**3. Results and discussion**

#### **3.1 Partial purification**

As can be seen in **Table 2**, after the precipitation with ammonium sulphate, it was not possible to recover the activity of the enzymatic extract in the fractions of 0–20 and 60–80%. In the other fractions, it was not possible to obtain a considerable purification factor (greater than 1). Thus, it was found that the use of ammonium sulphate as a precipitating agent was not efficient in the precipitation of the target protein (tannase), since this salt may have caused the denaturation of the enzymes, under the experimental conditions evaluated.

In the precipitation using ethanol, it was found that in the 50 to 70% saturation it was not possible to verify enzymatic activity and in the concentrations of 80 and 90% a reduction in it. In purification, the most desirable is that the proteins/ contaminants are decreased and the activity of the target protein is concentrated or not decreased. The use of organic solvents as a precipitating agent may have negatively influenced the activity of the enzyme, as already demonstrated by several authors [39–41]. The ethanol and ammonium sulphate might have caused denaturation through a conformational change in the enzyme tertiary structure.


*VA – Volumetric activity; TP – Total protein; AE – Specific activity; PF – Purification factor. The experiments were performed in triplicate and the mean standard deviation values were presented. Values followed by the same letter did not statistically differ in the Scott-Knott test at 5% probability.*

#### **Table 2.**

*Partial purification of tannase from* S. cerevisiae *CCMB 520.*

In reference [42], tannase was obtained and purified from *Aspergillus niger*. and The precipitation method using ammonium sulphate (50–70%) resulted in a purification factor of 4.89. Whereas in reference [43], after partial purification of tannase obtained from *Aspergillus niger* MTCC 2425, through precipitation with ammonium sulphate (75%) were obtained a purification factor around 1.4. In reference [44] tannase from *Aspergillus nomius* GWA5 was purified after three steps, using acetone and two chromatographic processes and the authors obtained the following purification factors: 1.59 (acetone fraction), 3.21 (molecular exclusion) and 4.48 (ion exchange).

From the data, we can evidence that the tannase application in integral pitanga juice did not change the evaluated parameters, indicating that it would be within

*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520*

Through the results obtained for the total phenolic contents, presented here in **Table 5**, we can infer that in all tests these compounds increased when compared to their respective controls. The assay 8 (4.5% and 180 minutes) stood out statistically significantly among the others, reaching 3630 mg Eq. AG/L (285.59 mg/100 g). The phenolic compounds are substances involved in the prevention processes of

In [47] after evaluating phenolic compounds in red pitanga found levels around

The results obtained experimentally for total phenolics were evaluated through F Test (Fisher's Test) and Analysis of Variance (ANOVA) (**Table 6**). The regression

chronic diseases, including diabetes, cancer, heart disease and Alzheimer's, and knowledge about their presence in different fruit can contribute to the development of production, consumption, rural diversification and income generation [46].

257 mg/100 g. Whereas in [45] found levels of 95.90 mg/100 g for the pitanga

**Assay Total phenolics (mg Eq. AG/L)**

 3630.00 106,066 d 4230.00 318,20 b 4142.50 53,033 b 3555.00 141,42 d 3842.50 53,033 c 3567.50 159,099 d 4317.50 88,39 b 4855.00 35,36 a 3955.00 106.066 c

C1 2655.00 70.71 f C2 2467.50 17.68 f C3 2630.00 35.36 f C4 2830.00 35.36 f C5 2467.50 17.68 f C6 2642.50 194.45 f C7 3205.00 176.78 e C8 3567.50 123.74 d C9 3242.50 17.68 e **Before application** 2663.33 115.47 f

*The experiments were performed in triplicate and the mean standard deviation values were presented. Values*

*Doehlert matrix results for total phenolics in Pitanga juice before and after application of partially purified*

*followed by the same letter did not statistically differ in the Scott-Knott test at 5% probability.*

*tannase from* Saccharomyces cerevisiae *CCMB 520.*

the pre-established national standards.

*DOI: http://dx.doi.org/10.5772/intechopen.96103*

*3.2.2 Total phenolics*

hydroalcoholic extract.

**After application**

**Controls (white)**

**Table 5.**

**25**

After carrying out the 30 kDa membrane separation process, was possible to verify a higher degree of compaction, resulting from the internal encrustation caused by smaller particles that were adsorbed on the tube walls, thus providing a result that characterized a partial purification (factor of purification above 1), with no statistically significant difference between the two fractions obtained (retained and permeated).

#### **3.2 Biotransformation of integral Pitanga juice by partially purified tannase from** *Saccharomyces cerevisiae* **CCMB 520**

#### *3.2.1 Physico-chemical analysis*

The physical–chemical results are shown in **Table 3** and the Standard of Identity and Quality for the pitanga juice are in **Table 4**. The samples of the integral pitanga juice before and after partially purified tannase application comply with the standards required by current Brazilian legislation [45].


*Sample 0: before application; Samples 1 to 9: after application. Values followed by the same letter did not statistically differ in the Scott-Knott test at 5% probability*.

#### **Table 3.**

*Physico-chemical parameters of integral Pitanga juice before and after application of partially purified tannase from* Saccharomyces cerevisiae *CCMB 520.*


#### **Table 4.**

*Standard of identity and quality for Pitanga juice.*

*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520 DOI: http://dx.doi.org/10.5772/intechopen.96103*

From the data, we can evidence that the tannase application in integral pitanga juice did not change the evaluated parameters, indicating that it would be within the pre-established national standards.

#### *3.2.2 Total phenolics*

In reference [42], tannase was obtained and purified from *Aspergillus niger*. and The precipitation method using ammonium sulphate (50–70%) resulted in a purification factor of 4.89. Whereas in reference [43], after partial purification of tannase obtained from *Aspergillus niger* MTCC 2425, through precipitation with ammonium sulphate (75%) were obtained a purification factor around 1.4. In reference [44] tannase from *Aspergillus nomius* GWA5 was purified after three steps, using acetone and two chromatographic processes and the authors obtained the following purification factors: 1.59

After carrying out the 30 kDa membrane separation process, was possible to verify a higher degree of compaction, resulting from the internal encrustation caused by smaller particles that were adsorbed on the tube walls, thus providing a result that characterized a partial purification (factor of purification above 1), with no statistically significant difference between the two fractions obtained (retained

**3.2 Biotransformation of integral Pitanga juice by partially purified tannase**

juice before and after partially purified tannase application comply with the

 3.40 a 11,85 a 1.67 a 3.40 a 12.10 a 1.67 a 3.40 a 12.55 a 1.57 a 3.40 a 12.25 a 1.55 a 3.30 a 12.00 a 1.62 a 3.40 a 10.35 a 1.38 a 3.20 a 12.20 a 1.66 a 3.40 a 12.20 a 1.73 a 3.40 a 11.70 a 1.74 a 3.40 a 12.40 a 1.70 a

**Samples pH Total Soluble Solids (°Brix) Total acidity (g/100 g, citric acid)**

*Sample 0: before application; Samples 1 to 9: after application. Values followed by the same letter did not statistically*

*Physico-chemical parameters of integral Pitanga juice before and after application of partially purified tannase*

**Legislation (BRAZIL, 2016) Minimum Maximum** pH 2.50 3.40 Total Soluble Solids (°Brix) 6.00 — Total acidity (g/100 g, citric acid) 0.92 —

The physical–chemical results are shown in **Table 3** and the Standard of Identity and Quality for the pitanga juice are in **Table 4**. The samples of the integral pitanga

(acetone fraction), 3.21 (molecular exclusion) and 4.48 (ion exchange).

**from** *Saccharomyces cerevisiae* **CCMB 520**

standards required by current Brazilian legislation [45].

and permeated).

*Saccharomyces*

*3.2.1 Physico-chemical analysis*

*differ in the Scott-Knott test at 5% probability*.

*from* Saccharomyces cerevisiae *CCMB 520.*

*Standard of identity and quality for Pitanga juice.*

**Table 3.**

**Table 4.**

**24**

Through the results obtained for the total phenolic contents, presented here in **Table 5**, we can infer that in all tests these compounds increased when compared to their respective controls. The assay 8 (4.5% and 180 minutes) stood out statistically significantly among the others, reaching 3630 mg Eq. AG/L (285.59 mg/100 g).

The phenolic compounds are substances involved in the prevention processes of chronic diseases, including diabetes, cancer, heart disease and Alzheimer's, and knowledge about their presence in different fruit can contribute to the development of production, consumption, rural diversification and income generation [46].

In [47] after evaluating phenolic compounds in red pitanga found levels around 257 mg/100 g. Whereas in [45] found levels of 95.90 mg/100 g for the pitanga hydroalcoholic extract.


The results obtained experimentally for total phenolics were evaluated through F Test (Fisher's Test) and Analysis of Variance (ANOVA) (**Table 6**). The regression

*The experiments were performed in triplicate and the mean standard deviation values were presented. Values followed by the same letter did not statistically differ in the Scott-Knott test at 5% probability.*

#### **Table 5.**

*Doehlert matrix results for total phenolics in Pitanga juice before and after application of partially purified tannase from* Saccharomyces cerevisiae *CCMB 520.*


#### **Table 6.**

*Analysis of variance applied to the data shown in Table 5.*

was statistically significant (Fcal 9.18 > 9.01 Ftab) and the lack of fit indicated a good agreement (Fcal 1.28 < 18.51 Ftab) between the fitted model and the experimental data. Furthermore, the quality of the fit was also confirmed through coefficient of determination (R2 = 0.94), and it implied that just 6% of the response variability was not explained by the model.

The model equation after regression, for the increase of phenolic compounds, was obtained (Eq. (3)):

$$\begin{aligned} \text{Total phenions } (\text{mgEQAG/L}) &= 39442.50 \cdot (\pm 5459.69) - 4545.83 \cdot EE \cdot (\pm 805.25) \\ &+ 161.11 \cdot EE^2 \cdot (\pm 49.45) - 217.19 \cdot T \cdot (\pm 40.56) \\ &+ 0.36 \cdot T^2 \cdot (\pm 0.093) + 12.29 \cdot EE \cdot T \cdot (\pm 2.71) \end{aligned} \tag{3}$$

From the **Figure 2**, we found that only the interaction (positive effect) was statistically significant in the experimental field studied. **Figure 3** shows the response surface and contour curves obtained as a function of enzyme application time and tannase concentration, where it indicated that the increase in the variables under study increased the phenolic compounds. While, by decreasing the two variables, there was also an increase in phenolic compounds. This result can be seen in the positive interaction term obtained in Eq. (3) and **Figure 2**.

#### *3.2.3 Antioxidant activity*

Studies have shown that the consumption of fruits and vegetables reduces the risk of chronic diseases such as cancer, cardiovascular diseases and stroke [48]. This may be due to the presence of several secondary metabolites, these being related to various biological activities, including antioxidant activity.

The results of the total antioxidant activity are shown in **Table 7**, where it can be seen that test 8 (69.41%), as well as for phenolics (**Table 5**), was the one that presented values statistically superior to the other tests. We also found that all tests in the presence of the enzyme were superior to their respective controls. This demonstrates that the tannase from *S. cerevisiae* CCMB 520 acted on the compounds present in the integral pitanga juice, biotransforming them and increasing their biological activity.

The results obtained experimentally for the total antioxidant activity were evaluated by Test F and ANOVA (**Table 8**). The regression was statistically significant (Fcal 20.61 > 9.01 Ftab) and the lack of fit indicated a good agreement (Fcal 10.33 < 18.51 Ftab) between the adjusted model and the experimental data. The fit of the

**Figure 3.**

**27**

**Figure 2.**

*planning of the Doehlert design.*

*Response surface and contour plot to total phenolic content, according to the Doehlert design. The three-dimensional plot shows partially purified tannase concentration and application time.*

*Pareto chart for the effects of the variables on the total phenolic content of Pitanga juice, according to statistical*

*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520*

*DOI: http://dx.doi.org/10.5772/intechopen.96103*

*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520 DOI: http://dx.doi.org/10.5772/intechopen.96103*

**Figure 2.**

was statistically significant (Fcal 9.18 > 9.01 Ftab) and the lack of fit indicated a good agreement (Fcal 1.28 < 18.51 Ftab) between the fitted model and the experimental data. Furthermore, the quality of the fit was also confirmed through coefficient of determination (R2 = 0.94), and it implied that just 6% of the response

**Variation Source Sum of squares Degree of freedom Mean square Fcal Ftab** Regression<sup>a</sup> 1324618 5 264923.60 9.18 9.01

Lack of Fit 33750 1 33750 1.28 18.51

*Statistically significant at 95% confidence interval. Fcal – calculated F value; Ftab – tabulated F value. R<sup>2</sup> = 0.94.*

Residual 86563 3 28854.14

Pure Error 52813 2 26406.25

The model equation after regression, for the increase of phenolic compounds,

*Total phenolics mgEQAG* ð Þ¼ *=L* 39442*:*50 � �ð Þ� 5459*:*69 4545*:*83 � *EE* � �ð Þ 805*:*25

From the **Figure 2**, we found that only the interaction (positive effect) was statistically significant in the experimental field studied. **Figure 3** shows the response surface and contour curves obtained as a function of enzyme application time and tannase concentration, where it indicated that the

increase in the variables under study increased the phenolic compounds. While, by decreasing the two variables, there was also an increase in phenolic compounds. This result can be seen in the positive interaction term obtained in Eq. (3) and

Studies have shown that the consumption of fruits and vegetables reduces the risk of chronic diseases such as cancer, cardiovascular diseases and stroke [48]. This may be due to the presence of several secondary metabolites, these being related to

The results of the total antioxidant activity are shown in **Table 7**, where it can be

The results obtained experimentally for the total antioxidant activity were evaluated by Test F and ANOVA (**Table 8**). The regression was statistically significant (Fcal 20.61 > 9.01 Ftab) and the lack of fit indicated a good agreement (Fcal 10.33 < 18.51 Ftab) between the adjusted model and the experimental data. The fit of the

seen that test 8 (69.41%), as well as for phenolics (**Table 5**), was the one that presented values statistically superior to the other tests. We also found that all tests in the presence of the enzyme were superior to their respective controls. This demonstrates that the tannase from *S. cerevisiae* CCMB 520 acted on the compounds present in the integral pitanga juice, biotransforming them and increasing their

various biological activities, including antioxidant activity.

<sup>þ</sup> <sup>161</sup>*:*<sup>11</sup> � *EE*<sup>2</sup> � �ð Þ� <sup>49</sup>*:*<sup>45</sup> <sup>217</sup>*:*<sup>19</sup> � *<sup>T</sup>* � �ð Þ <sup>40</sup>*:*<sup>56</sup> <sup>þ</sup> <sup>0</sup>*:*<sup>36</sup> � *<sup>T</sup>*<sup>2</sup> � �ð Þþ <sup>0</sup>*:*<sup>093</sup> <sup>12</sup>*:*<sup>29</sup> � *EE* � *<sup>T</sup>* � �ð Þ <sup>2</sup>*:*<sup>71</sup>

(3)

variability was not explained by the model.

*Analysis of variance applied to the data shown in Table 5.*

Total 1411181

a

**Table 6.**

*Saccharomyces*

was obtained (Eq. (3)):

**Figure 2**.

*3.2.3 Antioxidant activity*

biological activity.

**26**

*Pareto chart for the effects of the variables on the total phenolic content of Pitanga juice, according to statistical planning of the Doehlert design.*

#### **Figure 3.**

*Response surface and contour plot to total phenolic content, according to the Doehlert design. The three-dimensional plot shows partially purified tannase concentration and application time.*


**Figure 4.**

**Figure 5.**

**29**

*statistical planning of the Doehlert design.*

*DOI: http://dx.doi.org/10.5772/intechopen.96103*

*Pareto chart for the effects of the variables on the total antioxidant activity of Pitanga juice, according to*

*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520*

*Response surface and contour plot to total antioxidant activity, according to the Doehlert design. The three-dimensional plot shows partially purified tannase concentration and application time.*

*The experiments were performed in triplicate and the mean standard deviation values were presented. Values followed by the same letter did not statistically differ in the Scott-Knott test at 5% probability*.

#### **Table 7.**

*Doehlert matrix results before and after the application of partially purified tannase from* Saccharomyces cerevisiae *CCMB 520.*


a *Statistically significant at 95% confidence interval. Fcal – calculated F value; Ftab – tabulated F value. R<sup>2</sup> = 0.97.*

#### **Table 8.**

*Analysis of variance applied to the data shown in Table 7.*

model was measured by the coefficient of determination (R2), which had a value of 0.97 suggesting that 97% of the total variation in residual antioxidant activity was explained by the adjusted model. It is worth mentioning that this is the first report

*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520 DOI: http://dx.doi.org/10.5772/intechopen.96103*

#### **Figure 4.**

*Pareto chart for the effects of the variables on the total antioxidant activity of Pitanga juice, according to statistical planning of the Doehlert design.*

#### **Figure 5.**

*Response surface and contour plot to total antioxidant activity, according to the Doehlert design. The three-dimensional plot shows partially purified tannase concentration and application time.*

model was measured by the coefficient of determination (R2), which had a value of 0.97 suggesting that 97% of the total variation in residual antioxidant activity was explained by the adjusted model. It is worth mentioning that this is the first report

**Assay Antioxidant activity – DPPH (%) Antioxidant activity – μMTrolox/L**

 57.56 1.78 d 757.00 35.56 64.96 6.091 b 803.67 40.069 61.34 0.59 c 835.33 4.71 64.71 1.54 b 892.00 29.63 65.55 0.48 b 915.33 9.43 64.71 1.90 b 863.67 11.79 62.35 1.19 c 852.00 23.57 69.41 1.43 a 952.00 28.28 59.16 0.71 d 778.67 14.14

C1 50.00 1.31 e 607.00 25.93 C2 42.017 2.38 f 448.67 47.14 C3 42,10 1.31 f 450.33 25.93 C4 45.46 2.97 f 517.00 58.93 C5 42.27 0.12 f 453.67 2.36 C6 41.76 0.59 f 443.67 11.79 C7 50.42 1.90 e 615.33 37.71 C8 49.07 0.71 e 588.67 14.14 C9 46.97 1.54 e 547.00 30.64

*The experiments were performed in triplicate and the mean standard deviation values were presented. Values*

*Doehlert matrix results before and after the application of partially purified tannase from* Saccharomyces

**Variation Source Sum of squares Degree of freedom Mean square Fcal Ftab** Regression<sup>a</sup> 101.01 5 20.20 20.61 9.01

Lack of Fit 2.48 1 2.48 10.33 18.51

*Statistically significant at 95% confidence interval. Fcal – calculated F value; Ftab – tabulated F value. R<sup>2</sup> = 0.97.*

*followed by the same letter did not statistically differ in the Scott-Knott test at 5% probability*.

Residual 2.95 3 0.98

Pure Error 0.47 2 0.24

51.26 2.38 e 632.00 47.14

**After application**

*Saccharomyces*

**Controls (white)**

**Before application**

cerevisiae *CCMB 520.*

Total 103.96

*Analysis of variance applied to the data shown in Table 7.*

**Table 7.**

a

**28**

**Table 8.**

on the application of tannase in integral pitanga juice and its effect on total antioxidant activity and phenolic contends.

The second order equation (Eq. (4)) that describes the experimental data is presented:

$$\begin{array}{c} \text{TAA} \ (\forall \text{i}) = \text{146.71} \cdot (\pm \text{16.29}) - \text{22.79} \cdot \text{EE} \cdot (\pm \text{2.40}) - \text{0.64} \cdot \text{EE}^2 \cdot (\pm \text{0.15}) - \text{0.10} \\ \quad \cdot T \cdot (\pm \text{0.12}) - \text{0.0020} \cdot T \cdot (\pm \text{0.12}) - \text{0.0020} \cdot T^2 \cdot (\pm \text{0.00028}) + \text{0.15} \\ \quad \cdot \text{EE} \cdot T \cdot (\pm \text{0.0081}) \end{array}$$

(4)

moderate positive correlation between variables, by increasing phenolic

*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520*

The use of tannase for the release of phenolic antioxidants has become interesting for various types of food matrix. This is because most of them can release the phenolic compounds without requiring a pre-treatment such as the action of the pectinase or cellulase, or variation in temperature or pH [52]. The biotransformation of bioactive compounds is also an interesting alternative that deserves attention, since it precludes the use of toxic compounds such as organic solvents in the extraction. In these processes, bioactive compounds are obtained from natural sources by microorganisms through their secondary metabolism or by exogenous enzymatic action [53, 54]. According to [55], the bioconversion by enzyme as well as whole cell biocatalyst has tremendous importance in industry owing to escalated yields, low impurity profiles, environmental safety, and process reproducibility. The values found after tannase application, in relation to phenolic compounds and antioxidant activity, were due to the conversion of substances present in integral pitanga juice. These data demonstrate the action of tannase obtained from *S. cerevisiae* CCMB 520 in the biotransformation of this food matrix, suggesting that the enzyme has biotechnological potential in the production of foods with better

This is the first work to report application of tanase in integral pitanga juice. The purpose of the present study was to produce and apply tannase obtained from *S. cerevisiae* CCMB 520. From the results presented, we found that is possible, through enzymatic treatment, to increase the functional quality of integral pitanga juice, once there was an increase in total antioxidant activity, which is associated with an

The results suggest that the partially purified tannase of *Saccharomyces cerevisiae* CCMB 520 can potentially be used for industrial biotechnological application, as in the biotransformation of juices, to obtain a product with greater biological activity (functional property). It is worth mentioning that after the application of partially purified tannase, the juice remained with its physico-chemical characteristics within the Standard of Identity and Quality, according to the current legislation.

We thank Federal Education, Science and Technology Institute of Pernambuco for the granted scholarships, and the National Council for Scientific and Technological Development (Grant n. 469406/2014-3) for the granted scholarships and

compounds, antioxidant activity is increased.

*DOI: http://dx.doi.org/10.5772/intechopen.96103*

nutraceutical properties.

**Acknowledgements**

financial support.

**31**

**Conflict of interest**

The authors declare no conflict of interest.

increase in total phenolic compounds.

**4. Conclusions**

From **Figure 4**, it appears that the time in its linear term was not statistically significant in the experimental field studied.

Wherein: TAA = Total antioxidant activity.

The **Figure 5** illustrates the response surface and contour curves regarding the relationship between application time and tannase concentration. Corroborating with the data obtained for phenolic compounds, it was found that increasing or decreasing the independent variables, together, increases the response variable.

In reference [49] was evaluated samples of aqueous, ethyl acetate and butanolic extracts from pitanga fruits, where the author observed total antioxidant activity in the highest concentration (1000 μg / mL): 35.6, 86.1 and 88.7%, respectively.

Several patent filings have demonstrated the application of tannase in juices with the aim of increasing antioxidant activity. The Indiana patent application 613/KOL/ 2005, in [50], which describes a 37% increase in gallic acid content and an 8% increase in antioxidant activity after tannase application in the pomegranate juice. The Brazilian patent application BR 10 2015 001163–6, in reference [51] describes a total antioxidant activity of 98.20%. The results obtained by other researchers corroborate those presented in the present study. In this work, an increase in antioxidant activity of around 18.15% was possible.

Considering that the antioxidant activity is largely attributed to the presence of phenolic compounds, Pearson's correlation was calculated to verify the existence of a relationship between the two independent variables. The **Figure 6** illustrates a

**Figure 6.** *Pearson's correlation between antioxidant activity and total phenolics.*

*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520 DOI: http://dx.doi.org/10.5772/intechopen.96103*

moderate positive correlation between variables, by increasing phenolic compounds, antioxidant activity is increased.

The use of tannase for the release of phenolic antioxidants has become interesting for various types of food matrix. This is because most of them can release the phenolic compounds without requiring a pre-treatment such as the action of the pectinase or cellulase, or variation in temperature or pH [52]. The biotransformation of bioactive compounds is also an interesting alternative that deserves attention, since it precludes the use of toxic compounds such as organic solvents in the extraction. In these processes, bioactive compounds are obtained from natural sources by microorganisms through their secondary metabolism or by exogenous enzymatic action [53, 54]. According to [55], the bioconversion by enzyme as well as whole cell biocatalyst has tremendous importance in industry owing to escalated yields, low impurity profiles, environmental safety, and process reproducibility.

The values found after tannase application, in relation to phenolic compounds and antioxidant activity, were due to the conversion of substances present in integral pitanga juice. These data demonstrate the action of tannase obtained from *S. cerevisiae* CCMB 520 in the biotransformation of this food matrix, suggesting that the enzyme has biotechnological potential in the production of foods with better nutraceutical properties.

#### **4. Conclusions**

on the application of tannase in integral pitanga juice and its effect on total

The second order equation (Eq. (4)) that describes the experimental data is

TAA %ð Þ¼ <sup>146</sup>*:*<sup>71</sup> � �ð Þ� <sup>16</sup>*:*<sup>29</sup> <sup>22</sup>*:*<sup>79</sup> � EE � �ð Þ� <sup>2</sup>*:*<sup>40</sup> <sup>0</sup>*:*<sup>64</sup> � EE2 � �ð Þ� <sup>0</sup>*:*<sup>15</sup> <sup>0</sup>*:*<sup>10</sup>

From **Figure 4**, it appears that the time in its linear term was not statistically

The **Figure 5** illustrates the response surface and contour curves regarding the relationship between application time and tannase concentration. Corroborating with the data obtained for phenolic compounds, it was found that increasing or decreasing the independent variables, together, increases the response variable. In reference [49] was evaluated samples of aqueous, ethyl acetate and butanolic extracts from pitanga fruits, where the author observed total antioxidant activity in the highest concentration (1000 μg / mL): 35.6, 86.1 and 88.7%, respectively.

Several patent filings have demonstrated the application of tannase in juices with the aim of increasing antioxidant activity. The Indiana patent application 613/KOL/ 2005, in [50], which describes a 37% increase in gallic acid content and an 8% increase in antioxidant activity after tannase application in the pomegranate juice. The Brazilian patent application BR 10 2015 001163–6, in reference [51] describes a total antioxidant activity of 98.20%. The results obtained by other researchers corroborate those presented in the present study. In this work, an increase in

Considering that the antioxidant activity is largely attributed to the presence of phenolic compounds, Pearson's correlation was calculated to verify the existence of a relationship between the two independent variables. The **Figure 6** illustrates a

� *<sup>T</sup>* � �ð Þ� <sup>0</sup>*:*<sup>12</sup> <sup>0</sup>*:*<sup>0020</sup> � *<sup>T</sup>* � �ð Þ� <sup>0</sup>*:*<sup>12</sup> <sup>0</sup>*:*<sup>0020</sup> � *<sup>T</sup>*<sup>2</sup> � �ð Þþ <sup>0</sup>*:*<sup>00028</sup> <sup>0</sup>*:*<sup>15</sup>

(4)

antioxidant activity and phenolic contends.

� EE � *T* � �ð Þ 0*:*0081

significant in the experimental field studied. Wherein: TAA = Total antioxidant activity.

antioxidant activity of around 18.15% was possible.

*Pearson's correlation between antioxidant activity and total phenolics.*

presented:

*Saccharomyces*

**Figure 6.**

**30**

This is the first work to report application of tanase in integral pitanga juice. The purpose of the present study was to produce and apply tannase obtained from *S. cerevisiae* CCMB 520. From the results presented, we found that is possible, through enzymatic treatment, to increase the functional quality of integral pitanga juice, once there was an increase in total antioxidant activity, which is associated with an increase in total phenolic compounds.

The results suggest that the partially purified tannase of *Saccharomyces cerevisiae* CCMB 520 can potentially be used for industrial biotechnological application, as in the biotransformation of juices, to obtain a product with greater biological activity (functional property). It is worth mentioning that after the application of partially purified tannase, the juice remained with its physico-chemical characteristics within the Standard of Identity and Quality, according to the current legislation.

#### **Acknowledgements**

We thank Federal Education, Science and Technology Institute of Pernambuco for the granted scholarships, and the National Council for Scientific and Technological Development (Grant n. 469406/2014-3) for the granted scholarships and financial support.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Gustavo Monteiro<sup>1</sup> , Maria Araújo<sup>2</sup> , Paula Barbosa<sup>3</sup> , Marcelo Mello<sup>1</sup> , Tonny Leite<sup>1</sup> , Sandra Assis<sup>4</sup> and Amanda Sena<sup>1</sup> \*

**References**

1000-2

Janeiro; 2003.

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1 Federal Education, Science and Technology Institute of Pernambuco, Barreiros, Brazil


\*Address all correspondence to: amandareges@barreiros.ifpe.edu.br

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*Biotransformation of Pitanga Juice by Tannase from* Saccharomyces cerevisiae *CCMB 520 DOI: http://dx.doi.org/10.5772/intechopen.96103*
