CITRUS PECTINESTERASE INHIBITOR IN GRAPE LEAF

Transcription

1 KEW AND VELDHUIS: PECTINESTERASE Kramer, A. and I. C. Haut. Ripeness and Color Studies with Raw and Canned Peaches. Proc. Amer. Soc. Hort. Sci. 51: Showalter, R. K., S. A. Harmon, B. B. Brantley, D. W. Newsom, and J. F. Pittman. Changes in Congo Water melons After Harvest. Proc. Assn. Sou. Agr. Workers Vogele, A. C. Effect of Environmental Factors upon the Color of the Tomato and the Watermelon. Plant Physi ology 12: Zechmeister, L. and A. Polgar. The Carotenoid and Provitamin A Content of the Watermelon. Jour. Biol. Chem istry 139: CITRUS PECTINESTERASE INHIBITOR IN GRAPE LEAF EXTRACT Theo. J. Kew and M. K. Veldhuis U. S. Fruit h- Vegetable Products Laboratory1 Winter Haven Freshly prepared citrus juices are normally cloudy. Preservation of this cloud is of con cern to the producers of frozen concentrates and chilled juices. It has long been recognized that activity of the enzyne pectinesterase (PE) is generally associated with cloud insta bility in orange juice and a correlation has been observed (4). Complete or partial inactivation or inhibition of the PE is therefore desirable. Current methods for inactivating the en zyme involve the use of heat, which may affect the fresh flavor of juice products. of enzyme activity by means other than heat might be advantageous. Bell and Etchells (1) found an inhibitor of the enzyme polygalacturonase and cellulase in a water extract of grape leaves. Etchells, Bell, and Williams (3) used the extract to control soft ening of cucumber pickles during fermenta tion. Their work suggested the search for an inhibitor of PE from similar sources. Experimental Methods Raw Materials. Leaves of domestic musca dine grapes (Vitis rotundifolia, Michx.) were obtained from the vineyard of Dr. Charles Demko, Altoona, Florida. Varieties included Topsail, Stuckey, Tar Heel, and Thomas x Munsoniana. Leaves were also obtained from wild grape (Vitis munsoniana, Simpson) wide ly distributed in central Florida. The leaves were transported to the labora tory promptly after picking and rinsed in tap water. They were then dipped in dilute hydro chloric acid solution and rinsed again with tap water to remove spray residue. A final rinse lone of the laboratories of the Southern Utilization Re search and Development Division, Agricultural Research Serv ice, U. S. Department of Agriculture. v with distilled water was followed by drying in a current of air. Part of the leaves were used to prepare an extract and part were frozen for future use. Preparation of extract: The stems were re moved and 40 g. of leaf tissue were com minuted with 400 g. distilled water for 3 minutes in a high speed blender. The mixture was strained through four layers of cheese cloth, then centrifuged in 50 ml. tubes for 10 minutes at 1400 r.p.m. with a tip radius of 8 inches. Sediment was discarded. The super natant extract was used in this study and here after referred to as GLE. Partial purification of the wild grape leaf extract was effected by concentrating 5 vol umes to 1 under vacuum, and extracting re peatedly with ethyl ether. The active principle was returned to water solution, for comparison with the crude extract, by evaporation of the ether in the presence of distilled water. Pectinesterase activity. PE activity was de termined by the method published by Mac- Donnell, Jansen, and Lineweaver (7) and further described by Bissett, Veldhuis, and Rushing (2), except that a more concentrated solution of pectin (2%) was used to facilitate manipulation of concentrations. In this method the reaction mixture is adjusted to ph 7.5 and diluted to 40 ml., after which the rate of reac tion is measured. Dilution is normally made with distilled water. For the study of its inhibi tory effect, GLE was added in place of part of the water. It was therefore not necessary to individually dilute each control sample with out inhibitor to compare activities. Activities were expressed as pectinesterase units per milliliter of single strength or reconstituted orange juice multiplied by 10,000 (PEu/ mklo4). Cloud retention by visual observation. Por tions of reconstituted orange juices were placed in 1 x 8 inch test tubes fitted with

2 294 FLORIDA STATE HORTICULTURAL SOCIETY, 1960 Table.1 Effect of grape leaf extracts on pectinesterase activity of orange.juice Reduction Variety of grape Activity in activity PEu/mbclO^ % (juice w/o extract) 14#6 Stuckey 10,3 29 Topsail (old leaves) Topsail (young leaves) Tar Heel (young leaves) Thomas x Munsoniana 11* Wild grape (Vitis munsoniana) Table 2 Inhibitory effect on cloud loss by grape leaf extract used in the manufacture of 423 Brix frozen concentrated orange juice. Concentrate With GLE With GLE Percentage retention of cloud after storage Hours at 77 F# 5 S o 64 Days at 40 F TABIB3. Effect of time of contact between GLE and orange juice on the inhibition of PE activity Contact time Enzyme activity Minutes Table 4" Effect of varying the volume of grape leaf extract added to orange4 juice GLE per 6 ml, juice Activity ml S *3 13*0 9* screw caps. For preservation against microbial spoilage, two drops of toluene and sufficient sodium benzoate to make the mixture 1% were added to each tube. The tubes were sealed, stored at 40 F., and observed periodically under standardized lighting conditions for cloud stability. Cloud retention by the Loeffler method. The method described by Loeffler (5) for determining the cloud index of canned orange juice was adapted for use as follows. Recon stituted concentrate was centrifuged as above and the supernatant strained through 100- mesh screen to remove floating solids. Transmittance was measured with an Evelyn color imeter using a No. 720 filter. Measurements made before and after storage were used to calculate the percentage of cloud retained by each sample, the cloud density of the sample before storage being taken as 100%. Tests for inhibitory effect of extract. Single strength orange juice was mixed with an equal volume of GLE, and its PE activity compared with another portion of the same juice. Concentrated juices were treated with GLE in two ways. In the first, extract was used in lieu of one of the 3 volumes of water required to reconstitute 42 Brix concentrate to approxi mately single strength. Effect of the extract was determined by comparing the PE activity of treated concentrate with that of concentrate reconstituted solely with water. In the second, in which the addition of GLE might simulate its use in the manufacture of concentrate, 60 Brix concentrate was diluted to 42 Brix with extract. This, along with a control of 60 Brix concentrate adjusted to 42 Brix with water, was canned and stored at 0 F. The inhibitory effect of the extract was determined after storage of the concentrates at 40 or 77 F., by comparing the cloud retentions of the two concentrates. Table 5 Effect of heating GLE prior to treating orange juice Inhibitor Enzyme activity No inhibitor Unheated GLE Boiled GLE Autoclaved GLE

3 KEW AND VELDHUIS: PECTINESTERASE 295 % PECTIN Fig. 1. Non-competitive inhibition shown by the double reciprocal plot of PE activity vs. substrate concentration. The inhibitory effect of GLE on PE in vitro was also studied. A dry, salt-free PE prepara tion, free of suspended solids, was dissolved in dilute salt solution and treated with extract prepared from grape leaves which had been held in frozen storage for a year. This extract was added in the proportions of 1:1 and 3:1 of extract to PE solution. In order to eliminate any effect of suspended solids, part of the extract was filtered through a thick pad of filter aid and paper to remove any leaf frag ments or precipitates. Inhibition was deter mined by measuring the PE activity before and after treatment of the PE solution with, the regular and with the filtered extracts. Type of Inhibition. The method of Lineweaver and Burk (6) was followed in de termining the type of inhibition. A series of PE activities in a sample of reconstituted orange concentrate was run, by the method cited above (2), in which the pectin content of the reaction substrate was varied. Parallel determinations were made on juice with and without added GLE. A graph was made of the reciprocal of PE activities against the re ciprocal of the pectin concentrations. The best fitting line for the normal juice and for the juice plus GLE were drawn by the method of least squares, and extrapolated to zero concentration. Characteristics of these lines indicate whether or not the inhibitor competes with the substrate for the enzyme. Relationship of time to inhibitory effect. PE activity assays were made on orange juice immediately after mixing with equal volumes of GLE and at intervals up to 24 hours. Relationship of concentration to inhibitory effect. Holding eveiything but the volume of GLE constant, 6 ml. volumes of orange juice were treated with increments of GLE from 0 to 14 ml., and PE activities determined. Heat stability of grape leaf extract. A por tion of GLE was divided into three parts, one of which was autoclaved at 15 lbs. pressure for 15 minutes, another was rapidly brought to a boil and cooled, and the third was unheated. Each of these was added to an equal volume of orange juice and the PE activity determined. Results and Discussion The effect of GLE on cloud retention was tested by visual observation for leaf extracts of 3 varieties of grapes: Topsail, Stuckey, and wild grape. GLE was added to the test sam ples during reconstitution of concentrated juice, so no dilution effect was involved. All 6 tubes without GLE clarified within one month at 40 F. All 6 tubes containing added GLE showed no clarification after a month, and only 3 clarified in 4 months. Extracts from the leaves of all 5 varieties of grapes were effective in reducing the PE activity of reconstituted frozen concentrated orange juice, as shown in Table 1. Extracts from the leaves of cultivated grapes were mixed with equal portions of the same lot of reconstituted orange juice. The activity of this juice was 14.6 PEu/mlxlO4 before treat ment with GLE, while after treatment the activity was reduced in each case. The per centage reduction ranged from 19 to 43%. At another time leaves from wild grapes were included in the study. Another lot of reconsti tuted juice with different PE activity was treated with the wild grape extract and a re duction of 41% in its activity was observed. There is some indication that old leaves may produce a stronger inhibitor than do young leaves. Leaves from all varieties of grapes studied yielded pectinesterase inhibitor in their water extracts. An extract of leaves of an entirely different kind of plant, the oak (Quercus nigra,

4 296 FLORIDA STATE HORTICULTURAL SOCIETY, 1960 Linn.), did not reduce the PE activity of orange juice with which it was mixed. When GLE was used in lieu of one of the three volumes of water required to reconsti tute 42 Brix concentrate, the enzyme activi ty was reduced from 15.7 to 7.7 PEu/mlxlO4, or 51%. When GLE was used to reduce high dens ity concentrate to 42 Brix simulating possible commercial practice, there was a reduction in rate of cloud loss, as indicated in Table 2. Concentrate, held at room temperature of 77 F. for 7 hours, retained 64% of its cloud when it contained GLE and only 36% when cut back with water. When held at 40 F., cloud re tention after 4 and 5 days was more than doubled by the presence of GLE. When the water solution of PE was treated with an equal volume of GLE, activity was reduced from 32 to 23 PEu/mgxlO4, or 28%. When treated with 3 volumes of GLE, activi ty was reduced to 15 PEu/mgxlO4, or 53%. In another experiment in which the PE solution was treated with regular GLE and with fil tered GLE in equal volumes, activity was re duced from 31 to 24 units by the regular, and and from 31 to 25 units by the filtered GLE. It has been established by MacDonnell, Jansen, and Lineweaver (8) that PE as it exists in orange juice is not in solution, but bound to suspended matter. Its activity, therefore, can be influenced by mechanisms which pro mote or repress its solubilization. These re sults, which show that this inhibition is ef fective in filtered solutions, indicate that the mechanism of action of GLE is independent of adsorption of PE on suspended matter. The inhibitory effect of GLE on PE in orange juice was not influenced by prolonged contact times, as indicated in Table 3. There was no marked change in enzyme activity between assays started promptly and those made up to 24 hours after addition of GLE. Pectinesterase activity decreased with in crease in the volume of GLE added to a fixed volume of orange juice as shown in Table 4. The greatest reduction was found to be when the volume of GLE to orange juice was in the ratio of 5 to 3. Partial purification of one sample of GLE by removal of non-ether-soluble materials re sulted in no appreciable loss of inhibitoiy ef fect. The partially purified water clear extract reduced the enzyme activity of orange juice from 17.2 to 9.6 PEu/mlxlO4, or 44%. This suggests that a purified product could be readily prepared. It also suggests that the active ingredient of GLE is fairly stable. The stability of GLE toward heat is illus trated in Table 5. Quickly boiling the extract reduced its effect by 17.5%, or from 23% inhibition of PE activity to 19%; while autoclaving reduced its inhibitory effect by 53%. Some insight into the nature of the inhibitor was obtained by studying the relationship of inhibited and non-inhibited PE activities with pectin concentration in the reaction substrate. A non-competitive inhibitor may combine with an enzyme independently of the presence of the substrate, such as by heavy metals re acting with sulfhydryl groups resulting in the elimination of functional groups. A compe titive inhibitor competes with the substrate for active groups of the enzyme and the rela tive affinities of the inhibitor and substrate for the enzyme govern the degree of inhibition. When the reciprocals of inhibited and noninhibited enzyme activities are plotted against reciprocals of pectin concentrations, a line may be drawn for each reaction. When inhi bition is non-competitive, both the slopes and intercepts of the two lines differ. When inhibition is competitive, only the slopes differ. Data obtained by the method of Lineweaver and Burk were plotted as shown in Figure 1. Location and slope of the lines indicate that GLE inhibitor is non-competitive. This differs from the action of material found in grape leaves by Bell and Etchells (1) which inhi bited polygalacturonase and was shown to be competitive in nature. The existence of an inhibitor for PE in a plant extract has been demonstrated. This fundamental information is of sufficient value to warrant publication at this time. The addi tional information presented on the stability and non-competitive nature of GLE should be of value if a more comprehensive study of the material or its mechanism of action should be undertaken. Summary Water extracts of grape leaves were found to inhibit the PE activity and to improve the stability of cloud in orange juice and in frozen concentrated orange juice. The inhibitory fac tor was found in the leaves of four varieties of cultivated grapes and in wild grapes, but not in oak leaves. At an extraction ratio of

5 SAENZ: OKRA parts water to 1 of grape leaves, the ex tract was found to exhibit its greatest inhibi tory effect when mixed with juice in 5 : 3 proportions. The inhibitor was fairly stable when quickly boiled and cooled, but lost most of its effectiveness when autoclaved. It was demonstrated that the inhibitor was non-com petitive, i.e., did not compete with the sub strate for reaction with pectinesterase. The active principle could be extracted from water solution with ethyl ether, a characteristic which should facilitate purification and identi fication. Acknowledgment The authors gratefully acknowledge the as sistance of W. Clifford Scott in the prepara tion of the manuscript. LITERATURE REFERENCES 1. Bell, T. A., and Etchells, J. L. Pectinase inhibitor in grape leaves. Botan. Gaz., 119: (1958). 2- Bissett, O. W., Veldhuis, M. K., and Rushing, N. B. xv heat treatment temperature on the storage life of Valencia orange concentrates. Food Technol., 7: Etchells, J. L, Bell, T. A., and Williams, C. F. Inhi bition of pectinolytic and cellulolytic enzymes in cucumber 12r-m204a208nSn958?CUPPernOn9 9raP6 leaves# Food Tecnno1-/ 4. Kew, T. J. Changes in commercial frozen orange TtiT^ ^T^T^ "" F'ld,3fr308i3!4^94if 6. Lineweaver, H., and Burk, D. The determination of 658^666 n934)c'atlon constants> J* Amer- Chem- Soc-/ 5* 7. MacDonnell, L. R., Jansen, E. F., and Lineweaver, H. 6-e389r40iertne945?f range Pectlnesterase. Arch. Biochem., SOME APPLICATIONS OF OKRA IN THE FOOD INDUSTRIES1 William Saenz The Marine Laboratory University of Miami Miami Okra, also known as GUMBO, GOMBO and OCRA is given the scientific name of Hibiscus esculatus L., and belongs to the fam ily Malvaceae. It was first described by Abul- Abbas el-nebati, a Spanish botanist who found it in Egypt in The plant was grown in the Nile Valley and was used as food by the Egyptians. Reports indicate that as early as 1658 it was introduced in Brazil, and the first mention of the vegetable in North America was in Philadelphia in By 1781 the plant was being cultivated in Virginia and since then it has spread to many other states. Until 1925 okra was typically a vegetable of the Southern States. Since then, however, it has appeared with increasing frequency in the northern markets, and preservation by canning and freezing has increased its popu larization. The use of okra as a foodstuff is well known. In this paper some interesting properties and some less well known industrial applications are discussed. The John A. Manning Paper Co. of Troy, New York, a large consumer of vegetable gums as deflocculants in paper manufacture, com- ^ontribution No. 299 from The Marine Laboratory, Uni versity of Miami. missioned the Arthur D. Little research or ganization to find a substitute for karaya gum. The supply of this gum from India was not stable, and it was imperative to find a suitable alternate material in the Western Hemisphere. As a result of over seven years of investi gation with various agricultural products de hydrated okra proved to be suitable. The defloculant properties of the mucilagenous ma terial obtained from okra even surpassed those of karaya gum. Chemical constitution. The chemical constitution of the okra gum has not been completely determined. Since the whole okra pod is dehydrated it is assumed that approximately 50 per cent of the gum is made up of cellulosic material. The other half, the portion that imparts the ropiness in aqueous solutions, is believed to be a polymer of uronic acids and sugars, of which one at least is rhamnose. The elemental analysis is carbon 40.4 per cent, hydrogen 6.1 per cent, nitrogen 2.4 per cent, ash 6.6 per cent; cal cium is a major constituent of the ash. Ref erences in the literature state that some mucilaginous materials incorporate calcium and nitrogen in their structural formulas. Although the function of these elements is not yet known it is believed that they have a role in the rheological properties of their water solutions. Electron microscope examina tion of okra dispersions revealed a branched chain of over 4 microns in length.