Round 1

Reviewer 1 Report

Lactic acid bacteria, as an important and common probiotic, is very beneficial to human health. This article describes the effects of Carao on the activity characteristics of lactic acid bacteria. It's a meaningful study. However, this article did not find that Carao significantly enhance the activity of lactic acid bacteria, and the graphic writing is not standardized, it is recommended to reject the draft.

Moderate editing of English language required

Author Response

Comments and Suggestions for Authors

Lactic acid bacteria, as an important and common probiotic, is very beneficial to human health. This article describes the effects of Carao on the activity characteristics of lactic acid bacteria. It's a meaningful study. However, this article did not find that Carao significantly enhance the activity of lactic acid bacteria, and the graphic writing is not standardized, it is recommended to reject the draft.

Answer: The viability under different conditions, immunomodulatory capacity, and probiotic potential of L. plantarum as affected by carao were tested and investigated. The addition carao to the medium under different experimental conditions, including rich and minimal culture media and a gastrointestinal digestion process of skimmed milk, did not affected substantially the growth of Lactobacillus plantarum, but prolongs its viability. The administration of Lactobacillus plantarum with carao in mice did not induce a proinflammatory response at a systemic level. Still, it did induce an increase in the production of the immunoregulatory cytokines.

The manuscript gives insights on a well study probiotic on novel functional ingredient carao.

Comments on the Quality of English Language

Moderate editing of English language required

Answer: English language was improved.

Reviewer 2 Report

This paper consists of a thorough study of the health benefits associated with Lactobacillus plantarum and carao (Cassia grandis), as well as their potential synergies when used together.

Lactobacillus plantarum is known for its diverse positive effects, including aiding lactose digestion, lowering cholesterol, and improving intestinal health. Meanwhile, carao is noted for its rich nutritional content and potential dietary applications, although a deeper dive into its specific nutrients and health-promoting properties could enrich our knowledge.

The paper also outlines the interaction between carao and L. plantarum. Results indicate that carao did not compromise the viability or immunomodulatory capacity of L. plantarum. In fact, carao appeared to enhance several attributes of L. plantarum, such as acid and bile tolerance, suggesting potential benefits for fortified dairy products.

Nonetheless, this paper offers valuable insights into the health-promoting properties of L. plantarum and carao, as well as their combined potential for enhancing gut health and overall well-being.

It is a well written paper based on relative bibliography.Material and Methods are well described .Resistance to bile salts,tolerancer to pH, Tolerance to NaCl,Viability of lactobacillus plantarum ,Viability of lactobacillus plantarum under gastrointestinal digestion were considred ,followed by studies on the effect of carao on Experimental infection with Y. enterocolitica O9 IP383 in BALB/c mice .Yet, measurement of cytokines and IgA was performed .Results were evaluated statistically and thoroughly discussed.

The paper provides a compelling exploration of the health benefits associated with Lactobacillus plantarum and carao (Cassia grandis), capturing the interest of readers intrigued by probiotics and natural remedies ,scientists working in the field and industrials.

My suggestion is to ACCEPT and publish it in its present form.

Author Response

Comments and Suggestions for Authors

This paper consists of a thorough study of the health benefits associated with Lactobacillus plantarum and carao (Cassia grandis), as well as their potential synergies when used together.

Lactobacillus plantarum is known for its diverse positive effects, including aiding lactose digestion, lowering cholesterol, and improving intestinal health. Meanwhile, carao is noted for its rich nutritional content and potential dietary applications, although a deeper dive into its specific nutrients and health-promoting properties could enrich our knowledge.

The paper also outlines the interaction between carao and L. plantarum. Results indicate that carao did not compromise the viability or immunomodulatory capacity of L. plantarum. In fact, carao appeared to enhance several attributes of L. plantarum, such as acid and bile tolerance, suggesting potential benefits for fortified dairy products.

Nonetheless, this paper offers valuable insights into the health-promoting properties of L. plantarum and carao, as well as their combined potential for enhancing gut health and overall well-being.

It is a well written paper based on relative bibliography.Material and Methods are well described. Resistance to bile salts,tolerancer to pH, Tolerance to NaCl,Viability of lactobacillus plantarum ,Viability of lactobacillus plantarum under gastrointestinal digestion were considred ,followed by studies on the effect of carao on Experimental infection with Y. enterocolitica O9 IP383 in BALB/c mice .Yet, measurement of cytokines and IgA was performed .Results were evaluated statistically and thoroughly discussed.

The paper provides a compelling exploration of the health benefits associated with Lactobacillus plantarum and carao (Cassia grandis), capturing the interest of readers intrigued by probiotics and natural remedies ,scientists working in the field and industrials.

My suggestion is to ACCEPT and publish it in its present form.

Answer: Thank you for your comments.

Reviewer 3 Report

This manuscript is almost similar with another publication of the same authors, the "Marcia, J.; Aleman, R.S.; Montero-Fernández, I.; Martín-Vertedor, D.; Manrique-Fernández, V.; Moncada, M.; Kayanush, A. Attributes of Lactobacillus acidophilus as Effected by Carao (Cassia grandis) Pulp Powder. Fermentation 2023, 9, 408. https://doi.org/10.3390/fermentation9050408", referred to L. acidophilus.

There is no reference to the exact strain of L.plantarum used

Results and discussion are altogether make the understanding of the new findings [by authors] difficult.

Authors say "The carao's pulp was manually separated from the fruit, dried, and ground" I wonder how sterile is this process? I would like to had the authors culture the grounded carao and present the results. When we use an ingredient as prebiotic it must be a prebiotic only - otherwise the findings are questionable.

The aim of the study, lines 86-88 must be more clear

Line 76-78 "Carao has been shown to have interesting functional properties that make it suitable for use in food products, mainly as a symbiotic in fermented beverages". The word synbiotic must be changed to prebiotic.

In sub-chapters 2.2, 2.3 authors referred to "the strain". What srain??? no name, no characteristics. In 2.4 they referred to "the isolated strains" - that means more than one strain!

Explanations like the 1st paragraph of chapter 3.1 "what are bile acids" or 3.3 about "salt" is a common knowledge - no need for these

Author Response

This manuscript is almost similar with another publication of the same authors, the "Marcia, J.; Aleman, R.S.; Montero-Fernández, I.; Martín-Vertedor, D.; Manrique-Fernández, V.; Moncada, M.; Kayanush, A. Attributes of Lactobacillus acidophilus as Effected by Carao (Cassia grandis) Pulp Powder. Fermentation 2023, 9, 408. https://doi.org/10.3390/fermentation9050408", referred to L. acidophilus.

There is no reference to the exact strain of L.plantarum used

Answer: stain was added.

Results and discussion are altogether make the understanding of the new findings [by authors] difficult.

Authors say "The carao's pulp was manually separated from the fruit, dried, and ground" I wonder how sterile is this process?

I would like to had the authors culture the grounded carao and present the results. When we use an ingredient as prebiotic it must be a prebiotic only - otherwise the findings are questionable.

Answer: the process was sterile because the carao was added to the broths or medium, which were sterilized in an autoclave.

The aim of the study, lines 86-88 must be more clear

Answer: The aim of the study was improved.

Line 76-78 "Carao has been shown to have interesting functional properties that make it suitable for use in food products, mainly as a symbiotic in fermented beverages". The word synbiotic must be changed to prebiotic.

Answer: Sentence was changed.

In sub-chapters 2.2, 2.3 authors referred to "the strain". What srain??? no name, no characteristics. In 2.4 they referred to "the isolated strains" - that means more than one strain!

Answer: Sentences was changed.

stain was added.

Explanations like the 1st paragraph of chapter 3.1 "what are bile acids" or 3.3 about "salt" is a common knowledge - no need for these

Answer: Sentences were changed.

Reviewer 4 Report

In this research article the authors investigate the effect of carao on the viability and functional properties of a Lp. plantarum strain. This manuscript should undergo major revisions to be suggested for publication. The abstract is not suitable for this manuscript since no reference to the in vivo experiment with Y. enterocolotica infection was made. The results are not adequately presented here (please see below for details). Accordingly, the introduction section is mainly focused on lactose intolerance, the antioxidant, and protective effects of carao, topics that do not correspond to the experimental procedures performed in this work. Although the authors did present its immunomodulatory properties, no reference to pathogen-induced immunomodulation was made. The materials and methods section should be reconstructed. More specifically, the authors should revise the presentation of the viability tests that were performed on Lp. plantarum with or without the addition of carao. I would suggest to the authors to present the methods similarly to the presentation of results. For example, in figure 1 the authors include results from sections 2.2 and 2.6 which is confusing. It should be noted however, that the results derived from these two sections are not appropriately clustered in the same graph – since in 2.2 the strain is grown in MRS and in 2.6 in skimmed milk. Another major concern is that no information on the strain used is included in the manuscript. The strain name and origin must be included in the materials and methods section. The manufacturers of the materials used must be included, as well.

 Specific points:

Lines 23-24: the effect on what?

Lines 24-25 contradict lines 26-27, please revise.

Lines 101-104: it is not clear whether results were obtained after incubation for 8, 4, or 24 hours.

Lines 107-109: this experimental design is questionable. I expect that the bacterial population will follow the classic exponential growth curve. Why was this included here?

Line 119: why was TSB used here and not MRS. The composition of LPSM must be included in the text.

Lines 128-129: the antimicrobial activity was tested against which bacteria?

Lines 145-151: please include the approval number designated by the ethics committee for this work.

Lines 155-169: was milk used or yoghurt?

Figures 1-4: I do not understand the use of “*” here. Were only the annotated bars derived from three replications? An explanation about how the authors defined the significance of results should be included in the legend (what different letters indicate and how significance was measured).

Line 247: workhorse?

Line 337: what is the minimal medium used here? LPSM? Also, in the materials and methods it is outlined that TSB was used and not MRS.

Figure 5A: what was the negative control included here? Also, from these curves, it is clearly shown that carao negatively affects bacterial growth compared to positive control.

Figure 6: please change the bars for Ca 5%, they are almost invisible.

Sections 3.6, 3.8 please use the full name of the pathogen when referring to it (Y. enterocolitica instead of yersinia or yersinias).

Line 381: p > 0.05

Figure 6, 10: how were the error bars generated? They all seem to have the same size. Similarly, for the non-infected and infected groups in Figures 11, 12.  

Figure 7: I am curious how the data for non-infected mice and for infected mice receiving water or milk are significantly different.

Figure 11: letters should be added for the infected group too.

Lines 559-561 contradict lines 561-562 – please revise for clarity.

Author Response

In this research article the authors investigate the effect of carao on the viability and functional properties of a Lp. plantarum strain. This manuscript should undergo major revisions to be suggested for publication. The abstract is not suitable for this manuscript since no reference to the in vivo experiment with Y. enterocolotica infection was made. The results are not adequately presented here (please see below for details). Accordingly, the introduction section is mainly focused on lactose intolerance, the antioxidant, and protective effects of carao, topics that do not correspond to the experimental procedures performed in this work. Although the authors did present its immunomodulatory properties, no reference to pathogen-induced immunomodulation was made. The materials and methods section should be reconstructed. More specifically, the authors should revise the presentation of the viability tests that were performed on Lp. plantarum with or without the addition of carao. I would suggest to the authors to present the methods similarly to the presentation of results. For example, in figure 1 the authors include results from sections 2.2 and 2.6 which is confusing. It should be noted however, that the results derived from these two sections are not appropriately clustered in the same graph – since in 2.2 the strain is grown in MRS and in 2.6 in skimmed milk. Another major concern is that no information on the strain used is included in the manuscript. The strain name and origin must be included in the materials and methods section. The manufacturers of the materials used must be included, as well.

Answer: Thank you for the comments. Abstract, objective of study, results wording were improved as for keynotes on figures.

 Specific points:

Lines 23-24: the effect on what?

Answer: Sentences were changed.

Lines 24-25 contradict lines 26-27, please revise.

Answer: Sentences were changed.

Lines 101-104: it is not clear whether results were obtained after incubation for 8, 4, or 24 hours.

Answer: Sentences were changed.

Lines 107-109: this experimental design is questionable. I expect that the bacterial population will follow the classic exponential growth curve. Why was this included here?

Answer: Sentences were changed.

Line 119: why was TSB used here and not MRS. The composition of LPSM must be included in the text.

Answer: It was to mimic gastrointestinal conditions. The composition of LPSM was included.

Lines 128-129: the antimicrobial activity was tested against which bacteria?

Answer: Sentences were changed. the growth of L. plantarum was studied.

Lines 145-151: please include the approval number designated by the ethics committee for this work.

Answer: Approval number designated by the ethics committee for this work was added.

Lines 155-169: was milk used or yoghurt?

Answer: Sentences were changed.  Fermented milk was used.

Figures 1-4: I do not understand the use of “*” here. Were only the annotated bars derived from three replications? An explanation about how the authors defined the significance of results should be included in the legend (what different letters indicate and how significance was measured).

Answer: Figures notes were improved.

Line 247: workhorse?

Answer: Wording was changed.  

Line 337: what is the minimal medium used here? LPSM? Also, in the materials and methods it is outlined that TSB was used and not MRS.

Answer: Wording was changed to correct this confusion.

Figure 5A: what was the negative control included here? Also, from these curves, it is clearly shown that carao negatively affects bacterial growth compared to positive control.

Answer: Wording was added in the material and methods sections to correct this confusion. Sucrose was used as positive control and broth without carao was considered the negative control.

Sections 3.6, 3.8 please use the full name of the pathogen when referring to it (Y. enterocolitica instead of yersinia or yersinias).

Answer: Wording was changed.

Line 381: p > 0.05

Answer: P value was changed.

Figure 7: I am curious how the data for non-infected mice and for infected mice receiving water or milk are significantly different.

Answer: The main objective of the study was to compared treatments (Carao and no carao). The literature explains that fermented milk will have an effect in cytokine production and it would differ from water.

Figure 11: letters should be added for the infected group too.

Answer: If there are no letter it means that no significant difference was detected.

Lines 559-561 contradict lines 561-562 – please revise for clarity.

Answer: Wording was changed.  

Round 2

Reviewer 1 Report

This article has been revised in response to comments and is recommended for acceptance

 Minor editing of English language required

Author Response

This article has been revised in response to comments and is recommended for acceptance

 Minor editing of English language required

Answer: English editing was done.

Reviewer 3 Report

There is no response point-by point. please see below

This manuscript is almost similar with another publication of the same authors, the "Marcia, J.; Aleman, R.S.; Montero-Fernández, I.; Martín-Vertedor, D.; Manrique-Fernández, V.; Moncada, M.; Kayanush, A. Attributes of Lactobacillus acidophilus as Effected by Carao (Cassia grandis) Pulp Powder. Fermentation 2023, 9, 408. https://doi.org/10.3390/fermentation9050408", referred to L. acidophilus.

no response - no comment on it

Results and discussion are altogether make the understanding of the new findings [by authors] difficult.

no changes have been made

Authors say "The carao's pulp was manually separated from the fruit, dried, and ground" I wonder how sterile is this process? I would like to had the authors culture the grounded carao and present the results. When we use an ingredient as prebiotic it must be a prebiotic only - otherwise the findings are questionable.

Answer: the process was sterile because the carao was added to the broths or medium, which were sterilized in an autoclave. 

I want to see written that carao was sterile

The aim of the study, lines 86-88 must be more clear

Answer: The aim of the study was improved.

The paragraph on the purpose of a study begins with "the aim of the study is/was to investigate..." please change it

Author Response

This manuscript is almost similar with another publication of the same authors, the "Marcia, J.; Aleman, R.S.; Montero-Fernández, I.; Martín-Vertedor, D.; Manrique-Fernández, V.; Moncada, M.; Kayanush, A. Attributes of Lactobacillus acidophilus as Effected by Carao (Cassia grandis) Pulp Powder. Fermentation 2023, 9, 408. https://doi.org/10.3390/fermentation9050408", referred to L. acidophilus.

Answer: The manuscripts describe the continuation of carao’s probiotic properties of L. plantarum. Also, this research explores immunomodulatory capacity of L. plantarum as affected by carao.

Results and discussion are altogether make the understanding of the new findings [by authors] difficult.

Answer: Results and discussion were separated.

Authors say "The carao's pulp was manually separated from the fruit, dried, and ground" I wonder how sterile is this process? I would like to had the authors culture the grounded carao and present the results. When we use an ingredient as prebiotic it must be a prebiotic only - otherwise the findings are questionable.

Answer: carao was sterile was written.

The aim of the study, lines 86-88 must be more clear

Answer: The aim of the study was improved.

“the viability under different conditions, immunomodulatory capacity, and probiotic po-tential of L. plantarum as affected by carao were tested and investigated.”

Round 3

Reviewer 3 Report

* Line 480 - parernthesis have to close

* Lactobacillus plantarum is now re-named as Lactiplantibacillus plantarum. I think it is better to change the name throughout the text

I have no other comments