Probiotic lactobacillus strain

The L. fermentum ME-3 strain was previously isolated from the gastrointestinal tract of healthy Estonian children [22]. The strain was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures GmbH) under the registration number, DSM 14241. The L. fermentum ME-3 strain used in this study was confirmed by molecular identification with polymerase chain reaction that employed an internally-transcribed spacer and 16S rRNA sequencing [23]. The strain’s safety was confirmed previously in an animal model [16] and in healthy Estonian adults (registered trials ISCRNT43435738 and ISCRNT53154826).

Activity for bile hydrolysation

Enzymatic deconjugation of bile acids by the bile salt hydrolase (BSH) activity of L. fermentum ME-3 was tested according to Lim et al. [24]. L. fermentum ME-3 strains were cultured overnight on de Man, Rogosa, Sharpe (MRS; Oxoid, UK) agar in microaerobic conditions at 37 °C. For control cultures, we used three reference strains (L. acidophilus 821–3, L. plantarum 299v, and L. plantarum BAA793). After incubation, cultures were standardised to McFarland 3.0. For testing BSH activity, sterilised paper disks were impregnated with the cultures and placed on MRS agar plates supplemented with 4 % (w/v) tauro-deoxycholic acid sodium salt (TDCA, Sigma, USA) and 0.37 g/l CaCl 2 . The plates were anaerobically incubated at 37 °C for 72 h, and the diameters of the precipitation zones around the disks were measured. BSH activity was then calculated by subtracting the disc diameter (DD, mm) from the inhibition zone diameter (IZD, mm), and dividing by two, as follows: BSH activity = (IZD-DD)/2. The activity of three consecutive cultures of each strain was expressed as median (CI 95 %) value.

Kefir preparation

The probiotic kefir containing L. fermentum ME-3 (trademark “Hellus”, 2005) was developed at Tere AS in Estonia. Briefly, kefir was prepared from pasteurised (92–95 °C for 5 min) cow’s milk with 2.5 % fat content; L. fermentum ME-3 was added as an adjunct starter. Commercial starter cultures — including kefir grains and mesophilic aromatic cultures of Lactococcus lactis subsp cremoris, Lactococcus lactis subsp lactis biovar diacetylactis, Leuconostoc sp, and Lactococcus lactis subsp lactis (Chr. Hansen, Denmark) — were added to the incubation tank. For production of “Hellus” kefir, freeze-dried L. fermentum ME-3 cultures were added to the kefir milk to a concentration of 1 × 106 colony-forming units (cfu)/ml. The kefir milk fermentation process lasted approximately 20–24 h, until achieving the required pH 4.5. After fermentation, the kefir was cooled to 8-10 °C in a heat exchanger.

The final kefir product possessed a homogenous consistency, creamy colour, shiny smooth surface, fresh and acidic taste, and mild yeast-like aroma. The average nutritional value of 100 g kefir was as follows: energy content 217 kJ/52 kcal, 3.1 g protein, 4.2 g carbohydrate, and 2.5 g fat. The content of L. fermentum ME-3 in the probiotic kefir was 4 × 107 cfu/ml. Similar kefir produced without the probiotic adjunct served as a control.

Subjects

This randomised, double-blind, placebo-controlled, parallel-design, two-armed study (registered trial ISRCTN49744186) was conducted in South Estonia, according to the guidelines laid down in the Declaration of Helsinki 1996–2000. The study was approved by the Ethics Committee of the University of Tartu (protocol number 210/T-3; 18.01.2011). All participants signed the written informed consent at enrolment and were given the option to withdraw from the study at any time.

Subject inclusion criteria were a desire to participate; age 35–65 years; blood lipid fractions: total cholesterol >5.2 mmol/l, LDL-C >3.4 mmol/l, and/or TG >1.7 mmol/l, in addition, subjects had to be willing to maintain a stable diet and physical activity level. Exclusion criteria included a history of gastrointestinal disease; food allergy or acute infection; use of any antimicrobial agent within the preceding 2 months; use of any regular concomitant medication within the preceding 2 weeks, including non-steroidal anti-inflammatory drugs and antioxidant products; pregnancy or breastfeeding; any serious organ or systemic disease; high blood pressure (>140/95 mmHg); eating disorder; extensive exercise; genetic hyperlipidaemia; drug or alcohol abuse; active weight loss >5 kg in the prior 3 months; participation in other studies within the last 30 days or during the study; and not wishing to participate. Also, subjects were excluded when they had used any concomitant treatment within the preceding 2 months that could influence evaluation of the efficacy and tolerability of the investigational study product, including lipid-lowering drugs (e.g., statins, bile acid sequestering agents, cholesterol absorption inhibitors, nicotinic acid) or dietary supplements (e.g., omega-3 fatty acids, calcium, oat fibre, niacin, green tea extract, plant sterols, soy protein, psyllium seed husk, or probiotics/prebiotics).

Participants were randomly assigned to receive either probiotic or placebo using a computer-generated randomisation list prepared by the independent statistician. The 164 eligible subjects were randomised to receive either probiotic treatment (PG; n = 83) or control kefir (CG; n = 81; Fig. 1).

Fig. 1 Flow chart of the study subjects. Legend: Randomised, double-blind, placebo-controlled, parallel-design, two-armed study (registered trial ISRCTN49744186). Participants were randomly assigned to receive either probiotic or placebo using a computer-generated randomisation list prepared by the independent statistician. The 164 eligible subjects were randomised to receive either probiotic treatment (PG; n = 83) or control kefir (CG; n = 81). The measurements were performed at 4 weeks with 137 participants (n = 71 PG and n = 66 CG) while 27 subjects withdrew. At 8 weeks 76 participants (n = 43 PG and n = 33 CG) were followed; while 51 subjects withdrew Full size image

The sample size was calculated for a two-armed intervention investigation that included 4 weeks of probiotic kefir consumption. We assumed a predicted change of 0.2 U in the LDL-C level between the probiotic and control groups during the treatment period. The planned sample size of 83 subjects provided sufficient power (0.90) to detect a significance level of P < 0.05 with the Student’s t-test.

Study protocol

Each group consumed 200 ml/day of kefir without (CG) or with (PG) the probiotic L. fermentum ME-3. The total amount of L. fermentum ME-3 consumed was 8 × 109 cfu/day. The trial lasted for 8 weeks. The weekly food diary was not performed but the appearance of gastrointestinal complaints was registered (Fig. 1). Measurements were carried out at the start of the study (baseline values) with 164 participants that met the inclusion criteria. We conducted measurements at 4 weeks with 137 participants (n = 71 PG and n = 66 CG) while 27 subjects withdrew (n = 12 PG and n = 15 CG) due to respiratory infections, intestinal complaints, and loss of contact. At 8 weeks 76 participants (n = 43 PG and n = 33 CG) were followed; while 51 subjects withdrew due to the infections, loss of contact and motivation (Fig. 1).

Clinical investigations

Subjects in both groups habitually consumed a Western-type diet, typically rich in potatoes, vegetables, meat, eggs, but also characterised with a high content of fibre (black bread) and several dairy products. The trial code was broken after 8 weeks. At each measurement time-point (baseline, 4 and 8 weeks), the subjects underwent a clinical examination. Plasma samples were collected after an overnight fast and abstinence from any medications, tobacco, alcohol, tea, and coffee. Each participant was evaluated for anthropometric indices. Body mass index (BMI) was calculated as the weight (kg) divided by squared height (m2) [25]. A sphygmomanometer was used to measure blood pressure, after subjects rested for 5 min in the sitting position. After the blood pressure measurement, blood samples were collected. Markers for determining cardiovascular health were selected based on suggestions by the NDA of the European Food Safety Authority [26]. At baseline, the two groups did not significantly differ in the selected anthropometric, clinical, and biochemical indices (Table 1). Similarly, at the end of the trial (8 weeks), the PG comprised 36 women and 7 men, with mean BMI 26.5 kg/m2, and mean age 50.4 y; the CG comprised 30 women and 3 men, with mean BMI 27.1 kg/m2, mean age 50.8 y. The two groups showed no significant differences. All volunteers were considered clinically healthy according to the tested baseline and reference values (Nordic Reference Interval Project; NORIP). Significant reductions in serum LDL-C and TG were considered the primary outcome measures for lowering the CVD risk score.

Table 1 Clinical data of study participants Full size table

The obtained faecal samples were stored in a domestic refrigerator at 4 °C for no more than 2 h before transportation to the laboratory. There, the samples were frozen at −80 °C until analyses for determining the L. fermentum ME-3 prevalence and counts.

Measurement of oxidized LDL

Oxidized LDL (oxLDL) was measured with an ELISA kit (Cat. No. 10-1143-01, Mercodia AB, Uppsala, Sweden) [17]. This solid-phase, two-site enzyme immunoassay was based on the direct sandwich technique, in which two monoclonal antibodies were directed against separate antigenic determinants on the oxidized apolipoprotein B molecule [27]. During incubation, oxLDL in the sample reacted with the anti-oxLDL antibodies bound to the microtitration wells. After washing, a peroxidase-conjugated anti-human apolipoprotein B antibody recognised the oxLDL bound to the solid phase. After a second incubation and washing, the bound conjugate was detected in a reaction with 3,3′,5,5′-tetramethylbenzidine. The reaction was stopped by adding acid, and the colour intensity (U/l) was measured spectrophotometrically at 450 nm.

Metabolic indices (plasma glucose, total cholesterol, LDL-C, HDL-C, and TG) were analysed with standard laboratory methods and certified assays in the clinical laboratory of the Tartu University Clinics, Estonia. As references, we used indices for routine laboratory tests proposed by the Nordic Reference Interval Project (NORIP, http://www.tandfonline.com/doi/pdf/10.1080/00365510410006324#.Vhpmx27-VCE).

Molecular assessment of L. fermentum ME-3

Bacterial DNA was extracted from faecal samples with a QIAamp DNA stool mini kit (QIAgen, Hilden, Germany) with some modifications. Faeces (0.22 g) was suspended in 200 μl TE buffer (10 mM Tris, 10 mM EDTA pH 8, 20 mg/ml lysozyme, and 200 U/ml mutanolysin), and incubated for 1 h at 37 °C. To these samples, we added 0.3 g of 0.1-mm zirconia/silica beads and 1.4 ml ASL solution from the stool mini kit. The tubes were then agitated for 3 min at 5000 rpm in a mini-bead beater (Biospec Products Inc., USA). The protocol was completed as described by the manufacturer (QIAgen, Germany).

To establish a quantitative assay, we created plasmids that contained the PCR-amplified region of target bacteria with the pGEM-T vector system (Promega, Madison, WI). The PCR amplicon derived from L. fermentum ME-3 was inserted into the plasmid vector, and the recombinant vector was transformed into chemically competent E. coli. Plasmids were purified with a MaxiPrep kit (Qiagen), and then, serially diluted, and finally, measured in a spectrophotometric analysis (Quibit™, Invitrogen) [28]. The target DNA was quantified by comparing with serial, 10-fold dilutions (101 to 105 plasmid copies) of previously quantified plasmid standards. Comparisons between the plasmid standards and samples were run in triplicate.

Real-time PCR was performed with the ABI PRISM 7500 HT Sequence Detection System (Applied Biosystems, USA) equipped with optical grade 96-well plates. For detection of L. fermentum ME-3, the reaction mixture (25 μl) for the TaqMan assay contained 2× TaqMan Universal PCR Master Mix (PE Applied Biosystems, USA), 25 pmol primers (ME31 forward: 3′-CTTTTTACCGCCAAAAGCAG-5′; ME-3 reverse: 3′-AGCCCTTGTCGGTGATATTG-5′), 10 pmol of TaqMan probe (5′-FAM-TTGACACAGACTCGAGCAGT-Tamra-3′), and 200 ng of extracted DNA. The thermocycling program included an initial cycle of 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s and 60 °C for 1 min. Data was analysed with Sequence Detection Software, version 1.6.3 (Applied Biosystem, USA). L. fermentum ME-3 quantities were expressed as the log 10 number of gene copies/g faeces.

Statistical analysis

Statistical analyses were performed with the R program, v. 3 · 0 · 2 (A Language and Environment, http://www.r-project.org) and GraphPad Prism, version 4 · 00 for Windows (GraphPad Software, San Diego, CA). All data were expressed as the mean and standard deviation (SD; Tables 1 and 2) or as the medians with two-sided confidence intervals for counts of BHS and L. fermentum ME-3. Sex and temporal colonisation with probiotic bacteria were compared between groups with Fisher’s exact test (Table 1, Fig. 3). Baseline and intervention data (BL 1–4 and BL 2–8 weeks) were compared with a paired t-test or with a Wilcoxon signed-rank test, according to the data distribution (Table 2). The change (mean ± SD) of individual biochemical indices from baseline of particular participants to 4 and 8 weeks in the probiotic and control groups was assessed with the paired t-test (Fig. 2). The extent of changes between probiotic and control groups (the PG and CG groups) was compared with the t-test. Differences were considered statistically significant when P was <0.05 (Table 2).

Table 2 The impact of consumption of probiotic or control kefir for 4 and 8 weeks on biochemical indices of blood sera in healthy subjects Full size table