Rotina 420r

Firstly, 50 g of thyme or pomegranate peel, as detailed above, was dissolved in water at a ratio of 1:20 (w/v) and incubated at 90 °C for 15 min. The rosemary and echinacea were dissolved in water at a ratio of 1:50 (w/v) and incubated at 90 °C for 1 min. Next, for all beverages, 500 mL of MilliQ water at room temperature was added to the samples, and the bottles were cooled at 30 °C. The prepared beverages were inoculated a rate of 1% of each strain grown for 48 h at 30 °C. The pH was measured before and after fermentation to confirm its decrease (from pH values above 5 to values below 4.4, depending on the beverage) as a result of the fermentation process (Supplementary Table S1).
Selected samples were transferred to 50 mL screw-cap polyethylene centrifuge tubes and centrifuged (Hettich Rotina 420R, Sérézin du Rhône, France) at 3500× g for 2 min to clarify the extracts. The supernatants were transferred to new tubes and pasteurized in a water bath at 85 °C for 10 min. Next, the samples were aseptically stored at −80 °C for further analyses.

In the morning, the day after the intracoronary imaging procedures, fasting venous blood samples were collected in 5 mL ethylenediamine tetraacetic acid tubes, centrifuged for 20 min in 20 °C at 3000×g (Rotina 420R, Hettich zentrifugen), aliquoted and stored in − 80 °C freezer until miR analyses. Additional blood samples were analyzed for total cholesterol, total triglycerides, LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), lipoprotein (a), Apolipoprotein-B, Apolipoprotein-A1, glycated hemoglobin A1c, hemoglobin, and creatinine by standard in-hospital procedures at the Department of Medical Biochemistry, St. Olavs Hospital. At the time of inclusion, information on anthropometry and CVD risk factors were collected from the hospital medical records. This included sex, age, body mass index (kg·m⁻²), diabetes mellitus, systolic- and diastolic blood pressure, smoking status, other comorbidities, medications, previous CVD (CAD, stroke, peripheral arterial disease, and/or aortic disease), heredity for CVD [first-degree relative with CVD before the age of 55 years (father) and 65 years (mother)], hyperlipidemia, and medically treated hypertension. The two latter conditions were defined as previously being diagnosed with this condition or not by a general practitioner or in an outpatient clinic before enrollment in the present study.

In order to determine the encapsulation efficiency (EE), Vivaspin2 centrifugal concentrators with a MWCO of 30,000 (Sartorius AG, Göttingen, Germany) was used to separate the non-encapsulated peptides from the liposomes. Aliquots of 1 mL of both coated and uncoated liposomes were placed in Vivaspin2 centrifugal concentrators and centrifuged two times for 30 min at 4000× g (Rotina 420 R, Hettich GmbH, Tuttlingen, Germany). Experiments were made in triplicate. The concentration of free SP that passed through the Vivaspin2 filter was determined using HPLC analysis with the conditions described above.

For VFA and BCFA determination, 10 mL of each sample (liquid phase) was mixed with 30 mL of a 16% formic acid solution (1:3, v/v) and was kept at 4 °C for 72 h for precipitation. Then, samples were centrifuged three times at 4000× g for 15 min at 15 °C (Rotina 420R (Hettich Lab Technology, Tuttlingen, Germany)). Another centrifugation at 14000 rpm for 1 h at 4 °C was performed. The supernatant was sampled and stored at −20 °C [40 (link)] until chromatographic analysis as described in Exp. 1. Ammonia N was determined in the same centrifuged sample used for fatty acids after being thawed at room temperature and then diluted 2:13 (v/v) with distilled water (2:13, v/v). Ammonia N was determined via spectrophotometry [50 ].