Maize Starch Sample Preparation for Aqueous Size Exclusion

Chapter 14

Downloaded by UNIV MASSACHUSETTS AMHERST on August 1, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch014

Maize Starch Sample Preparation for Aqueous Size Exclusion Chromatography Using Microwave Energy Gregory A. Delgado, David J. Gottneid, and Robert N. Ammeraal American Maize-Products Company, 1100 Indianapolis Boulevard, Hammond, IN 46320 A convenient method for solubilizing maize starch is needed for characterizing and comparing starches from different genetic varieties of maize as well as their enzyme hydrolysis products. A water-based system for solubilization of starch was developed for size exclusion chromatography (SEC). Samples from four different commercial types of maize starch were prepared for SEC using microwave energy. A microwave method had the advantage of speed and convenience and it did not require addition of any reagent other than water while the chromatography yielded results comparable to other methods of preparation. A convenient method for s o l u b i l i z i n g starch i s needed for characteri z i n g and comparing starches from different genetic v a r i e t i e s of maize as well as their enzyme hydrolysis products. This method i s needed especially for performing size exclusion chromatography (SEC). SEC allows one to compare the r e l a t i v e d i s t r i b u t i o n of intact polymeric components of starches. A major concern about any s o l u b i l i z a t i o n method i s that disruption of the granular structure should proceed while minimizing degradation of the starch polymeric structure. Current methods of s o l u b i l i z a t i o n include u t i l i z i n g solvents, such as dimethyl sulfoxide (DMSO) ( 1 ) , or sodium hydroxide or autoclaving. The autoclave step i s usually followed by sonication i n either neutral or alkaline aqueous systems ( 2 ) . A new method for s o l u b i l i z a t i o n of starch has been developed and i s reported here. This method u t i l i z e s a neutral aqueous system and microwave energy. This microwave method has been used to compare starches from seven different genetic v a r i e t i e s of maize. Materials and Methods Materials.

Seven starches from different genetic v a r i e t i e s of maize

0097-6156/91/0458-0205$06.00A) © 1991 American Chemical Society

In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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were obtained from American Maize-Products Company. They included four commercial starches: waxy, common, Amylomaize V and Amylomalze VII. These starches have approximate apparent amylose contents of 0, 25, 50 and 70%, respectively. Starches from three other genetic v a r i e t i e s of corn were also used. They were d u l l waxy (duwx), d u l l horny (duh) and amylose extender d u l l (aedu). The approximate apparent amylose contents of these starches are 0, 30 and 50%, respectively. Chromatographic System. SEC was performed on a Hewlett-Packard 1090 Chromatograph. Two Waters l i n e a r U l t r a hydrogel columns (crosslinked methacrylate, 7.8 mm diameter χ 300 mm length) were connected i n series preceded by a guard column. The columns were maintained at 45°C The mobile phase was 0.1M sodium n i t r a t e i n water pumped at a flow rate of 0.8 m i l l i t e r s per minute. Temperature of the r e f r a c t i v e index detector was maintained at 4 0 ° C The i n j e c t i o n volume was 100 m i c r o l i t e r s . Commercial standards were used. Low molecular weight materials were obtained from Sigma. Higher molecular weight standards were obtained from Polymer Laboratories. They are derived from p u l l u l a n , and, as such, were used only as general indicators of molecular weight range rather than as precise standards. Standard solutions were prepared (0.1 wt. per volume) i n 0.1M sodium n i t r a t e aqueous mobile phase with 0.2% sodium azide added as a preservative. A l l solutions were passed through a 0.45 micron f i l t e r before i n j e c t i o n . Starch s o l u b i l i t y was determined by measuring t o t a l r e f r a c t i v e index response from SEC-HPLC chromatography. Detector response was compared to that of a standard soluble 5 DE waxy maize hydrolysate (Lo-Dex 5) of known concentration. Insoluble material was removed by f i l t r a t i o n . The mass of t o t a l s t a r t i n g material was measured gravimetrically. S o l u b i l i t y i s reported as the percent of soluble starch to the t o t a l s t a r t i n g dry s o l i d s . Autoclave/Sonication Method for S o l u b i l i z a t i o n . S o l u b i l i z a t i o n of maize starches by autoclaving followed by sonication i n both an aqueous and an a l k a l i system was performed according to the method of Jackson et a l . (2). Solvent (DMSO) Method for S o l u b i l i z a t i o n . A modified method for s o l u b i l i z i n g starch i n solvent was used (1). Maize starch samples were prepared i n a test tube by dissolving 16 mg. of starch i n 4 ml. of 90% dimethyl sulfoxide s o l u t i o n . A suspension was formed by mixing with a vortex mixer for 5 sec. It was then allowed to stand at room temperature for 30 min. It was again vortex-mixed for 5 sec. Then i t was placed i n a b o i l i n g water bath for 5 min. and allowed to cool to room temperature. It was again vortex-mixed for 5 sec. and centrifuged for 5 min. at 3000 χ g. Aliquots of 100 u l . were then c a r e f u l l y removed from the top of the supernatant l i q u i d and chromâtographed. Microwave Energy Method For S o l u b i l i z a t i o n . A microwave oven was preheated (3). A 0.1% starch sample was prepared by suspending 5.5 mg. of starch (dry basis) i n 5.0 ml. of deionized water. The sample was prepared i n a teflon microwave digestion bomb (model no. 4782,


14. DELGADO ET AL.

Maize Starch Sample Preparation

207

Parr Instrument Company). The bomb was placed i n a microwave oven and subjected to the desired radiation power l e v e l for the appropriate time i n t e r v a l . The bomb was then cooled i n a cold water bath. After the solution had reached room temperature i t was f i l t e r e d thru a 0.45 micron f i l t e r . The solution was then ion exchanged by adding ion exchange r e s i n and f i l t e r e d again thru a 0.45 micron f i l t e r . A sample of f i l t r a t e (100 u l . ) was then chromatograjihed by SEC.


Results and Discussion Our i n i t i a l objective was to evaluate the amylose component of different genetic v a r i e t i e s of starch. The exclusion volume of the p a r t i c u l a r column system was selected to end at 13 to 14 minutes; the early peak i n the chromatogram i s actually excluded material. This peak represents higher molecular weight materials and probably contains amylopectin. Molecular weight approximations were l i m i t e d to the highest pullulan molecular weight of 853,000 at a retention time of about 18 minutes. Sample Preparation Observations. The observations outlined below were made: A. During the autoclave/sonication method: 1) a gel was observed at the bottom of the test tube after b o i l i n g i n the aqueous system and i t persisted even after autoclaving, 2) sonication for longer time intervals showed increased dispersion of the g e l , 3) microscopy revealed both heat swollen and unswollen intact granules i n the sediment after centrifugation, 4) gelatinized starch was present after b o i l i n g i n the a l k a l i system; i t dispersed after autoclaving. B. When the solvent (DMSO) method for s o l u b i l i z a t i o n was used, after centrifugation: 1) a g e l - l i k e sediment formed at the bottom of the test tube and 2) the supernatant was c l e a r . C Increased c l a r i t y of solution and increased ease of f i l t r a t i o n were observed as the heating time and microwave power output were increased during the s o l u b i l i z a t i o n of starch using microwave energy. Autoclave Method. Amylomaize V was s o l u b i l i z e d i n an autoclaved aqueous system (a 10 ml. aliquot of a 0.5% by weight starch solution) and was sonicated at different time i n t e r v a l s . As sonication time increased, the r e l a t i v e r a t i o of high molecular weight material to low molecular weight material i n the soluble phase increased. This suggests that precipitated higher molecular weight material was being s o l u b i l i z e d by sonication. Table I shows that s o l u b i l i z a t i o n i n an aqueous system increased with increasing sonication time. S o l u b i l i z a t i o n i s also enhanced as the apparent amylose content of the starch decreases.


208

BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES Table I.

Effects of Sonication Time: % Solubilized


Sonication Time 30 seconds 80 seconds

Waxy 56.5% 79.0%

Starch Type Common 47.4% 75.3%

Amy V 39.5% 46.5%

When amylomaize 5 i s autoclaved and sonicated i n a l k a l i , the concentration of s o l u b i l i z e d components increased. As the a l k a l i concentration increased, a s h i f t i n the d i s t r i b u t i o n of components occurred with the appearance of more low molecular weight material. This may be due to degradation of the starch polymer. Solvent Method, unlike the autoclaved aqueous system, s o l u b i l i t y of the components i n 90% DMSO/water increased as the apparent amylose content increased. The lower molecular weight f r a c t i o n i s more easily s o l u b i l i z e d i n DMSO than i s the higher molecular weight fraction. O v e r a l l , this method yielded low percentages of s o l u b i l ity. Only 32.5% (by weight) was s o l u b i l i z e d for the most soluble starch, Amylomaize VII. Microwave Method. Common maize starch was heated by microwave for 6 minutes at different power l e v e l s . As the wattage increased, the concentration of s o l u b i l i z e d polymers also increased. At 1200 watts, 96% of the starch was s o l u b i l i z e d . However, the peak corresponding to the higher molecular weight f r a c t i o n was reduced, suggesting that degradation occurred. A l l of the following microwave method results were obtained by i r r a d i a t i n g starch samples with 720 watts of microwave energy. As the microwave heating i n t e r v a l increased, the amount of starch s o l u b i l i z e d increased. This was generally true for a l l the starches used i n this study. In the case of starch from corn with the d u l l horny mutation, as the microwave heating i n t e r v a l increased there was a s i g n i f i c a n t s h i f t toward a higher concentration of lower molecular weight materials. Chromatographic analysis of common and d u l l horny starches yielded s i m i l a r component d i s t r i b u t i o n patterns at approximately 70% s o l u b i l i t y (Figure 1). Waxy starch, which i s e s s e n t i a l l y a l l amylopectin, had an increase i n s o l u b i l i z a t i o n as the duration of heating with microwave energy was increased. The longest exposure time, however, revealed a reduction i n the higher molecular weight peak suggesting degradation. Starch obtained from d u l l waxy corn i s also comprised of amylopectin. The physical properties of this starch are s i g n i f i c a n t l y d i f f e r e n t , however, from those of conventional waxy starch. Chromatograms of d u l l waxy starch also show a s h i f t toward lower molecular weight materials with longer exposure to microwave energy, similar to waxy starch. Waxy and d u l l waxy starches yielded similar molecular weight d i s t r i b u t i o n at similar l e v e l s of solubilization. The d i s t r i b u t i o n patterns of components for a l l of the starches studied contained an early amylopectin peak (Figure 1) or shoulder (Figure 2). At a retention time of 19-20 minutes a peak i s evident which includes the higher molecular weight amyloses and intermediate


14.

DELGADOETAL.


209

WEIGHT % DISTRIBUTION 3.5 ι


2.5

0.5

14.2

16.2

18.2 20.2 RETENTION TIME (min.)

22.2

Figure 1. High-performance size exclusion chromatographic p r o f i l e s of common and d u l l horny maize starch s o l u b i l i z e d by microwave energy; Ο common, + d u l l horny.

WEIGHT % DISTRIBUTION 5

14.2

16.2

18.2 20.2 RETENTION TIME (min.)

22.2

Figure 2. High-performance size exclusion chromatographic p r o f i l e s at 62.7% s o l u b i l i z e d amylomaize V, 74.0% s o l u b i l i z e d amylomaize VII and 92.0% s o l u b i l i z e d amylose extender d u l l (aedu) maize starch s o l u b i l i z e d by microwave energy; A amy V, Φ amy V I I , Q aedu.


210

BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES

Table I I .

Microwave Energy Method for


Advantages

Solubilization

Disadvantages

Short Analysis Time

Indirect Temperature Control

Low Equipment Cost

Indirect Pressure Control

Low Energy Cost High S o l u b i l i z a t i o n No Special Reagents

Table I I I .

Comparison of S o l u b i l i z a t i o n Methods

Method Features

AC(Water)

AC(Akali)

DMSO

Preparation Time

135 min.

135 min.

105 min.

Equipment Needed

Hot

Steps Degradation

Plate

Hot

Plate

Microwave 90 min.

Hot Plate

Microwave Oven Digestion Bomb

Autoclave

Autoclave

Vortex Mixer

Sonicator

Sonicator

Centrifuge

Centrifuge

Centrifuge

Waterbath

Waterbath

8

9

10

4

No

Yes

No

No



14.

DELGADOETAL.


211

molecular weight branched molecules. This i s followed by a shoulder or broad d i s t r i b u t i o n of the lower molecular weight amyloses and smaller branched molecules. While there was s h i f t toward production of lower molecular weight fractions with overheating, the heating time at which this degradation begins i s different for each of the starches studied. Starch obtained from com with the amylose extender d u l l (aedu) mutation also contains approximately 50% apparent amylose. The aedu starch did not show a s h i f t i n molecular weight d i s t r i b u t i o n to a higher concentration of low molecular weight material as the duration of heating with microwave energy increased. This i s evident by a high concentration of material about the middle of the d i s t r i b u t i o n (Figure 2 ) . These features are unique when compared to Amylomaize V and Amylomaize VII molecular weight d i s t r i b u t i o n s , (Figure 2 ) . In general, as the apparent amylose content of starches increased, s o l u b i l i z a t i o n with microwave energy increased. This i s evident with 60% of the Amylomaize V starch s o l u b i l i z e d after only 4 minutes of heating time. The microwave energy method for starch s o l u b i l i z a t i o n for size exclusion chromatography had many advantages (Table I I ) . A minimum amount of time i s needed for sample preparation and equipment required i s r e l a t i v e l y inexpensive. A high degree of s o l u b i l i t y can be achieved but no special reagents or solvents are needed. The main disadvantages are that the temperature and pressures within the digestion bomb are controlled only i n d i r e c t l y . Excessive heating appears to damage the starch polymers. The autoclave/sonication method yielded a desired high s o l u b i l i t y but i t s disadvantages outweighed this advantage. It required a long analysis time and i t had a high equipment cost. High energy input and a s p e c i a l reagent were needed. The solvent (DMSO) method had a moderate analysis time and energy need. But, again, there was a high equipment cost and a special solvent was needed. This method's main disadvantage was that i t could not produce a high s o l u b i l i t y i n a short analysis time. Table III compares features of the four s o l u b i l i z a t i o n methods reviewed. A new microwave energy method was presented that i s convenient, quick and effective for the s o l u b i l i z a t i o n of starch. Current methods for s o l u b i l i z a t i o n were reviewed and the microwave method was compared to them. The microwave method was also used for comparing s o l u b i l i z a t i o n of starches from different genetic v a r i e t i e s of maize. It should be noted that i n d i v i d u a l methodologies must be varied depending on the type of starch and the apparent amylose and amylopectin content.

Literature Cited 1. 2. 3.

Kobayashi, S.; Schwartz, S. J.; Lineback, D. R.; Cereal Chem. 1986, Vol. 63, No. 2, 71-74. Jackson, D. S.; Choto-Owen, C., Waniska, R. D.; Rooney, L. W. Cereal Chem. 1988, Vol. 65, No. 6, 493-496. Schiffman, R. F. Microwave World 1987, Vol. 8, No. 1, 7-9, 14-15.

RECEIVED October 3, 1990


Maize Starch Sample Preparation for Aqueous Size Exclusion

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