Regulation of Cultivation Temperature on Biomass and Activity of Bifidobacterium breve B2798

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript examines how the cultivation temperature affects the biomass production and a key metabolic enzyme in the cells of the lactic acid-producing bacterium Bifidobacterium breve B2798. The findings of this study are important to the large-scale production of the prebiotic bacterium. Screening of the cultivation temperature for Bifidobacterium breve B2798 revealed that 38^oC was the optimum cultivation temperature based on viable cell count. The problem with the elevated cultivation temperature is that key glycolytic enzymes could be inactivated by elevated temperature. It appears that a shorter period of cultivation of bacterial cells is preferable to a longer period of cultivation time. It was also shown that the enzyme fructose-6-phosphate phosphoketolase showed maximal activity between 37-41^oC. The conclusions from this study should help in the large scale cultivation of Bifidobacterium breve B2798 cell biomass. The manuscript is generally written clearly and well organized. The references cited appear appropriate and are recent. The manuscript requires revision prior to being acceptable for publication. On page 9 (line 2), correct the spelling of phosphoketolase and on line 8 substitute “does” for “do”. On page 13 (lines 9-10), correct sentence construction to improve clarity. In the References, the journal style needs to be followed. Sometimes the article name is presented in uppercase (references 16 & 24) while other times in lowercase. Also, sometimes the journal name is abbreviated in some references while the full name is given in others.

Author Response

Comments 1: [The manuscript requires revision prior to being acceptable for publication. On page 9 (line 2), correct the spelling of phosphoketolase and on line 8 substitute “does” for “do”.]

Response 1:We agree with this comment.The spelling of phosphoketolase has been corrected on page 9 (line 2) of the manuscript, and "does" has been replaced with "does" on line 8.

Comments 2: [On page 13 (lines 9-10), correct sentence construction to improve clarity.]

Response 2:We agree with this comment.The study results show that within the temperature range of 37.0℃ to 39.0℃, Bifidobacterium breve B2798 exhibits good cell growth and stable cell activity. This test aimed to further verify these findings through high-density fermentation to achieve precise temperature control.

Comments 3: [Sometimes the article name is presented in uppercase (references 16 & 24) while other times in lowercase.]

Response 3:We agree with this comment.Unified as required，for example:

14 Madrid, R. E., & Felice, C. J. (2005). Microbial Biomass E Crit Rev Biotechnol, 25(3), 97-112.

16 Pang, X., Zhang, S., Lu, J., Liu, L., & Lv, J. (2017). Identification and Functional Validation of Autolysis—Associated Genes in Lactobacillus bulgaricus ATCC BAA-365. Frontiers In Microbiology, 8, 1367. https:// doi.org/ 3389/fmicb.2017.01367

Comments 4: [sometimes the journal name is abbreviated in some references while the full name is given in others.]

Response 4:We agree with this comment.Unified in all references，for example:RSC Adv=Royal Society of Chemistry Advances.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Throughout the manuscript, please keep the name of microorganism in italics wherever mentioned

Under the materials and methods section

2.2, The conditions for anaerobic fermentation were not specified, i.e; what pH maintained; volume of media to study and if conducted in controlled bioreactors, its gaseous nature; acid/base addition; how long was the fermentation time; endpoint to measure production.

2.4, under dry cell weight, typo “broths” not froths

2.6, please cite the manufacturer and model no of biochemical analyzer used to measure lactic acid

Under 3.1.5, stimulation test of cell activity

How was the strain stored? Was any protectant added to store at 4 C or just small volume sampled out and stored to test its activity? At which phase/point, a certain volume was drawn out for study?

In 3.2.2, First statement looks like incomplete and please rephrase it

Under this section, please mention the dry cell or wet cell weight in grams or percentage where appropriate. So that it can be easily comparable for readers

Author Response

Comments 1: [2.2, The conditions for anaerobic fermentation were not specified, i.e; what pH maintained; volume of media to study and if conducted in controlled bioreactors, its gaseous nature; acid/base addition; how long was the fermentation time; endpoint to measure production.]

Response 1: Thank you for pointing this out. I agree with this comment.Modified in 2.2, please refer to the attachment for details.

Comments 2: [under dry cell weight, typo “broths” not froths.]

Response 2: Thank you for pointing this out. I agree with this comment.Modified in 2.4, please refer to the attachment for details

Comments 3: [2.6, please cite the manufacturer and model no of biochemical analyzer used to measure lactic acid]

Response 3:Thank you for pointing this out. I agree with this comment. Added in the text.

Comments 4:[2.6, please cite the manufacturer and model no of biochemical analyzer used to measure lactic acid]

Response 4: Thank you for your question. The strain was stored at 4 ℃ without adding any protective solution, and was directly stored in the culture medium to evaluate its culture activity. Secondly, a certain amount of culture medium should be taken at the end of logarithmic phase and stable phase respectively to evaluate storage activity.

Comments 5: [In 3.2.2, First statement looks like incomplete and please rephrase it.]

Response 5Thank you for pointing this out. I agree with this comment. Modified in the main text.

Author Response File: Author Response.docx