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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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 :
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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.
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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
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Chapter 1 Review of literature
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
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Chapter 1 Review of literature
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
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Chapter 1 Review of literature
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.,
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Chapter 1 Review of literature
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
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Chapter 1 Review of literature
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
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Chapter 1 Review of literature
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).
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Chapter 1 Review of literature
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
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Chapter 1 Review of literature
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
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Chapter 1 Review of literature
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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
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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
http://www.myespresso.co.nz/media/catalog/product/cache/1/image/500x500/5e06319eda06f020e43594a9c230972d/2/0/200871175254_1.jpg
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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
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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.
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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
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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
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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
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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).
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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
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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
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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
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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.
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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.
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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
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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).
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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).
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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).
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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
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Chapter 1 Review of literature
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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
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Chapter 1 Review of literature
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).
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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,
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Chapter 1 Review of literature
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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.
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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
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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
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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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Chapter 1 Review of literature
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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
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Chapter 1 Review of literature
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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.
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Chapter 1 Review of literature
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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.
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