The substrate used for the production of microbial amylase is starch.
There are two main
methods of amylase production that utilizes the substrate (starch), namely:
Solid State Fermentation (SSF) and Submerged Fermentation (SmF).
Industrially important enzymes have traditionally been obtained from submerged fermentation because of the ease of handling and greater control of environment factors such as temperature and pH. The cost of enzyme
production in submerged production is high. The contents of synthetic media are
very expensive and these contents might be replaced with more economically
available agricultural by products for the reduction of cost of the medium.
SSF is a technique for growing microorganisms such as fungi and bacteria on a moist solid substrate. The use of agricultural wastes makes solid state fermentation an attractive alternative method Researchers have reported amylase production in solid state fermentation with wheat bran and rice husk as substrates.
Bacterial Amylases
Bacillus
species – B.
subtilis, B. cereus, B. coagulans, B. stearothermophilus,
Pseudomonas, Serratia, Clostridium etc
Bacteria produce more of α-amylase by either through solid state fermentation (SSF) and submerged fermentation
(SmF).
Media can be natural raw
materials such as wheat bran, CSL, corn starch, which contain trace elements,
vitamins, growth factors etc. Organic and inorganic nitrogen may be added if
required. Stillage from alcohol production from grain or soluble/hydrolyzed
starch supplemented with ammonium salts and buffer can be used. Hydrolyzed soy
bean cake, pea nut cake, casein act as sources of nitrogen and mineral salts. Wheat
bran supplemented with salts may be used.
Incubation is done at
25-37oC for 2-6 days, in tray method of solid state fermentation and
24-48 oC for 24-48 hours for submerged method. pH can increase
during fermentation, so maintained using buffers or media components like urea,
peptone etc.
Bacterial α-amylases
require minimum purification. After fermentation, extracellular enzyme is harvested
and recovered using precipitation, concentrated by dialysis and purified using different
chromatographical/adsorption techniques. Other contaminating enzymes are
removed by selective adsorption or inactivation.
α-amylases from B. subtilis and especially B. stearothermophilus, are thermostable
with industrail applications.
Fungal
Amylases
Fungi like Rhizopus,
Mucor rouxii, Penicillium spp, Candida spp, etc can be used for amylase production.
Aspergillus spp are widely used in both solid state fermentation (SSF) and submerged
fermentation (SmF) processes for amylase production.
Starchy
grain mash is inoculated with the molds. Amylases are produced by
saccharification of mashed substrate and this process is termed Amylo process.
Aspergillus oryzae
is used in solid state fermentation and Aspergillus
niger is generally used in submerged fermentation. Moistened, steamed
rice/wheat bran is used for amylase production from Aspergillus oryzae by tray method. In which
the mold is grown on thin layers of the medium in trays. Alternatively, drum
method can also be used, where the bran is filled loosely in a rotating drum. Maximum
yield is obtained in about 48 hours at 30oC in tray method with
proper humidity and adequate ventilation.
Moisture
content is critical in SSF and it depends on the substrate thickness and
incubation temperature, mainly. Incubation can be done from overnight to 4 days.
This depends on the organism employed. Fungal α-amylase accumulates largely in the
stationary phase and even during autolysis.
After
fermentation, amylases are extracted from the fermented mycelium and purified
using precipitation, chromatography and finally concentrated. Contaminating
enzymes in the fermented broth may be removed in the purification process.
SSF
can also be carried out in deep tanks with flour/starch as raw material
supplemented with inorganic salts. Addition of stillage, corn steep liquor and
yeast extract is found to increase the yields.
i. High volumetric productivity is obtained.
ii. Relatively higher concentration of the enzyme is produced.
iii. Less effluent is generated.
iv. Low cost of production is required.
v. It is easy to use.
vi. Raw starch is efficiently digested
Applications
Amylases have diverse
industrial applications in
- food industry for products like bread and sweeteners, brewing and distilling for fermentable sugars
- detergent industry to enhance stain removal, the textile industry for desizing fabrics
- paper industry to reduce viscosity.
- for producing biofuels like ethanol from starch
- in medicinal and analytical chemistry.
Food Industry
- Baking: Amylases break down
starch in dough into sugars, causing the dough to rise and improving the
texture and flavor of baked goods.
- Sweeteners: In the production of sweet syrups, especially high-fructose corn syrup, amylases convert starch into simpler sugars. By proper control of the type and proportion of enzymes used (α -amylase, amyloglucosidase, maltase), syrups of any desired proportions of glucose, maltose, and dextrins may be produced.
- Fruit Juices: They are used in the fruit juice industry.
- Microbial amylases are used for modifying starch in vegetable purees, and in treating vegetables for canning.
- Other applications of fungal and bacterial amylases are in processing of cereal products for food dextrin and sugar mixtures and for breakfast foods, for preparation of chocolate and licorice syrups and for recovering sugars from scrap candy of high starch content.
- Fungal amylases are also used for starch removal for flavoring extracts and for fruit extracts and juices, and in preparing clear, starch-free pectin.
Brewing and
Fermentation
- Alcohol Production: Amylases convert the
starch in grains (like barley) into fermentable sugars for yeast to
produce alcohol in beverages and biofuels.
- Maltose Production: Amylases are used to produce maltose, which is important in brewing and other food applications.
Textile Industry
- Desizing: Starch-based sizing agents are applied to yarns to strengthen them for weaving. After weaving, amylase is used to efficiently remove the starch, preparing the fabric for dyeing and finishing.
Detergent and
Laundry
- Stain Removal: Amylases are
incorporated into detergents and laundry products to break down
starch-based stains on fabrics.
Other
Industries
- Biofuel Production: Amylases are key in the
production of fuel alcohol from starch.
- Pharmaceuticals and Medicine: They have applications
in medicinal and clinical contexts, including the production of specific
digestive aids.
- Fine Chemicals: Amylases can be used in the production of various fine chemicals.
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