Transcript Food Biotechnology

Food Biotechnology
Dr. Tarek Elbashiti
5. Process Developments in Solid-State
Fermentation for Food Applications
• Solid-state (substrate) fermentation (SSF) has
been defined as the fermentation process
occurring in the absence or near absence of free
water.
• SSF processes generally employ a natural raw
material as the carbon and energy source.
• SSF can also employ an inert material as the
solid matrix, which requires supplementing a
nutrient solution to contain necessary nutrients
as well as a carbon source.
• The solid substrate (matrix), however, must
contain enough moisture.
• Solid substrates should generally have a
large surface area per unit volume (in the
range of 103–106 m2/cm3), to allow for
ready growth on the solid–gas interface.
• Several agro crops such as cassava and
barley, and agro-industrial residues such
as wheat bran, rice bran, sugarcane
bagasse, cassava bagasse, various oil
cakes (e.g., coconut oil cake, palm kernel
cake, soybean cake, ground nut oil cake),
• fruit pulps (e.g., apple pomace), corn cobs,
sawdust, seeds (e.g., tamarind, jack fruit), coffee
husk and coffee pulp, tea waste, and spent
brewing grains are the most often, most
commonly used substrates for SSF processes.
• There are several other important factors which
must be considered for development of SSF
processes.
• These include physico-chemical and biological
factors such as pH of the medium; temperature
and period of incubation; age, size and type of
the inoculum; nature of the substrate; and type
of microorganism employed.
SIGNIFICANCE OF SSF
• It is cost effective due to the use of simple
growth and production media comprising
agro-industrial residues, and it uses small
amounts of water and therefore releases
considerably less effluent, thus reducing
pollution concerns.
• SSF processes are simple, use low
volume equipment (lower cost), and are
effective in that they provide high product
titres (concentrated products).
• Further, the aeration process (availability
of atmospheric oxygen to the substrate) is
easier.
• There is increased diffusion rate of
oxygen into moistened solid substrate,
supporting the growth of aerial mycelium.
• SSF can be effectively used at smaller
volumes, which makes it suitable for rural
areas.
SSF PROCESSES FOR FOOD
APPLICATIONS
• Many kinds of fermented foods, such as
single-cell protein (SCP) probiotics, flavoring
products, beverages, pigments, and peptide
sweeteners, are produced largely by SSF.
• In addition to these, several enzymes,
organic acids, and exopolysaccharides have
been produced using SSF.
• Fermented foods can be stored for longer
periods and used for food supply during the off
season.
• Fermentation also contributes to the digestibility
and enhances the nutritional value of the
product.
• It can also increase fibre digestibility.
• However, there are chances of accidental
contamination of SSF food products by
mycotoxins, which are a class of unwanted
compounds and could be accidentally present or
produced in SSF products.
Historical Developments
• Historically, SSF processes have been used
since ancient time for food applications.
• SSF dates back to 6000 BC when Babylonians
made beer from natural yeast.
• Egyptians used this technique for bread making
in 2600 BC, using brewer’s yeast.
• Cheese making with Penicillium roquefortii was
recorded in Asia before the birth of Christ. Koji
processing reported to be migrated from China
to Japan in the seventh century.
• Miso, tempeh, tamari sauce, soy sauce, angkak, natto, tou-fu-ru, and minchin are some of
the other ancient fermented foods known for
centuries, which are prepared through SSF.
• Tempeh and tamari sauce are soybean
products, the former is an Indonesian food
fermented by Rhizopus species and the latter is
a Japanese food produced by using Aspergillus
tamari.
• Soy sauce, a brown, salty, tangy sauce, is
obtained from a sterile mixture of wheat bran
and soybean flour, fermented initially by lactic
acid bacteria, followed by alcoholic fermentation
and ripening.
• In the eighteenth century, SSF was used
to make vinegar from apple pomace.
• The beginning of the twentieth century
marked the use of SSF for the production
of enzymes and organic acids using
molds.
• The period of the 1970s saw a major
focus on production of SCP.
New Developments
• It has been used particularly for the
production of enzymes, organic acids,
pigments, SCP, exopolysaccharides, and
aroma compounds.
SSF PROCESSES FOR FOOD
ENZYMES
• Almost all the microbial enzymes can be
produced using SSF systems.
• Industrially important food enzymes, which
include alpha amylase, glucoamylase, lipase,
protease, pectinase, inulinase, glutaminase, and
tannase, have been widely studied.
• Enzyme production in SSF has often resulted in
higher yields in comparison to SmF.
• Studies conducted to examine the reasons why
SSF produced higher enzyme yields than SmF
have been unable to explain it fully, although
some of the characteristics of SSF provide
conditions for the microbes more like the habitat
from which they were isolated, and this may be
the major reason for higher enzyme production.
• Most of the SSF processes for the production of
food enzymes involve agro-industrial residues
as the substrate, although inert materials such
as polyurethane foam are also used Table 4.1.
1. Amylases
• α–Amylase (endo-α-1,4-D-glucan
glucohydrolase, EC- 3.2.1.1) is an extracellular
enzyme, which randomly cleaves the 1,4-α-Dglucosidic linkages in the interior of the amylose
chain.
• Amylases which produce free sugars are termed
as saccharogenic amylases, and those which
liquefy starch without producing free sugars are
known as starch-liquefying amylases.
• β-Amylase (α-1,4-glucan maltohydrolase, EC3.2.1.2) is an exoacting enzyme, which cleaves
the nonreducing chain ends of amylose,
amylopectin, and glycogen molecules, yielding
maltose.
• Glucoamylase [also known as
amyloglucosidase, glucogenic enzyme, starch
glucogenase, and gamma amylase (exo-α-1,4D-glucan glucanohydrolase, EC- 3.2.1.3)]
produces single glucose units from the
nonreducing ends of amylose and amylopectin
in a stepwise manner.
• Unlike α- and β-amylase, most glucoamylases
(GA) are able to hydrolyse the α-1,6 linkages at
the branching points of amylopectin, although at
a slower rate than 1,4-linkages.
• Thus, glucose, maltose, and limit dextrins are
the end products of GA starch hydrolysis.
• Extensive studies have been done on the
production of amylases, in particular for α-amylase
and GA in SSF, employing several microorganisms
and various kinds of agroindustrial residues,
among which starchy substrates have been
preferred (Table 4.1)
• Reports on microbial production of β-amylase are
scanty and very few in SSF.
• A mutant strain of Bacillus megaterium, B6 mutant
UN12, was described for the comparative
production of beta amylase in SmF and SSF.
• The starchy wastes used as substrates were from
maize, potato, rice, rice husk, tamarind kernel,
cassava, water chestnut, wheat, and wheat bran.
wheat bran gave the highest yields.
• Although the sources of α-amylases are fairly
extensive, the principal commercial preparations
are derived from some bacterial and fungal
species.
• The Bacillus species is considered the most prolific
producer of α-amylase by SSF.
• B. amyloliquefaciens and B. licheniformis are
considered potent species for thermophilic αamylase.
• Irrespective of enzyme properties (such as
temperature, pH optima, and range), SSFs are
typically performed at mesophilic temperatures
such as 30°C using agro-industrial residues for 24–
96 h.
• GA production in SSF was first
demonstrated in 1914.
• An extensive study was carried out on the
production of GA in solid cultures by a
strain of A. niger.
• The study included screening of various
agro-industrial residues, including wheat
bran, rice bran, rice husk, gram flour,
wheat flour, corn flour, tea waste, and
copra waste, individually and in various
combinations.
• Apart from the substrate particle size,
which showed profound impact on fungal
growth and activity, substrate moisture and
water activity also significantly influenced
the enzyme yield.
• Enzyme production in trays occurred in
optimum quantities in 36 h in comparison
to the 96 h typically required in flasks.
• Supplementation of wheat bran medium
with yeast extract increased glucoamylase
synthesis by the fungal culture.
2. Lipases:
• Microbial lipases (glycerol ester hydrolases, EC
3.1.1.3) catalyse a wide range of reactions,
specifically hydrolysis and interesterification.
• They also catalyse alcoholysis, acidolysis,
esterification, and aminolysis.
• Microbial lipases are used for the production of
desirable flavor in cheese and other foods, and
for the interesterification of fats and oils to
produce modified acyl glycerols, which cannot
be obtained by conventional esterification.
• Aspergillus lipases are highly selective for short
chain acids and alcohols.
• Candida rugosa lipase is more selective for
propionic acid, butyric acid, butanol, pentanol,
and hexanol.
• The production of flavor esters by the lipases of
Staphylococcus warneri and S. xylosus has
been reported.
• Mucor miehei and Rhizopus arrhizus lipases are
more selective for long chain acid and acetates.
• Several agro-industrial residues and inert
supports have been used to produce
lipases in SSF.
• These include peanut press cake, coconut
oil cake, wheat bran, rice bran, babassu
oil cake, olive oil cake, sugarcane
bagasse, and amberlite.
• Enzyme production in SSF has been
reported to be superior in comparison to
SmF
3. Proteases
• Proteolytic enzymes find wide applications in
food and other industries.
• They account for nearly 60% of the industrial
market in the enzyme technology.
• Proteases are produced extracellularly by fungi
and bacteria such as A. oryzae, A. flavus, R.
oligosporus, Penicillium citrinum, P.
chrysosporium, Bacillus subtilis, B.
amyloliquefaciens, and Pseudomonas species.
• In recent years, different types of proteases such
as acid, neutral, and alkaline proteases are
being produced by SSF.
• Proteases production is generally inhibited by
carbon sources, indicating the presence of
catabolic repression of the biosynthesis.
• It is interesting to note that although a number of
substrates such as wheat bran, rice bran, and oil
cakes have been employed for cultivating
different microorganisms, wheat bran has been
the preferred choice.
• Acid protease production was increased when
the gas had 4% CO2 (v/v) and it was directly
related with the fungus metabolic activity as
represented by the total CO2 evolved.
4. Pectinases
• Pectinases are a group of hydrolytic enzymes,
usually referred as pectolytic enzymes, which find
important applications in the food and beverages
industries, in addition to various other industries.
• Pectolytic enzymes degrade pectic material and
reduce the viscosity of a solution, making it easier
to handle.
• They are used industrially in fruit processing to
facilitate pressing and to help in the separation of
the flocculent precipitate by sedimentation,
filtration, or centrifugation in the extraction of the
clarified juice.
• Pectinases are used for the elimination of pectin in
coffee and tea processing plants, and maceration
of vegetable tissue.
• Pectinases can be obtained from several fungi,
bacteria, actinomycetes, and yeast,
• Among these, A. niger is considered as the most
important for production in SSF.
• Various agro-industrial residues such as apple
pomace, citrus waste, orange and lemon peels,
soy bran, sugar cane bagasse, wheat bran,
coffee pulp, cocoa pulp, and cranberry and
strawberry pomace are used as the substrates.
• Citrus pulps or peels could be used as inducers
for enzyme synthesis.
• In the case of pectinases, generally enzyme
yields are higher and the enzyme is said to be
more stable (over a wider range of pH and
temperature) in SSF in comparison to SmF.
SSF PROCESSES FOR ORGANIC
ACIDS
• Production of organic acids such as citric acid for
food application by SSF has been employed since
olden times.
• For example, citric acid production by SSF (the Koji
process) was first developed in Japan, and is the
simplest production method.
• SSF can be carried out using several raw materials.
• Generally, the substrate is moistened to about 70%
depending on the water-holding capacity of the
substrate.
• The initial pH is normally adjusted to 4.5–6.0 and
the temperature of incubation can vary from 28 to
30°C.
• One of the important advantages of the SSF
process is that the presence of trace elements
does not affect citric acid production negatively
as it does in SmF.
• Commercially, citric acid is produced mainly
using the filamentous fungus A. niger, although
Candida sp. has also been used, employing
both molasses- and starch-based media.
• In SSF, production has been obtained using
crops and crop residues such as apple pomace,
grape pomace, coffee husk, cassava bagasse,
beet molasses pineapple waste, and carrot
waste as substrates by A. niger.
• Citric acid production is largely dependent on
the microorganisms, production techniques, and
substrates employed.
• Generally the addition of methanol
increased citric acid production in SSF.
• It has been proposed that overproduction
of citric acid was related to an increased
glucose flux through glycolysis.
• At low glucose fluxes, oxalic acid could
accumulate.
• Table 4.2 shows SSF for citric acid
production using agro-industrial residues
by different strains of A. niger.
• Another important organic acid required for food
applications is lactic acid, which has been
produced in SSF using fungal as well as
bacterial cultures.
• The commonly employed cultures belong to
Rhizopus sp. and Lactobacillus sp., e.g., R.
oryzae, L. paracasei, L. helveticus, and L. casei.
• Different crops such as cassava and sweet
sorghum, and crop residues such as sugarcane
bagasse, and carrot-processing waste served as
the substrate.
• A comparative study involving fungal
strains of R. oryzae to evaluate L-(+) lactic
acid production in SmF and SSF showed
that SSF was superior in production level
and productivity.
• Fermentation yields were 77%
(irrespective of media) and yields were
93.8 and 137.0 g/l in SmF and SSF,
respectively.
• The productivity was 1.38 g/l per hour in
SmF and 1.43 g/l per hour in SSF.
PRODUCTION OF AMINO ACIDS
• The production of L-glutamic acid in SSF with
sugarcane bagasse was used as solid inert
substrate and a bacterial strain of
Brevibacterium sp. was used for the
fermentation.
• The study not only showed good L-glutamic acid
synthesis in solid culturing but also
demonstrated that with media and process
parameters manipulation, bacterial strains can
also be successfully cultivated in SSF.
• Yield as high as 80 mg glutamic acid per g dry
fermented matter was obtained.
MUSHROOM PRODUCTION
• Mushrooms have entered the new era of food
technology as a common universally accepted
nutritive food.
• Their commercial cultivation involving SSF has
rapidly spread globally, due to their innumerable
applications.
• They are a rich source of protein, carbohydrates,
vitamins, and minerals.
• Folic acid content in mushrooms has been found
to be higher than in liver and spinach.
• In addition to their nutritive value, mushrooms
also possess medicinal properties.
• They demonstrate antibacterial, antifungal, and
antiprotozoal activities, due to the presence of
polyacetylene compounds.
• Today more than 2000 species of mushrooms
are known, although only about 20 are cultivated
commercially.
• Button mushrooms, Japanese mark forest
mushrooms, Chinese mushrooms, oyster
mushrooms, and winter mushrooms are some of
the various types of popular mushrooms being
cultivated world wide.
• However, button mushrooms alone account for
about 60% of total world production.
Button mushrooms (Agaricus
bisporus)
• Mushroom cultivation involves SSF at three
different stages, namely composting, spawn
manufacture, and the growth of mushroom on
the moist substrate.
• A temperature range of 22–27°C, substrate
moisture content of 55–70%, and a pH of 6.0–
7.5 are generally considered as the most
suitable conditions.
• Animal manure from horses and chickens, and
agro-industrial residues such as wheat straw,
paddy straw, barley straw, rice bran, saw dust,
banana, maize stover, tannery waste, wool
waste, and sugarcane bagasse are used as
substrates.
• Spawn or inoculum production involves the
growth of mycelia of mushroom on cereal grains
such as rye, wheat, sorghum, and millet.
• A mixture of sawdust and coffee husk was found
quite suitable for spawn preparation for Agaricus
bisporus, Pleurotus sp., Lentinus edodus,
Flammulina velutipes, and Volvariella volvacea.
• Depending upon the nature of the substrate,
optimum conditions were moisture content at
40–60%, a pH of 6.5–7.0, and an incubation
temperature of 25°C.
• The final product spawn should be stored at 2–
5°C.
Pleurotus sp.
• The development of the fruiting body requires a
lower temperature than the optimum for mycelial
growth; it also requires proper ventilation, which
helps in releasing the accumulated carbon
dioxide, which retards the formation of fruiting
bodies.
• At this stage, substrate moisture should be
generally higher than previous stages, (i.e.,
compost formation and spawn preparation).
• High relative humidity (80–95%) is also a
desirable condition in order to control the heat,
mass, and gaseous exchange.
• After harvesting of the fruiting body, the leftover
solid residue can be used as manure or as
animal feed, depending upon the raw material
used as substrate.
PRODUCTION OF PIGMENTS
• Pigments are the normal constituents of the cells
or tissues that give color.
• Pigments play a vital role in the food industry, to
make food decorative and appealing.
• Natural pigments contain provitamin A, and have
anticancer activity, and other desirable
properties such as stability to heat, light, and pH.
• There are pigments which are chemo-synthetic
counterparts of regular food components, that
are referred as natural-identical.
• Microbial production of pigments has usually
been carried out in SmF, though SSF processes
have also been applied.
• Monascus pigments have good properties
as food colorants possessing reasonable
light and chemical stability, tinctorial
strength, and water solubility when
complexed with appropriate agents.
• Monascus pigments may be used as
substitutes for traditional food additives,
such as nitrites for the preservation of
meats, and as a potential replacement for
synthetic food dyes.
• Monoscus anka and M. pupureus are cultivated
in SSF for red pigment production (Table 4.3).
• Steamed rice is used as the substrate, although
oats, wheat, and barley have also been used.
• The culturing period is approximately three
weeks.
• Certain sugars, amino acids, and metals have
been found important for the production, and
yields are typically 10-fold higher in both SSF
and SmF.
• Pigment formation could be inhibited by the
presence of glucose in the fermentation
medium, but could be increased by limited
aeration in SmF.
• It was also observed that an increase in the
partial pressure of CO2 increases the pigment
production.
• For isolating the pigment from fermented matter,
simple extract with solvents such as ethanol is
effective.
PRODUCTION OF AROMA
COMPOUNDS
• One of the significant applications of SSF in food
applications involves production of food aroma
compounds.
• There are two main advantages of SSF as an
alternative technology for the production of
aroma compounds:
(1) release of the products from the microbial
membranes is facilitated by the higher
concentration in liquid phase, and
(2) sometimes the solid substrates or byproducts
can be used directly in SSF without any
pretreatment of the starting substrates.
• Rhizopus oryzae cultivation of tropical agroindustrial residues results in the production of
volatile compounds such as acetaldehyde and
3-methyl butanol.
• Neurospora sp. And Trichoderma viride produce
a fruity odour and coconut aroma in SSF with
pregelatinized rice and agar medium,
respectively.
• Methyl ketones are produced on a commercial
scale from coconut oil using A. niger, and the
yields are as high as about 40%.
• Ceratocystis sp. produces a large range of fruity
or flower-like aromas (peach, pineapple,
banana, citrus, and rose), depending on the
strain and cultivation conditions.
• Among these, C. fimbriata has been extensively
studied for the production of aroma compounds
in SSF.
• Wheat bran, cassava bagasse, coffee husk, and
sugarcane bagasse were used as the substrate.
• Production of aroma compounds in SSF using
naturally occurring substrates offers potential
benefits in production of food and fruity aroma
compounds for human consumption at low cost.
VITAMINS
• SSF has been used for the formation of water soluble
vitamins such as vitamin B-12, vitamin B-6, riboflavin,
thiamine, nicotinic acid, and nicotinamide.
• Rhizopus oligosporus, R. arrhizus, and R. stolonifer
formed riboflavin, nicotinic acid, nicotinamide, and vitamin
B-6.
• The final concentrations of these substances depended on
the different strains involved and on the fermentation time.
• Isolates of R. oligosporus were generally the best vitamin
producers.
• The molds did not produce physiologically active vitamin
B-12.
• Citrobacter freundii and Klebsiella pneumoniae showed the
best capabilities for physiologically active vitamin B-12
production.