Evaluation of the Therapeutic Impact of Probiotic-Enriched Orange and Pomegranate Juices on Some Health Parameters in Diarrhea-Induced Laboratory Rats

This research endeavor was undertaken to craft a functional beverage by blending orange and pomegranate juices, enriched with the inclusion of therapeutic probiotic strains, specifically Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus rhamanous. A comprehensive assessment was conducted to investigate the physicochemical, microbial, and sensory characteristics of the resulting beverage. Furthermore, we explored the potential health benefits of this fortified beverage within a rat model afflicted with E. coli infection, focusing on its influence on blood parameters and lipid profiles in male rats. The research findings revealed the presence of four distinct bacterial strains, namely Staphylococcus hominis, Staphylococcus aureus, E. coli, and Enterococcus gallinarum, in natural juices obtained from local markets in Tikrit. Regarding the physicochemical properties and bacterial counts in the manufactured juices, the results indicated a decline in pH levels and total sugar content over time, coupled with an increase in total acidity during refrigerated storage of functional juices. Additionally, the enumeration and viability of fortifying bacteria surpassed the prescribed probiotic threshold in the tested juice varieties. Sensory evaluation outcomes demonstrated the overall acceptability of the manufactured juices. The research findings demonstrated that E. coli-induced diarrhea led to a significant elevation (P<0.05) in key blood parameters, including white blood cell and platelet counts (12.63 and 747 mm^3/liter), along with a notable reduction in red blood cell counts and hemoglobin concentration (6.0 and 13.1 mm^3/liter, respectively). However, oral administration of functional juices resulted in a decrease in white blood cell and platelet counts and an increase in red blood cell counts and hemoglobin concentration across all measured parameters. Regarding lipid profiles, the diarrheal group exhibited elevated levels of detrimental lipid markers, including cholesterol, triglycerides, and low-density lipids (73.59, 71.61, and 14.8, respectively), accompanied by reduced high-density lipoproteins concentration (31.5). Conversely, treatment with oral doses of functional juices led to a reduction in these adverse lipid indicators for all treatment groups.


Introduction
The term "functional food" typically refers to food and beverages that offer health benefits beyond the basic nutrients inherent in typical dietary items.Conventional nutrients encompass vitamins and minerals, whereas functional foods are typically enriched with components capable of positively impacting human health.These foods often resemble staples of regular diets, including dairy products, bread, and certain beverages, as they incorporate bioactive compounds like vitamins, minerals, dietary fiber, and probiotic bacteria that promote the balance of gut microbiota [1,2].
The proliferation of functional foods in recent years can be attributed to the growing awareness of nutritional health and the increasing life expectancy, resulting in a larger elderly population.Escalating costs associated with conventional medical treatments have also prompted consumers to seek alternative remedies [3].While probiotics have traditionally been incorporated into dairy products, the food industry is now exploring other suitable options.Consequently, the production of fermented fruit juices emerges as a promising compromise, offering an ideal medium for the delivery of probiotic bacteria.These probiotic strains are known to generate a multitude of biologically active compounds, including vitamins, antioxidants, and amino acids.When integrated into fruit juices, they hold the potential to synergistically enhance health benefits derived from both sources [4].These juices or beverages play a pivotal role in promoting cardiovascular health and reducing sugar and cholesterol levels.Additionally, their rich fiber content contributes to digestive system health, bolstering the body's immune defenses.They exert various positive effects, including the production of antibacterial or bacteriocin-like compounds and defensin, modulation of intestinal microbiota, pH regulation, competition for nutrients, prevention of intestinal carcinogenesis, inhibition of cholesterol absorption, and the displacement of pathogenic bacteria [5,6].Furthermore, these functional drinks exhibit antioxidant properties, aid in cancer prevention, and enhance joint function [7,8].have underscored the significance of functional beverages in promoting cardiovascular health, aiding in weight management, and serving as adjunctive measures to mitigate the effects of aging.Consequently, the present study sought to explore alternative substrates for the development of non-dairy probiotic-rich functional food items.Specifically, our focus was on the production of functional fruit juices fortified with probiotics and prebiotics, with an emphasis on synergizing these components.Subsequently, we aimed to evaluate their efficacy in addressing diarrhea induced by E. coli bacterial infection [9,10].

Ethics approval
All study animals were treated and handled in accordance with the necessary biosafety and security protocols.Before beginning this study, the Ethics Committee of the College of Agriculture, Tikrit University, Ministry of Higher Education and Scientific Research, Iraq (Number 43 A.P. on Sep, 2022) accepted the guidelines for the Care and Use of Laboratory Animals and the Rats guide.

Isolation and diagnosis of bacterial species used in the study
The bacterial species under investigation were isolated and identified from samples collected from various natural juices available in the local markets of Tikrit city.Subsequently, these bacteria were cultivated on a variety of culture media, including Mannitol Salt Agar, MacConkey Agar, and Nutrient Agar.Comprehensive microscopic examinations, phenotypic assessments, and cultural tests were conducted to facilitate their accurate identification.The Vitek2 Compact system was employed as a diagnostic tool to confirm the identity of these bacterial strains.In parallel, the probiotic bacterial strains Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus rhamnosus were sourced from Al-Shifa Medical Center in Salah al-Din Governorate, and their cultivation was carried out on MRS medium.The diagnostic and purification processes for these probiotic species were also validated using the Vitek2 Compact system device.

Growth at different pH concentrations
In the present scientific investigation, Liquid MRS medium was meticulously prepared, varying its pH levels within the range of 1, 2, 3, 4, 6, and 8, employing precise adjustments via the utilization of 0.1 M HCl and 0.1 M NaOH solutions to achieve the desired pH values.Subsequently, the prepared Liquid MRS medium was apportioned into 10 mL test tubes.In each individual test tube, inoculation was carried out by introducing liquid cultures of probiotic bacteria.These inoculated test tubes were subjected to anaerobic incubation conditions, maintaining a temperature of 37°C, over a duration of 24 hours.Subsequent to the incubation period, an assessment for turbidity within the test tubes was performed to render judgment regarding the test outcome, with the presence of turbidity indicating a positive result.This experimental methodology was employed to investigate and ascertain the growth and metabolic activity of the probiotic bacteria within the Liquid MRS medium under the specified conditions [11].

Growth in different concentrations of bile salts
The experimental assessment was conducted through the inoculation of test tubes containing MRS broth, the composition of which was configured by dissolving various concentrations of bile salts (0.1, 0.3, 0.5) into a liter of the medium.Inoculation was achieved by introducing a 1% inoculum from liquid cultures of probiotic bacteria identified as members of the Lactobacillus genus.Subsequently, the inoculated tubes were subjected to an anaerobic incubation regimen at a controlled temperature of 37°C, with a duration of 24 hours.The determination of test positivity hinged upon the visual observation of turbidity within the liquid medium, wherein the presence of turbidity was construed as an indicator of a positive test outcome.This experimental protocol was employed to investigate and ascertain the response of probiotic bacteria from the Lactobacillus genus to varying concentrations of bile salts in the MRS broth under the specified incubation conditions [12].

The investigation delved into the assessment of antimicrobial activity
The evaluation was executed employing the pit diffusion method to gauge the inhibitory activity of the subject bacterial species against the bacterial strains isolated from the juice samples, consistent with the procedures outlined previously [13].

Manufacture of functional juices fortified with therapeutic bacteria
Premium-grade fruits were selected, subjected to juicing, and subsequently filtered.Inulin was judiciously introduced at a rate of 4%.The resulting juice was aseptically dispensed into 80 mL glass containers.Subsequently, the juice underwent pasteurization 1325 (2024) 012030 IOP Publishing doi:10.1088/1755-1315/1325/1/0120304 within a precisely controlled water bath, attaining a temperature of 85°C and maintaining it for a duration of 10 minutes.Post-pasteurization, the juice was swiftly cooled to a temperature of 37°C and subsequently inoculated with probiotic strains.The liquid matrix was fortified with probiotic bacteria, constituting 3.75% of its composition.Each type of juice was subjected to seven distinct treatment conditions, each treatment having two replicate samples.These inoculated samples were incubated anaerobically at a rigorously maintained temperature of 37°C for distinct durations of 24 and 48 hours.Following the stipulated incubation periods, the samples were transitioned into refrigeration storage conditions at 4°C, where they were meticulously preserved for varying intervals spanning a duration of 4 weeks.This comprehensive experimental regimen was undertaken to explore the impact of these conditions on the fermentative and probiotic characteristics of the juices [14].

Sensory tests of juices fortified with therapeutic bacteria
In the scope of this research, sensory sterilization was systematically conducted on probiotic juice samples that had undergone fermentation over varying time intervals and were subsequently subjected to cold storage conditions.The sensory assessment encompassed a comprehensive evaluation, encompassing key attributes such as appearance, flavor, color, taste, and overall acceptability.This rigorous sensory analysis was executed to ascertain the impact of different fermentation durations and refrigeration storage on the organoleptic characteristics and consumer acceptability of the probiotic-enriched juice formulations.

Physicochemical and microbial tests for juices fortified with therapeutic bacteria
Physicochemical analyses were conducted on the manufactured juice samples, in accordance with the methods described in the work of [15].These analyses encompassed the following parameters: 1. pH Estimation: The pH of the juice samples was determined utilizing a calibrated pH meter to measure the hydrogen ion concentration.

Total Acidity:
The total acidity of the juices was quantified employing the normalization method, with the results expressed as lactic acid equivalents.

Sugar Content:
The sugar content within the juice samples was assessed through the utilization of the phenol-sulfuric acid method, which provides a precise determination of sugar concentrations.

Probiotic Cell Counts:
The quantification of probiotic bacterial populations within the juices entailed colony counting using the plate counting method.Colonies of the specific probiotic bacterial strains employed in the juice formulations were enumerated on solid MRS (de Man, Rogosa, and Sharpe) medium.These plates were incubated under anaerobic conditions at 37°C for varying intervals spanning a duration of 4 weeks, facilitating the tracking of probiotic cell counts over time.These rigorous physicochemical tests were instrumental in characterizing the composition and stability of the probiotic-fortified juices.

Vital Experiment
The experimental cohort consisted of 55 male rats, specifically of the Dawley-Albino Sprague strain, characterized by an average age range of 8 to 9 weeks and an average body weight ranging from 146 to 148 grams.These rats were meticulously reared and maintained in stainless steel cages under controlled environmental conditions.The welfare of the animals was vigilantly attended to, ensuring they received adequate nourishment, proper lighting, effective ventilation, a constant ambient temperature set at 25 degrees Celsius, and a relative humidity level ranging from 45% to 70%.In strict adherence to established methodologies, inspired by the work of [16], the research subjects were randomly apportioned into eleven distinct groups, each comprising three replicates.This systematic division into groups served as the basis for executing a rigorous and comprehensive investigation, which aimed to discern the impact of various experimental conditions on the physiological parameters under scrutiny.
1. C1 Group: This served as the control group, representing the baseline condition for the study.
2. C2 Group: This group consisted of rats that were intentionally infected with E. coli bacteria but received no treatment.
3. T1 Group: Rats within this group were subjected to E. coli infection and subsequently treated with orange juice enriched with Lb. acidophilus bacteria.
4. T2 Group: Similar to the T1 group, rats in this category were infected with E. coli bacteria and then treated with orange juice, but enriched with Lb. plantarum bacteria.
5. T3 Group: Rats in this group underwent E. coli infection and were subsequently treated with orange juice fortified with Lb. rhamnosus bacteria.
6. G1 Group: Rats within this group were infected with E. coli bacteria and subsequently treated with pomegranate juice fortified with Lb. acidophilus bacteria.
7. G2 Group: Similarly, rats in this category were infected with E. coli bacteria and treated with pomegranate juice, which was enriched with Lb. plantarum bacteria.
8. G3 Group: Rats in this group experienced E. coli infection and received treatment with pomegranate juice fortified with Lb. rhamnosus bacteria.9. P1: Group of rats infected with E. coli bacteria and treated with Lb.acidophilus probiotic.
10. P2: Group of rats infected with E. coli bacteria and treated with the probiotic Lb.plantarum.
11. P3: Group of rats infected with E. coli bacteria and treated with the probiotic Lb.rhamnosus.
In our study, rats were deliberately induced with E. coli-induced diarrhea and subsequently treated with daily oral doses of therapeutic bacteria-fortified juices, administered at a 2 ml dose, in comparison to the standard McFarland solution at a rate of 1.5 x 10^8 cells per day.The experimental period spanned 28 days.Following the conclusion of the experiment, rats underwent a 20-hour fasting period before being anesthetized with chloroform.Subsequently, blood samples were collected for specific health assessments.Parameters such as the total red blood cell count, total white blood cell count, hemoglobin percentage (HCT), and platelet percentage were determined using a Complete Blood Picture device.Additionally, fat levels were quantified according to the method described in [17], utilizing standard solution kits from the French company Biolabo.

statistical analysis
Statistical analysis was executed employing the established statistical software program [18], utilizing the Completely Randomized Design (CRD) methodology.The determination of statistical significance among the mean values of the factors influencing the observed traits was achieved by employing the Duncan test [19] as the chosen post hoc test.A significance threshold of 0.05 was employed to ascertain the presence of statistically meaningful differences among the assessed factors and their impact on the observed traits.This robust statistical approach facilitated the rigorous evaluation of experimental data, enhancing the reliability and validity of the findings in the study.

Tests for selecting the types of probiotic bacteria
The findings from the pH tolerance assessment, as presented in Table (1), reveal that all strains of probiotic bacteria, including Lactobacillus acidophilus, Lb. plantarum, and Lb.rhamnosus, exhibited the capacity to proliferate and sustain growth within environments characterized by pH levels of 3, 4, 6, and 8.However, these same strains demonstrated a notable incapacity for growth when subjected to pH conditions of 1 and 2. These empirical results are in concordance with the anticipated outcomes, aligning seamlessly with the expectations as outlined in previous sections of this study [20,21].The test outcomes, as depicted in Table (2), elucidate that the probiotic strains, namely Lb. acidophilus, Lb. plantarum, and Lb.rhamnosus, exhibited growth capacity within bile salt concentrations of 0.1% and 0.3%, while their growth was notably inhibited in the presence of 0.5% bile salt concentration.These empirical findings corroborate the assertions made earlier in the research, reaffirming the anticipated responses of the probiotic bacteria to varying concentrations of bile salts [22].

Inhibitory activity of probiotic bacteria against bacteria isolated from juice by method of diffusion in pits
Table (3) provides an overview of the inhibitory susceptibility exhibited by the probiotic strains employed in this investigation, specifically Lb. acidophilus, Lb. plantarum, and Lb.rhamnosus, concerning various bacterial strains isolated from natural juices available in the local markets of Tikrit city.The target bacterial strains included E. coli, Staphylococcus hominis, St. aureus, and Enterococcus gallinarum.The inhibitory potential of the probiotic strains was assessed by measuring the diameters of inhibition zones, and it was found to be contingent upon both the probiotic strain and the specific bacterial species under scrutiny.Notably, for Lb.acidophilus, the inhibition zone diameters recorded were as follows: 17 mm against Staphylococcus hominis, 16 mm against St. aureus, 15 mm against E. coli, and 15 mm against Enterococcus gallinarum.These results provide valuable insights into the varying inhibitory capacities of the probiotic strains against different pathogenic bacteria, shedding light on potential therapeutic applications in the context of natural juice preservation and health considerations, In the case of the probiotic strain Lb. plantarum, the investigation yielded inhibition zone diameters of 20 mm, 15 mm, 14 mm, and 16 mm, respectively, when challenged by the bacterial species Staphylococcus hominis, St. aureus, E. coli, and Enterococcus gallinarum.Meanwhile, the probiotic strain Lb. rhamnosus exhibited inhibition zone diameters measuring 15 mm, 20 mm, 15 mm, and 22 mm, respectively, against the aforementioned bacterial species.Notably, the largest observed inhibition zone diameter, measuring 22 mm, was achieved by Lb. rhamnosus against Enterococcus gallinarum, underscoring its potent inhibitory effect against this particular bacterial strain.Conversely, the smallest inhibition zone diameter, measuring 14 mm, was recorded for Lb.plantarum when tested against E. coli, indicating a relatively lesser inhibitory impact against this specific bacterium.These findings underscore the diverse and strain-specific inhibitory capabilities of the probiotic strains, highlighting their potential utility in combatting pathogenic bacteria within the context of natural juice preservation and health-related applications.

Sensory test results
Tables ( 4) and ( 5) present the outcomes of the sensory assessment conducted on orange and pomegranate juices fortified with probiotics and inulin.In the case of orange juice, the sensory evaluation indicated a positive consumer reception, with discernible improvements in terms of taste, color, aroma, flavor, and overall acceptability when compared to the control sample.These findings collectively suggest that the incorporation of probiotics and inulin into the orange juice was well-received and yielded a product that aligns with consumer preferences.Similarly, for pomegranate juice, the sensory analysis revealed a favorable response among consumers, denoting significant enhancements in terms of taste, color, aroma, flavor, and overall acceptability when contrasted with the control sample.These observations signify that the addition of probiotics and inulin to pomegranate juice led to an improved sensory profile, further endorsing its appeal and marketability among consumers.The process of lactic acid fermentation, as employed in this study, was found to have a non-detrimental impact on the sensory attributes of both fruit and vegetable juices.Remarkably, the sensory properties of these juices were not compromised and retained their desirability.Furthermore, the utilization of Lb. plantarum as a probiotic culture exhibited notable potential in the production of health-oriented fruit beverages.This particularly holds significance for individuals adhering to vegetarian dietary preferences or those afflicted with lactose intolerance, as it offers an alternative source of probiotics distinct from traditional dairy-based products.Consequently, Lb. plantarum emerges as a viable candidate for the development of wholesome and palatable fruit drinks, catering to the diverse needs and preferences of consumers in pursuit of probiotic-rich dietary choices [23].
As observed in the research by [24], the inclusion of inulin as a prebiotic agent in juice formulations holds the potential to augment the sensory attributes of these beverages.This enhancement encompasses aspects such as flavor enrichment, modulation of sweetness levels, and the mitigation of any undesirable taste notes within the juice.Similarly, findings presented by [25] in their study indicate that the sensory characteristics of color, flavor, aroma, and overall acceptability exhibited no statistically significant differences (at p < 0.05) between juices fortified with probiotics and their respective control counterparts.Furthermore, the probiotic-fortified juices garnered superior scores in terms of color and overall acceptability when compared to the control juices.These insights collectively underscore the favorable sensory outcomes achievable through the incorporation of inulin and probiotics, thus contributing to the development of enhanced and well-received juice products.

Physicochemical and microbial tests for juices fortified with therapeutic bacteria
Tables (6) and (7) present the results derived from the comprehensive physicochemical testing of juices that were both manufactured and fermented with varying strains of probiotic bacteria, namely Lb. acidophilus, Lb. plantarum, and Lb.rhamnosus.These juices underwent fermentation at a controlled temperature of 37°C for distinct durations of 24 and 48 hours, followed by preservation under refrigerated conditions for a period spanning 4 weeks.The findings indicate a discernible decrease in the pH levels of the manufactured juices as the duration of refrigeration increased.Simultaneously, there was an observable increase in the levels of acidity, coupled with a reduction in the percentage of sugars present in comparison to the control sample.For instance, in the case of orange juice treatment T1, pH values were recorded as ( (4.40, 4.35, 4.16, 3.88).These observations highlight the dynamic changes in pH, acidity, and sugar content during the refrigerated preservation of the juices, reflecting the intricate interplay between fermentation and storage conditions.The total acidity values for orange juice over a four-week period displayed variations among the different treatment groups.In the case of treatment T1, the total acidity percentages were recorded as (0.40, 0.47, 0.50, 0.92)%, respectively, for the consecutive weeks.Treatment T2 exhibited values of (0.46, 0.55, 0.72, 1.52)%, while treatment T3 recorded percentages of (0.41, 0.49, 0.67, 1.21)%.On the other hand, treatment T4 displayed values of (0.22, 0.40, 0.77, 1.07)%, treatment T5 exhibited percentages of (0.43, 0.58, 0.79, 0.92)%, treatment T6 showed (0.37, 0.37, 0.57, 0.87)%, and treatment T7 recorded (0.41, 0.43, 0.67, 0.79)%.Furthermore, the total sugar content in orange juice exhibited varying trends across the four weeks.Treatment T1 recorded sugar percentages of (30.7, 30

b
Similar letters horizontally for each treatment by weeks mean that there is no significant difference at the 0.05 probability level; T1 Control sample, T2 Orange juice added with inulin and the probiotic Lb.acidophilus and fermented for 24 hours, T3 Orange juice added with inulin and the probiotic Lb.plantarum and fermented for 24 hours, T4 Orange juice added with inulin and the probiotic Lb.rhamnosus and fermented for 24 hours, T5 Orange juice added with inulin and the probiotic Lb.acidophilus and fermented for 48 hours, T6 Orange juice added with inulin and the probiotic Lb.plantarum and fermented for 48 hours, T7Orange juice added with inulin and the probiotic Lb.rhamnosus and fermented for 48 hours In the context of physicochemical assessments conducted on pomegranate juice fortified with the specified probiotic strains, notable variations in pH values emerged over a four-week period.For instance, in treatment T1, the pH values for pomegranate juice were recorded as (4.07, 4.06, 3.97, 3.57) across the consecutive weeks.Treatment T2 exhibited pH values of (4.06, 4.02, 3.83, 3.57), while treatment T3 displayed values of (4.08, 4.05, 3.91, 3.55).Similarly, treatment T4 recorded pH values of (4.06, 4.05, 3.90, 3.55), treatment T5 exhibited values of (4.06, 4.01, 3.80, 3.50), and treatment T6 displayed (4.06, 4.04, 3.88, 3.55).Notably, treatment T7 was recorded with pH values of (4.09, 4.04, 3.88, 3.55).These observations illuminate the dynamic pH fluctuations experienced by pomegranate juice during the four-week storage period, indicative of the influence of probiotic fortification and preservation conditions on this critical physicochemical attribute.The results of total acidity assessments conducted on pomegranate juice, across the four-week period, revealed distinct trends among the treatment groups.Specifically, in treatment T1, the total acidity percentages for pomegranate juice were recorded as (0.73, 0.78, 0.88, 1.66)%, respectively, for the consecutive weeks.Treatment T2 exhibited values of (0.81, 0.84, 0.92, 1.02)%, while treatment T3 displayed percentages of (0.32, 0.80, 0.82, 0.97)%.In contrast, treatment T4 recorded total acidity values of (0.78, 0.81, 0.88, 1.21)%, treatment T5 exhibited (0.83, 0.85, 0.92, 0.97)%, treatment T6 displayed (0.77, 0.89, 0.90, 1.20)%, and treatment T7 recorded (0.67, 0.67, 0.87, 1.55)%.Furthermore, the analysis of total sugar content in pomegranate juice over the four-week period demonstrated variations among the treatment groups.Treatment T1 recorded sugar percentages of (28, 27.1, 26.1, 26)%, while treatment T2 exhibited values of (23.5, 20.3, 20, 19.6)%.Treatment T3 displayed sugar percentages of (24.8, 20.3, 20, 19.7)%, while treatment T4 recorded values of (24.9, 22, 21.7, 21.1)%.In contrast, treatment T5 exhibited sugar percentages of (23, 21.1, 20, 20)%, treatment T6 displayed (24.1, 21.7, 21.3, 21)%, and treatment T7 recorded (24.3, 21.5, 21.1, 20.7)%.These findings illuminate the dynamic shifts in both total acidity and sugar content in pomegranate juice during the four-week preservation period, elucidating the multifaceted effects of probiotic fortification and storage conditions on these essential physicochemical attributes.The observed decrease in pH levels and total sugar content, coupled with an increase in the percentage of total acidity in the tested juices, can be attributed to the metabolic activities of probiotic bacteria.Probiotic bacteria have a propensity for metabolizing reducing sugars, which serve as ample carbon sources, and this metabolic process culminates in the production of lactic acid.Consequently, the accumulation of lactic acid within the juices leads to a subsequent reduction in pH values, as reported in previous studies such as [26].[27]also corroborated these findings, elucidating that lactic acid bacteria species play a pivotal role in enhancing the utilization of sugars, ultimately resulting in a lowering of pH levels and an elevation in acidity during the fermentation process.These insights underscore the mechanistic basis behind the observed physicochemical changes in the tested juices as a consequence of probiotic fermentation.
The findings presented in this study align with prior research conducted by [28], which similarly documented a decline in pH levels over the course of the preservation period in fermented orange juice.This pH reduction was attributed to the production of lactic acid, a characteristic metabolic outcome of probiotic fermentation.the acidity of the fermented orange juice was recorded at (3.87) after 72 hours of refrigerated preservation.These consistent outcomes underscore the reproducibility and reliability of the observed pH dynamics associated with probiotic fermentation in fruit juices, shedding further light on the influence of preservation conditions and probiotic metabolism on juice physicochemical attributes.The outcomes of the present study exhibit concordance with the findings of [29], affirming the suitability of Lb. plantarum as a fortifying agent for orange juice.reported that the population of Lb. plantarum reached approximately 10^9 colony-forming units per milliliter (CFU/mL) after 48 hours of cryopreservation, reinforcing its efficacy as a potential fortifying microorganism.Furthermore, their study emphasized that fortification of pomegranate juice with bacterial strains from the Lactobacillus genus could impart a protective effect against microbial spoilage.This protective influence was corroborated in our study, where it was observed that the levels of reducing sugars in the fortified juices exhibited a notable reduction of approximately 20% (from 56.0 g/L) and 23% (from 92.9 g/L) between the third day and the fourth week of storage.Concurrently, the levels of lactic acid showed a significant increase, culminating in the highest recorded value of 3.75 g/L during the fourth week of storage.These findings collectively underscore the potential of Lb. plantarum and Lactobacillus fortification to enhance the preservation and physicochemical attributes of fruit juices, signifying their utility in the juice processing industry.
The findings of this study align with prior research conducted by [30], which similarly reported a decrease in the content of total sugars during refrigerated preservation, with reductions of up to 20% attributed to the presence of Lb. acidophilus bacteria.Further observations showed a continuing decline, reaching 11.8% after the third week of preservation in juices fortified with Lb. acidophilus bacteria.These trends are in concordance with the results reported by [23], who also noted a decrease in sugar content with increasing IOP Publishing doi:10.1088/1755-1315/1325/1/01203013 preservation time in juices subjected to fermentation with Lb. plantarum bacteria.These consistent outcomes underscore the reproducibility of the impact of probiotic bacterial activity on sugar levels during juice preservation, shedding further light on the role of specific bacterial strains in influencing the physicochemical attributes of fermented juices.

Stability of probiotic bacteria in fortified orange and pomegranate juice with inulin added during refrigerated storage
Table (8) provides insights into the impact of cold storage on the viability of probiotic bacteria within orange and pomegranate juices fortified with Lb. acidophilus, Lb. plantarum, and Lb.rhamnosus strains.These juices were stored at a temperature of 4°C for a duration of four weeks.The cell counts of the respective bacterial species employed in the study were tracked within treatment T1 orange juice throughout the cold storage period and on days 2, 3, 10, 20, and 30.During this monitoring period, cell counts of (2.92, 2.96, 1.52, 2.2, 1.6) x 10^7 CFU/ml were recorded, respectively.Treatment T2 exhibited cell counts of (2.64, 2.68, 1.44, 1.2, 0) x 10^7 CFU/ml, while treatment T3 recorded (1.76, 1.68, 1.4, 0, 0) x 10^7 CFU/ml.Treatment T4 displayed cell counts of (4.88, 4.92, 2.83, 2.1, 1.0) x 10^7 CFU/ml, while treatment T5 showed (4.76, 4.68, 1.70, 1.4, 0) x 10^7 CFU/ml.Finally, treatment T6 exhibited cell counts of (2.04, 2.12, 1.4, 0.7, 0) x 10^7 CFU/ml.These data illuminate the dynamic changes in probiotic bacterial populations during the cold storage of fortified juices and highlight variations in bacterial viability over the designated storage period.The cell counts within pomegranate juice were diligently monitored over the course of refrigerated storage on days 2, 3, 10, 20, and 30.In treatment T1, cell counts were recorded at (2.76, 2.68, 1.37, 1.3, 1.2) x 10^7 CFU/ml, respectively, during this storage period.Conversely, treatment T2 exhibited cell counts of (2.48, 2.44, 1.43, 0, 0) x 10^7 CFU/ml, and treatment T3 recorded (1.16, 1.12, 1.3, 0, 0) x 10^7 CFU/ml.Meanwhile, treatment T4 displayed cell counts of (4.76, 4.28, 2.36, 2.9, 1.7) x 10^7 CFU/ml, and treatment T5 exhibited (4.40, 4.16, 1.88, 0, 0) x 10^7 CFU/ml.Finally, treatment T6 recorded cell counts of (1.56, 1.60, 1.2, 0, 0) x 10^7 CFU/ml.These recorded values encapsulate the dynamic trends in probiotic bacterial populations within pomegranate juice during the refrigerated storage period, highlighting the variations in bacterial viability across the designated time points.Similar letters horizontally for each treatment by weeks mean that there is no significant difference at the 0.05 probability level In accordance with [31], probiotic strains displayed remarkable resilience within fermented juices, retaining their viability over a three-week period under cryopreservation conditions.Likewise, [23] reported that the population of probiotic Lb. plantarum cells reached 10^8 × 3.36 CFU/mL.Notably, they observed a decline in the numbers of fortifying bacteria over time during cold storage, suggesting the potential influence of storage duration on bacterial populations within these beverages.[32,33] reported no significant variance in the growth and vitality of Lb. rhamnosus bacteria in the presence of inulin.Conversely, other studies have indicated a positive impact of inulin supplementation on the vitality of these cells.[34], for instance, demonstrated that probiotics exhibited robust growth across all treatments, with the inclusion of 2% inulin, without any discernible alteration in the properties or quality of the juice.These findings underscore the variability in the effects of inulin on probiotic cultures and highlight the potential benefits of such supplementation.[35] conducted a study revealing that pomegranate juice containing Lb. plantarum maintained consistently high levels of bacterial cell counts over a three-week period of refrigerated storage, with a particularly significant increase observed during the fourth week of refrigerated storage.This phenomenon could be attributed to the noticeable surge in probiotic bacteria numbers after the initial week, suggesting an adaptive response of booster bacteria to the prevailing environmental conditions within the juice, subsequently facilitating their growth and proliferation.The decline in cell numbers observed as the preservation period advanced may be attributed to alterations in pH levels and an increase in acidity, a phenomenon corroborated by studies such as [36,37].

The Impact of Oral Administration of Functional Juices on Hematological Parameters in Diarrhea-Induced Laboratory Rats
Table (9) reveals a statistically significant increase (P<0.05) in the total white blood cell count and platelet count within the group of rats afflicted with diarrhea induced by E. coli bacteria, measuring (16.4 and 747) mm^3, respectively, in comparison to the control group's readings of (10.83) mm^3 and (731.0)mm^3, respectively.Conversely, the findings also highlight a noteworthy reduction in red blood cell count, hemoglobin concentration, and the percentage of red blood cells, registering values of (6.0) mm^3, (12.9) g/dl, and (35.6)%, respectively, in contrast to the control group's values of (7.3) mm^3, (13.4) g/dl, and (36.6)%, respectively.Oral administration of probiotic-fortified juices to the group of infected rats yielded substantial alterations in hematological parameters.Notably, there was a significant reduction in the total white blood cell count and platelet count, while conversely, the counts of red blood cells and hemoglobin levels exhibited a marked increase when compared to the group of infected and untreated rats.Specifically, treatment T1 demonstrated notable efficacy in lowering the white blood cell count to (12.7) mm^3, treatment G3 exhibited proficiency in decreasing platelet count to (580) mm^3, treatment G1 showed effectiveness in elevating red blood cell count to (7.18) mm^3, and treatment G2 recorded a significant increase in hemoglobin levels, reaching (14.7) g/dl.[38] elucidated that the elevation observed in hematocrit (HCT), red blood cell (RBC) count, and hemoglobin (Hb) levels following the administration of probiotics to animals with diarrhea can be attributed to the concurrent reduction in pH due to the fermentation activities of probiotic bacteria.This shift in the intestinal environment towards acidity is conducive to improved nutrient absorption, particularly the B-Complex vitamins, which enhances digestion and the provision of essential nutrients, including proteins and vitamins.The resultant increase in vitamin B production further augments immune system efficiency and promotes the synthesis of organic acids [39].The findings are in accordance with those of [40], who demonstrated that probiotics exhibit the capacity to enhance the red blood cell count and facilitate their restoration to normal levels through various mechanisms.Among these mechanisms, a pivotal role is played by the reduction in pH attributed to the probioticinduced fermentation processes.Furthermore, the production of vitamin B-complex by probiotics has been identified as a significant contributor to the improvement of red blood cell quality, regulation of blood cell size, and control of hemoglobin concentration, as highlighted by [41].This alignment with prior research underscores the beneficial impact of probiotics on hematological parameters.Furthermore, beyond the previously discussed effects, probiotics demonstrate the capacity to mitigate lipid-related issues by inhibiting the production of detrimental cholesterol.They exert influence over the body's utilization of cholesterol found in cells and tissues, thereby enhancing the efficiency of the circulatory system and promoting overall blood health.These outcomes are consistent with prior research findings, as indicated by [42], highlighting that probiotics not only combat various pathogens, including bacteria, viruses, and fungi, but also contribute to the reduction of blood lipids.Consequently, they play a role in optimizing the size of red blood cells and maintaining their presence within well-balanced parameters.

Clinical Impact of Functional Juices on Lipid Profile in Diarrhea-Induced Laboratory Rats
In Table (10), the research findings unveil a substantial alteration in lipid parameters among rats afflicted with diarrhea when subjected to various orange and pomegranate juices fortified with probiotics.At a confidence level of (P<0.05), a noteworthy escalation is observed in markers indicative of detrimental lipids.Specifically, the levels of total cholesterol, triglycerides, and LDL cholesterol displayed marked increments, registering (73.59 mg/dL, 71.61 mg/dL, and 14.8 mg/dL), respectively.In contrast, the control group exhibited comparatively lower readings, standing at (68.41 mg/dL, 62.68 mg/dL, and 10.8 mg/dL), correspondingly.Notably, HDL cholesterol levels declined significantly, with the experimental group recording (31.12 mg/dL) in contrast to the control group's reading of (40.0 mg/dL).n this study, the incorporation of probiotic-fortified juices yielded favorable outcomes in terms of lipid profile parameters.Across all treatment groups, a consistent reduction in markers of detrimental fats, including cholesterol, triglycerides, and LDL cholesterol, was evident.Treatment T3 particularly excelled in mitigating cholesterol levels, registering a remarkable decline to 49.55 mg/dL.Furthermore, treatment G1 demonstrated exceptional efficacy in reducing triglyceride concentration to 46.78 mg/dL.Notably, treatment G3 exhibited superiority in decreasing LDL cholesterol by 6.1 mg/dL, while treatment T1 excelled in augmenting HDL cholesterol concentration, reaching a level of 69.3 mg/dL.These findings underscore the potential of probiotic-enriched juices as a dietary intervention to ameliorate lipid profiles and promote cardiovascular health.Various letters in one column show significant differences at the 0.05 probability level; C1 Control sample, C2 includes rats infected with E.coli bacteria, T1 Includes rats infected with E.coli bacteria and treated with orange juice to which the probiotic Lb.acidophilus is added, T2 Includes rats infected with E.coli bacteria and treated with orange juice to which the probiotic Lb.plantarum is added, T3 Includes rats infected with E.coli bacteria and treated with orange juice to which the probiotic Lb.rhamnosus is added, G1 Includes rats infected with E.coli bacteria and treated with pomegranate juice to which the probiotic Lb.acidophilus is added, G2 Includes rats infected with E.coli bacteria and treated with pomegranate juice to which the probiotic Lb.plantarum is added, G3 Includes rats infected with E.coli bacteria and treated with pomegranate juice to which the probiotic Lb.rhamnosus is added, P1 Includes rats infected with E.coli and treated with the probiotic Lb.acidophilus, P2 Includes rats infected with E.coli and treated with the probiotic Lb.plantarum, P3 Includes rats infected with E.coli and treated with the probiotic Lb.acidophilus The observed increase in cholesterol values can be attributed to heightened activity of the enzyme cholesterol acyltransferase, responsible for cholesterol absorption, which is stimulated by insulin deficiency due to oxidative stress on pancreatic beta cells induced by active oxygen species.Consequently, we witness an elevation in cholesterol levels absorbed by the intestines [43].Alternatively, this increase may be linked to disruptions in fat metabolism resulting from stress-induced formation of unsaturated fatty acids and peroxides, consequently inhibiting the excretion and secretion of bile salts and steroid substances.Furthermore, it is noted that certain irregularities occur in the absorption and digestion processes within the intestine [44].Numerous research studies have highlighted several mechanisms through which probiotic bacteria can effectively lower blood cholesterol levels.One of the proposed mechanisms is the enzymatic hypothesis, in which probiotics, specifically the BCH enzyme, deconjugate bile acids This deconjugation process renders bile acids less soluble and impedes their absorption in the intestines, ultimately leading to their excretion in feces.As a compensatory response, the body synthesizes new bile acids from cholesterol, thereby reducing the overall serum cholesterol level [45].The inhibition of cholesterol synthesis in the liver can be attributed to various mechanisms associated with probiotic bacteria.This includes the production of short-chain fatty acids, such as propionic acid, by certain probiotic strains [46].Additionally, probiotics may facilitate the return of cholesterol from the bloodstream to the liver.Furthermore, during their growth stages, probiotic bacteria can incorporate cholesterol into their cell membranes Another mechanism involves the precipitation of cholesterol with unconjugated bile acids [47].These multifaceted processes collectively contribute to the regulation of cholesterol levels.

Conclusions
In conclusion, the findings of this study highlight the feasibility of producing nutritionally enriched fermented functional juices with favorable sensory attributes, serving as a promising substitute for probiotic sources traditionally reliant on dairy products.Furthermore, it was observed that E. coli-induced diarrhea led to significant elevations in white blood cell counts, platelet percentages, and reductions in red blood cell counts.Additionally, this condition triggered increased levels of cholesterol, triglycerides, and LDL, coupled with a decline in HDL levels.Notably, oral administration of probiotic-fortified juices to rats demonstrated a constructive impact on mitigating all the assessed adverse health indicators.
for each treatment by weeks mean that there is no significant difference at the 0.05 probability level; T1 Control sample, T2 Pomegranate juice added with inulin and the probiotic Lb.acidophilus and fermented for 24 hours, T3 Pomegranate juice added with inulin and the probiotic Lb.plantarum and fermented for 24 hours, T4 Pomegranate juice added with inulin and the probiotic Lb.rhamnosus and fermented IOP Publishing doi:10.1088/1755-1315/1325/1/01203012 for 24 hours, T5 Pomegranate juice added with inulin and the probiotic Lb.acidophilus and fermented for 48 hours, T6Pomegranate juice added with inulin and the probiotic Lb.plantarum and fermented for 48 hours, T7 Pomegranate juice added with inulin and the probiotic Lb.rhamnosus and fermented for 24 hours

Table 1 .
Probiotic Bacterial Growth at Varied pH Levels + Positive for the test, -Negative for the test

Table 2 .
Probiotic Bacterial Growth in Various Bile Salt Concentrations + Positive for the test, -Negative for the test

Table 3 .
Inhibitory Effectiveness of Probiotic-Fortified Juices Against Pathogenic Bacteria

Table 4 .
Sensory Evaluation of Probiotic and Inulin-Enriched Orange Juice A Orange juice contains inulin and the probiotic Lb.acidophilus, B Orange juice contains inulin and the probiotic Lb.plantarum, C Orange juice contains inulin and the probiotic Lb.rhamnosus

Table 7 .
Physicochemical Analysis of Probiotic-Enriched Pomegranate Juice Preserved Under Refrigeration

Table 8 .
Microbial Counts of Probiotic Bacterial Species in Refrigerated Juices Over Time

Table 9 .
Effect of Probiotic-Fortified Juices on Hematological Parameters in Diarrhea-Induced Rat Model various letters in one column show significant differences at the 0.05 probability level C1 Control sample, C2 includes rats infected with E.coli bacteria, T1 Includes rats infected with E.coli bacteria and treated with orange juice to which the probiotic Lb.acidophilus is added, T2 Includes rats infected with E.coli bacteria and treated with orange juice to which the probiotic Lb.plantarum is added, T3 Includes rats infected with E.coli bacteria and treated with orange juice to which the probiotic Lb.rhamnosus is added, G1 Includes rats infected with E.coli bacteria and treated with pomegranate juice to which the probiotic Lb.acidophilus is added, G2 Includes rats infected with E.coli bacteria and treated with pomegranate juice to which the probiotic Lb.plantarum is added, G3 Includes rats infected with E.coli bacteria and treated with pomegranate juice to which the probiotic Lb.rhamnosus is added, P1 Includes rats infected with E.coli and treated with the probiotic Lb.acidophilus, P2 Includes rats infected with E.coli and treated with the probiotic Lb.plantarum, P3 Includes rats infected with E.coli and treated with the probiotic Lb.acidophilus

Table 10 .
Effect of Probiotic-Fortified Juices on Lipid Profile Parameters in Diarrhea-