Production of Sustainable Postbiotics from Sugarcane Straw for Potential Food Applications

Round 1

Reviewer 1 Report

Review comments on “Production of sustainable postbiotics from sugarcane straw for potential food applications”

 

General comments

 

It is research that studying the sustainable postbiotics extracts produced through Saccharomyces cerevisiae fermentation using sugarcane straw as substrate and the authors exhibited relevant biological properties with potential use as food and nutraceutical ingredients. I think the authors did a lot of work on the postbiotics from the sugarcane but is there possible to show some process pictures in the manuscript? And for applied science, and feedback from the application is more important than the data, I hope the authors can add some information about the product feedback. And the manuscript should take a major revision before the final consideration. Please see the comments below,

 

1. For chemical detection, hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids are found with the application of HPLC, but is that possible to use a standard chemical to label the detected chemical? I think you can make a confirmation after that; otherwise, it is still a similar match.

2. It showed a good result on the IC 50 (mg/mL) and ORAC, I think you should show the result images in your results, they are better than the numbers;

3. what’s the conversion of sugarcane to the products? Is that possible to calculate the ratio?

4. Please update the figure, they are in poor design, for example, I can not find which one in three in Figure 4, and the design is not professional. Where is the indicator for the bar?

5. I think you may need a pathway for the process to show the different parts of sugarcane use, and then the readers can know better.

 

 

Author Response

First, at all, we would like to thank all the suggestions given by the reviewers under supervision of the editor, author’s have carefully undertaken the revision requests for your reconsideration. All the changes made in the manuscript are highlighted at blue color directly in text and the identified in the reviewer’s letter with the line number.

 

Reviewer 1

General comments

It is research that studying the sustainable postbiotics extracts produced through Saccharomyces cerevisiae fermentation using sugarcane straw as substrate and the authors exhibited relevant biological properties with potential use as food and nutraceutical ingredients. I think the authors did a lot of work on the postbiotics from the sugarcane but is there possible to show some process pictures in the manuscript? And for applied science, and feedback from the application is more important than the data, I hope the authors can add some information about the product feedback. And the manuscript should take a major revision before the final consideration. Please see the comments below,

1. For chemical detection, hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids are found with the application of HPLC, but is that possible to use a standard chemical to label the detected chemical? I think you can make a confirmation after that; otherwise, it is still a similar match.

The description of each external standard used for the quantification of all mentioned compounds within the different classes was added to the material and methods section and can be read now on lines 233-239.

2. It showed a good result on the IC 50 (mg/mL) and ORAC, I think you should show the result images in your results, they are better than the numbers.

Because the manuscript already has higher quantity of images authors decided not to add results of antioxidant activity besides the ones already presented. The most common way to present antioxidant activity results is through IC50 or concentration in equivalents and because that we decided to show the results this way.

3. what’s the conversion of sugarcane to the products? Is that possible to calculate the ratio?

Straw biomass is a complex structure mainly composed of cellulose (40-50 %), hemicellulose (25-30 %) and lignin (15-20 %). In addition, other components are present in minor amounts, such as inorganic compounds. The composition of the sugarcane straw used in this work was not determined, as this was not an objective of the work. As such, the conversion of specific substrates into specific products is not possible to be calculated.

4. Please update the figure, they are in poor design, for example, I cannot find which one in three in Figure 4, and the design is not professional. Where is the indicator for the bar?

Figure 4 caption was improved including the indications of each bar that were missing and the repeating information of y- and x-axis was removed. Now this figure is represented as Figure 5.

5. I think you may need a pathway for the process to show the different parts of sugarcane use, and then the readers can know better.

A scheme was added to the manuscript, as Figure 1 to better clarify the reader about the process (Lines 156-173).

Reviewer 2 Report

This study produced sustainable postbiotics extracts by S. cerevisiae fermentation using  sugarcane straw as substrate, which exhibited significant biological properties with potential use  as food and nutraceutical ingredient. The topic is interesting and adding to the research trend of postbiotics.

The manuscript needs extensive linguistic and syntax reusing  sugarcane straw as substrate, which exhibited significant biological properties with potential use  as food and nutraceutical ingredient. The topic is interesting and adding to the research trend of postbiotics.vision. For example the word (through) is wrongly written as (trough) ten times allover the text.

 

L14. Change (In the last years to (in recent years).

L15-17.   Change (and in this work, we propose the use of sugarcane straw, as sugar and biological components source 16 and as a sustainable alternative.) into (In this work, we propose using sugarcane straw as a source of sugar and biological components and a sustainable alternative.).

L17. Change ( as substrate) into ( as a substrate)

L18. Change ( as fermentative microorganism) into ( as a fermentative microorganism)

L18-19. Change ( To release the fermentable sugars, sugarcane straw went through a previous saccharification process) into (Sugarcane straw underwent a precedent saccharification step to release the fermentable sugars.)

L20. Change ( characterized on their total content) into ( characterized for their total content)

L22. Change (with predominance) into (with the predominance)

L24. Change (were the predominant) into ( were most prevalent)

L24-26. Change into (The current work evaluated the potential use of this postbiotic extract for food applications, its antioxidant activity, gut microbiota modulatory effect, and intestinal anti-inflammatory potential.)

L26-28. Change into (The resultant extracts showed considerable antioxidant activity and the capacity to reduce the pro-inflammatory mediators (i.e., interleukin 6, 8 and tumor necrosis factor-alpha) in Caco-2 cells.)

L28-30.  Change into (During the fecal fermentability assay, no modulatory effect was observed on the main beneficial bacteria, such as Lactobacillus and Bifidobacterium.)

L30.  Change (But a significant increase of short-chain fatty acids,) into (Nevertheless, a significant increase in short-chain fatty acids)

L31-32. Change (showed potential) into (showed the potential)

L32-35. Change the last sentence in the Abstract section into (Finally, sustainable postbiotic extracts produced by S. cerevisiae fermentation using sugarcane straw as substrate exhibited relevant biological properties with potential use as food and nutraceutical ingredients.

 

L63 correct (sacarose) into (saccharose) and (microrganisms) into (microorganisms)

L65-68. Change the sentence into (However, there is a need to find new substrate sources that are abundant, cheap, and renewable, e.g., the agricultural and industrial by-products, which represent a significant opportunity to produce postbiotics rich in other compounds with enhanced bioactivities at reduced costs   [7])

L69, Change (the use of) into (using) and (production such) into (production, such)

L70, Change (Recently it) into (Recently, it)

L74, Change (of the biorefinery) into (of biorefinery)

L75, Change (high added) into (high-added)

L75-76, Change (in a sustainable manner) into (sustainably).

L78, Change (in food industry) into (in the food industry) and (related with) into (related to)

L79, Change (costs making) into (costs, making)

L80, Change (The use of) into (Using)

L81, Change (an economic) into (an economical).

L81-83, Change the sentence into (Sugarcane (Saccharum officinarum L.) is a crop that produces a higher quantity of by-products discarded after harvesting, including straw residues [12])

L84-86, Correct the sentence into (Straw may have two final destinations: it can be used for land cover or burned to supply energy to industries.)

L88-90, Simplify the sentence into (After hydrolysis, the lignocellulosic material releases monosaccharides which microorganisms could use in alcohol fermentation [15])

L92, Change (known by their prebiotic) into (known for their prebiotic)

L94-95, Correct (This work have 94 explored and demontrated) into (This work has explored and demonstrated).

L97, Change (as substrate) into (as a substrate)

L101, Change (characterized focusing) into (characterized, focusing)

L102, Change (ingredient) into (ingredients).

L115, Correct (and grinded) into (and ground)

L116-117, Change the sentence (After that, the sugarcane straw were sieved for 116 10 min, with an amplitude of 100 and on a sieve 900 μm (Retsch GmbH, Haan, Germany).) into (Subsequently, it was sieved on a 900 µm sieve (Retsch GmbH, Haan, Germany), with an amplitude of 100 for 10 min.).

L118, Change (grams of straw) into (grams of straw powder). Form this point on the material is a powder and should be corrected all-over the text to be accurate.

L119, Correct (composed by) into (composed of)

L122-125, change the three sentences into (This supplement was added to the sodium citrate buffer solution, containing 4.31 g/L of citric acid (C6H8O7) 123 and 8.68 g/L sodium citrate (NA3C6H5O7), the pH was adjusted to 5.0 with NaOH (1 M) and autoclaved at 121 °C for 10 min..

 But the ratio of these two solutions should be clarified.

L126, Change (during) into (for)

L126-127, The ratio E/S should be mentioned.

L128, Correct the word (experimental) into (experimental)

L129, Change (Along the process samples) into (Along the process, samples)

L129-130, Change the sentence into (Along the process, samples were collected after  0, 24 and 48 h, and the total sugars decay was determined along the saccharification process.)

L138, (and incubated at 30 °C, for …….h). Mention the time duration of the incubation.

L143, Change (pH measurements and total sugar content.) into (pH and total sugar content measurements.)

L143-144, Change (mechanical cell lysis together with growth broth took place trough sonication) into (sonication was applied for mechanical cell lysis).

L145, Change (during 10 min) into (for 10 min)

L147, Change (was store) into (was stored) and (and freeze-dried) into (before freeze-drying)

L154.Change (plated) into plating

L162, Change (were added) into (were combined)

L223, The title of the section 2.5.1. Should be (Impact of the postbiotic extract on gut microbiota) while the previous title can be the first sentence under this title.

 

 

L272, the title of section2.5.3. should be simplified into (Cytotoxicity evaluation)

L291, Simplify the title of 2.5.4. into (Immunomodulatory effect) 

L663-664, The sentence should be modified as follows; (The sublethal concentration was defined  through the cytotoxicity assay before conducting the Caco-2 inflammatory assay.)

L666-667, modify the sentence into (The extract showed toxicity for concentrations above 3.125 mg/mL  (metabolic inhibition above 30 %) after 24-h treatment (Figure 5).

 

Illustrations

The title of Figure 1 (Figure 1. Total sugar content (■) (mg/mL, average ± SD) and S. cerevisiae growth (■) 335 (Log cycles, average ± SD) along 72 h fermentation process of sugarcane straw after saccharification.) is missing information. The title should indicate the material in which these determination were taken and under which conditions. The Figure should be self-explained.

 

Table 1. The title may be better in the next form (Levels of the major biochemical constituents (g/100g DW; means ± SD) in sugar cane straw powder extract before and after S. cerevisiae fermentation.)

Please unify the units of determination into g/100g DW and do not mention the units in the first column.

Table 2. LC-ESI-UHR-QqTOF-MS data of phenolic compounds identified in straw extract after saccharification and with and without S. cerevisiae fermentation. (the results of with and without S. cerevisiae fermentation are not clear in the table. In which columns or rows? This should be clairified)

The definitions of the terms appearing in the first row should be explained under the table (Formula H, m/z Theoretical mass, m/z Measured mass , error, MS/MS fragment)

 

Table 3. The definitions of ABTS IC50, DPPH IC50 and ORAC should be explained under the table. The units μmol TE/g should be explained.

Figure 2. The line of y-axis is absent and should be added and that of x-axis is very faint and its color should be intensified.

Figure 2. The title should be a little modified into (The changes in pH value changes of simulated gut microbiota fermentation (five donors) along 48 h in the presence of inoculum control (IC, ■), fructooligosaccharides (FOS, ■), and straw extract after fermentation with S. cerevisiae (■). Different letters mean significant 531 differences (p < 0.05) between samples.). The inoculum control should be sufficiently explained under the figure.

Figure 3. Since y-axis is the same for all the four histograms, and also x-axis, the legends for each should be mentioned once,  at the left of figures (Bacterial cell viability) and under the figure (time).

Figure 4, needs also reorganization to avoid unnecessary repetition in the information appearing on the histogram.

Figure 5. The title should be reformulated into (The metabolic inhibitory action of the fermented sugarcane straw extract on the intestinal Caco-2 cell line, as a base for estimating its cytotoxicity profile. The dotted line shows the 30% cytotoxicity threshold. Values expressed are the mean ± standard deviation.),

 

Figure 6. The line of y-axis should be added and that of x-axis should be intensified in color. The title of the figure can be improved as explained in the previous ones.

Discussion needs further comparisons with recent similar work on postbiotics.

The authors are advised to compared their work with recent works on postbiotics such as: (Lipolytic postbiotic from lactobacillus paracasei manages metabolic syndrome in albino wistar rats. Osman et al. 2021).

 

 

 

 

 

 

Author Response

First, at all, we would like to thank all the suggestions given by the reviewers under supervision of the editor, author’s have carefully undertaken the revision requests for your reconsideration. All the changes made in the manuscript are highlighted at blue color directly in text and the identified in the reviewer’s letter with the line number.

This study produced sustainable postbiotics extracts by S. cerevisiae fermentation using sugarcane straw as substrate, which exhibited significant biological properties with potential use as food and nutraceutical ingredient. The topic is interesting and adding to the research trend of postbiotics.

The manuscript needs extensive linguistic and syntax reusing sugarcane straw as substrate, which exhibited significant biological properties with potential use as food and nutraceutical ingredient. The topic is interesting and adding to the research trend of postbiotics vision. For example, the word (through) is wrongly written as (trough) ten times all over the text.

Authors would like to thanks’ the reviewer corrections and input in manuscript improvement. The wrongly written word “trough” was corrected to “through” in the entire manuscript.

L14. Change (In the last years to (in recent years).

It was correct now in line 16.

L15-17. Change (and in this work, we propose the use of sugarcane straw, as sugar and biological components source 16 and as a sustainable alternative.) into (In this work, we propose using sugarcane straw as a source of sugar and biological components and a sustainable alternative).

The sentence was corrected as suggested (Lines 17-19).

L17. Change (as substrate) into (as a substrate)

Corrected on line 19.

L18. Change (as fermentative microorganism) into (as a fermentative microorganism)

Corrected on line 20.

L18-19. Change (To release the fermentable sugars, sugarcane straw went through a previous saccharification process) into (Sugarcane straw underwent a precedent saccharification step to release the fermentable sugars.)

Corrected on line 21-22.

L20. Change (characterized on their total content) into (characterized for their total content)

Corrected on line 22.

L22. Change (with predominance) into (with the predominance)

Corrected on line 24.

L24. Change (were the predominant) into (were most prevalent)

Corrected on line 26.

L24-26. Change into (The current work evaluated the potential use of this postbiotic extract for food applications, its antioxidant activity, gut microbiota modulatory effect, and intestinal anti-inflammatory potential.)

Corrected on lines 26-29.

L26-28. Change into (The resultant extracts showed considerable antioxidant activity and the capacity to reduce the pro-inflammatory mediators (i.e., interleukin 6, 8 and tumor necrosis factor-alpha) in Caco-2 cells.)

Corrected on lines 29-31.

L28-30. Change into (During the fecal fermentability assay, no modulatory effect was observed on the main beneficial bacteria, such as Lactobacillus and Bifidobacterium.)

Corrected on lines 31-33.

L30. Change (But a significant increase of short-chain fatty acids,) into (Nevertheless, a significant increase in short-chain fatty acids)

Corrected in line 33.

L31-32. Change (showed potential) into (showed the potential)

Corrected in line 35.

L32-35. Change the last sentence in the Abstract section into (Finally, sustainable postbiotic extracts produced by S. cerevisiae fermentation using sugarcane straw as substrate exhibited relevant biological properties with potential use as food and nutraceutical ingredients.

Corrected in lines 36-38.

L63 correct (sacarose) into (saccharose) and (microrganisms) into (microorganisms)

Corrected in line 65.

L65-68. Change the sentence into (However, there is a need to find new substrate sources that are abundant, cheap, and renewable, e.g., the agricultural and industrial by-products, which represent a significant opportunity to produce postbiotics rich in other compounds with enhanced bioactivities at reduced costs   [7])

Corrected in lines 67-70.

L69, Change (the use of) into (using) and (production such) into (production, such)

Corrected in lines 69-71.

L70, Change (Recently it) into (Recently, it)

Corrected in line 72.

L74, Change (of the biorefinery) into (of biorefinery)

Corrected in line 76.

L75, Change (high added) into (high-added)

Corrected in line 77.

L75-76, Change (in a sustainable manner) into (sustainably).

Corrected in line 78.

L78, Change (in food industry) into (in the food industry) and (related with) into (related to)

Corrected in line 80.

L79, Change (costs making) into (costs, making)

Corrected in line 80.

L80, Change (The use of) into (Using)

Corrected in line 82.

L81, Change (an economic) into (an economical).

Corrected in line 83.

L81-83, Change the sentence into (Sugarcane (Saccharum officinarum L.) is a crop that produces a higher quantity of by-products discarded after harvesting, including straw residues [12])

Corrected in lines 83-85.

L84-86, Correct the sentence into (Straw may have two final destinations: it can be used for land cover or burned to supply energy to industries.)

Corrected in lines 86-87.

L88-90, Simplify the sentence into (After hydrolysis, the lignocellulosic material releases monosaccharides which microorganisms could use in alcohol fermentation [15])

Corrected in lines 89-91.

L92, Change (known by their prebiotic) into (known for their prebiotic)

Corrected in line 93.

L94-95, Correct (This work have 94 explored and demonstrated) into (This work has explored and demonstrated).

Corrected in line 96.

L97, Change (as substrate) into (as a substrate)

Corrected in lines 98-99.

L101, Change (characterized focusing) into (characterized, focusing)

Corrected in line 102.

L102, Change (ingredient) into (ingredients).

Corrected in line 103.

L115, Correct (and grinded) into (and ground)

Corrected in line 116.

L116-117, Change the sentence (After that, the sugarcane straw were sieved for 116 10 min, with an amplitude of 100 and on a sieve 900 μm (Retsch GmbH, Haan, Germany).) into (Subsequently, it was sieved on a 900 µm sieve (Retsch GmbH, Haan, Germany), with an amplitude of 100 for 10 min.).

Corrected in lines 117-118.

L118, Change (grams of straw) into (grams of straw powder). Form this point on the material is a powder and should be corrected all-over the text to be accurate.

Corrected in line 119.

L119, Correct (composed by) into (composed of)

Corrected in line 120.

L122-125, change the three sentences into (This supplement was added to the sodium citrate buffer solution, containing 4.31 g/L of citric acid (C6H8O7) 123 and 8.68 g/L sodium citrate (NA3C6H5O7), the pH was adjusted to 5.0 with NaOH (1 M) and autoclaved at 121 °C for 10 min. But the ratio of these two solutions should be clarified.

The sentence was rewritten and the ratio of biomass to solvents was added, in lines 122-125.

L126, Change (during) into (for)

Corrected in line 127.

L126-127, The ratio E/S should be mentioned.

The ratio of enzyme per gram of cellulose was added on lines 126-127.

L128, Correct the word (experimental) into (experimental)

Corrected in line 129.

L129, Change (Along the process samples) into (Along the process, samples)

Corrected in line 130.

L129-130, Change the sentence into (Along the process, samples were collected after 0, 24 and 48 h, and the total sugars decay was determined along the saccharification process.)

Corrected in lines 130-131.

L138, (and incubated at 30 °C, for …….h). Mention the time duration of the incubation.

Corrected in line 142.

L143, Change (pH measurements and total sugar content.) into (pH and total sugar content measurements.)

Corrected in line 145.

L143-144, Change (mechanical cell lysis together with growth broth took place trough sonication) into (sonication was applied for mechanical cell lysis).

Corrected in line 145.

L145, Change (during 10 min) into (for 10 min)

Corrected in line 146.

L147, Change (was store) into (was stored) and (and freeze-dried) into (before freeze-drying)

Corrected in line 149.

L154.Change (plated) into plating

Corrected in line 155.

L162, Change (were added) into (were combined)

Corrected in line 180.

L223, The title of the section 2.5.1. Should be (Impact of the postbiotic extract on gut microbiota) while the previous title can be the first sentence under this title.

Corrected in lines 243-245.

L272, the title of section2.5.3. should be simplified into (Cytotoxicity evaluation)

Corrected in line 293.

L291, Simplify the title of 2.5.4. into (Immunomodulatory effect) 

Corrected in line 310.

L663-664, The sentence should be modified as follows; (The sublethal concentration was defined through the cytotoxicity assay before conducting the Caco-2 inflammatory assay.)

Corrected in lines 692-693.

L666-667, modify the sentence into (The extract showed toxicity for concentrations above 3.125 mg/mL (metabolic inhibition above 30 %) after 24-h treatment (Figure 5).

Corrected in lines 696-697.

 

Illustrations

The title of Figure 1 (Figure 1. Total sugar content (■) (mg/mL, average ± SD) and S. cerevisiae growth (■) 335 (Log cycles, average ± SD) along 72 h fermentation process of sugarcane straw after saccharification.) is missing information. The title should indicate the material in which these determinations were taken and under which conditions. The Figure should be self-explained.

The figure 1 title was changed to “Total sugar content (■) (mg/mL, average ± SD) and S. cerevisiae growth (■) (Log cycles, average ± SD) measured in cell broth along 72 h fermentation process of sugarcane straw that went through saccharification.” for better clarification on where samples were collected (now Figure 2).

Table 1. The title may be better in the next form (Levels of the major biochemical constituents (g/100g DW; means ± SD) in sugar cane straw powder extract before and after S. cerevisiae fermentation.) Please unify the units of determination into g/100g DW and do not mention the units in the first column.

Table 1 title were rewritten to “Levels of the major biochemical constituents (mg/g DW; means ± SD) in sugarcane straw powder extract before and after S. cerevisiae fermentation.”

Units were unified to mg/g DW.

Table 2. LC-ESI-UHR-QqTOF-MS data of phenolic compounds identified in straw extract after saccharification and with and without S. cerevisiae fermentation. (the results of with and without S. cerevisiae fermentation are not clear in the table. In which columns or rows? This should be clarified)

This title had an error, because initially we had in the same table compounds I&D and quantification data, and the title was not rewritten properly. Now it can be read “LC-ESI-UHR-QqTOF-MS data of phenolic compounds identified in straw extract after saccharification.”

The definitions of the terms appearing in the first row should be explained under the table (Formula H, m/z Theoretical mass, m/z Measured mass , error, MS/MS fragment)

The required information was added on lines 420-424.

Table 3. The definitions of ABTS IC50, DPPH IC50 and ORAC should be explained under the table. The units μmol TE/g should be explained.

This table was wrongly identified. It can be read that this is table 4 and not table 3. An explanation of the terms was included under the table 4 (L533-534).

Figure 2. The line of y-axis is absent and should be added and that of x-axis is very faint and its color should be intensified.

The Figure 2 quality was improved. Now as Figure 3.

 

Figure 2. The title should be a little modified into (The changes in pH value changes of simulated gut microbiota fermentation (five donors) along 48 h in the presence of inoculum control (IC, ■), fructooligosaccharides (FOS, ■), and straw extract after fermentation with S. cerevisiae (■). Different letters mean significant 531 differences (p < 0.05) between samples.). The inoculum control should be sufficiently explained under the figure.

Figure 2 title was changed as suggested, and the figure axis was improved.

Figure 3. Since y-axis is the same for all the four histograms, and x-axis, the legends for each should be mentioned once, at the left of figures (Bacterial cell viability) and under the figure (time).

Figure 3 was corrected was suggested by the reviewer, now as Figure 4.

Figure 4 needs also reorganization to avoid unnecessary repetition in the information appearing on the histogram.

Figure 4 was corrected was suggested by the reviewer, now as Figure 5.

Figure 5. The title should be reformulated into (The metabolic inhibitory action of the fermented sugarcane straw extract on the intestinal Caco-2 cell line, as a base for estimating its cytotoxicity profile. The dotted line shows the 30% cytotoxicity threshold. Values expressed are the mean ± standard deviation.)

Figure 5 title was revised and presented now as Figure 6.

Figure 6. The line of y-axis should be added and that of x-axis should be intensified in color. The title of the figure can be improved as explained in the previous ones.

Figure 6 title and image quality was revised and presented now as Figure 7.

 

Discussion needs further comparisons with recent similar work on postbiotics.

The authors are advised to compare their work with recent works on postbiotics such as: (Lipolytic postbiotic from lactobacillus paracasei manages metabolic syndrome in albino wistar rats. Osman et al. 2021).

As suggested recent works on postbiotics were added to the discussion on lines 520-524, 760-763.

Round 2

Reviewer 2 Report

The manuscript has been greatly improved.