Temperature and pH Optimization for Protease Production Fermented by Yarrowia lipolytica from Agro-Industrial Waste

Round 1

Reviewer 1 Report

This paper investigates the effects of pH, temperature and waste species on the synthesis of protein hydrolase by Y. lipolytica. The study is detailed and well-structured. Under appropriate conditions, the effectiveness of the substrate being transformed can be improved.

1. Lines 112-119： “To prepare the fermentation conditions in solid substrate, 10 grams of dehydrated 112 and sterilized sample were placed in Erlenmeyer flasks. ……Samples of enzymatic pro- 118 duction were taken every 12 hours.” Other culture conditions for fermentation can be suggested in detail, such as oxygen content, fermentation vessels, fermentation time, etc.

2. Line 115： “H2O (0.5);” should be replaced by“H₂O (0.5);“.

3. Line 146： “Effect of pH on enzymatic activity” I'm not quite sure if this article is exploring the effect of pH on protease production by the strain or the effect of pH on the protease performing its function.

4. Line 153: “Effect of temperature on enzymatic activity”。 In this part I have a similar confusion as in question 3.

5. Line 262：The figure shows solid state fermentation and how the culture process avoids contamination by other stray bacteria.

6. Line 372：“In a fermentation process, controlling the moisture content in the substrate is a crucial 372 factor to consider, as it affects the efficiency of solid-state processes”。 The water content during fermentation affects protein production and what is the water content at the end of fermentation for this experiment.

7. Lines 320 and 411：Both parts mention that different nutrient sources affect enzyme synthesis, but the specific reasons are not clear enough. It is recommended to further analyze the specific causes in relation to the characteristics of each substance.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

I am very grateful you for the invitation to review manuscript fermentation-2510839 by López-Trujillo and coauthors "Temperature and pH Optimization for Protease Production Fermented by Yarrowia lipolytica from Agroindustrial Waste”. This study aimed to evaluate the impact of pH and temperature of the substrate on the production of proteolytic enzymes using a non-conventional yeast strain of Y. lipolytica. The work is interesting but needs several adjustments to increase the quality of the material.

 

Comments:

- Title: Where is the optimization? Maximum activity was only verified in relation to two analytical conditions. Review this description.

- Review the optimization denomination throughout the work.

- Abstract, Line 13: “to add value” it's very generic.

- Lines 13-14: The sentences are confused. The authors must make clear the biological treatment to produce enzymes and consequently the modification of by-products.

- Line 16: Mention the Y. lipolytica strain.

- Lines 17-18: Specify the by-products used.

- Lines 19-21: Specify parameters used as responses.

- Line 21: “yields” or “production”?

- Lines 23-25: The conclusion presented does not correspond to the objectives presented in lines 15 and 16. The authors should be more specific about the conclusion.

- Authors should standardize throughout the text the use of the nomenclature waste, residues, by-products, and others.

- Line 26: Change the repeated keywords by different words from the title.

- Line 29-30: Include recent data regarding the enzyme and protease market.

- Line 30-33: Extraction of proteases from plants also uses “natural” material to obtain proteases. The advantage of biotechnological processes should be better and more deeply presented.

- Lines 41-42: The sentence regarding environmental impact is very generic. Not every biotechnological process is environmentally correct.

- Lines 45-52: Please include relevant data on generation of by-products and problems of accumulation and improper treatment.

- Line 52: Include the abbreviation for solid-state fermentation in the first presentation of the word.

- Line 53-60: And what about the disadvantages? What barriers need to be overcome to effectively apply SSF on an industrial scale?

- Line 53-60: Please insert other advantages associated with SSF and published in previous works, such as:

https://doi.org/10.3390/pr10020381

https://doi.org/10.1016/j.heliyon.2022.e09173

among others.

- Lines 62-64: In addition to pH and temperature, other parameters are important. Please highlight other commonly studied variables.

- Line 65: Specify that it is in relation to the optimum pH of the microorganism.

- Line 73: Observe the previous comment and specify better.

- Introduction: Better highlight the by-products used, including an overview of the verified components.

- Line 86: Change “microorganism.” to “microorganism”.

 - Line 90 and throughout the text: Standardize the use of units (l; L; ml; mL and others).

- Line 109: Modify the redundant words (Additionally, additional).

-  Line 112 and throughout the text: Standardize the use of g and grams units.

- Line 115: Check the correct form of chemical formulas KH₂PO₄ (1.0); MgSO₄ 7 H₂O (0.5); KCl (0.5).

- Line 117 and throughout the text: Change “2 x 107 spores/g” to “2 x 10⁷ spores/g”.

- Lines 125,135 and others: Standardize the numbering of the rpm.

- Line 165: one more term was added to the work, referring to by-products. Authors should standardize terminology throughout the text.

- Line 170: Change “40%. soybean (40.30±1.8%),” to “40%, soybean (40.30±1.8%),”.

- Line 175: Change “residue.. For example” to “residue. For example”.

- Lines 172-178: This information must be directed to the discussion item or unify items 3 and 4.

- Lines 181-113: This information must be directed to the discussion item or unify items 3 and 4.

- Lines 185-188: This information must be directed to the discussion item or unify items 3 and 4.

- Lines 189-196: This information must be directed to the discussion item or unify items 3 and 4.

- Line 204: corn gluten meal? This information is new.

- Lines 202: Check if the term “Invasion” is the most appropriate in this theme.

- Itens 3.2 e 3.3 have the same name. What's the difference?

- 3.4. Effect of Temperature on Enzymatic Activity: it is important to clearly define what is optimal temperature and stability temperature.

- Lines 311-319: The authors must deepen the discussions regarding the cultivation of Yarrowia and comparisons with other studies and by-products used, including the emphasis on the components of the by-products that favor the production of proteases and the like.

- Discussion about by-products is generic and not very deep.

- Lines 337-338: The terms of the work are confusing, since the optimization of parameters is mentioned (but not including optimization), the influence of parameters on extraction (but no extraction was studied, only the effect of pH on enzymatic activity). The authors should make the information clearer.

- Lines 340-349: This is a repetition of results presented in the previous item.

- Lines 350-352: Authors should highlight studies using Yarrowia.

- Lines 373; 376 and others: Standardize the use of the terms solid-state processes, solid-state fermentation, solid-state cultivation process, and others.

- Line 381, 4.3 Effect of temperature on enzymatic activity: No relevant discussion is presented in this item, just a repetition of the presentation of results.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have included my comments.