Review Reports - Probiotic Properties of Lactobacillus fermentum InaCC B1295 Encapsulated by Cellulose Microfiber from Oil Palm Empty Fruit Bunches

Round 1

Reviewer 1 Report

The manuscript entitled Probiotic properties of Lactobacillus fermentum InaCC B1295 encapsulated by cellulose microfiber from oil palm empty fruit bunches is interesting, informative and novel. The authors extracted the CMF and prepared a hydrogel and microencapsulated the probiotic Lactobacillus fermentum. The microencapsulated probiotic was characterized for various parameters including viability, acid treatment. The probiotic has good anti-oxidant activity also.However the manuscript contains minor errors which has to be corrected

Line 72-73 check the facts is it 1kg dry biomass from 4kg of empty fruit bunches

Line 99-L.fermentum is the bacteria

Line 138 which hour culture of L.fermentum inoculated in microencapsulation?

Line 565 these experiments demonstrated that

Author Response

Dear respected Reviewer 1

We send a response to reviewer 1 and hope that our response is in accordance with your suggestions. All suggestions have been accommodated and written in our paper.

With best regards

Usman Pato, PhD
Professor in Food Science
Faculty of Agriculture
Riau University
Pekanbaru, Indonesia

Response to Reviewer 1 Comments
The manuscript entitled Probiotic properties of Lactobacillus fermentum InaCC B1295 encapsulated by cellulose microfiber from oil palm empty fruit bunches is interesting, informative and novel. The authors extracted the CMF and prepared a hydrogel and microencapsulated the probiotic Lactobacillus fermentum. The microencapsulated probiotic was characterized for various parameters including viability, acid treatment. The probiotic has good anti-oxidant activity also.However the manuscript contains minor errors which has to be corrected

Line 72-73 check the facts is it 1kg dry biomass from 4kg of empty fruit bunches
Response: This statement has been corrected as suggested.

Line 99-L.fermentum is the bacteria
Response: The statement has been corrected according to the reviewer's suggestion

Line 138 which hour culture of L.fermentum inoculated in microencapsulation?
Response: The time of inoculation in the microencapsulation has been added to the paper as suggested

Line 565 these experiments demonstrated that
Response: The statement has been changed according to the reviewer's suggestion

Author Response File: Author Response.pdf

Reviewer 2 Report

1. The author used oil palm empty fruit bunch as a new material for the application to probiotic encapsulation. It's a good idea since palm oil is an important industry in the author's country (e.g. Indonesia). Additionally, the empty fruit bunch is a kind of byproduct, so it matches some issues of the recycling economic.

Author Response

Dear respected Reviewer 2

We send a response to reviewer 2 and hope that our response is in accordance with your suggestions. All suggestions have been accommodated and written in our paper.

With best regards

Usman Pato, PhD
Professor in Food Science
Faculty of Agriculture
Riau University
Pekanbaru, Indonesia

Response to Reviewer Comments

1. The author used oil palm empty fruit bunch as a new material for the application to probiotic encapsulation. It's a good idea since palm oil is an important industry in the author's country (e.g. Indonesia). Additionally, the empty fruit bunch is a kind of byproduct, so it matches some issues of the recycling economic.
Response: We concur with the reviewer's assessment, and as Indonesia has the largest palm oil production area in the world, solid waste from palm oil production is also quite prevalent in Indonesia.

2. The experiment design was not adequate. (1) What did the 28 day storage stand for? For the storage of probiotic product at 4 or 25 degree celsius, it should be couple months, even years for the stability. (2) Based on the current experiment design, we could just know the characteristic of the encapsulated LFB1295, however, the exactly effect of CMF on the LFB1295 against experiment stress was not investigated.
Response: (1) The phrase "the 28 day storage stand for" refers to the 28 days that LFB1295 cells that have been enclosed in CMF hydrogel made from oil palm fronds (CMFH-OPF) were kept both at room temperature and in the refrigerator. The purpose of the study is to produce functional foods that can be stored at room temperature or cold temperatures depending on the storage temperature of the processed food products with viability above 107 CFU/g and stable probiotic properties for a maximum storage of 28 days using LFB1295, which is encapsulated with CMFH-OPF. (2) The precise impact of CMF on the LFB1295 during experimental stress was not yet examined in this study. We solely looked into how CMF as an encapsulant affected viability, bile and acid tolerance, probiotic safety, and antioxidant activity of LFB1295. The reviewer's recommendations serve as important feedback for our future research.

3. Minor comments: (1) Please add the properties of CMF-OPEFB in the abstract. (2) line 29 and Table 8, please check the unit, is the g/mL for DPPH scavenge correct? (3) line 140, using distilled water to wash bacteria cells may cause damage because of the osmosis pressure, (4) Figure 4, it seems like incomplete?
Response: (1) Properties of CMF-OPEFB in the abstract has been added in the abstract as per suggestion. (2) The unit unit for DPPH scavenge should be μg/mL. (3) The washing of lactic acid bacteria (LAB) cells with distilled water is based on a number of earlier investigations. As long as the washing and centrifugation procedures were completed rapidly to prevent LAB cell injury, we believe that the use of distillate water had no appreciable impact on the decline in LAB viability. (4)Since the original document only contained 3 figures, we indicate that there is not a Figure 4 in this paper. Figufre 2 and 3 are consolidated into one figure as a result of comments from other reviewers, leaving just two figures in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

This work is good, however, the main default is the missing of control with free cells. The authors Just studied the properties of encapsulated cells without comparison of the original, or what did this immobilization process improved or achieved over the original free cells or other immobilized cells or processes. These comparisons should be highlighted to improve the Ms. Beside I have some other comments as below:

Ø L17; correct to (aimed to In vitro characterize….) and delete in in vitro at L19

Ø L21, delete (acid and bile …….days)

Ø L24 correct to of encapsulant……

Ø L34, correct to (the encapsulated ) ….delete by CMF hydrogel from OPFB

Ø L36 delete but

Ø L37, correct to ( . The inhibition ……)

Ø L61-62 Rephrase, what do you mean by harmfull metaboloites and nutrient to maintain cells

Ø L72073, rephrase how it come 4 kg is extracted from 1 kg??

Ø Rephrase the incomplete sentence at L80, for instance…etc

Ø L97 add the full name of FB195 as the first mention

Ø I hae confused by using LFB 195 and InaCC1295 along the manuscript, the authors should clarify the differences and if no please unify

Ø L114 and Line 115, I could not understand the potential of LFB1295 and the maintained of LFB1295 encapsulated HR

Ø Clarify LFB1295 at L14, 138 and differences as the authors used as cells, encapsulated or Indonesia fermented mild

Ø What do you mean by mild at 124

Ø What do you mean by encapsulated and culture of LFB195 ? is it same as Lactobacillus fermentum

Ø Enlarge fig .1 and add real/live picture of the encapsulated probiotics developed in this study and correct the formatting of Lactobacillus fermentum

Ø Results add full name of CMF-OPEFB at L 264 as first mention in this part

Ø Combine Fig 2 and 3

Ø It is recommended to delete Table 1 and add these data at the Ms text

Ø The column of average at Tables 2, 3 and 4 should be deletes as the authors used different factors and conditions

Ø Tables 2,3, and 4 are better to be combined in one table as table 4

Ø Table 4 should be shortened and similar data should be combined for example production of amines from L-histidine, tyrosine, ornithine or Lysin should be combined in one row

Ø What does the encapsulated probiotics archived over the other immobilized cells or free cells should be clearly discussed in this manuscript…

Ø The comparison with control free cells is the main fault of this work, please clarify and compared to highlight the obtained data by encapsulation

Ø What do you mean by T2 and T1 at table 7,

Ø The average column at Table 6 and 7 and 8 should be deleted and correct the footnote

Ø L427 what do you mean by 5-15 m??

Ø L444 correct to [20] add the authors names

Ø The aim of each experiment should be clear at the beginning of each section

Ø References

Ø The authors are advised to correct all scientific names for example see L612-613, 628, 638, 651,653, 659, 662,679,684,713,717,722, etc

Ø The reference no 30 is not English, provide correctly

Author Response

Dear respected Reviewer 3

We send a response to reviewer 3 and hope that our response is in accordance with your suggestions. All suggestions have been accommodated and written in our paper.

With best regards

Usman Pato, PhD
Professor in Food Science
Faculty of Agriculture
Riau University
Pekanbaru, Indonesia

Ø L17; correct to (aimed to In vitro characterize….) and delete in in vitro at L19
Response: The sentence section has been corrected according to the reviewer's suggestions

Ø L21, delete (acid and bile …….days)
Response: These words have been deleted

Ø L24 correct to of encapsulant……
Response: The Word and particle have been replaced according to suggestions

Ø L34, correct to (the encapsulated ) ….delete by CMF hydrogel from OPFB
Response: The sentence section has been corrected according to the reviewer's suggestions

Ø L36 delete but
Response: “but” had been deleted

Ø L37, correct to ( . The inhibition ……)
Response: As per the recommendation, the corrections have been improved.

Ø The introduction part should be rephrased, reorganized to be in logical flow for easier follow and understanding, the subsequent paragraphs should be connected not disconnected as current especially from L55-118
Response: The introduction has been rephrased, and the paragraphs have been restructured so that they relate systematically with one another.

Ø L61-62 Rephrase, what do you mean by harmfull metaboloites and nutrient to maintain cells
Response: This statement has been changed to more clearly convey its meaning.

Ø L72-73, rephrase how it come 4 kg is extracted from 1 kg??
Response: This sentence has been improved according to the reviewer's suggestion

Ø Rephrase the incomplete sentence at L80, for instance…etc
Response: This sentence has been rephrased so that it becomes a complete sentence

Ø L97 add the full name of FB195 as the first mention
Response: The full name of FB195 as the first mention has been added

Ø I have confused by using LFB 195 and InaCC1295 along the manuscript, the authors should clarify the differences and if no please unify
Response: The abbreviation Lactobacillus fermentum InaCC B1296 has been uniformly written as LFB1296 in all parts of this paper.

Ø L114 and Line 115, I could not understand the potential of LFB1295 and the maintained of LFB1295 encapsulated HR
Response: For clarity, this statement has been changed.

Ø Clarify LFB1295 at L124, 138 and differences as the authors used as cells, encapsulated or Indonesia fermented mild
Response: The LFB1295 used in the study was isolated from Indonesian fermented milk not 'mild' as written in the initial submitted paper. These LBF1295 cells were encapsulated with CMF hydrogel from oil palm frond.

Ø What do you mean by mild at 124
Response: The word "mild" should be "milk" and has been corrected in this paper

Ø What do you mean by encapsulated and culture of LFB195? is it same as Lactobacillus fermentum
Response: LFB1295 stands for Lactobacillus fermentum InaCC B1296 which is the lactic acid bacteria used in this study. Active cultures of LFB1295 were obtained by incubation at 37°C for 24 hours. LFB1295 cells were then separated from the supernatant by centrifugation. Then the cell-free supernatant of LFB1295 was encapsulated with CMF hydrogel from OPF.

Ø Enlarge fig .1 and add real/live picture of the encapsulated probiotics developed in this study and correct the formatting of Lactobacillus fermentum
Response: Figure 1 has been enlarged and improved upon the reviewer's suggestions.

Ø Results add full name of CMF-OPEFB at L 264 as first mention in this part
Response: "CMF-OPEFB" has been written in full "cellulose microfiver-oil palm empty fruit bunch" at the beginning of the mention as suggested

Ø Combine Fig 2 and 3
Response: Figures 2 and 3 have been combined as per suggestion

Ø It is recommended to delete Table 1 and add these data at the Ms text
Response: Table 1 has been deleted and replaced with just a statement as written in the revised paper.

Ø The column of average at Tables 2, 3 and 4 should be deletes as the authors used different factors and conditions
Response: The column of average at Tables 2, 3 and 4 have been deleted according to suggestion.

Ø Tables 2,3, and 4 are better to be combined in one table as table 4
Response: Tables 2,3, and 4 have been combined in one table as per suggestion (become Table 1 in the revised paper)

Ø Table 4 should be shortened and similar data should be combined for example production of amines from L-histidine, tyrosine, ornithine or Lysin should be combined in one row
Response: The words’ L-histidine, tyrosine, ornithine or Lysin” have been combined in one row acoording to suggestion.

Ø What does the encapsulated probiotics archived over the other immobilized cells or free cells should be clearly discussed in this manuscript…
Response: The explanation of immobilized cells or free cells has been clearly discussed in this paper

Ø The comparison with control free cells is the main fault of this work, please clarify and compared to highlight the obtained data by encapsulation
Response: The comparison of free cells in our previous study with encapsulated cells of LFB1295 in the present study is described in the "Discussion" section of this paper.

Ø What do you mean by T2 and T1 at table 7,
Response: The symbols T2 and T1 at Table 7 have been removed as suggested

Ø The average column at Table 6 and 7 and 8 should be deleted and correct the footnote
Response: The average column at Table 6 and 7 and 8 have been deleted and corrected the footnote

Ø L427 what do you mean by 5-15 m??
Response: The number range "5-15m" should be "5-15μm" and has been fixed in this paper

Ø L444 correct to [20] add the authors names

Response: Reference has been corrected and added the author's name

Ø The aim of each experiment should be clear at the beginning of each section
Response: The aim of each experiment have been clearly stated at the beginning of each section as suggested

Ø References
Ø The authors are advised to correct all scientific names for example see L612-613, 628, 638, 651,653, 659, 662,679,684,713,717,722, etc
Ø The reference no 30 is not English, provide correctly
Response: Correction of the writing of the reference has been made according to the reviewer's suggestion

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Major comment

1. The 28-days storage time is not long enough for most of the probiotic products, for example, powder, tablet or capsule products. Unless the author want to apply the encapsulated LFB1295 to some refrigerated fermented milk product (the storage time around 21-28 days). However, there was no related experiment to prove this point.

Minor comment

1. The properties of CMF-OPEFB is not added in the revised abstract.

Author Response

Response to Reviewer 2 Comments

Comments and Suggestions for Authors

Major comment

The 28-days storage time is not long enough for most of the probiotic products, for example, powder, tablet or capsule products. Unless the author want to apply the encapsulated LFB1295 to some refrigerated fermented milk product (the storage time around 21-28 days). However, there was no related experiment to prove this point.

Response: Based on the findings of our investigation, it was determined that 28 days of storage treatment at both room temperature and low temperature was the best period of time to keep LFB1295 encapsulated CMFH-OPEFB viable at a minimum level of 10⁷ CFU/g. Less than 10⁷CFU/g of viable bacteria were present after storage for longer than 28 days (unpublished data). While a probiotic needs between 10⁷ and 10⁹ CFU/g to carry out its therapeutic effects. The long-term objective of this research is to use encapsulated LFB1295 in a variety of non-fermented functional food products, including jams and other probiotic foods manufactured from different tropical fruits, and sold at room temperature or cold temperatures with a shelf life of fewer than 28 days..

Minor comment

The properties of CMF-OPEFB is not added in the revised abstract.

Response: As recommended, the amended abstract now included the characteristics of CMF-OPEFB.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have addressed almost all comments to great extent.

Author Response

Comments and Suggestions for Authors

The authors have addressed almost all comments to great extent.

Response: I want to thank you very much for your suggestions for improving our paper on behalf of all the authors.

Author Response File: Author Response.docx