Functional Kimchi Beverage Enhanced with γ-Aminobutyric Acid (GABA) Through Serial Co-Fermentation Using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
This study develops a GABA-enriched plant-based beverage using Leuconostoc citreum S5 and Lactobacillus plantarum KS2020 as raw materials through serial co-fermentation, which holds significant promise for the further development of functional plant-based beverages. However, some details in the article need to be improved and some content need to be refined, which are suggested to be accepted after modifications. Some specific comments are provided below:
Line 105: Please explain why 3-7% MSG was chosen.
Line 248-249: Please add error bars for viable cell count in Figure 1.
Figure 2 is missing data for Day 3.
Line 335-336: Please cite references to support the claim that mannitol can remain after fermentation, contribute to acid formation, and lead to possible quality changes.
Line 394-396: Please add a reference supporting the claim that residual MSG in fermented products can lead to a greasy taste. Was a sensory evaluation experiment conducted?
Line 462: Please add the reasons for the decrease in the a* value of kimchi beverage base in 2nd fermentation time.
Author Response
Thank you very much for the valuable comments.
Comments 1: Please explain why 3-7% MSG was chosen.
Response 1: We confirmed through a preliminary experiment that GABA conversion was fully achieved up to a concentration of 3% MSG. The GABA production was dependent upon the MSG content so that higher MSG content was used to obtain efficiently the higher GABA production during lactic acid fermentation.
Comments 2: Please add error bars for viable cell count in Figure 1.
Response 2: Thank you for pointing this out. The error values are minimal, with a maximum of 0.2 and generally below 0.1, making the error bars appear within the symbol size.
Comments 3: Figure 2 is missing data for Day 3.
Response 3: This experiment was conducted to identify the overall trend of sugar consumption from day 1 of 1st fermentation to day 5 of 2nd fermentation, rather than focusing on the values observed on day 3 of fermentation.
Comments 4: Please cite references to support the claim that mannitol can remain after fermentation, contribute to acid formation, and lead to possible quality changes.
Response 4: As you mentioned, we have cited the reference supporting the claim (Line 335):
Zioga, E.; Holdt, S. L.; Gröndahl, F.; Bang-Berthelsen, C. H. Screening approaches and potential of isolated lactic acid bacteria for improving fermentation of Saccharina latissima. BMC Biotechnol. 2025, 25, 2. https://doi.org/10.1186/s12896-024-00926-6
Comments 5: Please add a reference supporting the claim that residual MSG in fermented products can lead to a greasy taste. Was a sensory evaluation experiment conducted?
Response 5: Thank you for pointing this out. As we did not conduct a sensory evaluation, expressions related to the taste of MSG were removed as suggested. (Line 381)
Comments 6: Please add the reasons for the decrease in the a* value of kimchi beverage base in 2nd fermentation time.
Response 6: It is assumed that the slight decrease in the a* value is due to the dilution effect caused by the addition of MSG, distilled water, and starter at the beginning of 2nd fermentation. (Line 439)
Thanks to the reviewers' insightful feedback, the readability and quality of this paper have greatly improved.
Reviewer 2 Report
Comments and Suggestions for Authors
The authors studied preparation of fermented kimchi beverage through co-fermentation using Leuconostoc citreum and Lactiplantibacillus plantarum. Number of viable CFUs was measured for each strains at different stages of fermentation. Dynamics of releasing and consumption of sugars and mannitol as well as final concentration of organic acids were estimated. Dextran production by Leu. citreum was measured. Moreover, the beverage was enhanced with GABA via addition of monosodium glutamate during co-fermentation. The paper is well-written, results are clearly described. It is suitable for publication in the Fermentation journal and should be interesting for readers. However some points need to be corrected or improved.
L54: “non-protein” -> non-proteinogenic
LL115-120: it is not completely clear how the authors distinguish between Leu. citreum and Lb. plantarum? As I understood both strains can grow on MRS agar, but colonies counted on this medium were assigned only as Lb. plantarum. Please clarify the techniques.
L124: “13,000 rpm”. Please specify speed in “g”
LL131-132: “Organic acids analysis…was performed” or “Organic acids content…. was analyzed” should be used, but not “Organic acids analysis… was analyzed…”
L146: “…and centrifuged.” Please specify the speed.
L147: “chilled ethanol”. Please specify ethanol concentration
L173: “filtrate filtered through a 0.45 μm syringe filter”. Please rephrase “filtrate filtered… filter”
L171: Did the authors analyze content of all free amino acids or only GABA?
L216: “…viable cell count of Leu. citreum S5 was positively correlated with higher sucrose contents.” It is required to calculate the correlation values together with a statistical test to determine if there is a correlation and its strength.
L267: “…that Lb. plantarum can be utilized in mannitol…” -> “Lb. plantarum can utilize mannitol” or “mannitol can be utilized by Lb. plantarum”
L295: “levan, and fructan”. Levan is also fructan, because its monomer is fructose. Please rewrite it.
L295: “The results of measuring dextran content are presented in Figure 3A”. Delete the sentence; just cite the Figure 3A in the following text.
L314: see previous comment
LL348-380: this text should be shortened because key part is GABA production but not pH changing during this process.
LL401-403: It is irrelevant to the study being described in this paper
LL446-447: “…leaving lactate in the form of lactic acid, which contributed to an increase in the pH…”. Why do the authors think that lactic acid increase the pH? Please clarify this point.
LL466-474: please move it to the Conclusitons
LL488-489: “palatable taste”. As I understood there were no taste and flavor testing. These data must be added to Materials and methods as well as Results and Discussion sections. If such data is not available, the authors should avoid describing taste and flavor of the beverage.
Figure 4: Please correct “ABc” and “ABa” on the Figure 4B
Figure 5: actually I don't see much difference in GABA production in M3 and M5. Why did the authors prefer M5?
Table 4: It will be useful to provide additionally photo (e.g. as supplementary material) of the beverage samples in cuvettes for simple visual estimation of colors
Author Response
Thank you very much for the valuable comments.
Comments 1: “non-protein” -> non-proteinogenic
Response 1: As you mentioned, I have corrected it to non-proteinogenic. (Line 54)
Comments 2: it is not completely clear how the authors distinguish between Leu. citreum and Lb. plantarum? As I understood both strains can grow on MRS agar, but colonies counted on this medium were assigned only as Lb. plantarum. Please clarify the techniques.
Response 2: Thank you for pointing this out. Although both strains grow on MRS agar, their colonies cannot be distinguished on the same plate. Therefore, Leu. citreum was differentiated by using a sugar-supplemented medium, where dextran formation was visible, allowing it to be distinguished from Lb. plantarum colonies for viable cell count measurements.
Comments 3: “13,000 rpm”. Please specify speed in “g”
Response 3: It has been corrected to 15,710× g. (Line 124)
Comments 4: “Organic acids analysis…was performed” or “Organic acids content…. was analyzed” should be used, but not “Organic acids analysis… was analyzed…”
Response 4: We have revised the phrase to “Organic acids content… was analyzed”. (Line 131)
Comments 5: “…and centrifuged.” Please specify the speed.
Response 5: It has been corrected to 24,948× g. (Line 146)
Comments 6: “chilled ethanol”. Please specify ethanol concentration
Response 6: It has been corrected to chilled 99.9% ethanol. (Line 147)
Comments 7: “filtrate filtered through a 0.45 μm syringe filter”. Please rephrase “filtrate filtered… filter”
Response 7: We have revised the phrase to “the filtrate obtained through a 0.45 µm syringe filter was used as the test solution”. (Line 125)
Comments 8: Did the authors analyze content of all free amino acids or only GABA?
Response 8: We measured only glutamic acid and GABA among free amino acids.
Comments 9: “…viable cell count of Leu. citreum S5 was positively correlated with higher sucrose contents.” It is required to calculate the correlation values together with a statistical test to determine if there is a correlation and its strength.
Response 9: We have modified the expression to "viable cell count of Leu. citreum S5 increased with higher sucrose content." (Line 218)
Comments 10: “…that Lb. plantarum can be utilized in mannitol…” -> “Lb. plantarum can utilize mannitol” or “mannitol can be utilized by Lb. plantarum”
Response 10: We have revised the phrase to “Lb. plantarum can utilize mannitol”. (Line 268)
Comments 11: “levan, and fructan”. Levan is also fructan, because its monomer is fructose. Please rewrite it.
Response 11: Thank you for pointing this out. We have rewritten it. (Line 295)
Comments 12: “The results of measuring dextran content are presented in Figure 3A”. Delete the sentence; just cite the Figure 3A in the following text.
Response 12: Thank you for pointing this out. We have rewritten it. (Line 298)
Comments 13: see previous comment
Response 13: Thank you for pointing this out. We have rewritten it. (Line 314)
Comments 14: this text should be shortened because key part is GABA production but not pH changing during this process.
Response 14: Sharing the same perspective, we shortened the text as suggested. (Line 346-367)
Comments 15: It is irrelevant to the study being described in this paper
Response 15: We agree with this comment.
Comments 16: “…leaving lactate in the form of lactic acid, which contributed to an increase in the pH…”. Why do the authors think that lactic acid increase the pH? Please clarify this point.
Response 16: We have revised the phrase to “…leaving lactic acid in the form of lactate”. (Line 427)
Comments 17: please move it to the Conclusions
Response 17: We agree with this comment. (Line 449-465)
Comments 18: “palatable taste”. As I understood there were no taste and flavor testing. These data must be added to Materials and methods as well as Results and Discussion sections. If such data is not available, the authors should avoid describing taste and flavor of the beverage.
Response 18: As we did not conduct a sensory evaluation, references to the beverage's taste and flavor were omitted as per the suggestion. (Line 462)
Comments 19: Please correct “ABc” and “ABa” on the Figure 4B
Response 19: AB represents the condition, while c and a indicate fermentation times; therefore, ABc and ABa refer to two-way statistics simultaneously.
Comments 20: actually I don't see much difference in GABA production in M3 and M5. Why did the authors prefer M5?
Response 20: Since M5 contains a higher MSG concentration, a precursor of GABA, it is likely to convert more MSG into GABA. This is reflected in the larger GABA spot observed in M5 on day 5.
Comments 21: It will be useful to provide additionally photo (e.g. as supplementary material) of the beverage samples in cuvettes for simple visual estimation of colors.
Response 21: We have attached a corresponding image in Figure S3 of the supplementary material.
Thanks to the reviewers' insightful feedback, the readability and quality of this paper have greatly improved.
Reviewer 3 Report
Comments and Suggestions for Authors
Dear Author,
Please see below my comments for paper " Functional kimchi beverage enhanced with γ-aminobutyric 2 acid (GABA) through serial co-fermentation using Leuconostoc 3 citreum S5 and Lactiplantibacillus plantarum KS2020".
Materials and methods: provide the source of origin for material being used in the study.
Results: overall work done in the study is presented well. including a sensory study using control vs test samples and comparing the acceptability of the product must be discussed in details.
Thanks
Author Response
Thank you very much for the valuable comments.
Comments 1: provide the source of origin for material being used in the study.
Response 1: Thank you for pointing this out. As you mentioned, we have added the origin of the materials in the Materials and Methods section.
Comments 2: overall work done in the study is presented well. including a sensory study using control vs test samples and comparing the acceptability of the product must be discussed in details.
Response 2: We agree with this comment. We are currently in the planning and preparation stage of the sensory evaluation experiment, and we will quantify and document the results after its completion.
Thanks to the reviewers' insightful feedback, the quality of this paper have greatly improved.
