Hemp Seed Protein Hydrolysate Enriched with γ-Aminobutyric Acid and Peptides by Microbial Bioconversion

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments:

In this study, hemp seed protein was used to produce a multifunctional protein ingredient, and the bioconversion was optimized through alkaline protease hydrolysis and fermentation by Lactiplantibacillus plantarum. I have checked the manuscript, overall, the work is well written and has the potential to present a relevant topic.

In addition, I feel that the results are interesting and can motivate further studies in the field. In my opinion, the manuscript in the form presented is acceptable for publication. Therefore, worth to be recommend for publication within the domain of Fermentation.

Minor revision: Some suggestions for the improvement of the work:

- The conditions for enzymatic hydrolysis (pH, time, temperature), as well as the fermentation conditions, should be better explained. For example, why was the fermentation carried out for 5 days?

- The discussion needs further improvements as well. Especially compare the results in this study with most recent related literature and do a critical evaluation on the trends/similarities/differences

The topic is relevant and timely, which stimulates the interest of readers in this expanding field. However, the authors need to better explore some points, such as:

• What is the expected GABA yield in industrial production? Specifically, is the result obtained in this study (GABA content of 33.98 mg/g) compatible with industrial production? Additionally, compare this process in terms of GABA yield with other processes, such as germination.
• What is the recommended GABA concentration to achieve the desired health effects?
• Considering the chosen raw material, what is its potential compared to other sources in GABA production?

Other questions also need to be addressed, such as:

• What strategies and/or food sources could GABA be added to?
• What impacts could GABA have during food processing, and what strategies could protect it?
• What strategies could be used for the concentration and purification of this component on an industrial scale? The answers to these questions should be present in the introduction, discussion, and conclusion of the manuscript.

Another important point is to clarify the reason for choosing to produce a protease from Bacillus subtilis instead of using commercial proteases such as Alcalase, Neutrase, Brauzin, or Flavourzyme, which have costs and activities that are interesting from an industrial perspective.

 

- Conclusions

The conclusion needs to be rephrasing. The authors should write this section in comprehensive and suitable style. The conclusion as it stands is too much a summary of the results. Conclusions in a manuscript are meant to highlight major findings, linked to your objectives and to give a take home message to the readers. Therefore, it should answer the following questions: What is new? Why is it important? What are the limitations and potential consequences? In addition, the authors should indicate future research recommendations.

Author Response

We deeply appreciate the kind work of the editor and reviewers who provided detailed comments on our manuscript. We have revised the manuscript according to your suggestions and have written and sent you a response as shown below. Thank you.

 

1. Minor revision: Some suggestions for the improvement of the work:

- The conditions for enzymatic hydrolysis (pH, time, temperature), as well as the fermentation conditions, should be better explained. For example, why was the fermentation carried out for 5 days?

- The discussion needs further improvements as well. Especially compare the results in this study with

most recent related literature and do a critical evaluation on the trends/similarities/differences

Answer: Thank you for the reviewer's kind comments. The GAD enzyme required for GABA conversion is most active during the stationary phase. As the fermentation enters the death phase, enzyme activity declines, leading to limitations in GABA conversion. In the 8% and 10% MSG conditions, a noticeable decrease in viable cell counts and MSG depletion was observed after the third day, indicating that GABA conversion reaches its limit in lactic acid bacteria fermentation beyond 5 days. Therefore, further fermentation was not conducted beyond this point (line 315-322). Additionally, we have revised the discussion to include comparisons with recent studies on GABA production and other functional material to provide a more thorough evaluation (line 273-275, 330-335).

The topic is relevant and timely, which stimulates the interest of readers in this expanding field. However, the authors need to better explore some points, such as:

• What is the expected GABA yield in industrial production? Specifically, is the result obtained in this study (GABA content of 33.98 mg/g) compatible with industrial production? Additionally, compare this process in terms of GABA yield with other processes, such as germination.

Answer: Thank you for the reviewer's kind comments. The GABA yield achieved in this study was 67.96%, which is higher than the GABA content naturally produced or generated through fermentation in other studies. This indicates that the GABA content (33.98 mg/g) obtained in this process is compatible with industrial production and can achieve higher yields compared to other methods, such as germination. (line 330-333)

• What is the recommended GABA concentration to achieve the desired health effects?

Answer: Thank you for the reviewer's kind comments. In the KFDA it is reported that the consumption of 80mg GABA is effective on the decrease of blood pressure. One of the well-known effects of GABA, such as muscle enhancement, is observed when 100 mg is consumed. In our study, this amount can be achieved by consuming 3.0 mL of the fermented product. (line 333-335)

• Considering the chosen raw material, what is its potential compared to other sources in GABA production?

Answer: Thank you for the reviewer's kind comments. The hemp seed protein used in this study contains a rich nutrient with protein, mineral, and vitamins as plant by-product, which allows for GABA production even without the addition of MSG (unpublished results). This suggests that hemp seed protein may yield the highest GABA content compared to other protein sources under the same fermentation conditions. Additionally, hemp grain production can yield up to 2839 lbs/acre, which is comparable to the average yield for soybeans. Utilizing byproducts from hemp seed processing has significant potential for value-added applications, making it an attractive raw material for GABA production.

 

Other questions also need to be addressed, such as:

• What strategies and/or food sources could GABA be added to?

Answer: Thank you for the reviewer's kind comments. The fermented product optimized in this study contains functional compounds that support protein supplementation, muscle enhancement and reduction of blood pressure. Therefore, it is likely to be suitable for elderly individuals or those engaged in physical activity, as a functional food ingredient.

• What impacts could GABA have during food processing, and what strategies could protect it?

Answer: Thank you for the reviewer's kind comments. GABA is a relatively stable compound and does not show the significant loss or impact other ingredients during food processing. However, GABA may involve the non-enzymatic browning reaction, resulting in the loss of GABA. Thus, browning reaction can be inhibited by various chemical-physical methods.

• What strategies could be used for the concentration and purification of this component on an industrial scale? The answers to these questions should be present in the introduction, discussion, and conclusion of the manuscript.

Answer: Thank you for the reviewer's kind comments. While the optimization of fermentation in this study led to a high production yield of GABA, the data were not intended for the industrial-scale concentration and purification of GABA alone. The focus of this research was to develop a functional protein-enriched material. However, concentration of GABA in fermented hemp seed protein could be performed by sequential method including centrifugation, microfiltration and reverse osmosis.

When we consider the commercial purification of GABA in culture broth, it is necessary to prepare the defined culture broth to facilitate the downstream process.    

Another important point is to clarify the reason for choosing to produce a protease from Bacillus subtilis instead of using commercial proteases such as Alcalase, Neutrase, Brauzin, or Flavourzyme, which have costs and activities that are interesting from an industrial perspective.

Answer: Thank you for the reviewer's kind comments. The choice to produce protease from Bacillus subtilis was made to apply the additional benefits of microbial fermentation, which go beyond simple protein hydrolysis. This is discussed in detail in the introduction (line 1), where we explain how microbial fermentation can offer advantages not provided by commercial proteases such as Alcalase, Neutrase, Brauzin, or Flavourzyme with higher activity and convenience. (line 53-56)

 

- Conclusions

The conclusion needs to be rephrasing. The authors should write this section in comprehensive and suitable style. The conclusion as it stands is too much a summary of the results. Conclusions in a manuscript are meant to highlight major findings, linked to your objectives and to give a take home message to the readers. Therefore, it should answer the following questions: What is new? Why is it important? What are the limitations and potential consequences? In addition, the authors should indicate future research recommendations.

Answer: Thank you for the reviewer's kind comments. We have revised the conclusion to address the study's objectives, limitations, and potential implications, while also highlighting key findings.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

1.     The abstract should include specific details about the methods used for bioconversion and the significance of the results in the context of current research.

 

2.     The introduction should include a brief discussion on the current limitations in hemp seed protein utilization and how this study addresses those limitations.

 

3.     The discussion should consider discussing the implications of the findings in the context of broader applications in the food industry. It would be beneficial to include a critical analysis of the study's limitations and how future research could build upon the current work.

 

4.     The conclusion should discuss the potential impact of this research on the field and any recommendations for future studies.

 

5.     The figures should have the error bars, and the tables should have difference analysis.

 

6.     The manuscript is well-written, but a thorough proofreading for grammar, punctuation, and clarity is recommended. Consider engaging a professional editor for language polishing to ensure the manuscript meets the journal's standards.

 

Comments on the Quality of English Language

no

Author Response

We deeply appreciate the kind work of the editor and reviewers who provided detailed comments on our manuscript. We have revised the manuscript according to your suggestions and have written and sent you a response as shown below. Thank you.

 

1. The abstract should include specific details about the methods used for bioconversion and the significance of the results in the context of current research.

 Answer: Thank you for the reviewer's kind comments. The method used for bioconversion was fermentation, specifically sequential batch fermentation. We have added these details to the abstract. (line 9-11)

2. The introduction should include a brief discussion on the current limitations in hemp seed protein utilization and how this study addresses those limitations.

 Answer: Thank you for the reviewer's kind comments. The major protein in hemp seed, edestin, is not easily soluble under neutral or acidic conditions, which limits its use in food processing. Most studies address this by hydrolyzing the protein with commercial enzymes. However, this study aims to overcome these limitations through fermentation, which not only improves solubility but also generates additional valuable metabolites, creating high-value-added materials. (line 44-48)

3. The discussion should consider discussing the implications of the findings in the context of broader applications in the food industry. It would be beneficial to include a critical analysis of the study's limitations and how future research could build upon the current work.

 Answer: Thank you for the reviewer's kind comments. The optimized conditions in this study were designed specifically to enhance protein hydrolysis and GABA production. However, microbial fermentation also generates various other metabolites, which were not fully analyzed in this study due to the complexity and volume of the data. Further research will focus on analyzing these compounds, and we plan to publish those findings in future studies. (line: 273-275)

4. The conclusion should discuss the potential impact of this research on the field and any recommendations for future studies.

 Answer: Thank you for the reviewer's kind comments. As with the previous question, this study did not analyze the other compounds produced through fermentation. we plan to publish those findings in future studies. (line: 376-378, 389-393)

5. The figures should have the error bars, and the tables should have difference analysis.

 Answer: Thank you for the reviewer's kind comments. We have revised the figures to include error bars and updated the tables to include statistical analyses.

6. The manuscript is well-written, but a thorough proofreading for grammar, punctuation, and clarity is recommended. Consider engaging a professional editor for language polishing to ensure the manuscript meets the journal's standards.

Answer: Thank you for the reviewer's kind comments. We have thoroughly revised the manuscript for content.

Reviewer 3 Report

Comments and Suggestions for Authors

Yun-Ho Park and co-authors discussed the utilization of a series of co-fermentation processes, using Bacillus subtilis HA and Lactiplantibacillus plantarum KS2020, to convert industrial by-products, specifically hemp seed protein (HSP), into multifunctional components rich in γ-aminobutyric acid (GABA) and peptides. The article innovatively applies a two-step co-fermentation process, where Bacillus subtilis and Lactiplantibacillus plantarum jointly ferment to transform the industrial by-product hemp seed protein (HSP) into high-value multifunctional components enriched with GABA and peptides. The study confirmed that utilizing specific fermentation strains and optimized fermentation conditions can significantly enhance the content of GABA and beneficial peptides in HSP. This article possesses substantial significance and potential value in industrial applications, health nutrition, and environmental protection. However, before publication, I suggest authors make some minor revisions to the paper:

 1. Research Background: Although the paper provides an innovative two-step fermentation method, the authors should offer a more detailed explanation of the background and hypotheses. For instance, a more comprehensive review of existing literature could be provided, explaining why Bacillus subtilis and Lactiplantibacillus plantarum were chosen in this study, and discussing the roles of these strains in previous research.

2. Experimental Methods: In the Production section, the authors need to describe the fermentation process more comprehensively, including strain density (OD), fermentation concentration, medium pH, and other parameters in greater details. While some crucial parameters are discussed in the results section, it is suggested to introduce these in the methods section.

Also, the rationale for selecting fermentation conditions (such as using 5-10% MSG) is unspecified. The authors could provide relevant literatures support or preliminary experimental data to substantiate the rationale for those parameters.

3. Control Experiments: The study solely describes the effects of the two-stage serial co-fermentation process of Bacillus subtilis and Lactiplantibacillus plantarum. Did the authors try Bacillus subtilis or Lactiplantibacillus plantarum individually for fermentation as control results? It would be beneficial to observe and compare the effects of solo-fermentation with co-fermentation on HSP degradation and GABA production.

Author Response

We deeply appreciate the kind work of the editor and reviewers who provided detailed comments on our manuscript. We have revised the manuscript according to your suggestions and have written and sent you a response as shown below. Thank you.

1. Research Background: Although the paper provides an innovative two-step fermentation method, the authors should offer a more detailed explanation of the background and hypotheses. For instance, a more comprehensive review of existing literature could be provided, explaining why Bacillus subtilis and Lactiplantibacillus plantarum were chosen in this study, and discussing the roles of these strains in previous research.

Answer: Thank you for the reviewer's kind comments. In response, we have added a more detailed explanation regarding the necessity of the sequential co-fermentation using Bacillus subtilis and Lactiplantibacillus plantarum. This revision aims to provide a clearer rationale for their selection in our study. (line: 48-49, 56-59, 72-76)

2. Experimental Methods: In the Production section, the authors need to describe the fermentation process more comprehensively, including strain density (OD), fermentation concentration, medium pH, and other parameters in greater details. While some crucial parameters are discussed in the results section, it is suggested to introduce these in the methods section.

Also, the rationale for selecting fermentation conditions (such as using 5-10% MSG) is unspecified. The authors could provide relevant literatures support or preliminary experimental data to substantiate the rationale for those parameters.

Answer: Thank you for the reviewer's kind comments. we have revised the Materials and Methods section to include explanations regarding the initial cell density (OD) and the pH of the medium. Furthermore, we have also clarified the rationale behind comparing different concentrations of HSP and MSG. Specifically, the choice of MSG concentration was based on a preliminary experiment where we observed that Lactobacillus strains converted MSG into GABA effectively at MSG concentrations below 5%. Therefore, we decided to test concentrations above 5% in this study to further explore this conversion efficiency. (line: 100-115)

3. Control Experiments: The study solely describes the effects of the two-stage serial co-fermentation process of Bacillus subtilis and Lactiplantibacillus plantarum. Did the authors try Bacillus subtilis or Lactiplantibacillus plantarum individually for fermentation as control results? It would be beneficial to observe and compare the effects of solo-fermentation with co-fermentation on HSP degradation and GABA production.

Answer: Thank you for the reviewer's kind comments. In this study, the two-stage co-fermentation process using Bacillus subtilis and Lactiplantibacillus plantarum was specifically optimized to enhance both protein hydrolysis and GABA production. Individually, B. subtilis is primarily responsible for peptide enhancement via hydrolysis, while L. plantarum is essential for GABA production. The GABA production in the HSP was confirmed by single stage fermentation using Lb. plantarum. However, conducting fermentation with each strain individually would not achieve the synergistic effects observed with co-fermentation, as B. subtilis also plays a supporting role in the GABA production process when used sequentially with Lb. plantarum.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I didn't see error bars in many figures. 

Comments on the Quality of English Language

no

Author Response

We deeply appreciate the kind work of the editor and reviewers who provided detailed comments on our manuscript. We have revised the manuscript according to your suggestions and have written and sent you a response as shown below. Thank you.

 

I didn't see error bars in many figures. 

Answer: Thank you very much for your insightful comments. The error bars were included, but they were not easily visible due to their small range. To address this, I have adjusted the figure and the Y-axis scale to make the error bars more noticeable.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript can be accept for publication.

Comments on the Quality of English Language

no