High-Value Utilization of Tea Forest Resources: Breeding Eurotium cristatum Strains to Enhance Lovastatin Yields in Anhua Dark Tea
and Peixue Ling 2,4,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript has very significant information and sound scientific contribution. However the introduction contains the results of the research also, which should not be there. The conclusion needs to be inline with the results presented in the abstract. The language needs minor improvement.
Comments on the Quality of English LanguageThe English language needs minor improvements and sentence synthesis should be improved.
Author Response
Point 1: The manuscript has very significant information and sound scientific contribution. However the introduction contains the results of the research also, which should not be there. The conclusion needs to be inline with the results presented in the abstract. The language needs minor improvement.
Response 1: Thank you so much for your suggestion. The results of the research has been deleted. The introduction has been corrected. And the details can be seen in the revised manuscript. We do hope that it is now acceptable to the respected reviewer.
Point 2: The English language needs minor improvements and sentence synthesis should be improved.
Response 2: Thank you so much for your suggestion. The english and sentence synthesis have been revised. The details can be seen in the revised manuscript. We do hope that it is now acceptable to the respected reviewer.
Reviewer 2 Report
Comments and Suggestions for AuthorsComments:
I have gone through the manuscript.
My comments and observations are as follows:
Abstract
1. Statement may be corrected “Protoplast fusion obtained some Eurotium cristatum strains with higher lovastatin production.”
2. “Protoplast fusion occurred between protoplasts of inactivated Eurotium cristatum and Monascus.” May be rewritten as “Experiments were carried out to induce fusion between inactivated Eurotium cristatum and Monascus”…What is inactivated here?
3. Fusants, may be replaced by fusion product
4. “Compared with wild Eurotium cristatum, the bred strain could improve lovastatin yield in loose dark tea, and the maximum increase was more than three times”…….Repetition may be avoided.
Introduction
1. The tea plant is an important economic forest…statement may be corrected.
1. “The tea plant is an important economic forest [1, 2]. The leaves of tea plants could be used to manufacture many tea products. Hence, the economic value of tea forests will be improved. Tea is an important beverage with a long history. There are six categories of tea in China based on their manufacturing processes and special characteristics, such as green tea, white tea, yellow tea, oolong tea, black tea, and dark tea.”..In these statements, there is no clear-cut linkage and flow of language.
2. “The lipid-lowering effect and anti-obesity activity of dark tea were related to tea polysaccharides, tea polyphenols, tea amino acids, and some active small molecule substances.”
3. “There are many types of microorganisms in dark tea? Hence, the lipid-lowering effect of dark tea is a strong effect by microorganism species?...by these statements what message authors want to convey?
4. “Fuzhuan brick tea is one of the important and special dark?? due to its manufacturing processes and dominant fungus.Fungus is present in tea??
5. “After protoplast fusion, a high strain recombination frequency has frequently occurred.”…what do author mean by this?
1. A little lovastatin was produced by wild Eurotium cristatum (Quantity and reference?). A protoplast fusion between Eurotium cristatum JH1205 and Monascus CICC5031 was used for breeding recombinants to enhance lovastatin yield and anti-obesity activity in loose dark tea (Reference) what was the findings? . More than 99% inactivation ratio of parental protoplasts was obtained by 60 C water bath treatment. Compared with Eurotium cristatum JH1205, more than 41% of fusant increased lovastatin production (Reference) . And four strains (A4, A36, A54, and A76) with more than triple 76 lovastatin production yields were obtained. Among them, A54 and A76 were more similar to Eurotium cristatum due to their yellow colony morphology. Tea samples from different tea forests in Anhua country produced loose dark tea fermented by wild Eurotium cristatum and A36, A54, and A76 fusant. ..Findings of this manuscript should not be mentioned here. Objectives of hypothesis of your study should be mentioned here.
2. The tea sample location and strain type strongly affected lovastatin yield in loose dark tea. Strain A76 was suitable for further strain improvement and was applied to produce dark tea with a higher lipid-lowering effect. .. Findings of this manuscript should not be mentioned here. Objectives of hypothesis of your study should be mentioned here.
3. Introduction of your manuscript should end with objectives of your experiments/study.
Material and methods
1. Monascus CICC5031..what was the rationale behind selection of this strain? This details may be mentioned in the introduction part?
2. The release of protoplasts was monitored using a light microscope…what does it mean?
3. Preparation of protoplasts from Eurotium cristatum and Monascus…Isolation of …
1. The release of protoplasts was monitored using a light microscope…meaning?
1. The protoplasts of Eurotium cristatum and Monascus were inactivated by heating treatment, and the survival rate was lower than 0.1%...what was heating treatment?
2. Section 2.4 may be rewritten to present the process and steps in proper order.
3. “The original tea samples from four special tea production areas 132 were selected to produce loose dark tea” why samples were collected from 4 regions….is there any difference in tea leaf properties?
4. Section 2.8: Details of HPLC procedure may be given…which column was used…column length/dia, pressure…etc.
5. Section 3.1 “Plenty of protoplasts induce the successful fused rate of fusant.” What do authors mean?
6. Some conditions were performed to gain the maximum number of Eurotium cristatum and Monascus protoplasts… Experiments were performed not conditions??
7. Table 1…..The data are one time observation or experiments have been repeated? Any statistical test to support your findings that age of culture influences the protoplast yield?
8. It was found that the optimal concentration of lywallzyme and snailase were 2.0% 167 and 0.5%?? which experiment was performed to conclude these findings? Other concentration of enzymes have been used…what was result? Any statistical test to support your findings (table 1)?
9. In Fig 1….the density of protoplast in B is less, while as per table the protoplast are more in B?
10. What should be order of result presentation…Enzyme optimization? Age of mycelia?
11. Table 2: Inactivation ratio or %?
12. After inactivation and fusion, the protoplast solution was coated with PDA medium…we cannot coat solution? Recheck what author mean by this statement?
13. Strains constructed by protoplast fusion presented various colony morphologies on the PDA medium….mention morphologies?
14. Some of them failed to produce red pigment, whose morphologies were more similar to Eurotium cristatum. Based on the different morphologies, putative recombinant strains were collected to cultivate further. …What is significance of red pigment here? If selection is based on morphology then what is significance of inactivation?
15. And the lovastatin yields of A76 fusants were enhanced compared with wild Eurotium cristatum strain 206 JH1205. B 92 fusant were selected to analyze lovastatin production in fermentation broth by HPLC…Lovastatin yield may be given in a table to compare the parental strains and fused individuals, it will make easier to compare.
16. Fig 3. What is presented by the error bar?
17. Figure 4. The organ of lovastatin production?
Statistical data analysis is mission.......data are not presented in scientific manner.
There are too many corrections...I could read up to Fig4 only.
Comments on the Quality of English LanguageLanguage of the manuscript require extensive editing.
Sequence /order of experiments presented in inappropriate.
Author Response
Point 1: “Protoplast fusion occurred between protoplasts of inactivated Eurotium cristatum and Monascus.” May be rewritten as “Experiments were carried out to induce fusion between inactivated Eurotium cristatum and Monascus”…What is inactivated here?
Response 1: Thank you so much for your suggestion. This sentence has been rewritten. The details are as follows:
Experiments were carried out to induce fusion between inactivated Eurotium cristatum JH1205 and Monascus CICC5031.
Point 2: Statement may be corrected “Protoplast fusion obtained some Eurotium cristatum strains with higher lovastatin production.”
Response 2: Thank you so much for your suggestion. This sentence has been rewritten. The details are as follows:
Some Eurotium cristatum strains with high lovastatin production were obtained using the proto-plast fusion method.
Point 3: Fusants, may be replaced by fusion product.
Response 3: Thank you so much for your suggestion. The “fusants” could represent the fused strains produced by the protoplast fusion method, which is better than the “fusion product”.
Point 4: “Compared with wild Eurotium cristatum, the bred strain could improve lovastatin yield in loose dark tea, and the maximum increase was more than three times”…….Repetition may be avoided.
Response 4: Thank you so much for your suggestion! This sentence has been changed to “Compared with wild Eurotium cristatum, the bred strain could improve lovastatin yield in loose dark tea”.
Point 5: “The tea plant is an important economic forest [1, 2]. The leaves of tea plants could be used to manufacture many tea products. Hence, the economic value of tea forests will be improved. Tea is an important beverage with a long history. There are six categories of tea in China based on their manufacturing processes and special characteristics, such as green tea, white tea, yellow tea, oolong tea, black tea, and dark tea.”..In these statements, there is no clear-cut linkage and flow of language.
Response 5: Thank you so much for your suggestion! The sentence has been corrected. We do hope that it is now acceptable to the respected reviewer. The details are as follows:
The tea plant is an important economic forest resource for high-value tea products made from its leaves [1, 2]. Tea is an important beverage with a long history. There are six categories of tea in China based on their manufacturing processes and special characteristics, such as green tea, white tea, yellow tea, oolong tea, black tea, and dark tea.
Point 6: “There are many types of microorganisms in dark tea? Hence, the lipid-lowering effect of dark tea is a strong effect by microorganism species?...by these statements what message authors want to convey?.
Response 6: Thank you so much! This sentence has been rewritten. The details are as follows:
Dark tea is a type of post-fermentation tea fermented by microorganisms.
Point 7: “Fuzhuan brick tea is one of the important and special dark?? due to its manufacturing processes and dominant fungus.Fungus is present in tea??
Response 7: Thank you so much! Fuzhuan brick tea is one of the most important and special dark teas. This error has been corrected. The other same mistake in the manuscript has also been corrected. The Eurotium cristatum is the dominant fungus involved in the fermentation of Chinese Fu-zhuan brick tea. In other Chinese dark teas (such as Yunnan Pu-er tea, Guangxi Liubao tea, Anhua Tianjian tea, and so on), the concentration of Eurotium cristatum is even lower. Hence, Chinese Fuzhuan brick tea is special.
Point 8: “After protoplast fusion, a high strain recombination frequency has frequently occurred.”…what do author mean by this?
Response 8: Thank you so much! This sentence has been corrected. The details are as follows:
After protoplast fusion, a high strain recombination frequency has commonly occurred due to the two genomes, which come from their parents [32].
Point 9: A little lovastatin was produced by wild Eurotium cristatum (Quantity and reference?). A protoplast fusion between Eurotium cristatum JH1205 and Monascus CICC5031 was used for breeding recombinants to enhance lovastatin yield and anti-obesity activity in loose dark tea (Reference) what was the findings? . More than 99% inactivation ratio of parental protoplasts was obtained by 60 C water bath treatment. Compared with Eurotium cristatum JH1205, more than 41% of fusant increased lovastatin production (Reference) . And four strains (A4, A36, A54, and A76) with more than triple 76 lovastatin production yields were obtained. Among them, A54 and A76 were more similar to Eurotium cristatum due to their yellow colony morphology. Tea samples from different tea forests in Anhua country produced loose dark tea fermented by wild Eurotium cristatum and A36, A54, and A76 fusant. ..Findings of this manuscript should not be mentioned here. Objectives of hypothesis of your study should be mentioned here.
Monascus CICC5031..what was the rationale behind selection of this strain? This details may be mentioned in the introduction part?
Response 9: Thank you so much! The English has been revised. This sentence has been corrected. The details are as follows:
A small quantity of lovastatin (< 10 μg/mL) was produced by wild Eurotium cristatum [8]. In order to improve lovastatin yield in Eurotium cristatum strains and in-crease the anti-obesity activity in loose dark tea (fermented with Eurotium cristatum), a protoplast fusion was used between Eurotium cristatum JH1205 and Monascus CICC5031 (lovastatin production greater than 100 μg/mL) for breeding recombinants. A 99% inactivation ratio of parental protoplasts was used for screening potential fusants. The influence of fermentation conditions on lovastatin production was also studied. Furthermore, the influence of the source of tea samples on the lovastatin con-centration of loose dark tea was investigated.
Point 10: The release of protoplasts was monitored using a light microscope…what does it mean?
Response 10: Thank you so much for your suggestion! This sentence has been corrected. The details are as follows:
The concentration of the obtained protoplasts was calculated using a blood-counting chamber.
Point 11: The protoplasts of Eurotium cristatum and Monascus were inactivated by heating treatment, and the survival rate was lower than 0.1%...what was heating treatment?
Response 11: Thank you so much! The heating treatment was done in a 60 °C water bath. This has been added in the Material and methods section. The details are as follows:
The protoplasts of Eurotium cristatum and Monascus spp. were inactivated by heating treatment (60 °C water bath), and the survival rate was lower than 0.1%.
Point 12: Section 2.4 may be rewritten to present the process and steps in proper order.
Response 12: Thank you so much for your suggestion! This part has been rewritten. The details are as follows:
The tea sample (20 g) was sterilized at 121 â—¦C for 20 min and inoculated with 2 mL Eurotium cristatum JH1205 strain or fusants. The inoculated tea sample was incubated at 28 â—¦C for 10 days. The loosely fermented dark tea was stored in a ventilated and dry environment. The ascocarp was separated from the loose tea and fermented on degreased gauze. The ascospore was separated from the ascocarp and broken by ultrasonics at 600 W for 10 min. The conidium was formed in a hyperosmotic solution. A 2 mL seed solution was added to 100 mL PDA medium containing 15% sucrose and 10% sodium chloride, followed by stationary culture at 28 °C for 8 days. After fermentation, the conidium was separated from the mycoderm by degreased gauze and dried in a baking oven.
Point 13: “The original tea samples from four special tea production areas 132 were selected to produce loose dark tea” why samples were collected from 4 regions….is there any difference in tea leaf properties?
Response 13: Thank you so much! The tea leaf properties are strongly affected by climatic and geological conditions. The climatic and geological conditions of the four selected regions are different. Hence, the influence of the source of tea samples on the lovastatin concentration of loose dark tea was investigated.
Point 14: Section 2.8: Details of HPLC procedure may be given…which column was used…column length/dia, pressure…etc.
Response 14: Thank you so much for your suggestion! The details have been added in Section 2.8. The details are as follows:
The type of column is the EC-C18 chromatography column (2.1×50 mm, impacted PEEK-lined stainless steel columns), which was purchased from Agilent Technologies Co., Ltd. (Santa Clara, CA, USA).
Point 15: Section 3.1 “Plenty of protoplasts induce the successful fused rate of fusant.” What do authors mean?
Response 15: Thank you so much! The sentence has been deleted. We do hope that it is now acceptable to the respected reviewer.
Point 16: Some conditions were performed to gain the maximum number of Eurotium cristatum and Monascus protoplasts… Experiments were performed not conditions??
It was found that the optimal concentration of lywallzyme and snailase were 2.0% and 0.5%?? which experiment was performed to conclude these findings? Other concentration of enzymes have been used…what was result? Any statistical test to support your findings (table 1)?
Table 1…..The data are one time observation or experiments have been repeated? Any statistical test to support your findings that age of culture influences the protoplast yield?
Response 16: Thank you so much! The experiment conditions have been added in Section 2.2. The details are as follows:
To produce more protoplasts, the age of mycelia (from 4 to 12 days) and the concentration of lywallzyme and snailase (Table 1) were optimized. The concentration of the obtained protoplasts was calculated using a blood-counting chamber. All experiments were repeated three times, and the average concentration of protoplasts was calculated.
Table 1. Concentration of enzyme
|
lywallzyme |
snails |
|
1% |
0.3% |
|
1% |
0.5% |
|
1% |
0.7% |
|
1.5% |
0.5% |
|
2% |
0.5% |
|
2.5% |
0.5% |
Point 17: In Fig 1….the density of protoplast in B is less, while as per table the protoplast are more in B?
Response 17: Thank you so much! Fig. 1 is used to present the diameter of protoplasts. It can’t be used to calculate the number of protoplasts. The number of protoplasts was calculated using a blood-counting chamber.
Point 18: What should be order of result presentation…Enzyme optimization? Age of mycelia?
Response 18: Thank you so much! The presentation order of enzyme optimization and age of mycelia in the result is appropriate due to its relation of equivalence.
Point 19: Table 2: Inactivation ratio or %?
Response 19: Thank you so much! This is the inactivation ratio calculated by the following formula:
%Inactivation ratio = (1- (No. of growing colonies on PDA)/(No. of initial protoplasts)) × 100%
Point 20: After inactivation and fusion, the protoplast solution was coated with PDA medium…we cannot coat solution? Recheck what author mean by this statement?
Response 20: Thank you so much! This sentence has been corrected. The details are as follows:
After inactivation and fusion, the protoplast solution was incubated for 5 d at 28 °C.
Point 21: Strains constructed by protoplast fusion presented various colony morphologies on the PDA medium….mention morphologies?
Response 21: Thank you so much! The 92 fusants were screened by their different colony morphologies. And this sentence has been changed to “The fusants constructed by protoplast fusion presented various colony morphologies on the PDA medium.”
Point 22: Some of them failed to produce red pigment, whose morphologies were more similar to Eurotium cristatum. Based on the different morphologies, putative recombinant strains were collected to cultivate further. …What is significance of red pigment here? If selection is based on morphology then what is significance of inactivation?
Response 22: Thank you so much! In this work, the fusants obtained would be used to ferment dark tea. Eurotium cristatum could grow well on tea leaf, while Monascus does not grow well on tea leaf. The fusants that produced yellow pigment could be target strains. And the pigment is one important type of morphology.
Point 23: And the lovastatin yields of A76 fusants were enhanced compared with wild Eurotium cristatum strain 206 JH1205. B 92 fusant were selected to analyze lovastatin production in fermentation broth by HPLC…Lovastatin yield may be given in a table to compare the parental strains and fused individuals, it will make easier to compare.
Response 23: Thank you so much! The lovastatin yield of all 92 fusants is presented in “Table 4. The increased level of lovastatin production in fusants compared with Eurotium cristatum JH1205” to compare the parental strains and fused individuals. Figure 1 is used to confirm that fusant and Eurotium cristatum could produce lovastatin compared with standard lovastatin.
Point 24: Fig 3. What is presented by the error bar? Statistical data analysis is mission.......data are not presented in scientific manner. There are too many corrections...I could read up to Fig4 only.
Response 24: Thank you so much! The error bars are standard deviations. Which has been added to the revised manuscript. The statistical analysis section has been added to the Materials and methods section. The details can be seen in the revised manuscript. We do hope that it is now acceptable to the respected reviewer.
Point 25: Figure 4. The organ of lovastatin production?
Response 25: Thank you so much! The organ means ascocarp, ascospore, conidium, and mycelium from Eurotium cristatum.
Reviewer 3 Report
Comments and Suggestions for AuthorsPlease see the attachment.
Comments for author File: 
Comments.pdf
Minor editing of English language required
Author Response
Point 1: It would be interesting to discuss briefly whether the hypolipidemic effects of fermented tea are owing to plant or fungus, and to what extent.
Response 1: Thank you so much for your suggestion. Dark tea is a post-fermented tea fermented by microorganisms. And Eurotium cristatum is the dominant fungus involved in Fuzhuan brick tea. Research shows that the lipid-lowering effect of Fuzhuan brick tea is related to many compounds, such as tea polysaccharides, tea polyphenols, tea amino acids, and some active small-molecule substances, such as lovastatin (always lower than 20 μg/g). Those indicate that the hypolipidemic effects of Fuzhuan brick tea may be related to tea leaves. Research shows that Eurotium cristatum extracts also have hypolipidemic effects. Hence, the hypolipidemic effects of Fuzhuan brick tea may be related to both plants and fungi. These discussions have been added to the revised manuscript.
Point 2: . Citrinin is one of by-products by Monascus spp. fermentation with known nephrotoxicity. Is the substitution of Monascus spp. by Eurotium cristatum occur to be useful and even necessary, to which the reviewed manuscript could contribute.
Response 2: Thank you so much! Eurotium cristatum has been found for over 30 years. And a large number of studies have shown that Eurotium cristatum has a variety of health benefits. In recent years, through analysing the genome DNA of Eurotium cristatum, researchers have found that Eurotium cristatum can not produce Citrinin.
Point 3: Lines 19, 56, 58, 88, 91, 92, 108, 124, 125, 165, 168, 171, 172, 176, 184, 187, 191, 202, 220, Table 2: "Monascus" should be replaced by "Monascus spp.", if the species or strains are not available.
Response 3: Thank you so much for your suggestion. The “Monascus” has been replaced by "Monascus spp." The details can be seen in the revised manuscript.
Point 4: . Line 124: Subtitle of the section "2.5. Production of lovastatin by Eurotium cristatum and Monascus" should be better replaced by "Eurotium cristatum and Monascus spp. submerged inoculum preparation" or similar title.
Response 4: Thank you so much for your suggestion! This sentence has been changed.
Point 5: . Line 60: "the lovastatin yield in other Aspergillus is very little". Taking in mind previous point 4, if other representatives of Aspergillus genus are meant here, the authors should remember several relatively active lovastatin producers as Aspergillus niger, Aspergillus terreus, Aspergillus flavus, Aspergillus tamari, Aspergillus flavipes.
. Line 59: "other Aspergillus can also produce lovastatin, such as Eurotium cristatum". However, earlier fundamental and current works (see, e.g., [Butinar L, Zalar P, Frisvad JC, Gunde-Cimerman N (2005) The genus Eurotium – members of indigenous fungal community in hypersaline waters of salterns. FEMS Microbiol Ecol 51:155–166. https://doi.org/10.1016/J.FEMSEC.2004.08.002]; [Moura, M.A.F.e., Gomes, D.C., Takahashi, J.A. (2023). Applications of Fungi Secondary Metabolites in the Food Industry. In: Carocho, M., Heleno, S.A., Barros, L. (eds) Natural Secondary Metabolites. Springer, Cham. https://doi.org/10.1007/978-3-031-18587-8_24], and Refs. therein) tend to consider fungi under study as different genera, i.e. Aspergillus spp., Eurotium spp. So, the sentence should be corrected.
Response 5: Thank you so much for your suggestion! The sentence has been corrected. And the References have been added. The details are as follows:
Except for Monascus spp. and Aspergillus terreus, other Aspergillus can also produce lovastatin, such as Eurotium cristatum, Aspergillus niger, Aspergillus terreus, Aspergillus flavus, etc. [25, 26].
- Butinar, L.; Zalar, P.; Frisvad, J.C. ; Gunde-Cimerman, N. The genus Eurotium–members of indigenous fungal community in hypersaline waters of salterns. FEMS Microbiol. Ecol. 2005, 51, 155-166.
- Moura, M.A.F.e.; Gomes, D.C.; Takahashi, J.A. Applications of fungi secondary metabolites in the food industry. Natural Secondary Metabolites. 2023, 22, 739–776.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe comments have not been addressed properly. There is no statistical test used in the study.
Comments on the Quality of English LanguageThe language of the manuscript still requires a lot many corrections.
Author Response
Point 1: The comments have not been addressed properly. There is no statistical test used in the study.
Response 1: Thank you so much for your suggestion. The statistical analysis has been added to the revised manuscript. The details are as follows:
2.9 Statistical analysis
For all experiments, samples were randomly selected and repeated three times. All statistical analyses were performed using SPSS (Statistical Product and Service Solutions) 22.0 statistical software. Values were presented as the mean ± standard deviation. For multi-group comparisons, p values were derived from a one-way analysis of variance (ANOVA). The p values < 0.05 were considered statistically significant.
Point 2: The language of the manuscript still requires a lot many corrections.
Response 2: Thank you so much for your suggestion! The grammatical of manuscript have been revised, and the details could be seen in revised manuscript. We do hope that it is now acceptable to the respected reviewer.
