Enzymic d
reparation and Extr ction of I
PECTINIC ACIDS H. S. OWENS, R. M. McCREADY, AND w. D. MACLAY Western Regional Reseatch Laboratory,
U. 9. D e p a r t m e n t of Agriculture, Albany, Calif.
The presence of a pectin esterase in citrus f r u i t peel has made it possible to develop an efficient procedure for the m a n u f a c t u r e of a series of pectinic acids employing the enzyme in situ. The partially de-esterified pectins extracted f r o m the raw material f o r m very viscous solutions indicative of long-chain molecules. S u c h materials,
which differ f r o m pectic materials now available commercially, m a y find wide usage wherever water-soluble oil-repelling films are desired. Various salts differ in solubility characteristics and offer other possibilities for use. The conditions for the preparation of a series of pectic substances are described a n d several uses suggested.
A
products appear to be even more fibrous than pectins isolated from the same peel after blanching, The products seem to have different characteristics from those prepared by previous investigators and may open new fields for the utilization of pectic materials. Citrus fruits and apples are the only sources of pectin and pectin esterase that we have examined. The wastes from these materials alone could yield over 50,000,000 pounds of pectic substances yearly. If the demand were great, other sources, such as pea hulls and sugar beets, would warrant investigation. I n the work reported here the juice was removed from citrus fruits by burring and from the apples by means of a cider press. The peels and pomace, containing 18, 17, 20, and 25% of total solids from oranges, lemons, grapefruit, and apples, respectively, were stored in a constant-temperature room at -35" C. Just before the kinetic studies were begun, the materials were quickly thawed, ground in a meat grinder, and blended with water in a Waring Blendor under carefully controlled conditions.
LTHOUGH pectin has been known for 120 years, only recently have serious attempts been made to prepare derivatives for other purposes than gelling agents. Activity has been stimulated by the fact that low-methoxyl pectins are showing considerable promise in certain fields. Further interest has been due to the increase in pectin manufacturing facilities, which will make possible, in the postwar period, a variety of pectic products beyond the normal.requirements of jelly manufacturers. A complete survey of the technical literature is beyond the scope of this paper but the following references indicate the trend of developments. Paul and Grandseigne (16) and Mehlitz ( I d ) prepared low-solids gels by allowing pectin esterase (pectase) (8) to act on pectin dispersed in milk or fruit juices, or in water containing sugar and a calcium salt. The de-esterification catalyzed by the enzyme yields a pectic derivative which reacts with the calcium in the dispersion and forms a gel; these gels undergo marked syneresis and have been used very little. Platt (16) described a method for treating a pectin extraction liquor, during and after extraction, a t low pH in order to produce a pectin having a long setting time. It is now known that the treatment of pectin solutions a t low p H values lowers their methoxyl content and increases their setting time. Variations of the acid method have been used by Olsen and Stuewer (l4), Baker and Goodwin (5), Hills, White, and Baker (4),and Kaufman, Fehlberg, and Olsen ( 7 ) in the preparation of pectic materials which form gels with low concentrations of sugar. The method of preparation requires from 12 to 48 hours and acidresistant equipment. Hills et al. also developed a method for de-esterifying pectin by use of pectin esterase from tomatoes, but the product obtained has a low calcium tolerance and low gel strength. An alkaline method developed in this laboratory (11) overcomes the disadvantages of the acid method and has a high rate of reaction. The product is useful for many foods, such as puddings, pie iillings, desserts, and salads. Baier and Wilson (8) and Wilson (18) have prepared a fibrous sodium pectate by treating citrus peel with alkali, followed by extraction with alkaline phosphate solutions. This material has been suggested for latex creaming, for paper coatings (2), and as a n agar substitute (1,
la).
The last-mentioned method depends upon a de-esterification of pectin in situ by the pectin esterase naturally occurring in the raw citrus materials and not upon an alkaline de-esterification as proposed by the orig-inal wbrkers. The pH region of maximum activity is far removed from that for the pectinase (6),and the
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DE-ESTERIFICATION
Weighed quantities of the blanched peels were titrated with standard alkali by means of a glass electrode assembly to determine the amount of alkali required to adjust each blended slurry to a definite pH value. This amount of alkali was added rapidly to weighed portions of fresh peeled pomace blended in water; then measured portions of 0.5 N sodium hydroxide were added at such a rate that the pH was kept within $0.3 unit of the desired value. Time was noted to the nearest second, and temperature was maintained within *0.5" C. No isolation of the product was necessary to establish the rate curves, although it was done on most samples as a further check. When the reaction had proceeded until enough alkali had been added to yield the desired information, the pH of the slurry was adjusted to 3 with 1:l sulfuric acid, sodium hexametaphosphate (Calgon) was added, and the mixture was heated to boiling with steam, The time of the hot extraction was 15 minutes. The hot extract was filtered by suction with the aid of Hyflo Supercel. The filtrate was cooled and poured into a n equal volume of 95% ethanol. The precipitate was strained on cheesecloth, hardened in ethanol, dried in vacuo at 60-70" C., and ground to pass a 60-mesh screen. The ash content of the pectins so prepared averaged about 18% and was primarily partially hydrolyzed products from the sodium hexametaphosphate. Methyl ester analyses were run by Hinton's method (5).
INDUSTRIAL AND ENGINEERING CHEMISTRY
October, 1944
Viscosities were determined on 1% solutions of unpurified lowmethoxyl pectins at pH 7 in a Stormer viscometer, calibrated with National Bureau of Standards oils No. H-1 and D, glycerolwater solutions, and water, at 25 * 0.5" C. The values reported have only relative significance because of the ash content. For each gram of pectin, 250 mg. of sodium hexametaphosphate were added to reduce the viscosity to a minimum and reprodu:ible value. This reduction was apparently due in part to a reduction in the concentration of polyvalent ions, such as calcium, and in part to a reduction of the electroviscous effect of pectin.
I
u 7
8
9
10
PH
Figure 1. Effect of pH on Reciprocal of Half-Reaction Time a.t 25" C. Open circles refer to a theoretical ourve based on the Michaelis equation for undissociated ampholytes.
The pH of maximum activity of the pectin esterase in situ was determined a t 25" C. with 500 grams of fresh orange peel in 1.5 liters of water as described earlier. A plot of the reciprocal of the "half-reaction time" against pH is shown in Figure 1. Half-reaction times have been used only for convenience and do not imply that the order or the mechanism of the reaction was the same throughout its course. Diffusion phenomena become more important as the reaction proceeds because most of the pectin is located in the peel particles. I n the case of orange peel, half reaction time was obtained when 35 ml. of 0 5 N alkali were added per 500 grams of peel. Figure 1 is based on averages of two or more rate curves such as those shown in Figure 3. The results indicate a maximum near 9 under the conditions of this experiment. Alkaline deesterification is appreciable at pH 10, but the results obtained a t the lower pH values are not so seriously affected because the rate of the alkaline reaction is decreased markedly. I t is interesting to note that the shape of the curve is similar to that deduced by Michaelis (13) for the fraction of undissociated ampholyte as a function of pH on the acid side of the isoelectric region. If it is assumed that the enzyme has 100% activity at pH 10, half-activity is obtained a t pH 7. Using this as the pK2 value for the enzyme and substituting the corresponding Kz value in the equation,
Kz
= Kz
pH technique described earlier. A plot of the reciprocal of halfreaction time against temperature' in Figure 2 shows an almost linear increase in the rate of enzymic de-esterification with increase in temperature above 16" C. At 0" C. experimental errors were greater. Ninety-five per cent of the activity was destroyed when the esterase in orange peel was heated at pH 2 at 70" C. for 15 minutes. The effect of kind of fruit was determined with a iatio of 125grams of raw material to 1.5 liters of water a t 25" C. and pH 7. The results are shown in Figure 3. Various factors such as amounts of pectin and enzyme, salt concentration, and pH maximum for the enzyme could cause the differencesobserved. EXTRACTION
t - /
' 6
937
+ [H+]
where p = fraction of ampholyte undissociated
KZ = Kw/Kbae the theoretical curve shown by the open circles in Figure 1 is obtained. The coincidence between the theoretical and experimental curves would indicate that, as the activity of the enzyme increases, it is changed from the anionic to the undissociated form. It would also imply that the isoelectric point is above pH 7. A similar theoretical treatment applied to the action of intestinal aminopeptidase by Wilson (19) indicates that this enzyme is also more active when in the undissociated form. The influence of temperature was studied a t pH 7 with 125 grams of fresh orange peel in 1.5 lihers of water, using the constant
The extraction of low-methoxyl pectins from the zource material presents a problem, because such metallic ions as calrium will precipitate them. Calcium and magnesium account for nearly one third of the metallic ions in the ash of citrus fruith, some of the calcium is known to be pr-rent in the form of pectates in the intercellular tissue. Accordingly it is necessary to add reagents which form calcium complexes to aid in the disintegration of the plant material and thus allow the extraction medium to permeate the cellular structures more freely. A very acidic medium could be used, but the purpose was to retain the most fibrous structure possible. Another possibility that was examined in an effort to improve the yield is solubilization (9) of the pectic materials with a suitable detergent.
TEMP., 'C.
Figure 2. Effect of Temperature on Reciprocal of Half-Reaction Time a t pH 7
In the extraction experiments reported here, fresh, ground citrus peel (orange unless otherxise stated) was used. After grinding, the pectic materials in the peel were partially deesterified by maintaining the pH at 8 for 5 minutes a t 25" C. The pH of the dispersion was adjusted to between 2 and 3. The dispersion was heated to 80" C. for 5 minutes to inactivate the enzymes and then filtered. The treated peel was stored at temperatures near freezing before use, although storage of dried peel would be more feasible commercially. An amount equivalent to 500 grams of fresh peel was dispersed in 1.5 liters of water, extracted with or without special extraction agents at various acid pH values a t 90"C. and above for 15 minutes, and filtered. The cooled filtrate was poured into two volumes of ethanol. The precipitate was dehydrated in ethanol and further dried at 60-70" C. in vacuo. Quantities obtained in similar tests varied as much as 15% with the extraction technique used. The methoxy1 contents of the products were between 3 and 4%. It is possible, however, by reducing the amount of alkali consumed 1 The energy of activation was calculated from the variation in the rate constant with change in temperatures. Such calculations give 6 X I O J calories, which is near the value for the energy of activation found for other hydrolytic enzyme8 (17) and can be compared t o 11 X 103 calories found for alkaline de-esterification of pectin (11).
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INDUSTRIAL AND ENGINEERING CHEMISTRY
in the de-esterification to prepare pectins having higher niethoxyl contents. Products containing as much as 9.8% M e 0 have been made. I
1
I
POSSIBLE USES
/+
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T I M E IN Y l N
Figure 3. Kate of De-esterification w i t h Different Fruits a t pH 7 and 25’ C. Curve 1, grapefruit; 2, lemon; 3, orange; 4, apple.
Of the special extraction agents tried, only sodium hexametaphosphate and sodium tetraphosphate extracted the maximum amount of pectic materials. Detergents, oxalate, pyrophosphate, and metaphosphate extracted one quarter to one half; acid alone extracted only one tenth of the pectic materials. The yield reached a maximum a t 6 grains of hexanietaphoiphate per 500 grams of peel when 13 grams of pectin were extracted. The yields and viscosities of low-methoxyl pectins extracted from treated peel in 15 minutes a t 95’ C. and at different, pH value.: follow: PH
a
3 4 5 6
aluminum, or other effective metallic salts. These procedures and the subsequent washings and dryings are standard techniques in t,he pectin field and need no amplification.
I
1 2 H
Yield, Grams 10 13 13 15 15
Vol. 36, No. 10
q , Celltipoihen
28 28 25
16
7
The pH appears to have little influelice on the yieid, but a t pH 5 and 6 there is marked degradation of the pectin. This influence of pH on viscosity makes it possible to prepare materials of diverse chain lengths. With an extraction period of 60 minutes at pH 3 the yield was 15 grams but the viscosity of a 1 ?’ & solution decreased to 16 centipoises.
Solutions of pectins prepared in t,his manner have unusua,lly high viscosities compared to those of pectins extracted by the usual methods. A solution of the most fibrous low-methoxyl pectin from orange peel had a viscosity of 36 centipoises, whereas a similar solution of the most fibrous pectin extracted from the same orange peel wit,h sodium hexametaphosphate had a viscosity of 22 centipoises. The reason for t,his high viscosity is being investigated. The enzymatically prepared material apparently has a longchain molecule which should form good films and fibers. Fibers which show good orientation have been prepared from metallic salts of the pectic substance. Films formed from them are flexible and fairly strong if they contain glycerol as a plasticizer. Since these films are softened by water (unless they contain a, high percentage of metallic salt), they would have only limited usefulness as such, but they can be used to coat, oil containers of various t>ypes,in a manner similar to fibrous sodium pectate (d), or for other surfaces that must be oil repellent. They have fair adhesive qualities and can be used to laminate cardboard and other paper products. They also appear to be useful in certain fireproofing compositions. Low-solid gels have been prepared from the calcium salt of pectins having lower methoxyl contents. Like the fibrous sodium calcium pectate gel (1, IO), this gel has been used in nutrient media for bacteria and molds in plaw of agar. The metallic salts, except those of the alkali metals, are insoluble in water, but the organic amine salt,s t>endto be soluble. The latter may be useful in certain types of emulsions and agricultural sprays ( 2 ) . Many other possibilities for the application of these fibrous niaterials are the same as t,hose given by Haier and Wilson for fibrous sodium pectat’e ( 2 ) . The method presented here, however, permits the production of pectins having high as well as low inet,hoxyl contents. Consequently, products can be prepared which are less sensitive to precipitation by metallic ions, such lis valciiini, and are thus more adaptable t,han fibrous pect8icacid. iCKNOW LEDGMENT
\Ve express our appreciation to Ruth Frenchman for the methoxyl det,erminat,ions, t,o Harry Lotzkar for the viscosity measurement,s, and to Samuel Waishrot for carrying out sever:tl of the extractions. LITERATURE CITED
METHOD OF MANUFACTURE
The equipment ordinarily used for the isolation of pectin can be utilized for this series of pectic substances. The changes in the usual procedure permit determination of the amount of alkali required to neutralize the acids in the slurry of peel or pomace and the amount of alkali required to obtain the desired degree of de-esterification. The 1:3 ratio of peel to water is satisfactory. After the slurry of peel and water is prepared, the alkali required to neutralize the acids and give the desired pH may be added. Then the amount of alkali required to produce the de-esterification may be added while the pH is kept constant. A somewhat more practical method is to add all the alkali rapidly and allow the reaction to proceed until the pH has dropped to 7. The reaction is stopped by adding acid, such as sulfur dioxide or sulfuric acid, to pH 3 4 . One pound of sodium hexametaphosphate per 100 pounds of peel is added, the pH is readjusted to 3-4, and the slurry is heated to boiling for 10-15 minutes, pressed, and filtered. After vacuum-pan concentration the filtered liquid may be used for many purposes. To obtain a solid product the concentrated filtrate is precipitated with alcohol, calcium, iron,
Baier and Manchester, Food Industries, 15, 94 (July, 1943). Baier and Wilson, IND.ENG.CHEM.,33, 287 (1941). Baker and Goodwin, U. S. Patent 2,133,273 (1938). Hills, White, and Baker, PTOC. Inst. Food Tech., 1942, 47. Hinton, “Fruit Pectins”, p. 33, New York, Chemical Publishing Co., 1940. Joslyn, Plant Physiol., 15, 675 (1940). Kaufman, Fehlberg, and Olsen, Food Industries, 14, 57 (Dec.. 1942); 15, 58 (Jan., 1943). Lineweaver and Ballou, Proc. Fed. Am. SOC.Biol., 2, 66 (1943). McBain, in “Advances in Colloid Science”, Val. I, p. 99, New York, Interscience Publishers, 1942. McCready, Owens, and Maclay, Science, 97,428 (1943). McCready, Owens, and Maclay, unpublished data. Mehlita, Biochem. Z.,256, 145 (1932). Michaelis, “Hydrogen-Ion Concentration”, pp. 60-7, Baltimore, Williams & Wilkins Co., 1926. Olsen and Stuewer, U.S. Patent 2,132,577 (1938). Paul and Grandseigne, Bull. asaoc. chim. sum. dist., 46, 283,
.
245 (1929).
Platt, U. S. Patent 2,020,572 (1936). Stearn, ETgeb. Enzgmjorsch., 7, 1 (1938). Wilson, C. W., U. S. Patent 2,132,065 (1938). Wilson, P. W., “Respiratory Enzymes”, pp. 203-5, Publishing Co., 1939.
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