Correlation between Microbial Population and Oxidative Stability of the Yogurt-Based Tzatziki Salad
, Vassilis Athanasiadis 1,*, Theodoros Chatzimitakos 1, Chrysanthi Salakidou 1, Vassilis G. Dourtoglou 2 and Stavros I. Lalas 1
Round 1
Reviewer 1 Report
Dear authors
thank you for the submitted manuscript.
It is my opinion that this is a well designed research, and the significance and importance of the study is significant.
My comments are quite minor:
Line 150 (font size)
Figures: generally all figures would benefit from de-cluttering (perhaps in the legends use S1m S2, etc rather than the whole recipe
I recommend acceptance of the manuscript in its current form subject to minor editorial modifications.
Author Response
We would like to thank the reviewer for his/her comments. We have changed the font size and the figure legends, as suggested.
Reviewer 2 Report
Manuscript ID: oxygen-1783086
Title: Correlation between Microbial Population and Oxidative Stability of the Yoghurt-based Tzatziki Salad
Authors: Eleni Bozinou, Vassilis Athanasiadis, Theodoros Chatzimitakos, Chrysanthi Salakidou, Vassilis G. Dourtoglou, Stavros I. Lalas
Overview and general recommendation:
The aim of the manuscript was examine the stability of tzatziki salad against oxidation.
In the manuscript, the authors investigated the effect of the type of oil, yoghurt and other functional additives used on oxidative changes occurring in the oil extracted from the Tzatziki sauce. According to the reasoning presented by the authors, the bacteria introduced into the sauce with the addition of yoghurt had the greatest impact on reducing oxidative damage.
Unfortunately, on the basis of very limited research carried out by the authors, it is not possible to draw such conclusions. The rate of oxidation processes is influenced by many factors, but the basic ones are the oil content in the product, the pH of the product, the content of water and antioxidant components. The fat content in the tested sauces is very low (9% of added oil and a few% of milk fat from yoghurt). Therefore, in my opinion, it is not a product whose oxidative stability should be measured with the Rancimat test. These sauces cannot be compared to maynoise, the fat content of which is usually over 70%. Changes that may occur will be of a more hydrolytic nature, changes may be observed in the amount of free fatty acids, which will accelerate the oxidation process, therefore their amount as well as the pH of the products as well as the acid value should be examined. In addition, the total number of microorganisms is not enough to state that probiotic bacteria are responsible for the effect of increasing the oxidative stability of sauces, it should be determined what bacteria are in the product. It should be noted that the production of Tzatziki sauce does not involve pasteurization, and the microbial load is also introduced with herbs and spices.
Considering all the above reservations and the fact that the manuscript contains incorrect assumptions, I cannot recommend its publication.
Below I give also some concerns that might help Authors in the future
Comments:
Line 17 – “Different samples of tzatziki salad were prepared and stored for 16 or 27 days.” - Why did you decide to store some samples for 11 days longer than the other when planning your experiment?
Introduction – to little information about possibilities to limit the oxidation changes
Methodology - all equipment used should have the name, manufacturer and country of origin specified, including food processors (line 76).
Samples S and M - it is not entirely clear why the authors divided their experiment into two completely independent parts. There is no explanation for the ingredients used, nor information as to why other additives than those traditionally used in Tzatziki sauces were used.
Line 86 – “stomacher.” – not clear?
Statistical analysis – it is always good to give details about the tested which was performed - e.g. Tukey's test
Line 120 – “…despite its high quantity of oil…” - High compared to what type of product? Mayonnaise (mentioned by the authors) has over 70%, while in the recipe in tables 1 and 2 we only have 9%.
Line 114 – “…which in turn can result into differences in the composition…” – please add the composition of what ingredients? Fatty acids?
Statistical analysis – there are no data in tables or figures, it is not known whether the values differ significantly
Author Response
The aim of the manuscript was examine the stability of tzatziki salad against oxidation.
In the manuscript, the authors investigated the effect of the type of oil, yoghurt and other functional additives used on oxidative changes occurring in the oil extracted from the Tzatziki sauce. According to the reasoning presented by the authors, the bacteria introduced into the sauce with the addition of yoghurt had the greatest impact on reducing oxidative damage.
Unfortunately, on the basis of very limited research carried out by the authors, it is not possible to draw such conclusions. The rate of oxidation processes is influenced by many factors, but the basic ones are the oil content in the product, the pH of the product, the content of water and antioxidant components.
We agree with the reviewer that the rate of oxidation is influenced by the oil content, the pH, the water content and antioxidant components. However, this was not the aim of our study. We did not aim to examine how the different parameters affected the oxidation process, but define the parameters that are responsible for the oxidative stability of the product. This oxidative stability was known by consumers and validated by our experiments.
The fat content in the tested sauces is very low (9% of added oil and a few% of milk fat from yoghurt). Therefore, in my opinion, it is not a product whose oxidative stability should be measured with the Rancimat test.
According to the reviewer, the fat content of the product is low, and he/she is not suggesting the use of Rancimat method. We would agree with the reviewer, if this was the case, but there seems to be a misunderstanding. The fat was extracted from the tzatziki samples and then analyzed with the Rancimat methods. Therefore, we believe that this method is highly suitable for a fat extract.
These sauces cannot be compared to maynoise, the fat content of which is usually over 70%. Changes that may occur will be of a more hydrolytic nature, changes may be observed in the amount of free fatty acids, which will accelerate the oxidation process, therefore their amount as well as the pH of the products as well as the acid value should be examined. In addition, the total number of microorganisms is not enough to state that probiotic bacteria are responsible for the effect of increasing the oxidative stability of sauces, it should be determined what bacteria are in the product. It should be noted that the production of Tzatziki sauce does not involve pasteurization, and the microbial load is also introduced with herbs and spices.
All text was corrected, so as no comparison with mayonnaise was made throughout, as suggested. The CFU in the samples was examined and following a statistical correlation analysis, it occurred that there is a correlation between the oxidative stability and the number of CFU. This is not an assumption, this is a result, validated by statistical analysis. Determination of the type of bacteria present in the samples was not deemed necessary, since the main bacteria contained would be Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These are the main bacteria contained in the yoghurt, used for the preparation of the tzatziki and the main activity would be attributed to them. Although no pasteurization step was used for the preparation of tzatziki, it would be inaccurate to assume that the microbial content of yoghurt and herbs/spices is the same, since the later contain significantly lower number of bacteria, due to inherent antibacterial properties. Therefore, the number of bacteria derived from the other ingredients would be disproportionately lower, compared to the lactic acid bacteria derived from the yoghurt. Due to the above, we did not opt for further examination of such a parameter.
Considering all the above reservations and the fact that the manuscript contains incorrect assumptions, I cannot recommend its publication.
Below I give also some concerns that might help Authors in the future
Comments:
Line 17 – “Different samples of tzatziki salad were prepared and stored for 16 or 27 days.” - Why did you decide to store some samples for 11 days longer than the other when planning your experiment?
The samples stored for 27 days were prepared with the main aim to examine the oxidation degree of the tzatziki, containing different ingredients. Therefore, a longer storage time was studied. On the contrary, samples stored for 16 days were prepared with the main aim to examine the CFU and the IP, in order to shed light on whether there is any correlation between the two factors.
Introduction – to little information about possibilities to limit the oxidation changes
Additional information was added in the introduction section.
Methodology - all equipment used should have the name, manufacturer and country of origin specified, including food processors (line 76).
A new section (2.3) was added, listing all instruments used in the study.
Samples S and M - it is not entirely clear why the authors divided their experiment into two completely independent parts. There is no explanation for the ingredients used, nor information as to why other additives than those traditionally used in Tzatziki sauces were used.
The first part of the study, aimed to examine whether tzatziki is an oxidative stable product, and if this oxidative stability is attributed to the different ingredients used. Therefore, different ingredients were examined. In the second part of the study, we aimed to examine whether there is correlation between the CFU and the oxidative stability of the samples. Therefore, we examined the recipes of the commercially available tzatziki sauces, so as to have a better insight into the commercial products as well. Relevant comment were added in the text.
Line 86 – “stomacher.” – not clear?
Information on the instrument was added in section 2.3
Statistical analysis – it is always good to give details about the tested which was performed - e.g. Tukey's test
The information was added in the text.
Line 120 – “…despite its high quantity of oil…” - High compared to what type of product? Mayonnaise (mentioned by the authors) has over 70%, while in the recipe in tables 1 and 2 we only have 9%.
The sentence was corrected, as suggested
Line 114 – “…which in turn can result into differences in the composition…” – please add the composition of what ingredients? Fatty acids?
Examples of the differences were added in the text, as suggested.
Statistical analysis – there are no data in tables or figures, it is not known whether the values differ significantly
Statistically significant differences were added in Table 3.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
Dear Authors
The manuscript “Correlation between microbial population and oxidative stability of the yoghurt-based Tzatziki salad" investigates the stability against oxidation of this food using different recipes”.
The novelty of this study is good since the works on this topic are not numerous.
The materials and methods are quite well written, but the discussion of the results is scarce.
Other observations are the following:
- -Tocopherol analysis: did you filter the sample before injection? Which type of filter?
And which was the temperature of the column for the analysis?
- - In table 3 add ANOVA results
- - Add a discussion for results on lines 143-147.
- - Pasteurized samples should contain a lower quantity of microorganisms, how do the authors explain this result?
In my opinion, the manuscript needs minor revisions, in particular, the discussion should be improved.
Best regards
Author Response
Dear Authors
The manuscript “Correlation between microbial population and oxidative stability of the yoghurt-based Tzatziki salad" investigates the stability against oxidation of this food using different recipes”.
The novelty of this study is good since the works on this topic are not numerous.
The materials and methods are quite well written, but the discussion of the results is scarce.
Other observations are the following:
- Tocopherol analysis: did you filter the sample before injection? Which type of filter?
And which was the temperature of the column for the analysis?
Yes, samples were filters using PVDF syringe filters. The column temperature was 30oC. This information was added in the manuscript, as suggested.
- In table 3 add ANOVA results
Statistically significant differences were marked in Table 3.
- Add a discussion for results on lines 143-147.
A possible explanation was added, as suggested.
- Pasteurized samples should contain a lower quantity of microorganisms, how do the authors explain this result?
Both types of yoghurt used for the preparation of tzatziki were commercially available. This means that both products were pasteurized, so as to adhere to legislation. Therefore, the difference in the CFU is attributed to the different type of yoghurts. To avoid confusion, the word “pasteurized” was deleted from the manuscript.
In my opinion, the manuscript needs minor revisions, in particular, the discussion should be improved.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Manuscript ID: oxygen-1783086 Second Review
Title: Correlation between Microbial Population and Oxidative Stability of the Yoghurt-based Tzatziki Salad
Authors: Eleni Bozinou, Vassilis Athanasiadis, Theodoros Chatzimitakos, Chrysanthi Salakidou, Vassilis G. Dourtoglou, Stavros I. Lalas
Overview and general recommendation:
I would like to thank the authors for the scientific exchange of views. The authors reviewed my minor remarks but disagreed with me on the key remarks in my opinion.
I would like to point out that, it is obvious that only extracted fat is used for the Rancimat test, I had no doubts about the correctness of the analysis. My doubts relate to the assumption that with such a small amount of oil in the sauce, the changes are not of the nature of the fat oxidation itself. Additionally, in such a short time of storage, the changes taking place in the form of oxidation should also not be significant (unless the oil is very unstable, but this is not what we are dealing with here). I tried to present my point of view in the review, but the authors decided that this was not the purpose of their research. Unfortunately, considering the fact that the manuscript is to be published in the Oxygen journal, in which the published articles should be thoroughly prepared in terms of changes related to oxidation, I decided to maintain my opinion on the manuscript.
As always, I leave the final decision to the Editor.
Author Response
We would like to thank the reviewer for his/her comments, as well as apologize, since our aim was not to offend him/her when we stated the aim of our research. The aim of our study was defined before carrying out the experiments and this statement was not used to disprove the opinion of the reviewer. As regards the scientific part, the reviewer suggests that the hydrolytic rancidity takes place in the tzatziki samples, instead of oil-phase oxidation, due to the short-time of storage. We believe that both phenomena may occur during storage, albeit to a different extent. Although oxidation of the oil-phase is a slower process, compared to hydrolytic rancidity, it would not be advised to examine tzatziki salad after 1 month of storage, since it is meant to be eaten within a shorter period (usually less than 1 month), after preparation. Therefore, we believe that experiments after 1 month of storage would be of no value.
As regards tzatziki samples, if hydrolytic rancidity took place, off-flavor compounds would also be produced. Considering that the oxidation products (either from hydrolytic or oxidative deterioration) are highly detectable due to their extreme odor, the unpleasant flavor is easily detectable even if the fat phase content is relatively low. However, this neither is typical for tzatziki salads, nor occurred in our case. This could be attributed to the addition of garlic and cucumber that contain natural antioxidant compounds that either hinder or lower the rate of rancidity’s progress.
Moreover, hydrolytic rancidity would result in decrease of the pH of tzatziki salad. This would result in increases syneresis of the yogurt in the tzatziki salad, which was not apparent in our case. The fact that neither of the above were recorded during our preliminary experiments (and to our final experiment), we decided not to focus our study on hydrolytic rancidity. To further strengthen our notion, we performed additional experiments, measuring the pH of tzatziki samples within 16 days of storage and we found that it decreases from 4.3 to 4.1, which is not indicative of hydrolytic rancidity, since a similar decrease in the pH can be observed in yogurts.
We hope that the addition of the above information in the manuscript will help readers have a better overview of the topic.
Author Response File: Author Response.pdf
