THE PECTOLYTIC ACTIVITY OF EXTRACT 5 OF TYPES OF ONIONS Thai: for the Dome. of M. S. MICHIGAN STATE COLLEGE Henry Paul Moloche, Jr. I953 thu: This is to certifg that the thesis entitled The Pectolytic Activity of Extracts of Types of Onions. presented In] Henry Paul Meloche, Jr. has been accepted towards fulfillment of the requirements for Master of Science daycein Bacteriology . I 7 a //X ifw/w I ”I. ; Major professor llate AURUSt 10; 1953 THE PECTOLYTIC ACTIVITY OF EXTRACTS OF TYPES OF ONIONS By Henry Paul Egloche, Jr. A THESIS Suhmitted to the School of Graduate Studies of Michigan State College of Agriculture and.Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 1953 ACKNOWLED GMEI‘IT S The author is particularly grateful to Dr. F. W. Fabian of the Department of Bacteriology and Public Health, without those guidance and moral support this work would have been impossible. The author also wishes to acknowledge Dr. Ralph N. Costilow of the Department of Bacteriology and Public Health, Dr. Samuel Hosen of the Department of Zoolog, and Mr. Lawrence S. White of the Department of Bacter- iolog and Public Health, for their helpful suggestions, criticisms, and “V1000 n u/‘t‘v‘q’ L}()uJU I x.) TABLE OF CONTENTS I. INTRODUCTION . . . . . . . II. REVIEW OF LITERATURE . . . Pectolytic Enzymes . . . . Glycosidic enzymes . Esterases . . . . . . Physical and Chemical Characteristics of C O .nzm B O O O O O O O O O O O O O O O O Pectin polygalacturonase Pectinpmethylestorase . . . . . . . Pectolytio enzymes of fungal origin Mold Growth.on Onions III. EXPERIMENTAL METHODS O O 0 e e e e e e e Pectolytic Polygalacturonase-like Activity Produced'by Holds on onion ktrQCt O O O O O O O O O 0 O 0 0 O O O O O 0 Cultures used . . . . Extraction of onion cell sap Inoculation . . . . . RAGE :NNH 0" ON 4:: 12 12 12 12 12 Polygalacturonase-like enzyme activity determination 13 Action of a Commercial Fungal Pectase on Onions Pectinesterase . . . . . . . . . . Pectinesterase extraction . . . . . . . Determination of pectinesterase activity . . IVe REMTS . O O O O O O O O O O O O O O O O O O O O O The Elaboration of Polygalacturonase-like enzymes 13 in 1a 1a 16 16 V. VI. The Influence of a Comercial Fungal Enzyme Preparation on the Tissue of Bermda, Yellow and White Onions . . . Pectinesterase Activity of Onions DISCUSSION 0 e e e e e o e e s WANDCONCLUSIONS...... ' BIBLIocamn O O 0 e e e e o e e e PACE .8 32 36 iv TABLE 1. 2. 3. It. 5. 6. LIST OF TABLES pH changes, mat formation, and polygalacturonase-likae activity from the growth of various molds in bermda onion .xtract O O O O O O O O O O O O O O O O O O O O 0 pH changes, mat formation, and polygalacturonase—like activity from the growth of various molds in yellow onionextract..................... pH changes, mt formation, and polygalacturonase—liloe activity from the growth of various molds in white onionextract................-..... pH and polygalacturonaseeulike activity from various molds inoculated into white and yellow onion extracts at a rate of 1000 spores per 25 ml etextract. . . . . . . . The effect of standard injections of Pectinol I on bermuda, finite and yellow onion tissue . . . . . . . . Time and accumlative amounts of 0.1 11 NaOH required to neutralize pectic acid liberated by pectinesterase in yellowand white onion extracts .. . . . . . . . . . P13 17 18 19 26 27 LIST OF IIGURES HOUR] m. 1. pH changes and elaboration of PC-like ensyme expressed in units of percent less of viscosity of a standard pectin suspension by two molds on three onion extracts . . 20 2. pH changes and elaboration of PG-like enzyme expressed in units of percent less of viscosity of a standard pectin suspension by two nolds on three onion extracts . . 21 3. pH changes and elaboration of POI-like enzyme expressed in units of percent less of viscosity of a standard pectin suspension by two nolds on three onion extracts . . 22 ’4. Comparative pectinesterase activity curves from 50 ll aliquots of white and yellow onion extracts . . . . . . . 30 INTRODUC TION In recent years a great deal of work has been done on the pecto- lytic enzymes because of their great interest, and because of their importance to the food industry. These enzymes catalize the hydro- lysis of pectic substances. These substances make up the primary constituents of the middle lamellarportion of the cell wall, which act as the cementing material between cells in the higher plants. It is obvious that any degradation of this material would result in the softening, or in extreme cases, in the complete disintegration of plant tissue. The present study was undertaken to determine the pectolytic activity of various comon fungal air contaminants which are known to infect onions. In addition, studies were made as to the resistance of various types of onions to structural degradation by polygalacturonase- like enzymes. REVIEW OF LITERATURE Many authors have studied the role of pectolytic activity in the destruction of plant tissue. Jones (13) studied the enzyme pectinase produced by Bacillus caroutvorus and certain other soft rot organisms. He noted that the enzyme produced by this organism was capable of soft- ening carrots very quickly by dissolving the middle lamella and leaving the cells free. Wenzel and Fabian (29) found Aspergillus and Penicil- lium molds on garlic and attributed the elaboration of pectolytic 2 enzymes by these fungi as the cause of spoilage in genuine Kosher dill pickles. Misekow and Fabian (21) confirmed the presence of pectolytic enzymes in moldy garlic elaborated by Penicifilgiium ganesceg. Fabien and ravine (7) found that an enzyme elaborated by 92329.23; BELLE. in dextrose broth would soften freshened salt stock pickles in a short period of time. Fabian and Johnson (8) demonstrated the degradation of poetic materials in softened pickles by ruthenium red stains. White and Fabian (30) demonstrated the elaboration of pectolytic enzymes by a number of molds growing on black-raspberry extract and they demon.- strated the breakdown of the black-raspberry fruit by a fungal enzyme preparationp-Pectinol A. This constitutes only a portion of the liter- ature dealing with higher plant degradation under the influence of enzymes working on pectic materials, but it does demonstrate the im- portance of the study of these enzymes to the food industry. Pectglytic Enmes All of the known pectic enzymes belong to the class known as hydro- lases. However, these enzyme may be differentiated into two distinct groups; vi ., glycosidases and esterases. Individual enzymes in each class are identified by their specific action. Glycosidic enzymes. One group of pectolytic enzymes is that which catalyzes the hydrolysis of the alpha 14¢ glycosidic linkages which Join the galacturonic acid units to form pectic substances. Specific members of this group are protopectinase, pectin-polygalacturonase, and pectic acid depolymerase. Protopectinase is the name given to the enzyme which dissolves protopectin. Davidson and Willaman (6) attributed to this enzyme the action of maceration, or separation of the cells of the plants. Ac- cording to Kertesz (11+) the enzyme had been demonstrated only by its action on plant tissues, and a chemical definition of its reaction would not be possible until the nature of protopectin itself is clar- ified. He (II-t) also pointed out that there was a growing tendency to consider protopectinase to be identical with pectin-polygalacturonase. Pectin-polygalacturonase is the enzyme which catalyzes the hydro]:- ysis of pectic substances resulting in the fomation of smaller poly- uronides and free galactmnic acid. This enzyme which has been found in various microorganisms and in barley malt was previously known as pectinase and pectolase, but it is now known as polygalacturonase or pectinppolygalacturonase. It is also often designated by the abbre- viation "PG". Pectic acid depolymerase has been recently found in tomato macer- ates and in salt extractions from these macerates. McColloch and Kertesz (19) found that this enzyme differs from polygalacturonase in that it has a fairly sharp optimum at pH n.5, these authors also re- ported that the enzyme is activated by sodium chloride. Kertesz (11+) pointed out that pectic acid depolymerase was almost without action on pectinic acids, but was very active on pectic acid. McColloch and Iertesz (19) found that pectic acid depolymerase depended upon the si- multaneous presence of a de-esterifying enzyme. II Esterases. Only one pectolytic enzyme is recognized as a member of the esterases. This enzyme catalyzes the hydrolysis of the methyl ester group which is found on both pectin and pectinic acids. It was previously known as pectase and pectin-methoxylase but is now known as pectinesterase or pectin-methylesterase. It is also designated by the abbreviations 'PE" and "PM". Physical and Chemical Characteristics 2f Pectolytic Enzymes All enzymes, being proteinaceous in nature, are influenced in their activity by physical and chemical conditions. A study of these factors is necessary to completely characterize any particular enzyme, and generally these characterizations together with the nature and tin products of the reaction are used to distinguish similar enzymes. m-polyflacturonase. For years, most workers considered pectic substances as they existed in the hifier plants to be the true substrate of PG. However, Jansen and MacDonnsl (12) found that only the de-esterified portions of pectic substances were susceptible to the action of polygalacturonase. Kertesz (11+) emphasized that pectic acid was the true substrate of this same. In addition, Jansen and MacDonnel (12) reported that the initial rates of glycosidic hydrolysis on all pectinic acids containing 8.1 percent methoxyl or less were identical. However, the greater the methoxyl content, the sooner the speed of the reaction departed from the initial linear line. The same authors (12) suggested that at least two adjacent free carbonyl groups were necessary for polygalacturonase activity. Pectinppolygalacturonase has been found to have a pH optimum at about 3.5 by Fish and Dustman (9). Varying optima.have been found by other workers through.the years. Kertesz (14) suggested that these variations were due to advances in the use of substrates and.methods as more knowledge of the nature of the reaction was amassed. Recent authors such as Bell, Etchells and Jones (2); Jansen and Mac Donnell (12); and Lineweaver, Jang, and.Jansen (16) used a buffer at pH M.0 in their studies of this enzyme. Consequently, Kertesz (1N) assumed the optimum to be pH 3.5 to ”.0. Pectin-polygalacturonase like most proteinaceous material is quite susceptible to heat. However, in a dry form this enzyme is rather stable. matus (l7) reported.that when the dry enzyme was heated at 80, 9O and.lOO°C. for three hours, a loss of activity of 7, 30 and 85 percent respectively was noted. However, in a water suspension, ‘ this enzyme is quite stable. The same author (16) reported.that there is complete loss of activity of pectinppolygalacturonase when heated to 55°C. for 30 minutes or when heated at pH h.0 for 3 hours at 50°C. Kertesz (1“) stated that when pectinase suspensions were stored over- night they usually lost 20 to “0 percent of their activity even if the enzyme suspension was maintained at low temperatures. Matus (17) found that the addition of 80 mg percent of either sodium alginate, gelatin, glycerol, or the glycerol and glycol esters of pectinic acid increased the resistance of polygalacturonase to heating. Kertesz (1”) noted that sucrose also exerted a protective action for PG against heating. 6 Pectinpmethylesterase. Salts exert an activating influence upon pectinpmethylesterase and there is a direct relationship between.the valence of the cation of the salt and the activity of the PE. Line- weaver and Ballou.(15) working with alfalfa pectinesterase found that the activity of PE at pH 5.7 was about 30 times greater in the presence of 0.2 M monovalent cations or 0.02 M divalent cations than in the ab- sence of cations, If this is representative of cation activation of PE in general, it would indicate that a cationic valency increase of one would activate the enzyme ten—fold. These same authors (15) hypothesized that cations (a) prevent inhibition of pectinesterase by the pectin carboxyl groups by forming cation-carboxyl complexes and (b) that the form of the enzyme existing in alkaline solutions has a decreased tendency to form.an active complex with the carboxyl groups. Hills and Mottern (10) in working with tomato pectase reported that at pH 6.0 or above the presence of 2 to 5 percent added.NaCl caused.a 10 fold increase in the rate of filtration of the enzyme suspension through medium grade filter paper. This would indicate that added salts increase the dispersibility of the enzyme in question. The pH activation of pectinpmethylesterase is closely associated with the concentration of the added salt. McColloch and Kertesz (18) found that the activity of pectin-methylesterase of plant origin was nearly zero in salt free solutions at pH 1t.0 and increased rapidly as the pH was raised to 8.0. In general, the activity of pectin-methyl- esterase was definitely affected by the pH and salt concentration of the reaction mixture as well as by the cation component of the salt 7 as pointed out by Lineweaver and Ballou (15). Kertesz (1M) postulated that salts lowered.the optimum pH of pectin-methylesterase and that they extended the activity of this enzyme into the lower pH ranges. {At pH values approaching 8.0, de-esterification takes place by saponp ification. It is difficult to detenmine whether or not pectin-methylesterase possesses a true pH optimum. There is literature indicating that a definite optimum does exist, but Kertesz (1%) indicated that the valid- ity of the data reported was questionable and implied that better techniques would have to be worked out for the determination of pectin- methylesterase of plant origin before a definite optimum could be es- tablished. Hills and Mottern (10) studying tomato pectin-methylesterase found that the de-esterification reaction was of zero order for the initial #0 to 50 percent of the hydrolysis, but deviated thereafter from.a zero order reaction. The de-esterification does not correspond to a first order reaction over any portion of the curve. Pectolytic enzymes g§_§gggal.origin. The enzymes elabonlted by molds and other microorganisms, which are classified as pectolytic en- zymes, are in reality a mixture of various enzymes. Fish and Dustman (9) working with Pectinol A.found that the enzyme preparation after purification by the removal of sugar brought about the hydrolysis of sucrose, starch gnd.maltose at 37.500 at pH 3.3. In addition it con, tained both PG and PM. They concluded that any determination of pecto- lytic glycosidase activity in a medium.in which an organism.was growing 8 could not be interpreted as galacturonase activity, but could only be interpreted.as galacturonase—like activity. As indicated earlier in this review, the primary source of poly; galacturonase is in microorganisms; the exception being malted barley. However, pectin-methylesterase is found both in microorganisms and in the higher platns. It is interesting to note that pectolytic estersse activity differs depending upon its source. Calesnick, Hills, and Willaman (5) found that fungal pectase differs quantitatively from higher plant pectases in respect to physical characteristics. These authors (h) found that fungal pectinesterase essentially free from galacturonase, was active in the pH range 2.0 to 6.5 with optimum activity at pH 9.0. Pectinesterase in solution at its natural pH of 6.25, remained stable through a two week period at 5 to 6 G and at 23 C while 62 C completely inactivated the enzyme in 30 minutes at pH 3.5. Optimum salt concentrations varied with the pH. At the opti- mum, smaller quantities of NaCl and CaCl2 were required for full acti- vation. The pectase was more sensitive to calcium than to sodium ions. Phaff (22) studying the effect of various organic sources of car- bon on the pectolytic activity of Penicillium chrysogenum concluded that the polygalacturonase and pectin-methylesterase production of this or- ganism was adaptive depending upon the presence in the mediun.of certain structural groupings. This same author (22) also found that there was no relationship between growth and.amount of enzyme elaborated. Phaff stated that it seemed that the product of the reaction (galacturonic acid) apparently had a greater stimulatory effect upon the production 9 of PG and PE than did the substrate in the case of Pencillium chryso- gg_gm, Pitman and Cruess (23), studying the hydrolysis of pectin by molds, yeasts and bacteria found that the greatest hydrolytic action was exerted by the molds studied—-Penicillium glaucum and a Ehythium sp. Their results also indicated that as long as a sugar such as dextrose waa»present in the medium, Penicillium glaucum would not at- tack the pectins as rapidly as it would in the absence of the sugar,‘ and that as the sugar was utilized the rate of pectin hydrolysis in- creased. Mold Growth 93 Onions According to Weiss and O'Brien (28), there are many molds which infect onion bulbs. Among the molds listed are Penicillimm sp., Eu£g£_ sp., Aspergillus sp., Alternaria sp., Fusarium sp. and Botrytis cineria. These organisms are parasitic on both white and colored onions. A review of the literature (1”) showed.that all of these organisms are producers of pectic enzymes. An analysis of the onion gives some insight as to its ability to support the growth of organisms which.may be parasitic upon it. Jacobs (11) listed the following approximate analysis of the edible portion of the onion. ‘Water, 87.5 percent; protein, l.h percent; fat, 0.2 per- cent; ash, 0.58 percent; total carbohydrate, 10.3 percent; sugars, 6.7 percent; and starch, 0.5 percent. It is interesting to note that onions are rich in some vitamins. The same author (11) listed.the analysis of vitamins as vitamin A.value (International Units), 50; thiamine, 0.03 mg; 10 riboflavin, 0.02 mg; niacin, 0.1 mg; and ascorbic acid, 9.0 mg. This author (11) did not identify the type of onions studied. The analysis of vitamins was made on 100 gr samples. Thtscdata demonstrate! that, in general, onions have sufficient nutritive value to support the growth of most molds. Onions are rich in pectic substances. Conrad (5) found a sample of onions to contain “.8 percent pectic substances on a dry weight basis. It appears that onions have a defense mechanism against invasion of microorganisms. It has been observed in this work that molds, pri- marily Aspergillus 5139;, will grow upon the dried skin of onions when incubated in a moist area at room. temperature. However, there will be no breakdown of the tissue even after six weeks incubation unless there is a break in the cuticle of the bulb. It is generally observed that under norml conditions of storage there is little tendency for some onions to mold; and, if contamination does take place, it is localized and does not seem. to do too mch dam- age to the bulb so far as can be observed mcroscopically. However, the literature is meager when dealing with the factors controlling mold growth on onions. There are different degrees of resistance to mold growth exhibited by different types of onions. 'elker and Lindegreen (26) reported that colored onion varieties were not only highly re- sistant to ggylototrichum circinans (a non-presite on onions), but that they were also usually relatively free from infection by Botgztis allii which is a normal onion pathogen. lspergillus niger, however, 11 attacked both colored and white bulbs. Walker (25) attempted to ex- plain this phenomenon when he noted that the epidermal cells of the fleshy scales of colored varieties of onions contain phenolic pigment compounds in the cell sap. However, he noted that the pigment was destroyed as soon as the mold penetrated the host cuticle, and before the hyphae penetrated the cell lumen containing the phenolic compounds. Walker and.Link.(27) tested the effect of 21 phenolic compounds in standard broth against the growth of organisms including the three studied by Walker and Lindegreen (26). They found that Aspergillus Else; was most resistant to the influence of the phenolic compounds tested, followed by Botgytis allii.and leletotrichum.circinans in de- creasing order of resistance. EXPERII-IEE‘ITAL METHODS Mammals-like Activity Producgjd ‘31 1% i3; Onion Extract Cultures u_.s_e_d_. Cultures identified as (Fusarium ogspgrum, Botgtis cinerea, gternaria humiqojl‘a, lspergillus n_i_._gg_1_'_, Penicillium sp. and M sp. were grown for It weeks on potato-dextrose agar slants (Difco.). Spore suspensions of these cultures were then 11306“!- lated into extracts of three types of onions-abermda, yellow and white. Extraction _o__f_ M 9313; gap. whole, sound onions were peeled and macerated in a Waring blender. The macerate was then transferred to several layers of cheese-cloth and the cell sap was squeezed-out by hand. Twenty ml of the cell sap was then placed into each of a series of 125 ml Erlenmeyer flasks. Five flasks of cell sap from each type of onion were prepared for each culture studied. The flasks were plugged with cotton, sterilised by autoclaving, and stored at refrig- eration temperature until used. Alcohol precipitation tests indicated that no appreciable amount of pectin was present in the extracts. inoculation. .i spore suspension of each mold culture was pre- pared by adding sterile distilled water to each slant. This suspension was then transferred to a sterile dilution bottle and standardised by use of a haunocytometer counting chamber to contain 500 spores per .1. One ml of this suspension was inoculated into each of a series of 13 flasks. Four flasks of sterile extract of each type of onion were used for each mold. flue cultures were incubated at 30 i l C. Wacturonase-ngg m activity determinam. The method used in the determination of the P. G.-like enzyme activity was a modi- fication of the method of Bell, Etchells and Jones (1). Due to the high viscosity of the pectin"I suspension employed, a 1.5 percent sus- v pension was used in place of the 3 percent suspension. At 1.5, 2.5, 3.5 and #5 days incubation one flask was removed from the incubation for each mold and type of onion. The cell sap was observed for the presence of mat formation, layered withbluene, and the pH was measured. Two 5 ml portions were withdrawn from each flask. One portion was heated to 80 C for 10 minutes in a water bath to serve as the control, and the other was inoculated into 25 ml of pectin in phthalate buffer at pH “.0. The control was also inoculated into the buffered pectin solution. These samples were then incubated for 7 days at 30 t 1 0. At the end of the incubation period, the dropping times through a 25 ml pipette were recorded. The percent less of viscosity was determined by using the formula: percent less of viscosity = L15 x 100, where 28 represents the dropping time of the heated control, 3 and A represents the dropping time of the unheated sample. Action of a Commercial 11% Pectase on Onions A 10 percent Pectinol solution was prepared by dissolving 10 g of Pectinol it" in distilled water and diluting to 100 ml. 1. number * Pectin sample 13-890, supplied by California Fruit Growers Exchange, Ontario, California. " Supplied by Rohm and Bass 00., Philadelphia, Pennsylvania. in of whole, sound bemuda, yellow and white onions were selected and weighed. lach onion was injected with 10 mg of Pectinol for each gram of weight via hypodermic syringe. The onions were observed for six days and any degradation of the tissue in the area of the injection was noted. W Pectineeterase extraction. The pectinesterase extracts from the plant tissue were prepared in the following mnner. rive-hundred as of both white and yellow onions were frozen, permitted to thaw and mac- erated in a Waring blender with 2 percent NaCl. The ’macerate- was then ’ transferred to eight thicknesses of cheese-cloth, and the enzyme ex- tract was squeezed out by hand. The extract appeared to be clear . enough that filtering through filter paper was unnecessary. The ex- tract was then transferred to a 500 ml volumetric flask, and made up to volume by the addition of distilled water, layered with toluene and stored at refrigeration temperature until used. getermination of pectinesterase activity. The substrate was prepared according to the method of Bell, Etchells, and Jones (3).. A 50 ll aliquot of the onion extract was added to the substrate after both enzyme and substrate had reached a temperature of 30 C. The temperature was nintained throughout the experiment by means of a thermostatically controlled water bath. he enzyme activity was deter- mined by maintaining the mixture at a pH of 7.5 throughout the experiment. * Pectin, sample MFG-l, supplied by California Fruit Growers Exchange, Ontario, California. 15 The pH was measured with a Dance titration-pH meter, and was adjusted with 0.1 N NaOH. The pectinesterase activity was determined for Texas white and yellow onions. RESULTS T333 Elaboration oi; Mlacturcnasg-lcike Enzymes The results of the study “of the elaboration of the PG-like enzymes by six molds in extracts of three different types of onions are presented in Tables 1, 2, and 3 and in Figures 1, 2, and 3. In addition datum-e presented on the pH changes occuring in the ex- tracts and the relative amount of visible growth present. There appeared to be a relationship between the appearance of visible growth and a change in pH (Tables 1, 2, and 3). a change in the pH of the extract was noted either at the same time as the appear- ance of visible growth, or followed the growth by one day. Therefore, the first pH changes noted in figures 1, 2, and 3 were an indication of the presence of macroscopic growth. Figure 1 shows the relationship of pH changes, and the change in the PG-like activity produced by Botmig cinerea and l‘usarium ogsmrum when grown in the extracts of three types of onions. Bctrytis cinerea produced no change in pH throughout the duration of the experiment except when growing on the bermuda extract wherein a slight decrease was noted at 3.5 and 14.5 days. With all three extracts PG.like activity was noted to be similar and sufficient enzyme was pro- duced on each extract to cause a loss of viscosity in the maximum range measurable by this method. 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R I m.m m I man 0 I m.m 3350 Season 2. + o6 .8. + me o I Tm o I ma 5a: flame. gunmen; seam soul ma scam and... mm seam and... mm .3390 iii! flOflvdfigfld HO 03TH. may .3935 mouse cause an and! spots» no Aawoam an» Hour. hugged aadumncaaosammhacm and .mouananom via .33 mm Bottxlis 9199139. Fusarium oxysporum pH ‘7. loss of viscosity pH , IOO _ 3 Bermuda OnIon Bermuda OnIon L 0”0 -« / ‘ 8C) “' I ‘i 6 r- / . - 6 .——»—~L.\l so , _ I I I l I — 40 »— I 4 +- ’0 I 7 4 l _ I, . — 20 l- o ’ ‘1 (5’ 1” I 8. IOO l . 8 Yellow Onion ' Yellow OnIon J I _ ‘°/' -+ 8C) e i I, ' 6J*-__-_‘F___._JL‘.___"J 6() f;--"”"(/”._-‘\. 6 I 4 4C) — ’9 l 4 *- é / -1 4 I 0’ I 4 2‘: L. / / o I "— ‘ 2 ’l 0 fl ‘9’ P) s we 8 Whlle Onlon While Onlon ’ o“ 80 ~ 7* [I — ' . -4 6 _ / I “l / ' a 40 ~ ,v0 4 - / ,0 e 4 l / __ I 1 20 ——pH / _ ” —-°/o loss of viscosity 2"x__.I_er I l l ——I 9—1 v 1 J O l 2 3 4 5 O O l 2 3 4 52 Time (days) Firm?“- 1. 1." era-Ingres and elaboration n‘: ‘I..‘..li'.ce «I. prrrS‘sl in unit? if p r;~nt less of viadusity or n stanzari grctin sulgv31ion by th molds an three onion extracts. Allerncrio humicolc ' Penicillium sp. pH °/. loss of viscosity pH 8 , Ioo _ Bermuda OnIon Bermuda Omon P 0 ~ so - 0” ‘~o..’° “ / 6 - fp" I l’ 4 6 L l 7 I K I, 4 4° 7’ 4 L ‘ 4 I I . /° - 20 _ 2< ” o —— 2 e , IIO’.) . a Yellow OnIon Yellow Omon “ u so _ ’ d / o’m’o’ I 6 " ‘ 6 — so ’ _ I _ I, 4 40 _’I 4 ~ « 4 I I / ~ 20 H ‘- ’0 I a // 2GP 0 ““" 2 e . . loo e the Omen ’0\While Onion I- , \ -l I \o/ 6” T 6 I so I - I l a / I 0"‘13 1 410 H 4 -— / l 4 '4 / /’ -—°/o loss of viscosity 2 £” I i 1 1 o v 1 1 1 2 0 I 2 3 4» 5 O l 2 3 5 Time (dcys) Figure P. I! :Inmwnr and ulnboraticn of 13—1152 enzfi;v .‘ 7.: r51. "6 .1 I-\ ‘ in Jail: cf f a staaxari Lost 3 three onion extrarts. pflfgflnt 1335 :5 v.5;cszty suspensién by two moLds on 21 Aspergillus niger Mucor 5p. f-J h) pH °/o loss of viscosity pH 3 . Ioo Bermuda Onion Bermuda Onion L 0 _ Oa-o-Ow‘o__ go - ’0‘~ o’/ 0.... 4 6 6C) 1 4C) ~3/ I '4 4 I 20 II .. 2 o L 2 e lOO , 8 Yellow Onion Yellow Omcn so _ ° ‘ . /°‘~0--c” ‘ 6 —, -4 I u 20 4 .. l f l 2 o e 2 e . . loo 3 WhIle Oman 0 While Onion _ O’-O~-o” o .— /’ 813 r ‘/0‘-—43_u—CY"' I _. 60 v 6 I ---d 4~O ” II “ 4 20 —pH _ —-°. loss of viscosity 1 L 1— i 2 5 C) O I 2 3 .4 5 Time (days) pH changes and elaboration of is-like enzyme styreesud in units of percent less of viscosity a” a standard pectin suspension by two molds on three onion extracts. 23 Consequently, the maximum range of percent less of viscosity was consid- ered to be in the range 75 - 85 percent in this study. Any variations within this range were considered to be of no significance. ' Changes in pH produced by Pusarium M were similar in all three extracts. However, the W mold did not produce PG-like activity rapidly. The onion extracts resulted in no appreciable loss of viscosity of the pectin suspension until after 3.5 days of incuba- tion. Only with the white onion extract was a mximum reduction in the viscosity of the pectin suspension noted after 14.5 days of growth of this mold. Figure 2 demonstrates the relationship of pH change and change in the percent less of viscosity of a standard pectin suspension over a period of time produced by Alternaria humicola and Penicillium sp. It can be seen that the pH changes produced by thernria humicola were quite similar in the three extracts. No change was noted during the first, 2.5 days. At this time there was a slight increase of pH follow. ed by a decrease. The PO-like activity resulting from the growth of Alternarighumiccla was similar in all three extracts. At the end of ll.5 days of incubation of the inoculated extracts, the PG-like activity of all three extracts was sufficient to reduce the viscosity of the standard pectin suspension by 70 - 80 percent. Growth of Penicillium sp. resulted in pH changes in the three em- tracts similar to these promiced by Alternaria humicola and Pusarium ogspgrws. here appeared to be a slight increase in pH at 2.5 days on the yellow extract, while the initial change in 1H on the other extracts 2h occurred at 3.5 days. he significance of this variation is doubted since an examination of Tables 1, 2, and 3 shows that initial obvious at formation occurred at 2.5 days in all cases wherein W sp. was involved. Penicillium sp. differed from the other three organisms discussed so far in its ability to elaborate PO-lilne enzyme very rapidly on all three extracts. This mold elaborated sufficient enzyme to cause a man- im percent loss of viscosity at the end of 1.5 days. After 2.5 - 3.5 days growth of Penicillium sp. a decrease in the percent less of vie- cosity from 80 to approximately 70 was noted. However, this was not believed to be significant. Figure 3 shows the relationship of pH change and elaboration of PG-lilee enzyme for Aspergillus gigs}; and binge; sp. grown on the three substrates. Asmrgillus ML produced a rapid change in the pH of all three extracts which corresponded to rapid mat formation. ‘nie general trend of pH change was similar for all three extracts. However, the pH of the bermda and yellow extracts were in the range 6.6 - 7.2 at the end of the experiment, while the final pH of the white extract was 1$.8. However, after 5.5 days of incubation the In of the white ex.- tract was found to be about equal to that of the other two. The elaboration of PG-lika enzymes on all three substrates was rapid. The extracts in which Aspergillus gigs; was grown caused perceit loss of viscosity in the maxim range at the end of 1.5 due. The activity of the PG-like enzymes remained high throughout the duration of the experiment. 25 Mm sp. caused similar pH changes in Bermuda and yellow onion extracts. However, the pH of the whitez'e‘xotgact did not increase as rapidly after 3.5 - ’4.5 days of incubation as did the pH of the other two extracts. The significance of this is doubtful, since mat formation was similar on all three extracts (Tables 1, 2, and 3). Is in the cases of Penicillium sp. and Aspergillus $1525, 1113.393; sp. elaborated a maximum measurable titer of rank. enzyme at the end of 1.5 days, and this titer was mintained throughout the duration of the experiment. No significant difference in enzyme production by this mold in the three onion extracts was noted. Table 1+ gives the results of‘ an additional experiment run in order to establish any effect of the size of the inoculum on growth and en. zyle production by three of the molds used. The technique used in this experiment was identical to the one described previously except that 1000 spores instead of 500 spores were ineculated into 25 ml of extract On comparison of the results of the experiments with the 500 and 1000 spore inoculums of Penicillium sp., Aspgrgillus piggy, and Mm sp., it may be noted that pH changes and enzyme production were quite similar. Therefore, it my be concluded that increasing the inoculum tVO-fold did not appreciably influence the activity of these three molds. Tissue 9_f_ Bermuda, Yellow, and White Onions. The results of this experiment are shown in Table 5. It can be seen from this data that the ability of Pectinol M to degradate onion tissue was most pronounced in the white onion, somewhat less pronounced 26 rank pH and polygalacturonase—like activity from various molds inoculated into white and yellow onion extracts at a rate of 1000 spores per 25 ml of extract. Time of incubation 3 day- 5 daw- 6 day- ? den cultures pH PG. pH POI. pH PG* pH PG. Yellow onion extract Penicillium sp. 5.2 71$ 5.3 63 4.7 58 M9 61 Asmrillus niger 3.5 80 6.3 77 7.0 I 7.3 I White onion extract Penicillium sp. 5.6 78 u.o 73 3.8 70 3.9 6? lspergillus niger 3.14 77 4.1 71+ 6.1 71 7.2 68 M1100! ”e 5e“ 77 5e? 79 605 .76 6’7 75 't PG—like enzyme activity expressed in units of the percent less of viscosity of a standard pectin suspension. X indicates contamination of the standard pectin suspension. TABLES The effect of standard injections of Pectinol M on Bermuda, White and Yellow onion tissue. W Tissue Hermda onion Yellow onion White onion T1131? Tissue degradation 1 a - + 2 n - H 3 - - 4+ h + .. «H 5 + .. ++ 6 -H' I H Legend: - indicates no change of the tissue. + indicates localized graininess of the tissue. ++ indicates localized softness of the tissue. in the Bermuda tissue, and that Pectinol u had no influence on the tissue of the yellow onion under the conditions of the experiment. a preliminary experiment using heavy inoculation of the Pectinol It with a white and yellow onion of approxintely the same weight resulted in the tissue of the white onion becoming very soft within one week while the yellow tissue did not breakdown for approximately 3 weeks. Pectinesterase Activity 1; Onions The data of the pectinesterase activity of yellow and white onion extracts d'e presented in Tables 6. Figure it presents typical data fa.- each extract. It may be noted that the curveof the white extract rises quite sharply and equalizes in a short period of time in compar- ison to the curve of the yellow extract which approaches a linear re- lationship. These particular types of curves were found in each of TABLE 6 Time and accumulative amounts of 0.1 N NaOH required to neutralize pectic acid liberated by pectinesterase in yellow and white onion extracts. Accumulative ml of Accumulative ml of NaOH used HaOH used 1.1.1.1 1e Trial 2* Trial 3“ W Hinutes Yellow lhite Yellow White Minutes Yellow White 0 17.6 17.2 1n.6 11.8 o 16.7 22.9 5 1.9 u.7 2.7 h.1 u 2.2 5.9 10 h.9 9.3 6.3 8.u 8 3.2 9.8 15 6.6 12.9 7.9 12.7 12 u.u 12.h 20 9.1 16.3 10.2 16.u 16 6.3 13.7 25 11.u 19.7 12.7 20.6 20 7.6 1 .0 30 12.0 22.8 iu.o 23.u 2h 8.8 33 1u.6 26.0 16.8 26.2 28 9.9 1n.5 16.u 28.h 18.3 28.9 32 11.1 R5 17. 30.9 19.5 30.7 36 11.6 1h.7 5o 19. 33.3 20.6 32.3 #0 12.5 55 21.3 35.6 21.6 33.9 an 12.9 1u.9 60 23.2 36.8 23.2 .7' “8 13.5 70 25.5 39.1 25.u 35.7 52 13.6 80 28.2 No.8 27.3 35.u 56 1 .8 15.1 90 30.3 n2.0 28.9 36.7 on 1 .2 15.1 100 32.3 u3.0 30.7 37.1 80 1n.5 110 3h.2 31. 96 1n.9 120 36.2 nu.0 33. 37.5 112 15.5 133 37.6 "5.0 3 .5 37.9 1 39.0 u5.0 35.6 37.9 150 no.1 36.u 160 u1.2 37.2 170 “2.3 37.7 180 :3.2 38.3 190 .1 200 un.7 38.8 210 115.1 39.1 225 ”5.9 2ho 86.6 255 M7.o 27o u7.2 285 87.7 300 ‘47-? 1' In trials 1, and 2 the substrate was prepared according to the method of Bell, ltchells, and Jones (2). Fifty m1 aliquots of the extracts were used. H In trial 3 the substrate was so prepared that the addition of 100 ml of extract would give a final percentage of 0.“ with respect to the pectin present. m1 of 0.1 N NaOH .eaonaaue noamo aoaaoa and means we noocdaac Ha om seam sconce haaaaoon.eeeaeoucn«aoem eraacanaaoo com on... cam SN 02 £516.33....16-.. -2... .. .r 1..-... ...:.........6.. 731...... x. dOddO BOHHON a +611 3:5 SE: in 6.0. 2 m; 1 an i l... . . . 127i ceil- l .e- ._ {veil flunk. s. k ..1.v|ellu".llu. e newsman 3 .: chowdm cm .I 1 .I r. 55.5... .- 115! dill-Vale!) b. e.) .11 .151 sll ....... p.13. .‘i'lll'l'1 3 m ‘ r-I HO?“ R 1’0 3° Tm 31 three trials, from two different extractions. This emphasizes conclup sively that the titer of pectinesterase is much higher in the white onion extract than in the yellow onion extract. DISCUSSION The six molds studied all produced PG—lihe enzyme( s) but hey were divided into two groups acwrding to the speed at which this enzyme was produced. gternaiia humi_cci_l_a_, Fusarium ogspgrium and Botgtis cinerea produced the enzyme slowly while Penicillium sp., lspergillus n_i_.g_e_r_, and £339; sp. elaborated the enzyme rapidly. Although the latter groups were noted, in general, to grow more rapidly than the former, there was no obvious correlation of visible growth with enzyme production. With three of the molds, PG-like activity was evident prior to the appearance of visible growth. Similar observations have been mde by other workers ' (22, 30 and 31). ' Since very little or no pectin was present in the onion extracts 1 used for the growth of the molds, the Pan-like enzymes produced were probably of the constitutive group. This, plus the fact that some of the molds produced large amounts of PG-like activity with very little growth my be important factors in the ability of a mold to infect plant tissue. In addition, there is a possibility that mold spores may contain pectolytic enzymes since bacterial spores have recently been shown to have enzymtic activity (23). This possibility should be investigated. Carolus (16) stated that there was a direct relationship between intensity of coloration and the resistance to mold infection of onions. Those onions which were the least colored (white) were most susceptible and those which were most colored (yellows, reds) were most resistant to mold infection. However, it was shown here that each of the six 33 molds tested produced about the same amount of PG-like enzyme in extract of white and yellow onions. Also, when controlled amounts of a com- mercial fungal enzyme preparation were inoculated into white, yellow, and bermuda onions, the breakdown of tissue was most rapid in the whit onions and slowest in the yellow onions. Therefore, there must be some other factor besides Pflhlike enzyme concentration which accounts for the resistance of colored onions. On measuring pectinesterase activity of the onions, it was found that the white onions contained significantly higher quantities of this enzyme than did the yellow. Since Jansen and MacDonnel (12) have dele- onstrated that the action of PC is roughly proportional to the degree of deesterification of pectin, this factor may account for the differ- ence in the susceptibility of the white and yellow onions to mold in.- fection and tissue degradation. This should be f‘uther investigated. SUMMARY AND CONCLUSIONS According to the conditions set forth in this experiment the fol- lowing facts were indicated: 1. 2. 3. 5. Six Iold cultures showed growth and PG-lih enzyme elaboration in varying degrees on three types of onion extracts. Each culture behaved similarly in regard to pH change and enzyme elaboration on all three types of onion extracts. The molds could be placed into two min groups according to their ability to elaborate PG—like enzyme: (a) those cultures which elabor- ated the enzyme rapidly, 111.. Penicillium sp., Aspergillus gig; and Mm sp.; and (b) those cubtures which elaborated the enzyme slowly, 25° Alternaria humicola, Fusarium ogsmrum, and Botmis cinerea. Penicillim sp. produced a minmm measurable titer of enzyme in 1.5 days but showed no visible growth for 2.5 days. Botgztis cina'ea produced a maxim measurable titer of the enzyme in 2.5 - 3.5 days but showed signs of growth in only one extract at the end of lL5 days. ~ There appeared to be no relationship between visible growth and enzyme prochction. Inoculation of Penicillium sp., Aspgrgillus m and M sp. in- to white and yellow extracts in ratios of 500 and 1000 epores result- ed in no significant difference either in the pH changes or in the elaboration of PG-like enzyme by the organisms. 35 6. White onions appeared to be the most susceptible to the action of Peotinol u and yellow onions appeared to be the most resistant. 7. White onions appeared to contain a significantly higher titer of pectinesterase than did tb yellow onion5. 8. There is believed to be a direct relationship between susceptibility to Pectinol M, resistance to mold infection and pectinesterase con- tent in white and yellow types of onions. 1. 5. 6. 7. 10. 11. 12. BIBLIOCELAPHY Bell, T. A., Etchells, J. L, and Jones, I. D. Softening of comercial cucumber salt stock in relation to poly. galacturonase activity. Food Tech., 2, 157-163, 1950. Bell, T. 8., Etchells, J. I.., and Jones, I. D. Pectinesterase in the cucumber. Arch. Biochem., 11_, 181-15111, 1951. Calesnick, E. J., Hills, G. B., and lillaman, J.J. Properties of a comercial fungal pectase preparation. Arch Biochem., _2_9_, lee-alto, 1950. Carolus, n. L. Department of Horticulture, Michigan State College, personal communication. 1953. Conrad, C. M. » mfg-icon Journal of Botany, 13, 531, 1926. As quoted in Kertesz (l . Davidson, 1‘. B., and Willaman, J. J. Biochemistry of plant diseases. 11. Pectic enzymes. Bot. Gaz., £10 329‘361s 19270 ‘ Fabian, 1'. l., and Flaville, L. I. Isolation and identification of a mold, Oosmra lactis, as the causative agent of two cases of pickle spoilage. Fruit Products Jam. 2.8.. 297-298. 19%. Fabian, r. L, and Johnson, I. A. Experimental work on cucumber fermentation. X. Zymologioal studies of the cause of soft cucumbers. Tech. Bull. 157, 9-16, Miche Agre hp. Stae. 1938. Fish, V. B., and Dustman, R. B. The enzyme activities of Pectinol A on pectin and other substances. J. Am. Chem. Soc., 61, 1155-1157, 19115. 3111.. Ce He. and. MOtt.m. He He Praperties of tomato pectase. Jour. Biol. Chem., 168, 651-663, 19 7. Jacobs, )1. B. Food and Food Products, XXVII Vegetables and Mushrooms by I. A. Lee, second edition, Volume II, Interscience Publishers, New York, 1951. Jensen, 1:. 1., and Mac Donnel, In R. Influence of methoxyl content of pectic substances on the action of polygalacturonase. Arch. Biochem., §, 97-111, 1945. 13. 1h. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2h. 37 Jones. Le Re Pectinase, the cytolytic enzyme produced by Bacillus carotovo:1;u_s_ and certain other soft rot organisms. New York Agr. Exp. Sta. (Geneva) Tech. Bull. 11, 1909. Kertesz, Z. I. Th__e_ Pectic Substances Interscience Publishers, New Yorlz, 1951. Lineweaver, H., and Ballou, G. A. The effect of cations on the activity of alfalfa pectinesterase (pectase). Arch. Biochem., g, 373-387, 19%. Lineweaver, H., Jang, B., and Jansen, B. 1'. Specificity and purification of polygalacturonase. Arch. Biochem., go. ’137. 1949. Matu', Je Ber. schweiz. bot. Ges., E, 319, 19%., as quoted in Kertesz (11+). McColloch, R. J., and Kertesz, Z. I. Pectic Enzymes VIII. A comparison of fungal pectin-methylesterase with that of higher plants, especially tomtoes. Arch. Biochas., 11, 217-229, 1947. McColloch, B. J., and Kertesz, Z. I. Heat resistant pectolytic factor from tomatoes. Arch. Biochem., 11, 197 , 1948. 11000110011, R. J., and Kertesz, Z. 1. Recent developments of practical wignificance in the field of pectic enzymes. Food Tech., 3, 91l-96,191+9. Misekow, R. T., and Fabian, F. W. Pectolytic enzymes of garlic, Food Res., 1g, 1-5, 1953. Pfaff, H. J. The production of exocellular pectic enzymes by Penicillium chgsogemnn. 1. On the formation and adaptive nature of polygaizcturonase and pectinesterase. Arch. Biochem., 11, 67- 81, 19 . Pitman, G. A., and Cruess, I. V. The hydrolysis of pectin by various microorganisms. Ind. Eng. Chem., 21, 1291-1295, 1929. Stewart, B. T., and Halvorsan, H. 0. Studies on the spores of aerobic bacteria. I. The occurrence of alanine racemase. Jour. Bact., 65, 160-166, 1953. 25. 26. 27. 28. 31. I 38 Walker, J. 0. Disease resistance to onion smudge. Jour. Agr. Res., 211;, 1019- iouo, 1923. Walker, J. C., and Lindegreen, C. C. Further studies on the relation of onion scale pigmentation to disease resistance. Jour. Agr. 3.3., :39, 504.51% 1921;. Walker, J. 0., and Link, K. P. Toxicity of phenolic compounds to certain onion bulb parasites. Bate GQZe. 6. “ha-”81+. 1935. Weiss, L, and O'Brien, M. J. (Compilers) Plant Disease Survey, Special Publication, No. 1, part IV, UeSeDeAe Publicatione Wenzel, F. L, and Fabian, F. I. Experimental work on cucumber fermentation. Mich. Agr. Exp. Sta. Tech. Bull. 199, 1945. White, L. s., and Fabian, 1'. w. The pectolytic activity of molds isolated from black raspberries. Jour. of Applied Microbiology, Sept., 1953. (in publication) White. Le Se. and. Fabian. Fe We Personal communication, 1953.