Evaluation of some analytical methods for determination of calcium oxalate in Amorphophallus muelleri flour

Abstract

The presence of calcium oxalate is the major obstacle in flour processing of Amorphophallus muelleri tuber since the calcium oxalate can induce skin irritation and is harmful to kidneys. The development of a rapid analytical method to detect calcium oxalate in Amorphophallus flour is required. This research was intended to evaluate the use of some analytical methods (FTIR, SEM-EDS, XRD, XRF, and titration methods) in calcium oxalate detection in Amorphophallus muelleri flour prepared from different treatments (soaking in water (W), solution of sodium bisulfite 1000 ppm (B), solution of sodium chloride salt 3% (S), solution of sodium bisulfite 1000 ppm and sodium chloride salt 3% (BS)). Results showed that the presence of oxalate in Amorphophallus flour can be detected in the FTIR spectra from the C=O group at a wavenumber of 1610 cm-1. SEM images confirmed that calcium oxalate in Amorphophallus flour existed as raphide crystals in which their quantity can be estimated by the EDS feature of SEM. The presence of calcium oxalate crystals in Amorphophallus flour can be differentiated from other salts present in the flour by XRD. XRF can be used as a rapid analytical tool to detect the presence of calcium oxalate in Amorphophallus flour. The potassium permanganate titration technique can be used as a reference method for other rapid analytical methods in detecting calcium oxalate in Amorphophallus flour.

Keywords:
calcium oxalate crystal; analytical methods; Amorphophallus muelleri flour

1 Introduction

Most people in South East Asia consume rice as their staple food. However, consuming rice may not be suitable for some persons since it is classified as high glycemic index food which can induce high blood sugar (Kim et al., 2003). Moreover, older people should be more aware of the negative impact of daily and high rice consumption (Golozar et al., 2017). People with diabetes and other degenerative diseases should also avoid rice consumption since high blood sugar may induce organ or cell damage (Al-Ishaq et al., 2019). Therefore, alternative food with a low glycemic index should be introduced as a new staple food for people of South East Asia.

Amorphophallus tubers have been known as a source of food in Japan and China. People of Japan and China has consumed shirataki noodles prepared from the tuber as part of their daily food. However, food product from the tuber has not been widely known for people in South East Asia. Amorphophallus tuber contains a high percentage (up to 55% on a dry basis) of a precious carbohydrate substance namely glucomannan (Yanuriati et al., 2017). Glucomannan is classified as a soluble dietary fiber compound that contains tons of functional benefits for human health. It has the ability to lower the risk of developing hemorrhoids and small pouches in the colon (diverticular disease). It shows to lower cholesterol levels (Keithley et al., 2013) and helps control blood sugar levels (Shah et al., 2015). Therefore, food products developed from glucomannan of Amorphophallus tuber will be beneficial for health.

Most Amorphophallus tubers cannot be consumed directly since they contain calcium oxalate, an anti-nutrient compounds, that can irritate skin and is harmful to kidneys (Singh et al., 2018; Chairiyah et al., 2016; Siener et al., 2021). Therefore, a processing step should be carried out to remove the calcium oxalate from the flour of Amorphophallus tuber before it can be further processed to be a food product. Calcium oxalate can be removed from the flour of Amorphophallus tuber by means of separation techniques such as water soaking (Coronell Tovar et al., 2019) producing safe Amorphophallus flour (Witoyo et al., 2021; Kumar et al., 2017; Witoyo et al., 2020).

To date not so many analytical methods have been developed to evaluate calcium oxalate content in food. The most common methods to determine the calcium oxalate are titration and spectrophotometric methods (Mishra et al., 2017; Alavi & West, 1983; Ilarslan et al., 1997; Burrows, 1950; Fiske & Adams, 1931) and chromatographic method (Minocha et al., 2015; Huang & Tanudjaja, 1992). These methods require a long time for sample preparation and need a higher amount of toxic chemicals. Therefore, the development of a quick analytical method to estimate the calcium oxalate content in food samples specifically in Amorphophallus flour is required. This research was aimed to evaluate the use of some analytical methods in the determination of calcium oxalate in Amorphophallus muelleri flour.

2 Materials and methods

2.1 Materials

Amorphophallus muelleri tuber was purchased from a local farmer in the district of Subang, province of West Java, Indonesia. Sodium bisulfite and sodium chloride salt were purchased from the local market. All chemicals for analysis were analytical grade.

2.2 Sample preparation

Samples of Amorphophallus muelleri flour were prepared by the following procedure. Amorphophallus muelleri corms were washed, peeled, shredded, and soaked in different solutions including water (W), sodium bisulfite 1000 ppm (B), salt of sodium chloride 3% (S), sodium bisulfite 1000 ppm, and salt of sodium chloride 3% (BS) for 30 min. Subsequently, the sample was rinsed with 10 liters of water and dried finally dried in an oven at 55 °C for 12 h (UM500; Memmert, Germany). The dried sample was ground by a grinder (HR2115; Philips, Indonesia) and sieved by a laboratory test sieve (D-42757; Retsch Gmbh, Germany) with an aperture of 150 µm. The control sample (C) was prepared from the tuber without soaking in any solutions. All samples were prepared from triplicate treatments.

2.3 FTIR analysis of samples

The FTIR spectra of samples were assayed by using an FTIR Spectrometer ALPHA II (Bruker instrument, Billerica, MA-USA). For each sample, reading was taken three times. The FTIR spectra were processed according to the method of Goodacre et al. (1998) with some modifications. All data were baseline corrected by using Origin Pro Software 2016.

2.4 Morphological properties and mineral analysis by scanning electron microscope- energy dispersive X-ray spectroscopy (SEM-EDS) method

The morphological properties of samples were observed by a Scanning Electron Microscope (SEM) (JEOL JSM IT300, Japan). The mineral content of samples was estimated by surface map analysis using the Energy Dispersive X-Ray Spectroscopy (EDS) feature of the SEM. The sample was mounted on a metal stub then it was coated with gold. An accelerating voltage of 2 kV was used during observation.

2.5 The crystalline structure analysis

The crystalline structures of samples were assayed by using XRD (X-ray Diffraction,, Bruker, Germany) technique using a method of Nakorn et al. (2009) with modifications. The diffractogram of the sample was reported in the 2θ range of 5 to 70°.

2.6 X-ray fluorescence analysis of samples

The calcium content of samples was also evaluated by using the XRF method (XRF Portable Thermo Scientific, type of Niton XL3t 500 analyzers, Thermo Scientific, USA).

2.7 Determination of calcium oxalate by titration method

Calcium oxalate in the sample was determined by the protocol of Mishra et al. (2017) with some modifications. H₂SO₄ of 0.5 N with an amount of 30 mL was added into the sample of 0.5 g in a test tube, then it was heated in a water bath (water bath shaker type 1086; Gesellschaft für Labortechnik (GFL), Germany) at 100 °C for 15 min. The sample then was filtered with Whatman filter paper of number 41 and rinsed with an aqua distillate of 30 mL. Then, 10 mL of filtrate was mixed with 40 mL of H₂SO₄ 0.5 N and heated at 100 °C for 5 min. The sample then was immediately titrated with KMnO₄ 0.05 N until the titration endpoint was reached as indicated by light red color. Prior to analysis, the KMnO₄ 0.05 N was standardized. C₂H₂O₄.2H₂O of 0.0405 gr was mixed with 30 mL of H₂SO₄ 0.5 N in a test tube, subsequently, it was heated at 100 °C for 5 min. The standard sample then was titrated with KMnO₄ 0.05 N until the titration end point was reached as indicated by light red color. The calcium oxalate content was calculated according to the following Equation 1:

$\text{Calcium oxalate content}\left[\text{\%}\right]=\frac{\begin{array}{l}\text{titration volume}\left(\text{mL}\right){\text{x Normality of KMnO}}_4\text{x}\\ \text{Molecular weight of Calcium oxalate x dilution factor}\end{array}}{\text{weight of sample}\left(\text{gr}\right)}$ (1)

3 Results and discussion

3.1 FTIR spectra of sample

The FTIR spectra of all samples are presented in Figure 1. According to Derrick et al. (1995), the presence of oxalate salts in a sample can be identified by the FTIR technique. The identity peak of oxalate functional groups including C=O appears at a wavenumber of 1600-1700 cm^-1 and C-O shows at a wavenumber of 1200 cm^-1. The result of this experiment showed that the identity peak of oxalate in four different Amorphophallus flour samples including the C=O group appeared at 1610 cm^-1 and the peak C-O was observed at 1322 cm^-1 (Figure 1). Nurlela & Arizal (2020) reported that the C=O groups in the glucomannan structure (Figure 2) show two identity peaks at a wavenumber 1739 cm^-1 and 1736 cm^-1. Nurlela & Arizal (2020) also noted that the presence of the C-O group was seen at a wavenumber of 1230 cm^-1 and 1247 cm^-1. Based on the FTIR spectra of oxalic acid (Figure 3), the group of O-H, C=O, and C-O can be identified at wavenumber 3424 cm^-1, 1685 cm^-1, 1263 cm^-1, and 1126 cm^-1, respectively (Spectral Database for Organic Compounds, 1999). Shifting of FTIR peaks might be occurred due to the manifestation of gradual changes in the IR frequency associated with a specific chemical bond under the influence of molecular interactions (Ryu et al., 2010).

Figure 1
FTIR spectra of Amorphophallus muelleri flour from different treatments (Untreated (C), soaked in water (W), soaked in sodium bisulfite solution (B), soaked in sodium chloride solution (S), soaked in sodium bisulfite-sodium chloride solution (BS).

Figure 2
Chemical structure of glucomannan (M: Mannose, G: Glucose) (Behera & Ray, 2016).

Figure 3
The FTIR Spectra of Oxalic Acid (Spectral Database for Organic Compounds, 1999).

Figure 1 indicates that the peak intensity of the control sample was the highest among the samples. Meanwhile, the peak intensity of Amorphophallus flour from BS treatment was the lowest one. These results implied that the oxalate content of the BS sample was the lowest among the samples. In terms of preparation of Amorphophallus flour with low oxalate content, the BS treatment can be suggested as the best practice to remove oxalate from the flour compared to other treatments such as soaking in sodium chloride solution (Rofi’ana et al., 2018) and soaking in sodium metabisulfite solution (James et al., 2013). Moreover, a rapid quantitative analysis to determine the oxalate content in Amorphophallus flour might be applied by using an FTIR technique if the calcium oxalate standard is available (Sarifudin et al., 2021).

3.2 Scanning electron micrograph of samples

The SEM images of Amorphophallus muelleri flour samples are shown in Figure 4. Some particles with irregular shapes are observed which could be the agglomerates of flour components including glucomannan, protein, amyloplast, and starch (Takigami et al., 1997). Needle-shaped or raphide crystals are detected in the images of all treatments as indicated by arrows in the image. The length of the raphide is about 150 µm with a width of about 5 µm. Images of samples C and W show a higher number of the raphide crystals compared to those of samples B, S, and BS. Chairiyah et al. (2016) reported four shapes of calcium oxalate crystal found in amorphophallus tuber including styloid, prism, druse, and raphide shapes. Moreover, the raphide shapes of calcium oxalate can be seen by the microscope technique (Ramos et al., 2020). Therefore, in tandem with the microscopy technique, an image of SEM can be an early quick analytical technique to observe the presence of calcium oxalate in the Amorphophallus flour sample (Takigami et al., 1997).

Figure 4
Micrograph of Amorphophallus muelleri flour from different treatments (Untreated (C), soaked in water (W), soaked in sodium bisulfite solution (B), soaked in sodium chloride solution (S), and soaked in sodium bisulfite-sodium chloride solution (BS) in different magnification levels (1000X and 5000X).

3.3 EDS analysis of samples

The elemental analysis was performed by using an energy-dispersive X-ray spectrometer (EDS) which is a feature of SEM analysis. The reported trace elements are minerals that are concerned in this study including calcium and sodium as shown in Table 1. As expected, the percentage of calcium in the control sample was the highest among the samples. This result indicated that the calcium mineral in Amorphophallus flour is contributed by calcium oxalate. Treatments S and BS left sodium mineral residue in the sample. In terms of calcium oxalate removal, treatment S was found as the most effective one as indicated by the lowest value of calcium. Overall, the result followed the treatments in that the sodium residue were higher in B, S, and BS samples compared to that of C and W samples, whereas the calcium residues of C and W samples were higher than that of B, S, and BS samples (Nurlela & Arizal, 2020; Rofi’ana et al., 2018; Witoyo et al., 2020). Therefore, EDS analysis can be used to evaluate the presence of calcium oxalate in the Amorphophallus flour sample.

Table 1
EDS analysis of Amorphophallus muelleri flour from different treatments (Untreated (C), soaked in water (W), soaked in sodium bisulfite solution (B), soaked in sodium chloride solution (S), soaked in sodium bisulfite-sodium chloride solution (BS).

Sample	Na [%]	Ca [%]
C	0.00 ± 0.00*	1.76 ± 0.06
W	0.00 ± 0.00	1.34 ± 0.05
B	0.21 ± 0.06	1.2 ± 0.06
S	10.58 ± 0.16	0.32 ± 0.03
BS	8.64 ± 0.16	0.65 ± 0.04

• ^*
  Average ± standard deviation.

3.4 The crystalline structure of samples

X-ray diffractogram patterns of samples are presented in Figure 5. Overall, all samples exhibit X-ray diffractogram pattern of A-type starch as indicated by the presence of its identity peaks at 2$\theta$ of 17°, 18.1° and 23.3° (Li et al., 2013; Buléon et al., 1997). The percentage of starch is about 10-30% in Amorphophallus flour (Supriati, 2016). The presence of calcium oxalate crystal can be detected in the X-ray diffractogram of all samples. Identity peaks of calcium oxalate are shown by the X-ray diffractogram at 2$\theta$ of 15.0°, 15.4°, 24.5°, 30.2°, 31.6°, 36.1°, 38.4°, 40.1°, and 43.8° (Ahmed et al., 2012; Orlando et al., 2008). Lastly, the presence of sodium chloride residue from treatment S and BS is also detected by the identity peaks of NaCl crystal at 2$\theta$ of 27.4°, 31.7°, 45.5°, 56.5°, and 66.2° (Nickels et al., 1949). Based on this result, XRD can be suggested as a rapid analytical tool to detect the calcium oxalate in the Amorphophallus flour sample.

Figure 5
X-ray diffraction patterns of Amorphophallus muelleri flour from different treatments (Untreated (C), soaked in water (W), soaked in sodium bisulfite solution (B), soaked in sodium chloride solution (S), soaked in sodium bisulfite-sodium chloride solution (BS).

3.5 X-ray fluorescence analysis of samples

The result of the XRF analysis for the determination of calcium is presented in Figure 6. As expected, the control sample exhibited the highest calcium content (8.87%). Even though the results of calcium content determination by XRF were different from those of EDS, however, their trends were similar. The difference could be caused by the different analytical parameters among the analysis. The X-ray fluorescence is a fast, low cost, and non-destructive method for determining the concentration of elements in a sample (Peruchi et al., 2014) This technique has been used for screening inorganic nutrients in soybean (Otaka et al., 2014); wheat flour (Peruchi et al., 2014); and cassava (Udoro et al., 2020). Therefore, XRF can be used as an analytical tool to determine calcium oxalate in the Amorphophallus flour sample.

Figure 6
Calcium content of Amorphophallus muelleri flour from different treatments (Untreated (C), soaked in water (W), soaked in sodium bisulfite solution (B), soaked in sodium chloride solution (S), soaked in sodium bisulfite-sodium chloride solution (BS) using XRF method.

3.6 Calcium oxalate content by titration method

The results of calcium oxalate determination by using the titration method are shown in Figure 7. As expected, the control sample contained the highest percentage of calcium oxalate residue (13.5%). Meanwhile, treatments of S and BS produced Amorphophallus flour with low calcium oxalate content of 6.2 and 6.4%, respectively. Potassium permanganate titration is the most common method to determine calcium oxalate content in food samples (Karamad et al., 2019; Naik et al., 2014). However, this method is limited due to time and chemical consumption. Therefore, other methods are developed in order to overcome the limitation of the titration method in calcium oxalate determination such as capillary electrophoresis (Trevaskis & Trenerry, 1996) and high-performance liquid chromatography (Martz et al., 1990). Despite many limitations of the potassium permanganate titration technique, this method might be used as a comparison method in detecting calcium oxalate in the Amorphophallus flour by other rapid analytical methods. This is because the method can determine total oxalic content in the Amorphophallus flour sample including soluble oxalic acid and non-soluble form of calcium oxalate (Karamad et al., 2019). Prior to the permanganate titration, all oxalic contents in the sample are being solubilized by a strong acid solution i.e. sulfuric acid. The titration method uses the redox principle in which the oxidating agent, i.e. KMnO₄, oxidizes the soluble oxalate through titration. The deviation between replicates usually comes from the titration endpoint. In fact, in nature oxalate compounds can be found in dissolved and undissolved forms. Dissolved oxalate is usually formed when oxalate bind with potassium (K⁺) ions. In contrast, the undissolved form of oxalate will be formed if oxalate compounds bind with calcium (Ca²⁺) ions (Chairiyah et al., 2016). Therefore, in this context, the titration method could be a more sensitive method compared to the other instrumental methods.

Figure 7
Calcium oxalate content of Amorphophallus muelleri flour from different treatments (Untreated (C), soaked in water (W), soaked in sodium bisulfite solution (B), soaked in sodium chloride solution (S), soaked in sodium bisulfite-sodium chloride solution (BS).

4 Conclusions

Some analytical instruments have been evaluated in the determination of calcium oxalate in Amorphophallus muelleri flour. The identity peak of oxalate in Amorphophallus muelleri flour can be identified by FTIR technique including C=O group which appeared at a wavenumber of 1610 cm^-1. Needle-shaped or raphide crystals of calcium oxalate in Amorphophallus flour can be observed by SEM. The peaks identity of calcium oxalate crystals in X-ray diffractogram were observed at 2$\theta$ of 15.0°, 15.4°, 24.5°, 30.2°, 31.6°, 36.1°, 38.4°, 40.1°, and 43.8°. XRF can be used to estimate the calcium oxalate content in Amorphophallus flour based on the determination of calcium minerals. The potassium permanganate titration technique can be used to determine total oxalic in the Amorphophallus flour sample including soluble oxalic acid and non-soluble form of calcium oxalate. Treatment S was found as the most effective method to remove oxalate one as indicated by the lowest value of calcium content by all analytical methods.

Acknowledgements

The authors acknowledged Antonius Sukarwanto and Mukson for providing technical assistance during sample preparation.

• Practical Application: The investigated methods can be used to determine the calcium oxalate content in Amorphophallus muelleri flour in glucomannan industry.

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Publication Dates

• Publication in this collection
  08 July 2022
• Date of issue
  2022

History

• Received
  23 Feb 2022
• Accepted
  10 June 2022