3 1293 0064m llllllllll‘fl LIBRARY Michigan Stat: University This is to certify that the thesis entitled THE APPLICATION OF ANTHOCYANINS AS COLORANTS FOR MARASCHINO TYPE CHERRIES presented by MARK RICHARD MCLELLAN has been accepted towards fulfillment of the requirements for M.S. degree in FOOD SCIENCE (M M. (L/ y 1// Major professor Date April 25, 1978 0-7639 ‘3 4".' . AUG 1"3‘312!’ "r‘ ‘3 (:37 @9333 6/ JUN 0472003 THE APPLICATION OF ANTHOCYANINS AS COLORANTS FOR MARASCHINO TYPE CHERRIES BY Mark Richard McLellan A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1978 ABSTRACT THE APPLICATION OF ANTHOCYANINS AS COLORANTS FOR MARASCHINO TYPE CHERRIES BY Mark Richard McLellan Anthocyanins were extracted from dark, tart cherries and used as colorants in the manufacture of maraschino type cherries. Studies were initiated to determine the effects of added metal salts such as SnClZ, a K2C03 dip as a surface wax treatment, and varying storage temperatures on the stability of anthocyanin colored, maraschino type cherries. The addition of SnCl2 appeared to have no effect on the rate of degradation of pigment. The K2C03 dip caused a lighter shade of red due possibly to an intracellular pH alteration although the antho- cyanin concentration remained equal to that of the other treatments. The storage phase of this study shows the color to be relative-' 1y stable for an estimated period of six to nine months, depending on variations from.ambient temperature. These studies indicated that maraschino fruit can be produced using anthocyanins as the colorant although their color is somewhat different from that obtained using organic dyes. to my wife, Deanne ii ACKNOWLEDGMENTS I am sincerely grateful to Dr. J.N. Cash for his guidance, sug- gestions, and support throughout the course of this study and the prep- aration of this manuscript. I would also like to thank the other members of my guidance committee, Drs. R.L. Andersen, P.C. Markakis, and MlA. Uebersax. Special thanks to Dr. R.L. Anderson for his contribution of various cherry varieties for use in this project. Appreciation is also extended to R.C. Warren & Company Inc., Traverse City, Michigan for their generous supply of brined Napoleon cherries. Acknowledgment is extended to the Michigan Association of Cherry Producers for their partial financial support of this work. Finally, I am most grateful to my wife, Deanne, for typing the final manuscript and I extend my deepest appreciation for her encourage- ment and sacrifices during the course of my studies. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . LIST OF FIGURES . . . . . . . . . INTRODUCTION . . . . . . . . . . LITERATURE REVIEW . . . . General Anthocyanin Chemistry . . . Anthocyanin Interactions: Degradation and Stability . . . . . . . . Anthocyanin Extraction and Measurement Anthocyanins as Food Colorants . . . METHODS AND MATERIALS . . . . . . Comparison of Anthocyanin Sources . Modification Analysis . . . . . . Juice Preparation . . . . . . . Brined Cherry Preparation. . . . . Coloring and Processing . . . . . Storage Conditions . . . . . . . RESULTS AND DISCUSSION. . . . . . . Comparison of Anthocyanin Sources . . Modification Analysis . . . . . . Maraschino Cherry Coloring . . . SUMMARY AND CONCLUSIONS . . . . . . iv Page vi vii 13 l7 l9 19 20 21 21 22 22 24 24 25 25 46 Page APENDICES . O O I O O O O O I O O O O O O O O 48 IA. F statistics for the respective sources of variation for each type of measure made . . . . 48 IB. The Bonferroni t-statistics for the Hunter "a" value data on the treatment main effect. . . . 49 IIA. The Bonferroni t-statistics for the Hunter "L" value data treatment combination means . . . . 50 113. The Bonferroni t-statistics for the Hunter "a/b" value data treatment combination means . . . 51 IIC. The Bonferroni t-statistics for the Hunter "b" value data treatment combination means . . . . 52 III. Reaction rate constant K (days‘l) for the pigment exposed to the various treatments . . . . 53 IV. Calculation of the Bonferroni t-statistics . . . . 54 LIST OF REFERENCES I O O O O O O O O O 0 O O O O 55 Cited References. . . . . . . . . . . . . . . 55 General References . . . . . . . . . . . . . . 58 LIST OF TABLES Table Page 1. Comparison of cherry selections for anthocyanin content . . . . . . . . . . . . . . . . 24 vi Figure 10. 11. 12. l3. 14. 15. 16. 2-Phenyl-benzopyrylium (flavylium) structure . Six common Structures of anthocyanins at various pH levels . LIST OF FIGURES anthocyanidins . . Origin of the carbon atoms in the flavanoid skeleton Thermal decomposition ofxnalvin . . Bisulfite bleaching of anthocyanins . Anthocyanin degradation by phenol oxidase . Hunter L, a, b, Color Solid. The change time for The change time for The change time for The change time for The change time for The change time for The change time for The change time for in Hunter "L" value at 99°F each treatment in Hunter "L" value at 65°F each treatment . . . . in Hunter "L" value at 35°F each treatment . . . . in Hunter "a" value at 99°F each treatment . . . . in Hunter "a" value at 65°F each treatment . . . . in Hunter "a" value at 35°F each treatment . . . . in Hunter "a/b" value at 99°F with each treatment . . . . in Hunter "a/b" value at 65°F with each treatment . . . . vii with with with with with with Page 11 12 16 28 29 30 32 33 34 35 36 Figure 17. 18. 19. 20. 21. 22. 23. 24. The change time for The change time for The change time for The change time for The change in Hunter "a/b" value at 35°F with each treatment . . . . . . in Hunter ”b" value at 99°F with each treatment . . . . . . in Hunter "b" value at 65°F with each treatment . . . . . . in Hunter "b" value at 35°F with each treatment . . . . . . in the absorbance difference at 99°F with time for each treatment. . . . . The change in the absorbance difference at 65°F with time for each treatment. . . . . The change in the absorbance difference at 35°F with time for each treatment. . . . . The change in the reaction rate with temperature for each treatment 0 O O . O C O . viii Page 37 38 39 40 42 43 44 45 INTRODUCTION The deepest color contrasts in the predominantly green sur- roundings of nature are the red and blue shades, most commonly due to the class of flavanoid compounds called anthocyanins. Marquart, in 1835, first used the term.anthocyanin (from.the Greek: anthos, flower and kyanos, blue) to describe these water soluable pigments, which are widely distributed in nature (Harborne, 1967). In the past, the antho- cyanins' relative instability has caused them to be regarded as inter- esting objects for laboratory study but with very few uses in practical applications. More recently, these pigments have been considered as possible alternatives to the red dyes in the food industry. The recent banning of several red colorants, coupled with the growing concern over coal tar dyes, has led to a greater interest in the practical applications of anthocyanins as food colorants. In Michigan, the growing of sweet cherries for brining and subsequent dyeing to produce the maraschino cherry is a typical area where the introduction of a reliable and stable, natural red colorant would be a welcome innovation. At present, the maraschino cherry industry utilizes some 12,000 lbs of Red dye #40, however the future of this dye is uncertain and so the need for practical alternatives is a paramount concern. The objectives of this study were as follows: (1) to apply 1 the use of natural colorants to the production of maraschino type cherries and (2) to investigate the supposed protective effect of some metals on a naturally occuring anthocyanin system, (as opposed to a pure anthocyanin extract). In addition, the methods used in this study could lead to some interesting comparisons between a tristimulas method of color measurement and a 'direct' method of anthocyanin measurement . LITERATURE REVIEW General Anthocyanin Chemistgz Anthocyanins are glycosidic forms of anthocyanidins (agly- cones), whose basic skeletal structure is derived from the flavylium nucleus (Z-phenyl-benzopyrylium; Figure l). Figure l. 2-Phenyl-benzopyrylium (flavylium) Although the flavylium shown in Figure l is in its oxonium form, the positive charge is actually delocalized over the entire structure. Sixteen anthocyanins are known to exist but the six shown in Figure 2 occur in foods more often than any of the others. Although the occurence of free aglycones in nature has been reported from time to time (Harborne, 1960), it is much more the exception than the rule be- cause they almost always occur in glycosidic forms. PETUNIDIN PEONIDIN MALVIDIN Figure 2. Six common anthocyanidins Swain (1965) notes a total of six different classes of antho- cyanin glycosides: 3-monosides, 3-biosides, 3-triosides, 3-monoside- 5-glucosides, 3-bioside-5-glucosides, and 3-bioside-7-glucosides. Harborne (1967) and Markakis (1974) have combined the 3-bioside-S- glucosides and the 3-monoside-5-glucosides into one class designated as the 3,5-diglycosides, thereby listing a total of five glycosidic classes. In addition to the various glycosides, anthocyanins may also have acyl groups attached to the molecule. Due to their lability, it has been very difficult to determine the structure of the acylated anthocyanins but it has been shown that most of the acylated antho- cyanins isolated thus far have their acyl substituent attached to the sugar in the 3-position (Harborne, 1967) and this substituent is nearly always p-coumaric acid although caffeic and ferulic acids do appear occasionally (Harborne, 1967). There are further structural limitations noted in these antho- cyanin pigments. The monoglycosides appear to always be attached in the three position, except in the case of 3-deoxyanthocyanins in which the sugar is usually attached at the five position. There is no re- cord of a B ring hydroxyl having glycosidic substitution. If the mole- cule has a second position glycosylated, it is nearly always in the five rather than the seven position and when the five or seven position is glycosylated the sugar is always glucose (Harborne, 1967). The most obvious characteristic of these pigments is the fact that they absorb light in part of the visible range of the spectrum (400 to 800 mu) and are observed to be colored. The absorption of light causes the electrons to be excited. The more firmly bound these electrons are, the higher will be the energy requirement to produce excitation and the shorter will be the wavelengths at which light will be absorbed. The wavelengths not absorbed are reflected back and it is these wavelengths which make up the observed color. This color characteristic can be altered depending upon the physiochemical environment in which the pigment is viewed. The degree of hydrcxylation and methoxylation not only determines the difference between anthocyanidins but also the color of each. Increasing the number of phenolic hydroxyls tends to increase the blueness of the pigment with a hypsochromic effect. Methylation of the hydroxyl groups tends to increase the redness of the pigment with a bathochromic effect (Harborne, 1967). A change from.3 to 3,5-glycosylation has virtually no effect upon the visible maxima but the 3,5-dig1ycoside does appear to be slightly fluorescent in solution as compared to the 3-glycoside (Harborne, 1958). Anthocyanins behave much like pH indicators. This amphoteric nature is characteristic of these pigments and allows them to act as either an acid or a base, depending upon the pH of the media (Figure 3; Markakis, 1960). Red cation, Colorless, uncharged, Blue anion, low pH isoelectric point high pH Figure 3. Structures of anthocyanins at various pH levels. (R=glycosyl) 0n the acidic side of the isoelectric point, the color is red; at the isoelectric point, the pseudobase (shown in the center of Figure 3) is colorless and at pH's higher than the isoelectric point, the neg- atively charged tautomers are blue (Markakis, 1960). During the biosynthesis of the flavanoid structure, the A ring is formed by head to tail condensation of three acetyl (malonyl) units while the B ring and carbons 2, 3, and 4 originate from an intact phenylpropane unit (Figure 4; Grisebach, 1965). The phenylpropane compounds that most effectively serve as precursors for the B ring and its three associated carbon atoms are L-phenylalanine, cinnamic acid, and p-hydroxycinnamic acid (Grisebach, 1965). ‘ ll ‘ (6) (0) O phenylpropane unit A.carboxylgroup of acetate (malonate) I methyl group of acetate (malonate) Figure 4. Origin of the carbon atoms in the flavanoid skeleton. Anthocyanin Interactions: Degradation and §tabilit1 The flavylium nuclei of anthocyanins are highly reactive as a result of their electron deficiency and these compounds therefore readily undergo undesirable structural and color changes under a var- iety of processing and storage conditions employed in the fruit industry. In 1943, Beattie g£_a;; showed that storage effects on straw- berry juice were more noticeable in containers with air in the headspace than in containers fully filled. This early indication of interactions between oxygen and anthocyanins has been confirmed by numerous other works. Nebesky et a1. (1949) implicated heat and oxgyen as the most specific accelerating agents responsible for deterioration of color during storage while Daravingas and Cain (1965) and Starr and Francis (1968) reinforced findings that plant pigment loss can be attributed to the presence of oxygen. As with many chemical reactions, the rate of anthocyanin degradation can be shown to be directly proportional to temperature. Many investigations have established that anthocyanins are unstable to heat (Kertesz and Sondheimer, 1946; Mackinney and Chichester, 1952; Meschter, 1953 ) although the reactions involved in the thermal deg- radation of anthocyanins have not been completely elucidated. Hy- drolysis of the protective 3-glycosidic linkage may occur but no ex- perimental evidence has been brought forward to completely support this suggestion although some progress has been made recently by Hrazdina as cited by Jurd (1972). He found that heating the anhydro bases of malvidin, cyanidin, and peonidin 3,5-diglycosides at pH 7 partially converted them to their corresponding chalcones and caused the loss of the B ring to give the same colorless compound identified as the coumarin glucoside (Jurd, 1972). An example of this type of reaction is shown in Figure 5. (A) Malvin HC) 0 ‘3 + Cl \ OGI. Gli) (B) Chalcone (C) Coumarin glucoside Figure 5. Thermal decomposition of Malvin Powers and Esselen (1942) and Nebesky g£_§l. (1949) reported that the effects of light were of only minor importance in the deg- radation of anthocyanins in fruit juices as compared to the deleterious changes caused by heat and oxygen. Under extream conditions of expo- sure, light will act as an accelerating agent for reactions which would occur even if no light were present. The effects of sunlight on the flavylium salt-chalcone equilibrium in acid solutions has been studied by Jurd (1969) but in-depth studies of the long range effects of var- ious lighting conditions on the anthocyanin molecule have not been conducted. Sondheimer and Kertesz (1953) presented evidence for an in- direct ascorbic acid induced destruction of anthcyanins. Markakis ggiflg (1957) reviewed the evidence for ascorbic acid-oxygen interaction in terms of increased anthocyanin degradation and found that in the presence of oxygen , pigment alone or ascorbic acid alone is relatively un- stable, with the pigment being the more unstable of the two. In the absence of oxygen, the combined presence of both pigment and ascorbic acid leads to faster degradation than when each was alone. The inclu- sion of oxygen in the pigment ascorbic acid system exerts a synergistic effect which yields an even higher rate of degradation. Some degree of stabilization of the anthocyanin pigments can be achieved by metal or co-pigment complexes. Co-pigment complexes are anthocyanins combined with organic substances while metal complexes involve metal chelation with the anthocyanin molecule. Jurd and Asen (1966) studied the formation of metal and co-pigment complexes of cy- anidin 3-glucoside and found thatflavanol glycosides, such as quer- citrin and the gallates were among the most effective co-pigments and 10 noted that they were highly pH sensitive as are the metal chelates. An aluminum/co-pigment combination was found to under go a marked in- crease in intensity and noticeable bathochromic shift to lower energy and longer wavelength. Jurd and Asen (1966) demonstrated that some co-pigments required the presence of a metal to aid and possibly par- ticipate in the "co-pigmentation". Scheffeldt and Hrazdina (1978) noted that the resulting color intensification, due to co-pigmentation of anthocyanins under physiological conditions, enhances the possi- bility of their use as food colorants. Stannic Chloride (SICIZ‘Hmbeenre- ported as an effective chemical in stabilizing color in the pH range of 3.0 to 3.8 when determined by a Hunter Color Difference Meter on strawberry puree (Sistrunk and Cash, 1970). In 1973, Starr and Fran- cis reported that metallic ions, such as copper, aluminum, tin, etc., exerted very little protective effect on the color of cranberry juice cocktail. Experimental evidence indicates that anthocyanins are sen- sitive to the presence of sugars and their degradation products. Meschter (1953) and Tinsley and Bockian (1960) found that sugars and their breakdown products accelerated the degradation of anthocyanins. Daravingas and Cain (1965) in work involving the processing and stor- age of raspberry preserves noted that there was a significant increase in pigment degradation as the concentration of the sugar syrup was increased. As has been the case with other anthocyanin degradation reactions, the rate of breakdown due to the presence of sugars is highly sensitive to pH, oxygen availability, and temperature. Sulfur dioxide ($02) is one of the few allowed chemical ad- ditives recognized as safe (GRAS) by FDA and used extensively in the 11 fruit processing industry (Woodroof and Luh, 1975). It had been be- lieved that sulfite bleaching involved chalcones (Ribereau-Gayon, 1959), however, Jurd (lHflb) reported that sulfite bleaching reactions, at low concentration, (20 ppm) of anthocyanins involve equilibrium reactions, such as those shown in Figure 6. In the maraschino cherry industry where large concentrations of SO2 and lime are used for an- thocyanin destruction, these reactions are irreversible and the re- action mechanisms are not known. Figure 6. Bisulfite bleaching of anthocyanins. Several types of enzymes have been reported to be active in the degradation of anthocyanins. Huang (1955) reported on fungal glycosidases (anthocyanases) which may hydrolyze the glycosidic bond to yield an anthocyanidin, which could rapidly decolorize due to its instability. Peng and Mmrkakis (1963) found that anthocyanins alone are rather poor substrates for a phenol oxidase separated from mushrooms but in the presence of a better substrate, such as catechol, the anthocyanins proceed to decolorize rapidly. A probable mechanism was suggested as shown in Figure 7. Polyphenol oxidase and perox- idase have been implicated in anthocyanin degradation in a number of products and several investigations have been conducted to isolate 12 and characterize these enzymes in various plant tissues (Cash g£_§1;, 1976; Pifferi and Gultrera, 1974; Grommeck and Markakis, 1964; Tate et al., 1964; Reyes and Luh, 1960; Weurm n and Swain, 1953). It has been shown that several enzymes will decolorize anthocyanins but specific anthocyanases have not been isolated in a pure state and their existance is somewhat doubtful. ossaAoeo OZY Auruocvmm / OH ENZYME NONENZYMIO 0A HzotA‘ (1 AN THOOYANIN C) Figure 7. Anthocyanin degradation by phenol oxidase. It has been suggested that degradation of the anthocyanin molecule can occur in at least two possible sequences and possibly more pathways are yet to be found. As previously mentioned (Jurd, 1972), the anthocyanin molecule is thought to be partially converted to its corresponding chalcone and through subsequent decomposition, to a coumarin glycoside. Another pathway suggested by Adams (1972) and summarized by Markakis (1974) involves hydrolysis of the gly- cosidic bond yeilding a glycoside and unstable anthocyanidin. The anthocyanidin is in equilibrium with it's carbinol and diketone fonms which can degrade to various acids and aldehydes. Jurd (1972) has noted the possibility of decolorization through flavylium.condensation 13 reactions involving ascorbic acid, amino acids, sugars, sugar de- rivatives, and other cellular constituents. Markakis 3931(357) has used radiochemical studies to show that the typical red-brown pre- cipitate of anthocyanin solutions is comprised of the anthocyanins in degraded form. This polymerized form.of degraded pigment is a result of condensation of chalcones formed from the hydrolysis of the pyry- lium.ring. Anthocyanin Extraction and Measurement The classical solvent for anthocyanin extraction is a low boiling alcohol, such as ethanol or methanol containing small amounts of hydrochloric acid (0.1 to 1.0 Z v/v). Although this is still the most common solvent system used for extractions, arguments have arisen concerning the false conclusions possibly derived from data when using this system. Von Elbe and Schaller (1968) have found that wrong proportions of pigments and the presence of anthocyanins which do not occur naturally, resulted from drying of anthocyanin samples with small amounts of hydrochloric acid present. For this reason, some neutral solvents such as n-butanol, acetone-water-methanol mix- tures, cold acetone, and sulfur dioxide solutions have been offered as good alternatives. Fuleki and Francis (1968a)quantitatively ex- tracted anthocyanins from cranberries by macerating the frozen fruit with methanolic HCl, filtering the residue and repeatedly washing the residue with solvent until virtually all of the anthocyanin pig- ments were removed. Philip (1974) described an anthocyanin recovery system from grape wastes which utilized methanolic tartaric acid 0.1 to 1.0 Z. Neutralization of part of the acidity was achieved by 14 addition of potassium hydroxide which yielded potassium hydrogen tartrate as a precipitate. Purification of anthocyanins can be achieved by various means. Four of the more common examples are chromatography, ion exchange, chemical precipitation, and electrophoresis. The chromatographic methods, although valuable in their own right as methods for identi- fication of anthocyanins, are not practical in terms of purification of large quantities. Electrophoresis, as introduced into the area of anthocyanin applications by Markakis (1960), utilizes the ionic character of the pigments for purification and identification. Fuleki and Francis (l968b)compared some ion exchange resins and lead acetate as well as a polyamide column for their purification properties. Of these materials, it was their conclusion that the ion exchange resin, Amberlite CG-SO, gave the best overall perfonmance. Identification of anthocyanins is now mainly based upon chromatographic and spectrophotometric methods. Total anthocyanin content can be determined by the molar absorbtivity and absorbance at 1 max or in the case of a mixture of anthocyanins, respective weighted averages can be used (Fuleki and Francis, 1968a). Harborne's work (1967) included methods for analysis by paper chromatography and lisuilthe R values on Whatman #1 paper for most of the known f anthocynidins and anthocyanins separated with the following four solvents: (l) n-butanol-acetic acid-water (4:1:5); (2) n-butanol- 2N HCl (1:1); (3) 1% HCl; (4) acetic acid-cone. HCl-water (15:3:82). Fuleki and Francis (1967) introduced BBFW (1-butanol-benzene-formic acid-water, 100:19:10:25) which was reported to have exceptional re- solution in its separation of anthocyanins. Harborne's report 15 (1958) on spectral methods of characterizing anthocyanins is a stan- dard reference for anthocyanin spectroscopy. Characterization is based on the wavelength of absorbtion maxima, observed shifts due to glycosidation, interaction effects due to addition of aluminum chlo- ride, and ultraviolet absorbtion. The qualitative characteristics of an anthocyanin pigmented sample have been described in terms of: absorbtion at a predeter- mined wavelength maxima with no corrections made, absorbtion dif- ference at two different pH values as first suggested by Sondheimer and Kertesz (1948), actual milligrams of pigment per gram of fruit as described previously (Fuleki and Francis, 1968a), full scan of the U. V. and visible range of the spectrum for absorbtion,and finally, the L, a, and b values from.the Hunter Color Difference Meter. The first three examples mentioned are actually attempts to determine the anthocyanin content of a sample, with the third being considered the more accurate. A full scan of the U. V. and the vis- ible range of the spectrum requires intricate interpretatioiand un- less one is well versed in the matter and has a working knowledge, it is hard to interpret an accurate description of the color being measured. The final method mentioned is designed to close the gap between instrumental measurements and visual perception. Figure 8 displays the Hunter L, a, b color solid. The "a" value describes the comparison of red-to-green color dimension, the ”b" value similarly compares the yellow-to-blue color dimension. Lightness or darkness of the sample is denoted by the "L" value. 16 L=IOO WHITE YELLOW BLACK Figure 8. Hunter L, a, b, Color Solid. l7 Anthocyanins as Food Colorants There have been few examples of anthocyanins being used in research or pilot studies as food colorants. At the University of the West Indies, Trinidad, roselle (Hibiscus sabdariffay has been studied as a possible food colorant. Esselen and Sammy (1973) commented on its present uses in various food products, not merely as a food colorant but also as a flavor component. A study concerning the keeping quality of dried antho- cyanins from roselle was initiated using various drying techniques such as spray, foam mat, and vacuum.drying. Francis (1975) reported on various suggested sources of anthocyanins in a symposium.concerning utilization of plant pigments. Among the likely candidates were grape juice lees, wine grape skins, special grape varieties grown for pigment content, Miracle fruit, and cranberries. Shewfelt and Ahmed (1977) claimed that anthocyanin extract from.red cabbage showed good characteristics as a coloring agent for dry beverage mixes. Jurd (1964a)reported on a number of synthetic benzopyry- lium compounds, structurally related to natural anthocyanins and po- tentially useful as color additives for fruit drinks and juices. In dilute, slightly acid solutions, the color of the benzopyrylium.dyes are remarkably similar to those of the coal tar dyes and also to the natural pigments. Furthermore, he notes that the color of these benzopyrylium.compmnxb have, in general, good stability in an acidic environment and are about three times as intense as those of the 18 coal tar dyes at the same concentration. MATERIALS AND METHODS The source of pigment for this project was a dark tart cherry selection, designated as MC-lS, which came from the Michigan State University cherry breeding program. These cherries were harvested approximately one week after peak maturity and subsequently frozen at ~10° F until ready for use. The brined Napoleon cherries were obtained from R.C. warren & Company Inc., Traverse City, Michigan and consisted of a combina- tion grade 1-2-3, 22 mm.and greater. Comparison of Anthocyanin Sources The anthocyanin content of MC-lS was compared with that of both the sweet and tart cherry varieties, Stella, Suda, English Mo- rello, and valera. The comparison was based on the anthocyanin content as determined by the difference between spectrophotometric absorbance of anthocyanin containing solutions at pH 2.1 and 3.5. The wavelength used to compare the absorbance of the various sources at the two pH levels was 515 nm, which was the wavelength of maxi- mum absorbance for the anthocyanin extracts. In order to adjust the pH of the anthocyanin solutions for spectrophotometric measurements, two buffers were made up using 0.5N citric acid for the pH 2.1 buffer and 0.1N citric acid for the pH 3.5 buffer. Both buffers were subsequently adjusted with 19 20 2N NaOH to obtain the exact pH level required. For each cherry se- lection, 250 grams of fruit were combined with 150 m1 of 0.25M.tar- taric acid and macerated for three minutes in a waring blender. Blended samples were filtered through Whatman #1 filter paper and one ml of filtrate was diluted to 50 ml using buffers at the ap- propriate pH. The diluted samples were allowed to equilibrate in the dark under refrigeration for approximately thirty minutes. The ab- sorbance at 515 nm was measured on a Bausch & Lomb Spectronic 70 spectrophotometer, using the appropriate buffers as blanks. The dif- ference in absorbance was obtained by subtracting the absorbance at pH 3.5 from.the absorbance at pH 2.1 and this difference was taken as a measure of the relative anthocyanin content. Modification Analysis As noted in the literature review, co-pigmentation and metal chelation have a significant effect on the appearance of anthocyanins, therefore a preliminary experiment was conducted to observe the ef- fect of aluminum and tin on the rate of apparent color change in the cherry juice extracts. The juice extract was obtained by macerating a mixture of 1000 grams of MC-lS and 2000 ml of 0.25M.tartaric acid. This blend was subsequently filtered through Whatman #1 filter pa- per in a Buchner funnel. One percent solutions containing AlCl 3 and SnCl were added to juice samples as follows: 2 (1) Control (juice with no metal added) (2) Control with 0.01% AlCl added 3 (3) Control with 0.01% SnCl2 added (4) Control with 0.0057. SnCl2 added Four replicates of each sample were prepared and all sample replicates 21 were allowed to equilibrate overnigit in the dark under refrigeration before the zero time reading was taken. All sample replicates were then held at 500 C in a water bath and periodically read on the Hunter Color Difference meter over a 456 hour period. Juice Preparation Forty pounds of frozen MC-15 cherries were heated to 1750 F, held for three minutes, and pressed for their juice. The 9.5 liters of juice obtained were diluted with 14.5 liters of 0.25M malic acid to give a final volume of 24 liters of juice. The malic acid buffer was used to lower the pH which would exert a stabilizing affect on the anthocyanins. The 24 liters were divided into four lots of 6000 ml each. Two of the four lots had sufficient 1% SnCl2 solution added to bring the final concentration to 0.0025% SnCl the other two lots were 2; not treated with any metals. Brined Cherry Preparation Thirty-five pounds of commercially brined cherries were soaked for twenty-four hours in a moderately hard water solution. The cherries were then brought to a boil twice, in two changes of water, to bring the concentration of sulfur dioxide down to 75 to 150 ppm. The cherries were sorted in order to eliminate any badly bruised fruit and then divided into four equal lots. Two of these lots were subjected to a ten second dip in a boiling 0.2M potassium carbonate solution, in an attempt to alter the surface wax of the cherry and facilitate the transfer of anthocyanins and sugar into the fruit (Tullberg and Angus, 1972). 22 The four lots of cherries were colored by combining them with juice according to the following flow chart. Lot 1 & 2.‘=:::::::L0t #1 - Control / No DIP - L011 #2 - Control + 0.0025% SnCl BRINED CHERRIES \Lot 3 a 4 :4 5 , I- 6 65° F 2 l a . :I: ' U‘GONTROL 4 I-CONTROL-I- 00025:; $010.: 13-02»! ch03 DIP e-ozu «zoo3 DlP-I- oooasz. SNCLg 2 30 60 90 - DAYS - Figure 10. The change in Hunter "L" value at 65°F with time ' for each treatment. STD DEV. a 0,243 30 l4 a I2 I l I.” D .l < a > :4 a: m '5 6 ‘ o 35 ° F I ' D'CONTROL 4 I—couraou- ooozsxsm e-ozm cho3 DIP e-ozu K2603 Dips-09025:. $1301.; 2 so so so - DAYS — Figure 11. The change in Hunter "L" value at 35°F with time for each treatment. 311) DEV. = 0.243 31 SnCl as well as between the control and the potassium carbonate 2 treatment. The differences between the control plus SnCl2 and the potassium carbonate dip plus SnCl were also significant at all of 2 the three temperatures used in this study (Appendix 13). Figures 12 through 14 graphically illustrate the change in Hunter "a" values for the time, temperature, treatment combinations. The Hunter "a/b" value is generally regarded as a measure of the hue of the samples. Zero and negative values indicate green- ness, positive numbers less than 1 indicate yellowness, and relatively large numbers indicate redness. Figures 15 through 17 show the con- sistency with which the Hunter'h/b" values change with time for every treatment-temperature combination. Some significant differences are noted at zero and thirty days but the important point of these graphs is to illustrate the common change in hue with time (Appendix IIB). The Hunter "b" value is a measure of the blueness to yel- lowness of a sample and in this case, where the sample is all red, the usefulness of the value is not directly applicable; however, in some instances "b" values tend to indicate changes from red to purple hues. Figures 18 through 20 illustrate the change in Hun- ter "b" values with time for each treatment-temperature combination. A significant difference was noted between the control and the po- tassium carbonate treatments at every temperature (Appendix IIC). The anthocyanin content of the cherries was determined using the pH differential method. The analysis of variance conducted on the anthocyanin data indicated that there was no significant inter- action between treatments and either time or temperature. The data - HUNTER ”a“ VALUE — 32 I4 l2 8 a l 6 4 99° F Ell-CONTROL I-CONTROL-I- 00025:: $010.; 2 e-DIZM cho3 DIP e-ozu «ch3 DlP-rODOZSXSNOLg so so so — D A Y s — Figure 12. The change in Hunter ”a" value at 99°F with time for each treatment. STD DEV. = 0.516 33 I4 IO I In D 2‘ s > ID a: .‘i' 6 2 D I I 4 65°F D-CDNTRDL I-OONT'ROL+ (100259; Sues 2 e-ozu «zoo3 DIP a-ozu K2003 DlP-I- Doom 3»ch so so so - DAYS — Figure 13. The change in Hunter "a" value at 65°F with time for each treatment. STD DEV. a 0.516 34 I4 12 A I + I0 I I In :3 2’ > s I‘D a: .‘i‘ z 6 :> a: l 4 D'CONTROL 3 5° F I-CONTROU- 0.0025% $00.2 A-OZM K2603 DIP z A-OZM K2003 DIP+OOOZS%SNGLg 3O 60 90 - DAYS -- Figure, 14. The change in Hunter "a" value at 35°F with time for each treatment. STD DEV. = 0.516 35 I4 I2 O'CONTROL IO 99, F I-GONTROL-I- 0.0025% Snag I e-02M K2003 DIP IE e-Dzu K2603 DlP-I- Doom 80:ch § 8 ~ “at 5 I-'6 Z 3 I k A 4” I 2 so so so - DAYS- Figure 15. The change in Hunter "a/b" value at 99°F with time for each treatment. STD DEV. 30.173 36 l4 I2 Ell-CONTROL Io 65° F I'OON1'ROL+0.0025%SNGI.¢ e-ozu cho, DIP g e-OZM K2003 DIP-I- coca-5% 800ch § 8 To \- 3m m . :2 s 2: :3 I :I: I 4| 2. 30 60 90 - DAYS - Figure 16. The change in Hunter "a/b" value at 65°F with time for each treatment. STD DEV. = 0.173 37 I4 I2 U‘CONTROL 35° F I-CONTROL-I- 0.0025% Snag '0 e-02M K2003 DIP ‘ A-OZM K2003 DIP-I- 0.0025% SflGLg m 2 :PB e I 5° E s z a T 4 30 60 9O -ILAYS- Figure 17. The change in Hunter "a/b” value at 35°F with time for each treatment. STD DEV. = 0.173 38 I4 I2 D'CONTROL IO 99, F I-DDNTPDL-I- 0.0025% 8010.; e-DzIII «ch3 DIP l A-OZM cho,DIP-I-Doozsxs~otg UJ : a .J § :9 95 s p. I! :3 I A I 4 l 2 30 60 90 - DAYS- Figure 18. The change in Hunter "b" value at 99°F with time for each treatment. STD DEV. = 0.203 l4 l2 39 o—cDIIrrRoI. 65° F l-DDNTRoL-I- (100252 3.:ch 45-62»! «cha DIP e-ozu K2603 DIP+ODOZ§%SNGLg l l 30 60 90 - DAYS '- Figure 19. The change in Hunter "b" value at 65°F with time for each treatment. STD DEV. = 0.203 40 I4 I2 Ill-CONTROL, Io 35° F l-CONTRoL-I- 000259; 8.0.; e-ozm cho, DIP ' e-ozu K,DD,DIP+ooozszs~ctg m I 3 s < > .o {5 s '- 2 :3 I l p /\ I 30 60 90 - DAYS '- Figure 20. The change in Hunter "D” value at 35°F with time ' for each treatment. STD ng. - 0.203 41 indicates that none of the treatments had any significant effect in delaying pigment degradation. The results presented in Figures 21 through 23 indicate that degradation rates are nearly identical in all samples, regardless of the treatment at each temperature. Figure 24 shows the reaction rates for each treatment at the three tempera- tures used in the storage study. No significant difference was noted at any temperature between the reaction rates for the treatments. ABSORBANCE DIFFERENCE G -5I5nm— 42 °'° ' CI-DDNTPDI. 996 F .‘CONTROL'I' 0.0025% SNCLg A- QZM K2003 DIP 0.5 _ “'02" K2003 DIP'I'QOWSNCLg .0 u .0 no I .0 t . so so so — DAYS — Figure 21. The change in the absorbance difference at 99°F with time for each treatment. STD DEV. = 0.0506 ABSORBANCE DIFFERENCE @‘5I5nm 43 0.6 .. O‘CONTROL 65 o F I'CONTROL+ 0.0025% 300.2 6- am K2003 DIP 0 5c A-OZM K2003 DIP-POOOZSXSNCLg .0 0.3 0.2 P .0 T . so so so — DAYS — Figure 22. The change in the absorbance difference at 65°F with time for each treatment. STD DEV. = 0.0506 ABSORBANCE DIFFERENCE o 5I5nm— .0 m I .0 U I .0 °~2 " CI-DDNTPDL 35° F I-cou'rmu- 0.0025% Snag e-ozm K2003 DIP _ e-ozu I<,co3 DIP+QoozszsucL2 so so so - DAYS - Figure 23. The change in the abosrbance difference at 35°F with time for each treatment. STD DEV. = 0.0506 45 0 030 U‘CONTROL ‘ ' - .‘CDNTROL'I’ 0.0025% SNOLz 0902M K2003 DIP .‘OZM K2003 DIP‘I' 0.0025% SNCLg 0.025 .. LIJ I- < c: 0.020 - Z 9 |_ 2 0.0I 5 r LIJ D: 0.0I0 P I I I 35 65 99 DEG REES FAHRENHEIT Figure 24. The change in the reaction rate with temperature for each treatment. SUMMARY AND CONCLUSIONS The application of anthocyanins to the coloring of a mare- schino type cherry was successful and showed good promise as an al- ternative source of color. It was found that the intensity and tone of the anthocyanin colorant can be altered and certainly, with some diligence, the proper hue can be achieved. The products produced in this project were considerably darker than the present maraschino cherry but methods to alter the color hue such as the proper con- centration of anthocyanins, balancing the sulfur dioxide levels, and micro-environmental modifiers i.e. potassium.carbonate could be uti- lized to attain the desired end product. The attractive label of "all natural" might then be applied possibly securing further markets for the sales of maraschino type cherries. There is no doubt that the extended shelf life will require certain modifications in the way the product is handled, both during processing and during storage. Consideration of refrigerated stor- age would greatly increase the shelf 1ife, as shown by our storage studies. Future emphasis should be placed on streamlining the color- ing step and determining the best way to apply the color in order to reduce the anthocyanin degradation. The addition of stannous chloride had no apparent protective effect on the anthocyanins. The degradation rate was not signif- icantly affected by the treatments used in this study although the 46 47 observed color characteristics, as measured by the Hunter Color Dif- ference Meter, showed some varying effects by adding metal salts. Observations of apparent trends seem to indicate diminishing differences between treatments under stress conditions with time. The bathochromic shift, caused by the addition of stannous chloride, could account for much of the early significant differences measured by the Hunter Color Difference Meter. As the pigment was degraded, anthocyanin molecules, which were complexed with the stannous chloride, would undergo degra- dation and change the extent of the wavelength shift; thus causing less significant differences between treatments. Future studies might consider developing further the appli- cation of anthocyanins to new products. Modification of anthocyanins to fit the needs of the food industry are certainly not exhausted and leave much to be studied. APPENDICES 48 :m: nouns: mo muoowmo came nooauoouu ecu no women you mH xfiooomn< mom .muou osHmP H Hosoa assessmesmem ems s posse assessmeswam sea as homn.a kfihh¢.N mam.o ooh.o ¥¥O¢H.N ma 0 x m x < skeom.o *kOOh.o m0¢.o *kNoH.oH me.H c U N m moa.o kkoom.oN kkmmw.u HoN.H thHH.N m 0 x < omo.o *kmoo.¢ How.H mam.o mmm.H o m x 4 *komw.om fikomm.o¢¢ ktOHw.m¢H *¥¢~¢.wm *komo.mm m ADV oEHH «snom.aw sso~¢.om ssNH~.o «snm~.mo mha.a N Amy ousumuoaaoa o~m.~ *kmmo.~m *kooh.©HH %¥o~N.o¢ *kmom.HNH m A mo Donsom mowumqumum m .ovma museums me some some you coaumwum> mo moonsom o>auooemou any new mofiumaumum m .H xwoooon< mom m ooomoawuowfimooou.m.z ooomouwwowwm NmmIr ooomoumqowwm Nmmass road «no them? "No «swore «as macaw made new oumoonumo Esammcuoe m> «doom moan Houucoo Any new ousoonnmo asammmuoe m> aonuooo ANV macaw moan Houuaoo m> HouuooD adv .Afiuofiue mv mummuuooo Hmoowoeuucu Icon.woaaoaaow can so some on coo mumou u Hoouuomcom ouomouonu .Amv uoommo cams unoauoouu can uo>ao>ow oowuomuouou oz .uoommo mama unoaumouu onu no mumo moam> :m: nouns: ozu mommowumfiumumuumfioouuomoom ooh . mH NHszmm< 50 .uomoamwowfim uoo one xmfiuoumo no he oozoaaom no: muooesz oo.~ "ouoz Ho>oa moomowmwowfim fine I s amped ooomoqwaowum Nam I as «Hose a moose u> «dose a mouse as «Hoom a flouuooo e> macaw a moo~e m> Naosm a moose s> macaw a douuooo ob Noose a mouse as Noose e mooue s> «doom a Houuooo m> macaw a moose s> «Home a moose s> «doom a Houuooo m> mH.~I mu.oI om.HI ss~¢.qc asaH.nI an.~ s~m.~ mm.aI HH.o am.oI NH.NI ss~o.¢a sswm.¢n ssqe.¢u mn.o Nu.o o~.~ mm.oI m~.~ mm.HI aa.~- mm.NI ssoa.mu No.H nm.~ No.H mm.oI mo.~ smm.~ sa¢.~I ssm~.¢I «mm.~- em.a 5H.o aH.H mama mono momm .momoa coaumouoaoo unoaumoou memo souuooo Houuooo Houuooo mass om Houuooo douuooo Houuooo mess co Houuoou acuuooo Houucoo name om aonuooo Houuooo Houuooo ammo o o3m> :4: nouns: one you mowumfiumumnu Moouuomcom can. .4”: NHQZEA¢ 51 .uomoamwowan no: one xmwuoumm no he oesoaaom no: muonasz «ouoz ~o>o~ ooomowmaomwm flmm Is ADPDH oooooumwowfim Nam use me.o me.o- m~.o «Hose a momma mm.o o¢.~ NH.G N m¢.cu mm.o cH.HI Hoom a NH.o mo.o ~o.~- macaw e mcome nm.o Ne.~ oH.H ~ w~.o o¢.o mw.o doom a mm.e me.o- se.a- «Hose a mcome mn.a Pmm.~ s~m.~ N ssfio.qu «swo.¢u m¢.ou doom a Ne.H ssHm.mI ssmm.mu «doom w noomx mc.H *4mu.m ksmm.m ssqc.on om.~a oo.HI «goon a mama homo momm .momoe :oHumofiQEDo unusumouu as Noose e mcome as aouuooo m> as «noun a noose s> Honuooo m> as «seem a n8ND. s> douuooo m> as macaw e MOONM m> aouuooo m> aonuooo Houuoco Houuooo ammo om Honuoco Houuaoo Houuooo sass co aouuooo Houuooo Houuooo ammo om Houucoo Houucoo Houucoo mhmv o sumo D=Hm> :nxm: nouns: one now mofiumaumumuu Hoouuomcom one . mHH fiQZEmAw 52 .uomoamaswam no: one xmfiuoumm no he oozoaaom uo: mucoasz "ouoz ao>o~ cosmoawaomam Nmm I s ~D>DH cocoowmaowwm Rea I as ~Hosm a moose s> Noose e me.- an. an. smm.~- ssmo.m- Nm.a- N moo~e as sem.~ an. Ho.~ seem a Acheson s> mm.- mH.H me. macaw e mooue s>n~awsm e %*NH.mI «mo.~I ssmm.mu N co M m> m~.H ~m.- esm. seem a Hoousoo m> s~.- «m. moo. «Hose a mcums. as «seem a mm.H- aso.~- *Hm.~- N : mommw Mn ssmma.m Ismo.m we.a so A e no u o Hm.- he.e ssoe.m «seem a mouse up «noun a as.z- seam.m- same.m- mooue a> «smmm.o mm.a an. «Hoom a acuuooo n> e.ma m.ne momm .mdmg GOHumfifiAEOU Hagumwhu MUNU .UHH anzmmm< Houuooo flouuooo Houuoou mess om aouuoou Houuooo Houuooo ammo co Houuooo douuooo Houuooo mass on Houuooo Houuooo Houuooo show 0 osHm> :n: nouns: can now moaumwumumuu Hoouuowcomoea 53 «see o\co was momww as mnmwo.o Hmsmoo.o Neaaoo.o «Hose sane new oumoonuou sawmmmuom HeNNo.o mmosoo.o smmoo.o see «assesses asesmsupm eHaNo.o mmosoo.o mseoo.o Noose sans essence mmouo.o sseoo.o emwoo.o Acousoo mama mono momm uooaumoue .muooauoouu msoauo> one Do oomooxo uooawfio ago now Aanmhmov M announce Dump oowuooom .HHH anzmmm< 54 .m e To mEA n\xwo u V\~o>oa onsooooua m I sues mounou 0 one has one so can on uowua wouooaom oomgvcoocoeooow noov Hmoowsuuouooo one node: .momoa mmoam mwmmuwmwm .mufiumfiumumuu Hoouuomoom emu mo oowumasoamo .>H NHszmm< LIST OF REFERENCES CITED REFERENCES Adams, J.B. 1972. 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