Triticum durum (Desf.) and Oryza sativa L. are, after the primacy of maize, the two most produced crops worldwide and they contribute to more than a half of human dietary calories. Counting a production between 9 and 24 MMT in 2014, Europe contends the lead as the largest cereal waste producer together with USA and industrialized Asia (FAO, 2011); moreover, Italy is one of the major European cereal producers. According to FAO, one-third of the edible parts of food produced for human consumption gets lost or wastes globally, this amount corresponds to 1. 3 
year with a global cost of $750 billion every year (FAO, 2011) . Until a few decades ago, food wastes were considered neither a cost nor a benefit and they were only used as animal feed, landfilling or composting. Faced with the issue of food waste management, which is now particularly relevant with the COVID-19 pandemic crisis, the exploitation of industrial wastes becomes a priority, as suggested by the European "Circular Economy Action Plan" (European Commission, 2015) . A key point stressed by the Commission in the European Parliament in 2015 refers to food waste as possible raw materials for reuse and reinjection into the economy: organic wastes, for example, could return to the soil as sustainable fertiliser. Moreover, the circular economy approaches to biomolecules production fits with the United Nations 2030 Agenda for Sustainable Development which declared the 17 Sustainable Development Goals (European Commission, 2019; Galanakis, 2020) where 'sustainability' and 'circularity of processes' are the key words for sustainable development in many industrial and social sectors. In fact, the 2030 Agenda stresses the need of reducing J o u r n a l P r e -p r o o f postharvest losses, of moving on to processing and retailing, and of valorizing by-products by recapturing bioactive compounds from food processing by-products.
According to Cordell (2017) , natural product research, must necessarily consider sustainability in its development, and beside its sustainable aspect, the recovery of high value compounds must be economically interesting to be applied in industry. Conventional extraction processes are expensive, time-consuming, not sustainable (as they require amounts of energy and solvents) and quite laborious (Cvjetko, 2018), therefore, in past recent years, several green alternatives which are safer, more efficient and in some cases economic, have been considered. Ultrasounds assisted extraction (UAE), considered one of the 10 most popular emerging technologies (Galanakis, 2013) , can be a valuable tool to increase the extraction yield of biomolecules from agricultural waste while minimizing their environmental impact. In mechanism of action is due to hydrophobicity, decrease of negative surface charge and occurrence of local rupture and pore formation which cause irreversible changes in the cell membranes. The antioxidant and antimicrobial activities of the extracts obtained in our research have been therefore evaluated to find possible applications of cereal by-products in the dermo-functional and cosmetic industries, as natural preservatives, but other exploitations in the food sector as fortification agents and antioxidant additives may be considered (Galanakis, 2018) . Starting to the fact that WHO or any regulatory bodies stress on the importance of qualitative and quantitative methods for characterizing botanicals (quantifying biomarkers and chemical markers and fingerprinting their profiles) with the aim of achieving the standardization of botanicals and guaranteeing a reproducible safety and efficacy (Khan et al., 2019) , we chemically characterized the obtained extracts with various approaches. Briefly, the main goals of the present study were to J o u r n a l P r e -p r o o f investigate the contribution of the sonication effect during chemical hydrolysis in order to apply a more sustainable and effective extraction method from durum wheat and rice by-products, compared to those commonly used, to standardize the obtained extracts and to propose a possible industrial application.
Triticum durum (Desf.) bran was provided by Molino Grassi (Parma -Italy) and obtained after decortication of durum wheat caryopsis; Oryza sativa L. bran was provided by Grandi Riso S.p.a (Pontelangorino, Ferrara -Italy) and obtained after the rice husking phase. All plant materials derived from a homogenous batch and the supplied samples were about 1 kg. After being received, all plant materials were stocked at -20°C until used. Prior to any extraction, all plant materials were milled through a 2 mm sieving ring of a Variable Speed Rotor Mill (Fritsch, Germany).
All the solvents and reagents employed for analyses were chromatographic grade. Trans-ferulic acid (FA) standard, Trolox, DPPH (1,1-diphenyl-2-picrylhydrazil), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), methanol, ethyl acetate, ethanol, formic acid, acetic acid, toluene, natural products-polyethylene glycol reagent 
In order to extract free phenolics from cereal by-products matrixes, 5 g of each bran were placed into a volumetric flask 
Bound phenolic compounds were extracted as reported by Verma et al. (2009) from all UAE residues, which have previously had the free phenolics removed. Briefly, 2.5g of each matrix was hydrolyzed with 50 mL of 2M sodium hydroxide at room temperature for 1 h (alkaline hydrolysis, AH). After AH, the pH of the mixture was adjusted to 3 with 6M HCl. Bound phenolics were then extracted three times with ethyl acetate. The ethyl acetate extracts were evaporated to dryness under a stream of nitrogen at room temperature and the residue was dissolved again in ethyl acetate, centrifuged 10 minutes at 4000 rpm. All samples were evaporated to dryness under a stream of nitrogen at room J o u r n a l P r e -p r o o f temperature and finally weighed to calculate the extraction yields. Bound phenolics were also extracted coupling sonication to alkaline hydrolysis (ultrasound-assisted alkaline hydrolysis, UAAH) as described by Gonzales et al. (2014) , thus following the same procedure described above for the alkaline conditions but for 30 min instead of 60.
After the extraction, all samples were evaporated to dryness under a stream of nitrogen at room temperature and finally weighed to calculate the extraction yields. were put on the plate using the automatic sampler. The mobile phase was: chloroform:/ethyl acetate/methanol (7:2.5:0.5, v/v/v) + 1% acetic acid. Plates were captured at 254 nm and 366 nm before and after derivatization with NP/PEG in order to highlight the presence of phenolic compounds with TLC Visualizer (CAMAG).
HPTLC bioautographic assay was employed to determine antioxidant compounds of the extracts using DPPH radical and ABTS, following the method described by Rossi (2011) . Each extract was applied to HPTLC plates as described for HPTLC analyses. After development, plates were sprayed with DPPH (i) and ABTS (ii).
After development plates were sprayed with a methanolic solution of 2,2-diphenyl-1-picryl-hydrazyl radical (2 mg/mL) and photographed at visible light after 30 min;
(ii) After development, plates were sprayed with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) solution obtained after the reaction of 2 mM ABTS and 70 mM of potassium persulfate (K2S2O8) and
immediately photographed at visible light.
The analyses of phenolic acid extracts for the identification and the quantification of FA and p-CA acids were performed using a Waters modular HPLC system (MA, model 1525) coupled to a diode array detector-DAD (model 2998) and a FinniganMAT LCQ (TermoQuestCorp./FinniganMAT; San Jose, CA, USA) mass spectrometer module linked to an injection valve with a 20 μL sampler loop, following the method described by Robbins and Bean (2004) .
The separation of phenolic acids was achieved with a Luna C18 column (Phenomenex, 250 x 4.6 mm; particle size 5 m); the mobile phase consisted of methanol (B) and 0.1% aqueous formic acid (A) as the binary solvent system. The solvent gradient in volumetric ratios was as follows: 5-30% B over 50 min, held at 30% B for an additional 15 min; at column was thermostatically controlled at 30 • C. The mass experiments were carried out on a FinniganMAT LCQ (TermoQuest Corp./FinniganMAT; San Jose, CA, USA) mass spectrometer module, equipped with an ion trap mass analyzer and an ESI ion source electrospray, in negative ion mode. For ESI-MS and MS 2 experiments, the parameters were set as follows: the capillary voltage was 3.5 kV, the nebulizer (N2) pressure was 20 psi, the capillary temperature was 300°C, the auxiliary gas (N2) fow was 9 L/min, and the skimmer voltage was 40 V. The mass spectrometer was operated in the negative ion mode in the m/z range 100-1000. Injection volume was set to 20 µL. Dedicated JASCO software (ChromNAV ver 2.02.01) was used to calculate peak area by integration. Following chromatogram recording, sample peaks identification was carried out by comparison of UV spectra and retention time (RT) with those of the pure standard. Each tested extract was prepared in a methanolic solution (methanol/water, 80:20, v/v) at the concentration of 0.5 mg/mL. Four different concentration of trans-ferulic acid and p-coumaric standard were prepared in methanol (5 -500 µg/mL) and each solution was injected in triplicate. The obtained calibration graphs allowed the determination of trans-ferulic and p-coumaric acids concentrations. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated following the approach based on the standard deviation of the response and the slope as presented by
European Medicine Agency (1995).
The Folin-Ciocalteu spectrophotometric assay was used to determine total phenolics content of all extracts with a ThermoSpectronic Helios-γ spectrophotometer and performed according to previously described methods (Singleton et al., 1999; Tacchini et al., 2018) . The mean of three readings was used and the total phenolics content was expressed as mg of gallic acid equivalents (GAE)/g of dry extract. Bound phenolics percentage was calculated with the following formula: bound phenolics % = (TPCbound x 100) / (TPCbound + TPCfree). 
The ABTS, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), radical scavenging activity was performed using the method of Horswald and Andlauer (2011). The same concentrations prepared for the previously described DPPH assay were used for both extracts and Trolox. 7 mmol/L aqueous solution of ABTS (10 mL) and 51.4 mmol/L aqueous solution of K2S2O4 (0.5 mL) were mixed to obtain a radical cation solution that has been adjusted spectrophotometrically to 0.7 ± 0.05 at 734 nm. After 6 min of incubation in the dark at room temperature, microplates were analyzed with a microplate reader (Biorad, 680 XL, Hercules, California, USA) and the absorbance was read at J o u r n a l P r e -p r o o f providing 50% radical inhibition, and calculated as described for DPPH. All experiments were assessed in triplicate and values were reported as mean ± SD.
The antimicrobial susceptibility of the bacteria was determined on Mueller-Hinton agar (Oxoid S.A. Madrid, Spain)
using the disk diffusion method. Six different antimicrobial agents, widely used in human clinical, were studied: ampicillin (10 µg/disk), penicillin (10 µg/disk), chloramphenicol (30 µg/disk), kanamycin (30 µg/disk), ciprofloxacin (5 µg/disk) and doxycycline (30 µg/disk). Staphylococcus aureus reference strain ATCC 25923 was used as a quality control. Each antimicrobial agent has been tested against all bacteria strains and incubated overnight at 37°C. The measurement and interpretation of growth inhibition diameters was performed following the CLSI guidelines for human antimicrobial susceptibility tests for human pathogens (CLSI, 2015).
The antimicrobial activity test was performed on the most interesting extracts in terms of FA content because of its already well-known antimicrobial properties described in literature by Borges 
Starting from the interesting antioxidant and antimicrobial activity results obtained, one extract has been selected to be inserted in a cosmetic formulation, in order to propose a practical application of cereal by-products in the cosmetic market. We decided to formulate the finished products using durum wheat bran UAAH extract in two concentrations:
0.3% (F1) and 0.5% (F2).
The phenolics extract was included in cosmetic formulations (oil-in-water emulsion) with the following INCI A common oil-in-water base protocol was used for all formulations. Phase 1 (hydrophilic) was prepared by weighting each raw material and heating the phase up to 70°C. Phase 2 (lipophilic) was prepared and heated up to 40°C and subsequently added to phase 1 by pouring the lipophilic phase into the hydrophilic one with the use of a turboemulsifier mixer (Silverson   East Longmeadow, MA, USA). Finally, the emulsion obtained was divided into aliquots to add phase 3 (extract). The final pH was adjusted within the range 5-5.5 to which all preservative systems work best and to make the preparations suitable for topical application. The stability of the cosmetic products was evaluated at both ambient temperature and at 40°C in oven for 1 month.
The PCL assay, based on the methodology of Popov and Lewin (1994) , The extract for ACL measurements was centrifuged (5 min at 16,000 g) prior to analysis. The antioxidant assay was carried out in triplicate for each sample, and 20 μL of the diluted extract (1:40, v/v) HPLC-grade methanol (ACL), was sufficient to correspond to the standard curve.
All results were means of three independent experiments. The presented data show mean values  SD. The results were evaluated for statistical significance using Student's t-test for two groups (GraphPad Software, San Diego, California, USA) considering a significant difference of P < 0.05 and univariate analysis of variance for more than 2 groups with Statistica 6.0 software (StatSoft, Inc., Tulsa, OK, USA) and Tukey's post hoc test to investigate significant differences (P < 0.05) between the tested samples.
The yields obtained with the various extraction method applied are reported in Table 1 : free phenolics extracts (UAE) gave the highest extraction yields (around 17-20%) due to the hydroalcoholic solvent used which enable the extraction of other molecules with a low extractive specificity. Bound phenolics (AH and UAAH) yields are much lower but more selective for our phenolic target. In contrast to what was previously verified with maize germ (Burlini et al., 2020) , no significant differences in yields have been obtained coupling sonication to alkaline hydrolysis (P> 0.05).
A slightly higher percentage of yield was found by extracting the rice (0.54%) than durum wheat bran (0.41-0.48%). contributed to the extract's activity. The lipophilic fraction of rice bran is, in fact, higher than that characterizing wheat bran (Jiang and Wang, 2005) and it is the food source with the highest phytosterol content, class of molecules that have demonstrated to be antioxidants (Lerma-Garcia, 2009; Wang et al., 2002) . In relation to the different activity, it should also be noted that the relative amount of p-CA is much greater in the rice bran extracts and may contribute in the radical scavenging variation. Regarding bound phenolic extracts, durum wheat bran exhibited the best results both with DPPH (IC50 of 3.61 ± 0.09 µg/mL) and ABTS (IC50 of 1.16 ± 0.1 µg/mL); in particular, DPPH activity of UAAH durum wheat extract is significantly higher than AH (P<0.05). Moreover, the same result is comparable to that obtain with Trolox (DPPH: 3.43 ± 0.32 µg/mL) suggesting that the association of ultrasounds to alkaline hydrolysis can be a valid technique to effectively obtain antioxidant extracts from cereal wastes. Similar results have been obtained with rice bran extracts, although with slightly lower activity than durum wheat bran: bound phenolics obtained with AH and UAAH gave IC50 respectively of 55.00 ± 5.37 µg/mL and 38.01 ± 0.52 µg/mL with DPPH and IC50 respectively of 10. The antimicrobial susceptibility test was used to evaluate the susceptibility of the bacteria strains against six antimicrobial agents which are commonly used to treat human infections with S. aureus and S. epidermidis. Figure 4 shows bacteria growth diameters which has been used to decide whether the bacterium was susceptible (S), intermediate (I) or resistant (R) to each antimicrobial agent. In Table 6 . The antimicrobial activity test defined wheat and rice brans potentially good antimicrobial sources. They could be use as active ingredients in cosmetic or dermo-functional products with antimicrobial activity or as additives for microbiological stability and therefore as natural preservatives in finished preparations.
In order to propose a practical application of the most active extract, a cosmetic product has been formulated (O/W emulsions) and tested for its stability both at ambient temperature and at 40°C. The high quantity of FA, characterized by a deep yellow color, which was contained in durum wheat extract, caused a slight alteration of final preparation's color, in particular with 0.5% of extract concentration. However, a slight color variation did not affect the pleasantness of the finished product and it was not indication of a depletion of the product but enhance its naturalness. Its fragrance was characteristic and pleasant.
The cosmetic products showed excellent stability at room temperature while a slight alteration occurred at 40° C, with a slight surfacing of the oily phase, probably due to the reduced quantity of product taken for the investigation.
Furthermore, in both stability tests, a slight modification in the pH has been noted ( Table 7) which was in any case within the suggested limits for cosmetic formulations (pH: 3.5-6).
Since previous studies demonstrated that the type of cosmetic formulation, the pH and storage conditions significantly influence the antioxidant activity of a functional ingredient (such could be natural extracts), it is necessary to evaluate the antioxidant activity of a finished product in relation to that of the single active ingredient (Ziosi et al., 2010) .
Therefore, the antioxidant activity has been tested and compared with PCL method which can provide evaluation of both cosmetic formula and extract activities, consenting an effective comparison. The PCL results are reported in Table   8 The overall advantages of this study concern the timing of extraction and the quality of the extracts obtained in terms of phenolic molecules and in vitro biological activity. As far as the working time is concerned, in fact, associating sonication with alkaline hydrolysis allows to reduce the extraction time from 60 to 30 minutes, thus halving the working hours to obtain the product. Despite the speed, the extracts obtained were standardize with respect the quantification of 
Cereal by-products are already well-known sources of interesting phytochemicals but, although their extraction has been the subject of numerous literature studies, precisely because of the great application potential of these matrices, the extraction techniques used up to now require long extraction times and methods that are still not sustainable. The overall results of the present study confirm the possibility to apply a more sustainable and effective method for the release of bound phenolic acids from cereal by-products. In particular, durum wheat and rice bran by-products are interesting sources of phenolic acids, mostly in bound form, whose extraction can be improved by combining 
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
This work was supported by POR FSE 2014/2020 (Emilia Romagna region, Italy), project code: E79D16003170007.
The 
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 