Innovative Fermented Soy Drink with the Sea Buckthorn Syrup and the Probiotics Co-Culture of Lactobacillus Paracasei ssp. Paracasei (L. Casei® 431) and Bifidobacterium Animalis ssp. Lactis (Bb-12®)
, Roxana Elena Goroftei Bogdan 4, Monica Boev 1,3,*, Denisa Batîr Marin 1,3,*, Ana Yndira Ramos-Villarroel 5 and Alina-Viorica Iancu 3,6,7
Round 1
Reviewer 1 Report
1. Add city and country name to (Dr. Oetker) in line 122.
2. What is the rest of the product (soymilk) characterization?
3. What is the percentage level of soluble solid contents of syrup? What else does it contain (total solid, protein, sugars, etc.)?
4. Figures' qualities are not good, they should be revised.
5. It's better to write the full name of TA on the graphs.
6. There are no statistically difference parameters on the graphs (except Table 1), the authors should add p values and difference letters to each parameter.
Author Response
Response to Reviewer 1 Comments
Thank you for the time given to the review and we respond promptly to the comments, because we consider that all observations are objective and well founded.
Point 1: Add city and country name to (Dr. Oetker) in line 122.
Response 1: I have added the information.
Point 2: What is the rest of the product (soymilk) characterization?
Response 2: I have added the information.
Point 3: What is the percentage level of soluble solid contents of syrup? What else does it contain (total solid, protein, sugars, etc.)?
Response 3: According to the information declared by the manufacturer, the sea buckthorn syrup was obtained from a natural extract from fresh fruits of the sea buckthorn (Hippophae rhamnoides) as follows: 1 l of sea buckthorn syrup is obtained from 2 kg of fresh sea buckthorn fruits, the syrup being hypocaloric. We chose the following syrup in this study due to it being a natural and hypocaloric syrup, as to obtain a novel drink with natural ingredients. Thusly these being the only information presented by the manufacturer, we considered that its not necessary we declare them in the article. If you see fit, we can add this information as well.
Point 4: Figures' qualities are not good; they should be revised.
Response 4: I have reviewed the qualities of figures.
Point 5: It’s better to write the full name of TA on the graphs.
Response 5: I have written the full name of the TA.
Point 6: There are no statistically difference parameters on the graphs (except Table 1), the authors should add p values and difference letters to each parameter.
Response 6: The experiments were made in triplicate and the standard deviation was calculated for each analyzed parameter, the standard deviation was added to all the presented graphics for all parameters, due to the quality of the figures not being good enough, it’s difficult to observe the standard deviation. During this revision we have improved the quality as to observe all the necessary data.
For all suggestions for corrections which were introduced in the attached word file, I have modified the text where it was suggested. All changes have been marked in red.
Reviewer 2 Report
The manuscript describes a study based on an interesting idea, which is the incorporation of sea buckthorn syrup into a fermented soy drink with 2 probiotic bacteria. The study is valid but it has several obscure points as well as formal problems, and it needs to be significantly improved. Detail:
- Update the taxonomy of Lactobacillus casei
- The genus and species of microorganisms must always be written in italics. Check References
-The identification of the strains used must always be written in the title and text. If you just write genus and species it would appear that all strains of those genera and species give the same results.
- The Introduction is too long. I suggest summarizing the text between lines 64 and 88, at least
- line 64: clarify what "LABs" means
-There is a strong doubt regarding the concept of "co-culture" used by the authors. When speaking of a co-culture it is understood that the 2 strains develop together in the substrate. If this were the case, it should be clarified how they were inoculated to carry out the fermentation, that is, the ratio between the two. If the 2 strains grow mixed, a differential and/or selective counting medium is required to allow them to be counted separately, as shown in Figure 1. The counting method for both strains in what is supposed to be a mixture of both is not detailed. Microbiologically, this is complicated unless on MRS Agar the 2 strains develop very different colonies. Was it done like this? It is only mentioned that MRS Agar was used and that plates of between 25 and 250 colonies were used. Were different versions of MRS Agar used for lactobacilli and bifidobacteria? I don't understand.
- Figure 3: clarify in the title what "TA" means
- Figure 4: add the storage temperature in the title
- Figure 7: clarify in the title what "WHC" means
- Figure 9: the title is incomplete. It must be indicated when the phenolic content was measured or of what thing said content was measured. Remember that a Figure must give all the information to understand it without having to resort to the text of the manuscript.
- Discussion: it repeats a lot what was already written in Results. Summarize, eliminating repetition
- line 387: talk about bacterial population. A total count of both strains added? In a culture medium, two culture media....? .
- line 428: update taxonomy of S. thermophilus
- lines 575-6: are you referring to drinks produced with both strains separately? or the drink resulting from co culture? I don't understand
Author Response
Response to Reviewer 2 Comments
Thank you for the time given to the review and we respond promptly to the comments, because we consider that all observations are objective and well founded.
Point 1: Update the taxonomy of Lactobacillus paracasei
Response 1: I have updated the taxonomy to Lactobacillus paracasei and for Bifidobacterium animalis subsp. lactis. We have also checked the website of Christian Hansen for the correct name of the used strains.
Point 2: The genus and species of microorganisms must always be written in italics. Check References
Response 2: I have modified in the entire manuscript.
Point 3: The identification of the strains used must always be written in the title and text. If you just write genus and species, it would appear that all strains of those genera and species give the same results.
Response 3: I have modified it in the title and the abstract and the entire manuscript.
Point 4: The Introduction is too long. I suggest summarizing the text between lines 64 and 88, at least
Response 4: The introduction must state the importance of developing this novel drink and due to the other reviewer appreciating the Introduction chapter as being well structured, we have decided to not bring any modifications and we hope that you will also agree with our decision.
Point 5: line 64: clarify what "LABs" means
Response 5: I have clarified what LAB means in line 64-lactic acid bacteria.
Point 6: There is a strong doubt regarding the concept of "co-culture" used by the authors. When speaking of a co-culture it is understood that the 2 strains develop together in the substrate. If this were the case, it should be clarified how they were inoculated to carry out the fermentation, that is, the ratio between the two. If the 2 strains grow mixed, a differential and/or selective counting medium is required to allow them to be counted separately, as shown in Figure 1. The counting method for both strains in what is supposed to be a mixture of both is not detailed. Microbiologically, this is complicated unless on MRS Agar the 2 strains develop very different colonies. Was it done like this? It is only mentioned that MRS Agar was used and that plates of between 25 and 250 colonies were used. Were different versions of MRS Agar used for lactobacilli and bifidobacteria? I don't understand.
Response 6: We thank you for the observations, which were of a big help. Sadly, when we redacted the manuscript, we omitted the writing of these details of high importance. The drinks were inoculated with 0.1 mL each of inocula of lactic acid bacteria and bifidobacteria. In these experiments, the initial population of each organism in the soymilk was 5 log CFU·mL-1. To determine the cell concentration, a UV-Vis Jenway 6506 spectrophotometer (Bibby Scientific Ltd., Staffordshire, UK) was used to determine the optical density (OD) of a cell suspension, in sterile saline solution (0.85% NaCl w/v), at a wavelength of 600 nm. A standard curve was constructed, giving the relationship between the number of bacteria and optical density. The linear equation was found by fitting straight line. This equation was used to find out the approximate amount of cell suspension to be used for preparing the product.
Bifidobacteria were enumerated using MRS-NNLP agar. MRS-NNLP agar consisted of MRS agar (Sigma-Aldrich, St. Louis, MO) supplemented with nalidixic acid-15 mg·L-1, neomycin sulphate-100 mg ·L-1, lithium chloride-3·0 g·L-1, paromomycin sulphate-200 mg·L-1. The information above has been added in the chapter: Materials and Methods.
Point 7: Figure 3: clarify in the title what "TA" means
Response 7: TA means titratable acidity and I made the modification, as per your suggestion, in the manuscript.
Point 8: Figure 4: add the storage temperature in the title
Response 8: We have added the storage temperature.
Point 9: Figure 7: clarify in the title what "WHC" means
Response 9: WHC means water holding capacity. These abbreviations (TA and WHC) were mentioned in the chapter Materials and Methods.
Point 10: Figure 9: the title is incomplete. It must be indicated when the phenolic content was measured or of what thing said content was measured. Remember that a Figure must give all the information to understand it without having to resort to the text of the manuscript.
Response 10: We have modified the title of the figure.
Point 11: Discussion: it repeats a lot what was already written in Results. Summarize, eliminating repetition
Response 11: We have eliminated repetitions.
Point 12: line 387: talk about bacterial population. A total count of both strains added? In a culture medium, two culture media....? .
Response 12: We have made the necessary corrections in line 387. As well, we mention that the correct method of determination for the lactobacilli and bifidobacteria numbers was added in the Materials and Methods chapter.
Point 13: - line 428: update taxonomy of S. thermophilus
Response 13: For the S. thermophilus-deleted salivarius from the text of manuscript (because the quoted researchers have declared the following taxonomy: Streptococcus thermophilus).
https://doi.org/10.1016/j.lwt.2009.01.014
Point 14: lines 575-6: are you referring to drinks produced with both strains separately? or the drink resulting from co culture? I don't understand
Response 14: It referring to the drink resulting from the co-culture.
For all suggestions for corrections which were introduced in the attached word file, I have modified the text where it was suggested. All changes have been marked in red.
Round 2
Reviewer 2 Report
The manuscript has significantly improved its presentation. A few considerations remain to review and adjust. - Regarding the taxonomic update of L. casei, it must be said that the current one is Lacticaseibacillus casei/paracasei. The web pages of the market are usually outdated in terms of microbial taxonomy. I suggest writing in the title "Lacticaseibacillus paracasei ssp. paracasei (L. casei R 431)" - line 716: "paracasei" - It is necessary to add in some places of the text the identification of the strains used (lines 145, 219....). Review
