The biological effect of skim milk fortified with Lactobacillus plantarum bacteria on biochemical markers in obese rats

Introduction

The epidemic of obesity is among the most complicated and pervasive public health threat of the 21st century, and the World Health Organization reports a double in the rates of obesity worldwide. Obesity is not just a risk factor for higher weight, it is a key factor in a group of metabolic diseases called metabolic syndrome. This syndrome involves severe physiological disturbances, most prominently: hyperlipidaemia (high levels of total cholesterol, triglycerides and low-density lipoprotein (LDL)); insulin resistance; and oxidative stress (damage to liver cells, and consequently the development of non-alcoholic fatty liver disease (Boutari, 2022 & Mantzoros) [2] . In recent years, the gut microbiome has been identified as a critical regulator of energy balance and metabolism in its host. Research has already demonstrated that people and animals that are obese have a "microbial imbalance" or gut dysbiosis -a decline in beneficial intestinal bacteria, and an increase in strains that extract more energy from food and induce low-level inflammation in the body (Fan, 2023 & Pedersen) [3] . With this realization, probiotics have become an attractive and safe alternative therapeutic/preventative strategy to reduce obesity-related complications, which is termed microbial modification. One of the promising bacterial strains is the Lactobacillus plantarum with its unique metabolic properties that have attracted a great deal of scientific interest. This strain has been shown to have a high gastrointestinal survival ability and good intestinal mucosa adhesion ability. This bacterium has anti-obesity properties due to several mechanisms, including the production of bile salt hydrolase, which decreases cholesterol absorption in the intestine, and the fermentation of fibre and production of short-chain fatty acids (SCFAs) including butyric acids and propionic acids. These acids are important for enhancing the sensitivity of insulin, controlling hunger hormones, and decreasing fat buildup in the liver (Wang et al., 2024) [11] . But the most significant hurdle in using probiotics is getting enough living and viable bacterial cells to the large intestine. This is where the "food matrix" comes in. In obesity cases, skim milk is an excellent and thoughtful choice because it provides both a protein-rich environment (casein and whey) to protect against stomach acid and bile salts and is fat-free (zero-fat), reducing the caloric and fat content of patients or laboratory animals and therefore considered a food medium without additional calories or fat for individuals with obesity disorders (Khorshidian et al., 2023) [6] . In addition, skim milk has lactose as a prebiotic, which encourages the activity and growth of the L. plantarum bacteria in the intestine.

While previous studies have explored the use of functional carriers of beneficial bacteria by demonstrating benefits, no studies have examined the exact synergistic effect that the use of skim milk has on blood and liver functional parameters in models of induced obesity in relation to the use of Lactobacillus plantarum. Thus, the research problem is the importance of searching for safe and functional dietary alternatives to control metabolic disorders related to obesity. Based on this, this study involves the study and evaluation of the effect of feeding skim milk with Lactobacillus plantarum on blood biochemical parameters (total lipid profile, fasting blood glucose), and liver function enzymes (AST and ALT) in laboratory rats induced as obese rats to see the efficacy of feeding skim milk with Lactobacillus plantarum as an adjunctive and mitigating treatment of obesity complications.

Materials and Methods

Experimental Animals and Housing

This study was performed with 40 male albino rats weighing 180-200 g at the beginning of the experiments. The seedlings were randomly divided into five treatments, each having eight replicates to eliminate significant differences between the treatments at the beginning of the experiment based on biomass. Rats were kept in sterile plastic cages at room temperature in a normal light/dark cycle (12 hours/12 hours) environment. The rats were allowed to have free access to water and normal pellet food during the one week acclimatization before the experiment was started (Al-Asadi et al., 2023) [1] .

Bacterial Strain and Skim Milk Preparation

A ready-made purified bacterial culture of Lactobacillus plantarum (ATCC 14917) was used. For 24 hours, the bacteria were activated in the MRS broth at 37°C. Bacterial cells were then centrifuged at 5000 rpm for 10 minutes, and washed 2 times with physiological saline (PBS). The milk used for the sample was skim milk, which was pasteurized at 85°C for 30 minutes and then cooled to 40°C. To get the final count of 10⁹ CFU/ml, the activated bacteria were transferred into the milk. The plate pouring method (Shori et al., 2022) [10] was used to confirm the level of the bacteria.

Induction of Obesity and Diet

In the initial phase, rats were split into two groups: the negative control group: was fed a normal diet (12% fat) and the obesity group: was fed a diet consisting of 12% fat. The obesity group: received a ready-made diet that was used as a high-fat diet (HFD) to induce obesity. This period was 8

weeks until obesity was detected by comparing rat weights and performing body mass index (BMI) calculations (Garcia-Mazcorro et al., 2023) [4] .

Experimental Design

The rats were induced to obesity and then randomly allocated into five groups (8 rats in each group) and the treatment was continued for another 6 weeks, as follows: Group 1 (G1): Negative control -standard diet. Group 2 (G2): High-fat diet (HFD) -Positive control. Group 3 (G3): Daily oral dose of unfortified skim milk at a concentration of 2 ml/rat + standard diet. Group 4 (G4): Unfortified skim milk 2ml/rat per day + HFD. Group 5 (G5): Daily oral dose of skim milk fortified with L. plantarum bacteria at a concentration of 10⁹ CFU/ml + HFD.

Blood Sampling

Rats were deprived of food for 12 hours at the end of the 6th week of treatment. After anaesthetised with ether blood samples were obtained by cardiac puncture (5 mL in test tube without anticoagulant). The serum was centrifuged at 3000 rpm for 15 mins and stored at -20°C for biochemical analysis (Zhang et al., 2024) [11] .

Biochemical Analysis

Commercial kits and enzymatic-colorimetric method were used to measure the following parameters: Lipid profile: total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL-C). LDL-C calculation: The calculation is done mathematically using the Friedewald formula: LDL-C = TC -HDL-C -(TG/5). The glucose oxidase method is used to measure fasting blood glucose (FBG). Enzymes (ALT, AST) activity was used to evaluate liver cell integrity (Hamed et al., 2023) [5] .

Statistical Analysis

The data were statistically analyzed using the SPSS statistical software (version 26). The difference was compared among groups with One-way ANOVA and the means were compared with Duncan's Multiple Range Test.

Results are presented as mean ± S.E. and the difference was regarded as significant when (P < 0.05).

Results and Discussion

Table 1 shows the changes in serum lipid levels of obese rats fed skim milk with addition of Lactobacillus plantarum.

The findings revealed that the total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL-C) levels were significantly higher (P < 0.05) in the G3 and G4 groups as compared to the healthy control groups (G1 and G2) while the high-density lipoprotein (HDL-C) level was significantly lower (P < 0.05) in the G3 and G4 groups. This breakdown of lipid profile is directly related to eating a high fat diet with fat accumulation. However, in group 5 (G5) who were treated with probiotic enriched skim milk, there was significant improvement with a reduction in total cholesterol to 108.50 mg/dL, triglycerides to 92.40 mg/dL, LDL-C to 48.52 mg/dL and an increase in HDL-C to 41.54 mg/dL. The therapeutic effect of lipids lowering is attributed to the ability of L. plantarum strains to modify the gut microbiome and production of short-chain fatty acids (SCFAs) like acetic and propionic acids. The role of these acids in inhibiting cholesterol synthetic enzymes in the liver and decreasing the gene expression of the genes responsible for lipogenesis is very important (Zhao et al., 2022) [13] . In addition, these bacteria contribute to increased excretion of bile acids in feces, forcing the liver to utilize cholesterol from the blood to synthesize new bile acids, thus lowering their blood levels (Zheng et al., 2023) [14] . Table 2 shows the effect of skimmed milk fortified with Lactobacillus plantarum on blood liver function markers (AST and ALT) in obese rats. There was liver damage and impairment in groups G3 (obesity induced) and G4 (obesity induced), as reflected by the significant increase in liver enzymes; the mean AST level was 90.22 U/L and 92.49 U/L, respectively; and the mean ALT level was 78.66 U/L and 79.30 U/L, respectively. These markers were significantly lowered in G5 (P < 0.05) and were 62.88 U/L for AST and 43.51 U/L for ALT as compared to G3 and G4.

It is a good predictor of non-alcoholic fatty liver disease (NAFLD) mediated by lipotoxicity and oxidative stress from obesity. These enzymes leak into the blood stream if the liver cell membranes are damaged. L. plantarum protects the liver through various mechanisms such as the reduction of hepatic fat, the activation of the antioxidant enzyme system and the decrease of inflammatory cytokine concentrations that cause liver damage and apoptosis (Park et al., 2020) [9] . Table 3 shows that L. plantarum fortified skim milk supplementation had no significant effect on blood glucose and kidney function markers in obese mice. The fasted blood glucose (FBG) was high in obese group G3 (150.27 mg/dL) and G4 (152.45 mg/dL). Meanwhile, L. plantarum showed a significant effect in reducing the FBG level to 110.03 mg/dL in group G5. This is a significant improvement in the insulin sensitivity of cells and a decrease in insulin resistance, as a result of the control of systemic metabolic pathways and low-grade inflammatory condition of the organism which affects insulin sensitivity of cells (Zhao et al., 2022) [13] .

Regarding sort function, the dossier revealed a important decrease in creatinine levels in group 5 (G5), arriving 0.69 mg/dL, distinguished to the contaminated rats camp 3 (G3), that written 0.80 mg/dL. This decrease indicates the act of probiotics in asserting glomerular filtration rate (GFR) and lowering oxidative stress on sort fabric (Lin and others. 2021). As for urea levels, the results show a surprising variation; group 5 (G5) written an important increase of 73.58 mg/dL, distinguished to the not cooked corpulent group (G3), that recorded 43.47 mg/dL. Scientifically, likely the upgraded creatinine index, this increase in urea concede possibility not certainly develop kind failure or damage. Rather, it is more likely the result of two pertain determinants: raised total protein content from graze milk, accompanying raised bacterial effervescence of amino acids in the intestines for one additional microorganisms. This leads to greater levels of liquid that the liver subsequently converts to urea. This end demands further metabolic research to question allure exact pathways. Additionally, L. plantarum microorganisms grant permission play a role in lowering kind fabric damage (Kim and others. 2025) [7] .

Conclusion

Using glance milk as a a ship that carries airplanes for Lactobacillus plantarum bacteria is a very productive working food strategy. This study manifested that this collaboration not only supplies a fat-free digestive medium but also energetically adjusts the metabolic disorders guide obesity by threatening hurtful lipid levels, reconstructing insulin sympathy, enhancing kind function, and lowering liver stress.

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