The document discusses the production of enzymes, specifically focusing on upstream processing technologies for secondary metabolite production through microbial fermentation methods. It details the historical background of enzymes, their classification, and the biotechnological processes involved in enzyme production, including solid-state and submerged fermentation techniques. Additionally, it addresses the formulation, extraction, and purification of enzymes, emphasizing the optimization of conditions for maximum yield, particularly for α-amylase from Aspergillus oryzae using solid-state fermentation of edible oil cakes.

1. Subject: UPSTREAM PROCESSING
TECHNOLOGY
TOPIC -SECONDARY METABOLITE
PRODUCTION: PRODUCTION OF
ENZYMES

2. What are
enzymes
 Enzymes are proteins and are the biocatalysts synthesized by living
cells
 They classified into Oxidoreductases, transferases, hydrolases,
lyases, isomerases and ligases etc…
 Enzymes have been used ever since mankind discovered ways to
process food

3. History
 The first enzyme produced industrially was a fungal
amylase in 1896, in United States. It was used as a
pharmaceutical agent to cure digestive disorders.
 In 1901 Eduard Bucher won the Nobel prize in
biochemistry for proving the existence of enzymes
 A German scientist (Otto Rohm) demonstrated in 1905
that extracts from animal organs (pancreases from pig
and cow) could be used as the source of enzymes-
proteases, for leather softening.

4. BIOTECHNOLOGICAL PROCESS
OF ENZYME PRODUCTION
1) Screening
Choosing an appropriate micro-organism for the desired enzyme
2)Modification
Possible application of genetic engineering to improve the microbial strain
3)Laboratory Scale Pilot
To determine the optimum conditions for growth of
micro-organism
4 )Pilot Plant
Small scale fermenter to clarify optimum conditions
5)Industrial Scale Fermenter

5. Different organisms contribution in
the production of enzymes
a)Fungi – 60%
b)Bacteria – 24%
c)Yeast – 4%
d)Streptomyces – 2%
e)Higher animals – 6%
f)Higher plants – 4%

6. FLOW CHART

7. Regulation of enzyme productions
A maximal production of microbial enzymes
can be achieved by optimizing the
fermentation conditions
Fermentation
 Surface cultures ( solid-substrate cultures)
 Submerged cultures ( liquid cultures)

8. Solid state fermentation
 Solid state fermentation has been defined as “the
fermentation process occurring in the absence or near
absence of free water utilizing the solid substrate”.
 It is a bio-molecule manufacturing process used in the
food, pharmaceutical, cosmetic, fuel and textile
industries. These bio-molecules are mostly metabolites
generated by microorganisms grown on a solid support
selected for this purpose.
 This technology for the culture of microorganisms is an
alternative to liquid or submerged fermentation, used
predominantly for industrial purposes

9. Benefits of SSF
 Simple and cost effective
 Less effluent release, reduce pollution
 Aeration is easy
 Resembles the natural habitat of some fungi and bacteria
 SSF utilizes solid substrate, thus nutrient rich waste materials can be easily
recycled as substrate
 Substrate are used very slowly and steadily so the same substrate can be
used for longer fermentation period
 SSF is best suited for fermentation techniques involving fungi and
microorganism that require less moisture content

10. Submerged fermentation / liquid
fermentation
 Submerged fermentation is the techniques of cultivation of
microorganism in liquid broth which breaks down the nutrient to
release the desired bio-active compound into solution.
 In this method, selected microorganism are grown in closed
vessels containing a broth rich in nutrients and high
concentration of oxygen.
 In SMF substrate are utilized quite rapidly hence need to be
constantly replaced or supplemented with nutrients
 Bacteria that requires high moisture content or high water
activity are best suited for submerged fermentation.

11. Substrates
Submerged fermentation
(SMF)
Solid state fermentation
(SSF)
 Soluble sugar  Wheat bran
 Molasses  Rice and wheat straw
 Liquid media  Fruit and vegetable waste
 Fruit and vegetable juices  Paper pulp
 Sewage / waste water  Bagasses

12. Enzyme Formulation
WHY FORMULATION :
Primary task of formulation is to minimize losses in
enzymatic activity during transport, storage and use.
Secondary purposes include, prevention of microbial
contamination, to avoid the precipitation or haze
formation, minimizing formation of sensitizing dust or
aerosols and improving color and odor

13. Different formulations depending on
applications
 T-granulates
physical strength and minimum dust. High shear granulation and coating techniques. Detergent
industry.
 BG/SG granulates
Smaller particle size, easy incorporation into flour, safety. Spray drying & Fluidized bed drying.
Bakery industry.
 Micro granulates
Fluidized bed Drying for finer particle size distribution and safety (Non-dusting) in food industry
 CT-Granulates
(coated-Tough) for heat sensitive enzymes to prevent denaturation. Feed Industry.
 Immobilized Enzyme
High productivity at low cost. Enzyme immobilized on a carrier or in a matrix, enhancing stability
and preventing leakage into substrate during application. Starch, Oil & fat industry.
 Liquid Formulations
Liquid product formulated and stabilized with polyols like glycerol, sorbitol, MPG, sugar, salts
to decrease water activity

14. PACKAGING
One should use tight bottles PACKAGING and
stoppers to prevent access to moisture and should
not release any traces of heavy metals or other
enzyme-inactivating substances into the enzyme
solution or suspension. In some cases, enzymes
must be protected from light and packaged in brown
glass bottles

15. Solid state fermentation

17. Microorganism, culture conditions,
and substrates
 The fungal strain A. oryzae is obtained from the Microbial Type
Culture Collection
 The strain is maintained on yeast extract,malt extract ,agar and
Czapek Dox agar, respectively. Potato–dextrose agar is also
used for the growth and maintenance of the cultures. The
cultures are grown at 30 °C for seven days and then stored at 4
°C.
 The powdered edible oil cakes, namely groundnut, coconut,
and sesame oil cakes are selected as substrates
 The substrates are powdered and sieved using standard sieves
to eliminate the foreign materials and stored in aseptic
conditions

18. Substrates

19. Inoculum preparation and solid-
state fermentation
 Spores of 7-day-old fungal cultures are scrapped using an inoculation
loop and aseptically transferred to sterile distilled water containing 0.1%
Tween-80(polysorbate 80).
 Exactly 1 ml of spore suspension is used as inoculum for the entire
fermentation.
 About 5 g of dry oil cake is taken into a 250-mL flask, containing 2 ml of
mineral salts solution, containing 2 g of potassium di-hydrogen
phosphate, 5 g of ammonium nitrate, 1 g of sodium chloride and 1 g of
magnesium di-hydrogen sulfate in a liter of distilled water to adjust the
required moisture level
 All the contents are mixed, autoclaved at 121 °C for 20 min, and cooled.
 Spore suspensions are inoculated on the sterile solid substrate and
incubated in a solid-state fermenter maintained at 37 °C.

20. Development of a pilot-scale solid
state fermenter
 600L SSF is used for optimization
 A fan is fixed at the center of the chamber to maintain the
temperature. A relative humidity (RH) sensor is placed inside the
chamber .A compressor and water supply are connected to spray
water inside the chamber whenever RH decreases
 Under optimum conditions, the microorganisms or cells are able to
perform their desired functions. Temperature and RH inside the
chamber is monitored and controlled using sensors and controllers
 The bioreactor has four trays, an RH sensor, a temperature
controller, and a control system networked together. The pH
electrode is also set and working in the range of 0 to 13 can be used.
When pH change exceeded the set range, 0.1 N HCl or 0.1 N NaOH
can be added based on the measured value

21. Enzyme extraction
 A known quantity of fermented substrate is mixed with double
distilled water along with 0.1% Tween 80. The contents are shaken
in a rotary shaker and then centrifuged at 7000 rpm at 4 °C for 10
min
 The reaction mixture consist of 1.25 ml of 1% soluble starch, 0.25
ml of 0.1 M acetate buffer (pH 5.0), 0.25 ml of distilled water, and
0.25 ml of crude enzyme extract.
 After 10-min incubation at 50°C, the liberated reducing sugars
(glucose equivalents) is estimated using the dinitrosalicylic acid
(DNS) method. The intensity of color develops and measured at
540 nm
 One unit (IU) of α-amylase is defined as the amount of enzyme
releasing one μmol glucose equivalent per minute

22. Enzyme purification
 The crude enzyme is saturated up to 50% using ammonium
sulfate and incubated at 4 °C overnight for precipitation of
proteins. The sample is centrifuged for 15 min (7000 rpm at 4
°C) in a refrigerated centrifuge
 The supernatant-containing residues can be discarded and the
precipitate is used as an enzyme source

23. Reference
Optimization and scale-up of α-amylase production
by Aspergillus oryzae using solid-state
fermentation of edible oil cakes
M. Balakrishnan1*, G. Jeevarathinam1 , S. Kiran
Santhosh Kumar1 , Iniyakumar Muniraj2 and
Sivakumar Uthandi2*

24. THANK YOU