Planetary micro mill pulverisette 7

Carotenoid pigment extraction was performed according to Digesù et al. [44 (link)] with few modifications. Briefly, the freeze-dried samples of seeds and leaves were milled (Pulverisette 7 Planetary Micro Mill; Classic Line, Fritsch GmbH, Idar-Oberstein, Germany) with an agate jar and balls and stored at −20 °C until analysis. Seeds (0.5 g) and leaves (0.05 g) of each accession were extracted in a screw-capped tube by adding 2 mL of extraction buffer (hexane/acetone, 80:20 v/v) and 300 μL of butylated hydroxytoluene (BHT) (0.1% w/v) as an antioxidant and stirred in the dark for 16 h. Samples were then centrifuged for 10 min at 4000 rpm, the supernatants placed in glass tubes, and the residues were extracted once again by adding 2 mL of extraction buffer and stirring in the dark for 2 h. The organic layers were collected and filtered with a gyroscope filter for syringe PTFE (porosity of 0.45 mm). Then, 2 mL of extract was evaporated to dryness under vacuum. Finally, the dry residues were redissolved in 200 μL of MeOH: DCM (45:55 v/v) for the analysis.

Walnuts were cultivated in Apulia (Italy) and gently provided by a local farmer. Powders of walnut shells were obtained using a procedure reported in the literature [12 (link)]. Dried walnut shells were crushed into small fragments using a hammer and sieved with a 1 mm sieve (sample HM). The dried fragments of walnut shell, each smaller than 1 mm in size due to hammer milling, were further reduced in size by means of a planetary ball miller (Fritsch Pulverisette 7 planetary micro mill) equipped with two 12 mL capacity stainless steel reactors. Each reactor was filled with approximately 2 g of dried biomass (d/w). Additionally, 10 stainless steel spheres with a diameter of 5 mm and 3 stainless steel spheres with a diameter of 7 mm were included within each reactor. The milling protocol consisted of 8 cycles, each lasting 5 min at a rotation speed of 450 rpm. Following each cycle, a 10-min pause was implemented to prevent biomass heat build-up, thus avoiding putative structural modifications of extracted compounds. Following the completion of milling cycles, the temperature remained below 50 °C.
The resulting biomass was sieved with a 0.5 mm sieve. HM and BM samples were then subjected to Soxhlet extraction [50 (link)] for 12 h using EtOH, during which the temperature approached the boiling point of ethanol (78 °C).

Cell walls were characterised without fractionation using two-dimensional (2D) solution-state NMR (Kim and Ralph, 2010 (link); Mansfield et al., 2012 (link)). Straw (2-mm pieces) was pre-ground using a Mixer Mill MM400 (Retsch; 30/s vibrational frequency for 90–120 s). Samples were extracted three times with water, three times with 80% ethanol and once with acetone, then allowed to dry. The pre-ground extracted samples were ball-milled using a Fritsch Planetary micro mill Pulverisette 7 vibrating at 600 rpm with zirconium dioxide (ZrO₂) vessels containing ZrO₂ ball-bearings (10 mm × 10) with 5-min milling and 5-min cooling per milling cycle (cycle number depended on the amount of sample). The ball-milled samples were subjected to digestion (72 h × 2) to obtain ‘enzyme lignin’ (EL) by Cellulysin^® cellulase from Trichoderma viridae (Calbiochem), at 35°C in acetate buffer (pH 5.0). The ELs were dissolved into DMSO-d₆/pyridine-d₅ (4:1 v/v) and subjected to NMR on a Bruker Biospin AVANCE-III 700 MHz spectrometer equipped with a 5-mm QCI ¹H/³¹P/¹³C/¹⁵N cryoprobe with inverse geometry (proton coil closest to the sample). 2D-¹H–¹³C HSQC spectra were acquired using Bruker’s pulse program (hsqcetgpsip2.2). Bruker’s Topspin 3.2 (Mac) software was used to process spectra. The central DMSO peak was used as internal references (δ_C: 39.51, δ_H: 2.49 ppm).