Assisted Isolation of Camelliagenin B from Camellia oliefera Seed Cake Meal and Microbial Transformation by Bacillus subtilis ATCC 6633, Bacillus megaterium CGMCC 1.1741, and Streptomyces gresius ATCC 13273

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study uses 3 microorganisms for the microbial transformation of camelliagenin B to new metabolites. These metabolites are characterized using advanced techniques. The review of the subject is relevant, using both recent and older appropriate scientific sources in a structured way. The text is clear and scientifically sound. The experimental design is appropriate to obtain and characterize the derivatives and the results can be reproducible based on the details given. The tables and figures, as well as, the interpretation of the results and the conclusions are consistent and supported by the cited references. The ethics statements and data availability are adequate.

The paper is satisfactory in style and presentation. I recommend that the paper should be accepted upon its revision by the author to satisfy the some of the attached comments/ suggestions.

Comments & Suggestions

Page 2, 2nd paragraph. The taxonomical names of the three species should be in italics

Table 1 & par. 2.4. Why is the second substrate mentioned as Soybean Meal when it only contains pure chemicals and not any soy products?

It could be useful for the readers to give some details/references or software used in the Materials and methods for the techniques used (i.e. for HMBC, NOESY, HSQC, etc), also the full name of abbreviated terms should be used when first mentioned in the text

Some double spaces need to be corrected (i.e page 6-3rd line, last paragraph 2nd line, page 10-6th & 11th line, etc) and some dots added at the end of some paragraphs

Author Response

Dear Reviewer,

Thank you very much for taking the time to review this manuscript. We acknowledge and appreciate your invaluable suggestions. Hopefully, our manuscript will be eligible for acceptance after incorporating the comments from the respected reviewers.  Please find the detailed responses below and the corresponding revisions/corrections highlighted in red in the resubmitted files.   

Comments & Suggestions

Comment 1: Page 2, 2nd paragraph. The taxonomical names of the three species should be in italics.

 Response 1: We appreciate your valuable insights and apologize for the mistakes. As recommended, we have changed the taxonomical names of the microbial strains  to italics and highlighted the change in the revised manuscript [page 2 para.2].

Comment 2: Table 1 & par. 2.4. Why is the second substrate mentioned as Soybean Meal when it only contains pure chemicals and not any soy products?

 Response 2: We apologize for causing confusion. We have added the name and the amount of the soy products in the revised manuscript.

 Revised text: [Table no. 1]

Table 1. Composition of culture medium

Potato Dextrose (PD)		Soybean Meal (SM)
Peeled potatoes	200g	Glucose	20g
Glucose	20g	Yeast powder	5g
KH2PO4	3g	NaCl	5g
MgSO4.7H2O	1.5g	K2HPO4	5g
Distilled water	1L	Soybean meal	5g
		Distilled water	1L

 Comment 3: It could be useful for the readers to give some details/references or software used in the Materials and methods for the techniques used (i.e., for HMBC, NOESY, HSQC, etc), also, the full name of abbreviated terms should be used when first mentioned in the text

 Response 3: We sincerely appreciate the reviewer’s constructive suggestion. As advised, we have added the details of the software used in the Materials and Methods section to make it easier for readers. Additionally, we have added the full names of abbreviated terms first mentioned in the text.

 Revised text: Structure verification of the compounds was elucidated using High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) collected on the Agilent 1260-6530 QTOF spectrometer. One- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR) based on Heteronuclear Single Quantum Coherence (HSQC), Heteronuclear Multiple Bond Correlation (HMBC), Nuclear Overhauser Effect Spectroscopy (NOESY) were obtained with a Bruker DRX-600 spectrometer in chloroform-d and pyridine-d₅ using Tetramethylsilane (TMS) as the internal standard, and the chemical shifts are expressed in parts per million (δ) and coupling constants (J) in Hertz [page no. 2, section 2.1].

Comment 4: Some double spaces need to be corrected (i.e page 6-3rd line, last paragraph 2nd line, page 10-6th & 11th line, etc) and some dots added at the end of some paragraphs

 Response 4: Thank you for pointing out the mistakes. As recommended, the corrections have been made in the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

This study explored the microbial transformation of camelliagnin B from Camellia oleifera seed cake meal to develop novel metabolites with potential health benefits. This study is interesting for its utilization of green synthesis methods and its emphasis on sustainability. I have made comments regarding this study. Consequently, I recommend the publication of this article in Fermentation after incorporating the following suggestions:

Abstract:

Its well-written and structured. I missed the mention of some bioactive compounds identified in the study, perhaps mentioning the most relevant ones for health.

1-Introduction

The introduction is well-written, but it's necessary to insert the reference in the paragraph ("Recently, it has gained significant attention…").

2- Materials and methods

In section 2.1

"Defatted seed cake into a fine powder and sieved through an 80-mesh sieve for uniformity." The text is unclear; was the fine powder obtained and sieved by the authors, or did the company donate already processed material? Make this clearer in the text.

"These strains were inoculated on potato dextrose agar (PDA) slant culture medium and then stored at 4℃." This storage method for microorganisms is confusing. This culture medium (PDA) is recommended for fungi, yet this study works with bacteria. This storage temperature of 4℃ using slant culture medium is not adequate for maintaining these cultures.

It is necessary to insert the meaning of the acronyms throughout Section 2.

In section 2.2

"Each time, 5g of the substrate was hydrolyzed and repeated for each variable." The text is unclear; how many repetitions were made? This description needs to be clearer.

"and then dissolved in acetone for further filtration." What is analytical grade (PA) acetone, or a solution used?        

In section 2.4

"each bottle was divided into 50." What is the unit of measure for 50?

Table 1: Position the table after its first mention in the text.

 

3. Results and discussion

In section 3.1

Obtain 540 mg of hydrolyzed extract (a combination of glycosides and impurities)." The text needs revision to present the information more clearly and with better textual cohesion.

"(Table. 2)": The parenthesis and bold formatting are unnecessary here, as this is part of the running text. The data shown in the table should be explored more thoroughly in the text, and even minor data should be mentioned.

"The purification process was conducted using AB-8 macroporous resin with 75% elution and a 10 g/mL liquid-to-solid ratio." Some of this information, such as the elution percentage used, is not shown in the methodology. Another piece of information that is a result is the mobile phase ratio used, considering different ratios were employed.

"this method yielded 2.13g of tea saponin extract": In Table 2, the time to obtain 2.13g of saponin is 10 hours and does not correspond to the information shown in Figure 1.

Reference [24] is from 2018; therefore, the affirmation "recent literature" is not entirely adequate.

In section 3.2

Standardize the bolding of compounds and the chemical names of identified compounds.

Figures: Position the figures after their first mention in the text.

4. Discussion

It's well-written and structured. It's suggested that the expression "per se" be removed from the text: "suppress biological properties such as per se anti-inflammatory." This expression is colloquial and can compromise the formality and elegance of the academic text; using only "such as" is sufficient.

5 Conclusion

I suggest avoiding going into too much detail about past work in conclusion, because the focus of the conclusion should be on the results and implications of the current study. The introduction is the most appropriate place to discuss the research group's previous experiences with the subject of study, especially if those experiences provide the necessary context.

References

 The article makes good use of up-to-date and relevant scientific references.

Comments on the Quality of English Language I suggest a specialized English review.

Author Response

Dear Reviewer,

Thank you very much for taking the time to review this manuscript. We acknowledge and appreciate your invaluable suggestions. Hopefully, our manuscript will be eligible for acceptance after incorporating the comments from the respected reviewers. Please find the detailed responses below and the corresponding revisions/corrections highlighted in red in the resubmitted files.   

Comment 1:

Abstract: It’s well-written and structured. I missed the mention of some bioactive compounds identified in the study; perhaps it would be helpful to mention the most relevant ones for health.

Response 1: Thank you for your appreciation. We have mentioned the names of the compounds identified in the study, named as 1a-1j in the revised manuscript [page no. 1, Line 5].

Comment 2: 1-Introduction

The introduction is well-written, but it's necessary to insert the reference in the paragraph ("Recently, it has gained significant attention…").

Response 2: We appreciate your advice and apologize for the mistake. As advised, we have inserted the references and marked them in red, in the revised manuscript [page no. 2, para. 2, line 2].

Comment 3:

2- Materials and methods

In section 2.1

"Defatted seed cake into a fine powder and sieved through an 80-mesh sieve for uniformity." The text is unclear; was the fine powder obtained and sieved by the authors, or did the company donate already processed material? Make this clearer in the text.

Response 3: We thank the reviewer for this insightful suggestion. As recommended, we have implemented the suggestion in the revised manuscript, highlighted in red. We apologize for creating confusion.  

Revised text: The seed cake was defatted to remove the oil and then ground into a fine powder, which was sieved through an 80-mesh sieve to ensure uniformity. [page no. 2, section 2.1].

Comment 4:

"These strains were inoculated on potato dextrose agar (PDA) slant culture medium and then stored at 4℃." This storage method for microorganisms is confusing. This culture medium (PDA) is recommended for fungi, yet this study works with bacteria. This storage temperature of 4℃ using slant culture medium is not adequate for maintaining these cultures.

Response 4: We sincerely appreciate the reviewer's careful attention to the composition of our culture medium. We would like to clarify that our study specifically utilized potato dextrose broth (PDB) - a liquid medium without agar - rather than the solid PDA medium as initially described. This modified formulation was intentionally designed for optimal bacterial cultivation based on several key considerations. The liquid PDB, composed of potato infusion and glucose, provided essential carbohydrates from the potato extract to support initial microbial growth. In contrast, the glucose served as a readily utilizable carbon source for rapid biomass accumulation. Furthermore, the addition of KH₂PO₄ (potassium phosphate) and MgSO₄·7H₂O (magnesium sulfate heptahydrate) to the culture medium serves several essential functions in supporting microbial growth and biotransformation processes. KH₂PO₄ acts as both a buffering agent to maintain optimal pH stability. Meanwhile, MgSO₄·7H₂O provides magnesium ions that serve as vital cofactors for numerous enzymes involved in cellular metabolism, including those responsible for glycoside hydrolysis and oxidative transformations of saponins. This combination of minerals creates optimal conditions for microbial growth and enzymatic activity, enabling efficient bioconversion of tea saponins into target metabolites.

In our study, slant cultures stored at 4°C were used exclusively for short-term working stocks (≤2 weeks), with fresh subcultures prepared as needed. For long-term preservation, microbial strains were maintained using cryopreservation at –80°C in 20% glycerol (for Bacillus subtilis and B. megaterium) or lyophilization (for Streptomyces griseus ATCC 13273). The revision has been made in the revised manuscript [page no. 2, section 2.1].   

Comment 5:

In section 2.2

"Each time, 5g of the substrate was hydrolyzed and repeated for each variable." The text is unclear; how many repetitions were made? This description needs to be clearer.

"and then dissolved in acetone for further filtration." What is analytical grade (PA) acetone, or a solution used?        

Response 5: We appreciate the reviewer's valuable feedback regarding the need for greater precision in describing our experimental procedures. Below, we provide the clarifications and corresponding revisions to the manuscript text. The following clarification has been made in the revised manuscript.

Revised text: The 5 g substrate quantity represents the amount used per individual hydrolysis process, where initially 5 g of tea saponins was dissolved in 50 mL of a methanol-hydrochloric acid (HCl) solution, maintaining a methanol-to-water ratio of 3:1. The concentration of HCl was varied to 1.5, 2, 2.5, and 3 mol/L, while the hydrolysis time was set to 4, 4.5, 5, 5.5, and 6 h at a temperature of 80 °C [page no. 3, section 2.3]. Yes, all the chemicals used were HPLC or analytical grade.

Comment 6:

In section 2.4

"each bottle was divided into 50." What is the unit of measure for 50?

Table 1: Position the table after its first mention in the text.

 Response 6: We appreciate the reviewer's attention to this critical methodological detail. The number "50" refers to 50 mL aliquots per bottle, which was our standard working volume for the biotransformation experiments.

Revised text: Each flask was split into 50 mL, sterilized at 121 °C for 20 minutes, and then incubated at 180 rpm/min at 28 °C for 24 hours [page no.4, section 2.4].

The contents of Table 1 have been incorporated into the revised manuscript to enhance clarity for readers [page no. 4, Table no 1].

Comment 7:

1. Results and discussion

In section 3.1

Obtain 540 mg of hydrolyzed extract (a combination of glycosides and impurities)." The text needs revision to present the information more clearly and with better textual cohesion.

Response 7: We appreciate the reviewer's request for clarification. The following text has been modified to improve clarity.

Revised text: Initial small-scale experiment, optimal condition was observed : 5g of tea saponin dissolved in a methanol hydrochloride (HCl) solution with a concentration of 2mol/L (methanol: water = 3:1) and hydrolyzed at a temperature of 80℃ for 5.5h resulting in an average yield of 540 mg of hydrolyzed extract (a mixture of aglycones and residual impurities) [page no. 4, section 3.1, Line 2]. 

Comment 8:

 "(Table. 2)": The parenthesis and bold formatting are unnecessary here, as this is part of the running text. The data shown in the table should be explored more thoroughly in the text, and even minor data should be mentioned.

Response 8: We appreciate the reviewer's constructive feedback. The updated version now offers a comprehensive analysis of the hydrolysis optimization process (1)remove unnecessary formatting, (2) thoroughly discuss all data in Table 2 (including minor trends),

Revised text: The hydrolysis of tea saponin was systematically studied under varying conditions to determine the optimal parameters for maximum yield. Initial small-scale experiments using 5g of tea saponin dissolved in a methanol hydrochloride (HCl) solution with a concentration of 2mol/L (methanol: water = 3:1) and hydrolyzed at a temperature of 80℃ for 5.5h yielded an average of 540mg of crude extract (consisting of a mixture of aglycone and residual impurities). It was observed that the hydrolysis of a single 50 g of tea saponins produced the highest yield of 11.7893 g (23.58%), demonstrating that larger quantities enhance reaction efficiency. It was revealed that the HCl concentration has a significant impact on the hydrolysis process. Reducing HCl to 1.5 mol/L decreased the yield to 13.81%, while increasing it to 2.5 mol/L increased the yield to 26.61% (13.3061 g). However, excessive concentration of HCl (3.0 mol/L) led to a decline in yield (19.23%), likely due to degradation. Reaction time also played a crucial role; hydrolysis for 5 h at 2.5 mol/L HCl yielded 24.95%, whereas extending it to 6 h reduced the yield to 19.50%, suggesting that prolonged heating may induce degradation. Overall, the optimal condition of hydrolysis was observed to be 50 g of tea saponins, 2.5 mol/L HCl, and 5.5 h at 80℃. When applied to a large-scale batch (750 g of tea saponin), the optimized method produced 154.5 g of hydrolyzed product, confirming scalability. These findings underscore the importance of precise control over reaction parameters to optimize hydrolysis efficiency while minimizing degradation. Table 2 presents the optimization of acid hydrolysis of tea saponins [page no. 4, section 3.1].

Comment 9:

"The purification process was conducted using AB-8 macroporous resin with 75% elution and a 10 g/mL liquid-to-solid ratio." Some of this information, such as the elution percentage used, is not shown in the methodology. Another piece of information that is a result is the mobile phase ratio used, considering that different ratios were employed.

Response 9: We acknowledge the reviewer's valid concern about the purification process. To address this, we have revised the protocol as follows:

Revised text:

In the methodology, the samples were separated and purified using silica-gel (100 mesh to 200 mesh, 385g) and (200 mesh to 300 mesh) column chromatography. The fraction eluted with ethanol was collected, concentrated, and dried for further analysis. The extracts were visualized and identified using TLC with CH₂Cl₂ and CH₃OH as mobile phases at different ratios: 250:1, 100:1, 50:1, 30:1, 20:1, 10:1, and 5:1. A large yellow-brown substance was noted at the 30:1 ratio [page no. 4, section 2.3: Purification and separation of Camelliagenin B].

In the result, the purification process employed a sequential chromatographic approach to isolate the target compounds. The initial fractionation using silica gel column chromatography (50:1 solvent ratio, 12 mL/min flow rate) yielded 14 fractions containing the target component, as identified by TLC analysis. After excluding three fractions with lower content, the remaining 11 fractions were further purified by preparative HPLC under optimized conditions: (flow rate 12mL/min, 60% acetonitrile, 40% (0.2%) formic acid water, and the target substance was 18 minutes). As a result, this method yielded 2.13 g yield of tea saponin extract from C. oleifera seed cake meal, allowing us to isolate camelliagenin B (1). [page no. 4, section 3.2: Purification and separation of Camelliagenin B].

Comment 10:

"this method yielded 2.13g of tea saponin extract": In Table 2, the time to obtain 2.13g of saponin is 10 hours and does not correspond to the information shown in Figure 1.

Response 10: We sincerely appreciate the reviewer's careful reading and valid observation regarding the apparent discrepancy between the purification yield and timeline data. We want to clarify this point as follows: The 2.13 g yield refers specifically to purified camelliagenin B obtained from the final prep-HPLC step (60% ACN/40% 0.2% formic acid, 12 mL/min, 18 min retention time), not the initial hydrolysis product. This is distinct from: The 154.5 g crude hydrolysate obtained after acid hydrolysis (Table 2—the 20.7 g from small-scale optimization trials. The 10-hour duration in Table 2 reflects the total hydrolysis time for bulk processing (50 g batches × 5.5 h optimal conditions). Figure 1 represents a schematic illustration of the summary process of the extraction and preparation of compound 1, camelliagenin B. The revision has been made in the updated manuscript.

Comment 11:

Reference [24] is from 2018; therefore, the affirmation "recent literature" is not entirely adequate.

In section 3.2

Standardize the bolding of compounds and the chemical names of identified compounds.

Figures: Position the figures after their first mention in the text.

Response 11: We sincerely appreciate the reviewer’s meticulous attention to detail. We removed the descriptor "recent literature" for the 2018 reference and replaced it with: "as reported in the literature”  [page no. 5, section 3.2, para 2, line 1].

Comment 12:

4. Discussion

It's well-written and structured. It's suggested that the expression "per se" be removed from the text: "suppress biological properties such as per se anti-inflammatory." This expression is colloquial and can compromise the formality and elegance of the academic text; using only "such as" is sufficient.

Response 12: Thank you for your invaluable suggestions. As recommended, the “per se” has been removed from the text and replaced with “such as” [page no. 15, discussion, para 4, line 3] to maintain the formality and elegance of the academic writing.

Comment 13:

5 Conclusion

I suggest avoiding going into too much detail about past work in conclusion, because the focus of the conclusion should be on the results and implications of the current study. The introduction is the most appropriate place to discuss the research group's previous experiences with the subject of study, especially if those experiences provide the necessary context.

Response 13: We sincerely appreciate the reviewer’s constructive suggestion regarding the focus of the Conclusion section. We agree that the conclusion should prioritize summarizing the key findings and implications of the current study rather than revisiting past work. We removed the detailed discussion of prior work. We condensed the conclusion to emphasize: the novel biotransformation products obtained in this study, the significance of microbial modifications to camelliagenin B, and future research directions based on current results. We relocated the contextual background on our group’s prior work to the previous section, where it better supports the motivation for this study.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript describes an interesting technology for isolation of Camelliagenin B from Camellia oliefera seed cake meal and microbial transformation by Bacillus subtilis ATCC 6633, Bacillus megaterium CGMCC 1.1741, and Streptomyces gresius ATCC 13273. The authors conclude that these findings emphasize the capability of those strains to transform into a sustainable and environmentally friendly method for generating bioactive compounds from C. oleifera seed cake meals

The introduction is almost correct and describes the raw material and its properties as a precursor for some compounds, but it does not mention the possible metabolic way from microorganisms for biotransformation.
There are no references in the paragraph “Due to its eco-friendly approach ……”.
The following paragraph is recommended to be rewritten in the third person; if it is included, it should not describe the aim of the work.

The methodology 
It is weak in the “2.4. Biotransformation section”. The experiments are not considered to be triplicate.

Table 1. Soybean content is not reported

In the results sections
It is mentioned that “Compared with the substrate dC 81.58, dC 71.72 (C-3) displacement moved to the low field.” Which substrate is mentioned? In general, the substrate used to develop the strains does not impact the metabolic characteristics of the strains; two culture media were proposed, and the effect of the bioconversion of camelliagenin was not mentioned. Is a significant effect on strain selection
The results are descriptive of proposal structures, but the possible metabolic pathway that produces those changes in the compound is not mentioned.
There is no information about the reproducibility of the results. 

In the discussion part
It is a general discussion indicating only the complexity of the microorganisms and their enzymes. It does not include support for the microorganism's bioconversion capability according to metabolism or some reports about the theme.

The conclusion section discusses some results, and it is recommended that it be concise.

Author Response

Dear Reviewer, 

Thank you very much for taking the time to review this manuscript. We acknowledge and appreciate your invaluable suggestions. Hopefully, our manuscript will be eligible for acceptance after incorporating the comments from the respected reviewers.  Please find the detailed responses below and the corresponding revisions/corrections highlighted in red in the resubmitted files.  

Comment 1:

The introduction is almost correct and describes the raw material and its properties as a precursor for some compounds, but it does not mention the possible metabolic way from microorganisms for biotransformation.
There are no references in the paragraph “Due to its eco-friendly approach ……”.
The following paragraph is recommended to be rewritten in the third person; if it is included, it should not describe the aim of the work.

 Response 1: We are grateful for your positive remark and invaluable suggestions. As suggested, we have included the possible metabolic mechanism from microorganisms for biotransformation in the revised manuscript.

Revised text: This can be attributed to the ability of microorganisms to employ diverse metabolic pathways for biotransforming compounds, primarily through enzymatic hydrolysis, oxidation, glycosylation, and deglycosylation. In case of biotransforming tea saponins into their more bioavailable forms, the enzyme, such as β-glucosidases, cleaves sugar moieties, modifying the aglycone’s structure, producing sapogenins [21]. Oxidative enzymes catalyze hydroxylation or ring-opening reactions, further diversifying saponin derivatives. Certain fungi (e.g., Aspergillus, Rhizopus) and bacteria (e.g., Bacillus, Streptomyces) can selectively perform deglycosylation of tea saponins into their metabolites with improved pharmacological properties [22]. Furthermore, microbial glycosyltransferases may introduce new sugar units, altering solubility and bioavailability [23-24] [page no. 1, para 2, line 3].

We have also cited references in the paragraph as mentioned by the esteemed reviewers, [page no 1, para 2, line 5, ref [25-27]. Also, the following paragraph has been corrected as recommended.

Comment 2:

The methodology

It is weak in the “2.4. Biotransformation section”. The experiments are not considered to be triplicate.

Table 1. Soybean content is not reported

Response 2: We appreciate the reviewer’s attention to methodological rigor. While the initial screening experiments were performed as single runs due to the large number of strains tested (n=30), we confirmed the reproducibility of key results (e.g., catalytic activity of B. subtilis ATCC 6633, B. megaterium CGMCC 1.1741, and S. griseus ATCC 13273) through repeated TLC analyses of the same biotransformation extracts, which showed consistent band patterns. Parallel experiments were established: The experiments were carried out with a two-stage fermentation period. Culture controls included medium with no substrates, while substrate controls involved sterile medium with no microorganism. Also, during the initial screening of 30 microbial strains, three concentrations of camelliagenin B (5, 9, and 10 mg/mL) were tested to evaluate substrate tolerance and conversion efficiency. Among these, only B. subtilis ATCC 6633, B. megaterium CGMCC 1.1741, and S. griseus ATCC 13273 demonstrated significant biotransformation activity at the highest concentration (10 mg/mL), suggesting their superior catalytic capacity under high-substrate conditions; therefore, 10 mg/mL concentration was selected for fermentation.

Revised text: Among 30 microbial strains screened, only B. subtilis ATCC 6633, B. megaterium CGMCC 1.1741, and S. griseus ATCC 13273 strains showed significant catalytic activity towards the substrate, grown in potato dextrose (PD) and soybean meal (SM) medium in a two-stage fermentation protocol. During the stage I fermentation period, the medium was divided into 250 mL round-bottom flasks. Each flask was split into 50 mL, sterilized at 121 °C for 20 minutes, and then incubated at 180 rpm/min at 28 °C for 24 hours. The seed solution (1.6 mL) from the 24-hour stage I culture was transferred into fresh medium to initiate stage II fermentation. The mixture was then incubated under the same conditions, followed by the addition of 1 mL camelliagenin B dissolved in ethanol as a substrate separately into the stage-II culture medium. Control groups were prepared as substrate-free culture (microorganisms without camelliagenin B in a sterile culture medium) and strain-free culture (camelliagenin B without microorganisms in a sterile culture medium).  The cultures were further incubated for 4 days, respectively, and then extracted three times with ethyl acetate. The organic layer was filtered and concentrated under reduced pressure using a rotary evaporator. The sample was spotted on a silica gel thin-layer chromatography (TLC) plate. It was further analyzed by HPTLC (20.0 g, 15 mm × 400 mm), employing a stepwise gradient of dichloromethane/methanol (CH₂Cl₂/MeOH) from 100:1 to 5:1 as the elution system. Subsequent separation was conducted using preparative RP-HPLC with a mobile phase comprising 60%-70% CH₃CN in water. The soybean content is now mentioned in the revised manuscript to enhance clarity for readers. [page no 4, section 2.4].

Comment 3:

In the results sections

It is mentioned that “Compared with the substrate dC 81.58, dC 71.72 (C-3) displacement moved to the low field.” Which substrate is mentioned? In general, the substrate used to develop the strains does not impact the metabolic characteristics of the strains; two culture media were proposed, and the effect of the bioconversion of camelliagenin was not mentioned. Is a significant effect on strain selection

The results are descriptive of proposal structures, but the possible metabolic pathway that produces those changes in the compound is not mentioned.

There is no information about the reproducibility of the results.

Response 3: We acknowledge the reviewer’s observation. While PD (potato dextrose) and SM (soybean meal) media were selected based on their established use for microbial growth and secondary metabolite production previously in our lab, our primary goal was to identify strains capable of transforming camelliagenin B, irrespective of medium-specific effects. Notably, all three active strains (B. subtilis, B. megaterium, S. griseus) exhibited consistent biotransformation profiles across both media (TLC validation), suggesting that the catalytic potential was strain-dependent rather than medium-driven. Future studies will explicitly compare the effects of media on yield and pathway regulation.

We agree that pathway clarification strengthens mechanistic insights. The possible metabolic pathway that produces changes in the compounds is discussed in the results and discussion section, with references added in the revised manuscript and marked in red [page no. 11]. While the initial screening experiments were performed as single runs due to the large number of strains tested (n=30), we confirmed the reproducibility of key results (e.g., catalytic activity of B. subtilis ATCC 6633, B. megaterium CGMCC 1.1741, and S. griseus ATCC 13273) through repeated TLC analyses of the same biotransformation extracts, which showed consistent band patterns. The reproducibility of the hydrolysis optimization results was systematically ensured through multiple experimental approaches; initial small-scale optimization was conducted using 5 g of tea saponin under varied HCl and time conditions. The optimized conditions were then validated by scaling up to 50 g of tea saponins (2.5 mol/L HCl, 5 h). Finally, a full-scale up was tested utilizing 750 g of tea saponin under the optimized conditions.    

 Comment 4:

In the discussion part
It is a general discussion that highlights the complexity of microorganisms and their enzymes. It does not include support for the microorganism's bioconversion capability, as described by metabolism, or some reports related to the theme.

The conclusion section discusses some results, and it is recommended that it be concise.

Response 4: We appreciate the reviewer’s insightful critique. As recommended, we have clarified the metabolic basis of our biotransformation results, supported by strain-specific enzymatic evidence and literature cited in the revised manuscript, marked/highlighted in red. [page no 11, section 4, para 2, line 7]. As advised by the respected reviewers, we have revised the conclusion section to be concise.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript lacks a clearly stated objective. It is recommended to explicitly include a concise aim at the end of the introduction to clarify the purpose and direction of the research.

 

Heavy and sometimes repetitive writing. It makes the work difficult to reproduce.

 

No mention is made of substrate-free cultures (strains without camelliagenin) or substrate-free cultures without microorganisms. This is essential to validate that the products are derived from biotransformation.

 

“Different times and concentrations of HCl were evaluated” Replicates and basic statistical analysis (e.g., ANOVA or comparison of means) were required to justify which condition was "optimal." At this stage, statistical analysis would help objectively validate the most efficient conditions for releasing camelliagenin B.

 

Lack of functional evaluation: Although a number of bioactive compounds are isolated, no activity (anti-inflammatory, antioxidant, etc.) is demonstrated.

 

Section 3 is titled “Results and Discussion.” But then they include a section 4 titled “Discussion,” which creates confusion.

Section 3 describes the results along with interpretations, as if it were already the complete discussion. In section 4, they repeat information, re-explain pathways, and again discuss the contribution of microbial strains. If you choose the “Results and Discussion” format together, there should not be a subsequent “Discussion” section.And if you are going to separate the two, then section 3 should be called just “Results,” and the analysis should go in section 4.

 

The discussion would benefit from a more in-depth interpretation of the structure–function relationships and how the identified modifications may influence pharmacological activity, bioavailability, or metabolic stability.

 

A comparative discussion between microbial and chemical transformation methods would strengthen the rationale for using biocatalysis.

Author Response

Dear Reviewer,

Thank you very much for taking the time to review this manuscript. We acknowledge and appreciate your invaluable suggestions. Hopefully, our manuscript will be eligible for acceptance after incorporating the comments from the respected reviewers.  Please find the detailed responses below and the corresponding revisions/corrections highlighted in red in the resubmitted files.  

Comment 1: The manuscript lacks a clearly stated objective. It is recommended to explicitly include a concise aim at the end of the introduction to clarify the purpose and direction of the research. Heavy and sometimes repetitive writing. It makes the work difficult to reproduce. No mention is made of substrate-free cultures (strains without camelliagenin) or substrate-free cultures without microorganisms. This is essential to validate that the products are derived from biotransformation.

Response 1: We acknowledge the reviewer’s observation, and we agree to your insightful suggestion. We apologize for the mistake of not mentioning the control groups. As recommended, we have mentioned a concise aim at the end of the introduction to clarify the purpose and direction of the research.

Revised text: Therefore, this study explores the microbial biotransformation of Camelliagenin B obtained from Camellia oleifera seed cake meal, an underutilized industrial by-product. Using three microbial strains, Bacillus subtilis ATCC 6633, Bacillus megaterium CGMCC 1.1741, and Streptomyces gresius ATCC 13273, the study investigates multiple catalytic reactions and structural modifications of tea saponins with enhanced pharmacological properties. Through NMR-based structural analysis, ten novel derivatives were identified, demonstrating the potential of microbial fermentation as an alternative, sustainable, and efficient strategy to the conventional chemical synthesis method. This research work not only contributes to the valorization of agricultural waste but also advances the discovery of bioactive compounds with therapeutic applications. Also, this initiative reflects our ongoing efforts on the biotransformation of saponins in oil crops  [page no 2, introduction].

 Also we have mentioned “substrate-free culture (microorganisms without camelliagenin B in a sterile culture medium) and strain-free culture (camelliagenin B without microorganisms in a sterile culture medium)” in the revised manuscript  [page no 4, section 2.4, line 4].

Comment 2: “Different times and concentrations of HCl were evaluated” Replicates and basic statistical analysis (e.g., ANOVA or comparison of means) were required to justify which condition was "optimal." At this stage, statistical analysis would help objectively validate the most efficient conditions for releasing camelliagenin B.

Response 2: We are grateful for the reviewer’s insightful suggestion. In this study, we first tested different reaction times and HCl concentrations to identify general trends in yield, like seeing that 2.5 mol/L HCl and 5.5 hours gave higher amounts of product. However, we did not repeat each test multiple times or analyze the data statistically. In future work, we plan to repeat the top-performing conditions several times and use statistical tests to justify that the optimal conditions. For now, the current dataset was only analyzed descriptively based on yield comparisons across conditions. 

 Comment 3: Lack of functional evaluation: Although a number of bioactive compounds are isolated, no activity (anti-inflammatory, antioxidant, etc.) is demonstrated.

Response 3: We appreciate the reviewer’s important observation regarding functional evaluation. Tea saponins are pentacyclic triterpenoids (PTs), known to exhibit broad bioactivity across various models. Their mechanisms often differ markedly from those of other natural products due to their unique structural and target-binding properties. Our aim is to identify potential HMGB1 inhibitors; therefore, we did not conduct the regular bioassay. However, our future work on PTs focuses on evaluating the interaction mechanisms of PTs and their derivatives with HMGB1, utilizing in-silico analysis.

Comment 4: Section 3 is titled “Results and Discussion.” But then they include a section 4 titled “Discussion,” which creates confusion.

Section 3 describes the results along with interpretations, as if it were already the complete discussion.

In section 4, they repeat information, re-explain pathways, and again discuss the contribution of microbial strains. If you choose the “Results and Discussion” format together, there should not be a subsequent “Discussion” section. And if you are going to separate the two, then section 3 should be called simply “Results,” and the analysis should be placed in Section 4.

Response 4: We sincerely appreciate the reviewer’s advice, and we apologize for causing any confusion. As recommended, we have combined the results and discussion sections to improve the clarity. 

Comment 5: The discussion would benefit from a more in-depth interpretation of the structure–function relationships and how the identified modifications may influence pharmacological activity, bioavailability, or metabolic stability.

Response 5: We acknowledge your constructive suggestion and agree with it. As recommended, we have included an in-depth interpretation in the discussion section.

Comment 6: A comparative discussion between microbial and chemical transformation methods would strengthen the rationale for using biocatalysis.

Response 6: We are grateful for the esteemed reviewer’s invaluable suggestions. As suggested, we have included a comparison between microbial and chemical transformation methods, highlighting how microbial biotransformation is an advantageous and efficient method for producing more bioavailable compounds.

Revised text: Overall, from a broader perspective, these findings highlight several key advantages of microbial biotransformation over conventional chemical synthesis. The process exhibits exceptionally high regioselectivity, strong stereoselectivity, and a range of reaction types, particularly in handling complex natural compounds with multiple reactive sites [63]. Compared to chemical methods, microbial biotransformation offers a more environmentally friendly approach, minimizing issues of molecular rearrangements and isomerization [64,65] [page no 12, section 4, para 3].

 

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors heeded the recommendations for improving their article

Author Response

Dear Reviewer, 

We appreciate your valuable comments and suggestions.

Comments and Suggestions for Authors

The authors heeded the recommendations for improving their article

[Response]: We sincerely appreciate the time and effort you have dedicated to reviewing our manuscript, as well as your valuable comments and suggestions for improvement. We have carefully considered all the feedback and have revised the manuscript accordingly. Thank you