Effects of Cultivation Substrate Differences on Quality Formation and Polysaccharide Composition Characteristics of Tremella fuciformis

Round 1

Reviewer 1 Report

This work is a comprehensive study aimed at investigating the effect of five different substrates (MZK, BNM, LZK, HB, ZL) on a wide range of characteristics of the valuable medicinal fungus Tremella fuciformis. The authors applied a multidisciplinary approach, including the analysis of agronomic traits, nutritional value, textural and taste properties, as well as an in-depth untargeted metabolomic analysis, which allowed them not only to identify differences but also to propose the underlying metabolic mechanisms. The topic is relevant for optimizing mushroom cultivation and improving the quality of the final product. The work contains a large amount of experimental data and interesting observations. The use of standardized protocols (phenol-sulfuric acid, TPA, electronic tongue, UPLC-MS/MS) and rigorous statistical methods (PCA, OPLS-DA) enhances the reliability of the obtained results.

Minor comments

1. It is not always clear how many biological replicates were used for each measurement (except for metabolomics, where n=3 is specified). For agronomic traits and texture analysis, the number of replicates needs to be clearly stated.

2. Figure S2. Please enlarge the font in the figure.

Author Response

Reviewer 1

Major comments

This work is a comprehensive study aimed at investigating the effect of five different substrates (MZK, BNM, LZK, HB, ZL) on a wide range of characteristics of the valuable medicinal fungus Tremella fuciformis. The authors applied a multidisciplinary approach, including the analysis of agronomic traits, nutritional value, textural and taste properties, as well as an in-depth untargeted metabolomic analysis, which allowed them not only to identify differences but also to propose the underlying metabolic mechanisms. The topic is relevant for optimizing mushroom cultivation and improving the quality of the final product. The work contains a large amount of experimental data and interesting observations. The use of standardized protocols (phenol-sulfuric acid, TPA, electronic tongue, UPLC-MS/MS) and rigorous statistical methods (PCA, OPLS-DA) enhances the reliability of the obtained results.

Detailed comments

Minor comments

Comment 1: It is not always clear how many biological replicates were used for each measurement (except for metabolomics, where n=3 is specified). For agronomic traits and texture analysis, the number of replicates needs to be clearly stated.

Response: We thank the reviewer for this valuable suggestion. The number of biological replicates has now been clarified for all experiments in the revised manuscript. Specifically, measurements of agronomic traits and texture analyses were conducted with a minimum of three independent biological replicates. Corresponding descriptions have been added to the Materials and Methods and other relevant sections (Lines 180, 195, 235, 399, and 451) and highlighted in red. Furthermore, we have carefully reviewed the entire manuscript to ensure that the number of replicates is consistently and clearly reported throughout.

Comment 2: Figure S2. Please enlarge the font in the figure.

Response: Thank you for your helpful suggestion. We have increased the font size in Figure S2 to improve readability and overall clarity. The revised figure has been updated accordingly in the supplementary materials.

Reviewer 2 Report

The authors describe the effect of growing Tremella fuciformis on different substrates. They begin by discussing the importance of T. fuciformis which has nutritional and medicinal uses and contains polysaccharides that are active in immune modulation, have antioxidant properties and help regulate blood sugar. They outline the effects of various other factors on cultivation and point out that previous studies concentrated only on the primary constituents of the fruiting bodies and on yield. The Material and Methods section is detailed and should permit repetition by anyone wishing to do so. The English is of a very high standard and the results and discussion are clear and logical.

The authors describe how growth on different substrates affects yield, size and shape of fruiting bodies etc. They also analyse nutritional components and the texture and flavours of fruiting bodies. They use LC-tandem MS to carry out metabolomic analysis, identifying up- and downregualtion of metabolites when grown on 4 of the substrates, relative to growth on cottonseed hulls. PCA showed that growth substrate had a major effect on metabolomic characteristics. They further compare the levels of constituent amino acids and their derivatives. When describing differences in flavour of fruiting bodies (sweet, umami etc) the authors discuss the differences in metabolites that could contribute towards these differences in flavours. KEGG enrichment analysis was used to identify pathways that were up- or downregulated on different substrates and the monosaccharide makeup of constituent polysaccharides was discussed.

In the discussion the authors discuss the implications of differences observed and in the context of the literature. There were no significant differences in fiber content. They suggest that nutrients had been fed into particular pathways to yield different metabolites etc. in fruiting bodies from different substrates and pointed out possible ways in which the polysaccharide, monosaccharide and other metabolite makeup might alter the physicochemical properties. 

The study provides data regarding the effect of growth substrate on the yield and quality of fruiting bodies but also constitutes a template for future analyses of nutritional and medicinal foods and the effects of culture methods.

I recommend publication

I only spotted one minor grammatical error: 166: "polysaccharides"  should be "polysaccharide".

The main aim of the research is to establish how growth on different substrates affects the quality of the fruiting bodies of Tremella fuciformis. The quality was assessed in terms of substituent polysaccharides (some with known medicinal properties); the relative abundance of constituent proteins and the amino acids making up those proteins; the yield; the shape and texture of fruiting bodies and the taste. 

While other articles have assessed the levels of primary nutrients in fruiting bodies grown on different substrates, the authors assess a wide range of compounds and explain why some of these are important: whether they have medicinal or nutritional merit or if they contribute to beneficial or adverse flavor of fruiting bodies. This is of interest to the T. fuciformis industry but also to lab scientists as the authors carry out metabolomics and outline the biological pathways that appear to be up- or downregulated when the organism is grown of different substrates. This could allow researchers to establish which signaling pathways are at play on different substrates, how these pathways interact and how they relate to the transcriptional program, operating in the organism. 

Specifics about the source of flavours, the pathways by which metabolites are produces or consumed and the benefits of various substances, present in the fruiting bodies. While this overlaps with the work of previous authors, the authors look at a wider range of metabolites. Their methods also provide a template for similar assessments in the future.

The methodology is sound and needs no improvement. The use of an electronic tongue to assess sourness, sweetness, saltiness, bitterness and umami flavour is intriguing, as is the assessment of texture of fruiting bodies.

The conclusions are consistent with the evidence and are presented in the context of data from the literature. They do address the main question, though they could be emphasized further. 

The references are appropriate. There are a number of self-citations and most of the citations are for Chinese authors but this is because the field is highly specialized and involves a foodstuff/medicinal product that is common mainly in China and the authors have worked for some time on the organism in question.

I found the tables and figures clear and easy to interpret. They aptly present the relevant data.

Author Response

Reviewer 2

I only spotted one minor grammatical error: 166: "polysaccharides" should be "polysaccharide".

The main aim of the research is to establish how growth on different substrates affects the quality of the fruiting bodies of Tremella fuciformis. The quality was assessed in terms of substituent polysaccharides (some with known medicinal properties); the relative abundance of constituent proteins and the amino acids making up those proteins; the yield; the shape and texture of fruiting bodies and the taste. 

While other articles have assessed the levels of primary nutrients in fruiting bodies grown on different substrates, the authors assess a wide range of compounds and explain why some of these are important: whether they have medicinal or nutritional merit or if they contribute to beneficial or adverse flavor of fruiting bodies. This is of interest to the T. fuciformis industry but also to lab scientists as the authors carry out metabolomics and outline the biological pathways that appear to be up- or downregulated when the organism is grown of different substrates. This could allow researchers to establish which signaling pathways are at play on different substrates, how these pathways interact and how they relate to the transcriptional program, operating in the organism. 

Specifics about the source of flavours, the pathways by which metabolites are produces or consumed and the benefits of various substances, present in the fruiting bodies. While this overlaps with the work of previous authors, the authors look at a wider range of metabolites. Their methods also provide a template for similar assessments in the future.

The methodology is sound and needs no improvement. The use of an electronic tongue to assess sourness, sweetness, saltiness, bitterness and umami flavour is intriguing, as is the assessment of texture of fruiting bodies.

The conclusions are consistent with the evidence and are presented in the context of data from the literature. They do address the main question, though they could be emphasized further. 

The references are appropriate. There are a number of self-citations and most of the citations are for Chinese authors but this is because the field is highly specialized and involves a foodstuff/medicinal product that is common mainly in China and the authors have worked for some time on the organism in question.

I found the tables and figures clear and easy to interpret. They aptly present the relevant data.

 

Comment 1: I only spotted one minor grammatical error: 166: "polysaccharides" should be "polysaccharide".

Response: We sincerely appreciate the reviewer’s detailed and encouraging feedback. The minor grammatical error on line 166 has been corrected and highlighted in red, changing “polysaccharides” to “polysaccharide.” We also thank the reviewer for the positive overall evaluation of our work.

Reviewer 3 Report

The experimental article titled "Effects of Cultivation Substrate Differences on Quality Formation and Polysaccharide Composition Characteristics of Tremella fuciformis" is dedicated to a relevant research area, focusing on studying the impact of substrate compounds on key factors affecting the quality formation and polysaccharide composition characteristics of Tremella fuciformis.

The concept and results are interesting. The topic is of practical relevance. The manuscript is clearly structured, and the material is presented logically and consistently. The introduction concisely and scientifically substantiates the relevance and feasibility of the study. The experimental design is appropriately planned for the research objectives. The authors have obtained a significant amount of material, which is well presented and graphically illustrated.  The references cited are mainly recent publications. The concluding remarks summarise the main findings.  The article is of high quality and meets all the journal's requirements.

However, I would recommend taking a few minor comments into account.

Some technical and academic points that need minor refinement:
-line 38: According to the international botanical nomenclature, the author of the mushroom species should be given at the first mention in the introduction. Tremella fuciformis Berk.
-a clarification should be provided regarding the statistical analysis.
The number of repetitions of the experiments is not indicated, which is important for assessing the reliability and statistical significance of the results obtained. It is recommended to indicate how many times each experiment was repeated.
-With all the data and various analyses and methods performed, I did not have enough final conclusion on an important question that lies on the surface. The authors conducted a thorough study, but one question remained out of attention - what substrate do the authors recommend for Tremella fuciformis cultivating? It is important to emphasize what is unique about the work and highlight the data that were obtained for the first time.

Author Response

Reviewer 3

Major comments

The experimental article titled "Effects of Cultivation Substrate Differences on Quality Formation and Polysaccharide Composition Characteristics of Tremella fuciformis" is dedicated to a relevant research area, focusing on studying the impact of substrate compounds on key factors affecting the quality formation and polysaccharide composition characteristics of Tremella fuciformis.

The concept and results are interesting. The topic is of practical relevance. The manuscript is clearly structured, and the material is presented logically and consistently. The introduction concisely and scientifically substantiates the relevance and feasibility of the study. The experimental design is appropriately planned for the research objectives. The authors have obtained a significant amount of material, which is well presented and graphically illustrated.  The references cited are mainly recent publications. The concluding remarks summarise the main findings.  The article is of high quality and meets all the journal's requirements.

However, I would recommend taking a few minor comments into account.

Detailed comments

Comment 1: Some technical and academic points that need minor refinement:
-line 38: According to the international botanical nomenclature, the author of the mushroom species should be given at the first mention in the introduction. Tremella fuciformis Berk.

Response: We thank the reviewer for pointing out this important nomenclature issue. The species name has been corrected to include the author citation at its first mention in the Introduction, and it now reads Tremella fuciformis Berk., in accordance with international botanical nomenclature standards (Line 38). The revision has been highlighted in red in the corresponding section.

 

Comment 2: -a clarification should be provided regarding the statistical analysis.
The number of repetitions of the experiments is not indicated, which is important for assessing the reliability and statistical significance of the results obtained. It is recommended to indicate how many times each experiment was repeated.

Response: We thank the reviewer for this important comment. We have now clarified the statistical analysis in the Materials and Methods section. Specifically, all experiments were conducted with a minimum of three independent biological replicates to ensure reliability and allow appropriate statistical evaluation. This information has been added to the manuscript, and the relevant text has been highlighted in red in the corresponding section (Line 180, Line 195, Line 235, Line 399 and Line 451).

Comment 3: -With all the data and various analyses and methods performed, I did not have enough final conclusion on an important question that lies on the surface. The authors conducted a thorough study, but one question remained out of attention - what substrate do the authors recommend for Tremella fuciformis cultivating? It is important to emphasize what is unique about the work and highlight the data that were obtained for the first time.

Response: We thank the reviewer for this valuable comment and for recognizing the thoroughness of our study. Based on our comprehensive analyses, fruiting bodies cultivated on LZK consistently exhibited the highest overall quality, including superior dry matter accumulation, biological efficiency, textural firmness, and nutrient content, indicating that LZK provides favorable conditions for T. fuciformis cultivation.

To address the reviewer’s suggestion, we have emphasized in the Conclusion section the unique aspects of our work, highlighting that this study is the first to systematically integrate agronomic traits, nutritional quality, textural and sensory analysis, and untargeted metabolomics to elucidate the mechanisms by which cultivation substrates influence fruiting body quality. The relevant additions have been highlighted in red in the revised manuscript (Line 542 and Lines 549-552).