This is to certify that the thesis entitled The Effect of Various Holding Periods on the Catalase, Peroxidase, and Ascorbic Acid Oxidase Activity of Several Varieties of Blanched Ca i P as. nn ng e presented by William Henry Case has been accepted towards fulfillment of the requirements for m. 5. degree inJQILiJ‘eBLture Major professor Date Aneust 16. 191.8 no: Ermcr OF VARIOUS I-DLDING PERIODS on THE GAIALASE, PEROXIDASE, AND ASCORBIC ACID OXIDASE ACTIVITY OF SEVERAL VARIETIES or BLANCHED CANNING PEAS By WILLIAM HENRY CASE N A THESIS Submitted to the School of Graduéte Studies of Michigan State College of Agriculture and Applied Science in partial mlfillment of the requirements for the degree of MASTER OF SCIFJICE Department of Horticulture August 191:8 ACKNOWLEDGMENTS 111s writer wishes to acknowledge his indebtedness to Dr. R. E. Marshall and Mr. W. F. Robertson for their wholehearted assistance throughout the course of this experiment and in the preparation of the manuscript. Appreciation is also extended to Dr. E. H. Lucas, Miss Ardith Lickfeldt, and Mr. A. L. Feather for their assistance. 33?: $721.1 THE MOT OF VARIOUS HOLDING PERIODS ON THE CATALASE, PEROXIDASE, AND ASGORBIC ACID OXIDASE ACTIVITY OF SEVERAL VARIETIES OF BLANCH-ED CANNING PEAS INTRODUCTION In recent years a great deal of attention has been given to the nutritive aspects of processed foods. The results of work done on dehydrated foods during'World.War II have stimulated a reexami- nation of the common canning practices and.procedures. In l9h2, (h, 9) the National Canners Association and the Can manufacturers Institute set up a jointly sponsored cooperative program with the objectives of establishing the nutritive values of commercially canned foods, determining the factors influencing their nutritive values, improving their nutritive properties, and investigating how well the commercially canned feeds meet the human physiological needs. This program.is still in Operation. One aspect of the nutritive value of canned foods now being investigated is the role that the various enzymes play in the reten- tion of ascorbic acid during various handling and.processing opera- tions. In an effort to determine whether or not there are varietal differences in the enzyme activity of canning peas, a series of ex- periments were conducted and carried out during the l9h8 canning season for peas. 1‘9!- "Jl.'.- t... . ' - REVIEW’OFILITBRATURE "Enzymes in feed.products are like ants in the kitchen, the greatest problem is how to get rid of them".(3) mzymes are organic catalysts produced by living organisms and cells (8, 18). Emma are considered by most authorities to be either a protein or to-be in colloidal combination or association with a pro— tein.(8, 18). most enzymes are specific in.nature, are thermolabile; and.are sensitive to acids, alkalies, temperature changes, metal salts, pH, light, and radiation. Enzymes catalyze hydrolytic, oxidation-re- duction, and fermentation reactions (8). Catalase (8) is one of the most widely distributed enzymes since it is fOund in nearly every living cell. Peroxidase (8) is an enzyme that is widely distributed in plants and also occurs in some animal products. Ascorbic acid ox» ideas is found in plants and.particularly in the common vegetable plants (8). The three above mentioned enzymes catalyze oxidationpre- duction reactions. Oxidation-reduction enzymes may'be classified ac-r cording to the following‘bases: l. the chemical nature of the sub- strate, 2. the components of complete oxidation.systems, and 3. the chemical nature of the prosthetic group (8). According to Green (8), peroxidase and catalase are oxidation enzymes with a Fe porphyrin prosthetic group; and ascorbic acid.oxidase may be a copper protein. Both.ascorbic acid oxidase and.peroxidase are known to oxidize ascor- bic acid. Enzymatic action is involved in the determination of food (2, ‘3, S). Kany methods have been tried.by'man in his efforts to pre- serve food for future use. Mbdern methods are successful but not com— pletely satisfactory (2). much of the "Hay-like" and other undesir- able odors and flavors of unblanched dehydrated.and frozen vegetables -3— are attributed to enzymatic oxidation by Cruess and Joslyn (5) and Balls (2). Certain vitamin losses and discolorations in dehydrated and frozen vegetables may be caused by enzyme activity. The import- ance of enzymes in canned foods has not received the attention that it has in frozen and dehydrated foods. This lack of attention is mainly due to the fact that the heat process in canning inactivates the enzymes; therefore, enzymes aren't usually directly connected with the deterioration and spoilage of canned foods. In recent years, however, in investigating the losses in nutritive value of canned foods, attention has been directed toward the extent and causes of nutritive losses during handling and processing operations (6, 9, 10, ll, 13). Results of these studies by Jackson, et a1 (9), Lamb, et a1 (11), and others have indicated that considerable losses of ascorbic acid and carotene may take place during handling operations if mea- sures aren't taken to inhibit or destroy enzyme activity early in the processing operation. Blanching is the most common and practical means at present for inhibiting or inactivating enzyme action in vegetables. Many stud- ies have been conducted on blanching methods and their effect on nu— trient losses, and also on the proper times and temperatures to in- activate enzymes (5. 6, 9, 11). In studying the adequacy of the blanching process, tests have been made to determine at what temper- ature and time the most common enzymes were inactivated. Catalase, peroxidase, and ascorbic acid oxidase are the enzymes most often chosen to determine the adequacy of the blanch (S, 6, 111). Peroxi- dase has been most frequently selected as the test enzyme because it is generally the most difficult of the three enzymes to inactivate ('5, -h- 6, 11;). There seems to be an indication that different blanching re- quirements may be necessary to inactivate this enzyme in the same vegetable under different environmental conditions (6). In general, all three enzymes were found to be inactivated by a blanch of 200°F. for 3 minutes; and in most‘products, 190-19503; for 3 minutes was adequate (5, 6)- mmanmil. MATERIALS AND mums In an effort to duplicate commercial conditions and practices as much as possible, these experiments were carried out at the col- lege canning factory during the regular pea canning operations. Pride, CannermKing, Shasta, Bonneville, and Perfection were the varieties of sweet (wrinkled-seeded) peas used in this experi- ment. All varieties were grown under the same conditions on the col- lege farm. The peas were cut with a mower equipped with vine lifters and a windrow attachment, loam by hand onto flat bed wagons, and hauled approximately one half mile from the field to be vined. Imme- diately after vining, the peas, in lug boxes, were hauled to the can- ning factory. The time from mowing until the vined peas reached the factory was ' approximately 2 to 2% hours. On receipt at the canning factory, the peas were weighed and run through an air-blast cleaner to remove extraneous and foreign ma- terial. The cleaned peas were evenly distributed among six lug boxes, each of which held approximately 30 pounds of cleaned pass. The con— dition and maturity of the peas were noted. Then the temperatures of the peas in the lug boxes and of the air were noted, and the peas were divided into two lots of three lugs each. One lot was immediate- ly blanched, and the other lot was set aside in the shade of the can- ning factory porch for approximately four hours. At the end of the holding period, the tauperature of the peas in the lugs and the air temperature were again taken before the peas were blanched. The peas were run over a riffle to remove any pebbles, then through a split pea. pod remover and washer and were elevated to the blancher by a gooseneck elevator. The peas were blanched at ZOOOF. for two minutes, spray cooled in water for a few seconds, and run --6- over an inspection belt before the sample was removed. The sample was collected in a stainless steel bucket from the hcpper at the end of the inspection belt. Care was taken to get a representative sample of each lot of peas. The temperature of the sample peas in the bucket was noted, and a representative aliquot was taken for measuring the extent of enzyme inactivation. At the end of one-half hour, the temperature was recorded and another sample remov- ed for the enzyme tests. A third sample was taken at the end of two hours, and the tanperature was recorded. A sample of the unblanched peas was collected just before the peas were blanched for use as a measure of the fresh activity. The remainder of the peas were run through the line, filled into number 10 cans along with a salt-sugar brine, exhausted for 8% minutes to 171 to l76°F.,closed, and then retorted for 55 minutes at 240017. A random can was selected from this group for use as the con- trol sample. The activities of catalase, peroxidase, and ascorbic acid on- idase were assayed by the following methods. The percent inactivation of catalase was measured by a modification of the Thompson method (17), except that the method of preparation of the sample was modi- fied so that a larger, more representative sample of the lot could be used. A 20 gram sample of the vegetable material was weighed accu- rately into a Waring blender bowl containing 100 milliliters of dis- tilled water and 12 grams of precipitated calcium carbonate. The sam- ple was then blended until thoroughly macerated for approximately two minutes. The blended sample was then filtered through four thick- nesses of cheesecloth, and the filtrate was refiltered through four -7- thicknesses of cheesecloth into a 1.00 milliliter beaker. From here on the procedure followed the method by Thompson. Originally it was planned to use the Balls-Hale method (1) for determining the perendase activity, but the difficulty of getting consistent and reproducible results with it in another experiment led to the adoption of the Lucas-Bailey method (12). The preparation of the sample for the Lucas-Bailey method was modified to make it suit- able for use on fresh and blanched vegetables. The sample was prepar- ed in the same way as for the catalase assay; in fact, the same fil- trate was used in both assays. The Lucas-Bailey method was further modified in that the filtrate from the sample of raw, unblanched peas was diluted 100 to 1 before assaying.- p The method used for assaying the ascorbic acid oxidase activ- ity is essentially the method used by Robinson (16). It is a modifi- cation of the Robinson and Stats indophenol-leene extraction method for ascorbic acid (15). Due to inexperience and the lack of specific procedure for this method, very poor and unreliable results were ob- tained. After considerable trial and error, the writer believes that the method will henceforth give fairly reliable results. " ' ' RESULTS The pea canning season at East Lansing began June 26 and last- ed through July 9 of this year. The first variety canned was Glacier, but no tests were run. On June 29, the Thomas Laxton variety was cann- ed, and a trial run of tests was made for another experiment. Since the data on Thomas Laxton was incomplete, it is not included in this paper. The regular series of tests were started July 1, with the Pride variety and continued to the end of the season as each succeed- ing variety was harvested. There were two plantings of Canner King variety harvested on separate dates. The Canner King variety was used in another experiment of which only two treatments were comparable to the treatments reported in this paper. All varieties of peas were blanched at 200°F. for 2 minutes, then cooled to lOSOF. 1‘. 5° under water sprays. The normal. blanching temperature for peas in this plant is l9h°F. for 2 3/1; minutes. As may be noted from Table I, there were no consistent differ- ences in the percent inactivation of catalase or peroxidase, except tint from 2 to 6 percent catalase activity was observed, irrespective of variety, in the samples blanched without delay with no holding period after blanching. The degree of oatalase inactivation was con- siderably less for Shasta and Bonneville varieties when the peas were held for 1; hours prior to and two hours following blanching. These two varieties were considerably more mature than the other varieties tested. In general, there was more variability in the degree of cats- lase inactivation after blanching than in the degree of peroxidase inactivation. No results are included for the ascorbic acid oxidase assays due to the writer's lack of experience with the method of assay and r _9_ mechanical difficulties in obtaining a calibration curve for the calorimeter used to measure the light transmission values. The neces- sary adjustments and modifications of the method have been made at this time, but the data taken on peas cannot be interpreted since there is no correlation between the results obtained under the orig- inal method and the present modifications. a>wwm H. was none Homoawzzmw tzo mm>me> wozzmezmze omeml weHOXT novel menoxl omen: mononT omen: monoxw omen: pascal owner monoxT Home Homes Home Momma Home Menus Home Momma Home Hanna Home .Hamuo .IJWII:IIJNIIIIIJNIIIJfiIdNIIIIllmfiilfilmfilllIIImWIIfilmWIII:I1mWIIfiImfiIIIlllmfillllmfil. H. mesoeon swasoce aonw mownwsm weapon 0 surname oo.m om.o om.m ow.H om.w woo oo.w ww.m om.r Poo ou.a woo mo apogee woo we.» 3b 33 woo woo web 3% 3% 3.0 3% 3; HNO Sheaves Hoo om.o oq.H woo woo om.< woo ow.r HH. mwmsosoa emcee r woman. aon% mownwnm weapon 0 Eflflafl H8 00.“ H8 “.8 0%.? @005? P8 gee Hmo spouses om.m we.» eo.m woo 0H.u oo.w Poo ©©.w . . o zoao" wmmm overcame ma wooed. wow u suscaou one even Masaawosoww tween ooowoa we Hemow..wm . DISCUSSION In discussing the results obtained in this series of experi- ments, the first point to be considered is the adequacy of the blanch- ~ ing treatment used. The blanching temperature of 20001:. and time of 2 minutes was chosen to insure an adequate blanch so that any differ- ences observed among the holding treatments could not be attributed to an inadequate blanch. For all practical purposes, the blanch can be considered adequate since the peroxidase assays indicated practi- cally 100 percent inactivation for all treatments and varieties. This is in agreement with the findings of Cruess and Joslyn (5) and Davis, et a1 (6). The Observed catalase activity in the samples blanched.without delay with no holding period after blanching may possibly be due in part to the error in the method and in part to the possibility that the catalase was not yet mlly inactivated. Balls (2) states that a high initial temperature followed by lower temperature may serve to inactivate an enzyme. It may be that this happened in these experi- ments with catalase. Balls (2) further states that enzymes may be "destroyed" by heat and yet reappear on cooling. The increased activity of catalase in the samples of Shasta and Bonneville varieties that were held four hours before blanching and two hours after blanching may be an exam- ple of the regeneration of catalase. This apparent regeneration oc- curred only in the two varieties that were most mature at harvest and consequently contained larger peas than the other varieties. The larger size peas might possibly have had some residual catalase en- zyme that was only temporarily inactivated by blanching. Though no results were obtained from the ascorbic acid oxidase .12... assays, it may be assumed that the blanching treatment was adequate to insure the inactivation of the ascorbic acid clc’ldase since Davis, et a1 (6) found that ascorbic acid oxidase was inactivated for most products in about a third of the time that it took to inactivate per- oxidase. Greater differences in treatments and more regeneration might have been obtained in these experiments if a lower blanching temper- sture had been selected. SW AND OOLGGLUSIONS The problem of the losses of vitamins and other nutrients dur— ing the processing of canned foods is the object of investigation at this and other institutions. The role that enzymes play in these loss- es is one aspect of the problem now being investigated. This experi- ment was initiated in an effort to learn whether there are any signif- icant differences in enzyme activity between varieties of canning peas. Pride, Ganner King, Shasta, Bonneville, and Perfection canning varie- ties of peas, grown on the college farm, were used in this experiment. Customary methods were employed in harvesting, vining, and cleaning the peas. Approximately six lugs of well mixed peas were di- vided into two lots, one of which was set in the shade for four hours before processing3and the other lot was immediately blanched at 200°F. for 2 minutes and then water-cooled. Samples were taken of the un- blanched, blanched, and canned peas. Catalase and peroxidase assays were run immediately on all samples, and at the end of one-half and two hour holding periods for the blanched samples. These assays were repeated for the lot of peas that was held four hours in the shade. The results obtained indicated that there is no significant differences between treatments or varieties under the conditions of this experiment. It is concluded that for the varieties of peas used in this experiment, there are no apparent differences in enzymatic ac- tivity of the adequately blanched peas and very little apparent re- generation of enzymes even under conditions of delay seldom encount- ered in commercial canning. LITERATURE CITED 1. Balls, A. K. and Hale, W. S. Determination of 6:Efiroxidase in agrlmll'hlral prOdIICtSO Joure A. O. A. 0. 5.11530 1933. 2. Balls, A. K. Control of enzymatic action in foods. Proc. Institute of Foods Technologists for 19h3. 165-169. 3. Balls, A. K. Enzyme actions and food.quality. Food technology. 10 ZIJS’Zflo 19,47. h. Glifcorn, L. E. The nutritive value of canned foods. I. Introduction and sampling procedure. Jour. of Nutrition. 28:101’1050 191th. 5. Cruess,‘W. V. and Joslyn, M. A. Significance of enzyme reaction mdehydration of vegetables. Proc. Institute of Food Technol- ogists for l9h2. 99-110. 6. Iths, Me Be,Eidt, Ce Co, I‘iaCArthur, Me, and StraCh-an, Ce Ce Factors affecting the quality of dehydrated vegetables. Proc. Institute of Food Technologists for l9h2. 90—98. 7. Ezell, B. D. and Gerhardt, F. The use of ascorbic acid as a substrate in oxidase measurements. Jour. Agr. Research. 60:89-99. 19h0. 8. Green, D. E. lechanisms of Biological Oxidations. Cambridge University Press, London. lBlpp. l9hl. 9. Jackson, J. M., Feaster, J. F., and Pilcher, R.'w. The effect of canning procedures on vitamins in foods. Proc. Institute of Food Technologists for l9h5. 81-90. 100 HOhl, Le Ac, Swanburg, J03DaVid, Jo, and Ramsay, Re “Cling of blanched vegetables and fruits for freezing. Food Research. 12:h8h-h95. 19h7. L1. 1%, Fe Ce, Pressley, A0, and 211011, To Nutritive Value Of canned foods. XXI Retention of nutrients during commercial production of various canned fruits and vegetables. Food Research. 12:273-287. l9h7. 12. Lucas,F l. H., and.Bailey, D. L. 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