Encapsulation of Lactobacillus casei (ATCC 393) by Pickering-Stabilized Antibubbles as a New Method to Protect Bacteria against Low pH
, Gert-Jan W. Euverink 3,* and Albert T. Poortinga 4,*Round 1
Reviewer 1 Report
I think this manuscript could be published after the following issues are addressed.
- The presence of the bacteria inside the water droplets in the multiple emulsions should be confirmed with higher resolution microscopy imaging. It is claimed at line 193 that in Figure 2 "in all panels bacteria can be observed in the water droplets". It is possible to just identify what may be spherical shapes inside the water droplets shown in panel a. But aren't Lactobacillus casei rod-shaped bacteria that are 2 to 4 micrometres long? Higher resolution microscope images of the emulsion droplets are required to support this claim.
2. The presence of water inside the inner pores of the antibubbles needs to be confirmed and explained. The water-in-oil-in-water emulsions were freeze dried to remove both the water and oil phases to give a powder containing capsules with small pores insides them in which the bacteria was encapsulated in maltodextrin. The powder was then dispersed in water. How does the water penetrate into the inner pores to re-hydrate them? It is claimed at line 206 that "There is no aqueous connection between the inner droplets and the outer phase". How do we know that the inner pores contain water and how does water reach the inner pores?
Author Response
- The presence of the bacteria inside the water droplets in the multiple emulsions should be confirmed with higher resolution microscopy imaging. It is claimed at line 193 that in Figure 2 "in all panels bacteria can be observed in the water droplets". It is possible to just identify what may be spherical shapes inside the water droplets shown in panel a. But aren't Lactobacillus casei rod-shaped bacteria that are 2 to 4 micrometres long? Higher resolution microscope images of the emulsion droplets are required to support this claim.
We have produced new antibubbles and used another microscope to image the bacteria after all the steps of the production process. We replaced the old images with these new images and added an image at higher magnification. We think that the bacteria are now much clearer to see.
- The presence of water inside the inner pores of the antibubbles needs to be confirmed and explained. The water-in-oil-in-water emulsions were freeze dried to remove both the water and oil phases to give a powder containing capsules with small pores insides them in which the bacteria was encapsulated in maltodextrin. The powder was then dispersed in water. How does the water penetrate into the inner pores to re-hydrate them? It is claimed at line 206 that "There is no aqueous connection between the inner droplets and the outer phase". How do we know that the inner pores contain water and how does water reach the inner pores?
These are very relevant questions. In part these questions were addressed in our earlier work on antibubbles (references 9 and 11). We also added the following text at the end of section 3.1 to explain: ‘Inside the inner droplets Brownian motion of the bacteria could be observed. This leads us to believe that the inner droplets are in fact droplets, i.e. they are liquid. We expect that during reconstitution of the antibubble powder the dry cores inside the antibubble quickly rehydrate since water vapor can pass through the gas shell and that this makes the cores liquid again.’
Reviewer 2 Report
The paper focused the attention on the use of pickering-stabilized antibubbles as encapsulation system for L.casei, probiotic microrganism.
Starting from a W/O/W emulsion stabilized by silica particles, the water phase was enriched with solute (maltodextrin and sucrose) that became glassy upon drying and in which the oil phase as well as the water are removed by freeze-drying.
The formation of antibubbles is obtained after the reconstitution in water of the freeze-dried material.
They investigate the use of different oil phases, indicating that only decane and cyclohexane were able to form antibubbles.
They also investigate the survival of probiotics under different conditions showing good results for encapsulated bacteria after incubation at low pH for 1 hour.
General comments:
The article is in general well written. There are few typographic errors, which should be corrected during the revision.
Line 55: the sentence is not really clear, reformulate it
Line 56: extra space
Line 115 there is an extra parenthesis at the end of the sentence.
Figure 1 caption: I suggest to use letters (A-B-C instead of left, middle, right)
Also the last sentence refers to arrows that are not visible
Line 154 : center the equation
Figure 3 caption: extra space and typographic error
There are however some issues listed below:
Page 6
- You show in table 1 that 4 systems were obtained, but then only images of DMS system are reported. Did you observe also the other systems? If yes, there were any differences in the size or appearance?
Page 7
- Why the entrapment efficiency is reported only for DMS system?
- 3.3: I think that you should make more clear that due to the previous results the investigation of the survival at low pH was made only for two systems (DM and DMS) even if in the graph (Fig 4) is not really clear which system is considered as encapsulation.
- Line 267: you mentioned that analysis at microscopy after the addition of bile to the system did not show severe damage, but you did not show any figure (is it related to figure 2? – if not I suggest to add ‘data not showed’).
- Why did you evaluate only the survival at low pH when you already treated the systems with bile extract?
In my opinion, the article needs to be improved before being published.
Author Response
Line 55: the sentence is not really clear, reformulate it
We changed the sentence ‘Therefore, antibubbles have the potential for encapsulation and controlled-release purposes and antibubbles are seen to gain interest’ into ‘Antibubbles thus have the potential to be used as encapsulates with good barrier properties and the possibility of a fast and complete triggered release. This would make antibubbles an ideal type of encapsulate and hence antibubbles are gaining interest’.
Line 56: extra space
This has been changed.
Line 115 there is an extra parenthesis at the end of the sentence.
This has been removed.
Figure 1 caption: I suggest to use letters (A-B-C instead of left, middle, right)
Also the last sentence refers to arrows that are not visible.
This was changed.
Line 154 : center the equation
This has been done.
Figure 3 caption: extra space and typographic error
This has been changed.
There are however some issues listed below:
Page 6
- You show in table 1 that 4 systems were obtained, but then only images of DMS system are reported. Did you observe also the other systems? If yes, there were any differences in the size or appearance?
Other systems were also observed under the microscope but no differences in the appearance of the emulsions and antibubbles were detected. This is probably because the presence or absence of sugar has a small effect on the viscosity of the aqueous phase and hence on the emulsification process. We have added a note on this to the text (around line 193).
Page 7
- Why the entrapment efficiency is reported only for DMS system?
This was done for several reasons. First, because the DMS system gave the best survival of bacteria after encapsulation and therefore we think this is practically the most interesting system. Second, since use of maltodextrin alone did not lead to a change in the encapsulate structure we expect the entrapment efficiency to be similar for this formulation. Also, we were mainly interested to get a rough indication of the encapsulation efficiency just to check if the bacteria were predominantly encapsulated and therefore we were satisfied with knowing the entrapment efficiency for one formulation.
- 3: I think that you should make more clear that due to the previous results the investigation of the survival at low pH was made only for two systems (DM and DMS) even if in the graph (Fig 4) is not really clear which system is considered as encapsulation.
We have added a line (around line 267) mentioning that formulations CM and CMS were not included in the survival at low pH study because of their low number of live bacteria.
We have added the line ‘Note that the encapsulated formulations indicated with ‘Maltodextrin’ and ‘Maltodextrin + Sucrose’ are referred to as DM and DMS, respectively’ to the caption of Fig. 4 to clarify the point made by the reviewer about Fig. 4.
- Line 267: you mentioned that analysis at microscopy after the addition of bile to the system did not show severe damage, but you did not show any figure (is it related to figure 2? – if not I suggest to add ‘data not showed’).
In line 267 we state that the incubation at low pH did not do visible damage to the structure of the antibubbles. This data is indeed not shown and hence we have added ‘data not shown’
Line 304 and following describe that adding bile salt does influence the structure of the antibubbles since it leads to escape of the inner droplets (that contain the bacteria). This is shown in Fig 2e.
- Why did you evaluate only the survival at low pH when you already treated the systems with bile extract?
Since the presence of bile releases the bacteria from the antibubbles, we expect no protective effect of the antibubbles anymore in the presence of bile. Since our study aims at studying the properties of antibubbles for the encapsulation of bacteria we were not interested in the survival after release of the bacteria from the antibubbles.
Round 2
Reviewer 1 Report
The concerns I had with the original manuscript have been dealt with satisfactorily. I am happy to recommend publication.
Author Response
Test
Reviewer 2 Report
The authors have answered to my questions in an adequate way and some issues were clarified.
However, I include a few suggestions for the next version.
Figure 1. You said that you adapt the caption of the figure but it was not done.
Please change it and add the arrows that indicate the processing step that converts the structures, otherwise remove the last sentence.
Figure 3. Also here check the caption of the figure and the label of the last bar (sucrose is not spelled correctly)
Also you mention that you have added the line:
‘Note that the encapsulated formulations indicated with ‘Maltodextrin’ and ‘Maltodextrin + Sucrose’ are referred to as DM and DMS, respectively’ to the caption of Fig. 4 but it was not done.
Please add it in the new version.
Typographic errors:
Revise the ‘introduction’, the text has a different style.
Line 84: there is an extra dot at the end of the sentence
Line 94: missing dot at the end of the sentence
Check also the style of the bibliography.
Author Response
Thank you for your comments. We discovered that the manuscript contained some of the old figures and captions. This may be due to us being unfamiliar yet with the system of MDPI. We will email the revised manuscript to the editor and request that it will be send to you.
We also took a close look at the references and made several changes such as adding dots and changing the style of dashes.
With respect to the other typographic errors (in lines 84 and 94 and the difference in style of the Introduction: we also see these in the submitted version, but these errors are not present in the document that we upload. These errors seem to be the result of some conversion by the software.
