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Effects of citric acid esterification on digestibility, structural and physicochemical properties of cassava starch
Published Date 15 November 2015, Vol.187:378–384,doi:10.1016/j.foodchem.2015.04.076 Title Effects of citric acid esterification on digestibility, structural and physicochemical properties of cassava starch
Author
Ji-Qiang Mei a
Da-Nian Zhou a
Zheng-Yu Jin b
Xue-Ming Xu b
Han-Qing Chen a,,
aSchool of Biotechnology and Food Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China
bState Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China
Received 15 February 2015. Revised 31 March 2015. Accepted 2 April 2015. Available online 23 April 2015.
Highlights
•
Citric acid esterification can increase the SDS and RS contents of cassava starch.
•
Citric acid esterification does not change the crystalline pattern of cassava starch.
•
Citric acid esterification can improve the freeze-thaw stability of cassava starch.
•
Citric acid esterification changes the thermal properties of cassava starch.
Abstract
In this study, citric acid was used to react with cassava starch in order to compare the digestibility, structural and physicochemical properties of citrate starch samples. The results indicated that citric acid esterification treatment significantly increased the content of resistant starch (RS) in starch samples. The swelling power and solubility of citrate starch samples were lower than those of native starch. Compared with native starch, a new peak at 1724 cm−1 was appeared in all citrate starch samples, and crystalline peaks of all starch citrates became much smaller or even disappeared. Differential scanning calorimetry results indicated that the endothermic peak of citrate starches gradually shrank or even disappeared. Moreover, the citrate starch gels exhibited better freeze–thaw stability. These results suggested that citric acid esterification induced structural changes in cassava starch significantly affected its digestibility and it could be a potential method for the preparation of RS with thermal stability.
Keywords
Cassava starch
Citric acid
Degree of substitution
Digestibility
Structure
Physicochemical property
1 Introduction
Starch is the main component of cereal grains and the most important dietary source of energy for humans. It is composed of essentially linear amylose and highly branched amylopectin with α-d-glucopyranose as the structural unit (Zhang, Sofyan, & Hamaker, 2008).
For nutritional purposes, starch is generally classified into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) in terms of the rate and extent of its digestion (Englyst, Kingman, & Cummings, 1992). RDS induces a rapid increase in blood glucose and insulin levels after ingestion. SDS prolongs the release of glucose, thus preventing hyperglycaemia-related diseases. RS reduces starch availability for digestion and produces short-chain fatty acids in the large bowel through fermentation, which is beneficial to colon health and protection against colorectal cancer (Lehmann & Robin, 2007). Furthermore, resistant starch has been categorized into four classes: physically inaccessible starch (RS1), granular starch (RS2), retrograded amylose or high amylose starches (RS3), and chemically modified starches (RS4) (Eerlingen & Delcour, 1995). Consequently, starch ingredients containing high levels of SDS and RS can improve the nutritional function of foods.
In recent years, many types of chemically modified starches have been prepared by acid hydrolysis, oxidation, etherification and cross-linking (Santacruz, Koch, Svensson, Ruales, & Elisson, 2002). Esterification is one of the most important chemical modification methods for starch. Acetic, citric and formic acids have been often used for chemical modification of starch (Klaushofer, Berghofer, & Steyner, 1978). Compared with other substances, citric acid is nutritionally harmless, and the rate of digestion of esterified starch by pancreatin is decreased with increasing degree of substitution (DS) by citrate (Klaushofer et al., 1978). Citric acid, when heated, will dehydrate to form an anhydride, which will react with starch to form a starch-citrate adduct. Further heating can result in additional dehydration of citric acid and give rise to cross-linking (Wing, 1996). Xie and Liu (2004) reported that 78.8% resistant starch was obtained when citric acid (40% of starch dry weight) was reacted with normal corn starch at 140 °C for 7 h. Shin et al. (2007) showed that the SDS fraction of citric acid-treated rice starch increased to 54.1% after heat treatment.
Cassava starch contains less amylose as compared to wheat, potato and maize starches, and it has some superior qualities like bland taste and flavor, high paste clarity and less tendency to retrograde (Raja, 1995). Esterification of cassava starch for preparing low DS acetates and citrates was investigated by Agboola, Akingbala, and Oguntmein (1991). However, the effect of citric acid esterification on the structural and physicochemical properties of cassava starch has not been reported.
The aims of the present study were to investigate the influence of different citric acid concentrations on the esterification reaction of cassava starch, and the digestibility, structural and physicochemical properties of citrate starch samples with different DS were compared.
2 Materials and methods
2.1 Materials
Cassava starch was obtained from the Guangxi Hongfeng Starch Company (Guangxi, China). α-amylase type VI-B from porcine pancreas (EC 3.2.1.1, A3176) and amyloglucosidase (EC 3.2.1.3) were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA) and Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China), respectively. All other chemicals were of analytical grade (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China).
2.2 Preparation of citrate starch samples
Citrate starches were prepared based on the method described by Klaushofer et al. (1978) with some modifications. Citric acid (10%, 20%, 30%, 40% of starch dry weight) was dissolved in 12 mL distilled water, then the pH of the solution was adjusted to 3.0 with 10 M NaOH, and finally diluted to the final volume of 24 mL. The citric acid solution (24 mL) was mixed with 20 g cassava starch in a stainless steel tray and conditioned at room temperature for 8 h. The tray was then placed in a forced-air oven and dried at 50 °C for 24 h to a moisture level of 5–10% (w/w). The mixture was ground and dried at 130 °C for 5 h in a forced-air oven. The dry mixture was washed three times with distilled water and centrifuged to remove unreacted citric acid. Finally, the washed starch was air-dried at 45 °C and ground again.
2.3 Determination of in vitro digestibility of starch samples
The in vitro digestibility of starch samples was determined according to the method of Englyst et al. (1992) with a slight modification. Starch sample (200 mg) was dispersed in distilled water (5 mL) in 50-mL screw-capped polypropylene centrifuge tube and mixed well. After mixing, the tube was placed in a boiling water bath for 20 min. The sample was stirred with magnetic stirring bars during heating. After cooking, the tube was placed in a 37 °C water bath until it is cooled. Then phosphate buffer (10 mL, 0.2 mol/L, pH 5.2) and five glass balls (10 mm in diameter) were added to centrifuge tube. After equilibration at 37 °C for 5 min, the enzyme solution (5 mL) was added to the sample tube, followed by incubation in a water bath at 37 °C with shaking (170 rpm).
Aliquots (0.5 mL) were taken at intervals of 20 and 120 min and mixed with 4 mL of 80% ethanol to deactivate the enzymes. The mixed solution was centrifuged at 2000 rpm for 10 min, and the glucose content in the supernatant was measured using the 3, 5-dinitrosalicylic acid (DNS) method (Miller, 1959). The percentage of hydrolyzed starch was calculated by multiplying a factor of 0.9 with the glucose content (Zhang et al., 2011). Each sample was analyzed in triplicate. The percentages of RDS, SDS, and RS in the samples were calculated by the following equations:
where, G20 and G120 are the amounts of glucose released within 20 and 120 min of hydrolysis, respectively, and FG is the amount of free glucose in starch and TS is total starch weight.
2.4 Determination of degree of substitution (DS)
The amount of citric acid esterified to the starch was analyzed by the method of Klaushofer, Berghofer, & Pieber (1979), which was based on the reaction of citric acid and Cu2+ that formed a stable complex during titration with a solution of copper sulfate. DS was calculated based on the average number of substituent groups per anhydroglucose unit. Briefly, the starch sample (450 mg) was dispersed in 2 mL deionized water and dissolved in 50 mL of 1 M KOH. The solution was then boiled in a water bath for 10 min. After the solution was cooled to 25 °C, the pH was adjusted to 8.5 with 5 M acetic acid. The solution was added to sodium borate buffer (25 mL, pH 8.5H) containing indicator (0.3 g; Murexide:sodium sulfate = 1:500, w/w) and diluted to 300 mL with deionized water. The solution was titrated with 0.05 M copper sulfate solution until the color of the indicator (red-violet) disappeared. DS was calculated by the following equation:
where W (% by weight of substituent) = [bound citrate (g)/sample (g) − bound citrate (g)] × 100, and M = molecular weight of the citric acid substituent which was 175.1. Each sample was analyzed in triplicate.
2.5 Swelling power and solubility
Swelling power (SP) and solubility (S) were measured according to a modified method of Schoch (1964). A starch sample (0.5 g) was suspended in the centrifuge tube containing 50 mL distilled water and kept in a shaking water bath at 55 °C, 65 °C, 75 °C, and 85 °C for 30 min, respectively. The centrifuge tube was then cooled rapidly to 25 °C. After centrifugation at 2000 rpm for 15 min, the supernatant was collected and dried at 105 °C for 2 h and the remnant was weighed, and the precipitate was weighed. The solubility (%) was determined as the weight ratio of the dried supernatant to the dry starch. Swelling power (g/g) was determined as the weight ratio of the precipitate in the tube to the dry starch.
FT-IR was measured according to the method of Li, Ward, and Gao (2011) and Xie, Hu, Jin, Xu, and Chen (2014). All infrared spectra were obtained on a Nicolet 6700 spectrometer (Thermo Electric Corporation, Waltham, MA, USA). A spectral resolution of 4 cm−1 was employed and 64 scans were acquired for each spectrum. Spectra were baseline-corrected and deconvoluted by drawing a straight line at 1200 and 800 cm−1. The absorbance ratio of 1047 cm−1/1022 cm−1 was obtained from the deconvoluted spectra using Omnic version 8.0 software (Thermo Fisher Scientific Inc. Waltham, MA, USA). The ratio of 1047 cm−1/1022 cm−1 from deconvoluted FT-IR spectrum has been used to express the amount of ordered crystalline to amorphous domains in starches.
2.7 Differential scanning calorimetry (DSC)
The thermal properties of starch samples were determined by DSC (Q200, TA Instruments, New Castle, DE, USA) according to the method described by Xie et al. (2014) with a slight modification. Approximately 3 mg anhydrous starch sample was mixed with 6 μL deionized water and hermetically sealed in an aluminum pan. Then the pan was equilibrated at 4 °C for 24 h. After equilibration, the pan was heated from 20 °C to 130 °C at a rate of 10 °C/min. An empty pan was used as a reference. Onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and gelatinization enthalpy (ΔH) were calculated using DSC software (TA Instruments, New Castle, DE, USA). Experiments were conducted in triplicate.
2.8 X-ray diffraction
X-ray diffraction analysis was performed with an X-ray diffractometer (D/MAX 2500V, Rigaku Corporation, Japan), operating at 40 kV and 40 mA with Cu Kα radiation. The starch samples were dried at 40 °C for 6 h, and then scanned at 2θ values from 3° to 30° at room temperature (Xie et al., 2014). The degree of relative crystallinity was calculated with MDI-Jade 6.0 software (Material Date, Inc. Livermore, California, USA) according to the method of Nara and Komiya (1983).
2.9 Freeze–thaw stability
The freeze–thaw stability was determined by the method of Lee and Yoo (2011). An aqueous suspension of starch (5 g/100 g) was heated at 95 °C for 30 min under constant mild agitation, and then cooled to room temperature in an iced water bath. The paste of 15 g was taken and placed in a centrifuge tube and subjected to successive freeze–thaw cycles by freezing at −18 °C for 24 h and thawing at 30 °C for 1.5 h, followed by centrifugation at 2000 rpm for 30 min. After centrifugation, the supernatant eliminated from the gel was weighed, and the extent of syneresis was expressed as the percentage of liquid separated per total weight of sample in the centrifuge tube. In the present study, 3 freeze–thaw cycles were performed. Each sample was determined in triplicate.
2.10 Statistical analysis
Results are expressed as the mean ± standard deviation of triplicate experiments. Data were analyzed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test using SPSS 17.0 Statistical Software Program (SPSS Incorporated, Chicago). A value of P < 0.05 was considered statistically significant.
3 Results and discussion
3.1 Effect of different citric acid concentrations on the digestibility and DS of starch samples
When citric acid is heated, it will dehydrate to yield an anhydride. The citric anhydride can react with starch to form citrate starch. In the present study, citric acid of 10–40% based on starch dry weight was applied for esterification reaction. As shown in Fig. 1A, as citric acid concentration increased, the RDS content of citrate starch samples significantly decreased, whereas the SDS and RS contents markedly increased. When the citric acid concentration was increased from 10% to 30%, the RDS content was decreased from 61.03% to 16.23%, while SDS and RS contents increased from 8.22% and 30.75% to 14.24% and 69.53%, respectively. The increase in RS content of citrate starch was due to the increased citric acid concentration, thus more hydroxyls in starch molecules were replaced by citric anhydride to form citrate starch, which was composed of the cross-linked structure. This structure could resist enzyme hydrolysis, thus resulted in an increase in RS content (Xie & Liu, 2004). The increase in SDS content may be attributed to the introduction of the citric acid molecules, resulting in the increase of the space steric hindrance that delayed the enzyme contact with starch. However, further increase in the concentration of citric acid from 30% to 40% resulted in the increase in RDS content and the decrease in SDS and RS contents, which was because that space steric formed by too much citric acid hindered the further contact between starch and citric acid. Compared to native starch, when 30% citric acid was added, the RDS content was decreased by 69.84%, while SDS and RS contents were increased by 6.22% and 63.61%, respectively.
Fig. 1. Effect of citric acid concentration on the digestion property (A) and degree of substitution (B) of the starch samples.
The effect of different citric acid concentration on the degree of substitution of citrate starch was also investigated in this study. As shown in Fig. 1B, when citric acid concentration was increased from 10% to 30%, DS was increased from 0.058 to 0.178, however, when it was further increased to 40%, the DS was decreased to 0.129. These results indicated that the variation in DS was consistent with the SDS and RS contents.
3.2 Swelling power and solubility of starch samples
The effects of different temperatures on swelling power and solubility of the native and citrate starch samples are presented in Fig. 2. As shown in Fig. 2, for all the starch samples, the swelling power increased as the temperature increased, but the swelling power of citrate starch samples was lower than that of native starch, and the starch samples with different DS had different swelling power. Moreover, at a certain temperature, the swelling power of citrate starch samples was decreased with the increase in DS. These results indicate that the citrate substituent may alter the associations between amylose and amylopectin and each component with itself in the starch granules, thus preventing the starch granules from swelling (Xie & Liu, 2004).
Fig. 2. Swelling power (A) and solubility (B) of native and citrate starch samples with different DS.
The solubility of native and citrate starch samples increased with the increasing temperature, which was in agreement with the swelling behavior (Fig. 2B). These results are due to the lixiviation of amylose from starch granules and to high temperatures the external long chains of amylopectin also contribute to those solubility values (Carmona-Garcia, Sanchez-Rivera, Méndez-Montealvo, Garza-Montoya, & Bello-Pérez, 2009).
As shown in Fig. 2B, at a temperature below 65 °C, the swelling power and solubility of native starch had no significant change. However, when the heating temperature was beyond 65 °C, the swelling power and solubility were significantly increased, when the temperature ranged from 65 °C to 85 °C, they were increased from 6.65 g/g and 3.23% to 22.66 g/g and 23.83%, respectively, which might be due to that the starch was gelatinized at a higher temperature.
3.3 FT-IR spectroscopy
The FT-IR spectrum of starch has been shown to be sensitive to changes in structure on a molecular level (short-range order), such as starch chain conformation, crystallinity and retrogradation (van Soest, Tournois, de Wit, & Vliegenthart, 1995). Irudayaraj and Yang (2002) reported that the absorbances at 3381 and 2931 cm−1could be attributed to O–H and C–H bonds stretching, respectively. The absorbance at 1350 cm−1 has been attributed to the bended modes of O–C–H, C–C–H, and C–O–H (Bellon-Maurel, Vallat, & Goffinet, 1995). Absorbance at 1022 cm−1 is assigned to the vibration of C–O–H deformation (van Soest et al., 1995), and absorbance at 930 and 857 cm−1 are assigned both for C–H bending (Irudayaraj & Yang, 2002).
Fig. 3A presents the FT-IR spectra of native and citrate starch samples. Compared with native starch, a new peak at 1724 cm−1 was appeared in all citrate starch samples. This new absorption peak can be attributed to the characteristic ester group from citric acid in the structure of citrate starch. The band of 1724 cm−1 was associated with the stretching vibration of CO bond from the acetyl group (Chatel, Voirin, & Artaud, 1997). The peak signal was weak in the citrate starch samples with DS of 0.058, however, with the increase in DS, the intensity became stronger, which was in agreement with the contents of SDS and RS in the starch samples.
Fig. 3. FT-IR spectrum (A), deconvoluted FT-IR spectrum (B) and ratio of 1047 cm−1/1022 cm−1(C) of native and citrate starch samples with different DS.
Fig. 3B shows the deconvoluted FT-IR spectra of native and citrate starch samples. The IR absorbance band at 1047 cm−1 was sensitive to ordered or crystalline structure and the band at 1022 cm−1 was associated with amorphous structure in starch. Thus, the absorbance ratio of 1047 cm−1/1022 cm−1 can express the degree of order in starch (Capron et al., 2007 and van Soest et al., 1995). In our deconvoluted FT-IR spectra, we calculated the R values (R = the intensity of the band at 1047 cm−1/the intensity of the band at 1022 cm−1), which was presented in Fig. 3C. As shown in Fig. 3C, with the increase in DS, the ratio of 1047 cm−1/1022 cm−1decreased, when DS ranged from 0 to 0.178, R values decreased from 0.81 to 0.66, indicating that the citrate substitution altered chain packing and generated more amorphous regions. Xie, Liu and Cui (2006) reported that the ratio of 1047 cm−1/1022 cm−1 of citrate starch decreased with an increase of reaction time under a condition of low moisture content.
3.4 X-ray diffraction and relative crystallinity
X-ray diffractometry has been widely used to reveal the characteristics of the crystalline structure of starch granules (Zobel, Young, & Rocca, 1988). The X-ray diffraction patterns and relative crystallinities of native and citrate starch samples are shown in Fig. 4. Starch can be classified into A, B, and C types based on the X-ray diffraction pattern. Native cassava starch showed the A-type pattern with major peaks at 2θ = 15°, 17°, 18°, and 23°. This was not consistent with expected B-type pattern (Yuan, Zhang, Dai, & Yu, 2007). As shown in Fig. 4, the crystalline peaks of all citrate starch samples became much smaller or even disappeared in comparison with native starch, and the higher DS, the lower intensity of diffraction peaks in starch samples. But citric acid esterification did not dramatically alter the crystalline pattern of starch samples.
Fig. 4. X-ray diffractograms and relative crystallinity of native and citrate starch samples with different DS. The values in bracket indicate relative crystallinity (%).
Starch crystallinity has been shown to be influenced by: (1) amylopectin content, (2) average amylopectin chain length, (3) orientation of the double helices (within the crystallites) to the X-ray beam, and (4) crystallite size (Tester & Morrison, 1990). In the present study, we found that the relative crystallinity calculated from X-ray diffraction patterns ranged from 37.64% to 15.54%, which was contrary to the change trend of DS. When citric acid penetrated the starch granules through channels and cavities, it could disrupt the crystalline structure of granules due to a concentrated solution of citric acid. The reaction should occur both in the amorphous region and crystalline region. Substitution of citric acid groups on starch chains could form a highly cross-linked starch and thus limit starch chain mobility. But in the present study, the citrate starch samples with the lower relative crystallinity contained higher SDS and RS contents, suggesting that the decrease in digestibility may be attributed to the formation of cross-linked structure, rather than the crystalline structure.
3.5 Thermal properties of citrate starch samples
Fig. 5 shows the DSC thermograms of native and citrate starch samples. The gelatinization onset (To), peak (Tp), conclusion (Tc) temperatures and gelatinization enthalpy (ΔH) of starch samples are summarized in Table 1. As shown in Fig. 5, endothermic peak gradually shrank or even disappeared with the increase in DS, indicating that citric acid esterification reaction affected the starch crystallinity and eventually increased the starch amorphous region, which was consistent with the results of X-ray diffraction (Fig. 4). As shown in Table 1, the To, Tp, Tc and ΔH for the native starch were 57.80 °C, 67.08 °C, 77.29 °C, and 10.53 J/g, respectively. However, gelatinization parameters of citrate starch samples did not be determined when the DS was more than 0.129. Compared with the native starch, when DS was increased from 0 to 0.096, the To, Tp, Tc, and ΔH of citrate starch sample were significantly decreased to 53.69 °C, 59.66 °C, 67.72 °C, and 0.86 J/g, respectively. These results suggest that partial esterification can reduce the gelatinization parameters of citrate starch. The ΔH value represents the number of double helices which constitutes the crystalline region that unravel and melt during gelatinization (Cooke & Gidley, 1992). The decrease in ΔH was due to the citrate substitution that altered the chain packing and generated more amorphous regions, which could be proved by the decreased values of R and relative crystallinity (Liu et al., 2014).
Fig. 5. DSC thermograms of native and citrate starch samples with different DS.
Table 1. Gelatinization parameters and syneresis (g/100 g) of native and citrate starch samples with different DS.
Starch
Gelatinization parameters
Syneresis (g/100 g)
To (°C)
Tp (°C)
Tc (°C)
ΔH (J/g)
1 cycle
2 cycle
3 cycle
NS
57.80 ± 0.21a
67.08 ± 0.88a
77.29 ± 0.36a
10.53 ± 0.44a
38.24 ± 0.92a
44.78 ± 1.02a
49.22 ± 1.26a
DS = 0.058
53.33 ± 0.11b
61.81 ± 0.48b
71.79 ± 0.39b
3.71 ± 0.08b
30.17 ± 0.89b
35.12 ± 0.48b
37.65 ± 0.87b
DS = 0.096
53.69 ± 0.31b
59.66 ± 0.66c
67.72 ± 0.68c
0.86 ± 0.04c
23.36 ± 1.09c
26.66 ± 0.72c
30.31 ± 0.44c
NS indicates native starch. DS indicates degree of substitution.
The values followed by the different superscripts in the same column are significantly different (P< 0.05).
3.6 Freeze–thaw stability
Table 1 shows the percentage syneresis of native and citrate starch samples during 3 freeze–thaw cycles. The syneresis (%) values of the citrate starch gels were much lower than that of the native starch gel, and also decreased with an increase in DS. Superior freeze–thaw stability after citric acid treatment suggested that citrate starch samples would have suitable properties for use in the food industry. But when DS was more than 0.129, citrate substitution prevented the starch granule from swelling and gelatinization. So we could not obtain the syneresis. According to a previous study, substitution with citrate molecules increased the stability of cooked pastes of starch towards retrogradation (Agboola et al., 1991).
4 Conclusions
In the present study, in order to compare the digestibility, structural and physicochemical properties of citrate cassava starch samples with different DS, citric acid (10–40% of starch dry weight) was used to react with cassava starch at 130 °C for 5 h. The results indicated that citric acid esterification treatment significantly increased the contents of RS and SDS fractions in starch samples, and 69.53% RS and 14.24% SDS were obtained when DS was 0.178. Moreover, the variation in the contents of SDS and RS was consistent with DS. The swelling power and solubility of citrate starch samples were lower than those of native starch and they decreased with the increase in DS. Compared with native starch, a new peak at 1724 cm−1 was appeared in all citrate starch samples, and crystalline peaks of all citrate starches became much smaller or even disappeared, and the relative crystallinity was decreased from 37.64% to 15.54%. DSC analysis indicated that the endothermic peak of citrate starch samples gradually shrank or even disappeared as the DS increased. But citric acid esterification did not change the X-diffraction pattern of citrate starch samples. In addition, the citrate starch gels exhibited better freeze–thaw stability with a significant decrease in syneresis (g/100 g) compared to native starch when DS was no more than 0.096. These results suggested that citric acid esterification induced structural changes in cassava starch significantly affected its digestibility and it could be a potential method for the preparation of RS with thermal stability.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (No. 31171650 and 31230057), and Key Program of the National Technology R&D Program of China for the 12th Five-Year Plan (No. 2012BAD37B02 and 2012BAD37B01).
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O bond from the acetyl group (Chatel, Voirin, & Artaud, 1997). The peak signal was weak in the citrate starch samples with DS of 0.058, however, with the increase in DS, the intensity became stronger, which was in agreement with the contents of SDS and RS in the starch samples.
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