Fermentability of Cornstarch RELATION TO STARCH STRUCTURE RALPH W. KERR AND NORBERT F. SCHINK Corn Products Refining Company, Argo, Ill. 2A to 2E, we must qualify our N MANUFACTURING Further extension of our knowledge constatement by adding that all products from cornstarch cerning the composition and constitution reducing constituents of a corn for the fermentation indusof the starches is of industrial importance sirup are not necessarily fertries, various observations have in the production of many articles of commentable. In sirup 2E, for exbeen made which are significant merce made from starch. Recent attempts ample, the nonreducing dextrins to the chemistry of starch. have been reduced from 29.6 to One problem comes up in the have been made, for example, to improve 18.6 per cent by being split into production of a cornstarch sirup corn products supplied to the brew-era. substances now reducing in nawith the highest percentage of In this paper the authors point out the ture, but the percentage fermentfermentable dry solids. Starch theoretical significance of their results able has not increased. can be converted with acid into in a particular study in this field. These It is currently believed that a sugar which is almost comthe action of dilute acid on pletely fermentable. The use results claim to support the views on starch is manifold. While the of a solid, particularly a refmed starch chemistry, previously given by this hydrolytic action predominates sugar, is not desirable in certain laboratory, that the starches are heteroin the main, some synthetic acinstances; while a limited acid geneous and not composed of a single type tion is claimed ( 1 ) for producing conversion produces a noncrysof common molecule. From an applicacondensation products from tallizable sirup but not, as yet, dextrose; in addition a minor a sirup with a high order of fertion of principles given and an extension part of the dextrose may enter mentability. Our work has been of the work outlined, further improveinto side reactions of a destrucchiefly confined to enzymolysis ments in the manufacture of a highly fertive nature or into combination of cornstarch, with and without mentable corn sirup would be anticipated. with impurities. acid treatment; observations To answer the argument that which appear to have a general the results reported by us in theoretical significance in starch experiments where acid hydrolysis is invoked could be exchemistry are given. The results reported are representative plained on the basis of secondary reactions in the presence of of many similar experiments, both laboratory and plant scale, acid, we heated pure dextrose solutions in concentrations over a period of several years. shown in experiments 1 and 2E, respectively, under the conEffect of Acid Treatment Combined with Malt ditions of acid treatment given for 2E. The percentage ferConversion mentable was determined a t the beginning and end of the 2hour and 40-minute heating periods (used in making 2E). A If starch is converted by an extract of malt diastase, a sirup negligible drop from 94.9 to 94.7 per cent was found for the is readily obtainable, of which the dry solids are 70 per cent more dilute, but the more concentrated fell from 94.9 to fermentable or more. We examined such a sirup (No. 1, 89.4 per cent fermentable. The mean of these two, 2.75 per Table I) for its dextrose equivalent-that is, the total reduccent, indicates the probable loss in fermentable substance in ing value by the Fehling test calculated as dextrose-and the extreme case studied, due both to formation from dexfor its true dextrose content as determined by the Sicherttrose of new nonfermentables of higher molecular weight and Bleyer method (1.2). I t s maltose equivalent was calculated to the entry of dextrose into other side reactions. as the reducing sugars and reducing dextrins other than dextrose, expressed as maltose. The percentage of nonreHence, even for an acid conversion as extensive as that used in experiment 2E, it is not likely that we can explain the conducing dextrins was estimated by deducting the true dexstancy of fermentability and a simultaneous decrease of 9 per trose content plus the maltose equivalent from the total solids. Comparison of this analysis with the percentage of fermentcent in nonreducing dextrins as compared to experiment 1 as a fortuitous balance between conversion of original nonferable solids, as determined by the method of the American mentables into reducing substances which are fermentable, on Society of Brewing Chemists, seems to show that only the the one hand, and a re-forming of new nonfermentables, on the shorter chain carbohydrates are readily fermentable (that is, the chains short enough to show some reduction to Fehling other. It would appear, therefore, from experiments 2A to 2E that solution). the ability to reduce, which is probably related t o chain However, if we partially acid convert this enzyme-made length (6, 7), is not the only requisite to render a product of sirup at a p H of 1.5 with hydrochloric acid, a dilution of starch hydrolysis suitable for fermentation. Configuration 12' BB., and a temperature of 102' C. to produce sirups 1418
I
November, 1941
INDUSTRIAL AND ENGINEERING CHEMISTRY
1419
OB CORNSTARCH PRODUCTS TABLEI. ANALYSESAND FZIRMZINTABILITY
Sirup No. 1 2A
2B 2c
2D
2E
3A
3B 4A 4B 6 6 7A 7B SA SB 8C 9A
BB
Method of Production Straight malt conversion Malt conversion as in 1, then acid conversion Malt conversion as in 1 then acid conversion Malt conversion as in I’ then acid conversion Malt conversion as in then acid conversion Malt conversion as in 1: then acid conversion Acid conversion to 37 D E then malt Acid conversion to 64 D: E:: then malt Straight acid conversion Straight acid conversion Acid to 53 D E then fungus enzyme Acid to 68 D: E:’then malt Acid to 67.8 D. E then malt soybean Acid to 58.9 D. E:: then malt a-amylase Straight malt conversion (as in 1) SA acid oonversion (as 2C) SB malt conversion SA acid conversion (as 2 D or 2E) QA malt conversion
++
++ + +
Dextrose Equivalent (D.E.)
46.1 49.0 50.5 52.5 56.6 69.1 51.5 67.2 37.1 64.7 64.2 64.2 62.4 63.0 47.2 53.2 53.6 61.6 63.6
is probably an important factor as well. The conclusion can beddrawn that a portion of the cornstarch possesses a configuration different from the rest of it. If the steps in the production of sirups in series 2 are reversed-for example, if we first acid convert the starch to produce sirups 4A and 4B, and follow with a malt diastase conversion-sirups 3A and 3B are made. But in no case is a better fermentability obtained than that for sirup 1. We may conclude that the configuration which resists attack by malt and yeast enzymes is resistant to hydrolysis by acid also, in the sense that it is destroyed only in the last stages of acid hydrolysis. The experiments described above are sufficient to cast some doubt on the more simplified structures for starch proposed within the past decade. For example, the configuration of the “starch molecule” is probably neither so simple as proposed by Haworth (6) nor so symmetrical as proposed by Staudinger (IS),.if indeed there is but a single type of starch molecule. Neither can the chemistry of starch be explained by supposing that it is a conglomeration or mixture of chains of assorted lengths, the longest ones being that part of starch which resist enzymolysis. For on that basis, in producing the sirups of series 2, the longest chains have been partially converted or split into sections which are now short enough to show a reducing value but are, none the less, nonfermentable. Neither does Freudenberg’s explanation (3) agree with the above considerations. He proposed that the starch molecule is made up of diverse radicals-that is, a nonfermentable nucleus of a cyclic Schardinger dextrin with fermentable side chains. It is difficult to see how these cyclic nuclei could be broken by acid into relatively short reducing substances and at the same time be resistant to further enzymolysis, if the linkages within the cycle are simply that of maltose. It is reasonable to conclude, on the other hand, especially from experiments 1 and 2, that, as the nonreducing dextrins of sirup 1 are broken down into lesser products which are still nonfermentable, the molecular magnitude of the progenitor of the dextrins in sirup 1 might be even still larger than these nonreducing dextrins. It would then seem just as logical to conclude that the earlier views were more nearly correct: Starch is a mixture of at least two substances, one of which is more resistant both to diastatic enzymes and to acids. The differencesin structure are more fundamental than is currently believed and, for the more resistant portion, involve the union or joining of many dextrose units. Several other theories for starch composition and structure
Dextrose,
% 8.2
12.5 15.8 20.3 24.6 49.5 21.35 48.6 19.75 44.85 39.6 39.3 38.0 39.4 8.5
18.0 18.6 34.9 35.6
Maltose Equivalent
62.2 59.8 56.8 52.8 52.4 32.1 49.60 30.5 28.45 32.5 40.36 40.8
40.0 37.1 63.4 67.7 57.4 43.7 45.9
Dextrins,
% 29.6 27.7 27.4 26.9 23.0 18.6 29.05 20.9 51.8 22.65 20.05 19.9 22.0 23.6 28.1 24.3 24.0 21.4 18.5
Total Reduolng Sugar, %
70.4 72.3 72.6 73.1 77.0 81.4 70.95 79.1 48.2 77.35 79.96
Fermentables
% 7.1.2 71.9 70.4 71.4 71.4 73.3 65.2 71.4 40.4 68.7
68.4
80.1
68.9
78.0 76.6 72.9 75.7 76.0 78.6 81.5
66.2 69.7 75.3 74.2 74.6 74.5 75.6
which are out of harmony with our conclusions are not so vulnerable to the arguments just offered The purpose of this paper is to present experimental data to aid in deciding which theories are probably correct. Freudenberg recently proposed (4) that the structure of starch is essentially a branched chain, with one branch attached by a 1,6-glucosidic linkage for about every twenty dextrose units, and the latter joined normally through 1,4-aglucosidic linkages. That 1,6-glucosidic linkages probably do occur in starch is becoming fairly evident. But that the experimental findings in this connection should be construed to mean that all starches possess only one molecular configuration, principally a central chain with 1,6-a-glucosidic linked side chains, is not so certain Kerr and Trubell (8) anticipated and pointed out the dubious value of such concepts, grouped by them as the “multiple radical theory of starch structure”, into which category the branched-chain structure mi$ht be placed. Their arguments may be extended to show that the structure proposed by Freudenberg does not explain all of the behavior exhibited by starches. In particular, the 1,6-glucoside linkage, joining the side branch to the main chain as proposed by Freudenberg, would present a block to the enzyme in diastatic conversion. For example, @-amylase,starting from the nonaldehydic end of either the main chain or the side chain and splitting off successive maltose units, would be stopped when it arrived at the first point of branching, since a maltose configuration does not exist at this point. Likewise, for the a-amylase constituent of malt diastase, which splits starch into polysaccharides, difficulty would be encountered a t the point of branching. For example, suppose the product which fits the a-amylase factor is a hexasaccharide, as claimed by Hanes (6). Then this amylase would cease its activity before passing the point a t which branching occurs, unless we make the unusual assumption that the enzyme is not limited to any one type of configuration and may split off any hexasaccharide. From our knowledge of the “limits of conversion” of these two amylases, either used independently or together, some difficulty would be encountered in attempting to calculate the point a t which branching occurs, even in the simplest case of a short main chain and one sicle branch. Even more difficult, however, would be an interpretation of the results of an experiment to be given below. I n using a natural mixture of the amylases as in malt diastase, it should be evident that when enzymic activity ceases,
1420
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
the products resulting are maltose and a residue of the main chain to which are attached, a t branching points, residual stubs consisting of one dextrose unit or a t most two. For as long as the branch (or the main chain up to the first point of branching) is longer than two dextrose units, the p-amylase should be able to split off another maltose unit before ceasing its activity. Let us suppose that an acid conversion is applied to this limit dextrin, and that the equivalent of one or two moles of dextrose is broken away from this dextrin, which would correspond to a reduction in weight of some 4 to 8 per cent dextrin based on the total weight of original starch. A large share of the balance of the dextrin should again become susceptible to the P-amylase factor, which should then be able to convert it into fermentable products and materially increase the fermentability of the final sirup. Experiments 8 and 9 do not show that such is the case. On the contrary, they indicate no significant benefit to further malt diastase activity by acid converting a limit malt conversion within the limits discussed. We have made the tacit assumption that if the Freudenberg 1,6 branched chain represents the true picture, then in acid conversion the end or ends opposite the terminal aldose group are the more susceptible to acid hydrolysis, such as has been supposed in diastatic hydrolysis. This assumption would be necessitated by experiment 4A, followed by 3A. Just as in diastatic conversion, when a configuration is encountered in the hydrolysis of a given molecule which can act as an enzyme block, the residual fragment is left by the hydrolytic agent a t this point in favor of other molecules not yet hydrolyzed so far as a block. I n experiment 3A the total weight of all the probably blocked chains (dextrins) which could result from original starch has apparently been preserved throughout its acid conversion (in 4A). These results would not have been found had the initial hydrolysis by acid proceeded in an indiscriminate fashion on a branchedchain molecule. But experiments 2 (A to E) show and 4B plus 3B confirm this result in a measure: Vhen starch has been converted to the point where the limit dextrins represent the main bulk of the polysaccharides remaining, the mechanism of acid hydrolysis can hardly be pictured as an orderly removal of successive dextrose units from these dextrins. To explain our hydrolysis results on the basis of a 1,6 branched side-chain structure for starch, let us assume further that only the initial action of the acid is a more or less orderly attack until the side chain is reached, and that thereafter the action is indiscriminate, any glucosidic bond being open t o attack. We still cannot explain why in experiment 9A, when about 7 per cent of these limit dextrins have been cleaved by the acid into reducing substances, a subsequent malt action should not produce more fermentables. Assuming a purely indiscriminate attack by acid in this experiment, simple probability would BO assort the broken fragments that pamylase should be able to produce more maltose, at least to the extent of about half of this 7 per cent dextrin reduction, or around 3 per cent. In addition, about 3 per cent more maltose should form from the action of the malt on dextrin cleavage products, which although cleaved by the acid, are still too long in chain length t o exhibit reducing power and are still estimated as dextrins (the drop of 3 per cent in dextrins from sirup 9A to 9B). Yet the fermentability of the final sirup, 9B, is practically identical with the original, limit malt conversion. Our results do not necessarily support Freudenberg’s latest theory of starch structure. They rather argue for the concept that starch is heterogeneous ( 8 ) in the sense that some molecules, mainly maltose anhydrides, are readily hydro-
Vol. 33, No. 11
lyzed by diastase to maltose and by acid t o dextrose, maltose, and the latter’s homologs; on the other hand, other molecules are built on a different pattern which are resistant to the diastases and, except for the few 1,4-or-glucosidic linkages which they may contain, are hydrolyzed less readily by acid a t random points. Effect of Added E n z y m e Components Other T h a n Malt Another theory of starch structure which comes to light periodically, particularly in the patent literature, follows mainly from observations on the enzymolysis of starch. The theory has many variations but few have worked out the theory to a definite end. It is assumed that there are varied types of linkage in the starch molecule; some are split only by one particular amylase, while another type of amylase is required to split other linkages. The conclusion arrived a t by proponents of such theories is usually the same; that is, by the proper combination of component amylases in an enzyme mixture, starch can be completely hydrolyzed to a t least disaccharides, possibly even completely to dextrose. Such a result would be of great technical importance. Examples of this type of theory are found in the well known work of Ling and of Pringsheim, both of whom claimed to have degraded starch to very low saccharides. For example, Pringsheim (10, 11) claimed that starch could be completely converted to dextrose by the proper combination of a- and &amylase, both supposedly components of malt diastase. Recently a patent was issued (9) for increasing the fermentability of a mash by supplementing malt diastase with soybeans, known to be a rich source of @-amylase; on the other hand, another recent patent (9)would lead one to infer that a more unusual type of degradation, resulting in high fermentability in the end product, could be accomplished by conversion with a fungus enzyme after preliminary acid conversion of starch. However, our attempts have failed t o obtain a more substantial reduction in the resistant residues of starch remaining after a preliminary acid conversion by the addition of enzyme-containing products which would increase either the a-or &amylase ratio above that existing in malt diastase. Furthermore, the use of certain fungus enzyme preparations which, in addition to containing dextrinogenic amylases that may or may not be similar t o the or-amylase of malt, are distinguished from malt principally by the or-glucosidase they contain, appear no more effective in disrupting the resistant configurations in acid-converted starch. They rather seem t o break away dextrose units, in piecemeal fashion, from the nonreducing, nonfermentable residues and thereby increase both the true dextrose and the D. E. (dextrose equivalent) of the final sirup. If for practical considerations a D. E. (for example, 64 to 65) is desired in the final sirup, then an acid-converted sirup with a lower D. E. is selected as substrate for the fungus enzyme than would be used as substrate for a malt diastase. The result is that in the former case we have stopped the action of the acid in the very range where in acid conversion the reagent is building up the true dextrose content by splitting a higher proportion of more resistant linkages; and this work has been passed on to the a-glucosidase factor while other factors in the fungus enzyme preparation are completing the hydrolysis of the less resistant fraction. The net result is that the final sirup a t 64 to 65 D. E. contains approximately the same amount of limit dextrins and the same fermentability as the three other sirups mentioned. To illustrate, sirups were made by converting cornstarch with hydrochloric acid t o various dextrose equivalents. To one of these sirups a t 53 D. E., 0 05 per cent of a commercial
INDUSTRIAL AND ENGINEERING CHEMISTRY
November, 1941
fungus-diastase preparation was added, and conversion was continued until the D. E. reached 64.2 per cent, a t which time the analysis for sirup 5 was run. This sirup corresponds to a commercial product now sold as a sweetening agent and to the brewing trade for its fermentable content. Sirup 6 was produced by converting a 58 D. E. acid-made sirup with 0.5 per cent of a commercialmalt diastase extract to the same final D. E. and is also representative of a commercial run. Sirups 7A and 7B are experimental sirups made by converting acidmade sirups with 0.3 per cent of the same malt diastase preparation. In addition 0.9 per cent (dry basis) of ground soybean meal was added in 7A as a source of /3-amylase; in 7B 0.3 per cent of a commercial enzyme preparation high in a-amylase content was added instead of the soybean meal. No significant difference is noted in the fermentability of these sirups or in the total percentage of nonreducing dextrins left after conversion. We must conclude, therefore, that a t least up to the limit of conversion shown, which is dictated by practical considerations, little advantage appears in the use of excessive proportions of any one diastatic component with a view to fracturing the malt-resistant nonfermentable residues of cornstarch. However, the slow though continued rise in the dextrose content of conversions with a-glucosidase-containing preparations, apparently a t the expense of limit dextrins, opens an interesting line for speculation on the latter's configuration. That the configuration is open (and not an enzyme-closed synthetic cycle) and that the linkages are in the main a-glucosidic, seems to be indicated. Further work on the nature of these linkages is in progress.
Fermentation Tests Fermentation tests were run in all cases as given in the method of the American Society of Brewing Chemists (1937) except that the yeast was supplied by the Fleischmann Yeast Company. While fermentation appeared less vigorous than with brewer's yeast, nevertheless the Fleischmann yeast gave the same end point after a certain time, and results could be more exactly duplicated. Specific gravities were determined for estimating the percentage fermentables with a pycnometer rather than a Westphal balance, since the former appeared to give the degree of accuracy demanded in the work, An abstract of the method follows: Ferment 250 cc. of a 10 per cent solution of the sample with 5 grams of compressed yeast at 15-25' C. for 48 hours or until fermentation is complete. In the case of refined sugars and sirups such as corn sirup, before fermentation add to the solution 0.8 gram KzHP04, NH4HZPOd,and 0.5 gram Difco yeast extract. Fermention asks are eauimed with water seals and shaken several times a day. - When fermentation is complete, dealcoholize 100 cc. of the filtrate by distilling after dilution with 50 cc. of water. Transfer the residue quantitatively to a 100-cc. flask, cool, and make u to 100 cc. at 20" C. Determine the s ecific gravity from whici the "real extract" is determined from Pyato tables:
18'
fermentable extract (dry basis) = ( p
- n) 100 P
where P = real extract of 10% dilution before addition of nutrients p = real extract of 10% dilution before fermentation n = real extract of 10% dilution after fermentation
Enzymic Conversions When applied to untreated starch (such as in sirup l), diastase conversions were carried out in a manner comparable to manufacturing practice. Thoroughly washed cornstarch
1421
was made up to a gravity of 12' BB. On the basis of dry starch present, 1 per cent of a malt diastase preparation known as Exsize (Pabst), with a Linter value between 250 and 275, was added. The pH was adjusted to 6.0-6.2, and the mass brought up to 165-170' F. and held there for 30 minutes. The liquor was cooled to 120" F., the pH adjusted to 4.8-5.0 with hydrochloric acid, and 2 per cent more Exsize added. The conversion was held a t this temperature for 20 hours, a t which point it had reached its limit. The liquors were then brought to a boil and clarified by filtration. When enzymic conversions were superimposed on acid conversions of cornstarch, for malt diastase the liquors were adjusted to pH 4.8-5.0 with sodium hydroxide. Usually 1 per cent added Exsize is sufficient to convert to the latter's limit at 12-18' BB. and 120" F. in 16 hours. The same conditions were used when soybean meal was added to the malt as a source of P-amylase. However, when an enzyme preparation known as Amyliq (Wallerstein) was added as a source of aamylase, a compromise pH of 5.5-6.0 was used; we have found excellent liquefaction and dextrinization of cornstarch with this enzyme at pH values between 6 and 7. The terms a- and p-amylase have been used by us in the sense used by Hanes, the former to denote the dextrinogenic factors, the latter to denote the saccharogenic. The enzyme employed, supposedly of fungus origin, is known as Clarase-700 (Takamine). For these conversions the acid liquors were neutralized to 5.5 pH and concentrated to 27' BB. A conversion temperature of 130" to 135" F. was used for 48 hours. Comparable results were obtained with another enzyme preparation supplied by Rohm & Haas. Most commercial enzyme preparations appear to be so well buffered that no additional buffer was required to maintain pH a t the desired point in the above conversions.
Conclusions 1. The percentage fermentable of a sirup made by the diastatic conversion of cornstarch is not readily increased by a comparatively extensive acid conversion of this sirup. 2. A partial preconversion of starch by acid does not produce a substrate which is more readily freed of nonreducing, nonfermentable products by subsequent malt diastase conversion. 3. Inasmuch as the acid conversion decreases the nonreducing dextrin content by splitting the latter into reducing but malt-diastase-resistant and nonfermentable products, a t least two fundamentally different chemical configurations must exist in cornstarch. While both fractions possibly contain only a-glucoside linkages, probably only one is composed essentially of the 1,4-glucoside or maltose-type linkage.
Liteoature Cited (1) Berlin, If., J . Am. Chem. SOC.,48, 1107 (1926). (2) Dale, J. K., and Langlois, D. P., U. S. Patent 2,201,609(1940). (3) Freudenberg, K., Ann. Rev. Biochem., 8, 81 (1939). (4) Freudenberg, K.,Naturwissenschaften, 28,264 (1940); Ber., 73B, 60,609 (1940). (6) Hanes, C. S., New Phytologist, 36, 189 (1937). (6) Haworth, W.N., and Machemer: H., J. Chem. SOC.,1932,2270, 2272. (7) Kerr, R. W., J . Am. Chem. Soc., 62, 2735 (1930). (8) Kerr, R.W., and Trubell,0. R.,CereaE Chem., 18, 630 (1941).
(9) McPherson, W. K., and Christensen, L. M., U. S. Patent
2,219,368(1940). (10) Pringsheim, H., and lei bow it^, Ber., 58, 1262 (1925). (11) Ibid., 59,991 (1926). (12) Sichert and Bleyer, 2.anal. Chem., 107,328 (1936). (13) Staudinger, H., Naturwissenschaften, 25, 673 (1937). PR~BENTED before the Division of Sugar Chemistry at the 1Olst bleeting of the American Chemical Sooiety, St. Louis, Mo.
