Effect of the Encapsulation Process on the Viability of Probiotics in a Simulated Gastrointestinal Tract Model Medium

- Check that all the names of the bacteria and the Latin words are written in italics, including those in the figures.

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- Correct numbers in chemical formulas (subscript).

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- Check that all the methodology is written in the past, for example in line 83.

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- In materials and methods, the microscopy technique used is not described.

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Line 183-206.

- Section 3.3 belongs to the methodology as well as the scheme in figure 11.

Moved to the Materials and Methods section

Line 112-155.

- There is a lack of description of what is observed in the micrographs, what is seen, what the numbers mean, adding arrows, etc., For example, in figure 7, what is seen in the center? The bar is not visible.

Figures were updated. Descriptions were added.

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The pure sodium alginate capsule was found to be porous as shown in Figure 8. Sodium alginate capsules were obtained hard to the touch, with a strong multilayer shell, and have an irregular shape and heterogeneous structure. This porosity can lead to moisture and oxygen permeation, potentially reducing the shelf life and stability of the encapsulated probiotics. Therefore, gelatin was added to the sodium alginate to fill the porous structure as shown in Figure 9.

- It would look neat, orderly, if the images had a colored margin to separate from the text.

Images are corrected

New images of better quality were added (Figures 8, 9). Figure 10 was corrected (scale line)

- Figure 8 has no description

This figure was deleted

 major revision

Figure 5-6: You should explain what kind of image difference there is between the left and right. Did you try to zoom in on the surface or where? AFM imaging conditions are not included in the method.

Figures were updated. Descriptions were added.

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Sodium alginate capsules were obtained hard to the touch, with a strong multilayer shell, and have an irregular shape and heterogeneous structure. This porosity can lead to moisture and oxygen permeation, potentially reducing the shelf life and stability of the encapsulated probiotics.

 Line 231-234: Although it says "smooth surface", Fig. 7 shows wrinkles when gelatin is added. Doesn't the addition of gelatin cause the capsules to shrink and wrinkle easily? Since it is a common example, you should put a reference etc. Should be a little more specific.

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When encapsulated with aqueous gelatin solution and aqueous sodium alginate solu-tion in the ratio of 1:1, capsules are obtained as homogeneous in structure, spherical in shape and with an average size of 2.7 mm, with a homogeneous surface and high elas-ticity.

Line　238-239：There are many ways to measure diameter in a microscope. It should be more specific (e.g. Martin diameter).

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At encapsulation in the ratio with 0.5:1, 2:1, 3:1, 4:1 aqueous solution of gelatin and aqueous solution of sodium alginate, the capsules are formed soft in consistency, in-homogeneous in structure, with a rough surface, not spherical in shape. Figure 10 shows capsules obtained from different ratios of gelatin and sodium alginate.

Figure　7: Unclear number on scale bar. can not see

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Figure 10

Line　246-249　The reason for "best option" is unclear.　 What does Figure 8 mean in the manuscript? (What is different from Figure 7?) Unclear.　Have you evaluated the mechanical properties of the capsules?

Figure 8 was deleted.

Added new text about the elasticity of capsules:

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Next, the elasticity of capsules is studied. Elasticity is one of the factors that contribute to the overall quality and performance of encapsulated probiotics [41, 42]. The graph shows that the elasticity of capsules obtained at temperatures 20 °C and 30 °C is higher than at temperatures of 40 °C and 50 °C. That is, if we talk about the organoleptic analysis, the capsules obtained at temperatures of 20 °C and 30 °C when added to sour-milk products will be felt as unpleasant solid particles, and capsules obtained at temperatures of 40 °C and 50 °C will be felt as soft, elastic balls and, accordingly, more pleasant in organoleptic sensations.

Line 342-343 10^9 CFU/mL-Unclear   

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Line 234　”2.7×10-3 m” superscript check

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• It is necessary to improve the manuscript by physical and chemical characterization of the capsules. The proposal is FTIR analysis of the capsules (FTIR of sodium alginate without the addition of gelatin and sodium alginate with gelatin addition). Please discuss the obtained results.

Added new text about the elasticity of capsules:

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Next, the elasticity of capsules is studied. Elasticity is one of the factors that contribute to the overall quality and performance of encapsulated probiotics [41, 42]. The graph shows that the elasticity of capsules obtained at temperatures 20 °C and 30 °C is higher than at temperatures of 40 °C and 50 °C. That is, if we talk about the organoleptic analysis, the capsules obtained at temperatures of 20 °C and 30 °C when added to sour-milk products will be felt as unpleasant solid particles, and capsules obtained at temperatures of 40 °C and 50 °C will be felt as soft, elastic balls and, accordingly, more pleasant in organoleptic sensations.

• Figure 8. It is necessary to enter the scale in µm.

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Figure 10

• Figure 10a and b. It is necessary to enter the scale in mm.

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Figure 2

• Line 233, with a homogeneous smooth surface and high density. Please provide the density value if you mentioned it.

Added new text about the elasticity

Line 341-348:

Next, the elasticity of capsules is studied. Elasticity is one of the factors that contribute to the overall quality and performance of encapsulated probiotics [41, 42]. The graph shows that the elasticity of capsules obtained at temperatures 20 °C and 30 °C is higher than at temperatures of 40 °C and 50 °C. That is, if we talk about the organoleptic analysis, the capsules obtained at temperatures of 20 °C and 30 °C when added to sour-milk products will be felt as unpleasant solid particles, and capsules obtained at temperatures of 40 °C and 50 °C will be felt as soft, elastic balls and, accordingly, more pleasant in organoleptic sensations.

The manuscript is not consistent.  Explain why the results are not shown:  Viability of Р. Асidiрrорiоniсi and Р. thоеnii cells depending on the time of cell release from capsules in a simulated gastrointestinal tract model medium? In the first part of the results you presented: Viability of Р. асidiрrорiоniсi, Р. frеudеnrеiсhii, Р. thоеnii cells depending on incubation time in simulated stomach medium! While in the second part of the results you omitted Viability of Р. Асidiрrорiоniсi and Р. thоеnii cells depending on the time of cell release from capsules in a simulated gastrointestinal tract model medium. If you selected existing bacteria, to make the manuscript consistent, all results should be displayed for the selected bacteria.

Explanation was added

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While all three probiotic strains demonstrated resistance to the acidic stomach environment (pH 2.0), P. freudenreichii and P. thoenii exhibited higher cell release rates than P. acidipropionici. P. freudenreichii, in particular, displayed the highest release, making it a potentially more effective probiotic for delivery to the intestinal tract.

All three probiotic strains exhibited the ability to survive the simulated gastrointestinal conditions. This suggests that encapsulation in gelatin-alginate capsules provides a protective barrier, enabling probiotic delivery to the intestines. The data also indicates a sustained release pattern, with continued cell release beyond the initial exposure to acidic conditions. This extended release may contribute to prolonged probiotic activity in the intestines, which is desirable for probiotic supplements.