Enhancement of Cellulase Production by Penicillium oxalicum Using Traditional Chinese Medicine Residue and Its Application in Flavonoid Extraction

Comments 1: Clarity on Substrate "Mixture": The title and introduction mention a "mixed traditional Chinese medicine residue substrate." However, the results section (2.2, 2.3, 2.4, 2.5) primarily focuses on three individual residues (SR, AR, FR) and then selects FR alone for optimization and application. This is contradictory.

Suggestion: Revise the title to accurately reflect the work. For example: "Enhancement of cellulase production by Penicillium oxalicum using traditional Chinese medicine residue and its application in flavonoid extraction." Alternatively, if a true mixture was used in some experiments, this must be clearly stated and justified in the methods and results.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the title to”Enhancement of cellulase production by Penicillium oxalicum using traditional Chinese medicine residue and its application in flavonoid extraction”

Comments 2: Statistical Rigor: The presentation of statistical analysis needs significant improvement. Error bars are mentioned in Table 1 and the Methods, but their meaning is unclear. Phrases like "insignificant difference" (line 6, pg. 6) are not sufficient.

Suggestion: For all quantitative results (e.g., enzyme activities, weight loss, flavonoid yields), clearly state the results as mean ± standard deviation (n=3). For key comparisons (e.g., optimized vs. non-optimized yield, enzyme-treated vs. control extraction), perform proper statistical tests (e.g., Student's t-test) and report p-values to objectively state significance. The ANOVA results for the RSM model should be included in the main text or as a supplementary table to validate the model's significance.

Response 2: Agree. We have, accordingly, revised the description to provide a more quantitative and statistically rigorous presentation of the data. The original phrase "insignificant difference" on line 6, page 6 has been modified to: "The predicted result (2.82 U/mL) and actual result (2.75 IU/mL) showed no significant difference (p > 0.05)." This change can be found in the revised manuscript on page 7, lines 204-205.

And, we have, accordingly, modified the captions for all relevant figures and tables to clearly indicate that the quantitative results are presented as Mean ± Standard Deviation (SD) from three independent replicates (n=3). The updated captions for Figure 2, Figure 4, Table 1 and Table 2 now include "(Mean ± SD, n=3)". These changes can be found on page 5, lines 148 and 150, page 6, line 191 and page 7, line 219, respectively.

We sincerely apologize for this oversight. Due to the long revision history and multiple submissions of this manuscript, the ANOVA results for the RSM model were inadvertently lost during data transfer between versions. To rectify this error and ensure the completeness and statistical validity of our work, we will re-conduct the key experiments necessary to regenerate the complete ANOVA data for the RSM model. We will incorporate these crucial results into the revised manuscript upon completion of the experiments.In the revised manuscript, to enhance time efficiency during the experimental redesign, we employed Central Composite Design (CCD) methods instead of the originally described Box-Behnken Design. The experimental design and corresponding results are presented in Table 2, with the ANOVA analysis detailed in Table 3. Furthermore, the contour plot and response surface plot have been provided as supplementary figures (please refer to Page 6-7, Lines 186-208).

Comments 3: The discussion would be strengthened by directly comparing your optimized cellulase activity (2.79 IU/mL) with yields reported in other studies using agricultural residues or Penicillium species. This contextualizes your achievement and highlights the efficiency of using TCMR.

Similarly, when discussing the 55.1% increase in flavonoid extraction, compare the absolute yield (8.91%) with yields from other studies extracting flavonoids from similar sources. Is this a high yield? This adds weight to your application claim.

In the Results and Discussion, residues are referred to as SR, AR, FR, and sometimes as “traditional Chinese medicine residues” or “TCMR.” For clarity, please standardize the terminology and define the abbreviations in the Abstract and first use in the main text, then use them consistently throughout.

Response 3: Agree. We have, accordingly, revised the manuscript to address these points. Firstly, in the discussion section, we have strengthened the comparison of the optimized cellulase activity (2.75 IU/mL) with yields reported in other studies using agricultural residues, which now more prominently highlights the efficiency of utilizing Traditional Chinese Medicine Residues (TCMRs). This change can be found on page 9, lines 277-283. Furthermore, following the reviewer's suggestion, we have reinforced the argumentation for the flavonoid extraction data to enhance its persuasiveness, as detailed on page 9, lines 288-298. Finally, to improve clarity and consistency, we have standardized the terminology in the abstract by defining the abbreviation for Traditional Chinese Medicine Residues (TCMRs) upon its first use and employing it consistently thereafter. All aforementioned modifications have been highlighted in red within the revised manuscript for easy identification.

Comments 4: Mechanistic Insight: The discussion on why FR performed best is somewhat speculative. The FTIR data shows changes, but the interpretation is vague.

Suggestion: Consider adding a sentence or two hypothesizing why FR might be a superior substrate. Does it have a more easily degradable cellulose/hemicellulose ratio? Lower lignin content? Or, as you allude to, do the specific flavonoids in Forsythia act as better induces or less potent antimicrobials? This deeper insight would be valuable.

Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript by adding citations to relevant references, which provide a mechanistic explanation for the superiority of Fermentation Residue (FR) as a substrate in the culture medium. This change can be found in the revised manuscript on page 8, lines 237-236.

Comments 5: Abstract:

The final sentence is very long. Consider breaking it into two for better readability.

The term "cellulase cocktail" is introduced in the abstract before being defined. Consider using "crude cellulase" or "the produced cellulase" for clarity to a broad audience.

Response 5: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript by splitting the final sentence of the abstract into two shorter sentences for improved clarity, which can be found on page 1, lines 23-26. Additionally, the term "cellulase cocktail" has been replaced with the more standard and precise term "crude cellulase" on page 1, line 22.

Comments 6: Introduction:

The introduction is comprehensive but quite long. Consider tightening some paragraphs to improve flow (e.g., the paragraph on TCMR treatment methods could be more concise).

Line 3, Pg 3: "Chymosin" is likely a typo for "Chitinase".

Response 6: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the paragraph concerning TCMR treatment methods in the introduction section to present the information in a more concise manner. The revised paragraph, which has been streamlined and no longer contains any mention of "Chitinase," can be found on pages 2 to 3, lines 90-103.

Comments 7: Results:

Figure 1: Label the parts (A, B, C) directly on the figure or in the caption for clarity.

Figure 2 & 4: The figures are referenced as (a), (b), (c) in the text but labeled as (A), (B), (C) in the caption. Please make consistent. Ensure axis labels are clear and units are present.  ？

Figure 3: The FTIR spectra are crowded and the labels ("SR before", "SR after" etc.) are difficult to read. Consider separating into individual graphs or improving the labeling. The caption for Figure 3-B is missing.

Table 1: A good addition. Please ensure the "n" value for the replicates is stated in the table footnote or caption.

Response 7: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the figures and table accordingly. Specifically, we have directly labeled subparts (A, B, C) on Figure 1, and ensured the labels (A, B, C) in Figures 2 and 4 are consistent with their in-text citations. Furthermore, Figure 3 has been split into two separate panels (3-A and 3-B) for clarity, with Figure 3-B enlarged to make its labels clearer, and a dedicated title for Figure 3-B has been added. Finally, we have confirmed that the number of replicates 'n' is now clearly stated in the caption of Table 1. These changes can be found on pages 4 to 6, lines 132, 147-149, 170-173, and on page 5, line 150, respectively.

Comments 8: Materials and Methods:

4.2:      Add drying temperature, and clearly state whether fermentation medium used a specific residue (SR/AR/FR) or a pre-mixed TCMR and, if so, at which steps.

4.3: The composition of the fermentation medium lists "TCMR 20 g". Was this always FR after the optimization study? Or was it a generic TCMR mix initially? Please clarify. 

4.3: (“Fermentation medium”) mentions that P. oxalicum spore suspension was inoculated, but the spore concentration (e.g., 10⁶ spores/mL) and the volume of inoculum added are not specified. Please add these details to ensure reproducibility.

4.6: The enzymatic hydrolysis step for flavonoid extraction says "at 55 °C for 90 min". However, the results in 2.5 show that AR required 120 min to reach its maximum. Please clarify the protocol – was it a fixed time for all, or was time also a variable? This needs to match the results section.

The manuscript reports cellulase activity in IU/mL, but the definition of the “International Unit” used is not explicitly given. Please provide the exact definition (e.g., “One unit of activity is defined as the amount of enzyme required to release 1 μmol of glucose equivalents per minute under assay conditions”) in the Materials & Methods section to avoid ambiguity.

Response 8: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript to provide greater methodological clarity. Specifically, we have clarified that the fermentation medium was prepared using specific herbal residues (SR/ AR/ FR) in sections 4.3, 4.4, 4.5, and 4.6, as indicated on page 10, lines 336-338. Furthermore, we have specified that following the optimization study, the term "TCMR" in the fermentation medium context refers specifically to FR, which is now stated on page 10, line 356. Additionally, the inoculation details have been explicitly provided, stating that 2 mL of a Pseudomonas oxalicum spore suspension with a concentration of 1.8×10⁶ spores/mL was used, as detailed on page 10, lines 345-353. Moreover, we have noted that both the enzymatic hydrolysis temperature and time for flavonoid extraction are variable parameters that can be optimized based on specific experimental conditions, which is explained on page 11, lines 381-384. Finally, the definition of the International Unit (IU) used throughout the study has been explicitly stated on page 10, lines 345-347 for consistency and clarity.

Comments 1: It is recommended to revise the bibliography and supplement it with more recent references on the topic of the article. It may be appropriate to exclude older publications on the value of enzymes, with all due respect to the authors of these references.

It is recommended to carefully proofread the text and reduce the percent match value from 28% to an acceptable level. 3. In the materials and methods, it is recommended to provide the component composition of the raw material to confirm that it contains cellulose, hemicelluloses, and lignin, which are described in Section 3.2.

It is recommended to exclude the word "dissolution" throughout the text when referring to cellulose. The word "hydrolysis" should be used.

When describing the SEM results, it is not recommended to indicate that the authors observed cellulose erosion, since unfractionated plant material was used as the sample.

It is recommended to swap the description of the IR spectrum and SEM.

It is recommended to provide more commentary on the difference in mass loss rates (Table 1).

It is a pity that the manuscript does not include data on the concentration of reducing substances in solutions after enzymatic hydrolysis

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have comprehensively revised the manuscript to enhance its quality and clarity. Firstly, without compromising the analytical framework, we have updated the literature by replacing outdated publications on enzyme value with recent references, except for those pertaining to experimental methods. Secondly, we have selectively proofread the highlighted sections of the manuscript while maintaining the overall structure. Furthermore, in the Materials and Methods section, we have cited new references to substantiate that the raw materials indeed contain cellulose, hemicellulose, and lignin, as specified on page 10, lines 336-338. Additionally, the term "dissolution" has been consistently replaced with "hydrolysis" throughout the text. Moreover, the descriptions of FTIR and SEM have been reordered, and the SEM results have been reinterpreted, as detailed on page 5, lines 152-169. We have also expanded the discussion on the differences in mass loss rates, which can be found on page 4, lines 144-146. Finally, in the discussion, we have emphasized the value of reducing sugar quantification for evaluating conversion efficiency and indicated its role as a key monitoring indicator in subsequent pilot-scale studies, as stated on page 9, lines 309-312.