This document discusses the various applications of microbial enzymes in the food industry. It outlines several key enzymes produced by microorganisms that are used in food processing, including α-amylases, glucoamylases, proteases, lactase, lipases, phospholipases, and cellulases. These enzymes are widely used in industries like baking, brewing, dairy, and more to improve qualities like flavor, texture, and shelf life of foods. Microbial enzymes are preferable to plant and animal enzymes for industrial use due to properties like stability, consistency, and low cost of production.

1. JIMMA UNIVERSITY
INSTITUTE OF HEALTH
FACULTY OF HEALTH SCIENCES
SCHOOL OF MEDICAL LABORATORY
DEPARTEMENT OF CLINICAL CHEMISTERY
ADVANCED ENZYMOLOGY

2. -
Applications of Microbial Enzymes in Food Industry
Done by: Reshad Nuredin

3. outline
• Introduction to Applications of Microbial Enzymes in Food Industry
• α- Amylases
• Glucoamylases
• Proteases
• Lactase (β-galactosidase)
• Lipases
• Phospholipases
• α-Acetolactate decarboxylase
• Asparaginase
• Lipoxygenase
• Cellulase
• Xylanases

4. Applications of Microbial Enzymes in Food
Industry
Introduction
▪ Microbial enzymes play a major role in food industries.
▪ Various microorganisms and microbial enzymes are used
in food industries.
▪ Microbial enzymes are more stable than plant and animal
enzymes,
▪ hence they possess wide preference.
▪ . Microbial enzymes can be produced through
fermentation techniques in a cost effective manner with
less time and space requirement.

5. Intro……
• Microbial enzymes possess high consistency and
process modification and optimization can be done
very easily.
• Many of these enzymes find numerous applications
in various industrial sectors
• amylolytic enzymes find applications in food,
detergent, paper and textile industries.
• . It is used for the production of glucose syrups,
crystalline glucose, high fructose corn syrups,
maltose syrups etc.

6. Intro……
• In detergent industry, it is used as an additive to
remove starch based stains.
• In paper industry, it is used for the reduction of
viscosity of starch for appropriate coating of
paper.
• In textile industry, amylases are used for warp
sizing of textile fibers.
• Similarly, enzymes like proteases, lipases,
xylanases etc. have wide applications in food
sectors.
(Raveendran S et al,2017)

7. α- Amylases
• α- Amylases (E.C. 3.2.1.1) are starch degrading
enzymes capable of hydrolyzing α-1, 4 glycosidic
bonds of polysaccharides and results in the
production of short chain dextrins.
• α- Amylases finds wide applications in food industry
like baking, brewing, starch liquefaction as well as a
digestive aid .
• It is widely used in baking industry for improving the
bread quality.

8. Conti……
• . During the baking process, α- amylases are
added to dough of bread and results in the
conversion of starch to smaller dextrins which
are subsequently fermented by yeast.
• It improves the taste, crust colour and toasting
qualities of bread.
• Enzymatic conversion of starch involves three
steps - gelatinization, liquefaction And
saccharification.

9. Conti……
• Enzymatic conversion of starch involves three steps -
gelatinization, liquefaction. And saccharification.
• Gelatinization involves formation of a viscous
suspension by dissolution of starch granules.
• Liquefaction is process, which reduces the
viscosity and involves partial hydrolysis.
• Glucose and maltose are further produced by
saccharification
• This requires highly thermo-stable enzymes .

10. Glucoamylases
• Glucoamylases (E.C.3.2.1.3) are exo-acting enzymes.
• It catalyzes the hydrolysis of polysaccharide starch
from the non-reducing end.
• It is widely distributed in all living organisms.
• This enzyme is produced mainly by Aspergillus niger
and Aspergillus awamori.
• Majority of glucoamylases are stable at low
temperature
• At higher temperature, it loses its activity due to
conformational change.

11. Conti…….
• It is widely used for the production of high glucose
syrups as well as high fructose syrups.
• It improves bread crust colour and improves quality
of high fiber baked products.
• It converts the starch present in flour to maltose and
fermentable sugars.
• Glucoamylase plays an important role in the
production of sake and soya sauce.
• It is used for the production of light beer.

12. Conti……
• It improves bread crust colour and improves quality
of high fiber baked products
• Fermentation by yeast leads to dough rise.
• This enzyme is also used for the production of
glucose which upon fermentation with
Saccharomyces cerevisieae yields ethanol
• Glucoamylase metabolizes dextrins and convert
them to fermentable sugars with reduced calorific as
well as alcohol content in the beer.

13. Protease
• Proteases are widely used in detergent,
pharmaceutical followed by food industries.
• It represents 60% of industrial enzymes on the
market .
• Proteases are enzymes which catalyze the hydrolysis
of peptide bond.
• based on the site of action on polypeptide chains,
Proteases are divided into two:
• exo-peptidases and endo-peptidases.

14. Conti……
• The exo-peptidases act on the ends of polypeptide chains
• Endopeptidases act randomly in the inner regions of
polypeptide chains.
• proteases are widely used in food industry for various
applications such as brewing, tenderization of meat,
coagulation of milk and as a digestive aid.
• It also used to improve the flavor, nutritional value,
solubility and digestibility of food proteins.
• As well as to modify their functional properties
coagulation and emulsifying etc.

15. Conti….
• Another major application of protease is associated
with dairy industry.
• Naturally occurring protease contribute significantly
for the flavor characteristics of the cheese.
• It is used for acceleration of cheese ripening.
• Used to modify the functional properties of milk
properties
• To reduce the allergenic properties of milk products.

16. Lactase (β-galactosidase)
• Hydrolysis of lactose is an important biotechnological
application in food industry.
• The enzyme β-galactosidase catalyzes the hydrolysis
of lactose.
• The enzyme β-galactosidase can be obtained from
numerous biological systems including plants,
animals and microorganisms.
• β-galactosidase from yeasts with a pH optima of 6.5-
7.0 are generally used for the hydrolysis of lactose in
milk of whey.

17. Conti…….
• In case of acidic whey hydrolysis, fungal β-
galactosidase with pH optima of 3.0-5.0 is
suitable.
• In industrial applications, two major classes of
β-galactosidase are of prime importance.
• They are cold active and thermo-stable.
• Lactase in being used with milk and milk
based products to reduce the lactose
intolerance in people.

18. Conti……
• In case of fermented foods, the monomeric
forms of lactose could ferment more easily
compared to unhydrolyzed lactose.
• Another major application of lactase is the
lactose hydrolysis in whey.
• Whey is a by-product from cheese production.
• And its main components are lactose, proteins
and minerals.

19. Lipase
• Lipases are enzymes which catalyze the
hydrolysis of long chain triglycerides.
• Lipase is naturally present in the stomach and
pancreas of humans and other animal species.
• In order to digest fats and lipids.
• Microbial lipases are produced by bacteria,
fungi and yeast.
• enzymes contribute approximately 90 % of
global lipase market.

20. Conti…..
• The enzyme lipase finds application in various
industries.
• Including food, biofuel, detergents and animal
feed industries.
• lipases find major application in dairy, baking,
fruit juices, beer and wine industries.
• Commercial lipases are mainly used for flavor
development in dairy products and processing
of other foods contains fat.

21. Conti……
• Lipases are used in the dairy industry to
improve the characteristic flavor of cheese.
• This is done by acting on the milk fats to
produce free fatty acids upon hydrolysis.
• Different types of cheese can be made by
using lipases from various sources.
• For example: Romano cheese using kid/lamb
pre-gastric lipase and,

22. Conti…..
• Camembert cheese using lipase from Penicillium
camemberti
• and cheddar cheese using Aspergillus niger A. oryzae
• Lipase catalysation could improve the texture and
softness.
• Lipases are also used as flavor development agents in
butter and margarine.
• Lipases are used to increase the shelf life of various
baking products.

23. Conti….
• In alcoholic beverages such as wine, the aroma of
can be modified using lipase.
• Lipases are used to improve the quality of coca
butter.
• . Coca butter has a melting point of 37°C due to the
presence of palmitic and stearic acids and could
easily melt at 37 °C.
• Lipases also find application as a biosensor in food
industry.

24. conti,…..
• Lipases also find application as a biosensor in food
industry.
• Microbial lipases such as lipase from C. rugosa have
many applications which cannot be met by chemical
synthesis.
• This lipase finds application in the production of ice
cream, single cell protein, carbohydrate esters and amino
acid derivatives.
• lipase could also be used in the processing of different
waste streams that are released from food industries .

25. Phospholipase
• Phospholipases selectively break down
phospholipids into fatty acids and other lipophilic
substances.
• Phospholipase A1 (PLA1), Phospholipase A2(PLA2),
and lysophospholipase which act on the carboxylic
ester bonds of phospholipids.
• Phospholipases C (PLC) and D (PLD), which modifies
polar head group are also known as
phosphodiesterase's, recognize the phosphodiester
linkage.

26. Conti…….
• Phospholipases are widely used in food industry.
• the most important one is in the production of oils,
dairy industry and in the manufacture of several
bakery items.
• Phospholipases are also find applications in the
degumming of various vegetables oils, cheese
manufacture and bread manufacture .
• The PLA 2 commercialized by DSM Food
Specialties(Netherlands) with the trade name
Maxapal ® A2 has been reported by Zhao et al.

27. Esterase
• In aqueous solution, esterases are able to facilitate
the splitting of esters into acid and an alcohol.
• Esterases play a prominent role in the food industry
and alcoholic beverages industries;
• they have been mostly used for the modification of
oil and fat in various fruit juices and to produce
fragrances and flavors respectively .
• Feruloyl esterases, an important candidate from
esterases family.
• It breaks the ester bond between ferulic acid and
different polysaccharides in plant cell wall.

28. Conti…..
• Since feruloyl esterases hydrolyses
lignocellulosic biomass they are inevitable
waste management.
• By breaking plant biomass potential bioactive with
beneficial health effects are released due to the
deesterification of dietary fiber.

29. Lipoxygenase
• It involve in the dioxygenation of polyunsaturated
fatty acids in lipids containing a cis, cis-1, 4-
pentadiene.
• These enzymes are non-heme iron containing
enzymes.
• LOXes are used in aroma generation in food industry
and also used in bread making.
• Soya bean lipoxygenases (LOX) is the most-studied
lipoxygenase enzyme.

30. Conti….
• Bacterial LOXs possess different specificity towards
fatty acid.
• LOX from Nostoc sp.oxygenates at specific site in
linoleic acid.
• But the LOX from Anabaena sp. exhibits variable
specificity.
• The LOX from Acaryochloris marina, exhibit as a
fusion protein with hemedomain (N-Terminal) and
LOX29 domain (C-terminal).

31. Conti…..
• The main applications of LOXs in doughs are based
on their ability to bleach the flour pigment
carotenoid, with the co-oxidation of the pigment
carotenoids with fatty acids.
• Lipoxygenases are also employed to improve
tolerance to mixing and different handling properties
of dough.
• This effect is due to the thiol group oxidation of
gluten which may lead to redistribution of different
disulphide bonds formation of tyrosine cross-linking
and subsequent strengthening of the gluten.

32. Cellulases
• Cellulase are enzymes act on polymeric cellulose and
hydrolyze β -1, 4 linkages to liberates glucose units.
• The three major classes of cellulases are endo- (1, 4)-
β-D-glucanase (EC.3.2.1.4), exo-(1, 4)-β-D-
glucanase(EC.3.2.1.91) and β-glucosidases
(EC.3.3.1.21).
• The application of cellulases improves the extraction
methods, clarification and stabilization of juices .
• Cellulases can also reduce the nectars viscosity and
puree from fruits such as apricot, mango, plum,
papaya, pear and peach .

33. Conti…..
• Cellulases are used for the extraction of
flavonoids from flowers and seeds.
• The preferences of cellulase mediated extraction
over conventional methods are due to higher yield,
less he.at damage and low processing time.
• Cellulases are utilized for the extraction of phenolic
compounds from grape pomace. β-glucosidases in
combination with pectinase alter the structure,
flavor, aroma of fruits and vegetables .

34. Xylanases
• Xylanases are produced by microorganisms to
cleave xylans, a major constituent of
hemicellulose.
• Three major enzymes, endoxylanases,
exoxylanases and _ xylosidases,
• They act synergistically and are required for the
breakdown of xylan backbone in hemicellulose.
• Endo xylanases (EC 3.2.1.8) cleaves the -1,4 bonds
of xylan back bone.

35. Conti…….
• Exoxylanases (EC.3.2.1.37) hydrolyses - 1,4 bonds of
xylan from the non-reducing ends and releasing
xylooligosaccharides.
• - xylosidases cleaves the xylobiose and xylo
oligosaccharides to release xylose.
• The major enzyme functions of xylanases are
performed by a catalytic module .
• And few classes possess an additional carbohydrate
binding module (CBM) for binding to substrates.

36. Pectinase
• Pectinases are enzymes which catalyzes
hydrolysis of glycosidic bonds in pectic
polymers.
• Pectic substances found in tomato, pineapple,
orange, apple, lemon pulps and in orange peel and
other citrus fruits act as natural substrate for this
enzyme.
• Pectinases can be produced from natural as well
recombinant microbes with attempts made to
increase its thermo-stability and yield .

37. Conti……
• Pectinases find multitude industrial applications such as
in paper bleaching, food industry, remediation etc .
• Pectinase supplemented juices have a better cleared
appearance and filterability than enzyme depleted
counterparts.
• The gelatin and pectin of such juices would greatly
increase the viscosity and turbidity of juices and removal
of such haze is the most costly part of juice generation.
• The use of biogenic enzymes such as pectinases in juices
would act almost 9 times better than mechanical
maceration to get good results.

38. Glucose oxidase
• Glucose oxidase (EC.1.1.3.4) belongs to a large group
of enzyme family called oxidoreductases.
• The glucose oxidase production was reported from
various microorganisms and it was first discovered in
Aspergillus niger and Penicillium glaucum.
• Aspergillus niger species is widely used for
production of glucose oxidase and their strains can
produce higher amount of glucose oxidase.

39. Conti……
• Other major genus reported to produce glucose
oxidase are Penicillium, Gliocadium,
Scopulariopsis, and Gonatobotyrs.
• Penicillium adametzii is a widely used fungus for the
production of extracellular glucose oxidase.
• Although many species of bacteria and fungi are
reported to produce this enzyme.

40. Laccase
• Laccases (EC 1.10.3.2) comes under the cluster of
oxidases which is representing the largest subgroup of
multi copper enzymes.
• The commonly known as blue oxidases used for studying
their potential to oxidize phenolic compound and
therefore applied in several industrial sectors.
• These enzymes have different specificity for
substrate and a wide range of oxidizable substrates
which further depends on the type of microbial
sources producing the enzyme.

41. Conti…..
• Laccase used for modification of colour appearance
of food and beverage industries.
• Laccases are used for wine stabilization in food
processing industry as alternative to physical and
chemical adsorbent.
• this enzyme is used in cork stopper manufacturing
industries for using wine bottles .

42. Catalase
• Catalase (EC 1.11.1.6) tetrameric protein with and it
is found in aerobic organisms.
• These enzymes are mainly produced from the extract
ion of bovine liver,fungal and microbial sources such
as from Aspergillus niger and Micrococcus luteus.
• Catalase with glucose oxidases are often used
together for food preservation.
• Ough (1975) used a glucose oxidase/Catalase cocktail
for elimination of oxygen in wine before bottling.

43. Conti….
• This enzyme is applied in milk processing industry to
eliminate peroxide from milk.
• It is used for removing glucose from egg white in
baking industry and in food food wrappers to prevent
oxidation to control perishability of food.
• This enzyme has limited use in cheese production.

44. Peroxidase
• Peroxidases (EC 1.11.1.7) are oxido-reductases proteins
that contain iron (III) protoporphyrin IX as the prosthetic
group.
• It catalyses the reduction of peroxides and oxidation of
wide range of inorganic and organic compounds.
• Peroxidase catalyzes the wide ranges of substrates using
H2O2 or other peroxides.
• This enzyme is used in food industry for producing flavor,
color and texture and improving nutrition quality of food.

45. Conti….
• In the food industry the thermal inactivation of
peroxidases are used to measure the efficiency of
blanching treatment, which will further enhance the
shelf life of food.
• While the other effect of peroxidases are undesirable
like browning of fruits and off flavors of vegetables.

46. ASPARAGINASE
• Asparaginase, as the name implies, catalyses the
breakdown of the asparagine to subsequent acid
derivative aspartic acid and NH 3 and can be
considered as the asparagine-depleting enzyme.
• Asparagine is a nonessential amino acid to humans
• whereas it is an essential amino acid for cancerous
cells.
• Thus,the depletion of asparagine critically affects the
growth of cancerous cells, which forms the basis of
this enzyme as anticancer agent.

47. DEBITTERING ENZYMES – NARINGINASE
• Naringinase (EC 3.2.1.40) is mainly responsible for the
breakdown of naringin, the principle bitter flavanone
glycoside found in citrus fruits .
• Fungal sources of naringinase are found to be more
predominantly used than the bacterial ones due to
increased yield.
• Naringinase has a major role in food processing as a
debittering enzyme supplemented to fruit juices.
• The use of naringinase is also noted in tomato pulp
preparation, kinnow peel waste treatment and prunin
preparation .

48. α-ACETOLACTATE DECARBOXYLASE
• α-Acetolactate decarboxylase greatly aids in the fast
maturation of beer.
• This enzyme can be produced from natural microbes
such as Brevi bacillus brevis or from recombinant
Saccharomyces cerevisiae.
• Enzyme-based removal of α-acetolactate and α-
aceto-α-hydroxybutyrate assists in overcoming the
rate-limiting step of beer maturation.

49. References
• Couto SR, Sanromán MA. Application of solid-state
fermentation to food industry- A review. J Food Engg.
2006;76:291-02.
• Blanco CA, Caballero I, Barrios R, Rojas A.
Innovations in the brewing industry:
light beer. Int J Food Sci Nutr. 2014; 6
• Davies G, Henrissat B. Structures and mechanisms of
glycosyl hydrolases. Structure. 1995; 3:853 – 59
• Puri, M. Updates on naringinase: Structural and
biotechnological aspects. Appl Microbiol Biotechnol.
2012;93:49-60

50. • Hamid M, Khalil-ur-Rehman. Potential applications of
peroxidases. Food Chem. 2009; 115:1177–86
• Aravindan R, Anbumathi P, Viruthagiri T. Lipase
applications in food industry. Indian J Biotechnol.
2007;6:141-58
• Cumbee B, Hildebrand DE, Addo K. Soybean flour
lipoxygenase isozymes effects on wheat flour
dough rheological and bread making properties. J
Food Sci. 1997;62:281-83