*4.4. Sample Lyophilization*

After biometrical measurements and sex determinations were carried out, the organisms were sacrificed separating the bells by the manubrium bearing the oral arms, with the help of a Teflon knife, avoiding metal cross-contamination. Bells and oral arms were grouped to provide four different pools (*n* = 200 each), as reported in Table 3. Each pool was weighed, cleansed thoroughly with distilled water, minced, and lyophilized by an Alpha 1-2/LD Plus freeze dryer (Martin Christ, Osterode, Germany), for 72 h at −55 ◦C using a chamber pressure of 0.110 mbar. Then, the freeze dried pools were weighed, and stored at −20 ◦C until use. For each pool, a moisture content of ~95% *w*/*w* and a yield of 5% *w*/*w* were assessed.


**Table 3.** Sample pools considered for the present study.

### *4.5. Gross Energy Assessments*

To measure the gross energy content, a benchtop isoperibol calorimeter (Parr® 6200 Oxygen Bomb Calorimeter, Parr Instrument Company, Moline, IL, USA) was employed.

Approximately 1 g of each powdered pool was placed in a 1108 model oxygen bomb. To determine the gross energy densities of bombed samples, the calorimeter chamber was previously calibrated for the heat of combustion of 1 g of benzoic acid (26.46 kJ g<sup>−</sup>1), under controlled and reproducible operating conditions. In fact, the known amount of heat produced by the combustion of the calibration standard determined the energy equivalent (W) per change in water temperature between initial and postcombustion of the sample ( ΔT). Therefore, the energy content of each sample (ES) was calculated as follows

### ES = W × ΔT/exact sample weight

Each sample pool was run in triplicate. Results were expressed as Kcal 100 g<sup>−</sup>1, dw.

## *4.6. Crude Protein*

The crude protein content was determined using the AOAC Official Method 976.05 (automated Kjeldahl method) [99]. Approximately 1 g of each powdered pool was separately digested by the SpeedDigester K-439 (Büchi, Switzerland) and then analyzed by the KjelMaster System K- 375 (Büchi, Switzerland) and equipped with a scrubber of gases and vapors (Scrubber K-415, Büchi, Switzerland). For the calculation of the % protein in a sample, the obtained % nitrogen was multiplied by a conversion factor of 6.5. Each determination was conducted in triplicate.

### *4.7. Total Phenolic Content*

As a preliminary part of method development, an initial group of samples was subjected to two different extraction procedures: (i) with PBS (phosphate-buffered saline), pH 3.5 and (ii) with 80% methanol [74]. Aliquots (1.0 g) of each lyophilized sample pool were added with 16 mL of PBS and shaken for 2 h at 4 ◦C, in one case; with 16 mL of methanol and shaken for 16 h at 4 ◦C, in the other case. Successively, samples were homogenized at 9000 rpm and 4 ◦C for 30 min. To 1.0 mL of supernatant, 5.0 mL of Folin–Ciocalteau reagen<sup>t</sup> and 5.0 mL of sodium carbonate (20%) were added. The resulting solution was kept in the dark for 2 h, and later analyzed at the UV–Vis spectrophotometer, model UV-2401PC (Shimadzu, Milan, Italy). The wavelength of absorbance was set at 760 nm. A 5-point calibration plot was built up by using solutions of gallic acid in methanol in the range 50 to 2000 μg mL−1. Each point corresponded to three replicates.
