipitated by lime and therefore their loss would not be reflected in the C.J.P. determination. Enzymatic Degradation of Sucrose in Stored Beets Enzyme Analysis The reducing sugars produced in stored beets occur either as a result of mold activity or enzymatic degradation of sucrose by endogenous enzymes. Prolonged storage at tem— peratures above 5 to 8 C caused reducing sugars to accumu- late. This response to temperature was diametrically Opposite that commonly found in other plants; i.e., potatoes (Pressey, 1968). Acid invertase activity in fresh beet tissue was very low and is often assumed to be zero (Bacon, 1961: ‘Vaughan and MacDonald, 1967). However invertase activity 128 could be induced by aging beet disks in sterile aerated water. Avigad (1968) found that, due to its easily revers- ible nature, the sucrose synthetase in beet root was capable of producing reducing sugars. pH Profile of Sucrase Activity in Root Homogenates In a preliminary study* the amount of sucrose inver- sion in a brei preparation over the pH range of 4.0 to 8.5 was determined. Two hundred grams of fresh beet tissue was homogenized in a Virtis Blender at 10,000 RPM for one minute. The grinding medium contained EDTA, 10-4M; NaCN, 10-3M in phOSphate buffer, 10-3M at pH 7, and was cooled to 0 C prior to use. Ten m1 of the homogenate was added to test tubes 2 containing 10 ml of a 2 x 10- M buffer (pH 4 to 5 acetate: pH 6 to 7 citrate-PO pH 8 to 9 Tris) and 0.05 molar 4: sucrose. The samples were mixed and allowed to equilibrate at 30 C for 10 minutes. Ten m1 of the reaction mixture were removed and two m1 of 1 percent neutral lead acetate were added to stOp the enzymatic reaction and clarify the solu- tion. The increase in reducing power one hour later was measured using the tetrazolium method (Carruthers, 1955). *The preliminary investigation reported in this sec- tion was conducted in the Research Laboratory of the British Sugar Corporation in Norwich, England. The work was a con- tinuation of a study of enzymatic inversion in factory dif— fusers previously undertaken by J. V. Dutton and D. Grierson of the British Laboratory. 129 Results were eXpressed as relative activity with the peak activity at pH 5 taken as 10. ~All samples were run in quadruplicate with boiled controls at each pH. The acid invertase activity at pH 5.0 was very low as reported previously (Figure 28). However the striking increase in hydrolytic activity at pH 7 was totally unex- pected. As yet no characterization of this peak has been made, but it might be due to the reversal of sucrose syn— thetase (Avigad, 1968). Distribution ongydrolytic Activity in the Beet Root To determine the localization of the two enzymes in the beet root the homogenate was filtered through nylon bolting and the filtrate centrifuged at 30,000 g for 20 minutes. The resulting supernatant was a pale yellow. The inversion activity was determined on the homogenate and supernatant at pH 5 and 7.2. The activity at pH 7.2 occurred primarily in the supernatant while the activity at pH 5 was presumably attached to the cell wall (Table 43). Previous workers have reported that invertase was associated with the cell wall in beet disks (Vaughan and MacDonald, 1967). The possible importance in sugar tranSport across cell membranes has been implied, but never proved (Steward, 1965). Vaughan and Mac- Donald (1967) found no alkaline invertase activity in fresh beet discs. The invertase activity which develOped with 130 Relative Activity Figure 28. Effect of pH on the hydrolysis of sucrose by a beet root homogenate. 131 Table 43. Distribution of inversion activity at pH 5 and 7.2 between supernatant and cell wall material pH 5 Percent Supernatant . . . . . . . . . . . . . . . . . . 32 Cell Wall 0 O O O 0 O O O O O O O O O O O O O O 68 pH 7 Supernatant . . . . . . . . . . . . . . . . . . 69 cell wall 0 O O O O O C O O O O O O O O O O O O 3]- aseptic washing appeared in the cell wall and then moved into the cytOplasm and had a pH Optimum at 5 (MacDonald, 1968). Possible4Importance of Hydrolytic Activity at pH 5 and pH 7 In an attempt to determine the possible relative importance of these two enzymes in beet metabolism their activity was measured in beets stored at 3 and 11 C. -Assum— ing that the enzyme was Operating at maximum velocity 13 yi££2_if the milligrams of reducing sugar produced by the activity at pH 5 was less than or equal to the rate of carbon dioxide evolution it could be assumed that this enzyme was probably of secondary importance in sucrose breakdown in storage. Sections of beet root were passed through a grater to produce thin slices less than 1 millimeter thick and approximately two millimeters wide. Ten grams of these slices were placed in 150 milliliter beakers containing 132 50 ml of phOSphate buffer 2 x 10-2M at pH 7.2 or acetate 4M sodium cyanide 10-3M buffer 2 x lO-ZM pH 5.0, EDTA 10‘ and sucrose 0.05 M. The assays were run at 30 C. All determinations were run in duplicate with boiled controls. The entire experiment was repeated twice and the results given are an average of the two runs. The hydrolytic activity at pH 5 was approximately . twice as great in beets stored at 11 C as in beets stored g at 3 C (Table 44). The Opposite was true for the hydrolytic activity of pH 7.2. This may indicate that the accumulation _ of reducing sugars at warm temperatures was due to increased invertase activity and not to the enzyme active at pH 7.2. The activity at pH 7.2 doubled at low storage temperatures but reducing sugars did not (see Figure 12). The lower activity at pH 5 in the 3 C storage may be due to the Table 44. Effect of storage temperature on the enzymatic inversion of sucrose at pH 5 and 7 Storage Temperature pH 5 pH 7 Mg Kg-l/Hr 11 C* 66 76 3 C* 39 140 3 C** 33 129 *Assayed at 30 C. **Assayed at 3 C (different lot of beets). 133 presence of a temperature dependent inhibitor. Such an inhibitor having a molecular weight of 18,000 to 23,000 was found by Pressey (1968) in cold stored beets. When the assay was carried out at 3 C, the same as the low storage temperature, the activity was 33 and 129 mg/kg/hr (Table 44). Carbon dioxide evolved due to respi— ration at 5 C was approximately 10-15 mg/kg/hr. Since 1 mg carbon dioxide released in respiration equals 0.648 mg sucrose, the carbon dioxide evolved at 5 C (10-15 mg/kg/hr) was derived from 6.5 to 9.8 mg of sucrose. If it is assumed that the acid invertase activity attached to the cell wall was not involved in supplying the hexose sugars as sub- strates for respiration, the soluble invertase could supply 33 x 0.32 or 10.6 mg/kg-hr of hexose sugars. Therefore the relatively low invertase activity was theoretically great enough to supply the substrate for respiration in the beet root. Effect ofy§everal Preharvest Sprays on Hydrolytic Activity at pH 5 and 7.0 Using the same thin slice assay, the activity of the two enzymes was determined in beets treated prior to harvest with pyrocatechol, vanadium sulfate and CCC. Pyrocatechol and vanadium sulfate reduced the activity of both enzymes. CCC reduced invertase activity slightly at pH 5 but had no effect on the activity at pH 7 (Table 45). 134 Table 45. Effect of preharvest sprays on the degradation of sucrose by two enzymes assayed at pH 5 and pH 7 pH 5 pH 7 Mg Kg-l Pyrocatechol 23 78 Vanadium sulfate 22 94 CCC 26 142 Control 39 140 The accumulation of reducing sugars was slight in relation to the potential hydrolytic activity contained in the beet root. The enzymatic hydrolysis of sucrose in stored beets therefore appeared to be highly controlled if the beets were not allowed to mold or wilt. Summary of Factorsggnfluencing the Direct Loss of Sucrose in Storage The rate of respiration in the beet root could be maintained at a very low level by rapidly cooling the beets immediately after harvest and keeping injury at a minimum. Wilting increased respiration losses drastically. The res- piration in the beet was apparently very susceptible to regulation by high levels of carbon dioxide in the root. Gassing covered piles with carbon dioxide may be a method of reducing reSpiration losses. Low levels of carbon 135 monoxide also had a very inhibition effect on respiration in intact roots. Thermal induction of the beet root in storage in- creased the rate of reSpiration in the early part of the storage period. Measurements of total reSpirational losses by carbon dioxide analysis and dry matter loss indicated that carbo- hydrates other than sucrose comprise a considerable, but variable portion, of the substrate for respiration. The hydrolysis of sucrose in the beet root occurred via two pathways. Acid invertase with a pH Optimum of 5 occurred in the beet at very low levels of activity. Hydro- lytic activity at pH 7 is approximately 4-5 times the pH 5 activity and appeared to be due to the reversal of sucrose synthetase. The low activity of invertase was apparently large enough to supply hexose sugars for the carbon dioxide evolved in respiration. Storage at 11 C doubled the hydrolytic activity at pH 5 but decreased the activity at pH 7 to 50 percent of that in 3 C storage. Pyrocatechol reduced the activity at pH 7 by 50 percent over the control. Vanadium sulfate reduced the pH 7 activity by 33 percent but CCC had no effect. All preharvest Sprays decreased the activity at pH 5. The hydrolysis of sucrose in the beet root was apparently a highly controlled process since no substantial amount of reducing sugar accumulated if the beet root was maintained in a normal condition free from molds. ASSESSMENT OF QUALITY IN STORED BEETS The most common method for evaluating the quality of fresh beets is by determining the percent sucrose and clear juice purity. These determinations can be made rapidly and used to estimate the amount of bagged white sugar which can be recovered from a ton of beets. Carruthers (1961a, 1961b, 1961c) prOposed an alter- native method whereby the clear juice purity could be esti— mated by correlation with the sodium, potassium, amino acid, and betaine content of the clear juice. The correlation equation was as follows: Purity = 100.9 - (0.00143 (2.5 sodium + 3.5 potas— sium + 10 amino nitrogen + betaine)) This method appears to have merit both in evaluating fresh beets (Henry 35 a1., 1961) and factory juices (Car- ruthers _£H_1., 1963). Since the compounds used to calcu- late the impurity index do not change appreciably in storage, the index would be of little apparent value in assessing the quality of stored beets, in which changes in other substances assume major prOportions. 136 137 Use of theIImpurity Index in Evaluating Stored Beets A comparison of apparent impurities, total analyzed impurities (TAI) and impurity index from the eXperiment in 1967 on date of harvest is given in Table 46. In general the total analyzed non-sucrose constituents tend to corre- late very well with the apparent impurities. The difference between the two (unknown impurities) is approximately a con- stant 2500 mg/kg at harvest and during storage. However the impurity index remains essentially constant in storage and therefore the difference between the apparent impurities and the impurity index increases. Therefore the impurity index is of little value in assessing the technological value of stored beets. Formula for Correcting Sucrose Determinations for Raffinose and Invert The estimated RSPT often increased in short term storage (Dexter, 1966: Larmer, 1937; McCready, 1966). There— fore a formula was needed to correct the sucrose determina- tion for Optically active compounds in the clear juice to produce a more accurate estimate of the sucrose concentra- tion in the beet and in the clear juice. Raffinose has 1.6 times the Optical activity of sucrose (sp. act. 104 vs 66.5). At a high concentration of raffinose, considerable error can be introduced into sucrose determinations. Even small corrections to the sucrose 138 Table 46. Comparison of total impurities with the total analyzed impurities and the impurity index on the evaluation of three varieties stored 65 and 130 days at 3 C (1967) a . Unaccountedb Total Unknown Impurity for Variety Impurities TAI Impurities Index Impurities Mg Kg’l At Harvest 2 6671 4096 2575 3930 2741 3 6626 3860 2766 3763 2863 5 6419 4008 2411 4221 2198 Stored 65 Days 2 7606 5177 2429 3837 3769 3 7574 4621 2953 3757 3817 5 7211 4777 2434 3900 3311 Stored 130 Days 2 7570 5043 2527 3758 3812 3 8480 5757 2723 3534 4946 5 6914 4847 2067 3891 3023 aUnknown Impurities = Total - TAI. bUnaccounted for Impurities = Total - Impurity Index. 139 determination may make considerable difference in the clear juice purity. Reducing sugars primarily glucose and fruc- tose occur in the beet roughly equal concentrations and can therefore be called invert sugars. This combination of glu- cose and fructose has a specific activity of -40. In cases of high invert production in storage invert sugars may also contribute to errors in the determination of sucrose. Since specific rotation is based on the weight of the Optically active compound in Solution the following relationships can be made. 1 mg raffinose = ég45 (mg sucrose) = 1.59 (mg sucrose) 40 mg sucrose 66.5 x 2 = 0.302 (mg sucrose) 1 mg invert = Therefore 1 mg of raffinose would appear as 1.59 mg sucrose using polarimetric methods. A similar relationship can be made for invert. As a result of the above relation- ships the following equation can be derived. 1.59 mg/ml Raff.-0.302 mg/mlInvert Corr % s = App C-J.% S - 10 x Density (mg/m1) Assuming there is no loss of raffinose or invert in sample clarification* and that the raffinose and invert *This assumption is not entirely true (Appendix C). Some reducing sugars are lost by alkali decomposition. Since fructose is the most reactive the actual correction should no doubt be more positive than the -40 used here. 140 concentration per gram of sugar is a constant in the beet and in the clear juice the following relationship is also valid. Corr C.J. % S Corr A S on Beets = App A S on beets X App C,J; %,5 These equations were used to correct all sucrose determinations reported in the following section. The magnitude of the corrections on fresh and stored beets from several eXperiments are given in Table 47. The corrections are obviously substantial both at harvest and after storage. It is apparent that any storage eXperiments evaluated by RSPT measurements must be corrected for raffinose and invert. Base-Acid Balance in the Clear Juice During the processing campaign the lime salts normally increase significantly and require the addition of considerable amounts of sodium carbonate to reduce calcium salts and prevent acidic conditions in the evaporators. However, the addition of sodium carbonate results in a con- siderable loss of sucrose into the molasses (approximately 5 lbs of sucrose per pound of sodium carbonate added). The changes in the acid—base balance of the clear juice may be an important supplement to clear juice purity and RSPT cal- culations, particularly in evaluating stored beets. 141 Table 47. Magnitude of corrections required due to errors in polarimeter reading caused by raffinose and invert* Five Varieties (1967) 3: Stored at 3 C for E At Harvest 65 Days 130 Days 5.; g s CJP RSPT s CJP RS PT 3 CJP RSPT % % lb % % 1b % % lb 1 0.26 1.51 13.7 0.41 2.32 21.6 0.27 1.63 14.3 2 0.25 1.51 13.3 0.50 2.91 26.3 0.36 2.18 19.1 3 0.27 1.56 14.0 0.33 1.89 17.3 0.36 2.19 19.2 4 0.28 1.70 14.7 0.54 3.07 28.2 0.30 1.90 16.0 5 0.23 1.37 12.3 0.43 2.51 22.5 0.19 1.12 9.8 Variety-Temperature Experiment (1968) 5‘ Stored 100 Days at .3 At Harvest 3 C 11 C H g S CJ P 16 PT S CJ P RS PT S CJ P f6 PT % % 1b % % 1b % % lb 5 0.18 1.13 9.8 0.45 2.76 23.8 0.10 0.65 5.4 6 0.32 2.17 17.0 0.76 5.33 40.1 0.38 2.67 20.1 7 0.24 1.42 12.9 0.74 4.39 39.2 0.25 1.55 13.2 .' *All corrections in these experiments were negative. 142 During storage, the cations, sodium and potassium, and the anion chloride remain constant since they are not metabolized by the beet. The amino acids remain fairly constant but in general decline slightly during storage. A large part of the acids in thin juice which contribute to high lime salts in stored beets are derived from the alkaline decomposition of invert sugars during lime defeca- tion. The literature reports that in general one invert sugar produces two acids each with a molecular weight of 90 (McGinnis, 1951). However, our studies of alkaline decompo- sition of glucose and fructose indicate that the molecular weight is closer to 120 (Appendix C). In other words, the invert sugars produced from one molecule of sucrose would produce three molecules of acid upon decomposition. However the alkaline decomposition of invert produces a yellow color due to the presence of browning reaction products. These must be reduced by the addition of sulfur dioxide. The SO2 produces more acid in the juice which must also be neutral— ized with sodium carbonate. Therefore in the following calculations 90 will be used as the milliequivalent (meq) weight of the acids derived from invert to partially com- pensate for the required SO addition. 2 The base-acid balance for five varieties grown on high (150#/A) and low (24#/A) nitrogen and stored for 130 days was calculated using the following equation. 143 mg K mg Na _ mg Amino Acids _ mg invert _ mg C1 = . 39.1 + 22.9 140 90 35 RESIdPa} Alkalinity (all values are in mg/lOOS) Since a slight excess of base is required to main- tain alkalinity during evaporation this relationship will be designated as residual alkalinity. Since K, Na, Cl and amino acids are essentially con— stant during storage, the fluctuations in invert sugar will determine the amount of acid which must be neutralized as Ca Salts or by the addition of NaZCOB' Table 48 indicates the magnitude of the changes in residual alkalinity during storage. Table 48.. The residual alkalinity of five varieties stored at 3 C for 130 days At After Treatment Harvest Storage (meq/lOOS) (meg/1003) Variety 1 +7.4 —13.0 Variety 2 +4.4 +1.6 Variety 3 +1.4 -5.8 Variety 4 +7.6 -6.2 Variety 5 -2.2 -7.6 Variety 5 @ 11 C —2.2 -29.0 Average all varieties grown on: 150# N/A +1.2 -9.2 24# N/A +5.8 -6.4 filth?! if: y. . . .; ...., I... yflflj . . 0 .1. . «lie. - 144 Although the Optimum residual alkalinity for maximum processing efficiency is not known, beet variety, agronomic and storage practices obviously all provide a practical means for manipulating this balance. Utilizing the Residual Alkalinity in the Calculation of the RSPT Many factory chemists assume a loss of 5 pounds of sucrose into the molasses for each pound of Na2C03 added in order to produce the prOper base-acid balance. Assuming this 5 pound loss to be accurate, the following relationship between sucrose lost by Na2C03 addition and the base—acid balance of the clear juice can be made (Table 49). Therefore 0.26 percent of the total sucrose in the beet is lost for each meq of Na2C03 added to correct the residual alkalinity of the clear juice to a value of +11.* In two eXperiments the correction in the RSPT at harvest was only 2 to 3 pounds (Table 50 and Table 51). However in long term storage where reducing sugars accumulated the correc- tion was considerable. *Previous workers have indicated that an effective alkalinity of 2.24 or a K + Na - Amino nitrogen balance of 16 should allow efficient factory Operation. The beet con- tains approximately 2 meq of Cl and up to 3 meq of invert/ 100 S can normally be tolerated without causing difficulty in processing (Dexter g£_aI,, 1969: Frakes, 1969:.Anderson- Smed, 1963). Therefore 16 - 2 - 3 = 11. 145 Table 49. Calculation of sucrose losses due to sodium carbonate addition Assume 5 lbs sucrose lost/# Na CO added 2 3 or 5#/454,000 mg Na CO 2 3 or 5#/8566 meq Na CO 2 3 or 5.8 x 10-4#/meq Na CO 2 or 0.26 gm sucrose/meq. 3 Therefore 0.26 percent of the total sucrose will be lost for each meq Na CO3 added/100 gm sucrose. 2 or 5.2 x meq/100 S base deficit x percent sucrose = lbs sucrose lost per ton of beets due to sodium carbonate addition. Also, since 53 mg Na CO 2 3 90 mg invert = 1 meq, = l meq and %%-x 5# = 2.9# loss to molasses/lb of invert sugar 2.9# + L# = 3.9# of sucrose lost per pound of invert sugar. 146 0.0 000H0>< 0.00m 0.00m m.0 m.0am 00.m 0.aa 0 0.a¢m 0.0¢m 0.m 0.¢0m 0m.m m.0 ¢ m.00m 0.00m ¢.0 0.00m 00.m 0.0 m a.mmm m.m0m m.0 0.00m 00.m 0.0 m 0.0mm a.a0m m.a 0.m0m ¢n.m ¢.¢a I a 0009 0ma 00H000 0.m 000H0>< 0.00m m.¢>m 0.0 a.mam ¢0.a a.0 0 b.0hm a.mcm ¢.a a.¢mm ¢¢.0 m.a ¢ m.0hm 0.0mm 0.¢ 0.mmm m¢.a 0.0 m 0.m0m ¢.00m 0.m a.0am a0.0 a.m m m.a0m a.00m ¢.m 0.0mm ¢0.a 0.¢ a mng 00 00a000 0.m 000H0>< 0.0mm 0.00m 0.0 0.mmm m0.m 0.m 0 0.00m 0.00m ... a.0om 0a.0 ¢.0 ¢ m.amm 0.a0m m.¢ 0.0mm mm.a a.0 m m.amm m.mnm 0.m m.mam 00.0 0.m m m.m0m ¢.¢0m m.a ¢.¢mm 0m.0 ¢.a a um0>amm 04 pa na na B\Qa X 002 6000 9000 Dmoa 0moaoom umoa 00aa500m 00cmamm mu0aam> HHOU .050 amuoa 0moaosm mOOmmz Uaoam£ um m0au0aam> 0>a0 00 9000 0:» c0 mCOauO0HHOO 0Oomamn 0a0010mmn 00 000000 .00 0anma c 1| ..r- C. ,. .J.«n.r14 a1.,-ll \I- I I ‘ . v"! 147 0.mmm m.0¢m 0.¢ 000a0>¢ 0.mmm m.mmm 0.m m.¢om m0.a a.m 0.m m 0.mca m.aom 0.m a.mmm m¢.a a.m m.a m a.mcm 0.mmm m.¢ a.00m a0.a m.0 0.m m 0 ca 06 mmmo cca omeoum 0.mmm m.m¢m m.a mmmam>< o.mmm m.mcm m.a a.mam m¢.a a.m m.¢ c m.ama m.ama ... 0.6mm .... ... a.¢a m ¢.¢0m a.mmm m.o a.mcm mm.c o.c a.m m o m um mama ooa 600000 c.mmm o.¢0m m.a 0060054 m.omm m.¢mm a.m 0.mmm ¢o.a o.¢ o.0 m v.4mm m.mmm m.o 0.mmm om.o a.a 0.0 m m.amm m.mcm a.a m.mom ¢m.c m.a a.0 m um0>amm um 0a 0a 0a 93a om 00: 8000 8000 umoa 000a050 umoa 00aa500m 00cmamm mu0aum> HHOU .Udm HMUOB meHUSm MOUNMZ CHU‘lwmmm A000av 0 0a 0cm m um 00maoum H0000 0cm um0>am£ um m0au0aam> 00ana mo 8000 0:0 :0 mCOauomaaoo 00cmamn Cavalmmmn 00 000000 .a0 0anB 148 Quality Evaluation of Fresh and Stored Beets on the Basis of Recoverable Sugar* In the following results, corrections for raffinose, reducing sugars, and the residual alkalinity were applied as discussed previously. Recoverable Sugar Yields at Harvest The RSPA (Recoverable Sugar Per Acre) increased substantially during the 1967 harvest season (Table 52). The weather was dry until just prior to the October 26 har— vest when approximately 2 inches of rain fell. As a result the percent sucrose decreased for this harvest due to water uptake but the increased tonnage more than compensated to increase the RSPA with delayed harvest. Varieties 2 and 5 yielded more RSPA when grown on low nitrogen (24 pounds per acre) compared to high (150 pounds per acre) nitrogen (Table 52). Variety 5 which is extremely susceptible to luxury consumption of nitrogen (page 68) showed a substan- tial yield increase under low nitrogen fertilization. lOO-CJP))] *RSPT = 20 [(%»S - 0.3)(1 ‘ 1°667 ( CJP 149 Table 52. Increase in RSPA yields of three varieties at harvest grown on high (150#/A) and low (24#/A) levels of nitrogen during 1967 Harvest Date Variety October 6 October 26 November 6 Average pounds/acre 2H 5768 6426 6655 6283 2L 6082 6529 2792 6468 Average 5925 6478 6724 3H 5977 5820 6766 5988 BL 5733 5700 6282 5905 Average 5855 5760 6224 SH 6349 6375 7398 6707 SL 6763 7438 7670 7290 Average 6556 6907 7534 Effect of Harvest Date on Storage Characteristics In 1967 beets from three harvest dates were stored for 65 and 130 days at 3 C (Table 53). The RSPT data were rather erratic and the responses in storage are difficult to eXplain. The early harvested beets exhibited a sharp drop in RSPT after 65 days of storage but after 130 days averaged the same as the freshly harvested beets. This trend was true for all varieties and nitrogen treatments harvested on October 6. Although the loss of RSPT was greatest in the beets harvested on November 6 compared to October 26, the increased tonnage which occurred during this period resulted in a greater net RSPA after storage for the late harvested beets. 151 Table 53. Effect of harvest date on the percent sucrose, clear juice purity, and RSPT at harvest and after storage at 3 C (1967) Harvest Sucrose CJP RSPT RSPT % ‘% lb lb At Harvest October 6 16.2 93.5 273 6279 October 26 16.0 94.0 272 6582 November 6 16.6 94.0 285 7097 Stored 65 Days October 6 15.5 92.3 257 5911 (-368)a October 26 16.0 93.1 266 6437 (—145) November 6 16.4 93.8 279 6947 (-150) Stored 130 Days October 6 16.2 93.7 275 6324 ( +46) October 26 15.4 92.6 247 5977 (-605) November 6 15.5 92.4 245 6101 (-996) a O 0 O Parentheses indicate loss in storage. 152 The date of harvest eXperiment was repeated in 1968 with somewhat different results. The late harvested beets lost only 10 pounds of RSPT after 100 days of storage at 3 C as compared to 16 and 32 pounds for the earlier harvests (Table 54). Although the losses incurred during storage were very different in the two years the bagged sugar pro- duction for the campaign.wou1d have been substantially increased both years by delaying harvest. Delayed harvests would also facilitate the lowering of pile temperatures by ventilation due to the normally lower air temperatures. The primary disadvantage of delayed harvest is the possibility of inclement weather conditions which could prevent harvest— ing altogether. Vajna (1960) reported that although earlier harvested beets respired at a greater rate the sugar losses in storage were lower. Comparison of Varieties in Storage on the Basis of RSPT Three varieties were harvested on October 6, Octo- ber 26, and November 6, 1967 and stored for 65 and 130 days at 3 C. The October 6 harvest gave uneXplainable results as previously mentioned and will not be discussed further. Losses in RSPT were negligible for the first 65 days of storage and no differences between varieties existed (Table 55). After 130 days of storage the inferior keeping quality of variety 3 was evident. 152 The date of harvest eXperiment was repeated in 1968 with somewhat different results. The late harvested beets lost only 10 pounds of RSPT after 100 days of storage at 3 C as compared to 16 and 32 pounds for the earlier harvests (Table 54). Although the losses incurred during storage were very different in the two years the bagged sugar pro- duction for the campaign would have been substantially increased both years by delaying harvest. Delayed harvests would also facilitate the lowering of pile temperatures by ventilation due to the normally lower air temperatures. The primary disadvantage of delayed harvest is the possibility of inclement weather conditions which could prevent harvest- ing a1t0gether. Vajna (1960) reported that although earlier harvested beets reSpired at a greater rate the sugar losses in storage were lower. Comparison of Varieties in Storage on the Basis of RSPT Three varieties were harvested on October 6, Octo- ber 26, and November 6, 1967 and stored for 65 and 130 days at 3 C. The October 6 harvest gave uneXplainable results as previously mentioned and will not be discussed further. Losses in RSPT were negligible for the first 65 days of storage and no differences between varieties existed (Table 55). After 130 days of storage the inferior keeping quality of variety 3 was evident. 153 Table 54. Effect of harvest date on the percent sucrose, CJP and RSPT in storage (1968) at 3 C Harvest Sucrose CJP RSPT % 96 lb At Harvest September 1 16.4 94.7 292 October 1 15.3 97.3 284 November 1 16.2 94.5 288 Stored 50 Days September 1 15.8 93.7 275 (17) October 1 15.2 94.0 266 (18) November 1 15.9 93.6 276 (12) Stored 100 Days September 1 15.0 93.5 260 (32) October 1 15.5 93.4 268 (16) November 1 16.4 92.6 279 ( 9) a .. . . Parentheses indicate loss in storage. 154 Table 55. The decline in RSPT of three varieties harvested October 26, November 6 and stored for 65 and 130 days at 3 C (1967) Days Stored Loss in Variety Harvest 65 130 130 Days pounds October 26 Harvest 2 267 263 251 16 3 273 271 240 33 5 274 264 251 23 November 6 Harvest 2 281 273 257 24 3 290 285 221 69 5 283 279 257 26 The keeping quality of variety 5 was not affected by harvest date, but the other varieties lost considerably more RSPT when harvested late. However the incidence of surface mold after 130 days of storage was much greater in varieties 2 and 3 and this may account for the greater losses (see invert discussion on page 52). Variety-Nitrogen Interaction Five varieties grown on 150 and 24 pounds of nitro- gen per acre were harvested on October 26, 1967 and stored for 130 days. When beets were grown on low nitrogen, the RSPA at harvest was greater for varieties 2, 3, and 5 but lower for varieties l and 3 (Table 56). The much higher 155 Table 56. Recoverable sugar per acre yield at harvest and after 130 days of storage for five varieties grown on 24 and 150 pounds nitrogen per acre and stored at 3 C At Harvest Stored 130 Days Loss in Storage Nitrogen Nitrogen Nitrogen Variety 24#/A 150#/A 24#/A 150#/A 24#/A 150#/A pounds 1 6731 7178 5587 6214 1194 964 2 6856 6536 6444 6235 412 301 3 5889 6220 5308 5473 581 747 4 7200 6659 6421 6089 779 570 5 7949 6612 7148 6264 801 348 Average 753 586 RSPA for variety 5 under low nitrogen fertilization.wou1d indicate that this line is extremely sensitive to excessive nitrogen fertilization. .Since this variety has been used in many of the nitrogen studies in Michigan the results of these studies may have exaggerated the detrimental effects of exces— sive nitrogen application. The effect of nitrogen fertilization on the storage losses (RSPA) varied between varieties but the beets grown on low nitrogen lost 167 pounds more sugar on the average. Variety 2 was definitely the superior storing variety at both nitrogen levels. Although the RSPA losses in storage for variety 5 grown on low nitrogen were among the highest, 156 its overall RSPA yield after storage was 700 pounds per acre greater than any of the other varieties tested. The result was a greater sugar yield after 130 days of storage than any other variety at harvest. Therefore, though sugar losses were greater total production was increased by storing high quality beets. Table 57 gives the average RSPT over three harvest dates for three varieties grown on high and low nitrogen. Although variety 5 stored relatively better when grown on high nitrogen, it had a greater RSPT after 130 days of stor— age when grown on low nitrogen (Table 57). In contrast, variety 2 had similar losses for both nitrogen levels, but again it had a greater RSPT after 130 day storage when grown on low nitrogen. Variety 3 appeared to have a relatively lower loss when grown on low rather than high nitrogen. It is apparent that there is a great difference in the reSponse of varieties to nitrogen in terms of RSPT at harvest and also in the preservation of RSPT in storage. The average loss in RSPT was the same for both nitrogen levels, however, to evaluate a variety or agronomic practice the yield and the losses in storage must be evaluated to determine the best variety from a total economic standpoint. Preharvest Sprays None of the preharvest foliar Sprays (MH, GA, CCC, VaSO Pyrocatechol) affected the loss of RSPT in storage. 4! 157 .0mmnoum :0 mmoa mmumoewcfl mononucwnmmm Acme 44m AHNV mom Hem AHHV omm lame «am new Akmv omm Rome mom 06m mlmav mmm mamav chm mmm mom mmN mom omN mom ¢9N mocsom th mum mmm mom fiom hum How 0mm hwm mnm wow mmm mmmum>4 mmN m mmm m Nmm m mmmum>4 <\#oma 4\#¢N muw0nm> ammonuflz mmmnm>¢ «\¢omH «\#¢m mum0um> ammonuflz womam>< ¢\#Oma <\#¢m mumflum> a0000m> ammonuez whoa OMH GOHOum mama m6 pmnoum umw>nmm um Anomav o m um mmmuoum CH mm0um0Hm> 00050 00 80mm 00 mmoa mnu co coflumneaeuum0 cmmouuec 0o uom00m one .5mm magma 158 Previously Wittwer and Hansen (1951) reported maleic hydrazide reduced sucrose losses. D. J} Wort (1968) also reported that Va SO and pyrocatechol reduced sucrose losses 4 in the first 30 days of storage. Modified Atmosphere The loss of RSPT in modified atmOSphere storage* was significantly decreased in comparison to the samples stored in air (Table 58). The amount of sucrose conserved was much higher but the accumulation of impurities caused a greater loss in purity in the modified atmosphere. However other combinations of carbon dioxide and oxygen may alleviate the decreased purity problem. The 1000 ppm ethylene increased sugar losses, presumably by increased reSpiration (Dilley, 1969). Proportion of Recoverable Sugar per Ton Loss in Storage Accounted for by the Direct Loss of Sucrose In Tables 59 and 60 the loss of total sucrose is compared to the loss in RSPT. In all varieties, except 2, the loss of sucrose by reSpiration and conversion accounts for approximately 50 percent of the total loss in RSPT. In variety 2 the loss due to increased impurity content was much greater than the loss by respiration and transforma- tions. *5%.02, 5% CO 90% N2 1 1000 ppm ethylene. 2’ 1:43“ ..1' -. w ' 1m: "1 PO I‘m 159 .mz gem .moo gm .mo Xm« .mmmHODm G0 mmoa 80mm u mmmosucmummm Ham 4.60 a.mm umm>umm um Acme Hem e.ma m.am Anmv «om m.ma 0.aa ANHV mum a.ma 4.4m «mumnmmosua omflmflcoz lame mom a.mH «.mm Akmv wow o.mH a.mm alone Hem «.ma m.mm Houucoo 0: x x. a: .x. x a: .x. .x. emmm m mbo Emma m who ammm m 960 mumnmmosue wmmuo>< Ema coca Ema o mamameum Amomav o m um memo o¢ Hmu0m Bmmm cam wmonosm .muausm woesfl Hmmao co mumsmmOEum ©0000©oe 0o uom00m .mm manna 160 .IEvmwla so am. as: mmm cam mum awn m we hm: ma- Nam mmm mmm mom 4 mm km- ma- omm mom new Hmm m em man a: 5mm mom mum mam n me me: am. owm mom mmm «mm H unmuumm mna cou\mnH was cou\mna was cou\mna mmoq Emm 8mg wwOHUDm Ema QmOHUDm Ema ”WOHUSm humHHMNV dance 00 Hmuoa Hmuoa Hmuoa unmoumm mm mmoq mmouosm mmmuoum :0 mmoq mama 0ma pwuoum umm>nmm u< mmflum0um> m>00 CH mmouosm 0o lemmas o m um memo oma cmuoam mmoa on mac mommoH Bmmm 0o cofluuomoum .mm manna 3.44.14..- 1...»: . 1..-...3 lg _ 16]. vs Hm mm com won on mm 00 mow man How bmm b mm mm 00 mma 5mm no em ma baa mmm VNN mum 0 mm mm ma men ¢m~ mm m N wow mom «hm mom m mmoq mna scu\mna and cou\mna mmoa mad cou\mQH mQH cau\mna and Gou\mAH huo0um> Emma 00 ammm wmouosm Emma wmouozm ammm 0o a¢ma mmouusm 90mm mmouonm 90mm wmouosm unmoumm Hmuoa Hmuoe acouumm Hmuoa dance dance mm mmoq . o mmo mm mmoa . o mmo mmouusm .0 A mmouuam .0 A umm>umm u< o OH O m “an mama ooa cmuoum Amomav U 00 new m um mmmp ooa cmuoum mm0uo0um> owns» :0 mmouusm 0o mmoH 0» mac mommoH 90mm 00 newuuomoum .oo OHAMB 162 Of the 8 pounds of RSPT lost by variety 5 at 3 C only 2 pounds or 25 percent was by the direct loss of sucrose. Varieties 6 and 7 correSponded very closely to the 1967 results. Increasing the storage temperature increased the direct loss of sucrose more than the indirect loss. These results indicate the need for future research not only in the area of direct sucrose losses by respiration but also in the area of non-sucrose accumulation. Respira- tion losses can be reduced by controlled atmOSphere storage or inhibitors, but the usefulness of these methods may be negated by the RSPT losses due to lower clear juice purity. Summary of Recoverable Sugar gields at Harvest and After Sporage Estimates of recoverable sugar are valuable measures of quality in fresh beets. After storage the estimate appears to be less accurate but is still an excellent guide- line if the prOper corrections for Optically active compounds and for the decline in residual alkalinity are made. Delayed harvest increased the RSPT and RSPA yields. The effect of harvest date on sucrose losses in storage varied considerably from year to year. ~Although high quality beets showed greater losses in storage, net yields per acre were increased by storing high quality beets. Low nitrogen fertilization increased RSPA yields at harvest and after storage in three out of five varieties. The effect of nitroqen on storage losses depended strongly 163 on variety. Four out of five varieties lost more RSPA in storage when grown on 24 lbs of nitrogen per acre as com- pared to 150 lbs/acre. However the RSPA yields after stor- age were still higher in the low nitrogen beets. Variety appeared to be the most significant prestorage affecting recoverable sugar losses in stonage. Slightly over 50 percent of the RSPT lost in storage was by reSpiration. The remainder was due to a loss in purity. Storage at warmer temperatures increased respira- tory losses as well as the losses resulting from the accu- mulation of impurities. .Although the results differed between varieties the prOportion of sucrose lost by reSpira- tion is approximately the same at 3 and lC>C. Storage in modified atmospheres containing 5 percent oxygen, 5 percent carbon dioxide and 90 percent nitrogen significantly reduced losses in RSPT. Preharvest applications of maleic hydrazide, gib- berellic acid, CCC, vanadium sulfate, and pyrocatechol had no effect on the recoverable sugar losses in storage. SUMMARY AND CONCLUSIONS The effect of various agronomic and storage prac- tices on the physical and biochemical factors affecting the loss of sucrose during storage or in subsequent processing were studied. The changes in the major impurities during storage were determined and their effect on processing evaluated. Loss of sucrose in storage occurred either directly via respiration and sugar conversions or indirectly by changes in the chemical composition of the beet which decreased recovery in the factory. Slightly over 50 percent of the total RSPT losses were the result of the direct loss of sucrose in storage. Of these direct losses, 90 percent was by reSpiration. The sucrose lost by conversion to reducing sugars, raffinose, and other oligosaccharides accounted for only a small pro— portion of the total sucrose lost in storage. The remainder of the RSPT losses were the result of a decrease in clear juice purity. Therefore any future attempt to reduce stor- age losses must consider, not only reSpiration losses but also the losses due to the accumulation of melassigenic non- sucrose compounds. 164 165 The rate of reSpiration in storage was determined by carbon dioxide evolution or loss of dry matter. The results from both methods indicated that in addition to sucrose, ether carbohydrate components were acting as substrates for carbon dioxide evolution. ReSpiration losses immediately after harvest were substantially reduced by preventing injury, desiccation, reducing storage temperatures and con- trolled atmOSphere storage. The respiration rate of the beet root was markedly reduced by high levels of carbon dioxide and carbon monoxide. The results of this study indicate that the direct loss of sucrose via respiration could be reduced on an economically feasible commercial scale by (l) reducing harvesting and handling injury and (2) covering the pile with a material capable of increasing the carbon dioxide content, and of preventing desiccation. In processing, the amount of sucrose lost to molas- ses increases as the period of storage of commercial beets is extended. In this study the amount of sucrose lost to molasses was estimated using the percent sucrose and clear juice purity. The clear juice purity normally decreased during storage due to a decline in sucrose content and an increase in the non-sucrose components of the clear juice. Under the ideal storage conditions used in this study the TAI (raffinose, reducing sugars, amino acids, chlorides, sodium, and potassium) comprised approximately 65 percent of the total impurities in the clear juice at harvest and during storage below 4 C. Storage at warm temperatures . .l..:,.u.lu.flu..n..)l 1111i . . . - 1.. ”ll. 166 caused the accumulation of impurities not analyzed in this study. However the increase in total impurities was essen— tially identical at both cold (3 C) and warm (10 C) storage temperatures. Beets stored at higher temperatures contained more acids (reducing sugars, amino acids) and were therefore theoretically more melassigenic. Variety played a major role in determining the chemical composition of the beet at harvest. The interac- tion of variety and nitrogen fertilization were significant for amino acid, sodium, potassium and chloride content. Substantial differences existed between varieties in rela- tion to the RSPT losses in storage, the accumulation of raffinose and the stability of marc. Harvest date and tOp- ping had only minor effects on the chemical composition of the beets in storage. In general agronomic practices played a minor role compared to storage practices in controlling the chemical composition of the beet. During low temperature storage (below 4 C) the major non-sucrose component accumulating was raffinose. High levels of raffinose caused a considerable overestimation of sucrose by polarimetric methods. Since raffinose cannot be degraded during processing it accumulates in the factory and is a primary nuisance to processing. Raffinose accumulation was a function of temperature but the amount accumulated was variety dependent. Although all varieties in this study accumulated raffinose, substantial differences in sensitiv- ity to temperature existed between varieties. 167 Reducing sugars accumulated during storage primarily as a result of mold or desiccation. The accumulation of reducing sugars in storage as a result of these factors was many times greater than that due to variety or agonomic practices. Invertase apparently played a secondary role in the hydrolysis of sucrose in healthy beets. Reducing sugars have a threefold detrimental effect on processing. Not only is (1) sucrose lost directly by hydrolysis to reducing sugars, but (2) reducing sugars are degraded to acids and (3) contribute to the formation of highly colored clear juices due to non-enzymatic browning reactions. The excess acids produced in processing must be neutralized with sodium carbonate to prevent acid inversion. In commercial piles the accumulation of reducing sugars occurs to a much greater extent than that found in this study. However, on a commer- cial scale, the accumulation of reducing sugars could be reduced considerably by eliminating freezing and thawing on the pile surface, and preventing excessive pile heating. In the absence of acids derived from invert, the amino acids are the major contributors of acidity to factory juices. The amino acid content at harvest was primarily a function of the rate of applied nitrogen. The degree of luxury consumption of nitrogen was strongly variety depen- dent. In storage at temperatures below 7 C the amino acid content normally declined, while above 7 C the content may increase. Storage in atmOSpheres of low oxygen (5%) and 168 high carbon dioxide (5%) caused a substantial accumulation of amino acids. Since the major factors controlling the amino acid content of the root are nitrogen fertilization and variety, control of the amino acid content through breeding and prOper agronomic practices should be relatively easy. The quantity of anions absorbed by the beet root during growth was equal to the cations. This equilibrium relationship of ions is also nearly ideal for efficient factory processing. However due to preferential elimination of ions during juice clarification, the formation of acids by alkaline decomposition of reducing sugars, and the deam- ination of amides produces excess acids in the processing juices from stored beets. Therefore any method used to evaluate the quality of stored beets must consider the melassigenic nature of the method used to amend the com- position of process juices from stored beets. Since five pounds of sucrose is lost for each pound of sodium carbonate added, agronomic and storage practices which increase the acidity of process juices must be avoided (high nitrogen fertilization, warm storage temperatures, Sprouting, molds). Not only is the loss of sugar in the factory due to changes in the chemical composition of the clear juice, it is also related to the physical integrity of the root. How- ever under ideal storage conditions, the marc remained rea- sonably stable to extraction temperatures in the 70—80 C 169 range and showed evidence of resynthesis. The percent marc was variety dependent and influenced greatly by environment (rainfall and nitrogen fertilization). Marc stability in storage was influenced most by variety and storage temper- ature. APPENDICES CCC: CJP: EDTA: GA: Invert s MH-30: mg/lOO s mg/kg = APPENDIX A ABBREVIATIONS USED 2-chloroethyl-trimethylamonium chloride Percent clear juice purity Ethylenediamine tetraacetic acid Gibberellic acid ugars: equimolar quantities of glucose and fructose Maleic hydrazide : Milligrams per 100 gms of sucrose Milligrams per kilogram fresh.weight Reducing sugars: Sugar containing a free aldehyde group RSPA: RSPT: RDS: %S: TAI: capable of being oxidized Recoverable sugar per acre (bagged sucrose) Recoverable sugar per ton (bagged sucrose) Refractometric dry solids—~total soluble dry matter Percent sucrose on beets Total analyzed impurities, includes raffinose, reducing sugars, amino acids, sodium, potassium, chlorides. 170 U.- APPENDIX B DEVELOPMENT OF ENZYME METHOD FOR THE DETERMINATION OF RAFFINOSE A method was develOped to determine rapidly the amount of raffinose in the clear juice via a coupled enzyme system composed of galactose oxidase and peroxidase. Galac- tose oxidase is a metalloenzyme containing one gram atom of c0pper per mole (Amaral, 1963) which will oxidize the C6 position of galactose as well as some of its derivatives and polymers. The enzyme has a pH Optimum of 7.0. Galactose oxidase will react with free or bound (as in raffinose) galactose to produce D-galacto—hexodialdose and hydrogen peroxide (Avigad, 1961). The hydrogen peroxide can then be reduced to water by peroxidase concurrent with the oxidation of an appropriate chromagen to produce a detectable color. Four mg of galactose oxidase (20 units, 2.5 mg per- oxidase and 2.5 mg of O—tolidine were mixed and diluted to 50 m1 (Avigad, 1961). Due to the high pH (9.2) of the clear juice samples to be analyzed 1 ml of a 0.1 M phosphate buffer pH 7 was added to the reaction mixture. Although more dilute buffers were tried this concentration gave the most linear results. 171 172 The reaction was initially stOpped by addition of 6 ml of 0.1 M glycine buffer pH 9.7. However the sensitiv— ity of the procedure for low concentrations of raffinose could be increased by stOpping the reaction with 2 ml 0.05M EDTA by complexing with the c0pper ion. In the initial studies 2 m1 of the enzyme prepara- tion, 1 ml of 0.1 M phosphate buffer gave a linear absor- bance at 425 mg for raffinose standards over the range of 0 to 0.6 mg. The reaction was linear for up to 90 minutes. A 60 minute incubation time was used for all routine analy- sis. This length of time was sufficient to develOp readable color at the lower concentrations. The volume of the clear juice samples was limited to 0.25 ml due to irregular color develOpment which occurred when larger volumes were used. This problem could be elim- inated by deionizing the sample with a mixed bed resin (Bio Rad AG 501 - x 8, 20-50 mesh). The final procedure used for all routine analysis was as follows: - 2 ml enzyme reagent mixture (Worthington Biochemical, Freehold, New Jersey). Reagent was made to volume with 0.05 M phosphate buffer pH 7.0 - 0.25 ml sample containing 0-0.3 mg raffinose - incubate at 30 C for 60 minutes - 3 ml 0.05 M EDTA added to stOp reaction - read at 425 mg on Beckman DB Spectometer. 173 Chromatographic analysis of clear juice samples from many treatments did not reveal the presence of free galactose or galactinol which could act as substrates for the assay. APPENDIX C INVERT DEGRADATION During the clarification of sugarbeet diffusion E juice the pH is raised to approximately 11.5 with calcium oxide and the mixture heated to 75 C to decompose the reducing sugars present. Glucose and fructose under these conditions break down to form acids. It was necessary to determine the average molecular weight of these acids in order to predict the amount of sodium carbonate to be added to neutralize the acids during the processing of stored beets. Mixtures of glucose, fructose and sucrose were heated at various temperatures up to 80 C in a three—necked reaction flask containing 0.125 N sodium hydroxide pH 11.5. The reaction mixture was heated with an electric mantle con- trolled by a rheostat. An aliquot of the mixture was taken at the beginning and end of the alloted heating period and the net loss in reducing sugar and gain in acid were deter- mined. The equivalent weight of the acid produced was calculated as the grams of reducing sugar decomposed divided by the equivalents of acid produced. 174 175 Although the results were quite variable ranging from 110 to 130 grams per equivalent the results were well above those of 90 reported previously in the literature (Silin, 1964; McGinnis, 1950). Carruthers (1968) indicated that in extensive work by his laboratory, the average value was shown to be approximately 120. The chemically more reactive fructose began to decompose at 58 C in 15 percent sucrose or 10 C lower than glucose. It is therefore very important not to heat the limed—pressed juice sample above 60 C in the determination of clear juice purity if the assumption that no invert is decomposed in this process is to be valid. This assumption will be assumed valid in all calculations but due to the light yellow color of the clear juice samples it was apparent that some variable prOportion of the reducing sugars was lost during clarification. BIBLIOGRAPHY BIBLIOGRAPHY Allen, P. J., and J. S. D. Bacon. 1956. Oligosaccharides formed from sucrose by fructose-transferring enzymes of higher plants. Biochem. J. 63:200-206. Amaral, D. L. Bernstein, D. Morse and B. Horecken. 1963. Galactose oxidase of Polyporus circinatus: a c0pper enzyme. J. Biol. Chem. 238:2281. Andersen, E., and E. Smed. 1963. The chemical composition of sugarbeets and the effective alkalinity and the sugar loss in molasses. Paper presented to the 12th Cen. Ass. S.I.T.S. Atterson, A., A. Carruthers, J. V. Dutton, D. Hibbert, J. F. T. Oldfield, M. Shore and H. J. Teaque. 1963. Chemical changes occurring in sugarbeets during storage, freezing, wilting. 16th Ann. Tech. Conf. B.S.C. Avigad, B., D. Amaral, C. Asensio and B. Horecker. 1961. Galactdialdolase production with an enzyme from the mold Polyporus circinatus. Biochem. BiOphys. Res. Comm. 4:474. Avigad, G. 1968. Study of enzymatic degradation of sucrose in sugarbeet tissues, to provide information of improved procedures in the handling of sugarbeets for processing into sugar and other useful products. Final Report Project #URrA(10)50-25. The Hebrew Univ. of Jerusalem Dept. of Biol. Chem. Jerusalem, Israel. Bacon, J. S. D. 1961. The develOpment of invertase activity in washed slices of beet tissue. Biochem. J. 79:200. Bacon, J. S. D., I. R. MacDonald and.A. H. Knight. 1965. The develOpment of invertase activity in slices of the root of Beta vulgaris L, washed under aseptic conditions. Biochem. J. 94:175. Barbour, R. D., and C. H. Wang. 1961. 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Olson. 1958. II. The effect of fertilizer treatment on the calcium, potassium, raffinose, galactinol, nine amino acids and total amino acid content of three varieties of sugar beets grown in the Red River Valley of Minnesota. ASSBT 10:272-280. , M. D. Finkner and R. F. Olson. 1959. Effects of storage on raffinose content of sugar beets. I. Vari— etal changes occurring during storage. ASSBT 10:481-488. , J. F. Swink, C. W. Doxtator, R. F. Olson, and P. C. Hanzas. 1959. Changes in raffinose content and other characteristics of sugar beet varieties during six different harvest dates. ASSBT 10:459—566. , D. W. Grimes, and G. M. Herron. 1964. Effect of plant spacing and fertilizer on yield, purity, chemical constituents and evapotranSpiration of sugar beets in Kansas. II. Chemical constituents. ASSBT 12:699-714. Frakes, M. G. 1969. Personal communication. 180 Frydman, R. B., and E. F. Neufield. 1963. Synthesis of galactosylinositol from peas. Biochem. and Biophysical Res. Comm. 12:121-125. Gaskill, J. O. 1950. Effects of wilting, drought and temperature upon rotting of sugarbeets during storage. ASSBT 6:653-659. Goodban, A. E., J. Benjamin Stark and H. S. Owens. 1953. Content of sugarbeet processing juices. J. Agr. Food Chem. 1:261-264. Hac, L. R., A. C. Walker and B. B. Dowling. 1950. The effect of fertilization on the glutamic acid content of sugarbeets in relation to sugar production. ASSBT 6: 401—411. Haddock, J. L., D. C. Linton and R. L. Hurst. 1956. Nitrogen constituents associated with reduction of sucrose percentage and purity of sugarbeets. ASSET 9:110-117. , P. B. Smith, A. R. Downie, J. T. Alexander, B. E. Easton and V. Jensen. 1959. The influences of cultural practices on the quality of sugarbeets. ASSBT 10:290-301. Hackett, D. P. 1960. Respiratory inhibitors. Encyl. Plant Physiol. 12(2):30. Hangyal, K. 1966. Changes in the technical value of sugar- beet with various types of storage. Zucker, 19:279-285; 303-310. Henry, J., R. Vandewijer and R. Pieck. 1960. The technical value of sugarbeets in 1960. Presented to the 14th Annual Technical Conf. of the B. S. Co. Joslyn, M. A. 1962. The chemistry of protopectin: A critical review of historical data and recent develOp- ments. Advances in Food Research. 11:1-106. Kertesz, Z. I. 1951. The Pectic Substances. Interscientific Pub. Inc., New York. Khelemskii, M. Z., N. T. Poedinok and M. L. Pelts. 1963. Influences of the temperature factor on variation in the quality of beet during storage. Trudy CINS 11:31. 181 Khelemskii, M. 2., E. A. Vorobeva and M. L. Pelts. 1963. Influence of beet storage period on the activity of enzymes and the content of trisaccharides, organic acids and amino acids. Trudy CINS 11:18. , N. T. Poedinok and M. L. Pelts. 1963. .Effect of the temperature factor on the change in beet quality during storage. Trudy Tsentv. Nauch.-Issled. Inst. Sahar. Prom. 11:31—44. Khualkovkii, T. P. 1964. Melassigenic coefficients of non-sugars in standard molasses as a function of their effect on molasses viscosity and sugar solubility. Sakar. Prom. 38:580-583. Kursanov, A. L., and O. A. Pavlinova. 1967. Sugar accumu- lation as a function of growth processes in sugarbeet roots. Fiziologiya Rastenii 14:21-28. Lang, A. 1956. Induction of flower formation in biennial hyoscyanmus by treatment with gibberellin. Naturwis- senschaften 43:284-285. Larmer, F. C. 1937. Keeping qualities of sugar beets as influenced by growth and nutritional factors. J. Agr. Res. 54:185-98. MacDonald, I. R. 1968. Personal communication. McCready, R. M. 1966. Polysaccharides of sugarbeet pulp a review of their chemistry. ASSBT 14:260-270. ,.A. E. Goodban, R. Ratner, and A. Ulrich. 1966. Sugarbeet and purified juice quality in relation to non- sugar constituents. ASSBT 14:91—96. , and J. C. Goodwin. 1966. Sugar transformations in stored sugarbeets. ASSBT 14:197-205. McGinnis, R. A. 1951. Beet-Sugar Technology. Reinhold Publishing Corp. . 1954. Round-table discussion of "lime-salts." Milner, Y., and G. Avigad. 1964. The UDP-glucose: D- fructose glucosytransferase system from sugarbeet roots. Israel J. Chem. 2:316. 182 Moore, S., and W. M. Stein. 1954. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211:907. Moreno, A., and C. E. Cardini. 1966. A raffinose-sucrose transglactosidase from wheat germ. Plant Physiol. 41:909-910. Nelson, R. T., and R. R. Wood. 1950. Respiration and Spoilage studies employing a modification of method develOped by Stout and Fort. ASSBT 6:660-663. , and R. K. Oldemeyer. 1952. Preliminary studies applicable to selection for low respiration and resistance to storage rots of sugarbeets. ASSBT 7:400—406. Neumann, N. P., and J. O. Lampen. 1967. Purification and prOperties of yeast invertase. Biochemistry 6:468-475. Oldfield, J. F. T., J. V. Dutton, D. Grierson, R. K. Heaney and H. J. Teaque. 1968. Effect of draft and of inver- sion on the quality of juice in beet diffusers. 19th Ann. Tech. Conf. BSC. Owens, H. S., E. A. McComb, and G. W. Deming. 1954. Composition and percentage of marc in some varieties of sugarbeets. ASSBT 9:267-271. , J. B. Stark, A. E. Goodban and H. G. Walker, Jr. 1955. Application of composition knowledge to beet sugar technology. Agr. and Food Chem. 3:350-353. Ogden, D. B., R. F. Pinker, R. F. Olson and P. C. Hanzas. 1958. The effect of fertilizer treatment upon three different varieties in the Red River Valley of Minnesota for: stand, yield, sugar, purity and non-sugars. ASSBT 10:263—271. Pack, D. A. 1924. The storage of sugarbeets. Facts about sugar XIX, No. 8, 174-180; No. 9, 208-209: No. 10, 232- 235; No. 11, 251-253. . 1926. The effect of moisture on the loss of sugar from sugarbeets in storage. Journal Agr. Res. 32:1143-1152. Palmer, J. M. 1966. The influence of growth regulating substances on the develOpment of enhanced metabolic rates in thin slices of beet-root storage tissue. Plant Physiol. 41:1173-1178. 183 Pavlinova, O. A., M. F. Prasolova, and E. A. Ivanova. 1967. Mechanism of maleic acid hydrazide on growth and sugar accumulation in sugar beets. Fiziologiya Rastenii 14: 992-996. Pressey, R. 1968. Invertase inhibitors from red beet, sugarbeet, and sweet potato roots. Plant Physiol. 43:1430-1434. Pridham, J. E., and W. Z. Hassid. 1965. Biosynthesis of raffinose. Plant Physiology 40:984—986. Rorabaugh, G., and L. W. Norman. 1956. The effect of . various impurities on the crystalization of sucrose. ' ASSBT 9:238-252. Rounds, H. 1950. Second carbonation studies based on commercial application of the "effective alkalinity" concept. ASSBT 6:551-564. Rounds, H. G., G. E. Ruch, D. L. Oldemeyer, C. P. Parrish and F. N. Rawlings. 1958. A study and economic appraisal of the effect of nitrogen fertilization and selected vari- eties on the production and processing of sugarbeets. ASSBT 10:97-116. Schales, O., and S. S. Schales. 1941. A simple and accurate method for the determination of chloride in biological fluids. J. Biol. Chem. 140:879. Schneider, F., and D. Schleplake. 1963. The behavior of non-sugars during sugar extraction. Zucker. 16:503-509. Silina, N. P. 1964. Effect of potassium and sodium salts on molasses sugar content. Sakhar. Prom. 38:728-731. Silin, P. M. 1964. Technology of beet-sugar production and refining (Translated from the Russian, Israel Program for Scientific Translations LTD.) U.S. Dept. of Commerce. . 1965. Evaluation of the technological prOperties of sugarbeet for selection. Priklad. Biokhim. Mikro— biologo 1:90-94. Silina, N. P. 1966. Melassigenic coefficients of individual non-sugars. Sakhar. Prom. 40:15-18. Snyder, F. W., and S. H. Wittwer. 1959. .Some effects of gibberellic acid on stem elongation and flowering in sugarbeet. ASSBT 10:553-561. 184 Snyder, F. W., and N. E. Tolbert. 1966. Influences of nitrogen nutrition and season on photosynthetic incor- poration of C1402 into sucrose and other soluble com- pounds of the sugarbeet. Bot. Gaz. 127:164-170. Sorokina, T. I. 1963. The influence of sugar content on the level of sugar losses in beets during storage. Sakhar. Prom. 37:925. Stark, J. B., and R. M. McCready. 1968. The relationship of beet molasses composition to true purity. Part 1 Composition. ASSBT 15:61-71. , , and A. E. Goodban. 1968. The relation- ship of beet molasses composition to true purity. Part 2 Statistical evaluation. ASSBT 15:73-84. Steward, F. C. 1965. Plant Physiology: A Treatise. Vol IB: Photosynthesis and Chemosynthesis. Academic Press, New York. Stout, M. 1949. Relation of oxidation-reduction potential, respiration and catalase activity to induction of repro- ductive develOpment in sugarbeets. Bot. Gaz., pp. 438- 449. , and C. H. Smith. 1950. .Studies on the respiration of sugarbeets as affected by bruising, by mechanical harvesting, severing into tOp and bottom halves, chemical treatment, nutrition and variety. ASSBT 6:670—679. . 1954. Some factors that affect the respiration rate of sugarbeets. ASSBT 8:404—409. . 1957. ReSpiratory losses from sugarbeets soon after harvest. ASSBT 9:350—353. , and J. D. Spikes. 1957. ReSpiratory metabolism of sugarbeets. ASSBT 9:470-475. , and F. V. Owen. 1959. Effect of gibberellic acid on rate of bolting of annual beets. .ASSBT 10:302-310. . 1959. Some effects of gibberellic acid on the physiolOgy of sugar beets. ASSBT 10:306-310. Straus, J. 1962. Invertase in cellwall of plant tissue cultures. Plant Physiol. 37:342-348. ,Sugarbeet Research, 1964 Report. 1964. Determination of revoverable sugar using formula prOposed by Great Western Research Laboratory, Denver. USDA-ARS Bluebook, CR-4-64. Pg. 155. 185 Swink, J. F., and R. E. Finkner. 1956. Galactinol-weight relationships in breeding for resistance to the sugar— beet nematode. ASSBT 9:70-73. Ulrich, A. 1954. Growth and develOpment of sugarbeet plants at two nitrogen levels in a controlled tempera- ture greenhouse. ASSBT 8:325-338. . 1955. Influence of night temperature and nitro— gen nutrition on the growth, sucrose accumulation and leaf minerals of sugarbeet plant. Plant Physiology 30: 250-257. Vajna, S. 1960. Sugar Beet Storage, Translated by Michael Schalit, Storage of sugarbeets a literature survey, The Great Western Sugar Company Research Laboratory: Rpt No. RL 65-005 May, 1965. , and T. Vajna. 1965. Determination of sugar losses in sugarbeets during storage. Z. Zuckerind 15:387. Vajna-Papp, M. 1964. New measurements of the respiration rate and sugar loss in stored beet. Zeitsch. Zuckerind. 89 : 67-73 0 Vandewijer, R. 1962. The treatment of clear first carbona- tion juice. 15th Ann. Conf. of the BSC. Vukov, K., and L. Barany. 1964. Influences of storage on alterations of the technological quality of sugarbeets. Cukoripar 17:19. Vaughan, D., and I. R. MacDonald. 1967. Development of soluble and insoluble invertase activity in washed storage tissue slices. Plant Physiol. 48:456-458. , and . 1967. Invertase develOpment in storage tissue disks of Beta vulgaris; its nature, extent and location. J. EXpt. Botany 18:578-586. Wang, C. H., and R. D. Barbour. 1961. Carbohydrate metabolism of sugarbeets. II. Catabolic pathways for acetate, glyoxylate, pyruvate, glucose and gluconate. ASSBT 11:44-454. Walker, A. C., L. R. Hac, A. Ulrich and F. J. Hills. 1950. Nitrogen fertilization of sugarbeets in the woodland area of California. I. Effects upon glutamic acid content sucrose concentration and yield. ASSBT 6:362- 371. " . 3g mu- ‘.'..L 186 Walker, H. G., E. S. Rorem and R. M. McCready. 1960. Compositional changes in diffusion juices from stored sugar beets. ASSBT 11:206-214. Wallenstein, H. D., K. Bohn, G. Kagelmann and G. Willer. 1962. Results of beet storage eXperiments in 1957-1961. Zuckererzeugung 6, suppl. No. 2,1. , and G. Kagelmann. 1966. Control of mass change during sugarbeet storage by determining the content of invariable constituents particularly potassium. Zeitsch. Zuckerind. 91:189-194. Wittwer, S. H., and C. M. Hansen. 1951. The reduction of storage losses in sugarbeets by preharvest foliage Sprays of maleic hydrazide. Agron. J. 43:340-341. l Wohlert, W. 1962. Respiration tests on different varieties of beta-beets. Zuckererzeugung 6:303. E Wood, R. R., R. K. Oldemeyer and H. L. Bush. 1956. Inheritance of raffinose production in the sugarbeet. ASSBT 9:131-138. , H. L. Bush, and R. K. Oldemeyer. 1958. The sucrose-Sodium relationship in selecting sugarbeets. ASSBT 10:133—137. Woolley, D. G., and W. H. Bennet. 1959. Glutamic acid content of sugarbeets as influenced by soil moisture, nitrogen fertilization, variety and harvest date. ASSBT 10:624-630. Wort, D. J. 1968. Personal communication. University of British Columbia, Vancouver, Canada. Zhadan, V. Z., and M. Z. Khelemski. 1962. Dependence of sugar losses in respiration of beet on storage tempera- ture. Sakhar. Prom. 36(10):55.