*2.1. Metabolites and Phenolic Compounds Detected in Test Substances by HPLC-ESI-MS/TOF*

The metabolites detected by HPLC-ESI-MS/TOF in the analyzed substances are presented, as a heat map, in Figure 2. Colors are based on the relative abundance (logarithmic scale) of the metabolites detected, where red represents high abundance and green represents low abundance. Overall, among all substances examined, the analysis evidenced

the presence of a total of 54 metabolites already known in the literature. In particular, the "Water-extract" showed 50 metabolites, which is the highest number recovered; "EtOAcextract" and "Nitric-extract" contained 36 and 35 metabolites, respectively; finally, only 25 metabolites were detected in the "MetOH-extract". Some marked differences were observed among the substances; in particular, a higher abundance of free amino acids, such as phenyalanine, proline, serine, tyrosine and valine, was evidenced in the "Water-extract" and "MetOH-extract" over the "EtOAc-extract" and "Nitric-extract". Really high relative abundances in some metabolites were also observed; in particular, 2-Hydroxyisocaproic acid, 3-(4-Hydroxyphenyl) propionic acid and 4-aminobenzoic acid in "MetOH-extract", docosahexaenoic acid in "EtOAc-extract" and the phenylalanine in the "Water-extract" and "MetOH-extract".

The most important phenolic acids detected by HPLC-ESI-MS/TOF in the substances analyzed are presented in Table 1. Their abundance is expressed in mg/kg of each substance. The most abundant phenolic compound detected in all the analyzed substances was benzoic acid, whose amount ranged from a minimum of 0.87 mg/kg in "Nitricextract" to a maximum of 3.57 mg/kg in "EtOAc-extract". In order of abundance, vanillin (0.21–2.04 mg/kg) and syringic acid (0.16–1.21 mg/kg), which had the highest concentrations of "MetOH-extract", were detected. The p-coumaric (4-hydroxycinnamic acid) acid was another phenolic compound recovered in all the substances; its abundance ranged from a minimum of 0.27 mg/kg in the "Water-extract" to a maximum of 0.88 mg/kg in the sample "Nitric-extract". The "Nitric-extract" also reported the highest concentration of 1-2-Dihydroxybenzene (0.86 mg/kg). Few phenolic compounds were detected just in one substance; among these, the 3-(4-hydroxy-3-methoxyphenyl) propionic acid and ellagic acid were detected only in "Nitric-extract", while sinapic acid only in the "Water-extract".

**Table 1.** Concentration of phenolic compounds detected in the tested substances (mean value ± standard deviation).


#### *2.2. In Vitro Preliminary Tests*

Results from the *in vitro* preliminary tests evidenced an inhibitory effect on the growth of the pathogens examined only for the waste shrimp extracted with nitric acid, named "Nitric-extract". Additionally, none of the control solutions (each solvent used for the preparation of the respective extract) inhibited mycelial growth. In the agar diffusion test, "Nitric-extract" at concentrations of 100, 75 and 50% showed an inhibitory effect on all strains of fungal and oomycete pathogens, while at concentration of 25%, an inhibitory effect was still observed only on *Ph. nicotianae* T2.C-M1A, *F. sacchari* CBS 145949, *A. alternata* 646, *P. digitatum* P1PP0, *P. commune* CECT 20767, *C. gloeosporioides* C2, *F. proliferatum* CBS 145950, *Pl. tracheiphilus* Pt2 and *Ph. nicotianae* T3-B-K1A, in order of significance (Table 2 and Figure 2). The diameter of inhibition halos was directly proportional to the concentration of the extract (Table 2). Significant differences in the inhibitory effects of the extracts were noticed among fungal and oomycete species as well as between species of the same genus

and even between strains of the same species (Table 2). At the maximum dose, which is 100% of the extract concentration, the highest inhibitory effect was on *Pl. tracheiphilus* Pt2; at 75% concentration, the highest inhibitory activity was on *Pl. tracheiphilus* Pt2 and *Ph. nicotianae* T2.C-M1A; at 50%, on *Ph. nicotianae* T2.C-M1A; and at the lowest dose (25% extract concentration), on *Ph. nicotianae* T2.C-M1A as well as on three typically post-harvest pathogens, i.e., *F. sacchari* CBS 145949, *A. alternata* 646 and *P. digitatum* P1PP0.

**Figure 2.** Heat map representing the relative abundances of metabolites detected in different shrimp extracts.


**Table 2.** Inhibitory effect of different concentrations (from 25 to 100%) of shrimp nitric-extract on the mycelium growth of 12 fungal and three oomycete plant pathogens, determined with the agar diffusion test by measuring the diameter of the inhibition halo around the wells. The incubation period was three days for fungi and 15 days for oomycetes.

<sup>1</sup> In a horizontal direction, for each pathogen, values with different bold letters are statistically different according to Tukey's honestly significant difference (HSD) test (*<sup>p</sup>* ≤ 0.05). <sup>2</sup> In the vertical direction, for the concentrations 25% Nitric-extract, 50% Nitric-extract, 75% Nitric-extract, 100% Nitric-extract, values with different letters (in *italic* and within brackets) are statistically different according to Tukey's honestly significant difference (HSD) test (*p* ≤ 0.05).
