2.4. Clinical Trial—Study Design & Objective

The study was performed as an open-label, single-center pilot study between September and December 2020 at the study site of the Nutritional CRO BioTeSys GmbH (Esslingen, Germany). The clinical trial was conducted as a proof-of-concept study to determine the impact of a food supplement consisting of a probiotic strain and n-3 PUFA on the formation and uptake of SPM and their anti-inflammatory potential over a 4-week intake phase. The primary hypothesis was that intake of the food supplement leads to a significant elevation of circulating levels of SPM and/or SPM precursors. EPA and DHA uptake and tolerability of the study product were also observed. All subjects gave their informed consent for inclusion before they participated in the study. The study protocol was reviewed by the ethics committee of Landesärztekammer Baden-Württemberg without concerns (approval number: F-2020-096). The study was registered in the German Clinical Trials Register (DRKS00023304). During an intervention period of 4 weeks, study participants received the study product. At baseline (visit 1), after 24 h (visit 2), after 1 week (visit 3), after 2 weeks (visit 4) and after 4 weeks (visit 5) of supplementation, different outcome measures were evaluated. The study was conducted in orientation to the guidelines for Good Clinical Practice (GCP) and the Declaration of Helsinki regarding the treatment of human subjects in a study.

Overall, 79 people were pre-screened for eligibility, wherefrom 41 subjects were screened to ascertain their eligibility. Healthy women between 35 and 60 years with a body mass index (BMI) between 25 and 35 kg/m², non-smoker, were eligible to participate in the study. Further, subjects with low habitual consumption of fatty fish and seafood (defined as a frequency of twice per month or less) with an n-3 PUFA index in erythrocytes of <6.0% (determined at screening) were selected. The n-3 PUFA index is the sum out of EPA and DHA expressed as a percentage of total fatty acids in erythrocyte membranes. A total of 19 subjects were included in the study and all completed the study successfully, as shown in Figure 1.

Study flow diagram.

The main exclusion criteria for study participation were history or presence of any severe medical disorder potentially interfering with the study (e.g., malabsorption, chronic gastrointestinal diseases, heavy depression, diabetes, acute cancers within last 3 years except basal cell carcinoma of the skin), subject under prescription for medication or taking dietary supplements possibly interfering with this study (such as n-3 PUFA, probiotics, anti-spasmodic, laxatives and anti-diarrheic drugs or other digestive auxiliaries, use of PPIs, bismuth salts and/or H2-antagonists, fibers etc.) within 2 weeks prior to study start or during the study, intake of antibiotics in the last 2 months, significant changes in lifestyle or medication (within last 3 months), subjects consuming food or drinks claimed as ‘probiotic’ or ‘prebiotic’ more than once weekly, consumption of more than 3 portions of fruits and vegetables (sum) per day, subjects with stool frequency of ≤ 2 stools per week or the unwillingness to abstain from fish consumption or foods/oils high in n-3 PUFA during the study intervention. Furthermore, subjects were asked to keep their nutrition and lifestyle habits unchanged during study participation.

The study product consisted of 2 billion colony forming units of Bacillus megaterium DSM 32963, 477 mg n-3 PUFA lysine salt, 180 mg hyaluronic acid, 30 mg coenzyme Q10, 20 mg lecithin from sunflower, 30 mg vitamin C, 4 mg zinc, 30 μg selenium, 20 μg biotin, 7 μg vitamin D3, 1 μg vitamin B12 (content per 2 capsules) (Evonik Operations GmbH, Darmstadt, Germany). The capsules were coated for targeted delivery of the ingredients into the large intestine. Two capsules were taken daily (one in the morning and one in the evening), unchewed with water. To allow for standardization during the study visits, subjects were instructed to take the evening capsule 12 h prior to blood sampling. Compliance of product intake was calculated from returned capsules.

Blood sampling was performed at each visit after an overnight fast of at least 10 h for the determination of n-3 PUFA and LM that were measured in fasting plasma samples collected at baseline and end of the intervention.

Analysis of LM in plasma was performed as described in Section 2.2.

Analysis of EPA and DHA in plasma (each visit) and erythrocytes (baseline and end of intervention) was performed with gas chromatography at Omegametrix GmbH (Planegg, Germany). In brief, the fatty acid composition was analyzed using the HS-Omega-3 Index^® methodology as previously described [29]. Fatty acid methyl esters were generated by acid transesterification and analyzed by gas chromatography using a GC2010 Gas Chromatograph (Shimadzu, Duisburg, Germany) equipped with an SP2560, 100-m column (Supelco, Bellefonte, PA, USA) using hydrogen as carrier gas. Fatty acids were identified by comparison with a standard mixture of fatty acids characteristic of erythrocytes and plasma. A total of 26 fatty acids were identified and quantified. Results are given as a percentage of total identified fatty acids.

Throughout the study intervention, the subjects documented any adverse events and concomitant medication in diaries. Furthermore, safety blood routine parameters and vital signs were determined. Tolerability was assessed after 1, 2, and 4 weeks of intervention.

The study was performed as an exploratory proof of concept study. Data obtained in this study were listed and summarized with descriptive statistics or frequency tables as appropriate. Changes versus baseline were evaluated applying paired t-test statistic or Wilcoxon signed rank test in case of non-normal distribution of data sets. Non-normality was evaluated with the Shapiro-Wilk test (p < 0.05). All statistical tests were performed two-sided, with a significance level of 0.05. Statistical analysis and graphs were generated using GraphPad Prism (Version 5.04, GraphPad Software, Inc., San Diego, CA, USA). Within figures, mean levels with a 95% confidence interval (CI) are depicted.