Pectinase Production from Cocoa Pod Husk in Submerged Fermentation and Its Application in the Clarification of Apple Juice
, Patricia Beatriz Gruening de Mattos 1, Hervé Louis Ghislain Rogez 2 and Carlos Ricardo Soccol 1Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript of Rozendo et al., produced a pectinase from cocoa pod husk via the submerged fermentation using Aspergillus niger and applied it the apple juice for clarification purposes. The realm of biotechnology and the application of organisms to produce highly relevant chemicals/enzymes is hot topic and therefore this manuscript is worthy of investigation and deserves recognition in this field of research. The approach of the authors is well performed, the article is well written and the applied methodology merits publication in Fermentation. However, before considering this article for publication, there are some major and minor comments which are ought to be addressed:
- Please be consistent in the manuscript: use a spacing between number and unit. Sometimes a spacing is used, sometimes not.
- Lines 54-55: why is the degradation by micro-organisms considered a serious environmental problem? The problem is often not in the degradation itself but with pests associated with the disposal of waste material on site. Or emissions related to the burning of waste biomass.
- Please mind the subscripts in the chemicals provided in section 2.3.
- Please remove brackets in Line 134.
- Line 144, there is a line under “°”.
- Something is not grammatically correct in Line 148.
- Line 172: how was the juice extracted from the suspension? Please elaborate.
- Line 183: what was the pore size of the filter material?
- Line 223: how was the lignin, hemicellulose and cellulose content determined? This is not mentioned in the materials and methods section.
- Lines 224-229 are a repetition of the materials and methods section. The authors can just refer to the composition of the medium listed in the materials and methods section.
- Please mind significant figures throughout the text (and keep them consistent for the same parameters).
- The authors present a model depicted in Equation 3. Is there a statistical evaluation of this model? Was a lack-of-fit test determined? Were all parameters significant (it seems so since all the terms are included in the model).
- Once an abbreviation is used in the text alongside the full name of the abbreviation, just the abbreviation can be used throughout the text. Not necessary to explain the abbreviation multiple times in the manuscript.
- Do the authors have any idea regarding the composition of the extracted apple juice?
Comments on the Quality of English LanguageSee comments above.
Author Response
REVIEWER 1
The manuscript of Rozendo et al., produced a pectinase from cocoa pod husk via the submerged fermentation using Aspergillus niger and applied it the apple juice for clarification purposes. The realm of biotechnology and the application of organisms to produce highly relevant chemicals/enzymes is hot topic and therefore this manuscript is worthy of investigation and deserves recognition in this field of research. The approach of the authors is well performed, the article is well written and the applied methodology merits publication in Fermentation. However, before considering this article for publication, there are some major and minor comments which are ought to be addressed:
Response: Authors want to thank reviewer for the revision and insightful comments. We think that with inclusion of reviewer’s suggestions/comments the revised version is significantly improved.
- Please be consistent in the manuscript: use a spacing between number and unit. Sometimes a spacing is used, sometimes not.
Response: Yes, thank you. The spacing between number and unit was revised throughout the manuscript.
- Lines 54-55: why is the degradation by micro-organisms considered a serious environmental problem? The problem is often not in the degradation itself but with pests associated with the disposal of waste material on site. Or emissions related to the burning of waste biomass.
Response: Yes, thank you. About CPH disposal in the environment and its associated problems, we have added the following text: “Their disposal in the environment can cause serious environmental problems, such as degradation by microorganisms [7], mainly fungus that are capable to degrade these biomass. In favorable climatic conditions, CPH can become a breeding ground for harmful fungal pathogens like Marasmius perniciosus, Phytophthora palmivora, and P. megakarya. The Phytophthora species cause pod rot, or black pod disease, which affects susceptible cocoa plant genotypes by forming small dark lesions on the pods that quickly spread across the surface and into the internal tissues, including the beans and pulp. In the 1980s, Brazil experienced a severe outbreak of the witches’ broom fungus (Moniliophthora perniciosa), which resulted in the death of thousands of trees and a significant reduction in almond production for nearly twenty years. This phytosanitary issue is thought to have been exacerbated by improper disposal and management of cocoa waste. Mismanagement of CPH can compromise the quality of the next harvest and the health of the plants, leading to substantial economic losses (Vásquez et al. 2019).” Please, see: lines 54-66.
- Please mind the subscripts in the chemicals provided in section 2.3.
Response: Yes, subscripts in the chemicals were revised.
- Please remove brackets in Line 134.
Response: Brackets were removed. Please, see line 143.
- Line 144, there is a line under “°”.
Response: The line under “°” was removed. Please, see line 152.
- Something is not grammatically correct in Line 148.
Response: The sentence was corrected. Thank you. Please, see line 157.
- Line 172: how was the juice extracted from the suspension? Please elaborate.
Response: Thank you. The text was revised. Please, see lines 178-180.
- Line 183: what was the pore size of the filter material?
Response: A qualitative filter paper Whatman Grade 1 (pore size 11 µm) was used. The information was added to the text. Please, see line 190.
- Line 223: how was the lignin, hemicellulose and cellulose content determined? This is not mentioned in the materials and methods section.
Response: The determination of CPH composition was determined according to the National Renewable Energy Laboratory (NREL) procedures (Sluiter et al., 2012, 2008c, 2008a, 2008b). Please, see lines 94-99.
- Lines 224-229 are a repetition of the materials and methods section. The authors can just refer to the composition of the medium listed in the materials and methods section.
Response: Yes, thank you. The information of medium composition was removed. Please, see lines 232-235.
- Please mind significant figures throughout the text (and keep them consistent for the same parameters).
Response: Yes, thank you very much for this observation. All figures of the manuscript were revised and placed near the related data and are now consistent with parameters.
- The authors present a model depicted in Equation 3. Is there a statistical evaluation of this model? Was a lack-of-fit test determined? Were all parameters significant (it seems so since all the terms are included in the model).
Response: Thank you very much for these comments and suggestions. The statistical analysis of results was carried out with the use of Software Statistica Ultimate Academic (StatSoft, South America). The Analysis of Variance and the graphic of Predicted versus Observed results were included in Supplementary Material. The information and discussion is presented in the manuscript. Please, see lines 307-316.
- Once an abbreviation is used in the text alongside the full name of the abbreviation, just the abbreviation can be used throughout the text. Not necessary to explain the abbreviation multiple times in the manuscript.
Response: Yes, the reviewer is right. Thank you for pointing out these mistakes. All full names and related abbreviations were revised.
- Do the authors have any idea regarding the composition of the extracted apple juice?
Response: Yes, thank you. The composition of apple juice in terms of sugars was added to the text. “An apple juice with 8.13 °Brix (81,13 g/L total sugars), organic acids ((9.5 µmol/mL of galacturonic acid) and pH 4.21 was employed in enzymatic clarification using produced pectinase CauPec”. Please, see lines 370-372.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study used cocoa pod husk (CPH) and its extracted pectin as a potential substrate for the production of pectinase and to test the enzyme in the clarification process of apple juice. The authors optimized the fermentation medium conditions, measured the enzyme kinetics of pectinase, purified and concentrated the pectinase, and greatly improved the activity of the pectinase. Finally, the authors also carried out clarification test of apple juice with this pectinase, and the effect is equivalent to that of the commercial enzyme. The results suggest a low-cost way to prepare pectinase. This study fits the scope of the journal. The authors conducted a systematic study, and the results can provide inspiration and reference for researchers in the field of food microbial fermentation. It is suggested that the article may be accepted for publication after careful revision.
Language: The language of this article should be carefully revised by a native English speaker.
Title: I suggest revise the title
Abstract:
L16, please add the strain number
Introduction:
L32, please add the international number of the enzyme (EC number)
L34, L36, L38, L41, L66, et al., please check the citation format for the entire manuscript.
L42, revise this sentence.
L56, add a reference
L58-60, revise this sentence.
L60-61, add a reference
L57-71, Please re-structure the content of this paragraph. In the first half, the author points out that CPH contains a high amount of pectin, which can be used to extract pectin and produce pectinase, but in the second half, it is about the current utilization of CPH, and these utilization have no relationship with pectin and pectinase.
Materials and methods
L86: As the core material of this test, the physical and chemical characteristics and main component indexes of CPH should be given.
L87: Why only choose 1.18-2.00 mm and smaller than 0.84mm? Please explain it
L103-104: 0.84, MgSO4, , et al., please check for these small formatting errors
L126, table 1, the decimal point of a number, please revise them. And check the whole manuscript on the decimal point.
L-137, delete”-CCRD”.
L143, please provide the volume of flasks, the volume of fermentation liquid, the amount of inoculation, etc
L144, “during” change to “for”,
L148-149, revise the sentence
L195, L199, L206, L212, please provide the manufacturers of the instruments
Results and discussion
L308-315: What is the author's purpose in listing these citations? Is it to reflect that the enzyme activity of their fermentation is significantly higher than that reported in the literature?
L325: pH eventually dropped to a minimum of 2.62? What might be the acid that the bacteria metabolizes? Authors should cite relevant literature to discuss it.
L342: what is MF? What is UF?
Comments on the Quality of English LanguageModerate editing of English language is required.
Author Response
REVIEWER 2
This study used cocoa pod husk (CPH) and its extracted pectin as a potential substrate for the production of pectinase and to test the enzyme in the clarification process of apple juice. The authors optimized the fermentation medium conditions, measured the enzyme kinetics of pectinase, purified and concentrated the pectinase, and greatly improved the activity of the pectinase. Finally, the authors also carried out clarification test of apple juice with this pectinase, and the effect is equivalent to that of the commercial enzyme. The results suggest a low-cost way to prepare pectinase. This study fits the scope of the journal. The authors conducted a systematic study, and the results can provide inspiration and reference for researchers in the field of food microbial fermentation. It is suggested that the article may be accepted for publication after careful revision.
Response: Authors want to thank reviewer for the careful revision and detailed comments that could improve the revised version of our paper.
Language: The language of this article should be carefully revised by a native English speaker.
Response: Yes, thank you. The manuscript was completely revised.
Title: I suggest revise the title
Response: Yes, thank you for the suggestion. The title was modified to “Pectinase production from cocoa pod husk in submerged fermentation and its application in the clarification of apple juice”
Abstract:
L16, please add the strain number
Response: The number of the strain was added. Please, see line 15
Introduction:
L32, please add the international number of the enzyme (EC number)
Response: Yes, thank you. The EC number was added to the text. Please, see line 31.
L34, L36, L38, L41, L66, et al., please check the citation format for the entire manuscript.
Response: All citations were revised. Thank you.
L42, revise this sentence.
Response: The sentence was revised. Please, see lines 41-43.
L56, add a reference
Response: Thank you. A reference was cited.
L58-60, revise this sentence.
Response: Thank you. The sentence was revised. Please, see lines 68-70.
L60-61, add a reference
Response: The reference Vandeberghe et al., 2022 was included.
L57-71, Please re-structure the content of this paragraph. In the first half, the author points out that CPH contains a high amount of pectin, which can be used to extract pectin and produce pectinase, but in the second half, it is about the current utilization of CPH, and these utilization have no relationship with pectin and pectinase.
Response: Thank you for this suggestion. Paragraph was re-structured and divided into two parts for a better understanding and fluidity. Some references were added. Please, see lines 68-72 and lines 73-81.
Materials and methods
L86: As the core material of this test, the physical and chemical characteristics and main component indexes of CPH should be given.
Response: Thank you. CPH was characterized according to official methodologies. Please, see lines 95-98.
L87: Why only choose 1.18-2.00 mm and smaller than 0.84mm? Please explain it
Response: Yes, this is an important detail that is linked to a good homogenization and mass transfer of nutrients during fermentation. Please, see lines 98-99.
L103-104: 0.84, MgSO4, , et al., please check for these small formatting errors
Response: Yes, thank you. The formatting errors were corrected Please, see lines 114 and 115.
L126, table 1, the decimal point of a number, please revise them. And check the whole manuscript on the decimal point.
Response: Thank you. The whole manuscript was revised.
L-137, delete”-CCRD”.
Response: Ok, it was removed.
L143, please provide the volume of flasks, the volume of fermentation liquid, the amount of inoculation, etc
Response: The volume of Erlenmeyer flask was informed. Please, see lines 155-156.
L144, “during” change to “for”,
Response: Ok, it was changed.
L148-149, revise the sentence
Response: Thank you. The sentence was revised. Please, see lines 160-161.
L195, L199, L206, L212, please provide the manufacturers of the instruments
Response: The manufacturers were informed in lines 207, 212, 218, 224.
Results and discussion
L308-315: What is the author's purpose in listing these citations? Is it to reflect that the enzyme activity of their fermentation is significantly higher than that reported in the literature?
Response: Yes, the information about previously reported pectinase activities and references were added to the text to highlight the enzymatic activity that was reached in this work using CPH as substrate. So, a sentence was added to the text to discuss it. Please, see lines 321-322.
L325: pH eventually dropped to a minimum of 2.62? What might be the acid that the bacteria metabolizes? Authors should cite relevant literature to discuss it.
Response: Thank you for this observation. The fungal strain A. niger NRRL 2270 is a known producer of citric acid.
L342: what is MF? What is UF?
Response: The abbreviations are: MF – microfiltration and UF – ultrafiltrafiltration. The information was added in section 2.6, line 163.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors addressed most of the comments raised by the reviewer and therefore the reviewer accepts this manuscript for publication in Fermentation.
