**Abstract**

The pitanga (*Eugenia uniflora* L.) is a native species to Brazil and widely used by Brazilian industry, mainly in food, to juice, ice cream, soft drinks, jellies and liqueurs production. The fruit contains a high concentration of anthocyanins, flavonoids and carotenoids, which make it a promising source of antioxidant compounds. The objective of this work was to produce and purify tannase from *Saccharomyces cerevisiae* CCMB 520, to apply in the integral pitanga juice and to verify its physical and chemical effects. The tannase was produced under submerged fermentation in bench bioreactor. After the fermentation process the enzyme was partially purified. The partially purified tannase was applied in the integral pitanga juice using Doehlert statistical design. The effect of the enzymatic application was analyzed by means of phenolic compounds contents and antioxidant activity. Physical–chemical analyzes were carried out to investigate the Standard Identity and Quality of the juice. The best results for partial purification were obtained by ultrafiltration. After application, the total phenolics content was 4855 mg Eq. AG/L, and for the antioxidant activity was 952 μMTrolox/L (69.41%). It has been found that it is possible by means of enzymatic treatment to improve the functional quality of the integral pitanga juice.

**Keywords:** antioxidant activity, bioconversion, *Eugenia uniflora* L., experimental design, tannin acyl hydrolase

#### **1. Introduction**

Tannin is a term widely used to characterize the second largest class of phenolic compounds, which, like the others, has the primordial and essential function of protecting plant tissues against attack by insects, fungi or bacteria. Tannins have a high molecular weight (500 to 3000 Da), are considered antioxidants and combine with cellulose and pectin, in addition to precipitating alkaloids and proteins [1]. These compounds occur naturally in a wide variety of vegetables, and can be found in the roots, leaves, fruits, seeds and barks. They are considered secondary metabolic products of great economic and ecological interest and have a wide value in the interactions between the plant and its ecosystem. Such compounds are responsible for the astringency of many fruits and vegetable products, due to the precipitation of salivary glycoproteins, which causes the loss of lubricating power [2, 3].

Classically, according to the chemical structure, tannins are classified into two groups: hydrolyzable and condensed. The current and most accepted classification divides the tannins into four groups (**Figure 1**): gallotannins, ellagitannins, condensed tannins and complex tannins [5]. Gallotannins are the simplest tannins and are formed by units of gallo or di-gallo esterified to a nucleus of glucose or other polyhydroxy alcohol. The molecules are usually composed of a glucose nucleus and 6 to 9 gallo groups. The most common is tannic acid [6]. Ellagitannins are esters of hexahydro-xidifenic acid (HHDP), and during its hydrolysis, the HHDP group dehydrates and spontaneously lactonizes to form ellagic acid. Condensed tannins are oligomeric and polymeric proanthocyanidins containing flavan-3-ol (catechin) or flavan-3,4-diol (leucoanthocyanins). The basic structure of complex tannins, on the other hand, consists of a unit of galotannin or ellagitannin and catechin [7, 8].

The application of tannase in juices rich in hydrolyzable tannins is done to decrease the concentration of these in this food matrix, since the high content of this compound is responsible for the appearance of turbidity, bitter taste and astringency, characteristics which are often undesirable. However, the hydrolysis of gallotannins causes nutritional and sensory changes in the juice, since with the release of the gallo group occurs a retarding effect on the oxidation of ascorbic acid,

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

The pitanga (*Eugenia uniflora* L.), belonging to the Mirtaceae family, is native to Brazil, specifically in the South and Southeast regions, and has adapted favorably to the edaphoclimatic conditions of the Brazilian Northeast, mainly in the State of Pernambuco, with about 300 hectares cultivated [26]. It is widely used by the Brazilian industry for the production of juice, preparation of ice cream, soft drinks, jellies and liquors because it has a high economic potential, attracting the consumer for its high concentration of metabolites such as anthocyanins, flavonols and carotenoids, which make this fruit a promising source antioxidant compounds [26, 27]. The natural antioxidants present in the diet increase the resistance to damage caused by oxidation, thus presenting a significant impact on human health [27]. Based on this information, the tannase obtained from *Saccharomyces cerevesiae* CCMB 520 was applied in this study with purpose of biotransforming the integral pitanga juice polyphenols and, in this way, modifying their biological activity.

Tannic acid, gallic acid, bovine serum albumin and rodhanine were purchased at Sigma Aldrich (Sigma Chemical Co., St. Louis, MO, USA). All other chemicals used

The yeast species *Saccharomyces cerevisiae* CCMB 520 was kindly provided by the Culture Microorganisms Collection of Bahia (*Coleção de Cultura de Micro-organismos da Bahia* - CCMB), of State University of Feira de Santana, Bahia State, Brazil. The sample was kept on plates containing Yeast Malt (YM) and left to rest in YM medium, at pH 6.8, in order to be activated; subsequently, it was incubated in

A 48-hours culture grown in YM medium (Merck, Darmstadt, Alemanha) was used to prepare the inoculum at pH 6.8 and 28 °C in B.O.D incubator (Cienlab, Campinas, Brazil). After the 48-hours period, culture fragments were inoculated in 0.85% saline solution to generate a suspension presenting optical density OD600nm:

Enzyme production was performed in 7.5 L Bioreactor containing 2.5 L of submerged fermentation medium - Czapek-Dox broth (g/L) base: NaNO3 (7.5), KCl (1.25), MgSO4.7H2O (1.25), FeSO4.7H2O (0.025), K2HPO4 3H2O (2.5), yeast extract (25) and tannic acid (150); media were sterilized at 121 °C for 15 minutes. Tannic

**2.4 Enzyme production and extracellular tannase obtainment**

in the experiment were of high-quality analytical grade.

**2.2 Microorganism and its maintenance**

also increasing its antioxidant action [24, 25].

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

**2. Material and methods**

B.O.D at 28 ° C for 48 hours.

**2.3 Inoculum preparation**

0.8 at 0.9.

**19**

**2.1 Reagents**

Hydrolyzable tannins can be easily hydrolyzed, either chemically or enzymatically. Tannin Acyl Hydrolase (TAH), also known as tannase (EC 3.1.1.20), is an enzyme capable of hydrolyzing tannins, leading to the release of glucose and gallic acid or ellagic acid [9]. Some are still able to perform a transesterification reaction for the production of propyl gallate [10].

TAH is a glycoprotein esterase formed predominantly by a gallic acid esterase and a depsidase. Tannase can be separated into two esterases, a specific esterase for aliphatic esters such as methyl gallate, and another depsidase that hydrolyzes depsidic bonds like m-digallic acid as shown [11]. However, the proportion between the two activities can vary according to the cultivation conditions [12]. Tannase is a biocatalyst produced by vegetables, animals, bacteria, filamentous fungi and yeast. Tannins of yeast are effective only in the decomposition of gallotannin, while bacterial and filamentous fungi are efficient in the hydrolysis of gallotannins and ellagitannins [13].

Tannase is versatile since it can be widely used in the food, pharmaceutical and chemical industries, and even in bioremediation [14]. Among the possible applications we can mention: preparation of instant teas [15], additive for animal feed [16, 17], production of gallic and ellagic acid [18, 19], synthesis of esters and effluent treatment [9, 20], beverage manufacturing (juices, beers and wines) [21] and clarification of juices [22, 23].

**Figure 1.** *Main chemical structures of the tannins [4].*

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

The application of tannase in juices rich in hydrolyzable tannins is done to decrease the concentration of these in this food matrix, since the high content of this compound is responsible for the appearance of turbidity, bitter taste and astringency, characteristics which are often undesirable. However, the hydrolysis of gallotannins causes nutritional and sensory changes in the juice, since with the release of the gallo group occurs a retarding effect on the oxidation of ascorbic acid, also increasing its antioxidant action [24, 25].

The pitanga (*Eugenia uniflora* L.), belonging to the Mirtaceae family, is native to Brazil, specifically in the South and Southeast regions, and has adapted favorably to the edaphoclimatic conditions of the Brazilian Northeast, mainly in the State of Pernambuco, with about 300 hectares cultivated [26]. It is widely used by the Brazilian industry for the production of juice, preparation of ice cream, soft drinks, jellies and liquors because it has a high economic potential, attracting the consumer for its high concentration of metabolites such as anthocyanins, flavonols and carotenoids, which make this fruit a promising source antioxidant compounds [26, 27]. The natural antioxidants present in the diet increase the resistance to damage caused by oxidation, thus presenting a significant impact on human health [27].

Based on this information, the tannase obtained from *Saccharomyces cerevesiae* CCMB 520 was applied in this study with purpose of biotransforming the integral pitanga juice polyphenols and, in this way, modifying their biological activity.

## **2. Material and methods**

#### **2.1 Reagents**

Classically, according to the chemical structure, tannins are classified into two groups: hydrolyzable and condensed. The current and most accepted classification divides the tannins into four groups (**Figure 1**): gallotannins, ellagitannins, condensed tannins and complex tannins [5]. Gallotannins are the simplest tannins and are formed by units of gallo or di-gallo esterified to a nucleus of glucose or other polyhydroxy alcohol. The molecules are usually composed of a glucose nucleus and 6 to 9 gallo groups. The most common is tannic acid [6]. Ellagitannins are esters of hexahydro-xidifenic acid (HHDP), and during its hydrolysis, the HHDP group dehydrates and spontaneously lactonizes to form ellagic acid. Condensed tannins are oligomeric and polymeric proanthocyanidins containing flavan-3-ol (catechin) or flavan-3,4-diol (leucoanthocyanins). The basic structure of complex tannins, on the other hand, consists of a unit of galotannin or ellagitannin and catechin [7, 8]. Hydrolyzable tannins can be easily hydrolyzed, either chemically or enzymatically. Tannin Acyl Hydrolase (TAH), also known as tannase (EC 3.1.1.20), is an enzyme capable of hydrolyzing tannins, leading to the release of glucose and gallic acid or ellagic acid [9]. Some are still able to perform a transesterification reaction

TAH is a glycoprotein esterase formed predominantly by a gallic acid esterase and a depsidase. Tannase can be separated into two esterases, a specific esterase for aliphatic esters such as methyl gallate, and another depsidase that hydrolyzes depsidic bonds like m-digallic acid as shown [11]. However, the proportion between the two activities can vary according to the cultivation conditions [12]. Tannase is a biocatalyst produced by vegetables, animals, bacteria, filamentous fungi and yeast. Tannins of yeast are effective only in the decomposition of gallotannin, while bacterial and filamentous fungi are efficient in the hydrolysis of gallotannins and

Tannase is versatile since it can be widely used in the food, pharmaceutical and chemical industries, and even in bioremediation [14]. Among the possible applications we can mention: preparation of instant teas [15], additive for animal feed [16, 17], production of gallic and ellagic acid [18, 19], synthesis of esters and effluent treatment [9, 20], beverage manufacturing (juices, beers and wines) [21]

for the production of propyl gallate [10].

ellagitannins [13].

*Saccharomyces*

**Figure 1.**

**18**

and clarification of juices [22, 23].

*Main chemical structures of the tannins [4].*

Tannic acid, gallic acid, bovine serum albumin and rodhanine were purchased at Sigma Aldrich (Sigma Chemical Co., St. Louis, MO, USA). All other chemicals used in the experiment were of high-quality analytical grade.

#### **2.2 Microorganism and its maintenance**

The yeast species *Saccharomyces cerevisiae* CCMB 520 was kindly provided by the Culture Microorganisms Collection of Bahia (*Coleção de Cultura de Micro-organismos da Bahia* - CCMB), of State University of Feira de Santana, Bahia State, Brazil. The sample was kept on plates containing Yeast Malt (YM) and left to rest in YM medium, at pH 6.8, in order to be activated; subsequently, it was incubated in B.O.D at 28 ° C for 48 hours.

#### **2.3 Inoculum preparation**

A 48-hours culture grown in YM medium (Merck, Darmstadt, Alemanha) was used to prepare the inoculum at pH 6.8 and 28 °C in B.O.D incubator (Cienlab, Campinas, Brazil). After the 48-hours period, culture fragments were inoculated in 0.85% saline solution to generate a suspension presenting optical density OD600nm: 0.8 at 0.9.

#### **2.4 Enzyme production and extracellular tannase obtainment**

Enzyme production was performed in 7.5 L Bioreactor containing 2.5 L of submerged fermentation medium - Czapek-Dox broth (g/L) base: NaNO3 (7.5), KCl (1.25), MgSO4.7H2O (1.25), FeSO4.7H2O (0.025), K2HPO4 3H2O (2.5), yeast extract (25) and tannic acid (150); media were sterilized at 121 °C for 15 minutes. Tannic

acid (sterilized through membrane 0.45 μm) and inocolum were added to the fermentation medium after the Bioreactor cooled down to room temperature. The initial pH, fermentation time, rotation and incubation temperature, of the fermentation process, were 7, 24 h, 112 rpm and 27 °C, respectively. The fermentation broth was centrifuged (Thermoelectron, Langenser, Germany) at 1000 rpm for 15 minutes at 4 °C. The supernatant was frozen at 20 °C and used for further tests. this period, the reaction medium was centrifuged at 10,000 rpm for 20 minutes at 4 °C. The precipitate was ressuspended in 0.04 M sodium citrate buffer, pH 5.0, in the same volume of crude extract added during the precipitation process. Soon afterwards, enzyme activity and total protein tests were performed as previously described. After partial purification, tannase was used in the bioconversion of

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

The pitanga fruits (*Eugenia uniflora* L., 2000 g) were harvested in the orchard that is located near the Federal Institute of Education, Science and Technology of Pernambuco, Campus Barreiros, Brazil. They were collected between March and April, selected and cleaned in chlorinated water at 50 ppm for 15 minutes. Then were carried out, rinsing, pulp removal and crushing in an industrial blender. The integral pitanga juice was sifted and stored under freezing for further studies on the

The statistical Doehlert [32] using two variables – partially purified tannase concentration (%, v/v) and application time (minutes) - was herein applied to investigate the best condition for antioxidant capacity increase. The enzyme extract concentration was assessed at three levels (4.5, 6.0 and 7.5%), whereas the application time was assessed at five levels (160, 180, 200, 220 and 240 minutes), which

For each percentage of partially purified tannase, a control was performed,

System behavior was explained through the following quadratic equation

β11, β22, β<sup>33</sup> = quadratic coefficients, β12, β13, β<sup>23</sup> = interaction coefficients, A, B,

**Experiment Partially purified tannase (%, v/v) Application time (minutes)**

*Doehlert matrix (real and coded) used to optimize tannase application in the bioconvertion of integral Pitanga*

 7.5 (0.866) 180 (�0.5) 7.5 (0.866) 220 (0.5) 6.0 (0) 160 (�1.0) 6.0 (0) 200 (0) 6.0 (0) 200 (0) 6.0 (0) 200 (0) 6.0 (0) 240 (1.0) 4.5 (� 0.866) 180 (�0.5) 4.5 (� 0.866) 220 (0.5)

<sup>Y</sup> <sup>¼</sup> *<sup>β</sup>*<sup>0</sup> <sup>þ</sup> *<sup>β</sup>*1A <sup>þ</sup> *<sup>β</sup>*2B <sup>þ</sup> *<sup>β</sup>*3C <sup>þ</sup> *<sup>β</sup>*11*A*<sup>2</sup> <sup>þ</sup> *<sup>β</sup>*22*B*<sup>2</sup> <sup>þ</sup> *<sup>β</sup>*33*C*<sup>2</sup> <sup>þ</sup> *<sup>β</sup>*12AB <sup>þ</sup> *<sup>β</sup>*13AC <sup>þ</sup> *<sup>β</sup>*23BC <sup>þ</sup> *<sup>ε</sup>*

Wherein: Y = experimental response, β<sup>0</sup> intercept, β1, β2, β<sup>3</sup> = linear coefficients,

(1)

are presented in their actual values and codified in **Table 1**.

C = independent variables, and ε = experimental error.

integral pitanga juice.

application of the enzyme.

**2.8 Enzimatic biotrasformation**

exchanging it for distilled water.

(Eq. (1)):

**Table 1.**

*juice.*

**21**

**2.7 Preparation of integral pitanga juice**

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

### **2.5 Enzyme activity and protein content**

Tannase activity was estimated by using ethanolic rhodanine and tannic acid as substrate [28]. The reaction medium consisted of 250 μL substrate (0.05%, w/v) in 0.05 mol/L citrate buffer (pH 5.0) and of 250 μL enzyme extract. The substrate and the enzyme extract remained in contact for 5 minutes at 30 °C. Enzyme reaction was stopped through the addition of 300 μL ethanolic rhodanine (0.667%, w/v). After spending 5 minutes at 30 °C, the reaction medium was added with 200 μL of 0.5 mol/L potassium hydroxide in order to form a chromogen violet staining. After five more minutes at 30 °C, the obtained volume of each reaction was diluted in 4 mL of distilled water. The control tubes (enzyme extract addition at the end of the reaction) were simultaneously used. After the samples were subjected to 10 more minutes at 30 °C, the experiment proceeded in spectrophotometer (Novainstruments, Piracicaba, Brazil) at 520 nm and molar extinction coefficient was 648.15 L/mol cm. Tannase activity (U/mL) was expressed by the amount of enzyme required to produce 1 μmol of gallic acid per minute under assay conditions. Protein content was set according to the Bradford method [29]. Bovine serum albumin was used as standard. All tests were performed in triplicate and the mean values (different from <5%) were calculated.

#### **2.6 Partial purification by different methods**

#### *2.6.1 Ammonium sulphate precipitation*

The crude enzyme extract was fractioned by ammonium sulphate precipitation at percentage saturation ranged of 0–20, 20–40, 40–60, 60–80% (w/v), respectively [30]. At each saturation, the solution was left to stand for 2 hours. The sample was dialyzed against distilled water for 4 hours at 4 °C and the precipitate was collected by centrifugation (5000 rpm for 20 minutes at 4 °C). The precipitates were solubilized in 0.04 mol L<sup>1</sup> sodium citrate buffer (pH 5) and subjected to analysis of enzyme activity and total protein as previously described.

#### *2.6.2 Ultrafiltration membrane (30 KDa)*

The crude culture filtrate (10 mL) was added to the membrane and subsequently centrifuged at 4000 rpm for 60 minutes at 4 °C, and then the retained and permeated material were collected. The volumes obtained were separately reconstituted to the initial volume (10 mL). Soon afterwards, enzyme activity and total protein tests were performed as previously described.

#### *2.6.3 Ethanol precipitation*

The fractional precipitation followed the methodology from [31] with modifications. The solvent was cooled to a temperature of 0 °C and then added dropwise to the crude extract until you reach the desired concentrations of the same (50 to 90%, v/v). The mixture remained in contact for 1 hour at a temperature of 18 °C. After

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

this period, the reaction medium was centrifuged at 10,000 rpm for 20 minutes at 4 °C. The precipitate was ressuspended in 0.04 M sodium citrate buffer, pH 5.0, in the same volume of crude extract added during the precipitation process. Soon afterwards, enzyme activity and total protein tests were performed as previously described. After partial purification, tannase was used in the bioconversion of integral pitanga juice.

#### **2.7 Preparation of integral pitanga juice**

acid (sterilized through membrane 0.45 μm) and inocolum were added to the fermentation medium after the Bioreactor cooled down to room temperature. The initial pH, fermentation time, rotation and incubation temperature, of the fermentation process, were 7, 24 h, 112 rpm and 27 °C, respectively. The fermentation broth was centrifuged (Thermoelectron, Langenser, Germany) at 1000 rpm for 15 minutes at 4 °C. The supernatant was frozen at 20 °C and used for further tests.

Tannase activity was estimated by using ethanolic rhodanine and tannic acid as substrate [28]. The reaction medium consisted of 250 μL substrate (0.05%, w/v) in 0.05 mol/L citrate buffer (pH 5.0) and of 250 μL enzyme extract. The substrate and the enzyme extract remained in contact for 5 minutes at 30 °C. Enzyme reaction was stopped through the addition of 300 μL ethanolic rhodanine (0.667%, w/v). After spending 5 minutes at 30 °C, the reaction medium was added with 200 μL of 0.5 mol/L potassium hydroxide in order to form a chromogen violet staining. After five more minutes at 30 °C, the obtained volume of each reaction was diluted in 4 mL of distilled water. The control tubes (enzyme extract addition at the end of the reaction) were simultaneously used. After the samples were subjected to 10 more

minutes at 30 °C, the experiment proceeded in spectrophotometer

analysis of enzyme activity and total protein as previously described.

(Novainstruments, Piracicaba, Brazil) at 520 nm and molar extinction coefficient was 648.15 L/mol cm. Tannase activity (U/mL) was expressed by the amount of enzyme required to produce 1 μmol of gallic acid per minute under assay conditions. Protein content was set according to the Bradford method [29]. Bovine serum albumin was used as standard. All tests were performed in triplicate and the mean

The crude enzyme extract was fractioned by ammonium sulphate precipitation at percentage saturation ranged of 0–20, 20–40, 40–60, 60–80% (w/v), respectively [30]. At each saturation, the solution was left to stand for 2 hours. The sample was dialyzed against distilled water for 4 hours at 4 °C and the precipitate was collected by centrifugation (5000 rpm for 20 minutes at 4 °C). The precipitates were solubilized in 0.04 mol L<sup>1</sup> sodium citrate buffer (pH 5) and subjected to

The crude culture filtrate (10 mL) was added to the membrane and subsequently centrifuged at 4000 rpm for 60 minutes at 4 °C, and then the retained and permeated material were collected. The volumes obtained were separately reconstituted to the initial volume (10 mL). Soon afterwards, enzyme activity and total protein tests

The fractional precipitation followed the methodology from [31] with modifications. The solvent was cooled to a temperature of 0 °C and then added dropwise to the crude extract until you reach the desired concentrations of the same (50 to 90%, v/v). The mixture remained in contact for 1 hour at a temperature of 18 °C. After

**2.5 Enzyme activity and protein content**

*Saccharomyces*

values (different from <5%) were calculated.

**2.6 Partial purification by different methods**

*2.6.1 Ammonium sulphate precipitation*

*2.6.2 Ultrafiltration membrane (30 KDa)*

were performed as previously described.

*2.6.3 Ethanol precipitation*

**20**

The pitanga fruits (*Eugenia uniflora* L., 2000 g) were harvested in the orchard that is located near the Federal Institute of Education, Science and Technology of Pernambuco, Campus Barreiros, Brazil. They were collected between March and April, selected and cleaned in chlorinated water at 50 ppm for 15 minutes. Then were carried out, rinsing, pulp removal and crushing in an industrial blender. The integral pitanga juice was sifted and stored under freezing for further studies on the application of the enzyme.

#### **2.8 Enzimatic biotrasformation**

The statistical Doehlert [32] using two variables – partially purified tannase concentration (%, v/v) and application time (minutes) - was herein applied to investigate the best condition for antioxidant capacity increase. The enzyme extract concentration was assessed at three levels (4.5, 6.0 and 7.5%), whereas the application time was assessed at five levels (160, 180, 200, 220 and 240 minutes), which are presented in their actual values and codified in **Table 1**.

For each percentage of partially purified tannase, a control was performed, exchanging it for distilled water.

System behavior was explained through the following quadratic equation (Eq. (1)):

$$\mathbf{Y} = \beta\_0 + \beta\_1 \mathbf{A} + \beta\_2 \mathbf{B} + \beta\_3 \mathbf{C} + \beta\_{11} \mathbf{A}^2 + \beta\_{22} \mathbf{B}^2 + \beta\_{33} \mathbf{C}^2 + \beta\_{12} \mathbf{A} \mathbf{B} + \beta\_{13} \mathbf{A} \mathbf{C} + \beta\_{23} \mathbf{B} \mathbf{C} + \varepsilon \tag{1}$$

Wherein: Y = experimental response, β<sup>0</sup> intercept, β1, β2, β<sup>3</sup> = linear coefficients, β11, β22, β<sup>33</sup> = quadratic coefficients, β12, β13, β<sup>23</sup> = interaction coefficients, A, B, C = independent variables, and ε = experimental error.


#### **Table 1.**

*Doehlert matrix (real and coded) used to optimize tannase application in the bioconvertion of integral Pitanga juice.*

Each 10 mL of pitanga juice in Erlenmeyer flasks was added partially purified tannase at the proportions cited in **Table 1** and incubated in a shaker at 120 � 1 rpm at 30 °C, optimal temperature of the tannase from *Saccharomyces cerevisiae* CCMB 520 [33]. After the enzymatic application was done, according to the preestablished time, the enzyme was denatured at 70 °C, for 10 minutes.

**2.12 Statistical analysis**

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

performed in random order.

**3. Results and discussion**

**3.1 Partial purification**

The results were analyzed in the SISVAR software - Variance Analysis System [37] and the means were compared through the Scott-Knott test at 5% probability level. In addition, the results were assessed through Analysis of Variance (ANOVA) in the Statistica Version 10.0 software (StatSoft, Inc., Tulsa, USA) [38] to find the variables presenting statistically significant effects on enzyme application (p < 0.05), as well as the model fitting the experimental data. All assays were

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

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

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 denatur-

ation through a conformational change in the enzyme tertiary structure.

**Stage VA (U/mL) TP (mg/mL) SA (U/mg) PF** Crude extract 3.17 0.60 5.23 1.00 Retained (30 KDa) 21.080 0.67 31.66 6.040 a Permeate (30 KDa) 19.56 0.67 29.010 5.54 a Ammonium sulphate (0–20%) — — —— Ammonium sulphate (20–40%) 2.41 0.54 4.46 0.85 Ammonium sulphate (40–60%) 1.10 0.80 1.30 0.24 Ammonium sulphate (60–80%) — — —— Ethanol (50%) — — —— Ethanol (60%) — — —— Ethanol (70%) — — —— Ethanol (80%) 0.19 0.28 0.66 0.085 Ethanol (90%) 0.27 0.37 0.72 0.093 *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*

enzymes, under the experimental conditions evaluated.

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

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

**Table 2.**

**23**

## **2.9 Physico-chemical analysis of the pitanga juice**

The physical–chemical evaluation is necessary since bioconversion cannot influence the loss of quality with respect to the pre-established minimum standards for the Standard of Identity and Quality of a specific product, in this case the integral pitanga juice.

#### *2.9.1 pH*

The pH was determined directly in the same with the aid of a previously calibrated pHmeter, after filtration [34].

### *2.9.2 Total soluble solids (°Brix)*

Total Soluble Solids (°Brix) was determined by a Reichert digital refractometer by dropping two drops of the sample onto the surface of the properly calibrated apparatus.
