Study on the Structure and Bioactivity of Ganoderma lucidum Polysaccharides under Cassava Stalk Stress

Round 1

Reviewer 1 Report

Title:  Study on the structure and bioactivity of Ganoderma lucidum 2 polysaccharides under cassava stalk stress

The paper “Study on the structure and bioactivity of Ganoderma lucidum 2 polysaccharides under cassava stalk stress”  investigated the effect of using cassava stalk as a carbon source for the cultivation of Ganoderma lucidum, a type of mushroom, and the subsequent production of its polysaccharides. The composition, molecular weight distribution, and antioxidant activity of the polysaccharides were analyzed using various techniques. The study also evaluated the effect of these polysaccharides on the growth of Lactobacillus rhamnosus LGG, a probiotic bacterium. These findings provide support for using cassava stalk as a carbon source in G. lucidum cultivation.

Here are my comments and required corrections:

Abstract: In light of the conclusion, the first part of the abstract could describe cassava stalk as a novel source of carbon to be used for Ganoderma lucidum.

Please correct the term "monosaccharoses".

The introduction section lacks a clear statement of the research problem and why it is important. It would be helpful if the authors could provide more context on why the growth of Lactobacillus rhamnosus LGG is important and what knowledge gaps this study aims to fill.

Overall, the conclusion provides a good summary of the study's findings. However, it could be improved by providing more context for the significance of the results. For example, the authors could discuss how the identified polysaccharides and their properties could be applied in industry or medicine, or how this research contributes to our understanding of the effects of carbon sources on G. lucidum growth and polysaccharide production. Additionally, the authors could briefly address the limitations of the study and suggest directions for future research.

I recommend some moderate changes to the English language in order to improve the clarity and readability of the manuscript. There are a number of grammatical errors and awkward sentence constructions that could be addressed to make the manuscript more professional.

I encourage you to consider engaging a professional editor or native English speaker to assist with these language improvements, as it would greatly enhance the overall quality of the manuscript.

Thank you for your attention to these suggestions.

Author Response

Responses to the comments of Reviewer 1:

The paper “Study on the structure and bioactivity of Ganoderma lucidum 2 polysaccharides under cassava stalk stress” investigated the effect of using cassava stalk as a carbon source for the cultivation of Ganoderma lucidum, a type of mushroom, and the subsequent production of its polysaccharides. The composition, molecular weight distribution, and antioxidant activity of the polysaccharides were analyzed using various techniques. The study also evaluated the effect of these polysaccharides on the growth of Lactobacillus rhamnosus LGG, a probiotic bacterium. These findings provide support for using cassava stalk as a carbon source in G. lucidum cultivation.

Here are my comments and required corrections: Abstract: In light of the conclusion, the first part of the abstract could describe cassava stalk as a novel source of carbon to be used for Ganoderma lucidum.

Response: Thanks for your suggestion. The revised abstract starts and ends with the respective statements: “..the cassava stalk is considered a promising carbon source for G. lucidum.” and “This study provided essential data support for cassava stalk as a carbon source in G. lucidum cultivation.”

Please correct the term "monosaccharoses".

Response: It was revised: "monosaccharoses" was modified to "monosaccharides".

The introduction section lacks a clear statement of the research problem and why it is important. It would be helpful if the authors could provide more context on why the growth of Lactobacillus rhamnosus LGG is important and what knowledge gaps this study aims to fill.

Response: The revised “Introduction " contains the following major additions: “However, there are quite a few reports on GLPs promoting the growth of L. rhamnosus, which limits the more comprehensive application of G. lucidum polysaccharides. …The structure and biological activity of GLPs with different cassava stalk stresses are characterized by gas chromatography-mass spectrometry, near-infrared spectroscopy, and gel chromatography. The results are expected to be of great significance for cassava stalks to be used as a carbon source in the cultivation of G. lucidum to realize value-added utilization and to provide basic data support.”

Overall, the conclusion provides a good summary of the study's findings. However, it could be improved by providing more context for the significance of the results. For example, the authors could discuss how the identified polysaccharides and their properties could be applied in industry or medicine, or how this research contributes to our understanding of the effects of carbon sources on G. lucidum growth and polysaccharide production. Additionally, the authors could briefly address the limitations of the study and suggest directions for future research.

Response: The respective parts were added to “Results and Discussion " (see the last paragraph before “Conclusions” in lines 323-328:

“Noteworthy is that L. rhamnosus LGG is the most researched and applied probiotic strain, balancing and improving gastrointestinal function and enhancing human im-munity (Michels et al., 2022). Therefore, it has been widely applied to developing func-tional products such as yogurt, milk, juice drinks, etc. (Sezer et al., 2022). Given the above findings, G. lucidum polysaccharide is a lucrative food additive to enhance the functionality of products containing rhamnosus.”

Besides, the revised “Conclusions” lists the following limitations of this study (lines 345-346).

“However, the mechanism of action on the metabolites of L. rhamnosus LGG requires clari-fication, which is envisaged in a follow-up study.”

I recommend some moderate changes to the English language in order to improve the clarity and readability of the manuscript. There are a number of grammatical errors and awkward sentence constructions that could be addressed to make the manuscript more professional. I encourage you to consider engaging a professional editor or native English speaker to assist with these language improvements, as it would greatly enhance the overall quality of the manuscript.

Response: The revised manuscript was polished by the authorized editing agency with a native English speaker, being also double-checked with Grammarly Premium and AJE Digital Editing tools.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have reported an interesting work, the novelty and the idea behind the work is good. 

I have few comments

1. The authors need to do a modification of the introduction, the introduction seems to not touch on crucial aspects that need to be addressed. In fact, the last paragraph of the intro should highlight the objective and novelty of the work, this is lacking. Pls add

2. a clear discussion, relating the obtained results to the existing studies is lacking, this has to be connected

3. There are some places where the infomration is unclear, please go through thoroughly and revise. For example

2.4. Determination of GLPs content 113 The polysaccharide content of G. lucidum fruiting body was determined by phenol- 114 sulfuric acid method with reference to the method of Liu et al (Liu et al., 2013; Liu et al., 115 2020). 0.1 mg/mL glucose solution was prepared with glucose as the standard. 0 mL, 0.1 116 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL and 0.8 mL glucose solutions were put 117 into a 15 mL glass test tube with a stopper, distilled water was added to make up to 1mL 118 to prepare standard glucose solutions of different concentrations, then 1.0 mL 6% phenol 119 solution and 5.0 mL sulfuric acid was added in turn respectively, shake for 20 s, mixed 120 well, soaked in water bath at 100 ℃ for 15 min, quickly cooled to room temperature, and 121 the absorbance value(A) was measured at 490 nm. The standard curve was established 122 according to the absorbance values of different concentrations of glucose solution (C), 123 A=0.01C+0.0017(R2=0.9994). 124 Determination of GLPs content: 10 mg GLPs were added into a 10 mL centrifuge 125 tube, and dissolved with 3 mL distilled water, shake for 20 s and mixed well, soaked in 126 boiling water for 2 h, cooled to room temperature, and filtered by 0.45m organic mi- 127 croporous filter membrane, and volumed with distilled water to 100 mL in volumetric 128 flask, and the test solution of GLPs was obtained. According to the above steps, the ab- 129 sorbance value was measured at 490 nm, and the polysaccharide content was calculated 130 according to the curve. 

In this section the heading and subheading are the same???

4. Why did the authors choose cassava? this has to be reasoned. 

5. The paper should be clearly oriented and the flow organized for clarity. 

Author Response

Responses to comments of Reviewer 2:

The authors have reported an interesting work, the novelty and the idea behind the work is good.

I have few comments

1. The authors need to do a modification of the introduction, the introduction seems to not touch on crucial aspects that need to be addressed. In fact, the last paragraph of the intro should highlight the objective and novelty of the work, this is lacking. Pls add

Response: The revised “Introduction " contains the following major additions: “However, there are quite a few reports on GLPs promoting the growth of L. rhamnosus, which limits the more comprehensive application of G. lucidum polysaccharides. …The structure and biological activity of GLPs with different cassava stalk stresses are characterized by gas chromatography-mass spectrometry, near-infrared spectroscopy, and gel chromatography. The results are expected to be of great significance for cassava stalks to be used as a carbon source in the cultivation of G. lucidum to realize value-added utilization and to provide basic data support.”

 a clear discussion, relating the obtained results to the existing studies is lacking, this has to be connected

Response: We did our best in the revised version to relate the obtained results to the existing studies. In particular:

Lines 233-235

The monosaccharide composition of GLPs differed from that described by Huang et al. (2010) and Liu et al. (2023) due to the different carbon sources for cultivating G. lucidum.

Lines 245-256:

All six GLPs had broad absorption peaks in the range of 3359-3398 cm⁻¹, corresponding to O-H stretching vibration (Liu et al., 2018), C-H stretching vibration with a weak absorption peak of alkyl at approximately 2924-2929 cm⁻¹ (Liu, 2018), and C=O stretching vibration at 1620-1637 cm⁻¹ (Zhang, 2015). That of 1400-1410 cm⁻¹ was caused by the variable angle vibration of -CH. The peak near 1048-1084 cm⁻¹ was the common resonant absorption peak of the absorption peak of pyranose lactone and hydroxyl groups, which was due to the asymmetric stretching vibration of the C-O-C ether bond on the sugar ring, which constituted the characteristic absorption peak of sugar and the typical infrared spectrum signal of dextran (He et al., 2010). In addition, 903-914 cm⁻¹ featured a typical absorption peak of pyran glucan and β-glycosidic linkage (Virkki et al., 2005), while 876-905 cm⁻¹ was a typical absorption peak of β-D-Glc infrared spectrum (Zhang, 2015), and 866-914 cm⁻¹ was a typical absorption peak of β-D-Gal infrared spectrum (Zhang, 2015).

Lines 322-328:

Noteworthy is that L. rhamnosus LGG is the most researched and applied probiotic strain, balancing and improving gastrointestinal function and enhancing human immunity (Michels et al., 2022). Therefore, it has been widely applied to developing functional products such as yogurt, milk, juice drinks, etc. (Sezer et al., 2022). Given the above findings, G. lucidum polysaccharide is a lucrative food additive to enhance the functionality of products containing rhamnosus.

3. There are some places where the infomration is unclear, please go through thoroughly and revise. For example

2.4. Determination of GLPs content 113 The polysaccharide content of G. lucidum fruiting body was determined by phenol- 114 sulfuric acid method with reference to the method of Liu et al (Liu et al., 2013; Liu et al., 115 2020). 0.1 mg/mL glucose solution was prepared with glucose as the standard. 0 mL, 0.1 116 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL and 0.8 mL glucose solutions were put 117 into a 15 mL glass test tube with a stopper, distilled water was added to make up to 1mL 118 to prepare standard glucose solutions of different concentrations, then 1.0 mL 6% phenol 119 solution and 5.0 mL sulfuric acid was added in turn respectively, shake for 20 s, mixed 120 well, soaked in water bath at 100 ℃ for 15 min, quickly cooled to room temperature, and 121 the absorbance value(A) was measured at 490 nm. The standard curve was established 122 according to the absorbance values of different concentrations of glucose solution (C), 123 A=0.01C+0.0017(R2=0.9994). 124 Determination of GLPs content: 10 mg GLPs were added into a 10 mL centrifuge 125 tube, and dissolved with 3 mL distilled water, shake for 20 s and mixed well, soaked in 126 boiling water for 2 h, cooled to room temperature, and filtered by 0.45m organic mi- 127 croporous filter membrane, and volumed with distilled water to 100 mL in volumetric 128 flask, and the test solution of GLPs was obtained. According to the above steps, the ab- 129 sorbance value was measured at 490 nm, and the polysaccharide content was calculated 130 according to the curve.

In this section the heading and subheading are the same???

 Response: We aplogize for this unclear passage and duplicated subheading. It was revised as follows (see lines 122-138)

2.4. Determination of GLPs content

The polysaccharide content of the G. lucidum fruiting body was determined by the phenol‒sulfuric acid method with reference to the method of Liu et al. (2013, 2020). A 0.1 mg/mL glucose solution was prepared with glucose as the standard. Glucose solutions (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mL) were put into a 15 mL glass test tube with a stopper; distilled water was added to make up to 1 mL to prepare standard glucose solutions of different concentrations, then 1.0 mL 6% phenol solution and 5.0 mL sulfuric acid were added in turn, shaken for 20 s, mixed well, soaked in a water bath at 100 ℃ for 15 min, quickly cooled to room temperature. The absorbance value (A) was measured at 490 nm. The standard curve was established according to the absorbance values of different concentrations of glucose solution C as follows: A=0.01C+0.0017 with R²=0.9994.

Ten milligrams of GLPs were added to a 10 mL centrifuge tube, dissolved in 3 mL of distilled water, shaken for 20 s, mixed well, soaked in boiling water for 2 h, cooled to room temperature, filtered through a 0.45 m organic microporous filter membrane, and brought to 100 mL with distilled water in a volumetric flask. The test solution of GLPs was obtained. According to the above steps, the absorbance value was measured at 490 nm, and the polysaccharide content was calculated according to the curve.

 Why did the authors choose cassava? this has to be reasoned.

Response: The revised introduction describes the main reasons for this (see lines 36-46):

“Cassava (Manihot esculenta Crantz) is an essential crop in tropical areas and plays a significant role in economic growth in tropical areas. However, the value-added utilization of cassava stalks after harvesting has always been a complex problem in the green development of the industry. Cassava stalks are as high as 7.5-10.5 t/ha, and the utilization rate is less than 10%. Most of them are burned on the spot or manually moved to the fields and abandoned by the roadside, resulting in an enormous waste of resources and environmental pollution. Cassava stems contain 40-50% cellulose and 25-30% starch (Molina & El-Sharkawy, 1995; Jiang et al., 2015), which are very beneficial to the growth of edible fungi. Cassava stalks have been successfully used to cultivate Auricularia auricula (Huang et al., 2016) and Pleurotus geesteranus (Deng et al., 2013) and have achieved good results.”

 The paper should be clearly oriented and the flow organized for clarity.

 Response: Thanks for your solid suggestion. We added the respective flowchart to the attached graphical abstract.

Author Response File: Author Response.pdf