Lovastatin Production by Wild Eurotium cristatum Isolated from Fuzhuan Brick Tea Produced Using Forest Resources in Auhua
Round 1
Reviewer 1 Report
The idea of enhancing the functional properties of dark tea, specifically by increasing the levels of a lipid-lowering molecule, such as lovastatin, is interesting. However, there is no consensus on the required levels of lovastatin to achieve this purpose, even if the literature is over 20 years old (Kinlay, 2002, Boccuzzi, 1998). Perhaps a reinforcement of the therapeutical strategy should be presented.
Another area that must be considered is the variability in lovastatin production both in strains and according to culture conditions. This phenomenon is also observed in Monascus or Aspergillus. These problems should be addressed in the discussion. Which would be the required level of lovastatin in the tea leaves to promote dark tea as a lipid-lowering food?
The references that support the health benefits of lovastatin could be shown before entering the production details to justify all the effort.
The taxonomic classification should be spelled correctly and include the taxonomic categories (P. 2, second paragraph).
p5, end of first paragraph: confusing. Why would the lovastatin producer strain grow faster and why would it suggest they could grow on tea leaves?
P. 6, before figure 4. The production curve is a typical secondary metabolite one. If you say no more lovastatin is produced due to nutrients depletion, you should show evidence.
You state that lovastatin production in Eurotium is low compared to Monascus or Aspergillus, but you do not show data. I find it similar to some reports.
Both the abstract and the first paragraphs of the introduction should be rewritten to clarify the purpose of the study.
For example: The tea plant forest in China represents an important economic source of income. The consumption of the different tea varieties may be enhanced by promoting and improving their health benefits. This can be achieved by introducing species like Eurotium cristatum, which occur naturally in tea fermentation, and are high producers of biologically active compounds, such as the lipid lowering compound lovastatin. Therefore, several strains........These results offer the means to give the dark tea producers a new marketing strategy to expand the scope of tea forests resources application.
In several paragraphs there is a problem with the concordance of singular names and the verbs. For example, p 2 line 3 Research shows instead of researches show,. At the end of the second paragraph antiobesity metabolites are mostly polysaccharides and polyphenols.
Be consistent on the units: rpm or r/min
There are several details, English should definitely be revised
Author Response
Response to Reviewer 1 Comments
Point 1: The idea of enhancing the functional properties of dark tea, specifically by increasing the levels of a lipid-lowering molecule, such as lovastatin, is interesting. However, there is no consensus on the required levels of lovastatin to achieve this purpose, even if the literature is over 20 years old (Kinlay, 2002, Boccuzzi, 1998). Perhaps a reinforcement of the therapeutical strategy should be presented.
Response 1: Thank you so much for your suggestion! We cite some references in the Introduction to explain the lipid-lowering effect of dark tea is related to many compounds, including lovastatin. Dark tea is one of the drinks with lipid-lowering effects. It can not replace commercial lipid-lowering drugs. One commercial lipid-lowering drugs usually contain 20 mg of lovastatin. As an adjuvant drink, drinking tea could help healthy humans control blood lipid levels, especially for humans with high-fat diets. These results could confirm by mouse or zebra fish model experiments.
Point 2: Another area that must be considered is the variability in lovastatin production both in strains and according to culture conditions. This phenomenon is also observed in Monascus or Aspergillus. These problems should be addressed in the discussion. Which would be the required level of lovastatin in the tea leaves to promote dark tea as a lipid-lowering food?
Response 2: Thank you so much for your suggestion! The lovastatin yields in Eurotium cristatum are related to the strain type and its fermentation parameters. In our study, 6 wild Eurotium cristatum strains were selected to produce lovastatin. The influences of fermentation parameters on lovastatin production by Eurotium cristatum H20 strain were studied in detail. And the highest lovastatin yield is 7.24 μg/mL in the fermentation broth. The lovastatin yields in wild Monascus range from 10 μg/mL to 500 μg/mL in the fermentation broth. Researchers found that in diet-induced obese animals, body weight gain and fat accumulation can be prevented by feeding them dark tea extracts containing lovastatin (always lower than 20 μg/g).
Point 3: The references that support the health benefits of lovastatin could be shown before entering the production details to justify all the effort.
Response 3: Thank you so much for your suggestion. The logical sequence of the Introduction has been modified. The details are as follows:
Lovastatin effectively reduces human cholesterol levels and has been widely used as a lipid-lowering drug [1, 2]. It could limit cholesterol synthesis by inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase [3-5]. Lovastatin, as a secondary metabolite, was produced by numerous fungal species [6-8]. The lovastatin was always isolated from Monascus and Aspergillus terreus because they could produce more lovastatin. Except for Monascus and Aspergillus terreus, Eurotium cristatum can also produce lovastatin.
Point 4: The taxonomic classification should be spelled correctly and include the taxonomic categories (P. 2, second paragraph).
Response 4: Thank you so much! The introduction of Eurotium cristatum has been modified. The details are as follows:
Eurotium cristatum is the dominant fungus involved in the Fermentation of Fuzhuan brick tea, which functions to lower blood lipid levels [9-12]. Based on its secreted amylase and oxidase, tea leaves could transform into many compounds that benefit human health, such as anti-obesity, anti-diabetic, gastrointestinal protective, and so on.
Point 5: p5, end of first paragraph: confusing. Why would the lovastatin producer strain grow faster and why would it suggest they could grow on tea leaves?
Response 5: Thank you so much! The grow rate of wild Eurotium cristatum strains could be obtained based on its length of colony diameter. The details are as follows:
The length of colony diameter of strains producing lovastatin was larger than other strains which could not produce lovastatin. This indicated that these Eurotium cristatum strains could grow faster and accumulate more lovastatin.
Point 6: P. 6, before figure 4. The production curve is a typical secondary metabolite one. If you say no more lovastatin is produced due to nutrients depletion, you should show evidence.
Response 6: Thank you so much! Figure 4 is a typical secondary metabolite production curve, similar to wild Monascus-producing lovastatin. We cite references to explain why the lovastatin concentration remained the same at the end of fermentation. The details are as follows:
After that time, the lovastatin concentration remained the same. These results indicated that lovastatin was no longer produced in the late fermentation process, maybe due to the consumption of nutrient substances in the fermentation broth, similar to Monascus producing lovastatin in the late fermentation process [34]. In the late fermentation process, they found that carbon and nitrogen sources concentration was reduced by more than 95%.
- Zhang, J.; Wang, Y.; Lu, L.; Zhang, B.; Xu, G. Enhanced production of Monacolin K by addition of precursors and surfactants in submerged fermentation of Monascus purpureus 9901. Biotechnol. Appl. Bioc. 2014, 61, 202-207.
Point 7: You state that lovastatin production in Eurotium is low compared to Monascus or Aspergillus, but you do not show data. I find it similar to some reports.
Response 7: Thank you so much! The date and references have be added. The details are as follows:
Compared with wild Monascus (in the range of 10 μg/mL to 500 μg/mL in fermentation broth), the wild Eurotium cristatum strains produced lower lovastatin [6, 8].
Point 8: Both the abstract and the first paragraphs of the introduction should be rewritten to clarify the purpose of the study.
Response 8: Thank you so much! The abstract and introduction have be modified. We do hope that it is now acceptable to the respected reviewer.
Point 9: There are several details, English should definitely be revised.
Response 9: Thank you so much! The English have been revised. We do hope that it is now acceptable to the respected reviewer.
Reviewer 2 Report
1. The manuscript has grammatical errors and needs improvement.
2. Put significant letters on data presented in the Figure 1, 3-6.
3. Italicize “Eurotium cristatum” throughout the manuscript including references
4. In the 2.6. section “Determination of lovastatin”, the authors measured lovastatin production by using HPLC method. Add the type of Column and appropriate reference?
5. Superscript o (degree) throughout the text
6. Section 3.3 line 6; f in figure 4 should be capital. figure 6A,
7. In HPLC assay, what was the standard for determination and qualification of lovastatin?
8. Add Statistical analysis section to the end of Materials and methods and explain about type of analysis
9. Conclusion is too long, present the main outcome of the study in the conclusion, but do not repeat the results presented in the results section.
Comments for author File: 
Comments.pdf
Minor editing of English language required.
Author Response
Response to Reviewer 2 Comments
Point 1: The manuscript has grammatical errors and needs improvement.
Response 1: Thank you so much for your suggestion! The grammar of the manuscript has been revised, and we do hope that it is now acceptable to the respected reviewer.
Point 2: Put significant letters on data presented in the Figure 1, 3-6.
Response 2: Thank you so much for your suggestion! Figures 1, 3, and 4 contain one figure, and significant explanatory text was added to its legend. Figure 5-6 contains two figures, and significant letters (A or B) are presented in the Figure. We do hope that it is now acceptable to the respected reviewer.
Point 3: Italicize “Eurotium cristatum” throughout the manuscript including references
Response 3: Thank you so much for your suggestion! This has been corrected. The details can be seen in the revised manuscript.
Point 4: in the 2.6. section “Determination of lovastatin”, the authors measured lovastatin production by using HPLC method. Add the type of Column and appropriate reference?
Response 4: Thank you so much for your suggestion! The type of Column and appropriate reference have been added. The details are as follows:
The lovastatin was detected by the reported HPLC method [8]. The lovastatin extracts were filtered through a 0.2 μm filter membrane and then measured by an HPLC system. The analytical spectrophotometric detector was set at 238 nm. The type of column is the C18 chromatography column.
Point 5: Superscript o (degree) throughout the text
Response 5: Thank you so much for your suggestion! The “o (degree)” is superscript. It looks so different may due to its typeface (Palatino Linotype).
Point 6: Section 3.3 line 6; f in figure 4 should be capital. figure 6A,
Response 6: Thank you so much for your suggestion! This has been corrected. The details are as follows:
In the first few days, the concentration of lovastatin in the fermentation broth was linearly increased with increasing fermentation time (Figure 4).
Further, an increase in temperature reduced lovastatin yield (Figure 6A).
Point 7: In HPLC assay, what was the standard for determination and qualification of lovastatin?
Response 7: Thank you so much for your suggestion! The reported HPLC method detected the lovastatin, and a reference was added. The details are as follows:
Compared with strand lovastatin, the error of retention time of lovastatin in fermentation broth should be lower than 5%.
Point 8: Add Statistical analysis section to the end of Materials and methods and explain about type of analysis.
Response 8: Thank you so much for your suggestion! The statistical analysis section has been added to the Materials and methods. The experiments of “Lovastatin production by liquid fermentation”, “Extraction of lovastatin” and “Determination of lovastatin” were repeated three times, and their average value was calculated. And the error bar was the standard deviation, explained in the legend (Figure 1, Figure 3-6). The details are as follows:
Compared with strand lovastatin, the error of retention time of lovastatin in fermentation broth should be lower than 5%. All samples were detected three times, and their average value was calculated.
Point 9: Conclusion is too long, present the main outcome of the study in the conclusion, but do not repeat the results presented in the results section.
Response 9: Thank you so much for your suggestion! The conclusion has been rewritten. We do hope that it is now acceptable to the respected reviewer.
Point 10: Minor editing of English language required.
Response 10: Thank you so much for your suggestion! The manuscript's grammar has been revised, and we hope it is now acceptable to the respected reviewer.
Reviewer 3 Report
manuscript ID: forests-2420701
title: Lovastatin production by wild Eurotium cristatum isolated from Fuzhuan brick tea produced using forests resource in Auhua
I am not convinced that supplementation with statins from a natural source, which is tea, is a fully thought-out idea. The activities of statins are strictly dose dependent. Another concern is the side effects of statins, including musculoskeletal problems and adverse effects on glycemia, which some studies suggest may promote type 2 diabetes. For medical reasons, some patients cannot be treated with statins at all. Therefore, intensive research is being conducted on the development of drugs that lower plasma cholesterol levels that are not statins.
The above opinion is not intended to undermine the sense of this study. However, it is important for the Authors to be aware of the risks that an uncontrolled source of statins in the diet could bring, and to discuss the results in a broader perspective. Unfortunately, the discussion in this paper concerns only the results of the experiments. Definitely, the discussion should be deepened.
The work is methodically simple but clearly presented. Unfortunately, there is no separate section on statistics in the work. Moreover, I could not find a word on repetition numbers and statistical analysis. Are error bars standard deviation?
Another disadvantage of this work is the too long, verbose conclusions. It contains details and numerical values, comparisons, that should be presented in the Results and Discussion section. Good conclusions are a few accurate sentences.
I also have a few minor comments about the experiments and the manuscript. I have listed them below.
The Authors wrote in the Abstract: "The economic value of the tea forest is mainly related to tea products made from tea leaves" and in the Introduction "The tea plant is an important economic forest". What are the tea forests? I admit I do not know this concept. The combination of these words also does not occur in everyday language. It would be good if the Authors explained it in the text, also in relation to the thematic areas of the "Wood Science and Forest Products" Section.
Why were the experiments carried out at 28 degrees, when the most effective (in terms of levastatin production) turned out to be cultivation at 32 degrees?
I would use the words: "spectrophotometric detector" instead of "spectromonitor detector" in the description of the HPLC methodology.
I guess Fu-brick tea is short for Fuzhuan brick tea. Unfortunately, there is no explanation of this abbreviation in the text.
The work is careful in terms of editing. I found only the Latin name (Caenorhabditis elegans, p. 2) not written in italics and an inconsistency in the notation of the numerical value and the degree. I was taught not to use spaces in this case.
To sum up, the work should be supplemented in the methodological part with a description of the statistical analysis, the discussion should be significantly deepened, and the conclusions should be limited to a few most important observations.
June 11th, 2023
Author Response
Response to Reviewer 3 Comments
Point 1: I am not convinced that supplementation with statins from a natural source, which is tea, is a fully thought-out idea. The activities of statins are strictly dose dependent. Another concern is the side effects of statins, including musculoskeletal problems and adverse effects on glycemia, which some studies suggest may promote type 2 diabetes. For medical reasons, some patients cannot be treated with statins at all. Therefore, intensive research is being conducted on the development of drugs that lower plasma cholesterol levels that are not statins. The above opinion is not intended to undermine the sense of this study. However, it is important for the Authors to be aware of the risks that an uncontrolled source of statins in the diet could bring, and to discuss the results in a broader perspective. Unfortunately, the discussion in this paper concerns only the results of the experiments. Definitely, the discussion should be deepened.
Response 1: Thank you so much for the invaluable comment. The discussion of dark tea safety has been added to the Results. The details are as follows:
The safety of a drink with lovastatin should be considered. Compared with wild Monascus (in the range of 10 μg/mL to 500 μg/mL in fermentation broth), the wild Eurotium cristatum strains produced lower lovastatin [6, 8]. The red yeast rice fermented by Monascus has been made into many health foods, including red yeast rice powder, red yeast rice capsules, red yeast rice, and so on. And the lovastatin concentration in dark tea (always lower than 20 μg/g) was lower than in red yeast rice. Hence, dark tea is a safe drink for humans. And there is no report about the health risks of dark tea due to its little lovastatin.
Point 2: The work is methodically simple but clearly presented. Unfortunately, there is no separate section on statistics in the work. Moreover, I could not find a word on repetition numbers and statistical analysis. Are error bars standard deviation?
Response 2: Thank you so much for your suggestion. The statistical analysis section has been added to the Materials and methods. The experiments of “Lovastatin production by liquid fermentation”, “Extraction of lovastatin,” and “Determination of lovastatin” were repeated three times, and their average value was calculated. And the error bar was the standard deviation, explained in the legend (Figure 1, Figure 3-6).
Point 3: Another disadvantage of this work is the too long, verbose conclusions. It contains details and numerical values, comparisons, that should be presented in the Results and Discussion section. Good conclusions are a few accurate sentences.
Response 3: Thank you so much for your suggestion. The conclusion has been modified. We do hope that it is now acceptable to the respected reviewer.
Point 4: The Authors wrote in the Abstract: "The economic value of the tea forest is mainly related to tea products made from tea leaves" and in the Introduction "The tea plant is an important economic forest". What are the tea forests? I admit I do not know this concept. The combination of these words also does not occur in everyday language. It would be good if the Authors explained it in the text, also in relation to the thematic areas of the "Wood Science and Forest Products" Section.
Response 4: Thank you so much for your suggestion. The abstract has been modified. The “tea forest” was replaced by “tea plant forest” and has been explained in the text in detail. The details are as follows:
The tea plant forest contains tea gardens or “wild tea” trees grove, and its acreage is over 10 km2.
Point 5: Why were the experiments carried out at 28 degrees, when the most effective (in terms of levastatin production) turned out to be cultivation at 32 degrees?
Response 5: Thank you so much for your suggestion. The Eurotium cristatum always grows well at 28 degrees. Lovastatin is a secondary metabolite for Eurotium cristatum. The optimum temperature for various secondary metabolite production and Eurotium cristatum growing could be different.
Point 6: I would use the words: "spectrophotometric detector" instead of "spectromonitor detector" in the description of the HPLC methodology.
Response 6: Thank you so much for your suggestion. The words have been replaced. The details are as follows:
The analytical spectrophotometric detector was set at 238 nm.
Point 7: I guess Fu-brick tea is short for Fuzhuan brick tea. Unfortunately, there is no explanation of this abbreviation in the text.
Response 7: Thank you so much for your suggestion. All “Fu-brick tea” were replaced by Fuzhuan brick tea. The details can be seen in the revised manuscript.
Point 8: The work is careful in terms of editing. I found only the Latin name (Caenorhabditis elegans, p. 2) not written in italics and an inconsistency in the notation of the numerical value and the degree. I was taught not to use spaces in this case.
Response 8: Thank you so much for your suggestion. The Latin name (Caenorhabditis elegans) has been corrected in italics. The notation of the numerical value and the degree have been checked in the revised manuscript. The details are as follows:
Fuzhuan brick tea and Eurotium cristatum water extract decreased lipid droplet size and fat accumulation in Caenorhabditis elegans, zebrafish model, or high-fat diet-treated mice [13-15].
Point 9: To sum up, the work should be supplemented in the methodological part with a description of the statistical analysis, the discussion should be significantly deepened, and the conclusions should be limited to a few most important observations.
Response 9: Thank you so much for your suggestion. Some detail has been added to the results. The details can be seen in the revised manuscript. We do hope that it is now acceptable to the respected reviewer.
Round 2
Reviewer 2 Report
Accept
Author Response
Thank you so much for your acceptation！
Reviewer 3 Report
In the revised version of the manuscript, there are explanations and descriptions that I requested in the review. Now the work presents its assumptions in a more understandable way.
My doubts are raised primarily by the imprecise language, the use of many colloquialisms, simplifications and language errors, especialy in new sentences. In some sentences, the quality of the language is below average, so the work should be corrected by a specialist. Moreover, before doing so, the Authors should put a lot of effort into making some parts of the text more scientific.
There are quite a lot of imprecise sentences in the work, containing mental shortcuts or simplifications. The form of some sentences is very colloquial.
In the new sentence, ”Based on its secreted amylase and oxidase, tea leaves could transform into many compound that benefit human health, such as anti-obesity activity, anti-diabetic activity, gastrointestinal protective, and so on” the Authors did not specify what kind of oxidase it was. They also did not write what compounds are transformed into health-beneficial ones. It is sloppy to write that "tea leaf" can turn into many ”compound” (and not compounds).
In the new sentence, ”All samples were detected three times, and their average value was calculated” the phrase ”their average value” should be exchanged. Authors calculated ”the amount of lovastatin” (precisely) and not ”sample’s average value”.
The sentences like:
”And there is no report about the health risks of dark tea due to its little lovastatin”;
”And there is no report about dark tea impairing human’s health”;
”And the lovastatin production capacity in various Eurotium cristatum strains is absent”;
”And their biosynthetic pathway in Eurotium cristatum strains may have the same parts”;
”And the highest lovastatin yield was observed in the H20 strain with a slow grown rate, no wrinkle, rough edges in the colony, and little brownness at the center”;
should be repaced by the more formal vesions that would be precise and typical for scientific papers. In the last sentence, the Authors should also use a term ”slow growth rate” instead of ”slow grown rate”.
In the new sentence below the charts ("Error bars was standard deviation") there is an error, the word "was" should be replaced with "were" or another ("Error bars were/are/represent/depict standard deviation values").
The Authors used capital letters within the sentence; ”Eurotium cristatum is the dominant fungus involved in the Fermentation of Fuzhuan brick tea, which functions to lower blood lipid levels [9-12]”
I would wonder if the sentence "Lovastatin, as a secondary metabolite, was produced by numerous fungal species [6-8]" should not be presented in the Present Tense, since this ability is typical of these organisms.
In the new refrerence ”Li, C.; Tang, Y.; Wang, Y.; Yuan, X.; Zhang, B.; Wu, Z.; Tian, H. A novel environment-friendly adhesive based on recycling of broussonetia papyrifera leaf forestry waste protein. Forests. 2022, 13, e291” the name of the species should be witten in Latin, strarting with a capital letter, i.e. Broussonetia papyrifera.
The Authors wrote: ”The type of column is the C18 chromatography column”. When describing the column, I would give more precise data: length/diameter, grain size, or type/series and the company.
June 28, 2023
My comments about the language are included in the review.
Author Response
Dear reviewer
Thank you so much for your suggestion. Some sentences have been modified. We do hope that it is now acceptable to the respected reviewer. The details are as follows:
- The sentence “Based on its secreted amylase and oxidase, tea leaves could transform into many compound that benefit human health, such as anti-obesity activity, anti-diabetic activity, gastrointestinal protective, and so on.” has been changed to “Based on its secreted amylase and oxidase (such as polyphenol oxidase, cytochrome oxidase), tea leaves could transform into many compounds that benefit human health, such as anti-obesity activity, anti-diabetic activity, gastrointestinal protective, and so on.”
- The sentence “All samples were detected three times, and their average value was calculated.” has been changed to “All samples were detected three times, and the average concentration of lovastatin was calculated.”
- The sentence “And there is no report about the health risks of dark tea due to its little lovastatin.” has been changed to “And a large number of studies showed that dark tea has a variety of health benefits [27].”
- The sentence “And there is no report about dark tea impairing human’s health.” has been changed to “Evidence from thoses studies indicated that dark tea can increase human’s health behavior [15].”
- The sentence “And the lovastatin production capacity in various Eurotium cristatum strains is absent.” has been changed to “While the lovastatin production by various Eurotium cristatum strains was a lack of research.”
- The sentence “And their biosynthetic pathway in Eurotium cristatum strains may have the same parts.” has been deleted.
- The sentence “And the highest lovastatin yield was observed in the H20 strain with a slow grown rate, no wrinkle, rough edges in the colony, and little brownness at the center.” has been changed to “And the highest lovastatin yield was observed in the H20 strain with a slow growth rate, no wrinkle, rough edges in the colony, and little brownness at the center.”
- All the sentence “The error bars was standard deviation.” have been changed to “The error bars were standard deviation.”
- The sentence “Eurotium cristatum is the dominant fungus involved in the Fermentation of Fuzhuan brick tea, which functions to lower blood lipid levels [9-12].” has been changed to “Eurotium cristatum is the dominant fungus involved in the fermentation of Fuzhuan brick tea, which functions to lower blood lipid levels [9-12].”
- The sentence “Lovastatin, as a secondary metabolite, was produced by numerous fungal species [6-8].” has been changed to “Lovastatin, as a secondary metabolite, is produced by numerous fungal species [6-8].”
- The sentence “The type of column is the C18 chromatography column.” has been changed to “The type of column is the EC-C18 chromatography column (2.1×50 mm, impacted PEEK-lined stainless steel columns), which purchased from Agilent Technologies Co. Ltd.”
- All reference have been modified. The details could be seen in revised manuscript.
