Entrapment of Lactobacillus acidophilus into alginate beads for the effective treatment of cold restraint stress induced gastric ulcer

Abstract

Lactobacillus acidophilus (LAB) loaded alginate floating beads (FBs) were developed with an intent to (i) preserve their viability during manufacture and upon exposure to adverse physiological conditions existing in the stomach, (ii) achieve an increased stay of the system in the stomach for improved pharmacodynamics and to provide for their effective establishment within the gastric mucosa. In vitro characterization of developed beads was performed in terms of entrapment efficiency, buoyancy, and surface as well as cross sectional morphology and viability studies of LAB in a gastric environment. The developed system was evaluated and was found to be significantly better in an experimental model of cold restraint stress (CRS) induced gastric ulcer model in terms of ulcer index, hemorrhagic streak length, histopathological and biochemical markers and their cross talk with reactive oxygen/nitrogen species. The present study emphasizes the advantages and future potential of probiotic loaded FBs in gastric disorders.

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1. Introduction

Probiotics, meaning “life” are rapidly becoming a popular and important tool for preserving our natural health. They have been defined in several ways, depending on our understanding of the mechanism(s) of action of their effects on the health and well-being of humans. The most commonly used definition is that of Fuller:¹ “Probiotics are live microbial feed supplements that beneficially affect the host by improving its intestinal microbial balance.” Lactobacillus and Bifidobacterium species constitute a significant proportion of probiotic cultures used in developed countries that may undergo antagonistic interactions with pathogenic bacteria.²

Mucosal inflammations and alterations in gut microbiota are often refractive to conventional treatments involving the employment of anti-inflammatory and immunosuppressant drugs and this has led to a search for alternative therapies based on the use of probiotics.^3,4

A mechanistic study suggests that probiotics influence the protein expression in the stomach wall cells, leading to an increase in the formation of new blood cells and increased healing of the ulcer.⁵ In addition, probiotics form short chain fatty acids which serve as important nutrients for the mucosal cells and also help to improve the blood supply to the gut wall.⁶Lactobacillus also stimulates certain cells of the immune system, which improve the body defense mechanism. Epidemiologic and experimental studies suggest that the consumption of fermented milk products and lactic acid bacteria (LAB) decrease the incidence of various gastrointestinal disorders. In the last few decades the use of probiotic bacteria has gained considerable attention as a safe and accessible form of treatment for gastrointestinal diseases.^7,8

Use of probiotics in clinical or supplemental therapy is however limited, because:

1. Most probiotics are fastidious, noncompetitive, and sensitive to their environment, hence repetitive large doses of probiotic are required⁹ to elicit physiological effect; and the effect is observed only for the time for which the probiotic is being administered.

2. Orally administered probiotics during their passage from the mouth to the intestine face adverse physiological conditions, like acidic pH, mechanical stresses, digestive enzymes, and bile acids, limiting their establishment in the gut mucosa.

3. Stability of the probiotic is another parameter of concern during their manufacture and storage.

To act as probiotics, the bacteria must arrive in the intestine alive and in sufficient number. Further, a local therapeutic action with a long duration is desirable.¹⁰ The significance of survival of probiotics in the GI-tract, their translocation and colonization and the fate of probiotic derived active components indicate a need and scope of packaging them into a suitable delivery system to increase the viability of the probiotics, both outside and inside the body.¹¹

A floating drug delivery system (FDDS) forms the most promising delivery system for local gastric effects. It ensures a prolonged and continuous release of the probiotic in the stomach allowing sufficient time for its adhesion and establishment onto the gastric mucosa.¹² In the light of these considerations, the main objective of our study was to encapsulate the LAB in FBs by orifice ionic gelation method and then to evaluate the survival upon encapsulation and of encapsulated cells under gastrointestinal condition (pH 1.2). Developed system was evaluated in experimental model of cold restraint stress induced gastric ulcer in terms of ulcer index; hemorrhagic streak length; oxido-nitrosative stress; mucus content and histopathological examination.

2. Materials and methods

2.1 Materials

Probiotic (Lactobacillus acidophilus) was procured as a gift sample from Ranbaxy Gurgaon, India (NLT 200 Billion CFU g⁻¹). All other chemicals or reagents used in the study were of AR or GR grade.

2.2 Establishment of cell count of the probiotics used

An exactly weighed quantity of probiotic (10 mg) was suspended in 0.1% peptone water and hydrated for 30 min. The suspension was vortexed and serially diluted with peptone water to obtain 2000–4000 colony forming units (CFU) ml⁻¹. Accurately measured 0.1 ml of this dilution was mixed with 30 ml of sterile De Man Rogosa Sharpe (MRS-agar) media and plated by the pour plate method (n = 8). The plates were incubated anaerobically at 37 °C for 48 h and the colonies thus formed were counted.

The extent of viability of the probiotic after 6 h of anaerobic incubation in (i) simulated gastric fluid (SGF; pH 1.2); (ii) triple distilled water and (iii) peptone water (0.1% v/v) was also confirmed, as explained above.

2.3 Preparation of Lactobacillus acidophilus loaded calcium alginate floating beads

Calcium alginate beads were prepared by the orifice ionic gelation method.¹³ The measured amount (20 mg probiotic) of the probiotic was suspended in water. The solution was dispersed in sodium alginate solution (3% w/v) containing HPMC (alginate: HPMC = 9 : 1 w/w). The gas forming agent calcium carbonate was added to the solution in weight ratio ranging from 0.25 : 1 to 0.75 : 1 (gas forming agent: alginate w/w). The mixture was degassed under bath sonicator (20–30 min) to remove any entrapped air. The resulting solution was dropped through a 26 G syringe needle into 1% w/v calcium chloride solution containing 10% v/v acetic acid. The solution containing suspended beads was allowed to stir for 1 h to improve mechanical strength and for completion of the reaction to produce gas at room temperature. Carbonate salts are insoluble at neutral pH while its cation (calcium ion) is released in the presence of acid. The beads were prepared aseptically using sterile solutions, vehicles and container. The formed beads were separated and freeze-dried overnight using freeze dryer maintained at a temperature of −40 °C. Product was lyophilized further for 6 h at −70 °C. Final weight of the formed beads was noted and 2.11 ± 1.23 g of beads were formed when 20 mg of free probiotic was loaded in to these beads (n = 6).

2.4 Characterization and evaluation of floating beads

2.4.1 Determination of particle size. The particle size of calcium alginate beads was determined using particle size analyzer (Malvern instruments limited, Malvern UK). The determinations were made in triplicate and mean diameters were recorded.

2.4.2 Drug entrapment efficiency (DEE) of probiotic floating beads. The measured amount of beads were triturated in a sterile mortar using a small quantity of sterile peptone water and making the final volume up to 10 ml under aseptic conditions. Appropriate serial dilutions were prepared, which were plated on MRS-agar using the pour plate method. The plates were incubated anaerobically at 37 °C for 48 h and the number of colonies formed was counted. Thereafter % entrapment was calculated using the formula:

2.4.3 Determination of buoyancy. The floating ability of the beads was determined using USP type II dissolution test apparatus. Fifty beads were placed in the vessel containing 500 ml of SGF dissolution media maintained at 37 ± 0.5 °C and stirred at 100 rpm¹⁴ for 24 h. The number of beads settling down after 24 h was measured by visual observation and the percentage of beads that remain floating was determined.

2.4.4 Determination of porosity. Beads were filled in a 10 ml graduated measuring cylinder up to the mark. Then the cylinder was tapped 500 times and the volume was noted. Initial volume or the bulk volume was 10 ml in all cases and the final volume gave the tap volume of the beads.¹⁵ All determinations were made in triplicate. The porosity was calculated according to the following equation and mean % porosity and standard deviation were recorded.

V _b = Bulk volume of the beads = 10 ml, V_p = True/Tap volume of the beads, V = v = V_b − V_p = void volume of the particles (spaces between the particles)

2.4.5 Surface and internal morphology by scanning electron microscopy (SEM). The external and internal morphology of the freeze-dried FBs were studied by scanning electron microscopy. Samples were coated with gold film under vacuum to modify the conducting materials and investigated.

The internal morphology of the beads was examined by cutting them in half with a steel blade.

2.4.6 Viability study of probiotic loaded floating beads in SGF. Viability of probiotic entrapped within the beads was carried out in SGF under aseptic conditions. A measured amount of beads/free probiotic was incubated anaerobically at 37 °C in test tubes containing 10 ml of SGF. At intervals of 2, 4, and 6 h of incubation, all the beads from tubes at each time point were removed, washed with peptone water and immediately assayed for cell count, Tubes containing free probiotic were centrifuged at respective time points and pellets from each tube were used for cell enumeration after suitable dilution. Supernatant from each tube of probiotic FBs was also analyzed for cell count at respective time points for determining the release of probiotic from the developed beads in SGF.

2.5 In vivo studies

2.5.1 Cold restraint stress (CRS) induced gastric ulcers. Female Wistar rats, not more than 250 g, bred in the Central animal house, Panjab University, Chandigarh, India were used. Animals caged together and kept under natural light/dark cycle, were given food and waterad libitum. They were deprived of food but were allowed free access to water 24 h before the start of experiment. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC), Panjab University, Chandigarh, India (reference no. 972/DUI/PU; dated 08 Feb 2010). Animals were divided into nine groups; each group consisting of 5 animals. Group I comprised of naive control (24 h fasted animals). The remaining 40 animals were immobilized by strapping the fore and hind limbs on a wooden plank and kept for 3 h, at a temperature of 4 ± 1 °C.¹⁶

Further, the animals were divided into groups II–IX (Table 1). Groups II and III constituted CRS control groups, Groups IV and V were the CRS group that received free probiotic (10⁷ CFU per oral, suspended in 1% carboxy methyl cellulose), groups VI and VII were the CRS group that received cimetidine (10 mg kg⁻¹) orally, and groups VIII and IX constituted of CRS groups receiving probiotic FBs (equivalent to 10⁷ CFU per oral, suspended in 1% carboxy methyl cellulose). All animals were sacrificed by cervical dislocation, their stomach was isolated and cut along the longitudinal axis, washed with ice cold saline and assessed for ulcers and hemorrhagic streaks. Suitable portions of stomach were preserved in formalin solution for histopathological examination and the remaining portion were used for mucus content and oxido-nitrosative stress determination. Ulcer index was calculated by adding the total number of ulcers plus severity of ulcers. Severity of ulceration was judged based on the scale¹⁷ at two different time points of 2 h and 10 h post CRS and administration of a suitable treatment. The sum of the respective lengths of various hemorrhagic streaks (l) was also measured and used as another parameter for assessing the extent of ulcers. Groups II, IV, VI and VIII constituted the 2 h data points while groups III, V, VII and IX were sacrificed 10 h after CRS induction and suitable treatment. Isolated stomach from the sacrificed animals were cut along the longitudinal axis, washed with ice cold saline and observed for ulcers and hemorrhagic streaks length (l).

Table 1 Treatment schedule of different groups in CRS study at two different time points of sacrifice

Time of sacrifice	Group No.	Treatment received
2 h	I	Naive Control
	II	CRS
	IV	CRS + Free Probiotic (10^7 CFU)
	VI	CRS + Cimetidine (10 mg kg^−1)
	VIII	CRS + FBs-PB (Equivalent to 10^7 CFU)
10 h	III	CRS
	V	CRS + Free Probiotic (10^7 CFU)
	VII	CRS + Cimetidine (10 mg kg^−1)
	IX	CRS + FBs-PB (Equivalent to 10^7 CFU)

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2.5.2 Oxido-nitrosative stress in stomach homogenates. Removed stomachs were rinsed with ice cold saline and weighed. A 10% w/v stomach homogenate was prepared in 0.1 M phosphate buffer saline (pH 7.4), which was further used for lipid peroxidation,¹⁸catalase,¹⁹superoxide dismutase assay,²⁰protein estimation²¹ and measurement of nitrosative stress.²²

2.5.3 Histopathological examination. Stomachs were opened along the greater curvature, and quickly fixed in buffered formalin solution for histopathological examination of gastric lesions.

2.5.4 Estimation of gastric wall mucus. Gastric wall mucus was determined according to the method of ref. 23. The glandular segments from stomachs were scraped with a blunt spatula, weighed and incubated in tubes containing 0.1% alcian blue solution (0.16 M sucrose in 0.05 M sodium acetate, pH 5.8) for 2 h. The alcian blue binding extract was centrifuged and the absorbance of supernatant was measured at 598 nm. The quantity of alcian blue extracted (μg g⁻¹ of glandular tissue) was then calculated by using molecular extinction coefficient of alcian blue (E^1%_1cm140) determined experimentally.

2.6 Statistical analysis

Raw data obtained from in vitro studies is expressed as mean ± S.D (standard deviation).

The in vivo results are expressed as mean ± SEM (standard error of mean). The intergroup variation was measured by one-way analysis of variance (ANOVA) followed by Tukey's test. Statistical significance was considered at p < 0.05.

3. Results

3.1 Establishment of cell count of the probiotics used

Cell count of the probiotic used was confirmed by pour plate method and the results were compared with the labeled values of the received sample. Viable count determinations were repeated eight times (n = 8) and the mean viable count was found to be (133 ± 12) ×10⁶CFU mg⁻¹. The clamed count was 200 × 10⁶CFU mg⁻¹.

3.2 Viability of probiotic in different media after 6 h incubation

Purpose of the experiment was to observe the adverse effect of the gastric pH on the viability of the probiotic bacteria used in the study. After 6 h of an anaerobic incubation viable count in peptone water was (701 × 10⁵ CFU mg⁻¹) more than that in triple distilled water (629 × 10⁵ CFU mg⁻¹) and SGF. A log 2 times reduction of viable count was observed in SGF (358 × 10³CFU mg⁻¹), as compared to the peptone water, due to the highly acidic nature of the medium, which can kill the probiotic bacteria.

3.3 Characterization and evaluation of floating beads

3.3.1 Particle size. The average particle size of the probiotic FBs was invariably between 1.02–1.08 mm (Table 2). Incorporation of varying agents or varying the ratio of calcium carbonate to alginate did not result in any significant difference in particle size. Arithmetic increase in particle size with an increase in the amount of gas forming agent was however observed. Further the particle size for B₃ batch ratio (1: 0.75: sodium alginate: CaCO₃) was significantly larger (p < 0.05) than those for B₁ batch (1: 0.25: sodium alginate: CaCO₃). This may be due to the incorporation of more pores by the higher proportion of gas forming calcium carbonate.

Table 2 Characterization and in vitro evaluation of different batches of Lactobacillus acidophilus loaded floating beads (n = 3)

Batch	Polymer : CaCO3	Particle size/(mm)	Buoyancy (%)	Porosity (%)	DEE^a (%)
a n = 5.
B1	1 : 0.25	1.02 ± 0.11	47.33 ± 0.67	68.67 ± 2.03	74.37 ± 4.21
B2	1 : 0.50	1.04 ± 0.020	80.00 ± 1.15	76.33 ± 1.45	88.13 ± 3.11
B3	1 : 0.75	1.08 ± 0.015	88.00 ± 1.15	85.00 ± 2.08	75.00 ± 1.38

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3.3.2 Buoyancy. Floating ability of the prepared beads was evaluated in SGF pH 1.2. An increase in buoyancy from 47.33 to 88.00% was observed, when weight ratio of calcium carbonate to alginate was increased from B₁ batch to B₃ batch (Table 2).

3.3.3 Drug entrapment efficiency (DEE). Entrapment efficiency for various formulations was found to vary from 74.37% to 88.13% with the B₂ batch showing significantly (p < 0.05) higher entrapment (Table 2).

3.3.4 Porosity. An increase in porosity of FBs was observed from 68.67% to 85.00% when the weight ratio of calcium carbonate to alginate was increased from 0.25 : 1 to 0.75 : 1 (Table 2). Increasing the amount of gas forming agent increases the production and entrapment of CO₂ within the beads thus increasing their tap volume.

3.3.5 Surface and cross sectional characterization by scanning electron microscopy (SEM). The surface and cross sectional pictures of probiotic loaded FBs are shown the Fig. 1. Probiotic loaded FBs show wrinkled surface due to the released carbon-dioxide from the surface of beads (Fig. 1a). The closer views of the surface of the beads show growth of LAB along the edges (Fig. 1b). The cross sectional views of the beads revealed many closed channel or pores (Fig. 1c).

Fig. 1 (a) Surface picture of probiotic loaded beads; (b) Closer views (higher magnification) of probiotic loaded FBs showed rod shaped LAB; (c) a cross sectional picture of probiotic loaded beads.

3.3.6 Viability study of probiotic FBs in SGF. Results (Fig. 2a) show the decrease in bacterial count after exposure of free probiotic or probiotic beads to SGF at 37 °C at different time points. However, the rate constant of death/kill at all time points was significantly less for the probiotic loaded into beads as compared to the free probiotics (Table 3). This confirmed that encapsulation of probiotic bacteria within FBs protected them from the harsh acidic conditions in gastric fluids. Results show the superiority of the FBs system for maintaining the viability of LAB in gastric environment.

Where ‘K’ represents rate constant of death/kill, ‘a’ initial number of bacteria in the medium and ‘(a − x)’ the number of organisms in the same volume after exposure for time ‘t’.

Table 3 Rate constant of death/kill of free probiotic and probiotic loaded FBs in SGF (n = 4)^a

Time (h)	K value
	Free probiotic	Probiotic floating beads
a All values are significantly (p < 0.05) different at each time point.
2	−0.1135	−0.2147
4	0.1072	−0.0857
6	0.3766	0.0033

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Fig. 2 (a) % Survival of bacteria after exposure to SGF at 37 °C at different time intervals (n = 4) and (b) Viable counts of probiotic loaded FBs in SGF at 37 °C at different time intervals (n = 4).

Fig. 2b shows the release pattern of probiotic from the developed FBs.

3.4 In vivo studies

Studies were performed for two time points of 2 h and 10 h, representing the normal gastric transit time of 1.5 to 2 h, which will coincide for free probiotic, while the 10 h time point signifies a prolonged stay being achieved with FBs. Opening the stomach at different time points post administration of probiotic FBs, it could be observed that significant beads (22%) were retained with in the stomach even at 10 h.

3.4.1 Ulcer index and hemorrhagic streak length. A significant ulcer index of 17.6 ± 3 was observed in the CRS groups (Fig. 3a and 3b). Treatment of CRS rats with cimetidine, free probiotic, and FBs of probiotic significantly reduced the number as well as the extent of gastric mucosal ulcers (Fig. 3a and 3b). Probiotic loaded FBs showed significantly better reduction as compared to free probiotic group and cimetidine treated group at 10 h interval. A sustained release of probiotic from FBs close to the gastric mucosa for prolonged times may allow its adherence to the gastric mucosa, giving probiotics enough time and space to colonize thus showing a significantly better effect.

Fig. 3 Effect of free probiotic, cimetidine and probiotic loaded FBs in CRS model of gastric ulcer in rat. (a) ulcer index and (b) hemorrhagic streak length. ^ap < 0.05 as compared to group I, ^bp < 0.05 as compared to group II and III, ^cp < 0.05 as compared to group V, ^dp < 0.05 as compared to group VII.

3.4.2 Histopathological examination. Histopathological study of gastric mucosa was performed after staining with haematoxylin and eosin at the end of the 10 h study. Fig. 4 (a) shows normal healthy mucosa of naive control animal with the mucosal glands maintaining their identity. The CRS induced group showed mucosal hyperemia and hemorrhagic necrotic lesions with edema covering the entire glandular area of the stomach (outlined), indicating acute ulceration, as shown in Fig. 4b. In addition, gastric mucosal damage with dilation and exfoliation of gastric epithelial cells and disruption of the mucosal layer was observed and the glands were also found to lose their identity. After treatment with free probiotic, the mucosa showed slight recovery but a significant mucosal damage was still obvious, coupled with inflammation inside the mucosal cells (Fig. 4c). The cimetidine (10 mg kg⁻¹) treated group showed better recovery of gastric mucosa as compared to the free probiotic treated group, with most of the mucosa regaining its identity except the edges, as shown in Fig. 4d. The probiotic FBs treated group, except for some minor inflammation, showed better recovery than cimetidine and free probiotic. Most of the mucosa regained its identity and maintained its architecture, confirming better anti-ulcerative effect of probiotic FBs, as shown in Fig. 4e.

Fig. 4 Histological micrographs of rat stomachs at the end of 10 h study in CRS model of gastric ulcer in rat. (a) Naive control, (b) CRS, (c) free probiotic treated, (d) cimetidine treated, (e) probiotic loaded FBs treated.

3.4.3 Mucus content determination. Treatment with the free probiotic, cimetidine, and probiotic loaded FBs significantly (p < 0.05) attenuated the reduction in mucus levels. Probiotics are reported to stimulate the mucus secretion and increase transmucosal resistance in gastric mucosa.⁵ Further, the elevation of mucus levels was significantly more in the probiotic loaded FBs treated group as compared to the free probiotic treated group (Fig. 5).

Fig. 5 The effect of free probiotic, cimetidine and probiotic loaded FBs on mucosal content in a CRS model of gastric ulcer in rat. Group I: Naive control; Group II and Group III: CRS (sacrificed 2 h and 10 h after CRS induction); Group IV and Group V: CRS induced groups received free probiotic (10⁷ CFU) (sacrificed 2 h and 10 h after CRS induction); Group VI and Group VII: CRS induced groups received cimetidine (10 mg kg⁻¹ body weight) (sacrificed 2 h and 10 h after CRS induction); Group VIII and Group IX: CRS induced groups received probiotic loaded FBs (equivalent to 10⁷ CFU) (sacrificed 2 h and 10 h after CRS induction).^ap < 0.05 as compared to group I, ^bp < 0.05 as compared to group II and III, ^cp < 0.05 as compared to group V, ^dp < 0.05 as compared to group VII.

3.4.4 Oxido-nitrosative stress. We determined LPO, catalase, SOD and nitrite levels, for the free probiotic, cimetidine, and the probiotic loaded FBs and CRS induced rat stomach homogenates, as the markers of oxido-nitrosative stress and compared with the values obtained for the naive control.

The elevated TBARS levels were attenuated upon treatment with probiotic, cimetidine, and also with the FBs of probiotic. Very interesting observation was a lack of significanct difference between naive and FBs treated group i.e LPO levels return to the normal naive level (Fig. 6a) upon treatment with probiotic FBs.

Fig. 6 The effect of free probiotic, cimetidine and probiotic loaded FBs on lipid peroxides (a), superoxide dismutase (b), catalase levels (c) and nitrite levels (d) in CRS model of gastric ulcer in rat. Group I: Naive control; Group II and Group III: CRS (sacrificed 2 h and 10 h after CRS induction); Group IV and Group V: CRS induced groups received free probiotic (10⁷ CFU) (sacrificed 2 h and 10 h after CRS induction); Group VI and Group VII: CRS induced groups received cimetidine (10 mg kg⁻¹ body weight) (sacrificed 2 h and 10 h after CRS induction); Group VIII and Group IX: CRS induced groups received probiotic loaded FBs (equivalent to 10⁷ CFU) (sacrificed 2 h and 10 h after CRS induction).^ap < 0.05 as compared to group I, ^bp < 0.05 as compared to group II and III, ^cp < 0.05 as compared to group V, ^dp < 0.05 as compared to group VII.

Similarly, probiotic loaded beads were significantly (p < 0.05) more effective than cimetidine and free probiotic in restoring the SOD activity (Fig. 6b). A significant (p < 0.05) decrease in catalase activity was observed in the CRS treated group as compared to naive control. Administration of cimetidine, probiotic and probiotic loaded FBs significantly increased the catalase levels (Fig. 6c). A significant increase in nitrite levels was, however, observed in the CRS group as compared to the naive control. Significantly higher nitrite levels in CRS induced group suggest their involvement in the pathogenesis of stress induced ulcers. A similar increase in the serum nitrite levels has been reported by Demirbilek et al. in CRS rats.²⁴ The administration of free probiotic and cimetidine lower nitrite levels but probiotic loaded FBs produced a significant effect (Fig. 6d).

4. Discussion

The present study emphasizes the advantages and future potential of probiotic loaded beads in the treatment of gastrointestinal disorders. We developed probiotic loaded FBs for post-induction protective effect against CRS induced gastric ulcers and to assess the effects of formulation variables on bead characteristics, entrapment and viability in SGF. The study indicated that 0.50 : 1 w/w ratio of calcium carbonate and sodium alginate yields beads with a significantly better DEE as compared to the other two formulations (B₁ and B₃). Internal ionotropic gelation effect of divalent Ca²⁺ of calcium carbonate on alginate results in stronger gels, such that the developed beads show significant entrapment.²⁰ As the concentration of gas forming agent (calcium carbonate) increases, the entrapment efficiency increases; however, a high proportion of gas-forming agent (B₃ batch) can make the beads highly porous and fragile¹³ due to which the beads are unable to retain the drug efficiently. Thus, when concentration of calcium carbonate was increased from 0.50 : 1.00 to 0.75 : 1.00 (calcium carbonate : alginate), a decrease in entrapment efficiency was observed (Table 2).

The porosity and floating properties of the beads were increased with increase in the gas content of the polymer matrix. This could be due to the increasing quantity of the gas forming agent (CaCO₃) used in their formulation, which would result in an increase in pore size as well as in the number of pores/area of the formulated FBs, as is apparent from scanning electron microscope pictures of the cross sections of respective FBs. Chemical reaction between calcium carbonate and acetic acid results in the release of CO₂. During the formation of beads calcium carbonate effervesces, releasing carbon dioxide, which is entrapped in the gel network (HPMC-alginate), producing a formulation that remains buoyant for prolonged periods. So the higher the calcium carbonate quantity, the more CO₂ that will be produced, such that more and/or larger pores will be formed. As expected, the % viability of free probiotic was significantly less compared to that loaded into FBs (Fig. 2a), thus confirming that encapsulation of probiotic bacteria within FBs protects them from the harsh acidic conditions in gastric fluids. The viability of probiotics is reported to be influenced strongly by their physiological and chemical environment.^25,26 Even though LAB is reported to tolerate the acidic pH of the stomach,²⁷ yet there was a considerable loss of viability in SGF at 6 h (based on the fed state of stomach the transit time can be up to 6 h).

CRS induces gastric mucosal damages and the possible reasons assigned for the same are:

(i) lipid peroxidation, oxidation of some critical cellular proteins, and depletion of antioxidants, indicating production of ROS during gastric ulceration; (ii) activation of superoxide dismutase (SOD), which in turn favors endogenous accumulation of hydrogen peroxide; (iii) generation of oxygen ion at an enhanced rate during stress, as evidenced by increased SOD activity; (iv) transition metal ions play an important role in the generation of stress-ulcer; and (v) the hydroxyl ion is generated at a higher rate from an oxygen ion and hydrogen peroxide through the metal-catalyzed Haber–Weiss reaction and accounts for the major oxidative damage in stress-induced gastric ulceration.²⁸ Cold stress leads to a significant decrease in mucus content²⁹ and an increase in the prostaglandin levels³⁰ of rat stomach. Gastric mucus is an important protective factor for the gastric mucosa. Moreover, mucus is capable of acting as antioxidant and thus can reduce the mucosal damage mediated by oxygen free radicals. Mucosal damage increases gut permeability to macromolecules and facilitates the translocation of noxious materials such as carcinogens, endotoxin and other bacterial toxins to the bloodstream.³¹

The changes in LPO, catalase, SOD, nitrite, mucus content levels, and ulcer index, as well as hemorrhagic streaks induced by stress, were attenuated to normal values by probiotic loaded FBs. The beads seem to be adhering to the gastric mucosa, which may be due to the use of HPMC/sodium alginate in the preparation of FBs; both of these agents are reported to be mucoadhesive.³² A sustained release of probiotic from FBs close to the gastric mucosa for prolonged times may allow its adherence to the gastric mucosa, giving probiotics enough time and space to colonize, thus showing a significantly better effect. Histopathological studies also indicate that most of the mucosa regains its identity and its architecture is also maintained upon treatment with probiotic FBs, confirming the better anti-ulcerative effect of the latter.

5. Conclusion

The developed FBs not only efficiently protect the entrapped probiotic cells, but also help effectively deliver and retain viable bacteria in the stomach. This while ensuring a prolonged and continuous release of the probiotic in the gastric mucosa allows them to wade over the adverse gastric conditions. Pharmacodynamic results suggest the involvement of oxidative-nitrosative stress in CRS induced gastric ulcer. Treatments with FBs of probiotic confirm its therapeutic effectiveness against gastric ulcer post induction probably attributed to its antioxidant, regeneration of mucosal cells, and immunological/anti-inflammatory response. The present study emphasizes the advantages and future potential of probiotic loaded beads in the treatment of gastrointestinal disorders, until recently the probiotics have been largely propounded for their prophylactic and preventive effects. The present study establishes the therapeutic efficacy of the developed system against ulcers post-induction, thus suggesting an efficient line of treatment.

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