Effects of Microbial Inoculants on the Fermentation and Preservation of Triticale Silages at High and Low Moisture Levels

Round 1

Reviewer 1 Report

In the presented research authors had tested two novel inoculants of Lactobacillus strains (KCC-51 and KCC-52) on a single triticale cultivar Joseong. Small grain silage made is lower in quality than corn or sorghum silage, but if cut at the right stage and properly ensiled, triticale silage quality may also be pretty good. Furthermore, many changes occur once green forage with different moist is placed in a silo. I would like to see (maybe in the future research) how would you produce stable feed with rich nutritional value and energy, on more than one triticale cultivar as there are a lot of agronomic diversity among triticale cultivars. Anyway, I think that these results are worth of publishing in such a form.

 

Author Response

Reviewer comments 1

 

We thank the reviewers for their critical and judicious evaluation of our manuscript and providing constructive suggestions for improving the quality and presentation of the manuscript. We have carefully considered the comments of the reviewers and revised the manuscript thoroughly considering all the points. Pointwise response to the reviewer's comments is given below

 

In the presented research authors had tested two novel inoculants of Lactobacillus strains (KCC-51 and KCC-52) on a single triticale cultivar Joseong. Small grain silage made is lower in quality than corn or sorghum silage, but if cut at the right stage and properly ensiled; triticale silage quality may also be pretty good. Furthermore, many changes occur once green forage with different moist is placed in a silo. I would like to see (maybe in the future research) how would you produce stable feed with rich nutritional value and energy, on more than one triticale cultivar as there are a lot of agronomic diversity among triticale cultivars. Anyway, I think that these results are worth of publishing in such a form.

Thank you for your positive comments on our research article, also we thank your valuable suggestion on production of stable feed with rich nutritional value and energy using different cultivar of triticale. Definitely, we strongly agreed with reviewer comments and will do the same in future research work. It is laboratory level in-vitro experiment and we have planned to conduct field level experiment for making triticale silage with different cultivar at different stages with lactic acid bacteria.

Author Response File: Author Response.docx

Reviewer 2 Report

The work is interesting and can be published after appropriate changes, if they were done correctly. I have marked below many comments, which need to be addressed before potential resubmission.

As a general remark, the manuscript is badly written and needs extensive language editing by an expert.

Abstract. 
Data are missing from the abstract, there are only generalistic statements therein. The authors must rewrite the abstract by adding data from their results therein.

M&M.
The coordinates of the lands from which samples were collected, must be provided.
How many biological samples were collected and how many technical samples were tested from each biological sample? Which was the total number of samples examined? These must provided in detail.
The units are joined to the figures - this is wrong.
Please maintain uniformity: 8 but six - this is wrong.
Determination is the wrong word (see the general comment)
Analysis between results of repeated measurements (technical samples) must be carried out and the findings must be reported.

Results
Tables 1-3 must be moved to supplementary material.
Figures: use thinner lines in graphs.

Discussion
The nutritional effects for various species of farmed animals must be discussed; this should be included in a separate subsection.

General comments
If the authors have work with feeding animals with this ensilaged feed, it must be included in this manuscript. If they do not have such work, they must: a) write an undertaking that no such work exists and that they commit to not publish it in a future paper for the next 2-3 years and b) explain why they did not carry out work with feeding animals with ensilaged feed.

 

Author Response

Reviewer comments -2

 

We thank the reviewers for their critical and judicious evaluation of our manuscript and providing constructive suggestions for improving the quality and presentation of the manuscript. We have carefully considered the comments of the reviewers and revised the manuscript thoroughly considering all the points. Pointwise response to the reviewer's comments is given below.

As a general remark, the manuscript is badly written and needs extensive language editing by an expert.

 

Sorry for this inconvenience caused, we have carefully checked and edited the whole manuscript accordingly, and language of the manuscript was edited by renewed professor and native speaker.

 

Abstract. 
Data are missing from the abstract; there are only generality statements therein. The authors must rewrite the abstract by adding data from their results therein.

 

Thank you for your comment; we have included some essential data in the abstract according to reviewer suggestion. 

 

M&M
The coordinates of the lands from which samples were collected must be provided. How many biological samples were collected and how many technical samples were tested from each biological sample? Which was the total number of samples examined? These must provide in detail.

 

Thanks for your valuable comments

For bacteria isolation, we collected a triticale sample at different places of the same land and transferred to the laboratory and then numerous bacteria.

For silage productions; we harvested triticale at heading stage and transferred to the laboratory.

 We used five biological replicates for each group of silage productions; used technical replicates varied among experimental analysis. For example, nutrient and fermentative metabolites analysis, we used two technical replicates for each sample. But it differed for microbial counting, we used three technical replicated for microbial enumeration. Now all the details are given as footnote in figure and tables.

 

The units are joined to the figures - this is wrong.

 Yes, we agreed with the reviewer comment; we have modified all figures according to reviewer suggestion (Fig.1 and Fig.2)

 

Please maintain uniformity: 8 but six - this is wrong.

Yes, we made uniformity for all figures according to reviewer comments. 

 

Determination is the wrong word (see the general comment)

Thank you for your valuable suggestion, we have replaced determination by quantification (Line No 148).

 
Analysis between results of repeated measurements (technical samples) must be carried out and the findings must be reported.

We used five biological replicates for each group of silage productions; used technical replicates varied among experimental analysis. For example, nutrient and fermentative metabolites analysis, we used two technical replicates for each sample. But it differed for microbial counting, we used three technical replicated for microbial enumeration. Now all the details are given as footnote in figure and tables.

Results

Tables 1-3 must be moved to supplementary material, Figures: use thinner lines in graphs.
Thanks, asper reviewer suggestion we have provided these tables as supplementary tables and used thinner lines for figures.

Discussion

The nutritional effects for various species of farmed animals must be discussed; this should be included in a separate subsection.

Thank you for valuable comments; we have given the nutritional effects of triticale on farmed animals in introduction part instead of a discussion section for avoiding repetition. Triticale is a breed plant between wheat (Triticum) and rye (Secale). It has rich crude protein content and high resistance against many diseases and other environmental factors, (Natasa et al., 2018). Triticale has been used as a primary meal for the poultry, ruminants and non-ruminants due to its nutritional values [24]). Replacement of corn silage with triticale and wheat at 10% of the diet dry matter (DM) did not affect DM intake, but decreased milk yield and its components compared to corn silage. At the same time, milk fat from alternative forage diet had a higher level of 4:0, 6:0 and 18:0 and the lower concentration of total trans-fatty acids. Digestibility of neutral and acid detergent fiber was increased in triticale silage diet. Emission of methane was higher for triticale and CO₂ was reduced by both triticale and wheat silages, concluded that triticale or wheat double cropped with corn many be providing significant cropping strategy [25]. Triticale is one of the available options  for swath grazing to extend the fall grazing  and produce as silage or hay for beef cattle producers in Canada[26]. Ensiling of pre-mature cereal of barley, wheat, triticale, and rye with LAB could serve as alternative forages [27]. Triticale has an excellent impact on higher meat production as well as  gives good responses for dairy and sheep milk production in Algeria[28].

 

General comments
if the authors have work with feeding animals with this ensilaged feed, it must be included in this manuscript. If they do not have such work, they must: a) write an undertaking that no such work exists and that they commit to not publish it in a future paper for the next 2-3 years and b) explain why they did not carry out work with feeding animals with ensilaged feed.

Thank you for your valuable comments. We did not do an animal experiment and did only in-vitro laboratory level experiment. In future, we have planned to conduct a field-level investigation using same triticale at different stages and moistures with lactic acid bacteria, that time we will perform the animal experiment, particularly dairy and meat cattle’s. Few reports have been available on the improvement of nutritional values of triticale silages using LAB; recently many researchers have been focused on silage production using triticale plant and LAB due to its crude proteins and positive fermentative capabilities, respectively. In this aspect, recently, we are involving in LAB based triticale silages production for making standard feed for animals with enriched nutrients.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript titled "Effects of Microbial Inoculants on the Fermentation and Preservation of  Triticale Silages at High and Low Moisture levels" presents interesting six table and three figure. However, there are some problems and should be rewritten with major revision.

General comment:

1. Newly isolated two LAB (KCC-51 and 52) were applied throughout this research. However, basic taxonomic information (such as morphology, optimal temperature for growth, nutrition requirement, G+C content and similarities of 16S rRNA sequence compare with type strain) of newly isolated LABs were not indicated. Authors should indicate the several information for readers. 

2. Some of LAB inoculated research paper were published until now. However, advantageous point of strain KCC-51 and 52 as inoculant for high quality silage production were quite obscure. Authors should carefully discuss by using numeric data with related references.

Minor comment:

1. L118: "10gram" -> "10 gram"

2. Fig. 1: "S..aureus ..." -> "S. aureus..."

3. Fig. 1: "growth" -> "cell growth"

Author Response

Reviewer comment 3

We thank the reviewers for their critical and judicious evaluation of our manuscript and providing constructive suggestions for improving the quality and presentation of the manuscript. We have carefully considered the comments of the reviewers and revised the manuscript thoroughly considering all the points. Pointwise response to the reviewer's comments is given below

Newly isolated two LAB (KCC-51 and 52) were applied throughout this research. However, basic taxonomic information (such as morphology, optimal temperature for growth, nutrition requirement, G+C content and similarities of 16S rRNA sequence compare with type strain) of newly isolated LABs were not indicated. Authors should indicate the several information for readers. 

 

Yes we agreed with reviewer comments and thanks for valuable suggestions, asper reviewer suggestion we have provided details of morphology, optimal temperature for growth, nutrition requirement, G+C content and similarities of 16S rRNA sequence in supplementary table 1. Please see the supplementary table 1

 

Some of LAB inoculated research paper was published until now. However, advantageous point of strain KCC-51 and 52 as inoculant for high quality silage production were quite obscure. Authors should carefully discuss by using numeric data with related references.

 

Thanks for your valuable suggestion and we strongly agreed with reviewer comment on L. paracasei and L. rhamnosus as an inoculant for high-quality silage production. Reports have been available on the improvement of nutritional values of plant-based silages production using L. paracasei and L. rhamnosus; recently many researchers have been focused on silage production using triticale plant and LAB due to its crude proteins and positive fermentative capabilities, respectively. In this aspect, recently, we are doing research work on LAB based triticale silages production for making standard feed with enriched nutrients. This data suggested that addition of L. paracasei and L. rhamnosus these strains produced higher amounts of lactic acid (it’s essential for silage quality) in fermented triticale silage. It is an in-vitro laboratory level experiment and needs large scale production at field level and animal performance studies. We have planned to conduct a field-level investigation using same triticale at different stages and moistures with lactic acid bacteria in future.

 

 We have provided the available details of L. paracasei and L. rhamnosus in various field and plant-based silage productions with relevant literature. Please see the details. Dominant species involved in the fermentation process of silage are Lactobacillus, Pedioccous, and Lactococcus, the genus of lactobacillus such as L. plantarum, L.brevis, L.casei, L. rhamanosus, L. curvatus, L. gasseri, L. pentosus and the genus of pedococcus including P. pentosaceus, P. acidilactici, P. damnosus, P. confusa were noted in the silage samples [1,8,9]. L. casei, L. paracasei and L. rhamnosus are phylogenetically and phenotypically closely related [10,11]. These species can colonize a variety fermented food products representing dominant component of non-starter LAB in ripened cheeses and traditional fermented milk products[12-15] such as fermented vegetables[16], and kimchi[17]. Also, these strains have been used as starter culture in milk fermentation as adjunct cultures for the intensification and proven their probiotics features [18,19]. Furthermore, L. paracasei and L. rhamnosus could be considered as excellent additives for plant-based silage production with enriched nutrients for ruminant and non-ruminants [20-23].  

Minor comment:

L118: "10gram" -> "10 gram"2. Fig. 1: "S..aureus ..." -> "S. aureus..."3. Fig. 1: "growth" -> "cell growth

Thanks for your valuable suggestions, we have made the all the changes according to reviewer comments and made red color.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have made a good work and have revised correctly. Therefore, the resubmitted manuscript has been improved significantly and it shows better the merits of the study.

Most of the points raised in the previous evaluation have been answered correctly by the authors, who have a really good effort to answer the points.

The only point that was not covered was the following: "The coordinates of the lands from which samples were collected must be provided."

Therefore, in the new version of the manuscript you only need to provide the coordinates and also correct some spelling errors in English language.

I return an opinion for minor revision and the authors should respond to that. 

Author Response

We thank the reviewers for their critical and judicious evaluation of our manuscript and providing constructive suggestions for improving the quality and presentation of the manuscript. We have carefully considered the comments of the reviewers and revised the manuscript thoroughly considering all the points. Pointwise response to the reviewer's comments is given below

The authors have made a good work and have revised correctly. Therefore, the resubmitted manuscript has been improved significantly and it shows better the merits of the study.Most of the points raised in the previous evaluation have been answered correctly by the authors, who have a really good effort to answer the points.

Thank you very much for your positive comments and valuable suggestions on our research article

The only point that was not covered was the following: "The coordinates of the lands from which samples were collected must be provided. “Therefore, in the new version of the manuscript you only need to provide the coordinates and also correct some spelling errors in English language. I return an opinion for minor revision and the authors should respond to that. 

Thank you for your suggestion; we have included coordinates of the lands used for sample collection in method section (Line No: 95 and 122). Triticale sample was collected at different places of the same land from Jangsoo, South Korea (Latitude: 35.6185318 and longitude: 127.5107881) and carefully rechecked language of the paper. The changes were made with the red color font.

 For methods, we used previously published our protocol for most of the experiments. Thus, we included relevant literature in method section for avoiding repetition.

Reviewer 3 Report

Dear Authors

Revised manuscript was attained to acceptable level for “Applied science” by the revision from authors.

Author Response

Revised manuscript was attained to acceptable level for “Applied science” by the revision from authors.

Thank you very much for your positive comments and valuable suggestions on our research article