Probiotic Characteristics of Ligilactobacillus salivarius AS22 Isolated from Sheep Dung and Its Application in Corn-Fox Tail Millet Silage

Round 1

Reviewer 1 Report

In the manuscript "Probiotic characteristics of Lactobacillus salivarius AS22 Isolated from Sheep Dung and Its Application in Corn-Fox tail Millet Silage," Balasubramanian et al. describe the isolation of several LAB strains from sheep dung and evaluate their suitability for sillage of a mixture of corn and foxtail millet. While the manuscript has some interesting results, the English language within it needs to be thoroughly corrected. Moreover, several modifications are needed before potential publication:

1) Line 51-53: Genus Lactobacillus has been taxonomically reclassified since last year (doi.org/10.1099/ijsem.0.004107). Please take this into account and use only currently valid names of the species throughout the manuscript.
2) Line 135: Authors should present the exact name of the DNA extraction kit they used.
3) Line 138: Please cite the manuscript from which primers 27F and 1492R originate.
4) Line 140: The authors should explain why they isolated the strains at 28°C but measured the growth curve at 37°C.
5) Line 170: What are KCC-37 and KCC-38?
6) Line 175: Unit microgram refers to the mass, not concentration. Please specify the used concentration.
7) Line 196: Please specify a unit for 1x10^5 (per bag, per gram?).
8) Line 197: Silage fermentation was done under anaerobic conditions, but all LAB characterizations were done aerobically. Why?
9) Line 220: The authors analyzed the amount of acetic acid in the culture, but they neglected to mention that the MRS medium contains 5 g/L of sodium acetate. Thus, they do not measure how much acetic acid LAB produce but rather how much acetic acid remains in the media after cultivation. It follows that just because they detect acetic acid in the media, it does not mean that the strain is heterofermentative.
10) Line 237: Table 2 should be converted into Figure, and it should mention at which temperature the cultivation was done. Furthermore, the OD value of 2 is very low for LAB (they usually reach OD = 7-10) and suggests that the authors did not measure OD of decimal dilutions once the OD value rose above 1 (at this point, OD no longer linearly follows cell density). Is this correct?
11) Line 286: The authors provide no proof that the strain produces bacteriocins. As such, they should not claim it does.
12) Lines 341-357 should be moved to the Introduction section of the manuscript.
13) Table 5: The authors should also present the exact p-values they obtained.

Minor remarks:
14) Line 23: Panicumitalicum > Panicum italicum
15) Line 39: Hordeum Vulgare > Hordeum vulgare
16) Line 39: Loliummultiforum > Lolium multiforum
17) Line 69-71: ferulateesterases > ferulate esterases
18) Line 88: Pediococcu spentosaceus and Lactiplantibacillu splantarum > Pediococcus pentosaceus and Lactiplantibacillus plantarum
19) Line 110: Experimental section > Materials and Methods
20) Line 317: Reference 44 is misplaced.

Author Response

In the manuscript "Probiotic characteristics of Lactobacillus salivarius AS22 Isolated from Sheep Dung and Its Application in Corn-Fox tail Millet Silage," Balasubramanian et al. describe the isolation of several LAB strains from sheep dung and evaluate their suitability for sillage of a mixture of corn and foxtail millet. While the manuscript has some interesting results, the English language within it needs to be thoroughly corrected. Moreover, several modifications are needed before potential publication:

• Line 51-53: Genus Lactobacillus has been taxonomically reclassified since last year (doi.org/10.1099/ijsem.0.004107). Please take this into account and use only currently valid names of the species throughout the manuscript.

Answer: Thank you very much for your suggestion on binomial nomenclature. We modified the text according to the new valid names of the species throughout the manuscript.

In addition, included the following statement in the introduction section for better clarity

“The genus Lactobacillus has been reclassified recently based on core genome phylogeny, clade-specific signature genes, pairwise average amino acid identity, physiological characters and the ecological criteria of the bacteria. Based on this polyphasic approach, the genus Lactobacillus has been classified into 25 genera including the emended genus Lactobacillus (host-adaptive organism, Paralactobacillus and Lactobacillus delbrueckii group) and other 23 novel genera such as, Lapidilactobacillus, Companilactobacillus, Bombilactobacillus,  Amylolactobacillus, Holzapfelia, Lacticaseibacillus, Loigolactobacilus, Schleiferilactobacillus, Agrilactobacillus, Lactiplantibacillus, Ligilactobacillus, Liquorilactobacillus, Dellaglioa, Latilactobacillus, Acetilactobacillus, Fructilactobacillus, Limosilactobacillus, Paucilactobacillus, Furfurilactobacillus, Lentilactobacillus, Secundilactobacillus, Levilactobacillus and Apilactobacillus [Zheng et al., 2020]”.

2) Line 135: Authors should present the exact name of the DNA extraction kit they used.

Answer: Qiagen’s DNA purification kit (Qiagen, Germany) was used in this study.

3) Line 138: Please cite the manuscript from which primers 27F and 1492R originate.

Answer: As suggested by the reviewer, cited the following reference.

Rejiniemon, T. S., Hussain, R. R., and Rajamani, B. (2015). In-vitro functional properties of Lactobacillus plantarum isolated from fermented ragi malt. South Ind. J. Biol. Sci. 1, 15–23.

4) Line 140: The authors should explain why they isolated the strains at 28°C but measured the growth curve at 37°C.

Answer: Initial screening experiments were performed at 28 °C, and later we found optimum growth between the temperature 20 and 30 °C. Based on these preliminary results (Table 3 in previous version and table 2 in this revised version), 28 °C was selected to determine the growth curve and the typo error was corrected.

5) Line 170: What are KCC-37 and KCC-38?

Answer: These two strains (Lactobacillus plantarum KCC-37 and Pediococcus pentosaceus KCC-38) have been reported previously and the organism name was updated in this revised manuscript to avoid confusion.

Reference

Mayakrishnan, V.; Soundharrajan, I.; Kim, D.; Priya, K.; Choi, K. In-vitro assessment of the probiotic potential of Lactobacillus plantarum KCC-24 isolated from Italian rye-grass (Lolium multiflorum) forage. Anaerobe 2015, 32, doi:10.1016/j.anaerobe.2015.01.003.

Ilavenil, S.; Vijayakumar, M.; Kim, D.H.; Valan Arasu, M.; Park, H.S.; Ravikumar, S.; Choi, K.C. Assessment of probiotic, antifungal and cholesterol lowering properties of Pediococcus pentosaceus KCC-23 isolated from Italian ryegrass. Journal of the science of food and agriculture 2016, 96, 593-601, doi:10.1002/jsfa.7128.

6) Line 196: Please specify a unit for 1x10^5 (per bag, per gram?).

Answer: Updated the required unit, per gram.

7) Line 197: Silage fermentation was done under anaerobic conditions, but all LAB characterizations were done aerobically. Why?

Answer: Silage fermentation, growth and organic acids productions were performed under anaerobic conditions. The isolated strains were inoculated in MRS broth in 1000mL glass bottle containing butyl rubber stoppers with aluminum crimp seals, and then air was evacuated using vacuum machine.

8) Line 220: The authors analyzed the amount of acetic acid in the culture, but they neglected to mention that the MRS medium contains 5 g/L of sodium acetate. Thus, they do not measure how much acetic acid LAB produce but rather how much acetic acid remains in the media after cultivation. It follows that just because they detect acetic acid in the media, it does not mean that the strain is heterofermentative.

Answer: As described by the reviewer, the remaining acetic acid in the media after cultivation was studied. So, we do not claim the selected organism was “heterofermentative” and modified the text accordingly.

9) Line 237: Table 2 should be converted into Figure, and it should mention at which temperature the cultivation was done. Furthermore, the OD value of 2 is very low for LAB (they usually reach OD = 7-10) and suggests that the authors did not measure OD of decimal dilutions once the OD value rose above 1 (at this point, OD no longer linearly follows cell density). Is this correct?.

Answer: As suggested by the reviewer, Table 2 was converted as a Figure. The cultivation temperature was 28 °C and was corrected in the method section. As described in Table 2 (previously it was table 3), the optimal temperature for the growth was between 20 and 30 °C, hence, 28 °C was selected for this study. The culture was performed below 30 °C; this may be another one reason of less optical density at 600 nm. The culture was directly taken and the absorbance of the culture was read at 600 nm and as stated by the reviewer, either OD of decimal dilutions was not performed or plated on MRS agar plates to count the exact number of cells. We consider this suggestion in further works on Lactobacillus species.  

10) Line 286: The authors provide no proof that the strain produces bacteriocins. As such, they should not claim it does.

Answer: As suggested by the reviewer, the following statement was included and no claim on bacteriocins or bacteriocins likes substances.

“The antibacterial activity was mainly due to the synthesis of organic acids. For further work we plan to characterize bacteriocin from this organism”

10) Lines 341-357 should be moved to the Introduction section of the manuscript.

Answer: Thank you very much for the suggestion, as suggested by the reviewer this section was moved to introduction and rearranged reference numbers.

12) Table 5: The authors should also present the exact p-values they obtained.

Answer: As suggested by the reviewer “p” value was incorporated in Table 3.

Minor remarks:
13) Line 23: Panicumitalicum>Panicum italicum

Answer: Corrected as, Panicum italicum

14) Line 39: Hordeum Vulgare>Hordeum vulgare

Answer: Corrected as, Hordeum vulgare

15) Line 39: Loliummultiforum>Lolium multiforum

Answer: Corrected as, Lolium multiforum

16) Line 69-71: ferulateesterases > ferulate esterases

Answer: Corrected as, ferulate esterases

17) Line 88: Pediococcu spentosaceus and Lactiplantibacillu splantarum>Pediococcus pentosaceus and Lactiplantibacillus plantarum

Answer: Corrected as, Pediococcus pentosaceus and Lactiplantibacillus plantarum

18) Line 110: Experimental section > Materials and Methods

Answer: Corrected as, Materials and Methods

19) Line 317: Reference 44 is misplaced.

Answer:  Corrected the format error and placed reference 44 in appropriate place.

 

Reviewer 2 Report

The authors isolated Lactobacillus salivarius AS22 from sheep dung and characterized its probiotic properties. The application of ensiling on corn-foxtail millet was investigated as well. The authors proved A22 has probiotic traits through bile and acid tolerance tests, antibacterial, antifungal, and antioxidant properties. Microbial profile also provided evidence for A22 being able to exert a potential effect on silage quality.

 

General points

1. The English -including sentence structure, spelling, diction, and grammar- should be edited extensively to be error-free, concise, and professional.
2. Lactobacillus salivarius has been frequently isolated from the mammalian digestive tract, with its probiotic characteristic published and researched for years (Neville, BA.,  O’Toole, PW., Future Microbiology, 2010). Furthermore, its application in silage is well known (Guo, L. et al., frontiers in Microbiology. 2020). Although a positive outcome, this study cannot be considered innovative or particularly insightful - the authors should differentiate from other published papers by explaining the unique value of this research.

 

Specific points

1. The Materials and Methods sections should clearly specify conditions and include references.
2. Line173, nu?
3. Line 182, /100 should be X100.
4. Line 211, should either be Mean ± SD or Mean ±  SEM; also need to specify the number of repetitions.
5. Table 3, should quantify +’s and what they mean.
6. Results 3.1, should describe the identification method of the scientific name of AS22; should be consistent with either “selected bacterial strand” or “isolated AS22” to prevent confusion.
7. In 3.5 antibacterial activity, the cell number of indicator strains should be indicated. Its antibacterial property could be due to its acidity.
8. Line 298, need stronger evidence to support this statement.
9. Legends for all tables and figures are ambiguous and unclear.

Comments for author File: Comments.docx

Author Response

 

The authors isolated Lactobacillus salivarius AS22 from sheep dung and characterized its probiotic properties. The application of ensiling on corn-foxtail millet was investigated as well. The authors proved A22 has probiotic traits through bile and acid tolerance tests, antibacterial, antifungal, and antioxidant properties. Microbial profile also provided evidence for A22 being able to exert a potential effect on silage quality.

 

General points

1. The English -including sentence structure, spelling, diction, and grammar- should be edited extensively to be error-free, concise, and professional.

Answer: Extensive revision was carried out, typo, spell and grammar errors solved.

 

1. Lactobacillus salivarius has been frequently isolated from the mammalian digestive tract, with its probiotic characteristic published and researched for years (Neville,BA., O’Toole,PW., Future Microbiology, 2010). Furthermore, its application in silage is well known (Guo, L. et al., frontiers in Microbiology. 2020). Although a positive outcome, this study cannot be considered innovative or particularly insightful - the authors should differentiate from other published papers by explaining the unique value of this research.

 

Answer: In this revised manuscript, we cited these two references. The first paper reviewed Lactobacillus salivarius and its application as a probiotic. The later one used various Lactobacillus species for the preparation of silage using naturally available LAB from the fodder. In this study, the selected Lactobacillus salivarius was isolated from the sheep dung and produced various organic acids and meets probiotic properties. As suggested by the reviewer, these statements were incorporated in the revised manuscript.  

 

Specific points

1. The Materials and Methods sections should clearly specify conditions and include references.

Answer: Included references in materials and methods section.

1. Line173, nu?

Answer: Removed this typo error in this revised version.

1. Line 182, /100 should be X100.

Answer: We corrected this mistake as written as X100.

1. Line 211, should either be Mean ± SD or Mean ± SEM; also need to specify the number of repetitions.

Answer: Three different experiments were performed and included this statement in the revised manuscript.

1. Table 3, should quantify +’s and what they mean.

Answer: Corrected the typo error.

1. Results 3.1, should describe the identification method of the scientific name of AS22; should be consistent with either “selected bacterial strand” or “isolated AS22” to prevent confusion.

 

Answer: Thank you very much for your suggestion. This organism was isolated from the sheep dung, so we preferred to use “isolated AS22” throughout the manuscript.

 

1. In 3.5 antibacterial activities, the cell number of indicator strains should be indicated. Its antibacterial property could be due to its acidity.

 

Answer: Cell number of indicator strain (1 × 10⁸ CFU/mL) was incorporated in this revised manuscript. We strongly agree with the reviewers on “acidity and antibacterial property of cell free extract”.

1. Line 298, need stronger evidence to support this statement.

Answer: Included reference to support this statement

1. Legends for all tables and figures are ambiguous and unclear.

 

Answer: In this revised manuscript, the table and figure legends are very clear and explanatory. 

 

 

 

 

 

 

 

Round 2

Reviewer 2 Report

1. Unfortunately the English - including sentence structure, spelling, diction, and grammar - was not edited extensively. I strongly advise thorough revision by native English speakers before publication. There were still errors in the manuscript, some examples are illustrated as follow:

a. Line 43, can able to → are able to

b. Line 44, better not express as [at very low]

c. Line 48, delete rich

d. Line 49, delete content

e. Line 68, is → are; LAB is a plural noun.

f. Line 71, it → they

g. Line 71, quality silage → silage quality

h. Line81, An enzyme, …. esterases? cleave → cleaves

i. Line 110, faces → feces

j. Line 130, the grammar of the sentence “Application of bacteria inoculant and glucose with rice straw improved silage quality and described previously.” should be corrected.

k. Line 272, could be grew → could grow

l. Line 268, Growth kinetics of LAB → Growth kinetics of isolated LAB

m. Line 365-366, 369-370, please use plural verbs

n. Line 375, can be corrected as "have been reported to produce…. and inhibit".

2. In order to distinguish this paper from other publications, the authors should compare and contrast the A22 strain with another known same/different species of bacteria in discussion rather than briefly referencing it in the introduction.

3. The authors should provide a quantitative definition of what the ‘+’ means in Table 3 instead of roughly estimating the levels. Please define the ratio of OD600 in relation to bacterial growth in order to precisely indicate what positive reaction, improved growth, and maximum enzyme activity mean.

4. Please elaborate on the enzyme activity assay protocol in the materials and methods section.

Author Response

We thank the reviewer for giving valuable comments about our research paper which is very helpful to improve the quality of the presentation of the manuscript. We have gone through the whole manuscript according to reviewer suggestions and modified the same. All changes in the manuscript have been made with red color fonts

1. Unfortunately the English - including sentence structure, spelling, diction, and grammar - was not edited extensively. I strongly advise thorough revision by native English speakers before publication. There were still errors in the manuscript, some examples are illustrated as follow: Line 43, can able to → are able to b. Line 44, better not express as [at very low]c. Line 48, delete rich d. Line 49, delete content e. Line 68, is → are; LAB is a plural noun.f. Line 71, it → they g. Line 71, quality silage → silage quality h. Line81, An enzyme, esterases? cleave → cleaves i. Line 110, faces → feces.

Thanks and sorry for the careless mistake and errors in the manuscript. We carefully checked the whole manuscript with the help of English speaking professor and online software Grammarly (subscribed). All changes in the manuscript have been made with red color fonts.

2. Line 130, the grammar of the sentence “Application of bacteria inoculant and glucose with rice straw improved silage quality and described previously.” should be corrected.

Thanks for your kind suggestion; we have deleted these lines from the manuscript. The whole introduction part has been revised carefully.

3. Line 272, could be grew → could grow l. Line 268, Growth kinetics of LAB → Growth kinetics of isolated LAB m. Line 365-366, 369-370, please use plural verbs. Line 375, can be corrected as "have been reported to produce…. and inhibit".

We have revised grammar mistakes and errors according to the reviewer suggestion  (Figure 3,4, and 5)

4. In order to distinguish this paper from other publications, the authors should compare and contrast the A22 strain with another known same/different species of bacteria in discussion rather than briefly referencing it in the introduction.

Thanks and we have agreed with the reviewer comments and revised them accordingly. Now we have discussed our current data other reports obtained from the same species

Line numbers: 279-284; 299-304; 310-314; 322-330; 339-348; 364-368;;395-402;

5. The authors should provide a quantitative definition of what the ‘+’ means in Table 3 instead of roughly estimating the levels. Please define the ratio of OD600 in relation to bacterial growth in order to precisely indicate what positive reaction, improved growth, and maximum enzyme activity mean.

  Thanks, now we have revised as per reviewer suggestion : Growth was analyzed at 600 nm using a UV-visible spectrophotometer (>1.0 OD at 600 nm** referred as ++; <1.0 OD at 600 nm referred as + ;) and enzyme activity was assayed by spectrophotometer method (<50 U/mL* referred as +; >100 U/mL** referred as ++). Carbohydrate fermentation test: strong fermentation referred as ++; week fermentation referred as +.

6. Please elaborate on the enzyme activity assay protocol in the materials and methods section.

Yes, we have cited a reference for the protocol protease and cellulose enzyme activities quantification; The protease and cellulose activities were determined in the cell free supernatant[25]

Ho, J.C.K.; Yin Sze, L. Isolation, identification and characterization of enzyme-producing lactic acid bacteria from traditional fermented foods. Bioscience Horizons: The International Journal of Student Research 2018, 11, doi:10.1093/biohorizons/hzy004.

Round 3

Reviewer 2 Report

Plea check again and revise some English mistakes, for example Line 71, delete “to”, and Line 358, please use past tense to illustrate the data (eg. shows --> showed).

Author Response

We thank the reviewer for giving valuable comments about our research paper which is very helpful to improve the quality of the presentation of the manuscript. We have improved the experimental design and its protocols significantly. Most of the experimental used in this study is already been published previously. Therefore, we have cited a reference for the experimental protocol. The conclusion of the manuscript was revised according to the reviewer suggestion. We have revised the whole manuscript according to reviewer suggestions and modified the same. All changes in the manuscript have been made with red color fonts

 

1. Please check again and revise some English mistakes, for example Line 71, delete “to”, and Line 358, please use past tense to illustrate the data (eg. shows --> showed).

We have gone through the whole manuscript according to reviewer suggestions and modified the same. All changes in the manuscript have been made with red color fonts.

Most of the experimental protocols used in this study is already been published previously. Therefore, we have cited a reference for the experimental protocol

The conclusion of the manuscript was revised according to the reviewer suggestion