The Bacteriocin-like Inhibitory Substance Producing Lacticaseibacillus paracasei LPa 12/1 from Raw Goat Milk, a Potential Additive in Dairy Products

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The manuscript describes application of a bacterial strain as an additive in dairy products. The authors used animal model to assess the suitability of this bacterial strain and performed some experiments to evaluate the survival of this strain in two different products. The manuscript needs to be revised thoroughly considering following aspects.

 

1.     Language of the article needs extensive editing.

 

2.     The abstract is very vague. First it does not provide the rationale of this study clearly. Why was this strain used as a milk additive? Which strains are used currently and why do we need another strain? Write briefly about this rationale. Then the work plan or research strategy is very poorly described and does not provide a clear picture of this study. Introduction of any foreign particle/bacteria can lead to increased immune response, including phagocytosis. So why it was emphasized here?

 

3.     Remove firs two keywords. Some specific terms (not used in title) can be added.

 

4.     L46-47: The two sentences provide contradictory information. If Firmicutes is not abundant (despite 20% occurrence) then state which group dominates here.

 

5.     L59-65: These lines indicate that the strain was isolated earlier. It is important to mention, how it was identified in the previous study.

 

6.     L86: So, the strain was fed for 30 days? The earlier lines indicate 45 days.

 

7.     L115-L117: State clearly if this composition has been copied from the product label.

 

8.     L139: The labelling is not clear.

 

9.     What was the rationale to count amylolytic streptococci in cow yogurt samples?

 

10.  L154: What was the source of this strain? Any ref?

 

11.  L165-167: Write method clearly

 

12.  L170: Why against Listeria?

 

13.  L177-178: This is very confusing. Present data in exponent. It seems the count reduced to less than half (from 10 exp 9 to 1o exp 4). Can it be considered as colonization?

 

14.  L182: The terms 30EG and 45 EG have not been presented completely.

 

15.  L190: Argue how this organism can reach to lever.

 

16.  Captions of the tables should provide complete details about the terms presented in the tables.

 

17.  L228-231: These are meaningless unless given as footnotes of the tables (which is not clear here). Also correct it with other tables.

 

18.  L259: Data is not understandable.

 

19.  L324: Provide reference (for commensalism). Consider L 326 where it states the ‘some’ strains are beneficial. Authors need to correlate it with the definition of commensals.

 

20.  In discussion, add some points if the organism can act on casein and can have a role in yogurt making.

Comments on the Quality of English Language

At many places, meaningless sentences appear. Along with verb confusion, choice of words need to be considered.

Author Response

Rev 1 (red)

First of all, I would like to thank so much  for your effort to review the manuscript. Comments done contributed in its improvement. In red are involved improvements also regarding the English language.

2)To innovating Abstract

Goat milk starts to be gradually the preferred milk by consumers worldwide, Slovakia involving. It is also demanded as functional and/or nutraceutical drink as it is rich in bioactive components. However, also new, beneficial autochthonous strains are searched to enrich goat dairy products. Among individual representatives are involved also lacticaseibacilli. Bacteriocin-producing, beneficial strain Lacticaseibacillus paracasei LPa 12/1 was isolated from raw goat milk. This study has been focused on showing its potential to be a new additive in dairy products. No mortality was found checking safety of LPa 12/1 strain using BALB/c mice. The strain has reached higher counts in ewe-goat milk yoghurt (up to 6.1 cfu/g log 10) than in cow milk yoghurt (almost 5.0 cfu/g log 10). Cow milk yoghurts remained in better consistency after LPa 12/1 strain supplementation in comparison with ewe-goat milk yoghurts, although specific organoleptic tests were not performed. However, LPa 12/1 has survived in both types of yoghurts in sufficient amounts. Thermo-stable bacteriocin-producing strain LPa 12/1 applied in yoghurts in encapsulated form seems to be a suitable to supplement dairy products.

3. In Keywords, two words were deleted and replaced by the words postbiotic, enrichment
4. L46-47: I agree, Iam apologizing for dissmissing and now I hope it is more clear: Using next-generation sequencing method, useful bacteria of the phylum Firmicutes have been occurred in tested Slovak raw goat milks with abundance 20.5 % [5]. The phylum Actinobacteria reached abundance 62.8 % [5]. Although Firmicutes was not dominating phylum in tested raw goat milks, genera involved in the phylum Firmicutes were detected there. Moreover, using the standard microbiological method, individual representatives of the detected genera were isolated from raw goat milks [7].

5.L59-65: Here it is indicated how strain was identified: Its taxonomical species allocation was performed based on MALDI-TOF identification score evaluation (2.004) as reported previously [7].   

6. L86: Maybe it was not understood clearly. Strain was applied for 30 days, but experiment lasted 45 days meaning 15 days to the end of the experiment it was not applied.
7. L115-117: According to the product label, they contain commercial yoghurt culture; energy 309 KJ/74 kcal, fat 4.6 g of which saturated fatty acids participated with 3.6 g, carbohydrates were involved in 4.1 g, sugar value of which was 3.4 g; proteins content formed 4.1 g, and salt 0.07 g.

It was used in style for manuscript.

8. L139: It was copied from label. And the content was declared for 100 g as indicated.  Components content involving commercial culture was declared for 100 g of product: energy 500 KJ, fat 9.0 g of which saturated fatty acids formed 5.2 g.Carbohydrates content was 4.5 g,sugar value of which was 4.1 g. Proteins content formed 3.4 g, and salt 0.1 g.

9. Rationale for amylolytic streptococci was: among commercial culture was dairy Streptococcus species strain, dairy species of streptococci are amylolytic. It was the reason and dairy streptococi amount was indicated as amylolytic.
10. The source of strain was indicated in original manuscript.

The principal indicator-Enterococcus avium EA5 (the most sensitive strain) was used in the test (our strain isolated from feces of piglet),

11. It was improved, hoping now it is more clear.
12. Against Listeria because they are frequent contaminants of raw milks.
13. I think now it is more clear. Normally when you use amount/dose 10⁹ cfu/ml, in animal organism/tract count 10⁴ is ok, it is colonization because there could be different interactions with other microbiota. Counts of lactobacilli/LAB could be like this.
14. L182: LPa 12/1 strain contributed in the total LAB increase on day 30 compared with day 0/1 (difference 0.59 log cycle) and on day 45 (^abp<0.001). Fecal amylolytic streptococci were present in high counts (Table 1). Coliforms count was reduced significantly in the experimental group on day 30 (30EG) comparing with the experimental group on day 45- 45EG (^abp<0.05).
15. I dont understand how you mean it.
16. Everything was there, but I controlled.
17. It was controlled. When in microbiota counting is not significant difference, to use log difference is normal/common and used because in microbiology also difference in log mathemathical difference is relevant.
18. When in microbiology is not significant difference, but there is difference in log cycle it is mathematical difference, there is really difference when among samples are differences up to 1.0 or more log cycle, there is reduction.
19. It was involved. The species Lacticaseibacillus paracasei is bacterium that operates by commensalism [15]. Commensal lactobacilli are good candidates for development as probiotics [15]. Some of these species posses beneficial/probiotic properties among which also a bacteriocin-production can be involved [16]. The species Lacticaseibacillus paracasei is the most frequently isolated species from dairy environment among the other lacticaseibacilli.
20. Some bacteria can influence proteolysis of casein by different way and so influence processing of product/yoghurt.. In case of LPa 12/1 strain its optimal technological properties such as sufficient clotting capacity in milk were previously described.

Reviewer 2 Report

Comments and Suggestions for Authors

 The manuscript entitled “The bacteriocin-producing strain Lacticaseibacillus paracasei 2 LPa 12/1 from raw goat milk, a potential additive in dairy prod- 3 ucts “The given information is in this manuscript is useful for researchers and academia and article would be great contribution in related discipline.  The room for improvement is always there and I have suggested some minor revisions. Furthermore, the discussion section is required related citation to improve the discussion. I can extend my services to further review the incorporation of the corrections in article again.

·         Title: reconsider as per your aim and objective –

·         Mention numerical values- Even increase tendency in phagocytic activity in blood of mice was noted after LPa 27 12/1 strain application. Encapsulated form of LPa 12/1 strain producing thermo-stable bacteriocin 28 seems to be a suitable to supplement dairy products

·         L-53- Rewrite- When those species strains can produce bacteriocins 53 (antimicrobial proteinaceous substances) and also posses additional beneficial properties 54 (bile and low pH tolerance, sufficient adhesive ability, lactic acid production, diacetyl, 55 hydrogen peroxide or useful enzymes, etc.), they can play important role in food fermen- 56 tation process, in biopreservation and or in prevention of contamination

·         How they  were acclimatized? Mice were placed at Institute of Par- 82 asitology of the Slovak Academy of Sciences in Košice (Slovakia), approved by Slovak 83 Veterinary Administration as well as Ethic Committee of the institute (Ro-1604/19-221/3

·         Skim milk was used as wall materials???????? en broth culture was mixed with skim milk (Simandl company, Czech Republic) 105 in small flasks (in ratio 1:1). They were placed for freezing at - 80 ˚C

·         L-126-CREATI G CONFUSION- The LPa 12/1 strain was selected on MRS agar enriched with rifampicin (100 µg/ml), LAB 126 were determined on MRS agar and also amylolytic streptococci were checked using M17 127 agar (Difco). Yoghurt samples (one g) were taken and mixed (Stomacher-Masticator, IUL, 128 Spain) with peptone water (Merck, ratio 1:9), diluted and spread on the selected cultiva- 129 tion media (ISO) as formerly indicated. Moreover, pH was measured using Checker-pH 130 tester (Hanna instruments Inc., Woonsocket, USA).

·         168- REPHRASE- Precipitate was treated with enzymes such as α-chymotrypsin, trypsin, proteinase 168 and pepsin, Merck) and again inhibitory activity was checked against EA5 strain but also 169 against Listeria monocytogenes LM 7223 (Veterinary Institute in Olomouc, Czech Republic

·         L-258 PLEASE CROSS HECK THIS INCREASE IN VALUE- The count of LPa 12/1 strain in dry frozen form immediately before its addition in 258 yoghurts reached 6.6 x 107 cfu/g respectively 7. 82 cfu/g (log 10

·         Abstract- Mention numerical value- .

·         Any scientific ;ogic????????/ n the other hand, cow milk yoghurts retained better consistency 342 during 14 days in comparison with ewe-goat milk yoghurts

·         Any sensory evaluation of yogurt??????????

·         Please add more references in discussion section

·         Cite the following latest references.

·         Qureshi, A. W. Evaluation of Bacteriocin activity of Lactic Acid Bacteria (LAB) Isolates of Milk and Yogurt.

·         Krishnamoorthi, R., Srinivash, M., Mahalingam, P. U., Malaikozhundan, B., Suganya, P., & Gurushankar, K. (2022). Antimicrobial, anti-biofilm, antioxidant and cytotoxic effects of bacteriocin by Lactococcus lactis strain CH3 isolated from fermented dairy products—An in vitro and in silico approach. International Journal of Biological Macromolecules, 220, 291-306.

·         Qureshi, Asma Waheed. "Evaluation of Bacteriocin activity of Lactic Acid Bacteria (LAB) Isolates of Milk and Yogurt."

·         Biadała, A., Szablewski, T., Cegielska-Radziejewska, R., Lasik-Kurdyś, M., & Adzahan, N. M. (2023). The Evaluation of Activity of Selected Lactic Acid Bacteria for Bioconversion of Milk and Whey from Goat Milk to Release Biomolecules with Antibacterial Activity. Molecules, 28(9), 3696. How can you justify that your study is feasible /economical for the field?

·         Please avoid repetition-

·         Please check reference style throughout MS

·         Italic all the scientific names,

·         Remove grammatical mistakes

·         Need to rewrite the conclusion

·         Recheck Legends description is as per figure number and discussion-

·         I urge the authors to improve the English language for better flow of literature

Comments on the Quality of English Language

I urge the authors to improve the English language for better flow of literature

Author Response

2. (blue)

Thanks a lot for your reviewing. If the other improvement than asked from Rev.1, it will be in bleu.

Title: in this case, Iam not sure what you mean. Iam sorry.

Thermo-stable bacteriocin-producing strain LPa 12/1 applied in yoghurts in encapsulated form seems to be a suitable to supplement dairy products.

When those species strains produces bacteriocins (antimicrobial proteinaceous substances) and also posses additional beneficial properties such as bile and low pH tolerance, sufficient adhesive ability, lactic acid production, diacetyl, hydrogen peroxide or useful enzymes production, etc., they can play important role in food fermentation process, in biopreservation and/or in prevention of contamination............

The animals were acclimatized for 15 days after the stress of transport, and when they did not show signs of illness they were used in the experiment. Experiments on laboratory mice were carried out in the accredited vivarium at the Institute of Parasitology SAS. The animals were handled in accordance with the "Requirements for the handling of animals" specified in the Slovak Law no. 377/2012 on veterinary care.

The animal study protocol was conducted in accordance with current European and Slovak national legislative requirements for animal use, optimality of animal use and cruelty of the procedures. The project was approved by the Animal Care Ethics Committee at the Institute of Parasitology SAS (No. EK-PaU-2/2019, 2 April 2019) and the State Veterinary and Food Administration of the Slovak Republic (No. Ro-1604/19-221/3, 13 June 2019).

Before LPa12/1 strain application, samples of yoghurts were diluted in peptone water and controlled for possible contamination by spreading dilutions on Mac Conkey agar (Difco) to control Enterobacteriae. Yoghurts were absent of Enterobacteriacae. LAB were counted on MRS agar (Merck) and control was provided also on M17 agar (Difco) because of commercial culture -Streptococcus spp. (both counts reached 5.1 cfu/g). Then sampling of yoghurts was performed after 24 h, on day 7, on day 10 and on day 14 (which is also declared expiration time for this type of yoghurt). The cells of 

applied LPa 12/1 strain was selected on MRS agar enriched with rifampicin (100 µg/ml), LAB were determined on MRS agar. Amylolytic streptococci were checked using M17 agar (Difco). Yoghurts (one g) were sampled and mixed (Stomacher-Masticator, IUL, Spain) with peptone water (Merck, ratio 1:9). Then diluted and spread on the selected cultivation media (ISO) as formerly indicated. Moreover, pH was measured using Checker-pH tester (Hanna instruments Inc., Woonsocket, USA). Initial pH was 3.94. Yoghurts were maintained in the fridge during whole testing period

The enzymes such as α-chymotrypsin, trypsin, proteinase and pepsin (Merck) were used to treat precipitate. Then again inhibitory activity was checked against EA5 strain. Moreover also Listeria monocytogenes LM 7223 (Veterinary Institute in Olomouc, Czech Republic) was used as indicator (as frequent contaminant of milk).

Conclusion was controlled. legends and also what was indicated as appropriate.Added references:  E.g. Antimicrobial, anti-biofilm, and cytotoxic effect of bacteriocin -lactococcin produced by dairy origin strain Lactococcus lactis CH3 reported Krishnamoorthi et al. [18]. Also Biadala et al. [19] informed about antibacterial activity of selected LAB for bioconversion of milk and whey from goat milk. Enrichment of products by beneficial strains can be influenced. Also Biadala and 2 others.

Reviewer 3 Report

Comments and Suggestions for Authors

the following manuscript submitted "The bacteriocin-producing strain Lacticaseibacillus paracasei LPa 12/1 from raw goat milk, a potential additive in dairy products" to the Applied Sciences; 

Missing statistical data in the manuscript.  Authors should include and check it in the manuscript. 

Authors should include or cite recent information in the manuscript 

Extensive English correction is required and plagarism to be checked by authors.  

Authors should check all tables and figures with proper statistical data.

 

 

 

Comments on the Quality of English Language

the following manuscript submitted "The bacteriocin-producing strain Lacticaseibacillus paracasei LPa 12/1 from raw goat milk, a potential additive in dairy products" to the Applied Sciences; 

Missing statistical data in the manuscript.  Authors should include and check it in the manuscript. 

Authors should include or cite recent information in the manuscript 

Extensive English correction is required and plagarism to be checked by authors.  

Authors should check all tables and figures with proper statistical data.

Author Response

Rev 3. green but also bleu, red,Thanks for your notes.

SD was involved. Statistic was involved there also in original manuscript version. Recent information was cited. English was checked. Tables were checked for data.

Everything is involved in green or others colours according to detail other reviewers requests.

Responses also for the other reviewers: First of all, I would like to thank so much  for your effort to review the manuscript. Comments done contributed in its improvement. In red are involved improvements also regarding the English language. Goat milk starts to be gradually the preferred milk by consumers worldwide, Slovakia involving. It is also demanded as functional and/or nutraceutical drink as it is rich in bioactive components. However, also new, beneficial autochthonous strains are searched to enrich goat dairy products. Among individual representatives are involved also lacticaseibacilli. Bacteriocin-producing, beneficial strain Lacticaseibacillus paracasei LPa 12/1 was isolated from raw goat milk. This study has been focused on showing its potential to be a new additive in dairy products. No mortality was found checking safety of LPa 12/1 strain using BALB/c mice. The strain has reached higher counts in ewe-goat milk yoghurt (up to 6.1 cfu/g log 10) than in cow milk yoghurt (almost 5.0 cfu/g log 10). Cow milk yoghurts remained in better consistency after LPa 12/1 strain supplementation in comparison with ewe-goat milk yoghurts, although specific organoleptic tests were not performed. However, LPa 12/1 has survived in both types of yoghurts in sufficient amounts. Thermo-stable bacteriocin-producing strain LPa 12/1 applied in yoghurts in encapsulated form seems to be a suitable to supplement dairy products..

In Keywords, two words were deleted and replaced by the words postbiotic, enrichment

3. L46-47: I agree, Iam apologizing for dissmissing and now I hope it is more clear: Using next-generation sequencing method, useful bacteria of the phylum Firmicutes have been occurred in tested Slovak raw goat milks with abundance 20.5 % [5]. The phylum Actinobacteria reached abundance 62.8 % [5]. Although Firmicutes was not dominating phylum in tested raw goat milks, genera involved in the phylum Firmicutes were detected there. Moreover, using the standard microbiological method, individual representatives of the detected genera were isolated from raw goat milks [7].

5.L59-65: Here it is indicated how strain was identified: Its taxonomical species allocation was performed based on MALDI-TOF identification score evaluation (2.004) as reported previously [7].  

L86: Maybe it was not understood clearly. Strain was applied for 30 days, but experiment lasted 45 days meaning 15 days to the end of the experiment it was not applied.

6. L115-117: According to the product label, they contain commercial yoghurt culture; energy 309 KJ/74 kcal, fat 4.6 g of which saturated fatty acids participated with 3.6 g, carbohydrates were involved in 4.1 g, sugar value of which was 3.4 g; proteins content formed 4.1 g, and salt 0.07 g.

It was used in style for manuscript.

8. L139: It was copied from label. And the content was declared for 100 g as indicated.

 Components content involving commercial culture was declared for 100 g of product: energy 500 KJ, fat 9.0 g of which saturated fatty acids formed 5.2 g.Carbohydrates content was 4.5 g,sugar value of which was 4.1 g. Proteins content formed 3.4 g, and salt 0.1 g.

9. Rationale for amylolytic streptococci was: among commercial culture was dairy Streptococcus species strain, dairy species of streptococci are amylolytic. It was the reason and dairy streptococi amount was indicated as amylolytic.
10. The source of strain was indicated in original manuscript.

The principal indicator-Enterococcus avium EA5 (the most sensitive strain) was used in the test (our strain isolated from feces of piglet)

11. It was improved, hoping now it is more clear.
12. Against Listeria because they are frequent contaminants of raw milks.
13. I think now it is more clear. Normally when you use amount/dose 10⁹ cfu/ml, in animal organism/tract count 10⁴ is ok, it is colonization because there could be different interactions with other microbiota. Counts of lactobacilli/LAB could be like this.
14. L182: LPa 12/1 strain contributed in the total LAB increase on day 30 compared with day 0/1 (difference 0.59 log cycle) and on day 45 (^abp<0.001). Fecal amylolytic streptococci were present in high counts (Table 1). Coliforms count was reduced significantly in the experimental group on day 30 (30EG) comparing with the experimental group on day 45- 45EG (^abp<0.05).
    15. I dont understand how you mean it.
    16. Everything was there, but I controlled.
    17. It was controlled. When in microbiota counting is not significant difference, to use log difference is normal/common and used because in microbiology also difference in log mathemathical difference is relevant.
    18. When in microbiology is not significant difference, but there is difference in log cycle it is mathematical difference, there is really difference when among samples are differences up to 1.0 or more log cycle, there is reduction.
    19. It was involved. The species Lacticaseibacillus paracasei is bacterium that operates by commensalism [15]. Commensal lactobacilli are good candidates for development as probiotics [15]. Some of these species posses beneficial/probiotic properties among which also a bacteriocin-production can be involved [16]. The species Lacticaseibacillus paracasei is the most frequently isolated species from dairy environment among the other lacticaseibacilli.
    20. Some bacteria can influence proteolysis of casein by different way and so influence processing of product/yoghurt.. In case of LPa 12/1 strain its optimal technological properties such as sufficient clotting capacity in milk were previously described .

15.  Rev.2, it will be in bleu. Title: in this case, Iam not sure what you mean. Iam sorry.

    Thermo-stable bacteriocin-producing strain LPa 12/1 applied in yoghurts in encapsulated form seems to be a suitable to supplement dairy products.

    When those species strains produces bacteriocins (antimicrobial proteinaceous substances) and also posses additional beneficial properties such as bile and low pH tolerance, sufficient adhesive ability, lactic acid production, diacetyl, hydrogen peroxide or useful enzymes production, etc., they can play important role in food fermentation process, in biopreservation and/or in prevention of contamination............The animals were acclimatized for 15 days after the stress of transport, and when they did not show signs of illness they were used in the experiment. Experiments on laboratory mice were carried out in the accredited vivarium at the Institute of Parasitology SAS. The animals were handled in accordance with the "Requirements for the handling of animals" specified in the Slovak Law no. 377/2012 on veterinary care.The animal study protocol was conducted in accordance with current European and Slovak national legislative requirements for animal use, optimality of animal use and cruelty of the procedures. The project was approved by the Animal Care Ethics Committee at the Institute of Parasitology SAS (No. EK-PaU-2/2019, 2 April 2019) and the State Veterinary and Food Administration of the Slovak Republic (No. Ro-1604/19-221/3, 13 June 2019). Before LPa12/1 strain application, samples of yoghurts were diluted in peptone water and controlled for possible contamination by spreading dilutions on Mac Conkey agar (Difco) to control Enterobacteriae. Yoghurts were absent of Enterobacteriacae. LAB were counted on MRS agar (Merck) and control was provided also on M17 agar (Difco) because of commercial culture -Streptococcus spp. (both counts reached 5.1 cfu/g). Then sampling of yoghurts was performed after 24 h, on day 7, on day 10 and on day 14 (which is also declared expiration time for this type of yoghurt). The cells of applied LPa 12/1 strain was selected on MRS agar enriched with rifampicin (100 µg/ml), LAB were ..... etc. see in manuscript

Reviewer 4 Report

Comments and Suggestions for Authors

In this manuscript, the authors test the safety of Lacticaseibacillus paracasei LPa 12/1 through bacterial counts in different parts of mice and phagocytic activity in blood of mice, I think these results are not enough to verify the safety of this strain. The authors should afford more evidences. Meanwhile, the bacterial counts are meaningless, the authors need to afford at least the 16S rRNA sequencing results of the microbiota. Table 5 and Table 6, 16S rRNA sequencing results are needed. The most important, there are no information about the molecular mass, protein or DNA sequence, and the possible structure of the bacteriocin that might be produced by Lacticaseibacillus paracasei LPa 12/1 as the author mentioned. The identification of the bacteriocin is necessary. Whether the bacteriocin is the exact antimicrobial agent of Lacticaseibacillus paracasei LPa? Table 5 and 6, there is no sample repeat and SD analysis. Besides, the English writing is poor, the authors should prepare the manuscript carefully. In summary, the results in this manuscript are too preliminary to be published.

Comments on the Quality of English Language

The English writing is poor, the authors should prepare the manuscript carefully.

Author Response

Thanks so much for your effort to review manuscript. English was improved. Manucript was improved according to 4 reviewers requests or comments were noted and explained. I tried to involve your kind comments in the manuscript;

According to our meaning, it is not requested and also financially non possible to accompagne every research with sequencing. Moreover, when strain LPa 12/1 was confirmed by validated (but not sequencing) method which works well and sufficiently. The manuscript was not focused on purification of substance from LPa 12/1 strain. So this substance is not detaily described here, it could be for the individual manuscript. Regarding the safety using animal model is requested also by EFSA, so for this level safety confirmation is clear.

Becuase not detail asking were done, I decided to involve here also answers/responses to others reviewers., if space will be enough:

Thanks a lot for your reviewing. If the other improvement than asked from Rev.1, it will be in bleu.

Title: in this case, Iam not sure what you mean. Iam sorry.

Thermo-stable bacteriocin-producing strain LPa 12/1 applied in yoghurts in encapsulated form seems to be a suitable to supplement dairy products.

When those species strains produces bacteriocins (antimicrobial proteinaceous substances) and also posses additional beneficial properties such as bile and low pH tolerance, sufficient adhesive ability, lactic acid production, diacetyl, hydrogen peroxide or useful enzymes production, etc., they can play important role in food fermentation process, in biopreservation and/or in prevention of contamination............

The animals were acclimatized for 15 days after the stress of transport, and when they did not show signs of illness they were used in the experiment. Experiments on laboratory mice were carried out in the accredited vivarium at the Institute of Parasitology SAS. The animals were handled in accordance with the "Requirements for the handling of animals" specified in the Slovak Law no. 377/2012 on veterinary care.

The animal study protocol was conducted in accordance with current European and Slovak national legislative requirements for animal use, optimality of animal use and cruelty of the procedures. The project was approved by the Animal Care Ethics Committee at the Institute of Parasitology SAS (No. EK-PaU-2/2019, 2 April 2019) and the State Veterinary and Food Administration of the Slovak Republic (No. Ro-1604/19-221/3, 13 June 2019).

Before LPa12/1 strain application, samples of yoghurts were diluted in peptone water and controlled for possible contamination by spreading dilutions on Mac Conkey agar (Difco) to control Enterobacteriae. Yoghurts were absent of Enterobacteriacae. LAB were counted on MRS agar (Merck) and control was provided also on M17 agar (Difco) because of commercial culture -Streptococcus spp. (both counts reached 5.1 cfu/g). Then sampling of yoghurts was performed after 24 h, on day 7, on day 10 and on day 14 (which is also declared expiration time for this type of yoghurt). The cells of applied LPa 12/1 strain was selected on MRS agar enriched with rifampicin (100 µg/ml), LAB were determined on MRS agar. Amylolytic streptococci were checked using M17 agar (Difco). Yoghurts (one g) were sampled and mixed (Stomacher-Masticator, IUL, Spain) with peptone water (Merck, ratio 1:9). Then diluted and spread on the selected cultivation media (ISO) as formerly indicated. Moreover, pH was measured using Checker-pH tester (Hanna instruments Inc., Woonsocket, USA). Initial pH was 3.94. Yoghurts were maintained in the fridge during whole testing period

The enzymes such as α-chymotrypsin, trypsin, proteinase and pepsin (Merck) were used to treat precipitate. Then again inhibitory activity was checked against EA5 strain. Moreover also Listeria monocytogenes LM 7223 (Veterinary Institute in Olomouc, Czech Republic) was used as indicator (as frequent contaminant of milk).

Conclusion was controlled. legends and also what was indicated as appropriate.

Added references:  E.g. Antimicrobial, anti-biofilm, and cytotoxic effect of bacteriocin -lactococcin produced by dairy origin strain Lactococcus lactis CH3 reported Krishnamoorthi et al. [18]. Also Biadala et al. [19] informed about antibacterial activity of selected LAB for bioconversion of milk and whey from goat milk. Enrichment of products by beneficial strains can be influenced. Also Biadala and 2 others.

In Keywords,  two words were deleted and replaced by the words postbiotic, enrichment

4. L46-47: I agree, Iam apologizing for dissmissing and now I hope it is more clear: Using next-generation sequencing method, useful bacteria of the phylum Firmicutes have been occurred in tested Slovak raw goat milks with abundance 20.5 % [5]. The phylum Actinobacteria reached abundance 62.8 % [5]. Although Firmicutes was not dominating phylum in tested raw goat milks, genera involved in the phylum Firmicutes were detected there. Moreover, using the standard microbiological method, individual representatives of the detected genera were isolated from raw goat milks [7].

5.L59-65: Here it is indicated how strain was identified: Its taxonomical species allocation was performed based on MALDI-TOF identification score evaluation (2.004) as reported previously [7].   

6. L86: Maybe it was not understood clearly. Strain was applied for 30 days, but experiment lasted 45 days meaning 15 days to the end of the experiment it was not applied.
7. L115-117: According to the product label, they contain commercial yoghurt culture; energy 309 KJ/74 kcal, fat 4.6 g of which saturated fatty acids participated with 3.6 g, carbohydrates were involved in 4.1 g, sugar value of which was 3.4 g; proteins content formed 4.1 g, and salt 0.07 g.

It was used in style for manuscript.

8. L139: It was copied from label. And the content was declared for 100 g as indicated.

 Components content involving commercial culture was declared for 100 g of product: energy 500 KJ, fat 9.0 g of which saturated fatty acids formed 5.2 g.Carbohydrates content was 4.5 g,sugar value of which was 4.1 g. Proteins content formed 3.4 g, and salt 0.1 g.

9. Rationale for amylolytic streptococci was: among commercial culture was dairy Streptococcus species strain, dairy species of streptococci are amylolytic. It was the reason and dairy streptococi amount was indicated as amylolytic.
10. The source of strain was indicated in original manuscript.

The principal indicator-Enterococcus avium EA5 (the most sensitive strain) was used in the test (our strain isolated from feces of piglet)

11. It was improved, hoping now it is more clear.
12. Against Listeria because they are frequent contaminants of raw milks.
13. I think now it is more clear. Normally when you use amount/dose 10⁹ cfu/ml, in animal organism/tract count 10⁴ is ok, it is colonization because there could be different interactions with other microbiota. Counts of lactobacilli/LAB could be like this.
14. L182: LPa 12/1 strain contributed in the total LAB increase on day 30 compared with day 0/1 (difference 0.59 log cycle) and on day 45 (^abp<0.001). Fecal amylolytic streptococci were present in high counts (Table 1). Coliforms count was reduced significantly in the experimental group on day 30 (30EG) comparing with the experimental group on day 45- 45EG (^abp<0.05).
15. It was controlled. When in microbiota counting is not significant difference, to use log difference is normal/common and used because in microbiology also difference in log mathemathical difference is relevant.
16. When in microbiology is not significant difference, but there is difference in log cycle it is mathematical difference, there is really difference when among samples are differences up to 1.0 or more log cycle, there is reduction.
17. It was involved. The species Lacticaseibacillus paracasei is bacterium that operates by commensalism [15]. Commensal lactobacilli are good candidates for development as probiotics [15]. Some of these species posses beneficial/probiotic properties among which also a bacteriocin-production can be involved [16]. The species Lacticaseibacillus paracasei is the most frequently isolated species from dairy environment among the other lacticaseibacilli.
18. Some bacteria can influence proteolysis of casein by different way and so influence processing of product/yoghurt.. In case of LPa 12/1 strain its optimal technological properties such as sufficient clotting capacity in milk were previously described .

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised draft has not been improvised to a satisfactory level. Language of the article is difficult to follow (Such as in L15, 18, 20-21, 36-37).

 

 

1.     L30: There is no evidence stated regarding thermostability of bacteriocin in abstract

 

2.     The rationale for particularly estimating amylolytic streptococci has still not added in the paper

 

3.     It was asked that authors should state how the organism can enter in liver. Consider S. Typhi model, authors can speculate if the organism adopts the same route inside the body

 

4.     L260-261: Which number is correct?

 

5.     Discussion section states a lot about bacteriocin production; however, authors need to provide convincing proofs to confirm the bacteriocin nature of the inhibitory substance. Otherwise, it is written as ‘bacteriocin-like inhibitory substance (BLIS)’.

 

6.     In conclusion, write about limitation of safety analysis.

 

7.     L66: states about the identification through MALDI, however, results or discussion sections do not provide any information about it.

 

8.     Likewise, NGS method has been referred in introduction but has not been used in this study.

Comments on the Quality of English Language

Article needs extensive rephrasing to avoid verb confusion, redundant words and to bring meanings to many sentences.

Author Response

Rev. 1 Thanks again that you showed effort for improving our manuscript.

1. L30: There is no evidence stated regarding thermo-stability of bacteriocin in abstract.

 Thermo-stable bacteriocin-like producing strain LPa 12/1 applied in yoghurts in encapsulated form seems to be a suitable to supplement dairy products.

This was written in the results part, I think it is evidence:  Regarding the thermo-stability test, precipitate remained active after 2 weeks storage at -20 ˚C reaching inhibitory activity 100 AU/ml testing against EA5 strain as well as after treatment at 60 ˚C for 1 h (against EA5 strain- 100 AU/ml).

The sentence L15: it was changed, I think it is more correct now:

Based on safety testing results of LPa 12/1 strain using Balb/c mice, bacteriocin-like producing strain Lacticaseibacillus paracasei LPa 12/1 resulted  safe.

L18: LPa 12/1 strain in encapsulated form seems to be a suitable form for its potential application in dairy products.

L20-21: However, sufficient amounts of LPa 12/1 strain was counted in both types of yoghurts. The pH of yoghurts was not negatively influenced.

Among individual species representatives in raw goat milk are involved also lacticaseibacilli.

This study has been focused on its potential to be a new additive in dairy products.

36-37: Goats milk is attractive for consumers  because of its content components.

2. Commercial culture contained amylolytic Streptococcus, dairy streptococci are amylolytic. It was indication to check amylolytic streptococci.

I dont know why is problem with amylolytic streptococci there are in dairy products and obligatory streptococci in animals healthy tract are amylolytic, so therefore in context with dairy products and checking was specified as amylolytic.

4. The gut microbiota and liver have a bidirectional relationship. Based on it, bacterial products and metabolites from intestinal microbiota can pass through the intestinal barrier and reach the liver through the portal circulatory system. In case of „bad“ microbiota they can contribute to liver diseases through several mechanism. On the other side it is explanation also for beneficial bacteria to be occurred in liver with beneficial influence.
5. L260-261. The count of LPa 12/1 strain in freeze dried form reached 6.6 x 10⁷ cfu/g respectively 7. 82 cfu/g (log 10).

Answer: Both are correct, one number is in order form and the second one as log 10. So both are the same and correct. It is microbiological expression.

It is better expressed, I think now. But bacteriocin will be -it is in continuing study processing.

6. Answer:Although LPa 12/1 strain safety testing was provided using Balb/c mice model, bacteriocin-producing strain Lacticaseibacillus paracasei LPa 12/1 did not cause mortality in Balb/c mice.

Point 7.  L66: states about the identification through MALDI, however, results or discussion sections do not provide any information about it.

Answer: Some of reviewers wished to have information about LPa 12/1 strain identification. So, there is clearly inticated  it was done with appropriate reference from our previous  published study. It is not result from this manuscript, it is clearly indicated.

Point 8: As previously reported [5], using next-generation sequencing method, useful bacteria of the phylum Firmicutes have been occurred in tested Slovak raw goat milks with abundance 20.5 % [5]. The phylum Actinobacteria reached abundance 62.8 % [5]. Although Firmicutes was not dominating phylum in tested raw goat milks, genera involved in the phylum Firmicutes were detected there. Moreover, using the standard microbiological method, individual representatives of the detected genera were isolated from raw goat milks [7].

It is the same, it was previously done in our study not in this one, reference was involved there.

 

Reviewer 4 Report

Comments and Suggestions for Authors

I do not see the point to point responses for me.

Comments on the Quality of English Language

The quality of English needs to be improved.

Author Response

General: it is from previous answers:According to our meaning, it is not requested and also financially non possible to accompagne every research with sequencing. Moreover, when strain LPa 12/1 was confirmed by validated (but not sequencing) method which works well and sufficiently. The manuscript was not focused on purification of substance from LPa 12/1 strain. So this substance is not detaily described here, it could be for the individual manuscript. Regarding the safety using animal model is requested also by EFSA, so for this level safety confirmation is clear.

As you wish to answer/response point by point I would like to make points from your general summary as I received it.

New responses: I am sorry,  you did not find previous answers. Iam involving them also now. And also new: In spite of the fact that I did not find pointly done comments, I would like to answer you pointly.

Thanks for your effort:

Answer: Safety of LPa 12/1 was tested in mice-but it was not main point in this manuscript, main point was LPa 12/1 strain and its potential and one evidence was also safety. Here nothing more was involved,  LPa 12/1 is susceptible to antibiotics, non hemolytic etc. so more evidences here are not focused on involving. Regarding the safety using animal model is requested also by EFSA, so for this level safety confirmation is clear.

Answer regarding sequencing:  To our meaning, it is not requested and also financially non possible to accompagne every experimental (small experiments) research with sequencing. Moreover, when strain LPa 12/1 was confirmed by validated  method but not sequencing) -meaning by rifampicin labeled strain clearly differs its count from others LAB as described in the manuscript) , that one method works well and sufficiently confirms labeled strain presence.

Answer: The manuscript was not focused on purification of substance from LPa 12/1 strain. So this substance is not detaily described here, it could be for the individual manuscript.

Yes, bacteriocin is exact agent of LPa 12/1 strain because supernatant was exponed for 10 min at 80 ˚C and treated with EDTA as indicated in the manuscript to  eliminate antimicrobial effect of  other organic substrances.

Table 5 and 6 were improved -green and also SD analysis are involved already in revision 1 it was involved and it was in double repetition.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

In title, write ‘bacteriocin like inhibitory substance’. Remove ‘strain’

 

Write ‘bacteriocin like inhibitory substance’ throughout the manuscript. It can be abbreviated as BLIS.

 

L30: Revise to state about the data of thermostability in abstract.

 

Whatever reason authors providing in the response letter for investigating amylolytic organisms, they need to provide this rationale in the manuscript.

 

As suggested earlier on original draft and R1 drat, write in discussion about the possible entry of the strains to liver.

Comments on the Quality of English Language

There are numerous language errors that authors have not corrected even in this third draft.

Author Response

Here I responsed  questions and notes again. English was checked. I think I hope if everything now  will be  OK, it will be better after final agreement to submit  this version to check English by your system. So first to submit revised version and if it will be necessary then I will submit it finally for English checking using your system. Thanks for kindness and understanding.

Reviewer 1

1.Thanks again for your effort and kidness to improve manuscript. According to your advice, in title the word strain was deleted. I also added in title ...bacteriocin-like inhibitory substance producing LP17L/1… ….. . I hope it is understood well because  the mns is not totaly about substance, it is about beneficial strain which can produce bacteriocin-inhibitory substance (it is benefit of it) and it is about this strain potential. When I will use in title as you adviced .... The bacteriocin-like inhibitory substance Lacticaseibacillus paracasei LPa 12/1 from raw goat milk, a potential additive in dairy products…it is not correct because the strain has potential to be additive in dairy products, here not its bacteriocin-like inhibitory substance has potential as additive. I am apologizing but it could totally change the sense. Therefore, I used The bacteriocin-like inhibitory substance producing Lacticaseibacillus paracasei LPa 12/1 from raw goat milk, a potential additive in dairy products. Now it can be with the word …producing…

2.Write bacteriocin-like inhibitory substance throughout the mns. It was done.

3.L30:Revise to state about the data of thermostability in abstract.

R:  This bacteriocin-like inhibitory substance producing strain LPa 12/1 in encapsulated form applied in yoghurts seems to be a suitable to supplement dairy products.

 4.Whatever reason authors providing in the response letter for investigating amylolytic organisms, they need to provide this rationale in the manuscript.

R:Iam sorry but it was also involved in previous revision (R2) See

2.3. Surviving and stability of encapsulated strain LPa 12/1 in ewe-goat milk yoghurt

LAB were determined on MRS agar. Amylolytic streptococci were checked using M17 agar (Difco). Commercial culture contained amylolytic Streptococcus spp., dairy streptococci are amylolytic. It was indication to check amylolytic streptococci.  There it was in lines 140-142.

  As suggested in discussion..explanation in liver......

R:To find LPa 12/1 and/or microbiota in liver could be explained due the fact that the gut microbiota and liver have a bidirectional relationship. Based on it, bacterial products and metabolites from microbiota can pass through the intestinal barrier and reach liver through the portal circulatory system. So in case of beneficial bacteria they  can provide  beneficial influence in liver.

Reviewer 4 Report

Comments and Suggestions for Authors

1.      Why the author conclude that the antimicrobial agent produced by LPa12/1 is a kind of bacteriocin or a bacteriocin-like agent? As the author described that the precipitate treatment with α-chymotrypsin did not affect the antimicrobial activity. The authors did not describe how the antimicrobial activity is when treated with trypsin, proteinase and pepsin as you have mentioned in Line 172. If the antimicrobial agent retained its activity after proteinase treatment, it is possibly not a bacteriocin or a bacteriocin-like agent.

2.      Line 328. Please afford the picture of the inhibition zone or data to prove your description about the inhibitory activity and stability of the antimicrobial agent that you mentioned as bacteriocin, although I do not think there is enough evidence to prove that it is a kind of bacteriocin or bacteriocin-like agent.

3.      Line 345-347. There is no evidence showing that bacteriocin-like substance produced by L. paracasei LPa 12/1 strain showed narrow antimicrobial spectrum. Which indicator bacteria have you checked?

Comments on the Quality of English Language

The English writing is still needed to be edited and corrected carefully.

Author Response

Here I responsed  questions and notes again. English was checked. I think I hope if everything now  will be  OK, it will be better after agreement to submit  this version to check English by your system. So first to submit revised version and if it will be necessary then I will submit it finally for English checking using your system. Thanks for kindness and understanding.

Reviewer 4

Thanks again for your comments.

1. Iam apolozing becuase of confusion regarding proteolytic test treatment of BLIS. Inhibitory activity of BLIS was lost (inactivated) after precipitate treatment with, trypsin, proteinase K, pepsin and α-chymotrypsin (tested against the indicator E. avium EA5) which confirms its proteinaceous nature.
2. I also involved picture. You are not familiar to believe that our substance is bacteriocin-like. As I mentioned earlier that are preliminary or only starting results regarding bacteriocin study with this strain. What it is important, it is its beneficial effect in yoghurts as it is safe strain. So, if you dont want to have bacteriocin-like inhibitory effect here what do you recommend , to delete in title bacteriocin-like inhibitory substance  because Rev. 1 recommended to delete  the word from title  and replace by the use....bacteriocin-like inhibitory substance-producing LPa12/1  or? I continue in testing and I think now i tis better evidence for activity in which we  are continuing.

You mean to use only LPa12/1 strain from raw goat milk, potential addivie for dairy products? or......

I add some information (inhibitory activity) in the text but I would like also to underline that study regarding bacteriocin is processing it was as a first information to have this species strain with this kind effect in e.g. yoghurts.

See my explanation to him/her:

According to your advice, in title the word strain was deleted. I also added in title ...bacteriocin-like inhibitory substance producing LP17L/1… ….. . I hope it is understood well because  the mns is not totaly about substance, it is about beneficial strain which can produce bacteriocin-inhibitory substance (it is benefit of it) and it is about this strain potential. When I will use in title as you adviced ... The bacteriocin-like inhibitory substance Lacticaseibacillus paracasei LPa 12/1 from raw goat milk, a potential additive in dairy products…it is not correct because the strain has potential to be additive in dairy products, here not its bacteriocin-like inhibitory substance has potential as additive. I am apologizing but it could totally change the sense. Therefore, I used…. The bacteriocin-like inhibitory substance producing Lacticaseibacillus paracasei LPa 12/1 from raw goat milk, a potential additive in dairy products. Now it can be with the word …producing…

Also picture was involved.

3. I involved indicator strains but I would like to say that we continuing in testing.

In addition, the following indicator bacteria were used: 10 vancomycin resistant strains of Enterococcus faecium (VRE2, VRE3, VRE5, VRE 9, VRE 10, VRE 11, VRE 12, VRE 13, VRE15, VRE16, kindly provided by Dr. Bírošová from Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Slovakia), 11 fecal strains of E. faecalis from dogs (EE211- EE221) and 5 fecal strains of Staphylococcus chromogenes from cows (kindly provided from Dr. Trosciancyzk Aleksandra, University of Life Sciences in Lublin, Faculty of Veterinary Medicine, Poland). Moreover, Listeria monocytogenes LM 7223 (Veterinary Institute in Olomouc, Czech Republic) was used as indicator (as frequent contaminant in milk). Altogether, 28 indicator bacteria were tested.

Using the qualitative method, inhibitory zone size due to bacteriocin-like inhibitory substance (BLIS) produced by LPa 12/1 strain measured 30 mm in average. Inhibitory activity of BLIS (precipitate) LPa 12/1 was indicated up to now against 13 out of 28 indicator strains reaching inhibitory activity 100 AU/ml. The growth of EA5 indicator was inhibited with inhibitory activity 100 AU/ml. Vancomycin-resistant E. faecium strains of food-origin- VRE9, VRE3, VRE 2, VRE10 and VRE12 were inhibited with activity 100 AU/ml. Also E. faecalis strains EE211, EE215, EE220 and EE221 were inhibited using BLIS LPa 12/1 (100 AU/ml). Three out of five S. chromogenes strains (Sch141, Sch143 and Sch 147) were inhibited (100AU/ml). Inhibitory activity of BLIS was lost (inactivated) after precipitate treatment with, trypsin, proteinase K, pepsin and α-chymotrypsin (tested against the indicator E. avium EA5) which confirms its proteinaceous nature.