Optimization of the Production and Characterization of an Antifungal Protein by Bacillus velezensis Strain NT35 and Its Antifungal Activity against Ilyonectria robusta Causing Ginseng Rusty Root Rot

Round 1

Reviewer 1 Report

The objective of this study was to optimize the media and fermentation conditions of Bacillus velezensis NT35 for increasing secondary metabolite production against Ilyonectria robusta, which causes ginseng rusty root rot, and to further isolate, purify, and characterize the antifungal protein. The fermentation conditions were optimized using a Plackett–Burman design (PB test) and response surface methodology (RSM). The most protein precipitation concentration was determined through (NH4)2SO4 grading precipitation, then the purified single protein was obtained via DEAE-Sepharose Fast Flow and Superdex 75 10/300 540 GL. Finally, the authors determine that the purified protein has extremely high stability to changes in temperature, pH, and UV radiation, as well as strong antifungal activity in in vitro assays. Furthermore, the isolated protein was identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) and its secondary structure predicted and analyzed. Microscopic observation showed that mycelia treated with the isolated antifungal protein changed significantly: the hyphal branches increased, twisted, and partially expanded into a spherical or elliptical shape; the hyphae broke; and the hyphal cells became vacuoles. The results are adequately presented in the respective Tables and Figures, however, the writing of the manuscript must be substantially improved in order to be perfectly understood.

First, the authors already in the abstract must make it clear from which plant they isolated the Bacillus velezensis NT35 strain used in this study, as well as the plant species that is infected by the fungus Ilyonectria robusta. Panax ginseng C.A. Mey. or Panax quinquefolius var. ginseng (C.A. Mey.) Regel & Maack, Chinese ginseng is a small herbaceous plant of the Araliaceae family and the Panax genus, whose root is used in traditional medicine. Panax quinquefolium L. or Panax quinquefolius var. americanus Raf. is North American ginseng.

Second, the sentence included between lines 276 and 279 is difficult to understand, please clarify what it means. The meat extract provides the culture medium with a source of carbohydrates, nitrogen and vitamins for microbial growth. On the other hand, in common varieties of corn, protein content can range from 8 to 11 percent of kernel weight.

Third, when the secondary structure of the isolated protein was predicted and analyzed, the authors mention β-sheets in the text while in Table S6 they indicate β-turns. Both forms constitute different types of non-regular secondary structure in protein. β-turns are the most common form of turns—a type of non-regular secondary structure in proteins that cause a change in direction of the polypeptide chain. β-sheets consist of β-strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet.

Fourth, the sentences included between lines 514 and 518 are difficult to understand, please clarify what this mean. The authors begin by saying that the fermentation fluid of the B. velezensis HN-2 strain ‘exhibited strong antifungal activity against Xoo’. What does Xoo mean? To the best of my knowledge, Xoo refers to the bacterial strain Xanthomonas oryzae pv. oryzae. Therefore, the second sentence is acceptable, although it should be improved as follows: 'B. velezensis HN-2 secondary metabolite surfactin is the main active substance of HN-2, which can induce’ X. oryzae pv. oryzae to produce polyhydroxyalkanoates (PHAs).

Fifth, a correct wording must avoid the abusive use of terms such as ‘our’ (line 528), possessive form of the first-person plural, or ‘we’ (528, 536, 541, 554, and 569), a pronoun with which the person who speaks or writes refers to himself or herself plus another person or persons. Please rewrite these sentences.

Finally, scientific names must be spelled correctly, always in italics, and using the appropriate abbreviations. In the same way, the Figures and Tables must be explanatory by themselves.

The manuscript is well written and presented, therefore, if these matters as well as the specific comments detailed below are properly included and corrected, in my opinion it can be considered for publication in Fermentation.

 

Specific comments

- Lines 14-15: Panax ginseng C.A. Mey. 

- Line 16: P. ginseng.  

- Line 25: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

- Line 36: (Panax ginseng C.A. Mey.)

- Line 37: Panax genus.

- Lines 71, 72 and 495: S. aureus (in italics)

- Line 87: on nutrient agar (NA) medium

- Line 91: PDA medium

- Line 101: The optical density at 600 nm (OD₆₀₀) of

- Line 116: H₃BO₃

- Line 121: Response Surface Methodology (RSM)

- Line 122: Plackett–Burman (PB test) Experimental Design

- Line 124: Delete (PB test)

- Line 140: via RSM

- Line 164: (10⁵ spores·mL^-1)

- Line 183: (DEAE-Sepharose Fast Flow Column, GE Healthcare, Chicago, IL, EE. UU.)

- Lines 190-191:  Delete the entire parentheses.

- Lines 193-194: (Superdex 75 Increase 193 10/300 GL, GE Healthcare, Chicago, IL, EE. UU.)

- Line 199: via N-(2-Hydroxy-1,1-bis(hydroxymethyl) ethyl) glycine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE)

- Line 207: desorption/ionization

- Lines 244 and 249: ultra-depth of field microscope

- Line 256: 10⁸ cfu·mL^-1

- Line 259: Growth curve of the Bacillus velezensis NT35 strain.

- Line 302: of the Bacillus velezensis NT35 strain.

- Line 304: PB Test

- Line 336: Use the formula (K₂HPO₄) as always and therefore delete the IUPAC name (dipotassium hydrogen phosphate).

- Line 375: Effects of different liquid fermentation conditions on the Bacillus velezensis NT35 strain.

- Lines 398, 401, and 549:  MALDI-TOF-MS

- Line 402: molecular mass (Mr)

- Line 407: the BLAST program protein database

- Line 408: ProtParam software

- Lines 412-413:  Escherichia coli (in italics)

- Line 423: Through the online analysis software TransMembrane prediction using Hidden Markov models (TMHMM Server Version 2.0),

- Line 426: Using the online analysis software Self-Optimized Prediction Method with Alignment (SOPMA),

- Lines 427-428: where h is the α-helix, e is the extended polypeptide chain (β-strands), t is expressed as β-turns, and c represents ….

- Line 441: I. robusta

- Line 453 and 455: Ilyonectria robusta

- Line 462: Bacillus coagulans (in italics)

- Lines 464-465: Delete ‘through screening the nutrient conditions of W. cibaria strain JW15 using a Plackett–Burman design’. It's redundant.

- Line 466: JW15

- Lines 467, 468, 472, 474, 478, 479, and 483: RSM

- Line 473: Bacillus amyloliquefaciens (in italics)

- Lines 475 and 494: E. coli (in italics)

- Line 480: Bacillus subtilis (in italics)

- Line 494: Pseudomonas aeruginosa (in italics)

- Line 496: Bacillus anthracis (in italics)

- Line 498: Ralstonia solanacearum (in italics)

- Line 506: R. solanacearum (in italics)

- Lines 514-518:  A study found that the n-butanol extract of the B. velezensis HN-2 strain fermentation liquid exhibited strong antibacterial activity against Xanthomonas oryzae pv. oryzae, and the main active ingredient in the n-butanol extract was obtained through separation and purification. B. velezensis HN-2 secondary metabolite surfactin is the main active substance of HN-2, which can induce X. oryzae pv. oryzae to produce polyhydroxyalkanoates (PHAs).

- Line 519: Bacillus methylotrophicus (in italics)

- Lines 533-534: which can significantly inhibit growth mycelium of Alternaria solani causal agent of early blight in stem, foliage and tubers of potatoes.

- Line 563: (Streptomyces lunalinharesii A54A)

- Line 564: R. solani (in italics)

- Line 565: The authors state that mycelial changes may be caused by antifungal NPs (nanoparticles, nanoproteins, nanoproducts, ???) produced by S. lunalinharesii. It would be more appropriate to name them antifungal secondary metabolites, right?

- Line 566: S. lunalinharesii (in italics)

- Line 574: Biofungicides, please. In addition, despite the high stability shown by the 1-4-2F protein and the possibility of producing large quantities for commercial exploitation, sometimes the commercial formulations for dissemination in crops are fragile, short-lived, ineffective and difficult to register or obtaining authorization for use by the competent National Organizations. The in vitro results are not determinative to approach the suggested investigations and developments, therefore extra vitrum experiments are necessary and essential.

 

Supplementary Materials

Figure S2. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) results. (A) Identification results of antifungal protein 1-4-2F; (B) Amino acid sequence homology analysis of antifungal proteins.

Figure S3. Bioinformatic analysis of antifungal protein 1-4-2F. (A) Transmembrane structure prediction; (B) Secondary structure prediction, where h is the α-helix, e is the extended polypeptide chain, t is expressed as β-turns, and c represents random coils.

Table S1. Plackett–Burman experimental design number and measured value. A, cornmeal, 10–20 g·L^-1; B, maltose, 10–20 g·L^-1; C, glucose, 10–20 g·L^-1; D, (NH₄)₂SO₄, 15–35 g·L^-1; E, beef extract, 15–35 g·L^-1; F, yeast extract powder, 15–35 g·L^-1; G, K₂HPO₄, 10–20 g·L^-1; H, KCl, 10–20 g·L^-1; and I, Na₂CO₃, 10–20 g·L^-1.

Table S3. Experiment design and results of response surface analysis. The inhibition zone values (mm) are expressed as mean ± standard deviation (SD).

Table S6. Alpha helix

Author Response

Response to Reviewer 1 Comments

 

Point 1: First, the authors already in the abstract must make it clear from which plant they isolated the Bacillus velezensis NT35 strain used in this study, as well as the plant species that is infected by the fungus Ilyonectria robusta. Panax ginseng C.A. Mey. or Panax quinquefolius var. ginseng (C.A. Mey.) Regel & Maack, Chinese ginseng is a small herbaceous plant of the Araliaceae family and the Panax genus, whose root is used in traditional medicine. Panax quinquefolium L. or Panax quinquefolius var. americanus Raf. is North American ginseng.

Response 1: Thanks to the reviewer for valuable comments on the article, we have already corrected which plant they isolated the Bacillus velezensis NT35 strain used in this study, as well as the plant species that is infected by the fungus Ilyonectria robusta in the abstract.

Line 14-16: “A biocontrol Bacillus velezensis strain, NT35, was isolated from the rhizosphere soil of ginseng, and its sterile fermentation liquid had a significant inhibitory effect against Ilyonectria robusta, which causes rusty root rot in Panax ginseng.”

Line 35-36: “Ginseng (Panax ginseng C. A. Mey) is a perennial herbal medicinal plant belonging to the Araliaceae family and the Panax genus.”

 

Point 2: Second, the sentence included between lines 276 and 279 is difficult to understand, please clarify what it means. The meat extract provides the culture medium with a source of carbohydrates, nitrogen and vitamins for microbial growth. On the other hand, in common varieties of corn, protein content can range from 8 to 11 percent of kernel weight.

Response 2: Thanks to the reviewer for valuable comments on the article, I’m very sorry for the inconvenience caused by our inappropriate description, we have checked and rephrased..

Line 645-653: ”Secondly, according to the growth of the bacterium cells, beef extract is better than blank control, but beef extract is also both a carbon source and a nitrogen source, which may lead to inaccurate experimental results, so it is not suitable as a nitrogen source. The bacterial growth of (NH₄)₂SO₄ is slightly higher blank control and (NH₄)₂SO₄ is an am-monium salt required by the culture medium. Therefore, tryptone in the basic medium was finally replaced and screened using (NH₄)₂SO₄, which is more suitable as a nitrogen source. Therefore, the combination of an inorganic nitrogen source (NH₄)₂SO₄ and an organic nitrogen source yeast extract powder was selected as the nitrogen source for the next test.”

 

Point 3: Third, when the secondary structure of the isolated protein was predicted and analyzed, the authors mention β-sheets in the text while in Table S6 they indicate β-turns. Both forms constitute different types of non-regular secondary structure in protein. β-turns are the most common form of turns—a type of non-regular secondary structure in proteins that cause a change in direction of the polypeptide chain. β-sheets consist of β-strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet.

Response 3: Thanks to the reviewer for a valuable comments on the article. This was a mistake in our writing and it has been corrected.

Line 841-849: “the protein secondary structure was predicted and analyzed, where h is the α-helix, e is the extended polypeptide chain (β-strands), t is expressed as β-turns, and c represents random coils.”

 

Point 4: Fourth, the sentences included between lines 514 and 518 are difficult to understand, please clarify what this mean. The authors begin by saying that the fermentation fluid of the B. velezensis HN-2 strain ‘exhibited strong antifungal activity against Xoo’. What does Xoo mean? To the best of my knowledge, Xoo refers to the bacterial strain Xanthomonas oryzae pv. oryzae. Therefore, the second sentence is acceptable, although it should be improved as follows: 'B. velezensis HN-2 secondary metabolite surfactin is the main active substance of HN-2, which can induce’ X. oryzae pv. oryzae to produce polyhydroxyalkanoates (PHAs).

Response 4: Thanks to the reviewer for a valuable comments on the article. We have revised the sentences between lines 514 and 518 as suggested.

Line 989-994: “A study found that the n-butanol extract of the B. velezensis HN-2 strain fermentation liquid exhibited strong antifungal activity against Xanthomonas oryzae pv. oryzae, Xoo, and the main active ingredient in the n-butanol extract was obtained through separation and purification. B. velezensis HN-2 secondary metabolite surfactin is the main active sub-stance of HN-2, which can induce X. oryzae pv. oryzae to produce polyhydroxyalkanoates (PHAs).”

 

Point 5: Fifth, a correct wording must avoid the abusive use of terms such as ‘our’ (line 528), possessive form of the first-person plural, or ‘we’ (528, 536, 541, 554, and 569), a pronoun with which the person who speaks or writes refers to himself or herself plus another person or persons. Please rewrite these sentences.

Response 5: Thanks to the reviewer for a valuable comments on the article. We have rewritten these sentences.

 

Point 6: Finally, scientific names must be spelled correctly, always in italics, and using the appropriate abbreviations. In the same way, the Figures and Tables must be explanatory by themselves.

Response 6: Thanks to the reviewer for a valuable comments on the article. We have carefully checked and corrected the spelling of scientific names, using italics.

Line 971: “Clostridium perfringen, and S. aureus.”

Line 156-157: “B. velezensis strain NT35 was cultured on nutrient agar (NA) medium.”

Line 257: “(FeSO₄, KI, CuSO₄, H₃BO₃ and ZnSO₄) with a concentration of 5 g·L-1 were also used.”

Line 262: “2.4. Fermentation Optimization via Response Surface Methodology (RSM).”

Line 263: “2.4.1. Plackett–Burman (PB test) Experimental Design.”

Line 281: “2.4.3. Fermentation Optimization via RSM.”

Line 326-327: “The 80 μL conidia suspension (10⁵ spores·mL^-1) of I. robusta strain CBLJ-3 was uni-formly spread on a PDA medium plate.”

Line 344-345: “After using buffer A to equilibrate the weak anion exchange column (DEAE-Sepharose Fast Flow Column, GE Healthcare, Chicago, IL, EE. UU.) with 5 column volumes.”

Line 442-444: “After using buffer A to equilibrate the weak anion exchange column (Superdex 75 In-crease 193 10/300 GL, GE Healthcare, Chicago, IL, EE. UU.) with 5 column volumes.”

Line 449-451: “The purity of the target protein was determined via N-(2-Hydroxy-1,1-bis(hydroxymethyl) ethyl) glycine-sodium dodecyl sulfate poly-acrylamide gel electrophoresis (Tricine-SDS-PAGE).”

Line 587: “Microstructure changes in the purified antifungal protein against ginseng rust rot pathogen were observed via ultra-depth of field microscope (Keyence Co., Ltd., Shanghai, China).”

Line 631 “Figure 1. Growth curve of the Bacillus velezensis NT35 strain.”

Line 689-691: “Figure 2. Effects of different components of fermentation media on the inhibitory and bacterial concentration of the fermentation filtrate of of the Bacillus velezensis NT35 strain.. (A) Carbon sources; (B) Nitrogen sources; (C) Inorganic salts; (D) Trace elements.”

Line 729-730: “cornmeal, K₂HPO₄, and (NH₄)₂SO₄ can change within a certain range so that the number of viable bacteria in the fermentation broth can reach the highest level.”

Line 776 “Figure 4. Effects of different liquid fermentation conditions on the Bacillus velezensis NT35 strain.”

 

Point 7: The authors state that mycelial changes may be caused by antifungal NPs (nanoparticles, nanoproteins, nanoproducts, ???) produced by S. lunalinharesii. It would be more appropriate to name them antifungal secondary metabolites, right?

Response 7: Thanks to the reviewer for a valuable comments on the article. We have carefully checked and corrected this mistake.

Line 1098-1100: “An actinomycete (Streptomyces lunarinharesi A54A) co-cultured with the plant pathogen R. solani can produces secondary metabolites with antifungal activity.”

 

Point 8: Biofungicides, please. In addition, despite the high stability shown by the 1-4-2F protein and the possibility of producing large quantities for commercial exploitation, sometimes the commercial formulations for dissemination in crops are fragile, short-lived, ineffective and difficult to register or obtaining authorization for use by the competent National Organizations. The in vitro results are not determinative to approach the suggested investigations and developments, therefore extra vitrum experiments are necessary and essential.

Response 8: Thanks to the reviewer for the valuable comments on the article. In this study, the antifungal effect of 1-4-2F protein have been proved by in-dish treatment and in vitro inoculation of ginseng root. We will accept the suggestion of reviewer and carry out pot and field experiments to further determine the effect of protein on disease control.

 

Supplementary Materials

Figure S2. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) results. (A) Identification results of antifungal protein 1-4-2F; (B) Amino acid sequence homology analysis of antifungal proteins.

Figure S3. Bioinformatic analysis of antifungal protein 1-4-2F. (A) Transmembrane structure prediction; (B) Secondary structure prediction, where h is the α-helix, e is the extended polypeptide chain, t is expressed as β-turns, and c represents random coils.

Table S1. Plackett–Burman experimental design number and measured value. A, cornmeal, 10–20 g·L^-1; B, maltose, 10–20 g·L^-1; C, glucose, 10–20 g·L^-1; D, (NH₄)₂SO₄, 15–35 g·L^-1; E, beef extract, 15–35 g·L^-1; F, yeast extract powder, 15–35 g·L^-1; G, K₂HPO₄, 10–20 g·L^-1; H, KCl, 10–20 g·L^-1; and I, Na₂CO₃, 10–20 g·L^-1.

Table S3. Experiment design and results of response surface analysis. The inhibition zone values (mm) are expressed as mean ± standard deviation (SD).

Table S6. Alpha helix

Response 8: Thanks to the reviewer for the valuable comments on the article. We have revised and corrected those above you said in the manuscript.

Reviewer 2 Report

comments

1- the title  very long  Media optimization using response surface methodology and 2 purification and characterization of a novel antifungal protein  isolated from Bacillus velezensis strain NT35 against Ilyonectria robusta

2- line 37 genus  how italic line 412 Escherichia  coli. must be italic

3- line 86   2.1. Strain Culture change to source of culture strain

4- Discussion based on the result  out the result in most 

5- toxicity experiment must be add to confirm the safe of isolated protein

6- Fig. 9 contains chlamydospores, as a result of antifungal activity must be pointe to it in the text

7-  antifungal activity performed on one fungus how. 

Author Response

Response to Reviewer 2 Comments

 

Point 1: the title very long Media optimization using response surface methodology and 2 purification and characterization of a novel antifungal protein isolated from Bacillus velezensis strain NT35 against Ilyonectria robusta

 

Response 1: Thanks to the reviewer for valuable comments of the article. The title has been revised according to the comments of the fourth reviewer and has been marked in the article..

Line 2-5: ”Optimization the production and characterization of an antifungal protein by Bacillus velezensis strain NT35 and its antifungal activity against the Ginseng rusty root rot pathogen Illyonectria robusta.”

 

Point 2: line 37 genus how italic line 412 Escherichia coli. must be italic

 

Response 2: Thanks to the reviewer for valuable comments of the article. The italic have been corrected.

Line 826-827: ” more than 20 h in yeast cells, and more than 10 h in Escherichia coli.”

 

Point 3: line 86 2.1. Strain Culture change to source of culture strain

 

Response 3: Thanks to the reviewer for a valuable comment. We have changed 2.1. Strain Culture to source of culture strain.

Line 155: “2.1. source of culture strain.”

 

Point 4: Discussion based on the result out the result in most

Response 4: Thanks to the reviewer for a valuable comment. We have rewriten the discussion .

Line 906-1140: “Adjusting the nutritional and physical environment of secondary metabolites is a major step in the optimization of fermentation conditions. In our study, by measuring the growth curve of the strain NT35, it was shown that at 9 hours, the most abundant bacteria were 10⁸cfu/mL and had strong activity (Figure 1). In order to further explore the optimization of its fermentation conditions, the antifungal activity against I. robusta was detected by changing the composition of the fermentation medium, as well as the change of bacterial concentration, indicating that the combination of cornmeal, inorganic nitrogen source (NH₄)₂SO₄ and organic nitrogen source yeast extract was selected , K₂HPO₄ as candidate carbon sources, nitrogen sources, and inorganic salts for further optimization of strain NT35 (Figure 2). A study showed that the optimal nutrient conditions of Weissella cibaria strain JW15 were screened using a PB design. The optimal medium significantly increased the biomass production of JW15 by 1.98-fold. The dry cell weight was 1.67 times higher than that of the RSM medium [20]. Another study showed that the fermentation variables of Bacillus sp. RKY3 were designed according to the PB design and response surface method; the production of protease in the optimized medium increased by 2.3-fold overall, and the enzyme activity increased significantly by 522 U·mL^-1 [21]. In a study, the culture medium of Bacillus amyloliquefaciens strain HM618 was optimized based on the response surface method, and the surfactin level was increased by 1.152 g·L^-1 against Botrytis cinerea, Rhizoctonia solani, and E. coli [22]. Similarly, in our study, based on the single factor screening results, through the PB test, cornmeal, yeast extract, Na₂CO₃, and (NH₄)₂SO₄ significant factors were selected for the next step of the steepest climbing test (Table S1). The best dosage was explored through the steepest climbing test, and the results showed that yeast extract (25 g·L^-1), cornmeal (15 g·L^-1), K₂HPO₄ (15 g·L^-1), and (NH₄)₂SO₄ (25 g·L^-1) had the greatest antifungal effect (Table S2). Therefore, this combination is selected for the next step of RSM design. And last, through the RSM test, the response surface plot and contour plot were obtained (Figure S1). The optimized fermentation medium and basal medium were tested for antifungal activity, and the antifungal activity of the optimized fermentation medium was 40.46% higher than that of the basal medium (Figure 3). Although the optimized fermentation medium was obtained, the inoculum size, pH, temperature and rotational speed were not screened, so the physical environment of the optimized fermentation broth was studied. The optimal inoculum size finally screened out was 6%, the optimal initial pH was 7.0, the optimal temperature was 34°C, and the optimal rotational speed was 180 rpm (Figure 4). The antifungal activity of the optimized fermentation medium was significantly improved, which provided a basis for the subsequent separation of the fermentation filtrate.

The main goal is clarifying the mechanism of microbial biocontrol to isolate, purify, and identify antifungal metabolites as antibiotic substances that can inhibit target cells by interfering with the biosynthesis of the peptidoglycan layer by blocking cell wall formation [23]. For example, B. subtilis strain LFB112 can produce a bacteriocin BLIS with a molecular weight of 6.3 kDa, which exhibits inhibition activity against the pathogens E. coli, Salmonella pullorum, Pseudumonas aeniginasa, Pasteurella multocida, Clostridium perfringen, and S. aureus [24]. Yoshida et al. found that B. amyloliquefaciens RC-2 produced bacteriocin A2 against Bacillus anthracis and several other phytopathogenic fungi [25]. Two rhizosphere-associated B. velezensis isolates (Y6 and F7) exhibit strong antifungal activity against Ralstonia solanacearum and Fusarium oxysporum, among which lipopeptides play distinct roles [26]. A study isolated and screened a fungal-antagonistic endophytic B. velezensis FZ06 from fresh Pu-erh tea tree leaves, and used this as the starting strain to extract and isolate its antifungal products against B. velezensis FZ06. When purified, it has been identified as an antifungal lipopeptide, and has a good inhibitory effect on typical food spoilage bacteria and toxin-producing fungi [27]. In addition, the crude lipopeptides (iturins, fenycins, and suractins) of B. velezensis strain FJAT-46737 had an inhibitory rate of 96.2% against R. solanacearum causing tomato bacterial wilt [28]. In another study, a strain with high antipathogenic activity was screened out from 77 strains isolated from sea mud in the Arctic Ocean: B. velezensis. The production of active metabolites of the strain was improved by optimizing the medium composition and fermentation conditions, and the structure of the main metabolites was identified. The results show that it has a certain growth-promoting effect on plants. The metabolites of the strain contain macrolactin A, which has obvious antagonistic effects on a variety of pathogenic bacteria and fungi. Experiments on cucumber seedlings have shown that the metabolites of the strain had a protective effect on cucumber wilt [29]. A study found that the n-butanol extract of the B. velezensis HN-2 strain fermentation liquid exhibited strong antifungal activity against Xanthomonas oryzae pv. oryzae, and the main active ingredient in the n-butanol extract was obtained through separation and purification. B. velezensis HN-2 secondary metabolite surfactin is the main active substance of HN-2, which can induce X. oryzae pv. oryzae to produce polyhydroxyalkanoates (PHAs) [30].

By adding different saturation concentrations of (NH₄)₂SO₄ to screen the saturation concentration showing the best antifungal activity, it is concluded that 30% (NH₄)₂SO₄ can be used as the optimal concentration for the next step of separation and analysis (Figure 5A). After filtration by Superdex G-25 column, two absorption peaks were obtained. After antifungal detection, the absorption peak 1F with more significant activity was selected for the next step of ion exchange chromatography (Figure 5B). Five absorption peaks were obtained after DEAE-Sepharose Fast Flow column chromatography, among which 1-4F and 1-5F had antifungal activity, and 1-4F with stronger activity was selected for subsequent gel filtration chromatography (Figure 5C). Finally, after Superdex 75 10/300 GL gel chromatography, an active absorption peak was obtained, so Tricine-SDS-PAGE for this absorption peak showed only one band, indicating that the isolated and purified protein was a single antifungal Proteins can then be analyzed by mass spectrometry (Figure 5D, 6). The antifungal activity of the purified antifungal protein was measured, and it was shown that the growth rate of rust fungus was different after different concentrations of protein were added to PDA medium, indicating that the higher the protein concentration added, the more inhibited the growth of rust mycelium. According to the LC-MS/MS results, the amino acid sequence of protein 1-4-2F had 100% similarity to the hypothetical protein from B. velezensis YAU B9601-Y2 (Accession No: AFJ62117). A total of 17 unique peptides were detected in the protein with a molecular weight of 10.176 kDa, and 89 amino acids were detected with an isoelectric point of 9.08. This protein encodes an unknown protein, and so we speculated that a new antifungal protein may be isolated. We screened the searched proteins, and stipulated that the minimum number of matching peptides was 4; a score value > 70 and a sequence coverage > 20% were statistically significant (Figure S2). Transmembrane structure prediction of the antifungal protein revealed lack of transmembrane structure. The secondary structure predicts that α-helix contains 32.58%, extended polypeptide chain contains 32.58%, β-turns contains 17.98%, and random coils contains 16.85%. More alpha helix structures can enable the protein to maintain its structure and perform its functions (Figure S3). Aspergillus pachycristatus is useful for the production of the antifungal echinocandin B, but its secondary metabolites are unknown. Research by constructing mutants of secondary metabolic genes, evaluating the secondary metabolites produced by wild-type and mutant strains, and exploring secondary metabolism through metabolic networks reveals the presence of a series of unexplored secondary metabolites [31]. An actinomycete (Streptomyces lunarinharesi A54A) co-cultured with the plant pathogen R. solani can produces secondary metabolites with antifungal activity [32]. Similarly, although the protein in this study has no functional domain due to the small capacity of the provided B. velezensis protein database, it is not matched to the protein in the NCBI (https://www.ncbi.nlm.nih.gov/taxonomy). This indicates that the protein in this study is an unknown new protein, and this protein can exhibit strong antifungal activity against I. robusta after optimizing its fermentation conditions, and isolating and purifying it. Therefore, protein 1-4-2F can be further studied via cloning, prokaryotic expression, and gene knockout experiments. This lays the foundation for the construction of biocontrol engineering bacteria and the development and application of biopesticides.

A study found that Bacillus methylotrophicus NJ13 isolated from ginseng has an aseptic fermentation liquid that has an obvious inhibitory effect on I. robusta, and the antifungal protein obtained after separation and purification has thermal stability, pH stability, and ultraviolet stability [17]. The antifungal activity of the active substances in the fermentation product of strain B. velezensis FZ06 can be well maintained under heat treatment conditions in a 100 °C water bath for 30 min and can be kept stable for more than 28 days in an environment of 4 °C; it also has a good pH value in the range 2–10. The acid and alkali tolerance of the antifungal active substances in the fermented product can be extracted with anhydrous methanol, and no antifungal activity has been detected in the extraction residue [27]. Similarly, in this study, the stability test of the purified protein shows that it has a wide range of antifungal activity at temperature and pH, and it can still maintain a high activity after incubation at 20 °C-100 °C for 30 minutes, and it can still maintain high activity at pH 4-10. It can maintain the activity, and has no obvious effect on the antifungal activity of the protein under ultraviolet light irradiation, but the protein completely loses its activity after being treated with proteinase K and chloroform (Figure 7). It shows that this protein has extremely high stability, which can provide a theoretical basis in the next study.

A study found that B. subtilis ZD01 strain with a strong antagonistic effect on potato early blight was screened, and the secondary metabolite antimicrobial peptide of B. subtilis ZD01 was identified as the main antifungal substance, which can significantly inhibit growth mycelium of Alternaria solani causal agent of early blight in stem, foliage and tubers of potatoes. This leads to mycelium bending, surface wrinkling, local swelling, and other deformities, and extracellular secretions increase significantly [33]. In this study, it was found that after treatment of I. robusta with antifungal proteins, the growth of mycelia could be inhibited, and mycelium showed the phenomenon of increased branching, twisted mycelia, an enlarged top, and broken mycelia by microscopic observation. Therefore, the antifungal protein isolated and purified from B. velezensis NT35 is expected to become a new biological control agent to replace chemical fungicides.”

 

Point 5: toxicity experiment must be add to confirm the safe of isolated protein

 

Response 5: Toxicity test is very necessary to ensure the safety of the protein, and is also a prerequisite for the future development and application of the substance. This paper is in the basic research stage which the purpose is to isolate and purify the active substance, and to determine its antifungal effect, and no application experiment has been involved. We will accept the reviewer's good advice to determine its toxicity and confirm its safety before carrying out application studies.

 

Point 6: Fig. 9 contains chlamydospores, as a result of antifungal activity must be pointe to it in the text

Response 6: Thanks to the reviewer for a valuable comment. We have redone the microscopic observation experiment and supplemented the microscopic observation of spore morphology.

Line 892: “Figure 9 has changed.”

 

Point 7: antifungal activity performed on one fungus how.

Response 7: Thanks to the reviewer for a valuable comment. We demonstrated the inhibitory stability of antifungal proteins against the target pathogen Ilyonectria robusta causing ginseng rusty root rot through differernt physcial conditions in stability studies in Figure 7, and demonstrated the antifungal activity of different concentrations of antifungal proteins against fungi through toxicity assays in Figure 8.

 

Reviewer 3 Report

The work entitled “Media optimization using response surface methodology and purification and characterization of a novel antifungal protein isolated from Bacillus velezensis strain NT35 against Ilyonectria robusta”, authors are Mengtao Li, Hao Tang, Zongyan Li, Yu Song, Lin Chen, Chao Ran, Yun Jiang, and Changqing Chen, is devoted to estimation of antagonist (antifungal) activities of biocontrol bacteria Bacillus velezensis and investigation of biological properties of this bacterium. A lot of growth experiments was performed to select the medium for better growth of B. velezensis NT35 and its highest antifungal activity against the causative agent of ginseng rusty root rot Ilyonectria robusta. A novel protein with antifungal activity was isolated and identified. Its antifungal activity was measured and its stability in ranges of temperature and pH was studied. Additionally, stability of the protein under the action of ultraviolet irradiation was estimated.

 

Specific comments

1) In lines 68-75, it was written that another B. velezensis strain (RP137) was earlier used to optimize the production of antibacterial compounds, and other antibacterial substances were described in B. velezensis. It compromises the novelty of the presented research since just another strain of the same species is studied in this work, and it causes doubts that differences in growth and optimized fermentation media between different strains of one species are significant. To avoid such compromises, it is recommended to compare medium optimization for the NT35 and a novel protein from the NT35 with media optimization and antibacterial substances from other B. velezensis strains.

2) Lines 206-208 – Details of MALDI TOF MS procedure are required. Was a matrix used? How was the sample preparation performed?

3) In Discussion, it is recommended to compare (in terms better, higher, similar to, etc.) the antifungal activity and stability properties of the novel protein isolated from the B. velezensis ТЕ35 with antifungal agents from other biocontrol microorganisms.

Summary

Minor revision.

Author Response

Response to Reviewer 3 Comments

 

Point 1: In lines 68-75, it was written that another B. velezensis strain (RP137) was earlier used to optimize the production of antibacterial compounds, and other antibacterial substances were described in B. velezensis. It compromises the novelty of the presented research since just another strain of the same species is studied in this work, and it causes doubts that differences in growth and optimized fermentation media between different strains of one species are significant. To avoid such compromises, it is recommended to compare medium optimization for the NT35 and a novel protein from the NT35 with media optimization and antibacterial substances from other B. velezensis strains.

Response 1: Thanks to the reviewer for valuable comments on article, we have seriously corrected it,

we have rewritten this section on lines 139-153.

 

Point 2: Lines 206-208 – Details of MALDI TOF MS procedure are required. Was a matrix used? How was the sample preparation performed?

Response 2: Thanks to the reviewer for valuable comments on article,I’m really sorry that I wrote the wrong procedure of mass spectrometry as MALDI-TOF-MS, and now correcte it as follow.

Line 457-482: “The electrophoretic band was cut out and the target proteins were identified via LC-MS/MS (Beijing Protein Innovation CO., Ltd., Beijing, China) analysis.

Put the cut glue into a clean 1.5 ml centrifuge tube, add 1 ml ddH₂O to the tube, wash for 10 minutes, remove the water, repeat once. Add 1ml of Kodye destaining solution to the tube, wash for 10 minutes, remove the destaining solution, and repeat once (configuration of in-gel digestion and destaining solution: 50% acetonitrile, 25 mM NH₄HCO₃). Finally, acetonitrile was added for dehydration until the colloidal particles were completely white, and the acetonitrile was vacuum-dried. After draining, add 10 mM DTT to allow the particles to absorb completely, put them in a 56-degree water bath, and incubate for 1 hour. After incubation, remove excess DTT liquid, add 55 mM IAM, and incubate at room temperature in the dark for 45 minutes. After incubation, remove excess IAM liquid, add 25 mM ammonium bicarbonate, wash for 10 minutes, and repeat once. Centrifuge to remove ammonium bicarbonate, add destaining solution to wash for 10 minutes, and repeat once. Acetonitrile was dehydrated until the colloidal particles completely turned white, and the acetonitrile was vacuum-dried. 1 μg·μL^-1 enzyme stock solution was diluted 15 times with 25 mM NH₄HCO₃, and added to the dehydrated micelles to allow the micelles to fully absorb. Then add 25 mM NH₄HCO₃ to cover the micelles, put them in a 37 °C water bath, and digest overnight. After overnight, digestion was terminated by adding FA at a final concentration of 0.1%. Take a 10 μL sample on the machine and use a mass spectrometer for detection. The dried peptides were reconstituted with 0.1% formic acid (FA) solution and separated, and the separated peptides were ionized with an ESI ion source, and then entered into a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, America) for DIA mode Detection, and then use Mascot Version 2.3.01 to search the Uniprot_human (Number of sequences: 172097) database to identify the protein.”

 

Point 3: In Discussion, it is recommended to compare (in terms better, higher, similar to, etc.) the antifungal activity and stability properties of the novel protein isolated from the B. velezensis NT35 with antifungal agents from other biocontrol microorganisms.

Response 3: Thanks to the reviewer for valuable suggestions on article, we have rewriten and improved the discussion section as follow.

Line 906-1140: “Adjusting the nutritional and physical environment of secondary metabolites is a major step in the optimization of fermentation conditions. In our study, by measuring the growth curve of the strain NT35, it was shown that the most abundant bacteria were 10⁸cfu/mL and had strong activity at 9 hours (Figure 1). In order to further explore the optimization of its fermentation conditions, the antifungal activity against I. robusta was detected by changing the composition of the fermentation medium, as well as the change of bacterial concentration, indicating that the combination of cornmeal, inorganic nitrogen source (NH₄)₂SO₄ and organic nitrogen source yeast extract was selected , K₂HPO₄ as candidate carbon sources, nitrogen sources, and inorganic salts for further optimization of strain NT35 (Figure 2). A study showed that the optimal nutrient conditions of Weissella cibaria strain JW15 were screened using a PB design. The optimal medium significantly increased the biomass production of JW15 by 1.98-fold. The dry cell weight was 1.67 times higher than that of the RSM medium [20]. Another study showed that the fermentation variables of Bacillus sp. RKY3 were designed according to the PB design and response surface method; the production of protease in the optimized medium increased by 2.3-fold overall, and the enzyme activity increased significantly by 522 U·mL^-1 [21]. In a study, the culture medium of Bacillus amyloliquefaciens strain HM618 was optimized based on the response surface method, and the surfactin level was increased by 1.152 g·L^-1 against Botrytis cinerea, Rhizoctonia solani, and E. coli [22]. Similarly, in our study, based on the single factor screening results, through the PB test, cornmeal, yeast extract, Na₂CO₃, and (NH₄)₂SO₄ significant factors were selected for the next step of the steepest climbing test (Table S1). The best dosage was explored through the steepest climbing test, and the results showed that yeast extract (25 g·L^-1), cornmeal (15 g·L^-1), K₂HPO₄ (15 g·L^-1), and (NH₄)₂SO₄ (25 g·L^-1) had the greatest antifungal effect (Table S2). Therefore, this combination is selected for the next step of RSM design. And last, through the RSM test, the response surface plot and contour plot were obtained (Figure S1). The optimized fermentation medium and basal medium were tested for antifungal activity, and the antifungal activity of the optimized fermentation medium was 40.46% higher than that of the basal medium (Figure 3). Although the optimized fermentation medium was obtained, the inoculum size, pH, temperature and rotational speed were not screened, so the physical environment of the optimized fermentation broth was studied. The optimal inoculum size finally screened out was 6%, the optimal initial pH was 7.0, the optimal temperature was 34°C, and the optimal rotational speed was 180 rpm (Figure 4). The antifungal activity of the optimized fermentation medium was significantly improved, which provided a basis for the subsequent separation of the fermentation filtrate.

The main goal is clarifying the mechanism of microbial biocontrol to isolate, purify, and identify antifungal metabolites as antibiotic substances that can inhibit target cells by interfering with the biosynthesis of the peptidoglycan layer by blocking cell wall formation [23]. For example, B. subtilis strain LFB112 can produce a bacteriocin BLIS with a molecular weight of 6.3 kDa, which exhibits inhibition activity against the pathogens E. coli, Salmonella pullorum, Pseudumonas aeniginasa, Pasteurella multocida, Clostridium perfringen, and S. aureus [24]. Yoshida et al. found that B. amyloliquefaciens RC-2 produced bacteriocin A2 against Bacillus anthracis and several other phytopathogenic fungi [25]. Two rhizosphere-associated B. velezensis isolates (Y6 and F7) exhibit strong antifungal activity against Ralstonia solanacearum and Fusarium oxysporum, among which lipopeptides play distinct roles [26]. A study isolated and screened a fungal-antagonistic endophytic B. velezensis FZ06 from fresh Pu-erh tea tree leaves, and used this as the starting strain to extract and isolate its antifungal products against B. velezensis FZ06. When purified, it has been identified as an antifungal lipopeptide, and has a good inhibitory effect on typical food spoilage bacteria and toxin-producing fungi [27]. In addition, the crude lipopeptides (iturins, fenycins, and suractins) of B. velezensis strain FJAT-46737 had an inhibitory rate of 96.2% against R. solanacearum causing tomato bacterial wilt [28]. In another study, a strain with high antipathogenic activity was screened out from 77 strains isolated from sea mud in the Arctic Ocean: B. velezensis. The production of active metabolites of the strain was improved by optimizing the medium composition and fermentation conditions, and the structure of the main metabolites was identified. The results show that it has a certain growth-promoting effect on plants. The metabolites of the strain contain macrolactin A, which has obvious antagonistic effects on a variety of pathogenic bacteria and fungi. Experiments on cucumber seedlings have shown that the metabolites of the strain had a protective effect on cucumber wilt [29]. A study found that the n-butanol extract of the B. velezensis HN-2 strain fermentation liquid exhibited strong antifungal activity against Xanthomonas oryzae pv. oryzae, and the main active ingredient in the n-butanol extract was obtained through separation and purification. B. velezensis HN-2 secondary metabolite surfactin is the main active substance of HN-2, which can induce X. oryzae pv. oryzae to produce polyhydroxyalkanoates (PHAs) [30].

By adding different saturation concentrations of (NH₄)₂SO₄ to screen the saturation concentration showing the best antifungal activity, it is concluded that 30% (NH₄)₂SO₄ can be used as the optimal concentration for the next step of separation and analysis (Figure 5A). After filtration by Superdex G-25 column, two absorption peaks were obtained. After antifungal detection, the absorption peak 1F with more significant activity was selected for the next step of ion exchange chromatography (Figure 5B). Five absorption peaks were obtained after DEAE-Sepharose Fast Flow column chromatography, among which 1-4F and 1-5F had antifungal activity, and 1-4F with stronger activity was selected for subsequent gel filtration chromatography (Figure 5C). Finally, after Superdex 75 10/300 GL gel chromatography, an active absorption peak was obtained, so Tricine-SDS-PAGE for this absorption peak showed only one band, indicating that the isolated and purified protein was a single antifungal Proteins can then be analyzed by mass spectrometry (Figure 5D, 6). The antifungal activity of the purified antifungal protein was measured, and it was shown that the growth rate of rust fungus was different after different concentrations of protein were added to PDA medium, indicating that the higher the protein concentration added, the more inhibited the growth of rust mycelium. According to the LC-MS/MS results, the amino acid sequence of protein 1-4-2F had 100% similarity to the hypothetical protein from B. velezensis YAU B9601-Y2 (Accession No: AFJ62117). A total of 17 unique peptides were detected in the protein with a molecular weight of 10.176 kDa, and 89 amino acids were detected with an isoelectric point of 9.08. This protein encodes an unknown protein, and so we speculated that a new antifungal protein may be isolated. We screened the searched proteins, and stipulated that the minimum number of matching peptides was 4; a score value > 70 and a sequence coverage > 20% were statistically significant (Figure S2). Transmembrane structure prediction of the antifungal protein revealed lack of transmembrane structure. The secondary structure predicts that α-helix contains 32.58%, extended polypeptide chain contains 32.58%, β-turns contains 17.98%, and random coils contains 16.85%. More alpha helix structures can enable the protein to maintain its structure and perform its functions (Figure S3). Aspergillus pachycristatus is useful for the production of the antifungal echinocandin B, but its secondary metabolites are unknown. Research by constructing mutants of secondary metabolic genes, evaluating the secondary metabolites produced by wild-type and mutant strains, and exploring secondary metabolism through metabolic networks reveals the presence of a series of unexplored secondary metabolites [31]. An actinomycete (Streptomyces lunarinharesi A54A) co-cultured with the plant pathogen R. solani can produces secondary metabolites with antifungal activity [32]. Similarly, although the protein in this study has no functional domain due to the small capacity of the provided B. velezensis protein database, it is not matched to the protein in the NCBI (https://www.ncbi.nlm.nih.gov/taxonomy). This indicates that the protein in this study is an unknown new protein, and this protein can exhibit strong antifungal activity against I. robusta after optimizing its fermentation conditions, and isolating and purifying it. Therefore, protein 1-4-2F can be further studied via cloning, prokaryotic expression, and gene knockout experiments. This lays the foundation for the construction of biocontrol engineering bacteria and the development and application of biopesticides.

A study found that Bacillus methylotrophicus NJ13 isolated from ginseng has an aseptic fermentation liquid that has an obvious inhibitory effect on I. robusta, and the antifungal protein obtained after separation and purification has thermal stability, pH stability, and ultraviolet stability [17]. The antifungal activity of the active substances in the fermentation product of strain B. velezensis FZ06 can be well maintained under heat treatment conditions in a 100 °C water bath for 30 min and can be kept stable for more than 28 days in an environment of 4 °C; it also has a good pH value in the range 2–10. The acid and alkali tolerance of the antifungal active substances in the fermented product can be extracted with anhydrous methanol, and no antifungal activity has been detected in the extraction residue [27]. Similarly, in this study, the stability test of the purified protein shows that it has a wide range of antifungal activity at temperature and pH, and it can still maintain a high activity after incubation at 20 °C-100 °C for 30 minutes, and it can still maintain high activity at pH 4-10. It can maintain the activity, and has no obvious effect on the antifungal activity of the protein under ultraviolet light irradiation, but the protein completely loses its activity after being treated with proteinase K and chloroform (Figure 7). It shows that this protein has extremely high stability, which can provide a theoretical basis in the next study.

A study found that B. subtilis ZD01 strain with a strong antagonistic effect on potato early blight was screened, and the secondary metabolite antimicrobial peptide of B. subtilis ZD01 was identified as the main antifungal substance, which can significantly inhibit growth mycelium of Alternaria solani causal agent of early blight in stem, foliage and tubers of potatoes. This leads to mycelium bending, surface wrinkling, local swelling, and other deformities, and extracellular secretions increase significantly [33]. In this study, it was found that after treatment of I. robusta with antifungal proteins, the growth of mycelia could be inhibited, and mycelium showed the phenomenon of increased branching, twisted mycelia, an enlarged top, and broken mycelia by microscopic observation. Therefore, the antifungal protein isolated and purified from B. velezensis NT35 is expected to become a new biological control agent to replace chemical fungicides.”

 

Reviewer 4 Report

This is classical piece of work of which many already is published in the literature. I need to be presented in similar simple manner that literature is published. Please see and you may consider the point raised in the attached sheet of comments.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 4 Comments

 

Point 1: Abstract

• First sentence (line14) does not agree with what is said in the material and methods lines 87-89. How did you isolate that bacterium from the rhizosphere?
• Line 15 “sterile fermentation liquid” very ambiguous and not scientific statement !!! Use more microbiological scientific terminology such as “culture filtrate” sterilized by Millipore filtration (give origin of the pore ..) or some time called “cold sterilization”
• Lines 14-16 a conclusion statement out of place for an abstract
• Lines 16-19 should be reading, The fermentation media composed of….
• Line 18, ‘optimal inoculum was 6% ? very strange. Otherwise, what was the bacterial cell count or density of the inoculum??
• Lines 19-22 indicate as if that protein is already known in the background published literature and here the researchers are looking for that specific protein in the culture filtrate of this bacterium, which is NOT. The present work on a new strain. This is different from what is in lines 68-71 by the isolate and active component(s).
• Statement in lines 24-27 blows up all this presentation and probably the work behind it. So, were you looking for such protein to start with from the new strain? But what is isolated by all means is different protein. It is NOT the same protein. What is “hypothetical similarity”??

Response 1: Thanks to the reviewer for valuable comments on the article, we have checked and corrected it.

• First sentence (line14) does not agree with what is said in the material and methods lines 87-89. How did you isolate that bacterium from the rhizosphere?

The laboratory selected a ginseng rhizosphere soil in Nong'an County, Changchun City, Jilin Province, and obtained a strain of bacteria by gradient dilution with sterile water. After functional testing, it showed that it had an antagonistic effect, and named it “NT35”.

Line 14: “A biocontrol Bacillus velezensis strain, NT35, was isolated from the rhizosphere soil of ginseng.”

Line 156-157: “B. velezensis strain NT35 was isolated from ginseng rhizosphere soil, and it was purified on nutrient agar (NA) medium and stored at 4 °C.”

• Line 15 “sterile fermentation liquid” very ambiguous and not scientific statement !!! Use more microbiological scientific terminology such as “culture filtrate” sterilized by Millipore filtration (give origin of the pore ..) or some time called “cold sterilization”

Line 15: “sterile filtrate were obtained through a 0.22 μm filter.”

• Lines 14-16 a conclusion statement out of place for an abstract

We have checked and deleted it.

• Lines 16-19 should be reading, The fermentation media composed of….

Line 16-19: “In order to obtain the best sterile filtrate, the medium fermentation conditions of the strain NT35 were optimized by using response surface methodology (RSM), and the best composition was obtained, therefore the fermentation media composed of yeast extract powder 2.5%, cornmeal 1.5%, K₂HPO₄ 1.5%, and (NH₄)₂SO₄ 2.5%.”

• Line 18, ‘optimal inoculum was 6% ? very strange. Otherwise, what was the bacterial cell count or density of the inoculum??

In line 19, optimal inoculum was 6% is means that the optimal inoculation rate is the highest concentration at 9 h determined by the growth curve below, which is 10⁸ cfu·mL^-1, and this concentration is used as the standard, this part has been showed in line 600-602.

• Lines 19-22 indicate as if that protein is already known in the background published literature and here the researchers are looking for that specific protein in the culture filtrate of this bacterium, which is NOT. The present work on a new strain. This is different from what is in lines 68-71 by the isolate and active component(s).

We described the isolation and purification results of the NT35 antifungal protein 1-4-2F in this study in lines 20-23 of the abstract, and in lines 142-145 in the introduction, we explained that the purified metabolites of the strains have antifungal properties the characteristic of activity by the cited references. The two are different studies, and the isolated antifungal substances are not the same substance.

• Statement in lines 24-27 blows up all this presentation and probably the work behind it. So, were you looking for such protein to start with from the new strain? But what is isolated by all means is different protein. It is NOT the same protein. What is “hypothetical similarity”??

Thanks to the reviewer for valuable comments on the article, we precipitated the protein by (NH₄)₂SO₄ precipitation, and separated and purified a single antifungal protein by different chromatographic methods. The obtained antifungal protein was analyzed by LC-MS/MS, and this protein is a hypothetical protein through alignment with amino acid sequence database, that means it is a unknown function protein, but doesn't mean it's a new protein.

 

Point 2: Introduction

• Very wordy with so much information that need not to be included per example, lines 37-52 has no value and needs to delete.
• Line 55-56 is not exact citation to the information in the reference.
• Lines 56-59 also not exact citation and very ambiguous
• Lines 62-65 not related to present work.
• Lines 68-72 again reflect the problem of misunderstanding the microbiological activity resaerch. Line 68, you do not use the organism to optimize. You manipulate the growth condition of certain organism to optimize the production of …
• Lines 72-74. Why did you not target that protein???
• Line 76 another miss use of the scientific nomenclature. The “culture supernatant” is not ‘culture filtrate??
• Lines 81-84 should be.. to manipulate the growth conditions, nutrition and physical factors to optimize the .. production by this bacterium.

Response 2: Thanks to the reviewer for valuable comments on the article, we have checked and corrected those in introduction.

• Line 55-56 is not exact citation to the information in the reference.

Line 125-128: ” In one study, Wu et al. isolated and purified the antifungal protein from the supernatant of Bacillus to inhibit the growth of Staphylococcus aureus.”

• Lines 56-59 also not exact citation and very ambiguous

Line 128-131: ” In another study, Alkaline protease was isolated and purified from Bacillus sp. ZJ1502, and its enzymatic properties were studied, which laid a foundation for further under-standing of the enzymatic properties of Bacillus sp.”

• Lines 62-65 not related to present work.

Thanks to the reviewer for valuable comments on the article, we have checked and deleted.

• Lines 68-72 again reflect the problem of misunderstanding the microbiological activity resaerch. Line 68, you do not use the organism to optimize. You manipulate the growth condition of certain organism to optimize the production of …
• Lines 72-74. Why did you not target that protein???

This is another study cited where iturin A is a nonribosomally synthesized antifungal substance, which is a lipopeptide not a protein. But the protein we isolated is an antifungal substance synthesized by ribosomes, and the two are not the same type of antifungal substance.

• Line 76 another miss use of the scientific nomenclature. The “culture supernatant” is not ‘culture filtrate??

Line 142-145: ” One study investigated the antifungal potential of the culture supernatant of B. velezensis AR1.”

• Lines 81-84 should be.. to manipulate the growth conditions, nutrition and physical factors to optimize the .. production by this bacterium.

Line 150-153: “The objective of this study was to manipulate the growth conditions, nutrition and physical factors to optimize the media and fermentation conditions of B. velezensis NT35, and to further isolate, purify, and characterize the antifungal protein from sterile filtrate.”

 

Point 3: Material and methods

• Lines 87-89 so the bacterium was not isolated by the researcher.
• Lines 91-92. 1 liter of PDA contain 500ml of extract from cooking the 200 g of potato slices. It is not as stated adding the 200g potato. Then 20g of glucose or dextrose is added.
• Line 92. So actually, the LB medium was not used as it is, modified. However, notice what is the Yest Extract for in the medium.
• Yeast extract is not as a source of carbon in this media.
• Line 106 media preparation to optimize the .. production
• Lines, 111 and 113, and any where in this work, how did you reach to using a universal amount of 10g per liter of those ingredient as a substitute to carbon and/ or nitrogen. The C and/or N in those ingredients varies according to its composition. So the comparison has no similar constant background of each or bot of C and /or N.
• Line 121 another mix-up in scientific terminology, Fermintation optimization.
• Line 145 it is manipulation of.. or just fermentation conditions,,, of or name it what is it
• Lines 153 – on. Crude culture filtrate protein. Then where is detecting activity in that protein before jumping to a certain moiety??
• Line 90 the statement is not sound. A single colony…
• Lines 103, 121, 122, 123, 230, 304, 212, 319, 338, and in other lines and heading using “Flashy” names of techniques which are not perfectly used or adhered to. Also, it is not necessary to be used here in this very classical elementary microbiology work. Use simple commonly used and understood by general readers and researchers.
• Line 148. Inoculum is usually described by number of cells per unit volume of density.
• Line 164. Well size, diameter??
• Line 179. The eluted protein fraction not the peak of the curve
• Line 199. So there were several eluted protein fractions and tested. where is that??
• To the line there is something missing, reaching up to “the active peak. Chosen”
• Line 231. Indoor Toxicity. what was the outdoor one.. not good term
• Line 353. Optimized production of active protein ….
• Line 355 Inoculum added in percentage. This should have been more exact?.
• Line 375 not on .. fermentation broth???
• Line 378, what is meant by: (NH4)2SO4 was graded and precipitated???

 

Response 3: Thanks to the reviewer for valuable comments on the article, we have checked and corrected.

• Lines 87-89 so the bacterium was not isolated by the researcher.

First of all, the bacterium strain NT35 was isolated from rhizosphere soil of ginseng before by our laboratory which is the Laboratory of Integrated Management of Plant Diseases, Jilin Agricultural University. And this strain has been proved that it can control the disease of ginseng rusty root rot.

• Line 92. So actually, the LB medium was not used as it is, modified. However, notice what is the Yest Extract for in the medium.

Yest Extract is the most ideal biological medium raw material and the main raw material in the fermentation industry. Its efficacy is equivalent to 8 times that of yeast, and it can greatly increase the production rate of strains and the yield of fermentation products.

(Line 235-238: “The basic fermentation medium was composed of NaCl (5 g·L^-1), tryptone (10 g·L^-1), and yeast extract (10 g·L^-1), at pH 7.0. The optimized medium was changed based on the composition of carbon source (yeast extract), nitrogen source (tryptone), inorganic salt (NaCl), and trace elements of the Luria-Bertani (LB) medium.”

• Lines, 111 and 113, and any where in this work, how did you reach to using a universal amount of 10g per liter of those ingredient as a substitute to carbon and/ or nitrogen. The C and/or N in those ingredients varies according to its composition. So the comparison has no similar constant background of each or bot of C and /or N.

In the study, carbon and nitrogen sources are components which have the advantages of convenience, easy availability, and low cost.

The purpose of this experiment is not to explore the content of carbon and nitrogen source elements contained in different types of carbon sources and nitrogen sources to determine the optimal concentration screening, but to explore the different types of carbon sources and nitrogen sources effect on bacterial growth. influence to determine the optimal mass concentration.

• Line 121 another mix-up in scientific terminology, Fermintation optimization.

Line 281: “2.4.3. Optimization via RSM.”

• Line 145 it is manipulation of.. or just fermentation conditions,,, of or name it what is it

Line 304: “2.5. Fermentation Conditions.”

• Lines 153 – on. Crude culture filtrate protein. Then where is detecting activity in that protein before jumping to a certain moiety??

The obtained crude protein solution is precipitated by (NH₄)₂SO₄ to precipitate the protein in the sterile filtrate, but the activity of the crude protein solution precipitated by different saturations of (NH₄)₂SO₄ is different, so we detect the activity of the protein by adding how much saturation of (NH₄)₂SO₄.

• Line 90 the statement is not sound. A single colony…

Line 240-241: “Strain NT35 was inoculated into 250 mL of LB medium and grown at 28 °C and 160 rpm to make a fermentation seed liquid.”

• Lines 103, 121, 122, 123, 230, 304, 212, 319, 338, and in other lines and heading using “Flashy” names of techniques which are not perfectly used or adhered to. Also, it is not necessary to be used here in this very classical elementary microbiology work. Use simple commonly used and understood by general readers and researchers.

Line 247: “2.3. Single-Factor Optimization.”

Line 262-263: “2.4. Response Surface Methodology (RSM); 2.4.1. Plackett–Burman (PB) Test Design.”

Line 572: “2.10. Toxicity Test.”

Line 692: “3.3. PB Test.”

Line 483: “2.9. Determination of Antifungal Stability.”

Line 707: “3.5. RSM Test.”

Line 732: “3.6. RSM Graphical Analysis.”

• Line 148. Inoculum is usually described by number of cells per unit volume of density.

The number of cells per unit volume of density in the seed liquid in the method is determined by the growth curve in the results, so it is not described in the method.

Line 307-308: “The optimal concentration of seed liquid was inoculated into the medium at 2%, 4%, 6%, 8%, and 10% (v/v).

• Line 164. Well size, diameter??

Line 327-328: “Punch 6 holes in the culture medium with a hole punch.”

• Line 179. The eluted protein fraction not the peak of the curve

Line 340-342: “The antifungal activity of each peak was determined based on the inhibition zone via the agar punching method and collected a large number of peaks with antifungal activity for further separation.”

• Line 199. So there were several eluted protein fractions and tested. where is that??
• To the line there is something missing, reaching up to “the active peak. Chosen”

Thank you for your question. I have explained this question in the result line 863-880.

Line 445-448: “The eluted peaks were collected and dialyzed against Tris-HCl at 4°C. After lyophiliza-tion, the antifungal activity of each peak was measured, and the active peak protein was prepared and collected on a large scale.”

• Line 231. Indoor Toxicity. what was the outdoor one.. not good term

Line 572: “2.10. Toxicity Test.”

• Line 353. Optimized production of active protein ….

Line 749: “3.7. Optimized production of active antifungal protein.”

• Line 355 Inoculum added in percentage. This should have been more exact?.

Inoculum added in percentage is mean that add 2-10 mL seed liquid to 100 mL medium.

• Line 375 not on .. fermentation broth???

Line 776-777: “Figure 4. Effects of different liquid fermentation conditions on the Bacillus velezensis NT35 strain. (A) Inoculum size; (B) pH; (C) Temperature; (D) Rotational speed.”

• Line 378, what is meant by: (NH4)₂SO₄ was graded and precipitated???

When a neutral salt such as (NH₄)₂SO₄ is added to the protein solution, because the affinity of the neutral salt to water molecules is greater than that of the protein, the hydration film around the protein molecule is weakened or disappeared, and the solubility of the protein is reduced. At the same time, due to the addition of the neutral salt, the protein solution The ionic strength of the protein has changed, and the surface charge of the protein is largely neutralized, which further leads to a decrease in the solubility of the protein, and causes the protein molecules to aggregate and precipitate. Different proteins will form precipitates in different concentrations of salt due to their different properties such as chargeability and hydrophilicity. Therefore (NH₄)₂SO₄ was graded and precipitated means adding different concentrations to precipitate protein, so as to select the optimal concentration of (NH₄)₂SO₄.

Line 779-780: “The antifungal activity of crude protein treated with different saturated concen-trations of (NH₄)₂SO₄ was detected.”

 

Point 4: Results

• In general, needs to be condensed and avoid repetition of the chemicals and other treatments.
• Fig. 2, In A and B there is a heterogeneous comparison between organic and inorganic components in the medium. The consistency of the components of the ones which are not tested in the background should have been made constant??
• Fig 3, same note above.] in previous section. Also, the caption should be inclusive by giving the content of the media, keeping in mind all table, figures meant to stand alone for the reader to have full information about each one of them.
• Fig. 4, What kind of medium kept constant under this variable??
• Fig 5, A, why the “supernatant” has such a low reading when it is same as the crude culture filtrate. What is the difference between them??
• Not clear why pick the peak 1-4A with less discrete inhibition zone (D) over the ones which show more discrete inhibition zones in (B &C)
• Fig. 6, very faint to almost not present band???
• Line 235 incubated rather than cultured.
• Fig. 8, The caption does not show What kind of media? Also, the interaction seems to be fungi-stasis effect.
• Fig, 9, The picture are not of good quality and what is meant to be observed is not very clear.

Discussion

• Lines 458-459. The whole presentation needs to be turned around according to the basic concept of this work with the microorganisms. It is to optimize the production od certain product or activity by an organism by manipulating the nutritional and physical environment around that organism.
• There is a lot of Introduction, review of the literature not needed to be included in the discussion. For example see lines 460 and on..??
• The whole discussion needs to be re-written being specific and discussing the work done and results. See lines 489 -491 it is not dealt with in the work??
• So as in lines 519-520 and so on

 

Response 4: Thanks to the reviewer for valuable comments on the article, we have checked and corrected.

• Fig. 2, In A and B there is a heterogeneous comparison between organic and inorganic components in the medium. The consistency of the components of the ones which are not tested in the background should have been made constant??

Firstly, I’m very glad you asked this question. The consistency of the components of the ones which are not tested in the background should have been made constant.

• Fig 3, same note above.] in previous section. Also, the caption should be inclusive by giving the content of the media, keeping in mind all table, figures meant to stand alone for the reader to have full information about each one of them.

Line 747-748: “Figure 3. The diameter of the inhibition zone of different fermentation media on Ilyonectria robusta. (A) basal medium (B) optimized medium.”

• Fig. 4, What kind of medium kept constant under this variable??

For example, when the inoculum size is variable, the temperature, rotation speed and pH remain the same as those used in the previous growth curve determination.

• Fig 5, A, why the “supernatant” has such a low reading when it is same as the crude culture filtrate. What is the difference between them??

         Not clear why pick the peak 1-4A with less discrete inhibition zone (D) over the ones which show more discrete inhibition zones in (B &C)

 “supernatant” refers to the liquid after the fermentation filtrate without adding (NH₄)₂SO₄ is centrifuged at 4°C to remove precipitate, and we have added in line 326-332, and the crude protein refers to the addition of (NH₄)₂SO₄ to the sterile filtrate to precipitate the protein, and obtain it by centrifugation and dialysis.

(B) The crude protein was separated by G-25, and two elution peaks could be separated, and 1F with stronger activity was selected by detecting its activity for the next experiment, we have added in line 788-789.

(C) Then weak ion exchange chromatography (DEAE-Sepharose Fast Flow) was performed on 1F, and 5 peaks were obtained. Through plate activity detection, it can be concluded that although 1-4 is not the peak with the highest absorption peak, it is the only one that has antifungal activity. So choose 1-4F, we have added in line 793-794.

(D) Gel filtration chromatography (Superdex 75 10/300 GL) was performed on 1-4F, and three peaks were obtained, which were detected by plate activity, and only 1-4-2 had antifungal activity. Therefore 1-4-2F was chosen for LC-MS/MS analysis, we have added in line 797.

• Fig. 6, very faint to almost not present band???

After the purified protein was subjected to Tricine-SDS-PAGE, the bands showed very faintly in the gel imager, because the molecular weight was very small and it was a single band, We have adjusted the picture clarity.

• Fig. 8, The caption does not show What kind of media? Also, the interaction seems to be fungi-stasis effect.

The method of the article describes that different concentrations of protein are added to the PDA medium, and fungi cakes are pasted in the center of the medium to observe the growth of Ilyonectria robusta. The results show that the higher the protein concentration, the stronger the inhibitory effect on the Ilyonectria robusta.

Line 879: “Figure 8. Inhibition of I. robusta by antifungal protein 1-4-2F.”

• Fig, 9, The picture are not of good quality and what is meant to be observed is not very clear.

Response 6: Thanks to the reviewer for a valuable comment. We have redone the microscopic observation experiment and supplemented the microscopic observation of spore morphology.

Line 892: “figure 9 has changed.”

 

Point 5：Discussion

• Lines 458-459. The whole presentation needs to be turned around according to the basic concept of this work with the microorganisms. It is to optimize the production od certain product or activity by an organism by manipulating the nutritional and physical environment around that organism.
• There is a lot of Introduction, review of the literature not needed to be included in the discussion. For example see lines 460 and on..??
• The whole discussion needs to be re-written being specific and discussing the work done and results. See lines 489 -491 it is not dealt with in the work??
• So as in lines 519-520 and so on

 

Response 5: Thanks to the reviewer for valuable comments on the article, we have checked and corrected.

• Lines 458-459. The whole presentation needs to be turned around according to the basic concept of this work with the microorganisms. It is to optimize the production od certain product or activity by an organism by manipulating the nutritional and physical environment around that organism.

Line 907-908: “Adjusting the nutritional and physical environment of secondary metabolites is a major step in the optimization of fermentation conditions.”

• There is a lot of Introduction, review of the literature not needed to be included in the discussion. For example see lines 460 and on..??
• The whole discussion needs to be re-written being specific and discussing the work done and results. See lines 489 -491 it is not dealt with in the work??
• So as in lines 519-520 and so on

Thanks to the reviewer for valuable suggestions on article, we have rewritten and improved the discussion section as follow.

Line 907-1140: “Adjusting the nutritional and physical environment of secondary metabolites is a major step in the optimization of fermentation conditions. In our study, by measuring the growth curve of the strain NT35, it was shown that at 9 hours, the most abundant bacteria were 10⁸cfu/mL and had strong activity (Figure 1). In order to further explore the optimization of its fermentation conditions, the antifungal activity against I. robusta was detected by changing the composition of the fermentation medium, as well as the change of bacterial concentration, indicating that the combination of cornmeal, inorganic nitrogen source (NH₄)₂SO₄ and organic nitrogen source yeast extract was selected , K₂HPO₄ as candidate carbon sources, nitrogen sources, and inorganic salts for further optimization of strain NT35 (Figure 2). A study showed that the optimal nutrient conditions of Weissella cibaria strain JW15 were screened using a PB design. The optimal medium significantly increased the biomass production of JW15 by 1.98-fold. The dry cell weight was 1.67 times higher than that of the RSM medium [20]. Another study showed that the fermentation variables of Bacillus sp. RKY3 were designed according to the PB design and response surface method; the production of protease in the optimized medium increased by 2.3-fold overall, and the enzyme activity increased significantly by 522 U·mL^-1 [21]. In a study, the culture medium of Bacillus amyloliquefaciens strain HM618 was optimized based on the response surface method, and the surfactin level was increased by 1.152 g·L^-1 against Botrytis cinerea, Rhizoctonia solani, and E. coli [22]. Similarly, in our study, based on the single factor screening results, through the PB test, cornmeal, yeast extract, Na₂CO₃, and (NH₄)₂SO₄ significant factors were selected for the next step of the steepest climbing test (Table S1). The best dosage was explored through the steepest climbing test, and the results showed that yeast extract (25 g·L^-1), cornmeal (15 g·L^-1), K₂HPO₄ (15 g·L^-1), and (NH₄)₂SO₄ (25 g·L^-1) had the greatest antifungal effect (Table S2). Therefore, this combination is selected for the next step of RSM design. And last, through the RSM test, the response surface plot and contour plot were obtained (Figure S1). The optimized fermentation medium and basal medium were tested for antifungal activity, and the antifungal activity of the optimized fermentation medium was 40.46% higher than that of the basal medium (Figure 3). Although the optimized fermentation medium was obtained, the inoculum size, pH, temperature and rotational speed were not screened, so the physical environment of the optimized fermentation broth was studied. The optimal inoculum size finally screened out was 6%, the optimal initial pH was 7.0, the optimal temperature was 34°C, and the optimal rotational speed was 180 rpm (Figure 4). The antifungal activity of the optimized fermentation medium was significantly improved, which provided a basis for the subsequent separation of the fermentation filtrate.

The main goal is clarifying the mechanism of microbial biocontrol to isolate, purify, and identify antifungal metabolites as antibiotic substances that can inhibit target cells by interfering with the biosynthesis of the peptidoglycan layer by blocking cell wall formation [23]. For example, B. subtilis strain LFB112 can produce a bacteriocin BLIS with a molecular weight of 6.3 kDa, which exhibits inhibition activity against the pathogens E. coli, Salmonella pullorum, Pseudumonas aeniginasa, Pasteurella multocida, Clostridium perfringen, and S. aureus [24]. Yoshida et al. found that B. amyloliquefaciens RC-2 produced bacteriocin A2 against Bacillus anthracis and several other phytopathogenic fungi [25]. Two rhizosphere-associated B. velezensis isolates (Y6 and F7) exhibit strong antifungal activity against Ralstonia solanacearum and Fusarium oxysporum, among which lipopeptides play distinct roles [26]. A study isolated and screened a fungal-antagonistic endophytic B. velezensis FZ06 from fresh Pu-erh tea tree leaves, and used this as the starting strain to extract and isolate its antifungal products against B. velezensis FZ06. When purified, it has been identified as an antifungal lipopeptide, and has a good inhibitory effect on typical food spoilage bacteria and toxin-producing fungi [27]. In addition, the crude lipopeptides (iturins, fenycins, and suractins) of B. velezensis strain FJAT-46737 had an inhibitory rate of 96.2% against R. solanacearum causing tomato bacterial wilt [28]. In another study, a strain with high antipathogenic activity was screened out from 77 strains isolated from sea mud in the Arctic Ocean: B. velezensis. The production of active metabolites of the strain was improved by optimizing the medium composition and fermentation conditions, and the structure of the main metabolites was identified. The results show that it has a certain growth-promoting effect on plants. The metabolites of the strain contain macrolactin A, which has obvious antagonistic effects on a variety of pathogenic bacteria and fungi. Experiments on cucumber seedlings have shown that the metabolites of the strain had a protective effect on cucumber wilt [29]. A study found that the n-butanol extract of the B. velezensis HN-2 strain fermentation liquid exhibited strong antifungal activity against Xanthomonas oryzae pv. oryzae, and the main active ingredient in the n-butanol extract was obtained through separation and purification. B. velezensis HN-2 secondary metabolite surfactin is the main active substance of HN-2, which can induce X. oryzae pv. oryzae to produce polyhydroxyalkanoates (PHAs) [30].

By adding different saturation concentrations of (NH₄)₂SO₄ to screen the saturation concentration showing the best antifungal activity, it is concluded that 30% (NH₄)₂SO₄ can be used as the optimal concentration for the next step of separation and analysis (Figure 5A). After filtration by Superdex G-25 column, two absorption peaks were obtained. After antifungal detection, the absorption peak 1F with more significant activity was selected for the next step of ion exchange chromatography (Figure 5B). Five absorption peaks were obtained after DEAE-Sepharose Fast Flow column chromatography, among which 1-4F and 1-5F had antifungal activity, and 1-4F with stronger activity was selected for subsequent gel filtration chromatography (Figure 5C). Finally, after Superdex 75 10/300 GL gel chromatography, an active absorption peak was obtained, so Tricine-SDS-PAGE for this absorption peak showed only one band, indicating that the isolated and purified protein was a single antifungal Proteins can then be analyzed by mass spectrometry (Figure 5D, 6). The antifungal activity of the purified antifungal protein was measured, and it was shown that the growth rate of rust fungus was different after different concentrations of protein were added to PDA medium, indicating that the higher the protein concentration added, the more inhibited the growth of rust mycelium. According to the LC-MS/MS results, the amino acid sequence of protein 1-4-2F had 100% similarity to the hypothetical protein from B. velezensis YAU B9601-Y2 (Accession No: AFJ62117). A total of 17 unique peptides were detected in the protein with a molecular weight of 10.176 kDa, and 89 amino acids were detected with an isoelectric point of 9.08. This protein encodes an unknown protein, and so we speculated that a new antifungal protein may be isolated. We screened the searched proteins, and stipulated that the minimum number of matching peptides was 4; a score value > 70 and a sequence coverage > 20% were statistically significant (Figure S2). Transmembrane structure prediction of the antifungal protein revealed lack of transmembrane structure. The secondary structure predicts that α-helix contains 32.58%, extended polypeptide chain contains 32.58%, β-turns contains 17.98%, and random coils contains 16.85%. More alpha helix structures can enable the protein to maintain its structure and perform its functions (Figure S3). Aspergillus pachycristatus is useful for the production of the antifungal echinocandin B, but its secondary metabolites are unknown. Research by constructing mutants of secondary metabolic genes, evaluating the secondary metabolites produced by wild-type and mutant strains, and exploring secondary metabolism through metabolic networks reveals the presence of a series of unexplored secondary metabolites [31]. An actinomycete (Streptomyces lunarinharesi A54A) co-cultured with the plant pathogen R. solani can produces secondary metabolites with antifungal activity [32]. Similarly, although the protein in this study has no functional domain due to the small capacity of the provided B. velezensis protein database, it is not matched to the protein in the NCBI (https://www.ncbi.nlm.nih.gov/taxonomy). This indicates that the protein in this study is an unknown new protein, and this protein can exhibit strong antifungal activity against I. robusta after optimizing its fermentation conditions, and isolating and purifying it. Therefore, protein 1-4-2F can be further studied via cloning, prokaryotic expression, and gene knockout experiments. This lays the foundation for the construction of biocontrol engineering bacteria and the development and application of biopesticides.

A study found that Bacillus methylotrophicus NJ13 isolated from ginseng has an aseptic fermentation liquid that has an obvious inhibitory effect on I. robusta, and the antifungal protein obtained after separation and purification has thermal stability, pH stability, and ultraviolet stability [17]. The antifungal activity of the active substances in the fermentation product of strain B. velezensis FZ06 can be well maintained under heat treatment conditions in a 100 °C water bath for 30 min and can be kept stable for more than 28 days in an environment of 4 °C; it also has a good pH value in the range 2–10. The acid and alkali tolerance of the antifungal active substances in the fermented product can be extracted with anhydrous methanol, and no antifungal activity has been detected in the extraction residue [27]. Similarly, in this study, the stability test of the purified protein shows that it has a wide range of antifungal activity at temperature and pH, and it can still maintain a high activity after incubation at 20 °C-100 °C for 30 minutes, and it can still maintain high activity at pH 4-10. It can maintain the activity, and has no obvious effect on the antifungal activity of the protein under ultraviolet light irradiation, but the protein completely loses its activity after being treated with proteinase K and chloroform (Figure 7). It shows that this protein has extremely high stability, which can provide a theoretical basis in the next study.

A study found that B. subtilis ZD01 strain with a strong antagonistic effect on potato early blight was screened, and the secondary metabolite antimicrobial peptide of B. subtilis ZD01 was identified as the main antifungal substance, which can significantly inhibit growth mycelium of Alternaria solani causal agent of early blight in stem, foliage and tubers of potatoes. This leads to mycelium bending, surface wrinkling, local swelling, and other deformities, and extracellular secretions increase significantly [33]. In this study, it was found that after treatment of I. robusta with antifungal proteins, the growth of mycelia could be inhibited, and mycelium showed the phenomenon of increased branching, twisted mycelia, an enlarged top, and broken mycelia by microscopic observation. Therefore, the antifungal protein isolated and purified from B. velezensis NT35 is expected to become a new biological control agent to replace chemical fungicides.”

Round 2

Reviewer 1 Report

The manuscript has been greatly improved with the incorporation of the suggestions made by the different reviewers. However, it still requires minor corrections before acceptance for publication in Fermentation. Among them, it is worth highlighting the incorrect denomination of some microbial species and the lack of specification of the products and methodologies used.

Specific comments

- Line 36: C.A. Mey. (no italics) 

- Line 37: genus (no italics). The species Panax genus does not exist.

- Line 54: Bacillus coagulans (in italics)

- Line 55: L. rhamnosus (in italics)

- Lines 82, 86 and 93: B. velezensis (in italics)

- Line 88: antifungal (Delete the hyphen).

- Line 92: I. robusta (in italics)

- Line 98: Source (The s in capital letter)

- Lines 169, 224, 225, 231, 232, 233, and 621: min (instead of minutes)

- Line 223: double distilled water (ddH₂O)

- Line 228: DL-Dithiothreitol (DTT)

- Line 230: iodoacetamide (IAM)

- Lines 232-233: NH₄HCO₃ (If you have chosen the formula as a way of referring to this compound, do not use its common name here, ammonium bicarbonate).

- Line 239: formic acid (FA)

- Lines 240-241: 0.1% FA

- Line 241: electrospray ionization (ESI)

- Line 243: Waltham, Massachusetts, EE. UU.)

- Line 243:  Data Independent Acquisition (DIA) mode Detection

- Lines 505-511: This sentence is impossible to understand. Please rewrite and clarify.

- Line 515: RSM

- Line 542: Salmonella pullorum is not a bacterial species. Its identification and serotyping corresponds to the scientific name of Salmonella enterica subsp. enterica serovar Pullorum.

- Line 542: The species Pseudomonas (Pseudauninas, Pseuduinonas, Preudamanas) aeniginasa does not exist. It is actually the species Pseudomonas aeruginosa.

- Lines 563 and 565: pv. (no italics)

- Line 593: α-helix

- Line 599: The species Streptomyces lunarinharesi does not exist. The correct scientific name is: Streptomyces lunalinharesii.

Supplementary Materials

Caption: The authors have changed the title of the manuscript but not the heading of the supplementary materials.

Author Response

Response to Reviewer 1 Comments

Point 1: Specific comments

- Line 36: C.A. Mey. (no italics) 

- Line 37: genus (no italics). The species Panax genus does not exist.

- Line 54: Bacillus coagulans (in italics)

- Line 55: L. rhamnosus (in italics)

- Lines 82, 86 and 93: B. velezensis (in italics)

- Line 88: antifungal (Delete the hyphen).

- Line 92: I. robusta (in italics)

- Line 98: Source (The s in capital letter)

- Lines 169, 224, 225, 231, 232, 233, and 621: min (instead of minutes)

- Line 223: double distilled water (ddH₂O)

- Line 228: DL-Dithiothreitol (DTT)

- Line 230: iodoacetamide (IAM)

- Lines 232-233: NH₄HCO₃ (If you have chosen the formula as a way of referring to this compound, do not use its common name here, ammonium bicarbonate).

- Line 239: formic acid (FA)

- Lines 240-241: 0.1% FA

- Line 241: electrospray ionization (ESI)

- Line 243: Waltham, Massachusetts, EE. UU.)

- Line 243:  Data Independent Acquisition (DIA) mode Detection

- Lines 505-511: This sentence is impossible to understand. Please rewrite and clarify.

- Line 515: RSM

- Line 542: Salmonella pullorum is not a bacterial species. Its identification and serotyping corresponds to the scientific name of Salmonella enterica subsp. enterica serovar Pullorum.

- Line 542: The species Pseudomonas (Pseudauninas, Pseuduinonas, Preudamanas) aeniginasa does not exist. It is actually the species Pseudomonas aeruginosa.

- Lines 563 and 565: pv. (no italics)

- Line 593: α-helix

- Line 599: The species Streptomyces lunarinharesi does not exist. The correct scientific name is: Streptomyces lunalinharesii.

 Response 1: Thanks to the reviewer for valuable comments on the article, we have already checked and corrected those. On lines 528-531, we have rewritten and explained the meaning of this passage.

Point 2: Supplementary Materials

Caption: The authors have changed the title of the manuscript but not the heading of the supplementary materials.

Response 2: Thanks to the reviewer for valuable comments on the article, we have checked and rephrased.

 

Reviewer 4 Report

As you have worked out the procedure of isolating such protein, then it would have been good to be taken to the next stage of further characterization of the protein structure. 

Author Response

Response to Reviewer 4 Comments

Point 1:

Comments and Suggestions for Authors

As you have worked out the procedure of isolating such protein, then it would have been good to be taken to the next stage of further characterization of the protein structure. 

Response 1: Thank you very much for your valuable suggestion. We are designing experiments and shall carry out the study of characterization of the protein structure in the next step according to the opinions expert said.