Microencapsulation of Bifidobacterium breve to Enhance Microbial Cell Viability in Green Soybean Yogurt

Round 1

Reviewer 1 Report

Comments to authors

 

Fermentation-2272100

Title: Microencapsulation of Bifidobacterium breve to Enhance Microbial cell Viability in Green Soybean Yogurt

In this study, the author developed the microcapsules loaded with Bifidobacterium breve for quality enhancement of Green Soybean Yogurt. There appears to be interesting data in this paper and is potentially worth publishing. However, there are some comments and questions as follows:

Major comments

1.     The language of the manuscript needs a drastic revision as some of the information presented is vague just due to language issues. I have mentioned some language errors in the comments and some have been highlighted in the attached file. Besides these, the authors should revise the language of the whole manuscript very carefully.

2.     The comparison between the previous progenies and the results of the work in this research is very vague and should be addressed.

3.     The microencapsulation process in this study is complex. If the author draws or simulates the preparation process, it will help the reader to understand better.

4.     The introduction part needs a few improvements. The authors should tell the readers the need to perform this study. There are a lot of grammatical errors and the prepositions should also be used carefully. Improve the introduction with good connections among paragraphs. The application and properties of alginate and lactate in microencapsulation should be reviewed in the introduction part.

5.     In section 2, the treatment plan is not good. The author should also compare the encapsulation of bacteria in the sodium alginate and calcium lactate separately, then the comparison will be applicable.

6.     Why do the authors have not conducted the release profile test of encapsulated and free bacteria? Please analyze the release profile of encapsulated bacteria. This (https://doi.org/10.1080/02652048.2019.1618403) is a reference for the authors in this regard.

Minor comments

1.     The paragraphs of the entire manuscript should be consistent and not so long. Long paragraphs may confuse the readers and be hard to understand.

2.     Lines 57-58 should be revised; it does not make any sense.

3.     Lines 58-59: rewrite the sentence.

4.     Line 67-69: the phrase is confusing; the author should revise it.

5.     Line 71: use correct prepositions.

6.     Line 76: “and that these properties would influence….” use the correct language, please.

7.     Line 80-81: The sentence seems to be incorrect.

8.     Line 121-122: Correct the sentence, please.

9.     Line 150-152: Please rewrite the phrase, it seems to be incomplete.

10.  Line 166-168: The sentence is not structured, check the grammar.

11.  Line 181: change the word “newly” to freshly.

12.  Line 218-219: The author has used the word “ethanol” twice.

13.  Line 226, 229: 2.3, 2.4 what?

14.  SD error bars should be added to Figures.

15.  Line 278-285: The sentences should be moved to the introduction section.

16.  Conclusions should be shortened reporting the most salient findings.  They should not read like the summary of the study conducted.

 

Comments for author File: Comments.docx

Author Response

Response to Reviewer 1 Comments

 

The authors thank Reviewer for his/her kind and constructive comments to improve the quality and clarity of our manuscript.

 

Major comments

 

Point 1: The language of the manuscript needs a drastic revision as some of the information presented is vague just due to language issues. I have mentioned some language errors in the comments and some have been highlighted in the attached file. Besides these, the authors should revise the language of the whole manuscript very carefully.

 

Response 1: The authors have checked and revised the language of the whole manuscript very carefully. Moreover, we also further check the grammar, spelling, punctuation and phrasing to improve our manuscript through MDPI Language Editing Services. Please find attached a confirmation certificate.

 

Point 2: The comparison between the previous progenies and the results of the work in this research is very vague and should be addressed.

 

Response 2:

Line 305-311: In order for the report of experimental results relate to other previous research, we rearrange the sentence as follow:

“A similar result was reported by Rappai et al. [23], who stated that the microencapsulation efficiency of Pediococcus pentosaceus DM101 increased when the concentration of sodium alginate increased from 0.5 to 2.0% (w/v). The characteristic structure of alginate, which is a linear copolymer of 1,4-linked β-D-mannuronic acid and α-L-guluronic acid in which homopolymeric stretches of guluronic acid residues cooperatively bind calcium ions to form a three-dimensional gel structure, also known as the egg-box model, is related to the formation of calcium alginate gels [24].”

 

Line 342: The additional information for more discussion is given as follow:

“….This result is supported by the findings of Chean et al. [29] who found that the MEE of Lactiplantibacillus plantarum 299v microencapsulation increased with increasing calcium chloride concentrations from 0.5 to 2.0%. (77.16 to 92.88%).”

 

Point 3: The microencapsulation process in this study is complex. If the author draws or simulates the preparation process, it will help the reader to understand better.

 

Response 3: A diagrammatic representation of the emulsion based microencapsulation process has been included in the manuscript as Figure 1.

 

 

Figure 1. Diagrammatic representation of the emulsion-based microencapsulation process.

 

Point 4: The introduction part needs a few improvements. The authors should tell the readers the need to perform this study. There are a lot of grammatical errors and the prepositions should also be used carefully. Improve the introduction with good connections among paragraphs. The application and properties of alginate and lactate in microencapsulation should be reviewed in the introduction part.

 

Response 4:

Line 66: The sentence “During fermentation, proteins are hydrolyzed, the pH decreases, the viscosity increases, and bacterial metabolites are developed, all of which contribute to the flavor and possibly the health-promoting properties of yogurt.” has been removed to reduce the length of  content and replaced with the following sentence: “However, it is very important that probiotic strains retain their viability and function-al activity to confer the above-mentioned benefits on the host. Reports regarding the survival and viability of probiotics indicated that the viability of probiotic bacteria is frequently low in yogurt, producing lower quantities than the daily recommended in-take [6].”

 

Line 75: Further explanation is given in the second paragraph as follow: “…These reasons are promoting the demand for soy-based yogurt product containing probiotics in adequate amounts to confer a health benefit on the host [7].”

Line 98: Further explanation is given in the third paragraph as follow: “Therefore, several Bifidobacterium strains included in fermented food products, lose their viability during storage and in the digestive tract of humans.”

 

The application and properties of alginate in microencapsulation have been stated in the fourth paragraph Lines 121-123. The additional information on calcium lactate is given as follows: “Petraitytė and Šipailienė [14] reported that calcium lactate has no toxic effect on en-capsulated probiotics, showing an improvement in the encapsulation efficiency of L. plantarum.”

 

Lines 124-128: Further explanation of the research objective is given in the last paragraph of the Introduction as the following sentences:

“There are few studies about the viability of bifidobacterial in soy-based yogurt production and there is no published information about the production of green soy-bean yogurt (GSY) containing encapsulated B. breve. The viability of probiotics in GSY could be reduced during storage and gastrointestinal tract conditions could result in the death of B. breve.”

 

Point 5: In section 2, the treatment plan is not good. The author should also compare the encapsulation of bacteria in the sodium alginate and calcium lactate separately, then the comparison will be applicable.

 

Response 5: Each encapsulated material, sodium alginate and calcium lactate has been conducted separately to determine the optimum concentration (1.5, 2.0, and 2.5%). We found that the best concentration of sodium alginate and calcium lactate is 2.0%. However, we need to confirm the previous result in terms of interaction of the concentration levels of both materials.

 

Point 6: Why do the authors have not conducted the release profile test of encapsulated and free bacteria? Please analyze the release profile of encapsulated bacteria.

This (https://doi.org/10.1080/02652048.2019.1618403) is a reference for the authors in this regard.

 

Response 6: The authors wish to thank the reviewer for this recommendation to improve the quality of our manuscript. From the reference journal, the result of the release profile test of microencapsulated Bifidobacterium bifidum in simulated intestinal fluid (SIF) showed that the sodium alginate microspheres were able to release B. bifidum to confer the desired health benefits. Their report also confirms that sodium alginate microcapsule improved constantly viable cells (more than 10⁷ log CFU/g) after 60 min and 120 min which was similar to our present study. However, authors realize that the release profile of microencapsulated B. breve in SIF should be analyzed in terms of probiotics encapsulated in GSY for future studies

 

Minor comments

 

Point 1: The paragraphs of the entire manuscript should be consistent and not so long. Long paragraphs may confuse the readers and be hard to understand.

Response 1:

Introduction

Line 79: The third paragraph is separated into 2 sections.

Results and Discussion

Line 446: The second paragraph of “3.3. Particle Morphology” is separated into 3 sections.

Line 557: The fifth paragraph of “3.4. Quality Changes of Green Soybean Yogurt (GSY) During Refrigerated Storage” is separated into 2 sections.

 

Point 2: Lines 57-59 should be revised; it does not make any sense.

Response 2: The sentence was rephrased to:

“Yogurt containing adequate amounts of viable probiotic microorganisms has increased health benefits for the host.”

 

Point 3: Lines 58-59: rewrite the sentence.

Response 3: This sentence has been removed and replaced with the following sentences;

“Yogurt containing adequate amounts of viable probiotic microorganisms has increased health benefits for the host.”

 

Point 4: Lines 73-77: the phrase is confusing; the author should revise it.

Response 4: The sentence was revised to “Despite this, alternatives such as plant-based yogurt are a desire for people with lac-tose intolerance and allergy to cow's milk protein, as well as vegetarians. These rea-sons are promoting the demand for soy-based yogurt product containing probiotics in adequate amounts to confer a health benefit on the host [7].”

 

Point 5: Line 80: use correct prepositions.

Response 5: The preposition “to” is changed to “for” and the sentence was rephrased to “To be beneficial for the host, probiotic bacteria in foods must survive gastrointestinal transit and reach the small intestine in sufficient quantities.”

 

Point 6: Line 85: “and that these properties would influence….” use the correct language, please.

Response 6: The sentence was corrected to “…and these properties would influence commercial feasibility…

 

Point 7: Lines 90-92: The sentence seems to be incorrect.

Response 7: The sentence was revised to “By preventing the colonization of dangerous bacteria, the intestinal flora is improved, protein digestion is enhanced, and the immune system is stimulated.”

 

Point 8: Lines 143-145: Correct the sentence, please.

Response 8: The sentence was revised to “The B. breve TISTR 2130 culture in a glycerol stock with approximately 6 log CFU/ml of cell viability was stored at -20 °C until use [9].”

 

Point 9: Lines 172-175: Please rewrite the phrase, it seems to be incomplete.

Response 9: The sentence was rephrased to “The seeds (500 g) were further ground and mixed in 1,500 mL of heated distilled water at 87 - 90°C using the medium speed of a blending machine (HR2602, Philips, Ningbo, China) until homogeneous (approximately 5 min).”

 

Point 10: Lines 189-192: The sentence is not structured, check the grammar.

Response 10: The sentence is corrected to “The beads were harvested by filtering them through No. 4 filter paper, transferring them to a sterile Petri dish, and then storing them in a 10°C refrigerator. In the microcapsules, the cell concentration was approximately 8 log CFU/g.”

 

Point 11: Line 208: change the word “newly” to “freshly”.

Response 11: The word “newly” has been replaced by “freshly”.

 

Point 12: Line 244: The author has used the word “ethanol” twice.

Response 12: The second “ethanol” has been removed, and the sentence is corrected to “The samples were dehydrated in ethanol solutions at concentrations of 30%, 50%, 70%, and, finally, 99.8% (v/v) (RCI Labscan, Chiang Mai, Thailand).”

 

Point 13: Line 251, 254: 2.3, 2.4 what?

Response 13: The numbers 2.3 and 2.4 have been removed from sentences.

 

Point 14: SD error bars should be added to Figures.

Response 14: SD error bars in both Figures 3 and 4 have already been added. Due to the data of SD error are very low level, the bar in both figures are too small to see. However, we also add the phase of “n=3, bars indicate the standard deviation.” in the caption

 

Point 15: Line 278-285: The sentences should be moved to the introduction section.

Response 15: The sentence “Therefore, calcium alginate gels have been widely employed in a variety of biotechnology disciplines, including the functional food, pharmaceutical, and medical industries.” is moved to the Introduction Lines 112-114.

 

Point 16: Conclusions should be shortened reporting the most salient findings.  They should not read like the summary of the study conducted.

Response 16: The conclusion is summarized from the overall results and shortened report as follow:

“Microencapsulation of B. breve TISTR 2130 in 2.0% (w/v) sodium alginate and 2.0% (w/v) calcium lactate beads was successfully optimized, with the highest MEE. The optimized encapsulated B. breve TISTR 2130 was efficient in protecting the probiotics from simulated gastrointestinal fluids, which displayed a higher cell viability than free cells. The amount of viable probiotic bacteria in GSY after 10 days of storage was greater than the minimum recommendation of the International Dairy Federation (6 log CFU/mL). These encapsulation results prove this microencapsulation method to be a viable alternative, substantially maintaining the stability of B. breve TISTR 2130 in both passage through the gastrointestinal tract and during refrigerated storage. Moreover, encapsulated B. breve can also be applied to other probiotic bacteria and food products that have the potential to be incorporated into functional foods for balancing the microflora in the digestive tract of humans and conferring health benefits on the host.”

 

 

Kind regards,

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript deals with the encapsulation of bifidobacteria and their application in green soybean yoghurt. For bacteria encapsulation, the authors use the ionic gelation method, and alginate serves as a wall material.

The manuscript is well written. All experimental data are compared to previously published and discussed. However, the encapsulation of probiotics using alginate has been extensively studied, there are a lot of scientific papers on this topic. Therefore, the authors should revise the introduction and finally highlight the novelty of this work.

Author Response

Response to Reviewer 2 Comments

 

The authors thank Reviewer for his/her kind and constructive comments to improve the quality and clarity of our manuscript

Point 1: The manuscript is well written. All experimental data are compared to previously published and discussed. However, the encapsulation of probiotics using alginate has been extensively studied, there are a lot of scientific papers on this topic. Therefore, the authors should revise the introduction and finally highlight the novelty of this work.

Response 1:

The authors thank Reviewer 2 for his/her appreciation of our study. The additional information in the part of the Introduction meant to highlight the novelty of this work are given as follow:

 

Line 66: The sentence “However, it is very important that probiotic strains retain their viability and functional activity to confer the above-mentioned benefits on the host. Reports regarding the survival and viability of probiotics indicated that the viability of probiotic bacteria is frequently low in yogurt, producing lower quantities than the daily recommended intake [6].”

 

Line 73: The sentence is revised to highlight the research objective as follow:

“Despite this, alternatives such as plant-based yogurt are a desire for people with lactose intolerance and allergy to cow's milk protein, as well as vegetarians. These reasons are promoting the demand for soy-based yogurt product containing probiotics in adequate amounts to confer a health benefit on the host [7].

 

Line 124: Further explanation of the research objective is given with the following sentence:

“There are few studies about the viability of bifidobacterial in soy-based yogurt production and there is no published information about the production of green soybean yogurt (GSY) containing encapsulated B. breve. The viability of probiotics in GSY could be reduced during storage and gastrointestinal tract conditions could result in the death of B. breve.”

 

Line 617: The novelties of the study are provided in the “Conclusion” section as follows:

“The amount of viable probiotic bacteria in GSY after 10 days of storage was greater than the minimum recommendation of the International Dairy Federation (6 log CFU/mL). These encapsulation results prove this microencapsulation method to be a viable alternative, substantially maintaining the stability of B. breve TISTR 2130 in both passage through the gastrointestinal tract and during refrigerated storage. Moreover, encapsulated B. breve can also be applied to other probiotic bacteria and food products that have the potential to be incorporated into functional foods for balancing the microflora in the digestive tract of humans and conferring health benefits on the host.”

 

Kind regards,

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The article has been improved as per suggestions, I recommend it for publication.

Reviewer 2 Report

The authors improved the manuscript according to the comments. Thank you.