DEVELOPING INCREASED COLOR IN PAPRIKA PEPPER (CAPSICUM ANNUUM L.) Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY ZEMEDU WORKU 1971 v'k“ " LIBRA Q y University This is to certify that the thesis entitled DEVELOPING INCREASED COLOR IN PAPRIKA PEPPER (Capsicum Annuum L.) presented by Zeme du W0 rku has been accepted towards fulfillment of the requirements for PhoDo degree in Horticulture Date 0&2? [77/ 0-7639 ABSTRACT DEVELOPING INCREASED COLOR IN PAPRIKA PEPPER (CAPSICUM ANNUUM L.) By Zemedu Worku Color additives Of plant origin are ingredients of many food products. Improving the color of paprika pepper (Capsicum annuum L.) fruits is of value to producers and processors alike. Therefore studies were conducted to determine the influence of plant nutrients and growth regulators on color develOpment of two cultivars of paprika. Treatments consisted of 2 levels of N, P, K, Mg, Mn and Cu used in greenhouse and field tests. Ethephon, (2-Chloro- ethyl) phosphonic acid, malathion, 0,0-dimethyl dithiophos- phate of diethyl mercaptosuccinate and SADH, Succinic acid 2,2-dimethyl hydrazide were sprayed on plants at four con- centrations at different stages after anthesis. Standard maturity indexes were established and used in harvesting fruit. The pericarps were dried and ground. Samples were extracted with acetone and a dilution of 1/2500 or 1/5000 color content was determined at H60 mu. Capsanthin one of the major pigments was isolated by extraction with petroleum ether and acetone (1:1), Zemedu Worku saponified with KOH (20%) and divided between methanol in water and petroleum ether into epiphase and hypophase frac- tions. The pigments were separated with thin-layer chromatography. Capsanthin was quantitatively measured spectrophotometrically at “76 mu and quantitative differ- ences between treatments were evaluated. Color stability in storage was tested by two methods: (1) Equal concentration of color from each treatment was dispersed on salt (NaCl) and exposed to 70°C until the color faded and the time required recorded; and (2) A sample from each treatment was exposed for 0, 20, HO and 80 hours at 70°C. The remaining color analyzed and the loss determined. Ripe shriveled fruits showed a higher color concentra— tion than ripe non-shriveled fruits but lost their color during storage more rapidly. Application of K or Cu significantly increased the total extractable color of the fruit pericarps. Neither N, P, Mn nor Mg had an influence on color. There were indi— cations that increasing the N, P or Mn levels in the fruit may have decreased color but the variation was not significant. Flower formation was arrested with 500 ppm ethephon and leaves of plants treated with 200 and 500 ppm ethephon turned yellow. Ethephon stimulated ripening, increased total extractable color and capsanthin of paprika fruits pericarps. Treatment influenced the two cultivars Zemedu Worku differently. Paprika-505 contained more total extractable color and capsanthin and lost it more slowly than Paprika- D.Z. with ethephon treatment as compared to the controls. Malathion treatment increased color in the fruit pericarps with increasing concentration. SADH did not influence color significantly. The color loss Of the fruit pericarps in storage (70°C) was slower with SADH than ethephon, non- treated control or malathion treatments, respectively. DEVELOPING INCREASED COLOR IN PAPRIKA PEPPER (CAPSICUM ANNUUM L.) By Zemedu Worku A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1971 ACKNOWLEDGMENTS The author is deeply grateful to Dr. R. L. Carolus for encouragement and help throughout the program. The author is also very appreciative to Dr. S. Honma and Dr. R. C. Herner for giving their time and invaluable advice during the course of this program of study. Dr. C. M. Harrison's, member of the guidance committee, willingness to help at any time inspired the author. Thanks are extended to Dr. R. L. Donahue for serving on the committee. Many thanks are extended to Dr. A. L. Kenworthy, K. C. Sink and Kalamazoo Spice Extraction Company for the use of laboratory facilities, to Mr. N. Natarella and M. E. Hopping for suggestions and assistance in the laboratory, Mrs. Geri Burkhardt and others for their extraordinary kindness. The financial support of Kalamazoo Spice Extraction Company and in part Michigan State University Horticulture Department is gratefully acknowledged. The author is particularly indebted to Mr. K. R. Sandelin for his personal interest in the author's well- being and encouragement throughout the program. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . 3 Pigments . . . . . . . . . . . 3 Plant Nutrients . A Ethephon (2-chloroethyl) phosphonic acid 5 Malathion (O, 0- -dimethy1 dithiophosphate of diethyl mercaptosuccinate) . 7 SADH (succinic acid, 2 ,2-dimethyl hydrazide) 7 MATERIALS AND METHODS . . . . . . . . . . . IO Nutrient Experiments . . . . . . . . 10 Greenhouse Experiment (1969) . . . . . . 11 Field Experiment (1969) . . . . . . . 13 Color Determination . . . . . . . l3 Nutrient Analysis . . . . . . . 13 Greenhouse Experiment (1970) . . . . . . 13 Growth Regulators . . . . . . . . . 15 Isolation of Capsanthin . . 15 Quantitative Determination of .Capsanthin of Paprika- -505 . . . . . . . . . . . 17 Ripening Index . . . . . . . . . . 20 Experiment I . . . . . . . . . . 20 Experiment II . . . . . . . . . 20 Color Stability . . . . . . . '. 23 Salt Dispersion (NaCl) . . . . . . 23 Assay of Color . . . . . . . . . 23 RESULTS . . . . . . . . . . . . . . . 25 Ripening Index . . . . . . . . . . . . 25 Color Stability . . . . . . . . . 27 Effect of Nutrients on Color . . . . . . . 27 Greenhouse Experiments . . . . . . . . 27 Correlation Analyses . . . . . . . 29 iii Ethephon Experiment (1971) . . . Experiment I . . . . . . Experiment II . . . . . . Experiment III . . . . . . Capsanthin . Color Stability . Malathion Experiment (1970) SADH Experiment (1970) . Ethephon Experiment (1971) . . . . . Color Stability . . . . . Capsanthin . . Ethephon, Malathion and SADH Experiment (1971) Interaction Between Chemicals, Concentration and Time . . . Interaction Between Chemicals .and Concentrations . Interaction Between Chemicals and Time. Color Stability . . GENERAL DISCUSSION . . . . . . . . . SUMMARY . . . . . . . . . . . . . . LITERATURE CITED iv Table 10. 11. LIST OF TABLES Level Of nutrients in pounds per acre . Arrangement of treatments in greenhouse experiment . . . . . . . Arrangement of treatments in field experi- ment . . . . . . . . . Effect of stage of harvest on total extractable color content of Paprika-505 (1970) . . . . . . . . . . . Effect of nutrients on color content of Paprika-D.Z. fruit pericarps, greenhouse experiment (1969) . . Effect of nutrients on color content of Paprika— —505 fruit pericarps, greenhouse experiment (1970) . .7 . . . . . Correlation of color content with various plant nutrients . . . . . . Effect of nutrients on color content of Paprika—D.Z. fruit, field experiment (1969) . . . . . . . . . . . Effect of ethephon treatments on color con— tent Of Paprika-505 (1970) . . . . . Effect of ethephon treatment applied two and three weeks after anthesis on Capsanthin content of Paprika—505 fruits (1970) Effect of ethephon treatments on color stability of Paprika- -505 stored at 70°C (1970) . . . . . . . Page 11 12 12 26 28 28 3O 31 3“ A0 A2 Table Page 12. Effect of ethephon on reciprocal of extractable color content (l/E A60) of Paprika-505 stored at 70°C (1970) . . . A2 13. Second order reaction rate constant (K2) of ethephon treatments to Paprika- -505 after treated at 70°C . . . . . . A2 1A. Effect of malathion treatments on color con- tent of Paprika—505 (1970) . . . . . . A7 15. Effect of SADH applied two weeks after anthesis on color content of Paprika—505 (1970) . . . . . . . . . . . . A8 16. Effect of SADH treatments on color stability of Paprika-505 stored at 70°C (1970) . . A9 17. Effect of ethephon treatments at different stages after anthesis on color content of Paprika-D.Z. (1971) . . . . . . . . 51 18. Effect of ethephon treatments on color stability of Paprika-D. Z. stored at 70°C (1971) . . . . . . . . . 51 19. Effect of ethephon on capsanthin content of Paprika—D.Z. (1971) . . . . . . . . 5A 20. Effect of ethephon, malathion and SADH on color content of Paprika—D.Z. (1971) . . 56 21. Effect of chemical concentration on color content of Paprika-D.Z. (1971) . . . . 65 22. Effect of application time on color content of Paprika-D.Z. (1971) . . . . . .. . 65 23. Effect of ethephon, malathion and SADH on color stability of Paprika—D. Z. stored at 70°C (1971) . . . . . . . . . . 68 vi Figure LIST OF FIGURES Steps for isolation of capsanthin with thin layer chromatograph on silica gel after phase separation . . . . . . Standard curve of capsanthin prepared by diluting the initial 0.5 m1 capsanthin to 200 times . . . . . . . . . Increase of capsanthin with increased con- centration of ethephon . . . . . Color deterioration curve of Paprika-505 stored at 70°C. The quarter—life periods, t 1/A, from the graph were determined . . Color loss of Paprika-D.Z. fruit pericarps sprayed with 0, 50, 100, 200 and 500 ppm ethephon A and 6 weeks after anthesis stored at O, 20, A0 and 80-hours (70°C) Interactions of chemical, concentration and time on color increase of Paprika-D.Z. Interactions of chemicals and concentra- tions applied on color increase of Paprika-D.Z o o o o o o o o Interactions of chemicals and time of application on color increase of Paprika—D.Z. . . . . . . . Interaction between chemical, concentration and time on color loss of Paprika-D.Z. . vii Page 18 21 38 A3 52 57 60 62 66 INTRODUCTION Color additives of plant origin are important as ingredients of many food products. Among plant sources, pepper (Capsicum sp. L.) provides coloring matter for sausage and other meat products, salad dressings, pre- cooked food, condiment mixtures, catsup and other processed foods. In 1968 the United States imported more than 12.8 million pounds of ground and unground paprika with Ethiopia supplying part of this market. Paprika for food cOloring is sold by unit weight and it's market value determined by the pigment content. By improving the color, the value to the farmer will be increased. There are many carotenoids involved in the formation of red color in pepper with capsanthin comprising about 35% of the total. During drying, extracting and storing of the pericarps, the capsanthin and its derivatives undergo oxidation resulting in substantial color loss. Variety and stage of harvest are important factors con- tributing to color retention. The observations reported herein were conducted to determine whether paprika pepper fruit pericarps exhibit quantitative and qualitative differences in extractable color resulting from the application of ethephon, mala- thion, SADH and plant nutrients (N, P, K, Mg, Mn and Cu). REVIEW OF LITERATURE Pigment Red color of pepper provides coloring matter for foods (31). Many carotenoids are involved in the formation of the red color of pepper (13,17,56). Capsanthin (CA0H58O3) first isolated and identified by Zechmeister and Von Cholnoky (81) is the major carotenoid and accounts for 35% of the total (17). Brown (8) reported the red pigment from pimento was identical to capsanthin isolated from Japanese chillies and Hungarian paprika but quite different from the red pig- ment found in other fruits. It has a molecular weight of 586.86 (81) and has a cyclopentane structure (A,5). The pigment is soluble in most organic solvents and oxidizes during processing and storage. Light, air and temperature are important factors in its oxidation (A9). Philip and Francis (57) have reported the conversion of hydroxyl to ketone groups during oxidation of capsanthin. The carotenoid content increases markedly during the ripening process. Leonard gt a1. (50) reported an increase of about 32% of carotene when pepper fruits turn red on the plant. A similar increase was obtained when the green peppers were detached and left in the Open for three days until they turned red (A7). Fruit ripening is influenced by temperature, oxygen, carbon dioxide and growth regulating chemicals. Spencer also indicated that in pears, avocado and banana, top quality was obtained when ripened at 20°C (70). Burg (9) reported that temperatures above 35°C inhibit the produc- tion of ethylene, the lack of which delays ripening of many fruits. Plant Nutrients The relationship between the levels of plant nutrients and fruit color have been shown by many workers. Weeks gt gt, (77,78) Observed a decrease in anthocyanin derived fruit color of apple with an increase of N in the leaves. Shear and Horsfall (66) reported a decrease in the percent of red fruits of apple as the level of applied nitrate increased and a delay in apple fruit maturity. Manson (52) reported a decrease of 9% in color with a high level of N and Stembridge gt gt. (72) reported similar trends with peaches. Francis and Atwood (33) reported that high levels of P application resulted in decreased pigment content of cranberries. Panditn and Andrew (55) reported a negative correlation between the P level of tomato and lettuce leaf tissue and days to maturity. Rosié (60) indicated that maturity of paprika was enhanced more by P or PK than by NPK. Weeks gt gt. (77,78) showed that increasing levels of K in apple leaves was associated with improvement in fruit color. Fisher and Kwong (30) reported an increase of apple fruit color when K was added to an orchard low in K (0.5% K in leaf). Stembridge gt gt. (72) found that the amount of peach fruit color was directly related to K application. Bailey and Cochran (3) reported that pimento pepper plots which received K in the form of K2SOA produced fruits with thicker walls and better color than those receiving KCl. Fruits from plots receiving 8% K20 in the fertilizer con- sistently produced thicker fleshed fruits than those receiving A% K20 or none. Application of Cu as CuSOu to muck soil improved the color of onion scales (A6). How- ever, Francis and Atwood (33) did not find an increase of cranberry pigment when Cu or Mn were applied with or with- out other fertilizer materials. Ethephon The compound ethephon is (2—chloroethy1) phosphonic acid. The compound is also referred to as Amchem 68-2A0 (l). Edgerton and Blanpied (28) reported that aqueous solutions of ethephon are a direct source of ethylene. Cooke and Randal (1A) have demonstrated a breakdown of ethephon to ethylene in aqueous solution. Burg (10) has indicated that plant responses to auxins can be attributed to the action of induced ethylene production. Ethylene appears to be a ripening hormone (9,37) and enhances color (15,80). Several other reports also indicate that ethephon enhanced fruit maturation and ripening (2,25,28,3A,37,A2, 59). Russo gt gt. (62) showed a 50% loss of banana chloro- phyll 3 days after ethephon application. The effectiveness of this chemical was found to be comparable to 100 ppm of ethylene gas. Uniform early ripening of tomato fruits is possible after treatment with ethephon, both in the field and green- house (35,38,39,A0,67). Ethephon (100 ppm) sprayed on pep— per fruits as they changed color ripened one week earlier as compared to controls. There were a greater number of red pepper fruits at 250 and 500 ppm than at 100 ppm (7,68). Eck (2A) has reported that ethephon (600 ppm pre- harvest spray) to Early Black cranberry increased antho- cyanin development. Citrus varieties sprayed with 100 to 500 ppm ethephon developed more carotenoids in the fruit rinds (80). Ethephon applied 3 to A weeks before optimum maturity to apples at 100 to A00 ppm increased the rate of development of red color pigmentation (75). Lockwood and Vines (51) working with detached pimento fruits found that treatment with 3000 ppm Of ethephon for 1 minute at A0°C increased reSpiration within 20 hours. Similar increases in respiration after ethephon treatment have been shown for Valencia orange (5A) and for orange and Clematine tangerines (3A). Malathion The compound 0,0—dimethyl dithiOphosphate Of diethyl mercaptosuccinate (malathion) is an important, broad spectrum insecticide for controlling insects of fruits, vegetables, flowers and ornamentals. Shawa (65) has shown that malathion increased the extractable pigment in Howes and Early Black cranberry. Devlin gt gt. (19,20) in a similar study with Early Black cranberry found an increase in color and concluded that treatment 2 weeks before harvest with 1600 ppm gave best color and was within the restriction limits allowable on cranberries. The enhancement of cran- berry fruit color due to malathion sprays was also reported by Eaton gt gt. (22), who indicated a significant increase in anthocyanin when applied 2 or 3 weeks before harvest. SADH The effectiveness of SADH or Alar—85 (succinic acid, 2,2—dimethyl hydrazide) on growth and accumulation of color on many horticultural crops has been reported. Wilber and Nakayama (79) noted that spraying Chile pepper with SADH significantly reduced stem elongation. Foliar application of SADH modified the effect on days to fruit maturity of peaches (72). Chaplin and Kenworthy (11) reported that SADH applied 1 to A weeks after full bloom would advance the ripening of sweet cherries from 1 to 2 weeks. In another study the fruit maturity of sweet cherries was advanced by A to 5 days as compared to the control (11) with the same acceleration of ripening reported with all concentrations used. Ryugo (63), however, reported that SADH did not advance maturity of sweet cherries but induced early development of pigments. 0n the other hand, Eck (2A) has reported a delay in anthocyanin development in cran- berry due to SADH treatment. The application of SADH in combination with gibberellin to apple counteracted the ripening effect of the auxin (29). Dilley and Austin (21) have reported that SADH applications delayed the onset of the respiratory climacteric of apple and suggested that this in effect will delay maturity. The influence of SADH sprays on color of many kinds of fruits are reported. Unrath gtht. (7A) applied SADH to sour cherries and found that treatment advanced color for— mation 7 to 10 days as compared with untreated fruits. Crocker (16) also showed a significant increase in color of sour cherries from SADH spray. Ryugo (63) working with three cultivars of sweet cherries found a marked increase of color when SADH was applied 2 weeks after full bloom. Chaplin and Kenworthy (11) showed that foliar sprays to sweet cherries increased fruit color when applied 2 or A weeks after full bloom. IIII‘."I[(:(II||‘[((.‘O'I‘r'Ildul (I3 t, DEVELOPING INCREASED COLOR IN PAPRIKA PEPPER (Capsicum Annuum L.) The objectives of these experiments were to study the effect of plant nutrients (N, P, K, Mg, Mn, Cu), and growth regulators (ethephon, malathion, SADH) on color development of paprika. Paprika-D.Z. was selected because it is one of the most successfully grown cultivars in Ethiopia and Paprika-505 because of its early maturity. MATERIALS AND METHODS Paprika-505 (Peto Seed Company) and Paprika—D.Z. (Agr. Exp. St., Haile Sellassie I. University, Debre Zeit, Ethiopia) were used for the investigations. Seedlings were started in the greenhouse and experiments were conducted in both the greenhouse and the field. The data were subjected to variance analysis and evaluated by Duncan Multiple Range or least-significant-difference tests. Nutrient Experiments Paprika plants were fertilized with two levels and combinations of nitrogen, phosphorus, potassium, magnesium, manganese and copper as shown in Table l. The greenhouse IO 11 experiments consisted of 1A and 13 treatments and were replicated 3 and A times in 1969 and 1970 respectively (Table 2). Likewise, the field experiment consisted of 12 treatments and were replicated A times (Table 3). Table l.—-Level of nutrients in pounds per acre. Greenhouse Field High Low High Low Level (H) Level (L) Level (H) Level (L) Nitrogen 100 25 100 25 Phosphorus 100 25 100 25 Potassium 150 50 150 50 Magnesium 25 10 25 0 Manganese 25 10 25 0 Copper 25 10 25 0 Nutrient Carriers MgSOu ° 7H20 NHuNO 3 NHAHZPOA Manganese Replex KNO3 Magnesium Replex KHQPOM COpper Replex Greenhouse Experiment (1969). Seedlings were transplanted on May 23 to 10 inch pots filled with leached bank sand. The nutrient treatments per pot were applied at 3 times normal rate and were replicated three times. The nutrients were applied at three times. The first application of N, P and K was made on May 23, two days after transplanting. The second was on June 13. Mg, Mn and Cu were applied on June 25. The fruits were harvested when completely shriveled, from August 29 to October 3 and kept in a cold 12 Table 2.--Arrangement of treatments in greenhouse experiment. Levels Treatments N P K Mg Mn Cu 1 H H H H H H 2 H — - - - - 3 - H - - - - A - - H - - 5 - - - H - - 6 - — - - H - 7 _ _ _ _ _ .. 8 _ _ _ - _ - 9 - H H H H H 10 H - H H H H 11 H H — H H H 12 H H H - H H 13 H H H H - H lA H H H H H - Table 3.--Arrangement of treatments in field experiment. 1 — - - O O O 2 - H H O O O 3 - - H O O O A - H - O O O 5 H — — O O O 6 H — H O O O 7 H H — O O O 8 H H H O O O 9 H H H H O O 10 H H H O H O 11 H H H O O H 12 O O 0 O O O H = High level; — = Low level; 0 = None. 13 room at A0°F until harvest was completed. Due to limited number of fruits, only color, nitrogen and phosphorus were determined. Field Experiment (1969). The experiment was conducted at the Southwestern Michigan Sodus Experiment Farm at Sodus, Michigan on Oshtemo sandy loam soil of pH 6.2. Plots were each 22.5 square feet replicated four times. Seedlings were transplanted on June 11 and nutrients were applied as a side dressing on June 17. Fruits were harvested on October 10, 1969 when fully ripened but not shriveled. The pericarps were dried after removing seeds and stems and ground to pass a A0 mesh sieve. Color Determination. Two gram samples of pericarps from each treatment were extracted in 100 ml acetone until white. Six hours Of extraction in darkness with occasional shaking at room temperature was adequate. Aliquots of a l to 5000 dilution of the extract were transferred into a cuvet and absorbence Of the sample was determined at A60 mu as described by Rosebrook gt gt. (61) using acetone as a blank in a Beckman B spectrophotometer. Nutrient Analysis. Nutrient elements were determined from dried ground fruit pericarps; N by modified KJeldahl, K by the flame photometer, and P, Mg, Mn, and Cu spectro- graphically following procedures outlined by Kenworthy (A3). Greenhouse Experiment (1970). Paprika-505 plants were transplanted on April 30 in a randomized block arrangement ‘II lllllllvllvlil '..I.lll 'I 1(IE‘I[{(“II[( {{‘(I‘tt{((lr.I 1A with four replicates. Nutrients were applied on May 2, 15, 26 and June 2A. Fruits were harvested when shriveled. Fruit color, nitrogen, phosphorus, potassium, magnesium, manganese and copper were determined. 15 Growth Regulators The chemicals (2-chloroethyl) phosphonic acid (ethephon), 0,0-dimethyl dithiophosphate of diethyl mer- captosuccinate (malathion) and succinic acid 2,2—dimethyl hydrazide (SADH) were used on pot grown plants fertilized with plant nutrient solutions. In order to obtain maximum fruit set, the first blooms were removed. Treatments were applied at different stages after anthesis. Hereafter, in the text anthesis refers to the time of second flower open- ing after removal of the first flowers. Concentrations were varied from 50 to 500 for ethephon, 1000 to 8000 for SADH, and 500 to A000 ppm for malathion at a pressure of approxi— mately A0 psi. Sprays were applied to runoff with a hand sprayer. A surfactant (Tween-20) was applied at 0.5 ml per liter of ethephon solution. Observations were made of the effects of treatments on flowering, maturity and ripening. Pericarps from matured shriveled fruit were prepared for analysis. Isolation of Cgpsanthin. In order to determine a quanti- tative change of the capsanthin pigment due to treatment, isolation was made following the method of Philip and Francis (56). Paprika-D.Z. (150 g) was extracted in 1000 ml of 1:1 solution of petroleum ether (b.p. 30-60°C) and acetone. The extract was filtered with suction in a funnel with a glass fiber filter, evaporated to dryness and 16 saponified for 10 hours with 20% KOH in 50 ml methanol. The saponified pigments were extracted with ethyl ether and washed with distilled water until the pH was equal to that of distilled water. A small quantity of anhydrous sodium sulfate was added to remove all water and the solvent evaporated to dryness. The crude pigment was partitioned by transferring to a separatory funnel in 500 ml each of 95% methanol in water and petroleum ether. The methanol (lower) layer containing capsanthin was separated and the solvent was evaporated to dryness in A test tubes. The dry pigments in the test tubes were dissolved in 2 m1 (100%) methanol. Three of the test tubes were flushed with nitrogen, sealed and covered with aluminum foil and stored at -20°C for future use. Initial capsanthin purification was made on silica gel of 0.500 mm thickness using a solvent system consisting of 3.5, 10, 20 and 66.5% of ethanol, benzene, acetone and petroleum ether, respectively. The front was allowed to move 10 cm (16 minutes). The fraction between 6-7 cm was removed, extracted with 10 ml of methanol (100%), filtered and evaporated to dryness. The dry pig- ments were dissolved with 1 ml of methanol and 1000 ul of the extract was thin-layered on silica gel using the same solvent system. A 7 cm move was allowed (21-23 minutes). The fraction at 5 cm was removed, dissolved in methanol, filtered in a glass fiber filter, washed and evaporated to 1? dryness. Steps are shown in Figure l. The dry capsanthin was dissolved in 10 m1 ethanol and frozen. Quantitative Determination of Capsanthin of Paprika-505. A 0.5 ml quantity of purified capsanthin was added to 19.5 ml ethanol and 1 m1 taken from this capsanthin solution and A m1 Of ethanol was added. The capsanthin diluted 200 times was scanned at A76 mu in a Bausch and Lomb spectronic 20 Colorimeter as given by Polgar and Zechmeister (58). The Optical density obtained was 0.280. Quantitative determina- tion of capsanthin was made by setting a standard curve using the following equation. 0.280 Xh586.8 10.3 x 10H x 1.2 A = —E— x be; c = 2.85 ug/lO m1 MWT A = Absorbence; b = Cell length; 0 Extinction coefficient. Concentration; E = The molecular extinction coefficient of capsanthin in ethanol at A76 mu is 10.310'” (Polgar and Zechmeister, 58). The molecular weight of capsanthin is 586.86 (Zechmeister and Cholnoky, 81). One—gram ground samples of paprika were extracted in 100 m1 acetone, filtered, evaporated and dissolved with 5 m1 acetone. A 25 ml aliquot Of extract from each treatment and capsanthin standard were thin- layered on silica gel with the same solvent system used for purification of capsanthin. Two runs were made at 5 and 10 cm moves for better separation. The fraction corresponding with the standard capsanthin was scraped off, placed in 18 Figure l.-—Steps for isolation of capsanthin with thin layer chromatograph on silica gel after phase separation. A. Second step B. Third step Arrow indicates capsanthin band. l9 20 test tubes, extracted in acetone, filtered and evaporated. The dried capsanthin of each treatment was dissolved in 3 m1 acetone and optical density was read at A76 mu. The values obtained were 0.09A2, 0.1308, 0.1805 and 0.2328 for the control, 100, 200 and 500 ppm ethephon, respectively. These values were plotted on the standard curves (Figure 2) computed and the quantity of capsanthin for each treatment was obtained. Ripening Index. In order to determine the best time to harvest paprika pepper fruits to Obtain maximum extractable color, two experiments were conducted. Experiment I. Date of bloom was recorded and three to four fully ripened fruits were picked at 58, 60 and 62 days after anthesis. Experiment II. Fruit set at the same time were picked at A different ripening stages. These stages were: a) fully ripe but succulent, b) fully ripe and partially shriveled, c) fully ripe and about 75% shriveled, and d) fully ripe and completely shriveled. In the second experiment, the fruit from the first two harvests were kept frozen until the last harvest was made. Fruits of the last stage of harvest were left on the plant until the fruits were shriveled. Pericarps were separated from the seeds and stems, dried and ground. One-gram samples from each treatment were extracted with acetone and the total extractable color was determined. 21 Figure 2.-—Standard curve of capsanthin pre— pared by diluting the initial 0.5 m1 capsanthin to 200 times. A. Paprika-505 B. Paprika-D.Z. 22 0.400 I 0.300- 0.200 I 0.|00 l I .4 2.8 4.2 5.7 0.600 I OPTICAL DENSITY AT 476 mp. 0.400 I 0.200 L O |.2 2.4 3.6 4.8 CAPSANTHIN (#6) L. 23 Color Stability. To determine the rate of color deteriora- tion two methods were used. Salt Dispersion (NaCl). This method was adapted from that used by the Kalamazoo Spice Extraction Company, Kalamazoo, Michigan. Ten grams each of ground sample from fully ripened shriveled and fully ripened unshriveled fruits were extracted with 100 m1 acetone. Color at 1/5000 dilu- tion was determined at A60 mu. ii The mean value of color units dispersed were equalized to a concentration of 0.2A1 unit. Accordingly 10, 10, 10 ml for shriveled, 12.2, 12.2 and l2.A ml extracts for unshriveled were dispersed on 100 g of salt (granules). Aluminum pans having a diameter of A.5 inches at the top and 3.5 inches at bottom were used. Samples were placed in an oven at a temperature of 70°C until the red color had faded. The end point for this test was determined when the red color had dispersed with some yellow remaining. Time required for color to disappear was recorded and used to evaluate color stability. In Assay of Color. A 1 g ground sample from each treat- ment was weighed and placed in an aluminum pan 1.5 inch in diameter. Initial color of each treatment was determined. Pans were placed in an oven at 70°C and samples were removed at intervals Of 10, 20, A0 and 80 hours. Total extractable color was determined and evaluated using two methods. 2A Percent loss of color with time. x - aIx-x) Cf|I-‘ Second order reaction rate - constant, K2 = as proposed by Chen and Gutmanis (12). a = initial extractable color content; (a—x) = existing color content at time t; x - decrease in color content during time t; t = storage time in hours. RESULTS Ripening7Index One of the problems of comparing color differences of pepper fruit between treatments was the lack of a standard ripening index. Fruit of paprika pepper is often considered ripe after it has passed its pale green stage. Differences in total extractable color from pale green to fully ripe and shriveled stages are high. During these stages of ripening rapid changes take place within the fruit. Therefore, it was difficult to develop a parameter for comparing fruit color between treatments. These tests were designed to develop a standard for a ripening index. This standard can be based on physical changes such as ripeness and color of fruit. Such a standard should be of practical value to the grower where color of the fruit becomes a price deter- mining factor on the market. Two approaches were followed: (1) harvesting fruits on the basis of the number of days after anthesis; (2) harvesting fruits on the basis of the different stages of ripening and physical changes of fruits as described in materials and methods. The results of using a ripening index based on number of days from anthesis had optical densities of 0.A85, 0.602 and 0.555 for 58, 60 and 62 days respectively. 25 26 In this experiment one important observation was made. Fruits having an equal number of days from anthesis to harvest may not be at the same stage of ripening. This may be affected by environmental differences such as light, temperature and air. Based on this study, fruits picked 60 days after anthesis indicate a higher color content than fruits picked 62 days after anthesis. Differences of such magnitude affect the true value in comparing any treatment. A test was made with a ripening index based on stage of ripeness shown in Table A. Table A.~—Effect of stage of harvest on total extractable color content of Paprika-505 (1970). Harvestl Optical Density of 1/2500 Dilution at A60 mu Stages Rep. 1 Rep. 2 Mean 1 0.552 0.508 0.530a 2 0.6A0 0.6A0 0.6A0b 3 0.680 0.66A 0.6720 A 0.710 0.708 0.709d Means followed by different letters are significantly different at 1% level according to Duncan's Multiple Range Test. 11. Fully ripened but not shriveled. 2. Fully ripened and partially shriveled. 3. Fully ripened and about 75% shriveled. A. Fully ripened and completely shriveled. In Table A the mean increase in total extractable color was 21, 27 and 3A% for stages 2, 3 and A respectively as compared to stage 1. 27 ~This method of indexing appears to be more reliable than considering the number of days after anthesis. The rate of change in color during the first two stages of ripening is greater than in the later stages. The final stage can be recognized with the fruits becoming shriveled, starting from the tip of the fruit, and having a dark red color. This stage should not be confused with fruits that have shriveled under water stress and lack the dark red color. Color Stability. Differences in color stability of ripened shriveled and ripened unshriveled fruits were determined using the salt dispersion method. In this test, the differ- ence in time required for color to disappear was found to be 50 hours for unshriveled and 27 hours for shriveled fruits at 70°C. This test indicates a decrease in color stability with an advance in ripening. Effect of Nutrients on Color Greenhouse Experiments. The values in Tables 5 and 6 are the means of 3 and A replicates respectively. Only the data on color and the effects of N and K are presented due to limited fruit sample material. Fruit containing the high levels of N, P, Mn and Mg were delayed 5 to 8 days in ripening as compared to fruit treated with low levels of all the elements, or high levels 28 Table 5.--Effect of nutrients on color content of Paprika- D.Z. fruit pericarps, greenhouse experiment (1969). Treatments Optical Density N% K% N P K Mg Cu Mn 1/5000 Dilution - - H - - - 0.A08 1.59 A.1A - - - - - - 0.3938 1.72 A.07 H H H H H - 0.392a 1.55 3.11 H - H H H H 0.390ab 1.6A 3.39 - H H H H H 0.390ab l.Al 3.98 H H H - H H 0.389ab 1.60 3.03 H H - H H H 0.380abc 1.78 2.37 - H — - - - 0.379abcd 1.55 3.22 H H H H H H 0.377abcd 1.53 2.97 . - — - - H - 0.372abcde l.A8 3.A2 i — — — - - H 0.361 bcde 1.57 3.21 I - — - H - - 0.357 cde 1.69 3.26 I H H H H - H 0.350 de 1.5A 3.10 H - - - - - 0.3A7 e 1.8A 2.A9 Table 6.-—Effect of nutrients on color content of Paprika- 505 fruit pericarps, greenhouse experiment (1970). Treatments Optical Density Mn Cu N P K Mg Cu Mn 1/5000 Dilution N% P% K% Mg% ppm ppm - - H - - - .560a 1.33 0.256 u.u0 0.07 36 7.2 H H H H H H .526 b 1.97 0.363 3.67 0.12 51 9.8 H - H H H H .516 be 1.79 0.30M u.05 0.08 5H 10.3 - - - — H - .u96 cd 1.29 0.289 3.37 0.07 35 10.0 H H H H H — .u86 cde 1.62 0.352 u.11 0.10 35 9.8 H H H — H H .u80 cde 1.45 0.377 3.79 0.09 66 9.6 - H H H H H .u76 de 1.31 0.3A7 3.61 0.09 51 10.2 - H — - - - .u6u de 1.23 0.u31 3.35 0.07 A0 7.0 - - - - - - .u60 def 1.39 0.299 2.75 0.07 37 6.7 - - - - — H .u50 efg 1.35 0.268 3.03 0.07 58 7.u H - - — - - .u30 efg 2.19 0.273 3.13 0.07 39 6.5 - - - H - - .u06 fg 1.33 0.289 3.20 0.09 37 6.5 - H - - - - .38u g 1.65 0.395 2.80 0.08 5A 10.3 Means followed by different letters are significantly different at the 5% level according to Duncan's Multiple Range Test. H = High level; - = Low level. 29 of K or a high level of Cu with a low level of the other elements. From these results (Tables 5 and 6), it appears that plant nutrients are important in color development. Fruits from the plots treated with the low level of all elements in 1969 was as high in color content as any other. How— ever, in 1970 fruits from plots treated with the high level of elements was second highest in pigment. In both experi— ments, the pigment content was higher when K application was high and other elements were low. Also, it was found that fruits from plots treated with low Mn, P, N and Mg had more color than those treated with higher levels. Color obtained was lower from the plants treated with high levels of N but low levels of P, K, Mg, Mn, and Cu. Plants treated with a high level of K but low levels of other elements gave the highest fruit color. Correlation Analyses. Correlation coefficients were calculated to evaluate the relationship between mean color content and concentration of N, P, K, Mg, Mn and Cu in the pericarps (Table 7). There was no significant correlation between N, P, Mg and Mn concentration of the pericarps to total extractable color. However, N, P and Mn tend to show a negative correlation in the 1970 experiment, and also N in 1969. Similarly, no significant correlation was found between Mg concentration and color content but tended to show a positive trend. The 1970 result showed the 30 correlation coefficient between color content and Cu to be positive and was very near significance at the .05% level. Similarly the data of 1969 and 1970 showed the mean color content to have a highly significant positive correlation with K concentration of the pericarp. Other factors being equal, extractable color of fruit pericarp tissue increases with increasing K level. Table 7.--Correlation of color content with various plant nutrients. 1969 Experiment (Paprika-D.Z.) H 5 Colora -0.26 0.58* 1970 Experiment (Paprika—505) 1:1. E I 0.2 101 9.1.1. Colorb -0.03 -0.19 0.80** 0.18 —0.07 0.51 *Indicates significance at the .05 level. **Indicates significance at the .01 level. a I aMean of 3 replications. bMean of A replications. The effect of plant nutrients on color of paprika also g was investigated in the field. Fruits used for various 1 analyses were fully ripe but not shriveled. Analyses for color, N, P, K, Mg, Mn and Cu were made. Results of the test are shown (Table 8). .0202 I o mao>oa 3oq I mHo>mH swam u m 31 m.mz 6.3: mo. Hm.o mH.m ma.m msfi.o o o m m m m m.ms m.:m mo. mm.o :m.m os.H mma.o o o o o o o m.H: o.am ac. om.o mo.m mm.H mmfi.o o o o I m I m.mm m.mm so. mm.o so.m mo.m Hmfi.o o m o m m m a.mm m.=m we. :m.o mo.m mo.m mmH.o o o o I I I 0.0m o.mm No. mm.o oa.m oa.m amH.o o o o m I m H.mm E.H: so. am.o :m.m mo.m mma.o o o o I m m a.sm a.am No. mm.o :H.m Hm.H mmfi.o o o o m I I m.mz m.om mo. Hm.o mfi.m oo.m mmH.o o o o I I m m.mz p.0m so. om.o mm.m om.a mom.o o o o m m I m.mm m.mm mo. Hm.o mm.m mm.H aom.o o o o m m m o.mm m.Hm mo. :m.o mm.m mo.m aom.o m o o m m m Esp 56p a R R a cofipsfifio ooom\a so 22 m: x m 2 so :2 ms msgosamonm Sdfimmmuom comomuaz mpHmCOQ awoapoo mucoEumopB .Amwmav pcoefipoaxo UHOHQ .pHSLm .N.QImxHLomm mo pampcoo Loaoo co mpCOHLpsc mo poommmII.w magma 32 No significant differences in fruit color were found among treatments (Table 8). However, the addition of N, P and K in this particular test increased the color content of paprika slightly when compared with the non-treated control. An increase of 10% in color was obtained when a treatment containing higher levels of N, P and K was com— pared to the non—treated control. Copper, Mn and Mg appear to be very important elements in color development of paprika. Treatments containing Mn and Mg did show a decrease in color content in spite of the higher levels of N, P and K. This experiment suggests that Cu may have a positive and Mn and Mg a negative effect on pigment development. The concentration of N, P, K and Mg in fruits was higher with a treatment containing Cu than without it. In general, lower color content was found in the field experi- ment when compared to the greenhouse experiment. This may be partly due to harvest before fruits were shriveled because of an early frost in October. 33 Ethephon Experiment (1970) The influence of ethephon applied at different stages on color development of pepper were observed in two green- house experiments conducted in 1970. Experiment I. The results (Table 8, Experiment I) were obtained from plants that were transplanted on March 28 and sprayed twice on June 20 and 27 four and five weeks after anthesis. The fruits were picked when completely shriveled on July 29, 32 days after the last spray application. As shown in Experiment I (Table 9), the effectiveness of ethephon in enhancing color development is marked. There is an increase of 62 and 81% in color content from 100 and 200 ppm treatments, respectively over the control. Experiment II. Results in Experiment II (Table 9), were obtained in the greenhouse from plants transplanted on April 20. Ethephon was applied 3 and A weeks after anthesis on June 27 and July A and fruits were harvested on August 9 when completely shriveled 35 days after the last spray. Differences in ripening were observed. Fruits treated with 200 ppm begin to develop color 6 days after the second application while fruits treated with 100 ppm took 10 days. Fruits from control plants began to color 7 and 12 days later than those treated with 100 and 200 ppm respectively. Treatment with 200 ppm caused some flower abscission and Table 9.--Effect 3A of Paprika-505 (1970). of ethephon treatments on color content Concentrations Experiment I Optical Density of 1/2500 Dilution at A60 mu Rep. 1 Rep. 2 Rep. 3 Mean None 0.335 0.315 0.305 0.3183 100 ppm 0.A92 0.515 0.A85 0.507b 200 ppm 0.5A5 0.5A5 0.598 0.5830 Experiment II Optical Density of 1/2500 Concentrations Dilution at A60 mu Rep. 1 Rep. 2 Rep. 3 Mean None 0.320 0.335 0.3A5 0.333a 100 ppm 0.590 0.6A0 0.630 0.620b 200 ppm 0.7A0 0.750 0.730 0.7AOC Experiment III Optical Density of 1/2500 Concentrations Dilution at A60 mu Rep. 1 Rep. 2 Rep. 3 Rep. A Mean None 0.280 0.285 0.298 0.293 0.289a 100 ppm 0.3A0 0.360 0.368 0.3A5 0.353b 200 ppm 0.u03 0.A10 0.u21 0.A08 0.A100 500 ppm 0.A38 0.Au8 0.A35 0.A55 0.AAAd Means followed by different letters are significantly different at 1% level according to Duncan's Multiple Range Test. 35 accelerated the rate of leaf abscission as compared to 100 ppm; and 3 days after the second spray, leaves of all treated plants with 200 ppm ethephon quickly turned yellow. In this test Ethephon had a more marked effect on color than in the first experiment and ripening occurred 3 to A days earlier. This may be due to the three extra days given after treatment and the higher average outside temperature of 70.6°F as compared to 66.8°F for Experiment I. In this test, differences in color were found to be 86 and 119% greater for 100 and 200 ppm respectively as compared to the non-treated control. A greater flower drop was observed for 200 ppm than 100 ppm of ethephon. Experiment III. In this experiment, the plants were grown in a warmer greenhouse than those in the other experiments. Plants were transplanted on March 15, 25 days later than those in Experiment I and II (Table 9). However, treatments were applied on June 27 and July A, two and three weeks after anthesis at the rate of 100, 200 and 500 ppm ethephon. Fruits were completely shriveled when picked A0 days after the last spray on August 13. The results obtained in Experiment III were similar but had a more marked effect on color than observed in the previous tests. The increase of color in this experiment may be attributed to higher outside temperatures averaging 36 71.1°F and a delay in harvest of 8 and 5 days after the last treatment as compared to Experiment I and II, respec- tively. Ethephon increased color at all concentrations studied with increases of 22, A6 and 53% for 100, 200 and 500 ppm respectively over the control. At the higher concentration (500 ppm), inhibition of flowering and yellowing of leaves were evident after the second spray. At the same time ripening was enhanced by A and 6 days as compared to 200 and 100 ppm ethephon. Color obtained in this experiment cannot be compared with that of the previous two experiments because fruits were not given an equal number of days from the time of ethephon treatment to harvest and plants were not grown under the same conditions and time. However, the results of these experiments (Table 9) shows that the higher temperature that prevailed in Experi— ment II and III may be important in color development of paprika. The data of all three experiments indicate that ethephon enhanced ripening and increased the color of ripened pepper fruits. Capsanthin. Many pigments are involved in paprika pepper color. Curl (18) reported over 30 pigments with Capsanthin accounting for 35% of the total. In order to determine the origin of the quantitative increases in extractable color found in this experiment, carotenoids were extracted and analyzed quantitatively. There was 37 apparently no synthesis of a new pigment due to treatment. However, a quantitative increase of capsanthin due to ethephon was found (Fig. 3—A). The Rf value of capsanthin on thin layer silica gel at room temperature was 0.5A to 0.56. This value is in close agreement with that of Philip and Francis (56) who reported an Rf value of 0.53 using the same solvent system. However, when a methylene dichloride ethyl acetate (A:l) solvent system was used (Stahl, 71), the purified capsanthin standard further separated into two fractions. This further breakdown of standard capsanthin may be partially due to the oxidation of capsanthin during the preliminary purifica— tion on thin layer silica gel. Such a breakdown was appar- ent with the same solvent system (Philip and Francis, 57) as shown in Figure 3—A. The degradation of paprika color during drying and storage due to light and temperature have been reported (Lease and Lease, A8, and De La Mar and Francis, 18). Philip and Francis (57) have reported that oxygen is the agent changing the hydroxyl groups to ketone groups of capsanthin as shown below: OH 3 CAPSANTHIN OH/ 0 \ OH 0 ,_o és-xflo Knvrrocnnoul a cunnruon 6 0 ° 3 on o— 0 run IlTA-APO-I'-CAROTENAL 2n «annuum I OH”? CHAIN COIPOUND' 38 Figure 3.--Increase of capsanthin with in- creased concentration of ethephon. A. Paprika-505.. a to d = 5, 10, 20 and 30 ul, respectively, capsanthin standard. e to h = 25 ul of e, 0; f, 100; g, 200 and h, 500 ppm ethephon. B. Paprika-D.Z. a to c = 5, 10 and 20 01, respectively, capsanthin standard. a to h = 10 ul of e, 0; f, 100; g, 200 and h, 500 ppm ethephon. 39 A0 Apparently, capsanthin is highly oxidized under normal conditions. Therefore, extraction and thin-layering should be carried out in an inert atmosphere and at 0°C (Smith, 69). The quantitative difference in capsanthin between treatments were determined by dissolving the capsanthin 5: fraction in acetone. Values obtained at A76 mu were com- I puted from a capsanthin standard using equation A = MET x bc. Figure 3-A and Table 10 show that Ethephon has increased capsanthin content. I %' Table 10.--Effect of ethephon treatment applied two and three weeks after anthesis on Capsanthin con- tent of Paprika-505 fruits (1970). Increase mg Cap- Concentration Mg Capsanthin/g santhin for Treatment None 1.8A -- 100 ppm 2.38 0.5A 200 ppm 2.82 0.98 500 ppm 3.82 1.98 . "-' (min: The mean increases of capsanthin in the pericarp were 29, 70 and 102% for 100, 200 and 500 ppm ethephon respec- tively as compared with the control. The unit increases in mm! capsanthin for different treatments were higher as compared to the unit increases in total extractable color as shown in Table 9, Experiment III which suggests that ethephon may increase capsanthin and its derivatives which are responsi- ble for red color in pepper. A1 Color Stability. Color stability is another parameter of quality in paprika which is used by processors. Ethephon treatments have increased color content of Paprika-505 with each increased concentration. Therefore, it was of interest to determine if there was any relationship between increase and stability of color due to ethephon treatment. One-gram ground samples from each lot was exposed to 70°C for 0, 20, A0, and 80 hours. All determinations were made in duplicate from the bulk sample of the replicates. As shown in Table 11 the untreated fruit lost the most color with a larger retention of color with increased con— centrations of ethephon. These results confirm the report of Chen and Gutmanis (12) who indicated that the rate of color loss diminishes with time in storage at 70°C. The average estimated times for 25% color loss during the 80 hour period were 37, AA, A8, and 58 hours for control, 500, 100 and 200 ppm ethephon, respectively as determined from Figure A-A. According to Chen and Gutmanis (12) the per- cent loss calculated for a given time will not give a true picture of the overall color stability of the sample. Therefore, the second order reaction rate—constant, K2 was calculated from Table 11 as shown in Table 13 using equa— tion K2 = % ' §T§§;7-and in Figure A-B from Table 12 as proposed by Chen and Gutmanis (l2). _ t . X Rate constant, K2 - t ET§:§7u A2 Table ll.--Effect of ethephon treatments on color stability of Paprika-505 stored at 70°C (1970). Optical Density of 1/5000 Dilution at A60 mu Concentrations % % % O-hr 20-hr Loss AO-hr Loss 80-hr Loss None 0.325 0.269 17.2 0.238 26.7 0.195 no.0 100 ppm 0.375 0.315 16.0 0.28A 2u.2 0.2u1 35.7 200 ppm 0.u10 0.355 13.u 0.323 21.2 0.263 35.8 500 ppm 0.u57 0.398 13.5 0.368 19.u 0.323 30.0 Table l2.--Effect of ethephon on reciprocal of extractable color content (l/E A60) of Paprika-505 stored at 70°C (1970). Concentrations O-hr 20-hr AO-hr 80-hr None 3.08 3.71 A.2A 5.13 100 ppm 2.69 3.17 3.52 A.1A 200 ppm 2.Au 2.81 3.09 3.80 500 ppm 2.19 2.51 2.71 3.09 Table l3.-—Second order reaction rate constant (K2) of ethephon treatments to Paprika-505 after treated at 70°C. Concentrations O-hr 20-hr AO-hr 80-hr None -- 12.80 A5.20 167.20 100 ppm -- 10.20 3u.u0 120.00 200 ppm —- 7.60 26.u0 100.u0 500 ppm -- 6.60 21.20 72.80 A3 Figure A.-—A. Color deterioration curve of Paprika-505 stored at 70°C. The quarter-life periods, t l/A, from the graph were determined and given in the text. B. A second-order reac- tion plot of color deterioration in Paprika—505; reciprocal of extractable color content vs. storage time. AA mmm . . I , .0 waww m a . 8 W m m m. .w s a. /. m D. _ . _ ~ \ / U E . _ NIIIII ~II IIII IIIIIIIIIIII . l O 0 mm . u . .u s a . / 6 H E _ . _ \u ~HII\ IIIIIIIIIIIIII . a \m sm fl IIIIIIIIIIIIIIIII / W . . s. I. . 1 O I .\ r. g .............. / 4 T .\ M "\~ W m / / WU.— .\ .1 1 1 , . 0 A x \mm. m I / xfizR A .\ \x H\ m m m B I x ./ m \ \ s" . m " Iv . S P \. ks. \u u u n . P . w (0 O 0 0 0 0 9 9 9 9 0 0 0 0 0 I I I I 5. 4. 3 2 I 5. 4. 3. 2 0 O O 0 0 1281. 2 >tmzuo .2056 1209. 2 Emzmo .2055: A5 a = initial extractable color content; (a—x) = existing color content at time t; X = decrease in color content during time t; t = storage time in hours. The results indicate that color loss was not propor- tional to the square of the existing color content as pro- posed by Chen and Gutmanis (12). Salt Dispersion (NaCl). The color stability of dif- ferent rates of ethephon treatments at a temperature of 70°C were determined. The initial optical density of Paprika-505 at 1/5000 dilution of the acetone extracts were 0.2A6, 0.305, 0.335 and 0.A50 for control, 100, 200, 500 ppm ethephon, respectively. Colors were equalized to the same concentration by dispersing 18.3, 1A.8, l3.A and 10 ml of extract from control, 100, 200 and 500 ppm of ethephon on 100 g NaCl. Time required for color to fade from the control sample was 96 hours. It took 9, 1A and 23 hours additional for 100, 200 and 500 ppm of ethephon treated fruit pigment to fade. A6 Malathion Experiment (1970) According to recent findings malathion (0,0—dimethyl dithiophosphate of diethyl mercaptosuccinate) an insecti- cide, may serve as a dual purpose chemical in some fruit crops. Reports of Devlin gt gt. (19), Eck (2A) and Devlin gt gt. (20) have shown an increase of color in cranberries from malathion sprays. Peppers are frequently injured by aphids. They feed on plants and are more serious on younger than on older plants. They are also a vector for virus disease, which can destroy the entire crop. Malathion is an effective chemical to control this insect. With this background, it was of interest to determine if malathion had any effect on pepper color. A study was conducted with transplanted seedlings of Paprika-505 in a greenhouse experiment. The plants were sprayed twice, at weekly intervals two weeks after anthesis. Fruits were harvested at a completely shriveled stage at one time. Pericarps were separated from seeds and stems, dried, ground, extracted and total color determined. The color of pepper pericarp tissue increased signifi- cantly with 1000 and 2000 ppm malathion (Table 1A). Observations indicated that the treatment did not cause a reduction in fruit set. A7 Table 1A.--Effect of malathion treatments on color content of Paprika—505 (1970). Concentrationsoptical Density of 1/2500 Dilution at A60 mu Rep. 1 Rep. 2 Rep. 3 Mean None 0.535 0.510 0.532 0.526a 1000 ppm 0.555 0.580 0.605 0.580b 2000 ppm 0.715 0.655 0.702 0.6910 Means followed by different letters are significantly different at 1% level as determined by Duncan's Multiple Range Test. A8 SADH Experiment (1970) To determine the effect of SADH on pigmentation of pepper two experiments were conducted in the greenhouse. Treatments were applied to whole plants and branches of plants at the rates of 3000 and/or 6000 ppm. Treatments, for the whole plant test, were applied two weeks while for the branch treatment at four weeks after anthesis. The treatments were applied twice at weekly intervals. As shown by the data (Table 15), SADH treatment appar- ently resulted in a decrease in total extractable color due to a decrease in capsanthin and its derivatives. Table 15.--Effect of SADH applied two weeks after anthesis on color content of Paprika-505 (1970). Optical Density of 1/5000 Dilution at A60 mu Concentrations Rep. 1 Rep. 2 Rep. 3 Mean None 0.A33 0.A30 0.390 0.A18 3000 ppm 0.395 0.A05 0.360 0.387 6000 ppm 0.375 0.380 0.3A5 0.367 Further evidence of the effect of SADH was observed in an experiment in which the color of the fruit from treated branches averaging 0.A53 was not significantly different than the 0.A56 optical density observed in con— trols. While there was a decrease in color content, there appears to be a slight increase of color stability (Table A9 16). The percent loss of color was less at the higher rate of SADH application for the 80—hour period. Table 16.-—Effect of SADH treatments on color stability of Paprika—505 stored at 70°C (1970). Optical Density of 1/5000 Dilution at A60 mu Concentrat1°ns O-hr 10-hr 20—hr AO-hr 80—hr % Loss None 0.A13 0.363 0.359 0.331 0.285 30.9 3000 ppm 0.380 0.3A5 0.323 0.306 0.275 27.6 6000 ppm 0.365 0.322 0.319 0.301 0.275 2A.6 50 Ethephon Experiment (1971) The influence of ethephon on Paprika-D.Z. was tested in the greenhouse at 70 to 75°F DT in a split plot design. Seedlings transplanted on July 2A, 1971 were sprayed with ethephon twice, at weekly intervals, four or six weeks after anthesis. Treatments on plants sprayed with 500, 200, 100, 50, and 0 ppm four weeks after anthesis started to ripen A, 10, 12, 1A, and 17 days respectively after the second spray. Fruits sprayed twice at weekly intervals six weeks after anthesis started to ripen 6, 8, 10, 12 and 1A days after the first spray with 500, 200, 100, 50 and 0 ppm ethephon respectively. Flower and bud formation was arrested after the second spray application with 500 ppm ethephon during either period. Color content as a result of different treatments were compared from pericarps of fruits picked at the com— pletely shriveled stage. A significant difference (t.05 = 0.105) in color content was obtained due to concentrations of ethephon application (Table 17). The data showed no significant difference between time of application. Color Stability. Results of duplicate determinations from bulk samples is shown in Figure 5. The values are transformed to percent loss (Table 18). The observed results during the three periods of heat treatment indicated a loss of color with increased rate of ethephon application. The color retention was lower from 51 Table 17.--Effect of ethephon treatments at different stages after anthesis on color content of Paprika-D.Z. (1971). Concen- Weeks Optical Density of 1/5000 In- trations After Dilution at A60 mu crease Anthesis Rep 1 Rep 2 Rep 3 Rep A Mean % None 0.A09 0.367 0.A32 0.398 0.A02 —- 50 ppm A 0.A09 0.A56 0.A20 0.A09 0.A2A 5.2 50 ppm 6 0.A56 0.A2A 0.A56 0.AAA 0.AA5 9.7 100 ppm A 0.A75 0.A88 0.A20 0.A20 0.A5l 10.9 100 ppm 6 0.A20 0.516 0.A98 0.A32 0.A67 13.9 200 ppm A 0.A56 0.AAA 0.A75 0.A56 0.A58 12.2 200 ppm 6 0.538 0.A56 0.A95 0.A56 0.A86 17.7 500 ppm A 0.550 0.620 0.585 0.638 0.598 32.9 500 ppm 6 0.585 0.523 0.509 0.620 0.57A 30.0 L.S.D. (.05 level) .0A6 L.S.D. (.01 level) .066 Table 18.--Effect of ethephon treatments on color stability of Paprika-D.Z. stored at 70°C (1971). Weeks Initial Concentrations After Value Percent LOSS A Optical 20-hrs. AO-hrs. 80-hrs. nthesis Density None — 0.398 11.5 19.8 3A.6 50 ppm A 0.A03 15.1 23.1 A3.9 50 ppm 6 0.A32 16.2 29.A A5.1 100 ppm A 0.A32 18.5 28.8 A6.9 100 ppm 6 0.A62 12.5 2A.6 A3.l 200 ppm A 0.A50 19.5 30.2 52.2 200 ppm 6 0.A69 16.2 35.6 51.8 500 ppm A 0.585 27.8 38.9 65.6 500 ppm 6 0.538 21.9 A7.2 62.1 52 Figure 5.-—Color loss of Paprika-D.Z. fruit pericarps sprayed with 0, 50, 100, 200 and 500 ppm Ethephon A and 6 weeks after anthesis stored at 0, 20, A0 and 80-hours (70°C). OPTICAL DENSITY AT 460 my. 0600 0550 Q m 0 O 0450 0400 0350 0300 0250 0200 I I I I I 53 ETHEPHON o NONE A 50 ppm \ o IOOppm 4 D 200 ppm O 500 ppm 20 4O 60 HOURS AT 70°C 5A treatments applied at four rather than six weeks after anthesis (Figure 5 and Table 18). This experiment sug- gests that ethephon enhances the respiration rate of the cultivar Paprika-D.Z. and further supports the report of Lockwood (51) that ethephon increases the respiration rate of pepper. Capganthin. The capsanthin was isolated with phase separation and thin-layer chromatography. The content for each treatment was determined from the standard (Figure 2-B). Values presented in the table are from the bulk sample of each treatment (Table 19). Table l9.--Effect of ethephon on capsanthin content of Paprika-D.Z. (1971). Mg Capsanthin/ Increase mg concentrations g of fruit Capsanthin for Treatment None 1.89 —- 50 ppm 1.97 0.09 100 ppm 2.08 0.19 200 ppm 2.26 0.37 500 ppm 2.52 0.63 In this study, as in the previous one, there is an increase in capsanthin content due to ethephon treatment. The increase was found to be A.6, 10.1, 19.5 and 33.9 per- cent for 50, 100, 200 and 500 ppm ethephon, respectively, as compared to the non-treated control. Capsanthin under- goes oxidation (57) which is increased by ethephon (51). The cultivar Paprika-D.Z. is more sensitive to ethephon 55 application and results in a lower level of increase in total extractable color and capsanthin than Paprika-505. 56 Ethephon, Malathion and SADH Experiment (1971) The color content of the pericarps was increased significantly (Table 20) by the application of 200 and 500 ppm ethephon. Likewise, a significant increase in color was found when 2000 and A000 ppm malathion were applied six weeks after anthesis. The chemical SADH did not influence color development. Table 20.--Effect of ethephon, malathion and SADH on color content of Paprika-D.Z. (1971). Chemicals Ethephon Malathion SADH Levels Weeks After Anthesis None A 6 A 6 A 6 1l 0.A22 0.u2u 0.Au5 0.u23 0.A20 0.A12 0.A15 2 0.A70 0.A39 0.A51 0.A32 0.399 0.380 0.A15 3 0.A80 0.A58 0.A22 0.A57 0.A12 0.AOA 0.A15 A 0.A93 0.A8A 0.AA6 0.A60 0.A27 0.A02 0.A15 L.S.D. (.05 level) 0.0A0; L.S.D. (.01 level) 0.050 lLowest level Interaction between chemicals, concentration and time of application. This investigation indicated that the chemical, the concentration and the time of application had different influences on pigmentation (Figure 6). The increases in color with 100, 200 and 500 ppm at four weeks and 500 ppm ethephon applied at six weeks were significantly higher (t 01 = 0.0A9) than the increase found from SADH Figure 6.--Interactions of chemical, concen- tration and time on color increase of Paprika-D.Z. l. 50, 500 and 1000 ppm ethephon, malathion and SADH, respectively. 2. 100, 1000 and 2000 ppm ethephon, malathion and SADH, respectively. 3. 200, 2000, and A000 ppm ethephon, malathion and SADH, respectively. A. 500, A000, and 8000 ppm ethephon, malathion and SADH, respectively. 58 m_mmI.—.Z< mmhm< mxmm; 201.5sz (NOIIVHINBONOOI "IVOIWEHO 59 treatments applied six weeks after anthesis. The color increase from 2000 and A000 ppm malathion applied at six weeks was significantly higher (t 0 = 0.037) than the 5 increase obtained from the SADH treatment. The second order interaction indicated a decrease in color when 100, 200 and 500 ppm ethephon or 1000, 2000, A000 and 8000 SADH were applied six weeks after anthesis. Interaction between chemicals and concentrations. The increase of color at 200 and 500 ppm ethephon was signifi- cantly greater than all levels of SADH but not to malathion treatments (t = 0.0A5). Similarly, the difference in .05 increase of color between 500 ppm ethephon and 500, 1000 and 2000 ppm malathion was significant. Mean increase of color at A000 ppm malathion was higher than the increase found from 1000 and 2000 ppm SADH treatments (Figure 7). Most of the interaction of chemicals with concentrations is due to a decrease in color when 2000 and A000 ppm SADH were applied. Interaction between chemicals and time. There was a highly significant difference between chemicals and time of application (Figure 8). The data indicated a signifi- cant difference in color (t = 0.02A) when ethephon was 01 applied at four and six weeks as compared with SADH. Also, ethephon applied at four weeks resulted in a significantly higher color than malathion. The decrease in color from ethephon and SADH applied six as compared to four weeks 60 Figure 7.—-Interactions of chemicals and con- centrations applied on color increase of Paprika-D.Z. 61 Ema oooo oooe ooom ooo. oooe ooou ooo. oom 00m 00m 00. on I owed /// / W 65.0 7 50.0 wood 5% n. 20.12.22 7% 20:3th I 000.0 000.0 80100 ESVBHONI 62 Figure 8.--Interactions of chemicals and time of application on color increase of Paprika- D.Z. 63 909.324 Kuhn? mxwmz, :95 § 2055.445). Wl/A zozouzm RU 30.0 I 0N0.0 I 0¢0.0 I 000.0 00 80100 BSVBHONI 6A after anthesis is the major cause for the interaction (Figure 8). The effect of concentration and time of appli- cation was indicated in Tables 21 and 22. The increase of color at 500 ppm was significantly higher than that for 50 ppm ethephon. There were no significant differences in increase of color between concentration of application for malathion and SADH. Likewise, there was no significant difference in color content between time of application for any one chemical (Table 22). Color Stability. The stability of color in storage (70°C) at different times was tested and evaluated (Figure 9). The data presented is from the fruits that have received treat— ments at four weeks after anthesis. The values (optical density) were transformed to percent loss (Table 23). There was a significant difference between treatments during either period. The mean color loss is highest for ethephon treatment followed by non-treated control plant fruit pericarps. The chemical, SADH treatments showed the least in color loss in either period (Table 23). 65 Table 2l.-—Effect of chemical concentration on color con— tent of Paprika-D.Z. (1971). Optical Density Chemicals Conc. l Conc. 2 Conc. 3 Como. A Mean Ethephon 0.A23 0.A55 0.A69 0.A89 0.A59 Malathion 0.A3A 0.AA2 0.AAO 0.A53 0.AA2 SADH 0.Al6 0.390 0.A08 0.A15 0.A07 L.S.D. (.05 level) 0.038 L.S.D. (.01 level) 0.051 Table 22.—~Effect of application time on color content of Paprika-D.Z. (1971). Optical Density Chemicals Weeks After Anthesis A 6 Mean Ethephon 0.A66 0.A5l 0.A59 Malathion 0.AA1 0.AA3 0.AA2 SADH 0.A1A 0.A00 0.A07 L.S.D. (.05 level) 0.018 L.S.D. (.01 level) 0.024 66 Figure 9.--Interaction between chemical, con- centration and time on color loss of Paprika-D.Z. l. 50, 500 and 1000 ppm ethephon, malathion and SADH, respectively. 2. 100, 1000 and 2000 ppm ethephon, malathion and SADH, respectively. 3. 200, 2000 and A000 ppm ethephon, malathion and SADH, respectively. A. 500, A000 and 8000 ppm ethephon, malathion and SADH, respectively. 67 __—— ETHEPHON ---- MALATHION —-— SADH F0000 _0.|00 ,—0.200 -0.300 00 -0.400 ~0500 r0.600 80 68 Table 23.--Effect of ethephon, malathion and SADH on color stability of Paprika-D.Z. stored at 70°C (1971). Storage Time 20 Hours Levels Percent Loss Ethephon Malathion SADH None 1 30.0 15.0 18.5 38.5 2 51.5 31.5 25.0 38.5 3 A9.0 30.5 22.5 38.5 A A3.0 31.5 2A.5 38.5 Mean Loss A0.9 27.1 22.6 38.5 L.S.D. (.05 level) lA.7 L.S.D. (.01 level) 16.0 Storage Time A0 Hours 1 A0.5 A1.5 38.5 AA.0 2 61.0 A3.0 39.0 AA.0 3 58.5 A5.5 3A.0 AA.0 A 61.0 39.0 3A.0 AA.0 Mean Loss 55.3 A2.3 36.A AA.0 L.S.D. (.05 level) 18.2 L.S.D. (.01 level) 19.8 Storage Time 80 Hours 1 67.5 63.5 56.0 66.0 2 7A.5 66.0 62.0 66.0 3 73.5 66.0 55.5 66.0 A 78.0 59.0 55.0 66.0 Mean Loss 73.A 63.6 57.1 66.0 L.S.D. (.05 level) 13.2 L.S.D. (.01 level) 13.7 GENERAL DISCUSSION The carotenoid content of paprika fruit pericarp increases with maturation. This increase in color during fruit maturation and ripening may be due to either the disappearance of chlorophyll and synthesis or accumulation of carotenoids or to both (70). Paprika fruits harvested when ripe and completely shriveled had the highest color. Leonard gt_gt, (50) reported a thirty fold increase in carotene when pepper fruits turned from green to red. Fruits harvested at this completely shriveled stage had a high initial color but a shorter period of color retention in storage. The high rate of color loss as the fruit advances in ripening is associated with a rapid increase in respiration (70). Regardless of the cultivar, this study as well as that reported by Lease and Lease (A8), affirm that a basic understanding of the proper index for harvest- ing is essential to obtain maximum extractable and poten- tially stable color during processing and storing. The effect of plant nutrients on color of paprika fruits was examined. The highest color was found in the fruit from plants grown in the soils that had received high levels of K and Cu. In contrast, plants grown with high levels of N but low levels of P, K, Mg, Mn and Cu, the 69 70 color was lower compared to those grown in high levels of N, P, K, Mg, Mn and Cu. The effect of N on carotenoid develOpment depends upon the source of N and type of crop (36). Kobayashi gt gt. (AA,A5) reported that increasing the K level in relation to N improved the fruit color of grapes. Weeks gt gt. (77,78) found similar results with apple fruits. Schertz (6A) indicated that the chlorophyll content of potato leaves from plots that had received additional K contained less than the leaves of plants to which no potash was added. In turnips grown in sand cul— tures, deficient in S, N and K resulted in a decrease in carotene content while a deficiency of P showed no decrease (6). Trudel (73) reported that the carotenoid content of K-deficient tomato fruit was lower than that of normal fruit and suggested that K may regulate the amount of geranyl— gernayl pyrophosphate (GGPP). Chlorophyll and carotenoids are synthesized via GGPP and other intermediates and sug- gests that upon ripening of tomato fruits an intermediate synthesizes the carotenoid but not the chlorophyll (73). This indicates that K plays an important role in carotenoid formation during tomato ripening. Low levels of color content were found in fruits when the plants were fertilized with high levels of Mn and Mg. With cv. Paprika—505, where ethephon was applied the total extractable color was the highest for all treatments as compared with the non—treated control. The percent 71 increase in capsanthin content induced by 100, 200 and 500 ppm ethephon was higher than from the pericarp of unsprayed plants. Similar increases in total extractable color and capsanthin content for cv. Paprika-D.Z. was found. How- ever, the percent increases in total extractable color obtained was less than from cv. Paprika-505. Color retention decreased as concentration of ethephon increased. Such differences might be accounted for by the differences in response of the cultivars to ethephon sprays or to the environment that prevailed. High temperatures during maturation enhances ripening and increases respiration. Goodwin (36) reported that carrot require an optimum temperature range of 60-70°F for optimum carotene produc- tion in roots, and also that in tomato bagging maturing fruits on the vine reduced the carotenoid content. Ethephon application increases respiration of pepper (51). Many reports indicate that application of antioxi- dants to stored peppers reduce color loss (l2,A8,76). Although, the reports were related to experiments con- ducted on stored pepper, it may be practical to apply antioxidants in the field before and after ethephon appli- cation to prevent color loss during ripening, drying, processing and storage. On the basis of the evidence 72 found in this study, it appears quite possible to increase the color of paprika with ethephon spray. Regardless of the cultivar or concentration, no sig- nificant response to color development was found from SADH sprays. However, some reduction occurred in both cultivars. The observed color content from sprayed and unsprayed branches may indicate that the influence of SADH may be translocated in the plant. The result of this experiment as well as the reports of Martin and Williams (53), Edgerton and Greenhalgh (26) may indicate that SADH is highly mobile within the plant. The high concentrations (2000 and A000 ppm) of applied malathion increased significantly the color content of fruit pericarps. A definite difference in color content between the two cultivars was found for either concentration of malathion applications. The increase in color for cultivar Paprika-505 was higher than for Paprika-D.Z. Difference in the response of the cultivar to malathion and/or environ- ment may be related to the variation. Comparing the three chemicals, ethephon had greater effect on increasing the color than malathion. The chemi- cal SADH did not increase color development but had a slight reverse effect. Conversely, SADH treatment resulted in better retention in storage than either ethephon or malathion. SUMMARY Tests were conducted to determine the effect of plant nutrients and growth regulators on color content of paprika fruit pericarps. 1. Analyses of ripe shriveled fruits indicated a higher concentration of color than in ripe fruits that had not shriveled. Ripe and completely shriveled fruits lost color in storage (70°C) more rapidly than ripe fruits that were not shriveled. Differences in color content were correlated with a variation in nutrient supply. Color in the pericarp was positively correlated with K and/or Cu concentra- tion in the fruits. There were indications that increasing the N, P or Mn levels in the fruits resulted in decreasing the color content but the magnitude of the variation was not significant. Wide variation in color of the fruit pericarp due to application of ethephon, malathion and SADH was found. ethephon and malathion increased the color content of the fruit pericarps with increasing concentration. The chemical SADH did not influence the color content sig— nificantly but tended to reduce it. 73 7A The color loss of the paprika in storage (70°C) was slower with SADH than with ethephon or non—treated control and malathion treatments, respectively. Variation between two cultivars in total extractable color, capsanthin contents and retention of color in storage (70°C) due to ethephon treatments were evident. 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