Industrial production of amylases and proteases

1. DAVANGERE UNIVERSITY
DEPARTMENT OF FOOD TECHNOLOGY
SHIVAGANGOTHRI,DAVANGERE- 577002
SEMINAR ON :- ‘INDUSTRIAL PRODUCTION OF AMYLASES AND PROTEASES’
SEMINAR GUIDE :- SADASHIV S O Asst. professor.
Dept. of food technology, davangere
university
PRESENTATION BY :- MAHAMMAD ZEESHAN P
Dept. of food technology
4TH Semester
REG.NO:- FT181008

2. INDEX
SL.NO CONTENTS
1 INTRODUCTION
2 AMYLASES
3 PROTEASES
4 PRODUCTION OF AMYLASES AND PROTEASES
5 DIFFERENCES BETWEEN SUBMERGED AND SOLID STATE FERMENTATION
6 APPLICATIONS OF AMYLASES AND PROTEASES
7 CONCLUSION
8 REFERENCES

3. INTRODUCTION
Enzymes are the proteins which acts as catalyst , these lowers the energy required for the
reaction
Without being used up in the reaction. Many food and pharma industries for the production of
cheese, bread, alcohol and Medicines etc.., By employing the fermentation techniques. The industrial
production of these enzymes is by using bacteria and yeast (bacterial enzymes are mostly prefered).
Various bacteria like Bacillus subtilis, and Bacillus polymyxa, are referred for amylase production and
Bacillus licheniforms, pseudomonus, clostridium and fungi like Aspergillus flavus, Aspergillus niger,
Aspergillus oryzae for the production of proteases. By using ‘SUBMERGED AND SOLID STATE
FERMENTATION’ Methods.
KEY WORDS:- Enzyme, bacteria, Solid and submerged fermentation.

4. AMYLASES
 Alpha amylases are referred as endo 1,4 α-D- glucan , glucanohydrolase which randomly splits the
1,4 α glucoside linkages between the adjacent glucose units in linear amylase chain.
 Various bacteria and fungi produce amylases .Although the enzymes produced by these two are not
identical .
 Amylases breakdown starchy materials i.e. CHO molecules .
(large chain CHO’s)
Polysaccharides Glucose
(Starch) (Monosaccharides)
 Bacteria used are Bacillus subtilis and Bacillus polymyxa

5. Starch
Dextrose Amylase
Maltose
Glucose
 These bacteria produces amylase when they are subjected to lot of starchy materials and no
glucose and fructose.
 If the glucose is present readily in the medium then the m-org. do not produce amylase but
they utilize the readily available glucose.
 If the glucose is absent in the medium , M-org. produce amylase to act on starchy materials
to get utilizable molecules such as glucose.

6.  In that time we extract the produced amylase from the media.
 Optimum temperature required is 27-35 ̊ C and PH maintained at 6.8-7.0 .
 Amylases are extracellular and endo acting .
(act outside) (cleave at the middle of starch chain)
 And these enzymes are extracted by the Precipitation, salting out, sedimentation, chromatographic
techniques and dialysis.
 Then the product is made into pellet, powder depending on the type of application.

7. CULTURE MEDIUM FOR AMYLASES (BACTERIAL)
INGREDIENT QUANTITY (g/l)
Ground soya bean meal 18.5
Brewers yeast fraction 15
Distillers dried soluble 7.6
Enzymatic casein hydrolysate 6.5
Lactose 47.5
Magnesium chloride 0.4
Antifoam 0.5

8. PROTEASES
 Enzymes involved in the degradation of proteins are called as proteolytic enzymes / proteases.
 Proteolytic enzymes are produced by various type of M-org. such as licheniforms, pseudomonus,
clostridium, and fungi like Aspergillus flavus , Aspergillus niger and Aspergillus oryzae.
 Industrially available proteases are produced by M-org. are usually a mixture of endopeptidase
and exopeptidase.
A. PROTEIN (endopeptidase) POLYPEPTIDASE
B. POLYPEPTIDASE (exopeptidase) AMINO ACIDS

9. A.
 Catalyzes the breaking of peptide bond within the polypeptide chain.
 Cannot breakdown peptides into monomers.
B.
 Catalyzes the removal of an amino acid at the end of polypeptide chain.
 Release a single amino acid or dipeptide from peptide chain.
 Complex mixture of true proteinases and peptidases are usually called proteases.

10. CULTURE MEDIUM FOR PROTEASES (BAC. LICHENIFORMS)
COMPONENT AMOUNT (g/l)
STARCH HYDROLYSATE 50
SOYA BEAN MEAL 20
CASEIN 20
SODIUM HYDROGEN PHOSPHATE 3.3

11. METHODS OF FERMENTATION
1. SUBMERGED FERMENTATION
2. SOLID SURFACE FERMENTATION

12. SUBMERGED FERMENTATION

13. SUBMERGED FERMENTATION
Submerged fermentation is the cultivation of microorganisms in liquid nutrient broth. Industrial
enzymes can be produced using this process. This involves growing carefully selected micro
organisms (bacteria and fungi) in closed vessels containing a rich broth of nutrients (the
fermentation medium) and a high concentration of oxygen. As the microorganisms break down the
nutrients, they release the desired enzymes into solution. Due to the development of large-scale
fermentation technologies, the production of microbial enzymes accounts for a significant
proportion of the biotechnology industries total output. Fermentation takes place in large vessels
(fermenter) with volumes of upto 1,000 cubic metres. The fermentation media sterilises nutrients
based on renewable raw materials like maize, sugars and soya.

14. Most industrial enzymes are secreted by microorganisms into the fermentation medium in
order to break down the carbon and nitrogen sources. Batch-fed and continuous
fermentation processes are common. In the batch-fed process, sterilized nutrients are added
to the fermenter during the growth of the biomass. In the continuous process, sterilized
liquid nutrients are fed into the fermenter at the same flow rate as the fermentation broth
leaving the system.
This will achieve a steady-state production. Parameters like temperature, pH, oxygen
consumption and carbon dioxide formation are measured and controlled to optimiZe the
fermentation process.

15. Firstly, in harvesting enzymes from the fermentation medium one must remove insoluble
products, e.g. microbial cells. This is normally done by centrifugation. As most industrial
enzymes are extracellular (secreted by cells into the external environment), they remain in the
fermented broth after the biomass has been removed. The biomass can be recycled as a
fertilizer, but first it must be treated with lime to inactivate the microorganisms and stabilize it
during storage. The enzymes in the remaining broth are then concentrated by evaporation,
membrane filtration or crystallization depending on their intended application. If pure enzyme
preparations are required, they are usually isolated by gel or ion exchange chromatography.
Certain applications require solid enzyme products, so the crude powder enzymes are made into
granules to make them more convenient to use.

16. Sometimes liquid formulations are preferred because they are easier to handle and dose
along with other liquid ingredients. Enzymes used in starch conversion to convert glucose
into fructose are immobilised, typically on the surfaces of inert granules held in reaction
columns or towers. This is carried out to prolong their working life as these enzymes normally
go on working for over a year.

17. SOLID STATE FERMENTATION

18. SOLID STATE FERMENTATION
Solid-state fermentation (SSF) is another method used for the production of enzymes.
Solid-state fermentation involves the cultivation of microorganisms on a solid substrate, such as grains,
rice and wheat bran. This method is an alternative to the production of enzymes in liquid by submerged
fermentation. SSF has many advantages over submerged fermentation. These include, high volumetric
productivity, relatively high concentration of product, less effluent generated and simple fermentation
equipment.
There are many substrates that can be utilized for the production of enzymes by SSF.

19. These include wheat bran, rice bran, sugar beet pulp and wheat and corn flour. The selection of
substrate depends on many factors, which is mainly related to the cost and the availability of the substrate.
Other factors include particle size and the level of moisture. Smaller substrate particles have a larger surface
area for the proliferation of the microorganisms, but if too small the efficiency of respiration will be impeded
and poor growth and hence poor production of enzymes will result. Larger particles provide more efficient
aeration and respiration, but there is a reduction in the surface area. A compromise must be reached, regarding
the particle size of the substrate for a particular process. SSF requires moisture to be present on the substrate,
for the microorganisms to produce enzymes. As a consequence the water content of the substrate must also be
optimized, as a higher or lower presence of water may adversely affect the microbial activity. Water also has
implications for the physicochemical properties of the solid substrate. Enzymes of industrial importance have
been produced by SSF. Some examples are proteases, pectinases, glucoamylases and cellulases.

20. TYPES OF FERMENTATION PROCESS
1. BATCH FED FERMENTATION
Batch reactors are simplest type of mode of reactor operation. In this mode, the reactor is
filled with medium and the fermentation is allowed to proceed. When the fermentation has
finished the contents are emptied for downstream processing. The reactor is then cleaned, re-
filled, re-inoculated and the fermentation process starts again.

21. 2. CONTINUOUS FERMENTATION
Continuous reactors: Fresh media is continuously added and bioreactor fluid is continuously
removed. As a result, cells continuously receive fresh medium and products and waste products and
cells are continuously removed for processing. The reactor can thus be operated for long periods of
time without having to be shut down. Continuous reactors can be many times more productive than
batch reactors. This is partly due to the fact that the reactor does not have to be shut down as
regularly and also due to the fact that the growth rate of the bacteria in the reactor can be more
easily controlled and optimized.
In addition, cells can also be immobilized in continuous reactors, to prevent their removal and thus
further increase the productivity of these reactors.

22. Continuous reactors are as yet not widely used in industry but do find major application in
wastewater treatment. Fed batch reactor is the most common type of reactor used in industry. In this
reactor, fresh media is continuous or sometimes periodically added to the bioreactor but unlike a
continuous reactor, there is no continuous removal. The fermenter is emptied or partially emptied
when reactor is full or fermentation is finished. As with the continuous reactor, it is possible to
achieve high productivities due to the fact that the growth rate of the cells can be optimized by
controlling the flow rate of the feed entering the reactor.

23. DIFFERENCES BETWEEN SUBMERGED AND SOLID SURFACE
FERMENTATION
SUBMERGED
• Fermentation may be carried out as
batch or continuous.
• Medium added in large vessel.
• Aeration and agitation is necessary.
• Surface area to volume height ratio is
very less.
• Less space is required.
SOLID SURFACE
• As batch only.
• Only on flat surfaces.
• Usually by passing sterile air and no
agitation.
• Ratio is very high.
• More space required.

24. • Entire media is utilized by M-org.
• Less contamination.
• High power consumption.
• Automation and use of computer is
easy.
• Less labor required.
• wastage of fermentation media.
• More contamination may occur.
• Less power consumption.
• Difficult to use.
• More labour required.

25. APPLICATIONS OF AMYLASES AND PROTEASES
BAKING INDUSTRY:-
 As a flavor enhancer and ant stalling agent to improve bread quality.
 It improves crust colour , taste and toasting qualities.
 Used as a Glazing agent for the production of rice cakes and powdery foods.
STARCH INDUSTRY:-
 Starch liquification which converts starch into glucose and fructose syrup.
BEVARAGE INDUSTRY:-
 Clarification of juice to improve yield and make the process cost effective.
 For the production of ethanol.
 For the production of HFCS and HGS.

26. BAKING INDUSRY:-
 To reduce mixing time , to improve dough consistency, and to regulate gluten strength in bread.
DAIRY INDUSTRY:-
 To improve flavor characteristics of cheese and to accelerate cheese ripening.
 For the coagulation of milk.
BEVARAGE INDUSTRY:-
 Improves the fermentation of beer even in low Ph.
MEAT INDUSTRY:-
 As brewing and tenderization of meat.
 To improve flavor, nutritional value, solubility and digestibility of food proteins.
PROTEASES

27. CONCLUSION
Amylases and proteases are also be produced by plants, animals and other fungi, but the
enzymes produced by bacteria are more preferable because they are more stable and have better
characteristic property than the other resources. For the production of enzymes the culture
medium in the fermenter should not contain any smaller molecules such as glucose and must
contain high amount of starch substance . If even small quantities of glucose present in medium it
can reduces the productivity of enzymes. Our main intention is to produce enzymes. When there is
no glucose is available for the bacteria ,then these bacteria produce enzymes to break up the
starch substances in order to utilize them. In that time we recover and utilize the enzymes.

28. REFERENCES
 Parmjit S Panesar, Satwinder S. Marwaha, Harish k. chopra, 2013. Enzymes in food processing,
New delhi 110016, I.K International publishing House Pvt. Ltd.
 Naceur M’Hamdi, Jamel jabali, Cyrine Darej. Jan 2014 , Different enzymes and their production,
102.
 https://www.researchgate.net/publication/296324109