Fresh cassava pulp was obtained from the Korat Flour Industry Co., Ltd, Nakhon Ratchasima, Thailand. Fresh CDGs were obtained from the Thai ethanol power Pub Co., Ltd, Khon Kaen, Thailand. These were dried in a hot air oven at 55°C to 60°C for 2 d and then were ground to pass through a 1.0 mm mesh sieve before being stored at 4°C until further use. Prior to extraction, DCP and CDG were analyzed for dry matter, crude protein, ash, and ether extract according to the standard methods of AOAC [16]. The contents of total soluble and IDF were determined using the total dietary fiber Kit (K-TDFR-100A, Megazyme International Ltd., Wicklow, Ireland). Briefly, the samples (1 g) were treated with thermostable α-amylase and then incubated at 60°C with protease and amyloglucosidase to eliminate starch and protein components. The IDF was obtained by filtration and the residue was washed with warm distilled water. The SDF was precipitated with 95% ethanol and filtered. Total dietary fiber (TDF) was calculated as the sum of SDF and IDF. The chemical compositions of DCP and CDG are shown in Table 1.

Dietary fiber from DCP and CDG were treated with various concentrations of NaOH at levels of 2%, 4%, 6%, and 8% using a completely randomized design with 4 replications of each. The extraction procedure was slightly modified from Daou and Zhang [8]. A dried sample (1.0 g) was pretreated with 5.0 mL NaOH solution at different concentrations (2%, 4%, 6%, and 8%), the mixture was soaked for 1 h at room temperature, then centrifuged, and the residue was washed to pH 7.0 with distilled water. The sample was suspended in phosphate buffer (pH 6.0) ratio 1:30, and α-amylase (EC 3.2.1.1, Megazyme, Ireland) was added, then the mixture was incubated at 95°C in a boiling water bath for 1 h. The sample was allowed to cool at room temperature. The dietary fiber was precipitated in 95% ethanol at 60°C for 1 h, then was cooled to room temperature, and centrifuged. After centrifugation, the residue was washed with 78% ethanol, 95% ethanol and acetone, respectively, and finally dried at 55°C to 60°C overnight.

The DFCP and the DFCDG were analyzed to determine the amounts of TDF, SDF, and IDF by using a total dietary fiber Kit (K-TDFR-100A, Megazyme International Ltd., Ireland), according to the manufacturer’s instructions.

The infrared spectra were collected using attenuated total reflectance (ATR)-FTIR spectroscopy with single reflection ATR sampling module and coupled with a DTGS detector over the measurement range from 4,000 to 400 cm⁻¹. The measurements were performed with a spectral resolution of 4 cm⁻¹ with 64 scans co-added (Bruker Optics Ltd, Ettlingen, Germany). OPUS software was used for data acquisition and the spectra evaluation. The spectral changes of the functional groups were performed at the integral area of each peak such as cellulose, hemicellulose, lignin, and starch by using OPUS software.

The FTIR spectra were exported to the Unscrambler X 10.5 (CAMO, Oslo, Norway) for using PCA analysis. The spectral data were preprocessed by taking the 2nd derivative with Savitzky-Golay method (3rd polynomial, 9 smoothing points), normalization with extended multiplicative signal correction and PCA were performed for the determination of a significant variation between the spectra sets. In this study, PCA was used to compare the FTIR spectra of dietary fiber sources under treatment with different conditions of NaOH solution (2%, 4%, 6%, and 8%). The output of PCA can be presented as the sources of variability of data which were concentrated into the principal component (PC). The spectra were processed using the second derivative and vector normalized by the Savitzky–Golay method, and using the third polynomial and nine smoothing points setting [17].

The experimental design was completely randomized design. Determinations for analysis were made using a total of 4 replicates. Statistical analyses of dietary fiber contents (TDF, SDF, and IDF) and peak area proportions from integrating FTIR spectra were performed using SPSS software version 18.0 [18]. Data were analyzed using one-way analysis of variance followed by Tukey’s tests. Orthogonal polynomials were also used to assess linear, quadratic, and cubic effects of NaOH levels. Values were statistically different at p<0.05.

The DCP and CDG were treated with different concentrations of NaOH solution (2%, 4%, 6%, and 8%) to determine the optimal conditions for dietary fiber extraction. The contents of TDF, SDF, and IDF after extraction are shown in Table 2. The DFCP consisted of an increase in TDF (cubic, p = 0.032) and IDF (linear, p = 0.001) in response to increased NaOH levels. It revealed that DFCP derived from treated DCP with NaOH at concentrations of 6% and 8% produced significantly greater amounts of TDF and IDF than 2% NaOH (p<0.05). In addition, the DFCDG also comprised an increase in TDF (cubic, p = 0.009) and IDF (quadratic, p<0.001). The DFCDG obtained by treatment CDG with 4%, 6%, and 8% NaOH produced higher contents of TDF and IDF than 2% NaOH (p<0.05). However, the SDF content in both DFCP and DFCDG showed no significant differences (p>0.05) between treatments.

This study showed that the optimum levels of NaOH for the extraction of dietary fiber from DCP and CDG were 6% to 8% and 4% to 8%, respectively. The IDF represented a major component in both dietary fiber sources. In general, some hemicellulose such as β-glucans, pectin, and gums are defined as SDF, whilst cellulose and lignin are defined as IDF [1,19]. Cellulose is the main structural constituent in plant cell walls, and also in cassava by-products [1,5,6]. Previous studies have shown that defatted rice bran treated with 0.6% NaOH solution can produce the maximum yield and purity of TDF, SDF, and IDF [8]. Samanta et al [9] demonstrate that the extraction agricultural by-products with NaOH solution resulted in recovery of more than 90% of original xylan in plant materials. The results were similar to the highest xylan recovery from sugarcane bagasse [20]. Harun and Geok [21] stated that rice straw treated with NaOH obtained the highest glucan and lower lignin composition.

The band assignment of FTIR spectra of isolated dietary fiber is shown in Table 3. The spectral features of DFCP and DFCDG are shown in Figures 1 and 2. In this study, FTIR spectra were used to detect the extracted dietary fiber treated with NaOH solution in the spectral region of 4,000 to 400 cm⁻¹. A total of 428 and 434 FTIR spectra of DFCP and DFCDG were analyzed. The fingerprint regions of specific interest in this study were between 1,700 and 800 cm⁻¹, although many absorption bands associated with various NaOH solutions from vibrational modes in the wavelength region are also present in DFCP and DFCDG. The FTIR spectra measurements were carried out to reveal the molecular characteristics of functional groups of dietary fiber such as cellulose, hemicellulose and lignin. This technique can give information about the functional groups of C–O, C–O–C glycoside, and C–C from cellulose, hemicellulose, lignin, starch, and glucose in extracted dietary fiber [22–24].

The semi-quantitative analysis of FTIR spectra was car ried out by using OPUS software. The presented FTIR spectra were used to average the single spectra of each sample. The wavenumber of the FTIR spectra was determined to be in the region of 3,500 to 800 cm⁻¹ for the peak area integration, and the total area of integrated peaks was defined as 100%. The peak area units were expressed as relative proportions of the components in DFCP and DFCDG. The results of the semi-quantitative analysis of DFCP and DFCDG using FTIR spectra in term of proportions (%) of the functional groups are presented in Table 4. The results showed that DFCP comprised of an increase in C–H bending of crystalline versus amorphous structure of cellulose (cubic, p<0.001), C–O stretching of hemicellulose (cubic, p = 0.001) and C–O–C glycoside, C–O and C–C stretching of cellulose (cubic, p = 0.029) in response to increased NaOH levels. The DFCP derived from treated DCP with 6% NaOH yielded significant proportions of C-H bending of crystalline cellulose and C–O–C glycoside, C–O and C–C stretching of cellulose compared to 2% and 4% NaOH (p<0.05), but there was no significant difference when compared to 8% NaOH treatment. While the proportions of the components in DFCDG with peak area integration revealed an increase in C–H stretching (linear, p = 0.002), C–H bending of crystalline cellulose (linear, p = 0.015), C–O–C glycoside, C–O and C–C stretching of cellulose (cubic, p = 0.033), C–O stretching of starch (linear, p<0.001) and vibration of the pyranose ring (quadratic, p = 0.004). The DFCDG derived from treated CDG with 4% NaOH resulted in a significant yield of C–O–C glycoside, C–O and C–C stretching of cellulose compared to 2% NaOH (p<0.05). However, no significant differences were observed in crystalline structure of cellulose in response to the concentration of 2% and 4% NaOH. These results were similar to the chemical composition of DCP treated with 6% NaOH and CDG treated with 4% NaOH, which showed higher functional groups of cellulose. It has been well-established previously that IDF commonly includes cellulose [7].

In this study, the DCP and CDG treated with NaOH solutions at a concentration of 2%, 4%, 6%, and 8% were identified by their spectral distribution by using PCA. The PCA scores were plotted to characterize the sample relationships between the spectra and the dietary fiber extraction treatments. The FTIR characterizes chemical structure by identifying the functional groups present in each sample.

The results of the PCA scores from DFCP are presented in Figures 3. The variability of PC-1 and PC-2 accounts for 58% and 13%, respectively. The scores plot of DFCP derived from DCP treated with 6% and 8% NaOH appear clearly in the negative PC-1 score plot, while the scores plot of 2% and 4% NaOH treatments are clearly separated in the positive PC-1 score plot. The highest negative loading plot in PC-1 was observed in the C–O–C stretch of cellulose (centered at 1,170 cm⁻¹), C–O vibrations of cellulose (centered at 1,035 cm⁻¹), C–O and ring stretching (centered at 1,000 and 985 cm⁻¹), which was oppositely correlated with the positive score plots in DFCP treated with 2% and 4% NaOH group from the second derivative spectrum. While the treatment using 6% NaOH shows the scores plot differ from 8% NaOH and almost appear on the positive side of PC-2. The positive loading plot in PC-2 reveals O–H bending of adsorbed water (centered at 1,603 cm⁻¹), C–H bending of crystalline cellulose (centered at 1,407 cm⁻¹), C–O and C–O–C stretching (centered at 1,089 and 1,016 cm⁻¹), and C–O stretching of starch (centered at 981 cm⁻¹).

The score plot of the FTIR spectra of DFCDG is presented in Figures 4. The variation of spectra in PC-1 and PC-2 accounts for 30% and 23%, respectively. The scores plot of DFCDG derived from CDG treated with 2% and 4% NaOH appear in the negative PC-1 score plot, while the scores plot of 6% and 8% NaOH appears separately in the positive PC-1 score plot. The negative loading plot in PC-1 reveals C–H bending (centered at 1,472 cm⁻¹), C–O and ring stretching (centered at 1,000 cm⁻¹), and vibrations of the pyranose ring, glucose ring stretch (centered at 954 cm⁻¹). The DFCDG derived from CDG treated with 4% NaOH show the scores plot differs from that of 2% NaOH and almost appears in the positive PC-2 score plot. The positive loading plot in PC-2 reveals O–H bending of adsorbed water (centered at 1,620 cm⁻¹), C–H bending of crystalline cellulose (centered at 1,316 cm⁻¹), and C–O stretching and C–C stretching of cellulose (centered at 1,078 and 1,018 cm⁻¹).

The results of DCP and CDG using different NaOH solu tions indicate that PCA analysis of FTIR spectroscopy reveals differences in DCP and CDG treated with 6% and CDG 4% NaOH solutions, respectively. These results are related to the semi-quantitative analysis by integral area obtained from the spectra. The results also show the main components of cellulose in both dietary fiber sources. These results are consistent with those of Uthumporn et al [25], who found the predominant content of NSPs extracted from sago palm flour were cellulose, hemicellulose, pectin, and lignin by using FTIR. Chirinang et al [26] reported that the FTIR spectrum of dietary fiber from cassava pulp appears in the band of 1,031 to 1,005 cm⁻¹. This band is the fingerprint of polysaccharides. These results show that FTIR spectroscopy can be used as a very reliable and quick tool for evaluating and monitoring dietary fiber.