Liver fibrosis is the second stage of liver disease with few specific treatments currently available. Probiotics, specifically bifidobacterium longum can be used to alleviate liver fibrosis through several mechanisms such as reducing oxidative stress and inflammation, leading to restoring liver function and enhancing its histological parameters. However, probiotics viability can be reduced during gastrointestinal transit, diminishing treatment efficiency. Therefore, encapsulating them in matrices of carbohydrates (e.g., alginate and chitosan), and proteins (e.g., whey protein) may increase their viability and might positively affect their treatment efficacy. As a result, we hypothesized that encapsulation in alginate-whey protein matrix with a chitosan coat would enhance the delivery and hepatoprotective effects. Therefore, in this research we assessed the effect of bifidobacterium longum encapsulated with alginate-whey protein with chitosan coating on liver function tests, oxidative stress parameters, inflammatory gene expression and histological parameters in liver tissue. 48 male Male Wistar rats were categorized into 6 control and treatment groups: Normal control (NC), sham-operated control (SHC), BDL control (BDL + vehicle), free B.longum probiotic (BDL + FP), free microcapsule (BDL + FC), and encapsulated B. longum probiotic (BDL + CP). Free and encapsulated B. longum was administered at a dose of 3× \({10}^{9}\) Colony forming units (CFU) per day for 7 days before and 21 days following induction of cholestasis by bile duct ligation (BDL) surgery. After the treatment, all rats were euthanized, and their blood samples and liver tissue were collected for analysis. Liver function tests were assessed in blood plasma, while liver tissue was used for oxidant/anti-oxidant status measurement, pro- and anti-inflammatory gene expression, and histological properties. Our results showed that encapsulation of B. longum with alginate-whey protein and chitosan coating can provide a microcapsule with encapsulation efficiency of 76.6%. Encapsulation of B. longum also enhanced probiotic's viability in simulated gastric juice reduced from mean of log 10 to log 9.6 while free probiotics viability reduced from log 10 to log 3.5 and enhanced its release in the intestine. Also, Rats treated with encapsulated B. longum exhibited lowered ALT, AST and LDH serum activity, reduced expression of pro-inflammatory cytokines (IL-6, 0.283-fold, and TNF-α, 0.25-fold) and α-SMA gene, an indicator of fibrosis by 0.33-fold, and of anti-inflammatory (IL-10) cytokines' increased expression by 4.9-fold. Encapsulated B. longum enhanced anti-oxidative parameters such as CAT, SOD, TAC, GSH and reduced oxidative stress parameters such as NO, TOS and MDA levels significantly. Treatment with encapsulated B. longum also improved histological parameters such as fibrosis, necrosis and ductal hyperplasia based on Metavir scoring system. Encapsulated B. longum can be used as an efficient probiotic delivery system which provides high encapsulation yield and protection against probiotic cells in simulated gastrointestinal conditions. This treatment, as shown by inflammatory gene expression and assessment of oxidative parameters, can reduce inflammation and oxidative stress, leading to enhanced liver function and lowered liver enzyme activity in serum. These alterations improved key histological indices, leading to reduced fibrosis scores, necrosis, and ductal hyperplasia.
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