Cordyceps cicadae NTTU 868 Mycelium with The Addition of Bioavailable Forms of Magnesium from Deep Ocean Water Prevents the Aβ40 and Streptozotocin-Induced Memory Deficit via Suppressing Alzheimer’s Disease Risk Factors and Increasing Magnesium Uptake of Brain

Round 1

Reviewer 1 Report

The work is not impressive, and the paper does not fall under the scope of Journal. The authors emphasized that deep ocean water is the main source of inorganic salts, and minerals, and HEA production of C. cicadae NTTU 868, but failed to investigate the active ingredients that are necessary and sufficient in the water for improving rat’s memory by targeting the required biomarkers. The data is poorly organized and is not enough for publication.

Author Response

Thanks for your review. Cordyceps cicadae was originally unable to significantly improve Alzheimer’s disease. However, this study is the first study to investigate how DOW not only increased the anti-inflammatory effect of C. cicadae, but also modulated the magnesium uptake of brain. DOW-CC enhanced the MAGT1 expression due to high organic magnesium, which can reverse Aβ40-induced cortex magnesium deficiency and further repress Aβ40 accumulation. The AD improvements were contributed from the synergistic effect of HEA, polysaccharides, and organic magnesium of DOW-CC.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

I must congratulate You for the almost perfect performance and presentation of the results in relation to the Cordyceps cicada fungus under influence of deep ocean water in fermentation for the possible application in AD therapy. 

I am just more interested in the results that could be possible added to the discussion section in order to compare this specific fungal species with the similar two such are C. militaris  and C. sinensis since both species are thoroughly used in traditional  practice in Asia. 

Cordyceps cicadae (Mig.) Massee is one of the oldest and well-known traditional Chinese medicine (TCM), with its uses recorded as far back as the 5th century A.D. 

please compare your results with the similar results for the more popular species   

look at the reference 

Can Cordyceps cicadae be used as an alternative to Cordyceps militaris and Cordyceps sinensis? – A review - ScienceDirect

WinstonNxumalo^ab1Ahmed AbdelfattahElateeq^acYanfangSun^a1

Author Response

Thanks for your suggestion, we have revised the discussion as follow: 

Studies have argued that DOW can promote the growth of fungal mycelium and regulate the production of functional components [15]. This study examined the cultivation of C. cicadae using UPW, DOW, and MgCl₂. Compared with using UPW, cultivating C. cicadae with DOW can increase mycelium growth and HEA content but may reduce intracellular and extracellular polysaccharide content. Regarding the mycelium growth, C. cicada were similar to other fungi such as Monascus purpureus, Antrodia cinnamomea, Cordyceps militaris, etc., and can be significantly increased by DOW [12, 14-16, 36]. According to our past research, different minerals in DOW may affect the growth of C. militaris and the production of cordycepin [16]. MgCl₂ was regarded as the most compound in the DOW. However, MgCl₂ and DOW performed the opposite trend on the regulation of mycelium production. MgCl₂ largely reduced the production of mycelium and adenosine, increased HEA content, and reduced the production of intracellular polysaccharides. The lower intracellular polysaccharides production may result from the decreased mycelium production. Although the polysaccharide content is reduced, it may also change the polysaccharide composition. The crude polysaccharides isolated from DCC still have the ability to slightly improve memory deficit and anti-inflammatory effect. It shows that the polysaccharide composition of DCC has an improvement effect on the development of AD.

Past studies have pointed out that the chloride ion in DOW would reduce the production of cordycepin and mycelium. C. militaris mycelium was increased by MgNO₃ but not by MgCl₂ [16]. Therefore, in this study, the main factor that MgCl₂ reduces the production of C. cicadae mycelium and adenosine may not be caused by Mg²⁺, but by Cl^-. It is the first study to prove that DOW enhanced mycelium and HEA productions of C. cicadae. Although the production of polysaccharides was slightly decreased (p > 0.05), the co-fermentation product still performed the synergistic functional effect. This study also found that the mycelium of C. cicadae can absorb Mg²⁺ in DOW and increase the content of Mg²⁺ in cells. Mg²⁺ was absorbed in cells and converted into organic ions, and the absorbed Mg²⁺ should be the important factor for increasing magnesium uptake in brain (Table 2)

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript is extremely interesting and innovative in terms of topics, but needs to be corrected and supplemented before being published:

• Please correct the English language in the manuscript
• Please provide information about the microbiological purity of the DOW
• Please provide the centrifugation speed as the "g" value throughout the manuscript
• Please provide accurate information about the composition of potato dextrose broth (line 85). What was the pH of this medium? What was the dose of C. cicadae NTTU 868 inoculum? Under what conditions was C. cicadae NTTU 868 grown in this medium ??
• Line 86: what does the sentence"with 10% of seed age 3 days at 23 ° C for 10 days" mean?
• Can keeping the sample at -20oC be called “incubation”? (line 101)
• Line 133: does "C. cicadae NTTU 868 fermented product" mean "C. cicadae NTTU 868 mycelium"? I believe that in these studies, simply three forms of C. cicadae NTTU 868 biomass were obtained, differing in their chemical composition resulting from the different chemical composition of the culture medium. In my opinion, the mansuccript should be redrafted taking this into account.
• Why was the p <0.05 coefficient used in the statistical analysis, after all, p <0.01 is usually used in medical research?
• Please verify the results of the statistical analysis, because looking at the values of the standard deviation, the statistical analysis seems to have a different interpretation (e.g. figure 5).
• Lines 422-430: this discussion of the results is very simplified. I suggest extending it taking into account the presence of micronutrients in DOW and their influence on the biosynthetic activity of C. cicadae NTTU 868. In my opinion, the data from table 1 may also suggest the inhibitory effect of an excess of Mg ions. I think the nutritional requirements of C. cicadae NTTU 868 are well known and should be discussed in this manuscript.
• It is a pity that the magnesium content in the biomass of C. cicadae NTTU 868 was not investigated and the salt form of this element was not determined. This seems significant for this research. .
• In my opinion the title of the manuscript should be reworded - instead of "Cordyceps cicadae NTTU 868 Mycelium Fermented by Deep Ocean Water ..." it should be "Cordyceps cicadae NTTU 868 Mycelium with the addition of bioavailable forms of magnesium ..."

Author Response

The manuscript is extremely interesting and innovative in terms of topics, but needs to be corrected and supplemented before being published:

1. Please correct the English language in the manuscript

Ans: Thanks for your review. We have corrected the English language in the manuscript.

1. Please provide information about the microbiological purity of the DOW

Ans: Thanks for your review. DOW was treated by RO and dialysis to remove sodium salt. After concentration. DOW was adjusted to the optimal concentration and then autoclaved. Therefore, there was no microbial contamination in the DOW.

1. Please provide the centrifugation speed as the "g" value throughout the manuscript

Ans: Thanks for your review. We have revised the unit of centrifugation speed in the manuscript (line 104 page 3).

1. Please provide accurate information about the composition of potato dextrose broth (line 85). What was the pH of this medium? What was the dose of C. cicadae NTTU 868 inoculum? Under what conditions was C. cicadae NTTU 868 grown in this medium ??

Ans: Thanks for your review. We have revised the information in the manuscript (line 85 page 2).

1. Line 86: what does the sentence"with 10% of seed age 3 days at 23 ° C for 10 days" mean?

Ans: Thanks for your review. We have revised the information in the manuscript (line 85 page 2).

1. Can keeping the sample at -20oC be called “incubation”? (line 101)

Ans: Thanks for your review. We have revised this word in the manuscript (line 104 page 3).

1. Line 133: does "C. cicadae NTTU 868 fermented product" mean "C. cicadae NTTU 868 mycelium"? I believe that in these studies, simply three forms of C. cicadae NTTU 868 biomass were obtained, differing in their chemical composition resulting from the different chemical composition of the culture medium. In my opinion, the mansuccript should be redrafted taking this into account.

Ans: Thanks for your review. I am sorry for making the confusion. About the materials, C. cicadae NTTU 868 fermented product means the mixture including mycelium and filtrate. The medium of C. cicadae NTTU 868 means the biomass. Mycelium and filtrate were homogenized for the preparation of animal test samples. We have revised these words in the manuscript.

1. Why was the p <0.05 coefficient used in the statistical analysis, after all, p <0.01 is usually used in medical research?

Ans: Thanks for your review. In this study, the Ducan test was used for statistical analysis due to the emphasis on comparison between groups. In medical research, the test sample is usually only compared with the control group, so it performs p<0.05, p<0.01, and p<0.001 in statistical analysis. However, this study focused on the comparison among  the different Cordyceps groups, DOW, HEA, and PS groups. It is easy to show the significant differences of p<0.05, when the compared groups have different statistical English labels. The English alphabets are arranged in order as the data value increases. label a and label b have a difference of p<0.05; label a and label c indicate a difference of p<0.01.

1. Please verify the results of the statistical analysis, because looking at the values of the standard deviation, the statistical analysis seems to have a different interpretation (e.g. figure 5).

Ans: Thanks for your review. We have verified the results of the statistical analysis. The result of statistical analysis is correct. because many values are very close. Aβ group has significant differences (p<0.05) with Vh, D, UCC, DCC, and MgCC but not HEA and PS groups (p>0.05). Many values of the groups such as D, UCC, MgCC, HEA, PS are very close to Vh group, therefore, there is no significant difference (p>0.05) among these groups.

1. Lines 422-430: this discussion of the results is very simplified. I suggest extending it taking into account the presence of micronutrients in DOW and their influence on the biosynthetic activity of C. cicadae NTTU 868. In my opinion, the data from table 1 may also suggest the inhibitory effect of an excess of Mg ions. I think the nutritional requirements of C. cicadae NTTU 868 are well known and should be discussed in this manuscript.

   Ans: Thanks for your suggestion. We have added the discussion in the manuscript.  (Line 431-459, Page 12)

1. It is a pity that the magnesium content in the biomass of C. cicadae NTTU 868 was not investigated and the salt form of this element was not determined. This seems significant for this research.

Ans: Thanks for your suggestion. We have added the result of magnesium content in the biomass in Table 1 of the manuscript. However, the salt form will be further investigated in future study.  (Line 237, Page5)

1. In my opinion the title of the manuscript should be reworded - instead of "Cordyceps cicadae NTTU 868 Mycelium Fermented by Deep Ocean Water ..." it should be "Cordyceps cicadae NTTU 868 Mycelium with the addition of bioavailable forms of magnesium ..."

Ans: Thanks for your suggestion. We have revised the title.

Author Response File: Author Response.pdf

Reviewer 4 Report

1. Comparison of the yield of biomass, HEA and polysaccharides under different conditions of fungus cultivation.

Using the results obtained only on the 10th day of cultivation of the producer, the authors conclude about what conditions are more effective for obtaining a high yield of biomass, HEA and polysaccharides. However, correct conclusions are possible only on the basis of a comparison of their maximum values. To establish them, it is necessary to determine the content of products several times during submerged cultivation.

I believe that it is necessary to exclude conclusions about the effectiveness of cultivation conditions for obtaining biomass, HEA, extracellular and intracellular polysaccharides, since I consider them unsupported. Based on my comments, the title of table 1 should be corrected.

2. The authors give the values of the yield of the investigated products per 1.5 liters culture medium (Table 1). However generally accepted yield of product in similar studies counts on 1 liter.
3. Received UPW samples contain more polysaccharides than DOW ones. In the discussion, the authors point out only this fact. However, the qualitative composition of polysaccharides obtained on media of various compositions may differ significantly. As a consequence, the biological activity of samples may change.

Comments for author File: Comments.docx

Author Response

1. Comparison of the yield of biomass, HEA and polysaccharides under different conditions of fungus cultivation.

Using the results obtained only on the 10th day of cultivation of the producer, the authors conclude about what conditions are more effective for obtaining a high yield of biomass, HEA and polysaccharides. However, correct conclusions are possible only on the basis of a comparison of their maximum values. To establish them, it is necessary to determine the content of products several times during submerged cultivation.

Ans: Thanks for your review. The determination of cultivation time is important. However, the similar result had been published in our previous study. The biomass and polysaccharides can reach the maximum value on the 8th and 10th day. Therefore, the culture time at 10 days was used as the culture time.

Hung, Y. P.; Wang, J. J.; Wei, B. L.; Lee, C. L., Effect of the salts of deep ocean water on the production of cordycepin and adenosine of Cordyceps militaris-fermented product. AMB Express 2015, 5, (1), 140.

I believe that it is necessary to exclude conclusions about the effectiveness of cultivation conditions for obtaining biomass, HEA, extracellular and intracellular polysaccharides, since I consider them unsupported. Based on my comments, the title of table 1 should be corrected.

Ans: Thanks for your review. We have revised the title of Table 1 (Line 606-618, Page 16) . We also checked the result and conclusion about the biomass, HEA, extracellular and intracellular polysaccharides. (Line 606-618, Page 16)

1. The authors give the values of the yield of the investigated products per 1.5 liters culture medium (Table 1). However generally accepted yield of product in similar studies counts on 1 liter.

Ans: Thanks for your review. The value has been re-calculated and transferred to the value for mg/L (Line 237, Page5).

1. Received UPW samples contain more polysaccharides than DOW ones. In the discussion, the authors point out only this fact. However, the qualitative composition of polysaccharides obtained on media of various compositions may differ significantly. As a consequence, the biological activity of samples may change.

Ans: Thanks for your suggestion. We have revised the discussion (Line 441, Page12)

Round 2

Reviewer 1 Report

N/a

Author Response

Thanks for your review.

Reviewer 3 Report

I can see that the authors have corrected the manuscript taking into account the comments of the reviewers. I now believe that this manuscript may be released for publication.

Author Response

Thanks for your review.