Inorganic Selenium Transformation into Organic Selenium by Monascus purpureus
, Hui Dang 1,*, Yuyao Zhang 1, Mengjie Yang 1, Wei Zhang 1, Yu Zhao 1, Haisheng Zhang 1, Hua Ji 1 and Baoshan Zhang 2,*Round 1
Reviewer 1 Report
This manuscript describes the production of organic selenium from inorganic selenium by M. purpureus to enrich the manufacturing of functional selenium-rich foods. The authors revealed a novel biological route-selenium-rich M. purpureus fermentation for converting inorganic selenium into organic selenium. The results will contribute to make a robust industrial production of organic selenium by M. purpureus and provide the basic information of selenium fixation in microbe.
Prior to the publication, the following points should be considered.
1. Page 5, line 166
I thought that the information of ZORBAX SB-C18 column and injection volume were not correct. Please carefully check these points and correct the information.
2. Page 10, line 360
For morphological assessment, SeCys2, MeSeCys, SeMet and Se (Ⅳ) were analyzed. What kind of analysis did you do? In addition, authors described four standard reagents mentioned above, but the holding periods were five components. What did you mean?
3. Page 10, line 360
If you use the HPLC for analysis, authors should show the HPLC chromatogram of crude extraction as well as standard compounds.
Author Response
Dear Reviewer:
On behalf of all authors, I would like to thank the Foods editorial staff for handling and the reviewer for comments our manuscript entitled: “Inorganic Selenium Transformation into Organic Selenium by Monascus purpureus”.
We have revised our manuscript as suggested by the reviewer. Modifications in the revised text are marked in red.
1.Comment: Page 5, line 166: The information of ZORBAXSB-C18 column and injection volume were not correct.
1.Reply: Due to a caret format issue, the unit cannot be depicted correctly. The following fixes have been made:
For HPLC analysis: a ZORBAX SB-C18 chromatographic column (150 mm × 4.6 mm, 5 μm) was used with a column temperature of 25 ℃, flow rate of 1.0 mL/min, injection volume of 20 L, and UV detector wavelength at 237.0 nm.
2.Comment: Page 10, line 360: For morphological assessment, SeCys2, MeSeCys, SeMet and Se( IV ) were analyzed. What kind of analysis did you do? In addition, authors described four standard reagents mentioned above, but the holding periods were five components. What did you mean?
2.Reply: In Method 2.7, an additional explanation is provided to better explain the determination and analysis procedure. Only four standard substances were measured in the experiment, but the results showed a characteristic peak of five substances in sample. We analyzed the possible state of the fifth substance on the basis of the literature and indicated that further processing was needed to determine the specific form of the five.The following modifications are specific:
2.7 Selenium conversion rate of Se-rich M. purpureus: The sample solution (1.0 mL) was diluted to 100 mL using distilled water. The diluent (20 mL) was centrifuged at 5000 rpm for 30 minutes after stand at 4 ℃ for 1 h. The mixture contained 10 mL supernatant, 12.5 mL 6 mol/L HCl, and 2.5 mL of a 0.1 g/mL K3[Fe(CN)6] solution diluted to 50 mL with distilled water[23,24]. The blank solution and sample solution were injected into an ICP-QES, the response values of the measuring elements and the internal standard elements were determined, and the concentration of the element was measured in the digestion solution based on the standard curve. The determination of selenium form refers to the method of Vacchina and Liu et al[25,26]. The ICP-MS used were an Agilent 7500cx (for HPLC-ICP-MS measurements). Chromatographic separations were carried out using a Model 1200 HPLC pump (Agilent, Wilmington, DE, USA) as the delivery system. The outlet of the column was directly connected to the nebulizer of the ICP-MS by means of PEEK tubing. Injections were performed using a Rheodyne valve with a 100-μL sample loop.
3.5.1 Selenium conversion rate of Se-rich M. purpureus: The benchmark for the morphological assessment in this trial was a mixture of four standard reagents: SeCys2, MeSeCys, SeMet, and Se (IV) （Figure 9 A）. Figure 9 B depicts the appearance of five distinctive peaks in the determination. The holding periods of the five components were 3.02, 3.63, 4.29, 6.17, and 10.21 minutes, respectively. SeMet is the major form of selenium in the Se-rich M. purpureus, with traces of SeCys2 and MeSeCys. For the characteristic peak that appears at 4.29 minutes, according to literature analysis[37], the substance may be Se (VI), and further testing and verification are needed in the future.
3.Comment: Page 10, line 360: If you use the HPLC for analysis, authors should show the HPLC chromatogram of crude extraction as well as standard compounds.
3.Reply: We appreciate your rigorous suggestion. We added Figure 9 to section 3.5.1's analysis. The following are specific modifications:
Figure 9. HPLC chromatogram of crude extraction as well as standard compounds
The provided suggestions are highly appreciated, and we patiently anticipate completing this endeavor.
Kind regards,
Nan Sun
August 31, 2023
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The study focuses on enhancing organic selenium production using Monascus purpureus, commonly used in food production. The researchers optimized selenium-enriched culture conditions for M. purpureus using different doses of sodium selenite. The best conditions included a selenium content of 7.5 mg/100 mL, pH 6.8, temperature of 30 ℃, and rotation speed of 180 rpm. Under these conditions, M. purpureus yielded mycelia with a maximum selenium concentration of around 239.17 mg/kg, with 93.45% being organic selenium. Monacolin K production reached 70.264 mg/L, and external selenium was utilized at a rate of 22.99%.
Abstract: it could be beneficial to include more specific details about the research design, and make the abstract more informative.
The introduction provides a clear and concise overview of the research problem and its significance, effectively setting the context for the study
Materials and methods: Consider providing more information about the validity and reliability of the methods used to enhance the rigor of the study. Following is the specific comments:
Line 76 and line 83 and 93, 96, 98, : please replace the word (Bacterial) with (fungal)
Line 85: please replace (onto) by (into)
In section 2.3. How did you add selenium and its concentrations ant the time of addition?
Line 106: what do you means by triangular bottle?
Line 120: did you determined the selenium content in culture broth or in mycelium and spore?
Section 2.5.1. Why do you adjust the pH by lactic acid? Lactic acid is an organic acid in may be interfere with the nutritional value of the medium, I think HCl is commonly used for this purposes.
In the same section, the pH was adjusted at 5, 6, 7, 8, 9 while the optioned optimal pH (as mentioned in abstract) was 6.8. How do you get it?
In section 2.7 Selenium conversion rate of Se-rich M. purpureus. the method is not completed, what is the measurement wavelength ant the model of instrument used?
Line 162: please replace (into) with (to)
Line 162: what is the conditions of ultra sonication used like frequency and duration?
Line 163: please replace (rotation) by (centrifugation)
Line 171: what do you mean by Basal culture conditions?
Line 174: How did you assayed the color change rate in inoculated rice?
Results are well presented with acceptable tables and figures.
Please transfer the paragraph starting from line 193 to 195 from result section to material and method section
The add sodium selenite may be reduced by microbes to nano zero valent red selenium (as you mentioned in comment on fig 2) which may interfere with the pigment red color. How did you ensure that selenium was converted into organic selenium and not into nano selenium in culture media or in fungal cells? Especially you are determined in abstract the percentage of organic selenim content in fungal biomass by 93.45%.
Author Response
Dear Reviewer:
On behalf of all authors, I would like to thank the Foods editorial staff for handling and the reviewer for comments our manuscript entitled: “Inorganic Selenium Transformation into Organic Selenium by Monascus purpureus”.
We have revised our manuscript as suggested by the reviewer. Modifications in the revised text are marked in red.
1.Comment: Line 76 and line 83 and 93, 96, 98,:replace the word (Bacterial) with (fungal)
1.Reply: Thank you for your suggestion. We have completed replacing the matching words. The following modifications are specific:
To get the activated strain, M. purpureus was inoculated on PDA solid medium at 30 ℃until red fungal colonies with evident folds and hyphae formed. Subsequently, the newly grown colonies were picked and inoculated onto fresh PDA solid media at 30 ℃until the colonies became obvious[18].
Activated M. purpureus was cultured with 50 mL of PDB for 5 d at 30 ℃ and 180 rpm to obtain the seed culture medium. The seed culture solution was centrifuged at 4000 rpm for 10 min. The supernatant was discarded and the precipitate was washed twice with 0.9% sterile physiological saline (w/v). The sediment was diluted with sterile physiological saline to obtain a fungal suspension at a concentration of 108 CFU/mL.
Cell dry weight was used to express fungal biomass, and the detection method was based on that described by Khoshr[20]. The M. purpureus culture solution was centrifuged at 3500 rpm for 10 min and washed three times with sterile water. After drying at 80 ℃ to a constant weight, the precipitates were weighed. Fungal biomass was calculated using Equation (1).
Fungal biomass = （1）
2.Comment: Line 85: replace (onto) by (into)
2.Reply: We have completed replacing the matching words. The following modifications are specific:
Activated M. purpureus was inoculated into PDB medium and cultivated at 30 ℃ and 180 rpm.
3.Comment: In section 2.3. How did you add selenium and its concentrations ant the time of addition?
3.Reply: The corresponding instructions have been added to Method 2.3. The following modifications are specific:
Sodium selenite was added to the culture medium to adjust the content of selenium element to 0.0, 2.5, 5.0, 7.5, 10.0, 15.0 mg/100 mL respectively. Activated M. purpureus was inoculated into PDB medium and cultivated at 30 ℃ and 180 rpm.
4.Comment: Line 106: what do you means by triangular bottle?
4.Reply: We made a canonical substitution for this noun, replacing (triangular container) with (erlenmeyer flasks). The following modifications are specific:
Activated M. purpureus was added to the liquid medium and incubated for nine days under optimal culture conditions, and the cenobium floating above the liquid surface was captured. The cenobium was placed in a dish, rinsed with sterile saline, and the rinse solution was placed in an erlenmeyer flask.
5.Comment: Line 120:did you determined the selenium content in culture broth or in mycelium and spore?
5.Reply: We appreciated your considerateness and had added determination method to procedure 2.4. The following modifications have been made:
After filtering the washing solution with sterile cotton and washing with sterile water 2–3 times, the fat-free cotton was placed in an oven at 65 ℃ and baked to a constant weight. The dried cotton was gently tapped to obtain spores. Spores (1.0 g) were accurately weighed, 10 mL sterile saline was added, and a spore suspension was obtained by shaking. The cenobium with removed spores was baked at 65 ℃ to a constant weight, and the dried mycelium was obtained with a scraper. The mycelia (1.0 g) was weighed accurately, 10 mL of sterile saline was added, and a mycelium suspension was obtained by shaking. The mycelium and spore suspensions are stored at 4 ℃, and the selenium content was determined by ICP-OES.
6.Comment: Section 2.5.1: Why do you adjust the pH by lactic acid?
6.Reply: In the experiment, we utilized HCL, and the reporting procedure had a few typing mistakes. It has been promptly fixed. The following changes are more specific:
After adjusting the pH of PDB with HCL or sodium hydroxide to 5, 6, 7, 8, and 9 correspondingly, M. purpureus was cultured at 30 ℃ and 180 rpm for 15 days. This experiment explored the effects of medium pH on the growth process of M. purpureus, including changes in fungal biomass, yellow pigment, red pigment content, and selenium content.
7.Comment: In the same section, the pH was adjusted at 5, 6,7,8,9 while the optioned optimal pH (as mentioned in abstract) was 6.8. How do you get it?
7.Reply: In the previous experiment, single-factor studies were conducted with various pH values (5, 6, 7, 8, and 9), and it was found that pH 7 performed the best under a single variable scenario. After that, a number of elements were incorporated in the RSM test to ultimately improve the pH 6.8 result. (This outcome was described in more depth in Results and Discussion 3.4).
As shown in table 2, the F-value of the model determined from the test data was 131.98 (P < 0.0001), demonstrating the exceptional significance of the model. The developed regression equation was well-suited, with good fitting and reliability, which was beneficial to the growth of M. purpureus. The P of the mismatched term was 0.5505, which was not significant, R2 = 0.9993. Se-rich M. purpureus was most significantly affected by the interaction between culture temperature and nutrients (P < 0.001). However, the interaction between culture temperature and rotation speed had no significant effect on the metabolism of inorganic selenium by M. purpureus (p > 0.05). The results indicated that the culture mediums adjusted to pH 6.8 at 30 ℃ and rotated at 180 r/min were optimal for growing M. purpureus in a Se-rich environment.
8.Comment: In section 2.7 Selenium conversion rate of Se-rich M. purpureus. The method is not completed, what is the measurement wavelength ant the model of instrument used?
8.Reply: We used HCL in the experiment, and there were some clerical errors in the reporting process. It has been timely corrected. Specific modifications are as follows:
2.7 Selenium conversion rate of Se-rich M. purpureus
The sample solution (1.0 mL) was diluted to 100 mL using distilled water. The diluent (20 mL) was centrifuged at 5000 rpm for 30 minutes after stand at 4 ℃ for 1 h. The mixture contained 10 mL supernatant, 12.5 mL 6 mol/L HCl, and 2.5 mL of a 0.1 g/mL K3[Fe(CN)6] solution diluted to 50 mL with distilled water[23,24]. The blank solution and sample solution were injected into an ICP-QES, the response values of the measuring elements and the internal standard elements were determined, and the concentration of the element was measured in the digestion solution based on the standard curve.
The determination of selenium form refers to the method of Vacchina and Liu et al[25,26]. The ICP-MS used were an Agilent 7500cx (for HPLC-ICP-MS measurements). Chromatographic separations were carried out using a Model 1200 HPLC pump (Agilent, Wilmington, DE, USA) as the delivery system. The outlet of the column was directly connected to the nebulizer of the ICP-MS by means of PEEK tubing. Injections were performed using a Rheodyne valve with a 100-μL sample loop.
9.Comment: Line 162: replace (into) by (to)
9.Reply: We have replaced the statement in question and the issue is no longer present.
10.Comment: Line 162:what is the condition of ultra sonication used like frequency and duration?
10.Reply: The unit cannot be displayed properly due to a caret format issue, which has been rectified was as follows:
Monacolin was extracted and determined based on Liu's[27] method. Briefly, C2H5OH (30 mL of 75%) was added to 10 mL of the sample, and the mixture was extracted by ultrasound (20 kHz) at 50 ℃ for 30 min.
11.Comment: Line 163: replace (rotation) by(centrifugation)
11.Reply: We have completed replacing the matching words. The following modifications are specific:
After centrifugation at 4000 rpm for 10 min, the supernatant was filtered with an ultrafiltration membrane (0.45 μm) and the volume was fixed to 10 mL. For HPLC analysis: a ZORBAX SB-C18 chromatographic column (150 mm × 4.6 mm, 5 μm) was used with a column temperature of 25 ℃, flow rate of 1.0 mL/min, injection volume of 20 L, and UV detector wavelength at 237.0 nm. The mobile phase for the gradient elution analysis was a 75% acetonitrile solution (pH 2.5 adjusted with phosphoric acid).
12.Comment: Line 171:what do you mean by Basal culture conditions?
12.Reply: We have specified the cultivate conditions. The following modifications are specific:
- purpureuscultured for five days with Se enrichment under optimal conditions was further cultured for 15 days under 30 ℃and 180 rpm in PDB medium without sodium selenite. Changes in selenium content in the mycelia and the culture medium were measured. At the same time, M. purpureus cultured with selenium was inoculated onto cooked rice and cultured under optimal fermentation conditions for 15 days.
13.Comment: Line 174: How did you assayed the color change rate in inoculated rice?
13.Reply: We have supplemented the relevant reference literature and specific methodologies.Specific modifications are as follows:
The rice color and coloring rates were measured. Color value determination methods refer to Embate and Bhat et al[28,29]. The sample was crushed by a crusher and passed 250μm~380μm sieve. Accurately weigh 0.2g (accurate to 0.001g) of the sample to be crushed and mixed evenly. The solution temperature should be allowed to return to ordinary temperature after removing the sample. The solution was diluted to 100 mL with 70% ethanol solution, and thoroughly mixed. It was filtered using filter paper and collected in a stoppered colorimetric tube. The filtrate was extracted 5.0 mL into a 50 mL volumetric flask, diluted to 50 mL with 70% ethanol solution, and thoroughly mixed. The 70% ethanol solution was used as a reference, and the absorbance of the sample was measured at 505 nm.
14.Comment: The add sodium selenite may be reduced by microbes to nano zero valent red selenium(as you mentioned in comment on fig 2) which may interfere with the pigment red color. How did you ensured that selenium was converted into organic selenium and not into nano selenium in culture media or in fungal cells? Especially you are determined in abstract the percentage of organic selenim content in fungal biomass by 93.45%.
14.Reply: In order to determine the conversion of inorganic selenium, organic selenium, and their respective forms in the experiment, the products were analyzed for their main selenium species. The separation of organic and inorganic selenium from the products involved grinding, homogenization, centrifugation, and cyclohexane extraction. After digestion and acid removal steps, total selenium and inorganic selenium were quantified using ICP-OES under optimized conditions while total organic selenium was calculated by the subtraction method. To elucidate the form of organic selenium present in the product, HPLC-MS analysis was employed.
Thank you for your careful reading and recommendation of this paper, we have made corresponding modifications to the article. We hope to cooperate with you to complete this paper consummately.
Kind regards,
Nan Sun
August 31, 2023
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The manuscript is acceptable for publication in the current form
