Department of Carbohydrates and Cereals, UCT Prague
Czech Chemical Society
UNIVERSITY OF CHEMISTRY AND TECHNOLOGY PRAGUE
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Proceedings of the 12**" International Conference on Polysaccharides-Glycoscience Prague 19"'-21'* October 2016
ISBN 978-80-86238-59-3 ISSN 2336-6796
12th hiternational Conference on Polysaccharides-Glycoscience 2016 O P T I M I S A T I O N O F D I S S O L U T I O N C O N D I T I O N S O F P O T A T O S T A R C H
A R T U R S Z W E N G I E L
and
MARTA
F O RA Q U E O U S S E C
BEDNAREK'^
"Department oj Food Scienee and Nutrition, Institute of Food Technology of Plant Origin, Poznaii University of Life Sciences, Wojska Polskiego 28, 60-637 Poznan, ^'Department of Food Science and Nutrition, Institute of Food Technology of Plant Origin. Poznah University of Life Sciences, Wojska Polskiego 28, 60-637 Poznah, Poland artursz@up,poznan.pi,
[email protected]
Abstract
Dissolution conditions for normal potato starch for the purpose of aqueous S E C analysis were optimized using response surfoce methodology, fwo steps were combined—namely organic (5 % L i C l in DMSO) and aqueous stages. Three variables W'cre controlled: temperature, time, and volume of water added after the organic stage. The recoverv' of the sample, the molecular mass distribution, and radius of gyration were analyzed using multivariate statistics. The best results were obtained when the sample was dissolved in D M S O / L i C l at 80 °C lor 4 days with gentle stirring (80 rpm). The sample, after further dilution with DMSO, was directly injected into the S E C equipment. The calculated sample recover>^ was 98 % after aqueous S E C . Degradation of the sample during the dissolution process and retrogradation before the sample injection was not observed. This method additionally ensures mild dissolution conditions but requires a long period of time for sample preparation. I h e weight-average molecular mass (M„), polydispersity index {MJM,X and radius of gyration were calculated lor normal potato starch as 1.48 x 10' g/mol, 1.6, and 76 nm, respectively.
Introduction
Starch is commonly used in food products as a functional component and is the major source of energy for most individuals. It is important to understand the relationship between starch functionality and its molecular properties (weightaveraged molecular mass (Mw) and structure) ^ Ihe structural, physicochemical, and functional properties of potato starches have been studied extensively, and variation in starch parameters is probably considerably higher in different cultivars of potatoes than in other botanical starch sources". Starches isolated from dilTerent potato cultivars exhibit signitlcant variation in the physicochemical, morphological, thermal, and rheological properties ~\t has been suggested that the selection of potato cultivars may allow chemically modified starches to he replaced by native potato starches with unique properties". Industry has appreciated the ioiportatice of the average molecular mass and the radius of g\ration of starch for various reasons in the production of beverage thickeners, resistant starch, and enteral nutrition solutions, as well as in nonfood processing applications, for example the recoveiy of alumina from bauxite^ Water is a common solvent for starch (pressure cooking); other widely used solvents include sodium or potassium hydroxide and dimethyl sulfoxide (DMSO)'"^. Size exclusion chromatography ( S E C ) analysis of starch in aqueous media is difficult because the solubility of starch is limited in neutral aqueous solutions'\s for removing supermolecular starch structures are critical for S1:C, largely due to the fact that entangled amylose/annlopectin molecules cannot be separated and will affect the gyration radius and molecular mass deteniiined by the light scattering technique''', f-or this reason, DMSO is the most frequently used polar aprotic solvent for S E C analysis' Hydrogen-bond disrupters ( L i C l , L i B r ) are widely added to improve the solubility of p o l y s a c c h a r i d e s ' M o r e o v e r , interactions between column media and starch can occur, so lowmolecular additives (NaNOi, L i B r ) are added to the eluent'^' \ rhe aim of the present study was to determine the most optimal condition for complete dissolution of normal potato starch for the purposes of aqueous S E C . The central composite experiment was carried out with a two-step procedure for dissolving the starch samples. The sample recoverv, weight-average molar mass (/V/,,), and gyration radius {R^) were calculated, and the influence of the dissolution parameters was identified. Multidimensional statistics (cluster analysis, PCA, response surface methodology) were used for data analysis.
Materials and methods
A commercial native normal potato starch, Superior Standard (PPZ Trzemeszno, Poland), was used. PuUulan standard were purchased from Shodex (Japan). DMSO, NaNO^,, and L i C l were procured from Sigma-Aldrich Chemie GmbH (Germany). A two-step procedure was used to dissolve the normal potato starch using a central composite (response surface) experiment. Table I. Three variables were controlled—namely, temperature, time, and volume of water. The first dissolution step was carried out with DMSO containing 5% L i C l . Starch samples of 30 mg were dissolved in 5 ml DMSO/LiCl, and the temperature and time were adjusted; the samples were constantly stirred at 80 rpm in Reacti-Therm^heating and stirring modules (fhermo fisher Scientific, USA). The samples were further diluted with DMSO and SI-C was performed; the final concentration of the sample was 1.5 mg/mL. The second dissolution step was a continuation of the first. Water was added to 1.25 mL of solution irom the step and the samples were additionally incubated at 100 °C with gentle stirring (80 rpm); the volume of water and time were adjusted (Table I). The samples were further diluted with water to obtain a concentration of 1.5 mg/mL at the end and %EC assays were performed again.
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12th International Conference on Polysaccharides-Glycoscience 2016 1 he supplementar}' experiment was performed. The influence of time incubation at 80 °C on sample recovery in S E C was determined. 30 mg of normal potato starch was dissolved in DMSO containing 5% L i C l and the samples were thermostated for 6, 9, 24 h and 3, 6, 8 days. The samples were diluted after these times with DMSO to a final concentration of 1.5 mg/mL and S E C was performed. The samples were separated with S E C without nitration; the injection volume was 100 J L I L . S E C equipment (Malvern, T X , USA) with triple detection (Viscotek 305 T D A ) was used for starch separation. A conventional dual-cell refractometer, viscometer, and light scattering (low-angle light scattering, L A L S , and right-angle light scattering R A L S ) detectors were employed to act in concert. The S E C analysis was performed using three aqueous S E C columns (Shodex OHpak SB-800HQ series) with a guard S B - G type column (Showa Denko, Japan). The chromatography parameters were described by Kurzawska et al. (2014), where 0.1 M aqueous NaN03 solution was applied as an eluent at a 0.3 niL/min flow rate''\e calculations were perfbrmed using OmiSEC 4.7 software (Malvern, T X , USA). The experiments were conducted in triplicate. Parameters obtained from the S E C separations were the mean of three independent trials. Principal component analysis (PCA) was performed to reduce the dimensionality of the data. Hierarchical cluster analysis was performed using the Ward amalgamation rule with the Euclidean distance (d) measure. Ihe tree plots were scaled to a standardized scale (dlink/dmax*100)^\e models, including linear and quadratic main effects, were calculated to express the impact of variables on sample recovery, weight-average molar mass (M„.), and gyration radius (R^r). Statistical analysis was performed using Statistica version 10 (StatSoft, Inc., O K , US).
Results and discussion
The experiment was conducted using the parameters presented in fable I . Run 19 was eliminated because the time of dissolution was 0 minutes and the starch sample formed suspension. The temperature for runs 16 and 18 were too high, and nonenzymatic browning occurred (observable to the naked eye). Generally, three different temperatures were used during the first dissolution step: 80 °C, 108 °C, and 135 °C. The solutions of normal potato starch in D M S O / L i C l were diluted with DMSO and injected into the S E C equipment. The molar mass distribution (A/„, M„, M-), polydispersity index (M^v/M„), and radius of gyration (R^) were estimated. The starch sample recovery (%) was obtained from the integrated R I signal with cin/dc = 0.160 niL/g'^^. Ihe second dissolution stc]) was peribrmed because water is the most obvious solvent, corresponding to a real food environment; unfortunately, despite this, it gives incomplete dissolution' \ Table I Design of central composite (response surface) experiments on starch dissolution tactors/blocks/runs: 4/1/26 (constant mixing speed, 80 rpm), center points per block ( C )
during
two-step
process,
Step 1: Step 2: additional dissolution in D M S O / L i C l * dissolution with water (100 °C)** Temperature ( C ) Time (min) Water (mL) Time (min) 0.00 80 60 60 1 2 80 60 0.00 180 3 80 60 3.75 60 4 80 60 3.75 180 5 80 0.00 60 180 80 6 0.00 180 180 7 80 3.75 60 180 80 8 180 3.75 180 9 135 60 0.00 60 10 135 60 0.00 180 11 135 60 60 3.75 12 135 60 3.75 180 13 135 60 180 0.00 14 135 0.00 180 180 15 135 3.75 180 60 16 135 3.75 180 180 17 53 2.50 120 120 18 163 120 2.50 120 19 108 0 2.50 120 20 108 240 2.50 120 21 108 120 0.00 120 22 108 3.75 120 120 23 108 2.50 0 120 24 108 2.50 120 240 25 (C) 108 2.50 120 120 26(C) 108 120 2.50 120 * 5 mL of DMSO containing 5% of L i C l was added to 30 mg of starch sample ** water was added to 1.25 mL of solution from step 1 and incubated at 100 °C; water was added additionally at the end to obtain a concentration of 1.5 mg/mL Run
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12th International Conference on Polysaccharides-Glycoscience 2016 It was assumed that in the first stage, molecular dispersion of the starch would be obtained and that the water introduced in the second stage would give the opportunity to inject the sample into the S E C columns in a solution close to aqueous phase. The samples were diluted with water at the end to obtain the same concentration of starch per volume (1.5 mg/1 m L ) . The DSMO remaining (20%) after the second stage delayed the process of starch retrogradation, especially of amylose. It was reported previously that the aggregation of molecules gives inaccurate results in physical and structural a n a l y s e s ' T h e same parameters and sample recovery were estimated with S E C , as mentioned above, following the second dissolution stage. 0.5 M L i C l was used also as the solvent for amylose and potato starches by Radosta et al. (2001)1 fhe samples were shaken and swelled in the solvent for 24 h at 80 °C: they were additionally dissolved by stirring for 5 h at 120 °C under a nitrogen atmosphere was performed in their experiment. The measured sample recover}' was between 79% and 100% for commercial potato starch and debranched amylose form normal potato starch, respectively. PCA was performed for the samples from the first dissolution step; the eight parameters were represented by two principal components (PCs) (Fig. l A ) . The variables M,„ M,,, M-, and form a group of vectors that shows a positive correlation between these parameters. The MJM„ ratio was negatively correlated with molar mass averages and R^,. The sample recovery was positively correlated with temperature of dissolution, but dissolution time affected the molar mass distribution; the highest values were detected with longer dissolution time and moderate temperature. The three groups were circled in a score plot (Fig. I B ) . The group characterized by the lowest sample recovery (I) was dissolved at 53 °C and 80 °C for 120 and 60 minutes, respectively. The samples dissolved at 80 °C for 180 minutes were located together with samples dissolved at 108 °C—group I I , with the highest M„, A/,,., M- and R^r. Samples located in group I I I had the highest recover>' but lower molar masses and higher polydisperity indices, which indicates depolymerization of macromolecules when the samples were dissolved at 135 °C. fhese observations suggest that dissolution at lower temperatures with prorogated dissolution time may be the most effective. The average values of M„ were 1.13 xlO^\7 x 10"', and 1.18 x \{f g/mol for group I , I I , and I I I respectively. The average sample recovery for the samples in group I (28%) was 2.3, which is 2.8 times lower than the recovery^ calculated for groups I I and I I I . The radius of gyration was in range of 66-76 nm for all samples. The lowest average polydispersity index (1.9) was seen in group I I , with a value 3.9 and 9.0 times higher than in group I I I and I , respectively. It has been reported in the literature that amylopectin A/,v values are highest (3.7 x lO^ g/mol) for the waxy potato starch. The A/,v of amylopectin of amylose-containing starches is in the range from 2.3 x lO'^ to 2.6 x 10^ g/mol. Calculated /?g values range from 127 to 146 nm. The same authors determined M,v of amylose to range from 1.1 x 10^ to 2.4 x 10' g/mol and R^ to be between 19 and 36 nm'"^. Our results fall within these values as the partial coelution of amylopectin and amylose was observed and the parameters were estimated as average value for both fractions. Moreover, we assume that the molecular density of amylopectin can increase during aqueous S E C analysis (smaller R^ values). Since the samples were dissolved in DMSO/LiCl during aqueous S E C , the solvation layer was changed from DMSO to water. However, the conformations of high molecular mass amylose in DMSO and water are similar '. It has been reported that the solubility of corn amylopectin decreases when the water content in the DMSO increases '. Additionally wheat amylopectin in DMSO solutions has an oblate ellipsoid conformation, whereas in water it is aggregated to yield a more spherical shape"". Lee at al. (2014) also reported that an excess amount of water caused aggregation of corn amylopectin molecules"', fhe association of amylopectin was not observed (an enormous viscometric peak was not recorded; data not presented), probably because NaNO;, was used in the eluent to limit the interactions between the column media and starch; we presume that this low-molecular additive also limited interaction between molecules during S E C .
Fig. I . P C A of the loadings plot (A) and score plot (B); S E C results for the first step runs of starch dissolution in D M S O / L i C l using the parameters of the response surface experiment, Table I ; the supplementary variables are indicated by superscript (*) principal components P C I and PC2 were computed using only the active variables
Ihe similarity of the parameters set used in experimental runs in the two-step starch dissolution process (Table I) can be seen. The cases were explored using cluster analysis. The clustering was achieved with Ward's algorithm and the results are presented in Fig. 2. The profile similarity of the parameters can be seen. Flierarchical tree analysis (Fig. 2) shows that it is possible to distinguish two large groups, cutting the tree diagram at 70 %. It is possible to extract smaller groups from the
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12th hiternational Conference on Polysaccharides-Glycoscience 2016 dendrogram, cutting the diagram at a lower value that indicates that the parameters used during the two steps of dissolution process varied.
Fig. 2. Results of cluster analysis; parameter variability of the two dissolution steps was compared, the tree's scale was normalized to dlink/dmax*100 (d: distance; 1: linkage; max: maximum of linkage and Euclidean distance). Amalgamation rule: Ward's method; distance metric: Euclidean
PCA was also performed for the sample following the second dissolution step. The results presented in Fig. 3 were obtained after S E C chromatography. Belbre S E C , the starch samples were sequentially dissolved in D M S O / L i C l and the process was continued when water was added. M„, M„, M-, and R„ correlated positively with the volume of water added during the second dissolution step (Fig l A ) . It can be assumed that the retrogradation of starch occurred in water (positive correlation of and volume of water) and that the aggregation of molecules can give inaccurate results in physical and structural analyses', the sample presented as group I I (Fig. 3B). However, the aggregation of amylopectin was not observed in this dissolution step (an enormous viscometric peak was not recorded; data not presented). The sample recovery was positively correlated with sample incubation time during the second step and with temperature during the First step. The polydispersity index was higher in the group I (Fig. I B ) , where a small volume of water was added (Fig. l A ) . Presumably, the amylose after retrogradation had similar molecular mass as amylopectin and MJM„ was lower in group I I . The variable "Time ( D M S O / L i C l ) " was poorly explained by the two first principal components. For groups I and I I respectively, the average values of M„. were 2.40 x 10 and 4.12 X 10^' g/mol; for M,.//V/„, 3.3 and 1.8; for R„. were 77 and 88 nm; and for sample recovery, 48 and 57%. o Group: I
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Fig. 3. P C A of loadings plot (A) and score plot (B); S E C results after the two-step starch dissolution in D M S O / L i C l and water according the parameters of the response surface experiment (Table I ) ; the supplementary variables are indicated by superscripts (*), the principal components ( P C I and PC2) were computed using only the active variables
The linear relationship between variables was examined above with PCA. The linear ( L ) and quadratic (Q) effects are seen below using response surface models. The significant effects are shown in Figure 4. The inlluence of the dissolution parameters following the two steps of starch dissolution on sample recovery, M,, and R,^, was examined. In considering the sample recovery (Fig. 4 A ) , a significant negative quadratic effect of dissolution time in D M S O / L i C l was seen. This means that too high and too low dissolution times were negative; other parameters had positive linear effects. The time of sample incubation with water added during the second step had significant negative quadratic etTect on M„.; the other effects were on the borderline of statistical significance (Fig. 4B). The linear effect of dissolution time with water and volume of water had an iniluence on R^ (Fig. 4C). These observations are complementary to the results and the discussion presented during the interpretation of the PCA plot (Fig. 3).
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12th International Conference on Polysaccharides-Glycoscience 2016
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Time ( D M S O - L i C i ) (Q)
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Fig. 4. Pareto charts of standardized effects estimated with response surface methodology (A: sample recovery (%); B: M„ (g/mol); C : Rf. (nm)); vertical lines indicate the minimum magnitude of statistically significant linear ( L ) and quadratic (Q) effects. Effects were calculated for S E C results after the two steps of starch dissolution in D M S O / L i C l and water, following the parameters of the response surface experiment
In this connection, an additional experiment was carried out. It was assumed that i f the additional dissolution step with water leads to negative effects, it is necessary to perform the dissolution in D M S O / L i C l at 80 °C and then inject the sample diluted with DMSO into the S E C equipment. However, it was noted that during incubation of the sample at 80 °C (step 1, Table I), the maximal sample recovery v^as not higher than 66 %. I h e iniluence of incubation time on sample recovery, M„., and R^ at 80 °C was tested. The normal potato starch dissolved in D M S O / L i C l in 6 hours to 8 days. The S E C chromatograms of the R I and L S detectors are presented in Fig. 5. Sample recovery for samples incubated for 6, 9, and 24 h and 3, 6, and 8 days was calculated, and the recovery values were found to be 39 %, 48 %, 53 %, and 95 %, 96 %, 98 %, respectively. The best results were obtained for the third and sixth day of incubation; degradation of the sample was recorded after 8 days of incubation. The average values of the estimated parameters from the recorded S E C runs of the samples incubated for 3 and 6 days (with R S D (%)"given in brackets) are: M,, = 1.48 x 10^ g/mol (2.8), A/„/M, = 1.57 (4.4), 76 nm (1.2). The dissolution process at 80 °C for 4 days (with an additional one-day safety margin) was repeated three times and the results were not statistically dilTerent from samples dissolved for 3 and 6 days ( A N O V A was performed, p > 0.05). It has previously been noted that elongated dissolution time in 90 ^'C DMSO and exposure to shear is also necessar> to obtain fully solubilized com starch. It has also been observed that solubility stopped increasing after 67 h " . The dissolution conditions estimated above for normal potato starch are not universal for all types of starch samples (Radosta et al., 2001); the dissolution conditions were therefore adjusted for each potato starch^.
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Fig. 5. S E C chromatograms of normal potato starch dissolved in D M S O / L i C l at 80 °C for 6 hours to 8 days; A: signals from the refractive index (RI) detector; B: signals from the low-angle light-scattering ( L A L S ) detector
Conclusions
The application of aqueous S E C to starch is limited because it is not possible to obtain complete dissolution in water. Additionally, the process of retrogradation occurs easily in water. The analysis of dissolution parameters after the two steps of starch dissolution on sample recovery showed significant negative quadratic effect of dissolution time in DMSO/LiCl. The time incubation of the sample with added water during the second step had significant negative quadratic effects on M,,. The linear effect of dissolution time with water and the volume of water had an effect on R^r. The dissolution in DMSO/LiCl at 80 °C was tested in detail because the additional dissolution step with water gives negative effects. The calculated sample recovery alter aqueous S E C analysis indicated that dissolution of samples at 80 °C for 4 days in DMSO/LiCT allows appropriate sample dispersion and recovery close to 100 %. The advantage of this method is the lack of degradation of the sample during dissolution, retrogradation before sample injection, and mild dissolution conditions. The authors thank Katarzyna Raczyng for help with sample preparation for some of the SF.C measurements.
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12th International Conference on Polysaccharides-Glycoscience 2016 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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