Sugar Determination in Starch Hydrolyzates By Yeast Fermentation and Chemical Means ALFRED S. SCHULTZ, ROBERT A. FISHER, LAWRENCE ATKLV, AND CHARLES N. FREY The Fleischmann Laboratories, Standard Brands Incorporated, New York, N. Y.
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Two bottles are used for a “standardization curve”, one containing 0.5 pram of c . P. grade dextrose, the other 1 gram. An amount of the unknown which contains between 0.5 and 1 gram of fermentable sugar is weighed or pipetted into another reaction bottle. If the unknown is low in sugar, 0.5 gram of dextrose may be superimposed upon it. The total volume in each bottle should now be adjusted to 40 ml. by the addition of distilled water. When these preparations are complete, 10 mi. of a 10 per cent suspension of yeast are pipetted into each bottle. The bottles are then immediately connected to the gasometers. Fermentation is assumed to be complete when no more carbon dioxide is evolved during a 15-minute period. The difference between the amounts of carbon dioxide produced by 0.5 and 1 gram of pure dextrose is used to calculate the sugar content of the unknown. Dextrose is used as a standard throughout because it has been established that the fermentation of maltose hydrate or dextrose by baker’s yeast will produce identical volumes of carbon dioxide.
HE quantitative analysis of mixtures containing dex-
trose, maltose, and dextrins resulting from starch decomposition is one of the most difficult problems in organic analytical chemistry. h majority of the analytical methods are based upon the chemical reducing power of these hydrolyzed starch products, and are subject to certain errors due to lack of specificity. In the last two decades, however, methods have been devised which utilize the remarkable selectivity exhibited by certain cultures of yeast and bacteria in respect to their fermentative action upon sugars (3, 4, 7, 9, 11). These biological methods appear to be more specific than any purely chemical procedure. A direct comparison between biological and chemical analyses has not heretofore been made. I n the present communication the authors have attempted such a comparison in the determination of dextrose and maltose in a series of commercial starch hydrolyzates and in the successive steps of a n acid hydrolysis of starch. The latter work showed that certain sugar curves which have stood for 45 years as a classic picture of the relative composition of starch solutions during acid conversion may be in error. The biological method used was developed at this laboratory, and depends upon the measurement of carbon dioxide evolved during fermentation of sugar by yeast. The chemical method chosen is of the copper reduction type which Sichert and Bleyer (8) adapted for the analysis of commercial starch sirups and malt extracts.
Chemical Method Barfoed (2) in 1873 formulated a copper acetate reagent that is not reduced to any great extent by disaccharides. This reagent was adapted for quantitative use by Steinhoff (fO),who substituted sodium acetate for acetic acid and estimated cuprous oxide volumetrically with iodine and thiosulfate. I n the present work, the authors have used Sichert and Bleyer’s (8) modification of Steinhoff’s method, worked out for the analysis of acid or enzymically hydrolyzed starch products. In brief, to determine dextrose, they reduce copper held in an acetate complex in a sugar solution by heating the reagent beaker for 20 minutes in a boiling water bath. Precipitated cuprous oxide is filtered on an asbestos mat in a Gooch crucible. hfter being washed with hot water (60’ to 80” C.), the mat is transferred to the reaction beaker and stirred in an acidified ferric alum solution until the oxide is completely dissolved. The ferrous ion produced by reaction with cuprous oxide is titrated with standardized 0.1 N potassium permanganate. Dextrose and maltose are determined together as dextrose equivalent with the more alkaline Fehling’s solution in a similar manner. Milligrams of dextrose, value A , equivalent to milliliters of 0.1 N potassium permanganate can be found in Sichert and Bleyer’s copper acetate table. The dextrose equivalent, value B, is given by Sichert and Bleyer’s Fehling’s solution table. Thus maltose = 2(B - A ) . In place of the boiling water bath, the authors found it more convenient to use an Arnold steam sterilizer, adjusting the time of heating to 11 minutes by testing with pure sugars, so that they could use Sichert and Bleyer’s own tables.
Biological Method
The yeast fermentation method used is based upon the procedure outlined by Schultz and Kirby ( 7 ) , who determined fermentable sugars by measuring carbon dioxide evolved. Measurements are made with the fermentometer designed by Schultz, Atkin, and Frey (6), which consists essentially of a series of twelve 120-ml. wide-mouthed reaction bottles connected by means of pressure tubing to twelve 260-ml. gas burets or “gasometers”. The bottles are shaken at 110 cycles per minute in a cradle immersed in a 30” C. constant-temperature water bath. Fleischmann enriched baker’s yeast is used to ferment the common sugars-i. e., dextrose, fructose, sucrose, and maltose. A special yeast, Fleischmann Hi-B1 No. 2019, is used to ferment all the above sugars except maltose. The difference iq the total sugars fermented by the two yeasts therefore represents maltose. That part of the dextrin or starch subject‘to a t t a c k by 8-amylase may be determined by adding a source of p-amylase such as soy, barley, or STARCH HYDROLYZATES TABLE I. ASSAYOF COXMERCIAL wheat flour to the baker’s yeast ferTotal mentation. Sugar, Dextrose Subtracting from the total fermentFermen- Fermen- Chemical, Maltose Hydrate ables thus obtained the quantity of dextation, tation, copper FermenDifferProduct Yeast Ia yeast I1 b acetate tation Chemical ence trose and maltose previously determined in the original product, one,!s provided % % % % % % with a measure of the pamylaseA Malt sirup 60.8c 5.8 6.1 55.0 56.3 1.3 attackable substances”. B Acid and malt sirup 60.0 29.6 28.8 30.4 32.4 2.0 Each bottle, before the addition of C Malt sirup 55.0 2.7 4.2 52.3 59.5 4.2 sugar and yeast, contains the following D Corn sirup 34.8 16.5 17.5 17.9 32.1 14.2 E Dried low-converyeast nutrients: sion malt sirup 13.8 2.8 3.5 11.0 23.0 12.0 0 . 5 ml. of hfgS04.7H?O (150 mg. per ml.) 0 . 5 mi. of KC1 (100 mg. per ml.) 2 . 5 ml. of phosphate buffer (pH 6 . 0 to 6 . 5 ) containing per liter: 180 grams of NHaH2P04 72 grams of (N,Ha)?HPOa 0 . 2 gram of nicotinic acid
F Driedcornsirun G Dried low-conGersion malt sirup a
6 C
8-Amylase Convertible, Fermentation
% 1.9
6.7
7.6 28.7
38.0
18.6
19.8
19.4
35.0
15.6
36.5 38.5
16.3
6.3
6.5
10.0
24.4
14.4
56.3
Fleischmann enriched baker’s yeast, ferments common sugars. Fleischmann Hi-Bi No. 2019, ferments common sugars, but not maltose. 411 produots analyzed on “as is” basis.
496
ANALYTICAL EDITION
August 15, 1943
Experimental A group of seven commercial starch hydrolyzates which are in common use in the baking industry was analyzed by both the yeast fermentation and chemical methods. The results of these assays are presented in Table I.
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1
2
3
4
5
6
I
8
9
IO
II
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MlUlTERS OF 1-103 CONt HCI
FIGURE1. ACID HYDROLYSIS OF CORNSTARCH P e r cent suzars on d r v basis of starch A . Dextrose by yeast fermentation method B . hl altose by chemical method C. Maltose b y yeast fermentation method
The data on the acid hydrolysis of starch given in Figure 1 were obtained in the following manner:
497
limited conditions-namely, when the product under consideration contains a very small proportion of reducing dextrin. Sichert and Bleyer (8) analyzed a sirup of this type, and found that it contained 89.5 per cent dextrose (dry basis) and only 1.7 per cent maltose and 1.3 per cent dextrin. This is obviously not typical of a commercial starch sirup. For many years it has been customary to use the term “maltose” to describe both maltose and dextrins which reduce Fehling’s solution, and also mixtures of the two. Khenever the analytical method is not specific for maltose, some qualifying phrase should be used, such as “maltose equivalent”, “maltose plus nialto-dextrins”, etc. This distinction should be emphasized as much as possible because the use of “maltose” alone may be misleading. The availability of methods specific for maltose makes anything else obsolete. Rolfe and Defren ( 2 , 6) produced curves designed to describe the composition of a starch mixture in terms of dextrose and “maltose” a t any time during acid conversion (Figure 2 ) . They determined sugars by a combination of copper reduction and polarimetric methods, which apparently could not distinguish between true maltose and reducing dextrins. The results of the authors’ attempt to reproduce Rolfe and Defren’s curves are shown in Figure 1. Since the data obtained for dextrose content were nearly identical for both methods, only the fermentation curve for this sugar is reproduced. The “maltose” maximum attained by chemical analysis of the authors’ starch conversion series was 43 per cent which is lower than Rolfe and Defren’s maximum of 47 per cent, but close enough to show the similarity between the two methods used. The contrast between these maxima obtained by chemical means and the highest point on the true
A group of 100-ml. volumetric flasks, each containing 5 grams of cornstarch suspended in 50 ml. of distilled water to which varying amounts of dilute hydrochloric acid had been added, was autoclaved for 2 hours at a presure 9.1 kg. (20 pounds) in excess of atmospheric pressure. When the suspensions had been cooled, neutralized, and diluted to volume, they were analyzed for dextrose and maltose by each of the tlvo methods described.
Discussion The results of analyses of commercial starch hydrolyzates (Table I) by chemical and by fermentation methods do not agree, particularly in the determination of maltose. The difference is evidently due to the interference of malto-dextrin, or reducing dextrin, formed by the inconiplete hydrolysis of starch. Fehling’s solution cannot be used to distinguish maltose or dextrose from these other reducing substances. One may obtain a measure of the quantity of such reducing dextrins in a product by fermenting it with baker’s yeast in the presence of P-amylase, as previously described. A comparison of columns 4 through 7 in Table I will show a general relationship between the amount of starch or dextrin which can be converted to fermentable sugar by P-amylase and the reducing power these substances are able t o exert upon Fehling‘s solution. The difference between the results of the two methods for product A is 1.3 per cent and the amount of P-amylase-convertible starch is 1.9 per cent. I n product G, however, in which the P-amylase-convertible starch is 56.3 per cent, the difference betiyeen the two methods rises to 14.4 per cent. The cheniical method also tends to produce higher values for dextrose and this may be attributed to the slight reducing action caused by maltose on the copper acetate reagent. This is especially noticeable when maltose is present in large quantities. Sichert and Bleyer’s chemical method is claimed to be adequate for use in analyzing products of starch decomposition. It is satisfactory for this purpose, however, only under
DEGREES &Ip
FIGURE 2. ACID HYDROLYSIS OF STARCH BY ROLFE AND DEFREN (5) Per cent sugars on d r y basis of starch by chemical-polarimetric method -1.Dextrose B. Maltose
maltose curve, 25.2 per cent as determined by fermentation analysis, is a graphic demonstration of the wide difference between the two types of methods. This is in substantial agreement with work done in 1925 by Kanji and Beazeley (4), who, using a combined yeast fermentation-specific rotation method of their own, obtained a maximum of 29 per cent maltose in analyzing a potato starch conversion.
Summary A number of commercial starch hydrolyzates were analyzed for dextrose and maltose by measuring carbon dioxide
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INDUSTRIAL AND ENGINEERING CHEMISTRY
produced by yeast fermentation, and by the Sichert and Bleyer chemical method. Both methods gave satisfactory results in determining dextrose, but only the biological method, because of its high specificity, could be relied upon in analyzing for maltose. The chemical method is unreliable for the determination of maltose in the presence of the reducing dextrins which occur in both acid and enzymehydrolyzed starch products. P-rlrnylase can be used to give an indication of the amount of such reducing dextrins. The two methods applied to the analysis of the course of acid hydrolysis of starch showed that the longstanding work of Rolfe and Defren is in error, owing to the lack of specificity of Fehling’s solution for the determination of maltose in the presence of reducing dextrin.
Vol. 15, No. 8
Literature Cited (1) Barfoed, c,, z,
Chem., 12, 27 (1873),
(2) Browne, C. 8., and Zerban, F. W., “Sugar Analysis”, 3rd ed., New York, John Wiley & Sons. 1941. (3) Harding, V. J., and Nicholson, T . F., B i o c h m . J.. 27, 1082 (1933). (4) Nanji, D. R., and Beaaeley, R. G. L., J . Soc. Chem. Ind., 45, 215T (1926). ( 5 ) Rolfe, G. W., and nefren, G., J . Am. Chem. Soc., 18, 869 (1896). (6) Schultz, A. S., Atkin, L., and Frey, C. N., IND. ENC.CHEM., ANAL.E D . ,14, 35 (1942). (7) Schultz, A. S., and Kirby, G. W., Cereal Chem., 10, 149 (1933). (8) &chert, K., and Bleyer, B.. 2. anal. Chem., 107,328 (1936). (9) Somogyi M., J . Bioi. Chem., 119, 741 (1937). (10) Steinhoff,G., 2. Spiritusind., 56, 64 (1933). (11) Voorst, F. T . van, Chem Wsekblad, 35, 338 (1938).
Gas-Absorption Apparatus LUTHER BOLSTAD
AND
RALPH E. DUNBAR, North Dakota Agricultural College, Fargo, Pi. Dak.
D
U R I S G a series of acetylation studies with ketene it became necessary to devise a compact, flexible, and efficient type of absorption apparatus that would provide intimate and prolonged contact of the gas with the liquid reactant. After testing several arrangements of the traditional absorption equipment, the piece of apparatus shown in Figure 1 was constructed and has been found to be thoroughly satisfactory. The arrangement can be used for the absorption of other gases under similar conditions. The use of the glass-bead column was suggested by Sham ( I ) but this equipment is sufficiently different to justify description. -4 6-cm. piece of 6-mm. glass tubing, A , is sealed endwise to a 10-cm. piece of 12-mm. glass tubing, B. Four small oblong holes, C , of approximately 4-mm. diameter, are blown in the larger tube just above the point of sealing. A 6-cm. length of 20-mm. glass tubing, D, is then sealed t o B , just above the four openings. D should not reach to the lower level of A bv some 1 or 2”cm., in order to provide the correct circulating operation when the completed equipment is in use. This arrangement provides two concentric tubes, A and D, with B extending above the other t w o as shown. The upper end of R should be attached to an efficient reflux condenser during operation, by means of a rubber or g r o und-glass connection. The space between A and D is then nearly filled with FIC~T-RE 1 solid glass beads
and enough constrictions are formed at the lower end of D to hold. the glass beads in place. B is inserted through the opening of a, one-hole stopper that closes a 125-ml. filtering flask, E . The accompanying illustration shows all these essential features. Proportionate changes in dimensions may be made, so that this equipment can be used with any available size or type of filtering flask. The gas to be absorbed is then introduced through the side arm, F , of the filtering flask. The lower end of D should dip just below the surface of the liauid absorbent. As the Dressure within the flask increases, the liquid is forced up A and-D, until a few bubbles enter the glass-bead column. Here a percolating effect is produced, as gas and liquid ascend through the glass beads. A large portion of the resulting solution returns through A . As the volume of the liquid increases, owing to the absorption of gas, the same relative positions of liquid to absorption apparatus may be maintained by raising the glass tubes through the supporting stopper. This piece of equipment differs materially from that designed by Shaw ( 1 ) in the following major respects. It was constructed to handle relatively large volumes of liquids or solutions and highly concentrated gases in small volumes, rather than small volumes of liquids and large volumes of dilute gases. It has been employed in the preparation of numerous organic acetates in yields of better than 90 per cent of the theory, and in quantities of better than 40 grams of the final acetate. The absorption apparatus has operated continuously for 20 hours or more without further adjustment, except the raising of the absorption tube in relation to the liquid in the filtering flask. The rate of flow of ketene was 0.07 mole per hour (3 to 4 liters of mixed pyrolysis gases), although any slower perceptible rate of flow would be absorbed with the same quantitative efficiency. The average back pressure during operation is 50 to 60 mm. of mater, which is readily counterbalanced by any closed traditional gas generator. -4further advantage of this equipment is that precipitates, produced by reaction between gas and liquid, do not clog or hinder the operation, since there are no small openings to become clogged. I n case the gases employed are less soluble than ketene, the length of the glass-bead column may be extended to permit longer contact between the gas and liquid solvent. The entire piece of equipment is relatively cheap and can be constructed by any efficient glass blower from materials readily available in any laboratory.
Literature Cited (1) Shaw, J. A,, IND.EKG.CHEM., ANAL.E D . ,6,479 (1934).
