Antihyperlipidemic and Probiotic Characteristics of Novel Lactobacillus Strains Isolated from Traditional Iranian Dairy

Abstract

Background and Objective: This study aimed to investigate the antihyperlipidemic effects of potent probiotic Lactobacillus strains isolated from traditional Iranian dairy in a high-fat diet rat model. Material and Methods: Lactobacillus strains were isolated from tarkhineh samples and screened for significant in-vitro cholesterol and triglyceride-decreasing activities, with key probiotic characteristics. Seven strains were selected based on their high in-vitro lipid-decreasing activity, substantial resistance to simulated gastric and intestinal conditions, resistance to 0.5% phenol and 15 mg l-1 lysozyme, adhesion capacity to Caco-2 cells, antibiotic susceptibility profiles, and antagonistic activity against human pathogens in male Wistar rats fed a high-fat diet. The lipid-decreasing activity was assessed in male Wistar rats (n = 7 per group) over 6 weeks, with the probiotic mixture administered daily at a dose of 2 × 109 CFU ml-1 rat-1. Results and Conclusion: Using 16S rDNA analysis, these strains were identified as Lactobacillus casei, Lactobacillus fermentum, Lactobacillus kefiri, Lactobacillus alimentarius, Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus brevis. The lipid-decreasing activity was assessed in male Wistar rats. Compared to the high-fat diet control group, the probiotic-supplemented diet decreased serum total cholesterol by 16.6% and LDL-cholesterol by 56.7% (p < 0.05). Particularly, the probiotic mixture resulted in a 13.2-fold increase in the HDL-c/LDL-c ratio (from 0.132 to 1.75), compared to high-fat diet controls (p < 0.01). Mechanistically, the probiotic diet increased fecal cholic acid excretion by 4.7-fold (from 1.17 to 5.54 µmol g-1) (p < 0.05) and decreased hepatic steatosis. Treatment attenuated high-fat diet-induced upregulation of lipogenic genes (PPAR-γ, ACC, FAS, and C/EBPα) and restored the expression of AMPKα. These results indicated that supplementation with lactobacilli from homemade dairy was effective in improving dyslipidemia, suggesting these products could be a promising source of novel probiotics. Keywords: Probiotics, Traditional dairy, Lactobacillus spp., Tarkhineh, Hyperlipidemia Introduction   High levels of serum lipids are significant risk factors for cardiovascular disease (CVD). Individuals with dyslipidemia have a threefold higher risk of heart attack, compared to those with normal lipid levels. This condition is often related to dietary habits, particularly the consumption of unhealthy diets. Diets high in fat, especially saturated fatty acids (SFA), can increase blood total cholesterol (TC) and triglyceride (TG) levels, which increases the risk of atherosclerosis, coronary heart disease, and stroke [1, 2]. Medications such as statins and fibric acid derivatives are commonly used to manage blood lipid levels. However, these conventional drugs can include adverse effects, including muscle pain, liver damage, neurological disorders, increased blood sugar, and miscarriage. Therefore, there is an increasing need for safer and further cost-effective dietary interventions to address imbalanced serum lipids. Probiotics have emerged as a potential solution for managing dyslipidemia, and their use in functional foods is a promising area of research. Probiotics have been shown to significantly assimilate cholesterol and TG from culture media. Bacteria such as Lactobacillus, Lactococcus, and Bifidobacterium spp. are commonly detected in fermented dairy products [3-5]. Traditional fermented foods contain unique microbial communities that are shaped by local production methods and environmental conditions [6]. Within the lactic acid bacteria (LAB) family, Lactobacillus spp. are the most common members. These bacteria are often isolated from various Iranian traditional fermented foods. One such food is tarkhineh, a dried fermented mixture of yoghurt, cracked wheat, and vegetables, which offers a stable acidic matrix conducive to LAB preservation. The dry acidic nature of tarkhineh allows for the long-term preservation of its milk proteins [7]. While numerous studies have screened probiotics for cholesterol-decreasing characteristics, most isolates originate from well-characterized sources such as commercial yogurts or dairy products with standardized fermentation [8]. The wide microbial diversity of traditional, regionally distinct Iranian fermented foods, such as tarkhineh, as a source of novel strains with potentially superior or unique functionalities, is largely uninvestigated. Moreover, tarkhineh offers a unique ecological niche as a stable acidic matrix with high solid contents. This environment imposes selective pressure on the microbiota, favoring highly robust and acid-resistant Lactobacillus strains [7, 9].  These environmental stressors suggest that isolates from tarkhineh may possess superior functional traits such as enhanced resistance to gastric conditions and greater bile-salt hydrolase activity, compared to strains from conventional sources [10]. Furthermore, studies of novel isolates depend solely on in vitro screening, failing to provide the necessary comprehensive in vivo validation. The precise molecular mechanisms underlying the antihyper-lipidemic effects, particularly regarding the modulation of key hepatic lipogenic genes, are often not fully clarified for novel isolates. Therefore, this study aimed to fill this critical gap by providing a full characterization of novel Lactobacillus strains from tarkhineh, followed by a robust,  mechanistic in vivo validation of their efficacy in a high-fat diet (HFD) rat model. Materials and Methods 2.1 Isolation and Preliminary Identification of Lactobacilli Samples of tarkhineh (n = 22) were collected during July and August 2024 from various rural areas of Lorestan Province, Iran, including Dowlatabad and Mahrouw Villages in Aligoudarz County (33°09′N 49°24′E) and Dehnow and Emamabad Villages in Dorud County (33°29′58″N 49°03′11″E). To prevent microbial contamination and changes to the primary microbiota, samples were prepared under hygienic conditions and stored at 4 °C. For isolation, 5 g of each sample was mixed with 50 ml of sodium citrate (Merck, Germany) and homogenized using a stomacher. The samples were serially diluted up to 10⁻⁷ in sterile saline solution and cultured on de Man, Rogosa, and Sharpe (MRS) agar (HiMedia, India). After anaerobic incubation (Gas-pack system) at 37 °C for 48 h, colonies were inoculated into 10 ml of MRS broth and incubated at 37 °C for 24 h. The isolates were screened through primary morphological assessment using a fluorescent microscope (Zeiss Axioskop, Germany) and standard biochemical assessments, including catalase, oxidase, and carbohydrate fermentation. Screened lactobacilli were preserved at -70 °C in glycerol and skim milk. 2.2 Bile Salts and Low pH Tolerance The ability of isolates to tolerate bile salts was assessed as described by Saboori et al. [11]. Each strain (2% v/v) was grown in MRS broth containing 0.3% (w/v) bile salts (0% as a control). Cultures were incubated at 37 °C for 6 h, and optical density (OD) was measured at 600 nm. For acid tolerance, strains were cultured overnight in MRS broth adjusted to pH values of 2.0, 2.5, and 6.4 (as a control). The OD values at 600 nm were recorded at the beginning and end of cultivation. Tolerance was calculated using methods described by Liu et al. [12]. 2.3 In-vitro Cholesterol and Triglyceride Decreasing Activities The cholesterol-decreasing ability of strains was assessed using the method by Liu et al. [12]. Strains were grown in MRS broth at 37 °C for 24 h and then inoculated into MRS broth supplemented with cholesterol and 0.3% bile salts (MRS-CHOL). A control sample of uninoculated MRS-CHOL was also prepared. After 24 h of incubation at 37 °C without shaking, the bacterial broth was centrifuged, and cholesterol content in the cell-free supernatant was assessed. Strains that lowered cholesterol by more than 50% were selected for triglyceride (TG) decreasing assessments. These strains were grown overnight in MRS broth and then transferred to MRS-TG broth. This was prepared by mixing 20 ml of 2% polyvinyl alcohol solution with 50 ml of triglycerides (Merck, Germany) and adding the mixture to MRS broth (3% v/v). The pH was adjusted to 6.5, and the medium was sterilized. After 72 h of incubation at 37 °C, the TG quantity in the cell-free supernatant was assessed using a commercial kit (Cayman, USA) [13]. 2.4 Cell Survival in Simulated Gastrointestinal Juice To screen for tolerance to gastrointestinal stress, isolated lactobacilli were cultured in MRS broth and incubated anaerobically overnight at 37 °C. The suspension was centrifuged at 6000× g for 7 min, and the cells were washed twice with sterile phosphate-buffered saline (PBS, pH 2.0). Aliquots were cultured on MRS agar and incubated anaerobically at 37 °C for 0, 1, 2, and 3 h. The number of viable bacteria was assessed as colony-forming units per milliliter (CFU ml-1). For assessment under simulated conditions, overnight cultures of isolates were centrifuged at 6000× g for 5 min, washed with 50 mM PBS (pH 6.5), and dissolved in 3 ml of PBS buffer. One-ml aliquots of each isolate (containing 9-log CFU ml-1 of bacteria) were mixed with 9 ml of simulated gastric juice (7 mM KCl, 45 mM NaHCO₃, 125 mM NaCl, and 3 g l-1 pepsin at pH 2.5). The mixtures were incubated at 37 °C for 3 h and then centrifuged at 4000× g for 7 min. The pellets were washed three times with PBS and resuspended in simulated intestinal juice (pH 8.0) containing 0.15% (w/v) bile salt and 0.1% (w/v) pancreatin. Then, the suspensions were incubated at 37 °C for another 3 h, and the viable bacteria were counted and reported as Log CFU ml-1. 2.5 Phenol and Lysozyme Tolerance Phenol tolerance is critical for lactobacilli survival in the gastrointestinal tract (GIT), as phenol can be produced by gut microbiota. Bacterial cultures grown for 24 h were cultured in MRS broth containing 0.5% phenol (Sigma, USA). The ODs were measured at 580 nm using a microplate reader (Thermo Fisher Scientific, USA). Lysozyme tolerance was assessed as described by Zafar et al. [14]. Bacterial cells were collected (4000× g, 5 min), washed, and resuspended in PBS. Then, 10 µl of cell suspensions were transferred to PBS solutions containing 15 mg l-1 lysozyme (0% as a control). After 2 h of incubation at 37 °C, ODs were measured at 600 nm to estimate survival rates. 2.6 Safety Assessment 2.6.1 Antibiotic Susceptibility The antibiotic susceptibility of isolated lactobacilli was assessed based on the method of Saboktakin et al. [15]. Strains (1.5 × 10⁸ CFU ml-1) were cultured on MRS agar. Antibiotic discs were transferred onto the surface, set at room temperature (RT) for 10 min for diffusion, and then incubated at 37 °C overnight. The areas of inhibition around each disc were assessed. 2.6.2 DNase and Hemolytic Activities To assess DNase activity, isolates were streaked on DNase agar (HiMedia, India) and incubated at 37 °C for 72 h, and then clear zones were assessed. Staphylococcus aureus was used as a positive control. Hemolytic activity was assessed on blood agar containing 5% v/v sheep blood. Isolates were cultured on the media and incubated at 37 °C for 48 h. Blood lysis zones were characterized as α-hemolysis (green zones), β-hemolysis (clear zones), or γ-hemolysis (no zones). Only isolates showing γ-hemolysis were considered safe [16]. 2.7 Antibacterial Activity Assessment Antibacterial spectra were assessed using agar diffusion assay [17]. The indicator bacteria were five human pathogens of Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922, Listeria monocytogenes CMCC 54002, Bacillus subtilis ATCC 11060, and Staphylococcus aureus ATCC 25923. These pathogens were overlaid on Mueller-Hinton (M-H) agar plates with 7-mm diameter wells. Freshly prepared cell-free supernatants of each isolate were filtered (0.2-µm filter), poured into the wells, and incubated at 37 °C overnight. Then, diameters of the inhibitory zones were assessed. 2.8 Assessment of the Isolates' Adhesion Ability To assess the ability of strains to adhere to Caco-2 cells, an in vitro model for intestinal epithelia was assessed using the method of Greene and Klaenhammer [18]. Caco-2 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Sigma, USA) supplemented with 1% penicillin-streptomycin and fetal bovine serum (FBS) in a 5% CO₂ atmosphere at 37 °C for 20 days. Cells were transferred to tissue culture plates and grown to a monolayer. The plates were washed with sterile PBS to remove the media, particularly penicillin-streptomycin. Overnight cultures of bacterial strains were centrifuged (5000 rpm, 5 min), washed, and resuspended in DMEM to a concentration of 10⁸ bacteria ml-1. This suspension was added to Caco-2 monolayer cells and incubated at 37 °C for 1 h. Unbound bacteria were removed by washing with PBS. Then, Caco-2 cells were lysed with Triton X-100 (0.1% v/v), and bacterial counts were carried out on MRS agar. Adhesion capacity was calculated using the Eq 1:   Adhesion capacity (%) = (A / B) × 100                                                           Eq.1   Where A was the number of attached bacteria, and B was the total number of bacteria added to each well. 2.9 Molecular Identification and Phylogenetic Analysis Genomic DNA was extracted from overnight bacterial cultures using a commercial kit (Pars Azmoun, Iran). A conserved region of the 16S rDNA gene was amplified using 27F-1492R primers as described by Nami et al. Amplicons were electrophoresed on a 2% agarose gel. The amplified fragments were purified using the PureLink PCR purification kit (Thermo Fisher Scientific, USA) and sequenced by BGI Biotechnology (Shenzhen, China). Sequences were compared to the GenBank database via BLAST similarity search; isolates with over 98% similarity to reference sequences were assigned to the same species. A phylogenetic tree was constructed using MEGA7 software (Biodesign Institute, USA) with the Kimura 2-parameter model and the neighbor-joining method [19]. 2.10 In vivo Study A total of 24 male Wistar rats (age, 6 weeks; weight, 150 g ±15) were purchased from the Pasteur Institute, Tehran, Iran. All procedures were carried out based on the ARRIVE guidelines and approved by the ethical committee of Islamic Azad University (IR.IAU.ARAK.REC.1399.021). Experiments were based on the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH, USA). Rats were acclimated for 1 week with commercial foods (Specialty Feeds, USA) and water to minimize environmental stress. The animals were housed in a controlled environment (23 °C, 55% relative humidity, 12-h light/dark cycle). The rats were randomly allocated into four groups of six animals, with similar initial body weights. The groups received the following diets for 35 days: ND, Normal diet HFD, High-fat diet HFD and Pro, HFD with a potential probiotic mixture  (2 × 10⁹ CFU ml-1 d-1) HFD and Lov, HFD with lovastatin (15 mg kg-1 d-1) The HFD was composed of 78.3% commercial diet, 5% lard oil, 5% corn germ oil, 5% sucrose, 5% dried egg yolk, 1% cholesterol, 0.5% sodium deoxycholate, and 0.2% propylthiouracil. All administrations were carried out via intragastric gavage. The inclusion of propylthiouracil was a deliberate methodological choice to establish a robust model of complex, resistant dyslipidemia. The probiotic dose was selected based on prior studies demonstrating efficacy in rodent models [12, 20]. Blood samples were collected under ketamine-xylazine anesthesia on Days 0 and 35. Serum was extracted, and TC, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and TG levels were assessed using commercial kits (Pars Azmoun, Iran). At the end of the experiment, rats were euthanized using Forane (isoflurane). Livers were extracted for histological studies using hematoxylin and eosin (H&E) staining and for lipid analysis using a method described by Folch et al. Feces were collected on Days 33, 34, and 35 for cholic acid analysis. 2.11 Real-time Polymerase Chain Reaction The expression levels of six lipid metabolism-linked genes were assessed using quantitative real-time PCR (qRT-PCR) [21]. Primers were generated for peroxisome proliferator-activated receptor-γ (PPAR-γ), adenosine 5′-monophosphate-activated protein kinase-α (AMPKα), CAATT/enhancer-binding protein α (C/EBPα), hormone-sensitive lipase (HSL), fatty acid synthetase (FAS), acetyl-CoA carboxylase (ACC), and the internal control GAPDH (Table 1). Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, USA), purified with a Qiagen RNeasy mini kit (QIAGEN, Germany), and quantified by spectrometry (Eppendorf, Germany). The cDNA was synthesized using the RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific, USA). The qRT-PCR reactions were carried out with SYBR Premix Ex Taq II kit (Takara, China) on a PRIMEPRO48 real-time qPCR thermal cycler (Antylia Scientific, USA). Results were calculated using the 2_ΔΔCT method and are shown as fold changes. 2.12 Statistical Analysis Data were analyzed using SPSS software v.18 (IBM, USA) through a one-way analysis of variance (ANOVA). Differences between the means were reported using the Duncan test at a 95% confidence level (p < 0.05). All experiments were carried out in triplicate. Results and Discussion 3.1 Isolation and Screening of Potential Probiotic Lactobacilli A total of 83 Gram-positive, catalase-negative, coccoid bacterial isolates were collected from tarkhineh samples, all of which showed a carbohydrate fermentation profile characteristic of the Lactobacillus genus. These isolates were used in a bile-salt tolerance prescreening, from which 36 strains showing an inhibition rate below 50% were selected for acid tolerance assessment. Of these, 19 isolates demonstrated survival rates higher than 85% at pH values of 2.5 and 2.0. High levels of cholesterol and triglycerides in the diet and serum are major risk factors for cardiovascular and metabolic diseases. Since pharmacotherapy can cause adverse effects, it is important to find natural and non-toxic substances to decrease TC, LDL, and TG levels. Lactobacilli, which have been used in fermented dairy products for many years and are generally considered safe (GRAS) [22], have been shown to effectively decrease lipids in animal and clinical research [23, 24]. In this study, seven potential probiotic lactobacilli strains were isolated from Iranian traditional dairy products. These strains showed enhanced cholesterol and triglyceride-decreasing activity, increased tolerance to simulated gastric and intestinal juice, improved adhesion to human intestinal cells, and expanded antibacterial spectrum under in vitro conditions. 3.2 In-vitro Cholesterol and Triglyceride Assimilations Of the 19 acid-tolerant strains, seven strains showed cholesterol-decreasing rates exceeding 50%. Of these, three strains demonstrated triglyceride (TG)-decreasing rates of more than 40%. As shown in Table 2, the highest cholesterol-decreasing rate (67.41% ±0.20) was observed for Strain L-42, while the highest TG-decreasing rate (51.15% ±0.57) belonged to Strain L-3. In vitro, the L-42 and L-11 strains showed cholesterol-decreasing rates of 67.41 and 64.27%, respectively. These rates were higher than those of L. fermentum F1 (48.87%) [25], L. acidophilus L2-16 (64.11%) [26], Pediococcus acidilactici LAB 5 (62%) [27], L. sake C2 (53.2%) [28], B. longum (34.2%) [29] and L. helveticus MG2-1 (51.74%) [28]. There are a few investigations on the ability of potential probiotics in decreasing TG. The rate for the L-3 strain was higher than that L. acidophilus L2-73 reported in the Gao et al. study [26]. The authors observed that cholesterol and TG intake varied greatly in strains within the same species. This suggested that cholesterol sequestering was specific to individual strains rather than a characteristic of the entire species [30]. 3.3 Resistance to Simulated Gastrointestinal Conditions, Phenol, and Lysozyme All seven selected strains, except L-51 (69% survival), showed a survival rate above 80% after sequential exposure to simulated gastrointestinal fluids. The strains were highly resistant to simulated gastric fluid, with over 94.8% survival after 2 h and over 90% survival for most strains after 4 h. In simulated intestinal fluid, six isolates survived above 90%. Overall, the strains were relatively more tolerant to acid than bile. There was no significant difference between strains in their tolerance to simulated fluids, which was similar to the initial pH and bile salt assessments. All bacteria tolerated 0.5% phenol after overnight incubation (OD > 1.000) and showed significant resistance to 15 mg l-1 lysozyme after 2 h of exposure (Table 3). Probiotics must be able to tolerate gastrointestinal stressors such as acidity, bile, phenol, lysozyme, and pepsin. The current research showed that lactobacilli isolated from fermented dairy demonsterated strong tolerance to simulated gastrointestinal environments. 3.4 Safety Profile of Antibiotic Resistance, DNase, and Hemolytic Activity The isolated lactobacilli showed various resistance to the assessed antibiotics (Table 4). Chloramphenicol, novobiocin, and penicillin were effective against all the isolates. In contrast, most isolates were resistant to rifampicin. All seven lactobacilli were negative for DNase activity and showed no hemolytic activity (γ-hemolysis) on blood agar, indicating that they were safe. 3.5 Antagonistic Activity against Pathogens Six of the seven lactobacilli showed moderate antipathogenic activities; strain L-42 did not demonstrate antagonistic activity. The metab