How Pre-Harvest Inactivated Yeast Treatment May Influence the Norisoprenoid Aroma Potential in Wine Grapes

Round 1

Reviewer 1 Report

The manuscript by Crupi et al. is very interesting article about influence of pre-harvest inactivated yeast treatment on the norisoprenoid aroma potential in wine grapes. The experimental design is appropriate and the text is clearly written. 

However, the manuscript needs some technical corrections. Titles of books or book chapters are not properly written. Make all corrections in References according to Author Guidelines.

Author Response

Response to Reviewer 1

The manuscript by Crupi et al. is very interesting article about influence of pre-harvest inactivated yeast treatment on the norisoprenoid aroma potential in wine grapes. The experimental design is appropriate and the text is clearly written.

Thank you very much about your positive judgement.

 

However, the manuscript needs some technical corrections. Titles of books or book chapters are not properly written. Make all corrections in References according to Author Guidelines.

According to your suggestion, we have revised the “References” section and corrected it appropriately.

References

 

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2. Razungles, A.; Bayonove, C.L.; Cordonnier, R.E.; Baumes, R.L. Etude des Carotenoides du Raisin à Maturitè. Vitis 1987, 26, 183-191.
3. Razungles, A.; Bayonove, C.L.; Cordonnier, R.E.; Sapis, J.C. Grape carotenoides: changes during the maturation period and localization in mature berries. J. Enol. Vitic. 1988, 39 (1), 44-48.
4. Crupi, P.; Milella, R.A.; Antonacci, D. Simultaneous HPLC-DAD-MS (ESI+) determination of structural and geometrical isomers of carotenoids in mature grapes. Mass Spectrom. 2010, 45(9), 971-980.
5. Mendes-Pinto, M. M.; Silva-Ferreira, A. C.; Caris-Veyrat, C.; Guedes de Pinho, P. Carotenoid, chlorophyll, and chlorophyll-derived compounds in grapes and port wines. Agric. Food Chem. 2005, 53(26), 10034-10041.
6. Silva-Ferreira, A. C.; Monteiro, J.; Oliveira, C.; Guedes de Pinho, P. Study of major aromatic compounds in port wines from carotenoid degradation. Food Chem. 2008, 110, 83-87.
7. Crupi, P.; Coletta, A.; Milella, R.A.; Palmisano, G.; Baiano, A.; La Notte, E.; Antonacci, D. Carotenoid and Chlorophyll derived compounds in some wine grapes grown in Apulian region. Food Sci. 2010, 75, S191-S198.
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9. Crupi, P.; Alba, V.; Masi, G.; Caputo, A.R.; Tarricone, L. Effect of two exogenous plant growth regulators on the color and quality parameters of seedless table grape berries. Food Res. Int. 2019, 126, 108667.
10. Baumes, R. Wine aroma precursors. In Wine chemistry and biochemistry; Moreno-Arribas, M.V., Polo, M.C., Eds.; ()Springer: New York, 2009, pp. 251-274.
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12. Baumes, R.; Wirth. J.; Bureau, S.; Gunata, Y.; Razungles, A. Biogeneration of C13-norisoprenoid compounds: experiments supportive for an apo-carotenoid pathway in grapevines. Chim Acta 2002, 458, 3-14.
13. Oliveira, C.; Silva Ferreira, A.C.; Mendes Pinto, M.; Hogg, T.; Alves, F. Carotenoid compounds in grapes and their relationship to plant water status. Agr. Food Chem. 2003, 51, 5967-5971.
14. van Breemen, R.B. Liquid chromatography/mass spectrometry of carotenoids. Pure App. Chem. 1997, 69, 2061-2066.
15. Emenhiser, C.; Simunovic, N.; Sander, L.C.; Schwartz, S.J. Separation of geometrical carotenoid isomers in biological extracts using a polymeric C30 column in reversed-phase liquid chromatography. Agr. Food Chem. 1996, 44, 3887-3893.
16. Ferrari, S. Biological elicitors of plant secondary metabolites: Mode of action and use in the production of nutraceutic. In Bio-farms for nutraceuticals: Functional food and safety control by biosensors; Giardi, M.T., Rea, G., Berra, B., Eds.; (Landes Bioscience and Springer Science + Business Media LLC: New York, 2010, pp. 152-166.
17. Bektas, Y.; Eulgem, T. Synthetic plant defense elicitors. Plant Sci. 2015, 5, 1-17.
18. Portu, J.; López, R.; Baroja, E.; Santamaría, P.; Garde-Cerdán, T. Improvement of grape and wine phenolic content by foliar application to grapevine of three different elicitors: Methyl jasmonate, chitosan, and yeast extract. Food Chem. 2016, 201, 213 - 221.
19. Šuklje, K.; Antalick, G.; Buica, A.; Coetzee, Z.A.; Brand, J.; Schmidtke, L.M.; Vivier, M.A. Inactive dry yeast application on grapes modify Sauvignon Blanc wine aroma. Food Chem. 2016, 197, 1073 - 1084.
20. Gutiérrez-Gamboa, G.; Marín-San Román, S.; Jofré, V.; Rubio-Bretón, P.; Pérez-Álvarez, E.P.; Garde-Cerdán, T. Effects on chlorophyll and carotenoid contents in different grape varieties (Vitis vinifera L.) after nitrogen and elicitor foliar applications to the vineyard. Food Chem. 2018, 269, 380 - 386.
21. Betz, M.; Schindler, C.; Schwender, J.; Lichtenthaler, H. K. Jasmonic acid induced changes in carotenoid levels and zeaxanthin cycle performance. In Plant lipid metabolism; Kader, J.C., Mazliak, P. Eds.; 1995. Springer: Dordrecht, 1995, pp 353-355.
22. Lu, Y.; Jiang, P.; Liu, S.; Gan, Q.; Cui, H.; Quin, S. Methyl jasmonate- or gibberellins A3-induced astaxanthin accumulation is associated with up-regulation of transcription of b-carotene ketolase genes (bkts) in microalga. Haematococcus pluvialis Biores. Technol. 2010, 101, 6468-6474.
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24. Melendez-Martinez, A.J.; Britton, G.; Vicario, I.M.; Heredia, F.J. HPLC analysis of geometrical isomers of lutein epoxide isolated from dandelion (Taraxacum officinale F.Weber exWiggers). Phytochemistry 2006, 67, 771-777.
25. Martì, M.P.; Busto, O.; Guash, J. Application of a headspace mass spectrometry system to the differentiation and classification of wines according to their origin, variety and ageing. Chromatogr. A 2004, 1057, 211-217.
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27. Bureau, S.M.; Razungles, A.J.; Baumes, R.L.; Bayonove, C.L. Effect of vine or bunch shading on the carotenoid composition in Vitis Vinifera L. berries. I. Syrah grapes. Enol. Sci. 1998, 53(2), 64-71.
28. González-Gómez, D.; Lozano, M.; Fernández-León, M.F.; Bernalte, M.J.; Ayuso, M.C.; Rodríguez, A.B. Sweet cherry phytochemicals: identification and characterization by HPLC-DAD/ESI-MS in six sweet cherry cultivars grown in Valle del Jerte (Spain). Food Compos. Anal. 2010, 23, 533-539.
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30. Gómez-Plaza, E.; Mestre-Ortuño, L.; Ruiz García, Y.; Fernández-Fernández, J.I.; López-Roca, J.M. Effect of benzothiadiazole and methyl jasmonate on the volatile compound composition of Vitis vinifera L. Monastrell grapes and wines. J. Enol. Vitic. 2012, 63, 394-401.
31. Lucini, L.; Baccolo, G.; Rouphael, Y.; Colla, G.; Bavaresco, L.; Trevisan, M. Chitosan treatment elicited defence mechanisms, pentacyclic triterpenoids and stilbene accumulation in grape (Vitis vinifera L.) bunches. Phytochemistry 2018, 156, 1-8.
32. Kogkou, C.; Chorti, E.; Kyraleou, M.; Kallithraka, S.; Koundouras, S.; Logan, G.; Kanakis, I.; Kotseridis, Y. Effects of foliar application of inactivated yeast on the phenolic composition of Vitis vinifera L. cv. Agiorgitiko grapes under different irrigation level. J. Wine Res. 2017, 9, 23-33.
33. Paladines-Quezada, D.F.; Fernández-Fernández, J.I.; Bautista-Ortín, A.B.; Gómez-Plaza, E.; Bleda-Sánchez, J.A.; Gil-Muñoz, R. Influence of the use of elicitors over the composition of cell wall grapes. Proceedings of the In Vino Analytica Scientia, Salamanca, Spain, 2017.

Author Response File: Author Response.docx

Reviewer 2 Report

This study investigates the influence of pre-harvest foliar application of inactivated wine yeast on the carotenoid content of wine grapes. Carotenoids may serve as precursors of norisoprenoids, which are key aroma compounds of wines. In this context, the focus of this work lies on the examination of two grape varieties. The results are clearly presented and properly discussed and concluded. Overall, the manuscript is well-written and could be considered for publication after some minor modifications, according to the following comments:

 

Title (Line 3): Consider replacing “can” with “may”, since this statement has not been confirmed by measuring the actual norisoprenoid content. It is only deduced with reference to the content of their precursors (carotenoids).

Line 24: Please remove “derivatives” after “violaxanthin”.

Line 41: Remove the comma after “(i.e., C13-norisoprenoids)” and omit “a” after “having”.

Line 56: Replace “have proposed” with “has proposed”.

Line 57: It is suggested to use “kg” instead of “Kg”; please correct accordingly throughout the text (e.g. Lines 157, 309, 314 etc.)

Line 78: Replace “Ha” with “ha”.

Lines 90, 93, 94, 96: Please provide the city next to the country for each company, as indicated in other cases, e.g. J.T. Baker (Deventer, Holland) (Line 89).

Line 109: Replace “added of” with “containing”.

Line 110: Insert “the” before “presence”.

Line 112: Replace “added” with “diluted”.

Line 123-135: Please provide the conditions used for recording the MS² spectra, according to the data provided in Table 1.

Line 132: Replace “~3.5” with “ca. 3.0” and “volume” with “volumes”.

Line 141: Change “others” to “other” and use “structures” instead of “structure”. Is Table S1 correct? It seems that Table 1 lists the identified compounds.

Line 151: Please make sure that Table S1 accompanies the manuscript as a supplementary material.

Line 167: Remove “a” before “principal…”.

Line 175: Replace “among” with “including”.

Line 204: Remove “to” after “alongside”.

Line 459: Replace “make” with “making”.

Line 508: Insert “were” before “presented”.

Line 513: Replace “cleavage origins” with “serve as”.

Table 1: Please indicate which is the base peak (most abundant) ion for each compound in the column of “MS² product ions”, as it would be a useful information for a reliable identification.

Author Response

Response to Reviewer 2

This study investigates the influence of pre-harvest foliar application of inactivated wine yeast on the carotenoid content of wine grapes. Carotenoids may serve as precursors of norisoprenoids, which are key aroma compounds of wines. In this context, the focus of this work lies on the examination of two grape varieties. The results are clearly presented and properly discussed and concluded. Overall, the manuscript is well-written and could be considered for publication after some minor modifications, according to the following comments:

We want to thank the reviewer for the positive comment. We have tried to edit and improve the manuscript as suggested.

Title (Line 3): Consider replacing “can” with “may”, since this statement has not been confirmed by measuring the actual norisoprenoid content. It is only deduced with reference to the content of their precursors (carotenoids).

Yes, we agree. The new title will be: “HOW PRE-HARVEST INACTIVATED YEAST TREATMENT MAY INFLUENCE THE NORISOPRENOID AROMA POTENTIAL IN WINE GRAPES”

Line 24: Please remove “derivatives” after “violaxanthin”.

We have deleted the word “derivatives”.

Line 41: Remove the comma after “(i.e., C13-norisoprenoids)” and omit “a” after “having”.

Ok, corrections done.

Line 56: Replace “have proposed” with “has proposed”.

We have corrected the verbal form.

Line 57: It is suggested to use “kg” instead of “Kg”; please correct accordingly throughout the text (e.g. Lines 157, 309, 314 etc.).

You are right. We apologize. Every time “Kg” appears in the text and figures it has been replaced with “kg”.

Line 78: Replace “Ha” with “ha”.

Correction done.

Lines 90, 93, 94, 96: Please provide the city next to the country for each company, as indicated in other cases, e.g. J.T. Baker (Deventer, Holland) (Line 89).

More accurate details have been added: “HPLC grade hexane and acetone were purchased from J.T. Baker (Deventer, Holland). LCMS grade water, methanol, and tert-butyl-methyl-ether were purchased from Chromasolv (Exacta+Optech Labcenter S.p.A., Modena, Italy). Sodium hydroxide (NaOH) 0.1 N and bromothymol blue were purchased from Sigma Aldrich (Merck Life Science S.r.l., Milano, Italy). β-apo-8’-carotenal and 3-tert-butyl-4-hydroxyanisole (BHA) were purchased from Fluka (Germany Exacta+Optech Labcenter S.p.A., Modena, Italy). -carotene and magnesium carbonate basic were purchased from Sigma-Aldrich (Merck Life Science S.r.l., Milano, Italy); (allE)-lutein and zeaxanthin were obtained from Extrasynthese (Genay, France), whereas (9’Z)-neoxanthin, violaxanthin, 5,6-epoxide-lutein, and (9Z)-β-carotene were obtained from CaroteNature (Münsingen, Switzerland) and used as HPLC reference standards.”

Line 109: Replace “added of” with “containing”.

Line 110: Insert “the” before “presence”.

Line 112: Replace “added” with “diluted”.

We have corrected these points as indicated.

Line 123-135: Please provide the conditions used for recording the MS2 spectra, according to the data provided in Table 1.

MS condition has been inserted as such: “Positive electrospray mode was used for ionization of molecules with capillary voltage at -4000 V and skimmer voltage at 40 V. The nebulizer pressure was 15 psi and the nitrogen flow rate was 5 L/ min. Temperature of drying gas was 350 °C. In the full scan mode, the monitored mass range was from m/z 100 to 1200. MS2 was performed by using helium as the collision gas at a pressure of 4.6 x 10-6 mbar. CID spectra were obtained with an isolation width of 4.0 m/z for precursor ions and a fragmentation amplitude of 0.6 V for epoxyxanthophylls, and of 1.0 V for the other carotenoids.”

Line 132: Replace “~3.5” with “ca. 3.0” and “volume” with “volumes”.

Corrections done.

Line 141: Change “others” to “other” and use “structures” instead of “structure”. Is Table S1 correct? It seems that Table 1 lists the identified compounds. Line 151: Please make sure that Table S1 accompanies the manuscript as a supplementary material.

Changes done. You are right, it was just a typing mistake. Anyway, Table S1, regarding some validation parameters of the method, was already added as supplementary material.

Line 167: Remove “a” before “principal…”.

Line 175: Replace “among” with “including”.

Line 204: Remove “to” after “alongside”.

Line 459: Replace “make” with “making”.

Line 508: Insert “were” before “presented”.

Line 513: Replace “cleavage origins” with “serve as”.

We have accepted all the suggested modifications.

Table 1: Please indicate which is the base peak (most abundant) ion for each compound in the column of “MS2 product ions”, as it would be a useful information for a reliable identification.

Thank you very much for your useful suggestion. Table 1 has been modified as such:

Table 1. HPLC-DAD-MS (ESI⁺) characteristics of carotenoids in grapes.

 

peak	compound	k’	lmax (nm)	% (III/II)a	DB/DIIb	[M+H]+(m/z)	[M]·+(m/z)	MS2 product ions m/z
1	violaxanthin like structure	2.78	418;440;470	84		601.5	600.1	583.5, 565.5, 509.5, 491.5, 221.1
2	violaxanthin	2.92	416;440;468	86		601.5	600.1	583.5, 565.5, 509.5, 491.5, 221.1
3	(8'R)-neochrome	3.01	400;422;450	88		601.5	600.1	583.2, 565.3, 509.5, 221.1
4	(9'Z)-neoxanthin	3.06	414;436;464	86		601.5	600.1	583.2, 565.3, 509.5, 221.1
5	(8'S)-neochrome	3.12	400;422;450	88		601.5	600.1	583.2, 565.3, 509.5, 221.1
6	5,6-epoxylutein	3.19	416;440;468	90		585.4	584.2	567.1,  493.1, 221.1
7	luteoxanthin	3.33	399;422;448	94		601.5	600.1	583.2, 221.1
8	lutein like structure	3.42	(425);446;474			568.9	567.9	550.9, 532.9, 476.4, 429.4
9	Z lutein like structure	3.47	328;(412);436;464			568.9	567.9	550.9, 532.9, 476.4, 429.4
10	(8'S)-auroxanthin	3.64	380;402;426	98		601.5	600.1	583.5, 565.5, 509.5, 491.5, 221.1
11	chlorophyll b	3.70	258; 314; 342; 466; 600; 650
12	(allE)-lutein	3.75	(422);446;472	40		568.9	567.9	550.9, 532.9, 476.4, 429.4
13	zeaxanthin	4.00	(425);452;476	22		568.9	567.9	550.9, 532.9, 476.4, 429.4
14	(9Z) or (9’Z)-lutein	4.16	330;(422);440;468	50	0.075	568.9	567.9	550.9, 532.9, 476.4, 429.4
15	chlorophyll a	4.31	336;(385);(417);432; 618; 665
IS	b-apo-8'-carotenal	4.50	460
16	pheophytin b	5.46	(417);436;527;600;654				885
17	pheophytin a	5.59	410;506;536;666				871
18	b-carotene	5.98	(430); 452; 478	25		536.9	535.9	444.2, 430.3, 399.3
19	(9Z)- b-carotene	6.25	342;(424);446;474	17	0.03	536.9	535.9	444.2, 430.3, 399.3

 

 ^a %III/II is the ratio of the height of the longest-wavelength absorption peak, designated III, and that of the middle absorption peak, designated as II, taking the minimum between the 2 peaks as baseline [8]; ^b Q ratio which is the quotient between the cis peak band and the band II (normally l_max.) [21].

The fragments, corresponding to the base peak in MS² spectra, were subscribed.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript under appreciation is about the impact pre-harvest foliar application of inactivated yeast extracts on carotenoids, in wine grapes of Apulian Negroamaro and Primitivo varieties.

The manuscript is interesting and provides novelty and the results are important for future application.

The following comments are to be taken into account by the authors:

1. The introduction must be improved by adding a discussion and relevant references about the extraction protocols and the determination methods of these compounds.
2. Lines 143-151: Please also provide relative standard deviation, % RSD.
3. Lines 504-505: The only carotenoids that could be confidently identified are the eight compounds available as standards reported in subsection 2.2 (β-apo-8’-carotenal, β-carotene, (allE)-lutein, zeaxanthin, (9’Z)- neoxanthin, violaxanthin, 5,6-epoxide-lutein and (9Z)-β-carotene). The other compounds are tentatively identified as the authors’ state in lines 140-142. Therefore the phrase “Fifteen carotenoids, among which (allE)-lutein and β-carotene together with their 9Z isomers and 5,6-/5,8-epoxyxanthophylls, were confidently identified” is not accurate and should be corrected accordingly.

Author Response

Response to Reviewer 3

The manuscript under appreciation is about the impact pre-harvest foliar application of inactivated yeast extracts on carotenoids, in wine grapes of Apulian Negroamaro and Primitivo varieties. The manuscript is interesting and provides novelty and the results are important for future application.

Many thanks for your positive evaluation of our manuscript.

The following comments are to be taken into account by the authors:

The introduction must be improved by adding a discussion and relevant references about the extraction protocols and the determination methods of these compounds.

The following discussion has been introduced in the introduction: “There is no standard extraction procedure for carotenoids in foodstuffs; generally, in the case of grapes, aprotic and poor polarity organic solvents, such as acetone [2] or diethyl ether/hexane 1:1 [13], are employed to extract carotenes and xanthophylls simultaneously. Reversed-phase HPLC coupled with various detection techniques (e.g. DAD and MS) is currently the method of choice for carotenoid analysis [14]. In particular, C30 stationary phase has the highest separation selectivity including structural and geometrical isomers [15].”

Lines 143-151: Please also provide relative standard deviation, % RSD.

We have added RSD for assessing the intraday and interday repeatability. Values have been reported in the new Table S1.

Lines 504-505: The only carotenoids that could be confidently identified are the eight compounds available as standards reported in subsection 2.2 (β-apo-8’-carotenal, β-carotene, (allE)-lutein, zeaxanthin, (9’Z)- neoxanthin, violaxanthin, 5,6-epoxide-lutein and (9Z)-β-carotene). The other compounds are tentatively identified as the authors’ state in lines 140-142. Therefore the phrase “Fifteen carotenoids, among which (allE)-lutein and β-carotene together with their 9Z isomers and 5,6-/5,8-epoxyxanthophylls, were confidently identified” is not accurate and should be corrected accordingly.

Actually, your observation is right even though different compounds features were matched to identify them. Therefore, we have proposed to edit the phrase as “….were tentatively identified…”.

Author Response File: Author Response.docx