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How to cite this article: Yavari Maroufi L, Rashidi M, Tabibiazar M, Mohammadi M,

Pezeshki A, Ghorbani M. Recent Advances of Macromolecular Hydrogels for Enzyme
Immobilization in the Food Products. Advanced Pharmaceutical Bulletin, doi:
10.34172/apb.2022.043

Recent Advances of Macromolecular Hydrogels for Enzyme

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Immobilization in the Food Products

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Leila Yavari Maroufi1, Mohsen Rashidi2, Mahnaz Tabibiazar1, Maryam Mohammadi3,
Akram Pezeshki3, Marjan Ghorbani4,5*

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1
Department of Food Science and Technology, Faculty of Nutrition and Food Science, Tabriz
University of Medical Sciences, Tabriz, Iran.
2
Department of Pharmacology, Faculty of Medicine, Mazandaran University of Medical
Sciences, Sari, Iran.

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3
Faculty of Food Science and Technology, Faculty of Agriculture, University of Tabriz, Tabriz,
Iran an
4
Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
5
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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Corresponding Authors: Marjan Ghorbani: ghorbani.marjan65@yahoo.com

Recent advances of macromolecular hydrogels for enzyme immobilization

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in the food products

Abstract
Enzymes are one of the main biocatalysts with various applications in the food industry.
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Stabilization of enzymes on insoluble carriers is important due to the low reuse, low operational
stability, and high cost in applications. The immobility and the type of carrier affect the activity
of the immobile enzyme. Hydrogels are three-dimensionally cross-linked macromolecular
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network structures designed from various polymers. Hydrogels can provide a matrix for an
immobile enzyme due to their extraordinary properties such as high water absorbing capacity,
carrier of bioactive substances and enzymes, biocompatibility, safety, and biodegradability.
Therefore, this study mainly focuses on some enzymes (Lactase, Lipases, Amylases, Pectinase,
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Protease, Glucose oxidase) that are of special importance in the food industry. These enzymes
could be immobilized in the hydrogels constructed of macromolecules such as kappa-
carrageenan, chitosan, arabic gum, pectin, alginate, and cellulose. At last, in the preparation of
these hydrogels, different enzyme immobilization methods in macromolecular hydrogels, and
effect of hydrogels on enzyme activity were discussed.
Keywords: Hydrogels, Enzyme immobilization, Food industry

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1. Introduction
Enzymes are a type of biocatalysts, widely applied in several applications in the food industry,
such as baking, beverages, meat, dairy, fats and oils, as an effective, safe and eco-friendly
alternative for food production. Enzymes have been used as food preservatives for long years,
and nowadays they are enabling a variety of food industries to give the quality and stability of
their products, along with better production efficiency. They provide clean, environment
friendly and specific methods for biochemical reactions in moderate conditions.1–3 However,

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the use of enzymes is limited due to their high cost and low reusability. Moreover, the lack of

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proper mechanism to protect enzymes against protease attack, occurring in almost all biological
systems, is another major hurdle to achieve optimal activity.4 Additionally, the low operational
stability of some enzymes during any biochemical reaction is problematic. Therefore, enzyme
stabilization is the main objective of enzyme technology. The attainment of stable and active

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enzymes is a highly challenging effort. In order to overcome these limitations, the
immobilization of enzymes with functional efficiency is useful to solve the enzyme problems
and decrease the costs. The immobilization method involves the inclusion of enzymes in
matrices or binding them on various surfaces.5,6 The immobilization of enzymes on

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hydrophobic supports is a general method. There are various chemical catalyst carriers to
immobilize enzymes, one of which is the use of hydrogel matrixes, hydrogels may be used as
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appropriate carriers for enzymes.7 The ideal carrier matrix should have the following
properties: (a) to be economical, (b) inertness, (c) stability, (d) physical strength, (e) ability to
enhance enzyme specificity/activity, (f) regenerability, (g) ability to reduce product inhibition,
and (h) ability to prevent nonspecific adsorption and bacterial contamination. Immobilization
usually stabilizes the enzyme structure, allowing the hydrogels’use under harsh environmental
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conditions (pH, temperature,and presence of organic solvents). Hydrogels are water-insoluble
three-dimensional hydrophilic polymer networks that possess all the mentioned ideal carrier
properties with a high ability to retain water and other liquids.4 Therefore, the aquatic
environment of hydrogels can reduce the denaturation of enzymes and help their catalytic
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function.8,9 Hydrogels, as smart materials, can respond to many environmental stimuli,

including temperature 10,11, pH 12–14 by showing changes in structure, shape and interaction
with their loaded substrates. They have several applications, such as drug delivery, release 15,16,
enzyme trapping, releasing17 and biosensor.18 In this study, some important enzymes in the
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food industry immobilized in hydrogels, various natural polymers used in the preparation of
hydrogels, methods of enzyme immobilization in matrix hydrogels and hydrogels effect on the
activity of enzymes are discussed. Figure 1 summarized the different enzymes which can be
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immobilized on hydrogels with different methods.

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Fig 1. The scheme summarized the different enzymes which can be immobilized on
hydrogels with different methods.
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2. Enzymes
The enzymes play a variety of roles in the food industry. Some of these roles are listed in Table
1.
2.1. β-Galactosidase (Lactase)
β-Galactosidase is from the hydrolase family of enzymes. It is an enzyme usually used to
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hydrolyze lactose in dairy products. Lactose is the predominant disaccharide in milk and dairy
products, that some people are unable to consume due to sensitivity. The β-Galactosidase
enzyme by lactose hydrolysis, makes the consumption of dairy products possible for people
with lactose intolerance. Also recently, this enzyme has been used to produce oligosaccharides,
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known as prebiotic products. Therefore, the use of the β-Galactosidase enzyme facilitates the
production of useful products in the food industry. Since enzymes have low stability, their
immobilization and stabilization on suitable carriers are essential. Immobilization of enzymes
is an easy procedure with several benefits, including enzyme reusability, persistent process,
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increased stability under operation and storage state.19–25

2.2. Lipases
One of the widely used biocatalysts is lipase. Lipases are from the hydrolase family of enzymes.
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They are effective enzymes with various applications in medicine, pharmaceuticals, cosmetics,
detergents, paper production and the food industry. They are good catalysts for the production
of food additives and ingredients. They have great potential for synthesizing short-chain esters
to be used in the food industry as flavor modifiers or fragrance compositions. Lipases play an
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important role in the dairy industry, including hydrolyzing milk fat, accelerating cheese ripen,
increasing the flavor of cheese and lipolyzing butter. In the lipid industry, lipases can be applied
to retailor animal and vegetable oils. The industrial use of lipases is limited due to the high cost
of their production, the lack of long-term stability and difficulty in recycling them; thus,
immobilizing them on suitable matrices, such as hydrogels, can be very effective.26–28
2.3. Amylases
Amylases are from the hydrolase family of enzymes. They are widely found in microbial, plant,
and animal sources and are one of the important industrial enzymes with many applications in

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the food and beverage industries. Amylase is the essential enzyme in the bread industry, which
breaks down damaged starch in wheat flour into small dextrins and strengthens the dough,
resulting in improved bread volume. Further, small oligosaccharides and sugars such as glucose
and maltose, produced by this enzyme increase Millard responses responsible for browning the
shell and creating an attractive cooked taste.26,31
2.4. Pectinase
Pectinase is from the hydrolase family of enzymes. The enzyme is used in processing pectin,

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the main component in the middle lamella of the plant cell wall. Pectinases are widely used in

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the food industry, such as extracting and clarifying wine and fruit juices, macerating fruit,
reducing the viscosity of fruit juices, extracting vegetable oil, fermenting coffee and tea, and
valorizing industrial wastes; due to these extensive applications, they make up 25% of the
world's enzymes. Though, like many other industrial enzymes, pectinase has a limited yield

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and low efficiency in its economic generation.32–34
2.5. Protease
The protease enzyme belongs to the family of hydrolases. The origin of protease enzymes is
plant, animal, and microbial. Protease or peptidase is an enzyme that hydrolyzes peptide bonds,

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which is the main commercial and industrial enzyme. The proteases represent the largest group
of commercially available enzymes worldwide, accounting for 60% of the industrial enzymes
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market due to their wide range of applications in food, beverage, detergent, medical diagnosis,
leather industries, as well as research and development activities. In the food industry, it is
widely used in producing cheese by coagulating milk, improving the digestibility and
nutritional value of biscuits, pastries, wafers, cookies through protein hydrolysis, producing
gluten-free pasta and producing functional products. Therefore, its immobilization in the
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hydrogel matrix reduces costs and makes it easy to be widely used in the food industry.35–37
2.6. Glucose oxidase
Glucose oxidase is an oxidoreductase, that catalyzes the oxidation of glucose to gluconic acid
and hydrogen peroxide. It has many uses; for example, it scavenges oxygen in the food industry
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effectively, catalyzes the reaction of glucose and oxygen that generates glucuronic acid, and
successfully removes oxygen from food and beverages to prolong their shelf life.38

Table 1. Application of immobilized enzymes in food industry.

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Enzyme Application in food industry Ref

β- β-Galactosidase is an enzyme widely used in dairy products to 19–21

Galactosidase hydrolyze of lactose, makes it possible consumption of dairy

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(Lactase) products for people who are lactose intolerance, used to produce
oligosaccharides, that are known as prebiotic products
26–28
Lipase play an important role in the dairy industry, including hydrolyze
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milk fat, accelerating cheese ripen, increase the flavor of cheese

and lipolysis of butter. In lipid industry, lipases can be applied to
retailoring of animal and vegetable oils
31
Amylase Amylase is most important enzymes in the bread industry, which
breaks down damaged starch in wheat flour into small dextrins
and strengthens the dough, resulting in improved bread volume

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32,33
Pectinase extracting and clarifying wine and fruit juices, fruit maceration,
reducing the viscosity of fruit juices, extraction of vegetable oil,
coffee and tea fermentation and valorization of industrial wastes
35–37
Protease production of cheese by coagulating milk, improving the
digestibility and nutritional value of biscuits, pastries, wafers,
cookies through protein hydrolysis, the production of gluten-free
pasta and the production of functional products

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38
Glucose oxygen scavenger, catalyzes the reaction of glucose and oxygen

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oxidase and remove oxygen from food and beverages to prolong their
shelf life

3. Hydrogel matrixes as enzyme carriers

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Hydrogels are three-dimensional, polymeric and hydrophilic networks. They are formed from
both synthetic and natural hydrophilic polymers, that are water-insoluble, able to swell, absorb,
and retain major amounts of water. Over the years, researchers have defined hydrogels in many
different ways. The most commonly used definition is that the hydrogel is a water-swollen and

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cross-linked polymeric network, produced by the simple reaction of one or more
monomer/polymer/cross-linker units. One more description is that it is a polymeric material
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that exhibits the ability to swell and retain a large amount of water in its three-dimensional
network, however, will not dissolve in water.39,40 Hydrogels have good biocompatibility and
can provide a suitable microenvironment41 and they are widely used in different fields
including drug delivery systems, tissue engineering, protein and cell immobilization,
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agriculture and horticulture and food industry.42,43 In recent years, development of responsive
hydrogels has been observed in various field. In particular, hydrogels of polymers such as
chitosan, alginate, kappa-carrageenan, etc. have been used as supports for enzyme
immobilization. Some studies have reported the immobilization of various enzymes including
lipase, lactase, protease, and amylase on polymer-based hydrogels. Enzymes immobilization
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on soft and solid supports, such as hydrogels, is an efficient procedure amongst diverse enzyme
immobilization techniques. Because of retaining a large amount of water inside the three-
dimensional network, they provide efficient physiological conditions for enzyme activity. The
aqueous environment of polymeric hydrogels can reduce the denaturation of enzymes and help
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enzymatic functions. Therefore, it can be expected to maintain enzyme activity due to the
immobility in the hydrogel polymer matrix.44 As shown in Table 2, the various polymer based
hydrogels was used in this field.
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3.1. Kappa-carrageenan based hydrogel

Kappa-carrageenan, thermo-reversible gel, is a linear, negatively charged sulfated
polysaccharide extracted from marine red algae. Kappa-carrageenan is widely used in food,
cosmetics, and drug controlled release and encapsulation due to high biodegradability and
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biocompatibility. In the food field, due to gelling capabilities in the presence of counter-ions,
especially K, it is used for many applications.45–47 Z. Zhang et al.48,49 immobilized β-
Galactosidase enzymes into kappa-carrageenan-based hydrogel beads. As shown in studies
conducted by them, the immobilization of β-galactosidase enzyme into carrageenan-based bead
hydrogels improved enzyme activity at pH and medium temperature conditions; the
physicochemical origin of this effect was attributed to the ability of K ions used to cross-link
the polysaccharide chains to increase the stability and activity of β-galactosidase.

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3.2. Chitosan based hydrogel

Chitosan-based hydrogels have received substantial interest recently in enzyme
immobilization, drug delivery, agriculture, biomedicine and food industry. Chitosan (CS) is a
nontoxic natural polymer produced by the deacetylation of chitin and compound of
glucosamine (70%) and acetylglucosamine (30%) units with a molecular weight ranging from
∼50 to ∼1000 kDa. It is the second most abundant polysaccharide in nature after cellulose.50,51
Chitosan due to beneficial hydrophilic, cationic, and biodegradable properties, applied in

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several fields, such as agricultural, food, and pharmaceutical industries.52 This natural polymer

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has a high potential to produce gels, films, fibers and particularly hydrogels.53–57 Facin et al.
(2015), Wolf et al. (2019) and Wolf and Paulino (2019), and Ricardi et al. (2018) immobilized
β-Galactosidase enzyme. Pereira et al. (2017) immobilized lipase enzyme in the chitosan-based
hydrogel and showed that chitosan-based hydrogels can be useful for carrying the enzymes.

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3.3. Arabic gum based hydrogel
Acacia gum, also known as Arabic gum, is an edible gum extracted from the trunks and
branches of Acacia Senegal and Acacia Seyal rich in low-viscosity soluble fibers. A type of
natural amorphous, non-toxic, water-soluble, odorless, colorless. and tasteless polysaccharide;

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it has been widely applied from ancient times to the present for different purposes in
pharmaceutical, food, and other industries.63 Its molecular structure has a complex mixture of
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glycoproteins and sugars acting as active sites on immobilization processes. It includes mainly
of polysaccharides arabinose and galactose, calcium, magnesium, and potassium salts.64–66
Wolf et al. (2018) and Wolf and Paulino (2019) immobilized β-Galactosidase enzymes in the
Arabic gum-based hydrogel and showed Arabic gum-based hydrogels to be good solid matrices
for the β- galactosidase enzyme immobilization, able to be used for hydrolysis of lactose in
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dairy foods.
3.4. Pectin based hydrogel
Pectin is a frequently used thickening and gelling agent in several food and non-food industries
with high consumer acceptance. It is a natural heteropolysaccharide extracted from the skin of
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apple and citrus fruits. Pectin can be applied in various food applications, being a gelling agent,
emulsifier, stabilizer, glazing agent, and fat replacer.67 Predominantly, it consists of α-1,4-
linked galacturonic acid-based units. Pectin due to its unique properties including
biocompatibility, degradability, and great transparency, can be used as a matrix to carry useful
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materials such as enzymes.40,68 Cargnin et al. immobilized β-Galactosidase enzyme in the

pectin-based hydrogel and indicated it to be excellent solid supports for the immobilization of
enzymes. The immobilization of β-Galactosidase in pectin-based hydrogels can be used in the
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hydrolysis of lactose of dairy products for lactose-intolerant individuals25; also, Hasanah et al.
(2019) immobilized lipase enzyme in the pectin-based hydrogel.
3.5. Alginate based hydrogel
Alginate is a natural polymer. It, due to its properties such as non-toxicity, biocompatibility,
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low cost, gelation, chemically compatibility, availability, and degradability, is a suitable

polymer for many scientific studies.70 In the food industry, it is a favorite ingredient, food
additive, and carrier of effective ingredients in alginate gel encapsulation. Alginate, as an
anionic polysaccharide, can be modified using chemical and physical reactions to be a good
candidate for three-dimensional (3D) scaffolding derivatives such as hydrogels, microspheres,
microcapsules, sponges, foams, and fibers.71–73 Fabra et al. (2019) and Traffano-Schiffo et al.
(2017, 2018) immobilized β-Galactosidase enzyme; M. Mohammadi et al. (2019), Martín et al.
(2019), and de Oliveira, da Silva, et al. (2018) immobilized pectinase enzyme; also, J. H. Kim

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et al. (2017) immobilized lipase enzyme in the alginate-based hydrogel; they showed that in
order to better maintain the activity of the enzyme in the alginate matrix, alginate alone was
not enough and the presence of effective compounds such as trehalose was important.
3.6. Cellulose based hydrogel
Cellulose is the most abundant polymer in nature, it is found in natural plants and fibers
including cotton and linen. Cellulose is the starting material for a wide range of uses in the food
industry as food additives and gelling agents.82 Cellulose based hydrogels are important due to

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their biocompatibility, non-toxicity and natural originality; they have potential to be used in

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dye or metal ion adsorption, drug delivery, and enzyme support.83–85 S. Park et al. (2015) and
Jo et al. (2019) immobilized lipase enzyme in cellulose-based hydrogel and showed that
cellulose hydrogel could be applied as a support for lipase and suitable for the immobilization
of enzymes

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3.7. Polyacrylamide based hydrogel
Polyacrylamide (PAAm), including acrylamide (AM), is a type of synthetic polymers that have
several advantages, such as good flexibility, biocompatibility, and high solubility in water. It
is used widely in liquid–solid separation in water and waste treatment, paper making,

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processing of minerals in mining, and oil recovery enhancement. Cross-linked polyacrylamide
is used in the food industry as coating, films, and gelling agents. PAAm is a greatly utilized
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synthetic polymers in hydrogel production due to its great hydrophilicity. It also can be applied
to immobilization of enzymes88–90 ; for example, Mulko et al. (2019) successfully immobilized
alpha amylase enzyme in the PAAm-based hydrogel.
3.8. Polyvinyl alcohol (PVA) based hydrogel
PVA is a non-toxic, soluble (in water), semi-crystalline plastic, synthetic, and biocompatible
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polymer. It is a linear synthetic polymer produced by polyvinyl acetate hydrolysis. Due to its
great properties, such as solvent resistance, mechanical efficiency, water high solubility. and
eco-friendly, it is widely used in the preparation of hydrogels.92,93 The internal network of
polyvinyl alcohol hydrogel has free water, crystalline and swollen amorphous PVA domains;
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it creates a porous structure and can be effective for various applications, including enzyme
immobilization.94,95

Table 2. Different types of hydrogels for immobilization of enzymes.

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Group of polymers of hydrogel Hydrogel base Enzyme REF

Kappa-carrageenan β-Galactosidase 48,49

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β-Galactosidase 58–61

Chitosan
Lipase 62
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Arabic gum β-Galactosidase 60,66

β-Galactosidase 25

biopolymers Pectin
Lipase 69

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β-Galactosidase 74–76

Alginate Lipase 81

Pectinase 77–80

Cellulose Lipase 86,87

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Polyacrylamide 91
Alpha amylase

Synthetic polymers 96
Protease

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Polyvinyl alcohol (PVA)
97
Glucose oxidase

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4. Enzyme immobilization method in hydrogel
There are different methods to immobilize enzymes. As can realize from Fig.2, these methods
are generally divided into two types: physical and chemical. In the former, there is a weak
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interaction among the enzyme and the carrier substance, while in the latter, there is a strong
interaction due to the presence of covalent bonds. These immobilization methods are very
important since the stability and long-term use of the enzyme depend on them. Common
methods of enzyme immobilization include adsorption, encapsulation, entrapment, covalent
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attachment, and cross-linking.98–100 In addition, each of these methods has advantages and
disadvantages that are briefly listed in Table 3.
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Fig. 2. Different enzyme immobilization methods.

4.1. Entrapment and Encapsulation

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The caging of enzyme can be achieved by any of the following strategies: (1) by inclusion if
enzyme within a highly cross-linked polymer matrix, (2) by enzyme dissolution in a
nonaqueous phase, or (3) by separating enzyme from a bulk solution by using a semipermeable
microcapsule. In this method the enzyme is not bound to the support matrix unlike other
methods. When an enzyme is trapped inside a matrix, it is said to be encapsulated.
Encapsulation is a physical method with advantages such as being inexpensive and easy;
however, its most imperative benefit for enclosing is that no chemical change of the enzyme is

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required, not causing significant changes in the structure and activity of the enzyme. For this

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method, there are porous and gel-like matrices. Hydrogels, with their hydrophilic and very
porous polymer network, can be the most suitable structure for this method that is more
efficient than free enzyme. Enzymes are physically encapsulated in the hydrogel network
during the sol-gel transition that is a comparatively mild process, tending to protect the

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structural integrity and activity of the enzymes. The only drawback that has been mentioned in
these studies is the leakage of the enzyme out during storage in aqueous solutions17,101; in recent
studies on immobilization of enzymes in hydrogel, the encapsulation method has been used.
There are various methods of enzymes entrapment like fiber entrapping, gel entrapping,

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microencapsulation, etc. In C. rugosa, when the lipase enzyme was entrapped in chitosan
hydrogel, it showed enhanced enzyme activity and entrapment efficiency. It also prevented
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friability and leaching. This is mainly because the support matrix is biocompatible and
nontoxic; receptive to chemical modifications because of its hydrophilic nature it has high
affinity toward proteins.102
4.2. Adsorption
In this method, the enzyme molecules adhere to the surface of the carrier matrix by a
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combination of hydrophobic interactions and the formation of various salt linkages per
molecule of enzyme. Adsorption immobilization is a physical method that results from van der
Waals and other noncovalent interactions, including hydrophobic interactions and hydrogen
bonding electrostatic linkages among the support and the attached enzyme.93 Adsorption
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immobilization method is a naive, inexpensive, and reversible technique of enzyme

immobilization. The dsorbed enzymes are usually resistant to proteolysis and aggregation
because of their hydrophobic interaction with interfaces.102 Other benefits of this technique are:
it supports the lowest activation, or no preactivation at all is required so that no reagent is
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needed; it shields against aggregation, proteolysis, and main interactions, which could disrupt
enzyme and carrier potentials, and no working enzymes can be supplanted with new ones. The
drawback of this technique is that the binding or linking forces among the enzyme and the
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carrier are weak from being established via hydrogen bonding, hydrophobic interactions, and
ionic and van der Waals bonding forces. J. H. Kim et al. (2017), Pereira et al. (2017), and N.
S. Mohammadi et al. (2020), using the adsorption method, immobilized lipase in hydrogel
matrices.
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It was reported that when Yarrowia lipolytica lipase was immobilized on octyl-agarose and
octadecyl-sepa hydrogel beads supports by physical adsorption, resulted in greater stability,
higher yields, better process control, and quite economical as compared to free lipase. This was
mainly because of the hydrophobicity of octadecyl-sepa beads that increases the enzyme and
support affinity.102
4.3. Covalent attachment and cross-linking
Other technique is covalent attachment and cross-linking in which covalent bonds, in general,
are generated due to chemical reactions between enzymes and supported materials. This

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method is mainly depends on the formation of covalent bond between the enzyme and the
support material. Covalent bond formation between the enzyme and the matrix happens
through the side chain amino acids like histidine, arginine, aspartic acid, etc. Covalent bonds
can prevent enzyme leakage and improve the stability and reusability of enzymes; however,
there is a high risk of enzyme denaturation, possibly modifying enzymes chemically. Covalent
bond formation between the enzyme and the matrix happens through the side chain amino acids
like histidine, arginine, aspartic acid, etc. However, the reactivity depends on the presence of

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different functional groups such as carboxyl group, amino group, indole group, phenolic group,

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sulfhydryl group, thiol group, imidazole group, and hydroxyl group. It requires, however, only
low amounts of enzymes to be immobilized, and enzyme catalytic activity may be lost to some
extent104,105; for instance, Pereira et al. (2017) used covalent attachment method for
immobilizing lipase in chitosan-based hydrogel and showed this method to be performed by

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adding glutaraldehyde and binding between free aldehyde groups and amine groups (NH2)
lipase, performing better than physical methods. Maintenance of immobilized enzymes
structural and functional properties is very important which can be played by a cross-linking
agent. Glutaraldehyde is one such cross-linking agent, due to its solubility in aqueous solvents

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and can form stable inter- and intrasubunit covalent bonds, popularly used as bifunctional
cross-linker. an
5. Enzyme activity and release
Enzymes are applied as biocatalysts in the food industry. They are applied due to their different
properties, such as selectivity, non-toxicity, usage of mild reaction conditions, and lack of
secondary reactions. However, their use is limited due to low operational stability, low storage
stability, and non-reusability. Therefore, the development of stable and recyclable enzymes for
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industrial applications is significant research effort.106,107 Polymeric hydrogels have recently
emerged as a new matrix to immobilize enzymes, which can improve enzymatic activity and
stability, and make them possible to be reused, and reduce costs. Hydrogels, due to their porous
structure and water absorption properties, create a suitable environment for enzymatic activity
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and reduce enzymatic denaturation.8 The studies in this field clearly show that the
immobilization of enzymes in hydrogels improves and even increases enzymatic activity as
compared to the free state. For example, the encapsulation of lactase enzyme in carrageenan,
chitosan, alginate, and pectin-based hydrogels have increased enzymatic activity and stability
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in different temperature and pH conditions.25,61,76,108 In a study conducted by Mulko et al. the

retention of alpha-amylase using PAAm-based hydrogels reached 97.5%, indicating the ability
of this hydrogel to protect enzymes, making them reusable.91 Also, in a study conducted by de
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Oliveira et al. the immobilization of pectinase in alginate-based hydrogel showed high

operational stability and maintained more than 80% of its initial activity after the third cycle of
reuse.79
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Table 3. The advantages and disadvantages of different enzyme immobilization

techniques.

Technique Advantagess disadvantages

Limitation of enzyme loading, catalysis carried

Encapsulation/
Protection of enzyme activity, persistent action out at interphase enzyme/substrate, mass
Entrapment
transfer limitations

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Easy and cheap, without the need to use reagents,

Adsorption Low stability, poor bonding on supports
great catalytic activity
Powerful bonding, inhibition of enzyme leakage,
Covalent Limitation of enzyme mobility, reduce of
high thermal stability, increased operational
attachment enzyme activity, conformational restriction
stability, compatible with special process
Loss of the enzyme activity, reduce of
Powerful bonding, prevention of leakage, reduce of
Cross-linking diffusion rate, weak mechanical properties,
desorption, easy to reuse

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limitation of mass transfer

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6. Conclusion
Hydrogels are extensively applied in the food industry since they consist of safe and degradable

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hydrophilic polymers. In recent years, significant progress in design of enzyme immobilization,
support matrix with different pore size, and surface modific ations are developed . Designing
ideal support material by modifying specific structural features required for a target enzyme is

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now possible by new simulations. The current review has provided a universal overview of the
potentials of hydrogels for immobilizing enzymes to be applied in the food industry. β-
Galactosidase, lipase, pectinase, amylase, protease, and glucose oxidase enzymes are widely
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applied in the food industry, and their use is limited due to the low stability and high cost.
Hydrogels provide a suitable environment for enzyme activity and reduce enzyme denaturation
due to their water absorption properties. Therefore, the immobilization of enzymes in
polymeric hydrogels is a very effective approach in using them, leading to the optimal use of
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enzymes and cost reduction. It is our view that the future holds significant promise with
increased usage of immobilized enzymes in pharmacological, clinical, food, biotechnological,
and other industrial fields. Moreover, as the structure of enzyme and the mechanism of action
is known, controlled immobilization methods can be developed in future.
ed

Conflict of Interest
The author declares no conflict of interest.
pt

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