Dynamics of Microbiota in Three Backslopped Liquid Sourdoughs That Were Triggered with the Same Starter Strains
Round 1
Reviewer 1 Report
please see the attachment
Comments for author File: 
Comments.pdf
Author Response
We want to thank the reviewer for the positive comments and for the opportunity they give us to improve the manuscript.
The manuscript has been revised using the “track changes” function.
Here are the answers to the comments.
Reviewer 1: Specific comments:
- Paragraph 2.2: in line 112 or 124-133 the authors should specify the number of cells inoculated (in cell/g). It is very important to specify the initial level of strain inoculation since the paper is focused on the dynamic of microbiota in three sour-doughs.
The numbers of bacteria and yeasts cells inoculated as starter strains were measured after inoculum directly in the dough, and have already been reported in Figure 1. The first column in Figure 1, indicated as “S”, refers to the cell number just after the inoculum, and it is now explained in the caption. Might be it was not clear in the text, so I have included the following phrase in section 2.2: “With regard to the cell number of inoculated strains, it was detected on each dough after inoculum. For all samples the cell concentration was of the order of 107 UFC g-1 for bacteria, and 105 UFC g-1 for yeast strains. Values were reported in Figure 1 and indicated as “S”.
- Line 132: please add more details in the sentence “All the other fermentation steps..” What are the other fermentation steps?
Thank you for the suggestion. May be it was not clear. The sentences have been changed .
- Paragraph 2.4.: please add the references for the determination of pH and total titratable acidity.
The method for the determination of pH and TTA has been reported in full, and now something more has been added. I think there is no need for a bibliographic reference which would report the same, as it is written in the section.
- Line 202: please add more detail on Basic BLAST database and reference.
The BLAST database was mentioned at line 197 and 202, in the M&M section. The reference number has been inserted. The details about BLAST have been included in the references list.
- Line 172: please add the references for DNA extraction protocol and for the primers used Lac1 and
DNA extraction was performed following the procedure described by Promega and using the extraction kit provided by Promega. The phrase has been modified in section 2.6.1. The bibliographic references have been added for the procedure and the primers.
- Line 204: the authors write “Standard ANOVA procedure was applied to the dataset of acidity and cell counts” but in the results I didn’t find the evidence of this analysis. Please clarify this point.
Thank you for the suggestion. Now in the figure 1 and 2 the LSD intervals have been reported and the text in the captions has been corrected.
- Line 226 or in the Results section: please explain because you have chosen SD1 for the bakery trials.
Thank you for the suggestion. The phrase has been changed in the “Results” section, to explain that the first sourdough studied, i.e. SD1, was transferred to the bakery and used to make bread, and this gave us the chance to study the sourdough propagated in the bakery.
- For me the main criticism of the paper is the experimental design. Why did the authors decide to use different temperatures and different times for the fermentation process? It is well recognized that the temperature is an important selection parameter, and the diversity found in the microflora evolution and in the number of microbial species identified could be due precisely to these differences in temperature used, in addition to differences in inoculum levels. In fact it is normal that “bacteria dominated over yeast cells across samples” as written by the authors in line 234 since bacteria are inoculated at level of 107 CFU g-1 and yeasts 105 CFU g-1. Please clarify and explain better this point.
Thank you for the remarks. We know that the temperature is an important factor conditioning the microbial growth; in this work we wished to maintain a very high number of starter cells in sourdough, for this reason we have experimented the two temperatures, in order to see which one favored the growth of starter strains. This argument has now been included in the manuscript.
Regarding the inoculum level, we agree with the reviewer. Commonly, bacteria dominate over yeast in sourdoughs, in this work the high number of inoculated cells could be the main reason. The sentence has been changed in the text.
- Please improve the discussion of the results with the other data reported in literature, because it seems a summary of the data.
Thank you for the suggestion. The discussion has been modified.
- Please add the section of the Conclusions.
Ok. The section has been added.
- Check the references section according the journal’s guidelines.
Ok. Done.
Reviewer 2 Report
The manuscript written by Tolu et al. describes the dynamics of the microbiota in three liquid sourdoughs differently prepared. The authors used two approaches
Many studies have been focused on sourdough and several aspects have been examined, microbiological ones included.
The introduction is well done.
Materials and methods are quite complete.
The results are clearly exposed. I also appreciate the supplemental material.
There are some observations to make:
- Lines 101-102 MRS was added with maltose and yeast extract, why? MRS is already specific for Lactobacillus
- Lines 111-112: which is the initial load (CFU/ml) of the inoculum?
- In the preparation of sourdoughs, in my opinion, a sample without the starter inoculation should be interesting for the comparison with other samples
- Raw sequences must be deposited at NCBI in the Sequence Read Archive (SRA) and the Bioproject number must be indicated
- Lines 439-441, the strain identification could be done using the colonies isolated on plates
- In the conclusion (lines 521-527), the authors could expose which is the best protocol to prepare the sourdough on the basis of microbial dynamics, differences in pH and TTA
In my opinion, the manuscript can be published after these revisions.
Author Response
We want to thank the reviewer for the positive comments and for the opportunity they give us to improve the manuscript.
The manuscript has been revised using the “track changes” function.
Here are the answers to the comments.
Reviewer 2: Specific comments:
- Lines 101-102 MRS was added with maltose and yeast extract, why? MRS is already specific for Lactobacillus
Yes, MRS is specific for Lactobacillus, but maltose and fresh yeast extract are commonly added to the substrate when Lactobacilli originate from sourdough. The fresh yeast extract indicated in the text refers to compressed yeast, or bakers’ yeast, which was prepared following a specific protocol, and the reference has now been added in the text. The first authors reporting the use of maltose and fresh yeast extract were Kline and Sugihara in the 1971 (Microorganisms of the San Francisco Sour Dough bread process, Appl. Microbiol. 21 459–465) and the medium was called SDB (sour dough bacteria). Since then many other papers reported the use of such medium, for instance: Minervini et al. (2010) (Robustness of Lactobacillus plantarum starters during daily propagation of wheat flour sourdough type I. Food Microbiol. 27, 897-908]
- Lines 111-112: which is the initial load (CFU/ml) of the inoculum?
The initial loads of bacteria and yeasts cells, inoculated as starter strains, were measured after inoculum directly in the dough, and have already been reported in Figure 1. The first column in Figure 1, indicated as “S”, refers to the cell number just after the inoculum, and it is now explained in the caption. Might be it was not clear in the text, so I have included the following phrase in section 2.2: “With regard to the cell number of inoculated strains, it was detected on each dough after inoculum. For all samples the cell concentration was of the order of 107 UFC g-1 for bacteria, and 105 UFC g-1 for yeast strains. Values were reported in Figure 1 and indicated as “S”.
- In the preparation of sourdoughs, in my opinion, a sample without the starter inoculation should be interesting for the comparison with other samples
The reviewer suggests comparing the inoculated sourdoughs with a sourdough spontaneously fermented, it could be interesting, but our purpose was to study the microbiota after starter addition and at different growing conditions. The microflora evolution in spontaneous sourdoughs is completely different, the timing is different, so, it’s not easy to compare the results. We have chosen to study the microflora evolution in the sourdoughs triggered with starter strains.
- Raw sequences must be deposited at NCBI in the Sequence Read Archive (SRA) and the Bioproject number must be indicated
Thank you for the suggestion. Some sequences have already been deposited and the accession number has been indicated in the manuscript. Others sequences will follow (it's a very time-consuming work).
- Lines 439-441, the strain identification could be done using the colonies isolated on plates.
We agree with the reviewer. The identification of a strain can be done with PCR techniques (polymerase chain reaction), using strain-specific primers or RAPD method (random amplified polymorphic DNA), or other molecular biology methods, but this would have been a different work. Moreover, the analysis of colonies on plates can give limited information, as not all the microorganisms can be recovered from the plates, especially if they are at low number. We used a different method to obtain more information on the microbiota, on the complexity of the microflora.
- In the conclusion (lines 521-527), the authors could expose which is the best protocol to prepare the sourdough on the basis of microbial dynamics, differences in pH and TTA.
Thank you for the suggestion. A new section has been added for the conclusions with more considerations about the results obtained.
Reviewer 3 Report
1. Has the antagonism between Lactobacillus plantarum and yeast been considered. Have antagonistic tests been performed between strains.
2. What is the purpose of adjusting the pH of dough to 5.5 in SD2 and SD3? Does it affect the final flavor of the products? Why SD2 and SD3 did not transferred in the bakery.
3. The second row of the fungal part of Table 1, why the number of Candida lambica in SD1 Bak was lower than other groups?
4. It is recommended that the initial colony count of the flour be measured.
5. It is recommended to use natural fermented dough and commercial starter cultures as the control.
6、It is recommended to conduct sensory and flavor tests on the products made of dough, so as to prove whether the characteristics of start culture and dough are excellent.
7 The purpose of the experiment was not very clear in the paper.
8. L340-342: Why is Dipodascus australiensis found only in SD3?Maybe the expression "just" is incorrect?
9. L346: From Table 1, we can see that Wickerhamomyces anomalus , Candida santamariae differences are also significant, please discuss.
10. L354-355: "In SD1 the L. plantarum predominated the first three days, therefore its abundance decreased to very low values at day 22 "Explain the reason for this statement.
Author Response
We want to thank the reviewer for the positive comments and for the opportunity they give us to improve the manuscript.
The manuscript has been revised using the “track changes” function.
Here are the answers to the comments.
Reviewer 3: Specific comments:
- Has the antagonism between Lactobacillus plantarum and yeast been considered. Have antagonistic tests been performed between strains.
Thank you for the suggestion. The antagonism between the L plantarum strain and the yeast strains was not experimented in laboratory. All strains were isolated from natural sourdoughs, so this species commonly coexist in sourdough. The L. plantarum was tested for the acidifier capacity. The Saccharomyces strain was chosen based on the resistance to the acidity (previous studies). As reported in the paper the equilibrium among the strains is dependent on many factors, external factors, as the fermentation temperature, and internal factor, as the fermentation capacity.
- What is the purpose of adjusting the pH of dough to 5.5 in SD2 and SD3? Does it affect the final flavor of the products? Why SD2 and SD3 did not transferred in the bakery.
The pH of the dough was lowered with the purpose to favor the growth of lactic acid bacteria e to inhibit the growth of other species, especially those sensible at low pH.
We do not know if the flavor was affected, I presume it was, but in this work we were not interested to the sensorial aspects. It could be the subject for another project, considering that lower fermentation temperatures can increase the flavor production.
Only the sample SD1 was transferred to the bakery because was the first sourdough processed, and the bakery used it for baking purpose, so, it was not possible to transfer the other sourdoughs. We have reported it in the manuscript, in M&M and in the Results sections.
- The second row of the fungal part of Table 1, why the number of Candida lambica in SD1 Bak was lower than other groups?
The Candida species disappeared when SD1 was propagated in the bakery, as can be observed in figure 1 and in Figure 5. We don’t know why, probably is due to a different environment and to the house microbiota, in fact other strains disappeared, as the L plantarum, and some strain appeared, as the F. sanfranciscensis.
- It is recommended that the initial colony count of the flour be measured.
The work took much time, so, several batches of semolina were used, milled all in the same mill. Moreover, the number of viable cells in the flour is very low (periodically we made the cell count in some sample of flour), so, to know the number of cells would have been not very useful. While, it would have been more interesting the metataxonomic analysis of flour, but a lot of samples means a lot of money, and it was not possible. Could be the subject for a next study, as we have observed in this paper that the contaminant species originated from the flour .
- It is recommended to use natural fermented dough and commercial starter cultures as the control.
Our purpose was to study the microbiota of sourdough inoculated with our starter strains. Strains that were collected from natural sourdoughs produced in Sardinia. As we said to the other reviewer, the microflora evolution in spontaneous sourdoughs is completely different, the timing is different, so, it’s not easy to compare the results. Could be interesting to compare commercial starter, but it depends from the purpose of the research.
6、It is recommended to conduct sensory and flavor tests on the products made of dough, so as to prove whether the characteristics of start culture and dough are excellent.
We did not understand very well the question. I agree with the reviewer, the “flavor” is an interesting and an important subject for the sourdough and the baking products. But, the aim of the research was to study the microflora. If the reviewer means “to study the flavor of the sourdough”, this was not our purpose. If the reviewer means “to study the flavor of the bread made using the sourdoughs”, the work has been done and it will be the subject of the next manuscript.
7 The purpose of the experiment was not very clear in the paper.
Thank you for the suggestion. We have introduced new sentences into the text, hoping now the purpose would be clearer.
- L340-342: Why is Dipodascus australiensis found only in SD3? Maybe the expression "just" is incorrect?
The species Dipodascus australiensis was a contaminant species which was revealed in SD3 at relatively low abundance. May be the text was not clear, so, we have modified the sentence, indicating the relative abundance for the SD3 sample and removing the total relative abundance value.
- L346: From Table 1, we can see that Wickerhamomyces anomalus , Candida santamariae differences are also significant, please discuss.
Ok. The sentence has been modified.
- L354-355: "In SD1 the L. plantarum predominated the first three days, therefore its abundance decreased to very low values at day 22 "Explain the reason for this statement.
Thank you for the suggestion. The sentence has been modified in the results section, and a new comment has been added in the discussion section, to speculate on the decrease of the L plantarum.
Round 2
Reviewer 1 Report
The authors have greatly improved the manuscript following my suggestions and so for me now it is suitable for the publication in present form.
Reviewer 3 Report
Thank you for your explanation of the questions. The manuscript is better now.
