Application of Raman Spectroscopy to Rapid Discrimination of Autochthonous Lactic Acid Bacteria Isolated from Goat Cheese

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

As this is a methodological article or a proposal for a new technique, it should be clear or include some clarifications. For example, in the following text:

“Overnight culture cells were centrifuged at 12,000×g for 10 min at 4 °C and washed twice with sterile saline solution (0.8% w/v sodium chloride)”

What do we mean by “overnight culture”? Is it a solid or liquid culture? What culture medium is it? Based on the following steps, it would seem to be a liquid culture medium. However, later it states the following:

“8 measurements per strain from at least 8 different colonies with an exposure time of 5 s in the range 100–2000 cm⁻¹”

So, if we are talking about colonies, is it a solid medium? Or, from a solid medium, do we transfer one colony to a liquid medium to obtain an overnight culture? And do we repeat this with 7 more colonies to achieve what the text describes?

In the same text:

“Overnight culture cells were centrifuged at 12,000×g for 10 min at 4 °C and washed twice with sterile saline solution (0.8% w/v sodium chloride).”

It is important to specify the culture conditions (time, temperature, oxygen levels), because changes in these conditions could alter the proportions of carbohydrates, DNA, and proteins.

Figure 1 is missing the controls, whether from the three tested species, and we expect to see the three products described in the methodology. How do they ensure the product is as expected, just by size? Wouldn't sequencing be necessary if a control strain weren't available?

It's not specified what happens to the remaining strains. Do they not amplify the product? Do they amplify it to a different size? Another question arises: Did they only include the Lactiplantibacillus strains in the PCR?

The terminology is inconsistent; in some sections it's listed as RAPD-PCR, in others as multiplex RAPD-PCR, and in others simply as multiplex. Furthermore, based on the theoretical framework, is it RAPD-PCR or multiplex PCR?

The conclusions state that the technique allows for the differentiation of lactic acid bacteria species, but lactic acid bacteria are actually a broad group, and they were only able to distinguish between three species. The conclusion should be more specific.

Section 3.2.3 is missing. This section refers to Lactiplantibacillus species, because in section 3.2.2 they refer to Lentilactobacillus species, and on page 7 the subtopic is the same, but they are already mentioning Lactiplantibacillus.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The rapid and accurate discrimination of lactic acid bacteria is important for microbial identification, probiotic screening, and the development of functional foods. In this manuscript, the authors applied Raman spectroscopy combined with chemometric analysis to differentiate LAB strains isolated from goat cheese at the genus and strain levels. They further used PCA/LDA and spectral band analysis to classify L. plantarum, L. parabuchneri, and L. mesenteroides. Overall, the study is relevant and the topic fits the scope of the journal. The manuscript contains useful experimental data and shows potential practical value. I therefore recommend minor revision before it can be considered for publication.

1. The authors should provide stronger validation of the PCA/LDA classification model, such as cross-validation, an independent test set, or quantitative metrics including accuracy and a confusion matrix.

2. Additional experiments or analyses on the reproducibility of Raman spectra under biological and technical replicates would strengthen the reliability of the strain-level discrimination.

3. The discussion should be more cautious when interpreting Raman peak ratios as direct evidence of intracellular biochemical composition, since these assignments remain indirect without independent validation.

4. The manuscript would benefit from careful editing for formatting consistency and reference style. In addition, more recent related studies should be included to better support the novelty of the work.

Author Response

Please, see in attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors made the necessary corrections. The authors improved the manuscript