Evaluation of Sensory Properties and Short-Chain Fatty Acid Production in Fermented Soymilk on Addition of Fructooligosaccharides and Raffinose Family of Oligosaccharides

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes a study aimed at fermenting soymilk with a mixture of 2 supposedly probiotic bacteria and different concentrations of fructose and raffinose (synbiotic). The fermentation under different conditions is then evaluated from a sensorial point of view, checking also the development of acidity, bacterial development, changes in sugar concentrations and SCFA production. It is a classic study of a fermentation with an adequate analytical methodology. However, some conceptual errors appear and there is missing information. The results are presented in an adequate manner but also need improvements. The observations are detailed:

There is a conceptual confusion in my opinion. It is correct to verify the production of SCFA during fermentation given its importance for intestinal health but there is an error in considering that they are postbiotics. According to the consensus obtained by the ISAPP as to what should be considered a postbiotic and what should not. In 2021, ISAPP defined postbiotics as a "preparation of inanimate microorganisms and/or their components that confers a health benefit on the host" (Salminen et al., Nature Reviews/Gastroenterology & Hepatology 18, 649 (2021)). Since the word "components" gave rise to various confusions, ISAPP experts generated other publications to clarify the scope of the word "postbiotic". See: Vinderola et al., Foods 2022, 11, 1077 (see Fig. 2); Vinderola G., Anales de Microbiota, Probioticos y Prebioticos 4(2), 2023, and Vinderola et al., Front. Microbiolog. 14:1324565 (2024). According to all this, SCFA in isolation should not be considered postbiotics and should not be called as such. In this sense, it is necessary to adapt the text, giving it its importance but not giving it the character of postbiotics.

- lines 106-7: it is said that the 2 strains used are probiotics. What is this statement based on?
- 2.6: it is missing to express in which units the concentration of viable cells was measured
- Figures, titles: write "during 48 h of fermentation". Indicate the temperature at which the fermentation was done.
- 3.3: improve the subtitle. Add "concentrations" after "...sucrose"
- lines 283-6: it is missing to indicate the units for the reported counts
- Fig. 4, title: the units of the viable counts used in the Figure are not CFU/ml but log CFU/ml. It is missing to indicate the temperature and time of the fermentation to indicate after how much time the counts were measured. The genus and species are missing in italics
- 3.6: SCFAs are butyric, propionic and acetic acids. However, these were not directly quantified. Instead, methyl esters of SCFAs were quantified. Explain better why the latter were chosen to be determined and whether their health value is equivalent to that of SCFAs
- Fig. 5: it says "SCFA concentration...." but in reality methyl esters were measured. Replace "concentration" with "production"
- Table 1: the temperature and duration of fermentation are missing since the sensory parameters were determined after fermentation

Author Response

Response to Reviewers-MAJOR REVISIONS

Manuscript ID: fermentation-3479400
Type of manuscript: Article
Title: Evaluation of Sensory Properties and Short Chain Fatty Acids Production in Fermented Soymilk on Addition of Fructooligosaccharides and
Raffinose Family of Oligosaccharides

1. Reviewer 1
2. The manuscript describes a study aimed at fermenting soymilk with a mixture of 2 supposedly probiotic bacteria and different concentrations of fructose and raffinose (synbiotic). The fermentation under different conditions is then evaluated from a sensorial point of view, checking also the development of acidity, bacterial development, changes in sugar concentrations and SCFA production. It is a classic study of a fermentation with an adequate analytical methodology. However, some conceptual errors appear and there is missing information. The results are presented in an adequate manner but also need improvements. 

Response: Thank You for your careful observations and valuable comments. In accordance with your recommendations, we have made every effort to improve the manuscript.

 

2. There is a conceptual confusion in my opinion. It is correct to verify the production of SCFA during fermentation given its importance for intestinal health but there is an error in considering that they are postbiotics. According to the consensus obtained by the ISAPP as to what should be considered a postbiotic and what should not. In 2021, ISAPP defined postbiotics as a "preparation of inanimate microorganisms and/or their components that confers a health benefit on the host" (Salminen et al., Nature Reviews/Gastroenterology & Hepatology 18, 649 (2021)). Since the word "components" gave rise to various confusions, ISAPP experts generated other publications to clarify the scope of the word "postbiotic". See: Vinderola et al., Foods 2022, 11, 1077 (see Fig. 2); Vinderola G., Anales de Microbiota, Probioticos y Prebioticos 4(2), 2023, and Vinderola et al., Front. Microbiolog. 14:1324565 (2024). According to all this, SCFA in isolation should not be considered postbiotics and should not be called as such. In this sense, it is necessary to adapt the text, giving it its importance but not giving it the character of postbiotics.

Response: Thank you for your insightful remarks and keen observations. We have carefully reviewed our manuscript in accordance with your insightful recommendations after thoroughly going through this paper (Vinderola et al., 2024, https://doi.org/10.3389/fmicb.2023.1324565). The clarification by ISAPP experts clears that ‘a bacterial metabolite or a mix of metabolites does not comply with the definition of a postbiotic’. Therefore, they have recommended to describe ‘Short Chain Fatty acids’ as ‘microbial metabolites’ instead of ‘postbiotics’, owing to the fact that-short chain fatty acids, are not living things and so cannot have an ‘after-life.’ Therefore, in our manuscript, SCFAs have been described as ‘metabolites’ instead of ‘postbiotics’ after the revision.

Reference: Vinderola, G., Sanders, M. E., Cunningham, M., & Hill, C. (2024). Frequently asked questions about the ISAPP postbiotic definition. Frontiers in Microbiology, 14, 1324565.

 

3. Lines 106-7: it is said that the 2 strains used are probiotics. What is this statement based on?

Response: Thank You for your valuable comment. In our study, 2 bacterial species are used to carry out co-culture fermentation in soymilk. L. rhamnosus JCM1136 is a type strain where its probiotic characteristics have already been established (https://www.jcm.riken.jp/cgi-bin/jcm/jcm_number?JCM=1136).

1. confusa 30082b is a probiotic strain procurred from National Centre for Cell Science (NCCS) Pune, India (http://ncmr.nccs.res.in/ncmr/catalogue#cataloguesearch) (Sasi et al., 2023; Joglekar et al., 2023; Hasan et al., 2024). The probiotic characteristics of both the strains have already been established. Both the strains are categorised in the “Generally Recognized As Safe” (GRAS).
2. Joglekar, A., Nimonkar, Y., Bajaj, A., & Prakash, O. (2023). Resolution of inter/intraspecies variation in Weissella group requires multigene analysis and functional characterization. Journal of Basic Microbiology, 63(2), 140-155.
3. Hasan, M., Tripathi, K., Harun, M., Krishnan, V., Kaushik, R., Chawla, G., ... & Kumar, M. (2024). Unravelling the effect of extraction on anthocyanin functionality and prebiotic potential. Heliyon, 10(11).
4. Sasi, M., Kumar, S., & Dahuja, A. (2024). Development of probiotic containing soy-based functional foods with Weissella

 

 

4. 6: It is missing to express in which units the concentration of viable cells was measured.

Response: Thank you for your careful observation and valuable comment. The unit in which the concentration of viable cells was measured is CFU/ml. As per your suggestion, the unit is incorporated in the section 2.6 (Line No. 175-176).

 

5. Figures, titles: write "during 48 h of fermentation". Indicate the temperature at which the fermentation was done.

Response: Thank You for your keen observations and comment. Soymilk fermentation and incubation of MRS plates were performed at 37°C. The suggestion has been incorporated in the manuscript (Line No. 258; 277; 302; 326; 353; 373).

 

6. 3: improve the subtitle. Add "concentrations" after "...sucrose"

Response: Thank You for your insightful remark. The word ‘concentrations’ is added as per your valuable suggestion (Line No.279).

 

7. Lines 283-6: It is missing to indicate the units for the reported counts

Response: Thank You for your thoughtful observation and the comment. The units for the reported counts are CFU/ml and currently incorporated in the manuscript wherever missing in the section 3.4 as per your suggestion (Line No. 311-314).

 

8. 4, title: the units of the viable counts used in the Figure are not CFU/ml but log CFU/ml. It is missing to indicate the temperature and time of the fermentation to indicate after how much time the counts were measured. The genus and species are missing in italics.

Response: Thank You for your careful observations and suggestions. All the corrections have been done in the manuscript. 

1. The unit given was CFU/ml but is now corrected to log CFU/ml (Line No. 322).
2. Fermentation and co-culture incubation were performed at 37°C for 24 hours. Detail added (Line No. 325-326).
3. The genus name and species names are italicized (Line No. 324).

 

9. 6: SCFAs are butyric, propionic and acetic acids. However, these were not directly quantified. Instead, methyl esters of SCFAs were quantified. Explain better why the latter were chosen to be determined and whether their health value is equivalent to that of SCFAs.

Response: We really appreciate your thoughtful insights.

SCFAs like propionic, and butyric acids are highly polar and volatile, making them difficult to analyse directly. SCFAs often exhibit poor peak resolution due to their high polarity and strong interactions with the column. Derivatization into methyl esters increases their volatility, making them more suitable for gas chromatography (GC) analysis. Estimating SCFAMEs instead of free SCFAs allows for more accurate, sensitive, and reproducible analysis, especially when using gas chromatography-based techniques.

 

10. 5: it says "SCFA concentration...." but in reality, methyl esters were measured. Replace "concentration" with "production".

Response: Thank You for your careful observation and suggestion. Correction done in Fig 5 and replaced the word "concentration" with "production" in the legend (Line No. 350).

 

11. Table 1: the temperature and duration of fermentation are missing since the sensory parameters were determined after fermentation

Response: Thank You for your thoughtful comment. The suggestion has been incorporated in the legend of Table 1 (Line No. 372-373).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

This study investigated the fermentation process of Lacticaseibacillus rhamnosus JCM1136 and Weissella confusa 30082b with the addition of fructooligosaccharides (FOS) and raffinose family oligosaccharides (RFOs). Parameters such as pH, titratable acidity, probiotic cell count, sugar content (including raffinose, stachyose, glucose, galactose, fructose, and sucrose), and short-chain fatty acids (propionate and butyrate) were analyzed. Sensory evaluation of the fermented beverages was conducted with 25 participants.

 

 

The following suggestions are offered for revision.

1. All figure legends should have a brief, succinct title. (Figure 1, 2, 3)

 

2. There is no Table S2 in the manuscripts. Please check the correctness.

 

3. The table titles of table S2~7 shall be rewritten.

 

4. Methyl propionate and methyl butyrate do not belong to the category of short-chain fatty acids. It is misleading to suggest that these esters were present in the fermentation process. (Line 34~34 and section 3.6)

 

5. The preparation products for sensory analysis shall be presented in the method.

 

Line 4

It should be Fructooligosaccharides.

 

Line 124

How to prepare 2% and 3% RFO?

 

Line 129~130

stirred at 250g

Please check the correctness.

 

Line 140 (section 2.4)

The authors used Raffinose/Sucrose/D-Glucose Assay Kit (K-RAFGL) for the determination the concentration of glucose, sucrose and RFOs.

How can the concentrations of stachyose and raffinose be determined individually?

 

Line 153

Glucose Oxidase / Peroxidase

 

Line 156 (section 2.5)

How can the concentrations of galactose be determined?

 

Line 220~221

In our study (Fig. 1a), the pH change of synbiotic soymilk was monitored after 0, 12, 24, 36, and 48 h fermentation.

However, the data presented in Figure 1A show that the pH values were measured every 3 hours.

 

Line 220~231

The representation of significant figures should be consistent.

For example, 6.0±0.2, 4.2±0.1.

 

Line 253~254

The concentration of raffinose for soymilk supplemented with 2% FOS and 3% FOS decreased by 4.22-fold and 4.7-fold, respectively.

The description is not consistent with Figure 2A. Please check the correctness.

 

Line 254~256

Similarly, the concentration of stachyose decreased by 1.08-fold and 1.16-fold, respectively (Table S4).

Is the description about supplemented with 2% FOS and 3% FOS? The data are shown in Figure 2A, but not Table S4.

The description is not consistent with Figure 2A.

Please check the correctness.

 

Line 272~274

the sucrose content decreased after 24 h of fermentation by 1.29-fold in 3% FOS and 1.93-fold in 2% RFOs in synbiotic soymilk (Fig. 3d).

The description is not consistent with Figure 3D. Please check the correctness.

 

Line 280

Change in galactose concentration

It should be sucrose.

 

Line 290~291 and Line 30

MRS broth with 3% FOS instead of glucose showed a lesser viable count of 6.2 ×10⁷ CFU/ml.

The description is not consistent with Figure 4. Please check the correctness.

 

Line 313

0.51±0.08 mg/mL. However, it is 0.51±0.02 mg/mL in Table S5.

Please check the correctness.

 

Line 324 (Figure 5)

during 0 and 24 h of fermentation.

However, only 24 h fermentation data were shown. The figure legend of figure 5 should be rewritten.

 

Line 403

It should be Streptococcus salivarius subsp. Thermophiles.

 

Line 431

b-glucosidase should be b-fructosidase.

 

Line 438 (Figure 7)

(1) It should be Melibiose.

(2) Stachyose is a tetrasaccharide sugar.

(3) Verbascose is a pentasaccharide sugar.

Please check the correctness.

 

Table S7 ANOVA for colour, mouthfeel, consistency, taste, flavour and overall liking.

It should be Table S6.

Author Response

Response to Reviewers-MAJOR REVISIONS

Manuscript ID: fermentation-3479400
Type of manuscript: Article
Title: Evaluation of Sensory Properties and Short Chain Fatty Acids Production in Fermented Soymilk on Addition of Fructooligosaccharides and Raffinose Family of Oligosaccharides

Reviewer 2

The following suggestions are offered for revision.

1. All figure legends should have a brief, succinct title. (Figure 1, 2, 3)

 Response: We are grateful for your valuable comment. In accordance with your recommendations, titles for each figure legend have been added to the manuscript as follows:

Fig 1: Change in pH of synbiotic soymilk (Line No. 256)

Fig 2: Change in oligosaccharides concentration in synbiotic soymilk (Line No. 274)

Fig 3: Change in monosaccharides and sucrose concentration in synbiotic soymilk (Line No. 299)

Fig 4: Measurement of Viable count of bacteria in synbiotic soymilk (Line No. 321)

Fig 5: Concentration of methyl propionate (MeP) and methyl butyrate (MeB) esters in synbiotic soymilk (Line No. 350-351)

Fig 6: Principal Component Analysis (PCA) biplot as a sensory assessment tool for synbiotic soymilk (Line No. 378-379)

Fig 7: Scheme for RFOs and FOS utilization by probiotic bacteria (Line No. 472).

 

2. There is no Table S2 in the manuscripts. Please check the correctness.

 Response: Thank You for your careful observation and valuable comment. Table No. S2 was not mentioned in the manuscript unintentionally. As per your suggestion, it has been incorporated in the section 3.1 (Line No. 254).

 

3. The table titles of table S2~7 shall be rewritten.

 Response: We appreciate your insightful remarks.

The table titles have been rewritten as follows:

Table S2: ANOVA Results for Synbiotic Soymilk, MRS Broth, and pH and TA-Synbiotic soymilk at different time points

Table S3: DMRT Multiple Comparisons of means, SE(m), and CV for Synbiotic Soymilk Viability, and pH, TA-synbiotic soymilk

Table S4: ANOVA Results for FOS, RFOs, and SCFA concentrations at 0h and 24h

Table S5: Mean, Standard Deviation, CV, and DMRT comparisons at α=0.05 for FOS, RFOs, and SCFA Concentrations

Table S6: ANOVA Results for Sensory Attributes: color, mouthfeel, consistency, taste, flavor, and overall liking

Table S7: DMRT Multiple Comparisons of means at α=0.05, SE(m), and CV for sensory attributes

 

4. Methyl propionate and methyl butyrate do not belong to the category of short-chain fatty acids. It is misleading to suggest that these esters were present in the fermentation process. (Line 34~34 and section 3.6)

Response: Thank you for your insightful remarks and keen observations.  

Methyl propionate and methyl butyrate are esters derived from SCFAs, so their presence could still be relevant in fermentation studies. Since FAMEs are directly derived from SCFAs, their concentrations reflect the SCFA content in the fermentation sample.

SCFAs like propionic, and butyric acids are highly polar and volatile, making them difficult to analyse directly. Derivatization into methyl esters increases their volatility, making them more suitable for gas chromatography (GC) analysis. Also, SCFAs often exhibit poor peak resolution due to their high polarity and strong interactions with the column. Estimating SCFAMEs instead of free SCFAs allows for more accurate, sensitive, and reproducible analysis, especially when using gas chromatography-based techniques. Therefore, fatty acids are commonly derivatized into their methyl ester or trimethylsilyl ester derivatives for GC-MS analysis.

 

5. The preparation products for sensory analysis shall be presented in the method.

 Response: Thank You for your thoughtful comment. Details of the sample preparation have been briefly added in the method section-2.9 (Line No. 215-219).

6. Line 4 It should be Fructooligosaccharides.

Response: We appreciate your careful observation. Correction done in the title (Line No. 3).

 

7. Line 124 How to prepare 2% and 3% RFO?

 Response: Thank You for your comment.

For 2% (w/v) RFOs: Add 2 g of RFOs per 100 mL of soymilk.

For 3% (w/v) RFOs: Add 3 g of RFOs per 100 mL of soymilk.

The concentration of RFOs in unfermented soymilk (control), unfermented soymilk with known concentration of RFOs added (at 0 hour) and soymilk fermented for 24 hours with known concentration RFOs added were estimated in this study.

 

8. Line 129~130 stirred at 250g. Please check the correctness.

 Response: Thank You for your careful observation and valuable comment. We entered the unit incorrectly as "g" instead of "rpm." The manuscript has now been updated to reflect the correction (Line No.135).

 

9. Line 140 (section 2.4)

The authors used Raffinose/Sucrose/D-Glucose Assay Kit (K-RAFGL) for the determination the concentration of glucose, sucrose and RFOs. How can the concentrations of raffinose be determined individually?

 Response: Thank You for your careful observations and valuable comments.

The concentration of raffinose was measured Megazyme Raffinose/D-Galactose Assay Kit (K-RAFGA).

 

10. Line 153 Glucose Oxidase / Peroxidase

 Response: Thank You for your careful observation and valuable comment. Correction done.

 

11. Line 156 (section 2.5) How can the concentrations of galactose be determined?

 Response: Thank You for your careful observation and valuable comment. The concentration of free D-galactose was measured by Megazyme Raffinose/D-Galactose Assay Kit (K-RAFGA):

 

12. Line 220~221

In our study (Fig. 1a), the pH change of synbiotic soymilk was monitored after 0, 12, 24, 36, and 48 h fermentation. However, the data presented in Figure 1A show that the pH values were measured every 3 hours.

Response: Thank You for your careful observation and the comment. Correction done in Fig 1a.

 

13. Line 220~231

The representation of significant figures should be consistent. For example, 6.0±0.2, 4.2±0.1.

 Response: We appreciate your insightful feedback. As per your suggestions, significant figures have been kept consistent in the results for pH in the section 3.1 (Line No. 233, 236, 237, 238, 240, 241,242, 243).

 

14. Line 253~254

The concentration of raffinose for soymilk supplemented with 2% FOS and 3% FOS decreased by 4.22-fold and 4.7-fold, respectively. The description is not consistent with Figure 2A. Please check the correctness.

 Response: We appreciate your insightful remarks and thorough observations. Due to a calculation error, the description has been now updated. Rather than using fold change, the findings are now shown in the original values (Line No. 264-272).

 

15. Line 254~256

Similarly, the concentration of stachyose decreased by 1.08-fold and 1.16-fold, respectively (Table S4). Is the description about supplemented with 2% FOS and 3% FOS? The data are shown in Figure 2A, but not Table S4. The description is not consistent with Figure 2A. Please check the correctness.

 Response: We appreciate your insightful remarks and thorough observations.  ‘Table S4’ denotes the level of significance of fermentation treatment. The direct results are given in Figure 2A only.

16. Line 272~274

The sucrose content decreased after 24 h of fermentation by 1.29-fold in 3% FOS and 1.93-fold in 2% RFOs in synbiotic soymilk (Fig. 3d). The description is not consistent with Figure 3D. Please check the correctness.

 Response: We appreciate your insightful remarks and thorough observations. The description has now been updated (293-297).

 

17. Line 280 Change in galactose concentration. It should be sucrose.

 Response: Thank You for your careful observations and valuable comments. Correction done (Line No. 305).

 

18. Line 290~291 and Line 30

MRS broth with 3% FOS instead of glucose showed a lesser viable count of 6.2 ×10⁷ CFU/ml. The description is not consistent with Figure 4. Please check the correctness.

 Response: We appreciate your insightful remarks and thorough observations. In order to provide readers with greater clarity, the results are presented in log CFU/ml in Figure 4 but in CFU/ml in the paper. Because of this, it appears that the description and Figure 4 are inconsistent.

 

18. Line 313

0.51±0.08 mg/mL. However, it is 0.51±0.02 mg/mL in Table S5. Please check the correctness.

 Response: We appreciate your insightful remark. Typing error has been resolved (Line No. 341).

 

19. Line 324 (Figure 5)

during 0 and 24 h of fermentation. However, only 24 h fermentation data were shown. The figure legend of figure 5 should be rewritten.

 Response: Thank You for your careful observation. Correction done in the legend of Fig 5 (Line No. 353).

20. Line 403

It should be Streptococcus salivarius subsp. Thermophiles.

Response: We appreciate your thorough observation and valuable comment. Correction done in the manuscript (Line No. 389, 435).

21. Line 431

b-glucosidase should be b-fructosidase.

 Response: Thank You for your careful observation and valuable comment. β- fructosidase added (Line No. 464).

22. 22. Line 438 (Figure 7)

(1) It should be Melibiose (2) Stachyose is a tetra saccharide sugar (3) Verbascose is a pentasaccharide sugar. Please check the correctness.

 Response: Thank You for your careful observations and valuable comments. Correction done in Figure 7.

22. Table S7ANOVA for colour, mouthfeel, consistency, taste, flavour and overall liking. It should be Table S6.

Response: We appreciate your insightful remarks and thorough observations. According to our supplementary file for tables and figures, ANOVA for colour, mouthfeel, consistency, taste, flavour and overall liking should be Table S7. Therefore, Table S6 has been removed from section 3.6 in the manuscript (Line No. 356).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily responded to the comments made and the manuscript now appears much improved.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors response well, so I have no more suggestion.