Preparation and Antioxidant Activity In Vitro of Fermented Tremella fuciformis Extracellular Polysaccharides

Round 1

Reviewer 1 Report

The manuscript "Preparation and Antioxidant Activity In Vitro of Fermented 1 Tremella fuciformis Extracellular Polysaccharides" is well structured and written.

However some of the methods could be describe more precisely (i.e phenol-sulfuric acid method; DNS method etc.).

T. fuciformis is widely cultivated and studied organism as well as its EPSs, so the optimization of the parameters of the system for the maximum EPS production is more or less routinely and there is low novelty level. The study would be of greater value if a comparison was made between the antioxidant activity of the isolated EPSs from different strains and then to optimize the system parameters to produce the maximum amount of the exopolysaccharides with the best antioxidant activity.

Author Response

Dear reviewer,

Thanks for your patience and helpful suggestions. Below are the  point-by-point response to the reviewer’s comments. Attached is the revision.

Reply to reiewer 1:

Line 10-14 17, 20-21, 26, 35, 38, 41, 42, 43, 47,48, 62, 69, 75, 76, 87, 88, 90, 108, 125, 128, 161, 216, 251, 263-265, 278, 293, 295, 301, 302, 352,444... we have improved the English of the manuscript.

1.However some of the methods could be describe more precisely (i.e phenol-sulfuric acid method; DNS method etc.).

  Reply: Line 95-105

  The description of the DNS method and phenol–sulfuric acid method have been added: “The crude exopolysaccharide mixture obtained was separated using the ethanol precipitation method. The total sugar content was estimated using the phenol–sulfuric acid method with slight modifications[33]. Added 1 mL of a phenol solution at a concentration of 6% (v/v) to 1 mL of fermentation supernatant. After vortexing, 5 mL of concentrated sulfuric acid was added to the mixture, which was then vortexed again, and then left to stand at room temperature for 30 min, then the absorbance at 490 nm in each tube was determined by UV–visible spectrophotometer. Glucose (50–500 μg/mL) was used as a standard to calculate total sugar content. The reducing sugar content was determined by the DNS method[34]. For the measurement, 2 mL of DNSA reagent was pipetted into a test tube containing 1 mL of fermentation supernatant and kept at 95°C for 5 min. After cooling, 7 mL of distilled water was added to the solution and the absorbance of the resulting solution was measured at 540 nm using a UV–VIS spectrophotometer. The reducing sugar content was calculated from the calibration curve of standard D–glucose (200–1000 mg/L).”

33. Chen, S.; Huang, H.; Huang, G. Extraction, derivatization and antioxidant activity of cucumber polysaccharide. Int J Biol Macromol 2019, 140, 1047-1053.
34. Huang, F.; Hong, R.; Yi, Y.; Bai, Y.; Dong, L.; Jia, X.; Zhang, R.; Wang, G.; Zhang, M.; Wu, J. In vitro digestion and human gut microbiota fermentation of longan pulp polysaccharides as affected by Lactobacillus fermentum fermentation. Int J Biol Macromol 2020, 147, 363-368.

 

2. T. fuciformis is widely cultivated and studied organism as well as its EPSs, so the optimization of the parameters of the system for the maximum EPS production is more or less routinely and there is low novelty level. The study would be of greater value if a comparison was made between the antioxidant activity of the isolated EPSs from different strains and then to optimize the system parameters to produce the maximum amount of the exopolysaccharides with the best antioxidant activity.

Reply: It’s true T. fuciformis is widely cultivated and studied organism as well as its EPSs. However, the overall objective of this work was to increase the capacity of exopolysaccharide from fermented T. fuciformis for possiple application in medicine and food industry. Thus, three fermented strains，seven fermentation parameters, purification conditions and the antioxidant activity of extracellular polysaccharide fractions (EPS) were investigated in this work. Compared with previous T. fuciformis polysaccharide studies, a comprehensive discussion was made both to fermentation parameters and purification process, particularly separation assay of macroporous adsorption resin was applied, which is easy to industrial amplification. Also four polysaccharide fractions were isolated and purified as Tf1–a, Tf1–b, Tf2, and Tf3. A significant correlation on (r>0.8) among the concentration and antioxidant activities of TEPS major fractions Tf1–a and Tf1–b was observed. We believe these results provide valuable references for other researchers.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

1.       Line 11. It should read as “…..liquid, fermented strains…….”

2.       Line 26. …had excellent antioxidant activity in vitro. Please provide the values for the antioxidant activities in order to support the “excellent” antioxidant activity.

3.       Line 116. Reference for the DNS method is needed.

4.       Line 128-130. Please go into more depth regarding the resins. Since the resin's characteristics will have an impact on how well EPS is refined.

5.       Line 283-284. Please talk about how the molecular structure and type of the pigment affect the resin's ability to decolor.

6.       From Table 4. Is there a Maillard browning reaction occurring as the temperature rises? Because both amine and carbonyl compounds are present in the sample. The initial pigments in the sample may have increased as a result of this.

7.       Line 341-343. As stated in Line 334, FRAP is not for "free radical scavenging," but rather for "reducing capacity." Therefore, only the hydroxyl radical and the DPPH radical can be characterized as the method for scavenging free radicals.

8.       The primary active substances in charge of each mechanism (such as free radical scavengers and reducing power) should be presented to link with the antioxidant like structure-activity relationship when discussing antioxidant activity. Do the fractions still include any phenolic compounds and antioxidant peptides?

9.       Line 427. Because heat treatments and other aspects of food processing can undoubtedly affect the residual and effective activity, please be concerned about the safety issue and the carry-through property of the fraction.

Author Response

Dear reviewer,
Thanks for your patience and helpful suggestions. Below are the  point-by-point response to the reviewer’s comments. Attached is the revision.

Reply to reiewer 2:

Line 10-14 17, 20-21, 26, 35, 38, 41, 42, 43, 47,48, 62, 69, 75, 76, 87, 88, 90, 108, 125, 128, 161, 216, 251, 263-265, 278, 293, 295, 301, 302, 352,444... we have improved the English of the manuscript.

1. Line 11. It should read as “…..liquid, fermented strains…….”

Reply: Line10-14

This sentence was replaced by “This study was aimed to increase the capacity of fermented Tremella fuciformis extracellular polysaccharides (TEPS) for possible functional food applications. Thus, strain varietes, fermentation parameters and purification conditions, also in vitro antioxidant activities of purified EPS fractions were investigated. An EPS high-yield strain Tf526 was selected and the effects of seven independent fermentation factors (time, temperature, initial pH, inoculum size, shaking speed, carbon, and nitrogen source) on the EPS yield were evaluated. ”

2. Line 26. …had excellent antioxidant activity in vitro. Please provide the values for the antioxidant activities in order to support the “excellent” antioxidant activity.

Reply: Line23-27 

The description of antioxidant activity values has been added：“In general, the antioxidant activities of the Lf1-a and Lf1-b were lower compared with Vc, but the FRAP assay, DPPH scavenging activity, and hydroxyl radical scavenging activity analysis still revealed that Tf1-a and Tf1-b  possess significant antioxidant activities in vitro. At the concentration of 3 mg/mL, the reducing power of Lf1-a and Lf1-b reached 0.86 and 0.70, the maximum DPPH radical were 54.23 ± 1.68% and 61.62 ± 2.73%, and the maximum hydroxyl radicals scavenging rates were 58.76 ± 2.58% and 45.81 ± 1.79%, respectively. Moreover, there were significant correlations (r>0.8) among the concentration and antioxidant activities of TEPS major fractions Tf1-a and Tf1-b. Therefore, it is expected that Tf1-a and Tf1-b polysaccharide fractions from fermented TEPS may serve as active ingredients in functional foods.”

3. Line 116. Reference for the DNS method is needed.

Reply: Line95-105

  The description of the DNS method and phenol–sulfuric acid method have been added: “The crude exopolysaccharide mixture obtained was separated using the ethanol precipitation method. The total sugar content was estimated using the phenol–sulfuric acid method with slight modifications[33]. Added 1 mL of a phenol solution at a concentration of 6% (v/v) to 1 mL of fermentation supernatant. After vortexing, 5 mL of concentrated sulfuric acid was added to the mixture, which was then vortexed again, and then left to stand at room temperature for 30 min, then the absorbance at 490 nm in each tube was determined by UV–visible spectrophotometer. Glucose (50–500 μg/mL) was used as a standard to calculate total sugar content. The reducing sugar content was determined by the DNS method[34]. For the measurement, 2 mL of DNSA reagent was pipetted into a test tube containing 1 mL of fermentation supernatant and kept at 95°C for 5 min. After cooling, 7 mL of distilled water was added to the solution and the absorbance of the resulting solution was measured at 540 nm using a UV–VIS spectrophotometer. The reducing sugar content was calculated from the calibration curve of standard D–glucose (200–1000 mg/L).”

33. Chen, S.; Huang, H.; Huang, G. Extraction, derivatization and antioxidant activity of cucumber polysaccharide. Int J Biol Macromol 2019, 140, 1047-1053.
34. Huang, F.; Hong, R.; Yi, Y.; Bai, Y.; Dong, L.; Jia, X.; Zhang, R.; Wang, G.; Zhang, M.; Wu, J. In vitro digestion and human gut microbiota fermentation of longan pulp polysaccharides as affected by Lactobacillus fermentum fermentation. Int J Biol Macromol 2020, 147, 363-368.

4.Line 128-130. Please go into more depth regarding the resins. Since the resin's characteristics will have an impact on how well EPS is refined.

Reply: Line 114-124, 138-139 Table1Chemical and physical properties of the macroporous resins employed

Line 114-124

The description of the macroporous adsorption resin's characteristics has been added: “It is well known macroporous adsorption resins (MARs) are efficient matrices for enrichment of bioactive substances from plant resources due to their high selectivity and adsorption capacity, as well as stability and resistance to degradation by osmotic shock and oxidation. They are a group of polymers containing a permanent network of pores independent of the state of swelling of the resin thus display much better solvent tolerance than gel-type resins. The adsorption performance of MARs is closely related to its polarity. Non-polar resins with strong hydrophobic pore surface, without any functional groups, are suitable for adsorbing non-polar substances; medium-polar resins with both hydrophilic and hydrophobic surface properties, containing an ester group, are suitable for adsorbing both non-polar and polar substances; and polar resins adsorb polar substance mainly through electrostatic interactions. Static adsorption tests of polysaccharides were performed using different resins (DM130, HPD600, DA201, HPD100, AB-8, XAD1180N, and A-722MP), which had different polarity and particle sizes and their chemical and phasical properties, including polarity, particle size, surface area, and pore diameter are shown in table1[35-38]”

Line1387-139

Table1 Chemical and physical properties of the macroporous resins employed

Resins	Particle size (mm)	Surface area (m2/g)	Average pore diameter (nm)	Polarity
A-722MP	0.30 - 1.22	650 - 700	20 - 50	Polar
DA-201	0.30 - 1.25	≥200	10 - 13	Polar
HPD-600	0.30 - 1.25	550 - 600	8 - 9	polar
AB-8	0.30 - 1.25	450 - 530	13 - 14	Weak-polar
DM130	0.30 - 1.25	500 - 550	9 - 10	Weak-polar
HPD-100	0.30 - 1.25	650 - 700	8.5 - 9	Non-polar
XAD-1180N	0.35 - 0.60	150 - 900	4 - 9	Non-polar

 

35. Hu, Z.; Zhou, H.; Li, Y.; Wu, M.; Yu, M.; Sun, X. Optimized purification process of polysaccharides from Carex meyeriana Kunth by macroporous resin, its characterization and immunomodulatory activity. Int J Biol Macromol 2019, 132, 76-86.
36. Hou, M.; Hu, W.; Xiu, Z.; Shi, Y.; Hao, K.; Cao, D.; Guan, Y.; Yin, H. Efficient enrichment of total flavonoids from Pteris ensiformis Burm. extracts by macroporous adsorption resins and in vitro evaluation of antioxidant and antiproliferative activities. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2020, 1138.
37. Mou, Y.; Li, J.; Zhou, K.; Yu, R.; Xu, D.; Luo, H.; Lai, D.; Zhou, L. Enhanced Production of Palmarumycins C12 and C13 in Mycelial Liquid Culture of the Endophytic Fungus Berkleasmium sp Dzf12 with In situ Macroporous Resin Adsorption. Tropical Journal of Pharmaceutical Research 2015, 14, 407-414.
38. Rajesh, N.; Kumar, A.S.K.; Kalidhasan, S.; Rajesh, V. Trialkylamine Impregnated Macroporous Polymeric Sorbent for the Effective Removal of Chromium from Industrial Wastewater. J Chem Eng Data 2011, 56, 2295-2304

 

5.Line 283-284. Please talk about how the molecular structure and type of the pigment affect the resin's ability to decolor.

Reply: Line281-288  

The description of “the molecular structure and type of the pigment affect the resin's ability to decolor” has been added: “Macroporous resin is always apolymeric adsorbent with large internal surface areas and rigid network. The adsorptio mechanism of macroporous resin is primarily based on the van der Waals forces, hydrogen bonds, molecular sieving effect, dipole–dipole forces (“polar” interactions), and cation–anion interactions (“ionic” interactions)[51]. A–722MP resin is a strong basic anion exchange resin, and exhibited higher adsorption ratio to pigments of TEPS than weakly polar or non-polar resins. Thus, it can be speculated that the polysaccharide pigments may be mainly polar substances with negative charges. Additionally, owing to its higher surface area and bigger average pore diameter A-722MP exhibited the highest decoloration ratio. Therefore, A–722MP resin could adsorb pigments and be used for decolorization of polysaccharides.”

51. Kammerer, J.; Carle, R.; Kammerer, D.R. Adsorption and Ion Exchange: Basic Principles and Their Application in Food Processing. J Agric Food Chem 2011, 59, 22-42.

 

6.From Table 4. Is there a Maillard browning reaction occurring as the temperature rises? Because both amine and carbonyl compounds are present in the sample. The initial pigments in the sample may have increased as a result of this.

Reply: As we know, Maillard reactions are initiated by a condensation of amino groups on protein, peptides, and amino acids with carbonyl groups on reducing sugars, resulting in Schiff base formation and rearrangement to Amadori or Heyns products[1], and some studies have shown that Maillard reactions usually take place at around 110 ℃[2-5]. During the fermentation in this study, the content of reducing sugar was gradually reduced, and the main product of the fermentation supernatant after alcohol sedimentation was polysaccharides, and the decolorization temperature of the A–722MP resin was selected from 20 to 50 ℃. Therefore, the Maillard reaction is difficult to occur during the experiment, and the pigment in the sample didn’t increase obviously because of the increase in temperature.

1. Hellwig, M.; Henle, T., Baking, Ageing, Diabetes: A Short History of the Maillard Reaction. Angewandte Chemie-International Edition 2014,53(39), 10316-10329.
2. Lan, X.;  Liu, P.;  Xia, S.;  Jia, C.;  Mukunzi, D.;  Zhang, X.;  Xia, W.;  Tian, H.; Xiao, Z., Temperature effect on the non-volatile compounds of Maillard reaction products derived from xylose-soybean peptide system: Further insights into thermal degradation and cross-linking. Food Chem. 2010,120(4), 967-972.
3. Smejkal, Q.;  Fiedler, T.;  Kurz, T.; Kroh, L., Simplified Kinetics and Colour Formation in Sucrose Solutions Based on A-Dicarbonyl Compounds. International Journal of Food Engineering 2007,3(4).
4. Wang, Y.;  Xie, J.;  Zhang, C.;  Xu, Y.; Yang, X., Effect of lipid on formation of Maillard and lipid-Maillard meaty flavour compounds in heated cysteine-xylose-methyl linoleate system. Flavour Fragrance J. 2022,37(5), 274-284.
5. Sun, F.;  Cui, H.;  Zhan, H.;  Xu, M.;  Hayat, K.;  Tahir, M. U.;  Hussain, S.;  Zhang, X.; Ho, C.-T., Aqueous Preparation of Maillard Reaction Intermediate from Glutathione and Xylose and its Volatile Formation During Thermal Treatment. J. Food Sci. 2019,84(12), 3584-3593.

 

7.Line 341-343. As stated in Line 334, FRAP is not for "free radical scavenging," but rather for "reducing capacity." Therefore, only the hydroxyl radical and the DPPH radical can be characterized as the method for scavenging free radicals.

Reply: Line348-350

The description of the method has been corrected as “the FRAP assay used to measure the reducing power of the antioxidants. Additionally, DPPH radical, and hydroxyl radical scavenging rates are widely used to measure the free radical scavenging ability of polysaccharides, and they are also indicators of the potential antioxidant capacity of polysaccharides.”

 

8. The primary active substances in charge of each mechanism (such as free radical scavengers and reducing power) should be presented to link with the antioxidant like structure-activity relationship when discussing antioxidant activity. Do the fractions still include any phenolic compounds and antioxidant peptides?

  Reply: Line365-378, 398-405, 443-449

The discussion of the relationship between the primary active substances in charge of each mechanism and polysaccharide structure has been supplemented in the conclusions on antioxidant activities. The purified polysaccharide fractions does not contain phenolic compounds and antioxidant peptides since Sephacryl S100.

It has been previously reported that the reducing properties are generally associated with the presence of reductones, which can break free–radical chain by donating a hydrogen atom. Reductones are also reported to react with certain precursors of peroxide, thus preventing peroxide formation. Tseng et al. found the introduction of the sulfate group might lead to the diminution of hydroxyl groups and the steric conformation of polysaccharides, which decreased the electron cloud density of active hydroxyl groups and prevented some active sulfate groups from binding to the metal ion, and it resulted in the decrease of the reducing power[59]. In this assay, TEPS with high donating–hydrogen abilities showed excellent reducing power and Lf1–a might donate more electrons or act as a more efficient electron donor in our FRAP assay. Yang et al. found the sugar content of polysaccharides was reduced after phosphorylation modification, so it was speculated that the reducing ability might be related to the phosphorylated structure and sugar content of polysaccharide[60]. The higher sugar content polysaccharide would contain, the stronger reducing ability it would show[61]. From the perspective of sugar content, the TEPS was speculated that the excellent reducing ability might be related to the sugar content of polysaccharide. Additionally, the reduction ability of polysaccharides may be related to the carboxymethyl structure, and some studies have shown that carboxymethylated Daucus carota polysaccharide had lower reducing ability than unmodified ones[62].

It was reported that the effect of antioxidant on DPPH radical scavenging was due to their hydrogen donating ability[67]. The sulfated derivatives showed excellent scavenging activity on DPPH radical, which might be attributable to its strong hydrogen–donating ability by activating the hydrogen atom of the anomeric carbon[68]. Chen et al. used the chlorosulfonic acid–pyridine method to chemically modify Codonopsis polysaccharides. Through in vivo antioxidant experiments, they found that the 2,2‐diphenyl‐1‐ picrylhydrazyl (DPPH)‐free radical scavenging activity of sulfated Codonopsis polysaccharides could reach 85.71%, which is higher than the free radical scavenging activity of Codonopsis polysaccharide without sulfate modification[69]. The results demonstrated that Lf1–a is more capable of donating hydrogen than Lf1–b.

The hydroxyl radical scavenging ability of polysaccharides may be related to the glycosidic bond structure. Ge et al. Reported that there are 1,4 and 1,3,4 glycoside bonds in T.fuciformis polysaccharide’s structure, and it showed good antioxidative and hydroxyl radical scavenging abilities[28]. 

Molecular weight play an important role in antioxidant activity, within a certain range of molecular weight, the smaller the molecular weight, the better the antioxidant activity of polysaccharides[71]. Meanwhile, studies have shown that the higher molecular weight component of laminaria japonica polysaccharide, the stronger the antioxidant activity[72]. In other studies on T.fuciformis polysaccharides (TFP), the results showed that the larger molecular weight TFP-1 component had stronger reducing power than the smaller molecular weight TFP-2 and TFP-3 components, which was consistent with the experimental results in this study[55]. The structure of T. fuciformis polysaccharides is different from other fungal polysaccharides in that it is composed of a mannan α‐glycosidic bond as the main chain and contains side chains of xylose and glucuronic acid. The polysaccharides with β‐(1, 3)‐glucan as the main chain and β‐(1, 6) branched chains play an important role in exerting biological activity. Additionally, from many studies, it can be found that polysaccharides containing glucuronic acid residues or glucuronic acid are more biologically active, the antioxidant activity of silver fungus polysaccharides was positively correlated with the glucuronide content[73,74]. The antioxidant activity of TEPS may be related to these factors, including the composition of monosaccharides, the type of glycosidic bond, the groups on the α‐D‐mannose backbone, the molecular weight, the glucuronide content. Certainly, accurate characterization of high molecular weight branched polysaccharides are necessary to better investigate conformational relationships as polysaccharides' chemical structure and chain conformation have a profound effect on their activity. The detailed chemical structure and in vivo bioactivity of TEPS fractions are still under investigation.

28. Ge, X.Y.; Huang, W.W.; Xu, X.Q.; Lei, P.; Sun, D.F.; Xu, H.; Li, S. Production, structure, and bioactivity of polysaccharide isolated from Tremella fuciformis XY. Int J Biol Macromol. 2020, 148, 173-181.
29. Zheng, Q.W.; He, B.L.; Wang, J.Y.; Huang, S.S.; Zou, Y.; Wei, T.; Ye, Z.W.; Guo, L.Q.; Lin, J.F. Structural Analysis and Antioxidant Activity of Extracellular Polysaccharides Extracted from Culinary-Medicinal White Jelly Mushroom Tremella fuciformis (Tremellomycetes) Conidium Cells. Int J Med Mushrooms 2020, 22, 489-500.
30. Tseng, Y.-H.; Yang, J.-H.; Mau, J.-L. Antioxidant properties of polysaccharides from Ganoderma tsugae. Food Chem 2008, 107, 732-738.
31. Yang, W.J.; Zhang, Y.Q.; Tang, A.; Ruan, Q.Q.; Huang, G.L. Preparation and antioxidant activity of phosphorylated polysaccharide from purple sweet potato. Chem Biol Drug Des 2021, 98, 828-834.
32. Chen, L.; Huang, G. Antioxidant activities of phosphorylated pumpkin polysaccharide. Int J Biol Macromol 2019, 125, 256-261.
33. Qi, K.; Xia, G.D.; Huang, G.L.; Huang, H.L. Extraction, chemical modification, and antioxidant activities of Daucus carota polysaccharide. Chem Biol Drug Des 2021, 98, 1098-1103.
34. Chen, Y.; Xie, M.-Y.; Nie, S.-P.; Li, C.; Wang, Y.-X. Purification, composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of Ganoderma atrum. Food Chem 2008, 107, 231-241.
35. Chen, Y.; Zhang, H.; Wang, Y.X.; Nie, S.P.; Li, C.; Xie, M.Y. Sulfated modification of the polysaccharides from Ganoderma atrum and their antioxidant and immunomodulating activities. Food Chem 2015, 186, 231-238.
36. Liu, C.; Chen, J.; Li, E.T.; Fan, Q.; Wang, D.Y.; Li, P.; Li, X.P.; Chen, X.Y.; Qiu, S.L.; Gao, Z.Z.; et al. The comparison of antioxidative and hepatoprotective activities of Codonopsis pilosula polysaccharide (CP) and sulfated CP. Int Immunopharmacol 2015, 24, 299-305.
37. Li, M.; Ma, F.; Li, R.; Ren, G.; Yan, D.; Zhang, H.; Zhu, X.; Wu, R.; Wu, J. Degradation of Tremella fuciformis polysaccharide by a combined ultrasound and hydrogen peroxide treatment: Process parameters, structural characteristics, and antioxidant activities. Int J Biol Macromol 2020, 160, 979-990.
38. Zha, S.; Zhao, Q.; Zhao, B.; Ouyang, J.; Mo, J.; Chen, J.; Cao, L.; Zhang, H. Molecular weight controllable degradation of Laminaria japonica polysaccharides and its antioxidant properties. J Ocean Univ China 2016, 15, 637-642.
39. Camesano, T.A.; Wilkinson, K.J. Single molecule study of xanthan conformation using atomic force microscopy. Biomacromolecules 2001, 2, 1184-1191.
40. Chen, H.X.; Zhang, M.; Xie, B.J. Quantification of uronic acids in tea polysaccharide conjugates and their antioxidant properties. J Agric Food Chem 2004, 52, 3333-3336.

Line 1488-152, 332-339

The purified polysaccharide fractions does not contain phenolic compounds and antioxidant peptides since Sephacryl S100.

Sephacryl High Resolution chromatography resins is a highly versatile size exclusion chromatography resins that offers a wide range of fractionation capabilities. The matrix of Sephacryl High Resolution resins is a cross-linked copolymer of allyl dextran and N,N’-methylene bisacrylamide. This crosslinking gives good rigidity and chemical stability according to the product description.

The separation mechanism of size-exclusion chromatography is dependent on the molecular size of the analytes and the pore size of the size exclusion chromatography stationary phase. On filtration through a bed of starch particles, high and low molecular weight compounds also behave differently[56]. Larger molecules excluded from the pores of the packing material are eluted more rapidly than smaller molecules, whereas the smaller molecules with greater access to the pores elute more slowly. The smallest molecular weight dextran had the longest retention time; the dextran with the largest molecular weight eluted first. Thus, the molecular weight of Lf1-a is greater than that of Lf1-b (Figure 4b).

 Thus, the purified polysaccharide fractions does not contain phenolic compounds and antioxidant peptides since Sephacryl S100.

56. Porath, J.; Flodin, P. Gel filtration: a method for desalting and group separation. Nature 1959, 183, 1657-1659.

 

9.Line 427. Because heat treatments and other aspects of food processing can undoubtedly affect the residual and effective activity, please be concerned about the safety issue and the carry-through property of the fraction.

Reply: In this study, the separation and purification of Tremella fuciformis polysaccharide was mainly to obtain Tremella fuciformis polysaccharide. The properties of Tremella fuciformis polysaccharide were relatively stable, and the activity of it was not affected at 20-50℃. In addition, the temperature of separation and purification is 20-50℃, under which Maillard browning reaction is difficult to occur. Therefore, it is difficult to produce carcinogens due to Maillard browning reaction. The activity of the protein may be affected by 20-50℃. If the activity of the protein is studied in the future, other separation and purification methods will be selected.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I accept the corrections made by authors. 

Reviewer 2 Report

All points raised by reviewer were addressed and answered point-by-point. So, the manuscript can be accepted for publication.