PYT production in basal media
The PYT production was tested using two types of inoculum; spore (Sp-I) and vegetative (Ve-I). The results showed, slow and gradual increase in PYT production using Sp-I, with the maximum activity of 66 ± 3.3 IU/mL on 9th day, (i.e., 216 h) (Fig. 1). The glucose concentration also showed a gradual depletion, with complete utilization by 10th day (i.e., 240 h). In fact, using Ve-I, higher PYT activity of 86 ± 4.3 IU/mL was observed in a lower production time of 6 days (i.e., 144 h) with glucose depletion in 7 days (i.e., 168 h). Thus, the productivity [units of enzyme produced per day (IU/mL/day)] increased from 7.3 IU/mL/day obtained using Sp-I to 14.3 IU/mL/day using Ve-I. Thus, the type of inoculum has marked effect on the production of PYT and all further studies were therefore carried out using Ve-I.
Stability studies of PYT
The conditions for optimum activity and stability of the PYT was assessed by carrying out studies with respect to temperature and pH. Optimum temperature studies ranged from 30 to 70 °C. It was observed that, the optimum temperature for maximum PYT activity is 50 °C (Additional file 1: Figure S1). On considering the activity at 50 °C to be 100%, we observe that at 35 °C, the activity reduces to 30% of maximum activity and at 60 °C to 60% of maximum activity. Temperature stability studies were then carried out, which showed 100% stability in 1 h from 30 to 50 °C (Additional file 1: Figure S1) while 20% activity was reduced at 60 °C, 50% at 65 °C and 100% at 70 °C. Studies with varying pH interestingly showed that the enzyme showed high activity at a low pH value of 2.5 (Additional file 1: Figure S2). Considering the activity at pH 2.5 to be 100%, only 50 and 10% activity was observed at pH 4.5 and 7.0, respectively. It may be noted that the PYT showed overall broad pH stability from pH 2.5–9 (Additional file 1: Figure S2). Experiments showed that the enzyme activity was retained for 12 h in the pH range studied. The pH and temperature stability profile was also determined under poultry gut conditions as described in methods. High efficacy of phosphate release was shown by the PYT in simulated gastric fluid in pH ranging from 2.0 to 4.5 (Additional file 1: Figure S3).
Biodegradation of OpP using PYT
The ability of crude PYT, (100 IU) produced using basal media, to degrade CPyF was studied both under normal conditions (35 °C, pH 7.0) as well as under optimum enzyme conditions (50 °C, pH 2.5). HPLC analyses, showed a single major peak for the sample containing only CPyF (control sample) at 35 °C, pH 7.0 (Fig. 2a) as well as at 50 °C, pH 2.5 (Fig. 2c) and having a retention time of 3.62 min with a relative area of 97%. On the other hand, for a PYT treated sample at 35 °C, pH 7.0, multiple peaks were observed. Notably, it was observed that there was decrease in relative area by 72% at the retention time of CPyF (Fig. 2b). A similar study with PYT at 50 °C, pH 2.5, the relative peak area seen at retention time of CPyF showed an even higher decrease in relative area by 91% (Fig. 2d). The positive result for CPyF suggested degradation studies with other OpPs, namely, MCP and MP by PYT would be useful. HPLC analysis with MCP having a retention time of 5.5 min for the control sample (Additional file 1: Figure S4a) at 35 °C, pH 7.0 showed that a higher unit of PYT (250 IU) to obtain 53% degradation in 4 h (Additional file 1: Figure S4b) when compared to CPyF (PYT 100 IU, 72% degradation) (Fig. 2) in 2 h. Better degradation results with MP (HPLC retention time of 9.5 min for the control sample, Additional file 1: Figure S4c) in comparison with MCP were obtained by a higher decrease in peak area (77%) on treatment with PYT (250 IU) (Additional file 1: Figure S4d) in 4 h. Thus, all the above results with CPyF, MCP and MP corroborate the finding that PYT has the ability to effectively act on OpP and degrade them.
Application of PYT on harvested green chilli (Capsicum annuum L)
Phytase shows dephosphorylation action by breaking the phospho-ester bond to release phosphate from substrate (Joshi 2014). As per food safety and standards authority of India (FSSAI), limit of CPyF on vegetables is 0.2 ppm (FSSAI notification 2011). Detoxification of CPyF is achieved by cleaving the phospho-ester bond, generating TCP as the major product along with DETP (Chen et al. 2012; Hanley et al. 2000; Bicker et al. 2005), which are both water soluble. Human studies show that, both the metabolites are considered as urinary markers of CPyF exposure and are easily excreted through urine within 12 h. Studies on rat show that, TCP and DETP are the predominant urinary metabolites of CPyF catabolism (Bicker et al. 2005). In the present study, the applicability of PYT applied on post-harvest chillies to degrade water insoluble CPyF was therefore studied with respect to the formation of TCP and DETP water soluble degradation products.
LC–MS analysis of standard CPyF showed that it eluted with a retention time (RT) of 5.56 min and m/z of 349.90 while DETP and TCP eluted at 0.72 and 3.48 min with m/z values of 169.17 and 198, respectively. In PYT untreated sample (control), a single peak was detected after LC–MS analysis with a RT of 5.56 min and m/z of 349.90 indicating it to be CPyF (Fig. 3a). In PYT treated sample, 3 peaks (Fig. 3b–d) were observed at RT of 0.72, 3.48 and 5.56 min. MS analysis of these peaks showed m/z values of 169.17, 198 and 349.90, respectively. On comparing with the standards, the 3 peaks were ascertained to be DETP, TCP and CPyF, respectively. The percent degradation of CPyF using PYT from A. niger NCIM 563 can vary depending on pH and temperature prevailing at the field. Peak area analysis shows 8% degradation of CPyF at 35 °C and pH 7.0 in 2 h using PYT (80 IU). To increase the degradation, higher units of phytase or reaction time may thus be required. On using higher units of PYT (250 IU) to degrade CPyF on green chilli our results in fact showed that 90% degradation was possible in 12 h (Additional file 1: Figure S5a, b). It is reported that TCP is not fetotoxic and teratoxic in either rat or rabbits at dosage levels of 100 ppm. TCP is shown to have moderate toxicity to salmonoids at LD50 value of 1.8 ppm (Marino et al. 1999). Studies of TCP showed that a minimum concentration of 0.6 ppm when exposed for 24 h is toxic during the multiple developmental stages of zebra fish (Suvarchala and Philip 2016). Our observation is that PYT can degrade CPyF present on raw agricultural products. Thus, development of a potential new way that prevents toxic OpP from entering the food chain by forming easily removable metabolites could become possible.
Media optimization for enhanced PYT production in shake flask
For the 12 PBD runs the experimentally obtained maximum response values of PYT activity (IU/mL) obtained on the 6th day (i.e., 144 h) are reported in Additional file 1: Table S3. Run number 10 showed a maximum PYT production value of 132 ± 6.6 IU/mL. Regression analysis of the response values obtained for the PBD runs yielded a best fit linear model, viz.,
$$\begin{aligned} {\text{Phytase activity }} = { 111}.0 9 - 30. 6 7 { } \times \left[ {{\text{NaNO}}_{ 3} } \right] + 4 5. 3 3\times \left[ {\text{GrCf}} \right]{-} 1 6 4 4. 4 4 { } \times \left[ {{\text{MnSO}}_{ 4} \cdot{\text{H}}_{ 2} {\text{O}}} \right] \, \hfill \\ \, {-} 1 90.0 \times \left[ {{\text{CaCl}}_{ 2} \cdot 2 {\text{H}}_{ 2} {\text{O}}} \right] \hfill \\ \end{aligned}$$
(1)
The suitability of the model was further corroborated by ANOVA tests. Thus, the obtained model F-value of 11.35 implies the model is significant and that there is only a 0.35% chance that the model F-value could occur due to noise. The values of Prob > F was less than 0.05, for the four variables showing their significance. The coefficient of determination R2 = 0.87 provided a satisfactory measure for the variability in the observed response that could be explained by the model. The Pred R2 of 0.61 is in reasonable agreement with Adj-R2 of 0.79. The adeq precision, a measure of the signal-to-noise ratio, is found to have a high value of 9.72 and this indicated the signal strength to be strong. All the above tests confirm that the model Eq. (1) can be used to navigate the design space. In fact, using the above model, it was found that, the above four factors accounted for 86.68% of the total contribution to the estimates of the response values. The remaining variables then accounted for only 13.32% and thus PBD identified them to be less significant. The ANOVA results were complemented by the fact that four out of the 10 factors studied in the PBD, namely, NaNO3, GrCf, MnSO4·H2O and CaCl2·2H2O were significant on comparing their t-values using a Pareto chart (Additional file 1: Figure S6).
The maximum contributory factors identified by PBD for PYT production were further optimized by a BBD of experiments (29 runs) generated by DES employing three chosen levels for each variable. Additional file 1: Table S4 gives the BBD for the four significant variables along with the experimentally obtained PYT activity. We observe that the optimization studies showed that run number 25 remarkably improved the PYT activity (160 ± 8.0 IU/mL). The wide variation in activity reported in all the runs bring out the process sensitivity to the experimentally chosen conditions and shows the usefulness of having carried out this systematic optimization study. The response data of BBD runs was regressed successfully using actual factors and interestingly showed linear dependency without interacting terms, namely,
$${\text{Phytase activity}} = 2 8 6. 7 4 - 4 9. 1 7\times \left[ {{\text{NaNO}}_{ 3} } \right]{-} 1 20. 8 3\times \left[ {\text{GrCf}} \right]$$
(2)
The ANOVA analysis of the above model satisfied the statistical tests with an obtained model F-value of 27.95 implying that, the model is significant with only a 0.01% chance that the model F-value could arise due to noise. The value of correlation coefficient (Pred R2 = 0.62) for PYT production suggested a good agreement between the observed and model predicted response values. The coefficient of determination (R2 = 0.68), suggests that 68% of the variability in the data was explained by Eq. 2. The obtained signal-to-noise ratio value of 15.04 brought out the presence of an adequate signal.
The final formulation of ten variables in the range studied in PBD and BBD showed that the highest activity of 160 ± 8.0 IU/mL was obtained in 132 h with 100 mL media comprising of (g%): 4.0 glucose; 0.4 NaNO3; 0.075 MgSO4·7H2O; 0.075 KCl; 0.015 FeSO4·7H2O; 0.015 Tween 80; 1.0 GrCf; 0.35 dextrin; 0.02 MnSO4·H2O and 0.1 CaCl2·2H2O. To validate the formulation, a time course experiment for PYT production was carried out using this optimized condition. The results obtained after showed that a 1.86 fold enhancement in PYT activity from 86 ± 4.3 IU/mL to 160 ± 8.0 IU/mL with glucose completely utilized in 5.5 days (i.e., 132 h) was achieved by adopting the outlined hybrid media optimization approach (Fig. 4). The effect of varying the phosphate concentration by addition of sodium phytate and KH2PO4 using the optimized media formulation with 1% GrCf was thus studied. Results showed that the PYT activity decreased to 79 IU/mL on addition of sodium phytate (0.004 g%) and to 151 IU/mL on addition of KH2PO4 (0.002 g%). This may be compared to PYT activity of 160 IU/mL obtained using the optimized media with 1% GrCf and suggests that it optimally provides the requirements of phosphate.
Scale-up studies of PYT production in batch fermenters
The optimized media formulation was studied in scaled-up larger volume batches using fermenters to ensure maintenance of the PYT production throughput. The results of fermentation studies carried out with 2 and 10-L media scaled-up volume batches are discussed below. It is expected that when operating with larger volumes, the dissolved oxygen (DO) is an important factor to consider and this would depend on both the aeration rate and the agitation speed. For the present studies, we chose to keep the aeration rate constant at 0.5 vvm while varying the initial agitation speeds (400, 500, 600 rpm). The behavior in time of DO and pH were simultaneously monitored. For the 400 rpm run, a decrease in DO was observed till 36 h. 50% DO was maintained by gradual increase in rpm and it was thus maintained at the higher rpm. The pH of the media gradually decreased from 5.0 to 2.3. For the 2-L batch study with an initial rpm of 400, the maximum activity of 97 ± 4.8 IU/mL was obtained in 132 h. PYT production of 122 ± 6.1 IU/mL and 158 ± 7.9 IU/mL was achieved at initial rpm of 500 and 600 respectively, in 132 h. Thus, PYT production was successfully scaled up to 2-L production from the shake flask experiment (158 ± 7.9 IU/mL at 600 rpm) (Fig. 5a).
Successful production at 2-L scale, paved way to studying the feasibility of enzyme production in a 10-L volume fermenter to bring out the process biotech potential. Similar to studies in the 2-L scale and varying the initial agitation speed (400, 500 and 600 rpm), the DO and pH were monitored in 10-L scale. DO and pH pattern were again observed, but with higher decreasing rates than in the 2-L scale. Remarkably, the maximum PYT activity of 164 ± 8.2 IU/mL was maintained but it is important to note that the maximum activity was obtained in a much shorter time of 96 h at 500 rpm (Fig. 5b).