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Article
Peer-Review Record

Upcycling of Whey Permeate through Yeast- and Mold-Driven Fermentations under Anoxic and Oxic Conditions

Reviewer 1: Ehsan Mahdinia
Reviewer 2: Anonymous
Received: 23 December 2020 / Revised: 19 January 2021 / Accepted: 21 January 2021 / Published: 26 January 2021
(This article belongs to the Special Issue Biomass Conversion: Fermentation Chemicals and Fuels)

Round 1

Reviewer 1 Report

This study has looked into value-added ethanol and organic acids from whey lactose through fermentation; specifically for whey lactose-based beverages. It is a very original idea and may introduce a new avenue to putting whey streams into use besides other routes. Whey and lactose run offs are still a huge problem for the dairy industry. The experiments have been designed well and carried out with prejudice. Also, the results have been presented and discussed to the fullest extent and quite well. Thus. the study has significant scientific merit and for that deserves publication. I look forward to the sensory studies following this!

The manuscript reads and flows almost flawlessly with only a few typos and no grammatical errors. No convoluted sentences; no overcomplications; only some unconventional wordings here and there, which is acceptable. So a final proofing will suffice.

However, a few minor revisions must be addressed before the manuscript sees publication:

-The authors use aerobic vs anaerobic conditions terminology instead of oxic vs anoxic conditions ( they have actually used both in line 175!). Aerobic or anaerobic are referred to the metabolism and fermentation process in general not the fermentation conditions. Although this is a common misconception, authors should use the correct terminology for the conditions.

-The NRRL and/or ATCC species designations must be provided for as many of the stains as available.

-0.45 micron filtration that was used for sterilizing the medium mix prior to incubation does not actually sterilize: refrain from "sterilization" and note only filtration. The incubations are still fine since endospores don't matter in this case.

-The instrumentations (models, phases and other specifics) of the GC, ICP and HPLC methods used in the analyses must be detailed. This is essential to ensure replicability.

-The authors have not given any sensory perspective to any of the organic acid profiles they found in this study; especially tartaric acid which can be quite bold in high concentrations. It is not fair to save everything for the future studies and authors should at least give a few sentences in conclusions to pave the path for future studies on sensory.

Author Response

To whom it may concern,

Thank you for your time in reviewing this manuscript. We have taken your comments into consideration and have accompanied our response to each comment in red below. In addition to these comments, we have also resized the anoxic and oxic cell count graphs, to better reflect the standard deviation bars. While this adds to the total page count, we believe it increases the clarity for the reader.

Please let us know your thoughts.

Best regards,

Justin Marcus

-The authors use aerobic vs anaerobic conditions terminology instead of oxic vs anoxic conditions (they have actually used both in line 175!). Aerobic or anaerobic are referred to the metabolism and fermentation process in general not the fermentation conditions. Although this is a common misconception, authors should use the correct terminology for the conditions.

Response: Edits were made to the use of aerobic/anaerobic so that: oxic/anoxic were used when in reference to environment or condition of a fermentate; while aerobic/anaerobic was used when in reference to the action of fermentation. These differentiations were also added to the Keywords section.

-The NRRL and/or ATCC species designations must be provided for as many of the stains as available.

Response: In section 2.1 (Materials and Methods, Microorganisms), NRRL, ATCC or FSL (Food Safety Lab) numbers were given for each species. More information on FSL isolates was provided for reader’s access of the database.

-0.45 micron filtration that was used for sterilizing the medium mix prior to incubation does not actually sterilize: refrain from "sterilization" and note only filtration. The incubations are still fine since endospores don't matter in this case.

Response: “Sterilization” was replaced by the word “filtration” in section 2.2 (Materials and Methods, Experimental Design).

-The instrumentations (models, phases and other specifics) of the GC, ICP and HPLC methods used in the analyses must be detailed. This is essential to ensure replicability.

Response: Additional information regarding instruments used to collect data was provided in section 2.3 (Materials and Methods, Data Collection).

-The authors have not given any sensory perspective to any of the organic acid profiles they found in this study; especially tartaric acid which can be quite bold in high concentrations. It is not fair to save everything for the future studies and authors should at least give a few sentences in conclusions to pave the path for future studies on sensory.

Response: Additional information was added to the first and last paragraph of the Conclusion to address sensory impacts. In the first paragraph we note the importance of balancing the organic acids that these fermentations produced, and comment that this should be an area of research and future optimization.

In the final paragraph about tartaric acid, a sentence was added to address this acid’s impact on fermented beverages. A study from Food Chemistry concluded that wines with <2g/L of tartaric acid did not have noticeable sensory impacts on the beverage. With the highest tartaric acid levels seen as 0.83 +/- 0.76g/L and 0.63+/-0.09g/L (aerobic and anaerobic respectively), we believe tartaric acid would not have a significant impact on this beverage’s sensory properties. This citation has been added to the manuscript.

Reviewer 2 Report

  1. Double check the author guideline and Make sure the format is correct, for example. 1) Figure title: Please use Figure 1. Figure 2....instead of Graph 1.2. 2) Put Figure title to the bottom. 3). Put all figure and table in appropioate position in the text. The auother currently cluster all figure and table in the middle of text, which is very inconvient to read. 4) Figure legend, please use different shape or shade. the Color of legend is very difficult to tell because some colors are very close to each other. 
  2. Author should briefly introduce the yeast and molds used in this experiment in introduction to give readers some basic background and previous researches on similar applications
  3. Line 91-92. Please list out more information of the deproteinized whey protein (such as protein, fat, moisture, lactose/carbohydrate, ash level). 
  4. Line 92-93. Why diammonium phosphate and Fermaid K are added to the why permeate. Can yeast and mold only ferment lactose without those nutrients?
  5. Line 102. It is not very easy for some readers to picture how the Luer lock lookes like in your experiment. It will be much helpful if author has a diagram depicted how the anaerobic fermentation set up looks like, and how to prevent oxygen out while sampling. 
  6. For results and discussion. The authors did not discuss on the correlation between parameters linked to each other. For example, density changes should be linked to what metabolite the fermentation produced.  

Author Response

To whom it may concern,

Thank you for your time in reviewing this manuscript. We have taken your comments into consideration and have accompanied our response to each comment in red below. In addition to these comments, we have also resized the anoxic and oxic cell count graphs, to better reflect the standard deviation bars. While this adds to the total page count, we believe it increases the clarity for the reader.

Please let us know your thoughts.

Best regards,

Justin Marcus

 

-Double check the author guideline and Make sure the format is correct, for example. 1) Figure title: Please use Figure 1. Figure 2....instead of Graph 1.2. 2) Put Figure title to the bottom. 3). Put all figure and table in appropriate position in the text. The another currently cluster all figure and table in the middle of text, which is very inconvenient to read. 4) Figure legend, please use different shape or shade. the Color of legend is very difficult to tell because some colors are very close to each other. 

 

Response: Graphs were updated to be called Figures with accompanying number in the caption. Tables and figures where moved to be placed immediately after the first section they are called out in the text. Figure’s were updated to use shapes as data points instead of colors. All figures were changed to black and white.

 

-Author should briefly introduce the yeast and molds used in this experiment in introduction to give readers some basic background and previous researches on similar applications

 

Response: In paragraph 3 of the introduction, several sentences were added to describe each species used, and what environment/industry it is usually found in, with accompanying citations.

 

-Line 91-92. Please list out more information of the deproteinized whey protein (such as protein, fat, moisture, lactose/carbohydrate, ash level). 

 

Response: In the second paragraph of section 2.2 (Materials and Methods, Experimental Design), the composition of deproteinized whey powder used was added.

 

-Line 92-93. Why diammonium phosphate and Fermaid K are added to the why permeate. Can yeast and mold only ferment lactose without those nutrients?

 

Response: In the second paragraph of section 2.2 (Materials and Methods, Experimental Design), a sentence was added to explain both of these compounds were added as a nitrogen source. Due to the protein being extracted from deproteinized whey powder, several species would have difficulty continuing cellular processes without this nutrient.

 

-Line 102. It is not very easy for some readers to picture how the Luer lock looks like in your experiment. It will be much helpful if author has a diagram depicted how the anaerobic fermentation set up looks like, and how to prevent oxygen out while sampling. 

 

Response: Additional information was added to the third paragraph of section 2.2 (Materials and Methods, Experimental Design), to describe how Luer locks connect to an accompanying syringe.

 

-For results and discussion. The authors did not discuss on the correlation between parameters linked to each other. For example, density changes should be linked to what metabolite the fermentation produced.  

 

Response: In the first paragraph of section 3.1 (Results, Density), additional information was added to explain that secondary metabolites are produced as sugars are decomposed, changing the composition of the fermentate.

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