PECTIN STUDIES Relation of combining Weight to Other Properties of Commercial Pectins’ AICSEL G. OLSEN, REINHOLD F. STUEWER, ELLIS R. FEHLBERG, AND NEAL M. BEACH General Foods Research Laboratories, Hoboken, N. J., and Fairport, N. Y .
tion proceeds beyond this range, the setting time is again rapidly decreased. Pectins of low combining weight show abnormal viscosities within certain pH ranges and in the presence of salts. The viscosity of pectin solutions may be used as an index of grade, provided differences due to salt and pH effects are eliminated. The so-called optimum pH for jellies is a fictitious value useful only when considered in connection with narrowly limited conditions of jelly making. It has no general significance. A buffered acid solution which permits grading of any commercial pectin at pH 3.1 is described.
The recent theories regarding the molecular structure of pectin are critically reviewed. The properties of six different pectins and several different methods of evaluating pectin are compared. The grade of pectin and the true viscosity of its solutions are functions of the molecular chain length. The setting time of pectin jellies and the effect of salts are functions of the combining weight of pectin. As pectins undergo progressive demethylation, the combining weight decreases proportionally and the setting time increases to a maximum i n the range of combining weight of 550-575. If demethyla-
A
FEW years ago pectin was considered only as a phytochemical substance useful in the manufacture of jellies and jams. With the increasing number of medicinal uses and the many other suggested applications in connection with such different subjects as ice cream, cheese, bakery products, steel tempering, etc., the interest in pectin and pectin chemistry has greatly increased. Various methods for determining the value of pectins and pectin extracts have been proposed from time to time. These include jelly grade and viscosity, and more recently emphasis has been put on rate of setting, sugar tolerance, and acid requirement. The latter qualities may vary, and can be varied, entirely independently of the property measured as grade or viscosity. The relation of molecular size to the properties of pectin is readily apparent. It should also be apparent that the degree of acidity of a molecule of 100,000-200,000 molecular weight may profoundly affect its behavior. Nevertheless, little has been done in the past to interpret pectin behavior in terms of the properties of different pectinic acids. (In this paper the terms “protopectin,” “pectin,” and “pectic acid” are used as general terms in the classical sense. Pectinic acid designates well-defined purified pectins of high molecular weight and varying acidity. Polygalacturonic acid is reserved for welldefined completely demethylated pectinic acids.) Sloep ($3) titrated pectic acid and found the equivalent weight to be 205. Bonner (4) used several different methods and obtained figures from 201 to 207. The latter also prepared a pectinic acid with a combining weight intermediary 1
For previous articles in this series, see literature citations 16, 17, $4.
between so-called neutral pectin and pectic acid. The acid characteristics of pectin and the existence of several pectinic acids were recognized by both Fremy (8) and by Chodnew (6). I n this connection it seems worth while to recall that in 1844 Chodnew prepared rather carefully a series of salts of pectic acid from turnips. These included calcium, barium, lead, silver, and copper salts. His results for the silver salts agree closely with those of Fremy and with some of those reported by Regnault (19). We have recalculated Chodnew’s data on the basis of present atomic weights, and although his salts were prepared with different samples and a t different times, all his results indicate an equivalent weight for pectic acid between 206 and 207. This is a remarkable agreement with the most modern work and indicates the degree of reliance which can be placed upon these early pectin studies. Both investigators prepared different pectins and attempted to show their relation to each other by the salts formed. Fremy’s “metapectin,” with 19.4 per cent lead oxide ash corresponds to a pectinic acid with a combining weight of about 470; Chodnew’s “pectinic acid,” which he said was “pure pectin” with 23.2 per cent silver oxide ash, was in fact a pectinic acid with a combining weight of about 400. Later workers failed to see in their pectin extracts the significant differences observed by Fremy and by Chodnew. The different types of pectin which these two early investigators found and described were attributed to the errors of their crude procedures, when as a matter of fact the failure to duplicate these results should have been attributed to a lack of vision and understanding on the part of the later investigators.
1015
1016
INDUSTRIAL AND ENGINEERING CHEMISTRY
The possibility of the existence of pectins of different acidity did not again become apparent until Fellenberg (7) published his studies on pectin; even then little attention was paid to the degree of acidity. Major attention was given to the importance of the percentage of methoxyl. However, even on the basis of the Fellenberg formula it was apparent that there were seven possible pectinic acids, and the more recent work suggesting long-chain molecules makes the possible number practically unlimited. A major difficulty preventing systematic study of pectinic acids has been the absence of a suitable method for preparing them. Although differences in degree of methylation were recognized, pectin preparations were accepted as prepared, and one studied either such pectin or its completely demethylated pectic acid derivative. Since the latter was usually obtained by alkaline treatment of the pectins, it invariably had lost, to a large degree, the colloidal properties associated with the original long-chain molecule. This was disclosed particularly by the relative viscosities obtainable with solutions of pectic acid salts as compared with solutions of the original pectin. A recently patented procedure (18) affords a means of preparing pectinic acids of any desired average combining weight and thereby permits the study of those properties of pectin which are determined by the degree of acidity. The procedure briefly comprises the treatment of the raw material-for instance, apple pomace or citrus peel-with sufficient dilute hydrochloric acid barely to moisten the material and result in a p H of about 0.7. When it is kept a t about 40” C., a gradual demethylation results without apparent reduction of the size of the molecular complex. Samples can be removed a t desired intervals during a period ranging from a few hours to a week, and the pectin extracted in the usual manner but preferably a t a relatively low temperature. After clarification of the extract, the pectin is precipitated and dried. Further purification of the pectin preparation can be accomplished in the usual way. Equivalent weight and methoxyl determinations show a gradual change in acidity of the different samples with duration of the treatment, The jelly strengths and viscosities are exceptionally high, proving that the size of the pectin molecule has not been appreciably reduced as invariably occurs when demethylation is accomplished by the usual alkaline treatment of pectin. Both combining weight and molecular size must, of course, be considered as average values resulting from the presence of molecules differing in both size and degree of acidity. This, however. need not affect the interpretation of the results. The recent excellent work of Schneider and eo-workers (20,21, 2.2) has verified the fact that pectins are long-chain molecules composed largely of partially methylated galacturonic acid units, and that the specific viscosity (of a pectin derivative in a nonpolar solution) as well as the jelly strength are directly related to the molecular size. According to Schneider, a molecular weight of 150,000-200,000 indicates a very strong pectin, 90,000-1 15,000 molecular weight indicates a moderately good pectin, and a molecular weight of 50,000 or less shows poor quality. The fact that demethoxylation with alkali results in pectic acid salts of low molecular weight may explain why attempts to remethylate pectic acid to obtain jelly-forming pwtin have not been successful. The work reported by Buston and Nanji (6) in 1932 has not been successfully repeated, and it is apparent that a change from pectic acid of low molecular weight to a pectin of high molecular weight requires something more than a successfulmethylation. Present knowledge makes it apparent that pectins may differ both in chain length and in degree of methylation. The former, as clearly indicated by Schneider, determines such properties as grades and viscoPity; the latter, as will be more
VOL. 31, NO. 8
fully explained in the present paper, determines such characteristics as setting time, acid requirement, sugar tolerance, reactivity with metallic ions, etc. Pectins also differ in what Schneider calls “ballast material”-that is, pentosans, galactans, etc.-carried along with the pectin and usually analyzed as part of the pectin. These substances may modify the pectin characteristics.
Equivalent Weight of Pectin and Degree of Methylation Much attention has been given to the determination of the percentage methoxyl in pectin. However, the method for methoxyl determination requires considerable time and skill. As Sloep (93)and Bonner ( 4 ) pointed out, either pectic acid or pectins (pectinic acids) may be routinely titrated with standard alkali using phenolphthalein as the indicator. The combining or equivalent weight thus obtained is a reliable means of determining free acid groups and is therefore also a measure of the degree of methylation. The method is exceedingly simple and rapid. The current practice in this laboratory is as follows: A 10-gram sample of pectin is suspended in 60 per cent alcohol containing 5 per cent concentrated hydrochloric acid by volume and stirred for 10 minutes; sufficient solution is used to make a thin slurry. The slurry is then transferred t o a Buchner funnel, and the pectin is washed with the acid-alcohol solution until no more color is removed and the spent liquor shows no test for metals. Then the pectin is washed with 60 per cent alcohol until the spent liquor shows no test for chlorides with silver nitrate. After one wash with 95 per cent alcohol, the pectin is air-dried and then dried in a vacuum oven for 16 hours at 60-65’ C. After removal from the vacuum oven, the sample is cooled in a desiccator, and three 1.000-gram samples are transferred to 400cc. beakers. Two cubic centimeters of alcohol are added t o each sample of pectin to wet it, then 300 cc. of distilled water are quickly added with constant stirring. The pectin suspension is left with occasional stirring for a half hour and is then titrated with standardized 0.1 N sodium hydroxide to a faint pink, with phenolphthalein as the indicator. The equivalent weight is calculated as usual. The equivalent weight of the corresDondina Dectic acid can be conveniently determined on the same sampl; by subsequently adding an excess of standard sodium hydroxide solution and back-titrating after the sample has stood for 2-3 hours. Regular practice is to add 20 cc. of 0.5 N sodium hydroxide to the titrated solution and leave it at room temperature 2 hours, then add 20 cc. of 0.5 N hydrochloric acid or the exact equivalent of the added sodium hydroxide, and complete the titration with 0.1 N sodium hydroxide. The equivalent weight of the pectic acid is calculated from the total cubic centimeters of 0.1 N sodium hydroxide used in both titrations. Bonner (4) reported high values for some dried pectic acid samples. This difficulty, which he ascribed to formation of anhydrides during drying, has not been encountered here. Direct titration has always yielded reliable results. On the basis of 201 for the equivalent weight of the pectic acid ion, the methoxyl content of different pectinic acids is readily calculated from the combining weight. The relation, considered on the basis of eight galacturonic acid units, is given in Table I, and the calculated percentage of carbon dioxide corresponding to each is also included. I n this connection, Myers and Baker (14) reported carbon dioxide values ranging from 19.8 to 21.0 per cent and averaging close to 20.4 per cent for pectin with high methoxyl content. There is no reason except common usage for calculating the relations in Table I on the basis of Fellenberg’s eight galacturonic acid units. The work of Link and co-workers (2, 1%’) indicated a t least eight to ten units in the polygalacturonic acid residues obtained from a mild hydrolysis of pectic acid. For the present purpose it does not matter whether the calculations are made on the basis of an eight-unit fraction or on the approximately thousand units in one of Schneider’s mole-
INDUSTRIAL AND ENGINEERING CHEMISTRY
AUGUST, 1939
cules. In either case the actual values obtained must be viewed as averages. The various formulas suggested by Ehrlich, by Nanji, Paton, and Ling, and by Myers and Baker have served their purpose; but they are clearly out of harmony with the properties of pectin as now understood and need no longer be seriously considered. In this connection, attention should be called to the fact that the formula proposed by Schneider presupposes an equivalent weight (combining weight) of 176; yet the equivalent weights reported by Sloep and by Bonner, and repeatedly determined by us with pectins from various sources, all come close to the Bonner figure of 203. For this reason the conclusion of Schneider that the pectic acid molecule contains only chains of galacturonic acid units cannot be wholly accepted. Bonner (3) considered pectic acid as made up of long chains of galacturonic acid “with a n occasional galactose and arabinose unit scattered along the chain.” Although this view was rejected
also an ash equivalent to one calcium and one sodium. Such a preparation would analyze as follows : equivalent weight, 204; methoxyl, 0.54 per cent; ash (CaO and Na2C03), 1.9 per cent. However, ash is normally brought down to a fraction of one per cent and therefore cannot account for the apparent inactivity of an average of three carboxyl groups out of every thirty-two. The preliminary assumption is made here that an average of one group in thirty-two may remain methylated even after the usual alkaline treatment. The question of whether pectinic acids are anything more than partially methylated polygalacturonic acids must therefore remain unsettled for the present. Pectinic acids of high quality and with average combining weight ranging from 800 to below 300 have been prepared by an acid treatment of the raw material as described by Olsen and Stuewer (18). These show an increasing sensitivity towards metal ions and a decreasing sugar tolerance in jellies, and are characterized by minimum setting rate in the range of 600-550.
OF COMBINING WEIGHTOF PECTINIC TABLEI. RELATION ACIDSTO PERCENTMETHOXYL
Calcd. CHaO/
COOH 7:l 6:2 5:3 4:4 3:5 2:6 1:7 0:s
Com-
Mol. bining Weight/n* Weight
1706 1692 1678 1664 1650 1636 1622 1608
1706 846 559 416 330 273 232 201
--
yo Methoxyl-
Calcd.
J3etd.a
12.7 11.0 9.2 7.4 5.6 3.8 1.9 0
10:6 ‘(910)
10.1 (560) 7.3-7.4(402-420) 5.7 (340) 0 : 5 6 (240)
...
Calcd.
%
co2
20.6 20.8 21.0 21.15 21.3 21.5 21.7 21.9
The,CHaO values were obtained for pectinic acids with the oombining weights indicated in parentheses. All were metal-precipitated and subsequently washed with acidified 50 per cent alcohol to remove ash constituents and other impurities. = actual number of galacturonic acid anhydride units per molecule a
*
~
8
by Schneider, it appears to be more in agreement with the equivalent weight observations. The average occurrence of two galactose units for each thirteen galacturonic acid units would give an equivalent weight of about 201 for the pectic acid; and such a molecule with nine of each thirteen units methylated would have an equivalent weight of about 685. The methoxyl would be 10.2 per cent and the carbon dioxide would be 20.9 per cent. The latter is in close agreement with the highest figures for carbon dioxide reported by Myers and Baker (14). Schneider ignores the question of equivalent weight but otherwise gives detailed analytical determinations for ten nitrated pectins. The figures obtained for carbon, hydrogen, methoxyl, and nitrogen would be equally applicable to a pectin containing 13 per cent galactose, but such a pectin nitrated to the extent indicated by Schneider’s analyses should yield only about 15 per cent carbon dioxide, whereas the average of about 17 per cent carbon dioxide found by Schneider agrees with a molecule containing only galacturonic acid anhydride units. Much, therefore, hinges upon the reliance to be placed on the carbon dioxide determinations, and whether, under the conditions of the nitration procedure, the possibility remains for oxidation of primary alcohol groups in the 6-position, in which case all analytical differences would be eliminated. Link and co-workers ( l a ) have shown that it is difficult to prepare pectic acid completely free from methoxyl. The residual methoxyl of well-purified samples was in the neighborhood of 0.5 per cent. This is in agreement with our own experience. Ash is also difficult to remove completely. However, in order to explain the discrepancy between the experimental value of about 203 and the theoretical value of 176, it would be necessary to postulate not only one residual methoxyl for every thirty-two galacturonic acid units, but
1017
Viscosity of Pectin Solutions The viscosity of pectin solutions has long served as a practical indication of the strength of pectin extracts. With a relation assumed between molecular chain length, viscosity, and jellying power, the viscosity measurement suggests itself as a logical substitute for the more cumbersome jelly strength determinations. The use of viscosity as an index to pectin grade is, however, not a fully reliable guide except for routine daily comparisons between extracts prepared in the same manner-for instance, in normal factory operation-or for comparison of solutions of pectins known to be prepared by the same procedure. Pectins of varying combining weight vary greatly in sensitivity to metallic ions. Although the presence of calcium as an impurity may have little effect on the viscosity of a pectin with a combining weight of 1000, it may render one with a lower combining weight abnormally viscous. As Table I1 shows, even sodium chloride in dilute solution has a remarkable effect within certain p H ranges on the viscosity of pectins of low combining weight. Therefore, prior to viscosity determinations, pectin preparations must TABLE11. VARIATIONOF VISCOSITYOF 0.8 PERCENTPECTIN SOLUTIONSWITH PH, WITH AND WITHOUT SALTADDITION A
Pectin sample: Combining weight: Solution: PHa
2.7 2.9 3.0 3.1 3.2 3.5 3.6 4.0 4.4 a
b
0.1 N Water NaCl 8ec.h 8ec.b
373
...
357
409 385 385
... 370 ... .350 . . ,... .. 348 . .. 351 329 355
C 360
B
580
415
-----,
Water 8ec.h
609
594
.... ..
529
. .. ...
550
0.1 N
NaCl 8ec.b 12 X.108
... ... 976 .460 .. 443
Water 8ec.h
0.1 N NaCl 8ec.b
576 978
Gei
915
... .395 ..
640
400 410
36
X 103
...
380
...
368
Pectin solutions were adjusted with NaOH t o the pH indicated. Modified Ostwald viscometer, solutions a t 25’ C.
be carefully washed free of such ash constituents as are removable by washing with acidified 50 per cent alcohol, subsequent to which the remaining acid must be removed by washing with neutral alcohol. The solutions prepared with the washed and dried pectins must be adjusted to a definite pH. A 1 per cent solution of such an acid-washed pectin will have a p H between 2.5 and 2.8; but as Table I1 indicates, the acidity should be adjusted to pH 4.4-4.5 with dilute sodium hydroxide, since the pectins with low combining weight exhibit a tendency towards abnormally high viscosities in the vicinity of p H 2.9.
1018
INDUSTRIAL AND ENGINEERING CHEMISTRY
Where mere routine comparisons are to be made, any type of viscosity apparatus may be used. In the present study both the Ostwald and the Engler viscometers were employed. the solutions being maintained a t 25” C . With powdered pectins the solutions are preferably prepared one hour prior to the determination. If taken immediately, the viscosity will be slightly higher and will be observed to drop gradually for several hours, the rate of viscosity loss following an asymptotic curve.
60
50
L4-,
5
0
X
c 40
w p/
l-
SO
2cl
Y
3
-
20 -
\
’ 1
PECTIN.COMB. W E I G H T
~NO.1-------1200 0
+
10
- _ - _ _ _7 _6 0 N0.3 - - - - - - - 5 8 0 V 0 . 4 ------- 660
\. e N0.2
20
40
60
ao
loo
120
T I M E MINUTES FIGURE1. EFBECTOF COMBININQ WEIGHTON SETTING TIME
Copper is gradually taken up by pectin solutions and caubes an increase in viscosity. Hence such solutions should not be left standing in the viscometer. With pH and salt effects eliminated, the need for special procedures such as that suggested by Schneider et ol. does not appear essential for commercial routine comparisons; and the correlation observed between grade and viscosity is ns good as that which Schneider observed between viscosity of iiitrated pectin in nonpolar solution and jelly strength (Figuic 2, Table 111).
Determination of Setting Time The setting times of pectins differ greatly. With any given pectin the setting time is influenced by the method of preparing the jelly, by the acidity, by the sugar concentration, and to some extent by the salts present. With any given set of conditions pectins will vary in accordance with the degree of demethylation. Highly methylated pectins are quick setting, moderately demethylated pectins are slow setting, and a t still lower methoxyl content the pectins again become quick setting. Commercial pectin samples tested in this laboratory have ranged in combining weights from 1550 to 400. The setting time is easily observed in a qualitative way by merely noting the set of a jelly at intervals after pouring. A convenient way of obtaining a quantitative result is as follows: The apparatus consists of a cylindrical aluminum vessel inches high, with a diameter of 31/4 inches at the top and 21/* inches at the bottom. (A cheap aluminum cocktail shaker serves the purpose very well.) The aluminum vessel, surrounded by a water bath has a 6/16-inchoutlet through the bottom and extending through the bottom of the water bath. As in the Engler viscometer this outlet is plugged by a tapered ‘/*-inch dowel. Three or more such vessels may be placed in the same water bath, thus permitting the simultaneous testing of several pectins. The water bath is maintained at 60” C. The jellies may be prepared by any standard procedure-for instance, that described by Stuewer, Beach, and Olsen @&-and are at once 5114
VOL. 31, NO. 8
poured into t’ e aluminum vessel. The exact time i s noted and one-ounria glasses are filled at exact intervals by lifting the plug. Sam1 les are taken in this manner until the jelly shows a definite cur lling or set. The glasses of jrlly are left 16-24 hours, and thc je ly strength of each is determined at 20” C. The setting +,meis clefined as the time represented by the sample which reg; terj t!,e first definite drop in jelly strength. Figure 1 shows a fypIc9.1 set of such data.
Pectin Grade In the determination of pectin yields, the qualitative evaluation is fully as important as the quantity of pectin obtained In fact, for all commercial purposes a quantitative determination of pectin necessarily involves grade determinations. The yields are reported, not as per cent of pectin, but as per cent of 100-grade pectin. Thus, if a given lot of pectin yields 12 per cent of 175-grade pectin and another yields 10 per cent of 280-grade pectin, the yields are reported as 21 per cent 100-grade and 28 per cent 100-grade, respectively. Inasmuch as pectins have been largely used for jellies, the grade is a quantitative expression of the sugar-carrying capacity of the pectin, specifically “the number of pounds of sugar carried by one pound of pectin in a standard 65 per cent jelly.” The characteristics of a standard jelly have been described by Jameson (10) who discusses the requirements in terms of firmness, texture, clearness, syneresis, color, and flavor In the actual grading of pectin the firmness is the major characteristic measured, but texture may greatly affect the impression of firmness conveyed, particularly where jellies are compared by the so-called finger test. The same firmness may be registered on a mechanical device, yet one jelly may be elastic and the other stiff but salvy; these diffcwnces in texture necessarily influence the “feel” of the jelly to the finger testef. The net effect is to favor the jelly prepared with a small amount of high-strength pectin over its numerical equivalent of low-strength pectin. Tarr (26) and Baker (1) greatly aided the quantitative study of pectin by devising their simple but accurate device for measuring jelly strength. Several others have been suggested since then (see, for example, citation 11). However, in the absence of any official standard for pectin such as the bloom values for gelatin, any device such as the TarrBaker jelly meter and other mechanical devices in more or less current use must be checked against an accepted commercially graded pectin-for instance, a 100-grade pectin; and readings must then be converted on the basis of such tests. Otherwise, amounts of pectins which result in jelly strength readings of the same magnitude must be used in a strictly comparative manner. The varying jelly strengths obtained with the same amount of several different pectins cannot be employed directly as a comparative index of grade. As Olsen (16) pointed out, the log of the jelly strength is a function of the log of the pectin concentration. As an example of how “grading” can miscarry if the definition of grade is taken to mean merely that “number,” the following may be taken as an illustration: Grams 100-Grade Pectin in 555 Grams Jelly 2.0 2.6
3.3
Grams Sugar
330 330 330
Jelly Strength (Tarr-Baker) 29 50 80
Sugar/PeoBin Ratio (Grade 9 ) 166 128 100
In each case the jelly would be called acceptable by some people, and on the basis of the sugar/pectin ratio the pectin might be graded either 166, 128, or 100; yet obviously, from the standpoint of commercial usage the last is the only correct figure. Considering the fact that an accepted official “standard jelly” does not exist, the variation found among commercial 100-grade pectins is small. Actually each set of figures given above could be used as a standard basis of comparison b y acceptance as a fact that 2.0 grams of 100-
AUGUST, 1939
INDUSTRIAL AND ENGINEERING CHEMISTRY TABLE 111.
COMPARISON O F
1019
DIFFERENT TYPESO F P E C T I N S AVAILABLECOMMERCIALLY Grade Comparisons of Pectins ceived
CombinNo.
m
Weight
Type of Pect.n
-
Setting Time
E ---
hl ethoxyls--
Calcd.
Actual
%
%
Engler Viscosity of 1% So1n.b
DH 2.9 DH 4.5
Min. 5 15
Sec.
Grade of Washed PectinC
Commercia1 finger test
60 % sol. solids at optimum pH
Synthetic juice at PH 3.1, 65
a8
Re-
Myers and Baker Grade,d 69.4% sol. solids
,“Optimum pH”60% 69.4% sol. sol. solids solids
See.
7 250 170 105 104 96 162 2.9 3.0 Apple rapid set 1200 9.3 274 131 10 6 158 84 90 2.7 3.0 168 85 2 Citrus rapid set 761 10.7 189 264 474 9 4 250 250 257 2.6 3.0 373 100 3 Apple slow set 577 9.1 438 120 141 82 162 10.1 151 143 80 100 2.6 2.7 9 2 4 Citrus slow set 560 278 120 486 265 434 504 278 294 2.6 2.9 5 Gra efruit slow set 8 8 9.8 518 350 Curdled 10 376 350 346 2.7 484 6 7 5 7.8 AppTe rapid set 715 420 (?I a Calculated methoxyl figures are based on ‘Table I relation; the actual values were determined by L.Baur. b Viscosities are on acid- and alcohol-washed pectins. The “grade of washed pectin” is obC Pectins 1,,2,,and 4, as received, were mixed with sugar; the pectin content was 58, 53, and 56 per cent, respectively. tained b y dividing regular grade b y % pectin/100. d The Myers and Baker grades are the ratio of sugar to pectin at a jelly strength of 50. The amounts of pectin to give a jelly strength of 50 were in the order-2.48, 3.05, 0.845, 2.48, 0.92 gram; it w a . not determined for pectin 6 because of curdling.
1
grade pectin will give a reading of 29, 2.6 grams a reading of 50, and 3.3 grams a reading of 80. Inasmuch as the instrument reading is both accurate and convenient a t 50, Stuewer, Beach, and Olsen (24)suggested that jellies be compared on the basis of the amounts necessary to give a jelly strength of 50; a standard 100-grade pectin is one that gives a reading of 50 when 2.6 grams are used in 555 grams of jelly prepared as described. Commercially produced pectins vary from about 125 to over 350 grade. Myers and Baker (14) reported “grades” of over 500, but they used the ratio of sugar to pectin in an approximately 70 per cent jelly a t a jelly strength of 50 as grade. Obviously this is an entirely different unit from the current commercial grade unit. 700
I
’
/
600
/ /
I I
/
Baker (IS), who showed that the acid requirement is shifted by the addition of various salts. The optimum pH, as previously shown ( 2 4 , is also shifted by changing percentage of solids and percentage of pectin, and by modifying the procedure for preparing the jelly. I n our work it has often been noted that pectins which in plain water jellies (GO per cent soluble solids) would show an optimum at 2.5-2.7 and fail to gel above p H 3.0, would nevertheless form perfect jellies at p H 3.0 or above if fruit juice was used instead of the water, the proper adjustment being made so the soluble solids still remained at 60 per cent. This suggests that optimum pH is not definite even under comparable jelly-making procedures. Various buffer salts have been used along with different acid concentrations in order to simulate this effect of natural fruit juice, but no simple mixture was effective. However, a mixture whose composition was based on the average analysis of a series of strawberry juices has resulted in dupliis, elimination of socation of the fmit juice effect-that called optimum pH. The composition of this mixture, to make one liter of synthetic juice, is as follows: Kapok KaCsHs07.HzO
2
400
2 300
t
4
5
61 ., 35 3 5 grams
(NasCsHs0~)~.11Hz00 . 9 8 5 1 , 19 Caa(CeHs07)2.4Hz0
Citric Mg(CzH3Oz)z aoid, anhydrous Suorose Water
2 . 0 2 grams 25.00 40.00 T o make 1 liter
This solution has a p H of 3.17 and 7.9 per cent soluble solids. In the preparation of jellies with 65 per cent soluble solids to be used for grade determinations, allowance is made for the 7.9 per cent soluble solids in the synthetic juice.
/ ’e
200
I
100 100
/I 150
Comparison of Different Type Pectins I
200
I
250
I
300
350
PECTIN GRADE VARIATION OF VISCOSITY WITH GRADEOF PECTINAT PH 2.9 AND 4.5
FIGURE 2.
As Tarr (25, 26) and Ogg (15) pointed out independently, the apparent strength of pectin is tremendously affected by the p H of the jelly mixture. For this reason jellies for grading purposes are commonly prepared with varying amounts of acid and the grade is determined at the optimum. The role played by acids in jelly formation and means of avoiding the necessity of determining the optimum have been discussed by Olsen (17) and by Stuewer, Beach, and Olsen (84). The effects of salts on jellies have received some attention, for instance, by Halliday and Bailey (9) and by Myers and
In the present study five different commercially prepared pectins and one laboratory prepared pectin were compared and graded by the commercial finger test method; by two modifications of the Stuewer, Beach, and Olsen method with the Tarr-Baker jelly meter; and finally by the method described by Myers and Baker (14)) also with the Tarr-Baker jelly meter. In the latter case the grade is the ratio of sugar to pectin at a jelly strength of 50; but in the case of the Stuewer, Beach, and Olsen jellies the grades were calculated on the basis that 2.6 grams of 100 grade gives a reading of 50 on the jelly meter. The results are shown in Table 111. Comparison of the grade given for 60 per cent jellies a t the optimum pH with that obtained with 65 per cent soluble solids and p H 3.1 shows that the latter is completely satisfactory and eliminates the necessity of determining the optimum acidity. On the other hand, it is apparent that the Myers-Baker method of using the sugar/pectin ratio in a 70
1020
INDUSTRIAL AND ENGINEERING CHEMISTRY
per cent jelly a t a jelly strength of 50 results in figures that are almost twice as high as those used in commercial grading. Because of the high sugar concentration, difficulties were also experienced with rapid-setting pectins with low sugar tolerance, such as pectin sample 6 (Table 111). Both combining weights and percentage methoxyl were determined on each of these six pectins. The viscosity of one per cent solutions was determined at two acidities, and the setting times were compared at p H 3.1. The results are presented in Table 111.
Discussion of Results I n Figure 2 the viscosity values listed in Table I11 are plotted against the grade. A fair correlation exists a t p H 4.5, and the correlation a t pH 2.9 is almost as good except for the 350-grade pectin. The abnormally high viscosity of this sample a t p H 2.9 has nothing to do with the grade but must be attributed to the low equivalent weight, which, as Table I1 indicates, results in abnormal increases in viscosity a t acidities near p H 3.0. A reduction in combining weight from 1200 to about 500 is accompanied by increasing setting time, beyond which a rapid decrease in setting time occurs. Although only one sample below 500 is included in this study, other unpublished data substantiate this relation between combining weight and setting time. The grade figures show general agreement between finger tests and the Stuewer, Beach, and Olsen grades, whether carried out a t the optimum or with synthetic juice at p H 3.1. The Myers and Baker method, as expected, results in erroneous values. The high sugar concentration used by Myers and Baker also tends toward very rapid setting and failure with pectins of low sugar tolerance, such as sample 6. The methoxyl figures are in general agreement with the values expected from the combining weights, except in the case of sample 1 which is alcohol-precipitated. When pectin extracts are concentrated and then precipitated by alcohol, nonpectin substances are precipitated with the pectin. This may amount to as much as 25 per cent of the total and therefore upsets calculations made from the combining weight determinations. Metal-ion pectin precipitates are, as a rule, less contaminated by organic impurities. Hence we note closer agreement in the other samples, all of which were metal-ion-precipitated. I n determining jelly strength and grade a t the optimum pH, a series of jellies with varying p H was prepared with each pectin. Of the six pectins listed, only two formed a jelly a t p H 3.0, both much weaker than a t the optimum. Three of the pectins failed to set even a t 2.9; yet by using the highly buffered synthetic juice, all registered the full grade value a t pH 3.1. This once more proves that an “optimum pH” does not exist, except for special narrowly defined conditions of jelly making. There is a minimum acid requirement for jelly making, but when the total ion concentration is large, the actual hydrogen-ion concentration is less significant. I n nonbuffered jellies the hydrogen-ion concentration is a measure of the optimum amount, and too high hydrogen-ion concentration interferes with proper setting of the jelly. However, with more completely buffered mixtures, the amount of acid can be greatly increased and the hydrogen ion a t the same time decreased below the so-called optimum; yet the pectin will form jellies of maximum strength. The only conclusion that one is warranted in drawing from these facts is that the conditions under which maximum jelly strength is exhibited are those under which the pectinic acid is least soluble, provided conditions are such as to permit the necessary solution and other handling of the jelly mixtures within the time limit set by the rate of gelation. This is in
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harmony with the often observed increase in jelly strength when small increments of calcium are added to a mixture deficient in either acid or sugar. The effect of acid and sugar are complementary, and to some extent one can be used to replace a deficiency in the other.
Literature Cited Baker, G. L., IND. ENG.CHEM.,18,89-93 (1926). Baur, L., and Link, K. P., J. Biol. Chem., 109, 293-9 (1935). Bonner, J., Botan. Rev., 2,475-97 (1936). Bonner, J., Proc. Acad. Sci. Amsterdam, 38, 346-54 (1935). Buston, H. W., and Nanji, H. R., Biochem. J., 26, 2090-6 (1932). (6) Chodnew, A., Ann.,51, 355-95 (1844). (7) Fellenberg, T. von, Biochem. Z., 85, 118-61 (1918). (8) Fremy, E., Ann. chim. phys., [3] 24, 5-58 (1848). (9) Halliday, E. G., and Bailey, G. R., IND.ENG. CHEM., 16, 595-7 (1924). (10) Jameson, E., Ibid., 17, 1291-2 (1925). (11) Leers, H., and Lockmuller, K., Kolloid Z., 42, 154-63 (1927); Fellers, C. R., and Griffiths, F. P., IND. ENQ. CHEM., 20, 857-9 (1928); Fellers, C. R., and Clague, J. A., IND.ENQ. CHEM.,Anal. Ed., 4, 106-7 (1932); Spencer, G., J. P h y s Chem., 33, 1987 (1929). (12) Morell, S., Baur, L., and Link, K. P., J. Bid. Chem., 105, 1-13 (1934). (13) Myers, P. B.. and Baker, G . L., Del. Agr. Expt. Sta., Bull. 144, (1926). (14) Ibid., 168, 1-46 (1931); 187, 1-39 (1934). (15) Ogg, W. G., djssertation, Cambridge, 1924 [r6sumb in T. N. Norris’ “Principles of Fruit Preservation,“ pp. 29-40 (1933)l. (16) Olsen, A. G., IND. ENG.CHEM.,25, 699 (1933). (17) Olsen, A. G., J. Phys. Chem., 38, 919-30 (1934). (18) Olsen, A. G., and Stuewer, R., U. S. Patent 2,132,577 (Oct. 11, (1) (2) (3) (4) (5)
1838).
Regnault, J . Pharm., 24,201 (1838). Schneider, G. G., and Boch, H., Angew. Chem., 51, 94-7 (1938). Schneider. G. G.. and Boch, H., Ber., 70, 1617-30 (1937). Schneider, G. G., and Fritohi, U., Ibid., 70, 1611-17 (1937). Sloep, A. C., thesis, Delft, p 78 (1928). 1241 Stitewer. - -, - - . , R... Beach. N. M.. and Olsen. A. G.. IND.ENQ. CHEM.,Anal. Ed., 6, 143-6 (1934). (25) Tarr, L. W., Del. Agr. Expt. Sta., Bull. 143, 1-37 (1923). (26) Ibid.,142, 1-33 (1926). (19) (20) (21) (22) (23) ~
PRISENTED before the Division of Agricultural and Food Chemistry a t t h e 96th Meeting of t h e American Chemical Society, Milwaukee, Wis.
Courtesg, U.S. Department of Agriculture
PIERCING ROQUEFORT CHEESETO ADMITAIR MOLDGROWTH
AND
PROMOTE
