Determining Fermentation Conditions to Enhance Antioxidant Properties and Nutritional Value of Basil Seeds Using Lactobacillus plantarum

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors, your research and results are very interesting. I have suggestions for improving the paper, before its publication:

1) In the introduction section, was it not clear what is the gap in knowledge that encourages authors to carry out this research? as well as the hypothesis.

2) Methodology section needs information:

a) Sample preparation was based on what methodology? already published? if yes, you need to reference it; or was developed by the authors. It is important to make it clear to the reader why these fermentation times and temperatures were defined. In much of the methodology section, the authors had this concern.

b) the caption of table 1 is general and does not say "nothing". Figure and table captions must be self-explanatory; It took me a long time to understand the data in this table. To improve.

c) statistical analysis needs to be described in greater detail; for I know not what the residue of error is; whether the data had a normal distribution; if any data needed to be transformed to normalize, before performing the anova test.

d) looking at your design, could other statistical analyzes not explain your results better? a regression analysis? or a comparison of means using orthogonal contrasts. Reflect on this.

4) in tables 2 and 3 and figure 2; we have data, I believe, presented in the form of averages; but there is no illustration of statistical differences in these data.....wasn't it done? or there is no difference; this needs to be clear. Format the tables so that they show the essence of the study.

5) Many data presented in tables and figures that do not seem to differ; I recommend these that could be in "supplementary material" and thus reduce the pollution of the tables.

6) in figure 3 an asterisk was added; but it was not made clear what they should show.....improve your subtitles.....

7) review all your tables and figures; example fig 7. I can't see anything; when I get closer, the writing becomes distorted; showing poor resolution of images.

8) I liked your discussion, it is short and direct.

9) conclusion must answer the objectives: to think.

10) review the formatting according to the journal’s standards; as well as references.

Author Response

REBUTTAL FOR REVIEWER(S) COMMENTS

We thank all reviewers for their helpful comments & critiques and have responded below in a point-by-point reply. For convenience, responses are color-coded for each reviewer, with corresponding changes in the manuscript main text indicated with the same color scheme:

                  Reviewer #1: red

 

 

 

Reviewer1:

 

Question:

In the introduction section, was it not clear what is the gap in knowledge that encourages authors to carry out this research? as well as the hypothesis.

 

Response: Thank you for taking the time to review our manuscript and for your valuable comments and suggestions. We changed the context based on the comments.

Based on your suggestion, we have refined the content to better convey our objectives.Please find the changes below:

Fermentation is a traditional method that imparts unique aroma, flavor, and texture to food, enhances digestibility and water-soluble vitamins content, degrades anti-nutritional factors, converts phytochemicals such as polyphenols into more bioactive and bioavailable forms, produces antioxidant components which able to decrease/eliminate Reactive oxygen species (ROS) and oxidative stress, and enriches the nutritional quality of food

 

 Lactic acid bacteria (LAB), particularly probiotics can contribute to proliferation of the nutritional quality (e.g., increasing Vitamin levels) and show a positive effect on addressing oxygen radicals during fermentation process

One of the medicinal plant rich in high dietary fiber and protein is Basil seeds (Ocimum basilicum L.) belong to the Lamiaceae family and are increasingly common in Asian and Central American countries (Majdinasab et al., 2020).

Therefore, this research aims to optimize the environmental conditions of basil seed fermentation including varying the amounts of inoculum water and fermentation time. The hypothesis is that fermenting and optimizing basil seeds fermentation can result in a functional diet enriched with antioxidant components and enhanced nutritional value, thereby promoting health.

 

 

 

2) Methodology section needs information:

1. a) Question: Sample preparation was based on what methodology? already published? if yes, you need to reference it; or was developed by the authors. It is important to make it clear to the reader why these fermentation times and temperatures were defined. In much of the methodology section, the authors had this concern.

 

Response: Thank you for this important point. We followed established protocols from other studies for the entire process, including meshing, sterilization, the amount of starter, and the duration of fermentation. However, to determine the appropriate amount of water, we relied on the physical properties and water-holding capacity of the seed powder. After conducting preliminary tests, we found that due to the gummy nature of basil seeds, it was not feasible to add less water than the amount we selected. We added the explanation in the context and the references in first paragraph of methodology shown below.

 

(this amount of water was chosen based on the physical properties and water holding capacity of basil seeds)

 

After inoculating 5% (ml/ml) of activated L. planetarum into the sterilized-basil media for the final bacteria concentration of approximately 1 × 10⁸ cfu/mL, samples were incubated at 37 °C for 24, 48, 72, and 96 hours [26].

 

1. b) Question: the caption of table 1 is general and does not say "nothing". Figure and table captions must be self-explanatory; It took me a long time to understand the data in this table. To improve.

 

Response: Thankyou for addressing this important point. We added information to table 1. Description of fermented and unfermented basil samples. The aliphatic characteristics indicate the ratio of basil powder (g) to water (mL), and the numbers determine fermentation time(hr).

 

1. c) Question: statistical analysis needs to be described in greater detail; for I know not what the residue of error is; whether the data had a normal distribution; if any data needed to be transformed to normalize, before performing the ANOVA test.

 

Response: Thankyou for this point. We conducted outlier identification and the Shapiro-Wilk normality test using R software. The results are presented in the following figures, confirming that the data followed a normal distribution. Outlier identification figures for DPPH, TPC, and ABTS are shown below, respectively.

 

 

 

1. d) Question: looking at your design, could other statistical analyzes not explain your results better? a regression analysis? or a comparison of means using orthogonal contrasts. Reflect on this.

 

Response: Thankyou. We performed the outlier statistical analysis and after comparison, found no significant differences. Therefore, we used one-way ANOVA to compare all the data together. The following description below has been added to the statistical analysis method section.

 

The analytical procedures were performed using R software. Results are presented as mean ± standard error of the mean (SEM; n = 3). Shapiro-Wilk and outlier test were used to assess the normality of data distribution and homogeneity of variance, respectively. The samples were analysed using a one-way analysis of variance (ANOVA) at a significance level of p < 0.05. All experiments were conducted in triplicates. The figures were created using GraphPad Prism 8.0 (GraphPad Software, San Diego, USA).

 

 

4) Question: in tables 2 and 3 and figure 2; we have data, I believe, presented in the form of averages; but there is no illustration of statistical differences in these data..... wasn't it done? or there is no difference; this needs to be clear. Format the tables so that they show the essence of the study.

 

Response: Thankyou for this point. A significant difference in pH was observed between time 0 and all subsequent time points, as indicated by the letters 'a' and 'b'. However, no significant differences were noted among the other time points (24, 48, 72, and 96 hours). Regarding the Total Microbial Count (TMC), no significant differences were observed between the groups at any time point.

 

 

Samples/Time	0		24 hours		48 hours		72 hours			96 hours
	pH	TMC1	pH	TMC1	pH	TMC1	pH	TMC1		pH	TMC1
A	6.05a±0.07	8.92±0.85	5.19b±0.03	11.98±0.81	5.22b±0.13	11.99±0.11	5.24b± 0.01		12.20±0.058	5.24b± 0.08	12.89±0.55
B	6.23a± 0.16	8.92±0.85	5.22b± 0.07	11.79±0.91	5.22b± 0.02	11.82±0.03	5.21b± 0.25		12.00±0.12	5.22b± 0. 42	12.48±0.70
C	6.21a± 0.04	8.92± 0.85	5.12b± 0.04	11.61± 1.04	5.14b± 0.02	11.80± 0.32	5.19b± 0.01		12.44±0.14	5.20b± 0.218	12.62± 0.61
D	6.27a± 0.05	8.92± 0.85	5.16b± 0.02	11.75±2.04	5.17b± 0.01	11.90± 0.21	5.18b± 0.06		12.12±0.25	5.19b± 0.11	12.24± 0.16
E	6.24a± 0.01	8.92± 0.85	5.11b± 0.04	12.26± 1.05	5.12b± 0.01	12.25± 0.33	5.13b± 0.02		12.40± 0.33	5.22b± 0.27	12.72±0.761
F	6.31a± 0.02	8.92± 0.85	5.17b± 0.05	12.38± 0.79	5.18b± 0.06	12.46± 0.33	5.19b± 0.01		12.70± 0.44	5.22b± 0.11	13.15± 0.84
G	6.29a± 0.03	8.92± 0.85	5.17b± 0.01	12.73± 0.95	5.22b± 0.04	12.48± 0.12	5.16b± 0.07		12.50± 0.83	5.26b± 0.12	12.48± 0.82
1 TMC unit is cfu/mL; The difference between the two groups is illustrated in letters (a) and (b). Samples with no letters are not significantly different

 

5) Question: Many data presented in tables and figures that do not seem to differ; I recommend these that could be in "supplementary material" and thus reduce the pollution of the tables.

 

Response: Thank you for the insightful comment, we added table 4 as a supplementary data.

 

 

Supplementary:

Free radical scavenging ability of extracted fermented and unfermented basil samples (DPPH and ABTS•) and TPC

Samples name	DPPH%	TPC (mg GA /100g)	ABTS mg GA / g
A-24	43.99 ± 1.87 bc	1479.81± 25.9 g	51.77± 2.51 efgh
A-48	45.38 ± 0.721 bc	1638.35± 14.3 cdefg	54.92± 2.04 cdefg
A-72	41.45 ± 1.59 c	1606.46± 52.6 defg	51.00± 1.13 efghi
A-96	40.76 ± 1.4 c	1652.97± 64.7 cdefg	46.45± 3 hij
A-c	11.43 ± 3.25 d	731.56± 50.2 h	5.049± 1.07 k
B-24	44.78 ± 0.547 bc	1606.46± 10.1 defg	55.24± 1.06 cdef
B-48	49.30 ± 1.01 abc	1727.56± 16.3 abcde	53.83± 2.79 cdefg
B-72	44.82 ± 2.39 bc	1645.66± 62.4 cdefg	53.44± 1.46 cdefg
B-96	44.80 ± 2.26 bc	1681.64± 15.8 bcdef	50.49± 1.64 fghi
B-c	15.49 ± 5.76 d	750.58± 135 h	4.66± 0.77 k
C-24	46.73 ± 0.569 abc	1560.25± 22.6 efg	56.01± 1.75 cde
C-48	48.07 ± 1.72 abc	1743.65± 34.8 abcde	54.08± 0.588 cdefg
C-72	46.25 ± 1.77 abc	1667.89± 119 bcdefg	58.06± 0.889 bc
C-96	44.09 ± 1.85 bc	1705.62± 29 bcdef	51.39± 4.15 efghi
C-c	13.34 ± 6.36 d	758.77± 81.5 h	5.62± 0.77 k
D-24	44.78 ± 1.04 bc	1510.81± 51 fg	52.9± 0.969 cdefg
D-48	46.59 ± 0.367 abc	1650.05± 44.1 cdefg	54.66± 2.06 cdefg
D-72	43.12 ± 0.504 c	1512.86± 117 fg	52.42± 1.9 defg
D-96	42.92 ± 2.26 c	1746.58± 127 abcde	46.25± 0.578 ij
D-c	14.35 ± 5.38 d	813.47± 68.2 h	3.89± 0.77 k
E-24	42.34 ± 4.52 c	1641.86± 66.8 cdefg	53.96± 1.17 cdefg
E-48	48.83 ± 1.41 abc	1765± 20 abcd	54.47± 1.44 cdefg
E-72	45.54 ± 1.12 bc	1666.14± 22.6 bcdefg	52.48± 1.16 defg
E-96	43.83± 1.45 c	1723.76± 4.92 bcde	49.72± 1.17 ghij
E-c	11.11± 3.38 d	735.95± 38.6 h	4.27± 0.77 k
F-24	45.88± 0.31 abc	1643.61± 41.2 cdefg	57.29± 1.79 cd
F-48	48.93± 1.47 abc	1785.48± 38.6 abcd	55.56± 1.83 cdef
F-72	49.06± 2.11 abc	1830.53± 27.3 abc	55.43± 2.44 cdef
F-96	41.89± 1.52 c	1625.77± 38 defg	44.71± 0.509 j
F-c	15.71± 7.24 d	815.81± 77.9 h	3.89± 0.77 k
G-24	49.32± 1.26 abc	1781.97± 38.2 abcd	63.65± 0.588 a
G-48	55.38± 2.54 a	1919.74± 60.4 a	63.14±  0.868 ab
G-72	53.49± 2.2 ab	1858.02± 59 ab	64.68± 1.47 a
G-96	46.52± 1.11 abc	1728.44± 57.7 abcde	51.32± 1.28 efghi
G-c	12.83± 5.97 d	785.68± 6.2 h	4.66± 0.77 k

 

 

6) Question: in figure 3 an asterisk was added; but it was not made clear what they should show.....improve your subtitles .....

 

Response: Thank you for pointing this out. We added information to Figure 3: The Aliphatic characteristics show the Ratio of basil powder (g): water (mL), and the numbers indicate fermentation time (hr).

• We complete information of figure 2, 4, 5 and 6: The Aliphatic characteristics show the Ratio of basil powder (g): water (mL), and the numbers indicate fermentation time (hr).

 

• Also add information to figure 7: The Aliphatic characteristics show the Ratio of basil powder (g): water (mL), and the numbers indicate fermentation time (hr). Data are expressed as mean ± SD, n = 3; The stars are used to indicate levels of significance at four recognized thresholds. A p-value less than 0.05 = *, less than 0.001= *** and less than 0.0001 = ****.

 

 

7) Question: review all your tables and figures; example fig 7. I can't see anything; when I get closer, the writing becomes distorted; showing poor resolution of images.

 

Response:

• Thank you for the important point. The figure 7 was replaced, also shown here below.

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The work fermentation-3174571 is devoted to the study of the effect of fermentation conditions of basil seeds using Lactobacillus plantarum on the antioxidant and nutritional value of the seeds.

The work describes the methodology in detail, carefully conducted research and statistical analysis, so the reliability is beyond doubt. The work deepens and expands knowledge about the fermentation of basil seeds. The work corresponds to the journal Fermentation, as well as the Special Issue

Probiotics, Prebiotics and Their Use as Innovative Ingredients in Food Technology.

Notes:

1) It is recommended to change the title of the work, since optimization requires the involvement of mathematical design of experiments and the construction of a mathematical model. In this case, the dependence of fermentation on the quantitative ratio of seeds: water was studied

2) The results of the work show that the more water, the better the fermentation. It is necessary to either expand the range and add fermentation with a higher water content, or introduce a justification that a further increase in humidity will lead to non-solid-state fermentation.

3) It is necessary to justify why solid-phase fermentation was chosen rather than liquid-phase fermentation

4) In the conclusions, the appearance of the term SSF is unclear, SSF was not discussed in the work. SSF is usually used when enzyme preparations and microorganisms are used, in this case, fermentation was carried out using only Lactobacillus plantarum.

Author Response

REBUTTAL FOR REVIEWER(S) COMMENTS

We thank all reviewers for their helpful comments & critiques and have responded below in a point-by-point reply. For convenience, responses are color-coded for each reviewer, with corresponding changes in the manuscript main text indicated with the same color scheme:

 Reviewer #2: blue

Reviewer2:

1) Question: It is recommended to change the title of the work, since optimization requires the involvement of mathematical design of experiments and the construction of a mathematical model. In this case, the dependence of fermentation on the quantitative ratio of seeds: water was studied.

 

Response: Thank you for making this point. We choose title following some previous publications.

Please find some articles did optimizing of time, pH, temperature to enhance functional properties.

Sharma et al. (2020) Optimized the fermentation condition to obtain maximum functional properties (Proteolytic activity, Antioxidant activity and ACE inhibition %) was investigated using response surface methodology.

Also, Lin et al. (2024) investigated the effect of optimizing ginseng fermentation to produce functional cosmetics 

Also we changed the title” Determining Fermentation Conditions to Enhance the Antioxidant Properties and Nutritional Value of Basil Seeds Using Lactobacillus plantarum”.

 

2) Question: The results of the work show that the more water, the better the fermentation. It is necessary to either expand the range and add fermentation with a higher water content or introduce a justification that a further increase in humidity will lead to non-solid-state fermentation.

 

Response: Thank you for this very important point. We chose solid-state fermentation (SSF) because it is conducted on a solid substrate with sufficient moisture content but without free water. However, based on your suggestion, we adjusted our approach to focus on fermentation in general. The primary aim of our study is to investigate the effect of fermentation conditions on the antioxidant properties and nutritional value of basil seeds. We began by examining the impact of varying water levels on fermentation, starting with the lowest concentration. Our goal was to determine whether lower moisture content could enhance health properties or if higher moisture content would be more beneficial, as no prior research has addressed this question.

 

In Introduction: A biologically safe method that is conducted on a solid substrate without free moisture but with sufficient water content is one of the fermentation method [17][18][19]. Environmental conditions, such as water content and fermentation time, are crucial in determining the final properties of fermented products [15][20][21]

In Discussion: Increasing the number of LAB during fermentation can also enhance the efficiency (%) of antioxidant properties and TPC of extraction.

 

3) Question: It is necessary to justify why solid-phase fermentation was chosen rather than liquid-phase fermentation

Response: Thank you. We chose solid-phase fermentation because the aim of the study was to investigate fermentation conditions on the production of antioxidant compounds and nutritional value. We corrected the method to fermentation.

4) Question: In the conclusions, the appearance of the term SSF is unclear, SSF was not discussed in the work. SSF is usually used when enzyme preparations and microorganisms are used, in this case, fermentation was carried out using only Lactobacillus plantarum.

Response: Thank you for addressing this point. We corrected SSF to the fermentation phrase.

In conclusion section: Our study found that fermenting basil seeds with L. plantarum significantly increased the total polyphenol content and enhanced radical scavenging ability.

 

 

 

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The review of the manuscript was sufficient, it clarified doubts, made corrections, as well as improved the text and therefore I recommend publication.