Sterile tube

Resting blood samples were collected by venipuncture from an antecubital vein in sterile tubes (BD Vaccutainer, Franklin Lakes, NJ) coated with ethylenediaminetetra acetic acid (EDTA) as an anticoagulant. Plasma was separated by centrifugation at 1300 rcf for 10 minutes at 4°C. Samples were stored at −80°C until analysis. Muscle biopsy samples were obtained using the Bergstrom needle biopsy technique [30] (link) with the addition of manual suction from the vastus lateralis following local anaesthetization (2% lidocaine). For each experimental visit both muscle biopsies (fasted and post-prandial) were obtained from the same leg from separate incisions approximately 2–3 cm apart. Repeated biopsies from separate incisions have been shown to be unaffected by previous biopsies to the same leg [31] (link), [32] (link). Adipose tissue biopsies were also obtained using a Bergstrom needle with the addition of manual suction following local anaesthetization (2% lidocaine). Adipose tissue biopsies were obtained from the abdomen with the incision made approximately 5 cm lateral to the umbilicus and the needle being directed laterally once through this incision. Both muscle and adipose tissue were immediately blotted, snap-frozen in liquid nitrogen, and stored at −80°C until analysis.

Blood samples were collected in sterile tubes (BD Systems, Franklin Lakes, NJ, USA) and placed on ice immediately. A small sample of whole blood was transferred into EDTA-treated tubes (BD Systems) to be analyzed for hematocrit and hemoglobin, from which plasma volume shifts were calculated using the Dill and Costill [11 (link)] equation to account for hemoconcentration of the blood. The remaining whole blood specimens were centrifuged at 3000× g at 4 °C to separate sera. Testosterone, sex hormone-binding globulin (SHBG), and albumin were assessed with commercial analytical kits (DPC Inc., Los Angeles, CA, USA; Abcam, Cambridge, MA, USA). Additionally, free testosterone and bioavailable testosterone were calculated using procedures from Vermeulen et al. [12 (link)].

The probiotic bacteria described in Table 1 were pre-inoculated in MRS broth for 48 h at 37 °C in anaerobic conditions before the setup of the experiment. Maitake extract powder was added to mMRS and then sterilized together before inoculation, to a final concentration of 2% w/v.
The consortium was prepared in a sterile tube, mixing the selected probiotic in order to achieve an OD at 600 nm of 0.1. After the homogenization, the proper volume of the consortium (around 200 μL) was inoculated in sterile tubes (BD, Milano, Italia) with a final volume of 10 mL. Each tube contained only mMRS as a control or mMRS plus Maitake extract. After the inoculum, the tubes were placed in an anaerobic jar and then incubated for 48 h at 37 °C. The growth was evaluated as optical density at 600 nm.