The Production of Bioactive Hydroxytyrosol in Fermented Beverages: The Role of Must Composition and a Genetically Modified Yeast Strain

Round 1

Reviewer 1 Report (New Reviewer)

1 Title, I recommend the change ‘The production of bioactive hydroxytyrosol……’, please delete the second ‘the’.

2 Abstract, the bioactive properties actually contain the antioxidant activity, the expression ‘bioactive properties and antioxidant activity’ should be modified. Similar problem to ‘present in olives, olive oil and wine’, olives contain olive oil.

3 Introduction, Line 29-30, ‘Recently, these authors have estimated the dietary intake of free HT in the European adult population at 0.13–6.82 mg day’, Please add related references.

4 Table 1, Pleases redrew and format the table as three-line table

5 Figures, all figures 1-3 must be placed in ‘Results’ section, but not in ‘Material’ section. Furthermore, the figure 2 and figure 3 are actually the same, the OD600 value reflect the cell numbers in deed. Only one figure should be involved.

6 Figure 4 should be replaced, deleting the number such as 225, 226,. Similarly, the Figure 4 should be placed in the ‘Results’ section

7 Line 366 ‘show in figure show in figure show in figure show in figure show in figure 8.’ Please revise.

8 Line 392, Figure 8, the title of Figure 8 should give more detail information, such as giving some footnotes to explain figure. And giving the information of abscissa and ordinate. What is the meaning ‘Same letter means p< 0.05 between compounds’, please revise

9 Table 2 and table 3, lack statically difference analysis.

10 What the difference of the current work and Rebollo-Romero et al. [17]? Please discuss them in the manuscript.

Author Response

Answer to Reviewer 1

1 Title, I recommend the change ‘The production of bioactive hydroxytyrosol……’, please delete the second ‘the’.

We changed the title as recommended.

2 Abstract, the bioactive properties actually contain the antioxidant activity, the expression ‘bioactive properties and antioxidant activity’ should be modified. Similar problem to ‘present in olives, olive oil and wine’, olives contain olive oil.

We eliminate the words antioxidant activity. Please consider that olives and olive oil are two separate categories of foods. Data are compiled in these categories in FoodEx2 database, EFSA guidelines and so on. Therefore, we kept both foods in the text.

3 Introduction, Line 29-30, ‘Recently, these authors have estimated the dietary intake of free HT in the European adult population at 0.13–6.82 mg day’, Please add related references.

We refer to the previous reference, we have included again at the end of the phrase to clarify.

4 Table 1, Pleases redrew and format the table as three-line table

The table was redrawn as suggested.

5 Figures, all figures 1-3 must be placed in ‘Results’ section, but not in ‘Material’ section. Furthermore, the figure 2 and figure 3 are actually the same, the OD600 value reflect the cell numbers in deed. Only one figure should be involved.

We changed the position of the figures from M&M section to Results section as recommended. Figure 3 from the previous version showing the growth by OD600 was removed.  

6 Figure 4 should be replaced, deleting the number such as 225, 226,. Similarly, the Figure 4 should be placed in the ‘Results’ section

Figure 4 in the previous version is now Figure 3 as a consequence to  the comment number 5.  In the current version it is placed in the Results section. Numbers 225 and 226 which were referring to lines number have been deleted.

7 Line 366 ‘show in figure show in figure show in figure show in figure show in figure 8.’ Please revise.

We removed the phrase.

8 Line 392, Figure 8, the title of Figure 8 should give more detailed information, such as giving some footnotes to explain figure. And giving the information of abscissa and ordinate. What is the meaning ‘Same letter means p< 0.05 between compounds’, please revise

Figure 8 in the previous version is now Figure 6. Now the footnotes detail the meaning of every statistical difference found for a better understanding: “Statistical differences on HT and tyrosol production for the different conditions (Sugar and YAN) at the day of the maximum production of each strain. Same letter means p< 0.05 between different concentrations of sugar (b,f,a,c) or YAN (d,e,g) for the same strain and compounds; and between the same concentration of sugar or YAN for the different strains and the same compound”. Additionally, the information in abscissa and ordinate have been included.  

9 Table 2 and table 3, lack statically difference analysis.

The detail of the statical analysis has been included in the Tables footnotes for clarity purposes.  

“Table 2: HT (ng mL -1) and tyrosol (mg L -1) concentrations at two different concentrations of YAN (210 mg L -1 and 300 mg L -1) at 200 g L -1 of sugar for QA23 and modified strain. Same letter means p< 0.05 for the day of the maximum compound production between the same concentrations of YAN for the different strains and the same compound (a,d); and between the different concentration of YAN for the same strain and the same compound (f,c,g).”

“Table 3: HT (ng L ^-1) and tyrosol (mg L ^-1) concentrations at two different concentrations of sugar (100 g ^{L -1} and 240 g L ^-1) at 210 mg L ^-1 of YAN for QA23-WT and QA-ME strain. Same letter means p< 0.05 between the same concentrations of sugar for the different strains and the same compounds(a,e), and between the different concentration of sugar for the same strain and the same compound (c,d,g,h).”

10 What the difference of the current work and Rebollo-Romero et al. [17]? Please discuss them in the manuscript.

There are several differences between the two works. First, the strains are different (in this manuscript we use a modified strain while Rebollo et al explored commercial ones). Second, the experimental design varies as the present work explores the influence of the sugar content of the must. Rebollo et al explored the precursor tyrosine while in the present work the whole YAN is considered.

 We included the following (Line 369-283): “These results are according with the ones shown by Rebollo-Romero et al. [17], in which it was added two different concentrations of the precursor tyrosine to the syn-thetic must, 10 mg L ^-1 and 60 mg L ^–1. The higher tyrosine concentration did not result in better HT production. Rebollo-Romero et al. [17], only studied how the tyrosine influenced the final HT concentration. Comparatively, we have gone one step further and studied how YAN (including tyrosine among others amino acids and ammonium sulphate) may affect the production of HT. In our conditions, the lesser YAN concen-tration produced the highest HT concentration. Moreover, we not only focus on the YAN but also how glucose affects the HT production, including a modified strain. With these in regard, Nisbet et al [6] detailed that fusel alcohols are 15% hexose derived, being another contributor to tyrosol production (Figure 5). Related to this, our results show increases of tyrosol but also of HT with the highest sugar content. Indeed, Gallardo-Fernández et al [7] found that HT was mainly formed from another source different from the initial tyrosine in the must by using isotopically labelled precursors. Our results suggest that a high content of sugar could be a more relevant factor than YAN to HT production as it is shown in Figure 6.”

Reviewer 2 Report (New Reviewer)

The current manuscript entitled "The production of the bioactive hydroxytyrosol in fermented beverages: role of must composition and a genetically modified yeast strain” investigated the effects of glucose levels and yeast assimilable nitrogen (YAN) on HT production during alcoholic fermentation. For the fermentation of synthetic must, commercial Saccharomyces cerevisiae QA23 and its genetically modified strain were both used. Two YAN concentrations (210 and 300 mg L-1) and two glucose concentrations (100 and 240 g L-1) were tested for each strain. Under varied YAN and sugar concentrations, both the wild type (QA23-WT) and genetically modified (QA23-ME) strains produced HT and tyrosol. In comparison to QA23-WT, QA23-ME generated noticeably larger amounts of HT, particularly at lower YAN levels.

Comments:

1. Notable variations in HT generation under different circumstances are mentioned in the results. Making sure these differences are statistically significant is crucial, though. Figure 8, the statistical letters need to be revised.

2. To confirm the specificity of HT and tyrosol assays, the study would benefit from the inclusion of control studies. For example, including samples with inactive yeast or without any additional yeast might aid in determining if the chemicals found are, in fact, generated by the strains of yeast being studied.

3. Under varying YAN and sugar circumstances, the study shows temporal changes in HT production, with distinct peak production days reported for QA23-WT and QA23-ME. It would be interesting to discuss the underlying metabolic mechanisms causing these temporal fluctuations and how they connect to the particular traits of the yeast strains that were employed.

4. The notable rise in tyrosol production by QA23-ME, especially on day 5, the issue of what metabolic pathways are at work and if tyrosol and HT synthesis may interact. A more thorough examination of the mechanics behind this finding and how compounds are synthesized during fermentation.

5. Page 10, Lines 355-2366, This paragraph's repeated use of terms like "show in figure show in figure" makes it unclear and inconsistent. This reduces the text's overall readability and makes it harder for the reader to follow the argument. This section needs to be revised by the author to remove unnecessary repetitions and increase clarity. The link between YAN concentration, sugar content, and HT and tyrosol formation is covered in the paragraph. It does not offer a convincing explanation for this data, even if it implies that increased HT synthesis was caused by a decreased YAN concentration.

6. Although the genetic modification of the wine strain QA23 is mentioned in passing in the current paper, it is not detailed enough or provides enough detail about the changes performed to the genome. It would be helpful if the author could elaborate on the precise modifications made to the yeast genome, such as the reasoning for the integration of E. coli HpaBC genes and the choice of the ARO4 gene.

7. The statistical letters in both Table 2 and 3 should be revised.

Minor editing of English language required

Author Response

R2

The current manuscript entitled "The production of the bioactive hydroxytyrosol in fermented beverages: role of must composition and a genetically modified yeast strain” investigated the effects of glucose levels and yeast assimilable nitrogen (YAN) on HT production during alcoholic fermentation. For the fermentation of synthetic must, commercial Saccharomyces cerevisiae QA23 and its genetically modified strain were both used. Two YAN concentrations (210 and 300 mg L-1) and two glucose concentrations (100 and 240 g L-1) were tested for each strain. Under varied YAN and sugar concentrations, both the wild type (QA23-WT) and genetically modified (QA23-ME) strains produced HT and tyrosol. In comparison to QA23-WT, QA23-ME generated noticeably larger amounts of HT, particularly at lower YAN levels.

Comments:

1. Notable variations in HT generation under different circumstances are mentioned in the results. Making sure these differences are statistically significant is crucial, though. Figure 8, the statistical letters need to be revised.

Figure 8 in the previous version is now Figure 6. The footnotes detail now the meaning of every statistical difference found for a better understanding. Additionally, the information in abscissa and ordinate have been included.   The footnote has been rephrased as follow: “Statistical differences on HT and tyrosol production for the different conditions (Sugar and YAN) at the day of the maximum production of each strain. Same letter means p< 0.05 between different concentrations of sugar (b,f,a,c) or YAN (d,e,g) for the same strain and compounds; and between the same concentration of sugar or YAN for the different strains and the same compound”. Additionally, the information in abscissa and ordinate have been included.  

2. To confirm the specificity of HT and tyrosol assays, the study would benefit from the inclusion of control studies. For example, including samples with inactive yeast or without any additional yeast might aid in determining if the chemicals found are, in fact, generated by the strains of yeast being studied.

First of all, thank you for your consideration, first the musts were sterilized and then inoculated with the selected strain. On the other hand, a sample was taken from day 0, i.e. sterilized must without inoculation, and none of the compounds were detected. Finally, the intracellular yeast cell medium was analyzed, and tyrosol and hydroxytyrosol were detected, although the data are not shown.

We have also included this sentence for clarification (Lines 273-275): “To confirm the HT and tyrosol was produced by yeast; the must without inoculation was analysed (day 0 of fermentation), revealing that HT and tyrosol were not present.”

3. Under varying YAN and sugar circumstances, the study shows temporal changes in HT production, with distinct peak production days reported for QA23-WT and QA23-ME. It would be interesting to discuss the underlying metabolic mechanisms causing these temporal fluctuations and how they connect to the particular traits of the yeast strains that were employed.

We have included figures 4 and 5, showing the pathways leading to formation of tyrosol and hydroxytyrosol as a base of metabolic mechanims. Depending on the different condition in the synthetic must the yeast has different behavior. In a medium with different concentrations of sugar, the results show that no matter what concentration of sugar it has, there is a peak of HT and tyrosol concentration in the middle days of the fermentation. However, with low YAN concentration, the production is mostly at the end of the fermentation; on the other hand with more YAN is early produced. The fluctuations of the concentration are explained in line 294-326.

4. The notable rise in tyrosol production by QA23-ME, especially on day 5, the issue of what metabolic pathways are at work and if tyrosol and HT synthesis may interact. A more thorough examination of the mechanics behind this finding and how compounds are synthesized during fermentation.

As we have included in the following in the comment 6, one of the modifications of the yeast is to enhance the tyrosol production.

5. Page 10, Lines 355-2366, This paragraph's repeated use of terms like "show in figure show in figure" makes it unclear and inconsistent. This reduces the text's overall readability and makes it harder for the reader to follow the argument. This section needs to be revised by the author to remove unnecessary repetitions and increase clarity. The link between YAN concentration, sugar content, and HT and tyrosol formation is covered in the paragraph. It does not offer a convincing explanation for this data, even if it implies that increased HT synthesis was caused by a decreased YAN concentration.

We rephrased the paragraph as follows (Line 369-283): “These results are according with the ones shown by Rebollo-Romero et al. [17], in which it was added two different concentrations of the precursor tyrosine to the syn-thetic must, 10 mg L -1 and 60 mg L –1. The higher tyrosine concentration did not result in better HT production. Rebollo-Romero et al. [17], only studied how the tyrosine in-fluenced the final HT concentration. Comparatively, we have gone one step further and studied how YAN (including tyrosine among others amino acids and ammonium sulphate) may affect the production of HT. In our conditions, the lesser YAN concen-tration produced the highest HT concentration. Moreover, we not only focus on the YAN but also how glucose affects the HT production, including a modified strain. With these in regard, Nisbet et al [6] detailed that fusel alcohols are 15% hexose derived, be-ing another contributor to tyrosol production (Figure 5). Related to this, our results show increases of tyrosol but also of HT with the highest sugar content. Indeed, Gal-lardo-Fernández et al [7] found that HT was mainly formed from another source dif-ferent from the initial tyrosine in the must by using isotopically labelled precursors. Our results suggest that a high content of sugar could be a more relevant factor than YAN to HT production as it is shown in Figure 6.”

6. Although the genetic modification of the wine strain QA23 is mentioned in passing in the current paper, it is not detailed enough or provides enough detail about the changes performed to the genome. It would be helpful if the author could elaborate on the precise modifications made to the yeast genome, such as the reasoning for the integration of E. coli HpaBC genes and the choice of the ARO4 gene.

We include de following paragraph (Line 218-241):

“The genetic modifications carried out on QA23 strain were the multiple integration of a bacterial hydroxylase and reductase complex, namely HpaBC, under the control of strong constitutive promoters PTEF1 and PPGK1 into the genome using Ty1Cons2 sequences as homologous recombination targets, and the single integration of allelic variant ARO4K229L, henceforth ARO4*, under the control of TDH3 promoter in chromosome locus X-3. Such modifications would confer QA23 strain the ability to hydroxylate tyrosol as a result of the activity of HpaBC complex and at the same time, the tyrosine-insensitive variant ARO4* would increase metabolic flux towards tyrosol. These modifications were performed according to previous works (Muñiz-Calvo et al., 2020). Briefly, genes hpaB and hpaC and bidirectional promoter PTEF1-PGK1 were PCR-amplified from plasmids p426GPD-hpaB, p425GPD-hpaC and pCfB2628 respectively (Muñiz-Calvo et al., 2020), while ARO4* with TDH3 promoter was amplified from plasmid p423GPD-ARO4*. In parallel, vectors bearing the Ty1Cons2 and X-3 sequences for homology recombination from the EasyCloneMulti and EasyClone vector set pCfB2988 and pCfB257, were prepared by sequential treatment with enzymes AsiSI (SfaAI) (Thermo Fisher Scientific, Waltham, MA, USA) and BsmI (New England Biolabs, Ipswich, MA, USA). After purification, PCR products were cloned into pre-treated vectors by USER™ method (New England Biolabs). Ligation product was transformed into E. coli and successful cloning of both vectors was verified by Sanger sequencing (EUROFINS genomics, Ebersberg, Germany). Prior to yeast transformation, the resulting integrative vectors pCfB2988 HpaBC and pCfB257 ARO4* were linearized by FastDigest NotI (Thermo scientific, Vilnius, Lithuania) and the fragments containing the desired sequences to integrate were purified from agarose gel. Yeast cells were transformed with 1 – 1.5 µg of the linear fragment from the integrative vectors by the PEG/LiAc method and plated on selective agar medium according to strain auxotrophic markers.”

7. The statistical letters in both Table 2 and 3 should be revised.

The detail of the statical analysis has been included in the Tables footnotes for clarity purposes.  “HT (ng mL ^-1) and tyrosol (mg L ^-1) concentrations at two different concentrations of YAN (210 mg L ^-1 and 300 mg L ^-1) at 200 g L ^-1 of sugar for QA23 and modified strain. Same letter means p< 0.05 for the day of the maximum compound production between the same concentrations of YAN for the different strains and the same compound; and between the different concentration of YAN for the same strain and the same compound“

Round 2

Reviewer 1 Report (New Reviewer)

The statistical difference analysis of Tables 2 and 3 MUST be revised, it is not clear at present. Please refer to ‘ Antioxidants 2022, 11, 1543. https://doi.org/10.3390/antiox11081543’

Comments for author File: Comments.docx

Author Response

The footnotes of Table 2 and 3 include a sentence following the example provided by reviewer in antioxidants. Please consider that statistical analysis relevant for the discussion is highlighted despite there are more statistical differences.

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

The manuscript was improved after authors revision and the current version is now acceptable for publication.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

1.      L21,23 the -1 required superscript, and check the full text, there are multiple errors.

2.      L115, 201 S. cerevisiae need italics.

3.      The results presented in this paper are too simple and should reflect more raw data, such as HPLC-MS

4.      Through the experimental design, the author explored the relationship between different substances and the content of active substances, which is very meaningful, but the depth of the whole study is not high, the discussion is not deep enough, and there are no mechanical studies.

5.      The results should be stated separately so that the results is clearer.

6.      There are many formatting errors in the text, which should be checked in full, such as NH4Cl.

7.      The data in Table 2 should be further analyzed, such as principal component analysis.

Reviewer 2 Report

The authors in their original research article evaluated the biosynthesis of tyrosol and hydroxytyrosol by S. cerevisiae. The wild-type strain and its genetically engineered strain were compared in cultures with two different yeast assimilable nitrogen concentrations and different sugar concentrations. The authors presented only the results of chromatographic analyses in two tables. In my humble opinion, this is not enough for such an article to be published in Fermentation. The novelty of the work should be emphasized. I wonder if any routine analyses of cultures in synthetic musts were carried out. Why do the authors focus only on one analysis? 

When preparing a revised version of the manuscript, please also take care of editorial corrections - the authors did not use italics for microorganism names, as well as, sub- and superscripts in chemical formulas.