Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 sThetudy aimed to compare traditional kefir inoculated with kefir grains and other products inoculated by second fermentation or backslopping (sometimes known as the Russian kefir method) and determine whether this production method offers advantages. The authors could include in the introduction whether Argentina produces kefir industrially, and if so, by what process. In this study, as illustrated in Figure 1, fermentations apparently occurred in open tanks (loss of CO2 to the environment), and the type of packaging is not described in the storage case. The alcohol contents of the products were not determined.

The authors refer to the second fermentation inoculum (double fermentation product) as "starter," which creates some confusion with commercial starters. Essentially, these fermentation products also serve or function as starters, but other authors refer to these products as backslopping, and in this article, all three terms are used. In the case of starter-based processes, are the authors referring to commercial starters or backslopping? It is suggested that the terms be standardized and used appropriately, and that they be specifically used when using commercial starters.

Some comparisons between this procedure have been made with publications using commercial starters (Baars et al. 2025 used raw milk, 2% commercial inoculum, and fermentation at 24°C for 36 hours). The claims that double-fermentation inoculums allow for better process control need to be more emphatically demonstrated, as in some parts of the text, the authors state that there was "slower" fermentation (line 276, page 7, without performing kinetics), and in others, the double-fermentation products achieved higher acidity (G1F10). Indeed, with commercial starters, due to the limited number of strains, process control is easier because predictability is greater. However, with cultures with greater biodiversity, this predictability decreases. Therefore, it remains to be demonstrated more convincingly (line 302, page 8) that double-fermentation provides greater predictability and control.

Kefir microbiota varies depending on the origin of the grains, milk composition, incubation conditions (temperature, time, and aeration), inoculum percentage, and other factors. Depending on these conditions, the microbiota of the grains and product can vary greatly. Therefore, choosing a 30°C/24h temperature determines the type and percentage of each group that will predominate in the culture and product. In traditional, home-grown kefir, the average ambient temperature is below 30°C. This justifies the choice of this higher temperature, which would certainly favor bacteria. Also, when removing the grains, a greater presence of LAB is expected, rather than yeast, which are typically found primarily on the outer surface of the grain.

The determination of biofilm production was based on a method that assesses adhesion to surfaces, and in the case of the process chosen for sensory analysis (G1F10), during storage, it was not the inoculum with the highest production. Biomass measurement or EPS extraction could result in different results. But It is important to emphasize that although probiotic microorganisms were not studied, the inhibition of pathogenic species, as demonstrated in the study, in itself confers a health benefit, whether through acidity or other mechanisms such as bacteriocins. Biofilm production also contributes with beneficial effects.

In Figure 4 change log(CFU/ml) by log(CFU/mL)

The percentage of more recent citations could be increased (approximately 33% are old). The doi of the citation Han et al.2018 is incorrect.

The paper may be published with a major revision.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

A relevant study was conducted on fermentation strategies to improve Argentinian kefir quality, specifically evaluating the impact of double fermentation on physicochemical, microbial, and functional properties. The inclusion of the fermentation sample preparation scheme in the Methods section is helpful for understanding the experimental design. However, the abstract should be revised to avoid the use of sample codes, as they make the text more difficult to read.

Section 2.2: Please clarify where these materials were used—whether for fermentation or another type of experiment. If they were applied in fermentation, the description of the fermentation process should be expanded to make this clear.

Section 2.3: Consider separating the evaluation of grains and the fermented product. It may also be useful to include pH and Brix measurements alongside TTA.

Line 142: Please specify exactly which proteins were used and provide the manufacturer’s details.

Section 2.5: Please provide the calculation formula used.

Line 203: Please clarify how turbidity was measured

Section 3.3: Do you consider it appropriate to perform FTIR analysis in a food system where the protein and carbohydrate regions overlap? What scientific value and accuracy does this analysis provide in this context?

Figure 4 and 5: The presentation of results in this form is unusual; it would be clearer and more conventional to present them in a table.

Section 3.6: The presented data are not clearly visible and should be improved for clarity.

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Information was introduced that hadn't been included in the original version, such as data normalization for spider graphs. I believe that most of it made the text clearer. The suggestions were fully accepted, and the paper can be published.

Reviewer 2 Report

Comments and Suggestions for Authors

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