Non-Saccharomyces Yeast Strains, Aromatic Compounds and Sensory Analysis of Italy and Negra Criolla Pisco from the Moquegua Region of Peru

Round 1

Reviewer 1 Report

General comments:

 

The submitted manuscript suffers from some shortcomings, such has the number of samples analyzed, the number of isolates identified, the volume of pisco produced for volatile and sensory analysis, and no replicates performed. Therefore, it if important that authors make clear the scope of the work, the number of isolates, number of samples analyzed, etc. stating that this is exploratory research. Despite the low number of samples and lack of replicates, the methods used for yeast identification, yeast phenotypic characterization, and pisco analysis seem correct.

 

 

Detailed comments:

 

-       There seems to have no sense to include literature references in the paragraph describing the aims of the present study (lines 69-76). Please reconsider removing the references from here and include a new paragraph citing the mentioned works.

-       In Material and Methods (please correct the English, where stands “Y” should be “and”, please answer to the following doubts:

o   What was the volume of wine and then pisco produced from 20 Kg of grapes?!

o   The volume of wine produced is very small. In wine analysis it is known that the smaller the volume, the greater the variability. That is also why is so important to make replicates to minimize this variability.

o   Why only one sample of 10 g of grapes was used for isolation and culture of isolates? Why did you not perform duplicates?! 

o   Why did you only select 34 colonies (24 from I and 10 from NC)? Despite the lack to use duplicates, the number of isolates seems very small. It is common to select around 30 isolates from each plate.

 

 

-       Results and Discussion

o   From the 34 colonies isolated, did you only identify 6 species?

o   In line 287, there seems to be a mistake. Instead of NC grapes, isn´t I grapes?

o   In page 7, legend of Figure 1, letter C is missing.

o   In Figure 1, it is not clear how many isolates of each species were monitored? Did you test the 34 isolates? Please specify?

o   Relating the phenological tests, did you perform them in all 34 isolates? It is not clear from the results… For instance relating “Ethanol tolerance”, it is clearly stated on line 376 and 377, from literature review that “The ethanol tolerance of M. pulcherrima depends on the strain and it ranged from 6% to 12% (51)”, but Table 2 only shows 6 species (and not the 34 strains). Did you test only one strain of each species?

o   Relating the volatile compounds showed in Table 3, did you find any significant difference (if any) based on the zones? In M&M (line 79), the authors refer that grape samples were collected from 3 zones located in Moquegua valley.

 

-       The Conclusions drawn seem not very clear. In fact, there were differences encountered in the pisco volatile composition and sensory analysis, but from the analysis one does not conclude that is related to the different microbiota encountered, since the grapes were different. Only if the same must fermented with the NSYSs isolates alone or in co-inoculation was used, the influence of the microbiota could be partially evaluated.

 

 

In Material and Methods please correct the English, where stands “Y” should be “and”.

 

Author Response

Material and Methods

Point 1: In Material and Methods (please correct the English, where stands “Y” should be “and”, please answer to the following doubts:

Response 1: In the material and methods section “Y” has been changed by “and”

 

Point 2: What was the volume of wine and then pisco produced from 20 Kg of grapes?

Response 2: We produced 12 L of wine and 2.5 L Pisco from 20 Kg grapes

Point 3: The volume of wine produced is very small. In wine analysis it is known that the smaller the volume, the greater the variability. That is also why is so important to make replicates to minimize this variability?

Response 3: We done replicates in order to minimize the variability. This information has been included in the manuscript.

 

Point 4: Why only one sample of 10 g of grapes was used for isolation and culture of isolates? Why did you not perform duplicates?

Response 4: We done triplicates in order to minimize the variability. This information has been included in the manuscript.

 

Point 5: Why did you only select 34 colonies (24 from I and 10 from NC)? Despite the lack to use duplicates, the number of isolates seems very small. It is common to select around 30 isolates from each plate.

Response 5: The number of colony from 3 plates were randomly selected based on color and morphological differences.

 

Point 6: Why did you only select 34 colonies (24 from I and 10 from NC)? Despite the lack to use duplicates, the number of isolates seems very small. It is common to select around 30 isolates from each plate.

Response 6: The number of colony from 3 plates were randomly selected based on color and morphological differences.

 

Results and Discussion

Point 7: From the 34 colonies isolated, did you only identify 6 species?

Response 7: Yes.

 

                                         

Point 8: In line 287, there seems to be a mistake. Instead of NC grapes, isn´t I grapes?

Response 8: Thank you for your correction, it has been included in the manuscript.

 

 

Point 9: In line 287, there seems to be a mistake. Instead of NC grapes, isn´t I grapes?

Response 9: Thank you for your correction, it has been included in the revised manuscript.

 

 Point 10:  In page 7, legend of Figure 1, letter C is missing.

Response 10: The title of Fig 1 has been changed to more clearly including C letter.

 

 Point 11:  In Figure 1, it is not clear how many isolates of each species were monitored? Did you test the 34 isolates? Please specify?

Response 11: We evaluated the arbohydrate fermentation and polygalacturonase activity of isolated and identified non-Saccharomyces yeast strains from Italy (Metschnikowia pulcherrima, Pichia terricola and Nagan-ishia vaughanmartiniae) and Negra Criolla (Aureobasidium pullulans, Vishniacozyma heimaeyensis and Vishniacozyma carnescens) grapes from the Moquegua region. This information has been included in the revised manuscript.

 

 Point 12:  Relating the phenological tests, did you perform them in all 34 isolates? It is not clear from the results… For instance relating “Ethanol tolerance”, it is clearly stated on line 376 and 377, from literature review that “The ethanol tolerance of M. pulcherrima depends on the strain and it ranged from 6% to 12% (51)”, but Table 2 only shows 6 species (and not the 34 strains). Did you test only one strain of each species?

Response 12: The phenological test was carried out in the isolated and identified non-Saccharomyces yeast strains from Italy (Metschnikowia pulcherrima, Pichia terricola and Nagan-ishia vaughanmartiniae) and Negra Criolla (Aureobasidium pullulans, Vishniacozyma heimaeyensis and Vishniacozyma carnescens) grapes from the Moquegua region. This information has been included in the revised manuscript.

 

 

 Point 13:  Relating the volatile compounds showed in Table 3, did you find any significant difference (if any) based on the zones? In M&M (line 79), the authors refer that grape samples were collected from 3 zones located in Moquegua valley.

Response 13: The volatile analysis was carried out using the total grape collected (18).

 

Conclusions

Point 14:  The conclusions drawn seem not very clear. In fact, there were differences encountered in the pisco volatile composition and sensory analysis, but from the analysis one does not conclude that is related to the different microbiota encountered, since the grapes were different. Only if the same must fermented with the NSYSs isolates alone or in co-inoculation was used, the influence of the microbiota could be partially evaluated.

Response 14: The conclusion has been changed in the revised manuscript as folowing:

Italy and Negra Criolla grapes collected from vineyards in the Moquegua region of Peru showed different non-Saccharomyces yeast strain (NSYS) microbiota. In addition, the NSYSs showed differences in their metabolisms, including carbohydrate fermentation, polygalacturonase activity, ethanol tolerance, sulphite production and nitrogen consumption. Pisco elaborated from I and NC variety grapes also showed differences in volatile profiles and sensory descriptors.

This work represents an initial approach to the use of NSYSs alone or in combination as starters in sequential fermentation to improve the sensory quality and typicity of Moquegua Pisco, which deserves further study. The NSYSs isolated from I and NC grapes could contribute to the volatile compounds found in the resultant Piscos.

 

 

 

 

Reviewer 2 Report

The work present relevant information about aromas formation. It´s very important to link the chemical composition associates with grape microbiota withe the distillate final aroma. Hoever, there are fewer clear aspects:

What is the final alcohol content of all distillates? It will be the same considereing the same conditions (lines 196, 197)!!

On the other hand, table 3 line 462 refer that distillates are produces under indsutrial conditions. Did industrial copper still heve only 20 L.

The phenylethyl alcohol (Pa), for exemple, is clearly a tail compound and if the distillate do not have the same tails conditions the compounds have naturally different concentrations!!

Author Response

Material and Methods

Point 1: What is the final alcohol content of all distillates? It will be the same considereing the same conditions (lines 196, 197)

Response 1: All distillates contained 42 % ethanol v/v. For more clearly the manuscript has been changed as following:

The must was then distilled in a 20 L capacity copper still, the head was cut at 0.8% of total volume of the must and the Pisco was cut using 42% final alcohol grade. The Pisco obtained was transferred to a glass container for analysis of volatile compounds and for sensory assessment.  

 

Point 2: On the other hand, table 3 line 462 refer that distillates are produces under indsutrial conditions. Did industrial copper still heve only 20 L.

Response 2: The title of the Table 3 was changed in the revised manuscript as following:

Table 3: Minimum, maximum, mean and standard deviation values for volatile compounds in pisco made from Italia and Negra Criolla grape varieties.

 

Point 3: The phenylethyl alcohol (Pa), for exemple, is clearly a tail compound and if the distillate do not have the same tails conditions the compounds have naturally different concentrations

Response 3: All Pisco was cut using 42% final alcohol grade in order to obtain homogenous samples.