Bacillus Probiotic Strains Induce Gonadal Maturation and Sex Differentiation in Red Abalone Haliotis rufescens Using a Plant-Based Diet

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall Assessment

The manuscript addresses an important and novel topic in aquaculture nutrition and reproductive management. The use of Bacillus probiotics to enhance phytoestrogen bioavailability and influence gonadal maturation in red abalone is innovative, with strong implications for sustainable feed strategies. The study is generally well-structured, and the experimental design appears robust. However, several aspects require clarification, deeper discussion, and some restructuring to strengthen the manuscript.

 

Major Comments

1. Novelty and Mechanistic Clarity
• The study claims a novel role of probiotic-liberated phytoestrogens, but the direct evidence for phytoestrogen involvement is indirect. Consider tempering claims or adding molecular/biochemical evidence (e.g., phytoestrogen quantification, receptor assays).
• More discussion is needed on alternative explanations (e.g., other soy bioactives, microbiota modulation beyond enzymatic activity).
2. Experimental Design
• The diets are described as “proprietary,” but more transparency is needed. Journals usually require at least a proximate composition analysis (amino acid profile, lipid quality, fiber, etc.).
• Since both plant and fish diets contained Bacillus, the observed differences may stem not only from soy phytoestrogens but also from ingredient quality. Please clarify how these confounding effects were controlled.
3. Statistical Analysis
• The use of multiple ANOVAs and chi-square tests is appropriate, but please provide exact sample sizes per treatment at the final time point (n for males vs. females). Were any mortalities excluded from analysis?
• For sex ratio differences, confidence intervals should be provided to better demonstrate statistical robustness.
4. Figures and Tables
• Figures (e.g., VGI, sex ratios, correlations) are informative but could be clearer. Some legends are too brief—expand explanations of abbreviations (VGI, F:M ratios).
• Consider merging Table 2 (reproductive performance) with relevant figure captions to avoid redundancy.
5. Discussion
• While phytoestrogen liberation is discussed extensively, the ecological/evolutionary significance for abalone should be addressed: how might plant-derived estrogens compare to natural algal diets in the wild?
• The potential risks of phytoestrogen supplementation (e.g., endocrine disruption, reduced male fertility in later generations) should be acknowledged.
6. Conclusions
• Currently written in a very strong tone. Suggest softening claims to “suggests,” “indicates,” or “provides evidence” unless direct biochemical confirmation is available.

 

Minor Comments

• Abstract: streamline sentences; currently too long and detailed. Focus on what was done, key findings, and significance.
• Keywords: add terms like abalone aquaculture, reproductive endocrinology, sustainable feeds for better indexing.
• Methods: Include how probiotic viability in feed was confirmed post-processing (CFU counts after drying).
• References: Several are self-citations; balance with more external sources for context.
• Formatting: Ensure consistency in species names (Haliotis rufescens italics throughout).
• Grammar: Some sentences are long and complex—shorten for clarity.

 

Author Response

Comment 1: Novelty and Mechanistic Clarity

The study claims a novel role of probiotic-liberated phytoestrogens, but the direct evidence for phytoestrogen involvement is indirect. Consider tempering claims or adding molecular/biochemical evidence (e.g., phytoestrogen quantification, receptor assays).

More discussion is needed on alternative explanations (e.g., other soy bioactives, microbiota modulation beyond enzymatic activity).

 Response 1: Thank you for this important point. We agree that direct biochemical evidence for phytoestrogen involvement is currently indirect in our study, and we have clarified this in the revised manuscript.

 Published work demonstrates that certain Bacillus strains possess β-glucosidase activity that converts isoflavone glycosides (poorly bioavailable) into aglycones (bioavailable), increasing estrogenic activity in vitro. For example, Bacillus subtilis natto rapidly deglycosylates daidzin and genistin to daidzein and genistein and increases ERβ transactivation in reporter assays (Kuo et al., 2006). Long-term fermented soybean products rich in Bacillus also modify the gut microbiota and produce physiological effects consistent with increased isoflavone bioavailability (e.g., improved bone and glucose parameters in ovariectomized rats).

By contrast, previous abalone feeding trials that used soybean-containing diets but without probiotic enhancement did not consistently reproduce phytoestrogen-specific effects (Wu et al.; Meusel et al.), suggesting that soybean alone is not always sufficient to generate the responses we observed. Taken together, these literatures make our interpretation plausible: probiotic activity (β-glucosidase and related enzymes) can increase aglycone isoflavone availability and therefore increase estrogenic signaling — but the evidence in our present abalone trial is indirect.

Accordingly, we have softened mechanistic claims throughout the revised manuscript (e.g., “suggests” / “is consistent with” rather than “demonstrates”) and added a new paragraph to the Discussion explicitly acknowledging alternative explanations, including (a) other soy bioactives (saponins, phytates, peptides) that could influence gonadal physiology; (b) direct modulation of the host microbiome by our Bacillus strains (which can alter hormone metabolism, bile acids and energy balance); and (c) host enzymatic activity (abalone glycosidases) that might also convert glycosides to aglycones. We cite Kuo et al. for enzyme-mediated deglycosylation and Yang/Zhang for in vivo fermented-soy + Bacillus effects in mammalian models, and we cite Wu/Meusel for abalone studies that did not include probiotics.

Finally, we explicitly acknowledge that definitive demonstration requires biochemical and multi-omic confirmation (quantification of isoflavone aglycones/equol in diet and abalone tissues, in vitro receptor/transactivation assays, host transcriptomics of steroid/estrogen pathways, and microbiome sequencing). We have added a short “Future work” section listing these experiments and recommended methods (HPLC-MS quantification of daidzein/genistein/equol; ERα/ERβ reporter assays; 16S/shotgun metagenomics; gonad + hepatopancreas RNA-seq). These planned analyses are now described in the revised Discussion (lines 304-314) and in the response letter.

 

Comment 2: Experimental Design

The diets are described as “proprietary,” but more transparency is needed. Journals usually require at least a proximate composition analysis (amino acid profile, lipid quality, fiber, etc.).

Since both plant and fish diets contained Bacillus, the observed differences may stem not only from soy phytoestrogens but also from ingredient quality. Please clarify how these confounding effects were controlled.

 

Response 2: Thank you for this important observation. We have clarified the dietary formulations to ensure transparency while protecting proprietary details. In the revised manuscript, we now provide a new table (Table 1) with the proximate macronutrient composition of the experimental diets (protein, carbohydrates, lipids, and fiber), together with the main protein sources (fishmeal, soybean meal, vegetable meals) and vitamin/mineral mix. The suppliers/manufacturers of these ingredients (fishmeal from Procesadora Mar de Ensenada S.A. de C.V., soybean meal from Soyarin®, wheat flour from De la Rosa®, vitamin/mineral premix Brovel S.A. de C.V.) have been specified. For comparison, the proximate composition of fresh Macrocystis pyrifera was included using published values from Rodríguez-Montesinos and Hernández-Carmona (1991) for the Ensenada region where the kelp was collected for the present study.

To control potential confounding effects between plant- and fish-based diets, both formulations were designed equivalent in their proximate profiles (21% protein, 55% carbohydrates, 5% lipids), differing only in their primary protein source (soybean meal vs. fishmeal). This allowed us to specifically evaluate the effect of soybean meal. Both experimental diets were supplemented with the same Bacillus strains at identical concentrations (2 × 10⁶ CFU/g), ensuring that observed differences were attributable to the protein source rather than probiotic presence.

The revised description of diet formulation and proximate composition is provided in the Materials and Methods section 2.3.1 (lines 118-128) and summarized in Table 1 of the manuscript.

 

Comment 3: Statistical Analysis

The use of multiple ANOVAs and chi-square tests is appropriate, but please provide exact sample sizes per treatment at the final time point (n for males vs. females). Were any mortalities excluded from analysis?

For sex ratio differences, confidence intervals should be provided to better demonstrate statistical robustness.

 Response 3: Thank you for this helpful suggestion. We have clarified sample sizes and survival in the revised manuscript. Each dietary treatment began with 99 abalone (n = 297 total). At the final sampling point, the numbers were: fish diet (n = 93), kelp diet (n = 96), and plant diet (n = 96). Mortalities were removed but not replaced, and statistical analyses (ANOVA, chi-square) were conducted using the actual number of animals per treatment and sex at each time point. Survival ranged from 93.9% to 97.0%, with no significant differences among treatments, and therefore did not introduce detectable bias.

To improve transparency, exact sample sizes for males and females at each time point remain indicated in the figure panels (“n = …”), and we have now also summarized initial and final sample sizes per treatment in the Materials and Methods section 2.6 (lines 179-182). In addition, 95% confidence intervals for sex ratio estimates, and the initial n and final n (for each treatment) have been added to Table 3 (previously Table 2) in the survival row, as recommended.

  

Comment 4: Figures and Tables

Figures (e.g., VGI, sex ratios, correlations) are informative but could be clearer. Some legends are too brief—expand explanations of abbreviations (VGI, F:M ratios).

Consider merging Table 2 (reproductive performance) with relevant figure captions to avoid redundancy.

 Response 4: Thank you for this constructive suggestion. We agree that the figure legends needed clearer explanations. In the revised version, all figure captions have been expanded to define abbreviations (e.g., VGI = Visual Gonad Index, F:M = female-to-male ratio) and to provide more context on sample sizes and statistics. However, we believe that Table 2 (now Table 3) provides an important stand-alone summary of reproductive performance across treatments, which allows readers to quickly compare results without overloading the figures. For this reason, we have retained Table 3 but clarified its role as a complementary summary.

 

Comment 5: Discussion

While phytoestrogen liberation is discussed extensively, the ecological/evolutionary significance for abalone should be addressed: how might plant-derived estrogens compare to natural algal diets in the wild?

The potential risks of phytoestrogen supplementation (e.g., endocrine disruption, reduced male fertility in later generations) should be acknowledged.

 Response 5: We appreciate this valuable comment. In the revised manuscript, we have added a paragraph to the Discussion that addresses the ecological and evolutionary significance of using plant-derived phytoestrogens in abalone diets. Specifically, we compare soy phytoestrogens with the bioactive compounds naturally present in diverse algal diets, and we acknowledge potential risks, such as endocrine disruption or reduced male fertility in subsequent generations. This new text has been inserted in the Discussion Section 4.4 (lines 378-394).

New paragraph: From an ecological and evolutionary perspective, abalone in the wild consume a wide variety of macroalgae rich in polysaccharides, carotenoids, phlorotannins, and sterols that can influence physiology and reproduction [57]. In contrast, soybean meal introduces high concentrations of isoflavones ranging from 1.2 to 4.2 mg·kg⁻¹ dry matter [70]. These compounds provide an estrogenic chemical signal not normally encountered in natural abalone diets, which may help explain the female-biased sex ratios observed in this study. However, the ecological relevance and long-term consequences of this effect remain uncertain. Phytoestrogen supplementation has been associated in other taxa with endocrine disruption, altered sex differentiation, and reduced male fertility across generations [66,67]. Similar risks could emerge in abalone if reproductive pathways are overstimulated or if a persistent female bias compromises fertilization success.

Finally, the lipid composition of formulated diets differs from that of wild kelp, with higher total lipids and atered sterol profiles that may increase substrate availability for steroidogenesis and transport of lipophilic hormones [71], which has been documented in mollusks [72]. Taken together, these compositional differences suggest that (a) soy-bean-derived phytoestrogens act through pathways distinct from natural algal bioactive compounds, and (b) dietary lipids and sterols may interact with these compounds to further modulate reproductive physiology. Future studies should therefore quantify isoflavones in feeds and tissues, characterize sterol and lipid classes, and evaluate their combined impact on endocrine gene expression, fertility, and multigenerational reproductive performance to ensure that the benefits of probiotic–soy supplementation do not come at the cost of long-term reproductive capacity.

 

Comment 6: Conclusions

Currently written in a very strong tone. Suggest softening claims to “suggests,” “indicates,” or “provides evidence” unless direct biochemical confirmation is available.

 Response 6: Thank you for this valuable suggestion. We have revised the Conclusion to use more cautious language (“suggests,” “is consistent with,” “provides evidence”) and to avoid definitive mechanistic claims, since biochemical confirmation is still pending. The revised text emphasizes the promising nature of the findings while highlighting the need for further molecular and multigenerational studies.

 Minor Comments

 1- Abstract: streamline sentences; currently too long and detailed. Focus on what was done, key findings, and significance.

 Response 1: We appreciate this helpful suggestion. The Abstract has been revised to streamline overly long sentences and focus on the study design, main findings, and significance. Mechanistic details have been reduced, and the tone has been softened to emphasize that the results suggest potential effects rather than provide definitive proof. The revised Abstract is now shorter, clearer, and aligned with the recommendations.

New abstract: This study examined the effects of supplementing plant- and fish-based feeds with Bacillus probiotic strains on the reproductive performance of red abalone Haliotis rufescens, using fresh giant kelp Macrocystis pyrifera as a reference diet. Over 180 days, abalone fed the plant–probiotic diet reached higher female gonadal maturation, with 56% of females attaining the maximum Visual Gonad Index (VGI 3) and showed a female-biased sex ratio of 1.5:1 compared with other treatments. These results suggest that probiotics can improve nutrient utilization from soybean meal and may enhance the bioavailability of phytoestrogens and other bioactive compounds, contributing to reproductive outcomes. Although the mechanisms remain to be confirmed, this approach provides a promising strategy to reduce reliance on fishmeal and wild macroalgae while supporting faster reproductive cycles in abalone aquaculture. Future research should focus on biochemical validation, molecular pathways, and multigenerational trials to ensure the long-term safety and sustainability of probiotic–plant-based feeds.

 2- Keywords: add terms like abalone aquaculture, reproductive endocrinology, sustainable feeds for better indexing.

Response 2: Thank you for the suggestion. We have revised the keywords to improve indexing. The terms abalone aquaculture, reproductive endocrinology, and sustainable feeds have been added. To keep the list concise and avoid overloading, we removed gut microbiota and sex-specific maturation.

3- Methods: Include how probiotic viability in feed was confirmed post-processing (CFU counts after drying).

Response 3: Thank you for this important methodological clarification. We have added a detailed description of probiotic viability confirmation methodology in section 2.3.2 (lines 138-146). The post-processing analysis confirmed that our thermal processing conditions maintained probiotic viability within the target range, supporting the effectiveness of our feed preparation protocol.

 4- References: Several are self-citations; balance with more external sources for context.

Response 4: We appreciate this observation. Some self-citations were initially included because our previous studies used the same methodology, adding Bacillus probiotics in spore form directly into diets rather than pre-fermenting soybean meal, making them the most relevant comparisons. This approach differs from most published work that relies on pre-fermented soybean meal, and we highlight it as a cost-effective strategy with potential commercial scalability.

To balance the references and provide broader context, we have added several recent external sources addressing the use of Bacillus fermentation to improve soybean meal digestibility and bioactivity in aquafeeds (Chen et al., 2024; Boonmee et al., 2024; Zhang et al., 2021; Siddik et al., 2024; El-Dakar et al., 2023). These references strengthen the background and discussion by situating our findings within the wider field of soybean meal enhancement strategies while maintaining the distinction of our spore-based approach.

5- Formatting: Ensure consistency in species names (Haliotis rufescens italics throughout).

Response 5: Thank you for this observation, consistency was ensured.

 6- Grammar: Some sentences are long and complex—shorten for clarity.

Response 6: Thank you for this important observation regarding sentence clarity. We have systematically reviewed and shortened complex sentences throughout the revised manuscript to improve readability while maintaining scientific precision.

Reviewer 2 Report

Comments and Suggestions for Authors

The presented material seems interesting and requires partial revision.
The authors need to make minor adjustments to improve the article.
The comments are below.
1. It is necessary to provide a more detailed description of paragraph 2.3. Composition and preparation of feed enriched with probiotics, describe the raw materials used for fish meal, who is the manufacturer.
2. Also, who is the manufacturer of soybean flour, soybean meal, wheat flour?
3. It is necessary to provide a more detailed justification for why treatment with plant-based probiotics showed more moderate results (line 249)
4. Provide a more detailed justification for why Bacillus Sp1 and Sp3 strains were used?
5. It is necessary to indicate possible options for practical application of the obtained results in the conclusion

Author Response

Comments 1: It is necessary to provide a more detailed description of paragraph 2.3. Composition and preparation of feed enriched with probiotics, describe the raw materials used for fish meal, who is the manufacturer.

Comment 2: Also, who is the manufacturer of soybean flour, soybean meal, wheat flour?

Response 1 & 2: Thank you for this important observation. We have clarified the dietary formulations to ensure transparency while protecting proprietary details. In the revised manuscript, we now provide a new table (Table 1) with the proximate macronutrient composition of the experimental diets (protein, carbohydrates, lipids, and fiber), together with the main protein sources (fishmeal, soybean meal, vegetable meals) and vitamin/mineral mix. The suppliers/manufacturers of these ingredients (fishmeal from Procesadora Mar de Ensenada S.A. de C.V., soybean meal from Soyarin®, wheat flour from De la Rosa®, vitamin/mineral premix Brovel S.A. de C.V.) have been specified. For comparison, the proximate composition of fresh Macrocystis pyrifera was included using published values from Rodríguez-Montesinos and Hernández-Carmona (1991) for the Ensenada region where the kelp was collected for the present study.

The revised description of diet formulation and proximate composition is provided in the Materials and Methods section 2.3.1 (lines 118-129) and summarized in a new table (Table 1 of the revised manuscript).

Comments 3: It is necessary to provide a more detailed justification for why treatment with plant-based probiotics showed more moderate results (line 249)

Response 3: Thank you for this observation. The introductory paragraph of the Discussion serves as an overview of key findings, with detailed mechanistic justifications provided in subsequent sections. Specifically, the moderate male reproductive responses compared to females are thoroughly analyzed in Section 4.2 (lines 318-334), where we discuss the differential energy requirements between oogenesis and spermatogenesis, and the greater responsiveness of female reproductive pathways to phytoestrogenic stimulation. This structure allows readers to first grasp the overall findings before exploring the underlying biological mechanisms in detail.

Comment 4: Provide a more detailed justification for why Bacillus Sp1 and Sp3 strains were used?

Response 4: The selection of Bacillus strains Sp1 and Sp3 was based on their complementary enzymatic profiles documented in our institutional strain collection and previous laboratory studies. Strain Sp1 (B. velezensis) demonstrates extensive proteolytic activity on wheat flour proteins, while strain Sp3 (B. amyloliquefaciens) shows superior degradation capacity for soybean meal proteins and exceptional performance in complex carbohydrate hydrolysis, with moderate α-galactosidase production essential for oligosaccharide breakdown. This complementary enzymatic activity is documented in Table 2, where Sp1 shows highest activity (+ + +) on wheat flour and soy protein concentrate, while Sp3 excels in soybean meal degradation (+ + +) and carbohydrase activity. The synergistic combination of these strains was designed to maximize nutrient liberation from the plant-based matrix. Both strains maintain GRAS status and have been characterized in our previous aquaculture applications, providing a solid foundation for their safe and effective use in abalone feeds.

Comment 5: It is necessary to indicate possible options for practical application of the obtained results in the conclusion

Response 5: Thank you for this important suggestion. We have significantly enhanced the conclusion section to include specific practical applications of our research findings for the aquaculture industry (lines 403-408). The revised conclusion now details concrete applications that practitioners can implement: (1) programming reproductive cycles rather than relying solely on natural seasonal patterns, enabling year-round breeding management; (2) potentially obtaining higher quality larvae from fully mature females, which may enhance settlement and survival rates; (3) reducing dependency on marine-derived ingredients by replacing fishmeal with probiotic-supplemented soybean meal; and (4) achieving operational stability through reduced reliance on seasonally variable wild macroalgae, providing more predictable feed costs and consistent nutritional quality. These practical applications directly address commercial aquaculture needs while supporting the transition toward more sustainable production practices that reduce pressure on marine ecosystems.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been sufficiently improved to warrant publication in Microbiology Research.