Formulation and Evaluation of Probiotic Tonu Pet Gut Gel for Gastrointestinal Health in Companion Animals

1. Background of Veterinary Gastrointestinal Health

Regarding companion animals, such as dogs and cats, GI function plays a vital role in determining their overall wellbeing. Apart from breaking down the food and absorption of nutrients, the digestive tract is responsible for maintaining the healthy environment of microorganisms that have a direct effect on the body's immunity, metabolism, and ability to fight infections. The diseases associated with GI dysfunction include diarrhea, constipation, malabsorption syndromes, inflammatory bowel diseases, and poor feed efficiency.¹

The emergence of diseases related to the GI tract has been on the rise due to alterations in diet, stress, use of antibiotics, environmental influences, and lifestyle issues that result from domestication. The fact that the commercial food used by pets is usually very nutritious, but does not contain sufficient amounts of natural microflora that would promote healthy functioning, often leads to dysbiosis, a condition where there is an imbalance between beneficial and non-beneficial microorganisms in the gut. Scientists and veterinarians are currently exploring safe ways of healing the gut.²

2. Concept of Probiotics in Animal Health

Probiotics are defined as live microorganisms that confer health benefits on the host when administered in adequate amounts. Some examples of probiotics include lactobacilli, bacilli, enterococci, and bifidobacteria. These microbes exhibit various beneficial effects, such as occupying the gut environment, competing against pathogens, producing antibiotics, and stimulating the immune system..^3.4.5

Probiotics are very popular within the field of veterinary practice as::

• An aid to restore normal gut flora after an administration of antibiotics
•  A way to reduce incidences of diarrhea among young livestock
•  An approach that facilitates efficient absorption and digestibility of nutrients
• Reducing intestinal infections caused by pathogens

Due to the high degree of stability, resistance to heat, and capability to endure harsh environments of the gastrointestinal tract, probiotics like Bacillus subtilis, Bacillus coagulans, and Bacillus clausii are highly useful. In addition to this, the ability of Lactobacillus acidophilus to produce lactic acid, which decreases pH levels and prevents the proliferation of pathogenic bacteria, has been studied in depth.

Using multiple types of probiotics ensures synergistic effects that promote increased bacterial diversity and provide greater gastrointestinal protection.

3. Importance of Gastrointestinal Microbiota Balance

Billions of bacteria which live in association with the host form an ecological community known as gut microbiota. There is a perfect balance between good and bad bacteria within a healthy animal body. Balance of bacteria is required for:

• Normal metabolism and digestion

• Production of vitamins like vitamin K and vitamin B complex

• Normal development of the immune system

• Protection against invading pathogens

• Maintenance of integrity of the intestinal lining

Development of dysbiosis occurs due to excessive proliferation of pathogenic bacteria when this equilibrium is disturbed. Symptoms of dysbiosis include bloating, diarrhea, vomiting, loss of appetite, and weight loss. Long-term health of the gut relies on balancing gut microorganisms.

Probiotic supplementation is a scientifically-proven technique for restoring and maintaining intestinal balance.

4. Need for Advanced Probiotic Delivery Systems

The use of probiotics is highly effective; yet, stability concerns, low survival in acidic gastric conditions, and inadequate transportation into the intestines often limit their efficiency. The conventional approach of probiotics in powdered or capsule form faces obstacles such as:

• Reduced survival during storage periods

• Susceptibility to environmental conditions of humidity and temperature

• Absence of action throughout the stomach passage

• Limited palatability for the targeted animal species

• In companion animals, inconsistent dosing

More advanced delivery systems, like gels, suspensions, and mucoadhesive systems, have been developed to overcome these limitations. Amongst these, gel-based approaches provide several advantages, such as prolonged gastric retention, greater palatability, better administration, and enhanced stability.

Probiotics can be encapsulated within a gel matrix, which ensures their controlled release and improved surviva

5. Rationale for Gel-Based Probiotic Formulation^6.7

Gel preparations are semisolid formulations with the ability to retain large quantities of water or aqueous solutions because of being based on the polymeric three-dimensional network. Due to the advantageous physical properties, they are widely used in veterinary medicine.

The following advantages contributed to choosing gel as the form of drug administration in this research:

• Better resistance of probiotic microorganisms;

• Consistent administration of the drug;

• Easy absorption due to the gel consistency by animals;

• Convenient oral administration;

• Controlled release of active microbes; and

• Better mucosa adherence.

Oral gels represent very convenient means of medication administration to pets in veterinary practice because it may be administered in an oral gel form either directly or with food.

6. Role of Xanthan Gum in Gel Formulation

One of the most widely used gelling agents, thickeners, and stabilizers in pharmaceuticals is xanthan gum, a natural polysaccharide. It is fermented by Xanthomonas campestris and is well known for its excellent viscosity properties even in minute amounts.

The features of xanthan gum are:

• High viscosity in minute amounts

• pH and temperature stability over a wide range

• Shear-thinning and pseudoplastic nature

• Non-toxic and biocompatibility properties

• Excellent ability to suspend active components

It is necessary for the probiotic gel formulation as it ensures uniform distribution, protects microbial cells from any environmental stress, and guarantees accurate delivery.

In addition, it improves the texture and mouthfeel of the product, which makes it more appealing to animals.

7. Role of Additional Excipients in the Formulation

The following excipients are included in the formula along with xanthan gum for improved stability, flavor, preservation, and functionality.

Glycerine

It acts as a plasticizer and moisturizer. It adds smoothness to the gel consistency and boosts its viscosity. Moreover, it ensures that the formula does not dry up by retaining moisture

Potassium Sorbate

Potassium sorbate is widely used as a preservative. It enhances the shelf life of the probiotic gel by inhibiting the growth of yeast, fungus, and bacteria.

Dextrose

It makes the product more palatable and supplies energy to the probiotic bacteria.

Flavor Agent (Chicken Flavor)

Flavoring agents play an important role in enhancing the palatability of veterinary products. Since chicken flavor has excellent palatability in dogs and cats, it is commonly sele

8. Selection of Probiotic Strains

The following four strains of probiotics have been used in the formulation:

1. B. Subtilis

2. B. Clausii

3. B. Coagulans

4. L. Acidophilus

These probiotics were used due to their complementary functioning:

- Spores produced by bacilli are helpful for their survival under adverse conditions.

- L. Acidophilus will help in producing lactic acid and act against harmful organisms.

- A combination of strains will provide broad-based GI protection.

Use of multiple strains will be beneficial for the formulation's purpose

9. Significance of Companion Animal Nutrition and Gut Health ^8.9

The health and nutrition of pets have increasingly gained recognition due to the humanization of the former. The two preventive healthcare techniques for pets that have been made known by their owners include the use of probiotics and functional foods.

Currently, in veterinary medicine, preventative care is preferred to curative care. One of the most important aspects in this approach involves keeping the gut healthy through the use of probiotics.

In addition, some of the most common problems presented by clients in clinics include gastrointestinal problems. It is essential, therefore, to formulate probiotics that are not only effective but also palatable.

AIM AND OBJECTIVE :

The present study entitled “Formulation and Evaluation of Tonu Pet Gut Gel for Gastrointestinal Health in Companion Animals” was designed with the following objectives:

1. To develop a stable and palatable probiotic-based oral gel formulation for companion animals to support gastrointestinal health.
2. To select and incorporate suitable probiotic strains, namely Bacillus subtilis, Bacillus clausii, Bacillus coagulans, and Lactobacillus acidophilus, for synergistic gut health benefits.
3. To formulate an appropriate gel base using xanthan gum and optimize its concentration for desired viscosity and consistency.
4. To incorporate suitable excipients such as glycerin, potassium sorbate, dextrose, and flavoring agents to enhance stability, palatability, and preservation of the formulation.
5. To evaluate the physicochemical properties of the prepared gel, including appearance, homogeneity, pH, and organoleptic characteristics.
6. To determine the microbial viability and count of probiotic organisms in the formulated gel.
7. To assess the stability of the formulation under different storage conditions over a defined period.
8. To overcome limitations of conventional probiotic dosage forms by improving ease of administration, stability, and acceptance in companion animals.
9. To develop a formulation that ensures effective delivery of viable probiotics to the gastrointestinal tract for improved gut microbiota balance.
10. To provide a basis for future development of advanced veterinary probiotic gel delivery systems.

MATERIALS AND METHODS

1. Materials

The materials used for the formulation of probiotic Tonu Pet Gut Gel included probiotic strains, gelling agent, humectant, preservatives, sweetening agents, flavouring agents, and purified water.

1.1. Active Ingredients

• Bacillus subtilis
• Bacillus clausii
• Bacillus coagulans
• Lactobacillus acidophilus

1.2 Excipients

• Xanthan gum (gelling agent)
• Glycerin (humectant)
• Potassium sorbate (preservative)
• Dextrose (sweetener)
• Chicken flavour (palatability enhancer)
• Distilled water (vehicle)

2. Method of Preparation of Tonu Pet Gut Gel

The probiotic gel was prepared using a dispersion and cold mixing method to maintain viability of probiotic microorganisms.

2.1 Preparation of Gel Base^11,10,12

1. Approximately 300 mL of distilled water was taken in a stainless steel vessel.
2. Water was heated to 40–50°C.
3. Xanthan gum was slowly sprinkled into the vortex to avoid lump formation.
4. Glycerin was added simultaneously to enhance hydration and smooth dispersion.
5. The mixture was stirred for 30 minutes at moderate speed until a uniform gel base was formed.

2.2 Preparation of Excipient Phase

1. In a separate beaker, 100 mL of distilled water was taken.
2. Potassium sorbate, dextrose, and chicken flavour were dissolved completely.
3. The solution was filtered if required to remove undissolved particles.

2.3 Preparation of Probiotic Suspension (Thermolabile Step)

1. Bacillus subtilis, Bacillus clausii, and Bacillus coagulans were dispersed in 80–100 mL of sterile distilled water.
2. The mixture was gently stirred to avoid cell damage.
3. Lactobacillus acidophilus was added separately due to its sensitivity and incorporated later into the gel base.

2.4 Incorporation into Gel Base

1. Approximately half of the gel base was removed temporarily.
2. Lactobacillus acidophilus was added into the remaining gel under slow stirring.
3. The removed gel was reintroduced gradually.
4. Additional xanthan gum (0.3–0.5%) was added to restore viscosity if required.

2.5 Final Mixing

1. Probiotic suspension was added slowly into the gel base.
2. Excipient solution was added with continuous stirring.
3. Final mixing was carried out for 45–60 minutes.
4. The gel was allowed to stand for deaeration.

2.6 Packaging

The final gel was filled into sterile laminated tubes and sealed under hygienic conditions.

3. Formulation batches :     

Table no. 1 : Formulation Batches

Each gel contain 60 gm total volume

4. Evaluation Parameters and Methods^13.14.15.16

1. Organoleptic Evaluation

Visual evaluation, as well as sensory evaluation, was performed for all formulated gels (TG1-TG10).

Procedure:

Appearance, color, smell, and texture of the gels were carefully observed. A small quantity of the gel was placed on the glass slide and observed under natural light conditions. Odor evaluation was done through direct sensory evaluation. A small quantity was rubbed between the fingers to observe the homogeneity and smoothness.

2. pH Determination

The pH of each sample was determined to be suitable for the oral cavity and animal digestive system.

Procedure:

1. One gram of gel was accurately weighed.

2. Ten milliliters of distilled water was used to disperse the sample.

3. The sample was allowed to stand for two minutes.

4. pH was measured using a digital pH meter which was calibrated.

5. Before measuring, the electrode was washed using distilled water.

3. Viscosity Measurement

Viscosity was determined to evaluate gel consistency and flow behavior.

Procedure:

1. Viscosity was measured using a Brookfield viscometer.
2. Appropriate spindle (e.g., spindle No. 62 or suitable for gel) was selected.
3. Gel sample was placed in the sample container.
4. Spindle was rotated at a fixed speed (e.g., 20 rpm).
5. Readings were recorded after equilibrium was reached.

4. Spreadability Test

Spreadability can be defined as the ease with which gel spreads.

Procedure:

1. Two glass slides were placed.

2. Gel in a specified quantity was put between them.

3. A fixed load was applied to the top glass slide.

4. Time required for the top slide to cover a specified distance was recorded.

5. Spreadability was calculated using the traditional method (S = M x L/T).5.

5. Homogeneity Test

This test ensures uniform distribution of ingredients.

Procedure:

A small quantity of gel was pressed between thumb and index finger and spread on a glass slide. It was observed for the presence of lumps, aggregates, or phase separation under light6.

6. Extrudability Study

This test measures the difficulty of extruding from a tube or canister.

Procedure:

1. A flexible tube was filled with gel.

2. Standard pressure or force was applied.

3. The quantity of gel extruded in a given period of time was measured.

7. Swilling index :

Procedure:

1. A known mass of the gel was put into a petri dish.

2. It was then treated with a buffer solution (pH 6.8/7.4).

3. Excess fluid on its surface was removed periodically.

4. Mass gain was measured after some tim

8. In Vitro Release Study^17.18

The release of the probiotic bacteria from the gel matrix is determined through this experiment.

Procedure:

1. The dialysis membrane was loaded with gel in accordance with the given dose.

2. The dissolution medium (buffer solution) was immersed into it.

3. The temperature was maintained at 37 ± 0.5°C.

4. Specimens were collected at predetermined time intervals.

5. Fresh medium was employed in exchange for each specimen.

6. Samples were tested for the probiotic bacteria released using a suitable microbiological technique.

9. Stability Study

Long term stability testing ensures the stability of the formulation.

METHOD:

1. Airtight containers were used for storing the formulations.

2. The samples were stored in two different environments which were:

- Room temperature

- 75% humidity and 40 °C ± 2 °C

3. Samples were analyzed at intervals of 0, 1, 2 and 3 months.

4. Viscosity, microbial viability, and pH changes were observed.

10. Microbial Viability Test (CFU Count) ^19.20,21

This tests the capacity of the probiotic bacteria to withstand such an environment. Method:

1. The sample of gel was diluted using sterile saline.

2. The serial dilution process took place.

3. Nutritional Agar (or MRS Agar) was employed.

4. The plates were allowed to incubate for 24 - 48 hours at 37°C.

5. The colony forming units per gram of substance (CFU/g) was recorded.

RESULT AND DISCUSSION :

1. Organoleptic Evaluation

Table no 2: Organoleptic Properties

Organoleptic evaluation of the formulations TG1-TG10 showed consistency in terms of cream to light cream colors and semisolid characteristics in all the preparations, thus showing high compatibility and chemical stability. Homogeneity was proven by the lack of phase separation and particulate matter. Due to the increased amount of xanthan gum, glycerin, and flavoring agent content in the gels with high viscosity, the distinctive smell present in the low-polymerized formulations gradually diminished. As the viscosity increased, the texture of the formulations changed from soft to thickened in TG1-TG3 and TG8-TG10, respectively. Overall, all the formulations were acceptable, but TG4-TG7 had the best formulation characteristics.

2. Post Formulation Characterization :

Tablet no 3 : Poat Formulation Characterization

The study of physicochemical properties resulting from the chemical composition of TG1-TG9 formulations of probiotic gel revealed a direct correlation between them. The effects caused by glycerin as a humectant, as well as by an increase in the content of xanthan gum, are responsible for such alterations in pH, viscosity, spreadability, homogeneity, extrudability, and swelling index.

All samples exhibited a pH in the range of 6.6-7.0, which is a biologically adequate value and within the range required for gastrointestinal administration in pets. Probiotic viability and ensuring its physiological compatibility depend on a near-neutral pH. A slight difference between pHs might occur due to differences in the hydration of polymer chains and excipients' reactions to the aqueous medium. However, the difference is insignificant, and it does not influence the physicochemical characteristics of the formulation.

An increase in the amount of xanthan gum led to an increase in viscosity from TG1 (2100 cP) to TG9 (4500 cP), which is expected due to the presence of additional hydrogen bonds, entanglements of polymer chains, and gel formation. The t. An excessive viscosity level may hinder administration, emphasizing the importance of achieving the proper viscosity value.

The spreadability trend showed a decreasing pattern from TG1 to TG9. This inverse correlation of viscosity with spreadability is a well-established phenomenon in semisolid formulations. Higher viscosity gels resist flow owing to higher internal resistance, whereas low viscosity products facilitate easy spreadability for proper dispersion of the gel at the desired site.

All formulas had excellent homogeneity, implying a well-distributed probiotic strain inside the gel matrix. Good homogeneity is necessary in formulations to ensure dosage uniformity and consistency in terms of biological efficiency. This is especially important in probiotics, where microbial distribution directly influences the biological effect of the formula.

An analysis of extrudability showed that formulas having low viscosity were easily extruded compared to high viscosity formulas. Formulations TG1-TG3 were easily extrudable, whereas formulations TG8-TG9 required high pressure to extrude them. This finding was directly associated with the rigidity of the gel. While high viscosity enhances structural stability, it can pose challenges during administration, which can lead to reduced compliance among patients. Formulas with medium viscosity present an ideal option to balance ease of administration and stability.

The swelling index rose steadily from 120% in TG1 to 210% in TG9, signifying a higher swelling capacity when the xanthan gum content was increased. Xanthan gum's hydrophilicity enabled it to absorb water and swell. The high swelling property is beneficial in controlled delivery systems since it promotes diffusion of probiotic strains and maintains their presence inside the gastrointestinal system for a more extended period.

3.Optimization :

The optimum formula zone lies in the middle range of glycerin and xanthan gum, based on the CCD response surface analysis. This range would be perfect for selecting the final formula because of its optimum values regarding spreadability, swelling capacity, viscosity, and probiotics'stability.^23.24.

Figure no 1 : 3D response Plot

The synergistic effect of glycerin (B) and xanthan gum (A) on the overall acceptability of probiotic gel formula is illustrated in the 3-D response surface graph. Interaction effects between the two variables can be noticed clearly from the curved (quadratic) nature of the surface. Acceptability attains its peak value of approximately 0.92 as the content of xanthan gum rises from 1.0% to around 1.3% and glycerin from 5.0% to approximately 6.0%. Further increase leads to unacceptable levels of viscosity and low spreadability due to which acceptability drops off. The contour map suggests that TG5-TG6 are ideal formulations for further testing.

Figure no 2 counter plot

The Central Composite Design (CCD) method has been employed for formulating a substance via the interaction between Xanthan Gum (A) and Glycerin (B), maximizing the "Overall Desirability" aspect. From a visual perspective, concentric ellipses are present, creating a "bullseye-like" effect. There is a particular stationary point where this product attains the optimum functionality. Referring to the legend titled the "Optimum Region," the maximum "Overall Desirability" score ($\approx0.90 - 0.92$) is attained while the value of Xanthan Gum remains at 1.25-1.35%, and that of Glycerin lies at 5.8 - 6.2%. With an increase in the distance from the inner-most central region (red color) towards the outer regions (blue & green colors), there is a considerable reduction in the quality level, falling below $0.50$, since these ingredients diverge from the center of the graph^25,26

Given result observed that TG6 batch is optimized final formulation for further evaluation

4.In Vitro Drug Release Study :

Table no. 4: parameter and their condition

Table no 5 : Cumulative % drug Release

Figure no 3 graphical representation of release profile

The in vitro drug release study of the optimized probiotic gut gel formulation showed a sustained and controlled release pattern over a period of 120 minutes. The cumulative drug release increased gradually from 12.35% at 5 minutes to 99.12% at 120 minutes, indicating effective release of probiotic organisms from the gel matrix. The graph demonstrates that the formulation is capable of providing prolonged delivery of probiotics in the gastrointestinal environment.

During the initial stage of the study, the formulation exhibited a slower release profile, with approximately 35.62% drug release observed within 15 minutes. This controlled initial release may be due to the hydration and swelling of xanthan gum present in the gel. Upon contact with the dissolution medium, the polymer forms a viscous barrier around the formulation, which controls diffusion of probiotic organisms. Such behavior is beneficial because it prevents sudden release and supports gradual availability of probiotics in the gut.

A significant increase in drug release was observed between 15 and 60 minutes. The formulation released 56.78% at 30 minutes and 85.91% at 60 minutes. This phase indicates active swelling and relaxation of the polymeric network, allowing easier diffusion of probiotics into the surrounding medium. The sustained release behavior confirms the effectiveness of xanthan gum in controlling release from the gel system.

In the final stage, the release profile approached completion, reaching 96.23% at 90 minutes and 99.12% at 120 minutes. The slower release during this phase may be attributed to diffusion of the remaining probiotic content from deeper regions of the swollen gel matrix.

Overall, the release graph confirms that the developed probiotic gut gel possesses suitable sustained-release characteristics for gastrointestinal delivery in companion animals. The optimized formulation successfully maintained controlled release, which may improve probiotic stability, enhance intestinal retention, and support prolonged therapeutic activity in the gastrointestinal tract.

5. Microbial count :

Table no 6 microbial count

Results of the microbial viability assessment of the optimized TG6 probiotic gut gel formulation revealed outstanding probiotic strain viability in the formula. Specifically, viable cell counts of Bacillus clausii, Bacillus coagulans, Bacillus subtilis, and Lactobacillus acidophilus were within the recommended USP standard probiotic microbial viability limit that requires at least 1 × 10? CFU/g. Of all strains, Lactobacillus acidophilus was noted to have the highest probiotic viability count at 10.5 × 10? CFU/g.^27.-30.

These high counts of probiotics can be linked to the use of xanthan gum and glycerin in maintaining a favorable moisture level for the survival of probiotics in the gel formula. Moreover, the right pH and proper storage conditions in the preparation of TG6 probiotic gel formula were also factors contributing to the probiotics' high counts. This is in light of the fact that spore forming Bacillus strains were included in the formulation, providing additional support towards better probiotic viability.

Overall, the optimized TG6 probiotic formula proved to be a good carrier for probiotics with acceptable microbial viability and meeting USP standards.

6. Stability Study :

Table no 7 stability data