Round 1

Reviewer 1 Report

        In this article, different LAB strains were used to ferment the black mulberry juice through single strain fermentation and multi-strain fermentation. Based on the so-called the optimal inoculation ratio, fermentation conditions were investigated as well. The fermentation increased the SOD content and total phenols concentration in BMJ. Then the optimal fermentation process for FBMJ was determined by uniform design and RSM. The antioxidant effect of FBMJ on C2C12 cells was studied. This article is accord with the topics of Fermentation and may attract some peer interests. However, there are some revisions needed before the acceptance for the publication in Fermentation. Besides, the language was strongly suggested to be revised by professional English edition or native English speakers.

Major comments

Figure 1: A LAB cell counts-time course is needed.

Tables 5-7 are not appropriate in the results section. The experiment design should be displayed in the “Materials and Methods” section or supplementary material.

2.3: What type of incubator was used for Lactic acid bacteria culture in the experiment? Flask or bioreactor? What’s the volume of the incubator? What’s the liquid volume in the incubator? Are the fermentations aerobic, anaerobic or microaerobic? Such information is very significant for LAB fermentation.

Minor comments

Line 10, 204, 205, 214: Please use italics for strain names, and check all through the manuscript to reach such writing standard.

Line 217-219: The illustration is confused, at least, L. plantarum can produce lactic acid, please check more literatures.

Table. 4: The strain names of N. casei and C. animalis subsp. Lactis are clerical errors. There should be a space between abbreviation of generic name and species name.

Figure 3: The labels “MJ” and “FMJ” are clerical errors.

Line 359-363: Why did 0.5 mg/g BMJ display higher cell viability than the other two concentration? It is strange that there is no dosage-correspondence. Is this an experiment error or something else? Please try to explain the result.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

General comments:

The manuscript deals with the production of a fermented black mulberry juice prepared with probiotic, determining the optimum technology to achieve the maximum active substance content using response surface methodology.

However, the scientific merit of the paper is very poor because there are many papers dealing with the evaluation of the probiotic properties of different lactic acid bacteria including strains of Lactobacillus. In addition, there are many probiotic products containing different Lactobacillus strains that are currently commercialized. Then, the authors have to explain what is the contribution of their work to the development of the extensive accumulated knowledge about probiotic microorganisms. In addition, the authors should explain the advantages of using the LAB strains tested in their work with respect to other LAB present in other commercial probiotic products.

The material and methods section is incomplete and must be rewritten to provide more information about the analytical and statistical methods used in the manuscript. On the other hand, the results section must be rewritten and some affirmations are speculative since some important response variables to characterize a probiotic beverage (organic acids concentration and total viable counts of probiotic bacteria) were not measured.

 Chemical composition of the substrate (black mulberry juice) and product (probiotic-fermented black mulberry juice) is not provided and some important culture variables (culture pH, biomass concentration and sugars consumption) were not measured in the fermentations. The measurement of these three important variables could give information to explain the trend observed in the different cultures and avoid giving the speculative affirmations to explain the results obtained. So that, the fermentations were incomplete and the analysis of the results is incorrect and incomplete.

I strongly recommended that all authors carefully revise the manuscript to correct spelling errors and technical language. There are sufficient authors to make an appropriate and efficient revision of the paper.

For these reasons, the paper would not be accepted for publication in the Microorgnisms journal.

Other considerations are as follows:

Abstract: line 10: Morus nigra must be written in lowercase letter.

Abstract: line 13: The abbreviation RSM should not be used in the abstract.

Abstract: lines 15–16: the species of the different bacteria must be written in lowercase letter.

Abstract: line 17: Define SOD in this line and not in line 20.

1. Introduction.

1.1. Lines 54–55: Avoid repetition of “black mulberry juice”. If you produced a fermented black mulberry juice, it is clear that you used the black mulberry juice as a substrate.

1.2. Lines 55–58: Why did the authors screen 6 strains with high acid production capacity? It would be preferable to use probiotic strains with a low acid production capacity to obtain a fermented low acid beverage, or use yeast strains able to assimilate the organic acids produced by LAB in the fermentations.

2. Materials and methods.

2.1. Lines 90–92: Why was the juice digested with enzymes? In a single fermentation, the juice is used without any enzyme digestion because the use of enzymes increase the production cost of the probiotic beverage.

2.2. Lines 92–93: Thermally treated fruit juices can present disagreeable cooked and less fruity odors as observed by many researchers (Alonso et al., 2010; 2011). Did the authors consider this fact? How did the authors avoid this drawback?

2.3. Line 94: Why was the juice fermented for 18 h? Did the authors perform kinetic experiments to optimize the fermentation time?

2.4. Line 94: Replace “18 hours” by “18 h”. Please replace “hours” with “h” through the manuscript.

2.5. Line 98: Why was the total soluble solid (TSS) content adjusted to 18.5°Brix?

2.6. Lines 98–99: Why was the fermentation performed at 37°C and for 20 h?

2.7. Line 105: Why was the SOD value used as the response value? Why were other important variables (acid production capacity, high TPC, and high sensory score) not considered in this study?

2.7. Lines 113–122: The range of variables, soluble solid content (18.5, 19, 19.5, 20, and 20.5 °Brix), inoculum volume (1, 3, 5, 7, 9 and 12×10⁶ CFU/mL), fermentation temperature (27, 32, 37, 42, and 47 °C), and fermentation time (3, 6, 9, 12, 15 ,18, 21and 24 h), tested to investigate their effects on SOD activity (Y1) and TPC (Y2), were not in concordance with the values shown in Table 2.

2.8. Line 124: It is not necessary to say that the pH value was measured with a pH meter. So that, this sentence should be deleted.

2.9. Line 128: Which slight modifications did the authors use to measure the Total phenol concentration (TPC)?

2.10. Lines 130–132: Please explain how did the ten sensory-trained people rated the appearance, color, smell, taste, and texture of FBMJ? Did they use a five- or ten-point scale? How was this scale prepared? How were the samples for testing prepared? Which conditions were used to conduct the sensory analysis of FBMJ?

2.11. Lines 182–184: Which data were statistically compared? What statistical method was used to compare means and obtain the statistical significance of differences between groups?

2.12. Lines 190–192: Which enzymes lactic acid bacteria produce to biotransform the functional components (anthocyanins and polysaccharides) in mulberries?

3. Results. This section seems to be a mixture of the results obtained by the authors and a bibliographic review of similar works. However, this section should be used to show the results obtained by the authors in their research and the extensive literature review should be avoided, since in the majority of the cases, the inclusion of these reviews (e.g. lines 199-201, 208-211, 214-219, etc.) does not give information related to the results obtained by the authors.

So that, I strongly recommend rewriting this section by deleting those information related to the literature review and only showing the results obtained by the authors.

3.1. Lines 188–194: These sentences should be included in the Introduction section rather than in the Results section.

3.2. Line 196: Was really the highest values of pH used as a variable to select the strains for the mixed fermentation?

3.3. Line 199: Superoxide dismutase and its abbreviation (SOD) is constantly used in a join way through the manuscript. Please use one or the other.

3.4. Line 204: Please write L. reuteri and L. mesenteri in italics. This error is constantly repeated through the manuscript with different strains. Please replace “mesenteri” with “mesenteroides”.

3.5. Lines 223–224: Rewrite the sentences “Among them, the pH value of Weissella sp. fermentation broth is 3.38±0.01, and L.acidophilus is 3.38±0.01.” by “Among them, the pH value of Weissella sp. and L. acidophilus fermentation broth was 3.38±0.01.”

3.6. Lines 224–226: The authors said “They all have strong acid-producing ability, which is the reason why its lower sensory evaluation, high acidity destroys the sweet-acidity of the fermentation broth”. However, the initial pH of the black mulberry juice was 3.43±0.01 and the pH values in both the Weissella sp. and L. acidophilus fermentation broths were 3.38±0.01. This indicated that the pH dropped in both cultures by 0.05 pH units. Therefore, why did the authors say that Weissella sp. and L. acidophilus have a strong acid-producing ability? This affirmation is speculative since the authors did not measure the concentrations of organic acids. Why did the authors measure the organic acids production in the different fermentations? This could be an important response variable to be measured in the fermentations that could directly support the affirmations made by the authors rather than the results obtained by other researches, which probably did not use the same strains as those used by the authors.

3.7. Lines 227–229: Based on the above comment, it is not clear why the strains L. paracasei, L. casei, L. fermentum, L. delbrueckii, L. plantarum, and B. animalis subsp. lactis were determined as fermentation strains to ferment black mulberry juice. What were the response variables used to select these strains? This must be clearly discussed considering the results showed in Table 3.

3.8. Lines 231–232: This affirmation is speculative and should be experimentally demonstrated because there are a high number of papers showing that production of antibacterial compounds (e.g. bacteriocins) by a lactic acid bacterium strain could inhibit the growth of other related bacteria.

3.9. Lines 250–253: This information is not necessary in the Results section.

3.10. Line 255: I don’t understand what does the word “Subfifigures” mean.

3.11. Lines 257–258: How did the authors know that the lactic acid bacteria in the FBMJ took 6 h to reach the logarithmic growth phase and 12 h to the stable phase? Did the authors measure the growth of LAB?

3.12. Lines 260–261: According to Figure 1A, the SOD activity showed a slow decreasing trend after 15 h and not after 168 h. Please correct.

3.13. Lines 261–263: Please justify the selection of a fermentation time of 15 h by comparing statistically the TPC production at 15 and 18 h.

3.14. Lines 268–272: Please provided only a description of the results obtained, the other information is not needed in this section. In addition, the authors stated that “When the temperature is high, the intolerance of bacteria leads to the decrease of enzyme metabolism ability and activity[25], which affects the biotransformation of phenolic substances in the system. At the same time, phenolic substances are easy to be degraded in the environment of high temperature”. I suppose that the authors reviewed this information as a previous step before designing the experiments. So that, the authors should know that high temperatures affect the productions of SOD and TPC. Why did the authors test the effects of high temperatures on SOD and TPC production?

3.15. Line 274: The term “inoculation amount” is incorrect, the correct form is “inoculum concentration”. Please correct in Figure 1C and in the text.

3.16. Lines 274–275: Why did the authors affirm that “when the inoculation amount reaches 6×10⁶ CFU/mL, the growth and metabolism rate of lactic acid bacteria is moderate”? This affirmation is speculative because the authors did not measure the growth of LAB in the different cultures.

3.17. Lines 285–286: This affirmation is incorrect because TPC production increased between 18.0 to 19.5 ºBrix and decrease afterwards (Figure 1D).

3.18. Lines 286–287: This affirmation is speculative: “When the TSS content is adjusted to 19.5 °Brix, the fermentation of lactic acid bacteria was complete”.

There are many errors and speculative information through the manuscript that makes it very difficult to read and understand.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear Editor

This article "Development of probiotic-fermented black mulberry (Morus nigra L.) juice and its antioxidant activity in C2C12 cells” was revised and has a novelty and I recommend it for publication after consideration of the following comments.

·         Line 17: please say the complete words in the first time of expression. Not in line 20.

·         The statistical design used in this research should be mentioned as detail for example alpha, the factorial and axial repeat etc. Also factors and responses ought to mention. Center point of the study?

·         In the final introduction paragraph please consider statistical design. And express the treatments used in the study.

·         Please write materials as Company Name (City, Country), especially for chemical analysis assessment which used in the study.

·         Table 1: Why is the sum of numbers in each row different in percentage and why is the sum of all rows not based on 100%?

·         Table 1: What was the basis for choosing such percentages?

·         Please bring the treatment combination obtained from the RSM software in a combined table below table 2. Please pay attention to the following articles for a better understanding of my request and take ideas from them and cite them in your entire article, both in terms of the characteristics of the RSM plan and the types of tables and figures.

·         https://doi.org/10.1016/j.lwt.2021.110850

·         https://doi.org/10.1007/s11694-020-00567-1

·         https://doi.org/10.1111/jfpp.14563

·         Line 69 and 127: Method Folin-Ciocalteu is not a suitable method for measuring total phenol due to the interference of protein compounds, organic acids and reducing sugars with Folin's reagent and resulted to overestimation, and it would be better if it was measured with device methods like HPLC. Explain what measures you have taken to remove these interfering factors.

·         Table 3: why did not have “Sensory Evaluation (Score)” for Black mulberry juice.

·         Explain how method Sensory evaluation score has been evaluated accurately and in full detail in the article.

·          Table 4: Why was the basis of optimization only based on SOD Activity and more and better responses such as total phenol were not used?

·         Despite Table 4, Table 1 is no longer needed. In addition, the treatment combination resulting from the factors considered in RSM is more important, which I explicitly emphasized in the previous suggestion.

·         In Formula 1, 2, and 3 etc: please pointe to R², Adjuasted-R², CV, and Adeq Precision.

·         Line 397 and etc.: In the throughout the text of the article, the average number is sufficient and there is no need to state the standard deviation or standard error.

·         Discussion text must grammar improve and in some cases it is very weak and maybe there is no discussion at all. Discussion is very concise and please rewrite it comprehensively.

·         Conclusion is very general, try to make it more scientific, comprehensive and concise in detail, especially.

The article has many flaws in express and concept of English, it is suggested to be revised in a scientific and native way.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

General comments:

In the revised version of the manuscript, the authors address my referees’ comments. However, there are some comments that were not appropriately addressed by the authors and remain unclear in the revised version of the manuscript.

These comments are shown below:

1. Introduction.

1.1. Lines 45–46: “The fermentation process produces organic acids and other bioactive compounds that help extend the shelf life of fresh juices [9]”.

Reviewer comment: The fermentation process does not produce organic acids and other bioactive compounds. In fact, both the organic acids and other bioactive compounds are produced by the lactic acid bacterium and not by the fermentation process.

1.2. Lines 59–60: Why did the authors screen 6 strains with high acid production capacity? It would be preferable to use probiotic strains with a low acid production capacity to obtain a fermented low acid beverage, or use yeast strains able to assimilate the organic acids produced by LAB in the fermentation.

Response 9: In consideration of the fact that domestically distributed fermented drinks cannot contain alcohol in concentrations exceeding the national standard, yeast is not considered the fermentation strain, and strains with an appropriate pH value are instead used for the fermentation process. If the pH value is too low, the resulting flavor of the juice will be compromised. The juice pulp was already very sour.

Reviewer comment: In this answer, the authors did not explain why they screen 6 strains with high acid production capacity. As I indicated in the first review, it would be preferable to use probiotic strains with a low acid production capacity to obtain a fermented beverage with low acid content.

Point 11: 2.2. Lines 98–99: Thermally treated fruit juices can present disagreeable cooked and less fruity odors as observed by many researchers (Alonso et al., 2010; 2011). Did the authors consider this fact? How did the authors avoid this drawback?

Response 11: Considering the unpleasant appearance of some components and the destruction of nutrient components, the pretreatment, enzyme deactivation, and fermentation process were all performed by pasteurization.

Reviewer comment: I disagree with this response. Why didn´t the authors assayed different temperature values to conduct the appropriate thermal treatment of the juice for enzyme deactivation and for avoiding the destruction of nutrients of the juice? This could be a great influence in the development of disagreeable cooked and less fruity odors rather than in the fermentation process since the initial pH of the black mulberry juice (3.43±0.01) is considerably low, so that the possibility of a further contamination of the substrate during the fermentation could be reduced. How did the authors assure that nutrients were not lost with the pasteurization condition used (70 ºC)?

Point 12: 2.3. Line 94: Why was the juice fermented for 18 h? Did the authors perform kinetic experiments to optimize the fermentation time?

Response 12: This section has supplemented the data in the text 3.3.

3.3 Fermentation Kinetics and Specific Rate of FBMJ

Reviewer comment: According to the above graphical representation of the fermentation kinetics of FBMJ, there are different doubts that remains unclear.

For example:

1. The pH-time course is not showed. However, the authors stated that (lines 375-377 in the revised paper) “As can be seen from Figure 3A, after 16 h, due to the reduced sugar content, the low pH inhibited the growth of the bacteria and the specific growth rate of Lactobacillus decreased”.

2. If the culture pH drops from the initial pH of the black mulberry juice (3.43±0.01) to 3.38±0.01, that means only 0.05 units of pH,  what lower pH value did the authors refer to? 

According to the initial pH of the black mulberry juice (3.43±0.01), the growth of the Lactobacillus strain should already be inhibited from the beginning of the culture. However, according to the data shown in Figure 3A, the strain consumed approximately 36 g/L of reducing sugars. Although some LAB strains (Streptococcus gordonii. S. salivarius, and Lactobacillus casei) are considered to be extremely aciduric bacteria (Fajardo et al., 2008; Fozo et al., 2004; Svensater et al., 1997), it is very difficult to think that at low pH values, LAB are incapable of consuming high amounts of nutrients, since low pH values limit nutrient transport in LAB (Fajardo et al., 2008; Hutkins and Nannen, 1993; Poolman and Konings, 1988). These bacteria are capable of increasing the levels of long-chained, mono-unsaturated membrane fatty acids in their membrane in response to acidification (Fozo et al., 2004; Svensater et al., 1997). This may be a mechanism commonly used by these LAB to survive apprpiately in acidic environments (Fozo et al., 2004).

So that, the high consumption of reducing sugars by Lactobacillus strain in FBMJ at pH values between 3.43±0.01 to 3.38±0.01 is very doubtful. The authors should provide a consistent response for this behavior.   

3. How was the lactic acid concentration shown in Figure 3A measured? The authors stated that they did measure the lactic acid concentration in the fermentation medium and, in addition, the analytical method used to determine the lactic acid concentration was not described in the Materials and Methods section.

4. Why did not the authors show the graphical representation of the fermentation kinetics of FBMJ in the previous paper?

5. After 15 h of fermentation, the maximum SOD value was obtained and the bacterial density almost reached its maximum value. After 15 h, the SOD began to decrease. So that, I do not understand why did the authors select a fermentation time of 18 h because a fermentation time of 15 h is sufficient to stop the fermentation.

6. I do not understand why was Figure 3B included in the revised paper. In fact, this Figure does not contribute anything to the discussion of the results because it is obtained from Figure 3A.

Hutkins, R.W., Nannen, N. L. (1993). pH homeostasis in lactic acid bacteria. Journal of Dairy Science, 76, 2354–2365.

Fajardo, P., Rodríguez, I., Pastrana, L., Guerra, N. P. (2008). Production of a potentially probiotic culture of Lactobacillus casei subsp. casei CECT 4043 in whey. International Dairy Journal, 18,  1057–1065.

Fozo, E. M., Kajfasz, J. K., Quivey, R. G. (2004). Low pH-induced membrane fatty acid alterations in oral bacteria. FEMS Microbiology Letters, 238, 291–295.

Poolman, B., Konings, W. N. (1988). Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport. Journal of Bacteriology, 170, 700–707.

Svensater, G., Larsson, U. B., Greif, E. C., Cvitkovitch, D. G., Hamilton, I. R. (1997). Acid tolerance response and survival by oral bacteria. Oral Microbiology and Immunology, 12, 266–273.

Point 19: 2.9. Line 128: Which slight modifications did the authors use to measure the Total phenol concentration (TPC)?

Response 19: Plant-derived polyphenols exist in the form of free phenols and bound phenols; most are bound phenols. pH is an important factor that affects the non-covalent interactions between phenols, proteins, and polysaccharides. Our team used acetic acid-sodium acetate buffer to adjust the pH value to 3.5 when the liquid absorbance was the highest.

During the pretreatment, the absorbance value of the samples decreased significantly (by more than 30%) after the oligosaccharides were removed by alcohol precipitation, which may be attributed to the reducibility of the free aldehyde groups (such as isomaltose and galactose oligosaccharides). Therefore, some degree of interference was eliminated by adjusting the pH and removing the oligosaccharides by the pretreatment of the mulberry juice samples.  

Reviewer comment: Why did not the authors measure the Total phenol concentration (TPC) by using a HPLC method? With this procedure, the interference caused by of protein compounds, organic acids and reducing sugars with Folin's reagent could be avoided (Pérez-Gregorio et al., 2011).

Pérez-Gregorio, M.R., Regueiro, J., Alonso-González, E., Pastrana-Castro, L.M., Simal-Gándara, J. (2011). Influence of alcoholic fermentation process on antioxidant activity and phenolic levels from mulberries (Morus nigra L.). LWT - Food Science and Technology, 44, 1793–1801.

Point 29: 3.6. Lines 224–226: The authors said “They all have strong acid-producing ability, which is the reason why its lower sensory evaluation, high acidity destroys the sweet-acidity of the fermentation broth”. However, the initial pH of the black mulberry juice was 3.43±0.01 and the pH values in both the Weissella sp. and L. acidophilus fermentation broths were 3.38±0.01. This indicated that the pH dropped in both cultures by 0.05 pH units. Therefore, why did the authors say that Weissella sp. and L. acidophilus have a strong acid-producing ability? This affirmation is speculative since the authors did not measure the concentrations of organic acids. Why did the authors measure the organic acids production in the different fermentations? This could be an important response variable to be measured in the fermentation that could directly support the affirmations made by the authors rather than the results obtained by other researches, which probably did not use the same strains as those used by the authors.

Response 29: I am very sorry that I failed to measure the data at that time. We can only rely on the pH value as the basis. We have recognized this problem from the opinions of experts and will actively correct it in the future scientific research work.

Reviewer comment: I disagree with this response mainly considering the affirmations made by the authors in the revised version of the manuscript “Among them, the pH value of the fermentation broth produced by Weissella sp. and L. acidophilus were both 3.38. These two strains exhibited a strong acid-producing ability, which underlies their lower sensory evaluation, as high acidity destroys the sweet-acidity of the resulting fermentation broth.”

In fact, the authors only rewrite one sentence. So that, I continued asking, why did the authors say that Weissella sp. and L. acidophilus have a strong acid-producing ability? This affirmation is speculative since the authors did not measure the concentrations of organic acids produced by both strains. This important response variable must be measured in the fermentations with LAB (organic acids producers) and could be used to support the affirmations made by the authors rather than the results obtained by other researchers, which probably did not use the same strains as those used by the authors.

In addition, the affirmation “as high acidity destroys the sweet-acidity of the resulting fermentation broth” is also speculative. Did the authors demonstrate this affirmation? What high acidity did the authors refer to?

The culture pH dropped only from 3.43±0.01 (initial pH of the black mulberry juice) to 3.38±0.01 in both the Weissella sp. and L. acidophilus fermentation broths. Thus, the pH of the black mulberry juice could also be considered low enough to destroy the sweet-acidity of the resulting fermentation broth.

The authors also said, “We have recognized this problem from the opinions of experts and will actively correct it in the future scientific research work”. I do not understand this response. Why will this be corrected in future experiments and not now? It is not serious.

The determination of organic acids must be taken into account before carrying out the experiments with LAB, which are mainly producers of organic acids.

So that, the scientific merit of the paper remains to be very poor.

I strongly recommended that all authors carefully revise the manuscript to make an appropriate and efficient technical revision of the paper. In the revised paper, there are also many errors and speculative information through the manuscript that makes it very difficult to read and understand.

For these reasons, the paper would not be accepted for publication in the Fementation journal.