# Formulation Optimization and Evaluation of ProbioticLactobacillus sporogenes-Loaded Sodium Alginate with Carboxymethyl Cellulose Mucoadhesive Beads Using Design Expert Software

Authors: Himanshu K. Solanki; Dushyant A. Shah
Journal: Journal of Food Processing
 (2016)

---

## Abstract

The present study deals with the formulation optimization of sodium carboxymethyl cellulose-alginate mucoadhesive beads containing probioticLactobacillus sporogenes through ionotropic gelation using 32 factorial design. The effect of sodium carboxymethyl cellulose-alginate concentration on the probiotic entrapment efficiency (PEE, %), viability in simulated gastric fluid (log CFU/g), and mucoadhesion over 8 hr (%) was optimized. The optimized beads containing probioticLactobacillus sporogenes showed entrapment efficiency of 93.7
±
1.97%, viability of probiotic in simulated gastric fluid (log CFU/g) of 9.34, mucoadhesion of 71.75
±
1.38%, and mean diameter of 1.21
±
0.11 mm. The beads were also characterized by SEM, FTIR, and XRD. The swelling and degradation of these beads were influenced by pH of the test medium. Finally, stability tests performed at room temperature (25~28°C) highlighted a bacterial viability of about 91% and 86% after 1 and 2 months, respectively. The advantageous properties of probioticLactobacillus sporogenes-loaded mucoadhesive beads make them suitable for incorporation in functional food and/or pharmaceutical products.


---

## Body

## 1. Introduction

Probiotics are microorganisms that are introduced orally in the gastrointestinal tract (GIT) and are able to positively contribute to the activity of the intestinal microflora and to the health of its host. A variety ofin vitro properties, including adhesion and resistance to pH, have been investigated to determine the suitability of candidate bacterial strains as a probiotic [1]. Probiotics can provide beneficial effects on the human body by keeping the healthy gut microflora, inhibiting the growth of pathogenic bacteria, relieving constipation, stimulating the immune system, synthesizing vitamins and antimicrobial agents, and improving the absorption of calcium, when there are enough probiotics in colon [2].In order to provide health benefits for probiotic bacteria [3] it has been recommended that they must be present at a minimum level of 106 CFU/g of food product [4] or 107 CFU/g at point of delivery or be eaten in sufficient amounts to yield a daily intake of 108 CFU [5].It is believed that the efficiency of probiotic health benefits can be enhanced by coupling the application of probiotics with a selective prebiotic growth substance, thus favouring colonization of probiotics in the human gut [6]. Hence, a combined application of pro- and prebiotics, a concept referred to as synbiotics, [7, 8] also emerged in the field of probiotic microencapsulation.The selection and optimization of a suitable microencapsulation process should be carried out such that the viability of encapsulated cells is maintained during both processing and storage. Thus, the selected method should be able to produce microspheres with the necessary physical/chemical attributes while causing minimal damage to cell integrity and viability and be easy to scale up with acceptable processing costs [9].Many studies showed the potential of microencapsulation to improve probiotic survival during storage in food products or gastrointestinal transit as summarized in several reviews [10–12].To our knowledge, nowadays no pharmaceutical studies were described for the encapsulation of probiotics associated with prebiotics BioEcolians (α-gluco-oligosaccharides) for therapeutic use while probiotics encapsulation is a process widely used in food applications in order to protect microorganisms against stomach acidity [12].The most common encapsulation material is sodium alginate due to its simplicity, nontoxicity, biocompatibility, and low cost [13, 14]. Alginate, the monovalent form of alginic acid belonging to a polysaccharide extracted from algae, consists of β-D-mannuronic and ∞-L-guluronic acids. The various amounts and sequential distribution of β-D-mannuronic and ∞-L-guluronic acids in chain can affect functional properties of alginate as a supporting material. Alginates undergo ionotropic gelation in aqueous solution in the presence of divalent cations like Ca2+, Ba2+, and so forth and trivalent cation like Al3+, due to an ionic interaction between the carboxylic acid groups located on the polymer backbone and these cations [15]. Alginates have mucoadhesive property, but the cross-linked alginates are usually fragile [16]. Therefore, to formulate various cross-linked alginate mucoadhesive microcapsules for controlled drug delivery, blending with mucoadhesive polymers is one of the most popular approaches. Again, blending with suitable polymers can improve the drug encapsulation and stability [17], which is found lower in alginate microcapsules, prepared by ionotropic gelation. Various mucoadhesive microcapsules of gliclazide using sodium alginate and mucoadhesive polymers such as sodium carboxymethyl cellulose, carbopol 934 P, and hydroxyl propyl methyl cellulose by ionotropic gelation was formulated by Prajapati et al. [18].Nevertheless, it is found that no attempt has been taken to formulate probioticLactobacillus sporogenes-loaded alginate-based microcapsule or bead system using methyl cellulose as a mucoadhesive polymer.The objectives of this research were first to optimize the conditions of microencapsulation of the probiotic strainsL. sporogenes using the extrusion method to obtain beads and then to evaluate the survival of the encapsulated cells after the technological processing, under simulated gastrointestinal conditions and during storage at room temperature (24–28°C) over a period of 2 months.


## 2. Materials and Methods

### 2.1. Materials

Lactobacillus sporogenes (ATCC 11748) were purchased from Unique Biotech Ltd., Hyderabad, India, and MRS agar and glucose yeast extract (GYE) agar medium, from Hi-media, Mumbai, India. Sodium alginate was procured from Chemdyes Pvt. Ltd, Rajkot, India, and sodium carboxy methyl cellulose (sodium CMC) was procured from Astron Chemicals, Ahmedabad, India. BioEcolians (α-gluco-oligosaccharides) were provided by Solabia group.


### 2.2. Microencapsulation Procedures

All glassware and solutions used in the protocols were sterilized at 121°C for 15 min. The microencapsulation procedure employed in the present study used a slightly modified version of the extrusion technique [13]. In brief, 2.5 to 3.5% sodium alginate was mixed in distilled water containing 0.5–1% sodium carboxymethylcellulose; 2% BioEcolins; and 1010 CFU/mL ofL. sporogenes. The cell suspension mixture and sodium alginate were injected into a 5% CaCl2 solution using a sterile syringe. The droplets immediately formed gel spheres. The distance between the syringe and the CaCl2 solution was 3 cm. The beads were then separated by filtration using Whatman filter paper, transferred to a sterile petri dish, and stored in a refrigerator until further use.


### 2.3. Experimental Design for Optimization

Factorial design is an experimental design technique, by which the factor involved and their relative importance can be assessed. In the present study, two-factor, three-level factorial design (32) was employed for optimization with concentration of sodium alginate in % (A) and concentration of sodium CMC in % (B) as the prime selected independent variables, which were varied at three levels, low level (−1), medium level (0), and high level (+1). The entrapment efficiency (EE, %), viability in SGF (log CFU), and mucoadhesion over 8 hr (%) were selected as responses. Design-Expert Version 9.0.4.1 software (Stat-Ease Inc., USA) was used for the generation and evaluation of the statistical experimental design. The matrix of the design including investigated responses, that is, entrapment efficiency (EE, %), viability in SGF (log CFU), and mucoadhesion over 8 hr (%) are shown in Table 1. The effects of independent variables upon investigated responses were modeled using following quadratic mathematical model(1)
Y
=
b
0
+
b
1
A
+
b
2
B
+
b
3
A
B
+
b
4
A
2
+
b
5
B
2
,where Y is the response, b
0 is the intercept, and b
1
,
b
2
,
b
3
,
b
4
,
b
5 are regression coefficients. A and B are individual effects; A
2 and B
2 are quadratic effects; A
B is the interaction effect. One-way ANOVA was applied to estimate the significance (p
<
0.05) of the model and individual response parameters. The surface response plots and contour plots were analyzed to reveal the effect of independent factors on the measured responses. Numerical optimization was performed using the desirability approach to develop optimized formulation. To check the validation of these generated mathematical models for optimization of the formulation, and overlay plot were analyzed.Table 1
Experimental plan and observed response values from randomized run-in factorial design layout.
Formulation batch code
Independent variable
% entrapment efficiency (Y
1)
Viability in SGF over 2 hr (log CFU/g) (Y
2)
% mucoadhesion over 8 hr (Y
3)
Sodium alginate (A)
Sodium CMC (B)
Coded value
Actual value
Coded value
Actual value
D1
−1
2.5
−1
0.5
90.88 ± 1.4
8.48 ± 0.3
54 ± 0.81
D2
0
3
−1
0.5
92.33 ± 1.2
8.58 ± 0.4
56.35 ± 0.57
D3
1
3.5
−1
0.5
94.81 ± 1.2
8.45 ± 0.2
58.85 ± 0.57
D4
−1
2.5
0
0.75
91.57 ± 1.6
8.42 ± 0.3
61.75 ± 1.15
D5
0
3
0
0.75
93.39 ± 1.3
8.56 ± 0.2
62.95 ± 0.57
D6
1
3.5
0
0.75
95.44 ± 1.2
8.26 ± 0.2
65.65 ± 0.57
D7
−1
2.5
1
1
92.66 ± 1.2
8.85 ± 0.2
68.85 ± 0.58
D8
0
3
1
1
93.74 ± 2.0
9.34 ± 0.2
73.75 ± 0.57
D9
1
3.5
1
1
95.55 ± 1.8
9.28 ± 0.22
76 ± 1.00


### 2.4. Percentage Yield (%w/w)

The percentage yield of prepared batches were calculated using the weight of final product after proper drying with respect to the initial total weight of probiotic and polymers used for preparation of beads. The percentage yield was calculated from the following formula:(2)
Percentage  Yield
=
P
r
a
c
t
i
c
a
l
m
a
s
s
o
f
b
e
a
d
s
T
h
e
o
r
e
t
i
c
a
l
y
i
e
l
d
×
100
.


### 2.5. Probiotic Entrapment Efficiency (PEE)

To determine Entrapment efficiency of beads, 100 mg of dried beads were weighed and added to sterile conical flask containing simulated intestinal fluid (pH 6.8). The added beads in conical flask were kept in orbital shaker at a period of 2 h for mechanical disintegration in the simulated intestinal fluid. 1.0 mL aliquot of the mixture was removed and adequate dilutions were made and subjected to a viable colony count (expressed in number of colony forming units (CFU)) ofL. sporogenes count by pour plate method. The entrapment efficiency value was calculated using (3) and was reported in percent:(3)
Entrapment  efficiency
%
=
log
10
⁡
N
log
10
⁡
N
0
×
100
,where N is the number of colonies formed from the beads, and N
0 is the number of free cells added to the biopolymer mixture and number of colonies formed [19].


### 2.6. Determination of Bead Size

The particle sizes of the prepared beads were determined by measuring diameters of 20 dried beads at ×100 magnifications using an optical microscope (Microtech, India) fitted with a calibrated micrometer scale. The mean diameters of beads were calculated and presented with standard deviations (Table4).


### 2.7. Surface Morphology Analysis

The surface morphology of the formulated beads containingL. sporogenes was analyzed by SEM (JEOL, JSM-6360A). Beads were platinum coated by being mounted on a stub using double-sided adhesive tape and under vacuum in an auto fine coater (JEOL, JFC-1600) to make them electrically conductive and their morphology was examined by SEM.


### 2.8. Fourier Transform-Infrared (FTIR) Spectroscopy

FTIR spectra were recorded using KBr pellet in a FTIR spectrophotometer (Model: Bruker Alpha FTIR Spectrophotometer).Lactobacillus sporogenes, sodium alginate, sodium carboxy methylcellulose, calcium chloride, andLactobacillus sporogenes-loaded microparticles were each separately finely grounded with KBr. The pellet was placed in the sample holder. Spectral scanning was taken in the wavelength region between 4000 and 400 cm−1 at a resolution of 4 cm−1 with scan speed of 1 cm/s.


### 2.9. X-Ray Diffraction Study

X-ray powder diffractograms ofLactobacillus sporogenes, sodium alginate, andL. sporogenes-loaded calcium alginate beads were recorded on an X-ray diffractometer (Instrument Details: Manufacturer: Panalytical Model: Xpert PRO MPD Anode: Copper K-alpha Wavelength: 1.5405 Angstorm Power: 45 KV and 40 mA Detector: Xcelerator with Diffracted Beam). The samples were scanned between 2
θ
=
3
° and 60° at the scan rate of 4°/min.


### 2.10. Determination of Viability of Encapsulated Probiotic in Simulated Gastric Fluid

For assessing the viability of probiotic inin vitro conditions (simulated gastric fluid), 0.1 g of beads were taken from each formulation batch and introduced into 10 mL of simulated gastric fluid. For one hour of incubation interval in simulated gastric fluid for 2 h, 1.0 mL aliquot was removed and serial dilutions were carried out using anaerobic solution. The serial dilutions (108) were subjected to a viable colony count (expressed in number of colony forming units (CFU)) ofL. Sporogenes by pour plate method on GYE agar media. The plates were incubated for 72 hrs in the B.O.D incubator at 37°C and the colonies formed were counted by using colony counter.


### 2.11. Evaluation of Swelling Behavior

Swelling behavior evaluation of sodium alginate beads containingLactobacillus sporogenes was studied by measuring the percentage swelling index of the formulated beads [20] (Figure 8). A known weight (50 mg) of beads was placed in a conical flask containing 10 mL of simulated gastric fluid (pH 1.2) for a period of time (10, 30, 60, 90, and 120 min) and during each time interval in simulated gastric fluid, the weight of the beads was determined after being blotted with a piece of filter paper to remove the excess water on the surface and was transferred to fresh medium (simulated gastric fluid) in order to continue to swell. After 120 min, the beads were transferred to a conical flask containing 10 mL simulated intestinal fluid. The weight of the beads was determined for a period of time (150, 180, and 210 min). Fresh SIF was replaced at each time interval. The mass of dry beads was determined at time zero and the mass of wet beads was measured at each sampling point on a weighing balance (Precisa XR 205SM-DR) accurate to ±0.001 g. The percentage of swelling index was calculated using the following: (4)
Swelling  index
%
=
w
e
i
g
h
t
o
f
b
e
a
d
s
a
f
t
e
r
s
w
e
l
l
i
n
g
-
D
r
y
w
e
t
o
f
b
e
a
d
s
D
r
y
w
e
t
o
f
b
e
a
d
s
×
100
.


### 2.12.In Vitro Wash-Off Test for Mucoadhesion

The mucoadhesive properties of sodium alginate beads containingLactobacillus sporogenes were evaluated byex vivo wash-off method [21]. Freshly excised pieces of goat intestinal mucosa (2 cm × 2 cm) (collected from slaughterhouse) were mounted on glass slide (7.5 cm × 2.5 cm) using thread. About 20 beads were spread onto the wet tissue specimen, and the prepared slide was hung onto a groove of disintegration test apparatus. The tissue specimen was given a regular up and down movement in a vessel containing 900 mL of simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8), separately, at 37
±
0.5°C. After regular time intervals, the machine was stopped and the number of beads still adhering to the tissue was counted. The mucoadhesion value was calculated using (5) and was reported in percent:(5)
Mucoadhsion
%
=
N
o
.
o
f
m
i
c
r
o
b
e
a
d
s
a
p
p
l
i
e
d
-
N
o
.
o
f
m
i
c
r
o
b
e
a
d
s
l
e
a
c
h
e
d
o
u
t
N
o
.
o
f
m
i
c
r
o
b
e
a
d
s
a
p
p
l
i
e
d
×
100
.


### 2.13. Stability Studies of Free and EncapsulatedL. sporogenes

The stability studies were conducted on the free and encapsulatedL. sporogenes-loaded selected optimized formulation F-O at room temperature (24–28°C). The stability studies were done at the intervals of 0, 30, and 60 days. The parameters studied were entrapment efficiency and viability ofL. sporogenes.


### 2.14. Statistical Analysis

Statistical optimization was performed using Design-Expert Version 9.0.4.1 software (Stat-Ease Inc., USA). All measured data are expressed as mean ± standard deviation (S.D.) of replicated determinations. Data were subjected to one-way analysis of variance (ANOVA). Statistical significance was set atp
≤
0.05.


## 2.1. Materials

Lactobacillus sporogenes (ATCC 11748) were purchased from Unique Biotech Ltd., Hyderabad, India, and MRS agar and glucose yeast extract (GYE) agar medium, from Hi-media, Mumbai, India. Sodium alginate was procured from Chemdyes Pvt. Ltd, Rajkot, India, and sodium carboxy methyl cellulose (sodium CMC) was procured from Astron Chemicals, Ahmedabad, India. BioEcolians (α-gluco-oligosaccharides) were provided by Solabia group.


## 2.2. Microencapsulation Procedures

All glassware and solutions used in the protocols were sterilized at 121°C for 15 min. The microencapsulation procedure employed in the present study used a slightly modified version of the extrusion technique [13]. In brief, 2.5 to 3.5% sodium alginate was mixed in distilled water containing 0.5–1% sodium carboxymethylcellulose; 2% BioEcolins; and 1010 CFU/mL ofL. sporogenes. The cell suspension mixture and sodium alginate were injected into a 5% CaCl2 solution using a sterile syringe. The droplets immediately formed gel spheres. The distance between the syringe and the CaCl2 solution was 3 cm. The beads were then separated by filtration using Whatman filter paper, transferred to a sterile petri dish, and stored in a refrigerator until further use.


## 2.3. Experimental Design for Optimization

Factorial design is an experimental design technique, by which the factor involved and their relative importance can be assessed. In the present study, two-factor, three-level factorial design (32) was employed for optimization with concentration of sodium alginate in % (A) and concentration of sodium CMC in % (B) as the prime selected independent variables, which were varied at three levels, low level (−1), medium level (0), and high level (+1). The entrapment efficiency (EE, %), viability in SGF (log CFU), and mucoadhesion over 8 hr (%) were selected as responses. Design-Expert Version 9.0.4.1 software (Stat-Ease Inc., USA) was used for the generation and evaluation of the statistical experimental design. The matrix of the design including investigated responses, that is, entrapment efficiency (EE, %), viability in SGF (log CFU), and mucoadhesion over 8 hr (%) are shown in Table 1. The effects of independent variables upon investigated responses were modeled using following quadratic mathematical model(1)
Y
=
b
0
+
b
1
A
+
b
2
B
+
b
3
A
B
+
b
4
A
2
+
b
5
B
2
,where Y is the response, b
0 is the intercept, and b
1
,
b
2
,
b
3
,
b
4
,
b
5 are regression coefficients. A and B are individual effects; A
2 and B
2 are quadratic effects; A
B is the interaction effect. One-way ANOVA was applied to estimate the significance (p
<
0.05) of the model and individual response parameters. The surface response plots and contour plots were analyzed to reveal the effect of independent factors on the measured responses. Numerical optimization was performed using the desirability approach to develop optimized formulation. To check the validation of these generated mathematical models for optimization of the formulation, and overlay plot were analyzed.Table 1
Experimental plan and observed response values from randomized run-in factorial design layout.
Formulation batch code
Independent variable
% entrapment efficiency (Y
1)
Viability in SGF over 2 hr (log CFU/g) (Y
2)
% mucoadhesion over 8 hr (Y
3)
Sodium alginate (A)
Sodium CMC (B)
Coded value
Actual value
Coded value
Actual value
D1
−1
2.5
−1
0.5
90.88 ± 1.4
8.48 ± 0.3
54 ± 0.81
D2
0
3
−1
0.5
92.33 ± 1.2
8.58 ± 0.4
56.35 ± 0.57
D3
1
3.5
−1
0.5
94.81 ± 1.2
8.45 ± 0.2
58.85 ± 0.57
D4
−1
2.5
0
0.75
91.57 ± 1.6
8.42 ± 0.3
61.75 ± 1.15
D5
0
3
0
0.75
93.39 ± 1.3
8.56 ± 0.2
62.95 ± 0.57
D6
1
3.5
0
0.75
95.44 ± 1.2
8.26 ± 0.2
65.65 ± 0.57
D7
−1
2.5
1
1
92.66 ± 1.2
8.85 ± 0.2
68.85 ± 0.58
D8
0
3
1
1
93.74 ± 2.0
9.34 ± 0.2
73.75 ± 0.57
D9
1
3.5
1
1
95.55 ± 1.8
9.28 ± 0.22
76 ± 1.00


## 2.4. Percentage Yield (%w/w)

The percentage yield of prepared batches were calculated using the weight of final product after proper drying with respect to the initial total weight of probiotic and polymers used for preparation of beads. The percentage yield was calculated from the following formula:(2)
Percentage  Yield
=
P
r
a
c
t
i
c
a
l
m
a
s
s
o
f
b
e
a
d
s
T
h
e
o
r
e
t
i
c
a
l
y
i
e
l
d
×
100
.


## 2.5. Probiotic Entrapment Efficiency (PEE)

To determine Entrapment efficiency of beads, 100 mg of dried beads were weighed and added to sterile conical flask containing simulated intestinal fluid (pH 6.8). The added beads in conical flask were kept in orbital shaker at a period of 2 h for mechanical disintegration in the simulated intestinal fluid. 1.0 mL aliquot of the mixture was removed and adequate dilutions were made and subjected to a viable colony count (expressed in number of colony forming units (CFU)) ofL. sporogenes count by pour plate method. The entrapment efficiency value was calculated using (3) and was reported in percent:(3)
Entrapment  efficiency
%
=
log
10
⁡
N
log
10
⁡
N
0
×
100
,where N is the number of colonies formed from the beads, and N
0 is the number of free cells added to the biopolymer mixture and number of colonies formed [19].


## 2.6. Determination of Bead Size

The particle sizes of the prepared beads were determined by measuring diameters of 20 dried beads at ×100 magnifications using an optical microscope (Microtech, India) fitted with a calibrated micrometer scale. The mean diameters of beads were calculated and presented with standard deviations (Table4).


## 2.7. Surface Morphology Analysis

The surface morphology of the formulated beads containingL. sporogenes was analyzed by SEM (JEOL, JSM-6360A). Beads were platinum coated by being mounted on a stub using double-sided adhesive tape and under vacuum in an auto fine coater (JEOL, JFC-1600) to make them electrically conductive and their morphology was examined by SEM.


## 2.8. Fourier Transform-Infrared (FTIR) Spectroscopy

FTIR spectra were recorded using KBr pellet in a FTIR spectrophotometer (Model: Bruker Alpha FTIR Spectrophotometer).Lactobacillus sporogenes, sodium alginate, sodium carboxy methylcellulose, calcium chloride, andLactobacillus sporogenes-loaded microparticles were each separately finely grounded with KBr. The pellet was placed in the sample holder. Spectral scanning was taken in the wavelength region between 4000 and 400 cm−1 at a resolution of 4 cm−1 with scan speed of 1 cm/s.


## 2.9. X-Ray Diffraction Study

X-ray powder diffractograms ofLactobacillus sporogenes, sodium alginate, andL. sporogenes-loaded calcium alginate beads were recorded on an X-ray diffractometer (Instrument Details: Manufacturer: Panalytical Model: Xpert PRO MPD Anode: Copper K-alpha Wavelength: 1.5405 Angstorm Power: 45 KV and 40 mA Detector: Xcelerator with Diffracted Beam). The samples were scanned between 2
θ
=
3
° and 60° at the scan rate of 4°/min.


## 2.10. Determination of Viability of Encapsulated Probiotic in Simulated Gastric Fluid

For assessing the viability of probiotic inin vitro conditions (simulated gastric fluid), 0.1 g of beads were taken from each formulation batch and introduced into 10 mL of simulated gastric fluid. For one hour of incubation interval in simulated gastric fluid for 2 h, 1.0 mL aliquot was removed and serial dilutions were carried out using anaerobic solution. The serial dilutions (108) were subjected to a viable colony count (expressed in number of colony forming units (CFU)) ofL. Sporogenes by pour plate method on GYE agar media. The plates were incubated for 72 hrs in the B.O.D incubator at 37°C and the colonies formed were counted by using colony counter.


## 2.11. Evaluation of Swelling Behavior

Swelling behavior evaluation of sodium alginate beads containingLactobacillus sporogenes was studied by measuring the percentage swelling index of the formulated beads [20] (Figure 8). A known weight (50 mg) of beads was placed in a conical flask containing 10 mL of simulated gastric fluid (pH 1.2) for a period of time (10, 30, 60, 90, and 120 min) and during each time interval in simulated gastric fluid, the weight of the beads was determined after being blotted with a piece of filter paper to remove the excess water on the surface and was transferred to fresh medium (simulated gastric fluid) in order to continue to swell. After 120 min, the beads were transferred to a conical flask containing 10 mL simulated intestinal fluid. The weight of the beads was determined for a period of time (150, 180, and 210 min). Fresh SIF was replaced at each time interval. The mass of dry beads was determined at time zero and the mass of wet beads was measured at each sampling point on a weighing balance (Precisa XR 205SM-DR) accurate to ±0.001 g. The percentage of swelling index was calculated using the following: (4)
Swelling  index
%
=
w
e
i
g
h
t
o
f
b
e
a
d
s
a
f
t
e
r
s
w
e
l
l
i
n
g
-
D
r
y
w
e
t
o
f
b
e
a
d
s
D
r
y
w
e
t
o
f
b
e
a
d
s
×
100
.


## 2.12.In Vitro Wash-Off Test for Mucoadhesion

The mucoadhesive properties of sodium alginate beads containingLactobacillus sporogenes were evaluated byex vivo wash-off method [21]. Freshly excised pieces of goat intestinal mucosa (2 cm × 2 cm) (collected from slaughterhouse) were mounted on glass slide (7.5 cm × 2.5 cm) using thread. About 20 beads were spread onto the wet tissue specimen, and the prepared slide was hung onto a groove of disintegration test apparatus. The tissue specimen was given a regular up and down movement in a vessel containing 900 mL of simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8), separately, at 37
±
0.5°C. After regular time intervals, the machine was stopped and the number of beads still adhering to the tissue was counted. The mucoadhesion value was calculated using (5) and was reported in percent:(5)
Mucoadhsion
%
=
N
o
.
o
f
m
i
c
r
o
b
e
a
d
s
a
p
p
l
i
e
d
-
N
o
.
o
f
m
i
c
r
o
b
e
a
d
s
l
e
a
c
h
e
d
o
u
t
N
o
.
o
f
m
i
c
r
o
b
e
a
d
s
a
p
p
l
i
e
d
×
100
.


## 2.13. Stability Studies of Free and EncapsulatedL. sporogenes

The stability studies were conducted on the free and encapsulatedL. sporogenes-loaded selected optimized formulation F-O at room temperature (24–28°C). The stability studies were done at the intervals of 0, 30, and 60 days. The parameters studied were entrapment efficiency and viability ofL. sporogenes.


## 2.14. Statistical Analysis

Statistical optimization was performed using Design-Expert Version 9.0.4.1 software (Stat-Ease Inc., USA). All measured data are expressed as mean ± standard deviation (S.D.) of replicated determinations. Data were subjected to one-way analysis of variance (ANOVA). Statistical significance was set atp
≤
0.05.


## 3. Result and Discussion

### 3.1. Optimization

In the development of pharmaceutical dosage forms, an important and crucial issue is to search an optimal formula for designing a product of desired quality by minimum trials within a short period [22]. Optimization by means of computer-aided statistical experimental design methodologies has been successfully applied in the literature for the development of various pharmaceutical dosage forms [23, 24]. The statistical optimization technique encompasses designing a set of experiment that will reliably measure the response variables, fitting mathematical models to the data, conducting appropriate statistical tests to select best possible model, and determining the values of independent formulation variables to produce optimum response. Among various statistical design, factorial design is one of the popular experimental designs that have been employed in the formulation optimization [25, 26].Optimization of the concentrations of the formulation factors was performed to obtain probiotic microparticles with enhanced viability ofL. sporogenes and adequate release profile in different regions of the gastrointestinal tract. Although several responses were monitored to select the optimal values of the formulation factors, the main concern was given to the viability ofL. sporogenes in simulated gastric fluid. This implication is accounted on the fact that the probiotics must first survive the deleterious effect of the gastric pH in order to reach the intestine where the bioadhesive properties of the delivery system are among the key factors for achieving prolonged residence time and effective colonization of the probiotic.For the 32 factorial designs, 9 trial formulations were suggested by Design-Expert 9.0.4 software (Stat-Ease Inc., USA) for two independent variables: % amount of sodium alginate (A) and % amount of Sodium CMC (B), which were varied at three levels: low level (−1), medium level (0), and high level (+1). The % entrapment efficiency (Y
1), log CFU viability in SGF (Y
2), and % mucoadhesion over 8 hours (Y
3) were evaluated as dependent variables (responses) in the current investigation. According to this trial plan, alginate sodium CMC beads containingL. sporogenes were prepared through ionotropic gelation. Overview of matrix of the design including investigated responses is presented in Table 1.The values of investigated responses measured for all trial formulations were fitted in the 32 factorial designs to get model equations for responses analyzed in this investigation. These models were evaluated statistically by applying one-way ANOVA (p
<
0.05). The result of the ANOVA is shown in Table 2.Table 2
Summary of ANOVA for the response parameters in 32 factorial designs.
Source
Sum of squares
df
Mean square
F value
p value Prob. > F
Significance
For entrapment efficiency (%)
Model
21.62
2
10.81
99.23
<0.0001
Significant
A
19.05
1
19.05
174.83
<0.0001
B
2.57
1
2.57
23.63
0.0028
Residual
0.65
6
0.11
Cor. total
22.27
8
For viability in SGF (log10 CFU)
Model
1.13
5
0.23
12.84
0.03
Significant
A
0.06
1
0.06
3.53
0.16
B
0.30
1
0.30
17.00
0.03
A
B
0.05
1
0.05
3.08
0.18
A
2
0.08
1
0.09
4.77
0.12
B
2
0.34
1
0.34
19.40
0.02
Residual
0.05
3
0.018
Cor. total
1.18
8
For mucoadhesion (%)
Model
448.86
2
224.43
188.54
<0.0001
Significant
A
42.14
1
42.14
35.40
0.0010
B
406.73
1
406.73
341.68
<0.001
Residual
7.14
6
1.19
Cor. total
456.03
8The model equations relating various measured responses became(6)
Entrapment  Efficiency
%
Y
1
=
80.71944
+
3.56333
×
A
+
2.62000
×
B
,
Viability  in  SGF
l
o
g
10
CFU
Y
2
=
+
5.77658
+
4.28986
×
A
-
11.38256
×
B
+
0.92991
×
A
B
-
0.81776
×
A
2
+
6.59816
×
B
2
,
Mucoadhesion
%
Y
3
=
+
23.63889
+
5.30000
×
A
+
32.93333
×
B
.It can be noted that all coefficients of these above three mathematical models had statistical significance (p
<
0.05). The influences of main effects (factors) on responses investigated were further elucidated by response surface methodology. Response surface methodology has been reported as an effective tool for optimization of a dosage form to investigate the combined effect (interaction) of investigated factors on the desired responses [25–27]. In the response surface methodology, the three-dimensional response surface graph is very useful in learning about the main and interaction effects of the factors, whereas two-dimensional contour plot gives a visual representation of the response [26].


### 3.2. Effect of Formulation Variable on % Entrapment Efficiency(
Y
1
)

The three-dimensional response surface plot and corresponding two-dimensional contour plot relating entrapment efficiency are presented in Figures1(a) and 1(b), respectively. The R
2 (0.9707) was high indicating the adequate fitting of the linear model. The polynomial equations can also be used to draw conclusions considering the magnitude of coefficient and the mathematical sign it carries, that is, positive. The % entrapment efficiency of all the batches was within 90.88 to 95.55% (Table 1). It was observed that PEE (%) of these beads containingL. sporogenes was increased with the increment of sodium alginate and sodium CMC amount in the polymer blend. The increased PEE (%) with the increasing amount of sodium alginate and sodium CMC in these beads may be due to the increase in viscosity of the polymer solution with the increasing amount of polymer addition, so that it might have prevented drug leaching to the cross-linking solution. In addition, the increasing amount of sodium alginate and sodium CMC in polymer-blend might have elevated the cross-linking by CaCl2 through availing more numbers of sites for ionic cross-linking.Figure 1
Effect of formulation variable on % entrapment efficiency presented by (a) contour plots and (b) response surface plots.
(a)
(b)Among the nine batches D9 showed highest % probiotic entrapment efficiency, and this may be due to a high level of sodium alginate and sodium CMC which allows the probiotic to get entrapped more as compared to formulation containing a low level of sodium alginate and sodium CMC (Figure5).


### 3.3. Effect of Formulation Variables on Log CFU Viability ofL. sporogenes in SGF (
Y
2
)

Response surface plot and corresponding two-dimensional contour plot relating viability in SGF are presented in Figures2(a) and 2(b), respectively.Figure 2
Effect of formulation variable on viability in SGF (log CFU) presented by (a) contour plots and (b) response surface plots.
(a)
(b)TheR
2 (0.9554) was high indicating the adequate fitting of the quadratic model. The polynomial equations can also be used to draw conclusions considering the magnitude of coefficient and the mathematical sign it carries, that is, positive or negative. According to the values of regression coefficients, A, B (Table 2), the viability ofL. sporogenes in microparticles obtained was mostly affected by the sodium alginate (A), while the sodium CMC (B) concentration had the lowest influence on this response. The best response for the probiotic viability was obtained at sodium CMC concentration of 1% W/V in the concentration range of alginate 3% W/V and CaCl2 5% W/V, however, with no significant effect on the probiotic viability when increased sodium alginate concentration up to 3.5 was used. The viability ofL. sporogenes of all batches was within 8.48 to 9.34 log CFU (Table 1).In fact, microencapsulatedL. sporogenes showed that more than 108 CFU/mLL. sporogenes capsules survived after 120 min and were resistant to simulated gastric conditions. The results showed that microencapsulation in alginate significantly (p
<
0.05) improved the survival rate ofL. sporogenes.Chandramouli et al. (2004) and Iyer and Kailasapathy (2005) have shown that only the microencapsulated probiotics were able to maintain viability in gastrointestinal conditions [28, 29]. Microencapsulation of probiotics in alginate beads has previously been tested for improving viability of probiotic bacteria in simulated gastric conditions [30] although some authors have reported the effect of alginate encapsulation on survival of lactic acid bacteria in simulated gastrointestinal conditions [31–33].


### 3.4. Effect of Formulation Variables on Mucoadhesion(
Y
3
)

TheR
2 (0.9990) was high indicating the adequate fitting of the linear model.Response surface plot and corresponding two-dimensional contour plot relating mucoadhesion are presented in Figures3(a) and 3(b), respectively.Figure 3
Effect of formulation variable on mucoadhesion (%) presented by (a) contour plots and (b) response surface plots.
(a)
(b)The mucoadhesion was significantly (p
<
0.05) increased as the concentration of sodium alginate and sodium CMC increased over the range of 2.5 to 3.5% and 0.5 to 1.0%, respectively. Formulation D9 (containing concentration of sodium alginate 3.5% and sodium CMC 1%) exhibited maximum mucoadhesive strength. The result also showed that both sodium alginate and sodium CMC significantly increased the viscosity as well as the mucoadhesive property. Sodium CMC has an abundance of hydroxyl and ether groups along their length, which are responsible for the mucoadhesive properties. Increasing the concentration of the sodium CMC in the formulation increased the bonds forming groups, thus increasing the mucoadhesive force of the formulations. Mucoadhesion behavior of alginate was due to the low surface tension (31.5 mN/m) of the alginate, which is lower than the critical surface tension of the mucin (38 mN/m), resulting in good spreading and adhesion.The three-dimensional response surface graph relating to mucoadhesion (Figure3) indicates the increase in mucoadhesion observed with increasing amount of sodium CMC (A) and amount of sodium alginate (B).The influences of main effects (factors) on responses (here, % encapsulation efficiency, viability in SGF, and % mucoadhesion over 8 hours) were further elucidated by response surface methodology. Response surface methodology is a widely proficient approach in the development and optimization of drug delivery devices. The purpose of the response surface methodology is to understand as fully as possible the effect of factors and their levels in a model, over the whole of the experimental domain and to predict the response inside the domain. Moreover, it can be used for optimizing a formula (i.e., maximizing one or more of the responses, keeping the formulation variable setting within a satisfactory range), carrying out simulations with the model equations and plotting the responses. The three-dimensional response surface graph is very useful in learning about the main and interaction effects of the independent variables (factors), whereas two-dimensional contour plot gives a visual representation of values of the response. The three-dimensional response surface plots and corresponding contour plots were presented to estimate the effects of the independent variables (factors) on each response investigated.A numerical optimization technique using the desirability approach was employed to develop new formulations with desired response (optimum quality). The desirable ranges of the undependable variables (factors) were restricted to2.5
≤
A
≤
3.5% and 0.5
≤
B
≤
1.0%, whereas the desirable ranges of responses were restricted to 90
≤
Y
1
≤
95%, 8.8
≤
Y
2
≤
9.3 log CFU, and 70
≤
Y
3
≤
80%. The optimal values of responses were obtained by numerical analysis using the Design-Expert 9.0.4.1 software and one of them was selected based on the criterion of desirability. In order to evaluate the optimization capability of the mathematical models generated according to the results of full 32 factorial design, optimized sodium alginate-sodium CMC mucoadhesive beads containingL. Sporogenes were prepared by ionotropic gelation technique using these optimal process variable settings proposed by the design (Table 3). The selected optimal process variable setting used for the formulation of optimized formulation was A
=
3.27% and B
=
1.00%. Optimized sodium alginate-sodium CMC mucoadhesive beads containingL. Sporogenes (F-O) were evaluated Y
1, Y
2 and Y
3.Table 3
Results of experiments for confirming optimization capability.
(a)
Code
Factor
A (%)
B (%)
F-O
3.27
1.00
(b)
Responsea
Predicted value
Observed value
% errorb
Y
1 = entrapment efficiency (%)
95.02
93.7 ± 1.97
1.39
Y
2 = viability in SGF after 2 hr exposure (log CFU)
9.3
9.34 ± 0.3
−0.438
Y
3 = mucoadhesion over 8 hr (%)
73.81
71.75 ± 1.38
3.60
aObserved response values: mean ± S.D. (n
=
3).
bError (%) = [difference between predicted value and actual value/predicted value] × 100.Table 4
Mean diameter of calcium alginate/CMC beads containingL. sporogenes.
Formulation code
Mean diameter (mm)a
D1
1.15 ± 0.03
D2
1.3 ± 0.04
D3
1.39 ± 0.03
D4
1.17 ± 0.07
D5
1.22 ± 0.04
D6
1.41 ± 0.02
D7
1.19 ± 0.04
D8
1.24 ± 0.02
D9
1.50 ± 0.04
amean ± SD.The optimized mucoadhesive beads containingL. sporogenes (F-O) showed Y
1 of 93.7
±
1.97%, Y
2 of 9.34, and Y
3 of 71.75
±
1.38% with small percentage error values (1.26, −0.538, and 2.54, resp.). Percentage error evaluation is helpful in establishing the validity of generated model equations to describe the domain of applicability of optimization model. The percentage error values reveal that mathematical models obtained from the full 32 factorial designs were well fitted (Figure 4).Figure 4
The desirability plot: (a) indicating desirable regression ranges and the overlay plot and (b) indicating the region of optimal process variable settings.
(a)
(b)Figure 5
Depicts the scanning electron microscopy (SEM) images of dried beads ofLactobacillus sporogenes at different magnification, (a)Lactobacillus sporogenes-loaded batch D9, (b) cross section OfLactobacillus sporogenes-loaded batch D9, (c) after dissolution ofLactobacillus sporogenes-loaded batch D9, and (d) cross section of after dissolutionLactobacillus sporogenes-loaded batch (D9).
(a)
Lactobacillus sporogenes-loaded beads batch F-O
(b)
Cross section ofLactobacillus sporogenes-loaded optimized batch
(c)
After dissolution of optimized batch
(d)
Cross section of after dissolutionLactobacillus sporogenes optimized batch


### 3.5. Bead Size

The average size of the formulated dried beads containingLactobacillus sporogenes ranged from 1.15
±
0.03 to 1.50
±
0.04 mm.Increasing bead size was found with increasing amount of sodium alginate and sodium CMC. This could be attributed to increase in viscosity of the polymer blend (sodium alginate and sodium CMC) solution with incorporation of both the polymers in increasing ratio that in turn increased the droplet size during addition of the polymer blend solution to the cross-linking solution.


### 3.6. Surface Morphology Analysis

It can be inferred from the images that beads obtained were spherical in shape with smooth surfaces. The complete roughness of beads was observed under higher magnification. Reduction of beads’ size after dissolution was observed, soL. sporogenes might be released from the surface and gel matrix of sodium alginate. It might be due to erosion of beads during dissolution.


### 3.7. FTIR Spectroscopy

The FTIR spectra of probioticL. sporogenes sodium alginate, sodium CMC, BioEcolian, and optimized ionotropically gelled sodium CMC-alginate beads containingL. sporogenes are shown in Figure 6.Figure 6
The FTIR spectra ofL. sporogenes (a), sodium alginate (b), sodium CMC (c), BioEcolian (d), and sodium alginate sodium CMC beads containingL. sporogenes (F-O) (e).In the FT-IR spectrum of sodium alginate powder, the various distinct peaks of alginate evident are those of the hydroxyl group at 3429 cm−1, carbonyl at 1614 cm−1, and carboxyl and carboxylate groups between 1000 and 1400 cm−1. The absorption band around 2880, 1656, 1421, and 1080 cm−1 corresponds to the stretching of -CH, COO-, -CH, and C-O-C, respectively.The IR spectra of sodium carboxy methyl cellulose exhibited presence of characteristic absorption bands corresponding to asymmetric and symmetric carboxylate anions at, respectively, 1609 cm−1 and 1426 cm−1, a broad band at 3412 cm−1 corresponding to stretching vibration of hydroxyl group, and a peak at 1059 cm−1 corresponding to alkyl substituted ether. The FTIR spectra of BioEcolian (GOS) showed characteristic peaks around 3500–3200 cm−1 for OH stretching, 2147 cm−1 for CH stretching, and 1650 cm−1 for CO stretching, 1025 cm−1.In the FTIR spectrum of optimized ionotropically gelled sodium CMC-alginate beads containingL. sporogenes, various characteristic peaks of sodium alginate, sodium CMC, andL. sporogenes appeared without any significant shifting of these peaks. In short, the sodium CMC–alginate beads containingL. sporogenes prepared with sodium CMC-alginate polymer-blend had significant characters ofL. sporogenes in the FTIR spectrum, suggesting absence of any interaction between the probiotic,L. sporogenes, and the polymer blend used.


### 3.8. X-Ray Diffraction Study

In order to confirm the physical state of the probioticL. sporogenes in the beads, X-ray diffraction studies of the probioticLactobacillus sporogenes (a),L. sporogenes-containing beads (b) were carried out and the diffractograms shown in (Figures 7(a) and 7(b)).Lactobacillus sporogenes exhibited multiple characteristic sharp peaks at 2θ varying from 5° to 50° which were due to their crystalline nature. From the X-ray diffraction data ofL. sporogenes-loaded beads, it was seen that no crystalline state ofL. sporogenes was detected inL. sporogenes-loaded beads. This means thatL. sporogenes was not present in the crystalline form in the bead matrix, but in an amorphous state. This clearly indicates that changes in the crystalline state of theL. sporogenes occurred during the preparation of beads by this ionotropic gelation method.Figure 7
XRD spectra of (a)L. sporogenes and (b)L. sporogenes-loaded beads.
(a)
(b)Figure 8
Swelling behaviour of optimized sodium CMC-alginate beads containingL. sporogenes in 0.1 N HCl (pH 1.2), and phosphate buffer (pH 7.4) [mean ± S.D., n
=
3].


### 3.9. Swelling Behavior

The swelling index of ionotropically gelled optimized sodium alginate CMC beads containingL. sporogenes was found lower in 0.1 N HCl, pH 1.2 in comparison with that of in phosphate buffer, pH 6.8, initially. This occurred due to shrinkage of alginate at acidic pH. Maximum swelling of these beads was noticed at 2-3 h in phosphate buffer, pH 6.8, after which erosion and dissolution took place. It has been previously reported that the swelling of calcium alginate-based beads in presence of Ca2+ ion-capturing agent depends on the progressive displacement of Ca2+ ions within calcium alginate-based beads [21]. It has been also reported that the swelling enhanced of calcium alginate-based beads by the presence of phosphate ions (in phosphate buffer), which act as Ca2+ sequestrant [34]. Therefore, the swelling behaviour of these beads containingL. sporogenes in phosphate buffer, pH 6.8, could be explained by the ion exchanging between Ca2+ ions of the ionotropically cross-linked beads and the Na+ ions present in phosphate buffer with the influence of Ca2+ sequestrant phosphate ions. This could result in disaggregation in the sodium alginate CMC matrix structure leading to matrix erosion and dissolution of the swollen beads. This phenomenon clearly suggests that the ionotropically gelled optimized sodium alginate CMC beads containingL. sporogenes are able to swell slightly in the stomach and begin to swell more when these beads subsequently move to the upper intestine, where theL. sporogenes is to be absorbed.


### 3.10. Stability Study of Free and EncapsulatedL. sporogenes

The final challenge related to the development of probiotic-loaded microcapsules was represented by their stability during storage. When probiotics are exposed to moisture, oxygen, and heat, irreversible damage to microbial cells occurs. The stability of probiotics is particularly important for the pharmaceutical products, as they are often stored for longer periods than the dairy products [35]. Products in powder form, even capsules or tablets, generally have displayed longer stability than the liquid preparations when stored at low temperature, showing a tendency to decrease the viable count throughout the storage period. Actually, the data reported in literature [36, 37] evidence a decrease in survival of probiotic strains when stored for up to 6 months at room temperature or in the refrigerator. Anyway the decrease of the viability of the samples stored in the refrigerator is significantly lower than that of products stored at room temperature. In this research stability of probiotics bacteria in FDP and formulated in microcapsules during storage, room temperature (24–28°C) was investigated over 2 months (Figure 9). There was not much variation in beads at these temperature conditions. There were no significant changes in entrapment efficiency, physical stability, and survival of encapsulatedL. sporogenes for the selected optimized formulation F-O after 30 days and 60 days at room temperature (24–28°C). The results of stability study after two months are given in Table 5.Table 5
Results for stability study.
Time in days
Shape of beads
% probiotic entrapment efficiency
Survival  ofL. sporogenes (log CFU/g)
L. sporogenes free cell
EncapsulatedL. sporogenes beads
Initial
Round
93.59  ± 0.4
8.68 ± 0.32
8.59 ± 0.24
After 30 days
Round
92.5 ± 0.5
6.88 ± 0.30
7.85 ± 0.23
After 60 days
Round
92.09  ± 0.2
5.36 ± 0.24
7.41 ± 0.37Figure 9
Shows the stability of free and encapsulatedL. sporogenes during 60 days of storage at room temperature (24–28°C).The cell survival after 30 days was 6.88 and 7.85 of the initial log CFU in free and encapsulatedL. sporogenes, respectively. The viability ofL. sporogenes free cells showed a decrease to 5.36 log CFU after 60 days, throughout the storage period at room temperature (24–28°C), whereas viability for alginate encapsulatedL. sporogenes beads showed little decrease to 7.41 after 60 days throughout the storage period at room temperature (24–28°C).


## 3.1. Optimization

In the development of pharmaceutical dosage forms, an important and crucial issue is to search an optimal formula for designing a product of desired quality by minimum trials within a short period [22]. Optimization by means of computer-aided statistical experimental design methodologies has been successfully applied in the literature for the development of various pharmaceutical dosage forms [23, 24]. The statistical optimization technique encompasses designing a set of experiment that will reliably measure the response variables, fitting mathematical models to the data, conducting appropriate statistical tests to select best possible model, and determining the values of independent formulation variables to produce optimum response. Among various statistical design, factorial design is one of the popular experimental designs that have been employed in the formulation optimization [25, 26].Optimization of the concentrations of the formulation factors was performed to obtain probiotic microparticles with enhanced viability ofL. sporogenes and adequate release profile in different regions of the gastrointestinal tract. Although several responses were monitored to select the optimal values of the formulation factors, the main concern was given to the viability ofL. sporogenes in simulated gastric fluid. This implication is accounted on the fact that the probiotics must first survive the deleterious effect of the gastric pH in order to reach the intestine where the bioadhesive properties of the delivery system are among the key factors for achieving prolonged residence time and effective colonization of the probiotic.For the 32 factorial designs, 9 trial formulations were suggested by Design-Expert 9.0.4 software (Stat-Ease Inc., USA) for two independent variables: % amount of sodium alginate (A) and % amount of Sodium CMC (B), which were varied at three levels: low level (−1), medium level (0), and high level (+1). The % entrapment efficiency (Y
1), log CFU viability in SGF (Y
2), and % mucoadhesion over 8 hours (Y
3) were evaluated as dependent variables (responses) in the current investigation. According to this trial plan, alginate sodium CMC beads containingL. sporogenes were prepared through ionotropic gelation. Overview of matrix of the design including investigated responses is presented in Table 1.The values of investigated responses measured for all trial formulations were fitted in the 32 factorial designs to get model equations for responses analyzed in this investigation. These models were evaluated statistically by applying one-way ANOVA (p
<
0.05). The result of the ANOVA is shown in Table 2.Table 2
Summary of ANOVA for the response parameters in 32 factorial designs.
Source
Sum of squares
df
Mean square
F value
p value Prob. > F
Significance
For entrapment efficiency (%)
Model
21.62
2
10.81
99.23
<0.0001
Significant
A
19.05
1
19.05
174.83
<0.0001
B
2.57
1
2.57
23.63
0.0028
Residual
0.65
6
0.11
Cor. total
22.27
8
For viability in SGF (log10 CFU)
Model
1.13
5
0.23
12.84
0.03
Significant
A
0.06
1
0.06
3.53
0.16
B
0.30
1
0.30
17.00
0.03
A
B
0.05
1
0.05
3.08
0.18
A
2
0.08
1
0.09
4.77
0.12
B
2
0.34
1
0.34
19.40
0.02
Residual
0.05
3
0.018
Cor. total
1.18
8
For mucoadhesion (%)
Model
448.86
2
224.43
188.54
<0.0001
Significant
A
42.14
1
42.14
35.40
0.0010
B
406.73
1
406.73
341.68
<0.001
Residual
7.14
6
1.19
Cor. total
456.03
8The model equations relating various measured responses became(6)
Entrapment  Efficiency
%
Y
1
=
80.71944
+
3.56333
×
A
+
2.62000
×
B
,
Viability  in  SGF
l
o
g
10
CFU
Y
2
=
+
5.77658
+
4.28986
×
A
-
11.38256
×
B
+
0.92991
×
A
B
-
0.81776
×
A
2
+
6.59816
×
B
2
,
Mucoadhesion
%
Y
3
=
+
23.63889
+
5.30000
×
A
+
32.93333
×
B
.It can be noted that all coefficients of these above three mathematical models had statistical significance (p
<
0.05). The influences of main effects (factors) on responses investigated were further elucidated by response surface methodology. Response surface methodology has been reported as an effective tool for optimization of a dosage form to investigate the combined effect (interaction) of investigated factors on the desired responses [25–27]. In the response surface methodology, the three-dimensional response surface graph is very useful in learning about the main and interaction effects of the factors, whereas two-dimensional contour plot gives a visual representation of the response [26].


## 3.2. Effect of Formulation Variable on % Entrapment Efficiency(
Y
1
)

The three-dimensional response surface plot and corresponding two-dimensional contour plot relating entrapment efficiency are presented in Figures1(a) and 1(b), respectively. The R
2 (0.9707) was high indicating the adequate fitting of the linear model. The polynomial equations can also be used to draw conclusions considering the magnitude of coefficient and the mathematical sign it carries, that is, positive. The % entrapment efficiency of all the batches was within 90.88 to 95.55% (Table 1). It was observed that PEE (%) of these beads containingL. sporogenes was increased with the increment of sodium alginate and sodium CMC amount in the polymer blend. The increased PEE (%) with the increasing amount of sodium alginate and sodium CMC in these beads may be due to the increase in viscosity of the polymer solution with the increasing amount of polymer addition, so that it might have prevented drug leaching to the cross-linking solution. In addition, the increasing amount of sodium alginate and sodium CMC in polymer-blend might have elevated the cross-linking by CaCl2 through availing more numbers of sites for ionic cross-linking.Figure 1
Effect of formulation variable on % entrapment efficiency presented by (a) contour plots and (b) response surface plots.
(a)
(b)Among the nine batches D9 showed highest % probiotic entrapment efficiency, and this may be due to a high level of sodium alginate and sodium CMC which allows the probiotic to get entrapped more as compared to formulation containing a low level of sodium alginate and sodium CMC (Figure5).


## 3.3. Effect of Formulation Variables on Log CFU Viability ofL. sporogenes in SGF (
Y
2
)

Response surface plot and corresponding two-dimensional contour plot relating viability in SGF are presented in Figures2(a) and 2(b), respectively.Figure 2
Effect of formulation variable on viability in SGF (log CFU) presented by (a) contour plots and (b) response surface plots.
(a)
(b)TheR
2 (0.9554) was high indicating the adequate fitting of the quadratic model. The polynomial equations can also be used to draw conclusions considering the magnitude of coefficient and the mathematical sign it carries, that is, positive or negative. According to the values of regression coefficients, A, B (Table 2), the viability ofL. sporogenes in microparticles obtained was mostly affected by the sodium alginate (A), while the sodium CMC (B) concentration had the lowest influence on this response. The best response for the probiotic viability was obtained at sodium CMC concentration of 1% W/V in the concentration range of alginate 3% W/V and CaCl2 5% W/V, however, with no significant effect on the probiotic viability when increased sodium alginate concentration up to 3.5 was used. The viability ofL. sporogenes of all batches was within 8.48 to 9.34 log CFU (Table 1).In fact, microencapsulatedL. sporogenes showed that more than 108 CFU/mLL. sporogenes capsules survived after 120 min and were resistant to simulated gastric conditions. The results showed that microencapsulation in alginate significantly (p
<
0.05) improved the survival rate ofL. sporogenes.Chandramouli et al. (2004) and Iyer and Kailasapathy (2005) have shown that only the microencapsulated probiotics were able to maintain viability in gastrointestinal conditions [28, 29]. Microencapsulation of probiotics in alginate beads has previously been tested for improving viability of probiotic bacteria in simulated gastric conditions [30] although some authors have reported the effect of alginate encapsulation on survival of lactic acid bacteria in simulated gastrointestinal conditions [31–33].


## 3.4. Effect of Formulation Variables on Mucoadhesion(
Y
3
)

TheR
2 (0.9990) was high indicating the adequate fitting of the linear model.Response surface plot and corresponding two-dimensional contour plot relating mucoadhesion are presented in Figures3(a) and 3(b), respectively.Figure 3
Effect of formulation variable on mucoadhesion (%) presented by (a) contour plots and (b) response surface plots.
(a)
(b)The mucoadhesion was significantly (p
<
0.05) increased as the concentration of sodium alginate and sodium CMC increased over the range of 2.5 to 3.5% and 0.5 to 1.0%, respectively. Formulation D9 (containing concentration of sodium alginate 3.5% and sodium CMC 1%) exhibited maximum mucoadhesive strength. The result also showed that both sodium alginate and sodium CMC significantly increased the viscosity as well as the mucoadhesive property. Sodium CMC has an abundance of hydroxyl and ether groups along their length, which are responsible for the mucoadhesive properties. Increasing the concentration of the sodium CMC in the formulation increased the bonds forming groups, thus increasing the mucoadhesive force of the formulations. Mucoadhesion behavior of alginate was due to the low surface tension (31.5 mN/m) of the alginate, which is lower than the critical surface tension of the mucin (38 mN/m), resulting in good spreading and adhesion.The three-dimensional response surface graph relating to mucoadhesion (Figure3) indicates the increase in mucoadhesion observed with increasing amount of sodium CMC (A) and amount of sodium alginate (B).The influences of main effects (factors) on responses (here, % encapsulation efficiency, viability in SGF, and % mucoadhesion over 8 hours) were further elucidated by response surface methodology. Response surface methodology is a widely proficient approach in the development and optimization of drug delivery devices. The purpose of the response surface methodology is to understand as fully as possible the effect of factors and their levels in a model, over the whole of the experimental domain and to predict the response inside the domain. Moreover, it can be used for optimizing a formula (i.e., maximizing one or more of the responses, keeping the formulation variable setting within a satisfactory range), carrying out simulations with the model equations and plotting the responses. The three-dimensional response surface graph is very useful in learning about the main and interaction effects of the independent variables (factors), whereas two-dimensional contour plot gives a visual representation of values of the response. The three-dimensional response surface plots and corresponding contour plots were presented to estimate the effects of the independent variables (factors) on each response investigated.A numerical optimization technique using the desirability approach was employed to develop new formulations with desired response (optimum quality). The desirable ranges of the undependable variables (factors) were restricted to2.5
≤
A
≤
3.5% and 0.5
≤
B
≤
1.0%, whereas the desirable ranges of responses were restricted to 90
≤
Y
1
≤
95%, 8.8
≤
Y
2
≤
9.3 log CFU, and 70
≤
Y
3
≤
80%. The optimal values of responses were obtained by numerical analysis using the Design-Expert 9.0.4.1 software and one of them was selected based on the criterion of desirability. In order to evaluate the optimization capability of the mathematical models generated according to the results of full 32 factorial design, optimized sodium alginate-sodium CMC mucoadhesive beads containingL. Sporogenes were prepared by ionotropic gelation technique using these optimal process variable settings proposed by the design (Table 3). The selected optimal process variable setting used for the formulation of optimized formulation was A
=
3.27% and B
=
1.00%. Optimized sodium alginate-sodium CMC mucoadhesive beads containingL. Sporogenes (F-O) were evaluated Y
1, Y
2 and Y
3.Table 3
Results of experiments for confirming optimization capability.
(a)
Code
Factor
A (%)
B (%)
F-O
3.27
1.00
(b)
Responsea
Predicted value
Observed value
% errorb
Y
1 = entrapment efficiency (%)
95.02
93.7 ± 1.97
1.39
Y
2 = viability in SGF after 2 hr exposure (log CFU)
9.3
9.34 ± 0.3
−0.438
Y
3 = mucoadhesion over 8 hr (%)
73.81
71.75 ± 1.38
3.60
aObserved response values: mean ± S.D. (n
=
3).
bError (%) = [difference between predicted value and actual value/predicted value] × 100.Table 4
Mean diameter of calcium alginate/CMC beads containingL. sporogenes.
Formulation code
Mean diameter (mm)a
D1
1.15 ± 0.03
D2
1.3 ± 0.04
D3
1.39 ± 0.03
D4
1.17 ± 0.07
D5
1.22 ± 0.04
D6
1.41 ± 0.02
D7
1.19 ± 0.04
D8
1.24 ± 0.02
D9
1.50 ± 0.04
amean ± SD.The optimized mucoadhesive beads containingL. sporogenes (F-O) showed Y
1 of 93.7
±
1.97%, Y
2 of 9.34, and Y
3 of 71.75
±
1.38% with small percentage error values (1.26, −0.538, and 2.54, resp.). Percentage error evaluation is helpful in establishing the validity of generated model equations to describe the domain of applicability of optimization model. The percentage error values reveal that mathematical models obtained from the full 32 factorial designs were well fitted (Figure 4).Figure 4
The desirability plot: (a) indicating desirable regression ranges and the overlay plot and (b) indicating the region of optimal process variable settings.
(a)
(b)Figure 5
Depicts the scanning electron microscopy (SEM) images of dried beads ofLactobacillus sporogenes at different magnification, (a)Lactobacillus sporogenes-loaded batch D9, (b) cross section OfLactobacillus sporogenes-loaded batch D9, (c) after dissolution ofLactobacillus sporogenes-loaded batch D9, and (d) cross section of after dissolutionLactobacillus sporogenes-loaded batch (D9).
(a)
Lactobacillus sporogenes-loaded beads batch F-O
(b)
Cross section ofLactobacillus sporogenes-loaded optimized batch
(c)
After dissolution of optimized batch
(d)
Cross section of after dissolutionLactobacillus sporogenes optimized batch


## 3.5. Bead Size

The average size of the formulated dried beads containingLactobacillus sporogenes ranged from 1.15
±
0.03 to 1.50
±
0.04 mm.Increasing bead size was found with increasing amount of sodium alginate and sodium CMC. This could be attributed to increase in viscosity of the polymer blend (sodium alginate and sodium CMC) solution with incorporation of both the polymers in increasing ratio that in turn increased the droplet size during addition of the polymer blend solution to the cross-linking solution.


## 3.6. Surface Morphology Analysis

It can be inferred from the images that beads obtained were spherical in shape with smooth surfaces. The complete roughness of beads was observed under higher magnification. Reduction of beads’ size after dissolution was observed, soL. sporogenes might be released from the surface and gel matrix of sodium alginate. It might be due to erosion of beads during dissolution.


## 3.7. FTIR Spectroscopy

The FTIR spectra of probioticL. sporogenes sodium alginate, sodium CMC, BioEcolian, and optimized ionotropically gelled sodium CMC-alginate beads containingL. sporogenes are shown in Figure 6.Figure 6
The FTIR spectra ofL. sporogenes (a), sodium alginate (b), sodium CMC (c), BioEcolian (d), and sodium alginate sodium CMC beads containingL. sporogenes (F-O) (e).In the FT-IR spectrum of sodium alginate powder, the various distinct peaks of alginate evident are those of the hydroxyl group at 3429 cm−1, carbonyl at 1614 cm−1, and carboxyl and carboxylate groups between 1000 and 1400 cm−1. The absorption band around 2880, 1656, 1421, and 1080 cm−1 corresponds to the stretching of -CH, COO-, -CH, and C-O-C, respectively.The IR spectra of sodium carboxy methyl cellulose exhibited presence of characteristic absorption bands corresponding to asymmetric and symmetric carboxylate anions at, respectively, 1609 cm−1 and 1426 cm−1, a broad band at 3412 cm−1 corresponding to stretching vibration of hydroxyl group, and a peak at 1059 cm−1 corresponding to alkyl substituted ether. The FTIR spectra of BioEcolian (GOS) showed characteristic peaks around 3500–3200 cm−1 for OH stretching, 2147 cm−1 for CH stretching, and 1650 cm−1 for CO stretching, 1025 cm−1.In the FTIR spectrum of optimized ionotropically gelled sodium CMC-alginate beads containingL. sporogenes, various characteristic peaks of sodium alginate, sodium CMC, andL. sporogenes appeared without any significant shifting of these peaks. In short, the sodium CMC–alginate beads containingL. sporogenes prepared with sodium CMC-alginate polymer-blend had significant characters ofL. sporogenes in the FTIR spectrum, suggesting absence of any interaction between the probiotic,L. sporogenes, and the polymer blend used.


## 3.8. X-Ray Diffraction Study

In order to confirm the physical state of the probioticL. sporogenes in the beads, X-ray diffraction studies of the probioticLactobacillus sporogenes (a),L. sporogenes-containing beads (b) were carried out and the diffractograms shown in (Figures 7(a) and 7(b)).Lactobacillus sporogenes exhibited multiple characteristic sharp peaks at 2θ varying from 5° to 50° which were due to their crystalline nature. From the X-ray diffraction data ofL. sporogenes-loaded beads, it was seen that no crystalline state ofL. sporogenes was detected inL. sporogenes-loaded beads. This means thatL. sporogenes was not present in the crystalline form in the bead matrix, but in an amorphous state. This clearly indicates that changes in the crystalline state of theL. sporogenes occurred during the preparation of beads by this ionotropic gelation method.Figure 7
XRD spectra of (a)L. sporogenes and (b)L. sporogenes-loaded beads.
(a)
(b)Figure 8
Swelling behaviour of optimized sodium CMC-alginate beads containingL. sporogenes in 0.1 N HCl (pH 1.2), and phosphate buffer (pH 7.4) [mean ± S.D., n
=
3].


## 3.9. Swelling Behavior

The swelling index of ionotropically gelled optimized sodium alginate CMC beads containingL. sporogenes was found lower in 0.1 N HCl, pH 1.2 in comparison with that of in phosphate buffer, pH 6.8, initially. This occurred due to shrinkage of alginate at acidic pH. Maximum swelling of these beads was noticed at 2-3 h in phosphate buffer, pH 6.8, after which erosion and dissolution took place. It has been previously reported that the swelling of calcium alginate-based beads in presence of Ca2+ ion-capturing agent depends on the progressive displacement of Ca2+ ions within calcium alginate-based beads [21]. It has been also reported that the swelling enhanced of calcium alginate-based beads by the presence of phosphate ions (in phosphate buffer), which act as Ca2+ sequestrant [34]. Therefore, the swelling behaviour of these beads containingL. sporogenes in phosphate buffer, pH 6.8, could be explained by the ion exchanging between Ca2+ ions of the ionotropically cross-linked beads and the Na+ ions present in phosphate buffer with the influence of Ca2+ sequestrant phosphate ions. This could result in disaggregation in the sodium alginate CMC matrix structure leading to matrix erosion and dissolution of the swollen beads. This phenomenon clearly suggests that the ionotropically gelled optimized sodium alginate CMC beads containingL. sporogenes are able to swell slightly in the stomach and begin to swell more when these beads subsequently move to the upper intestine, where theL. sporogenes is to be absorbed.


## 3.10. Stability Study of Free and EncapsulatedL. sporogenes

The final challenge related to the development of probiotic-loaded microcapsules was represented by their stability during storage. When probiotics are exposed to moisture, oxygen, and heat, irreversible damage to microbial cells occurs. The stability of probiotics is particularly important for the pharmaceutical products, as they are often stored for longer periods than the dairy products [35]. Products in powder form, even capsules or tablets, generally have displayed longer stability than the liquid preparations when stored at low temperature, showing a tendency to decrease the viable count throughout the storage period. Actually, the data reported in literature [36, 37] evidence a decrease in survival of probiotic strains when stored for up to 6 months at room temperature or in the refrigerator. Anyway the decrease of the viability of the samples stored in the refrigerator is significantly lower than that of products stored at room temperature. In this research stability of probiotics bacteria in FDP and formulated in microcapsules during storage, room temperature (24–28°C) was investigated over 2 months (Figure 9). There was not much variation in beads at these temperature conditions. There were no significant changes in entrapment efficiency, physical stability, and survival of encapsulatedL. sporogenes for the selected optimized formulation F-O after 30 days and 60 days at room temperature (24–28°C). The results of stability study after two months are given in Table 5.Table 5
Results for stability study.
Time in days
Shape of beads
% probiotic entrapment efficiency
Survival  ofL. sporogenes (log CFU/g)
L. sporogenes free cell
EncapsulatedL. sporogenes beads
Initial
Round
93.59  ± 0.4
8.68 ± 0.32
8.59 ± 0.24
After 30 days
Round
92.5 ± 0.5
6.88 ± 0.30
7.85 ± 0.23
After 60 days
Round
92.09  ± 0.2
5.36 ± 0.24
7.41 ± 0.37Figure 9
Shows the stability of free and encapsulatedL. sporogenes during 60 days of storage at room temperature (24–28°C).The cell survival after 30 days was 6.88 and 7.85 of the initial log CFU in free and encapsulatedL. sporogenes, respectively. The viability ofL. sporogenes free cells showed a decrease to 5.36 log CFU after 60 days, throughout the storage period at room temperature (24–28°C), whereas viability for alginate encapsulatedL. sporogenes beads showed little decrease to 7.41 after 60 days throughout the storage period at room temperature (24–28°C).


## 4. Conclusion

In this investigation, ionotropically gelled carboxymethylcellulose (CMC) Ca-alginate mucoadhesive beads containingL. sporogenes were successfully developed and optimized using 32 factorial designs. The primary response, viability of the encapsulatedL. sporogenes in simulated gastric conditions, pointed to the formulation factor with the lowest influence, that is, sodium alginate and sodium CMC. In this respect and in conjunction with the other experimental responses as well, it was concluded that the optimal formulation ofL. sporogenes-loaded carboxymethylcellulose-Ca-alginate microparticles should be prepared using 3.27% w/v sodium alginate, 1.00% w/v sodium CMC, and 5% w/v CaCl2. The optimized beads demonstrated goodp entrapment efficiency, good mucoadhesivity with the biological membrane, and suitable controlled probiotic release pattern over prolonged period, which could possibly be advantageous in terms of advanced patient compliance with reduced dosing interval. The technique for the preparation of CMC-calcium alginate beads containingL. sporogenes was found to be simple, reproducible, easily controllable, economical, and consistent. Besides, the excipients such as sodium alginate, sodium CMC, and calcium chloride used for the formulation of these beads were cheap and easily available.



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*Source: 6041671-2016-01-17.xml*