new production process of the antifungal chaetoglobosin a using cornstalks

b r /Cheng Jianga, Jinzhu Songa, Junzheng Zhangb, Qian Yanga, ∗ Q1 aHarbin Institute of Technology, School of Life Sciences and Technology, Harbin, Heilongjiang, People’s Republic of Chi

h tt p : / / w w w b j m i c r o b i o l c o m b r /

Cheng Jianga, Jinzhu Songa, Junzheng Zhangb, Qian Yanga, ∗

Q1

aHarbin Institute of Technology, School of Life Sciences and Technology, Harbin, Heilongjiang, People’s Republic of China

bHarbin Institute of Technology, School of Chemical Engineering and Technology, Harbin, Heilongjiang, People’s Republic of China

Article history:

Received8January2016

Accepted28November2016

Availableonlinexxx

AssociateEditor:MiguelJ

Beltran-Garcia

Keywords:

ChaetoglobosinA

Cornstalk

Purification

Stability

Chaetomium globosum

ChaetoglobosinAisanantibacterialcompoundproducedbyChaetomium globosum,with potentialapplicationasabiopesticideandcancertreatmentdrug.Theaimofthisstudywas

toevaluatethefeasibilityofutilizingcornstalkstoproducechaetoglobosinAbyC globosum

W7insolid-batchfermentationandtodetermineanoptimalmethodforpurificationofthe products.TheoutputofchaetoglobosinAfromthecornstalkswas0.34mg/g,anditscontent

inthecrudeextractwas4.80%.Purificationconditionswereoptimizedtoincreasethe con-tentofchaetoglobosinAinthecrudeextract,includingtheextractsolvent,temperature,and

pHvalue.Theoptimumprocessconditionswerefoundtobeacetoneastheextractant,under roomtemperature,andatapHvalueof13.Undertheseconditions,aproductionprocessof theantifungalchaetoglobosinAwasestablished,andthecontentreached19.17%.Through furtherverification,cornstalkscouldreplacecropsfortheproductionofchaetoglobosinA usingthisnewproductionprocess.Moreover,thepurifiedproductsshowedgreatinhibition againstRhizoctonia solani,withchaetoglobosinAconfirmedasthemaineffectiveconstituent (IC50=3.88␮g/mL).Collectively,theseresultsdemonstratethefeasibilityofusingcornstalks

tosynthesizechaetoglobosinAandthattheproductionprocessestablishedinthisstudy waseffective

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/)

Introduction

strongcytotoxicitytovariouskindsofcells,includinganimal,

∗ Corresponding author.

E-mails:microbio207@gmail.com,yangq@hit.edu.cn(Q.Yang)

such as Setosphaeria turcica, Rhizopus stolonifer, and Conio-thyrium diplodiella.7–9Despitethesebroadeffects,development

http://dx.doi.org/10.1016/j.bjm.2016.11.008

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/)

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Pleasecitethisarticleinpressas:JiangC,etal.NewproductionprocessoftheantifungalchaetoglobosinAusingcornstalks.Braz J Microbiol.

antagonistthatiswidelyusedforbiologicalcontrol,16andhas

isgenerallyleftinthefieldsafterharvestorisevenburnt18;

thus,thevastavailabilityofthisresourceshowsitspotential

Alternatively,useofacomplexculturesystemislikelyto

Finally,thecostofthecrudeextractusingcornstalksasa

effectiveantibacterialactivity

Microorganisms and culture conditions

C globosumW7wasobtainedfromtheMicrobialGenetic

perinch)usingaplantpulverizer(Beijinglightmedical equip-mentCo.Ltd.,Beijing,China).Thecornstalksusedinthisstudy

(nitrogencontent26.17%)witharatioof20/1(w/w),sothatthe

20/1

Extraction effects of different solvents

temper-ature(20–25◦C)for24hwith50mLoforganicsolventperflask,

dif-ferentsolventsweremethanolat50◦C,ethanolat60◦C,ethyl acetateat50◦C,acetoneat40◦C,dichloromethaneat40◦C,

Determination of optimal operating temperature

wereplacedatdifferenttemperatures(−20◦C,0◦C,room tem-perature, 40◦C,50◦C, 60◦C, 80◦C, 100◦C,150◦C) foreither

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performedintriplicate,and thenthe remainingamount of

The influence of pH value on the crude extract

triplicate

Preliminary purification of the crude extract

Basedontheresultsoftheextractioneffecttests,the

asthecontrol

Owingtoitsnegligibleeffectforextractingchaetoglobosin

Chaetoglobosin A detection

Gas chromatography–mass spectrometry (GC–MS) analysis of the crude extract

to280◦Catarateof3◦C/minandheldfor5min,withatotal

5min.Thecharacteristicionsandretentiontimesofthetarget

(http://www.organchem.csdb.cn/scdb/main/mssintroduce

Biocontrol efficiency of the second purified extract against Rhizoctonia solani

R solani(CGMCC3.2888)waschosentotestthebiocontrol

dis-solvedinethanolwasaddedpriortofiltrationforsterilization

0.5,2.5, 5.0,25,and 50␮g/mL).Aslice (0.8cm)ofR solani

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Pleasecitethisarticleinpressas:JiangC,etal.NewproductionprocessoftheantifungalchaetoglobosinAusingcornstalks.Braz J Microbiol.

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2

0

60

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50

40

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20

10

0

Extraction solvents

Acetone

Dichloromethane

Chlorof

orm N-he xane

Weight of crude extract Weight of chaetoglobosin A

Purities of chaetoglobosin A

Fig 1 – Effects of different solvents on the extraction of

chaetoglobosin A All extractions were performed on the

fermentation residue of 1 g of cornstalk medium.

inhibitoryconcentration(IC50)value(3.88␮g/mL)wasverified

proce-dures.Allofthetestswereperformedintriplicate

Extraction of chaetoglobosin A

A.Accordingtopreviousstudies,methanol,21ethylacetate,22

solutionisacidic(pH<4)usingextensivepHindicatorpaper

organicacids,includingaceticacid,butyricacid,succinicacid,

Optimal handling temperature

chaetoglobosinAwereplacedat−20◦C,0◦C,40◦C,50◦C,60◦C,

80◦C,100◦C,and150◦Cfor1hor24h,respectively.Theinitial

thesamplesweretreatedat80◦C,100◦C,and150◦Cfor1h,

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0.5

0.4

0.3

0.2

0.1

0.0

–20 0 RT 40 50

Temperature (ºC)

60 80 100 150

Heat for 1 h Heat for 24 h

** ** **

**

**

** **

Fig 2 – Thermostability of chaetoglobosin A placed at

various temperatures for 1 h or 24 h The initial amount of

chaetoglobosin A in every sample was 0.5 mg Asterisks

indicate a statistically significant difference(*p< 0.05) in the

amount of chaetoglobosin A remaining between the control

temperature (room temperature, 20–25 ◦ C) and the others.

Two asterisks indicate a highly significant difference

(**p< 0.01).

The effect of acid or alkali treatment on the crude extract

aftereithertreatmentfor1h,whichindicatedthatdirect

inwater

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0.4

0.2

0.0

0 1 2 3 4 5 7 9

pH value

10 11 12 13 14 Ctrl

Weights Purities

*

*

**

Fig 3 – pH stability of chaetoglobosin A The initial amount

of chaetoglobosin A in each sample was 0.5 mg Asterisks indicate a significant(*p< 0.05) or highly significant

(**p< 0.01) difference between the treatment groups and control.

combina-tionwithethylacetate.Owingtoitsweakstabilityinacidand lye,thetypicalprocessof␤-lactamantibioticsinadjustingpH

Purification of the crude extract

abilitytoeffectivelyremovetheseimpurities.Fig.4showsthe resultsofthesetests

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Pleasecitethisarticleinpressas:JiangC,etal.NewproductionprocessoftheantifungalchaetoglobosinAusingcornstalks.Braz J Microbiol.

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4

2

0

Weights of curde extract Weights of chaetoglobosin A

Purities of chaetoglobosin A

Handling compounds

Control

N-he xane

Sodium h ydro xide

Methanol w ater

Sodium h ydro xide

and methanol-w

ater

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20

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12

8

4

0

**

*

**

**

**

Fig 4 – Effects of different compounds on the purification of

chaetoglobosin A The initial amount of chaetoglobosin A

in every sample was 0.5 mg Asterisks indicate a significant

difference between the results of the treatment groups and

control(*p< 0.05;**p< 0.01).

Establishment of the complete production process

wholeprocessisshowninFig.5.Afteraseriesofpurification

ofagriculturalantibioticproducts

Cornstalks

Fermentation process Fermentation

products

Acetone extraction Crude extract

Deacidification

Degreasing

1st purified extract

2nd purified extract

Operating temperature

40 ºC

Fig 5 – Production process of chaetoglobosin A products.

Rectangles represent compounds, and ovals represent processes.

increased

C globosumwasculturedonPDAandsubjectedtothenewly

Biological activities of products against R solani

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Table 1 – Chaetoglobosin A production by different strains ofChaetomium.

Substrate Strain Extractionsolvent Yielda Contentb Study

Corn-sucrose C globosumDAOM240349 Ethylacetate 33.13mg/L Nodata 22

a YieldreferstotheyieldofchaetoglobosinAintheculturesystem

b ContentreferstothecontentofchaetoglobosinAinthecrudeextract

astheevaluationcriterionforaninhibitioneffect,becausethe

compo-nentinthecrudeextract,8insomecases,theuseofdifferent

againstR solaniisshowninFig.6

Inthistest,waterwassetastheblankcontrol,andethanol

astatisticallysignificant difference (p<0.05)in theaverage

wassetasthesolventcontrolinthecalculationofthemycelial

areaofthetreatmentset,anddiistheinitialcolonyareaofthe

aver-ageareasofR solanimyceliadecreasedsignificantly(p<0.05),

higherconcentrations,theinhibitioneffectoccurredearlier.In

theregressioncurveY=a×log10(X)+b,determinedbyprobit

differenttimepoints(Table2 Atthe48thhour,theIC50value

60

A

40

0

–40

–80

80

60

40

20

0

40

20

0

24 48 72

Culture time (h)

2)

2)

96 120

0 24 48 72

Culture time (h)

96 120

Control

0 µg/mL 0.5 µg/mL 2.5 µg/mL 5.0 µg/mL

25 µg/mL

50 µg/mL

Inhibition ratio of crude extract Inhibition ratio of standard Colony areas of ethanol control Colony areas of crude extract Colony areas of standard

Fig 6 – Inhibitory efficiency of the crude extract (20%

content) againstRhizoctonia solani.(A) Inhibitory efficiency

of chaetoglobosin A at different final concentrations under different culture times (B) Verification of the predicted IC 50

value of chaetoglobosin A using ethanol as the solvent, and comparison of the effect of the ethanol standard under the same concentration (3.88 ␮g/mL).

R solani decreased significantly, and the inhibition ratio

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Pleasecitethisarticleinpressas:JiangC,etal.NewproductionprocessoftheantifungalchaetoglobosinAusingcornstalks.Braz J Microbiol.

Table 2 – Inhibitory regression curves and predicted IC50 values of chaetoglobosin A againstR solaniat different culture

times.

Culturetime(h) Regressioncurvea R2 IC50(␮g/mL)

a XrepresentstheconcentrationofchaetoglobosinA,andYindicatestheinhibitionratioagainstR solani.

wasnotstatisticallysignificant(p>0.05),whichindicatedthat

5days)

Conclusion

Acknowledgements

Q2

financialsupportofthisstudyundertheNationalHigh

Tech-nologyResearchandDevelopmentProgram(2011AA10A205)

and“TwelfthFive-YearPlan”NationalScienceandTechnology

ProgramonRuralArea(2014BAL02B00)

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