Elicitation kinetics of phenolics in common bean (Phaseolus vulgaris) sprouts by thermal treatments

Phenolics are plant secondary metabolites with numerous health benefits, produced via the phenylpropanoid pathway in plants in response to environmental conditions. In this study, the mathematical relationship between thermal elicitation (25°C, 30°C, 35°C, and 40°C) of phenolic compounds through the accumulation of oxidative stress markers (hydrogen peroxide [H2O2], malondialdehyde [MDA], catalase [CAT], and guaiacol peroxidase [GPX]) and activation of phenylpropanoid triggering enzymes (phenylalanine ammonia‐lyase [PAL] and tyrosine ammonia‐lyase [TAL]) was kinetically modeled at different sprouting stages of common bean. The rate of H2O2 and MDA formation showed an increasing trend with an increase in sprouting temperature. However, activation rates of CAT, GPX, PAL, and TAL were highest at 30°C, after which there were significant reductions. Also, activation rate of PAL was lower as compared with TAL, which was further established with its low activation energy Ea value of 150 kJ/mol compared with TAL (221 kJ/mol). Also, activation energy values for total phenolic acids (30.4 kJ/mol) and flavonoids (64.0 kJ/mol) showed that they required less energy for formation during sprouting, compared with anthocyanins (209 kJ/mol), with the activation energy results obtained from their estimated kinetic rate constants and production percentages. Thus, manipulation of sprouting temperature can increase the potential use of common beans as natural functional foods with improved health benefits.

such as antioxidative, anti-inflammatory, antidiabetic, anticancer, and antimutagenic effects (Curran, 2012). Thus, demand exists for minimally processed plant foods that have high phenolic contents.
Common beans (Phaseolus vulgaris) are pulse foods that have become dietary components of human populations since ancient times (Gan et al., 2016). The wide consumption of common bean is not only due to its protein, fiber, resistant starch, and B-vitamin content but also due to the presence of bioactive secondary metabolites such as phenolics (Gan et al., 2016). However, depending on cultivar, growing conditions, geographical location, and growth stage, the concentration of phenolics differs among common bean varieties (Sutivisedsak et al., 2010). In order to increase phenolic yield, technologies including genetic engineering, tissue culture, and sprouting have been applied in plant foods. Genetic engineering is limited by its high cost and low consumer acceptance, whereas tissue culture has been shown to cause cell damage as well as resulting in lower yield (Owolabi et al., 2018). However, although sprouting is a cheap, simple, and consumer acceptable household process, it is a slow process and has low yield compared with genetic engineering and tissue culture (Swieca & Baraniak, 2014), thus the need to develop technologies that can increase the rate of PPP during sprouting for enhanced phenolic accumulation.
One such approach is the process of elicitation. Elicitation is the induction of physiological processes in plants, in the presence of an elicitor/stressor. During elicitation, applied elicitors/stressors (e.g., temperature, chemicals, light, and ultrasound) induce wounds, osmotic disruptions, and membrane weakening, leading to increased production of oxidative stress markers (e.g., reactive oxygen species such as hydrogen peroxide [H 2 O 2 ] and OH radicals) and subsequent stimulation of the PPP for biosynthesis of phenolics as antioxidative protectants (Liu et al., 2019). For instance, a previous study by Swieca and Baraniak (2014) reported on 40 C-elicited lentil sprouts with total phenolic acid and flavonoid contents of 23.70 and 2.50 mg/g, respectively, compared with control sprouts (25 C) which possessed 19.8 and 1.84 mg/g contents, respectively. However, no comparative investigation on triggering of PPP, phenolics accumulation, and antioxidative properties of common bean at the sprouting developmental phase has been reported. A good understanding of the optimum elicitation conditions during sprouting will be very significant in designing processing systems for optimizing phenolic synthesis and producing phenolic-enriched sprouts. Thus, the objectives of this study were to establish the kinetic relation between thermal elicitation and stress markers, catalysis of PPP stimulating enzymes, and phenolic concentrations of common bean at different sprouting stages by kinetic modeling.

| Common bean seeds
Common bean cultivar "Kabulengeti" was provided by the Council for Scientific and Industrial Research Center of Zambia. This cultivar has high yield stability, high tolerance to diseases and pests, and possesses protein and lipid contents of 24.38% and 1.38%, respectively. However, it has low levels of total phenolic acids (3.76 mg CE/100 g) and flavonoids (2.24 mg RE/100 g). Therefore, it was chosen with this study as a model for common bean cultivars with low concentrations of phenolic compounds.

| Elicitation processes
Common bean seeds (100 g) were disinfected (1% sodium hypochlorite in 500 ml), for 15 min and rinsed afterwards to obtain neutral pH. For thermal elicitation, disinfected seeds were soaked in distilled water (1:3 w/v, 25 C, 24 h; Mubarak, 2005 Alexieva, Sergiev, Mapelli, and Karanov (2001). Approximately 0.1 g of sample flour was homogenized with 1 ml of mixed solution (0.25 ml of 0.1% trichloroacetic acid [TCA], 0.5 ml of 1 M potassium iodide [KI], and 0.25 ml of 10 mM potassium phosphate buffer, pH 7.0) at 4 C for 10 min. Afterwards, the obtained mixture was centrifuged (10,000 g, 15 min, 4 C); the supernatant was recovered and kept in the dark for 30 min and its absorbance measured at 390 nm with a microplate reader. A control was prepared with H 2 O instead of KI.
Lipid peroxidation was determined by measuring accumulation of malondialdehyde (MDA) as described by Dhindsa, Plumb-Dhindsa, and Thorpe (1981). Approximately, 0.2 g of sample was homogenized in 2 ml of 5% TCA for 10 min and centrifuged at 13,500 g for 15 min at 25 C. Afterwards, 1 ml of supernatant was mixed with 1 ml of 0.5% (v/v) thiobarbituric acid (in 20% (v/v) TCA). The obtained mixture was heated at 96 C for 30 min, cooled in ice bath and centrifuged at 9,500 g for 10 min. After centrifugation, absorbance of the recovered supernatant was measured at 532 nm, and the value for nonspecific absorption at 600 nm was measured and subtracted. MDA concentration was expressed as nmol MDA per g of dry weight.

| Determination of antioxidant enzymes
Extract preparations and protein content determination Total protein assay. Protein contents of enzyme extracts were measured according to the method of Bradford (1976). Bovine serum albumin was used as the reference protein to calibrate the assay.
Catalase (CAT) and guaiacol peroxidase (GPX) assays were performed on the same day of extraction. Enzyme extraction was performed in triplicate.
CAT and GPX assays. For CAT activity, the reaction mixture (0.05 ml of enzyme extract, 0.95 ml of 10 mM H 2 O 2 in 100 mM sodium phosphate buffer, pH 7) was incubated at 30 C for 1 min, and decomposition of H 2 O 2 was measured at 240 nm. One unit (1 U) of CAT activity was defined as the amount of CAT capable of decomposing 1.0 μmol H 2 O 2 per min under the conditions of the assay .
GPX assay was performed as described by Burguieres, McCue, Kwon, and Shetty (2007). The reaction mixture (0.1 ml of enzyme extract, 2 ml of 8 mM guaiacol in 100 mM sodium phosphate buffer-pH 6.4) was incubated at 30 C for 1 min. Afterwards, 1 ml of 24 mM H 2 O 2 was added, and the change in absorbance was measured at 460 nm. Enzymatic extractions were performed in triplicate.

| Determination of PAL and TAL activities
For PAL activity, the reaction mixture (300 μl of enzyme extract, 1.2 ml of 0.02 M L-phenylalanine and 2 ml of PAL extracting buffer) was incubated at 30 C for 60 min. Afterwards, the reaction was terminated by adding 0.5 ml of 10% TCA, centrifuged (15,000 g, 10 min, 4 C) and absorbance of supernatant measured at 290 nm; 1 U was defined as the amount of PAL that produced 1.0 μmol trans-cinnamic acid per min under conditions of the assay (Assis, Maldonado, Munoz, Escribano, & Merodio, 2001).
Activity of tyrosine ammonia-lyase (TAL) was determined by incubating reaction mixture (100 μl of enzyme extract and 0.9 ml of 0.02 M L-tyrosine) for 60 min at 30 C. Next, the reaction was stopped by adding 0.5 ml of 10% TCA, centrifuged (15,000 g, 10 min, 4 C) and absorbance of supernatant read at 310 nm. One unit (1 U) was defined as the amount of TAL that produced 1.0 μmol p-coumaric acid per min under conditions of the assay (Assis et al., 2001). 2.5 | Assessment of phenolic compounds and antioxidant capacities 2.5.1 | Extraction of phenolic compounds Methanolic extraction of phenolics was carried out as described by Marathe, Rajalakshmi, Jamdar, and Sharma (2011). Approximately 1 g of sample flour was extracted for 20 min with 15 ml of 80% aqueous methanol. Pooled triplicate extractions were centrifuged (5,000 rpm, 10 min), and supernatant organic solvents were removed at 35 ± 3 C by using a rotary vacuum evaporator. The obtained extract was freeze-dried and dissolved in 80% aqueous methanol prior to ultraviolet (UV)-vis quantification.

| Determination of total flavonoids content
Total flavonoids were analyzed as described by Hairi, Sallé, and Andary (1991), and expressed as rutin equivalent in mg/100 g of sample flour. A standard curve for quantitative measurement was prepared with rutin (0-50 μg ml −1 ).

| Kinetic analysis
Kinetics of the experimental data were determined by the dimensionless normalized concentration At = Ao at the different sprouting temperatures and time; where A t and A o are the concentrations of phenolic compounds in elicited and untreated samples, respectively. According to Chen and Ramaswamy (2002), the first-order kinetic model is suitable for studying activities of enzymes and accumulation of biochemical components. Thus, kinetics of all investigated biomolecules, enzymes, and antioxidant capacities were calculated according to the following first-order equation: Furthermore, the Arrhenius equation was used to evaluate the temperature dependence of the reaction rate k: where k O is the pre-exponential factor h −1 , E a is the activation energy of the enzymatic reaction, R is the gas constant, and T is the absolute temperature.

| Statistical analysis
Statistical significance was assessed by one-way analysis of variance According to Kumar et al. (2013), exposure of plant cells to temperatures above threshold levels increases cellular damage and respiratory activities, leading to an increased accumulation of free radicals (e.g., H 2 O 2 ) as metabolic end-products. Also, the determination coefficients (R 2 ) were very high at 0.99 (except 0.58 at 25 C), implying that the experimental data for H 2 O 2 fit well the first-order kinetic model under the temperatures tested.
Similar to H 2 O 2 accumulation, an increase in elicitation temperatures of sprouts increased the rate of MDA production. As shown in  Similarly, treatment at 30 C for 96 h of sprouting yielded the highest percentage formation of total anthocyanins by 637%, compared with its initial concentration of 1.64 mg/100 g. This observation at 30 C was 1.18, 1.47, and 2.87 fold higher than total anthocyanin T A B L E 2 First-order kinetic parameters for activation of phenylpropanoid triggering enzymes during thermal elicitation of common bean sprouts percentages observed with 35 C, 40 C, and 25 C treatments, respectively, at the same sprouting time. The enhanced percentage production of phenolics at 30 C is in synchrony with optimum activation rates of PAL and TAL at the same temperature, compared with other evaluated temperatures. Thus, increased activation rates of phenylpropanoid triggering enzymes with a 30 C elicitation resulted in the production of minimally processed phenolic-enriched common bean sprouts.

| Accumulation kinetics of total phenolic acids, flavonoids, and anthocyanins
Also, as shown in Table 3, the rate of formation k (h −1 ) of total phenolic acids, flavonoids, and anthocyanins was dependent on elicitation temperatures. The highest rate formation for phenolic acids, flavonoids, and anthocyanins was estimated as 6.2200 × 10 −2 , 6.4400 × 10 −2 , and 3.3800 × 10 −2 , respectively. At temperatures more than 30 C, there were reductions in k (h −1 ) formations for all evaluated phenolic compounds.
These kinetic parameters are in synchrony with phenolic percent- It signifies that during thermal elicitation of common bean sprouts, less energy was required for the formation of free radicals and lipid peroxidation compared with activation rates of antioxidant enzymes.
Therefore, the demand for phenolic signaling as an alternative defense mechanism against accumulated stress markers (H 2 O 2 and MDA).
In response to accumulation of H 2 O 2 , MDA, CAT, and GPX, the activation energies for activities of phenolic triggering enzymes were calculated as 150 and 221 kJ/mol for PAL and TAL, respectively. Low E a (kJ/mol) observed for PAL explains its estimated low activation rate and high end-product formations, compared with TAL. Thus, less energy was required by PAL to catalyze the deamination of phenylalanine into trans-cinnamic acids. Also, the activation energies calculated for total phenolic acids, flavonoids, and anthocyanins were 30.4, 64.0, and 209 kJ/mol, respectively. This confirms the higher formation rates of phenolic acids and flavonoids, compared with anthocyanins, as also seen in Figure 1 and Table 3.

| CONCLUSION
This study showed that biochemical processes leading to the accumulation of phenolics along the sprouting phase of common bean are temperature dependent. Increasing sprouting time increased the kinetic rates of all investigated biochemical and enzyme mechanisms.
From this study, it was established that, although the production rates of H 2 O 2 and MDA were increased at high sprouting temperatures, this trend was not reflected by activation kinetic rates of antioxidant enzymes, phenylpropanoid triggering enzymes, and final accumulation rates of phenolic compounds. Thermal elicitation of common bean sprouts at 30 C resulted in a strong correlation between oxidative stress markers, activation of phenylpropanoid triggering enzymes, and final accumulation rates of phenolic compounds. Thus, optimization of sprouting conditions is a potential alternative for the functional food industry to produce phenolic-enriched common bean sprouts with improved health benefits.

ACKNOWLEDGMENT
The authors gratefully acknowledge the International Fund for Agricultural Development (IFAD) for providing financial assistance through IFAD project grant 2000000974.

CONFLICT OF INTEREST
The authors declare no conflict of interests.

AUTHOR CONTRIBUTIONS
Josephine Ampofo design the study, performed the experiments, analyzed the data, and wrote the manuscript. Hosahalli Ramaswamy provided supervision, reviewed, and edited the manuscript. Michael Ngadi did the supervision, funding, concept testing, and reviewing of the manuscript. All authors contributed to the final review and editing of the manuscript.

ETHICS STATEMENT
This article does not contain any studies with human and animal subjects.

DATA AVAILABILITY STATEMENT
Due to technical limitations, the full dataset is unable to be published at this time. However, it is available upon request from the first author. Note: Data are presented as mean ± SD of three independent experiments. Abbreviations: ANTH, total anthocyanins; CAT, catalase; E a , activation energy; GPX, guaiacol peroxidase; H 2 O 2 , hydrogen peroxide; MDA, malondialdehyde; PAL, phenylalanine ammonia-lyase; PPP, phenylpropanoid pathway; R 2 , coefficient of determination; TAL, tyrosine ammonia-lyase; TFC, total flavonoids contents; TPA, total phenolic acids.