Antimicrobial potential of pyroligneous extracts – a systematic review and technological prospecting

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brazilian journal of microbiology49S(2018)128–139

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Antimicrobial

potential

of

pyroligneous

extracts

–

a

systematic

review

and

technological

prospecting

Juliana

Leitzke

Santos

de

Souza

a

_,

_Victoria

_Burmann

_da

_Silva

_Guimarães

b

_,

Angela

Diniz

Campos

c

_,

_Rafael

_Guerra

_Lund

a,b,∗

a_Universidade_Federal_de_Pelotas,_Programa_de_{Pós-Graduac¸ão}_em_Bioquímica_e_{Bioprospecc¸ão,}_Pelotas,_RS,_Brazil

b_Universidade_Federal_de_Pelotas,_Faculdade_de_Odontologia_de_Pelotas,_Laboratório_de_{Microbiologia}_Oral,_Programa_de_{Pós-Graduac¸ão} emOdontologia,Pelotas,RS,Brazil

c_Empresa_Brasileira_de_Pesquisa_{Agropecuária,}_Embrapa_Clima_Temperado_(CPACT),_Laboratório_de_Fisiologia_Vegetal,_,_Monte_Bonito, RS,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28February2018 Accepted1July2018

Availableonline14August2018 AssociateEditor:AdalbertoPessoa

Keywords: Foodpreservation Antimicrobial Infections Preservative Pharmaceutical

a

b

s

t

r

a

c

t

Pyroligneousextractisappliedindiverseareasasanantioxidant,anantimicrobial,and ananti-inflammatoryagent.Thediscoveryofnewcost-effectiveantimicrobialagentsof naturaloriginremainsachallengeforthescientificcommunity.Thisstudyaimedto con-ductasystematicreviewandatechnologicalforecastingoftheexistentevidenceregarding theuseofpyroligneousextractasapotentialantimicrobialagent.Studieswereidentified throughaninvestigationofvariouselectronicdatabases:PubMed,SciFinder,WebofScience, Scopus,Scielo,Googlescholar,andProQuest.PatentsweresearchedthroughINPI,Google patents,Espacenet,Patentsonline,USPTO,andWIPO.Theliteratureonantimicrobial activ-ityofpyroligneousextractarelimitedgiventheshortdurationofstudiesandvariabilityin studydesign,useofpyroligneouspreparations,andreportsonresults.However,evidence suggeststhepotentialofpyroligneousextractasanaturalantimicrobialagent.Themost studiedactivitywastheroleofPEasafoodpreservative.However,pyroligneousextracts arealsoeffectiveagainstpathogenicbacteriaintheoralmicrofloraandtreatmentof candi-dalinfections.Furtherresearchisneededusingstandardizedpreparationsofpyroligneous extractstodeterminetheirlong-termeffectivenessandabilityasantimicrobialagents.

Introduction

Pyroligneousextract(PE),alsocalledpyroligneousacid,liquid smoke,orwoodvinegar,isacrudecondensateproducedfrom the distillationofsmokegeneratedin woodcarbonization.

∗ _{Corresponding}_author_at:_Postgraduate_Program_in_Dentistry,_School_of_Dentistry,_Federal_University_of_Pelotas_(UFPel)_–_Rua_Gonc¸alves Chaves,457/504,96015-000Pelotas,RS,Brazil.

E-mail:rafael.lund@gmail.com(R.G.Lund).

This extract is a complex mixture of compounds derived fromthechemicalbreakdownofwoodcomponentsthrough thecondensationofvaporsandgasesgeneratedduringthe pyrolysisofalimitedamountofoxygen.1_PE_is_a_complex_and

highly oxygenated aqueous liquid fraction; it results from

https://doi.org/10.1016/j.bjm.2018.07.001

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129

thethermochemicalbreakdownorpyrolysisofplantbiomass components,suchascellulose,hemicellulose,andlignin.2,3

PEfindsapplicationindiverseareas,actingasan antiox-idant, antimicrobial, and anti-inflammatory agent. PE also actsasasourceofvaluablechemicalsandimpartsasmoky flavor and antimicrobial protection in food.3 _PE _has _been

reportedtopossessextremeantifungalactivityagainst sev-eral plant pathogenic fungi4 _and _a _termiticidal _activity.5

Several researchershavereported the antibacterial activity of PE against several pathogenic bacteria, including plant pathogens.6_Recently,_the_medicinal_use_of_PE_has_been_studied

intensivelyintheﬁeldoforientalmedicalscience,wheresome naturalresourceshavebeenusedforinvestigatingbiological activities.7_However,_the_{antimicrobial}_potential_of_PE_against

humanandanimalpathogenicmicroorganismshasnotbeen elucidated.Therefore,thepresentworkiscarriedoutto eval-uate the antimicrobial potential of PE againsthuman and animalpathogenicmicroorganismsandorganizeitspresent status.PE,aby-productofcharcoal-makingandoften consid-eredaswaste,wasselectedbecauseofitsavailabilityinRio GrandedoSul,Brazil.

Systematicreviewsare consideredthegoldstandard for evidence and used to evaluate the beneﬁts and harms of healthcareinterventions.8_Such_reviews_are_much_more_likely

toyieldvalidconclusions.9_The_levels_of_evidence_of_the

stud-iesarerankedaccordingtothedegreeofconﬁdence,whichis relatedtothemethodologicalquality.Thus,systematicreview

oftheliteratureoccupiesthetopofthepyramid,followedby randomizedclinicaltrials,cohortstudies,case-control,case series,casereports,andlastly,expertopinionandresearchin animalsorinvitro.10_This_study_aimed_to_search_the_various

electronicdatabasesforarticlesandonlinesystemsofpatents andincludedassaysofPEantimicrobialpotentialforhumans andanimals.

Material

and

methods

Eligibility criteria: The inclusion criteria comprised articles andpatentsthatinvestigatedtheantimicrobialactivityofPE againstpathogenicmicroorganismsofhumansandanimals. Informationsourcesandsearch

Thissystematicreviewwasconductedaccordingtothe guide-lines ofthe CochraneHandbookforSystematic Reviewsof Interventions,11_following_the_four-phase_ﬂow_diagram_of_the

PreferredReportingItemsforSystematicReviewsand Meta-Analyses(PRISMA)Statement.12 _This_report_is_based_on_the

PRISMAStatement.Thefollowingdatabaseswerescreened: MedLine (PubMed),SciFinder, Web ofScience, Scopus, Sci-elo,Google scholar, and ProQuest.For patents, thesources searched comprised the following: INPI, Google patents, Espacenet,Patentsonline,USPTO,andWIPO.

Table1–Searchstrategy.

Searchstrategy

Pubmed

#1(“woodvinegar”OR“pyroligneousacid”OR“pyroligneousextract”OR“pyroligneous”OR“liquidsmoke”)

AND

#2(“Anti-InfectiveAgents”[MESH]“Antimicrobialactivity”OR“Antibacterialactivity”OR“Antifungalactivity”OR“Anti-InfectiveAgents”

OR“Agents,Anti-Infective”OR“AntiInfectiveAgents”OR“AntiinfectiveAgents”OR“Agents,Antiinfective”OR“Microbicides”OR

“AntimicrobialAgents”OR“Agents,Antimicrobial”OR“Anti-MicrobialAgents”OR“Agents,Anti-Microbial”OR“AntiMicrobialAgents”)

Webofscience

#1Topic:(“woodvinegar”)ORTopic:(“pyroligneousacid”)ORTopic:(“pyroligneousextract”)ORTopic:(“pyroligneous”)ORTopic:(“liquid

smoke”) AND

#2Topic:(“antimicrobialactivity”)ORTopic:(“Anti-InfectiveAgents”)

Scopus

#1(“woodvinegar”)OR(“pyroligneousacid”)OR(“pyroligneousextract”)OR(“pyroligneous”)

AND

#2(“antimicrobialactivity”)OR(“Anti-InfectiveAgents”)

SciFinder

#1(“antimicrobialactivityofWoodVinegar”)OR#2(“antimicrobialactivityofpyroligneousacid”)OR#3(“antimicrobialactivityof

pyroligneousextract”)

Scielo

#1(“woodvinegar”)OR(“pyroligneousacid”)OR(“pyroligneousextract”)OR(“pyroligneous”)OR(“liquidsmoke”)

AND

#2(“antimicrobialactivity”)OR(“Anti-InfectiveAgents”)

GoogleScholarandProQuest

#1(woodvinegar)OR(pyroligneousacid)OR(pyroligneousextract)OR(pyroligneous)OR(liquidsmoke)

AND

#2(antimicrobialactivity)

Patentsdatabases:INPI,Googlepatents,Espacenet,Patentsonline,USPTOandWIPO.

#1(woodvinegar)OR(pyroligneousacid)OR(pyroligneousextract)OR(pyroligneous)OR(liquidsmoke)

AND

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Records identified throught databases of articles and patents searching: PubMED (16), Scopus (8), Web of science (9), Scifinder (27),

Google scholar (34), Proquest (3).

INPI (0), Google patents (0), Espacenet (7), Patents on line (44), USPTO (11) and WIPO (7).

93 articles indentificated

93 articles screened after duplicates removed

29 studies assessed for elegiblility

29 studies included in qualitative syntesis

Indentification

Screening

Eligibility

Included

60 articles excluded after reading title and astract.

12 articles excluded after reading full text: 1 insecticide against insects 1 protozoan parasite

1 agent accelerating the growth of plants 4 agent for fungi of wood

3 weed control agent 2 font of phenol

2 fulltext articles excluded for no extractable data.

1 review study.

69 patents indentificated

37 patents screened after duplicates removed

37 patents assessed for elegiblility

25 patents excluded after reading title and astract.

25 patents excluded after reading full text: promoter for natural environment for soil, fishes, and plants.

Fig.1–Flowchart.

ThesearchstrategyisdescribedinTable1,andthefocused questionisasfollows:Whatistheantimicrobialpotentialof PEagainsthumanandanimalpathogenicmicroorganisms?

Studyselectionanddatacollection

Studycharacteristics,demographicinformation,enrollment criteria, microorganisms tested, antimicrobial assay types, duration, results, control and groups, and sample size (Tables2and3)wereextractedindependentlyfortwo review-ers (J.L.S.S. and V.B.S.G.). Missing information was sought from authors and/or inventors. The full text papers and patents were assessed independently and in duplicate by the reviewers. Any disagreement onthe eligibilityof stud-iesincludedwasresolvedthroughdiscussionandconsensus, andincaseofdisagreement,athirdreviewer(R.G.L.)decided whetherthearticleshouldbeincluded.Alltitlesandabstracts of articles and patents initially found were analyzed and selectedinaccordance tothe eligibilitycriteria.No restric-tions were considered regarding the language and year of publication. The reference lists of studies included were hand-searchedforadditionalarticles.Fullcopiesofall poten-tiallyrelevantstudies wereidentiﬁed. Studiesthat metthe inclusion criteriaor forwhich insufﬁcientdatawere avail-able in the title and abstract to make a decision were selectedforfullanalysis. Authorsofthe studies were con-tactedincaseofmissingdata(e.g.,dataprovidedingraphs); these studies were only included if the authors provided themissinginformation.Dataextractionwasconductedby consensusbetweenthetworesearcherswhoconductedthe collection.

Assessmentofriskofbias

Theriskofbiasforalltheincludedstudieswasassessedbased on The Cochrane Collaboration’s tool,11 _and _the

method-ologicalqualitywasadaptedfromanothersystematicreview ofantimicrobialmonomersusedindentalmaterials.13 _The

parametersused forthe evaluationofmethodology assays

werediscussedbytheresearchersinvolved,andjudgmentwas carriedoutbygroupdiscussion.Assessmentofriskofbiaswas conductedusingReviewManager5.3software.

Results

Resultsofsearches

Afterdatabasescreening[Pubmed(16),Scopus(8),SciFinder (27), WebofScience(9), Scielo(0), Googlescholar(34), and ProQuest(3)]andremovalofduplicates,89studieswere iden-tiﬁed.Aftertitleandabstractscreening,29studiesremained, andthisnumberwasreducedto14aftercarefulexamination ofthefulltexts.Thelastelectronicsearchwasconductedon December4th,2017.Fig.1showsaﬂowchartsummarizingthe selectionprocessofarticlesandpatents.

Ofthe89articlesinitiallyrecoveredfromalldatabases,75 articleswereexcludedbecausetheywerenotrelatedtothe antimicrobialactivityofPEandfailedtosatisfytheselection criteria.Atotalof60articleswereexcludedafterreadingthe titleandabstract,and12wereexcludedafterscreeningthefull textbecausetheytestedpropertiesotherthanantimicrobial pathologyforhumansoranimals.Onestudytestedan intesti-nalprotozoanparasitethatcausesdiarrheainbothhumans anddomesticanimals14_;_one_article_used_an_agent

accelerat-ingthegrowthofplantsandthedevelopmentofroots.15_PE

hasbeenstudiedasarepellentandinsecticideagainstinsect peststocrops,5_as_an_{antimicrobial}_agent_for_wood_fungi,_and

asaweedcontrolagent.4,6,16–19 _Two _studies_used_the_PE_as

phenolsourceandtested theantibacterialactivity of sepa-ratecomponents.20,21_In_addition,_two_articles_were_excluded

astheresearchersfeaturednoaccesstofulltextversions,22,23

andonearticlewasareviewstudy.24

In the patent databases, INPI (0), Google patents (0), Espacenet(7),patentsonline(44),USPTO(11),andWIPO(7), thesearchstrategyinitiallyretrieved69patents.Afterremoval ofduplicates,thisnumber wasreducedto37.Atotalof25 patentswereexcludedafterreadingthetitlesandabstracts (Fig.1)astheywerenotrelatedtoPE.Oftheremaining12

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patents,1patentwasexcludedbecauseitreportedapromoter ofenvironmentalpropertiesinsoil,plants,andﬁsh.25_Twelve

patentswereincludedintheanalysis. Studycharacteristics

Table2describesthe microorganismstested, the sourceof PE,concentrations,methodologiesusedandtheapplication areaforeachstudyfromarticles,andpatentsselectedinthe search.Table3showsthedataofthepatentsincludedinthis review.

Allarticleswere publishedbetween1998and2014.Most studiesshoweddifferentcellulosesourcestoburnand gen-erate different types ofPE for its test as an antimicrobial agent.Severalsourceswerecommercialextracts,1,7,26–33_and

others were experimentalextracts.19,34,35 One study tested microbialstrainsisolated fromdogsand cats.26 _One_article

analyzedfourstrains ofpathogenic Candidaalbicans,which wereisolatedfrompatientssufferingfromurinarytract infec-tion(twostrains),vaginitis,andonychomycosis(oneeach),7

andonetestedanotherC.albicansstrain.30_One_study_tested

PE as an agent to prevent viral epidemics in agricultural andhumanenvironments.27 _Various_studies_reported_PE_as

afoodpreservative.28,30,31,33,36,37_Only_one_in_vivo_study_used

Salmonella-infectedBalb/cmousemodel.35_All_in_vitro_studies

reportedthebacterialorantifungalactivityofPE.The meth-odsusedforthetestsincludeddiskdiffusionandminimum inhibitoryconcentration.Thegrowthproﬁleofthebacteria wasexaminedviatime-killandviralinactivationassays.27

Regardingpatentdocuments,thedatashowed11patents deposited from1981 to2009.Antibacterial, antifungal, and preservativeproprietiesofPEwere foundinthese patents, which claimed PE incorporation into additives for the treatmentof animal feedstuffs,38 _{ﬁber-reinforced}_cellulosic

food casing,39 _compositions _for_food _{preservation,}40–42

car-bonﬁber,43 _cosmetic_composition,44_{biodeodorizing} _agent,45

antimicrobial compositions,46 _{pharmaceutical} _composition

forsymptomsofatopicdermatitis,47_and_compositions_for_oral

microbes.48

Discussion

Fromtheliteraturereviewed,mostresearchershavereported promising results for PE as an antimicrobial agent. The most studied activity for PE was for food preserva-tive, with seven articles28–33 _and _ﬁve _patents _presenting

related results.38,39,41,42,48 _The _PE _was _effective _for _the

following microorganisms important for the food indus-try: Salmonella enteritidis, Salmonella typhimurium, Salmonella muenster,Salmonellaseftenburg,Escherichia coli,Staphylococcus aureus,Pseudomonasputida,Pseudomonasaeruginosa, Lactobacil-lusplantarum,Listeriainnocua,Listeriamonocytogenes,Aeromonas hydrophila,Yersiniaenterocolitica,Saccharomycescerevisiae,and

Aspergillusniger.Table2showsallthestrainstestedineach study.PEwasusedasantimicrobialinfoodpreservationand hasdemonstratedabilitiestoreduceorinhibitpathogenicand spoilageorganisms.Comparingresultsofstudiespresented difﬁcultygiventheirvariousdifferences.Thesestudiesused differentmethodologiesfortheantimicrobialactivityandfor

preparationofPE.Somestudiesprovidednoinformationon the preparations.Mostcommercial extractsweretestedfor useasfoodpreservative;onewasforviralepidemics,twofor antibacterialactivity,andanotherforantifungalinfections.

Su ˜nenetal.(1998)testedsevencommercialpreparations of PE used in food industry in Spain against L. monocyto-genes and other pathogenic microorganisms.30 _In_2001, _the

sameauthortestedfourothercommercialpreparationsofPE usedintheSpanishfoodindustryandevaluatedtheir antimi-crobialproperties atlowtemperature againstA.hydrophila, Y.enterocolitica,andL.monocytogenes.All fourextracts effec-tively eliminatedorsuppressedthe growthofA. hydrophila

after21days.31_Another_study_on_food_preservative_examined

theeffectsofselectedPEsonthecontrolofL.monocytogenes

infrankfurters.Treatments withPEreduced andcontrolled

L. monocytogenes growth in the most permissive franks for 10 weeks.32 _Milly_et _al. ₍₂₀₀₈₎_used_PE _fractions_applied_on

ready-to-eatmeatproductstocontrolthegrowthofinoculated

L.innocuaM1.28

In2012,VanLooetal.investigatedtheantibacterialactivity ofeightcommercialPEsamplesagainstS.enteritidis,S.aureus,

andE.coli,demonstratingthatthecommercialsmokes inhib-itedthegrowthofthesefoodbornepathogens.36

Haradaetal.(2013)determinedthemaximuminhibitory dilutions of bamboo PE against104 E. coli, 112 Staphylococ-cuspseudintermedius,and58P.aeruginosastrainsisolatedfrom dogsandcats.Theresultsindicatedthatbamboopyroligneous acidexertssigniﬁcantlyinhibitthegrowthofrepresentative bacterialpathogensfromcompanionanimals,although inhi-bitiondifferedamongspecies.26

Bamboo PE-inactivated picornavirus and encephalomy-ocarditis virus showed that phenol is the sole germicidal component,andthataceticacidaugmentedthephenol inac-tivatingactivity.TheseﬁndingssuggestthatbambooPEisa potentiallyusefulagenttopreventviralepidemicsin agricul-turalandhumanenvironments.27

Ibrahim et al. (2013) tested the PE, concentrated PE, and dichloromethane extracts of CPA, namely, DCM A and B, against four pathogenic strains of C. albicans. The resultsexhibitedsigniﬁcantinhibitionzones.Theresultsalso revealedthatextractDCMBofCPAshowedthemost signiﬁ-cantpotentialasananti-candidalagent.7_In_2014,_the_author

concludedthat RhizophoraapiculataPEmayalsobeabroad antimicrobialagentagainstpathogenicbacteria.49

Otherauthorsinvestigatedtheeffectsoftemperatureon antimicrobialpropertiesoftwocommercialPEfractionsand PEderivedfrompecanshells,againsttwocommonfoodborne pathogens,ListeriaandSalmonella.Understandinghow stor-age temperatureaffectsthe efﬁcacy ofantimicrobialsisan importantfactorthatcancontributetoreducinghighlevels andcostsofantimicrobialsandultimatelyimprovefoodsafety forconsumers.33

Threepapersreportedthedevelopmentofnew experimen-talPE.Theytesteddifferentkindsofcelluloseasfeedstock: walnut treebranches,Eucommiaulmoides,olivebranch, and rice hull. Wei et al. (2010) prepared and collected PE by pyrolizingwalnuttreebranchesatthreetemperatureranges: 90◦C–230◦C, 230◦C–370◦C, and 370◦C–450◦C. All the PEs exhibited antibacterialactivity.Thehighlevel of antibacte-rialactivityofWP3indicatedthatpyroligneousacidscollected

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 S (2 0 1 8) 128–139

Table2–Demographicdatafromarticlesandpatentsconsideredinthisstudy. Microorganismtested Pyroligneousextract

source

Concentrations Methods Application

area Assays Samplesize

(pergroup)/ repetitionof assays

Period Positivecontrol Negativecontrol

30 _Bacillus_cereus_(CECT_495),_Bacillus

subtilis(CECT38),S.aureus(CECT 239and976),L.monocytogenes

(CECT932),L.inocua(CECT4030),

Brochothrixthermosphacta(CECT 847),L.plantarum(CECT220),L. brevis(CECT216),L.coryniformes

(CECT982),L.lactisssp.cremoris

(CECT697),L.lactisssp.lactis(CECT 185),Leuconostoccarnosum(CECT 4024),Carnobacteriumdivergens

(CECT4016),E.coli(CECT533,471, 405),S.tyhimurium(CECT443),S. enteritidis(CECT556),Y. enterocolitica(CECT559),P. aeruginosa(CECT378),Vibrio vulniﬁcus(CECT529)and

Rhodotorularubra(CECT1159). StrainsofC.albicans,S.cerevisiae

andP.aeruginosa022werefrom theirowncollection(Spanish NationalCollectionofType CulturesValencia,Spain).

Sevencommercial smoke condensates.1 0.05,0.1,0.2and 0.4%forL1,L4,S2 andS3;0.2,0.3,0.4, 0.6and0.8%forL2; 0.5,1,2,4,8%forL3 and0.5,1,1.5%for S2. Agardilution methods

n=2/Twice. 24and48h TSAandMRSagar withoutsmoke inoculatedwiththe workingcultures.

No Asfood

preservative.

31 _L._{monocytogenes}_(932),_A._hydrophila (839)andY.enterocolitica(559) (SpanishNationalCollectionof TypeCultures,Valencia,Spain).

Fourcommercial smoke

condensates.1

1%forthedried extract,0.4%forL1, 0.6%forL2and4% forL3. Brothand agardilution methods n=1/three times 0,1,2,7,14 and21days InoculatedTSB withoutsmoke extractsservedas positivecontrols. Non-inoculated ﬂaskswereusedfor sterilitycontrol.

Asfood preservative.

29 _S._muenster,_S._seftenburg,_S.

typhimurium,E.coli8677,andP. putida;L.plantarumandL.innocua

M1;S.cerevisiaeandA.niger.

Ninecommercial liquidsmokes.1 Fractions(v/v)were 0.5%,0.75%,1.0%, 1.5%,and2.0%to 10.0%. Brothoragar dilution methods n=3/three times

24h Petridisheswithno smokeextractsthat wereinoculated.

No. Asfood

preservative.

32 _L._{monocytogenes}_Scott_A-2 (serotype4b,clinicalisolate),V7-2 (serotype1/2a,milkisolate),39-2 (retailfrankfurterisolate),and 383-2(groundbeefisolate).

Twocommercial liquidsmokes.1

Dippedfor5,15,30, 60,and90swith liquidsmokeextract.

Directspiral plating methods

n=3 10weeks Frankfurtersnot dipped.

No. Asfood

preservative.

28 _L._innocua_M1,_a_strain_of_Listeria resistanttotheantibiotics streptomycinandrifampicin.

Fourcommercial liquidsmokes.1

2% Directspiral

plating methods

n=15 2and4weeks Threesamplesof eachmeatproduct.

No. Asfood

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133

–Table2(Continued)

Microorganismtested Pyroligneousextract source

36 _S._Enteritidis_(PTA_13A),_E._coli 0157:H7(ATCC43888),S.aureus

(ATCC25923andATCC6538),and twomethicillin-resistantS.aureus

(MRSA).L.monocytogenes174 (serotypel/2a),L.monocytogenes

163(serotype4b),S.typhimurium 29,S.typhimuriumLT2(ATCC 19585),andS.aureusCol(MRSA).

Eightcommercial liquidsmoke extracts. 96%–0.375%. Broth microdilution method n=3 24h Controlcontaining PBSsolution. No. Asfood preservative. 27 _{Picornavirus,}_{encephalomyocarditis} virus. Twocommercial liquidsmokes.

Notinformed. Viral

inactivation assay

Notinformed. 6h Notinformed. Notinformed. Asanagent

for preventing viral epidemicsin agricultural andhuman environments. 26 ₁₀₄_E._coli,₁₁₂_S._{pseudintermedius}

and58P.aeruginosastrainsisolated fromdogsandcats.

Onecommercial liquidsmokes. Serialdilutionsof BPA(i.e.1/2,1/3,1/4, 1/5,1/6,1/7,1/8,1/9, 1/10,1/11,1/12,1/13, 1/14,1/15,1/16,1/17, 1/18,1/19,and1/20). Maximum inhibitory dilution/agar method n=1 18h E.coli,E.faecalis,P. aeruginosa,S.aureus andS. pseudintermedius

wereusedasquality controls.

No. For

antibacterial infectionsin animals.

7 _Four_strains_of_pathogenic_C.

albicanswhichwereisolatedfrom patientswithsufferingfrom urinarytractinfection(two strains),vaginitisand onychomycosis(oneeach).

Fourextractofliquid smokes. Between0.39and 100.00mg/mL. Diskdiffusion method; brothdilution method; time-kill assay

n=3 24and48h Notinformed. No. Asan

antifungal agent especiallyto treatcandidal infections. 1 _B._cereus,_B._subtilis,_B._spizizenni,_S.

aureus,MRSA,S.epidermidis, Streptococcuspyogenes,S.faecalis, Citrobacterfreundii,E.coli,Erwinia sp.,K.pneumonia,P.mirabilis,P. aeruginosa,Salmonellatyphiand Yersiniasp.(IndustrialBiotechnology ResearchLaboratoryCulture Collection.

Fourextractofliquid smokes. Between0.39and 100.00mg/mL. Diskdiffusion method; brothdilution method; time-kill assay n=3 24h Chloramphenicol (Sigma,Germany)at theconcentrationof 30␮g/mLwasused asapositivecontrol. Commercial antibioticdiskGFA (Whatman,England) with6.0mm. Asan antimicrobial agentagainst pathogenic bacteria.

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 S (2 0 1 8) 128–139 –Table2(Continued)

Microorganismtested Pyroligneousextract source

33 _L._{monocytogenes}₂₀₄₅_(Scott_A, serotype4b,fromDr.Martin Weidemann,DepartmentofFood Science,CornellUniversity,Ithaca, NY),L.monocytogenes10403S (serotypel/2a,fromDr.Aubrey Mendonca,DepartmentofFood ScienceandHumanNutrition, IowaStateUniversity,Ames),L. innocuaATCC33090,L.innocuaMl ATCC33091,S.typhimuriumLT2 ATCC19585,andS.heidelbergATCC 8326(AmericanTypeCulture Collection,Manassas,VA).

Threecommercial liquidsmokesfrom Mesquite,Hickory andpecanshell.

Eightserialdilutions rangedto48and 0.375%(v/v). Brothmicro dilution/agar method n=3 24h Controlscontaining onlyPBSplus bacteriawere included. No Asfood preservative.

19 _S._aureus,_E._coli,_Bacterium_proteus,

Bacteriumprodigious,andAerobacter aerogenes. Walnuttree branches. Concentrationsof 40,20,10,5,2.5,1.25 and0.625mg/mL. Diskdiffusion and(EC50).

Notinformed. Notinformed. Notinformed. Notinformed. Asnatural

germicide. 34 _S._aureus,_E._coli,_B._prodigious,_B.

subtilis,A.aerogenes,Pseudomonas sp.andothersnon-human pathogenic. Eucommiaulmoides Oliv.Branch. Concentrationsof 50,25,12.5,6.25, 3.125,1.5625, 0.781mg/mL. Diskdiffusion method. n=1/three times

48h Sterilewater. No. Asgermicide.

35 _Salmonella_enterica_serovar typhimurium(ATCC#14028)(S. typhimurium)AmericanType TissueCultureCollection(ATCC, Manassas,Va.,U.S.A.).

Rice(Oryzasativa

L.)hull. Concentrationsof 0.1%,0.5%,and1.0% (v/v). Diskdiffusion method.

n=3 24h Notinformed. Notinformed. As

antimicrobial ﬂavor formulations for applicationto humanfoods andanimal feeds. Forinvitroassayand

(1.0%,v/w)forinvivo assay. Salmonella-infected Balb/cmouse model. Threegroups of10mice each. 12/12hfor 48h

PBS-treatedcontrol. Vancomycin (20mg/mL).

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135

–Table2(Continued) Patentsdata 38 _Bacillus_{stearothermophilus}_ATCC_7953,_S._typhimurium

ATCC14028,C.albicansATCC10231,Aspergillusﬂavus

ATCC9643,AspergillusnigerATCC9642,Chaetomium globosumATCC6205,PenicilliumfuniculosumATCC 11797,ChaetomiumglobosumATCC6205,Gibberella zeaeATCC24688,TrichodermavirideQM9123,Bacillus cereus,Enterobacteraerogenes,Serratiamarcescens, Pseudomanassp.,Proteussp.,andEnterobactersp.

Cellulosicﬁbermaterials,mainlyhard woodﬁbers,suchashickory,mapleand otherhardwoods.

Notinformed. Diskdiffusionmethod Asantimicrobial

formulationsfor applicationtoanimal feedstuffs.

39 _P.sub.2B_Penicillium_P.sub.2C_No_easily_recognizable conidialstate;maybelonginMyceliaSteriles P.sub.2DPenicilliumP.sub.2EPenicilliumP.sub.4

TrichodermsP.sub.5PaecilomycesP.sub.9Paecilomyces

P.sub.11APenicilliumP.sub.11BPenicilliumP.sub.12A

AspergillusP.sub.12BPenicilliumS.sub.1Fusarium

S.sub.2PenicilliumS.sub.3MonocilliumS.sub.4

PenicilliumS.sub.5PenicilliumS.sub.6Penicillium

R.sub.1PenicilliumR.sub.2PenicilliumR.sub.3

PenicilliumV.sub.1PenicilliumV.sub.2Penicillium Penicillium(eitherP.sub.2DorP.sub.12B)Aspergillus glaucus(source:T.LaBuzaU.ofMinnesota)A.niger

ATCC1004.

RoyalSmokeAA.sup.(a);RoyalSmoke A.sup.(a);RoyalSmokeB.sup.(a);Royal Smoke16.sup.(a);CharsolC-12.sup.(b); CharsolC-10.sup.(b);CharsolX-11.sup.(b); CharsolC-6.sup.(b);CharsolC-3.sup.(b); SmokaromaCode–12.sup.(c);Code– 10.sup.(c);Code–S.sup.(c);Code– 6.sup.(c).

(a)GrifﬁthLaboratories,Inc.12200South CentralAvenue,Alsip,I.(b)RedArrow ProductsCo.,P.O.Box507,Manitowoc,WI. (c)MeatIndustrySuppliers,Inc.770 FrontageRoad,Northﬁeld,IL.

89wt.% Numberofviablemolds.

Antimycoticaction.

Asafoodpreservative.

41 _L._{monocytogenes} _ZESTI_SMOKE_(Code₁₀₎_Hickory

Specialties,Inc.ofBrentwood,Tenn.

Aceticacidinaconcentrationofabout 6.5–8.0%;carbonyl1.0–8.0%;0.1–1.0%;and water83–92.4%(w/v).

Wienerssprayed. Asfoodpreservative.

43 _B._subtilis,_S._aureus,_S._epidermidis,_E._coli,

MidoriMinorikin,Serratia,Salmonella.

Woodvinegarandbamboovinegar,

T=500–900◦C

Notinformed. Diskdiffusionmethod. Hospitaltextileindustry.

44 _E._coli. _Wood_vinegar_is_Quercus_(by_Caicos)

T=80–150◦C

Between0.5%and5.0%. Detectionofbacteriain thewoodvinegar.

Cosmeticindustry.

45 _E._coli,_Salmonella_sp.,_S._aureus,_Vibrio_sp. _Not_informed. _Between_3.0%_and_5.0%. _Tested_as_to_whether_the

growthinhibitionon.

Newnaturalbio deodorantcomposition. 42 _E._coli_8677,_S._seftenberg,_L._innocua_M1,_L.

monocytogenes,S.cerevisiae,A.nigerspores.

ZESTI-SMOKECode1OandZESTI-SMOKE CodeV.MastertasteofCrossville, Tennessee.

Between0.5%and5.0%. Minimuminhibitory concentrations.

Asafoodpreservative.

46 _E._coli,_Salmonella_sp.,_Bacillus_sp.,_{Staphylococcus}_sp.,

Vibriosp.,Aspergillussp.,Fusariumsp.,Tricodermasp.

andCandidasp.(CandidakruseiATCC6258,Candida parapsilosisATCC22019,CandidaglabrataATCC90.03,

C.albicansATCC64550andATCC90028).

Notinformed. Between100␮Lto1600␮L. Minimuminhibitory

concentrations.

Asantisepticisaddedto thefood.

47 _Trichophyton_rubrum. _Puriﬁed_wood_vinegar_is_acetic_acid_2–4%

byweight,formicacid0.050.15wt.%, Propionicacid0.050.15wt.%.

1–5%ofweightofthetotalweightofthe medicament.

Halotest. Amedicamentofatopic dermatitiscontaining reﬁnedwoodvinegar. 48 _{Streptococcus}_mutans,_Porphrymonas_gingivalis,

fusobacteriumnucleatinssp.polymorphum.

ZESTI-SMOKECode1OandZESTI-SMOKE CodeV.MastertasteofCrossville, Tennessee.

Between0.01%and50.0%. Minimuminhibitory concentrations.

Asoralantimicrobial.

40 _L._{monocytogenes.} _ZESTI-SMOKE_Code_1O_and_ZESTI-SMOKE

CodeV.MastertasteofCrossville, Tennessee.

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136

Table3–Patentsdata,antibacterialcompositionsandclaimsrelatedtopyroligneousextractsantimicrobialactivity.

Patent Country Title Year Antibacterialcomposition Claimed

US430829338 _United_States _{Antimicrobial}_treatment_and_preservation_of animalfeedstuffs

1981 Pyroligneousacidandpyroligneousacid complexes.

Preservativeagentsforthetreatmentof animalfeedstuffs.

US437718739 _United_States _Liquid_smoke_impregnated_ﬁbrous_food casing

1983 Liquidsmoke. Aﬁbrousreinforcedcellulosicfoodcasing withtheimpregnatedliquidsmoke providingantimycoticquality. US504317441 _United_States _Meat_processing_with_Listeria_monocytogene

re-inoculationcontrolstage

1991 Liquidsmokederivativeproduct containingaminimumofcarbonyland phenol.

Compositionsforantimicrobialtreatment offoodproducts.

JP2000160476(A)43 _Japan _Production_of_carbon_ﬁber_and_carbon_ﬁber producedthereby

2000 Extractofmugwort(Artemisiaprinceps)

andoneofpyroligneousacidfromwood orbamboo.

Carbonﬁberthathasantimicrobial activity.

KR20030005075(A)44 _Korean _Cosmetic_composition_containing pyroligneousacidsolution

2003 Thecosmeticcompositioncontains0.5to 5.0%byweightofapyroligneousacid solution,basedonthetotalweightofthe composition.

Acosmeticcompositioncontaininga pyroligneousacidsolutionwith antimicrobialactivityandantioxidant activityforprotectingtheskin. KR20030014052(A)45 _Korean _Natural_{biodeodorizing}_agent_composition ₂₀₀₃ _The_Bacillus_strain_has_a_ﬁnal

concentrationof0.5x10not7to1x10not 7,basedon3to5%pyroligneoussolution.

Abiodeodorizingagentcompositionwith anexcellentantimicrobialactivityagainst putrefactiveorpathogenicbacteriaanda long-lastingdeodorizingeffect.

US20050175746A142 _United_States _Low_ﬂavor_{anti-microbials}_derived_from smokeﬂavors

2005 Derivativesofliquidsmoke. Compositionsforantimicrobialtreatment offoodproducts.

KR20060109757(A)50 _Korean _{Silver-ionized}_wood_vinegar_having_enhanced antimicrobialactivityandusethereoffor improvingorpreventingdiseasecausedby pathogenicbacteria

2006 Thesilver-ionizedwoodvinegaris preparedbyionizingsilverinwood vinegarwithelectrolysis.

Compositionsforantimicrobialactivity.

KR102007004286847 _Korean _{Pharmaceutical}_composition_for_ameliorating symptomsofatopicdermatitiswithoutskin irritationComprisingreﬁnednontoxicwood vinegarhavingnoharmfulmaterials

2007 Reﬁnedwoodvinegar24wt.%ofacetic acid,0.05–0.15wt.%offormicacid, 0.50–0.15wt.%ofpropionicacid.

Pharmaceuticalcompositionfor amelioratingsymptomsofatopic dermatitisandimprovingantimicrobial activity.

US20070212310A148 _United_States _{Antimicrobial}_smoke_ﬂavor_for_oral microﬂoracontrol

2007 Compositionsthatincludelowﬂavor antimicrobialliquidsmokederivatives.

Compositionsandmethodsforinhibiting thegrowthoforalmicrobesinasubject. US2009001109640 _United_States _{Preservatives}_for_food ₂₀₀₉ _Combination_of_N_long_chain_alkyl_of_di

basicaminoacidalkylesteracidsalt biocideswithliquidsmokecompositions.

Compositionsforantimicrobialtreatment offoodproducts.

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athighertemperaturefeaturestrongerinhibitioneffectson bacteria.19

PEofE.ulmoidesolivebranchwascollectedatdifferent tem-peratureranges:90◦C–200◦C,200◦C–340◦C,and340◦C–520◦C. TheresultsshowedthatthemaximumamountofthePEwas collectedattherange200◦C–340◦Candalsoshowedthemost anti-pathogenicactivities.Afterthepreliminaryanalysis, phe-nolswereconsideredtheactivecomponentsofbacteriostatic activity.34

A previously characterized rice hull PE was tested for bactericidal activity against S. typhimurium using the disk-diffusionmethod.Theinvivoantibacterialactivityofricehull smokeextract(1.0%,v/v)wasalsoexaminedina

Salmonella-infectedBalb/cmousemodel. Thecombinationofricehull smokeextractandvancomycinactedsynergisticallyagainst the pathogen. The beneﬁcial results suggest that the rice hullPEpossessesthepotentialtocomplementwood-derived smokesasantimicrobialﬂavorformulationforapplicationin humanfoodsandanimalfeeds.35

TechnologyprospectingforPEasanantimicrobialagent: thepatentsearch

StudiesonPEinpatentswereolderthanarticles.In1981,PEs incorporatingselectiveadditiveswereusedasantifungaland antibacterialpreservativeagentsforthetreatmentofanimal feedstuffs.38_In_1983,_PE_was_used_in_a_ﬁbrous_reinforced

cel-lulosicfoodcasingwithPEtoprovideantimycoticqualityin thecasingwithoutseparatingantimycoticagent.39_In_1991,_a

PEderivativeproductwasappliedtowienerspost-peelingand beforepackagingtoinhibitL.monocytogenesreinoculationand extendtheshelflifeofthewienerswithoutadverselyaffecting theirtasteand/oredibility.41_In₂₀₀₄_and_2008,_other_patents

developedmethodsandcompositionsforantimicrobial treat-mentoffoodproducts.40,42

TheantimicrobialpropertyofPEwasusedforthe devel-opmentof carbon ﬁber bysoaking carbon ﬁbers in aheat treatment solution mainly comprising PE from wood or bamboo.43_PE_was_also_used_as_{biodeodorizing}_agent

compo-sitioncontainingaculturesolution;thePEsolutionshowed an excellent antimicrobial activity against putrefactive or pathogenicbacteriaandalong-lastingdeodorizingeffect.45

Two studies developed antimicrobial products for skin; acosmetic composition containinga PE solution featuring anantimicrobialactivityandantioxidantactivityasamain component was obtained and considered suitable for skin protection.44 _A _{pharmaceutical} _composition _for

ameliorat-ing symptoms of atopic dermatitis comprising reﬁned PE wasproventoavoidsideeffects,suchasskinirritationand badsmell, byremovingharmfulmaterialsandtoxicityand improvingantimicrobialactivity.47

One patent developed a silver-ionized PE to enhance antimicrobial activity of PE against pathogenic bacteria:

E.coli,Salmonellasp.,Bacillussp.,Staphylococcussp.,Vibriosp.,

Aspergillussp.,Fusariumsp.,Tricodermasp.,andCandidasp.46 AnotherpatentusedPEagainstpathogenicmicroorganismsof oralcavityandprovidedcompositionsandmethodstoinhibit thegrowthoforalmicrobesandpromoteoralcare.48

The examination of PE as an antimicrobial agent, the broad spectrum of its properties, with and without enhancing additives,was evaluated against heat-resistant, spore-forming, aerobicbacilli, gram-negative bacillus asso-ciatedwithavian,andhumanenteritis.Varioussaprophytic molds (mycelial fungi) are associated with animal feeds, spoilage and, in several instances, human and animal mycotoxicoses. In each instance, our ﬁndings indicate that PE effectively and irreversibly reduces natural and/or experimentalmicrobial contaminantsassociatedto animal feedstuffs.38

CellulosicsourcesofPE

The cellulose sources reported in the studies included in thissystematicreviewcomprisedwoodsofhickory,mesquite, apple, pecan, moso bamboo (Phyllostachys pubescens), Rhi-zophoraapiculata,walnuttreebranches,andE.ulmoidesolive. Branchandricehullandﬁvestudiespresentednoinformation aboutthesecellulosesources.

FutureprospectsforPE

Furtherinvivostudiesarerequiredforthedevelopmentofnew productsusingPEandtheinvestigationofitspossibleuseas anantimicrobialagentagainstresistantpathogenic microor-ganisms and development of pharmaceutical medicines. Manypathogenicmicroorganismsaretestedforuseasfood preservative;thisextractdemonstratedaremarkable antimi-crobialpotentialbutwasnotidentiﬁedininvivostudiesfor humansorclinicalassays.

Conclusion

In conclusion, the evidence suggests that PE features an antimicrobialactivityagainstpathogenicmicroorganismsfor humans andanimals.Itsuseisprolongedandsafeinfood products.However,onlyonestudywasconductedonanimals, andnoclinicalcasewasfound.

Funding

sources

TheauthorswouldliketothanktheBrazilianresearchsupport agenciesCAPESandFAPERGSfortheﬁnancialsupport(PqG– Grant#17/2551-0001067-1).

Conﬂicts

of

interest

Theauthorsdeclarethattheyhavenoconﬂictofinterest.

Acknowledgments

The authors wish to thank the Coordination for the Improvement of Higher Education Personal (CAPES, Brazil) for the granting of Doctorate’s scholarship for the ﬁrst author.

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