Enzyme immobilization involves confining the enzyme molecules to a distinct phase from the one wherein the substrates and the products are present

1. Enzyme immobilization And Its
Applications In Food Industry
Presented by: Nadia Bashir
Registration Id: J-14-D-226-A
Seminar Incharge : Dr. Julie D.
Bandral

2. Enzymes
• Macromolecules that catalyse chemical reactions, both biosynthetic and
degradative
(Singh, 2012)
• Highly specific and versatile
(Manay and Shadaksharaswamy, 2008)
• Ordinarily proteins and some of them may depend on the co-operation of
non-protein substance for their activity
(Singh, 2012)
• Catalyse reactions under mild conditions of moderate temperature (37°C) and
pH close to neutrality
(Elnashar, 2010)

3. • Increase the rate of reaction by a factor of about 105– 108
(Singh, 2012)
• Able to catalyse reactions in both aqueous solutions as well organic media
(Carrea and Riva, 2000)
• Widely used in
a. Food processing (baking, dairy products, starch conversion)
b. Beverage processing (beer, wine, fruit and vegetable juices)
c. Textiles, pulp and paper, detergents, pharmaceuticals industry
(Homaei et al., 2013)
….Enzymes

4. Mechanism Of enzyme catalysis
Fig 1: Effect of enzyme catalysis on the activation energy of a system
•Enzymes reduce the
activation energy barrier
(energy difference) between
ground state and transition
state thereby enhance the
rate of reaction
Elnashar, 2010

5. Disadvantages Of Enzymes
• Lack of long-term operational stability and shelf-storage life
• Complicated recovery and reuse
• Product contamination
• Enzyme costs too high
(Homaei et al., 2014)

6. The Solution: Immobilization
 Protection from degradation and deactivation
 Ability to stop reaction rapidly by removing enzyme from
reaction Solution (or vice-versa)
 Enhanced stability of enzymes
 Renders enzymes less sensitive to environment
 Avoids product contamination with enzyme
(Torres-Salas et al., 2013)

7. The Solution: Immobilization
What is Enzyme Immobilization
• Process of confining enzyme molecule in a certain defined region of space with
retention of their catalytic activities and which can be used repeatedly and
continuously
(Edet et al., 2013)
• Technique of confining enzyme molecule to a distinct phase over which a
substrate is passed and converted to products
(Singh, 2012)
• Immobilized enzyme is an enzyme attached to an inert, organic or inorganic,
insoluble material such as calcium alginate or silica with restricted movement
(Mohamad, 2015)

8. Advantages of enzyme immobilization
• Multiple or repetitive use of a single batch of enzymes
• Immobilized enzymes are usually more stable
• Ability to stop reaction rapidly by removing enzyme from reaction solution
• Product is not contaminated with enzyme
• Easy separation of enzyme from product
• Allows development of a multi-enzyme reaction system
• Reduces effluent disposal problems
(Nisha et al., 2012)

9. Disadvantages of enzyme immobilization
• Loss in activity of enzymes
• Reduced activity per unit volume
• Diffusion limitation
• Additional cost
(Hanusova et al., 2013)

10. History of Enzyme immobilization
Year Development
1919 Enzyme immobilization first reported by
Nelson and Griffin
Observed “Yeast Invertase” adsorbed on
charcoal catalysed the hydrolysis of sucrose
1953 immobilization of several enzymes on
diatomized polyaminostyrene observed by
Grubhofer and Schelth
1950s and 1960s Several covalent methods of enzyme
immobilization developed
1969 First successful industrial application of
immobilised enzymes carried out by Chibita
and co-workers
1971 Term “immobilized enzyme” adopted for the
first time at Enzyme Engineering Conference
Till today Several enzymes namely penicillin G acylase,
invertase, lipases, proteases, etc. used as
catalysts in various large-scale processes
( Homaei et al., 2016)

11. Properties of the ideal carrier for enzyme immobilization
• Materials used for confinement or immobilization of enzymes are called “Carrier matrices”
Desirable properties of support/matrix are:
 Should be physically resistant to compression
 Enhance enzyme specificity
 Should be inert towards enzymes
 Should be easy to obtain/ derive/ regenerate
 Should be resistant to microbial attack
 Should be readily available at low cost
 Should have large surface area and high permeability
 Should possess sufficient functional groups for enzyme attachment under non-denaturing conditions
 Should be water insoluble.
(Homaei et al., 2016)

12. CLASSIFICATION OF CARRIERS
Carrier
Chemical
In Organic Organic
Synthetic Biopolymers
Morphological
Porous Non-porous
Homaei et al., 2016
Fig 2: Classification of carriers

13. Methods of immobilization
• A number of immobilization methods are available to improve enzyme
activity ranging from reversible physical adsorption and ionic linkages to
stable covalent bonds
(Mohamad, 2015)
• Enzyme immobilization methods can be classified in two ways as:
 Reversible and irreversible methods on basis of separation of enzyme
from support
 Support binding and entrapment methods on basis of type of chemical
reaction used for binding
(Homaei et al., 2016)

14. Classification of Enzyme immobilization methods
Enzyme immobilization
methods
Reversible
Adsorption Ionic linkage Via disulfide
bonds
Irreversible
Covalent
Binding
Entrapment Crosslinking
(Homaei et al., 2016)
Fig 3: Classification of immobilization methods into two broad categories: irreversible and reversible methods

15. Classification of Enzyme immobilization methods
Immobilised enzyme
Attachment to
support
Covalent
Cross-
linking
Covalent
Binding
Non-covalent
Chelation/
Metal binding
Adsorption
Physical Ionic
Encapsulation/
entrapment
In fiber In gel Micro-capsules
Fig 4: Classification of immobilization methods according to the binding capacity of the carrier material
(Homaei et al., 2016)

16. Irreversible Enzyme Immobilization Methods
• Biocatalyst attached to support cannot be detached
without destroying either biological activity of enzyme or
that of support
• Includes:
1. Covalent Binding
2. Cross linking
3. Entrapment
(Costa et al., 2005)

17. Covalent Binding
• Most widely used method of enzyme immobilization
• Involves formation of covalent bonds between chemical groups
in enzyme and to chemical groups on support or carrier
• Covalent association of enzymes to supports occurs via side
chain amino acids like arginine, aspartic acid, histidine
• Such groups are not essential for catalytic activity of enzyme
• Employed when there is a strict requirement for absence of
enzyme in the product
(Singh, 2009)

18. ……Covalent Binding
Advantages:
• Strong binding
• No leakage of enzymes
Disadvantages:
• Alters conformational
structure and active site of
enzyme leading to major loss
of activity
Fig 5: Covalent bond between the biocatalysts
and a carrier
(Costa et al., 2005)

19. Cross Linking
• Also known as Copolymerization
• Enzymes joined to one another with help of bi- or
multifunctional reagents like glutardialdehyde,
glutaraldehyde, glyoxal, diisocyanates, toluene
diisocyanate, etc.)
Advantage:
 Cheap and simple technique
Disadvantage:
 Polyfunctional reagents used for cross linking
enzyme may denature or structurally modify
enzyme leading to loss of catalytic properties
Fig 6: Biocatalysts immobilized by means of cross
linking ( Costa et aI., 2005)
(Albayrak and Yang, 2002)

20. Physical Entrapment
• Enzymes are physically trapped into a film, gel, fiber, coating, or
microencapsulation
• Polymeric network allows substrate and products to pass through but
retains enzyme
• Enzyme or active molecule mixed with a polymer and followed by
crosslinking of polymer to form a lattice structure that traps enzyme
• Matrices commonly used for entrapping enzymes are polyacrylamide
gel, collagen, gelatin, starch cellulose, silicone and rubber
(Costa et al., 2005)

21. Physical Entrapment
Advantages
• Extremely large surface area between
substrate and enzyme within a relatively
small volume
Disadvantages
• Occasional inactivation of enzyme
during microencapsulation
• Leakage of enzyme from polymer
matrix
•Requirement of smaller pore size to
retain the enzyme
• Physically entrapped enzyme tends to
be diffusion limited Fig 7: Enzyme encapsulation in a matrix (a) , fiber (b), or capsule (c)
(Nisha et al., 2012)

22. I. Adsorption
 Simplest method of preparing immobilized enzymes
 Enzyme is attached to support material by non-covalent linkages like ionic,
hydrophobic interactions, hydrogen bonding
 Type of binding depends on surface chemistry of support and on type of amino
acids exposed at surface of enzyme molecule
 Materials used for adsorption are activated charcoal, Alumina, Ion exchange
resins
(Brady and Jordan, 2009)
Reversible Enzyme Immobilization Methods

23. ....Adsorption
• Advantages
 Simple in operation
 Little effect on conformation/activity of
enzyme
 Possibility of regenerating inactive enzyme by
addition of fresh enzyme
• Disadvantage
 Desorption of enzyme from support because of
changes in environment medium such as pH,
temperature, solvent, and ionic strength
(Costa et al., 2005)
Fig 8: Biocatalysts bound to a carrier by adsorption

24. …Methods of Reversible Enzyme Immobilization
II. Ionic Binding
• Based on ionic binding of enzyme molecules or
active molecule to solid supports containing ionic
charges
• Difference between ionic binding and physical
adsorption is strength of interaction
• Interactions are much stronger for ionic binding
but less strong than covalent binding
• Reversed by changing temperature, polarity and
ionic strength conditions
Fig 9: Biocatalysts bound to a carrier by
ionic binding (Costa et al., 2005)
(Nisha et al., 2012)

25. …Methods of Reversible Enzyme Immobilization
III. Immobilization via disulfide linkages
• Involves formation of disulfide (-S-S-) bonds with support
• Enzymes bearing exposed non-essential thiol (-SH) groups can be immobilized onto thiol-
reactive supports provided with reactive disulfides or disulfide oxides under mild
conditions
Advantages
 Reversibility of bonds formed between activated solid phase and thiol-enzyme
 Bound protein can be released with an excess of a low-molecular-weight thiol like
dithiothreitol [DTT]
 Possibility of reusing polymeric support after inactivation of enzyme
(Ovsejevi et al., 2013)

26. …Methods of Reversible Enzyme Immobilization
III. Affinity Binding:
• Exploits specificity of enzyme to its support under different physiological
conditions
• Can be accomplished in two ways-
i. Matrix is precoupled to an affinity ligand for target enzyme
ii. Enzyme is conjugated to an entity that develops affinity toward the matrix
Advantages
• Enzyme is not exposed to any harsh chemicals conditions
• Conformational changes during immobilization are minimal
• Retention of high activity by immobilized bio molecule
(Sardar et al., 2000)

27. Application of Immobilized Enzymes
Present applications of immobilized enzymes are :
1. Biomedical/ Therapeutic Application
• Immobilized enzymes are presently used in diagnosis and treatment of various diseases
• Used in biosensors and ELISA for detection of various analytes from complex substrates like stool,
blood etc.
(Malhotra and Chaubey, 2003)
• Immobilised enzymes are preffered in biomedical applications over free as free enzymes are consumed
by cells and are not active for prolonged use
(Nisha et al., 2012)
2. Wastewater Treatment/ Bioremediation:
• Effluents generated by various industries are threat to environment because of their carcinogenic
nature
• Enzymes are used to degrade industrial effluents like dye stuffs

28. ....Application of Immobilized Enzymes
• Enzymes commonly used in wastewater treatments are peroxidases, laccase, azo reductases
• Immobilized lipase is used for hydrolysis of oils and fats present in waste water generated from food
industry
(Nisha et al., 2012)
3. Biodiesel Production
• Biodiesel is mono alkyl esters of long chain fatty acids
• Produced from triglycerides (vegetable oil, animal fat) with esterification of alcohol (methanol, ethanol)
in the presence of the catalyst
• Conventional methods for the production of biodiesel are costly and require high energy requirement
for production of catalyst
• Immobilized enzymes can be used in biodiesel production with aim of reducing production cost by
reusing enzyme
(Salis et al., 2008)
4. Other Applications
• Immobilized enzymes like proteases and cellulases are used on a large scale for production of
detergents.
• Immobilised proteases are used to remove stains of blood, egg, grass and human sweat
(Mahmoud, A. R. D. and Helmey, 2009)

29. Applications of Enzyme immobilization in food
industry
• Application of immobilized enzymes in food industry is
increasing gradually
• Immobilized enzymes are of great value in analysis and
processing of food samples
(Khan and Alzohairy, 2010)

30. Immobilized enzymes—In the anti-microbial packaging of
foods
• Microbial contamination of food occurs mainly at their surface due to post process
handling necessitating use of anti-microbial agents
• Antimicrobial active packages are those which are in contact with food and
prevent surface growth of pathogenic micro-organism thereby extending its shelf
life
(Fucinos et al., 2012)
• Immobilization of antimicrobials on food package rather than coating it on surface
of wrapper is more effective in preventing microbial contamination
• Immobilization reduces amount of anti-microbial reagent required to achieve
antimicrobial effect as well as prolongs their activity
• Enzymes like papain, lysozyme exhibit anti-microbial properties
(Manohar et al., 2015)

31. Design of a papain immobilized antimicrobial food package with curcumin as a cross
linker
• Manohar et al. 2015 studied the anti-microbial activity of papain covalently immobilised to
LDPE (low density polyethylene), HDPE (high density polyethylene), LLDPE (linear low
density polyethylene) and PCL (polycaprolactam) with curcumin as the photocrosslinker
• Anti-microbial activity was determined against Acinetobacter sp. and Staphyloccocus sp.
• Antimicrobial action of Papain crosslinked (PCC) LDPE was tested on beef samples against
both bacterial strains
RESULTS:
• After 30 days, free enzyme retained 87% of its original activity, while immobilized enzyme
retained more than 90% of its activity
Table 1: The weight percentages of curcumin and papain immobilized on the four polymers

32. ...Design of a papain immobilized antimicrobial food package with curcumin as a cross linker
...Results:
• Papain cross linked to LLDPE exhibited
best antibiofilm properties against
Acinetobacter sp and Staphylococcus
aureus when compared to other three
polymers
• Films with immobilised enzyme were
able to reduce amount of carbohydrate
and protein contents in biofilms formed
by these organisms Fig 10: Population (Log CFU/cm2) of (A) Acinetobacter sp. and (B) S.aureus
biofilms formed on bare, Curcumin Crosslinked and Papain Curcumin
Crosslinked polymers after 24 hours of incubation
Fig 11: Protein content of (A) Acinetobacter sp. and (B) S.aureus biofilms formed on bare, Cucumin crosslinked (CC) and
Papain curcumin crosslinked (PCC) polymers after 24 hours of incubation

33. ......Manohar et al., 2015...Design of a papain immobilized antimicrobial food package with curcumin as a cross linker
Fig 12 :Exopolysaccharide content of (A) Acinetobacter sp. and (B) S.aureus biofilms
formed on bare, CC and PCC polymers after 24 hours of incubation
Fig 13 : Effect of wrappers formed of LDPE, CC-LDPE and PCC-LDPE on meat samples,
inoculated with A) Acinetobacter sp. and B) S.aureus after 7 days of storage at 4°C.
....Results:
•Meat wrapped with Papain
curcumin cross linked LDPE and
stored at 4°C showed 9 log
reductions of these organisms at
end of seventh day as compared
to samples wrapped with bare
polymer

34. Immobilized enzymes—In the analysis of food samples
(biosensor)
Biosensors :
•Analytical devices integrated with bio molecules as sensing
element
•Utilize sensitivity and selectivity of bio molecules toward
their corresponding analyte in conjunction with
physiochemical transducers to convert complex bio analytical
signals into simple easy to use signals
Has three major components –
1. Bio receptor/bio-element (enzyme, antibody, DNA) that
interacts with analyte
2. Immobilization surface (conducting polymers,
nanomaterial, sol-gel films) for immobilization of
biomolecule
3. Transducer unit for conversion of biochemical reaction
product into a recognizable signal
(Jugovic et al., 2016)
Fig 14: Schematic diagram of Biosensor

35. ......Immobilized enzymes—In the analysis of food samples ( biosensor)
• Use of biosensors in food analysis is rapidly increasing because of their following
advantages over conventional methods:
i. Low cost
ii. Simplicity
iii. Short duration of analysis
Widely used in
 Detection of calcium in milk
 Evaluation of antioxidant capacity of tea and orange juices
 Determination of sulphite in food and beverages
 Analysis of ethanol, glucose and lactate in wine
 Determination of polyphenols in beers
(Jugovic et al., 2016)

36. Development of cholesterol biosensor based on immobilized
cholesterol esterase and cholesterol oxidase on oxygen
electrode for the determination of total cholesterol in food
samples
• Cholesterol belongs to the sterol group of fats
• Present in egg yolk, dairy products, goat meat etc
• Increased concentration of cholesterol in the blood i.e.
hypercholesterolemia is mainly responsible for increased risk of
cardiovascular diseases
• Cholesterol is most frequently determined analytes in clinical as well as in
analysis of food samples
(Foster et al., 2000)

37. ...Development of cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on oxygen
electrode for the determination of total cholesterol in food samples
• Basu et al. 2007 carried out a study in which study cholesterol biosensor was developed using co-
immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOX) on oxygen
electrode
• Electrode consisted of gold cathode and Ag/AgCl anode
• Enzymes were immobilized by cross-linking with glutaraldehyde and Bovine Serum Albumin
(BSA)
Results:
1. Optimum pH and temperature of the sensor was found to be, 6 and 25 °C respectively.
Fig15: Optimization of pH performed at 25±2 °C by making use of
0.2 M Citrate–phosphate buffer (pH 4, 5, 6), sodium phosphate
buffer (pH 7 and 8), glycine–NaOH buffer (pH 9), containing 33.33
mg/dl of cholesterol palmiate.
Fig16: Temperature profile determined at pH 6.0 (at
varying temperatures of 10±2 to 50±2 °C), containing
33.33 mg/dl of cholesterol palmiate.

38. ......Basu et al. 2007...Development of cholesterol biosensor based on immobilized cholesterol esterase and cholesterol
oxidase on oxygen electrode for the determination of total cholesterol in food samples
....Results:
2. Immobilized enzymatic layer retained its activity over 30 times of use
3. Concentration of cholesterol found in different samples by biosensor method
was found to be close to values obtained by colorimetric method
Table 2: Determination of cholesterol in different real food
samples (10 times diluted except duck egg) by the developed
cholesterol biosensor.
Fig 17: Correlation between the total cholesterol
values determined by the Libermann–Burchard
method and the present method using coupled
cholesterol esterase and cholesterol oxidase system

39. Immobilized enzymes—In the clarification of juices
•Industrial juice clarification is chiefly accomplished by application of
pectinolytic enzymes.
(Pinelo et al., 2010)
• Pectin -main polysaccharide in plant cell wall responsible for juice
turbidity .
• Pectinolytic enzymes hydrolyze pectin
(Jayani et al., 2005)
• Most widely occurring enzymes are Polygalacturonase (PG), Pectin
methylesterase (PME)
(Jia et al., 2009)

40. .....Immobilized enzymes—In the clarification of juices
• Despite excellent catalytic properties of pectinase native
enzymes exhibit some draw backs
 Poor stability under operational conditions
 Difficulty of product recovery
 Impossibility of multiple reuses in an industrial process
• Application of immobilized pectinase is an alternative to
conventional processes for clarification of fruit juice
(Sheldon et al., 2005)

41. Immobilization of commercial pectinase on celite and its
application in juice clarification
• Chauhan et al. 2015 carried out
studies on clarification of pineapple
juice using immobilised pectinase
enzyme
• Different concentration of
commercial pectinase
(polygalacturonase) was
immobilised on celite through
adsorption using 2.5 per cent
concentration of glutaraldehyde as
cross-linking agent
Results:
 Immobilized enzyme retained
almost 50 per cent of activity after
third cycle of reuse
Table 3: Reusability of immobilised polygalactouronase

42. ....Immobilization of commercial pectinase on celite and its application in juice clarification
Fig 19: Effect of incubation time on activity of free and
immobilized
Fig 18: Effect of incubation temperature on activity of free and
immobilised polygalactouronase
...Results:
• Free enzyme showed maximum activity
with polygalacturonic acid (PGA, 0.9%) as
substrate at 40ᵒC temperature, pH 5.5 and
incubation time of 20 min
• Immobilized enzyme showed maximum
activity with PGA (0.9%) as substrate at
temperature of 45ᵒC, pH 5.5 and
incubation time of 15 min Fig 20: Effect of pH on activity of free and immobilized polygalactouronase

43. .....immobilization of commercial pectinase on celite and its application in juice clarification
Fig 21: Effect of enzyme concentration on clarification of pine apple juice
....Results:
• For clarification of 1 mL of pineapple juice, 20 mg of
immobilized enzyme per mL of juice at 45ᵒC
temperature and 1 hour of holding time was
optimized
Table 4: Effect of incubation temperature on juice clarification
Table 5: Effect of incubation time on juice clarification
Fig 20: Difference in the clarity of treated (with immobilised enzyme)
and untreated (control) pineapple juice

44. Immobilized enzymes—In the production of
lactose free milk
•Need for lactose free milk:
•Lactose free milk is regular milk, with the addition of the
lactase enzyme
•β-galactosidase enzyme hydrolyzes milk sugar lactose into
glucose and galactose
(Ghatak et al., 2013)
•Lactase enzyme is produced by walls of small intestines.
• Deficiency of this enzyme leads to lactose intolerance.
•Lactose intolerance is a condition in some people
characterised by decreased ability to digest lactose, a sugar
found in milk products.
•Symptoms of lactose intolerance include abdominal
pain, bloating, diarrhoea, nausea
(Heyman, 2006)

45. ......Immobilized enzymes—In the production of lactose free milk.
• Presence of high lactose content in milk leads to the production of
inferior quality ice-cream and condensed-milk due to
crystallization of lactose
(Ghatak et al., 2013)
• High concentration lactose in dairy wastes is major cause of
environmental pollution as it increases B.O.D. of water
(Panesar, 2011)
• Hydrolysis of lactose in milk makes it suitable for lactose intolerant
people, improves processing characteristics of milk and reduces
environmental pollution

46. Immobilization of β-galactosidase from Enterobacter cloacae: Characterization
and its use in the continuous production of low lactose milk
• Ghatak et al. 2013 carried out studies on production of
lactose free milk using immobilised β-galactosidase enzyme in
a jacketed bed column reactor (height 150mm, internal dia
22mm).
• β-galactosidase isolated from Enterobacter cloacae and
immobilised using alginate gel (2%)
Results:
• Maximum activity of enzyme was observed in 2 % Barium
alginate
Fig 21: Schematic diagram of experimental
set up used for production of lactose free
milk
Table 6: Effect of alginate concentration on immobilized β-
galactosidase activity

47. ....Ghatak et al., 2013: Immobilization of β-galactosidase from Enterobacter cloacae: Characterization and its use in the continuous
production of low lactose milk
Fig 22: Effect of reaction and storage temperature on
immobilized β-galactosidase activity
Table 8: Repeated use of β-galactosidase immobilised in Ba-
aliginate without cross linking
Table 7: Hydrolysis of milk lactose using immobilised β-galactosidase
in a continuous packed bed column reactor
•Immobilised enzyme exhibited maximum
activity at a concentration of 39.33 IU/mg at a
temperature of 50°C and pH 9.
•Immobilised enzyme retained 54.47 per cent
of activity after third run.
•Maximum conversion of lactose (46.34%)
was observed using milk (lactose content
4.2%) as substrate at 8 hour operation

48. Immobilized enzymes—In cheese ripening
• Cheese ripening or cheese maturation is a process in cheese making.
• Responsible for the distinct flavour, aroma and texture of cheese.
• Usually takes 6 months to 2 years depending on the cheese variety.
• Characterized by a series of complex physical, chemical and microbiological
changes
• Various methods can be used to accelerate cheese ripening like
I. Elevation of ripening temperature
II. Use of modified starters (lactic culture)
III. Addition of adjunct cultures (yeasts, molds) can be used to accelerate cheese
IV. Addition of enzymes like rennet, lipases
(Anjani et al., 2007)

49. ..............Immobilized enzymes—In cheese ripening
• Addition of exogenous enzymes is one of the simplest and most
specific of all the methods
• Direct addition of enzymes to milk during cheese making is
undesirable because of
a. Loss of enzyme in whey
b. Poor enzyme distribution
c. Reduced yield
d. Poor quality cheese
• Addition of encapsulated enzymes eliminates these problems
• Immobilised enzyme in microcapsules is physically separated
from its substrate in the milk and curd mixture during cheese
making

50. Application of encapsulated enzymes to accelerate cheese ripening
• Kailasapathy and Lam, 2005
determined the suitability of gellan, k-
carrageenan and a high-melting-fat-
fraction of milk fat (HMFF) to
encapsulate protease enzymes
(Flavourzyme) and demonstrated
their impact in accelerating Cheddar
cheese ripening
Results:
• Rates of enzyme entrapment were
48.2 per cent, 55.6 per cent, and 38.9
per cent for gellan, k-carrageenan and
HMFF, respectively.
• k-Carrageenan and gellan capsules
showed higher retention (90.0% and
91.5%) than milk fat capsules (73.5%).
Table 9:Retention of enzyme capsules in cheese curd
Table 10: Encapsulation efficiency of Flavourzyme in
different capsules

51. ...Application of encapsulated enzymes to accelerate cheese ripening
• Moisture content of cheeses with added gum capsules was higher than control cheeses
• Cheese treated with encapsulated enzyme showed higher rates of proteolysis than
control cheese throughout the ripening period
• Rate of proteolysis was greater with cheeses made incorporating k-carrageenan capsules
containing protease
Table 10 : Chemical composition of experimental cheese Table 11: Changes in b-casein content of ripening
cheese
*** C=control, K= k-carrageenan, G=gellan, F=HMFF
l, m and h are levels low, medium and high, respectively, of enzyme applications

52. .....Kailasapathy and Lam, 2005...Application of encapsulated enzymes to accelerate cheese ripening
• Little effect of type of
encapsulating material on
Cheese texture and
sensory quality.
• Lower enzyme losses
from gum gel capsules
(5.62% and 8.66% for
gellan and k-carrageenan
gums, respectively,
compared with 17.93% for
HMFF capsules)
• Lower sensory scores
noted for cheese treated
with enzymes
encapsulated in k-
carrageenan mainly
responsible because of β-
casein degradation
responsible for cheese
structure
Table 13 : Sensory scores of cheeses ripened for 5 months
Table 12 :Changes in textural properties of cheeses ripened for 5 months

53. Immobilized enzymes—In the production of flavour esters
Short-chain aliphatic esters
 Are small volatile molecules having fruity and pleasant aromas and flavor
 Ethyl and hexyl esters are among important and versatile components of natural
flavors and fragrances
 Ethyl valerate has a typical fragrance compound of green apple and hexyl acetate has a
pear flavour
 Widely used in food, cosmetic and pharmaceutical industries
(Rodriguez-Nogales et al., 2005)

54. ....Immobilized enzymes—In the production of flavour esters
• Mostly produced artificially or extracted from natural sources at high
cost
• Demand for natural flavors is growing
• Such esters can be ‘naturally’ produced using biocatalysts such as
lipases and esterases
(Brault, 2014)
• Production of these esters using free lipases is low due to lipase
inhibition by short-chain acids
• Such inhibition can be overcome by immobilization of enzyme
• In addition immobilized lipases can be recycled, reducing catalyst
costs
(Chaabouni et al., 2006)

55. Production of flavour esters by immobilized Staphylococcus simulans
lipase in a solvent-free system
• Chaabouni et al., 2006 produced flavour
esters (hexyl acetate and ethyl valerate)
using immobilised lipases
• Lipase from Staphylococcus simulans was
immobilised on CaCO3, Celite 545, Glass
beads and Carboxy-Methyl Sephadex
• Esterification reactions were carried out in
screw-capped flasks containing 3 g of
substrates mixture (acid and alcohol) taken
at various acid to alcohol molar ratio at 37°
C and stirred at 200 rpm using different
concentration of immobilised lipase
Results:
• CaCO3 was selected as the most suitable
support for immobilization of lipase
• Immobilized lipase retained its activity over
for five cycles and ten cycles of use in hexyl
acetate and ethyl valerate respectively.
Table 14: Immobilization of Staphylococcus simulans lipase
on different supports
)
Fig 23: Effect of repeated use of the immobilized lipase on the
ethyl valerate (□) and hexyl acetate (▄) synthesis

56. ....Production of flavour esters by immobilized Staphylococcus simulans lipase in a solvent-free system
Fig. 24: Effect of different amounts of immobilized Staphylococcus simulans lipase on the specific initial reaction rate
(mmol/h mg protein) (▲) and the conversion (∆) during the ethyl valerate synthesis and the hexyl acetate synthesis.
..Results:
•Optimum concentrations of immobilised lipase for hexyl acetate (41% conversion ) and ethyle
valerate were 100 IU and 200 IU respectively at 37°C
•Specific initial reaction rate decreased with increase in conc of lipase because of formation of
lipase aggregates that do not expose active site of enzyme to substrate

57. Immobilized enzymes—In wine processing
(haze removal)
Haze
 Is an aesthetic problem in white wines
 Serious quality defect for consumers
 Optical phenomenon caused by presence of small
suspended particles that divert light from its regular course
 Formed during storage when grape proteins of bottled
white wines become insoluble/ precipitate out
(Vincenzi et al., 2011)

58. ....Immobilized enzymes—In wine processing (haze removal)
• Haze can be corrected by removing grape proteins surviving
winemaking process
• Adsorption on bentonite is commonly used technique for
removal of unstable haze forming proteins from wine
• Bentonite fining causes significant variation in concentration of
micro-elements
• Is non-specific removing various molecules or aggregates involved
as aroma and flavour compounds apart from protein
• Affects organoleptic properties of wine
• Use of proteolytic enzymes is an alternative to bentonite fining
(Sauvage, et al., 2010)

59. Immobilised native plant cysteine proteases: packed-bed reactor for
white wine protein stabilisation
• Benucci et al., 2016 conducted
studies on white wine protein
stabilisation using immobilised
native plant cysteine proteases
(packed-bed reactor)
• Two enzymes namely stem
bromelain (EC 3.4.22.32) and
papain (EC 3.4.22.2) were
immobilised on commercial
chitosan beads by a direct
mechanism at pH 3.2
• Seven different unfined
monovarietal white wines
(Moscato di Terracina, Malvasia
del Lazio, Malvasia di Candia,
Chardonnay, Manzoni bianco,
Riesling, Sauvignon blanc) were
used as samples
Fig 26: Sketch of the packed-bed reactor apparatus, 1-unprocessed
white wine, 2-peristaltic pump, 3- packed-bed reactor containing
immobilised bromelain or immobilised papain, 4. butterfly valve, 5:
enzymatically processed white wine;

60. .....Immobilised native plant cysteine proteases: packed-bed reactor for white wine
protein stabilisation
Results:
• Immobilised bromelain was more effective in terms of decreasing both hazing potential and
total protein than that of immobilised papain
Fig 28: Hazing potential (ΔNTU Index) of wines before and after
enzymatic treatment in packed-bed reactor containing immobilised
bromelain (PBR-br) or immobilised papain (PBR-pa),with the
corresponding turbidity removal yield
Fig 27: Total protein content (mg/L) of wines before and after
enzymatic treatment in packed-bed reactor containing immobilised
bromelain (PBR-br) or immobilised papain (PBR-pa), with the
corresponding protein removal yield

61. ........Benucci et al., 2016...Immobilised native plant cysteine proteases: packed-bed reactor for white wine protein stabilisation
• Little effect of enzymatic
treatment on the mineral
content of wines as compared
bentonite finings
• Significantly different Sensory
parameters in bentonite-fined
wine with respect to the
unprocessed wine
• No significant difference in
sensory parameters of the
enzymatically treated sample
with respect to unprocessed
wine
Table 15: Mineral components of unprocessed Sauvignon blanc
(Control wine), wine enzymatically treated in a packed bed
reactor containing immobilised bromelain (PBR-br) and
bentonite-fined wine

62. Immobilized enzymes— In active packaging/ In
package processing (Debittering of fruit juices)
• In active packaging system active agents are embedded into or on the surface
of food packaging materials
• Such active agents enhance nutritive value, economics, and stability of food,
as well as enable in-package processing
(Wong and Goddard, 2014)
• Most of citrus juices (grape fruit) are bitter in taste
• Naringin (4, 5, 7,-Trihydroxy flavanone-7-rhamnoglucoside) is mainly
responsible for bitterness in citrus juices
• Processed grapefruit juice have naringin content above 50 ppm
(Puri and Banerjee, 2000)
• Presence of bitter taste affects the commercial acceptance of citrus juice

63. …….Immobilized enzymes— In active packaging/ In package processing (Debittering of fruit juices)
• Improvement in sensory properties and stability of citrus fruit juice can be
achieved by decreasing naringin level in processed juices
• Treatments like adsorptive debittering, chemical methods, and treatments
with ion-exchange resin based on polystyrene/divinyl benzene polymers
can be employed to reduce naringin level in citrus juices
• These treatments have several drawbacks
• An alternative to these treatments is hydrolysis of naringin by naringinase
enzyme
(Del Nobile et al., 2014)
• Naringinase, an enzyme containing α-rhamnosidase and β-glucosidase
activity hydrolyzes naringin tasteless aglycone naringenin
(Soares and Hotchkiss, 1998)

64. Naringinase immobilization in polymeric films intended for food packaging applications
• Del Nobile et al., 2014 developed a food-
grade active film capable of reducing
naringin content of grapefruit juices when in
direct interaction with the product
• Active film was based on a crosslinked PVOH
containing immobilised naringinase
• Naringinase isolated from Penicillium
decumbens was immobilised on commercial
Polyvinyl acohol in presence of
glutaraldehyde (50% aqueous solution) as
crosslinking agent and glacial acetic acid as
catalyzer
• PVOH cross linked film without immobilised
naringinase was used as control
Results:
• Concentration of naringin in sample in
contact with active film containing
immobilised naringinase was considerably
reduced after 6 h
• No effect on the concentration of naringin in
sample kept in contact with bare film
remained almost constant for about 100 h
Fig 29: Naringin concentration in the buffer solution plotted as a function of
time (о) =Bare PVOH film, (●) = PVOH film with immobilised naringinase

65. Immobilized enzymes—In the production of
syrups/sweetners
• Sucrose- commonly used sweetner in canned fruits, fruits, fruit drinks and carbonated
beverages
• Hydrolysis of sucrose produces an equimolar mixture of glucose and fructose called invert
sugar
(Yadavilli et al., 2013)
• Invert sugar is much sweeter than glucose and sucrose
• Posses desirable functional properties like
 High osmotic pressure
 High solubility
 A source of instant energy
 Prevents crystallization of sugar in food products
(Kurup et al., 2005)

66. ....Immobilized enzymes—In the production of syrups/sweetners
• Sucrose hydrolysis carried out by 2 methods
I. Acid hydrolysis: Hydrochloric acid at 75–80 °C
II. Enzyme hydrolysis: Invertase (EC.3.2.1.26) at 30–40°C
• Acid hydrolysis is expensive and generates by-products
• Enzyme hydrolysis is an alternative to acid hydrolysis because
• Immobilised enzyme-based processes operate at lower temperatures
• Produce fewer emissions and by-products
• Decreases overall production costs due to reutilization of biocatalyst
(Vitolo, 2004)

67. Use of immobilised pea invertase for production of invert sugar...
El-Sayed et al., 2015 prepared invert sugar from enzymic hydrolysis of sucrose
• Free invertase was isolated from pea pods and immobilised by two methods viz., Co-valent
binding and entrapment
• Enzyme was covalently linked to chitosan in presence of glutral dehyde as cross-linking
agent
• Entrapment of enzyme was carried out in sodium alginate beads.
• Free and Immobilized invertase activity was determined by measurement of the liberated
reducing sugar (fructose and glucose)
Results:
• Covalent binding was not suitable for immobilization of invertase
• The resultant of entrapping beads showed complete immobilization of all added enzyme
(100%) with high activity (1108×103 μg fructose) and immobilization efficiency 739.2
Table 16: Specific activity and immobilization efficiency of immobilized invertase

68. ...El-Sayed et al., 2015 ...Use of immobilised pea invertase for production of invert sugar..
...Results:
• Sucrose hydrolysis was found to be maximum
immobilised enzyme concentration of 0.02
mg/reaction mixture beyond which it decreased
because increased enzyme concentration of
product inhibits further hydrolysis
• For sucrose hydrolysis optimum temperature and
pH was found to be 45°C and 5 respectively
Fig 30: Effect of different immobilized enzyme concentrations on
sucrose hydrolysis
Fig 32: Effect of different temperatures on immobilized
invertase activity
Fig: Effect of different pH on immobilized invertase activity
Fig 31: Effect of different pH on immobilized invertase activity

69. Summary
• Enzyme instability, combined with high cost associated with their isolation and purification,
restricts general use of enzymes
• Immobilization enables separation of enzyme catalyst easily from reaction mixture, lowers
costs of enzymes dramatically and enhances their stability
• Enzyme immobilization is confinement of an enzyme to a phase (matrix/support) other than
the substrates and products
• Immobilized enzymes are preferred over their free counterpart due to their prolonged
availability reducing redundant downstream and purification processes
• Wide variety of methods can be use for enzyme immobilization ranging from reversible
physical adsorption and ionic linkages to stable covalent bonds

70. .......Summary
• Immobilised enzymes find wide applications in food industry
• Used to obtain different types of sugar syrups, lactose free milk, clarified and debittered
juices and wines
• Employed for production of different active packaging material like oxygen scavenging, anti-
microbial films
• Commercialization of immobilized enzymes is still at a slower pace because of their costs
and storage problems
• Research should be carried out to overcome current limitations related to immobilization
techniques expanding horizon for all-round application
• In future, immobilized enzymes can play a vital role in various industries including
pharmaceuticals, chemicals, food and fuel industry