Bioproduction of Nordihydroguaiaretic and Ellagic Acid from Creosote Bush Leaves (Larrea tridentata) Using Solid-State Fermentation with Aspergillus niger GH1
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsManuscript ID: fermentation-3569446
Title: Bioproduction of Nordihydroguaiaretic and Ellagic Acid from Creosote Bush Leaves (Larrea tridentata) Using Solid-State Fermentation with Aspergillus niger GH1
The authors studied the solid-state fermentation (SSF) of Larrea tridentata biomass using the fungal species Aspergillus niger GH1. The effect of pH and MgSO4 on the production of nordihydroguaiaretic acid and ellagic acid was studied and optimised by a chemometric approach based on the Box-Behnken design. The fermentation extracts were analysed by different techniques, such as HPLC-MS and in vitro antioxidant assays (DPPH and ABTS).
The paper is easy to read, the results are quite interesting and the topic is appropriate for the journal Fermentation. However, the novelty of the study should be better emphasised and the structure of the paper should be improved, as well as the description of some of the methods and the discussion of the results. Therefore, the manuscript needs a major revision before it can be accepted for publication. As follows:
Major comments:
- Authors must add line numbering to facilitate the review process.
- Introduction section – lines: “SSF offers sustainable and cost-effective bioprocess, enabling the direct utilization of undervalued plants and/or agro-industrial wastes to produce high-value compounds [10].”. The authors emphasised the advantages of solid-state fermentation; however, for a critical discussion of the state of the art in the Introduction section, the main limitations of this type of fermentation should be briefly described.
- The introduction section should be significantly improved. The convergence of environmental sustainability and resource valorization has catalyzed substantial research interest in biological processes that effectively harness agricultural residues. This is a fundamental aspect that should be better emphasised by the authors in order to make the concept of the technological application of this type of study for the conversion of biomass in modern biorefinery models more robust. From this point of view, the authors should mention the following relevant and recent studies on the complete and selective valorisation of all biomass components of residual biomasses based on the use of hydrolytic enzymes: https://doi.org/10.1016/j.cattod.2024.114941, https://doi.org/10.1002/cplu.202200189.
- In the Introduction section, a focus on the chemistry of lignocellulosic biomasses should be added. In particular, the recalcitrant nature of lignocellulosic biomass presents crucial challenges for bioconversion processes, primarily due to the complex architectural arrangement of its three principal components, namely cellulose (35-55%), hemicellulose (15-35%), and lignin (15-30%), creating a naturally resistant matrix that has evolved to withstand biological degradation. In modern biorefinery schemes, a sustainable and efficient pretreatment step is often combined with enzymatic hydrolysis of cellulose and hemicellulose based on the use of fungal enzymes. This aspect should be discussed by the authors, and the following relevant and recent work on the strategic combination of different fractionation technologies and enzymatic hydrolysis should be reported by the authors: https://doi.org/10.1021/acssuschemeng.2c06356.
- Introduction section – lines: “This study aimed to evaluate the SSF creosote bush leaves to produce NDGA and EA, and to assess the antioxidant activity of the resulting extracts.”. The authors should better highlight the innovative aspects of the present study and the advancement of knowledge with respect to results previously reported in the scientific literature.
- Section 2.2. –
- Lines: “By combining the WAC values with the moisture and dry matter content, the maximum moisture content of the material was calculated [25].” – Authors should add the equations used for calculating different parameters.
- Lines “…concentrated H2SO4 for 3 h, the samples were filtered and transferred to a 50 mL volumetric flask…” – Authors should specify the concentration of the acid and the material of the filter used in the subsequent filtration step.
- Lines “The lipid determination was carried out by gravimetry using the Soxhlet method [27],…” – Add a brief and clear description of the method. The use of alcohol in the Soxhlet method does not ensure the selective extraction of lipids but also other organic extractives, such as phenols, pigments, etc. How exactly do the authors quantify the extracted lipids?
- From a chemical point of view, what did the authors consider as a fiber content? A brief and detailed description of the method and equations used for their quantification must be reported to ensure reproducibility.
- How was the quantification of the starting total amount of bioactive compounds performed by the authors? The method and equations should be added to the manuscript.
- Section 2.4 –
- Lines: “3 g of plant material was placed in sterile Petri dishes and 7 mL of culture medium with inoculum…”. The chemical composition of the culture medium must be added.
- In the Box-Behnken experimental design, no reproducibility tests were considered. Usually, the center point (combination 0, 0) is replicated at least 3 times. Authors must improve the DoE accordingly.
- Table 2 –
- The sum of fat, fiber, protein, ash and total sugars is far from completing the mass balance of the initial dry feedstock. What are the remaining fractions of the dry biomass from a chemical point of view? The authors need to improve the chemical characterisation of the starting material.
- What is the content of water and ethanol extractives of creosote bush leaves? It is fundamental to quantify this fraction in order to calculate the extraction efficiency of the proposed biological method.
- Section 3.2 –
- Why did the authors select only pH and MgSO4 as independent variables in the Design of Experiments approach? Other important parameters can affect the performance of an SSF, such as temperature, moisture content, particle size and distribution, mixing mode and frequency, other inorganic salts, etc. The authors must discuss this choice before the sentence reported in the discussion section “Coronado-Contreras (2020) [44], in prior study conducted in our group, established optimal conditions for NDGA production via SSF employing A. niger GH1 and utilizing creosote bush leaves as support, this study identified pH and MgSOâ‚„ concentration as key factors influencing NDGA accumulation (6-12 mg/g NDGA) through Plackett-Burman ex-perimental design.”.
- Why did the authors select 0.26, 0.76 and 1.26 g/L as values of the DoE?
- Section 4 (Discussion) –
- The discussion of the experimental results reported in Figures 1 and 2 needs to be better discussed by the authors. For example, a comment for each run should be added as well as a critical discussion of some comparison, especially with respect to the control test.
- What is the role of Aspergillus niger GH1 in the extraction of nordihydroguaiaretic acid and ellagic acid from creosote bush leaves? Just a destructuration of the lignocellulosic matrix to facilitate the extraction of NDGA and EA by solvent extraction with ethanol/water (50:50 v/v)? Authors should discuss all these aspects in depth.
- Lines: “Coronado-Contreras (2020) [44], in prior study conducted in our group, established optimal conditions for NDGA production via SSF employing A. niger GH1 and utilizing creosote bush leaves as support, this study identified pH and MgSOâ‚„ concentration as key factors influencing NDGA accumulation (6-12 mg/g NDGA) through Plackett-Burman experimental design. The present study employed a Box-Behnken design with the same variables, with the objective of process optimization, and achieved a maximum of 7.39 mg/g NDGA.” – The authors reported that in their previous study on the same biomass and fungus they obtained a maximum NDGA production of 12 mg/g, while in the present study they obtained a maximum value of 7.39 mg/g. So, what are the innovative aspects and improvements in knowledge and technical progress of this study compared to the state of the art?
- What is the chemical composition and fate of the solid substrate at the end of the SSF? Can it be further valorised by biotechnological approaches?
Minor comments:
- Abstract – lines: “This study investigated the solid-state fermentation of creosote bush (Larrea tridentata) leaves using Aspergillus niger GH1 to enhance the extraction of nordihydroguaiaretic acid and ellagic acid.” - Add acronyms of nordihydroguaiaretic acid and ellagic acid the first time the two molecules are mentioned.
- Introduction section – lines: “These attributes position creosote bush as a valuable source for applications in various industries, particularly in the development of functional ingredients and therapeutic agents.”. From the industrial scale-up perspective, this biomass’s productivity (in terms of tons/hectare/year) should be added together with the typical extractives content expressed as wt% on a dry basis.
- Introduction section - The authors may add the chemical structure of the two molecules of interest in the present study, such as nordihydroguaiaretic acid and ellagic acid.
- Section 2.1 - Authors should add the geographical coordinates of the field where the harvest took place.
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 1 Comments |
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Summary |
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Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.
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Point-by-point response to Comments and Suggestions for Authors |
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Major comments Comments 1: [Authors must add line numbering to facilitate the review process.] |
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Response 1: Line numbers were added to the manuscript.
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Comments 2: [Introduction section – lines: “SSF offers sustainable and cost-effective bioprocess, enabling the direct utilization of undervalued plants and/or agro-industrial wastes to produce high-value compounds [10].”. The authors emphasised the advantages of solid-state fermentation; however, for a critical discussion of the state of the art in the Introduction section, the main limitations of this type of fermentation should be briefly described.] |
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Response 2: The main disadvantages of the SSF process were added in lines 60-63. Although the SSF process offers several advantages, it also presents significant challenges, the main ones being the scaling and control of operational parameters such as pH, temperature and oxygen gradients within the bioreactor.
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Comments 3: [The introduction section should be significantly improved. The convergence of environmental sustainability and resource valorization has catalyzed substantial research interest in biological processes that effectively harness agricultural residues. This is a fundamental aspect that should be better emphasised by the authors in order to make the concept of the technological application of this type of study for the conversion of biomass in modern biorefinery models more robust. From this point of view, the authors should mention the following relevant and recent studies on the complete and selective valorisation of all biomass components of residual biomasses based on the use of hydrolytic enzymes: https://doi.org/10.1016/j.cattod.2024.114941, https://doi.org/10.1002/cplu.202200189.] Response 3: We agree with your comment. Accordingly, we have addressed this point by considering the literature proposed by the reviewer. A short text has been added on line 67-73 highlighting the current trend of moving to a circular economy system in which undervalued or underutilized lignocellulosic materials represent an opportunity to obtain products with potential applications. The global imperative is to shift from a linear, fossil resource-dependent economy to a circular model valorizing low-value lignocellulosic waste. Annually, 181.5 billion tons of lignocellulosic biomass are produced globally; however, only 8.2 billion tons are currently utilized, representing a substantial untapped resource for diverse applications, such as the production of valuable compounds through enzymatic processes [13,14].
Comments 4: [In the Introduction section, a focus on the chemistry of lignocellulosic biomasses should be added. In particular, the recalcitrant nature of lignocellulosic biomass presents crucial challenges for bioconversion processes, primarily due to the complex architectural arrangement of its three principal components, namely cellulose (35-55%), hemicellulose (15-35%), and lignin (15-30%), creating a naturally resistant matrix that has evolved to withstand biological degradation. In modern biorefinery schemes, a sustainable and efficient pretreatment step is often combined with enzymatic hydrolysis of cellulose and hemicellulose based on the use of fungal enzymes. This aspect should be discussed by the authors, and the following relevant and recent work on the strategic combination of different fractionation technologies and enzymatic hydrolysis should be reported by the authors: https://doi.org/10.1021/acssuschemeng.2c06356.] Response 4: We have attended this comment. A new paragraph on line 73-80 highlights the relationship between lignocellulosic conformation, pretreatments, and enhanced hydrolysis efficiency. Plant biomass, formed by complex polysaccharides, presents a challenge for microbial enzymatic degradation. To improve enzymatic hydrolysis, pretreatments of the plant material are frequently used, including physical methods such as particle size reduction (milling), ultrasound, and microwaves, alongside chemical treatments. These pretreatments aid in disrupting the plant cell wall, thus enhancing hydrolysis efficiency. However, careful selection of the pretreatment method is essential to avoid the formation of non-desirable by-products during lignocellulosic biomass processing [15].
Comments 5: [Introduction section – lines: “This study aimed to evaluate the SSF creosote bush leaves to produce NDGA and EA, and to assess the antioxidant activity of the resulting extracts.”. The authors should better highlight the innovative aspects of the present study and the advancement of knowledge with respect to results previously reported in the scientific literature.] Response 5: In the introduction section on line 96-101 the authors added innovative aspects of the study. This study aimed at the implementation of a SSF optimization process employing creosote bush leaves as a substrate to produce NDGA and EA, building on previous research by our group that identified key parameters for NDGA production in a related SSF system. This approach is particularly relevant considering the limited recent literature concerning the application of SSF to creosote bush biomass.
Comments 6 [Section 2.2] Response 6: Section 2.2 was revised.
Comments 7: [Lines: “By combining the WAC values with the moisture and dry matter content, the maximum moisture content of the material was calculated [25].” – Authors should add the equations used for calculating different parameters.] Response 7: Agree. the equations corresponding to the WAC and moisture and dry matter calculation have been added on line the equations corresponding to the maximum humidity of the support have been added
Where Xs,3 is the maximum moisture content that the substrate can support (%), M1 is the mass of dry material (g), Xs1 is the percentage of total solids divided by 100 and M3 is the value of WAC
Comments 8: [Lines “…concentrated H2SO4 for 3 h, the samples were filtered and transferred to a 50 mL volumetric flask…” – Authors should specify the concentration of the acid, and the material of the filter used in the subsequent filtration step.] Response 8: The text was improved because it was somewhat confusing. When we refer to "concentrated Hâ‚‚SOâ‚„," we mean that we used the undiluted acid, that is, reagent grade at 96% purity. After 3 hours of sample hydrolysis with the concentrated acid, water was added to dilute it slightly and allow the liquid to be filtered with Whatman filter paper without the risk of it being destroyed. Once the liquid samples were filtered, they were transferred to a 50 mL volumetric flask, and the volume was brought up to 50 mL with distilled water, here the improved text: To determine the total sugars, the samples were previously hydrolyzed; 10 mg of each sample was hydrolyzed with 2 mL of concentrated (96% v/v) H2SO4 for 3 h, Following hydrolysis, the resulting liquid was diluted with distilled water, filtered through Whatman No. 1 paper, and the filtrate was adjusted to a final volume of 50 mL with distilled water in a volumetric flask.
Comments 9: [Lines “The lipid determination was carried out by gravimetry using the Soxhlet method [27]…” – Add a brief and clear description of the method. The use of alcohol in the Soxhlet method does not ensure the selective extraction of lipids but also other organic extractives, such as phenols, pigments, etc. How exactly do the authors quantify the extracted lipids?] Response 9: In line 143-148 We have added a brief description of the Soxhlet method used for the determination of lipids in the sample. We did not use alcohols; we used one of the most common solvents for extracting lipids with the Soxhlet technique, which was hexane. Being a non-polar solvent, it has an affinity for lipids, fats, oils, etc., present in the sample. While hexane may co-extract some non-lipidic compounds (e.g., certain phenols), its primary selectivity for lipids makes it a common and effective choice for this application. Here the text added and the equation that explain how we calculated the lipids%: Briefly, 50 mL of hexane was used as solvent, 4 g of sample were deposited on dry Whatman no 1 filter papers at constant weight, lipid extraction was carried out for 6 hours with an Soxhlet equipment. After extraction and recovery of the solvent by distillation, the residue was dried at 40 °C for 48 hours. The lipid content was determined by gravimetric analysis comparing the weight of the filter paper before and after the process according to the following equation:
where Wp1 represents the weight of dry Whatman filter paper before Soxhlet extraction, Smpl represents the weight of the dry sample, Wp2 represents the weight of the Whatman filter paper after the Soxhlet extraction.
Comments 10: [From a chemical point of view, what did the authors consider as a fiber content? A brief and detailed description of the method and equations used for their quantification must be reported to ensure reproducibility.] Response 10: We consider as fiber all the biomass obtained after acid and alkaline hydrolysis called crude fiber, i.e. polysaccharides resistant to hydrolysis such as cellulose, hemicellulose, lignin, etc. in line 153 A brief description of the method, as well as the equation used to report the fiber content, was added. Briefly, a 0.5 g defatted sample obtained in the lipid determination step was sequentially hydrolyzed in 200 mL beakers with 1.25% (v/v) Hâ‚‚SOâ‚„ and 1.25% (v/v) NaOH at 100°C for 30 min each. After each hydrolysis, the residue was filtered through dry Whatman No. 1 filter paper at constant weight and rinsed twice with hot distilled water. The final residue was dried at 40°C for 48 h, and fiber content was determined gravimetrically using the following equation:
where Wf represents the final weight (g) of dry Whatman filter paper with sample af-ter the determination, Wi represents the initial weight (g) of the dry Whatman filter paper at constant weight, Sw represents the weight (g) of the sample used.
Comments 11: [How was the quantification of the starting total amount of bioactive compounds performed by the authors? The method and equations should be added to the manuscript.] Response 11: Attended the information was added on line 206 of page 5. The quantification of NDGA and EA was performed using a calibration curve (0-1000 ppm) for both standards.
Comments 12: [Section 2.4 –] Response 12: Section 2.2 was revised.
Comments 13: [Lines: “3 g of plant material was placed in sterile Petri dishes and 7 mL of culture medium with inoculum…”. The chemical composition of the culture medium must be added.] Response 13: Comment addressed a description of the culture medium was added. The culture medium consisted of water and MgSOâ‚„ for all treatments; only the concentration of MgSOâ‚„ and the pH values varied as showed in table 1: (distilled water, MgSO4 concentrations and pH values varied according to treatment as shown in Table 1).
Comments 14: [In the Box-Behnken experimental design, no reproducibility tests were considered. Usually, the center point (combination 0, 0) is replicated at least 3 times. Authors must improve the DoE accordingly.] Response 14: It is important to clarify that Table 1 shows the condensed matrix. In line 181 it was clarified that the treatments of the Box-Behnken experimental design were carried out in triplicate: All treatments were performed in triplicate. Consequently, the central point was made at least three times. Comments 15: [Table 2 –] Response 15: Table 2 was revised.
Comments 16: [The sum of fat, fiber, protein, ash and total sugars is far from completing the mass balance of the initial dry feedstock. What are the remaining fractions of the dry biomass from a chemical point of view? The authors need to improve the chemical characterisation of the starting material.] Response 16: While it is true that the sum of the parameters is far from 100% by about 30%, that 30% could be compounds that are not detected by the analyses performed, and proximate analyses are generally always far from 100%. We will be working hard to improve our chemical analysis.
Comments 17: [What is the content of water and ethanol extractives of creosote bush leaves? It is fundamental to quantify this fraction in order to calculate the extraction efficiency of the proposed biological method.] Response 17: At least according to what has been reported in the literature, Creosote Bush leaves do not contain ethanol. Unfortunately, we do not have the reagents or equipment to measure water.
Comments 18: [Section 3.2 –] Response 18: Section 3.2 was revised.
Comments 19: [Why did the authors select only pH and MgSO4 as independent variables in the Design of Experiments approach? Other important parameters can affect the performance of an SSF, such as temperature, moisture content, particle size and distribution, mixing mode and frequency, other inorganic salts, etc. The authors must discuss this choice before the sentence reported in the discussion section “Coronado-Contreras (2020) [44], in prior study conducted in our group, established optimal conditions for NDGA production via SSF employing A. niger GH1 and utilizing creosote bush leaves as support, this study identified pH and MgSOâ‚„ concentration as key factors influencing NDGA accumulation (6-12 mg/g NDGA) through Plackett-Burman ex-perimental design.”.] Response 19: In lines 276-287, it is mentioned why pH and MgSO4 were selected as independent variables in the experimental design. The selection of pH and MgSO4 concentration as independent variables for NDGA accumulation was based on a previous study conducted by Coronado-Contreras (2020) [38] within our research group. That study evaluated NDGA production by SSF using creosote bush leaves and employed a Plackett-Burman experimental design to identify factors influencing NDGA accumulation. The factors evaluated were temperature, pH, humidity, inoculum (spores/g), and the concentrations of NaNO3 (g/L), MgSO4 (g/L), and KCl (g/L). The results indicated that only the concentration of MgSO4 and pH had a significant influence on NDGA accumulation. Therefore, the present study focused on evaluating only the concentration of MgSO4 and pH as independent variables to optimize the process using a Box-Behnken experimental design. The other factors (NaNO3 (6 g/L), KCl (0.26 g/L), temperature (30 °C), humidity (70%), inoculum (1x106 spores/g) were kept constant based on the previous findings.
Comments 20: [Why did the authors select 0.26, 0.76 and 1.26 g/L as values of the DoE?] Response 20: These values were selected based on previous work by Coronado-Contreras, 2020 (results in the process of publication). In this previous work, the best value for magnesium sulfate was 0.76. For this reason, in the present study, it was decided to evaluate a level above and below the aforementioned value.
Comments 21: [Section 4 (Discussion) –] Response 21: This section was revised.
Comments 22: [The discussion of the experimental results reported in Figures 1 and 2 needs to be better discussed by the authors. For example, a comment for each run should be added as well as a critical discussion of some comparison, especially with respect to the control test.] Response 22: On line 347 to 354 the discussion was improved. Box-Behnken design treatments significantly altered tannin release compared to controls, suggesting that the SSF process did influence the release of hydrolyzable and condensed tannins (Figure 3). Specifically, all treatments (1-9) exhibited elevated levels of hydrolyzable tannins, with treatment 8 demonstrating the highest accumulation of hydrolyzable tannins (36.17 ± 0.01 mg GAE/g). Similarly, many treatments, except for treatment 3, showed increased condensed tannin levels compared to controls. Notably, treatments 9 and 8 yielded the highest condensed tannin concentrations, measuring 27.07 ± 0.70 mg CE/g and 24.32 ± 1.06 mg CE/g, respectively.
On line 368 to 377 the discussion was improved. The observed variations in tannin accumulation across the different Box-Behnken de-sign treatments highlight the sensitivity of tannin release to the manipulated variables. treatment 8, characterized by a pH of 5.5, resulted in the highest levels of both hydrolyzable and elevated condensed tannins (treatment 8 condensed tannins were statistically similar to treatment 9). Conversely, treatment 3, conducted at a pH of 4.5, displayed minimal condensed tannin accumulation. This observation suggests a potential positive correlation between the pH of the fermentation medium and the release of condensed tannins (as visually represented in Figure 4). This aligns with the reported microbial resistance of condensed tannins [52], where higher pH may facilitate their release. Consequently, high pH conditions may favor the release of condensed tannins.
Comments 23: [What is the role of Aspergillus niger GH1 in the extraction of nordihydroguaiaretic acid and ellagic acid from creosote bush leaves? Just a destructuration of the lignocellulosic matrix to facilitate the extraction of NDGA and EA by solvent extraction with ethanol/water (50:50 v/v)? Authors should discuss all these aspects in depth.] Response 23: In line 355 to 367 was added a discussion of the role of A. niger GH1 in the SSF process The complex cell wall matrix of plant biomass, primarily composed of structural polysaccharides (cellulose, pectin) and proteins, can associate with various phenolic compounds, including tannins, phenolic acids, flavonoids, and lignans [47]. These interactions are known to impede the efficient extraction and release of such compounds. To address this limitation, the application of microbial enzymes, particularly those derived from filamentous fungi, has been explored to facilitate cell wall degradation and enhance the liberation of cell wall-bound compounds [48,49]. The genus Aspergillus spp. is widely employed in this context due to its capacity to produce a diverse array of hydrolytic enzymes capable of degrading various lignocellulosic cell wall components, such as cellulases, and xylanases, among others [50]. Specifically, A. niger GH1 has been reported to produce these hydrolytic enzymes and, notably, tannin-degrading enzymes like ellagitannase and tannase, which are considered key enzymes in the release of phenolic compounds of interest, such as ellagic acid and other phenolic compounds [51].
Comments 24: [Lines: “Coronado-Contreras (2020) [44], in prior study conducted in our group, established optimal conditions for NDGA production via SSF employing A. niger GH1 and utilizing creosote bush leaves as support, this study identified pH and MgSOâ‚„ concentration as key factors influencing NDGA accumulation (6-12 mg/g NDGA) through Plackett-Burman experimental design. The present study employed a Box-Behnken design with the same variables, with the objective of process optimization, and achieved a maximum of 7.39 mg/g NDGA.” – The authors reported that in their previous study on the same biomass and fungus they obtained a maximum NDGA production of 12 mg/g, while in the present study they obtained a maximum value of 7.39 mg/g. So, what are the innovative aspects and improvements in knowledge and technical progress of this study compared to the state of the art?] Response 24: Unfortunately, we were unable to optimize the process. This could be due to several factors: For example, the Creosote Bush leaves used in the previous study were not the same as those used in this study; the content of these compounds varies depending on the geographic area where they were collected, weather conditions, soil, and other factors. In addition, other factors that were not evaluated could influence the fermentation process, such as dipotassium phosphate and ferrous sulfate (essential salts for optimal growth of Aspergillus species). The working group has extensive experience in the analysis of these compounds. Sometimes, the quantities do not reflect significant biological activity. For now, the novelty of our work is demonstrating that the fermentation process is efficient and attractive for the release of these compounds compared to traditional methods. Furthermore, these compounds were shown to exhibit significant antioxidant activity. We will be working to demonstrate that these compounds have broad applications in the pharmaceutical, agricultural, and food industries.
Comments 25: [What is the chemical composition and fate of the solid substrate at the end of the SSF? Can it be further valorised by biotechnological approaches?] Response 25: We do not know the chemical composition of biomass. For the time being, the final disposal of the biomass is to produce organic fertilizer for plants. It is definitely an area of opportunity to employ biotechnology techniques and properly treat this biomass.
Minor comments Comments 1: [Abstract – lines: “This study investigated the solid-state fermentation of creosote bush (Larrea tridentata) leaves using Aspergillus niger GH1 to enhance the extraction of nordihydroguaiaretic acid and ellagic acid.” - Add acronyms of nordihydroguaiaretic acid and ellagic acid the first time the two molecules are mentioned.] Response 1: The summary was reviewed and corrected.
Comments 2: [Introduction section – lines: “These attributes position creosote bush as a valuable source for applications in various industries, particularly in the development of functional ingredients and therapeutic agents.”. From the industrial scale-up perspective, this biomass’s productivity (in terms of tons/hectare/year) should be added together with the typical extractives content expressed as wt% on a dry basis.] Response 2: In line 80- 83 a brief data of the distribution of creosote bush was added, and in line 88 of page was added the typical extractive content present in the leaves: The creosote bush, an undervalued perennial shrub native to the arid regions of North America, is distributed across approximately 19 Mha of the continent, specifically in central and northern Mexico and the southern United States [16], is recognized for its robust survival in harsh environments [17]. This resilience is attributed to its rich phytochemical profile, featuring bioactive compounds. Historically, creosote bush has been employed in folk medicine, reflecting its potent biological activities, including antioxidant, antimicrobial, and antiviral properties [18,19]. Furthermore, this extensive distribution, coupled with the fact that approximately 50% of its leaf dry weight is extractable matter, positions creosote bush as a valuable source for applications in various industries, particularly in the development of functional ingredients and therapeutic agents.
Comments 3: [Introduction section - The authors may add the chemical structure of the two molecules of interest in the present study, such as nordihydroguaiaretic acid and ellagic acid.] Response 3: The images corresponding to the structures of the ellagic acid and NDGA molecules have been added to the introduction section.
Figure 1. Ellagic acid structure.
Figure 2. Nordihydroguaiaretic acid structure. Comments 4: [Section 2.1 - Authors should add the geographical coordinates of the field where the harvest took place.] Response 4: Creosote bush was collected from Saltillo, Coahuila (25°33'18.3"N 100°55'34.7"W). The plant material was disinfected in a 10% (v/v) water-sodium hypochlorite (NaOCl) solution. Subsequently, it was dried at 50°C for 72 h. Leaves and flowers were manually removed and ground to a particle size less than 2 mm. The ground plant material was stored in plastic bags protected from light at room temperature. |
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have presented their research work in a MS entitled, "Bioproduction of Nordihydroguaiaretic and Ellagic Acid from Creosote Bush Leaves (Larrea tridentata) Using Solid-State Fermentation with Aspergillus niger GH1". The work looks good, but changes are needed before it is considered for publication in the Fermentation journal.
- In the abstract, give an opening/background statement.
- What is the novelty of this work? It must be reflected in the abstract and introduction sections.
- Fig.1: In the X-axis, what treatments are being referred here to? It should be added.
- The values in Table 2 should be consistently rounded. For example, some values have two decimal places (e.g., 5.11 ± 0.08%), while others have none (e.g., 5.0 ± 0.00%). Standardize the format.
- In Table 3, list compound families in a more structured format. The term "Methoxyflavonols" appears twice—ensure consistency with nomenclature.
- Rephrase "being NDGA the major compound" to "with NDGA being the major compound."
- In conclusion, the major result values are not mentioned. It should be added.
Author Response
Response to Reviewer 2 Comments |
Comments 1: [In the abstract, give an opening/background statement.] |
Response 1: Attended in the abstract section a short statement about creosote bush was added to give more information about creosote bush and why it is of interest for use in a solid-state fermentation process. Creosote bush (Larrea tridentata), a shrub distributed across approximately 19 Mha of arid North American regions, has traditional applications in folk medicine due to the presence of bioactive molecules such as nordihydroguaiaretic acid (NDGA) and ellagic acid (EA).
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Comments 2: [What is the novelty of this work? It must be reflected in the abstract and introduction sections.] |
Response 2: In the abstract section on line 15 the author added the novelty of the work: This study investigated the implementation of a solid-state fermentation (SSF) optimization process employing creosote bush leaves as substrate using Aspergillus niger GH1 to improve NDGA and EA extraction, this study was based on previous research by our group that identified key parameters for NDGA production in a related SSF system In the introduction section on line 96 the authors added innovative aspects of the study. This study aimed at the implementation of a SSF optimization process employing creosote bush leaves as a substrate to produce NDGA and EA, building on previous research by our group that identified key parameters for NDGA production in a related SSF system. This approach is particularly relevant considering the limited recent literature concerning the application of SSF to creosote bush biomass.
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Comments 3: [Fig.1: In the X-axis, what treatments are being referred here to? It should be added.] Response 3: On the X-axis the treatments refer to those shown in table 1 of the condensed experimental matrix with C being the control.
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Comments 4: [The values in Table 2 should be consistently rounded. For example, some values have two decimal places (e.g., 5.11 ± 0.08%), while others have none (e.g., 5.0 ± 0.00%). Standardize the format.] Response 4: Table 2 was modified.
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Comments 5: [In Table 3, list compound families in a more structured format. The term "Methoxyflavonols" appears twice—ensure consistency with nomenclature.] Response 5: The term methoxyflavonols appears twice because they are different compounds but belong to the same family and their retention time is different, so they are listed separately in table 3.
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Comments 6: [Rephrase "being NDGA the major compound" to "with NDGA being the major compound.”] Response 6: The sentence was modified.
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Comments 7: [In conclusion, the major result values are not mentioned. It should be added.] Response 7: The main results of the research were added in the conclusions section.: Creosote bush leaves proved to be viable for use as a support/substrate to perform a SSF due to physical tests demonstrating that they possess a WAC value of 3.62 ± 0.02 and support a maximum humidity above 70%, the microorganism A. niger GH1 was able to grow and invade the plant material. Although it was not possible to optimize the SSF process, the evaluated conditions allowed the extraction and characterization of a total of six-teen different compounds, highlighting the presence of NDGA and EA, which were quantified in treatment 8. The fermentation extracts of treatment 8 also exhibited significant in vitro antioxidant activity, with inhibition percentages of 55.69% in the DPPH assay and 84.84% in the ABTS assay. This demonstrates that the compounds present in the extracts possess biological activities that can be beneficial and used in various industries. To achieve optimal yields and further enhance the production of these valuable compounds, future research should focus on elucidating the specific parameters required for optimizing the SSF process using creosote bush leaves. |
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsManuscript ID: fermentation-3569446
Title: Bioproduction of Nordihydroguaiaretic and Ellagic Acid from Creosote Bush Leaves (Larrea tridentata) Using Solid-State Fermentation with Aspergillus niger GH1
The authors properly implemented the required revision. In the present form, the manuscript can be accepted for publication.
Comments for author File: Comments.pdf