Perspectives of Solid State Fermentation for Production of Food Enzymes

American Journal of Biochemistry and Biotechnology 4 (4): 354-366, 2008
ISSN 1553-3468
© 2008 Science Publications

Perspectives of Solid State Fermentation for Production of Food Enzymes
1Cristobal Noe Aguilar, 2 Gerardo Gutiérrez-Sánchez, 3PLilia A. rado-Barragán, 1Raul Rodríguez-Herrera.
1José L. Martínez-Hernandez and 1Juan C. Contreras-Esquivel
1Department of Food Research, Universidad Autónoma de Coahuila, 25280, Saltillo, Coahuila Mexico
2Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, USA
3Biotechnology Department, Universidad Autónoma Metropolitana,
Iztapalapa, O9340, Mexico, D.F.

Abstract: Food industry represents one of the economic sectors where microbial metabolites have
found a wide variety of applications. This is the case of some enzymes, such as amylases, cellulases,
pectinases and proteases which have played a very important role as food additives. Most of these
enzymes have been produced by submerged cultures at industrial level. Many works in the literature
present detailed aspects involved with those enzymes and their importance in the food industry.
However, the production and application studies of those enzymes produced by solid state
fermentations are scarce in comparison with submerged fermentation. This review involves production
aspects of the seven enzymes: tannases, pectinases, caffeinases, mannanases, phytases, xylanases and
proteases, which can be produced by solid state fermentation showing attractive advantages.
Additionally, process characteristics of solid state fermentation are considered.

Key words: Food enzymes, production, solid state culture

INTRODUCTION
important to consider that to evaluate the quality of

foods, some analytical methods are based in enzymatic

Employment of enzymes in food industry is based
reactions, such is the case of the soluble sugar content
on three basic aspects: i) the control of quality of
analysis, which is executed in units that possess such
certain foods, ii) the modification of the properties of
enzymes in electrodes or absorbed in membranes
some food additives and of the foods itself and, iii) the
(enzymatic biosensors). The enzymatic reaction usually
production of enzymes used as food additives. In the
liberates a product, which is then related to the
first case, presence or absence of some enzymatic
concentration of initial substrate provided by the
activities has a great impact in the quality of the final
analyzed sample.
product. Examples are: Alkaline-phosphatase as

The second aspect refers to the use of enzymes to
evaluation of the effectiveness of the process of milk
modify the physico-chemical and reological properties
pasteurization; the catalase is an index of microbial
of the foods, such as amylases, lipases and pectinases.
contamination in certain foods, the activation in situ of
The enzymes used as biocatalysts for the production of
the pectinesterase allows to obtain firmer fruits and
some aminoacids can also be considered as an example
vegetables with more attractive sensorial attributes for
of this particular aspect[5]. These last aspects, involve
the consumers[1,2], the peroxidase and the lipoxigenase
production processes of enzymes, which have
are used to evaluate the efficiency of the blanching
applications in the food industry. The processes include
process of some vegetables[3]. Baduí[4] mentioned that
the extraction of enzymes from agroindustrial and
presence of some other enzymes have been proposed to
fishing wastes, vegetable and animal tissues and
be used in the control of quality of the foods, such it is
microbial sources. The latter being one of the most
the case of the invertase in the beer pasteurization; the
important production systems in the food industry, due
amylase like index of excessive heating in honeys, the
to the fact of enzymes are obtained in shorter times
esterases are an index of fungal contamination, the N-
without chemical compounds production that then can
acetyl- -D-deshydrogenase for the destruction of
represent potential problems like environmental
Salmonella in pasteurized eggs and the deshydrogenase
contamination and health; they also possess very
for the microbial contamination of the milk. It is
homogeneous characteristics.
Corresponding Author: Cristobal Noe Aguilar, Food Research Department, School of Chemistry, Universidad Autónoma de
Coahuila, Saltillo, 25280, Coahuila, México
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At industrial level, enzyme production is a growing

In the western world the SSF has been fewer
field of biotechnology. Annual world sales figures are
studied that the SmF and SLF. The most important
close to 109 dollars with increasing numbers of patents
differences among these systems are the relatively low
and research articles related to this field[6]. Most
content of humidity in the means, the formation of
enzyme manufacturers produce enzymes using
gradients of temperature, nutrients and products and
submerged fermentation (SmF) or liquid surface (SLF)
also, sporulation mechanisms as well as the production
fermentation techniques with enzyme titers in the rage
of enzymes and secondary metabolites as the
of grams per liter[7]. Such levels are a prerequisite if
rifamycin[25], citric acid[26] and aromas[27].
specific compounds are to be considered as

Substrates from agricultural or industrial wastes
commodities because product recovery costs are
(wheat straw or barley, sugar cane bagasse, coffee pulp,
inversely proportional to concentration in a
grape wastes, copra pasta, among other) or inert
fermentation broth. There is however a significant
materials (as resins of ionic exchange, acreolite or
interest in using solid-state fermentation (SSF)
poliurethane foam) can be used. The pretreatments of
techniques to produce a wide variety of enzymes,
these materials is really few, generally a milled
mainly from mold origin, as indicated by the growing
previous and wash. Of these characteristics some
number of research papers in the literature and the
advantages and disadvantages of the SSF in comparison
marketing and development by a small but visible
to the SmF are derived and presented next[28].
number of fermentation industries[6]. Among the

advantages for SSF processes it is often cited that
Advantages:
enzyme titers are higher than in SmF, when comparing

the same strain and fermentation broth. However, there
• The culture media are simple. Some substrates can
is a scarcity regarding physiological studies comparing
be used direclty as a solid media or enriched with
SSF and SmF when trying to explain why
nutrients
microorganisms produce higher titers in the first kind of
• The product of interest is concentrated, that which
process as compared to the second. This lack of
facilitates its purification
information makes difficult any assessment regarding
• The used inoculum is the natural flora of the
the value of one process versus the other thereby
substrates, spores or cells
hindering the fundamental approach to process
• The low humidity content and the great inoculum
optimization and design for SSF technique in areas such
used in a SSF reduce vaslty the possibility of a
as strain improvement, solid substrate engineering and
microbial contamination
process control.
•

Some of the better known examples are the
The quantity of waste generated is smaller than the
amylases[8-11],
Glucoamylases[9,11],
cellulases[10,11],
SmF
pectinases[12-16], caffeinases, lipases[18,19] and proteases
• The enzymes are low sensive to catabolic
production[20,21].
repression or induction[29]

Considering
the
contributions
mentioned

previously, in this work the generalities of the SSF
Disadvantages:
process are reviewed and the catalytic characteristics

and some of the advances achieved in the production of
• The used microorganisms are limited those that
six enzymes with potential application in the food
grow in reduced levels of humidity
industry for SSF are presented.
• The determination of parameters such as humidity,

pH, free oxygen and dioxide of carbon, constitute a
MATERIALS AND METHODS
problem due to the lack of monitoring devices

• The scale up of SSF processes has been little
Solid State Fermentation (SSF): Term solid state
studied and it presents several problems
fermentation (SSF) is applied for the processes in which

insoluble materials in water are used for the microbial

Some authors have described the types of bio-
growth[22], In the fermentative processes of this type,
reactors more used in SSF[30]. The used simpler
the quantity of water should not exceed the capacity of
bioreactors are the trays that consist on placing fine
saturation of the solid bed in which the microorganisms
layers of solid substrate 5-10 cm on a horizontal area,
grow[23]. Water is essential for the microbial growth and
the air flux in these trays is not forced through the
in SSF and it is present in thin layers and in occasions,
substrate tray but if around the trays and frecuently the
absorbed inside the substrates[24].
temperature is controlled in a room. The following type

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Am. J. Biochem. & Biotech., 4 (4): 354-366, 2008

in complexity is the bio-rector of packed channel,
SmF[7,28,32,34-38]. In these works, attractive advantages
which is characterized to have the static substrate on a
are indicated, such as the high enzymatic production
plate perforated through the one which a current of air
(up to 5.5 times more than in SmF), the nature
is drived. The typical design of these reactors consists
extracellular of the enzymes and the stability to wide
on a cylinder of stainless steel that it can have a better
pH and temperature ranges[32,33] reported a tannase
control of temperature and humidity when a wet air is
productivity of 6.667 and 1.275 UE/Lh for SSF and
provided. Both systems are static.
SmF respectively and a maximum of intracellular and

The dynamic bio-rectors used in SSF can rotational
extracellular tannase activity respectively 18 and 2.5
or upset drum being. The first one consist on an
times higher in SSF that in SmF.
inclined cylinder or horizontal with or without agitation

At the moment, the biggest commercial
that it is rotated on their axial axis to increase the air
applications of tannases are in the manufacture of
flux in the channel. The second one presents a
instantaneous tea or of acorn liquor and in the gallic
configuration different to the first one, possesing palette
acid production[39-41], which is used as an important
or helical agitators to move the channel and sometimes,
intermediary compound in the food and pharmaceutical
an air flux is forced through the packing. In the
industry, respectively for the synthesis of propylgallate
dynamic bio-reactors the agitation can be intermittent
and trimethoprim[42]. Also, the tannase is used as
or continue, but this factor represents some
clarifying agent in some wines, juices of fruits and in
disadvantages for some processes like damage in fungal
refreshing drinks with a coffee flavor[43].
structures. Furthemore, the control of the temperature

In the case of the wines, it is important to state that
gets complicated.
the main tannins present are catechins and epi-
RESULTS
catechins, which can get a complex with galacto-

catechins and others galloyl-derivated. The amount of
Production of Enzymes by SSF: In studies where the
catequin in white wines is around of 10-50 mg L?1,
enzyme concentration obtained from SmF, SLF and
while in other wines it can reach 800 mg L?1[44]. Fifty
SSF have been compared, it has been observed that in
percent of the wine color is due to the presence of
SSF the titles are higher and also, the produced
tannins, however, if these compounds are oxidized to
enzymes are stable to wider ranges of temperature and
quinones by contact with the air, an undesirable
pH. This has been evidenced for enzymes such as the
turbidity could be formed, affecting the wine quality.
amylase[31], pectinase[14,15], tannase[32] and protease[21].
The use of tannases can solve this problem.
In these studies it has demonstrated that SSF presents

Tannins are present in low quantitites in beer,
attractive advantages for the production of microbial
especially as anthocianins. However, when beer
enzymes. More recent studies in SSF involve the
proteins are present in high quantities, an undesirable
production of enzymes with potential utilization in the
turbidity is presented by complexing with these tannins.
food industry. Such enzymes are a) tannin acyl
This problem could be solved with the employment of
hydrolase, b) protopectinase, c) caffeinase, d)
tannases.
mannanase, e) phytase, f) xylanase and g) protease, on

The tannery effluents contain high amounts of
those which next some of the most important
tannins, mainly polyphenols, which are dangerous
characteristics are revised, including the reaction that
pollutants, for this reason the use of the tannase
catalyze and the possible industrial applications.
represents an inexpensive and effective treatment for

the removal of these compounds.
Tannase: Tannase or tanin acyl hydrolase (EC,

3.1.1.20) catalyzes the hydrolysis reaction of the ester
Pectinases: Most of pectic-polymers are comprised of
bonds present in the hydrolizable tannins and gallic acid
smooth homogalacturonan and ramified hairy regions.
esters. Its industrial production is only by a microbial
Smooth regions consist of a linear homogalacturonan
way using SmF, where the enzymes are mainly
backbone,
while
hairy
regions
consist
of
produced intracellularly, implying additional costs in its
rhamnogalacturonan backbone with side-branches of
manufacture[33]. Recently, tannase is commercialized by
varying length[45]. Detailed information on elaborate
Biocon (India), Kikkoman (Japan) ASA Specilaeznyme
models on pectin structure can be found in some
GmbH (Germany) and JFC GmbH (Germany) with
reviews[46,47]. Traditional breaking down of pectic
different catalytic units depending of the product
substances was through the use of well-know pectic
presentation. However several studies have reported
enzymes (pectinesterase, polygalacturonase and pectin
interesting advantages between the tannase produced by
lyase) able to degrade only the smooth region.
SSF in comparison with that produced by
Recently, several new enzymes have been reported

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which degrade parts of hairy region such as
nitrogen source in solid state fermentation (SSF) using
rhamnogalacturonan hydrolase, rhamnogalacturonan
two different supports, polyurethane foam (PUF) and
lyase and xylogalacturonase,[45]. Properties and
sugarcane bagasse. Results in this study demonstrated
characteristics of common and new pectic enzymes
that caffeine can be used in SSF as sole nitrogen source
are discussed by Contreras-Esquivel et al.[47] and
if sucrose is used as the carbon source. If a simpler
Aguilar et al.[48].
nitrogen source (ammonium sulfate, urea) is added to

Common pectinolytic enzymes products have been
the medium containing caffeine, this N-source will
commerciallized for more than two decades[49] and can
repress the caffeine consumption. If saccharose is still
be produced either by SmF and SSF[50,51]. Production of
present when the simple nitrogen source has been
common pectic enzymes from Aspergillus niger and
degraded, caffeine will be used as N-source. Caffeine at
others fungi has been reported in solid state cultures
a concentration of 8 g L?1 has not effect the fungal
employing agricultural by-products such as: cassava
growth when a simple N-source is present in the solid
fibrous waste[52], wheat bran[53], apple pomace[54-56],
medium. The main difference between the two solids
corn barn[57], citrus wastes[15,58-60], coffee pulp[15], sugar
used as supports in SSF was the required time to
cane baggase[14,61-63] and raw starch from cassava tuber.
complete the fermentation. With sugarcane bagasse as
SSF represents an interesting alternative for the
support, substrates in the impregnated medium were
production of mould pectic enzymes, because these
degraded ca 1.6 times faster than with PUF as support.
enzymes are synthetized in a high concentration[64].
Theophylline, the major product from caffeine

The pectinolytic fungal enzymes are of commercial
degradation was completed. However, the major
interest to the food, textil, pulp and paper and
advantage of using PUF as a support, over sugar cane
wastewater treatment areas[65]. In food technology,
bagasse, is the possibility to determinate the fungal
pectic enzymes are industrially employed in the
biomass.
extraction, clarification and concentration of fruit

The literature about this enzymes is still scarse, but
juices, in the clarification of wines, in the extraction of
is very attractive the ability of certain fungal strains to
oils, flavours and pigments from plant materials.
degradate the caffeine, which could be used to
Furthermore, pectic enzymes have been employed for
minimize the concentration of this molecule in foods
carried out extraction and modification of pectin chain.
and feeds.
Great amounts of agroindustrial wastes rich in

polysaccharides, such as pectic substances are produced
Mannanases:
?-D-mannanases
are
hydrolytic
at worldwide. Some of these wastes are used for the
enzymes, which act on hemicellulose chains (called
production of pectin. Purified pectinolytic enzymes
mannans) whose composition and structure chemistry is
have been proposed for extracting and modifying of
formed by units of D-manose. It is important to clarify
pectin. Currently, pectin is extracted at industrial scale
that xylan is another type of hemicellulose composed
by physicochemical methods, but lately new
by monomeric units of the D-xilose. Also, it should be
biotechnological (microbial and enzymatic) alternatives
remembered that the hemicellulose is not a
are being developed.
homopolymer, consequently its chemical structure not

is formed by only type of sugar, but rather its
Caffeinases: Among the enzymes presented in this
monomeric units are two to four different residuals of
rewiew, the caffeinases are the less studied. These
sugar forming a heteropolysaccharide[66]. In this
enzymes are methyl-releasing from caffeine molecule
context, this review will refer as substrate for the
to give teophilline and methanol as products, then
mananases to that hemicellulose type with a
teophilline is transformed in 3-methylxantine and
composition of D-manose.
xantine. Some metabolic studies have been carried out

The
mannanases
or
(1-4)-D-mannan-
in humans, bacteria and filamentous fungi[18] trying to
manohydrolases are those enzymes that produce
elucidate the biodegadation pathway of caffeine.
oligosaccharides through the hydrolysis of the

Caffeine is an alkaloid contained in some
connections
(?1-4)-D-manopyranosil
into
beverages, fruits and agroindustrial wastes. The
hemicellulose chains. Most of the mannanase
presence of caffeine in agroindustrial wastes represents
production studies involve microbial SmF, but it is
a limitant for its use as animal feed. Several attempts
known that this enzyme can be extracted from
have been made to eliminate this molecule from foods
crustaceans, leguminous seeds; and some spineless[67].
and feeds, but the results have been poors.

Microbial
production,
purification
and

Hakil et al.[17] studied the ability of
characterization of mannanases have been studied in
Aspergillus tamarii V12A25 to use caffeine as sole
SmF using microorganisms such as Escherichia coli[68],

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Bacillus
pulmilus[69],
Thielvia
terrestris[70],
acetylesterases, arabinofuranosidases, glucuronidases
Trichoderma harzianum[71], Pseudomonas sp. P.T.5[72],
and other esterases.
Caldocellum saccarolyticus[76], T. reesei C-30[73],

In the nature, some microorganisms metabolize
Bacillus sp. KK01[74]. Venegas et al.[75] demonstrated
several energy sources due to the capacity to produce
that mannanases from A. oryzae and A. niger can be
different xylanase classes, besides the degradation of
produced in SSF using coffee pulp and coconut copra
the cellular wall of the plants in coordination with other
cake as solid substrates. The produced mannanases
hidrolases permits the colonization processes. These
were also extracted, purified and characterized. These
microbial xylanases have found biotechnological
results confirmed that SSF is a highly attractive process
applications in the paper and food industries, as well as
for mannanase production using agroindustrial
in the treatment of solid residuals[80]. When xylanases
byproducts.
are free of cellulases, these preparations are used for the

The mannanase production at industrial level is still
bleaching treatment of the pulp in the production and
limited, but it is known that these are being used in the
recycling of the paper. This application brings
bleached of the paper pulp diminishing the use of
environmental implications of relevance; since it is
chlorine until in 25%[76] and they can be applied in the
avoided or it diminishes the use of toxic compounds
instantaneous coffee manufacture to reduce the
during these processes without alter the quality of the
viscosity of the extract during the extraction process[72].
paper obtained. The use of xylanases of microbial

origin has substituted the use of chlorine-compounds in
Phytases:
Phytase
(EC,
3.1.1.8)
is
a
these processes[81,82].
phosphomonoesterase that hydolyses phytic acid and

Join with other hemicellulases, the xylanases
several phosphoesters contained in foods, such as
allows the obtaining of oligosaccharides from xylans
wheat, canola oil and mung beans. The products of the
(from agricultural wastes) which can be used as food
hydrolysis reaction of phyitic acid are inositol and
additives and edulcorants[83]. Also, xylanases and
phosphoric acid. Microbial phytases are of interest for
cellulases are used for the texture modification of
biotechnological applications, especially for the phytate
bakery masses and also, for the improvement of the
reduction in foods and feedstuffs. Some attempts for
diets of corral birds, since the enzymatic hydrolysis of
industrial phytase production in SSF have been carried
arabinoxylans (of forages and cereals) increases the
out. However, the SmF system is the main process used
nutritious efficiency of the diet.
for the production of this enzime.

Commercial xylanases preparations are obtained

Phytase production by Aspergillus carbonarius in
from different microorganisms and diverse culture
SSF using canola oil as substrate, was studied by Al-
systems, which are applied according to the desired
Asheh and Duvnjak[78]. They showed that phytase
enzymatic profile. New fungal xylanases have
production of was associated with fungal growth,
interesting properties such as thermal, electrostatic, pH
reaching a maximum activity after 72 h of culture.
and catalytic stabilities[84,85-87].

By other hand, Ebune et al.[79] reported that

These new enzymes have been produced by SSF
phosphate concentration influenced phytase production
process due to it offers an alternative that is worthwhile
of A. ficuun in a solid state fermentation process. Lower
to study with more detail, to produce isoenzymes with
concentrations of this compound favoured the enzyme
the mentioned properties. Likewise, it should be
production. Addition of Tween 80 and sodium oleate
considered the study of the xylanases expression in
strains obtained from parasexual cross where it is
increased the rate of phytase production and hydrolysis
carried out the genetic recombination for the obtaining
of phytic acid, while the Triton X-100 addition had a
of new strains with characteristics highlighted in the
negative effect on these microbial processes. Glucose
levels and in the xylanase productivity[88].
concentrations of up to 5.2% increased the biomass

formation, production of phytase and the rate of phytic
Proteases: Proteases have an important position in the
acid hydrolysis, while glucose concentgrations higher
enzyme industry as they have a determinant role in the
than 9.8% had an adverse effect on these processes.
microbial and human physiological needs as well as the

great commercial market applications. Since they are
Xylanases: Xylanases are a complex system of
indispensable for living organisms, proteases occur in a
enzymes required to complete degradation of xylan, a
wide
diversity
of
plants,
animals
and
heterogeneous polysaccharide. Endoxylanases hydrolise
microorganisms[89]. Papain, bromelain, keratinases and
the xylose chains of xylan, while the different lateral
ficsin represent some of the plant proteases[90];
groups of the molecule are hydrolyzed by a mixture of
however, the use of plants as a source of proteases is

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governed by factors no easily controlled such as land
properties make bacterial proteases most advantageous
availability and climatic conditions as well as its
for the detergent and leather industry[98-102]. Also,
excretation is a time-consuming process. Pancreatic,
alkaline proteases from Brevibacyterium linens have
trypsin, chymotrypsin, pepsin and rennin are the most
been used in the dairy industry and proteases from
important proteases of animal origin. However, their
Bacillus thermoprotelyticus are used for the enzymatic
production depends on the availability of livestock for
synthesis of aspartame.
slaughter[91]. Therefore microbial proteases are

There is a long list of bacterial proteases
preferred above enzymes from plant and animal sources
commonly used in the food industry and they are
since they present most of the desired characteristics for
mostly produced by submerged fermentation, however
biotechnological applications[92].
fungal protease production is an attractive source for

In early days, proteases were classified according
proteases. Fungi can elaborate a wider variety of
to their source (animal, plant or microbial), catalytic
enzymes than bacteria and a clear example of this
action (endo or exopeptidases), the molecular size,
statement are the acid, neutral and alkaline proteases
charge or substrate specificity, however a more rational
produced by Aspergillus niger. The fungal proteases are
system is now based on a comparison of active sites,
active over a wide pH range (4-11) and exhibit board
mechanisms of action and three-dimentional structure.
substrate specificity. Fungal enzymes are actually
Four mechanistic classes are recognized by the
produced by solid state fermentation (SSF) and the
International Union of Biochemistry and within these
advantages of fungal enzyme production in SSF
classes, six families of proteases are recognized to date:
over submerged state fermentation (SmF) system
serine proteases (EC 3.4.21), serine carboxy proteases
have been extensively discussed by Viniegra-
(EC 3.4.16), cystein proteases (EC 3.4.22), aspartic
González, et al.[6,103].
proteases (EC 3.4.23), metallo proteases I (EC 3.4.24)

Fungal acid proteases have an optimal pH rang
and metallo carboxy proteases (EC 3.4.17)[93].
from 4-4.5 and they can be stable at pH values from

Proteases are by far the most important enzymes in
2.5-6.0. In general, fungal neutral proteases are
the food industry used in food proteins modification.
metalloproteases inhibited by chellating agents,
Proteases have been used in ancient technology to
however they can supplement the action of plant,
improve palatability and storage stability of the
animal and bacterial proteases reducing bitterness in
available protein sources; consequently, proteases have
food protein hydrolysates and food protein
a long history of applications in food products and they
modifications. Currently, most detergent proteases are
are used in baked goods, brewing, cereals,