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Value-AdditioninFood
ProductsandProcessing
ThroughEnzymeTechnology
Edited by
Mohammed Kuddus
Professor of Biochemistry, College of Medicine, University of Hail, Kingdom of Saudi Arabia
Cristobal Noe Aguilar
Bioprocesses and Bioproducts Research Group, Food Research Department, School of Chemistry,
Universidad Auto´noma de Coahuila, Saltillo, Coahuila, Mexico

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Contributors
Sabu Abdulhameed, Department of Biotechnology and
Microbiology, Kannur University, Kannur, Kerala, India
Nor Hasmaliana Abdul Manas, School of Chemical and
Energy Engineering, Faculty of Engineering, Universiti
Teknologi Malaysia, Skudai, Johor, Malaysia
Clementz Adriana, Instituto de Procesos Biotecnológicos
y Químicos (IPROBYQ), Consejo Nacional de Inves-
tigaciones Cientíﬁcas y Técnicas (CONICET), Facultad
de Ciencias Bioquímicas y Farmacéuticas, Universidad
Nacional de Rosario (UNR), Santa Fe, Rosario,
Argentina
Cristóbal N. Aguilar, Bioprocesses and Bioproducts
Research Group, Food Research Department, School of
Chemistry, Universidad Autónoma de Coahuila, Salt-
illo, Coahuila, Mexico
Ricardo D. Aguilar-García, Tecnológico Nacional de
México Campus Instituto Tecnológico de Ciudad
Valles, Ciudad Valles, San Luis Potosí, Mexico
Pedro Aguilar-Zárate, Tecnológico Nacional de México
Campus Instituto Tecnológico de Ciudad Valles,
Ciudad Valles, San Luis Potosí, Mexico
A.F. Aguilera-Carbó, Departament of Animal Nutrition,
Autonomous Agrarian University Antonio Narro, Salt-
illo, Mexico
Taiwo O. Akanbi, School of Environmental and Life
Sciences, College of Engineering, Science and Envi-
ronment, University of Newcastle, Ourimbah, NSW,
Australia
Diana Laura Alva-Sánchez, Laboratorio de Bio-
rremediación, Facultad de Ciencias Biológicas, Uni-
versidad Autónoma de Coahuila, Torreón, Coahuila,
Mexico
Carmen Alvarez-Ossorio, AZTI, Food Research, Basque
Research and Technology Alliance (BRTA), Parque
Tecnológico de Bizkaia, Derio, Spain
Eduarda Nataly de Andrade Soares, Center for Exact
Sciences and Technology, Federal University of Ser-
gipe, São Cristóvão, Sergipe, Brazil
Uday S. Annapure, Department of Food Engineering and
Technology, Institute of Chemical Technology (ICT),
Mumbai, Maharashtra, India
Roberto Arredondo-Valdes, Nanobioscience Research
Group, Universidad Autónoma de Coahuila, Saltillo,
Coahuila,Mexico
K.B. Arun, Rajiv Gandhi Centre for Biotechnology,
Thiruvananthapuram, Kerala, India
Shailendra Kumar Arya, Department of Biotechnology,
University Institute of Engineering and Technology,
Panjab University, Chandigarh, Punjab, India
J.A. Ascacio-Valdés, Bioprocesses and Bioproducts
Research Group, Food Research Department, School of
Chemistry, Universidad Autónoma de Coahuila, Salt-
illo, Coahuila, Mexico
Kassandra T. Ávila-Alvarez, Nanobioscience Research
Group, Universidad Autónoma de Coahuila, Saltillo,
Coahuila, Mexico
Ramiro Baeza-Jiménez, Research Center in Food and
Development A.C., Delicias, Chihuahua, Mexico
Marcela Pavan Bagagli, Biosystem Engineering, Campus
Avaré, Federal Institute of Education, Science and
Technology of São Paulo (IFSP), Avaré, São Paulo,
Brazil
Nagamani Balagurusamy, Laboratorio de Bio-
rremediación, Facultad de Ciencias Biológicas, Uni-
versidad Autónoma de Coahuila, Torreón, Coahuila,
Mexico
Carlos Bald, AZTI, Food Research, Basque Research and
Technology Alliance (BRTA), Parque Tecnológico de
Bizkaia, Derio, Spain
Margarida Maria Barros, Department of Breeding and
Animal Nutrition, AquaNutri Laboratory, School of
Veterinary Medicine and Animal Science, São Paulo
State University (UNESP), Botucatu, São Paulo, Brazil
Colin J. Barrow, Centre for Chemistry and Biotechnology,
Deakin University, Geelong, VIC, Australia
xv

Soorej M. Basheer, Department of Molecular Biology, Dr
PK Rajan Memorial Campus, Kannur University,
Nileswaram, Kerala, India
Brenda Bezus, Research and Development Center for
Industrial Fermentations (CINDEFI, UNLP; CCT-La
Plata, CONICET), La Plata, Buenos Aires, Argentina
Parameswaran Binod, Microbial Processes and Technol-
ogy Division, CSIR-National Institute for Interdiscipli-
nary Science and Technology (CSIR-NIIST),
Trivandrum, Kerala, India
German Bolivar, MIBIA Group, Biology Department,
Faculty of Natural and Exact Sciences, Universidad del
Valle, Cali, Valle del Cauca, Colombia
A. Javier Borderías, Institute of Food Science Technology
and Nutrition (ICTAN-CSIC), Products Department,
Madrid, Spain
Tamara Bucher, School of Environmental and Life
Sciences, College of Engineering, Science and Envi-
ronment, University of Newcastle, Ourimbah, NSW,
Australia
Juan Buenrostro-Figueroa, Research Center in Food and
Development A.C., Delicias, Chihuahua, Mexico
Luis A. Cabanillas-Bojórquez, Centro de Investigación en
Alimentación y Desarrollo A.C., Culiacán, Sinaloa,
Mexico
Filipe Carvalho, Department of Bioengineering and IBB,
Institute for Bioengineering and Biosciences, Instituto
Superior Técnico, Universidade de Lisboa, Lisbon,
Portugal; DREAMS and Faculty of Engineering, Uni-
versidade Lusófona de Humanidades e Tecnologias,
Lisbon, Portugal; Associate Laboratory i4HBdInstitute
for Health and Bioeconomy at Instituto Superior Téc-
nico, Universidade de Lisboa, Lisboa, Portugal
Pedro Luiz Pucci Figueiredo de Carvalho, Department of
Breeding and Animal Nutrition, AquaNutri Laboratory,
School of Veterinary Medicine and Animal Science,
São Paulo State University (UNESP), Botucatu, São
Paulo, Brazil
Sebastián Cavalitto, Research and Development Center
for Industrial Fermentations (CINDEFI, UNLP; CCT-
La Plata, CONICET), La Plata, Buenos Aires,
Argentina
Ivana Cavello, Research and Development Center for
Industrial Fermentations (CINDEFI, UNLP; CCT-La
Plata, CONICET), La Plata, Buenos Aires, Argentina
Deniz Cekmecelioglu, Department of Food Engineering,
Faculty of Engineering, Middle East Technical Uni-
versity, Ankara, Turkey
Heena Chandel, Department of Biotechnology, School of
Basic Sciences, Indian Institute of Information Tech-
nology Una, Una, Himachal Pradesh, India
Mónica L. Chávez-González, Nanobioscience Research
Group, Universidad Autónoma de Coahuila, Saltillo,
Coahuila, Mexico
Sreeja Chellappan, Department of Molecular Biology, Dr
PK Rajan Memorial Campus, Kannur University,
Nileswaram, Kerala, India
S. Chozhavendhan, Department of Biotechnology, V.S.B
Engineering College, Karur, Tamil Nadu, India
Juan Carlos Contreras-Esquivel, Food Research
Department, School of Chemistry, Universidad Auton-
oma de Coahuila, Saltillo, Coahuila, Mexico
J.D. Corona-Flores, Department of Planning, Autono-
mous Agrarian University Antonio Narro, Saltillo,
Mexico
Iris R. Cuellar-Rincón, Tecnológico Nacional de México
Campus Instituto Tecnológico de Ciudad Valles,
Ciudad Valles, San Luis Potosí, Mexico
Romanini Diana, Instituto de Procesos Biotecnológicos y
Químicos (IPROBYQ), Consejo Nacional de Inves-
tigaciones Cientíﬁcas y Técnicas (CONICET), Facultad
de Ciencias Bioquímicas y Farmacéuticas, Universidad
Nacional de Rosario (UNR), Santa Fe, Rosario,
Argentina
P.N. dos Santos, Laboratory of Food Biotechnology,
Institute of Science and Technology, Federal University
of Jequitinhonha and Mucuri Valleys, Diamantina,
Brazil
Montalvo González Eﬁgenia, Tecnológico Nacional de
México, Instituto Tecnológico de Tepic, Laboratorio
Integral de Investigación en Alimentos, Tepic, Nayarit,
Mexico
Deilson Elgui de Oliveira, Botucatu Medical School,
Department of Pathology, São Paulo State University
(UNESP), Botucatu, São Paulo, Brazil; Institute of
Biotechnology, São Paulo State University (UNESP),
Botucatu, São Paulo, Brazil
Shibitha Emmanual, Department of Zoology, St. Joseph’s
College, Thrissur, Kerala, India
Tovar-Pérez Erik Gustavo, CONACyTeUniversidad
Autónoma de Querétaro, Facultad de Ingeniería, San-
tiago de Querétaro, Querétaro, Mexico
Alaina Alessa Esperón-Rojas
, UNIDA, Tecnologico
Nacional de Mexico Campus Veracruz, M.A. de
QUevedo, Col. Formando Hogar, Veracruz, Mexico
xviContributors

Pedro Fernandes, Department of Bioengineering and IBB,
Institute for Bioengineering and Biosciences, Instituto
Superior Técnico, Universidade de Lisboa, Lisbon,
Portugal; DREAMS and Faculty of Engineering, Uni-
versidade Lusófona de Humanidades e Tecnologias,
Lisbon, Portugal; Associate Laboratory i4HBdInstitute
for Health and Bioeconomy at Instituto Superior
Técnico, Universidade de Lisboa, Lisboa, Portugal
Ángel Fernández-Sanromán, Department of Chemical
Engineering, University of Vigo, Vigo, Spain
Luciana Francisco Fleuri, Department of Chemical and
Biological Sciences, Biosciences Institute, São Paulo
State University (UNESP), Botucatu, São Paulo, Brazil
Alexis García Figueroa, GIPAB Group, School of Food
Engineering, Faculty of Engineering, Universidad del
Valle, Cali, Valle del Cauca, Colombia
Shivani Singh Gaur, Department of Food Engineering and
Technology, Institute of Chemical Technology (ICT),
Mumbai, Maharashtra, India
Andressa Genezini dos Santos, Department of Chemical
and Biological Sciences, Biosciences Institute, São Paulo
State University (UNESP), Botucatu, São Paulo, Brazil
Daniela A. Gonçalves, CEB - Centre of Biological Engi-
neering, University of Minho, Braga, Portugal
Mayela Govea-Salas, Nanobioscience Research
Group, Universidad Autónoma de Coahuila, Saltillo,
Coahuila, Mexico
Tharuka Gunathilake, Centre for Chemistry and Bio-
technology, Deakin University, Geelong, VIC, Australia
Sugriv Shyamlal Gupta, Department of Food Engineering
and Technology, Institute of Chemical Technology
(ICT), Mumbai, Maharashtra, India
Erick Paul Gutiérrez-Grijalva, Cátedras CONACYT-
Centro de Investigación en Alimentación y Desarrollo
A.C., Culiacán, Sinaloa, Mexico
J. Basilio Heredia, Centro de Investigación en Alimenta-
ción y Desarrollo A.C., Culiacán, Sinaloa, Mexico
Ayerim Yedid Hernández-Almanza, Laboratorio de
Biorremediación, Facultad de Ciencias Biológicas,
Universidad Autónoma de Coahuila, Torreón, Coahuila,
Mexico
Javier Ulises Hernández-Beltran, Laboratorio de
Biorremediación, Facultad de Ciencias Biológicas,
Universidad Autónoma de Coahuila, Torreón, Coahuila,
Mexico
Inty Omar Hernández-De Lira, Laboratorio de Bio-
rremediación, Facultad de Ciencias Biológicas, Uni-
versidad Autónoma de Coahuila, Torreón, Coahuila,
Mexico
Pui Khoon Hong, College of Computing and Applied
Sciences, Faculty of Industrial Sciences and Technol-
ogy, Universiti Malaysia Pahang, Kuantan, Pahang,
Malaysia
Chieh Chen Huang, Department of Life Sciences, Na-
tional Chung Hsing University, Taichung City, Taiwan
Jone Ibarruri, AZTI, Food Research, Basque Research
and Technology Alliance (BRTA), Parque Tecnológico
de Bizkaia, Derio, Spain
Anna Ilyina, Nanobioscience Research Group, Uni-
versidad Autónoma de Coahuila, Saltillo, Coahuila,
Mexico
Bruno Iñarra, AZTI, Food Research, Basque Research
and Technology Alliance (BRTA), Parque Tecnológico
de Bizkaia, Derio, Spain
Paula Jauregi, AZTI, Food Research, Basque Research
and Technology Alliance (BRTA), Parque Tecnológico
de Bizkaia, Derio, Spain; Ikerbasque, Basque Founda-
tion for Science, Bilbao, Spain
Edmundo Juárez-Enríquez, School of Chemistry,
Autonomous University of Chihuahua, Chihuahua,
Mexico
Asmita Deepak Kamble, Department of Biological
Sciences, Sunandan Divatia School of Science,
NMIMS Deemed to be University, Mumbai, Mahara-
shtra, India
Anupreet Kaur, Department of Biotechnology, University
Institute of Engineering and Technology, Panjab Uni-
versity, Chandigarh, Punjab, India
Jyoti Kaushal, Department of Biotechnology, University
Institute of Engineering and Technology, Panjab Uni-
versity, Chandigarh, Punjab, India
Meliane Akemi Koike, Department of Chemical and
Biological Sciences, Biosciences Institute, São Paulo
State University (UNESP), Botucatu, São Paulo, Brazil
Guerra Laureana, Instituto de Procesos Biotecnológicos y
Químicos (IPROBYQ), Consejo Nacional de Inves-
tigaciones Cientíﬁcas y Técnicas (CONICET), Facultad
de Ciencias Bioquímicas y Farmacéuticas, Universidad
Nacional de Rosario (UNR), Santa Fe, Rosario,
Argentina
Anahí Levario-Gómez, School of Chemistry, Autono-
mous University of Chihuahua, Chihuahua, Mexico
García-Magaña María de Lourdes, Tecnológico Nacio-
nal de México, Instituto Tecnológico de Tepic, Labo-
ratorio Integral de Investigación en Alimentos, Tepic,
Nayarit, Mexico
Aravind Madhavan, Rajiv Gandhi Centre for Bio-
technology, Thiruvananthapuram, Kerala, India
Contributorsxvii

M.C.R. Mano, Chemical Technology Development
Center, State University of Mato Grosso do Sul,
Naviraí, Brazil
Romero-Garay Martha Guillermina, Tecnológico
Nacional de México, Instituto Tecnológico de Tepic,
Laboratorio Integral de Investigación en Alimentos,
Tepic, Nayarit, Mexico
José L. Martínez-Hernández, Nanobioscience Research
Group, Universidad Autónoma de Coahuila, Saltillo,
Coahuila, Mexico
Gincy Marina Mathew, Microbial Processes and
Technology Division, CSIR-National Institute for
Interdisciplinary Science and Technology (CSIR-
NIIST), Trivandrum, Kerala, India
A. Mejía-López, Department of Food Science and Tech-
nology, Autonomous Agrarian University Antonio
Narro, Saltillo, Mexico
Karime de M. Moctezuma-Dávila, Universidad Autón-
oma de San Luis Potosí, Facultad de Estudios
Profesionales Zona Huasteca, Ciudad Valles, San Luis
Potosí, Mexico
G. Molina, Laboratory of Food Biotechnology, Institute of
Science and Technology, Federal University of Jequi-
tinhonha and Mucuri Valleys, Diamantina, Brazil
Helena M. Moreno, Veterinary Faculty, Department of
Food Technology, Madrid, Spain
O. Moreno-Sánchez, Department of Food Science and
Technology, Autonomous Agrarian University Antonio
Narro, Saltillo, Mexico
J.Y. Méndez-Carmona, Bioprocesses and Bioproducts
Research Group, Food Research Department, School of
Chemistry, Universidad Autónoma de Coahuila, Salt-
illo, Coahuila, Mexico
Liliana Gabriela Mendoza-Sánchez, UNIDA, Tecnolo-
gico Nacional de Mexico Campus Veracruz, M.A. de
QUevedo, Col. Formando Hogar, Veracruz, Mexico
Diana B. Muñiz-Márquez, Tecnológico Nacional de
México Campus Instituto Tecnológico de Ciudad
Valles, Ciudad Valles, San Luis Potosí, Mexico
Preetha Nair, Department of Biotechnology, Mount Car-
mel College Autonomous, Bengaluru, Karnataka, India
Erika Nava-Reyna, CENID RASPA, Instituto Nacional de
Investigaciones Forestales Agrícolas y Pecuarias,
Gomez Palacio, Durango, Mexico
Clarisse Nobre, CEB - Centre of Biological Engineering,
University of Minho, Braga, Portugal
Paula Kern Novelli, Department of Chemical and Bio-
logical Sciences, Biosciences Institute, São Paulo State
University (UNESP), Botucatu, São Paulo, Brazil
Hysla Maria Albuquerque Resende Nunes, Center for
Exact Sciences and Technology, Federal University of
Sergipe, São Cristóvão, Sergipe, Brazil
Emilio Ochoa-Reyes, Research Center in Food and
Development A.C., Delicias, Chihuahua, Mexico
Ashok Pandey, Center for Innovation and Translational
Research, CSIR-Indian Institute of Toxicology
Research (CSIR-IITR), Lucknow, Uttar Pradesh, India
B.N. Paulino, Faculty of Pharmacy, Federal University of
Bahia, Salvador, Brazil
Mercedes M. Pedrosa, Food Technology Department,
National Agricultural and Food Research and Tech-
nology Institute (INIA-CSIC), Madrid, Spain
M. Rajamehala, Department of Biotechnology, Viveka-
nandha College of Engineering for Women (Autono-
mous), Namakkal, Tamil Nadu, India
Aizi Nor Mazila Ramli, College of Computing and Applied
Sciences, Faculty of Industrial Sciences and Technology,
Universiti Malaysia Pahang, Kuantan, Pahang, Malaysia;
Bio Aromatic Research Centre of Excellence, Universiti
Malaysia Pahang, Lebuhraya Tun Razak, Gambang,
Kuantan, Pahang Darul Makmur, Malaysia
Rodolfo Ramos-González, CONACYTeUniversidad
Autónoma de Coahuila, Saltillo, Coahuila, Mexico
O.N. Rebolloso-Padilla, Departament of Animal Produc-
tion, Autonomous Agrarian University Antonio Narro,
Saltillo, Mexico
Anna María Polania Rivera, MIBIA Group, Biology
Department, Faculty of Natural and Exact Sciences,
Universidad del Valle, Cali, Valle del Cauca, Colombia;
GIPAB Group, School of Food Engineering, Faculty of
Engineering, Universidad del Valle, Cali, Valle del
Cauca, Colombia
L. Rodríguez-Gutiérrez, Department of Statistics and
Calculus, Autonomous Agrarian University Antonio
Narro, Saltillo, Mexico
X. Ruelas-Chacon, Department of Food Science and
Technology, Autonomous Agrarian University Antonio
Narro, Saltillo, Mexico
Denise Santos Ruzene, Center for Exact Sciences and
Technology, Northeastern Biotechnology Network,
Federal University of Sergipe, São Cristóvão, Sergipe,
Brazil
A. Sabu, Department of Biotechnology & Microbiology,
Dr Janaki Ammal Campus, Kannur University,
Thalassery, Kerala, India
V. Sanjuvikasini, Department of Biotechnology, Viveka-
nandha College of Engineering for Women (Autono-
mous), Namakkal, Tamil Nadu, India
xviiiContributors

David San Martin, AZTI, Food Research, Basque
Research and Technology Alliance (BRTA), Parque
Tecnológico de Bizkaia, Derio, Spain
M. Ángeles Sanromán, Department of Chemical
Engineering, University of Vigo, Vigo, Spain
Brenda Lohanny Passos Santos, Northeastern Bio-
technology Network, Federal University of Sergipe, São
Cristóvão, Sergipe, Brazil
Sowmya R. Sathyanarayana, Department of Food Engi-
neering and Technology, Institute of Chemical Tech-
nology (ICT), Mumbai, Maharashtra, India
Christopher J. Scarlett, School of Environmental and Life
Sciences, College of Engineering, Science and Envi-
ronment, University of Newcastle, Ourimbah, NSW,
Australia
Elda P. Segura-Ceniceros, Nanobioscience Research
Group, Universidad Autónoma de Coahuila, Saltillo,
Coahuila, Mexico
Hugo Sergio García, UNIDA, Tecnologico Nacional de
Mexico Campus Veracruz, M.A. de QUevedo, Col.
Formando Hogar, Veracruz, Mexico
Archita Sharma, Department of Biotechnology, Univer-
sity Institute of Engineering and Technology, Panjab
University, Chandigarh, Punjab, India
S. Shruthi, Department of Biotechnology, Vivekanandha
College of Engineering for Women (Autonomous),
Namakkal, Tamil Nadu, India
Daniel Pereira Silva, Center for Exact Sciences and Tech-
nology, Northeastern Biotechnology Network, Federal
University of Sergipe, São Cristóvão, Sergipe, Brazil
Raveendran Sindhu, Microbial Processes and Technology
Division, CSIR-National Institute for Interdisciplinary
Science and Technology (CSIR-NIIST), Trivandrum,
Kerala, India
Gursharan Singh, Department of Medical Laboratory
Sciences, Lovely Professional University, Phagwara,
Punjab, India
Harinder Singh, Department of Biological Sciences,
Sunandan Divatia School of Science, NMIMS Deemed
to be University, Mumbai, Maharashtra, India
Aldo Sosa-Herrera, Laboratorio de Biorremediación,
Facultad de Ciencias Biológicas, Universidad Autón-
oma de Coahuila, Torreón, Coahuila, Mexico
María Alejandra Sánchez-Muñoz, Laboratorio de Bio-
rremediación, Facultad de Ciencias Biológicas, Uni-
versidad Autónoma de Coahuila, Torreón, Coahuila,
Mexico
Antonela Taddia, Institute of Biotechnological and
Chemical Processes (IPROByQ) CONICET, Faculty of
Biochemical and Pharmaceutical Sciences, National
University of Rosario, Rosario, Santa Fe, Argentina
Alicia Guadalupe Talavera-Caro, Laboratorio de Bio-
rremediación, Facultad de Ciencias Biológicas, Uni-
versidad Autónoma de Coahuila, Torreón, Coahuila,
Mexico
Castro-Jácome Tania Patricia, Tecnológico Nacional de
México, Instituto Tecnológico de Tepic, Laboratorio
Integral de Investigación en Alimentos, Tepic, Nayarit,
Mexico
José A. Teixeira, CEB - Centre of Biological Engineering,
University of Minho, Braga, Portugal
Juan M. Tirado-Gallegos, School of Animal Sciences and
Ecology, Autonomous University of Chihuahua, Chi-
huahua, Mexico
Cristina Ramírez Toro, MIBIA Group, Biology Depart-
ment, Faculty of Natural and Exact Sciences,
Universidad del Valle, Cali, Valle del Cauca, Colombia
Clara A. Tovar, Department of Applied Physics, Faculty
of Sciences, University of Vigo, Ourense, Spain
Gisela Tubio, Institute of Biotechnological and Chemical
Processes (IPROByQ) CONICET, Faculty of Bio-
chemical and Pharmaceutical Sciences, National Uni-
versity of Rosario, Rosario, Santa Fe, Argentina
Manuel A. Uranga-Soto, Centro de Investigación en
Alimentación y Desarrollo A.C., Culiacán, Sinaloa,
Mexico
Sibel Uzuner, Department of Food Engineering, Faculty of
Engineering, Izmir Institute of Technology,_Izmir,
Turkey
Quan Van Vuong, School of Environmental and Life
Sciences, College of Engineering, Science and Envi-
ronment, University of Newcastle, Ourimbah, NSW,
Australia
Madan L. Verma, Department of Biotechnology, School
of Basic Sciences, Indian Institute of Information
Technology Una, Una, Himachal Pradesh, India
M. Vijay Pradhap Singh, Department of Biotechnology,
Vivekanandha College of Engineering for Women
(Autonomous), Namakkal, Tamil Nadu, India
Nur Izyan Wan Azelee, School of Chemical and Energy
Engineering, Faculty of Engineering, Universiti
Teknologi Malaysia, Skudai, Johor, Malaysia; Institute
of Bioproduct Development (IBD), Universiti Tekno-
logi Malaysia, UTM Skudai, Johor, Malaysia
Contributorsxix

Bo Wang, School of Behavioural and Health Sciences,
Australian Catholic University, North Sydney, NSW,
Australia
Jorge E. Wong-Paz, Tecnológico Nacional de México
Campus Instituto Tecnológico de Ciudad Valles,
Ciudad Valles, San Luis Potosí, Mexico
Mirella Rossitto Zanutto-Elgui, Department of Chemical
and Biological Sciences, Biosciences Institute, São Paulo
State University (UNESP), Botucatu, São Paulo, Brazil
Jaime Zuﬁa, AZTI, Food Research, Basque Research and
Technology Alliance (BRTA), Parque Tecnológico de
Bizkaia, Derio, Spain
xxContributors

Foreword
Currently, the growth rate of world population is around 1.0%, which could add about 80 million more people per year. As
a consequence, the increase in demand for the food will be directly proportional. But, with decrease in land area available
for food crops cultivation, water shortage, and other problems associated with climate change, there will be huge chal-
lenges in meeting this demand for food production. One other task is to achieve sustainable production under the
aforementioned limitations. In this context, the best available alternative is to meet the demand not in terms of increasing
the quantity of the food produce, but by improving the nutrient content of the available food produce. Such a technology
would help to meet the energy and nutrient needs of the increasing human population with available food production. This
would also aid in overcoming the dependence on resource intensive agriculture, which could damage the health of global
environment. So, is there such technology available is the question in front of us at this critical juncture.
Dr. Kuddus and Dr. Noe Aguilar, renowned scientists in international arena in food technology and editors of this book
onValue-Addition in Food Products and Processing Through Enzyme Technology, critically analyzed this question and
have presented a promising solution, which also is based on sustainability. Outwardly the solution they suggest, the use of
enzyme technology might appear as old. But, the innovative way they chose the different chapters for the book reﬂects
their insight in dealing the question. The book chapters widely cover the innovations in enzyme-mediated food processing
to improve their nutritive value and as well the application of enzyme technology for value addition in the processed foods,
such as nutraceuticals and functional foods to meet the energy needs of the growing population. Chapters on bio-
prospection of novel enzymes and future perspectives give the reader the possibility of exploring more on this in future.
Further, authors from 10 different countries representing North America, South America, Europe, Asia, and Australia
pooled their expertise and have contributed in a collaborative manner, which is additional strength of this book.
This book is a boon for students and young researchers who are interested in updating their knowledge on the emerging
technologies as well as to use the available literature as basis for their research projects. Further, the contents of this book
make it suitable for adoption in graduate programs on food and nutrition technology in international universities. The
concepts and the technology discussed in the book could be a key factor, leading to mind change in research and
administrative personnel of the food industry to develop functional, nutritive, and value-added food products through
innovations and by employing modern tools to search for novel enzymes and novel products.
My hearty congratulations to editors, Dr. Kuddus and Dr. Aguilar, and all contributing authors of the book chapters. I
am conﬁdent that this book will have a strong positive impact on all readers.
Prof. Nagamani Balagurusamy
Autonomous University of Coahuila
Torreón, México
August 24, 2021
xxiii

Preface
Food is the fuel for life. The relationship between population and food supply is not new. Today, the food crisis is a major
problem in the world. Application of enzymes in food industry is well known, and lots of research is going on consistently
to fulﬁll food crisis worldwide. Food biotechnology along with enzyme technology could provide an efﬁcient solution for
global food security. Scientists have already identiﬁed potential uses of enzymes in food industry, from bioconversion to
genetic engineering. The lack of knowledge of scientiﬁc progress and most recent technologies in enzyme engineering and
technology contributes signiﬁcantly slow progress of foods production and value addition in the food products. In this
perspective, the aim of this book is to offer an updated review regarding potential impact of new enzymes and enzyme
technology on the food sector. The book brings together the novel sources and technologies regarding enzymes in
value-added food production, foods processing, food preservation, food engineering, and food biotechnology.
This book includes a comprehensive reference in the most progressiveﬁeld of enzyme technology in value-added food
production by using various low cost metabolites or wastes and will be of interest to professionals, scientists, and aca-
demics related to food science and food biotechnology. This book covers 39 chapters that provide an updated knowledge
of food processing and production through enzyme technology. It will help to increase food production to cope food crisis
problems in the world. The chapters highlighted the potential impact of enzymes and various low cost metabolites or
wastes along with its signiﬁcant applications in various food sectors. These chapters also present future perspectives of
enzyme technology for value-added food production and processing.
In conclusion, this book is an updated reference in the most progressiveﬁeld of food production and processing by
using enzyme technology that will be useful for the food scientists and academics involved in food science and technology.
In the last, but not least, we would like to thank all the authors who have eagerly contributed their chapter in this book. We
also express our sincere gratitude to Elsevier for providing this opportunity.
Mohammed Kuddus, Hail, KSA
Cristobal N. Aguilar, México
August 2021
xxv

About the editors
Dr. Mohammed Kuddus, Professor of Biochemistry, College of Medicine, University of Hail, Kingdom of Saudi
Arabia: After completing PhD in Enzyme Biotechnology, he worked in different R&D projects funded by various sci-
entiﬁc agencies. Prof. Kuddus’main research area includes biochemistry, enzyme technology, molecular biology, and
microbial biotechnology. He has more than 15 years of integrated teaching and research experience and published more
than 65 research articles in peer-reviewed international journals along with 6 books and 20 book chapters. Also, he has
published 40 abstract at International/National conferences and symposia and received best poster presentation awards. He
supervised 6 PhD thesis and 12 PG/UG dissertations. He has been serving as an editor/editorial board member for 20 and
reviewer for more than 40 international peer-reviewed journals, along with research grant reviewer for scientiﬁc body of
the United States, Italy, South Africa, India, and Saudi Arabia. He has also been awarded SERC Young Scientist Project
from the Department of Science and Technology, Govt of India, and Young Scientist Project from International Foun-
dation for Science, Stockholm, Sweden.
xxi

Dr. Cristobal Noe Aguilar, Professor of Food Science and Biotechnology, Universidad Autònoma de Coahuila,
Mexico: Prof. Aguilar is member level III of the National System of Researchers of Mexico (S.N.I.). He has awarded with
several prizes including: Outstanding Researcher Award 2019 (International Bioprocessing Association, IBA-IFIBiop
2019), Prize of Science, Technology and Innovation Coahuila 2019, National Prize of Research 2010 of the Mexican
Academy of Sciences, the Prize-2008 of the Mexican Society of Biotechnology and Bioengineering, National Prize
AgroBio-2005, and the Mexican Price in Food Science and Technology from CONACYT-Coca Cola México 2003. From
2014 he is member of the Mexican Academy of Science. Prof. Aguilar is member of the International Bioprocessing
Association, Mexican Academy of Sciences, Mexican Society for Biotech and Bioeng, Mexican Association for Food
Science and Biotechnology. He is associate editor of Heliyon-Microbiology (Elsevier) andFrontiers in Sustainable Food
Systems(Frontiers), and member of several editorial boards of International Scientiﬁc Journals.
xxiiAbout the editors

Chapter 1
Enzyme technology for production of
food ingredients and functional foods
J.Y. Me´ ndez-Carmona, J.A. Ascacio-Valde´ s and Cristo´ bal N. Aguilar
Bioprocesses and Bioproducts Research Group, Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo,
Coahuila, Mexico
1. Introduction
It is well known that a balanced diet contributes to maintaining optimal health. The growing population demand for the
consumption of ingredients and foods that prevent diseases, in addition to optimizing the physical and mental state, has
gained part of the territory in the food market. In this area, the intake of foods and ingredients called functional has shown
important beneﬁts in the health of regular consumers. The consumption of functional ingredients or functional foods
provides a remarkable number of vitamins, minerals, fatty acids (FAs), dietaryﬁber, a wide range of antioxidants, and
bioactive compounds. These biologically active compounds obtained from various natural sources have been used, due to
their therapeutic properties, as allies in the treatment of different afﬂictions. The methods applied for the recovery of these
bioactive compounds have been improving in terms of performance, simplicity, sustainability, and reproducibility. In
particular, the enzymes used from plant, animal, and microorganism sources have demonstrated the ability to obtain safe
compounds for food enrichment and obtain ingredients and foods that meet the requirements to be classiﬁed as functional.
In this sense, the use of enzyme technology to catalyze bioprocesses will be critical. For this reason, the development of
new, more efﬁcient, and robust enzyme systems is of importance for the scientiﬁc and industry area involved in the
production of functional foods. Our research group works in the development of new enzyme technology that drives us
toward cleaner and more environmentally friendly commercial bioprocesses for food industry. In this chapter, we provide
information related to the topic,ﬁrst describing the most outstanding examples of food and functional food ingredients,
while in the background, we describe the innovations and innovative bioprocessing strategies based on enzyme tech-
nology, describing the use of the most relevant enzymes in the topic to guide the improvement of the relevant beneﬁts for
human health of functional foods and food ingredients.
2. Functional ingredients and functional foods
In the year 1980, Japan originated the deﬁnition of functional food that alluded to processed foods that provide a speciﬁc
beneﬁt to the organism beyond simple nutrition. For 1991 Japan included the term FOSHU for Foods for Speciﬁc Sanitary
Uses. These foods are classiﬁed as“Food for special dietary use”in the“Nutrition Improvement Law”referring to foods
that are consumed in the regular diet to improve personal health, in addition to being presented as conventional foods
(Madhujith and Wedamulla, 2020). Despite the great acceptance of the population in recent decades, there is currently no
legal deﬁnition for functional foods, since foods provide multiple beneﬁts to the body. Furthermore, functional foods are
understood as natural or industrialized foods that, when consumed regularly in adequate quantities as part of a varied diet,
provide speciﬁc health beneﬁts (Granato et al., 2017). On the other hand, functional ingredients are foods that by virtue of
their natural composition improve the nutritional, physicochemical, and structural qualities of the foods to which they have
been included.
Functional foods have become allies for the conservation and improvement of health, as well as to avoid diseases.
There are different deﬁnitions to refer to this type of food (Table 1.1). The importance of standardizing a concept of
Value-Addition in Food Products and Processing Through Enzyme Technology.https://doi.org/10.1016/B978-0-323-89929-1.00002-0
Copyright©2022 Elsevier Inc. All rights reserved.
1

functional foods lies mainly in the protection of the population through complete, concise, and clear information. Pre-
venting the distortion of the meaning of functional foods and the ambiguous labeling of foods would favor the conscious
choice of products based on the health and nutrition needs of the consumer and thus preserve scientiﬁc legitimacy in these
consumers and government ofﬁcials (Martirosyan and Singh, 2015).
3. Bioactive ingredients for the formulation of functional foods
Butnariu and Sarac (2019)establishﬁve ways in which a food can acquire functional properties: (1) by eliminating
compounds with adverse effects on the consumer (e.g., allergen components); (2) increase the amount of a speciﬁc
compound naturally present in the food to the point of encouraging positive health effects (e.g., increase the content of a
macro- or micronutrient to strengthen the recommended intake); (3) add a biological compound that is generally not found
in many foods, and that is not indispensable as a nutrient but due to its beneﬁcial effects has been used (e.g., nonvitamin
antioxidants); (4) replace a compound present in excess (usually a macronutrient) by a health-promoting compound; (5)
increase the stability of a compound characterized by providing multiple beneﬁts or signiﬁcantly reduce the risk of disease
development. Functional foods can be classiﬁed according to the type of food and/or active compound that they naturally
contain or have been added to.
TABLE 1.1Several deﬁnitions of functional foods.
Agency/organization Deﬁnition of functional food References
International Food Information Council Foods that may provide health beneﬁts
beyond basic nutrition.
Vorage et al. (2020)
Institute of Food Technologists (IFT) Foods and food components that provide
health nutrition (for the intended popula-
tion); examples may include conventional
foods, fortiﬁed, enriched and enhanced
foods, and dietary supplements.
Kuesten and Hu (2020)
17th International conference organized by
the US Department of Agriculture (USDA)
and Agricultural Research Service (ARS)
Natural or processed foods that contain
known or unknown biologically active com-
pounds which provide a clinically proven
documented health beneﬁt for the preven-
tion, management, or treatment of chronic
diseases in effective nontoxic amounts.
Martirosyan and Singh (2015)
Foundation for Healthy Food A functional food can be a natural product
that contains useful biological components,
or a food obtained through a technological
intervention that increases its level of bio-
logically active compounds.
Butnariu and Sarac (2019)
Academy of Nutrition and Dietetics Functional foods cover a variety of foods.
Minimally processed, whole foods along
with fortiﬁed, enriched, or enhanced foods
can all be functional foods. Generally, these
foods have a potentially beneﬁcial effect on
health when consumed on a regular basis
and at certain levels.
Klemm (2020)
European Commission Concerted Action A food can be regarded as ‘functional’ if it
satisfactorily demonstrates to improve bene-
ﬁcially one or more target functions in the
body, beyond the adequate nutritional ef-
fects in a way that is relevant to either an
improved state of health and well-being
and/or reduction of risk of disease.
Ye et al. (2018)
American Dietetic Association (ADA) Foods that provide additional health bene-
ﬁts that may reduce disease risk and/or pro-
mote optimal health.
Vorage et al. (2020)
2Value-Addition in Food Products and Processing Through Enzyme Technology

3.1 Probiotics
According to the deﬁnition established by the International Scientiﬁc Association of Probiotics and Prebiotics (ISAPP),
probiotics are living microorganisms that provide multiple beneﬁts to the health of the host, as long as they are supplied in
appropriate amounts (Hill et al., 2014). Regarding to consumer safety, the United State Food and Drug Administration, at
the strain level, designed them as Generally Regarded as Safe, while the at European Food Safety Authority has classiﬁed
them as a Qualiﬁed Presumption of Safety (Martín and Langella, 2019). Lactic acid bacteria likeLactobacilli,Enterococci,
Biﬁdobacterium,andLeuconostocspp. and yeast such asSaccharomycesspp. are commonly used in the preparation
of functional foods and fermented foods in order to contribute to the maintenance of the intestinal microbiota
(Ashaolu, 2020).
The multiple beneﬁts attributed to probiotics are triggered by various mechanisms of action where they not only stand
out for increasing the stability or recovery of the intestinal microbiota of the host when it has been altered, but also
participate in the production of antimicrobial compounds, reduce the risk of developing allergies, modulate the expression
of genes of the commensal, as well as contribute to release functional proteins that reduce the adhesion of pathogenic
agents (Shariﬁ-Rad et al., 2020).Ashaolu (2020)highlights an important aspect to consider in obtaining probiotics, the
ability to adhere to the gastrointestinal tract without being affected by the physiological conditions of the environment. In
this sense, the effectiveness to annul pathogenic microorganisms is directly related to the modulation of the immune system
and more speciﬁcally with the faculty of probiotics to contribute to stimulate the synthesis of immunoglobulins type A
(IgA) supporting immunomodulation. Result of in-depth study and technological advances in the scientiﬁcﬁeld, recently
commensal bacteria, have been isolated, identiﬁed, and modiﬁed, which have been deﬁned as the next generation of
probiotics. Nonetheless, its safety has not been considered proven due to its short period of appearance (Martín and
Langella, 2019).
3.2 Prebiotics
Prebiotics compounds are substrates that are speciﬁcally used by host-dwelling microorganisms to confer functional effects
on health (Gibson et al., 2017). Due to microbial selectivity, these nondigestible dietary compounds contribute to main-
taining, improving, and restoring host health. Some examples of these compounds that travel to be selectively fermented in
the colon are fructooligosaccharides, lactulose, and xylooligosaccharides (XOSs) as well as their hydrolyzates, to mention
a few (Granato et al., 2020).
3.3 Synbiotic
The ISAPP deﬁned the symbiotic term for the combination of living microorganisms and substrates used selectively by
host microorganisms that provide multiple health beneﬁts (Swanson et al., 2020). So, a product called symbiotic must
contain a mixture of probiotic and probiotic microorganisms that act independently to stimulate a health beneﬁt. It is
important to note that in order to label a product as symbiotic it must have evidence of joint operation and not only for each
individual component. In addition, ISAPP stipulated two subsets of symbiotics: complementary and synergistic. Theﬁrst
are distinguished since the probiotic microorganisms to be used are chosen based on the beneﬁts offered to the host. On the
other hand, prebiotics are used to stimulate the growth and functions of the members of the native microbiota and thus
confer the positive effect on the health of the consumer. Regarding synergistic symbiotics, they are formulated by selecting
a microorganism for its beneﬁcial properties, while the substrate is chosen according to it, to aid the growth and activity of
the selected microorganism. Some of the main combinations tested for their effects as synbiotics are microorganisms such
asLactobacillus casei, Lactobacillus acidophilus, andBiﬁdobacterium lactisusing as substrates inulin, fructooligo-
saccharides, galactooligosaccharides (GOSs), and XOSs (Granato et al., 2020). Regarding the characterization of synbiotic
products for commercial purposes, a rigorous description of identity, safety, purity, and potency of the living microor-
ganism is necessary in order to comply with the regulatory standards applicable in the country of commercialization
(Swanson et al., 2020).
3.4 Antioxidants
Nature has enriched different food sources with substances that help to slow or inhibit the oxidation of other molecules,
preventing various types of cell damage. These molecules called antioxidants have been used as food additives as well as
nutritional supplements, thanks to the beneﬁcial effects they have been shown on human health (Granato et al., 2020;
Enzyme technology for production of food ingredients and functional foodsChapter | 13

Krishna, 2020). Antioxidants can be classiﬁed according to their nature, mechanism of action, and source of procurement.
In addition, they have different mechanisms of action against reactive oxygen species (ROS) and free radicals, and
neutralize or divert ROS by catalytic systems (Jamshidi-Kia et al., 2020). To ensure that a food or ingredient has
antioxidant qualities, it is necessary to evaluate the antioxidant activity that alludes to the rate of reaction between a
particular antioxidant and a speciﬁc oxidant. On the other hand, antioxidant capacity quantiﬁes the amount removed from a
speciﬁc free radical by a sample (Gulcin, 2020).
Plants as a source of antioxidants, mainly polyphenols, vitamins, and carotenoids, include a wide range of fruits,
vegetables, herbs, and spices. Regular consumption of these foods and ingredients in a balanced diet improves health due
to its high nutrient content. Fruits and vegetables have an important range of antioxidant compounds that include
plant-speciﬁc primary and secondary metabolites. B-complex vitamins, as well as amino acids and fatty and nonfat acids,
are part of primary metabolisms. Nonetheless, organic compounds such as sulfur, alkaloids, and phenolic compounds, to
mention a few, constitute the secondary metabolisms that conform the majority of antioxidants (Jideani et al., 2021).
Currently, one of the main scientiﬁc challenges involves the extraction of antioxidant compounds present in the
aforementioned plant sources, and thus, to enrich foods to obtain functional foods or ingredients.
3.5 Polyunsaturated fatty acids
Polyunsaturated fatty acids (PUFAs) are biomolecules characterized by having more than one double bond in their
structure. There is an important variety of PUFAs such as linolenic acid (LA, 18:2, n-6), alpha-linolenic acid (ALA, 18:3,
n-3), eicosapentaenoic acid (EPA, 20:5, n-3), docosahexaenoic acid (DHA, 22:6, n-3), and long-chain PUFA (LCPUFA)
that have been studied due to the demonstrated effects on the maintenance of physiological functions, including the
treatment of metabolic, inﬂammatory, and oxidative pathological processes (Davinelli et al., 2021;Granato et al., 2020).
Due to the inability of the human organism to synthesize PUFAs, the consumption of foods rich in these biomolecules has
become indispensable to ensure the synthesis of FAs, and with it the correct integral functioning of the human body. In this
area, to increase the content of FAs available in food, the industry has focused on adding PUFAs extracted from natural
sources asﬁsh, eggs, seed oil, and cereals, for the ingredient procurement and functional food development.
Various bioactive compounds are used as functional ingredients or for the development of functional foods (Fig. 1.1).
Their choice varies according to the physiological needs they wish to meet, together with economic, reproducible, and
accessibility factors, among others.
FIGURE 1.1Example of some bioactive compounds used for the development of functional foods.
4Value-Addition in Food Products and Processing Through Enzyme Technology

4. Functional foods formulation
The impact that an adequate diet has on the state of health of the human being, in addition to improving the quality of life
during old age, is not new. That is why the sector dedicated to food development has focused on the development of
products that contribute to maintaining and preventing noncommunicable diseases. However, producing foods enriched with
bioactive compounds obtained from natural sources, preserving the organoleptic properties of consumer-like food, remains a
challenge for the food industry. Based on the fact that functional foods can be developed by adding compounds already
present in the food to increase its content or add a new ingredient to the food matrix, various phases have been established
that detail this process: (1) the deﬁnition of a target health beneﬁt and its progress, (2) identiﬁcation and/or selection of
functional ingredients and assessment of physiological effect, (3) development of a suitable food matrix, (4) bioaccessibility
and/or bioavailability tests, (5) human studies, (6) approval by regulatory authorities, and (7) product announcement and
commercialization (Keservani et al., 2020a,b). Regardless of the modiﬁcations that may have these phases according to the
type of ingredient or functional food to be obtained or formulated, the approval of theﬁnal product for its subsequent
commercialization regulated by ofﬁcial authorities of each country is essential to maintain consumer safety.
5. Regulations of functional foods
The growing awareness of the population, coupled with the interest in preserving health through food, is directly related to
the increase and spread of the international market of ingredients and functional foods. The market has registered a revenue
of 300 billion USD in 2017 and is expected to increase to 440 $B in 2022 (Farid et al., 2019). However, the global market
share for functional foods varies from region to region (Fig. 1.2). An important area for the commercialization of functional
foods focuses on the complete and speciﬁc regulation of health claims (labeling, presentation, advertising campaigns).
With the initial development of ingredients and functional foods, Japan was the only country that implemented a speciﬁc
regulatory process to approve products that had such functional qualities (Kaur and Das, 2011). The accelerated growth of
the food industry focused on the development of ingredients and functional foods worldwide that has occurred in recent
decades has required the work of regulatory agencies to certify those products that claim to be classiﬁed as functional,
without neglecting consumer safety. It is also necessary to establish the objective of the products on the market, the
packaging requirements, the nutrition information, the Good Manufacturing Practice System, the safety review systems for
the ingredients of the product, as well as the intellectual property regulations of each country (Farid et al., 2019). Despite
regulatory standards, there is no international regulatory framework that standardizes these claims (Domínguez Daíz et al.,
2020). This represents a difﬁculty at the international level, since each country has different legal standards that regulate
functional ingredients and functional foods (Table 1.2).
FIGURE 1.2Global functional foods market share by region worldwide (Farid et al., 2019).
Enzyme technology for production of food ingredients and functional foodsChapter | 15

6. Enzymes in production of functional foods
The development of bioprocesses using enzymes for the production of ingredients and functional foods has been deeply
studied given the ability of these compounds to enhance the bioactive molecules present in food, provide solubility,
stability, and even deactivate some food-speciﬁc antinutritional properties. The fermentation through microorganisms and
enzymatic hydrolysis or transformation induced by commercial enzymes obtained from various sources, functional foods
have been developed that provide one or more health beneﬁts. Some of the most important enzymes used in the production
of functional foods are peptidases, lipases, tannases, carbohydrate-modifying enzymes,
L-asparaginase, and phytases,
among others (Chourasia et al., 2020).
6.1 Lipases
Lipases (EC 3.1.1.3) are hydrolases capable of catalyzing the hydrolysis of triacylglycerols (TAGs) into glycerol and FAs
(Melani et al., 2020). Lipases are enzymes that play vital roles in lipid metabolism. Also called TAG hydrolyzes have the
ability to catalyze the hydrolysis of TAG. In recent decades, its use has increased due to multiple applications and
versatility as biocatalysts for the production of esters, PUFAs, and long-chain FAs. Thanks to the various properties of oils
and fats obtained by the action of lipases, foods formulated from these enzymes can be classiﬁed as functional (Castejón
and Señoráns, 2020;Chourasia et al., 2020;Priji et al., 2016). The sources for obtaining this enzyme vary considerably,
ranging from plants, animals, to microorganisms that include fungi and bacteria. Lipases obtained from microbial sources
are preferred because of their low procurement costs, their simplicity with respect to genetic manipulation, ease in the
process of isolation and production, as well as a diverse range of biochemical activities. On the other hand, they have
shown greater stability compared to enzymes derived from plants and animals (Adetunji and Ademola, 2021;Mehta et al.,
2021). This represents a potential alternative that has been used for the production of enzymes from bioprocesses that are
more economical and energetically viable. However, one of the great challenges continues to be the quantity and quality of
compounds to be extracted compared to other technologies.
In the food processing industry, lipases are commonly used for the formulation and improvement of dairy products and
derivatives (such as cheeses), bakery products, processing of fats and oils for the production of modiﬁed acylglycerols, and
in the fruit juice industry (Melani et al., 2020). In the case of dairy products, lipase is used to modify the lengths of the FA
chain and enhance theﬂavor of cheeses through the hydrolysis of milk fat. In addition, it can be applied to accelerate the
maturation of the cheese, as well as to provide speciﬁcﬂavors mainly to soft cheeses (Chandra et al., 2020). The functional
properties of lipases have given them great popularity in the industrial application related to food and in the development of
other commercial products such as medicines and cosmetics, among others. The versatility and potential use of these
enzymes are sufﬁcient factors to continue developing innovative technologies that promote the production and use of
lipases as a source of ingredients and functional foods.
TABLE 1.2Regulation of functional foods in some countries.
Country Regulatory agency References
Japan Japanese Ministry of Health, Labour, and Wel-
fare (MHLW)
Iwatani and Yamamoto (2019)
United States Food and Drug Administration (FDA) Grochowicz et al. (2019)
European Union European Commission (EC) Grochowicz et al. (2019)
Canada Health Canada Brown et al. (2020)
India Food Safety and Standards Authority of India Keservani et al. (2014)
China Chinese Food and Drug Administration
(CFDA)/State Administration for Market
Regulation
Bagchi (2019)
Korea Ministry of Food and Drug Safety (MFDS) Farid et al. (2019)
Taiwan Taiwan Food and Drug Administration (TFDA) Farid et al. (2019)
Turkey Republic of Turkey Ministry of Food, Agricul-
ture and Livestock
Gok and Ulu (2019)
6Value-Addition in Food Products and Processing Through Enzyme Technology

6.2 Proteases
Proteases refer to a complex group of proteolytic enzymes that hydrolyzes peptide bonds. The classiﬁcation of the protests
depends on various factors such as the nature and chemical properties of the catalytic site, the type of reaction where the
molecule acts as a catalyst, and the evolution of the protease structure and its relationship (Gurumallesh et al., 2019).
Another interesting classiﬁcation is based on the active site of the molecule cataloging them in: cysteine, serine, aspartic,
asparagine peptide lyases, threonine, glutamic, and metalloproteases (Fernández-Lucas et al., 2017). Proteases can be
obtained from plant, animal sources, as well as microorganisms and have promising potential in functional food
production.
In the area of food, proteases have been used to modify proteins, rectify the taste of food, improve the storage stability
of available protein sources, as well as improve digestibility and decrease the allergenic load (Hamza, 2017;Tavano et al.,
2018). The use of protease in food production includes making cheese, bakery products, hydrolyzed soybeans, and
tenderizing meat (Singh et al., 2019). In the case of proteases, there is an extensive study of the production of these
enzymes by microorganisms and their application for the production and improvement of food. An example of this is the
Flavourzyme produced by selectedAspergillus oryzae. This enzyme in combination with Protamex is used to convert meat
by-products into meat-ﬂavored broth that is later added in meat processing, thus reducing the amount of salt added in the
ﬁnal products. In the case of beverages, Neutrase is used to maintain a constant growth of yeast, resulting in an
improvement in the performance and quality of the beer (Chew et al., 2019). Nevertheless, in the case of enzymes produced
from microorganisms, the safety assessment of the producing strain is important. Not only is it sufﬁcient for the enzyme to
be accepted as safe for the health of the consumer, but it must also be the microorganism that is the product of that enzyme.
Within the legal framework, the Joint FAO/WHO Experts Committee on Food Additives (JECF) are among the bodies that
provide such certiﬁcation (Tavano et al., 2018).
Some enzymes of plant origin such as papain have gained commercial importance, thanks to the outstanding activity in
various operating conditions, in addition to the important proteolytic activity that has shown against various protein
substrates. An example of this is the application of papain as an alternative for cheese making, since it has been shown to
be useful in the formulation of semisoft and creamy cheeses. However, it is important to consider that the clotting rate of
this enzyme is not uniform with the use of different types of milks (Fernández-Lucas et al., 2017).
6.3 Carbohydrate-modifying enzyme
Carbohydrases are enzymes responsible for carbohydrate catalyst. In the food industry, enzymes such as amylases,
inulinases, galactosidases, glucosidases, pectinases, glucosyltransferases, and fructosyltransferases originate beneﬁcial
biological compounds for the formation or enrichment of functional foods (Chourasia et al., 2020).
Research on lactic acid bacteria has been deeply studied for its beneﬁcial properties to human health. An example of
this is theb-galactosidase enzyme that stands out for its application in the dairy industry since it helps to facilitate the
metabolism of lactose, thereby alleviating the symptoms of lactose-intolerant consumers.b-galactosidase has been used for
the production and recovery of GOSs (Fara et al., 2020). GOS intake has been linked to immunomodulatory effects due to
direct interaction with immune cells improving the population of health-promoting bacteria (Pujari and Banerjee, 2020).
Obtaining enzymes with carbohydrate activity from microorganisms is not only a more affordable source, but also a more
environmentally friendly technology.Lactobacillus paracaseiBGP1 has been used to ferment GOS extracted from various
plant species, resulting in a potential alternative source for the production of useful prebiotic ingredients in the preparation
of functional foods (Palacio et al., 2020). As an example of functional foods, prototypes of beverages enriched with GOS in
addition to antimicrobial properties have been developed from ferments with lactic acid bacteria and apple by-products.
The results show a positive acceptability in theﬁnal product, in addition to postulating new beverage prototypes with
prebiotic and antimicrobial effects (Zokaityte et al., 2020).
The formulation of packaging that preserves food and, in turn, made from environmentally friendly materials is one of
the methods that has gained great popularity. In this area,Fernandes et al. (2020)studied the preparation of edible prebiotic
ﬁlms from whey protein enriched with GOS and XOSs, obtaining semipermeable packaging for the coating of fresh fruits
and vegetables and bakery foods. With this postulate, it is clear the wideﬁeld of study that concerns the elaboration and/or
enrichment of functional foods from enzymes obtained from diverse biotechnological sources. Like GOSs and XOSs are
part of the most abundant oligosaccharides. XOSs are of great relevance in the commercial development of oligosac-
charides. The enzymatic production of these compounds is highly feasible since during enzymatic hydrolysis they
signiﬁcantly decrease the production of undesirable reactions and secondary products (Bhatia et al., 2019). XOSs are
present in natural sources such as fruits, vegetables, honey, milk. In addition, some agro-industrial wastes such as wheat oat
Enzyme technology for production of food ingredients and functional foodsChapter | 17

bran, rice straw, wheat, and maize are potential sources of XOSs in quantity and production (Bhatia et al., 2019;Tan and
Norhaizan, 2020). The formulation of functional foods from XOSs increases the functional and nutrient compounds
available in conventional food. An example of this is symbiotic soy milk added with XOSs and inoculum of Weissella
cibaria FB069 (FSMXW) made for the purpose of studying the effects on the proliferation of cancer cells in the colon
compared to other fermented soybean products (Le et al., 2020).
6.4 Tannase
Tannase, like other enzymes, are widely distributed in nature in plants, animals, and microorganisms. Its mode of action is
dividing the ester and depside bonds of the hydrolyzable tannins to release glucose and gallic acid. As a result, tannase is
one of the main sources of gallic acid. In the food industry, it is used for the production of instant tea, in addition to being
used to improve the quality of beverages such as fruit juices, beer, and wine (Kumar and Sreekumar, 2019). The production
of tannase from microorganisms is one of the most widely used and studied techniques for its accessibility. Fungi such as
Aspergillussp.,Aspergillus fumigatus, andAspergillus versicolor, in addition to bacteria such asLactobacillus plantarum,
Lactobacillus paraplantarum,Pseudomonas aeruginosa, andKlebsiella pneumoniaehave shown potential to produce this
enzyme adequately (Dhiman et al., 2018;Govindarajan et al., 2019). It has been reported the use of tannase produced by
microorganisms and used for food production, speciﬁcally for enzymatic extraction from green tea using tannase obtained
fromAspergillus nigerFJ0118 (Shao et al., 2020).
6.5L-asparaginase
The enzymeL-asparaginase is found in most algae, plants, microorganisms such as bacteria and fungi, and some animals. It
has been widely studied for its role as a potent anticancer. Among the microorganisms that have reported
L-asparaginase
production, the following stand outErwinia aroideae, Pseudomonas aeruginosa, Aspergillus tamari, Vibro succinogenes,
Aspergillus terreus, Pseudomonas stutzeri y Staphylococcussp. (Muneer et al., 2020). In the foodﬁeld, its use stands out to
eliminate or reduce acrylamide, a compound present in foods rich in carbohydrates that have been cooked with high
temperatures, and that presents probable carcinogenic activity in consumers (Muneer et al., 2020).
6.6 Phytases
Phytase acts as a catalyst of the phytate hydrolysis reaction that is not digestible by the human organism and is found in
greater quantity in foods of plant origin. The enrichment of foods with phytase for the production of ingredients and
functional foods can increase the performance and nutritional value of products (Kumar and Sinha, 2018). Like other
enzymes, phytase is found in plants, animals, fungi, bacteria, and yeast. Numerous studies have focused on the production
of this enzyme fromﬁlamentous fungi such asAspergillus, Myceliophthora, Mucor, Penicillium, Rhizopus, andTricho-
derma(Jatuwong et al., 2020). This enzyme plays an elemental role in the digestive process for the prevention of the
antinutritional effects of phytates and phytic acid, in addition to enhancing the bioavailability of minerals and proteins,
resulting in better general health. In the food industry, phytase has been used to bread processing, improving the nutritional
value of plant-based, cereal bran fractionation, grains wet milling, isolation of plant proteins (Song et al., 2019).
7. Alternatives for the recovery or extraction of enzymes with possible applications
in the food ﬁeld
A wide variety of biocomposites used for the formulation of functional ingredients and functional foods are extracted from
natural sources or synthesized enzymatically. In addition, enzyme production is a favorite when it comes to large-scale
production due to certain properties highlighted as catalysts: speciﬁcity, selectivity, activity under smooth operating
conditions, high rotation number, and biodegradability. Glycoside hydrolases (GHs), glucosyltransferases (GTs), and
transglycosylases (TGs) are some most widely used enzymes for the production of non-digestible polysaccharides (NDPs)
(Vera et al., 2021). It is relevant to note that the origin of the enzyme molecule inﬂuences the synthesis of NDPs; this is a
factor that must be considered when choosing the type of food or ingredient to be developed.
The economic, ecological, and methodological implications in the synthesis of enzymes for commercial use have
contributed to industrial biocatalysis that has focused on the formulation of processes that are more respectful with the
environment, simple and sustainable while preserving the quality of the products obtained. In this sense, the production of
enzymes from some microorganisms represents an emerging biotechnological alternative. Solid-state fermentation (SSF)
8Value-Addition in Food Products and Processing Through Enzyme Technology

and submerged fermentation (SbF) are techniques used in the recovery of enzymes at an industrial level. The SSF has
demonstrated an efﬁcient production, in addition to high values in enzyme activity. However, the selection of the method
will inﬂuence the performance of the process efﬁciently and the production of the desired enzyme (Sepúlveda et al., 2020).
Despite the in-depth study of enzymatic production technologies such as fermentation with enzyme-producing microor-
ganisms using agro-industrial waste as substrate, it is necessary to evaluate the requirements previously established by the
regulatory authorities of each country.
8. Opportunities of enzyme technology for production of food ingredients and
functional foods
The modern food industry is undergoing a profound transformation that makes it sustainable and turns it into one of the key
activities for the promotion of health that humanity requires from the year 2020, date in which new challenges have been
obligatorily revealed before the need to face rads problems of humanity. Various sectors of the food industry are examples
of transformation by the inclusion of technological innovations using enzymes, including the development of food and
functional ingredients, processing, preservation and conservation innovations, packaging, input biotechnology, and waste
valorization, among others. Enzymes represent selective and speciﬁc biological catalysts that operate under mild
processing conditions and are biodegradable, and are required with new catalytic properties, including increased activity,
greater stability, and selectivity. To achieve this, strategies such as mutation techniques, bioprocess control and optimi-
zation, optimization of reaction conditions, and structural improvements of proteins through synthetic biology, among
other strategies, are implemented.
9. Conclusion
Functional foods and ingredients play a very important role today. The increase in scientiﬁc evidence related to diet and the
prevention of various diseases has raised awareness of the population regarding a healthier lifestyle. With this, the
preference and consumption of foods that offer multiple health beneﬁts has led to the tremendous growth of the industry
dedicated to the development of foods and functional ingredients. Today, food products that promise optimal health are
truly extensive. In this sense, it is necessary for the various agencies or regulatory bodies in each country to collaborate to
establish uniﬁed criteria that facilitate the regulation of food that day by day reaches the consumer’s hands. On the other
hand, the technologies applied for the development of ingredients and functional foods ingredients have evolved in order to
make industrial production more competent and efﬁcient. Enzyme technologies for the formulation of fortiﬁed foods are
among the oldest in human history. The postulate of new techniques from natural sources, such as enzymatic production
from microorganisms, represents an alternative under development that is cleaner and friendlier with the environment.
Nevertheless, this implies great challenges related to the quality of the products developed, in addition to their acceptability
by the consumer.
Acknowledgments
The authors thank the National Council of Science and Technology (CONACyT, Mexico) for the support of the scholarship awarded to Méndez-
Carmona J.Y. to study the master’s degree in Food Science and Technology at the Autonomous University of Coahuila (UAdeC).
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Enzyme technology for production of food ingredients and functional foodsChapter | 111

Chapter 2
Enzymes in probiotics and genetically
modiﬁed foods
K.B. Arun
1
, Aravind Madhavan
1
, Shibitha Emmanual
2
, Raveendran Sindhu
3
, Parameswaran Binod
3
and
Ashok Pandey
4
1
Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India;
2
Department of Zoology, St. Joseph’s College, Thrissur, Kerala, India;
3
Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Trivandrum,
Kerala, India;
4
Center for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow,
Uttar Pradesh, India
1. Introduction
Enzymes are catalysts produced by living organisms to speed up biochemical reactions. The catalytic potential of these
enzymes has been utilized for various industrial applications. Since centuries ago, with the utilization of bacteria and yeast,
enzymes were unintentionally used to prepare food products such as bread, cheese, beer, and wine (Chaudhary and Sagar,
2015;Leisola et al., 2009). The commercial use of enzymes starts with the usage of trypsin and protease in detergents. The
advancements in knowledge in various scientiﬁcﬁelds have boosted the usage of enzymes in industries which signiﬁcantly
reﬂects the market value of enzymes. The global enzyme market value is predicted to reach 8.7 billion USD in the year
2026. Among various sources of enzymes, microbial enzymes are the most preferred one as they are easy to produce and
can manipulate well to excel activity, speciﬁcity, and stability. These peculiarities make them suitable for application in
food, detergent, pharmaceutical, and agricultural sectors (Singh et al., 2016).
Food is one of the essential components that generate the energy required to maintain fundamental processes and
develop an organism. Human history is related to how they feed themselves, and from the period of hunting, we have
reached up to using genetically modiﬁed foods. Microbes and microbial enzymes play a critical role in the food industry.
Microorganisms are manipulated and are used in developing dairy and meat products, wine, medicines, and health sup-
plements (Eldarov and Mardanov, 2020;Pham et al., 2019;Deckers et al., 2020;Coffey et al., 1994). The past few decades
have witnessed the signiﬁcant role played by genetically modiﬁed organisms and the enzymes produced by them in the
food industry, resulting in the development of commercially feasible innovated food products (Hanlon and Sewalt, 2020).
Probiotics are live microbes that, when ingested, offer health beneﬁts, usually by enhancing or restoring gutﬂora. They
help in digestion, compete and remove pathogens, produce some essential nutrients, and are even involved in drug
metabolism (Nagpal et al., 2012;Maldonado Galdeano et al., 2019).Lactobacillus,Biﬁdobacteria, and certain species of
Saccharomycesare commonly used probiotics (Konuray and Erginkaya, 2018). Consumers are now aware of the health
beneﬁts of probiotics which have increased the market value of probiotic foods, and hence probiotics are widely used in the
food industry for developing functional foods. Enzymes from these probiotics are also used in various food industries. The
advancements in molecular biology have led to the development of recombinant probiotics with speciﬁc traits which
facilitate their use in the food industry.
This chapter focuses on current probiotic enzymes used in the food industry, advancements in the development of
genetically modiﬁed probiotics, and their applications in the food industry. Further, the prospects of genetically modiﬁed
probiotics and their enzymes in food application were discussed.
Value-Addition in Food Products and Processing Through Enzyme Technology.https://doi.org/10.1016/B978-0-323-89929-1.00006-8
Copyright©2022 Elsevier Inc. All rights reserved.
13

2. Probiotic enzymes for the food industry
Humans have been utilizing microorganisms since the ancient period for fermentation purposes. With their stability,
speciﬁcity, and activity, the microbial enzymes have signiﬁcantly reduced the time, space, and cost in the food industry.
Microbial enzymes are generally utilized for different applications for amylases, galactosidases, xylanases, lipases, pro-
teases, peptidases, peroxidases, phenoloxidases, ureases, and esterases in the food industry. Many of these enzymes are
produced by probiotic bacteria, which are considered safer organisms to use, and hence they are used or manipulated
genetically to manufacture enzymes to use in the food industry (Kaswurm et al., 2013;Ku, 2016). The enzymes from
probiotic sources that are used in the food industry are summarized inTable 2.1.
Amylase, proteinase, and lipase are the principal technical enzymes used in the food and animal feed industry.
Amylases from lactic acid bacteria are extensively used in the baking industry to modify the starch and ferment several
food products based on carbohydrates (Amapu et al., 2016). This starch-degrading enzyme is used for liquefaction and
brewing in various food industries as it breaks thea-D-1,4-glycosidic bonds and releases oligosaccharides from starch.
Lactobacillus fermentum,Lactobacillus plantarum,Lactobacillus manihotivorans, andLactobacillus amylovorusproduce
amylases (Padmavathi et al., 2018;Uma Maheswar Rao and Satyanarayana, 2003;Morlon-Guyot et al., 1998).
Proteinase enzymes from lactic acid bacteria catalyze the proteolysis of casein milk protein to release peptides by
hydrolyzing peptide bonds, and this process plays a signiﬁcant role in determining the texture and aroma of milk-related
products (Konkit and Kim, 2016). Proteases also catalyze the conversion of whey, a by-product formed during cheese
manufacturing, to protein-rich hydrolyzate, thus reducing the waste. They are also used to remove proteinetannin complex
and water-soluble proteins from fresh beer (Wang et al., 2013). Proteases and peptidases fromLactobacillusspecies
catalyze the release of peptides and amino acids from protein present in the meat tissue during fermentation. These peptides
and amino acids are typicalﬂavors of fermented food products. The peptides are also reported to have various biological
activities (Cao et al., 2019;Fernández et al., 2016).
Lipases are involved in lipids’catabolism, and especially they catalyze the conversion of triglycerides to fatty acids and
glycerol. This lipolytic process also favors the aroma of dairy products.LactococcusandBacillussp. are excellent sources
of technical enzymes with broad applications in food industries (Konkit and Kim, 2016).b-Galactosidase catalyzes the
hydrolysis ofb-galactosides into monosaccharides (such as lactose to glucose and galactose), which is yet another essential
enzyme use in the food industry, especially to synthesize lactose-hydrolyzed products to meet the needs of lactose-
intolerant people. These enzymes are also used in the food industry to reduce the crystallization in food products
caused by the hygroscopic nature of lactose (Saqib et al., 2017). Prebiotics are oligosaccharides which when consumed,
promote the growth of probiotic bacteria in the gut (Arun et al., 2019;Davani-Davari et al., 2019). The prebiotic
galactooligosaccharides are synthesized by the action ofb-galactosidase obtained from various probiotic species.
a-Galactosidase resistant to protease activity extracted fromBacillus coagulanscan be used in the food industry to remove
nondigestible rafﬁnose and stachyose from food products (Zhao et al., 2018).
Antinutrients present in food such as phytate make the vital minerals unavailable for their action through their chelating
property. Food industries make use of phytase enzymes to degrade the phytate present in food. Some probiotics such as
LactobacillusandBacillusspecies have been identiﬁed to have better phytase activity which is being used in food in-
dustries (Priyodip et al., 2017). Phosphatases hydrolyze phosphoproteins, including milk casein, to release peptides and
amino acids during cheese production (Magboul and McSweeney, 1999). Laccases (oxidize phenolic and nonphenolic
compounds) are utilized in the food industry to stabilize beverages by discerning the removal of phenolic and amine
compounds (Arevalo-Villena et al., 2017;Callejón et al., 2017). Acetolactate decarboxylase (converts acetolactate to
acetoin) is used for the maturation of beer (Al-Maqtari et al., 2019). The presence of pectins makes fruit juice turbid and
viscous which hinders fruit juice processing. Polygalacturonases are used in juice processing to break the glycosidic bonds
in pectin molecules which signiﬁcantly reduces the processing time (Swain and Ray, 2010). Esterases such as feruloyl
esterase and cinnamoyl esterase are utilized to develop functional foods as they enhance the release of polyphenols and
increase their bioavailability (Palaniswamy and Govindaswamy, 2016;Hole et al., 2012).
Thus, enzymes of probiotic origin were widely used in various applications in the food industry. With the advance-
ments in molecular biology techniques, probiotic bacteria were engineered to produce enzymes with speciﬁc traits. The
following section will deal with engineered probiotics used in food industries.
3. Engineered probiotics for food applications
The awareness of the health beneﬁts of probiotics has led to identifying new probiotic strains, and continuous research is
being made to engineer these strains with better activities. The most commonly used probiotic strains in food industries are
14Value-Addition in Food Products and Processing Through Enzyme Technology

TABLE 2.1Enzymes produced by probiotic bacteria and their use in food industry.
Probiotics Enzyme
Food industry
application References
Lactobacillussp. Amylase Fermentation Hattingh et al. (2015)
Bacillus subtilis Amylase Sacchariﬁcation and starch
liquefaction
Al-Maqtari et al. (2019)
Biﬁdobacterium breve
Biﬁdobacterium dentium
Biﬁdobacterium infantis
Biﬁdobacterium pseudolongum
Biﬁdobacterium thermophilum
Amylase Degradation of starch Ryan et al. (2006)
Lactobacillus fermentum Amylase Preparation of high-energy
food
Nguyen et al. (2007)
B. subtilis Lipase Bread manufacturing Mnif et al. (2012)
Lactobacillus casei
Lactobacillus plantarum
Lactobacillus rhamnosus
Lipase Flavor enhancement in
cheese
Chandra et al. (2020)
Lactobacillus paracasei Lipase Flavor enhancement in
cheese
Stefanovic et al. (2018)
B. infantis b-Galactosidase Lactose hydrolysis Saqib et al. (2017)
Biﬁdobacterium longumstrains
Lactobacillus pentosus
b-Galactosidase Galactooligosaccharides
production
Hsu et al. (2007)
Biﬁdobacterium biﬁdum b-Galactosidase Galactooligosaccharides
production
Osman et al. (2010)
L. plantarum b-Galactosidase Galactooligosaccharides
production
Kittibunchakul et al. (2020),
Gobinath and Prapulla (2014)
Lactobacillus acidophilus
Lactobacillus bulgaricus
Lactobacillus helveticus
Lactobacillus keﬁranofaciens
Lactobacillus lactis
Lactobacillus sporogenes
Lactobacillus thermophilus
Lactobacillus delbrueckii
b-Galactosidase Galactooligosaccharides
production
Panesar et al. (2010)
Bacillus coagulans a-Galactosidase Removes nondigestible
oligosaccharides from food
Zhao et al. (2018)
L. plantarum
L. fermentum
L. pentosus
Protease Meat processing Cao et al. (2019)
Bacillus amyloliquefaciens Protease Brewing of beer Wang et al. (2013)
Bacillus licheniformis Xylanase Processing fruit juice and
dough-raising in bakery
Bajaj and Manhas (2012)
Lactobacillus sakei
Lactobacillus curvatus
L. plantarum
L. pentosus
Protease and lipase Enhancing quality, smell
and taste by stabilizing the
meat
proteolysis and lipolysis
Neffe-Skocinska et al. (2016)
B. subtilis
B. coagulans
Phytase Phytate degradation,
fermentation
Hong (2011),Seung-Hun et al.
(2006)
B. longum Phytase Phytate degradation Sun et al. (2019)
Pediococcus acidilactici Phytase Dephytinization Sharma and Shukla (2020)
Continued
Enzymes in probiotics and genetically modiﬁed foodsChapter | 215

LactobacillusandBiﬁdobacterium. They are used for the production of dairy-based foods such as cheese, quark, yoghurt,
ice cream, chocolate mousse, fermented milk, and desserts, and also in nondairy-based food products such as vegetable-
and fruit-based drinks, soy-based drinks and desserts, cereal-based products, and meat products (Song et al., 2012). They
are also used to produce or deliver nutrient supplements or bioactives with health beneﬁts (Bron and Kleerebezem, 2018).
The activity of probiotics is lost during assimilation into food products or after consumption, as they are less tolerant to
temperature, acidity, oxygen, and salt. To overcome the drawbacks of naturally occurring probiotics that limit them from
using for industrial applications, these probiotic organisms are extensively engineered to make themﬁt for industrial
applications (Table 2.2).
The current probiotic strains can be engineered to enhance their stress tolerance in industrial and gut environments, to
bestow beneﬁcial properties to the food, and to increase the yield of desired probiotic enzymes. The amylase production in
Bacillus subtiliswas improved by overexpressing the PrsA lipoprotein and SPnprE signal peptide (Chen et al., 2015). The
heterologous coexpression of PrsA inB. subtilisenhanced the production ofa-amylase (Quesada-Ganuza et al., 2019).
A recombinant strain ofB. subtiliswas engineered by overexpressinga-amylase gene fromBacillus stearothermophilus,
with optimized signal peptides and overexpressed chaperones yield more active amylase enzyme (Yao et al., 2019).
RecombinantLactobacillus caseidisplayinga-amylase (fromStreptococcus bovis) anchored using PgsA anchor protein
fromB. subtilisexhibits better hydrolytic property (Narita et al., 2006). Chemical and physical mutagenesis combined with
genome shufﬂing ofB. subtilisresulted in identifying new strains with enhanced cellulase activity (Ega et al., 2020). The
advanced CRISPR/Cas9-mediated gene integration technique was adopted by García-Moyano et al. to develop
recombinantB. subtilisto enhance protease enzyme production (García-Moyano et al., 2020).
When heat shock proteins are overexpressed,Lactobacillus paracaseiwas reported to be more heat tolerant than the
wild-type strains (Desmond et al., 2004). The catalase gene fromB. subtiliswas introduced toBiﬁdobacterium longumto
make it tolerant to oxidative stress (He et al., 2012). In another study, recombinantB. longumheterologously expressed
superoxide dismutase and catalase fromStreptococcus thermophilusandL. plantarum, respectively. The heterologous
expression of these two proteins protect the recombinant strain from oxidative stress. A similar study was also done in
Lactobacillus rhamnosus(Zuo et al., 2014a;An et al., 2011).L. caseiwas engineered to become resistant to bile salt and
oxidative stress by coexpressing catalase fromLactobacillus sakei
and bile salt hydrolase fromL. plantarum(Wang et al.,
2011).B. longumoverexpressing ahpC (alkyl hydroperoxide reductase subunit C) increases its tolerance to oxidative stress
as the enzyme detoxiﬁes hydrogen peroxide (Zuo et al., 2014b).
Kang et al. have engineeredB. subtilisto exhibit peptidoglycan hydrolase protein (p75) fromL. rhamnosususing the
coat protein CotG as an anchor. The p75 enzyme in the modiﬁed strain was active over a wide pH range, even at high
temperatures (Kang et al., 2020). The CotG and CotC were used to display trehalose synthase inB. subtilisspores, and
these strains give better trehalose yield (Liu et al., 2019c). The phytase protein fromEscherichia coliwas anchored to
B. subtilisspores, improving enzyme activity (Mingmongkolchai and Panbangred, 2019).
Liu et al. have successfully increased the trehalose production by expressing a mutant trehalose synthase and deleting
two maltose transporters inB. subtilis. These recombinant strains release an active trehalose synthase that catalyzes
TABLE 2.1Enzymes produced by probiotic bacteria and their use in food industry.dcont’d
Probiotics Enzyme
Food industry
application References
L. acidophilus
Lactobacillus brevis
L. casei
L. paracasei
L. fermentum
L. rhamnosus
L. delbrueckii
L. plantarum
Phytase Phytate degradation,
various fermentation food
products
Priyodip et al. (2017),Fischer
et al. (2014),
Zamudio et al. (2001),Lavilla-
Lerma et al. (2013),Onipede et al.
(2014)
L. plantarum Phosphatase Phosphoprotein
hydrolyzing
Magboul and McSweeney (1999)
L. paracasei Phosphatase Phosphoprotein
hydrolyzing
Bhagat et al. (2019)
P. acidilactici Laccase Amine degradation Callejo´n et al. (2017)
16Value-Addition in Food Products and Processing Through Enzyme Technology

TABLE 2.2Engineered probiotic strains and its application.
Probiotics Method Application References
Bacillus subtilis PrsA lipoprotein and SPnprE signal
peptide overexpressed
Improveda-amylase activity Chen et al. (2015),
Quesada-Ganuza
et al. (2019)
B. subtilis Coexpression of PrsA Improved a-amylase activity Quesada-Ganuza
et al. (2019)
B. subtilis Overexpressing AmySA with optimized
signal peptide and overexpressing
chaperones
Increaseda-amylase production Yao et al. (2019)
Lactobacillus casei Displayed amylase fromStreptococcus
bovisanchored using PgsA from
B. subtilis
Improved hydrolytic activity Narita et al. (2006)
Lactococcus lactis Overexpression of lipase from
Burkholderia cepacia
Improved lipolytic activity Raftari et al. (2013)
B. subtilis Mutagenesis and genome shufﬂing Enhanced cellulase production Ega et al. (2020)
B. subtilis CRISPR/Cas9-mediated gene
integration
Protease production improved Garcı´a-Moyano et al.
(2020)
Lactobacillus
paracasei
Overexpression of GroESL heat shock
protein
Improved heat and solvent toleranceDesmond et al. (2004)
Biﬁdobacterium
longum
Expressing catalase gene from
B. subtilis
Enhance resistance to oxidative stressHe et al. (2012)
B. longum Coexpressing SOD and KAT Resistant to oxidant stress Zuo et al. (2014a)
Lactobacillus
rhamnosus
An et al. (2011)
L. casei Coexpressing bile salt hydrolase and
KAT
Resistant to bile salt and oxidant stressWang et al. (2011)
L. lactis Deletion of prophage-related fragment Multistress tolerance Qiao et al. (2020)
Lactobacillus
salivarius
Biﬁdobacterium breve
Expression of the listerial betaine
uptake system BetL
Improved osmo tolerance, cryo toler-
ance, baro tolerance, and chill toler-
ance; better resistance to gastric juice;
and spray- and freeze-drying
Sheehan et al. (2006,
2007)
B. subtilis Displayed peptidoglycan hydrolase
protein fromL. rhamnosus
Improved hydrolase activity at wide
range of temperature at 50

C
Kang et al. (2020)
B. subtilis Displayed trehalose synthase protein Better trehalose production Liu et al. (2019c)
B. subtilis Displayed phytase protein fromE. coliImproved phytase activity Mingmongkolchai and
Panbangred (2019)
B. subtilis Expressing mutant trehalose synthase,
and deletedmalPandamyEmaltose
transporter genes
Better trehalose production Liu et al. (2019b)
B. subtilis Pglvpromoter mutation Better trehalose production Liu et al. (2019a)
B. subtilis Integrated Pgracpromoter Enhanced b-galactosidase activity Tran et al. (2020)
L. lactis Express trehalose synthase fromE. coliIncrease freeze-drying and gastric
tolerance
Termont et al. (2006)
L. lactis Trehalose 6-phosphate phosphatase
fromPropionibacterium freudenreichii
Better trehalose production under
acidic condition
Carvalho et al. (2011)
L. lactis Overexpression of rbsA, rbsB, msmK,
and dppA
Improved activity under acidic
conditions
Zhu et al. (2019)
B. longum Overexpression of ahpC Improves oxidative stress tolerance Zuo et al. (2014b)
Continued
Enzymes in probiotics and genetically modiﬁed foodsChapter | 217

trehalose production from unutilized maltose (Liu et al., 2019b). Pglv, the maltose utilization promoter ofB. subtilis, was
mutated for the improved activity of trehalose synthase (Liu et al., 2019a). Tran et al. have integrated the Pgracpromoter in
B. subtilisto improve theb-galactosidase activity (Tran et al., 2020).Lactococcus lactisexpressing interleukin 10 (IL-10)
lost its sensitivity and viability during freeze-drying and gastric conditions, respectively. Termont et al. have integrated the
trehalose synthase gene fromE. coliin the IL-10 expressingL. lactis. This enhanced the production of trehalose in the
recombinant strain, which helps it to retain its sensitivity and viability (Termont et al., 2006). Trehalose 6-phosphate
phosphatase fromPropionibacterium freudenreichiiwas incorporated inL. lactisfor enhancing trehalose production
under acidic conditions (Carvalho et al., 2011).
The overexpression of four ATP-binding cassette transporter genes rbsA, rbsB, msmK, and dppA signiﬁcantly in-
creases the activity ofL. lactisin acidic conditions (Zhu et al., 2019). The asparaginase production was improved by
expressing the P43 promoter and optimizing with Ribosome-binding-site (RBS) sequences inB. subtilis(Li et al., 2019).b-
Mannanase gene fromBacillus clausii, an alkaliphilic strain, was cloned and expressed inB. subtilis. The recombinant
strain exhibited better polysaccharide degradation (Zhou et al., 2018). Liu et al. have engineeredB. subtilisby deleting
eight proteases, sigma factor F, and surfactin. This new recombinant strain was transformed with a plasmid containing
PspovGpromoter fromBacillus naganoensisresulting in the enhanced production of pullulanase enzyme (Liu et al., 2018).
Alanine dehydrogenase ofBacillus sphaericuswas cloned inL. lactisto increase the production of
L-alanine, which is
used as a food sweetener (Hols et al., 1999). Malic acid is a crucial constituent used in food industries for improving
sweetness. Sun et al. have developed a recombinant strain ofL. lactiswith enhanced production of malic acid by deleting
lactate dehydrogenase and incorporating malate dehydrogenase fromActinobacillus succinogenesand pyruvate carbox-
ylase fromL. lactis(Sun et al., 2020). The lactate dehydrogenase deleted strains ofL. lactisoverexpressing mannitol 1-
phosphate dehydrogenase gene ofL. plantarumand the mannitol 1-phosphate phosphatase gene ofEimeria tenellawere
found to effectively utilize glucose for the synthesis of mannitol (Gaspar et al., 2004). Probiotic bacteria are engineered for
the production of vitamins riboﬂavin and folate. A mutant strain ofL. lactiswas identiﬁed by Sybesma et al. through
directed mutagenesis. This mutant is characterized by producing riboﬂavin and the same strain overexpressing GTP
cyclohydrolase I synthesizes folate (Sybesma et al., 2004).
TABLE 2.2Engineered probiotic strains and its application.dcont’d
Probiotics Method Application References
B. subtilis Improving P43 promoter and
optimizing with synthetic RBS
sequence
Improved L-asparaginase production Li et al. (2019)
B. subtilis b-Mannanase gene fromBacillus
clausii
Enhanced enzyme activity Zhou et al. (2018)
B. subtilis Expressing PspovGpromoter from
Bacillus naganoensisin recombinant
strain with deletions proteases
(aprE, nprE, nprB, epr, mpr, bpr, vpr,
and wprA), sigma factor F (spoIIAC),
and surfactin (srfAC)
Pullulanase production Liu et al. (2018)
L. lactis Overexpressedalanine dehydrogenase
ofBacillus sphaericus
Synthesize
L-alanine Hols et al. (1999)
L. lactis Lactate dehydrogenase was deleted;
malate dehydrogenase and pyruvate
carboxylase overexpressed
Malic acid synthesis improved Sun et al. (2020)
L. lactis LDH gene deletion followed by
overexpressing mannitol 1-phosphate
dehydrogenase ofLactobacillus
plantarumand mannitol 1-phosphate
phosphatase gene ofEimeria tenella
Improved mannitol production Gaspar et al. (2004)
L. lactis Directed mutagenesis and overexpres-
sion of GTP cyclohydrolase I
Synthesize B2 and B11 vitamins Sybesma et al. (2004)
18Value-Addition in Food Products and Processing Through Enzyme Technology

4. Genetically modiﬁed food: current perspectives and future prospects
The practice of genetic engineering had been carried out by humans from time immemorial, particularly by the crossing of
organisms carrying enviable qualities (Karalis et al., 2020). This mainly helps in raising new organisms giving better yield
and disease tolerance. With the advancements in science, genetic engineering techniques have improved, which can
implement modiﬁcations even at the DNA level to develop genetically modiﬁed organisms and food. Industries have
beneﬁted from genetically engineered organisms, signiﬁcantly enhancing yield in minimum time with less waste and
thereby reducing the cost of production. Food industries widely use genetically modiﬁed microbes and their enzymes for
enhanced food production and the preparation of fortiﬁed/functional foods with health beneﬁts. Probiotic microbes and
their enzymes have vast applications in various sectors of food industries. However, naturally occurring probiotics have
many limitations that hinder their wide usage in the industry and consumption.
The current perspective is to modify these probiotics to overcome the drawbacks and to avail of the beneﬁts. As
discussed in earlier sections, engineered probiotics are tolerant to harsh industrial conditions of industries. Furthermore, the
same is found to survive better in gastric and bile juice environments while consumed by humans. The various tools
adopted to modify these microbes include constitutive and induced expression of genes of interest, optimizing signal
peptides for better secretion, anchoring speciﬁc proteins on the cell surface, plasmid assisted modiﬁcation, crossover,
deletion and CRISPR/Cas9 techniques, and directed and transposon mutagenesisdthe genome modiﬁcation results either
in forming cis or transgenic organisms. Cis-modiﬁcation usually has DNA from the same species, while trans-modiﬁcation
has DNA from different species. Even though there are apprehensions for all types of genetic modiﬁcations, the cis type
modiﬁcation is considered to be safer.
The gut microbiome plays a signiﬁcant role in managing health, and genetically modiﬁed probiotics can act as
antagonists and immune modulator in various diseased conditions. Since this triggers an immune response, some pop-
ulations may experience severe allergies after consumption (Mazhar et al., 2020). In general, probiotic microorganisms are
considered safe and have been given the generally regarded as safe status by WHO (Zommiti et al., 2020). However, the
selection, identiﬁcation, and modiﬁcation of probiotics for industrial food application should be vigilant and strict
experiments must be performed to conﬁrm its safety. The genetically modiﬁed organisms are reported to create an
imbalance in the environment as the wild type. Even though the usage of genetically modiﬁed organisms is controversial,
in future, the use of these organisms will be unavoidable to meet the food requirements of the population. With the
reduction in land-water sources and maintaining nutrition status, these engineered organisms should be used. Hence, new
molecular biology techniques should be identiﬁed to implement the desired traits without affecting humans and the
environment.
Acknowledgment
Raveendran Sindhu acknowledges Department of Science and Technology for sanctioning a project under DST WOS-B scheme.
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Zuo, F., Yu, R., Feng, X., Khaskheli, G.B., Chen, L., Ma, H., et al., September 6, 2014a. Combination of heterogeneous catalase and superoxide dismutase
protectsBiﬁdobacterium longumstrain NCC2705 from oxidative stress [Internet] Appl. Microbiol. Biotechnol. 98 (17), 7523e7534. Available from:
http://link.springer.com/10.1007/s00253-014-5851-z.
Zuo, F., Yu, R., Khaskheli, G.B., Ma, H., Chen, L., Zeng, Z., et al., September 2014b. Homologous overexpression of alkyl hydroperoxide reductase
subunit C (ahpC) protectsBiﬁdobacterium longumstrain NCC2705 from oxidative stress [Internet] Res. Microbiol. 165 (7), 581
e589. Available
from:https://linkinghub.elsevier.com/retrieve/pii/S0923250814000886.
Enzymes in probiotics and genetically modiﬁed foodsChapter | 223

Chapter 3
Extremozymes in food production and
processing
A´ngel Ferna´ndez-Sanroma´n and M. A´ngeles Sanroma´n
Department of Chemical Engineering, University of Vigo, Vigo, Spain
1. Introduction
Food products represent complex mixtures of organic and mineral compounds. As such, reproducibly transforming raw
livestock and agricultural feedstock into high-quality products crucially depends uponﬁne-tuning their chemical
composition during this transformation process (Compton et al., 2018). Similarly, improved conservation depends on
preservation of the physicochemical properties of the product for longer periods of time. A plethora of different operations
are used in the food industry for these production and processing purposes, e.g., pasteurizing, freezing, drying,ﬁltering,
and cooking (Chemat et al., 2017). Many of these processes require heating, cooling, cleaning, and sterilizing systems
operating at large scale, making of the food processing industry one of the largest consumers of energy and water resources
in the manufacturing sector (Herrando et al., 2020;Meneses et al., 2017;Xu and Szmerekovsky, 2017).
Food production and processing is a dynamic area of research aimed at meeting with current customer demands: faster
production rates, expansion of product shelf life, higher quality, faster and more convenient food (Pereira and Vicente,
2010). Furthermore, the development of new green techniques has become a priority, in line with the agreement reached by
195 nations in 2015 at the COP21 climate conference in Paris (European Commission, 2016). These challenges have led to
a myriad of emergent green foodeprocessing methods that outperform conventional approaches in terms of nutritional
value retention, production efﬁciency, and time and energy consumption. Examples of such innovations are ultrasound-
assisted processing, supercriticalﬂuid extraction and processing, microwave processing, controlled pressure process,
and pulse electricﬁeld (Chemat et al., 2017).
Another trend in this environmentally friendly context is the increasing application of enzymes, biodegradable
proteinaceous catalysts that speed up chemical reactions with minimal by-product formation and the speed, efﬁciency,
substrate, and product selectivity required by industrial processes (Ravindran and Jaiswal, 2016). Indeed, under mild
conditions, enzymatic reactions are favored over competing chemical treatments (James and Simpson, 1996).
These unique properties are the root of a global market that reached $9.9 billion in 2019 and is estimated to expand to
$17.2 billion by 2027, which represents a 7.1% CAGR (Compound Annual Growth Rate) between 2020 and 2027 (https://
www.grandviewresearch.com/industry-analysis/enzymes-industry). The food industry is a large segment of this market,
with a value of $2 billion in 2018 and expectations to reach $3 billion by 2026 considering its CAGR of 5.6% (https://
www.alliedmarketresearch.com/food-enzyme-market). Overall, microbial enzymes represent 35% of the industrial
enzymes, between the 50% represented by fungi and yeast enzymes and the 15% represented by animal and plant enzymes
(Liu and Kokare, 2017). Microbial enzymes are highly valued, particularly in the food industry, because they are more
stable and easily produced and puriﬁed than their animal and vegetal counterparts (Mishra et al., 2020). This is proved by
the $2 billion current value of the food industry enzyme market, underpinned by the extensive application of enzymatic
processes to produce dairy, baking, beverages, brewing, fats, oils, meat, and functional foods (Zhang et al., 2018). Here,
enzymes can be introduced as processing aids in a wide range of operations (e.g., coagulation, ripening, cell rupture, and
hydrolysis), where they contribute to improve the quality, appearance, nutritional value, freshness, aroma, aspect, and
conservation of the food products (Ermis, 2017).
Value-Addition in Food Products and Processing Through Enzyme Technology.https://doi.org/10.1016/B978-0-323-89929-1.00032-9
Copyright©2022 Elsevier Inc. All rights reserved.
25

Furthermore, the growing prospects for this food processing enzyme market (5.6% CAGR) encourage active research
of novel enzymes and enzymatic technologies better suited to particular food processing applications. Such novel enzymes
will offer new functions or properties: from catalysis of new biochemical reactions to improved pH and operational
temperature range, high salinity, or solvent tolerance. Bacteria represent a vast source of functional biodiversity wherein to
look for catalysts with such properties. Fortunately, the advent of improved omics and bioinformatics and its blending in
approaches such as metagenome screening and genome mining are now making possible to study this biological diversity
with unprecedented depth, breadth, and throughput (Zhang and Kim, 2010;Adrio and Demain, 2014).
Nevertheless, a subset of microorganisms, extremophiles, might be of particular value for industrial processes,
considering that they live under extreme physicochemical conditions to which their enzymes are adapted, contrary to the
common mild conditions required for optimal standard enzymatic activity (Woodley, 2013;Elleuche et al., 2014)
(Fig. 3.1).
In this chapter, extremophiles and their biodiversity areﬁrstly introduced, including the adaptations that these mi-
croorganisms and, particularly, their enzymes (extremozymes), present. Afterward, several food extremozymes such as
lipase, esterase, carbohydrase (amylase and xylanase), and protease activities are revised.
2. Extremophiles
Extremophilic microorganisms optimally grow under environmental conditions that would be inhospitable for most living
forms. The range of environmental variables to which extremophiles have successfully adapted include: low (ﬁ2to20
ﬂ
C)
and high (55 to 121
ﬂ
C) temperatures, alkalinity (pH>8) and acidity (pH<4), high pressure (>500 atm), salinity (2e5M
NaCl or KCl), radiation (UVR resistance>600 J/m), heavy metals (arsenic, cadmium, copper, and zinc), and scarce
nutrients (Dumorné et al., 2017). Extremophiles are a functionally diverse group classiﬁed according to their preferred
environmental conditions (Raddadi et al., 2015). For example, thermophiles optimally grow at temperatures above 55
ﬂ
C,
FIGURE 3.1Wider scope of extremozyme catalysis.Boxes include ranges where the different functional categories of enzymes are operative. Salt
tolerance of halophilic enzymes is illustrated by showing the concentrations of KCl and NaCl wherein they can operate.Figure based on Elleuche, S.,
Schröder, C., Sahm, K., Antranikian, G., 2014. Extremozymes - biocatalysts with unique properties from extremophilic microorganisms. Curr. Opin.
Biotechnol. 29, 116e123.
26Value-Addition in Food Products and Processing Through Enzyme Technology

while psychrophiles live at temperatures below 15
ﬂ
C; xerophiles live in low water environments; halophiles are suc-
cessfully adapted to high salinity; acidophiles and alkaliphiles live in environments with pH<4 and pH>8, respectively;
and piezophiles thrive under high pressure. The required adaptations to these extreme environmental conditions are not
necessarily mutually exclusive. Thus, some microorganisms have evolved to withstand more than one extreme environ-
mental variable. For example, thermoacidophiles thrive in conditions of both high temperature and acidity.
Cellular function critically depends on the stability and functionality of macromolecules. The physical and chemical
environmental factors to which extremophiles are exposed demand biochemical modiﬁcations tailored to the surrounding
conditions. The example of thermophiles is illustrative of this point. Temperatures above 55
ﬂ
C trigger a cascade of
biochemical modiﬁcations: increase of membraneﬂuidity, denaturation of proteins and nucleic acids, and changes in the
solubility of gases (e.g., O
2and CO2). Adaptive mechanisms of thermophiles go in the line of preserving stability and
functionality of their nucleic acids, proteins, and cytoplasmic membrane under such conditions. This includes a uniquely
adapted structure and biochemical composition of the cytoplasmic membrane, chemical and structural modiﬁcation of their
proteins, synthesis of heat shock proteins, and higher DNA and RNA stability (Morozkina et al., 2010). Furthermore,
nutritional requirements adapted to the typical nutrient scarcity of extreme environments and an efﬁcient nucleic acid repair
system are survival mechanisms shared with other types of extremophiles (Schröder et al., 2020). However, many of these
mechanisms are not yet fully understood at the most basic biochemical level.
For industrial application, one of the most interesting adaptive mechanisms of extremophiles is the unique structure and
functionality of their enzymes. Extremozymes have evolved in such a way that they can remain functionally active under
harsh conditions where enzymes of other organisms would have aggregated, precipitated, or denatured (Kumar et al.,
2018). Unique functional properties of extremozymes ultimately rely on their protein structure. This explains observations
of how changes in just one amino acid can have a profound effect in adaptation to harsh conditions if that change abruptly
transforms the previous protein structure (Sharma et al., 2017). Many structural modiﬁcations are involved in extremozyme
stability and functionality, and they differ depending on the particular conditions to which they are adapted. For example,
thermophilic enzymes show dense and compact structures, with a tight hydrophobic core and superior accessibility to the
active site (Han et al., 2019;Elleuche et al., 2014). Meanwhile, xerophilic enzymes are able to compete for hydration in
low water environments due to their greater surface charge and increase molecular motion, which contribute to the
maintenance of a tight hydration shell (Karan et al., 2012). This shows that the structural mechanisms underpinning
extremozyme stability are different in distinct functional groups of extremophiles. Furthermore, it proves that the structures
of such enzymes have been speciﬁcally optimized by natural selective pressures to catalyze biochemical reactions under
certain harsh conditions, hence representing more suited biocatalysts for many industrial processes than their
nonextremophilic counterparts (Fig. 3.2).
Therefore, in the context of food industry chemical processes, extremozymes might represent naturally optimized
catalysts with unparalleled properties and performance. Among these enzymes, cold-tolerant, acid-tolerant, alkali-tolerant,
and salt-tolerant extremozymes are the most promising, with application in a variety of processes. Cold-tolerant, acid-
tolerant, alkali-tolerant, and salt-tolerant extremozymes stand out as the most used in industrial applications. In the
food industry, extremozymes can be useful in a variety of processes. For instance, cold-active enzymes are operative in
processes performed at low temperatures and can be easily inactivated by mild heat treatment (Zhang et al., 2018).
Thermophilic enzymes can be useful in high-temperature processes, such as fermentation of grains to produce distilled
spirits or the hydrolysis of starch for glucose and fructose production (Akanbi et al., 2020). Another example would be the
application of salt-tolerant enzymes in the production of salty foods such as pickles, sauerkraut, and Asianﬁsh sauce
(Daoud and Ben Ali, 2020). A more complete description of the applications of extremozymes is presented in the following
sections, but these examples clearly illustrate how extremozymes meet many industrial needs while representing a more
efﬁcient and environmentally friendly alternative than chemical catalysts.
However, we are far from a complete catalogue of extremophiles and their corresponding extremozymes. For this
purpose, the unique environmental conditions wherein these organisms grow are difﬁcult to replicate and hinder the
application of classical microbiology techniques. Due to these limitations, the advent of new technologies has been
transformative for extremozyme research, making technically possible to bioprospect for new enzymatic activities in
environments with conditions analogous to those characterizing a particular industrial food process. Among these
advances, sequence and function-based metagenomics, as well as improved direct enzyme exploration approaches, stand
out (Fig. 3.3):
eSequence-based metagenomics screening and genome miningheavily rely on sequencing technologies and bioinfor-
matics data processing pipelines toﬁnd the presence of DNA/RNA coding for the target enzyme (Lorenz and Eck,
2005;Goodwin et al., 2016). The main advantage of these techniques for extremophile research is that they allow
Extremozymes in food production and processingChapter | 327

recovery, analysis, and interpretation of the genetic material directly extracted from niches without microorganism
cultivation, problematic for extremophiles (Krüger et al., 2018). With the DNA/RNA sequence of all the microorgan-
isms within an environment, it is possible to query for homologous sequences to already described enzymes. Hits with
signiﬁcant homology are likely to show an analogous enzymatic function and hence they can be considered candidates
for further exploration given their potentially improved adaptation to harsh physicochemical conditions. Despite its
throughput and power, a main caveat of this approach is its inherent bias to the discovery of already described enzy-
matic activities.
eFunction-based metagenomics assaysare a solution toﬁnd the genes coding for novel extremozymes, without simi-
larity to previously discovered enzymes (Ngara and Zhang, 2018). Different screening strategies are performed for
this purpose, mainly phenotypic detection, heterologous complementation of host strains, and induced gene expression
(Madhavan et al., 2017). The main problem is that functional metagenomics screening is fraught with technical chal-
lenges and hence it is not a robust technology (Uchiyama and Miyazaki, 2009). Furthermore, once found a gene coding
for a novel extremozyme, it is usually required to improve its performance by protein engineering (Lutz and Iamurri,
2018) and/or directed evolution (Packer and Liu, 2015) before it can be efﬁciently implemented in industrial
applications.
eDirect enzyme explorationimplies: (i) designing a functional assay that reports the presence and activity of a target
enzymatic function under the desired physicochemical conditions and (ii) applying it to samples from relevant environ-
mental niches (in this case, extreme environments) that will harbor a wide variety of microorganisms (Boehmwald
et al., 2016). The advantage of this technique is that only those enzymes with feasible industrial application are
detected. This, however, requires screening through a robust enzymatic assay. Properly designing and carrying out
FIGURE 3.2Unique extremozymes structural and chemical adaptive mechanisms.Boxes highlight the adaptive mechanisms characteristic of each
functional extremophile category. The overall effect is presented to highlight the global result of all these modiﬁcations.
28Value-Addition in Food Products and Processing Through Enzyme Technology

such analyses is time consuming. Consequently, theﬁeld is moving forward by trying to optimize this approach
through high-throughput screening pipelines similar to those used by the pharmaceutical industry toﬁnd promising
drugs (Sarmiento et al., 2015).
The three previous approaches have already been successful inﬁnding extremozymes with interesting applications in
the food industry, as comprehensively revised byKhan and Sathya (2018). Nevertheless, despite the promising applica-
bility of the constantly expanding catalogue of extremozymes, few of them have actually reached the enzyme market
(Sarmiento et al., 2015). One major gap is that production and puriﬁcation of these enzymes from their natural hosts are
quite challenging. Extremophiles are characterized by low growth requirements and unconventional growing conditions
that make microorganism cultivation operationally challenging and in many cases quite expensive. For example, this
would be the case of cultivation of hyperthermophiles, with enormous heating requirements (Aguirre et al., 2018).
FIGURE 3.3New approaches for extremozyme discovery.Sequence-based metagenomics, genome mining, function-based metagenomics, and direct
enzyme exploration require a sample from an extreme environment with conditions similar to those for which an optimized biocatalyst is being looked for.
ORFs, Open reading frames.
Extremozymes in food production and processingChapter | 329

Therefore, bridging the gap to homologous expression of extremozymes (i.e., producing and purifying these enzymes
by extremophile cultivation) will still require to improve extremophile culture efﬁciency and simplify downstream
processing, thus reducing costs and energy consumption to the required levels (Deive and Sanromán, 2017). The current
solution to this problem for industrial-scale extremophile production is heterologous expression, i.e., cloning of the target
extremozyme gene in mesophilic hosts and production and puriﬁcation of the enzyme in these microorganisms. The most
common hosts includeEscherichia coli,Saccharomyces cerevisiae,Bacillus subtilis, andPichia pastoris(Wang et al.,
2019;Sanchez et al., 2019), microorganisms for which cultivation and cloning techniques are optimized. This standardized
approach also has some important pitfalls that can result in insufﬁcient and/or nonfunctional extremozyme production.
First, differences in codon usage could result in low protein yield (Khan and Patra, 2018). And second, expression in a
different host and environmental conditions could also result in improper protein folding and hence extremozyme
disfunction (Elleuche et al., 2014).
In the next sections the diversity of extremophilic microorganisms able to produce amylase, xylanase, lipase, esterase,
and protease and their potential application for food production and processing are reviewed.
3. Amylase
a-Amylases belong to the carbohydrase group, which includes other different enzymes such as glucoamylases, glucosidases,
lactase, pectinase, galactosidases, invertases, xylanase, and inulinases. These carbohydrases have in common their ability
to catalyze the breakdown of carbohydrates into simple sugars. Thus,a-amylase (a-1,4-D-glucan 4-glucanohydrolase,
EC 3.2.1.1) is an endo-acting hydrolase that randomly cleaves thea-1,4-glycosidic linkages in starch (Cheng et al., 2017).
Since the 1950s, fungal amylases have become a suitable alternative to conventional acid hydrolysis of starch to
manufacture sugar syrups containing speciﬁc mixtures of sugars that could not be produced by chemical methods. Besides,
amylase has application as brewing and baking agents. In baked goods (bread, cake, biscuits, etc.), starch is the main
constituent, which may play a role as emulsiﬁer, thickener, gelling agent, and water binder. Thus, the presence of
a-amylase in doughs during the baking process entails the hydrolysis of starch to yield small dextrins that can be sub-
sequently metabolized by the yeasts at the fermentation, affecting the viscosity, improving the textures, and loaf volumes
(Deive and Sanromán, 2017). In the enzymatic liquefaction and sacchariﬁcation of starch, it is requested to operate at high
temperatures, being adequate the use of thermostable amylase. It is recognized thata-amylases represent around 30% of
the commercial enzyme market worldwide (Cheng et al., 2017). However, the wide number of industrial processes in
which the amylases could be used extend the demand of novel extremophilic amylases.
Nowadays, the industrial source ofa-amylase is the bacteriaBacillus licheniformis, and the production of thermoto-
lerant or thermophilic amylases by different microorganisms has been well documented (Table 3.1). In addition, the use of
cold-adapted amylase is also beneﬁcial for its high speciﬁc activities and efﬁciency at low and moderate temperatures
(D’Amico et al., 2006). Several investigations have been conducted to isolate microorganisms able to produce cold-
adapted amylases with application to starch liquefaction. For example, the bacteriumAeromonas veroniiNS07 was
isolated from farm soils being able to produce a psychrophilic amylase. Several factors were studied in the cold-adapted
amylase production, conﬁrming that sucrose’s addition enhances the secretion of amylase activity compared to other
carbon and nitrogen sources. This enzyme exhibited high amylolytic activity in a temperature range from 0 to 60
ﬂ
C with
maximum activity at 10
ﬂ
C. Besides, this cold-adapted amylase was active in a pH range of 4.0e6.0. However, the maximal
stability was obtained at a pH of 4 in sodium citrate buffer keeping the activity after 1 day, which is ideal for performing
the starch bioprocessing (Samie et al., 2012).
As mentioned before, amylases used at industrial scale come mainly from bacterial sources (around 83% of the total
production) due to a higher thermal stability. Nevertheless, thermophilic amylases’production extends their application in
several sectors such as baking food, where in 2015 the market size was near 70,000 tons (Machado de Castro et al., 2018).
Based on amylases’industrial application, thermostable and acid stablea-amylases, active at the temperatures of gelati-
nization and liquefaction (around 105 and 85
ﬂ
C, respectively), are desired to reduce these processes’costs. Therefore, there
is a need and continual search for more thermoactive and thermostable amylases to meet the requirements for speciﬁc
applications in the food industry (Dey et al., 2016;Sidhu et al., 1997). In the literature, numerous examples of thermophilic
and hyperthermophilic microorganisms adapted to thrive at high temperatures and isolated from different hot environments
are reported:Bacillus thermoleovorans,Bacillus amyloliquefaciens,B. licheniformis, Geobacillus stearothermophilus,
Thermusspp.,Thermococcussp.,Pyrococcus furiosus,Pyrococcus woesei, etc. All of these are able to produce new and
robusta-amylases active at high temperature (until 100
ﬂ
C) (Cheng et al., 2017;Duy and Fitter, 2005;Koch et al., 1991;
Wang et al., 2008)(Table 3.1).
30Value-Addition in Food Products and Processing Through Enzyme Technology

TABLE 3.1Several examples of enzymes produced from extremophilic microorganisms and the optimal operational
conditions.
Extremozyme Microorganism
Temperature
8C pH Additional comments References
Amylase Aeromonas veroniiNS07 10 4 Amylase activity enhanced
the presence of metal ions
inhibited by EDTA, urea,
and SDS
Samie et al. (2012)
Amylase Pyrococcus woesei 100 6.5 e7.5 Hyperthermophilic enzyme
with activity after auto-
claving at a pressure of
2 bars at 120

C for 5 h
Koch et al. (1991)
Amylase Pyrococcus furiosus 100 5.5 Thermal activation of the
enzyme is due to slight
changes in interdomain
distances
Laderman et al.
(1993)
Amylase ThermococcusHJ21 95 5 Stable enzyme in a broad
pH range from pH 5.0 to
9.0
Wang et al. (2008)
Amylase Bacillus
stearothermophilus
70e80 5.0 e6.0 Enzyme stabilized by
Ca
2þ
,Na
þ
, and bovine
serum albumin
Vihinen and
Mantsala (1990)
Amylase BacillusstrainHUTBS62 90 4.4 Enhanced activity with
Co
2þ
,Cd
2þ
,Mn
2þ
, and
Mg
þ2
and inhibited by
Ca
2þ
,Zn
2þ
, and Cu
þ
Al-Quadan et al.
(2011)
Amylase Pyrococcussp. ST04 95 5.0 Gene from Pyrococcussp.
ST04 cloned and expressed
inEscherichia coli
Jung et al. (2014)
Amylase Bacillus subtilisDR8806 70 5.0 B. subtilisDR8806 in
E. coli
Emtenani et al.
(2015)
Amylase Alkalilimnicolasp. NM-
DCM-1
55 10.5 Immobilized in agar-agar
double its half-life at
50e60

C
Mesbah and Wiegel
(2018)
Xylanase Thermus brockianus 95 6 Xylanase-encoding gene
Xyn10 expressed inE. coli
BL21
Blank et al. (2014)
Xylanase Aureobasidium pullulans
NRRL Y-2311-1
50 4 Provides increased water
absorption, development
time and stability of the
dough, reduced dough
softening degree, and mix-
ing tolerance index
Yegin et al. (2018)
Xylanase Bisporasp. MEY-1 95 4 Xylanase-encoding gene
Xyl10E expressed inPichia
pastorisGS115
Wang et al. (2017)
Xylanase Dictyoglomus
thermophilum
80 7.0 e7.5 Expressed inE. coliXL.
High pressure
100e300 MPa increase
enzyme reaction
Li et al. (2015)
Xylanase Microcella alkaliphila
JAM-AC0309
65 8 No effects of metals only
inhibited by Hg
2þ
and
Cu
2þ
ions
Kuramochi et al.
(2016)
Continued
Extremozymes in food production and processingChapter | 331

TABLE 3.1Several examples of enzymes produced from extremophilic microorganisms and the optimal operational
conditions.dcont’d
Extremozyme Microorganism
Temperature
8C pH Additional comments References
Xylanase Thermus thermophilus
F1208
75 6.5 Xylanase-encoding gene T-
Xyn expressed inE. coli
Wu et al. (2019)
Xylanase Streptomyces thermovulga-
risTISTR1948
65 6.5 Stable across a broad pH
range 4.0e11.5
Boonchuay et al.
(2016)
Xylanase Microbial community in a
hot spring sediment from
the Lobios Hot Spring
located in the province of
Ourense, Spain
80 6.6 Xylanase-encoding gene
XynA3 expressed inE. coli
BL21
Knapik et al. (2019)
Xylanase Ruminococcus ﬂavefaciens55 6.0 Xylanase-encoding gene
XylCMS expressed in
E. coli. Activity stimulated
by NaCl 1e5M
Ghadikolaei et al.
(2019)
Lipase Alkalibacillus salilacus40 8.0 High salt concentrations
until 30% NaCl
Samaei-Nouroozi
et al. (2015)
Lipase Xanthomonas oryzae 70 9.0 High stability in methanol,
n-hexane, n-heptane, chlo-
roform, and toluene
Mo et al. (2016)
Lipase Bacillus licheniformis 90 9.0 Enhanced activity with
Ca
2þ
,Cd
2þ
, and Zn
þ2
and
some organic solvents
Ugras‚(2017)
Lipase Bacillus atrophaeus 70 9.0 Optimal activity at 4M
NaCl
Ameri et al. (2017)
Lipase Rhodotorulasp. 35 8.0 Enzyme optimal stability
at20

C and pH 5.0
Maharana and Singh
(2018)
Lipase Pseudomonassp. 10 7.0 Stimulated by 50 Mm Cu
þ2
and various organic
solvents
Maharana and Ray
(2015)
Lipase Anoxybacillussp. 55 9.5 Bivalent metal ions, PMSF,
CMC, NBS, and SDS
inhibited enzyme activity
Burcu Baki (2017)
Esterase Ureibacillus
thermosphaericus
70e80 8.0 High stability in organic
solvents and inhibition by
PMSF
Akanbi et al. (2020)
Esterase Geobacillus
thermodenitriﬁcans
60 7.0 e8.5 b-Mercaptoethanol, DTT,
n-hexane, and Triton
X-100 had an activating
effect. Other organic
solvents had a negative
effect
Curci et al. (2019)
Esterase Unidentiﬁed eAntarctic
desert soil
20 11.0 Optimal activity recorded
toward p-nitrophenyl
propionate
Hu et al. (2012)
Esterase Erythrobacter seohaensis60 10.5 Halotolerant activity e
highest hydrolytic activity
in 0.5M NaCl
Huo et al. (2017)
Esterase Janibactersp. 80 8.0 e9.0 Strong activation by
mixture of Na
þ
, and K
þ
Castilla et al. (2017)
32Value-Addition in Food Products and Processing Through Enzyme Technology

Into the bargain,a-amylases should be acidophilic besides thermostable to fulﬁll industrial requirements in starch
bioprocessing. During sacchariﬁcation, liqueﬁed starch is converted into glucose or maltose using amylase and other starch
enzymes in a pH range of 4.2e4.0, conditions where most of the currently used industrial amylases demonstrate low
amylolytic activity and stability (Al-Quadan et al., 2011). To overcome these drawbacks, the starch slurry pH must be
raised to 5.7e6.0, and CaCl
2is added (Wang et al., 2008). However, these issues have a negative effect on the following
conversion stages of the liqueﬁed product. Thus, pH must be returned to about 4.5 for glucoamylases to catalyze the
conversion into glucose efﬁciently and promote the formation of high pH by-products such as isomalitol (Wang et al.,
2008). In addition, Ca
2þ
ions are a strong inhibitor of glucose isomerase, and these ions should be removed before the
isomerization of glucose to fructose. These facts request additional steps such as pH adjustments or ion-exchange process
that increases the cost of high-fructose syrup production.
Consequently, the identiﬁcation and exploitation of thermoacidophilic Ca-independent amylases are of great demand in
the starch food sector in order to reduce production costs and simplify the process (Emtenani et al., 2015). In this sense,
severala-amylases have been puriﬁed and characterized, such as a Ca-independenta-amylase produced byBacillussp.
KR-8104 isolated from the soil that exhibited the maximum activity at a broad range of pH (4.0e6.0) at high temperatures
(75e80), maintained the activity with/without Ca
2þ
ions, thus avoiding the pH adjustments, and the addition/removal of
Ca
2þ
ions steps, which is a great advantage for their utilization at industrial scale (Sajedi et al., 2005).
Although the screening of thermoacidophilic Ca-independent amylases following conventional isolation methods is
still an active researchﬁeld, the natural isolates present several limitations for their commercial use, for example, their low
productivity and in several cases, high production costs related to extreme operational conditions (high temperatures that
reduce the oxygen solubility). Therefore, as already described inSection 2above, the alternative is the production by
bioengineering procedures using mutagenesis and/or recombinant DNA technology (Sidhu et al., 1997).
TABLE 3.1Several examples of enzymes produced from extremophilic microorganisms and the optimal operational
conditions.dcont’d
Extremozyme Microorganism
Temperature
8C pH Additional comments References
Esterase Dictyoglomussp. 75 8.0 Stable in media with high
content of different organic
solvents
Zarafeta et al. (2016)
Protease Thermomonas haemolytica 55 9.0 Signiﬁcant inhibition by
PMSF, partial by EDTA,
EGTA, SDS, urea, and
Zn
2þ
and Cu
2þ
. Activation
by Ca
þ2
,Mg
2þ
, and Mn
2þ
Perfumo et al. (2020)
Protease Aeribacillus pallidus 60 9.0 Complete inhibition by
PMSF and DIFP
Mechri et al. (2017)
Protease Bacilluslicheniformis 70 10.0 Elevated organic solvent
resistance
Hadjidj et al. (2018)
Protease Planococcussp. 35 10.0 Retention of activity from
5to35
ﬂ
C
Chen et al. (2018)
Protease Pseudoalteromonas arctica40 9.0 Displayed proteolytic
activity from 0 to 40
ﬂ
C.
Activity enhanced by
Cu
þ2
. No negative effect
by the presence of surfac-
tants and other chemicals
Park et al. (2018)
Protease Geobacillus toebii 95 13.0 Optimum activity at 30%
NaCl. Activity stimulated
by Ca
2þ
,Mg
2þ
, DTNB,b-
mercaptoethanol, and SDS
Thebti et al. (2016)
Protease Melghiribacillus
thermohalophilus
75 10.0 Inhibition by DFP and
PMSF
Mechri et al. (2019)
Extremozymes in food production and processingChapter | 333

Table 3.1shows several examples ofa-amylases produced by genetic manipulation using conventional mesophilic
hosts such asE. coli, B. licheniformis, B. subtilis,orP. pastoris, avoiding a high temperature in the production bioprocess.
Thus, different mutated variants showed that replacement of Asn112 or Asn129 to aspartic acid and Asn312 to serine
increased the half-life markedly compared toBacillussp. KR-8104a-amylase previously mentioned. The removal of
deamidating residues enhanced the enzyme stability due to the role of electrostatic interactions such as salt bridges and
hydrogen bonding in protein thermostability (Rahimzadeh et al., 2012). In other studies, the thermostability ofa-amylase
was improved by deleting amino acid residues and/or by combination to substitutions employing site-directed mutagenesis
(Gai et al., 2018;Suzuki et al., 1989). For example, the properties ofa-amylase fromBacillus stearothermophiluswas
improved by deleting amino acids Arg179 and Gly180. This constructed deletion mutant exhibited higher half-life and acid
resistance with lower calcium requirements to maintaina-amylase activity that fulﬁlls the food industrial requirements
(Gai et al., 2018). As reviewed (Nielsen and Borchert, 2000), the stability at low pH can also be improved by conventional
protein stability engineering techniques such as helix capping, deamidating residues, and cavityﬁlling. Other alternatives
are the overexpression of molecular chaperones or signal peptide optimization. In this sense, by screening optimal signal
peptides, chaperones overexpression, and performing random mutagenesis strategy the heterologous expression of
B. stearothermophilusa-amylase inB. subtiliswas improved, being possible its production in a 3-L bioreactor (Yao et al.,
2019).
4. Xylanase
Xylanases (endo-b-1,4-xylanase, EC 3.2.1.8) are hydrolyases capable of breaking down hemicellulose into a mixture of
xylooligosaccharides (XOSs) of different sizes. Xylanases are useful for various food applications due to their ability to
reduce the viscosity of wheat, barley, corncobs, and other hard to digest foodstuffs. The addition of xylanase improves the
dough’s rheological properties, such as softness, ductility, and elasticity, and the bread’s speciﬁc volume and crumb
hardness (Camacho and Aguilar, 2003). From an economic standpoint, carrying out industrial processes at elevated
temperature has many advantages, including improved substrate solubility and decreased reaction mixture viscosity.
Generally, prior to xylanase application, substrates are at high temperature (higher 50
ﬂ
C) and/or acid/alkali pH. It is of
great interest to explore more active and stable xylanases under these conditions.
Xylanase can be produced by different living organisms such as bacteria, algae, fungi, yeast, protozoa, gastropods, and
arthropods (Saﬁtri et al., 2017). In the literature, it has been reported in several studies in which numerous hot springs and/
or hydrothermal systems were examined in order to obtain novel species, that were screened and the produced thermostable
xylanase characterized (Lischer et al., 2020).
Among thermophilic bacteria that can survive at high temperatures 50e65
ﬂ
C,B. licheniformisTS10, isolated from Hot
Springs Tanjung Sakti Lahat, has the highest xylanolytic index of 0.63. The fermentation process of thermostable xylanase
production byB. licheniformisTS10 has been optimized using oil palm empty fruit bunches as a substrate. In a temperature
range of 50e80
ﬂ
C, it was detected that xylanase activity produced increased with the temperature at pH 6 and the substrate
concentration of 4% (Saﬁtri et al., 2017).
Kumar and Shukla (2018)determined thatThermomyces lanuginosusVAPS24 showed the highest xylanase activity
from 15 thermophilic fungi isolated, achieving production levels around 131 U/mL at laboratoryﬂask scale. Its ther-
mostability at 80
ﬂ
C could be enhanced by the addition of polyols such as sorbitol, mannitol, or glycerol (average 1.4-fold),
which change the microenvironment of produced xylanase showing shielding effect against its deactivation. Besides, this
enzyme showed a broad pH stability range with optima of 5.0e9.0, making it attractive for application in acid/alkali
industrial bioprocesses. Similarly, puriﬁed xylanase from the thermophilic fungusT. lanuginosusCBS 288.54 that
showcased molecular weight ofw26.2 kDa exhibited an optimal temperature around 75
ﬂ
C with a neutral optimum pH
being also stable in a range of 6.5e10 (Li et al., 2005).
Xylanase production byT. lanuginosuswas lower in 5 L stirred tank bioreactor; however; the maximum level,
115.37 U/mL, was achieved after 3 days of culture, which reduces the production time and consequently the energy
consumption, resulting in a more cost-effective process. This reduction of activity level may be explained to the early
accumulation of toxic substances and operational problems related to aeration, agitation, and foaming. Thus, xylanase
activity decreased with an increase in agitation speed in the bioreactor, highlighting theﬁne compromise between the
oxygen and shear demand for homogenization of the cultivation broth, and the negative impact of shear on growth and
production. Four aeration levels from 0.5 to 2.0 vvm were evaluated in 3 L bioreactor, determining the aeration effect on an
acidophilic and extreme halophilic xylanase production byAureobasidium pullulansNRRL Y-2311-1. Results demon-
strated that xylanase production was higher when the aeration increases until a maximum operating at 1.5 vvm. However,
the production levels decreased considerably at 2.0 vvm due to the foam formation that generates insoluble substrate and
34Value-Addition in Food Products and Processing Through Enzyme Technology

cell mass retention into the foam (Yegin et al., 2017). Therefore, these problems could be attenuated applying strategies
tested with otherﬁlamentous fungi asTrichodermaby operation in fed-batch cultures or reducing mycelial fragmentation
by selecting gentler shear bioreactors (Patel et al., 2009).
Other extracellular alkali-thermostable xylanases were identiﬁed and puriﬁed from different fungi cultures using rice
straw as substrate.Thielaviopsis basicolaMTCC 1467 produced xylanase with a molecular mass of approximately 32 kDa
with high xylanolytic activity at pH 5 that remains the majority of its activity in a range of 5e10 (Goluguri et al., 2016).
ThermophilicHumicolaspp. are recognized for their capacity to produce alkali-thermostable xylanases; among them,
Humicola insolensY1 isolated from a forest soil sample, and exhibited maximum xylanolytic activity at pH 6.0e7.0 and
70
ﬂ
C(Du et al., 2013). However, the recombinant enzymes produced during heterologous expression inP. pastoris
showed higher stability, retaining more than 90% of activity, in an extensive pH range (5.0e10.0) at 60
ﬂ
C. This is due to
increased number of alkaline residues located on the protein surface and around the catalytic center of the xylanase (Mamo
et al., 2009).
Concerning xylanase’s application, several investigations conﬁrmed that its addition could play a signiﬁcant role in
dough rheology and bread quality. Thus, the addition into bread formulations of extremophilic xylanase produced by
A. pullulansNRRL Y-2311-1 (1.25 U/gﬂour) provided remarkable enhancements on dough handling led to signiﬁcantly
greater bread speciﬁc volume and minor crumbﬁrmness than the commercial counterparts improving the bread quality
(Yegin et al., 2018). On the other hand, the recombinant xylanases produced during heterologous expression inP. pastoris
exhibited better performance in mashing than Ultraﬂo (Novozyme) with a higher reduction of theﬁltration rate and
viscosity under simulated conditions (Du et al., 2013). These extremophilic xylanases provide an opportunity to extend
their use in different food industry processes compared to commercial and conventional xylanases produced by no
extremophilic microorganisms.
Thermostable xylanasesﬁnd application in the food industry to enhance theﬂavor of bread through starch and xylan
hydrolysis and subsequent release of sugars by combination with amylases. But thermostable xylanases can also enhance
textures, being this the reason for their application in the biscuit industry. Moreover, during the bread processing, xylanases
incorporate into the bread XOSs, which can act as prebiotics by enhancing the growth of several beneﬁcial gut micro-
organisms and simultaneously inhibiting proliferation of harmful pathogens present in the gastrointestinal tract (Verma
et al., 2019). The XOS properties play an increasingly important role in formulated foods, being the conversion by
xylanase the general method used for industrial XOS production. However, the implementation of this enzymatic process
at commercial scale is limited because of a number of disadvantages, including thermal and acid sensitivities. Therefore,
the demand for functional xylanases under high temperature and acidic pH is prevailing in this bioprocessing industry
(Wu et al., 2019).
Halophilic xylanases show immense applications in the processing of seafood and clariﬁcation of juices and wine
(Verma and Satyanarayana, 2020). Halophilic xylanase from the Arctic Mid-Ocean Ridge’s hot sediments was isolated and
puriﬁed, showing the ability to function under harsh conditions. This xylanase exhibited a pronounced salt-dependent
increase in activity up to 1 M NaCl, keeping its activity in the presence of NaCl at 80
ﬂ
C in a range of 1e3M
(Fredriksen et al., 2019). Other halophilic xylanases were considerably stimulated in a wide range (1e5 M NaCl). Thus,
these extreme xylanases are tolerant to salt and its presence in the medium stimulated their activity.
Several molecular techniques, such as genome-walking PCR (GWPCR), recombination methods, or site-directed
mutagenesis, are considered powerful tools for improving and adapting enzymes’characteristics to speciﬁc industry ap-
plications (Kumar and Shukla, 2018). In this context,Sunna and Bergquist (2003)used a modiﬁed GWPCR technique to
retrieve xylanase genes from a hot pool“KP”in Kuirau Park, Rotorua, New Zealand, environmental DNA sample, using
E. coliINVaF
0
One-Shot cells as the bacterial host for all DNA cloning and expression studies. They reported the cloning,
sequencing, and expression of a novel xylanase. Complete 16S rDNA nucleotide sequences of RFLP selected plasmids
revealed similarity to cultured and noncultured bacterial and archaeal representatives. As reported in previous in-
vestigations (Wang et al., 2012), xylanase thermostability could be further improved by mutagenesis strategies, such as
introducing an extra disulﬁde bridge into the N-terminal region ofT. lanuginosusxylanase.
Thus, there are numerous examples in which modern recombinant DNA technology had been used to increase
expression level and improve enzyme activity. Although xylanases could be expressed in both homologous and heter-
ologous host organismsP. pastorisis the optimal host for the expression of heterologous proteins including thermostable
xylanases; whileE. colishows limitations due to the formation of inclusion bodies (Basit et al., 2018). Nevertheless,
several xylanases have been successfully cloned and expressed in a heterologous hostE. coli.For example, xylanase
(Xyn10A)-encoding gene from the hyperthermophilic bacteriumThermotoga thermarumor xylanase (Xyn30Y5) gene
from alkaliphilicBacillussp. 30Y5 was successfully expressed inE. coli, exhibiting optimal enzymatic activities at
temperatures above 70
ﬂ
C(Lai et al., 2021;Verma et al., 2019).
Extremozymes in food production and processingChapter | 335

5. Lipases and esterases
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) can act on the lipidewater interface, which is crucial to have access to
their water-insoluble fatty substrates (Javed et al., 2018). Their main enzymatic activity is the hydrolysis of carboxylic ester
bonds, which sequentially breaks down triglycerides into diglycerides, monoglycerides, andﬁnally fatty acids and glyc-
erol. Nevertheless, lipases are versatile and can also catalyze other chemical reactions such as interesteriﬁcation, esteri-
ﬁcation, alcoholysis, acidolysis, and aminolysis. Similarly, esterases (EC 3.1.1.1) catalyze cleavage of the ester bond
between an alcohol and a carboxylic acid in water and its reverse reaction (esteriﬁcation or transesteriﬁcation) in
nonaqueous media. However, they differ with lipases on their kinetics and preference for short-chain fatty acids, which are
partially soluble in water (Delgado-García et al., 2018).
The versatility of both enzymes, together with their substrate speciﬁcity, chemo-, enantio-, and regioselectivity, ex-
plains that they are highly appealing to the food industry. In fact, the microbial lipase market grows at a 6% CAGR and it is
expected to reach $590.2 million by 2023 (Stergiou et al., 2013;Chandra et al., 2020). Food industry is an important
segment of such market, wherein lipases/esterases can be applied to the production of a wide range of foods including
meat,ﬁsh, egg, oil, dairy, baking, alcoholic beverages, cocoa butter substitutes, and human milk fat substitutes (Guerrand,
2017;Negi, 2019). More generally, what is pursued with lipases and esterases is the modiﬁcation of fats to develop
organoleptic and nutritional qualities. As these enzymes often require mild conditions (Aravindan et al., 2007), extrem-
ophilic microorganisms are emerging as a source of new-generation lipases and esterases that can be applied to a wider
range of industrial processes. New metagenomics approaches, already described inSection 2, are enabling the identiﬁ-
cation and characterization of novel lipases and esterases (Gu et al., 2015;López-López et al., 2014;Ribera et al., 2017).
To translate theseﬁndings to industrial application, efforts are being made as well for the production and puriﬁcation of
these enzymes (Gutiérrez-Arnillas et al., 2020).
All this work has resulted inﬁnding extremophilic lipases in a variety of niches, such as hot lakes, saltworks, hy-
drothermal vents, geothermal springs, Arctic soils, heated seaﬂoor volcanos, and hot springs (Al-Dhabi et al., 2020;
Gutiérrez-Arnillas et al., 2016;Ramle and Abdul Rahim, 2016;Sahoo et al., 2020). The range of environmental conditions
to which these lipases have adapted results in a varied catalogue of enzyme properties, suitable for different food pro-
cessing operations (Table 3.1). Numerous alkaliphilic lipases have been discovered so far. Recently, an alkaline lipase from
novel halotolerant isolated and identiﬁed asBacillus gibsoniishowed under alkaline pH (9.0) the optimum activity at 60
ﬂ
C
(Sonkar and Singh, 2021). Similarly, a thermoalkaliphilic lipase fromAeribacillus pallidus, insulated from a geothermal oil
ﬁeld, exhibited a pH optimum of 10.0, with high stability in pH range of 9e11, and maintained its original activity after 1 h
at 40
ﬂ
C(Ktata et al., 2019). Recently,Verma et al. (2020)performed a combined experimental and computational
methodology to isolate and identify extremophilic microbial lipases from Thar Desert of Rajasthan (considered as saline
habitats). A total of 30 soil and water samples were collected from different places with temperature ranging between 43
and 50
ﬂ
C, pH from 7.95 to 10.00, and salinity in the range of 70e95&.Bacillus tequilensis(F7) was isolated and pro-
duced lipase exhibited substantial activity and stability at 60
ﬂ
C and pH 9 (Verma et al., 2020). Its structural modeling
showed a highly conserveda/bhydrolase fold at the sequence and structural level except for the N-terminal region.
Besides, the residue Glu128 was different from the template structure and showed salt bridge interaction between Glu128
and Lys101, which contribute to preserving the stability and activity of lipase at high temperatures and alkaline pH
conditions.
Therefore, the molecular-level characterization of extremophilic lipases displayed that they can be used directly or
engineered to make them prospective candidates for a broad range of applications based on their chemo-, regio-, and
enantioselective properties (Verma et al., 2021).
Fermentation, cheese manufacture, bakery, and meat tenderizing are some of the applications of psychrophilic lipases
(Negi, 2019). Another application of these extremophilic enzymes is the synthesis of food additives such as antioxidants,
foodﬂavors and coloring, phytosterol and sugar esters, and conjugates of bifunctional compounds (Akanbi et al., 2020).
Particularly promising is the use of these enzymes for puriﬁcation or enrichment in omega-3 polyunsaturated fatty acids,
essential fatty acids highly valued for their nutraceutical properties. LipBL enzyme from halophilicMarinobacter lip-
olyticusSM19, expressed inE. coliand highly stable in the presence of organic solvents, showed a signiﬁcantly improved
performance extracting eicosapentaenoic acid (EPA) fromﬁsh oil (Pérez et al., 2011). Not only EPA, but also docosa-
hexaenoic acid (DHA) selectivity was shown by lipase of thermophilicT. lanuginosus, which was used to concentrate and
separate these fatty acids from anchovy oil (Akanbi et al., 2013). Remarkably, cold-adaptedCandida antarcticahas two
lipases with industrial application. First, lipase A can be used to produce high-purity DHA concentrate from thraus-
tochytrid or tuna oil (Akanbi et al., 2013). Its commercial product (Lipozyme 435) was successfully used to produce
36Value-Addition in Food Products and Processing Through Enzyme Technology

human milk fat analogues (Wei et al., 2020). On the other hand, commercial lipase B of this microorganism (Novozym
435) was used, in combination with Lipozyme TL IM, as a biocatalyst system for the synthesis of infant formula fat
analogues rich in DHA and arachidonic acid (Wei et al., 2020).
Discovery of new extremophilic esterases has been also reported in different habitats (Adõgüzel, 2020;Curci et al.,
2019;Kumar et al., 2012;Ribera et al., 2017;Wang et al., 2016;Wu et al., 2013)(Table 3.1). As in the case of lipases, the
potential applications of esterases in food processing are varied. For example, thermophilic EST2 fromAlicyclobacillus
acidocaldariusshowed a higher esterase activity, key for foodﬂavor development in dairy products, than standard EstA
fromLactococcus lactis(Mandrich et al., 2006). Recently, it has been proved that the addition of this enzyme to cheese
increases lipolysis up to 30% and, due to its increased esterase activity, it intensiﬁesﬂavor compound release compared to
control cheese (De Luca et al., 2019). Other extremophilic esterases, such as esterase lip3 identiﬁed in an Arctic meta-
genomics library (De Santi et al., 2016), have been suggested as promising for cheese ripening processes.
6. Proteases
Proteases (EC 3.4) hydrolyze peptide bonds between amino acids in proteins yielding peptides and/or amino acids. They
can be further subclassiﬁed into exopeptidases (EC 3.4.11e3.4.19) and endopeptidases (EC 3.4.21e3.4.24, 3.4.99). The
main difference between both is that while exopeptidases can only cleave peptide bonds involving the carbon or nitrogen
terminal amino acids, endopeptidases can act on nonterminal residues (Gurumallesh et al., 2019). As a whole, proteases
represent 60% of the global enzyme market, and in the food industry, they are the second most used class of enzyme
(Akanbi et al., 2020;Kumari et al., 2015). Apart from being extensively used, they have varied applications in the food
industry, including meat tenderization, curdling of milk, beverage clariﬁcation, debittering, texturization, deproteinization,
andﬂavor production (Zhang et al., 2018).
Extremophilic proteases, as well as other extremozymes, present the following interesting features for the food in-
dustry: higher speciﬁcity, lower risk of adverse reactions, lower risk of contamination with mesophilic microﬂora, and
convenient selective thermal inactivation if psychrophilic or thermophilic enzymes are used (Białkowska et al., 2016). In
the particular case of proteases, alkaliphilic enzymes are particularly useful, as they are required to produce protein hy-
drolysates from casein, whey, soy, and meat (Gupta et al., 2002). But alkaliphilic enzymes can be used in other processes
as well. For example, SEB Tender 70 hydrolyzes collagen to improve meatﬂavor (Singhal et al., 2012).
Cold-adapted enzymes are also promising tools because they can operate at low temperatures, which greatly reduce by-
product formation. Furthermore, they can be inactivated easily without high energy input. These properties are particularly
interesting for beer and bakery production, as well as cheese ripening (Białkowska et al., 2016). But their applications go
beyond that. For example, a protease fromPlanococcus maritimusXJ2 showed optimal activity at 40
ﬂ
C and 9.0 pH, as
well as salt tolerance. Due to the conserved activity of this enzyme at lower temperatures, the use of psycrophilic
P. maritimusas a starter culture for low-saltﬁsh sauce incubated at 21
ﬂ
C signiﬁcantly increased amino acid nitrogen
content, as well as the amounts of desirable volatiles such as alcohols, ketones, acids, esters, and pyrazine (Gao et al.,
2020). An interesting case is the cold-adapted metalloprotease fromEnterococcus faecaliswhich is safe for oral admin-
istration and hence could be applied directly to improve the stability and solubility of health foods (Furhan, 2020). Another
example is the collagenolytic protease MCP-01, which demonstrated superior performance than commercially used ten-
derizers papain and bromelain. Concretely, 10U reduced meat shear force by 23% and increased its myoﬁbrillar frag-
mentation index to 91.7% at 4
ﬂ
C, while maintaining both moisture and color of the meat (Zhao et al., 2012). However,
thermophilic proteases can be useful as well for this same application. Thermophilic aspartic protease fromRhizomucor
mieheiexpressed inP. pastorisshowed optimal activity at pH 5.5 and 55
ﬂ
C and its application resulted in lower shear force
compared to papain-treated samples (Sun et al., 2018).
Finally, halophilic proteases would be of great value to enhance saline fermentation processes, which can be required to
produce some protein-rich food (mainly,ﬁsh and meat-based products, but also others such as soy sauce). Characterization
of a halophilic and alkalithermophilic serine protease fromAlkalibacillussp. NM-Da2 revealed that this enzyme exhibited
optimal activity at 56
ﬂ
C, 9.0 pH, and 2.7M NaCl (Abdel-Hamed et al., 2016). These properties make this enzyme suitable
and attractive for a wide range of applications. Remarkably, its stability at high salt concentration and its performance in
organic solvents make it an attractive candidate for catalyzing reactions commonly performed in nonaqueous conditions.
There are even more food industrial processes that could still beneﬁt from extremophilic protease application.
Therefore, the biodiversity of extremophiles is being actively screened to fetch microorganisms producing these enzymes,
adapted to harsh conditions and hence better adapted to diverse processes (Jabalia et al., 2014). New omics approaches are
being fruitful for this purpose, as demonstrated by the work ofPerfumo et al. (2020). Here, screening of Antarctic glacier
Extremozymes in food production and processingChapter | 337

foreﬁeld-bacteria for novel cold-active enzymes with comparative genomics identiﬁed the strain 94-6B ofPsychrobacter
sp. as a source of a protease that was later heterologously expressed inE. coli. Its characterization revealed an optimal
temperature between 20 and 30
ﬂ
C and stability in the presence of common inhibitors.
7. Conclusions
The food industry represents a large and expanding segment of the enzyme market that demands environmentally friendly
catalysts operative under harsh physicochemical conditions. Extremophiles, microorganisms that thrive under different
harsh conditions, have recently stood out as a vast source of enzymes with stabilities and functions meeting these current
demands, which could help to develop more efﬁcient and sustainable processes contributing to the expansion of a bio-
based economy. Although there are some examples of extremophilic lipases, esterases, carbohydrases, and proteases
with potential and current application in food industry applications, few extremozymes have actually reached the market.
Metagenomics has revolutionized bioprospection of novel enzymes. Both function and sequence-based methodologies
allow the identiﬁcation of extremozymes with unprecedented throughput in spite of the lack of cultivability of these
microorganisms. The development of new enzymatic assays, more robust and with higher throughput, is also allowing
researchers to test more rapidly the presence of interesting enzymatic functions in different environments. By harnessing
these innovative approaches, the vast catalogue of extremozymes keeps growing, as well as their potential applications in
the food industry. In the future, considering the fast pace at which omics technologies, bioinformatics, and protein
engineering are evolving, we should expect an increased ratio of the discovery of extremozymes meeting food industry
needs.
Nevertheless, large-scale production of extremozymes is currently fraught with difﬁculties and this hinders the sys-
tematic application of these enzymes in industrial processes. Neither cultivation of extremophiles in bioreactors nor
heterologous expression in mesophilic hosts is currently optimized. In fact, these are and probably will be the main
bottlenecks for extremozyme application unless novel extremophilic culture methods, improved molecular and genetic
engineering tools, as well as more efﬁcient mass production processes are developed. Although costly and not immediately
rewarding, current work in thisﬁeld will be crucial to build a solid foundation that paves the way for future easier and
faster production of extremozymes.
Aﬁnal key aspect for the success of extremozyme-based bioprocesses in the food industry is social acceptance. The
growing use of enzymes in food processes is coherent with current chemophobia, i.e., social aversion for chemistry-based
products. Enzymes are natural products, and as such, they are considered as a more“natural”option by customers.
However, as we move forward, approaches such as protein engineering and heterologous expression entail the use of
enzymes derived from genetically modiﬁed microorganisms, which mightﬁnd public rejection. Therefore, clearly deﬁning
the potential safety risks of these technologies and promoting an adequate education about these products will be crucial
for the systematic implementation of extremozymes.
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The General concluded his sentence for him. "—Had not your
foresight placed it in safety and out of their reach: that's understood.
Well, Sir,—what then?"
"But, on the contrary, General, it is in imminent peril! The carts
conveying it have stuck fast, not a mile ahead: the bullocks are
foundered and cannot proceed; and I have ridden back to request
that you supply me with fresh animals."
"Look at me, Sir, and then pray look about you."
"I beg your pardon——"
"You ought to. Am I a bullock-driver, Sir, or a muleteer? And in this
country"—with a sharp wave of his hand—"can I breed full-grown
mules or bullocks at a moment's notice to repair your d——d
incompetence? Or, knowing me, have you the assurance to tell me
coolly that you have lost—yes, lost—the treasure committed to you?
—to confess that you, who ought to be a day's march ahead of the
main body, are hanging back upon the rearmost company of the
rearguard?—and come to me whining when that company is actually
engaged with the enemy? Look, Sir"—and it seemed to some of the
28th that their General mischievously prolonged his address to give
the Assistant-Paymaster a taste of rearguard work, for Soult's heavy
columns were by this time pressing near to the entrance of the defile
—"Observe the kind of strife in which we have been engaged since
dawn; reflect that our tempers must needs be short; and
congratulate yourself that, if this mountain be bare of fresh bullocks,
it also fails to supply a handy tree."
The little man waited no longer on the road, along which French
bullets were beginning to whistle, but clambered on his horse, and
galloped off with hunched shoulders to rejoin his carts.
The rearguard, galled now by musketry and finding that, for all their
floundering, the enemy were creeping past the rocky barrier below,
retired in good order but briskly, and so, in about twenty minutes,
overtook the two treasure-carts and their lines of exhausted cattle.
Plainly this procession had come to the end of its powers and could

not budge: and as plainly the officers in charge of it were at
loggerheads. Paget surveyed the scene, his brow darkening
thunderously: for, of the guns he had sent forward to overtake the
reserve, two stood planted to protect the carts, and the artillery-
captain in charge of them was being harangued by the fuming
Assistant-Paymaster, while the actual guard of the treasure—a
subaltern's party of the 4th (King's Own)—stood watching the
altercation in surly contempt. Now the 28th and the King's Own
were old friends, having been brigaded together through the early
days of the campaign. As Paget rode forward they exchanged
hilarious grins.
"Pray, Sir," he addressed the artilleryman, "why are you loitering
here when ordered to overtake the main body with all speed? And
what are you discussing with this person?"
"The Colonel, Sir, detached me at this officer's request."
"Hey?" Paget swung round on the Assistant-Paymaster. "You dared
to interfere with an order of mine? And, having done so, you forbore
to tell me, just now, the extent of your impudence!"
"But—but the bullocks can go no farther!" stammered the poor man.
"And if so, who is responsible? Are you, Sir?" Paget demanded
suddenly of the subaltern.
"No, General," the young man answered, saluting. "I beg to say that
as far back as Nogales I pointed out the condition of these beasts,
and also where in that place fresh animals were to be found: but I
was bidden to hold my tongue."
"Do you admit this?" Paget swung round again upon the Assistant-
Paymaster.
"Upon my word, Sir," the poor man tried to bluster, "I am not to be
cross-examined in this fashion. I do not belong to the reserve, and I
take my orders——"
"Then what the devil are you doing here? And how is it I catch you
ordering my reserve about? By the look of it, a moment ago you

were even attempting to teach my horse-artillery its business."
"He was urging me, Sir," said the artillery-captain grimly, "to
abandon my guns and hitch my teams on to his carts."
The General's expression changed, and he bent upon the little man
in blue a smile that was almost caressing. "I beg your pardon, Sir: it
appears that I have quite failed to appreciate you."
"Do not mention it, Sir. You see, with a sum of twenty-five thousand
pounds at stake——"
"And your reputation."
"To be sure, and my reputation; though that, I assure you, was less
in my thoughts. With all this at stake——"
"Say rather 'lost.' I am going to pitch it down the mountain."
"But it is money!" almost screamed the little man.
"So are shot and shells. Twenty-eighth, forward, and help the guard
to overturn the carts!"
Even the soldiers were staggered for a moment by this order.
Impossible as they saw it to be to save the treasure, they were men;
and the instinct of man revolts from pouring twenty-five thousand
pounds over a precipice. They approached, unstrapped the tarpaulin
covers, and feasted their eyes on stacks of silver Spanish dollars.
"You cannot mean it, Sir! I hold you responsible——" Speech choked
the Assistant-Paymaster, and he waved wild arms in dumbshow.
But the General did mean it. At a word from him the artillerymen
stood to their guns, and at another word the fatigue party of the
28th climbed off the carts, put their shoulders to the wheels and
axle-trees, and with a heave sent the treasure over in a jingling
avalanche. A few ran and craned their necks to mark where it fell:
but the cliffs just here were sharply undercut, and everywhere below
spread deep drifts to receive and cover it noiselessly. After the first
rush and slide no sound came up from the depths into which it had
disappeared. The men strained their ears to listen. They were

listening still when, with a roar, the two guns behind them spoke
out, hurling their salutation into Soult's advance guard as it swung
into view around the corner of the road.
II
In a mud-walled hut perched over the brink of the ravine and
sheltered there by a shelving rock, an old Gallegan peasant sat
huddled over a fire and face to face with starvation. The fire, banked
in the centre of the earthen floor, filled all the cabin with smoke,
which escaped only by a gap in the thatch and a window-hole
overlooking the ravine. An iron crock, on a chain furred with soot,
hung from the rafters, where sooty cobwebs, a foot and more in
length, waved noiselessly in the draught. It was empty, but he had
no strength to lift it off its hook; and at the risk of cracking it he had
piled up the logs on the hearth, for the cold searched his old bones.
The window-hole showed a patch of fading day, wintry and sullen:
but no beam of it penetrated within, where the firelight flickered
murkily on three beds of dirty straw, a table like a butcher's block,
and, at the back of the hut, an alcove occupied by three sooty dolls
beneath a crucifix—the Virgin, St. Joseph, and St. James.
The alcove was just a recess scooped out of the adobe wall: and the
old man himself could not have told why his house had been built of
unbaked mud when so much loose stone lay strewn about the
mountain-side ready to hand. Possibly even his ancestors, who had
built it, could not have told. They had come from the plain-land near
Zamora, and built in the only fashion they knew—a fashion which
their ancestors had learnt from the Moors: but time and the
mountain's bad habit of dropping stones had taught them to add a
stout roof. For generations they had clung to this perch, and held
body and soul together by the swine-herding. They pastured their
pigs three miles below, where the ravine opened upon a valley
moderately fertile and wooded with oak and chestnut; and in
midwinter drove them back to the hill and styed them in a large pen
beside the hut, in which, if the pen were crowded, they made room
for the residue.

The family now consisted of the old man, Gil Chaleco (a widower
and past work); his son Gil the Younger, with a wife, Juana; their
only daughter, Mercedes, her young husband, Sebastian May, and
their two-year-old boy. The two women worked with the men in
herding the swine and were given sole charge of them annually,
when Gil the Younger and Sebastian tramped it down to the plains
and hired themselves out for the harvest.
But this year Sebastian, instead of harvesting, had departed for
Corunna to join the insurrectionary bands and carry a gun in defence
of his country. To Gil the Elder this was a piece of youthful folly. How
could it matter, in this valley of theirs, what King reigned in far-away
Madrid? And would a Spaniard any more than a Corsican make good
the lost harvest-money? The rest of the family had joined him in
raising objections; for in this den of poverty the three elders thought
of money morning, noon, and night, and of nothing but money; and
Mercedes was young and in love with her husband, and sorely
unwilling to lend him to the wars. Sebastian, however, had smiled
and kissed her and gone his way; and at the end of his soldiery had
found himself, poor lad, in hospital in Leon, one of the many
hundreds abandoned by the Marquis of Romana to the French.
News of this had not reached the valley, where indeed his wife's
family had other trouble to concern them: for a forage party from
the retreating British main guard had descended upon the cabin four
days ago and carried off all the swine, leaving in exchange some
scraps of paper, which (they said) would be honoured next day by
the Assistant-Paymaster: he could not be more than a day's march
behind. But a day had passed, and another, and now the household
had gone off to Nogales to meet him on the road, leaving only the
old man, and taking even little Sebastianillo. The pigs would be paid
for handsomely by the rich English; Juana had some purchases to
make in the town; and Mercedes needed to buy a shawl for the
child, and thought it would be a treat for him to see the tall foreign
red-coats marching past.

So they had started, leaving the old man with a day's provision (for
the foragers had cleared the racks and the larder as well as the sty),
and promising to be home before nightfall. But two days and a night
had passed without news of them.
With his failing strength he had made shift to keep the fire alight;
but food was not to be found. He had eaten his last hard crust of
millet-bread seven or eight hours before, and this had been his only
breakfast. His terror for the fate of the family was not acute. Old age
had dulled his faculties, and he dozed by the fire with sudden starts
of wakefulness, blinking his smoke-sored eyes and gazing with a
vague sense of evil on the straw beds and the image in the alcove.
His thoughts ran on the swine and the price to be paid for them by
the Englishman: they faded into dreams wherein the family saints
stepped down from their shrine and chaffered with the foreign
paymaster; dreams in which he found himself grasping silver dollars
with both hands. And all the while he was hungry to the point of
dying; yet the visionary dollars brought no food—suggested only the
impulse to bury them out of sight of thieves.
So vivid was the dream that, waking with a start and a shiver, he
hobbled towards the window-hole and stopped to pick up the
wooden shutter that should close it. Standing so, still half asleep,
with his hand on the shutter-bar, he heard a rushing sound behind
him, as though the mountain-side were breaking away overhead and
rushing down upon the roof and back of the cabin.
He had spent all his life on these slopes and knew the sounds of
avalanche and land-slips—small land-slips in this Gallegan valley
were common enough. This noise resembled both, yet resembled
neither, and withal was so terrifying that he swung round to face it,
aquake in his shoes—to see the rear wall bowing inwards and
crumbling, and the roof quietly subsiding upon it, as if to bury him
alive.
For a moment he saw it as the mirror of his dream, cracking and
splitting; then, as the image of the Virgin tilted itself forward from its
shrine and fell with a crash, he dropped the shutter, and running to

the door, tugged at its heavy wooden bolt. The hut was collapsing,
and he must escape into the open air.
He neither screamed nor shouted, for his terror throttled him; and
after the first rushing noise the wall bowed inwards silently, with but
a trickle of dry and loosened mud. His gaze, cast back across his
shoulder, was on it while he tugged at the bolt. Slowly—very slowly,
the roof sank, and stayed itself, held up on either hand by its two
corner-props. Then, while it came to a standstill, sagging between
them, the wall beneath it burst asunder, St. Joseph and St. James
were flung head-over-heels after the Virgin, and through the rent
poured a broad river of silver.
He faced around gradually, holding his breath. His back was to the
door now, and he leaned against it with outspread palms while his
eyes devoured the miracle.
Dollars! Silver dollars!
He could not lift his gaze from them. If he did, they would surely
vanish, and he awake from his dream. Yet in the very shock of awe,
and starving though he was, the master-habit of his life, the
secretive peasant cunning, had already begun to work. Never once
relaxing his fixed stare, fearful even of blinking with his smoke-sored
eyes, he shuffled sideways toward the window-hole, his hands
groping the wall behind him. The wooden shutter and its fastening
bar—a short oak pole—lay where he had dropped them, on the floor
beneath the window. He crouched, feeling backwards for them;
found, lifted them on to the inner ledge, and, with a half-turn of his
body, thrust one arm deep into the recess and jammed the shutter
into its place. To fix the bolt was less easy; it fitted across the back
of the shutter, its ends resting in two sockets pierced in the wall of
the recess. He could use but one hand; yet in less than a minute he
found the first socket, slid an end of the bolt into it as far as it would
go, lifted the other end and scraped with it along the opposite side
of the recess until it dropped into the second socket. He was safe
now—safe from prying eyes. In all this while—these two, perhaps
three, minutes—his uppermost terror had been lest strange eyes

were peering in through the window-hole: it had cost him anguish
not to remove his own for an instant from the miracle to assure
himself. But he had shut out this terror now: and the miracle had not
vanished.
A few coins trickled yet. He crawled forward across the floor,
crouching like a beast for a spring. But as he drew close his old legs
began to shake under him. He dropped on his knees and fell
forward, plunging both hands into the bright pile.
Dollars! real silver dollars!
He lay on the flood of wealth, stretched like a swimmer, his fingers
feebly moving among the coins which slid and poured over the back
of his hands. He did not ask how the miracle had befallen. He was
starving; dying in fact, though he did not know it; and lo! he had
found a heaven beyond all imagination, and lay in it and panted, at
rest. The firelight played on the heave and fall of his gaunt shoulder-
blades, and on the glass eyes of the Virgin, whose head had rolled
half-way across the floor and lay staring up foolishly at the rafters.
"Mother, open! Ah, open quickly, mother, for the love of God!"
Whose voice was that? Yes, yes—Mercedes', to be sure, his
granddaughter's. She had gone to Nogales ... long ago.... Yet that
was her voice. Had he come, then, to Paradise that her voice was
pleading for him—pleading for the door to open?
"Mother—Father! It is I, Mercedes! Open quickly—It is Mercedes, do
you hear? I want my child—Sebastianillo—my child—quick!"
The voice broke into short agonised cries, into sobs. The door
rattled.
At the sound of this last the old man raised himself on his knees. His
eyes fell again on the shining dollars all around him. His throat
worked.

Suddenly terror broke out in beads on his forehead. Someone was
shaking the door! Thieves were there trying the door: they were
come to rob him!

He drew himself up slowly. As he did so the door ceased to rattle,
and presently, somewhere near the windy edge of the ravine, a faint
cry sounded.
But long after the door had ceased to rattle, old Gil Chaleco stared
at it, fascinated. And long after the cry had died away it beat from
side to side within the walls of his head, while he listened and life
trickled from him, drop by drop.
"Thou shalt not be afraid for the terror by night." But he was
listening for it: it would come again....
And it came—with a rough summons on the door, and, a moment
later, with a thunderous blow. The old man stood up, knee-deep in
dollars, lifting both arms to cover his head. As the door fell he
seemed to bow himself toward it, toppled, and slid forward—still
with his arms crooked—amid a rush of silver.
III
Although crushed in the rear and broken inwards there, the hut
showed its ordinary face to the path as Mercedes reached it in the
failing daylight. She ran like a madwoman, and with short, distraught
cries, as she neared her home. Her eyes were wild as a hunted
creature's, her coarse black hair streamed over her shoulders, her
bare feet bled where the rocks and ice had cut them. But one thing
she did not doubt—would not allow herself to doubt—that at home
she would find her child. For two days she had been parted from
him, and in those two days ... God had been good to her, very good:
but she could not thank God yet—not until she clutched Sebastianillo
in her arms, held his small, wriggling body, felt his feet kick against
her breast....
The great sty beside the cabin was empty, of course: and the cabin
itself looked strange to her and desolate and unfriendly. For some
hours the snow had ceased falling, and, save in a snowstorm or a
gale, it was not the family custom to close door or window before
dark: indeed, the window-hole usually stood open night and day the
year round. Now both were closed. But warm firelight showed under

the chink of the door; and on the door she bowed her head, to take
breath, and beat with her hands while she called urgently—
"Mother! Quickly, mother—open to me for the love of God!"
No answer came from within.
"Mother! Father! Open to me—it is I, Mercedes!"
Then, after listening a moment, she began to beat again, frantically,
for at length she was afraid.
"Quick! Quick! Ah, do not be playing a trick on me: I want my child
—Sebastianillo!"
Again and again she called and beat. No answer came from the hut
or from the sombre twilight around her. She drew back, to fling her
full weight against the door. And at this moment she heard, some
way down the path, a man's footstep crunching the snow.
She never doubted that this must be her father returning up the
mountain-side, perhaps after a search for her. What other man—now
that her husband had gone soldiering—ever trod this path? She ran
down to meet him.
The path, about forty yards below, rounded an angle of the sheer
cliff, and at this angle she came to a terrified halt. The man, too, had
halted a short gunshot away. He did not see her, but was staring
upward at the cliff overhead; and he was not her father. For an
instant there flashed across her brain an incredible surmise—that he
was her husband, Sebastian: for he wore a soldier's overcoat and
shako, and carried a musket and knapsack. But no: this man was
taller than Sebastian by many inches; taller and thinner.
He was a soldier, then: and to Mercedes all soldiers were by this
time incarnate devils—or all but one, and that one a plucky little
British officer who had snatched her from his men just as she fell
swooning into their clutches, and had dragged and thrust her
through the convent doorway at Nogales and slammed the door
upon her; and (though this she did not know) held the doorstep,
sword in hand, while the Fathers within shot the heavy bolts.

The British had gone, and after them—close after—came the French:
and these broke down the convent door and ransacked the place.
But the Fathers had hidden her and a score or so more of trembling
women, nor would allow her to creep out and search for
Sebastianillo in the streets through which swept, hour after hour, a
flood of drunken yelling devils. So now Mercedes, who had left home
two days ago to watch an army pass, turned from this one soldier
with a scream and ran back towards the cabin.
In her terror lest he should overtake and catch her by the closed
door, she darted aside, clambered across the wall of the empty sty,
and crouched behind it in the filth, clutching at her bodice: for within
her bodice was a knife, which she had borrowed of the Fathers at
Nogales.
The footsteps came up the path and went slowly past her hiding-
place. Then they came to a halt before the hut. Still Mercedes
crouched, not daring to lift her head.
Rat, rat-a-tat!
Well, let him knock. Her father was a strong man, and always kept a
loaded gun on the shelf. If this soldier meant mischief, he would find
his match: and she, too, could help.
She heard him call to the folks within once or twice in bad Spanish.
Then his voice changed and seemed to threaten in a language she
did not know.
Her hand was thrust within her bodice now, and gripped the handle
of her knife; nevertheless, what followed took her by surprise,
though ready for action. A terrific bang sounded on the timbers of
the door. Involuntarily she raised her head above the wall's coping.
The man had stepped back a pace into the path, and was swinging
his musket up for another blow with the butt.
She stood up, white, with her jaw set. Her father could not be inside
the hut, or he would have answered that blow on his door as a man
should. But Sebastianillo might be within—nay, must be! She put her
hands to the wall's coping and swung herself over and on to the

path, again unseen, for the dusk hid her, and a dark background of
cliff behind the sty: nor could the man hear, for he was raining blow
after blow upon the door. At length, having shaken it loose from its
hasp, he stepped back and made a run at it, using the butt of his
musket for a ram, and finishing up the charge with the full weight of
one shoulder. The door crashed open before him, and he reeled over
it into the hut. A second later, Mercedes had sprung after him.
"Sebastianillo! You shall not harm him! You shall not——"
The door, falling a little short of the fire, had scattered some of the
burning brands about the floor and fanned the rest into a blaze. In
the light of it he faced round with a snarl, his teeth showing beneath
his moustache. The light also showed—though Mercedes neither
noted it nor could have read its signification—a corporal's chevron on
his sleeve.
"Who the devil are you?" The snarl ended in a snap.
Mercedes stood swaying on the threshold, knife in hand.
"You shall not harm him!"
She spoke in her own tongue and he understood it, after a fashion;
for he answered in broken Spanish, catching up her word—
"Harm? Who means any harm? When a man is perishing with
hunger and folks will not open to him——"
He paused, wondering at her gaze. Travelling past him, it had
fastened itself on the back wall of the hut, across the fire. "Hullo!
What's the matter?" He swung round. "Good Lord!" said he, with a
gulp.
He sprang past the fire and stooped over the old man's body, which
lay face downward on the shelving heap of silver. It did not stir. By-
and-by he took it by one of the rigid arms and turned it over, not
roughly.
"Warm," said he: "warm, but dead as a herring! Come and see for
yourself."

Mercedes did not move. Her eyes sought the dark corners of the
cabin, fixed themselves for a moment on the shattered image of the
Virgin, and met his across the firelight in desperate inquiry.
"What is this? What have you done?"
"Done? I tell you I never touched the man; never saw him before in
my life. Who is he? Your father? No: grandfather, more like. Eh? Am
I right?"
She bent her head, staring at the money.
"This? This is dollars, my girl: dollars enough to set a man up for life,
with a coach and lads in livery, and dress you in diamonds from head
to heel. Don't stand playing with that knife. I tell you I never
touched the old man. What's more, I'm willing to be friendly and go
shares." He stared at her with quick suspicion. "You're alone here,
hey?"
She did not answer.
"But answer me," he insisted, "do you live alone with him?" And he
pointed to the body at his feet.
"There was my mother," said Mercedes slowly, in her turn pointing to
the third bed of straw by the fire. "We journeyed over to Nogales,
she and I. Your soldiers came and took away our pigs, giving us
pieces of paper for them. They said that if we took these to Nogales
someone would pay us: so we started, leaving him. And at Nogales
your men were rough and parted us, and I have not seen her since."
The Corporal eyed her with the beginnings of a leer. She faced him
with steady eyes. "Well, well," said he, after a pause, "I mean no
harm to you, anyway. Lord! but you're in luck. Here you reach home
and find a fortune at your door—a sort of fortune a man can dig into
with a spade; while a poor devil like me——" He paused again and
stood considering.
"You knew about this?" She nodded towards the dollars. "You knew
how it came here, and you came after it?"

"I did and I didn't. I knew 'twas somewhere hereabouts; but strike
me, if a man could dream of finding it like this!"
"Yet you came to this door and beat it open!"
"You've wits, my girl," said the Corporal admiringly; "but they are on
the wrong tack. I mean no harm; and the best proof is that here I'm
standing with a loaded musket and not offering to hurt you. As it
happens, I came to the door asking a bite of bread. I'm cruel
hungry."
Mercedes pulled a crust of millet-bread from her pocket. The Fathers
at the convent had given it to her at parting, but she had forgotten
to eat. She stepped forward; the Corporal stretched out a hand.
"No," said she, and, avoiding him, laid the crust on the block-table.
He caught it up and gnawed it ravenously. "I think there is no other
food in the house."
"You don't get rid of me like that." He ran a hand along the shelves,
searching them. "Hullo! a gun?" He took it down and examined it
beside the fire, while Mercedes' heart sank. She had hoped to
possess herself of it, snatching it from the shelf when he should be
off his guard. "Loaded, too!" He laid it gently on the block and eyed
her, munching his crust.
"You'd best put down that knife and talk friendly," said he at length.
"What's the use?—you a woman, and me with two guns, both
loaded? It's silliness; you must see for yourself it is. Now look here:
I've a notion—a splendid notion. Come sit down alongside of me,
and talk it over. I promise you there's no harm meant."
But she had backed to her former position in the doorway and would
not budge.
"It's treating me suspicious, you are," he grumbled: "hard and
suspicious."
"Cannot you take the money and go?" she begged, breathing hard,
speaking scarcely above a whisper.

"No, I can't: it stands to reason I can't. What can I do in a country
like this with dollars it took two carts to drag here—two carts with
six yoke of bullocks apiece? And that's where my cruel luck comes
in. All I can take, as things are, is just so much as this knapsack will
carry: and even for this I've run some risks."
The man—it was the effect of hunger, perhaps, and exposure and
drunkenness on past marches—had an ugly, wolfish face; but his
eyes, though cunning, were not altogether evil, not quite formidably
evil. She divined that, though lust for the money was driving him,
some weakness lay behind it.
"You are a deserter," she said.
"We'll pass that." He seated himself, flinging a leg over the block and
laying the two guns side by side on his knees. "I can win back,
maybe. As things go, between stragglers and deserters it's hard to
choose in these times, and I'll get the benefit of the doubt. I've
taken some risks," he repeated, glancing from the guns on his knees
to the pile of silver and back: "pretty bad risks, and only to fill my
knapsack. But, now it strikes me——Can't you come closer?"
But she held her ground and waited.
"It strikes me, why couldn't we collar the whole of this, we two?
We're alone: no one knows; I've but to lift one of these"—he tapped
the guns—"and where would you be? But I don't do it. I don't want
to do it. You hear me?"
"You don't do it," said Mercedes slowly, "because without me you
can't get away with more than a handful of this money. And you
want the whole of it."
"You're a clever girl. Yes, I want the whole of it. Who wouldn't? And
you can help. Can't you see how?"
"No."
He sat swinging his legs. "Well, that's where my notion comes in. I
wish you'd drop that knife and be friendly: it's a fortune I'm offering
you. Now my notion is that we two ought to marry." He stood up.

Mercedes lifted the knife with its point turned inward against her
breast. "If you take another step!"
"Oh, but look here: look at it every way. I like you. You're a fine
build of a woman, with plenty of spirit—the very woman to help a
man. We should get along famously. One country's as good as
another to me: I'm tired of soldiering, and there's no woman at
home, s'help me!" He was speaking rapidly now, not waiting to cast
about for words in Spanish, but falling back on English whenever he
found himself at a loss. "I dare say you can fit me out with a suit of
clothes." His glance ran round the hut and rested on the body of the
old man.
Mercedes had understood scarce half of his words: but she divined
the meaning of that look and shuddered.
"No, no; you cannot do that!"
"Hark!" said he raising his head and listening. "What's that noise?"
"The wolves. We hear them every night in winter."
"A nice sort of place for a woman to live alone in! See here, my
dear; it's sense I'm talking. Better fix it up with me and say 'yes.'"
She appeared to be considering this. "One thing you must promise."
"Well?"
"You won't touch him"—she nodded towards her grandfather's
corpse. "You won't touch him to—to——"
"Is it strip him you mean? Very well, then, I won't."
"You will help me to bury him? He cannot lie here. I can give you no
answer while he lies here."
"Right you are, again. Only, no tricks, mind!"
He stowed the guns under his left arm and gripped the collar of the
old man. Mercedes took the feet; and together they bore him out—a
light burden enough. Outside the hut a pale radiance lay over all the

snow, forerunner of the moon now rising over the crags across the
ravine.
"Where?" grunted the Corporal.
Mercedes guided him. A little way down the path, beyond the wall of
the sty, they came to a recess in the base of the cliff where the
wind's eddies had piled a smooth mound of snow. Here, under a
jutting rock, they laid the body.
"Cover him as best you can," the Corporal ordered. "My hands are
full."
He stood, clasping his guns, and watched Mercedes while she knelt
and shovelled the snow with both hands. Yet always her eyes were
alert and she kept her knife ready. From their mound they looked
down upon the ravine in front and over the wall of the sty towards
the cabin. Behind them rose the black cliff.
"Hark to the wolves!" said the Corporal, listening: and at that
moment something thudded down from the cliff, striking the snow a
few yards from him; rolled heavily down the slope and came to a
standstill against the wall of the sty, where it lay bedded.
The round moon had risen over the ravine, and was flooding the
mound with light. The Corporal stared at Mercedes: for the moment
he could think of nothing but that a large, loose stone had dropped
from the cliff. He ran to the thing and turned it over.
It was a knapsack.
He did not at once understand, but stepped back a few paces and
gazed up at the crags mounting tier by tier into the vague
moonlight. And while he gazed a lighter object struck the wall over
head, glanced from it, went spinning by him, and disappeared over
the edge of the ravine. As it passed he recognized it—a soldier's
shako.
Then he understood. Someone had found the spot on the road
above where the treasure had been upset, and these things were
being dropped to guide his search. The Corporal ran to Mercedes

and would have clutched her by the wrist. The knife flashed in her
hand as she evaded him.
"Quick, my girl—back with you, quick! They're after the money, I tell
you!"
He caught up the knapsack. They ran back together and flung
themselves into the cabin. The Corporal bolted the door.
"King's Own," he announced, having dragged the knapsack to the
firelight. "If there's only one, we'll do for him."
He stepped to the window-hole, pulled open the shutter, laid the two
guns on the ledge, and waited, straining his ears.
"Got such a thing as a shovel or a mattock?" he asked after a while.
"I reckon you could make shift to cover up the dollars: there's a deal
of loose earth come down with them."
It took her some time to guess what he wanted, for he spoke in a
hoarse whisper. He listened again for a while, then pointed to the
treasure.
"Cover it up. If there's more than one, we'll have trouble."
She produced a mattock from a corner of the cabin and began,
through the broken wall, to rake down mud and earth and cover the
coins. For an hour and more she worked, the Corporal still keeping
watch. Once or twice he growled at her to make less noise.
He did not stand the suspense well, but after the first hour grew
visibly uneasy.
"I've a mind to give this over," he grumbled, and fell to unstrapping
his knapsack. "Here!"—he tossed it to her—"pack it, full as you can.
Half a loaf may turn out better than no bread."
She laid the knapsack open on the floor and set to work, cramming
it with dollars.
"Talking of bread," he went on by-and-by, "that's going to be a
question. My stomach's feeling at this moment like as if it had two

rows of teeth inside."
"Hist!" Mercedes rose, finger to lip. He turned again to the window-
hole and peered out, gun in hand, his shoulder blocking the recess.
A man's footsteps were coming up the path—coming cautiously.
Their crunch upon the snow was just audible, and no more.
Mercedes stole towards the window and crept close behind the
Corporal's back; stood there, holding her breath.
The man on the path halted for a moment, and came on again, still
cautiously.... There was a jet of flame, a roar; and the Corporal,
after the kick of his musket, strained himself forward on the window-
ledge to see if his shot had told.
"Settled him!" he announced, drawing back and turning to face her
with a triumphant grin.
But Mercedes confronted him with her father's fowling-piece in hand.
She had slipped it off the window-ledge from under his elbow as he
leaned forward.
"Unbar the door!" she commanded.
"Look here, no nonsense!"
"Unbar the door!" She believed him to be a coward, and he was.
"You just wait a bit, my lady!" he threatened, but drew the bolt,
nevertheless; when he turned, the muzzle of the fowling-piece still
covered him.
She nodded toward the knapsack. "Pick up that, if you will.... Now
turn your back—your back to me, if you please—and go."
He hesitated, rebellious: but there was no help for it.
"Go!" she repeated. And he went.
Above the cabin the path ended almost at once in a cul de sac—a
wall of frowning cliff. There was no way for him, whether he wished
to descend or climb the mountain, but that which led him past the
body of the man he had just murdered. He went past it tottering,

fumbling with the straps of his knapsack: and Mercedes stood in the
moonlit doorway and watched him out of sight.
By-and-by she seated herself before the threshold, and, laying the
gun across her knees, prepared herself to wait for the dawn. The
dead man lay huddled on his side, a few paces from her. Overhead,
along the waste mountain heights, the wolves howled.
Hours passed. Still the wolves howled, and once from the upper
darkness Mercedes heard, or fancied that she heard, a scream.
At noon, next day, two men—a priest and a young peasant—were
climbing the mountain-path leading to the hut. The young man
carried on his shoulder a two-year-old child; and, because the sun
shone and the crisp air put a spirit of life into all things untroubled
by thought, the child crowed and tugged gleefully at his father's
berret. But his father paid no heed, and strode forward at a pace
which forced the priest (who was stout) now and again into a run.
"She will not be there," he kept repeating, steeling himself against
the worst. "She cannot be there. When she missed her child——"
"She is waiting on her grandfather, belike," urged the priest. "They
left him with one day's food: so she told the Brothers. And they, like
fools, let her go with just sufficient for her own needs. Yet I ought
not to blame them for losing their heads in so small a matter. They
saved many women."
He told again how he—the parish priest of Nogales—had found Gil
the Younger and his wife dead and drunken, with their heads in a
gutter and the child wailing in the mud beside them. "Your wife had
given her mother the child to guard but a minute before she fell in
with the soldiers. A young officer saved her, the Brothers said."
"Mercedes will have sought her child first," persisted Sebastian; and
rounding the corner of the cliff, they came in sight of the hut and of

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