The Potential of Black Soldier Fly (Hermetia illucens L.) Larvae in Whole Wheat Bread Production: Effects on Physicochemical, Antioxidative, and Sensory Properties

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the application of black soldier fly (Hermetia illucens L.) larvae powder in whole wheat bread production, examining its effects on the physicochemical properties, antioxidative activity, and sensory characteristics of bread. The research demonstrates that bread supplemented with 10% black soldier fly larvae powder maintains acceptable sensory properties while enhancing nutritional value, thus providing scientific evidence for the application of insect protein in bakery products. 

The abstract is excessively long, exceeding the recommended length for scientific paper abstracts. Condensation is recommended.

The abstract fails to clearly identify the research's innovative aspects and does not sufficiently differentiate from existing studies on black soldier fly.

Numerical expression of results in the abstract lacks precision; terms like "significantly increased" or "significantly decreased" should include specific values or percentages.

Lines 47-61: The description of research background is overly broad and fails to quickly focus on the specific research problem.

Lines 62-68: The discussion on food neophobia lacks sufficient quantitative data support.

Lines 92-104: The statement of research objectives does not clearly explain expected results and hypotheses, lacking theoretical foundation.

The introduction lacks a systematic literature review of existing insect food research, particularly regarding bakery products. It also fails to adequately explain why whole wheat bread was selected as the research vehicle.

Lines 105-117: Material source description is insufficient, especially regarding rearing conditions and feed composition of black soldier fly larvae, which may affect nutritional content.

Lines 206-218: Antioxidant activity measurement relies solely on the PCL method; complementary methods such as DPPH and ABTS should be considered for verification.

Lines 313-347: The discussion of protein and fat content does not address how insect powder addition affects the bread's energy value.

Lines 348-389: Mineral analysis does not evaluate bioavailability of these minerals; reporting total content alone is limiting.

Lines 464-560: The fatty acid analysis overemphasizes the potential negative health impacts of SFA/PUFA ratio without adequately discussing the potential benefits of lauric acid.

Lines 562-615: Texture analysis lacks correlation with bread microstructure, such as scanning electron microscopy observations.

Lines 761-770: The conclusion is too brief and fails to summarize all key findings. Research limitations are not sufficiently discussed. Discussion of potential food safety issues is inadequate. Specific application recommendations and future research directions are lacking.

Author Response

Comments and Suggestions for Authors

The abstract is excessively long, exceeding the recommended length for scientific paper abstracts. Condensation is recommended.

Answer: We have shortened the abstract as recommended.

The abstract fails to clearly identify the research's innovative aspects and does not sufficiently differentiate from existing studies on black soldier fly.

Answer: We have revised the abstract to highlight the study's innovative aspects and its distinction from existing research.

Numerical expression of results in the abstract lacks precision; terms like "significantly increased" or "significantly decreased" should include specific values or percentages.

Answer: We have included specific values in the abstract as suggested.

Lines 47-61: The description of research background is overly broad and fails to quickly focus on the specific research problem.

Answer: We have reviewed and condensed the background to focus more directly on the research problem.

Lines 62-68: The discussion on food neophobia lacks sufficient quantitative data support.

Answer: We have added quantitative data to substantiate the discussion.

Lines 92-104: The statement of research objectives does not clearly explain expected results and hypotheses, lacking theoretical foundation.

The introduction lacks a systematic literature review of existing insect food research, particularly regarding bakery products. It also fails to adequately explain why whole wheat bread was selected as the research vehicle.

Answer: We have carefully considered this comment. We have revised the introduction to include a literature review of insect-based bakery products and to explain the selection of whole wheat bread.

Lines 105-117: Material source description is insufficient, especially regarding rearing conditions and feed composition of black soldier fly larvae, which may affect nutritional content.

Answer: Thank you for pointing this to us. We have added details on rearing conditions and feed composition in section 2.1.

Activity measurement relies solely on the PCL method; complementary methods such as DPPH and ABTS should be considered for verification.

Answer: We appreciate this suggestion. While we have previously compared PCL with DPPH and ABTS methods, we now use PCL exclusively for its automation, sensitivity, and ability to distinguish antioxidant types. Although we cannot implement additional methods at this time, we compare our results with published data to provide context for our findings.

Lines 313-347: The discussion of protein and fat content does not address how insect powder addition affects the bread's energy value.

Answer: We did not analyze fiber content, so we could not determine the exact caloric value. Our focus was on protein and fat content. However, adding black soldier fly larvae meal increases the bread's energy value due to its high fat content. The specific increase depends on inclusion rate and processing methods.

Lines 348-389: Mineral analysis does not evaluate bioavailability of these minerals; reporting total content alone is limiting.

Answer: Thank you for this comment. We recognize the importance of mineral bioavailability. Determining mineral composition is an essential first step before conducting detailed bioavailability studies. We have added a statement to this effect in the manuscript.

Lines 464-560: The fatty acid analysis overemphasizes the potential negative health impacts of SFA/PUFA ratio without adequately discussing the potential benefits of lauric acid.

Answer: We have revised the manuscript to provide a balanced discussion of both the beneficial and harmful effects of saturated fatty acids, as requested. Please see sections 3.4.1 and 3.4.2 for details.

Lines 562-615: Texture analysis lacks correlation with bread microstructure, such as scanning electron microscopy observations.

Answer: We do not currently have access to this equipment but plan to collaborate with a research unit to enhance future studies on bread texture with insect meal.

Lines 761-770: The conclusion is too brief and fails to summarize all key findings. Research limitations are not sufficiently discussed. Discussion of potential food safety issues is inadequate. Specific application recommendations and future research directions are lacking.

Answer: We have expanded the conclusions section as recommended. Thank you.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses an innovative topic: the use of Hermetia illucens larvae in whole wheat bread. The experimental plan is well-structured, covering proximate composition, mineral and fatty acid profiles, antioxidant properties, color, texture, and sensory evaluation. The work is relevant to the field of sustainable and alternative proteins. However, there are significant weaknesses in the characterization of the insect meal, in the nutritional discussion (especially regarding fatty acids), and in the sensory analysis design. Some interpretations are overly optimistic relative to the data.

Major Concerns

Characterization of the insect meal

The starting material (HI meal) is not adequately characterized. Table 2 reports only basic proximate composition (moisture, protein, fat), partly from supplier data. No information is provided on ash, dietary fiber/chitin, amino acid profile, or bioactive compounds. Rearing conditions (substrate, growth stage, environment) and post-harvest processing (drying parameters, storage, grinding) are not described, although these strongly affect composition. This weakens the reproducibility and robustness of the study. It is also unclear whether a single batch or multiple batches were used. Finally, only microbiological safety was assessed (Table 3), while other safety aspects, such as allergens, were not considered.

Experimental design

Only two inclusion levels (10% and 30%) were tested. In my opinion, the 30% substitution is extreme and technologically/sensorially unrealistic. Intermediate levels (5–20%) should be considered to identify more precisely the threshold of acceptability.

Nutritional aspects

While protein and mineral enrichment are demonstrated (Tables 2 and 4), the fatty acid profile worsens significantly with HI addition: SFA increase from 22.48% to 73.83%, PUFA decrease from 30.93% to 9.54% (Table 6), and the n-6/n-3 ratio and AI/TI indices shift toward unfavorable values. These findings are acknowledged but downplayed in the discussion. A more critical analysis of the potential health implications is required.

Sensory analysis

The sensory analysis was conducted with only 10 trained assessors aged 20–40 and explicitly without food neophobia. While this is acceptable for descriptive sensory evaluation, it is not adequate to support conclusions about consumer acceptability. Excluding neophobic participants removes an essential barrier to insect-based products and limits the external validity of the results.

Antioxidant results

The increase in TPC and antioxidant activity is statistically significant (Table 5), but quantitatively modest (e.g., TPC from 76.78 to 97.16 mg GAE/100 g). The discussion frames these results as strongly positive, but the practical nutritional impact should be discussed more cautiously.

Minor Concerns

Introduction

The introduction is well-referenced but somewhat redundant (e.g., lines 80-81)

Tables

Tables (e.g., 2, 4, 6) report values with two or even three decimal places (e.g., 32.36% moisture; 232.36 mg K/100 g). This level of precision is not justified by the analytical methods or the biological variability of the samples. It is recommended to round the results to a more appropriate number of significant figures (usually one decimal place for proximate composition, minerals, and fatty acids). Consistency across tables should also be ensured.

Discussion

The discussion of lauric acid and SFA is unbalanced, focusing mainly on potential benefits while downplaying established cardiovascular risks.

References

The manuscript is well-referenced, but the use of citations is sometimes redundant (e.g., [30] cited repeatedly within short sections: lines 320, 324 - 692, 702 - or [31]: lines: 304, 307). The discussion tends to emphasize positive findings from the literature while underplaying critical aspects such as lipid profile deterioration, safety, or consumer neophobia. The inclusion of recent reviews or EFSA opinions on edible insects would strengthen the background and discussion.

Recommendation

The manuscript presents valuable data and could make a useful contribution to the field, but it requires major revision before being considered for publication. In particular, the authors should:

• Provide a more complete characterization (or at least a critical discussion) of the HI meal.
• Address the nutritional drawbacks of the fatty acid profile more explicitly.
• Moderate conclusions on sensory and antioxidant results.
• Consider adding or discussing more realistic inclusion levels.

Author Response

Comments and Suggestions for Authors

Major Concerns

Characterization of the insect meal

The starting material (HI meal) is not adequately characterized. Table 2 reports only basic proximate composition (moisture, protein, fat), partly from supplier data. No information is provided on ash, dietary fiber/chitin, amino acid profile, or bioactive compounds. Rearing conditions (substrate, growth stage, environment) and post-harvest processing (drying parameters, storage, grinding) are not described, although these strongly affect composition. This weakens the reproducibility and robustness of the study. It is also unclear whether a single batch or multiple batches were used. Finally, only microbiological safety was assessed (Table 3), while other safety aspects, such as allergens, were not considered.

Answer: We have addressed this concern by updating the Methods and Materials section (2.1) with details on rearing and post-harvest processing. Nutritional information, including moisture, protein, fat, ash, and dietary fiber, is now included. We have also emphasized allergen testing and moved related results to the supplementary file. Although we were unable to provide an amino acid profile, bioactive compounds are discussed later in the manuscript.

Experimental design

Only two inclusion levels (10% and 30%) were tested. In my opinion, the 30% substitution is extreme and technologically/sensorially unrealistic. Intermediate levels (5–20%) should be considered to identify more precisely the threshold of acceptability.

Answer: We have clarified the study's aim in the introduction, hoping that this explains the rationale behind choosing higher addition levels. Also, the two inclusion levels were chosen intentionally to help identify additive amounts for future experiments.

Nutritional aspects

While protein and mineral enrichment are demonstrated (Tables 2 and 4), the fatty acid profile worsens significantly with HI addition: SFA increase from 22.48% to 73.83%, PUFA decrease from 30.93% to 9.54% (Table 6), and the n-6/n-3 ratio and AI/TI indices shift toward unfavorable values. These findings are acknowledged but downplayed in the discussion. A more critical analysis of the potential health implications is required.

Answer: We have taken this comment very seriously and have revised the manuscript accordingly, ensuring a balance between requests from Reviewer 1 and Reviewer 2, who wished an exploration of the beneficial and harmful effects of saturated fatty acids, respectively. For your reference, please see sections 3.4.1 and 3.4.2, where additions to the text are highlighted in red.

Sensory analysis

The sensory analysis was conducted with only 10 trained assessors aged 20–40 and explicitly without food neophobia. While this is acceptable for descriptive sensory evaluation, it is not adequate to support conclusions about consumer acceptability. Excluding neophobic participants removes an essential barrier to insect-based products and limits the external validity of the results.

Answer: We have carefully considered this comment and avoided referencing our findings to consumer acceptability, which may have given that impression, particularly in the abstract and conclusions. Thank you for pointing this to us. We are planning to conduct a consumer acceptability analysis in the upcoming experiments.

Antioxidant results

The increase in TPC and antioxidant activity is statistically significant (Table 5), but quantitatively modest (e.g., TPC from 76.78 to 97.16 mg GAE/100 g). The discussion frames these results as strongly positive, but the practical nutritional impact should be discussed more cautiously.

Answer: We have carefully addressed this comment and introduced necessary improvements to the text in section 3.3.2, all highlighted in red.

Minor Concerns

Introduction

The introduction is well-referenced but somewhat redundant (e.g., lines 80-81)

Answer: Thank you. Nonetheless, following the reviewer 1 suggestion, the introduction has been improved.

Tables

Tables (e.g., 2, 4, 6) report values with two or even three decimal places (e.g., 32.36% moisture; 232.36 mg K/100 g). This level of precision is not justified by the analytical methods or the biological variability of the samples. It is recommended to round the results to a more appropriate number of significant figures (usually one decimal place for proximate composition, minerals, and fatty acids). Consistency across tables should also be ensured.

Answer: We have standardized the values across the tables. The values now display 2 decimal places instead of 3.

Discussion

The discussion of lauric acid and SFA is unbalanced, focusing mainly on potential benefits while downplaying established cardiovascular risks.

Answer: We have taken this comment very seriously and have revised the manuscript accordingly, ensuring a balance between requests from Reviewer 1 and Reviewer 2, who wished an exploration of the beneficial and harmful effects of saturated fatty acids, respectively. For your reference, please see sections 3.4.1 and 3.4.2, where additions to the text are highlighted in red.

References

The manuscript is well-referenced, but the use of citations is sometimes redundant (e.g., [30] cited repeatedly within short sections: lines 320, 324 - 692, 702 - or [31]: lines: 304, 307). The discussion tends to emphasize positive findings from the literature while underplaying critical aspects such as lipid profile deterioration, safety, or consumer neophobia. The inclusion of recent reviews or EFSA opinions on edible insects would strengthen the background and discussion.

Answer: We have addressed and corrected the manuscript as recommended, where possible.

Recommendation

The manuscript presents valuable data and could make a useful contribution to the field, but it requires major revision before being considered for publication. In particular, the authors should:

Provide a more complete characterization (or at least a critical discussion) of the HI meal.

Address the nutritional drawbacks of the fatty acid profile more explicitly.

Moderate conclusions on sensory and antioxidant results.

Consider adding or discussing more realistic inclusion levels.

Answer: We hope we have successfully fulfilled the majority of the recommendations. Thank you.