Ravi Thesis

Crystallization and Drying studies of Biomaterials

A THESIS

SUBMITTED TO THE

UNIVERSITY OF MUMBAI

FOR THE

MASTER OF TECHNOLOGY DEGREE

IN

BIOPROCESS TECHNOLOGY

(PARTLY BY PAPERS PARTLY BY RESEARCH)

SUBMITTED BY

RAVIKANT VITHALRAO DEVAKATE

UNDER THE GUIDANCE OF

PROFESSOR B. N. THORAT

INSTITUTE OF CHEMICAL TECHNOLOGY


MATUNGA, MUMBAI - 400 019

JUNE 2007

CERTIFICATE

The work described in this thesis has been carried out by Mr. Ravikant Vithalrao

Devakate under my supervision at the Chemical Engineering Department,

Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai –

400 019. I certify that it is his bonafide work. The work described is original and

has not been submitted for any other degree of this or any other university.

Professor B. N. Thorat

Research Supervisor

Professor of Chemical Engineering

Chemical Engineering Department,

Institute of Chemical Technology,

University of Mumbai, Date: 30 June 2006

Matunga, Mumbai-400 019 Place: Mumbai

STATEMENT BY THE CANDIDATE

As required by the University Regulation No. R 2304, I wish to state that the

work embodied in this thesis titled “Crystallization and Drying studies of

Biomaterials” forms my own contribution to the research work carried out

under the guidance of Professor B. N. Thorat at the Chemical Engineering

Department, Institute of Chemical Technology, University of Mumbai, Matunga,

Mumbai – 400 019. This work has not been submitted for any other degree for

this or any other University. Whenever references have been made to previous

work of others, it has been clearly indicated as such and included in

bibliography.

Ravikant Vithalrao Devakate

(Research Student)

Certified by

Professor B. N. Thorat

Research Supervisor

Professor of Chemical Engineering

Chemical Engineering Department,

Institute of Chemical Technology,

University of Mumbai,

Matunga, Mumbai – 400 019

Date: 30th June. 2007.

Place: Matunga, Mumbai.

Acknowledgement

This thesis is the end of my journey in obtaining my degree at UDCT. I have not traveled

in a vacuum in this journey. There are some people who made this journey easier with

words of encouragement and more intellectually satisfying by offering different places to

look to expand my theories and ideas. I would like to express my gratitude to all those

who gave me the possibility to complete this thesis.

I would like to express my deep and sincere gratitude to my supervisor, Professor B. N.

Thorat. His wide knowledge and his logical way of thinking have been of great value for

me. His understanding, encouraging and personal guidance have provided a good

basis for the present thesis.

He challenged me to set my benchmark even higher and to look for solutions to

problems rather than focus on the problem. I learned to believe in my future my work

and myself. Thank you Professor.

I wish to express my warm and sincere thanks to Professor A. M. Lali, who introduced me

to the field of Chromatography. Their ideals and concepts have had a remarkable

influence on my entire career in the field of Biotechnology.

I would like to thank Professor S. S. Lele, BPT Coordinator, for providing research facilities

during my first year.

I am thankful to Department of Biotechnology (DBT), Govt. of India, for sponsoring me

fellowship and funds to carry out my research work.

My thanks to all my lab mates who always helped me and I will never forget whole BNT

group: Sunil (Chollar), Sachin (Chinu), Varsha (Kaku), Mahendra (Mamu), Vilas (Gotya),

Ganesh, Sanjay, Rekha, Neeraj, Ankush, Amol, Sushil, Ashok and Sagar. Trip to

Sindhudurg and Janjira with them will remain always memorable.

I am thankful to all my BPT classmates especially my room mate Yogesh (Partner),

Prashant (Bhai), Deepak D. (DP), Vivek (VickyPharma), Tushar (Chhava), Kalpesh (kalpya),

Shripad (Shree), ………. Trips to Ganapati Pule and Elephanta Caves with them will always

be memorable.

Special thanks to all my Hostel friends Mangesh (Mango), Vishwa, Tuks, Om and all my

M. Chem. friends. They made my stay at hostel more enjoyable.

I would also like to thank ABP group members: Gopal, Amit, Shailesh, Mohan, Shashank,

etc. We really had a good time with them in the lab.

I am thankful to Abijar for his help in explaining me the chromatographic techniques

and giving his valuable suggestions throughout my research work.

I would also like to gratefully acknowledge the support of some very special individuals.

They helped me immensely by giving me encouragement and friendship. I warmly

thank Sunil (Chollar), Ashutosh (Ashu) and Vilas (Gotya), for their valuable advice and

friendly help. Their extensive discussions around my work and interesting explorations in

operations have been very helpful for this study.

Trip to France with Vilas will always be memorable.

Special thanks to my parents and my relatives who have put in their efforts and prayers

for me to attain success in life. I am falling short of words to express my feelings towards

them. Their blessings and encouragements were beyond comparison.

………..Ravikant

...dedicated to My Parents

Index

1. INTRODUCTION

1.1. Biomaterials – An overview 1

1.1.1. Enzymes 1

1.2. Crystallization as a purification step 2

1.2.1. Protein crystallization 2

1.2.2. Factors affecting crystallization 5

1.2.3. Applications of protein crystallization 6

1.3. Introduction to Drying Techniques 8

1.3.1. Freeze Drying 9

1.3.2. Spray Drying 10

1.3.2.1.Prediction of enzyme activity retention 11

1.3.3. Heat Pump Drying 14

1.4. Objectives of the work 15

1.5. References 17

2. BROMELAIN – A LITERATURE OVERVIEW

2.1. Introduction to Proteases 21

2.2. Sources of Proteases 23

2.3. Classification of Proteases 24

2.4. Cysteine or thiol or sulfhydryl proteases 24

2.4.1. Mechanism of action of cysteine proteases 26

2.5. Bromelains 28

2.5.1. Composition of pineapple fruit 29

2.5.1.1. Enzymes found in pineapple juice 31

2.5.2. Biochemical properties of bromelain 32

2.5.2.1. Stability 32

2.5.2.2. Physical properties of bromelain 32

2.5.2.3. Chemical properties of bromelain 34

2.5.3. Activators, inhibitors and chemical modifications 37

2.5.4. Specificity, kinetic properties and enzymic mechanism 38

2.5.5. Applications of bromelain 40

2.6. References 42

3. MATERIALS AND METHODS

3.1. Materials and Chemicals 46

3.2. Methods of Analysis 46

3.2.1. Measurement of Enzyme Activity 46

3.2.2. Measurement of Protein Content 48

3.2.3. Measurement of reducing sugar 49

3.2.4. Measurement of water activity 50

3.2.5. Measurement of moisture content 51

3.2.6. Differential Scanning Calorimetry (DSC) 51

3.2.7. Fourier transformation infra-red spectroscopy (FTIR) 52

3.3. Experimental Methods 52

3.3.1. Preparation of clarified fruit bromelain extract 52

3.3.2. Crystallization of clarified fruit juice 53

3.3.2.1.Crystallization using Ammonium Sulfate 53

3.3.2.2.Crystallization using Acetone 54

3.3.2.3.Crystallization using ammonium sulfate and sodium chloride 55

3.3.3. Dialysis of the purified sample 56

3.3.4. Adsorptive Chromatographic separation 57

3.3.5. Drying of purified fruit juice. 58

3.3.5.1.Freeze Dryer 58

3.3.5.2.Spray Dryer 59

3.3.5.2.1. Experimental set – up 59

3.3.5.3.Heat Pump Dryer 62

3.4. References 64

4. RESULTS AND DISCUSSION

4.1. Crystallization of bromelain 66

4.1.1. Crystallization using ammonium sulfate 66

4.1.2. Crystallization using acetone 70

4.1.3. Crystallization using ammonium sulfate and sodium chloride 71

4.1.4. Effect of type of salt 72

4.2. Chromatographic purification of fruit bromelain 73

4.3. Drying of purified bromelain 74

4.3.1. Freeze drying 74

4.3.2. Spray Drying 75

4.3.2.1.Inactivation kinetics in spray dryer 77

4.3.2.2.Spray dryer performance 79

4.3.3. Heat Pump Drying 80

4.4. Product quality parameters 81

4.5. Product Characterization 83

4.5.1. Inactivation kinetics in aqueous solution 83

4.5.2. FTIR Study 85

4.5.3. DSC Study 86

4.5.4. Optimum pH 87

4.5.5. Optimum Temperature 88

4.5.6. Effect of time on reaction velocity 89

4.5.7. Time and Temperature stability 90

4.6. References 91

5. CONCLUSION 93

6. SCOPE FOR FUTURE WORK 94

SYNOPSIS

List of Tables

Table

No.

Title Page No.

1.1 Comparison among spray drying and freeze drying 13

2.1 Sale of enzymes in the market 22

2.2 Bromelain content of pineapple plants (press juice from hand

press)

30

2.3 Amino acid composition of stem and fruit bromelain 35

2.4 Comparison of BAEE and BAA hydrolysis catalyzed by SH

proteinases

39

2.5 Some food related uses of bromelains 41

4.1 Effect of percent saturation on bromelain and total Protein

recovery in precipitation

68

4.2 Effect of percent saturation on activity retention and protein

content in ammonium sulfate precipitation

69

4.3 Acetone fractionation in the range of 40 – 80% saturation 70

4.4 Results of Chromatographic Purification 73

4.5 Effect of freeze drying on product quality parameters 75

4.6 Effect of spray drying on product quality parameters 76

4.7 Spray Dryer Performance 79

4.8 Effect of heat pump drying on product quality parameters 80

4.9 Product quality parameters: Comparison with commercial

bromelain

81

List of Figures

Figure

No.

Title Page No.

1.1 Solubility curve of a protein 4

2.1 Catalytic mechanism of cysteine proteases 27

3.1 Standard plot for enzyme assay using tyrosine as a standard 47

3.2 Standard plot for Protein estimation using BSA as a standard 49

3.3 Standard plot for sugar estimation using glucose as a standard 50

3.4 Chromatography setup for Purification of Bromelain 57

3.5 Flow diagram of laboratory spray dryer 59

3.6 Heat pump drying system 63

4.1 Precipitation kinetics in the range of 40 – 60% saturation 67

4.2 Moisture Profile in Freeze Dryer 74

4.3 Effect of inlet air temperature on activity retention 77

4.4 Effect of outlet air temperature on activity retention 78

4.5 Profiles of bromelain inactivation in pH 7.4 aqueous solution 83

4.6 Arrhenius plots of apparent first-order rate constants obtained

for bromelain inactivation

84

4.7 Fourier Transform IR spectra of the 4000 – 500 cm-1 region of

freeze dried bromelain

85

4.8 DSC thermogram of freeze dried bromelain obtained at a

heating rate of 10ºC/min

86

4.9 The pH dependence of the proteinase activity of purified fruit

bromelin. Casein was used as substrate

87

4.10 Temperature dependence of the protease activity of purified

fruit bromelain. Casein was used as substrate

88

4.11 Effect of time on Reaction velocity 89

4.12 Time and Temperature stability of freeze dried bromelain 90

Chapter one

Introduction

Introduction

1.1 BIOMATERIALS – AN OVERVIEW

In general, it is assumed that a biomaterial is a substance which is related to a living

organism, its vital functions and the activities. Most frequently, it is a single cell or a system

of cells which form a living organism. However, this can be a structural part of a cell or an

organism which constitutes a compact entity; a component built into various structural parts

of the organism, or a substance which is formed or is subjected to transformations as a result

of processes in which living organisms are involved (Kudra and Strumillo, 1998).

In the literature the term ‘bio – product’ is regarded as equivalent to a ‘bio – material’ and

denotes a substance which is a product of biotechnological transformation with the use of

microorganisms, their active elements or biochemically active substances. Biomaterial can

also be defined as biologically active material.

Biopolymers are related to bio – material, bio – product and biotechnology. Biopolymer

covers proteins, nucleic acids, sugars, lipids, enzymes, etc.

1.1.1. Enzymes

Enzymes are proteins with catalytic activity allowing chemical reactions in a living cell to

occur at ambient temperature at a high rate. Every biotechnological process is a based on

enzymatic reactions. Enzymes catalyze thermodynamic reactions by altering the activation

energy of substrates, i.e. the energy necessary to complete a given chemical reaction. Thus,

they enhance reaction equilibrium. Besides the active form, the enzymes may be inactive

and can be activated when needed. The structure of enzymes is similar to the structure of

amino acids and proteins. The central region directly engaged in a biochemical reaction is

Crystallization and Drying studies of Biomaterials 1

Introduction

called the active centre. This region includes atoms or groups of atoms which control

enzymatic activity, e.g. ions of metals, lipids, hydrocarbons, etc. The enzymatic reaction

rate is affected by the factors including duration of the reaction, temperature, pH, enzyme

stability, substrate concentration and presence of activators. Because enzymes are proteins,

their structures change easily under the action of the above mentioned factors which may

reduce even completely their catalytic ability.

Based on mechanism of biochemical reaction, a large variety of enzymes are commonly

categorized into six basic groups: oxidoreductases, tranferases, hydrolases, lyases,

isomerases and ligases. Due to enzymatic treatment, the source materials are used

economically, processes are more efficient, production cycles are shortened, product’s

quality and durability are improved, and reaction rates can be easily controlled. For these

reasons, production of enzymatic preparations is one of the most profitable branches of

biotechnology.

1.2 CRYSTALLIZATION AS A PURIFICATION STEP

1.2.1 Protein Crystallization

Crystallization is an aspect of precipitation, obtained through a variation of the solubility

conditions of the solute in the solvent, as compared to precipitation due to chemical reaction.

A solution system remains at equilibrium until the point is reached where there is

insufficient solvent to maintain full hydration of the solute molecules. If more solute is

added to the solution, the system is no longer at equilibrium under this so-called

supersaturated state. The system will be thermodynamically driven toward a new


Introduction

equilibrium corresponding free energy minimum by forming a solid phase. The solid phase

can appear as either amorphous precipitate or crystals. The principle of crystallization is

similar for small molecules (e.g., salts and small organic compounds) and macromolecules

(e.g., proteins, DNA, and RNA). Three stages are common to all crystallizations: nucleation,

crystal growth, and cessation of crystal growth. In nucleation molecules or non-crystalline

aggregates (dimers, trimers, etc.) produce a stable aggregate with a repeating structure. The

nucleus must first exceed a specific size, called the critical size, before it is capable of

further growth. However, the formation of crystal nuclei from supersaturated solution does

not necessarily result in the subsequent formation of macroscopic crystals. Cessation of

crystal growth can occur for many reasons. The most obvious reason is that the equilibrium

between the crystalline and soluble form is achieved.

The solubility of a protein can be described by a phase diagram as shown in Figure 1.1.

The solubility curve, S, divides unsaturated from supersaturated areas. On the curve proteins

are in equilibrium between solution and crystal, they will not crystallize beneath S. The

supersaturated are is divided into three zones:

Zone 1 (Metastable zone): The solution may not nucleate for a long time but this zone will

sustain growth. It is frequently necessary to add a seed crystal.

Zone 2 (Nucleation zone): Protein crystals nucleate and grow.

Zone 3 (Precipitation zone): Proteins do not nucleate but precipitate out of solution.


Introduction

Figure 1.1 Solubility curve of a protein

The crystallization of proteins is usually more difficult than small molecule crystallization.

This is probably a result of the difficulty to obtain a high quality protein sample for

crystallization. Because precise protein–protein contacts are needed for the crystallization of

proteins, any factor that introduces heterogeneity into the protein sample may have

interfering effects on the crystallization. There are numerous factors that can introduce

heterogeneity into a protein sample including differential glycosylation on the protein

molecules, different conformations of the protein can be present, contamination in the form

of other proteins, protein denaturation, and protein degradation (Wicknick, 2001).

The basic strategy to crystallize proteins is to bring the system into a state of limited degree

of supersaturation. This can be done, for example, by removing the solvent, adding a

precipitating agent, or by altering some physical properties such as temperature. However,

there are dozens of other parameters that are involved in protein crystallization.


Introduction

1.2.2 Factors affecting Crystallization

Crystallogenesis is affected by a range of factors, in other words, crystallization is a

multifactorial process.

1. Purity of proteins: Proteins need not be absolutely pure to give crystals, crystals from

impure solutions, however, frequently give poor diffraction data. To get good quality

crystals, proteins need not only to be reasonable free of contaminating proteins but

“conformationally pure”. Denatured proteins are more disruptive to crystal growth than

unrelated proteins. An important purity issue is that all traces of protease must be

removed from the samples as incubation times are frequently long.

2. Substrates and co-factors: Crystallization is often enhanced by inclusion of ligands and

enzyme substrates. These stabilizes the quaternary structure of the protein and promote

lattice packing.

3. pH: The net charge on the surface of a protein is an important determinant of lattice

interactions. Hydrophobic interactions are not so important in crystal packing.

4. Temperature: Significant entropy changes occur on crystallization, so temperature has a

significant effect. In crystallization trials one normally evaluates several temperatures

over the stability range of the protein.

5. Protein Concentration: Changes in protein concentration affect the kinetics of approach

to supersaturation and can also affect crystal morphology.

6. Nature of crystallizing agent (Precipitant): The most widely used precipitants are

ammonium sulphate and potassium phosphate. Other common ones are polyethylene

glycol (PEG) 6000, acetone and ethanol. The nature of the precipitant affects the kinetics


Introduction

of approach to supersaturation. This approach is usually slower with PEG than with salts,

for instance.

1.2.3 Applications of protein crystallization

• Structure determination

Protein crystallization is mainly used for structure determination by X-ray crystallography

where large, high quality crystals are needed. Obtaining high quality diffractive crystals is

the bottleneck in protein structure determination. For structure determination purposes

protein crystals are most often crystallized by small-scale microdiffusion methods. Very

often crystallization conditions are found after setting up hundreds or thousands of

individual crystallization conditions employing the “trial and error” approach, as

crystallization is often considered as a necessary evil in the structure determination work.

The structural analysis is based on the principle that in a perfect crystal all the molecules

have the same conformation and orientation. When X-rays are focused through protein (or

any other pure substance) crystal, individual atoms diffract the rays. The number of

electrons in each atom determines the intensity of the scattering of X-rays and thus, the X-

rays striking particular atoms will all be diffracted in the same way. A diffraction pattern is

generated by the interference of individual X-rays. A series of diffraction patterns are taken

from several angles and these patterns represent the way atoms are arranged in the molecule

and can be used to determine the structure of the protein. There are currently more than

18000 protein structures in the Protein Data Bank (PDB) (Berman et al., 2000), which have

been determined by X-ray diffraction analysis.


Introduction

• Protein purification

Crystallization is an efficient protein purification method. However, purification by

crystallization is relatively rare even though the quality of the crystals is not crucial.

Chromatographic methods have replaced protein crystallization in protein purification

although the latter was commonly used earlier. However, protein purification by

crystallization has many advantages: high yield, high purity in one step, unlimited scale up

possibilities, and the product is highly concentrated protein crystal slurry ready for further

formulation. Some industrial proteins, like xylose isomerase (EC 5.3.1.5) (Visuri, 1987),

cellulase (EC 3.2.1.4) (Nilsson et al., 1998) and protease (Gros and Cunefare, 2001) have

been purified by crystallization in large – scale.

• Medical applications

A majority of small molecular weight drugs are produced in crystalline form because of the

high storage stability, purity and reproducibility of the drug properties (Hancock and

Zografi, 1997). There are hundreds of macromolecular therapeutic agents used in clinical

trials or approved as drugs. However, only insulin is produced and administered in a

crystalline form (Jen and Merkle, 2001). The crystallization of macromolecular

pharmaceuticals can offer significant advantages, such as: a) protein purification by

crystallization as presented above; b) high stability of the protein product compared with

soluble or amorphous forms; c) crystals are the most concentrated form of proteins, which is

a benefit for storage, formulation and for drugs that are needed in high doses (e.g.,

antibiotics); d) the rate of crystal dissolution depends on the morphology, size and additives;

thus, crystalline proteins may be used as a carrier – free dosage form.


Introduction

• Cross-linked protein crystals (CLPCs)

In many applications crystallized proteins are not suitable for use as such, as a result of their

fragility and solubility. In order to produce a crystalline protein matrix that is insoluble also

in other conditions than those used in crystallization, the crystals have to be chemically

cross-linked. Cross-linking of enzyme crystals brings about both stabilization and

immobilization of enzyme without dilution of activity. In general, chemical cross-linking of

protein crystals creates an active and microporous protein matrix that can be used in

catalytic and separation applications (Roy and Abraham, 2006).

1.3 INTRODUCTION TO DRYING TECHNIQUES

Enzymes are obtained from plants, animals and from microorganisms as a result of

fermentation processes (Poutanen, 1997). In the presence of water proteins can undergo a

variety of chemical and physical degradation reactions (Manning et al., 1989) and one of the

ways to achieve long term stability is to dry protein formulations (Pikal, 1990). The final

stage of downstream processing is the removal of water by dehydration or the stabilization

in solution.

Since most enzymes are not stable in solution, drying is often used to improve the stability

during storage. Drying of enzymes may be performed according to the following methods

(Strumillo et al., 1991): spray drying, vacuum drying, heat pump drying and freeze drying.

The choice of drying method depends on the quality parameters of enzyme, production

output, end applications and the cost of drying. Although freeze drying is usually favoured

for drying of high – valued and thermally labile enzymes in small quantities, spray drying is


Introduction

largely applied in large scale production of enzymes because of its much lower cost

(Yamamoto and Sano, 1992). The design of a proper drying process should guarantee a high

level retention of enzyme activity. Generally, enzyme activity after drying is a function of

the composition of the initial liquid to be dehydrated, the process parameters and the

physicochemical characteristics of the enzyme. So, the drying of each enzyme should be

considered on an individual basis (Sadykov et al., 1997).

1.3.1. Freeze Drying

Freeze drying (lyophilization) is increasingly used in the biotechnology and pharmaceutical

industries for preparation and storage of many therapeutic proteins, as well as labile

enzymes (Liapis and Bruttini, 1995).

The process consists of two major steps: freezing of a protein solution and drying of the

frozen solid under vacuum. The drying step is further divided into two phases: primary and

secondary drying. The primary drying removes the frozen water and the secondary drying

removes the non – frozen ‘bound’ water (Arakawa et al., 1993).

A glassy material is formed following crystallization of ice during the freeing process. As

water becomes ice, freeze concentration takes place and the uncrystallized mixture gets

concentrated and changes from a ‘viscous liquid’ to a ‘brittle glassy’ structure, i.e. the

material undergoes the so called “glass transition”. The temperature where this transition

occurs in the maximally freeze – concentrated mixture is known as Tg, termed as the ‘glass

transition of the maximally freeze concentrated solute’ (Levine and Slade, 1988). Thus, non


Introduction

– crystallizing solutes, such as proteins, remain with glassy matrix and become kinetically

frozen in.

Formation of a glass is marked by a drop in the rates of molecular diffusion, due to

increased viscosity that helps to reduce the degree of conformational distortion due to

drying (Pikal, 1990). Also, the lower initial temperature keeps unwanted reactions between

amino acid reactive groups to a minimum.

Thus, the principle advantages of lyophilization as a drying process are:

• Minimum damage and loss of activity in delicate heat-liable materials

• Speed and completeness of rehydration

• Possibility of accurate, clean dosing into final product containers

• Porous, friable structure

The principle disadvantages of lyophilization are:

• High capital cost of equipment

• High operating costs

• Long process time

1.3.2. Spray Drying

Spray drying is a convective drying technique that uses hot air to transfer heat and remove

the water evaporated. It is a short time process in the range of few seconds and if process

conditions are optimized and stabilizers are added, it can be suitable even for heat sensitive

enzymes (Carpenter and Crowe, 1989; Yamamoto and Sano, 1992).


Introduction

The process may be summarized in three phases: spray formation, drying and powder

separation. The well mixed co-current system with the air disperser is preferred to avoid

strong heat impact on drying of enzymes (Strumillo et al., 1991).

1.3.2.1. Prediction of enzyme activity retention

Design and choice of operating conditions for spray drying of heat – sensitive products, are

still largely empirical. Therefore, the prediction of activity retention via simulation is of

considerable importance for the drying of enzymes. To predict the degree of enzymes

inactivation during drying, the thermal inactivation kinetics determined at different water

content must be integrated with drying models (Banga and Singh, 1994; Liou et al., 1985).

The kinetic parameters of enzyme inactivation should be considered in relation to

temperature, moisture content and time of exposure to heat.

Generally the inactivation process of enzymes is assumed to be a first order reaction (Luyben

et al., 1982; Yamamoto and Sano, 1992) and the dependence of rate constants on temperature

is described by the Arrhenius equation (Meerdink and Van’t Riet, 1991; Sadykov et al.,

1997).

Several authors have studied the simulation of enzymes degradation during drying.

A theoretical description of inactivation of enzymes during spray drying has been done based

on literature data concerning inactivation behaviour of several enzymes during the spray

drying of milk (Wijlhuizen et al., 1979).


Introduction

Glucose oxidase, β – galactosidase and alkaline phosphatase retention during drying of a

single suspended droplet could be predicted on the basis of a model calculation which

included the inactivation rate constants, the water diffusivity and water activity as a function

of water content and temperature (Yamamoto and Sano, 1992). The model calculations and

the experimental results showed that lower temperature and small droplets allow higher

enzyme retention.

In a collaborative European research program, a model solution with sensitive tracers was

established to characterize and compare different spray driers with respect to their effect on

enzyme activity retention. Results indicated that α – amylase and peroxidase were stable

during spray drying while tyrosinase was significantly affected. Low outlet temperature was

found to be important for the preservation of enzymes.

Alkaline phosphatase activity, moisture content and bubble volume in spray dried condensed

milk were measured experimentally and compared with predictions from a mathematical

model, including bubble formation during spray – drying. Enzyme activity decreased with

increasing outlet air – temperature (Etzel et al., 1996).

The advantages of spray dryers are that this technique can

• handle heat sensitive, non – heat sensitive and heat – resistant pumpable fluids as feed

stocks from which a powder is produced.

• produce dry material of controllable particle size, shape, form, moisture content and

other specific properties irrespective of dryer capacity and heat sensitivity.

• provide continuous operation adaptable to both conventional and PLC control.


Introduction

• handle wide range of production rates i.e. any individual capacity requirements can be

designed by spray dryers.

• provide extensive flexibility in spray dryer design, such as drying of aqueous feed stocks,

drying of toxic materials, etc.

However, they also offer some limitations, such as

• high installation costs.

• lower thermal efficiency

• product deposit on the drying chamber may lead to degraded product or even fire hazard.

Table 1.1 gives the summary of comparison among the spray drying and freeze drying operations.

Table 1.1: Comparison among spray drying and freeze drying (Filkova et al., 2004)

Parameter Spray Dryer Freeze Dryer

Drying time Short Long

Powders Agglomerated or irregular Cake

Product quality Medium Good

Energy consumption Low High

Product capacity High Medium

Operation Continuous Batch

Installation Cost Medium High


Introduction

1.3.3. Heat Pump Drying

Drying systems incorporating a dehumidification cycle, called heat pump dryer (HPD), have

been developed that not only accelerate the drying process and preserve the quality of the

product by drying at low temperature but also conserve energy of the drying processes. The

heat pump recovers the sensible and latent heats by condensing moisture from the drying air.

Consequently, partial vapour pressure in the drying air decreases which increases the driving

potential of evaporated moisture. The recovered heat is recycled back to the dryer by heating

the drying air.

There are several advantages as well as limitations associated with heat pump – assisted

dryers. Some of these can be offset by using hybrid drying technologies. Following are some

of the advantages (Kiang and Jon, 2004):

• Heat pump drying (HPD) offers one of the highest Specific Moisture Extraction Rate

(SMER) often in the range of 1.0 to 4.0, since heat can be recovered from the moisture –

laden air.

• Heat pump dryers can significantly improve product quality by drying at low

temperatures. At low temperatures, the drying potential of the air can be maintained by

further reduction of the air humidity.

• A wide range of operating conditions typically in the range of – 20 ºC to 100 ºC (with

auxiliary heating) and relative humidity 15 to 80% (with humidification system) can be

generated.


Introduction

• Excellent control of environment for high value products and reduced electrical energy

consumption for low value products.

Among the limitations are the following:

• Higher initial capital cost and maintenance cost due to need to maintain compressor,

refrigerant filters and changing of refrigerant.

• Leakage of refrigerant.

• Marginally complex operation relative to simple convection dryer.

• Additional floor space requirement

1.4 OBJECTIVES OF WORK

The present work deals with the purification by precipitation followed by

crystallization or drying of plant protease named, Bromelain, from the fruit, stem and leaf

portion of pineapple plant (Ananas comosus, Family: - Bromeliaceae).

• To crystallize crude bromelain from pineapple extract using different precipitants

like ammonium sulfate, sodium chloride and acetone.

• To study precipitation kinetics using ammonium sulfate as precipitant.

• To purify bromelain from fruit portion of pineapple plant (Ananas comosus) using

ion exchange chromatography.

• To study enzyme inactivation kinetics in aqueous solution.

• To obtain purified bromelain in dry powder form using different drying techniques

such as freeze drying, spray drying and heat pump drying.


Introduction

• To see the effect of drying temperature on retention of enzyme activity and protein

content in all the three drying operations.

• To find the optimum pH and temperature for bromelain.

• To carry out FTIR study and DSC study of bromelain powder.


Introduction

1.5 REFERENCES

Arakawa, T., Prestrelski, S.J., Kenney, W.C., Carpenter, J.F. (1993). Factors affecting

short – term and long – term stabilities of proteins. Advance Drug Delivery Reviews, 10,

1 – 28.

Banga, J.R., Singh, R.P. (1994). Optimization of air drying of foods. Journal of Food

Engineering, 23, 189 – 211.

Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H.,

Shindyalov, I.N., Bourne, P.E. (2000). The protein data bank. Nucleic Acids Research,

28, 235 – 242.

Carpenter, J.F., Crowe, W.H. (1989). An infrared spectroscopic study of the interactions

of carbohydrates with dried proteins. Biochemistry, 28, 3916 – 3922.

Etzel, M.R., Suen, S.Y., Halverson, S.L., Budijono, S. (1996). Enzyme inactivation in a

droplet forming a bubble during drying. Journal of Food Engineering, 27, 17 – 34.

Gros, E.H., Cunefare, J.L. (2001). Crystalline protease and method for producing same,

US Patent 6207437.

Hancock, B.C., Zografi, G. (1997). Characteristics and significance of the amorphous

state in pharmaceutical systems. Journal of Pharmaceutical Science, 86, 1 – 12.


Introduction

Fikova, I., Huang, L.X., Mujumdar. A.S. (2004). Heat Pump – Assisted Drying, pp.215

– 256. In: Handbook of Industrial drying (Second Edition), A.S. Mujumdar (Ed.),

Mercel Dekker Inc., New York and Basel.

Kiang, C.S., Jon. C.K. (2004). Heat Pump Drying Systems. pp. 1103 – 1132. In:

Handbook of Industrial drying (Second Edition), A.S. Mujumdar (Ed.), Mercel Dekker

Inc., New York and Basel.

Jen, A., Merkle, H.P. (2001). Diamonds in the rough: protein crystals from a

formulation perspective. Pharmaceutical Research, 18, 1483 – 1488.

Kudra, T., Strumillo, C. (1998). Characteristics of Bio-materials, In Thermal Processing

of Bio-materials. T. Kudra, C. Strumillo (Ed.), 12-13. Amsterdam (The Netherlands):

Gordon and Breach Science Publishers

Levine, H., Slade, L. (1988). Principles of cryostabilization technology from

structure/property relationships of carbohydrate/water systems – A review. Cryo –

Letters, 9, 21 – 63.

Liapis, A.I., Bruttini, R. (1995). Freeze – drying, pp.309 – 343. In: Handbook of

Industrial drying (Second Edition), A.S. Mujumdar (Ed.), Mercel Dekker Inc., New

York and Basel.

Liou, J.K., Luyben, K.Ch.A.M., Bruin, S. (1985). A simplified calculation method

applied to enzyme inactivation during drying. Biotechnology and Bioengineering, 27,

109 – 116.


Introduction

Luyben, K.Ch.A.M., Liou, J.K., Bruin, S. (1982). Enzyme degradation during drying.

Biotechnology and Bioengineering. 24, 533 – 552.

Manning, M.C., Patel, K., Borchardt, R.T. (1989). Stability of protein pharmaceuticals.

Pharmaceutical Research, 6, 903 – 918.

Meerdink, G., Van’t Riet, K. (1991). Inactivation of a thermostable alpha – amylase

during drying. Journal of Food Engineering, 14, 83 – 102.

Nilsson, B.M., Laustsen, M.A., Rancke – Madsen, A. (1998). Separation of proteins. US

Patent 5728559.

Pikal, M.J. (1990). Freeze – drying of proteins. Part I. Process design. BioPharm, 3 (8),

18–27.

Poutanen, K. (1997). Enzymes: An important tool in the improvement of the quality of

cereal foods. Trends in Food Science and Technology, 8, 285 – 320.

Roy, J.J., Abraham, T.E. (2006). Preparation and characterization of cross – linked

enzyme crystals of laccase. Journal of Molecular Catalysis B: Enzymatic, 38, 31 – 36.

Sadykov, R.A., Pobedimsky, D.G., Bakhtiyarov, F.R. (1997). Drying of bioactive

products: Inactivation kinetics. Drying Technology, 15, 2401 – 2420.

Strumillo. C., Markowski, A.S., Adamiec, J. (1991). Selected aspects of drying of

biotechnological products, pp. 36 – 55. In: Drying 91, A.S. Mujumdar and I. Filkova

(Eds.), Elsevier Science publishers, Amsterdam.


Introduction

Visuri, K. (1987). Stable glucose isomerase concentrate and a process for the thereof.

US Patent 4699882.

Wicknick, J. A. (2001). Protein Crystallization, pp. 267 – 284. In: Handbook of

Industrial Crystallization (Second Edition), Allan S. Myerson (Ed.), Butterworth

Heinemann, USA.

Wijlhuizen, A.E., Kerkhof, P.J.A.M., Bruin, S. (1979). Theoretical study of the

inactivation of phosphatase during spray drying of skim – milk. Chemical Engineering

Science, 34 (5), 651 – 660.

Yamamoto, S., Sano, Y. (1992). Drying of enzymes: Enzyme retention during drying of

a single droplet. Chemical Engineering Science, 47, 177 – 183.


Chapter Two

Bromelain – A

Literature Overview

Bromelain – A literature overview

2.1. INTRODUCTION TO PROTEASES

The commercial exploitation of enzymes ranges from very high volume of low cost

enzymes for the industrial purpose such as detergent enzymes to highly purified low

volume high cost enzymes for medicinal and therapeutic purposes. The market is

generally divided as industrial enzymes, enzymes for medicinal use and enzymes for

analytical and diagnostic purposes. Although about 3000 enzymes have been isolated and

characterized and only 300 enzymes are commercially available. The vast majority of

enzymes used industrially are hydrolases. They constitute 85% of total enzymes,

whereas, remaining 15% being divided between oxidoreductases and isomerases. Of the

hydrolyzes, 70% hydrolyze proteins (proteases), 26% hydrolyze carbohydrates and 4%

hydrolyze lipids (Fowler, 1988). The industrial enzyme market consists of various

enzymes and are listed in the following Table 2.1.

Among the hydrolases, proteases are the single class of enzymes which occupy a pivotal

position with respect to their applications in both physiological and commercial fields.

Proteolytic enzymes catalyze the cleavage of peptide bonds in other proteins. Proteases

are degradative enzymes which catalyze the total hydrolysis of proteins. Advances in

analytical techniques have demonstrated that proteases conduct highly specific and

selective modifications of proteins such as blood clotting and lysis of fibrin clots and

processing and transport of secretory proteins across the membranes.



Table 2.1 Sale of enzymes in the market (Fowler, 1988)

Enzyme type Fraction of sales %

Bacillus protease 30-35

Glucoamylase 8-10

Bacillus amylase 10-12

Glucose isomerase 5-7

Calf rennet 10-12

Microbial rennet 2-4

Pectinase 4-5

Pancreatic, trypsin 2-4

Papain, bromelain 4-6

Lipase 2-3

Others (invertase, lactase, hemicellulase, 5-10

lysozyme, penicllin acylase)

Their involvement in the life cycle of disease-causing organisms has led them to become

a potential target for developing therapeutic agents against fatal diseases such as cancer

and AIDS. Proteases have a long history of application in the food and detergent

industries. Their application in the leather industry for dehairing and bating of hides to

substitute currently used toxic chemicals is a relatively new development and has

conferred added biotechnological importance. The vast diversity of proteases, in contrast

to the specificity of their action, has attracted worldwide attention in attempts to exploit



their physiological and biotechnological applications (Rao et al., 1998). Interest in

proteases has increased with the realization that they play key roles in rheumatoid

arthritis and cancer metastasis.

2.2. SOURCES OF PROTEASES

Since proteases are physiologically necessary for living organisms, they are ubiquitous,

being found in a wide diversity of sources such as plants, animals, and microorganisms.

The most familiar proteases of animal origin are pancreatic trypsin, chymotrypsin, pepsin

and rennin. The inability of the plant and animal proteases to meet current world

demands has led to an increased interest in microbial proteases. Microorganisms

represent an excellent source of enzymes owing to their broad biochemical diversity and

their susceptibility to genetic manipulation. Microbial proteases account for

approximately 40% of the total worldwide enzyme sales. Proteases from microbial

sources are preferred to the enzymes from plant and animal sources since they possess

almost all the characteristics desired for their biotechnological applications. Most

commercial proteases, mainly neutral and alkaline are produced by organisms belonging

to the genus Bacillus (bacteria), Aspergillus (Fungi) (Rao et al., 1998).

The use of plants as a source of proteases is governed by several factors such as the

availability of land for cultivation and the suitability of climatic conditions for growth.

Moreover, production of proteases from plants is a time-consuming process. Papain,

bromelain, keratinases and ficin represent some of the well-known proteases of plant

origin. Among the plant enzymes, papain is the single industrial product available in

large quantity.



2.3. CLASSIFICATION OF PROTEASES

According to the Nomenclature Committee of the International Union of Biochemistry

and Molecular Biology, proteases are grossly subdivided into two major groups, i.e.,

exopeptidases and endopeptidases, depending on their site of action. Exopeptidases

cleave the peptide bond proximal to the amino or carboxy termini of the substrate,

whereas endopeptidases cleave peptide bonds distant from the termini of the substrate.

Based on the functional group present at the active site, proteases are further classified

into four prominent groups, i.e., Serine proteases, Aspartic proteases, Cysteine proteases,

and Metalloproteases.

As the present work deals with one of the medicinally as well as industrially important

plant cysteine protease named as bromelain (from the pineapple fruit), the further

information is given only for the cysteine proteases.

2.4.CYSTEINE OR THIOL OR SULFHYDRYL PROTEASES

Cysteine proteases occur in both prokaryotes and eukaryotes. About 20 families of

cysteine proteases have been recognized. Distribution of cysteine proteases is very wide

in both plant and animal kingdoms, ranging from the bacteria (peptidase-B of

Clostridium) through many higher plant families: genera and species of these include

Carica papaya, the tropical paw-paw, source of papain; Bromelia penguin, source of

penguinain; Asclepia, milkweeds containing asclapain in stem and roots; Ficus, tropical

“fig” trees of several species, the source of ficins and Ananas comosus (Pineapple the

king of fruits), source of bromelain. In animals, the cathepsins and streptococcal



proteinase from bacteria are reasonably similar. Each of these several proteases differ in

degree relative to composition and activity: examples are the bromelains, papain and the

ficins, which contrast greatly in origin but nevertheless shows remarkable homology in

the amino acid sequences around the reactive, essential cysteine site of each (Murachi,

1964). It should be noted, however, that not all plant proteases are sulfhydryl enzymes.

Thus, solanain (from the berries of the horsenettle), arachain (from the cotyledon and

embryo of the peanut) do not require activation by sulfhydryl reagents and are not

affected by mild oxidizing agents. Several mammalian lysosomal cathepsins, and the

cytosolic calpains (calcium-activated) as well as several parasitic proteases (e.g.

Trypanosoma, Schistosoma) also belong to this class (Drenth et al., 1971). The activity of

all cysteine proteases depends on a catalytic dyad consisting of cysteine and histidine.

The order of Cys and His (Cys-His or His-Cys) residues differ among the families.

Generally, cysteine proteases are active only in the presence of reducing agents such as

HCN or cysteine (sulfhydryl containing reagents). Papain is the best-known cysteine

protease. Cysteine proteases have neutral pH optima, although a few of them, e.g.,

lysosomal proteases, are maximally active at acidic pH. They are susceptible to

sulfhydryl agents such as Para Chloro Mercury Benzoate (PCMB) but are unaffected by

(Di isopropyl Fluoro Phosphate (DFP) and metal – chelating agents. Papain was the first

recognized member of the class of proteolytic enzymes (cysteine proteases) that need free

sulfhydryl group for activity. These enzymes usually need an activator for activity which

has a function of releasing the blocked SH groups (Drenth et al., 1971). Like the serine

proteinases, catalysis proceeds through the formation of a covalent intermediate and

involves a cysteine and a histidine residue. The essential Cys25 and His159 (papain



numbering) play the same role as Ser195 and His57 respectively. The detail mechanism

of action for cysteine proteases is described below (Rao et al., 1998). All these plant

proteases have broader specificities than trypsin but peptides of positively charged amino

acids are preferred (Reed, 1966).

2.4.1.Mechanism of Action of Cysteine Proteases

The mechanism of action of proteases has been a subject of great interest to researchers.

Purification of proteases to homogeneity is a prerequisite for studying their mechanism of

action. Vast numbers of purification procedures for proteases, involving affinity

chromatography, ion-exchange chromatography, and gel filtration techniques, have been

well documented.

Cysteine proteases catalyze the hydrolysis of carboxylic acid derivatives through a

double-displacement pathway involving general acid-base formation and hydrolysis of an

acyl-thiol intermediate. The mechanism of action of cysteine proteases is thus very

similar to that of serine proteases. A striking similarity is also observed in the reaction

mechanism for several peptidases of different evolutionary origins. The plant peptidase

papain can be considered the archetype of cysteine peptidases and constitutes a good

model for this family of enzymes. They catalyze the hydrolysis of peptide, amide ester,

thiol ester, and thiono ester bonds. The initial step in the catalytic process (Figure 1)

involves the noncovalent binding of the free enzyme and the substrate to form the

complex. This is followed by the acylation of the enzyme, with the formation and release

of the first product, the amine. In the next deacylation step, the acyl-enzyme reacts with a

water molecule to release the second product, with the regeneration of free enzyme. The



presence of a conserved aspargine residue (Asn175) in the proximity of catalytic histidine

(His 159) creating a Cys-His-Asn triad in cysteine peptidases is considered analogous to

the Ser-His-Asp arrangement found in serine proteases.

Figure 2.1 Catalytic mechanism of cysteine proteases



2.5. BROMELAINS

The bromelains are proteases occurring as glycoproteins from the pineapple plant,

Ananas comosus L. (Merr.), as well as in related genera of the Bromeliaceae. Pineapple is

a perennial herb native to tropical America. Principal sources today include Hawaii,

Japan and Taiwan. The existence of bromelain was probably first established in 1891 by

Chittenden (Chittenden, 1891) who salted it out of the juice and studied its action in

considerable detail. Later on Heinicke and Gortner (1957) showed that the juice of the

stem of the pineapple plant is a rich source of proteolytic enzymes. The properties of the

bromelain lead to conclusion that it is similar to papain (Balls et al., 1941). Bromelain is

probably the first proteolytic enzyme of plant origin to be reported as glycoprotein

(Murachi et al., 1964). In 1950, the Pineapple Research Institute of Hawaii began a study

of the proteases of the pineapple plant. They found that not only all varieties of

commercial pineapples contain proteases, but that probably all species of genera of the

family Bromeliaceae contains similar, but probably slightly different, proteases.

Furthermore, they found that the proteases of the fruit, the leaves, and the stems

represented different mixtures of proteases. To avoid coining, several hundred new plant

protease names based on species name, Heinicke and Gortner (1957) suggested that the

name bromelin replace by bromelain and be the generic name for any protease obtained

from any member of the family Bromeliaceae. They further suggested that the individual

preparations be distinguished by prefixing the binomial latin name of the plant source and

the organ yielding the enzyme. Thus the name for the proteases from the fruit of

commercial pineapple plant would be as Ananas comosus (L.) Merr. Var. Cayenne fruit



bromelain. Similarly the enzyme from stem part is Ananas comosus (L.) Merr. Var.

Cayenne stem bromelain (Collins, 1960).

2.5.1. Composition of pineapple fruit

The Pernambuco variety of pineapple appears to be richer in enzyme than the Cayenne,

but the latter is the only variety in industrial use. The bromelain content of pineapple

plants is as shown in Table 2.2. Bromelain is well distributed all over in the pineapple

plant. The enzyme bromelain is present in the leaves and stalks as well as in the fruit. The

enzyme from fruit part was first described as bromelain and now called as Fruit

Bromelain (EC 3.4.22.33) and that from stalk (stem) part is designated as Stem

Bromelain (EC 3.4.22.32). It is evident that the bromelain follows the juice, not the solid

matter. The enzyme is remarkably stable toward heat, as shown by the fact that the press

juice heated to 600C (less stable than papain) and screened thereafter still contained a

large proportion of the original enzyme in the active state (Balls et al., 1941).

The juice of the pineapple contains various amounts of polysaccharides and polyuronides.

These may range from 30 – 70%. The polyuronides are partially responsible for the high

viscosity of fruit juice. Furthermore, when present in the enzyme preparations, they

account for some of the strong binding of inorganic cations (Collins, 1960).



Table 2.2: Bromelain content of pineapple plants (press juice from hand press)

(Balls et al., 1941)

Variety Material % juice

obtained

Milk units/ml

juice(not

activated)

Milk units/ml

juice(activated)

Cayenne

Green Leaves & stems 10 0.6 0.6

Green Shell 36 --- ---

Green Inside 69 --- ---

Ripe Shell 41 0.5 0.6

Ripe Inside 62 1.5 0.9

Very Ripe Shell -- --- 1.022

Very Ripe Inside -- --- 0.8

Pernambuco

Green Leaves & stems 45 --- 1.4

Green Shell 42 --- 2.2

Green Inside 57 --- 3.6

Ripe Leaves 53 --- 3.1

Ripe Shell 48 --- 2.1

Ripe Inside 60 --- 3.12



2.5.1.1. Enzymes found in pineapple juice

Different enzymes from the pineapple juice have been reported. About half of the protein

in pineapple flesh is accounted by the protease bromelain. The enzyme is not present at

all during the early stages of fruit development and then increases very rapidly and stays

at a high level up to onset of ripening, at which there is marked decrease in activity.

Pineapple is unique among fruits in having high concentration of protease in the ripe

fruit. Unlike papain, bromelain doesn’t disappears as fruit ripens (Table 2.2). Papain has

high levels of enzyme in the green stage, but become completely inactive when the fruit

is fully ripe (Felton, 1971).

Other non proteolytic enzymes reported from pineapple fruit are IAA oxidase,

peroxidase, phosphatase, catalase, and cellulase (Dull, 1971). The other proteolytic

enzyme reported from pineapple is carboxypeptidase. Several protease inhibitors from

pineapple stem bromelain have also been reported.

The economic advantages in the manufacture of bromelain over papain are apparent.

Since, the fruit part is the more valuable part of plant, the enzyme is produced from the

parts of the plant unusable for food and thus, enzyme is usually a byproduct from

pineapple industry. The waste parts, leaves, cores (stems) and the skin of the fruit are

used as source for the preparation of bromelain. As material obtained is a byproduct of

another industry and always obtained from a single variety of the plant, it would therefore

be unusually uniform. The quantity made could be easily adjusted to suit the market, and

manufacture would require very little addition to the existing facilities for pineapple

canning. However, there are disadvantages in the manufacture of bromelain in place of



papain. The material used must be factory waste, since other pineapple products are more

valuable. The amount of enzyme present is small, and it would not pay to destroy the

sugar or citric acid even in the waste in order to get the bromelain (Balls et al., 1941).

2.5.2. Biochemical Properties of bromelain

Two kinds of bromelain are available commercially, stem bromelain and fruit bromelain.

Commercial bromelains are slightly soluble in water and glycerol but insoluble in most

organic solvents, are active in pH range from 3 to 10 with optima between 5 to 8,

depending upon the protein it is acting upon. It is most active at temperature range of 50-

600C and remains stable up to temperature of about 700C, whereupon it is inactivated.

2.5.2.1. Stability:

The enzyme retains full activity against casein when kept at 50C for 24 hours over a range

of pH from 4 to 10 (Inagami and Murachi, 1963). The enzyme is stable in 25% (v/v)

methanol at 250C for 20 minutes, while it looses 33% caseinolytic activity in 20% (v/v)

ethanol at 370C for 20 minutes. A 50% loss of the activity is caused by heating the

enzyme solution at 550C for 20 minutes at pH 6.1 (El-Gharbawi and Whitaker, 1963).

Lyophilization causes 27% decrease in activity (Murachi et al., 1964).

2.5.2.2. Physical Properties of bromelain

There is still uncertainty about the exact molecular weight of stem bromelain, as it was

found from literature that different author has given a different molecular weight for their

purified preparations. Using a further purified preparation, reexamination of the



molecular weight has been done by polyacrylamide gel electrophoresis in the presence of

SDS and also by sedimentation equilibrium ultracentrifugation by Takahashi et al.

(1973). Measured values range from 25,600 to 28,100 Dalton and in practice it is

recommended to adopt a tentative value of 28,000 Dalton.

Physical properties of bromelain protein reported by Yamada et al. (1976) are listed in

Table 2.3.

Table 2.3 Physical Properties of bromelain

Properties Fruit Bromelain Stem Bromelain

Molecular weight 31,000 28,000

Isoelectric point 4.6 9.55

Absorbance at 280 nm of 1% solution 19.2 20.1

Molar extinction coefficient 59,500 56,300

Sedimentation coefficient 2.75 s 2.77 s

Carbohydrate content None 2.1 %

Amino terminal sequence Ala-Val-Pro-GIn Val-Pro-Gln

Carboxyl – terminal residue Gly Gly

Specific activity towards casein 11.6 6.86

Similarly, the molecular weight of fruit bromelain is also a subject of controversy: MW

of 18,000 as determined by Sephadex G-75 gel filtration was reported by Ota et al.



(1972) and 31,000 by polyacrylamide gel electrophoresis in the presence of SDS and by

Sephadex G-75 gel filtration by Yamada et al. (1976).

Murachi et al. (1964) have found the isoelectric point of their bromelain preparation to be

at about pH 9.5 by isoelectric focusing technique. Wharton (1974) also gave an evidence

of the alkaline isoelectric point of stem bromelain. At pH values of 4.0, 7.0, and 9.0,

migrations takes place towards cathode and at pH 9.5, it was slightly towards anode

indicating that the isoelectric point is near to pH 9.5.

In contrast to stem bromelain, the isoelectric point of fruit bromelain is considerably

lower. Yamada et al. (1976) reported an isoelectric point of pH 4.6 for their purified fruit

bromelain fraction FA2 by isoelectric focusing technique.

2.5.2.3. Chemical Properties of bromelain

In Table 2.3, the amino acid composition of stem bromelain reported by different

investigators (Feinstein and Whitaker, 1964; Murachi, 1964; Husain and Lowe, 1968) is

shown. The stem enzyme is basic and amino acid analysis shows a relatively high content

of lysine and arginine as compared to amino acid composition of fruit bromelain (Ota et

al., 1964). The principal amino terminal residue is valine (Takahashi et al., 1973) and the

carboxyl terminal is glycine (Ota et al. 1972).

Stem bromelain contains four methionine residues per molecule; whereas, no methionine

is present in papain (Murachi, 1964). The abundance of basic amino acids is more

apparent in stem bromelain than papain. This is in accord with the finding that stem

bromelain has an isoelectric point higher than that for papain, pH 8.75 (Smith et al.,



1954). Stem bromelain has only one cysteinyl and histidyl residue per molecule whereas

papain has two histidyl residues (Murachi, 1976).

Table 2.3: Amino Acid composition of Stem and Fruit Bromelain

Amino acid Stem Bromelain Fruit Bromelain

1 2 3 4 5 6

Lysine 14 17 17 12 5 8

Histidine 1 1 1 1 1 1

Arginine 8 8 9 6 5 9

Aspartic acid 23 23 23 16 17 32

Threonine 10 10 11 8 8 14

Serine 21 21 22 16 18 30

Glutamic acid 19 17 18 12 13 25

Proline 11 13 11 8 7 10

Glycine 27 24 26 19 18 35

Alanine 30 26 28 20 14 25

Half-cystine 9 9 7 5 6 9

Valine 16 16 18 12 11 20

Methionine 3 3 4 2 3 5

Isoleucine 17 17 18 12 9 10

Leucine 7 8 8 5 4 10

Tyrosine 14 16 16 11 13 16

Phenylalanine 8 8 7 5 4 8

Tryptophan 7 7 6 5 3 7

Total (245) (244) (250) (179) (161) (282)

Ammonia (amide) -- 21 28 19 24 --

Glucosamine 2 2 2 4 <0.2 0

Carbohydrate (%) 2.1 2.1 0.9 2.0 3.2 0



Sources of values are as follows.

Column 1: (Takahashi et al., 1973). Nearest integral number of residues per mole of

28,000 for component SB 1. Column 2: (Murachi, 1964) Number of residues per mole of

MW 33,000 reported earlier for step 6 preparation has been recalculated on the basis of

MW of 28,000. Column 3: (Ota et al., 1972). Number of residues per mole of 28,000 for

component I-1. Column 4: (Feinstein and Whitaker, 1964) for component II, taken

methionine as two residues per molecule. Column 5: (Ota et al., 1972). Number of

residues per mole of 18,000 for component A. Column 6: (Yamada et al., 1976). Nearest

integral number of residues per mole of MW 31,000 for component FA2.

The amino acid composition of fruit enzyme is similar to stem enzyme with the notable

exception that it contains much less lysine, and smaller alanine content relative to glycine

(Yamada et al. 1976). This difference is reflected by in the isoelectric points of the two

proteins (Ota, 1966). The amino terminal residue is alanine (Yamada et al., 1976).

Three separate laboratories studied that the stem bromelain contained a small amount of

carbohydrate. Murachi et al. (1964) were first to observe that stem enzyme appears to be

a glycoprotein. They reported a carbohydrate content of 2% in the purified enzyme,

whereas Ota et al. (1964) reported 1.46% carbohydrate. Feinstein and Whitaker (1964)

who isolated several proteolytically active components from bromelain, found 2 to 4

moles of carbohydrate per mole of purified enzyme. Scocca and Lee (1969) have reported

the same oligosaccharide content of 2:1:1:2 of mannose, fucose, xylose and glucosamine

respectively for their purified fractions bromelain II and bromelain III which are

electrophoretically distinct one. Papain, Chymopapain and ficin contain no carbohydrate,



while among the proteolytic enzymes from animal origin plasmin and enterokinase have

been reported to contain 1.5% hexose and 41.1% carbohydrate, respectively (Murachi,

1964). Murachi et al. (1967) found that a glycopeptide prepared by digestion of

bromelain with Pronase, contained mannose, fucose, xylose, and glucosamine in the ratio

3:1:1:4.

In contrast to stem bromelain, which contains carbohydrates in its molecule, Yamada et

al. (1976) reported that fruit bromelain (purified by them called FA2) contained neither

amino sugar nor neutral carbohydrates determined by four different methods and stated

that FA2 is not a glycoprotein. While, Ota et al. (1964) reported that their purified fruit

enzyme though devoid of glucosamine contains about 3% carbohydrate which cannot be

removed by purification procedures.

2.5.3. Activators, inhibitors and chemical modifications

The crude bromelain enzyme shows about 25% of their maximum activity against casein

without any addition of activating agent (Ota et al., 1964). The enzyme can be fully

activated in the presence of 0.005M cysteine, 2-mercaptoethanol, or dithiothreitol

(Murachi, 1970). Ota et al. (1964) suggested the use of mercaptoethanol or cysteine plus

EDTA for the maximum proteolytic activity. But, Murachi and Neurath (1960) reported

that the addition of EDTA besides cysteine was not essential for full activity. Cysteine

and cyanide activate the enzyme to about the same degree, while H2S and Na2S produce a

much lower degree of activation. The reason behind the low activation by sulfides is

might be that the oxidized sulfhydryl groups of bromelain are not readily accessible by

sulfides (Greenberg and Winnick, 1940).



Stem bromelain as well as fruit bromelain are reversibly inhibited by inorganic mercuric

ion, organic mercurials, and tetrathionate. Murachi and Neurath (1960) gave evidence

that the inhibition by mercuric ion was instantaneous and could be completely reversed

by the addition of excess cysteine, suggesting that the sulfhydryl groups of the enzyme

protein are essential for its catalytic activity. Irreversible inactivation occurs when stem

bromelain is reacted with N-ethylmaleimide, N-(4-dimethyl-3,5-dinitrophenyl)

maleimide (DDPM), momoiodoacetic acid and 1,3-dibromoacerone. These reagents

alkylate the essential sulfhydryl group of the enzyme protein. Chloromethyl ketone

derivative of N-tosyl-L-phenylalanine (TPCK) and 1-chloro-3-tosylamido-7-amino-2-

heptanone (TLCK) also alkylate the –SH group, resulting in inactivation of the enzyme.

Diisopropylphosphofluoridate (DFP) does not inhibit stem bromelain but it

alkylphosphorylates the enzyme-protein at pH 8.2 without inhibition of the proteinases

activity (Murachi, 1970). Murachi and Neurath. (1960) also reported that 0.001M DFP in

propanol neither inhibits the enzyme nor does it change its activation by cysteine

suggesting that, in contrast to other endopeptidases, no reaction with active site had

occurred. The results of them are in agreement with those reported by Heinicke, who

suggested that DFP is a specific inhibitor of sulfhydryl proteases.

2.5.4. Specificity, kinetic properties, and enzymic mechanism

The specificity of the stem bromelain has been examined on a number of substrates. It

hydrolyses proteins like casein and haemoglobin at high rates, while a synthetic

substrates of smaller molecular size, like Benzoyl Arginine Ethyl Ester (BAEE) has also

shown to be hydrolyzed at moderate rate (Inagami and Murachi, 1963). Although the



enzyme shows similarities in its specificity to that exhibited by papain, significant

differences have also been observed. In comparison with papain, the pineapple enzyme

hydrolyzed casein equally well; haemoglobin is hydrolyzed four times faster and BAEE

and Benzoyl Argininamide (BAA) much more slowly (Murachi and Neurath, 1960;

Inagami and Murachi, 1963).

Basic amino acyl residues are preferred, but the preference is less strict than in the case of

papain (Murachi, 1970). A preliminary experiment carried out by the Inagami and

Murachi (1963) indicates that arginine derivatives like BAEE and BAA are the best

substrates among various amino acid esters and amides, respectively (Table 2.4). Among

the three substrates (esters of the L-arginine derivatives) examined by Inagami and

Murachi (1963).

Table 2.4: Comparison of BAEE and BAA hydrolysis catalysed by SH proteinases

(Inagami and Murachi, 1963)

Enzyme Substrate Km (M) kcat (sec-1)

Bromelain BAEE 0.17 0.50

BAA 0.0012 0.0035

Papain BAEE 0.0020 12

BAA 0.040 10

Ficin BAEE 0.025 1.0

BAA 0.048 0.9

(BAEE - Benzoyl Arginine Ethyl Ester; BAA - Benzoyl Argininamide)



The fruit enzyme is more active against BAA and BAEE than the stem enzyme (Ota et

al., 1964). The pH optima for casein, denatured hemoglobin are at 8.3 and pH 8.0,

respectively.

2.5.5. Application of Bromelain

Bromelain is said to possess wide variety of uses both in industrial purpose as well as

medicinal purpose. Industrial uses include as detergents; for dehairing and tanning in

leather industries; cleaning agent; for processing of raw silk and spot remover in textile

industries; and food related applications are summarized in Table 2.5.

Proteases from plant origin such as papain and bromelain are used frequently

interchangeably in such applications, choice depending upon price and availability.

Bromelain has been proved to be having wide range of biological activities, including

anti-inflammatory, burn debridement, smooth muscle relaxation, skeletal muscle

relaxation, inhibition of blood platelet aggregation, enhancement of antibiotic absorption,

prevention of ulcers, sinusitis relief, cancer prevention, and prevention of epinephrine

induced pulmonary oedema, etc.

Bromelain's anti-inflammatory activity appears to be due to a variety of physiological

actions. Evidence indicates that bromelain's action is in part a result of inhibiting the

generation of bradykinin at the inflammatory site via depletion of the plasma kallikrein

system, as well as limiting the formation of fibrin by reduction of clotting cascade

intermediates. Bromelain has also been shown to stimulate the conversion of

plasminogen to plasmin, resulting in increased fibrinolysis. Bromelain might be capable



of selectively modulating the biosynthesis of thromboxanes and prostacyclins, two

groups of prostaglandins with opposite actions which ultimately influence activation of

cyclic-3, 5-adenosine and an important cell-growth modulating compound. It is

hypothesized that bromelain therapy leads to a relative increase of the endogenous

prostaglandins, PGI2 and PGE2 over thromboxane A2.

Bromelain is absorbed intact through the gastrointestinal tract of animals, with up to 40

percent of the high molecular weight substances detected in the blood after oral

administration. The highest concentration of bromelain is found in the blood one hour

after administration; however, its proteolytic activity is rapidly deactivated.

Bromelain is generally considered safe, even at high doses. Bromelain can cause an

allergic reaction (red or itchy eyes, sneezing, running nose, irritated throat) in people who

are sensitive to it.

Table 2.5: Some food related uses of bromelains (Mehrlich, 1978)

Food

commodity

Uses

Beer Chill-proof; improve flavor; retain foam

Bread dough Reduce kneading time; baked goods; increase fluidity of glutin

gels

Eggs yolks Partial digestion to prevent gel formation in frozen state

Fats Deodorize

Fish Solubilize the protein

Meat pickling Improved corned meat products

Meat smoking Release of casings after processing

Meat

tenderizing

Antimortem injections



2.6 REFERENCES

Balls, A.K., Thompson, R.R., Kies, M.W. (1941). Bromelain: Properties and Commercial

Production. Industrial Engineering Chemistry, 33, 950 – 953.

Chittenden, R.H. (1891). On the proteolytic action of bromelin, the ferment of pineapple

juice. Journal of Physiology, 15, 249 – 310.

Collins, J.L. (1960). “The Pineapple”, Interscience Publishers Inc., New York, p. 253 –

254.

Drenth, J.N. Jansonius, J.N. Koekoek, R., Wolthers, D.G. (1971). “Papain, X-ray

Structure”, in The Enzymes, Ed.-P.D. Boyer, Edition-III, Vol.3, Academic Press, New

York, p.485 – 499.

Dull, G.G. (1971). “The Pineapple: General”, in The Biochemistry of Fruits and their

products, Ed-Hulme, A.C., Vol. 2, Academic Press, London, p. 303 – 309.

El-Gharbawi, M., Whitaker, J.R. (1963). Fractionation and partial characterization of the

proteolytic enzymes of stem bromelain. Biochemistry, 2, 476 – 481.

Feinstein, G., Whitaker, J.R. (1964). Molecular weights of the proteolytic enzymes of

stem bromelain. Biochemistry, 3 (8), 1050 – 1054.

Felton, G.E. (1971). “Pineapple Juice”, in Fruit and vegetable juice processing

technology, Edition-II, Eds-Tressler, D.K. and Joslyn, M.A., The Avi Publishing

Company Inc, Westport, Connecticut, p. 180.



Fowler, M.W. (1988). “Enzyme Technology”, in Biotechnology for Engineers, Ed -

Scragg, A.H., Ellis Horwood Ltd., Britain, p. 171-182.

Greenberg, D.M., Winnick, T. (1940). Plant Proteases: I. Activation – Inhibition

Reactions. Journal of Biological Chemistry, 135, 761 – 787.

Heinicke, R.M., Gortner, W.A. (1957). Stem Bromelain – A new protease preparation

from pineapple plants, Economical Botany, 11, 225 – 234.

Hussain, S.S., Lowe, G. (1968). Amino acid sequence around the active site cysteine and

histidine residues of stem bromelain. Chemical Communication, 22, 1387 – 1389.

Inagami, T., Murachi, T. (1963). Kinetic studies of bromelain catalysis. Biochemistry, 2

(6), 1439 – 1444.

Mehrlich, F.P. (1978). “Bromelains” in Encyclopedia of Food Science, Eds-Peterson,

M.S. and Johnson, A.H., The Avi Publishing Company Inc., Westport Connecticut, p. 94

– 97.

Murachi, T. (1964). Amino acid composition of stem bromelain. Biochemistry, 3 (7), 932

– 934.

Murachi, T. (1970). “Bromelain Enzymes”, in Methods In Enzymology, Eds-Perlmann,

G.E., and Lovand, L., Vol. 19, Academic Press, New York, p. 273 – 285.

Murachi, T. (1976). “Bromelain Enzymes”, in Methods in Enzymology, Eds- Perlmann,

G.E. And Lorand, L., Vol. 45, Academic Press, New York, p. 475 – 485.



Murachi, T., Neurath, H. (1960). Fractionation and specificity studies on stem bromelain.

Journal of Biological Chemistry, 235, 99 – 107.

Murachi, T., Suzuki, A., Takahashi, N. (1967). Evidence for glycoprotein nature of stem

bromelain. Biochemistry, 6, 3730 – 3736.

Murachi, T., Yasui, M., Yasuda, Y. (1964). Purification and physical characterization of

stem bromelain. Biochemistry, 3, 48 – 55.

Ota, S. (1966). On a Minor Component of Proteolytic Enzymes Contained in the

Pineapple Fruit, Journal of Biochemistry, 59, 463-468.

Ota, S., Horie, K., Hagino, F., Hashimoto, C., Date, H. (1972). Fractionation and some

properties of the proteolytically active components of bromelains in the stem and the fruit

of the pineapple fruit. Journal of Biochemistry, 71, 817 – 830.

Ota, S., Moore, S., Stein, W. (1964). Preparation and chemical properties of purified stem

and fruit bromelains. Biochemistry, 3, 180 – 185.

Rao, M.B., Tanksale, A.M., Ghatge, M.S., Deshpande, V.V. (1998). Molecular and

biotechnological aspects of microbial proteases, Microbiology and Molecular Biology

Reviews, 62 (3), 597 – 635.

Reed, G. (1966). “Enzymes in Food Processing”, Academic Press New York, p. 128 –

130.



Scocca, J., Lee, Y.C. (1969). The composition and structure of the carbohydrate of

pineapple stem bromelain. Journal of Biological Chemistry, 244, 4852 – 4863.

Smith, E.L., Kimmel, J.R. and Brown, D.M. (1954). “Crystalline Papain II”, Journal of

Biological Chemistry, 207, 533 – 549.

Takahashi, N., Yasuda, Y., Goto, K., Miyake, T., Murachi, T. (1973). Multiple molecular

forms of stem bromelain. Journal of Biochemistry, 74, 355 – 373.

Wharton, C.W. (1974). The structure and mechanism of stem bromelain. Biochemistry

Journal, 143, 575 – 586.

Yamada, F., Takahashi, N., Murachi, T. (1976). Purification and characterization of a protei


Chapter Three

Material and Methods

Materials and Methods

3.1. MATERIALS AND CHEMICALS

Fresh pineapple fruits were purchased from a local supermarket (Mumbai, India). L –

Cysteine for biochemistry and Casein (acc. to Hammarsten) for biochemistry were obtained

from Sisco Research Laboratories (SRL) Pvt. Ltd., India. Trichloroacetic acid (TCA) was

purchased from S.D. Fine Chemicals Ltd. Mumbai. All other chemicals used were of

analytical grade and purchased from S.D. Fine Chemicals Ltd., Mumbai. Standard purified

bromelain sample was obtained from Hong Mao Biochemical Co. Ltd. Thailand. Bovine

Serum Albumin (BSA) and Tyrosine were purchased from Hi – Media Laboratory.

Dialysis Membrane having molecular weight cut off (MWCO) 12000 was also purchased

from Hi – Media Laboratory. Ion exchange resins were made available from Resindion

S.R.L., Italy as a free sample.

3.2. METHODS OF ANALYSIS

3.2.1. Measurement of Enzyme activity

Bromelain when assayed for proteolytic activity against casein shows specificity similar to

that of papain. The assay was based on the estimation of the amount of small molecular

weight digestion products (Trichloroacetic acid (TCA) soluble material) formed from

proteins due to proteolytic action of the enzyme (Arnon and Shapira, 1967).

A stock solution of 1 mg/ml in dilute HCl of Tyrosine was prepared. Serial dilutions of this

stock solution were prepared by using dilute HCl so as to obtain the solutions of different

concentrations of tyrosine ranging from 31.5 µgm/ml to 125 µgm/ml. The absorbance of

this prepared solution was measured at 280 nm using Chemito 2500 UV-VIS



spectrophotometer. A graph of absorbance at 280 nm versus tyrosine concentration

(μgm/ml) was plotted and is shown in Figure 3.1, which is used as a standard.

1

y = 0.0061xR2 = 0.9996

0

0.2

0.4

0.6

0.8

0 50 100 150 200Conc. of Tyrosine (ugm/ml)

Abso

rban

ce

Figure 3.1 Standard plot for enzyme assay using tyrosine as a standard.

Proteolytic activity of bromelain was measured by the method described by Dapeau,

(1976).

• Assay Method described by Dapeau (Dapeau, 1976)

The assay consisted of 5 ml of 0.75% casein prepared in 50 mM anhydrous disodium

Phosphate buffer and the pH was adjusted to 7 using 0.1 N HCl (slow addition) to avoid

destruction of matrix and was brought to 37 ºC by pre incubation for 10 min. To this

substrate a known volume of enzyme was added after diluting it to 1 ml with activating

buffer. The 30 mM Cysteine Hydrochloride monohydrate in 6 mM disodium EDTA was

used as activating buffer. Casein proteolysis was stopped after 10 min by addition of 5 ml

of TCA (Trichloroacetic acid) stock solution. 0.11 M Tri Chloro acetic acid and 0.22 M

sodium acetate prepared in 0.33 M acetic acid was used as a TCA stock solution. The

mixture was allowed to stand for 30 min at 37 ºC after addition of TCA. After cooling to



room temperature, the solution was filtered twice through Whatman No. 42 filter paper.

Absorbance of the filtrate was measured at 280 nm using 2500 UV-VIS.

Spectrophotometer.

“One unit of bromelain was taken as the amount of enzyme which while acting on the

casein substrate under specified conditions, produced one microgram of tyrosine per

minute under the specified conditions”.

3.2.2. Measurement of Protein content

Protein content was measured spectrophotometrically by using the Bicinchoninic method

of protein estimation (Smith et al., 1985). Sigma Bicinchoninic Acid Protein Assay Kit was

used for assay purpose. The principle of the bicinchoninic acid (BCA) assay is similar to

the Lowry (Lowry et al., 1951) procedure, in that both rely on the formation of a Cu2+

protein complex under alkaline conditions, followed by reduction of the Cu2+ to Cu1+. The

amount of reduction is proportional to the protein content. BCA forms a purple-blue

complex with Cu1+ in alkaline environments, thus providing a basis to monitor the

reduction of alkaline Cu2+ by proteins. To determine the protein concentration, 200 µl BCA

working Reagent was added to 25 µl of suitably diluted protein. The protein assay

containers were sealed with film and incubated at room temperature for 2 hours. The color

developed was measured at 565 nm using micro plate reader (Model 680, BioRad) after 2

hours. Bovine serum albumin (BSA) was used as the standard for protein assay. A stock

solution of 0.1 mg/ml BSA was prepared. Solutions of different concentrations were

prepared by diluting it with distilled water from this stock solution. The assay of these

prepared samples were carried out by the method mentioned above. A plot of absorbance at



565 nm versus BSA protein concentrations in micrograms per ml was plotted and used as a

standard, as shown in Figure 3.2.

y = 0.0007xR2 = 0.9943

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 200 400 600 800 1000 1200Conc (ugm/ml)

Abso

rban

ce

0.8

Figure 3.2 Standard plot for Protein estimation using BSA as a standard.

3.2.2. Measurement of reducing sugar

Crude juice, chromatographic fractions and dried samples were assayed for reducing sugars

by DNSA (Dinitrosalicylic acid) method (Miller, 1959). To determine the total reducing

sugar, 1 ml of the diluted sample was treated with 1 ml of DNSA reagent and the mixture

was kept for 10 minutes in boiling water bath. The resultant mixture was then cooled and

final volume was made up to 12 ml with the help of distilled water. The absorbance of this

was taken at 540 nm against a corresponding blank.

Glucose was used as a standard for reducing sugar assay. Solutions of different

concentrations were prepared by diluting it with distilled water from this stock solution. A

plot of absorbance at 540 nm versus glucose concentrations (mg/ml) was plotted and used

as a standard as shown in Figure 3.3.



y = 0.3503xR2 = 0.9996

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 0.2 0.4 0.6 0.8 1 1.2

Glucose Concentration (mg/ml)

Abs

orba

nce

Figure 3.3 Standard plot for sugar estimation using glucose as a standard.

3.2.3. Measurement of water activity

The Freeze dried, spray dried, heat pump dried and crystallized samples were subjected to

analyze water activity in a water activity meter (M/S Aqua Lab) in order to estimate free

moisture. The water activity was calculated at room temperature. The effect of temperature

on water activity was also studied.

Water activity is defined as,

wpp

wa = …………….(3.1)

Where,

p = partial pressure of water at given conditions

pw = vapour pressure of pure water at the same conditions



3.2.4. Measurement of moisture content

Moisture contents of crude juice, unbound fraction, washout and elute were analyzed using

loss on drying (LOD) method. 10 ml of sample in petri plates was subjected to vacuum

drying at a temperature of 50 ºC. Drying was carried out for sufficient time (approximately

12 hrs) and based on LOD moisture content was calculated.

Residual moisture content of dried formulations was measured with the Karl-Fisher method

(MacLeod, 1991) on Microcontroller based Digital Karl Fischer (Model No. MI 453, M/s

Polmon Instruments Pvt. Ltd., India). At least 100 mg of powder was mixed with dry

methanol and titrated with Karl Fisher reagent untill the end point was reached. The

samples were dispersed in methanol and the water content was determined. Methanol was

used as blank. The water content of every formulation was given as the average of

calculated water content of three independent batches.

3.2.5. Differential Scanning Calorimetry

Thermal analysis of freeze dried and spray dried powders was performed using differential

scanning calorimeter (DSC) (Perkin Elmer Pyris – 6) Prior to measurement, the dried

samples were transferred into vacuum desiccators and equilibrated for 3 – 4 days over P2O5

for ‘zero’ moisture content. Approximately 2 – 10 mg of powder was used. The sample

was sealed in an aluminium pan and an empty pan was used as a reference. Dried sample

were heated in the range of 40 to 300 ºC. Heating rate of 10 ºC/min was used. All glass

transition temperatures were reported as the midpoint temperature of the heat capacity step

associated to the glass transition with respect to the ASTM (1991).



3.2.6. Fourier transformation infra-red spectroscopy

Fourier transform infrared (FTIR) spectra were measured utilizing a Perkin Elmer 1600

series FTIR, and analyzed using a PE-GRAMS/32 1600 software, as described previously

(Liao et al., 2004). Briefly, a dry protein sample (approximately 0.5 mg protein) was mixed

with about 300 mg ground potassium bromide and compressed into a pellet. The spectra

were smoothed with a nine-point Savitsky – Golay function to remove any possible white-

noise. The baseline of the spectrum in the amide I region was leveled and zeroed, then the

spectrum of the sample was normalized for area in the region and the intensity of the α –

helical band was recorded.

3.3. EXPERIMENTAL METHODS

3.3.1. Preparation of clarified crude fruit bromelain extracts

The stalk (central core) of the pineapple fruit and the leaves were separated from fleshy

fruit part of pineapple. The fruit portion was then cut into small pieces and blended in

mixer. Approximately 400 – 500 ml of juice was prepared from one pineapple fruit. This

juice was filtered through a muslin cloth to remove the fibrous material. The resultant juice

still contained ruptured plant cells so in order to remove that it was subjected to filtration

under vacuum. The clarified crude juice was also subjected to centrifugation on a Remi R –

24 research centrifuge for twenty minutes at low temperature and 10,000×g. The clear

supernatant obtained as a clarified extract was stored in aliquots at 4 °C and used for

crystallization study. Each batch of bromelain extract from the pineapple gave different

bromelain content in the range of 1000 – 1500 casein hydrolyzing units per ml or 70,000 to

90,000 units per 100 gm of fruit part of pineapple.



3.3.2. Crystallization of clarified fruit juice

Crystallization of clarified juice was carried out by using different precipitants such as

ammonium sulfate, acetone and sodium chloride..

3.3.2.1. Crystallization using Ammonium Sulfate

Attempts were made to obtain a crystalline enzyme preparation by fractionation of fruit

bromelain with ammonium sulfate and sodium chloride.

The following is a preliminary description of the procedure which was found to yield crude

crystalline material.

A cryostat bath (M/s Asha Scientific Company, Mumbai) containing water was maintained

at 4 ºC and the sample undergoing fractionation was chilled upto 4 ºC. The protein

concentration was in the range of 15 – 20 mg/ml. Slow addition of precipitating agent with

efficient cooling and under constant stirring was carried out. After the last bit of salt was

dissolved, stirring was continued for 10 – 30 min to allow complete equilibration between

dissolved and aggregated proteins. Then, the solution was centrifuged (Remi R – 24

research centrifuge) at 10000×g for 10 to 15 min and the precipitate was collected. The

supernatant was decanted, its volume noted and the amount of salt required for the next cut

was calculated. The precipitate was dissolved in minimum quantity of suitable buffer and

was further subjected to dialysis as a process of buffer exchange.



For ammonium sulfate precipitation, the amount of ammonium sulfate added to the

solution in order to increase % saturation from S1 to S2 was calculated by using following

equation (Scopes, 1982):

2S0.3100

)1S2(S533g

−

−= …………….(3.2)

Where,

g = grams of ammonium sulfate to be added to 1 liter of a solution,

S2 = Final required % saturation of solution,

S1 = Initial % saturation of solution.

The equation is related by the assumption that 100 % saturation = 4.05 M.

3.3.2.2. Crystallization using Acetone

Crystallization using acetone as a precipitating agent was carried out using a method

described by Apte et al. (1979)

A 20 g of fresh tissue (callas or leaves) was homogenized in a mixer with 150 ml of 0.05 M

potassium phosphate buffer (pH 6.1). The suspension was further homogenized using a

homogenizer to ensure maximum cell disruption. The suspension obtained from leaves was

passed through a muslin cloth prior to the second homogenization to eliminate fibres. The

homogenate was centrifuged at 10000 rpm for 15 min in a Remi R – 24 research centrifuge

using SS – 34 rotor at 0 – 5 °C. The supernatant was taken for further purification.



Preliminary experiments, employing a gradient acetone fractionation, showed that the 40 –

80% fraction gives maximum enzyme activity. The supernatant was brought to 40%

saturation by the addition of pre – chilled acetone at 4 °C. The precipitate isolated by

centrifugation at 10000 rpm for 10 min was discarded. The supernatant was brought to

80% saturation and the precipitate obtained after centrifugation as above was collected and

dried in a vacuum desiccator.

A formula for calculating the amount of organic solvent to be added is given as:

y100x)(y1000v

−−= …………….(3.3)

Where,

v = volume of solvent to be added to one liter to take % from x to y,

x = initial % saturation,

y = final % saturation.

Effect of precipitant concentration on enzyme activity retention and residual protein

content was studied. Dialysis of the precipitate was also carried out.

3.3.2.3. Crystallization using ammonium sulfate and sodium chloride

Attempts were made to obtain a crystalline enzyme preparation by fractionation of stem

bromelain with ammonium sulfate and sodium chloride (Murachi and Neurath, 1960).

50 ml of Stem bromelain juice after adjusting to pH 7.5 with 1 N NaOH was centrifuged

for 30 minutes at 14,000 rpm. To the supernatant solution (50 ml), 12.5 g of ammonium



sulfate of pH 7.5 and room temperature was added and after 1 hour the mixture was

centrifuged. The precipitate was washed with a solution of 6.25 g of ammonium sulfate in

25 ml of water and the washed precipitate was dissolved in water to make a solution of 25

ml. At pH 7.5, 2.25 g of ammonium sulfate was added and the precipitate was collected by

centrifugation. The precipitate was dissolved in 100 ml of water, reprecipitated by adding

30 g of NaCl, washed with saturated NaCl solution, and then dissolved in 400 ml of 80%

saturated sodium chloride solution by adjusting to pH 9.0 with 1N NaOH. When 0.1 N

acetic acid was added slowly to lower the pH to approximately 8, a faint turbidity due to

crystalline material appeared which did not increase substantially by standing but could be

increased by further cautious addition of acetic acid to a yield of about 5% of the starting

material. A similar type of crude crystalline material was also obtained when stem

bromelain was treated in the same manner except that 0.01 M cysteine was employed

throughout the procedure instead of converting the enzyme into the mercury derivative.

For precipitation using sodium chloride, it is assumed that 100% saturation is 5 M.

3.3.3. Dialysis of the purified sample

To remove the salt from the precipitated sample it was further dialyzed against 50 mM

ammonium sulfate or phosphate buffer, pH 7.0 as a process for buffer exchange. The

dialysis was carried out by using the dialysis membrane available from Hi-Media

(Mumbai, India) Laboratory having MWCO (Molecular Weight Cut Off) of 12,000. The

enzyme activity units and protein content after dialysis were checked for the percentage

loss in the process.



3.3.4. Adsorptive Chromatographic separation

Chromatographic purification of bromelain was carried out on a preparative scale. The

column dimensions were 30 cm (L) x 2.5 cm (D). Acetate buffer was used as an

equilibrating buffer and for removal of unbound or weakly bound protein, sodium chloride

(1N) solution was used as elution buffer. The column was regenerated using 0.5N NaOH

solution.

The column setup for Chromatographic purification is as shown in Figure 3.4

Figure 3.4 Chromatography setup for Purification of Bromelain

Ion exchange resins (cation) were packed in a glass column of 25 mm internal diameter and

equilibrated with four column volumes of acetate buffer (25 mM, pH 4.0). Sufficient

quantity of clarified crude juice was passed in upward direction through the packed bed of

adsorbent. This was followed by the wash of equilibration buffer till no protein was

detected at 280 nm using an online spectrophotometer (Model No. U – 1100, Hitachi,

India). Finally the bound enzyme (protein) was eluted using 1N sodium chloride solution.



The eluting protein was determined at 280 nm on online spectrophotometer. The enzyme

activity and protein content for unbound fraction, washings and elution fraction were

carried out by using the methods mentioned in previous section to calculate the total

recovery of enzyme units with respect to the bound one.

The results of purification by Crystallization and Chromatography were compared on the

basis of specific activity.

3.3.5. Drying of purified fruit juice

The purified sample was subjected to drying. Three different types of dryers were used.

3.3.5.1. Freeze Dryer

A laboratory freeze dryer (M/s Ref-vac Consultancy, India) having condenser ice collection

capacity of 1.8 kg and temperature of –35 ºC was used. The drying chamber (0.4 m (L) ×

0.4 m (D)) was equipped with heating plates. 20 ml of purified Bromelain sample was

filled manually into the petri plate. An electronic weighing balance was kept inside the

drying chamber for online sample LOD measurement in order to study the drying kinetics.

The freezing step was performed by placing the petri plate in a deep freezer for 6 hr. at -23

ºC. Then, the petri plate was transferred into the drying chamber which was kept at room

temperature. The product temperature was recorded using thermocouple, which was placed

on the surface of the product. Pressure inside the chamber was recorded at different time

intervals. The shelf temperature was kept at 30 ºC and pressure inside the chamber was

kept at 24 Pa.



The powder obtained was dissolved in distilled water at proper dilution and was further

analyzed for activity retention and residual protein content. The activity retention and

protein retention were calculated on the basis of solid content. Activity of the freeze dried

samples was then expressed as a percentage of the activity of the initial sample.

3.3.5.2. Spray Dryer

3.3.5.2.1. Experimental Set – up

Figure 3.5 Flow Diagram of Laboratory Spray – dryer

CY-1 and CY-2: Cyclones 1 and 2

DC: Drying Chamber

T : Temperature probe for inlet air

T1 From Atm.

P

FEED AG

F

H2

H1

F To Atm

ASP

V

SC

T2

CY-1

C2

CY-2

C3

C1

HOT AIR

COMP

AIR

DC

1



T2: Temperature probe for outlet air

heavy particulates

r

re regulator

d 2.

A laboratory spray dryer (LU-222, M/s Labultima, India) of 1 lit/hr evaporation capacity

In the present work, two different set of operating parameters were used.

• In order to study the effect of air inlet temperature on the enzyme activity retention, the

C1: Collection bottle for collection of

C2 and C3: Collection pots for 1st and 2nd cyclones.

SC: Scrubber

ASP: Aspirato

F: Filter

P: Pressu

H1 and H2: Heater 1 an

V: Vacuum gauge

AG: Agitator

F: Feed Pump

was used. The dimensions of the drying chamber were 0.4 m (L) × 0.1 m (D). The spray

dryer operates in a co-current manner and has a two fluid spray nozzle with an orifice of

0.7 mm in diameter. The spray dryer was equipped with two cyclone collector for

collection of product. The air used for the heating purpose was passes through HEPA

filters.

inlet air temperature was varied in the range of 150 – 200 ºC while the outlet air



temperature was maintained at 40 ºC by varying the feed flow rate. The flow rate of

drying air was kept at 41.3 Nm3/h.

• In the second set of operating parameters, the effect of outlet air temperature on

enzyme activity retention was studied. In this case, inlet air temperature was kept at 160

ºC and the feed flow rate was varied in such a way that the outlet temperature was

varied in the range of 35 – 65 ºC. The flow rate of drying air was kept at 41.3 Nm3/h.

Effect of inlet and outlet air temperature on enzyme inactivation kinetics was studied.

Every experiment was carried out in duplicates, without any additive and in second,

Maltodextrin was used as an additive. However, the powder obtained was hygroscopic and

activity loss was high and hence, no further experiments were carried out with

Maltodextrin.

To study the spray dryer performance following parameters were defined:

Drying Ratio: Drying ratio was equal to powder solid content divided by feed solid

content.

contentsolidFeedcontentsolidPowderratioDrying = ……….. (3.4)

Productivity: Productivity (kg/h) was equal to feed rate divided by drying ratio.

ratio DryingrateFeedtyProductivi =

…………… (3.5)

Drying Rate: Drying rate (kg/h) was equal to productivity subtracted from feed flow rate.



……….. (3.6) Drying rate = Feed flow rate - Productivity

3.3.5.3. Heat Pump Dryer

Figure 3.6 shows an open loop heat pump drying system (HPD) used in the present study.

R134a was used as a refrigerant. The system used for the study consisted of heat bypass

coil for improving the performance of heat pump and hence, the specific moisture removal

rate in HPD. The heat pump system comprised of a 1.5 kWh heat load providing the

dehumidified air of 100 – 175 Nm3/hr. The temperature range obtained from this HP unit

was in between 30 – 45 °C and the corresponding relative humidity of air was in the range

of 10 – 20%.

The 20 ml of purified bromelain sample was dried in HPD for a period of 5 – 7 hrs. The

samples were collected after every 30 min time interval for determining its weight loss, in

order to find out the drying kinetics.



Desuperheater

External Condenser

Internal Condenser

Reboiler

Precooler Evaporator

Dryer

Ambient air

Expa

nsio

n V

alve

Com

pres

sor

Figure 3.6 Heat Pump Drying System



3.4. REFERENCES

Apte, P.V., Kaklij, G.S., Heble, M.R. (1979). Proteolytic enzymes (bromelains) in tissue

cultures of Ananas Sativus (Pineapple). Plant Science Letters 14, 57 – 62.

Arnon, R., Shapira, E. (1967). Antibodies to Papain. A selective fractionation according to

inhibitor capacity. Biochemistry 6, 3942-3950.

ASTM (1991). Standard test method for glass transition temperatures by differential

scanning Calorimetry or differential thermal analysis, 1356 – 1391.

Dapeau, G.R. Methods in Enzymology, vol. XLV (Lorand, L., ed.) pg. 471, Academic

press, New York (1976).

Liao, Y., Brown, M. B., Martin G. P. (2004). Investigation of the stabilisation of freeze-

dried lysozyme and the physical properties of the formulations. European Journal of

Pharmaceutics and Biopharmaceutics 58, 15 – 24.

Lowry, O.H., Rosebrough, N.J., Lewis Farr, A., Randall, A. (1951). Protein measurement

with the Folin phenol reagent. Journal of Biological Chemistry 193, 265 – 275.

MacLeod, Steven K. (1991). Moisture determination using Karl Fischer titrations.

Analytical Chemistry 63, 557 – 566.

Miller, G.L. (1959). Use of dinitro salicylic acid reagent for determination of reducing

sugar. Analytical Chemistry 31, 426 – 428.



Murachi, T., Neurath, H. (1960). Fractionation and specificity studies on stem bromelain.

The Journal of Biological Chemistry 235(1) 99 – 107.

Scopes, R. K. (1982). Separation by Precipitation, In Protein Purification: Principles and

Practice (Second Edition). R. K. Scopes (Ed.), 41 – 65,.Springer – Verlag New York.

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D.,

Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, O.C. (1985). Measurement of protein

using bicinchoninic acid. Analytical Biochemistry 150, 76-85.


Chapter Four

Results and Discussion


4.1 CRYSTALLIZATION OF BROMELAIN

4.1.1. Crystallization using Ammonium Sulfate

Ammonium sulfate precipitation was performed as the first step of bromelain purification.

Ammonium sulfate fractions i.e. supernatant and precipitate were collected at levels of

saturation of 20% (w/v) and assayed for specific protease activity before and after dialysis.

Fractions collected at 40 – 60% and 60 – 80% ammonium sulfate were found to have the

highest percent of protease activity and specific protease activity, with a recovery of 79.4%,

and accounted for 34% of the total protein concentration. The enzyme activity and specific

enzyme activity were found to be very less in the fractions obtained at lower saturation that

means proteases were soluble in 35% of saturation of ammonium sulfate, but not in 60% of

saturation of ammonium sulfate. Many other proteins in the supernatant were not soluble in

35% of saturation of ammonium sulfate and they were precipitated out of solution.

To more precisely define the ammonium sulfate fraction containing the highest amount of

specific protease activity, smaller ammonium sulfate fractions were collected over the

range of 40 – 80%. It was observed that the 40 – 70% ammonium sulfate fraction contained

68% of the protease activity whereas the 70 – 80% fraction contained < 5% of the protease

activity. An ammonium sulfate precipitation between 40 – 70% resulted in approximately 3

– fold increase in specific activity compared to the crude fruit juice.

Fractions of saturation 40 – 60% and 60 – 80% were showing highest activity and hence a

saturation of 40 – 70% was obtained and was found to contain most of the bromelain and

hence, the fraction of 40 – 70% was used for crystallization purpose.

Crystallization and Drying of Biomaterials 66


To more precisely define, the precipitation kinetics in the saturation range of 40 – 60%, the

saturation range is increased by 2%, and for every 2% increase in saturation, the precipitate

and supernatant is collected and analyzed for bromelain activity.

A second preliminary experiment shows that an increase in solid – phase activity could be

correlated with a fall in the supernatant activity. The kinetics of bromelain precipitation in

the saturation range of 40 – 60% is as shown in Figure 4.1.

0

4000

8000

12000

16000

20000

35 40 45 50 55 60 65% Saturation

CD

U

Supernatant Precipitate

Figure 4.1 Precipitation kinetics in the range of 40 – 60% saturation



Table 4.1 shows the percent recovery of bromelain and protein from crude fruit juice of

pineapple during each stage of supersaturation and Table 4.2 shows the percent recovery of

bromelain obtained in the saturation range of 40 – 70% and 70 – 80%.

Table 4.1 Effect of percent saturation on bromelain and total Protein recovery in precipitation

Saturation Units

(CDU)

Protein

Content

(mg)

Specific

Activity

(Units/mg

Protein)

Fold Purity % Yield

Crude 160721 2506.28 64.13 1.0 100%

0 – 20 % (P) 7393.166 476.1932 15.53 0.24 4.6%

0 – 20 % (S) 153022.384 2134.0868 71.71 1.12 95.21%

20 – 40 % (P) 27804.733 350.8792 79.24 1.23 17.3%

20 – 40 % (S) 124458.642 1783.2076 69.79 1.09 77.44%

40 – 60 % (P) 66056.331 401.0048 164.73 2.57 41.1%

40 – 60 % (S) 70302.146 1482.2824 47.43 0.74 43.74%

60 – 80 % (P) 61556.143 451.1304 136.45 2.13 38.3%

60 – 80 % (S) 8664.324 1124.276 7.71 0.12 5.39%

80 – 100 % (P) 6589.561 401.0048 16.43 0.26 4.1%

80 – 100 % (S) 4374.476 744.1274 5.88 0.09 2.72%

100 % – + (P) 5785.956 601.5072 9.61 0.15 3.6%



Table 4.2: Effect of percent saturation on activity retention and protein content in ammonium sulfate precipitation

Saturation Units

(CDU)

Protein

Content

(mg)

Specific

Activity

(Units/mg

Protein)

Fold Purity % Yield

20 – 40 %

(S)

124458.642 1783.2076 69.79 1.00 100%

40 – 70 %

(P)

86772.428 418.233 207.47 2.97 69.72%

70 – 80 %

(P)

5351.721 1042.672 5.13 0.074 4.3%

(P – Precipitate; S – Supernatant; CDU – Casein Digesting Unit)

For further study, precipitates of 40 – 60% and 60 – 80% were mixed and were subjected

for vacuum drying or air drying for crystallization purpose.

The advantages in using Ammonium sulfate as a precipitant over other precipitating agents

were that the enzyme obtained using Ammonium sulfate was much more stable and the

high salt concentration prevented proteolysis and bacterial action.



4.1.2. Crystallization using Acetone

Acetone precipitation was also performed as the first step of bromelain purification. The

supernatant and precipitate were collected at levels of saturation of 20% (w/v) and assayed

for specific protease activity before and after dialysis. Fractions collected at 40 – 80%

saturation contained the highest percent of protease activity and specific protease activity,

with a recovery of 42%, and accounted for 9.8% of the total protein concentration.

Table 4.3 gives the percent recovery of bromelain and protein from leaves of pineapple

during 40 – 80 % of supersaturation

Table 4.3 Acetone fractionation in the range of 40 – 80% saturation

Saturation Units

(CDU)

Protein

Content

(mg)

Specific

Activity

(Units/mg

Protein)

Fold Purity % Yield

Mature leaves

(20 gm)

9728.18 176.8 55.023 1.00 100

40 – 80 % 4085.84 17.45 234.15 4.26 42

Acetone fractionation of tissue extracts eliminates most of the pigmented matter at 40%

saturation. Proteins in this fraction showed a negligible proteolytic activity. Most of the

enzyme activity was recovered in 40 – 80% fraction. The total proteolytic activity of the

matured leaves of the plantlets was comparable, but much less than that observed in the

stem and fruit of the mature plant.



At 60 % saturation using acetone, the recovery obtained was 42%, which was very less as

compared to that obtained by using ammonium sulfate precipitation. However, acetone

fractionation when coupled with ammonium sulfate precipitation or gel filtration may give

maximum recovery (Apte et al., 1979).

These results indicate that cold acetone is probably a suitable effective agent for the initial

step of protease purification. The effectiveness of cold acetone as a purification agent for

proteolytic enzymes was also reported by Popova and Pishtiyski (2001). Michail et al.

(2006) also reported that cold acetone was a much better purification agent than other

precipitating agents as a first purification step.

4.1.3. Crystallization using Ammonium sulfate and Sodium Chloride

Crude crystalline enzyme was obtained by fractionation with ammonium sulfate and

subsequently with sodium chloride.

The specific activity of the crude crystalline product thus obtained was the same as, or only

slightly higher than, that of the starting material as assayed toward casein in the presence of

0.005 M cysteine, which was in good agreement with the observations of Murachi and

Neurath (1960).



4.1.4. Effect of type of salt

The nature of the salt is known to have a strong influence on protein solubility.

Experimental results show that the salting – out effect of salts mainly depends on the nature

of the anions. Salts exert their effect by dehydrating proteins through competition for water

molecules (Melander and Horvarth, 1977). Their ability to dehydrate depends primarily on

the square of the valence of the anion of the salt. Thus, salts with polyvalent anions are

more effective at salting-out than those containing univalent anions. Comparison of salting-

out ability of salts should be made on the basis of salt concentration rather than ionic

strength since the Hofmeister classification is based on salt concentration.

The precipitation potentials of the anions have the sequence

Sulfate > phosphate > chloride

which is consistent with the Hofmeister series and for cation the sequence would be,

NH4+>K+>Na+

Of the common inexpensive salts that are effective in causing precipitation, sodium,

potassium and ammonium sulfates, phosphates are attractive candidates (Arakawa and

Timasheff, 1984).



4.2 CHROMATOGRAPHIC PURIFICATION OF FRUIT BROMELAIN

Since the fruit bromelain was found to possess more activity than the one found in stem or

leaf portion of pineapple, it was decided to purify it using preparative chromatography

technique, like ion exchange chromatography.

The results of chromatographic purification are as shown in Table 4.4

Table 4.4: Results of Chromatographic Purification

Sample id Volume

(ml)

Enzyme

Activity

(CDU)

Protein

Content

(mg)

Reducing

Sugars

(mg)

Crude 172 160721.3115 2733.5714 2822.3123

Unbound 174 9508.196721 1957.5 2755.7865

Washout 312 11934.42623 757.7143 137.5963

Elute 222 135868.8525 237.8571 46.8969

Enzyme recovery with respect to load was found to be 84.54% and elution efficiency was

observed to be 97.56%. The specific activity of the crude juice was 58.79 Units/mg Protein

and that of Elute was 590.92 Units/ mg Protein. Thus the fold purity obtained was 10.05.

The unbound fraction mainly contained sugars which could have been further processed for

concentration of juice or sugar recovery.



4.3 DRYING OF PURIFIED BROMELAIN

4.3.1. Freeze Drying

The profile of Moisture content (db) with respect to time in freeze dryer is as shown in

Figure 4.2.

0

2

4

6

8

10

12

14

16

18

0 100 200 300 400 500 600Time (min)

Moi

stur

e co

nten

t (db

)

Figure 4.2 Moisture Profile in Freeze Dryer

Where,

Moisture content = kg moisture/ kg dry solid

When the water molecules sublime and enter the vapour phase, they also keep with them a

significant amount of the latent heat of sublimation (2840 kJ/kg ice) and thus the

temperature of the frozen product is again reduced.



The effect of freeze drying on product quality parameters such as residual activity, protein

content and reducing sugars is given in Table 4.5.

Table 4.5 Effect of freeze drying on product quality parameters (Ts = 30ºC)

Parameter Value

Yield 95 – 97%

% activity retention 92 – 97%

% residual protein 85 – 90%

% sugars recovery 50 – 55%

The stability of freeze dried product can be attributed to its ability to go through the drying

process without change in size, porous structure and shape.

4.3.2. Spray Drying

The effect of spray drying on product quality parameters such as residual activity, protein

content and reducing sugars is given in Table 4.6

The yield obtained was less in case of spray dryer as compared to that obtained in freeze

dryer. This may be due to poor collection efficiency. Carry over of fine particles along with

the air or the product deposition on the drying chamber May leads to lowder yield.



Table 4.6 Effect of spray drying on product quality parameters (To = 40 ± 2ºC)

Parameter Value

Yield 70 – 75%

% residual activity 75 – 80%



The less enzyme activity retention may be due to the degradation of the product because of

heating, or structural changes that are occurring during drying. Also, the powder obtained

from chamber showed negligible activity. The reason behind this may be that the deposited

powder in the drying chamber faced higher temperature for longer period of time, which

ultimately resulted in denaturation of enzyme.

The outlet temperature of the spray dryer was determined solely by the main effects of the

inlet temperature, feed flow rate and air flow rate. The outlet temperature of the spray dyer

is considered to be the most important factor in determining the residual activity of spray

dried heat sensitive materials.



4.3.2.1. Inactivation Kinetics in Spray dryer

y = 0.7932

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

130 140 150 160 170 180 190 200 210

Inlet Temperature (ºC)

% a

ctiv

ity re

tent

ion

• Effect of inlet air temperature:

Figure 4.3 shows the effect of inlet air temperature on activity retention of bromelain.

Figure 4.3 Effect of inlet air temperature on activity retention; To = 40 ºC

Inlet temperature was having negligible effect on enzyme activity retention. The enzyme

activity retention was found to be in the range of 70 – 75%. The inlet temperature was

varied in the range of 140 – 200 ºC and the outlet temperature was maintained constant by

adjusting feed flow rate and hence the temperature attained by the dry powder was not

more than 40 ºC in any run. Thus, the enzyme activity retention was more or less remains

constant. However, the particle size obtained was different, since feed flow rate affects the

particle size of product. These results are in good agreement with those reported by

Samborska et al. (2005).



• Effect of outlet air temperature

y = -0.0011x2 + 0.0846x - 0.8252R2 = 0.9866

0

0.2

0.4

0.6

0.8

1

30 40 50 60 70

Outlet Temp (deg C)

% A

ctiv

ity r

eten

tion

Figure 4.4 shows effect of outlet air temperature on activity retention of bromelain.

Figure 4.4 Effect of outlet air temperature on activity retention

The residual activity of spray dried bromelain was found to vary between 77% – 1% at

different outlet temperatures. At constant inlet air temperature when lower feed flow was

used, higher outlet temperature was achieved and hence, the enzyme activity was found to

decreased for each drying air temperature. At an outlet temperature of 65 ºC, the enzyme

almost showed zero activity. The reason behind this may be that the inactivation

temperature of the enzyme was reported to be 65 – 70 ºC. Feed flow rate has a remarkable

influence on particle size of powder.

Some authors reported that proteins are more resistant to thermal denaturation at lower

water content conditions. It is generally known that when protein substance is dried, its



thermal stability is markedly enhanced as a result of water evaporation (Samborska et al.,

2005). It means that higher evaporation rate leads to better enzyme activity preservation,

since the enhanced resistance to elevated temperature is reached faster.

The final relative bromelain activity after drying can be strongly correlated to outlet

temperature and moisture content in powders. In order to maintain the relative activity of

dried enzyme on the acceptable level, the outlet temperature should be kept below 45 ºC.

4.3.2.2. Spray Dryer Performance

Average drying ratio, productivity and drying rate are shown in Table 4.7.

Feed Rate

(ml/hr) Drying ratio

Productivity

(kg/hr)

Drying rate

(kg/hr)

75 13.72 5.79 73.71

120 13.61 9.35 117.85

150 13.49 11.79 147.21

180 13.42 14.21 176.85

Table 4.7: Spray Dryer Performance

Drying ratio was found to increase from 13.42 to 13.72 with a decrease in feed rate. It can

be seen that Productivity and drying rates were increased from 5.79 to 14.21 kg/hr

respectively with an increase in feed rate.



4.3.3. Heat Pump Drying

The effect of heat pump drying on product quality parameters such as residual activity,

protein content and reducing sugars is given in Table 4.8

Table 4.8 Effect of heat pump drying on product quality parameters (T = 38ºC)

Parameter Value

Yield 75 – 80%

% residual activity 75 – 80%



The less yield obtained in Heat pump dryer may be attributed to loss of fine powder which

was carried away along with the air during drying. Heat pump drying results in the

crystallization of bromelain powder. Though the powder was having comparable activity,

its stability was very poor and also, the moisture content was observed to be more. The

crystalline powder of bromelain looses its 50% of activity after 4 days, when it was kept at

4 ºC. The preparation of such enzymes in the crystalline state does not necessarily

guarantee long term stability.

The results are in good agreement with the findings of Pikal and Rigsbee (1997). They

have found that the insulin in the amorphous state is more stable than in crystalline form

during storage.



4.4 PRODUCT QUALITY PARAMETERS

The powder obtained by different drying techniques was analyzed for different product

quality parameters and these parameters were compared with that of standard commercial

powder sample obtained from Hong Mao Biochemical Co. Ltd. The results of which are as

shown in Table 4.9.

Table 4.9 Product quality parameters: Comparison with commercial bromelain

Quality

Parameter

Freeze

Drying

Spray

Drying

Heat Pump

Drying

Commercial

Powder

Enzyme Activity

(CDU/mg Powder)

9.39 8.47 8.68 132.5

Protein Content

(mg Protein/mg

Powder)

0.039 0.024 0.019 0.775

Reducing sugars

(mg sugar/mg

powder)

0.017 0.022 0.019 0.317

Specific Activity

(CDU/mg Protein)

482.31 462.47 457.14 170.96

Moisture Content

(gm water/gm

powder)

0.047 0.058 0.091 -------

TDS

(gm solid/gm

powder)

0.953 0.942 0.909 -------

Water Activity 0.514 0.466 0.482 --------



The enzyme activity of freeze dried powder was 9.39 CDU/mg powder, which was slightly

more than that obtained by spray and heat pump drying. But the enzyme activity of

commercial powder was 132.5 CDU/mg powder, which was very high as compared with

that obtained by freeze drying. The reason behind this may be that the enzyme subjected to

freeze drying was chromatographically purified fraction containing NaCl. NaCl was not

removed before freeze drying. Due to this reason the protein concentration in the powder

was very less and hence, subsequently the enzyme activity.

The specific activity of freeze dried protein was observed to be 482.31 Units/mg protein

and that of standard commercial powder was 170.96 Units/mg protein i.e. 1 mg protein

contains 482.31 casein digesting units (CDU). This proves that our enzyme is having much

more purity than the standard commercial available bromelain sample.

The enzyme activity can be increased by subjecting the eluted sample to dialysis as a

process of buffer exchange or gel chromatography or ultra filtration to as a step of

concentration prior to drying so as to remove the salt and concentrate the protein sample.

However, the selection of buffer in dialysis should be checked for pH and stability. The

enzyme should be stable in that buffer, which is proposed as future work.

The water activity of both spray dried and freeze dried powder was well below 0.8 which

concludes that the powder is stable.

The reducing sugar content of standard commercial powder was 0.317 mg/mg powder

which means that after concentration sugars were added so as to increase glass transition

temperature of protein formulation in order to avoid structural changes.



4.5 PRODUCT CHARACTERIZATION

4.5.1. Inactivation Kinetics in an aqueous solution

Figure 4.5 shows the profile of bromelain inactivation in aqueous solution at various

temperatures. The activity ratio to the initial value is plotted against time.

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200

Time (min)

Rel

ativ

e A

ctiv

ity

Figure 4.5: Profiles of bromelain inactivation in pH 7.4 aqueous solution. (♦ – 45ºC; ■ – 50ºC; ▲ – 55ºC; ○ – 60ºC)

The solid lines shown in the figures were calculated from the parameters obtained by

nonlinear regression analysis according to a first-order kinetic expression. The activation

energy calculated by assuming the linearity of the plot was 21.4 kcal/mol.

Figure 4.6 shows Arrhenius plot of apparent first order rate constant obtained for

bromelain inactivation.



y = -21.27x + 61.699R2 = 0.999

-6

-4

-2

2.9 3 3.1 3.2

1000/T (ºC-1)

ln K

Figure 4.6: Arrhenius plots of apparent first-order rate constants obtained for bromelain inactivation (pH 7.4).

Protein degradation in aqueous solution has been described by a first-order expression in

most of the studies published to date (Tsuda et.al., 1990). In this study, bromelain was

found to degrade in a similar pattern. Information on the activation energy of protein

degradation is very limited. The kinetics of enzyme inactivation in aqueous solution was

studied for bromelain. Inactivation of bromelain was found to follow simple first-order

kinetics and the rate constant obtained appeared to confirm to the Arrhenius relationship,

suggesting that the inactivation rate can be predicted by interpolating the relationship. The

rate constants obtained by nonlinear regression analysis provided a reasonable Arrhenius

relationship. The results obtained suggest that inactivation of enzymes in aqueous solution

can be modeled even if the profile is complicated and the dependence of kinetic parameters

on temperature can be determined.



4.5.2. FTIR study

An FTIR spectrum of freeze dried bromelain is as shown in Figure 4.7.

Figure 4.7: Fourier Transform IR spectra of the 4000 – 500 cm-1 region of freeze dried

bromelain

Characteristic C-N stretch vibration frequencies of monoalkyl guanidinium are assigned to

observed IR bands at 1655-1685 cm-1, 1615-1635 cm-1 and 1170-1180 cm-1. The band at

1630 – 1860 cm-1 shows presence of >C=O stretching groups (Amides at ~ 1650 cm-1). It

confirms the presence of amino acids which may contain amide group which contains

amide group as their side chain, i.e. aspargine and glutamine.

The spectra at 3280 – 3340 cm-1 show presence of CH stretching vibrations. The

experimental spectra of phenylalanine, proline, valine, leucine and isoleucine are in the

range of 900 - 3700 cm-1.



4.5.3. DSC study

The DSC thermogram of freeze dried bromelain is as shown in Figure 4.8.

Figure 4.8: DSC thermogram of freeze dried bromelain obtained at a heating rate of 10ºC/min

The freeze dried enzyme powder containing NaCl was subjected to Differential Scanning

Calorimetry (DSC) and it was found that the glass transition temperature of the powder is

61.16 0C. The thermogram is an endotherm indicating that the powder is amorphous and

the curve obtained is that of the glass transition temperature and the melting temperature is

104.83 ºC.

From DSC study, we can say that in order to avoid structural changes from plastic to

rubbery viscous state, the drying temperature should not be more than 60 ºC and hence, all

the drying operations were carried out below 60 ºC.



4.5.4. Optimum pH

The pH profile of protease activity of fruit portion of bromelain is as shown in Figure 4.9.

0

0.2

0.4

0.6

0.8

4 6 8 10pH

Abso

rban

ce (A

at 2

80 n

m)

12

Figure 4.9: The pH dependence of the proteinase activity of purified fruit bromelin.

Casein was used as substrate

The casein was not soluble below pH 6.0 and hence all assays were performed above pH

6.0. The optimum pH for fruit bromelain was found to be around 8.0 when casein was used

as substrate. This implies that the fruit portion contained protease with optimum pH on the

basic side, and hence, all the assays were carried out at pH of 8.0.

However, the optimum pH may vary depending on the substrate and the buffer in which the

substrate is prepared. Hence, a range of optimum pH is defined. The optimum pH range

defined for bromelain was 5.0 to 8.0.



4.5.5. Optimum Temperature

The enzyme was preincubated for a period of 10 minutes at different temperatures in

cystein activators and then was subjected to assay. The temperature profile of protease

activity of fruit portion of bromelain is as shown in Figure 4.10.

From Figure 4.10 it is clear that the enzyme is most active at a temperature of 60 ºC. This

was in good agreement with the findings of Liang et al. (1999). They have stated that the

optimum temperature of bromelain was in the range of 50 – 60 ºC and above 65 ºC, it gets

inactivated.

0

0.1

0.2

0.3

0.4

0.5

0 20 40 60 80 1Temp (deg C)

Abso

rban

ce (A

at 2

80 n

m)

00

Figure 4.10: Temperature dependence of the protease activity of purified fruit bromelain.

Casein was used as substrate.

The results are in good agreement with the observations of Chittenden, 1891. He has

reported that neutralized pineapple juice exerts its maximum digestive power at about 60º

C.



From Figure 4.10, one may conclude that the enzyme should be stored at 60 ºC, but the

fact is that though the enzyme is having maximum activity at 60 ºC, it remains active at

that temperature for a short period of time and thereafter, it gets deactivated.

Hence, storage temperature is different from optimum temperature. Optimum Storage

temperature is also discussed as another product characterization parameter.

4.5.6. Effect of time on reaction velocity

The enzyme sample activated by Cystein HCl was analyzed for its activity. The enzyme

was mixed with casein substrate and was allowed to react for different time intervals. The

reaction was stopped by adding TCA at different time intervals. The effect of reaction time

on the reaction velocity is as shown in Figure 4.11.

0

50

100

150

200

250

0 5 10 15 20 25 30 35Time (min)

CDU/

ml

Figure 4.11: Effect of time on Reaction velocity

The Casein degradation rate was very fast during the initial period of reaction. It increased

exponentially till 15 minutes and thereafter it was found to be almost constant. So, in order



to give sufficient time for completion of reaction, it was decided to keep reaction time

equal to 30 minutes.

4.5.7. Time and Temperature stability

The time and temperature stability of freeze dried bromelain is as shown in Figure 4.12.

0

20

40

60

80

100

0 24 48 96Time (hrs)

% A

ctiv

ity R

eten

tion

4 deg C Room Temperature 60 deg C

Figure 4.12: Time and Temperature stability of freeze dried bromelain

The powder which was stored at 4 ºC showed only 4 – 5% loss of activity at the end of 4

days; whereas, the powder stored at room temperature showed 50 – 60% activity loss at the

end of 4 days and the powder which was stored at 60 ºC looses its activity by 90% at a

faster rate within 4 days.

Thus, the inactivation rate was found to be a strong function of temperature of dry powder

as well as liquid solution.



4.6 REFERENCES

Apte, P.V., Kaklij, G.S., Heble, M.R. (1979). Proteolytic enzymes (bromelains) in tissue

cultures of Ananas Sativus (Pineapple). Plant Science Letters, 14, 57 – 62.

Arakawa, T., Timasheff, S. N. (1984). Mechanism of protein salting-in and salting-out by

divalent cation salts: balance between hydration and salt binding. Biochemistry, 23, 5912 –

5923.

Chittenden, R.H. (1893). On the Proteolytic action of Bromelin, the ferment of pineapple

juice. Journal of Physiology, 15, 249 – 310.

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bromelain. Food Research International, 32(10), 545 – 551.

Melander, W., Horvarth, C. (1977). Salt effect on hydrophobic interactions in precipitation

and chromatography of proteins: An interpretation of the lyotropic series. Archives of

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Michail, M., Vasiliadou, M., Zotos, A. (2006). Partial purification and comparison of

precipitation techniques of proteolytic enzymes from trout (Salmo gairdnerii) heads. Food

Chemistry, 97, 50 – 55.

Murachi, T., Neurath, H. (1960). Fractionation and Specificity studies on stem bromelain.

Journal of Biological Chemistry, 235(1), 99 – 107.



Pikal, M.J., Rigsbee, D.R. (1997). The stability of insulin in crystalline and amorphous

solids: observation of greater stability for the amorphous form. Pharmaceutical Research,

14, 1379 – 1387.

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of different purities. European Food Research Technology, 213, 67 – 71.

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amylase – The effect of process variables on the enzyme inactivation. Drying Technology,

23, 941 – 953.

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Chapter Five

Conclusion

Conclusion

• Dry solid formulation provides acceptable enzyme shelf life. Dried bromelain

powder results in more activity retention and longer shelf life than the crystallized

bromelain.

• The glass transition temperature of the studied enzyme, bromelain in its native

form, is about 60 ºC. Hence, to avoid structural changes drying was carried out

well below 60 ºC.

• In spray drying of bromelain, the yield obtained was very less; however, this

problem can be overcome by adding some bulking agents. Outlet temperature has

a significant effect on activity retention of bromelain in spray drying. Outlet air

temperature in between 35 – 40 ºC gave maximum activity retention.

• Heat pump drying of bromelain results in crystalline product having low

enzyme activity and small shelf life.

• Freeze dried bromelain has more enzyme activity and shelf life as compared

to the spray dried bromelain. However, the mode of drying and purification

should be decided by the end application of the product. If the end application of

the bromelain is for dehairing in leather industry or in meat tenderization then

precipitation using ammonium sulfate followed by spray drying would be an

economical combination. But if the end application is in pharmaceutical (as an

digestive aid or anti swelling agent, etc.), then in such cases chromatography

should be used as a purification method and freeze drying should be preferred as a

final step as it provides longer shelf life and high activity retention for bromelain.


Chapter Six

Scope for Future Work

Scope for future work

The stability study of the bromelain during dialysis in buffers having different pH

can also be studied. Their final examination can be considered as the next step of this

project work; since dialysis results in removal of salt which gives concentrated protein,

which ultimately will result in an increased enzyme activity.

The powder properties such as particle size, porosity and generation of adsorption

isotherms at various temperatures, sticky point temperatures and glass transition

temperatures for bromelain are very important parameters and have a scope for further

study. In freeze drying of the said enzyme, secondary drying was not considered, i.e.

moisture was removed by means of sublimation only. However, the enzyme can be

heated further to a temperature as high as 60 ºC, the glass transition temperature of

bromelain, to remove unfrozen moisture which will have some effect on drying time and

drying kinetics.

In crystallization, crystal size distribution plays an important role, which also

needs to be examined. Scale up in crystallization can also be studied.

Some physical properties of the enzyme need to be examined. Further work can

be done for thorough examination of Michelis Menton constant, which will explain

binding of enzyme with the substrate.


Synopsis

SYNOPSIS

OF THE THESIS TO BE SUBMITTED TO


IN PARTIAL FULFILMENT FOR THE DEGREE OF

MASTER OF TECHNOLOGY

IN

BIOPROCESS TECHNOLOGY

Title of the Thesis : Crystallization and Drying studies of Biomaterials

Name of Candidate : DEVAKATE RAVIKANT VITHALRAO

Name and Designation of Research Supervisor : Professor B. N. THORAT Professor of Chemical Engineering Place of Research Work : DEPARTMENT OF CHEMICAL ENGINEERING Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai - 400019

Registration Number

and Date : 433 / 01-10-2005

Date of Submission : 29/03/2007

Professor B. N. Thorat Ravikant Vithalrao Devakate

(Research Supervisor) (Research Scholar)

Introduction:

A biomaterial is regarded as a bio-product and denotes a substance which is a

product of biotechnological transformation with the use of biochemically active

substances such as enzymes, proteins, whole cells, microorganisms, etc. (Kudra and

Strumillo, 1998). Enzymes are proteins with catalytic activity allowing chemical

reactions in a living cell to occur at ambient temperature at a high rate.

Bromelain (EC 3.4.22.4) is a collective name for proteolytic enzymes or proteases

found in tissues including stem, fruit, and leaves of the pineapple plant family

Bromeliaceae. Isolation of the enzyme from pineapple fruit and its study has been

investigated since the beginning of 20th century. The enzymes occurring in the stem and

the fruit of Ananas comosus are the most studied (Doko et. al., 1991). The properties of

proteinase from pineapple are similar to the sulfhydryl containing enzymes (Balls, et al.,

1941). Besides bromelain (proteases), other enzymes reported from pineapple are IAA

oxidase, peroxidase, phosphatase and cellulase. Proteases accounts for the half of the

protein content in pineapple (Dull, G.G., 1971).

Two types of bromelains from pineapple are commercially available, stem

bromelain and fruit bromelain. One that is obtained from stem is called stem bromelain

and from fruit is called as fruit bromelain. It is most active in the pH range of 5 – 8 and

temperature range of 30-40 ºC. It remains stable up to 50 ºC.

The potential therapeutic value of bromelain is due to its biochemical and

pharmacological properties and hence, it is desired to obtain bromelain in its highest

purified form. Once the enzyme has been purified to the desired extent, the main aim is to

retain the activity. Dry solid formulations are often developed to provide acceptable

protein and enzyme shelf lives. Freeze drying, Heat pump drying and Spray drying

techniques can be employed to obtain the dry solid formulation of enzyme.

Crystallization is the other technique besides chromatography (Przybycien et al.,

2004). Crystallization is one of the way to reach a more stable, lower energy state from a

metastable supersaturated state by reducing the solute concentration. Crystallization goes

through three stages of nucleation, growth and cessation.

In the present work fruit bromelain was selected over stem bromelain because

unlike crude stem bromelain, which is used widely in industry, fruit bromelain is not

commercially available despite the large quantities of waste pineapple fruit portion at

pineapple canneries (Caygill, 1979). Second reason to choose bromelain is due to the

scant literature available on proteases derived from the plant source.

Objectives of work:

The present work deals with the purification followed by crystallization or drying

of plant protease named, Bromelain, from the fruit portion of pineapple plant (Ananas

comosus, Family: - Bromeliaceae).

• To purify bromelain from fruit portion of pineapple plant (Ananas comosus) using

ion exchange chromatography.

• To crystallize crude bromelain from pineapple extract using different precipitants.

• To obtain purified bromelain in dry powder form using different drying

techniques such as freeze drying, spray drying and heat pump drying.

• To see the effect of drying temperature on retention of enzyme activity and

protein content in all the three drying operations.

• To find the optimum pH and temperature for bromelain.

• To study the water activity of enzyme as a function of temperature.

• To carry out FTIR study and DSC study of bromelain powder.

Experimental:

Column mode Chromatography experiments:

Experiments were performed by using 2.5 cm id glass columns. The sample was

loaded through the column containing ion exchange resins (SP). The column was then

washed with equilibration buffer (sodium acetate) and the bound protein was then eluted

with the proper buffer (sodium chloride) at desired flow rate (5-6 ml/min). The crude,

unbound, washing and elution fractions were then assayed for the presence of proteolytic

activity.

Freeze Drying and Spray Drying:

The eluted sample was subjected to freezing at temperature below -25ºC for a

period of 6 hrs. Then the sample was subjected to freeze drying in a freeze dryer (M/s.

Ref – Vac Consultancy). The dimensions of the chamber were 0.4 m x 0.4 m.

The eluted sample was also subjected to the spray drying (1 lit/hr evaporation

capacity spray dryer, M/s Labultima). The spray dryer operates in a co-current manner

and is equipped with a two fluid nozzle.

Results and Discussion:

The specific activity of crude juice was 64.19 Units/mg proteins and after

chromatographic purification the specific activity was found to be 617.29 Units/mg

proteins. So the fold purity achieved in chromatographic purification was 9.61.

The effect of operating parameters during drying was studied to obtain product

quality parameters such as enzyme activity, total protein content, total dissolved solid,

specific activity, reducing sugars and purification factor. The recovery of enzyme activity

ranges from 90 to 95% and that of protein varies from 80 to 85 % with purification factor

of 2.0 to 2.5 in case of freeze drying. The drying time required to reduce the moisture

content from 4.72 (kg/kg solid.min) to 0.015 (kg/kg solid.min) was found to be 8 hrs.

Drying rate was as high as 1.6 kg/kg.min at a moisture content of 4.72 (DB) and it

drastically reduces to 0.282 kg/kg.min at a moisture content of 4.14 (DB).

The yield obtained in spray drying was less as compared to the freeze drying. The

moisture content of the product obtained was 0.087 (DB). The recovery of enzyme

activity ranges from 70 to 75% and that of protein varies from 45 to 50 %.

Water activity of the Freeze dried powder and spray dried powder was found to be

0.466 and 0.514 respectively, at a temperature of 25 ºC.

Crystallization with the help of precipitating agents like ammonium sulfate,

sodium chloride and acetone was also carried out. However, Lyophilized and Spray dried

enzyme extract showed a higher specific activity than crystalline bromelain obtained by

fractionation with ammonium sulfate, sodium chloride and acetone.

References:

1. Kudra, T. and Strumillo, C., 1998. Characteristics of Bio-materials, In Thermal

Processing of Bio-materials. T. Kudra, C. Strumillo (Ed.), 12-13. Masterdam (The

Netherlands): Gordon and Breach Science Publishers.

2. Doko, M.B., Bassani, V., Casadebaig, J., Cavailles, L. and Jacob, M., 1991.

Preparation of proteolytic enzyme extracts from Ananas comosus L., Merr. fruit

juice using semipermeable membrane, ammonium sulfate extraction,

centrifugation and freeze-drying processes. International Journal of Pharmaceutics

76, 199-206.

3. Balls, A.K., Thompson, R.R. and Kies, M.W., 1941.Bromelain – Properties and

Commercial Production. Industrial Engineering Chemistry 33, 950-953.

4. Dull, G.G., 1971. The Biochemistry of fruits and their products, vol. 2, 303-309.

Academic Press, London.

5. Przybycien, T.M., Pujar, N.S. and Steele, M.L. 2004. Alternative bioseparation

operations: life beyond packed-bed chromatography. Current Opinion in

Biotechnology 15, 469-478.

6. Caygill, J.C. 1979. Sulphydryl plant proteases. Enzyme and Microbial

Technology 1, 233-242.

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