Mg, K-containing microparticle: A possible active principle of a culture extract produced by a microbial consortium.

A synthetic microbial consortium called Effective Microorganisms (EM) consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects of EM∙XGOLD, a health drink produced by EM, on life cycle of Dictyostelium discoideum were described previously. Here, we report our attempt to identify the active principle, termed EMF, that brought about the observed effects. Throughout the purification processes, the presence of the active principle was monitored by promoted fruiting body formation. By liquid-liquid separation the activity was recovered in aqueous phase, which, after concentration, was further subjected to reverse-phase column chromatography. No activity was detected in any eluant, while almost all the activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures to the residual fraction did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, the residue revealed spherical particles of μm size range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence, on the surface of the particles, of magnesium and, to a certain extent, of potassium. In separate experiments, acid treatment and alkali neutralization of EM∙XGOLD completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticles to be an active principle of EM culture extract. How these particles exert their effect is currently under intensive investigation.

Introduction

Microbiome, the world of microorganisms, has attracted more and more attention in recent years. Metagenomic approach that deals with the collection of microorganisms in its entirety has uncovered a massive number of novel genes most of which are with unknown functions [1-3]. Of particular interest is the growing number of findings that a combination of microorganisms, or a microbial consortium, exhibits novel effects that could not be expected from purecultures [4-7]. Atarashi et al. showed that a set of selected 17 Clostridia strains is essential for highest Treg induction, with its subset being less effective [5]. A similar observation with a combined 11 bacterial strains has been reported in IFNγ+CD8 cell induction [6]. In these reports, synergistic cooperation contributed by constituent bacteria has been postulated for their maximum inducing activity. The exact mechanism, as well as molecular properties produced by these consortia, is yet to be identified. These trends, however, have definitely aroused renewed interests in microbial consortium, either naturally occurring or synthetic, and set up a stage for a novel applied microbiology.

EM is a short form of Effective Microorganisms and a synthetic microbial consortium which consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast. EM is also a registered trademark and a brand name owned by EMRO (EM Research Organization). Since its serendipitous discovery by T. Higa in 1970s [8], EM itself or its culture extract has proved to be beneficial in numerous fields such as agriculture, bioremediation, environmental cleanup and so forth [9-12]. By functional genomics and metabolome analyses, ECEM, one of EM products, has exhibited anti-inflammatory and immunostimulatory effects in mouse macrophage [13]. Furthermore, EM∙XGOLD (abbreviated as EMXG in this communication), another EM product brought to the market as a health drink, has been reported to be effective in sustaining human immunity function [14]. However, the molecular mechanism of those effects is still unknown.

Prompted by these reports and out of our genuine curiosity, we initiated a more in-depth examination of the effect of EM products on cellular activity with the use of cellular slime mold Dictyostelium discoideum as a model. We described in the foregoing paper various effects of EMXG on life cycle of this organism, including a novel finding, i.e. modulation of cAMP oscillation rhythm [15]. Here, we report our attempt to identify the active principle that brings about the observed effects. Several lines of evidence point to the fact that the activity resides in Mg, K-containing microparticle of several micrometer range. How these particles exert their effect is currently under intensive investigation.

Results

The active principle is water-soluble

ECEM was first subjected to liquid-liquid partition to see whether the activity is organic solvent-soluble or water-soluble. After extraction by ethyl acetate followed by n-butanol, the combined organic solvent soluble fraction and water-soluble fraction were assayed as described in Materials and Methods. As shown in Fig 1, most of the activity was recovered in water-soluble fraction. The lower activity observed in both organic solvent and the water soluble fractions was due to the inhibitory effect of dimethyl sulfoxide used for dissolving powder state of each fraction into water.

Fig 1

Liquid-liquid partition of EMF activity.

The activity was recovered in aqueous phase. C (Control without ECEM); ECEM (10% ECEM); Aq (Aqueous phase); Org (Organic phase). **: Significant difference in comparison to the Control by Steel test (p<0.01), a,b: different letters show significant difference by Steel-Dwass test(p<0.01).

The active principle was not found in the column eluates

Water-soluble fraction was then applied onto HPLC column and was eluted with a mixture of water-methanol (1:4) to give five fractions (S2 Fig). Each eluted fraction was subjected to activity assay, and none of the fractions showed the promoted fruiting body formation (Fig 2). Fraction 1 showed even an inhibitory activity. Here, possibility exists that the activity is separated into more than two fractions during the course of chromatography. To address this possibility, various combinations of eluted fraction were tested for activity assay. As seen from Fig 3, none of the combinations exhibited the promoted fruiting body formation, thus excluding the supposed possibility.

Fig 2

Chromatographic separation of aqueous phase in Fig 1.

Aqueous phase was separated into 5 fractions and insoluble residue (S2 Fig). C (Control without ECEM); ECEM (10% ECEM). **: Significant difference in comparison to the Control by Steel test (p<0.01).

Fig 3

Assay of combinatorial mixture of fractions in Fig 2.

C (Control without ECEM); ECEM (10% ECEM); All (All fractions combined); Three (Fr. 1, Fr. 2 and Fr. 4 are combined); Two (Fr. 3 and Fr. 5 are combined). **: Significant difference in comparison to the Control by Steel test (p<0.01).

The active principle was recovered in the insoluble residue

S2 Fig shows that water soluble fraction, in which the activity was associated, was divided into 5 column eluants and insoluble residue. Since no activity was found in any eluants and in any of their combinations, logical possibility suggests that the activity should be found in insoluble residue, if activity is not lost during the course of chromatographic separation. We addressed this possibility. The insoluble residue, however, was not solubilized, as its name stands, by such procedures as vortexing and sonication with occasional addition of dimethyl sulfoxide. As a final trial, insoluble residue suspension equivalent to 10% ECEM was mixed with agar and water, and autoclaved according to the conventional procedure for preparing agar plates. To our little surprise, agar plate finally obtained looked smooth without showing any blobs anticipated from insoluble residue. This enabled us to proceed further to the assay procedure. Fig 4 describes the result of activity assay experiment. As seen, it is clear that almost all activity was recovered in the insoluble residue. The result that the activity is associated with insoluble residue raises several possibilities as to the nature of activity in the residue. Whatever the mode of existence, various organic chemistry procedures were applied, such as esterification, acid hydrolysis for detection of polysaccharide and NMR. Nothing informative could be obtained by these trials. Finally, it was found that the residue dissolves in acidified methanol with concomitant formation of bubbles, suggesting the possibility of carbonate salt, the result rather resisting rational understanding.

Fig 4

Activity of fruiting body formation was recovered in the residual fraction.

C (Control without ECEM); ECEM (10% ECEM); Insol (Insoluble residue). **: Significant difference in comparison to the Control by Steel test (p<0.01), a, b: different letters show significant difference by Steel-Dwass test(p<0.01).

Views of insoluble residue under scanning electron microscope

The insoluble residue was viewed under scanning electron microscope. Fig 5 represents a typical image which reveals spherical particles with various sizes of μm range. Application of Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence, on the surface of the particles, of magnesium and, to a certain extent, of potassium (S3 Fig). One additional observation was the matrix-like structure revealed when the residue was autoclaved, the meaning of which remains an enigma (S4 Fig). Over all, the insoluble residue consists of spherical microparticles containing mainly magnesium and, to a certain extent, potassium. So, from now on, just for convenience, we call it Mg-particle.

Fig 5

Scanning electron micrograph of insoluble residue.

Acid treatment of EMXG

In order to unarguably assign the active principle to Mg-particle, it would be the best to isolate Mg-particle in a pure form, followed by activity assay. Various procedures were attempted, such as sedimentation and equilibrium centrifugation, to no avail. As described above, it was observed that the insoluble residue dissolves into acid with concomitant release of gas. This observation suggests that one of the major constituents of the insoluble residue is MgCO3, the inference apparently arguing against the reasonable understanding that the inorganic compound could hardly modulate the cellular function. If, however, the Mg-particle in the residue is effective in some way to promote fruiting body formation, the acid treatment would be expected to wipe away the activity, since the salt composed by weak acid will be destroyed by strong acid. We challenged this possibility. For this experiment, we used EMXG, a diluted form of ECEM, as a test material, since acidification of ECEM resulted in the formation of insoluble precipitate and made the experiment complicated. Thus, EMXG, into which pH electrode was inserted, was added diluted HCl until pH reached 1~2. Then, with the same set up, diluted NaOH was added dropwise till pH went up to 7~8. The neutralization step was needed because the agar, which was used for activity assay, was unable to solidify under acidic condition. Fig 6A shows what resulted in one of such experiments. It is clear that the activity of fruiting body formation is almost completely wiped away by acid treatment followed by alkali neutralization. To exclude the possible effect of sodium and chloride ions produced by these treatments, an equivalent amount of NaCl was added to the control, which revealed no effect on fruiting body formation. Furthermore, to ensure that no reformation of Mg-particle or any kind of activity reversal was occurred during a long period after neutralization, the same reaction mixture was assayed 2 months after the neutralization reaction. The result is shown in Fig 6B, which is similar to that in Fig 6A. Whatever the mode of action may be, the results of this experiment suggest that Mg-particle to be a most possible candidate for the active principle of EMXG.

Fig 6

EMXG was acidified with HCl followed by neutralization with NaOH.

The treated EMXG was assayed for fruiting body formation. A; Assay was performed right after acid-alkali treatment. B; The treated sample was kept at room temperature for 2 months and assayed for fruiting body formation. C (Control without ECEM); E20 (EMXG 20%); A-A (Acid treatment followed by alkali neutralization), **: Significant difference in comparison to the Control by Steel test (p<0.01).

Discussion

In the preceding paper, we described how EMXG affects the life cycle of Dictyostelium discoideum [15]. EMXG promoted the proliferation of amoeba cells, stimulated the differentiation process by formulating more of fruiting bodies and modulated the oscillation of cAMP with concomitant increase of cAMP level of the medium. Nothing is known, however, whether these multiple effects are caused by a single factor or by several different factors contained present in EMXG. As described, the follow-up procedure of active principle was through monitoring the accelerated fruiting body formation of Dictyostelium discoideum. Therefore, it would be appropriate to state that Mg-particles identified in this communication represent one of the active principles residing in EMXG and other EM-related products. Therefore, possibility cannot be ruled out that other factors active principles that are as distinct from Mg-particles is at work, exhibiting beneficial effects in a wide variety of fields. These aspects are yet to be subjected to further in-depth studies, the presence of other factors being clearly in the realm of possibility.

The result of acid treatment suggests the chemical form of Mg-particle to be the carbonate form of magnesium, most probably MgCO3. Based on the experiments described above, we propose the observed Mg-particle to be a most possible candidate for the active principles of ECEM and EM∙XGOLD. These lines of evidence prompted us to see whether authentic MgCO3 itself possesses the acceleration activity of fruiting body formation similar to the one observed with EMXG. Ten batches of MgCO3 with various degree of purity and impurities were supplied by a chemical company. Ten mg or so of each were included in agar plate for the assay. It was found that only one batch of MgCO3 exhibited remarkable stimulatory activity for fruiting body formation. How this particular one has the observed activity is at present in an enigma, research currently being carried out in regard to its specific crystal structure. In the current study, we did not focus on the role of potassium which was also associated with Mg-particle.

It is of great interest how inorganic materials exert any effect on biological cells. There were several preceding findings regarding the biological effect by inorganic materials. As a pioneering observation, Glass and Kennett reported that carbon material added to an agar plate or a liquid culture medium showed growth-enhancing effect on certain bacteria [16]. The plausible hypothesis for these observations was that these carbon materials act indirectly by adsorbing growth inhibitory substances from the medium. Following this observation, Matsuhashi et al. reported that carbon material such as graphite and activated charcoal, but not diamond, stimulated the growth of special bacteria, carbophilic strain of Bacillus [17]. Also, Matsuhashi et al. presented several lines of evidence which support the direct effect of carbon materials on living organisms [18]. They showed, through ways of effect-blocking experiments, that the effect is physical, not chemical [18]. More interestingly, the growth-enhancing signal was shown to propagate through distance, by way of the sound, termed by them biosonics [19]. Another report concerning the effect of inorganic material deals with carbon micro coil, abbreviated as carbon micro coil (CMC) [20]. CMC was produced by metal-catalyzed pyrolysis of acetylene at 700~800°C [20]. Ogawa found that CMCs have activation effect for skin cells and their collagen mRNA production [21]. For example, the addition of CMCs promoted the growth of Pam212 mouse keratinocyte, in dose dependent fashion, upto 1.6 times versus the control for the culture period of 6 days [21]. Also, CMC was shown to suppress the growth of HeLa cells by almost 80%. Interestingly, this effect was essentially wiped out when the coiled structure was sufficiently destroyed [22]. Furthermore, Komura reported that CMC, coupled with ultrasound exposure, inhibited the growth of human hepatocarcinoma and mouse sarcoma cells, and that this effect was through generation of hydroxyl radical (・OH) in the culture medium. Komura suggested that CMC is a potent sonosensitizer and applicable to cancer therapy in combination with ultrasound [23]. At the present time, it is rather hard to make up a unified theory regarding the possible mechanisms for the observations described. It is, however, of interest to note that the steric structure might be involved based on two independent observations. Matsuhashi et al. showed that the effect was not detected with diamond while graphite and activated charcoal was effective [17]. In the case of CMC, the effect disappeared when the coiled structure was destroyed [22]. Our observation that some authentic MgCO3 showed the stimulatory effect in fruiting body formation may have some relevance to this kind of inference.

The present communication described Mg-microparticle to be one of the active principles that exert the stimulatory effect on the growth, differentiation and cAMP oscillation rhythm in cellular slime mold, Dictyostelium discoideum. How this effect is exerted, i.e. what kind of molecules stands between Mg-particle and fruiting body formation prompts our further interest, and is under intensive investigation.

Materials and methods

EM, ECEM and EM∙XGOLD

EM represents a short form of Effective Microorganisms and is a registered trademark and a brand name owned by EMRO (EM Research Organization). EM is a synthetic microbial consortium consisting mainly of photosynthetic bacteria, lactic acid bacteria and yeast. ECEM is an extract from cultured EM. EM∙XGOLD (Abbreviated as EMXG in this communication) is a diluted form of ECEM and brought to the market as a health drink [14].

Assay for the active principle

During purification processes, active principle was monitored by the promoted fruiting body formation of Dictyostelium discoideum [15; S1 Fig].

Liquid-liquid partition

ECEM was extracted with ethyl acetate three times. The ethyl acetate layer was concentrated in vacuo to yield ethyl acetate soluble fraction. The water layer was extracted with n-butanol three times. The n-butanol and water layers were concentrated in vacuo, respectively, to give n-butanol soluble and water-soluble fractions. The ethyl acetate soluble and n-butanol soluble fractions were combined as organic solvent soluble fraction.

Column chromatography

Two hundred g of dried water-soluble fraction were dissolved in 10 mL of water-methanol (1:4) mixture, and the solution was filtered through filter paper to give the filtrate and the insoluble fraction (31.5 mg). The filtrate was subjected to preparative HPLC. The column was eluted with a mixture of water-methanol (1:4) to give five fractions (S2 Fig).

Electrommicroscopy

Insoluble residual fraction was resuspended in water, and a drop of it was placed onto a carbon tape attached to a sample stage, air fried and further subjected to gold deposition. The specimen was viewed with scanning electron microscope, LA101, JOEL. The atomic element was analyzed with Energy Dispersive X-ray (EDX) Spectroscopy equipment.

Acid treatment

EMXG was acidified by dropwise addition of HCl until its pH reaches around 2. Then, it was neutralized by addition of NaOH until its pH reaches 7 ~ 8.

Assay for the active principle.

Twenty droplets of AX2 amoeba cell suspension, containing ca. 8,000 cells, were spotted onto non-nutrient agar plate which contained a separated fraction to be assayed. Forty-eight hours later, number of fruiting body was counted under dissecting microscope, and averaged to give a number for one spot. Control was composed of agar and water. Positive control contained 10% ECEM. For monitoring the active principle during purification processes, agar plate contained the separated fraction in an amount equivalent to 10% ECEM.

(TIF)

Click here for additional data file.

Column chromatography of water-soluble fraction.

Two hundred g of dried water-soluble fraction were dissolved in 10 mL of water-methanol (1:4) mixture, and the solution was filtered through filter paper to give the filtrate and the insoluble (31.5 mg). The filtrate was subjected to preparative HPLC (column, JAIGEL-GS310 (20 mm x 500 mm, Japan Analytical Industry, Co. Ltd.). The column was eluted with a mixture of water-methanol (1:4) to give five fractions.

(TIF)

Click here for additional data file.

Energy Dispersive X-ray (EDX) spectroscopy.

The downward arrows point to signals of Mg and K. Other signals are from the background.

(TIF)

Click here for additional data file.

Scanning electron micrograph of insoluble residue after autoclave treatment.

Note the exposed mesh-like structures.

(TIF)

Click here for additional data file.

(XLSX)

Click here for additional data file.

30 Jun 2021

PONE-D-21-17352

Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortium

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Reviewer #1: The work Higashinakagawa et al. describes stimulatory effects of an extract from culture of Effective Microorganisms (ECEM) and a health drink containing ECEM (EM XGOLD) on cellular activity of model organism Dictyostelium discoideum. Higashinakagawa et al. show that inorganic material such as magnesium and potassium containing microparticles can be active substances of ECEM. However, no statement about possible mechanisms of this effect is included. Authors only discuss few papers regarding the biological effect of inorganic materials and write that this subject is currently under their investgation.

This simple manuscript is clearly written. The methodological data are well documented by nice micrographs, graphs and by statistic evaluation and I do not have not any major reservations. I believe this work will interest many workers in the field of cell biology and action of inorganic compounds on cellular functions as well as in nutrition supplements.

Reviewer #2: Higashinakagawa et al, Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortium.

Higashinakagawa et al reported a very interesting and intriguing study and explain how simple elements have shown significant biological activity, namely, as they called the Effective Microorganisms (EM) on life cycle of Dictyostelium discoideum. They carried out systematic and careful studies of such EM･XGOLD, which is a well-known and popular health drink in Japan. This study is a continuation from their report in 2019 that “The life cycle of Dictyostelium discoideum is accelerated via MAP kinase cascade by a culture extract produced by a synthetic microbial consortium.”

They used various methods including i) Liquid-liquid partition, ii) column chromatography, iii) electron microscopy, iv) elemental analysis using Energy Dispersive X-ray (EDX) Spectroscopy, v) acid treatment and alkali pH titrations to figure out the major component of the biological activity. Through their careful observations and detailed studies, they conclude that the Mg, K-containing microparticle biological effect could be due to MgCO3. Although intriguing, their careful data and systematic analysis justify their conclusion. They may have solved one of long-standing puzzles in the popular health drink in Japan.

Some minor points:

1) They use the term EM for Effective Microorganisms. This abbreviation is not necessary since there are only 2 words. EM commonly refers to Electron Microscopy. It is rather confusion to read EM but it refers to something totally different. The legendary Francis Crick advised this reviewer 30 years ago to avoid use all unnecessary abbreviations and acronyms, so the readers can understand the articles better and avoid confusions.

2) Please write out ECEM on line 83, page 5.

3) Please write out CMC (carbon microcoils) on line 238, page 9. CMC commonly refers to critical micelle concentration.

4) Please add more figure legend to Figure 5, provide more information about the microparticle, why the sizes cross a wide range. Do they all have similar elemental contents?

5) On lines 183-184, they wrote “The result was exactly as shown in Fig. 6”. Please replace the word exactly with “similar” since the results in Figure are not exactly, but similar. For example, in Panel A, E20 is ~33% and in Panel B, the E20 is ~38%. They are similar but not exactly identical.

6) On line 221, page 9, they wrote “we have no idea regarding the functional role and the state of being of potassium……”. Please change to “in the current study, we did not focus on the study of potassium”.

After they make these corrections and changes, this reviewer highly recommends publication in PLoS ONE.

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Reviewer #1: No

Reviewer #2: Yes: Shuguang Zhang

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Submitted filename: Referee report for PONE 6-2021.docx

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22 Jul 2021

Academic Editor:

Comment: There are several point mentioned and addressed by the reviewers that need to be corrected in a revised version. I also recommend careful language editing before resubmission.

Reply: Yes, we did it.

Comment: For details on the required changes please carefully address all points raised be the reviewers. Because it may not be clear from the comment from reviewer one, I like to add that the authors should add and discusse more carefully the state of the art regarding possible mechanisms.

Reply: In the revised manuscript, we discussed to some extent the state of art regarding the possible mechanisms.

Journal Requirements:

Comment: Thank you for stating the following financial disclosure:

Reply: We stated the financial disclosure ‘This study was supported in the form of a grant awarded from the University of Tsukuba to HK.’

Comment: At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

Reply: Our study was funded from a grant awarded from the University of Tsukuba to HK.

Comment: State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Reply: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Comment: If any authors received a salary from any of your funders, please state which authors and which funders.

Reply: We did not receive any salary from any funders.

Comment: If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Reply: We got funding from a grant awarded from the University of Tsukuba to HK.

Comment: Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Reply: We did so as you suggested.

Comment: We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Reply: We removed the sentence which includes “data not shown”.

Comment: We note that Figure(s) 5 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

Reply: Fig.5 and Supplementary Fig. 4 in the submitted manuscript are the raw photographic data which we obtained directly from the scanning electron microscope. However, in order not to invite unnecessary confusion or misunderstanding, we removed the bottom end of the photograph and inserted a scale bar of 1 μm to Fig.5 and Supplementary Fig. 4.

Comment: Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Reply: Done as requested.

Reviewers' comments:

Reviewer's Responses to Questions

Reviewer #2:

Comment 1: They use the term EM for Effective Microorganisms. This abbreviation is not necessary since there are only 2 words. EM commonly refers to Electron Microscopy. It is rather confusion to read EM but it refers to something totally different. The legendary Francis Crick advised this reviewer 30 years ago to avoid use all unnecessary abbreviations and acronyms, so the readers can understand the articles better and avoid confusions.

Reply 1: EM is not simply an abbreviation. EM is a registered trademark and a brand name owned by EMRO (EM Research Organization). EM refers to a synthetic microbial consortium and is used over 100 countries. We already used this word, EM, in our previous paper (Ref.15 of this manuscript). On visiting web site emrojapan.com, more information about EM is available. Accordingly, we changed line 263 -265 as follows.

“EM represents a short form of Effective Microorganisms and is a registered trademark and a brand name owned by EMRO (EM Research Organization). EM consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast.”

Comment 2: Please write out ECEM on line 83, page 5.

Reply 2: In compliance with the comment of Reviewer #2, we left out the term ECEM.

Comment 3: Please write out CMC (carbon microcoils) on line 238, page 9. CMC commonly refers to critical micelle concentration.

Reply 3: The term CMC for Carbon Micro Coil is an abbreviation made not by us, but was coined by its inventor, S. Motojima. Ref. 20 of this manuscript shows how it was termed. Accordingly, we changed line 238 (236 in the revised manuscript) as follows.

“carbon micro coil (CMC)[20].”

Comment 4: Please add more figure legend to Figure 5, provide more information about the microparticle, why the sizes cross a wide range. Do they all have similar elemental contents?

Reply 4: In compliance with the suggestion by Reviewer #2, we provided more information about the microparticle in the figure legend to Fig.5.

Comment 5: On lines 183-184, they wrote “The result was exactly as shown in Fig. 6”. Please replace the word exactly with “similar” since the results in Figure are not exactly, but similar. For example, in Panel A, E20 is ~33% and in Panel B, the E20 is ~38%. They are similar but not exactly identical.

Reply 5: In compliance with the suggestions by Reviewer #2, we replaced the word “exactly” with “similar” in the revised manuscript.

Comment 6: On line 221, page 9, they wrote “we have no idea regarding the functional role and the state of being of potassium……”. Please change to “in the current study, we did not focus on the study of potassium”.

Reply 6: In compliance with the suggestion by Reviewer #2, we changed the wording in the revised manuscript.

Question: PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Reply: No

Submitted filename: Response to Reviewers7.docx

Click here for additional data file.

6 Oct 2021

PONE-D-21-17352R1Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortiumPLOS ONE

Dear Dr. higashinakagawa,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 20 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Stefan Wölfl, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Thank you for the submission of the revised version. As the reviewers point out most of their major concerns have been addressed in the revision. There is however still one aspect that I do not fully understand at that makes the manuscript difficult to understand and unclear regarding the Intension of the work. The work is solely used on the trademark registered EM consortium of microorganisms from EM research organization. This is a commercial product and registered trademark. This needs to be clear in the manuscript and it will be necessary to stay potential conflicts of interest regarding the impact the work will have on the commercialization of EM XGold or EM(r).

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have addressed the reviewer's concerns and suggestions. They have made corrections and revised the manuscript. This reviewer now recommends publication of this very interesting paper.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

15 Oct 2021

October 15, 2021

PLoS One

Dear Dr. Stefan Wölfl:

Please find herewith our revised manuscript entitled “Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortium” by Toru Higashinakagawa, Haruhisa Kikuchi and Hidekazu Kuwayama.

In compliance with the Editor’s suggestion, we stated in Introduction that EM is a registered trade mark and a brand name owned by EMRO (EM Research Organization). We stated that this work was initiated based on genuine scientific curiosity, since many papers on EM thus far published described only phenomenological observations. We were truly curious to know how EM works in a more simple system like cellular slime mold. This point was stated in the third paragraph of the introduction in the revised manuscript.

The Editor also asked about the intension of our work. To answer this question, I will describe how this work started and then proceeded. Almost 10 years ago, Kuwayama, the 3rd author of the present paper, expressed his interest in EM•XGOLD which was/is freely available on the market. Kuwayama worked on cellular slime mold Dictyostelium discoideum for many years, and had been looking for any biological substances which may affect the life cycle of Dictyostelium discoideum. He had heard the rumor about EM•XGOLD and asked me to collaborate with him for looking at the effect of EM•XGOLD on the life cycle of this slime mold. I was then just retired from Waseda University, Tokyo, but still got a lab space at Tokyo Women’ Medical University. Thus, our joint work started in 2010. Since my place at TWMU did not have any facilities to grow Dictyostelium discoideum, most experiments were carried out in Kuwayama’s lab, 3 hours’ train ride away. In 2019, our first paper along this line was accepted in Journal of Molecular Microbiology and Biotechnology (JMMB, 29, 35-42, 2019). The JMMB paper described the differentiation promoting activity, namely promotion of fruiting body formation, in Dictyostelium discoideum, among other interesting effects. We found those observations quite interesting and proceeded to identify what the active principle of EM•XGOLD really is. At this point, we asked Kikuchi, the 2nd author of the present manuscript, to join our group. Kikuchi was/is an organic chemist and contributed to the identification of biologically active substances from a variety of bio-organisms including Dictyostelium discoideum. Thus, our joint work started for identifying the active principle of EM•XGOLD which lead to the present manuscript.

As described above, our work is genuinely based on the scientific curiosity. We have never intended to advertise EM•XGOLD to the society. Moreover, our result refers only to cellular slime mold Dictyostelium discoideum, never meaning to address humans. Accordingly, we do not think the information inherent in this manuscript would not affect the market situation.

We hope the present manuscript will meet the publication in PLOS One.

Thank you for your consideration. I look forward to hearing from you.

Sincerely,

Dr. Toru Higashinakagawa

International Center for Molecular, Cellular and Immunological Research

Tokyo Women’s Medical University

8-1 Kawada-cho, Shinjuku

Tokyo 162-8666, Japan

TEL: +81-3-3353-8112 ( ext. 24062 )

FAX: +81-3-3352-3088

E-mail: toru@waseda.jp

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

18 Oct 2021

Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortium

PONE-D-21-17352R2

Dear Dr. higashinakagawa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Stefan Wölfl, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

21 Oct 2021

PONE-D-21-17352R2

Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortium

Dear Dr. Higashinakagawa:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Dr. Stefan Wölfl

Academic Editor

PLOS ONE