Screening and Probiotic Properties of Lactic Acid Bacteria with Potential Immunostimulatory Activity Isolated from Kimchi

Round 1

Reviewer 1 Report

The authors have provided an in vitro study on the abitlity  of the cell free supernatants of 28 different strains of lactic acid bacteria isolated form the fermented food kimchi to induce nitric oxice production, induction of pro-inflammatory cytokines, phagocytosis and nuclear factor kappa beta induction in  RAW 264.7 cells. The most active strains were further tested for their tolerance to gastrointestinal stress factors, antibiotic resistance, hemolytic activity and adhesion to the intestinal cell line HT-29. After these selection processes, five strains showed probiotic potential.

I do not have questions or comments on the in vitro testing of the immmunostimulatory and proinflammatory functions of the cell free extracts of the probiotic candidate strains.

However, regarding the probiotic properties, especially antibiotic sensitivity and resistance, as well as in vitro adhesion properties the authors should revise the manusxcript.in vitro

1) The antibiotic sensitivity was tested using the strip method, and the MIC values determined by EFSA were used as criteria for resistance and sensitivity. However, EFSA recommends the use of serial dilution method to determine the MIC values, and therefore referring to the EFSA breakpoints is not approppriate, as these two mwthods have not been validated againsti each other. The authors should only refer to the values obtained by their test system and to the rsistance sensitivity criteria provided by the manufacturer of the antibiotic strips.

2) Lack of hemolysis is not a safety criterion for most of lactic acid bacteria. While hamolytic strains would be suspicious, also nonhaemolytic strains can be associated with opportunistic infections. The authors should refer to the latest list of QPS (Qualified Presumption of Safety) microorganisms published by EFSA and check, whether the species they have studied are listed there.

3) In vitro adhesion of bacteria to different cell lines can be highly variable depending on the testing conditions, and it is highly recommended that a known adhesive strain is included as both a positive control and a reference point. Comparing adhesion data from the literature (lines 357 - 360) to experimentally obtained is not approppriate. 

4) Cell free bacterial culture supernatants were used in the experiments with RAW 264.7 cells. The authors could speculate a little what kinds of molecules migh be present there (cell wall components?, cell debris?, teichoic acids ?)

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Overall, the work is well planned, the methods and results are well described and discussed. Few minor revisions in attachment are needed.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed my main concerns. Although I do not think that the MIC values of the antibiotic strip method and the serial dilution methods are fully comparable, there is no danger of a major discrepancy between these two methods. Therefore I do not require any more corrections or revisions.