Food industry assessment, trends and current issues

FOOD AND BEVERAGE CONSUMPTION AND HEALTH

FOOD INDUSTRY
ASSESSMENT, TRENDS
AND CURRENT ISSUES

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FOOD AND BEVERAGE CONSUMPTION
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FOOD AND BEVERAGE CONSUMPTION AND HEALTH

FOOD INDUSTRY
ASSESSMENT, TRENDS
AND CURRENT ISSUES

DORIS CUNNINGHAM

EDITOR

New York


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CONTENTS
Preface
Chapter 1

Chapter 2

Chapter 3

Chapter 4

vii
An Overview on the Main Microbial Enzymes in
the Food Industry

Cristiano José de Andrade,
Ana Elizabeth C. Fai B. de Gusmão,
Ana Paula Resende Simiqueli,
Evandro Antonio de Lima,
Guilherme Keppe Zanini,
Joelise de Alencar Figueira Angelotti
and Fabiano Jares Contesini
Hygiene of Conveyor Belts for Food Production:
Trends and Challenges
Sebastjan Filip, Martina Oder, Eva Stražar
and Rok Fink
Best Practices in Refrigeration Applications to
Promote Energy Efficiency: The Portuguese Case
Study
P. D. Silva, P. D. Gaspar, L. P. Andrade,
J. Nunes and C. Domingues
Winemaking in Developing Countries:
Challenges and Efficiency
Anatoliy G. Goncharuk, Dr. Habil
and Aleksandra Figurek

1

45

63

133

vi
Chapter 5

Index

Contents
Economic-Financial Performance from the Wine
Industry in the Crisis: The Case of the Region
of Castilla y León (Spain)
Francisco Andrés Díez-Zamudio
and Silverio Alarcón

157

193


PREFACE
This book provides an assessment of the food industry. It discusses trends
and current issues. Chapter One gives an overview of the main microbial
enzymes in the food industry. Chapter Two focuses on trends and challenges
for the hygiene of conveyor belts in food production. Chapter Three identifies
energy-saving opportunities (technological, organisational or behavioural) and
describes tailored energy-saving measures in the food and drink industry.
Chapter Four evaluates and compares the efficiency of winemaking in two
developing countries (Ukraine and Bosnia and Herzegovina) from the
perspective of their development. Chapter Five makes a comparison between
Denominations of Origins (DO) in Castilla y León in a determined period of
time of economical crisis of the wine industry.
Chapter 1 – Enzymes are one of the most important tools in the food

industry due to their applications for the obtainment of important compounds
such as sweeteners, flavors and bioactive compounds, among others. They can
also be applied in food modification, including galactose hydrolysis of dairy
products and juice clarification, as well as for recovery and recycling of
industrial byproducts. In the food industry, enzymes can be classified
according to the substrate from the food product, for instance (I) carbohydrateactive enzymes such as amylolytic, pectinolytic, cellulolitic and
hemicellulolitic enzymes, (II) enzymes applied to lipids and hydrophobic
compounds including lipases and esterases, (III) enzymes applied to proteins
present in food, such as several types of proteases. For instance, starch is a
polysaccharide that presents several applications in the food industry. Starch
can be degraded/modified by amylases for process improvement; α-amylases
randomly act on α-1,4-glycosidic bonds producing malto-oligosaccharides,
maltose or even glucose, while β-amylases act on non-reducing end of starch


viii

Doris Cunningham

producing maltose. Other amylases are also important for starch modification,
including isoamylase and pulullanases. Hemicellulases like xylanases can be
used to obtain sweeteners, such as arabinose. Regarding lipases and esterases,
several industrially relevant compounds can be obtained including mono- and
diacylglycerols, and polyunsaturated fatty acids (PUFAs). Fatty acids, in
particular short-chain fatty acids, have significant impact in the flavor of food
products. Lipases can be applied to release short chain fatty acids from
triglycerides in order to result in flavor enhancement or cheese ripening
processes. Proteases correspond to an important group of enzymes in the food
industry, since they can be used for the production of important bioactive
peptides that present biological activity, such as anti-oxidant and antihipertensive activities. Although there are several sources of enzymes for the

food industry use, microbial enzymes are highlighted since they can be
obtained through fermentation processes. In addition, several genetic
engineering techniques, such as recombinant production strains and protein
engineering have been studied in order to enhance enzymes performance as
well as improve their production. Therefore, the application of enzymes by
food industry covers a very wide range of food processing techniques, which
is fundamental for technological advances, resulting in more convenient and
healthier products, with extended shelf life.
Chapter 2 – Concerns for proper hygiene are greatest in food industry,
where poor sanitation practices can lead to food related illnesses. This is
especially important in technology where foodstuff is in contact to surfaces for
long period. Conveyor belts are the linking element in many segments of the
food industry and they are an important part of the process. Cleaning methods
can affect the amount of soil that is removed from the surface. Choosing the
right belt is vital for the efficiency and hygiene condition of the production
line. This chapter represent principles of conveyor belt for food industry,
HACCP and cleaning and disinfection of conveyor surface. Moreover, chapter
includes also results of testing different methods removing B. cereus from the
surface of polyurethane conveyor belts. Results of the research show that
hurdle technology of cleaning agent and ultrasound is the most efficient.
Therefore cleaning in place system can maximize the cleanability of the belts
by combining two or more methods for bacterial adhesion control.
Chapter 3 – The food and drink industry is the largest and most dynamic
manufacturing sector of the European Union. With its 286,000 companies
(mostly SMEs) and turnover share of 15%, it provides jobs for over 4 million
people. There is an acute need to replace energy-intensive processes in this
sector by new efficient ones. The major energy consumption originates from


Preface

ix

heating, cooling and drying processes, refrigeration, electrical drive systems,
among others. Refrigeration of food products can account more than 50% of
the energy consumption. This chapter identifies energy-saving opportunities
(technological, organisational or behavioural) and describes tailored energysaving measures. Best practices and measures for energy efficiency
improvement are disclosure, which can be applied to infrastructures, cooling
chambers, vapour compression refrigeration systems, compressed air systems,
steam generator/hot water systems, among others. Additionally, the use of
renewable energies and procedures for analysis of the electricity consumption
and power management are discussed. These best practices and energy
conservation measures may substantially improve the energy efficiency and
competitiveness of agrifood companies and will ultimately benefit the
consumers and society by reducing food price, food waste and carbon
emissions.
Chapter 4 – Purpose – The paper is devoted to evaluation and comparison
of the efficiency of winemaking in two developing countries (Ukraine and
Bosnia and Herzegovina) from the perspective of their development.
Design/methodology/approach – In the research, four models of Data
envelopment analysis (DEA), correlation and other tools of the data analysis
are used to analyse the efficiency of wineries in two developing countries.
Returns to scale, scale efficiency, super-efficiency and some other indicators
are examined. The research is based on the sample including 33 wineries from
Ukraine and Bosnia and Herzegovina. Findings – Having the same average
efficiency and number of leaders, medium and large wineries in Ukraine are
developing more efficiently than small wineries, whereas in Bosnia and
Herzegovina, contrary, a small wine business is more efficient. The authors
found the high potential growth of efficiency in Ukrainian (up to 28.9%) and
Bosnian wineries (up to 28.3%). The ways for its realization were suggested.

The cross-country efficiency analysis enabled us to find inter-country leaders
in the wine industry. They grouped inefficient wineries, calculated the
potential to reduce inputs and found main directions to improve efficiency for
each group. Research limitations/implications – The research is limited to a
single industry in only two developing countries. Future research can be
devoted to comparison of the efficiency of wineries in developed and
developing countries. The results can determine which countries can be
leaders in the global wine market in the future. Practical implication – This
study has a practical implication. Its results provide useful information for:
researchers of wine market in developing countries to understand the current
state, basic problems and efficiency levels of wineries in Ukraine and Bosnia


x

Doris Cunningham

and Herzegovina; domestic policy-makers to improve regulation of wine
industry to make it more competitive and efficient; wine producers in these
countries to find the benchmarks with the best practice to adapt them in own
business and increase its efficiency. Originality/value – This study on the
example of Ukraine and Bosnia and Herzegovina has shown that each such
country has its own conditions of doing the wine business. This is the first
paper that compares the efficiency of wine industry in Ukraine and Bosnia and
Herzegovina.
Chapter 5 – The wine sector in Castilla y León is composed of many
Denominations of Origins (DO) and other new wineries without DO, many of
these areas were created in the beginning of this century. However, some
appellations have a long tradition in the elaboration of wine, which are
recognized in Spain and also internationally, thus the industry has a relevantly

important role in the region. The aim of the study is to make a comparison
between DOs in Castilla y León in a determined period of time of economical
crisis. In order to analyse the different types of enterprises, it is utilized
economical-financial information. The data was collected from SABI database
(Sistema de Análisis de Balances Ibéricos, in English Iberian Balance Sheet
Analysis System) which gathers accounting information from Spanish firms
which present their financial statements. The methodology consists of an
analysis of 12 economic-financial ratios to examine the diagnosis of the wine
sector by years and the different DO selected from Castilla y León. All the
economical-financial information that is obtained showed how the wine sector
was affected by the economical crisis, which is expressed by the different
trends obtained. The profitability in the period time 2008-2013 presents a clear
decrease, which is corroborated by the literature in other sectors and wine DOs
in the same period. In the case of the DOs, it is possible to see a clear
difference between the areas with more tradition in wine production with a
specialization in certain types of wine and the areas with more concentration
of cellars. Additionally, the wineries without DO permit to see the
development of the sector in a new area. Finally, the regression analysis was
done by three different models; it is examined from less exactitude to the most
trustable. In this way it was possible to determine which financial ratios
influence more the profitability in this sector. In parallel it was done the same
regression only for Ribera del Duero, because it represents around the 45% of
all the autonomous community.


In: Food Industry
Editor: Doris Cunningham

ISBN: 978-1-63485-792-5
© 2016 Nova Science Publishers, Inc.

Chapter 1

AN OVERVIEW ON THE MAIN MICROBIAL
ENZYMES IN THE FOOD INDUSTRY
Cristiano José de Andrade1,
Ana Elizabeth C. Fai B. de Gusmão2,
Ana Paula Resende Simiqueli3,
Evandro Antonio de Lima4, Guilherme Keppe Zanini5,6,
Joelise de Alencar Figueira Angelotti7
and Fabiano Jares Contesini5,6*
1

Polytechnic School of the University of São Paulo (USP),
São Paulo, Brazil
2
Department of Basic and Experimental Nutrition, Institute of Nutrition,
Rio de Janeiro State University (UERJ), Rio de Janeiro, Brazil
3
National Agricultural Laboratory - Brazilian Ministry of Agriculture,
Livestock and Food Supply (LANAGRO-SP/MAPA), Campinas, Brazil
4
Brazilian Biosciences National Laboratory (LNBio), Campinas, Brazil
5
Institute of Biology, University of Campinas – Unicamp, Brazil
6
Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE),
Centro Nacional de Pesquisa em Energia e Materiais (CNPEM),
Campinas, Brazil
7

Faculty of Food Egineering – University of Campinas –
Unicamp, Campinas, Brazil
*

Corresponding author:


2

Cristiano José de Andrade, Ana Elizabeth C. Fai B. de Gusmão et al.

ABSTRACT
Enzymes are one of the most important tools in the food industry due
to their applications for the obtainment of important compounds such as
sweeteners, flavors and bioactive compounds, among others. They
can also be applied in food modification, including galactose hydrolysis
of dairy products and juice clarification, as well as for recovery and
recycling of industrial byproducts. In the food industry, enzymes
can be classified according to the substrate from the food product, for
instance (I) carbohydrate-active enzymes such as amylolytic, pectinolytic,
cellulolitic and hemicellulolitic enzymes, (II) enzymes applied to lipids
and hydrophobic compounds including lipases and esterases, (III)
enzymes applied to proteins present in food, such as several types of
proteases. For instance, starch is a polysaccharide that presents several
applications in the food industry. Starch can be degraded/modified by
amylases for process improvement; α-amylases randomly act on α-1,4glycosidic bonds producing malto-oligosaccharides, maltose or even
glucose, while β-amylases act on non-reducing end of starch producing
maltose. Other amylases are also important for starch modification,
including isoamylase and pulullanases. Hemicellulases like xylanases
can be used to obtain sweeteners, such as arabinose. Regarding lipases

and esterases, several industrially relevant compounds can be obtained
including mono- and diacylglycerols, and polyunsaturated fatty acids
(PUFAs). Fatty acids, in particular short-chain fatty acids, have
significant impact in the flavor of food products. Lipases can be applied
to release short chain fatty acids from triglycerides in order to result in
flavor enhancement or cheese ripening processes. Proteases correspond to
an important group of enzymes in the food industry, since they can be
used for the production of important bioactive peptides that present
biological activity, such as anti-oxidant and anti-hipertensive activities.
Although there are several sources of enzymes for the food industry
use, microbial enzymes are highlighted since they can be obtained
through fermentation processes. In addition, several genetic engineering
techniques, such as recombinant production strains and protein
engineering have been studied in order to enhance enzymes performance
as well as improve their production. Therefore, the application of
enzymes by food industry covers a very wide range of food processing
techniques, which is fundamental for technological advances, resulting in
more convenient and healthier products, with extended shelf life.

Keywords: food industry, microbial enzymes, amylases, lipases, proteases


An Overview on the Main Microbial Enzymes in the Food Industry

3

1. INTRODUCTION
In the past years, the advances in protein engineering technology have
led to significant increase of enzyme applications by industries. In general,
enzymes have high specificity, are more efficient and also show higher

catalytic activity compared to chemical catalysts. For commercial enzyme
production, a very wide range of sources are used. The production of microbial
enzymes is easier to control compared to the production of animal and plant
enzymes (Soares et al., 2012). In the food industry, an emblematic example of
the transition from animal enzymes to microbial enzymes is the production of
cheese involving curdling of milk, which traditionally extracts enzymes from
stomachs of calves, however, currently, can be produced by yeast (Kumar et
al., 2010). The production of microbial enzymes contains also fewer harmful
compounds, including phenolic and endogenous enzyme inhibitors. In this
sense, the food industry represents one of the major consumers of enzymes,
and focuses its use on debranching (higher solubility and clarification).
However, there is a trend toward researching on the enhancement of enzyme
activity and enzyme stability against heat and organic solvents (considered
the most critical parameters) by empirical and advanced computational
(Rosetta@home) approaches and also for new uses (Choi et al., 2015).
Currently, due to advances in biotechnology and biochemistry, enzymes
can be obtained in high purity form and can be used in the food industry to a)
reduce the amount of sulfur and increase flavors in wines; b) clarify juices; c)
increase the softness and durability of breads; d) reduce the alcohol content
and calorie in beers; e) stabilize beer; f) nutritional additives; g) embedded
texturing foods such as sausages (Kuraishi et al., 2001; Duran et al., 2002;
Sponher et al., 2015; Soares et al., 2012). Besides the ability to act in various
processes, the use of enzymes may be considered an environmentally correct
strategy, as it eliminates certain by-products that should be processed in the
food industry generating energy expenditure (Fernandes, 2010). Moreover,
these molecules are considered natural products and have been used as part of
the human diet for a long time and are, therefore, non-toxic and food
compounds and are preferred by consumers rather than the use of chemical
compounds for processing foods (James and Simpson, 1996; Duran et al.,
2002).

Enzymes are classified by the substrate that they act upon, (I)
carbohydrate-active enzymes such as amylolytic, pectinolytic, cellulolytic and
hemicellulolytic enzymes (II) enzymes applied to lipids and hydrophobic


4

Cristiano José de Andrade, Ana Elizabeth C. Fai B. de Gusmão et al.

compounds including lipases and esterases, (III) enzymes applied to proteins
present in food as several types of proteases.
The application of carbohydrate-active enzymes (I) by food industry
includes: Pectinases (Ia) widely used in juice production (extraction,
clarification and concentration). In addition, pectinases are applied for fruit
ripening, effluents treatment, fiber degumming, tea and chocolate productions,
animal nutrition and oil extraction. Lactases (Ib) (β-galactosidases) are
fundamental in the dairy industry hydrolyzing lactose in glucose and
galactose. Cellulases (Ic) are simultaneously applied with pectinases in juice
and wine industries. Cellulases also act as extraction aid in green tea, soy,
proteins, essential oils, etc. Amylases (Id) are the main enzymes used by
baking industry resulting in better bread color, volume and texture. Glucose
isomerase (Ie) reversible acts on glucose resulting in fructose; and also on
xylose resulting xylulose. Glucose isomerase is widely used for production of
high fructose corn syrup and researches described the potential use of glucose
isomerase on the production of ethanol from hemicelluloses. Invertase (If)
catalyses the hydrolysis of sucrose in glucose and fructose, enhancing the
sweetness, particularly in juice production (Soares et al., 2012). Glucose
oxidase (Ig) is usually applied to remove traces of glucose, in albumin and
powdered whole eggs (Bigelis and Lasure, 1987) and also for better storage
condition (oxygen sensitive materials) (Soares et al., 2012).

Lipases and esterases (II) are responsible for the enzymatic modification
of lipids and hydrophobic compounds. Their use in food industries
encompasses both the development of new products, such as low calorie food
and functional fatty acids, and quality improvement of well-known ones, like
bread and cheese, increasing palmitic acid at sn-2 (higher absorption
capability) and cocoa-butter-like lipid (Houde et al., 2003; Hasan et al., 2006).
One of the most important advantages of using lipases instead of
traditional chemical processes is the versatility of this group of enzymes,
since, besides their hydrolytic activity in mild conditions, they may also
catalyze esterification, interesterification and transesterification in low water
content or in nonaqueous media (Hasan et al., 2006; Kotogán et al., 2016),
except phospholipases that, due to their lack of affinity with free fatty acids,
does not catalyze synthesis (Gerits et al., 2014). Moreover, they usually
exhibit good chemioselectivity, regioselectivity and enantioselectivity, as well
as substrate specificity (Joseph et al., 2008).
Last but not least, proteases (III) (also known as peptidases, proteolytic
enzymes and peptide bond hydrolases) are hydrolytic enzymes that catalyze
the total hydrolysis of proteins into amino acids and represent one of the


An Overview on the Main Microbial Enzymes in the Food Industry

5

largest groups of enzymes (Tavano et al., 2013). Proteases are applied by the
food industry in the curdling of milk (cheese production), in which proteases
by Aspergillus niger var. awamori is commercially available). Proteases are
also applied in formulation of low allergenic infant food; flavor improvement
in dairy products; tenderization of meat; prevention of chill haze formation in
beer; production of fish meals, fish/meat/vegetable extracts, or hydrolysates,

among others (Ackaah-Gyasi et al., 2015; Li et al., 2013). Proteases represent
approximately 60% of the industrial enzymes market (Ryder et al., 2016;
Mayerhofer et al., 2015; Moreno et al., 2013; Rai and Mukherjee, 2010).
Out of the ≈4.000 enzymes currently widely known, 200 are from
microbial origin. Nevertheless, only 20 enzymes are produced at industrial
scale (Li et al., 2012). In addition, the market of enzymes is an oligopoly
composed by three companies (≈ 75%), Novozymes, DuPont and Roche. In
this sense, carbohydrases, proteases and lipases are the most used enzymes, in
particular in hydrolysis activity (Li et al., 2012). Therefore, there is a need for
researches on screening for novel microbial enzymes and their applications,
enhancement of activity enzymes, etc.

2. PRODUCTION OF MICROBIAL ENZYMES
Enzymes play an important role in many industrial processes. The
benefits provided by their use, both economically and environmentally, lead to
a great increase in demand for this type of molecule. Currently, the two main
production processes used to obtain enzymes are the Solid-State Fermentation
(SSF) and the Submerged Fermentation (SmF).

2.1. Solid-State Fermentation
In nature, several microorganisms secrete enzymes for their own needs.
Many of these enzymes have high added-value, since they can be employed
in processes in the food industry, which leads to the isolation of these
microorganisms for this purpose. The SFF is an industrial-scale production
method of enzymes by isolated microorganisms in an economically and
environmentally friendly manner, which makes this process widely endorsed
by the industry (Thomas et al., 2013). This technique is based in a simple
principle: cultivation of an isolated microorganism in controlled conditions of
certain parameters (i.e., temperature, relative humidity, oxygen concentration)

6

Cristiano José de Andrade, Ana Elizabeth C. Fai B. de Gusmão et al.

on a solid substrate in absence of free-water or near to it. Therefore, the
substrate must contain all the water and nutrients necessary for the
microorganism’s development or it must be an inert material that serves as a
support for nutrients (Singhania et al., 2009). This method does not result in a
sudden change in the microorganism’s habitat since it represents almost their
native growth condition. As a result, the production level of enzymes is higher
compared to the liquid culture medium method, reaching the necessary level to
meet the industrial demand. Moreover, by using this production method,
industries can mitigate problems inherent to their production processes, such
as waste destination. In SSF, industrial waste can be used for the production of
commercially important enzymes, adding value to these compounds that
would generate costs for proper disposal in addition to environmental
problems related to it. This is one of many reasons why several studies
have been conducted in order to improve and optimize SSF. For instance,
fungi and yeast are most suitable for use at the expense of bacteria due to the
physicochemical conditions that are found in SSF fermentation, as moisture
and water available for use. However, with the advancement in the
understanding of the nutritional needs of microorganisms, the composition of
the substrates, how the interaction between them is and advances in
bioreactors itself, it is possible to overcome certain barriers, as shown by
Sharma and Satyanarayana, (2012) with the production of α-amylase from a
strain of bacteria on a SSF system.
Several examples arise in this promising scenario for cost reduction and
utilization of industrial waste for production of enzymes. Rosés and Guerra
(2009) performed surface response methodologies and empirical modeling to

reach at optimal conditions of temperature, pH, moisture and the physical form
that the substrate should be (granulated with specific particle size) for the
cultivation of the filamentous fungus Aspergillus nidulans U0-01 in order to
produce α-amylase. The substrate used was sugarcane bagasse, which is an
important industrial waste found in countries that produce ethanol from
sugarcane. Currently, the bagasse does not have any industrial application
apart from being burned to generate electricity. Roses and Guerra, (2009),
showed that the optimum conditions for α-amylase production in that case
was: sugarcane bagasse particle size of approximately 7 mm, pH 6.0 and
temperature around 30ºC. In these conditions, they produced 457.82 EU/g of
dry suport Another example was elucidated by Coradi et al., (2013) in which
the lipase production system for the filamentous fungus Trichoderma
harzianum in a SSF system was for the first time described. Coradi et al.,
(2013) tested various industrial residues for lipase production in a SSF and


An Overview on the Main Microbial Enzymes in the Food Industry

7

reached the conclusion that a substrate consisting of a mixture of sugarcane
bagasse and castor oil cake in a 1:2 ratio supplemented with some other
nutritional sources generated the best rate of production of this enzyme by the
fungus T. harzianum in comparison with other substrates tested .. At that
time, the vast majority of the studies focused on the production of this class
of enzymes by other microorganisms in other fermentation system to be
discussed in the next section. Another example also involving lipases
production was published by Salgado et al., (201). The Aspergillus niger
strains MUM 03:58, Aspergillus ibericus MUM 03:49, and Aspergillus
uvarum MUM 08:01 were selected for lipase production by SSF with the main

residue found in the olive mill industry, called Two-phase olive mill waste
(TPOMW). The authors applied a Plackett–Burman experimental design in
order to evaluate the effect of substrate composition and time on lipase
production. It was observed that the highest amounts of lipase were produced
by A. ibericus on a mixture of Two-phase olive mill waste (TPOMW), urea,
and exhausted grape mark. The optimal condition for lipase production by this
species was found using 0.073 g urea/g of substrate and 25% of exhausted
grape mark resulting 18.67 U/g of lipolytic activity. Finally, knowing that the
use of industrial waste is both an economical and environmental solution,
Muthulakshmi et al., (2011) tested the production of proteases by SSF using
different industrial waste, such as cottonseed, rice straw and bran wheat. They
found that the bran wheat residue generated better results with respect to
enzyme production. Therefore, further studies were made in order to optimize
fermentation par ameters. The cultivation was done at pH 5.0 for 7 days at
30°C with a supplement of 3% KNO3 as the nitrogen source, inoculum size 3%
and 3% of substrate concentration that yielded 170 U/mg of protein using the
fungus Aspergillus flavus.

2.2. Submerged Fermentation
The SmF is performed in a liquid nutrient medium, where the
microorganism is submerged.. As it is held in liquid medium and not solid,
some microorganisms such as bacteria end up being favored because they
require greater degree of moisture to survive that is not present in solid
fermentation (Singhania et al., 2009; Thomas et al., 2013). Furthermore, the
extraction of the final product, enzymes or secondary metabolites, is easier and
less costly than the SSF method, in which it is necessary to extract the product
from the cultured microorganisms therein. However, the use of industrial


8

Cristiano José de Andrade, Ana Elizabeth C. Fai B. de Gusmão et al.

waste for production of enzymes by SmF is not as easy as SSF.Several studies
have been published comparing the production of certain enzymes by these
two methods of fermentation, suggesting that in each case, a preliminary study
is required to determine the most suitable one. A study published by Hashemi
et al., (2013) showed significant differences between the production of αamylases from Bacillus ssp. by SSF and SmF. It was observed that for solidstate fermentation, the optimum pH for enzyme activity was 4 and the
temperature was 70°C whereas for the enzyme produced by SmF the optimum
pH was around 3.0 at 60°C. Furthermore, it was observed that for the enzyme
produced by SSF, pH and temperatures led to lower enzyme activity in 45.8 ºC
and pH 5.5, while for the enzyme produced by SmF lowered activities were
found in 74.1°C of temperature and pH 5.5. This study shows the importance
of studying the enzyme production by fermentation processes because each
has specific characteristics that may be advantageous or not.

2.3. Enzyme Expression Systems and Engineering
Regarding productivity and production cost, with the steady growth in the
use of enzymes in various industrial processes as a sustainable solution (Adrio
et al., 2014), there is the need to create production systems that are compatible
with the requirements of industrial processes. Due to advances in
biotechnology, it was possible to create enzymatic production systems based
on enzyme’s gene cloning in microorganisms with specific characteristics of
protein expression, inhibiting resistance to temperature changes and pH
(Fernandes et al., 2010). There are many different microorganisms that can be
used for this purpose, so it is important to understand the differences between
then.The bacterium Escherichia coli has been widely used for enzyme
production for having the ability to grow to high cell densities in a short time,
accumulate about 50% of its dry weight in heterologous enzymes, grow in
cheap culture media and for being easy to handle. These characteristics make

it a good microorganism for the industrial production of enzymes, however,
there are some disadvantages, such as its inability to perform post-translational
modifications, which are responsible for correct protein folding and therefore
relates to the enzyme activity (Adrio et al., 2014). Because of this, it is
important to evaluate the enzyme amino acid sequence to make sure that E.
coli will be able to produce it in the active form. According to Morrow,
(2007), an alternative to the use of E. coli is the yeast Picchia pastoris, which
also has the ability to produce high levels of heterologous enzymes and may


An Overview on the Main Microbial Enzymes in the Food Industry

9

reach 30 g/L. Picchia pastoris is also capable of performing posttranslational
modifications, checking the correct folding of the heterologous enzymes.
Other widely used yeast is Saccharomyces cerevisiae, which like P. pastoris,
also carries out post-translational modifications and has high levels of enzyme
production.
Nevertheless, P. pastoris offers several advantages over S. cerevisiae
such as a responsive promoter that allows methanol tight control of
heterologous protein expression and greater capacity to integrate exogenous
DNA fragments to the genome, facilitating the construction of stable strains
capable of producing the enzymes (Adrio et al., 2014). One example of the
utilization of P. pastoris as an industrial enzyme productin system is with the
enzyme laccase. Laccases are used in the food industry for wine and beer
stabilization and therefore require a suitable production system that is capable
of supplying the industrial needs. Kittl et al., (2012) enabled the production of
a laccase from Botrytis aclada by cloning its gene in P. pastoris, reaching the
production of 0.495 g/L of the protein in the extra cellular medium which

was at the time the highest concentration of this protein obtained from a
heterologous expression system with P. pastoris. Lipases, enzymes used, for
example, for the manufacture of margarine from fat-oil blends, were produced
with P. pastoris under the control of the methanol responsive promoter as high
concentrations of 20 U/mL in the extracellular medium (Shi et al., 2010).
Due to advances in genetics and biotechnology, it is now possible not
only to improve the production of the enzymes as described above, but it is
also possible to improve various characteristics of the enzyme itself such as
stability, which is intrinsically related to the amount used in the process,
ability to act in a higher pH range without losing activity and greater
specificity for certain substrates, among other features. Two approaches can be
employed in order to improve enzymes features.
The first approach is (a) directed evolution and second (b) rational protein
design. The first is based on a set of techniques that allow the insertion of
random mutations in the genome of the host to be selected, aiming the
different clones of the microorganism that have obtained the best mutations
that somehow have improved the protein that was meant to be improved. In
this case, a vast knowledge of the enzyme itself is not necessary in order to
improve it, as the technique is based on the random insertion of mutations into
the sequence of the enzyme and subsequently selected, making this a versatile
technique. An example of this technique is with glucoamylases, enzymes
responsible for starch processing. The starch is liquefied at 105°C in the
presence of α-amylases but has to be cooled to 60°C so that the glucoamylases


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Cristiano José de Andrade, Ana Elizabeth C. Fai B. de Gusmão et al.

may be used. In order to eliminate this cooling step which is costly for

the industry, many researchers applied directed evolution technique in this
enzyme to increase its termostability. Another example is the application of
the enzyme glucose isomerase derived from Thermotoga neapolitana for
production of sweeteners. This enzyme has optimum activity in media with
neutral pH and at temperatures around 100°C, however, in the industry, the
isomerization of glucose occurs in an alkaline media and at about 50°C. With
the use of directed evolution, a more thermostable glucose isomerase, rather
than the parental one and active at alkaline pH and 50°C, was selected by
Sriprapundh et al., (2003).
The second approach used, the rational protein design, was only possible
due to advances related to the understanding of protein structures and their
functions, which was accompanied by the development of bioinformatics. This
technique is based on the rational insertion of changes in the protein’s
aminoacid sequence. To do that, previous bioinformatics analyzes has to be
done to indicate which changes could improve the characteristics of that
enzyme.. Unlike the first approach of directed evolution, it is necessary to
have thorough knowledge of amino acid sequence of the proteins, the structure
and functionality of each part of the enzyme that will be modified (Adrio et al.,
2014; Fernandes et al., 2010). An example of this technique was the
improvement of other glucose isomerase, when the rational insertion of two
point mutations (G138P and G274P) culminated in an enzyme that had a
higher specific activity, increased half-life compared to the parental and
greater thermostability (Zhu et al., 1999). Although these two techniques that
could generate improved enzymes, both are not mutually exclusive and can be
used in a complementary manner (Adrio et al., 2014).

3. MICROBIAL ENZYMES IN FOOD INDUSTRY
Although enzymes are easily found (in animals, plants, microorganisms),
there are many advantages of using microbial enzymes instead of animal and
plant enzymes such as better stability, higher productivity (shorter generation

times), easier scaling up and genetic manipulation, more robust production
conditions, less environment impact. Also, they are not affected by seasonal
fluctuations and can be produced using inexpensive media (Andualema and
Gessesse, 2012). In this sense, examples of microorganisms commercially
exploited as enzyme sources are fungi such as Aspergillus, Candida, Mucor,
Penicillium and Saccharomyces, and bacteria such as Bacillus, Klebsiella,


An Overview on the Main Microbial Enzymes in the Food Industry 11
Lactobacilli, and Streptomyces (Ackaah-Gyasi et al., 2015; Pant et al., 2015;
Ray et al., 2012; Abou-Elela et al., 2011). Thus, microbial enzymes that are
applied in food industry were classified in 3 groups (carbohydrate-active
enzymes, lipases and proteases) and described below.

3.1. Carbohydrate-Active Enzymes
Carbohydrates are the main components of food, and they can be found as
natural components or as added ingredients. Carbohydrates show many
different molecular structures, sizes, shapes and exhibit a variety of chemical
and physical properties. They can be modified by enzymatic process, and these
modifications are employed in food industry for improving their properties
and extending their uses (BeMiller and Huber, 2007; Park et al., 2008; Sathya
and Khan, 2014). The relationship between the major carbohydrates (foods)
and carbohydrate-active enzymes (cellulases, hemicellulases, amylases and
pectinases) is described below.

3.1.1. Cellulases
Cellulases is a collective term referring to a group of enzymes that
hydrolyze the β-1,4-glycosidic linkages in cellulose and produce as primary
products glucose, cellobiose and cello-oligosaccharides. Cellulolytic enzymes
consist of three major components: (i) endoglucanases (EC 3.2.1.4), (ii)

exoglucanases or cellobiohydrolases (EC 3.2.1.91) and (iii) β-glucosidases
(EC 3.2.1.21) (Lynd et al., 2002; Phitsuwan et al., 2013). The synergistic
action of these three enzymes is required for the complete hydrolysis of
cellulose to glucose. Endoglucanase randomly hydrolyses internal β-1,4
linkages of cellulose chains and creates new reducing and nonreducing ends.
Exoglucanase cleaves disaccharide cellobiose from the nonreducing end and in
some cases from the reducing end of the cellulose chain. These cellobiose
units and short-chain cellodextrins are hydrolyzed by β-glucosidase into
individual monomeric units of glucose (Singhania, 2011).
Microbial cellulases are inducible enzymes synthesized by a large
diversity of microorganisms during their growth on cellulosic material,
but bacteria and fungi appear to be the major sources (Kuhad et al.,
2011). Nowadays, the main commercial preparations of cellulases are
obtained from filamentous fungi, such as Aspergillus niger and Trichoderma
reesei. Other well-known cellulase-producing microorganisms include
species from Penicillium, Fusarium, Neurosposra, Humicola, Cellulomonas,


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Cristiano José de Andrade, Ana Elizabeth C. Fai B. de Gusmão et al.

Thermomonospora, Clostridium, Bacillus and Streptomyces (Bhat and Bhat,
1997; Sukumaran et al., 2005; Kuhad et al., 2011; Gupta et al., 2013; Behera
and Ray, 2016).
Cellulases have a wide range of potential applications in food industry.
One of these applications is in the beverage industry for the production of fruit
and vegetable juices. In this case, cellulases have been used together with
hemicellulases and pectinases, collectively known as macerating enzymes, for
extraction and clarification of juices (Bhat, 2000; Karmakar and Ray, 2011).

The combined use of cellulolytic, hemicellulolytic and pectinolytic enzymes in
juices facilitates the complete liquefaction of plant tissues. Thus, it is possible
to directly filter the juice from the pulp without any need for pressing and still
increase the efficiency of extraction (Lozano, 2006; Fleuri et al., 2015; Molina
et al., 2015). The addition of these enzymes in juices processing have also
helped to reduce viscosity, improve cloud stability and aromatic properties of
the fruit juices and their pulps during processing, and increase the release of
color components from the skins of fruits (Lozano, 2006; Jurutu and Wu,
2014).
Yu and Rupasinghe (2012) investigated the effects of enzymatic mash
maceration with commercial cellulases and pectinases as a pre-treatment
for making carrot juice. The authors observed that the supplementation of
pectinolytic enzymes with endoglucanases and β-glucosidases increased about
27% the carrot juice yield when compared with untreated juice. The enzymatic
pre-treatment reduced the viscosity and turbidity of the carrot juice, and, in
addition, increased the total soluble solids content (about 20%) and the
recovery of β-carotene (1.6 times).
Cellulases, mainly β-glucosidases, have also been used with hemicelulases
(arabinofuranosidase, α-L-rhamnosidase, β-xylosidase) in beverage industry to
increase the aroma and improve sensory characteristics in wines, musts and
fruit juices (Palmieri and Spagna, 2007; Pgorzelski and Wilkowska, 2007).
The aroma in wines and grape juices, for example, are due to the presence of
compounds like terpenes, norisoprenoids, aliphatic alcohols and phenolic
(Slegers et al., 2015). These compounds are usually present in a free
fraction, which contributes directly to must aroma, and in a bound fraction to
various sugars, which is non-volatile and flavourless. This bound fraction
is quantitatively more significant than the free fraction and represents a
significant reservoir of aromatic precursors, which can be released by acid or
enzymatic hydrolysis (Romo-Sánchez et al., 2014). The enzymatic hydrolysis
using β-glucosidases is considered a promising method for enhancing the

aroma because these enzymes can selectively and efficiently release the


An Overview on the Main Microbial Enzymes in the Food Industry 13
glycosidically bound compounds or aroma precursors without modifying or
rearranging aglycones (Ferner et al., 2016).
Li et al., (2013) studied the influence of β-glucosidase on volatile profiles
in mango wine, and observed that the addition of this enzyme accelerated the
release of volatile substances such as terpenols, acetate esters, benzene
derivatives and C13-norisoprenoids. These authors related that enzyme-treated
wines presented enhanced terpenols by up to ten times and acetate esters by up
to three times. Furthermore, enzyme treatment mitigated, by up to five times,
the formation of medium-chain fatty acids and ethyl esters to moderate levels.
Another field of application of cellulases in foods is the bakery industry.
The use of these enzymes has enabled increased volume and improved quality
parameters of bakery products. In addition, cellulases can replace the use of
chemical bread conditioners (Nadeem et al., 2009; Kuhad et al., 2011). Haros
et al., (2002) investigated the effects of carbohydrases, including cellulases, on
fresh wheat bread characteristics, and they observed that the breads treated
with cellulases showed an increase in the specific volume and required slightly
lower fermentation times to reach the optimum volume compared to the
control. According to these authors, the cellulase supplementation also
modified the texture of breads, reducing the crumb firmness of the fresh bread,
and provided an antistaling effect during storage.
Cellulases can also be used to improve extraction of oils and other
important compounds to food industry from plants. According to Sharma et
al., (2015), around 24% of the olive oil is not extractable using the existing oil
extraction technologies. However, when these authors applied one enzymatic
treatment with pectinase and cellulase (1:1) at 0.05%, it was possible to
increase the oil recovery in 11%, improve sensory quality (appearance and

clarity) and enhance total phenols content as compared with nonenzyme
treatment. In another study, Ribeiro et al., (2012) evaluated the development
of an enzymatic method of extraction of caffeine from the guarana seed
(Paulliniacupana) for use in energetic drinks as an alternative to the traditional
process, based on the extraction with a hydroalcoholic solution. Non-alcoholic
guarana extracts with low tannin concentration and high caffeine contents
were obtained with the use of cellulases in combination with hemicellulase
and α-amylase.

3.1.2. Hemicellulases
Hemicellulose refers to a group of homo – and heteropolysaccharides
consisting of xylose, mannose, glucose and galactose main chains with a
number of substituents resulting in a structurally complex polymer (Manju and


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Cristiano José de Andrade, Ana Elizabeth C. Fai B. de Gusmão et al.

Chadha, 2011). It includes xylans, mannans, xyloglucans and β-glucans
(Scheller and Ulvskov, 2010). Due to the heterogeneity and complex chemical
of the hemicellulose, its hydrolysis requires a significantly large number of
enzymes with diverse catalytic and modes of action. Hemicellulases include
endoxylanase (EC 3.2.1.8), β-xylosidase (EC 3.2.1.37), α-glucuronidase (EC
3.2.1.139), α-arabinofuranosidase (EC 3.2.1.55), arabinanase (EC 3.2.1.99),
acetyl xylan esterase (EC 3.1.1.72), feruloyl esterase (EC 3.1.1.73), βmannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), β-glucanase or
lichenase (EC 3.2.1.73), among others (Jurutu and Wu, 2013; Soni and Kango,
2013; Grishutin et al., 2006). These enzymes have been produced by
fermentation processes using fungi, yeast, actinomyces and bacteria (Polizeli
et al, 2005; Jurutu and Wu, 2013).

Xylans are the most abundant hemicelluloses, and consist of a backbone
of β-1,4-linked xylopyranosyl groups that are further decorated with different
side chain residues. Xylanases (endoxylanases and β-xylosidase) are central
enzymes in the degradation of xylans. Endoxylanases randomly cleave the
glycosidic linkages in the xylan backbone and β-xylosidase release xylose
monomer from cleavage of the nonreducing end of xylo-oligosaccharides and
xylobiose (Colins et al., 2005). Another hemicelulose generally found in
cereals, like barley, oat, rye, rice, sorghum and wheat gran, are the β-glucans.
These polysaccharides, that consist of cellotriosyl and cellotetraosyl residues
linked by β-1,3 and β-1,4-glucoside linkages, are hydrolyzed by β-glucanases
(or lichenases) and also endoglucanases (Grishutin et al., 2006).
One of the main fields of application of hemicellulases in food is the
bakery industry. Xylanases and β-glucanaseshave been employed in bread
making to break down the arabinoxylansand β-glucan present in flours and
improve some dough properties and bread quality. Kumar and Satyanarayana
(2014) demonstrated that the addition of xylanase produced by Bacillus
halodurans TSEV1 in whole wheat breads provides an increase in volume, a
greater absorption of water, an improvement in reducing sugar and protein
soluble contents, an increasing in shelf life and the liberation of
xylooligosaccharides. In another study, Li et al., (2014) studied the influence
of β-glucanase on the properties of dough and bread from 70% wheat and 30%
barley composite flour. According to these authors, bread with added βglucanase (0.04%) showed an increased specific volume (57.5%) and
springiness (21%), and reduced crumb firmness (74%) and staling rate. In
biscuit-making, xylanases allow cream crackers to be lighter and improve
some properties of the wafer, like texture, palatability and uniformity (Polizeli,
et al., 2005).