Fermentation of Clementine Juice with Lactobacillus salivarius spp. salivarius CECT 4063: Effect of Trehalose Addition and High-Pressure Homogenization on Antioxidant Properties, Mucin Adhesion, and Shelf Life
Round 1
Reviewer 1 Report (New Reviewer)
In my opinion, the manuscript entitled Fermentation of clementine juice with Lactobacillus salivarius spp. salivarius CECT 4063: effect of trehalose addition and homogenization on antioxidant properties, mucin adhesion and shelf-life by Burca-Busaga et al., is a good one. Introduction provides enough information, materials and methos are enough described and the results are commented and compared with the current state of the art. I have some comments as follows:
1. line 6, 1 must be superscript written.
2. Lines 13-18, are quite confusing and needs to be better clarified. Please try to rephrase it in two different sentences.
3. Line 75 – Hence, the objective of the present study was to evaluate instead of evaluating….
4. line 97, please use as described by Barrera et al., instead of in
5. Table 2: please leave all number with two decimals after point, as follows: 0.84 and correct the value of 0.747
6. For me it is quite confusing the significance of a-c and A-D values in table 2. For instance, it would be easier to mentioned that different a-c in a row or column means statistically significant differences between samples at the same fermentation time…..please consider it in all tables.
7. lines 312-313 – please mention where the results could be seen? In which table or figure.
8. line 330, presented instead of present.
9. Figure 2. The results weren’t analysed from the statistical point of view? Please mentioned the differences also on the figure. Thank you
10. lines 351-361 – please mentioned where readers can observe the values? It is easier to read and to understand the results if the authors also mentioned the figure or table where the results could be found.
11. lines 402-414 – considering that standard deviation of COL values (figure 3) are big, please consider to insert one more explanatory sentence in order to strengthen the results.
Author Response
REVIEWER 1
In my opinion, the manuscript entitled Fermentation of clementine juice with Lactobacillus salivarius spp. salivarius CECT 4063: effect of trehalose addition and homogenization on antioxidant properties, mucin adhesion and shelf-life by Burca-Busaga et al. is a good one. Introduction provides enough information, materials and methos are enough described and the results are commented and compared with the current state of the art. I have some comments as follows:
- line 6, 1 must be superscript written.
Change already made
- Lines 13-18, are quite confusing and needs to be better clarified. Please try to rephrase it in two different sentences.
In agreement with reviewer 1 comment about the information provided in these lines, authors have rewritten them so now the aim of the study is expressed in two different sentences and is somewhat clearer.
- Line 75 – Hence, the objective of the present study was to evaluate instead of evaluating….
Change already made
- line 97, please use as described by Barrera et al., instead of in.
Change already made
- Table 2: please leave all number with two decimals after point, as follows: 0.84 and correct the value of 0.747
The number of decimal places was established following the theory of error and significant figures, which states that “Experimental uncertainty (standard deviation) should be rounded to one significant figure (first value different to zero). The only exception is when the uncertainty has a leading digit of 1, when a second digit should be kept”. Then, the number of decimal figures in the mean value was adjust to match that of its standard deviation. This is why the number of decimal places is not always the same.
- For me it is quite confusing the significance of a-c and A-D values in table 2. For instance, it would be easier to mentioned that different a-c in a row or column means statistically significant differences between samples at the same fermentation time… please consider it in all tables.
Authors agree with reviewer 1 that this way of expressing the effect of processing variables in the tables is quite confusing. We have managed to express such differences in terms of rows and columns in tables 1, 2 and 3.
- lines 312-313 – please mention where the results could be seen? In which table or figure.
This result can be deduced from the values shown in table 2, more specifically by comparing the ability to scavenge the DPPH and ABTS free radicals measured at times 0 and 24 h. This has been made clear in the text.
- line 330, presented instead of present.
Apologies but since line 330 contains a figure instead of a text, authors do not really know which “present” should be replaced with “presented”.
- Figure 2. The results weren’t analysed from the statistical point of view? Please mentioned the differences also on the figure. Thank you!
Figure 2 is the graph of means and LSD intervals obtained from the multifactor ANOVA analysis of the experimental data for a confidence level of 95%. Authors do not consider it necessary to include the letters indicating the homogeneous groups since the length of the error bars is enough to indicate the existence (if they overlap) or not (if they do not overlap) of significant differences between hesperidin (HESP), narirutin (NAR) and didymine (DID) content in the juice samples measured before and after 24 h of fermentation at 37 °C.
- lines 351-361 – please mentioned where readers can observe the values? It is easier to read and to understand the results if the authors also mentioned the figure or table where the results could be found.
Those particular values are not shown in any figure or table, but can be deduced from the TPC, the TFC and the AO_ABTS values shown in table 2. In this particular case, the degradation of the antioxidant compounds was calculated from the values obtained after fermentation for 24 and 96 h as the difference between them referred to the value after fermentation for 24 h. This has been made clear in the text.
- lines 402-414 – considering that standard deviation of COL values (figure 3) is big, please consider to insert one more explanatory sentence in order to strengthen the results.
Certainly, the coefficient of variation for the bacterial adhesion to collagen is for some treatments higher than 25%. Authors ruled out that this could be due to experimental errors since the dispersion in the bacterial adhesion to mucin and bovine serum albumin (measured at the same time and by the same methodology) were not that high. In previous studies, also high values in the standard deviation of the binding potential of several lactobacillus strains with collagen were observed. As an example, Rokana et al. (2018) reported values of 0.73 ± 0.52% (coefficient of variation of 71%), 11.07 ± 3.54% (coefficient of variation of 32%) and 1.03 ± 0.74% (coefficient of variation of 72%) for the attachment potential to collagen of L. plantarum NCDC20, L. casei HM1 isolated from human milk and E. coli, respectively. These values have been added to the new version of the manuscript, together with an extended explanation about the high standard deviation for adhesion to COL values: “High deviations obtained for adhesion to COL values suggest that nonspecific interactions (such as hydrophobic interactions and aggregating interactions) might play a more important role in this case. Since such nonspecific interactions are reported to be largely pre-disposed by the composition of bacterial surface covering components (Rokana et al., 2018), slight modifications on the growing media composition or in the processing conditions might have a greater impact on them. Coupling between specific and nonspecific interactions would also explain that the ability of Lactobacillus salivarius spp. salivarius CECT 4063 to adhere to COL was in general terms higher than that to MUC.
- Rokana et al. 2018. Screening of cell surface properties of potential probiotic lactobacilli isolated from human milk. Journal of Dairy Research, 85(3), 347-354. doi:10.1017/S0022029918000432
Reviewer 2 Report (Previous Reviewer 1)
Dear authors
There is a new sentence in the introduction that discusses the purpose of the study. Please explain in more detail this new goal, because the introduction does not include such a statement. It has an impact on the whole manuscript, which is not considered in the next sections of the manuscript. Information, at least minimally about goal achievement, must be complete.
Did the authors perform previous experiments at a lower pH of the juice?
Author Response
REVIEWER 2
There is a new sentence in the introduction that discusses the purpose of the study. Please explain in more detail this new goal, because the introduction does not include such a statement. It has an impact on the whole manuscript, which is not considered in the next sections of the manuscript. Information, at least minimally about goal achievement, must be complete.
The interest in obtaining an impregnation liquid with the highest content in microorganisms with potential probiotic effect has been made clear in the introduction section. More specifically, the following information has been added: “In the manufacture of a probiotic snack by means of vacuum impregnation and further dehydration (Betoret et al., 2012), the higher the microbial concentration in the impregnation liquid, the greater the counts in the vacuum impregnated product. Then, the decrease in the microbial population undergone by the impregnated product during its further processing can be reduced in different ways”.
- Betoret et al. 2012. No invasive methodology to produce a probiotic low humid apple snack with potential effect against Helicobacter pylori. Journal of Food Engineering 110(2), 289-293. DOI: 10.1016/j.jfoodeng.2011.04.027
Did the authors perform previous experiments at a lower pH of the juice?
Yes, a previous study was performed aimed at evaluating Lactobacillus salivarius spp. salivarius and Lactobacillus acidophilus growth in mandarin and pineapple/grape juices whose pH had been adjusted by the addition of sodium bicarbonate to 5, 5.5 and 6 (Betoret et al., 2012). Precisely from this study it was possible to conclude that the optimum pH for the growth of Lactobacillus salivarius spp. salivarius in mandarin juice is 6.
- Betoret et al. 2012. No invasive methodology to produce a probiotic low humid apple snack with potential effect against Helicobacter pylori. Journal of Food Engineering 110(2), 289-293. DOI: 10.1016/j.jfoodeng.2011.04.027
Reviewer 3 Report (Previous Reviewer 2)
Well, with the changes several points are already understood. The PCA thing was just to understand time trends, formulations, and other factors, it was not necessary to reduce it to 3 or 4 factors and not to find statistical differences. Better to leave the results as they were before.
Author Response
REVIEWER 3
Well, with the changes several points are already understood. The PCA thing was just to understand time trends, formulations, and other factors, it was not necessary to reduce it to 3 or 4 factors and not to find statistical differences. Better to leave the results as they were before.
Thank you for the clarification. Following reviewer 3 recommendation, the results were left as they were in the original manuscript.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Dear authors
The manuscript deals with an interesting and valuable theme. I suggest publishing this manuscript after major revision.
Page 2, line 92: the sentence: „When needed, trehalose was incorporated to the juice formulation at a rate of 10% (w/w).“ in my opinion, does not correspond to the objectives of the study: „...the objective of the present study was evaluating the effect of trehalose addition (10% w/w)“ (page 2, line 73)
Page 4, line 149: „2,2-Diphenyl-21-Picrylhydrazyl“ - please correct the compound name.
Page 4, line 170: „temperature of 5 °C“ - please check the temperature.
„(p-value < 0.05)“: I recommend using the label (p< 0.05).
Table 1, 2, 3: „Mean value of 4 replications ± standard deviation.“ - please move this information under the table as a note.
Table 1, 2, 3: please unify the number of decimal places
Page 14, line 415, 417: “5.4 ± 0.5-log10 ...... 4.92 ± 0.08-log10” I recommend writing without a dash (e.g. 5.4 ± 0.5 log10).
Please correct format of citation 41: „41. Hashemi, S.M.B., Khaneghah, A.M., Barba, F.J., Nemati, Z., Shokofti, S.S., & Alizadeh, F. (2017). ......“
Reviewer 2 Report
The manuscript is well written and novel. Here are some observations to clarify some important points:
1. On page 2, line 81 mention the characteristics of the juice, whether it contains pulp or not, and say if it is fresh or how it is packaged.
2. On page 3, line 94 mention the time to which the pressure process was subjected.
3. On page 3, line 98 mention here or in the introduction, the reason for homogenizing at this point once the bacteria are already there, since it is well known that this process decreases cell viability.
4. On page 3, line 104 clarify whether the HPH samples were no longer subjected to this fermentation process, since they only mention the Non-HPH and I+HPH treatments.
5. On page 4, line 157 mention why those 3 flavonoids, if they are the most common in clementine or why.
6. In Figure 3, explain the reason for such high deviations from COL treatments
7. A little suggestion, organize the data in a principal component analysis (PCA) as a complement to figure 5, since they are a lot of data and it would be easier to visualize the trends per day and variable responses with a PCA
8. In the conclusions, mention the potential that any of these treatments would have at a commercial level if the addition of trehalose and the treatment of high pressures are recommended and the conditions, or the areas of opportunity that you see to be able to launch such a product to the market.
