Low Ester Pectin from Apple Pomace yiclclr 01' :ihi& y u d i t y prodH. H . JIOTTERX' A ~ CL-ILDE D H. HILLS y i ~ i i ~ ago s t1ii.s iict, ( h ) c4ficiently utilizing 1abor:itory undertook E a s t e r n R e g i o n a l Research L a b o r a t o r y , tlie catalyst (tomato p e c t : ~ ~ ) , the devclopmc.iit (it' a coniI . .%, U r p n r t m e n t oj ' 4 g r i c ~ l t 1 ~Philadrlphirl r~. 18. /'ti. :ind (c) ccirirdinntingthe ~('1)snicrcial prows.< for preparing rate step i i i t o :i unificd proclow estvr pectin. Initial contm. ' l h w ctudie- ctxter sideration n-as given to acid i siniplified procedlare is described f o r the preparatiol: :irou~itlthrc~esteps --ic.auliing, tie-esterification, a m e t h o d of low ester pectin from apple pomace, in which tonlato cxtractioii, nnd the enzymic \vhich had prcviously been pectaie is used as the de-esterification ratal?st. The esseilde-esterification process itself. studied by Bakcr and Goodtial step* in the process are polyphosphate cxlraction, filLEACHING.In the prop:tn.in (4). This method is time tration and concentration of the pectin extract, simultarlerittion of :I liquid pectiu con('011 s u m i n g, requires acid011sde-e$terificationand starch remo\al, enzyme inacti\acentr:tte from apple ponince, irc,*istaiit equipment, and tion, and precipitation and purification of the product. the p0mac.c is usually leached c : i i i ~ [ -time ~ drgradation of The proposed process is rapid and easily controlled and tvith cold water before cxt l i t > product. -4lkaline saponirequire9 only minor rhanpes in thc l l r u a ~procedure for traction t o remove sugars, firation \\-as discarded aftcr Iwrtin manufacture. salts, and coloring mnttcsr. (iiily slight c.onsiderntion, bcIn t h preparation 11f dry ( ~ i i s de-eitc*rification e by this pectiii this i,y less important because tlic precipitating agent I,
TABLE I. PECTINREMOVED BY LEACHIKG AXD EXTRACTIONS
Fraction Leach water Istextract 2nd extract 3rd extract
Jelly Units per G r a m Pomace 3 06
17 25
1.20 0 25
Vo of Total Jelly Units
Vol. 38, No. 11
simultaneously with the de-esterification reaction. The properties of three enzymes must be considered-pectase, diast,ase,and pectinase. [The nomenclature used for the pectic enzymes is that SOCIETY Committee on recommended by the AMERICANCHEMICAL Somenclature of Pectin ( I ) . 1 Pect,inase is a pectin-degrading enzyme present in small amounts in commercial diastase preparations and in tomato pectase extracts. The present study n a s concerned primarily with the selection of conditions of pH and temperature which give efficient pectase and diastase action yet minimize pectin degradation by pectinase and by heat. Tomato pectase was prepared from ground ripe tomatoes as previously described (8, fQ), and the extract was stored at 0" C. until used. The same extract was used for studies on tomato pectinase. The diastase (chiefly a-amylase) was a dried extract of mold mycelium sold under the trade name Clarase (supplicd by Takamine Laboratory, Inc.). Pectase activity vias determined by continuous electrometric titration a t constant pH in the presence of 0.05 A l sodium chloride and 0.002 M sodium oxalate (7). Diastase activity was determined by the Wohlgemuth method (21). Pectinase activity was determined by the rate of decrease in viscosity of a 1% solution of 180-grade apple pectin, the readings being limit,ed t o the initial 157, change in relative viscosity. Under these conditions the per cent decrease in viscosity was equal to the per cent decrease in jelly grade. This enables interpretation of pectinase action on the basis of per cent change in that property of pectin (grade) commonly used for determining its commercial value. The determination of pectinase in the presence of pectase required two different substrates in order to cover the entire pH range of activity: a pectic acid substrate for pH 4.0 and above, where pectase action would cause a change in substrate (pectin to pectic acid) and a drift in pH; and a pectin substrate a t pH 4.0 and below, to avoid abnormal viscosities caused by thixotrophy or by precipitation of pectic acid. w
e3
80
2
4
6
8
IO
3
12
PER CENT SODIUM TETRAPHOSPHATE (BASED ON WT. OF POMACE)
Figure 2.
Effect of Concentration of Sodium Tetraphosphate
Tomato pectinase (Figure 3) was active over the range p H 3.0 t o 6.5, with a narrow optimum a t pH 4.8. The pectic acid degradation curve was extended to p H values below 4.0 (dotted line) by multiplying the corresponding values for pectin by the ' rat'io between the two values a t pH 4.0. The rate of degradation of pectic acid at pH 4.0 was about three times the rate for pectin a t the same pH. This observation does not necessarily differ from that of Jansen and MacDonnell (IO)regarding the relative rates of glycosidic hydrolysis of pectin and pectic acid, since our enzyme extract was B mixture of pectase and pectinase. SUCCES~IVE Although it is apparent, that degradation by pectinase may be eliminated by conducting the de-esterification reaction a t pH 6.5 or above and by subsequently inactivating the enzyme by Calculated Grade of heating a t p H 3.0, it should be noted that the actual amount of D r j Pectin pectinase present in tomato pectase extrhcts and in diastase preparations is relatively small. The amount of pectinase in the various tomato extracts studied was sufficient to cause a decrease in viscosity of 0.9 to 1.27, per hour when 1 ml. m-as added to 200
INDUSTRIAL AND ENGINEERING CHEMISTRY
November, 1946
ml. of 1% pectin solution a t pH 4.0 and 30" C. This ratio of enzyme to substrate is the same as that used in the de-esterificat.ion of pomace extracts in 1 hour a t pH 6.5 and 30" C. The amount of pectinase contributed by the diastase ( C h a s e ) under these conditions would cause a loss of about 0.001% per hour, calculated on the ba6s of the ratio of enzyme to substrate used in starch removal. The diastase preparation has a much broader pH range than pectinase and may be used a t a pH at which the latter enzyme is inactive (6.5 to 7 . 5 ) . Although the relative activity of the dinstase in tliiq pH range is only 57 to 2870 of the maximum, the amount of diast:ise preparation required is small-about 3 to 10 granis per 100 gallons of apple pomace extract. I n the pH range 3.2 to 3.5, commonly used for starch hydrolysis of pomace extracts, diastase reacts at only 4 to 12% of its maximum velocity. Tomato pectase is only slightly less active at pH 6.5 than at the optimum pH of 7.5, but, because of the greater hest degradation of pectin at the latter pH at temperatures no higher than 40" C.. it appears desirable to conduct the de-esterification a t pH 6.5. Tomato pectase activity is also affected by salt concentration (?). Concentrated pomace extracts prepared by pol>-phosphate eztraction eont,ain sufficient cations in solution to girc full pectase activity, hut estracts prepared by hc:iting n-ith dilutcs mineral acids may require additional salt to piovide efficient peet nse action. The increase in pectase activity n-ith, increase i n suhstrate concentration ( 7 ) n-ould provide a 570 saving in quantity of enzyme in de-esterifying a concentrated (2y0)instead of a dilutc (0.8y0)pomace extract. The temperature a t which the de-esterification is conducted is extremely important, The principal considerations are the increase in pectase activity and the extent of heat degradation of the pectin a t higher temperatures. The Qlo value for tomato pectase is approximately 1.4 within the range studied ( 7 ) . At tcmperatures above 40" C. the inactivation of the enzyme bccomes a factor in the over-all rate for a given time interval. For that reason i t is better to compare the extent of reaction per unit volume of enzyme for a given time. On the basis of a 1-hour reac-
'rABI,E
11. EFFECTO F pH
ON
YIELDS
BY
POLYPHOSPHlTE
EXTRACTIOX OF APPLE POUCE
(90' C . , 50 min., 4Yc sodium tetraphosphate) Jelly Units per PH G r a m Pomace
2.5 3.0 3.4 4.0
17.20 17.30 17.45 16.90
TABLE 111. HEAT DEGR.4D.4TION O F P O M A C E EXTRACT .4T S'ARIOGS TEMPERATURES A N D pH VALUES Condition of Heating C. pH Control, not heated
30 40
F 5
50 GO 40
6 5 6 5 6 5 6.5
2;
Z,,.?J
TABLE
... 4 4
2
1 4 4 4
Iv. I K A C T I Y A T I O N
Condition of Heatingc. PH Control-not heated 4.0 4.0 GO 4.0 65 3 5 05 3.5
;;
Time, Hr.
Grade of Extract
1.82 1.75 1.55 1.44 1.30 1.55 1,39 1.26
Decrease in Grade %/Hod
... 0.95 3.7 10.4 28.6 3.7
Figure 3.
1155
pH- i c t i \ i t J Relations of Pectase, Pectinase, and Diastase
tion the observed amounts of de-esierification at temperaturc,s nf 30", io", SO", and 60" C. n'ere in the ratio of 1.0, 1.42, 1.68, :ind 1.36, respectively. The r:tte of heat degradation of a 1.82-grade pomace extract is recorded in Table 111in terms of per cent dccrease in grade per hour. Two facts are apparent: The rate of degradation was increased by an increase in pH from 6.5 to 7.5, and the amount of degradation a t p H 6.5 increased about 3.5-fold for each 10" C. rise in temperature. I n view of these results it does not appear economical to conduct the enzyme de-esterification at' a temperature above 40' C. or at a pH above 6.5. In general, it is advisable to heat-inactivate the pectinase in the de-esterifieation mixture, because the enzyme is precipitated with the low ester pectin and may be carricd through to the final dried product. The extent of pectinase inactivation in the presence of pectin was determined by heating mixtures of two parts of tomato pectase extract and three parts of pomace extract. Aliquots of the heated mixtures were tested for pectinase activity a t pH 4.0 on pectin substrate by the method previously described. The data (Table IV) indicate that temperatures of 65' C. or higher are required to destroy pectinase. An examination of the data for pH 4.0 and 3.5 indicates that lower pH values would be even more effective. Purification of the low ester pectin by washing with acidified alcohol eliminated about 95% of the pectiuase present. Thus either procedure, or a combination of both, may be used to eliminate pectinase from the final dried product. Pectase and diastase are effectively eliminated by conditions used for pectinase inactivation. I n fact, tomato pectase is readily destroyed by alcohol a t room temperature, SUGGESTED PROCEDURE FOR PREPARATION
A satisfactory procedure for the preparation of low ester pectin may be illustrated by the following example:
Eighty pounds of dried apple pomace were placed in a 200gallon stainless steel tank provided with steam coil and a propeller-type agitator. Hot t a p water was added to give a total 5.9 volume of 1.30 gallons, and the mixture was heated to 80" C. 7.7 Three pounds of sodium tetraphosphate and 830 ml. of dilute (1:5) sulfuric acid were added (final pH was 3.1). O F T O M A T O PECTIX.4sE I N PRESESCE The mixture was all&ved to stand for 60 minutes a t 80" C. and OF PECTIN then cooled to 60" C. by circulating cold Lvater through the steam coil in the extraction tank. The pectin extract \vas pressed from Time, 31in. yo Activity Retained the pomace by means of a hydraulic press and then filtered through a plate-and-frame filter press using a medium grade of .. 100.0 30 21.0 diatomaceous filter aid. The filtered extract (110 gallons) \vas 30 6.1 concentrated to 39 gallons in a forced-circulation vacuum evapo30 2.5 rator. 10 1.3 30 0 5 The pectin concentrate was placed in a 50-gallon tank equipped with an air-driven stirrer. .In aliquot of tlie pectin concentrate
INDUSTRIAL AND ENGINEERING CHEMISTRY
1156
EHZY ME- OEESTERlflED
/-•
2
Fipiirc 1.
3
4
PH
t
,
5
6
7
Comparison of Acid- and EnzjnieDe-esterified Pectins
was assayed for pcctiii nierliyl chtcr by tllc fu~~lJ\I'iilg iiioi1ifii;atioii of the previously described niethorl ( 8 ) : One hundred nil. (103 grains) of concentrate \vt:re weig a 600-nil. beaker and diluted to 500 ml. The solution 1 tralized t o pH 7.50, and 50 ml. of tomato pect,asc extr 7.5) ~vercadded. After 30 niinutes t,hc niixtu trated to pH 7.5; titration required 7.7 cc. of 0 the present experiment it, ~va8desired to hydro cnzyme IahliL incthyl eFt('r groups. This xiiiild requirt-
The pcctiii emcentrate \vas adjustcvl i u 1)Hti.30 a i d -10' ( : i i i i l 2 liters of tomato pectase extract (pH 630) and 10 grams of diastase (Clarase) were added. The niixture was stirred and tlit: pH maintained a t 6.50 by the continuous addition of 2.0 A\. S a O H from a large buret. The diaatasc~rtiaction reitched completion (negative starch-iodine test) in 30 minutes. Thv rlc,-citci,ification reaction v a s allo\wd to proceed until thc, requirctl amount, of 2.0 S S a O H (1420 nil.) liail 1) consumid (38 niiiiutes). The mixturc was immediately a ified to pH 4.0 11y adding 800 nil. of 1:5 sulfuric arid and t 1 heating to (i.5" (-'. for 20 minutrs. The low ester pectin was prwipitatA1 ivit 11 1.25 volumes of SOc/;: alcohol and pressctl on a hydraulic precc. The pressed prccipitatc as disintegrated ant1 covrred witli 10 gallons of 957; alcohol anti a l l o ~ e dt o stand for 2 hours. The. alcohol was again removed by pressing. The loiv ester pectin was dried 16 hours in a forced draft air oven at ground t o 60 mesh in a hammer mill. The yield or G.25Y6 of the weight of the apple pomace used. T h e product a n a l y x ~ d4.ac; nicthoxyl a n d wap calciilat(~tlto hc 3:3c; ( ~ ~ t v r i f i i ~ i l I.,
Vol. 38, No. 11
