Unlocking the Potential of Lacticaseibacillus rhamnosus 73 as a Ripening Agent in Semi-Hard Cheese After Freeze-Drying and a Six-Month Storage Period
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAuthors evaluated the reactivation capacity after freeze drying process ans as inoculant for milk and cheese ripening of a Lacticaseibacillus rhamnosus, in both processes the bacteria showed interesting results as a potential starter for dairy products, however there are some points not clear.
- If during ripening peptides were produced, why the authors did not show the changes in the concentration of protein?, as I understand, total protein was also determined
- How the authors determined that peptides have a low contain of aromatic amino acids?, did they compare with peptide standards?, did they determine the molecular weight by mass spectrometry (m/z) of the peptides?, how it was determined?
- Please put the first time month (m) in line 95, to know that m means month.
Author Response
Responses of the authors (AU)
AU: First of all, we sincerely appreciate the valuable comments and suggestions made, which helped us to improve the quality of this manuscript. We have carefully addressed the reviewers’ queries and used track changes to highlight the modifications on the manuscript.
Reviewer #1, (R#1)
Authors evaluated the reactivation capacity after freeze drying process ans as inoculant for milk and cheese ripening of a Lacticaseibacillus rhamnosus, in both processes the bacteria showed interesting results as a potential starter for dairy products, however there are some points not clear.
R#1: If during ripening peptides were produced, why the authors did not show the changes in the concentration of protein?, as I understand, total protein was also determined
AU: Thank you for this question. As detailed in the Materials and Methods section, all determinations were carried out at the end of the ripening period. In particular, protein content was determined using the Kjeldahl method, which measures total nitrogen without distinguishing its origin (i.e., whether from proteins, peptides, amino acids, etc.). In this method, the concentration of nitrogen is then multiplied by a conversion factor to estimate protein content. Therefore, even if the peptide profiles change during ripening, the total nitrogen—and consequently the protein content—remains unchanged. It would be interesting to study and identify peptides in future works, but we would need to consider more complex techniques such as MALDI-TOF and LC-MS.
R#1: How the authors determined that peptides have a low contain of aromatic amino acids?, did they compare with peptide standards?, did they determine the molecular weight by mass spectrometry (m/z) of the peptides?, how it was determined?
AU: Thank you for this question. Peptide analysis in cheese extracts was performed using HPLC with a UV detector. This method provides a peptide profile of the cheeses but does not allow for individual peptide identification. Based on the chromatographic conditions, the peptides can be broadly classified as hydrophilic or hydrophobic, as they are eluted at the beginning and end of the chromatographic run, respectively.
R#1: Please put the first time month (m) in line 95, to know that m means month.
AU: The clarification was added (line 100).
Reviewer 2 Report
Comments and Suggestions for AuthorsIntroduction
Please states that lactic acid bacteria are part of the common microbiota of raw milk and dairy environment; starter and adjunct cultures are particularly needed when milk is subjected to thermal treatment. Add relevant references.
L31-32: please change: “the most common goals of adjunct cultures”
L37-38: Lactiplantibacillus plantarum, and Lacticaseibacillus rhamnosus
L58: delete among others
L59: delete etc.
L59-60: delete the sentence “The mechanism of action of both is different”
Materials and Methods
L76: Please add information on the storage condition of the strain.
L88: were harvested by centrifugation?
L92: 2 mL of cell suspension were distributed in glass vials
L.83: replace “as per” with “according to”; same in line 93.
L94-95: vacuum-sealed; 6 m? months?
L97-100: Please consider to write a separate section on viable plate counts (analysis of the freeze-dried samples).
L112: were determined
L119: Please better explain the experimental plan for cheesemaking trials. “Semi-hard cheeses were made in triplicate”: You mean you prepared three cheese wheels for each condition? Or one cheese-making trial for each condition?
L120-121: add space between 6 and m (6 m)
Section 2.4: please consider to add a flow-chart of the cheesemaking process; add a figure of the cheese if available
L141-142: please add a reference about the practice of adding distilled water to cheese for pH measurement.
L144: total
Section 2.5: add information on how the results were expressed
Section 2.7: did you processed the samples before RP-HPLC? Extraction of the soluble fraction?
L167: add space between 6 m
L173 p < 0.05
Results and discussion
The results are clearly presented and easy to follow. However, sentence structure and overall readability should be improved.
L178: microbiological counts; add the microbial group analyzed e.g.: microbiological/viable counts of lactobacilli
L178-179: a point is missing to conclude the sentence. Moreover, I suggest to rephrase for better readability.
L179: p > 0.05; same in L180
L182-183: The positive effect of freeze-drying on the viability and activity of lactic acid bacteria during storage is well documented
L184: lactic acid bacteria
L186: can affect
L191: (e.g. glucose and fructose)
L192: (e.g. maltodextrin),
L203: Silva et al. [33]
L219: The results of carbohydrates and organic acids concentrations in the cultured milk
L224: (p > 0.05)
L227-228: was significantly lower in CM 6m compared to NIM (p < 0.05)
L231-233: Consider revising the sentence for clarity. For example:
"Determining the organic acids naturally present in milk [38] is important because some microorganisms can utilize them as an energy source for growth."
Avoid "lies in the fact that."
L233-234: with the production of flavor compounds
L234: change especially (particularly, mainly). Characterized by butter-like aroma (remove its)
L240: Hippuric acid is another organic acid naturally present in milk
L250 (Figure 1 caption): Different letters for the same compound indicate statistically significant differences (p < 0.05).
L252: Results of composition…..of cheese samples (NRA,…)…are reported in Table 1.
L254: for moisture, fat, and protein content
L255: the results of chemical composition and pH are similar to
L258: remove (a commercial and expensive medium)
L266-267: Although slight numerical differences in total LAB counts were observed between cheeses with and without the ripening agent, these differences were not statistically significant (p > 0.05)
L268: are in line with
L273-274; cheese matrix [43], not only by growing but also due to the concentration in the curd during the draining. Please rephrase
L274-276: Consider revising for clarity and completeness. For example:
"It is well established that bacteria in milk become physically entrapped within the curd matrix during coagulation, and only those located on exposed cut surfaces are released into the whey."
L281: , and salt content.
L284: change “It is interesting to mention”
L284-286: Relying solely on colony morphology for bacterial identification can be misleading. While the sentence “Nevertheless, molecular studies must be performed to confirm this hypothesis” is appreciated, the preceding observation does not provide sufficient evidence to support the hypothesis. I recommend deleting these sentences, as the morphological similarity alone is not adequate to suggest the presence of L73 without confirmatory data.
L292: cheese-milk?
L290: why the detection limit is 4 log cfu/g?
L296: Please consider to divide Table 1:
- Table 1: Physico-chemical parameters of cheeses
- Table 2: Viable counts of cheeses
Delete rows with “composition” and “microbiological counts”
L330: add a reference to support the sentence “Cheese is a more complex system in which matrix composition, microbiota, long ripening times, among other factors, could have influenced the final concentration of this acid.
L304-339: The section from lines 304–339 is very well-written, with clear structure and concise scientific language. I recommend revising the other results sections to match this level of clarity and organization, to improve the overall readability and consistency of the manuscript.
L382: add a reference
Author Response
Responses of the authors (AU)
AU: First of all, we sincerely appreciate the valuable comments and suggestions made which helped us to improve the quality of this manuscript. We have carefully addressed the reviewers’ queries and used track changes to highlight the modifications on the manuscript.
Reviewer #2, (R#2)
Introduction
R#2: Please states that lactic acid bacteria are part of the common microbiota of raw milk and dairy environment; starter and adjunct cultures are particularly needed when milk is subjected to thermal treatment. Add relevant references.
AU: This part was modified (lines 27-30, 500-501)
R#2 L31-32: please change: “the most common goals of adjunct cultures”
AU: The sentence was changed accordingly (line 34)
R#2 L37-38: Lactiplantibacillus plantarum, and Lacticaseibacillus rhamnosus
AU: It was modified as suggested (line 40)
R#2 L58: delete among others
AU: It was modified (line 60)
R#2 L59: delete etc.
AU: It was deleted as suggested (line 61)
R#2 L59-60: delete the sentence “The mechanism of action of both is different”
AU: That sentence was deleted (lines 61-62)
Materials and Methods
R#2 L76: Please add information on the storage condition of the strain.
AU: More details were added (lines 78-82)
R#2 L88: were harvested by centrifugation?
AU: The clarification was added (lines 91-92)
R#2 L92: 2 mL of cell suspension were distributed in glass vials
AU: It was modified as suggested (lines 96-97)
R#2 L.83: replace “as per” with “according to”; same in line 93.
AU: It was modified (lines 86, 98-99)
R#2 L94-95: vacuum-sealed; 6 m? months?
AU: We modified it (lines 99-100)
R#2 L97-100: Please consider to write a separate section on viable plate counts (analysis of the freeze-dried samples).
AU: A new section was added as indicated (lines 101-106)
R#2 L112: were determined
AU: It was modified accordingly (line 118)
R#2 L119: Please better explain the experimental plan for cheesemaking trials. “Semi-hard cheeses were made in triplicate”: You mean you prepared three cheese wheels for each condition? Or one cheese-making trial for each condition?
AU: We made nine cheeses in total: three cheeses “NRA”, three cheeses “RA -20” and three cheeses “RA 5”. This was clarified (line 125)
R#2 L120-121: add space between 6 and m (6 m)
AU: The space was added (line 127)
R#2 Section 2.4: please consider to add a flow-chart of the cheesemaking process; add a figure of the cheese if available
AU: A flow-chart and a figure of the cheeses were added (line 143-147, Figure 1)
R#2 L141-142: please add a reference about the practice of adding distilled water to cheese for pH measurement.
AU: The reference was added (line 152, 543-544)
R#2 L144: total
AU: It was modified (line 154)
R#2 Section 2.5: add information on how the results were expressed
AU: The information was added (lines 156-157)
R#2 Section 2.7: did you processed the samples before RP-HPLC? Extraction of the soluble fraction?
AU: That is correct, the information was added (lines 170-174)
R#2 L167: add space between 6 m
AU: It was added (line 183)
R#2 L173 p < 0.05
AU: It was modified (line 189)
Results and discussion
R#2 The results are clearly presented and easy to follow. However, sentence structure and overall readability should be improved.
L178: microbiological counts; add the microbial group analyzed e.g.: microbiological/viable counts of lactobacilli
AU: It was modified as suggested (lines 194-196)
R#2 L178-179: a point is missing to conclude the sentence. Moreover, I suggest to rephrase for better readability.
AU: This part was rephrased (lines 194-196)
R#2 L179: p > 0.05; same in L180
AU: It was modified as indicated (lines 195-197)
R#2 L182-183: The positive effect of freeze-drying on the viability and activity of lactic acid bacteria during storage is well documented
AU: It was modified as suggested (lines 199-202)
R#2 L184: lactic acid bacteria
AU: It was changed accordingly (line 203)
R#2 L186: can affect
AU: It was modified (line 205)
R#2 L191: (e.g. glucose and fructose)
AU: It was changed as suggested (line 209)
R#2 L192: (e.g. maltodextrin),
AU: It was modified (line 210)
R#2 L203: Silva et al. [33]
AU: It was changed (line 224)
R#2 L219: The results of carbohydrates and organic acids concentrations in the cultured milk
AU: The sentence was rephrased (line 241)
R#2 L224: (p > 0.05)
AU: It was modified (line 246)
R#2 L227-228: was significantly lower in CM 6m compared to NIM (p < 0.05)
AU: It was changed accordingly (line 250)
R#2 L231-233: Consider revising the sentence for clarity. For example:
"Determining the organic acids naturally present in milk [38] is important because some microorganisms can utilize them as an energy source for growth."
Avoid "lies in the fact that."
AU: The sentence was reformulated as suggested (lines 254-256)
R#2 L233-234: with the production of flavor compounds
AU: It was modified (lines 258-259)
R#2 L234: change especially (particularly, mainly). Characterized by butter-like aroma (remove its)
AU: The amendments were made (line 259)
R#2 L240: Hippuric acid is another organic acid naturally present in milk
AU: It was modified (lines 265-266)
R#2 L250 (Figure 1 caption): Different letters for the same compound indicate statistically significant differences (p < 0.05).
AU: It was changed as suggested (lines 276-277)
R#2 L252: Results of composition…..of cheese samples (NRA,…)…are reported in Table 1.
AU: It was modified (lines 279-280)
R#2 L254: for moisture, fat, and protein content
AU: The amendment was made accordingly (line 282)
R#2 L255: the results of chemical composition and pH are similar to
AU: It was modified (line 291)
R#2 L258: remove (a commercial and expensive medium)
AU: It was removed (line 294)
R#2 L266-267: Although slight numerical differences in total LAB counts were observed between cheeses with and without the ripening agent, these differences were not statistically significant (p > 0.05)
AU: It was modified as requested(lines 302-304)
R#2 L268: are in line with
AU: It was changed (line 305)
R#2 L273-274; cheese matrix [43], not only by growing but also due to the concentration in the curd during the draining. Please rephrase
AU: It was rephrased (lines 310-314)
R#2 L274-276: Consider revising for clarity and completeness. For example:
"It is well established that bacteria in milk become physically entrapped within the curd matrix during coagulation, and only those located on exposed cut surfaces are released into the whey."
AU: It was modified accordingly (lines 314-318)
R#2 L281: , and salt content.
AU: It was changed (line 330)
R#2 L284: change “It is interesting to mention”
AU: It was deleted (line 333)
R#2 L284-286: Relying solely on colony morphology for bacterial identification can be misleading. While the sentence “Nevertheless, molecular studies must be performed to confirm this hypothesis” is appreciated, the preceding observation does not provide sufficient evidence to support the hypothesis. I recommend deleting these sentences, as the morphological similarity alone is not adequate to suggest the presence of L73 without confirmatory data.
AU: That part was removed (line 333-336)
R#2 L292: cheese-milk?
AU: It was modified (line 342)
R#2 L290: why the detection limit is 4 log cfu/g?
AU: This was clarified in lines 338-339.
R#2 L296: Please consider to divide Table 1:
- Table 1: Physico-chemical parameters of cheeses
- Table 2: Viable counts of cheeses
AU: It was divided as suggested (Now Tables 1 and 2)
R#2 Delete rows with “composition” and “microbiological counts”
AU: They were removed
R#2 L330: add a reference to support the sentence “Cheese is a more complex system in which matrix composition, microbiota, long ripening times, among other factors, could have influenced the final concentration of this acid.
AU: It was added (line 380, 615-616)
R#2 L304-339: The section from lines 304–339 is very well-written, with clear structure and concise scientific language. I recommend revising the other results sections to match this level of clarity and organization, to improve the overall readability and consistency of the manuscript.
AU: We modified some paragraphs to improve the readability and consistency throughout the whole manuscript. For instance, lines 214-218, 233-235, 239-240, 245-246, 263-264, 440, 444, among others
R#2 L382: add a reference
AU: It was added (lines 433, 623-624)
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript has the quality to be published
Author Response
Reviewer #1 (R#1)
R#1: The manuscript has the quality to be published
Authors: We really appreciate the feedback and the time dedicated to revising our manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsL143: “A flow-chart of the cheesemaking is shown in Figure 1.” Please move this sentence before the description of the cheesemaking process (e.g. L129).
L175: 2.7
Author Response
Authors (AU): We really appreciate the feedback and the time dedicated to revising our manuscript. We used track changes to highlight the modifications on the manuscript.
Reviewer #2 (R#2)
R#2: L143: “A flow-chart of the cheesemaking is shown in Figure 1.” Please move this sentence before the description of the cheesemaking process (e.g. L129).
AU: This part was moved (lines 124-125,139-140)
R#2: L175: 2.7
AU: This part was modified (lines 170)
