Changes of Composition and Nutrition Value of Some Gluten Free (Pseudo)cereals by Addition of Carotenogenic Yeast Cell Homogenate

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall evaluation

The research aims to develop a fortified/functional, gluten-free food. This is an interesting goal, but the authors could have better explained why they chose to add Rhodotorula rather than another yeast.

The work has several limitations, listed below, and therefore cannot be accepted for publication. It may be resubmitted after major revision.

 

Major remarks

The composition (proteins, lipids, fiber, carbohydrates and simple sugars) of the different products developed in this trial is not reported.

Since yeasts are also producers of B vitamins, the Authors could also determine this class of compounds.

The two Lactobacillus utilized to evaluate the prebiotic properties of Rhodotorula toruloides extract, were from human gut or have isolated from other environment ? Since the experiment is aimed at producing a food with extra-nutritive properties, it is important that the possible prebiotic effect is tested on bacteria capable of growing in the human intestine.

Neither the statistical model nor the software used are described.

 

Table 1               For buckwheat and quinoa, the 10% supplementation level with RTH is not reported, while 5% is reported twice. Please correct.

Figure 1             Statistically significant differences should be indicated in the figure. Please use different letter or *

Table 2               In lines 185-185, Authors say that “Lipid-soluble metabolites, including ergosterol and carotenoids (torularhodin, torulene, and lycopene), were detected exclusively in samples enriched with RTh”. However in table 2, data of carotenoids content in pseudocereals not supplemented with Rth are not reported.

 

Table 2.              In addition to what is written in the previous paragraph, why are there no carotenoids present in the Oat + 5% Rth treatment?

 

Figure 2 A-B    Authors discuss differences in fatty acid profile, but are these differences significant ?

 

Table 4               This table appears to report the same data as Figure 2, so one of the two should be eliminated. Furthermore, although the table captions mention statistically significant differences, they are not reported in Table 4.

Figure 3             Figure 3 does not show statistically significant differences between means.

 

Table 5               This table does not provide an explanation of the symbols used. Furthermore, where is PI (Prebiotic effect) ?

 

Minor remarks

I don't understand why the Authors write (pseudo)cereal instead of pseudocereal.

Lines 38-39                   Please change “as non-digestible carbohydrates” in “as non-digestible carbohydrates sources”.

Lines 169                        The text does not specify that it is talking about figure 1.

Line 186                           See previous line, what table ?

Lines 203-204             What figure ?

Line 249                           What figure ?

Line 300                           What figure ?

Author Response

Dear Reviewer 1,

We would like to thank you for your careful reading of our manuscript and for the valuable comments and suggestions. We have revised the manuscript accordingly, and all changes have been highlighted (blue) in the revised version.

Below, we provide a detailed point-by-point response to all comments. Changes in the manuscript are highlighted in blue.

Comment 1: The research aims to develop a fortified/functional, gluten-free food. This is an interesting goal, but the authors could have better explained why they chose to add Rhodotorula rather than another yeast.

Response 1: The rationale for selecting Rhodotorula toruloides as a fortification agent has now been clarified in the revised manuscript (Introduction, page 3, lines 97-126).

Comment 2: The composition (proteins, lipids, fiber, carbohydrates and simple sugars) of the different products developed in this trial is not reported.

Response 2: To address this point, a table summarizing the basic nutritional composition (proteins, lipids, carbohydrates) of the cereal and pseudocereal raw materials used in this study has now been included in the revised manuscript (Table 5, page 16). These values were obtained from the manufacturer’s specifications and provide a general nutritional context for the developed products.

We acknowledge that the detailed proximate composition of the final fortified products was not experimentally determined within the scope of this study, which primarily focused on bioactive compounds and biological activity. This limitation has now been clarified in the manuscript (page 15-16, lines 477-482, table 5).

Commnet 3: Since yeasts are also producers of B vitamins, the Authors could also determine this class of compounds.

Response 3: We agree that yeasts, including Rhodotorula toruloides, may represent a potential source of B-group vitamins, which could further enhance the nutritional value of the developed products. However, the primary objective of the present study was focused on the evaluation of selected bioactive compounds, namely carotenoids, sterols, and lipid-related metabolites, together with their biological activity. Therefore, the determination of B vitamins was not included within the scope of this work. Nevertheless, this aspect represents an interesting direction for future research, and this has now been acknowledged in the revised manuscript (Discussion, page 15, lines 467-470).

Comment 4: The two Lactobacillus utilized to evaluate the prebiotic properties of Rhodotorula toruloides extract, were from human gut or have isolated from other environment? Since the experiment is aimed at producing a food with extra-nutritive properties, it is important that the possible prebiotic effect is tested on bacteria capable of growing in the human intestine.

Response 4: The strains used in this study were obtained from the Czech Collection of Microorganisms (CCM). Bifidobacterium bifidum CCM 3762 was originally isolated from human stool, while Lactobacillus plantarum CCM 7039T was isolated from fermented cabbage. Although L. plantarum originates from a plant-based environment, it is a well-known facultative species that is frequently found in the human gastrointestinal tract and is widely used as a model probiotic organism in studies evaluating prebiotic effects. Both selected strains are therefore relevant representatives of gut-associated microbiota and are commonly employed in in vitro assessments of prebiotic potential. This clarification has now been added to the revised manuscript (page 19-20, lines 636-639).

Comment 5: Neither the statistical model nor the software used are described.

Response 5: The information has now been added to the Materials and Methods section of the revised manuscript (page 20, lines 681-685).

Comment 6: Table 1 - For buckwheat and quinoa, the 10% supplementation level with RTH is not reported, while 5% is reported twice. Please correct.

Response 6: We thank you for carefully checking the data and for pointing out this error. The table has now been corrected to include the 10% supplementation level for buckwheat and quinoa, and the duplicated 5% values have been removed. The revised version of Table 1 is now presented in the manuscript.

Comment 7: Figure 1 - Statistically significant differences should be indicated in the figure. Please use different letter or *

Response 7: Statistically significant differences have now been indicated in Figure 1 using asterisks (*). The figure and its caption have been updated accordingly.

Comment 8: Table 2 - In lines 185-185, Authors say that “Lipid-soluble metabolites, including ergosterol and carotenoids (torularhodin, torulene, and lycopene), were detected exclusively in samples enriched with RTh”. However in table 2, data of carotenoids content in pseudocereals not supplemented with Rth are not reported.

Response 8: The carotenoids and ergosterol were also analyzed in non-fortified samples; however, these compounds were not detected in any of the samples without RTh supplementation. For clarity, “ND” (not detected) has now been added to Table 2 for all corresponding values. The table and its caption have been updated accordingly.

Comment 9: Table 2 - In addition to what is written in the previous paragraph, why are there no carotenoids present in the Oat + 5% Rth treatment?

Response 9: In the oat + 5% RTh sample, carotenoids were below the detection limit of the applied analytical method. This may be attributed to the relatively low level of supplementation combined with matrix-related effects, such as interactions with oat components or limited extractability of lipophilic compounds.

Comment 10: Figure 2 A - Authors discuss differences in fatty acid profile, but are these differences significant ?

Response 10: We thank you for this important comment. The fatty acid profile data were not subjected to statistical analysis, and the comparison was intended to be descriptive. To avoid any misinterpretation, the text has been revised to remove statements implying statistical significance (page 8, lines 236-239).

Comment 11: Table 4 - This table appears to report the same data as Figure 2, so one of the two should be eliminated. Furthermore, although the table captions mention statistically significant differences, they are not reported in Table 4.

Response 11: Table 4 and Figure 2 present different aspects of the fatty acid composition and are therefore not redundant. Figure 2 shows the overall distribution of major fatty acid groups (SFA, MUFA, and PUFA), while Table 4 provides detailed data on individual fatty acids. Table 4 has been renumbered as Table 3 in the revised manuscript.

Regarding the second point, we acknowledge that the reference to statistically significant differences in the table caption was incorrect. The fatty acid data were not subjected to statistical analysis and are presented in a descriptive manner. The table caption has now been revised accordingly (page 9, line 267).

Comment 12: Figure 3 - Figure 3 does not show statistically significant differences between means.

Response 12: The data presented in Figure 3 were not subjected to statistical analysis and are intended to provide a descriptive comparison. To avoid any misunderstanding, the figure and its caption have been revised to remove any implication of statistical significance (page 10, lines 288-290).

Comment 13: Table 5 - This table does not provide an explanation of the symbols used. Furthermore, where is PI (Prebiotic effect) ?

Response 13: The caption of Table 5 has now been revised to clearly explain all symbols used, including the definition of the prebiotic index (PI) and its corresponding ranges. In addition, missing explanations (e.g., symbol “x”) have been clarified.

Furthermore, the table has been carefully revised to correct inconsistencies and ensure accurate reporting of all supplementation levels. The updated Table 5 is now presented in the revised manuscript. Table 5 has been renumbered as Table 4 in the revised manuscript (page 11, lines 313-315).

The use of categorical symbols instead of numerical values was intended to improve clarity and facilitate comparison of prebiotic effects across multiple samples and bacterial strains, particularly by highlighting relative differences in bacterial growth.

Comment 14: I don't understand why the Authors write (pseudo)cereal instead of pseudocereal.

Response 14: The term “(pseudo)cereal” was used to collectively refer to both cereals (e.g., oat) and pseudocereals (e.g., quinoa and buckwheat), which were included as base ingredients in this study.

Comment 15: Lines 38-39 - Please change “as non-digestible carbohydrates” in “as non-digestible carbohydrates sources”.

Response 15: The text has been revised accordingly (page 1, line 39).

Comment 16: Lines 169 - The text does not specify that it is talking about figure 1.

Response 16: The text has been revised accordingly (page 4, line 169).

Comment 17: Line 186 - See previous line, what table ?

Response 17: The text has been revised accordingly (page 6, line 221).

Comment 18: Lines 203-204 - What figure ?

Response 18: The text has been revised accordingly (page 7, lines 225-226, 229 and 235).

Comment 19: Line 249 - What figure ?

Response 19: The data are not presented in a figure but are reported in the text of the Results section.

Comment 20: Line 300 - What figure ?

Response 20: The text has been revised accordingly (page 11, lines 328, 332 and 337).

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates the fortification of gluten-free oat, quinoa, and buckwheat porridges with a cell-free homogenate of the carotenogenic yeast Rhodotorula toruloides and evaluates the resulting changes in antioxidant capacity, lipid-soluble bioactives, fatty acid composition, approximate prebiotic potential, and cytotoxicity. The topic is relevant and timely, particularly in the context of developing value-added gluten-free functional foods. The study shows that supplementation with R. toruloides homogenate increased ergosterol and carotenoid contents, altered fatty acid profiles, and in some cases improved antioxidant activity and bacterial growth responses, while maintaining generally low cytotoxicity. The conclusion that 5% supplementation may represent a more balanced fortification level than 10% is, in general, supported by the presented data.

The manuscript has several strengths. It compares multiple cereal and pseudocereal matrices and includes both compositional and biological evaluations, which gives the work a broader functional perspective. The inclusion of buckwheat, quinoa, oat, and their blends is useful because it allows matrix-dependent effects to be observed. In particular, the data suggest that buckwheat-based samples had stronger antioxidant performance, while oat–quinoa blends showed promising approximate prebiotic behavior. Overall, the work has practical significance for the formulation of gluten-free cereal products enriched with natural microbial ingredients.

However, the manuscript still has important limitations that should be addressed before publication. First, the novelty is somewhat limited because the study is largely descriptive and focuses mainly on compositional improvement rather than mechanistic insight. Although the authors discuss possible interactions between yeast-derived compounds and cereal matrices, these interactions are not experimentally demonstrated. The discussion would benefit from a more critical explanation of why different cereal bases responded differently to the same enrichment treatment.

Second, some methodological aspects reduce the rigor of the study. A major concern is that β-glucan values in cereal–yeast mixtures were not measured directly, but instead were calculated from the individual components, with an assumed standard deviation of approximately 5%. This weakens the robustness of the quantitative comparison and should be clearly acknowledged as a limitation. In addition, the “prebiotic effect” is inferred only from growth responses of two strains, Lactobacillus plantarum and Bifidobacterium bifidum, which is not sufficient to support strong claims about prebiotic functionality. The wording should therefore remain cautious, for example by consistently using “approximate prebiotic potential.”

Third, the cytotoxicity evaluation is relatively limited. Only one cell model was used, and some 10% supplementation variants showed moderate cytotoxicity, especially in buckwheat and oat–buckwheat samples. Therefore, the safety discussion should be more careful and should avoid overgeneralization. The recommendation of 5% supplementation appears more reasonable, but validation in more realistic food systems, digestion models, and sensory settings would strengthen the practical relevance.

Finally, the manuscript requires substantial language editing. There are numerous grammatical errors, repetitions, inconsistencies in sample labeling, duplicated entries in tables, and figure/table cross-referencing problems that reduce clarity and professionalism.

In summary, the study has potential and addresses a meaningful application-oriented topic, but the current version requires significant improvement in methodological rigor, interpretation depth, and English presentation. 

Author Response

Dear Reviewer 2,

We would like to thank you for your careful reading of our manuscript and for the valuable comments and suggestions. We have revised the manuscript accordingly, and all changes have been highlighted (pink) in the revised version.

Below, we provide a detailed point-by-point response to all comments. Changes in the manuscript are highlighted in pink.

Comment 1: First, the novelty is somewhat limited because the study is largely descriptive and focuses mainly on compositional improvement rather than mechanistic insight. Although the authors discuss possible interactions between yeast-derived compounds and cereal matrices, these interactions are not experimentally demonstrated. The discussion would benefit from a more critical explanation of why different cereal bases responded differently to the same enrichment treatment.

Response 1: We agree that the original version of the manuscript lacked a deeper mechanistic interpretation of the matrix-dependent effects. In the revised version, the Discussion section has been expanded to provide a more critical explanation of the observed differences between cereal and pseudocereal matrices (page 14, lines 445-466). These revisions strengthen the discussion and better explain why different cereal bases responded differently to the same enrichment treatment.

Comment 2: Second, some methodological aspects reduce the rigor of the study. A major concern is that β-glucan values in cereal–yeast mixtures were not measured directly, but instead were calculated from the individual components, with an assumed standard deviation of approximately 5%. This weakens the robustness of the quantitative comparison and should be clearly acknowledged as a limitation. In addition, the “prebiotic effect” is inferred only from growth responses of two strains, Lactobacillus plantarum and Bifidobacterium bifidum, which is not sufficient to support strong claims about prebiotic functionality. The wording should therefore remain cautious, for example by consistently using “approximate prebiotic potential.”

Response 2: We acknowledge that the β-glucan content in cereal–yeast mixtures was not determined experimentally but estimated based on the values of individual components. In the revised manuscript, this has now been clearly stated in the Materials and Methods, Results and Discussion sections, including the assumption of an approximate standard deviation (~5%). Furthermore, this approach has been explicitly acknowledged as a limitation of the study, as it may affect the robustness of quantitative comparisons between samples.

Changes made: page 4, lines 169-170; page 14, lines 379-383

Regarding the evaluation of prebiotic properties, we agree that the assessment based on the growth response of only two strains (Lactobacillus plantarum and Bifidobacterium bifidum) does not allow for definitive conclusions about prebiotic functionality. Therefore, the wording throughout the manuscript has been revised to a more cautious interpretation. Specifically, terms such as “prebiotic effect” have been replaced with “approximate prebiotic potential” or “indicative prebiotic activity,” and the limitations of the experimental design have been clearly discussed. These revisions improve the transparency and scientific rigor of the study and ensure that the conclusions are appropriately balanced.

Changes made: page 1, line 18 and 24-25; page 10, lines 291, 295, 303, 307, 310-312,313; page 16 lines 416-419; page 19 lines 631-635

Comment 3: Third, the cytotoxicity evaluation is relatively limited. Only one cell model was used, and some 10% supplementation variants showed moderate cytotoxicity, especially in buckwheat and oat–buckwheat samples. Therefore, the safety discussion should be more careful and should avoid overgeneralization. The recommendation of 5% supplementation appears more reasonable, but validation in more realistic food systems, digestion models, and sensory settings would strengthen the practical relevance.

Response 3: We agree that the cytotoxicity evaluation is limited by the use of a single cell model and that the safety assessment should therefore be interpreted with caution.

In the revised manuscript, we have clarified that the cytotoxicity results are based solely on the Caco-2 cell model and do not fully represent the complexity of the intestinal environment. We have also revised the Discussion to more carefully interpret the observed moderate cytotoxicity at the 10% supplementation level, particularly in buckwheat and oat–buckwheat samples, and to avoid overgeneralization of safety conclusions.

Furthermore, we now emphasize that the 5% supplementation appears to be a more suitable level, balancing functional properties and cytocompatibility. Finally, we have included a statement highlighting the need for further validation in more complex models, including in vitro digestion systems, additional cell models, and sensory evaluation, to confirm the practical applicability of the proposed formulations.

Changes made: page 14 lines 428-434 and 437-444

Comment 4: Finally, the manuscript requires substantial language editing. There are numerous grammatical errors, repetitions, inconsistencies in sample labeling, duplicated entries in tables, and figure/table cross-referencing problems that reduce clarity and professionalism.

Response 4: We fully agree that the language quality and overall clarity of the manuscript required improvement. The manuscript has been thoroughly revised to improve language quality, consistency, and formatting. Grammatical errors, redundancies, and inconsistencies in sample labeling have been corrected, and all tables and figures have been carefully checked for accuracy, duplication, and proper cross-referencing.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been formally accepted for publication.