Development and Characterization of Pectin-Based Colon-Targeted Pellets Containing Lactobacillus Plantarum FNCC-0461

Lactobacillus plantarum FNCC-0461 is a strain of lactic acid bacteria derived from " dadih ", a traditional Indonesian food, with significant potential as a probiotic. Particularly, probiotics provide health benefits once a minimum cell viability of 7 log CFU is maintained in the distal ileum and colon. However, most of these live microorganisms are not resistant to harsh conditions of the gastrointestinal tract (GIT). Encapsulating probiotics in the form of pectin-based colon-targeted pellets offers a promising solution to enhance viability in the presence of extreme GIT conditions while facilitating release to a specific site in the colon. In addition, the pellets were produced through the extrusion-spheronization method using microcrystalline cellulose (MCC), lactose, and pectin. Optimizations were conducted by varying the spheronization speed and time, as well as adjusting the total pectin concentration and the type of coating polymer (cellulose acetate phthalate (CAP) or shellac) used for the pellet formulation. Subsequently, the morphology, particle size, moisture content, micromeritic properties, process yield, and viability of pellets were evaluated. The release of probiotics from coated and uncoated pellets was examined under simulated colon fluid conditions at pH 1.2, 6.8, and 7.4 for 24 h. The formulation containing MCC, lactose, and pectin in a ratio of 5:4:1, with a spheronization speed of 1500 rpm for 15 min, showed the best pellet characteristics and cell viability. The prepared pellets were spherical, with a particle size distribution of 0.914 ± 0.008 mm, a process yield of 88.71 ± 1.04 %, and viability of 7.50 x 10 7 cfu/g. Those coated with CAP had the highest probiotic release in simulated colon fluid, reaching 1.38 x 10 7 CFU/g at 24 h. This research proved that the CAP-coated pellet formulation indicated promising potential for colon-targeted delivery of L. plantarum, offering a means to protect probiotics viability.


INTRODUCTION
Probiotics are live microorganisms that improve intestinal microflora balance, inhibit pathogen growth, and stimulate the immune system when present in adequate amounts in humans (Qin et al., 2021;Zheng et al., 2017).For instance, L. plantarum FNCC-0461 isolated from "dadih", a traditional Indonesian food produced from the fermented milk of buffalo, exhibits probiotic potential (Maghfirotin Marta et al., 2019).This strain of lactic acid bacteria prevents the growth of pathogens such as Enterobacteriaceae, E. coli, and non-E.coli coliform (Liwan et al., 2020).Furthermore, it adheres strongly to the intestinal surface of rats, possesses pH resistance, remains relatively stable at pH 3.0 for 3 h, and grows in MRS media (de Man Rogosa Sharpe) containing 0.5% bile salt (oxgall) (Darmastuti et al., 2021;Utami et al., 2017).
For probiotics to provide health benefits, a cell viability of at least 10 7 CFU must be maintained while traversing the gastrointestinal tract (GIT) before colonizing the distal ileum and colon (Sun & Wicker, 2021).However, most probiotics encounter significant irresistible challenges during processing, storage, distribution, and navigation 94 Volume 35 Issue 1 (2024) through the GIT where there is exposure to stomach acid, digestive enzymes, and intestinal bile salts (Torp et al., 2022).This was evident in a previous result showing that the incubation of L. plantarum ZJ2868 in pH 2.5 for 3 h and a bile salt concentration of 0.1% for 1 h reduced the number of viable cells to 61.2% and 59.8%, respectively (Fu et al., 2022).Encapsulation of probiotics serves as a promising solution by enveloping the therapeutic material within a protective matrix to shield against harsh environmental conditions ( Raise et al., 2020).In this regard, colon-targeted delivery systems gained prominence as a means to enhance probiotic viability during transport across the GIT and provide a direct release in the colon (Kumar et al., 2020).Various strategies have been employed to achieve this type of delivery through the oral route, including time-dependent systems, osmotic pressure-dependent systems, coatings with pHsensitive polymers, and enzyme-based biodegradable, each with its inherent limitations (Prudhviraj et al., 2015;Wang et al., 2021).
A particular source recently explored the encapsulation of L. plantarum in a colon-targeted microsphere dosage form, primarily using pectin and starch as key excipients.The recorded observations indicated higher viability in encapsulated probiotics after exposure to simulated gastric fluid and bile salt solution, compared to free cells (Dafe et al., 2017).Another investigation, featuring a dissolution process, encapsulated L. plantarum in a colon-targeted microemulsion dosage form with alginate and Ca-EDTA as excipients.The results showed that the L. plantarum microemulsion shielded probiotic cells from simulated gastric fluid and facilitated direct release in simulated colon fluids (Qin et al., 2021).Both approaches had limitations associated with the complexity of the respective methodologies applied.Furthermore, relying on polymers with one release mechanism may reduce their effectiveness in colon-targeted probiotic delivery, considering the variability in digestive tract conditions influenced by the fasting state, medical history, gender, age, and concurrent drug administration (Shahdadi et al., 2019).Consequently, in this research, a combination of enzyme-based biodegradable polymers (pectin) and pH-sensitive polymers, namely shellac or cellulose acetate phthalate (CAP), was implemented as the optimal strategy for probiotic delivery to specific sites.It is important to note that shellac and CAP dissolve at pH values around 7 and over 5.8, positioning both as suitable candidates for colon-targeting formulations (Gately & Kennedy, 2017; Sampath Udeni Gunathilake et al., 2020).Pectin, a polysaccharide, resists enzymatic degradation in the small intestine but succumbs to the action of colon microbial enzymes, including pectinase, pectate lyase, and polygalacturonase (Noreen et al., 2017;Wang et al., 2021).
L. plantarum can be encapsulated into pellets, which offer distinct advantages over conventional solid dosage forms, such as ease of swallowing, size uniformity, free-flowing properties, and seamLess coating and packaging (Aponte et al., 2018).Therefore, this research aimed to formulate pectin-based pellets containing L. plantarum FNCC-0461, using CAP or shellac as coating polymers, to achieve colon-targeted probiotic delivery at specific sites for improved cell viability.The pellets were developed through the extrusion-spheronization method, followed by a well-established coating process in a pan (Singh et al., 2021).In the absence of existing literature regarding the selected topic, this research provided the first report on the production of pectin-based colon-targeted pellets encapsulating L. plantarum FNCC-0461 as probiotics.

Optimization of the extrusion-spheronization process
Optimization was conducted by adjusting independent variables, including spheronization speed (500, 1000, and 1500 rpm) and time (5, 10, and 15 min), each with three levels.The dependent variables assessed were shape, particle size, percentage recovery, and cell viability per gram.Specifically, nine experimental conditions were explored to optimize the extrusion-spheronization process for pellet preparation.

Preparation of uncoated pellets
To formulate pellets containing L. plantarum FNCC-0461, the extrusion-spheronization method was used.The materials required for this procedure included a 10 g dry mass comprising 10 9 CFU (equivalent to 0.3 g) of lyophilized L. plantarum, pectin, MCC, and lactose in the ratio (Table I).These components were thoroughly mixed, and 5.5 mL of distilled water was added to create a suitable dough mass for the pelletization process.Four different formulations (F1 to F4) were developed by varying the concentration of pectin as the enzyme-based biodegradable polymer and lactose as the bulking agent to achieve the desired pellet characteristics.The best formulation was selected for coating with a pH-sensitive polymer, such as CAP or shellac.Moreover, the uncoated pellets were dried at 40°C in an oven (Memmert, Germany) and characterized for morphology and shape, particle size, moisture content, yield, and viability, before being coated.

Pellets coating
CAP and shellac were used as pellet-coating polymers.Each was properly weighed to obtain a 10% concentration in the coating solution.A solvent medium consisting of a mixture of organic solvents (acetone: ethanol, 9:1) was applied, with the addition of 5% glycerol as a plasticizer.Furthermore, the peristaltic pump tube was rinsed using organic solvents before filling it with the coating solution, ensuring the absence of air bubbles, and the spray gun was connected.Pellets from the optimal formulation were conditioned in the coating drum to facilitate drying during the coating process.For every 5 g of pellets, the coating pan parameters were set as follows: drum speed of 20 rpm, blower temperature of 55°C, a flow rate of 1.5 mL/min, and one minute delay to attain complete solvent evaporation (Singh et al., 2021).This procedure was performed in three repetition cycles to achieve a good coating.CAP-coated pellet formulations were labeled as FC, and those coated with shellac were tagged as FS.

Pellets morphology
Visual observation of pellet shape, texture, and color was conducted under an optical microscope.The diameter of both uncoated and coated pellets was measured using an optical microscope equipped with a camera (Olympus, Japan), and Image J software was deployed for image analysis.Additionally, surface texture and morphology were examined using a scanning electron microscope (SEM, LEO 420i, England).

Micromeritic properties
Micromeritic properties play a crucial role in probiotic release behavior and these can be evaluated as follows:

The angle of repose and flow ability test
Flow rate and angle of repose were determined using a flowmeter (Erweka, Germany).The flow rate was calculated by dividing the sample weight with time, and the angle of repose was estimated by dividing the anti-tangent of the height value using the radius of the pile of preparations formed after the test.

Friability test
Friability was determined using a Roche friabilator (type TAR 10, Erweka, Germany), with 200 rotations at 25 rpm.This was expressed as a percentage of weight reduction in the test sample after the experiment compared to before.

Tapped and bulk density
Bulk and tapped density were measured using a bulk-density tester (Erweka, Germany).Bulk density was calculated by dividing the predetermined mass (50 g) of pellets by the volume formed in the cylindrical glass before turning on the device.Tapped density was estimated by dividing the mass of the pellet by the volume formed in the cylindrical glass after tapping for 10 min.

Hausner's ratio
This ratio was determined by dividing the tapped density value with the bulk density.

Particle size distribution
Particle size was deduced through the sieving method with a shaker (Retsch GmbH, Jerman) by weighing and placing 50 g of pellets on the top sieve.The vibration amplitude was set at 0.25 for 10 min.

Moisture content
The moisture content parameter was evaluated using a moisture analyzer (Adam, USA) by heating one gram of pellets at 115°C.

Process yield
Process yield was calculated by comparing the total weight of raw materials incorporated during pellet preparation to the weight of dry pellets obtained, using the following equation:

Process yield (%) = Wm x 100 Wt
Where Wm represented the weight of dry pellets obtained (g) and Wt was the total weight of the raw materials (g).

In vitro release research
In vitro drug release from CAP-coated, shellac-coated, and uncoated pellets, was investigated using a USP dissolution test apparatus I (Erweka GmbH, Germany) in a 500 mL medium at 37±0.5°C with a rotational speed of 100 rpm.In this research, approximately 1 g of pellets was placed into the dissolution basket.The dissolution research was conducted for 2 h in Simulated Gastric Fluid (SGF) at pH 1.2, 3 h in the Simulated Intestinal Fluid (SIF) at pH 6.8, and 19 h in Simulated Colon Fluid (SCF) at pH 7.4 with subjection to pectinase enzyme (totalling 24 h).At various time intervals, 5 mL of the sample was collected, and to maintain a constant volume of 500 mL, 5 mL of fresh dissolution medium was added to the dissolution chamber after each sampling.

Viability count of L. plantarum from pellets
The viability test was performed on the dissolution results as well as the uncoated and coated pellet samples obtained.Approximately 1 mL or 1 g of sample was dissolved in 10 mL of phosphate buffer, generating a 10 −1 stock solution (standard solution).Around 1 mL of the standard solution was mixed with diluent solution (0.1% buffered peptone water + 0.05% Tween 80) to reach 10 mL, amounting to 10 -2 dilutions.Serial dilution steps were carried out, producing 10 -3 and 10 -6 colony counts, which were used for dissolution research and pellet samples, respectively.Subsequently, 20 μl of the serial dilutions were inoculated into Petri dishes containing 10 -15 mL of de Man Rogosa Sharpe (MRS) agar medium.These dishes were incubated at 37°C under facultative anaerobic conditions for 48 h.To ensure accuracy, the procedure was conducted three times.L. plantarum colonies were counted, and the results were expressed as cfu/g (colony forming units per gram).

Statistical analysis
All data were analyzed for mean and standard deviations.Statistical analyses were conducted using the IBM Statistical Package for the Social Sciences (SPSS) free trial version.Three replicates were performed on each sample, and the results were averaged.Additionally, a one-way analysis of variance (ANOVA) was executed to compare the results of each group, where a p-value < 0.05 was considered statistically significant.

Optimization of the spheronization process
The process variables, namely spheronization speed and time, were optimized.Their effect was observed on shape, particle size, moisture content, percentage yield, and viability of pellets.In the first batch (C1), a spheronization speed of 500 rpm) and time of 5 min were used (Table II).The speed and duration of spheronization were known to affect the pellet shape (Chopra et al., 2013).Therefore, their optimization was crucial for formulating drugloaded pellets (Kaur et al., 2020).The results of the partial t-test conducted showed that both factors simultaneously exerted a significant negative effect on pellet size (Sig < 0.05).Specifically, a higher spheronization speed led to increased frictional force between the extrudate and the spheronizer lattice plate (crosshatch pattern), making the extrudate more prone to break into smaller pieces upon contact with the rotating spheronizer plate.This phenomenon-initiated collisions between the extrudate and the spheronizer plate, as well as among the extrudate particles during the process (Muley et al., 2016).Increasing the spheronization speed from 500 rpm to 1500 rpm, where the time was constant, led to a decrease in pellet size and an elevation in pellet viability.
An increase in spheronization time generated rodshaped pellets at lower speeds, while a spherical shape with a smooth surface was obtained at higher speeds.
Evaluation of the spheronization speed and time showed that only the speed variable had a significant effect on the shape of pellets produced (Sig.< 0.05), while time exerted no significant effect (Sig.> 0.05).These differences in dosage forms might be attributed to variations in the mechanical energy generated from the spheronization plate due to disparities in rotational speed.During spheronization, this mechanical energy was converted into kinetic energy, causing the extrudate to move in a fluidized manner on the plate mechanically, leading to a transformation into a spherical shape (Muley et al., 2016).As the extrudate experienced more frictional forces, its roundness increased over time, until an optimum spheronization time was reached, at which point the pellets became compact enough to resist further frictional forces (Sinha et al., 2007).
Multiple linear regression analysis revealed that spheronization speed and time did not affect the recovery percentage, as indicated by the significance of the F value (Sig.> 0.05).In contrast, this analysis showed that both variables simultaneously had a significant effect on the cell viability of the pellets (Sig.< 0.05).The conducted partial t-test confirmed that both variables had a positive effect on the pellet viability.This might be attributed to the dosage form generated, as more spherical or near-spherical pellets tended to offer a greater surface area for probiotic cell adsorption compared to other forms such as rods, cylinders with rounded ends, dumbbells, or ellipsoids.Following the attainment of the best results using a spheronization speed of 1500 rpm for 15 min (resulting in spheroid-shaped pellets with roundness of 0.90, circularity of 0.85, moisture content of 1.93 ± 0.05, percentage yield of 89.66 ± 1.58, average particle size of 1.264 ± 0.060 mm, and viability of 9.42 x 10 7 cfu/g), the process C9 was selected for further formulation (Table II).

Optimization and preparation of uncoated pellets containing probiotics
The formulation variables, specifically the weight ratios of pectin in the pellet, were optimized from 5% to 20% (w/w), and their effect was observed on the shape, particle size, yield percentage, water content, and viability.The selection of a maximum concentration of 20% (w/w) was based on prior research indicating that concentrations exceeding 20% could render the formed mass extremely difficult to pelletize (Akhgari et al., 2013).The optimal process variable based on previous results was used for the preparation of uncoated pellets.It is important to note that the pelletization process was significantly influenced by the excipient and its quantity in the pellet formulation.The consistency of the wet mass affected the surface and shapes of the pellets (Chopra et al., 2013).Formulations F1 to F4 with increasing pectin concentration from 5% to 20% showed a significant effect on pellet shape (Sig.< 0.05).
Those comprising lower pectin concentrations produced more spherical pellet shapes compared to the counterparts containing higher concentrations.This phenomenon was attributed to the swelling of pectin in the moist mixed mass, making it challenging to process using the extrusion-spheronization method for pellet production (Martins et al., 2017).As the amount of pectin in the formulation increased, the possibility of swelling elevated which reduced the suitability of the mixed mass for pelletization.This change tended to be initiated by numerous hydrogen bonds formed between distilled water and the carboxyl groups in the pectin structure, leading to difficulty in achieving the desired spherical shape during the spheronization process (Tho et al., 2002).
There was a significant correlation between pectin concentration and pellet size (Sig.< 0.05), indicating a positive and moderate relationship.This is proven by increasing the amount of pectin in the formulation which causes larger pellet sizes in F2 to F4 (Table III).The quality of pellets depends greatly on the characteristics of the mixed mass, which must exhibit plasticity and moisture absorption capacity.In this context, the formulation needs to provide sufficient mechanical strength for the production of pellets, influenced by the compatibility properties of the ingredients in the formulation (Palugan et al., 2015).However, pectin is known to have poor compatibility, capable of hindering the formation of a compact mass (Sande, 2005).Pectin swelling in the wet mass could reduce the quality of the resulting pellets when prepared through the extrusionspheronization method.To address this issue, a granulation liquid, such as a 10% citric acid solution (w/v), could be added.Although, the highest controllable pectin content in formulations is limited to 20% (w/w), as a greater content tended to cause over-expansion (Martins et al., 2017).
The correlation between pectin concentration and recovery percentage (Sig.< 0.05) was also significant, indicating a strong relationship.These results show that elevating pectin concentration from 5% to 20% led to an increase in the percentage of recovery from 78.97% to 91.07%.The effect of pectin concentration on cell viability in the pellet preparations was significant (Sig.<0.05) and negative (Table III).When the pectin content in the formulation increased from 5% to 20%, viability decreased from 8.18 x10 7 CFU/g to 2.00 x10 7 CFU/g.This decrease could be attributed to the resulting dosage forms, as more spherical or near-spherical pellets had greater surface areas for probiotic cell adsorption compared to those with ellipsoid or dumbbell shapes.After achieving the desired results (spheroid shape, moisture content: 2.08 ± 0.31, percentage yield: 88.71 ± 1.04, average particle size: 1.150 ± 0.029 mm, and viability of 7.50 x10 7 cfu/g), batch F2 was selected for the subsequent coating process.

Pellet coating Process
Following the formulation optimization, batch F2 was coated with either CAP or shellac.Both types of enteric-coated polymers were applied at a 10% concentration, along with a 5% plasticizer (glycerol).At the end of the process, CAP-coated pellets gained an additional weight of 0.496 g (9.92 %) to the initial 5.00 g.In contrast, shellac-coated pellets had an additional weight of 0.411 g (8.21 %).These pellet weights, approaching 0.675 g for a 5 g batch of pellets (equivalent to 13.5%), suggested that the use of 10% material concentration would provide an even coating, leading to a smooth surface.
Cell viability of pellets coated with either CAP or shellac decreased significantly compared to uncoated forms.The viability reduced from 7.5 x 10 7 CFU/g to 4.67 x 10 7 CFU/g for FC, while FS had the lowest viability at 1.48 x 10 7 CFU/g.This decrease might be attributed to the extreme conditions during the coating process, including a temperature of 50 ˚C and contact between probiotic cells and organic solvents in the form of an acetone and ethanol mixture, with a tendency to cause cell death.In FS, the lowest cell viability results obtained might be due to the presence of seven sesquiterpene acids in shellac, which possessed antibacterial activity (Lu et al., 2014).Five of these seven metabolites are known to have significant antibacterial activity against S. aureus, E. coli, and B. subtilis (Lu et al., 2018).

Pellet Morphology
Uncoated pellets were odorless, with a rigid and spherical shape, and appeared yellowish white.The application of pH-sensitive coating polymers such as CAP produced spherical, whitish, and odorless pellets.Meanwhile, coating with shellac led to transformation into spherical shapes, yellow to orange coloration, and odorless smell (Figure 1).The surface morphologies of FP, FC, and FS, (Figure 1(D,EF)) showed a predominance of spherical shapes, as revealed by SEM analysis.The FP was smooth, spherical, compact, and uniform on the outer surface.

Micromeritic property
The particle size distribution presented in Table IV, showed the sizes of FP, FC, and FS to be 0.913 mm, 0.984 mm, and 0.934 mm, respectively.It was evident that the coating process increased the diameter of FC and FS compared to FP.The size difference was in accordance with the results of the pellet coating thickness test, which showed a 9.9% and 8.8% increase for FC and FS, respectively, after the coating process.Moreover, the results of the flow characteristics, including tapped density, bulk density, angle of repose, Hausner ratio, and Carr's index values, did not indicate significant differences among the three pellet samples (Sig.> 0.05).
The angle of repose, Hausner ratio, and Carr's index values for all three groups showed that the preparations had very good flow properties.This was attributed to the circularity of the pellets, as increased circularity enhanced their flowability.The minor differences in density (tapped or bulk) might be due to the similarities in particle size and size distribution, as these properties were consistent (Sinha et al., 2007).According to Table IV, the friability test results from three sample groups were below 1%, indicating strong mechanical strength and compatibility in the pellets (Jubie et al., 2020).This phenomenon could be due to optimum drying temperature and time, rendering the pellets less friable and more hardened by reducing porosities and shrinkage.The moisture content of each pellet batch remained below 5 %, and the drying process, consistently performed at 40 ˚C for 24 h, ensured complete moisture removal after this time frame.

In vitro probiotic release and viability count
For FP, no bacterial colonies were observed in the viability test conducted during the dissolution research, spanning from SGF to SCF medium (over a period of 30 min to 24 h).This was attributed to the death of all probiotic cells contained in the pellet preparation due to the highly acidic conditions in the SGF medium at pH 1.5 for 2 h.These results were in line with research performed by Fu et al., where L. plantarum was found to be nonresistant to very low pH conditions, as probiotic cells reduce to 61.2 % viability at pH 2.5 (Fu et al., 2022).Colon-targeted pellet samples coated with CAP or shellac did not experience complete probiotic cell death as observed in FP (Figure 1).This showed that the combination of enzyme-and pH-based enteric polymers effectively preserved probiotic viability against a pH of 1.2 for 2 h, compared to pellet preparations relying solely on one release mechanism (enzyme-based), such as the uncoated pellets.
The mechanism of retaining probiotic release with pH-sensitive polymers is based on the relationship between their pKa values and the pH of the medium.In general, polyanion polymers are ideal excipients for inhibiting drug release at low pH and promoting release at higher pH (Ghaffar et al., 2020).According to the Henderson-Hasselbalch equation, the ionization of groups in the polymers is affected by the pH of the system and pKa.When the pH of the system is above the pKa value, ionization occurs.Conversely, once the pH of the system falls below the pKa value, the polymer remains un-ionized.Shellac is known to have a fairly high dissolution pH of around 7, making it a suitable candidate for use in colon-targeted formulations (Gately & Kennedy, 2017).Additionally, Cellulose Acetate Phthalate (Cellulose Acetate Phthalate, CAP) dissolves at a pH > 5.8 (Sampath Udeni Gunathilake et al., 2020).For the FC formulation, no probiotics were released within the first 2 h in SGF at pH 1.2 as well as between the 2 nd and 5 th h in SIF at pH 6.8, Cumulative probiotic release (%) (Figure 2).At the 6 th hour in SCF at pH 7.4, only 0.125 % of the probiotics were released due to the dissolution of the coating polymer in the SCF medium, enabling the degradation of pectin by the pectinase enzyme.By the 6th hour, most of the coating had degraded from the pellet surface, facilitating a more rapid release of the active substance compared to shellac-coated pellets.The influence of coating on active substance release can be explained by the Noyes-Whitney/Nerst-Brunner equation.Higher coating concentrations result in thicker diffusion membranes, decreasing the dissolution rate of the active substance.After 24 h, 29.551% of the probiotic was released from 1 g of pellets.Note: FP (uncoated pellets), FC (CAP-coated pellets), and FS (shellac-coated pellets)

Figure 2 .
Figure 2. Cumulative probiotic release of L. plantarum from uncoated pellets, CAP-coated pellets, and shellac coated-pellets at pH 1.2, 6.8, and 7.4, with data shown as mean ±SD.

Table I .
Composition of pellet formulations

Table II .
Micromeritic properties of pellets obtained from the optimization process Volume 35 Issue 1 (2024)

Table III .
Micromeritic and properties of pellets formulation