*2.2. Agri-Wastes*

Walnut fruits with green hulls (*Juglans regia* L., Figure 1) were harvested from trees grown in two different agroclimatic localities of Calabria, in the south of Italy, which were Montalto Uffugo (latitude: 39◦24020"88 N, longitude: 16◦9 031"68 E) and Zumpano (latitude: 39◦18042"84 N, longitude: 16◦17034"44 E). Altitudes, geographical and climatic conditions of gathering areas are summarized in Table 1. *Fermentation* **2021**, *7*, x FOR PEER REVIEW 3 of 16

**Figure 1.** Walnut fruits with green hulls (*Juglans regia* L.). **Figure 1.** Walnut fruits with green hulls (*Juglans regia* L.).

*2.3. Extraction of Glucans*

characterized by FT-IR, SEM and DSC.

*2.4. α- and β-Glucan Content*

mL) at 40 °C for 20 min.

The employed extraction procedure was a conventional method involving two stages

Preliminary treatments of this method involve sequential extractions with three sol-

vents in order of increasing polarity, to remove lipids and soluble material such as vita-

mins, polyphenols, monosaccharides, disaccharides, and others [21]. The flour (3 g) was

first extracted with acetone (3 × 30 mL each), then with methanol (3 × 30 mL each) and

finally with 70% aqueous ethanol (3 × 30 mL each). All the extractions were performed at

room temperature (t = 2 h). For starch precipitation, residue solid was added to a solution

of 20%, w/v sodium carbonate in distilled water (pH 10, 30 mL), and the obtained suspen-

sion was warmed at 55 °C in a water bath with stirring for 30 min and then was centri-

fuged (Model J2–21, Beckman Instrument Co., Mississauga, ON, Canada) at 5000× *g* for 30

min. The solid was removed, and the pH of the supernatant was added to HCl 2 M until

a pH of 4.5 was reached (isoelectric point of proteins), and centrifuged at 5000× *g* for 30

min to separate proteins [22,23], which were discarded. An equal volume of absolute eth-

anol was added to the supernatant in order to precipitate glucans. After 12 h at 4 °C, the

solution was centrifuged at 5000× *g* for 30 min. The precipitate was resuspended in etha-

nol, filtered, rinsed with ethanol and freeze-dried. The extractions were performed in trip-

licate on three samples of husk powder from each agroclimate locality and the results

were expressed as mean ± standard deviation. The crude glucans were measured and

β-glucan content of husks was determined in triplicate using β-glucan assay kit from

Megazyme Ltd., Bray, Co. Wicklow, Ireland [17]. The dry sample (100 mg) was weighed

into a 25 mL flask and 12 M ice-cold sulphuric acid solution (2 mL) was added. The solu-

tion was vortexed and left to incubate for 2 h in an ice-water bath, and then it was diluted

with distilled water (12 mL) and left for 2 h in a boiling-water bath (T = 100 °C). After

cooling the temperature, 10 M KOH solution (6 mL) and 200 mM sodium acetate buffer

(pH 5) were added. After centrifugation (1500× *g*, t = 10 min), an aliquot (0.1 mL) of the

supernatant was mixed with 0.1 mL of a mixture of *exo*-1,3-β-glucanase (20 U/mL) plus β-

glucosidase (4 U/mL) at 40 °C for 60 min. Finally, the content of glucose in the solutions

was determined by incubating the solution with glucose-oxidase/peroxidase (GOPOD, 3.0

extraction at pH 10 and 55 °C and acidic precipitation, respectively [12,18–20].


**Table 1.** Geographical and climatic conditions of the two gathering areas.

The whole fruits were collected in the period of October 2017, when the walnut fruits were mature while the husks (mesocarp) were green and firm. The husks were manually separated from the walnut, freeze-dried (Telstar freeze-dryer, mod. Cryodos) after freezing at −20 ◦C and then reduced to powder using a pestle and mortar. The powder was sieved through a 60-mesh (250 µm) screen and then kept in screw-cap vials under nitrogen at −20 ◦C, before any extraction. Three samples per area were used, and all the extractions were carried out in triplicate. The data were expressed on a dry matter basis as mean ± standard deviation.
