.1“ THERMAL INACTIVATION 0F PECTINASE IN CUCUMBER BRINE Thesis for the Degree of M. S. MICHIGAN STATE umvmsrw SUPARATH CHAVANA 1975 x 6&30/67» ABSTRACT THERMAL INACTIVATION OF PECTINASE IN CUCUMBER BRINE BY Suparath Chavana The filamentous fungi on cucumber flowers and fruits have been found to be the source of the enzyme which can cause softening during fermentation of cucumbers. Pectinases from fungi which have been reported to be most common on cucumber flowers and fruits were studied for resistance to heat denaturation to evaluate the adequacy of pasteurization as a treatment for recycled spent brine. The enzyme from genicillium ianthinellum was found to be the most heat stable. Temperature, pH, salt, and protein concentrations affected the heat stability of the enzyme. The enzyme was partially purified by Sephadex G-100 column chromatography. Two peaks of pectinase activity were found. The pectinase with greatest activity was iden- tified to be an endOpolygalacturonase. Penicillium lan- thinellum pectinase was shown to be less heat stable in commercial spent brine than in simulated spent brines. Based upon the heat stability of pectinases likely to occur in commercial pickle brines, it is concluded that Suparath Chavana a pasteurization treatment of 175°F (79.4°C) for 30 seconds is sufficient to assure pectinase inactivation. THERMAL INACTIVATION OF PECTINASE IN CUCUMBER BRINE BY Suparath Chavana A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1976 DEDICATION To my mother and father ii ACKNOWLEDGMENTS I wish to express my sincere appreciation to my advisor, Dr. R. F. McFeeters, for his help and guidance in the completion of this work. My gratitude is also extended to Dr. R. N. Costilow, Dr. L. E. Dawson, and Dr. T. Wish- netsky for serving as my committee members. I am very grateful for the AID scholarship which supported my study at Michigan State University. Thanks also go to Kasetsart University for permitting me a leave of absence to further my studies in this country. I am also indebted to Miss Linn Farabaugh for her diligent and skillful typing. Finally, my sincere thanks are extended to those whose help and suggestions during the preparation of this project have made this thesis possible. iii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . Vi LIST OF FIGURES . . . . . . . . . . . . Vii INTRODUCTION . . . . . . . . . . . . . 1 LITERATURE REVIEW. . . . . . . . . . . . 3 Softening in Relation to Pectin Degradation . . 3 Microbiological Studies of the Softening Spoilage . . . . . . . . . . . . . 5 Enzyme Activity Measurement. . . . . . . . 9 Reduction of Softening Enzymes. . . . . . 9 Influence of Salt (NaCl) on Pectinolytic Enzyme Activity . . . . . . . 10 Significance of Inactivation of the Softening Enzymes. O O O O I O O O O O O O O 11 MATERIALS AND METHODS . . . . . . . . . . 13 Materials. . . . . . . . . . . . . . 13 Methods . . . . . . . . . . . . . . 13 Assay of Pectinase . . . . . . . . . . 13 Determination of pectinase activity. . . . 15 Preparation of pectin solutions . . . . . 15 Calculation of pectinase activity units . . 16 Protein determination . . ,. . . . . . 16 Preparation of Pectinases. . . . . . . . 17 Heat Inactivation of Pectinases. . . . . . 18 Preparation of brines . . . . . . . . 18 Preparation of enzyme samples. . . . . . 19 Determination of decimal reduction time (D-values) . . . . . . . . . . . 19 iv Page Effect of pH on Heat Inactivation of Pectinases. . . . . . . . . . . . . 20 Effect of NaCl on Heat Inactivation of Pectinase . . . . . . . . . . . . . 20 Purification of Pectinase by Sephadex Chromato- graphy . . . . . . . . . . 21 Effect of Protein Concentration on Heat Inactivation of Pectinase. . . . . . 21 Mode of Action of Penicillium Janthinellum Pectinase . . . . . . . . . 22 Heat Inactivation of Penicillium Janthinellum Pectinase in Spent Br1ne . . . . . . . . 22 RESULTS. . . . . . . . . . . . . . . . 24 Protein Content of Pectinase Solutions. . . . . 26 Preliminary Studies on Heat Inactivation of Pectinases . . . . . . . . . . . . . 26 Heat Inactivation Studies . . . . . . . . . 27 Effect of pH . . . . . . . . . . . 27 Effect of NaCl Concentration . . . . . . . . 29 Purification and Heat Inactivation of Pectinase. . . . . . . . . . . . . . 30 Effect of Protein Content on Heat Inactivation of Pectinase from Penicillium Janthinellum. . . 31 Mode of Action of Penicillium Janthinellum Pectinase. . . . . . . . . . . . . . 32 Heat Inactivation of Penicillium Janthinellum Pectinase in Spent Brine. . . . . . . . . 33 DISCUSSION. . . . . . . . . . . . . . . 40 Effect of Various Factors on Heat Stability of Pectinase. . . . . . . . . . . . . 42 Mode of Action of Penicillium Janthinellum Pectinase. . . . . . . 46 Implications of Heath Stability of Pectinase for Commercial Cucumber Spent Brine Pasteurization . . . . . . . . . . . . 47 CONCLUS ION O O O O O O O O O O O O O O O 4 8 BIBLIOGRAPHY o o o o o o o o o o o o o o 50 ' Table 1. LIST OF TABLES Ten fungi most frequenty isolated from flowers, ovaries, and fruits of pickling cucumbers (Etchells et al., 1958a). . . Organisms that produce pectinases and their sourCeSo O O O O O O O O O O C Time required for maximum enzyme production for each species investigated . . . . Protein content of fungal pectinase concen- trations . . . . . . . . . . . Preliminary heat inactivation tests of pectinases from fungi frequently found in pickling cucumbers . . . . . . . . D-values of pectinases with the highest thermal-Stability o o o o o o o 0 Effect of NaCl concentrations on heat inactivation of pectinase from Penicillium ianthinellum at 75°C . . . . . . . The D-values of purified pectinase at 75°C. 0 O O I O O O O O I O 0 vi Page 14 25 26 28 29 30 32 LIST OF FIGURES Figure Page 1. Standard curves for pectin hydrolysis of pectinases from Penicillium janthinellum and Trichoderma viride at pH 4.0. . . . . 34 2. Effect of pH on heat inactivation of Penicillium janthinellum pectinase at 75°C and 78°C . . . . . . . . . . . 35 3. Effect of pH on heat inactivation of Trichoderma viride pectinase at 65°. . . . 36 4. Heat inactivation curves of Penicillium janthinellum pectinase without protein addition, 1 mg/ml BSA addition, and 3 mg/ml BSA addition. . . . . . . . . 37 5. Sephadex G-100 chromatoqraphy of pectinase from Penicillium janthinellum. . . . . . 38 6. Hydrolytic action of purified pectinase determined by viscosity assay and reducing sugar assay. . . . . . . . . . . . 39 vii INTRODUCTION It has been established that softening in cucumber pickles is caused by pectinase and cellulase which are mainly of fungal origin (Etchells et al., 1958a). The enzymes are produced by molds which occur on cucumber flowers and fruits. The actual mode of enzyme introduction into pickle brining tanks is primarily by way of heavily mold-laden cucumber flowers that remain attached to the cucumber fruit. These enzymes are not inactivated by the acidity and high salt content existing in the brine. Recently, recycling of spent brine has been sug- gested to help reduce the serious pollution problem in the industry (Durkee et al., 1973). Several investigators have reported that normal fermentations were obtained from recycled brine. It is important to treat spent brine to assure the destruction of softening enzymes which may carry over from cucumbers or which may be introduced during storage of the brine. Chemical treatment or pasteurization of spent brine has been recommended on a commercial scale based upon operating cost and practical use. The objective of this study was to investigate the heat inactivation of pectinases produced by filamentous fungi frequently found on pickling cucumbers. This infor- mation is needed to determine whether the pasteurization process in current use is adequate to assure the destruc- tion of pectinases which are most likely to be found in curing brine. LITERATURE REVIEW Softening in Relation to Pectin Degradation Fabian et al. (1932) were the first to suggest that pectic substances might be involved in pickle softening. The microsc0pic examination of both normal and softened pickles showed an absence of what was assumed to be pectic material between epidermal and parenchymatous cells in the softened pickles. Further, Fabian and Johnson (1938) measured the total calcium pectate-forming ability in both normal and soft pickles. They reported that spoilage can occur by way of the conversion of insoluble pectic sub- stances to more soluble forms during the pickling process. This change was accompanied by the loss of ruthenium red stainability. Etchells et al. (1955b) reported that the disintegration of cellulosic substances in the middle lamella was also responsible for cucumber softening. The chemical changes of the pectic and cellulosic substances, therefore, have been related to firmness and texture quality. The pectic enzymes, pectinesterase, polygalac- turonase, and pectate lyase, are involved in pectic degradation of fruits and vegetables. Pectinesterases (PE) catalyze de-esterification of pectin. Polygalacturonases (PG) catalyze the glycosidic hydrolysis of pectin and pectic acid. Pectate lyases split the glycosidic bond by trans- elimination of hydrogen from C—4 to C-5 positions of the aglycone portion of the substrate. Pectinase is frequently used to describe pectic enzyme mixtures, as has been recom- mended by the 1926 Committee on Pectin Nomenclature (Lineweaver and Jansen, 1951). Pectic enzymes have been demonstrated in a great number of fungi, bacteria, yeasts, and higher plants (Phaff and Joslyn, 1947; Bell et al., 1951; Demain and Phaff, 1957; Etchells et al., 1958a; Arima et al., 1964; Rombout and Pilnik, 1972; McMillan and Shei- man, 1974; and Pressey and Avants, 1975). In 1950, Bell et al. demonstrated that curing brine possesses polygalacturonase and pectinesterase activity. Good correlation was observed between the presence of poly- galacturonase in the brine and loss of cucumber firmness. Pectinesterase activity has not been shown responsible for cucumber softening. Cellulase has been reported to be present in cucumber brine by Jansen and Seegmiller (1954) and Bell et al. (1955). Bell and associates (1955) demon- strated the correlation of pectinolytic and cellulolytic enzyme activity with firmness of cucumber salt-stock. Bell (1951) reported the presence of a polygalacturonase in various parts of the cucumber plant and fruit. Additional studies by Pressey and Avants (1975) have shown the presence of a polygalacturonase in cucumbers. This enzyme has been identified as an ex0polygalacturonase. Microbiological Studies of the Softefiing Spo11age Initial studies on cucumber softening were con- cerned with a group of bacilli, isolated from the brines of softened pickles, as the causative agents (Aderhold, 1899; Kossowicz, 1908; and Jones, 1909). The conditions which favored softening of cucumbers were reported by several investigators (Rahn, 1913; Joslyn, 1928; Lesley and Cruess, 1928). The enzymes from the aerobic bacilli group, which were capable of softening salt-stock, were studied by Faville and Fabian (1949) and Fabian and Johnson (1938). Later, Nortje and Vaughn (1953) showed that the pectinolytic enzymes of Bacillus subtilis, Bacillus mesentericus fuscus, and Bacillus pumilis had high pH optima of 8.5 and 9.4 respectively. They also demonstrated that the Bacillus subtilis enzyme was relatively unstable in cucumber brines. The nature of the acid fermentation of cucumbers presented three important obstacles to consideration of aerobic bacilli as the source of softening enzymes. These were: (1) the salt content of the fermenting brines was too high to allow growth of these organisms, (2) the relatively low pH of the fermenting brines inactivated the enzymes from these aerobic bacilli, and (3) the low oxygen tension of the brine would have very likely inhibited the growth of aerobic bacilli. These factors led to the conclusion that pectinases from bacteria were not an important factor in cucumber softening (Etchells et al., 1958a). Yeasts were also considered as a possible source of cucumber softening. Though many species of film-forming yeasts have been reported to be responsible for de- esterification of pectin (Etchells and Bell, 1950; Bell and Etchells, 1956), they were not able to hydrolyze the glyco- sidic bonds of pectic materials (Bell and Etchells, 1956). Therefore, this group of organisms could not be a signifi- cant cause of softening. Although many species of molds are known to be potential sources of pectic enzymes, their aerobic nature would not enable them to develop well under the anaerobic conditions of commercial cucumber fermentation. Fabian and Faville (1949) first reported that softening, presumably caused by Geotricum candidum, occurred during removal of salt from pickles. Bell et a1. (1950) studied the nature of the softening of commercial salt-stock. They concluded that the softening of 74 samples of commercially brined cucum- bers, from 19 brining stations located in 9 states, was the result of pectinolytic activity with a pH optimum of 4.0. Enzyme activity was found in brines with a pH range of 3.2 to 3.9, a salt content from 10% to 21% and brine acidity of 0.2% to 1.2% calculated as lactic acid. The enzymes found in salt-stock brine were similar in properties to the purified fungal polygalacturonase which was able to soften firm, cured cucumber brine-stock. ‘ Etchells and co-workers observed serious outbreaks of spoilage from softening during 1947-1948 and in 1955. They suggested that the softening enzymes in cucumber brines were mainly of fungal origin. Softening could be found in many vats of brine stock despite early, vigorous lactic acid fermentation. Etchells et al. (1955a) reported that the softening enzymes are introduced into the brine by way of partially dried, heavily mold-laden cucumber flowers that may remain attached to the cucumber fruit. It was also found that the brine from vats filled with cucumbers having a high percen- tage of flowers had high polygalacturonase activity. The salt-stock from these tanks was either soft or inferior in firmness. To reduce the softening enzyme content of brines, these investigators recommended the original brine be drained off after 36 hours and replaced with fresh brine. Etchells et al. (1958a) studied the population and softening enzyme activity of filamentous fungi on flowers, ovaries, and fruits of pickling cucumbers throughout one growing season. During the 4 to 6 week harvest season, rather high fungal p0pulations and accompanying pectinolytic enzyme activity were observed on cucumbers. This was especially true for flowers collected from fields and those removed from the fruit at the brining station. Over 1,000 cultures were isolated in the study. From 1,032 isolates, 72 species in 34 genera were identi- fied. The remaining 68 isolates were placed in 10 uniden- tified groups. Further, the five most common isolated species in five genera, Penicillium, Ascochyta, Fusarium, Cladosporium, and Alternaria, which represented 60% of the isolations, were shown to produce both pectinolytic and cellulolytic enzymes. The ten most common isolates are shown in Table 1. Since molds would not survive long in the anaerobic conditions in brine, it was concluded that most of the pectinase found in brines would have formed on the cucumbers or flowers prior to brining. Table 1.--Ten fungi most frequently isolated from flowers, ovaries, and fruits of pickling cucumbers (Etchells et al., 1958a). Species Percent of Total Isolations Penicillium oxalicum 15.7 Ascochyta cucumis 15.4 Fusarium roseum 10.3 Cladosporium cladosporioides 10.1 Alternaria tenuis 8.5 Fusarium oxysporum 5.8 Fusarium solani 5.6 Mucor salvaticus 1.8 Penicillium janthinellum 1.6 Trichoderma viride 1.6 Enzyme Activity Measurement Phaff and Joslyn (1947) listed five general methods for measuring activity of polygalacturonase on pectin solu- tions: (a) increase in reducing substances measured as aldoses; (b) decrease in calcium pectate precipitate; (c) decrease in alcohol precipitate; (d) marked drOp in viscosity of pectin solution; and (e) decrease in Optical rotation. Except for measurement of viscosity changes, all of these methods require extensive hydrolysis of pectin. Since significant viscosity changes occur with the hydroly- sis of only a few bonds in the pectin molecule, this is the only approach which has been useful for the detection of low enzyme levels in brine. Bell et a1. (1955) developed a detailed viscometric procedure for measurement of a poly- galacturonase in brine. Reduction of Softening Enzymes Several approaches directed toward eliminating or significantly reducing the concentration of softening enzymes in commercial cucumber brines have been suggested (Bell et al., 1950; Etchells et al., 1955b). These are: (a) mechanical removal of flowers from the cucumbers, (b) development of new cucumber varieties with a minimum of retained flowers, (c) development of draining procedures to reduce enzyme content of brines before it can affect cucum- ber firmness, and (d) inactivation of the enzymes in the brine with specific, non-toxic inhibitors. The draining 10 procedure has received rather thorough investigation (Etchells et al., 1955b). Brines are commonly drained 36 hours after filling the tanks in the southern brining areas. Bell and co-workers (Bell and Etchells, 1958; Bell et al., 1960; 1962; 1965 and Etchells et al., 1958b) demon- strated the inhibition of pectinase and cellulase by using grape leaves and sericea plants. Reduction of enzyme activity in the fermenting brine was directly related to the concentration of the inhibitor. The exact chemical nature of this pectinase and cellulose enzyme inhibitor has not been determined. Influence of Salt (NaCl) on Pectinolytic Enzyme ActiVity Pallman et a1. (1946) observed that a number of chloride salts activated pectinase. Maximum activity was obtained at .017% NaCl regardless of whether sodium or potassium chloride was used. In 1950, Bell et a1. studied the effect of NaCl on the viscosity of pectin and on the activity of Aspergillus niger polygalacturonase upon pectin. They reported that increasing the salt concentration above 10% raised the relative viscosity of the pectin solution and reduced the enzyme activity as measured by the rate of loss in viscosity. Bell and Etchells (1961) studied the influence of NaCl on pectinolytic softening of cucumbers. Experimental packs of pasteurized cucumbers in 0-20% NaCl concentrations were treated with fungal pectinases from three sources under controlled conditions with respect to ll temperature, pH, acidity, salt concentration, and absence of microbial development. The organisms from.which the pectinases were derived were not given. The firmness of cucumber pickles increased according to a first order reaction with increasing salt concentration. This investi- gation substantiates the belief of the pickle operators that the use of low-salt-brining procedures for cucumbers can result in more softening than the use of higher brine strength. Significance of Inactivation of the Softening Enzymes Much of the salt used in the pickling industry is discarded as waste brine. There are an estimated 60 million gallons of spent brine produced in cucumber brining, based upon current practices (Palnitkar and McFeeters, 1975). Reusing the salt would help reduce the disposal problem of the pickling industry. There are three methods for recycling spent brine: (1) submerged combustion (Durkee et al., 1973), (2) chemical (NaOH) treatment of spent brine (Geisman and Henne, 1973a,b), and (3) heat treatment (Cranfield, 1973). Normal characteristics of salt-stock were obtained with recycled brine (Durkee et al., 1974; Palnitkar and McFeeters, 1975). The chemical or heat treat- ment procedures appear to be less costly than submerged combustion. Therefore, they are most likely to be chosen for commercial application. In doing this, it is important to assure the destruction of undesirable enzymes which may 12 be present in Spent brine. Palnitkar and McFeeters (1975) reported on the heat inactivation of Aspergillus niger pectinase in simulated brine. Further information on inactivation of pectinase from organisms which are most often found on cucumbers is needed because these enzymes would, most likely, be present in spent brine. MATERIALS AND METHODS Materials Fungi that produce pectinases were obtained from the Northern Regional Research Laboratory (NRRL) and from Dr. E. S. Beneke, Department of Botany and Plant Pathology, MSU. Organisms and the source of the cultures which were used are given in Table 2. Methods Assay of Pectinase Determination of pectinase activity. The enzyme was determined by a modification of the method described by Bell et al., 1955. Oswald-Fenske viscometers (model 300A) were used. One ml of enzyme solution was added to 5 m1 of 1% pectin solution, pH 4.0, which had been equilibrated at 30°C. The enzyme sample was prepared to contain 12% NaCl, 0.6% lactic acid and 0.1% Ca++ion. Calcium was added as CaClZ-ZHZO. The pH of the enzyme solutions was varied between 3.0 and 4.5 for studies of heat denaturation. Five m1 of enzyme-pectin mixture was transferred to the visco- meter. The mixture contained 0.83% pectin, 2% NaCl, 0.1% lactic acid and 0.017% Ca++ion. The pH ranged from 3.9 to 13 14 Table 2.--Organisms that produce pectinases and their sources. Organism Source Alternaria tenuis NRRL 2169 Cladosporium cladosporioides MSU Fusarium oxysporum NRRL 1943 Fusarium roseum MSU Fusarium solani NRRL 3078 Paecilomyces gp. MSU Penicillium digitatum MSU Penicillium frequentans MSU Penicillium janthinellum NRRL 2016 Penicillium oxalicum NRRL 790 Trichoderma viride MSU 15 4.1. After 10 minutes of reaction at 30°C, the flow time of the sample was recorded. Ten minutes was chosen for the standard reaction time because it provided good discrimina- tion of enzyme concentrations over the ranges that were studied. Preparation of pectin solutions. Citrus pectin (Sigma Chem. Co.) was dissolved in deionized water con- taining sodium lactate as buffer. The final volume contained 1% pectin in 0.15 M sodium lactate. The solution was adjusted to pH 4.0 with 2N HCl after dissolving the pectin. The blank sample was prepared to contain 12% NaCl, 0.6% lactic acid, and 0.1% Ca++ion. The flow time was measured in the same way as the sample with the enzyme. Due to the gradual decrease of viscosity of the pectin solution at pH 4.0, the viscosity of the substrate was checked daily and a factor was calculated to correct for the change in viscosity. The flow time for each enzyme sample was then adjusted by this factor. Calculation of this adjustment factor is shown below. Example of Correction: Blank reading of the reference sample: 56.0 seconds/ 10 min. Blank reading on a subsequent day: 54.6 seconds/ 10 min. Reading for an enzyme sample: 20.0 seconds/ 10 min. Therefore, correct reading is: 56 x 20.0=20.5 seconds 54 10 m1n. 16 A standard curve for the determination of enzyme concentration was constructed for a pectinase concentrate from each organism. The standard curve is the correlation between percent viscosity loss and relative units of enzyme. One unit of enzyme activity was that amount of activity in 0.1 ml of enzyme solution for each organism. Calculation of pectinase activity units. The flow time in seconds for blank sample was recorded (A) and for each enzyme-pectin sample (B) at 10 minutes of reaction. The average flow time of water (W) in the viscometer was 5.2 seconds, so this is applied in the equation and calcu- lated as follows: % loss 1n v1scos1ty at A _ B x 100 10 minutes of reaction = —_:7W Example of Calculation: Blank sample (A) = 58.3 seconds Enzyme sample (B) 21.8 seconds 68.7% loss in viscosity Therefore, 58.3-21.8 58.3-5.2 Protein determination. The Biuret B method which can determine the protein content in the range of 0.25 to 2.0 mg was employed (Chaykin, 1966). In this method, 1.5 m1 of Biuret reagent was mixed with 1.5 m1 of protein sample. In cases where the protein concentrations were less than 200 ug/ml, the Lowry method was employed (Lowry et al., 1951). 17 Preparation of Pectinase Each species of fungi was cultivated in the mineral broth media described by White and Downing (1951). The medium was prepared as follows: Stock reagents ml used/ liter of medium 10% NH4NO3 10‘ 10% MgSO4 3 1% Yeast extract (Difco Lab.) 10 1 M KH2P04 10 Pectin (Sigma) 5 g The pH was adjusted to 6.4 with 4 N NaOH before sterilizing at 121°C for 15 minutes. One liter of medium was placed in a Fernbach flask with a capacity of 3 liters using cotton as a stopper. After cooling to room tempera- ture, the medium was inoculated and placed on a rotary shaker for aeration of the culture. The shaking rate was 110 rpm. The culture was grown at room temperature (21° to 24°C) until sporulation. The activity of the enzyme was measured daily until no increase in activity occurred for 2 to 3 days. Generally, the cultures with moderate growth took about 14 days for maximum enzyme production (i.e., Penicillium janthinellum, Trichoderma viride). The cultures which sporulated faster, such as Penicillium oxalicum and Fusarium oxysporum, took approximately 10 days for maximum enzyme production. The time required for each organism is 18 shown in Table 3. The cell-free liquid was separated by centrifugation at 12,000 x g using a Sorval SS-3 centrifuge. The supernatant was vacuum filtered through a millipore membrane with a pore diameter of 0.45 u to remove any cells that remained after centrifugation. A concentration step was accomplished with an Amicon model 402 ultrafiltration cell. An Amicon UM-10 membrane with a MW cut-off of 10,000 was used. The cell was pressurized with nitrogen gas at 20 psi. The fold con- centration depended upon the activity of the enzyme. Each enzyme solution was concentrated until the activity was approximately equivalent to 1 mg/ml of Aspergillus niger pectinase (pectinol 10 M 6-5831, Rohm and Haas lot 3-2029). The enzyme solutions from Penicillium oxalicum and Penicil— lium janthinellum were concentrated approximately 2 and 100 fold respectively to obtain this level of activity. For Penicillium janthinellum pectinase, one unit of enzyme activity caused a 33% loss in the initial viscosity of a 1% pectin solution in 10 minutes under the reaction conditions used. The relationship between enzyme concentration and viscosity loss is non-linear. Therefore, a standard curve such as that shown in Figure l is required to accurately determine enzyme concentration. Heat Inactivation of Pectinases Preparation of brines. Stock brine solutions were prepared containing 18% w/v NaCl, 0.9% w/v lactic acid and 19 0.15% Ca++ion (Ca++ion_was added as CaCl ~H20) and adjusted 2 to the appropriate pH values with NaOH.' The pH meter used was Radiometer model PHM4d. Preparation of enzyme samples. One volume of enzyme concentrate was mixed with two volumes of stock- brine solution. Thus, the enzyme sample contained 12% w/v NaCl, 0.6% lactic acid, and 0.1% Ca++ion at the time of heating. These conditions were chosen to approximate the composition of commercial spent brines (Palnitkar and McFeeters, 1975). One-half ml of the enzyme sample was transferred into a 6 x 50 mm tube (Fisher Co.) and heated in a water bath. Samples were removed from the water bath at appro- priate time intervals and were quickly cooled in an ice bath. In general, measurements of pectinase activity were done within 3 hours after heating. However, it was found that holding time at 4°C for 24 hours caused no change in measurement of activity. In order to have sufficient enzyme for activity measurement, three identical tubes were heated and cooled at the same time. The heated enzyme from the three tubes was mixed and assayed for activity with the standard pectinase assay. After a preliminary screening, the pectinases most stable to heat inactivation were inves- tigated more thoroughly and the D-values were determined. Determination of decimal reduction time (D-values). From the heat inactivation data obtained, the logarithm of 20 the percent of the enzyme activity remaining was plotted vs. time of heating (Whitaker, 1972). The SIOpe of the straight line portion of the curve and the correlation coefficient were calculated with a linear regression program. The D-value is equal to the reciprocal of the lepe. The cor- relation coefficient obtained from each inactivation curve was greater than 0.9900 for Penicillium janthinellum pectinase and greater than 0.9800 for Trichoderma viride pectinase. Effect of pH on Heat Inactivation of Pectinases Aliquots of the enzyme sample were mixed with stock brine solutions of appropriate pH values. The pH of the enzyme-brine mixtures ranged from 3.0 to 4.5. Each sample contained 12% NaCl, 0.6% lactic acid, and 0.1% Ca++ion. Aliquots at each pH were heated in 6 x 50 mm tubes for apprOpriate time intervals and quickly cooled in an ice bath. The activity of both heated and unheated enzyme solutions was determined. The temperatures employed were 75° and 78°C. The D-value was calculated for heat inacti- vation at each pH and temperature. Effect of NaCl on Heat Inactivation of Pectinase The stock brine solutions were prepared to contain 12%, 18%, and 24% NaCl with 0.9% lactic acid and 0.15% Ca++ion, pH 3.5, and pH 3.7. ‘One voluem of the pectinase concentrate from Penicillium janthinellum was mixed with 21 two volumes of the stock brine. The mixture contained 8%, 12%, and 16% NaCl with 0.6% lactic acid and 0.1% Ca++ion respectively. D—values were determined at 75°C. Purification of Pectinase by Sephadex Chromatography G-100 Sephadex gel, with a fractionation range of 4,000 to 150,000 MW, was packed in a 2.5 x 57.5 cm column and equilibrated with 12% brine solution, pH 4.0. Five ml . of Pencillium janthinellum pectinase concentrate was mixed with 2.5 9 sucrose to increase the density. After mixing well, the enzyme solution was applied to the column and eluted with the equilibrium brine. The flow rate was main- tained at 40 ml/hr with an Isco pump. Five ml fractions were collected and assayed for enzyme activity and protein content. The fractions which contained significant enzyme activity were collected and concentrated on a UM-10 mem- brane to have approximately 10 units of enzyme activity per milliliter of concentrate. D-values were determined at 75° for each of the peaks from the column which had pectinase activity. Effect of Protein Content on Heat Inactivation of Pectinase The partially purified pectinase with the greatest activity (peak II in Figure 5) was studied for the effect of protein in the system on resistance to heat of the enzyme. One mg/ml of BSA (Bovine Serum Albumin) and 22 3 mg/ml BSA were added to the enzyme solutions at pH 4.0 and assayed for heat inactivation at 75°C. Mode of Action of Penicillium JanthinelIfim Pectinase The partially purified enzyme from peak II in Figure 5 was concentrated by ultrafiltration with a UM-lO membrane. Comparison of the change in viscosity of a polymeric substrate with the rate of reducing sugar forma- _ tion was used to evaluate the mode of enzymatic hydrolysis. The viscosity loss was measured in an Oswald-Fenske visco- meter using 1% pectin solution as substrate. The reducing sugar released was assayed by using 3, 5—dinitrosalicylic acid reagent (DNS) (Miller, 1959). The amount of reducing sugar was measured at ten minute intervals. The concentra- tion of reducing sugar was determined by using glucuronic acid as the standard. One ml of sample was mixed with 2 m1 of DNS and heated in a boiling water bath for five minutes. Absorbance measurements were made at 540 nm. Samples with high reducing sugar levels were diluted prior to addition of DNS reagent in order to be within the range of the standard curve (60-176 pg glucuronic acid). Heat Inactivation of Penicillium JanthineIIum Pectinase in Spent Brine Pasteurized commercial spent brine which contained 12.7% NaCl, pH 3.5 was used in this study. Ten ml of 23 enzyme concentrate was transferred into a dialysis membrane with an exclusion limit of 6,000—8,000 MW. The enzyme was placed in 150 ml of spent brine with agitation outside the membrane. The dialysis was done at 0°C for 12 hours. D-values of the dialyzed enzyme solutions at pH 3.5 and pH 3.7 were determined at 75°C. RESULTS The fungi most commonly found on cucumber flowers, ovaries, and fruits by Etchells et al. (1958a) were grown on mineral broth media with pectin as the carbon source. The organisms produced varied considerably in the time for maximum enzyme production and in the amount produced. Table 3 shows the time in days required for maximum enzyme production for each species investigated. The organisms are listed in order of decreasing amounts of enzyme produced. Fusarium oxysporum, which was sixth in terms of the number of isolations from cucumber flowers and fruits, produced the highest enzyme concentration under the culture condition used. Fusarium oxysporum and Penicillium oxalicum pectinases were concentrated about two fold to obtain a solution of sufficient activity for heat inactivation studies. The enzyme from the other organisms were concen- trated from 20 to 50 fold to obtain a similar level of activity. A different standard curve was required for each organism in order to accurately measure enzyme activity. Figure 1 shows the standard curves for Penicillium jan- thinellum and Trichoderma viride pectinases. 24 25 Table 3.--Cu1tivation time required for maximum enzyme production for each species investigated. (The organisms are listed in order of decreasing amounts of pectinase produced.) Organism Days of Cultivation Fusarium oxysporum 9 Penicillium oxalicum 10 Alternaria tenuis 13 Trichoderma viride l4 Paecilomyces sp. 13 Penicillium janthinellum 15 Fusarium roseum 7 Fusarium solani . 20 Cladosporium cladosporioides 10 Penicillium digitatum 18 Penicillium frequentans 15 26 Protein Content of Pectinase Solutions Table 4 shows the protein concentrations of the crude pectinase concentrates determined by the Biuret method. The crude pectinase concentrate from Penicillium janthinellum was found to have the highest protein content among the organisms investigated. The enzyme concentrates from Penicillium oxalicum and Cladosporium cladosporioides have the lowest protein contents. Table 4.--Protein content of fungal pectinase concentrates. Organism mg Protein/m1 Enzyme Solution Alternaria tenuis 2.0 Cladosporium cladosporioides 0.7 Fusarium oxysporum 1.4 Fusarium roseum 4.3 Fusarium solani 2.1 Paecilomyces 32' 4.7 Penicillium digitatum 2.5 Penicillium janthinellum 5.5 Penicillium oxalicum 0.5 Trichoderma viride 1.7 Preliminary Studies on Heat Inactivation of Pectinases The pH of each pectinase-brine mixture was 3.3 during the time of heat inactivation in preliminary experi- ments. The enzyme was equilibrated in 12% NaCl, 0.6% lactic acid, and 0.1% Ca++ion for 8 to 12 hours. The results of 27 preliminary screening tests for relative heat stability of pectinases are shown in Table 5. Penicillium janthinellum pectinase was the most heat stable of those studied. Penicillium oxalicum pectinase was somewhat less stable. The enzymes from Trichoderma viride, Fusarium oxysporum, Alternaria tenuis, and Fusarium solani were intermediate in stability. The remaining organisms were relatively unstable to heat. The six pectinases which were most resistant to heat were used for further heat inactivation studies. Heat Inactivation Studies After temperature equilibrium occurs, the loss of enzyme activity is first order for each pectinase. Figure 4 shows the heat inactivation curves for Penicillium janthinellum pectinase at 75°C. The D-values were calcu- lated from the slopes of the straight line portions of heat inactivation data. Table 6 shows the D-values of the heat stable pectinases at several temperatures. Penicillium janthinellum pectinase was again found to be the most stable of the enzymes investigated. The D-value was very sensitive to temperature changes. An increase from 75°C to 78°C resulted in over a four-fold decrease in the D—value. Effect of pH Figure 2 shows the effect of pH on heat inactivation of Penicillium janthinellum pectinase at 75° and 78°C. The heat stability of the enzyme was shown to be significantly influenced by both pH and temperature. By increasing the 28 Table 5. --Preliminary heat inactivation tests of pectinases from fungi frequently found on pickling cucumbers. Time for Organism Temperature Inactivation Activity (°C) (min.) (+/') Penicillium janthinellum 70 2 + 80 1 - Penicillium oxalicum 7O 2 + 70 3 - Trichoderma viride 60 2 + 70 l - Fusarium oxysporum 60 2 + 70 1 - Alternaria tenuis 60 2 + 70 1 ' Fusarium solani 6O 2 + 70 l - Cladosporium cladosporioides 6O 1 - Fusarium roseum 60 1 - Paecilomyces s2. 60 l - Penicillium frequentans 60 1 - Penicillium digitatum 60 1 - 29 Table 6.--D-values of pectinases with the highest thermal stability. (The D-values were obtained at pH 3.3, 12% NaCl, 0.6% lactic acid, and 0.1% Ca++ion.) Temperature D-value Organism (°C) (sec.) Penicillium janthinellum 78 4 75 18 Penicillium oxalicum 70 18 Fusarium solani 70 5 Fusarium oxysporum 70 4 Alternaria tenuis 65 25 Trichoderma viride 65 10 temperature 3°C, the D-value of the pectinase was decreased from 33 seconds to 8 seconds at.pH 3.7. The influence of pH on the stability of the pectinase was greater at 75°C than at the higher temperatures. The enzyme was the most stable to heat in the pH range of 3.5 to 3.7 at 75°C, and 3.5 to 4.5 at 78°C, respectively. Figure 3 shows the effect of pH on heat inactivation of Trichoderma viride pectinase. The enzyme was shown to be the most stable to heat at a pH range of 3.9 to 4.5. The greatest heat stability of pectinase from Penicillium janthinellum was at a slightly lower pH than that of Trichoderma viride pectinase. Effect of NaCl Concentrations Table 7 shows the effect of NaCl concentration on heat inactivation of Penicillium janthinellum pectinase. 30 Table 7.--Effect of NaCl concentration on heat inactivation of pectinase from Penincillium janthinellum at 75°C. (The enzyme solutions contained 0.6% lactic acid, 0.1% Ca++ion with variable NaCl concentrations.) D-values at 75°C % N°C1 H 3.5 H 3.7 (sec.) isec.) 8 26 31 12 29 33 16 30 37 At 16% NaCl concentration the D-values were greater than those with lower NaCl concentrations. The results were the same at both pH 3.5 and pH 3.7. When this enzyme was present in 16% brine solution, pH 3.5 and pH 3.7, the D-values at 80°C were 9 and 13 seconds respectively, while the D-values of this enzyme in 12% brine at both pH were 6 and 9 seconds at 78°C respectively. Increases in tempera- ture had less effect on inactivation of the enzyme in higher than in lower salt concentrations. Purification and Heat Inactivation of Pectinase The pectinase from Penicillium janthinellum was partially purified by a Sephadex G-100 column which was equilibrated with the brine solution containing 12% NaCl, 0.6% lactic acid, and 0.1% Ca++ion, pH 4.0. The protein content of each fraction was determined by the Lowry 31 method. The elution profile of the enzyme from the Sepha- dex G-100 column is shown in Figure 5. The two peaks of pectinases obtained by the Sepha- dex G-100 chromatOgraphy were different in concentration. The first peak was present in the fraction number 19-30 and had a lower concentration. The one with the highest con- centration was in fractions 30-43. After concentration, the heat inactivation of both purified pectinases was done. The enzyme from peak II was found to be more stable to heat than the enzyme from peak I. The D-values at 75°C and pH 4.0 were 15 seconds and 7 seconds respectively. The D-value for the crude enzyme was 19 seconds under these conditions. This indicated that the purified pectinase was slightly less stable to,heat than the crude enzyme. Effect of Protein Content on Heat Inactivation of Pectinase from PenicilIium Janthinéllum The heat stable pectinase obtained by the Sephadex G-100 chromatography was used to determine the effect of protein content on heat inactivation. Addition of BSA significantly increased the heat stability of the pectinase. Results are shown in Table 8. When the protein content in the system was increased with 1 mg/ml BSA and 3 mg/ml BSA, the D-values of the purified pectinase were increased by 7 seconds and 14 seconds, respectively. 32 Table 8.--The D-values of purified pectinase at 75°C. (The enzyme solutions contained 12% NaCl, 0.6% lactic acid and 0.1% Ca++ion, pH 4.0, with variable pro- tein concentrations.) - . Calculated D-values Sample mg/ml BSA add1t1on at 75°C (sec.) 1 o1 15 2 1 22 3 3 30 1Protein content was 0.78 mg/ml by the Lowry method. Mode of Action of Penicillium I ’Janthinéllum Pectinase Figure 6 illustrates the results of the comparison between the changes in viscosity of a polymeric substrate and the rate of chain cleavage. Such a comparison is use- ful in identifying whether the pectinase is an endo- or exo-splitting enzyme. The reaction mixture, containing 0.83% pectin, 1 unit/m1 of purified pectinase, 2% NaCl, 0.1% lactic acid, and 0.016% Ca++ion was assayed for vis- cosity loss concurrently with the measurement of reducing groups released. During the first 30 minutes of reaction, there was a marked decrease in viscosity, after which time reduction of the viscosity was very slow. Simultaneously, the reducing sugar was slightly increased. At the time when the viscosity stopped decreasing at a rapid rate (after 2 1/2 hours of reaction), the viscosity was reduced by 84% while only 3% of the glycosidic bonds were 33 hydrolyzed.s This comparison indicated an endocleavage pattern by the enzyme. Therefore, Penicillium janthinellum pectinase is an endopolygalacturonase as defined by Whit- aker (1972). Heat Inactivation of Penicillium Janthinellum Pectinase in Spentggrine The crude enzyme concentrate from Penicillium janthinellum was dialyzed with spent brine to contain 12.56% NaCl, pH 3.5 and pH 3.7 respectively. Heat inacti- vation of both enzyme solutions was done at 75°C. The calculated D-values were 14 seconds and 18 seconds at pH 3.5 and pH 3.7 respectively. The D-values in simulated brines were 29 and 33 seconds at pH 3.5 and pH 3.7 respec- tively. Therefore, pectinase from Penicillium janthinellum was less stable in spent brine than in the simulated brines. 34 100 l 80 . do / u; so .. U) O H >1 4.) --.-l 0) o . 8 “H 40 I- > 20 F o - ' . . . J o l 2 3 4 5 Enzyme concentration, units Figure 1. Standard curves for pectin hydrolysis of pectinases from Penicillium janthinellum (O-—O) and Trichoderma v1r1de (O—-O) at pH 4.0. seconds D-value, 4O 30 20 10 35 pH Figure 2. Effect of pH on heat inactivation of Penicillium janthinellum pectinase. Temperature75'C (0—0) and 78°C (O—O). seconds D-value, 30 20 10 36 0i. 1 1 I n 3.0 3.5 4.0 4.5 5.0 pH Figure 3. Effect of pH on heat inactivation of Trichoderma viride pectinase at 65°C. - log % initial enzyme remaining 37 A. 1 . 0 ' 10 1. .A 0 . 5 . 0 136 W 3'0 4'0 5‘0 Heating time, seconds Figure 4. Heat inactivation curves of Penicillium janthinellum pectinase without protein addition (i:-I), 1 mg/ml BSA addition (A-—A), and 3 mg/ml BSA addition (O-O). Unit Activity, 38 20 - 15 - 10 h -1 O 5F “0.5 0 .1 °..; I 0 10 20 30 40 Tube No. Figure 5. Sephadex G-100 chromatography of pectinase from Penicillium janthinellum. ActiVity (o-o), Frotein’Yi-—o). Enzyme Peak I, fraction no. 19730; Peak II, fraction no. 30-44. 'ureqoxd OW % or Reduction in Viscosity, Hydrolys1s, % 100 50 39 Figure 6. 100 200 300 Reaction Time, minutes Hydrolytic action of purified pectinase determined by viscosity assay (O——O) and reducing sugar assay (O-—.). DISCUSSION The purpose of this project was to determine whether the commercial pasteurization treatment at 175° (79.4°C) for 30 seconds is adequate to inactivate pecti— nases which are most likely to occur in spent brines. It is known that pectinases from filamentous fungi cause cucumber softening. However, the Specific species which have been the cause of commercial softening have not been identified. Therefore, the selection of pectinases for this study was based upon the investigation of Etchells et al. (1958a) in which the relative populations of fungi on cucumber fruits and flowers were surveyed. The p0pulations of organisms were determined by grinding samples of flowers or shaking fruit in saline solution, then counting colonies on apprOpriate agar plates. Differences in the amount of breakage of hyphae from different species can distort the absolute and relative p0pulations of these organisms in a particular sample. Therefore, this survey may not provide a completely accurate picture of the organisms present. In addition, the relative populations of organisms may vary significantly in different growing regions and in different seasons. 40 41 Ten of the most common fungal species found by Etchells et al. (1958a) are listed in Table 1. Based upon the pectinase activity produced by the five most common species and the fact that they accounted for most of the total population in field and brining station flowers, Etchells et al. (1958a) concluded that most of the pectinase activity found on flower samples was probably produced by these five organisms. However, identification of specific species as the cause of certain commercial softening out- breaks was not done. In this study, each of the species in Table 1 except Mucor salvaticus and Ascochyta cucumis was grown in a mineral medium with pectin as the carbon source. After concentration of the culture filtrate, pectinase from each organism was evaluated for its heat stability under condi- tions of salt, acid, and pH similar to those which are. likely to occur in commerical spent brine. In commercial brining of cucumbers for salt-stock pickles, the final NaCl concentration is usually in the range of 10 to 16%, the protein content of the brine is approximately 0.6 mg/ml and the final pH varies from 3.3 to 3.5 according to acid pro- duction (Palnitkar and McFeeters, 1975; McFeeters et al., unpublished). These factors, along with the concentration of unidentified brine components, may affect the stability of the undesirable pectinase. Of the organisms investi- gated, the enzyme produced by Penicillium janthinellum was 42 found to have the greatest heat stability (Table 6). There- fore, if a process is sufficient to inactivate this enzyme, it should also be adequate to inactivate the other less— stable enzymes. Consequently, the studies of the effect of various factors on heat inactivation of pectinases were pri- marily concerned with the Penicillium janthinellum pectinase. Effect of Various Factors on Heat Stability of Pectinase The heat stability of an enzyme is a function not only of termperature but also of pH, salt concentration, concentration of the enzyme and other proteins in the system. The pH was shown to have a great effect on the heat inactivation of Penicillium janthinellum pectinase (Figure 3). The pH of the enzyme-brine mixtures during heat inactivation was varied from pH 3.0 to pH 4.5 to cover the pH range of spent brine. The enzyme was most stable in the range of pH 3.5 to pH 3.7. The average pH of commercial spent brine sampled in the Spring from a Michigan processing plant was 3.5 (McFeeters et al., unpublished). Therefore, the probably pH at the time of heat treatment would be in the range of greatest thermostability of the pectinase. When the temperature was increased by 3°C the stability of the pectinase decreased approximately four-fold at pH 3.7. This Shows that if the undesirable enzyme is to be destroyed, time and temperature of heat inactivation must be strictly controlled. 43 Comparisons of the two curves obtained by heat inactivation of pectinases at 75° and 78°C (Figure 2) Shows that the changes in pH had more of an effect on the stability of pectinase at lower temperatures than at higher temperatures. Therefore, the pH of brine will have a greater influence on the treatment time if temperature below 78°C are used. Figures 2 and 3 Show the pH profile curves of Penicillium janthinellum and Trichoderma viride, respec- tively. In comparison, the effect of pH on heat inactiva- tion of both enzymes was quite different. The greatest heat stability of Trichoderma viride was at a Slightly higher pH range than that of Penicillium janthinellum and produced a broader curve. The effect of salt concentration on enzyme stability was also studied. The pectinase solutions contained 8% to 16% of NaCl which covers the range of salt concentrations most likely to be found in spent brines. The enzyme solu- tions were heated while the other factors were controlled. The stability of the pectinase was shown to be influenced by the addition of various concentrations of NaCl (Table 7). The greatest-D-values were obtained at the highest NaCl concentration at both 75° and 80°C. The changes in tempera— tures had less effect on the stability of the pectinase in higher NaCl concentrations than in lower NaCl concentra- tions. The D-value of 16% NaCl was decreased approximately three-fold by raising the temperature 5°C while at 12% 44 NaCl, the D-value was decreased approximately four-fold by raising the temperature 3°C. Although the pectinase was Shown to be the least heat stable at 8% NaCl, the final salt concentration of commercial spent brine is not likely to be found at this level. Bell et a1. (1950) reported a reduction in pecti- nase activity, as measured by viscosity loss, when the salt content of brine was increased. Bell and Etchells (1961) also reported decreased cucumber softening as the salt content of brines was increased from 10% to 20%. The pre- sent data Show increased heat stability of Penicillium janthinellum pectinase as salt is increased from 8% to 16%. It may be that high salt levels result in a decrease in the rate at which polygalacturonases hydrolyze bonds while at the same time stabilizing the structure of the enzymes. Since pectinases from different organisms were used in these investigations, further work is required to determine whether the reduction in activity observed by Bell and co-workers is a result of enzyme denaturation or Simply a reduction in the rate of hydrolysis as suggested by the present results. In general, an enzyme is more stable to temperature in a homogenate where its structure is protected by the presence of other colloidal material (protein, carbohydrate, pectin, etc.) than it is in a purified form (Whitaker, 1972).‘ BSA was used to increase the protein concentrations of the purified pectinase from Penicillium janthinellum. 45 The results in Table 8 Showed that protein had a protective effect on heat denaturation of the enzyme. An increase in the D-value by 7 seconds occurred with the addition of 1 mg/ml BSA. When 3 mg/ml BSA was added, the D-value was increased by an additional 7 seconds. During heat inacti— vation, the protein content of the crude Penicillium janthinellum enzyme solution (1.8 mg/ml) was greater than that of the purified pectinase concentrate (0.8 mg/ml). At 75°C, the D-value of the crude enzyme was greater than that of the purified enzyme by 3 seconds at pH 4.0. Protein concentration of the pectinase concentrate from different organisms was also determined. In Table 4, the enzyme concentrate from Penicillium janthinellum, which was the most heat stable pectinase, also contained the highest protein content. Penicillium oxalicum enzyme con- centrate, the second most heat stable pectinase, had the lowest protein content. Fusarium roseum enzyme concentrate, which had relatively high protein content, was shown to be much less stable to heat than either Penicillium janthinel- lum or Penicillium oxalicum pectinase. There appears to be no correlation between the thermostability of pectinases from different organisms and the protein concentration of the concentrates. Therefore, the differences in measured thermostability are primarily a result of differences in the molecular structure of the enzymes rather than in the protein concentration of the enzyme preparations. 46 Mode of Action of Purified Penicillium Janthinellum Pectinase The rate of viscosity reduction of the pectin solu- tion by the Penicillium janthinellum pectinase action had two major stages. There was a marked decrease in viscosity in the first stage with only small changes in viscosity after 2 1/2 hours of reaction (Figure 6). Simultaneously, the extent of hydrolysis, which was determined by reducing sugar formation, increased in a linear fashion. Several methods are available to distinguish between the exo- and endo- Splitting enzymes. (One of the most useful methods is to compare the rate of decrease in viscosity with rate of hydrolysis as measured by increase in reducing groups. An endOpolygalacturonase causes approximately 50% reduction in viscosity when only 3 to 5% of the glycosidic bonds have been hydrolyzed, while with an exopolygalacturonase approximately 10 to 15% hydrolysis of glycosidic bonds are needed to produce a 50% reduction in viscosity (Whitaker, 1972). From Figure 6, 3% of the glycosidic bonds were hydrolyzed while the viscosity loss was 84%, using Penicillium janthinellum pectinase.' It was concluded that Penicillium janthinellum pectinase was an endopolygalacturonase. 47 Implications of Heat Stability Of Pectinase fOr’COmmercial Cucumber Spent Brine Pasteurization Palnitkar and McFeeters (1975) reported that the D-valueS of Aspergillus niger pectinase at pH 3.6 were 34 seconds, 23.2 seconds, and 11.6 seconds at 80°, 86°, and 93°C respectively. Pectinase from Penicillium janthinellum, which was the most heat stable of those studied, had the greatest D—value (8 seconds at 78°C, pH 3.7). From this study of the effect of temperature on heat inactivation, the D-value of the enzyme was reduced an estimated four-fold by raising the temperature 3°C. Commercial pasteurization at 79.4°C for 30 seconds will give at least 3.7D or will decrease the undesirable pectinase by approximately 99.99%. In addition, the heat inactivation of the same pectinase in Spent brine showed that the enzyme was less stable in Spent brine than in simulated brine. The D-valueS of pectinase in the two brines differed by 16 seconds at the same pH. The commercial pasteurization of spent brine at 79.4°C (175°F) for 30 seconds might be more than required to destroy the undesirable enzyme which may be present in the spent brine. CONCLUSION Pectinase from Penicillium janthinellum was Shown to be the most stable to heat among the organisms investi— gated. Studies of the various factors which affect heat stability of this enzyme were made and results were observed: 1. A decrease in temperature of 3°C increased the D—value of the enzyme by a maximum of 4 times at the same condition. 2. pH significantly affected the heat stability of the enzyme. At pH 3.7, the enzyme Showed the greatest heat stability. -3. The heat stability of this enzyme increased with increasing NaCl concentrations and protein content of the system. 4. The enzyme was shown to be less stable in commercial Spent brine than in simulated brine. 5. The enzyme which was partialy purified by Sephadex G-100 chromatography showed two polygalacturonases with different molecular weights and relative activities. 6. The purified pectinase with the greatest activity was identified as an end0polygalacturonase. 48 49 The greatest D—value of the most heat stable pectinase was 8 seconds at 78°C. The commercial pasteuri- zation of spent brine at 79.4°C (175°F) for 30 seconds will give at least a 3.7 D process. This treatment is sufficient to assure the destruction of the enzymes studied if they were to occur in spent brines. BIBLIOGRAPHY BIBLIOGRAPHY Aderhold, R. 1899. Untersuchung fiber daS Einsafiren von Fruchten und Gemfisen. I. Das Einsafiren Gurken. Zentr. Bakteriol. Parasitenk. Abt. II, 5, 511-14; Landwirtsch Jahrb., 28, 69-131. Arima, K.; Yamasaki, M.; and Yasui, T. 1964. Studies on pectic enzymes of microorganisms, Part 1: Isola- tion microorganisms which Specifically produce one of several pectic enzymes. Agr. Biol. Chem., 28, 248-54. Bell, T. A. 1951. Pectinolytic enzyme activity in various parts of the cucumber plant and fruit. Bot. Gaz., 113, 216-21. Bell, T. A.; Aurand, L. W.; and Etchells, J. L. 1960. Cellulase inhibitor in grape leaves. Botan. Gaz., 122, 143-48. Bell, T. A., and Etchells, J. L. 1956. Pectin hydrolysis by certain salt tolerant yeasts. Appl. Microbiol., 4, 196-201. Bell, T. A., and Etchells, J. L. 1958. Pectinase inhibi- tor in grape leaves. Botan. Gaz., 119, 192-96. Bell, T. A., and Etchells, J. L. 1961. Influence of salt (NaCl) on pectinolytic softening of cucumbers. J. Food Sci., 26, 84-90. Bell, T. A.; Etchells, J. L.; and Jones I. D. 1950. Softening of commercial salt-stock in relation to polygalacturonase activity. Food Technol., 4, 157—63. Bell, T. A., Etchells, J. L., and Jones I. D. 1951. Pectinesterase in the cucumbers. Arch. Biochem. BiOphys., 31, 431-41. 50 51 Bell, T. A.; Etchells, J. L.; and Jones, I. D. 1955. A method for testing cucumber salt—stock brine for softening activity. U.S. Dept. Agr., ARS 72-5, pp. 1-15. Bell, T. A.; Etchells, J. L.; Singleton, J. A.; and Smart, W. W. G., Jr. 1965. Inhibition of pectinolytic and cellulolytic enzymes in cucumber fermentations by sericea. J. Food Sci., 39, 233-39. Bell, T. A.; Etchells, J. L.; Williams, C. F.; and Porter, W. L. 1962. Inhibition of pectinase and cellulase by certain plants. Bot. Gaz., 123, 220-23. Chaykin, S. 1966. Biochemistry Laboratory Techniques. John Wiley and Sons, Inc., N.Y., p. 17. Cranfield, D. 1973. Cucumber brining and salt recovery. Pickle Packer's International Seminar on Pickle Processing Wastes. Demain, A. R., and Phaff, H. J. 1957. Softening of cucum- bers during curing. J. Agri. and Food Chem., 3 (1), 60-64. Durkee, E. L.; Lowe, E.; Baker, K. A.; and Burgess, J. W. 1973. Field tests of salt recovery system for Spent brine. J. Food Sci., 33, 507-11. Durkee, E. L.; Lowe, E.; and Toocheck, E. A. 1974. ’Use of recycled salt in fermentation cucumber salt-stock. J. Food Sci., 33, 1032-33. Etchells, J. L., and Bell, T. A. 1950. Classification of yeast from commercially brined cucumber. Farlowia, .4, 87-112. Etchells, J. L.; Bell, T. A.; and Jones, I. D. 1955a. Studies on the origin of pectinolytic and cellulotic enzymes in commercial cucumber fermentation (Abs.). Food Technol., 3 (3), 14, 16. Etchells, J. L.; Bell, T. A.; and Jones, I. D. 1955b. Cucumber blossoms in salt-stock mean soft pickles. Quart. Public., Research and Farming, N.C. Agri. Expt. Sta., 33 (1-4), 14. Etchells, J. L.; Bell, T. A.; Monroe, R. J.; Masley, P. M.; and Demain, A. L. 1958a. Populations and softening enzyme activity of filamentous fungi on flowers, ovaries and fruit of pickling cucumbers. Appl. Microbiol., 3, 427-40. 52 Etchells, J. L.; Bell, T. A.; and Williams, C. F. 1958b. Inhibition of pectinolytic and cellulytic enzymes in cucumber fermentations by Scuppernong grape leaves. Food Technol., 33, 204-08. Fabian, F. W.; Bryan, C. S.; and Etchells, J. L. 1932. Experimental work on cucumbers. IV. Morphological studies on spoiled cucumber pickles. Mich. State Coll. Agr. Exp. Sta. Tech. Bull., 126, pp. 1-60. Fabian, F. W., and Faville, L. W. 1949. Isolation and identification of a mold, Oospora lactis, as the causative agent. Fruit Products J., 28, 10, 297-98. "’ Fabian, F. W., and Johnson, E. A. 1938. Experimental work on cucumber fermentation. Mich. State Coll. Agr. Exp. Sta. 157, pp. 1-31. Faville, L. W., and Fabian F. W. 1949. The influence of bacteriophage, antibiotics and Eh on the lactic fermentation of cucumbers. Mich. State Coll. Agr. Exp. Sta. Tech. Bull. No. 217, pp. 1-42. ' Geisman, J. R., and Henne, R. E. 1973a. Recycling food brine eliminates pollution. Food Engr., 33 (11), 119-21. Geisman, J. R., and Henne, R. E. 1973b. Recycling brine from pickling. Ohio report, 33, 76-80. Jansen, E. F., and Seegmiller, C. G. 1954. Personal communication. Jones, L. R. 1909. Pectinase, the cytolytic enzyme pro- duced by Bacillus carotovorous and certain other soft rot organisms. N.Y. Agr. Exp. Sta. (Geneva) Tech. Bull., 11, 289-368. Joslyn, M. A. 1928. Some observations on the softening of dill pickles. Fruit Products J., g (8): 19; ‘3 (9): l6. Kossowicz, A. 1908. Bacteriolgische Untersuchungen fiber das Weichwerden eingesauerter. Gurken Z. Land- wirtsch. Versuch. Deut. Oesterr., 33, 894-900. Lesley, B. E., and Cruess, W. V. 1928. The effect of acidity on the softening of dill pickles. Fruit Products J. and American Vinegar Industries, 1, (10), 12. 53 Lineweaver, H., and Jansen, E. F. 1951. Pectic enzymes. "Advances in Enzymology." Vol. II. Nord, F. F. (ed.), John Wiley and Sons, N.Y. pp. 267-93. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; and Randall, R. J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265-75. Macmillan, J. D., and Sheiman, M. I. 1974. Pectic emzymes. In "Food Related Enzymes," Whitaker, Jr., ed. American Chemical Society, Washington, D.C. pp. 101-27. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 33, 426-28. Nortje, B. K., and Vaughn, R. H. 1953. The pectolytic activity of Species of the genus Bacillus: qualita- tive studies with B. subtilis and B. pum1lis in relation to the soften1ng of oliveS and p1ck1es. Food Research, 33, 57-69. Pallman, H.; Matus, J.; Denel, H.; and Weber, F. 1946. Uber die aktivitat der pektinase. Rec. Trav. Chim. Pays. Bas., 33, 633. Palnitkar, M. F., and McFeeters, R. F. 1975. Recycling Spent brines in cucumber fermentations. J. Food Sci., 33, 1311-15. Phaff, H. J., and Joslyn, M. A. 1947. The newer knowledge of pectic enzymes. Wallerstein Lab. Comm., 33, no. 30, 133-37. Pressey, R., and Avants, J. K. 1975. Cucumber polygalac- turonase. J. Food Sci., 33, 937-39. Rahn, O. 1913. Bacterioloqical studies of brine pickles. Canner and Dried Fruit Packer, Nov. 20, 27. Rombouts, F. M.; and Pilnik, W. 1972. Research on pectic depolymerases in the Sixties. Critical Review in Food Technology, vol. 3, issue 1, CRC Press, pp. 1-25. Whitaker, J. R. 1972. Principles of Enzymology for the Food Sciences. Fennema, O. R., ed. Marcel Dekker InC., N.Y., pp. 321-24, 473. White, W. L., and Downing, M. H. 1951. Coccospora agricola goddard, its specific status, relationships and cellulolytic activity. Mycologia, 33, 656.