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BIOTECHNOLOGICAL APPROACHES

TO PRODUCTION OF

BIOACTIVES FROM COFFEE BY-PRODUCTS

A Thesis Submitted to the

UNIVERSITY OF MYSORE

In fulfilment of the requirements for the award of

DOCTOR OF PHILOSPHY in BIOTECHNOLOGY by

PUSHPA S. MURTHY

Under the supervision of

Dr. M. Madhava Naidu, Scientist

Department of Plantation Products, Spices and Flavour Technology Central Food Technological Research Institute Council of Scientific and Industrial Research

Mysore 570 020, India

April 2011

Mrs. Pushpa S. Murthy Scientist, PPSFT Department CFTRI, Mysore- 570 020

CERTIFICATE

I, Mrs. Pushpa S. Murthy, certify that this thesis is the result of research work done by

me under the supervision of Dr. M. Madhava Naidu, Scientist at the Plantation Products, Spices

& Flavour Technology Department, Central Food Technological Research Institute, Mysore. I am

submitting this thesis for the award of Doctor of Philosophy (Ph.D.) degree in Biotechnology by

the University of Mysore.

I further certify that this thesis has not been submitted by me for award of any other

degree/ diploma of this or any other University.

Signature of Doctoral Candidate

Date:

Signature of Guide

Date: Counter signed by

Signature of Head of Department

Acknowledgements

I owe my first and foremost, love and gratitude to almighty for every blessing he has

showered on me.

I thank CSIR and CFTRI, Mysore for giving an opportunity and providing me resources

to carry out my research successfully.

My thanks to Dr. M. Madhava Naidu my academic supervisor for his guidance and

constant encouragement throughout my research work.

I am grateful to Dr. P. Srinivas, Head, PPSFT, for helpful advice, guidance and support

during my research investigation.

I am indebted to Dr. V. Prakash, former Director of CFTRI for providing me constructive

suggestions and guidance during the formulation of my research plan and studies. I am also

grateful to present acting director of CFTRI, Dr. G. Venkateshwara Rao. I sincerely thank Dr.

B.R. Lokesh, Dr. A.G. Appu Rao, Dr.M.C. Varadaraj, Dr. Lalitha R. Gowda, Dr. H.K.

Manonmani, Dr. K. Srinivasan and Dr. T. R. Shamala for their valuable suggestions during the

registration and pre-thesis submission viva-voce presentations.

My heartfelt thanks to Dr. Ramasharma, Ms. Latha and Mr. Mandappa for their

essential help during purification of enzymes. My special thanks are to Mr. Mukund, CIFS for

all the help extended during Mass spectroscopy studies, Mr.Manjunath for carrying out NMR

studies.My thanks are also due to FOSTIS which provided valuable information during my

investigations. I extend my thanks to HRD Dept for being the catalyst in processing all the

required documents of PhD for the university.

I wish to place on record my immense thanks to CCRI, Coffee Board, for providing the

necessary coffee samples to carry out my work. My thanks are extended to Dr. K. Basavaraj,

Head, Quality Evaluation center, Coffee Board for carrying out organoleptic evaluation.

The Department of Biotechnology and the administration section of Mysore University is

acknowledged for all the service rendered from the time of enrollment to submission of the thesis.

I owe my sincere thanks to Mr. Rajesh, Mr. Vijyashankara, Ms. Subhashini, Ms. Priya,

Ms. Kavya and Ms. Rahath for their support during my work.

My humble thanks to the staff of PPSFT Department, Dr. K. Ramalakshmi, Dr. H.B.

Sowbhagya, Mrs. G. Sulochanamma, Mrs. H.J. Lalitha, Dr. S. Nagarajan, Dr. J. Puranaik,

Dr. L. Jagan Mohan Rao, Dr. B.B. Borse, Mr.Chandrashekar, Ms Hafeeza khanum, Mrs.

Somalatha, Mr. Mohammed Zia Ulla, Mrs. Anusuyamma and Mrs. Ningamma for all the co-

operation, motivation and help extended in various forms.

I would like to thank my lovable grandma, respected parents, supporting brother, sister and

their families for their blessings and wishes which have made me prosperous. My gratitude to dear

friend Manonmani for all the help extended.

I owe deep respect to my school teachers, lecturers, students, friends and all the ones who

have contributed to science of coffee, who taught me many things towards my educational journey.

This dream would not have been realized without the essential and gracious support of my

esteemed husband Mr. K. Srinivas Murthy, my little ones Yaju, Vibu and my maid Kempamma.

Pushpa S. Murthy

INDEX

Description Page Nos

List of Tables and Figures i

List of Abbreviations vi

Abstract viii

Synopsis ix

Chapter 1 Introduction and Review of Literature 1

1.1 Introduction 1

1.2 Review of literature 4

1.2.1 Coffee classification 5

1.2.2 Coffee cultivation, processing, production and export 7

1.2.3 Biotechnological management of coffee by-products 14

1.2.4 Production of enzymes by microorganisms and SSF 19

1.2.5 Bioactives from agro- industrial wastes 31

1.3 Conclusions 44

1.4 References

45

Chapter 2 Utilization of coffee by-products for the production of

enzymes (Amylase,Xylanase,Protease,Pectinase) by

Solid-state fermentation.

2.1 Introduction 59

2.2 Materials 62

2.3 Methods 68

2.3.1 Screening for microorganisms for enzyme production 68

2.3.2 Preparation of microbial inoculum 69

2.3.3 Studies on production of -amylase 69

2.3.4 Studies on xylanase production using coffee by-products by SSF

75

2.3.5 Studies on protease enzyme production utilizing coffee by-products by SSF

78

2.3.6 Studies on pectinase production by SSF using coffee by-products

82

2.4 Results and discussion 86

2.4.1 Screening of microbes for enzyme production 86

2.4.2 SSF for production of -amylase and optimization of the process parameters

89

2.4.3 Studies on Xylanase production by SSF 106

2.4.4 Studies on Protease enzyme by Aspergillus oryzae CFR 305 by SSF

121

2.4.5 Studies on pectinase production by Aspergillus niger

CFR 302

134

2.5 Conclusions 147

2.6 References 150

Chapter 3 Application of enzymes in coffee processing with emphasis on demucilage of coffee beans

3.1 Introduction 167

3.2 Materials 169

3.3 Methods 171

3.3.1 Proximate composition of robusta coffee pulp and mucilage

171

3.3.2 Solid-state fermentation for production of pectinase by A.niger

175

3.3.3 Decolorization of the extracted enzyme 177

3.3.4 Demucilisation of robusta coffee by natural

fermentation and with enzymes

178

3.3.5 Physical and organoleptic analysis of raw/green robusta coffee

180

3.4 Results and Discussion 183

3.4.1 Proximate composition of robusta coffee pulp and

mucilage

183

3.4.2 Solid-State fermentation for production of pectinase 186

3.4.3 Decolorization of crude enzyme using activated carbon 187

3.4.4 Effect of pH and temperature on decolorized crude pectinase activity

190

3.4.5 Demucilage of robusta coffee 191

3.4.6 Physical and organoleptic characteristics of robusta coffee

197

3.5 Conclusions 200

3.6 Re References 201

Chapter 4 Extraction, isolation and in-vitro studies of bioactive compounds from coffee by- products

4.1 Introduction 209

4.2 Materials 212

4.3 Methods 214

4.3.1 Extraction of chlorogenic acids (CGA) from coffee by-products

214

4.3.2 Determination of dietary fiber and its properties from coffee by-products

217

4.3.3 Extraction of anthocyanins from coffee pulp 221

4.3.4 In-vitro studies of CGA conserves, dietary fiber and anthocyanin derived from coffee by-products

225

4.4 Results and discussion 231

4.4.1 Extraction of CGA conserves from coffee by-products 231

4.4.2 Dietary fiber and its properties present in coffee by-products

234

4.4.3 Anthocyanins from coffee pulp 238

4.4.4 In-vitro studies of bioactive compounds (CGA conserve fiber and anthocyanins) derived from coffee by-products

247

4.5 Conclusions 257

4.6 References 258

Summary and conclusions 268

Publications and papers presented in symposia 271

List of Tables

Tabl

e No. Description Page No

1.1 Phenolic compounds obtained from agricultural by-products 33

1.2 Main groups of anthocyanidins 42

2.1 Details of the organisms which produced high enzyme yield 86

2.2 Summary on purification of -amylase from N. crassa by SSF 101

2.3 Effect of metals and chelators on -amylase activity 106

2.4 Influence of pre-treatments on coffee pulp and production of

xylanase

114

2.5 Partial purification of xylanase produced under SSF by

Penicillium sp

116

2.6 Effect of metals and chelators on xylanase activity 119

2.7 Treatment of lignocellulosic substrate and kraft pulp with

purified xyalanse

120

2.8 Influence of pre-treatment of coffee husk on the production of

protease

128

2.9 Summary of purification of protease produced under SSF by

Aspergillus oryzae.

129

2.10 Effect of metallic ions on protease activity 132

2.11 Effect of inhibitors on protease activity 133

2.12 Influence of pre-treatments of coffee pulp on the production

of pectinase

141

2.13 Purification of pectinase produced under SSF by Aspergillus

niger

142

2.14 Effect of metallic ions on pectinase activity 145

2.15 Effect of inhibitors on pectinase activity 146

3.1 Physico-chemical properties of robusta coffee pulp and

mucilage

185

3.2 Enzyme present in crude pectinase extracted from A.niger CFR

305

186

3.3 Effect of colour intensity of crude enzyme on adsorption by

charcoal

188

3.4 Physical and chemical changes occurred during robusta coffee

fermentation

195

4.1 Phenolic content of coffee by-products 231

4.2 Yield and chlorogenic acid composition present in coffee by-

products

232

4.3 Dietary fiber composition present in coffee by-products 235

4.4 1H Spectral Assignments for cyanidin 245

4.5 13C Spectral Assignments for cyanidin 246

4.6 Antioxidant activity of the dietary fiber from coffee by-

products

250

4.7 Antioxidant activity of anthocyanins from coffee pulp 251

List of Figures

Figure

No. Description

Page

No.

1.1 Coffee Plant 5

1.2 Coffee flowers blossomed in the estate 6

1.3 Coffee harvesting 7

1.4 Coffee pulping 8

1.5 Coffee drying 11

1.6 Green coffee beans 11

1.7 Coffee roasting and brewing 12

1.8 Coffee by-products obtained during coffee processing 15

1.9 Structure of chlorogenic acid 35

1.10 Basic structure of anthocyanins 41

2.1 Neurospora crassa 87

2.2 Penicillium sp 87

2.3 Aspergilllus oryzae 88

2.4 Aspergillus niger 89

2.5 Solid- state fermentation of coffee pulp for production of -amylase 90

2.6 Production of -amylase from coffee by- products 90

2.7 Optimisation of process parameters for production of - amylase 96

2.8 Effect of carbon and nitrogen sources on -amylase activity 98

2.9 Effect of pre-treatments on - amylase on coffee pulp 100

2.10 SDS- PAGE of purified - amylase from N. crassa 102

2.11 Effect of pH, temperature and substrate concentration on - amylase activity

104

2.12 Production of xylanase from coffee substrates under SSF 107

2.13 Optimization of process parameters on xylanase production by

Penicillium sp

110

2.14 Supplementation of carbon and nitrogen on production of xylanase

by SSF

113

2.15 SDS- PAGE of xylanase from Penicillium sp 116

2.16 Influence of temperature, pH and substrate concentration on

xylanase activity

118

2.17 Production of protease using coffee by-products in SSF 122

2.18 Effect of moisture, temperature, pH ,fermentation time and

substrate particle size on protease production on SSF

125

2.19 Effect of carbon and nitrogen sources on protease in SSF 127

2.20 Electrophoretic profile of protease from A.oryzae 130

2.21 Influence of pH and temperature on activity of purified protease 131

2.22 Pectinase production by A.niger CFR 302 in SSF 134

2.23 Optimisation of process parameters for pectinase production in SSF 138

2.24 Effect of Carbon and nitrogen sources on pectinase by SSF 140

2.25 Electrophoretic profile of pectinase, lane C-purified pectinase 143

2.26 Influence of pH and temperature on activity of pectinase from

A.niger

144

3.1 Pre-treatment of crude enzyme using activated charcoal 189

3.2 Optimization of pH and temperature on decolorized pectinase 190

3.3 Mucilage of the robust coffee fruit 191

3.4 Coffee fermentation tank and testing of mucilage of coffee 192

3.5 Physical and organoleptic characters of robusta coffee beans treated with pectinase

199

4.1 Chlorogenic acid conserve derived from coffee by-products 233

4.2 HPLC profile of CGA conserve obtained from coffee silver skin 234

4.3 Hydration properties of coffee fiber (Silver skin) at different granulometry

238

4.4 Anthocyanins extracted from coffee pulp

239

4.5 HPLC profile of the (a) purified anthocyanin from coffee pulp, (b) Purified extract of anthocyanin of coffee pulp after acid hydrolysis

240

4.6 Electrospray mass spectrum of (a) crude pulp (b) major fraction of HPLC with ion peak m/z (595) (C) minor fraction of HPLC (449)

243

4.7 Structure of cyanidin 3-rutinoside in coffee

245

4.8 Antioxidant activity of CGA conserve derived from coffee by-products by DPPH method

247

4.9 Antioxidant activity of CGA conserve derived from coffee by-products by hydroxyl radical scavenging assay

249

4.10 - Amylase inhibitory activity at different concentration of anthocyanins

254

4.11 - glucosidase inhibitory activity at different concentration of anthocyanins

256

List of Abbreviations

% : percent @ : at ~ : approximately 0C : Degree Centigrade A512nm : Absorbance at 512 nanometers A700nm : Absorbance at 700 nanometers A765nm : Absorbance at 765 nanometers AA : Ascorbic acid ABTS 2,2 : Azinobis-3-ethylbenzthiazoline-sulphonic acid

ANOVA : Analysis of variance BHA : Butylated hydroxyanisole BHT : Butylated hydroxytoluene Cm : Centimeter(s) DEAE : Diethyl amino ethyl DNS : Dinitrosalicylic acid DPPH : 2, 2-diphenyl-1-picrylhydrazyl EC : Enzyme Commission EDTA : Ethylene diamine tetra Acetic Acid ESI

: Electro-spray ionization F-C : Folin-Ciocealteu reagent g : gram h : hour(s) ha : hectares HPLC : High performance liquid chromatograph IC50 : concentration for 50% inhibition kDa : kilo Dalton kg : kilograms L : Liter(s) M : Molar m/z : mass to charge ratio MALDI : Matrix-assisted laser desorption ionization mg : Milligram Min : Minute(s) mL : Milliliter mm : Mllimeter(s) MS : Mass Spectrometry MW : Molecular weight N : Normality

nm : nanometer rpm : rotation per minute s : seconds SD : Standard deviation SDS : Sodium dodecyl sulphate

SDS PAGE : Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SmF : Submerged fermentation Sp. : Species SSF : Solid state fermentation TDF : Total dietary fibre TEAC : Trolox equivalent antioxidant capacity TFA : Trifluoroacetic acid V/v : Volume/volume W/v : Weight/volume : alpha : beta : gamma A : Difference in absorbance values max : Maximum wavelength mg : Microgram(s) L : Microlitre(s) : Molar absorbance coefficient Ugds-1 : Units per gram dry substrate U/mL : Units per mL :

Abstract

The interest in the application of biotechnological tools for exploitation of the food

processing wastes into value-added products has increased. The present study explored

utilization of coffee by-products for production of industrially useful enzymes such as amylase,

protease, xylanase and pectinase by solid-state fermentation utilizing fungal organisms,

Neurospora crassa, Aspergillus oryzae, Penicillium sp, and Aspergillus niger respectively,

followed by partial purification and characterization of the enzymes. The crude decolorized

pectinase obtained from coffee pulp was used in the fermentation step in robusta coffee

processing for demucilisation. The lab scale approach using this enzyme resulted in completion

of demucilage process in about of 2-3 h in case of robusta coffee compared to that of 48-72 h

period required in case of natural fermentation. The coffee by-products were also explored for

extraction of bioactive compounds such as chlorogenic acid, dietary fiber and anthocyanins.

The chlorogenic acid conserve from pre-treated coffee by-products were highest in case of

silver skin (25 %) followed by spent waste (19 %) and cherry husk (17 %). The total dietary fiber

(TDF) in coffee by-products was determined and maximum yield was obtained with silver skin

(80 %) followed by cherry husk (43 %) and pulp (43 %). The coffee pulp has cyandin-3-rutinoside

as the major anthocyanin. The in-vitro studies on bioactive compounds from coffee by-

products demonstrated antioxidant activity, - amylase and -Glucosidase inhibitory activity.

Chapter 1 Review of literature

1

1.1 INTRODUCTION

Biotechnological applications in the field of industrial residues management

promote sustainable development of the countrys economy. The objectives concerning

food processing by-products, waste and effluents is the recovery of fine chemicals and

the production of precious metabolites via chemical and biotechnological processes

(Federici et al., 2009). Indeed, after specific pre-treatments with physical and biological

agents followed by tailored recovery procedures, they might provide value-added

natural antioxidants, antimicrobial agents, vitamins, etc., along with macromolecules

(enzymes, cellulose, starch, lipids, proteins and pigments) of enormous interest to the

pharmaceutical, cosmetic and food industries. Several other compounds occurring in

the hydrolyzate obtained through by-products/ waste pre-treatment can be further

transformed into more sophisticated natural chemicals (such as pharmaceuticals,

flavours, vitamins and organic acids), macromolecules (such as biopolymers, lubricants

and microbial enzymes) and biofuels through tailored biotechnological processes

(Laufenberg et al., 2003, Wyman et al., 2003).

A more recent approach has involved the use of processing technologies to

fractionate potentially high value components from them, thereby turning waste

streams into products of interest (Laufenberg et al., 2003, Wyman et al., 2003,

DAnnibale et al., 2003). Other bioactive components are carotenoids, phytoestrogens,

natural antioxidants, such as phenolic compounds and functional compounds (Llorach et


2

al., 2002, Moure et al., 2001, Schieber et al., 2001). Phenolic and flavonoid compounds

have recently attracted much interest because they are potent antioxidants and exhibit

various other physiological activities including anti-inflammatory, antimicrobial, anti-

allergic, anti-carcinogenic and antihypertensive activities (Akkarachiyasit et al., 2009).

The recovery of such value added compounds from processing by-products and waste

has increased their availability.

In addition, relevant amounts of components of pretreated by-products and

waste remain unexploited and might compose an environmental problem (Wyman et

al., 2003). Thus, the currently adopted valorization steps, lead to the complete

exploitation of the by-product and waste biomass, with remarkable improvements of

the environmental and economic sustainability of the overall approach, such as

enzymatic pre-treatment and extraction/recovery (via precipitation, membrane or

chromatography technologies, as well as supercritical fluid extraction) of natural

chemicals, biomaterials and food ingredients (antioxidants, pigments, vitamins, gelling

agents, pectin, oligosaccharides, dietary fibers) followed by the biotechnological

conversion of some of the obtained chemicals/ bio-products into more sophisticated

tailored bio-compounds, such as flavourings, pharmaceuticals, secondary building blocks

etc., (Benoit et al., 2006).

Coffee is one of the internationally traded produce and it is the second largest

commodity in the world, next only to petroleum. Globally, coffee is cultivated on 11.6


3

million hectares and its production is 7.2 million MT. World average productivity is 505

kg/ha. Coffee is grown in about 80 countries across the globe of which 51 are

considered to be the major producers. India stands sixth in the world coffee production

and fifth in world coffee productivity with an average of 860 kg/ha which is higher than

that of Colombia (775 kg/ha.) and Brazil (535 kg/ha). In India, coffee occupies an

important position among the export commodities particularly in the plantation sector.

The Indian coffee Industry is also heading for the highest ever crop production by the

year 2010 and the estimated crop will be about 3 lakh metric tons.

Traditionally, coffee pulp and husk are large amounts of by-products obtained

during industrial processing of coffee bean had found only a limited application as

fertilizers, livestock feed, compost, etc. These applications utilized only a fraction of

available quantity and were not technically very efficient. Recent attempts have

focused on thier application as substrates in bioprocesses and vermicomposting (Pandey

et al., 2000).

In the back ground of this high crop production in the upcoming years, there is

an imperative need to counterpart this production with utilization and industrial

application of coffee by-products for development of nutraceuticals since coffee

industry throws out enormous amount of coffee by-products which are rich in

carbohydrates, proteins, pectins, bioactive compounds like polyphenols and fiber

(Pushpa et al., 2010). Agro wastes can also represent a resource of potentially useful


4

chemical substances after direct recovery of simple and complex carbohydrates that

could be used for fermentation processes (Crognale et al., 2006).

Efficient recovery of fine bioactive chemicals and/or production of value added

products such as edible mushrooms, ethanol, organic acids and enzymes appear to be

the new frontier in waste management. Explorations to value addition of coffee by-

products can be made with the integration of techniques and current bioengineering

principles in food processing and waste management, which attempts to conserve

environment along with improvement of country economy. Thus, in this thesis

utilization of the coffee industrial wastes have been the key targets in production of

industrially useful enzymes and isolation of bioactive compounds along with their

application with the topic entitled Biotechnological approaches to production of bio-

actives from coffee by-products.

1.2 REVIEW OF LITERATURE

Coffee is one of the worlds most popular beverages and has grown steadily in

commercial importance for last 150 years (Dalgia et. al., 2000). Coffee originated from

the Arabic word Quahweh. Today its popularity is identified in various terms such as

cafe (French), caffe (Italian), Kaffee (German), koffie (Dutch) and coffee (English) (Smith,

1985). The stimulatory effects of roasted coffee beans were well known to the natives

of Africa when the Arabs brought Coffea arabica seeds from Ethiopia to Yemen (Arabian

Peninsula) during the 13th century, and established the first plantations (Monaco et al.,


5

1977). The province of Kaffa in Ethiopia is considered to be the original habitat of

Arabica and Central Africa is reckoned to be the home of robusta. Today Brazil is the

largest producer and exporter of coffee in the world.

1.2.1 Coffee Classification

Coffee is an important plantation crop belonging to the family Rubiaceae,

subfamily Cinchonoideae and tribe Coffeae (Clifford et al., 1989). The Rubiaceae

members are largely tropical or subtropical with nearly 400 genera and 4800-5000

species. Botanically, coffee belongs to the genus Coffea of the family Rubiaceae. The

sub-genus Coffea is reported to comprise over 80 species, which are prevalent to Africa

and Madagascar (Bridson and Verdcourt, 1988).

Classification:

Kingdom

Plantae

Subkingdom Tracheobionta

Division Magnoliophyta

Class Magnoliopsida

Subclass Asteridae

Order Rubiales

Family Rubiaceae

Genus Coffea

Coffea arabica L. popularly known as arabica and C. canephora Pierre

commonly known as robusta are the only cultivated species and are responsible for

Fig 1.1 Coffee plant

http://plants.usda.gov/classification/output_report.cgi?3|S|Plantae|u|140|+63http://plants.usda.gov/classification/output_report.cgi?3|S|Tracheobionta|u|140|+63http://plants.usda.gov/classification/output_report.cgi?3|S|Magnoliophyta|u|140|+63http://plants.usda.gov/classification/output_report.cgi?3|S|Magnoliopsida|u|140|+63http://plants.usda.gov/classification/output_report.cgi?3|S|Asteridae|u|140|+63http://plants.usda.gov/classification/output_report.cgi?3|S|Rubiales|u|140|+63http://plants.usda.gov/classification/output_report.cgi?3|S|Rubiaceae|u|140|+63


6

over 99 % of global coffee produce. Coffee is a perennial plant and evergreen in nature.

It has a prominent vertical stem giving rise to horizontal primary branches in pairs

opposite to each other (Fig 1.1). Coffee has shallow root system, the feeder roots of

Arabica coffee penetrate relatively deeper in soil whereas, Robusta which has feeder

roots concentrated very close to the surface of the ground. The spread of the roots

depends on the type of the soil and cultural practices (Pushpa et al., 2001).

Coffee leaves are opposite decussate on suckers, but plagiotropic branches by

torsion, successive nodes with the leaves lie in one plane. The leaves are shiny, wavy,

and dark green in color with conspicuous veins. Coffee is a short day plant i.e., floral

initiation takes place during short day conditions of 8-11 h of day light. The group of

flowers, technically known as the inflorescence is a condensed cymose type subtended

by bracts (Fig 1.2). Pollination takes place within 6 hours after flowering.

Fig 1.2 Coffee flowers blossomed in the estate

of self-compatibility. The process of fertilization is completed within 24-48 h after

pollination. Seeds are elliptical or egg shaped, Plano convex possessing longitudinal

furrow on the plane surface. Seed coat is represented by the silver skin which is also

Arabica coffee is autogamous with

different degrees of natural cross-

pollination in contrast to Robusta

coffee, which is strictly allogamous

with an inbuilt ametophytic system


7

made up of scleroides. The size, thickness or numbers of pits in the walls of scleroides

are considered as important taxonomic characters in evaluating differences between

species. Germination takes place in about 45 days.

1.2.2 Coffee cultivation, processing, production and export

Coffee trees grow in tropical regions, between the tropic of Cancer and

Capricorn, that have abundant rainfall, year round warm temperatures averaging 70

degrees Fahrenheit, and no frost. They grow at altitudes ranging from sea level to 6,500

feet and above.

1.2.2.1 Harvesting

Fig. 1.3 Coffee harvesting Fig 1.3 Coffee harvesting

Once the fruits are harvested, sorting of the greens/ immature, overripe are carried out

and dried separately since they affect the final quality of coffee by producing foul flavor

(Pushpa et al., 2001).

It takes about five years for a coffee tree to bear its

first full crop of beans and will be productive for

about fifteen years. Only ripe fruits are harvested

by selective picking from each dominant variety

situated at particular elevation separately and

treated as an independent lot (Fig 1.3).


8

1.2.2.2 Coffee Processing techniques

Processing is a major activity in Coffee production converting the raw fruit of the

coffee plant into the coffee. The two basic methods of coffee processing which differ in

complexity and the quality of the resultant raw coffee and the liquor are wet method

and dry method.

1.2.2.3 Wet method

Coffee processed by the wet method is called washed or parchment coffee. In

the wet Process, the fruit covering the seeds/ beans are removed before they are dried

(Fig 1.4). The wet method requires the use of specific equipment and substantial

quantities of water.

Fig 1.4.Coffee pulping

Preparation of wet method requires reliable

pulping equipment and adequate supply of clean

water. Whatever be the method of demucilaging

adopted by using different pulpers (drum pulper,

disc pulper, vertical spiral drum pulper), the final

objective is to ensure complete removal of

mucilage from the parchment cover for production

http://www.google.co.in/imgres?imgurl=http://www.babble.com/CS/blogs/strollerderby/2008/11/01-07/coffee.jpg&imgrefurl=http://www.babble.com/cs/blogs/strollerderby/archive/tags/coffee/default.aspx&usg=__HBBkCFIQ5G4rV3rCrwHJkJZO1ik=&h=346&w=347&sz=76&hl=en&start=3&zoom=1&itbs=1&tbnid=DiE4aXKCPP95NM:&tbnh=120&tbnw=120&prev=/images?q=coffee&hl=en&tbs=isch:1


9

of high quality coffee (Pushpa et al., 2001). The purpose of fermentation is to break

down the mucilage layer on the parchment into simple non-sticky substances. The

mucilage of the coffee fruit is removed and digested by natural fermentation. Over

fermentation or under fermentation should be avoided because over fermentation

results in a loss of bean colour and poor cup and under fermentation would lead to

moisture absorption by the beans due to the sticky mucilage on the parchment and

cause mustiness in the final cup. The optimum temperature for fermentation is 30 -

35C. The coffee mass should be stirred 2 - 3 times during the fermentation period with

help of a Raker.

The degradation of mucilage takes approximately 24 - 36 h for arabica and 72 h

for robusta depending on the inherent concentration of pectinolytic enzymes ambient

temperature and elevation. Correct washing is ensured by hand feel where the

parchment will not stick to the hand after washing. In addition to the methods of

decomposition of mucilage mentioned above, there is mechanical way of removal of

mucilage. Use of this machine is advocated to completely/partially fermented beans

basically to achieve total washing of the beans and also reduce the quantum of water

usage and to get desirable flavour in coffee. After washing, soaking of the parchment

under clean water for a period of 12 h is practiced. The parchment is stirred

occasionally. Under water soaking removes diterpenes and polyphenolic substances


10

which are responsible for hardness in brew. Soaking improves visual appearance of the

beans and also quality.

The washed parchment is drained for excess moisture, conveyed to the drying

barbecues and spread evenly to a thickness of 5 centimeters, turned with wooden rakes

to ensure uniform drying and reduce parchment splits. However, slow drying and

avoiding over drying of coffee beans should be invariably followed. Strong solar

radiations should be avoided on the 3rd and 4th day during noon by providing artificial

overhead shade by a tarpaulin or stitched knitted clean bags. Latter the coffees are

dried until the moisture reaches around 10 %. Too thin layer of parchment leads to

rapid drying which causes splitting of parchment skin and shrunken beans. Improper

and uneven drying of parchment results in mottled roast.

1.2.2.4 Cherry or Dry method

The freshly harvested fruits are spread evenly to a thickness of about 8

centimeters on clean drying yard (Fig 1.5). The fruits are stirred and ridged once very

hour. The cherry is ensured dried when a fistful of coffee produce a rattling sound when

shaken (Pushpa et al., 2001). The coffees are dried to the prescribed moisture level.

The cherry coffee should normally be fully dry at the end of 12 to 15 days under bright

weather conditions. Dry cherry coffee should not be exposed to wet conditions to avoid

mould formation which adversely affect coffee quality. Each lot of cherry should be


11

bagged separately in clean dry gunny bags. Drying is an important operation in the

preparation of coffee.

Fig 1.5 Coffee drying

1.2.2.5 Green Coffee

Fig 1.6.Green coffee beans

The green coffee is composed of both volatile and non-volatile compounds. The major

components of green coffee are carbohydrates, protein, lipid, minerals, ash, caffeine,

chlorogenic acid, trigonelline, water etc. The consumable form of these green coffee

beans is obtained after the process of roasting (Clarke and Macrae, 1985).

The green coffee is obtained after all the above

processing (Fig 1.6). The green coffee is classified into

washed and unwashed based on the method of

processing, i.e. wet or dry process.

Proper drying contributes the quality in terms of

colour, shape and aromatic constituents. Drying

rate of parchment is dependent on the initial

moisture of the parchment, ambient air

temperature, humidity, thickness of the spread

and periodicity of stirring the coffee.

Fig 1.6 Green coffee beans

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12

1.2.2.6 Coffee roasting and brewing

The characteristic flavor and aroma of coffee result from a combination of

hundreds of chemical compounds produced by the reactions that occur during roasting

and brewing (Fig 1.7) (Castillo et al., 2002). This process can be divided into three

consecutive stages (i) drying, (ii) roasting or pyrolysis (iii) cooling. The first stage is

characterized by a slow release of water and volatile substances, during the first half of

processing. Bean color changes from green to yellow. Pyrolysis reactions take place

during the second stage, resulting in considerable changes in both physical and chemical

properties of the beans.

Fig 1.7 Coffee roasting and brewing

Coffee brewing is hetrophase ranging from smooth pure solution to emulsion

(Drip filter coffee, Nordic boiled coffee, Turkish style brew, Espresso, and cappuccinos).

Coffee processing is an art as well as science and involves a series of stages each of

which has a distinct purpose. To produce high quality coffees, it is imperative that all

stages are taken utmost care in accordance to the recommended procedures.

http://www.google.co.in/imgres?imgurl=http://www.dccoffeeproducts.com/inc/MCA.jpg&imgrefurl=http://www.dccoffeeproducts.com/bunnpodbrewers.html&usg=__x5vBj1yqGPJCT7BGAPmLHp-e3lQ=&h=500&w=500&sz=13&hl=en&start=21&zoom=1&itbs=1&tbnid=0e4pthpUC-Xo6M:&tbnh=130&tbnw=130&prev=/images?q=coffee+brewing&start=20&hl=en&sa=N&ndsp=20&tbs=isch:1


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1.2.2.7 Coffee production and export

Coffee is an important commodity and a popular beverage. Over 2.25 billion

cups of coffee are consumed in the world every day (Stefano ponte, 2002). Over 90 % of

coffee production takes place in developing countries, while consumption happens

mainly in the industrialized economies (Stefano ponte, 2002). Worldwide, 25 million

small producers rely on coffee for a living. Coffee is grown in about 80 countries across

the globe, of which 51 are considered to be the major producers (Anonymous 1996).

Brazil, Vietnam and Colombia account for more than half of worlds production. The

global coffee production per year on an average accounts to about 7.0 million metric

tons and India is one of the coffee producing countries with an average production of

3.0 lakh metric tons annually.

India is a producer of both arabica and robusta varieties of coffee in proportion

of 35 : 65. In India, coffee occupies an important position among the export

commodities particularly in the plantation sector. Production of coffee has risen from

18,000 tons during 1950s to 230,000 tons in 1998 - 99 (Anonymous 1996) and today

ranks 6th amongst the top coffee producing countries. According to Coffee Board of

India estimates, production in India during 2010 was 3.0 lakh tones from 2.89 lakh tones

in the previous year (2009). Italy, Germany and Russia are India's biggest overseas

coffee markets. Together, these three countries constitute over 40 per cent of India's

http://en.wikipedia.org/wiki/Commodity


14

total exports. However, the exports to other than traditional countries are knocked to

encourage more exports from India.

1.2.3 Biotechnological management of coffee by-products

Advances in industrial biotechnology offer potential opportunities for economic

utilization of agro-industrial residues. There is an increasing demand to replace

traditional chemical process involving microorganisms, which not only provide an

economically viable alternative but also more environmental friendly. Indian economy

is one of the most important agricultural-based economies in the world, producing

coffee, sugarcane etc. Almost every product is exported, which is definitely an excellent

contribution for its economic development. However, this greater production is

responsible for the generation of very high amounts of residues that cause serious

environmental problems (Pandey et al., 2000). There are several recent publications

describing bioprocesses that have been developed utilizing these raw materials for the

production of bulk chemicals and value-added fine products such as ethanol, single-cell

protein (SPC), mushrooms, enzymes, organic acids, amino acids, biologically active

secondary metabolites etc. (Pandey et al.,2000). Not only the application of agro-

industrial residues in bioprocess provides alternative substrates, but also helps solving

pollution problems. Biotechnological processes, specially the solid-state fermentation

(SSF) technique, have contributed enormously for such utilization.


15

1.2.3.1 Coffee industry residues

Industrial processing of coffee cherries is done to isolate coffee powder by

removing shell and mucilaginous part from the cherries. There are two methods i.e. dry

and wet processing. Depending upon the method of coffee cherries processing, i.e. wet

or dry process, the solid residues (sub-products) obtained are termed as pulp or husk,

respectively (Fig 1.8). Coffee pulp is the largest by-product obtained and represents 40

% of the coffee berry in wet form (Bressani et al., 1972). This large quantity of the

coffee pulp pose problems of disposal of coffee berry producers, due to putrefaction

and causes environmental pollution if not disposed after appropriate pretreatment

(Zuluaga, 1989). Due to its high organic matter content, coffee pulp can be utilized for

beneficial purposes.

A. Coffee pulp B. Cherry husk C. Silver skin D. Spent coffee

Fig 1.8 Coffee by-products obtained during coffee processing

In India, the coffee cherries are generally processed by wet and dry method,

resulting in coffee pulp and coffee husk, which is rich in organic nutrients. Although

A B C D


16

several bacteria, yeasts and fungi have been cultivated on coffee pulp and husk for

various purposes, filamentous fungi, especially basidiomycetes are the preferred choice

and have most widely been employed. Traditionally, coffee pulp and husk had found

only a limited application as fertilizers, livestock feed, compost, etc. These applications

utilize only a fraction of available quantity and are not technically very efficient. Recent

attempts have focused on their application as substrates in bioprocesses and

vermicomposting (Pandey et al., 2000).

SSF is a batch process using natural heterogeneous materials (Raimbault, et al

1981; Tengerdy, 1985), containing complex polymers like lignin (Agosin et al., 1989),

pectin (Oriol, 1988) and lignocellulose (Roussos, 1986). SSF has been focused mainly to

the production of feed, hydrolytic enzymes, organic acids, gibberelins, flavours and

biopesticides. Most of the recent research activity on SSF is being done in developing

nations as a possible alternative for conventional submerged fermentations, which are

the main processes in pharmaceutical and food industries in industrialized nations.

A novel approach for the production of natural aroma compounds using coffee

husk, effect of conservation method on caffeine uptake by Penicillium commune

V33A25, screening of filamentous fungi for the production of extra-cellular tannase in

solid-state fermentation, influence of carbon source on tannase production by

Aspergillus niger Aa-20 in solid- state culture, commercial production and marketing of


17

edible mushrooms cultivated on coffee pulp, coffee pulp in vermicomposting treatment

and extraction of polyphenols from coffee pulp have been explored (Sera et al.,2000).

1.2.3.2 Production of mushrooms

The nutritional and organoleptic properties along with therapeutic value of

mushrooms have paved way for improved methods for their cultivation all over the

world. First attempts on mushroom cultivation on coffee industry residues were made

by Fan et al., (2001). A systematic study on cultivation of L. edodes, Pleurotus sp and

Flammulina velutipes using different residues such as coffee husk, leaves and spent

ground, individually or in mixture are reported (Pushpa and Manonmani, 2006; Fan et

al 2001). SSF was carried out using coffee husk, coffee spent ground and a consortium

of the coffee substrates under different conditions of moisture and spawn rate. The

biological efficiency reached at 85.8, 88.6 and 78.4 % for treated coffee husk, spent

ground and mixed substrate, respectively. Results showed the feasibility of using coffee

husk and coffee spent as substrate without any pre-treatment for cultivation of edible

fungus in SSF and is one of the first steps in economical utilization of these otherwise

unutilized, or poorly-utilized residues.

1.2.3.3 Gibberellic acid

In a recent work, Machado et al., 2002 reported the production of gibberellins

(plant hormones) in SmF and SSF utilizing coffee husk as the carbon source. Five strains

of Gibberella fujikuroi and one of its imperfect states, Fusarium moniliforme were used


18

for comparison. Production of gibberellic acid (GA) reached 1100 mgkg1 dry coffee

husk as a sole substrate of fermentation. In the all fermented samples, SSF appeared

superior to SmF in the production of gibberellic acid.

1.2.3.4 Biological detoxification of coffee pulp and husk

Due to the presence of couple of anti- physiological and anti-nutritional factors,

coffee pulp and husk are not considered as suitable substrates for bioconversion

processes. Consequently, most of the pulp and husk remain unutilized or poorly-

utilized. If these toxic constituents could be removed, or, at least degraded to a

reasonably low level, it would open new avenues in their utilization as substrates for

bioprocesses. With this in mind, several authors have worked on detoxification of

coffee pulp and husk through various means.

SSF has been frequently used for the biological detoxification of coffee husk

using fungal strains (Brand et al., 2000). SSF was carried out by A. niger in glass column

fermenter using factorial design experiments and surface response methodology to

optimize bioprocess parameters such as substrate pH, moisture and aeration rate.

Results showed that moisture content of the substrate and aeration rate were

significant factors for the degradation of toxic compounds. The kinetic study on

degradation of toxic compounds was related with the development of the mould and its

respiration and also with the consumption of the reducing sugars present in coffee husk

(Raimbault, 1998).


19

1.2.3.5 Coffee pulp and Vermicomposting

Composting and vermicomposting is a cost effective technology which could be

used at industrial level for recycling the industrial wastes. These recycled products

enhance soil nutrients, provide better growth and possess commercial appreciation.

Coffee husk (CH) is suitable for compost and vermicompost. Though, coffee pulp

contains higher proportions of cellulose besides potash and lignin, it has excellent

moisture retaining capacity but is slow in decomposition. The high bacterial growth in

the earthworm intestines improves soil fertility and stimulates plant growth making

vermicasts as good organic manure and potting media (Sathyanarayana and Khan,

2008).

1.2.4 Production of enzymes by microorganisms and SSF

1.2.4.1 Microorganisms

Bacteria, yeasts and fungi can grow on solid substrates, and find application in

SSF processes (Ball and McCarthy. 1989). Filamentous fungi are the best adapted for

SSF and dominate in research works. Bacteria are mainly involved in composting,

ensiling and some food processes (Doelle et al., 1992). Yeasts can be used for ethanol

and food or feed production (Saucedo et al., 1992). But filamentous fungi are the most

important group of microorganisms used in SSF process owing to their physiological,

enzymological and biochemical properties. The hyphal mode of growth gives a major

advantage to filamentous fungi over unicellular microorganisms in the colonization of


20

solid substrates and for the utilization of available nutrients. The basic mode of fungal

growth is a combination of apical extension of hyphal tips and the generation of new

hyphal tips through branching. An important feature is that, although extension occurs

only at the tip at a linear and constant rate, the frequency of branching makes the

kinetic growth pattern of biomass exponential, mainly in the first steps of the vegetative

stage.

The hyphal mode of growth gives the filamentous fungi the power to penetrate

into the solid substrates. The cell wall structure attached to the tip and the branching of

the mycelium ensures a firm and solid structure. The hydrolytic enzymes are excreted

at the hyphal tip, without large dilution like in the case of submerged fermentation,

what makes the action of hydrolytic enzymes very efficient and allows penetration into

most solid substrates. Penetration increases the accessibility of all available nutrients

within particles. The fungal mycelium synthesizes and excretes high quantities of

hydrolytic exo-enzymes. The resulting contact catalysis is very efficient and the simple

products are in close contact to enter the mycelium across the cell membrane to

promote biosynthesis and fungal metabolic activities (Raimbault, 1981).

1.2.4.2 Substrates

In general, substrates for SSF are composite and heterogeneous products from

agriculture or by-products of agro-industry. The basic macromolecular structure (e.g.

cellulose, starch, pectin, lignocellulose, fibers etc.) confers the properties of a solid


21

substrate. The structural macromolecule may simply provide an inert matrix (sugarcane

bagasse, inert fibers, resins) within which the carbon and energy sources (sugars, lipids,

organic acids) are adsorbed. Preparation and pre-treatment represent the necessary

steps to convert the raw substrate into a suitable form for use, that include size

reduction by grinding, rasping or chopping, increase substrate availability by the fungus,

supplementation with nutrients (phosphorus, nitrogen, salts) and setting the pH and

moisture content, through a, mineral solution, cooking or vapour treatment for

macromolecular structure, pre-degradation and elimination of major contaminants.

Variety of agro, industrial and food processing substrates such as pineapple,

mixed fruit, maosmi waste, wheat rawa with raspberry seed powder, broiler matter,

corn stover, almond meal, apple pomace, corncob, barley husk, banana waste, soybean

cake, cacao jelly, sweet limerind, cassava, soybean, amaranth grain, eucalyptus

kraftpulp, coffee residues, hardened chickpeas, lignite, rubber or orange peels (Nigam

and Pandey., 2009) are used as substrates for SSF.

1.2.4.3 Solid- state fermentation (SSF)

SSF process can be defined as microbial growth on solid particles without the

presence of free water. The water present in SSF systems exists in a complex form

within the solid matrix either absorbed to the surface of the particles or less tightly

bound within the capillary regions of the solid. Free water will only occur once the

saturation capacity of the solid matrix is exceeded. However, the moisture level at


22

which free moisture becomes apparent varies considerably between substrates and is

dependent upon their water binding characteristics. For example, free water is

observed when the moisture content exceeds 40 % in maple bark and 50 - 55 % in rice

and cassava (Oriol et al., 1988). With most lignocellulosic substrates free water

becomes apparent before the 80% moisture level is reached (Moo-Young et al., 1983).

The moisture levels in SSF processes, which vary between 30 and 85 %, has a

marked effect on growth kinetics, as SSF is a well-adapted process for cultivation of

fungi on vegetal materials which are broken down by excreted hydrolytic enzymes. SSF

are aerobic processes where respiration is fundamental for energy supply but, because

respiratory metabolism is highly exothermic, severe limitation of growth can occur when

heat transfer is not efficient enough to avoid temperature increase. Enzymes

commercially available now are not economically comparable to the chemical

processes. Hence, any substantial reduction in the cost of production of enzymes will be

a positive stimulus for the commercialization of enzymatic production.

1.2.4.4 Production of enzymes

Approximately 90 % of all industrial enzymes are produced in submerged

fermentation (SmF), frequently using specifically optimized, and genetically manipulated

microorganisms. Microbial enzymes are widely used as aids in food processing

industries. However, the fields of new industrial and analytical applications are being

extended in recent years, making necessary to study more deeply this kind of enzymes.


23

Food enzymes have been traditionally produced by submerged fermentation of

substrates such as corn flour, soybean meal, products of protein-rich legumes such as

groundnuts, etc. Submerged fermentation is often viewed disadvantageously owing to

its high operation cost (Viniegra et al., 2003). Enzyme production by SSF using agro by-

products not only brings down the cost of production (both of fermentation and

downstream processing), but it also provides an alternative path for the effective and

productive utilization of such nutrient-rich agro residues. On the other hand, almost all

these enzymes could also be produced in SSF using wild-type microorganisms (Pandey et

al., 2001).

Interestingly, fungi, yeasts and bacteria that were tested in SSF in recent decades

exhibited different metabolic strategies under conditions of solid state and submerged

fermentation. The low estimated costs of SSF are due to traditional preferential claim of

SSF, viz. SSF utilizes complex, heterogeneous agricultural wastes as substrates and uses

low-cost technology regarding sterility and regulation demands. However, attempts to

reduce costs by using cheap substrates have hampered biotechnological progress in SSF

because of the strongly increased diversity in SSF research. There is no consensus on

the methods, the microorganisms or the substrates used, that would allow comparison

with other cultivation technologies. One great advantage of SSF has always been the

possibility of using substrates that are abundant, cheap, and not applicable to SmF.


24

a) Amylases

Amylases are among the most important enzymes and are of great significance

in present-day biotechnology. Amylases have most widely been reported to occur in

microorganisms, although they are also found in plants and animals. Two major classes

of amylases have been identified in microorganisms, namely -Amylase and

glucoamylase. In addition -amylase, which is generally of plant origin, has also been

reported from a few microbial sources. -amylase (endo -1, 4- -D-glucan

glucohydrolase, EC 3.2.1.1) are extra-cellular enzymes that randomly cleave the 1,4- -

D-glucosidic linkages between adjacent glucose units in the linear amylase chain. These

are endozymes that split the substrate in the interior of the molecules and are classified

according to their action and properties. - amylase may be derived from several

bacteria, yeasts and fungi (Rao et al., 1998).

The SSF process is a potential tool for achieving economy in enzyme production

and starch hydrolysis. Different patterns of enzymes induction were obtained when

beet pulp, corn cob, rice husk, wheat bran and wheat straw were used separately to

partially replace the nutrient content of the selected medium. - amylase was

maximally expressed in the effects of different carbon sources (glucose, maltose, xylose

and starch). Higher cell density and higher specific growth rate were obtained from

glucose but higher enzyme activity and higher specific enzyme activity were obtained


25

from starch. Increased production of the enzyme could be obtained by manipulating

the growth conditions and medium composition.

Enzyme application in pharmaceutical and clinical sectors requires high purity

amylases. Traditionally the purification of amylases from fermentation media has been

done in several steps, which include centrifugation of the culture (a step of extraction

may be required for solid media), selective concentration of the supernatant usually by

ultra-filtration, and selective precipitation of the enzyme by ammonium sulphate or

organic solvents such as ethanol and the the crude enzyme is subjected to

chromatography (usually affinity or ion-exchange chromatography) and gel filtration.

The properties of each -amylase such as thermo stability, pH stability, and Ca-

independency are important in the development of fermentation process (Ghildyal et

al., 1985). Most reports about fungi that produce -amylase have been limited to a few

species of mesophilic fungi, and attempts have been made to specify the cultural

conditions and to select superior strains of the fungus to produce on a commercial scale

(Gupta 2003). Fungal sources are confined to terrestrial isolates, mostly to Aspergillus

and Penicillium (Kathiresan, 2006). The fungal -amylases are preferred over other

microbial sources due to their more accepted GRAS (Generally Recognized as Safe)

status (Gupta 2003).


26

Amylases have potential application in a wide number of industrial processes

such as food, fermentation and pharmaceutical industries. A large number of microbial

-amylases have applications in different industrial sectors such as processed-food

industry (baking, brewing), preparation of digestive aids, production of cakes, fruit juices

and starch syrups (Couto and Sanroman, 2006). The use of -amylases in the pulp and

paper industry is for the modification of starch of coated paper, i.e. for the production

of low-viscosity, high molecular weight starch and also textile industry (Souza et al.,

2010).

b) Xylanases

Xylan, the major renewable hemicellulosic polysaccharide of plant cell walls,

accounts for approximately 10 - 35 % and 10 - 15 % of total dry biomass in angiosperms

and gymnosperms, respectively. Xylan is a heteropolymer consisting of a backbone of -

1, 4- linked D-xylopyranose residues with a-L-arabinofuranose, acetyl and glucuronic

acid side chains. Xylanase (endo-1, 4- -D-xylanohydrolase; EC 3.2.1.8), the xylan-

degrading enzyme has been reported mainly from bacteria, fungi, actinomycetes and

yeast (Sanghi et al., 2008).

The xylanolytic enzyme system carrying out the xylan hydrolysis is usually

composed of hydrolytic enzymes such as -1,4-endoxylanase, -xylosidase, -L-

arabinofuranosidase, -glucuronidase, acetyl xylan esterase, and phenolic acid (ferulic

and p-coumaric acid) esterase (Beg et al., 2001; Kuhud and Singh 1993).


27

Xylanases (EC 3.2.1.8) of microorganisms find immense biotechnological

applications in the food, feed and paper-pulp industries. Conversion of hemicellulose to

value-added products by xylanases holds strong promise for the use of a variety of

unutilized and underutilized agricultural residues for practical purposes. The cost of

enzyme is one of the main factors determining the economics of any process. Reducing

the costs of enzyme production by optimizing the fermentation medium and conditions

is the goal of this basic research for industrial applications (Park et al. 2002).

The use of abundantly available and cost-effective agricultural residues, such as

wheat bran, corn cobs, rice bran, rice husk and other similar substrates, to achieve

higher xylanase yields using SSF allows reduction of the overall manufacturing cost of

bio-bleached paper. This has facilitated the use of this environment friendly technology

in the paper industry. Xylanolytic enzymes from microorganisms have attracted a great

deal of attention in the last decade, particularly because of their biotechnological

potential in various industrial processes such as food, feed, pulp and paper industries

(Bajpai, 1999; Niehaus et.al., 1999). Xylanases have shown an immense potential for

increasing the production of several useful products in a most economical way.

Xylanase are also concurrently used along with cellulase and pectinase for clarifying

juices, liquefying fruits and vegetables (Biely 1985), and in the pre-treatment of forage

crops to improve the digestibility of ruminant feeds and to facilitate composting (Gilbert

and Hazelwood 1993).


28

c) Proteases

The use of plants as a source of proteases is governed by factors no easily

controlled such as land availability and climatic conditions. Pancreatic, trypsin,

chymotrypsin, pepsin and rennin are the most important proteases of animal origin.

However, their production depends on the availability of livestock for slaughter

(Neurath 1994). Therefore microbial proteases are preferred to above enzymes from

plant and animal sources since they present most of the desired characteristics for

biotechnological applications.

In early days, proteases were classified according to their source (animal, plant

or microbial), catalytic action (endo or exo peptidases), the molecular size, charge or

substrate specificity. However a more rational system is now based on a comparison of

active sites, mechanisms of action and three-dimensional structure. Four mechanistic

classes are recognized by the International Union of Biochemistry and within these

classes; six families of proteases are recognized to date: serine proteases (EC 3.4.21),

serine carboxy proteases (EC 3.4.16), cystein proteases (EC 3.4.22), aspartic proteases

(EC 3.4.23), metallo proteases (EC 3.4.24) and metallo carboxy proteases (EC 3.4.17)

(Whitaker, 1994).

Proteases are by far the most important enzymes in the food industry used in

food proteins modification. Proteases have been used in ancient technology to improve


29

palatability and storage stability of the available protein sources; consequently,

proteases have a long history of applications in food products and they are used in

baked goods, brewing, cereals, cheese, chocolate/cocoa, egg products, meat and fish,

wine, protein hydrolyzates, anti-nutrient factors removal. Also they are widely used in

the detergent, pharmaceutical, clinical diagnostic, leather, cosmetics and fine chemical

Industries (Fox et al., 1991; Macfarlane 1992). Protease market represents 60 % of the

worldwide sale of enzymes. However, the vast diversity of proteases produced in

contrast to the specificity of their action, has attracted great attention in attempts to

exploit their physiological and biotechnological applications.

There is a long list of bacterial proteases commonly used in the food industry

and they are mostly produced by submerged fermentation; however fungal protease

production is an attractive source for proteases. Fungi can elaborate a wider variety of

enzymes than bacteria and a clear example of this statement are the acid, neutral and

alkaline proteases produced by Aspergillus niger. The fungal proteases are active over a

wide pH range (Aguilar et al., 2008) and exhibit board substrate specificity. Fungal

enzymes are actually produced by solid- state fermentation (SSF) and the advantages of

fungal enzyme production in SSF over submerged state fermentation (SmF) system have

been extensively discussed by Viniegra-Gonzalez, et al (2003).

Work related to the fungal protease production and their application has been

reported. The most frequently used fungal strains are Aspergillus oryzae Rhizopus


30

oligosporum, Aspergillus flavus, Aspergillus niger, and have been successfully used in the

acid, alkaline and neutral protease production (Aguilar et al., 2008),. Presently, in order

to overcome the high prices of the industrial proteases, especially those used in the

food and pharmaceutical industries, several studies adopting fungal organisms in SSF,

the feasibility of these processes and their positive implications on the protease

production; however, studies on the proteolytic specificity and selected applications are

important (Aguilar et al., 2008).

d) Pectinases

Pectinolytic enzymes are widely distributed in higher plants and microorganisms

(Kashyap et al., 2001). Pectinases or petinolytic enzymes hydrolyze pectic substances

and share 25 % in the global sales of food enzymes. Pectinases are one of the most

widely distributed enzymes in bacteria, fungi and plants. In nature, microorganisms

have been endowed with vast potential. They produce an array of enzymes, which have

been exploited commercially over the years (Patil and Dayanand, 2006; Reid and Richard

2004). Pectinases are of great significance with tremendous potential to offer to

industry. Protopectinases, polygalacturonases, lyases and pectin esterases are among

the extensively studied pectinolytic enzymes (Jayani et al., 2005). Almost all the

commercial preparations of pectinases are produced from fungal sources (Singh et. al.,

1999).


31

Aspergillus niger is the most commonly used fungal species for industrial

production of pectinolytic enzymes (Naidu and Panda., 1998). Pectinolytic enzymes or

pectinases are a heterogeneous group of related enzymes that hydrolyze the pectic

substances, present mostly in plants. They are of prime importance for plants as they

help in cell wall extension and softening of some plant tissues during maturation and

storage (Souza et al., 2005). They also aid in maintaining ecological balance by causing

decomposition and recycling of waste plant materials. Plant pathogenicity and spoilage

of fruits and vegetables by rotting are some other major manifestations of pectinolytic

enzymes (Lang et al., 2000).

Pectinolytic enzymes are of significant importance in the current

biotechnological era with their espousal applications in fruit juice extraction and its

clarification, scouring of cotton, degumming of plant fibers, waste water treatment,

vegetable oil extraction, tea and coffee fermentations, bleaching of paper, in poultry

feed additives and in the alcoholic beverages and food industries (Jayani et al., 2005).

1.2.5 Bioactives from agro- industrial wastes

Residues from agriculture and the food industry consist of large and varied

wastes (Dey et al., 2003). Biotechnology can offer many viable alternatives to the

disposal of agricultural waste, producing new products in the process. The production

of industrial products using agro-industrial residues as substrates for bioprocesses,


32

Enzymes degrading agro-industrial residues, their production and bioconversion of agro-

industrial residues have been explored (Nigam and Pandey, 2009).

"Bioactive compounds" are extra nutritional constituents that typically occur in

small quantities in foods and are being intensively studied to evaluate their effects on

health. Many bioactive compounds have been discovered. These compounds vary

widely in chemical structure and function and are grouped accordingly. Agro-industrial

by-products are good sources of phenolic compounds, and have been explored as

source of natural antioxidants (Fki et al., 2005). The practical aspects that need to be

considered include extraction efficiency, availability of sufficient raw material, and

toxicity or safety considerations. The very complexity in the phenolic compounds profile

of these by-products has to be resolved to obtain the optimum antioxidant efficiency

(Balasundaram et al., 2006). The processing of plant foods results in the production of

by-products that are rich sources of bioactive compounds, including phenolic

compounds (Table 1.1, Schieber et al., 2001). The availability of phenolic compounds

from agricultural and industrial residues, their extraction and antioxidant activity have

been the subject of a review by Moure et al., (2001). Phenolic compounds with

antioxidant activity have been identified in several agricultural by-products, such as rice

hulls, almond hulls etc. Gorinstein et al., (2001) found that the total phenolics content in

peels of lemons, oranges, and grapefruit were 15 % higher than those in the unpeeled

fruits.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T6R-4H877SK-1&_user=1333940&_coverDate=12%2F31%2F2006&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1578248938&_rerunOrigin=google&_acct=C000052350&_version=1&_urlVersion=0&_userid=1333940&md5=f69d8b1442a553b8093114f848b42433&searchtype=a#bib79


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Table 1.1 Phenolic compounds obtained from agricultural by-products

By-product Phenolic compounds Levelsa Reference

Almond [Prunusdulcis (Mill.) D.A. Webb] hulls

Chlorogenic acid 42.52 4.50 mg/100 g fw

Takeoka and Dao (2002)

4-O-Caffeoylquinic acid 7.90 mg/100 g fw

3-O-Caffeoylquinic acid 3.04 mg/100 g fw

Apple peels Flavonoids 2299 mg CE/100 g dw Wolfe and Liu

(2003) Anthocyanin 169 mg CGE/100 g dw

Artichoke blanching waters

Neochlorogenic acid

11.3 g phenolics/100 mL Llorach et al.

(2002)

Cryptochlorogenic acid

Chlorogenic acid

Cynarin

Caffeic acid derivatives

Buckwheat (Fagopyrumesculentum Mench) hulls

Protocatechuic acid 13.4 mg/100 mg dw

Watanabe et al. (1997)

3,4-Dihydroxybenzaldehyde 6.1 mg/100 g dw

Hyperin 5.0 mg/100 g dw

Rutin 4.3 mg/100 g dw

Quercetin 2.5 mg/100 g dw

Dried apple pomace

Flavonols 673 mg/kg dw

Schieber et al. (2003)

Flavanols 318 mg/kg dw

Dihydrochalcones 861 mg/kg dw

Hydroxycinnamates 562 mg/kg dw

Dried coconut husk 4-Hydroxybenzoic acid ferulic acid

13.0 mg phenolics/g dry weight

Dey et al. (2003)

a Expressed on fresh weight (fw) or dry weight (dw) basis.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T6R-4H877SK-1&_user=1333940&_coverDate=12%2F31%2F2006&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1578248938&_rerunOrigin=google&_acct=C000052350&_version=1&_urlVersion=0&_userid=1333940&md5=f69d8b1442a553b8093114f848b42433&searchtype=a#tblfn11http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T6R-4H877SK-1&_user=1333940&_coverDate=12%2F31%2F2006&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1578248938&_rerunOrigin=google&_acct=C000052350&_version=1&_urlVersion=0&_userid=1333940&md5=f69d8b1442a553b8093114f848b42433&searchtype=a#bib120http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T6R-4H877SK-1&_user=1333940&_coverDate=12%2F31%2F2006&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1578248938&_rerunOrigin=google&_acct=C000052350&_version=1&_urlVersion=0&_userid=1333940&md5=f69d8b1442a553b8093114f848b42433&searchtype=a#bib120


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The peels of several other fruits have also been found to contain higher amounts

of phenolics than the edible fleshy parts. Similarly, Li et al (2006) have reported that

pomegranate peels contain 249.4 mg/g phenolics compared to just 24.4 mg/g phenolics

in the pulp. Apple peels were found to contain up to 3300 mg/100 g dry weight of

phenolics (Wolfe and Liu, 2003), while the lypholisate recovered from apple pomace

was found to contain about 118 mg/g of phenolics (Schieber et al., 2003).

Polyphenolic compounds are ubiquitous natural products with diverse structural

motif and chemical/ biological activity. Phenolic compounds, including their

subcategory, flavonoids, are present in all plants. A major tool to explore bioactive

compounds particularly from natural sources is their tedious and expensive routes of

extraction and isolation. Further to remove some toxic compounds, some elimination

steps have to be taken up in order to enrich the fraction of interest so that the required

activity can be anticipated without facing much of toxicity and also with more

consistency.

1.2.5.1 Chlorogenic acid

Chlorogenic acid is a hydroxycinnamic acid, a member of a family of naturally

occurring organic compounds. These are esters of polyphenolic caffeic acid and cyclitol

(-)- quinic acid. It is an important biosynthetic intermediate (Delgado and Lopez., 2003).

It is also one of the phenols found in coffee, bamboo (Hendry and Houghton, 1996), as

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T6R-4H877SK-1&_user=1333940&_coverDate=12%2F31%2F2006&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1578248938&_rerunOrigin=google&_acct=C000052350&_version=1&_urlVersion=0&_userid=1333940&md5=f69d8b1442a553b8093114f848b42433&searchtype=a#bib107http://en.wikipedia.org/wiki/Hydroxycinnamic_acidhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Caffeic_acidhttp://en.wikipedia.org/wiki/Quinic_acidhttp://en.wikipedia.org/wiki/Phenolhttp://en.wikipedia.org/wiki/Coffee


35

well as many other plants (Clifford et al., 2003). This compound, long known as an

antioxidant, also slows the release of glucose into the bloodstream after a meal.

Structurally, chlorogenic acid (CGA) is the ester formed between caffeic acid and

(L)- quinic acid (1 L- 1(OH), 3, 4/ 5- tetrahydroxycyclohexane carboxylic acid (Clifford,

2003). Isomerisation of chlorogenic acid have been reported with 3 isomerisations of

the quinic acid in position 3, (3 - CQA), 4 (4 - CGA) and 5 (5 - CQA). Isomerisation at

position 1 has not yet been reported. It is also an antioxidant and an inhibitor of the

tumor promoting activity of phorbol esters. Chlorogenic acid and caffeic acid are

antioxidants in vitro and might therefore contribute to the prevention of Type 2

Diabetes Mellitus (Johnston et al., 2003) and cardiovascular disease (Clifford 1999). It is

claimed to have antiviral (Paynter et al., 2006) antibacterial (Lincoln et al., 2000) and

antifungal (Jassim and Naji, 2003) effects with relatively low toxicity and side effects.

Potential uses are suggested in pharmaceuticals, foodstuffs, feed additives and

cosmetics.

Fig. 1.9. Structure of chlorogenic acid

http://en.wikipedia.org/wiki/Antioxidanthttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Bloodstreamhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Caffeic_acidhttp://en.wikipedia.org/wiki/Quinic_acidhttp://en.wikipedia.org/wiki/Carboxylic_acidhttp://en.wikipedia.org/wiki/Antioxidanthttp://en.wikipedia.org/wiki/Phorbolhttp://en.wikipedia.org/wiki/Antioxidantshttp://en.wikipedia.org/wiki/In_vitrohttp://en.wikipedia.org/wiki/Chlorogenic_acid#cite_note-9http://en.wikipedia.org/wiki/File:Chlorogenic-acid-from-CAS-2D-skeletal.png


36

Chlorogenic acid has been proven in animal studies in vitro to inhibit the

hydrolysis of the enzyme glucose-6-phosphatase in an irreversible fashion. This

mechanism allows chlorogenic acid to reduce hepatic glycogenolysis (transformation of

glycogen into glucose) and to reduce the absorption of new glucose. In addition, in vivo

studies on animal subjects have demonstrated that the administration of chlorogenic

acid lowers the hyperglycemic peak resulting from the glycogenolysis brought about by

the administering of glucagon, a hyperglycemiant hormone. The studies also confirmed

a reduction in blood glucose levels and an increase in the intrahepatic concentrations of

glucose-6-phosphate and of glycogen.

The chlorogenic acids (CGA) are an important group of non volatile compounds

in green coffee bean. They are composed of a family of esters between certain trans-

cinnamic acids, such as caffeic acid, ferulic acid, and quinic acid. Although 30 different

species of CGA have now been identified in green bean, the vast majority of the

compounds found belong to three classes: caffeoylquinic acids (CQA; 3CQA, 4CQA, and

5CQA), di- caffeoylquinic acids (di-CQA; 3, 4 di CQA, 3, 5 di CQA, and 4, 5, di CQA) and

feruloylquinic acids (FQA). There is increasing evidence that diets rich in plant foods can

reduce the risk of important human afflictions such as cancer and cardiovascular

disease. One mechanism implicated in this reduced disease risk is the protection

afforded by different antioxidants present in plant foods. The growing realization of the

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importance of plant antioxidants in human health and wellness has increased research

interest concerning the synthesis and accumulation of antioxidants in plants.

In addition to being found in coffee, these compounds are also found at

significant levels in plant foods such as apples, pears, tomato, potato, and eggplant.

Importantly, coupled with the fact that the dietary intake of CGA can be relatively high

in people consuming certain plant foods, this class of molecules, and/or their

degradation products, are thought to have significant bio-availability. Beside the

proposed utility of plant derived CGA for human health, these compounds are also very

important plant metabolites.

1.2.5.2 Dietary fiber

Agro wastes are great sources of dietary fiber, which includes cellulose,

hemicelluloses, lignin, pectin, gums, and other polysaccharides. The soluble and

insoluble dietary fiber fractions (SDF and IDF) are known to confer a wide range of

health benefits, including reduction in the risks of gastrointestinal diseases,

cardiovascular diseases, and obesity (Figuerola et al., 2005). There is a need for

supplementation of dietary fiber via fiber-rich foods as the normal daily intake of most

populations is still below the recommended Dietary Reference Intake of 14 g of dietary

fiber per 1000 kcal, or 25 g for adult women and 38 g for adult men. Hence, high-fiber

products are gaining popularity as functional foods with a low glycemic index and

hypocholesterolemic properties. However, high-fiber content in food is often


38

associated with undesirable sensory properties due to the inherent properties of fibers

being coarse and grainy.

Healthy foods such as high-fiber cereals are dry and have increasingly

undesirable organoleptic properties as fiber content is increased. The food industry has

developed processing methods and compound coatings that can effectively mask and

reduce fibrous mouth feel associated with dietary fiber. However, compound coatings

are essentially made up of fats and carbohydrates, which increase the caloric value of

the food upon intake. The diminutization of fibers to nano size may reduce or even

completely remove the undesirable organoleptic properties inherently and eliminate

the need for additional processing steps or high-calorie additives, which may defeat the

net purpose of high-fiber health functional foods (Lincoln et al., 2000).

Major agricultural waste fiber materials (FM), namely, oil palm trunk (OPT), oil

palm frond (OPF), buck wheat and okara are good sources for fibrous residues (FR) with

food-based applications in functional foods(Watanbe,1997). Agro waste materials could

be converted into valuable and functional materials, including food and drug carriers,

thus extending the life cycle of the agriculture by-products. However, processing of raw

materials to obtain DF concentrates may result in important losses of compounds with

antioxidant capacity.


39

Biomass waste such as agricultural residues is creating great environmental

concerns, with approximately 200 billion tonnes of lignocellulosic wastes being

produced annually (Larrauri et al., 1999: Mohanty et al., 2000). Agro wastes are great

sources of dietary fiber, which includes cellulose, hemicelluloses, lignin, pectin, gums,

and other polysaccharides (Wai et al., 2010).

1.2.5.2 Antioxidant Dietary Fiber

ADF could be used as a new food ingredient. In addition to the properties

derived from ordinary dietary fibers, a prevention of lipid oxidation in food products can

be expected from the presence of antioxidant polyphenols. On the other hand, the

potential combined actions of non-extractable proanthocyanidins and bioavailable

flavonoids of the ADF are quite promising in nutrition and health. ADF can be defined as

a product containing significant amounts of natural antioxidants associated with the

fiber matrix. The requirements to be considered as an ADF: (1) DF content, measured

by the AOAC method (Prosky et al., 1988), should be higher than 50 % on a dry matter

basis. (2) One gram of ADF should have a capacity to inhibit lipid oxidation equivalent

to, at least, 200 mg of vitamin E (measured by the thiocyanate procedure) and a free

radical scavenging capacity equivalent to, at least, 50 mg of vitamin E (measured by the

DPPH method). (3) The antioxidant capacity must be an intrinsic property, derived from

natural constituents of the material neither by added antioxidants nor by constituents

released by previous chemical or enzymatic treatments.

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The fiber-antioxidant complex could be considered as a natural way to deliver

antioxidant compounds to the hindgut bacteria preserving antioxidants from gastric

degradation. The presence of this complex could explain data showing that it is much

better for body health to consume the DF as part of whole fiber-rich foods (cereals,

legumes, vegetables or fiber-enriched functional foods) compared to the intake of only

purified fiber, tablets, pills and other medical preparations (Esposito et al.,2005).

1.2.5.3 Anthocyanins

Anthocyanins are flavonoid compounds responsible for the red/blue coloration

of many fruits and flowers (Stintzing and Carle, 2004). Anthocyanin structures are based

on the C15 skeletons of anthocyanidins (consisting of a chromane ring bearing a second

aromatic ring B in position 2) that are glycosylated and/or acylated at specific

hydroxylated positions (Delgado and Lopez, 2003). There are over 600 naturally

occurring anthocyanins, and most of them are either 3-glycosides or 3, 5-diglycosides.

Anthocyanins are considered secondary metabolites as a food additive with E number

163. Over 500 different anthocyanins have been isolated from plants. They are all

based on a single basic core structure, the flavyllium ion (Fig 1.10). As shown in Figure

1.10, there are 7 different side groups on the flavylium ion. These side groups can be a

hydrogen atom, a hydroxide or a methoxy-group.

http://en.wikipedia.org/wiki/Secondary_metabolitehttp://en.wikip

pushpa s. murthy - central food technological …ir.cftri.com/10749/1/pushpa_s_murthy.pdfmrs. pushpa...

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