Production and Recovery of Enzymes for Functional Food Processing

Full text

219
Chapter 12
Production and Recovery of Enzymes
for Functional Food Processing
Leonardo Sepúlveda, Ramón Larios-Cruz, Liliana Londoño, Ayerim Hernández,
Berenice Álvarez, Nathiely Ramírez, Cristian Torres, Alberto Neira, José L. Martínez,
Janeth M. Ventura-Sobrevilla, Daniel Boone-Villa and Cristobal N. Aguilar
12.1 Introduction
Enzymatic activities have been used in the production of
food and associated commercial activities since ancient
times. The earliest enzyme-like activities reported were
from the foods of the Babylonians and Sumerians, related
to the production of alcoholic beverages from barley,
using whole-cell enzymatic activities (Singh et al. 2016).
It is well known that enzymes are useful tools in
many industries, such as the pharmaceutical, textile,
chemical, paper and biotechnology industries, among
others (Sundarram and Murthy 2014; Llenque-Díaz et al.
2015; Singh et al. 2017), due its potential to reduce the
duration of the process, and the necessity of energy, cost,
and waste formation in the management of catalysis for
production processes (Choi, Han, and Kim 2015). As the
AU: Th is section
is uncl ear – do
you mea n ‘ and
to reduc e energy
expenditure,
wast e formation
and cos ts ….?
Functional Foods and Biotechnology Production and Recovery of Enzymes for Functional Food
Processing
12.1 Introduction 219
12.2 New and Traditional Sources of Enzymes 220
12.2.1 Amylase Production 220
12.2.2 Protease Production 221
12.2.3 Lipase Production 222
12.2.4 Cellulase Production 223
12.3 Recovery and Application of Enzymes 223
12.3.1 Proteases 223
12.3.2 Amylase 224
12.3.3 Lipases 224
12.3.4 Cellulases 225
12.4 Mechanisms of Action of Hydrolytic Enzymes in the Food and Pharmaceutical Industry for
Improving Functional Properties 225
12.5 Perspectives for Enzyme Use in Functional Food 226
12.5.1 Proteases 226
12.5.2 Amylases 227
12.5.3 Lipases 228
12.5.4 Cellulases 228
12.6 Conclusion 228
References 229
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Functional Foods and Biotechnology
food industry evolves, there is a need to produce improved
quality and quantity of foods, which have led to the search
for new and better strategies and novel approaches to the
existing techniques, with aim of designing and develop-
ing more nutritive, functional, and digestible products.
Enzymes therefore have relevance in the continuous
development of the food processing industry, due to the
variety of sources and the wide-ranging catalytic effects
that can exert beneficial effects (Fernández-Lucas,
Castañeda, and Hormigo 2017; Hmad and Gargouri 2017).
These enzyme tools for the food industry are important,
with a global market worth 1.5 billion dollars in 2016,
which promises continued growth (Fernández-Lucas,
Castañeda, and Hormigo 2017).
The hydrolases (enzymes that cleave chemical
bonds by adding a water molecule) are a very important
category of enzymes for the food industry. This group
includes proteases, lipases, amylases and cellulases that
modify proteins, lipids, amylose and cellulose, respec-
tively. One of the more important characteristics is the
selectivity inherent to enzymes, that make these catalytic
proteins capable of processing and transforming a com-
plex matrix of food to more simple biotransformed prod-
ucts, making it more convenient and useful to the industry
and for end-use by the consumer (Singh et al. 2016). The
use of enzymes in industrial food processing exerts
effects over coagulation, ripening, baking, brewing, cell
rupture, hydrolysis, and other modification in molecular
structures with the scope to develop improved production
processes and to enhance yield, design new quality pro-
cesses for existing products, increase the digestibility of
products and obtain new bioactive compounds suitable as
functional ingredients in a range of food products (Ermis
2017).
In the industrial sector, food processing enzymes
can be obtained either by extraction from animal or plant
tissues or by fermentation, using a wide variety of micro-
organisms (Dodge 2010). Microbial sources have gained
the preference of producers as a result of their cost effi-
ciency and the variety of enzymes which can be produced
for diverse applications (Al-Mazeedi, Regenstein, and
Riaz 2013), and now comprise 90% of the 260 different
enzymes available in just the EU market (Fraatz, Rühl,
and Zorn 2014). In this context, Submerged Fermentation
(SbF) and Solid-State Fermentation (SSF) are the two
available strategies used for the production of enzymes
at an industrial level (Singhania et al. 2010); although SSF
has shown higher values in terms of enzyme activity and
production efficacy, selection of the method relies on the
need for efficient process development and producing the
correct enzyme.
An important issue in all food production processes
is the safety of the raw material and the final product.
Genera lly Recognized As Safe (GRAS) is a reg ulatory con -
cept from the US Food and Drug Administration, coined
in 1958, that reflects the harmlessness of any substance
reasonably expected to become a component of food and/
or affect its properties (Sewalt et al. 2017). Industrial
enzymes can play a role in being GR AS when being part
of modified/potential foods and/or for modifying food
properties. Therefore, enzymes used in industrial food
processing must hold the GRAS recognition to ensure
safety in their use for food processing. Nevertheless,
enzymes, as food additives or modifiers, align perfectly
with the concept of GRAS for several reasons: 1) the his-
tory of safe use of enzymes from ancient times in empiri-
cal food processes before the activity of enzymes was
understood; 2) the majority of enzymes used in the food
industry are obtained from microorganisms, these “bio-
logical sources” being produced and conserved under
controlled conditions, and belonging to safe strains, that
match the requirements of GRAS recognition; 3) many
toxicological studies of these products have been carried
out in animal models that have shown no evidence of any
negative effect; 4) the use of an enzyme has no or neg-
ligible environmental impact due its natural degradation
into amino acids; 5) even the enzymes from genetically
modified strains are suitable and can clearly be shown
to have GRAS characteristics (Sewalt et al. 2017); 6) the
enzymes that are safely produced are even suitable to be
considered as “Halal” (meaning lawful or permissible) as
approved food additives, by achieving the requirements of
the Islamic religious rules of food use (Ermis 2017).
In the present chapter, the current state of the art
concerning the production, recovery, application and the
future perspectives in the use of hydrolytic enzymes in
the food industry will be summarized, with particular
respect to the design and production of functional foods.
12.2 New and Traditional
Sources of Enzymes
12.2.1 Amylase Production
Amylases are widely used in industries including the tex-
tile, food, detergent and fuel alcohol sectors (Sundarram
and Murthy 2014). The production sources of these
enzymes could be animals, plants, or microorganisms.
Among microorganisms, fungi and bacteria are the most
used. De Castro and Sato (2013) produced α-amylase from
Aspergillus oryzae LBA 01 under SSF. The use of wheat
bran as a fermentation substrate resulted in the highest
amylase activity when compared with other substrates
such as soybean meal and cottonseed meal, singly or in
combination. It was also shown that properties of wheat
bran enhanced α-amylase activity in the same way that
water absorption index and particle size did. The water
absorption index was the highest in wheat bran and the
distribution of different particle sizes was uniform. Those
AU: Plea se
consider
intr oducing a
para graph brea k
here – sho rter
paragraphs are
easie r to read
and understa nd
AU: Plea se con-
sider i ntroduc-
ing a pa ragraph
break here
AU: The E nglish
lang uage in
this s ection has
been co rrected
but woul d the
follow ing be more
appro priate? ‘mu st
compl y with the
class ificati on of
GRAS…..’
AU: Plea se
consider including
the EC nu mber
aft er each tri vial
enz yme name at
first mention
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Production and recovery oF enzymes For Functional Food Processing
properties were related to being beneficial for enzyme
activity. The optimal conditions of fermentation were
30 °C incubation temperature with 50% of humidity for the
substrates (De Castro and Sato, 2013). Enzyme produc-
tion by other microorganisms or using other techniques
could be different with regard to the fermentation condi-
tions (De Castro and Sato 2013).
Another example of SSF for the production of
α-amylase was from Bacillus subtilis, using wheat bran as
the substrate. Fermentation conditions were 37 °C and 48
h fermentation. After production of the α-amylase, charac-
terization of the enzyme was performed, leading to partial
purification; the results showed maximum specific activ-
ity at 40 °C (13.14 µmol/mg/min) and an optimal pH of
7.1 (8.74 µmol/mg/min at 40 °C) (Raul et al. 2014). Saha
et al. (2014) improved the production of α-amylase, using
Bacillus amyloliquefaciens MTCC 1207 under SSF, with
wheat bran as substrate. Their results showed a higher
specific α-amylase activity at 37 °C (14.25 ± 0.24 U/mg)
than at other temperatures tested. The presence of cer-
tain ions, such as calcium (Ca+2), chloride (Cl−) and
nitrate (NO3−), during fermentation enhanced the yield of
α-amylase, as did the presence of sugar alcohols such as
d
-inositol and
d
-mannitol. Aspergillus oryzae S2 has been
used to produce two α-amylases (AmyA and AmyB) under
SSF (Sahnoun et al. 2015). The results from this study
showed another α-amylase, AmyC, which was an isoform
of AmyB. Characterization of AmyC revealed an enzyme
with a molecular weight of 172 kDa with four 42 kDa sub-
units. It had a molecular weight higher than the other iso-
enzymes (52–68 kDa).
Hashemi et al. (2013) found the highest α-amylase
production at 37 °C with 3824 U L−1 from a filamen-
tous fungus (Aspergillus awamori) in a single bioreac-
tor. Elsayed et al. (2016) enhanced the production of
α-amylase under submerged fermentation (SbF) with a
scaled bioreactor and B. amyloliquefaciens NRRL B-14396
as the producer. They moved from a 250-mL Erlenmeyer
flask (50 mL of working volume) to a 3-L stirred tank
bioreactor Bioflow III (2 L of working volume) with two
operational approaches, batch and fed-batch, and two fed-
batch strategies (constant feeding and increased feeding).
Their results showed an increase in the production yields
directly proportional to the increase in the fermentation
volume, as well as a rise in the specific activity. Using the
strategy of increased feeding, they started with an activit y
titer of 1950 µkat/L in Erlenmeyer flask and reached 8160
µkat/L with the increased feeding strategy in the biore-
actor, demonstrating a relationship between the working
volume and the activity yield (Elsayed et al. 2016). Salman
et al. (2016) optimized the medium for the production of
α-amylase by SbF with B. subtilis RM16. The addition of
starch as the carbon source increased enzyme production
achieved relative to that obtained by the use of glucose,
maltose or sucrose. Other important parameters were the
incubation temperature (40 °C) and pH (8.0), as well as
the use of yeast extract as the nitrogen source. The pres-
ence of metal ions, such as Mg+2 and Ca+2, also enhanced
enzyme production, whereas the presence of Cu+2 inhib-
ited enzyme activity.
The residues of plants are other sources of amy-
lases, and fruits can contain this activity. Saini et al. (2016)
extracted amylase from apple with a specific activity of
the crude extract of 0.2 U/mg; after partial purification
with ammonium sulfate precipitation and gel filtration,
the specific activity reached 4.76 U/mg. Characterization
of the enzyme showed that optimal conditions of tempera-
ture and pH were 35 °C and 6.0, respectively. The addition
of ions such as Ca+2 , Mg+2, and Mg+2 increased the amylase
activity, whereasHg+2 inhibited the activity of the enzyme.
12.2.2 Protease Production
Proteases are enzymes capable of hydrolyzing peptide
bonds in proteins and are also useful in the food, phar-
maceutical, and chemical industries, among others.
Microorganisms are usually the sources for the produc-
tion of proteases but residues of plants and animals can
also be sources. Karataş et al. (2013) increased the pro-
duction of protease under SSF by Bacillus licheniform-
mis ZB-05. Between the eight different substrates used
(including wheat bran, maize oil cake, millet, lentil bran,
orange peel, banana peel, and apple peel), rice husk pro-
duced the highest quantity of protease activity (469,000
U/g). Nisha and Divakaran (2014) produced protease
under SbF with B. subtilis NS. The optimal conditions for
protease production were pH 9.0 (123.5 U/mL), 40 °C cul-
ture temperature (117.4 U/mL), medium supplemented
with glucose (199.01 U/mL), beef extract as the nitrogen
source (118.42 U/mL), magnesium chloride as the min-
eral additive (149.29 U/mL), and a sodium chloride con-
centration of 7 % (128 U/mL). Pant et al. (2015) reported a
similar process with B. subtilis, where the maximum pro-
tease activity was achieved with galactose and peptone as
the carbon and nitrogen sources, respectively (236.31 and
175.03 U/mL per min, respectively). After partial purifica-
tion of the enzyme, the highest stability of protease was at
pH of 7.4 (143.73 U/mL per min) and 40 and 50 °C.
Hussain et al. (2017) produced protease from a
microorganism isolated and identified as B. subtilis.
Wheat bran, an agroindustrial waste material, was capa-
ble of achieving the highest yield of protease (0.7 IU/mL).
Further optimization of the protease production indicated
that the optimal conditions included maltose as the carbon
source (7.93 IU/mL) and peptone as the nitrogen source
(9.66 IU/mL). After partial purification of the enzyme, the
optimum pH value was 8.0 and the temperature at which
maximum activity was achieved was 57 °C. The effect of
metal ions on alkaline protease activity depended on the
AU: Shou ld this
be ‘5 0% moistur e
conte nt’? If so,
pleas e correct at
other m entions in
this chapter
AU: Par agraph
break?
AU: Do you
mean t hat
chan ges in such
conditions could
aff ect enzy me
production??
AU: Plea se indica te
what th e error bars
denote – s tandard
error, st andard
deviation?
Alternatively,
please consider
delet ing the err or
bars.
AU: Plea se
consid er using the
actu al units , to
allow c omparis on
with t he B. subtil is
enzyme
AU: Diva lent and
tri valent ions
are usually pre-
sente d as Ca2+..
Plea se address
this t hroughout
the ma nuscript
AU: Can it t hen be
descr ibed as an
isoform of AmyB?
AU: Par agraph
break?
AU: Previously,
you have be en
using t he equiva -
lent of U/L i n this
chapt er. Please be
consistent
AU: Is th is correct ?
AU: Hav ing
dif ferent unit s of
enz yme activ ity
makes c ompari -
sons diffic ult
AU: Diva lent
and tr ivalent
ions ar e usually
prese nted as
Ca2 +.. Please
check us age
throughout
cha pte r.
AU: Do you
mean ‘e nzyme
acti vity’ or
‘enz yme
production’?
AU: Par agraph
break
AU:
Alpha-amylase?
AU: mg pro tein?
AU: Do you
mean ‘a ctivit y’
or ‘stability
AU: Would it
be appr opriate
to incl ude the
acti vities her e
too?
AU: New pa ra-
grap h break?
AU: Th is is
anot her activ ity
unit , which can
make it d ifficu lt
to comp are
resul ts. Pleas e
consider makin g
consistent.
AU: Th is is the
first m ention of
dif ferent ty pes
of prote ase –
please consider
descr ibing aci d
and alkaline pro-
teas es (and their
EC numb ers)
earlier.
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Functional Foods and Biotechnology
ion. The ions Ca+2 and Mg+2 increased the protease activ-
ity, whereas Hg+2, Cu+2, Zn+2, and Fe+2 inhibited enzyme
activity. Chatterjee et al. (2015) obtained a lkaline protease
by SSF of B.subtilis ATCC 6633, using wheat bran as the
substrate. Protease yield was maximal at 48 h of fermen-
tation, 40 °C incubation temperature, and the presence
of yeast extract (1 %) as the nitrogen source. The maxi-
mum stability of the protease with respect to temperature
was 50 °C (907.28 AU/mL), and to pH was between 7.0
(807.28 AU/mL) and 10.0 (750.9 AU/mL). The presence
of ions such as Ca+2 , Cl−, NO3−, and SO4−2 did not affect the
protease activity, nor did the addition of inhibitors such as
ethylenediaminetetraacetic acid and phenylmethanesul-
fonyl fluoride affect protease activity under the concentra-
tions used in the study.
Dey, Bhunia, and Dutta (2016) improved the opera-
tional conditions of a 2.2-L bioreactor to produce a pro-
tease from B. licheniformis NCIM-2042. The maximum
protease production was found at 2 vvm (airflow) and a
dissolved oxygen concentration maintained above 30 %.
de Castro et al. (2015) studied the production of a prote-
ase by SSF from Aspergillus niger LBA 02, using different
combinations of four substrates. Optimal fermentation
conditions were 50 % humidity and 30 °C for incubation
temperature. The use of single substrates (wheat bran,
soybean meal, cottonseed meal or orange peel) did not
favor enzymatic production, with the highest protease
activity being found with a mixture of equal parts wheat
bran and soybean meal after 48 h of fermentation, with a
protease activity of 262.78 U/g.
12.2.3 Lipase Production
Lipases are enzymes obtained from microorganisms,
preferably cultured in the presence of high-fat substrates.
These catalytic proteins are present in various agroindus-
trial residues which can be used as a source of carbon and
energy for the production of lipases from microorganisms
(Aceves-Diez and Castañeda-Sandoval 2012). The l ipolytic
bacteria Acinetobacter haemolyticus NS02-30 was isolated
from various oil-contaminated soils, olive pomace-soil,
and olive pomace. T he production of lipase was carr ied out
in 100 mL NB medium inoculated with 2 % inoculum of an
overnight culture and incubated in a 250-mL Erlenmeyer
flask for 24 h, incubated at 30 °C and shaken at 130 rpm.
The enzyme obtained had the ability to hydrolyze vari-
ous edible and waste oils, making it suitable for applica-
tions in the field for lipid degradation (Sarac and Ugur
2016). In another study, the fungus Rhizopus microspo-
rus was selected as a good lipase producer in SSF. The
culture medium included rice straw, rice bran, and olive
waste, which are agricultural byproducts, used as solid
substrates for SSF. Samples (10 g) of mixed substrates
were moistened with Czapek-Dox medium in a 500-mL
Erlenmeyer flask to reach a final moisture content of 70
% (w/v). The authors concluded that the highest recovery
yield (92.3 %) was obtained with 30 % (w/w) crude load at
pH 8.0. On the other hand, cultures of Fusarium solani
were used in a 250-mL Erlenmeyer flask containing 50 mL
sterilized olive mill wastewater. Different sources of nitro-
gen, in the form of (NH4)2SO4, yeast extract, or soybean
peptone, were investigated. After inoculation with 1 %
(v/v) of F. solani suspension, cultures were incubated aer-
obically for 5 days on a rotary shaker at 60 rpm at 30 °C.
Under these conditions, the lipase activity was 14 U/mL.
The results confirmed the potential application of the
strain for waste treatment, by its use in lipase production
(Jallouli and Bezzine 2016). Further isolates of bacteria
were screened for lipase production, using the rhodamine
B agar plate method. Bacteria were inoculated and incu-
bated at 28 °C for 2 days. In this study, eight variables,
including carbon sources, nitrogen sources and lipase
inducers were selected on the basis of their effect on the
increased secretion of lipase using a Plackett-Burman
experimental design. The results indicated that, under
these conditions, the activity obtained was 11.49 U/L (Kai
and Peisheng 2016).
Another focus for lipase production has been to
explore the most cost-efficient and optimal medium com-
position for the production of lipase from Pseudomonas
fluorescens NRLL B-2641 culture grown on sunflower
oil cake by applying response surface methodology
(Tanyol, Uslu, and Yönten 2015), using culture medium
variables such as 5 %–15 % (w/v) carbon, 0 % - 2 % (w/v)
peptone and 0 % - 1 % (w/v) ammonium sulfate. The pH
was adjusted to 6 and fermentations were carried out in
250-mL Erlenmeyer flasks containing 100 mL of fermen-
tation medium. After sterilization, each reactor was inoc-
ulated with 10% (v/v) of cell suspension and incubated at
30 °C with agitation at 150 rpm. The authors concluded
that, under these conditions, the maximum lipase activ-
ity achieved was 10.8 U/mL (Tanyol, Uslu, and Yönten
2015). A lipase was partially purified and characterized
from Geobacillus stearothermophilus AH22. This strain
was inoculated into a Luria-Bertani medium in a conical
flask and incubated at 55 °C with shaking at 150 rpm for
16 h. The lipase yield achieved was greater by more than
80 % when the process was carried out at pH 8 and 50 °C.
The enzyme described in this study could be applied to
treat lipid-rich industrial effluents or to synthesize vari-
ous useful chemical compounds (Ekinci et al. 2015). A
crude enzyme was obtained by SbF of Penicillium cyclo-
pium in 300-mL Erlenmeyer flasks containing 100 mL of
medium consisting of 1.5 g milled sunflower seed, 0.2 g
(NH4)2SO4 and 1.36 g KH2PO4, at pH 4.5. The medium
was inoculated with 1 x 106 spores/mL and incubated
at 28 °C for 4 days. The results showed that extraction
yields were over 90 % in comparison with another type of
lipase (A ntov, Ivetić, and Knežević Jugović 2016).
AU: Th is is
another different
unit.
AU: New pa ra-
grap h break?
AU: Plea se
DEFI NE THIS
ABBREVIATION
AU: Is th is
revisión correct?
AU: ‘Hu midity ’
or ‘mois ture
content’
AU: Plea se be
consis tent with
prese ntation
styl e – should it
be ‘/g protein’ ?
AU: do you mea n
that high-fat sub-
strates, rather
tan t he enzym es,
are pr esent in
agroindustr ial
residues?
AU: Wh ich
species?
AU: Wh ich
conditions gave the
highest activity?
AU: Do you me an
‘highest activity’
AU: New pa ra-
grap h break?
AU: Plea se indica te
which c ombina-
tions of v ariable s
gave th e highest
activ it y.
AU: Wh at was the
original pH?
AU: It is unc lear
what you m ean by
this h ighlight ed
section.
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Production and recovery oF enzymes For Functional Food Processing
12.2.4 Cellulase Production
Lignocellulosic residues can be used as a source of car-
bon and energy for obtaining hydrolytic enzymes, such
as cellulases, using microorganisms and a fermentation
system. These enzymes have broad applications in the
area of food and other bioprocesses (Llenque-Díaz et al.
2015). This section describes the most relevant results for
cellulase production, highlighting the fermentation condi-
tions for the production of these hydrolases from agroin-
dustrial residues. In one study of the potential of a mutant
T. viride strain HN1 for cellulase production from cheaper
agroindustrial waste, fermentation was carried out in
Vogel´s medium and the pH was adjusted to 5.5. Cultural
conditions such as substrate concentration, inoculum
concentration, fermentation period, initial pH, tempera-
ture, surfactants, and metal ions were optimized for cel-
lulase production. The optimal conditions to achieve the
objective were 6 % substrate, 5 days of fermentation, pH 5,
35 °C, 5 % of inoculum, and the presence of Tween 80 and
Mn+2(Iqtedar et al. 2015).
In another study, Streptomyces griseorubens JSD-1
was used for the pretreatment of rice straw. Furthermore,
the Plackett-Burman experimental design was used and
the independent variables were rice straw, wheat bran,
yeast extract, peptone, CaCO3, NaCl, MgSO4.7H2O and
Tween 20. Under these conditions, the maximum cellu-
lase activity was 269.53 U/mL. The results indicate that
the cellulases had great saccharification efficiency (above
88 %) (Zhang et al. 2016). In a separate study, the model-
ing and optimization of production of a lignocellulolytic
enzymatic cocktail by Cotylidia pannosa under SbF was
evaluated. Enzyme production was carried out at different
pH values and cult ure temperatures for an incubation time
of between 24 and 120 h and an agitation rate of 50–150
rpm, based on the central composite design. A maximum
enzyme activity of 20 U/L for cellulase was obtained when
the fungus was cultured under conditions of pH 5, 30 °C,
140 rpm agitation, and 72 h incubation, using 2 % wheat
bran as the substrate (Sharma, Garlapati, and Goel 2016).
In another study, two bacterial strains were isolated and
identified for the production of cellulase. Cellulase pro-
duction from a strain of Saccharomyces cerevisiae MTCC
4779 has been evaluated. The cellulase was produced by
inoculating S. cerevisiae in a 250 -mL Erlenmeyer fl ask con-
taining 100 mL sterilized Czapek-Dox medium, incubated
at 25 °C under agitation at 200 rpm for 72 h. The enzy-
matic extract obtained was used to hydrolyze the banana
(Musa acuminata ‘Cavendish’) pseudostem within 30 h,
showing potential to be a reliable catalyst for the pretreat-
ment of lignocellulosic biomass (Seenuvasan et al. 2017).
In another study, optimization of cellulase produc-
tion from isolated cellulolytic bacteria was evaluated.
Carboxymethylcellulose concentration, yeast extract, pH,
and incubation temperature were the significant variables
assessed by the Plackett-Burman design and further opti-
mized using a central composite design. The optimum
conditions produced an enzyme activity titer of 3.55 U/
mL, that was 2.8 times that from the un-optimized system
(Parkhey, Gupta, and Eswari 2017). Using natural humic
straw as a substrate, a microorganism was isolated from the
low-temperature area in the northeast of China that could
produce cellulase efficiently. The bacterium was identified
as a strain of Pseudomonas mendocina. For enzyme produc-
tion, an enriched mineral medium with the inoculum at
1 × 10−3 ~ 1 × 10−8 was used for 72 h at 30 °C. The effects of
temperature, pH and heavy metals on enzymatic produc-
tion were evaluated. The fermentation parameter values
that most favored enzyme production were pH 7.5, 28 °C
and the presence of Pb+2. Under these conditions, activity
titers reached values above 80 U/L (Zhang et al. 2016).
12.3 Recovery and
Application of Enzymes
The applications of enzymes to different industries, like
the cosmetics, pharmaceuticals, textiles, food, agricul-
ture or even the paper industry, have been rising in recent
years (Castellanos 2006; Amaro et al. 2015), due to their
ability to catalyze chemical reactions, in so many biologi-
cal processes. Incremental improvements in activity and
yield have resulted i n increased demand for the production
of this class of catalysts to satisfy the requirements of the
bioprocessing sector (Rojo et al. 2007; Gómez et al. 2016;).
Nevertheless, the most difficult part is not the production,
but the challenges represented by the subsequent down-
stream processing, in terms of the separation and purifica -
tion of the product (Rodrigues et al. 2017). It is estimated
that the expenditure to achieve an adequate purification
grade enzyme involves about 60 % of the total cost of the
enzyme preparation, without including the cost of the raw
materials (Lee 1992; Chi et al. 2009). Protein responses
to environmental changes, light, ionic potential, pH, and
temperature is superior to that of other catalyst products
(Shapovalova et al. 2016). For this reason, the search for
improved strategies for bioseparation, in order to obtain
high levels of purification which do not compromise their
enzymatic activity, is required. Furthermore, understand-
ing the uses each enzyme can have in different areas of
biotechnology is needed to achieve more versatile use of
these biocatalysts.
12.3.1 Proteases
Proteases represent a biological catalyst which func-
tions in the hydrolysis of peptide bonds in proteins to
release peptides and/or amino acids (Theron and Divol
2014). They represent an important group of enzymes
AU: Plea se
descr ibe what the
mutation i nvolved.
Au: Pl ease indic ate
the concentrat ion
of Tween 80 us ed
AU: Th is sentence
is inco mplete
for desc ribing
this s tudy –
which s trains?
Conditions?
Reference. Please
expa nd or delete
this h ighlight ed
sentence
AU: New pa ra-
grap h break?
AU: Plea se
includ e the units
AU: Plea se pro-
vide reference
for the c itation
Lee 1 992.
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Functional Foods and Biotechnology
for bioprocessing and are commercially relevant with
respect to a vast number of applications in the dairy, food,
leather and pharmaceutical industries (Kasana, Salwan,
and Yadav 2011). A large array of assay protocols is avail-
able in the literature. With the predominance of molecu-
lar approaches for the generation of better biocatalysts,
the search for newer substrates and assay protocols that
can be conducted at the micro/nano scales are becoming
important (Gupta et al. 2003b).
Enzyme purification represents a challenge due
to the dilute and labile nature of the mixed products in
the medium, and their separation from contaminants
usually involves the application of chromatographic tech-
niques such as gel filtration chromatography (Sandhya,
Nampoothiri, and Pandey 2005). For example, a serine
alkaline protease was isolated, purified and character-
ized from the culture filtrate of the thermophilic fungus
Thermomyces lanuginosus. This enzyme was precipitated
by iso-propanol and further purified by gel filtration
chromatography through Sephadex G and ion-exchange
column chromatography on diethyl amino ethyl (DEAE)-
cellulose, with a yield of 30.12 % and 13.87-fold purification
(Ghareib et al. 2014).
12.3.2 Amylase
In 1894, the first enzyme produced at an industrial scale was
an amylase, and, over the years, amylases have become one
of most used enzymatic families in bioprocessing (Pandey
et al. 2000; MacGregor et al. 2001). The production process
employs di fferent tec hniques, wit h the use of plant a nd micro-
bial models (Tallapragada et al. 2016) to purify the same
enzymes. Generally, methods used involve protein precipita-
tion with diverse solvents and sequential chromatographic
processes in their different modalities (Gupta et al. 2003a;
Mathew and Rathnayake 2014). To purify an amylase pro-
duced by a Bacillus sp. strain, precipitation was performed
with ammonium sulfate, obtaining a product with a molecu-
lar weight of 77.6 kDa (Quintero et al. 2010). However, for an
α-amylase from B. licheniformis, 40.4 % y ield of the α-amylase
with a molecular weight of 55 kDa was obtained by a fermen-
tative process on a laboratory scale, followed by ammonium
sulfate precipitation and fast protein liquid chromatography
(FPLC) (Ul-Haq et al. 2010). Using an amylase-rich source
in the form of roots of Paederia foetida, a plant used as a spice
in Thailand, the enzyme was purified through DEAE col-
umn chromatography and shown to have a molecular mass
by SDS-PAGE of 60 kDa (Sottirattanapan et al. 2017).
12.3.3 Lipases
Lipases are a group of enzymes that have wide industrial
applications, from their use in beverages, to improve the
aroma, to their use in leather products, where hydrolysis
of lipid-rich material occurs during the tanning process.
Nowadays, investigations on lipases are focused on struc-
tural elucidation, mechanism of action, characterization
and recovery, among other important aspects. Most of
the industrial lipases are enzymes from microorganisms,
which have versatility due to the catalysis of many organic
reactions, giving rise to important products with differ-
ent properties that can be used in various sectors, such as
medicine, agriculture and food.
There are different systems of separation of lipases
due the different production systems, and the method
selected depends on the final application. The most com-
mon purification method is ammonium sulfate precipita-
tion, where the precipitated lipase protein is separated,
dialyzed against distilled water and then lyophilized to
obtain a dry lipase powder. Many authors have reported the
use of this technique, like Kanwar, Gogoi, and Goswami
(2002), who obtained yield percentages of 59% for a
Pseudomonas lipase with 758 mg of total protein with an
activity of 14,750 U. Ionic and affinity chromatography are
other techniques widely employed, because of their high
loading capacities, which are mainly based on the estab-
lishment of strong electrostatic interactions between the
enzyme and the matrix of the chromatographic column.
Farooqui, Yang, and Horrocks (1994) reported the use of
heparin-Sepharose chromatography and their interactions
with lipases. Among other techniques are filtration, elec-
trophoresis and reverse micelle approaches (Ventura et
al. 2011). However, some of these techniques are not eas-
ily scaled up or are highly expensive, so alternatives have
been sought for the recovery of lipases. One method of
emerging potent ial involves two -phase systems, such as liq-
uid-liquid extraction. These methods allow rapid and selec-
tive separation of biologically active molecules, including
these enzymes. Duarte et al. (2015) reported variations of
aqueous systems, based on alcohols, polymers, copolymers
or surfactants (Ventura and Coutinho 2016). Separation in
the system is based on exclusion when two polymers are
mixed and affinities are changed; when this occurs, poly-
mers tend to separate into two different phases, due to ste-
ric exclusion. Variations in the extraction methods are due
mainly to the variables used, such as pH, temperature, addi-
tion of salts and quality and quantity of polymers, among
others (Barbosa et al. 2011). Duarte et al. (2015) reported
a two-phase liquid-liquid extraction system for separating
lipase activity between the upper and lower phases. Three
systems were proved to be relevant: polyethylene glycol
(PEG) 4000/sodium phosphate buffer, obtaining lipase
activity of 0.65 U mL−1, PEG 1500/polyacrylic acid (PA),
showing a greater activity of 0.5 U mL −1 , with activity being
restricted to the upper phase in both cases, and Triton
X-114/McIlvaine buffer, where the reported lipase activity
was 0.45 U mL−1 for the upper phase and 1.30 U mL−1 in the
lower phase at 28°C Duarte et al. 2015). Authors compared
AU: Plea se
consider a
para graph brea k
here
AU: Thou gh the
inclus ión of the
author ity name
at firs t mention
of a binom ial is
laudab le, you
would ne ed to
be cons istent
and do t he same
for al l binomials
thro ughout the
manus cript,
which h as not
been don e. I
would recom-
mend deletion
AU: Plea se con-
sider ex pressin g as
spec ific acti vity to
allow compar isons
with r esults fr om
other studies.
AU: Prev iously in
this c hapter, you
wrot e this as U/
Ml. It s hould be
consis tent. Ple ase
revie w usage in
chapt er and fix
where appropriate.
AU: Th is is lower
than 0 .65 U/l
Au: Do yo u mean
‘T he authors’,
mean ing Duar te
et al. ?
TNF_12_K338166_C012_docbook_new_indd.indd 224 03-Jan-20 21:18:06

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Production and recovery oF enzymes For Functional Food Processing
the enzymatic activity of the lipase isolated by the PEG/
sodium phosphate and PEG/PA systems, finding that the
PEG/sodium phosphate system showed the best results
in terms of both enzymatic activity and recovery percent-
age. For the Triton X-114 system, the KLip was 0.75 at 25 °C,
compared with the value of 0.68 observed at 28.0 °C. This
result indicated that, at 25 °C and pH 7.0, the enzyme had a
slightly greater preference for the micelle-rich phase than
at the lower temperature (Duarte et al. 2015).
12.3.4 Cellulases
These enzymes have high industrial value and are used
in different sectors, such as food, animal feed, agriculture
and biomass refining, among others. Due to the high costs
in the market, some solutions have been proposed for pro-
duction of large quantities of cellulases. Fermentation is
one of the preferential alternatives for cellulase produc-
tion, using SSF or SbF (Yang et al. 2017).
Some authors have reported different methods for
cellulase recovery. Wu et al. (2010) reported the recov-
ery of cellulase from Trichoderma reesei ATCC 26921,
using adsorption/desorption from commercially avail-
able acidic ion-exchange resins. The authors recovered
59.5 % of β-glucosidase activity, together with cellulases,
and they proposed that a possible way of separating
β-glucosidases from cellulases would be to immerse the
resins in solvents of a higher pH, but this may completely
denature the enzyme due to the extreme conditions. They
also indicated that, even under optimal conditions, only
about 35–40% of the total proteins were recovered, but the
recovery of the total activity of cellulases was almost 100%
in association with β-glucosidase in the recovery process.
Yang et al. (2017) used microfiltration membranes
with nanofiber-like attapulgite separation layers for the
purification of cellulase, concluding that, compared
with ZrO2 ceramic membranes and traditional plate fil-
ters (GLQ-SBK), attapulgite membranes achieved bet-
ter results. Tang et al. (2012) used ammonium sulfate
precipitation (30–75 % saturation), obtaining 1310.9 mg
of total protein (TP) with a total activity (TA) of 6460.3
(IU). Following dialysis and ultrafiltration, they recov-
ered 869.9 mg of TP and 6096.5 (IU) TA. Finally, using
Sephadex G-100, the authors recovered 13.8 mg TP and
1674.1 (IU) of TA, compared with a crude fermentation
product having 1896.4 mg of TP and 6548.6 IU of TA.
During the purification procedure, they purified cellulase
35-fold, compared with the crude fermentation product,
demonstrating that the strain used, Rhizopus stolonifer
var. reflexus TP-02, is valuable for both academic research
and industrial application. There are more separation
techniques being applied at the industrial level, based on
cost, efficiency and product quality, among other charac-
teristics, and it is still necessary to investigate different
separation methods to identify the most effective pro-
cesses in terms of enzyme recovery and purification.
12.4 Mechanisms of Action of
Hydrolytic Enzymes in the Food
and Pharmaceutical Industry for
Improving Functional Properties
Currently, the pharmaceutical and food industries have
using increasing numbers of biocatalysts in their process-
ing strategies. This is due to the fact that enzymes ensure
improved quality of the final products, reduce their price
and eliminate contaminants. These biocatalysts include
the use of enzymes from various microorganisms, which,
in most applications, increase the quality properties of the
formulated final products. Two of the enzymes which have
been studied in detail and which are used in the industry
are described below.
Phytase is a class of hydrolytic enzyme, belong-
ing to the group of acid phosphatases. These enzymes
catalyze phosphate hydrolysis from phytic acid into inor-
ganic phosphate and myo-inositol phosphate derivatives
(Meena et al. 2013). Phytase was first described by Suzuki
et al. (1907), who reported an enzyme capable of hydrolyz-
ing phytic acid, which came from rice bran (Mukesh et
al. 2011). After this discovery, the study of this enzyme
intensified, and it was found to be widely distributed in
nature. Phytases can be found in plants, certain animals
and microorganisms such as fungi, bacteria, and yeasts,
that produce it constantly to adapt to certain nutritional
matrixes (Monteiro et al. 2015).
Phytases are classified into three classes, depend-
ing on the position of the first dephosphorylation of the
phytate they carry out: 3-phytase, 4- and 6-phytase, and
5-phytase. Within each class, additional differences in
the mechanism of the hydrolysis of phytic acid can be
found. The 3-phytase (myo-inositol hexakisphosphate-
3-phosphohydrolase, EC 3.1.38) removes phosphate
from the phytate 3 position (Ribeiro Corrêa, de Queiroz,
and de Araújo 2015; Rodríguez-Fernández et al. 2015).
This enzyme is usually produced mainly by microor-
ganisms (bacteria and fungi), either intracellularly or
extracellularly. The 4- and 6-phytase (myo-inositol-
hexakisphosphate 4/6-phosphohydrolase EC 3.1.3.26)
hydrolyzes the phosphate ester in the L-6 (or D-4)
phytic acid position and is generally present in plant
seeds (Bhavsar et al. 2010; Ribeiro Corrêa, de Queiroz,
and de Araújo 2015).
The phytase enzyme, when added to animal feed,
carries out a hydrolytic interaction in the phosphomono-
ester bonds present in phytic acid, which is a highly
antinutritional compound, causing various physiological
disease states in non-ruminant animals, such as pigs and
poultry (Bilgiçli, Elgün, a nd Türker 2006). When phy tases
AU: Plea se clari fy
– high co sts of
what? Cellulases?
AU: Th is section
headi ng is very
long. Co uld it
be shor tened
slightly?
AU: Plea se
clar ify – do
you mea n that
the en zyme or
phyt ic acid came
from r ice bran?
TNF_12_K338166_C012_docbook_new_indd.indd 225 03-Jan-20 21:18:06

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Functional Foods and Biotechnology
are ingested, they interact at the stomach level with the
phytic acid contained in the feed, which is released dur-
ing digestion. This enzyme hydrolyzes various bonds of
the phytic acid molecule, releasing phosphate molecules
that are more easily assimilated by non-ruminant organ-
isms (Menezes-Blackburn, Gabler, and Greiner 2015).
The mechanism of action of this enzyme depends largely
on the source from which it was obtained. Of great impor-
tance industrially are 3-phytases produced by fungal
microorganisms (Liao et al. 2012).
On the other hand, cellulase activity is carried out
by various enzymes, which act on different positions
within the cellulose molecule. However, the purpose of all
cellulases is the release of monomers derived from cel-
lulose. According to the mechanism of action, we can find:
a) endoglucanases [1,4 (1,3; 1,4) -β-D-glucan 4-glu-
canohydrolases] randomly break the internal
β-glucosidic bonds of the molecule in the amor-
phous regions, causing a rapid decrease in relative
viscosity relative to the rate of increase of reducing
groups. The products, especially at the end of the
sequence of reactions, include glucose, cellobiose,
and cellodextrins of various sizes ( Izarra et al. 2 010).
b) exoglucanases can be categorized into two large
groups:
• cellobiohydrolases (1,4-β-D-glucan-
cellobiohydrolases, EC 3.2.1.91), which
degrade amorphous cellulose by quant itatively
eliminating cellobiose from the non-reducing
ends of cellulose. The rate of decrease in
viscosity relative to the increase in reducing
groups is much lower than in endoglucanases.
• exoglycohydrolases (1,4-β-D-glucanglu-
cobiohydrolases), which hydrolyze consecu-
tive glucose units from the non-reducing end
of the cellodextrins. The rate of hydrolysis
decreases as the length of substrate chain
decreases (Sriariyanun et al. 2016).
c) β-glucosidases (β-D-glucoside glucohydrolases)
cleave cellobiose to glucose, removing glucose
from the non-reducing end of small cellodextrin
molecules. In contrast to exoglycohydrolases,
the rate of hydrolysis of β-glucosidase increases
as the substrate size decreases, with cellobiose
being the most rapidly hydrolyzed substrate
(Gangwar, Rasool, and Mishra 2016).
12.5 Perspectives for Enzyme
Use in Functional Food
In a biolog ical and empi rical s ense, biotech nology has been
used in food processing for more than 8000 years, in the
production of bread, alcoholic beverages, yogurt, cheese,
and other foods produced using the enzymes inherent in
some microorganisms (Bagchi, Lau, and Ghosh 2010).
Enzymes are applied in various commercial fields, such as
food manufacturing, animal nutrition and the pharmaceu-
tical industry, and as tools for research and development.
At present, almost 4000 enzymes are known, of which 200
original microbial enzyme types are used commercially,
although only 20 enzymes are produced at an industrial
scale (Li et al. 2012). The food industries are continually
in search of enzymes that show catalytic specificity, ther-
mostability and high activity across a wide range of pH
and temperatures (Contesini et al. 2017).
Nowadays, food enzymes (Table 12.1) are used in
the baking industry, fruit juice release and cheese man-
ufacturing, as well as in wine making and brewing, to
improve the flavor, texture, and digestibility of the food
products, in order to meet the demands of consumers
(Li et al. 2012). However, the nutritional value and health
benefits of food is another important aspect to consider
and the development of functional foods with high added-
value, is required nowadays to cover this increasing
demand.
12.5.1 Proteases
Due to the importance of multifunctional protease
enzymes in the pharmaceutical, medical, food and bio-
technology industries, in recent years studies have
focused on the extraction, production, and recovery of
these enzymes from different sources such as plants,
animals, and microorganisms (De Castro and Sato 2013).
Proteases have been found to have various biochemical
characteristics, according to the source from which they
are obtained, whereby they can break certain fragments
of the proteins to generate peptides with specific proper-
ties, known as bioactive peptides.
Bioactive peptides are short sequences of approxi-
mately 2–30 amino acids in length, with a low molecular
weight, which have different activities or functionalities
such as antithrombotic, antioxidant, antihypertensive,
hypocholesterolemic, antiobesity, immunomodulatory,
anticancer, antiaging, anticariogenic, antimicrobial, anti-
inflammatory, mineral binding, regulation of glucose and
insulin homeostasis functionalities (Lafarga and Hayes
2014; Singh, Vij, and Hati 2014; Li-Chan 2015). As a con-
sequence, their consumption may be beneficial to human
health. Peptide bioactivity depends on the specificity of the
enzyme used, the protein source and any treatment prior
to hydrolysis that modifies the native protein structure (de
Castro and Sato 2015; Ozuna et al. 2015). Such peptides
can be used as nutraceuticals or active components in
functional foods (Udenigwe and Fogliano 2017). However,
the main challenge is to ensure that these bioactive pep-
tides do not lose their functionality during food processing
AU: EC num ber?
AU: Plea se
includ e the EC
number
AU: Plea se
check t hat
this a ddition is
correct
AU: Plea se
includ e EC
number
AU: Would ‘ wild-
typ e’ be more
appro priate th an
‘original’?
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Production and recovery oF enzymes For Functional Food Processing
and/or when they pass through the gastrointestinal tract.
Delivery strategies, therefore, need to protect bioactive
peptides from enzymic degradation and to enhance both
mucus and intestinal permeability (Gleeson, Ryan, and
Brayden 2016). It is also essential to ensure that these pep-
tides do not modify the sensory characteristics, mainly the
flavor, of the foods to which they are added.
In recent years, various technologies have been
developed and applied to reduce the bitter taste of these
peptides, in order to include them in food. Among these
technologies are spray-drying encapsulation with malto-
dextrin and cyclodextrin or other multifunctional mate-
rials, such as protein-tannic acid multilayer films, as
carriers and nanoencapsulation (Li-Chan 2015; Katouzian
and Jafari 2016; Lau et al. 2017). Furthermore, technolo-
gies for processing, such as microwaves, high hydrostatic
pressures and high-intensity ultrasound, have shown pos-
itive results (Ozuna et al. 2015). In addition to these tech-
nologies, it is necessary to find and characterize other
proteases that improve biologically relevant activity and
yield in the production of bioactive peptides but with a less
bitter taste sensation.
12.5.2 Amylases
Amylases are important enzymes used in biotechnology,
food, fermentation and other industries, mainly as the
AU: Plea se
check whether
this c hange is
appropriate
AU: Th is has
been st ated
previously.
Plea se be aware
of the r isks of
repetition
Table 12.1 Enzymes Used in Food Processing
Enzyme Functional Ingredient Bioactivity or Functionality Reference
Proteases Bioactive peptides Antithrombotic Tu et al. (2017)
Antioxidant Zhuang, Tang, and Yuan (2013)
Hypocholesterolemic Marques et al. (2015)
Antiobesity Jemil et al. (2017)
Immunomodulatory Chalamaiah et al. (2014)
Antimicrobial Luz et al. (2017)
Anti-inflammatory Moronta et al. (2016)
Antihypertensive Michelke et al. (2017)
Amylases Isomalto-oligosaccharides Prebiotic Sorndech, Sagnelli, and Blennow (2017)
Sugar alcohols (isomaltitol
and maltotriitol) Anti-dental plaque Niu et al. (2017b)
Lipases Fatty acids Medium-long-medium triacylglycerols Nunes et al. (2011)
Human milk fat substitutes Yu, Xu, and Xiao (2016)
Cocoa butter-like fat Yu, Xu, and Xiao (2016)
Cellulases Flour Hypoallergenic wheat flour Watanabe et al. (2000)
Proteases Bioactive peptides Antithrombotic Tu et al. (2017)
Antioxidant Zhuang, Tang, and Yuan (2013)
Hypocholesterolemic Marques et al. (2015)
Antiobesity Jemil et al. (2017)
Immunomodulatory Chalamaiah et al. (2014)
Antimicrobial Luz et al. (2017)
Anti-inflammatory Moronta et al. (2016)
Antihypertensive Michelke et al. (2017)
Amylases Isomalto-oligosaccharides Prebiotics Sorndech, Sagnelli, and Blennow (2017)
Sugar alcohols (isomaltitol
and maltotriitol) Anti-dental plaque Niu et al. (2017b)
Lipases Fatty acids Medium-long-medium triacylglycerols Nunes et al. (2011)
Human milk fat substitutes Yu, Xu, and Xiao (2016)
Cocoa butter-like fat Yu, Xu, and Xiao (2016)
Cellulases Flour Hypoallergenic wheat flour Watanabe et al. (2000)
AU: Plea se note
that , in Tables,
all abb reviatio ns
must be de fined
at firs t mention,
even if t hey have
alre ady been
define d in the
text , or the ful l
name b e given
with out the
abbreviation.
AU: Al l abbre-
viat ions need to be
define d or deleted
in Ta bles
TNF_12_K338166_C012_docbook_new_indd.indd 227 03-Jan-20 21:18:06

228
Functional Foods and Biotechnology
preferred carbohydrate-degrading enzyme for starch-
based industries (Ait Kaki El-Hadef El-Okki et al. 2017).
In combination with other enzymes, amylases can
produce a number of compounds based on starch, such as
isomalto-oligosaccharides (IMOs), which are composed
of glucose oligomers with α-D-(1,6)-linkages, with or
without α-(1→4) linkages, including isomaltose, panose,
isopanose, isomaltotriose, nigerose, kojibiose and higher
branched oligosaccharides. Currently, these compounds
are recognized for their properties such as low viscosity,
resistance to crystallization, reduced sweetness, and bifi-
dogenic effects (Sorndech, Sagnelli, and Blennow 2017).
IMOs are considered to be prebiotic, so their use in the
food industry as functional ingredients is interesting.
Among their beneficial effects are improved intestinal
health, mineral absorption, cholesterol regulation and
immunity, as well as the prevention of and resistance to
various diseases such as dental caries. IMOs are also
used as substitute sugars for patients with diabetes (Niu
et al. 2017a; Sorndech, Sagnelli, and Blennow 2017). The
current trend is toward the optimization of the produc-
tion of IMOs, using enzymes from microbial sources, to
increase yields.
Among other applications of amylases is the pro-
duction of maltitol and maltotriitol from hydrogenated
starch hydrolysates, which are recognized by their anti-
dental plaque effect, among others. Currently, these
compounds are produced by a chemical hydrogenation
process; however, the process yields are low. The use
of an enzymatic cocktail, including amylases, could
improve the production process of these compounds
(Niu et al. 2017b).
12.5.3 Lipases
Lipases have been obtained from various organisms,
such as animals, plants, and microorganisms, although
microbial lipases have received more industrial atten-
tion due to their greater stability, selectivity and broad
substrate specificity (Taskin et al. 2016). In the con-
text of lipase, in 2014, Novozymes™ reported that this
enzyme represented the second best-selling product
for the food industry. Food sectors such as dairy and
baking goods were the ones which required increased
production of lipases (Contesini et al. 2017). Rhizopus
lipases have been used in the synthesis of Medium-
Long-Medium (MLM) triacylglycerols, human milk
fat substitutes, cocoa butter replacements or oils with
a specific structure (Yu, Xu, and Xiao 2016). Santos
et al. (2013) reported the evaluation of the catalytic
properties of lipases from plant seeds for application
to oil hydrolysis to produce fatty acids concentrates,
and these authors indicated that the use of lipase from
dormant castor bean seeds has the potential to hydro-
lyze vegetable oils.
12.5.4 Cellulases
The application of cellulases to hydrolyze cellulose is an
environmentally friendly process. Three principal types
of cellulases confer the hydrolysis of cellulose, namely
endoglucanases, exoglucanases, and exoglucosidases
(Juturu and Wu 2014). They have a wide spectrum of
applications in industries such as food and brewed bev-
erage production, animal feed, detergent production and
laundry, textile processing and paper pulp. In food pro-
cessing industries, cellulases have been employed in the
extraction and clarification of fruit juice, to increase their
yields, with the use of cellulases having been found to
enhance the extraction of olive oil, as well as its quality
(Will, Bauckhage, and Dietrich 2000; Juturu and Wu 2014;
Sharma et al. 2016). Cellulases and xylanases are used in
the production of cr ispy bread and biscuits, decreasing the
need for liquid use during dough preparation (Juturu and
Wu 2014). Cellulases are also relevant for the modification
of sensory parameters of food, such as aroma, flavor, and
texture properties, which can be altered by the addition
of enzymes like pectinases and cellulases. Watanabe et
al. proposed a novel method for producing hypoallergenic
wheat flour suitable for patients allergic to wheat; in this
study, wheat flour was mixed with a cellulase solution and
the product was evaluated by an enzyme-linked immuno-
sorbent assay (Watanabe et al. 2000).
12.6 Conclusion
Enzymes are widely used in different processes, mainly
by the food and pharmaceutical industries, and are being
increasingly researched to obtain new products, such as
functional foods. In this context, the use of agroindus-
trial waste offers great potential to reduce the cost of
production and increase the use of enzymes for indus-
trial purposes. The wastes and by-products also allow the
production of important enzymes by the microbial route,
which also contributes to increasing the value of biore-
sources that are currently underutilized. The production
of microbial enzymes requires more research to evaluate
the influence of parameters such as pH, aeration, agita-
tion, concentration of carbon source, type of reactor and
fermentation, as well as the use of genetically modified
organisms. The efficiency of applied biocatalysis favors
the implementation of enzyme-based processes. New
research should be orientated to optimize the extraction
and purification parameters to obtain enzymes in large
amounts and of high purity.
AU: Plea se
check whether
this i s correct
AU: Shou ld this
be cited?
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Production and recovery oF enzymes For Functional Food Processing
References
Aceves-Diez, A.E., and L.M. Castañeda-Sandoval.
2012. “Producción Biotecnológica De Lipasas
Microbianas, Una Alternativa Sostenible Para La
Utilización De Residuos Agroindustriales.” Vitae,
Revista de La Facultad de Química Farmaceútica 19
(3): 244–47. http://www.scielo.org.co/pdf/vitae/
v19n3/v19n3a1.pdf.
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