Survival of Lactobacillus paracasei subsp. paracasei LBC 81 in Fermented Beverage from Chickpeas and Coconut in a Static In Vitro Digestion Model

Round 1

Reviewer 1 Report

There are lots of manuscript discussed the digestion condition for probiotic in vitro system, in this article can not found any difference. 

1.Why the author choice chickpeas and coconut beverage? and why the ratio was 10% coconut extract and 90% chickpea extract?

2.In line 90-91, which part of coconut was choice to this study? coconut milk?

3.In method 2.3., why the author design this "cell suspension in chickpea and coconut beverage?

4.In fig.1A, could the author explain FCCb is higher than SCSS? and there no difference among SCSS, FCCB and CSCCB.

5. In line 197-199, "all treatments were exposed only to pancreatic juice, it was not possible to ~~~", It didn't give any explain about it.

6.The change of carbohydrate during fermentation? and other composition should be mentioned.

Author Response

Reviewer 1 - Comments and Suggestions for Authors

 

Many manuscripts discussed the digestion condition for probiotic in vitro systems, in this article can not found any difference.

 

1.Why the author choice chickpeas and coconut beverage? and why the ratio was 10% coconut extract and 90% chickpea extract?

Response: Chickpea and coconut and their respective proportions 90% and 10% were used based on the studies by Rincon et al.(2020) and Mesquita et al.(2020). Recently Rincon et al. (2020) proposed the chickpea and coconut beverage as a new alternative vegetable beverage for cow's milk. They used chickpea because it contains a high protein content and it has low allergenicity. They used coconut because it increases the lipid content and gives aroma and flavor to the beverage, increasing its acceptability. In the study by Rincon et al (2020), different proportions of chickpea extract and coconut for the beverage composition were investigated. However, Mesquita et al.(2020) observed in the beverage with 90% chickpea extract and 10% coconut extract remarkable growth of L. paracasei LBC 81, with viable cells above 8 log CFU / mL. Thus, the authors chose to carry out the study with the 90% chickpea extract and 10% coconut extract beverage.

2.In line 90-91, which part of coconut was choice to this study? coconut milk?

Response: The used coconut parts were the husk (hard inner layer), and meat (solid endosperm) processed together with drinking water in a blender for 3 minutes. In item 2.1 coconut extract, for clarity, it was inserted “coconut (shell - inner hard coat, and meat – solid endosperm)”.

3.In method 2.3., why the author design this "cell suspension in chickpea and coconut beverage?

Response: Cell suspension in chickpea and coconut beverage was used as control 2, and there was no fermentation process. Therefore, the suspension of cells in chickpea and coconut beverage (control 2) was used to evaluate the effect of the beverage fermentation on the viability of L. paracasei LBC 81.

4.In fig.1A, could the author explain FCCb is higher than SCSS? and there no difference among SCSS, FCCB and CSCCB.

Response: In Figure 1A, the greater survival of L. paracasei LBC 81 in FCCB may be related to the lower pH value found in the beverage (4.10+0.03). It is possible that during the fermentation process of the coconut bean beverage, the strain has adapted to low pH conditions, which may have induced the activation of resistance mechanisms against the exposure to simulated gastric juice. Furthermore, it is observed that food was a protective factor for L. paracasei LBC 81 against the exposure to simulated gastric juice, given the lower survival of the strain found in SCSS. In item 3.1, the current explanation was inserted, after citing the pH values of FCCB, SCSS, and CSCCB.

5. In line 197-199, "all treatments were exposed only to pancreatic juice, it was not possible to ~~~", It didn't give any explain about it.

Response: When all treatments were exposed only to pancreatic juice, it was not possible to observe the protective effect of the food on the viability of L. paracasei LBC 81, as there was no significant difference (p > 0.05) between cell suspensions. Also, lower survival percentage was observed in the fermented beverage. When all treatments were exposed to gastric juice, it was possible to observe significant differences (p < 0.05), with higher percentages of strain survival in the fermented beverage and in the cell suspension. This confirms that the protective action of the chickpea and coconut beverage against L. paracasei LBC 81 is associated with previous exposure in an acidic medium. The current explanation was inserted after the mentioned passage.

6. The change of carbohydrate during fermentation? and other composition should be mentioned.

Response: In our study, we did not evaluate the use of carbohydrates from the chickpea and coconut beverage as a source of growth for L. paracasei LBC 81. However Mesquita et al. (2020) mentioned that oligosaccharides and polysaccharides present in chickpeas can contribute to the growth of L. paracasei LBC 81. As 90% of the beverage is made up of chickpea extract, and according to Faridy et al, chickpeas are mainly composed of carbohydrates, we assume that these may have played an important role as a physical barrier against the exposure to gastric juice. We also highlighted in our study that according to Jukante et al and Cortés-Giraldo et al., chickpea has free amino acids, including arginine, with the possibility that L. paracasei LBC 81 has transported arginine to its cytoplasm, neutralizing the acidic environment, providing greater resistance to exposure to gastric juice. We did not find any other chemical components of chickpea and coconut in the literature, which could be related to resistance under simulated gastrointestinal conditions.

Thank you for your comments!

Author Response File: Author Response.pdf

Reviewer 2 Report

The topic of the article falls within the thematic scope of the journal.

The objective of this study was to evaluate in a static in vitro digestion model the survival of Lactobacillus paracasei subsp. paracasei LBC 81 in fermented chickpea and coconut beverage. The Authors also determined whether refrigeration of the fermented beverage had an effect on the survival of L. paracasei under conditions simulating gastric / intestinal transit.

The topic is interesting. I have no objections to the section 1. Introduction, but the authors should mention that one of the aims was also to determine the impact of storage :)

I propose that the Authors complete section 2. Materials and Methods with a drawing that would show what the consecutive stages of the experiment consisted of. This would help the reader a lot.

I also propose to enter the results of the pH determination after 1 and 8 days of storage of beverages to Table 1.

All remarks are marked in the text of manuscript in the review mode.

After introducing these minor changes, in my opinion, the Editors may direct the manuscript for further processing in Fermentation.

Comments for author File: Comments.pdf

Author Response

The topic of the article falls within the thematic scope of the journal.

The objective of this study was to evaluate in a static in vitro digestion model the survival of Lactobacillus paracasei subsp. paracasei LBC 81 in fermented chickpea and coconut beverage. The Authors also determined whether refrigeration of the fermented beverage had an effect on the survival of L. paracasei under conditions simulating gastric / intestinal transit.

1. The topic is interesting. I have no objections to the section 1. Introduction, but the authors should mention that one of the aims was also to determine the impact of storage :)

Response: The authors agree to add as one of the study's objectives the impact of refrigerated storage of the chickpea and coconut fermented beverage on the survival of L. paracasei LBC 81, considering that it was evaluated. In the introduction, the objective was changed to “Given the above, the objective of this study was to evaluate in a static model of in vitro digestion the survival of Lactobacillus paracasei subsp paracasei LBC 81 in chickpea and coconut fermented beverage, as well as the impact of refrigerated storage of the fermented beverage in the survival of the strain.

2. I propose that the Authors complete section 2. Materials and Methods with a drawing that would show what the consecutive stages of the experiment consisted of. This would help the reader a lot.

            Response: A figure with the steps to carry out the experiments was created and added in section 2. Materials and Methods.

3. I also propose to enter the results of the pH determination after 1 and 8 days of storage of beverages to Table 1.

           Response: The pH values of the FCCB and CCCCB treatments on the 1 and 8 days of refrigerated storage were entered in Table 1.

4. All remarks are marked in the text of manuscript in the review mode.

           Response: The observations were verified, and corrections were made.

5. After introducing these minor changes, in my opinion, the Editors may direct the manuscript for further processing in Fermentation.

Thank you for your comments!

Author Response File: Author Response.pdf

Reviewer 3 Report

English need extensive revision.

Abstract. Please revise. For example in Lines 15-16 the method for determination of survival of cells is well-known.

Line 16. “The survival of L. paracasei in the fermented beverage was high” too general. Please provide details. High compare to what?

Lines 97-102. After the growth in MRS there is no washing. Is this appropriate? Usually washing takes place. Please explain.

Lines 151-155. The equation is probably wrong. There is two times log. For example logN1 but N1=logcfu/mL and therefore log(logcfu/mL). Please revise.

Figure 1 is very misunderstanding. In addition the use of % survival using logcfu is also not appropriate. However, at least for the need of reviewer please provide in the revision the actual logcfu/mL and not the % percentage.

Please also provide the initial logcfu/mL in each case.

Please also provide the logcfu/mL after the fermentation in the beverage.

All these values are necessary in order to complete the review.

I recommend to the authors to use or all of the following recent works in their introduction/discussion

1. https://doi.org/10.3390/fermentation7020090
2. https://doi.org/10.3390/fermentation6040113
3. https://doi.org/10.3390/app11073116
4. https://doi.org/10.1016/j.cofs.2015.11.012
5. https://doi.org/10.3390/fermentation6010030

Author Response

Reviewer 3 - Comments and Suggestions for Authors

1. English need extensive revision.

 Response: English has been revised.

2. Please revise. For example in Lines 15-16 the method for determination of survival of cells is well-known.

Response: The abstract was revised, and the respective excerpt was removed. The authors agree that the method used to determine survival is known in the literature; therefore, it is not necessary to describe it in the abstract. For greater clarity, the excerpt "The survival of L. paracasei was determined in log CFU / mL and expressed as a percentage of survival" was added to the abstract.

3. Line 16. “The survival of L. paracasei in the fermented beverage was high” too general. Please provide details. High compare to what?

Response: The respective excerpt of the abstract was modified by adding more details to the sentence and emphasizing that the highest survival percentages of L. paracasei obtained in the fermented beverage were compared to controls 1 and 2 of the study. The sentence is thus "The survival of L. paracasei in the fermented beverage, after exposure to gastric juice and sequential exposure to gastric and pancreatic juices, was 99.47+2.05% and 93.21+0.43 %, these values were higher than those found for controls 1 and 2”.

4. Lines 97-102. After the growth in MRS there is no washing. Is this appropriate? Usually washing takes place. Please explain.

Response: The preparation of the fermented beverage followed the protocol described by Mesquita et al. (2020) which after cultivation of the microorganism in MRS broth, the culture in its active form is added to the chickpea and coconut beverage, which is then incubated in a bacteriological incubator at 37ºC for 6 hours. For greater clarity, the work by Mesquita et al. (2020) was cited in section 2 on chickpea and coconut fermented beverage production.

5. Lines 151-155. The equation is probably wrong. There is two times log. For example logN1 but N1=logcfu/mL and therefore log(logcfu/mL). Please revise.

Response: The equation has been revised and reformulated, the word log before N has been removed, the equation remains that way Survival (%) = N1/N0 x 100%.

6. Figure 1 is very misunderstanding. In addition, the use of % survival using logcfu is also not appropriate. However, at least for the need of reviewer please provide in the revision the actual logcfu/mL and not the % percentage.

Response: In studies where the microorganism is exposed to some stressful situation, the survival percentage is presented. However, let's forward the requested log results.

Table 1 - Survival (log CFU/mL) of L. paracasei LBC 81 in the suspension of cells in saline solution (SCSS), fermented chickpea and coconut beverage (FCCB), and cell suspension in chickpea and coconut beverage (CSCCB), after exposure to simulated gastric juice, simulated pancreatic juice and sequential exposure to gastric and pancreatic juices.

 

 

 

 

 

	Treatment
Log CFU/mL		SCSS	FCCB		CSCCB
Gastric juice
N0*		9.05+0.06	9.31+0.13		9.95+0.03
N1**		4.41+0.011	9.26+0.17		9.27+0.22
Pancreatic juice
N0*		8.67+0.08	9.32+0.06		9.15+0.08
N1**		6.49+0.07	6.36+0.23		8.29+0.06
Sequential exposure to gastric and pancreatic juices
N0*		8.89+0.11	9.38+0.07		9.81+0.10
N1**		3.53+0.21	8.74+0.07		7.13+0.07

 

                         

 

gastrointesti

                      N_0*: (log cfu/mL) represents the viability before exposure to gastrointestinal stress conditions in vivo. N_1**: (log cfu/mL) represents the viability after exposure to gastrointestinal stress conditions in vitro.

 

Table 2 - Survival (log CFU/mL) of L. paracasei LBC 81 during refrigerated storage at 4 ºC of FCCB and CSCCB, after simulated gastrointestinal conditions.

 

	Treatment
Time (day)	FCCB* (N0)	FCCB* (N1)	CSCCB** (N0)	CSCCB** (N1)
Gastric juice
1	9.31+0.13	9.26+0.17	9.95+0.03	9.27+0.22
8	9.50+0.04	9.51+0.11	9.03+0.03	9.95+0.05
Pancreatic juice
1	9.32+0.06	6.36+0.23	9.15+0.08	8.29+0.06
8	9.55+0.08	6.89+0.70	9.21+0.05	6.30+0.36
Sequential exposure to gastric and pancreatic juices
1	9.38+0.07	8.74+0.07	7.81+0.10	5.13+0.07
8	9.56+0.04	9.20+0.30	8.06+0.09	7.00+0.02

 

7. Please also provide the initial logcfu/mL in each case.

    Response: They are described in table 1

8. Please also provide the logcfu/mL after the fermentation in the beverage.

Response: They are described in table 1

9. All these values are necessary in order to complete the review.

Response: The requested amounts were forwarded.

10. I recommend to the authors to use or all of the following recent works in their introduction/discussion

    Response: The works were cited in the introduction …/

1. https://doi.org/10.3390/fermentation7020090          --> 27
2. https://doi.org/10.3390/fermentation6040113          -->20
3. https://doi.org/10.3390/app11073116                      -->26
4. https://doi.org/10.1016/j.cofs.2015.11.012              --> 19
5. https://doi.org/10.3390/fermentation6010030          -->21

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript had corrected according to my comments.

Reviewer 3 Report

The authors replied in all my comments.

I think that the manuscript has been improved following all reviewers comments.