Supplementation of microencapsulated probiotics modulates gut health and intestinal microbiota

Abstract The beneficial effect of probiotics on host health is impaired due to the substantial loss of survivability during gastric transit caused by small intestinal enzymes and bile acids. Encapsulation helps to preserve the probiotics species from severe environmental factors. Lactobacillus paracasei, highly sensitive probiotic species to gastric acid, was encapsulated with polyacrylate resin. C57BL/6 male mice were equally divided into three groups; control group was fed with basal diet without any additives, the un‐encapsulated group was fed with 0.1% of a mixture of encapsulating material and L. paracasei, and encapsulated group was fed with 0.1% encapsulated L. paracasei (microcapsule) for 4 weeks. The result showed elevated fecal moisture percentage in the encapsulated group, but not in the un‐encapsulated group. Further study showed that the ratio of villus height to crypt depth in the small intestine was significantly higher compared to un‐encapsulated and the control group. Microencapsulated probiotics also remarkably increased intestinal mucin and secretory immunoglobulin A (sIgA) concentration, intestinal MUC‐2, and tight junction protein mRNA expression levels improving the intestinal barrier function of mice. In addition, microcapsules also reduced proinflammatory factor mRNA expression, while considerably increasing anti‐inflammatory factor mRNA expression. Microbiota metabolites, fecal LPS (Lipopolysaccharide) were downregulated, and acetate and lactate were upraised compared to control. Furthermore, glutathione peroxidase (GSH‐Px) and TAOC levels were increased and Malondialdehyde (MDA) was decreased improving antioxidant capacity. Microflora and bioinformatic predictive analysis of feces showed that encapsulated probiotics remarkably increased Lactobacillus proportions. Mice's intestinal health can thus be improved by using microencapsulated probiotics.


| INTRODUC TI ON
Intestine helps in food digestion and absorption of food-derived nutrients in the host (Okumura & Takeda, 2017). The three components of the intestine, named single-cell layer epithelium, microbiome, and immune system, together have a vital role in nourishing homeostasis in host health (Fay et al., 2017). Intestinal epithelial cells contribute to the maintenance of host-microbe symbiosis by controlling the nutrient uptake and protecting against stress (Bonis et al., 2021;Fay et al., 2017). Gut microbiota refers to the community of microorganisms that inhabit the digestive tract (Turner, 2018) and the microbial environment is dominated by bacteria, gram-positive Firmicutes, and gram-negative Bacteroidetes. The diversity of the microbiome is closely associated with intestinal health (Rinninella et al., 2019). The enteric microbiota, inhabiting the gastrointestinal tract, has a significant contribution to nutrient and drug metabolism, detoxification, and prevention of the pathogens' colonization along with the induction and regulation of essential components of the host innate and adaptive immune system (Jandhyala et al., 2015;Magne et al., 2020).
Meanwhile, the immune system organizes the principal aspect of the symbiotic relationship between the host and highly diversified microorganisms. But, the changes in the composition of the gut microbiota or disruption of the interaction between host microbes and the immune system affect intestinal health and can lead to the development of autoimmune diseases or disorders (Zheng et al., 2020). Thus, the maintenance of the gut microbiota is crucial to regulate immune homeostasis and impart health benefits to the host.
Probiotics are live microorganisms, when administered in sufficient numbers enhance the host's health. They play a substantial role in intestinal health by restoring gut microbiome composition and providing a favorable environment for the commensal bacteria that results in the treatment of many infections (Anselmo et al., 2016;Wang et al., 2021). But, the probiotics strain selection is limited as they lack stability during storage, transportation, and gastric transit. Higher temperature, oxygen level, and relative humidity (RH) are harmful to many probiotics (Yao et al., 2020). Similarly, when probiotics are ingested, it needs to face harsh environmental complexity in the gastrointestinal (GI) tract. Generally, they can survive at the pH range of about 6-7 (Yeung et al., 2016) but, gastric fluids are highly acidic (pH around 1-3) that can be deleterious to probiotic species (Sarao & Arora, 2017). In addition, high ionic strength and enzyme (pepsin) activity in the stomach (Yao et al., 2020;Yeung et al., 2016), bile acid, and digestive enzymes (lipases, proteases, and amylases) in the small intestine also affect the viability of probiotics (Han et al., 2021;Yao et al., 2020). As a result, there is a reduction in the number of bacteria reaching the hindgut. Thus, several methods have been documented to surmount these obstacles and enhance the ability to survive in the gastrointestinal tract, strengthen mucoadhesion characteristics, and elevate colonization (Terpou et al., 2019).
Microencapsulation protects the probiotics from environmental stress, GI tract insult and holds its structure in the upper GI tract before releasing it in the intestinal area, enhancing its efficacy Yeung et al., 2016). This technology has been widely used in clinical medicine for the controlled release of encapsulated drugs (Lopez-Mendez et al., 2021). However, the material used for microencapsulation should be selected wisely. Some encapsulating material may not break down on the targeted site and others can dissolve partially, preventing the complete release of probiotic species into the hindgut, which could excrete out without utilization (Lee et al., 2019). Considering these facts, we developed a microcapsule to deliver the probiotics in the intestine for its efficient utilization.
Lactobacillus paracasei was taken as a probiotic species encapsulated with Polyacrylate resin to prepare a microcapsule, used as a feed additive. L. paracasei is an extensively used probiotic strain, but it is highly sensitive to low pH (Shori, 2017). Polyacrylate resin is pHsensitive material that can dissolve in the intestinal juice of pH ≥7; thus, it can protect L. paracasei from gastric acid, release it on the lower GI tract, and enhance its potential as probiotics. Nevertheless, there is no research using this matter as encapsulating material for probiotics, although it is used as adhesive material. Thus, our objective was to enhance the survivability of L. paracasei probiotics by utilizing microencapsulation, which shields them from harmful effects of gastric acid and other gastrointestinal insults, while also studying the effect of the prepared microcapsules on the morphology, immune response, antioxidant capacity, and gut microbiota of mice.

| Microencapsulation of probiotics
First, starch pellets are used as a carrier and added to the coating pot which was preheated to 37°C. A probiotic bacterium known as L. paracasei GDMCC 1.649, which was isolated from the human gut, was used for microencapsulation. After a 10-min preheating period, the bacterial liquid (L. paracasei) was sprayed onto the pellets using a spray bottle. The purpose of this step was to ensure that the bacteria are evenly distributed on the surface of the starch pellets.
Once the bacterial liquid was applied, the enteric coating material was then sprayed onto the pellets. This coating material is pHdependent, it will remain intact as it passes through the stomach and will only dissolve in the intestines where the pH is higher. Polyacrylic resin is commonly used as an enteric coating material due to its pH sensitivity and ability to protect the bacteria until it reaches the intestines.
By encapsulating the bacteria in starch pellets and then applying an enteric coating, the survivability of the probiotics is increased.
This process protects the bacteria from the harsh acidic environment of the stomach and ensures that they can reach the intestines intact, where they can provide their beneficial effects.

| In vitro test of coated probiotics
The artificial gastric juice (Yuanye Bio-Technology Co Ltd, R30386) and small intestine fluid (Yuanye Bio-Technology Co Ltd, R30384) were prepared according to the manufacturer's instructions.
The coated and uncoated bacteria were inoculated into separate Petri dishes of the artificial gastric juice and then incubated at a temperature of 37°C for 1.5 h. Following this, the bacteria were moved from the artificial gastric juice to separate Petri dishes of the artificial small intestine fluid and incubated again at 37°C for another 1.5 h. After spending 1.5 h in the artificial small intestine fluid, the bacterial solution was diluted by a factor of 10,000. This diluted bacterial solution was then plated on a suitable agar medium and incubated under appropriate conditions to promote the formation of colonies. Finally, the number of monoclonal colonies that formed was counted, and a comparison between the number of colonies that formed from the coated and uncoated bacteria was performed to determine whether the coating provided any protection against the simulated digestive fluids.

| Animals, experimental design, and dietary treatments
A total of 30 C57BL/6 male mice (5 weeks) were obtained from the Animal Experiment Center of Guangdong Province (Guangzhou, Guangdong, China) and were caged individually, in a sterile and controlled environment with a temperature of a 24°C ± 2°C, relative humidity of 65% ± 10% and 12 h light 12 h/dark cycle. For 2 weeks, mice were provided with mouse feed and water ad libitum. After 2 weeks of adaptation, mice were assigned into three groups (n = 10) according to their weight: one group was blank control (Control) which received a basal diet without any additives, the second group was un-encapsulated L. paracasei (0.1% MELP), which received 1 g/ Kg of a mixture of L. paracasei GDMCC 1.649 (0.25 g/Kg) and coating material, polyacrylate resin (0.75 g/Kg), and the last group was 0.1% encapsulated L. paracasei (0.1% ECLP) which received 1 g/Kg of encapsulated L. paracasei. Here, encapsulated Lactobacillus was prepared by encapsulating L. paracasei GDMCC 1.649 with polyacrylate resin by Hefei Ansheng Pharmaceutical Technology Co., Ltd, Hefei, China. Feed was prepared with water by maintaining a pH of 3.5 to protect encapsulating material from disintegration in the presence of water. The animals were individually housed in cages. Each animal's body weight and feed intake were recorded weekly. The Nuclear magnetic resonance system (Body Composition Analyzer MiniQMR23-060H-I, Niumag, China) was used to measure the body composition.

| Fecal sample collection
For moisture content, fresh feces were collected every day for a week and then once a week for 3 weeks. Feces were collected instantly after ejection and placed on airtight tubes to prevent evaporation. The collected fresh feces were weighed in a tube, dried for 24 h at a 60°C dry oven, and reweighted to get dry weight. At the end, the moisture content was determined as (weight of feces before drying − weight of feces after drying)/weight of feces before drying × 100%. Similarly, all the feces were also collected regularly from the litter on 10 mL tubes to measure total dry fecal weight and use the feces for further studies. Feces collected in sealed tubes (10 mL) were dried for 48 h at 37°C to protect them from the loss of volatile nutrients that can be evaporated and lost at a higher temperature. After drying, feces were weighed to obtain dry fecal weight and stored at −80°C for later use.

| Sample collection and processing
After feeding for 28 days, all mice were sacrificed. Blood was harvested from an eyeball. Blood samples were collected in a 1.5 mL centrifuge tube, placed for 1 h at room temperature for clotting, and centrifuged (3500 rpm, 15 min, and 4°C). The serum of each sample was separated and stored at −20°C for subsequent detection and analysis. From the dissected mice, tissues and internal organs were observed and weighed. Intestinal tissues and their contents were stored at −80°C for future use. The parts of separated specimens of the intestinal section (duodenum, jejunum, and ileum) were fixed in a 4% paraformaldehyde solution for morphological study.

| Morphological study
The specimens placed in paraformaldehyde were embedded in paraffin wax, and slices were sectioned at 5 μm. The obtained sections were stained with hematoxylin and eosin (HE) by mounting in a glass slide. Afterward, the slides were observed under the microscope (Olympus, Tokyo, Japan) for villi length (V) and crypt depth(C). The measurement of V and C was done using Image (Image-Pro Plus 6.1 Media Cybernetics, Rockville, MD, USA). Finally, the ratio of V and C (V/C) was determined.

| Western blotting
The protein was extracted from intestinal tissues using RIPA lysis buffer (P0013B, Beyotime), and its concentration was determined using a BCA protein assay kit (23227, Thermo Scientific). The protein concentration was then adjusted to 20 μg/20 μL and denatured with protein loading buffer (LT 101, EpiZyme) by boiling it in water for 10 min. The western blot (WB) procedures followed a previous study . The primary antibody used was anti-Claudin (sc-166338, 1:1000, Santa Cruz) and β-Tubulin was used as the loading control. The proteins were visualized on a PVDF membrane using Protein Simple (Santa Clara, CA USA) and super ECL Enhanced Pico Light Chemiluminescence Kit (SQ 101, EpiZyme). The protein expression level was analyzed using ImageJ (National Institutes of Health, USA).
The obtained supernatant was used to detect different contents by following the kit instructions. Digesta pH was measured by inserting the sterile glass electrode of a pH meter (Thermoscientific™ Eutech Elite pH Spear) to a tube containing digesta, and the values were noted down.

| Determination of mRNA expression
The expressions of mRNA were determined by quantitative real-time PCR (q-PCR). For RNA extraction, intestinal tissues stored at −80°C were taken. Total RNA was extracted from intestinal sections (duodenum, jejunum, and ileum) using RNA extraction kit (Guangzhou Magen Biotechnology Co., Ltd, China) as per the manufacturer's instructions, and then determined using a Nanodrop spectrophotometer. Complementary DNA (cDNA) was synthesized by using 2 μg of total RNA by treating with DNase I (Takara Bio Inc., Shiga, Japan) to obtain a final volume of 20 μL by using Random Primer 9 (Takara Bio Inc., Shiga, Japan) and M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) by following the protocol of the company. Then, total cDNA was mixed with antisense primers, SYBR green Real-Time PCR master Mix and Nucleic acid-free water. The qPCR reaction had a final volume of 20 μL. The qPCR was carried out with the Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, USA). GAPDH was used as control, and the relative gene expression of mRNA was calculated by 2 −ΔΔCt . The primer sequences used for PCR are provided in Table 1.

| Detection of concentration of lactate and SCFAs in digesta
The determination of SCFA and lactic acid concentrations were done via HPLC (high-performance liquid chromatography). In brief, the fecal and jejunal contents were taken out from a refrigerator, thawed at 4°C, and mixed thoroughly. Around 0.2 g of the contents from each sample was diluted with double-distilled water at the ratio of 1:5 (w/v) and mixed using a vortex for around 20 min to break all the feces. The mixture was centrifuged for 15 min, 12,000× g at 4°C.
Around 400 μL of the supernatant was extracted via pipette after centrifugation. By the use of a disposable syringe, the supernatant was filtered via a 0.22 μm filter, placed in a glass sample bottle, and injected into a glass column of 4.6 × 250 mm dimension. As per the protocol of the company and the parameters of the machine, the test was carried out.

| 16S rRNA gene sequencing analysis
After 21 days of feeding, fresh fecal samples from four mice of control and encapsulated groups were collected on a well-sterilized 1.5 mL tube twice a day for 5 days, placed on liquid nitrogen, and stored at −80°C until DNA extraction. 16S rRNA sequencing was F I G U R E 2 Effect of encapsulated probiotic on growth performance, fecal output, and metabolism: Body weight gain (a), Cumulative feed intake (b), Dry fecal weight (c), Fecal moisture %, * represents significant difference of 0.1% ECLP with control and * with 0.1% MELP (d), Body composition analysis by QMR (e), Blood urea nitrogen (BUN) (f), Glucose (g). The data are presented as the mean ± SEM. *p < .05, **p < .01, (n = 8-10 per group). Here, * represents a significant difference in ECLP group compared to control group and * represents a significant difference compared to MELP group. Control group supplemented with basal feed without additives, 0.1% MELP group supplemented with 0.1% of a mixture of encapsulating material and Lactobacillus paracasei and 0.1% ECLP group supplemented with 0.1% of encapsulated L. paracasei. The sequencing library was generated using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA), and quality was assessed using a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina NovaSeq platform, and 250 bp paired-end reads were generated.

| The viability of L. paracaesi is enhanced in vitro through its encapsulation
The use of different materials to encapsulate probiotics has been found to offer protection against the harsh conditions of the gastrointestinal tract. In this study, both free and encapsulated probiotic cells were subjected to simulated gastric and intestinal fluids. Results showed that after approximately 3 h, there was a decrease in the number of nonencapsulated probiotics compared to encapsulate ones (Figure 1a-c). The use of polyacrylate resin as an encapsulating material increased the survival rate of the probiotics; without coating the survival rate was only 5.56%, 0%, and 5.57% while after coating, the survival rate increased to 38.89%, 27.78%, and 38.89%.

| Encapsulated probiotics increased fecal weight and moisture content
The effect of encapsulated and un-encapsulated Lactobacillus was studied on growth performance and fecal parameters. There were no significant differences (p > .05) in a body weight gain or feed intake throughout the experiment (Figure 2a,b). Dry fecal weight was significantly increased on the 6th and 14th days of the experiment in encapsulated group (Figure 2c). In addition, the fecal moisture percentage was also remarkably raised in the encapsulated group (p < .05) compared to the control (Figure 2d) from the 2nd day of the experiment. Similarly, on the 28th day, QMR was carried out, but no difference was observed in the lean and fat mass among the groups (Figure 2e). Blood urea nitrogen (BUN) and blood glucose also showed no notable difference (Figure 2f,g).

| Encapsulated probiotics improved overall histomorphometric parameter and barrier function of the intestine
The liver, muscle, and adipose tissue organ indices are displayed in
the level of total antioxidant capacity (TAOC) and glutathione peroxidase (GSH-Px) in blood serum. In addition, we observed the reduction of malondialdehyde (MDA) level (p < .05) in the encapsulated group (Figure 4m).

| Encapsulated probiotics regulated and promoted microbial metabolites
The feces stored in the refrigerator were used to determine the effect on the different fecal indices for microbiota study. The fecal lipopolysaccharide (LPS) and trimethylamine-N-oxide concentration (TMAO) were decreased (Figure 5a,b) in encapsulated Lactobacillus group compared to control with a significant difference. In addition, the pH of digesta was reduced in the jejunum, colon (p = .08), and cecum (p < .05) (Figure 5c). The value of urea nitrogen in feces was determined for 3-week period. We found the values were decreased for the whole period. For the first week, no significant difference was seen, but for the second and third weeks, there was a significant reduction (p < .05) in its level ( Figure 5d). Similarly, the level of fecal ammonia did not show any considerable variation for 3 weeks (Figure 5e). The effect of encapsulated Lactobacillus on peroxide production revealed an elevation in H 2 O 2 production for the same period (Figure 5f). To show the additional potential of encapsulated Lactobacillus to influence the gut microorganisms, the SCFA level was examined. Figure 5g-i shows lactic acid and SCFAs concentration in digesta of jejunum, colon, and cecum along with feces. Encapsulated Lactobacillus increased the levels of lactate and acetate in colon digesta, cecum digesta, and feces significantly. But, in the jejunum, the rise in lactate and acetate was seen but was not significant. Furthermore, the levels of propionic acid were close in both groups but were slightly higher in the treatment group.

| Encapsulated probiotics altered fecal microbiota and their predicted function
To study the effects of encapsulated Lactobacillus on fecal microbiota, feces were analyzed by 16S rRNA sequencing. We investigated that the consumption of encapsulated Lactobacillus decreased (p < .05) the observed species (Figure 6a). Similarly, the microbial diversity within the samples was analyzed by using the Shannon index, which determines the richness and evenness of microbial communities. We observed a significant reduction in the Shannon index Here, single bar for feces represents the feces collected on third week. The data are presented as the mean ± SEM. *p < .05, **p < .01, ***p < .001 (n = 6-8 per group). Control group supplemented with basal feed without additives and 0.1% ECLP group supplemented with 0.1% of encapsulated Lactobacillus paracasei.

F I G U R E 6
Effect of encapsulated probiotic on modulation of microbiota: Observed species (a), Shannon index (b), Nonmetric multidimensional scaling (NMDS) (c), Composition of gut microbiota by using KEGG enrichment analysis: Phylum (d), Order (e), Family (f), Genus (g), Species (h) where the size of the bubble represents the relative abundance of bacteria, the x-axis label shows log2 (FC) that represents fold change, which was calculated as first, the relative content of bacteria in the control group was divided by our treatment group to get first value (FC). By using excel, log2 (FC) was calculated to get log2 (Fold change). Bacteria that are up-regulated relative to the control has a positive log2 FC value, and downregulated relative to the control has a negative log2 FC value, left y-axis label represents the bacteria at a different level and a different color on the right side shows significance (p value), Prediction of metabolic function at KEGG (level 3) (i), left y-axis shows different predicted function, x-axis shows the log2 (FC) that represents fold change. Control group supplemented with basal feed without additives and 0.1% ECLP group supplemented with 0.1% of encapsulated Lactobacillus paracasei.
Lactobacillus and Bacteroides were dominant bacteria at the genus level. The relative abundance of Lactobacillus, Turicibacter, and Lachnoclostridium was elevated in the treatment group, and Bacteroides was slightly reduced. There was a statistically significant reduction of Romboutsia in the treatment group ( Figure 6g).

Additionally, we also observed a reduction of harmful bacteria like
Streptococcus, Helicobacter, Corynebacterium, etc. without statistical significance. At the species level, Lactobacillus sp. was dominant over others. Lactobacillus murinus was the dominant species in both groups, but it was significantly higher in the encapsulated group.
To further analyze the relative difference between the control and encapsulated Lactobacillus in terms of function prediction of microbiota in feces, a histogram with FC and p value was used to analyze their KEGG pathway based on 16s rRNA sequencing. The feces of mice had shown that encapsulated Lactobacillus group has a higher relative abundance of microbiota involved in fructose, mannose, and lipoic acid metabolism. We found those microbiomes related to antimicrobial resistance genes and biosynthesis of ansamycins were enriched. However, the pathways in cancer, salmonella infections, bacterial chemotoxins, and biofilm formation in Escherichia coli were reduced in the treatment group (Figure 6i).

| DISCUSS ION
The purposes of microencapsulation are to mitigate the poor viability of probiotics due to the harsh environment on the upper part of the GI tract, and to release probiotics at a controlled rate on the lower part for its beneficial action to the host. Previously, several microencapsulation methods and materials have been studied on different strains of probiotics to analyze their viability. Alginate-pectin microgel (Zhuge et al., 2020), chitosan-alginate (Lohrasbi et al., 2020), cellulose sulfate (Gunzburg et al., 2020), etc., as an encapsulating material protected different bacterial and fungal strains of probiotics from unfavorable gastrointestinal environment and improved the survival of the bacterial cell, intestinal delivery and release resulting to several health benefits. Moreover, some materials like pectin-encapsulated probiotics did not enhance the effect of probiotic supplementation (Lee et al., 2019). In our study, polyacrylate resin was used as encapsulating material that can hold its structure at lower pH and release probiotic strain completely on the hindgut.
The moisture content in feces, which determines the softness or firmness, was significantly increased throughout the experiment.
Our results were consistent with earlier studies on probiotic species (Gan et al., 2020;Saw et al., 2019). The production of lactic acid (Saw et al., 2019) may be the reason for a higher moisture content that helps to alleviate constipation and induce bowel movement.
The present study revealed that probiotic Lactobacillus species could improve intestinal morphology as we observed an increase in the V/C ratio on different intestinal tissues. They are the standard index for intestinal health and indicate enhancement in digestion and absorption by increasing the epithelium surface layer (Celi et al., 2017). Similarly, the intestinal epithelium layer represents the most important barrier against pathogenic molecules and bacteria.
The integrity of the intestinal epithelial cell layer is maintained by adherens junctions, tight junctions (TJ), and desmosomes. Claudin, occludin, and zonula occludens (ZO-1) are majorly studied tight junction proteins (Schneeberger & Lynch, 2004) and their expression was increased in the intestinal tissues. Our result is consistent with other studies on different probiotic species (Bao et al., 2021;Yi et al., 2017). Numerous studies strongly suggest that gut microbiota can influence TJ expression and assembly, and hence regulate transepithelial permeability (Allam-Ndoul et al., 2020).
The gut microbiota contributes to host physiology by producing a wide range of metabolites. LPS, also called endotoxins, and TMAO, are intestinal microbiome-derived toxins correlated with inflammation, cardiovascular disease (CVD), and other diseases on the host (Yamashita et al., 2021). Reduction of their level in the encapsulated group suggests that L. paracasei reduces the toxins production and the expression of markers of inflammation .
Similarly, short chain fatty acids (SCFAs) are other key metabolites of microbiota in the colon. Lactate, short-chain hydroxyl-fatty acid, is produced by several bacterial species and converted to SCFA by lactate fermenting bacteria (Russell et al., 2013;Silva et al., 2020). Similarly, an increase in Firmicutes and a decrease in Bacteroidetes phyla are correlated with increased absorption of nutrients (Jumpertz et al., 2011), which indicates that the supplementation of encapsulated Lactobacillus improves digestion by improving absorption. We also found upraised value in Firmicutes to Bacteroidetes ratio (0.99 and 1.43) after supplementing the encapsulated probiotic, which is believed to be the marker for obese animals. However, we did not find any difference in weight gain in mice between those groups. A recent study has presented that this biomarker is still difficult to associate with the weight and health of an individual (Magne et al., 2020). Interestingly, we observed a significant increase in soleus weight that could be due to the variation in the gut microbiota and metabolites produced by them that influence the skeletal muscle mass (Lahiri et al., 2019). But, for an accurate conclusion, further research is necessary. Raise of Lachnospiraceae family in encapsulated group could be beneficial to host as it is chiefly responsible for producing short-chain fatty acids (Pan et al., 2020). outlined that it has a beneficial effect on the host, including antimicrobial production, antagonist against pathogens, intestinal barrier, and can be developed as a potential probiotic. B. thetaiotaomicron helps in Carbohydrate metabolism, lipid metabolism, and enervates the production of proinflammatory cytokines, and finally helps to strengthen the host-microbiome ecosystem (Jandhyala et al., 2015).
As per the function prediction, the pathway associated with the antimicrobial resistance gene was increased. Biosynthesis of antibiotics may be the reason for the enrichment of the antimicrobial resistance gene. Here, the pathway related to the synthesis of ansamycins (antibiotic) was also enriched. Pathway associated with lipoic acid metabolism may elevate host antioxidant properties and anti-inflammation (Moura et al., 2015).
Epithelial cell of the intestine establishes a physical and chemical barrier for preventing antagonism between immune cells of host and gut microbes to protect the mucosa from inflammation (Okumura & Takeda, 2017). SIgA is an abundant antibody class found in the intestinal lumen, illustrated as the first line of defense to protect the intestinal epithelium from pathogens and enterotoxins and has a key role in immune protection, which was upraised in the feces of encapsulated groups (Mantis et al., 2011). Similarly, the increased mucin level and MUC-2 mRNA expression suggests that the encapsulated group could protect and safeguard the GI tract, as mucin is essential for epithelial lubrication, and MUC2 covers the intestinal tract and It is also stated that when enteric commensal bacteria contact gut epithelia, reactive oxygen species are rapidly generated (ROS) (Jones et al., 2012;Shandilya et al., 2022). Higher production of reactive oxygen species (ROS) than antioxidants leads to oxygen stress in host health. Due to such imbalance, there is a disturbance in a cell leading to damage of DNA, lipids, and proteins. During ROS production, several antioxidant enzymes like superoxide dismutase (SOD) and glutathione peroxidase (GPx) defend against oxidative stress for balancing the system. However, MDA activity is increased during oxidative damage (Mishra et al., 2015). In earlier studies, different In this study, we developed a novel microcapsule that keeps its structure in gastrointestinal transit and releases the probiotic species completely in the hindgut to improve the overall health performance of the host. We demonstrated that the encapsulation of probiotics with polyacrylate resin could upregulate the anti-inflammatory cytokines and downregulate proinflammatory cytokines. Additionally, it enhances intestinal barrier function, antioxidant ability, improves intestinal histomorphometry, and promotes microbial metabolites.
All these activities are associated with the change in the composition of gut microbiota. Our study suggests that the microcapsule developed can be applied to the commercial production of livestock and poultry. However, more research may be required to improve the efficacy of microcapsules.

ACK N O WLE D G E M ENTS
The authors acknowledge the valuable contribution of Hefei Ansheng Pharmaceutical Technology Co., Ltd, Hefei, China.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

E TH I C S S TATEM ENT
Animal care and procedures were performed as per the guidelines and were approved by the Animal Subjects Committee of South