Development of Antibacterial, Antioxidant, and UV-Barrier Chitosan Film Incorporated with Piper betle Linn Oil as Active Biodegradable Packaging Material

Round 1

Reviewer 1 Report

Author Response

Response to Reviewer 1 Comments

Dear Sir or Madam, thank you for very interesting paper, which should be published after minor revision.

1. However, the methods and results sections need improvement. The presentation of results must be improved.

Response: Thank for the reviewer’s valuable comment. We have made some modifications as yellow highlights in revised manuscript.

2. More literature, clear link to obtained results should be made, summarized and concluded in the conclusion.

Response: Thanks you for your helpful suggestions. We have added more references and careful interpretation in results and discussion section in revised manuscript.

3. Specific comments: Please provide further details on methods for antioxidant and antibacterial measurements.

Response: Thanks you for your helpful suggestions. We have made some modifications on methods for antioxidant and antibacterial measurements in revised manuscript.

4. In addition, the toxicity of the films or chemicals used as contact material should be assessed or mentioned.

Response: Thanks you for your valuable comment. We have added the supplier and purity of all chemicals as yellow highlights in revised manuscript. Herein, for film preparation, used chemicals such as chitosan, glycerol, and Tween 80 are nontoxic and safe for food preservation when listed in Generally Recognized as Safe (GRAS) by US food and Drug Administration in 2013 that food additives is recognized safe by qualified experts.

5. Please provide at least some literature data on the suitability of this material for packaging applications, are we allowed (healthy, toxicitcy, food safety...) to use chitosan as a direct food contact material? please elaborate? Greetings!

Response: Thanks you for your helpful suggestions. As reported, chitosan was considered as nontoxic and safe material for food preservation when listed in Generally Recognized as Safe (GRAS) by US food and Drug Administration in 2013 that food additives is recognized safe by qualified experts. Meanwhile Piper betle Linn is a traditional herb from Piperaceae family and additives such glycerol, and Tween 80 are nontoxicity. We provided information as yellow highlight in introduction section in revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Nguyen and coworkers Developed an antibacterial, antioxidant, and UV–barrier chitosan film incorporated with piper betle Linn oil. Various of techniques are used in this study. However, there is big problem with the data interpretation and experimental design. Please find below some comments/suggestions which might improve the quality of the manuscript.

1. The polydispersity (PDI) of chitosan used in this study should be added.
2. Line 107-108, the information of all the chemicals should be listed (company and purity).
3. The authors should interpret the ATR/FTIR more carefully. It is difficult to say there is interaction between chitosan and PBLO through small change of the absorption band at 1020 cm^-1.
4. Is the sample pCS in ATR/FTIR with glycerol? If it is not (seems so), the comparison in figure 1 is meaningless.
5. In figure 4, the spherical oil droplets maybe mainly be composed of glycerol (30%). Again, pCS sample is without glycerol. 0.4-1.2% of PBLO would not form so high volume percentage of oil droplets in the film. The difference size of such drops in the film is due to preparation method but not the amount of PBLO.
6. Why the chitosan coating on the orange surface begins to peel off but the pCS-0.4PBLO coating is not? It is not clear that why the adhesion for pCS-0.4PBLO on the orange surface is stronger than that of pCS.

Author Response

Response to Reviewer 2 Comments

We would like to express our gratitude for the Editor and Reviewer’s efforts to improve the quality of this manuscript. We have tried our best to address all issues indicated in the review report. In the revised version, we have highlighted the changes to our manuscript using the yellow color. Here, we would like to address the reviewer’s concerns as follows:

Reviewer #2:

Nguyen and coworkers Developed an antibacterial, antioxidant, and UV–barrier chitosan film incorporated with piper betle Linn oil. Various of techniques are used in this study. However, there is big problem with the data interpretation and experimental design. Please find below some comments/suggestions which might improve the quality of the manuscript.

1. The polydispersity (PDI) of chitosan used in this study should be added.

Response: Thanks you for your helpful suggestions. In fact, the polydispersity index should be provided for chitosan nanoparticle preparation instead chitosan film. As reported, two important parameters in chitosan film preparation were the the acetylation degree and molecular weight of chitosan which significantly effect on the physical and biological of chitosan film [Rong Huei Chen et al., Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane, Carbohydrate Polymers, 29 (4), 1996, 353-358]. Previous reports only provided the information about the acetylation degree and molecular weight of chitosan without the polydispersity index in for chitosan film preparation [Hossein Haghighi ets al., Comprehensive characterization of active chitosan-gelatin blend films enriched with different essential oils, Food Hydrocolloids 95,  2019, 33-42; Seyed FakhreddinHosseini et al. Bio-based composite edible films containing Origanum vulgare L. essential oil, Industrial Crops and Products 67, 2015, 403-413; Murat Kaya et al., Antioxidative and antimicrobial edible chitosan films blended with stem, leaf and seed extracts of Pistacia terebinthus for active food packaging, RSC Advances, 8, 2018, 3941-3950; Lijun Sun et al., Preparation and characterization of chitosan film incorporated with thinned young apple polyphenols as an active packaging material, Carbohydrate Polymers 163, 2017, 81–91; Jun Liu et al., Physical, mechanical and antioxidant properties of chitosan films grafted with different hydroxybenzoic acids, Food Hydrocolloids, 71, 2017, 176-186]. Herein, we provided the acetylation degree (75%-85%) and molecular weight of chitosan (50-190 kDa) in the manuscript.

2. Line 107-108, the information of all the chemicals should be listed (company and purity).

Response: Thanks you for your helpful suggestions. We made the addition of the formation of all chemicals. Pls see yellow highlight in material section in page 3 in revised manuscript.

3. The authors should interpret the ATR/FTIR more carefully. It is difficult to say there is interaction between chitosan and PBLO through small change of the absorption band at 1020 cm^-1.

Response: Thank for the valuable comment. We carefully discussed in ATR/FTIR analyses section in revised manuscript: “It was found that appreciable changes in chitosan ATR-FTIR spectrum by the addition of PBLO. For instance, the absorption band at 3624-3012 cm^-1 (O ̶ H and N ̶ H stretching vibration) became weaker and more flatted by the addition of PBLO. The decrease in the peak intensity at 1533 cm^-1 corresponding to C-N stretching vibration and 1402 cm^-1 (NH-CO stretching vibration) by the addition of PBLO along with the significant evolution of shoulder at 1584 cm^-1 due to the aromatic ring vibration of phenolic compounds [32]. Furthermore, the absorption band at 1020 cm^-1 (corresponding to C ̶ O stretching vibration) in the film formulation made from chitosan and PBLO shifted to higher frequency region and became more intense and compared to those in control chitosan spectrum when PBLO abundantly contained C-O groups in structure of phenolic compounds. These changes were likely ascribed to the hydrogen-bonding interaction between O ̶ H and N ̶ H groups in chitosan and hydroxyl of phenolic compound in PBLO [32]. Murat Kaya et al. [34] also found the possible hydrogen interactions arising between film functional groups and Berberis crataegina's seed oil evidenced by the slight shift in the C ̶ O stretching vibration peak to a higher frequency region when seed oil was incorporated into chitosan. “. Pls see yellow highlight in page 6-7 in revised manuscript.

4. Is the sample pCS in ATR/FTIR with glycerol? If it is not (seems so), the comparison in figure 1 is meaningless.

Response: We thank the reviewer for taking the time to consider our work with valuable suggestions offered. In the present work, we used glycerol as plasticizer for all films including chitosan with and without PBLO.

5. In figure 4, the spherical oil droplets maybe mainly be composed of glycerol (30%). Again, pCS sample is without glycerol. 0.4-1.2% of PBLO would not form so high volume percentage of oil droplets in the film. The difference size of such drops in the film is due to preparation method but not the amount of PBLO.

Response: Thank for the reviewer’s valuable comment. In this study, glycerol with 30% (w/w, based on chitosan) was used as plasticizer for all films which were prepared in the similar conditions. In fact, the existence of spherical-like droplets indicated the dispersion of lipid droplets on the surface of resulting films. This observation is coincide with the results reported in previous studies on chitosan-based film with essential oils as the lipid droplets appeared on the film surface or in film matrix [Hossein Haghighi ets al., Comprehensive characterization of active chitosan-gelatin blend films enriched with different essential oils, Food Hydrocolloids 95,  2019, 33-42; Seyed FakhreddinHosseini et al. Bio-based composite edible films containing Origanum vulgare L. essential oil, Industrial Crops and Products 67, 2015, 403-413; Murat Kaya et al., Antioxidative and antimicrobial edible chitosan films blended with stem, leaf and seed extracts of Pistacia terebinthus for active food packaging, RSC Advances, 8, 2018, 3941-3950]. Besides to this, no report mentioned the surface of chitosan-based film with the spherical droplets likely due to composed glycerol. The surface morphology at micro level of plasticized chitosan films with glycerol presented uniform, smooth, homogeneous without the appearance of spherical-like droplets [Antioxidative and antimicrobial edible chitosan films blended with stem, leaf and seed extracts of Pistacia terebinthus for active food packaging, RSC Advances, 8, 2018, 3941-3950; Lijun Sun et al., Preparation and characterization of chitosan film incorporated with thinned young apple polyphenols as an active packaging material, Carbohydrate Polymers 163, 2017, 81–91; Jun Liu et al., Physical, mechanical and antioxidant properties of chitosan films grafted with different hydroxybenzoic acids, Food Hydrocolloids, 71, 2017, 176-186]. The difference in droplet size as 0.4%-1.2% (v/v) incorporated-PBLO was discussed in revised manuscript as follows: “At lower PBLO content (0.4%, v/v), lipid droplets were scarcely observable since they abundantly concentrated on the surface of film with small droplet size. As increasing PBLO concentration to 1% (v/v), droplet size slightly enlarged and became visible. However, the film surface became more heterogeneous when some spherical-like droplets were embedded and trapped in the polymeric network. The slight increase in droplet size may due to the fact that flocculation and coalescence occurred at higher lipid content ⁴¹. Although droplet size of lipids was lower, the surface of chitosan film containing 1.2% (v/v) PBLO become more homogeneous as compared to that in inclusion of 0.4% (v/v) and 1% (v/v) PBLO. The decrease in lipid size likely due to well-embedded oil droplets in polysaccharide matrix resulting in the change in the shape of lipid droplets. This observation was coincide with what was seen gelatin–chitosan containing Origanum vulgare L. essential oil (OEO) as higher droplet size was found in film incorporated with 0.8%(w/v) OEO than that in 0.4 (w/v) and 1.2 (w/v) incorporated-OEO ⁴².”

6. Why the chitosan coating on the orange surface begins to peel off but the pCS-0.4PBLO coating is not? It is not clear that why the adhesion for pCS-0.4PBLO on the orange surface is stronger than that of pCS.

Response: Thank for the reviewer’s valuable comment. The coating efficiency of the coatings solution was evaluated by the two parameters including work of adhesion and spreading coefficient due to contact angle measurement of coating liquids over orange skin with known surface tension of coating solution according to the Wenzel equation [R.S. Hebbar, A.M. Isloor, A.F. Ismail, Chapter 12 Contact Angle Measurements, Membrane Characterization, Elsevier (2017), 220-225; Sitthiphong Soradech, Jurairat Nunthanid, Sontaya Limmatvapirat, Manee Luangtana-anan, Utilization of shellac and gelatin composite film for coating to extend the shelf life of banana, Food Control 73 (2017) 1310-1317] as follows:

                                                       (3)

(Cosθ-1)                                                   (4)

where γ₁ and θ are respectively the surface tension of coting solution and the contact angle of the film coating on the orange skin

Herein, the work of adhesion and spreading coefficient values of plasticized chitosan solution is observed at 0.5 mN/m and -84.72 mN/m, respectively. Meanwhile the increase in these values to 47.78 mN/m and -36.34 mN/m by the addition of PBLO, indicating an increment in coating efficiency. This is supported for the fact that the adhesion for pCS-0.4PBLO on the orange surface is stronger than that of pCS. Pls see the yellow highlight in discussion section of preservation application in revised manuscript.

Table S2. Surface tension, work of adhension, and spreading coefficient of chitosan solution with and without PBLO

Sincerely,

Thuong Thi Nguyen

Author Response File: Author Response.docx

Reviewer 3 Report

This manuscript presents a complex study on design, obtaining and evaluation of chitosan films containing different amounts of piper betle Linn oil. I consider the paper being of interest in view of the properties suitable for application in food packaging, but several modifications and completions should be performed in order to be published. My suggestions are as follows:

Page 3, line 99: I do not understand the meaning of this affirmation “the light of present work was high transparent of fil (…)”

Page 3, line 106: Authors mentioned that “Piper betle Linn oil was collected from Hau Giang province, Vietnam”. I suppose they collected the Piper betle Linn leaves, some information on obtaining the oil used in their study should be also provided.

Page 3, Section 2.3. Preparation of pCS-PBLO films – Thickness of the resulted films should be mentioned.

Table 1: Replace Composition with Area percent, the presented data in this Table referring to compounds (in terms of percentage peak area), as MS is a qualitative analysis, not quantitative. Please modify correspondingly in the Section 3.1. Chemical composition of piper betle Linn oil

Page 5, lines 221-222: This phrase has no meaning “It was found that no pronounced change in band positions by the addition of PBLO.”

Lines 227-228: As PBLO is abbreviation for piper betle Linn oil, authors should remove “oil” from “PBLO oil”

Line 230: Please mention the types of “inter-molecular interaction between functional groups”.

Page 6, Line 242: Replace crystalline with crystallinity in “decrease in crystalline of blend”. Same in line 249.

Page 7, line 289: Delete that from “indicated that the dispersion”.

Lines 290-291. I consider this affirmation out of context as leaves extract and oil have different compositions, therefore is obvious to present different morphologies. Moreover, no information on leaves extract was given in reference 23. "However, the spherical-like droplets were not found in chitosan film incorporated with piper betle Linn leaf extract 23."

Figure 4. Authors should suggest why film containing 1% PBLO present higher spherical dropplets than film with 0.4 and 1.2 PBLO

Page 8, line 328: Should be Table 2 instead Table 1 for mechanical properties.

Lines 327-328: The mentioned values correspond to simple chitosan film (reference), not chitosan-based blend films, please modify accordingly.

Line 329: Brackets should contain the decreasing percent of those mechanical properties, as values are indicated already in Table 2.

Page 9, lines 336-337. As following all testing standards, the samples used for mechanical testing should be conditioned before analysis. Therefore, the affirmation “likely due to the moisture content, which acted as efficient plasticizer in these films” is not correct. As authors did not mentioned the obtaining way of PBLO (necessary to be included in the manuscript), I think this is a vegetal oil, therefore their explanation of PBLO acting as a plasticizer should be concentrated on structure and composition of this vegetal oil, such as flexible long fatty acid chains and unsaturated bonds.

Page 10, Section 3.8. Swelling degree: A discussion on the crystalline/amorphous phases effect on swelling behaviour of studied films should be inserted.

Table 2 - Swelling degree should be also included in Table caption.

Line 376: Delete “there is”.

Page 11

Please explain the affirmation “the addition of PBLO significantly improved the antibacterial activity of chitosan film” (lines 393-394), but also “The formation of chemical interactions between functional groups (-NH2, -OH) of chitosan and phenolic compounds in PBLO may reduce active functional groups which directly interact with bacterial cell and thus decreasing bactericidal activity”. (381-383). There is a contradiction.

Table 3: What does GO abbreviation refer to?

Table 4 is missing in the manuscript.

Author Response

Response to Reviewer 3 Comments

We would like to express our gratitude for the Editor and Reviewer’s efforts to improve the quality of this manuscript. We have tried our best to address all issues indicated in the review report. In the revised version, we have highlighted the changes to our manuscript using the yellow color. Here, we would like to address the reviewer’s concerns as follows:

Reviewer #3:

This manuscript presents a complex study on design, obtaining and evaluation of chitosan films containing different amounts of piper betle Linn oil. I consider the paper being of interest in view of the properties suitable for application in food packaging, but several modifications and completions should be performed in order to be published. My suggestions are as follows:

1. Page 3, line 99: I do not understand the meaning of this affirmation “the light of present work was high transparent of fil (…)”

Response: Thank for the reviewer’s valuable comment. We have made a modification in revised manuscript “Interestingly, the light of present work was high transparent of film made from chitosan and PBLO in visible region as compared to chitosan film containing PBLLE in dark brownish which significantly affected to see-through property important for food packaging material as customer want to see packaged product before buying.”.

2. Page 3, line 106: Authors mentioned that “Piper betle Linn oil was collected from Hau Giang province, Vietnam”. I suppose they collected the Piper betle Linn leaves, some information on obtaining the oil used in their study should be also provided.

Response: Thank for the reviewer’s valuable comment. In this study, Piper betle Linn leaves collected from Hau Giang province were washed to remove impurities before grounding into powder by mechanical grinder. The extraction of PBLO was performed by the hydrodistillation. For extraction process, 100 g of resh leave powders were placed in a 1 L flask containing distilled water in a specified ratio. Following that hydrodistillation took place for 180 min using a Clevenger-type apparatus. The produced vapour was then subjected to condensation in the apparatus. The essential oil is separated from water by decantation, dehydrated with anhydrous magie sulfate and stored at 4 ºC for further analysis.

3. Page 3, Section 2.3. Preparation of pCS-PBLOfilms – Thickness of the resulted films should be mentioned.

Response: Thank for the reviewer’s valuable comment. We have made s modification in Preparation of pCS-PBLO films section in revised manuscript “The thickness of films was controlled by taking equal volume in film fabrication process to ensure ignorable variation and thickness values were recorded at 0.053±0.002; 0.055±0.003; 0.064±0.003; and 0.078±0.004, respectively for pCS, pCS-0.4PBLO, pCS-1PBLO, and pCS-1.2PBLO. Pls see yellow highlight in page 3 in revised manuscript.

4. Table 1: Replace Compositionwith Area percent, the presented data in this Table referring to compounds (in terms of percentage peak area), as MS is a qualitative analysis, not quantitative. Please modify correspondingly in the Section 1. Chemical composition of piper betle Linn oil.

Response: Thank for the reviewer’s valuable comment. We’ve made corrections in revised manuscript. Please see the yellow highlight.

5. Page 5, lines 221-222: This phrase has no meaning“It was found that no pronounced change in band positions by the addition of PBLO.”

Response: Thank for the reviewer’s valuable comment. We have made a modification in revised manuscript “It was found that appreciable changes in chitosan ATR-FTIR spectrum by the addition of PBLO. For instance, the absorption band at 3624-3012 cm^-1 (O ̶ H and N ̶ H stretching vibration) became weaker and more flatted by the addition of PBLO. The decrease in the peak intensity at 1533 cm^-1 corresponding to C-N stretching vibration and 1402 cm^-1 (NH-CO stretching vibration) by the addition of PBLO along with the significant evolution of shoulder at 1584 cm^-1 due to the aromatic ring vibration of phenolic compounds [32]. Furthermore, the absorption band at 1020 cm^-1 (corresponding to C ̶ O stretching vibration) in the film formulation made from chitosan and PBLO shifted to higher frequency region and became more intense and compared to those in control chitosan spectrum when PBLO abundantly contained C-O groups in structure of phenolic compounds. These changes were likely ascribed to the hydrogen-bonding interaction between O ̶ H and N ̶ H groups in chitosan and hydroxyl of phenolic compound in PBLO [32]. Murat Kaya et al. [34] also found the possible hydrogen interactions arising between film functional groups and Berberis crataegina's seed oil evidenced by the slight shift in the C ̶ O stretching vibration peak to a higher frequency region when seed oil was incorporated into chitosan.”. pls see yellow highlight in page 6-7 in revised manuscript.

6. Lines 227-228: As PBLO is abbreviation for piper betle Linn oil, authors should remove “oil” from “PBLO oil”.

Response: Thank for the reviewer’s valuable suggestion. We have deleted all “oil” from PBLO oil.

7. Line 230: Please mention the types of “inter-molecular interaction between functional groups”.

Response: Thank for the reviewer’s valuable comment. We have modified inter-molecular interaction between functional groups in to “hydrogen-bonding interaction between O ̶ H and N ̶ H groups in chitosan and hydroxyl of phenolic compound in PBLO” in revised manuscript.

8. Page 6, Line 242: Replace crystallinewith crystallinity in “decrease in crystalline of blend”. Same in line 249.

Response: Thank for the reviewer’s valuable suggestion. We have made modification in revised manuscript.

9. Page 7, line 289: Delete thatfrom “indicated that the dispersion”.

Response: Thank for the reviewer’s valuable suggestion. We have made modification in revised manuscript.

10. Lines 290-291. I consider this affirmation out of context as leaves extract and oil have different compositions, therefore is obvious to present different morphologies. Moreover, no information on leaves extract was given in reference 23. "However, the spherical-like droplets were not found in chitosan film incorporated with piper betle Linn leaf extract 23."

Response: Thank for the reviewer’s valuable comment. We mean that the spherical-like droplets were not found on the surface of chitosan film incorporated with piper betle Linn leaf extract when we did not surfactant/emulsifier (Tween 80) for the preparation of film with piper betle Linn leaf extract. However, lipid droplets may disperse between chitosan chains and well-embedded in compact polymeric structure, resulting in the increase in plasticizing effect evidenced by the increment in the elongation at break [Thuong, N.T.; Ngoc Bich, H.T.; Thuc, C.N.H.; Quynh, B.T.P.; Minh, L. Van Preparation and Characterization of Piper Betle Linn. Leaf Extract Incorporated Chitosan Films as Potential Active Food Packaging Materials. ChemistrySelect 2019, 4, 8150–8157]. In our another study, we only evaluated the the content of phenolic acids such as hydroxychavicol, and eugenol, and gallic acid in piper betle L. leaves extract and its antibacterial activity [Nguyen, L.T.T.; Nguyen, T.T.; Nguyen, H.N.; Bui, Q.T.P.,  Simultaneous determination of active compounds in  Piper betle  Linn. leaf extract and effect of extracting solvents on bioactivity . Eng. Reports 2020, 2, 2–9] and thus, we cannot provide more information to make clear this sentences.

11. Figure 4. Authors should suggest why film containing 1% PBLO present higher spherical droplets than film with 0.4 and 1.2 PBLO

Response: Thank for the reviewer’s valuable comment. We have made a modification in revised manuscript “At lower PBLO content (0.4%, v/v), lipid droplets were scarcely observable since they abundantly concentrated on the surface of film with small droplet size. As increasing PBLO concentration to 1% (v/v), droplet size slightly enlarged and became visible. However, the film surface became more heterogeneous when some spherical-like droplets were embedded and trapped in the polymeric network. The slight increase in droplet size may due to the fact that flocculation and coalescence occurred at higher lipid content [41]. Although droplet size of lipids was lower, the surface of chitosan film containing 1.2% (v/v) PBLO become more homogeneous as compared to that in inclusion of 0.4% (v/v) and 1% (v/v) PBLO. The decrease in lipid size likely due to well-embedded oil droplets in polysaccharide matrix resulting in the change in the shape of lipid droplets. This observation was coincide with what was seen gelatin–chitosan containing Origanum vulgare L. essential oil (OEO) as higher droplet size was found in film incorporated with 0.8%(w/v) OEO than that in 0.4 (w/v) and 1.2 (w/v) incorporated-OEO [42].”. Pls see yellow highlight in page 9-10 in revised manuscript.

12. Page 8, line 328: Should be Table 2 instead Table 1 for mechanical properties.

Response: Thank for the reviewer’s valuable comment. We have made change in revised manuscript.

13. Lines 327-328: The mentioned values correspond to simple chitosan film (reference), not chitosan-based blend films, please modify accordingly.

Response: Thank for the reviewer’s valuable comment. We have made a modification in revised manuscript.

14. Line 329: Brackets should contain the decreasing percent of those mechanical properties, as values are indicated already in Table 2.

Response: Thank for the reviewer’s valuable comment. We did not understand what you mean. In fact, we placed the brackets when evaluating the mechanical decrease “The incorporated-PBLO caused a considerable decrease in Ts (15.26-5.42 MPa) and Em (111.96-25.95 MPa)”.

15. Page 9, lines 336-337. As following all testing standards, the samples used for mechanical testing should be conditioned before analysis. Therefore, the affirmation “likely due to the moisture content, which acted as efficient plasticizer in these films” is not correct. As authors did not mentioned the obtaining way of PBLO (necessary to be included in the manuscript), I think this is a vegetal oil, therefore their explanation of PBLO acting as a plasticizer should be concentrated on structure and composition of this vegetal oil, such as flexible long fatty acid chains and unsaturated bonds.

Response: We thank the reviewer for taking the time to consider our work with valuable suggestions offered. The samples used for mechanical testing were conditioned before analysis and thus, we agree with you that “the increase in elongation at break is likely due to the moisture content, which acted as efficient plasticizer in these films” is not exact. We have made modification in revised manuscript “Notably, the film made from chitosan and PBLO became more flexible and mobile evidenced by increasing elongation at break (17-25%) compared to the film without PBLO. This behavior was partially due to the presences of lipid droplets in film matrix which hinder polymer chain-chain interaction and result in flexible domains in film [42,46]. Furthermore, phenolic components interacted with chitosan functional groups and weaken the intra molecular forces among polymeric chains resulting in the increment in mobility and flexibility of chitosan film [45]. Another possible explanation due to the presence of emulsifier like Tween 80, importantly contributed to plasticizing effect as synergistic effect between glycerol surfactants with small molecules which could stay between polymeric chains and increase the chain mobility [47].” Pls see yellow highlight in page 11 in revised manuscript.

16. Page 10, Section 8. Swelling degree: A discussion on the crystalline/amorphous phases effect on swelling behaviour of studied films should be inserted.

Response: Thank for the reviewer’s valuable comment. We agree with you that crystalline/amorphous phases effect on swelling behavior and we have made the addition of discussion as follows: “This result is coincided with XRD analyese as the addition of PBLO may convert semi-crystalline in chitosan film toward more amorphous structure”

17. Table 2 - Swelling degree should be also included in Table caption.

Response: Thank for the reviewer’s valuable comment. We have made modification in revised manuscript.

18. Line 376: Delete “there is”.

Response: Thank for the reviewer’s valuable comment. We’ve made corrections in revised manuscript.

19. Page 11: Please explain the affirmation “the addition of PBLO significantly improved the antibacterial activity of chitosan film” (lines 393-394), but also “The formation of chemical interactions between functional groups (-NH2, -OH) of chitosan and phenolic compounds in PBLO may reduce active functional groups which directly interact with bacterial cell and thus decreasing bactericidal activity”. (381-383). There is a contradiction.

Response: Thanks you for your helpful comment. In our previous report, we found that hydrogen bonding between chitosan matrix and flavonoid
constituents may induce a decrease in the interaction between bacterial
cells and phenolic molecules resulting to negligible antibacterial activity [Thuong T. N. et al., Enhanced antimicrobial activities and physiochemical properties of edible film based on chitosan incorporated with Sonneratia caseolaris (L.) Engl. Leaf extract, Progress in Organic Coatings 140 (2020) 105487]. In the present, we thus  think this possible reason for decrease in antibacterial activity of film made from chitosan and PBLO against S. typhi and S. aureus at higher PBLO concentration (1.2%, v/v) as compared to 0.4%, and 1% (v/v) incorporated-PBLO likely due to “The formation of chemical interactions between functional groups (-NH₂, -OH) of chitosan and phenolic compounds in PBLO may reduce active functional groups which directly interact with bacterial cell and thus decreasing bactericidal activity”. However, after your comment, we consider this is unreasonable and hence, we have deleted this sentence in revised manuscript.

20. Table 3: What does GO abbreviation refer to?

Response: Thanks you for your helpful suggestions. GO abbreviation refer to “growth over” and we have added this abbreviation in revised manuscript.

21. Table 4 is missing in the manuscript.

Response: Thanks you for your helpful suggestions. However, this manuscript is without including table 4. Hence, we did not understand what table 4 you mentioned.

Sincerely,

Thuong Thi Nguyen

Round 2

Reviewer 2 Report

As the authors pointed out in the revision, they used glycerol as plasticizer for all films including chitosan with and without PBLO. Please include such information in the ms. 

After that, I will recommend it for publication in Coatings.

Author Response

Response to Reviewer 2 Comments (Round 2)

As the authors pointed out in the revision, they used glycerol as plasticizer for all films including chitosan with and without PBLO. Please include such information in the ms.  After that, I will recommend it for publication in Coatings.

Response: Thank you for your helpful suggestions. We have added the information about the utilization of glycerol as plasticizer for all films including chitosan with and without PBLO in revised manuscript, line 319-321 as follows: “Herein, glycerol was additionally used as plasticizer for all the films and the successful film formation for all applied formulations was obtained as expected”.

Author Response File: Author Response.docx