ENCAPSULATION OF INVERTASE IN BARIUM ALGINATE BEADS

Abstract

Invertase (E.C. 3.2.1.26) was encapsulated in barium alginate beads. The gelation was carried out at 30 o C by stirring (360 rpm and 540 rpm) using alginate concentrations (5 g/L and 10 g/L), barium nitrate concentrations (0.1M and 0.2M), and three pH of alginate-invertase solution (4.0, 6.7 and 8.0). Invertase encapsulation yielded 75.2% and encapsulated invertase activity yielded 0.249 U/mL. These results were obtained using beads produced with 10 g/L alginate (SG800) and 0.1M barium nitrate at pH 4.0, 30 o C and 540 rpm. The kinetic constants for soluble and encapsulated invertase, calculated using Hanes-Woolf's plot, were K M = 52.9 mM, V max = 0.368 U/mL, (K M) encap = 7.51 mM and (V max) encap = 0.269 U/mL. The beads were reused three times batchwise without significant loss of invertase activity.

Full text

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Vitolo. World Journal of Pharmaceutical Research
ENCAPSULATION OF INVERTASE IN BARIUM ALGINATE BEADS
Michele Vitolo*
Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical
Sciences of University of São Paulo, Brazil.
ABSTRACT
Invertase (E.C. 3.2.1.26) was encapsulated in barium alginate beads.
The gelation was carried out at 30oC by stirring (360 rpm and 540 rpm)
using alginate concentrations (5 g/L and 10 g/L), barium nitrate
concentrations (0.1M and 0.2M), and three pH of alginate-invertase
solution (4.0, 6.7 and 8.0). Invertase encapsulation yielded 75.2% and
encapsulated invertase activity yielded 0.249 U/mL. These results were
obtained using beads produced with 10 g/L alginate (SG800) and 0.1M
barium nitrate at pH 4.0, 30oC and 540 rpm. The kinetic constants for
soluble and encapsulated invertase, calculated using Hanes-Woolf’s
plot, were KM = 52.9 mM, Vmax = 0.368 U/mL, (KM)encap = 7.51 mM
and (Vmax)encap = 0.269 U/mL. The beads were reused three times
batchwise without significant loss of invertase activity.
KEYWORDS: Immobilization, invertase, alginate, encapsulation, entrapment.
INTRODUCTION
Encapsulation is a type of immobilization in which a biocatalyst (enzyme, cells, and
organelles) is involved by a semipermeable membrane that allows low MW molecules to
cross it freely.[1] This is a gentle method because gelation between sodium alginate and
barium ion occurs under smooth aqueous conditions (temperature: 25oC – 40oC, pH: 4.5 –
6.5, and stirring: 20 – 100 rpm). Thus, the enzyme does not suffer any significant injuries at
the molecular level mainly due to the absence of physicochemical interaction between the
enzyme and the carrier. For example, by encapsulating invertase (an enzyme with a tertiary
structure level) in alginate beads, there is an improvement of the catalytic activity due to the
extemporaneous aggregation of invertase molecules in tetramers (a quaternary structural
level).[2] In addition, barium alginate beads can be produced using a low-tech apparatus and
World Journal of Pharmaceutical Research
SJIF Impact Factor 8.074
Volume 8, Issue 10, 222-233. Research Article ISSN 2277– 7105
Article Received on
15 July 2019,
Revised on 04 August 2019,
Accepted on 25 August 2019
DOI: 10.20959/wjpr201910-15771
*Corresponding Author
Michele Vitolo
Department of Biochemical
and Pharmaceutical
Technology, School of
Pharmaceutical Sciences of
University of São Paulo,
Brazil.

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the sodium alginate is a cheap commodity plentifully available in the market and sold by
several suppliers.[1][3]
Alginates are extracted mainly from algae species such as Macrocystis pyrifera, Laminaria
hyperborean and Ascophyllum nodosum.
Alginates are a group of linear polysaccharides (molecular weight ranging between 33,000
and 400,000 g/mol) consisting of homogeneous or heterogeneous arrangements of 1,4-linked
-D-mannuronic acid (M) and 1,4 -L-guluronic acid (G) residues. Their main
physicochemical properties – viscosity, sol/gel transition (jelly capability in the presence of
divalent cations) and water-uptake ability – are related to the amount and distribution pattern
of M-blocks and G-blocks along the polymer backbone.[1][3] The sol/gel transition property,
which occurs at room temperature, allows attaining a diversity of semisolid or solid structures
under mild conditions. Such structures have a large variety of industrial applications.
Alginates (alginic acid, sodium alginate, ammonium alginate, calcium alginate, and
propylene glycol alginate) have a large spectrum of applications in food industry (as
emulsifier, texturizer, stabilizer, thickener, among others), in pharmaceutical industry (as
emulsifier, film former, humectants, tablet binder and disintegrant, for instance), and in
chemical-pharmaceutical industry (as carrier for entrapping enzymes, cells and
organelles).[1][2][4] Alginates are labeled as biocompatible, non-immunogenic and nontoxic
compounds.[5] Thereby, they can be used in association with calcium or barium ions (calcium
alginates) in nonwoven dressings for the treatment of infected surgical wounds and as an aid
in epidermis fistulae healing.[6][3] Moreover, the alginate is becoming a reference material in
tissue engineering, envisaging the confection of 3D-microencapsulated human cells – for
instance, microencapsulation of pancreatic islets to treat type 1 diabetes.[7]
Sodium alginate (certainly one of the most widely investigated alginates in the
pharmaceutical and biomedical field) is water-soluble and forms stable viscous solutions
provided the pH of the solvent is above 3.6 and the ionic strength is low. Sodium alginate
gelation can be induced in the presence of divalent ions (such as Ca2+ or Ba2+) or by lowering
the solvent pH below the pKa of the alginate using lactones such as d-glucano--lactone. The
former procedure is useful for attaining alginate beads – applied to entrap enzymes, cells,
drugs and organelles-, whereas the latter is useful for preparing sheet pellicles for therapeutic
purposes.

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Alginate beads for applying in bioreactors must be spherical, resistant to shear force and
simultaneously permeable for low MW substances and impermeable for macromolecules.
Beads with these properties can be attained through a combination of factors such as
concentration of alginate and divalent cation, solvent pH, temperature, and agitation. Of
course, the number of combinations involving the cited factors would be practically limitless.
In the case of Ba2+-alginate, it has been noted that beads meeting the three characteristics
cited could be obtained using alginate and barium concentrations, respectively, at the
intervals 5-10 g/L and 0.1-0.2M. Additionally, the solvent pH between 4.0 and 8.0, agitation
up to 540 rpm, and temperature of 30oC also contributed to the confection of suitable beads.
Alginate concentration below 5 g/L and/or barium concentration below 0.1M produce non-
spherical beads.
Among the several biocatalysts useful in the chemical-pharmaceutical industry, invertase
(E.C.3.2.1.26) has undoubtedly a great use in sugar hydrolysis. Invertase – one of the first
enzymes to be identified (by Berthelot in 1860) and produced in large scale (since the
beginning of the last century) – has been largely used in industry (mainly in sucrose
hydrolysis) and more recently as medicine and reagent.[8][9]
This work studies the invertase entrapped in barium alginate beads aiming their use in
sucrose hydrolysis. The effects of alginate concentration (5 g/L and 10 g/L) and type (SG800:
M/G < 1; S1100: M/G > 1), pH of alginate solution (4.0, 6.7 and 8.0), agitation (360 rpm and
540 rpm) and barium nitrate concentration (0.1M and 0.2M) on the encapsulation of invertase
were studied. In addition, the soluble and encapsulated invertase activities were determined,
as well as their kinetic parameters (KM and Vmax). Reusability of encapsulated invertase was
also evaluated.
MATERIAL AND METHODS
Material
Sodium alginates [SATIALGINE® forms: SG800 (M/G=0.5; 400-490cP; granules  200;
and humidity  15%) and S1100 (M/G=1.2; 550-750cP; granules  160; and humidity 
15%)] were purchased from Sanofi Bio-Industries (Paris, France). Invertase (Invertin®), with
a protein concentration of 2.2 mg/mL, was purchased from Merck (Gernsheim, Germany).
All other reagents were of analytical grade.

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METHODS
Encapsulation
The alginate solution was prepared by dissolving 5 g or 10 g of SG800 or S1100 in 1 L of
deionized water (pH adjusted to 4.0, 6.7 or 8.0). Then, the solution was left resting at 4oC for
24 h. Two milliliters of Invertin® and 18 mL of the alginate solution were mixed, then
dropped into a 100-mL solution of 0.1M or 0.2M Ba(NO3)2 from a cylindrical reservoir
(inner diameter = 14 mm and height = 34 mm). The outlet extremity (internal diameter = 2
mm) was positioned 80 mm from the surface of barium nitrate solution. The gelation was
carried out under agitation of 360 or 540 rpm at 30oC. Then, the beads (mean diameter = 3
mm) were left to harden for 12 h in the barium solution. Finally, the beads were separated
through a sieve and washed with 20 mL of deionized water. Each batch of gelation led to 500
beads.
The yield of invertase encapsulated (YIE) was evaluated by the equation:
YIE = [(Vencap)  (Vsol)] x 100 (Eq. 1)
Where (Vencap) = Activity of immobilized invertase (mg RS/min.mL); (Vsol) = Activity of
soluble invertase (mg RS/min.mL); RS = Reducing sugars.
Invertase Activity Measurement
Soluble invertase
Two hundred milliliters of sucrose solution (100 g/L in 0.01M acetate buffer, pH 4.6) and 1
mL of Invertin® were added to an Erlenmeyer flask. The hydrolysis was carried out for 60
min at 35oC and agitation of 450 rpm. Aliquots between 0.1 mL and 0.3 mL were taken at
every ten min to monitor the reaction. The aliquots were transferred to Folin-Wu test tubes
containing 1.0 mL of alkaline Somogy's solution. The tubes were immersed in a boiling water
bath for ten min. Thereafter, the procedure was followed as described elsewhere.[10]
The initial invertase activity (Vsol) was calculated (always in triplicate) using the slopes of
reducing sugars (RS) versus reaction time plots (Figure 1 is an example of such a plot). One
invertase unit (U) was defined as the quantity of RS (milligrams) formed per minute under
the conditions of the test. The soluble invertase had an activity equal to 0.368  0.0015 U/mL
of reaction medium.

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Immobilized invertase
Five hundred barium-alginate invertase beads were suspended in 50 mL of deionized water
and left stirring for 1 h. After that, the beads were separated through a sieve and the washing
water was collected for soluble protein detection. Then, the beads were suspended in 200 mL
of buffered sucrose solution, and the reaction was carried out as described above. The initial
encapsulated invertase activity (Vencap) was calculated (always in triplicate) using the slopes
of reducing sugars (RS) versus reaction time plots (Figure 1). In all tests, the amount of
protein washed out from the beads was lower than 5 g/mL. The encapsulated invertase had
an activity varying from 0.148 to 0.249 U/mL and from 0.140 to 0.218 U/mL, respectively,
for SG800 and S1100.
ANALYTICAL METHODS
Titration of Ba2+ with EDTA
After separation of the barium-alginate beads, the residual Ba(NO3)2 solution was collected.
The remaining Ba2+ was then measured. Twenty milliliters of this solution was titrated with
0.1M EDTA solution in the presence of murexide as indicator. The titration was carried out at
30oC and pH 12.0. One milliliter of 0.1M EDTA is equal to 13.7mg of Ba2+.
Soluble protein measurement
Protein was determined based on the difference between UV absorbance measured at 215 and
225 nm using bovine serum albumin (BSA; Fraction V) as a standard. By using a 0.1 mg/mL
(w/v) of BSA solution, the linear correlation between Abs (Abs215nm – Abs225nm) and protein
concentration (P) (varying from 10 to 100 g/mL) was:
Abs = 5.90x10-3P – 7.0x10-4 (r = 0.9997) (Eq. 2)
Reducing sugars measurement
The reducing sugars (RS) were measured by spectrophotometer as described elsewhere.[11]
The absorbance (read at 540nm) was converted into RS, expressed as glucose, through a
standard curve (Eq. 3). A standard glucose solution (0.2 mg/mL) was used, from which 0.2-
1.0 mL aliquots were taken.
Ygluc = 2.63.Xgluc + 0.027 (r = 0.996) (Eq. 3)
Where Ygluc = absorbance and Xgluc = amount of glucose (mg).

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RESULTS AND DISCUSSION
The amount of Ba2+ consumed in function of the number of beads obtained was plotted as
shown in Figure 1. The minimal square linear regression equations are:
Y = 0.0250XS1100 + 0.198 (r = 0.9993) (Eq. 4)
Y = 0.0194XSG800 + 0.148 (r = 0.9996) (Eq.5)
Where Y = Ba2+ consumed (mg); XS1100 and XSG800 = Number of beads related to alginates
S1100 and SG800, respectively.
The linear correlation between these variables was observed in all kinds of beads
confectioned - SG800 or S1100 (5 g/L or 10 g/L) dropped in barium nitrate solution (0.1M or
0.2M) at a pH of 4.0, 6.7 or 8.0 and agitation of 360 rpm or 540 rpm. This result corroborates
that observed in a previous work that produced Ca2+-alginate beads.[1]
Figure 1: Number of beads in function of the Ba2+ consumed. The gelation conditions
were 10 g/L alginate [SG800 (●); S1100 (□)], 0.1M Ba(NO3)2, pH = 6.7, agitation: 360
rpm and 30oC.
Figure 2 shows the formation of reducing sugars (Y) in function of reaction time (X) for
soluble and encapsulated invertase. The minimal square linear regression equations are:
Ysol = 0.331 X + 0.518 (r = 0.998) (Eq. 6)
YSG800 = 0.242 X + 0.281 (r = 0.992) (Eq. 7)
YS1100 = 0.152 X + 0.341 (r = 0.997) (Eq. 8)

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Figure 2: Formation of reducing sugars in function of the reaction time catalyzed by
soluble invertase (●) and Ba2+-alginate invertase [SG800 () and S1100 (■)]. The beads
were obtained by jellifying 10 g/L of alginate with 0.2M Ba(NO3)2 at pH 8.0, 360 rpm
and 30oC.
The slope of each straight line represents the activity for soluble (0.331 U/mL) and
encapsulated invertase - Ba2+-SG800 (0.242 U/mL) and invertase-Ba2+-S1100 (0.152 U/mL).
By applying equation 1, the yield of invertase encapsulated (YIE) in beads of Ba2+-SG800
and Ba2+-S1100 - obtained under the gelation conditions cited in the caption of Figure 2 was
73.1% and 45.9%, respectively. YIE values for other gelation conditions are presented in
Tables 1 and 2.
The invertase encapsulation yield (YIE) and the bead invertase activity varied according to
the gelation conditions (pH, stirring, barium and alginate concentration) used during the
production of beads (Tables 1 and 2).
The YIE varied from 44.7% to 75.2% for SG800 (M/G <1) and from 42.3% to 55.9% for
S1100 (M/G >1), whereas the bead invertase activity varied between 0.148 U/mL and 0.249
U/mL for SG800 and between 0.143 U/mL and 0.218 U/mL for S1100. The most significant
fact relates to the types of alginates (SG800 and S1100), which differ in -L-guluronic acid
content (SG800 > S1100). Probably, the high amount of this monomer leads to a major
number of structures with an “egg-box” format along the polysaccharide chain, which, in
turn, can accommodate the barium ions more efficiently.[3] In addition, the pH  4.0
guarantees a net negative charge of alginate molecules (pKa: 3.38 - 3.65), making them more
avid for chelating Ba2+. Furthermore, at pH 4.0, the invertase molecules do not have electrical
charge (“Zwitterion” form; pHi = 4.0), which, in turn, could facilitate their encapsulation

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inside the beads, zeroing any possibility of chemical interaction with the barium-alginate
carrier. This, associated with an adequate stirring (540 rpm) and balanced reagent
concentrations (SG800 = 10 g/L and Ba(NO3)2 = 0.2M), led to the highest YIE (75.2%) and
invertase activity (0.249 U/mL).
By setting YIE  70%, only the beads produced with SG800 have surpassed that limit (5 g/L,
0.1M, pH 8.0 and 540 rpm; 10 g/L, 0.1M, pH 4.0 and 540 rpm; 10 g/L, 0.1M, pH 8.0 and 540
rpm; 10 g/L, 0.2M, pH 6.7 and 360 rpm; and 10 g/L, 0.2M, pH 8.0 and 360 rpm) (Tables 1
and 2). Under an industry point of view, to which the secrecy of the process is a desirable
target, the large variety of gelation conditions contributes to reach that aim.
Table 1: Encapsulated invertase activity (U/mL) in function of SG800 (5 g/L and 10
g/L), barium nitrate (0.1M and 0.2M) and pH (4.0, 6.7 and 8.0). The hydrolysis was
carried out in the presence of 500 Ba2+-SG800-invertase beads and 100 g/L of sucrose at
35oC, pH 4.6 and 450 rpm. The yields of encapsulated invertase (YIE), expressed as
percentage, were calculated by the equation 1 (Vsol = 0.331 U/mL).
SG800
(g/L)
Ba(NO3)2
(M)
pH
4.0
6.7
8.0
5
0.1
YIE (%)
a0.226/b0.217
0.201/0.172
0.209/0.233
68.2/65.6
60.7/52.0
63.1/70.4
0.2
YIE (%)
0.159/0.177
0.186/0.192
0.190/0.174
48.0/53.5
56.2/58.0
57.4/52.6
10
0.1
YIE (%)
0.203/0.249
0.153/0.194
0.148/0.233
61.3/75.2
46.2/58.6
44.7/70.4
0.2
YIE (%)
0.217/0.190
0.235/0.210
0.242/0.208
65.6/57.4
71.0/63.4
73.1/62.8
a Stirring = 360 rpm; b Stirring = 540 rpm.

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Table 2: Encapsulated invertase activity (U/mL) in function of S1100 (5 g/L and 10 g/L),
barium nitrate (0.1M and 0.2M) and pH (4.0, 6.7 and 8.0). The hydrolysis was carried
out in the presence of 500 Ba2+-S1100-invertase beads and 100 g/L of sucrose at 35oC,
pH 4.6 and 450 rpm. The yields of encapsulated invertase (YIE), expressed as
percentage, were calculated by the equation 1 (Vsol = 0.331 U/mL).
S1100
(g/L)
Ba(NO3)2
(M)
pH
4.0
6.7
8.0
5
0.1
YIE (%)
a0.174/b0.140
0.168/0.141
0.171/0.141
52.3/42.3
50.8/42.6
51.7/42.6
0.2
YIE (%)
0.174/0.164
0.170/0.157
0.173/0.147
52.6/49.5
51.4/47.4
52.3/44.4
10
0.1
YIE (%)
0.185/0.166
0.151/0.166
0.143/0.213
55.9/50.2
45.6/50.2
43.2/64.4
0.2
YIE (%)
0.166/0.192
0.218/0.167
0.152/0.167
50.2/58.0
66.0/50.5
46.0/50.5
a Stirring = 360 rpm; b Stirring = 540 rpm.
The kinetic constants (KM and Vmax) were determined using the conventional Hanes-Woolf
plot (Figure 3) for soluble and encapsulated Ba2+-SG800 invertase. The initial invertase
activity was obtained from initial sucrose concentration (S) varying from 5.5 mM to 45 mM.
The minimal square linear regression equations are:
(S/V)sol = 2.72S + 144 (r = 0.9993) (Eq. 9)
(S/V)encap = 3.38S + 25.4 (r = 0.9997) (Eq. 10)
The kinetic constants calculated using the equations 9 and 10 for soluble and encapsulated
invertase, respectively, were KM = 52.9 mM, Vmax = 0.368 U/mL, (KM)encap = 7.51 mM and
(Vmax)encap = 0.296 U/mL.
The KM of 52.9 mM for soluble invertase is in accordance with that found in the literature,
whose values vary between 40 and 166 mM.[12][13][14] The difference observed could be due to
the different origin of enzymes and assay conditions. The (KM)encap determined for Ba2+-
alginate-invertase (7.51 mM) was lower than that described in the literature, such as for
invertase entrapped in beads of polyacrylamide-gelatine (166mM)[12] or in polyethylenimine-
grafted poly(GMA-MMA) (29mM).[13] However, the (KM)encap of 7.51 mM was close to that
calculated for Ca2+-alginate invertase (7.2 mM).[14] The apparent high efficiency of invertase
entrapped either in Ba2+ or Ca2+ alginate beads – which requires a low substrate concentration
to reach the Vmax - over other carriers could depend on the high diffusion of substrate and
products through the bead membrane and/or aggregation degree of invertase molecules inside

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the bead (monomer, dimmer, trimmer or tetramer). The latter situation could lead to invertase
molecules aggregate in tetramer form, which is more active than the isolated invertase
molecule.[2]
Figure 3: Hanes-Woolf plot for soluble (●) and encapsulated invertase (■). The beads
were obtained by polymerizing 10 g/L of SG800 with 0.2M Ba(NO3)2 at pH 8.0, 360 rpm
and 30oC.
Reusability of Ba2+-SG800-invertase was analyzed by measuring its activity over three
successive batch sucrose hydrolysis (Figure 4).
Figure 4 – Reusability of Ba2+-alginate invertase beads for three successive batches [1st
(●), 2nd (□) and 3rd ()]. The beads were obtained by polymerizing 10 g/L of SG800 with
0.1M Ba(NO3)2 at pH 4.0, 540 rpm and 30oC.

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The minimal square linear regression equations are:
Y1st = 0.271X + 0.788 (r = 0.996) (Eq. 11)
Y2nd = 0.271X + 1.82 (r = 0.995) (Eq. 12)
Y3rd = 0.286X + 3.96 (r = 0.994) (Eq. 13)
Where Y1st, Y2nd and Y3rd are the RS formed (mg/mL) in the first, second and third batch,
respectively; X = reaction time (min).
As can be seen in Figure 4, there was no significant difference in invertase activity. However,
the initial RS concentration after the second batch is not zero, indicating an accumulation of
reducing sugars inside beads, which would probably increase as batches were tested
successively. Undoubtedly, the best approach for evaluating the reusability of encapsulated
invertase – and by extension any kind of immobilized enzyme – would be the continuous
process.[12]
CONCLUSION
The data obtained led to the conclusion that the barium consumption and the number of beads
produced are linearly correlated. In addition, the invertase encapsulation yield (YIE) varied
from 44.7% to 75.2% for SG800 (M/G < 1) and from 42.3% to 55.9% for S1100 (M/G > 1),
whereas the encapsulated invertase activity varied for SG800 (from 0.148 U/mL to 0.249
U/mL) and for S1100 (from 0.143 U/mL to 0.218 U/mL). The YIE over 70% was obtained
using SG800 alginate. The kinetic constants for soluble and encapsulated invertase were KM
= 52.9 mM, (KM)encap = 7.51 mM, Vmax = 0.368 U/mL and (Vmax)encap = 0.269 U/mL. Finally,
the barium-alginate beads were reused three times batchwise without significant loss of
invertase activity.
Funding: This work was supported by the National Council for Scientific and Technological
Development – CNPq (grant no. 303082/2015-1).
REFERENCES
1. Vitolo M. Calcium-alginate beads as carriers for biocatalyst encapsulation. World Journal
of Pharmaceutical Research, 2019. [in press].
2. De Queiroz AAA, Vitolo M, De Oliveira RC, Higa OZ. Invertase immobilization onto a
radiation, induced graft copolymerized polyethylene pellets. Radiation Physics and
Chemistry, 1996; 47: 873-80.

www.wjpr.net Vol 8, Issue 10, 2019.
233
Vitolo. World Journal of Pharmaceutical Research
3. Szekalska M, Pucilowska A, Szymanska E, Ciosek P, Winnicka K. Alginate: current use
and future perspectives in pharmaceutical and biomedical applications. International
Journal of Polymer Science, 2016. http://dx.doi.org/10.1155/2016/7697031.
4. Repka MA, Singh A. Alginic acid. In: Handbook of Pharmaceutical Excipients, London;
Pharmaceutical Press, 2009; 20-22.
5. Sachan KN, Pushkar S, Jha A, Bhattcharya A. Sodium alginate: the wonder polymer for
controlled drug delivery. Journal of Pharmacy Research, 2009; 2(8): 1191-99.
6. Wahl EA, Fierro TR, Peavy TR. In vitro evaluation of scaffolds for the delivery of
mesenchymal stem cells to wounds. BioMed Research International, 2015; 2015: Article
ID 108571.
7. Aljohani WJ, Wenchoo L, Ullah MW, Zhang X, Yang G. Application of sodium alginate
hydrogel. Journal of Biotechnology and Biochemistry, 2017; 3(3): 19-31.
8. Vitolo M. Effect of saline solution and osmotic pressure on invertase extraction from cell
wall of baker’s yeast. European Journal of Pharmaceutical and Medical Research, 2019;
6(5): 158-163.
9. Ozcan O, Yildirim RM, Toker OS, Akbas N, Ozulku G, Yaman M. The effect of
invertase concentration on quality parameters of fondant. Journal of Food Science and
Technology, 2019. https://doi.org/10.1007/s13197-019-03894-4.
10. Vitolo, M. Autolysis of baker’s yeast and partial purification of invertase in the presence
of surfactants. European Journal of Pharmaceutical and Medical Research, 2019; 6(5):
84-90.
11. Vitolo, M. Invertase activity of intact Saccharomyces cerevisiae cells cultured in
sugarcane molasses by batch fermentation process. World Journal of Pharmacy and
Pharmaceutical Sciences, 2019; 8(3): 126-137.
12. Emregul E, Sungur S, Akbulut U. Polyacrylamide-gelatine carrier system used for
invertase immobilization. Food Chemistry, 2006; 97: 591-97.
13. Arica MK, Bayramoglu G. Invertase reversibly immobilized onto polyethylenimine-
grafted poly(GMA-MMA) beads for sucrose hydrolysis. Journal of Molecular Catalysis
B: Enzymatic, 2006; 38: 131-38.
14. Arruda LMO, Vitolo, M. Characterization of invertase entrapped into calcium alginate
beads. Applied Biochemistry and Biotechnology, 1999; 81: 23-33.