Identification of Phytochemical Compounds, Functional Properties and Antioxidant Activity of Germinated Purple Corn Protein Concentrate and Its Gastrointestinal Hydrolysates

Round 1

Reviewer 1 Report

Review of Manuscript Number: ID: agronomy-1856693

Title:   “Identification of Phytochemical Compounds, Functional Properties and Antioxidant Activity of Purple Corn Protein Contrate Germinated and its Gastrointestinal Hydrolysates”

Journal: Agronomy

 

 

The authors of the manuscript in their research took up an interesting and important research topic. Observing the continuous development of civilization diseases, it is important to look for new ways to eliminate their effects. One of them may be just harnessing the health potential of Purple Corn Protein Concentrate. In conclusion, the manuscript is interesting.

I suggest major editorial changes:

3 line – “contrate” should be “concentrate”

18 line – “flavonoid content” should be “ flavanols (TFLC)”

51-52 lines - µg/g, DM should be µg/g DM

94 line – 2,20- Azino……… should be 2,2’-Azino……

123 line - How this extract was prepared should be reported

159 line- 250 µl sample - Indicate how the sample was prepared

159 line – 1 m/mL should be 1 mg/mL

170, 183 line- How this extract was prepared should be reported

- In the abstract, the authors use the term DW, please change to DM. It is about expressing given values in terms of the dry mass of a given sample. In the text (line 50-52) the authors use the term dry metter, which is correct. Standardize throughout the text.

-110 line- The particle size should be reported in µm, i.e. <500 µm

- 110-111 lines- Provide more details to the deffated process i.e. time that the solvent was removed

-131 line- The values ​​should be based on the dry weight of the sample, i.e. DM. Similarly, make changes in points from 2.4. to 2.7 and 2.11.1-2.11.3.

-279 line- 2.50 m should be 2.50 mL, Replace the + symbol with "plus"

-332-334 - There should be a dot after the title. This consideration should apply to the entire manuscript.  Complete the title with the abbreviations: (PCF) and (GH25 and GH35)

- linie 3.1.1. - Add a sentence indicating that in FIG. 1 are the results concerning the solubility of the tested material

- Fig 1- % protein solubility should be protein solubility (%)

- tab. 1 - Modify the title, i.e. include hydrolysates in the title and provide abbreviations

-372 line (Table 1) should be (Tabel 2)

- 449 and 481 line – Table 4 should be Table 3

- 501 i 520 lines- Figure 2a and b should be Figure 3a and b

-535 and 561 lines- Enter information that the results of the degree of PCPC hydrolysis are included in table 4

- 561 line – Tab. 5 should be Tab. 4

569 and 623 lines- Table 6 should be Table 5

623 line - …………corn protein contrates should be ……concentrates

Conclusion asks you to add the practical aspect of research.

 

 

Author Response

Reviewer # 1

The authors of the manuscript in their research took up an interesting and important research topic. Observing the continuous development of civilization diseases, it is important to look for new ways to eliminate their effects. One of them may be just harnessing the health potential of Purple Corn Protein Concentrate. In conclusion, the manuscript is interesting.

 

I suggest major editorial changes:

 

3 line – “contrate” should be “concentrate”

Answer: this mistake was corrected: “Identification of Phytochemical Compounds, Functional Properties and Antioxidant Activity of Purple Corn Protein Concentrate Germinated and its Gastrointestinal Hydrolysates

18 line – “flavonoid content” should be “ flavanols (TFLC)”

Answer: this error was corrected for: total flavonoid content (TFC), total flavonol content

51-52 lines - µg/g, DM should be µg/g DM

Answer: this mistake was corrected in all the manuscript: (RE)/100g DM) and TFL (7975.59 mg RE/100g DM). PCPCs presented better functional properties than the hydrolysates. PCPC25 presented a protein solubility percentage of 59.43% at pH 8.0; 27.77% water absorption capacity (WAC) and 24.94% oil absorption capacity (OAC). PCPCs showed high values of antioxidant activity. PCPC25 showed values of ABTS (570.97 µmol trolox equivalents (TE)/g DM) and FRAP (772.85 µmol TE/g DM). PCPCs hydrolysates were less active with ABTS (74.12 µmol TE/g DM) and FRAP (59.42 µmol TE/g DM) values…

 

94 line – 2,20- Azino……… should be 2,2’-Azino……

Answer: this mistake was corrected: 2,2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS)…

123 line - How this extract was prepared should be reported

Answer: this step was explained:

2.6. Total Polyphenol Content (TPC).

TPC was performed by UV-visible spectrophotometry using the method described by Samaniego et al. [28]. PCPCs and purple corn flour samples were used to obtain the extracts for analysis. PCPCs and purple corn flour were dissolved in the mix of methanol and water (70:30 v:v). This step was repeated several times until the plant material was exhausted. 1 mL of diluted extract

 

159 line- 250 µl sample - Indicate how the sample was prepared

 

Answer: this step was explained: 0.3 g of lyophilized PCPC was mixed with 5 mL of pH 1.0 buffer (KCl 0.2 N and HCl 0.2 N)

159 line – 1 m/mL should be 1 mg/mL

Answer: this mistake was corrected

170, 183 line- How this extract was prepared should be reported

Answer: this was added in the manuscript

2.5. Total Proanthocyanin Content (TPAC).

 TPAC was quantified by butanol acid assay. 250 µL of lyophilized sample (1mg/mL) was mixed with 3.25 mL of n-butanol-HCl in proportion (50:50, v:v)…

 

- In the abstract, the authors use the term DW, please change to DM. It is about expressing given values in terms of the dry mass of a given sample. In the text (line 50-52) the authors use the term dry metter, which is correct. Standardize throughout the text.

Answer: this comment was corrected in all the manuscript

 

-110 line- The particle size should be reported in µm, i.e. <500 µm

Answer: this error was corrected for:

a flour which was sieved (<500 µm).

- 110-111 lines- Provide more details to the deffated process i.e. time that the solvent was removed

 

-131 line- The values ​​should be based on the dry weight of the sample, i.e. DM. Similarly, make changes in points from 2.4. to 2.7 and 2.11.1-2.11.3.

Answer: this comment was attended in all the manuscript

-279 line- 2.50 m should be 2.50 mL, Replace the + symbol with "plus"

Answer: this was corrected for: 2.11. Antioxidant activity.

2.11.1. Ferric-Reducing Antioxidant Power (FRAP) Method.

The FRAP reagent was prepared by mixing (25 mL of 300 mM sodium acetate buffer at pH 3.60 plus 2.50 mL of 10 mM TPTZ diluted in 40 mM HCl plus 2.50 mL of 20 mM ferric chloride hexahydrate)…

 

-332-334 - There should be a dot after the title. This consideration should apply to the entire manuscript.  Complete the title with the abbreviations: (PCF) and (GH25 and GH35)

Answer: this was corrected for: 3.1. Functional Properties:

3.1.1 Protein Solubility (PS) of purple protein corn concentrate (PCPC) and Hydrolysates (GH25, DH25, GH35 and DH35). 

 

 

Table 1. Functional properties of purple corn protein concentrate (PCPC) , flour purple corn (PCF), and hydrolysates (GH25, DH25, GH35 and DH35), % water absorption capacity (WAC) and % oil absorption capacity (OAC).

 

Table 2. Quantification of total polyphenols content (TPC) total flavanol content (TFLC), total flavonoids content (TFC), total anthocyanins content (TAC) and total pro-anthocyanins content (TPA) of purple corn protein concentrate (PCPC) germinated and its gastrointestinal hydrolysates (GH25, DH25, GH35 and DH35).

Table 3. Identification of phenolic components of purple corn flour (PCF), PCPCs and its hydrolysates (GH25, DH25, GH35 and DH35).  

 

- linie 3.1.1. - Add a sentence indicating that in FIG. 1 are the results concerning the solubility of the tested material

Answer: this was corrected: Figure 1 shows the results of % protein solubility of the samples tested in the study.  PCPC25 presents higher values of protein solubility at pH 2.0; 6.0 and 8.0. At pH 10 it presents a high and…

 

- Fig 1- % protein solubility should be protein solubility (%)

Answer: this was corrected: Figure 1.  Protein solubility (%) of purple corn flour, purple corn protein concentrate (PCPC) germinated and its hydrolysates at different pHs (a)PCPC25 and gastric and duodenal hydrolysates; (b) PCPC35 and gastric and duodenal hydrolysates. PCF (non-germinated purple corn flour).

 

- tab. 1 - Modify the title, i.e. include hydrolysates in the title and provide abbreviations

Answer: this was corrected in all the manuscript: Table 1. Functional properties of purple corn protein concentrate (PCPC), flour purple corn (PCF), and hydrolysates (GH25, DH25, GH35 and DH35), % water absorption capacity (WAC) and % oil absorption capacity (OAC).

Table 2. Quantification of total polyphenols content (TPC) total flavanol content (TFLC), total flavonoids content (TFC), total anthocyanins content (TAC) and total pro-anthocyanins content (TPA) of purple corn protein concentrate (PCPC) germinated and its gastrointestinal hydrolysates (GH25, DH25, GH35 and DH35).

Table 3. Identification of phenolic components of purple corn flour (PCF), PCPCs and its hydrolysates (GH25, DH25, GH35 and DH35).  

 

 

-372 line (Table 1) should be (Table 2)

Answer: this error was corrected:

3.2. Quantification of Phenol Compounds of PCPCs and Gastrointestinal Hydrolysates.

The content of the phytocomponents of PCPCs and their gastrointestinal hydrolysates was determined (Table 2).

 

- 449 and 481 line – Table 4 should be Table 3

 

 

Answer: this error was corrected: 3.3. Identification of Phenolic Acid and Flavonol Compounds by UPLC- QDa. Table 3 shows the phenol acids and flavonoids identified

 

- 501 i 520 lines- Figure 2a and b should be Figure 3a and b

Answer: this error was corrected:  At the suggestion of reviewer 2, Figure 3 b was removed.

3.4. Analysis of Profile Protein of PCPC and Hydrolysates by Electrophoresis SDS.PAGE.

Figure 3 shows the protein profile analysis of purple corn flour, PCPC25, PCPC335…

-535 and 561 lines- Enter information that the results of the degree of PCPC hydrolysis are included in table 4

Answer: this error was corrected.

 

- 561 line – Tab. 5 should be Tab. 4

Answer: this error was corrected:

 

569 and 623 lines- Table 6 should be Table 5

Answer: this error was corrected:

 

623 line - …………corn protein contrates should be ……concentrates

Answer: this error was corrected:

Table 5. Antioxidant activity of purple corn protein concentrate (PCPC) germinated and its gastrointestinal hydrolysates (GH25, DH25, GH35 and DH35) by ABTS, FRAP and DPPH methods.

 

Conclusion asks you to add the practical aspect of research.

Answer: this suggest was attended: The characterization of the functional and biological properties of PCPC and its hydrolysates may be an important aspect for its use in the food industry as a functional ingredient.

Author Response File: Author Response.docx

Reviewer 2 Report

GENERAL COMMENTS FOR AUTHORS

1.      Abstract. English language should be revised and edited. There are grammatical and style errors in the manuscript. Check the use of abbreviations in the whole text (starting from abstract). umol should be substituted withh µmol (please check the whole manuscript)

2.      Introduction should be partially re-written according to the coments in the pdf of the manusctipt. Generally, particular points should be presented in particular paragraphs; for example: methods for obtaining PC and Pis; use of PC and Pis; Corn as the source of polyphenols and proteins etc. In present form, authors jump from one topic to another, than get back to the previous topic – it is confusing. Also, there are information that should be omitted (like extensive description of the composition or use of whey or soy protein isolates). Additional information should be inserted (importance of plant based proteins in terms of sustainibility and climate change; factors influencing gastrointestinal digestibility of PCs and PIs. Authors should focus and clearly emphasizeon the novelty importance and aim of their research.

3.      Materials. Needs additional editing (according to specific comments in the text); particularly formulation of the titles. Also, use of abbreviations needs to be revised (as specified in the comments).

Also, there is a methodological problem with the use of Folin-Ciocalteu reagent for determination of total phenols, since it intereferes with proteins. The authors havent cconsidered this – therefore this topic should be adressed or results should be omitted from the manuscript.

A subchapter about preforemd elemental analysis should be insertrd into Material and method section

4.      Results and Discussion. Specific comments were inserted into the pdf of the manuscript. Again, as in Introduction, authors need to be clearer and more focused and large parts need to be rewritten. For example, there should be one introductory sentence describing in short what has been conducted and with what purpose in particular phase of the research. The reference to the particular table or the figure where the results were presented should be given. Than, authosr should start commenting the results.

More importantly, obtained results should be commented more extensively, in terms of explaining observed changes ore comparing obtained results with the results of other authors. When refering to work of other authors, major conclusions should be emphasized and their relevance to this work (instead of extensive description of their metodology). Additional specific comments about have been given in the text.

Figure 1. Title/legend below the figure should contain explanation of all the abbreviations presented in the figure

Table 1. Title/legend below the figure should contain explanation of all the abbreviations presented in the figure

Table 4 should be renamed into Table 3

Figure 2 (a and b) should be Figure 3 and its content should be additionally explained in the legend of the figure. Consider omitting figure a from the manuscript (same set of data is presented in both parts of the figure

Table 5 should be renamed into Table 4

Table 6 should be renamed into Table 5           

Table 7 should be renamed into Table 6 or omitted

5. Conclusion should be rewritten (specific comments in the pdf should be taken into account)

ADDITIONAL SPECIFIC COMMENTS HAVE BEEN INSERTED INTO PDF VERSION OF THE MANUSCRIPT

Comments for author File: Comments.pdf

Author Response

Reviewer # 2

GENERAL COMMENTS FOR AUTHORS

1. English language should be revised and edited. There are grammatical and style errors in the manuscript. Check the use of abbreviations in the whole text (starting from abstract). umol should be substituted withh µmol (please check the whole manuscript).

Answer: The entire manuscript has been revised. The English language of the manuscript has been reviewed by a Native English colleague.

 

2. Introductionshould be partially re-written according to the coments in the pdf of the manusctipt. Generally, particular points should be presented in particular paragraphs; for example: methods for obtaining PC and Pis; use of PC and Pis; Corn as the source of polyphenols and proteins etc. In present form, authors jump from one topic to another, than get back to the previous topic – it is confusing. Also, there are information that should be omitted (like extensive description of the composition or use of whey or soy protein isolates). Additional information should be inserted (importance of plant based proteins in terms of sustainibility and climate change; factors influencing gastrointestinal digestibility of PCs and PIs. Authors should focus and clearly emphasizeon the novelty importance and aim of their research.

 

Answer: the introduction was rewritten

3. Materials. Needs additional editing (according to specific comments in the text); particularly formulation of the titles. Also, use of abbreviations needs to be revised (as specified in the comments).

Also, there is a methodological problem with the use of Folin-Ciocalteu reagent for determination of total phenols, since it intereferes with proteins. The authors havent cconsidered this – therefore this topic should be adressed or results should be omitted from the manuscript.

Answer: We worked with the extracts. In the manuscript it was added how the extracts were obtained from PCPC. The determined biocompounds were separated from the proteins by solvent extraction.

A subchapter about preforemd elemental analysis should be insertrd into Material and method section

4. Results and Discussion.Specific comments were inserted into the pdf of the manuscript. Again, as in Introduction, authors need to be clearer and more focused and large parts need to be rewritten. For example, there should be one introductory sentence describing in short what has been conducted and with what purpose in particular phase of the research. The reference to the particular table or the figure where the results were presented should be given. Than, authosr should start commenting the results.

More importantly, obtained results should be commented more extensively, in terms of explaining observed changes ore comparing obtained results with the results of other authors. When refering to work of other authors, major conclusions should be emphasized and their relevance to this work (instead of extensive description of their metodology). Additional specific comments about have been given in the text.

Answer: This paragraph was deleted of the section 3.6. Antioxidant Activity.

López-Martínez et al. [43] have described the antioxidant activity of 18 maize varieties from Mexico. The varieties with 100%DPPH and 100%ABTS activity were two varieties of purple corn (AREQ516540TL and Veracruz 42). These two varieties presented a high TAC content and their antioxidant activity was related to the presence of these components.

The figure 3 b was removed of the manuscript

Figure 1. Title/legend below the figure should contain explanation of all the abbreviations presented in the figure.

 

Answer: This comment was attended

Table 1. Title/legend below the figure should contain explanation of all the abbreviations presented in the figure.

Answer: This comment was attended

Table 4 should be renamed into Table 3

Answer: This mistake was corrected.

Figure 2 (a and b) should be Figure 3 and its content should be additionally explained in the legend of the figure. Consider omitting figure a from the manuscript (same set of data is presented in both parts of the figure

Answer: This mistake was corrected.

Table 5 should be renamed into Table 4

Answer: This mistake was corrected.

Table 6 should be renamed into Table 5 

Answer: This mistake was corrected.

 Table 7 should be renamed into Table 6 or omitted

Answer: This mistake was corrected.

 

5. Conclusion should be rewritten (specific comments in the pdf should be taken into account)

Answer: The conclusions were changed

 

ADDITIONAL SPECIFIC COMMENTS HAVE BEEN INSERTED INTO PDF VERSION OF THE MANUSCRIPT

The manuscript has been edited as a whole, taking into account the suggestions. English grammar has been checked, abbreviations have been checked and all formatting errors have been corrected. The introduction was rewritten in response to the suggestions and several bibliographic references were eliminated and new references were introduced in the introduction (24, 25 and 26). The overall objective was rewritten. The methodologies were restructured and the section for elemental analysis was added. Suggested changes in results and discussion were addressed. The conclusions were rewritten.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have conducted changes in the manuscript, however additional improvements are necessary:

1.      Extensive English editing is still necessary

2.      The use of abbreviations should be additionally checked. Sometimes authors explain them several times

3.      The order of methods in Material and methods section should fit the order in Results section It should be changed accordingly (for example Results start with methods that are the last described in Material and methods section)

4.      RESULTS:

a.      There is the problem with application of FC reagent in determining total polyphenols in protein isolates. It is not acceptable method for this purpose and therefore results should be omitted from the paper (it is also obvious from obtained results (as commented in the text).

b.      Table 3 and Figure 4 can be moved to supplementary; Table containing data about the presence of phenolic compounds in analysed samples (flour, PCPC or hydrolysate) should be inserted (because that is what authors comment in the text) and is more relevant to the topic (than details of analytical performance that are presented in Table 3 and Figure 4)

c.      Subtitles of Results section should be revised. They should clearly state presented results and not the technique/methodology applied. Please revise.

d.      Authors need consistency in naming Tables and Figures. Since there are lot of abbreviations, they can be used in titles and figures and tables (do not explain them in the title!) but add a legend under table/figure. Partially it has been done but more consistency is needed

e.      Parts of the data marked in table 5 should be removed, or authors should explain the relevance

f.       Section 3.7 should be removed from the manuscript. The manuscript contains enough data already – this section does not provide any new insight.

5.      CONCLUSIONS should be additionally improved. They should also focus on functional properties of analysed samples, not only polyphenols and antioxidants.

Aditional comments have been inserted into the pdf of the manuscript

Comments for author File: Comments.pdf

Author Response

Reviewer # 2

 

The authors have conducted changes in the manuscript, however additional improvements are necessary:

 

1. Extensive English editing is still necessary

Answer: The English was edited in the manuscript.

2. The use of abbreviations should be additionally checked. Sometimes authors explain them several times

Answer: all abbreviations were checked in the manuscript.

 

3. The order of methods in Material and methods section should fit the order in Results section It should be changed accordingly (for example Results start with methods that are the last described in Material and methods section)

Answer: the order of the methodologies was modified and was organized according to the results section. The order of the bibliographical references was changed according to the changes made in the manuscript.

 

4. RESULTS:

 

1. There is the problem with application of FC reagent in determining total polyphenols in protein isolates. It is not acceptable method for this purpose and therefore results should be omitted from the paper (it is also obvious from obtained results (as commented in the text).

Answer: Total polyphenols content (TPC) was removed from the manuscript. It was removed from the abstract, from materials and methods, from results and eliminated from the table of biocompounds. The number and order of the references was modified.  

 

1. Table 3 and Figure 4 can be moved to supplementary; Table containing data about the presence of phenolic compounds in analysed samples (flour, PCPC or hydrolysate) should be inserted (because that is what authors comment in the text) and is more relevant to the topic (than details of analytical performance that are presented in Table 3 and Figure 4).

 

Answer: We think that this section is important for the manuscript because the title contains this point: identification of phytochemical compounds.

 

1. Subtitles of Results section should be revised. They should clearly state presented results and not the technique/methodology applied. Please revise.

Answer: all subtitles were revised in the manuscript.

 

1. Authors need consistency in naming Tables and Figures. Since there are lot of abbreviations, they can be used in titles and figures and tables (do not explain them in the title!) but add a legend under table/figure. Partially it has been done but more consistency is needed

Answer: this mistake was corrected.

 

1. Parts of the data marked in table 5 should be removed, or authors should explain the relevance

Answer: antioxidant activity of standards was deleted.

 

3. Section 3.7 should be removed from the manuscript. The manuscript contains enough data already – this section does not provide any new insight.

Answer: the section 3.7 Analysis of correlation was removed of the manuscript.

 

5. CONCLUSIONS should be additionally improved. They should also focus on functional properties of analysed samples, not only polyphenols and antioxidants.

 

Answer: this comment was added. PCF and PCPCs showed high functional properties (%PS, %WAC, and %OAC) and antioxidant activity when using the ABTS, FRAP and DPPH methods…

 

Aditional comments have been inserted into the pdf of the manuscript

Answer: the comments of the PDF were added. Dry mass (DM) was reviewer suggested change 1

Author Response File: Author Response.docx