Enhanced Anti-Melanogenic Effect of Adlay Bran Fermented with Lactobacillus brevis MJM60390

Round 1

Reviewer 1 Report

Review comments of manuscript Applmicrobiol-1807222 entitled:

 

Enhanced Anti-melanogenic Effect of Adlay Bran Fermented with Lactobacillus brevis MJM60390

 

In this manuscript, adlay bran was fermented by Lactobacillus brevis MJM60390 (LBFAB), and the anti-melanogenic effect was investigated. The results demonstrated that LBFAB significantly suppressed melanin accumulation in mouse melanogenic B16F10 cells, and the activity was higher than non-fermented adlay bran (NFAB). The molecular mechanism study showed that LBFAB inhibited melanin synthesis by suppressing the gene expression of Mc1r, Mitf, Tyr, Trp-1, and Trp-2 genes. LBFAB also strongly decreased the expression of Tyr, Trp-1, and Trp-2 compared to NFAB. Furthermore, phenolic compounds were significantly increased in LBFAB compared with NFAB. Thus, LBFAB holds great potential as an anti-melanogenic agent and can be used to develop whitening cosmetics.

I think there are many important issues to be addressed before further review on the result of this manuscript, and I would like to address several points as follows:

 

Major comments:

1．On page 2, the information of Adlay Bran, including primitive family, genus, origin, and medicinal parts, should be provided in materials and methods.

 

2．On page 2, the same title of 2.2 and 2.3 should be checked and confirmed; it seems that the title of 2.3 is inconsistent with its content.

Besides, in the 2.2 part, the process of preparation of adlay bran extracts, such as liquid material ratio, extraction times, and drying conditions, should be described in detail.

 

3．In lines 99 to 100, “the formazan concentration was detected by Elisa reader”, does Elisa kit detect it?

 

4．On page 4, part 2.8, the preparation method and concentration of adlay bran extract solution for HPLC determination are not provided.

In lines 165 to 166, “The concentration of the phenolic compounds in the samples was calculated based on the curves of the standard compounds”, however, there is a lack of relevant methodological review, and at least the linearity of these compounds (gallic acid, p-coumaric acid, ferulic acid, and sinapic acid) should be provided. Besides, the HPLC profiles of LBFAB and NFAB under the same concentration and the reference substance's HPLC profiles should be supplemented.

 

5．In Fig 1, A and B in Figure 1 should indicate their respective meanings; the same problems also occurred in other Figs.

 

Author Response

Dear reviewers,

Thank you for your careful review on our manuscript. Your comments helped us improve the quality of the draft. We revised the contents according to your comments. We hope our revision are clear enough to answer our questions. The point-to-point answer to the reviewer’s comments are given below. 

Reviewer 1:

In this manuscript, adlay bran was fermented by Lactobacillus brevis MJM60390 (LBFAB), and the anti-melanogenic effect was investigated. The results demonstrated that LBFAB significantly suppressed melanin accumulation in mouse melanogenic B16F10 cells, and the activity was higher than non-fermented adlay bran (NFAB). The molecular mechanism study showed that LBFAB inhibited melanin synthesis by suppressing the gene expression of Mc1r, Mitf, Tyr, Trp-1, and Trp-2 genes. LBFAB also strongly decreased the expression of Tyr, Trp-1, and Trp-2 compared to NFAB. Furthermore, phenolic compounds were significantly increased in LBFAB compared with NFAB. Thus, LBFAB holds great potential as an anti-melanogenic agent and can be used to develop whitening cosmetics.

 

I think there are many important issues to be addressed before further review on the result of this manuscript, and I would like to address several points as follows:

1．On page 2, the information of Adlay Bran, including primitive family, genus, origin, and medicinal parts, should be provided in materials and methods.

à L87 Adlay (Coix lachrymal-jobi) bran was obtained from a local mill of Yeoncheon, the main place for production of adlay in Republic of Korea.

 

2．On page 2, the same title of 2.2 and 2.3 should be checked and confirmed; it seems that the title of 2.3 is inconsistent with its content.

à L101 2.3 Cell viability assay

 

Besides, in the 2.2 part, the process of preparation of adlay bran extracts, such as liquid material ratio, extraction times, and drying conditions, should be described in detail.

• The detail information was added in L87- 100

Adlay (Coix lachrymal-jobi) bran was obtained from a local mill of Yeoncheon, the main place for the  production of adlay in Republic of Korea. The adlay bran was mixed with water in a ratio of 20:80 (w:w) to make bran sludge and sterilized at 121ºC for 20 min.

Lactobacillus brevis MJM60390 was isolated from fermented vegetables in our previous study [33]. For seed culture, MJM60390 was inoculated to 50 mL of De Man, Rogosa, and Sharpe (MRS) broth and incubated anaerobically at 37ºC for 16 h. Then, the adlay bran sludge was inoculated with 0.1% (v/v) of the seed culture of L. brevis MJM60390 and incubated statically at 37 °C for 48 h (FAB). As a control, adlay bran sludge without inoculation of L. brevis MJM60390 (NFAB) was incubated under the same conditions.

After fermentation, the adlay bran was extracted with 70% ethanol (1:3, v/v) for 24 h at room temperature. The solid part was discarded after centrifugation (6,000 rpm for 20 min), and the supernatant was filtered with Whatman filter paper No. 1, evaporated, and freeze-dried at -80^oC for 24 h. The free-dried powder was preserved at -20 °C until further experiments.

.  

3．In lines 99 to 100, “the formazan concentration was detected by Elisa reader”, does Elisa kit detect it?

à It is not Elisa Kit, I used the wrong word here so I already changed Elisa to microplate

 

4．On page 4, part 2.8, the preparation method and concentration of adlay bran extract solution for HPLC determination are not provided.

In lines 165 to 166, “The concentration of the phenolic compounds in the samples was calculated based on the curves of the standard compounds”, however, there is a lack of relevant methodological review, and at least the linearity of these compounds (gallic acid, p-coumaric acid, ferulic acid, and sinapic acid) should be provided. Besides, the HPLC profiles of LBFAB and NFAB under the same concentration and the reference substance's HPLC profiles should be supplemented.

à L165-180, the detail information for the methodology was added. As required by the reviewer, we added the HPLC chromatogram for this analysis to the draft as figure 6. The linearity of these compounds were shown in Figure 6

 

5．In Fig 1, A and B in Figure 1 should indicate their respective meanings; the same problems also occurred in other Figs.

à Figures were reorganized. A, B were remarked in the figure legend. And all the figures were reorganized. Black bar indicated non-treated control, gray bar indicated a-MSH treatment control, dark gray indicated arbutin treatment (positive control), white bar indicated NFAB, and bar with diagonal indicated FAB.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

This article explores the potentiality and effectiness of fermented adlay bran (fermented by Lactobacillus brevis) (LBFAB) to suppress melanin accumulation in mouse melanogenic B16F10 cells through following and specifying the molecular mechanism of action.  Moreover, the study investigated the anti-melanogenic activity of LAB-fermented adlay bran in B16F10 cells correlating it to the phenolic compounds in adlay bran extracts. The objective of the fermentation process was to enhance the levels of the phenolic compounds. The study proved this enhancement, but there is still a need to prove that the enhanced levels of the phenolic compounds stands behind the anti-melanogenic activity of LAB-fermented adlay bran. It would have been helpful if the study included external controls from some free phenolic compounds.

Generally the article’s objective is a worthwhile and can output useful information and applications.

For simplification sake the studied material (fermented adlay bran) can be refrerred to as (FAB), while the unfermentented control can be designated as (NFAB). The appellation as such are a little bit confusing and distracting.

In Figure 1, there are two histograms A and B, which are not clarified in the title or in the legend. It may be concluded that A is referring to NFAB and B to FAB. Please check and identify.Instead of writing A and B at the top of the histograms, write down directly what each means. If A is NFAB, just replace A by it. And if B is FAB do the same.

Figure 2. The figure sections (A,B,C and D) should be better explained. The symbols above the columns are not visible and cannot be read. The images of the test tubes are redundant and can better be eliminated to give space to the other figures sections.

Figure 3 should also be better presented. In Figure 3B, x-axis what is concentration of ?. The columns in each section, seemingly represent different treatments. This should be explained clearly, as similar colors in the same figure should usually have the same reference, otherwise explained clearly.

Figure 4 and 5. needs also more clarifications and organization.

L259 change (B16F10 was) into (B16F10 cells were).

In the introduction the study should clarify why adlay bran was used for this study as source of phenolic compounds (richness, availability, feasibility??).

L68-70 (Adlay extracted by the supercritical fluid CO2 was reported to reduce melanin production [31]. However, there is no research on the anti-melanogenic effects of adlay bran and LAB-fermented adlay bran extracted with ethanol. )

The authors should explain scientific ground behind deciding to limit their work on Adlay bran and not at the whole adlay seed followingthe previous study of [31].

In the material and methods, an approximate chemical analysis should be conducted of adlay bran. It is understtod that it is not only phenolic compounds. Other components may exist. Also, after fermentation exhaustive chemaical analysis should be conducted. This is necessary so that the reader can understand and the character of the acting material.

The authors explained in the results that  the contents of the phenolic cmpounds increased by fermention as a result of transforming the conjugated phenolic compounds into free phenolic compounds. It would be meaningful if the study determined the content of the conjugated phenolic compound in the starting material at the zero time fermentation and after the fermentation. Relating the content of the free phenolic compounds to the total content of conjugated phenolic compounds in the starting material may indicate the efficiency of the fermentation process and if period of fermentation was enough to have all the phenolic compounds in the free form.  The data in Figure 2 should be qualified as the free phenolic compounds to exclude any confusion.

L 326-331 The authors confirm that the adlay phenolic compounds ( gallic acid, p-coumaric acid, ferulic acid, and sinapic acid significantly increased after fermention with Lactobacillus brevis and thus are responsible for the the greater antimelanogenesis effect of LAB-fermented adlay bran. To strengthen this conclusion a control of free phenolic compounds with approximately the same concentration in the extract may be conducted to confirm this conclusion.

The mechanism of action of the free phenolic compounds through the antimelanogenesis process needs to be further clarified.

 

4 July 2022

Author Response

Dear reviewers,

Thank you for your careful review on our manuscript. Your comments helped us improve the quality of the draft. We revised the contents according to your comments. We hope our revision are clear enough to answer our questions. The point-to-point answer to the reviewer’s comments are given below. 

Reviewer 2:

This article explores the potentiality and effectiness of fermented adlay bran (fermented by Lactobacillus brevis) (LBFAB) to suppress melanin accumulation in mouse melanogenic B16F10 cells through following and specifying the molecular mechanism of action.  Moreover, the study investigated the anti-melanogenic activity of LAB-fermented adlay bran in B16F10 cells correlating it to the phenolic compounds in adlay bran extracts. The objective of the fermentation process was to enhance the levels of the phenolic compounds. The study proved this enhancement, but there is still a need to prove that the enhanced levels of the phenolic compounds stands behind the anti-melanogenic activity of LAB-fermented adlay bran. It would have been helpful if the study included external controls from some free phenolic compounds.

Generally the article’s objective is a worthwhile and can output useful information and applications.

For simplification sake the studied material (fermented adlay bran) can be refrerred to as (FAB), while the unfermentented control can be designated as (NFAB). The appellation as such are a little bit confusing and distracting

• As required, “LBFAB” was changed to “FAB” through the article to avoid confusion.

In Figure 1, there are two histograms A and B, which are not clarified in the title or in the legend. It may be concluded that A is referring to NFAB and B to FAB. Please check and identify.Instead of writing A and B at the top of the histograms, write down directly what each means. If A is NFAB, just replace A by it. And if B is FAB do the same.

• As required, we clarified the sample name in the top of the figure. And So did in other figures.

Figure 2. The figure sections (A,B,C and D) should be better explained. The symbols above the columns are not visible and cannot be read. The images of the test tubes are redundant and can better be eliminated to give space to the other figures sections.

• The symbols indicated significant difference. This was clarified in the figure legend, and the symbol size was enlarged. The test tubes were removed and the figure.

Figure 3 should also be better presented. In Figure 3B, x-axis what is concentration of ?. The columns in each section, seemingly represent different treatments. This should be explained clearly, as similar colors in the same figure should usually have the same reference, otherwise explained clearly.

Figure 4 and 5. needs also more clarifications and organization.

• All Figures were clarifications and re Black bar indicated non-treated control, gray bar indicated a-MSH treatment control, dark gray indicated arbutin treatment (positive control), white bar indicated NFAB, and bar with diagonal indicated FAB.

L259 change (B16F10 was) into (B16F10 cells were)

à (B16F10 was)  was changed to (B16F10 cells were) (now is L278)

In the introduction the study should clarify why adlay bran was used for this study as source of phenolic compounds (richness, availability, feasibility??)

à We add the reason why we use adlay bran. Line 60-64, Adlay were widely planted in East Asia, including South Korea, and adlay bran is a by-product during the adlay milling process. It was reported that adlay bran contained higher contents of phytochemical compositions than hulled and polished fractions [23]. Despite containing abundant active ingredients, adlay bran is almost used as livestock feed or compost. So developing cosmetics using adlay bran is a good way to increase the value of waste material.

L68-70 (Adlay extracted by the supercritical fluid CO2 was reported to reduce melanin production [31]. However, there is no research on the anti-melanogenic effects of adlay bran and LAB-fermented adlay bran extracted with ethanol. )

The authors should explain scientific ground behind deciding to limit their work on Adlay bran and not at the whole adlay seed followingthe previous study of [31]

• It was reported that adlay bran contains more phytochemical components than hulled and polished adlay. That’s why we choose bran for study. We added the reference in Line 63.

In the material and methods, an approximate chemical analysis should be conducted of adlay bran. It is understood that it is not only phenolic compounds. Other components may exist. Also, after fermentation exhaustive chemaical analysis should be conducted. This is necessary so that the reader can understand and the character of the acting material.

• We agree with the reviewer’s points for many other components may contribute to the activity. However, due to the limitation of the analysis, we chose some important and representative compounds for analysis since these compounds have been known for whitening effects. Furthermore, in this study, we focused on the increase of activity and active compounds by fermentation rather than exploring the active compounds from adlay bran.
• We also discussed this point in Line 360 -363.

The authors explained in the results that  the contents of the phenolic cmpounds increased by fermention as a result of transforming the conjugated phenolic compounds into free phenolic compounds. It would be meaningful if the study determined the content of the conjugated phenolic compound in the starting material at the zero time fermentation and after the fermentation. Relating the content of the free phenolic compounds to the total content of conjugated phenolic compounds in the starting material may indicate the efficiency of the fermentation process and if period of fermentation was enough to have all the phenolic compounds in the free form.  The data in Figure 2 should be qualified as the free phenolic compounds to exclude any confusion

• We really appreciate the reviewer’s comments. In Table 2, it was clarified as “free phenolic compounds”
• For industrial application, simple extraction process is very important. That’s the reason why we use 70% ethanol for extraction. And we found the phenolic compounds were increased. These compounds should be free phenolic compounds because 70% ethanol can not extract conjugated phenolic compounds. Some bacteria were reported to transform conjugated form of phenolic acid to free form. So we speculated the increase of phenolic acid in the FAB was due to the transformation by bacteria during fermentation.
• We discussed this part in the discussion section (Line 343 -359)

L 326-331 The authors confirm that the adlay phenolic compounds ( gallic acid, p-coumaric acid, ferulic acid, and sinapic acid significantly increased after fermention with Lactobacillus brevis and thus are responsible for the the greater antimelanogenesis effect of LAB-fermented adlay bran. To strengthen this conclusion a control of free phenolic compounds with approximately the same concentration in the extract may be conducted to confirm this conclusion

• We changed “the main reason contributing” to “ one of the reason contributing….) in line 361~362.
• Since we used extract (a mixture containing many compounds) for the experiment, many factors should be considered: the concentration of some active compounds, many other unknown compounds, and the interactions between these compounds. We discussed this part in Line 366~361.

The mechanism of action of the free phenolic compounds through the antimelanogenesis process needs to be further clarified

• The mechanism studies for these phenolic compounds on the anti-melanogenesis have been elucidated in many other articles. And we cited them in this paper.
• These reference were listed here for your reference.
• Kim, Y.-J., Antimelanogenic and antioxidant properties of gallic acid. Biological and Pharmaceutical Bulletin, 2007. 30(6): p. 1052-1055.
• Su, T.-R., et al., Inhibition of melanogenesis by gallic acid: Possible involvement of the PI3K/Akt, MEK/ERK and Wnt/β-catenin signaling pathways in B16F10 cells. International journal of molecular sciences, 2013. 14(10): p. 20443-20458.
• Boo, Y.C., p-Coumaric acid as an active ingredient in cosmetics: A review focusing on its antimelanogenic effects. Antioxidants, 2019. 8(8): p. 275.
• Maruyama, H., et al., Biochemical characterization of ferulic acid and caffeic acid which effectively inhibit melanin synthesis via different mechanisms in B16 melanoma cells. Biological and Pharmaceutical Bulletin, 2018. 41(5): p. 806-810.
• Yoon, H.S., et al., Differential effects of methoxylated p-coumaric acids on melanoma in B16/F10 cells. Preventive Nutrition and Food Science, 2015. 20(1): p. 73.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Review comments of manuscript Applmicrobiol-1807222 entitled:

 

Enhanced Anti-melanogenic Effect of Adlay Bran Fermented with Lactobacillus brevis MJM60390

 

The author added necessary information on the preparation of the sample, active components detection by HPLC, and other errors were also revised in the manuscript. Thus, these modifications and improvements greatly improve the manuscript’s quality. Only a small question needs to be answered or explained by the author:

 

1．In part 2.2, “Preparation of adlay bran extracts”, NFAB The adlay bran was extracted with water in a ratio of 20:80 (w:w), and FAB was extracted with 70% ethanol (1:3, v/v) for 24 h at room temperature. Can you explain the inconsistency of extraction methods?

 

2．The UV absorption spectrum of phenolic compounds in Fig 6 is not clear enough to be identified. The linearity curves and linear range of phenolic compounds should be included in Tab 2. 

Comments for author File: Comments.docx

Author Response

Dear reviewers,

Thank you for your careful review on our manuscript. Your comments helped us improve the quality of the draft. We revised the contents according to your comments. We hope our revision are clear enough to answer our questions. The point-to-point answer to the reviewer’s comments are given below. 

 

Reviewer 1:

Review comments of manuscript Applmicrobiol-1807222 entitled:

 

Enhanced Anti-melanogenic Effect of Adlay Bran Fermented with Lactobacillus brevis MJM60390

 

The author added necessary information on the preparation of the sample, active components detection by HPLC, and other errors were also revised in the manuscript. Thus, these modifications and improvements greatly improve the manuscript’s quality. Only a small question needs to be answered or explained by the author:

 

1．In part 2.2, “Preparation of adlay bran extracts”, NFAB The adlay bran was extracted with water in a ratio of 20:80 (w:w), and FAB was extracted with 70% ethanol (1:3, v/v) for 24 h at room temperature. Can you explain the inconsistency of extraction methods?

• Both fermented and non-fermented adlay bran were extracted with 70% ethanol. We clearly mentioned this in line 97 to avoid confusion.
• We mix adlay bran with water to make medium for LAB fermentation, not for extraction. Line 93. Because adlay bran is powder, it contains no water. For fermentation, we need add water to the bran powder to make sludge, thus LAB can grow in the bran sludge.

2．The UV absorption spectrum of phenolic compounds in Fig 6 is not clear enough to be identified. The linearity curves and linear range of phenolic compounds should be included in Tab 2.

• The UV absorption spectrum of phenolic compounds in Fig 6 was changed to make more clear than before.

 

• The linearity curves and linear range of phenolic compounds were put in Table 2.

 

 

 

Reviewer 2 Report

The authors have adequately responded to the comments and the manuscript has been largely improved.

Author Response

Dear reviewers,

 

The authors have adequately responded to the comments and the manuscript has been largely improved.

• Thank you for your comment and encouragement!

Author Response File: Author Response.docx