Review Reports - Development of High-Glucosinolate-Retaining Lactic-Acid-Bacteria-Co-Fermented Cabbage Products

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Manuscript fermentation-3340478

The manuscript presents research to improve health benefits and nutritional characteristics through lactic acid fermentation using a bioreactor, focusing on glucosinolate retention. A consortium of lactic acid bacteria was used as the inoculum. The procedure's efficacy was evaluated by measuring glucosinolate retention.

Abstract

Bioreactor conditions should be included in the abstract.

Specific comments

L35 is enriched?

L39. Italic missing (check across manuscript)

L42. Please use full names and acronyms in parenthesis

L46 The expression is vague.

L68-69. The sentence is not generally true. Specify.

L79. The sentence is unclear. The study does not pretend to control cabbage overproduction. I guess it intends to extend the self-life of the fermented product. Maybe to address the effects of overproduction?

L83-86. Adequate verb tense?

L90. c

L91-92 There is no indication of a significant difference between fresh and fermented products in this case.

L96-98. Where are produced?

L101-103. Your study?

Figure 1 and others. Instead of grouping in the x-axis according to fresh or fermented, comparisons could be straightforward grouping by DPPH, ABTS, or any other parameter. Another question is whether the comparison of percentages using tests developed for normal distribution data is questionable. Other non-parametric tests are recommended.

L122. Please check the sentence's correctness. T test?

L126. Parenthesis necessary?

L133. The research…?

L139. GAE?

Figure 2. Similar comment to Figure 1.

L149 T test. Clarify sentence.

L153 Parenthesis necessary? How were flavonoids formed during fermentation?

L160, Your research?

L162 QE?

L176-177. T-test. Clarify sentence.

L182 Parenthesis necessary?

L204. The exponent can be indicated by the super index

Comments on the Quality of English Language

In general, it is acceptable. Some suggestions are included in the comments to authors.

Author Response

We would like to sincerely thank the reviewers for thoroughly reviewing our manuscript and providing another opportunity to improve it. It is only due to your constructive suggestions that we are able to comprehend our shortcomings and carry out a major revision of this manuscript. In this response, we have carefully addressed the comments and suggestions raised by the reviewers. The changes have been introduced to the main text, and the detailed changes point-by-point are shown below. Upon this revision, we hope that you will find our responses satisfactory and that the revised manuscript is now acceptable for publication.

Abstract

Bioreactor conditions should be included in the abstract.

We thank the reviewer for the comment, following information has been added:

Line 41:

fermentation process (35^oC, 24 hrs of fermentation at 5 rpm).

Specific comments

L35 is enriched?

We revised it as: is rich with…

L39. Italic missing (check across manuscript)

Lactiplantibacillus plantarum

L42. Please use full names and acronyms in parenthesis

Line 43:

1,1-diphenyl-2-picrylhydrazil (DPPH) and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays…

L46 The expression is vague.

Following information has been provided:

Line 48:

…14 days (from 90.34% to 66.49%).

L68-69. The sentence is not generally true. Specify.

We thank you for pointing out the concern, we have removed “on the surface” from the context.

Following change has been made to address the confusion:

Line 80:

To ensure the effective utilization of surplus cabbage

L83-86. Adequate verb tense?

We have done the grammar check, thank you.

L91-92 There is no indication of a significant difference between fresh and fermented products in this case.

The increase of DPPH after fermentation is significant and the decrease of ABTS+ was insignificant. Following change has been made to avoid confusion:

Line 91:

DPPH radical scavenging rate increased by 16.32% (p < 0.05).

L96-98. Where are produced?

Line 100:

…antioxidants, such b-carotene and vitamin E, from…

L101-103. Your study?

Following change has been made:

Line 103:

Zubaidah et al. investigated the changes in antioxidant activity…

We thank the reviewer for the comment, since we are comparing the changes in cabbage before and after fermentation, we will still present them together in the same graph. Thank you for your understanding.

L122. Please check the sentence's correctness. T test?

Following change has been made to avoid confusion.

Line 124:

…are significantly different according to an independent sample t-test.

L126. Parenthesis necessary?

We have removed the parenthesis.

L133. The research…?

Line 136:

The study investigated the changes…

L139. GAE?

GAE means Gallic Acid Equivalents here. We have revised the context as requested.

Line 142:

Gallic Acid Equivalents (GAE)/g…

Figure 2. Similar comment to Figure 1.

L149 T test. Clarify sentence.

Following change has been made to avoid confusion.

Line 152:

…are significantly different according to an independent sample t-test.

L153 Parenthesis necessary? How were flavonoids formed during fermentation?

We have removed the parenthesis.

Many flavonoids in plant tissues, including cabbage, are present in bound forms (such as glycosides or conjugates) that are not easily bioavailable. During fermentation, microbial enzymes (e.g., glycosidases) can break these bonds, releasing free flavonoids, thereby increasing the total flavonoid content.

L160, Your research?

The results were cited from Ref 18.

L162 QE?

We have revised the context as requested.

Line 164:

0.29 μg Quercetin Equivalents (QE)/mg

L176-177. T-test. Clarify sentence.

Line 176:

…significant according to an independent sample t-test depends on…

L182 Parenthesis necessary?

We have removed the parenthesis.

L204. The exponent can be indicated by the super index

Fixed.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript described study on development of a fermented cabbage using lactic acid bacteria. This topic is quite interesting, but more details should be provided and manuscript should be further improved for more clear and solid.

Line 108-110, DPPH and ABTS focused on different compounds when evaluating anti-oxidant activities. As shown in Figure 1, differences were seen in DPPH and ABTS results, authors should further explain the results.
Figure 2, mM GAE/g or mg GAE/g?
Line 189-199, in the scale up study, author described lower the inoculating rate from 3.0% to 0.3% and obtained the similar effect. Author should provide exact cell numbers of inoculation. Lactic acid bacteria is anaerobic bacteria, why need aeration?
Line 221, why 4 C for 21 days, please provide reference.
From Table 1, it seemed lactic acid bacteria would die out after 21 days, what will happen after 21 days? will these data stay stable afterwards? Authors wrote in Lin 247-249 that no significant decline happened after 21 days, please provide data.
What samples were used for storage studies? In current study, authors should have three different samples, lab-fermented, 5-L bioreactor fermented(0.3%), 5-L bioreactor fermented (3%). The three samples should be different.
What is the reasons for the quick drop of LAB, GS, Firmness in the first 7 days? What happened in the first week’s storage? Authors should explain the results.
Line 299-300, what is the cell number of each lactic acid bacteria after inoculation, at the beginning of fermentation?
Authors should give more information on the raw materials used in current study, including geographic and seasonal variability of the cabbage samples.
Section 3.3, authors please provide more information on the fermentation process of cabbage include treatment of cabbages, lab fermentation and scale-up fermentation, reference needed.
line 350, Na2(PdCl4) number should be subscript.
More discussion is needed on the role of lactic acid bacteria regarding increasing the phenolic compounds and flavonoid compounds in fermented products.
Author better separate results and discussion, current version caused difficulties for the readers in telling out which part is the result of this study and which parts are from other researches.

Author Response

Line 108-110, DPPH and ABTS focused on different compounds when evaluating anti-oxidant activities. As shown in Figure 1, differences were seen in DPPH and ABTS results, authors should further explain the results.

We thank the reviewer for the comment, following change has been made:

Lines 95~102:

In the processing of salted and dehydrated cabbage, some water-soluble antioxidants may have been lost, leading to a decrease in the ABTS+ radical scavenging capacity of the fermented cabbage. However, during the lactic acid fermentation process, extracellular polysaccharides with antioxidant properties are produced. Additionally, the enzymatic activity of the bacteria may facilitate the extraction of more fat-soluble antioxidants, such b-carotene and vitamin E, from the samples during ethanol extraction, resulting in an increased DPPH radical scavenging capacity.

Figure 2, mM GAE/g or mg GAE/g?

It’s mg GAE/g. Thank you for pointing out the issue.

Line 189-199, in the scale up study, author described lower the inoculating rate from 3.0% to 0.3% and obtained the similar effect. Author should provide exact cell numbers of inoculation. Lactic acid bacteria is anaerobic bacteria, why need aeration?

In this case, stirring is intended to ensure even fermentation of the cabbage by the lactic acid bacteria, rather than providing oxygen. We have removed “aeration” from the context.

We adjust the lactic acid bacteria concentration to 10⁷ CFU/ml for inoculation.

Line 312:

…lactic acid bacteria (10⁷ CFU/ml) was 0.3% and 3.0% (w/w)…

Line 221, why 4 C for 21 days, please provide reference.

It’s from [33] Wiczkowski, W., Szawara-Nowak, D., Topolska, J. Changes in the

content and composition of anthocyanins in red cabbage and its antioxidant capacity

during fermentation, storage and stewing. 2015, Food Chem. 167, p.115-123. (line 401

in M&M)

From Table 1, it seemed lactic acid bacteria would die out after 21 days, what will happen after 21 days? will these data stay stable afterwards? Authors wrote in Lin 247-249 that no significant decline happened after 21 days, please provide data.

What we claimed here is that we observed that the number of lactic acid bacteria showed no significant change until day 21. Sorry for the confusion. Since this product is intended for short-term sales, we conducted the experiment for only 21 days. (Ref. 33)

What samples were used for storage studies? In current study, authors should have three different samples, lab-fermented, 5-L bioreactor fermented (0.3%), 5-L bioreactor fermented (3%). The three samples should be different.

This is a 5-L bioreactor fermented (0.3%) sample. Following change has been made to avoid confusion:

Line 398:

A 5-L bioreactor fermented (0.3%) sample of lactic acid co-fermented cabbage were stored….

What is the reasons for the quick drop of LAB, GS, Firmness in the first 7 days? What happened in the first week’s storage? Authors should explain the results.

We have provided references and discussion in the context:

For LAB: (lines 233-235)

The decline may be due to that sauerkraut is not a nutrient-rich substrate, and in a 4°C storage environment, resulting in the death of some bacteria and a subsequent decrease in the viable lactic acid bacteria count [23].

For GS: (lines 240-245)

Palani et al. (2016) recorded the changes in the content of glucosinolates compounds in cabbage during fermentation and storage were investigated [24]. The results showed that the content of the glucosinolates glucobrassicin in fermented cabbage significantly decreased after being stored at 4°C for 5 days, while its main decomposition products, ascorbigen and indole-3-carbinol, showed an increasing trend [24].

Lines 258-260:

The firmness also decreased during storage because of the acidification, enzymatic activity and microbial metabolites.

Line 299-300, what is the cell number of each lactic acid bacteria after inoculation, at the beginning of fermentation?

We adjust the lactic acid bacteria concentration to 10⁷ CFU/ml for inoculation.

Line 296:

A 1.0% (v/v) inoculation (10⁷ CFU/ml) of lactic…

Authors should give more information on the raw materials used in current study, including geographic and seasonal variability of the cabbage samples.

Lines 268-270:

…Brassica oleracea var. capitata (Early Autumn cabbage), grown in the central and northern parts of Taiwan being particularly suitable due to their cooler temperatures during the early autumn months, purchased…

Section 3.3, authors please provide more information on the fermentation process of cabbage include treatment of cabbages, lab fermentation and scale-up fermentation, reference needed.

The process were modified from the method by previous research [25]: Song, L.,

Thornalley, P. J. Effect of Storage, Processing and Cooking on Glucosinolate Content

of Brassica vegetables. Food Chem. Toxi. 2007, 45, p.216-224.

https://doi.org/10.1016/j.fct.2006.07.021.

A 1.0% (v/v) inoculation (10⁷ CFU/ml) of lactic acid bacteria culture was added to 200 mL of MRS broth and incubated statically at 30°C for 24 hours. After incubation, the culture was centrifuged at 6,000 ×g to remove the broth, and the total weight of the remaining lactic acid bacteria was measured. The bacterial pellet was resuspended in 10 mL of 0.1% (w/v) peptone water by shaking, creating the lactic acid bacteria solution for fermentation. Each prepared lactic acid bacteria-peptone water mixture was then added to cabbage slices in a ratio that the inoculation amount of each bacterial strain was 1.0% (w/w) of the cabbage slices materials.

line 350, Na2(PdCl4) number should be subscript.

Fixed.

More discussion is needed on the role of lactic acid bacteria regarding increasing the phenolic compounds and flavonoid compounds in fermented products.

The discussion can be found in 2.2 and 2.3. Such as:

In vegetables, phenolic compounds often exist in glycoside form, bound to sugars. During fermentation, the β-glucosidase enzyme produced by lactic acid bacteria can convert some of these bound phenolic compounds into free forms, thereby enhancing the free phenolic content of the fermented product while reducing the bound phenol content [14].

Their results indicated that the total phenolic compound content increased by 0.70% after one month of fermentation, showing no significant difference before and after fermentation [15].

The glycosidases from lactic acid bacteria can degrade the flavonoid glycosides and some phenolic compounds in cabbage, producing free flavonoids, while esterase and tannase help break down flavonol-gallic acid complexes [17]. Additionally, pectinases from lactic acid bacteria can alter the cell wall structure, facilitating the release of more antioxidants. All of these enzymatic actions contribute to the increased total flavonoid content in fermented cabbage [18].

Author better separate results and discussion, current version caused difficulties for the readers in telling out which part is the result of this study and which parts are from other researches.

We thank the reviewer for the comment. The journal provides a fixed writing format, and we follow the journal's guidelines.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

1. Why didn't you store the fermented product more than 21 days?

2. Did you check any contaminant microorganism (such as mold, E.coli, etc.) during the cultivation or storage.

2. The results should be supported by more references than the authors used.

Author Response

Why didn't you store the fermented product more than 21 days?

We thank the reviewer for the comment. Since this product is intended for short-term sales, we conducted the experiment for only 21 days. (Ref. 33)

Did you check any contaminant microorganism (such as mold, E.coli, etc.) during the cultivation or storage.

Since fermented cabbage is an acidic food (pH 4.6), pathogenic bacteria cannot survive in it and it is considered a GRAS (Generally Recognized as Safe) product. Therefore, we did not detect the presence of pathogenic bacteria.

The results should be supported by more references than the authors used.

Thank you. We have incorporated more reference and discussion in our revision.

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Table 1, correct ”mM GAE/g”
DPPH and ABTS surely involved different anti-oxidants, and rely on different radicals. Here, sample compositions and type of anti-oxidants would both affect the results. But no evidence was shown in current study.
Authors claimed here is that we observed that the number of lactic acid bacteria showed no significant change until day 21, but the cell number changed from 7.54 to 3.14 from day 0 to day 14, and N.D. at day 21. Although it is a short-term product, reasonable explanations should be given for this rapid decline of lactic acid bacteria under 4 C storage, which is not normal.
ref 23 mainly discussed fermentation process, not storage. better use other reference.
Since accurately lower the inoculation from 3.0% to 0.3%, and it resulted in similar results, the results should be further discussed.

Author Response

Response to the reviewer 2:

Table 1, correct ”mM GAE/g”

mM GAE/g has been revised as mg GAE/g

DPPH and ABTS surely involved different anti-oxidants, and rely on different radicals. Here, sample compositions and type of anti-oxidants would both affect the results. But no evidence was shown in current study.

DPPH radical scavenging activity primarily measures the antioxidant capacity of compounds. It is commonly used to evaluate the ability of phenolic compounds, flavonoids, polyphenols, and other antioxidants to neutralize free radicals by donating hydrogen or electrons. This method is widely applied in assessing natural products, plant extracts, and synthetic compounds for their potential antioxidative properties.

ABTS radical scavenging activity primarily measures the antioxidant capacity of compounds, similar to DPPH. It is particularly effective for evaluating hydrophilic and lipophilic antioxidants, including phenolic compounds, flavonoids, polyphenols, vitamins (e.g., vitamin C and E), and carotenoids.

Lines 95-102:

Lines 259-261:

In Table 1, we observed a significant reduction in total flavonoid content, which may explain the decrease in DPPH activity from 91.32% to 76.19% after 21 days of storage.

Authors claimed here is that we observed that the number of lactic acid bacteria showed no significant change until day 21, but the cell number changed from 7.54 to 3.14 from day 0 to day 14, and N.D. at day 21. Although it is a short-term product, reasonable explanations should be given for this rapid decline of lactic acid bacteria under 4 C storage, which is not normal.

We thank the reviewer for the comment. The number of LAB showed a significant downward trend during 21 days of storage.

Lines 231-235:

The viable lactic acid bacteria count showed a significant downward trend during the first 0 to 7 days of storage, decreasing by approximately 3 log CFU/g. The decline may be due to that sauerkraut is not a nutrient-rich substrate, and in a 4°C storage environment, resulting in the death of some bacteria and a subsequent decrease in the viable lactic acid bacteria count owing to the low-temperature stress, membrane damage and nutrients depletion [23].

ref 23 mainly discussed fermentation process, not storage. better use other reference.

A new reference has been added: (lines 501-502)

Sionek, B., et al. 2024. The Impact of Physicochemical Conditions on Lactic Acid Bacteria Survival in Food Products. Fermentation 10(6):298.

Since accurately lower the inoculation from 3.0% to 0.3%, and it resulted in similar results, the results should be further discussed.

The possible reason may be the slow agitation, which provides a homogeneous environment for LAB growth.

Lines 202-204:

This result confirms that under reduced initial inoculum conditions, the bioreactor, due to its stirring capabilities, allows lactic acid bacteria to grow rapidly as a dominant strain in the fermentation environment.

Round 3

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

manuscript was improved after revision. I suggest accept after language editing.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The author discusses the potential of lactic acid fermentation technology in enhancing the nutritional and functional quality of cabbage, as well as addressing the issue of overproduction and expanding its market value. The structure of the paper is clear, and the writing is smooth.

My main concern is that the author's design of the control group is incomplete. When exploring the fermentation process, only the fresh and fermented groups were compared, with no other control groups included. Cabbage fermentation is a traditional process with wide applications in many regions, and the microorganisms involved are also diverse. Unfortunately, the author did not include comparisons with these widely used microorganisms or fermentation processes as controls.

The author should remove inappropriate descriptions (Line 56, Asian countries such as Taiwan). From both historical and practical perspectives, Taiwan cannot be regarded as a country. It is an inseparable part of China's territory, with a clear historical, geographical attributes, political status, and basis in international law.

Other issues.

Repetitive paragraph: “3.6. Total Glucosinolates Retention Rate Measurement.”, “3.7. Total Glucosinolates Retention Rate Measurement.”

Line 375, “H2SO4”, Notice the subscript of the formula.

Reviewer 2 Report

Comments and Suggestions for Authors

Taiwan is a province of the People's Republic of China, not the REPUBLIC OF CHINA.