International Journal of Biotechnology and Bioengineering Research.
ISSN 2231-1238, Volume 4, Number 4 (2013), pp. 299-306
© Research India Publications
http://www.ripublication.com/ ijbbr.htm
Effect of Enzymatic Hydrolysis on the Juice Yield from
Apple Fruit (Malus Domestica) Pulp
Shefali Srivastava1 and Sudhir Kumar Tyagi1
1
M.Tech.Student, Department of Biotechnology, Meerut Institute of Engineering and
Technology, Meerut, Uttar Pradesh, India.
Abstract
Fruit juice companies utilize a variety of different treatments and
enzymes to enhance fruit juice yield. The enzymes which are used are
designed to break down the cell wall within the fruits and release the
liquids and the sugars which make up the fruit content. Pectinases,
amylases and cellulases all break down different structures of the fruit
cells and thus have different activity. During breakdown of the fruit
cells a variety of polysaccharides are found within the juice extract
making the juice hazy, cloudy and thick which reduces its utilization
by customers and affect its market value. Pectinases can break down
these insoluble compounds releasing soluble sugars which clarify the
juice producing a clearer, sweeter liquid. The present paper illustrates
that juice yield and clarity are function of enzymatic hydrolysis
conditions. The process is industrially applicable to improve the yield
and quality of the juice. Thus, it can be said that all the above
mentioned findings can be very beneficial for the development of fruit
juice industries.
Keywords: Enzymes; Hydrolysis; Yield; Clarity.
1. Introduction
Enzymatic juice extraction from apples was introduced 35 years ago and today some 5
million tons of apples are processed into juice annually throughout the world. Where
enzymes are used in commercial production, juice is extracted as follows, after they
have been crushed; apples are usually left for 20–30 minutes so that enzyme inhibitors
in the pulp are oxidized. The pulp is then heated to 30 °C before pectinases are added
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(this compares with a temperature of 50–60 °C which is needed if enzymes are not
used). Typically, 130 mL of enzyme is added for every ton of apples. Enzyme
treatment takes anything from 15 minutes to 2 hours, depending upon the exact nature
of the enzyme, the dosage rate, the reaction temperature and the variety of apple used.
Some varieties, like Golden Delicious, are very difficult to break down. During
incubation, the pectinases degrade soluble pectin in the pulp, making the juice flow
more freely. Next, the apples are pressed. Yields of juice may be increased by up to
20% by enzyme treatment, depending upon the age and variety of apple used and
whether pre-oxidation is employed. Pectinase treatment is particularly effective with
mature apples and those from cold storage. These enzymatic reactions to the pectin
molecule not only increase yield, they clarify the juice. The cloud consists of small
particles made up of pectin/protein complexes suspended in the juice. These create a
haze. Adding enzymes that help break down the pectin changes two things. First, the
viscosity of the juice decreases. Second, the enzymes cause ions to form, resulting in
electrostatic aggregation which increases the size of the particles. The combination of
decreased viscosity and increased particle size causes a floc that settles out, and the
clarified juice can be removed. This project investigates some of the factors that affect
how fast enzymatic reactions occur.
2. Materials & Equipments
2.1 Materials
Fresh apple fruits (Malus domestica) of kinnaur variety, without any visual defects,
were purchased from easy day, govindpuram, ghaziabad (India).The fresh pulp so
prepared was used to extract juice. Enzyme source: Pectinase, e.g., Novozymes
Pectinex®, 1 ml. Dilute with an equal volume of distilled water immediately before
use. OPTIONAL: Cellulase enzyme, e.g., Novozymes Celluclast®, 1 ml. Dilute with an
equal volume of distilled water immediately before use. OPTIONAL (if starchcontaining purée is used): Amylase, e.g., Novozymes Termamyl® or
Amyloglucosidase, AMG, 1 ml. Dilute with an equal volume of distilled water
immediately before use Enzymes.
2.2 Equipment
Sharp knife, Balance for weighing out apple pieces, Paper coffee filters, Plastic wrap,
Disposable plastic spoons for stirring, Two 1 mL syringes or pipets, Two small
funnels, Two 100 mL graduated cylinders, Two 100 mL beaker (or clean glass jars),
Water bath (can be as simple as a styrofoam box or insulated chest, big enough to hold
the beakers), Distilled water, Timer clock, pH meter, Electronic weighing meter,
Calorimeter, Incubator.
Effect of Enzymatic Hydrolysis on the Juice Yield from Apple Fruit
301
3. Procedure and Methods
3.1 Procedure
1. Chop the apples into cubes that are roughly 5 mm on a side. (Use care with the
knife!) It is important to chop the apple into very small pieces—added surface
area helps the enzyme break down the pectin in the plant cell walls, releasing
more juice.
2. Use the balance to weigh equal amounts of chopped apple (about 50 g) into
each beaker.
3. Prepare the pectinase/amylase/cellulase enzyme. Label the beakers
accordingly.
4. Stir the chopped apple pieces in each beaker with a separate plastic spoon. Be
sure to wet all of the pieces. Cover the beakers with plastic wrap.
5. Put beakers into water bath at range of temperature for 2 hrs. The water should
come up to the level of the chopped apples, the beakers float and tip over. After
removing jars from water bath use a wooden spoon to gently stir/squeeze the
apple pieces in each.
6. Use the paper coffee filters in funnels to filter the juice from the apple
preparations into 100 mL graduated cylinders. Again, label the cylinders so you
can keep track of contents.
3.2 Methods
The enzyme activity in fruit juice extraction can be understood with the help of these
points:
1. 1 Preliminary experiment with different concentration of commercial enzymes
2. At different pH
3. At different temperature
4. Relative activity of enzyme on the basis of percentage transmittance/ juice
clarity (table 1)
3.2.1 Preliminary experiment with different concentration of commercial enzymes:
Preliminary experiments were performed for the selection of the ranges and levels for
the concentration of pectinase, amylase, cellulase. The different concentrations of the
pectinase (1-6mg/25g of pulp) were used for the treatment of apple pulp and the juice
yield and its quality in the form of viscosity and clarity were observed. (Table1)
3.2.2 Selection of relevant variables and experimental ranges: The initial step was
the selection of experimental ranges for the independent variables. The experimental
ranges for the independent variables were selected as temperature in the range of 3555°C for 2 hrs at pH-5 and at pH range (1-8) and temperature fixed at 450C.
3.2.3 Clarity: Juice clarity was measured according to the methods of Krop and
Pilnik 1974 and Ough and Crowell 1979. The juice was shaken and 10 ml portion of
juice was centrifuged at 3000 rpm for 10 min to remove pulp coarse cloud particles.
The clarity of the juice obtained was determined by measuring the transmittance at a
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Shefali Srivastava & Sudhir Kumar Tyagi
wavelength of 450 nm using UV- VIS spectrophotometer. Distilled water was used as
a reference. The percent transmittance was considered as a measure of juice clarity.
3.2.4 Viscosity: Clean and dried Ostwald capillary viscometer was used for the
measurement of viscosity. Double distilled water was used as a reference. Time
required to flow through the capillary section of the Oswald viscometer was noted
using a stopwatch for the reference and the sample at 20±2C (Table1)
4. Observations and Results
4.1 Preliminary experiment with different concentration of commercial enzymes:
pectinase
Table 1
Conc. of
Juice viscosity(cps)
clarity
pectinase(mg/25gpulp) yield%
%Transmittance(T)
1
72.3
1.28
20.6
2
74.8
1.20
24.2
3
78.8
1. 13
25.6
4
81.9
1.07
26.8
5
82.5
1.02
27.9
6
83.2
0.8
29.2
7
83.8
0.7
29.8
4.2 Measuring enzyme activity of various enzymes in extraction of Apple juice
Variables Types of enzyme and their combinations (table.2)
Constant Variety: Kinnaur Apple/50gram; Enzyme: Concentration: 10mg/10ml
(D.W.); Incubation Temperature: 40-45 Degree Celsius;Incubation time-2 hrs
Effect of Enzymatic Hydrolysis on the Juice Yield from Apple Fruit
303
Table 2
Enzyme
pectinase
amylase
celllulase
Amylase + Cellulase
Amylase+Pectinase
pectinase+ cellulase
Volume
22
17.2
19.2
22.5
23.7
22.5
Result
Maximum volume obtained by enzyme pectinase and in combination, it is obtained by
pectinase + amylase
4.3 Activity of pectinase at different pH used to observe the yield of fruit juice
from apples
Table 3
Serial No. pH Volume(ml)
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
23.2
24.2
23.5
25.7
27.6
27.4
24
22
Constant
Temperature
45
45
45
45
45
45
45
45
Result
Maximum activity of pectinase at pH 5.5 enhance the yield of apple juice upto 34ml /
50gm
4.4 Activity of cellulase at different pH used to observe the yield of fruit juice
from apples:
Table 4
Serial
No.
1
2
pH Volume(ml)
1
2
21
22
Constant
Temperature0C
45
45
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Shefali Srivastava & Sudhir Kumar Tyagi
3
4
5
6
7
8
3
4
5
6
7
8
23
26
25
23
20
18
45
45
45
45
45
45
Result
Maximum activity of cellulase at pH 4, enhance the yield of apple juice upto 26ml /
50gm
4.5 Activity of amylase at different pH used to observe the yield of fruit juice
from apples:
Table 5
Serial
No.
1
2
3
4
5
6
7
8
pH
Volume(ml)
1
2
3
4
5
6
7
8
20.2
21.2
21.8
22.7
24.2
25.8
25.4
24.5
Constant
Temperature
45
45
45
45
45
45
45
45
4.6 Activity of pectinase at different temperature used to observe the yield of
fruit juice from apple
Table 6
Serial
No.
1
2
3
4
5
6
Temperature0C Volume(ml) Constant
pH
30
19.2
5
35
21.4
5
40
23.5
5
45
25
5
50
24.6
5
55
24.3
5
Effect of Enzymatic Hydrolysis on the Juice Yield from Apple Fruit
305
Result
Maximum activity of pectinase at temperature (45-50 0C), enhance the yield of apple
juice upto 25ml / 50gm
4.7 Activity of cellulase at different temperature used to observe the yield of fruit
juice from apples
Table 7
Serial No. Temperature0C Volume(ml) Constant
pH
1
30
14.2
5
2
35
17.4
5
3
40
19
5
4
45
21.5
5
5
50
22
5
6
55
21.6
5
Result
Maximum activity of cellulase at temperature 50 0C, enhance the yield of apple juice
upto 22ml/50gm
4.8 Activity of amylase at different temperature used to observe the yield of fruit
juice from apples
Table 8
Serial
No.
1
2
3
4
5
6
Temperature Volume(ml) Constant
pH
30
8.9
5
35
10.2
5
40
13.6
5
45
16.3
5
50
17.7
5
55
17.4
5
Result
Maximum activity of cellulose at temperature 50 0C, enhance the yield of apple juice
upto 17ml/50gm.
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Shefali Srivastava & Sudhir Kumar Tyagi
5. Conclusion
The present study concluded that juice yield and clarity are function of enzymatic
hydrolysis conditions. This study also assesses how process variables of enzymatic
treatment of fruit pulp, particularly incubation temperature, incubation time(constant),
pH and pectinase/amylase/cellulase concentration affect the % yield and clarity of the
juice. The enzymatic hydrolysis of pulp improved the juice yield and quality, in terms
of Juice yield, viscosity, clarity (Table1). The process is industrially applicable to
improve the yield and quality of the juice. The results indicate that clarity of the juice
from untreated pulp to enzymatically treated one is increased. The optimum pectinase
concentration obtained was 7mg/25gm pulp. During comparative studies different
results were obtained-for cellulose maximum volume obtained at pH-4 and
temperature 500C for pectinase maximum volume obtained at pH- 5 and
temperature500C, for amylase maximum volume at pH-6 and temperature 500C
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