Invitro and Invivo Analysis of Human Milk Lactic Acid Bacteria Isolates for Their Anti-hypercholesterolemia Actions

Abstract

The aim of this study was to evaluate the cholesterol lowering ability of Lactic Acid Bacteria (LAB) isolated from human breast milk under in vitro and in vivo conditions. Six LAB isolates namely Lacticaseibacillus casei 1A, Lactobacillus gasseri 5A, Enterococcus faecium 2C, Limosilactobacillus fermentum 3D, Pediococcus acidilactici 1C, and Lactiplantibacillus plantarum 7A, were examined for their bile resistance, bile salt hydrolase activity, cholesterol assimilation and viability in cholesterol rich; DeMan Rogosa and Sharpe broth, simulated gastric, small and upper intestinal conditions. During in vivo experiments, two putative LAB isolates were orally gavage to BALB/c mice, fed with normal basal and cholesterol rich (HCD) diets, daily for a period of 4 weeks. Blood serum analysis including total serum cholesterol, triglycerides, high-density and low-density lipoprotein (LDL) cholesterol levels and total fecal LAB counts of the animals were determined. The isolates in study showed bile resistance and bile salt hydrolysis activity, while significant differences (P < 0.05) were seen in their cholesterol assimilation ability. L. gasseri 5A (195.67%) and L. plantarum 7A (193.78%) displayed highest cholesterol removal percentages, respectively. Animals in HCD, fed with L. gasseri 5A and L. plantarum 7A showed decreased levels of total cholesterol and LDL, compared to the control groups. In HCD group liver weight was increased, while fecal LAB counts were decreased. No changes were observed in behavior or body weight in all experimental groups. In conclusion, L. gasseri 5A and L. plantarum 7A isolated from human breast milk demonstrates significant hypocholesterolaemic actions in vitro and in vivo and might be considered a promising candidates for preventing hypercholesterolemia in man and animals.

Introduction

Hypercholesterolemia, a major risk factor for coronary heart disease (CHD) is strongly associated with elevated levels of low density lipoprotein (LDL) cholesterol [1]. According to the reports, elevated LDL cholesterol is a leading risk factor and one of the most closely linked markers of atherosclerotic cardio vascular disease. In 2021 approximately 3.81 million cardiovascular deaths were attributed to elevated LDL levels [2]. Although, chemical drugs like statins have been shown to lower the risk of heart attack and mortality rates, but their usage is linked to various side effects and symptoms, like myositis, myalgia, cognitive impairment, liver dysfunction, neuropathy, etc. [3, 4].

As an alternative therapeutic regimen to chemical drugs, several herbs, nutraceutical ingredients and functional foods have been used to lower serum cholesterol levels and to reduce the risk of CHD [5, 6]. Among these natural and safe ingredients, probiotic microorganisms have been widely are known for their potential to reduce the risk of cardiovascular disease (CVD) [7]. Probiotic microorganisms are defined as the beneficial microbes that can stimulate gut microbiota to modify the gastrointestinal environment, and provide health benefits to the host when consumed in adequate amounts [8]. Hypercholesterolemia is a health problem associated with the dysbiosis of the intestinal microbiota [8], and hence probiotic microorganisms possessing the potential to prevent dysbiosis can consequently prevent hypercholesterolemia [9]. Several Lactic Acid bacteria (LAB) are been widely known for their ability to lower serum LDL cholesterol, regulates blood pressure and improves blood glucose and lipid profiles [10, 11]. Cholesterol lowering ability of LAB species are strain dependent as certain strains of the same species might not show similar actions [12]. Different mechanism has been proposed regarding the ability of probiotic bacteria to reduce blood cholesterol levels, which includes deconjugation of bile acids, inhibition of hepatic cholesterol synthesis and/or direct binding or assimilation of cholesterol [13, 14].

The probiotic LAB isolates used in the study were previously isolated from breast milk of healthy mothers aged between 24 and 28 years with weaning infants aged up to 9 months [15]. The protective effect of human milk has been mainly attributed to its rich composition in beneficial bacteria and oligosaccharides playing a central role in the metabolism, physiology and immunity of infants [16]. Significant anti-hypercholesterolemia behavior including lower total cholesterol and LDL cholesterol among breast fed infants in their adolescence has been reported previously [17], that could be attributed to the LAB flora present in breast milk. Also, the higher plasma cholesterol levels in breastfed infants compared to those fed standard artificial formulas protects babies against the consequences of hypercholesterolemia in adult life [18, 19]. Oral supplementation of probiotics with cholesterol lowering property might be a useful natural and safe strategy for controlling elevated cholesterol levels, reducing the risk of coronary heart diseases and improving cholesterol metabolism‐related disorders.

While, a limited number of studies have reported the cholesterol lowering role of human breast milk originated LAB, hence, we aimed to investigate the cholesterol lowering potential of the selected human milk LAB isolates using invitro and invivo experiments.

Materials and Methods

Bacterial Isolates and Cultural Conditions

Probiotic LAB species isolated from human breast milk in a previous study [15]; including Lactiplantibacillus plantarum 7A, Lacticaseibacillus casei 1A, Lactobacillus gasseri 5A, Enterococcus faecium 2C, Limosilactobacillus fermentum 3D, and Pediococcus acidilactici 1C, were used. The isolates were deposited as probiotic strains at Razi type culture collection center, Iran with RTCC No 1290–1, 1296–1, 1305, 2347, 1303 and 1412–2, respectively. All LAB isolates were grown in deMan Rogosa and Sharpe (MRS) (Merck, Germany) medium under anaerobic conditions (anaerobic Jar, Sigma-Aldrich; with anaerobe atmosphere generation bag) at 37 °C for 24 h. For long term preservations, the freshly grown cultures in MRS broth supplemented with 20% (v/v) glycerol and maintained as frozen stocks at −80 °C.

Bile Salt Tolerance (BST)

Respective LAB cultures were screened for their bile tolerance by the method described earlier [16]. In brief; freshly growing LAB cultures corresponding to McFarland index 0.5 (1.5 × 10⁸ CFU/ml) were inoculated in MRS broth supplemented with 0.3, 0.7 and 1.0% w/v Oxgall (Sigma, UK) salt. MRS broth without Oxgall was used as control. After 8 h of incubations, the number of viable cells were enumerated by performing surface plating on MRS agar. The survival rate was calculated as log 10 values of CFU/ml. The experiments were performed in triplicate and mean values were calculated.

Qualitative Bile Salt Hydrolyses (BSH) Activity

BSH activity of the selected isolates was determined by placing sterile filter discs saturated with freshly grown LAB cultures on MRS agar plates supplemented with 0.3% (w/v) bile salts, 0.5% taurodeoxycholate (TDC) (Sigma, UK) and 0.037% (w/v) calcium chloride [17, 18], in triplicate. All plates were incubated at 37 °C for 72 h and later the diameters of the deconjugated bile acid precipitation zones (opaque halos) measured and BSH activity score recorded in millimeters.

Cholesterol Assimilation Assay

Cholesterol assimilation ability of the LAB isolates was investigated in cholesterol rich MRS broth medium, simulated gastric juice, and in simulate small and upper intestinal conditions. Filter sterilized water-soluble cholesterol (polyxyethylene cholesteryl sebacate, Sigma, UK) at final concentrations of 100 μg/mL was added to respective fluids (MRS-C, SG-C, SSI-C and SUI-C).

Cholesterol Assimilations in MRS Broth Media (MRS-C)

The isolates were tested for their ability to assimilate cholesterol in cholesterol rich MRS broth media by inoculating 1% v/v of freshly grown LAB cultures (0.5 McFarland) into cholesterol rich MRS broth media (MRS-C) prepared by adding 0.3% (w/v) Oxgall, and 0.2% (w/v) sodium thioglycolate (Sigma, USA). Control samples included MRS broth without cholesterol. After 4, 8 and 24 h of incubations at 37 °C, the samples were centrifuges (9000 g, 10 min) and the cholesterol content in the spent broth extracted and estimating the evaporated residues by enzymatic assay described earlier [19]. The percentage of assimilated cholesterol in the spent broths and control samples were calculated using following formula:

Cholesterol assimilation (%) = OD₆₀₀ of control—OD₆₀₀ of respective cell suspensions / OD₆₀₀ of control × 100.

Cholesterol Assimilations in Simulated Gastric and Intestinal Conditions

Simulated gastric (SG-C) and intestinal (SI–C) conditions were created by slight modifications in the methods described earlier [18, 20]. In brief, SG-C suspensions were prepared by inoculating 5% v/v of pepsin (Sigma, USA), 0.1 mg/L lysozyme, 0.05 mg/L porcine bile, and 0.5% sodium chloride in MRS broth. After adding cholesterol (final concentration of 100 μg/mL), the pH of the suspension was adjusted to 3.0 with 5 M Hydrochloric acid. Amount of 1% v/v of freshly grown LAB cultures (0.5 McFarland) were added and cholesterol removal was estimated after 30, 60 and 90 min as described above.

Simulated small intestinal (SSI) fluids were prepared by adding 1.5 g/L of Oxgall, 3.5 g/L glucose and 6 g/L of pancreatic from porcine (pancreatin, Pancrex, UK), to 1 M phosphate buffered saline (PBS), and pH adjusted to 6.0 by adding 2 M NaOH. Simulated upper intestinal (SUI-C) conditions were created by adjusting pH of the suspension to 8.0. The suspensions were enriched with the mentioned concentrations of cholesterol, and inoculated with 1% (v/v) of respective probiotic cultures. The tubes were incubated at 37 °C for 24 h and cholesterol content determined after 30, 60, and 90 min of incubation as described earlier.

Viability of LAB Isolates in Cholesterol Rich Suspensions

The survival of the LAB isolates in MRS-C, SG-C, SSI-C and SUI-C contents was determined by inoculating of actively growing bacterial cells (1% v/v) into the respective cholesterol rich mixtures and incubating at 37 °C, under anaerobic conditions. The numbers of viable cells (CFU/ml) were determined by performing standard plate count method after 4 h of incubations in MRS broth, after 60 and 90 min in SG-C and SSI conditions, respectively [21, 22]. Suspensions without cholesterol, inoculated only with the respective bacterial cultures were used as positive control. The survival rate was calculated as log 10 values of CFU/ml. The experiments were performed in triplicate and mean values were calculated. Resistance of the isolates under these conditions was estimated by enumerating the number of viable cells at specific time intervals and determining their R values using the following equation:

R = Average number of cells at specific time interval / Average number of cells at time zero.

According to the formula, R = 1 when, no effect on the growth and survival of bacteria is seen, while a ratio of 0.5 indicated a loss of 50% of the viability. Ratios above 1 represent bacterial growth.

In Vivo Experiments

Animal Ethics

All animal procedures applied in the present study were approved by the Animal Ethic Committee, Razi vaccine & Serum Research Institute, Iran. All animal experiments complied with the ARRIVE guidelines and was carried out in accordance with the EU Directive 2010/63/EU for animal experiments.

Animals and Diets

A total of 42 male BALB/c mice (six-week-old), weighing 22.5 ± 0.5 g was obtained from the Department of Laboratory Animal Research, Razi Vaccine and Serum Research Institute, Iran. All mice were acclimatized for 1‐week being housed in rooms set at 22 °C with 48% humidity. All animals were housed in a controlled environment (21 °C, 50% humidity and 12 h light cycle) and water provide Ad libitum.

Treatment Groups

Two isolates showing significant anti-cholesterol activity during in vitro analysis (L. gasseri 5A and L. plantarum 7A) were selected for in vivo cholesterol assimilation assay. The animals were divided randomly into two main groups (NBD and HCD), 21 in each group. Group A recieved normal basal diet (NBD) while,  high cholesterol diet (HCD) group recived normal basal diet enriched with 2% cholesterol and 0.4 % cholic acid 10 days prior to the start of the experiments. All the animals in study were acclimatized to the respective diets for a week before the experiment started. HCD animals received the mentioned cholesterols concentrations prior to the start of the experimental trials to simulate experimental hypercholesterolemia. The two groups (NBD and HCD) were further divided into 3 sub-groups (P1, P2 and C) 7 each; including animals fed with L. gasseri 5A (P1) and L. plantarum 7A (P2) and the control group that received no LAB (C). The respective bacterial cultures at the concentrations of 3 × 10⁸ CFU/ml (1.0 McFarland index) were administered to the animals via oral gavage (0.5 mL), once daily for a period of 28 days. The dosage rate of probiotics (3 × 10⁸ CFU/ml) was selected based on earlier studies on hypocholesterolaemic activity of LAB [23, 24]. The control groups (NBD-C and HBD-C) animals were administered saline alone. The body weight of the animals was measured once weekly, while all animals were observed for overall behavior, signs of toxicity and mortality daily, till the end of experimental period.

Liver Weight and Blood Serum Analysis

At the end of the 4-wk feeding trial, rats were fasted overnight, and anesthetized by diethyl ether. Liver of the animals in each experimental group were excised, rinsed and their weight recorded in mg. Blood was collected from the eyes of anaesthetized animals under sterile conditions in sterile tubes. Sera were collected via centrifugation (1,800 × g for 10 min at 4 °C), and evaluated for total cholesterol, triglycerides (TG), high-density and low-density lipoprotein (HDL and LDL) cholesterol, using commercial kits (Asan Pharm, Korea) according to manufacturer 's instructions.

Total LAB Counts in Fecal Samples

Fecal samples on day 1 and once every week were collected from the rectum by rectal stimulation [25]. Total LAB counts were determined by standard plate count on MRS-agar and incubation at 37 °C under anaerobic conditions for 48 h. Number of colony forming units were recorded in Log₁₀ CFU/gram of wet feces.

Statistical Analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) using Origin 7 software (MicroCal Software, USA), and the differences between treatments were estimated using Tukey’s test. Significance level was considered at P ≤ 0.05.

Results

Six LAB isolates were initially tested for their bile tolerance after being exposed to 3 different concentrations of bile salt. As seen in Table 1, significant differences were observed in the bile tolerance of the isolates (P < 0.05). The isolates were more resistant in 0.3% and 0.7% of the salt and showed higher survival rates in these conditions compared to 1% salt concentrations. L. plantarum 7A showed highest bile resistance followed by L. gasseri 5A and L. casei 1A, respectively. In contrast, L. fermentum 3D appeared least bile tolerant (P < 0.05) and compared to other isolates, it showed least growth at all used salt concentrations.

Table 1.

Viability (Log₁₀ CFU/ml) of selected LAB isolates in different bile salt concentrations

LAB isolates	Bile salt concentrations (%)
	0.3	0.7	1.0
L.plantarum 7A	8.51 ± 0.16a	8.16 ± 0.13a	6.97 ± 0.09b
L.casei 1A	7.39 ± 0.09c	7.13 ± 0.11c	6.71 ± 0.11cb
L.gasseri 5A	8.13 ± 0.07ba	7.88 ± 0.06ba	6.95 ± 0.09b
E.faecium 2C	8.01 ± 0.09b	7.78 ± 0.06b	7.57 ± 0.07c
L.fermentum 3D	7.06 ± 0.18c	6.74 ± 0.07d	6.37 ± 0.05a
P.acidilactici 1C	7.34 ± 0.12c	7.14 ± 0.09c	6.87 ± 0.04b

Values are expressed in ± SD (standard deviations) of three replicates. Superscript shown with different alphabetical letters within a column indicate significant differences between the LAB isolates (P < 0.05)

Figure 1 demonstrates the differences in BSH activity of the isolates as determined by diameter of the halos appearing around the filter discs. As seen, L. gasseri 5A showed the highest halo zone diameters (20.5 ± 1.2) followed by L. plantarum 7A (18.7 ± 0.9), while the difference between the two were not significant (p ˃ 0.05). However, these differences (BSH activity score) were more pronounced (P < 0.05) in L. casei 1A (16 ± 0.5), P. acidilactici 1C (12.2 ± 1.1) and E. faecium 2C (10.4 ± 0.4), respectively.

Fig. 1.

Bile salt hydrolysis activity of the mentioned bacterial isolates. The activity was recorded by measuring the diameter of precipitation zones in millimeters. 1A: L. casei; 2C: E. faecium; 3D: L. fermentum; 5A: L. gasseri; 7A: L. plantarum and 1C: P. acidilactici

Cholesterol assimilating potential of the LAB isolates was determined after exposing them to different cholesterol rich conditions. Table 2 shows the isolates potential to assimilate cholesterol in cholesterol rich MRS broth media after 24 h. According to results, highest cholesterol assimilations in these conditions were demonstrated by L. gasseri 5A (51.5 ± 0.59%), and L. plantarum 7A (49.61 ± 1.07%), with no significant difference observed. However, other isolates showed significantly lower level of cholesterol assimilations (P < 0.05). The results showed that cholesterol assimilations by the isolates were time-dependent, and with passage of time, cholesterol assimilations were enhanced (P < 0.05). For further investigations, only three isolates L. gasseri 5A, L. plantarum 1A and 7A that showed significant cholesterol assimilating percentages in MRS broth were selected.

Table 2.

Percentage cholesterol reduction by LAB strains in cholesterol enriched a) MRS broth (MRS-C)

LAB isolates	Cholesterol assimilation (%) in MRS-C
	4 h	8 h	24 h
E. faecium 2C	9.74 ± 0.98b	10.19 ± 0.13b	10.67 ± 0.13b
L. fermentum 3D	0.74 ± 0.98a	0. 78 ± 0.13a	0.82 ± 0.44 a
L. gasseri 5A	37.41 ± 0.86e	44.52 ± 1.32e	51.45 ± 0.59e
L. plantarum 1A	23.21 ± 1.32c	34.51 ± 1.12d	38.70 ± 1.21c
L. plantarum 7A	29.35 ± 1.11d	31.21 ± 1.10c	49.61 ± 1.07d
P. acidilactici 1C	11.22 ± 1.15b	14.37 ± 0.99b	14.23 ± 1.14b
Average cholesterol reduction	0–37%	0–44%	0–51%

Results are indicated in means (standard deviations) of three replicates. Tukey’s homogenous subsets generated from pairwise comparison are represented as a, b, c, d

Tables 3, 4 and 5 reflect the hypocholesterolemic effects of the mentioned isolates in simulated gastric (SG-C) and intestinal (small and upper intestine) conditions. Cholesterol assimilations by the respective isolates in simulated small (SSI-C) and upper intestinal conditions (SUI-C) were greater compared to simulated gastric conditions (P < 0.05). In SG-C conditions the cholesterol assimilation potential of the selected isolates showed a significant increase within 60 min while, with increasing time period the cholesterol reducing potentials of the isolates showed significant decrease and highest reduction was observed at 90 min. Under these acidic conditions, L.gasseri 5A showed highest cholesterol reducing potentials within 60 min, while L.plantarum 1 A were least effective in assimilating cholesterol under these conditions.

Table 3.

Percentage cholesterol reduction by LAB strains in cholesterol enriched simulated gastric (SG-C) condition

LAB isolates	Cholesterol assimilation (%) in SG-C
	30 min	60 min	90 min
L. gasseri 5A	14.74 ± 0.98b	25.19 ± 0.13b	7.67 ± 0.13b
L. plantarum 1A	0.74 ± 0.98a	0. 78 ± 0.13a	0.82 ± 0.44a
L. plantarum 7A	17.22 ± 1.15b	16.37 ± 0.99b	11.23 ± 1.14b
Average cholesterol reduction	0–17%	0–11%	0–6%

Results are indicated in means (standard deviations) of three replicates. Tukey’s homogenous subsets generated from pairwise comparison are represented as a, b, c, d

Table 4.

Percentage cholesterol reduction by LAB strains in cholesterol enriched simulated small intestinal (SSI-C)) conditions

LAB isolates	Cholesterol assimilation (%) in SSI-C
	30 min	60 min	90 min
L. gasseri 5A	23.41 ± 1.24e	31.52 ± 1.24d	45.45 ± 0.70d
L. plantarum 1A	16.21 ± 1.15b	16.51 ± 1.26b	30.70 ± 1.13c
L. plantarum 7A	23.35 ± 0.85e	29.89 ± 1.41d	48.61 ± 1.34e
Average cholesterol reduction	16–23%	16–31%	30–48%

Results are indicated in means (standard deviations) of three replicates. Tukey’s homogenous subsets generated from pairwise comparison are represented as a, b, c, d

Table 5.

Percentage cholesterol reduction by LAB strains in cholesterol enriched simulated upper intestinal (SUI-C) conditions

LAB isolates	Cholesterol assimilation (%) in SUI-C
	30 min	60 min	90 min
L. gasseri 5A	28.99 ± 3.10b	31.19 ± 2.09b	44.57 ± 1.12b
L. plantarum 1A	13.98 ± 0.59b	15.78 ± 1.13b	17.03 ± 0.88b
L. plantarum 7A	27.22 ± 0.99b	29.37 ± 2.21b	39.23 ± 2.44b
Average cholesterol reduction	13–28%	16–31%	17–44%

Results are indicated in means (standard deviations) of three replicates. Tukey’s homogenous subsets generated from pairwise comparison are represented as a, b, c, d

In simulated small intestinal conditions percentage of cholesterol reduction (30–48%) were significantly higher (p < 0.05) compared simulated upper intestinal conditions (17–44%). While, compared to other two LAB isolates, L. planatrum 1A showed negligible hypocholesterolemic effects (P = 0.001). Under the set alkaline conditions, the tested isolates showed enhancing cholesterol reducing effects that increased with enhancing time period.

Viability and survival rate of all six isolates in cholesterol rich media including MRS-C, SG-C, SSI-C and SUI-C, was tested in order to determine influence of growth rate on cholesterol assimilating potentials (Fig. 2). According to results, all tested isolates tolerated the harsh gastric and intestinal conditions with their R values ≥ 0.5 (indicating viability of equal to or above 50%). However, survival of the isolates appeared affected by gastric conditions where acidic conditions prevailed. Decrease was significantly evident under these conditions compared to simulated small and upper intestinal conditions where the viability was comparatively higher in the alkaline conditions. Cholesterol assimilations appeared to be growth independent in E. faecium 2C and L. fermentum 3D as although they showed significant (p < 0.05) viability in all simulated stress conditions but had the least cholesterol reducing percentages.

Fig. 2.

Viability of LAB cultures in cholesterol rich MRS broth (MRS-C after 4 h), simulated gastric (SG-C, after 60 min), small and upper intestinal (SSI-C and SUI-C, after 90 min). MRS: non-supplemented MRS broth after 4 h (control). The survival rate of the tested bacteria is estimated in R value. R values above 1 is considered survival and growth, lower than 1 indicates only survival, while below 0.5 shows cell growth inhibitions. Data are presented as means and standard deviation for three samples. p < 0.05 significantly different compared to control. 2C: E. faecium; 3D: L. fermentum; 5A: L. gasseri; 1A: L. case; 7A: L. plantarum and 1C: P. acidilactici. The data are shown as mean ± standard deviation. Different cases indicate significant differences between mean values

During in vivo analysis, no significant difference (P ≥ 0.05) were seen in the behavior of the BALB/c mice in different treatment groups, neither, any abnormal mortality was recorded. However, slight differences were seen in the body weight of BALB/c mice fed with normal basal diet or cholesterol rich diet (HCD) that was insignificant (P ≥ 0.05). Significant increase was recorded in the liver weight of the mice in HCD groups (P < 0.05) receiving high cholesterol diet (data not included). Evidently, the mean liver weight of animals in HCD control were higher (2.1 g), compared to NBD control group (1.3 g) animals and this differences were significant (P < 0.05). However, liver weight of the animals in HCD groups appeared effect by the LAB isolates. HCD animals fed with the respective LAB isolates (P1 and P2) showed liver weights almost comparable to NBD groups (P < 0.05).

Serum cholesterol levels of the HCD and NBD group mice are shown in Fig. 3a. Compared to NBD control group (46.5 ± 34 mg/dl), probiotic fed groups P1 and P2 (81.7 ± 22 and 72.9 ± 24 mg/dl), higher levels of total serum cholesterol were recorded in the sera of control group animals in HCD (98.2 ± 12 mg/dL), and the differences were significant (P < 0.05). However, the levels of cholesterol were significantly reduced in probiotic fed animals in HCD group (P < 0.01), compared to the control group. While, in NBD group this difference was not significant (P ≥ 0.05).

Fig. 3.

Serum lipid levels (mg/dl) in BALB/c mice fed with normal basal diet (NBD) and high cholesterol diet (HCD) supplemented with live cells of L. gasseri 5A and L. plantarum 7A, for 3 weeks. C: control group animals not fed probiotics; P1: animals fed with L.gasseri 5A; P2: L.plantarum 7A while, TC Total serum cholesterol; LDL Low density lipoproteins; HDL High density lipoproteins and TG Triglycerides. The results presented are the mean of triplicate experiments (± standard deviation)

The animals in HCD group not fed with the LAB isolates showed an abnormal blood lipid metabolism, and increased levels of TC, TG and LDL were recorded in their serum. In contrast, HCD mice fed with the probiotic LAB (P1 and P2) showed significant decrease in their TC, TG and LDL levels, compared to the control group (P < 0.05). However, no significant difference in HDL levels between NBD and HCD group animals were observed (P ≥ 0.05).

Total LAB counts in the feces of the animals in both NBD and HCD group was estimated (Fig. 4). Based on the obtained data, significantly higher LAB counts were observed in P1 and P2 groups fed with the respective LAB isolates, compared to the control group animals (P < 0.05) that did not receive the mentioned LAB isolates. While, these counts were more pronounced in NBD group compared to HCD group animals that showed lower total fecal LAB counts (p ˃ 0.05). In NBD group animals, P1 group animals fed with L. gasseri 5A showed slightly higher LAB counts compared to P2 group, however the differences were insignificant (p ˃ 0.05). In contrast, in HCD group animals the total fecal LAB counts were higher in P2 group animals than P1 group animals (P < 0.05). Differences in total fecal LAB counts in the control group animals (NBD-C and HCD-C) appeared stable during 28 days of observations, while a significant increasing pattern of growth was observe in the treatment groups receiving LAB (P < 0.05).

Fig. 4.

Total fecal LAB counts (Log CFU/g) of BALB/C mice fed with normal basal diet (NBD) and high cholesterol diet (HCD) supplemented with L. gasseri 5A (P1) and L. plantarum 7A (P2) for 3 weeks. C: control group animals not fed probiotics; P1: animals fed with L. gasseri 5A; P2: L. plantarum 7A while, TC Total serum cholesterol; LDL Low density lipoproteins; HDL High density lipoproteins and TG Triglycerides

Discussion

Cholesterol as a vital substance in body, is used by liver as a precursor to synthesize the primary bile acid, mainly chenodeoxycholic acid (CDCA) and cholic acid, which are then modified by intestinal bacteria to form secondary bile acid (lithocholic acid and deoxycholic acid) [26]. However, elevated serum cholesterol levels can lead to atherosclerosis and pose high risk of developing coronary diseases [27]. While, certain pharmacological agents are known to effectively reduce serum cholesterol levels but unwanted side effects such as gastro intestinal discomfort has been seen in many. In this context, several reports have recommended the use of probiotics as a safe alternative to chemically used drugs for lowering serum cholesterol levels.

Probiotics as live bacterial supplements, in adequate amounts could show beneficial health effects in the host. Several decades ago, it was stated that fermented milk enriched with probiotics can show hypocholesterolemic effect in humans [3]. Since then, a number of studies have highlighted the anti-hypercholesteremic effect of probiotic bacteria and signified their role as a promising and safe and cost effective therapeutic approach for combatting high serum cholesterol levels [4]. Significant anti-hypercholesterolemia behavior including lower total cholesterol and LDL cholesterol among breast fed infants in their adolescence has been reported previously [28], that could be attributed to the LAB flora present in breast milk. The higher plasma cholesterol levels in breastfed infants compared to those fed standard artificial formulas protects babies against the consequences of hypercholesterolemia in adult life [29, 30]. In this research, we aimed to investigate cholesterol reducing effects of several human milk LAB isolates, with a particular focus as biotherapeutics for the management of high cholesterol.

Probiotics mainly reside in host intestinal tract, where, they interact with bile salts and ingested cholesterol. Bile has crucial role in the emulsification and solubilization of lipids, facilitating hepatobiliary secretion of lipids, endogenous metabolites, and xenobiotics [30]. The capacity of probiotics to tolerate bile salts is strain specific, while a number of external factors such as pH, temperatures, other environmental factors and diet are known to affect the tolerance or vulnerability of a bacteria to bile concentrations. Consistent with these reports, although all studied probiotic strains showed resistance to all tested bile salt concnetrations, but their level of resistance differed. Depending on the food regime and health status, the bile acid concentration in the intestine of different persons ranges from 0.2 to 2% [31], with mean intestinal bile concentration of approximately 0.3% (w/v) [32]. It has reported that probiotic bacteria capable of tolerating 0.3–0.5% bile are able to easily colonize the host gut.

BSH activity in colonic bacteria led to enhance rate of excretion that alternatively reduce and control serum cholesterol levels [6]. Several Lactobacillus, Enterococcus and Bifidobacterium have been identified with bile salt hydrolase activity. Contrasting to non-deconjugating bacteria, the bacteria deconjugating bile salts appear to have the ability to assimilate cholesterol from the culture medium to a significant extend [6, 8]. Debating reports are present regarding the co-relation between bile salt tolerances and BSH activity, as although these bacteria reduce serum cholesterol levels, but, they simultaneously increase the level of undesirable de-conjugated bile salts [32, 33]. Comparable with the reports of others, in the present study the bile tolerant strains were able to assimilate cholesterol and a direct correlation between BSH activity score, bile tolerance and cholesterol assimilation was observed in the screened LAB isolates [7]. However, contrasting reports has also been reported that high bile tolerance strains do not essentially have high cholesterol assimilating ability [8].

The ability of probiotic bacteria to survive and assimilate cholesterol in gastrointestinal tract allows reduction of cholesterol absorption by enterocytes and consequently leads to the excretion of cholesterol [33]. In this research, we reproduced stress conditions mimicking simulated gastric and intestinal environments and assessed the survival and cholesterol assimilating property of the selected LAB bacteria under anaerobic conditions. While pH and pepsin are the main stress factors for a bacterium transiting through gastric conditions, in intestinal conditions pancreatin and bile salts can exert their adverse effects. The LAB strains showed significant cholesterol assimilations not only in MRS-c where only cholesterol and bile was present, but also resisted the conditions mimicking the gastro-intestinal tract where pH, and gastric enzymes were additionally present. These results show the significance of the selected LAB isolates harboring cholesterol reducing properties, as they not only could survive the harsh conditions of human digestive tract but also exerted their hypercholesteremic effects [34]. The significant viability of L. gasseri 5A and L. plantarum 7A in the intestine indicates that these strains might aid in the control of serum and liver cholesterol levels. These results were in accordance with the findings of other reseachers [35].

It has been reported that cholesterol assimilation by lactobacillus species is a growth dependent factor [8]. Similarly, other reports also stated growth- associated cholesterol assimilation by LAB species [36]. However, in this study we showed that this relation is not applicable for all LAB species, as E. faecium 2C with significant growth under stress conditions demonstrated negligible bile tolerance, BSH activity and cholesterol assimilating property. However, L. gasseri 5A possessing the highest cholesterol removing ability, also demonstrated maximum growth and viability under the stress conditions. Thus, it is not possible to assure a linear relation between growth rate and cholesterol assimilating ability of LAB bacteria and other factors besides active growth might play a role in lipid metabolism.

The isolates showing significant in vitro hypocholesterolemic effects were further assessed in a murine model in vivo, using BALB/c mice models, as in vitro cholesterol reducing activity could not be predictors of in vivo cholesterol lowering effects. Animal models like rats, mice, hamsters, guinea pigs and pigs are often used for evaluating the effect of probiotic bacteria on serum cholesterol levels [37]. These animal models show wide similarities with humans in terms of cholesterol and bile acid metabolism, plasma lipoprotein distribution, and regulation of hepatic cholesterol enzymes [38]. Additionally, these animals share an almost similar digestive anatomy and physiology, nutrient requirements, bioavailability and absorption, and metabolic processes with humans, making them useful experimental models for research applications [31]. In this research, we used BALB/c mice models for evaluating the role of our selected LAB strains in reduction of serum cholesterol levels. Total serum lipids including total serum cholesterol, HDL, LDL and TG levels in BALB/c mice fed with basal diet (NBD) or high cholesterol diet (HCD), was investigated. Intriguingly, probiotic fed animals in HCD groups showed decreased levels of TC and LDL when compared to the control groups and the LAB isolates were able to reduce both total serum cholesterol and LDL levels. In a previous report, the effect of L. gasseri SBT0270 on serum lipids, bile acids, and fecal microbiota in hypercholesterolemic rats have been demonstrated. Results showed that there is a relationship between the hypocholesterolemia effect of L. gasseri SBT0270 and its ability to suppress the reabsorption of bile acids into the enterohepatic circulation that enhanced acidic steroids excretion in feces of hypercholesterolemic rats [27]. Slight increase in HDL levels observed in this study are in agreement with other findings that showed that with a decrease in total cholesterol amounts a simultaneous increase in HDL levels are observed [39]. Total fecal LAB counts in different treatment groups indicated that as expected probiotic treatment groups have higher LAB counts especially in NBD-P1 and NBD-P2 group animals, while these counts were significantly affected in the presence of high cholesterol environments. Similarly, it has shown that probiotic-supplemented group resulted in an increase in Lactobacillus spp. count along with reduced pathogen count in the feces [40]. Although, presence of LAB in these animals might suggest the tolerance of the tested LAB isolates to harsh gastrointestinal conditions, a phenomenon also observed by others [41].

Conclusion

Our findings identified that two putative LAB isolates namely L. gasseri 5A and L. plantarum 7A display significant cholesterol lowering properties in vitro and in vivo. Both the isolates demonstrated high capability to resist bile salts and cholesterol in the media, and survived in cholesterol environment in simulated gastric and intestinal contents. The results of the study suggest that human milk is a potential source of probiotic LAB with hypocholesterolemic effects, while, studies are required to elucidate the mechanisms underlying these effects. Furthermore, human studies are recommended for affirming the role of selected LAB species as a promising probiotic candidate for prevention of hypercholesterolemia in humans.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Human and Animal Participants

All applied procedures related to animals were approved by the Animal Ethic Committee, Razi vaccine and Serum Research Institute, and the experiments were performed according to the animal care and welfare regulations.