Beetroot Stalk Extract as a Functional Colorant for Stirred Yogurt Beverages: Effect on Nutritional Value and Stability during Storage

Round 1

Reviewer 1 Report

The manuscript deals with the evaluation of the biological activity of water extract of beetroot stalks and its potential application as a colorant to raspberry-flavored stirred yogurt. The results demonstrated that the water extracts of fresh stalks are an important source of phenolics with a considerable betalains content, with high biological activities. Several analysis were carried out including total phenolic and flavonoid content,  phenolics determination by HPLC, betalains content, antioxidant activity (DPPH and ABTS), antimicrobial effect, stability as well as other sensorial and physical properties. The manuscript also includes Statistical.

The topic is suitable to be published in Fermentation, in my opinion, it could be published in Fermentation after some revisions.

The grammar should be revised and the english should be improved in all the manuscript. The experimental section and the results are very interesting, but they discussion should be improved. It is not clear the contribution of this study in the field. Please, add it in the introduction section.

Author Response

Response to Reviewer #1

The manuscript deals with the evaluation of the biological activity of water extract of beetroot stalks and its potential application as a colorant to raspberry-flavored stirred yogurt. The results demonstrated that the water extracts of fresh stalks are an important source of phenolics with a considerable betalains content, with high biological activities. Several analyses were carried out including total phenolic and flavonoid content, phenolics determination by HPLC, betalains content, antioxidant activity (DPPH and ABTS), antimicrobial effect, stability as well as other sensorial and physical properties. The manuscript also includes Statistical.

The topic is suitable to be published in Fermentation, in my opinion, it could be published in Fermentation after some revisions.

Response to Reviewer #1:

We appreciate your valuable effort and time and thank you for your constructive comments that enhanced the quality of the manuscript.

• The grammar should be revised, and the English should be improved in all the manuscript.

Response:  Thank you for your valuable comment. The manuscript has been carefully revised and improved.

• The experimental section and the results are very interesting, but they discussion should be improved.

Response: Thank you for your valuable comment. The discussion was improved as suggested.

• It is not clear the contribution of this study in the field. Please, add it in the introduction section.

Response: Thank you for pointing this out. The introduction has been updated and supported with evidence; the contribution to field has been included.

Author Response File: Author Response.pdf

Reviewer 2 Report

Overall comments:

The manuscript added different concentration of beetroot stalks water extract to yogurt, the properties changes of yogurt were monitored and measured. Authors need to present more scientific evidence for this research which appropriate for the journal of “Fermentation”.   

Major comments:

1) In introduction part: it not clearly explain the objective of this study, this part is too short, can you added some references which related to this study to explain detail for the background of this study.

 2) Can you highlight the innovation of this study?

 3) In 3.3 section: can you explain why the taste of adding BSE yogurt is good than control? The taste properties including which taste? Can you do detail for the taste experiment？For example, using electronic tongue equipment.

 4) In 3.6 section: what the antimicrobial component of BSE? The LAB count is very important for yogurt fermentation process, i think this is a critical negative effect for yogurt quality. Is there any way to overcome this problem?

5) Compare with pH results，on day 7, the pH was similar with each samples, it dose not match the trend with the results of microbial viable cell. 

Author Response

Response to Reviewer #2

The manuscript added different concentration of beetroot stalks water extract to yogurt, the properties changes of yogurt were monitored and measured. Authors need to present more scientific evidence for this research which appropriate for the journal of “Fermentation”.   

Response to Reviewer #2:

In advance, thank you for your valuable efforts and comments, which really improved the quality of the manuscript. The introduction has been revised and supported with evidence related to the journal of “Fermentation”.

Major comments:

• In introduction part: it not clearly explain the objective of this study, this part is too short, can you added some references which related to this study to explain detail for the background of this study.

Response: Thank you for pointing this out. The introduction has been updated as recommended.

• Can you highlight the innovation of this study?

Response: Thank you for your valuable comment. The innovation of the study is highlighted in the introduction: the use of a natural, sustainable, cost-effective and nutritious extract from beet stalks as a functional coloring agent for dairy products, which has not been studied yet. The highlighting of innovation of the study has been added to the conclusion as well.

 

• In 3.3 section: can you explain why the taste of adding BSE yogurt is good than control? The taste properties including which taste? Can you do detail for the taste experiment？For example, using electronic tongue equipment.

Response: Thank you for your valuable comment. Unfortunately, we are not currently able to perform a taste experiment with the electronic tongue; however, we will consider the experiment in our further investigation. Panelists reported a deep sweetness from the increase in BSE concentration; however, panelists expected an increase in raspberry concentration. The taste enhancement could be explained in several ways:

1- The color-taste synesthesia. Panelists related the increase in color intensity to the sweet taste and mouthfeel of the product (on day 1 and during storage).

2- The stalks are composed of simple sugars that could slightly increase the sweetness of the product. Besides, glutamic acid is part of the amino acid composition of beetroot and beet green, which can give the product a deep intense taste.

3- More importantly, the acidity of the treatments was lower than that of the control, which also increased the acceptability of the taste of the products. Therefore, the taste acceptability was correlated strongly with the acidity.

 

• In 3.6 section: what the antimicrobial component of BSE? The LAB count is very important for yogurt fermentation process, i think this is a critical negative effect for yogurt quality. Is there any way to overcome this problem?

Response: We totally agree with your valuable comment that viability is negatively affected. However, it should be mentioned that despite the negative effect on LAB count, the product successfully fermented and developed with a range of LAB log of 6-8, which is within the acceptable ranges of probiotics when consumed.

This negative effect could be overcome by encapsulating the extract with a suitable wall matrix that restrains and delays the release of antioxidants, which in turn reduces the antimicrobial properties of the extract and promotes LAB growth.

• Compare with pH results，on day 7, the pH was similar with each samples, it dose not match the trend with the results of microbial viable cell. 

Response: Thank you for your valuable comment. Growth of LAB results in an increase in total titratable acidity (calculated as lactic acid); however, pH was stable for all samples on day 7. Initially, growth of LAB increased acidity with slight differences between samples on day 7, all calculated as lactic acid, ignoring the weak acids. In addition, the amphoteric properties and buffering capacity of milk may neutralize the weak acids. Thus, the increase in calculated acidity is not necessarily accompanied by a decrease in pH.

Author Response File: Author Response.pdf

Reviewer 3 Report

I am very grateful you for the invitation to review manuscript fermentation-2621769 by Abdo and coauthors " Beetroot stalks extract as a functional colorant for stirred yogurt beverage: Effect on nutritional value and stability during storage”. The present study aimed to evaluate the biological activity of water extract of beetroot stalks and its potential application as a colorant to raspberry-flavored stirred yogurt. The work is interesting but needs adjustments to increase the quality of the material.

 

Comments:

- Abstract: Please indicate a better step-by-step about the work, including what was evaluated.

- Abstract: Despite highlighting performance at lower pH levels, this is not presented.

- Lines 50-55: Authors must highlight the number of stalks produced and available for use. Furthermore, it is important to highlight the concentration of pigment in this material.

- Line 53: Highlight the main bioactive components.

 - Introduction: Highlight the proportion of stalks in relation to beet production.

- Line 70-71: The indication and application of pathogens is not clear in this item. Furthermore, the objectives of the work are not mentioned throughout the text.

- Line 87: Define and better detail the “cut into small pieces”.

- Line 189: This does not represent common product storage. Most analyzes are carried out with the product in its original packaging, checking the release of liquid on the surface. The procedure adopted changes the product matrix and promotes greater liquid release.

- 3.1.4. Antioxidant activity: This item is only a presentation of results, and there is no discussion regarding potential antioxidant mechanisms or components.

- 3.1.5. Antimicrobial activity: The reason for determining this activity is unclear. Would it be to use the extract as an antioxidant or check the inhibition potential in the yogurt matrix?

- Line 269: Totally? This is not clear since the inhibition halo is determined.

- Line 278: Change “affect” to “affected”.

- Line 303: Correlate with the quantification of acids presented in the sequence.

- 3.5.3. Antioxidant activity: The discussion on antioxidant activity should be improved.

- Antioxidant activity: Specify the reason for the difference in relation to the principles of the methods and the characteristic of each antioxidant.

3.6. LAB viability:  The discussion must be carried out to inform whether the quantity of LAB with the presence of BSE makes production unfeasible due to the low count.

Author Response

Response to Reviewer #3

I am very grateful you for the invitation to review manuscript fermentation-2621769 by Abdo and coauthors " Beetroot stalks extract as a functional colorant for stirred yogurt beverage: Effect on nutritional value and stability during storage”. The present study aimed to evaluate the biological activity of water extract of beetroot stalks and its potential application as a colorant to raspberry-flavored stirred yogurt. The work is interesting but needs adjustments to increase the quality of the material.

 

Response to Reviewer #3:
Thank you for your valuable comments that have improved the quality of our manuscript. We appreciate your valuable time and effort.

1- Abstract: Please indicate a better step-by-step about the work, including what was evaluated.

Response: We appreciate your valuable comment. The abstract has been updated to be more informative.

2- Abstract: Despite highlighting performance at lower pH levels, this is not presented.

Response: Thank you for your valuable comment. The stability of betalains was included in the abstract.

3- Lines 50-55: Authors must highlight the number of stalks produced and available for use. Furthermore, it is important to highlight the concentration of pigment in this material.

Response: Thank you for your valuable comment. The quantity of beetroot stalks produced was added based on our previous investigation as mentioned in lines 65-67; the highest concentration of betalains was added in line 72.

4- Line 53: Highlight the main bioactive components.

Response: Thank you for pointing this out. The main bioactive components were included in lines 70-71.

 5- Introduction: Highlight the proportion of stalks in relation to beet production.
Response: Thank you for pointing this out. The ratio of stalks to beetroot production was included in lines 65-67.

6- Line 70-71: The indication and application of pathogens is not clear in this item. Furthermore, the objectives of the work are not mentioned throughout the text.

Response: Thank you for pointing this out. The reason for using the pathogens was added to lines 101-102; the objective of the study was added to the introduction.

7- Line 87: Define and better detail the “cut into small pieces”.

Response: Thank you for pointing this out. The estimated length of the stalk pieces was added in line 119.

8- Line 189: This does not represent common product storage. Most analyzes are carried out with the product in its original packaging, checking the release of liquid on the surface. The procedure adopted changes the product matrix and promotes greater liquid release.

Response: We completely agree with your valuable comment. However, syneresis and water-holding capacity are usually determined by draining or centrifuging the milk product to determine the amount of whey that separates from the 3D gel matrix (protein) of the yogurt [1] [2]. Since the product is liquid, it is difficult to accurately determine syneresis by checking the liquid release at the product surface. In addition, to avoid further release of whey, we chose to determine syneresis by draining rather than centrifugation.  

 

1. dos Santos, C.D.; Scherer, R.K.; Cassini, A.S.; Marczak, L.D.F.; Tessaro, I.C. Clarification of red beet stalks extract by microfiltration combined with ultrafiltration. J. Food Eng. 2016, 185, 35-41.
2. Soutelino, M.E.M.; Silva, D.B.d.; Rocha, R.d.S.; de Oliveira, B.C.R.; Esmerino, E.A.; Cruz, A.G.d.; Mársico, E.T.; Silva, A.C.d.O. Yogurt added with beetroot extract: physicochemical parameters, biological activities and sensory evaluation by CATA method. Int. J. Food Sci. Tech. 2022, doi: https://doi.org/10.1111/ijfs.16214.

9- 3.1.4. Antioxidant activity: This item is only a presentation of results, and there is no discussion regarding potential antioxidant mechanisms or components.

Response: We appreciate your valuable and constructive comment. The discussion in this section has been improved.

10- 3.1.5. Antimicrobial activity: The reason for determining this activity is unclear. Would it be to use the extract as an antioxidant or check the inhibition potential in the yogurt matrix?

Response: Thank you very much for your valuable comment. The reason for determining the antimicrobial activity of the extract was included in lines 289-293. As a strong antioxidant, BSE could have great antimicrobial activity, which could extend the shelf life of the products.

11- Line 269: Totally? This is not clear since the inhibition halo is determined.

Response: Thank you very much for your comment. The sentence has been corrected in lines 313-314.

12- Line 278: Change “affect” to “affected”.

Response: Thank you for your valuable comment. The word corrected as suggested in line 322.

13- Line 303: Correlate with the quantification of acids presented in the sequence.

Response: Thank you for pointing this out. The acidity scores were correlated with the total titratable acidity TTA in lines 353 and 357.

- 3.5.3. Antioxidant activity: The discussion on antioxidant activity should be improved.

Response: Thank you for your valuable comment; the section was improved.

14- Antioxidant activity: Specify the reason for the difference in relation to the principles of the methods and the characteristic of each antioxidant.

Response: Thank you for your valuable comment; the reason for the difference of the characteristics of each antioxidant was included in section 3.5.3.

15- 3.6. LAB viability:  The discussion must be carried out to inform whether the quantity of LAB with the presence of BSE makes production unfeasible due to the low count.

Response: Thank you for your valuable comment; the section has been improved. However, it should be mentioned that regardless of the reduction in LAB count, it is within the acceptable range for probiotics Log 6-8 during consumption throughout the storage period (14 days). Accordingly, BSE could be used as a colorant in the development of stirred yogurt. To improve the stability of the color, delay the release of the antioxidants and thus the high antimicrobial effect of the extract, which in turn promotes LAB growth, encapsulation with a suitable wall matrix could be a further investigation.

Author Response File: Author Response.pdf