STRUCTURE OF PYRODEXTRINS BERNADINE BRIMHALL Iowa Agricultural Experiment Station, Ames, Iowa
and unbranched (amylose) components of cornstarch, as well as amylodextrin, retrograded starch, and ordinary granular starch, have been dextrinized and the course of conversion has been followed by water solubility, reducing power, and digestibility with p-amylase. The results indicate that the linear portions of starch become branched during the heating process as demonstrated by loss of ability to retrograde, increased resistance toward &amylase, and percentage of tetramethylglucose upon hydrolysis of the methylated product. A possible mechanism for heat dextrinization is discussed.
Pyrodextrins are degradation products of starch obtained by roasting, either alone or in the presence of small amounts of catalytic agents. By varying the time and temperature of roasting and the catalyst, hundreds of different dextrins are produced for industrial consumption, especially in textile printing and finishing and as adhesives and envelope glues. The properties of a commercial water-soluble pyrodextrin are shown to be in harmony with a molecule containing approximately 65 glucose residues arranged so that there are four or five short branches of approximately five glucose units each. The branched (amylopectin)
T
and insoluble in 70 per cent methanol was relatively homogeneous and constituted 70 per cent of the original dextrin. It had a reducing value of 60, corresponding to an average molecular size of about 66 glucose units (17). (Molecular weights in terms of glucose units used throughout this paper were calculated from ferricyanide reducing values, 3). Subsequent experiments in most cases were carried out on the original dextrin as well as on this fraction. PERIODICACID OXIDATION.Oxidation of the dextrin with periodic acid (8) followed the same course as that of the original cornstarch:
H E most thorough studies of the dextrinieation process in recent years have been published by Katz (9,IO). Although not arriving at a definite conclusion as to chemical structure, he suggests that dextrins are formed by the breaking up of the starch chains, with accompanying anhydride formation, into smaller molecules terminated by units of the levoglucosan type. Certain experimental observations would tend to support such a structure: (a)During dextrin formation, water vapor is given off (9, 16); disappearance of the crystalline x-ray pattern indicates t h a t the loss of water is irreversible. (b) Levoglucosan may be formed from starch by dry distillation under more drastic conditions of heating than those used in dextrin manufacture. (c) Levoglucosan itself is nonreducing; the reducing power of pyrodextrins is considerably lower than that of corresponding dextrins prepared by other processes (18). (d) Levoglucosan types are stable toward alkali; with increasing conversion from starch to dextrin, the alkali labile value first rises to a maximum and then falls (8). However, in the light of recent developments leading to a more integrated concept of starch behavior and improved techniques for investigating it (7,13), a renewed examination of the pyrodextrins seemed warranted:
Time, Hours 0 0.5 1 2
HI04 Consumed, Mg. Dextrin Starch fraotion 0 0 10.6 11.9 16.1 15.3 16.8 18.0
'
Time Hour; 4 6
lo 22
HI04 Consumed, Mg. Dextrin starch fraction 19.7 18.8 22.7 20.5 25.2 25.5 27.2 27.3
This indicates that the hydroxyl groups on carbons 2 and 3 of the glucose units in the starch molecule were not changed by dextrinization. AMYLOSE CONTENT. The potentiometric iodine titration curve (1) for the dextrin resembles that of glycogen. I n other words, while the original cornstarch contained 21 per cent of the unbmnched component, the dextrin contains no linear moleculee long enough to give a blue color with iodine. SOLWILITYBEHAVIOR. Although the action of dry heat on starch can produce water-soluble dextrins of about 60-70 glucose units, the action of cold acid on unswollen starch gives rise to water-insoluble products (amylodextrins) containing as low as 20 glucose residues per molecule. The latter, consisting chiefly of linear molecules, will easily retrograde from solution while the pyrodextrins will not, as Katz (9) showed. The insolubility of acid-modified starch products as compared to pyrodextrins points strongly to a n entirely different molecular structure for the two. Branching of the pyrodextrin molecules would be one logical explanation for their greater solubility and resistance t o retrogradation. DIGESTIBILITY WITH &AMYLASE. The most striking property of the dextrin, characteristic of all pyrodextrins so far examined, is its resistance to attack by starch-digesting enzymes. Whereas
PROPERTIES OF A COMMERCIAL PYRODEXTRIN
STRUCTURALSIGNIFICANCE. The dextrin selected for study was a commercial product prepared by roasting cornstarch in the absence of a catalyticr agent until it was almost completely soluble in cold water-i. e., a British gum. It was light tan in color and had a ferricyanide reducing value (3) of 97 (maltose = 1900). Under the microscope the granules looked like those of the original starch and were still strongly birefringent. However, in aqueous glycerol, swelling occurred typically in concentric layers as described by Sjostrom (30). The crystalline A x-ray pattern of cornstarch had given way to a practically amorphous one. Since products of this type usually contain material which has been converted to a considerably greater or less extent than the main body of dextrin, a more homogeneous fraction w a obtained ~ by fractionation with methanol and water. Preliminary experiments indicated that the portion soluble in 30 per cent methanol 72
.
January, 1944
INDUSTRIAL AND ENGINEERING CHEMISTRY
starch is about 55 per cent converted to maltose by the action of @-amylase,the dextrin is only 22 per cent converted. The method of Newton, Farley, and Naylor (16) was used; the amount of digestion by soybean @-amylaseis determined by the difference in reducing power of the solution before and after enzyme action. Figure 1 compares the behavior of starch, the water-soluble pyrodextrin, and two other dextrins-5 and 45 per cent watersoluble, respectively-which were withdrawn a t earlier periods in the same conversion. The decrease in digestibility with increasing dextrinization is pronounced. The linkage responsible for resistance to @-amylase is quite stable. For example, the dextrin was converted both to the monoformate and the triacetate, then saponified to recover the original dextrin ( 2 2 ) , after which neither its enzyme digestibility nor its reducing power were found to be changed. When starch or dextrin is dissolved in formic acid and the solution precipitated by pouring into water or alcohol, a compound containing 16 per cent formyl is obtained, corresponding to one formyl group per glucose unit, probably on carbon 6 ( 5 ) . The enzyme from B. macerans ACTIONOF OTHERENZYMES. failed to produce any Schardinger dextrins (12) from the pyrodextrin. It has been suggested that this enzyme acts only when an uninterrupted chain of at least 6 or 7 glucose units is available at the end or ends of the molecule ( 4 ) . Starch itself can be made to yield 55 per cent of Schardinger dextrins. The pyrodextrin was also very resistant to the action of a-amylase which digested it only 3.5 per cent in terms of maltose, compared to 45 per cent for starch under the same conditions. END-GROUP ASSAY. The pyrodextrin fraction was methylated, using sodium and methyl iodide in liquid ammonia as a solvent. The product (methoxyl = 44.5 per cent) was hydrolyzed, and the resulting methylglucoses were separated by solvent extraction as described by Hassid and Dore ( 6 ) . Three separate determinations, starting with 6 grams of trimethyldextrin, yielded 0.512, 0.572,and 0.485 gram, respectively, of crystalline tetramethyl glucose, or 8.3 per cent. This corresponds to at least one nonreducing end group for every 12 glucose units in the dextrin molecule as contrasted to one in every 24-30 for starch itself. (In all cases the tetramethylglucose crystallized in characteristic
Figure 1. Digestibility of Pyrodextrins with Soybean @-Amylase at Different Stages of Conversion (Sample. digested at 40’ C. after heating at temperature indicated.
73
fashion with methoxyl values close to the theoretical 52.5 per cent. The amount of this product isolated, and hence the calculated degree of branching, should be considered as a minimum value, since there is considerable opportunity for loss during the separation.) STRUCTURAL SIGNIFICANCE.Although both ferricyanide reducing value and end group assay methods leave much to be desired from the stahdpoint of accuracy, they can be considered sufficiently reliable to provide a semiquantitative picture of the arrangement of glucose residues in the pyrodextrin molecule. Comparing the average molecular size of 66 glucose units with the results of end-group assay (one end group per 12 glucoses), each molecule is calculated to have between 5 and 6 nonreducing end groups (66 f 12 = 5.5),or between 4 and 5 branches (5.5 1 = 4.5). Meyer (14) used the results of 6-amylase digestion to calculate the length of the branches of the glycogen and amylopectin molecules. There is good evidence that this enzyme starts a t the nonreducing ends of the molecule and cleaves off maltose residues until a point of branching is reached, where it stops one or two glucoses short of the 6-or-glucosidic linkage. On this basis the branches of the pyrodextrin molecule appear to be very shortabout 5 glucose units each: A molecule of 66 glucose units with 6 nonreducing ends, 21 per cent digested by &amylase, would lose upon digestion a total of 66 X 0.21 = 14 glucose units, or 2.3 off each end. Adding to this the 1 or 2 units which remain attached to the branch point, a value of 5 units (2.3 1.5) per branch is obtained. This type of structure, based on the results of reducing power, end-group assay, and &amylase action, is in harmony with the other data presented above. It would be stable to acetylation and saponification and would behave like starch toward periodic acid. The branches would be too short for attack by the Schardinger enzyme, yet long enough to bring about increased solubility and prevent the molecular orientation necessary for retrogradation.
+
DEXTRINIZATION O F AMYLOSE AND AMYLOPECTIN
The branched (amylopectin) and unbranched (amylose) fractions of cornstarch were dextrinized separately to get a clearer picture of the changes which are produced in the unfractionated material. Waxy cornstarch has been shown (1) to be a natural source of almost pure amylopectin. Before dextrinizing, it was made into a paste, homogenized t o disrupt any remaining granule structure, then precipitated with methanol and dried. The product designated here as amylose was isolated from ordinary cornstarch as Schoch’s butanol-precipitated fraction (19). For comparison, a sample of amylodextrin was prepared (11) by treatment of unpasted cornstarch with cold aqueous 15 per cent sulfuric acid for 3 months; then it was purified by retrogradation. This material is composed essentially of unbranched molecules like amylose but of much shorter chain length (15 to 25 glucose units). French (4) described its formation as follows: “Since branch points in the starch chain and other structural irregularities are incapable of partici ating in crystallite formation, these amorphous regions are hysolyzed by (cold) acid with the production of easily soluble low-molecular-weight compounds which diffuse into the bulk of the solution. The organization of the crystallites protects them against rapid hydrolysis, since dilute acid is unable to penetrate the crystalline regions. Only the strai h t chains and straight portions of branched chains are fount in the original crystallites, and so by recovering the crystalline portions of the original starch granule it is possible t o obtain a purely straight chain amylodextrin.” The isolation of crystallites by selective removal of amorphous portions is not peculiar to starch; it has also been carried out on silk fibroin (13-4). These three materials-amyIopectin, amylose, and amylodextrin-along with the original granular cornstarch and retroy d e d cornstarch were dextrinized in 100-gram lots in a smalli aboratory unit with electrical temperature control. Samples. were withdrawn at frequent intervals during the course of conversion for analysis. Solubility was determined b y adding. 25 ml. of water to 0.1 gram of sample, allowing i t to stand with frequent shaking; for
'14
INDUSTRIAL AND ENGINEERING CHEMISTRY
2 hours a t room temperature, centrifugin and removing a 15-ml. aliquot of the supernatant liquor. &is was placed in a weighed beaker and evaporated t o dryness i; a 100" C. oven. Solubility is calculated as the percentage of dissolved substance with respect t o the total weight of solid initially present (13B).
Figure 2 shows the relative rate of solubility increase with time of heating a t 200' C., all samples bein,g in the dextrinizer for a constant time of 3 hours before this temperature waa reached. The amorphous amylopectin (curve I) dextrinized with great ease, requiring only one hour to attain complete solubility; the amylom (curve IV) was only 65 per cent soluble after 6 hours under the same conditions. Intermediate between these two w w granular cornstarch (curve 11) which is made up both of crystalb e and amorphous regions (approximately 21 per cent amylose and 79 per cent amylopectin). Its solubility curve, however, is much steeper than either of its fractions. When cornstarch was retrograded (granule structure destroyed by pasting and recrystallized by freezing), curve I11 was obtained for the product. Dextrinization is considerably slowed up, although the composition of the starch was not appreciably changed by retrograding. These samples represent a maximum divergence of types, including one amorphous (amylopectin) and three different crystalline modihations of starch as shown by their x-ray diagrams. Granular cornstarch shows an A type of pattern; retrograded starch and amylodextrin, a B pattern. The two differ in the size of their unit cells, but the crystallites in both cmes contain linear portions of molecules oriented parallel to one another. Amylose can also be obtained in the B form, but the sample used in this work had a V x-ray diagram (7),the crystallites being made up of closely packed cylinders of molecules in the helical configuration. The reducing power of the water-soluble pyrodextrin from amylopectin was only 42 as compared to 136 for the watersoluble pyrodextrin from cornstarch (cornstarch = 10). For determination of @-amylasedigestibility, the method used above (16)was modified in that 0.1-gram samples were dissolved in cold 0.5 N potassium hydroxide, then neutralized with hydrochloric acid before the enzyme was added. The decrease in digestr ibility upon dextrinization was markedly different for each of the four materials as recorded in Table I. If enzyme digestibility is used as a criterion of branching in the starch molecule, the above data would indicate that the un-
VoI. 36, No. 1
TABLEI. EFFECTOF DEXTRINIZATION ON A AMYLASE DIQESTIBILITY OF STARCH AND STARCH FRACTIONS
0
Sample Amylopeotin Cornstarch Amylose Amylodextrin Only 66% water soluble.
-(+Amylase Original material 56
66
82 81
Conversion, % MaltosWater-so!. pyrodextrin Difference 44 30 47" 11
11
26 35
70
branched fraction becomes branched during dextrinization. This possibility was checked by end-group assay on the pyrodextrin formed from amylodextrin. The results are as follows: Grams of tetramethylglyoose OCHs = 52.0%) from 6 grams methylated pyrodextrin O C L i 5 43.8%) Per cent of nonreducing en6 groups (0.941/6.3) Mol. size from reduoing power ( Iucose units) Nonreduoing end-groups per m o t (16 X 15%) Branches per mol. (2.25 1)
-
0.941 15 15 2.25 1.25
Thus, after dextrinization by heat, the branched character of the material is evidenced by the proportion of reducing to nonreducing end groups (1:2), the resistance t o &amylase digestion, and lack of ability t o retrograde, even though the molecular size of the amylodextrin was reduced only from 17 t o 15 glucose units. During the process the alkali labile value of the amylodextrin decreased progressively from 53 t o 20. MECHANISM
FOR HEAT DEXTRINIZATION
The molecules in retrograded amylodextrin and in the crystallites of starch granules are oriented together in essentially parallel fashion, as shown by their birefringence and crystalline x-ray patterns. These portions, in contrast to the amorphous and branched parts, undergo the greatest change upon dextrinization, especially with respect to enzyme digestibility. As heat is applied t o such crystallites, a glucosidic linkage in one molecule might conceivably become ruptured and the free end become attached to a hydroxyl group on an adjacent molecule. Since the chains are not free to wander about in the dry state and since the sixth carbon with its primary hydroxyl group is most exposed to such a reaction, it seems logical that branches could form at this position as indicated in Figure 3. The proposed mechanism does not account for the water vapor given off during dextrinination. The source of this water can be explained, however, by the observation of Meyer ( l a , pages 396 and 405) that analysis of airdried amylose shows the composition (CoH1oOs.H~0) =. He points out that the chains in the starch crystallite are not so straight or compactly oriented as in cellulose, and that there would be room between the starch chains for water molecules. He considers this water as indispensable for crystal structure since the x-ray diffraction pattern of the amylose disappears when it is completely dehydrated. Removal of the water by heat would account for loss of the x-ray pattern upon dextrinization, in accord with the results of Kotz (9). Meyer (13) regarded the starch granule as consisting of a mixture of branched and unbranched molecules, portions of which are oriented together in crystalline areas. He considered the compact crystallites t o be held together by a loose network of amorphous material; portions of molecules near points of branching, where parallel orientation of parts is hindered, would be expected to remain amorphous. Therefore, considering dextrinization from the standpoint of the granule as a whole, the amorphous molecules ( l a ) , being less rigidly held in Figure 2. Solubitity Changes in Starch Fractions with Time of position, can more easily adjust themselves to the Dextrinization I. Amylo IV. Amylow stress and strain produced in the granule by heat and V. Amylodsoctrla II. G r a n Z Z t a r c b b f water from the crystallites. However, UI. Retrograded oonutareh v
7s
INDUSTRIAL AND ENGINEERING CHEMISTRY
January, 1944
parts of a molecule may be amorphous and other parts, chiefly the longer branches, tied up within the crystallites. A strain on such a molecule would cause cleavage of its more rigid crystalline parts. The longer branches might thus be broken from the amylopectin fraction, leaving molecules with only short branches which are less digestible by p-amylase than the original. When amylopectin, separated from the starch granule, is dextrinized, its enzyme digestibility is decreased by a smaller amount because the branches are less involved in crystalline units and suffer less rupture. The crystalline portions of the granule would dextrinize as described above, producing branched molecules from linear portions of chains by the rearrangement suggested in Figure 3. The reducing value would not increase very rapidly because few new aldehyde groups are produced. The granules could conceivably retain their same outward appearance since the molecules would not be appreciably moved from their original positions with respect to one another. But the pyrodextrin, now consisting of easily hydrated branched molecules, would be very soluble and would IIQ longer retrograde like the material from which i t was formed.
OH €I
. .-0-
TABLE11. COMPARISON OF CHANGESIN FERRICYANIDE REDUCINQVALUE AND ALKALILABILEVALUE DURING HEATDEXTRINIZATION OF CORNSTARCH Canary Dextrin Conversion (Heat HC1) Hours Solubility, % ALV Rou heated 0 0.4 12.5 0.25 0.4 19 10.6 1.0 1.9 21.6 17.5 2 2.8 23.5 25.3 3 4.1 39 39 4 10.6 89 4.5 16.5 53 120 62 60 150 5 43 5.5 95-100 56.5 185 6 96-100 54 180 6.25 95-100 47.5 198 6.5 95-100 197 42 176 6.75 95-100
+
.-
..
British Gum Conversion. (Heat Alone} Solubility, % ALV Rcu 20 7.6 it:i 8.0 20 9.5 0.8 26 14 1.7 34 18.7 5.6 34 28 58.7 34 45 95-100 34 53 95-100 36 65.5 95-100 77 95-100 33 83 95-100 36 95 95-100 34 204
...
..
..
taneously with the other changes associated with dextrinization. It follows that dextrinization is intimately linked with loss of water of crystallization. According to the anhydride theorythat water of constitution is lost upon cleavage of the moleculesthe amorphous portions of the starch granule (making up roughly 75 per cent of the total weight based on the yield of amylodextrin) should begin t o dextrinize and give off water long before the crystallites rupture and lose their x-ray pattern. Dextrinization would proceed gradually over a relatively wide range of time and temperature; it would not under o the rapid, almost comdete change within narrow limits tvDic3 - - of granular starch (Figcre 2). 3. The dror, in alkali labile value a t the later starres of dextriniaation is difficult t o interpret as a conversion of aldghyde ends to levoglucosan ends, since the ferricyanide reducing value (primarily a measure of aldehyde groups) does not fall in like manner. This is illustrated in Table 11, which compares the changes in reducing value (Rc,) and alkali labile value, ALV @I), for two typical commercial dextrin conversions. 4. The increased resistance t o p-amylase digestion upon dextriniaation is not explained by the formation of levoglucosan terminal groups. These would scarcely influence the enzyme, which apparently begins its attack a t the opposite end of the molecule. I
h
__jt
OH H
LITERATURE CITED
I
n
Bates, French, and Rundle, J . A m . Chem. SOC.,65,142 (1943) Caesar and Cushing, IND. ENG.CHEM.,31,921 (1939). Farley and Hixon, IND. ENG.CHEM.,ANAL.ED., 13,616 (1941:. , ~Frenoh, , unpub. thesis, Iowa State Coll. Library, 1942. (5) Gottlieb, Caldwell, and Wixon, J, Am. Chem. Soc., 62, 5342:
(1940). (6) Hassid and Dore, Ibid., 59, 1507 (1937). (7) Hixon and Rundle, in Alexander’s “Colloid Chemietry”, VoL. V, New York, Reinhold Publishing Gorp., in press.
Figure 3.
Suggested Mechanism for Conversion of Linear
to Branched Portions of Starch Molecules during Heat
Dextrinization
As pointed s u t early in the paper, the idea of anhydride formation upon dextrinization is based on (a) the assumption that dextrinization is a dehydration process, along with the fact that more drastic treatment yields levoglucosan, and (b) the increase in alkali stability after a certain point in the conversion. It must be admitted that certain portions of the data presented in this manuscript can be used to support this mechanism as well as the branching concept suggested above. However, when considered as a whole, the evidence tends to favor the branching concept, especially in view of the following points: 1. Formation of terminal levoglucosan groups would not be a dehydration reaction but a shift of the 1,4-glucosidic bond to a n inner 1,6-glucosidic linkage involving merely the migration of a hydrogen atom. By analogy the branching concept postulates formation of 1,6 bonds between molecules rather than within the terminal glucose unit. 2. Katz and Weidinger (IO) showed that loss of water with disappearance of the crystalline x-ray pattern occurs simul-
(8) Jackson and Hudson, J. A m . Chem. SOC.,59,2049 (1937). (9) Katz, Reo. trav. chim., 53, 554 (1934). (IO) Katz and Weidinger, 2. physik. Chem., A l a , 100 (1939). (11) Klason and Sjoberg, Ber., 59,40 (1926). (12) . . McClenahan, Tilden, and Hudson, J . A m . C h m . SOC.,64,2139 (1942). (13) Meyer, “Natural and Synthetic High Polymers”, pp. 387-416, New York, Interscience Pub., 1942. (13A) Ibdd., p. 446. (13B) Ibid., $. 575. (14) Meyer and Fuld, Helv. Chim. Acta, 24, 375 (1941). (15) Newton, Farley, and Naylor, Cereal C h m . , 17, 343 (1940). (16) Radley, “Starch and Its Derivatives”, p. 155, New York, D . Van Nostrand Co.. 1940. (17) Richardson, Higginbotham, and Farrow, J . Text. Inst., 27,
T131 (1936). (18) Rolfe, Orig. Com. 8th Intern. Congr. Applied Chem., 13, 237
(1912).
(19) Schoch, J . A m . Chem. SOC.,64,2957 (1942). ( 2 0 ) Sjostrom, IND.
[email protected].,28, 63 (1936). (21) Taylor, Fletcher, and Adams, IND.ENG. CEBIX.,ANAL.ED.,
7, 321 (1935). ( 2 2 ) Zemplen, Ber., 69B,1827 (1936).
JOURNAL PAPER5-1094, Iowa Agricultural Experiment Station, Project 62V (supported in part by a grant from American Maize-Products Company>, The data represent part of a Ph.D. thesis, Iowa State College.
