Sustainability: Obtaining Natural Dyes from Waste Matrices Using the Prickly Pear Peels of Opuntia ficus-indica (L.) Miller

Round 1

Reviewer 1 Report

This paper is extremely interesting, of high research quality. In the present times, when there is a constant need to provide green solutions and alternatives, this research paper adequately describes an eco-innovative extraction method of natural dyes from Opuntia ficus-indica (L) Miller waste. All the methods are clearly presented so that, even to someone outside the research field, it is easy to understand what the research is about.  It is clear that all the authors are highly specialized in their research field and much attention has been paid to all the research aspects with a big thumbs up for the waste management and green method.

Author Response

“This paper is extremely interesting, of high research quality. In the present times, when there is a constant need to provide green solutions and alternatives, this research paper adequately describes an eco-innovative extraction method of natural dyes from Opuntia ficus-indica (L) Miller waste. All the methods are clearly presented so that, even to someone outside the research field, it is easy to understand what the research is about.  It is clear that all the authors are highly specialized in their research field and much attention has been paid to all the research aspects with a big thumbs up for the waste management and green method.”

Authors thanks Reviewer 1 for positive comments.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript submitted for publication in Agriculture refers to a solution for obtaining natural dyes from prickly pear peels resulting from Opuntia Ficus indica (l.) Miller. The subject by itself is exciting and may capture the attention of the scientific community.

Authors are declaring many goals for their experimental approach: 1. comparison between maceration and Naviglio extraction of peels; 2. characterization of the extracts by means of TLC and HPLC/DAD; 3. fractionation of the extracts through liquid-liquid extraction and fraction characterization; 4. characterization of extracts through determination of pH, specific gravity and dry residue; 5. dyeing fibers (wool and cotton) with the extract and study of the fixing modes with two types of mordants; 6. colorimetric and fastness properties of dyed fibers.

On my personal opinion, there are too many goals for a single publication and each aspect is quite poorly defended in the manuscript. Probably, the most discussed aspects in the manuscript refers to the way of dyeing the fibers and the color measurements.

Details with respect to TLC/HPLC separation are missing. An eluting system for TLC, more precisely isopropane/aluminium oxide 80/20 is confusing, as long as isopropane is a gas and aluminium oxide is a solid. The only HPLC chromatogram given in the manuscript allows identification of two compounds, namely betanin and isobetanin. Authors do not clarify how did they made identification, as long as in the Experimental section, the two compounds were not listed as available standards.

The argumentation that the Naviglio extraction system behaves better than a simple maceration is not obvious from the presented data. Fractionation through liquid-liquid extraction of the initial extracts produced by Naviglio and maceration modes, although mentioned  in the experimental part is not discussed at all.

Readers are not finding within the manuscript facts which are enounced in the title.

To conclude, on my personal opinion the manuscript should be rejected. Authors should rewrite and split the content of the manuscript in at least two different papers. One providing data about the advantages of the Naviglio extraction versus the classic maceration based on TLC and HPLC data, and another relating with the dyeing abilities of the resulting extracts.

Author Response

“The manuscript submitted for publication in Agriculture refers to a solution for obtaining natural dyes from prickly pear peels resulting from Opuntia Ficus indica (l.) Miller. The subject by itself is exciting and may capture the attention of the scientific community.

Authors are declaring many goals for their experimental approach: 1. comparison between maceration and Naviglio extraction of peels; 2. characterization of the extracts by means of TLC and HPLC/DAD; 3. fractionation of the extracts through liquid-liquid extraction and fraction characterization; 4. characterization of extracts through determination of pH, specific gravity and dry residue; 5. dyeing fibers (wool and cotton) with the extract and study of the fixing modes with two types of mordants; 6. colorimetric and fastness properties of dyed fibers.

On my personal opinion, there are too many goals for a single publication and each aspect is quite poorly defended in the manuscript. Probably, the most discussed aspects in the manuscript refers to the way of dyeing the fibers and the color measurements.”

Thank you for your comments and for pointing out the parts that were badly reported or difficult to interpret. We have corrected and added, point by point, the following:

“Details with respect to TLC/HPLC separation are missing. An eluting system for TLC, more precisely isopropane/aluminium oxide 80/20 is confusing, as long as isopropane is a gas and aluminium oxide is a solid. The only HPLC chromatogram given in the manuscript allows identification of two compounds, namely betanin and isobetanin. Authors do not clarify how did they made identification, as long as in the Experimental section, the two compounds were not listed as available standards.”

• the right solvent systems used for Chromatography on TLC has been included in line 172 to 174:

methanol-n-hexane (6:4, v/v), ethyl acetate-methanol-water (8:2:1, v/v) and chloroform-isopropanol (95:5, v/v). The spots were visualized by exposure to UV radiation (253 nm).

• the Sigma-Aldrich product used for the determination of betanin and isobetanin compounds has been added in line 78, in Materials and Methods, in the Chemical and Solvents subsection:

betanin (red beet extract diluted with dextrin).

• a part which explains the importance of the compounds recovered has been included in lines 154 to 157:

The fractions that were obtained through liquid-liquid separation carried out with solvents with increasing polarity allowed to extract several compounds of well-known biological interest and that could have a nutraceutical use: compounds such as fatty acids, small molecules like polyphenols and amino acids have been found.

• a part which explains, based on the literature, the identification made for betanin and isobetanin has been included in lines 320 to 325, in lines 327 to 330 and in lines 337 to 342:

Commercial betanin diluted with dextrin (which contains a mixture of betanin and isobetanin) purchased from Sigma-Aldrich represents one of the main betalainic sources used commercially for coloring purposes in North America and Europe. The comparison between the UV-VIS spectra obtained from of Opuntia ficus-indica (L.) Miller extracts and the commercial betanin solution allowed to identify its presence, as also reported in the literature [35].

35. Gonalves, L.C.P.; Trassi, M.A.D.S.; Lopes, N.B.; Dörr, F.A.; Santos, M.T. Dos; Baader, W.J.; Oliveira, V.X.; Bastos, E.L. A comparative study of the purification of betanin. Food Chem. 2012, 131, 231–238.

The thin layer chromatography (TLC) profiles of Opuntia ficus-indica (L.) Miller extracts (data not shown) revealed and confirmed the presence of natural dyes in the extract, the data were consistent with those obtained in the absorption spectra (Figure 5) and they are according with literature [36].

36. Bilyk, A. Thin‐Layer Chromatographic Separation of Beet Pigments. J. Food Sci. 1981, 46, 298–299.

Under the same conditions it was established that for the sample containing commercial betanin there are only two more important peaks, associated precisely with betanin and isobetanin; it has been calculated that betanin has a shorter retention time than isobetanin (Δt_R = 0.4 min) in the HPLC analysis, as reported in this work. This difference is explained by the fact that isobetanin has a stronger interaction with the non-polar stationary phase of the chromatographic column, as also shown in the literature [37,38].

37. Schwartz, S.J.; von Elbe, J.H. Quantitative Determination of Individual Betacyanin Pigments by High-Performance Liquid Chromatography. J. Agric. Food Chem. 1980, 28, 540–543.
38. Wybraniec, S. Formation of decarboxylated betacyanins in heated purified betacyanin fractions from red beet root (Beta vulgaris L.) monitored by LC-MS/MS. J. Agric. Food Chem. 2005, 53, 3483–3487.

• the caption of Figure 8, standards have been added to better clarify identification, so it has been modified in lines from 347 to 351:

Figure 8. HPLC chromatogram detected at 340 nm of molecules contained in aqueous extract of peel fruits of Opuntia ficus-indica (L.) Miller obtained by Extractor Naviglio®. Peaks 2 and 3 have been identified as pigment molecules such as betanin and isobetanin. Peaks 1, 4, 5 and 6 are identify such as luteolin, kaempferol, quercetin and isorhamnetin according to the standards available in the laboratory; the other peaks were unknown compounds.

“The argumentation that the Naviglio extraction system behaves better than a simple maceration is not obvious from the presented data. Fractionation through liquid-liquid extraction of the initial extracts produced by Naviglio and maceration modes, although mentioned in the experimental part is not discussed at all.”

• Authors thank Reviewer for comment. To overcame this lacking information a part that explains how obtaining richer extracts with the Naviglio method has been included in lines 289 to 294:

By comparing the spectra obtained for the extracts obtained through conventional maceration and the Naviglio methodology (data not shown for brevity) they showed that: the extracts obtained under the same conditions (solvent, time, pH, sample:solvent ratio) with the Naviglio methodology with respect to those obtained by maceration had a higher content of coloring molecules. So, it was concluded that the use of the Naviglio method allowed to obtain richer extracts in less time, as reported in literature for other applications [31–33].

31. Naviglio, D.; Pizzolongo, F.; Romano, R.; Ferrara, L.; Naviglio, B.; Santini, a An innovative solid-liquid extraction technology: use of the naviglio extractor for the production of lemon liquor. African J.Food Sci. 2007, 1, 42–50.
32. Naviglio, D.; Franchi, G.G.; Rossi, I.; Fiore, G.; Massarelli, P.; Nencini, C.; Santini, A. Preparation of an elixir from common juniper ( Juniperus communis L.) berries: the new Naviglio Extractor versus the traditional maceration technique . Food Manuf. Effic. 2009, 2, 41–47.
33. Salvatore, M.M.; Ciaravolo, M.; Cirino, P.; Toscano, A.; Salvatore, F.; Gallo, M.; Naviglio, D.; Andolfi, A. Fatty acids from paracentrotus lividus sea urchin shells obtained via rapid solid liquid dynamic extraction (Rslde). Separations 2019, 6.

“Readers are not finding within the manuscript facts which are enounced in the title.”

• Authors are grateful to Reviewer for this comment. Comments were added on the valuation of the work and method in lines 412 to 425:

This work highlights the possibility of using waste matrices such as those of prickly pear peels which within the agro-food chain represent an important cost in terms of disposal for companies. Therefore, using such scraps or more 'secondary raw materials' to obtain natural dyes, but not only, can represent a step towards reducing important costs for companies and increasingly directing them towards a circular economy. The use of the Naviglio extractor, an eco-innovative equipment, would allowed various companies working in the agro-food sector to obtain molecules with a high intrinsic value from the 'secondary raw materials' (waste o by-products) to be used in various sectors, such as that of natural dyes.

The compounds contained within the fractions obtained from liquid-liquid extractions could have an important role. Their recovery, also linked to the extractive method used, is of fundamental interest in the qualitative and qualitative evaluation that can be done. these bioactive compounds is valid choice for the use in the nutraceutical field and they could be an important step of innovation within the agro-food chain and an added value for companies willing to innovate their systems and productions; not lastly this process can be considered environmentally friendly.

“To conclude, on my personal opinion the manuscript should be rejected. Authors should rewrite and split the content of the manuscript in at least two different papers. One providing data about the advantages of the Naviglio extraction versus the classic maceration based on TLC and HPLC data, and another relating with the dyeing abilities of the resulting extracts.”

Author Response File: Author Response.docx

Reviewer 3 Report

The Authors presented the results in detailed. All necessary elements are included. Only one error should be improve: in subsection: Maceration extraction please provide the amount of water and EtOH (v/v) used for extraction. 

Author Response

“The Authors presented the results in detailed. All necessary elements are included. Only one error should be improve: in subsection: Maceration extraction please provide the amount of water and EtOH (v/v) used for extraction.”

Authors thanks Reviewer 3 for comments and for pointing out the parts that were badly reported or difficult to interpret. We have corrected and added the following:

• a part which explains the amount of water and EtOH (v/v) used for extraction has been included in lines 131 to 132 and in lines 138 to 139.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The manuscript submitted for publication in Agriculture refers to a solution for obtaining natural dyes from prickly pear peels resulting from Opuntia Ficus indica (l.) Miller. The subject by itself is exciting and may capture the attention of the scientific community.

Authors are declaring many goals for their experimental approach: 1. comparison between maceration and Naviglio extraction of peels; 2. characterization of the extracts by means of TLC and HPLC/DAD; 3. fractionation of the extracts through liquid-liquid extraction and fraction characterization; 4. characterization of extracts through determination of pH, specific gravity and dry residue; 5. dyeing fibers (wool and cotton) with the extract and study of the fixing modes with two types of mordants; 6. colorimetric and fastness properties of dyed fibers.

On my personal opinion, there are too many goals for a single publication and each aspect is quite poorly defended in the manuscript. Probably, the most discussed aspects in the manuscript refers to the way of dyeing the fibers and the color measurements.

Details with respect to TLC/HPLC separation are missing. An eluting system for TLC, more precisely isopropane/aluminium oxide 80/20 is confusing, as long as isopropane is a gas and aluminium oxide is a solid. The only HPLC chromatogram given in the manuscript allows identification of two compounds, namely betanin and isobetanin. Authors do not clarify how did they made identification, as long as in the Experimental section, the two compounds were not listed as available standards.

The argumentation that the Naviglio extraction system behaves better than a simple maceration is not obvious from the presented data. Fractionation through liquid-liquid extraction of the initial extracts produced by Naviglio and maceration modes, although mentioned  in the experimental part is not discussed at all.

Readers are not finding within the manuscript facts which are enounced in the title.

To conclude, on my personal opinion the manuscript should be rejected. Authors should rewrite and split the content of the manuscript in at least two different papers. One providing data about the advantages of the Naviglio extraction versus the classic maceration based on TLC and HPLC data, and another relating with the dyeing abilities of the resulting extracts.

Author Response

The replies to the comments are those previously reported since they are the same observations.