Determination of Gossypol in Hamid and Bt (Seeni 1) Cottonseed Oil using Fourier Transform Infrared Spectroscopy

This study was conducted to determine the gossypol content in Bt cottonseed (Seeni-1) oil by using Fourier Transform Infrared (FTIR) spectroscopy with an Attenuated Total Reflectance (ATR) element. The wavelengths used were selected by spiking refined, bleached deodorized palm oil (RBDPO) to gossypol concentrations of 0-5% and noting the regions of maximal absorbance. Absorbance values of the wavelength regions 3700-2400 & 1900-750 cm−1 and a partial least squares (PLS) method were used to derive calibration models for Hamid cottonseed oil, Seeni-1 cottonseed oil, and gossypol-spiked RBDPO. The coefficients of determination (R2) for the calibration models were computed for the FTIR spectroscopy results against those found by using the wet chemical method AOCS method Ba 8–78. The R2 was 0.8916, 0.9581, and 0.9374 for Hamid cottonseed oil, Seeni-1 cottonseed oil, and gossypol-spiked RBDPO, respectively. The standard error (SE) of the calibration was 0.053, 0.078, and 0.062, respectively. The calibration models were validated using the crossvalidation technique within the same set of oil samples. The results of FTIR spectroscopy as a useful technique determining gossypol content in crude cottonseed oil showed that there is a significant difference (p <0.05) in the amount of gossypol content in Hamid and Bt Seeni-1 cottonseed oils.

Gossypol is the anti-nutritional compound secreted by the plant as an insect repellent, the glandless cotton being more susceptible to insect attack (Mirghani & Che Man, 2003).
The toxicokinetics studies showed two forms of gossypol, viz. free and bound (Wedegaertner & Rathore, 2015). The bound form is produced via covalent bonds between gossypol and free epsilon amino groups from lysine and arginine (Soto-Blanco, 2008;Fernandez et al., 1995). Free gossypol is a physiologically active form, which is toxic to young ruminant animals than monogastric animals. The gossypol toxicity is moderate in most animal species (Alexander et al., 2008); for ruminant is less than for monogastric, since gossypol is bound to proteins in the rumen, i.e., unavailable for absorption. Free gossypol is lipophilic and readily absorbed by the intestine. Absorbed gossypol appears to have a long half-life in the body (Dalefield, 2017;Soto-Blanco, 2008). Alexander et al. (2008) reported that gossypol influences the reproductive organ and growth of the embryo. Gossypol causes infertility, and in men, it stops spermatogenesis at low doses (Coutinho, 2002).
Same toxicity and contraceptive to males were reported by Ye et al. (1983). Weather conditions and cotton species play a significant role in the production of total gossypol, e.g., positively correlated with rainfall rate and negatively correlated with temperature (Zhang et al., 2006).
Gossypium barbadense has high gossypol content than G.
hirsutum. The level of gossypol declines slightly during cotton storage (Soto-Blanco, 2008). The concentration of total gossypol in cottonseed (CS) may be higher than 14,000 mg/kg and 7,000 mg/kg of free gossypol (Alexander et al., 2008). After oil extraction, the available concentration could be up to 0.6%. However, in mechanical presser oil extracted and heat treatment, approximately 0.06% of gossypol could be available (Pons et al., 1953). Abdurakhim (2019) reported a study trying to control the content of gossypol using the biotechnological method by soaking the seed in biosolution that contains chicken droppings.
Bacillus thuringiensis (Bt) cotton is a genetically modified cotton that expresses an insecticidal protein (Kranthi, 2012). Bacillus thuringiensis produces insecticidal toxin protein (endotoxin) specific to lepidopteran insects. This bacteria were used in the traditional form of biopesticides or the modern form of genetically engineered (Ibrahim et al., 2010). Seeni-1 is a Bt transgenic cotton variety released in 2012 by the National Variety Release Committee, Sudan, and approved by the Biosafety Authority for commercial production to control the bollworms. Crop protection costs ca. 30-40% of total cotton production cost.
Fourier Transform Infrared (FTIR) spectroscopy is a method of an IR spectrometer, which measures an IR spectrum (Baker et al., 2014). Infrared passed across a sample, which absorbs some of the IR and some of it transmitted by the sample. The resulting spectrum represents the molecular absorption and transmission, creating a molecular fingerprint of the sample (Chen et al., 2015). Therefore, the IR spectra analysis can identify the unknown material, type, and concentration of molecules in the sample (Martens et al., 2018). An IR spectrum is formed because of the absorption of electromagnetic radiation that correlates to the vibration of specific sets of chemical bonds from within molecules. Therefore, Che Man & Mirghani (2000) used FTIR to determine moisture-content and iodine value (IV) of palm oil. The determination of functional groups in coal was successfully done using FTIR spectroscopy (Painter et al., 1985). Hence, Mirghani & Che Man (2003) reported that the FTIR spectroscopic technique is useful to substitute the standard wet chemical methods for gossypol's rapid and routine determination. This study's objective was to determine the gossypol concentration in Senni-1 oil and compare it to the gossypol in the traditional Sudanese cottonseed oil (CSO) by using the FTIR spectroscopy.

Materials
The cottonseed oil samples,  were stored on a disk as SPA format files for subsequent chemometric analysis (Alkhalf & Mirghani, 2017).

Statistical analysis
The relationships between each of the parameters

RESULTS AND DISCUSSION
The results obtained from chemical and FTIR analyses as means with standard deviation (SD) and relative SD (CV) for total gossypol contents in the calibration sets of cottonseed oil, Bt CSO, and gossypol-spiked CSO samples are shown in Table I   The band at 772 cm −1 to -CH out-of-plane bending (Yadav, 2005;Stuart, 2004). Figure 3 showed the spectrum of cottonseed oil-spiked gossypol that has might bands as in vegetable oils in addition to some other bands of gossypol; however, challenging to differentiate using the naked eye. Figure 4 shows the spectra of both types of CSOs, conventional and Bt CSOs. Both spectra look normal like other vegetable oils; however, the fast Fourier transform algorithm Cooley & Tukey (Cooley-Tukey FFT algorithm) (Arrondo et al., 1993) as part of the software with FTIR instrument was used to determine and quantify the amount of gossypol in both oil samples.

Development of calibration models
The best spectral region for gossypol determination was selected using variance and correlation spectra. The data from the selected regions with features of interest related to gossypol were abstracted by the PLS software for the calibration standards. The chemical analysis results to be uniquely representative of the samples to be analyzed (Fuller et al., 1988). The spectral regions that show the best correlation between the gossypol content and spectral response were set and selected to include all the data from 3700-2400 cm −1 and from 1900-750 cm −1 for determining the gossypol content in the cottonseed oil and Bt cottonseed oil samples as presented in Table II.
The data from these regions produced the highest R 2 and lowest SE of calibration and SECV for calibration and cross-validation. --R 2 = coefficient of determination; SEC = standard error of calibration; SECV = standard error of cross-validation * See Table I A regression was derived by plotting the gossypol contents in the Seeni-1 CSO as determined by the AOCS method against the calibration set ( Figure 5)