Effect of agro waste raw materials as carbon source in tannase production
The key ingredients from the agro-industrial waste materials which influence the biosynthesis of tannase were identified using traditional OFAT method. The production of tannase evaluated by employing diverse agro-industrial waste materials as sole carbon source and sugarcane bagasse exhibited the highest tannase production amongst the others (Fig. 1) followed by wheat bran and coffee husk at 3% (w/v) concentration. Tannase production observed in sugarcane bagasse, wheat bran and coffee husk were calculated as 34.81 U/ml, 23.87 U/ml and 19.69 U/ml respectively.

There are some reports fairly agreed with the present observations that the wheat bran, pomegranate rind, oil mill waste and palm kernel powder as best substrates for tannase production.^3,20-22 Paranthaman et al.²³ stated that the agro-industrial wastes sugarcane bagasse and rice straw were the better alternatives for the enhanced tannase production.

Table (1):
A four factor variables and levels of the CCD experiment.

Table (2):
Experimental design and corresponding responses of the RSM analysis.

RSM analysis
To determine the optimal value of the factors that affects tannase production, experiments were designed using uniform design method, which include the experiment with four factors (sugarcane bagasse, temperature, pH and incubation time) and the experimental design is given in Table 1. The tannase production was enhanced after the analysis of CCD data (102.7 U/ml and 103.1 U/ml at run 13 and 20 respectively, Table 2) when all the four factors were remained the same as of preliminary experiments (center point values). When the pH was lower and all the other factors i.e., sugarcane concentration, temperature and incubation time were maintained at 0 levels, augmented tannase production was observed more or less same as that of center point values (100.3 U/ml, run 5, Table 2).  Similarly, when temperature and incubation time were retained at +1 with pH at -1 level, increased tannase production was detected (96.4 U/ml, run 8, Table 2) which indicates that the lower pH favored tannase production. When we compared the runs where sugarcane concentrations were maintained at levels +1 and +α with varying pH, temperature (-1 and +1), it was found that the tannase production was lower (87.4 U/ml, 85.4 U/ml and 83.6 U/ml, run 6, 16 and 17 respectively. Table 2) which denotes that higher concentration of carbon source may affect the enzyme production. Similarly reduction in tannase production i.e., 81.1 U/ml and 84.9 U/ml were observed at – α and + α levels of temperature and with other three parameters at 0 levels which described the dependence of tannase production on temperature. The effects of the four variables on tannase production using ‘Design Expert11’ software was described below.

Y = 103.1 – 0.462*A + 1.40*B – 0.323*C – 2.217*D – 3.701*A² – 4.822*B² – 2.379*C² – 4.176* D² – 1.216*AB + 1.879*AC + 0.069*AD + 0.764 *BC – 1.311*BD + 0.386*CD

Where Y is the tannase production from S. marcescens IMBL5 and A, B, C and D are the values of the four independent factors such as sugarcane bagasse, temperature, pH, and incubation time respectively. The significance and competence of the regression model was verified by ANOVA (Table 3) authenticate that the model is significant for tannase production using agro waste material. In this study, B, D, AB, AC, BD, A², B², C² and D² are predicted as a significant model terms.

Table (3):
ANOVA results for the response surface quadratic model for tannase production.

The two dimensional contour plots showed in the Fig. 2A- 2F are the graphical representations of the analytic model showing the four factor interaction effect of tannase production. The contour lines characterize the interaction of any two variables upon the response when the remaining variables are maintained at optimum. The smallest ellipse region is known to be the vital response area and the intersection point of the minor and major axes of the ellipse is described as the optimal point. Fig. 2A demonstrate the effect of pH (x1) and incubation time (x2) on enzyme production while sugarcane bagasse and temperature are maintained at their optimal value. The elliptical contours in Fig. 2A designate the interface between two variables x1(pH) and x2 (incubation time) on tannase production. The perfect elliptical contours in Fig. 2B, 2D and 2F states the ample interaction on the responses where the effect of sugarcane bagasse (x1) and pH (x2), temperature (x1) and pH (x2), pH (x1) and incubation time (x2) were analyzed respectively.²⁴ Virtually a circular plot in Fig. 2C implies that the interaction between the represented variables, sugarcane bagasse (x1) and incubation time (x2) are less.²⁵ In Fig. 2E, the interaction effect of temperature and incubation time on tannase production was measured and the elliptical plot indicate the substantial interaction among the factors. RSM was effectively utilized to optimize the tannase production by A. oryzae using agro-waste pomegranate rind extract.²²

Validation of the analysis model
The predicted values obtained from the analytical model were 19.2 g/l, 40°C, 4.3 and 96 for x1, x2, x3 and x4 with the tannase production of 102.98 U/ml. This was correspond to the response obtained from experimental value of 100.72 U/ml with <2 % error which was 3.3 fold higher than that of the enzyme production through OFAT analysis.

Apple juice clarification
Microbial enzymes with stability in acidic pH range are suitable candidates for fruit juice clarification as most of the fruit juices are acidic.²⁶ The effect of tannase on the clarification of apple juice is shown in Fig. 3, here 1 ml (253 U/ml) of tannase enzyme was used for the clarification of 10 ml apple juice. Apple juice clarification up to 60% was observed in 3 hours of incubation with tannase enzyme and gelatin mixture. The enzyme mixture gave up to 80% juice clarification with five more incubation hours. The yield of clarified juice was also increased about 5% and visually the enzyme treated juice showed maximum clearance. The pectin present in fruits leads to colloid formation, which decreases the commercial value of juices. During the enzymatic clarification process, removal of the colloidal materials occur and this leads to the presence of more soluble materials.²⁷ The reduction of turbidity can be related to the degradation of cell wall components such as  tannins and pectin which indicate the efficiency of the clarification process.¹⁴

Detannification of apple juice
Tannins like plant polyphenols are astringent compounds and bitter in taste. The eco-friendly and healthy method of detannification by using the microbial tannase was showed in Fig. 4. About 45% of tannin content decrease was observed within 3 hr of incubation with enzyme-gelatin mixture. A gradual decrease in total tannin content represented the effective tannin degradation by tannase which evidenced the active clarification. Then, the tannin concentration was further decreased slowly with extending the incubation hours. Generally, addition of tannase into fruit juice could aid the tannin degradation but the addition of gelatin along with tannase gives commendable result. The tannase-gelatin treatment may be attributed the precipitation of tannins, which in turn the bitterness of the juice was reduced. The results were most consistent with previous reports.^28,29

Determination of reducing sugar in juice
The reducing sugar level in juices are the best indicative of the quality juices, and monitoring their levels during juice preparation could aid to improve the market quality. In the Fig. 5, the amount of reducing sugar was exponential and significantly increased by nearly 50 µg/ml after 5 h of incubation time. This may lead to increase the sweetness of the clarified juice. The percentage of reducing sugar observed was higher than that of the juice clarified using enzyme alone. Suryawanshi et al.³⁰ stated that fruit juice treated with mixture of enzymes increased the reducing sugar level.

Antiradical scavenging assay
The antiradical scavenging activity is a very significant characteristic feature of fruit juices and it is measured by DPPH scavenging assay which is a standard method to estimate the stable radicals. The scavenging activity of tannase clarified apple juice was presented in Fig. 6. Tannase from S. marcesens IMBL5 significantly enhanced the clarification and maximum scavenging activity was obtained for the tannase treated apple juice with 74.16 % and 83.67 % for ascorbic acid. Compared with the treated one, lower level of scavenging activity was noticed in the commercial apple juice with 66.08 %. Generally, the cloudy juices show greater ability to scavenge free radicals might be attributed to its high tannin content and so neutralizing the free radicals through transferring the hydrogen atom or an electron. Due to the free radical scavenging capacity, the phenolic compounds like flavonoids and tannins are denoted as the main group of antioxidant phytochemicals with remarkable properties.³¹ The results supports the findings of Abdullah et al.²⁹ and Jana et al.⁷ that the tannase treated fruit juices showed 78.44 % and 87.7 % of DPPH radical scavenging activity respectively.

Infrared spectra of a clarified apple juice
FTIR spectroscopy is a cost-effective more rapid method for the investigation of phenolic derivatives and carbohydrates in fruit juices and it can potentially provide facts about their presence and proportions. The IR spectrum of tannase clarified apple juice exhibited a diverse absorption peak patterns from 3390 to 590 cm^-1 was presented in Fig. 7. The presence of intermolecular bonded hydroxyl groups (OH) was identified by means of broad and deeply stretched peak at 3390.01 cm^-1 confirming the existence of a tannins polymerization.³² The region of aliphatic stretching is diagnosed as a methylation of hydrolysable and condensed tannins. The CH2 stretching of a peak at 2925.68 cm^-1 provide information about the methylene substituents.³³ A specific, strong peak at 1717 cm^-1 denotes the C=O — H bond interaction of catechin occurring in polymers include condensed tannins.³⁴ An aromatic – C = C – stretching are also detected in the spectral region of 1449.46 cm^-1 which is typically characterized by an envelope of strong bands with the combinations of C-O stretching, C-H aromatic bending and C-OH deformations. Generally, bands of main carbohydrates were spotted between 900 and 1400 cm^-1 spectral range. The absorption peaks patterned at 1092.15 and 1035.67 cm^-1 represented C–C stretching C–O modes may be ascribed to the band of glucose. The presence of minor peak at 976.23 cm^-1  revealed by the C–O–H bending vibrational mode of fructose.³⁵ The FT-IR result exhibited that tannin present in the apple juice was degraded efficiently by the tannase of S. marcescens strain IMBL5 with varied modification pattern and also the fruit juice quality and authenticity was firmly evaluated.

Chromatographic characterization of apple juice
HPLC system is an ample technique used to detect the presence of phenolic compounds and the rapid characterization of many nutritionally significant polyalcohol and major sugars found in food products. The chromatogram of tannase clarified apple juice was presented in Fig. 8. Phenolic compounds and sugar derivatives in the clarified juice were identified as gallic acid, catechin, caffeic acid, epicatechin, quercetin, sucrose, fructose and sorbitol in C18 column. The compounds spotted in the spectrum were identified through retention time and confirmed by the spectral data of the resultant peaks of previously reported chromatographic investigations of apples juices. The major peak was detected at the retention time of 2.6 min on a chromatogram indicates the presence of gallic acid, an important derivative of tannin degradation and it is also present in apple juice. This peak observation evidently designates the metabolic reaction of tannase on the juice clarification. The peak detected at the retention time of 6.43 min and 12.71 min are indicating the occurrence of phenolic compounds like quercetin and catechin respectively.^36,37 These peaks also confirms the presence of carbohydrates such as sucrose and fructose in the clarified apple juice.³⁸ A mild peak observation shown at retention time 15.19 min confirms the presence of the sugar derivative sorbitol. Another peak detected at 17.947 min indicating that the presence of caffeic acid.³⁹ Du et al.⁴⁰ stated that the caffeic acid, catechin and epicatechin are the leading polyphenols present in apple juice. A peak attained at 20.01 min evidently demonstrating the presence of epicatechin, the predominant byproduct of tannin degradation.⁴¹ Similar to our investigation, many other researchers have reported the study of polyphenols in apple juice in various centuries.^17,42-44 The chromatogram study indicate the occurrence of polyphenols and sugar molecules were identified evidently and the significance of tannase in apple juice clarification was also proved.