Assessment of Intestinal Immunity and Permeability of Broilers on Partial Replacement Diets of Two-Stage Fermented Soybean Meal by Bacillus velezensis and Lactobacillus brevis ATCC 367

Round 1

Reviewer 1 Report

This manuscript by Tsai and colleagues have evaluated the intestinal immunity and permeability of broilers on partial replacement diets of two-stage fermented soybean meal. It is an interesting topic. The experimental was well designed and data was well presented. While the following changes could improve the quality of the paper.

1. Title, please specified the bacteria were used to do the two-stage fermentation.
2. Lines 23-25, the writing of experimental design need to be improved. For example, the name of the bacterial could use the abbreviation due to it was used in the previous sentence.
3. Line 31, there is one more space before “Further”.
4. Line 32, correct“IL-6mRNAs”to“IL-6 mRNAs”.
5. Line 34, space needed “.FSBM B”. Please check throughout the manuscript.
6. Line 115, please explain why 6% FSBM has been used?
7. Lines 121-123, do not use the bold form for this sentence.
8. Line 228, correct “n=6” to “n = 6”. Please check other place with similar issue.
9. Line 239, correct “P < 0.05” to “P < 0.05”. Please check other place with similar issue.
10. It is better to do some Western blot based on the mRNA data. The protein changes will be more interesting.

Author Response

The authors appreciate the comments from the reviewers. The manuscript has been revised in accordance with their requests. We have tried our best to take all comments into account, incorporating them into the revised manuscript as indicated in our responses to the reviewer.

(Revisions related to reviewer’s comments are shown in blue in the revised manuscript)

 

Responses to Reviewer I 's comments:

 

This manuscript by Tsai and colleagues have evaluated the intestinal immunity and permeability of broilers on partial replacement diets of two-stage fermented soybean meal. It is an interesting topic. The experimental was well designed and data was well presented. While the following changes could improve the quality of the paper.

 

Q1.

Title, please specified the bacteria were used to do the two-stage fermentation.

Response: Many thanks for your further review and suggestion. We rewrite the article title as “Assessment of intestinal immunity and permeability of broilers on partial replacement diets of two-stage fermented soybean meal by Bacillus velezensis and Lactobacillus brevis ATCC 367”

 

Q2.

Lines 23-25, the writing of experimental design need to be improved. For example, the name of the bacterial could use the abbreviation due to it was used in the previous sentence.

Response: Thanks for responsion. We correct lines 22-25. Also, rewrite the simple summary and abstract.

 

Simple summary:

Fermented soybean meal (FSBM) by Bacillus velezensis (Bv) and Lactobacillus brevis ATCC 367 (Lb) reduces these negative factors and provides bioactive active peptides that are beneficial to intestinal repair and regulate the immune system of the intestinal tract.

 

Abstract:

Two-stage fermented soybean meal was prepared by Bv and Lb (FSBM_B+L), which has nearly 3 times higher soluble peptides than, and reduced galacto-oligosaccharide (GOS) content and allergen protein than soybean meal (SBM). The one-stage fermented by Bv (FSBM_B) and commercial lactic acid bacteria (FSBM_L), which had respectively highest soluble peptides and Lactic acid bacteria count were used to compare process differences. Ross308 broilers (n = 320) were divided into four groups: SBM, and practical replaced diet with 6% of FSBM_B+L, FSBM_B, and FSBM_L.

Q3&4.

Line 31, there is one more space before “Further”.

Line 32, correct “IL-6mRNAs”to “IL-6 mRNAs”.

Response: Thanks for notice and suggestion, we delete the extra space as “Further, the level of NF-κB and IL-6 mRNAs in FSBM_L increased, respectively, by 4 and 2.5 times.”

 

Q5.

Line 34, space needed “. FSBM B”. Please check throughout the manuscript.

Response: Thanks for suggestion. We found the less space at line 34 and rewrite as”. FSBM_B and FSBM_B+L could also upregulate MUC2 in the jejunum (P < 0.05).”

 

Q6.

Line 115, please explain why 6% FSBM has been used?

Response: Thanks for noticing. From near researches on broilers, the another try to partial replacing diets from 3% to 6% by FSBM. The result turns out that 6% show better immune response [4,5]. We thought the relationship between the FSBM content and immune response on broilers has fewer articles discuss so that we followed and extended the experiment from the previous study. Therefore, we have corrected and adding the paragraph as follows" In nearly study, a partial replacement diet from 3% to 6 % show better growth performance, immunity, and intestinal morphology [4,5]. Cheng's group using two-stage fermentation by reducing anti-nutrition factors with <6 kDa soy peptide content raised, which decrease the serum IgG and spleen IL-4 by partial replacement 10% broilers diets [18]. But, still need more discuss the relation from the FSBM content and the intestinal cell repair ability, which associate to immune response in broilers."

 

4. Sembratowicz, I.; Chachaj, R.; Krauze, M.; Ognik, K. The effect of diet with fermented soybean meal on blood metabolites and redox status of chickens. Ann. Anim. Sci. 2020, 20(2), 599-611.
5. Chachaj, R.; Sembratowicz, I.; Krauze, M.; Ognik, K. The effect of partial replacement of soybean meal with fermented soybean meal on chicken performance and immune status. J. Anim. Feed Sci. 2019, 28, 263-271.

 

Q7.

Lines 121-123, do not use the bold form for this sentence.

Response: Thanks for the suggestion, we amend the typeface and check ones again.

 

Q8.

Line 228, correct “n=6” to “n = 6”. Please check other place with similar issue.

Response: Thanks for notice and suggestion, we correct the similar pattern through whole study.

 

¹Each value represents the mean ± standard deviation (n = 3).

¹Each value represents the mean ± standard deviation (n = 3).

Each value represents the mean ± standard deviation (n = 4).

¹ Each value represents the mean ± standard deviation (n = 6).

¹Each value represents the mean ± standard deviation (n = 5).

Each value represents the mean ± standard deviation (n = 5).

Each value represents the mean ± standard deviation (n = 5).

Each value represents the mean ± standard deviation (n = 5).

 

Q9.

Line 239, correct “P < 0.05” to “P < 0.05”. Please check other place with similar issue.

Response: Thanks for notice and suggestion, we check the similar pattern through whole study and remove the wrong describe pertain on all of “P < 0.05” at chapter 3.3 as below:

 

3.3. Effect of Two-stage FSBM on Intestinal Morphology of 35-day-old Broilers

 

In the jejunum (figure 3a), we observed that SBM had a thin villus with a thickened, proliferated crypt compared to other groups. FSBM_B+L had a less proliferated crypt. In the ileum (figure 3b), four groups had similar villus apparent traits, but SBM had the same status as the jejunum on a proliferated crypt. According to the images we captured, the intestinal morphology results show in table 4. All three treatments significantly increased the villus height; only FSBM_B significantly decreased the crypt depth compared with other groups (P < 0.05). In the ileum, four groups had no significant difference in villus height and crypt depth. However, FSBM_B+L had a higher villus height/ crypt depth ratio (P > 0.05).

 

Q10.

It is better to do some Western blot based on the mRNA data. The protein changes will be more interesting.

Response: Thanks for the suggestion. We notice the Western bolt could better support our study. However, we could backward to the aim of the study. First, the NFκB pathway will affect the gut epithelial integrity which associates with permeability [7]. Second, the peptides may stimulate the immune response, including up-regulated cytokine expression, and Young [6] showed the strong connection between soy peptides and injury gut epithelial with mRNA cooperation with the ELISA results. If we could up-regulate the Treg while soothing the up-regulate of NFκB and other cytokines (IL-6, IL-1β), that will helpful for keep epithelial completeness. Still, very thanks for the advice, we will do better in-depth research efforts in this direction in the future.

 

6. Young, D.; Ibuki, M.; Nakamori, T.; Fan, M.; Mine, Y. Soy-derived di- and tripeptides alleviate colon and ileum inflammation in pigs with dextran sodium sulfate-induced colitis. A. S. N. 2012, 142(2), 363-368.
7. Lee, B.; Moon, K. M.; Kim, C.Y. Tight junction in the intestinal epithelium: Its association with diseases and regulation by phytochemicals. J. Immunol. Res. 2018, Article ID 2645465.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Figure 2a change “Grem (g)” as “Body weight (g)”

Figure 2b change “Grem (g) as “Body weigt gain (g)”

Figure 3 is not commented on

Line 240 “3.5. Effect of Two-stage FSBM on Relative mRNA Expression in Jejunum of 35d-old Broilers “ this chapter is the repetition of 3.3

 

Line 261 “Replacement of dietary SBM by FSBM improved the broiler’s BWG and FCR”  the discussion begins by commenting on insignificant data

Author Response

The authors appreciate the comments from the reviewers. The manuscript has been revised in accordance with their requests. We have tried our best to take all comments into account, incorporating them into the revised manuscript as indicated in our responses to the reviewer.

(Revisions related to reviewer’s comments are shown in blue in the revised manuscript)

 

Responses to Reviewer Ⅱ 's comments:

 

Dear authors

Thanks for your revised submission. There are following suggestion get improved:

 

Q1&2.

Figure 2a change “Grem (g)” as “Body weight (g)”

Figure 2b change “Grem (g) as “Body weigt gain (g)”

Response: Thanks for attention and suggestion. We change the y axis title so that more suitable for figures.

 

Q3.

Figure 3 is not commented on

Response: Very thanks for attention. We have the correct describe for figure 3 as follows “In the jejunum (figure 3a), we observed that SBM had a thin villus with a thickened, proliferated crypt compared to other groups. FSBM_B+L had a less proliferated crypt. In the ileum (figure 3b), four groups had similar villus apparent traits, but SBM had the same status as the jejunum on a proliferated crypt.”

 

Q4.

Line 240 “3.5. Effect of Two-stage FSBM on Relative mRNA Expression in Jejunum of 35d-old Broilers “ this chapter is the repetition of 3.3

Response: Very thanks for attention. We have the correct describe at chapter 3.3 as below:

 

3.3. Effect of Two-stage FSBM on Intestinal Morphology of 35-day-old Broilers

 

In the jejunum (figure 3a), we observed that SBM had a thin villus with a thickened, proliferated crypt compared to other groups. FSBM_B+L had a less proliferated crypt. In the ileum (figure 3b), four groups had similar villus apparent traits, but SBM had the same status as the jejunum on a proliferated crypt. According to the images we captured, the intestinal morphology results show in table 4. All three treatments significantly increased the villus height; only FSBM_B significantly decreased the crypt depth compared with other groups (P < 0.05). In the ileum, four groups had no significant difference in villus height and crypt depth. However, FSBM_B+L had a higher villus height/ crypt depth ratio (P > 0.05).

 

Q5.

Line 261 “Replacement of dietary SBM by FSBM improved the broiler’s BWG and FCR”  the discussion begins by commenting on insignificant data

Response: Thanks for the suggestion. Studies about partial replacement diet by FSBM before tend to focus on improved SBM digestibility to improve growth performance but ignore the possibility that fermented products could also improve the immune response, which also beneficial for broilers breeding. Our study inherited past views on fermented soybean meal by growth traits and discussed the impact of this fermented product on broiler chickens in the follow-up discussion. It is by no means only changing the growth traits. We rewrite the segment at discussion as below:

 

Past studies suggest that partial replacement of dietary SBM by FSBM improved the broiler's BWG and FCR due to increased protein digestibility [23]. Although fermentation with the soy peptide upgraded the content, only 3 to 6% partial replacement diet could reach a similar effect [5,24]. FSBM_B+L showed a better FCR through the entire feeding period compare to SBM, and we observed that the nutrient and peptides in FSBM_B+L were better absorbed and utilized by the chickens, increased the income over feed cost, which means FSBM_B+L has the potential to bring more economic value, at the same time make sure birds intake better protein recourse.

 

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

This paper presents a report on using a two-stage fermented soybean meal to be beneficial to digestive tract of broiler. This paper give no more important information, due to:

1 fermented soybean meal has been widely studied so far, and its beneficial effects on gut health has also been known. Thus, this ms may not contain new information.

2 In the methods, it is described that 50g soybean meal was fermented, how much fermented soybean meal were used in the broiler experiment? If more used, how were they prepared? If they had the same or similar fermentation properties?

3 In the introduction, peptides produced in fermentation is paid more attention. But this paper did not determine detail information of peptides, only provided data on TCA- soluble peptides. Thus, results can not illustrate function of fermented peptides.

4.Results in this paper is not meaningful enough, since growth performance did not differ each other. We need better performance and lower cost, not only better gut health.

Author Response

The authors appreciate the comments from the reviewers. The manuscript has been revised in accordance with their requests. We have tried our best to take all comments into account, incorporating them into the revised manuscript as indicated in our responses to the reviewer.

(Revisions related to reviewer’s comments are shown in blue in the revised manuscript)

 

Responses to Reviewer Ⅲ 's comments:

 

This paper presents a report on using a two-stage fermented soybean meal to be beneficial to digestive tract of broiler. This paper give no more important information, due to:

Q1.

Fermented soybean meal has been widely studied so far, and its beneficial effects on gut health has also been known. Thus, this ms may not contain new information.

Response: Thanks for the advice. FSBM mostly application on piglet diets as passing weaning period and the newer discover of FSBM were more tests in mice mode instead of broilers. The details about how FSBM improved broilers' performance and gut health still need more study to get improved. For instance, the latest study in broiler was published by Sembratowicz [4] and Cheng [18], which major focuses on other immune organ responses from the degradation of anti-nutrition factors. There have been many documents confirming that soy peptides can affect intestinal health. Still, the fermented condition needs to be used to produce soy peptides, and how much content needs replacement to chicken diets to promote chicken intestinal health effectively? Our study can provide more favorable evidence. We supplement the above arguments in the introduction and discussion, respectively.

 

Introduction:

In nearly study, a partial replacement diet from 3% to 6% show better growth performance, immunity, and intestinal morphology [4,5]. Cheng’s group using two-stage fermentation reducing anti-nutrition factors with < 6 kDa soy peptide content raised, which decrease the serum IgG and spleen IL-4 by partial replacement 10% broilers diets [18]. But, still need more discuss the relation from the FSBM content and the intestinal cell repair ability, which associate to immune response in broilers.

 

Discussion:

In Cheng’s groups [18], although the effect of gut immune response by partial replacement 10% FSBM cannot show clearly, still observed the downregulation of IL-4 and IL-10 in spleen and IgG levels in serum due to degradation of anti-nutrition factors, but didn’t description about the positive effect from their fermented soy peptide. FSBM_B plays the role of soybean meal made by Cheng, which also degrades anti-nutritional factors and increases peptide content. However, our experiments have shown that fermentation strains and fermentation conditions that strongly degrade allergic protein factors, fermentation products, and soybean peptides cannot be fully equipped to regulate the function of intestinal immunity.

 

4. Sembratowicz, I.; Chachaj, R.; Krauze, M.; Ognik, K. The effect of diet with fermented soybean meal on blood metabolites and redox status of chickens. Ann. Anim. Sci. 2020, 20(2), 599-611.
5. Cheng, Y.; Hsiao, F.S.; Wen, C.; Wu, C.; Dybus, A.; Yu, Y. Mixed fermentation of soybean meal by protease and probiotics and its effects on the growth performance and immune response in broilers. J. Appl. Anim. Res. 2019, 47(1), 339-348.

 

Q2.

In the methods, it is described that 50g soybean meal was fermented, how much fermented soybean meal were used in the broiler experiment? If more used, how were they prepared? If they had the same or similar fermentation properties?

Response: Thanks for the attention. In animal experiments, we fermented one time with four 5 kg packages in the same place using the same fermented condition to enrich the FSBM to 20 kg for addition. For future large-scale production, we used a space bag that provides 100 kg with the same traits corresponds to 50 g. We supplement the usage amount at the chapter 2.1 as follows “The TCA-soluble protein content was as per the method by Xie [21]. Then, 20 kg FBSM was processing for the animals’ trials after measuring the contents.”

 

Q3.

In the introduction, peptides produced in fermentation is paid more attention. But this paper did not determine detail information of peptides, only provided data on TCA- soluble peptides. Thus, results can not illustrate function of fermented peptides.

Response: Thanks for the suggestion. So far, to prove the function of peptides from FSBM, we have to separate the small molecular soluble peptides using in vitro tests like antioxidant activity (Handa et al. (2017)) or in vivo tests through molecular mechanisms. Cui [14] and Zhang [15] show the bioactivity of FSBM peptides in both in vitro and in vivo experiments in mice and piglets, respectively. As our study showed at figure 5, we used molecular mechanisms to support our investigation. Although down-regulates of cytokines were used to ascribe on anti-nutrition factors degradation, studies support that peptides from FSBM could affect the immune response under gut injury [6] like il-10, IL-1β, IL-6, etc. Also, Zhu’s group [40] using molecular mechanisms to support the FSBM in animal experiments, which also shows the TCA-soluble protein content without other in vitro bioactive-related results. But still, the reviewer gave a great comment that we could progress the in vitro experiment design in future studies to figure out how our two-stage fermented soy peptides improved the immune molecular mechanisms. Thus, we supplement the describe as below:

 

So far, the ability of soy peptides to repairer gut injury or balance could through in vitro study or stimulate with in vivo experiment [14,15]. But Zhu [40] gives a different way to prove the effect between FSBM soy peptides and animals’ immune response in the piglet. In our study, the FSBM_B+L and FSBM_B TCA-soluble proteins show a positive influence on immunity and tight junction, especially molecular mechanisms in the jejunum.

 

Handa, C.L; de Lima, F.S.; Guelfi, M.F.G.; Georgetti, S.R.; Ida, E.I. Multi-response optimisation of the extraction solvent system for phenolics and antioxidant activities from fermented soy flour using a simplex-centroid design. Food Chem. 2016, 197, 175-184.

 

6. Young, D.; Ibuki, M.; Nakamori, T.; Fan, M.; Mine, Y. Soy-derived di- and tripeptides alleviate colon and ileum inflammation in pigs with dextran sodium sulfate-induced colitis. A. S. N. 2012, 142(2), 363-368.
7. Cui, J.; Xia, P.; Zhang, L.; Hu, Y.; Xie, Q.; Xiang, H. A novel fermented soybean, inoculated with selected Bacillus, Lactobacillus and Hansenula strains, showed strong antioxidant and anti-fatigue potential activity. Food Chem. 2020, 333, 127527.
8. Zhang, Y.; Chen, S.; Zong, X.; Wang, C.; Shi, C.; Wang, F.; Wang, Y.; Lu, Z. Peptides derived from fermented soybean meal suppresses intestinal inflammation and enhances epithelial barrier function in piglets. Food Agr. Immunol. 2020, 31(1), 120-135.
9. Zhu, J.; Gao, M.; Zhang, R.; Sun, Z.; Wang, C.; Yang, F.; Huang, T.; Qu, S.; Zhao, L.; Li, Y.; Hao, Z. Effects of soybean meal fermented by L. plantarum, B. subtilis and S. cerevisieae on growth, immune function and intestinal morphology in weaned piglets. Microb. Cell. Fact. 2017, 16(1), 191-201.

 

Q4.

Results in this paper is not meaningful enough, since growth performance did not differ each other. We need better performance and lower cost, not only better gut health.

Response: Thanks for the suggestion. For our purpose, we tested the fermented condition to provide FSBM derived soy peptides. The large-scale experiment will carry out to assess growth performance. Still, with more replicate and birds per pen, 6% partial replacement by FSBM could saw a significant difference on Sembratowicz [4] and Chachaj [5]. On the other hand, the economic benefits were preliminary assessment through this study, FSBM_B+L show the better meat income and income over feed cost while having the similar feed cost with FSBM_B and FSBM_L. We supplement the feed cost analysis in table 6 and chapter 3.6 and the discussion, supporting further studies to adjust proportion in experiment design.

3. Results

3.6. Effect of Two-stage FSBM on Economic Benefits

An evaluation of the economic benefits of adding EP to the broiler diet is summarized in Table 6. The income over feed cost (IOFC) of the control, FSBM_L, FSBM_B and FSBM_B+L groups were 61.9, 59.9, 57.2 and 63.3 NT$/bird, respectively.

 

Table 6. Evaluation of the economic benefit of two-stage FSBM supplemented in diet

Item	Experimental diets
	SBM	FSBML	FSBMB	FSBMB+L
Feed cost, NT$/bird
1-35 days	46.6	49.0	49. 6	49.3
Meat income, NT$/bird
1-35 days	108.6	108.9	106.8	112.6
Income over feed cost, NT$/bird
1-35 days	61.9	59.9	57.2	63.3

Feed cost: Basing on the costs (NT$/kg) of the ingredients as follows: corn meal 10.1, soybean meal 15.0, fermented soybean meal 23.5, full fat soybean meal 18.2, Soybean oil 40.0, fish meal 42.0, monocalcium phosphate 14.5, Calcium carbonate 1.70, salt (NaCl) 4.5, DL-Methionine 110.0, L-Lysine-HCl 58.00, choline chloride, 50% 46, vitamin premix 210.0, and mineral premix 44.00. The fees for processing of basal ration per kg were 14.54 for grain mixture of control group, 14.97 for FSBM_L group, 14.92 for FSBM_B group, and 15.02 for FSBM_B+L group during 1-21 day, respectively. The fees for processing of basal ration per kg were 14.54 for grain mixture of control group, 14.97 for FSBM_L group, 14.90 for FSBM_B group, and 15.02 for FSBM_B+L group during 22-35 day, respectively.

 

Discussion:

Past studies suggest that partial replacement of dietary SBM by FSBM improved the broiler's BWG and FCR due to increased protein digestibility [23]. Although fermentation with the soy peptide upgraded the content, only 3 to 6% partial replacement diet could reach a similar effect [5,24]. FSBM_B+L showed a better FCR through the entire feeding period compare to SBM, and we observed that the nutrient and peptides in FSBM_B+L were better absorbed and utilized by the chickens, increased the income over feed cost, which means FSBM_B+L has the potential to bring more economic value, at the same time make sure birds intake better protein recourse.

 

4. Sembratowicz, I.; Chachaj, R.; Krauze, M.; Ognik, K. The effect of diet with fermented soybean meal on blood metabolites and redox status of chickens. Ann. Anim. Sci. 2020, 20(2), 599-611.
5. Chachaj, R.; Sembratowicz, I.; Krauze, M.; Ognik, K. The effect of partial replacement of soybean meal with fermented soybean meal on chicken performance and immune status. J. Anim. Feed Sci. 2019, 28, 263-271.

 

Overall, still very much thanks for the reviewer’s response. Many new findings on FSBM derived soy peptides are continue published through mice mode and gradually used in monogastric animals’ diets. The further FSBM should contain more functional properties so that improved better performance. Two-stage fermentation, which has more potential small molecular peptides, thus provides better absorption, intestinal immunity, and economic efficacy.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Thanks for your clarification.

Author Response

Thank you in advance for your time and effort on reviewing our work.

Reviewer 2 Report

The manuscript has been corrected as suggested by the Reviewers and therefore we can accept

Author Response

Thank you in advance for your time and effort on reviewing our work.