MSU RETURNING MATERIALS: Place in book drop to remove this checkout from LIBRARIES “ your record. FINES win be charged if book is returned after the date stamped beIow. (10”.- , q 9" I / 199» STABILIZATION 0F ORANGE JUICE CLOUD BY ENZYMIC INHIBITION By Mohammad Amir-uz-zaman A DISSERTATION Submitted to Michigan State University in partiaI fulfiITment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1985 ABSTRACT STABILIZATION OF ORANGE JUICE CLOUD BY ENZYMIC INHIBITION By Mohammad Amir-uz-zaman Stabilization of cloud of freshly extracted orange juice was attempted by means other than pasteurization such as chemical inhibition of the pectinesterase and freezing of juice. The three pectic enzymes viz. pectinesterase, polygalacturonase and pectinlyase were tested for their cloud destabilizing properties and it was found that pectinesterase was responsible for this serious quality defect of unpasteurized Juice. Polygalacturonase and pectin lyase did not naturally exist in the fresh extracted juice and the pectinesterase found in the juice alSo originated from the tissue and released into the juice due to pressure applied during extraction. Based on this, pectinesterase was extracted and partially purified from the peel and pulp of Valencia oranges. The inhibition of partially purified pectinesterase was studied with possible inhibitors. Polygalacturonase and polygalacturonic acid which inhibited 85% of the pectinesterase could make the juice cloud stable for more than a month. Greater concentration of EDTA was required to stabilize the cloud than that which effectively inhibited the enzyme. This might be because of occurrence of divalent cations in the juice, which were chelated by Mohammad Amir-uz-zaman EDTA, thereby making EDTA ineffective for the enzyme. Freshly extracted juice kept frozen was found to be cloud stable for at least a month, because of slow action of the pectinesterase in the frozen state. During the frozen state, total pectin decreased asla net result of decrease of high-methoxyl pectin and increase of low- methoxyl pectin. Similar study with frozen oranges showed that there was no appreciable change in the pectic substances in the juice portion during the frozen state of the fruits. To My Parents who blessed me from halfway around the world. ii ACKNOWLEDGMENTS The author would like to express his very sincere thanks and gratitude to Dr. Jerry N. Cash, Associate Professor, Department of Food Science and Human Nutrition, for his expert and imaginative constructive criticism, professional guidance and encouragement, financial assistance, his understanding and sincere friendship throughout the course of this study; Dr. Pericles Markakis, Professor, Department of Food Science and Human Nutrition, for acting as my guidance committee member and for all his friendly discussions; Dr. Mark A. Uebersax, Associate Professor, Department of Food Science and Human Nutrition, for acting as my guidance committee member and helpful suggestions; Dr. Everett S. Beneke, Professor, Department of Botany and Plant Pathology, for accepting the membership of my guidance committee and many advice; Dr. Jack Giacin, Professor, School of Packaging, for spending many of his restricted hours as a member of my guidance committee and valuable suggestions. The author wishes to express his gratitude to Dr. John Partridge, Assistant Professor, Department of Food Science and Human Nutrition for allowing the author to use the pH stat and the automatic fraction collector at his laboratory. The author further expresses his thankfulness to Bangladesh Agricultural Research Institute and Bangladesh Agricultural Research Council for sponsoring him for higher studies in the United States. Thanks also goes to all of the author's fellow graduate students and friends, particularly Charles R. Santerre, Touran Cheraghi and Dr. N.R. Nayini, for their help with insight and materials, useful discussions and thoughtful advice. Finally, the author wishes to express his appreciation and love for his wife Mona whose patience and understanding, tolerance and forebearance knew no bounds during the course of this study. iv TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES . LIST OF ABBREVIATIONS . INTRODUCTION. LITERATURE REVIEW . Composition of Citrus Juice Cloud . . Role of Pectic Enzymes in Juice Clarification Occurrence and Distribution of Pectinesterase in Citrus Fruits. . . . . . . . . . . . Assays for Pectinesterase . Occurrence of Polygalacturonase in Oranges. Methanol Determination. Extraction and Purification of Pectinesterase : Heat Inactivation of Pectinesterase and Pasteurization of Juice . Inhibition of Pectinesterase by Chemicals Pectic Constituents in Orange Juice . MATERIALS AND METHODS Optimization of Assay Conditions of Pectin- esterase. . Determination of PE Activity in Orange Juice. Polygalacturonase Activity in Orange Juice. Extraction and Purification of PE from Oranges: Cloud Stability of Reconstituted Orange Juice . Clarification of Juice and Release of Methanol. Inhibition of Pectinesterase by Inhibitors. Effect of Frozen Storage on Juice Cloud and Pectic Substances . . . . . . . . . RESULTS AND DISCUSSION. Selection of the Extraction Buffer. Optimum pH for Pectinesterase . Optimum Temperature . . . Page vii Buffer pH for Maximum Extraction of PE. . . . 78 Effect of Salt Concentration on PE Activity . . 8l Effect of Time Extraction . . . . . . . 84 PE Activity in Orange Juice . .. . . . . . . . 88 Extraction and Purification of PE . . . . . . . 9l Polygalacturonase Activity in Oranges . . . . . 110 Assay of Polygalacturonase and Pectolyase . . . ll6 Effect of Pectic Enzymes in Cloud of Recon- stituted Juice. . . . . . . . . 120 Methanol Formation in Orange Juice. . . . . . . l3O Inhibition of PE by Sugars. . . .,. . . . . . . I42 Inhibition by Pectolyase. . . . . . . . . . . 144 Inhibition by Polygalacturonase . . . . . . . . l44 Inhibition by Polygalacturonic Acid . . . . . . lSB Inhibition by EDTA. . . . . . . 167 Effect of Freezing Orange Juice and Whole Oranges on Cloud Stability. . . l80 Measurement of Pectin in Frozen Juice and Juice from Frozen Fruit . . . . . . . . . . . . . . l92 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . ZOl APPENDIX. . . . . . . . . . . . . . . . . . . . . . 206 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . 224 vi Table 0101-th \l 10 11 12 13 14 15 16 17 18 19 20 LIST OF TABLES Enzyme activity in different extracts. Properties of purified plant pectinesterases PE activity in orange juice. Enzyme activity in crude extract . Salting out of buffer extract. Effect of dialysis on the PE activity of an orange peel and pulp extract. . . Enzyme activity in final step. . . . . . Purification of PE from Valencia oranges/kg fruit. Standard curve data for assay of polygalacturo- nase . . . . . . . . . . . . . . . . Polygalacturonase activity in orange tissue. Assay of PGase from Rhizopus . Assay of pectolyase from Aspergillus japonicus Data for standard curve for methanol Inhibition of pectinesterase by sugars Inhibition of PE by pectolyase Inhibition of PE by polygalacturonase. [Mysfor orange juice to reach 60% T. Inhibition of PE by polygalacturonic acid. Days needed for clarification. Inhibition of PE by EDTA . vii Page 67 72 89 92 95 98 106 107 111 115 117 119 131 143 145 145 155 159 168 169 Table Page 21 Delay in clarification of orange juice by EDTA. 179 22 Effect of storage temperature on PE action. . . 188 23 Data for standard curve for pectin determina- tion. . . . . . . . . . . . . . . . . . . . . . 193 24 Activity of orange pectinesterase at different pHs . . . . . . . . . . . . . . . . . . . . . . 206 25 Effect of temperature on PE activity. . . . . . 207 26 Effect of pH on extraction of PE from orange tissue. . . . . . . . . . . . . . . . . . . . . 208 27 Effect of NaCl concentration on PE activity . . 209 28 Effect of time of extraction on PE activity . . 210 29 PE activity in the ion exchange fractions . . . 211 30 Protein contents in ion exchange fractions. . . 212 31 Effect of pectic enzymes on cloud of recon- stituted juice. . . . . . . . . . . . . . . . . 213 32 Clarification of unpasteurized orange juice . . 214 33 Release of methanol in unpasteurized orange juice . . . . . . . . . . . . . . . . . . . . . 215 34 Action of heat and pectic enzymes on orange juice cloud . . . . . . . . . . . . . . . . . . 216 35 Effect of polygalacturonic acid or orange juice cloud . . . . . . . . . . . . . . . . . . . . . 217 36 Effect of EDTA on orange juice cloud. . . . . . 218 37a Effect of freezing oranges and orange juice on cloud . . . . . . . . . . . . . . . . . . . . . 219 37b Effect of storage at 4°C on juice cloud . . . . 219 38 Effect of short freezing on juice cloud . . . . 220 39 Effect of freezing of juice and fruits on PE activity. . . . . . . . . . . . . . . . . . . . 221 viii Table Page 40 Pectic fractions in frozen juice. . . . . . . . 222 41 Change in pectic fractions in juice of frozen oranges . . . . . . . . . . . .,. . . . . . . . 223 ix LIST OF FIGURES Figure 1 2 3 9a 9b 9c 10 11 12 13 14 14a Effect of pH on PE activity. Effect of temperature on PE activity . Effect of buffer pH on PE activity in the extract. . . . . . . . . . . . . . . . . Effect of salt concentration on PE activity. Effect of time of extraction on PE activity. PE activity vs. elution fraction in ion- exchange chromatography of orange PE . Protein content of elution fractions in ion- exchange chromatography of orange PE . Standard curve for assay of PGase and pecto- lyase. . . . . . . . . . . . . . . . . Effect of PE on cloud of reconstituted juice Effect of PGase on cloud of reconstituted juice. Effect of pectolyase on cloud of reconstituted juice. . . . . . . . . . . . . Standard curve for methanol determination. Clarification of unpasteurized orange juice. Methanol release in clarifying orange juice. Correlation between % T and methanol release . Inhibition of PE by PGase. Enzymic breakdown of pectin. Page 71 75 80 83 85 102 104 113 122 124 126 132 135 138 141 147 149 Figure 15a Effect of heat and pectic enzymes on juice cloud. . . . . . . . . . . . . 15b Effect of heat and pectic enzymes on juice cloud. . . . . . . . . . . . . . . . 16 Inhibition of PE by polygalacturonic acid. 17a Effect of PGA (0.1% and 0.2%) on orange juice cloud. . . . . . . . . . . . . 17b Effect of PGA (0.3% and 0.4%) on orange juice cloud. . 18 Inhibition of PE by EDTA . 19a Effect of EDTA (0.005 M and 0.01 M) on juice cloud. . . . . . . . . . . . . . . . . . 19b Effect of EDTA (0.05 M and 0.075 M) on juice cloud. . . . . . . . . . . . . . . . . . . . 19c Effect of EDTA (0.1 M and 0.125 M) on juice cloud. . . . . . . . . . . . . . . . . . . . 20a Effect of freezing on juice cloud. 20b Effect of cool storage on juice cloud. 21 Effect of short freezing on juice cloud. 22 Effect of frozen storage on PE activity of juice. . . . . . . . . . . . . . . . 23 Standard curve for pectin determination. 24 Effect of freezing on pectic fractions of juice. . . . . . . . . . . . 25 Effect of freezing oranges on pectic fractions of juice . . . . . . . xi Page 152 154 162 164 166 171 174 176 178 182 184 186 191 195 197 200 LIST OF ABBREVIATIONS Abbreviation Full Nord EDTA Ethylenediaminetetraacetic acid PE Pectinesterase PGase Polygalacturonase PL Pectolyase PGA Polygalacturonic acid xii INTRODUCTION Citrus fruits and their products are capable of fulfil- ling a valuable role by supplying various needed nutrients. Some of these compounds, such as ascorbic acid, folacin and vitamin 8-6, are well known in the literature of human nutrition. Citrus juices, orange juice in particular, are among the most universally accepted and desirable foods throughout the world, due to their highly pleasant flavor characteristics, appealing aroma and significant nutritional value. Among the products of orange fruit, single-strength juice, frozen concentrated juice, dehydrated juice and high-brix frozen concentrates predominate. The single strength orange juices marketed at present are either canned or chilled. The canned single strength orange juices still dominate the market, but their share of the total market has declined because of improved quality and other facilities offered by the frozen concentrates. thallorange juice products, chilled orange juice probably comes nearest to fresh juice in flavor, aroma and texture. The flavor of freshly extracted orange juice is preserved in this type of product, because there is no heat treatment in its produc- tion. But this product has a very short storage life, loses cloud stability within a few days and becomes unmarketable. This destabilization of cloud in orange juice is a serious problem in citrus juice making and has been the subject of research for a number of years. Citrus juices extracted by the commonly used methods contain considerable quantities of insoluble solids, usually referred to as suspended matter. The method of extraction, screen- ing, filtering or centrifuging employed in the preparation of the juice causes variation of the amount of this material. When fresh juice stands for a few hours, a sharp line of demarkation is generally obtained between the coarse and the more finely divided insoluble particles. This material which remains as more or less permanent suspension is referred to as the cloud of the juice. Since most of the cloud is composed of pigmented material, it is also respon- sible in a large part for the characteristic color and general appearance of the juice. The cloud also carries much of the flavor of the juice and is therefore very important from the standpoint of overall quality. The cloud in fresh juice is uniformly dispersed and may remain so until destabilization is brought about by pectic enzyme action. When flocculation starts, the whole mass of cloud may appear to coagulate and precipitate, leaving the supernatent liquid clear or nearly so. The theory of the enzymic destabilization assumes that pectic substances, which serve to stabilize the suspension of finely divided insoluble matter in the juice, is reduced in molecular size and also partially demethoxylated by pectic enzyme action, thereby becoming more susceptible to cross-linking with calcium and other polyvalent metals. Once cross-linked, these substances may be converted into a solid gel which also occludes the other finely divided insoluble particles and takes them out of suspension. This settling out causes the juice to become a two-phase liquid with a clear supernatent at the top and a solid gel at the bottom. Numerous studies have been undertaken to elucidate the mechanism of cloud destabilization (Braverman, 1949; Joslyn and Pilnik, 1961; Bissett et al., 1953; Krop, 1974), but this complex reaction is still not fully understood. Though pectinesterase has been singled out to be the main enzyme responsible for the initiation of this complex set of reaction, other pectic enzymes and even non-pectic enzymes have been proposed from time to time as potent clarifiers of fruit juices. However, at present, the production of single strength orange juice and orange juice concentrates in the industry is based on heat inactivation of enzymes, i.e. treating the products at a certain temperature for a period of time to achieve the inactivation and/or destruction of the enzymes. Pasteurization of the juice at a temperature near 90°C is required to inactivate the enzymes, but the organo- leptic quality is seriously damaged by this heat treatment and the taste, aroma and flavor of the fresh orange juice is lost to a great extent in the processed products (Bissett et al., 1953; Kew and Veldhuis, 1960). However, the use of the high-temperature short-time method of pasteurization in which the juice is heated to a higher temperature for a fraction of a second can preserve the flavor and aroma of the juice (Eagerman and Rouse, 1976; Nath and Ranganna, 1977). In many developing countries these facilities of pasteurizing the orange juice effectively are lacking and so marketed juices are often ill-pasteurized and have poor shelf-stability. Alternate methods of cloud stabilization have been investigated by some researchers. For example, inhibition of the responsible enzyme by some chemical inhibitors, but a great deal is still unknown about this area. Little research has been conducted to determine the effect of chemical inhibitors on orange juice cloud. Inhibition of the enzyme by potential inhibitors in model systems and application of the results to freshly extracted orange juice may offer a viable alternative to pasteurization. Moreover, freezing the orange juice as soon as it is extracted to suppress the enzyme action may also be an effective way to stabilize the cloud. This research aimed at determining the enzyme, or enzymes, which are primarily responsible for cloud destabili- zation. Partial purification of the enzyme(s) and inhibition with chemical inhibitors was also examined. Effective inhibitors such as polygalacturonase, polygalacturonic acid and EDTA were tested, after screening in the model system, in freshly extracted orange juice. This research also attempted to study the behavior of the enzyme and its action on cloud stability and pectin constituents, during frozen storage of juice and whole fruits. LITERATURE REVIEW The quality of extracted citrus juices depends to a great extent on the actions of the enzymes which are released into the juice during the process of extraction. Several of these enzymes catalyze reactions that affect the taste and appearance of the juice adversely. The loss of cloud which is a serious quality defect in fresh and unpasteurized citrus juice is brought about by such an enzymic action. Research efforts to identify and characterize the reactions, to isolate and purify the enzymes and to develop methods to control the reactions are described here in this review. Composition of Citrus Juice Cloud Citrus juice cloud has generally been considered a heterogenous mixture of cellular materials and perhaps emulsoids, held in suspension by pectin. The soluble pectin forms a matrix which supportsand stabilizesthe particulate materials of the cloud. Yamasaki and his co-workers (1964, 1967) studied the water suspension of apple juice cloud obtained by ultra- centrifugation. A concept of juice cloud based on cloud structure and clarification mechanism emerged from their studies. They found apple juice cloud particulates to be 36% protein which was encapsulated with pectin. They hypo- thesized that with the action of pectic enzymes, the protective colloid layer of pectin is removed, thereby exposing the positive charge of the proteinaceous cOre which interacted with the negatively charged pectin of another cloud particle causing flocculation and coagulation. Scott et a1. (1965) analyzed the orange juice cloud chemically and indicated that it differed markedly in its gross composition from apple juice cloud. Lipids constituted one-fourth of the cloud, while the other three-fourths remained as ethanol, acetone and hexane-insoluble materials. About 80% of this insoluble material was identified as pectins while only 7% was reported as nitrogen, and cellulosic materials around 3%. Thus the amount of protein was limited and according to them, the Yamasaki hypothesis was not appli- cable in orange juice. Baker and Bruemmer (1969), however, re-examined the cloud insolubles and found that 47% of the pectin fraction reported by Scott et a1. (1965) was protein. They further showed that soluble pectin was not necessary for cloud support, as a stable suspension of orange juice particu- lates could be made in water. Indeed, in contrast to the role assigned to it in other fruit juices, they presented evidence which suggested that pectin might contribute to cloud collapse rather than to its stability by providing a handy substrate for the enzymes. Mizrahi and Berk (1970) determined that Shamouti orange juice cloud contains particles, between 0.05 and 2 mm in diameter which is comprised of crystals of hesperidin. The participation of hesperidin crystals as a general constituent of orange cloud has been questioned by Baker and Breummer (1972) and Lankveld (1973), as, perhaps, being peculiar to Shamouti oranges only. They maintained the concept of protein and pectin as the constituents of orange juice cloud. Rothschild and Karsenty (1974) mentioned that freshly prepared orange juice is supersaturated with bioflavinoids. Role of Pectin Enzymes in Juice Clarification Pectin is the intracellular cement of cell-wall tissue occurring in fruits and succulent vegetables. It is composed of anhydrogalacturonic acid units that exist in a chain-like combination with each unit connected through the 1,4 glycosidic linkages forming a polygalacturonic acid. Some of its carboxyl groups are esterified with methanol, some are neutralized with cations and some are free acids. However, it is not a homopolymer and has been found to contain various proportions of L-rhamnose in the main chain and arabinose, xylose, fucose and galactose in the side chains, according to Zitko and Bishop (1965) and Smit and Bryant (1967). Pectin is abundant in citrus fruits and is mostly located in the white spongy albedo portion of the peel. Enzymes that act on pectin and substances derived therefrom are ubiquitously distributed among plants and microorganisms, but are not present in animals. After many years of controversy in attempting to classify the various types of action, it would appear from the reviews of Rexova- Benkova and Markovic (1976) and Rombouts and Pilnik (1978) that there are three distinct types of reactions catalyzed by enzymes acting on the pectic substances. These are the pectinesterases, the pectin or pectate depolymerases and the lyases. Pectinesterases split methanol off from their substrate transforming pectin into low-methoxyl pectin and pectic acid. They appear in many fruits and vegetables and are particularly abundant in citrus fruits and tomatoes. Polygalacturonases split the glycosidic linkages in the galactouronan chain by hydrolysis, while pectin or pectate lyases split the same linkage by a process known as B-elimi- nation. All of these enzymes have been studied vis a vis their rolesin fruit juice clarification. Although this study is concerned with clarification of orange juice, it is useful to consider the research conducted with other fruit juices to enhance the concepts of cloud loss phenomenon. While clarifi- cation of apple and grape juices is a desirable process, the same thing in citrus juices is highly objectionable. Biochemically and chemically, though, they are based on the same reaction; action of pectic enzymes on pectin molecules occurring in the juice. 10 Polygalacturonase In the study of apple juice clarification, Yamasaki et a1. (1964) implicated joint PE and PGase action. Endo (l965a, 1965b, l965c, l965d) demonstrated that clarification of apple juice could be accomplished by the joint action of a purified endo PGase and a purified PE. Yamasaki et a1. (1964) reported a mechanism in which the joint action of PE and PGase stripped the normally negatively charged cloud particle of its pectin coat exposing a core of pectin polymers intermingled with other glycans and protein. The molecules of the latter, being positively charged at the pH of the juice, are neutralized by the negatively charged soluble pectin and pectinate. Being colloids the oppositely charged entities would precipitate. Endo (l965a) purified three endopolygalacturonases, one exopolygalacturonase and two pectinesterases from preparations derived from Coniotherium diplodiella and found that when added separately no single enzyme fraction was capable of clarifying apple juice satisfactorily. Endo (1965b) continued his research and showed that mixtures of the polygalacturonases or of the pectinesterases did not have any effect and only the combined action of at least one of the polygalacturonase fraction with one of the pectinesterase fractions could clarify the juice. In order to understand the mechanism of cloud loss phenomenon by joint PE and PGase action, he (l965c, l965d) showed that 11 hydrolysis of pectin accompanied by a decrease in viscosity of the juice, wasthe principal step during clarification. He separated the clarification process into three steps: 1) solubilization of insoluble pectin bound to the suspended particles; 2) decrease in viscosity due to hydrolysis of the soluble pectin, and 3) flocculation of the suspended particles. He opined that only a small percentage of the total glycosidic linkages present needed to be split to cause flocculation and resultant clarification. Earlier research conducted by Jansen et a1. (1945) showed that polygalacturonase, acting simultaneously with pectinesterase on pectin at pH 4.0, had a favorable effect on the rate of de-esterification and on maintenance of the rate, as compared with action of pectinesterase alone. However, they did not test this observation in clarification studies. Dahodwala et al. (1974) observed that action of pectic enzymes in fruit juice clarification varied with respect to soluble juice components and solution parameters such as pH, viscosity, ionic strength and physical hetero- geneity in different fruit preparations. They worked with polygalacturonase and pectinesterases from the protein extracts of Aspergillus niger and observed that the poly- galacturonase catalyzed the hydrolysis of the pectin molecule where the galactouronide unit had been deesterified by pectinesterase and this joint action of the two enzymes was responsible for the clarification of fruit juice. 12 Pectolyases Commercial pectin-degrading enzymes, especially those manufactured from special strains of Aspergillus, have frequently been found to possess pectin lyase as well as hydrolase activity. Though no report is found about the action of this enzyme on orange juice cloud, researches have been carried out regarding involvement of this enzyme in the clarification of other fruit juices. Ishii and Yokotsuka (1971, 1972, 1973) reported that purified pectin lyases from Aspergillus sojae initiated clarification both in apple and grape juice. However, they found that grape juice was more resistant to clarification by pectin lyases than apple juice. One reason for this was that apple pectin wasmore highly esterified and a better substrate for pectin than grape pectin. It can be mentioned here that Yamasaki et al. (1964) determined apple pectin to be 87-90% esterified while grape pectin was esterified to the extent of 45-50%. Pectinesterase Early in the development of the citrus juice processing, clarification of bottled and canned citrus juices and citrus beverages was recognized as resulting from the action of the pectic enzyme that had not been destroyed by heat pasteuri- zation. Pectinesterase was discovered as early as 1840 by Fremy who noted that the addition of carrot juice to a pectin solution caused formation of a gel and this gel was 13 produced by the enzymatic deesterification of pectin followed by precipitation of the resulting polygalacturonic acid as calcium pectate. Cruess (1914) was one of the earliest workers on citrus juices and he reported on a clarifying and clotting enzyme present in orange juice. He reported that fresh orange juice formed a jelly-like suspension a few hours after expression, and that after a few days the suspended matter coalesced and settled, leaving a clear supernatent liquid. He speculated that heating the juice to 85°C would destroy the enzyme and prevent the juice from clearing. Joslyn and Sedky (1940) observed that settling of cloud in citrus juices was always accompanied by decomposition of pectic substances and used the rate of clarification as a measure of the rate of action of the pectic enzyme. Braverman (1949) and Joslyn and Pilnik (1961) thought that the pectic enzyme pectinesterase was the causative agent of cloud loss in citrus juices. The latter persons further observed that this enzyme was firmly associated with the cell-wall fractions andvms located mainly in the peel, the rag and the juice sac tissue. Krop (1974) made an extensive study on cloud loss phenomena and observed that orange juice clarification dependedon the pectinesterase' and bivalent cations were indispensable for clarification. Joslyn and Pilnik (1961) also ascribed the cloud loss to deesterification of juice pectin by pectinesterase and subsequent precipitation of the low methoxy pectin as calcium pectinate or pectate. Krop and 14 Pilnik (1974b) confirmed the presumed role of calcium ions in juice clarification phenomena; they noted that cloud stability was preserved, in spite of normal pectinesterase activity, when calcium ions were previously precipitated as calcium oxalate. Earlier studies conducted by Rouse et a1. (1954), Stevens et al. (1950) and Bisset et a1 (1953) provided evidence for the important contribution of enzymic deesteri- fication of the pectin to cloud loss and singled out pectin- esterase as the causative enzyme. They also found out an interrelationship between heat treatment, inactivation of PE and cloud stability. Later on, several citrus researchers like Bissett and Veldhuis (1956), Huggart (1952), McCollach and Rice (1955) and Rouse et a1. (1957) also substantiated the effect of pectinesterase, its concentration on the stability of orange concentrates. Rouse et a1. (1961) showed the effect of two levels of pectinesterase on the stability of 42° and 50.70 Brix Valencia orange concentrates at 40°F. They found that pectinesterase at the level of 0.5 unit/gm did not effect cloud loss, while with 2 units of PE/gm of concentrate, the cloud loss was rapid. 50.7o Brix concentrate with similar PE activity, however, remained cloud stable. Versteeg et a1. (1978) isolated two pectinesterase isoenzymes from Navel oranges, but he limited his studies on the biochemical nature and kinetics of the isoenzymes. Rombouts et a1. (1979) also isolated two iso- enzymes from Navel oranges by chromatography in cross-linked 15 pectate. They synthesized cross-linked pectate with 0.46 degree of cross-linking and was able to get strong binding of both the isoenzymes to the pectate. They also concentrated on the biochemical characterization of these isoenzymes. Korner et a1. (1980) worked with Shamouti and Valencia oranges and found the existence of two different pectinesterase entities after chromatography in both the orange varieties. Peak A and Peak 8, as they called the isoenzymes, were found to destabilize the cloud of orange juice in a pattern similar to that obtained with natural juice. They assumed that the two pectinesterase chromatographic entities had similar catalytic properties. Another important research on citrus PE isoenzymes was conducted by Evans and McHale (1977). They also demonstrated the presence of the forms of pectinesterases in West Indian limes and Washington Navel oranges. In the orange, one pectinesterase was located almost exclusively in the peel while the other waslocated within the segment covers and juice sacs. The location of the two pectinesterases in limes had not been determined. Versteeg et al. (1980) isolated three molecular forms of pectinesterase from Navel oranges. They observed that there was electrophoretic evidence for the existence of five forms of pectinesterase in Navel oranges. This situation was not unique in the Navel variety, because one year earlier Versteeg (1979) reported the presence of as many as twelve forms of pectinesterase, in other orange 16 varieties like Salustiana,Shamouti as well as in lemon, grapefruit and mandarin. Versteeg et a1. (1980) observed that the third isoenzyme, which they called the high molecular weight pectinesterase, accounted for only about 5% of the total activity, but it was the most heat-stable out of the three forms and times and temperatures commonly used for orange juice pasteurization entirely were determined by this thermostable isoenzyme of PE. They also anticipated that this enzymic form might be largely responsible for gelation phenomena which were known to occur with concentrates produced by the cut-back process and which were not stored at, or below the rather critical temperature limit of -20°C. Occurrence and Distribution of Pectinesterase in Citrus Fruits McDonnell et a1. (1945) first isolated and characterized the pectinesterase occurring in oranges, lemons and grape- fruits. They reported that in all fruits, the flavedo had a higher activity per gram of wet weight than did the albedo. However, the specific activity on a protein nitrogen basis was not greatly different for the two parts of the peel. They also mentioned that while the juice sacs separated from orange juice obtained by mechanical reaming had an activity per gram of wet weight of approximately half that of the flavedo, the filtered juice had little or no activity. Rouse (1951) reported on the relationship of pectinesterase 17 activity in orange juice with water-insoluble solids and with increased pulp content. He found that juice samples prepared to contain increasingly higher amounts of pulp (juice sacs), also had correspondingly higher PE activities. Rouse (1953) determined pectinesterase activities of the flavedo, albedo, rag,juice sacs, seeds and juice for one variety of mandarin, 4 varieties of oranges, and 2 varieties of grapefruit and without exception, the highest pectin esterase activity had been found in the juice sacs, while the least amount of activity was found in the juice. The order of component parts for pectinesterase activity per gm of dry solids for mandarin and oranges from highest to lowest activity was found to be juice sacs, rag, flavedo, albedo, seeds and juice. In the grapefruit varieties the flavedo and albedo exhibited high activity. Rouse and Atkins (1954) showed that lemon and lime peel (flavedo plus albedo) had the highest PE activity followed by juice sacs and rag in that order. Rouse, Atkins and Huggart (1954) showed that PE activity in orange juice was directly proportional to pulp content. Rouse et a1. (1962) reported pectinesterase activity on five component parts of Valencia oranges during a 7-month maturation cycle for 2 seasons. They found that usually, PE activity for peel, membrane and juice sacs was least in December, the first month of the maturation study, when the Brix/acid ratio was least, than during the following 6 months. They had their greatest PE 18 activity in June, when the Brix/acid ratio was highest. The order of component parts for PE in most cases, from highest to lowest activity, was juice sacs, membrane, peel, seeds and juice. Rothschild et a1. (1974) investigated the pectinesterase activity in component parts of different citrus varieties like grapefruit and orange and reported that the highest activities in all varieties were found in pulp and rag, and the lowest in juices. Tahir et al. (1975) conducted a study to determine pectinesterase activity in various parts of Jaffa oranges during ripening. They reported highest activity in the juice sacs and lowest activity in the juice and the activity increased throughout the ripening period. Assays for PE Because PE hydrolyses pectin with the production of pectic acid and methanol, the enzyme concentration can be assayed by measuring the rate at which free carboxyl groups or methanol is released from the substrate. In the early days of research, PE activity was mostly measured by the formation of the pectinate gel from the pectin in the presence of calcium salts. Gradually, researchers came to know that this method was unsuitable, since the appearance of the gel was influenced both by enzyme activity and by the suitability of the prevailing conditions for gel formation. The quantitative expression 19 of such results was also difficult. Kertesz (1937) was one of the pioneers in titrating the free carboxyl groups to measure the PE activity. He used methyl red to indicate the pH (6.2) and added 0.1 N NaOH at frequent intervals to maintain the pH relatively constant for 30 minutes. Lineweaver and Ballou (1945) modified this method by the introduction of pH-meter instead of the indicator and employed the continuous titration with alkali to pH 6.2 instead of adding alkali every 5 minutes. This procedure has been further refined by Rouse and Atkins (1955) who introduced the automatic titrators to titrate the liberated acid groups with alkali at a constant pH. The method of recording the pH drop over a short pH interval was proposed by Somogyi and Romani (1964) to assay pectinesterase activity and they found that within certain limits, the drop of pH per minute was proportional to the amount of the enzyme. However, this method was suitable only to measure the initial reaction rates and at a pH not close to the pvaalue of pectin. The manometric assay of pectinesterase activity was used by Glasziou and Inglis (1958) who added sodium hydrogen carbonate to the reaction mixture and measured the develop- ment of carbon dioxide for determining PE activity. Zimmerman (1978) reported assay of PE by using the substrate 20 p-nitrophenyl acetate instead of pectin and followed the increase in absorbance at 400 nm/minute with increasing concentrations of enzyme. However, Versteeg (1979) commented on this method that some other esterase activity was measured instead of pectinesterase activity. Occurrence of Polygalacturonase in Oranges Polygalacturonases have been isolated from yeasts, molds, bacteria and even insects. Research efforts to find and isolate this enzyme from higher plant sources have been also highly successful. Luh et al. (1956) partially purified polygalacturonase from tomatoes that hydrolyzed 4% of the glycosidic bonds in pectic acid. Pressey et a1. (1971) found this enzyme in peaches. Pressey and Avants (1976) were able to isolate this enzyme from pears. The absence of the enzyme in all these unripe fruits, its appearance near the onset of ripening and the increase in its activity concomitant with the release of soluble pectin during ripening strongly suggested that it was implicated in pectin solubilization. Hobson (1962) tabulated the relative amount of the presence of this enzyme in various fruits and vegetables and pineapples contained only 1.6% of the amount present in tomatoes. There was, however, no mention of citrus fruit in that table. Indeed, until 1975, it was not clear whether citrus fruits contained polygalac- turonases, though they are so rich in pectic substances. MacDonnell et al. (1945) failed to find any PGase activity 21 in orange flavedo and reported that polygalacturonase did not exist in the extract. Several research reports appeared regarding occurrence of this enzyme in citrus, and it was concluded that destruction of pectic substances in citrus fruits was due to pectinesterase action followed by nonenzy- matic reactions. Rogers and Hurley (1971) indicated presence of PGase inhibitors in citrus fruit. Riov (1974) finally found that determination of polygalacturonase activity in citrus required the inhibition of another enzyme, uronic acid oxidase, which oxidized the reaction products of the PGase and caused the galacturonic acid to disappear. He was able to inhibit the oxidase by incorporating 1 mM sodium hydro- sulfite in the reaction mixture which did not cause any noticeable harm to PGase. Methanol Determination As each enzymic split of a methyl ester group releases one molecule of methanol, quantitative determination of methanol offers another way for the determination of pectin- esterase activity. Several color reactions and gas chro- matographic methods are available to determine methanol which have been used by researchers in this line. Clark (1932) reported the determination of methanol by determining methyliodide after reaction with hydrogen iodide. The colorimetric methods require an oxidation step in which methanol is oxidized to formaldehyde. Boos (1948) 22 reported determination of methanol by measuring formalde- hyde which produced a violet color with chromotropic acid reactant. He measured the colored compound at 580 nm and claimed it to be a rapid, accurate and Specific method for methanol in which several other aldehydes did not interfere. However, Beyer (1951) stated that in this method, the absorbance wasaffected by presence of ethyl alcohol. Wood and Siddiqui (1971) reported a sensitive method which could be used to measure methanol as low as 2 ug. They used pentane-2,4-dione to produce a color with formaldehyde and measured the absorbance at 412 nm. They claimed this method as direct, sensitive and specific for measuring methanol in biological fluids and stated that by this method, methanol released from pectin, either by saponifi- cation or by enzymolysis, might be sensitively determined without prior distillation steps. Dyer (1970) reported a gas-liquid chromatography method for determination of methanol content at concentra- tion as low as 0.003%. With this GLC determination, a standard curve must be prepared in which the sample concen- tration wasinclusive, since a nonlinear relationship existed with increasing methanol concentration. Bartolome and Hoff (1972) described a gas-liquid chromatographic method to determine methanol after converting it to methyl nitrite. Conversion was brought about by allowing the sample to react with nitrous acid in closed tube. 23 The direct gas chromatographic determination of methanol became feasible by the advent of special column filling materials suitable for gas-solid chromatography which prevented some of the drawbacks experienced by ordinary GLC, such as poor separation of low molecular polar compounds and assymmetrical peaks. These gas-chromatographic methods have been stated to be very sensitive and reproducible. Gessner (1970) developed a method for reacting nitrous acid with the alcohols in extracts of tissue homogenates and then analyzing the head space by GC. He claimed good reproducibility of values with concentrations of methanol at l mg/litre. Krop et a1. (1974) reported a gas chromato- graphic method of methanol determination after steam distillation of standards and samples with a detection limit of about 1 mg/litre. Versteeg (1979) gave a detailed information about a gas chromatographic alcohol assay. In this method, alcohols were converted to their volatile nitrite esters and the headspace of the solutions were sampled and injected. He claimed the method to be rapid, sensitive and reproducible and suitable for biological fluids without any pretreatment of the material. Termote et a1. (1977) increased the sensitivity and reproducibility of the method developed by Wood and Siddiqui by including a steam distillation step prior to oxidation and color development. Kauss et al. (1959) and Milner and Avigad (1973) determined radioactive methanol after enzymatic deesterification of pectin labelled with carbon-l4 methyl. 24 Extraction and Purification of PE McDonnell et a1. (1945) extracted pectinesterase from dried orange flavedo with borate acetate buffer at pH 8.2 by comminuting the extraction mixture in a Waring blender for 5 minutes followed by filtration. The enzyme was then precipitated by 60% saturated ammonium sulfate and dried at 5°C in vacuum. Lineweaver and Ballou (1945) extracted the enzyme from alfalfa by blending 1 part of dried alfalfa with 7 parts of 0.1 M sodium acetate solution followed by centrifugation, filtration and dialysis for partial purifi- cation. MacDonnell et a1. (1950) modified their early method by using the 40 to 80% ammonium sulfate fraction and adsorbing this fraction in filter paper pulp which was subsequently extracted with 0.1 M NaCl and chromatographed in a Celite 505 column. The second stage of purification was carried out by elution, salt fractionation, solubiliza- tion,dia1ysis and further chromatographed in a Ducil column which was eluted with l M NaCl containing 0.025 M NazHPO4. The eluate was then dialyzed and freeze dried. Rouse (1953) extracted pectinesterase from different parts of citrus fruits by comminuting the ground tissues in 2% sodium chloride solution and passing the slurry through a colloid mill with a clearance setting of 0.01 inch. Rouse et al. (1962), however, dropped the filtration step, while working with Valencia oranges, and used directly the slurry mixture 25 for assay of the enzyme. Hultin and Levine (1963) extracted the PE from banana pulp with successive portions of distilled water and sodium chloride solutions of varying concentrations. The extracts were then centrifuged and the supernatents were taken for assay of PE activity. Hultin et a1. (1966) further modified this extraction from bananas by including the steps of salt fractionation, dialysis and ion-exchange chromatography. The successive extractions of distilled water and salt solutions were brought to 15% saturation and 60% saturation with ammonium sulfate respectively, dialyzed against water and polyethylene glycol and then chromato- graphed on carboxymethyl cellulose ion-exchange column. 0n the basis of kinetic properties and pH-activity profile, they stated that there were four fractions of the enzyme in banana. Chang et a1. (1965) extracted the enzyme from papaya by chopping the fruit and making pulp by a pulper. The pulp was then pressed with addition of Hyflo-supercel and the press cake obtained therefrom was mixed thoroughly with 2 M NaCl and pressed again. The press juice was mixed with Hyflo-supercel again and dialyzed successively against sodium bisulfite and demineralized water. The dialyzate was then centrifuged and ly0philyzed. Pectinesterase was extracted by Hall (1966) from tomato pulp by using 5 percent sodium chloride solution,dialyzing the salt extract against water and after centrifugation, extracting the precipitate with 1 percent salt solution. Zauberman and Schiffmann-Nadel 26 (1972) extracted the enzyme from avocado by grinding the fruit with l M NaCl for 3 minutes in a blender and filtering the slurry. Pectinesterase was extracted and purified from tomatoes by Pressey and Avants (1972) by blending 100 gm of tissue with 100 ml of cold water and 2 m1 of 0.8 M sodium bisulfite, followed by centrifugation and suspension of the residue in 500 ml of l M NaCl solution. The suspension was further salt fractionated and the precipitate obtained between 30 and 75% saturation of ammonium sulfate was dissolved in 0.5 M NaCl, dialyzed against 0.15 M NaCl, centrifuged and applied in a column of DEAE-sephadex. The elution was carried out with 0.15 M NaCl at a rate of 12 ml/ hour. Manabe (1973) purified the enzyme from citrus fruit by chromatography on a DEAE-cellulose column followed by Sephadex column adsorption of the active portion. The final enzyme preparation was homogenous as determined by disc electrophoresis and the specific activity was 460 times higher than the original extract. Dahodwala et al. (1974) purified a crude fungal pectinesterase by removing a majority of pectinase with hydroxylapatite ion exchange chromatography. Pectinesterase from frozen pulp of banana was extracted by Brady (1976) with two buffers in succession increasing the ionic strength in the second buffer, followed by centrifugation, salting out and chromatography in DEAE- cellulose column. The eluates were further purified by isoelectric focusing and chromatography in Sephadex columns. 27 Evans and McHale (1978) extracted and purified the enzyme from limesand oranges by blending the fruit tissue in Tris buffer containing sodium chloride. After filtration, the solution was brought to 70% saturation with ammonium sulfate. The ppt. was collected by centrifugation and redissolved in the Tris buffer followed by dialysis and another salting out step with ammonium sulfate. The precipitate obtained between 40 and 65% saturation with ammonium sulfate was redissolved in the buffer and chromatographed in columns of G-75 Sephadex. The elution was carried out with the same buffer. The active fractions were pooled together and brought to 70% saturation with ammonium sulfate followed by centrifu- gation and dissolving the precipitate in the buffer. Further chromatography was done on columns of DEAE-Sephadex A-50 and elution by Tris buffer. Versteeg et a1. (1978) reported an elaborate procedure applied to purify and isolate two iso- enzymes of pectinesterase from Navel oranges. They used the borate acetate buffer with an extra 15 gm of sodium chloride per liter for homogenizing the tissue. The press liquid obtained after filtering the slurry was salt fractionated and the precipitate obtained between 30 and 75% saturation with ammonium sulfate by centrifugation was dissolved in water. This solution was dialyzed again 0.01 M sodium maleate buffer in 0.1 M sodium chloride. The dialyzate was chromatographed in Bio-Gel P-lOO and eluted with sodium maleate buffer. The active fractions were combined and 28 dialyzed against 0.02 M sodium succinate buffer, followed by cross-linked pectate chromatography where the elution was done by a linear gradient of NaCl. The two isoenzymes came apart and active fractions were pooled separately, dialyzed against 0.005 M sodium phosphate buffer and applied to CM-Bio Gel chromatography eluting the column with this buffer. Korner et a1. (1980) partially purified the enzyme by extracting the orange tissue with a 0.25 M NaCl solution, squeezing the slurry through layers of gauze and centri- fuging the extract to remove the solid particles. The supernatent was salt fractionated with solid ammonium hydroxide and the precipitate obtained between 30 and 80% saturation was collected by centrifugation and dissolved in 0.01 M acetate buffer. After dialysis against the same buffer, the enzyme solution was centrifuged again to remove any precipitate and the clear solution was chromatographed in CM-Sephadex column and eluted with a linear gradient of 0.0 M to 0.6 M NaCl. The fractions were assayed for protein and pectinesterase activity. Versteeg et a1. (1980) further reported the existence of a third isoenzyme of orange pectinesterase. They obtained the crude pectinesterase from Navel oranges by extraction, ammonium sulfate precipi- tation, dialysis and freeze-drying as described by Versteeg et al. (1978). The third isoenzyme was separated by gel filtration on Bio-Gel P-100 from the other two entities, which themselves were separated from each other by chromato- graphy on cross-linked pectate and finally purified on 29 CM Bio-Gel column. Pressey and Avants (1982) isolated two chromatographic entities of pectinesterase from tomatoes. The tissue was homogenized with 0.25 M NaCl and centrifuged. Both the supernatents and the pellets Were used subsequently for purification steps. Followed by salt fractionation at 80% saturation with ammonium sulfate, the precipitates were collected by centrifugation, dissolved in water, dialyzed against 0.15 M NaCl and concentrated by ultrafiltration. After chromatography on DEAE-Sephadex, the two entities were further purified by two successive chromatographic steps, the first on a Sephadex G-75 column and the second on a CM-Sephadex C-SO column. The elutions on all these columns were done with 0.5 M NaCl solution. Heat Inactivation of Pectinesterase and Pasteurization of Juice Citrus juice has got a number of delicate aroma and flavor compounds which may be lost or damaged if subjected to heat for a relatively long time. So citrus juices should be pasteurized as rapidly as possible. Single-strength orange juices are pasteurized not for microbial inactivation as the primary goal but for inactivating the enzymes, although these microorganisms are destroyed in the process. A temperature treatment of 65-660 was found sufficient to kill all the spoilage organisms by the earlier researchers, but the juices thus treated were subject to another type of 3O deterioration; they separated into a clear liquid with a sediment at the bottom. Kertesz (1939) was one of the pioneers in studying the heat inactivation of pectic enzymes. He reported that the pectin—methoxylase of tomatoes was completely inactivated by heating the juice to 80°C for 45 seconds and the sensi- tivity of the enzyme towards heat increased with decrease in pH. Thus inactivation was reported on heating for two minutes at about 70°C for pH 4.4, at 55°C for pH 2.5 and at 50°C for pH 1.1. At pH 6, no inactivation was found for a heating period of two minutes at the highest temperature used, 70°C. Joslyn and Sedky (1940) also investigated the effect of pH, temperature and time of heating on the inacti- vation of enzymes responsible for the clearing of citrus juices. They found that clearing of juices occurred at lower rates the higher the temperature or the longer the time of heating; that heating orange juice to 80°C for 1 minute at pH 4 inactivated the clearing enzymes and inacti- vation was more rapid at pH 2.5 than at pH 4.0. Stevens et a1. (1950) observed that raw frozen juice was always superior in flavor to pasteurized frozen juice, but subse- quently after six to twelve months storage, the pasteurized product was as equal or preferable due to slower deterioration than raw juice. They recommended a series of times and temperatures at different pHs. At pH 3.8 of orange juice, 89°C for 2 minutes or 94°C for 15 seconds; at pH 3.3, 85°C for 2 minutes or 90°C for 15 seconds and at pH 3.2 of 31 grapefruit juice, 84°C and 89°C. MacDonnell et a1. (1945), in a study of effect of temperature on orange pectinesterase, found that the enzyme started inactivation slowly at 40°C in the presence or absence of salt at pH 7.5 and lost about two-thirds of its activity in five minutes at 56°C. Pollard and Kieser (1951) extracted apple pectin- esterase and reported the heat inactivation of the purified enzyme. Heating for 5 minutes at 65°C and 68°C completely inactivated the enzyme but the inactivation pattern of the enzyme in the apple juice was completely different. They found the retention of 80% of enzyme activity even after 40 minutes' heating at 68°C and for complete inactivation, the apple juice had to be heated at 80°C for 15 minutes. Earlier, Holden (1946) reported that pectinesterase, extracted from tobacco leaves and heated at pH 8.0 for 5 minutes, showed 30% loss of activity at 70°C and complete inactivation at 80°C. Rouse and Atkins (1952) studied the heat inacti- vation of the enzyme in several citrus juices and observed that the enzyme present in grapefruit and orange juice was inactivated by heat at a lower temperature when the pH was decreased. They reported lower temperatures required to inactivate the enzyme in grapefruit juice than were required for the same in orange~juice because of pH differences in the two citrus juices; for grapefruit juice of pH 3.5, it was 93.5°C for 0.8 seconds, while for Valencia orange juice of pH 3.6, it was found to be 96°C for 0.8 seconds. 32 Rouse and Atkins (1953) further worked with different cultivars of oranges and grapefruits and attempted to explain the relationship of pH, time and temperature for heat inactivation of the enzyme in the citrus juices. With the help of graphs and tables, they established that lower the temperature, higher was the time needed for inactivation, and lower the pH of the juice, lower was the temperature needed. Atkins et a1. (1956) observed that heating to about 71°C prevented fermentation in orange juice, but 86 to 99°C was required to stabilize the cloud. They further stated that percentage inactivation of pectinesterase increased in the orange and grapefruit juices with the temperature of the heat treatment and this inactivation was not affected by degree of concentration of the juices. Based on a study of heat treatment of orange juice and orange concentrates, Bissett et a1. (1953), however, found that a treatment of 7l.l°C effected cloud stabilization in 6-fold concentrates, while less concentrated juices required treatment temperature of 87.8°C, even though enzyme inactiva- tion at any processing temperature was of the same order for all products regardless of concentration. Hultin and Levine (1963) studied heat treatment of three molecular forms of pectinesterase isolated from banana and found that the fraction extracted with water was repidly inactivated at 55°C, while the other forms extracted by increasing salt concentration and alkaline conditions were only slightly 33 inactivated at this temperature even after 20 minutes heating. They opined that the PE forms which were 'bound' to the tissue, were more heat-resistant than the one which was 'extractable'. Pressey and Avants (1972) conducted similar study with the four different molecular forms of the enzyme from tomatoes and found that the four isoenzymes had different heat stabilities, the one first eluted from the DEAE- Sephadex column was most susceptible to heat, while the isoenzyme which came off the column in the last stage was the least heat susceptible. Eagerman and Rouse (1976) developed the necessary pasteurization temperature, F and 2 values for pectinase inactivation for three varieties of orange juice and one grapefruit juice. However, since it was common commercial practice to blend juice from different varieties of oranges, they recommended the pasteurization conditions equal to those necessary to stabilize Valencia orange juice, which is equivalent of 1 minute at 194°F with z = 12.2, be used for all orange varieties. For grapefruit juice, their recommendation was 7°F Tower than the orange juice setting or a cut of 10 seconds in time. Nath and Ranganna (1977) studied the time/temperature relationship for thermal inactivation of the enzyme in mandarin orange juice. They obtained F value of 1 minute at temperature 197.5°F for juices at pH 3.6, while for the juice at pH 4.0, the value 34 was 1 minute at 201.5°F. These F values were equivalent to 1.85 D at the lower pH and to 2.28 D at the higher pH. Therefore, they recommended a 2 0 process at pH 3.6 and a 2.5 0 process at pH 4.0. Versteeg et a1. (1980) studied the heat stability of the three isoenzymes present in Navel orange juice and they found that the two isoenzymes of relatively low molecular weights were less heat resistant than the high molecular weight isoenzyme. However, the '2' value that is the raise in temperature necessary to observe a ten times faster heat inactivation was found to be higher for one of the two low molecular weight isoenzymes. Korner et a1. (1980) compared cloud stability of heat-treated juice with that of natural juice and heat-treated and enzyme-added juice. They found no destabilization of cloud in juice heated for 3 minutes at 98°C for 56 hours, while in the same period of time, the other two samples became separated into clear liquid at the top and a sediment at the bottom. Inhibition of Pectinesterase by Chemicals The use of pectinesterase inhibitors to preserve cloud in citrus juice dates back only 20 years from now. Before that, there had been virtually no research in this line. Even after the sixties, there had been only a few research papers published on this section of citrus juice technology. 35 Kew and Veldhuis (1960) reported that water extracts of grape leaves could inhibit the PE activity and improve the stability of cloud in orange juice and in frozen concen- trated orange juice. The inhibitory factor was found in the leaves of four varieties of cultivated grapes and in wild grapes, but not in oak leaves. They demonstrated that the inhibitor was non-competitive, i.e. did not compete with the substrate for reaction with pectinesterase, and the active principle could be extracted with ethyl ether. Chang et a1. (1965) studied the inhibitory effect of sugars and reported the inhibition of papaya PE by sucrose at the same concentrations that delayed gelation of papaya puree. The inhibitory effect was found linear with sucrose concentration up to 50% sucrose. Conditions for optimum PE activity, pH and salt concentration were not affected by inhibitory concentration of sucrose. Working with sucrose for inhibition of banana PE isoenzymes, Hultin et a1. (1966) found that fraction II was more sensitive at low sucrose levels than at higher levels but the other two isoenzymes showed the normal behavior of inhibition at higher sucrose levels. Lee and Wiley (1970) observed inhibition of 40% of the apple PE activity by the presence of 15% sucrose. Hall (1966) investigated the effect of tannic acid and several other phenolic compounds on tomato pectinesterase and reported that the degree of inhibition of tomato pectinesterase by tannic acid was dependent on the 36 concentrations of both the inhibitor and the pectin substrate. A tannic acid concentration of 5x10‘5 molarity gave complete inhibition with 0.05 percent pectin as substrate, while 1x10“3 molarity was required to inhibit 1 percent pectin. Thus the degree of inhibition was directly proportional to tannic acid concentration and the inhibition decreased with the increase of pectin substrate concentration. Earlier Williams (1960) reported tannic to be an extremely efficient inhibitor of pectic enzymes. Porter and Schwartz (1962) considered the active principle of grape leaf extract which was shown to inhibit pectinesterase of papaya to be a tannin. Dahodwala et a1. (1974) used polygalacturonic acid on tomato pectinesterase and fungal pectinesterase from Aspergillus niger to see its effect and observed that the presence of PGA inhibited the tomato enzyme but the fungal enZyme showed a small increase in activity in its presence. However they did not find any significant effect of pectinase on the activity of tomato pectinesterase. Baker and Bruemmer (1972) demonstrated that cloud of fresh orange juice was stabilized without heating by adding a commercial pectinase and observed that pectinases degraded orange juice particulates and stabilized the juice cloud by depolymerizing pectic substances to soluble pectates instead of insoluble pectates. Subsequently it was shown by Krop and Pilnik (1974a) that a yeast-polygalactouronase could stabilize orange juice cloud in presence of active 37 pectinesterase. They postulated that the stabilization mechanism was probably based on the destruction of poly- galacturonide formed by pectinesterase action to low molecular uronides and no insoluble calcium pectate could then be formed which would precipitate the cloud particles. Krop and Pilnik (1974b) also confirmed the presumed role of calcium ions in juice clarification phenomena; cloud was stabilized, inspite of normal pectinesterase activity, where calcium ions were previously precipitated as calcium oxalate. Markovic and Patocka (1977) experimented the inhibition of tomato pectinesterase and found that the organophosphates and carbamates did not inhibit the isolated enzyme. But this enzyme was effectively inhibited by iodine and this inhibition was found to be non-competitive and irreversible. However, this inhibition was found to be more rapid in case of crude enzyme than on the purified one. Termote et a1. (1977) studied product inhibition of orange pectinesterase as an approach to stabilizing cloud of orange juice and found that both chemically and enzy- matically prepared pectic acid hydrolyzates with an average degree of polymerization of 8 to 15 proved to be suitable, because their molecular weight.waslarge enough to inhibit pectinesterase but too small to cause juice clarification by coagulating themselves. They observed that the period of cloud stability of orange juices from various origins could be extended by a factor of 3 to 5 through the additions 38 1 mg per ml of hydrolyzate with a degree of polymerization of 12. Zimmerman (1978) used agar plate technique to assay tomato pectinesterase action on 5% (w/v) pectin, poured into agar plates. He found that sodium dodecyl sulfate inhibited the enzyme logarithmically from 1 to 50 pg, while at levels higher than 50 pg total inhibition was observed. Versteeg et al. (1978) measured the activities of the two PE isoenzymes isolated from Navel oranges in presence and in absence of polygalacturonic acid inhibitor and found one of the two isoenzymes wasweakly inhibited and the other was strongly inhibited by the inhibitor. Korner et al. (1980) found that polygalacturonic acid and EDTA at the concentrations of 0.1% and 10"2 moles/litre respec- tively had inhibitory effects on both the PE isoenzymes isolated from Shamouti and Valencia oranges. None of them, however, tested the effect of using these inhibitors in orange juice.7 Pectic Constituents in Orange Juice In freshly extracted juice, viscosity imparted by pectin is a desirable characteristic, commonly referred to as body. If this viscosity is lacking or is lost through destruction of the pectin colloid, juice becomes thin, watery and does not have a juice-like mouth feel. Fresh, single—strength or reconstituted juices should, therefore, 39 contain some soluble pectin for optimum quality. As already described in previous pages, most citrus juices will clarify when allowed to stand, if steps are not taken to stabilize cloud. This cloud, which is composed of mainly cell-wall fragments and cellular organelles, enhances the acceptability of the juice and provides most of the characteristic color and flavor. Pectin has the main role in this phenomenon, because clarification occurs when native pectinesterase lowers the ester content of juice soluble pectin until it becomes susceptible to precipitation as insoluble pectates. Since pectin is a cell-wall component, it follows that comparatively little would be present in juice expressed from fruit. Even at this low level, pectin influences juice quality by its contribution to viscosity, gelation and cloud stability. During the growth and maturation period citrus fruits may be exposed to freezing temperature which has been found to effect the quality and yield of the extracted juice by altering the composition of the pectic constituents, pectin- esterase activity and expression of juice from the fruit. This exposure might be long causing internal fruit injury or short when no such internal or external injury is caused. Researchers have reported a number of investigations regarding physical and chemical changes in citrus fruits damaged by freezing, but such changes occurring in fruits kept in the freezing temperature for a shorter period of 40 time has not been investigated by anybody. Also there has been hardly any research to investigate the effect of freezing the orange juice directly after its expression from the fruit and keeping it in the frozen state until it is thawed and consumed. Most of the researches regarding low temperature application to orange juice were on frozen concentrated orange juice or chilled orange juice. The author has failed to find any literature regarding freezing preservation of single strength orange juice and therefore reviews none. However, literature on pectin constituents in orange juice and whole fruits are reviewed here. Rouse (1953) studied the distribution of total pectin in component parts of citrus fruits and observed that the pulp content of citrus fruits consisted mainly of rag and juice sacs, both of which contained a high percentage of pectin. As pulp levels were increased in citrus juices, the level of pectin also increased and effected the degree of gelation and clarification. Dietz and Rouse (1952) applied a rapid method of estimating pectic substances of citrus juices by calorimetry and divided the pectic substances into three groups on the basis of their solubility in increasingly stronger reagents, viz: high-methoxyl pectin, low-methoxyl pectin and protOpectin. Atkins and Rouse (1953) used the same method except using ammonium oxalate in place of sodium hexametaphosphate to extract the low-methoxyl pectin in an attempt to study 41 the effect of methods of extraction on the pectin content of orange juice and found that juices obtained by the different methods were different in composition and pectin in the juice increased as the pulp increased. Rouse et a1. (1958) studied change in pectinesterase activity and pectic constituents of citrus juices extracted from frozen fruit and found that in case of pineapple oranges and seedy grapefruit the pectinesterase activity was decreased. The high-methoxyl pectin increased in the juice and the protopectin decreased and a very slight increase in the low-methoxyl pectin was observed. In case of Valencia oranges, however, different results were obtained. Pectinesterase activity remained low and decreased in juices from frozen fruits as in other fruits but the high- methoxyl pectin decreased, while the protopectin increased in the juice and the low-methoxyl pectin remained almost the same. Sinclair and Jolliffe (1961) reported seasonal changes in concentrations of pectic substances of the peel and pulp of Valencia oranges. They determined the total and water-soluble pectic substances as anhydrogalacturonic acid during growth and development under normal conditions. They reported a tremendous initial increase in the total and water soluble pectic materials in the early part of the season, while they decreased gradually through the remainder of the season. They also observed that the percentage methylation of the pectic substances of Valencia 42 orange peel rose rapidly to approximately 80 percent level and remained relatively constant during the rest of the season. Rouse et al. (1963) determined pectinesterase activity, three pectic fractions and other characteristics on component parts of pineapple oranges and Silver Cluster grapefruit picked prior to and after the December sub-freezing tempera- tures. They reported an increase in pectinesterase activity in the pulp and juice of both the fruits after the freeze. However, a different pattern was observed in case of pectin materials. In case of pineapple oranges, low-methoxyl pectin and protopectin remained almost the same while the high-methoxyl pectin decreased, while in the Silver Cluster grapefruit, the protopectin and the high-methoxyl pectin remained almost the same but the low-methoxyl pectin increased. Robertson (1981) reported a modified method of measuring the pectic substances in citrus juices by spectrophotometry. They used m-hydroxydiphenyl instead of carbazole for the development of color. The successive extractionsof the alcohol insoluble solids were done by water, ammonium oxalate and cold alkali. However, they found no significant difference when the chromogen was formed using either m-hydroxydiphenyl or using carbazole. MATERIALS AND METHODS Optimization of Assay Conditions of Pectinesterase from Valencia Oranges Chemicals Pectin Grade I from citrus fruit was purchased from Sigma Chemical Company. All other chemicals used in this experiment were reagent grade and obtained from the General and Biochemical Stores, Michigan State University. A freeze— dried preparation of pectinesterase from orange peel was procured from Sigma Chemical Company to verify the assaying process. Oranges Mature, ripe "Valencia" oranges, a cultivar of Citrus sinensis Osbeek were obtained through Food Stores of Michigan State University from Detroit Market. It was selected because of its higher juice yields, more soluble solids and consequently with superior processing quality. Procedure Assay of pure enzyme: 0.5 g enzyme preparation (250 mg protein) was dissolved in 1 litre distilled water, 43 44 0.1 mL of which was added to 20 mL 0.5% pectin and titrated with 0.016 M NaOH at pH 7.5 and 30°C in the pH-stat (Metrohm- Harisau,Sweden). It required 0.593 mL of NaOH for 10 minutes on the average and from this the enzyme preparation was found to contain 38 units of PE/mg protein. Thus the assay procedure was found very satisfactory. Selection of Extraction Medium: It was done and evaluated as detailed in the Results and Discussion section. Optimumng: Oranges were cut in halves and pressed on a press extractor. The juice was drained through a thick burlap cloth using pressure. Samples were immediately frozen with dry ice and kept frozen at -20°C, until further use. The press cakes, together with the peel segments were immediately transferred to the freezer and kept frozen for future use. After thawing at 4°C, pectinesterase was extracted from the preparation of peel and pulp by a borate-acetate buffer, pH 8.2 according to MacDonnell et al. (1945) with an extra 15 g sodium chloride per liter added, to improve extraction. A 10 9 portion of peel and pulp was extracted in 150 mL of buffer in a cold Waring blender for 5 minutes. During extraction, the cup was wrapped with an ice-packed plastic bag. After extraction, the slurry was filtered through 8-fold cheese cloth and the extract was kept on ice. The 45 peel and pulp were re-extracted with another 150 mL of buffer in the same way and the two extractions (280 mL) were pooled. Ten mL of a 1% (w/v) pectin solution were mixed with varying quantities of 0.02 M NaOH to obtain different pH values. The reaction mixture was then made to 20 mL with water to get a 0.5% (w/v) pectin solution. This mixture was placed in the reaction vessel of the pH-stat with the instrument set at 30°C. A 0.1 mL sample of the peel extract was added to the cup and the instrument was adjusted to give the correct pH of the reaction mixture. Then the reaction mixture was titrated automatically with 0.016 M NaOH solution for 10 minutes and the amount of the alkali needed for keeping the pH of the reaction mixture constant for this time was recorded. At each pH, a separate blank was run with buffer only and the actual quantity of alkali was calculated by deducting the blank value from the sample value. The enzyme activity in 1 mL of the extract was plotted against the pH of the reaction mixture to find the optimum reaction pH. Optimum Temperature: The reaction vessel in the pH-stat was adjusted to different temperatures by using a circulating water bath. The pH of the reaction mixture was kept at optimal pH 7.5. From the buffer extract mentioned in pH study, enzyme solution was added (0.1 mL) and the 46 reaction mixture was titrated automatically for 10 minutes with 0.016 M NaOH, as described earlier. The temperature of the reaction mixture was then plotted against the activity of the enzyme. Effect of the pH of the Extraction Buffer on the PE Activity Extracted from Peel and Pulp By mixing varying amounts of 0.2 M solution of boric acid and 0.2 M solutions of sodium borate, buffers of different pHs were made. In each buffer 40 9 [liter sodium acetate hydrated and 15 9 /liter of sodium chloride were added. Twenty g of orange tissue was extracted by two successive portions of 100 mL buffer of each pH, according to the method already described. One tenth mL of extract (volume 190 mL) from each buffer pH was added to the pH stat reaction mixture consisting of twenty mL 0.5% pectin solution adjusted to pH 7.5 and temperature of 30°C. The titration with 0.016 M NaOH solution was done and the enzyme activity in each extract was plotted against the buffer pH to find out the pH of the buffer which extracted maximum enzyme units. Effect of Salt Concentration on Enzyme Activity: NaCl was added to the standard pectin solution in varying amounts. The pH was adjusted to 7.5, temperature 30°C and 20 g. of tissue were extracted with 200 mL of borate-acetete buffer, pH 8.2, according to MacDonnell et a1. (1945). One tenth mL.of extract out of 180 mL.total volume was added to the reaction mixture. The enzyme activity was plotted against salt concentration to find the optimum salt concentration. 47 Effect of Time of Extraction on Enzyme Activity: Twenty 9 tissue were homogenized in 200 mL buffer in a Waring blender, each time for a different period of time and 0.1 mL from each extract (total volume 180 mL) was added to .5% pectin solution, pH adjusted to 7.5 and temperature 30°C. The enzyme activity from each extract was plotted against time of extraction. Determination of Pectinesterase Activity in Valencia Orange Juice Portion of frozen orange juice extracted from Valencia oranges was thawed at 4°C and 0.1 mL.was added to 20 mL.of 0.5% pectin solution adjusted to pH 7.5 and a temperature of 30°C. The reaction mixture was titrated with 0.021 M NaOH solution for 10 minutes. Enzyme activity was calculated as enzyme units per mL juice. A Oranges were sectioned and mildly pressed to yield juice which was collected in chilled test tubes. Enzyme activity of this juice was determined as described, except that 0.3 mL juice was used, in this case. Polygalacturonase Activity in Valencia Juice For this determination the method of Riov (1975) was followed. Eight gm juice was blended for 3 minutes in a Waring blender with 80 mL of 50 mM potassium phosphate buffer, pH 7.5 containing 6% (w/v) ammonium sulfate. The extract was 48 filtered through 8-f01d cheesecloth and centrifuged for 10 minutes in a refrigerated centrifuge (Damon/IEO Division) at 20,000 xg. The supernatent was decanted and (NH4)ZSO4 was slowly added to 80% saturation. The container holding the supernatent was held in an ice bath. After standing for 1 hour, the mixture was again centrifuged for 10 min at 20,000 xg. The supernatent was discarded and the pellet was dissolved in 8 mL.of 1% NaCl for dialysis against two changes of 1% NaCl. Polygalacturonase assay was carried out by the modified Willstatter-Schudel hypoiodite method (Jansen and MacDonnell, 1945). One unit of polygalacturonase was the amount of enzyme which catalyzed the liberation of 1 umole 0f galactu- ronic acid in 24 hours at 25°C and pH 4.0 (Riov, 1975). For the assay, 2 mL.0f the dialyzed solution were added to 100 mL of 2% polygalacturonic acid solution, with 37.5 mM sodium acetate and 1 mM sodium hydro-sulfite solution, adjusted to pH 4.00 and incubated at 25°C for 24 hours. Aliquots of 5 mL were removed and added to 0.9 mL.of 1 M Na2003 in a glass- stoppered Erlenmeyer flask, followed by 5 mL.of standard 0.1 N iodine. After standing for exactly 20 minutes, the reaction mixture was acidified with 2 mL.0f 2 M H2504 and the residual iodine titrated with standard sodium thiosulfate solution. The umole of reducing groups liberated was determined from a standard curve prepared from data obtained with galacturonic acid monohydrate. A blank was carried out 49 with 100 mL of 2% polygalacturonic acid under similar condi- tions of incubation and 5 mL was subsequently analyzed. For the purpose of determining this enzyme from the tissue, 20 g of peel and pulp were homogenized with 120 mL of the potassium phosphate buffer, pH 7.5. The same procedure was followed and finally the dialyzate was made volume to 12 mL with 1% NaCl. 2 mL.0f this dialyzate were added to 23 mL of 1% polygalacturonic acid with Na-acetate and Na-hydro sulfite, incubated under similar conditions and 5 mL aliquot were taken for analysis of reducing groups. For assay of pectate lyase, however, the reaction mixtures was adjusted to pH 5.5 and 8.00, and 2 mL dialyzate added to each and tested for presence of reducing groups. Extraction and Purification of Pectinesterase from Valencia Oranges Extraction from Tissue After thawing at 4°C, pectinesterase was extracted from the preparation of peel and pulp by the borate-acetate buffer as described earlier. Fifty 9 portions of the preparation were homogenized with 150 mL buffer for 5 minutes in a previously cooled Waring blender. The homogenized preparation was stirred for one hour, wrapping the beaker with plastic bags filled with ice, then packed in cheesecloth and pressed. The press cake was homogenized with 150 ml- buffer again and pressed one more time to yield the press 50 liquid (total volume 320 mL.extract). The enzyme activity of this press liquid was determined in the pH-stat, titrating automatically with 0.024 M NaOH solution at pH 7.5 and temperature 30°C. The protein content of the press liquid was determined by the Lowry et al. (1949) method, with bovine serum albumin as the standard. This method consisted of: Reagents: A: Solution of 10% Na2C03 in 0.5 N NaOH 8: Solution of 1% CuSO4,5H20 C: Solution of 2% K-tartrate Folin-phenol reagent: 5 mL of 2 N folin-phenol reagent was diluted to 50 mL with distilled water. Procedure: Fifteen mL of reagent A, 0.75 mL of reagent 8 and 0.75 mL of reagent C were mixed in a 50 mL Erlenmeyer flask. One mL of this mixture was added to 0.1 mL of extract in a 16x150 mm test tube, mixed thoroughly and incubated at room temperature for 15 minutes. Three mL.0f folin-phenol reagent were added to the tube, mixed and incubated at room temperature for 45 minutes. The absorbance was read at 540 nm in a Bausch and Lamb spectronic-70 spectrophotometer. A standard curve using a series of standard solutions containing 0 to 300 ugh bovine serum albumin/mL.were prepared and used for the determination of protein in sample solution. 51 Salt Fractionation Out of 320 mL extract, 100 mL was taken and 19.6 g. of solid ammonium sulfate was added to it (to 33% saturation) in small amounts under mild stirring c0ndition until all the salt dissolved. It was then allowed to stand overnight. All these operations were done at 0-4°C in an ice bath. The extract was centrifuged in a refrigerated centrifuge at 10,000 xg for 20 minutes and both the precipitate and the supernatent tested for enzyme activity. Since the precipitate contained some enzyme activity, concentration of ammonium sulfate this time was reduced to 17.6 g /100 mL.extract (30% saturation) and the steps were repeated. No activity was found in the precipitate and it was discarded. To the super- natent, 31.4 g' of ammonium sulfate/100 mL supernatent (to 75% saturation) was added, dissolved, stirred, kept overnight and centrifuged in the same way. The supernatent still contained some activity and therefore 80% saturation ammonium sulfate was tried. This time the precipitate contained all the enzyme activity while the supernatent contained none. Therefore the supernatent was discarded and the precipitate was dissolved in distilled water. Enzyme activity and protein content of this enzyme solution were measured. 52 Dialysis Dialysis bags were cleaned in boiling water with 100 9/ liter acetic acid and rinsed four times with boiling water. The bags were then treated with boiling water to which a small amount of EDTA had been added. They were then washed with four changes of boiling water to remove EDTA. These bags were used for dialysis 0f the enzyme solution obtained from above against four changes of distilled water at 4°C. Enzyme activity and protein content of the dialyzate were determined as described. Ion Exchange Chromatography Two 9 CM-Sephadex cation Exchanger C-50-120 purchased from Sigma Chemical Company were soaked in acetate buffer pH 6.00 for two days, then packed in a 2x50 cm column and washed several times with the buffer. Ten mL: of the dialyzate was placed on the column and eluted with 100 mL acetate buffer plus 100 mL of l M NaCl in a linear gradient. Flow rate was maintained at 1 mL/minute and 3 mL of the eluate were collected in each tube with an automatic fraction collector. The contents in each tube were analyzed for enzyme activity and protein. Salt Fractionation,,Centrifugation and Dialysis The contents of tube Nos. 25 to 57 were pooled, then ammonium sulfate to 80% saturation was added, the sample was centrifuged at 10,000 xg for 20 minutes and the precipitate 53 was dissolved in a minimum quantity of water for dialysis against four changes of distilled water. This solution was also analyzed for enzyme activity and protein then freeze-dried for future use. For purposes of this work, this enzyme preparation is referred to as semipurified PE. Assay_of Polygalacturonase and Pectolyase Two enzyme preparations viz. polygalacturonase from Rhizopus and pectolyase from Aspergillus japonicus were purchased from Sigma Chemical Company to examine their effects on cloud stability of reconstituted orange juice. The assay of these two enzymes was made by the modified Willstatter-Schudel hypoiodite method. The substrate was 2% polygalacturonic acid solution in water with 37.5 mM sodium acetate to dissolve polygalacturonic acid and_adjusted to pH 4.0 and pH 5.5 with alkali for assay of polygalacturonase and pectolyase, respectively. Except for the difference in substrate pH, all other procedures were the same for both enzymes. One unit of each enzyme was the amount of enzyme which catalyzed the liberation of 1 umole of galacturonic acid from polygalacturonic acid in 24 hours at 25°C. For polygalacturonase assay, 0.1 9 solid enzyme prepara- tion was dissolved in 20 mL water with 0.5 mL of this solution being added to 100 mL of 2% polygalacturonic acid (pH 4.0 adjusted) and incubated for 24 hours at 25°C. Then 5 mL of this mixture was analyzed for reducing groups as previously 54 described. A blank was carried through a similar procedure. For assay of pectolyase, 100 mL of 2% polygalacturonic acid (adjusted to pH 5.5) was added to 6 mL enzyme solution prepared by dissolving 5 mg of enzyme in 100 mL water. In the same manner, 5 mL of enzyme-acted solution was analyzed with a blank running in the same manner. Cloud Stability of Reconstituted Orange Juice PE-inactive frozen concentrated orange juice (°B 41.3) from Valencia oranges was purchased from a local market and reconstituted with distilled water to °B 12.2. The pH of the reconstituted juice was 3.7. Potassium metabisulphite was added to make 500 mg S02/1itre (Krop et al., 1974) and 0.1% Na benzoate was added (Baker and Bruemmer, 1972). One hundred mL of the reconstituted juice was distributed in glass jars. Semipurified pectinesterase enzyme, poly- galacturonase from Rhizopus and pectolyase from Aspergillus japonicus were added to each. Concentrations used were: 1) pectinesterase 1.2 units/mL, 1.0 unit /mL and 0.6 unit / ml; 2) polygalacturonase 0.3 unit /mL, 0.25 unit /mL and 0.2 unit /mL; 3) pectolyase 0.3 unit /mL, 0.25 unit /mL.and 0.20 unit /mL. The jars were stored at 4°C and 28°C. The cloud stability test was done every 2 days during the first week and then every 4 days up to 30 days of storage. Turbidity measurements were done according to the method of Krop et a1. (1974). At specified times, the jar was shaken, then 10 mL of sample was centrifuged for 10 minutes at 360 xg. Percent transmittance was determined at 660 nm 55 in a Bausch & Lomb Spectronic-7O spectrophotometer. The percent transmittance was considered a measure for the cloudiness and juice with 60% transmittance was considered as clarified. Clarification of Orange Juice and Release of Methanol Oranges were sliced in a slicer (Sunkist Co.) and pressed to obtain juice which was collected in a steel pail immersed in ice. One hundred ML' of juice were distributed in each of six glass jars; three at 28°C and three at 4°C. During a period of two weeks' storage, turbidity and methanol released were measured periodically. Turbidity was deter- mined by the method of Krop et a1. (1974) and methanol was determined by the method of Wood and Siddiqui (1971). For methanol determination, 10 mL.juice from each temperature storage were mixed with 20 mL distilled water and distilled at 65-70°C in a thermostatically controlled distillation apparatus. The distillates were collected in a flask immersed in ice. The distillates were mixed with concentrated sulphuric acid then diluted with distilled water to get a 1.0 N solution. One mL.of these distillates was cooled in test tubes in an ice-water bath with the blank which contained 1 mL of 1.0 N H2504. One tenth mL of 2% (w/v) potassium permangamate (Baker Analyzed reagent) was added, taking care the sides of the tubes were not 56 splashed. The solution was mixed by gentle swirling and the tubes were held in an ice-water bath for 15 minutes. Two tenths ml of 0.5 M sodium arsenite (Mallickrodt analytic reagent) in 0.12 N H2S04 (MCB reagent) was added, followed by 0.6 mL water and the thoroughly mixed solution was left at room temperature for 1 hour. Two mL.of 0.02 M pentane-2,4- dione (Sigma Chemical Co.) dissolved in a solution containing 2.0 M ammonium acetate and 0.05 M acetic acid was then added and shaken. The closed tubes were heated at 58-60°C for 15 minutes then cooled to room temperature. Absorbance was read at 412 mu in the spectronic-7O spectrophotometer and the methanol content was determined by using a standard curve. Inhibition of Pectinesterase by Inhibitors Sugars 'Sucrose: The semipurified PE enzyme kept in frozen storage was thawed at 4°C and tested for enzyme activity. A 25% (w/v) sucrose solution was made which served as the inhibitor. To one mL.of a 1% pectin solution taken in a measuring cylinder were added varying quantities of 25% sucrose solution to achieve different concentration of sucrose in the reaction mixture. The volume was made up to 20 mL.with water and put in the reaction vessel of the pH stat. The pH was adjusted to 7.5 by delivering small quantities of concentrated alkali. Then 1 mL.enzyme solution was added and the reaction mixture was titrated 57 at 30°C with 0.024 M NaOH solution for 20 minutes. For each concentration of sugar, duplicate readings were taken along with a blank which did not contain the enzyme. Glucose, Fructose and Maltose: In the same way, solu- tions containing 20% glucose, fructose or maltose were used to test the inhibition of the enzyme by these sugars. The conditions of assay, concentration of substrate and inhibi- tors were the same as for sucrose. Duplicate readings were taken for each concentration of inhibitors with a blank reading. Since all these sugars were found to partially inhibit the enzyme only at high concentrations, none of them was experimented with the orange juice. Pectolyase Pectolyase which was assayed according to the previously described method, was used to test the inhibition of the semipurified PE enzyme. Five mg of the solid enzyme were dissolved in 100 mL water. 0.1 mt, 0.2 mL and 0.3 mL of this enzyme solution were added to 10 mL of 1% pectin solution to get concentra- tion of pectolyase in the reaction mixture of 0.55 units/mL, 1.1 units/mL and 1:65 units/mL.respective1y. Then the volumes of the reaction mixtures were made to 20 mL and pH adjusted to 7.5 with water and dilute NaOH solution. Samples were incubated at room temperature for 24 hours 58 along with a blank in which no pectolyase was added. Assays of semipurified PE enzyme in these reaction mixtures were carried out at 30°C for 20 minutes in the pH stat. From the results obtained, it was found that even at a high concentration of 1.65 units of pectolyase/mL of reaction mixture, the pH enzyme was inhibited only to the extent of 19%. So this inhibitor was not further used in the orange juice. Polygalacturonase Polygalacturonase from Rhizopus, which was assayed as described, was used as the inhibitor of semipurified PE enzyme. One tenth 9 solid enzyme was dissolved in 1000 mL distilled water and increments from 0.1 mL through 0.6 mL were added to 10 mL of 1% pectin solution to get different concentrations of polygalacturonase in the reaction mixture. The volumes of the reaction mixtures were made to 19 mL and pH adjusted to 7.5. Then they were titrated against 0.024 M NaOH in pH-stat for 20 minutes at 30°C after the addition of 1 mL of semipurified PE enzyme. For inhibition of the orange juice pectinesterase enzyme by polygalacturonase 0.1 9 solid polygalac- turonase enzymes was dissolved in 200 ml distilled water and 0.4 mL, 0.5 mL and 0.8 mL out of this was added to 59 100 mL freshly extracted and sulfited orange juice to obtain enzyme concentration of 0.263 unit /mL juice, 0.33 unit /mL juice and 0.52 unit /mL juice respectively. The other portion of the orange juice was heated to 98°C for 3 minutes and cooled. Potassium metabisulfite and Na-benzoate were added like the unpasteurized portion. The juice was distributed in 100 mL.portions to glass jars. Semi- purified PE enzyme at 1.0 unit/mL was added to one set of samples. Polygalacturonase at 0.33 unit/mL was added to the second set, pectolyase at 0.40 unit /mL to the third set with nothing being added to the fourth set of samples. Each treatment jar was stored in duplicate at 28°C and 4°C. Polygalacturonic Acid Polygalacturonic acid from orange obtained from Sigma Chemical Company was added as an inhibitor. ’A 2% (w/v) and 5% (w/v) solution was made and aliquots were added to 10 mL of 1% pectin solution, volume made up to 20 mL.and pH adjusted to 7.5. For concentrations of 0.1% through 0.5%, 2% poly- galacturonic acid was used and for 0.75% and 1.25%, 5% solution was used. For inhibitor concentrations of 0.5%, 0.75% and 1.25%, the substrate used was 0.05% pectin instead of 0.5%. Blanks were run at each concentration of inhibitor without enzyme, and the values obtained were deducted from values obtained with enzyme. The titrations were done at 30°C for 20 minutes with 0.024 M NaOH solution in 60 the pH-stat. For action of polygalacturonic acid on orange juice cloud, a 10% (w/v) solution of polygalacturonic acid was made and 1 mL, 2 mL, 3 mL and 4 mL were added to 100 mL of freshly extracted juice to give concentrations of polygalacturonic acid of 0.1%, 0.2%, 0.3% and 0.4% respectively. The samples were then incubated at 28°C and 4°C for 32 days and every'week 10 mL sample was taken out for turbidity measurement. EDTA EDTA, disodium salt, dihydrate (Baker Analysed reagent) was tested as an inhibitor. A l M solution of this salt was made and from this solution, required amounts were added to 1 mL of 1% pectin solution, to give inhibitor concentrations in the reaction mixture of 0.05 M and 0.01 M. The volume of the reaction mixture was made up to 20 mL with water and alkali to get a pH of 7.5. Then 1 mL PE enzyme was added and the mixture was titrated as before for 20 minutes. For the inhibitor concentrations of 0.00125 M, 0.0025 M and 0.005 M, 0.1 M EDTA solution was used. Blanks were run for each concentration of inhibitor without adding enzyme and these values were deducted from runs with enzyme. For studying the effect of this inhibitor on juice cloud, calculated amounts of the salt were directly added to 100 ml.freshly extracted and sulfited (500 PPm $02 and 0.1% NA-benzoate) juice, stored for 1 month in glass jars at 28°C 61 and 4°C with turbidity measurements each week. The pH's of all juices were adjusted to 4.0. Other Compounds Potassium sorbate and sodium propionate, both reagent grade, were used at levels of 0.1%, 0.05% and 0.15% in the reaction mixture. To achieve these concentrations, a 1% (w/v) solution was made for both compounds and required quantities of solution were added to 1 mL 1% pectin solution, made to volume and pH adjusted. Ascorbic acid at the levels of 0.1%, 0.15% and 0.2% in the reaction mixture were made from a 2% solution. Sodium benzoate at levels of 1000 ppm, 500 ppm and 200 ppm in the reaction mixture were prepared from a 2% solution and galacturonic acid was used at levels .01 M, 0.5 M and 0.1 M the reaction mixture. Sodium acetate and acetic acid both were used at levels of 20 mN, 30 mM and 37.5 mM in the reaction mixture. Since none of these compounds showed any appreciable inhibition of the pectinesterase enzyme, they were not tested in the orange juice for cloud stabilization studies. Effect of Frozen Storage on Juice Cloud and Pectic Substances Freezing of Juice and Whole Fruits For this study, whole oranges and extracted juice with added sulfite and Na-benzoate were frozen, kept in the frozen state until thawed at 4°C, then tested for cloud stability and pectic 62 fractions. Juice extracted from frozen and subsequently thawed oranges were also sulfited, then studied for cloud and pectic substances. For this study, the juice and the fruits were kept frozen throughout the period of the experiment and samples were taken out from time to time. To study the effect of short-time freezing, thawed samples of orange juice and juice extracted from thawed fruits were sulfited and transferred to 0°C and kept there for a month to test the effect of freezing on the juice cloud. This was done after one week of freezing and two weeks of freezing. The pectic substances of the orange juice were analyzed according to the colorimetric method of Dietz and Rouse (1953). For this, about 20 g of juice were taken in centrifuge bottles and 60 mL of 95% ethyl alcohol was added to precipitate the pectic materials. Then this alcoholic mixture was heated in a water bath at 8522°C with occasional stirring. The solutions were boiled gently during the last half of the heating period. Then the tubes were centrifuged at 1000 xg for 10 minutes. The supernatants were decanted and discarded. Forty mL of 60% alcohol was added to the precipitate. After mixing thoroughly, samples were heated for 10 minutes in the water bath at 85:2°C, centrifuged and decanted as before. Forty mL distilled water were added to each centrifuge tube and stirred to evenly disperse the mixture. After standing for 10 minutes, tubes were stirred, then centrifuged and the supernatents were decanted to 100 mL 63 volumetric flasks. The water extraction was repeated once again and the supernatent decanted to the same volumetric flask. Five mL: of l N sodium hydroxyde were added to the extract and diluted to volume. In the same way, 40 mL.of 0.4% sodium hexametaphosphate were used for extraction of the residue and the supernatent of the two successive extractions were decanted to 100 ML volumetric flask. Five mL. of 1 N sodium hydroxide were added before making to volume. The remaining residues were extracted for 15 minutes at room temperature with 40 mL of 0.05 N sodium hydroxide, stirred, centrifuged, decanted to 100 mL flasks and made to volume. One mL aliquot from each extract were taken in large test tubes and 0.5 mL of 0.1% alcoholic carbazole was added, followed by 6 mL concentrated sulfuric acid with constant agitation. The tubes were rubber stoppered and allowed 15 minutes standing for development of color. The tube's contents were read at 525 mu wavelength, in the spectro- photometer which had been previously adjusted with a blank. The standard curve was prepared from data of working standard galacturonic acid solutions, prepared from a stock solution of concentration 100 ug of galacturonic acid anhydrous per mL, taking different amounts from it and making volume to 50 mL. 64 Pectinesterase Activity of Frozen Juice and Juice from Frozen Oranges Frozen juice and frozen fruits were thawed at 4°C over night and juice was extracted from the thawed fruits. One tenth mL juice was added to 1% pectin solution and made to 20 mL with distilled water and dilute alkali to get pH 7.5. The reaction was titrated for 10 minutes in the pH-stat set at 30°C against 0.024 M NaOH and the calculated enzyme units were plotted against duration of freezing. Frozen juice was assayed every week for 4 weeks and for frozen oranges, assays were done every 2 weeks for a period of 5 weeks. Effect of Freezing of Pectinesterase on Retarding Activiby For this study, the degree of esterification of 25 mL of 0.5% pectin solution with or without pectinesterase added to it was determined under three different temperatures: 1) freezing, 2) room temperature of 25°C, and 3) 0°C, after one week's storage. The degree of esterification was measured (Versteeg, 1979) by titrating the pectin solution with 0.1 N NaOH (x mL) and adding 50 mL.of excess alkali with incubation for one hour at room temperature. After incubation 50 mL of 0.1 N H2504 were added to each sample and the solution was titrated against 0.1 N NaOH solution (y mL). The degree of esterification = (y/x+y x 100) %. RESULTS AND DISCUSSION Selection of the ExtractingyBuffer In the first phase of this research, extraction of the pectinesterase enzyme from the orange peel and pulp was done in three different ways: 1) blending the tissue in l M NaCl for 3 minutes (Rouse and Atkins, 1955); 2) homogenizing the tissue in 0.25 M NaCl for 3 minutes according to Korner et a1. (1980); and 3) extracting the tissue in borate buffer with 15 9' extra sodium chloride per litre according to Versteeg et a1. (1978). In each case, 10 g: of tissue was blended with 200 mL.of buffer for 3 minutes and the slurry was filtered through 8-fold cheesecloth. Then 0.1 mL.0f this extract was added to 20 mL.of 0.5% pectin solution in the pH stat adjusted to pH 7.0 and titrated with 0.016 M NaOH solution for 10 minutes. The table below gives the net amount of alkali consumed for each enzyme solution and units of enzyme per mlsof extract. In this experiment, the pH was kept constant at 7.0 and the alkali used to titrate the liberated carboxyl groups was a direct measure of the pectinesterase activity. The temperature of the set-up was 30°C and one unit of pectinesterase was defined as the activity corresponding to the release of l umole carboxyl 65 66 groups per minute which was equivalent to l umole of sodium hydroxide used per minute. The amount of alkali given in Table l is the mean of duplicate readings from which the blank value has been subtracted. From the table, it was noticed more enzyme units were extracted if the NaCl concen- tration was increased at a relatively lower pH, but a higher pH of the buffer and consequently a higher pH of the extract results in more extraction of enzyme. MacDonnell et a1. (1945) studied the effect of pH and salt concentration on extraction of pectinesterase from orange tissue and they found that with 0.25 M NaCl, the optimum yield of the enzyme was obtained at pH 8. They also showed that at pH 4.5, an increase in the NaCl concentration from 0.1 M to 0.5 M materially promoted extraction, but further increase to 2 M had little effect on extraction. Again at higher pH of 8, higher salt concentration up to l M did not appreciably increase the yield of soluble enzyme. Lineweaver and Ballou (1945) studied the effect of cations on alfalfa pectinesterase and found that pectinesterase was activated by cations and their pH optima shifted to lower values in the presence of cations. They suggested that the increase in activity caused by salt at low pH or by raising the pH at low salt concentration was due to prevention of inhibition of the enzyme by cations. Nagakawa et a1. (1970) found that the concentration of salt affected activity particularly in the acidic pH. They showed that tomato pectinesterase 67 Table 1. Enzyme activity in different extracts. Extraction mL.of NaOH Enzyme Units/mL. pH of the Media Required Extract Extract 1 M NaCl 0.082 1.312 4.6 0.25 M NaCl 0.071 1.136 4.5 Borate buffer + 0.145 2.32 6.9 15 g. NaCl/litre 68 attained half maximal activity at pH 5 in presence of salt, while in the absence of salt, it was attained at a pH of 6.5. Our results agree fully with the previously reported findings. As the pH of the slurry was increased due to use of a different extracting medium, same or even lower salt concentration yielded a higher enzyme extraction. Again at approximately the same acidic pH of the slurry, more salt concentration in the extracting medium resulted in more yield of enzyme units. On the basis of these results and in order to avoid the necessity of adding alkali to maintain an alkaline pH a borate-acetate buffer was selected according to MacDonnell et a1. (1945). This buffer had sufficient capacity to maintain a suitable pH, even in the case of the comparatively large amounts of acid formed by desterification during the extraction and release of large amount of plant acids into the slurry. However, to impart proper ionic strength, an extra 15 g' of NaCl per litre was added to the buffer, according to Versteeg (1979). Optimum pH for PE Enzyme To find the optimum pH for the pectinesterase, varying amounts of 0.02 M NaOH solution were added to the reaction mixture to get a pH range from 4.50 to 9.151. One tenth mL extract was delivered to the reaction mixture and alkali 69 needed to titrate the released carboxyl groups for 10 minutes was recorded. The data were plotted on a linear scale with enzyme activity vs. pH (Figure 1). From Figure 1, it is clear that at pH 7.5, the activity of the enzyme was highest and above or below this pH, the activity declined. The pH-activity profile of this enzyme was a bell-shaped one, though at pH values higher than 7.5, the rate of decline is slower than that at pH values lower than 7.5. The result obtained from this experiment agreed with results reported by several authors on different fruits and vegetables (Table 2). From this table, it was observed that pectinesterase isolated from different plant materials had an optimal pH in the range 7 to 8, most of them again being 7.5. Versteeg (1979) in a kinetic study with the two isoenzymes of orange PE, which together accounted for 90% of the total PE activity, reported optimum pHs for PEI and PEII to be 7.6 and 8.00 respec- tively. The effect of pH on enzyme activity is a complex phenomenon, involving composition and structure of the enzyme, its acid-base behavior and inactivation or denatura- tion at extreme pHs. From the result of our experiment, it was also observed that the pH of the normal intracellular surroundings of the enzyme was far below the optimal pH and this lied in the ascending slope of the pH-activity profile. 70 Figure 1. Effect of pH on PE activity. The enzyme activity was measured by automatic titration of carboxyl groups liberated at 30°C for 10 minutes in an enzyme-acted solution of 0.5% (w/v) pectin. Relative Activity 100p O O l b O 1 20- 71 L Effect of pH on PE Activity Or- .Amnmpv amoumsm> soc» umuaoufl. Aeempv .Pe ea upmzuozma .a.z on m—.o .m.z cacaoh Amsmpv semen: om .m.z .e.z o.m emeeeo Am~a_v .pm pm Fst»*: on no ~.o m.~ mpaa< v_ Aofiapv mapppou .m.z om . mN.o m.m . m.n mom; Ahempv escape: use m—PF: .m.z .m.z mo.o m.n cumsoh Ameapv .Pe ea meeeu .¢.z .¢.z ~.o m.~ exeeee AmemPv .Pe ea ppmccooom: .m.z cmusoaos uoz ~.o . _.o m.~ mmcmso Aesopv sap.z a was om mm eaeeeees eoz o.~ epee< Auov :.5 mpim new» Away Amsuw_\Poev mocmsmmmm cowum>wuum=~ new» Poswuao Fonz —oswuqo Ia pmepaao mucsom .mmmmsmumocwuqu ucapa uwvasaa co mmpusmaosm .N «pan» .1 73 Therefore a natural control exists for its activity in its natural environment. Optimum Temperature. Keeping the pH of the reaction mixture in the pH-stat constant at 7.5, activity of the enzyme was measured at different temperatures by adjusting the temperature of the circulating water. From the graph (Figure 2) obtained by plotting the values, it is obvious that as the tempera- ture was raised from 33°C, the enzyme showed increased activity up to 63°C but above this temperature, the activity declined gradually. In Table 2, though optimum temperature has not been reported for orange pectinesterase, pectinesterases from other fruits and vegetables are found to have optimum temperature in the range 50 to 65°C. So our result of 63°C as the optimum temperature of pectinesterase isolated from Valencia orange is an acceptable one. From Figure 2 we can see that the rate of the enzyme-catalyzed reaction increased with rise in temperature within the temperature range in which the enzyme is stable and retains full activity. The increase in temperature within this range might hasten the three stages of the enzymic catalytic reaction viz. formation of the enzyme-substrate complex, conversion of this into the enzyme-product complex and 74 Figure 2. Effect of temperature on PE activity. The enzyme activity was measured by automatic titration of carboxyl groups liberated at pH 7.5 for 10 minutes in an enzyme-acted solution of 0.5% pectin (w/v) adjusted to different temperatures. 1001- on g V a: O 1 h a Relative Activit N 9 75 2‘0 4'0——6'0—To 100 Temperaturefc Effect of Temperature on PE Activity 76 dissociation of the product or the effect of the tempera- ture on the reaction was the resultant of its separate effects on these stages. Whatever might be the case, an increase of activity was obtained until the temperature was raised to 63°C, the 010 between 40°C and 50°C being 1.38 and that between 50°C and 60°C was 1.28. Thus it is seen that the rate of increase in activity was lower for a 10°C rise in temperature near the optimum temperature than that when a similar rise in temperature occurred but at a lower range. Thus as the temperature was raised, a certain proportion of the enzyme was inactivated even before reaching the optimum temperature, though the activity at this elevated temperature was higher because of acceleration of the stages of the enzymatic catalysis. So optimum temperature was the resultant of two processes: 1) the usual increase in reaction rate with temperature and 2) the increasing rate of thermal denaturation of the enzyme above a critical tem- perature. The temperature co-efficient or 010 is a good index to see effect of temperature on rate of enzyme-catalyzed reaction. The earlier reports of 010 values were by Bell et a1. (1951) who reported a 010 value of 1.34 for cucumber pectinesterase in the range 20 - 50°C, by Collins (1970) who reported 1.33 for pea pectinesterase. Hills & Mottern 77 (1947) reported 010 value of tomato pectinesterase as 1.44 while McColloch and Kertesz (1947) reported this value to be 1.47. All these values have been reported in the range 20-50°C. Our value of 1.38 agrees well with these findings. From Figure 2 again we see that inactivation of the enzyme at temperature higher than the optimum startedrather quickly and by 80°C, about 60% of PE activity'waslost at the end of 10 minutes reaction time. The enzyme wasalmost completely inactivated at 90°C after an exposure of 10 min. This result also agrees well with findings reported by other researchers. Lee and Wiley (1970) and Pollard and Kieser (1951) reported 80°C as the inactivation temperature of tomato pectinesterase while Arakji and Yang (1969) reported that 100°C was the inactivation temperature for cranberry PE. Buren et a1. (1962) found that at a temperature of 90°C, PE from snapbeans became completely inactivated after 10 minutes. All these reports are for an exposure time of 5 minutes to 15 minutes. In this experiment,90°C appears to be the inactivation temperature as only 2.65% activity was found during a lO-minute reaction time at this temperature. Though the author did not continue another 10 minutes to see the effect of 20 minutes exposure at 90°C, it was obvious from the experimental condition that no alkali would be added to the reaction mixture in this time. It means that there was hardly any enzyme activity left in the extract after 10 minutes exposure at 90°C. 78 Bufferng for Maximum Extraction of PE In order to find out the exact pH of the borate-acetate buffer which will extract the maximum enzyme units from the tissue, varying quantities of boric acid and sodium borate solution were mixed as per Gomori (1955). From Figure 3, it is seen that at a pH of 8.2, the borate-acetate buffer extracted the maximum enzyme units. At pH values higher and lower than that, the relative activity was found to be lower. MacDonnell et a1. (1945) extracted orange flavedo at different pHs with addition of NaCl 0.25 M and they found that at pH 8.0 the extraction was maximum in terms of PE units and at higher and lower values, the activity of the extract was lower. -0n the basis of this study, they recommended a borate-acetate buffer of pH 8.2 to be suitable for extraction. Miller and MacMillan (1970) purified pectinesterase from Clostridium multifermentans and reported that largest yields of pectin- esterase were obtained when the purification was conducted at lower temperatures and enzyme preparations were kept at pH 6.00. Versteeg (1979) reported incubation of the two pectinesterase isoenzymes in pectin solutions of pH 4.0 and 7.0 at a temperature of 30°C. He observed both the forms retained more stability at pH 7.0 than at 4.0. However he did not test pHs slightly lower and slightly Figure 3. 79 Effect of buffer pH on PE activity of the extract. The extracting buffer was adjusted to different pH values and the enzyme activity in the extract was measured by automatic titration of liberated carboxyl groups at pH 7.5 and 30°C for 10 minutes in the enzyme-acted solution of 0.5% pectin (w/v). 80 110V 100° to C? c? 9 Relative Activity 0) O \\ i .1 7.5 8.0 8.5 9.0 9.5 Buffer pH T7950 Effect of Buffer pH on PE Activity in The Extract 81 higher than 7.0. In view of these findings, our result obtained was highly acceptable since at different buffer pHs, the extract pH also changed and at extract pH 6.15 corresponding to a buffer pH 8.2, maximum enzyme activity was obtained from the tissue, and the enzyme activity was also stabilized and retained. Effect of Salt Concentration on PE Activity Lineweaver and Ballou (1945) made the significant study to see effect of cations on pectinesterase and found cations to activate pectinesterase and this activation was more pronounced in case of divalent cations than monovalent cations. In order to study the effect of NaCl concen- tration, different quantities of salt were added to the pectin solution and activity of the results was measured on these substrates. The data showed that 0.15 molar NaCl gave the highest activity while concentration higher and lower than this gave less activity. These values were obtained keeping the pH of the reaction mixture 7.5. The effect of salt concentration on enzyme activity at other pHs was not studied because pH 7.5 was selected as the assay condition on the basis of the study reported earlier. From Figure 4, it can be seen that the optimum concen- tration of sodium chloride for plant pectinesterase was in the range of 0.1 to 0.25 mol/litre and these optimum 82 Figure 4. Effect of salt concentration on PE activity. NaCl concentration was varied in the reaction mixture of 0.5% pectin (w/v) solution and enzyme activity was measured by automatic titration at pH 7.5 and 30°C for 10 minutes. 83 Relative Activity (:1 5F 4 Q a; PM? 0.05 ‘ 0"".15 ‘ 0725 Salt Conc.,Moies/L Effect of Salt Cone. on PE Activity 84 concentrations were usually determined at pH 7.00 or around 7.00. Lineweaver and Ballou (1945) studied the effect of NaCl at pH 6.00 and found at this pH, 0.20 molar concentra- tion gave the highest activity. Miller and McMillan (1970), however, found 0.05 M as the optimum salt concentration when the clostridial pectinesterase showed the highest activity at a pH 7.5. As discussed earlier, the effect of salt concentration becomes less and less in increasing the PE activity as the pH of the reaction medium goes higher and higher. Line- weaver and Ballou (1945) observed that the activity of pectinesterase isolated from alfalfa was about 30 times as great in presence of 0.2 M monovalent cations as in the absence of cations, but at pH 8.5 these concentrations of cations were practically without effect on the activity. In our study salt concentration could not drastically alter the rate of enzymatic reaction or the cations could effect the PE activity only slightly because of the relatively higher pH of 7.5 of the reaction mixture. Effect of Time of Extraction From Figure 5, it is seen that at 3.5 and 4.0 minutes extraction time, the enzyme activity was fairly constant but when the extraction (homogenization) was continued for 5 or 6 minutes the activity increased sharply, 85 Figure 5. Effect of time of extraction on PE activity. Orange peel and pulp was extracted with borate-acetate buffer, pH 8.2 for different times and enzyme activity was measured by automatic titration of 0.5% pectin solution (w/v) at pH 7.5 and 30°C for 10 minutes. 86 1001‘ cf '\ 901- a! o T Relative Activit s1 0 a) O r / a a 6 i. 1 Time, Minutes \ \ £1 mi Effect of Time of Extraction on PE Activity 87 again the difference between 5 and 6 minutes was very small. When the blending was done for 7 minutes, enzyme activity lowered a little bit because the heat generated during this time was sufficient enough to inactivate 4% of the enzyme, in spite of freezing the blender cup and wrapping it around with ice during the blending operation. Thus for extraction and assay of the pectinesterase enzyme in the subsequent steps of this study the following conditions were selected: pH of the reaction mixture: 7.5 Buffer pH: 8.2 Salt concentration in reaction mixture: 0.15 M Time of extraction: 5 minutes Extraction medium: Borate-acetate buffer Temperature: 30°C The temperature selected was on the basis of several authors, particularly MacDonell et a1. (1945) and Versteeg (1979), who chose this temperature even though it was not optimum for the enzymes isolated and studied by them. Moreover this temperature was selected because of least variation (less than 3%) in temperature-activity profile in the range 26°C to 33°C and impracticality of the assay at the optimum temperature of 63°C. 88 PE Activity in Orange Juice Orange juice expressed from sound, mature, ripe oranges was collected in a bucket, dipped in ice and 0.1 mL was added to 20 mL of 0.5% pectin solution in pH-stat, adjusted to pH 7.5 and temperature 30°C. Similarly, 0.3 mL orange juice expressed by mild hand pressure was also used for the determination of PE activity. In both cases, the reaction time was 15 minutes. Table 3 summarizes the results. From this table, it is observed that when the pressure is reduced to a minimum, the juice expressed contains very small enzyme activity. Several researches have been conducted to locate the distribution of this enzyme in the different parts of the fruit and it is a general consensus that all the enzyme activity found in the juice originates in the pulp. The juice itself, uncontaminated and pure, does not contain any pectinesterase activity. This has been adequately reviewed in the literature review chapter, page‘g , and the author does not feel it necessary to repeat it here. MacDonnell et a1. (1945) reported PE activity occurring in the flavedo and cell- sac of Navel oranges to be 0.058 units and 0.028 units per gram, respectively. However, they defined one pectinesterase unit as the quantity of enzyme which would catalyze the hydro- lysis of pectin at an initial rate of 1 milliequivalent of ester bonds per minute at 30°C and optimum pH. Rouse (1953) reported these values for Valencia flavedo, albedo, rag, 89 Table 3. PE activity in orange juice Observa- Quantity Blank Difference Mean Enzyme tion of 0.021 M (mL) (1111—) (mL) units/ml. NaOH used juice in 15 min (mL) Juice expressed by hydraulic press 1 0.092 0.029 0.063 2 0.077 0.024 0.053 0.055 0.77 3 0.078 0.028 0.050 Juice expressed by mild hand pressure 1 0.063 0.030 0.033 2 0.054 0.030 0.024 0.028 0.13 3 0.059 0.031 0.028 90 juice sacs, seeds and juice as 0.0095units/gram, 0.0066 units/g , 0.0141, 0.076, 0.004 units/g' and 0.0002 units/g respectively. He also noted that most of the enzyme units found in the juice were bound to the pulp particles. Rothschild (1974) compared the factory-line juices of two orange varieties and one grapefruit variety expressed by FMC juice extractors with those expressed in the laboratory by the hand reamer after removing the peel of the fruits. The PE activity in the factory line juice varied from 0.0016 units/g to 0.00285 units/g while the PE activity of the laboratory-made juices was 50% of this. Versteeg (1979) for the purpose of determining heat stability of pectinase extracted juice from Valencia orange on a rotary kitchen citrus press and found 1.23 units PE/mL.juice, calculated on the basis of one umole COOH-groups released in one minute. The result obtained in our experiment was calculated in the same way and it seems to be lower than the reported value of Versteeg (1979). This variation may be due to the differ- ence in the extent of maturity and ripeness of the fruits, the pressure applied for extraction which resulted in different pulp contents of the juice or some inactivation of the enzyme itself during storage and handling. From Table 3 and results obtained by researchers, it is clear that juice containing more pulp particles has more pectinesterase activity in it and that is the reason why factory-line juice has got more PE than laboratory-made juice 91 and the laboratory-made juice again is more PE-active than mild-hand-pressed one. In view of the result that juice contains comparatively negligible quantity of PE and all the PE Occurring in the juice originates in the tissue, hence we proceed to extract the enzyme from the peel and pulp for subsequent purification steps. Extraction and Purification of PE from Pulp and Peel 0.1 mL out of 320 mL extract obtained by homogenization of 50 9 tissue with 400 mL buffer and subsequent filtration was used for its PE activity in the pH-stat at standard assay conditions. The following table (Table 4) gives the results obtained. Thus for 320 mL extract which came from 50 g peel and pulp, total quantity of PE was 3947.52 units. In other words each gm of tissue contained 78.95 PE units. This result is more or less similar to our results reported in Table 24 where except varying the pH of the reaction mixture, all other conditions were similar. If the value obtained at pH 7.5 of this table is taken into consideration, the enzyme activity is calculated to be 70.7 units per gm of tissue. In the studies of effect of temperature and effect of buffer pH, the values obtained under similar conditions are 50 units/g and 62 units/g respectively vide Tables 25 and 26 (Appendix). However, in Table 27 (Appendix), the value obtained is 75 units/g and in the time of extraction study vide Table 28 (Appendix), this value under similar conditions is 65 units/g. In spite of all 92 Table 4. Enzyme activity in crude extract. Observation Quantity Blank Mean Enzyme 0f 0.024 M hnL) (mL) units/mL. NaOH used Diff. extract for 10 min - (m 1.) 1 0.531 0.033 2 0.598 0.042 0.514 12.336 3 0.517 0.029 Absorbance at 540 nm of 0.1 mL.extract = 0.38 93 precautions taken to preserve the activity of the enzyme, this variation was caused due to minor errors in weighing the tissues, extraction and centrifugation losses or error in adjusting the pH-stat at different times of experimen- tation. The delivery of relatively random quantities of alkali in the 10 minute reaction time might be also a reason for this variation. However, if this variation is ignored, it may be stated that extraction of different quantities of tissue with different amounts of extraction medium under a reasonable range of variation, does not cause wide fluctuation in the enzyme concentration of tissue, calculated on weight basis. Also there is no effect of varying the concentration of the titrating alkali within a reasonable limit. If we assume that, on the basis of all the results reported above, 75 units of PE is the average value per gram of peel and pulp, the enzyme concentration per kg of fruits becomes 38,000 units, considering the yield of tissue from whole oranges. The protein content of the buffer extract was measured by the method of Lowry et a1. (1951) and from this, a specific activity of 5.14 E. Units/mg protein was found. In case of Navel oranges, Versteeg et a1. (1978) reported PE concentration of 49,000 units/kg fruit and a specific activity of 7. Korner et a1. (1980) reported a specific activity of 39.4, 5.0 and 11.0 in pulp, flavedo and albedo of Shamouti orange respectively. Evans and McHale (1978) reported a value 94 of 23,400 units of PE per kg of Washington Navel oranges but they did not report the specific activity of the crude extract. Fractional Precipitation by Ammonium Sulfate This step was completed in two stages each at the lower level and the upper level of saturation of ammonium sulfate as mentioned in Materials and Methods. Table 5 summarizes the results obtained in these two levels. Ammonium sulfate was used because of its large solubility in water and absence of harmful effects on most enzymes. 0n the other hand, it has been found to have a stabilizing action on many enzymes. Though the separations are not very sharp and sometimes a percentage of the enzyme has to be sacrificed in discarded portions, but still the total enzyme recovery is almost complete. The addition of the salt was done slowly under constant mild stirring at cold conditions. I Since the precipitate obtained after fractionation by 35% saturated ammonium sulfate was found to contain some enzyme activity, the salt concentration was lowered to 30% saturation and the precipitate obtained thereby did not have any enzyme activity. The precipitate was rejected and to the supernatent salt to 80% saturation was added after trying 75% saturation. So, the enzyme got precipitated between 30% and 80% saturation. The protein contents of the initial and final enzyme solution were measured and it was f0und that after the initial salting out, the specific activity was 6.60 and that after 80% 95 Table 5.. Salting out of buffer extract. Observation 0.024 M Blank Mean Enzyme NAOH used, (mL) diff. units/mL (ml) (mL) supernatent by 30% saturated ammonium sulfate 1 0.483 0.029 0.436 10.464 2 0.449 0.031 Absorbance at 540 nm for 0.1 mL.= 0.255 by 80% saturated ammonium sulfate 1 0.605 0.033 0.561 13.464 2 0.580 0.030 Absorbance at 540 nm for 0.1 mL.= 0.21 96 saturation treatment rose to 10.36. Thus after the salt fractionation step, the purification factor doubled though about 15% of the enzyme activity had to be sacrificed due to inactivation during handling and loss in discarded portions.‘ Evans and McHale (1978) obtained the precipitate between 40 - 65% saturated ammonium sulfate in the process of enzyme purification from Washington Navel oranges and found a specific activity of 3 with loss of 20% of enzyme activity. They however did not report the purification factor. Brady (1976) reported a 4.2 times purification after salting out with ammonium sulfate and getting the fraction between 30 to 80% saturation, during purification of pectinesterase from banana pulp. Versteeg et a1. (1978) reported a purification factor of 4 with loss of 35% enzyme activity when they isolated the enzyme from navel oranges and took the fraction between 30 to 75% saturation. Korner (1980), however, used 30 to 80% fraction and reported a purification factor of 2 during purification of the enzyme from Shamouti pulp. So salt fractionation with ammonium sulfate was an important initial step in the purification of the pectin- esterase from oranges, which brought out precipitation of the enzyme protein at a particular range of saturation, by the process of dehydration i.e. by attracting water molecules to bind the dissolved ammonium sulfate molecules and thereby making the enzyme protein lose their solubility and precipi- tate out. Our obtained value of purification and loss of 97 enzyme activity after this step agreed well with the reported findings. The calculation of exact quantities of ammonium sulfate was done as per nomogram of Cooper (1977). Dialysis and Ion Exchange Chromatography Since ion-exchange methods require that the sample should be applied at low ionic strength and since the salt frac- tionated enzyme solution was of high ionic strength, this step of dialysis was undertaken to remove the inorganic salt. The removal of the contaminants of the cellophane dialysis tubing was done according to McPhie (1971) and Versteeg (1979). Dialysis was carried out at 4°C for 6 hours each for one exchange of water, to ensure that all the salt and other small molecules were dialyzed out into the water. After dialysis, the enzyme solution was again tested for activity and protein concentration. The results are shown in Table 6. From the results, it is Seen that dialysis caused removal of some protein and loss of some activity, but most of enzyme proteins did not pass out of the dialysis bag. The slight reduction of specific activity was due to relatively a little more loss in activity than the propor- tionate decrease in protein concentration in the dialysate. The harvest of enzyme units after the step was 8% less than the previous step. The relatively longer stay in the cold room in the stirred condition might be the reason of 98 Table 6. Effect of dialysis on the PE activity of an orange peel and pulp extract. Observation 0.024 M Blank Mean Enzyme NaOH used, (mL) Diff activity, mL (mL) units/mL solution Before dialysis 1 0.605 0.033 0.561 13.464 2 0.580 0.030 After dialysis 1 0.517 0.028 0.483 11.592 2 0.508 0.031 Absorbance for 0.1 mL = 0.18 99 denaturation of this 8% enzyme proteins. The dialysis was followed by ion-exchange step. Versteeg (1979) tabulated the isoelectric points of tomato, grape, banana and orange pectinesterase and it was seen that iso- electric points are well in the alkaline pH and ranged from 8.00 to 11.00. Since enzymes are proteins and proteins have a net positive charge at pH values below the isoelectric points, pectinesterase solutions at pH lower than 8.0 contain enzyme molecules with a net positive charge and behave like cations. Therefore, an ion exchanger which had negatively charged groups at neutral pH was required. Carboxymethyl sephadex is a cation exchanger carrying negative charges on its functional group and was therefore suitable for adsorbing the positively charged protein molecules. Korner et a1. (1980) also used carboxymethyl sephadex for the ion exchange chromatography and obtained excellent separation of the two isoenzymes of PE from Shamouti pulp after this step. As reported earlier, the maximum stability of the pectin- esterase enzyme in this study was obtained at pH 6.2, which was determined by varying the pH of the enzyme slurry after changing the pH of the extracting medium. On the basis of this finding, acetate buffer was chosen for this step which was anionic and therefore suitable for cation exchanges, by not interacting with the negative charge of the functional group. The buffer was prepared at pH 6.00 as per Gomori (1955). 100 The elution was done by the same buffer with change of the ionic strength of the buffer linearly with time. The eluants were collected in small test tubes and were analyzed for enzyme activity and protein as soon as possible. From the results obtained and shown in graphs (Figures 6 and 7), it can be seen that the enzymic proteins started elution when the molar concentration of sodium ions was about 0.4 and the peak of elution was at the concentration of 0.5 molar. Closely similar results were obtained by Korner et a1. (1980) when they eluted the PE from Valencia flavedo and Shamouti juice with acetate buffer, pH 5.00 with a linear gradient of NaCl in the range 0 - 0.4 M. They obtained elution of the enzyme at the concentration 0.3 moles of salt/ litre and the elution continued until the buffer contained 0.4 molar concentration of the salt. From the peaks of enzyme activity and protein, it is observed, the eluates contained purely enzyme proteins and as the enzyme activity went up in the fractions, the protein concentrations also went up and followed closely the pattern of enzyme activity in all the tubes. The non-enzymic proteins could not be eluted in the range of ionic strength used in the buffer and therefore the proteins occurring in the eluate were fairly homogenous in terms of enzyme activity. The two main peaks of enzyme activity and protein corresponded to the main isoenzymes, termed as peak A and peak 8 by Korner et a1. (1980) and as PEI and PEII by Versteeg et a1. (1978), who thoght them to Figure 6. 101 PE activity vs. elution fraction in ion-exchange chromatography. 10 mL of PE solution after dialysis were applied to CM-Sephadex C-50 column and elution was carried out by 100 mL acetate buffer, pH 6.00 plus 100 mL of 1 M NaCl in a linear gradient, flow rate being 1 mL/minute. 102 EamamofiEoEo. omcmcoxw :2 :_ c288”. co_S_m .m> >g>=o< Mn. 82:0 .oz .3 mm m... mm mm m. m + l \ i 4 \ Nd. .. _ \ .e . . _ 7 .. \ m j ‘31) J \ \ m... \ u. + N Meet in \\ m. 7 \ we \ \ L .. .I \ +\ \ + it. \ / .e \ \\ /_ OiN +N em enema mug/spun euMzug 103 Protein content of elution fractions in ion- exchange chromatography of orange PE. The contents of the enzyme-active fractions were analyzed by Lowry method of protein determination. Figure 7. as 104 65 \ 65 +\/ if ' m "\ 3 I i- -‘ to «‘8 6 2 .IO ,_ «In L a L L I j to N to 0) 59; F. '1' 8 O C! c O ‘3 d o O iuenIe 1w elfim‘weiuoo ugeioid 105 represent more than 96% of total PE activity. Apart from these two main peaks, three other small peaks also appeared in the enzyme activity profile of the eluate tubes. They could be the other isoenzymes as detected by Versteeg (1979) after pH gradient electrophoresis of crude PE from different citrus fruits. He recognized as many as twelve molecular forms of the enzyme out of which ten forms were present in grapefruit alone. The presence of the high molecular weight isoenzyme or other forms of PEI and PEII were, however, not further elaborated by him. Since this study does not concern with the presence or resolution of the isoenzymes,the tubes showing the enzyme activities were separated from the rest and the contents pooled together for the final steps of purification. Salt Fractionation and Dialysis Carried out the final steps as outlined in the Materials and Methods. Volume of precipitate solution was 20 m1” after dialysis it slightly increased to 20.2 mL. 0.1 mL.of this dialyzed enzyme solution was added to 20 mL.of 0.5% pectin and titrated in the pH-stat against 0.024 M NaOH solution. Table 7 outlines the results. Mean absorbance for protein using 1 mL.dialyzate = 0.1175, which corresponded to 0.072 mg of protein. Table £3 gives the yield of enzyme units after each step of purification and degree of purity associated with it. 106 Table 7 . Enzyme activity in final step. Observation Quantity Blank, Mean Enzyme of InL difference, units/mL. 0.024 M NaOH . mL. dialyzate used, mL 1 0.124 0.025 0.092 2.208 2 0.114 0.029 107 umepa_c .um>pommwv «an muempzm .Em momixom .mmnzu um.mp om.m oo.om mm.em~ c-.momn o~.mmmm co mcwpooa muespm xgaasmoumEocgu om.m~ mm.e pm.m~ m~.-¢ mme.muppp oF.N~_mm macesuxmucoH swam; mm.om Fo.~ mm.o_ upp.oemp mm_.~m~o~ op.om~— umcwmma memxpm_o =o_u=pom .uaa mummpam .sm m_.mm po.~ mm.o~ mn.mm- om.mmpm~ mm~.-~P .aumm New acmuucsmaam oumwpam .Ee mm.em mN._ oo.o oo.ompm mo.mmmmm comm .zumm Rom smmcaa sup: cop p ep.m o~.mm- mm.m~mmm mmmm cowuumsuxu souuew xue>Puuu Ausv . a upm_> :owueu_cvs=a overcoam =_muosm mu_:= .m auev m53~o> mgmum .uvzcm mx\momcmso mvucwpe> Ease we we copumu_mws:a . m «page 108 From a close look at the table, it is clear that as the enzyme solution went through the different steps, the enzyme activity was less due to loss of some of the activity. Despite utmost care in handling, application and storage during experimentation, this loss occurred. Adjustment of the pH in the reaction vessel of the pH-stat was done with very dilute solutions of sodium hydroxide and lactic acid, which is a weak acid. Whenever there was necessity of keeping the enzyme solution in storage, it was kept at 0°C. The stock solution was kept in the deep-freezer. The addition of acid or alkali to the reaction vessel was done slowly along the side of the vessel keeping the reaction mixture well stirred so that there was no zone of destruction on contact of the acid or alkali with the reaction mixture. There was no undue exposure to heat, light or shock and undue delay in going to the subsequent step Was avoided. The biggest losses of enzyme activity, as we can see, were in the salt franctionation steps. When solid salt was added gradually, it salted out the dissolved air from the solution as fine air bubbles and there was formation of froth at the surface. The evolution of air bubbles and occurrence of the froth might be the reason of denaturation of the protein and consequently loss of some enzyme activity during these steps. Another big loss occurred during the ion- exchange chromatography. The set-up was carried out in the cold room at temperature 4°C. The fluctuation of temperature 109 inside the room by frequent Opening of the doors might cause some denaturation. Some of the enzyme molecules might have been adsorbed to the column matrix so tightly that it could not be eluted by the ionic strength used in the buffer. In spite of the losses in the yield of the enzyme, the purification factor increased at each step because of removal of non-enzyme protein. After the salting out steps, the purification was 2 times higher. In the range of 30% to 80% saturated ammonium sulfate, mostly enzymic proteins got precipitated with a few non-enzymic proteins. Dialysis step alone did not have anything to do with purification though it resulted in 8% loss in activity because of not-too-rigid maintenance of 4°C inside the cold room. The biggest jump in purification happened in the ion-exchange step. The careful selection of the ion-exchanger, choice of eluting buffer with desired concentration of salt ions in a linear gradient caused purification of the enzyme nearly 5 times the original solution. Further purification of the enzyme was accomplished by selective precipitation of the enzymic protein by another salting out step and finally 20% enzyme was harvested, which was 6 times as pure as the original. Korner et a1. (1980) purified PE from Shamouti oranges using a similar method and they obtained 5 times purified enzyme and the yield was 58%. 110 Polygalacturonase Activity in Oranges For assay of polygalacturonase in the juice and in the tissue of Valencia oranges, the modified Willstatter-Schudel hypoiodite method was followed. For extraction and partial purification of this enzyme, the method of Riov (1975) was followed. The standard curve (Figure 8) was prepared by taking 5 mL aliquots from each of solutions of galacturonic acid monohydrate which corresponded to amounts of the solute as detailed in Table 9 and treating them the same way as the samples, as described in Materials and Methods. The regres- sion line based on this data was drawn (Figure 8). From the preparation obtained from juice as per method outlined in the Materials and Methods, both the blank and duplicate sample readings were 3.7 mL of sodium thiosulfate, which meant there has been no reduction of the iodine. In other words, no reducing power of galacturonic acid was present in the sample solution. Hence no polygalacturonase activity was present in the orange juice. Similar attempts were made to find polygalacturonase activity in the orange peel and pulp. Several combinations of tissue and extraction media were used and finally the author could trace the enzyme activity. 20 9 tissue were homogenized with 120 mL of buffer and after all the steps the dialyzate was made volume to 12 mL, 2 mL of which were added to 23 mL of polygalacturonic acid and then assayed the 111 Table 9,. Standard curve data for assay of polygalacturonase. Concentration Concentration Amount Difference Iodine* in stock of of with blank, solution solutions of galacturonic thio- mL. consumed, galacturonic acid, mg sulfate, mL. InL acid used, mg/25 mL Blank 0 0 3.8 0.00 7.5 1.5 3.6 0.2 0.143 15 3 3.4 0.4 0.286 20 4 3.3 0.5 0.3575 25 5 3.2 0.6 0.429 30 6 3.1 0.7 0.5005 35 7 2.9 0.9 0.6435 *10 mL thiosulfate a 7.15 mL 12 solution. 112 Figure 8. Standard curve for assay of PGase and pectolyase. This was prepared from the best-fitted data obtained with known concentrations of galacturonic acid according to Willstatter-Schudel hypoiodite method. 113 0.7 r 0.5L- 0.3 " iodine aoin. consumed.mL 2 4 6 Galacturonlc acid, mg 4‘ n 8 Standard curve for assay of polygalacturonase and pectin lyase 114 aliquot as mentioned in Materials and Methods. The results are tabulated in Table 10. From the standard curve (Figure 8% 0.024 mL.of 12 solution was found to be equivalent to 0.25 mg of galacturonic acid. This reading was made accurately by redrawing the lower portion of the standard curve on expanded scales. The calculation for PGase activity was done which is detailed here: 1 unit of PGase activity corresponded to l umole of galacturonic acid, i.e. 0.1941 mg of galacturonic acid 5 mL.aliquot contained 0.25 mg of galacturonic acid after 24 hour. Therefore 25 mL.contained 1.25 mg galacturonic acid which was formed due to catalysis by 2 mL.of enzyme extract. On this basis, 12 mL.enzyme solution contained enzymes to catalyze the formation of 7.5 mg of galacturonic acid. 20 g of tissue gave 12 mL.of enzyme solution. So one gram had the activity for 0.375 mg of galacturonic acid. Therefore the enzyme units per gram of tissue were 1.93. Polygalacturonase activity may be followed by measuring, among other means, the rate of decrease in viscosity of the reaction mixture or the rate of formation of reducing groups. Since the release of reducing groups could be directly correlated with the number of gTycosidic bonds hydrolyzed, this method was chosen and the time and temperature of incubation and the unit of PGase activity were followed as per Riov (1975). 115 Table 10. Polygalacturonase activity in orange tissue. Observation Blank, mL Sample Mean Iodine reading, difference, solution, mL mL reduced, , mL 1 3.7 3.65 2 3.7 3.7 0.033 0.024 3 3.7 3.65 Riov (1975) reported polygalacturonase activity in various citrus fruits and tabulated enzyme units in different parts of the fruit. The highest enzyme activity was found in grapefruit flavedo (1.70 units/g) followed by flavedo of Valencia oranges (1.55 units/g). He, however, used dinitrosalicylic acid to measure the reducing groups liberated, while iodine solution and sodium thiosulfate solution were used in this experiment for the same purpose according to Willstatter-Schudel hypoiodite method. The result obtained was 1.93 units/g of tissue and no activity in the juice. The slight difference between these two values might be due to state of ripeness of the fruit used. Thus the enzyme activity in the fruits was relatively low. Moreover, since there was no activity found in the juice, it was highly unlikely that this enzyme could account for the cloud destabilization in orange juice. However, poly- galacturonase isolated from Rhizopus and purified was pro- cured from Sigma Chemical Company and was used for subsequent 116 experimentation, instead of isolating it from the oranges, because of very low concentration of the enzyme occurring in the fruit. Even though there was no literature found mentioning occurrence of pectate lyase in any fruit or vegetable or any other plant source, the presence of this enzyme in the tissue extract was tested in the same way of measuring the reducing groups liberated as in PGase. However, the pH of the reaction mixture was maintained at 5.5 and 8.00, in view of the wide range of pH optima of the pectinlyase isolated from different microorganisms (5.0 - 9.5) found in the literature. There was no reducing group in the aliquots taken from the reaction mixtures and it was concluded that Valencia oranges did not have any appreciable amount of this enzyme. However, for further experimentation involving this enzyme, a purified pectolyase from a fungal source was procured from Sigma Chemical Company. Assay of Polygalacturonase and Pectolyase Purified polygalacturonase from Rhizopus and pectolyase from Aspergillus were purchased from Sigma Chemical Co. and assayed as detailed in Materials and Methods. The following Table summarize the results for the above two enzymes (Table 11). The standard curve (Figure 8) was used to find the quantity of galacturonic acid corresponding to the iodine solution consumed. 0.286 mLI2 solution corresponded to 117 m.m n.m N mm.pmp emm.o v.0 m.m ~.m F .45 we._om .sea_a gee: msxmuwcz mucmgmwmwu xup>+uuo .aE .uwuaumc com: .45 .mpqum .fls .xcepm mE>~cu copuapom H cowum>gmmno vet'scms wuampamorgu Ezpuom . amonga Ease enema co xmmm< .Fp m—nmh 118 3.2 mg of galacturonic acid, whose release was catalyzed by the PGase in 24 hours. By calculating all the way down to the solid enzyme powder used, it was found that there were 132,000 PGase units per gram of solid. However, according to the specification of the company, the solid contained 480 units of PGase per g“ and the unit of PGase was the quantity of enzyme which catalyzed the release of 1 umole of galacturonic acid per minute. According to that, there should have been 691,200 umoles released after 24 hours from use of 1 g of solid. But since enzyme activity fell with time due to fall of the substrate concentration or some inactivation at the temperature or pH of the reaction owing to instability, the progress curve or the calculation of the enzyme units after 24 hours did not become the simple multiple of the time in minutes, but much lower than that. ' The same was true for the pectolyase also. According to the company specification, one mg of solid enzyme contained 3 units, one unit being the enzyme able to catalyze the libera- tion of l umole of galacturonic acid per minute. As per this specification after 24 hours, one mg of solid enzyme prepara- tion should have liberated 4320 umoles of galacturonic acid, but as the enzyme activity lowered down with time, in this case also only 2330.06 moles were liberated after 24 hours (Table 12). 119 m.N n.m N ©0.0mm~ Nxm.c m.o m.~ m.m F EB... ms\mu_== .ae .umuzums mucosa _ com a can go soe>_eoe ea_e=_om NH cc.c 2 F m s .m eoeoe>comeo msx~cm .35 .umuom: muewpzmo_;u Eavuom .mzupcqmefl mapppmsmam< soc; mmexpouuaa mo xmmm< .NP mpnmh 120 Effect of Pectic Enzymes on Cloud of Reconstituted Juice The results obtained through a period of 30 days with reconstituted juice incubated with three different pectic enzymes at two temperatures have been shown in Figures 9a and 98. It was noticed that neither polygalacturonase nor pecto- lyase, at the concentrations used, were able to destabilize the cloud at any temperature. But pectinesterase was able to clarify the juice after 1 week's storage at 28°C at a concentration of 1.2 units/mt juice. The same temperature storage clarified the juice after 10 days and 21 days when the PE concentrations were reduced to 1.0 units/mL and 0.6 unit/mL, respectively. This clearly showed that the cloud stabilization was very much dependent on the amount of PE units present in the juice and 1.2 units of PE/mL juice, which have been found to be normal level of the enzyme by Versteeg (1979), clarified the juice after a week at 28°C and it took 27 days to do the same job at 4°C. Similarly, on comparison, all the treatments showed less destabilization at 4°C than at 28°C. Therefore, the temperature of storage had also a role to play. Cold temperature was able to slow down the destabilization, because it suppressed the action of the PE on pectin molecules, as would be shown later on. The polygalacturonase concentrations, though unable to clarify the juice at any temperature, increased the percent Figure 9a. 121 Effect of PE on cloud of reconstituted juice. Semi-purified PE at 1.2 units/mL, 1.0 unit/mL and 0.6 units/mL were added to juice (08 41.3), adjusted to pH 4.0, reconstituted from PE-inactive Valencia frozen concentrate and incubated at 28°C and 4°C. Turbidities were measured at 660 nm after centrifuging the juice for 10 minutes at 360xg. 96Transrnlttance ioor _* I 40 I” + L V+/+ 80 4/ ‘ \ PE 0.6, 28°C 4. 122 , re 1.2, za'c I 152 1.0, ze‘c ~---- PE 1.0. 4'0 A / 'K “‘ PE 0.6. 4.0 /A T , . 10 20 3‘0 40 Days Effect of PE on cloud of reconstituted juice Figure 9b. 123 Effect of PGase on cloud of reconstituted juice. Polygalacturonase at 0.30 unit/mL, 0.25 unit/mL and 0.20 unit/mL were added to juice (°B 41.3) adjusted to pH 4.0, reconstituted from PE-inactive Valencia frozen concentrate. Turbidities were measured at 660 nm after centrifuging the juice for 10 minutes at 360xg. 124 Penn 0.30, za'c 4O 35 '30 96Tranamittance \ "PGase 0.20. 4'c 10 20 30 40 Days Effect of PGase on cloud of reconstuted juice Figure 9C. 125 Effect of pectolyase on cloud of reconstituted juice. Pectolyase at 0.30 unit/mL, 0.25 unit/mL and 0.20 unit/mL (not shown in the graph) were added to juice (°B 41.3), pH adjusted to 4.0, reconstituted from PE-inactive Valencia frozen concentrate. Turbidities were measured at 660 nm after centrifuging the juice for 10 minutes at 360xg. %Transmlttance 126 30 1’ PL 0.30, 28‘0 28 " , ’l +—+ J + /-- PL 0.25, 28'0 26 - /+-—-+ ’ FL 0.30. 4°C / I A ' 4.4.4 44 \ 24 5. °.PL 0.25. 4°C 2 20 *- 10 20 30 40 Days Effect of pectolyase on cloud of reconstituted juice 127 transmittance particularly at 28°C and this increase was higher when the concentrations were higher. The pectolyase concentrations were even less active in increasing the transmittance and again the percent trans- mittances were higher at higher temperature than at lower temperature. Orange juice contains pectin and this pectin concentra- tion varies from 0.01 to 0.13% in orange juice depending on variety of fruit and its maturity. In this pectin, many of the carboxyl groups of galacturonic acid residues are esterified with methanol and the degree of esterification is expressed as the degree of esterification (0 - 100% DE) or methoxyl content (0 - 16.32% correspondingly). Orange juice contains both high-methoxyl pectins and low-methoxyl pectin, depending on whether the amount of methoxy groups is more than 7% or lower. There has been researches to find the relation between the degree of esterification and cloud destabilization. It is known that native pectinesterase lowers the ester content of juice soluble pectin until it becomes susceptible to precipitation as insoluble pectates. However, the finding of the critical ester content is very much difficult, because the juice contains pectin which exhibits a natural heterogeneity with respect to ester content in its molecules. However, after a careful study of the pectins fractionated according to ester content of pectins, Baker (1979) found the critical DE for precipitation 128 of pectins as pectates was between 14 and 21%. In view of the above information, it was postulated that the reconstituted juice normally contained pectin whose degree of esterification was above the critical range. As the concentrations of PE and the temperature increased, more or more deesterification took place until the DE reached the critical level when the clarification started. Polygalacturonase could not clarify the juice because it had no role in desterification of the pectin. However, since there were low-methoxy pectin molecules also occurring in the juice, the action of the enzyme was not completely stopped and it hydrolyzed the pectin molecules in those unesterified portions giving rise to higher oligogalac- turonides which also took part in the calciUm binding mechanism and settled down. Termote et a1. (1977) observed that oligogalacturonides with a degree of polymerization of 18 and higher were responsible for accelerated juice clari- fication as readily as pectic acid. The enzyme possibly catalyzed the hydrolytic cleavage of the low-methoxyl pectin forming higher polymer oligogalacturonic acid. Jansen et a1. (1945) made a detailed study of influence of methoxyl content of pectin on action of polygalacturonase and hypo- thesized that the enzyme required at least two adjacent free carboxyl groups in order to hydrolyze a glycosidic 129 bond associated with these free carboxyl groups. They also observed that the susceptibility of the pectin molecule to hydrolysis by polygalacturonase depends upon the methoxyl content and the extent of hydrolysis was an inverse linear function of the methoxyl content between 9.5 and 2.5% methoxyl and was complete when only the pectinic acids contained less than 2.5% methoxyl. The low-methoxyl pectin of the orange juice in this study might contain methoxyl groups in this range and therefore was susceptible to hydrolysis which ultimately gave rise to oligalacturonic acid of critical degree of polymerization. However, the formation of these polymers was quite slow and that is why even after 30 days, the polygalacturonase-treated juice showed a percent transmittance of 13 at the higher tempera- ture and the highest concentration used. . Since there was no appreciable polygalacturonase activity in the orange juice and this enzyme could not destabilize the cloud of the reconstituted juice even at a high concen- tration, it was postulated that this enzyme had no role in cloud destabilization in single-strength freshly extracted orange juice. Pectolyase also could not destabilize the cloud at the concentrations used. This enzyme cleaved the glycosidic linkage of the pectin molecules by a transelimination mechanism rather than by hydrolysis forming unsaturated methyl oligogalacturonates which did not participate in 130 cloud destabilization. Moreover this enzyme was found to be absent in orange tissue or orange juice. Methanol Formation in Orange Juice To get the working standards, a stock solution of 1 mg/mL of methanol in water was used. From this, different quanti- ties were taken and diluted to 100 mL with l N H2504 solution. Table 13 gives the data with these standards. A regression line was drawn from the data plotting absorbance values against concentrations of methanol to get the standard curve (Figure 10). The data for turbidity measurements of freshly extracted orange juice of 08 10.8, pH 3.6 and methanol released were used for drawing Figures 11 and 12. From Figure 11, it is seen that transmittance increased gradually at both temperatures and attained 60, the thresh- old value for clarified juice, on 4th and 5th day at 28°C and 4°C respectively. So in freshly extracted juice, low temperature did not play a big role in retarding the clarification process as in reconstituted orange juice, where the juice became clarified 3 weeks later with a comparable level of pectinesterase of 1.2 units/mL juice. The initial percentage transmittance of recon- stituted juice was lower than fresh juice because of the difference in °Brix. One explanation of delay in 131 Table 13, Data for standard curve for methanol determination. Working Standards Concentration of methanol Absorbance in 100 mL.of 1 N H2504 (ug/mL) 0 mL.from stock 0 0.00 solution 0.5 mL.from stock 5 0.08 solution 1 mL.from stock 10 0.17 solution 2 mL.from stock A 20 0.36 solution 3 mL.from stock 30 0.53 solution 4 mL.from stock 40 0.69 solution 132 Figure 10. Standard curve for methanol determination, drawn from the regression data obtained from known concentrations of methanol in water, according to Wood and Siddiqui (1970) method. Absorbance 0.7 0.6 V 133 I | L l 10 20 30 . Conc. of Methanol, (.ig/rnL4 21:“ Standard curve for methanolfl 0 Figure 11. 134 Clarification of unpasteurized orange juice. 100 mL freshly extracted juice from Valencia oranges were stored in glass jars at 280C and 4°C and turbidities were measured at 660 nm after centrifugation for 10 minutes at 360xg. 135 1001- . %Transmittance 4 L 8 12 16 Days of storage as. Ciarificatlon of unpasteurized orange juice 136 in clarification at low temperature might be that rela- tively longer time was taken by the enzyme to adjust itself in the reconstituted environment for catalyzing attacks on the substrate. 1 The methanol release curve (Figure 12) shows that at 28°C there were three distinct phases of methanol release throughout the storage, an initial slow phase, an inter- mediate fast phase and a final stationary phase. The sudden upsurge of methanol release in the fast phase might be due to the involvement of a certain autocatalytic step in the chain of chemical reactions with the attainment of a certain concentration of methanol or of a certain degree of esteri- fication in the pectin molecule. Versteeg (1979) observed that velocity of pectinesterase I on highly esterified pectin increased after some substrate was saponified because of increase in affinity for the changed substrate or creation of new points of attack by the pectinesterase in the pectin chain. The stationary phase was attained when a certain percentage of methoxy groups was saponified and the activity of the enzyme was also reduced and remained more or less constant. From Figure 12, it was also observed that in the initial slow phase, there was a lowering down of methanol release at 28°C, which might be due to some product inhibition, according to Krop et a1. (1974), who also observed a similar depression in the methanol release curve. Figure 12. 137 Methanol release in clarifying orange juice. Freshly extracted juice from Valencia oranges were stored at 28°C and 4°C and methanol release was determined after distillation, according to Wood and Siddiqui (1971). Methanol cone. in juice, pglmL 138 za'c ELM 30- ° ‘.° A / 47p 1% 4 a {2 To Days of storage Methanol release In clarifying orange juice 139 To study the relation of methanol formation and cloud destabilization, data of methanol released were plotted against percent transmittances for both the temperatures (Figure13). Since a linear relation was visualized between these two sets of values, coefficients of correlation were ‘calculated and it was found that at both temperatures a strong linear relationship existed between methanol and clarification of juice and 90-95% of the variation in transmittance values could be explained by the variation in amount of methanol formed. Furthermore, it was observed at 28°C, the juice became clarified when a level of 17.5 pg of methanol was attained per mL.of juice and this value at 4°C was 18 ug/mL.juice. However, Versteeg (1979) reported that 30 ug of methanol was found in orange juice when it started clarification by Pectinesterase I at a level of l unit/mL. Since the extent of clarification depended on initial percent transmittance, which again was dependent on °Brix, the difference in degree of Brix was perhaps the cause between these two discrepancies. After alkali saponification of juice according to Versteeg (1979), a value of 51 ug/mL. of methanol was obtained, which meant that potentially 51 pg of methanol/mL.juice could be released by the enzyme on complete deesterification of the pectin molecules. After two weeks storage, the naturally occurring enzyme released 45 ug and 36.00 g of methanol at 28°C and 4°C respectively. So the enzyme was 87% efficient at the higher temperature Figure 13. 140 Correlation between % T and methanol release. Methanol concentrations in freshly extracted orange juice during incubation at 28°C and 4°C were plotted against the corresponding % transmittance. Coefgicient of correlation: 0.93 at 28°C and 0.95 at 4 C. Methanol released.pglmL 48 Q a: I N 9; 141 ‘ \§ \ a . J. T7770 4'? 6‘0 Jr‘s—"‘50 %Transmittance Correlation between %T and methanol release 142 70% efficient at the lower temperature. Though the effi- ciency differed in these two temperatures, the amount of methanol released correSponding to a particular transmit- tance value, say 60% T, was more or less the same, as can be seen on the overlapping curves of Figure 13,except that the sigmoid nature of the curve at the higher temperature became more or less flattened at the lower temperature and became a linear one because of retarded enzymic action at low temperatures. Inhibition of Pectinesterase by Sugars Table 14 summarizes the results obtained with concentra- tions of different sugars. From the table it is found that fructose was the most effective of all sugars tested in inhibiting the enzyme followed by glucose, maltose and sucrose in that order. Closely similar results were obtained by Chang et a1. (1965) who found that sucrose and glucose both inhibited the papaya pectinesterase. With 20% and 30% sucrose concentration, they got about 30% and 40% inhibition of the enzyme and this inhibition was linear up to 50% sucrose concentration. Though no data were furnished for inhibition by glucose, they stated that the inhibition was more with glucose. However no other sugar was tried. 0n the basis of data obtained by changing the substrate concen- tration, they postulated that this inhibition was non-compe- titive and sugars lowered the water activity which was less 143 Table 14. Inhibition of pectinesterase by sugars. Sugar Concentration, % Relative activity of the reaction mixture (w/v) No inhibition 0.00 100 Sucrose 11.25 81.89 18.75 78.70 23.75 72.99 Glucose 11.25 77.10 18.75 62.61 23.75 49.62 Fructose 11.25 52.19 18.75 31.45 23.75 16.22 Maltose 11.27 88.23 18.75 ’ 78.98 23.75 59.87 144 suitable for the enzyme to carry on its catalytic activity. Hultin et a1. (1968) tested the inhibition of three chr0- matographic fractions of banana pectinesterase by sucrose at levels up to 50%, and found that the percent inhibition was different for the different fractions. Since all the sugars inhibited the pectinesterase in this experiment only at relatively high concentrations, none of them was tested in the orange juice. Inhibition by_Pectolyase From Table 15, it is Observed that the enzyme which contained endopectinlyase inhibited the pectinesterase only to the extent of 19% even at a high concentration of 1.74 units/mL. Baker (1980) observed that relatively large quantities of pectinlyase were required for cloud 'stabilization of orange juice because of competition between this enzyme and pectinesterase for the esterified pectin. This is in contrast to polygalacturonase because only the low-ester pectin has to be destroyed enzymically for cloud stabilization and polygalacturonase readily carried it out as will be seen in the subsequent discussions. Inhibition by Polygalacturonase From Figure 14 drawn on data of Table 16, it is seen that increasing the concentration of the polygalacturonase effectively inhibited the pectinesterase. Baker and 145 Table 15. Inhibition of PE by pectolyase. Units of pectolyase/mL Relative activity of PE reaction mixture 0 100 0.58 98.21 1.16 86.42 1.74 81.71 Table 16. Inhibition of PE by polygalacturonase. Units of polygalacturonase Relative activity /mL.of reaction mixture 0.00 100 0.066 73.64 0.132 44.41 0.198 31.07 0.264 22.44 0.33 16.11 0.396 10.93 Figure 14. 146 Inhibition of PE by PGase. Increments of poly- galacturonase from 0.066 unit/mL through 0.396 unit/mL were added to 0.5% (w/v) pectin solution and residual activity of PE (1 unit/mL) was measured at pH 7.5 and 30°C for 20 minutes. 147 10 80 401- \ . , 20 \o \ 0.132 ' 0.264 0.396 PGase conc., units/mL Relative activity of PE Inhibition of PE by PGase 148 Bruemmer (1972) showed a pathway of combined action of pectinesterase and polygalacturonase on high-methoxyl pectin to form ultimately oligogalacturonic acids which inhibited the pectinesterase action (Figure 14a). They postulated that high-methoxyl soluble pectin was converted to low-methoxyl pectin which again was attacked by poly- galacturonase to convert to short chain low-methoxyl pectin, further converting it to oligogalacturonic acids which actually inhibited the pectinesterase. Such results were also reported by Termote et a1. (1977) who found that oligalac- turonides with a degree of polymerization 7, 8, 9.2 and 10.00 inhibited the pectinesterase enzyme and the degree of inhibition was in the same order. In this study, results obtained fully supported these views. As the concentrations of the PGase increased, more and more oligogalacturonides in the effective range (DP 8 to 15) were formed and more and more inhibition of PE took place. These concentrations of PGase were tested in single strength orange juice. Figure 15a and 15b were drawn on the data and Table 17 was derived from these curves. From Figures 15a and 15b, it is obvious that unpasteurized juice and pasteurized juice with PE 1 unit/mL behaved in the same way in becoming clear, though in case of unpasteurized juice, the cold temperature retarded clarification for 1.5 days. This presumably was because Figure 14a. 149 Enzymic breakdown of pectin, possible pathways for breakdown of pectin by pectinesterase, polygalacturonase and polymethyl alacturonase, adapted from Baker and Bruemmer 1972). 150 cacao. B $69.35 2535 2:3 2:21 3033096 a. E 82303 t 588 3.853.130. 22:35 . Is. 1 Sage team 8.x. [a «2203 I 583 588 833.8 @328:- 12 358592133 Sago :05 mm/ \OSE 583 23.8 0.2m .— ._o On. 53322.". Figure 15a. 151 Effect of heat and pectic enzymes on juice cloud, A portion of orange juice was heated for 3 minutes at 98°C and cooled, to which were added separately PE 1 unit/mL, PGase 0.33 unit/mL and no enzyme. The unheated portion was stored as zgch. All incubations were done at 28°C and C. %Transmittance 100 90 30 152 Heated. PE 1 unit, za’c M ..__-..._...-.—-—-»a P/t . It: Hated. PE 1 unit, 4'0 2"“ " ./'N3/hm. "0 Enzyme. 4.0 _ Heated, PGase 0.33. 28‘0 ”X: I 1-4....———-———-- 4‘ 1 t. . 2-... 1Heated, PGase 0.93, 4‘0 / "1"”:ng x --.-.-Fe{::r/:~WI= x ' l ‘ . Heated, no enzyme, 4‘0 T I I ' - 4 Days Effect of heat and pectic enzymes on juice cloud Figure 15b. 153 Effect of heat and pectic enzymes on juice cloud. A portion of the freshly extracted juice was heated for 3 minutes at 98°C and to it was added pectolyase 0.40 unit/mL. To the unheated portion were added separately polygalacturonase 0.263 unit/mL, 0.33 unit/mL and 0.52 unit/mL. All incubations were done at 28°C and 4°C. 70 60 b 01 O - %Transmittance N H (0 154 No heat. PGase 0.263. 28‘0 o °°°°° 96‘. , ‘.‘\e ‘ . a i N ‘ Gas. 0-33' 2 c fa“. t t ° N° °°°° P 0.52. 28‘0 / . No Mange”. 40 28 , Heat d. Windy”. °' ' as 0.52. ‘c k - 1° _ o heat. P91al ‘ 4—1: Heated, Pectolyase 0.40, 4'0 I l I I ’1 7 14 21 28 35 Days Effect of heat and pectic enzymes on juice cloud 155 Table 17. Days for orange juice to reach 60% transmittance. Treatment Days needed Unpasteurized, 28°C ' 3.5 Unpasteurized, 4°C 5 0. 33 PGase, pasteurized, 28°C Did not attain 0. 33 PGase, pasteurized, 4°C Did not attain 0. 4O pectolyase, pasteurized, 28° C Did not attain 0. 40 pectolyase, pasteurized, 4° C Did not attain Pasteurized, PE 1 unit, 28° C 3.5 Pasteurized, PE 1 unit, 4° C 3.5 Unpasteurized, PGase O. 263, 28°C 29 Unpasteurized, PGase 0. 263, 4°C 40 (*) Unpasteurized, PGase 0.33, 28°C Beyond 40 Unpasteurized, PGase 0.33, 4°C No clarification Unpasteurized, PGase 0.52, 28°C No clarification Unpasteurized, PGase 0. 52, 4°C No clarification Pasteurized, no enzyme, 28° C No clarification Pasteurized, no enzyme, 4° C No clarification *Interpolated from the curve. 156 of ready attack of the substrate by the semipurified enzyme. Polygalacturonase and pectolyase did not have any role in clarifying the pasteurized juice, though percent transmit- tance showed a slow and gradual increase in polygalacturonase treated juices. Similar patterns were obtained with recon- stituted juice as already discussed. However, the percent transmittance in case of fresh juice were more at any temperature and any treatment than reconstituted juice because of initial percent transmittance owing to lower contents of soluble solids (°B 10.8). Polygalacturonase at all concentrations used stabilized the juice cloud at least up to about a month even at the high temperature, which was observed in case of 0.263 units/ mi“ The higher concentrations lengthened the cloud stability even more and at 0.52 units/mL, there was no clarification at all. The heat-treated juices also did not clarify, as the temperature and time was sufficient to inactivate the enzymes. Baker and Bruemmer (1972) obtained cloud stability for 34 days at 4°C using several commercial pectinases which contained, in addition to polygalacturonases, pectinesterases and polymethylgalacturonases. They stated that the success of the pectinases depended on the relative contents of PGase. The enzymes with high PGase activity and low PE or low PMGase activities were effective in stabilizing juice cloud, because the former degraded pectin to oligogalacturonic 157 acids which formed soluble pectates, while the latter two degraded pectin to low methoxyl pectin which formed insoluble pectates. Since they expressed the units of the polygalac- turonase as the amount of enzyme which reduced the viscosity of 1% polygalacturonic acid solution by 25% in 100 minutes, the results obtained by this experiment could not be compared with theirs in respect to effective concentration of PGase to get cloud stability. Krop and Pilnik (1974) and Versteeg (1979) reported stabilization of cloud of reconstituted orange juice by a purified yeast polygalacturonase. However, they observed no influence of the PGase in the activity of the pectinesterase when both were used in the reconstituted juice. On the contrary, a stimulation in pectinesterase activity was observed by them resulting increased methanol concentration. This was in contrast to our results obtained in the model system in which lower PE activity was observed in the presence of higher PGase concentration. The stabilization mechanism of juice cloud, according to Versteeg (1979), was due to hydrolytic breakage of longer chains of oligogalac- turonides by PGase to shorter chains, which was not any more inhibitory to pectinesterase. But Termote et a1. (1977), who conducted a detailed study on inhibition of pectinesterase by pectic acid hydrolyzates, obtained a 75% inhibition by use of decagalacturonic acid and moderate inhibition by use of oligogalacturonides of degree of 158 polymerization of 9, 8 and 7. Again according to pathway shown by Baker and Braummer (1972), it is seen that PE and PGase acting simultaneously on pectin, can degrade the substrate ultimately to oligogalactUronides of shorter degree of polymerization. Therefore, the possible explana- tion of lower PE activity in our model experiment in presence of PGase, might be the use of purified substrate which was rapidly attacked by the semipurified PE and pure PGase in succession ultimately forming oligogalacturonides of critical degree of polymerization which caused inhibition on the pectinesterase. As more and more pectinesterase was inhibited, less and less low-methoxyl pectin was formed, causing the production of lower amounts of oligogalacturo- nides which actually inhibited the PE. That might be the reason why proportionate increments of polygalacturonase gave less than proportionate increase in inhibition of pectinesterase. The mechanism of cloud stability in orange juice might be, however, different from this, where the polygalacturonide of the longer chains were hydrolyzed to shorter oligomers which did not precipitate with calcium ions, rather than inhibition of pectinesterase. Inhibition by Polygalacturonic Acid Table 18 summarizes the results of inhibition of semi- purified PE by different concentrations of polygalacturonic acid (PGA). The second set values were obtained by using 159 Table 18. Inhibition of PE by PGA. Concentration of PGA “Relative in reaction mixture, activity 9/100 mL 1st Set 0.00 100 0.10 43.05 0.20 23.75 0.30 18.33 2nd Set 0.00 100 0.50 13.39 0.75 12.16 1.25 7.12 160 0.05% pectin solution instead of 0.5% solution as the substrate. The curve drawn with these data (Figure 16) reveals that up to a concentration of 0.3% PGA, the rate of inhibition was very high and the relative activity fell sharply after which the decline in activity became very slow and it took 0.9% more PGA to have a decline of 8% in relative activity. Krop (1974) observed that orange pectinesterase was inhibited by pectic acid but it could not be used to stabilize cloud, as it would by itself cause clarification in concentration as low as 0.2 mg per mL.juice (Krop and Pilnik, 1974b). Termote et a1. (1977) obtained inhibition of pectinesterase with pectic acid and its hydrolyzates, but found it to destabilize the cloud even in presence of potential inhibitors of PE. All these findings were made in PE-inactive reconstituted orange juice. Versteeg et a1. (1978) reported that polygalacturonic acideas a competitive inhibitor of PE, inhibiting PEII more than PEI. They used 0.1 mg/lOO mL.reaction mixture of polygalacturonic acid for PEII and 0.5 mg/lOO mL.reaction mixture for PEI. Korner et a1. (1980) found 20% inhibition of both Peak A and Peak 8 of pectinesterase with use of 0.1% of polygalacturonic acid, but none of them reported its effect on single strength orange juice. When polygalacturonic acid was used in single strength orange juice, the results obtained were used to draw the curves (Figure 17a and 17b) from which again the 161 Figure 16. Inhibition of PE by polygalacturonic acid. For inhibitor concentrations of 0.1%, 0.2% and 0.3%, 0.5% pectin solution was the substrate for PE, while for inhibitor concentrations of 0.5%, 0.75% and 1.25%, 0.05% pectin was used. Relative activity 10 h N O 162 \ ‘ 4 .2 . 0.2 0.6 1.0 Polygalacturonic acid, glmL Inhibition of PE by polygalacturonic acid 1.4 Figure 17a. 163 Effect of PGA (0.1% and 0.2%) on juice cloud. To the freshly extracted juice (°B 10.8), was added 0.1% and 0.2% polygalacturonase acid from a stock solution of 10% PGA6 pH adjusted to 4.0 and incubated at 28°C and 4 C. 96Transmittance 164 100r PGA 0.196, za'c I /"=:- 3 PM 0.2%. 4'0 . O I «I» T i ' 35’ PG" °', [GA 0.2%. za-c 701- 551- I 40- 25 : . . . a. 10 20 30 40 Days Effect of Polygalacturonic acid on juice cloud 165 Figure 17b. Effect of PGA (0.3% and 0.4%) on juice cloud. A 10% PGA solution was used for 0.3% and 0.4% PGA concentration in the juice, pH adjusted to 4.0 and incubated at 28°C and 4°C. 96Transmittance 75F 60*- 45" 15" 166 + x PGA 0.3%. 28‘0 P A 0.3%. 4'0 ABA 0.4%, ze'c 44. PGA 0.4%, 4°C I n L 4 10 20 30 40 Days Effect of Polygalacturonic acid on juice cloud 167 following table (Table 19) for clarification of orange juice was done. 0.3 mg and 0.4 mg of PGA per 100 mL of orange juice delayed destabilization of orange juice for more than a month. In model experiment, it was found that 0.3 mg PGA brought about 85% inhibition of PE. Therefore it can be stated that PE having a residual activity of 15% could not clarify orange juice up to 30 days or more. Since PGA was a competitive inhibitor of PE, it bound the enzyme at the active sites, therefore, there was no formation of enzyme substrate complex. The pectin molecules were spared from enzymic action and destabilization did not take place. The polygalacturonic acid molecules were bound to the enzyme molecules and they themselves might not take part in forma- tion of cross-linkages with divalent cations. Hence, the orange juice became cloud stable. Inhibition by Ethylenediaminetetraacetic acid (EDTA) Table 20 summarizes the results obtained in inhibiting semipurified PE in model system by EDTA. It is known that EDTA can bind metal ions, specially the divalent cations to form a coordination complex by a chemical process known as chelation. Korner et a1. (1980) found 60% inhibition of the two PE fractions by using 0.01 moles EDTA/litre reaction mixture. Table 20 shows that EDTA at a concentration of 0.01 M inhibited the enzyme more than 90%. Even at a low concentration of 0.00251moles/litre, this coupound effectively 168 Table 19. Days needed for clarification. Concentration of PGA, -g/100 Temp., 00 Days mL.juice 0.00 28 3 days 0.00 4 4.5 days 0.1 28 3 days 0.1 4 4.5 days 0.2 28 5.5 0.2 4 14 0.3 28 30.5 0.3 4 did not clarify 0.4 28 did not clarify 0.4 4 did not clarify 169 Table 20. Inhibition of PE by EDTA. Concentration of EDTA, Relative moles/litre activity 0.00 100 0.05 1.39 0.01 2.77 0.005 6.94 0.0025 34.72 0.00125 97.00 170 Figure 18. Inhibition of PE by EDTA. PE solution (1 unit/mL) of reaction mixture) was added to 0.5% pectin solution and activity was measured in presence of different concentrations of the inhibitor. iOdK y 013E 0 Relative aftlvlt O N O 80[ 171 \+ . . . l + 0.01 0.02 0.03 0.04 0.05 Conc. of EDTA, moles/L Inhibition of PE by EDTA 172 inhibited the enzyme to the extent of 65%. From this result, it might be speculated that the enzyme might require metal ions for its activity and when this metal ion was found by EDTA, the enzyme was inhibited. Figures 19a, 19b and 19c show results when EDTA was used in fresh single-strength orange juice. From these: graphs, the following table (Table 21) was prepared. Therefore, the behavior of EDTA was different when used in orange juice. A comparison of these data with Table 20 would show that EDTA in the same concentration of 0.005 M which inhibited more than 90% of PE in the model system, could not stabilize the cloud even for a week. Likewise, all other concentrations of EDTA were much less effective. 0.075 M EDTA stabilized the cloud at 4°C for 18 days and at the same temperature, 0.125 M EDTA stabilized the cloud for more than a month. It is known that many enzymes require metal ions as a component part of the co-factor which influence the activity of the enzyme either by substrate binding or in the process of catalysis itself. This type of enzymes are inhibited by agents that are capable of binding the essential metal. Pectinesterase might be such an enzyme and as such was inhibited by EDTA which chelated the metal ion by coordinate bonding. But when EDTA was used in orange juice, a portion was apparently engaged to chelate the metal ions already present in the juice, specially the divalent cations like magnesium and calcium and less EDTA was available for enzyme 173 Figure 19a. Effect of EDTA (0.005 M and 0.01 M) on juice cloud. Calculated amount of EDTA salt, disodium, dihydrate, was added to fresh orange juice, pH adjusted to 4.0 and incubated at 28°C and 4°C. 96Transmittance 174 10 0.01M. 28b 0.005M. 28‘3 1, Days Effect of EDTA on juice cloud 175 Figure 19b. Effect of EDTA (0.05 M and 0.075 M) on juice ' cloud. Disodium salt of EDTA was used, pH of juice adjusted to 4.0 and temperatures of incubation were 28°C and 4°C. 176 o... .293... Soc 8.5. co (Em .o 82m. 239 mu om mp op o... .38.qu a 0.0m .2006 eouemwsue: 1,95 177 Figure 19c. Effect of EDTA (0.1 M and 0.125 M) on juice cloud. Disodium salt of EDTA was used to juice of 08 10.8, pH adjusted to 4.0 and C incubated at 28°C and 4 . 178 26.6 8.2 co wuum ~5z~=m mm .mm mpnap 212 Table 30. Determination of protein content in ion-exchange fractions by the Lowry method (from standard curve). Tube No. Absorbance Protein Protein content, content, mg/mLeluate mg/3 m1. eluate 25 0.00 0.00 0.00 26 0.02 0.01 0.03 27 0.02 0.01 0.03 28 0.03 0.016 0.048 29 0.035 0.019 0.057 30 0.04 0.023 0.069 *31 0.026 0.078 *32 0.031 0.093 33 0.06 0.036 0.108 *34 0.045 0.135 *35 0.050 0.150 36 0.09 0.054 0.162 *37 0.054 0.162 *38 0.052 0.156 39 0.085 0.051 0.153 *40 0.047 0.141 *41 0.043 0.129 42 0.065 0.038 0.114 *43 0.035 0.105 44 0.055 0.032 0.096 45 0.040 0.023 0.069 *46 0.019 0.057 47 0.03 0.016 0.048 *48 0.015 0.045 49 0.025 0.013 0.039 *50 0.021 0.063 51 0.05 0.029 0.087 *52 0.028 0.084 *53 0.025 0.075 54 0.04 0.023 0.069 *55 0.015 0.045 56 0.02 0.010 0.030 57 0.00 0.00 0.00 2-777 mg/ 10 mL.E solution *Determinations made from Figure 7. 213 Table 31. Effect of pectic enzymes on cloud of reconstituted juice. Treatment Tempegature % Transmittance on day C o 2 4 »5 8 14 18 22 30 No enzyme 4 21 21 22 22 22 22 22 22 22 28 22 23 23 23 23 23 23.5 23.5 23.5 PE 1.2 units/m1 4 22 22 22 23 23 25 28 so 55 28 22 23 30 49 79 92 97 97 97 PE 1.0 units/ml 4 22 22 22 23 23 24 25 42 57 28 22 23 25 32 49 79 81 83 87 p5 0.5 unit/ml 4 22 22 23 23 23 23 23 32 37 28 21 22 22 24 25 28 33.5 59 86 PGase 0.25 unit/ 4 23 23 23 23 24 24 24 25.5 27 ml 28 22 22 23 25 25.5 28 30 37.5 39 PGase 0.2 unit/ml 4 22 23 23 23 23 23 23 24 25 28 23 23 23 25 25 25 25 25 27 PGase 0.3 unit/ml 4 22 22 23 24 24 25 25 25 27 28 23 23 23 25 25 28 33 38 4o Pectolyase 0.3 4 22 23 23 23 25 *25 25 25 25.5 unit/m1 28 22 23 23 25 25 25.5 27 27 27 Pectolyase 0.25 4 23 23 23 24 24.5 25 25 25 25 unit/m1 28 23 23 23 24 25 25 25 25 27 Pectolyase 0.20 4 23 23 23 24 24 24 24 24.5 25 unit/m1 28 23 23 24 24 25 25 25 27 27.5 214 Table 32. Clarification of unpasteurized orange juice. Storage Percent transmittance on day 0 temp' C o 2 4 5 7 10 14 28 30 42 61 75 88 88 89 4 30 39 53 60 71 82 90 215 Table 33. Release of methanol in unpasteurized orange juice. Storage Day ‘Juice Final vol. Absorbance Amount temp, °C taken, of . of mL. distillate, methanol m1. 119/mL. juice 0 10 35 0.045 8.75 2 10 32 0.08 14.4 4 10 38 0.09 17.5 28 5 10 28 0.17 26.6 7 10 40 0.195 44 10 10 40 0.195 44 14 10 45 0.175 45 ‘7. 0 10 35 0.045 8.75’ 2 10 35 0.06 12.25 4 10 40 0.075 16.00 4 5 10 30 0.105 18.00 7 10 35 0.115 22.75 10 10 40 0.13 30.00 14 10 40 0.16 36.00 216 Table 34. Action of heat and pectic enzymes on orange juice cloud. Treatment Temperature Percent transmittance on day of sgorage - C 7 14 21 28 35 No treatment 28 92.5 94 95 96 97 4 72 76.5 80.5 89.5 90 Heat-treated 28 28 28.5 31 32 32 4 28 28 29 29 30 Heated, PGase 28 30 33 40 42 42 added 0.33 4 34 40 38 38 38 units/mL Heated, pecto- 28 30 30 32 34 36 lyase added 4 30 30 30 30 31 0.40 units/mL. No heat, PGase 28 39 45 53 59 65 added 0.263 4 36 41 45 51 58 units/mL. No heat, PGase 28 41 42.5 49 50 52 0.33 units/mL. 4 37 40 44 44 44 No heat, PGase 28 33 35.5 39 42 42 0.52 units/mL. 4 28 29 32 34 36 Heated, PE 28 91.5 94.5 96 97 97 added 1 unit/mL. 4 90 92 92 92 93 217 Table 35. Effect of PGA on orange juice cloud. Concentration Temp. of % Transmittance on day of PGA, g/ storage 100 mL juice °C 6 12 18 25 32 0.00 28 80 90 93 96 96 4 64 84 91 95 96 0.1 28 80 90 94 94 95 4 63 89 90 94 94 0.2 28 62 72 81 89 89 4 49 51.5 82 86 90 0.3 28 26 30 34 53 62 4 30 30 36 38 39 0.4 28 28 28 30 31.5 36 4 29 29 30 30 30 218 Table 36. Effect of EDTA on orange juice cloud. Concentration Temp. of % T on day * of EDTA storage (00) 5 7 12 14 18 25 32 0.005 M 28 - 85 - 87 - - - 4 - 73 - 90 - - - 0.01 M 28 - 83 - 90 - - - 4 - 55 - 91 - - - 0.05 M 28 87 - 87 - 90 89.5 89 ‘ 4 82 - 80 - 79.5 86 86 0.075 M 28 42 - 69.5 - 77.5 80 85 4 53 - 57 - 59 68 74 0.1 M 28 53 - 60 - 75 80 82 4 50 - 52 - 54 65 72 0.125 M 28 39 - 52.5 - 68.5 75 79.5 4 4o - 41 ; 42 43 43 *% T of fresh juice = 30 219 Table 378. Effect of freezing oranges and orange juice on cloud. Percent transmittance Days °f freezing Frozen juice‘ Juice from frozen fruit 0 31 31 6 30 32 12 37 32 19 44 35 25 54.5 40 32 61 45 39 68 49 Table 37b. Effect of storage at 4°C on juice cloud. Percent transmittance Days of storage at 4°C Extracted juice Juice from fruit 0 31 31 7 72 32.5 14 76.5 34 21 80.5 35 28 89.5 35 35 90 36 220 Table 38. Effect of short freezing on juice cloud. Percent transmittance Days of storage at 00c Frozen Juice . 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