a\ 1.2.3.34... .- 3.2}... 1.... a.:§.l.2s.:.§1v1..i..:..:..:..c3:3..a 12:11.33! “.2353... . . 5 . 3.4! ...:....;iv....z§.ii:5, § 5 . r . .. 0.54- In; .4523; E... . .. ‘ 1‘ . » . , . ,...‘ L. 1.3....WINW .. . . . n ‘ . ‘ “sysadl. IHESIS This is to certify that the thesis entitled PECTIC SUBSTANCES AND PECTIC ENZYMES OF FRESH AND PROCESSED MONTMORENCY CHERRIES presented hg Khalaf S. Al-Delaimy has been accepted towards fulfillment of the requirements for Ph.D. degree in Food Science dejé75flnkq I Major {arofessor ‘ Date 12/18/53 0-169 LIBRARY Michigan State University iiiiiiiiiiiiiiiil tin ii iii i e DATE DUE DATE DUE DATE DUE _ ion/Equal Opportunity Institution c:\drt rmative Act PECTIC SUBSTANCES AND'PECTIC ENZYMES OF FRESH AND PROCESSED MONTMORENCY CHERRIES By Khalaf Alsoori Al-Delaimy A THESIS Submitted to Michigan State university in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1963 "c;aqsio sz/hf To My Parents 11 ACKNOWLEDGEMENT I would like to take this Opportunity to express my deep gratitude to Professors Georg A. Borgstrom, Clifford L. Bedford and Pericles markakis for their constant help, advice and encouragement during the prOgress of this work. Thanks are also due to Professors Laurence G. Harmon, William B. Drew and Bernard S. Schweigert for their help in the preparation of this thesis. my deep appreciation to Miss Janet Gassman for typing this manuscript and for her valuable suggestions throughout the writing of this thesis. 111 TABLE OF CONTENTS INTRODUCTION . . . . . . . . REVIEW OF LITERATURE . . . . Pectic Substances . . . . Pectic Enzymes . . . . . Protopectinase . . . . Pectinesterase . . . . Polygalacturonase . . Polymethylgalacturonase Pectin-transeliminase METHODS AND MATERIALS . . . Assembling the Material . O O O O retermination of Pectic Substances in Total pectin . . . . . Water insoluble pectin Water soluble pectin . O O O 0 Determination of Pectic Enzymes in Cherries . Pectinesterase . . . . Polygalacturonase . . Determination of methanol RESULTS AND DISCUSSION . . . Pectic Substances . . . . Frozen cherries . . . Canned cherries . . . O O O 0 Content iv 0 O O 0 page 11 11 11,'12 12, 15 12 12 2o 20 20 22 22 22 22 22 2a 25 28 28 28 . 3L. TABEE OF CONTENTS CONT'D Pectic Enzymes . . . . . . . . . . . . . . . . Pectinesterase . . . . . . . . . . ... . . Pectinesterase activity in stored frozen cherries . . . . . . . . . . . . . . . . Pectinesterase activity of irradiated cherries during frozen storage . . . . . Pectinesterase activity in canned cherries Pectinesterase activity in fresh cherries methanol Content of Cherries . . . . . . . . . SUMMARYANDCOMLUSION ............. LITERATURE CITED . . . . . . . . . . . . . . . . ”mmm .................... page 37 37 37 39 42 "3 48 5O 59 LIST OF FIGURES Figure Page 1 Irradiation of cherries with accelerated electron by a resonant transformer . . . . . 2l 2 Standard curve for galacturonic acid deter- mination by the Carbazole method . . . . . . 23 3 Standard curve for methanol determination by Mehlitz and Brews , , . . ... . . . . . . 27 A Total pectin, water insoluble pectin and water soluble pectin content of stored frozen cherries harvested at various stages of maturity.................. 33 5 Total pectin, water insoluble pectin and water soluble pectin content of stored canned cherries harvested at various stages of maturity.................. 36 6 Pectinesterase (PE) activity of frozen cherries picked at various stages of maturity 38 7 Pectinesterase (PE) activity of irradiated cherries of various maturity during frozen storage.................. hi 8 Methanol content of mature cherries during frozenstorage............... 45 vi ABSTRACT PECTIC SUBSTAmES AND PECTIC EMYMES or FRESH AND PROCESSED MONTMORENCY CHERRIES by Khalaf Alsoofi Al-DEIaimy The purpose of this investigation was to study the effects of maturation and storage time on the pectic consti- tuents and pectic enzymes of fresh, frozen, and canned red tart cherries (Prunus cerasus L. var. Montmorency). Also, the effect of irradiation on the pectic enzymes of frozen Montmorency cherries picked at various stages of maturity was studied. The Carbazole-colorimetric method was used for the determination of pectic constituents in Montmorency cherries. The titrimetric method was used for measuring pectinesterase (PE) activity of fresh, frozen and irradiated (93, 186, 372 K rad) cherries. The polygalacturonase (PG) activity was measured by titrating reducing groups or by calorimetry by the method of Willaman and Davison (192A). The chromotropic acid-colorimetric method was used for the methanol content determination. It was found that the total pectin (TT), the water insoluble pectin (NIP), and the water soluble pectin (WSP) contents of the premature cherries were considerably higher than those of the mature and overmature cherries. There was no significant variation in the pectic components of mature and overmature fruits. The storage time of frozen and canned Khalaf Alsoofi Al-Delaimy - 2 cherries did_not considerably affect the content of pectic constituents. The T? content of the premature, mature and overmature canned cherries was practically the same during the first eight months of storage but decreased slightly after 12 months of storage. There was a small but contin- uous decrease in the WSP content of both premature and mature canned cherries during the storage. Overmature canned cherries did not show such a trend. The PE activity increased as the stage of maturity of Mbntmorency cherries advanced. In the frozen cherries the activity showed a slight increase at the end of the fourth month of storage but later it decreased as the storage time of frozen cherries was prolonged for 12 months. The PE activity of the irradiated cherries of various maturity during frozen storage followed the same general trend as the non-irradiated stored frozen fruits, but was slightly higher. The 372 K rad dose seemed to enhance the PE activity after the fourth month of frozen storage of the premature and mature cherries. This dose did not affect the PE activity of overmature fruits. No PG activity was detected in fresh or frozen cherries picked at various stages of maturity. The methanol content of cherries increased with time of frozen storage and reached a maximum level at the end of 12 months. Longer storage did not seem to further increase the methanol content. The levels of methanol obtained are far below those considered harmful to humans. INTRODUCTION About one half of the plant organic material formed each year on earth consists of the cell walls of higher plants. Yet little is known about the formation, composi- tion and metabolism of the plant cell wall. The typical cell wall of higher plants consists of three morphOIOgically distinct layers: namely, the middle lamella, the primary wall and the secondary wall. The middle lamella is composed of lignin, cellulose, xylans, pectic substances and other polysaccharides. The primary wall is heavily lignified but is predominantly car- bohydrates with pectin, uronic acid containing polysacchar- ides, xylans or mannan and with cellulose and hemicellulose in relatively lesser amounts. The secondary wall consists largely of cellulose with some other constituents as in the primary wall, but in smaller amounts. Pectic substances are a mixture of a galactan, the methylester of a galacturonan and an araban. Experimental evidence indicates that the pectin prOperties such as gela- tion, film formation and high viscosity are due to the galacturonan chain and that araban and galactan act as diluents (Whistler and Corbet, 1957). The nomenclature in the field of pectic substances which was adapted by the American Chemical Society (Kertesz, lghh, 1955) will be used throughout this thesis: pectins or pectinic acid designates the water soluble polygalacturonic l acids of varying degrees of methyl ester content and neutral- ization which show colloidal prOperties and are capable of forming, under certain conditions, gels with acid and sugar. Pectic acid is polygalacturonic acid of colloidal nature but essentially free of the methyl ester group. Protopectin is the water insoluble parent substance which occurs in the plant, and upon acid hydrolysis, yields pectins. The enzyme protOpectinase supposedly hydrolyzes protopectin into pectin and perhaps cellulose. Pectinesterase (PE) (also referred to as pectin-methylesterase or pectase) catalyzes the fis- sure of the natural or synthetic methylester of polygalac- turonic acids. gglygalacturonase (PG) (pectinase) cleaves hydrolytically the glycosidic bonds of polygalacturonic acids. Enzymatic degradation of pectic substances is considered to be one of the major causes of loss of the texture of red tart cherries (Prunus cerasus, L. var. Montmorency) as well as many other fruits and vegetables. The purpose of this study has been to investigate: l) the changes taking place in pectic constituents of cherries a) during maturation, b) during storage of canned and frozen fruit picked at different stages of maturity; 2) the activity of pectic enzymes of cherries a) during maturation, b) during storage of canned, frozen and frozen irradiated fruit picked at different stages of maturity; and 3) methyl alcohol content of fresh and stored frozen cherries. REVIEW OF LITERATURE Pectic Substances Pectic substances have been under investigation by researchers of various interests--botanists, plant patholo- gists, plant physiologists, biochemists, food chemists, industrial chemists, and others-~for a long time. These researchers have studied pectic substances from several points of view: their relation to the metabolic changes which take place in fruits and vegetables during maturation and senescence; their relation to plant diseases; their use in the setting of Jams and Jellies, and as emulsifiers; their importance in clarification or retention of "cloud" of Juices. The chemistry of pectic substances has been extensively reviewed by Kertesz (1951, 1959) and Deuel and Stutz (1958) and can be sumarized as follows: Pectic substances are composed mainly of galacturonic acid units which are united withcx-l, h-glycosidic linkages. A varying number of the carboxyl groups are esterified with methanol (degree of esterification). Pectic substances are widely distributed in plants in the natural state, the polymer is insoluble in water, and if dispersed in water they are negatively charged hydro- philic colloids. Pectins prepared from different sources are not entirely alike; other carbohydrate materials such as araban, galactan 3 or xylan may be present in variable amounts along with the polygalacturonic acid. The mechanical combination of these molecules is not clearly understood. Experimental evidence shows that many of the prominent prOperties of pectin such as gelation ability, film formation, high viscosity are derived mainly from the galacturonic acid chain and that araban and galactan act mainly as diluents. The prOperties of pectic substances are altered by partial or complete de- methylation which increases the number of free carboxyl groups. Commercial pectins form colloidal solutions and usually 2/3 to 3/h of the carboxyl groups are methylated. Molecular weights of 50,000 to 250,000 have been reported for pectin so the polymer may contain several hundred units. Pectic substances occur in higher plants in the middle lamella and in the primary wall of meristematic and paren~ chymous tissue cells (Bailey, 1938). They exist as water- insoluble pectin (Fremy, 18A8), now designated as protOpectin (Tschirch, 1909) and as water soluble pectins. The pectic substances present in the cell wall differ from those in the middle lamella in their solubility and histochemical properties (Joslyn and Deuel, 1963). Joslyn and Deuel (1963) stated that the quantity and composition of pectic substances vary with many factors, such as the origin of the specimen, the methods used in the preparation of the tissue, the methods of extraction and separation, the type and extent of purification, and the analytical procedures used in assay and characterization. 5 methods of extraction of pectins from plant tissue are limited. Most investigators have selected conditions for extractions that would yield the highest quantity of the pectin with the particular properties desired (Bender, 1959; and Kertesz, 1951). Pectins similar to those actually pre- sent in plant tissue have not been obtained because of the difficulties of avoiding degradation during extraction (Joslyn and Deuel, 1963). Only recently some procedures have been introduced for the characterization of pectic sub- stances in situ (Gee, 3131., 1958, 1959). The recently introduced methods for extraction of pectic substances are considerably less drastic than the older ones, and newer methods of analysis better characterize the pectins extracted (Bender, 1959; Berglund, 1950; Deuel, 1943; Does- burg, 1959; Henglein, l9fl7; Hills and Speiser, l9h6; and Owens §£_g;., 1952). The use of acids for the extraction of pectins from plant tissues, both for analytical and com- mercial production, is now well established, although the chemistry of this extraction is not well elucidated (Joslyn and Deuel, 1963). Qualitatively, detection of pectic substances in plant tissue was based on morphological and histological tech- niques which were not satisfactory (Kertesz, 1951). In 1955, NbCready and Reeve introduced a satisfactory histo- chemical qualitative test for pectin based on the reaction of the ester groups in pectin with aqueous alkaline hydro- xylamine at room temperature for two minutes. With the addition of ferric ions, this reaction produces hydroxamic acids, which are water insoluble, red-colored complexes. Quantitatively, pectic substances have been estimated by several procedures: 1) by the direct titration proced- ure as anhydrouronic acid (Gee gt_gl., 1958); 2) by the Carbazole colorimetric procedure (McComb and McCready, 1952; Sinclair and Jolliffe, 1960 and others) applied to the extracts prepared by the versene-pectinase procedure of McCready and McComb (1952); 3) by the measurement of calcium pectate (Carre and Haynes, 1922). The first two procedures are most widely used. A considerable amount of work has been conducted on the influence of auxins on the metabolism of pectic sub- stances in the wall of the plant cell (Ordin et a1., 1955; Cleland and Bonner, 1956; Glasziou and Inglis, 1958; Bonner, 1959; Albersheim and Bonner, 1959; Jansen et a1., 1960). Bonner (1959) and Albersheim and Bonner (1959) in their studies on avena coleOptiles, stated that the rate of synthe- sis of pectic substances increased in the presence of auxin. In addition, the flow of newly formed pectic material deviated from protopectin to water soluble pectin. Pectin transformation often seems to be responsible, to a large extent, for the softening of fruits and other plant materials during ripening and storage. The major change in stored fruit is the loss of water-insoluble protOpectin and pectates leading to an increase in soluble pectin consti- tuents and to a decrease in cohesion between cells and layers of cells in the tissue (Kertesz, 1951). Recently Jeslyn (1960) reported that histological, physiological and other factors control the firmness in texture of fruits and vegetables rather than the protopectin content. In apples, the firmness has been related more to cellulose than pectin contents or the kind of pectins present. Changes in hemicellulose, arabans, galactans, glucosans and particularly xylans seem to be more closely correlated with softening than pectic substances. Deuel and Stutz (1958) also stated that the enzymatic changes of hemicellulose play an important role in fruit ripening. The early study of Appleman and Conrad (1926) on pectic changes in peaches with relation to the softening of the fruits showed that the transformation of protOpectin to pectin seems to be the only pectic change that takes place during ripening. They stated that the increase in the amount of pectic substance began with the softening of the fruits and reached its maximum in the fully ripened ones. The total pectin and protopectin were practically constant at all stages of ripening, but both disappeared slowly in the over-ripened peaches. They concluded that the amount of soluble pectin formed from the insoluble protOpectin in the cell walls is closely parallel to the degree of soften- ing of the peaches during ripening. Postlmayer and Duh (1956) also found that pectic sub- stances of Halford cling peaches changed very slightly during maturation. But in the case of Pay Elberta freestone peaches, protopectin was converted into water soluble pectin, causing softening of the texture. The high retention of protopectin in Halford clings is apparently the major factor responsible for their inherent firm texture. A partial con- version of protOpectin into water soluble pectin occurred during heat processing of both varieties. On the other hand, Sinclair and Jolliffe (1961) found that their studies on oranges showed a tremendous initial increase in the total and water-soluble pectic materials during the rapid development of fruit growth in the early part of the season, and a gradual decrease through the rest of the season. McCready and McComb (195113) found that the degree of esterification drapped to below h0¢ after the pears and avocados ripened and the molecular sizes of all of the pectins decreased. This result, they added, suggested that pectic enzymes come in contact with the pectic substances during ripening and hydrolyze them to materials of lowered molecular size. The degraded materials are then less effective in maintaining firm.structure in fruits and less important in contributing to the consistency of processed foods. Woodmanaee gt_gl, (1959) recently reported that, con- trary to previously published observations, neither the degree of polymerization nor the degree of esterification of apple pectins change materially during ripening and storage. They found that in some apple and tomato varieties 9 the total pectin decreased significantly from the unripe to the overripe stage. They did not find a consistent result in the case of the soluble pectin. Conrad (1926) reported that unripened dewberry fruits contained an average of 1.00% total pectin and 0.15% soluble pectin on fresh weight basis, and the ripened fruits con- tained 0.70% and 0.51% respectively. In strawberries, on the other hand, he found that the percentage of pectic con- stituents of strawberries are more stable than those of raspberries. Lampitt and Hughes (1928) reported an average of .68% total pectin (as pectic acid on fresh weight basis). Lampitt and Hughes (1928) reported that the average total pectic substances of ripened raspberries is 0.71% (as pectic acid on a fresh weight basis). money and Christian (1950) found only O.h0% total pectin (as calcium pectate on a fresh weight basis). Iampitt and Hughes (1928) found that the average total pectin of ripened blackberries is 0.9A% as pectic acid on a fresh weight basis. More recently, MOney and Christian (1950) gave an average of 0.70% and 0.63% calcium.pectate in wild and cultivated blackberries, respectively. Parikh (1953) found that the total pectic content of Montmorency cherries, Rubel blueberries and El-Dorado black- berries decreased as the fruit matured. All fruits studied had sufficient amounts of pectin for Jellying except cherries. Gee and McCready (1957,) reported that Montmorency cherries became firmer when they were stored at temperatures 10 of 20°F or higher, but the texture did not change when the fruits were stored at -10°F. This is related to the chemi- cal changes of pectic substances. Gee and McCready (1957) found that the percentage of esterification of cherries stored at 20°C or higher is lower than those stored at -10°F. They attributed the change in texture to an enzymatic de- esterification and the formation of calcium.pectate gel from the calcium present in the fruit tissue combining with the carboxyl groups when the cherries are not frozen solid as they were at -10°F. Bedford and Robertson (1962) indi- cated that harvesting, pruning, spraying, handling and harvest time affect the yield and quality (such as firmness) cf both canned and frozen red cherries. These changes may be due to the enzymatic activity involved. The degree of pectin breakdown in apples and oranges depends on the variety and stage of maturity (Doesburg, 1951b). NbCready and MbComb (195ha) also suggested that changes in pectic substances due to enzymatic action may be largely responsible for the softening of brined cherries. This theory was substantiated later by Ross g§_gl, (1958), Yang 3241;. (1959), and watters M. (1963). Euch gt_gl, (1961) showed that red tart cherries allowed to stand before being canned, either with or without having been previously bruised, were much firmer after canning than were similar cherries canned immediately after harvest. They explained that during the aging a portion of the pectin did not change. The histolOgical examination revealed that 11 the cell walls of the aged cherries were more rigid and less easily separated from each other than the unaged. Several workers have estimated pectic substances in cherries on a dry weight basis (Conrad, 1926); on a fresh weight basis (as pectic acid) (Lampitt and Hughes, 1928); as calcium pectate (money and Christian, 1950) and more recently by Carbazole calorimetry and titrimetrically (as anhydrouronic acid) (Gee e£_al,, 1958). MbArdle and Nehemias (1956) found that gamma radiation induced softening of apple and carrot tissues as a result of changes in pectic substances. Gamma radiation caused a decrease in protOpectin and total pectic substances. Solu- ble pectin and pectates, on the other hand, increased. These changes were accompanied by depolymerization of the pectin, pectate and protOpectin molecules as indicated by a decrease in their relative viscosity. Pectic Enzymes The field of pectic enzymes is rather confusing due to inconsistent nomenclature, non-homogeneous substrates and poor methods of analysis. Protopectinase supposedly hydrolyzes protOpectin and pectin and other cell wall constituents (Davison and Willa- man, 1927). There is very limited knowledge about proto- pectins and their enzymes. Pectinesterase (PE) (Lineweaver and Ballou, l9h5) or pectin-methylesterase (Kertesz, 1937) or pectase (Willaman, 12 1927) catalyzes the fissure of the natural or synthetic methylester of polygalacturonic acids (Fellenberg, 1918). pectin -—RE—9 pectic acid -+ methanol Polygalacturonase (PG) (Kertesz, 1936) or pectinase cleaves hydrolytically the glycosidic bonds of polygalac- turonic acids. pectic acid .39—; oligOgalacturonic acids -—-—e» galacturonic acid Polygethylgalacturonase (PMG) or pectic depolymerase (PP) hydrolyzes esterified pectic substances and pectic acid producing polygalacturonates (Morell M” 1931}; Jansen, g£_gl,, 19h9; Roboz 3§_g;,, 1952; and Seegmiller and Jansen, 1952). PMG has been found only in fungi where its signifi- cance has not yet been determined. Pectin-transeliminase (PTE) (Albersheimug£_gl,, 1960; Albersheim and Killias, 1962) attacks only the methylester of pectic acid, resulting in unsaturated galacturonic acid groups. PTE was originally detected in a fungal preparation although Albersheim.and Killias (1962) have claimed that this enzyme is present in higher plants. This study will concern only PE and PG. Pectinesterase (PE). PE occurs in plant tissue mainly adsorbed on the cell walls (MacDonnell §£_g;,, 19h5; McColloch 313;” 1946; McColloch and Kertesz, 19117; Pollard and Kieser, 1951; Pollard and Kieser, 1951; Owen £2_§;,, 1952; Kertesz, 1951, 1955; Jansen gt_§l,, 1960) and in some fungi and bacteria, but apparently not in animal tissue. 13 The chemical action of PE was not understood until after Fellenberg (1918) recognized the presence of the methyl ester group in pectin. Glasziou and Inglis (1958) have presented evidence that tobacco pith contains two PE fractions, and artichoke tubers yield three fractions. More recently, Hultin and Levine (1963) Presented evidence that fractionation based on differential extraction, pH dependence, activation or inact- ivation by the anionic surfactant, sodium.dodecy1 sulfate, and differential temperature inactivation resulted in at least three molecular forms of PE in the pulp of the banana. PE was best extracted from.the pulp of the tomato (Mc- Colloch $121., 1946; McColloch and Kertesz, 1947; Pithawala 5£_21,, 1948) and from the insoluble particles of orange (MacDonnell g£_2l,, 1945) with about 0.25 M na01 or 0.1 M NaePOu (Pithawala M” 1948) at a pH near 8 (Owens 3331., 1952). Nagel and Patterson (1963) found that NaC1 concen- tration, pH, blending time, storage time, and method of filtration affect the extraction of PE in AnJou pears. They concluded that the extraction of PE was maximum in a pH range of 9 to 10 and a salt concentration of 4%. Tomato PE has been purified by repeated dialysis and extraction of the resultant precipitate (MbColloch g£_g},, 1946) or by fractional precipitation with (Nflu)2SQh (Mc- Colloch and Kertesz, 1947) and the orange PE by fractional precipitation, dialysis, adsorption and elution (MacDonnell 5333., 1950). It seems that the PE activity of higher 14 plants is greatly affected by the concentration and type of salt present in the reaction (Kertesz, 1951). It has been found that 3-indoleacetic acid promotes the activity of PE (Bryan and Newcomb, 1954) and promotes its binding to the cell walls of tobacco pith (Glasziou, 1957) but Jansen g§_g;, (1960) have reported that this substance did not influence the PE binding capacity of Avena coleOptile cell walls or tobacco pith tissue. Assay has been performed by three methods: a) measuring the gel-formation time (in min.) in the presence of Ca012 (Willaman, 1927); b) measuring carboxyl group formation from 0.5% to 1.0% solution of pectin 1n the presence of 0.15 to 1.5 M univalent cation by continuous titration at pH 7.5 (MacDonnell gt_gl,, 1945; Konovalova, 1961); c) determination of the free methanol (Holden, 1946; Pithawala and Savur, 1948; Mehlitz and Drews, 1960; Kenovalova, 1961). Optimum pH is dependent on the kind and concentration of cations (Lineweaver and Ballou, 1945) e.g., in .15 N Na+' the optimum pH is 7.5 whereas in .05 M Ca“ full activity is obtained from 5 to 8.5. It seems that as the concentra- tion of salt was decreased, the Optimum pH shifted toward the alkaline region (MacDonnell M” 1945; and Pithawala g§_§l., 1948). The dependency of PE activity on the pH- cation relation is complex. PE is very resistent to heat in its natural state, but the enzyme is rapidly inactivated at temperatures above 45°C when it is in solution (HacDonnell £t_gl,, 1945). 15 Lineweaver and Jansen (1951), NbCready and Seegmiller (1954) indicated that PE purified from higher plants is very specific; it hydrolyzes only the methyl and ethyl esters of polygalacturonides of at least ten units length. Fungal PE, on the other hand, hydrolyzes the methyl ester of galacturonic acid (macDonnell gt_gl,, 1950; MbCready and Seegmiller, 1954). More variability has been demonstrated. PE is not readily inactivated by such substances as cyan- ide, iodine, formaldehyde, etc. (MbColloch and Kertesz, 1947). ‘Pglygalacturonase (PG). PG causes the glycosidic de- polymerization of pectic substances and may be involved in fruit softening during ripening, especially post-harvest softening. This enzyme occurs in many bacteria, yeasts and molds as well as in certain fruits and vegetables, namely tomatoes (McCready g_t_a_1_., 1955; Foda, 1957; Robson, 1962), avocados (NbCready-gt_gl,, 1955; Robson, 1962), pears and pineapple (Robson, 1962). Some workers contend that there is but one PG (McColloch and Kertesz, 1948; MbCready and Seegmiller, 1954; MbCready g§_§;., 1955; and Phaff and Demain, 1956). Other workers, however, claimed to have detected two (Ayres g£_gl,, 1952; Ozawa and Okamoto, 1955), three (Dingle gt_§l,, 1953; Hath- way and Seakins, 1958), and even four (Schubert, 1952) different PG. Purr g£_g;, (1957) have finally proved the existence of at least two PG. Idneweaver g£_gl, (1949) stated that there are two purification methods for PG: one consists of adsorption 16 on alginic acid, elution and dialysis; the other is by acid treatment, fractionation with (NHu)230u and with lead ace- tate and dialysis. The following four methods have been used for assaying PG: a) measurement of reducing group foration by the Willstitter-Schudel hypoiodite method (Jansen‘g§_gl,, 1945; Deuel and Stutz, 1958); b) measuring the decrease in viscos- ity (by 0stwald viscosimeter in sec.) at 30°C after citrate buffer addition (pH 4.4) (Reid, 1951); c) colorimetric method (Foda, 1957) based on that of Willaman and Davison (1924); and d) by a diffusion method in agar substrate, preposed by Dingle gt_gl, (1933). Optimum pH is 4 to 5. PG from most sources is rapidly inactivated at 60°C or above (MbColloch and Kertesz, 1948). Only de-esterified pectin substances are hydrolyzable (Jansen and MbcDonnell, 1954) and no other polysaccharides are known to be hydrolyzable (Lineweaver g§_§1,, 1949). No co-factors are known and no reversibility has been demonstrated. Contrary to early reports (MbColloch and Kertesz, 1948), the course of hydrolysis of pectic acid by the tomato enzyme is identical with that from fungi and the avocado (McCready M” 1955; and Foda, 1957). Fremy (1848) was first to report the existence of an enzyme which in the presence of calcium ions converts pectin to a gel. Biedermann (1951) and Doesburg (1951a) stated that both pectase and pectinase are required to decompose pectin. 17 McColloch (1949) also reported that changes in pectic sub- Stances are due to both PE and PP. The pectinic acid is first demethylated by the action of PE, then PP depolymerizes the pectic acid. Solms and Deuel (1955) reported that the PE and PG degradation of pectic substances depends on the length of pectic molecules and the number and distribution of methyl ester groups. They concluded that free carboxyl groups next to an esterified group are essential for the action of PE. Two neighboring free carboxyl groups allow a higher reaction velocity than only one. The probable explanation for softening of cherries is that pectic substances are degraded to lower molecular weight material by pectic enzymes, probably produced by microorganisms (MbCready and MbComb, 1954a). It seems most likely that mold or yeast contamination of cherries or the container occurs before the brining procedure and the subsequent action of pectin-degrading enzymes. Due to the PE action of pectin de-esterification the Ca present in the brine reacts with the pectic acid, forming Ca pectate gel in cherries which results in firmness. Beneke gt__a_v_1_. (1954) reported that the apparent degrada- tion of molded Michigan strawberries was found to be associated with a greater pectinase content. Brekke.gt_gl. (1963) observed that two different mechan- isms, enzymatic and non-enzymatic, may be involved in the softening of brined cherries. They found that heat treatment 18 (165°to 170°F) served to inactivate pectic degrading enzymes and the addition of extra Calcium.chloride (2% or more by weight) to the brine served to inhibit either enzymatic or non-enzymatic softening. Commercial applications of pectic enzymes (such as pectolytic enzymes) have been used on a large scale in fruit Juice and wine technology (Langlykke‘g£_g&,, 1952). Pectinesterases have been used for the production of low ester pectins which are used in the manufacture of low-sugar Jams and Jellies (Kertesz, 1960). A flash heat treatment and cooling of many concentrated fruit Juices such as prunes, grapes and citrus inactivate the pectin~destroying enzymes and result in a natural color and fresh flavor. In the pickle industry, the "softening" of cucumbers is a well-known spoilage problem and results in losses to this industry. when information on this subject has been pub- lished (Bell, 1951; Bell gt_§l,, 1951, 1955. 1958; Etchells et_gl,, 1955, 1958). The cause of this softening is gen- erally known to be due to the hydrolysis of protopectin of the middle lamella of the cucumber and results in a mushy texture. Polygalacturonase and pectinesterase seem to be the responsible degrading pectolytic enzymes in cucumber pickling. The source of these enzymes is reported to be from the microorganisms grown on the flowers, leaves and vines which adhere to the cucumber (Bell, 1951; Bell et al., 1951, 1958). 19 Hamilton and Johnson (1961) were unable to demonstrate the ability of ninety-nine gram positive sporeforming bacteria and fifteen yeast cultures isolated from comner- cial cucumber salt stock brine to produce pectolytic enzymes. They stated that the pectic enzymes of both the cucumber tissue and filamentous fungi could be responsible for the pectolytic softening of pickled cucumbers. METHODS AND MATERIALS Assembling the Material In this investigation, red tart cherries grown at the Muchigan State University farm in East Lansing were used. Cherry samples were picked at three stages of maturity--two weeks prior to commercial harvest, at commercial harvest, and two weeks after commercial harvest. Upon arrival at the laboratory, aliquots were used to determine pectic consti- tuents, the activity of pectic enzymes and methanol content. The samples were then divided into three parts. The first part was canned and processed at 210°F for 15 minutes, then stored at 55 F. The second part (including pitted samples for methanol analysis) was frozen in sealed cans at -12°F. The last part was irradiated by a resonant transformer electron accelerator at the Michigan State university Agri- cultural Engineering Department. Each sample at each stage of maturity received three levels of radiation dosage (93, 186 and 372 K.rads). The radiation treatment was conducted on both sides of the fruit (Figure 1). All irradiated same ples were then frozen in sealed cans at -l2°F. A11 prepared materials were stored under the given conditions and held for further analysis. Determination of Pectic Substances in Cherries In the canned and frozen material at various stages of maturity water insoluble pectin, water soluble pectin and 20 Irradiation of cherries with accelerated electrons by a resonant transformer. Figure l. 22 total pectin were determined after storage for 0, 4, 8 and 12 months. All samples were extracted and prepared as alco- hol insoluble solids (AIS), stored in a freezer and then analyzed for pectic components. All results were expressed as percentages of anhydrouronic acid on a fresh weight basis. Figure 2 shows the standard curve for galacturonic acid determination by the Carbazole method used for pectic com- ponent analysis. Tbtal_pectic sgbstances. The Carbazole-colorimetric method develOped by McComb and McCready (1952) and the versene- pectinase extraction procedure developed by NbCready and McComb (1952) were used for the determination of total pectin in cherries. water insolublepectin (protopectin). The extraction pro- cedure introduced by Postlmayer gt_§l, (1956) and the Carbazole-colorimetric method developed by McCready and McComb (1952) were used for the determination of protopectin in cherries. nWater soluble_pectin. The difference between the total pectin and protOpectin contents is the water soluble pectin. Determination of Pectic Enzyme Activity in Cherries Pectinesterase (PE). The pectinesterase activity was measured by the titrimetric method as originally developed by Kertesz (1937) and later modified by Lineweaver and Ballou (1945), McColloch and Kertesz (1947), Owens g_t_:_a_1_. (1952), and Nagel and Patterson (1963) was used, with slight 23 .coseos oaoasaaso one as seaweedsauuoo pace oacoasposaam you o>azo pudendum .m shaman A.H\xv couumanaoosoo owed odooaspomasu on om 0: cm om 0H 0 q . fl d J 4 d q 41 (I1 . I OOH. .L com. .I com. I 00:. oom. °nm oagfoueqaosqv ; 24 modification. It is as follows: 20 g. of pitted cherries, either fresh or frozen, (after a partial defrost) were blended in a Waring blender with 50 ml. of 1.5 M NaCl for 3 minutes. The macerate was adjusted to pH 7.5 by a pH meter using a magnetic stirrer for continuous mixing. The volume was made to 100 ml. by the addition of 1.5 M NaCI and the mixture left in 40°F for about one hour to extract the en- zyme which was adsorbed on the cell wall material. The macerate was filtered through #2 Whatman filter paper in a Buchner funnel with a slight vacuum. Five m1. of the fil- trate (equivalent to 1 ml. cherry extract) was used to measure the PE activity. This crude preparation of the en- zyme was added to a mixture of 20 m1. of 1% high methoxy orange pectin and 5 m1. of 1.5 M NaC1 previously adjusted to pH 7.5, and placed in a 100 ml. beaker. The total volume of the reacting mixture was 30 ml., which contained 0.5 M NaCl. This beaker was partially immersed in a water bath adjusted to a constant temperature of 30°C. Immediately after adding the enzyme extract, the pH was readjusted to 7.5 and the time was noted. TWo-hundredths normal HaOH was added continuously to keep the pH level between 7.0 and 7.5. The volume of NaOH used was recorded at five minute intervals for 30 minutes. A pH.meter with glass electrode and magnetic stirrer was used. The pectinesterase activity was eXpressed as -COOH .aequiv./min./g. cherries. Polygalacturonase (PG). Fresh and mature pitted cherries 25 were used in these experiments. The titrimetric method based on changes on reducing value as presented by Jansen and MacDonnell (1945), Kertesz (1951) and Owens g§_gg, (1952) was used to measure the PG activity in cherries. The colorimetric method based on that of Willaman and Davison (1924) and modified by Foda (1957) and used by Hobson (1962) was also used to measure the activity of PG in cherries. mny workers believed that certain microorganisms grown on cherries may produce PG, which is supposedly responsible for pectin destruction and consequently the cause of the loss of firmness in fruits. Upon this suggestion, samples of mature freshly picked cherries were left under room conditions for a week to permit the natural microbial flora of cherries to populate; then the samples were analyzed by the above methods. Determination of Methanol Content in Cherries The method of Mehlitz and Drews (1960) for determina- tion of methanol content in juices was used with slight modification. In the pitted frozen cherries methanol con- tent was determined after 0, 1.5, 12 and 24 months of storage. Fifty grams of pitted cherries and 50 ml. boiling demineralized water were added to a 250 m1. distillation flash with a small amount of an anti-foam compound to pre- vent frothing. Immediately, in order to minimize further 26 methanol production, the mixture was distilled at about 100°C until about 66 ml. of distillate were collected and made up to 100 ml. with demineralized water. Five ml. of the diluted distillate were transferred to a test tube and 5 ml. KMhQu-H3P0h solution (a mixture of 2 ml. of a solution prepared from 6 ml. H3P0h (d 1.70) made to 100 ml. with demineralized water and 3 ml. of 5% KMnQu) were added. The mixture was stirred and left to stand for 15 minutes. Then 8 ml. of a 7% oxalic acid solution (containing 10 m1. of concentrated H2804) were added and the solution mixed. After the solution was decolorized, 2 ml. were transferred to a test tube to which was added 1 ml. of a solution consisting of l g. of chromotrOpic acid and 0.5 g. of crystallized sodium sulfide in 100 ml. of water (this solution generally keeps only 3 days) and 8 ml. of 81% sulfuric acid. The well-shaken mixture was placed in boiling water for #5 min. and later cooled to room temperatures. The red coloration obtained was measured in a Spectronic 20, at a wave length of 570 mu. The amount of methanol was deduced from a calibration curve based on known concentration of methanol (Figure 3). Absorbancy570 mu. .900 .800 .700 .600 .500 " .MOO ___l_____ _r___ 27 'T'" 0300 o/ I.- . !--_. ._ _ ‘ L _ - _ u | 50 100 150 mg. methanol/1. Figure 3. Standard curve for methanol determina- tion by Nehlitz and Brews method. RESULTS AND DISCUSSION Pectic Substances The alcohol insoluble solid (AIS) samples which were stored in a freezer (-l2°F) were used to determine total pectin (TT), water insoluble pectin (NIP) or protOpectin and water soluble pectin (WSP) content in fresh, frozen and canned cherries by the Carbazole-colorimetric method of MbComb and McCready (1952). A standard curve for galactur- onic acid determination by the Carbazole method (Fig. 2) was used to estimate the pectic components. All results were tabulated as grams anhydrouronic acid (AUS) per 100 grams fresh fruit (% AUA on a fresh weight basis). All the analyses were conducted, as far as possible, under similar conditions to minimize the variations in the results. Frozen cherries. Analyses were performed to determine the effect of the various stages of maturation (two weeks prior to commercial harvest (premature), at commercial harvest (mature), and two weeks after commercial harvest (overmature)) and storage time (0,h,8 and 12 months) on the total pectin (TT), water insoluble pectin (WI?) and water soluble pectin (WSP) contents of frozen cherries. The results are presented in Appendix Tables 1, 2 and 3 and are histographically summarized in Figure 4. The total pectic content of the premature cherries was considerably higher than in the mature or overmature fruits. 28 g _ 29 The total pectic content in the premature cherries stored for 0, 4, 8 and 12 months averaged .431, .466, .460 and .431% AUA, respectively, compared to .305. .308, .287 and .282% AUA in the mature fruits and .304. .335. .308 and .306% AUA in the overmature cherries. It is clear from these data that the total pectic content did not change drasti- cally during frozen storage at each stage of maturity. There was a slight increase in total pectin in the mature cherries after 4 and 8 months storage, but after 12 months storage this value decreased again to nearly the original content. In the mature fruits total pectic content de- creased slightly after the 8th month of storage and an insignificant further decrease occurred after the 12th month. There was only a slight increase in the total pectic content after the 4th month of storage. This value declined to approximately the original amount after the 8th and the 12th month in the overmature fruits. In the unripe cherries Conrad (1926) found 11.4% pectic substances (on a dry weight basis). In the corresponding ripe samples he found only 4.3%. This significant decrease in the total pectin from the unripe to the ripe cherries is not supported by the results of this investigation. Conrad (1926) found the Opposite result in strawberries where the percentage of pectic constituents did not decrease when the berries became overripe. McCready and NbComb (1954b) also reported that the anhydrouronic acid content of ripe peaches, pears and avocados were essentially the 30 same as those of unripe fruits. Woodmansee g£_g;, (1959), on the other hand, recently reported that, contrary to the previously published observations, the total pectin de- creased significantly from.the unripe to the overripe stages of some apple and tomato varieties, and they did not find a consistent result in the case of WSP. Lampitt and Hughes (1928) gave an average of .35% (.2& -'.545) pectic acid in cherries on a fresh weight basis. This result seems to be in general agreement with the total pectic substances found in the cherries used in this study. Money and Christian (1950) found later that in Morrella cherries an average of .16% calcium pectate was present while in red and white cherries the contents were .28% and .31%, respectively. mere recently, Gee g£_§l. (1958) investigated the pectic content of a number of fruits and found that the total pec- tin (estimated as percentage AUA of the mare, which was extracted, dried, ground to pass through a 40 mesh screen under ordinary laboratory conditions) of Mbntmorency cherries was 29.3% by the Carbazole method and 28.5% by the titri- metric method; the total pectin of Royal Anne cherries deter- mined by the above methods was 30.5% and 30.4%, respectively. It is rather difficult to compare the several findings of the previous workers with the results obtained in this investigation since the methods of analyses and the units expressing the results were different. The water insoluble pectin and the water soluble pectin 31 content of cherries at various stages of maturation through- out the stored frozen period are also presented in FigureL}. The data shows that there were inconsistent variations in the WIP and WSP during the various stages of maturation and throughout the storage time. The average WIP content in the premature cherries was considerably higher (.404% AUA) than that of the mature (.289% AUA) or of the overmature (.288% AUA) fruits. In the stored frozen cherries the WIP content of the premature fruits increased slightly after the 4th month (.427% AUA) and continued through the 8th month (.433% AUA). Then this value decreased to about its orig- inal (.401% AUA) at the end of the 12th month. In the mature cherries there was only a slight decrease in the WIP content between the 8th (.283% AUA) and the 12th month (.259% AUA). The WI? content of the overmature cherries was the same except for a slight increase at the 4th month (.314% AU'A). The average water soluble pectin content (the differ- ence between the total pectin and the water insoluble pectin content) of the premature cherries was higher (.027% AUA) than in the mature (0.016% AUA) or overmature (.016% AUA) fruits. Some workers (Conrad, 1926; Kertesz, 1951; Postl- mayer and Duh, 1956; Sinclair and Jolliffe, 1961) have found that WSP increased with maturation; others, however (Conrad, 1926; Woodmansee g£_§;,, 1959), have found no such increase. The type of fruit, the varietal difference, the 32 climatic and soil conditions, the physiological conditions of the trees and fruits, the microbial contamination of the fruits, the variations in the sampling, handling and method of analysis could contribute to the inconsistent variation of the results. Joslyn and Deuel (1963) stated that it is difficult to avoid the pectic degradation during extraction. Gee g§_§;. (1958) reported that there was a measurable change in the esterified carboxyls to free carboxyls if the nnrc was left for one week at room temperature. The AIS used in this investigation was left at room temperature for 48 hours only. Kertesz (1951) stated that the major change in stored fruit is the loss of water insoluble protopectin and pec- tates leading to an increase in WSP. This statement support- ed the findings of the early work of Appleman and Conrad (1926) and Postlmayer and Duh (1956) who reported that the amount of WSP formed from the insoluble protopectin was parallel to the degree of softening of peaches during ripening, but this was in contrast to the results found in this study on cherries. Enzymatic changes could obviously take place in frozen cherries during storage since pectin- esterase activity is indicated (p.37) and the production of methanol established (p. 45'). This might result in some degree of softening which could affect the commercial yield of long term stored cherries as well as dry matter charac- teristics. 33 .mpdnzpms mo humour msoanm> pm oepne>nmo meanneno nouoau concur no psopcoo :«ooeo oaosaom pops: one Cancun eaosnonca mops: ecapooa Hopoe .: enemam pmo>nmn Hedonuasoo umo>mo2 pno>smn Hmaosoasoo nevus execs m Haaomoasoo no on scape execs m 08H» wnfixodm «H m a 0 ma m a o «H m s o 185 scsoem \\:\w\ \\mH&\\\\\. «Om... \:\\- mixiu x \xVb. 1 00H. m 1 com. .1 00m. .L 00:. 2303 oaosaom § .1 - nausea dance 55er 0.33.32.“ I I oom. 31nd; used; '8 001/‘3 pics otuoanoapxquv 34 Shanned cherries. The results of the effect of the stage of Imaturity and storage time on total pectin, water insoluble jpectin and water soluble pectin content of canned cherries are presented in Appendix Tables 1, 2 and 3 and they are histographically summarized in Figure 5. The total pectic content in the premature canned cherries stored for 4, 8 and 12 months averaged .389%, .386% and 359% AUA, respectively, compared to .383%, .383% and .316% AUA in the mature canned fruits and .33756. .35956 and .316% in the overmature canned cherries. From this data the total pectic content of the premature and mature mater- ial after the fourth and the eighth months and of the over- mature fruits after the eighth month can be seen to be practically identical. The total pectin of the mature and the overmature fruits after the twelfth month were also the seam. The NIP and the WSP of canned cherries at various stages of maturation throughout the storage time are also Summarized in Appendix Table 3 and Figure 5. The WIP contents of the premature and mature canned cherries after the fourth month of storage were practically the same (.367% and .365% AUA, respectively). These values decreased slightly to .357% and .356% AUA after the eighth month of storage, and decreased further (.339% and .294%) after the twelfth month of storage. The results of NIP contents of the overmature canned cherries during storage were variable. 35 The WSP content of canned cherries which had been picked at various stages of maturity throughout the storage period were slightly variable. The premature canned cherries stored for 8 months had the highest value of .029% AUA and the mature canned cherries stored for 4 months had the lowest value of .018% AUA and the rest of the results were between these two values. In canned cherries, processed at 210°F for 15 minutes, heat may affect the pectic constituents in plants directly by changing the water insoluble pectins into water soluble ones in addition to causing other reactions which influence the texture change (Kertesz, 1951). The WSP formed by heat may be lost from the fruit to the water in the cans since only the drained pitted fruits of the canned cherries were used for this analysis. The fact that the WI? of the premature and mature cherries de- creased with storage time might indicate a transformation of the WIP to NSF; however, since there is no significant increase of NSF in both of these during storage, it is possible that the excess WSP formed is diffused out into the water inside the cans. Buchlg£_gl. (1961) presented data showing that red tart cherries allowed to stand up to 20 hours before being canned, either with or without having been previously bruised, were much firmer after canning than were similar cherries canned immediately after harvest. They stated that during the aging period some pectin was completely demethylated to 36 .thmSme no humans usednm> pm nepno>nwn meanness menace oopOpm no pcopcoo nausea eHosHon means one nausea oHozHomcfi mops: .aauoeo Hmuoa .m shaman pno>amn HedonoEEOO pme>nos pme>nmn HoaopeEEOo mound name: N Hdaomoaaoo pm 0» moama mxoez N 05“» wsfixoam «H m e «H w s «H m a fiscal esteem _ \\ \ \\\\s§s\\\ \\\\\\ fit 1 02. I oom. .I 00m. I|||IL. I II I 8:. nausea eHosHom .I oom. nausea Hmpoe nausea mangoes.“ WV 11nd} used; '8 con/'8 pros opuOJnoaquuv 37 pectic acids by pectinesterase and the histological examina- tion showed that the cell walls of the aged cherries were ‘more rigid and less easily separated from each other than the unaged. Gee g£_§;, (1961) showed that this phenomenon is not related to changes in pectic substances since the rigidity could be maintained even when all pectic acid was removed with neon. They believed that during the aging period some unknown compounds were formed that imparted sufficient rigidity to the cell walls of cooked cherries. Pectic Enzymes Pectinesterase (PE) The PE activity will be discussed separately for the frozen, irradiated and canned cherries. PE activity;in stored frozen cherries. The effects of degree of maturity and storage time of frozen cherries on PE activity are presented in detail in Appendix Table 5 and summarized in Appendix Table 6 and Figure 6. The PE activity of the premature cherries frozen for one month was less (.81 -CO0H "-equiv./min./g. fruit) than the PE activity of the mature (3 .24 -coon e-{l-equiv./inin./g. fruit) and the overmature (4.88 -000H .4Lequiv./min./g. fruit) cherries frozen for the same period of time. These results agree with those of Gee and MbCready (1957) who reported that the PE activity of ripe and frozen Montmorency cherries from Washington and Michigan was 4.00 -CO0H zV-equiv./min./g. fruit. 5.00 4.50 4.00 3.50 3.00 2.50 PE Activity (-coon A-equivalent/min./g. fresh fruit) 1.00 .50 4} = 2 wks prior to cml. harvest _ 43" at cml. harvest ~D'= 2 wks after cml. harvest p Are” {a ir l 4L— ! x .1 J J 1 J l ' 0 1 4 8 12 Storage Time (Months) Figure 6. Pectinesterase (PE) activity of frozen cherries picked at various stages of maturity. 39 These results seem to indicate that either the enzyme is not synthesized in the early stages of fruit maturation or there may be some unknown compounds present in the earlier stages of fruit maturation which could inhibit the activity of PE. There was only a slight increase of PE activity between the frozen cherries stored for one month and those stored for four months. After 8 months of storage the PE activity sharply decreased in the mature (1.97 -CO0H,6(-equiv./m1n./g. fruit) and in the overmature (2.76.17-equiv./min./g. fruit) cherries, but only a slight decrease took place in the pre- mature cherries (.84 -COOH xfi-equiv./min./g. fruit). There was a further decrease of the PE activity in the mature (1.60 -COOH x7-equiv./min./€. fruit) and the overmature (2.15 -COOH.Ay-equiv./min./g. fruit) frozen cherries after 12 months of storage. This decrease was not significant in the premature fruit (.80 -coon 47;equiv./min./g. fruit). The results also show that as the storage time in- creased (8 months or longer), the PE activity decreased. This result may suggest that under very low storage tempera- tures for a long period of time some proteins in frozen tissues may denaturate and, since enzymes are protein, it is possible that some of the PE are inactivated under these conditions. PE activity of irradiated cherries during frozen storagg. The results of PE activity of the irradiated (93, 186 and 372 K rad) cherries at various stages of maturation during 4O frozen storage is shown in detail in Appendix Table 6 and summarized in Table 7 and Figure 7. In general, the PE activity of the irradiated cherries at various stages of maturation during frozen storage follow the same trend as the non-irradiated stored frozen cherries, although the values for irradiated cherries increased slightly (Figures 6,7) in all cases where the PE activity of the premature cherries was less than in the mature cherries and the PE activity of the mature cherries was less than that of the overmature fruits. It seems that doses of 93 K rad and 186 K rad did not significantly influence the PE activity at various stages of maturity and during the frozen storage time. The 372 K rad dosage did increase the PE activity after 4 months storage only in the premature and mature cherries, but in the case of the overmature material this activity was slightly decreased. At the dose levels used here no enzyma- tic inactivation was expected because it has been found that doses of approximately a million reds are required for enzymatic inactivation. However, the doses used here have been suggested for pasteurization of fruit. It seems that the 372 K.rad dose activated pectin- esterase in the premature and mature cherries, but not in the overmature fruits. McArdle and Nehemias (1956) reported that gamma radiation caused a decrease in protOpectin and an increase in soluble pectin in apples and carrots and resulted in tissue softening. These changes were accompanied by depolymerization of the pectin. Therefore, it is possible PE Activity (-COOH/tL-equiva1ent/min./g. fresh fruit) 6.00 5.00 4.00 3.00 2.00 1.00 41 Time related to commercial harvest 0: 2 weeks prior £r=at harvest {}=2 weeks after Radiation dose (K rad) __ _ ... :: 18g \ -37 oi..- \ x . 0.,— {‘0 r- ‘0 1 l J L .1... i O 4 8 12 Storage Time (months) Figure 7. Pectinesterase (PE) activity of irrad- iated cherries at various stages of maturity during frozen storage. 42 that the increase of PE activity at the 372 K rad dose causes the transformation of protopectin to soluble pectin and results in a subsequent increase in the substrate avail- able for the enzyme. This explanation is valid for the premature cherries but not for the mature or overmature material since the premature fruit has the highest proto- pectin content. It should be noted that the entire volume of cherries did not receive the entire dose of 372 K rad. Desrosier and Rosenstock (1960) calculated that l Mev. beta particle penetrates approximately 0.5 cm. in water. The cherries used in this study had an average radius of 1 cm. and the average radius of the pits was 0.3 cm. If we assume that due to the density of the cherry flesh, the depth of pene- tration was only 0.2 cm., then the percentage of flesh irradiated would be 55.6% calculated as follows: 4 'n’ (l — 0.8)3 3- .512 x 100 -.- 55.6% § 11’ (1 - 0.2)3 '92 The fact that not all of the tissue was subjected to radia- tion is an important consideration in evaluating the pectinesterase activity. PE activity_in canned cherries. Uhpitted cherries were canned and processed at 210°F for 15 minutes and then stored at 55°F. This processing can completely inactivate pectin- esterase as well as other familiar enzymes. There was no detectable PE activity in cherries canned for 4, 8 and 12 43 months which dispelled any Opinion that PE may regenerate its activity in canned material after a certain length of storage. PE activity_of fresh chergigg, During the first phase of this investigation no determination of the PE activity of fresh cherries was performed. In 1963 fresh premature and mature cherries were analyzed immediately after picking. It was found that PE activity of premature cherries was .68 ~COOH sobequiv./min./t. fruit and mature cherries 1.21 -000H ,6/-equiv./min./g. fruit (Appendix Table 9). These cherries were also frozen (-12°F) and their PE activity determined a month later. No change in the PE act- ivity of either the premature or mature cherries was observed. Polygalacturonase (PG) Pectin-glycoside-splitting enzymes such as polygalac- turonase have not been widely reported as occurring in fruits with the exception of tomatoes (Kertesz, 1951; Foda, 1957; and Hobson, 1962), ripe avocados and pears (MbCready and McComb, 1954b; and Hobson, 1962) and Medlar pineapple (Hob- son, 1962). MbCready and McComb (1954b) reported that no PG activity was detected in unripe pears, avocados and unripse or ripe peaches. Hebson (1962) also could not detect PG activity in cranberries, persimmons, cucumbers, tangerines, carrots, melons or grapes. weurman (1954) report- ed that an inhibitor is present in some peach varieties, which may prevent the action of PG. 44 Cherries were assayed for PG to determine, if possible, whether its presence could be demonstrated in red tart cherries. The titrimetric method based on the increase of reducing power (Jansen and MacDonnell, 1945) and the colorimetric method based on that of Willaman and Davison (1924) were used for the estimation of PG activity in cherries. There was no detectable PG activity in fresh and frozen cherries at various stages of maturation. Another attempt was made to measure the PG activity in ripe cherries left exposed to the air for a week for natural microbial spoilage since MCCready and McComb (1954a) suggested that pectic degrading enzymes in cherries are probably produced by microorganisms (molds or yeasts); but these cherries were also negative for PG activity. Methanol Content of Cherries The methanol content of plant tissue can be considered a criterion for the PE activity measurement (Holden, 1946; Pithawala and Savur, 1948; Mehlitz and Drews, 1960; and Konovalova, 1961). Lineweaver and Jansen (1951) stated that PE hydrolyzes the methyl ester of polygalacturonic acids at least 1000 times as fast as it does any of approximately 50 non—galacturonic esters tested. PE hydrolyzes the ethyl ester of polygalacturonic acid at a rate of only 8% of that observed for methyl esters. The methanol content (as mg. methanol/100 g. fresh _ _ _ u _ 45 fruit) of mature cherries during frozen storage is presented in Appendix Table 8 and Figure 8. The recovery experiment on the method indicated an accuracy of 91% 15; therefore the values obtained for methanol were multiplied by the factor 1.1. Fresh cherries contained slightly less (4.82 mg./100 3. fruit) methanol than cherries stored frozen for 1.5 months (10.85 mg./100 g. fruit). The methanol content was nearly doubled after 12 months (16.83 mg. methanol/'lOO g. fruit) and 24 months (17.11 mg. methanol/ 100 g. fruit). These values are of the same order as those reported by Mehlitz and Drews (1960) who found a methanol content of tart cherry Juice of 13.0 mg./100 m1. Juice. These levels of methanol are far below those considered harmful to humans. Mehlitz and Brews (1960) reported that even drinking several liters of apple or grape Juice one would not ingest more than 0.5 g. methanol, whereas 20 g. of methanol are required for sensitive persons to show signs of poisoning. Therefore to ingest this amount one would have to eat more than 100 Kg. (220 lbs.) of cherries at one time. The methanol production in cherries seems to continue during frozen storage at -12°F until it reachesrxmm'maximum level after 12 months frozen storage. Subsequently, and up to 24 months of frozen storage, no significant increase was noticed. Further investigation of this problem would be profitable since methanol production is an indication of the continuous de-methylation of polygalacturonic acid by Methanol mg./100 g. fresh fruit 18 16 14 12 10 46 F { i 'I | L l I J 0 12 24 Storage Time (Months) Figure 8. Methanol content of mature cherries during frozen storage. 47 PE and the de-methylation of pectin is a prerequisite to further de-polymerization of pectic substances by the pectic glycosidic splitting enzymes. As a result, plant tissue is believed to lose its original texture and become mushy and undesirable. It is possible that the methanol did not continue to increase to any degree after 12 months in the frozen stored cherries because the accumulation of methanol in the sealed container may have suppressed further methanol formation. SUMMARY AND CONCLUSIONS The effectsof the degree of maturity and storage time of canned and frozen Mbntmorency cherries on the pectic con- stituents and pectic enzymes of this fruit were studied. Also, the effect of irradiation on the pectic enzymes of frozen Montmorency cherries picked at various stages of maturity was investigated. 1) 2) 3) 4) The following results were obtained: The total pectin, the water insoluble pectin and the water soluble pectin contents of the premature cherries were considerably higher than those of the mature and overmature fruits. There was a little variation in the pectic components between mature and overmature cherries. The time of storage of canned and frozen cherries had little effect on the total pectin, water insoluble pectin and water soluble pectin contents. The total pectic content of the premature, mature and overmature canned cherries was practically the same during the 4th and the 8th months of storage, but these values were slightly lower after 12 months of storage. There was a continuous but slight decrease in the water soluble pectin content of both premature and mature canned cherries during the storage periods. Overmature canned cherries did not show a definite trend in WSP content during storage. 48 5) 6) 7) 8) 9) 10) 49 Pectinesterase activity increased as the stage of matur- ity of the cherries advanced. Pednnesterase activity generally decreased as the storage time of frozen cherries was prolonged for 12 months with the exception of a slight increase after the 4th month of storage . The pectinesterase activity of the irradiated cherries of various stages of maturity during frozen storage followed the same trend as the non-irradiated, stored frozen fruits, but was slightly higher. An irradiation dose of 372 K rad seemed to enhance the pectinesterase activity after the 4th month of frozen storage of the premature and mature cherries. This dose did not affect the PE activity of overmature fruits. There was no detectable polygalacturonase activity in fresh, frozen or spoiled cherries at various stages of maturity. The methanol content of cherries increased as the time of frozen storage was prolonged, until it reached near the maximum level after 12 months. Longer storage resulted in only a slight increase in the methanol con- tent. The concentrations of methanol obtained are far below those considered harmful to humans. Literature Cited Albersheim, P., and Bonner, J. 1959. Metabolisms and hor- monal control of pectic substances. J. Biol. Chem. 234 . 3105. Albersheim, P., Neukom w., and Deuel, H. 1960. fiber die Bildung von ungesAttigten Abbauprodukten durch ein pektinabbauendes Enzym. Helvetica Chimica Acta 18 (173). 1422. 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Total pectin content of fresh, frozen and canned Mbntmorency cherries of varying degrees of maturity after different eriods of storage (5 anhydro- uronic acid (AUA? on a fresh fruit weight basis). % A133 95 AUA“ 5% AUA Treatm nt Absorbancy2 in in in Code I II Avg.fr.fr. AIS fr.fr. F P o .364 .362 .359 .362 .362 1.4 .339 .431 F H 0 .295 .297 .305 .303 .300 1.2 .281 .305 F A .297 .284 .301 .305 .297 1.2 .278 .304 F P .372 .374 .349 .352 .363 1.5 .340 .466 F H .303 .305 .297 .303 .302 1.2 .283 .308 F A .305 .299 .305 .303 .303 1.3 .284 .335 F P .362 .362 .352 .364 .360 1.5 .338 .460 F H .310 .312 .301 .299 .306 1.1 .287 .2 F A .301 .305 .305 .297 .302 1.2 .283 .30 F P .374 .37“ .352 .349 .362 1.4 .339 .43; F H .297 .305 .305 .297 .301 1.1 .283 .2 F A .305 .301 .301 .292 .300 1.2 .281 .306 C P 4 .290 .284 .286 .282 .285 1.6 .268 .389 C H 4 .292 .284 .276 .272 .281 1.6 .264 .383 C A 4 .27”: .274 .290 .292 .283 1.4 .266 .337 C P 8 .284 .284 .292 .252 .284 1.6 .266 .386 C H 8 .286 .222 .268 .2 .282 1.6 .264 .383 c A 8 .278 .2 .270 .288 .281 1.5 .264 .359 c P 12 .280 .284 .282 .282 .282 1.5 .264 .359 C H 12 .282 .284 .290 .284 .285 1.3 .268 .316 C A 12 .290 .282 .290 .282 .286 1.3 .268 .316 1 F = frozen; C = canned; P = 2 weeks prior to comercial harvest; H = at commercial harvest; A = 2 weeks after commercial harvest; 0, 4, 8, 12 months from harvest. 2 Absorbancy at 520 mm. for galacturonic acid used in the test. 3 Alcohol insoluble solids. 4 Anhydrouronic acid. 61 Table 2. water insoluble pectin (protopectin) content of fresh, frozen and canned Montmorency cherries of varying degrees of maturity after different per- iods of storage (% anhydrouronic acid (AUA) on a fresh fruit weight basis). % A185 % AUAfilg AUA Treatmint Absorbancya in in in Code I II Avg. fr.fr. AIS fr.fr. F P o .340 .342 .335 .340 .333 1.4 .318 .404 F H o .292 .2g8 .286 .280 .2 1.2 .266 .289 F A o .388 .2 o .276 .286 .283 1.2 .266 .288 F P 4 .335 .328 .344 .335 .335 1.5 .314 .427 F H 4 .286 .280 .276 .280 .281 1.2 .264 .288 F A 4 .276 .284 .290 .284 .283 1.3 .266 .314 F P 8 .349 .337 .335 .3 5 .339 1.5 .318 .433 F H 8 .280 .282 .284 .284 .282 1.1 .264 .26 F A 8 .278 .280 .286 284 .282 1.2 .254 .28 F P 12 .342 .344 .332 .330 .337 1.4 .316 .401 F H 12 .276 .272 .2 o .278 .277 1.1 .260 .259 F A 12 .280 .272 .268 —- .273 1.2 .256 .278 c P 4 .264 .260 .274 .276 .269 1.6 .253 .367 c H 4 .248 .276 .270 .274 .267 1.6 .231 .365 c A 4 .260 .264 .264 .276 .266 1.4 .2 9 .317 c P 8 .264 .258 .258 .268 .262 1.6 .246 .357 c H 8 .262 .260 .264 .256 .261 1.6 .243 .356 c A 8 .258 .258. .268 .272 .264 1.5 .24 .337 c P 12 .270 .272 .264 .258 .266 1.5 .249 .33 c H 12 .278 .258 .260 .262 .265 1.3 .249 .29 c A 12 .264 .272 .266 .260 .266 1.3 .249 .294 1 F = frozen; C = canned; P = 2 weeks prior to commercial harvest; H'= at commercial harvest; A = 2 weeks after commercial harvest; 0, 4, 8, l2-months from harvest. 2 Absorbancy at 520 mu. for galacturonic acid used in the test. 3 Alcohol insoluble solids. 4 Anhydrouronic acid. Table 3. Total pectin, water insoluble pectin, and water soluble pectin content of fresh, frozen and canned Mbntmorency cherries of varying degrees of maturity after different periods of storage (% anhydrouronic acid (AUA) on a fresh fruit weight basis). Code* Total Insoluble Soluble FPO .431 .484 .02; FHO . 0 .2 .0 FAQ .30: .288 .016 FP4 .466 .427 .035 FH4 .308 .288 .020 FA4 .335 .314 .021 FP8 .460 .433 .027 FH8 .2 26% .024 FA8 .30 .28 .020 FP12 .4gg .401 .030 FH12 .2 .259 .023 FA12 .306 .278 .028 024 .389 .367 .022 CH4 .383 .365 .018 CA4 .337 .317 .020 CP8 .386 .357 .029 CH8 .383 .356 .027 CA8 .359 .337 .022 CP12 .359 .33 .020 CH12 .316 .293 .022 CA12 .316 .294 .022 * F = frozen; C = canned; P = 2 weeks prior to commercial harvest; H = at commercial harvest; A = 2 weeks after commercial harvest; 0, 4, 8, 12 months from harvest. 63 Table 4. Pectinesterase activity of stored frozen Montmor- ency cherries picked at various stages of maturity. Treatment Time m1. .02N NaOH used under test conditions Codes (min.) I II III Ave. F P 1 5 .20 .22 .20 .21 10 .20 .22 .20 .21 15 .20 .21 .20 .20 20 .20 .21 .20 .20 25 .20 .21.20 .20 30 .20.21.20 .20 Ave. rate (m1. .02N’NaOH/min. ) .040 F H 1 5 .80 .82 .82 .81 10 .80 .82 .82 .81 15 .80 .82 .82 .81 20 .80 .82 .82 .81 25 .80 .82 .82 .81 30 .80 .82 .82 .81 Ave. rate (m1. .02N NaOH/min. ) .162 F A 1 5 1.25 1.22 1.20 1. 22 10 1.25 1.22 1.20 1. 22 15 1.25 1.22 1.20 1. 22 20 1.25 1.22 1.20 1. 22 25 1.25 1.22 1.20 1. 22 30 1.25 1 .22 1. 20 1. 22 Ave. rate (m1. .02N NaOH/min. ) .244 F P 4 5 .30 .28 .25 .28 10 .28 .28 .25 .27 15 e30 .26 e25 oz? 20 .32 .27 .25 .28 25 .30 .27 .25 .27 30 .30 .26 .25 .27 Ave. rate (m1. .02N NaOH/min.) .054 F H 4 5 .80 .82 .83 .83 10 .82 .82 .83 .83 15 .82 .82 .83 .83 2O .82 .82 .83 .83 25 . .82 .83 .83 30 .82 .82 .83 .83 Ave. rate (m1. .02N neon/min. ) .166 F A 4 5 1.25 1.20 1.23 1.23 10 1.25 1.20 1.23 1.23 15 1.25 1.20 1.23 1.23 20 1.25 1.20 1.23 1.23 25 1.25 1.20 1.23 1.23 30 1.25 1.20 1.23 1.23 Ave. rate (mo. .02N NaOH/min.) .246 Table 4 (cont.) Treatment Time m1. .02N NIOH used under test conditions Codes (min.) I II III Ave. F P 8 5 .20 20 22 21 10 .20 .20 22 .21 15 .20 .20 .22 .21 20 20 .20 .22 .21 25 .20 20 .22 21 30 2O 20 .22 .21 Ave. rate (m1. .02N NaOH/min.) .042 F H 8 5 .50 .48 .48 .49 1o .50 .48 .48 .49 15 .50 .48 .48 .49 20 .50 .48 .48 .49 25 .50 .48 .48 .49 30 .50 .48 .48 .49 Ave. rate (m1 .02N NaOH/min.) .088 F A 8 5 .69 .70 .68 .69 10 .69 .70 .68 .69 15 .69 .70 .68 .69 20 .69 .70 .68 .69 25 .69 .70 .68 .69 30 .69 ' .70 .68 .69 Ave. rate (1111. .02N NaOH/min.) .138 F P 12 5 .20 .19 .22 .20 10 .20 .19 .22 .20 15 “.20 .19 .22 .20 20 .20 .19 .22 .20 25 .20 .19 .22 .20 30 .20 ' .19 .22 .20 Ave. rate (1111. .02N NaOH/min.) .040 F H 12 5 .40 .40 .40 .40 10 .40 .40 .40 .40 15 .40 .40 .40 .40 2o .40 .40 .40 .40 25 .40 .40 .40 .40 3o .40 .40 .40 .40 Ave. rate (m1. .02N NaOH/min.) .080 F A 12 5 .54 .55 .54 .54 1o .54 .55 .54 .54 15 .54 .55 .54 .54 20 .54 .55 .54 .54 25 .54 .55 .54 .54 3o .54 .55 .54 .54 Ave. rate (n11. .02N NaOH/min.) .108 Table 5. Pectinesterase activity in frozen Montmorency cherr ies (-C OOH //-equ iv . /m1.n . /3 . ) Code“ 1 month 4 months 8 months 12 months FP .81 1.09 .84 .80 FH 3.24 3.32 1.96 1.60 FA 4.88 4.92 2.76 2.15 * F - frozen; P O 2 weeks prior to comercial harvest; H 8 at comercial hsrvest; A I 2 weeks after comrcial harvest. 66 Table 6. Pectinesterase activity of Montmorency cherries irradiated with 3 doses of Mev. electrons and subsequently stored frozen. Treatment Time m1. .02N NaOH used under test conditions Codes * (min . ) I II III Ave . IP 4./93 5 .40 .40 .39 .40 10 .40 .40 .39 .40 15 .40 .40 .39 .40 20 .40 .40 .39 .40 25 .140 .140 039 ouo 30 .40 .40 .40 Ave. rate (m1. .CRN NaOH/min. ) .068 IP 4/186 5 .40 .40 .40 .40 10 .40 .40 .40 .40 15 .40 .40 .40 .40 20 .40 .40 .40 .40 25 .40 .40 .40 .40 30 .40 .40 .40 Ave. rate (m1. .02N NaOH/min. ) .080 IP 4/372 5 .76 .76 .75 .76 10 .76 .76 .75 .76 15 .76 .76 .75 .76 20 .76 .76 .75 .76 25 .75 .76 .75 .75 30 .75 75 .75 .75 Ave. rate (1111. .02N NaOH/min.) .152 IH 4 3 5 1.00 .95 .97 .98 l/9 10 1.00 .95 .97 .98 15 1.00 .95 .97 .9 20 1.00 .95 .97 .9 25 1.00 .95 .97 .98 30 1.00 .97 .98 Ave. rate (m1. .02N Neon/lain.) .196 IH 4 186 5 1.05 1.05 1.02 1.04 / 10 1.05 1.05 1.02 1.04 15 1.05 1.05 1.02 1.04 20 1.05 1.05 1.02 1.04 25 1.05 1.05 1.02 1.04 30 1.05 1.05 1.02 1.04 Ave. rate (m1. .02N NaOH/min.) .208 IE 4 2 5 1.25 1.27 1.26 1.26 /37 10 1.25 1.27 1.26 1.26 15 1.25 1.27 1.26 1.26 20 1.25 1.27 1.26 1.26 25 1.25 1 .27 1.26 1.26 30 1 25 1 .27 1. 26 1.26 C [0 U1 [0 Ave. rate (m1. .02N NaOH/min. ) P:- Table 6 (cont.) Treatment Time m1. .mN NaOH used under test conditions Codes * (min . ) I II III Ave . IA 4 5 1.45 1.48 1.47 1.4 /93 10 1.45 1.48 1.50 1.48 15 1.45 1.48 1.50 1.48 20 1.45 1.48 1.50 1.48 25 1.45 1.481.50 1.48 30 1.45 1. 48 1. 50 1.48 Ave. rate (m1. .CQN NaOH/min. ) .296 IA 4/ 135 51.50 1.50 1.50 1.50 10 1.50 1.50 1.50 1.50 15 1.50 1.50 1.50 1.50 20 1.50 1.50 1.50 1.50 251.501.501.50 1.50 30 '1.50 1.50 1.50 ' 1 .50 Ave. rate (1111. .02N NaOH/min. ) .300 1414 3 5 1.321.35 1.361.34 15 1.32 1.35 1.36 1.34 20 1.32 1.35 1.36 .34 25 1.321.351.36 1:34 301.32 1.35 1.31.34 Ave. rate (m1. .mN NaOI-I/min. ) .26 IP 8/ 93 5 .35 .35 .35 .35 10 .35 .35 .35 .35 15 .35 .35 .35 .35 20 .35 .35 .35 .35 25 .35 .35 35 .35 30 .35 .35 35 .35 Ave. rate (ml. .02N NaOH/min. ) .070 I? 8 186 5 .35 .35 .35 .35 / 10 e35 e35 035 035 15 .35 .35 .35 .35 20 035 035 035 035 25 .35 .35 .35 .35 30 .35 .35 .3; .35 Ave. rate (m1. .02N neon in.) .070 IP 8 3 5 .35 .36 .35 .35 ’/ 72 10 .35 .36 .35 .35 15 035 036 035 035 20 .35 .36 .35 .35 25 .35 O36 O35 035 30 .35 .36 .35 .35 Ave. rate (m1. .02N NaOH/min.) .070 68 Table 6 (cont.) Treatment Time m1. .02N NaOH used under test conditions Codes" (min . ) I II III Ave . IH 8 5 .83 .85 .82 .83 /93 10 .83 .85 .82 .83 15 083 e e& .83 20 .83 .85 .82 .83 25.83 .85 .82 .83 30.83.85 .82 .83 Ave. rate (m1. .02N Neon/min.) .166 IH 8/186 5 .77 .76 .75 .76 10 .77 .76 .75 .76 15 .77 .76 .75 .76 20 .77 076 075 O76 25 .77 .76 .75 .76 30 ' .77 .76 .7 .76 Ave. rate (m1. .02N NaOH min.) .152 IH 8/372 5 .70 .72 .70 .71 10 .70 .72 .70 .71 15 .70 .72 .70 .71 20 .70 .72 .70 .71 25 .70 .72 .70 .71 30 .70 .72 .70 .71 Ave. rate (m1. .wN NaOH/min. ) .142 IA 8/93 5 1.00 1.00 1.00 1.00 10 1.00 1.00 1.00 1.00 15 1.00 1.00 1.00 1.00 20 1.00 1.00 1.00 1.00 25 1.00 1.00 1.00 1.00 30 1. 00 1. 00 1. 00 1.00 Ave. rate (1111.02.11 NaOH/min. ) .200 IA 8 86 5 1.00 .98 .96 .98 /1 10 1.00 .98 .96 .98 15 1.00 .98 .96 .98 20 1.00 .98 96 .98 25 1.00 .98 .96 .98 30 1.00.98 96 .98 Ave. rate (m1. .02N neon/mln.) .196 IA 8/372 5 .95 .97 .98 .97 10 .95 .97 .98 .97 15 .95 .97’ .98 .97 20 .95 .97 .98 .97 25 .95 .97 .98 .97 30.95.97 .98 .97 Ave. rate (m1. .02N NaOH/inin.) .194 59 Table 6 (cont.) ' ‘ Treatment Time 1111. .02N NaOH used under test conditions Codes * (min . ) I II III Ave . IP 12/93 5 .30 .30 .30 .30 10 .30 .'30 .30 .30 15 .30 .30 .30 .30 20 .30 .30 .30 .30 25 .30 .30 .30 .30 30.30.30 .30 .30 Ave. rate (m1. .02N NaOH/min.) .060 IP 12/186 5 .25 .25 .25 .25 10 .25 .25 .25 .25 15 .25 .25 .25 .25 20 .25 .25 .25 .25 25 .25 .25 .25 .25 30 ' .25 ' .25 .25 .25 Ave. rate (m1. .02N NaOH/inin. ) .050 I? 12," 372 5 .30 .25 025 027 10 .30 .25 .25 .27 15 .30 .25 .25 .27 20 .30 .25 .25 .27 25 .30 .25 .25 .27 30 .30 .25 .27 Ave. rate (ml. .02N NaOH/min. ) .054 IR x2’ 5 .47 .46 .45 .46 / 93 10 .47 .46 .45 .46 15 .47 .46 .45 .46 2o .47 .46 .45 .46 25.47.46 .45 .46 30 ."7 .46 .4 .46 Ave. rate (ml. .02N NaOH/min. ) .092 IH 12 186 .50 .47 .48 .48 / lg .50 .47 .48 .48 15 .50 .47 .48 .48 2o .50 .47 .48 .48 25.50 .47 .48 .48 30 .50 .47 .48 .48 Ave. rate (m1. .02N NaOH/min. ) .096 m 12 3 2 5 .57 .58 .58 .58 / 7 10 .57 .58 .58 .58 15 .57 .58 .58 .58 20 .57 058 058 ‘58 25 .57 .58 .58 .58 3o .57 .58 .58 .58 Ave. rate (m1. .02N NsOH/min.) .116 70 Table 6 (cont.) Treatment Time ml. .(BN NaOH used under test conditions Codes 4* (min. ) I II III Ave. IA 12 93 5 69.68 .67 .68 / 10 .69 6.8 .67 .68 15 .69 .68 .67 .68 20 .69 .68 .67 .68 25 .69 .68 .67 .68 30.69.68 .68 Ave. rate (ml. .02N NaOH/min. ) .136 IA 12/186 5 .68 .70 .69 .69 10 .68 .70 .69 .69 15 .68 .70 .69 .69 20 .68 .70 .69 .69 25 .68 .70 .69 .69 30.68.70 .69 Ave. rate (m1. .02N N30H6 in.) .138 IA 12 372 5 .60 .61 .60 .60 / 10 .60 .61 .60 .60 15 .60 .61 .60 .60 20 .60 .61 .60 .60 25 .60 .61 .60 .60 30 .60.61.60 .60 Ave. rate (m1. .02N Neon/min. ) .120 *I c irradiated: 93. 186, 372 (K rad) doses of radiation, p = 2 was prior to commercial harvest; H 8 at oonmercial harvest; A - 2 weeks after commercial harvest. 71 Tab1e 7. Pectinesterase activity,7/-equiv./‘min./g. in irradiated and stored frozen Montmorency cherries picked at various stages of maturity. Codes* 4 months 8 months 12 months IP/93 1.36 1.40 1.20 IP/372 3.04 1.40 1.08 111/93 3.92 3.32 1.84 IH/186 4.16 3.04 1.92 Iii/372 5.04 2.84 2.32 IA/93 5.91 11.00 2.72 1A/186 6.00 3.92 2.76 IA/372 5.36 3.88 2.40 * I 7- irradiated: 93, 186, 372 (K rads) doses of radiation; - P 8' eeks prior to commercial harvest; H 8 at comercial 2 w harvest; A = 2 weeks after commercial harvest. Table 8. Methanol content in pitted fresh and frozen Mont- morency cherries. Storage time No. of Absorbancy" mg. methanol/ at -12°F expts. - Ave. 100 g. fr.fr. *(monthij’ 0 I. .215 .21 .222 . 11. .266 .26 .276 .213 9'82 1.5 1. .266 .268 -- .274 10.85 11. .282 .276 .276 12 I. .444 .475 11. .uau .u1u .569 .ues 16.83 111. .103 .398 -— 21 1. .111 .462 -- 11. .438 .483 .423 .432 17.11 111. .u1u .409 -- * Absorbancy at 570 mu. of methanol derivative used in the test. 73 Table 9. Pectinesterase activity in fresh and frozen MOnt- morency cherries of the 1963 season. Treatment Time ml. .02N NaOH used under -COOH’equiv./ Code* (min.) the test conditions min./€. . I II III Ave. fresh fruit FPO 0 .17 .17 .17 .17 5 .17 .17 .17 .17 10 .17 .17 .17 .17 15 .17 .17 .17 .17 .68 20 .17 .17 .17 .17 25 .17 .17 .17 .17 30 .17 .17 .17 .17 Ave. rate (m1. .02N Na0H/min.).034 FHO 5 .30 .30 .30 .30 10 .30 .30 .30 .30 15 .30 .30 .30 .30 1.21 20 .30 .30 .30 .30 25 .30 .30 .30 .30 30 .30 .30 .30 .30 Ave. rate (m1. .02N Na0H/inin.).060 FP1 5 .17 .17 .17 .17 10 .17 .17 .17 .17 15 .17 .17 .17 .17 .68 2O .17 .17 .17 .17 25 .17 .17 .17 .17 30 .17 .17 .17 .17 Ave. rate (ml. .02N Na0Hfinin.).034 FH1 5 .30 .30 .30 .30 10 .30 .30 .30 .30 15 .30 .30 .30 .30 1.21 20 .30 .30 .30 .30 25 .30 .30 .30 .30 30 .30 .30 .30 .30 Ave. rate (m1. .02N NaOH/min.).060 *'F = frozen; P = prior to commercial harvest; H = at har- vest; 0,1 = time (months). W ROOM USE ONLY ROOM USE 0341’ ll!IIINHINHIIIHIWUllllllllllHlHlllWlJHIHHIIWI