# Quality Attributes of Cupuaçu Juice in Response to Treatment

Authors: Maria Francisca Simas Teixeira; Jerusa Souza Andrade; Ormezinda Celeste Cristo Fernandes; Nelson Durán; José Luiz de Lima Filho
Journal: Enzyme Research
 (2011)

---

## Abstract

Cupuaçu (Theobroma grandiflorum Schum) is an Amazonian
Basin native fruit whose fruit pulp is consumed as a juice which presents high
density, viscosity, and turbidity. Pectic enzymes, usually yielded by
microorganisms, are used to reduce the juice viscosity and turbidity. The
present study aims to evaluate the use of pectic enzymes when processing
cupuaçu juice. The cupuaçu juice was obtained by using
Aspergillus japonicus 586 crude enzyme extract and
incubation at 50°C with agitation (140 rpm) for one hour. Enzyme
activities were determined, and the juices were evaluated as to their yield,
turbidity, viscosity, and chemical composition. The juice produced by using
crude enzyme extract presented higher soluble solids, reducing sugars, and lower
viscosity and turbidity.


---

## Body

## 1. Introduction

Commercial preparations of pectic enzymes used in food industries are frequently
derived from filamentous fungi [1], mainly
strains of Aspergillus niger [2–4]. Species of this group
include fungi with asexual reproduction (deuteromycete), included in the class
Hyphomycetes, which are characterized by morphological structures typical of the
genus Aspergillus. In the Amazon, Aspergillus
japonicus 586, niger group, are preserved in the
mycology collection (collection of microorganisms) of the Universidade Federal do
Amazonas. They are thermotolerant species, isolated from various natural substrates,
such as wood shavings and parts (peel, pulp and seeds) and kept in a liquid medium
with pH 2–5 [5].In recent decades, the highlight of deuteomycetes is associated with the production
of bioactive compounds, such as enzymes (cellulases, pectinases, and proteases),
which present many advantages for being recognized as safe (GRAS). Highlighted by
the production of biocatalysts, fungal species are used in food industry and in
bioremediation processes, contributing, respectively, to lower the residue rate and
biodegradation need [6, 7].Pectinases have application in the food industry to degrade pectin. These enzymes are
used in the fruit juice extraction process on account of facilitating the
filtration, increasing yield, and reducing the viscosity and turbidity caused by
pectin [8–10]. Among the pectic enzymes, polygalacturonase and
pectinesterase bring about pectin depolymerization and de-esterification,
respectively. Traditionally, crude preparations containing several pectinases are
used by fruit juice and alcoholic beverage industries, although some processes
require only a few of them [6].The Amazon presents high fruit species diversity, some of them are already part of
traditional crops [10], yet most still come
from natural extractivism. Some are known worldwide, but most are only known and
consumed locally. Some fruits whose chemical composition is known arouse great
interest due to the presence of bioactive substances. Other fruits are preferred by
their very attractive flavor [11, 12]. Cupuaçu is a native fruit which is
cultivated in the Amazon [13, 14]. Its pulp has high consumer acceptance and
is used for the preparation of juices, ice creams, candies, cream, jam, dessert, and
liquors. Due to the consumer’s preference within and outside the Amazon, the
cupuaçu is a fruit that stands out as the most promising for industrialization.
Thus, the development of studies can help to encourage industrialization and,
especially, to give value to the Amazonian biodiversity.The pulp of cupuaçu presents a pseudoplastic behavior [15]. Among the traditional juices, the cupuaçu is
preferred by consumers in the Amazon. This juice is thick and viscous and has high
concentrations of colloids and solids particles. These features cause problems for
the industrialization of the juice. The flow of juice inside the tubular system of
industrial equipment is complicated, and the physical stability of the manufactured
product is low.The present study is undertaken so as to couch the pulp industrialization, the
increase on the demand for cupuaçu nectars, and the importance of microbial
enzymes for improving the process and especially the Amazonian biodiversity
valorization. This research aims to evaluate the effect of crude extract of enzymes
produced fromAspergillus japonicus 586 on the cupuaçu juice
physicochemical characteristics.


## 2. Material and Methods

### 2.1. Microorganism

In this study,Aspergillus japonicus 586 was selected randomly
from the collection (Culture Collection DPUA) maintained by the Federal
University of Amazonas (Universidade Federal do Amazonas (UFAM)), located in
Manaus, Amazonas, Brazil. The stock culture was obtained in test tubes (148
× 230 mm) containing Czapek yeast extract agar (CYA). The cultures
were kept for seven days at 30°C, and their viability was monitored every
24 hours [16].


### 2.2. Crude Enzyme Extract

A suspension of5
×
10
6 spores/mL of liquid medium containing
0.5% (w/v) citric pectin was obtained (aseptically) from the stock culture
in CYA. The pectinolytic enzymes were produced in the culture medium containing
(g/L of distilled water) KH2PO4 (2.0),
NH4SO4 (1.0), MgSO4·7H2O
(0.1), Na2HPO4·7H2O (0.9), yeast
extract (1.0), and citric pectin (5.0), and the pH was adjusted to 3.0 with a
dilute sulfuric acid solution. The medium was sterilized during 15 min at
120°C. After autoclaving, a spore suspension plus 5 mL of
polidimetilsilorano (1% w/v) aqueous solution was added to 5 L of
liquid culture medium to give a final concentration of 5
×
10
6 spores/mL. The fermentation was carried
out in Bioflo III fermenter (New Brunswick Scientific, USA) containing
5,000 mL of liquid culture medium using the following conditions:
temperature of 28°C, shaking of 140 rpm, and aeration rate of 0.6
v/v/min (volume of air/volume medium/minute) during 120 hours. The crude enzyme
extract was recovered by vacuum filtration through a 0.45 mm membrane
[5, 17].


### 2.3. Cupuaçu Pulp

Mature fruits were collected from crops cultivated in the vicinity of Manaus,
Amazonas, Brazil. The fruits were washed, and the shell was broken. The pulp was
the separated from the seed by hand with the aid of scissors and immediately
used in the experiments.


### 2.4. Treatments

In the experiment four juice samples (in duplicate) were obtained by varying the
liquid phase (crude enzyme extract or water) and process (incubated and not
incubated). To obtain the juice quantities of 100 g of pulp and
100 mL of water or crude enzyme extract were used. The trituration was
done for three minutes in a blender (high speed). The incubation was conducted
using 300 mL beakers (opened), Dubnoff shaker digital model NT 232, with
shaking of 140 rpm, temperature 50°C for one hour. Enzyme activity
was interrupted by heat treatment (immersion immediately after trituration to
control without incubation and after incubation for the others) for five minutes
in boiling water bath and cool immediately in ice water bath. Filtration (under
manual pressure) was done with gauze tissue (folded eight times) and with
quantitative recovery of juice and residue.


### 2.5. Physicochemical Characteristics

The volume (mL) of juice was measured, and the residue (filtration under
compression) was weighed after drying (constant weight) in an oven at 80°C.
The pH was determined by pH meter (Procyon-10) and acidity (expressed as
percentage of citric acid) by titration with 0.1 M NaOH (phenolphthalein
as indicator). Ascorbic acid was extracted with 0.5% oxalic acid and
measured by titration with 2,6-dichlorophenolindophenol [18]. The sugars were extracted with water and quantified by
the Somogyi-Nelson [19]. Reducing sugars
were determined prior to and total sugars following heated-acid hydrolysis. The
nonreducing sugars were determined by the difference found between total and
reducing sugars. On account of presenting very high absorbance (1.49 to 2.81) it
was necessary to dilute the juice (10 times) with water so as to perform the
turbidity analysis. To evaluate the turbidity, the volume of 5 mL of
juice was diluted into 50 mL of distilled water and the absorbance read
on a Perkin-Elmer spectrophotometer at 660 nm [20]. The findings were expressed as diluted-juice
absorbance units. The viscosity was determined by Ostwald viscometer reading
[21].


### 2.6. Endopolygalacturonase Activity

Endopolygalacturonase activity was measured viscosimetrically by the Tuttobello
and Mill method [21] by using
250 μL of the samples and 5.75 mL of
0.2% (w/v) citric pectin in 0.025 M acetate buffer (pH 5.0)
containing 1 mM ethylenediaminetetraacetic acid (EDTA). The blank was
prepared with 250 μL of buffer solution. The
incubation was conducted at 50°C during 10 and 60 minutes for the crude
enzyme extract and juices, respectively, and then cooled in an ice water bath.
The readings were made on the same Ostwald viscometer. Viscosimetric unit (U)
was defined as the enzyme quantity required to decrease the initial viscosity by
50%. The enzymatic activity was expressed as U/mL/min.


### 2.7. Exopolygalacturonase Activity

Exopolygalacturonase activity was determined using the 3,5-dinitrosalicylic acid
(DNS) reagent [22]. The reactive mixture
contained 250 μL of 0.5% (w/v) citric pectin
in 0.025 M acetate buffer (pH 5.0), 1 mM
ethylenediaminetetraacetic acid (EDTA), and 250 μL
of the crude enzymatic extract or 500 μL of the
juice. The incubation time was 10 (crude enzymatic extract) and 60 (juices)
minutes at 50°C. After incubation, 500 μL of
DNS solution was added to each tube and boiled for 5 minutes. After cooling,
5 mL of distilled water was added. The absorbance at 575 nm was
measured. One enzymatic unit (U) was defined as the enzyme quantity that
releases one μmol of reducing group per minute under the
assay conditions. The enzymatic activity was expressed in nmol/mL/min.


### 2.8. Pectinesterase Activity

Pectinesterase activity was evaluated by the pH decrease of the medium and by
titration of carboxylic groups released according to the modified methods [23, 24]. For the activity analysis the volumes of 2 mL of 1%
(w/v) citric pectin solution in 0.025 M Tris-acetate buffer (pH 6.5) and
1 mL of crude enzyme extract or 3 mL of juice were used. The blank
was prepared in the same way except that the samples were substituted by the
buffer solution. After incubation for 2 hours at 50°C the reaction was
stopped in a boiling water bath for 3 minutes. The samples were cooled in an ice
bath and titrated with 0.1 M NaOH solution. A pectinesterase unit (U) was
defined as the quantity of enzyme which liberates a microequivalent of
carboxylic group in one hour of reaction under the described conditions. The
results were expressed as U/mL/min.


### 2.9. Amylase and Cellulase Activity

To detect the presence of amylase and cellulase a solid medium was used and each
8 mm diameter “cup-plate” was completed with
100μL of the samples. The Petri plate was
protected by aluminum paper enclosing it (so as to prevent evaporation) and
incubated at 37°C for 18 hours. Following incubation and addition of
0.1 N iodine solution (amylases) or 0.1% Congo red (cellulases)
positive reaction was detected by a translucent halo display (measured in cm)
around each “cup-plate.” Enzyme extracts from Aspergillus
awamori (amylase positive) and celluzyme (Novo Nordisk) were used
as controls for amylase and cellulase, respectively [5, 25].


## 2.1. Microorganism

In this study,Aspergillus japonicus 586 was selected randomly
from the collection (Culture Collection DPUA) maintained by the Federal
University of Amazonas (Universidade Federal do Amazonas (UFAM)), located in
Manaus, Amazonas, Brazil. The stock culture was obtained in test tubes (148
× 230 mm) containing Czapek yeast extract agar (CYA). The cultures
were kept for seven days at 30°C, and their viability was monitored every
24 hours [16].


## 2.2. Crude Enzyme Extract

A suspension of5
×
10
6 spores/mL of liquid medium containing
0.5% (w/v) citric pectin was obtained (aseptically) from the stock culture
in CYA. The pectinolytic enzymes were produced in the culture medium containing
(g/L of distilled water) KH2PO4 (2.0),
NH4SO4 (1.0), MgSO4·7H2O
(0.1), Na2HPO4·7H2O (0.9), yeast
extract (1.0), and citric pectin (5.0), and the pH was adjusted to 3.0 with a
dilute sulfuric acid solution. The medium was sterilized during 15 min at
120°C. After autoclaving, a spore suspension plus 5 mL of
polidimetilsilorano (1% w/v) aqueous solution was added to 5 L of
liquid culture medium to give a final concentration of 5
×
10
6 spores/mL. The fermentation was carried
out in Bioflo III fermenter (New Brunswick Scientific, USA) containing
5,000 mL of liquid culture medium using the following conditions:
temperature of 28°C, shaking of 140 rpm, and aeration rate of 0.6
v/v/min (volume of air/volume medium/minute) during 120 hours. The crude enzyme
extract was recovered by vacuum filtration through a 0.45 mm membrane
[5, 17].


## 2.3. Cupuaçu Pulp

Mature fruits were collected from crops cultivated in the vicinity of Manaus,
Amazonas, Brazil. The fruits were washed, and the shell was broken. The pulp was
the separated from the seed by hand with the aid of scissors and immediately
used in the experiments.


## 2.4. Treatments

In the experiment four juice samples (in duplicate) were obtained by varying the
liquid phase (crude enzyme extract or water) and process (incubated and not
incubated). To obtain the juice quantities of 100 g of pulp and
100 mL of water or crude enzyme extract were used. The trituration was
done for three minutes in a blender (high speed). The incubation was conducted
using 300 mL beakers (opened), Dubnoff shaker digital model NT 232, with
shaking of 140 rpm, temperature 50°C for one hour. Enzyme activity
was interrupted by heat treatment (immersion immediately after trituration to
control without incubation and after incubation for the others) for five minutes
in boiling water bath and cool immediately in ice water bath. Filtration (under
manual pressure) was done with gauze tissue (folded eight times) and with
quantitative recovery of juice and residue.


## 2.5. Physicochemical Characteristics

The volume (mL) of juice was measured, and the residue (filtration under
compression) was weighed after drying (constant weight) in an oven at 80°C.
The pH was determined by pH meter (Procyon-10) and acidity (expressed as
percentage of citric acid) by titration with 0.1 M NaOH (phenolphthalein
as indicator). Ascorbic acid was extracted with 0.5% oxalic acid and
measured by titration with 2,6-dichlorophenolindophenol [18]. The sugars were extracted with water and quantified by
the Somogyi-Nelson [19]. Reducing sugars
were determined prior to and total sugars following heated-acid hydrolysis. The
nonreducing sugars were determined by the difference found between total and
reducing sugars. On account of presenting very high absorbance (1.49 to 2.81) it
was necessary to dilute the juice (10 times) with water so as to perform the
turbidity analysis. To evaluate the turbidity, the volume of 5 mL of
juice was diluted into 50 mL of distilled water and the absorbance read
on a Perkin-Elmer spectrophotometer at 660 nm [20]. The findings were expressed as diluted-juice
absorbance units. The viscosity was determined by Ostwald viscometer reading
[21].


## 2.6. Endopolygalacturonase Activity

Endopolygalacturonase activity was measured viscosimetrically by the Tuttobello
and Mill method [21] by using
250 μL of the samples and 5.75 mL of
0.2% (w/v) citric pectin in 0.025 M acetate buffer (pH 5.0)
containing 1 mM ethylenediaminetetraacetic acid (EDTA). The blank was
prepared with 250 μL of buffer solution. The
incubation was conducted at 50°C during 10 and 60 minutes for the crude
enzyme extract and juices, respectively, and then cooled in an ice water bath.
The readings were made on the same Ostwald viscometer. Viscosimetric unit (U)
was defined as the enzyme quantity required to decrease the initial viscosity by
50%. The enzymatic activity was expressed as U/mL/min.


## 2.7. Exopolygalacturonase Activity

Exopolygalacturonase activity was determined using the 3,5-dinitrosalicylic acid
(DNS) reagent [22]. The reactive mixture
contained 250 μL of 0.5% (w/v) citric pectin
in 0.025 M acetate buffer (pH 5.0), 1 mM
ethylenediaminetetraacetic acid (EDTA), and 250 μL
of the crude enzymatic extract or 500 μL of the
juice. The incubation time was 10 (crude enzymatic extract) and 60 (juices)
minutes at 50°C. After incubation, 500 μL of
DNS solution was added to each tube and boiled for 5 minutes. After cooling,
5 mL of distilled water was added. The absorbance at 575 nm was
measured. One enzymatic unit (U) was defined as the enzyme quantity that
releases one μmol of reducing group per minute under the
assay conditions. The enzymatic activity was expressed in nmol/mL/min.


## 2.8. Pectinesterase Activity

Pectinesterase activity was evaluated by the pH decrease of the medium and by
titration of carboxylic groups released according to the modified methods [23, 24]. For the activity analysis the volumes of 2 mL of 1%
(w/v) citric pectin solution in 0.025 M Tris-acetate buffer (pH 6.5) and
1 mL of crude enzyme extract or 3 mL of juice were used. The blank
was prepared in the same way except that the samples were substituted by the
buffer solution. After incubation for 2 hours at 50°C the reaction was
stopped in a boiling water bath for 3 minutes. The samples were cooled in an ice
bath and titrated with 0.1 M NaOH solution. A pectinesterase unit (U) was
defined as the quantity of enzyme which liberates a microequivalent of
carboxylic group in one hour of reaction under the described conditions. The
results were expressed as U/mL/min.


## 2.9. Amylase and Cellulase Activity

To detect the presence of amylase and cellulase a solid medium was used and each
8 mm diameter “cup-plate” was completed with
100μL of the samples. The Petri plate was
protected by aluminum paper enclosing it (so as to prevent evaporation) and
incubated at 37°C for 18 hours. Following incubation and addition of
0.1 N iodine solution (amylases) or 0.1% Congo red (cellulases)
positive reaction was detected by a translucent halo display (measured in cm)
around each “cup-plate.” Enzyme extracts from Aspergillus
awamori (amylase positive) and celluzyme (Novo Nordisk) were used
as controls for amylase and cellulase, respectively [5, 25].


## 3. Results and Discussion

### 3.1. Enzymatic Activities

Differences in the activities of pectinases were detected in the crude enzyme
extract produced byAspergillus japonicus 586, as well as in
the cupuaçu juices (Figure 1). The
pectic enzymes (pectinesterase and polygalacturonases) were detected in crude
extract from Aspergillus japonicus 586, and their activities
were affected by singled or combined carbon source different concentrations
[5]. Exopolygalacturonase activity was
also detected in the juice obtained without the addition of crude enzyme
extract. The incubation decreased the activity of exopolygalacturonase. On the
other hand, incubation during one hour at a temperature of 50°C increased
the activity of pectinesterase, which showed higher values, especially in the
juice from the crude enzyme extract. The activity of pectinesterase detected in
juices was higher compared to crude enzyme extract.Figure 1
Pectinase activities in the crude enzyme extract produced byAspergillus japonicus 586 and juices of
cupuaçu (Theobroma grandiflorum Schum).Positive reaction (90 mm halo) to the cellulase was detected in the crude
enzyme extract fromAspergillus japonicus 586. Amylase was
neither in the juice nor in crude enzyme extract. Qualitative detection of
cellulase may also indicate the possibility of reducing agroindustrial residue
and thus minimizing the negative effect on the environment, when being used in
large quantities needed by major agroindustries.Commercial products of pectic enzymes, containing pectinesterase,
polygalacturonase, and pectin lyase, have been used in wine and juice industries
to increase the yield and improve pigment, flavor, transmittance, and viscosity
quality [6]. On account of there being no
commercial enzyme available at the time the present experiment was carried out
we were obliged to use the traditional method of making cupuaçu juice by
using water.


### 3.2. Yield, Viscosity, and Turbidity

The major aim of this study is to evaluate the use of enzymes extracted from a
fungus isolated in Brazilian Amazon for processing the juice from a native plant
consumed by local people. In this research, the beneficial effect of crude
enzyme extract fromAspergillus japonicus 586 is indicated by
the results shown in Figure 2. In
addition to increasing the juice yield, the amount of waste to be discharged was
diminished. As the agroindustrial waste is polluting, the smallest volume to be
discharged into the environment implies a lesser need for bioremediation
processes. With the exception of juice extracted with water and subjected to
incubation, the others showed high yield in the range from 94 to 100%. Pulp
yield increase and waste reduction were also achieved with the use of commercial
pectinase when obtaining cupuaçu pulp [26].Figure 2
Effect of crude enzyme extract produced byAspergillus
japonicus 586 on the yield and characteristics of juices of
cupuaçu (Theobroma grandiflorum Schum).Produced with the ratio of 1 : 1 (pulp : liquid) and
subjected to filtration (with manual pressing), the juice still presented high
turbidity and had to be diluted so as turbidity could be assessed. The
incubation of the juice obtained with water caused an increase in turbidity and
a remarkable decrease in viscosity. However, during incubation the enzymes
present in crude enzyme extract caused a significant reduction in the turbidity
especially in the viscosity in the cupuaçu juice. The results also show
that the incubation process was essential to enzyme activity since optimum
temperature and time lapse are essential for the activity of any enzyme.The results shown in Figure2 indicated
that the incubation process is essential for the action of enzymes, reducing
viscosity and lowering turbidity and, therefore, altering the characteristics of
the final product. This is the most interesting result, confirming the
usefulness of this technique for the quality control analysis and improving the
industrial process.


### 3.3. Chemical Composition

The effect of the enzymes alone or in combination with incubation is shown by the
data presented in Table1. Taking just
incubation into account the juice obtained with the crude enzyme extract shown
to be sweeter. This characteristic is indicated by the lowest values of pH and
Brix/acidity ratio and the highest acidity. Moreover, in degradation of pectin
by the action of pectinesterase, there occurs the release of methanol and
H+. Thus, this ion also contributes to the decrease in pH [6].Table 1
Effect of crude enzyme extract produced byAspergillus
japonicus 586 on the chemical composition of juices
cupuaçu (Theobroma grandiflorum Schum).
Juice characteristics
Not incubated
Incubated
Juice (water)
Juice (enzyme extract)
Juice (water)
Juice (enzyme extract)
pH
3.0
2.9
3.0
2.9
Titratable acidity (%)
0.92
±
0.02
1.04
±
0.02
0.70
±
0.01
0.92
±
0.01
Ascorbic acid (mg%)
13.87
±
0.32
14.11
±
0.31
5.44
±
0.20
8.1
±
0.02
Soluble solids (°Brix)
6.6
±
0.13
6.6
±
0.13
6.8
±
0.13
7.1
±
0.14
Brix/acidity ratio
7.17
±
0.01
6.35
±
0.09
9.71
±
0.20
7.72
±
0.33
Reducing sugars (%)
0.49
±
0.03
0.58
±
0.03
0.61
±
0.01
1.09
±
0.08
Nonreducing sugars (%)
1.83
±
0.01
1.73
±
0.01
1.34
±
0.22
1.05
±
0.13However, incubation for one hour on recipient (beaker) open and under shaking
(140 rpm) at 50°C resulted in loss of volatile acids, with more
intensity in the juice obtained with water (3.67 mg/min) than in the one
with enzyme extract (2.0 mg/min). The incubation also caused oxidation of
ascorbic acid, and as this vitamin is more stable in acidic medium [18], the loss in the juices (more acidic)
obtained with crude enzyme extract was lower. The conversion of sugars by
hydrolysis is also favored by heating and acidity [19]. The occurrence of such conversion during incubation is
indicated by the increase and decrease of reducing and nonreducing sugars,
respectively.The methanol production was not evaluated. However, information about
toxicological aspects is necessary since this handmade method could be replaced
by industrial process. The methanol production is positively associated with
enzyme activities of pectinesterase and pectate lyase. Several studies have
found that methanol can be produced from the hydrolysis of methyl ester groups
in pectins by endogenous pectinesterases and occurs naturally at a low level in
fresh squeezed juices, stored and canned fruit juices, and most of the alcoholic
beverages. The addition of pectic enzymes can cause an increase in methanol
level during mashing, fermentation, and aging stages fruit wines and distilled
fruit spirits [27–29].As a consequence, this process probably intensifies the release of methanol and
increases the risk for consumers. Methanol is toxic to human through ingestion
and inhalation. Accidental intake of the compound will result in severe
intoxication due to accumulation of toxic metabolites. In the body, methanol is
metabolized in the liver and converted first to formaldehyde and then to
formate. Acute intoxication of methanol usually causes headache, vertigo,
fatigue, nausea, vomiting, blurred vision, blindness, and even death [30–32].The presence and amount of methanol were not determined in this study. The
incubation was conducted for one hour at 50°C in open beaker with stirring.
Later, the juices were subjected to heat treatment for five minutes in boiling
water, also in open beakers. Methanol is a volatile compound (the boiling point
is 65°C), and its absence in the controlled cashew apples wine may be due
to its loss by evaporation during pasteurization [33]. To avoid potential health hazard, the analysis of this
methanol content in the fresh juice or in the processed beverage is desirable
because the equipments for industrial process are closed. It is important to
note that the methanol is an undesirable product in juice processing and is a
common problem in the industrial beverages production.In the Amazon, cupuaçu pulp is frozen and stored under freezing. At the time
of consumption, this pulp is added with water and sugar to get the juice.
Currently this handmade process of obtaining juice tends to be replaced by the
industrial process [31, 32]. Thus, the use of pectic enzymes
produced by microorganisms, and especially selected in the Amazon, is a
suggestion to improve the process and also obtain the desirable characteristics
[9, 33] of the processed product. The results shown in Figure 2 indicated that the incubation process is
essential for the action of enzymes, reducing viscosity and lowering turbidity
and agroindustrial residues, therefore altering the characteristics of the final
product. This is the most interesting result, confirming the usefulness of this
technique for the quality control analysis and to improve the industrial
process.


## 3.1. Enzymatic Activities

Differences in the activities of pectinases were detected in the crude enzyme
extract produced byAspergillus japonicus 586, as well as in
the cupuaçu juices (Figure 1). The
pectic enzymes (pectinesterase and polygalacturonases) were detected in crude
extract from Aspergillus japonicus 586, and their activities
were affected by singled or combined carbon source different concentrations
[5]. Exopolygalacturonase activity was
also detected in the juice obtained without the addition of crude enzyme
extract. The incubation decreased the activity of exopolygalacturonase. On the
other hand, incubation during one hour at a temperature of 50°C increased
the activity of pectinesterase, which showed higher values, especially in the
juice from the crude enzyme extract. The activity of pectinesterase detected in
juices was higher compared to crude enzyme extract.Figure 1
Pectinase activities in the crude enzyme extract produced byAspergillus japonicus 586 and juices of
cupuaçu (Theobroma grandiflorum Schum).Positive reaction (90 mm halo) to the cellulase was detected in the crude
enzyme extract fromAspergillus japonicus 586. Amylase was
neither in the juice nor in crude enzyme extract. Qualitative detection of
cellulase may also indicate the possibility of reducing agroindustrial residue
and thus minimizing the negative effect on the environment, when being used in
large quantities needed by major agroindustries.Commercial products of pectic enzymes, containing pectinesterase,
polygalacturonase, and pectin lyase, have been used in wine and juice industries
to increase the yield and improve pigment, flavor, transmittance, and viscosity
quality [6]. On account of there being no
commercial enzyme available at the time the present experiment was carried out
we were obliged to use the traditional method of making cupuaçu juice by
using water.


## 3.2. Yield, Viscosity, and Turbidity

The major aim of this study is to evaluate the use of enzymes extracted from a
fungus isolated in Brazilian Amazon for processing the juice from a native plant
consumed by local people. In this research, the beneficial effect of crude
enzyme extract fromAspergillus japonicus 586 is indicated by
the results shown in Figure 2. In
addition to increasing the juice yield, the amount of waste to be discharged was
diminished. As the agroindustrial waste is polluting, the smallest volume to be
discharged into the environment implies a lesser need for bioremediation
processes. With the exception of juice extracted with water and subjected to
incubation, the others showed high yield in the range from 94 to 100%. Pulp
yield increase and waste reduction were also achieved with the use of commercial
pectinase when obtaining cupuaçu pulp [26].Figure 2
Effect of crude enzyme extract produced byAspergillus
japonicus 586 on the yield and characteristics of juices of
cupuaçu (Theobroma grandiflorum Schum).Produced with the ratio of 1 : 1 (pulp : liquid) and
subjected to filtration (with manual pressing), the juice still presented high
turbidity and had to be diluted so as turbidity could be assessed. The
incubation of the juice obtained with water caused an increase in turbidity and
a remarkable decrease in viscosity. However, during incubation the enzymes
present in crude enzyme extract caused a significant reduction in the turbidity
especially in the viscosity in the cupuaçu juice. The results also show
that the incubation process was essential to enzyme activity since optimum
temperature and time lapse are essential for the activity of any enzyme.The results shown in Figure2 indicated
that the incubation process is essential for the action of enzymes, reducing
viscosity and lowering turbidity and, therefore, altering the characteristics of
the final product. This is the most interesting result, confirming the
usefulness of this technique for the quality control analysis and improving the
industrial process.


## 3.3. Chemical Composition

The effect of the enzymes alone or in combination with incubation is shown by the
data presented in Table1. Taking just
incubation into account the juice obtained with the crude enzyme extract shown
to be sweeter. This characteristic is indicated by the lowest values of pH and
Brix/acidity ratio and the highest acidity. Moreover, in degradation of pectin
by the action of pectinesterase, there occurs the release of methanol and
H+. Thus, this ion also contributes to the decrease in pH [6].Table 1
Effect of crude enzyme extract produced byAspergillus
japonicus 586 on the chemical composition of juices
cupuaçu (Theobroma grandiflorum Schum).
Juice characteristics
Not incubated
Incubated
Juice (water)
Juice (enzyme extract)
Juice (water)
Juice (enzyme extract)
pH
3.0
2.9
3.0
2.9
Titratable acidity (%)
0.92
±
0.02
1.04
±
0.02
0.70
±
0.01
0.92
±
0.01
Ascorbic acid (mg%)
13.87
±
0.32
14.11
±
0.31
5.44
±
0.20
8.1
±
0.02
Soluble solids (°Brix)
6.6
±
0.13
6.6
±
0.13
6.8
±
0.13
7.1
±
0.14
Brix/acidity ratio
7.17
±
0.01
6.35
±
0.09
9.71
±
0.20
7.72
±
0.33
Reducing sugars (%)
0.49
±
0.03
0.58
±
0.03
0.61
±
0.01
1.09
±
0.08
Nonreducing sugars (%)
1.83
±
0.01
1.73
±
0.01
1.34
±
0.22
1.05
±
0.13However, incubation for one hour on recipient (beaker) open and under shaking
(140 rpm) at 50°C resulted in loss of volatile acids, with more
intensity in the juice obtained with water (3.67 mg/min) than in the one
with enzyme extract (2.0 mg/min). The incubation also caused oxidation of
ascorbic acid, and as this vitamin is more stable in acidic medium [18], the loss in the juices (more acidic)
obtained with crude enzyme extract was lower. The conversion of sugars by
hydrolysis is also favored by heating and acidity [19]. The occurrence of such conversion during incubation is
indicated by the increase and decrease of reducing and nonreducing sugars,
respectively.The methanol production was not evaluated. However, information about
toxicological aspects is necessary since this handmade method could be replaced
by industrial process. The methanol production is positively associated with
enzyme activities of pectinesterase and pectate lyase. Several studies have
found that methanol can be produced from the hydrolysis of methyl ester groups
in pectins by endogenous pectinesterases and occurs naturally at a low level in
fresh squeezed juices, stored and canned fruit juices, and most of the alcoholic
beverages. The addition of pectic enzymes can cause an increase in methanol
level during mashing, fermentation, and aging stages fruit wines and distilled
fruit spirits [27–29].As a consequence, this process probably intensifies the release of methanol and
increases the risk for consumers. Methanol is toxic to human through ingestion
and inhalation. Accidental intake of the compound will result in severe
intoxication due to accumulation of toxic metabolites. In the body, methanol is
metabolized in the liver and converted first to formaldehyde and then to
formate. Acute intoxication of methanol usually causes headache, vertigo,
fatigue, nausea, vomiting, blurred vision, blindness, and even death [30–32].The presence and amount of methanol were not determined in this study. The
incubation was conducted for one hour at 50°C in open beaker with stirring.
Later, the juices were subjected to heat treatment for five minutes in boiling
water, also in open beakers. Methanol is a volatile compound (the boiling point
is 65°C), and its absence in the controlled cashew apples wine may be due
to its loss by evaporation during pasteurization [33]. To avoid potential health hazard, the analysis of this
methanol content in the fresh juice or in the processed beverage is desirable
because the equipments for industrial process are closed. It is important to
note that the methanol is an undesirable product in juice processing and is a
common problem in the industrial beverages production.In the Amazon, cupuaçu pulp is frozen and stored under freezing. At the time
of consumption, this pulp is added with water and sugar to get the juice.
Currently this handmade process of obtaining juice tends to be replaced by the
industrial process [31, 32]. Thus, the use of pectic enzymes
produced by microorganisms, and especially selected in the Amazon, is a
suggestion to improve the process and also obtain the desirable characteristics
[9, 33] of the processed product. The results shown in Figure 2 indicated that the incubation process is
essential for the action of enzymes, reducing viscosity and lowering turbidity
and agroindustrial residues, therefore altering the characteristics of the final
product. This is the most interesting result, confirming the usefulness of this
technique for the quality control analysis and to improve the industrial
process.


## 4. Conclusion

The reductions in viscosity, turbidity, and the amount of waste are the major
positive contribution of the use of crude enzymatic extract yielded byAspergillus japonicus586 for the production of juice of
cupuaçu. Also, it stands out as a positive contribution to efforts to encourage
future research in the Amazon, concerning the isolation and selection of
microorganisms, and specially investigating the use of these processing enzymes in
sound and acceptable products from this and other fruits, which make up the
Amazonian biodiversity. The joint efforts, consequently, will contribute to
sustainable development and extractivism Amazonian biodiversity.



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*Source: 494813-2011-11-01.xml*