VOL. 14, 263-276 (1975)
BIOPOLY MERS
Ultrastructure of Acid- and Enzyme-Modified Cross-Linked Potato Starch G. HOLLINGER and R. H. AIARCHESSAULT, Ddpartement de Chimie, UniversitC de MontrCal, C.P. 6,910, MontrCal, QuC., Canada
Synopsis Purified potato starch, cross-linked with epichlorohydrin (65-5.4 anhydroglucose units per cross-link) by an iterative method that preserved the native crystalline structure of the starch granule, was exposed to enzymic ( B . subtilis a-amylase at 50°C) and acidic (3 N HCl a t 40°C) hydrolysis. The stability of the granules to digestion by enzymes, as measured by their weight loss after incubation with the enzyme solution, increased with the degree of cross-linking; by contrast, the stability t o acid attack decreased as a function of the degree of cross-linking. The morphology of these samples was studied by scanning electron microscopy. A lamellar structure, which was most evident after enzyme treatment, was obsecved in samples that had a weight loss of greater than 507,. The results reflect differences in accessibility, which are interpreted in terms of a statistical model of the ultrastructure with correlated fluctuations in density in the radial direction.
INTRODUCTION A number of papers on the morphology of starch using transmission electron microscopy (TERI) 1-4 have provided some insight into the ultrastructure of the starch granule. The study of starch by T E M requires selective staining techniques combined, in some cases, with acid or enzyme hydrolysis. This treatment enhances the contrast between accessible and nonaccessible regions. However, possible artifacts due t o sample preparation have made the interpretation of TEA4 pictures difficult. Scanning electron microscopy (SEN) provides a complementary method for the study of starch morphology. Unlike T E M and light microscopy, SEA1 provides a n almost three-dimensional picture of the surface of a specimen as a result of its high depth of field; an added bonus is t h a t fractured granules allow viewing of internal structure. Two major types of organization of the starch granule have been rep ~ r t e d : ~a?n~onion-like or concentric lamellar structure, which usually is not seen in the dry state; and a fibrillar texture, which is observed in fracture surfaces of acid-treated potato ~ t a r c h . ~ Dilute acid treatment is needed t o develop the lamellar structure of potato starch. Stronger acid treatment has revealed that these lamellae, 5000 8 thick, have subspacings of a few hundred angstroms.6 Unlike the 265
@ 1975 by John Wiley & Sons, Inc.
266
HOLLTNGER AND MARCHESSAULT
lamellae of cereal starches, those of potato starch are not affected by climinating the daily rhythm of light and t c r n p e r a t ~ r e . ~The ? ~ lamellation has been attributed to chemical differmces between the layers,j differences in the density of deposition of molecules of starch,6and differences in crystallinity.2 Sterling? and others4 have proposed a radial fibrillar structure. The existence of microfibrils in potato starch (bawd on the appearance of fracture surfaces of acid-treatcd potato starch) has been reported. On the basis of the fracture surfaces of untreated potato starch, Hall and Sayre* proposed that potato starch is composed of plates stacked one on top of the other. These fracture surfaces display radially oriented ridges similar to those described by Sterling? and are rcminiscent of the conchoidal fracture of glass. I n this paper, we continue a previously reportedg effort t o characterize epichlorohydrin cross-linked potato starch. The cross-linking was performed under conditions t o maintain the granular and radially symmetric organization. Comparison of the degree of cross-linking as measured by a swelling analysis and a chemical analysis yielded similar results, leading us t o conclude that the cross-links were distributed uniformly. By observing the changes in accessibility to acid and enzyme hydrolysis as a result of cross-linking, further information on the ultrastructure and cross-link distribution of the starch samples has been obtained.
EXPERIMENTAL The preparation and characterization of the crystalline granular epichlorohydrin cross-linked starch (purified J. T . Baker potato starch) samples have been described e l s ~ w h e r c . ~All samples were dried in a vacuum oven below 65°C for 11/2hr prior t o the following treatments.
Enzyme-Modified Crystalline Cross-Linked Potato Starch Partially purified Bacillus subtilis a-amylase having an activity of 2500 SKB* units per gramlo was used in the following preparation. A 0.15-g sample of the cross-linked material, dried a t 8.5"C for 4 hr under vacuum was incubated with 20 mg of enzyme in a neutral phosphate buffer solution containing 0.01 M KH2P04 and O.OU3 fM chloride ion. 2 ml of toluene were added as an antiseptic; the mixture was gently swirled and placed in a thermostated oven a t 50°C for 32 hr. 10 ml of the supernatant were removed, replaced by 10 ml buffer, 20 mg of enzyme, and 0.2 ml tolu-
* The SKBll unit of activity for a-amylase is defined as the number of grams of soluble starch which under the influence of an excess of a-amylase are dextrinized by 1 g of enzyme preparation. Dextrinized starch is arbitrarily defined by the change from a violet-staining starch to a red-brown-staining starch. For example, if 0.05 g of enzyme preparation dextrinizes 0.4 g of starch in 1.5min, the a-amylase activity is 0.4_X 60 q.t X 60(min) - ~ _ -_32. genz X t(min)
0.05 X 15
CROSS-LINKED POTATO STARCH
267
ene, and replaced in the oven for 32 hr more. The sample was cooled, acidified to pH 2.0, and allowed t o stand for 1 hr with occasional shaking t o inactivate the enzyme. After neutralization, the sample was thoroughly washed, filtered, exchangod in an acetone gradirnt, air-dried overnight, and dried under vacuum a t 50°C for 2 hr.
Acid-Modified Crystalline Cross-Linked Potato Starch Approximatcly 0.9 g of cross-linked starch was mixed with 20 ml of 3 N HCl and the mixture was placed in a thermostated oven a t 40°C for 50 hr. After this time the mixture was washed t o neutrality and exchanged in an acetone gradient, air-drird and vacuum-dried at 50"C and reweighed. An alternate sample, yielding similar results, was prepared by treating 0.9 g2 of dry starch with 3 ml of 2 N HC1 for 7 days a t room temperature (24°C) followed by the addition of 2 ml of 4 N HC1 a t 38°C for 18 hr, washing and cxchanging as before, and drying a t 85°C in a vacuum ovcn.
Microscopic Observations All acid hydrolyzed samples were observed in thc polarizing microscope. It was noted that the treated granules mounted in H 2 0 were unusually soft and elastically deformable. Viewed bctwcen crossed polaroids, the maltese cross was retained in spite of the considerable wcight loss. The enzyme- and acid-modified samples were mounted on cylindrical aluminium supports (stubs) with a pressure-Zensitive adhesive film, rendered electrically conductive with a 200-400-A coating of 60: 40 goldpalladium alloy, and vicwed in a Stereoscan scanning electron microscope.
RESULTS AND DISCUSSION Acid Hydrolysis Since mild acid hydrolysis degrades noncrystalline rrgions preferentially,lZ it provides a useful qualitative indication of the degree of order in the g r a n ~ l e . ~However, thc rate and extent of hydrolysis are dependent on the starch used, the concentration of acid, and the temperature of hydrolysis. I n this study, nongelatinizing conditions werc chosen, but the conccntration of acid was adjusted t o give significant weight loss in a convenient time span. The results of two different acid treatments are shown in Figure 1. Evidently, the more acidic conditions increased the extent of hydrolysis, but the results were similar for the two treatments. The more highly crosslinked samples suffered greater weight loss, which parallels their sorption, bed volume, and water retention behavior.1° It should be noted that these hydrolysis results were opposite t o those obtained by Shimizu13whose cross-linked starches showed increased resistance t o acid hydrolysis. This will be discussed below.
268
HOLLINGER AND MARCHESSAULT
wt.
a0
Loss,% 40
1 MDC
I
20
10
(a)
M DC
I
20
10
(bl Fig. 1. (A) Weight loss of crystalline cross-linked starch after hydrolysis in 3 N HC1 a t 40°C. The point a t 0.0 MDC corresponds to a chemical treatment that was identical to that of the first cross-linked sample, but without epichlorohydrin. (B) Weight loss of crystalline cross-linked starch after acid hydrolysis in 2 iV HCl. The blank was identical to that in (A).
Enzyme Hydrolysis The extent of hydrolysis of various starches was studied by Leach and Schoch with different commercially available amylases. l 4 The amylolysis of corn and sorghum starches occurred by a uniform erosion and fragmentation of all the granules. I n contrast to this type of action, there was selective attack on indidivual granules of potato starch, i.e., certain granules were completely degraded while others were still intact. Others have found that degradation occurs slowly a t the periphery of the potato starch granule, but very rapidly once hydrolysis reaches the hilum. l 4 < l 5 The results of the a-amylase hydrolysis of cross-linked starch, as observed in this study, are presented in Figure 2. The results show that the introduction of cross-links into the crystalline granular starch inhibits the digestion by enzymes. This decrease in digestibility was a result of the inability
10
20
Fig. 2. Weight loss of crystalline cross-linked starch after incubation with 10% (dry starch basis) of B. subtilis a-amylase a t 50°C. The enzyme activity was 2500 SKB. The point of MDC = 0.0 corresponds to a chemical treatment that was identical t o that used for the MDC = 0 sample in Fig. I A.
CROSS-LINKED POTATO STARCH
269
of the enzyme to penetrate the network adequately (accessibility) and the inability of the enzyme to hydrolyze a-D-glucosidic linkages in the vicinity of the branch points or cross-links (molecular fitting). The importance of molecular fitting can be deduced from studies of the enzymic hydrolysis of substituted starches such as hydroxyethyl substituted starch; hydrolysis was markedly depressed by substitution.
Electron Microscopy I n this study our starting observations by SEN (Figure 3) were on the cross-linked crystalline potato starches, all of which showed character-
Fig. 3. (A) J.T. Baker purified starch. (B) Cross-linked starch (18.5 cross-links/lOO AGU) showing numerous fractured granules. (C) J.T. Baker starch showing fracture surface. (D) Characteristic surface of potato starch.
270
HOLLINGER AND MARCHESSAULT
Fig. 4. (A) Acid-modified J.T. Baker starch mounted as a slurry in water; the granules were broken by sliding a glass slide back and forth over the aluminium supports. The specimens were allowed to dry in air. Note the depression a t the center of the granule, the onion layering, and radial striations. (B) Acid-modified potato starch mounted as described above showing a core. (C) Acid-modified potato starch showing layering and a central cavity. In part this cavitation results from drying the starch granule under vacuum a t 85°C. (D) Acid-modified cross-linked starch (MIX2 = 1.54). The lamellae are very evident.
istically smooth external surfaces independent of their NDC. * I n comparing Figure 3A and B, it was evident that the number of fragmented granules increased with increasing cross-linking. Somc of thc granules that were broken showed radial ridges (Figure 3C) similar to those described by
* MDC: molar degree of cross-linking defined as number of cross-links per 100 anhydroglucose units (ACU).
CROSS-LINKED POTATO STARCH
271
Sterling.? The most noteworthy feature of Figure 3C and D is the characteristically smooth surface. After acid hydrolysis treatment, the cross-linked granules also showed radial striations whenever a fractured granule was viewed (Figures 4A and B). These samples were mounted on the aluminum stubs as a water slurry and were broken by shearing the surface of the stub with a microscope slide, after which the sample was allowed t o air dry. The fracture surfaces show radial striations, but the overall impression from micrographs such as these is that the “radial ridges” are definitely not microfibrils but rather conchoidal fracture surfaces produced by the shearing stress, which fractures the granules. I n Figure 4B a “core” is apparent; it appears analogous t o the “heart” of an onion and, in terms of the lamellar structure, what is seen is simply an interface between two lamellae. The appearance of this core is indistinguishable from a starch granule of similar size. This alone does not imply that growth takes place by apposition (i.e., by deposition of layers of starch a t the periphery of the granule) though autoradiographic experiments support this ont tent ion,^ but it is a logical way t o account for this frequently observed morphological trait. The effect of hydrolysis in highlighting the lamellar structure is shown in Figure 4C and D where the concentric lamellae become more contrasted with increased cross-linking. At the highest weight loss (-90yo), the lamellae appear physically separated from one another (Figure 5 ) . I n Figure 6, the electron micrographs of enzyme-modified cross-linked crystalline starch are presented. I n Figure 6A, the uncross-linked sample has a very rough pitted surface, and a large pit or LLbore hole” seems t o be developing. The layered structure is evident in the borehole. A comparison of micrographs corresponding t o increasing levels of cross-linking suggests that enzyme digestion occurs first a t the periphery of the granule. Figure 6B shows a granule center that is highly eroded as well as a pitted surface and well-defined lamellae, giving the impression that the central region of the granule is more susceptible t o enzyme attack than the outer layers and the periphery of the granule.” This is in agreement with the rcsults obtained by other researcher~’~ l5 who state that hydrolysis proceeds slowly at the periphery of the granule but very rapidly in the central regions once enzymes have penetrated t o the hilum. It would seem that the enzyme molecules reach the hilum by slowly creating “canals” in the granule a t points where the molecular structure is accessible t o the enzymes, or where the structure of the granule is inherently weak. I n Figure 6C, the enzyme-modified potato starches show very clearly defined lamellat. I n Figure 7, conchoidal fractures are superimposed on the lamellar structure. The radial striations in Figure 7 are similar t o those in Figure 4 and suggest that these fracture surfaces should not be interpreted as indicative of a radial arrangement of microfibrils in the starch granule. I n comparing thc electron micrographs of enzyme-treated crosslinked starch, the degree of attack seems to decrease as the degree of cross-
272
HOLLINGER AND MARCHESSAULT
Fig. 5 . Acid-modified cross-linked starch (J1I)C = IX..?). The lamellse remain separate probably due t o stresses developed in the drying procedure.
linking increases (c.g., in Figure 7B, granules c.xhibit decreased internal and surface attack by the tmzymw). This must cwtainly be rclatcd t o a decrease in molecular fitting and acc ibility of intckrnal surfacc to th(. ('11zymcs. I n the cnzymic hydrolysis study, a tc,mpcraturc, just beloir thc gdatinization temperature was used; in addition, thc starch was cxtcnsivcly dried prior t o its incubation with thc c~nzymi~ solution. Thc l a t h will tend t o minimize. the importanw of acccssibility brcauw of the structural faults14 produccd by drying. The c.xtcnt of hydrolysis in tliih study IS limited by
CROSS-LINKED POTATO STARCH
273
Fig. 6. (A) Enzyme-modified potato starch showing pitted surface and a developing bore hole. (B) Enzyme-modified potato starch showing pitted surface and degraded interior. (C) Higher magnification of the lamellae in (B). ( U ) Enzyme-modified cross-linked potato starch (1ll)C = 7.7). Note well-defined lamellae and eroded central portion.
t h r molecular fitting of the cnzj mC t o thc glucosidic linkagc in the vicinity of the cross-link. I n agreemclnt with the model for homogeneously crosslinked starch, thc digestion was complctcly inhibited when the distance. between cross-links was about three. glucow residues. lXFor lightly crosslinked starch, the cross-links had littlc effect on enxymolysis w h m compared t o the uncross-linkcd starch. Diff cwnt accessibility behavior was found when hydrolysis of t h r crosslinked sample was performed with H30+, rathcr than a-amylase. Thc
274
HOLLINGER AND MARCHESSAULT
Fig. 7. (A) Enzyme-modified cross-linked potato starch (MDC = 7.7). Note central erosion and lamellae. (B) Enzyme-modified cross-linked potato starch (MDC = 18.5). The damage t o the granule does not show lamellations nor surface pitting.
difference is so striking that it does not apprar likely t o be due t o a simple loss of crystallinity with increasing cross-linking. We believe that the effect of cross-linking on the starch is analogous t o the effect of wet-crosslinking in rayon :19 it prevents molecular and structural rearrangement on drying, which would normally decrease accessibility after treatment under a given set of conditions. Instead, a more open and internally strained structure, which is more accessible, is produced. However, since the effect involves molecular movements in the range of only a few angstroms, it has not been considered significant by enzymologists.
Comparison of the Morphological Chan es for Acid- and Enzyme-Modified Potato S arch
f
The layer structure is noticeably’ enhanced by enzyme hydrolysis, as compared to acid hydrolysis (compare Figures 4, 5, with I;igures 6, 7). Since the acid treatment was less effectivr than thc rnzymr hydrolysis in highlighting the lamellac, it can be concluded that thcse lamellae reflect large periodic density fluctuations in the radial direction. Superimposed on these periodic density fluctuations arc smaller fluctuations (Figure 8), the H30+ ion is which represent disorder within t h r l a y ~ r s . Evidently, ~ capable of penetrating regions whose density is greater than the average density (Dav)of the whole starch granule and therefore hydrolysis occurred in regions having densities up to DHaO+ ( D H a O + is defined as the maximum density of rrgions that arr accessiblc to hydrolysis by acid). This results in poorly defined lamellac a t the resolution and magnification used in these studirs. By contrast, the enzyme can only hydrolyzr in rrgions with a
CROSS-LINKED POTATO STARCH
275
DENSlT Y
I
GRANULE
RADIUS
R BEYOND RESOLUTION OF S E M
Fig. 8. Radial density fluctuations in the potato starch granule: density vs. granule radius. The crosshatching indicates regions of density accessible to enzyme attack as well as H,O+ while the speckled regions are accessible t o acid only. This statistical model with correlated radial fluctuations explains the lamellar texture by assuming that tangential density fluctuations are much smaller than radial fluctuations
degree of ordcr or density bclotv D,,,hcncc. the grcatcr cnhancemcnt of thc lamellar structure. Because of t h e low resolution of the SElI, lamellac of a thicknoss of a fow hundwd angstroms or less are not visible. Othcr methods such as T E l I on thin sections, may possibly rwc.al tht.m. Tho sevcxrity of acid hydrolysis is bopnd t o affect the fineness of lamellation. Another striking diff erenee between thc two modes of h j drolytic attack seems t o be in the e x t m t of surface damage duc t o mzymolysis. Bore holes and surface pits were common in the enzyme-modified samples, whereas in the acid-modified samples a uniform attack over all parts of the granule prevailed. According t o thc mechanism of a-amylase hydrolysis in solution, the substrate is bound t o the mzyme surface. Aftw the initial hydrolysis, thc macromolecular fragmcnt undergoes rc.arrangement on t h r enzyme site. In a heterogeneous system, there is lrss opportunity for the enzyme to diffusc away from the substrate, thereby favoring repeated attack in the Sam(’ area, which is particularly visible. a t thc surface of the granules.
Structure of the Starch Granule Because of the molecular compl(.xity and poor crystallinity of starch, structural models are difficult t o imagint.. Th(. radial chain organization and concentric lamellac have suggested a rcdationship t o the classical folded chain structure of synthrtic polymCrs.20-21 I n this paper, wc propose a statistical model (Figure 8) for the outer lamellac of starch with “correlated” density fluctuations in the radial direction. This means t h a t one
276
HOLLINGER. AND MARCHESSAULT
could, for a given starch, derive some correlation function that would give the probability that two lamellac of equal average densky would be a distance T apart. For a structure that is composed of spherical concentric lamellac, the correlation function has t o be the same along all radii. Starch granules seldom possess this degree of regularity and, in addition, thc relatively thick lamellac. in 1;igure 8 lead one t o expect a good deal of disorder or sublamellar structure inside thc granule. The evident cohesiveness of the lamellae is consistent wit,h the small density fluctuations within large lamellae as shown in Figure 8; the same small fluctuations are t o be expected in the tangential direction also, s a w for the points where fractures occur (Figurc. 7A). Thc lamcllac in potato starch arc seldom concentric and an organization as shown in 1;igurcI 8 accounts for the potential faults and clcavage planes brought out by the cnzymc: treatment, as in Figure 7A. Figure 7A also shows how the lamellation is much mow a characteristic of the outcr shell rather than t h r central cow. This suggcsts a lesser organization in the central zone; hence, a greater suscctpt,ibility t o cnzymolysis or acid hydrolysis since cooperative crystallization generally leads to greater cohesive density.
References 1. Frey-Wyssling, A. & Buttrose, 11.S. (1961) Makromol. Chem. 44, 173-178. 2. Bancher, E. & Holzl, J. (1964) Sturkc 16,81-84. 3. Buttrose, 34. S. (1960) J. Ultrastruct. Rrs. 4,231-257. 4. Sterling, C. (1968) in Starch and its Derivativcs, 4th ed., Radley, J. A,, Ed., Chapman and Hall. 5. Badenhuizen, N. P. (1969) The Biogenrsis of the Starch Granule in Higher Plants, Appleton-Century-Crofts, New York. 6 . Frey-Wyssling, A. & Lluhlethaler, K . (1965) Ultrastructural Plant Cytology, Elsevier, The Netherlands. 7. Sterling, C. (1971) Stiirke 23,193-196. 8. Hall, 1). 31. & Sayre, J. G. (1970) Trxtilc RPS.J . 40(2), 147-1.57. 9. Hollinger, C. G., Kuniak, L. & Narchessault., 11. H. (1974) 13iopol:ymers 13, 879 (1974). 10. Hollinger, C. G. (1973) 1 1 . S ~thesis, . Dept. Chemistry, Universite de XIontr6al. 11. Sandstedt, 13. AI., Kneen, E. &Shlish L. (1939) Ccreal Chen:. 16,712-720. 12. Bat,tista, 0. A. (1950) Znd.Eng. Chem. 42,502-507. 13. Shimizu, T. (1961) KogyoKagak.c~%ashi64,1241;(1962) as quoted in Chenz. Abstr. 57,7502,10908. 14. Leach, H. W. & Schoch, T. J. (1961) Cereal Chem. 38,34-46. 15. Gallant, D., Guilbot, A. & 1Zercier C. (1972) Cereal Chcm. 49,354. 16. Banks, W., Greenwood, C. T. & LIiiir, 1). I). (1972) Sturko 24, 181-187. 17. Borch, J. (1970), Ph.1). thesis, State University of New York, College of Forestry, Syracuse, N.Y. 18. Robyt, J. F. & Whelan, W. J. (1968) in Starch and its Derivatives, Radley, J. A., E d . , Chapman and Hall. 19. Tovey, H. (1961) Textile Res. J . 31,185-252. 20. Muhlethaler, K. (196.5)Stark? 17,245. 21. Kainuma, K. &French, 1). (1972) Biopolymcrs 11,2241-2250.
Received June 27, 1974 Accepted July 29, 1974
BIOPOLY MERS
Ultrastructure of Acid- and Enzyme-Modified Cross-Linked Potato Starch G. HOLLINGER and R. H. AIARCHESSAULT, Ddpartement de Chimie, UniversitC de MontrCal, C.P. 6,910, MontrCal, QuC., Canada
Synopsis Purified potato starch, cross-linked with epichlorohydrin (65-5.4 anhydroglucose units per cross-link) by an iterative method that preserved the native crystalline structure of the starch granule, was exposed to enzymic ( B . subtilis a-amylase at 50°C) and acidic (3 N HCl a t 40°C) hydrolysis. The stability of the granules to digestion by enzymes, as measured by their weight loss after incubation with the enzyme solution, increased with the degree of cross-linking; by contrast, the stability t o acid attack decreased as a function of the degree of cross-linking. The morphology of these samples was studied by scanning electron microscopy. A lamellar structure, which was most evident after enzyme treatment, was obsecved in samples that had a weight loss of greater than 507,. The results reflect differences in accessibility, which are interpreted in terms of a statistical model of the ultrastructure with correlated fluctuations in density in the radial direction.
INTRODUCTION A number of papers on the morphology of starch using transmission electron microscopy (TERI) 1-4 have provided some insight into the ultrastructure of the starch granule. The study of starch by T E M requires selective staining techniques combined, in some cases, with acid or enzyme hydrolysis. This treatment enhances the contrast between accessible and nonaccessible regions. However, possible artifacts due t o sample preparation have made the interpretation of TEA4 pictures difficult. Scanning electron microscopy (SEN) provides a complementary method for the study of starch morphology. Unlike T E M and light microscopy, SEA1 provides a n almost three-dimensional picture of the surface of a specimen as a result of its high depth of field; an added bonus is t h a t fractured granules allow viewing of internal structure. Two major types of organization of the starch granule have been rep ~ r t e d : ~a?n~onion-like or concentric lamellar structure, which usually is not seen in the dry state; and a fibrillar texture, which is observed in fracture surfaces of acid-treated potato ~ t a r c h . ~ Dilute acid treatment is needed t o develop the lamellar structure of potato starch. Stronger acid treatment has revealed that these lamellae, 5000 8 thick, have subspacings of a few hundred angstroms.6 Unlike the 265
@ 1975 by John Wiley & Sons, Inc.
266
HOLLTNGER AND MARCHESSAULT
lamellae of cereal starches, those of potato starch are not affected by climinating the daily rhythm of light and t c r n p e r a t ~ r e . ~The ? ~ lamellation has been attributed to chemical differmces between the layers,j differences in the density of deposition of molecules of starch,6and differences in crystallinity.2 Sterling? and others4 have proposed a radial fibrillar structure. The existence of microfibrils in potato starch (bawd on the appearance of fracture surfaces of acid-treatcd potato starch) has been reported. On the basis of the fracture surfaces of untreated potato starch, Hall and Sayre* proposed that potato starch is composed of plates stacked one on top of the other. These fracture surfaces display radially oriented ridges similar to those described by Sterling? and are rcminiscent of the conchoidal fracture of glass. I n this paper, we continue a previously reportedg effort t o characterize epichlorohydrin cross-linked potato starch. The cross-linking was performed under conditions t o maintain the granular and radially symmetric organization. Comparison of the degree of cross-linking as measured by a swelling analysis and a chemical analysis yielded similar results, leading us t o conclude that the cross-links were distributed uniformly. By observing the changes in accessibility to acid and enzyme hydrolysis as a result of cross-linking, further information on the ultrastructure and cross-link distribution of the starch samples has been obtained.
EXPERIMENTAL The preparation and characterization of the crystalline granular epichlorohydrin cross-linked starch (purified J. T . Baker potato starch) samples have been described e l s ~ w h e r c . ~All samples were dried in a vacuum oven below 65°C for 11/2hr prior t o the following treatments.
Enzyme-Modified Crystalline Cross-Linked Potato Starch Partially purified Bacillus subtilis a-amylase having an activity of 2500 SKB* units per gramlo was used in the following preparation. A 0.15-g sample of the cross-linked material, dried a t 8.5"C for 4 hr under vacuum was incubated with 20 mg of enzyme in a neutral phosphate buffer solution containing 0.01 M KH2P04 and O.OU3 fM chloride ion. 2 ml of toluene were added as an antiseptic; the mixture was gently swirled and placed in a thermostated oven a t 50°C for 32 hr. 10 ml of the supernatant were removed, replaced by 10 ml buffer, 20 mg of enzyme, and 0.2 ml tolu-
* The SKBll unit of activity for a-amylase is defined as the number of grams of soluble starch which under the influence of an excess of a-amylase are dextrinized by 1 g of enzyme preparation. Dextrinized starch is arbitrarily defined by the change from a violet-staining starch to a red-brown-staining starch. For example, if 0.05 g of enzyme preparation dextrinizes 0.4 g of starch in 1.5min, the a-amylase activity is 0.4_X 60 q.t X 60(min) - ~ _ -_32. genz X t(min)
0.05 X 15
CROSS-LINKED POTATO STARCH
267
ene, and replaced in the oven for 32 hr more. The sample was cooled, acidified to pH 2.0, and allowed t o stand for 1 hr with occasional shaking t o inactivate the enzyme. After neutralization, the sample was thoroughly washed, filtered, exchangod in an acetone gradirnt, air-dried overnight, and dried under vacuum a t 50°C for 2 hr.
Acid-Modified Crystalline Cross-Linked Potato Starch Approximatcly 0.9 g of cross-linked starch was mixed with 20 ml of 3 N HCl and the mixture was placed in a thermostated oven a t 40°C for 50 hr. After this time the mixture was washed t o neutrality and exchanged in an acetone gradient, air-drird and vacuum-dried at 50"C and reweighed. An alternate sample, yielding similar results, was prepared by treating 0.9 g2 of dry starch with 3 ml of 2 N HC1 for 7 days a t room temperature (24°C) followed by the addition of 2 ml of 4 N HC1 a t 38°C for 18 hr, washing and cxchanging as before, and drying a t 85°C in a vacuum ovcn.
Microscopic Observations All acid hydrolyzed samples were observed in thc polarizing microscope. It was noted that the treated granules mounted in H 2 0 were unusually soft and elastically deformable. Viewed bctwcen crossed polaroids, the maltese cross was retained in spite of the considerable wcight loss. The enzyme- and acid-modified samples were mounted on cylindrical aluminium supports (stubs) with a pressure-Zensitive adhesive film, rendered electrically conductive with a 200-400-A coating of 60: 40 goldpalladium alloy, and vicwed in a Stereoscan scanning electron microscope.
RESULTS AND DISCUSSION Acid Hydrolysis Since mild acid hydrolysis degrades noncrystalline rrgions preferentially,lZ it provides a useful qualitative indication of the degree of order in the g r a n ~ l e . ~However, thc rate and extent of hydrolysis are dependent on the starch used, the concentration of acid, and the temperature of hydrolysis. I n this study, nongelatinizing conditions werc chosen, but the conccntration of acid was adjusted t o give significant weight loss in a convenient time span. The results of two different acid treatments are shown in Figure 1. Evidently, the more acidic conditions increased the extent of hydrolysis, but the results were similar for the two treatments. The more highly crosslinked samples suffered greater weight loss, which parallels their sorption, bed volume, and water retention behavior.1° It should be noted that these hydrolysis results were opposite t o those obtained by Shimizu13whose cross-linked starches showed increased resistance t o acid hydrolysis. This will be discussed below.
268
HOLLINGER AND MARCHESSAULT
wt.
a0
Loss,% 40
1 MDC
I
20
10
(a)
M DC
I
20
10
(bl Fig. 1. (A) Weight loss of crystalline cross-linked starch after hydrolysis in 3 N HC1 a t 40°C. The point a t 0.0 MDC corresponds to a chemical treatment that was identical to that of the first cross-linked sample, but without epichlorohydrin. (B) Weight loss of crystalline cross-linked starch after acid hydrolysis in 2 iV HCl. The blank was identical to that in (A).
Enzyme Hydrolysis The extent of hydrolysis of various starches was studied by Leach and Schoch with different commercially available amylases. l 4 The amylolysis of corn and sorghum starches occurred by a uniform erosion and fragmentation of all the granules. I n contrast to this type of action, there was selective attack on indidivual granules of potato starch, i.e., certain granules were completely degraded while others were still intact. Others have found that degradation occurs slowly a t the periphery of the potato starch granule, but very rapidly once hydrolysis reaches the hilum. l 4 < l 5 The results of the a-amylase hydrolysis of cross-linked starch, as observed in this study, are presented in Figure 2. The results show that the introduction of cross-links into the crystalline granular starch inhibits the digestion by enzymes. This decrease in digestibility was a result of the inability
10
20
Fig. 2. Weight loss of crystalline cross-linked starch after incubation with 10% (dry starch basis) of B. subtilis a-amylase a t 50°C. The enzyme activity was 2500 SKB. The point of MDC = 0.0 corresponds to a chemical treatment that was identical t o that used for the MDC = 0 sample in Fig. I A.
CROSS-LINKED POTATO STARCH
269
of the enzyme to penetrate the network adequately (accessibility) and the inability of the enzyme to hydrolyze a-D-glucosidic linkages in the vicinity of the branch points or cross-links (molecular fitting). The importance of molecular fitting can be deduced from studies of the enzymic hydrolysis of substituted starches such as hydroxyethyl substituted starch; hydrolysis was markedly depressed by substitution.
Electron Microscopy I n this study our starting observations by SEN (Figure 3) were on the cross-linked crystalline potato starches, all of which showed character-
Fig. 3. (A) J.T. Baker purified starch. (B) Cross-linked starch (18.5 cross-links/lOO AGU) showing numerous fractured granules. (C) J.T. Baker starch showing fracture surface. (D) Characteristic surface of potato starch.
270
HOLLINGER AND MARCHESSAULT
Fig. 4. (A) Acid-modified J.T. Baker starch mounted as a slurry in water; the granules were broken by sliding a glass slide back and forth over the aluminium supports. The specimens were allowed to dry in air. Note the depression a t the center of the granule, the onion layering, and radial striations. (B) Acid-modified potato starch mounted as described above showing a core. (C) Acid-modified potato starch showing layering and a central cavity. In part this cavitation results from drying the starch granule under vacuum a t 85°C. (D) Acid-modified cross-linked starch (MIX2 = 1.54). The lamellae are very evident.
istically smooth external surfaces independent of their NDC. * I n comparing Figure 3A and B, it was evident that the number of fragmented granules increased with increasing cross-linking. Somc of thc granules that were broken showed radial ridges (Figure 3C) similar to those described by
* MDC: molar degree of cross-linking defined as number of cross-links per 100 anhydroglucose units (ACU).
CROSS-LINKED POTATO STARCH
271
Sterling.? The most noteworthy feature of Figure 3C and D is the characteristically smooth surface. After acid hydrolysis treatment, the cross-linked granules also showed radial striations whenever a fractured granule was viewed (Figures 4A and B). These samples were mounted on the aluminum stubs as a water slurry and were broken by shearing the surface of the stub with a microscope slide, after which the sample was allowed t o air dry. The fracture surfaces show radial striations, but the overall impression from micrographs such as these is that the “radial ridges” are definitely not microfibrils but rather conchoidal fracture surfaces produced by the shearing stress, which fractures the granules. I n Figure 4B a “core” is apparent; it appears analogous t o the “heart” of an onion and, in terms of the lamellar structure, what is seen is simply an interface between two lamellae. The appearance of this core is indistinguishable from a starch granule of similar size. This alone does not imply that growth takes place by apposition (i.e., by deposition of layers of starch a t the periphery of the granule) though autoradiographic experiments support this ont tent ion,^ but it is a logical way t o account for this frequently observed morphological trait. The effect of hydrolysis in highlighting the lamellar structure is shown in Figure 4C and D where the concentric lamellae become more contrasted with increased cross-linking. At the highest weight loss (-90yo), the lamellae appear physically separated from one another (Figure 5 ) . I n Figure 6, the electron micrographs of enzyme-modified cross-linked crystalline starch are presented. I n Figure 6A, the uncross-linked sample has a very rough pitted surface, and a large pit or LLbore hole” seems t o be developing. The layered structure is evident in the borehole. A comparison of micrographs corresponding t o increasing levels of cross-linking suggests that enzyme digestion occurs first a t the periphery of the granule. Figure 6B shows a granule center that is highly eroded as well as a pitted surface and well-defined lamellae, giving the impression that the central region of the granule is more susceptible t o enzyme attack than the outer layers and the periphery of the granule.” This is in agreement with the rcsults obtained by other researcher~’~ l5 who state that hydrolysis proceeds slowly at the periphery of the granule but very rapidly in the central regions once enzymes have penetrated t o the hilum. It would seem that the enzyme molecules reach the hilum by slowly creating “canals” in the granule a t points where the molecular structure is accessible t o the enzymes, or where the structure of the granule is inherently weak. I n Figure 6C, the enzyme-modified potato starches show very clearly defined lamellat. I n Figure 7, conchoidal fractures are superimposed on the lamellar structure. The radial striations in Figure 7 are similar t o those in Figure 4 and suggest that these fracture surfaces should not be interpreted as indicative of a radial arrangement of microfibrils in the starch granule. I n comparing thc electron micrographs of enzyme-treated crosslinked starch, the degree of attack seems to decrease as the degree of cross-
272
HOLLINGER AND MARCHESSAULT
Fig. 5 . Acid-modified cross-linked starch (J1I)C = IX..?). The lamellse remain separate probably due t o stresses developed in the drying procedure.
linking increases (c.g., in Figure 7B, granules c.xhibit decreased internal and surface attack by the tmzymw). This must cwtainly be rclatcd t o a decrease in molecular fitting and acc ibility of intckrnal surfacc to th(. ('11zymcs. I n the cnzymic hydrolysis study, a tc,mpcraturc, just beloir thc gdatinization temperature was used; in addition, thc starch was cxtcnsivcly dried prior t o its incubation with thc c~nzymi~ solution. Thc l a t h will tend t o minimize. the importanw of acccssibility brcauw of the structural faults14 produccd by drying. The c.xtcnt of hydrolysis in tliih study IS limited by
CROSS-LINKED POTATO STARCH
273
Fig. 6. (A) Enzyme-modified potato starch showing pitted surface and a developing bore hole. (B) Enzyme-modified potato starch showing pitted surface and degraded interior. (C) Higher magnification of the lamellae in (B). ( U ) Enzyme-modified cross-linked potato starch (1ll)C = 7.7). Note well-defined lamellae and eroded central portion.
t h r molecular fitting of the cnzj mC t o thc glucosidic linkagc in the vicinity of the cross-link. I n agreemclnt with the model for homogeneously crosslinked starch, thc digestion was complctcly inhibited when the distance. between cross-links was about three. glucow residues. lXFor lightly crosslinked starch, the cross-links had littlc effect on enxymolysis w h m compared t o the uncross-linkcd starch. Diff cwnt accessibility behavior was found when hydrolysis of t h r crosslinked sample was performed with H30+, rathcr than a-amylase. Thc
274
HOLLINGER AND MARCHESSAULT
Fig. 7. (A) Enzyme-modified cross-linked potato starch (MDC = 7.7). Note central erosion and lamellae. (B) Enzyme-modified cross-linked potato starch (MDC = 18.5). The damage t o the granule does not show lamellations nor surface pitting.
difference is so striking that it does not apprar likely t o be due t o a simple loss of crystallinity with increasing cross-linking. We believe that the effect of cross-linking on the starch is analogous t o the effect of wet-crosslinking in rayon :19 it prevents molecular and structural rearrangement on drying, which would normally decrease accessibility after treatment under a given set of conditions. Instead, a more open and internally strained structure, which is more accessible, is produced. However, since the effect involves molecular movements in the range of only a few angstroms, it has not been considered significant by enzymologists.
Comparison of the Morphological Chan es for Acid- and Enzyme-Modified Potato S arch
f
The layer structure is noticeably’ enhanced by enzyme hydrolysis, as compared to acid hydrolysis (compare Figures 4, 5, with I;igures 6, 7). Since the acid treatment was less effectivr than thc rnzymr hydrolysis in highlighting the lamellac, it can be concluded that thcse lamellae reflect large periodic density fluctuations in the radial direction. Superimposed on these periodic density fluctuations arc smaller fluctuations (Figure 8), the H30+ ion is which represent disorder within t h r l a y ~ r s . Evidently, ~ capable of penetrating regions whose density is greater than the average density (Dav)of the whole starch granule and therefore hydrolysis occurred in regions having densities up to DHaO+ ( D H a O + is defined as the maximum density of rrgions that arr accessiblc to hydrolysis by acid). This results in poorly defined lamellac a t the resolution and magnification used in these studirs. By contrast, the enzyme can only hydrolyzr in rrgions with a
CROSS-LINKED POTATO STARCH
275
DENSlT Y
I
GRANULE
RADIUS
R BEYOND RESOLUTION OF S E M
Fig. 8. Radial density fluctuations in the potato starch granule: density vs. granule radius. The crosshatching indicates regions of density accessible to enzyme attack as well as H,O+ while the speckled regions are accessible t o acid only. This statistical model with correlated radial fluctuations explains the lamellar texture by assuming that tangential density fluctuations are much smaller than radial fluctuations
degree of ordcr or density bclotv D,,,hcncc. the grcatcr cnhancemcnt of thc lamellar structure. Because of t h e low resolution of the SElI, lamellac of a thicknoss of a fow hundwd angstroms or less are not visible. Othcr methods such as T E l I on thin sections, may possibly rwc.al tht.m. Tho sevcxrity of acid hydrolysis is bopnd t o affect the fineness of lamellation. Another striking diff erenee between thc two modes of h j drolytic attack seems t o be in the e x t m t of surface damage duc t o mzymolysis. Bore holes and surface pits were common in the enzyme-modified samples, whereas in the acid-modified samples a uniform attack over all parts of the granule prevailed. According t o thc mechanism of a-amylase hydrolysis in solution, the substrate is bound t o the mzyme surface. Aftw the initial hydrolysis, thc macromolecular fragmcnt undergoes rc.arrangement on t h r enzyme site. In a heterogeneous system, there is lrss opportunity for the enzyme to diffusc away from the substrate, thereby favoring repeated attack in the Sam(’ area, which is particularly visible. a t thc surface of the granules.
Structure of the Starch Granule Because of the molecular compl(.xity and poor crystallinity of starch, structural models are difficult t o imagint.. Th(. radial chain organization and concentric lamellac have suggested a rcdationship t o the classical folded chain structure of synthrtic polymCrs.20-21 I n this paper, wc propose a statistical model (Figure 8) for the outer lamellac of starch with “correlated” density fluctuations in the radial direction. This means t h a t one
276
HOLLINGER. AND MARCHESSAULT
could, for a given starch, derive some correlation function that would give the probability that two lamellac of equal average densky would be a distance T apart. For a structure that is composed of spherical concentric lamellac, the correlation function has t o be the same along all radii. Starch granules seldom possess this degree of regularity and, in addition, thc relatively thick lamellac. in 1;igure 8 lead one t o expect a good deal of disorder or sublamellar structure inside thc granule. The evident cohesiveness of the lamellae is consistent wit,h the small density fluctuations within large lamellae as shown in Figure 8; the same small fluctuations are t o be expected in the tangential direction also, s a w for the points where fractures occur (Figurc. 7A). Thc lamcllac in potato starch arc seldom concentric and an organization as shown in 1;igurcI 8 accounts for the potential faults and clcavage planes brought out by the cnzymc: treatment, as in Figure 7A. Figure 7A also shows how the lamellation is much mow a characteristic of the outcr shell rather than t h r central cow. This suggcsts a lesser organization in the central zone; hence, a greater suscctpt,ibility t o cnzymolysis or acid hydrolysis since cooperative crystallization generally leads to greater cohesive density.
References 1. Frey-Wyssling, A. & Buttrose, 11.S. (1961) Makromol. Chem. 44, 173-178. 2. Bancher, E. & Holzl, J. (1964) Sturkc 16,81-84. 3. Buttrose, 34. S. (1960) J. Ultrastruct. Rrs. 4,231-257. 4. Sterling, C. (1968) in Starch and its Derivativcs, 4th ed., Radley, J. A,, Ed., Chapman and Hall. 5. Badenhuizen, N. P. (1969) The Biogenrsis of the Starch Granule in Higher Plants, Appleton-Century-Crofts, New York. 6 . Frey-Wyssling, A. & Lluhlethaler, K . (1965) Ultrastructural Plant Cytology, Elsevier, The Netherlands. 7. Sterling, C. (1971) Stiirke 23,193-196. 8. Hall, 1). 31. & Sayre, J. G. (1970) Trxtilc RPS.J . 40(2), 147-1.57. 9. Hollinger, C. G., Kuniak, L. & Narchessault., 11. H. (1974) 13iopol:ymers 13, 879 (1974). 10. Hollinger, C. G. (1973) 1 1 . S ~thesis, . Dept. Chemistry, Universite de XIontr6al. 11. Sandstedt, 13. AI., Kneen, E. &Shlish L. (1939) Ccreal Chen:. 16,712-720. 12. Bat,tista, 0. A. (1950) Znd.Eng. Chem. 42,502-507. 13. Shimizu, T. (1961) KogyoKagak.c~%ashi64,1241;(1962) as quoted in Chenz. Abstr. 57,7502,10908. 14. Leach, H. W. & Schoch, T. J. (1961) Cereal Chem. 38,34-46. 15. Gallant, D., Guilbot, A. & 1Zercier C. (1972) Cereal Chcm. 49,354. 16. Banks, W., Greenwood, C. T. & LIiiir, 1). I). (1972) Sturko 24, 181-187. 17. Borch, J. (1970), Ph.1). thesis, State University of New York, College of Forestry, Syracuse, N.Y. 18. Robyt, J. F. & Whelan, W. J. (1968) in Starch and its Derivatives, Radley, J. A., E d . , Chapman and Hall. 19. Tovey, H. (1961) Textile Res. J . 31,185-252. 20. Muhlethaler, K. (196.5)Stark? 17,245. 21. Kainuma, K. &French, 1). (1972) Biopolymcrs 11,2241-2250.
Received June 27, 1974 Accepted July 29, 1974
