Identification and Genetic Diversity of Amylase Producing Lactic Acid Bacteria from Brown Rice (Oryza nivara) Wakawondu Cultivar Based on 16S rRNA Gene

Round 1

Reviewer 1 Report

The manuscript is about isolation and identification of two amylolytic lactic acid bacteria involved in a traditional fermented food. The manuscript provides very limited information but surprisingly long. The authors need to prove the role of isolates in this particular information.

 

In abstract:

1.     Do not italicize the terms, other than scientific names

2.     State clearly which fermentation is referred here.

3.     Write what is meant by 175 mU/mL.

4.     The values given in the OD and units appear like jargons. It is not possible to track which value refers to which experiment.

5.     Since the title refers to ‘diversity’ therefore, state clearly how many isolates were identified in this study

 

In Introduction:

1.     It is not conceivable to get the process of fermentation referred in introduction.

2.     The research void should be indicated in this section.

 

In Materials and Methods

1.     What is meant by ’washing water samples’?

2.     How was the fermentation process initiated?

3.     Do not italicized the words unnecessarily.

4.     The section 2.2.3 can be omitted as such basic information is not required in a research paper

5.     Section 2.3 can be condensed. It would be better to refer a standard protocol only.

6.     What is 1 ose of bacteria?

7.     Likewise, the section 2.5 needs to be condensed.

 

Results

1.     It is surprising to note that authors obtained only 2 amylolytic bacterial strains. Amylase production is a widely distributed trait among microorganisms

2.     Many figures and tables can be transferred to the supplementary file. Figures related to staining or cultural features need not to be presented in the main manuscript file.

3.     The section 3.2.2 and 3.2.3 describe unnecessary information

4.     Instead of providing the sequence in the result section in box form, refer accession number to the sequences

5.     Table 9 and 10 do not add any information.

 

Discussion

1.     The first line is in contradiction to the result section as ‘several isolates’ were not obtained in this study

2.     The results have not been given in the form of CFU per mL, so better to not to discuss it in this way

3.     Most of the part is about to discuss very basic information that is widely known by the scientific community. The mechanism of Gram staining or starch hydrolysis test or similar information do not add to the knowledge of the scientific community.

 

Author Response

REVIEWER 1

 

Comments and Suggestions for Authors

 

The manuscript is about isolation and identification of two amylolytic lactic acid bacteria involved in a traditional fermented food. The manuscript provides very limited information but surprisingly long. The authors need to prove the role of isolates in this particular information.

 

Response: We have improved our paper according to the reviewer's suggestions.

 

 

In abstract:

1. Do not italicize the terms, other than scientific names

Response: The terms have all been written vertically aside from the scientific names.

 

2. State clearly which fermentation is referred here.

Response: The term "fermentation" in abstract has been clearly stated.

 

3. Write what is meant by 175 mU/mL.

Response: "175 mU/mL" is the amylase activity value of lactic acid bacteria isolates.

 

4. The values given in the OD and units appear like jargons. It is not possible to track which value refers to which experiment.

Response: Each strain's OD and units values have been fixed in the abstract for the strain with the highest amylase activity.

 

5. Since the title refers to ‘diversity’ therefore, state clearly how many isolates were identified in this study

Response: The number of LAB isolated from fermented Wakawondu rice washing water has been included in the Abstract and also in the Research Results section.

 

 

In Introduction:

1. It is not conceivable to get the process of fermentation referred in introduction.

Response: The fermentation process of Wakawondu rice washing water has been included in the Introduction section.

 

2. The research void should be indicated in this section.

Response: Several studies related to LAB-producing amylase have been included in the Introduction section.

 

 

In Materials and Methods

1. What is meant by ’washing water samples’?

Response: The meaning of “washing water samples” is water from washing brown rice of the Wakawondu variety used as a sample to be fermented spontaneously for 3 days at room temperature. Furthermore, LAB was isolated from this fermented Wakawondu washing water and LAB strains which had high amylating activity were identified.

 

2. How was the fermentation process initiated?

Response: The fermentation process was carried out spontaneously without the addition of starter for 3 days at room temperature. A full description of the fermentation process has been included in the Methods section.

 

3. Do not italicized the words unnecessarily

Response: Words other than the organism's scientific name and other words that shouldn't be italicized have been changed in accordance with the reviewer's suggestions.

 

4. The section 2.2.3 can be omitted as such basic information is not required in a research paper

Response: This statement has been ommitted as suggested.

 

5. Section 2.3 can be condensed. It would be better to refer a standard protocol only.

Response: Section 2.3 have been condensed as suggested.

 

6. What is 1 ose of bacteria?

Response: The statement "1 ose of bacteria" has been replaced with 1 mL of bacteria culture.

 

7. Likewise, the section 2.5 needs to be condensed.

Response: Section 2.5 have been condensed as suggested.

 

 

Results

1. It is surprising to note that authors obtained only 2 amylolytic bacterial strains. Amylase production is a widely distributed trait among microorganisms

Response: 10 different LAB strains were actually the first ones to be discovered. Following more analysis, it was shown that the SBM3D and SBM4A strains exhibited the highest amylase activity. In order to identify the genus and species of these 2 isolates, further testing was done. The Abstract and Research Results of this publication both provide an explanation of this issue.

 

2. Many figures and tables can be transferred to the supplementary file. Figures related to staining or cultural features need not to be presented in the main manuscript file.

Response: Some tables have been eliminated, while others have been combined. This is done to take into account both Reviewers 1 and 2's suggestion at once.

 

3. The section 3.2.2 and 3.2.3 describe unnecessary information

Response: Only crucial and pertinent explanations were included in the paper and unnecessary information was deleted from Sections 3.2.2 and 3.2.3.

 

4. Instead of providing the sequence in the result section in box form, refer accession number to the sequences

Response: Corrections have been made according to the reviewer's suggestions.

 

5. Table 9 and 10 do not add any information.

Response: Tables 9 and 10 have been deleted as suggested.

 

 

Discussion

1. The first line is in contradiction to the result section as ‘several isolates’ were not obtained in this study

Response: Statements regarding this have been corrected as suggested.

 

2. The results have not been given in the form of CFU per mL, so better to not to discuss it in this way

Response: This statement has been corrected in the Discussion part as suggested.

 

3. Most of the part is about to discuss very basic information that is widely known by the scientific community. The mechanism of Gram staining or starch hydrolysis test or similar information do not add to the knowledge of the scientific community.

Response: At the suggestionof the reviewer, very basic material that is well known in the scientific community, such as the mechanism of the Gram stain, the starch hydrolysis assay, or other information of a similar nature, has been omitted.

 

Pekanbaru, 21 November 2022

Corresponding author

Prof. Usman Pato, PhD.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript, Wahyuni et al, reported two lactic acid bacteria (LAB) strains isolated from the washing of Wakawondu, a brown fermented rice. Two strains, named SBM3D and SBM4A, were confirmed to be gram-positive bacteria. The authors then described their colony morphologies, and performed several biochemical assays, including catalase production, starch hydrolysis, amylase activity, and sugar/acid fermentation. Finally, using 16s rRNA sequencing and alignment, these two strains were found to have a super close evolutionary relationship with two strains from the same species (P. pentosaceus), at a high sequence identity (almost 100%).

 

Wakawondu is a special fermented brown rice that is thought to have a nutrient advantage compared to regular unfermented white rice. This may be because of the pre-degradation by bacteria during fermentation. Thus, isolating LAB strains involved in this process is quite interesting and helpful to understand the detailed fermentation process. This will also benefit the potential applications of these strains in fermentation with other rice, or food. This manuscript is a straight forward report, describing the two strains isolated by the authors. They performed careful assays to understand the phenotypes, and provided careful notes. They also tried to identify their species based on 16s sequencing. Although several minor English edits may be needed, this manuscript is generally well written, and easy to follow.

 

Since the experiments in this study are simple and straightforward, all my concerns are minor, mainly about some clearances.

 

1.     Rewrite the last paragraph of introduction.

The introduction is in general good, but when I finished reading this section, I was lost about what is the question the authors want to answer, and what is their major conclusion or discovery. They have a good introduction about the background, and recent research progress. How about adding another paragraph like this: (please modify based on authors’ own style)

“Isolating LAB strains from the rice washing waster is interesting because…” (this kind of explanation is usually important to readers that are not in this field)

“In this study, we successfully isolate two bacteria, that are later proved to be Gram-positive. Then, we performed a series of biochemical assays, in order to….” (briefly summarize your experiments in this study)

“Finally, by sequencing and aligning their 16s RNA, we found…” (major conclusion and something surprising/interesting).

 

2.     Keywords

These words are fine. How about re-organizing their orders? Starting with “Wakawondu”, then “lactic acid bacteria”, “identification” “pediococcus pentosaceus”. I don’t think amylase is a good fit here.

 

 

3.     16s rRNA gene amplification.

This part needs more classification. For example, what are the primers used in this study? I guess some “common” primers were used here. So how about adding related references, and say “as described before…”

There are also some typo in 2.5.3 section. Like 950C???

I guess the author used the primer in 2.1 materials “5’ Forward….”. How about moving the primer information here, together with the “16s rRNA gene amplification”?

 

4.     16s rRNA sequencing and alignment.

I really don’t like the description of “similarity”, which should be replaced with “identity”. For example, how to define “100% similarity”? Identity is a much better word to describe, “what is the ratio of nucleotides that are the same, or not”. For example, 99% identity means 99% nt are exactly the same, which means a “high similarity”. This should be corrected in 3.8 section, Table 9, discussions, conclusions and many other places.

 

5.     Acknowledgment / author contributions / statements of conflict of interest.

Didn’t find these necessary sections in the current manuscript file. Please double-check with editors to make sure your manuscript follows publication policies.

 

 

 

Small typos:

1.     2.5.1, please replace letter “x” with multiply symbol “´”, which can be done by insert – advanced symbol in Microsoft Word.

2.     2.5.3, 950C, and some space.

3.     Generally, please go through the “Materials and Methods” sections and correct typos, space, non-standard symbols carefully again. I understand this part is annoying, and super important for other scientists if they want to learn from your research.

4.     Section 3.7 – “SBM4A” in the first line.

5.     Try to improve the resolution for figure 7 and 8. They look like screen capture. Maybe you can try to export as PDF at first.

Author Response

REVIEWER 2

 

Comments and Suggestions for Authors

 

In this manuscript, Wahyuni et al, reported two lactic acid bacteria (LAB) strains isolated from the washing of Wakawondu, a brown fermented rice. Two strains, named SBM3D and SBM4A, were confirmed to be gram-positive bacteria. The authors then described their colony morphologies, and performed several biochemical assays, including catalase production, starch hydrolysis, amylase activity, and sugar/acid fermentation. Finally, using 16s rRNA sequencing and alignment, these two strains were found to have a super close evolutionary relationship with two strains from the same species (P. pentosaceus), at a high sequence identity (almost 100%).

 

Wakawondu is a special fermented brown rice that is thought to have a nutrient advantage compared to regular unfermented white rice. This may be because of the pre-degradation by bacteria during fermentation. Thus, isolating LAB strains involved in this process is quite interesting and helpful to understand the detailed fermentation process. This will also benefit the potential applications of these strains in fermentation with other rice, or food. This manuscript is a straight forward report, describing the two strains isolated by the authors. They performed careful assays to understand the phenotypes, and provided careful notes. They also tried to identify their species based on 16s sequencing. Although several minor English edits may be needed, this manuscript is generally well written, and easy to follow.

 

Since the experiments in this study are simple and straightforward, all my concerns are minor, mainly about some clearances.

 

1. Rewrite the last paragraph of introduction.

The introduction is in general good, but when I finished reading this section, I was lost about what is the question the authors want to answer, and what is their major conclusion or discovery. They have a good introduction about the background, and recent research progress. How about adding another paragraph like this: (please modify based on authors’ own style).

Response: The introduction's final sentence has been revised in accordance with suggestions. The Introduction section has also been updated with new data and research findings.

 

“Isolating LAB strains from the rice washing waster is interesting because…” (this kind of explanation is usually important to readers that are not in this field)

Response: The Introduction section has been updated in response to comments highlighting the significance of isolating LAB with high amylase activity from this fermented product.

 

 

“In this study, we successfully isolate two bacteria, that are later proved to be Gram-positive. Then, we performed a series of biochemical assays, in order to….” (briefly summarize your experiments in this study)

Response: A summary regarding these two isolates has been included in this paper in the Abstract and Conclusion sections.

 

“Finally, by sequencing and aligning their 16s RNA, we found…” (major conclusion and something surprising/interesting).

Response: In accordance with the advice, we have completed the aforementioned assertion and added it to the Conclusion section.

 

2. Keywords

These words are fine. How about re-organizing their orders? Starting with “Wakawondu”, then “lactic acid bacteria”, “identification” “pediococcus pentosaceus”. I don’t think amylase is a good fit here.

Response: Keywords have been rewritten according to the reviewer's suggestions.

 

 

3. 16s rRNA gene amplification.

This part needs more classification. For example, what are the primers used in this study? I guess some “common” primers were used here. So how about adding related references, and say “as described before…”

Response: Primes used for this stydy was 27F 5' forward primer–AGAGTTTGATCMTGGCTCAG–3'primary reverse 1492R 5'–GGTTACCTTGTTACGACTT– 3' (Genetics Lab., 2021). This statement has been added to the Methods section.

 

 

There are also some typo in 2.5.3 section. Like 950C???

Response: The text of this document has been edited to remove a few typos like "950C" and others.

 

 

I guess the author used the primer in 2.1 materials “5’ Forward….”. How about moving the primer information here, together with the “16s rRNA gene amplification”?

Response: We have moved it according to the reviewer's suggestions

 

4. 16s rRNA sequencing and alignment.

I really don’t like the description of “similarity”, which should be replaced with “identity”. For example, how to define “100% similarity”? Identity is a much better word to describe, “what is the ratio of nucleotides that are the same, or not”. For example, 99% identity means 99% nt are exactly the same, which means a “high similarity”. This should be corrected in 3.8 section, Table 9, discussions, conclusions and many other places.

Response: The word "similarity" has been replaced by "identity" in all parts of our paper.

 

5. Acknowledgment / author contributions / statements of conflict of interest.

Didn’t find these necessary sections in the current manuscript file. Please double-check with editors to make sure your manuscript follows publication policies.

Response: According to suggestions, we have included acknowledgments, author contributions, and conflict of interest disclosures to our paper.

 

 

 

Small typos:

1. 5.1, please replace letter “x” with multiply symbol “´”, which can be done by insert – advanced symbol in Microsoft Word.

Response: We have improved our paper according to the reviewer's suggestions.

 

2. 5.3, 950C, and some space.

Response: We have improved our paper as suggested.

 

3. Generally, please go through the “Materials and Methods” sections and correct typos, space, non-standard symbols carefully again. I understand this part is annoying, and super important for other scientists if they want to learn from your research.

Response: We have corrected typos, spaces, non-standard symbols in the “Materials and Methods” sections according to the reviewer's suggestions

 

4. Section 3.7 – “SBM4A” in the first line.

Response: We have corrected the spelling of "SBM4" to "SBM4A"

 

5. Try to improve the resolution for figure 7 and 8. They look like screen capture. Maybe you can try to export as PDF at first.

Response: We have improved the resolution for figures 7 and 8 based on suggestions from reviewers.

 

Pekanbaru, 21 November 2022

Corresponding author

Prof. Usman Pato, PhD.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The revised version still lacks significant improvement. The comments made on the original draft have not been addressed. The paper describes basic information about two bacterial isolates, their microscopic, morphological and 16S rDNA sequence. No information has been given related to fermentation and fermentative potential of these isolates. The phylogenetic relatedness has serious flaws into it. Without external clade and without genebank accession numbers, it is hard to follow anything out of the figures given. Most of the data is not presentable in the manuscript. It is not conceivable to present Gram staining figures and morphological (similar) data in a manuscript submitted to a reputable journal.

 

Some minor comments for author’s consideration:

 

In abstract:

Write ’Ten lactic acid bacterial strains…’

Write ‘Two isolates…’

Replace ‘Fermentation time on’ with ‘Fermentation time of’

The so-called increase in OD and amylase activity from 12 to 27 h is not considerable. (OD from 0.856 to 0.886 and amylase from 175 to 176 U mL^-1)

State about phylogenetic relationship in terms of clades and not in % identity.

 

In Introduction

Rephrase ‘washing water can produce lactic acid bacteria’

The phrase ‘The majority result of fermentation of carbohydrate…’ is too general and swayed away from the facts.

The last paragraph of the introduction needs complete revisit. The recent advancement in the molecular taxonomy has been neglected.

 

In Methods

Do not begin a sentence with a numeral.

Still the dose/inoculum of bacteria is mentioned as ‘ose’

 

In Results

Authors have still given just basic information about morphological and cultural characteristics of the isolates (that too do not vary greatly from each other)

 

Discussion

Relies on extending the already well appreciated information.

 

 

Author Response

Response to the Comments and Suggestions for Authors from Reviewer 1.

 

The revised version still lacks significant improvement. The comments made on the original draft have not been addressed. The paper describes basic information about two bacterial isolates, their microscopic, morphological and 16S rDNA sequence. No information has been given related to fermentation and fermentative potential of these isolates. The phylogenetic relatedness has serious flaws into it. Without external clade and without genebank accession numbers, it is hard to follow anything out of the figures given. Most of the data is not presentable in the manuscript. It is not conceivable to present Gram staining figures and morphological (similar) data in a manuscript submitted to a reputable journal.

Response: We appreciate your feedback on how we can make our paper better. From this research, we have excluded the paper that provided basic details about two bacterial isolates, as well as their microscopic, morphological, and other general characteristics. In the Discussion section, we have also given details about fermentation and these isolates' potential for fermentation. In accordance with the advice of Reviewer 1, we also provided the findings and explanation of phylogenetic relatedness with external clade and genebank accession numbers. The Results and Discussion section has a brief presentation of morphological data and gram staining. We did everything we could to make our paper better so that it would satisfy Reviewer 1.

 

 

 

Some minor comments for author’s consideration:

 

In abstract:

Write ’Ten lactic acid bacterial strains…’

Response: Improvements have been made as per suggestion.

 

Write ‘Two isolates…’

Response: Writing has been corrected according to suggestion.

 

Replace ‘Fermentation time on’ with ‘Fermentation time of’

Response: Writing has been corrected according to suggestion

 

The so-called increase in OD and amylase activity from 12 to 27 h is not considerable. (OD from 0.856 to 0.886 and amylase from 175 to 176 U mL^-1)

Response: This statement has been corrected according to the reviewer's suggestion.

 

State about phylogenetic relationship in terms of clades and not in % identity.

Response: This paper has utilized phylogenetic relationships in terms of clades as per recommendations.

 

 

In Introduction

Rephrase ‘washing water can produce lactic acid bacteria’

Response: In response to comments, the sentence "washing water can produce lactic acid bacteria" has been rephrased.

 

The phrase ‘The majority result of fermentation of carbohydrate…’ is too general and swayed away from the facts.

Response: Sentences that start with "The majority consequence of carbohydrate fermentation" have been changed as recommended.

 

The last paragraph of the introduction needs complete revision. The recent advancement in the molecular taxonomy has been neglected.

Response: The final sentence of the introduction has been changed in accordance with the advice.

 

 

In Methods

Do not begin a sentence with a numeral.

Response: As indicated, sentences that start with a numeric have been rephrased.

 

Still the dose/inoculum of bacteria is mentioned as ‘ose’

Response: The word "ose" has been omitted in this paper.

 

 

In Results

Authors have still given just basic information about morphological and cultural characteristics of the isolates (that too do not vary greatly from each other)

Response: The Results section contains a brief description of the morphological and cultural traits of the isolates as suggested.

 

 

Discussion

Relies on extending the already well appreciated information.

Response: The discussion of the physiological and morphological characteristics of the LAB isolates has been condensed but added to the discussion of the fermentative capacity of these isolates. The genus and species of the two isolates from fermented Wakawondu rice washing water are also discussed, along with external clades and genebank accession numbers.

 

 

 

Pekanbaru, 25 November 2022

Corresponding author

 

 

Prof. Usman Pato

 

 

 

 

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

The discussion section related to phylogenetic relatedness needs improvement. For intance, the paragraph: 

"Determination of bacterial kinship using the current genotypic identification method using DNA produced a genetic material resistant to evolution. However, the use of DNA was hampered by the inadequate number of samples"

is technically wrong. Revise such sections to remove errors. 

The paper can be accepted after minor revision, particularly to remove language errors.

Author Response

Response to the Comments and Suggestions for Authors from Reviewer 1

The discussion section related to phylogenetic relatedness needs improvement. For intance, the paragraph: 

"Determination of bacterial kinship using the current genotypic identification method using DNA produced a genetic material resistant to evolution. However, the use of DNA was hampered by the inadequate number of samples"

is technically wrong. Revise such sections to remove errors. 

Response: In light of the comments "Determination of bacterial kinship using the current genotypic identification method using DNA produced a genetic material resistant to evolution. However, the use of DNA was hampered by the inadequate number of samples", it might be best to delete this remark if it isn't actually pertinent to the research findings. We are hoping that Reviewer 1 shares our viewpoint.

 

The paper can be accepted after minor revision, particularly to remove language errors.

Response: We sincerely hope that following this minor revision, our manuscript will be accepted. Thank you so much for your comments and suggestions on behalf of all the authors.

Pekanbaru, 26 November 2022

Corresponding author

 

Prof. Usman Pato

Author Response File: Author Response.pdf