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 *Corresponding author.

Email: noriznan@upm.edu.my

eISSN: 2550-2166 / © 2018 The Authors. Published by Rynnye Lyan Resources

  Food Research 3 (2) : 145 - 150 (April 2019)
  Journal homepage: http://www.myfoodresearch.com

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Modelling and simulation of heat penetration in palm fruitlets during thermal

treatment process

1,3Shehu, U.E., 1Baharuddin, A.S., 2Nawi, N.M. and 2*Mokhtar, M.N.

1Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, Serdang

43400, Selangor Malaysia
2Department of Agricultural and Biological Engineering, Faculty of Engineering, Universiti Putra

Malaysia, Serdang 43400, Selangor Malaysia
3Department of Agricultural and Bio-environmental Engineering, College of Engineering, Kaduna

Polytechnic, Kaduna, P.M.B. 2021, Kaduna Nigeria.

Article history:

Received: 7 June 2018

Received in revised form: 8

September 2018

Accepted: 27 October 2018

Available Online: 6

November 2018

Keywords:

Palm fruit,

Endogenous lipase,

Free fatty acid,

Heat treatment,

Sterilization,

Simulation.

DOI:
https://doi.org/10.26656/fr.2017.3(2).120

Abstract

Crude palm oil (CPO) is derived from the fleshy mesocarp of the oil palm fruits, Elaeis

guineensis. The mesocarp of the fruit contained an endogenous enzyme (lipase), which is

activated upon detachment or bruising of the fruit. The lipolytic activity of the enzyme

leads to the accumulation of free fatty acid (FFA) which is a major quality index of CPO.

Heat treatment is used to substantially inactivate the lipase in the mesocarp fibre of palm

fruitlets. However, the current practice of using steam has been faced with the challenges

of improper steam distribution within the vessel which in conjunction with the complex

matrix of the bunch and whole loading of the fruit bunch into the sterilizer brings about

the differential cooking of the fruit, thereby causing partial inactivation of the enzyme. A

COMSOL Multiphysics 5.3 was used to simulate heat treatment of the fruit using hot

water. The simulation results reveal that the surface temperature of the kernel will attain at

70°C within 50 mins when submerged in hot water at 70°C. A series of experiments were

also conducted to verify whether a temperature of 70°C will completely inactivate the

enzyme. The result shows that the residual activity of the lipase was inactivated

completely after 50 mins of heat treatment at 70°C. Since simulation result shows that the

surface temperature of the kernel will be 70°C when the fruit is heat treated at that

temperature, therefore, it implies that heat treatment of the fruit with hot water at 70°C

will be an effective heat treatment to inactivate the endogenous lipase of palm fruits.

1. Introduction

Heat treatment is one of the most common methods

of industrial raw material and food processing.

Biological materials such as wood, grains and fruits have

microscopic capillaries and pores which cause a mixture

of transfer mechanisms to occur simultaneously during

heat treatment (Kulasiri and Samarasinghe, 1996). Oil

palm fruit (Elaeis guineensis) from which crude palm oil

(CPO) is extracted being a biological material is made up

of a multiplex matrix consisting of cellulose fibre as its

building structure with lipid cells, water, enzyme (e.g.

lipase) and some microelements filling-up the pore

spaces. These compositions made the analysis of heat

transfer mechanisms through it a challenging task.

However, there is an understanding among researchers

that conduction is the most dominant heat transfer

mechanism within the fruits during heat treatment

processes. Though the rate of heat conduction in the fruit

is influenced by the internal parameters such as physical

and thermal properties and the external parameters such

as heating medium. The variations in this parameters are

responsible for the non-uniformity of heat transfer in the

fruit which is one of the main obstacles to the

industrialisation of heat treatment of palm fruits.

The activity of lipase in the mesocarp of palm fruit is

activated upon abscission or bruise that may occur

during handling and processing leading to accumulation

of free fatty acid (FFA) in the fruit. FFA is a major

quality index of CPO internationally and to some extent

determines the price of the commodity oil.  To curb this

menace, fresh palm fruits are sterilized with steam to

inactivate or stop the lipolytic activity of the enzyme and

ease the fruits stripping process (Tan et al., 2009).

However, during this process, the whole fresh fruit

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bunches (FFB) are loaded into cages and are subjected to

high-pressure steam 2.76 bars for about 70 to 90 mins

(Sivasothy et al., 2005). But due to variation in sizes of

FFB, in addition to the close-knitted matrix or

arrangement of spikelet and the depth of the inner layers

hinder penetration of heat. This may result in an

ineffective sterilization process which is responsible for

the high percentage of unstripped bunches (USB) and

contributes to partial inactivation of lipase leading to

enzymatic hydrolysis of CPO in subsequent unit

operations. Sivasothy et al. (2005) reported that an

attempt has been made to solve the problem of

ineffective penetration of heat in FFB through the

introduction of the continuous sterilization. This method

involves chopping or splitting the FFB into spikelet,

heating the chopped FFB and transferring it into a

sterilizer vessel by a conveyor.

Fatin and Rosnah (2014) reported that the processes

of the continuous sterilizer cause a lot of oil loss in

condensate and oil palm stalks. Junaidah et al. (2015)

also reported issues with current sterilization processes

to be high oxidation risks and over-sterilization that may

lead to poor bleachability of the resultant oil. The

process performance has also been reported to be limited

due to the inherent temperature gradient and difficult

process control (Zaror et al.,1993). In addition,

Sivasothy et al. (1992) reported that CPO quality is

influenced by heating parameters of the process. These

challenges, therefore, will definitely contribute to a great

extent of deterioration of CPO quality. Steaming

duration of the process has also been reported by

Junaidah et al. (2015) to have contributed to the

accumulation of FFA in CPO. This observation is

consistent with the report by Cong and Gapor (1985),

and Lau et al. (2006) who reported that the percentage of

FFA reflects the degree of hydrolysis of oil, which is

possibly caused by the presence of moisture under

prolonged heating (extended heating time). Apart from

the extended heating time, Junaidah et al. (2015) also

reported that at a constant time but increased

temperature, there is an increment in FFA. Hence, the

temperature of the sterilization steam and the duration of

heating are paramount to obtaining high-quality CPO in

terms of FFA content. The heating medium in both the

conventional/traditional sterilization process is steam,

which is faced with the challenges stated above. Hence,

the need to explore the use of liquid water as a heating

medium for the inactivation of the endogenous lipase in

palm fruits.

According to Wang et al. (2001), some heat

application methods have resulted in non-uniform

heating of fruit and caused a reduction in general quality

attributes (firmness, fruit colour, pitting, bruising, etc).

Heat transfer to the inner portions of food materials

during conventional heating is limited by their low

thermal conductivity, thus prolonged heating is

necessary in many cases (Lu et al., 2007). Heating times

to bring the fruit centre to the desired temperatures range

from 23 mins for cherry to 6 hrs for apple (Wang et al.,

2001). However, in bulk processing, the heating field to

which each individual commodity is exposed can hardly

be the same, leading to treatment differences among

products. Low specific heat capacity and poor heat

transferability of conventional heating media can cause

problems, making it difficult to obtain uniform heating

within each individual fruit.

The objectives of this study is therefore, to develop

from theory a heat transfer simulation model for palm

fruit immersed in heated water using COMSOL

multiphysics 5.3 and to use the model to determine the

heat penetration profile within the oil palm fruit with a

view to gain insight into appropriated temperature and

duration of heat treatment adequate for total inactivation

of the lipase.

2. Materials and methods

Heat penetration in palm fruitlets is a process of heat

transfer that combined both convective and conductive

forms of heat transfer. Heat is transfer by convective

from the medium in this case water to the surface of the

fruit and by conduction through the solid mass of the

fruit. To simulate this process in COMSOL the following

steps were followed.

2.1 Geometry modelling.

Based on the subject’s shape and the condition of the

problem, a model in 1D, 2D, 2D-axisymmetric or 3D

space coordinate system can be run in COMSOL 5.3.

The geometry of most fruits, such as cherries, apples and

oranges, can be characterized as a spherical shape.

However, Yunus et al. (2015) suggested that for

simulation purposes, the fruit can be modelled as an

ellipsoidal shape. Hence, the fruit was modelled as that

shape, and due to the rotational symmetry of an ellipsoid,

a 2D-axisymmetric space coordinate was chosen. The

dimensional parameters of the palm fruit used for the

geometry were taken from Owolarafe et al. (2007), as

shown in Table 1. It was assumed that the domains are

concentric (Figure 1a). The finalized geometry as shown

in Figure 1b has 2 domains (mesocarp and kernel), 7

boundaries, and 6 vertices.

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Property Value (mm)

Length 35.96

Width 20.15

Thickness 17.11

Table 1. Physical properties of palm fruit (Tenara)

Adapted from Owolarafe et al. (2007)

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eISSN: 2550-2166 © 2018 The Authors. Published by Rynnye Lyan Resources

The geometry was then meshed using the fine mesh

option of the software. The Completed mesh consists of

758 domain elements and 86 boundary elements (Figure

2).

2.2 Heat transfer model

The source of thermal energy in conventional

heating is hot air/or water and therefore, the temperature

distribution in fruits during heat treatment is a function

of radial position and treatment time. Thermal energy is

transferred from the heating medium to the fruit surface

(r = ro) by convection as described by the following

boundary heat flux equation (Wang et al., 2001):

Where k is the thermal conductivity of the fruit

(Wm−1K−1); and h is the surface heat transfer coefficient

(W m−2K−1).

The heat transfer module comes with a library of

heat transfer coefficient functions, r is the radial

coordinate originate from the fruit centre, ro is the fruit

radius (m), t is the treatment time (min), T is fruit

temperature (K), and Te is the heating medium

temperature (K).

The moment heat energy is transferred to the surface

of the fruit; it moves into the interior by conduction.

Fruit temperature as a function of time at any locations

within spherical fruit is governed by a general energy

balance equation given by Holdsworth (1997) as:

Where Cp is the specific heat of the fruit (J kg−1K−1); Q is

the heat generation within the fruit (Wm−3); and ρ is the

fruit density (kg m−3).

Relative to externally applied energy, the heat of

respiration is small over the period of heat treatment.

Thus, Q = 0. Dividing by ρCp and substituting α for k/

ρCp, Equation 2 becomes:

Where α is the thermal diffusivity (m2 s−1).

2.3 Physics settings

The physics menu contains two settings, which are

subdomain setting and boundary setting. Subdomain

setting is for each domain’s material properties, initial

conditions and etc. Boundary setting is for boundary

conditions in two aspects. Firstly, it can be used to set

boundary conditions on the interface of different

materials. Secondly, it can also be used to set boundary

conditions on the interface between the material and the

environment. The physics used for the simulation is the

built-in time-dependent heat transfer in solid.

In the setting window of the software under time-

dependent, there is an option to choose the default heat

transfer model, either fluid or solid can be chosen. In this

case, it was taken to be solid. In the area where the

domain can be set, all of the domains must be chosen

(kernel and mesocarp). The fruit (ellipsoidal shape

geometry) was set to an initial temperature of 26°C and

the environment (water) was set to temperatures of 35°C,

40°C, 45°C, 50°C, 60°C and 70°C. The temperature

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Figure 1. Model geometry of palm fruit in 2D Axisymmetric. a) Ellipsoidal geometry; b) symmetry of the model geometry

showing the two domains

(a)

Figure 2. The meshed geometry

(b)

(1)

(2)

(3)

148 Shehu et al. / Food Research 3 (2) (2019) 145 - 150

eISSN: 2550-2166 © 2018 The Authors. Published by Rynnye Lyan Resources

dependent properties of the heating medium (water) are

in-built in the software and shown in Table 2. While the

required properties of the fruit are shown in Table 3 were

entered into the appropriate fields in the physics window.

The governing equations were solved by default as a

time-dependent study with the recorded time step of 1

min between times 0 min and 90 mins.

2.4 Experimental

The experiment was conducted with fresh palm fruits

(tenara) to find out an appropriate water temperature and

duration of treatment that will completely inactivate the

endogenous lipase. In this study, the amount of FFA

accumulated per unit time after heat-treated fruits are

mashed is considered as the enzyme activity.

2.4.1 Materials

Fresh and ripe fruit bunches of the oil palm (Elaeis

guineensis Jacq. var. tenera) were obtained from the

University Putra Malaysia farm. The fruits bunches are

from palm fruit trees that are 10 years old. The fruits

were removed from the bunch immediately they arrived

at the lab. They are cleaned, surface-sterilized in 95%

ethanol for 10 mins, and washed in sterile distilled water

(Tang et al., 2017). The bruised and rotten fruits were

handpicked and the remaining sorted fruits were gently

mixed with hand to avoid bruise and to give a

homogeneous lot. The lot was randomly divided into 6

groups (A, B, C, D, E, F, and G), each with an equal

weight of 1200 g.

2.4.2 Heat treatments of fresh palm fruit

The 6 groups were allotted to the heat treatments i.e.

group A, B, C, D, E, and F were allotted heat treatment

of 35°C, 40°C, 45°C, 50°C, 60°C, and 70°C respectively

while group G served as control without any treatment

but was directly cooked in a boiling water for 30 mins.

Thermal inactivation experiments were conducted in a

water bath (model DH WSB01018 Daihan WiseBath,

South Korea) at temperatures of 35°C, 40°C, 45°C, 50°

C, 60°C, and 70°C. During the heat treatment, 2 Samples

(1 and 2) were withdrawn from the water bath at an

interval of 5 mins through 60 mins. A similar procedure

was carried on group G except there was no initial

inactivation heat treatment.

2.4.3 Lipase activity measurement

Immediately after the samples were withdrawn from

the water bath, the flesh (mesocarp) was peeled off from

each fruit of sample 1. The peeled mesocarp was mashed

in a mortal and incubated in an incubator at 37°C for 10

mins thereafter, the mash was wrapped in a polyethylene

bag and immersed in a boiling water for 30 mins to stop

the activity of the enzyme initiated as result of mashing

and incubation. While sample 2 was immersed in a

boiling water for 30 mins to completely inactivate any

residual enzyme thereafter the mesocarp was peeled

from the kernel and mashed. All experiments were

carried out in triplicate.

2.4.4 Detection of the product

This step refers to that process by which the product

of in vivo enzyme (lipase) activity is located, identified,

and the amount formed during a specific incubation

interval is quantified. In this case, the product is the free

fatty acid (FFA). To detect and quantify FFA liberated in

the mashed mesocarp, the oil was extracted from the two

samples (1 and 2). This was done by using a laboratory

scale screw press. The FFA liberated in sample 1 was

termed FFA1 and the one liberated in sample 2 was

termed FFA2 while the one liberated in group G was

termed FFAi

2.4.5 Determination of FFA content.

The FFA content of the crude palm oil (CPO)

extracted was determined using AOCS Method Ab 5–49

(AOCS, 2004). The percentages of FFA was calculated

as palmitic acid (molecular weight = 256.6) per 7 g of

CPO, considering palmitic acid to be the predominant

fatty acid present in palm oil. Hence,

Where N is the normality of NaOH (0.5 mol/L); V is the

volume of NaOH used; and W is the weight in (g) of the

sample.

The lipase activity (LA) was computed using

Equation 5 and the lipase activity relative to the initial

activity in an untreated fruit (RLA) using Equation 6.

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Property At 300 K At 325 K

Density (kg/m3) 997 987

Viscosity (Ns/m2) 8.5x10-4 5.3x10-4

Thermal conductivity (W/mK) 0.62 0.66

Heat capacity (J/kgK) 4180 4182

Diffusivity (m2/s) 2.0x10-9 2.0x 10-9

Table 2. Physical properties of liquid water

Table 3. Physical properties of a palm fruit

 Property
Part of

Palm Fruit
Value Unit

Density (ρ)
Mesocarp 993 kg/m3

Kernel 1203 kg/m3

Specific Heat (Cp)
Mesocarp 2.816 kJ/kgK

Kernel 2.291 kJ/kgK

Thermal Conductivity

(k)

Mesocarp 0.347 W/mK

Kernel 0.529 W/mK

Thermal Diffusivity

(α)

Mesocarp 1.24 m2/s x 10-7

Kernel 1.92 m2/s x 10-7

Adapted from Hadi et al. (2015)

(4)

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eISSN: 2550-2166 © 2018 The Authors. Published by Rynnye Lyan Resources

Where t is the mashing time + incubation time in mins.

Where LA0 is the lipase activity in the untreated fruit.

3. Results and discussion

3.1 Temperature distribution

Figures 3 shows a uniform temperature penetration

into the fruit when immersed in water at different

temperatures. From Figure 3, it can be seen that after 60

mins of heat treating the fruit in water at 70°C, the kernel

surface temperature attained 69°C, which approximately

equal to the 70°C temperature identified in Figure 4 as a

temperature that reduced the lipase activity substantially.

It is also evident in Figure 3 that the temperature of

domain 1 (mesocarp) is higher than the kernel surface

temperature, hence, the native lipase in the mesocarp was

inactive or denatured. The plots also indicate a deeper

heat penetration in y-axis compared with x and z-axis

even though the kernel surface temperature is the same at

all the axis.

The objective of the palm fruit sterilisation process is

to inactivate the native lipase in the mesocarp fibre of the

fruit which caused FFA accumulation in CPO. As it is

shown in Figure 5, heat treatment of palm fruit in hot

water at 70°C for 60 mins is enough to stop the activity

of the enzyme and will help to remove the heavy dirt,

pesticides and fungal spores on the freshly harvested

produce and also improve general product appearance

and maintain product quality.

4. Conclusion

A 2D-asymmetric time-dependent model using

COMSOL Multiphysics® software was developed to

simulate the temperature changes in the mesocarp of

palm fruit immersed in heated water. The geometrical

shape (ellipsoidal) used for the simulation fitted very

well with the actual shape of the fruit. Although this

modelling illustrates the basic heat transfer for a single

product based on the assumption of ellipsoidal shape, the

software was able to accurately simulate changes in

temperature in the fruit with time. The relatively low

temperature of 70°C compared with the 120°C steam

temperature used in conventional palm fruit sterilizers

and the shorter exposure time in hot water immersions

treatments make hot water immersion a viable

alternative. A comparative study between the convention

steam sterilization and hot water immersion in terms of

heat penetration and residual lipase activity as well as

energy consumption, oil extraction rate (OER) and

quality of CPO should be conducted.

Acknowledgements

The authors gratefully acknowledge the support of

this work by the Universiti Putra Malaysia through Putra

Grant (GP-IPS/2018/96350) and financial support to

Shehu U.E. by the Kaduna Polytechnic, Kaduna, Nigeria.

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Figure 3. Temperature distribution in the fruit immersed in

water at 70°C for 60 mins.

(6)

(5)

Figure 4. Relative residual lipase activity after heat treatment

of the fruit in hot water at different temperatures

Figure 5. Simulated temperature profile of a palm at different

hot water temperatures ( mesocarp temperature,

 kernel surface temperature)

35°C

150 Shehu et al. / Food Research 3 (2) (2019) 145 - 150

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 *Corresponding author.

Email: ufshahjadee@gmail.com

eISSN: 2550-2166 / © 2020 The Authors. Published by Rynnye Lyan Resources

  Food Research 4 (4) : 1066 - 1070 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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Effect of chemical agents, metallic salts on the stability of α-amylase, protease

and comparative analyses of enzyme activity of selected salad vegetables

1Rahman, M.M., 1.*Shahjadee, U.F., 2Khanam, F., 1Rupa, A.Z. and 2Azad, M.A.K.

1Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and

Industrial Research (BCSIR), Dhaka-1205, Bangladesh

2Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia -7003,

Bangladesh

Article history:

Received: 18 July 2019

Received in revised form: 30

January 2020

Accepted: 9 February 2020

Available Online: 9 March

2020

Keywords:

Salad vegetables,

Enzymes activity,

Chemical agent,

Metallic salts,

Stability

DOI:
https://doi.org/10.26656/fr.2017.4(4).259

Abstract

The present investigation was conducted to analyze the activity of enzymes (protease, α-

amylase, cellulase and urease) of selected salad vegetables (white radish, red radish, beet,

carrot, papaya, cucumber and tomato) as well as to determine the effect of chemical agents

and metallic salts on the stability of enzymes. Salad vegetables are one of the cheap

sources of adequate vitamins, minerals and enzymes. The protease, α-amylase and

cellulase activity were highest in papaya (4.11±0.21 U/g, 1.68±0.21 U/g and 0.26±0.13 U/

g) whereas the urease activity was not detected in papaya, cucumber and tomato. The

protease, α-amylase and cellulase activity of papaya were increased 24.83%, 42.26% and

57.69% than cucumber while the cellulase activity of carrot was increased 44.45% than

tomato. The activity of protease and α-amylase of beet were decreased 52.44%, 33.63%

while the cellulase activity of beet was increased 36.36% from radish. The toxicity

indicating urease enzyme activity was not detected in papaya, tomato and cucumber but

negligible in radish, beet and carrot which can be nullified. The activities of enzymes were

increased in the presence of metallic salts such as Ca2+, Mg2+ and Mn2+ while Fe2+, Zn2+

and Cu2+ inhibited the enzyme's activity moderately. Results revealed that in the presence

of higher concentrations of urea, EDTA and acetic acid, the activities of all the enzymes

were completely inhibited.

1. Introduction

Salad vegetables are an essential part of people’s diet

all around the world. They are usually consumed as raw

or cooking. Vegetable salad is a very common food

accompaniment. The vegetables that usually make up

these salad items include carrot, cucumber, tomato,

papaya, beet and radish (Itohan et al., 2011). Carrot,

cucumber, tomato, papaya, beet and radish are a versatile

vegetable that may be consumed raw, cooked or in

combination with other vegetables and meats in salads,

freshly prepared foods and frozen prepared foods. Fruits

and vegetables have health benefits for consumers, due

to their content of fiber, vitamins and antioxidant

compounds. However, for the antioxidant compounds,

many changes occur during harvesting, preparation

(fresh-cut fruits) and storage of these fruits. These

changes induce a pronounced loss of microbiological and

antioxidant qualities (Lindley, 1998). Fresh fruits and

vegetables provide mankind with an abundance of

benefits. These play an important role in health and have

the ability to prevent many diseases such as heart

disease, cancers and diabetes (Liu, 2004; Pandey, et al.,

2006). The highly soluble minerals like calcium,

phosphorus, iron, magnesium, copper, and potassium

contained in vegetables maintain the acid/base balance of

hydrogen concentration of body tissues. These help to

completely absorb the vitamins, proteins, fats and

carbohydrates of the food (Odoh and Okoro, 2012).

The World Health Organization (WHO) (1989)

recommends the consumption of at least five 100 g

portions of fruits and vegetables daily to stay healthy.

Health nutrition specialists recommend adding to

everyday diet as much as possible fruits and vegetables

although the general public has a limited understanding

of what the real benefits are, or how much should be

eaten. Current recommendations are that adults should

consume at least 400 g (or five servings a day) of fruits

and vegetables (WHO, 1989). The nutritional analysis of

edible fruit and vegetables plays a crucial role in the

assessment of nutritional significance. A comprehensive

survey was conducted by an extensive tour throughout

the selected locations during both winter and summer

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seasons from April 2010 to December 2012. In the

selected locations two or more vegetables are grown

every year (Khan et al., 2010). Vegetables are richer in

mineral substances as compared with fruits. Enzymes

enter into a vast number of biochemical reactions in

fruits and vegetables, they may be responsible for

changes in flavor, color, texture and nutritional

properties. In fruit and vegetable storage and processing,

the most important roles are played by the enzymes

classes of hydrolyses (invertase, protease, amylase,

cellulase) (Enacheshu, 1995). Salad vegetables are one

of the cheap sources of adequate vitamins, minerals and

enzymes.

Data available on the biochemical parameters of

radish produced in Bangladesh are quite scanty. Only

limited work has been done on the physiochemical

properties of tomato, papaya, cucumber and carrot. Some

works have been going on at Bangladesh Agricultural

Research Institute (BARI) with emphasis on the higher

production and development of new varieties and control

of the diseases. No detail work has been done on the

chemical composition and enzyme activities in tomato,

papaya, cucumber and carrot. Enzymes have become a

powerful catalytic tool in a wide variety of chemical

processes. Higher plants play an important role as a

source of many useful enzymes. Extensive use of

enzyme activity determination as an aid to the diagnosis

in hospital and laboratories all over the world is one of

the dramatic developments in modern medicine (Jahan et

al., 2011). The present study is, therefore, designed to

study the activities of different enzymes and also to

determine the chemical agents and metallic salts on the

stability of the enzymes of selected salad vegetables.

2. Materials and methods

2.1 Collection of samples

Samples were collected directly from the cultivated

field of the local area in the winter season.

2.2 Enzyme activity assay

Fresh healthy samples were washed thoroughly with

distilled water, cut into small pieces and peste in a

mortar at 4ºC with cold 0.1M phosphate buffer of

respective pH, for amylase 6.7, for protease citrate

buffer, 5.5, for urease 0.2M phosphate buffer, 7.0 for

cellulase 0.2M sodium acetate buffer, 5.2. The extracts

were filtered with few layers of cheesecloth and further

clarified by centrifugation at 10,000 rpm for 15 mins at

4ºC. The clear supernatant was collected and used as a

crude enzyme extract.

2.3 Assay of α-amylase activity

Amylase activity was assayed following the method

as described by Jayaraman (1981). A total of 1% starch

solution was used as the substrate (1.0 g in 100 mL of

0.1M phosphate buffer, pH 6.7). The amylase activity

was measured by estimating the release of maltose. The

amount of maltose released was calculated from the

standard curve prepared with maltose. One unit of

amylase activity was defined as the amount required for

liberating 1 mg of maltose in 15 mins at 37℃.

2.4 Assay of protease activity

Protease activity was assayed following the modified

method as described by Mahadevan and Sridhar (1982).

Haemoglobin was used as substrate. The protease

activity was measured by estimating the amount of

leucine released from haemoglobin. The amount of

leucine released was calculated from the standard curve

prepared with leucine. One unit of protease activity was

defined as the amount required for liberating 1 mg of

leucine in 30 mins at 37℃.

2.5 Assay of cellulase activity

Cellulase activity was measured following the

method described by Mahadevan and Sridhar (1982).

Carboxymethyl cellulose (CMC) was used as substrate.

Cellulase activity was measured by estimating the

reducing sugar released from the substrate using the

DNS method. One unit of cellulase activity was defined

as the amount of enzyme required for liberating 1 mg of

reducing sugar from CMC per minute at 37°C.

2.6 Assay of urease activity

Urease activity was assayed following the method as

described by Jayaraman (1981). A total of 3% urea

solution was used as substrate (1 g in 100 mL of 0.2M

phosphate buffer, pH 7.0). The Urease activity was

measured by estimating the release of ammonium. The

amount of ammonium released was calculated from the

standard curve prepared with ammonium. One unit of

Urease activity was defined as the amount required for

liberating 1 µg of ammonium in 15 mins at 55℃.

2.7 Effect of chemical agents and metallic salts

Treatment with CaCl2, urea, acetic acid, EDTA and

various metallic salts at different concentrations were

added to 5.0 mL of amylase, protease, cellulase and

urease enzymes extract solutions from salad vegetables

and incubated for 10 mins at 20℃. The mixtures were

again incubated with the substrate for 15 mins at 37℃

and the enzyme activities were assayed (Rahman, 2001).

2.8 Statistical analysis

Each experiment was replicated three times.

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Reported data represented as mean values ±SD obtained

from three individual experiments. Data were subjected

to analysis of variance using the Microsoft Excel

Program (Redmond, Washington DC, USA).

3. Results and discussion

3.1 Enzymes activity

Vegetables are very rich sources of essential

biochemicals and nutrients such as vitamins, minerals

and enzymes which are the major portion of the human

diet. The results of protease and α-amylase activity are

shown in Table 1. The protease activity of white radish,

red radish, beet, carrot, papaya, cucumber and tomato

were 1.99±0.20 (U/g), 2.44±1.00 (U/g), 1.16±0.16 (U/g),

2.55±0.19 (U/g), 4.11±0.20 (U/g), 1.18±0.21 (U/g) and

2.85±0.22 (U/g) respectively (Table 1). The maximum

protease activity was found in papaya 4.11±0.20 (U/g)

and minimum in beet 1.16±0.16 (U/g). The protease

activity of papaya 0.72-3.11 (U/g) which was slightly

lower than the investigation (Xiang-dong et al., 2006).

The papaya showed maximum α-amylase activity

than beet. Results of the present study are in accordance

with the findings with some researchers in the case of

vegetables (Ajayi et al., 2011). The cellulase activity of

white radish, red radish, beet, carrot, papaya, cucumber

and tomato were 0.14±0.07 (U/g), 0.072±0.012 (U/g),

0.22±0.11 (U/g), 0.18±0.07 (U/g), 0.26±0.06 (U/g),

0.11±0.03 (U/g), and 0.10±0.05 (U/g) respectively

(Table 1). Salad vegetables have a poor amount of

protein and carbohydrate contents. That’s why the

enzyme protease and α-amylase show fewer assay

results.

The highest cellulase activity was found in papaya

(0.26±0.06 U/g) and the lowest was in red radish

(0.072±0.012 U/g). Tomato showed lowest (9.09%)

cellulase activity than cucumber among the salad variety.

These findings are in close agreement with the

researchers who revealed the low cellulase activity in

tomato (Wilson, 2012). Besides, the highest cellulase

activity was 57.69% in papaya than cucumber. The

urease activity of white radish, red radish, beet, carrot,

papaya, cucumber and tomato were 0.067±0.10 (U/g),

0.061±0.25 (U/g), 0.081±0.10 (U/g), 0.018±0.09 (U/g)

respectively (Table 1). The urease activity of papaya,

cucumber and tomato were not detected. The α-amylase

activity of carrot was 0.18±0.15 (U/g). The cause may be

the presence of urease activity in carrot the utilization of

nitrogen fertilizer in cultivating soil. Besides this, the

results showed no presence of urease activity in carrot in

his research work (Richard and Michael, 1973). Urea

was found naturally in the environment and is also

artificially introduced, comprising more than half of all

synthetic nitrogen fertilizers used globally. For this

reason, the negligible amount of urease activity was

found in radish, beet and carrot (Nowak et al., 2003).

3.2 Effect of calcium on the activities of α-amylase and

protease

The effect of calcium as metallic salt on the activities

of α-amylase and protease is presented in Table 2. The

activities of enzymes were gradually increased with the

increasing concentration of calcium and in the presence

of 0.50 M Ca2+, the activities of α-amylase and protease

became 135.24% and 129.46% respectively.

3.3 Activities of the enzymes after treatment with

different concentration of urea

  Table 3 shows the relatives activities of the

enzymes after treatment with different concentration of

urea. The activities of the α-amylase and protease were

found to be decreased remarkably in the presence of urea

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Name of samples
Enzymes activity

Protease (U/g) α-amylase (U/g) Cellulase (U/g) Urease (U/g)

White Radish 1.99±0.20 1.47±0.20 0.14±0.07 0.067±0.10

Red Radish 2.44±1.00 1.10±0.21                 0.072±0.012 0.061±0.25

Beet 1.16±0.16 0.73±0.10 0.22±0.11 0.081±0.10

Carrot 2.55±0.19 1.56±0.18 0.18±0.07 0.018±0.09

Papaya 4.11±0.20 1.68±0.23 0.26±0.06 Not detected

Cucumber 1.18±0.21 0.97±0.20 0.11±0.03                     Not detected

Tomato 2.85±0.22 1.52±0.22 0.10±0.05 Not detected

Table 1. Protease, α-amylase, cellulase and urease activities of white radish, red radish, beet, carrot, papaya, cucumber and

tomato.

The values are mean ±SD of determinations made in triplicate

Relative activities (%)
Concentration of CaCl2 (Molar)

α-amylase Protease

0.000 100.00 100.00

0.001 104.36 105.66

0.005 107.18 108.34

0.010 113.35 112.68

0.050 117.38 116.38

0.300 130.24 123.65

0.500 135.34 129.46

Table 2. Effect of calcium on the activities of α-amylase and

protease

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and at 6M urea concentration. Protease and α-amylase

decreased by about 88% and 79% respectively.

3.4 Activities of α-amylase and protease after treatment

with different concentration of EDTA

The relative activities of α-amylase and protease in

the presence of different concentrations of EDTA are

presented in Table 4. The activities of α-amylase and

protease are abolished gradually with the increase of

EDTA concentrations and the enzymes lost their

activities completely in the presence of 0.50M EDTA.

3.5 Activities of α-amylase and protease after treatment

with different concentration of acetic acid

As shown in Table 5, the activities of the enzymes

were gradually decreased with the treatment of higher

percentage of acetic acid. It is found that α-amylase and

protease retained 22.43% and 19.17% after treatment

with 20% acetic acid. Overall, the effect of calcium,

urea, EDTA and acetic decrease the enzyme's activity

which is partially in agreement with Akand (2003) and

Haque et al. (2017)

3.6 Effect of metallic salts on the activities of α-amylase

and protease

Table 6 represents the effect of various metallic salts

on the activities of α-amylase and protease. The activities

of enzymes were increased remarkably in the presence of

Mn2+ salts while that was increased slightly in the

presence of Mg2+ salts. Other metallic salts such as K2+

and Na2+ produce little or no inhibitory on the activities

of the enzymes but the activities of all the enzymes

reduce significantly in the presence of Zn2+, Cu2+ and

Fe2+.

The activity of α-amylase and protease were

abolished completely by denaturing agents 8.0M urea.

These denaturing agents may destroy the secondary or

tertiary structure of the enzymes this results in complete

destruction of the active enzyme at higher concentration

(Rahman, 2001; Haque et al., 2017).

The activities of the enzymes increased significantly

in the presence of divalent cation Ca2+ and Mn2+

suggesting the involvement of these divalent ions in

maintaining the active conformation of the enzymes.

Other metallic salts such as K2+ and Na2+ produce little

or no inhibitory on the activities of the enzymes but the

activities of all the enzymes reduce significantly in the

presence of Zn2+, Cu2+ and Fe2+ (Akand, 2003; Haque et

al., 2017).

4. Conclusion

It was aimed to evaluate the enzymatic activity (α-

amylase, urease, cellulase and protease) of white radish,

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Concentration of urea  (Molar)
Relative activities (%)

α-amylase Protease

0 100.00 100.00

1 95.17 91.62

2 82.41 75.78

4 46.82 41.76

6 22.64 13.35

8 0.00 0.00

Table 3. Effect of urea on the activities of α-amylase and

protease

Relative activities (%)
Concentration of EDTA (Molar)

α-amylase Protease

0.000 100.00 100.00

0.001 85.34 76.69

0.005 67.29 59.82

0.010 53.22 47.34

0.100 34.46 27.64

0.300 15.60 13.36

0.500 0.00 0.00

Table 4. Effect of EDTA on the activities of α-amylase and

protease

Concentration of acetic acid (%)
Relative activities (%)

α-amylase Protease

0.00 100.00 100.00

2.50 90.16 89.53

5.00 75.33 75.89

10.00 46.65 47.20

20.00 22.43 19.17

30.00 0.00 0.00

Table 5. Effect of acetic acid on the activities of α-amylase

and protease

Test Salts
Concentration

(Molar)

Relative activities (%)

α-amylase Protease

None 100.00 100.00

 0.001 105.72 106.01

MgCl2 0.002 108.32 109.12

 0.001 85.24 93.26

ZnCl2 0.002 73.21 86.55

 0.001 86.71 71.84

CuCl2 0.002 76.36 53.64

 0.001 117.73 113.42

MnCl2 0.002 127.63 121.66

 0.001 100.00 99.72

NaCl2 0.002 99.78 99.98

 0.001 100.00 100.00

KCl2 0.002 100.00 100.00

 0.001 73.16 67.28

FeCl2 0.002 589.44 53.20

Table 6.  Effect of various metallic salts on the activities of α-

amylase and protease.

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red radish, beet, carrot, papaya, cucumber and tomato

with each other. It was found that the enzymes’ activity

was different in all the selected salad vegetables its play

an important role in the metabolic function of the human

body. Through this research work, the enzymatic activity

of salad vegetables was determined. Moreover, the

observations were shown that higher concentrations of

chemical agents and metallic salts inhibit enzymes

activity in a maximum potential level.

Conflict of Interest

The authors have no conflict of interest.

Acknowledgement

The authors gratefully acknowledge the research

facilities provided by Food Enzymology Section,

Institute of Food Science and Technology (IFST) of

Bangladesh Council of Scientific and Industrial Research

(BCSIR), Dhaka-1205.

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

ZIKIR AL-TAWHID
Al-Habib cUmar Ibn cAbd al-Rahman al-cAttas1



Kekerapan Bacaan Bil

3 kali الل ااولهاسهارَااد امَاحَا،امهااللهاَلاإ ااهَالَ اإ ااَلا 1

5 kali اللهاَلاإ ااهَالَ اإ ااَلا
2

25 kali للها
3

3 kali الل ااولهاسهارَااد امَاحَا،امهااللهاَلاإ ااهَالَ اإ ااَلا 4

1 kali اللهاَلاإ ااهَالَ اإ ااَلا
5

1
 Dengan ijazah daripada K.H. Amir Hamzah melalui sanadnya yang bersambung

kepada al-Habib
c
Umar Ibn

c
Abd al-Rahman al-

c
Attas.