Results

3.1. Proximate analysis

The proximate composition of the cassava flour was shown in Table 1. The moisture content of three cultivars of cassava flour ranged between 10.56 ± 0.01–10.85 ± 0.45%, which is within the range acceptable for effective storage of cassava flour. Total Protein of flour shows that flour from three cultivars of cassava had a protein content of 1.97 ± 0.00%, 2.15 ± 0.01%, and 2.18 ± 0.01% respectively. It was considered that the flour from three cultivars of cassava was the normal value of protein in cassava flour. Ash, total carbohydrate, and fat of the three cultivars of flour were no difference in the ash 1.53 ± 0.07–1.91 ± 0.00%, carbohydrate 84.27 ± 0.04–84.91 ± 0.46%, and fat 0.79 ± 0.02–0.95 ± 0.01%.

Table 1. Proximate analysis of flour from difference varieties of cassava flour.

^1/ Mean of percent dry substance (%) ± Standard deviation. ^2/ ns = not statistically significantly different. *, ** = statistically significant difference p≤ 0.05 and highly significant p≤ 0.01, respectively.

3.2. Total cyanide content, Acrylamide, Amylose, Gluten content and Viscosity

The results of amylose content, acrylamide, cyanide content, gluten content and viscosity of cassava flour revealed that amylose content at 19.93 ± 0.470%, and acrylamide and gluten content were not found in flour. The cassava flour's cyanide content at 2.93 ± 0.16 mg/kg dry basis did not exceed the Codex Alimentarius limit (10 mg/kg of dry weight) considered harmless to consumers. The viscosity of the flour was 6286.00 ± 1.52 mPa.s (Table 2).

Table 2. Amylose content, acrylamide, cyanide content, and viscosity from difference varieties of cassava flour.

^1/ Mean ± Standard deviation; ^2/ N = Not Detected. ^3/ ns = not statistically significantly different. *, ** = statistically significant difference p≤ 0.05 and highly significant p≤ 0.01, respectively.

3.3. Color of cassava flour

Three varieties of cassava flour samples were evaluated in a colorimeter (CR-400, Konika Minolta), it was found that the color values (L*, a* and b*) were non significantly different between samples. The characteristics of lightness L* value were between 97.65 ± 0.65–99.46 ± 0.40 which represented the whiteness of the flour. The average a* value (red-green degree) of flour samples was between 1.01 ± 0.34–1.75 ± 0.12. The flour derived from cassava varieties Pirun 4 had the highest redness. However, the three cassava varieties showed low redness in the flour. The b* averaged (yellow blue degree) between 14.73 ± 0.04–15.76 ± 1.20, indicating a slight yellowness of the flour. The overall color characteristics of each cassava flour are shown in Table 3.

Table 3. Color parameters of flour from three varieties of cassava.

^1/ Mean ± Standard deviation; *The CIE = L*, lightness; a*, redness; b*, yellowness. 2/ ns = not statistically significantly different. *, ** = statistically significant difference p≤ 0.05 and highly significant p≤ 0.01, respectively.

3.4. FTIR Analysis

The comparison of the differences between the flour of three varieties of cassava by the FTIR technique shows the spectral characteristics of the flour of peaks in the range of 4000-800 cm^-1 contains functional groups 3000–2800 cm⁻¹, 1834–1583 cm⁻¹ and 1200–800 cm^-1 Figure 1A.

In addition, principal components analysis (PCA) and loading plot analyzed the differences between the three varieties of cassava flour found that the three varieties were clearly different as shown in Figure 1B. Loading was distinguished by the difference in the peak of cassava flour variety Pirun 6 (P6) in 2928, 1638, 1149, 1076, and 997. While Pirun 2 (P2) and Pirun 4 (P4) show a dominant range of 1200–800, according to spectrum in the Figure 1A shows the difference in the peak in the ranges 3000–2800 cm⁻¹ and 1834–1583 cm⁻¹ of flour from cassava, Pirun 6. Moreover, the cluster results showed that flour from P6 was the most different from the other two varieties, while P2 and P4 were closely related, giving results consistent with PCA. The data analysis represents that the PC2 and PC3 regularly are the most different clustering of the three groups. The PC2 and PC3 loading of each cassava flour sample were shown that separation between PC2 and PC3 corresponded to total variance of 29% from PC2 and 13% from PC3 (Figure 1 B–C).

It was found that the cassava flour varieties Pirun 2 and Pirun 4 were similar in cluster analysis for FTIR but was a different group compared with Pirun 6 (Figure 2). The results show biochemical accorded with the proximate composition of each cassava flour sample.

Figure 1. Comparison of three varieties of cassava flour by FT-IR technique (A) average absorbance spectra (B) PCA analysis (C) loading plot.

Figure 2. Cluster analysis for FTIR spectra compares three varieties of cassava flour (P2=Pirun 2, P4=Pirun 4, P6=Pirun 6).

3.5. Microstructure of cassava flour

Microstructural analysis of flour samples using a scanning electron microscope (SEM) reveals that the morphology of different varieties of flour distribution of granules were found evenly. They were spherical and dome-shaped, ranging from 9 to 20 µm in size (Figure 3). There were no apparent differences, between species with respect to granule morphology.

From physico-chemical properties of three varieties of cassava flour, it was found that all of the flour were not different physico-chemical properties. Therefore, flour from cassava variety Pirun 6 was selected for use in the next experiments, because there was low cyanide, and fat content, and had high ash, protein, and viscosity values.

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Figure 3. Characteristics of cassava flour granules, Pirun 6 (A–C), Pirun 4 (D–F), Pirun 2 (G–I) were photographed under a scanning electron microscope (SEM) at 1000X (C, F, I) 3000X (B, E, H) and 8000X (A, D, G).

3.6. Effects on quality and sensory characteristics of Gluten-free muffin

3.6.1. The physical characteristics of Gluten-free muffin

A basic muffin formula as compared with gluten-free muffins containing cassava flour was tested for baking loss (%), firmness (N), and color. The results found that gluten-free muffins had higher bake weight loss than the basic muffin at 13.13 ± 0.56%. While the Firmness values were not significantly different between the two muffin formulations (Figure 4). The color values found that gluten-free muffins had higher red (a*) values than the basic muffin formula. But the gluten-free muffins containing cassava flour were a yellow color (b*) with no significant difference when compared with the basic muffins (Table 4).

Figure 4. Characteristics of muffins. Muffins using wheat flour (A and C) and muffins using cassava flour (B and D).

Table 4. Color values and physical characteristics of gluten-free muffins from cassava flour.

^1/ Mean ± Standard deviation; *The CIE = L*, lightness; a*, redness; b*, yellowness. ^2/ ns = not statistically significantly different. *, ** = statistically significant difference p≤ 0.05 and highly significant p≤ 0.01, respectively.

3.6.2. Sensory evaluation of Gluten-free muffin

The results of sensory characteristics of basic muffin formula and gluten-free muffins containing cassava flour showed that the texture appearance, color, taste, and crunchiness were not different. Although tasters rated the smell, wallowing sensation, and overall liking of gluten-free muffins containing cassava flour over basic muffins (Figure 5), indicating a sensory quality of gluten-free muffins comparable to that of the basic muffins.

Figure 5. Sensory evaluation values of gluten-free muffins from cassava flour.

In Thailand, the local varieties of edible cassava used in the food industry have many variations in the nutrient quality of the cassava root depending on the cassava variety used. A study of the physic-chemical properties of flour from three varieties shows that the moisture content of three cultivars of cassava flour was between 10.56 ± 0.01–10.85 ± 0.45%. Hasmadi et al. [21] reported that high-quality cassava flour usually contains moisture content ranging from 6.34% to 14.58%. Moisture is an important parameter in the storage of cassava flour. It was also found that total protein content of 1.97 ± 0.00%, 2.15 ± 0.01%, and 2.18 ± 0.01%, respectively. Hasmadi et al. [21] and Peprah et al. [22] reported that the protein content of cassava flour in the range of 1–3% on a dry basis. Ash, total carbohydrates, and fat of the three cultivars of flour were different. Amylose content at 19.93 ± 0.470%, and the viscosity was 6286.00 ± 1.52 times. According to [23,24] reported that the proximate analysis ranged from 8.79 to 9.35%, 0.55 to 26.23%, 0.34 to 2.01%, 0.32 to 8.24% and 0.10 to 17.86% for moisture, protein, fat, and ash respectively while carbohydrate ranged from 36.31 to 89.62%, amylose contents 18.47 to 25.35%. Results of color analyses of cassava flour samples found that the color values (L*, a* and b*) were non-significantly different between samples. The characteristics of lightness L* value were between 97.65 ± 0.65–99.46 ± 0.40 which represented the whiteness of the flour. The average a* value (red-green degree) of flour samples was between 1.01 ± 0.34–1.75 ± 0.12. The b* averaged (yellow blue degree) between 14.73 ± 0.04–15.76 ± 1.20 indicating a slight yellowness of the flour. Chisenga et al. [8] reported that the difference in color in flour was due to the composition of the cassava such as ash content, protein, pigment, and starch. The color of the flour comes from the root flesh and the reaction of the complex with mucilage and latex as well as starch-lipid, fiber-lipid, and protein-lipid interactions. As a result, the cassava flour has intense color [5,7]. The peak viscosity of flour from cassava Pirun 6 was 6286 ± 1.52 mPa.s, which was significantly higher than Pirun 4 (4640 ± 0.58 mPa.s) and Pirun 2 (3390 ± 0.57 mPa.s). The higher peak viscosity related with a higher degree of swelling of starch granules and also starch content [25]. The cluster analysis for FTIR spectra of all samples found that the three varieties of flour were clearly different. Cassava varieties Pirun 6 had higher regions than flour from cassava varieties Pirun 2 and Pirun 4 at wave number 2928 cm^-1 representing –C-H (CH₂) stretching. The peak of protein of flour from cassava varieties Pirun 6 was higher than Pirun 2 and Pirun 4 at wave number 1638 cm^–1 representing amide I (-C=O) stretching respectively. FTIR results showed proximate compositions of protein and fat content in flour from cassava varieties Pirun 6. The flour from cassava varieties Pirun 2, Pirun 4, and Pirun 6 quality traits varied among the cassava varieties, even within the same shape, these components of physico-chemical were still not consistent and the source of variation was due to differences in flour fiber, ash, fat, protein, total cyanide, and peak viscosity. Therefore, it could be said that the differences in chemical components would be a characteristic of each variety [8,26]. Microstructural analysis of flour samples using a scanning electron microscope (SEM) reveals that the morphology of different varieties of flour distribution of granules was found evenly. They were spherical, dome-shaped, polyhedral, and truncated in shape from flour granules squeezed together ranging from 9 to 20 µm in size [8,27]. It is considered a typical standard size of cassava flour. The shape of the flour granules has not identified the difference between species with respect to granule morphology [28]. The test of physical properties and sensory attributes of gluten-free muffins compared with wheat flour found that gluten-free muffins had higher bake weight loss than the basic muffin. That could be due to the limited moisture evaporation during baking by the enhanced water binding capacity [29]. The color values found that gluten-free muffins had higher red (a*) values than the basic muffin formula. This is because cassava flour affects the formation of color in gluten-free muffins such as the Maillard reaction (reaction between carbohydrates with protein in the product) and caramelization (the sugar contained in the product is exposed to heat) at the time of baking [30]. From this experiment, it can be concluded that the gluten-free muffins had overall preferences similar to basic muffins.^{[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]}

Conclusions

Edible cassava of the three varieties -Pirun 2, Pirun 4, and Pirun 6- eventually have the potential to be applied in the food industry. Several analyses of cassava flour's physico-chemical suitability and quality of flour for gluten-free muffins (functional properties) show positive results. The results found that different varieties influenced the proximate compositions of cassava flour, and significant differences were observed in ash, fat, protein, total cyanide, and peak viscosity. Significant differences were also observed for the spectral characteristics of the flour from cassava variety Pirun 6 show chemical components (functional groups) such as lipids, proteins, etc by the FTIR technique. The physical characteristics of gluten-free muffins showed the characteristics and color values of muffins using cassava flour had no significant difference when compared with muffins using wheat flour. The sensory evaluation of gluten-free muffins using cassava flour compared with wheat flour was acceptable by the sensory panelists. These studies may contribute to the development of gluten-free products based on cassava flour, aiming to substitute for wheat flour.