This document summarizes a study that immobilized the enzyme invertase extracted from yeast in calcium alginate beads. The immobilized and free enzyme were added to honey samples to study their effects. Total polyphenols, flavonoids, and antioxidant activity were measured and compared between honey alone and honey with added free or immobilized enzyme. The honey with free enzyme had the highest polyphenol content, while honey with immobilized enzyme had lower polyphenols than free enzyme but higher than honey alone. Flavonoid levels were similar for honey with free or immobilized enzyme and higher than honey alone. Antioxidant activity was lower in treated honeys compared to untreated honey. The size of calcium alginate beads

1. Jour of Adv Research in Dynamical & Control Systems, Vol. 10, 04-Special Issue, 2018
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ISSN 1943-023X
Received: 5 Mar 2018/Accepted: 10 Apr 2018
Immobilization of the Enzyme Invertase
Extracted From the Yeast Saccharomyces
Cerevisiae In Calcium Alginate In Order
To Improve The Physicochemical Quality
Of Honey
1, 2
* Baali Souad, 3
Benamia Fatiha, 1
Mehnane Mourad, 1
Alaimia Djamel, 3
Ziane Aissa
*Corresponding Author
1
Institute of Nutrition, Food and Agro-Food Technology, University of the brothers Mentouri, Constantine 1, BP, 325 Ain El Bey
Road, Constantine, 25017 (Algeria)
2
Laboratory of Environmental Engineering, Departement of Process Engineering, Faculty of Engineering Sciences, Badji Mokhtar
University -Annaba, B.P.12, Annaba, 23000 (Algeria).
3
Laboratory of Applied Organic Chemistry, Departement of Chemistry, Badji Mokhtar University -Annaba, B.P.12, Annaba, 23000
(Algeria).
Abstract—Immobilization of the enzyme invertase extracted from the yeast Saccharomyces Cerevisiae in
calcium alginate gel were studied. The incorporation of the free and immobilized enzymatic solution in a sample
of honey containing crystals to determine their influence on the content of total polyphenols, flavonoids and its
antioxidant activity or their influence on its physicochemical quality was studied. The total polyphenol assay
shows that the sample containing free enzyme honey has the highest content of 27.923 ± 0.001 μg gallic acid
equivalent per gram of sample (EAG /g S). This content is higher than that obtained in honey and is equal to
26.454 ± 0.001 µg EAG /g S. A polyphenolic content lower than that of the free enzyme is recorded for the
sample containing immobilized enzyme honey with 24.146 ± 0.001 EAG /g S. The results of the quantitative
flavonoid assay reveal that the sample containing enzyme containing honey has a considerable flavonoid content
of 68.949 μg equivalent of quercetin/ mg of sample. This content is almost equal to that obtained in honey with
the enzyme immobilized with 68.439 μg EQ / mg of sample. These levels are higher than that of honey alone and
is equal to 60.713 μg EQ / mg of sample. The average antioxidant activity measured by DPPH was lower in
treated honey than in untreated honey.
Keywords— Immobilization, Invertase, Honey, Saccharomyces Cerevisiae, Calcium alginate, Antioxidant
activity.
Introduction
Honey is a sweet liquid composed mainly of sucrose dissolved in water, to which is added the enzyme
Hcontaining bee saliva, glucose-invertase, which converts sucrose into two simple sugar molecules: fructose and
glucose. The presence of enzymes in honey has been known for many years. The main enzymes of honey have
been reported as invertase, glucose oxidase, and a mixture of α- and β-amylases, also known as diastase [1].
Honey enzymes generally do not influence the final food product and are present at relatively low concentrations.
However, enzymes play a vital role because the nectar is ripened into honey. Enzymatic reactions in nectar and
honey ripen in a broad array of compounds that contribute to the uniqueness and functionality of honey [2]. The
complicated carbohydrate profile of honey is partially the result of enzymatic action; research has shown that the
minor sugars in honey arise from the transglucosylation activity of - and -glucosidases [3]. One of the most
important roles of enzymes as natural biocatalysts is their ability to increase the rate of chemical reactions at the
cell level. Several studies have focused on the immobilization of invertase on various supports (porous glass,
resins, synthetic polymers, polysaccharides, alginate gel) and by different techniques [4].
The crystallization of honey is a natural and inevitable phenomenon. The consumer prefers in general, a liquid or
creamy honey, but especially not crystallized, the beekeeper will very often have to liquefy his honey before
putting it on the market. We propose in this study a simple method to keep the honey in liquid form, this proposal
consists in putting a quantity of the free enzyme invertase and immobilized in the honey to improve their
nutritional quality. The enzyme invertase is extracted from baker's yeast (Saccharomyces cerevisiae). This

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enzyme is immobilized by the encapsulation (inclusion) technique in the presence of a crosslinking agent in a
sodium alginate matrix in order to improve their catalytic activity.
Alginate, commercially available in the form of alginic acid salt, commonly known as sodium alginate, is a linear
polysaccharide isolated from several types of brown marine algae, hence its alginate name [5]. Alginate is a
collective limit for a family of polysaccharides produced by brown algae and bacteria. Alginic acid was
discovered, extracted, and first patented by Stanford. Alginate is one of the most versatilebiopolymers.
It is used in the agri-food sector and the pharmaceutical industry because it has many properties: thickener,
stabilizer, gelling agent [6]. This polysaccharide has been identified as a structural component of marine brown
algae, where it constitutes up to 40% of the dry matter and occurs mainly in intercellular mucilage and algal cell
wall as an insoluble mixture of calcium, magnesium, potassium, and sodium salts [7]. The alginates consist of β-
d-mannuronic acid (M) and α-1-guluronic acid (G) whose sequence varies (Figure 1).
β-D-mannuronate (M) α-L-guluronate (G)
Figure 1: Monomers of the alginate chain [8].
Encapsulation is often a relatively effective method to limit their degradation during processing and storage of
the product. It is the most widely used method today [9, 10, 11]. This method consists of physically retaining an
enzyme locked inside a matrix (gel, fiber or microcapsule) in a purely physical way; the enzyme is distributed in
a solution of the monomer or in an emulsion. The polymerization of the monomer or its setting in mass leads to
the formation of a network in which the enzyme is trapped [12]. Generally, alginate and carrageenan gels are the
products commonly used for immobilization of enzymes by inclusion [13]. Alginate is the most widely used
copolymer because it is an anionic, light and non-toxic polymer [10, 14].
Stages of formation of calcium alginate gels
There are three consecutive steps that lead to the association of calcium with alginate when the concentration of
calcium ions increases [15] (Figure 2). The first step is the interaction of Ca2+
ions with a simple guluronnate
chain to form a monocomplex. The second step is the propagation and formation of "eggbox" type dimers via the
association of these monocomplexes. The third and final stage is the lateral association of the "egg box" dimers,
which thus form multimers.
Figure 2: Ca-Alginate gel formation steps [8].
Step 1 - Coordination of Ca2 +
ions within a cavity created by a pair of guluronnate sequences along the alginate
chains;
Step 2 - Dimer "egg box";
Step 3 - Multimers "egg box" associated laterally [8].
Material and methods
A. Chemical reagents and standards
To perform experiments, several materials was provided. All chemicals were commercially available and used as
received. Bovine Serum Albumin 99% (BSA) and Sodium alginate were purchased from Sigma-Aldrich Chemie
GmbH (Steinheim, Germany). Enzyme invertase (β-D-fructofuranoside fructohydrolase, E 3.2.1.26) produced by
extraction from baker’s yeast (Saccharomces cerevisiae). Honey was obtained from the Wilaya of Souk Ahras,
Algeria.

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1,1 diphenyl-2-picrylhydrazyl (DPPH), ferric chloride and Folin-Ciocalteu reagents were purchased from Sigma
chemicals (Steinheim, Germany). Gallic acid and quercetin were obtained from Fluka (Buchs, Switzerland).
Sodium bicarbonate (NaHCO3), Sodium acetate (C2H3NaO2), Sodium hydroxide (NaOH), Acetic acid
(CH3COOH) (99%), Coomassie brilliant blue G250, Ethanol (95%), Orthophosphoric acid (85%), 3,5-
dinitrosalicylic acid (3,5-DNS),Tartrate of sodic potassium (NaKC4H4O6), Calcium chloride (CaCl2), Sucrose
(C6H12O6), Sodium carbonate (Na2CO3); Tri Aluminum Chloride (AlCl3). All the chemicals reagents and solvents
were of analytical grade and used as purchased without any further pretreatment. All solutions were prepared
using distilled water. All other chemicals were of analytical grade.
B. Immobilization of invertase
The method of preparation of the alginate beads is an adaptation of the method described by Loncle (1992) [16].
The enzymatic solution is mixed with a solution of sodium alginate (4%), the solubilized enzyme (0.22%) is then
polymerized in the presence of a cross-linking agent. The mixture is dispersed through a droplet orifice (syringe)
in a saline solution which is calcium chloride (0.3M CaCl 2) (extrusion dispersion) in order to obtain gel beads
having good mechanical properties and in which the enzyme is retained [17]. The formed beads are allowed to
stand for 30 minutes in calcium chloride (0.3M) at room temperature, and then filtered and rinsed several times
with acetate buffer (pH 4.8-0.1M). Afterwards, the beads are kept in a well closed bottle (or petri dishes sterilized)
at 4 ° C.
The diameter of drops formed by simple extrusion gravity is always greater than millimeter even with very thin
needles whose size ranges from a few millimeters to less than a micrometer (between 2000 and 4000 microns)
which gives it the status of "Microencapsulation" [18, 19]. The bead size of the network depends on the percentage
of alginate [20, 16].
Evaluation of the Activity of the immobilized Enzyme in honey
C. Determination of Phenolic Compounds
Determination of Total Polyphenols
The determination of the polyphenols is carried out using Folin-Ciocalteau reagent in the presence of Na2CO3. It
is a solution of phosphotungstic acid (H3PW12O40) and phosphomolybdic acid (H3PMo12O40) whose reduction by
the action of polyphenols including a mixture of complexes of salts of tungsten (W8O23) and molybdenum
(Mo8O23) of blue color. The intensity of the coloration is proportional to the amount of polyphenols present in
the sample [21, 22]. 0.2 ml of each treated and untreated honey sample was added to 1 ml of Folin-Ciocalteu
reagent 10 times diluted. The solutions were mixed and incubated for 4 minutes. After incubation, 2 ml of Na2CO3
sodium carbonate solution (7.5%) was added. The final mixture was shaken and then incubated for 2 hours in the
dark at room temperature. Absorbance of all extracts was measured by a spectrophotometer at 760 nm.
A series of four test tubes is prepared as follows: Blank tube: 1ml Folin + 2ml Na2CO3 + 0.2ml distilled water;
Tube 01: 1ml Folin + 2ml Na2CO3 + 0.2ml honey; Tube 02: 1ml Folin + 2ml Na2CO3 + 0.2ml honey + 0.2ml free
enzyme; Tube 03: 1 ml Folin + 2ml Na2CO3 + 0.2 ml honey + 0.2 ml immobilized enzyme.
The results are expressed in micrograms gallic acid equivalent (the standard) per milligram of sample (μg EGA /
mg S).
Determination of flavonoids
The determination of flavonoids in a sample can be demonstrated by the colorimetric assay according to the
aluminum trichloride (AlCl3) method cited by (Djeridane I., et al., 2006 [23, 24].
To quantify the flavonoids in our samples; 1 ml of each sample and the standard (dissolved in methanol) with the
appropriate dilutions was added to an equal volume of a solution of AlCl3 (2% in methanol).
The mixture was vigorously stirred and the absorbance at 430 nm was read after 10 minutes of incubation. The
quantification of flavonoids was made according to a linear calibration curve (y = ax + b) carried out by a standard
standard "quercetin" at different concentrations (1.75 - 40 μg / ml) under the same conditions as the 'sample.
A series of four test tubes is prepared as follows: Blank tube: methanol; Tube 01: 2ml AlCl2 + 2ml honey; Tube
02: 2ml AlCl2 + 2ml honey + 2ml free enzyme; Tube 03: 2ml AlCl2 + 2ml honey + 2ml immobilized enzyme.
The results are expressed in micrograms of quercetin equivalent (the standard) per milligram of the sample (μg
EQ / mg S).

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DPPH radical-scavenging assay
To evaluate the antiradical activity, we used the method of DPPH (2,2-diphenyl-1-pierylhydrazyl) according to
the protocol of Guilong yan et al., (2011) [25] modified by Hassan fahmi ismail et al., (2017) [26].
A methanolic solution of 0.2mM of DPPHis mixed with different samples studied, Put 2ml of each sample in a
test tube, add 2ml of methanolic solution of DPPH, and then incubate 30 minutes away from light at room
temperature. Read the absorbance at 517 nm against a blank.
In a series of test tubes, four tubes were prepared containing the following components: Blank tube: 2ml DPPH
+ 2ml methanol; Tube 01: 2ml DPPH + 2ml honey; Tube 02: 2ml DPPH + 2ml honey + 2ml free enzyme; Tube
03: 2ml DPPH + 2ml honey + 2ml immobilized enzyme. Repeat the same operations, replacing the sample studied
with ascorbic acid (standard) at different concentrations (3.9 - 500 μg / ml); the negative control is prepared by
mixing 2ml of methanol with 2ml of the DPPH solution.
D. Statistical Analysis
The experimental data of the assay and the evaluation of the antioxidant activity obtained were expressed by the
average of the results found. The correlation coefficient of the antioxidant properties was determined using the
Excel 2003 and 2013 programs. Mean percentage, standard deviation (SD) and range were calculated. Analysis
of variance (ANOVA) at a significance level of 0.05 was performed by the STATISTICA 7.0 software (Stat Soft,
Inc., Tulsa, OK, USA) followed by significant differences in Tukey post hoc test.
Results and discussions
E. Effect of bead size to immobilized efficiency
The extrusion speed is adjustable. From this velocity and the diameter of the needle, the syringe depends on the
size of the beads. This method makes it possible to obtain balls of homogeneous diameter, however it is relatively
long. The size of the alginate beads plays an important role in enzymatic catalysis, its influence has been verified
with different diameters. All these experiments were conducted at room temperature.
To have significant immobilization of the enzyme in the alginate gel, syringes of different volumes were usedto
determine the most significant immobilization efficiency.
F. Optical microscope characterization of alginate beads
Figure 3: Photos (x 40) of the enzyme immobilized in a calcium alginate matrix.
Observation of the alginate bead under microscope x40 allowed us to see a colored enzyme distributed inside a
small calcium alginate bead and the outer layer (figure 3). Immobilized enzyme efficiency with different bead
sizes is depicted as follow (figure 4).
Figure 4: Effect of volume of syringes to bead size of immobilized enzyme
a, b, c and d indicates a significant difference at the 0.05 threshold.
volume of syringes (ml)
50ml20ml10ml5ml
Diametr(mm)
a
c
d
b

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The protein content is determined by the same formula used for the soluble enzyme from the standard Bradford
Protein Calibration Curve (figure 5).
Figure 5: Immobilized enzyme efficiency at different diameter of the alginate beads (bead sizes)
a, b, c and d indicates a significant difference at the 0.05 threshold.
From the histograms of Figures 4 and 5, it is found that the bead of smaller diameter has the most significant
yield compared to the beads of a larger diameter. At a volume of 5, 10, 20 and 50 ml and a dilution of the enzyme
solution equal to 1/100, the immobilization rate of the observed enzyme is quite high.
This rate reaches an average of 98.54% for beads of 2 mm in diameter, and it is only 98.04% for beads 3.6 mm
in diameter, for this diameter only 2 beads of form spherical are produced, the rest of the immobilized enzyme
has no precise shape that is to say these do not have a homogeneous form.
The influence of the size of the beads on the rate of immobilization of the enzyme therefore clearly appears. Hulst
et al., (1984) [27] and Bradenberger and Widmer (1998) [28]. describe methods for obtaining beads of
homogeneous diameter and in very short times.These methods make it possible to envisage applications on an
industrial scale.
G. Influence of immobilized enzyme activity on honey quality
The idea of associating the immobilized enzyme as a catalyst can be of great interest for the production of
molecules of high added value and which make it possible to envisage applications on an industrial scale. To take
into account the constraints of the industry, we worked with a crude preparation of invertase, resulting from
Saccharoniyces cerevisiae, (the use of "raw" preparations is common in food enzyme technology because it
avoids the expensive processes of purification of enzymes). As a result, the study of the behavior of the enzyme
has been relatively complicated.
As we mentioned in the introduction, the crystallization of honey is an inevitable phenomenon. The consumer
prefers in general, a liquid or creamy honey, but especially not crystallized, so we must liquefy the honey before
putting it on the market. For this and to prove that this method is customary for liquefying honey, it must be
proven that adding a proportion of free enzyme and then immobilized enzyme in honey inevitably leads to an
improvement in their quality.
Total polyphenol content
TABLE 1
TOTAL POLYPHENOLS CONTENT, FLAVONOID CONTENT AND % OF INHIBITION OF FREE
RADICAL DPPH OFSAMPLES
Sample S (honey only) S2 (honey + FE) S3(honey + IE)
ppht content (a)
26,454 ± 0,001 27,923 ± 0,001 24,146 ± 0,001
FT content (b)
60,713± 0,001 68,949± 0,001 68,439± 0,001
I% 85,58± 0,001 80,06± 0,001 75,93± 0,001
(a) μg gallic acid equivalent per gram of sample.
(b) μg equivalent of quercetin per gram of sample.
ppht : total polyphenol
FT : total flavonoids
I% : percentage of free radical inhibition DPPH
S : sample (honey), FE :free enzyme, IE:immobilized enzyme
The values represent the average of 3 measurements ± SD (standard deviation).
94,5
95
95,5
96
96,5
97
97,5
98
98,5
99
Efficiency%
Diameter (mm)
a
d
2 2,8 3 3,6
c
b

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The percent inhibition of DPPH (I%) by the three samples is calculated by the following formula:
Ablank
AsampleAblankI 100)(% 
The total polyphenol content shows that the treated honey with free enzyme has the highest content of 27.923 ±
0.001 μg gallic acid equivalent per gram of sample. This content is higher than that obtained in untreated honey
and is equal to 26.454 ± 0.001 μg of gallic acid equivalent per gram of sample. A polyphenolic content lower
than that of the treated honey with free enzyme is found for the sample containing enzyme-immobilized honey
with 24,146 ± 0,001 μg of gallic acid equivalent per gram of sample, this content is very low compared to that of
the standard which is gallic acid (Table 1).
The results of the quantitative flavonoid assay (Table 1) reveal that the treated honey with free enzyme has a
considerable flavonoid content of 68.949 μg EQ / mg of sample. This content is almost equal to that obtained in
treated honey with the immobilized enzyme with 68.439 μg EQ / mg of sample. These are higher than that of
honey alone (untreated) and equal to 60.713 μg EQ / mg of sample.
Figure 6: Comparison of percentage of radical DPPH's inhibition by the various samples.
Ascorbic acid: standard, S: sample (honey only), S+FR : treated honey with free enzyme and S+IE: treated
honey with immobilized enzyme
From Figure 6, it can be seen that the difference was found between untreated and processed honey (treated). The
average antioxidant activity measured by DPPH was lower in treated honey than in untreated honey. The results
obtained were not consistent with previous results of antioxidant activity, as well as the total content of phenolics,
was strongly affected by transformation conditions [29, 30].Our results confirm those obtained by Nguyen Xuan
Nam et al., 2017 [31].
Conclusion
These results have fulfilled some important requirements for immobilization of invertase (β-D-fructofuranoside
fructohydrolase, E 3.2.1.26) because the enzyme preparation and enzyme immobilization protocols are simple.
The study showed that the process of treating honey with free and immobilized enzyme under controlled
conditions should not affect the quality of the honey, especially its antioxidant activity. The treatment was found
to lead to significant changes in the values of the analyzed parameters: total polyphenols and increased flavonoids
content
98,55%
85,58%
80,06%
75,93%
0
20
40
60
80
100
ascorbic
acid
S S+FR S+EI
Inhibition%

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