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Effect of enzymatic hydrolysis of pineapple fruit pulp on yield and analytical

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EFFECT OF ENZYMATIC HYDROLYSIS OF PINEAPPLE FRUIT PULP ON YIELD AND

ANALYTICAL PARAMETERS OF DERIVED JUICE
M. G. AZIZ2*, M. A. R. MAZUMDER2, M. H. ALI3, M. B. UDDIN2 and K. D. KULBE1
1
Division of Food Biotechnology, 2Department of Food Science and Technology BOKU, University of Natural
Resources and Applied Life Sciences, Vienna Muthgasse 20, Austria, 2Department of Food Technology and Rural
Industries, Bangladesh Agricultural University, Mymensingh, Bangladesh and Classis Food and Dairy Products Ltd.,
Mymensingh, Bangladesh. * Corresponding author.
ABSTRACT
This study is concerned with the hydrolysis of pineapple fruit pulp in the laboratory of Food Biotechnology,
Austria and Food Technology & Rural Industries, Bangladesh Agricultural University in 2008. The principal
aim of the study was to assess the hydrolysing effect of commercial pectinase (pectinase NS3000) and
cellulase (cellubrix) enzymes preparations on juice yield and composition. Enzyme dosages were optimized by
orthoginal test at constant temperature and incubation time in a laboratory incubator. Both pectinolytic and
cellulolytic enzyme treatment of pineapple pulp showed significant improvement of juice yield. Individual
effects yielded only 6% more juice whereas combined enzyme preparations yielded about 15 % more juice over
control. Besides juice yield, enzyme treatment was found advantageous in increasing total soluble solids and
clarification of juice. Enzymatic treatment for quality juice production might be an important tool for
liquefaction of fruits and vegetable, which are difficult to process into juice by mechanical means.

Keywords: Pectinase, Cellulase, Liquefaction, Polysaccharide components, Alcohol insoluble solids.

INTRODUCTION

Pineapple (Ananas cosmosus) is extensively seasonal fruit in Bangladesh. It ranks fourth among the most
cultivated fruit in the country. The fruit can be consumed fresh or processed in various forms. The most popular
processing products from pineapple in the world market are canned pineapples. But today pineapple juice is a
standard product in most supermarkets. Among the wide range of fruit juices available in the market, the
popularity of cloudy juices from tropical fruits is increasing both in Europe and in Asia. The essential problem in
all cloudy beverages is cloud loss (Baker and Cameron, 1999). In case of pineapple juice, cloud is known to be
unstable due to low hydrocolloid content (Wallrauch, 1992; Will et al., 1994) with higher amount of coarse cloud
compared to fine cloud (Will et al., 1999). Technological possibilities for the improvement of cloud stability of
pineapple juices are the addition of hydrocolloids and homogenization (Carle, 1998).

Nowadays enzymes are used to ensure optimal juice yield and quality (Pilnik and Voragen, 1991). The pulp of
fruits and vegetables is mainly composed of cells, which are surrounded by a cell wall (Roland, 1980). Cell wall
consists of a middle lamella, a primary wall and a secondary wall (Keegstra et al., 1973). These cell walls need
to be broken down to make juice recovery easier and these cell walls are the main substrate of pectinases and
cellulases. Application of exogenous enzymes leads to the degradation of fruit cell walls or the selective
extraction of some of their components, allowing the creation of new types of finished products and fruit
derivates (Grassin and Fauquembergue, 1996; Acar, 1999). At the same time, hydrolysis of the cell wall
constituents increases soluble components which offer a number of advantages in producing juice, such as high
yield, better colour, phenolic components and cloud stability (Buchert et al., 2005). Enzyme preparations for
extraction belong to the group of pectinases, hemicellulases and cellulases and are used during pulp maceration,
liquefaction or juice depectinization stages. Enzyme preparations for maceration enable the disintegration of pulp
tissues and consequently cell separation through the breakdown of the highly esterified middle lamella pectin.
Under the influence of cellulases a disintegration of the cell wall can finally occurr. Macerating enzyme
preparations like Pectinex Ultra SP-L and Cellubrix (Novozymes) are used to macerate the pineapple plant cell
wall and consequently increased juice yields (Janser, 1997). However, at present little information is available on
detailed characterization of these two enzyme preparations. These two preparations were developed for
maceration and liquefaction of fruits. So the effectiveness of these two enzymes in hydrolyzing pineapple fruit
pulp in improving juice quality needs to be investigated. The specific objectives of this study was: i)
characterization of pineapple fruit pulp polysaccharides; ii) characterization of commercial enzymes used in the
study; ii) optimization of enzyme dosae to assess juice yield and analytical parameters

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MATERIALS AND METHODS

This study was conducted in the laboratory of Food Biotechnology, Austria and Food Technology & Rural
Industries, Bangladesh Agricultural University in 2008. The fresh pineapples of Smooth Cayenne [Ananas
comosus (L) Merr.] variety produced in Costa Rica were bought from a market in Austria. Cellulolytic enzyme
preparation (Cellubrix) and pectinolytic enzyme preparations (Pectinase Ultra SP and Pectinase NS 3000) kindly
supplied by Novozyme, Switzerland were used for the enzymatic treatment of pineapple fruit pulp. All chemicals
were supplied by Sigma Aldrich, Austria.

Preparation of pineapple juice: After rinsing the fruit in tap water, the shell and core were removed using a
stainless steel knife and the flesh was cut into small pieces. Fruit slices were blended into puree by using kitchen
blender. The pulp was pasteurized at 850C temperature for 10 minute and stored under freeze conditions. It was
brought to room temperature and used for the experiments. Different dosages of cellulolytic (1.0, 1.5 and 2.0
g/kg) and pectinolytic (0.025, 0.03 and 0.035 g/kg) enzyme preparations were added into the pineapple purees
and incubated at 3180K for 60 minutes in a shaker at 160 rpm. Then they were heated at 3580K for 5 min to
inactivate the enzyme activities, cooled down immediately and filtered through 55mm filter paper under vacuum.

Preparation and hydrolysis of cell wall materials: The alcohol insoluble solids (AIS) was obtained after
washing the fruit pulp with 95% alcohol three times and a final wash with acetone. Sequential extraction of AIS
was carried out according to the method of Frügel et al. (2003). Little modification was done only in
centrifugation and pectic fraction isolation. Pectic a wes isolated by 50 mM NaOH, hemicellulose fraction by 4M
NaOH and residual pellet was cellulose fraction. At each step, samples were diafiltrated with 10 kD membrane
centrifuge tube at 2000 rpm. The retentate was washed with water two times and the supernatant volume was
made 50ml and then freeze-dried. The cell wall fractions were hydrolysed using sulphuric acid according to the
method of Frügel et al. (2003).

Assay of commercial enzyme preparations: The xylanase, cellulase and pectinase activities of the enzymatic
preparations were measured by assaying the xylose, glucose and galacturonic acid liberated from xylan, CMC
and polygalacturonic acid, respectively as described by Qin et al. (2005). The liberated sugars were assayed by
reaction with dinitrosalicylic acid (DNS) reagent (Miller, 1959). Endopolygalacturonase (Bailey and Pessa,
1990), pectin lyase (Manachini et al., 1988), β-glucosidase, β-xylosidase activities were measured using
polyglacturonic acid, apple pectin, X-Glu (5-bromo-4.4 chloro-3-indolyl-β-D-glucopyranoside) and X-Xylo (5-
bromo-4.4 chloro-3-indolyl-β-D-xylucopyranoside) respectively. Pectin methylesterase (Suutarinen et al., 2002)
was assayed by titration of the liberated carboxyl groups of apple pectin using an automatic titrator. The effect of
temperature on the activities of the enzyme preparation was studied at different temperatures (30-500C). The
effect of pH (3.6-5.0) on enzyme activity was also studied.

Yield and some analytical compositions: Juice yield was defined as the percentage of mass of pineapple juice
to the pineapple puree. The pH was determined on all juice samples using pH meter. 0Brix was determined using
a digital Pocket Refractometer, Pal-3. Rapid sediment of cloud was determined by following the method
described by Mensah-Wilson et al. (2000). Colour determination of pineapple juice was done by Hunter colour
meter. Three Hunter parameters, namely “L” (Lightness), “a” (redness and green) and “b” (yellowness and
blueness) were measured. Hue was expressed as tan-(b/a), while chroma was expressed as (a2+b2)1/2
(Rattanathanalerk et al., 2005). Data represent averages calculated from triplicate experiments.

Determination of anhydrous galacturonic acid (AGA) and neutral sugars: The AGA and other sugars
components were separated with a Dionex DX-500 Bio-LC system, using a Carbopac PA10 column (250mm ×
4mm) in combination with a Carbopac guard column (25mm × 4 mm) (Dionex Corp., Sunnyvale, CA). All
analyses were carried out at a temperature of 300C and a flow rate of 1ml/min. The neutral monosaccharides
were eluted isocratically using 15mm NaOH for 35 min. The AGA were eluted using a gradient reaching 170mM
sodium acetate and 100mM NaOH. The column was washed with 100mm NaOH for 15 min and re-equilibrated
with 15mm NaOH for 20 min before the next injection.

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RESULTS AND DISCUSSION

Polysaccharide compositions of pineapple pulp and juice

Alcohol insoluble solids (AIS) of the pineapple fruit mash was prepared and fractionated into pectin,
hemicellulose and cellulose (Table 1). The amount of AIS obtained from 100g pulp was 2.88 g of which pectin,
hemicellulose and cellulose contributed 19.2 %, 10.9 % and 20.10 % respectively (Table 2). The recovery of
fraction is only 50 %. This might be explained by the loss during extraction and purification steps. Pectic
polysaccharides (Rhamnose, arabinose, galactose and anhydrous galacturonic acid) form most of the cell wall
polysaccharides. The predominant sugar in the pectin fraction was anhydrous galacturonic acid, and the main
neutral sugar was galactose. The hemicellulose fraction was rich in galactose, glucose, arabinose and mannose.
Considerable amounts of anhydrous galacturonic acid and xylose was also present. Hemicellulose polymers were
mainly composed of glucuroarabinoxylans (GAXs) and, in small amounts, of xyloglucans (Smith and Harris,
1995; Femenia et al., 2007. The presence of glucoronic acid, arabinose and xylose in hemicellulose fraction
revealed the presence of GAXs. As expected, cellulose fraction consisted mainly of glucose, about 67% of total
sugar. Arabinose and galactose typical of hemicellulose were also present in the cellulose fraction, indicating
partial retention during sequential extraction.
Table 1. AIS from fruit pulp and juice.
Material Amount isolated Amount isolated
Pulp AIS 2880 mg/100g 1440 mg
Juice AIS 1446 mg/L 1200 mg

Table 2. Sequential extraction of pulp AIS.

Samples Amount taken (mg) Extracted (mg) (%) recovery % basis on recovery
AIS 200 - -
Pectin - 21.8 10.9 21.73
Hemicellulose - 38.4 19.2 38.24
Cellulose - 40.2 20.10 40.03
Total 200 100.4 50.2

There was no arabinose in pectic fractions whereas hemicellulose and cellulose fraction contained significant
amount of arabinose, however cellulose fraction contained no mannose. Galactose was found the predominant
neutral sugar components in the hemicellulose fraction, followed by glucose and arabinose. Glucose was the
most abundant neutral monosaccharide of the pineapple flesh cell wall, followed by xylose and then followed by
arabinose and galactose as reported by Smith and Harris (1995). In trifluoroacetic acid hydrolysates, they found
xylose was the most abundant neutral monosacharide, followed by arabinose and galactose.

Table 3. Acid hydrolysis of extracted components (Relative mass %).

Mono sugar components Pectic fraction Hemicellulose fraction Cellulose fraction
Arabinose - - 13.03
Xylose 2.74 12.48 6.79
Mannose 2.93 9.96 -
Galactose 42.56 52.68 12.66
Glucose 9.22 15.66 67.52
Galacturonic acid 42.56 9.22 -

From the results obtained by characterisation of the neutral sugar composition of pectins, hemicellulose and
cellulose fractions it became evident that AIR of pineapple mash contained less pectic material but higher
hemicellulose and cellulose materials. Therefore, during juice extraction by maceration or liquefaction of
pineapple mash, the enzyme preparations with high hemicellulose and cellulose activity are recommended.

Activity profile of enzyme preparations

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The hydrolytic activities of two kinds of three commercial enzyme preparations were investigated with respect to
temperature and pH. The temperature and pH dependence were checked only over polygalacturonase activity
(Fig.1).During enzymatic maceration, temperature and enzyme concentration were varied keeping the mash mass
constant. From fig. 1, it is clear that polygalacturonase activity was the highest between 40 0C and 50 0C. Ceci
and Lozano (1998) reported 50 0C was the well-defined breaking point where enzymes rapidly loss their activity.

1400 Cellubrix Pectinase Ultra SP Pectinase NS

Volumetric activity (µmol/min/ml

1200
1000
800
600
400
200
0
29 34 39 44 49 54
Temperature (C)
Fig. 1. Effect of temperature on polygalacturonase activityof three commercial enzyme prepations at pH 5
Polygalacturonase activities of the three different commercial enzymes are highly pH dependent as shown in
table 4. At pH 3.6, all of the enzymes showed about four times less activity than at pH 5.0. As the pH of
pineapple mash is around 3.5 the activity profile of the enzyme preparation was investigated extensively at pH
3.6 (Table 5). At pH 3.6 and room temperature, the activity profile of the enzyme preparations is very low.
Cellubrix practically showed no activity at this condition. Pectinase NS3000 contained higher activities than the
other enzymes. Enzyme preparations Pectinase NS3000 used showed good stability at pH 3.6. It is clearly
unexpected that Cellubrix had no cellulase activity at pH 3.6 and room temperature whereas pectinase NS3000
had good cellulase activity at this operating condition. Pectinase Ultra SP showed low polygalacturonase activity
at these conditions but both Pectinase Ultra SP and Pectinase NS had the same xylanase activities. No β-
glucosidase and β-xylosidase activity was found at this pH and temperature. Buchert et al. (2005) reported that
Pectinase Ultra SP contained no cellulase and glucosidase active at pH 3.5. When the incubation temperature
increased to 45 0C (as used for pineapple mash treatment), the activity of all enzyme preparations increased
significantly (Table 4).
Table 4. Polygalacturonase activity of enzyme preparations at 450C.

Protein content Volumatric activity Specific activity

Enzymes U/mL U/mg
mg/mL
pH 3.6 pH 5 pH 3.6 pH 5
Cellulase 116.17
2.7 9.5 0.023 0.081

Pectinase Ultra SP 67.25 261 1140 3.88 16.95

Pectinase NS3000 87.15 298 1266 3.42 14.53

Table 5. Activity profile of enzyme preparations at pH 3.6 and room temperature (200C).

Enzyme CE PME PG PL XYL β-GLU β -XYL

preparation Nkat/mL Nkat/mL Nkat/mL Nkat/mL Nkat/mL nkat/mL nkat/mL
Cellubrix 123 0 0 0 16 0 0
Pectinase
0 5 8 0 15 0 0
Ultra SP
Pectinase
53 22 44 0 15 0 0
NS3000
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Effect of enzyme treatment on juice yield
Both pectinolytic and cellulolytic enzyme treatment of pineapple pulp either indiviually or in combined gave
significant improvement of juice yield vs control (Table 6). From individual effects, it is shown that cellulloytic
enzyme preparation yielded 10 % more juice over control whereas pectinolytic enzyme preparations yielded only
6 % more juice. So it is evident that cellulolytic enzyme preparation is very important for pineapple juice
production. This is due to higher hemicellulose and cellulose content of pineapple cell wall materials. To
compare the effect of different pectinase enzyme preparations, pineapple mash was incubated with the three
different pectinases (Table 7).
Table 6. Effect of individual enzyme preparations.

Enzyme Yield (g/Kg) pH TSS Abs at 660 nm Abs at 420 nm RS

Control 552 3.85 12.0 1.11 2.0 0.0%
Pectinolytic (0.035g/Kg) 585 3.96 12.3 0.913 1.55 2.0%
Cellulolytic (2.0g/Kg) 600 4.03 12.5 1.0 1.85 6.5%

Table 7. Effect of different pectinase enzyme preparations.

*Pectinolytic enzyme Yield pH TSS Abs at l a b c Hue
Preparation (g/Kg) (0Brix) 660nm
Control 555 3.44 11.85 1.12 32.0 -2.3 2.2 3.2 -43.73
Pactinase NS3000 584 3.40 12.00 1.10 31.4 -2.1 1.1 2.4 -27.65
Pectinase Ultra SP 577 3.41 11.89 1.00 31.0 -2.1 1.4 2.5 -33.69
Pectinase from Sigma 565 3.44 11.80 0.98 31.0 -2.1 1.2 2.4 -29.74

* Enzyme dose used 0.035 g/Kg, incubation time 1 h and temperature 45 0C and l, a, b and c are Hunter colour
parameter

The highest juice yield was obtained by Pectinase NS3000. Besides pectinase activity, pectinase NS3000 had
high hemicellulase and cellulase activity (Table 5). Hence, the ability of pectinase NS 3000 to liquefy pineapple
pulps was higher, which was effective for higher juice yield and total soluble solid. The dosages of the combined
enzymatic preparations were determined by an orthogonal test and the results are presented in table 8. About
22% more juice (over control) could be obtained by combined cellulase and pectinase activities whereas this was
only 6-10% by the effect of individual enzyme preparations.

Table 8. The orthogonal test of the enzyme hydrolysis

Sl Pectolytic Cellulytic Yield pH TSS Absorbance Absorbance Rapid

No enzyme dosage (g enzyme (g kg- (0Bx) at 660 nm at 420 nm Sediment
. kg-1) Dosage 1
) (% of total
(gkg-1) height)
1 0.025 1.00 497.5 3.54 11.75 0.382 2.643 2.0
2 0.025 1.50 520.0 3.53 12.10 0.338 2.601 2.5
3 0.025 2.00 500.0 3.49 11.90 0.314 2.574 2.5

4 0.030 1.00 517.5 3.52 12.40 0.224 2.505 ND

5 0.030 1.50 535.0 3.67 12.50 0.174 2.456 ND
6 0.030 2.00 530.0 3.56 12.40 0.209 2.481 ND

7 0.035 1.00 543.5 3.60 13.10 0.175 2.430 2.5

8 0.035 1.50 545.0 3.57 13.15 0.240 2.487 3.5
9 0.035 2.00 574.0 3.59 13.20 0.215 2.589 3.0

ND= not determined

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Due to the presence of higher hemicellulose and cellulose content of pineapple cell wall materials, the synergistic
effect of different enzyme blends with higher hemicellulase and cellulase activities are very important for hydrolysis
of pineapple pulp. The cell wall of pineapple fruit has a middle lamella and a primary wall. The middle lamella
acts as an intercellular sticking substance and is mainly composed of pectin. Pectin of the middle lamella is the
main substrate of pectolytic enzyme preparation (Anastasakis et al., 1987; John et al., 2003). The primary cell
wall is a strong network composed of bundles of crystalline cellulose with embedded lignin. This amorphous
matrix is floating in an aqueous gel composed of different fractions of hemicelluloses and pectin. This makes the
primary cell wall probably more susceptible to cellulolytic enzyme degradation. The progressive degradation of
cellulose fibrils led to loss of wall strength and its breakdown into fragments making juice recovery easier (John
et al., 2003; Grassin and Fauquembergue, 1996). In the manufacture of pineapple juice, pulping makes high
viscous puree due to the presence of a natural gum which is responsible for increasing suspension pulp,
decreasing filtration capabilities and the foaming properties of the juice. This gum is a neutral polysaccharide
containing 70% sugars, mainly galactomannans (mannose and galactose ratio is 2:1) Chen chin and Yamamoto,
(1978). To facilitate separation, the pulps are treated with enzyme preparations that are able to degrade the
polysaccharide gel formed. When the cellulloytic or the pectinolytic enzyme preparations were used individually,
they had lower pectinase or cellulase activity and could not degrade pectin or cellulose effectively. By combining
pectin-degrading enzymes with cellulose-degrading enzymes, the synergistic effects of the two enzyme
preparations were more effective, almost completely degrading the gum and cell wall thus yielded higher juice.

The use of commercial enzymes showed the improvement of other quality parameters like total soluble solids.
With increasing enzyme dosage, total soluble solid (TSS) increased significantly. This effect was more easily
realized with increasing cellulase preparation, since enzymes solubilized polysaccharides of cell wall and
degraded the solubilized material into smaller fragments which subsequently contributed to the increase of TSS
of derived juice. The use of cell-wall degrading enzymes proved to be advantageous in extracting cellular
contents with high yield of soluble solids and a lower viscosity (Kilara, 1982; Sreenath and Nanjundaswamy,
1987 and Sreenath and Santhanam, 1992), which was also evident in our investigation by increasing total soluble
solids.

Colour of enzyme treated juice

The Hunter parameters are used to describe the visual colour change of fruit juice (Avila and Silva, 1999;
Rattanathakalerk et al., 2005). The L value represents the light-dark spectrum, a value is for the green-red
spectrum and b value represents the blue-yellow spectrum (Ranganna, 1986). Since the major color of cloudy
pineapple juice is yellow, the amount of this pigment in pineapple flesh is an excellent measure of quality
(Mehrlich and Felton, 1980). So the change in b value was used to describe the color destruction in the juice.
Water soluble colour (absorbance at 420 nm) was also assessed. The change in b value and water soluble colour
were not significantly different with respect to enzyme dosage (Table 7). The b value for control was 2.2 but it
was always around 1.6 for enzyme treated juice (Table 7). So a slight colour loss was observed due to enzyme
action. This b value was much lower than that of fresh juice or concentrate pineapple juice (b about 21) (Paciello,
2002). This was possibly due to freezing of the pulp that we used in the experiments. The yellowish colour of
pineapple is mainly due to carotenoids. Heat processing, freezing and thawing lead to cell disintegration, pigment
degradation and isomerization of carotenoids (Hodgson and Hodgeson, 1993; Bartolome et al., 1996).

Table 9. Colour parameters of enzyme treated pineapple juice.

Pectinolytic dose (g/kg) Cellulolytic dose L a b c Hue Chroma
(g/kg)
0.025 1.00 29.8 -1.6 1.4 1.9 -32.00 1.88
0.025 1.50 30.2 -2.0 1.6 2.8 -33.69 2.88
0.025 2.00 29.7 -2.0 1.5 2.2 -19.29 2.12
0.030 1.00 29.7 -1.6 1.6 1.8 -29.35 1.84
0.030 1.50 30.9 -1.8 1.4 1.9 - 23.96 1.87
0.030 2.00 30.5 -2.2 1.5 2.2 -17.65 2.24
0.035 1.00 28.9 -2.3 1.0 2.3 -7.43 2.34
0.035 1.50 29.4 -1.8 1.1 1.8 -3.12 1.8
0.035 2.00 30.4 -1.7 0.9 1.7 -10.00 1.72
*
l, a, b and c are Hunter colour parameter

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CONCLUSION

The polysaccharide content of pineapple fruit consists mainly of hemicellulose and cellulose. So, treatment of
pineapple pulp with higher dosage of cellulase enzyme along with low dosage of pectinase enzymes improved
the yield and quality of cloudy pineapple juice. On the other hand, pectinase NS 3000 is an enzyme preparation
with wide range of different enzyme activities which proved to be excellent for pineapple pulp maceration.

ACKNOWLEDGMENTS

This study was carried out with financial support from the Austrian Exchange Service (OEAD), Agency for
International Cooperation in Education and Research within the North South Dialogue Scholarship Programme.

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