MSU LIBRARIES RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES wi11 be charged if book is returned after the date stamped beIow. STUDIES ON THE OCCURRENCE, MEASUREMENT, AND CONTROL OF BITTERNESS IN CARROTS By Paul Hack Beasey AN ABSTRACT Submitted to the School for Advenced Graduate Studiea of kflchigan State University of Agriculture and Applied Science in.part1al fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 19S? Approved;.,;%:1:2dKl.£7:(\aauuufiLmz/ PAUL HACK BESSEI ABSTRACT Bitterness has recently become a serious problem for processors of stored carrots and has also been found in carrots on the fresh market. A series of studies was conducted to determine the factors involved in .its occurrence and measures usable for its control. Organoleptic, fluorescence and spectrophotometric ratings were used for bitterness evaluation. The first was based upon taste panel recog- nition of five intensities of bitterness ranging from 1- non-bitter to S- exceedingly bitter. A yellowish-green fluorescence in tiny spots in phloem tissues of bitter roots exposed to short wave ultra violet light was found and was correlated significantly at .05 (r - 0.6%) with bitterness by taste test. This phenomenon was adopted as a rapid bitter- ness evaluation technique using a 1-5, non-fluorescent to highly fluor- escent, rating scale. Spectrophotometric evaluations were based upon energy absorbmcy of petroleum ether extracts of carrots at 2:0, 265 and 290 mu. Early maturing varieties Nantes and Touchon harvested at the same chronological age as later maturing Danvers and Imperator became more bitter in cold storage. Host strains of the intermediate maturing Red Core Chantenay variety were similar in bitterness response to Nantes and Touchon. A progemr test of bitter and non-bitter selections from Red Core Chantenay resulted in non-significant differences in bitterness; however, the growing season may have reduced their susceptibility to bitterness. Short type Ohantenay am Chanticler, seeded May 27 became more bitter in storage than when seeded July 6. Early Chanticler was PAUL MACK BESSEI ABSTRACT apparently'nore mature than early'Chantenay'in that more flower stalks were formed. It also became more bitter further substantiating the inn terpretation that maturity enhances bitterness. Late sown Chanticler appeared less mature in shape than.Chantenay at harvest and became much less bitter during storage. In a comparison of long and short Chantenay strains fron.a single planting, bitterness differences did not correlate with types. Carrots from mineral soils were higher in soluble solids than car- rots fron muck soils, yet evidenced no differences in bittemess that could be ascribed to soil type. Applications of copper and manganese to field plots of carrots de- ficient in these two elements did not significantly affect bitterness. Deficiencies of capper and manganese did not induce bitterness. Mineral analyses of bitter carrots from an acid muck showed a higher content of iron but less manganese than.non-bitter carrots from an alkaline muck. Although not compared directly in the same study; early harvests resulted in more bitterness than late harvests. The cool growing season of 1956 was more influential in reducing bitterness susceptibility than any varietal, field or storage treatment. Injuries to roots during harvest and handling increased losses due to disease (Solerotinia species), but.did not increase the incidence of bitterness in storage. Conversely, injuries appeared to speed depletion of bitterness when present. PAUL MACK MY ABSTRACT Bitterness was three to five times as intense in carrots stored at hO’ F as in carrots at 32' F. Carrots stored in the presence of apple emanations developed bitterness within six weeks while similar carrots stored in atmospheres free from fruit emanations remained non-bitter. Imature carrots and carrots stored for three months treated with apple emmations and ethylene were not induced to become bitter. Controlled atmosphere treatments with reduced ongen (3 and 7 percent) and accumu- lated carbon dioxide (10 and 5 percent) retained their typical carrot flavor and developed no bitterness. Carrots stored under anaerobic con- ditions develOped no bitterness, but a fermented flavor and aroma. Bitterness appeared to increase to a peak during cold storage fol- lowed by a prolonged depreciation aided by ventilation and root injuries. In smary, bitterness susceptibility appeared related to early matm'ity and a warm growing season. The presence of ethylene, or a similar substance in the storage atmosphere facilitated the development of bitterness in storage. Fluorescence of bitter roots under ultra violet light was found and related to bitterness as a rapid evaluation technique. / STUDIES ON THE OCCURRENCE, MEASUREMENT, AND CONTROL OF BITTERNESS IN CARROTS By Paul Mack Bessey A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of ‘Doc'roa or PHILOSOPHY Department of Horticulture 1957 ACKNOJLE) ms The author expresses his sincere appreciation for the guidance, support, and encouragement received from Dr. Robert L. Carolus, Dr. Donald H. Dewey, and Dr. Harold 14. Sell throughout the course of this investigation. Thanks are extended also to those who aided in various phases of the work, especiallyu Dru C. L. Bedford - canning of carrot samples; Dr. F. J. McArdle - ionizing radiation treatment; Dr. J. A. Cook - special evaluations in one experiment and in organising another; Mr. .A. N. Heath and Gerber Products Company - consultation and the supplyb ing of certain experinental lots; Dr. E. J. Demo and his staff in .Agricultural Chemistry - certain nitrogen, sugar and mineral analyses; Dr. H. N. Fukui and Mr. A. B. Dodson, also of Agricultural Chemistry - fluorescence techniques and spectrophotometric analyses; and to the many others who willingly tasted carrots never knowing when a bitter one would "bite back". TABLE OF CONTENTS Chapter I mmm T10" O O O O O O O O O O O 0 II REVIWOFHTERATURE........ Off Flavors in Carrots . . . . . . Effects of Environment on Carrots Storage Relationships Biochemical Composition Studies III m‘m 0’ mm C O C O O O C IV METHODS OF ms EVALUATION Organoleptic Test Spectrophotometric Tests . . . Fluorescence Test V THE mCURREIBE OF BITERNESS IN CARROT VARIETIES AND WATIW AND STORACE STRAINS AS AFFECTED B! 8011., Introduction....... l95h3urvey ....... Methods and Materials . Results 1955 Trials . . . . . . . Methods and Materials . Results Discussion of the 1951; and 1955 Trials VI STUDY OF MITANCE OF CARROT BITTERNESS Methods and Materials Results andDiscussion. . . . . . . . . O Page 10 15 18 19 19 21 22 2h 2h 2h 25 29 29 30 3h 3? 37 38 Chapter VII BITTERNESS STUDY ON SEEDINO DATE AND DEVELOPMENT TRENDS THROUGHOUT THE STORAGE SEASON . . . . . . . Mbthods and Materials Results andDiscussion . . . . . . . . VIII HARVEST AND POST-HMRVEST HANDLING’STUDY Methods and Materials 0 O O C O O O 0 Results and Discussion . . . . . . . . IX CONTROLLED AND.MODIFIED STORAGE ATMOSPHERE Storage Atmosphere Study 1955-56 . . IMethods and Materials Results and Discussion Storage Atmosphere Study Methods and Materials Results and Discussion ‘1 IMINORIELEHENT NUTRITION AS INCWOTS O O 0 O O O O 0 Methods and Materials . Results and Discussion . II FLUORESCENCE STUDIES . . . Methods and Materials . Results and Discussion . III GENERAL.DISCUSSION . . . . Genetic Relationships . Environmental Factors . 1956-57 Post-Harvest and Storage Factors XIII SUMMARY XIV LITERATURE CITED . . . . . . . . . Page 65 65 67 7O 7O 73 79 79 8b 86 88 95 LIST OF TABLES Table l. Bitterness Ratings as Influenced by Various Facters . . . 2. Variation in the Soluble Solids Content of Carrots as Influenced by Source, Sterage Temperature ani PackageVentilation................... 3. Bitterness Ratings of Carrot Varieties and Strains, 1955’56eeeeeeeeeeeeeeeeeeeoaieee h. Bitterness Ratings of Fifteen Red Core Chantenay Carrot Strains Compared by Long and Short Root mes 1955.56 0 O O O O C O C O O O O O O O O O O O O O O S. Bitterness Ratings of Six Strains of Red Core Chantenay Carrots Stored in Two Locations in 1955-56 . . 6. Bitterness Ratings of Selected Inbred Carrots, 1956 crap 0 O O O O O O O O O O O O O O O O O O O O O O O 7. Storage Influence on Bitterness Ratings of Two Strains of Carrots Seeded on Two Dates, 1955 Crop . . . . . . . . 8. Dry Matter, Ash, Total Sugars and Nitrogen Composition of Two Strains of Carrots Seeded on Two Dates, May 27 am July 6,1955 O O O O O O O O O 0 O O O O O O O O O 0 9. Bitterness Ratings and Fungal Disease Infection of Red Core Chantenay Carrots as Affected by Harvest and Post- Harvest Treatments, 1956-57 . . . . . . . . . . . . . . . 10. Quality Factors in Stort Type Chantenay Carrots and Percentage Carbon Dioxide and Oxygen Atmosphere Composition as Affected by Storage Atmosphere Treatments 11. Influence on Carrots of Caubined Storage with Apples at 32, 36 am 75. F. O O O I O O O O O O I O O O O O O O 12. Influence of Storage Atmospheres on Bitterness Develop- mentinl’our Lots of Carrots, 1956-57 . . . . . . . . . . 13. Variety and Handling Treatment Influence on Bitterness Ratings of Stored Carrots, 1956-57 . . . . . . . . . . . 1114. Mineral Analysis of Carrots from Two Sources, 1955 Crop . Page 26 28 31 32 33 39 N2 115 no 55 57 62 63 LIST OF.APPENDIX TABLES Table 1. Ratings of Fluorescence of Transverse Sections and Bitterness by Taste of a Portion of the Cortex of Sixtybfour Individual Short Type Chantenay Carrots, IMarch l, 1956 . . . . . . . . . . . . . . . . . . . . Characteristics of Sixtyafour Short Type Chantenay carrOtvs, March 1, 1956 O O O O O O O O O O O O O I 0 Fluorescence, Bitterness and Aroma.Ratings of Ten Ingévidual Short Type Chantenay Carrots, March 28, 19 O O O O O I O O O O O O O O O O O O O O O O O 0 Characteristics of Ten Short Type Chantenay Carrots, mCh 28’ 1956 O O O O O O O O O I O O O O O O O 0 e Page 96 97 100 101 LIST OF FIGURES Figure l. 2. 3. 9. Rating Form for Organoleptic Bitterness Evaluation andExampleomearyTabulation. . . . . . . . . . . Typical Spectrophotometric Absorption Curves for Roxane Extracts of Bitter and Non-bitter Carrots . . . Storage Influence on Bitterness by Taste Ratings of Two Strains of Carrots Seeded on Two Dates Percent of Carrot Roots Infected by Rot and Surface Mold as Influenced by Harvest and Post-Harvest Treat- ments, after Five Months of Cold Storage, 1956-57 . . . Experimatal Setup for Apple Damnation Treatment for ImiucingBitternessinCarrots . . . . . . . . . . . . Location of Fluorescence in a Bitter Red Core Chantenay Carrot Under Excitation of 25371 Ultra Violet Light . . Typical Fluorescence Scale of 1- Non-Bitter, 2- Just Detectably Bitter, 3- Moderately Bitter, h- Strongly Bitter, and 5- Exceedingly 31th Carrets Under Ultra Violet Light Excitation at 2537A . . . . . . . . . . . Fluorescence Variations of a Longitudinal Section Filter Paper Impressi n of a Bitter Red Core Chantenw Carrot under (A) 2537 , and (B) 36601 Ultra Violet Excitation O O O O O O O O O O C O O O O O O O D O O O Identical Section of Fluorescent Carrot Under Ultra Violet Liglt (A) and Transmitted Light (B) Page 20 23 143 1:9 52 71 75 76 78 CHAPTER I INTRODUCTION Bitter compounds, flavor components of many foods, are recognized readily in coffee, chocolate, and grapefruit where they provide part or all of their characteristic flavor. Except for sugars, the principle ‘ flavoring component of the grapefruit is naringin, (Kesterson am Hendricksom, 1953) a bitter glyooside. when bitterness appears abnormally in other products, consumers abruptly reject them'because of poor quality. The tubers of most varieties of-potatoes, when exposed to light, are highly susceptible to greening. The chlorophyll that is developed in the exposed areas extends deeply into the tissues and is associated with the production of a glucosidal alkaloid, solanine (Henry, l9h9), which in high concentration is bitter and poisonous. Occasionally fruit of slicing cucumbers, particularly in greenhouse varieties, develops a bitterness which is more concentrated at stem end of the fruit. Most Dutch and Danish slicing cucumbers are sampled dur- ing the marketing process to check for bitterness. In this crap, bitter- ness often appears after a period of slow growth (Gram and Haber, 1953). The presence of elaterase, a relatively specific enzyme for the hydro- lysis of bitter glycosides of the Cucurbitaceae, has been reported in the cucumber and is thought to be related to bitterness. Genetic sus- ceptibility to bitterness has been found in an African cucumber intro- duction (Barham, 1953) . A genetic line which produces bitter tomatoes was reported by Bor- chers and Nevin (l95h) from a survey of plant introduction lines. The bitter principle was identified as an alkaloid and could be accurately measured by a chemical test. Hot, dry weather and long days that often accompany slow vegetative growth and bolting in lettuce often result in a bitter flavor. However, the problem is not as serious as the other morphological changes that occur which make the plant unsalable. One explanation for the market disadvantage of eastern Pascal celery, as compared to the western grown crOp, is the strong, bitterish flavor that is frequently present. He- frigerated storage of five to seven days, comparable to the shipping period of California celery, usually dissipates the strong flavor so that it becomes difficult to distinguish celery from the two places. Celery has often been stored or field blanched to remove color and ob- jectionable off-flavors. An Ontario report, however, has mentioned the development of a strong or bitter flavor in celery stored for market (Truscott, 19Sh). According to Gram and‘weber (1953) the bitter taste is associated with a high nitrogen content. In carrots, several off-flavors have been found. Pesticide resi- dues, particularly'benzene hexachloride, have resulted in disagreeable, usually musty flavors. Herbicide spray oils with low volatility have frequently left a disagreeable oily flavor. Green shoulders, resulting from chlorophyll formation with prolonged exposure to the sun during growth, give a strong, almost bitter flavor. in earthy flavor is occa- sionally found in carrots from.muck soils and is probably related to fungal infection. Carrots which have developed a seed stalk usually are strongly flavored with a greater concentration in the core. A strong carrot flavor, as distinguished from bitterness, commonly appears in some roots in most plantings. Bitterpit of apples, also known as stippin and Baldwin spot, is particularly troublesome with the varieties Baldwin, Northern Spy, Rhode Island Greening and York Imperial. Recent studies by German and.Mathis (1955) have related an_unbalanced supply of calcium to magnesium and potassium as the cause. Calcium.salts applied as sprays or soil inject- ions have given partially successful control. Bitter rot of apples, caused by the fungus Glanerella cinflata, is more of a problem than bitterpit of apples. An additional off-flavor, recognized as bitterness, and for which there has been no apparent explanation,is a problem in carrots for pro- cessing and fresh market. This type of bitterness is the subject of this study which involves varieties, maturity, nutrition, harvest.and post- harvest handling, storage temperatures and atmospheres. CHAPTER II REVIEW OF LITERATURE Off-flavors in Carrots When carrot bitterness studies were initiated by the author in the fall of 19514, the only published references pertaining directly to bit- ter flavors in the carrot were by Brown _e_t___a_l_. (19141;) and Hervey and Schroeder (19h9) . The former reported bitterness in apparently mature Chantenay carrots placed on the fresh market. Hervey and Schroeder in- dicated that yellows virus infection in carrots caused a bitter astrin- gent flavor which persisted into the canned product. Yamaguchi fl. (1955) showed by tasting that yellows infected ca‘rots could be readily distinguist from other bitter carrots. The off-flavor associated with green shoulders of carrots could be distinguished from the other two. In an experiment comparing reconstituted dehydrated carrots for Michigan, Illinois and Wisconsin muck and upland sources, Newcombe and Alderman (19%) reported that muck-grown carrots developed greater oxi- dative rancidity than upland carrots and possessed a decided off-flavor not present in the upland carrots. Barnell, Gooding and wager (1955) indicate that dehydration or storage of dehydrated carrot products is responsible for oxidative breakdown of fi-carotene tofi-ionone with accompanying off-flavors and odors. Weier (19%) showed complete carotene breakdown in 21; hours in carrot sections placed in moist air at 62' C. Carotene crystals dis- solved in oil droplets in the carrot and gave a positive test for alde- hydes when using Schiff's reagent. Reeve (l9h3) showed the disappear- ance of carotene into oil droplets under the microscope. Benzene hexachloride and lindane, both insecticides, and DD, a soil fumigant and nematocide, have resulted in off-flavors in carrots accord- ing to Hinreiner and Simone (1956) and Lear 3_t_a_1_. (1952;). Truscott (1951.), Yamaguchi 2.2.9.- (1955), Sondheimer 113;. (1955) and Atkin (1956) all described the flavor under consideration as bitter with a persistent effect in the mouth. Cracker (1916) in reviewing taste physiology, diagramed the lo- cation of bitterness detection in the mouth as concentrated on the sides and back of the tongue with a few bitter sensitive tastebuds on the tip of the tongue and on the soft palate. Effects of Environment on Carrots Bangs and DeBruyn (1951:) and Bangs, DeBruyn and Sheets (1955) showed temperature effects on carotene content and "degree of ripeness" of carrots. When gram at 8‘ C. roots had less carotene per gram dry weight than at 18‘ C. At the lower temperature total dry weight was lower, carrots were longer, more tapered and pointed at the tip than at the higher temperature. Degree of ripeness was indicated by the rela- tionship between carotene/dry weight ratio and root shape. Similar interpretations of growing temperature influence have been presented by Barnes (1936), Bremer (1931), Magruder M. (19110), Hiller 3331. (1935), Hansen (1915), Lents (19M) and saith 3151. (19kb). Drought and irrigation treatments by Yamaguchi _e_t__§_l_. (1955) and Atkin (1956) resulted in no bitterness differences in the several varieties tried. With the latter, irrigation led to a definite off- flavor distinguishable from bitterness. Yamaguchi £t_a_1_. (1955) indicated that maturity could have an effect upon bitterness. Several varieties of carrots harvested at market ma- turity, minimum size for bunching, had developed no bitterness while those harvested later at processing maturity, after full size was reached, had a stronger taste and were sometimes slightly bitter. Date of seeding was shown by Atkin (1956) to have little influence on subsequent bitterness. Harvest dates, October 2, November 2, and November 27 on the other hand, had a definite effect upon bitterness development of carrots immediately placed in refrigerated storage and held until processing in December and January. The earlier the harvest, the more bitter the carrots became. His data indicate a slight increase in bitterness from December to January in carrots from the first two harvests. Atkin found that carrots from the late harvest had developed no bitterness'by either December of January; In comparing large and small roots for bitterness.Atkin found slightly'more bitterness in small roots and concluded that maturity was not a factor in controlling bitterness. Seasonal differences in carrot composition have been noted by many workers including Miller gt_§1. (1935), smith gt_gl. (l9hh), Hansen (1916), Lmtz (19h9), Janes (19149), Booth and Dark (19169 and Yamaguchi 33:1. (1952) who agree generally that for the same varieties, carotene content in the winter is one third to one half that of summer carrots. .Aeration, reduced by a high water table was thought responsible for much of the poorer color in winter carrots according to'Hiller‘SELEI. smith 'gt_gl. (l9hh) found much higher carotene content in carrots from the south side of raised beds than from the north side in winter crop carrots. iHorris'gt_gl. (l9h6) in studying the seasonal variations in enzyme content of eleven varieties of carrots found ascorbic acid oxidase conp tents highest uhan growing conditions were most favorable. Peroxidase content in contrast was not markedly’influenced by season. Thiamin and ascorbic acid contents were not particularly affected by season accord- ing to Snith _e_t__a_l. (19M). Lantz (19119) also found no seasonal dif- ference for ascorbic acid. varieties and strains of carrots have been studied for bitterness response by both Yamaguchi 213.1.- (1955) and Atkin (1956). Bitterness has been present in all tested and no marked differences were reported. Ismaguchi, however, in progeny testing selections from bitter and non- bitter parents found strongly.flavored carrots appearing from.the bitter selections and only mild flavored roots from the non-bitter. Atkin comp mented on the extreme differuices that appeared from root to root within varieties and strains. Both suggested that breeding for bitterness imp munity or resistance was a likely approach to eliminating the problem. In a limited comparison.Atkin (1956) found more bitterness in car- rots from.muck soils than from.sands or loams. However, bitterness was found in carrots from.a11 soil types. Fertilization practices of carrot growers were surveyed by Atkin (1955). He found both bitter and nonpbitter carrots produced on fields which were both very heavily fertilized and very lightly fertilized and concluded that the quantity of fertilizer had little or no bearing on bitterness development. In a preliminary minor element study Atkin and Sayre (1955) mentioned the application of a “shotgun" treatment of minor elements in frit at O, 50 and 100 pounds per acre. The most bitterness appeared in the 0 treatment with progressively less at 50 and 100 pounds. The bitterness intensity at 100 pounds was still so high that the car- rots would not have been used in processing. Freeman. and Harris (1951) reported progressive increases in caro- tene content of carrots following increasing increments of fertiliser nitrogen on a.Nonroe silt loam soil with a low test for nitrogen and phOSphorus but high in potassium. Additions of phosphorus and potassium were non-significant. The addition of chloride (K01) depressed the carotene content while sulfate (xzsoh) did not have this effect. Carotene content, color and sweemess of carrots were improved by addition of copper sulfate to copper deficient organic soil according to Han-er (191:6). Kelly, flamers and Ellis (1952) showed improvement in growth and carotene content of carrots grown on boron deficient soil following boron application. Warington (191:0) found boron deficiency of carrot to result in tapering, poorly colored roots which appeared immature. Bernstein and were (1953) noted that carrots grown on saline soils were better flavored than those grown in low saline soils, and on a dry weight basis, contained 30 percait more sugar. may suggested that the influence of salinity on sugar content was probahably effected pre- ponderantly through the osmotic prOperties of the soil solution than any specific effect of the added salts. 10 Storage Relationships .Atkin found in several studies that carrots placed in refrigerated storage tended to become bitter while common storage carrots showed no bitterness. The usual practices in operating storages for carrots in New York State according to Tyler (l9hh) are as follows. Cold storages are Operated at about Bl-Bh‘ F. with a relative humidity of 8h-923. Common storages are cooled by ventilation with cool outside air so that temperatures are slower to come down to holding levels of 36-h5' F. Relative humidity ranges from 88-92fi. Carrots kept better and longer in cold storage. He noted that disease organisms causing the most stor- age losses were the same in the two types of storages. Sclerotinia sclerotiorum and other Sclerotinia species were most destructive,fol- lowed by Erwinia carotovora (soft rot) and Botgztis species. There was less rot where crates, bins and storage rooms were well ventilated. Rader (1952) found a similar disease situation in stored carrots. Newhall (1953) found that the most.important factor in reducing storage rots in carrots was rapid cooling to 31-32' F. immediately fol- lowing harvest. Carrots from wet ends of fields spoiled more quickly' than from drier areas. Mechanically harvested carrots deve10ped more spoilage than hand topped ones. Roots with aster yellows usually de- veloped a deep crown rot within a few months so were a bad storage risk. Washing was not recommended because of the danger of spreading several disease organisms. Carrots with excessive amounts of dirt on them as they came to storage were not predisposed to rot, nor did piling soil on several crates cause more rotting. There was no mention of bitterness. ll 'Wright, Rose and‘Whiteman (l95h) state that heat of respiration produced by topped carrots in BTU's per ton per 2h hours is 2130 at 32‘ F., 3h7O at hO' F. and 8080 at 60’ F. Their recommendations for best preservation of sugars and general quality of carrots is storage at 32° F. with relative humidity at 90-95 percent. smudies of changes in the carotene content of carrots during stor- age show differing trends. Langley, Richardson and Andes (1933) found no appreciable change in vitamin A values. ‘Werner (19h1) and Hansen (l9h5) found carotene content to remain stable followed by a decrease as they sprouted in the spring. Barnes (1936), and Lee and Tapley'(l9h7) found carotene content to decrease from the beginning of storage. Lents (19h9) and Lipton (1953) found that data expressed on fresh weight at analysis and on dry weight indicated an increase in carotene content, but when corrected to original fresh weight, there was a decrease in carotene content during storage. Increases in carotene following har- vest were reported by Anon. (1914b), Lachman (1914b), Brown (191:7), MoKillican (191.8), Wharton and Ohlson (19149), Eyes (191.9), Kelley _e_t__§_l. (1950), and Booth (1951). The latter found carotene content, regardless of variety, age or initial pigmentation, to increase by 11 percent in about sixty days and to decrease thereafter. According to Lipton, there is strong evidence that there is an increase in carotene concen- tration in carrots following harvest. Both Lipton and Booth suggested that the apparent increase in carotene was due to the presence at her- vest of unconverted precursors which are changed in the roots during storage to measurable carotenes. 12 Observations indicated that carrots which are stored with apples frequently become bitter. Therefore, it was considered advisable to review briefly the literature relative to apple emanations. iDifficul- ties resulting from the storage of carrots with apples have not been reported. However, apples have caused certain.problems in other com- modities stored with them. ‘Curtis and Rodney (1953) found that dormant nursery stock in cold storage could be damaged in.the presence of ethylene gas in the storage atmosphere at concentrations as low as 1 ppm. Cambial proliferation followed by death required about two months at 35' F., and occurred in about ten.days at 55’ F. The source of ethylene in one case seemed to be from an apple stor- age on the other side of a well insulated wall. Apples and many other fruits as they ripen produce ethylene and other volatiles. Gene (l93h) indicated from biological tests that one apple would produce a volume of about 1 ml. of ethylene, and that the amount would vary with variety and size of the fruit. This was OOH! firmed chemically by Hansen and Christensen in 1939. Snack (1916) demonstrated that stored apples influenced the rate of ripening of other apples stored with them. He indicated that although ethylene did have an effect, other unknown volatile materials from.apples were probably responsible for storage scald and other apple troubles. Several sources of ethylene other than ripening fruits have been established. Young, Pratt and Biale (1951) identified ethylene as a volatile product of the fungus Penicillium.digitatum. The same organism has been found to be a minor storage disease of carrots according to Rader (1952). 13 Certain diseased tissues have been shown by Williamson (1951) and Ross and Williamson (1951) to produce physiologically active emanations in greater amounts than those produced by comparable healthy tissues. Emanations were biologically evaluated for physiological activity by a pee seedling test and were presumed to be ethylene. The increased evolu- tion of volatiles was apparently a response to injury and occurred only as long as the infected tissue was alive. In ggzsalis floridana infected with potato virus Y, they found greater ethylene production at 70‘ F. than 80’ F. which corresponded with an increased incidence of necrosis. Necrotic lesions were induced in leaves of g. floridana and Nicotiana Tobacum hy treatment with phytotoxic chemicals, eg. copper sulfate; which also increased ethylene production. ‘Denny'(l935) showed that immature and maturing fruit, seeds in green pods, parts of flowers, leaves, stems and roots produced volatiles which caused epinasty of young potato plants similar to that obtained with low concentrations of ethylene. Pratt (l9Sh) gave direct chemical proof of ethylene production by detached leaves. Fiftybthree pounds of thistle leaves produced 16.8 ml. of ethylene in two experimental periods of four days each. The presence of unsaturated compounds with physio- logical action similar to ethylene in self-blanching celery was found by'Nelson and Harvey (1935). ‘Nonpself-blanching varieties did not have these compounds. In.summarizing the effects of ethylene on fruits, Thornton (l9h0) listed the destruction of chlorophyll thereby allowing the character- istic ripe color to predominate as in citrus and tomatoes. Respiration rate is speeded as well as certain metabolic changes, for instance the conversion of starch to sugar in the ripening of the banana. 1h Severe internal brown spotting of lettuce has resulted from storage with apples at 38 and hh’ F. according to Rood (1956). The ethylene fraction of the atmosphere was determined as the agent responsible for the damage. Tests with pure ethylene at 20 ppm. Produced severe symptoms at 38 and M? F. in a few days but not at 32’ F. lS Biochemical Composition Studies According to Atkin (1956) processors have observed that bitter carrots are lighter in color than non-bitter carrots. He divided ten lots of carrots from variety trials each into three sampels. One sample was canned at harvest and the other two following refrigerated and common storage respectively. Bittemess was found only in the sample stored under refrigeration, which was also less red in color than the other two as measured by a Hunter Color Difference Meter. Truscott (1951;) reported two off-flavors in processing carrots. One he described as hot and peppery, the other as bitter. Both were de- cidedly objectionable in the raw product. The hot and peppery flavor did not persist after cooking so was not considered a problan for pro- cessing carrots. The bitter flavor, however, did remain after cooking. By placing carrot slices in beakers of boiling water and evaluating a peculiar "flat" aroma arising, he felt that he was able to arrange the samples in order of bitterness intensity. The use of this test has not been reported in subsequent literature. Following overnight storage of the raw carrots in the laboratory at room temperature little bitterness could be found. From this experience, he suggested a practice of high temperature, ventilated conditioning of bitter carrots for several days before they were to be processed. Yamaguchi 333;. (1955), Sondheimer 333;. (1955) and Atkin (1956) all reported the bitter flavor to be found in phloem tissues, not the glen. 16 A syrupy bitter glycoside has been isolated from.leaves of wild car- rots by v. Gizyoki and Harms-s (1951), but was not identified. From seeds and stems of red carrots, Reeb (1923) reported a bitter glucoside which he called daucusin. Neither of the substances was crystalline so their identity remains questionable. ‘Dodson gt_gl. (1956) reporting on isolation and identification studies on a bitter principle isolated from the roots of bitter tasting carrots found it to have a molecular weight of 268. Percentage compo- sition of carbon and hydrogen were 63.9h and 5.68 respectively. An emperical formula of 01581305 was given to the compound. The ultra violet absorption Spectra was found to have a peak at 268 mp and minima at 2&2 and 287 mp. This is slightly in variance with the 265, 2&0 and 290 mp values given by Sondheimer‘gt_gl. (1955) for their tentative spectrophotometric procedure. Paper chromatographic Rf values in five solvents were the same for the bitter crystalline principle and for acetone extracts of bitter carrots. Spots were detected on filter paper by their fluorescence in ultra violet light. Blue fluorescence in roots of carrot seedlings was reported by Goodwin and Kavanagh (19h8) when tissues were exposed to 3650‘ ultra violet light. Zechmeister and Sandoval (l9h5) separated chromatographically a pale orange oily substance, phytofluene, from carrot root extracts. This substance was found to have a greeniSh fluorescence under ultra violet light. Another polyene which was colorless was reported by Porter and Zscheile (19h6) and was obtained in the same manner from carrots. l7 Goodwin and Kevanagh (1950, 1952) noted an increase in fluorescence intensityinsolutions atpalevelsotholOand abovewithanumber of fluorescent compomds, especially comarin derivitives. Sondheimer, Phillips and Atkin (1955) reported a high spectrophoto- metric absorption peak of bitter carrot extracts in the ultra violet range at 265 my and a lower, but always present peak at 290 mp. Extracts from non-bitter roots show little or no absorption from about 220 mp up to about hOO mp. They also found a bitter, orange residue upon evapora- tion of the solvent from patrolman either extracts of bitter carrots. Chemical analyses by Imguchi M' (1955) showed less ot-carotene, h.8 mg per 100 grams, in bitter carrots than non-bitter, 7.3 mg per 100 grams. B-carotene, total sugars, starch and protein showed negligible differences as did analyses for calcium, iron, phosphorus and vitamins C, El, 32 and niacin. They concluded that the larged-carotene difference between bitter and non-bitter carrots, plus petroleum ether solubility and magnesium oxide column adsorption of the bitter principle, suggested that bitterness may be caused by metabolic products of the carotenoids. 18 CHAPTER III STATEMENT OF PROBLEM This investigation involved studies on the occurrence, measurement, and possible methods of control of bitterness in fresh market and pro- cessing carrots. The role of variety, genetic selection, soil type, mineral nutri- tion, physiological age, harvest and poet-harvest handling, and storage treatment in relation to bitterness are aspects of the prdblen that will be evaluated. The primary objectives were the identification of conditions re- sulting in carrot bitterness and the development of usable controls or preventative measures. l9 CHAPTERIV METHODS OF BITTERNESS EVALUATION For the evaluation of bitterness in these experiments, organoleptic , spectrOphotonetric, and fluorescence tests were used. Organoleptic Test Mix was used in practically all of the studies conducted, either by a panel or by the writer. Carrots were selected at random from each treatment, washed, peeled, quartered and coarsely ground and mixed for taste testing. Spectromotometric analyses were made on the same samples. hcept for one preliminary experiment where salples were dried, all lots were handled in the fresh state at room temperature. Treatments were replicated and rated in comparison with reference sanlples by taste panels which ranged from one to thirty persons depending upon the experiment. A rating scale of 1 to 5 was devised with the following designa- tions: 1- not bitter; 2- Just detectably bitter; 3- moderately bitter; h- strongly bitter; and 5- exceedingly bitter. A rating fen, Figure l, was used and a sunnary sheet compiled. Each bitterness intensity level was given a weighted value. Ratings from all tasters were totaled and multiplied by their scale values. For each treatment these products were stunned and divided by the number of persons participating, as shown by the example in Figure 1. Sample A would be given an over-all rating of 2.0, or Just detectably bitter. Sample B, with an over-all rating 20 TASTE TEST RATING roan Name Date Experiment Not Most Sanple Bitter Bitter Gamente 1 2 3 h ‘ 5 I ‘ 1 B c D Instructions: Rate samples as to their bitterness in comparison with Sample I which has already been checked. Thank you. SUMMARY TABUHTION OF ORGANOLEPTIC RATINGS Bitterness Intensity Rating and Value Sailple Total Average 1 2 3 h S A h 18 6 h 32/16 2.0 B h 15 28 10 57/16 3.6 c 16 16/16 1.0 Figure 1. Rating Form for Organoleptic Bitterness Evaluation and Example of Summary Tabulation. of 3.6 would be quite bitter, ranging between moderate and strong. Sample 6 would be recognised by all tasters as non-bitter. This rating system is considerably different from that used by Sondheimer 3131. who rated bitterness on a l to 10 basis with l indi- cating the highest level of bitterness. A subsequent report by Atkin, referring to the same experimental data rated bitterness by taste rang- ing from 0 to 9 with the 0 on the non-bitter end. ‘ Spectrophotaaetric Tests The spectrophotometric procedure developed by Phillips (1951;) was used to supplement taste test evaluations in the early stuiies, but be- cause of :its elaborate extraction procedure, the laboratory evaluation capacity was limited to about eight samples a day. The basic steps were: (1) extraction with acetone in a blender, (2) centrifu- gation to remove all solids, (3) dehydration by combina- tion with anhydrous Nazsou, (1;) transfer of bitter prin- ciple to Skellysolve B by separation with a separatory funnel, (5) concentration by evaporation, (6) chromato- graphic separation on a 2:1 by weight MgO:Celite column under vacuum followed by washing with Skellysolve B, (7) removal of the upper portion of the column on which the bitter principle is adsorbed, (8) elution with spectro- grade methyl alcohol, (9) determination of transmittance values at 2148, 268, and 290 my, and (10) the plotting of points on plain graph paper (10 x 10 to the inch). Measureaent of the depression in cm. of the 268 mp value below a line drawn between the 21:8 am 290 mp values was fitted to a formula to give 'degrees of bitternessn (B‘). A B’ score below 5 to 7 was considered non-bitter, above 10 defi- nitely bitter. After Hay 1956 treatments were evaluated by using a modification of this method by Sondheimer, Phillips and Atkin (1955) which allowed lab- oratory capacity to be increased to approximately 50 samples a day. 22 Modified spectrOphotometric method 3 (1) Samples were pureed in a blender, (2) 5 gram ali- quots were placed in a 50 ml ground glass stoppered Erlenmeyer, then (3) shaken with to ml of spectra pads Skellysolve B, and (11) measured directly in a Beckman DU spectrOphotometer and later a Beckman DK2 Recording spectrOphotometer. (5) light absorption (optical den- sity) at 2110, 265, and 290 my was measured. Values were fitted to a formula which gives a bitterness reading in terms of "height of 265 mp peak“ as follows: o.d. at 265 my - o.d. at 290 + o.d. at 2140 my 2 x ml solvent grams of sample A height of 265 my peak of about 0.75 would be close to the taste threshold of bitterness. A typical bitterness absorption curve is shown in Figure 2. Fluorescence Test In January 1956 it was observed that bitter carrots fluoresced in short wave ultra violet light (25371) am that this phenomenon was cor- related (r - 0.61;) significantly with their bitter taste. Since fluor- escence correlated with taste testing about as well as the spectrophoto- metric methods did, fluorescence estimations were used as a third means of evaluation. Fluorescence appeared yellowish-green and was patterned in tiny spots present only in the phloem region which was also the site of bitterness by organoleptic and spectrophotometric observations. The system of rating devised paralleled the taste test rating on a l to 5 basis with 1 being non-bitter and non-fluorescent, and 5 being exceedingly bitter and highly fluorescent. Vertical sectioning of the roots gave the best representation of fluorescence concentrations and distribution. Optical Density 1.0 .94 .84 e7' Bitter Kym Non-Bi tter I U I T 1 U 1 I r—V ' r 21.0 260 Absorption Spectrum mp Figure 2. Typical Spectrophotometric Absorption Curves for Roxane Extracts from Bitter and Non-Bitter Carrots. 23 2h CHIPTER V THE OCCURRENCE OF BITTERNESS IN CARROT VIRIETIES AND STRAINS A3 AFFECTED BY SOIL, LOCATION AND STORAGE Introduction In this study the range of bitterness susceptibility within the varieties and strains of carrots was investigated. It had been noted that reports of bitterness primarily involved carrots stored for pro- cessing, of which Red Core Chantenay and its strains are the most impor- tant. This suggested that these strains should be surveyed as well as market types which could be used for either purpose. Most of the processing carrots and a large part of the fresh market carrots in.Hichigan are grown on muck soils. Lipton (1953) and Newcombe and Alderman (19hh) indicated that quality of carrots grown on mineral soils was generally superior to those grown on muck, therefore, bitter- ness responses of carrots from both soils were evaluated. It had also been observed that bitterness more often appeared in some storages than others which directed attention to the influence of different storage conditions. l95h Survey Methods and Materials Carrot varieties were available in the fall of 195k from one mineral and two muck soils. Eight varieties and strains were selected from each 25 source. Five of the University muck and mineral trial lines were identi- cal. Bulk lots were obtained at harvest and were held in a 32' F. stor- age. Eight 5-pound samples of each of the 21; lots were packed in 8-pound polyethylene bags, half of which were sealed and the remainder punched for ventilation. Duplicate samples were placed in two storage rooms at 32 and 110° F. In addition, samples from the muck trial near Grant, Michigan which had been held from harvest in two commercial storages were transferred in December to a 32‘ F. storage at East Lansing. All carrots remained in storage until April 7 to 16, 1955 when they were removed and evaluated with a hand refractometer for soluble solids content, and subsequently processed in No. 1 carrot enamel cans. Canning was used to eliminate differences that might be due to variations in 1mgth of storage resulting from use of the complicated original spectro- photometric evaluation procedure and in running statistically comparable taste tests. Bitterness evaluations were actually delayed until September 1956 when the rapid spectrophotometric test modified by Sondheimer _e_t;_g._l_. could be run on the camed samples. Results Carrots in ventilated polyethylene bags stored at 110’ F. gave the highest spectrophotometric bitterness ratings, as shown in Table 1. In the University mineral and muck trials, the early maturing varieties, Nantes and Touchon became much more bitter at 110‘ F. in punched bags than the late varieties Gold Spike and Imperator. The second-early Chantenay strains rated close to Nantes and Touchon in bitterness re- sponse . 26 TABIE 1 81W RATINE AS INFHIENCED BI VARIOUS FACTORS (Height of 265 mu peakl) ‘“ Source, Storage Temperatures Varieties 32‘ F. 110’ F. Averages 5. Strains Ventilated Sealed Ventilated Sealed A. Mineral Soil-H. 3.U. Nantes (a)2 .32 .20 h.h2 .18 1.23 Touchon (b) .55 .21; 2.91.; .142 1.014 Chantenay (c) . .30 1.90 . .80 Nantes .18 .21 2.21 .21 .70 Dmvers .50 e20 1.148 .39 06h Denvers (d) .60 .22 .70 .22 .1111 Long Chant. .52 .18 .61. .26 .110 001d 3311“ (e) .22 .12 .56 .08 .211 Avergges . 1114 . 21 1 . 86 . 26 . 69 Be Mk Son-H.860. Nantes (a) .27 .27 1.21 .22 .50 Touchon (b) .25 .10 2.21 .18 .68 Chantenay (c) .28 .33 .97 .18 .1111 Danvers (d) .20 .15 .53 .10 .211 Gold Spike (e) .31 .20 .55 .15 .30 Royal Chant. .11 .19 1.39 .23 .h8 Red Core Chant. .17 .22 1.20 .27 .116 Imperator .25 .28 .61 .28 . 36 Averages . 23 . 23 1.09 . 20 . 113 c- M son'mt Chanticler 1.86 .55 1.73 .61 1.19 Red Core Chant. .53 .118 2.18 .37 .89 Royal Chant. .55 .67 1.1.2 .32 .71. 3110“ Type Chute ow .60 1001 073 e714 Red Core Chant. .111 .113 1.25 .78 .72 Long Type Chant. .142 .78 1.10 .50 .70 Long Type Chant. .58 .66 1.09 .37 .68 Short Type Chant. .111; .29 .611 .39 .1114 _ ”Averages . 55 . 56 A. 36 . 51 . 76 Spectrophotanetric values are expressed as height of 265 mp absorption peak and for this experiment were determined on canned samples. Car- rots with values below 0.75 are probably not bitter to taste. Varieties with the same letter in the University mineral and muck trials came from identical seed sources. 27 Comparing the three sources, the highest over-all average was found in the carrots from the muck trial at Grant. The highest individual bitterness ratings, however, were in the early maturing varieties in the University mineral. soil trial which also included the widest differences in bitterness response among varieties. The lowest bitterness average was in carrots from the University muck trial. Carrots from the Grant muck stored by the Gerber company in the two counercial storagea produced a marked difference in bitterness. The average bitterness rating for the eight Chantm strains in storage A was 1.29 while in B the value was 0.72. Carrots and apples had been stored together in A. In B, carrots were stored alone. Bitterness re- sponse could not be separated on the basis of long and short types of Chantenay. Data in Table 2 on soluble solids show a general decrease where car- rots were stored at hO’ F. as compared to 32' F. At 32‘ F. the percent or soluble solids was lower in carrots from sealed than from ventilated bangs while at 110' F. there was little consistent difference. For the three sources, percent soluble solids was highest - 6.06 for carrots from the mineral soil, followed by 5.66 and 11.32 for the {himsity and Grant mucks respectively. The highest soluble solids v£Llues for carrots from the Grant muck were below the lowest from the University muck and mineral trials. Varietal differences in soluble solids from the mineral soil were “Qt consistent; but from the University muck soil, the late varieties, Gold Spike and Imperator averaged much higher in soluble solids than the earlier maturing varieties. 28 TABIEZ VARIATIQJ IN 11-13 SOLUBIE SOLIDS CONTENT OF CARROTS AS INFIHENCED BI some, STCRAE TEMPERATURE AND PACKME VENTILATION. (Percent) Source, Storage Temperatures Varieties 32' F. 110' F. Averages 8: Strains Ventilated Sealed Ventilated Sealed A. Mineral Soil-H.S.U. Nantes (a)1 6.75 6.20 5.80 5.75 6.12 Touchon (b) 6.30 6.05 11.140 5.65 5.60 Chantenay (c) 7.140 6.10 5.80 5.60 6.22 Nantes 7. 25 6. 20 6. 50 5. 55 6. 38 Danvers 6 . 90 6 . 80 6.10 5. 65 6. 36 Danvers (d) 6.25 6.85 5.00 5.35 5.86 Long Chant. 6.90 6.05 6.15 5.05 6.01.; Gold Spike (e) 6.60 5.90 5.50 5.60 5.90 Averages 6.79 6.27 5.66 5.52 6.06 Be ”Mk SOil-H.S.U. . Nantes (a) 5.90 5.62 8.88 5.05 5.36 Touchon (b) 6.10 6.12 5.80 5.65 5.92 Chantenay (c) 5.70 5.62 5.38 5.60 5.58 Danvers (d) 6.10 6.12 11.75 5.50 5.62 001‘1 Spike (0) 7.70 6.00 5.65 6.35 6.112 Royal Chant. 5.05 5.00 11.80 11.60 11.86 Red Core Chant. 5. 25 5. 25 5.75 5.15 5.35 Imperator 7.60 6.62 5.25 5.85 6.33 Averages 6. 18 5. 79 5. 28 5. h? 5. 66 0. Huck Soil-Grant Chanticler h. 85 1..oo 1.. 35 3. 7o 1.. 22 Red Core Chant. 11.50 11.75 14.35 11.50 11.52 Royal Chant. h.50 14.15 3.85 3.70 h.05 Short Type Chant. 5.15 M0 3.80 14.00 11.31. Red Core Chant. 11.35 11.00 3.70 3.70 3.9h Long Type Chant. 5.05 5.00 - - - Long Type Chant. 11.50 11.95 11.35 14.65 11.61 snort Type Chant. 14.90 11.90 h.2o 14.35 b.59 ._ Averages 14.72 11.52 11.08 14.08 14.32 1 Varieties with the same letter in the University mineral and muck trials came from identical seed sources. 29 From the muck trial at Grant, soluble solids differences between long and short Chantenay types were less than within the respective types. The long-short type characteristic is apparently not a valid indicator for soluble solids contents. Data for bitterness and soluble solids appear highly variable and not related. 1955 Trials Methods and Materials The 1955 trials involved chiefly processing types and included seed of several of the strains and varieties evaluated in 19511. Seventeen seed lots were planted at the University muck farm June 2, three at the Horticulture farm on Hillsdale sandy loam June 16 both in replicated, randomized, couplets block designs. Six more were planted in early May by the Gerber Products Company on muck soil near Grant, Michigan. Carrots from the University muck trial were dug by hand and mechani- <3&11y topped October ‘18. Diseased and damaged roots were discarded am samples of sound carrots placed in 10 pound onion bags. Carrots from this mineral soil were dug and tapped by hand November 3. Samples from both trials were stored under refrigeration at 32-35’ F. until bitterness “was evaluated during July 1956. There was some loss due to mold growth, but in general, the carrots were in acceptable condition. The carrots planted at Grant were mechanically dug and topped september 28, 1955. About one ton of each strain was stored at 32’ F. 111 each of the two commercial storages compared in 19511. Samples were I‘Qmoved to the carrot storage in East Lansing in January 1956. At that time, bitterness was detected in some lots. 30 Results No bitterness was detected in stored carrots from the mineral soil trial (Table 3). Their color and flavor were superior to that of any of the muck grown varieties. A low to moderate level of bitterness developed in the carrots from the University muck farm. The highest level of bitterness developed in the carrots from the muck trial at Grant. Variety and strain differences were apparent in both muck trials. Red Core Chantenay (Coreless) from Northrup, King was by far the most bitter in the group of six strains grown in both places. No strains were consistently low in bitterness from both muck trials. In the University muck trial, the two market varieties, Imperator and Gold Spike, were rilted as essentially non-bitter. Three Chantenay strains were rated as low. By comparing bitterness development by taste test in long and short Chantenay strains, it was found that the short type tended to be more bitter than the long in both muck trials (Table 11). Short strains grown on University muck averaged 2.62 by taste test against 2.15 for the long a"i-rains. The Grant muck showed 3.50 for the shorts and 2.62 for the lrungs. The range for bitterness went from slight to moderate in each St‘oup, however. Muck trial carrots placed in the two comercial storages again re- vealed a significant difference between storages (Table 5). Carrots in a‘I‘oorage A became markedly more bitter than in storage B with taste test ‘Values of 3.17 and 2.67 and spectrophotometric values of 2.95 and 2.52 x‘eopectiveily. As in 19511, carrots and apples had been stored together in storage A. Apples were not in the carrot room in storage B but were g‘i‘onred in adjoining rooms. 31 mm 3 131mm RATINGS or mow vmm'ms AND STRAINS, 1955-56 Bitterness Ratings University Trentm Variety Huck Spectro- or Taste Test Taste Test photometric Strain Seed Source 1-5 naung) 1-5 Ratingg Ht. 265 mu peak(2) Red Core Northrup,King 3.3 11.5 3.83 Chantenay - Long Type Voodruff 2. 7 2. 5 1. 59 Chantenay snort Type Woodruff 2.3 2.5 1.110 Chantenay Royal Northrupfiing 2.2 2.0 3.31 Chantenay Royal Corneli 2. 0 2 . 5 3.39 Chantenay Long Type Corneli 1.7 3.5 2.89 Chantenay Red Core Harris 3. 5 Chantenay Red Core Asgrow 3.0 Chantenay Chanticler Asgrow 2.7(3) Red Core Corneli 2.5 Chantenay Long Type Northrup,King 2.5 Chantenay IaOng Type Ferry-Horse 2 . 2 . Chantenay alert Type Ferry-Horse 2.0(3) Chantenay Chantenay Northrup,xing 1.7 InIsperator Ferry-Morse 1. 7 Inng Type 10mg Chant. Harris 1.3 Special Gold Spike Ferry-Morse 1.3(3) Averages 2 . 111 2 . 92 2. 73 \ #____ (1) Taste test values of l - non-bitter, 5 - exceedingly bitter. Pam]. d6. (2) Spectrophotometric values expressed as height of 265 mp absorption peak. taste. Carrots with values below 0.75 are probably not bitter to (3) These three varieties were also tested on Hillsdale sandy loam. No bitterness could be detected after storage by taste or spectrophoto- metric tests. 32 TABLE 11 3m RATINGS OF FIFTEEN RED CORE CHANTENAI CARROT STRAINS COMPARED a! mo AND sacs! 300': ms, 1955-56 Bitterness Root by Taste Averages Type Strain Source (1-5) (1-5) 2225 long Type Chantenay Ferry-Norse 2 . 2 Long Type Chantenay Corneli 1.7 Long Type Chantenay Northrup,ng 2.5 Long Type Chantenay Hoodruff 2 . 7 Long Chantenay Special Harris 1.3 Chanticler Associated 2 . 7 Royal Chantenay Corneli 2.0 Royal Chantenay Northrup,King 1.2 2.1 Short alert Type Chantenay Ferry-Horse 2 . 0 Short Type Chantenay floodruff 2.3 Red Core Chantenay Associated 3.0 Red Core Chantenay Harris 3. 5 Red Core Chantenay #2 Corneli 2. 5 Red Core Chantenay, Northrup,ng 3.3 Coreless Chantenay Northrup,King l. 7 2 . 6 33 TABLES BITl'ERNESS RATINCB or 51:: swims 0F RED com: cwmmr cmors s'rcm'n IN mo LOCATICNS IN 1955-56 Bitterness Ratings Taste reetfl) Height of 265 mp pend?) No. gymgfl ) gurus; Averages!- ’S‘torage; Averag_e_s___ 1 Royal 2 2 2.0 3.28 3.311 3.31 2 Long Type is 3 3.5 3.15 2.62 2.89 3 Royal 3 2 2.5 b.95 1.83 3.39 is Red Gore 5 h h.5 3.16 13.50 3.83 S Snort Type 2 3 2.5 1.1.]. 1.36 l.h0 . 6 Long Type 3 2 2.5 1.69 1.50 1.59 Averages 3.17 2.67 - 2.95 2.52a- .. (1) Taste test ratings on a 1-5 basis with l - non-bitter and S - exceed- ingly bitter. Panel of 6. (2) Spectrophotometric rating of bitterness in terms of height of 26S nip absorption peak with a bitterness taste threshold at about 0.75. Since these tests were obtained from dried carrot tissues, values are a little lower than would be expected from this procedure if run on fresh tissues. (3) Source of Chantenay strains: 1- Northrup,King; 2- Corneli 1951; 3- Corneli 3355; h- Northrup,King, Careless; s. Woodruff, so 1-5520; 6- woodmrr, 36 1.6510. * Statistical differences significant at the 5 percent level for stor- ages under the spectrophotometric test and closely approaching the 5 percent level for varieties under the taste test. 3h Discussion of the 1951; am 1955 Trials Variety and strain.differences in bitterness response were clearly present with the earliest maturing lines developing the most bitterness. Nantes and Touchon, which reach.market maturity about 10 days earlier than most strains of Red-Core Chantenay, definitely were more susceptible to becoming bitter. Imperator and Gold Spike, which require about a week longer to reach market maturity than Red Core Chantenay strains, consistently developed the least bitterness. Although the bitterness response of the Red Core Chantenay strains varied from almost nonpbitter to exceedingly bitter, most tended to be close to the response of the early varieties, in other words, quite bitter. One strain, Red Core Chantenathoreless from Northrup, King, was the most bitter of the Red Gore Chantenay strains in all three of the tests in which it was in— cluded. Chanticler, an Associated strain rated highly bitter in two trials. Three strains (Long Type Chantenay - Corneli, Chantenay - Northrup, King, and Long:ChantenayeSpecial - Harris) rated essentially nonpbitter in a single comparison (University muck trial - 1955). Al- though l95h trials showed no differences between averages of long and short type strains, in 1955 more bitterness was found in the short types. Atkin in 1956 cautiously indicated that muck soils produced more bitterness in carrots than mineral soils. This view was supported in general by observations of processors; however, data from this study 35 does not support this contention. Of all the 19524 comparisons, the most intense bitterness developed in carrots from mineral soil. In 1955, mineral grown carrots were not bitter while some muck carrots were ex- ceedingly bitter. In both the 195). and 1955 trials the highest levels of bitterness were found in the earliest harvested carrots regardless of soil type effects, with the conclusion that muck carrots are not necessarily more bitter than mineral grown carrots. This suggests that date of harvest is a more important factor. This stuiy indicated that mineral soil grown carrots contain more soluble solids than muck grown carrots, agreeing with the work of Lipton. here was a lack of correlation between sugar content and bitterness. . The 19514 temperature-packaging study revealed that only the carrots stored at 110‘ F. became bitter. Most all of the bitterness developed in carrots in polyethylene bags which had been punched for ventilation. Although carrots in the 32' F. room were essentially non-bitter, spec- trophotometric data showed generally higher values in punched bags, as found at 140‘ F. Two possibilities are apparent to explain the interaction. First, the reaction to form the bitter principle operates as a function of tunperature, with carrots in sealed bags inhibited from developing bitterness because of high 002 and/or low 02 content. Second, the in- cidence of bitterness could be a fxmction of presence of physiologically active volatiles in the storage atmosphere affecting carrots in vented bags but not those in sealed bags. 36 Varietal differences were also present in respect to the tempera- ture-ventilation interaction in that the earliest maturing varieties became most'bitter while the late varieties were almost free from bitter- ness. Commercial storage A resulted in more bitterness in processing carrots in both years of the study than storage B. The observed dif- ference in management between them was the inclusion of apples and carrots in the storage.i. .Apples were kept in the same building but not in the same room in B. It is suggested that the higher incidence of bitterness in.A was due to the presence of physiologically active volatiles emanating from the stored fruit. 37 CfldPTER VI STUDY OF INHERITINCE 0P GIRROTIBITTERNESS It has been reported by Atkin (1956) and observed by the writer that bitterness frequently varies markedly from root to root within the same variety and strain. This suggests the possibility of genetic dif- ferences which might be eliminated during a breeding progral. To deter- mine the presence of a genetic factor, both bitter and nonébitter roots would have to be selfed or grown in isolation and their progeny evalu- ated. Methods and Materials In the winter of 1955, bitter, long-type Chantenay carrots were screened for five intensities of bitterness. The rating of 1- non- bitter to S- exceedingly bitter was based on a taste evaluation of chips removed from the phloem region of the roots. Each intensity level included 20 roots which were planted in pots in the greenhouse. Those which survived were moved to a cold frame and later to the field. At flowering, bags were placed over each plant enclosing at least two umbels. Fly pupae were introduced as often as necessary for about two weeks to promote pollination. Seven plants from all five levels of bitterness produced viable seed. For evaluation of this first inbred generation, seed was sown on muck soil in May 1956 and the carrots harvested in.mid-September. After storage in a refrigerated root cellar at about 35° F. for four and a half months, bitterness evaluations were made. 38 Results and Discussion The results, Table 6, indicate that‘bitterness was not present to any appreciable extent. Despite the negative results, this experiment does not allow the conclusion that bitterness either is or is not gen- etically controlled. .As observed in other studies, weather during root development was apparently more influential in affecting bitterness than any'trsatment applied. Also, very little bitterness was detected in the entire commercial processing crop in 1956, and in addition, the carroty flavor was generally low and in some cases undetectable. Bork reported by‘Iamaguchi‘gt_§l. (1955) on conparison.of bitter and nonebitter progenies indicated that the bitter selections produced strong and slightly bitter roots while the nonsbitter selections pro- duced mild flavored roots. Thus it would appear that selection of roots on the basis of carroty flavor, strong versus mild, might produce dif- ferences in bitterness response. Strong flavored roots are objection- able, yet much of this flavor is dissipated in cooking by volatiliza- tion; on the other hand, carrots which are too mild, lack characteristic carrot flavor. It is possible that a low content of these flavoring materials may not only result in the develOpment of bitterness, but also affect keeping quality in storage, particularly favoring the development of Sglerotinia as evidenced by the poor flavor and high incidence of disease in the 1956 crop. 39 TIBLE 6 sxrrsausss RATINGS or ssLscrzn Insane cansors, 1956 030? (Rated February 1h, 1957 by the Author) Original Bitterness Inbred Bitterness R‘tEE89 Rating Plot Spectro- by Taste Number Taste Test Fluorescence photometric (l-S basis) 1956 (l-S basis) (l-S basis) (Rt 265 my peak) 1 h76 1.0 1.0 0.2h l h77 2.0 1.0 0.10 2 h78 1.0 1.0 0.20 2 hao 2.0 1.0 0.19 3 h81 1.0 1.0 0.2h h h82 2.0 1.2 0.1h S h83 1.0 1.0 0.12 Hilde hBh 1.0 1.0 0.31 a This was not an inbred, but was grown from locally harvested wild carrot seed planted and handled with the inbreds. to cm VII BITTENESS STUD! 0N SEEING DATE AND DEVEIDPMENT mums ‘fl'lROUG-IOUT THE STORAE SEASON The stage of physiological maturity at harvest has been suggested by Drewes (Reath, 1953) as a possible factor contributing to the sub- sequent development of carrot bitterness. see core Chantenay strains are usually used for processing while Imperator types are grown for the fresh market; however, both may be used for either purpose. Carrots for processing in the northern states are seeded as early as feasible and to obtain maximal yields, are left in the field until freezing weather. As Red Core Chantenay strains tend to mature earlier than Imperator types, they are frequently overmature at harvest. .Hsrket carrots, however, are seeded by schedule to provide a continuous supply of tender maturing roots during the season. Bitter- ness has been more frequently Observed in canning than fresh market car- rots, which may possibly be due to differences in their physiological ages. Methods and Materials Two strains of Red Core Chantenay carrots, Short Type Chantenay (Perry-Horse) and Chanticler (Associated), and Gold Spike were seeded on luck Ray 27 and July 6, 1955 in a complete randomised block design. Some bolting appeared in the May planted Chantenay and Chanticler varieties. All plants with flower stalks were removed prior to harvest on October 19. The carrots were dug and topped by machine and were sorted to elimi- nate diseased and badly damaged roots. Two bushel lots were stored at 32' P. from November 1 until July 10, 1956. Samples were removed at monthly intervals and were evaluated for bitterness by a panel and by Spectrophotometric analysis to obtain a pattern for bitterness develop- ment during the storage season. Fresh weight, dry weight and ash content were obtained at the first evaluation period. Total sugars and nitrogen content for each period were determined by the Agricultural Chemistry Department. Results and Discussion At harvest, all samples were comparatively non-bitter by taste test; however, Chanticler from the early planting had a tendency to develop more bitterness during the storage season than Start Type Chantenay (Table 7). By taste test the average bitterness value of mart Type Chantenay was 2.73 for the early and 2.15 for the late seeding, while Chanticler was 3.12 and 1.87 respectively. Spectrophotometric values in "degree of bitterness' from the analytical procedure by Phillips (199;) were h.57 and 5.21; for Short Type Chantenay and 6.21; and 1.97 for chanti- cler for early and late plantings. Monthly bitterness ratings by the taste panel gave relatively con- stant values through the storage season as shown in Table 7 and Figure 3. Carrots from early seedings of both strains were more bitter than from late seedings. Roots from late seedings of both strains were Just de- tectably bitter with Chanticler tending to be slightly less bitter than Chantenay; however, in roots from the early seedings, Chanticler was 1.2 TABLE 7 swam INFLUENCE on BITERNESS names OF Two STRAINS or cannon-s man on mo DATES, 1955 caop Bitterness Ratings - Taste Test (l-S Scale) (1) Chantenay Seeding Strain Date Dec. Jan. Feb. Mar. £11 Hay Averages alort Type Early 3.1 2.2 2.8 2.14 2.6 3.3 2.73 Short Type Late 2.5 1.8 2.2 2.0 2.0 2.1. 2.15 Chanticler Early 2.0 3.0 3.6 3.1 3.6 3.1t 3.12 Chanticler Late 1. 8 1.8 2.0 2.0 1.7 1. 9 l. 87 (l) Bitterness ratings by taste test: 1 - non-bitter, S - exceedingly bitter. Number of members in panel: Dec. 15, Jan. 11;, Feb. 214, Mar. 2h, April 15, and May 20. 1:3 5. -— —~-H-~~————Exceedingly ..__ Short Type Chantenay 31“” -- -- Chanticler & o Seeded May 27, 1955 § h.o-—— ASeeded July 6, 1955 rongly .. Bitter a: .0 g). 1:: ‘ v 1.5 g .Moderately a Bitter . u E . +3 .... etectably “3 ‘2 Bitter ; J,— 4 § Not Bitter Dec. Jan. Feb. Mar. April May Month of Evaluation Figure 3. Storage Influence on Bitterness by Taste Ratings of Two Strains of Carrots Seeded on Two Dates. 14h °°Mid ecu-ably more bitter except at the first taste evaluation period. 6"“ $11“ was tested for bitterness by the panel from January through May but. was not checked for other characteristics. Roots from the early muting developed a low level of bitterness, but those from the late pint-3.118 were practically free of bitterness. Emu seeded carrots contained considerably less sugars on a dry “19“: basis than the same strains seeded later, Table 8. Late seeded mmtfiualer had a higher sugar content in November than Slort Type Chante- “373 however, by May it had lost much more sugar than Snort Type Chante- nay- This difference suggests that late Chanticler was of a younger mMological age than late Short Type Chantenay and that it had a higher 1“." of respiration. me first 'seeding developed a small number of carrots withflower “We and carrots infected with aster yellows. Chanticler had more 3°96 ere, 2.71 to 0.614 percent, while Slort Type Chantenay had more yel- lows, 1.33 to 0.81 percent. TABIE 8 115 1333mm, ASH, 10m. sums AND NITROGEN COMPOSITION 01“ Two STRAINS or 0T8 was 0N Two DATES, m 27 AND JUL! 6. 1955 ;:-\ ‘ Analysis aiort Type Chantenay Chanticler _.——-\‘ Date Early Late Early Late Dry himcarter - z Nov. 10.15 10.16 9.55 11.30 “h ‘ z 1). wt. Nov. 9.85 8.12 8.37 7.1.6 ”h ‘ x 1*. wt. Nov. 0.96 0.85 0.75 0.83 Tot.“ Sugars - 1 D. Wt. Nov. 31.9 36.5 32.8 111.2 May 29.2 37.1 33.1 36.7 Nitrogen - S D. Ht. ' Nov. 2.611 2.16 2.58 2.01 May 2.88 2.21; 2.70 2.33 \_ ’46 CW VIII HARVEST AND POST-HARVEST HANDLING STUDY In the date of seeding study, it was noted that large, ovemature ”no“ were more severely danaged by mechanical harvesting than smaller, 1°" h‘fiture roots. Bitterness appeared related to size, maturity and man} at harvest. This handling experiment was designed to explore the reL ‘t-ionship between bitterness, injury, and the incidence of storage Maefiaes. Methods and Materials Long type Chantenay carrots were seeded on muck June 16, 1956, grown cmmtionally and harvested in different ways on October 11. Two lots “are dug and topped with a Chickeriag mechanical harvester, so operated ‘8 to cause heavy and extensive injury to the roots of one lot, and as 11 t"file damage as possible with another lot. A third lot was carefully d“: and topped by hand. The carrots were sorted to remove obviously diseased roots, then h‘fl-d in a 35' F. refrigerated root crop storage from October 18 to 25, uh“ the carrots were washed and subsequently stored at 32‘ F. These carrots were packed in bushel apple crates lined with polyethylene (Har- Cletl‘3“.1rg, 1956) to retard moisture loss and escape of volatiles. 0n Q0tober 30, additional lots from the same source were treated with Beta ms with a total radiation of 150,000 rep. (Roentgen equivalent physical). Carrots were spread on trays, irradiated, turned over, and irradiated on the other side, then packed in polyethylene crate liners and returned to the storage. h? Results and Discussion Bitterness evaluations were made October 26, January 11; and March 21 using m three measurement techniques. 1“Late tests, spectrophotometric and fluorescence analyses at the be- ginn'Lng of the study indicated no sign of bitterness. A low level of “”1““ bittemess as measured by fluorescence did appear in a few car- rots Q\lring storage but no further evidence of bitterness could be de- mtgd (Table 9). As proposed in the experiment, different harvest and post-harvest tr “inherits did influence the incidence of disease (Table 9 and Figure 11). “hr five months of storage, visible infection rates ranged from 5.8 to 78-1 percent. The primary organism was identified as a Sclerotinia apeeies described by Radar (1952) as one of the more serious storage “Senses of carrots. Rough machine harvesting was responsible for more infection than g“thine machine harvesting. Digging and topping by hand resulted in the least. infection. Irradiation of unwashed roots reduced disease incidence by ‘11 average of 10.6 percent while washing reduced it by 19.6 parent. The fluorescence observed was for the most part related to injury appfirently received from Beta irradiation used to destroy surface micro- organisms. Damage to the affected carrots appeared as dried blackened “3311a on one side of the roots. Only a few roots showed damage and it Kfi Euspected that they were not turned between irradiation treatments so ‘0’“ they received a double exposure. in examination of cross sections through the blackened area of the roots indicated that fluorescence was present in apparently sound tissue, centripetally located to the injury appearing as typical bitterness fluorescence spots. 148 TABLE 9 gm RATINCB AND FDNGAL DIssAas INFECTION or as) com: CHAN'rsNAI ”3° ‘33 118 mm BI Hiavss'r AND POST-HARVEST TREATMENTS, 1956-57 R Bitterness Ratings“) Visible Disease Infection Spectro- Roots Hm Phatometric Roots with Post‘at and Height f6; Fluorescen s with airface Total Trea 1‘Iarvest mp peak 2 1-5 Ratinng Rot Mold Infected flnts Jan. Mar Jan. Mar. 1 S L "awaited “2‘81: Harvested .10 .22 1.0 1.0 32.6 16.5 78.1 a trt—le Harvested .13 .16 1.0 1.0 26.2 33.8 60.0 and Harvested .08 .17 1.0 1.0 13.3 10.7 211.0 Igr‘diated °ugh Harvested .13 .16 1.5 1.600 311.3 30.6 611.9 Gentle Harvested .1). .13 1.0 1.60:) 35.2 18.0 53.2 “find harvested .28 .12 1.2 1.6 9.2 2.9 12.1 washed Rough Harvested .19 .13 1.2 1.0 15.0 1.3.7 58.? Gentle Harvested .18 .15 1.0 1.0 26.1 13.7 39.8 art'ad Harvested .12 .11: 1.0 1.0 1.2 11.6 5.8 \ (1) Bitterness evaluations at harvest by taste test, spectrophotometric and fluorescence analyses all indicated a lack of bitterness. (2) Height of 265 my peak values of carrots below 0.75 indicate an absence of bitterness to taste. (3 ) 1- non-bitter, non-fluorescent; 5- exceedingly bitter and fluorescent. (it) Several roots had black sides suggesting irradiation damage. These were the only roots with fluorescence in their respective lots and the fluorescence was concentrated on the black sides. Patent 01' 120095 Visibly Infected A R- Rough Harvesting I? by Machine g G— Gentle Harvesting by Machine 70 7 7 H- Hand Harvest 5. r i 60 i F F‘— 3' r of 5: . a) . 0 4‘3 Sc) 1. é: 03 L10 -L / 30 .. . ,I’ / V 3" / . . / . 20-7. +0) ,7], I} 03 , /g. / r 10.1- / I. / r// 7 . 0 ,2 / /',, 4 ‘ y/ // , R G H R 0 H R G H Unwashed Unwashed Washed Irradiated Figure )4. Percent of carrot Roots Infected by Rot and Surface Mold as Influenced by Harvest and Post-Harvest Treatments, after Five Months of Cold Storage, 1956-57. 149 cm Di cmonsn AND MODIFIED STORAE AmOSPHERE STUDIES In the fall of 1955 a 1.0 ton lot of bitter carrots which had tem- porn->113, been stored with Bartlett pears was examined. Ripening Bartlett pears have a high rate of respiration and are also a prolific source of “ht-3.1. muons, particularly ethylene (Hansen and Christensen, 1939). 1711.3 ‘tould tend to cause a build up of 002 and ethylene and a reduction of 02 within a self contained storage atmosphere. The carrots had the appsfirance and darkness of color of roots which might have been stored to:- Six months. Three weeks previously, however, these carrots were of n°“‘~‘bitter, acceptable processing quality. This observation indicated that bitterness might have a commotion "1th products given off by fruits and led into the following series of experiments. Storage Atmosphere Study 1955-56 1‘et-hods and Materials To study these factors in relation to bitterness, six treatments Were devised to evaluate the effects of controlled atmospheres with high (:02 and low 02 contents, and the influence of apple emanations. Six S-kilogram samples of short type Chantenay carrots were selected won a planting seeded July 6, 1955 and harvested on October 20. They we placed in large-mouth Bogallon jars designed for respiration stxxiies 51 and that! held in a 32‘ F. apple storage. The treatments were as follows: A - Sealed B - Controlled atmosphere - 3% 002 210$ 02 c - Controlled atmosphere - 71 002 :51 02 D - Apple ananations in a 220 Nil/minute air stream E - Air - 220 ml/minute stream F - Open to storage room atmosphere Treatment A was sealed so that ready exchange of 002 and 02 would act QQ cur. The controlled atmospheres in treatments B and C were main- W by flushing with N2 gas to lower 002 concentration and adding cc‘l‘pt‘essed air to raise 02 to the desired levels. In treatment D, pic- M in Figure 5, the air flow was regulated by adjusting the pressure on ‘n air flow manometer board. Jonathan apples were placed in a gallon 3" ”which was sealed, except for air tubes, and kept in a laboratory at ‘b‘hl‘t 75’ F. to speed respiration ani emanation rates. in air tube was D‘BSed from this jar into the cold room to discharge in a humidifying 3‘8 which also served to cool the air as it passed into the carrot cham- ber- Apples were replaced at the end of three weeks. The air treatment E ’ Or control, was similarly set up except for the elimination of the ‘Pple chamber. The final treatment F was left open to the atmosphere of the storage room vhich contained uncovered apples. The carrots were placed under the various treatments on October 29 an all treatments were checked daily for 002 and 02 content with a \W-Orsat gas analyzer. December 9, after a period of 113 days, the flax-rots were evaluated for bitterness. 52 \\\\\\ :\\\\\ Chamber Insulated 75‘? wall 1’1 We 5. Experimental setup for Apple finanation Treatment for Induc- ing Bitterness in Carrots. 53 Random samples were washed and peeled and portions of five individu- 111 ”Of-3 vars checked for soluble solids with a hand refractometer. The We material from the samples was then ground and part evaluated by a taste panel, the remainder dried and analysed spectrophotometrically. 0th” anValuations incltxled rating of amount of mold, darkening of the p ”I hid loss of weight during storage. “19 remaining unsmnpled carrots were left in open jars which were transferred to a carrot storage room at about 35' F. A final bitterness "uk‘l‘alon was made July 6 on stored carrots from the apple emanation and air- treatments. the test the practicability of a quick test for bitterness suscepti- . bill» by combined storage of apples and carrots, and experiment was set up ‘h-a run in December 1955. Six 1500~gram lots of carrots from the amfi aouroe as above were sealed with two crisp, ripe Jonathan apples in 8.quthid polyethylme bags at 32, 36 and 75’ F. Evaluations were made at the find of one and two weeks. Ethylene gas was used in an experiment started in February 1956, in ‘tiother attempt to induce bitterness development. Two 5-pom1d lots or hedimn to small roots, from the July 6 plantings used above, were I)l‘ued in wide-mouth 3-gallon respiration jars, and a third lot placed in a paper bag and stored at 32‘ F. as a check. To one jar, ethylene “‘3 introduced under pressure through a rubber tube to the bottom of “‘9 jar with air vented out through a glass tube at the top. The daily b“QQedure was to first flush the atmosphere of both jars for five min- thQ with compressed air, then to apply ethylene to one jar for 30 sec- QQ‘Q as at five pounds pressure. Ethylene and ventilation treatments were (i x§continusd February 26. Results and Discussion Where apple volatiles were added to a constant air stream passing “Wash an container of carrots, bitterness, which was rated by a taste "”1 as intense, developed within a six week period (Table 10). A 1°“? level of bitterness occurred in the container open to the apple Storage atmosphere but none of the other treatments in the controlled and mitied atmosphere shady caused the development of a detectable ”it”? :lavor. SpectrOphotometric analysis on the dried tissues in Jm‘i‘y substantiated these evaluations. A further evaluation in July °n 1"“‘Q-tathents D and E gave the same results by both the taste test and ”poo “Qphotometric analysis. N o bitterness appeared in carrots from any treatment which developed m” <>ne percent of 002, suggesting the possibility that added 002 may inhibit the development of bitterness. However, where apples or pears and earrots have been stored together, the C02 content would be expected to b9 above normal,- yet bitterness has appeared under such circumstances. 1““ , the inhibition of bittemess by 002 accmmlation is unlikely. In color of peel, a rough measure of freshness and quality, the e‘:"1‘ots exposed to apple manations became darker than those under a conatant air flow alone or controlled atmospheres. This condition was 811“ilar to that observed the previous September where carrots became bitter and dark in color in a pear storage. The brightest color of peel at“ the sweetest carrots were found in the two controlled atmosphere tbgfi‘tments where 002 content was above normal while darkest color and ohfist sweetness and soluble solids values occurred in the carrots a 1; 1‘ed under anaerobic conditions. SS TABIE lo CHAIM: rAcTOHs IN nicer mm CHANTBHY cARHOTs AND Penman CARBON ”1011138 AND onw AmospHms COMPOSITION As AFFECTED BY STORAGE Amos- Prm MNTS Storage Atmosphere Treatments mty Factors and A a c D E F Pliers compositionu) Sealed 35002 75002 Apple Air Open 10502 5‘02 Duane- --\ tions Bit‘e‘btzaess: 1-5 taste rating 1.0 1.0 1.0 5.0 1.0 3.0 3 Whig”, 1-6 rating 6(2) 1 l masked 3 11 $01le Solids: z 6.08 7.36 7.66 7.81. 7.60 6.78 “1°" of Peel: 1-6 ranking 6 1 2 5 3 h “amt Loss: S 0.50 0.15 0.25 0.25 0.25 1.00 "a“ S 1-5 rating 1 2 2 5 3 3 c °2 content. x 80 3 7 0.5 M. 0.3 02 c Ontent: i O 10 5 19-20 19-20 20-21 \ (l) The lowest value in the rating and ranking scales indicates the most 1‘ avorable characteristic. (2) Flavor and aroma of this sample were that of fermented, dead tissue. (3) Sweetness was present but could not be rated by the panel of six. A low incidence of an unidentified mold developed on the surface of carnots in all except the sealed treatment (A). The highest mold rating was in treatment. D which also developed the highest intensity of bitten-.33.. Since mold ratings for treatments E and F were both 3, and ““5111st ratings for these treatments were 1 and 3, respectively, it 1‘ thou-glut that the appearance of mold was not related to bitterness develoment. The concentrated apple emanations in treatment I) may have m‘ted mold growth giving it the highest mold rating. 1iifi’snlts of the test of combined storage of Jonathan apples and car- rots 111 polyethylene bags at 32, 36 and 75‘ I". (Table 11) in December 1955’ show that bitterness was not induced by any of the treatments tried ‘ The only marked flavor changes occurred in carrots from the 75' F' t1‘Qatment where nearly a quarter of the roots developed a fermented Ham)? in one week and all were slightly soured in two weeks. Several possibilities are suggested for the lack of bitterness de- mopluent although the carrots and apples used were from the same sources ‘ ‘3 in the previous tests. 1. By December, the precursor of the bitter principle in the carrot had been dissipated or converted to a more stable QQII‘DO‘Lmd so that bitterness induction was impossible. 2. Apples effect- ive in causing bitterness in Novanber could have passed their peak pro- duction of ethylene and therefore were ineffective. 3. The reaction to develop bitterness may take more than the two weeks allowed in this test. 1‘. within the polyethylene bags the atmospheres tended in the direction 01‘ high 602 content and migit be responsible for an inhibition of hi tterness. TABLE 1.]. 57 INFLUHICE (N CARI-101‘s 0F COMBINED ST C8 WITH APPLES AT 32, 36 and 75° F. 1 Mm Storage Temperature 32' F. 36° F. 75° 1'. One Week nun]? x i of Roots % of Roots S of Roots Nam or Swaet 14.1.7 63.? 116.2 05:01:13 50.0 27.3 23.1 ’1‘ 8.3 ) 9.0 23.1 (earthy (earthy) (fermen 3“th 0.0 0.0 73‘22) (slightl 3:3:th Slight Slight Moderate( ; mR\tingU-l) l 1 2 None apparent Turpentins- Apple-NH3-like some like ”fighaction Slight Slight Heavy( 5) Condition Hard, crisp Fairly firm Fairly firm Sldn soft Skin soft 17%“ 1‘ 3 Two Weeks 31% or Sweet 511.5 50.0 0.0 o I‘mg 36.21 (40.0 0.0 r 9.1 10.0 100.0 E (fl 1'.) (musty) (slightly sour) shifter 0.0 . 0.0 Outing Slight 811311. No record 11°16 Rating“) 1 2 M Slightmuaty. Sum-apple Shu'p-NH3-1ike 1: carroty ‘gndensatim Slight Very slight Moderate 91a Condition Firm Firm Soft u\ C ) Evaluations made by Dr. James A. Cook. 2) Since there was a low incidence of bittemess in the carrot popula- tion used for this experiment, the appearance of this specimen is (3) discounted. (la Ihese carrots also seemed more woody than any of the others. ) The mold intensity rating is based on a 1 to 5 scale with 1- no mold (5) find 5- severe rotting. The bag was also puffed indicating anaerobic respiration. 58 Emylene treated carrots tasted by a 17 member panel in February "are rated at 1.8, or questionably bitter, while carrots with daily Ventilation were rated 2. 3, or slightly bitter. The control lot held in storage was rated at 1.0, or non-bitter. when the three lots were “fled again by the writer at the end of March, no bitterness could be “meted in any of the three lots. Extensive surface pitting and dis- cdoratim, indicating injury, occurred to the ethylene treated carrots. 59 Storage Atmosphere Study 1956-57 The previous studies suggest that carrots from the 1955 crop might no 1021.893 be induced to become bitter after six to eiglt weeks of storage. Thar9130:”, the determination of the effect of ethylene on bitterness de- nun‘lent was delayed until a new crop of carrots was available. Tine objectives of this study were: l- to determine whether ethylene might, be a causal factor in bitterness development; 2- to repeat the ob- “WE-c1 effects of apple summations in inducing bitterness; 3- to deter- min 5 whether ventilation could be effective in inhibiting or preventing th ° QQvelopment of bitterness; l.- to detemine whether harvest injuries inf 3“ch bitterness susceptibility; S— to compare varieties for bitter- neaa response; and 6— to check the tendency for bitterness development in “Qatari! packaged emote. HQMs and Materials Long type Chantenay and Gold Pak carrots were seeded June 11, 1956 on Imack and dug by hand and machine on October 11. Those dug by hand were sorted carefully to eliminate any injured, diseased, or off type 13.30“ and were handled carefully to avoid further injury. The machine due roots were bruised intentionally in the harvesting process and were an‘ted to remove uninjured, diseased, or off type roots. The Gold Pak misty was also machine harvested but in the usual manner so that the mount of injury was considerably less than with the machine harvested 0'}wa variety. The harvested roots were held for seven days in a common storage, the” Stored in a refrigerated root crop storage at 35’ F. until placed ma” e~=Itr>erimental conditions December 17. Mil—nerd soil grown Colorado Imperator carrots were obtained from a ““1 store in mid-November. sprouts and dry leaf tissues attached to the cro‘m indicated that these carrots had been stored previously. The basic desigl of the experiment included four primary treatments: aI’D-13 emanation, ethylene gas, air or ventilation, and an open control, each e<>nsisting of: uninjured Chantenay, injured Chantenay, machine hang Q‘fied Gold Pak, and the western sample from the market. The experi- meat.“ set up was similar to that in the 1955 study “1th treatments rep:Lz‘lLQated twice with a total of eight jars. The purpose of the experiment was to store carrots in an atmOSphere corthfining about 100 ppm of ethylene which was based on a calculated eats‘l‘late of the ethylene concentration in the pear storage where bitter- ness had appeared in 1955. Following two weeks of unsuccessful attempts to 131‘ oduce a continuous flow of an atmosphere of this concentratim “hi-ch could be readily maintained and adjusted, it was decided to use an intermittent treatment. Ethylene was introduced to the constant air 1.1% at the rate of 3% ml per minute for five minutes each day. Delicious apples were used to test the effect of emanations which ‘Pe passed through the system by a controlled air flow of about 220 ml "91‘ minute. 61 Results and Discussion Bitterness was evaluated by fluorescence, taste, and spectrophoto- metric tests. The bulk lots of Chantenay and Gold Pak were evaluated one week previous to starting the treatments and were rated as non- bitter. The western carrots were not sampled prior to the test. The carrots were placed under treatment from December 17 to January 9 when five roots from each lot were removed and evaluated for bitter- ness; the remainder were left under treatment until February 5 when all jars were opened and stored in place. A second evaluation for bitter- ness was made February 9 on the first replication and March 18 on the second. Results in Table 12 show a slightly lower bitterness value for car- rots in the open control. This was due almost entirely to lower values for the Colorado Imperator. Circulation of air in the storage room and in the open containers was probably greater than circulation through the constant air flow treatments, thus effecting a more rapid dissipation of the bitter principle. Ratings by all three evaluation techniques showed a tendency that had been previously noted for a decrease in bitterness from the second to the third evaluation. A emery of the influence of variety and handling sub-treatments, Table 13, shows the locally grown Chantenay and Gold Pak carrots to be essentially free from bitterness while Imperator, grown and stored in Colorado were moderately bitter. Same individual roots from this lot were strongly bitter. SpectrOphotometric values for the harvest injured Chantenay carrots were slightly lower than for those carefully harvested by hand. This suggests that mechanical injury may reduce the amount of & O O o e NOOOO VI mQLor \n as s s a a a 0 \005—0 Ethylene S.T.C.-Hand 30 T .C a “Row Gdkiihk Hummlmu. “um” .38 .23 031‘ .20 TEElZ .w .W 2.91 1.h6 HHWMEWSNflMAMWMflEONMHRMfiDflHNMMINmmINS or CARROTS, 1956-57 Bitterness Ratingsfl) Storage ‘Variety and Spectro- Atmosphere Handling Taste Fluorescence photometric Trutment Treatment 1-5 Rating l-S Rating Ht 265 mu peak 2nd 3nd 2nd 3rd 2nd 3nd Apple S.T.C.-Hand .21 .26 Emanations S.T.C.4Rough .28 .11 001d Phk .39 .28 Western Imper. 2.68 1.81 Averages .89 .62 1.01 .5h 1“ ". SOVUJIO wrbOO U1 0 0 Air S.T.C.-Hand S.T.0.€Rough mmrw ‘Uestern Imper. Man” .U .% .28 .13 .U . 2.56 1.8h .92 .63 mmboo O a s wwooo N geese games genes 0 O O 0 80000 COntrOI Se T 00 s -11“ (0pm) 8. Too 0 .Rough Gold Pak manann hangs .23 .22 .22 .18 .% .N 1.11 1.28 .M .m Sboob . H 00 e a WU‘OOO m gbboo . HNHHH HMHHH ywHHy HwHHH HwHHH HNHHH rNHHH HNHHH HHHHH HHHHy HNHPH HNHHH HNHHH HuHHy PNHHH HNHHH (1) Taste, fluorescence and Spectrophotometric evaluations on Short Type Chantenay and Gold Pak on November 2 indicated no bitterness to be present. The western sample was not tested at that time. The second and third organoleptic tests were made by panels of 11 and 1h individuals respectively. TABIE 13 63 VARIETY AND HANDLING mm INFLUEME 0N emu-mass RATINGS 0? 3m CABROTS, 1956-57 Bitterness Ratingsu) Variety and Spectro- Hamiling Taste Fluorescence photometric Treatment l-S Rating l-S Rating Ht 265 mp peak 2nd 3rd 2nd 3rd 2nd 3rd Serge. Rm 101 1.0 1.0 1.1 .31 .25 8.120. Rough 1.2 1.1 1.1 1.0 .28 .16 001d Pak 1.3 1.1 1.0 1.0 .37 .28 Western Imperator 2. 8 1.8 2.7 2.6 2.32 1.60 (1) Taste, fluorescence and spectrophotometric evaluations on mart Type Chantenay and Gold Pak on November 2 indicated no bitterness to be present. The western sample was not tested at that time. The second and third organoleptic tests were made by panels of ll and 114 individuals respectively. .M‘ 6h potential bitterness by allowing more ready escape, removal, or conver- sion of the precursor of the bitter principle, assuming it to be present in the carrot at the time of harvest. .A possibility for the absence of induced bitterness may be due to the fact that the study was initiated so late in the storage life of the roots that the precursor of the bitter principle had been lost by volatilisation. However, short type Chantenay roots placed in an apple storage on Novanber 10, shortly after harvest, developed a low intensity of bitterness by taste, fluorescence, and spectrophotometric analysis in three weeks. The effect of season was also a factor, in that there was no bitter- ness reported in the commercial crop in.Michigan,for'l956. Two factors in the weather could have affected potential bitterness. First, the growing season was exceptionally cool through August with a possible deficiency in production of the precursor. Second, the harvest and early storage season was unseasonably warm and comparatively dry, pos- sibly resulting in field volatilization or reorganization of the pre- cursor of bitterness. 65 CHAPTER I MINOR ELEMENT NUTRITICN AS RELATED TO BITTERNESS IN CARROTS Banner (19116) found that carrots were moderately responsive to copper fertilization on copper deficient muck soil, and below a runimum level of copper, carotene and sugar contents of the roots were low. Manganese deficiency in carrots is a recognized problem in muck soils with symptoms appearing as interveinal chlorosis and yellowing of the younger leaves. Poliar sprays with 11:60:, at 2 and 11 pounds per acre quickly and effectively alleviate the symptoms. Manganese toxicity symptoms on many plants appear as iron defi- ciency symptoms and can be helped by foliar applications of iron salts or iron chelates. Methods and Materials Three studies to determine whether or not minor element deficiencies were related to bitterness were carried out in 1955 and 1956. Copper deficient muck soil plots treated with O, 6.25, 12.5, 25 and 50 pounds of copper per acre were planted with Red Core Chantenay carrots. At harvest, samples were obtained and stored in 50 pound onion bags at 32’ 1". until January 12, 1956 when a taste panel evaluation was made. At about this same time two samples of carrots, one bitter, the other non-bitter, were obtained from a comercial carrot storage where they had been held for the same length of time. The bitter sample had been grown on an acid muck and the non-bitter one had come from an alka- line muck in which marl was present. These samples were analyzed for minerals to reveal differences which might be associated with the de- veloment of bitterness. In a subsequent study, Long Type Chantenay carrots were seeded on June 16, 1956 in a muck field which was heavily limed to pH 6.2 in 1951;. Manganese deficiency symptoms had regularly appeared on several crops previous to 1956. The seeding was bani fertilised with 800 pounds per acre of 5-10-20. ‘ Manganese deficiency appeared in the carrot seedlings and was cor- rected in certain plots by 110801, spray treatments at both 3 and 6 pounds of elemental manganese to the acre. A foliar application of F030), at 11.2 pounds of iron per acre resulted in foliage damage wherein "burning" appeared. Growth of new leaves masked the damage within two weeks. The crop was harvested October 11, and placed in refrigerated storage at 32‘ l". on October 18. Bitterness was evaluated at harvest by tasting, and again Novuber 1, December 12 and January 27 by tasting, fluorescence and spectrophotometric analysis. 67 Results and, Discussion Carrots from all plots of the copper experiments were tasted at harvest and found free of bitterness. After three months of cold stor- age, a taste panel again rated all lots as non-bitter. Spectrophoto- metric evaluations made on the same samples the following July showed no essential differences. Mineral analyses of bitter and non-bitter carrots from a comercial cold storage room showed marked differences in content of manganese, iron and sodium (Table 111). The sample from the alkaline muck soil which was not bitter was higher in manganese but lower in iron and sodium than the bitter sample from the acid muck soil. Although these differences could not be considered as evidence that deficiencies of any of the elements were directly related to bitterness, the manganese-iron inversion sug- gested as promising further investigation on the influence of manganese nutrition. . In the subsequent evaluation of foliar treatments with manganese and iron salts, no consistent differences were found among spectrophoto- metric bitterness ratings of the stored carrots. Under the conditions of these experiments, deficiencies of neither copper nor manganese caused bitterness to develop in carrots. As already pointed out, this does not necessarily mean that a shortage of these elements in carrot nutrition will prevent bitterness. Some carrots from the same farm, but not in the experiment, although stored in the same refrigerated room as those in the copper study, be- came bitter indicating that storage conditions develOped which pranoted 68 TABIEIh 111mm. ANALYSIS 0? cmo'rs mow mo swacss, 1955 01209 (it dry weight) Blanents Non-bitter“) Bitter(2) 0a 0.25 0.25 Cu 0.0010 0.0011: Fe 0.0083 0.0112 x mm h.5h Mn 0.0151 ' 0.0036 Na 0.10 0.25 P 0.h6 0.39 Ga 0.10 0.09 y (1) Parent an alkaline muck soil. (2) F’x'om an acid muck soil. 69 bitterness. As no bitterness appeared in the carrots from the copper study, copper had no apparent effect on bitterness. The manganese and iron study was conducted in 1956, a year in which no bitterness was reported in commercial carrots in Michigan. 'Ihis indi- cated that the trend of the weather for the growing season was probably 01' greater importance than nutritional factors, and that regardless of the nutritional situation, the other coniitions required for bitterness development were not present. The possibility that a manganese deficiency may allow bitterness development under other seasonal conditions cannot be ruled out. 70 CHAPTER XI FLUORESCENCE STUDIES The possibility that the metabolic changes associated with the formation of the bitter principle in carrots might be related to the development of fluorescent substances in the roots (Zechmeister and Polgar, l9hh) led to an examination of tissues from bitter and non- bitter carrots under ultra violet light. Yellowish green fluorescent spots were concentrated in the phloem.tissue of bitter roots, Figure 6, but were not discernable in normal carrots. The area of fluorescence in the phloem was the site of the most intense bitterness by organo- leptic and spectr0photometric tests. is fluorescence appeared to have considerable promise as a fairly accurate rapid means of evaluating bitterness and perhaps aid in the interpretation of some aspects of the physiological response associated With the development of a bitter metabolite, its relation to bitterness was investigated. Methods and Materials After discovering that bitter carrots fluoresced under ultra violet excitation from a 25373.nineralight source, a correlation test was made With 6h muck grown short type Chantenay carrots which ranged from.non- bitter to exceedingly bitter. The size, shape, height of crown, kind and extent of injury, amount of mold present, relative proportion of 71 Figure 6. location of Fluorescence a Bitter Red Core Chutney Carrot we: mat-Mon of 2537 mm wont. nest. 72 xylem and phloem, shoulder color and surface fluorescence were measured and tested statistically for correlation with internal fluorescence and bitterness intensity evaluated by taste test. A,center slice removed from.each carrot was rated in a dark room under a 2537i ultra violet light for intensity of fluorescence according to a l to S, non-fluores- cent to highly Moment scale. An additional slice was removed from each carrot, peeled and organoleptically rated by the writer for inten- sity of bitterness using again a l to S, nonabitter to exceedingly bitter scale. A second correlation study was conducted on 10 roots from the same source evaluated for the above characteristics, but based upon longi- tudinal sections with fluorescence ratings upon five arbitrarily selected concentric levels of the phloem and xylem. Bitterness ratings were made by tasting perings from each of the fluorescence levels. Correlation values were determined upon summations of fluorescence and bitterness ratings for individual roots. Imprints of bitter carrots upon whatman #1 filter paper showed fluorescence under ultra violet excitation and could be used to quickly evaluate fluorescence without interference from reflections or the orange pigments of the carrots and provide a semi-permanent record of its intensity and distribution. Photographs were taken of patterns of fluorescence produced by'ultra violet sources of 2537 and 36603. Effects of potassium hydroxide, hydrochloric acid, sulfuric acid, when applied to bitter fluorescent carrots were observed. Photomicrographs were taken of hand sections of fresh bitter car- rots with white transmitted light and under 2537: ultra violet incident light showing the lecatien and extent ef flueresceat spots. 73 Results and Discussion Results of the correlation study of sixty-four roots (Appendix Table 1) showed the correlation coefficient between fluorescence of transverse sections and bitterness by taste test both rated on a l to 5 basis to be a non-significant 0.28. By sectioning a number of other carrots longitudinally and transversely, it was noted that fluorescence was not always uniformly distributed at a given transverse level. By sectioning roots longitudinally, more representative sections were obtained for fluorescence evaluation. Other observations (Appendix Table 2) from this study included a positive correlation between both injury and mold, and external fluores- cence but no correlation between mold or injury and bitterness. Roots with large cores and/or thin cortices averaged 3.9 on the bitterness scale by taste while roots with small cores and/0r thick cortices aver- aged 2.3 in bitterness. The large cored roots also tended to have much taller growing points which suggested an earlier initiation of bolting. Color of xylem or phloem and color of shoulder had no apparent effect upon bitterness; however, carrots with green shoulders tended to have a slightly higher than average external and a lower internal fluorescence rating. Carrots with purple shoulders showed no correlation with either internal or external fluorescence. There was no relationship between carrot diameter or length and bitterness. A small number of roots were bitter to taste but exhibited little or no fluorescence indicating the presence of more than one bitter principle. 7h Data from the second fluorescence4bitterness correlation study, (Appendix Table 3), showed an improvement in correlation coefficient from 0.28, when using transverse sections, to 0.6h when using longitudinal sections. Bitterness and fluorescence were shown to be located in the same morphological tissue, the phloem, and were both lacking in the core or xylem. .A tendency towards increasing bitterness and fluorescence from the outside to the cambial layer was recorded. No correlation was present between root size, and bitterness or fluorescence in either this or the previous study'(Appendix Table h). with molds, a correlation of 0.h5 was found with surface injuries, 0.39 with surface fluorescence, but only' 0.05 with bitterness. A correlation coefficient of -0.h6 was found between surface injury and bitterness, indicating the greater the injury, the less the bitterness. By'pressing cut carrots onto sheets of soft filter paper, Whatman #1, semi-permanent records (some of which have lasted over a year) of fluorescence intensity and distribution were obtained. A photograph of carrot imprints representing the five fluorescence intensity levels used in.raiing bitterness is shown in Figure 7. The root section at the upper left shows no fluorescence indicating freedom from bitterness, while the highly fluorescent root section at the lower right indicates an.exceed- ingly bitter flavor. Differences in fluorescence pattern produced by ultra violet sources of 2537 and 36601 are shown in Figure 8. Figure 7. Typical Plumes Nun-Bitter, 13- Just Detectebly Bitter, 3- Hoderate once Scale of 1- !: Bitter, heeedhnly Bitter t light new at. - may Bitter, and 5- cerrots under Ultra Viola 2537f. inal Section Filter aper arrot under (A) 2537 and 8. fluorescence Variations of a Longitud of a Bitter Red Core Chantenay 0 U (3; Ultra Violet Excitation. 77 Some of the yellow-green fluorescent spots in the phloem of bitter carrots under excitation of 25371 ultra violet light are missing under excitation at 36601 which suggests the presence of at least two fluor- escent compounds. A general over-all fluorescence is more common at 3660; and appears more intense in senescent carrots stored for a long to... Application of five percent potassium.hydroxide by drops or as a fine mist over cut bitter carrots enhanced fluorescence under 2537; ultra violet presumably'much in the same manner as Goodwin and Kavanagh (1950, 1952) found in their pH studies of coumarin compounds. Applicap tion of dilute hydrochloric acid and sulfuric acid reduced fluorescence materially; Potassium hydroxide, hexane, and water applied to fluores- cent spots caused thsm to spread. Photomicrographs, Figures 9A and 9B show fluorescent areas of a fresh, hand sectioned bitter carrot under incident ultra violet and transmitted white light. Fluorescence appeared to be concentrated in groups of cells thought to be oil glands and in areas beginning to break down. flute . manual section of a timeout carrot undo:- Ultra Violet to light (a). e E‘. i i a 79 CHAPTER III ENERAL DISCUSSION Bitterness in carrot roots depreciates the edible quality by caus- ing an unexpected biting flavor which persists through cooking and pro- cessing. The bitter principle has been morphologically located in the phloem and its intensity evaluated by small spots of yellowish-green fluores- cence under excitation frat short wave 253?: ultra violet light. In the discussion that follows, an attempt will be made to inter- relate the role of heredity and pertinent environmental factors during growth and storage to the development and possible control of bitterness. Genetic Relationships Differences among commercial varieties and strains of carrots in their susceptibility to bitterness was suggested by Atkin, 1956, but was not clearly supported by his experiments. In this study, using carrots of the same chronological age, the early maturing fresh market varieties Nantes and Touchon developed the most bitterness, while later maturing Danvers, Imperator, Gold Spike and Gold Pak showed the least bitterness. The intermediate Red Gore Chantenay strains varied in bitterness, but tended to fall closer in response to that of Nantes than Imperator. A similar trend in maturity was noted by Dickson (1957) in studies of temperature induced bolting response. 80 Earliness of maturity was thought by Bangs 3133;. (1955) to be due to a rapid attainment of the maximum contents of carotenoide and dry' matter and the typical mature shape for the variety. The precursor of the bitter principle may be a usual metabolic intermediary compound, accumulating near the end of the first season of growth. Briefly, the earlier the maturity of the carrot, the longer the synthesis of the precursor could continue if the plants remain in the field. In progeny tests of bitter and nonpbitter selections from Red Gore Chantenay, Yamaguchi'gt_al. (1955) found that bitter parents produced strongly flavored progeny and that mild flavored progeny were produced from non-bitter parents. In this work, in similar tests conducted from January 1955 through January 1957, no differences in progeny evaluations for bitterness were observed. However, this is not in contradiction of Immaguchi's observation since the over-all level of bitterness in the 1956 Michigan crOp was negligible, probably due to unfavorable weather for synthesis of the bitterness precursor. The development of non-bitter, strongly flavored progeny from bitter parents suggests that the precursor may be one of the oily flavoring components of carrots. Environmental Factors 5011 £223 Carrots for processing and fresh market are grown on many soil types, but most of the carrots in the northeastern states are produced on muck 80113. Atkin (1956) indicated from limited tests that carrots grown on muck soils were generally more bitter than those from upland loams. In his study, carrots from sandy soils showed a greater variation in bitterness than those from either mueks or loams but were lower in bitter- ness than those from.mucks. Analyses by Lipton.(l955) showed greater total sugars and carotene content in upland than in muck grown carrots. Newcombe and Alderman (191m pointed to a greater oxidative rancidity of dehydrated carrots from muck than from upland 80118., Results in this investigation supplement Lipton's observation that upland carrots tended to be of higher quality; yet in bitterness response, soil types appeared to be of little consequence. In one series of harvests, the most bitter carrots came from an upland trial and in another, from.muck soils. - Nutrition In a nutritional study related to bitterness in carrots, Atkin and Sayre (1955) reported that the level of bitterness in muck grown Red Core Chantenay carrots was reduced by an application of 50 or 100 pounds per acre of fritted minor elements. In this study, copper and manganese deficient carrots developed no difference in bitterness as compared with carrots amply supplied with these elements, indicating that deficiencies of neither are the direct cause of bitterness. However, the possible role of minor elements in the enzymatic activities resulting in the development of bitterness warrant further investigation. ‘EgzsiOIOgical Age Carrots seeded at varying times were shown by Atkin (1956) to dif- fer but little in bitterness response, and he found that early harvest, regardless of seeding date, was most influential in effecting bitterness occurrence. In this study, May 27 seeded carrots developed considerably more bitterness in storage than carrots seeded July 6 indicating the 82 possibility that at a certain stage of physiological maturity, the nets- belie processes in the root may be altered in some manner by harvesting and storage which results in the development of bitter products. Early and late seeded short type Chantenay were similar in bitterness response, while early seeded Chanticler was considerably more bitter. Late seeded Chanticler was decidedly less bitter than short type Chantenay sown at the same time. This suggested that short type Chantenay tended to mature earlier than Chanticler, thus allowing the late Chantenay seeding to catch up with the earlier one. Late seeded Chanticler in addition to being least bitter, accmnulated the most total sugars by the time of harvest and lost the most total sugars during storage, suggesting that it had the highest metabolic rate of any carrots in the test. Date of harvest was not directly involved in this study; however, it was noted both in 1951; and 1955 that the highest intensities of bitterness occurred in the earliest harvested carrots. As found both by Atkin (1956), and in this work, harvests of processing carrots as late as November, resulted in little or no bitterness. From a commercial point of view, September harvested carrots placed in cold storage have provided the largest total source of bitterness. Thus, a delay in harvest of carrots to be stored for a prolonged period appears Justified to reduce bitterness occurrence, despite a potential increase in suscepti- bility due to carrot maturity. To interpret this apparent divergence, over-maturity versus early harvest as a cause of bitterness, the evalu- ation of storage factors should be considered. 83 Seasonal Influence As noted by Ianaguchi 3331. (1955) marked differences in bitterness of comerical carrots from year to year are probably affected by weather. Koch (1997) of the Gerber Products Company stated that there has been no bitterness in their carrots in California since 1994. In 1956 the com- mercial carrot crop in Michigan exhibited an almost complete lack of bitterness which appeared to be related to a deficiency in the typical oily carrot flavor. It may not be a coincidence that in 1956 the peach crop in Central Michigan was also deficient in typical peach flavor and that the central Visconsin McIntosh apples showed the same defect (Roberts, 1957). A study by Biggs and Leopold (19514) on the influence of temperature on synthesis of menthol and mentholated esters in pepper- mint - essential oils similar to some of those in the carrot -- revealed that high temperatures (80‘ F. day, 70’ F. night) resulted in greater yields than lower temperatures. Although rainfall was near the long term average in East Lansing in 1956, temperatures during the growing season were considerably below nor- mal through August. During September, October and early November, tem- peratures were above average and precipitation was light. Even though conclusive evidence is lacking, it is suggested that the precursor of the bitter principle may be an essential oil or of hydrocarbon nature and is favored in its formation by comparatively high growing tempera- tures . 8h Post-Harvest and Storage Factors In uries Reference to injuries sustained by carrots in mechanical harvesting in relation to bitterness is missing from the literature. Observations in carrot harvesting by machine in 195:4, 1955 and 1956 showed that early planted, over-mature, and large roots tended to be bruised, scuffed, cut and cracked. Stuiies in 1956 on controlled injuries and correlation of factors in the fluorescence experiments indicated that injury tended to reduce bitterness. This may possibly be due to an acceleration of res- piration or other reactions which affected the precursor, or to the effect of increased volatilization of precursor or bitter principle from the injured root. §c_1erotinia rot infection was intensified by injury and affected storability, but not bitterness. Storage Atkin (1956) showed that carrots kept in refrigerated storage be- came bitter, while carrots held in air cooled cox-non storage did not. Among storages there are differences in temperatures, hmnidities, and atmospheric composition. Carrots in common storage reach a desirable holding temperature more slowly than in cold storages held at or near 32’ F., and consequently higher respiratory and disease losses occur. Common storages show no marked deviation from normal air, while cold storages involve a more or less self-contained atmosphere which permits depletion of oxygen and accumulation of carbon dioxide and volatile materials . 85 Controlled atmosphere tests in this study with depleted oxygen and accumulated carbon dioxide did not induce bitterness. At the end of six weeks, edible root quality was actually superior under controlled atmos- pheres. Anaerobic conditions resulted in a fermented flavor and odor, but no bitterness. Mature carrots stored in anaespheres containing pear or apple eme- nations became bitter in a few weeks. In atmospheres supplemented with apple emanations or ethylene, long stored and immature carrots did not become bitter, but darkened and developed surface pitting similar to that of fresh, mature carrots stored with fruit. Bitterness development in carrots appeared in a few weeks, followed by gradual depletion during storage. On the basis of this study, it is suggested that carrots for stor- age be seeded late to provide imature roots at harvest and harvested as late as possible to escape storage conditions condusive to bitterness development. Less bitterness should occur in late than in early matur- ing varieties when harvested at the same time. Carrots should not be stored with fruit or where fruit volatiles are present. Since it is possible that injured or diseased carrots may produce volatiles which have a bitterness inducing effect, it is suggested that all diseased or badly injured roots be discarded and that the remainder be handled carefully. CHAPTER XIII SW Bitterness has been found in carrots held in cold storage for later processing or packaging following full attainment of maturity in the field. Early maturing varieties, Nantes and Touchon became more bitter in storage than the late maturing Danvers, Imperator, Gold Spike and Gold Pak, when harvested at the same chronological age. Strains of Red Core Chantenay were intermediate in bittemess response, yet were higlfly variable and showed a marked interaction with seeding date. A progeny test of bitter and non-bitter parents showed no differences in a season in which full maturity was probably not reached. Minor element fertilizer tests with copper and manganese indicated that deficiencies were not a direct cause for bitterness and that ample copper nutrition did not affect bitterness. The effect of manganese was masked due to immaturity at harvest. Injuried received by roots in digging and topping did not increase subsequent‘bitterness but tended to decrease it probably through stimu- lated metabolism. Refrigerated storage of carrots with Bartlett pears and apples re- sulted in bitterness development within three weeks. Apple emanations passed over refrigerated roots caused bitterness while carrots in air free from volatiles retained acceptable quality. The induction of 87 bitterness in immature and long stored carrots with ethylene was unsucess- ful; however, discoloration and surface pitting were similar to such damage received in commercial storages. Controlled atmospheres of 3% 002: 101 02 and 7S COzzSS 02 resulted in retention of sweetness but no bitter- ness. Anaerobically stored carrots became fermented, but not bitter. Bitterness development in the absence of stored fruit may possibly be brought about by naturally formed volatiles from carrots , from disease organisms or from seepage of fruit emanations through storage room walls. A phenomenon of yellowish-green fluorescence under short wave 2537; ultra violet light was significantly correlated with bitterness in the phloem tissues of the carrot. Microscopic study indicated that the fluorescence was located in oil glands and in senescent areas. A quick bitterness rating test employing fluorescence was developed. 88 cm XIV LITERATURE CITED Anonymous. 19104. 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Department of Horticul- ture, University of Wisconsin, Madison, Nisconsin. Rood, P. 1956. Relation of ethylene and post-harvest temperature to brown spot of lettuce. Proc. Amer. Soc. Hort. Sci. 68: 296-303. Ross, A. F. and C. E. williamson. 1951. Physiologically active emanations from virus-infected plants. Phytopath.h1: 1131-1138. Rygg, G. L. 19119. Changes in the carotenoid content of harvested carrots. Proc. Amer. Soc. for Hort. Sci. 514: 307-310. Smith, Margaret C. , Emily Caldwell and Louise 0. Burlinson. 19111:. Some factors affecting the carotene, thiamin, and ascorbic acid content of carrots grown in Arizona. Aria. Agr. Exp. Sta. Mimeo Rpt. 66. August. Smock, R. M. 191:3. The influence of stored apples on the ripening of other apples stored with them. NJ. (Cornell) Agr. Exp. Sta. Bul. 799: 1-36. June. 93 Stock, R. M. and A. M. Neubert. 1950. Apples and apple products. Interscience Publishers, Inc. , NJ. Sondhei-er, E., w. F. Phillips, J. D. Atkin. 1955. Bitter flavor in carrots I. A tentative spectrophotometric method for the estima- tion of bitterness. Food Research 20: 659-665. Thornton, N. C. 19110. The facts about ”artificially“ ripened fruit. I and II. Food Industries 12 (7): 148 arri 12 (8): 51. Truscott, J. H. L. 19511. Bitter flavour in stored carrots. Ontario Dept. Agr. Hort. Exp. Sta. and Products Lab. Report 1953-511: 108-109. Tyler, L. J. 19111:. Vegetable storage diseases in New York. Plant Disease Reporter 28: 1113-155. Warington, Katherine. 19110. The growth and anatomical structure of the carrot (Daucus carota) as affected by boron deficiency. Ann Appl. Biol. 27: 17:183. Weier, T. E. 19111:. Carotene degradation in dehydrated carrots I. Cytological changes in carotene and fat droplets under conditions favorable for carotene degradation. Amer. J. Bot. 31: 3112-3116. Werner, H. O. 191:1. Dry matter, sugar and carotene content of morpho- logical portions of carrots through the growing and storage season. Proc. Amer. Soc. for Hort. Sci. 38: 267-272. wharton, Marion A. and Margaret A. Ohlson. 191:9. The availability to the rat of certain carotenes in raw and cooked vegetables. Mich. Agr. Exp.'Sta. Quarterly Bul. 32: 130. Williamson, C. E. 1950. Ethylene, a metabolic product of diseased or injured plants. Phytopath. 1:0: 205-208. Wright, R. C., D. H. Ross and T. M. Hhitemen. 199:. The comercial storage of fruits, vegetables and florist and nursery stocks. U.S.D.A. Agr. Handbook 66. September. Ianaguchi, M., F. D. Howard and L. B. McNelly. 1955. Observations on ., bitterness of California grown carrots. Plant Disease Reporter 39 (1:): 302-3011. April 15. ___ , Betty Robinson and J. H. MacGillivray. 1952. Some horticultural aspects of the food value of carrots. Proc. Anter. Soc. for Hort. Sci. 60: 351-358. 91: Young, R. E. , H. K. Pratt and J. B. Biale. 1951. Identification of ethylene as a volatile product of the fungus Penicillium digitatum. Plant Physiol. 26: 3011-310. Zechmeister, In, and A. Polgar. 191411. On the occurrence of a fluor- escing polyene with a characteristic spectrum. Science 100: 317-3180 and A. Sandoval. 191:5. The occurrence and estimation We 0 none in plants. Arch. Biochem.and Biophysics 8: 1:25- 1:30. CHAPTER IV APPENDIX 95 APPENDIX TABLE 1 mos or swan-resumes or mamas sacrrous AND arr-muss 31 man: or A PORTION or m coam 0F sun-Fons INDIVIDUAL sunn- m Gamma cursors, mu 1, 1956 Root Fluorescence“) Bitter- Root Fluorescence“) Bitter- No. Ext. Phloem ness lo. Ext. Hrloem ness 1 5 2 2 33 2 I: 2 2 2 3 3 31: 1 1 1 3 3 3 3 3S 3 2 2 h 1: 2 3 36 l l l 5 5 2 S 37 2 2 S 6 5 2 l: 38 1 1 2 7 1: 2 5 39 S 3 I: 8 2 1 1 1:0 2 1: 3 9 3 2 1: 1:1 5 2 2 10 2 2 3 1:2 . 5 3 3 11 2 3 1: 1:3 5 3 3 12 S h 2 1:1: 2 1 S 13 3 3 5 1:5 5 3 3 11: I: 2 1: 1:6 1: 3 3 15 2 3 5 1:7 3 2 3 16 1: 3 5 1:8 1: 3 2 17 S 3 5 1:9 1 2 1 18 , 1: S S 50 5 l: 1: 19 3 h 5 51 S 2 5 20 2 2 3 52 5 2 2 21 5 3 h 53 3 2 3 22 5 2 3 51: 1: 3 2 23 1: I: S 55 1: 3 2 21: 2 2 5 56 5 3 3 25 1 2 2 57 3 2 2 26 3 3 1 58 2 2 I: 27 5 3 1: S9 1: 2 S 28 2 2 I: 60 5 3 3 29 5 2 3 61 3 2 3 3o 3 3 1: 62 2 2 3 31 3 1 3 63 1: 2 1 32 I: h 5 61: 3 3 3 Correlation coefficient, r - .28 with statistical significance at .05 on phloem fluorescence and bitterness. (l) 1 to 5 Scales: 1 - non-fluorescent and non-bitter, 5 - highly fluor- escent and higily bitter. APPENDIX rant: 2 summaries or sun-mun seen me amateur cmors, MARCH 1, 1956 5) Cuts Gauges (8) Cortex Shoulder Color (1-5) (7) Core (1-5) (7) Hold (1-5) (6) Cracks (1-51 Bruises (1-5) Rel. Sire In Core: Scuffs Cortex (h) t Crown Hei (1-5) (3) 1 SEER. A Ti (2 mt Di- ameter T (cm) Len Eh (e5) Root No. 97 O0888800800880000038009-880 NNNNNNNNNNNNNNHNNHNMNNNNNN MJJJM—SNMMMMN N MHJNMMMJQN-fi-flm «t + HNNMJNMMHMNJNMMMIHNNMNNN—S’NH MMMNMJN NMN MAN MJd-s-flmn-J—fl—S'MMN gagggaaaaaaagaafiagaagaagaa + I '0’ HNMNNNJNNHNMMNNNHMNNJNMHMH +++|Ivl+rl§+++|II+++I+I++I + + +§ I+Il+l+++ +lll+ IIII+|I+II + I + * + a:e-cpunonuncrununc3c3ouU\one-e-u\e:ooe-caouuxgeunun (“.50 £0 inn—5.50 JUN-3:34 (“M-3.3.30 “Mm-3"“ "2 “a": v: u: n massedvas322222222: nmmnrmmz-cmumw can mun fimha mm mm mm 75’): m an“ “flu Emu“ (1-5) Rel. Sise mm: 1‘"? b (3) 0mm & m- LmahamurTwu Tm Root Nm (7) (3) (1-5) (1-5) (7) (1-5) (6) Wm“ (1-5) mun (h) (en) (1) (2) (an) 1 W 800888mmmooogeogoggoooao08880a NNNNNNNMNNNNNNNNFA‘MNNNNNNNNNNN N + madeMMMMM'l-AMNMMNMM-fi—flu‘MNMJMMMM-fl' JMNNMJJMMHMMNMN-fiMN—SMNNMMNNMMM—fl 41.3mm M—SM—QMMMM—QMJMMMM—‘fJMMMJJM—fi-fl-fl 40 HP d? 40 d? “J aggaaaaaaaaaaaaaait that + +I+I8It'd-iI++§§+I+++t+¢§i++§+++ I+t+§+|lI1++l+tx§+lifiv++++lv+l ounmmooo O 0.:sz O\\O omo (TIAMOQQ O‘QMO 00 (~15 :mmazaammmamaammxo mmznnxo MAN-1‘0 MMU‘ e a a a m e seassauasaaassaaaanaasaaeasome asassaaaaaaaasesassassasdaasna 99 .3955 A am «cook» a 0 «093.3 a o «.333on peace as .omceao amen .- m sandman $5.3” u H Naoaou CL 63.66% 3:33.113: o room ooom poaog handgun Soon we on? 5 gunmanop a a moon Ho omega.» u m .32- on a a N30: 3v .aasnfi gaseous as asses .. m .55 ea _. N Earn 3 .930 .398vo owned .- M1. No.30 no scab" non—.283 ado—H a u: Naoodt 335. a me. .506an 3.5 I m: Newshoeo a an «Noumea one coco no mean degeaom CC .33» eased no assessed .. m .8385 Se. Hosea .. a .8335 cacao canoe Nausea No cameos 3&8: cacao 5 .H no mood: 83o 35.3 so: any A3 Cog . I- ngpfi “ mug . .0. nag “mg I ++ ”Raga Adv 0 fl N an m a mu N + + in mt: .3 o N m m m m an m . . ace. NH no a N a N m m a? m .. + 1: NH No 8 N m an m a an N . + Rm N G o N m an m a .3. m + .. o5 m6” 8 o N m N a m an. N - 1 Rm mg: mm o N N an N m an N + .. N.m m6 mm o N m a... m m .3. m .. . Rm 3 am Am-av a: E 5 g £38 Amé 3v 5 $5 8:: Gé causes 835 notes Ts Mm.“ 3 ES ca; 6: .8336 notes 28 3o: 38 chasm . £8 pause: tease e38... fines seem aoaoo G :H 3.3 .Hom 83.8 aqua :3 “cadence I N H349 "HE 100 APPENDIX TABDE.3 monsscmxcz, surnames no man mums or m mm saom ms emu cmnors, macs 28, 1956 (1 to 5 ratings“- ) J Root fi§§?$3§°§y°f 3331.333“. 323% No. Layers“) of Layers“) Taste on Arena 1 2 3 b 5 1 2 3 h 5 WhOIB Root 1 l 2 2 2 1 2 1 3 3 1 3 1 2 2 2 3 h 1 3 2 3 h 2 5 3 3 2 2 2 2 1 1 3 S 5 1 S S h l 2 2 2 1 2 2 3 h 1 3 1 5 1 2 2 1 2 1 2 2 1 3 l 6 1 2 3 3 1 1 2 3 5 1 h h 7 1 2 2 2 1 l 1 1 2 1 2 3 8 2 h h h 1 2 3 ’4 5 1 5 5 9 1 2 2 2 1 1 3 h 5 1 5 h 10 2 2 2 2 1 2 2 3 h 1 h 3 (1) Ratings of 1 are most favorable, 5 least favorable. (2) Layer 1 is outside peeling; layer 11 is tissue about the cenbium; layer 5 is xylem tissue or core. Correlation coefficients significant to .05 by t test: l-Suuation of layers of phloem (1 to h) for fluorescence and bitterness r - 0.6115; 2-Sumnstiou of layers of phloem and bitterness of whole root r - 0.588; Bitterness of whole root and aroma r - 0.712; Bitterness on mation of layers and on whole root r - 0.9111. 101 .mm «Nan .uoaogaoou com Adv N m m N n mu N ea 45 2 S N m N N m an a fin a mm m N m m H N mu N m.m m.m . mm m m m *N N N Na N Wm 3 2. N m a N N N Na 4 N.m d .5 o m N *m m 4 3+ m .3 fl NNN m m a H N m an 3 n.m mAN :3 a N 4 m N m mu N m3 NN 8H m N m *N N N mm 3 mi NH NE N m m *m 4 4 mu N NA .3 03 N Movaoo Amaav Inigo emu.“ “mm g “mm. .mww “um. ”my.“ Luna gummy h“ an“ .38 II?! 4cm 33 98m 33% .NN .852 .885“. 2353... E 985 2B .8 Sag—ago 4 Manda Nanmgmd EC .2". USE ONLY “tr WICHIGQN star: UNIV. LIBRQRIES mllWill”WHIWNWIHWIUIIWEINIHIWI 31293000771745