INTRODUCTION:

Cholesterol is an element that occurs naturally in the body of humans that promotes cell wall construction and hormone production. The lining of the gastrointestinal tract and liver can generate around 80% of the freshly produced (endogenous) cholesterol needed by the body¹, providing only 20% for nutritional (exogenous) cholesterol. Still, elevated cholesterol levels can lead to hypercholesterolemia.

This causes cholesterol accumulation in blood vessels, resulting in blocks or narrowing of the arteries that feed blood to the cardiovascular system and brain. Hypercholesterolemia is the primary precursor for CVD (cardiovascular disease)².

Humans have used probiotics to enhance their health and assimilate various nutritional components. Probiotics provide several advantages, including reduced risk of infections caused by microbial agents, especially in the gastrointestinal tract, resistance to and management of diarrhea, blood pressure regulation, cholesterol decrease, allergy suppression, phagocytosis, and leucocyte stimulation³.

Several studies have looked into probiotics' ability to reduce lipid levels and their hypocholesterolemic effects⁴. Probiotic bacteria produce bile salt hydrolase (BSH), which assists in reducing blood cholesterol. BSH effectiveness is considered while choosing probiotics ⁵. Research has shown that changes in the bacterial lipid composition at the cellular level indicate changes in the content of the fermentation medium ⁶. After having been eliminated from the media, the bacterial cell membrane's fatty acid content changed due to cholesterol absorption ^{7, 8}. For decreasing cholesterol levels in the blood, numerous oral marketed probiotics, such as Lactobacillus acidophilus, have been recommended ⁹.

Numerous investigations on biological activity, therapeutic uses, and pharmaceuticals have documented the benefits of probiotics. The study of exopolysaccharide boosted anti-colon cancer action, suggesting that exopolysaccharide and its nanocrystalline material form could be created by the probiotic Lactobacillus brevis for use in humans ¹⁰. According to additional research, Enterococcus faecium, a probiotic, has a broad spectrum of potency versus both Gram +ve and -ve bacteria, making it a viable biotherapeutic agent. As such, it can be investigated further for potential uses in treating pathogenic bacterial illnesses ¹¹.

The probiotic Lactobacillus brevis was extracted from cow's milk and examined for its probiotic features in the other research. The findings indicate that the substance have an inhibitory impact on Gram-negative as well as Gram-positive bacteria. Its prospective usage in animal models of diabetes treatment might clear a path to improve the management of type 2 diabetes in human beings ¹².

The BSH enzyme, which is found in many different types of bacteria that comprise the human gastrointestinal microbiota, acts a significant role in the metabolism of bile acids. It catalyses the hydrolysis of bile acids linked with taurine and glycine into free bile acids and residues of amino acid, which are less easily reabsorbed than their conjugated cousin. As a result, stool excretes higher amounts of free bile acids. Consequently, deconjugation of bile salt decreases blood cholesterol because it increases the need for cholesterol for the new generation of bile acids to replace those pushed out in stool. Increased conversion of cholesterol to bile and loss of bile salt are caused by decreased bile salt transport to the liver feedback inhibition ¹³.

In the present research, we used a number of in vitro tests to figure out Lactobacillus reuteri's efficacy as a cholesterol-lowering agent. As an outcome, our goal was to assess multiple strategies for eliminating cholesterol. Additionally, we wanted to produce more data that would add to our understanding of the hypocholesterolaemic consequences of lactobacilli in order to develop new multifunctional pharmacological forms, such as tablets. Therefore, in order to optimize the activity of cholesterol assimilation, the current study aimed to apply the Plackett–Burman design (PB) to examine the relevant aspects for Lactobacillus reuteri's culture procedures and media.

MATERIALS AND METHODS:

Probiotic and growth medium:

The Lactobacillus reuteri NRRL B-14171 strain was selected from the Agricultural Research Service's (the U.S. Department of Agriculture's in-house research arm) culture collection. The strain underwent a 24-hour aerobic incubation at 30°C after being sub-cultivated in 10 millilitres of De Man, Rogasa, and Sharpe broth (MRS) broth (Fluka No. 93780) ¹⁴. The turbidity of the proliferation of bacteria was measured using the 0.5 McFarland standard, which involved use a standard inoculum of 100 microliters (1x10⁷ cells/ml) of bacterial population and incubating it for 24 hours at 30°C under static circumstances ^15,16.

Cholesterol assay:

The ability of the probiotic Lactobacillus reuteri to utilize cholesterol from MRS broth was examined. After adding 100 g/mL of cholesterol-PEG 600 to MRS broth, it was incubated for 24 hours at 30°C. After the time of incubation, the cells were processed, and the enzymatic approach was used to determine what quantity of cholesterol was still present in the broth. To perform this, mix the filtrate well, extract 10 microns, add 1 milliliter of the enzymatic reagent, and incubate in a water bath at 37ºC for 10 minutes.

Total cholesterol minus residual cholesterol equals removal cholesterol. The standard curve and reference solutions were created in the same way as previously described. After that, the residual cholesterol is calculated using figure 1 of the standard curve (y = 0.0004x + 0.028) ¹⁷.

Figure 1: Cholesterol standard curve

Plackett–Burman design (PBD) for cholesterol assimilation using Lactobacillus reuteri:

The most useful factors that have a major impact on the absorption of cholesterol were found using PBD. By examining the impacts of each variable without conducting several trials, PBD may effectively eliminate ineffective variables and screen a large number of variables with reliability ^18-23. PBD was utilized in this experiment to determine the importance of numerous factors in preventing the absorption of cholesterol. Regarding Lactobacillus reuteri, To optimize the percentage of cholesterol assimilation (CA%), PBD demonstrated great efficiency and accuracy. To test and measure these five factors, five different trials were conducted: pH value, inoculum size, bill salt concentrations, incubation length, and cholesterol concentrations. Each independent variable was set at two levels the highest level (1) and the lowest level (–1) as illustrated in table 1. The Minitab software (V18, Minitab Inc., State College, PA, USA) was used. All trials were prepared in screw cap glass tube 20 ml containing 10 ml medium. All experiments were done in triplicate and the average value was calculated. The CA% were assayed of each run. Plackett-Burman screening design depends on the first order model equation 1:

Y = β₀ + Σ β_iX_i

In this model, Xi is the variable, β0 is the model intercept, βi is the variable estimate, and Y is the response (% of cholesterol uptake). By utilizing basic regression analysis to calculate the p-value, the significance of the variables was ascertained.

Table 1: Factors and levels used in PBD

No.	Factors	Level -1	Level 1
1	Cholesterol conc.(mg/dl)	100	200
2	Incubation Period (h)	24	48
3	Bill salt Conc.(%)	0.5	1.0
4	pH value	4.0	6.5
5	Inoculum size (µl)	100	200

Qualitative assay of BSH activity:

The BSH measurement method was modified from Dashkevicz and Feighner; Ahn et al. ^24,25. We used soft MRS agar (pH 5.6; Oxoid), which contained bacteriological agar (7.5 g/l; Oxoid), CaCl₂ (0.37 5 g/l; Lach-Ner, Neratovice, Czech Republic), bile salts (0.1, 0.2, 0.3, 0.4, and 0.5% w/v; Ox Bile, Himedia, India), and MRS broth (52.5 g/l; Oxoid).
Petri dishes containing agar were incubated in an anaerobic atmosphere at 37 °C for 48 hours. After growing for 18 hours, 10 μl of Lactobacillus reuteri was punctured into MRS agar to inoculate it. The visible halos surrounding the punctures indicate positive BSH activity. Then, the diameters of the halos were measured to assess the outcome. As the negative control, Lactobacillus reuteri cultured on MRS agar without bile salts were employed. Three repetitions of the measurements were made.

Quantitative assay BSH activity:

The following two steps were taken in order to calculate BSH activity:

As a substrate for BSH, 4 mM sodium thiocholate was incubated in a water bath for 5 minutes at 37 ^oC in the first step. Subsequently, 80 μl of 0.1 M sodium phosphate buffer pH 6.5 (including 20 mM ɑ-mercaptoethanol and 1mM EDTA) was combined with 20 μl of crude enzyme added to the substrate. After that, the reaction was incubated for 30 minutes at 37 °C. After adding 50 μl of 15% trichloroacetic acid (TCA) for fifteen minutes to halt the enzymatic action, the precipitated protein was extracted using a high-speed microfuge. The supernatant was taken off in order to calculate the BSH activity test.

Second phase: The reaction mixture was placed in a boiling water bath for 15 minutes after 20 μl of supernatant and 80 μl of distilled water were combined in a test tube and each tube was filled with 1.9 ml of ninhydrin reagent. The absorbance of the cooled sample was measured at 570 nm. The BSH activity (unit/ml) was the amount of enzyme that released one micromole of glycine or taurine from the substrate per minute ²⁶.

Scanning Electron Microscopy (SEM):

The Hassanein et al. ²⁷ protocol was employed in order to produce Lactobacillus in MRS broth either with or without cholesterol. Applying SEM, the adherence of cholesterol towards lactobacilli cells was examined. The cell was prepared as previously described and re-suspended for four hours in fixative (4%, v/v, glutaraldehyde, Sigma–Aldrich). Lactobacillus reuteri cells were centrifuged, and then the cell was immersed in ethanol at concentrations of 30, 50, 70, and 90%. This process was repeated to ensure dehydration. Distilled water was used twice to clean the cell. The cells were dried, then put on a SEM specimen stub, sprayed in gold for five minutes, then examined with an QUANTA FEG250 SEM.

RESULTS AND DISCUSSION:

Multifactorial statistical PBD for cholesterol assimilation:

For the purpose of to measure the impact of five independent factors, including cultural requirements, on the assimilation of cholesterol by Lactobacillus reuteri in 24 practical runs, Resolution IV, a 2-level Plackett-Burman design was created. The generated runs, as well as the mean of the triplicate results in cholesterol assimilation percentage (CA%) for each run, are compiled in Table 2. Over the course of the 24 distinct runs of the PBD experiment, a significant range in the percentage of cholesterol assimilation by the Lactobacillus reuteri strain was observed. There was a range of 51 to 97% variance. At run 22 (cholesterol concentrations of 100 mg/dl, incubation time of 24 hours, bill salt concentrations of 0.5%, pH 6.5, and inoculum size of 200 µl) at 30 ^oC, the greater CA% (97%) was attained. Despite the fact that run 14 (cholesterol concentrations 200) produced the lowest CA% (51%) (cholesterol concentrations 200 mg/dl, incubation period 24 h, bill salt concentrations 1%, pH 6.5 and and inoculum size 100 µl) at 30 ^oC.

ANOVA analysis:

The model coefficients R2, adjusted R2, and anticipated R2 were determined in order to establish the model's significance. The calculated values for the R2 found to be 64.79%, showing that the model could explain the data in PBD. Additionally, the predicted R2 and corrected R2 values were 37.40% and 55.01%, respectively. PBD calculated ANOVA is summarized in Table 3. Analysis of variance is used to identify the significant factors affecting the CA%. The probability of error caused by the respective variable is represented by the p-value, where p-value < 0.05 indicates a significant effect on the model and p-value > 0.05 indicates an insignificant effect on the model. it have been found that cholesterol concentrations, incubation period, pH value and inoculum size showed a significant effect (p-value < 0.05) on the CA%, while the other factor and the interactions between it showed p-value > 0.05. These results were confirmed by the pareto chart and normal plot of standardized effects as shown in figure 2 and 3.

For the data means acquired throughout the optimization trial runs, a principal impact graphic was created (Fig. 4). The main effect plots and the residual normal probability plot in Figure 5 demonstrated the association between the factors and their responses expressed as a percentage of the total.

The five potential mechanisms by which lactobacilli remove or reduce cholesterol in vitro from media include assimilation of cholesterol during development, binding of cholesterol to cellular surfaces, disruption of cholesterol micelle, deconjugation of bile salt, and BSH activity. In order to offer extra nutraceutical benefits, the strain under study may be employed as possible health supplement cultures in fermented dairy products ²⁸.

Statistical PBD is situated on using a less number of experiments rather than concurrently connecting the effects of numerous factors and determining the optimum factor for ideal conditions ²⁹. El-Waseif et al. ³⁰ noted that the viable parameters for cholesterol removal were examined for the five-probiotic strains investigated. Incubation time and inoculum size are the greater essential factors affecting cholesterol removal. Therefore, the ideal cholesterol concentration is 100 μg/ml, the growth time is 48 h, bile salts are 0.5%, the probiotic dose is 200 μl, and the initial pH is 6.

In another research project, the experimental factorial design was utilized to maximize the conditions for cholesterol assimilation using Lactobacillus paracasei ATCC 25302. The model indicated that 120 µg/ml, 96 hours of incubation, 0.1% bile salts, 200 µl of probiotics, and an initial pH of 6.5 were the ideal values for cholesterol assimilation. Up until 120 µg/ml, when the maximum cholesterol assimilation (93%) was reached, the assimilation of cholesterol increased as the concentration of cholesterol increased ³¹.

The factors under consideration were chosen based on their significance as determined by the in vivo cholesterol absorption mechanism. Reduced cholesterol assimilation led to a further rise in cholesterol levels because low cholesterol assimilation is likely the outcome of irreversible or inactive structures formed by high cholesterol assimilation. Factors that were researched and employed to transform an optimal strain into a tablet-like medicinal form. The potential applications of probiotic microorganisms under ideal conditions for the treatment of physiological diseases are highlighted by our research.

Table 2: The Plackett-Burman layout for CA % using Lactobacillus reuteri

Runs	Cholesterol conc.(mg/dl)	Incubation period (h)	Bill salt conc. (%)	pH value	Inoculum size (µl)	Mean Response (%)
1	200	48	1	4	200	79
2	200	48	0.5	6.5	100	66
3	100	48	0.5	4	100	88
4	100	48	1	6.5	100	80
5	100	24	0.5	4	100	73
6	100	48	1	4	200	83
7	200	24	1	4	100	52
8	100	24	1	6.5	200	95
9	200	24	1	4	100	55
10	100	48	0.5	4	100	72
11	200	48	1	4	200	82
12	100	48	1	6.5	100	77
13	200	48	0.5	6.5	200	91
14	200	24	1	6.5	100	51
15	100	24	0.5	4	100	73
16	200	48	0.5	6.5	200	89
17	100	24	1	6.5	200	81
18	200	24	1	6.5	100	86
19	100	48	1	4	200	91
20	200	24	0.5	4	200	66
21	200	48	0.5	6.5	100	93
22	100	24	0.5	6.5	200	97
23	100	24	0.5	6.5	200	96
24	200	24	0.5	4	200	63

Table 3: Analysis of Variance

Source	DF	Adj SS	Adj MS	F-Value	P-Value
Regression	5	2856.5	571.31	6.62	0.001
Cholesterol conc.	1	737.0	737.04	8.55	0.009
Incubation Period	1	442.0	442.04	5.13	0.036
Bill salt Conc.	1	126.0	126.04	1.46	0.242
pH values	1	651.0	651.04	7.55	0.013
Inoculum size	1	900.4	900.37	10.44	0.005
Error	18	1552.4	86.25
Lack-of-Fit	6	296.9	49.49	0.47	0.816
Pure Error	12	1255.5	104.63
Total	23	4409.0

Figure 2: Pareto graph showing the different factors tested and their standardized estimates for CA%

Figure 3: Normal plot of the standardization effects

Figure 4: The main effects plot for response

Figure 5: The residual normal probability plot

Qualitative assay of bile salts hydrolases activity:

Agar plates were used to measure Lactobacillus reuteri's BSH activity. As seen in figure (6), precipitation zones' diameters were 2, 4, 6, 6, and 9 mm, and the bile salt concentrations were 0.1, 0.2, 0.3, 0.4, and 0.5, respectively. Different bile salts can lead to different Lactobacillus species performing different BSH activities. After testing twelve Lactobacilli isolates for the generation of BSH, Lafy and Alash ³² discovered that only three isolates showed BSH output precipitation zones (cholic acid) surrounding the filter paper on the plate. The diameter of the zones was 16, 14, and 11 mm, respectively. Specifically, bile salts formed from glycine hydrolyze faster than bile salts obtained from taurine ^33,34. Corzo and Gilliland ³⁵ demonstrated in their investigation that three strains of Lactobacillus acidophilus hydrolyzed cholylglycine sodium salt more quickly than cholyltaurine.

Figure 6: The precipitation zone of BSH activity of Lactobacillus reuteri grown on MRS with bile salt concentrations (0.1, 0.2, 0.3, 0.4 and 0.5).

Quantitative assay of bile salts hydrolases:

Afterwards the qualitative test, Lactobacillus reuteri's ability to produce BSH was measured numerically. A BSH activity of 18.83 U/ml was observed. The concentration of released amino acids was calculated using a standard curve of absorbance at 570 nm that was made using glycine.

BSH activity demonstrated by varying levels of amino acids released in broth that contained either a combination of sodium taurocholate and sodium glycocholate, oxgall, or sodium taurocholate alone ²⁸. According to Lafy and Alash ³², a quantitative method (the ninhydrin method) was used to evaluate the formation of BSH by all Lactobacillus isolates. Except for two, every Lactobacillus isolate used in this study shown a range of TCA deconjugation activity (1.21 – 17.25) U/ml. The generation of BSH was higher in the Lb8 and Lb6 isolates. According to De Smet and Verstraete ³⁶, BSH entirely degraded all of the conjugated bile salts of oxgall without the help of additional phospholipids, cholesterol, or reaction products like taurine, glycine, or cholic acid.

Scanning electron microscopy:

Lactobacillus reuteri was a small, rod-shaped, full bacterium with a wrinkled surface that was grouped singularly when it was grown in the MRS, as seen in Fig. 8. Furthermore, the morphology of the strain changes to a slender, long rod-like structure with a concave surface and changing length when high cholesterol concentrations are present (Fig. 8B). Additionally, Kimoto et al. ³⁷ found that Tween 80 improves Lactococcus lactis's ability to tolerate bile. Furthermore, it was discovered that the peptidoglycans found on bacterial cell walls, which are composed of amino acids with the ability to bind cholesterol, have certain chemical and structural properties that lead to a physical phenomena whereby cholesterol is attached to bacterial cells.

Consequently, it might be because of tween-80's pro-solvent, stabilizing, and protecting effects on Lactobacillus cell membranes, which might enable Lactobacillus to continue its regular metabolism in a medium with high cholesterol concentrations ³⁸. The application of eco-friendly probiotics as reducing agents rather than risky chemicals, for synthesis nanoparticles has several advantages than chemical synthesis ^39-42.

CONCLUSION:

The hypocholesterolemic processes in the Lactobacillus reuteri strain, confirmed using the multifactorial statistical Plackett-Burman Design (PBD) to improve cholesterol uptake conditions. The greatest level of cholesterol assimilation (97%) occurred at the level of cholesterol 100 mg/dl, incubation time 24 hours, bill salt concentrations 0.5%, pH 6.5, and inoculum size 200 µl at 30 ^oC in MRS broth. The diameter of the precipitation zones was (2, 4, 6, 6 and 9 mm) for bile salt levels of (0.1, 0.2, 0.3, 0.4, and 0.5%). The quantitative values of BSH enzyme activity were found to be 18.83 U/mL for sodium thioglycocholate substrate. The SEM results probiotic strain in cholesterol-containing medium had a nearly empty and smooth morphology. The findings suggested that lactobacilli might eliminate cholesterol in vitro via a variety of pathways and may have similar hypocholesterolemic effects in vivo.

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Received on 20.03.2025 Revised on 12.07.2025

Accepted on 22.09.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):193-200.

DOI: 10.52711/0974-360X.2026.00029

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