Antifungal Microbial Agents for Food Biopreservation—A Review

35 Pages·2017·0.78 MB·English

microorganisms
Review
Antifungal Microbial Agents for Food
Biopreservation—A Review
MarciaLeyvaSalas1,2,JérômeMounier1,FlorenceValence2 ,MonikaCoton1,AnneThierry2
andEmmanuelCoton1,*
1 LaboratoireUniversitairedeBiodiversitéetEcologieMicrobienne(LUBEMEA3882),UniversitédeBrest,
TechnopoleBrest-Iroise,29280Plouzané,France;[email protected](M.L.S.);
[email protected](J.M.);[email protected](M.C.)
2 UMR1253ScienceetTechnologieduLaitetdel’Œuf,INRA,AgrocampusOuest,65ruedeSaintBrieuc,
35000Rennes,France;fl[email protected](F.V.);[email protected](A.T.)
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditors:RégineTalonandMoniqueZagorec
Received:9May2017;Accepted:24June2017;Published:8July2017
Abstract: Food spoilage is a major issue for the food industry, leading to food waste, substantial
economic losses for manufacturers and consumers, and a negative impact on brand names.
Among causes, fungal contamination can be encountered at various stages of the food chain
(e.g., post-harvest, during processing or storage). Fungal development leads to food sensory
defects varying from visual deterioration to noticeable odor, flavor, or texture changes but can
also have negative health impacts via mycotoxin production by some molds. In order to avoid
microbialspoilageandthusextendproductshelflife,differenttreatments—includingfungicides
andchemicalpreservatives—areused. Inparallel,publicauthoritiesencouragethefoodindustry
tolimittheuseofthesechemicalcompoundsanddevelopnaturalmethodsforfoodpreservation.
Thisisaccompaniedbyastrongsocietaldemandfor‘cleanlabel’foodproducts,asconsumersare
lookingformorenatural,lessseverelyprocessedandsaferproducts.Inthiscontext,microbialagents
correspondingtobioprotectivecultures,fermentates,culture-freesupernatantorpurifiedmolecules,
exhibitingantifungalactivitiesrepresentagrowinginterestasanalternativetochemicalpreservation.
This review presents the main fungal spoilers encountered in food products, the antifungal
microorganisms tested for food bioprotection, and their mechanisms of action. A focus is made
inparticularontherecentinsitustudiesandtheconstraintsassociatedwiththeuseofantifungal
microbialagentsforfoodbiopreservation.
Keywords: antifungal; bioprotection; biocontrol; food; post-harvest; fungi; lactic acid bacteria;
propionibacteria;Bacillus;fermentates;molecules
1. Introduction
Today, food losses are a major concern worldwide especially with an ever-growing world
populationandthefactthatapproximatelyone-thirdofallfoodproducedforhumanconsumptionis
eitherlostorwasted[1]. Eachyear,anestimated1.3billiontonsoffoodarelostorwastedasstatedby
theFoodandAgricultureOrganizationoftheUnitedNations[2].Whenconsideringeachfoodcategory,
theselossescorrespondto40–50%ofrootcrops,fruits,andvegetables;35%offishandseafood;30%of
cereals; and 20% of meat, oil seed, and dairy products [2]. In developing countries, post-harvest
lossratesarehighwith30–40%occurringduringthepost-harvestandprocessingstage[3],whilein
industrialized countries similar loss percentages (30%) occur at the retail or consumer levels [1].
The reasons for this massive global food loss are diverse, but microbial spoilage, which affects
organolepticproductquality(aspect,texture,taste,andaroma),playsamajorrole. Amongspoilage
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microorganisms,fungiareamajorissueatanystageofthefoodchainbecauseoftheirabilitytogrow
indifferentandevenharshenvironments[4]. Beyondtheirnegativeimpactonfoodquality,some
fungal generasuch as Aspergillus, Penicillium, Alternaria, and Fusarium have theability toproduce
secondarymetabolitesthatcanhaveatoxiceffectonhumansandanimalsandarethereforenamed
mycotoxins. Moreover, mycotoxins are able to withstand various food processing steps and can
thus lead to food safety concerns [5]. Fungi, mainly under an airborne spore form (either sexual
or asexual), can settle and grow at different stages of a product life, i.e., at the field, post-harvest,
duringprocessing,storageandhandlingstepsbytheproducer,wholesaler,retailer,andconsumer.
Theimpactofcontaminationateachlevelcanobviouslyleadtoeconomiclossesatboththeproducer
and consumer levels [1]. Moreover, producer losses can be reinforced by a negative brand image
inducedbyconsumerdissatisfaction.
In this context, it is crucial to reduce food losses by controlling fungal contamination at all
stages of food process chains. Three main stages can be defined to group fungal contamination
factors: (i)thefield,wherewater,soil,andairarenaturalfungalniches;(ii)rawmaterials—suchas
post-harvestcrops,meats,andmilk—wherefungaloccurrenceisrelatedtofoodmanagementduring
harvestorcollecting,transportation,storage,andpackaging[4]and(iii)duringfoodprocessingwhile
manufacturingdairy,bakery,dry-ripened,anddrinkproducts. Forfieldcrops,fungalcontaminationis
usuallycontrolledbyusingsyntheticfungicidesassociatedwithsomecropmanagementpracticessuch
ascroprotation,useofresistantcultivars,andtillage[6]. Concerningrawmaterials,fungicidesare
widelyappliedtoprotectpost-harvestfruitsandvegetables,butothertreatmentssuchasdisinfection
with ozone, chlorine, acidified hydrogen peroxide, pH change with sodium bicarbonate, surface
sterilizationusingirradiationorthermaltreatments,aswellaswaxingwithactivecoatingscontaining
fungicidal agents and preservatives are also used. Moreover, different packaging techniques are
also used to protect crops from mechanical damage, which is the most common entry point of
microbialinfections[7,8]. Duringfoodprocessingandrawmaterialstorage,goodhygienepractices,
ahazardanalysiscriticalcontrolpoint(HACCP)system,efficientequipmentandairdecontamination,
andcontrolofairpressurizationtopreventairflowfromthedirtiesttocleanestareas—cleanroom
technologies (aseptic or ultraclean technologies)—can also be applied to prevent contaminations.
Moreover, hurdle technologies are also applied according to the given process and raw materials.
They can include thermal treatments such as cooling or heating by pasteurization, sterilization,
andheatingatultra-hightemperatures(UHT);waterremovalbydrying,freeze-drying,smoking;water
activityreductionbyadditionofhighsugarandsaltconcentrations;pHmodificationbyimmersion
in vinegar or other acids; and the use of non-thermal preservation such as modified atmosphere
packagingtoreduceO andincreaseCO levels;andfinallyhigh-pressuretreatmentandhighelectric
2 2
fieldpulsetocontrolorinactivatemicroorganismsandsporesespeciallyinfoodsandbeveragessuch
as yogurts, soups, sauces, liquid eggs, fruit and vegetable juices, raw milk and whey, soft drinks,
andalcoholicbeverages[9,10]. Moreover,chemicalpreservatives—includingbenzoate,propionate,
sorbate,nitrate,nitrite,andsulfites—areusedtoavoidfungalspoilage[11].Theonlymicrobialderived
antifungalcompoundusedasfoodpreservativeisnatamycin(E235). ItisproducedbyStreptomyces
nataliensisandbelongstothegroupofpolyenemacrolideantimycotics.Theuseofnatamycinoncheese
asasurfacetreatmentisapprovedbymostcountriesanditsadditionintootherfoodsdependson
legislation[12].
Thepotentialadverseenvironmentalandhealtheffectsofcertainfungicidesandpreservatives
have led to more natural methods. Concerning crops, undesirable concentrations of residual
pesticides on harvested products, regulations, acquired resistances by fungi, and environmental
impact have led to biocontrol agent development (reviewed for fruits and vegetables by [13] and
for cereals by [14] currently a fast-growing sector, is also in need of natural solutions to reduce
croplosses.Severalproductscomposedofbacteria,fungi,andyeastsarecurrentlycommercialized
worldwide[15–18].Concerningrawmaterialsandprocessedfoods,preservativesarewidelyused.
However,whiletheiruseisregulatedinmanycountries,ingestionofsomeofthemraisesquestions
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abouttheirpotentialadversehealtheffects,especiallywhenconsideringtheirchronicconsumption
over years [11]. Moreover, fungal spoiler resistances have been previously reported in some
cases [19,20]. In this context, there is a strong societal demand, supported by public authorities,
forlessprocessedandpreservative-freefoods,suchas‘cleanlabel’products,andtheuseofnatural
alternatives. Suchalternativesmainlycorrespondtotheuseofessentialoils[21–23]andantagonistic
microorganismsaspreservationtools[24–29].Theuseofnaturaloraddedmicroorganisms,fermentates
(i.e.,aningredientproducedbythefermentationofavarietyofrawmaterialsbyamicroorganism),
or their metabolites to extend food shelf-life and/or increase food safety (through antibiosis
of pathogenic or spoilage microorganism) is referred to as ‘biopreservation’ or ‘biocontrol’ [30].
Thisalternativeapproachiscomplementarytohurdletechnologies. Biopreservationbylacticacid
bacteria(LAB)iscurrentlytheprincipalalternativetopreservativesinfoodandiswidelystudied
becauseoftheroleandlonghistoryofuseofLABinfermentedfoods,theirabilitytoproduceantifungal
metabolites,andtheirGRAS(GenerallyRegardedasSafe)andQualifiedPresumptionofSafety(QPS)
status in the USA and EU, respectively. The use of antifungal LAB for food biopreservation was
previouslyreviewedin2013byCrowley[24].Otherantifungalmicroorganismstopreventpost-harvest
fungalspoilagehasbeenreviewedbySharmaetal.(2009)andLiuetal.(2013)[29,31].Sincethen,there
havebeensignificantadvancesintheknowledgesurroundingtheuseofantifungalmicroorganisms.
Therefore,theobjectiveofthisreviewistoreporttheadvancesonLABapplicationsasantifungalagents
(overthelastthreeyears)aswellasotherantifungalmicroorganisms,namelynon-LABbacteria,yeasts,
andmoldsthatcanbeusedatthepost-harveststage,forrawmaterials,orduringfoodprocessing.
Cropbiocontrolwillnotbeconsideredinthisreviewandthereaderisreferredtotherecentreviewfor
cerealsbyOliveiraetal.2014[14],Punja&Utkhede,2003[32],Milindetal.2016[33],andNguyenetal.
2017 [34]. It will detail the main fungal spoilers encountered in different raw or processed foods,
antifungal microorganisms that can be used for food bioprotection, and current knowledge about
theirmechanismsofaction. Finally,advancesandconstraintsassociatedwiththeuseofantifungal
biopreservativesinfoodandthefutureareasofresearchrequiredinthisfieldwillbediscussed.
2. FungalSpoilers
2.1. QualityandSafetyIssues
Foodproductsaresusceptibletofungalspoilageduetotheirnaturalnutrientrichcomposition.
This susceptibility does however depend on various factors, namely (i) the food matrix nature
(livingmaterialornot,liquidorsolid),itscomposition(nutrientcontent,solutetype),andbiological
(e.g.,itsnaturalmicrobiota),physical,andchemicalparameters(wateractivity,pH);(ii)management
during harvesting of fruits and vegetables (maturity, handling) and raw material storage
(temperature,hygrometry,andduration);(iii)technologicalprocessesappliedduringmanufacture
(e.g.,heating,drying,salting,fermentation,preservativeaddition)includingcleaning/disinfection
steps; and(iv)storageconditionsaftermanufacturing(type, atmosphereandextentofpackaging,
temperature,relativehumidity). Foodmatrixintrinsiccharacteristicsandalsoassociatedextrinsic
factors will control the development of certain fungal genera or species. This usually leads to the
selectionordominanceofoneormorefungalspeciesonagivenmatrix.
Regardingquality,theimpactoffungalcontaminationscanleadtovisualand/orotherdefects.
Regarding visual defects, the most obvious is product aspect with apparent fungal growth due
to noticeable presence of thallus or yeast colonies that eventually leads to rotting. The presence
of black, white, green, pink, or yellow spots can also be associated with fungal development [4].
Thisusuallyleadstoeliminationoftheentireproductattheindustrialorconsumerlevel.Accordingto
thenatureofthecommodityandthecontaminanttype,theimpactmightbelessevidenttoobserve,
butfungalmetabolismcanleadtovariousorganolepticdefectsincludinggasproduction,off-flavors,
and texture changes. One of the best known off-flavors associated with fungal contamination
correspondstoanimal-likearomadevelopmentlinkedtovolatilephenols(4-ethylphenol,guaiacol,
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orcatechol)inwineandciderbyBrettanomycesspp.[35,36].Inwine,moldshavealsobeenshowntobe
responsiblefortheproductionofearthy/moldy(e.g.,geosmin,methylisoborneol)off-flavors[37,38].
Gas production have also been observed. For example, the presence of bubbles was observed in
packagedmeatinoculatedwithKazachstaniapsychrophila[39]. Kazachstaniaservazziihasbeenassociated
with severe package swelling of prepared fresh pizzas [40] while yogurt has been shown to be
contaminatedbyfermentativeyeasts(e.g.,Meyerozymaguilliermondii)thatledtotoplidbulging[41].
Moreover,duetoenzymaticlipolytic,proteolytic,andamylolyticactivities,producttexturecanalsobe
affected. Forexample,Yarrowialipolyticacanproduceoff-flavors,stimulatetheformationofbiogenic
amines,andnegativelyaffectcheesetexture[42].
Regardingsafetyconcerns,fungalpresenceandgrowthmayalsoleadtohealthhazardsassociated
with the presence of mycotoxins that can be produced by species belonging to the Aspergillus,
Penicillium, Fusarium, and Alternaria genera. This aspect is of importance as about 25% of raw
materials produced by agriculture worldwide are estimated to be contaminated by fungi and
mycotoxins [43,44]. Some mycotoxins can induce toxic, carcinogenic, and mutagenic reactions in
humansatlowconcentrations[5];aflatoxins,ochratoxinA,patulin,fumonisins,zearalenone,T-2and
HT-2 toxins, and deoxynivalenol are considered to be the most significant mycotoxins. The main
problemisthepresenceofmycotoxinsinprocessedfoodsasthepresenceofthemycotoxigenicspoilers
can no longer be detected, but these metabolites are usually resistant to technological treatments.
Thishasledtoregulationsonmycotoxinlimitsinvariouscountries[45].Amongimportantmycotoxins
infoodspoilage,aflatoxinsandochratoxinAareproducedbyseveralmembersofAspergilluswhile
OTAcanalsobeproducedbysomePenicilliumspecies. Aspergillusflavus,themainaflatoxinproducer,
isacommonspoilerofvariouscommodities(fruitsandvegetables,spices,cereals,bread,andnuts
such as peanuts, almonds and pistachios). Fumonisins are produced by Fusarium and Aspergillus
species. For example, Aspergillus awamori strains infecting onions and Aspergillus niger found in
coffeebeansandgrapesproducedfumonisinsandochratoxins. Moreover,afurtherpotentialriskis
associatedwithproductsderivedfromrawmaterialscontainingthesemycotoxins[5]. Patulin,which
isnon-carcinogenicbutimmunotoxicandneurotoxicinanimals,canbeproducedbyseveralAlternaria,
Paelomyces,Aspergillus,andPenicilliumspecies. Forexample,patulincanbeproducedbyPenicillium
expansum, a common apple spoiler, and then be transferred to juices and other derived products
(e.g.,babyfood,applesauce)[5]. FungifromtheAlternariagenusarecommononpost-harvestfruit
decay. They can produce different mycotoxins such as alternariol, alternariol monomethyl ether,
altenuene,altertoxinsI,II,III(ATX-I,-II,-III),andtenuazonicacidthathaveamutageniceffect[46].
Sofar,themycotoxinriskisconsideredforasinglemycotoxin,however,mycotoxincontamination
rathercorrespondstomulti-occurrenceduetothesimultaneouspresenceofseveralmycotoxigenic
speciesand/orofspeciesabletoproduceseveralmycotoxins[47].
2.2. IsolationandIdentificationofFungalSpoilers
Beforedevelopingbiopreservationorbiocontroltools(i.e.,selectingantifungalmicrobialagents)
to control fungal spoilers, it is fundamental to determine the main fungal contaminants and their
occurrence in a given food. This mainly relies on their isolation and accurate identification at the
species level, which will consequently also provide information on their mycotoxigenic potential.
Inthecontextoffungalspoilage,contaminationofagivenfoodproductisusuallylinkedtoalimited
numberofspeciesand,therefore,culture-dependentmethodsarethemostappropriate. Thefirststep
correspondstoisolationand/orenumerationofthesemicroorganismswhichcanbeperformednotonly
fromthecontaminatedfoodbutalsofromotherpotentialcontaminationsourceslikeairandsurfaces.
Theuseofspecificmediaisaclassicalsteptoisolateanddeterminetotalcountsaswellasmorphotype
observation. Afterisolationoftheobservedcontaminant,identificationisperformedusingdedicated
methods. Althoughmoldscanbepresumptivelyidentifiedfromtheirmorphology(e.g.,byPitttest),
molecularmethodshavebecomethegoldstandardforfungalidentification. PCRamplificationand
sequencingoftheD1/D2domainofthelargesubunitofribosomalRNA(26S)andoftheinternal
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transcribedspacer(ITS)areroutinelyperformedforyeastandmolds,respectively[48–51]. However,
concerningmolds,ITSisnotalwaysinformativeenoughforsomespecies,andothergenetargetscanbe
sequencedtocompleteidentificationatthespecieslevel. Forexample,theβ-tubulingeneiscommonly
usedforPenicilliumandAspergillusspp.,actingeneforCladosporiumspp.,andtheelongationfactor
geneforFusariumspp. Itismoreandmorecommontouseacombinationoftaxonomicallyrelevant
genestofacilitateidentificationandpotentiallydescribenewspoileragents.
It is worth noting that the constitution of fungal spoiler culture collections would be of great
interesttostudyantifungalcultures. Thesecollectionsareespeciallyofinterestiftheycontainvarious
strains of a given species. Indeed, most studies only focus on one strain of a given fungal spoiler,
raisingquestionsabouttherepresentativenessoftheselectedtargetstrain.
2.3. FungalSpoilersandFoodChains
Thediversityoffungiandyeastasfoodspoilershasbeenexploredindifferentstudiesandfood
products. Notably,fungalspeciesarenotalwaysdetrimentalforfoodproductionandsomespeciesare
evencrucial,especiallyinfermentedfoodstoobtainthetypicalorganoleptictraitsofthefinalproduct,
e.g.,Saccharomycescerevisiaeinfermentedbeverages,Penicilliumcamembertiinmold-ripenedcheeses,
orAspergillusoryzaeinsoysauce. Moreover,itshouldbekeptinmindthatafungalspeciesthatis
expectedandoftendeliberatelyaddedinsomefermentedfoodscanbeaspoilerinanotherproduct.
Forexample,Penicilliumroqueforti—aspeciesessentialforblue-veinedcheeseproduction—canspoil
gratedcheeseorfreshcheese.
Whenconsideringfoodspoilagebyfungi,twomainfoodcategorieshavebeenproposedbyPitt
&Hocking,namely(i)freshorperishablefoods(subcategorizedinlivingandnon-livingcells)and(ii)
storedorprocessedfoods[4]. Foracompleteviewoffungalspoilageoftheseproducts,oneshould
refertotheseauthors’books,whichareconsideredasthereferenceinthedomain. Post-harvestfungal
spoilageofcropsismostoftenreferredtoas‘post-harvestdisease’, andspoilersarereferredtoas
‘pathogens’,thereforethesetermswillalsobeusedhereafter.
2.3.1. FreshorPerishableFoods
Concerningfoodsandrawmaterialsofplantorigin,fruitsandvegetablesaremainlysusceptible
to fungal contamination during the ripening stage because of changes in pH, skin, carbohydrates,
anddefensesthatinducefavorableconditionsforfungalspoilers. Fungiinducevisiblesymptoms
onpost-harvest cropsincluding discoloration andtissuelesion formation.Theirpresenceleadsto
spoilageofavarietyoffruitsincludingcitrus, pome, berry, stone, tropical, andsolanaceousfruits.
Fungalpathogensoffruitsandvegetableshavebeenlargelydocumented[5,8,52].Tubersandother
vegetables—including bulbs, crucifers, cucurbits, and legumes—are less affected than fruits by
fungalpathogens,partlybecauseoftheirpHmakingthemamoresuitableenvironmentforbacterial
pathogens[52]. FungalspeciesbelongingtothePenicillium,Botrytis,Monilinia,Rhizopus,Alternaria,
Aspergillus,Fusarium,Geotrichum,Gloeosporium,andMucorgeneraareresponsibleformanyofthemost
importantpostharvestdiseases[29].
Forfoodsandrawmaterialsofanimalorigin,freshmilkandfisharelesssusceptibletofungal
spoilage but highly affected by bacterial spoilage. Concerning meat, fungal spoilage can occur,
especially during refrigeration, even if bacterial spoilage is predominant [53]. Despite a need for
more studies, Cladosporium, Penicillium, and Aureobasidium species have been reported to provoke
blackspots,whileThamnidiumspp. cancause‘whiskers’oncarcasses. YeastbelongingtoCryptococcus,
Candida,andYarrowiagenerahavebeenreportedonaerobicpackagedfoodandcanprovokeoff-flavors
andaspectdefects,suchasslimeorspots[54].
2.3.2. StoredandProcessedFoods
Stored and processed foods include a wide variety of food products, in which water activity
(a )determinesviabilityandfunctionalityofmicrorganisms. Mostofthemcannotmultiplybelow
w
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0.900a ,whilexerophilicfungicansurviveat0.755–0.605a [55]. Finalmoisturecontentandwater
w w
activityarelinkedtothetechnologicalprocessesused(e.g.,fermentation,drying,salting,evaporation,
andfreeze-drying)[10]. Forintermediateandhighwateractivityproducts,themainfoodcategories
concernedbyfungalspoilagearedairyproducts(yogurt,cream,butter,cheese... ). Theseproducts,
usuallykeptrefrigerated,canbeaffectedbybothyeasts,especiallyCandidaspp.,Yarrowialipolyticaand
M.guilliermondii,andmolds,mainlyPenicillium,MucorandCladosporiumspp.[49]. Processedmeats
(chilled meats, bacon), which exhibit an intermediate moisture content, are commonly spoiled by
Penicillium,Aerobasidium,Cladosporium,andEurotiumspp.,butalsoDebaryomyceshansenii,Y.lipolytica
andCandidaspp. Forlowwateractivityfoodproductssuchascereals,nuts,spices,driedmilk,dried
meats(driedbeef,biltong)and,fermentedmeats(drycuredham,salami,andfermentedsausage)[4].
Eurotium,Aspergillus,andPenicilliumspp. arethemainspoilers. SpoilingyeastssuchasD.hanseniiand
Y.lipolyticahavealsobeenfoundindriedmeats[56]andCandidaspp. infermentedmeat[4]. Someof
themostxerophilicspeciesareWallemiasebi,Eurotiumrepens,Eurotiumhalophilicum,Xeromycesbisporus,
andtheyeastZygosaccharomycesrouxiifoundinconcentratedfoods(e.g.,fruitconcentrates,jamsand
confectionery),cerealsandspices,[55,57,58]. Recently,Aspergilluspenicillioidesfoundindriedfish[59]
wasshowntobethemostxerophilicfungalspeciesregisteredtodateasitwasabletogerminateat
0.585a [60].
w
Heattreatments,especiallywhenappliedtoacidicfoods,usuallyallowforfungaldestruction.
However, ascospores of some species (Byssochlamys nivea) are resistant and can cause spoilage in
fruit puree or fruit juices [61]. Noteworthy, contamination of heat-treated products can occur as
post-contaminationbynon-heat-resistantspecies. Thisisthecaseofbakeryproducts,withbreads
beingparticularlysusceptibletofungalspoilage. ThemostfrequentlyfoundgeneraarePenicillium[62]
Aspergillus,Wallemia,andEurotium[63].Insaltedfoods,Wallemiasebi,Aspergillusspp.andEurotiumspp.
arethemainspoilersassociatedwithsaltedfish[64].
3. AntifungalMicroorganismsinFood
Aspreviouslystated,toanswerthestrongsocietaldemandforlessprocessedandpreservative-free
foods,biopreservationhasreceivedgrowinginterestforimprovingfoodqualityandsafety[14,65].
In past years, many strains from various microbial species that harbor antifungal properties have
beenidentified.Theyhavebeenisolatedfromvarioussources,suchasfruits,vegetables,cereals,milk,
meat,andotherfood-relatedproducts. Theisolationofnewbioprotectivecultureshasrecentlybeen
extended to other environments, such as deep-sea [66] and Antarctic soil samples [67] in order to
discovermicroorganismspotentiallyproducingnewantifungalmetabolites.
Screeningstepsarerequiredtofindefficientantifungalmicroorganismsasantifungalactivity
levels and the spectrum of the inhibited fungal targets greatly vary depending on the considered
species,andfromstraintostrainwithinaspecies. Forexample,upto75%ofvariationwasobserved
betweenfivestrainsofLactobacilluscaseitestedfortheirpotentialtoinhibitthegrowthoffourspoilage
molds [68]. In another study, only a few L. plantarum isolates among 88 screened showed a wide
spectrumoffungiinhibition[69]. Inanotherexample,among55yeastisolates(Aureobasidiumpullulans,
Cryptococcusmagnus,Hanseniasporauvarum,Candidazeylanoides,Candidasake,Rhodotorulamucilaginosa,
andPseudozymaaphidis)fromthesurfaceofgrapevarieties,58%wereabletoinhibitA.tubingensisgrowth
invitroand27from37strainsofA.pollulanswereantifungalindicatingastrain-dependenttrait[70].
3.1. ScreeningandValidationMethods
3.1.1. InVitroScreening
Candidate bacteria, yeasts, and molds are generally first isolated from various biotopes and
purifiedinordertobetestedfortheirantifungalproperties.Thesecandidatesarethentestedagainst
oneorseveralfungaltargets. Asstatedinthepreviouschapter,thechoiceoffungaltarget(s)iscrucial,
sincetheyshouldberepresentativeofthedominantspeciesassociatedwithagivenfoodproductand
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sincetheantifungaleffectsobserveddependonthefungalspecies. Forexample,theinhibitionofthree
berrycontaminants—namelyBotrytiscinerea,PenicilliumdigitatumandPenicilliumitalicum—byfour
antifungalyeastsvariedaccordingtothetarget[71].Also,growthinhibitionofseventargetsfrom
thePenicillium,Aspergillus,andCladosporiumgeneravariedforthe88Lactobacillusplantarumstrains
tested[69].
Severalapproaches,rangingfromclassicalmethodsinagarplatestohighthroughputassaysin
multiwell-plates,havebeendevelopedtoscreenmicroorganismsfortheirabilitytoinhibitspoilage
fungi. Inthespot-on-lawnassay,culturesorcell-freesupernatantsofthetestedstrainsarespotted
over an agar plate, over which a second layer containing the fungal target is poured. In the agar
diffusion method, the tested agent is spotted over an agar layer inoculated with the fungal target.
Inboththeseassays, formationofaninhibitionhaloafterincubationaroundthespotsrevealsthe
antifungal activity. A dual culture technique has also been used to test the antagonistic effect of
apotentialantifungalmoldagainstatargetmold,inwhichagarplugscontainingthetargetandthe
antifungalmolds,respectively,wereinoculatedattheoppositesidesoveranagarplate[70,72].Another
screeningapproachcorrespondstotheinvestigationoftheenzymaticactivitiesrequiredtodegrade
thecellwallofthefungaltargetasshownbyTokpahetal. (2016)thatscreenedbacteriaforcontrol
ofaricepathogen,Magnaporthegrisea. Inthiscontext,activityofcellwalldegradingenzymes—such
ascellulases,proteases,chitinases,andglucanases—wasassessedbyplatingbacteriaonagarmedia
containingsubstratesoftheseenzymeswhereactivebacteriainducedclearingzones[73].
For high-throughput screening purposes, agar plate methods have been recently adapted in
24-wellplates. Thefungaltargetcanbeincludedinanagarmediumandpouredineachwellover
afirstagarlayerspottedwiththepotentialantifungalstrains[74]. Alternatively,sporesofthetarget
moldareinoculatedonthesurfaceofthefirstagarlayercontainingthepotentialantifungalstrains[63].
Most invitro screening experiments of LAB antifungal activities have been performed using
syntheticmedia,suchasthedeMan,Rogosa,andSharpe(MRS)agarmedium.ThecompositionofMRS
canstronglyimpacttheexpressionofantifungalactivitybyLABbecauseitcontainsacetate,whichmay
reinforceLABantifungalactivityandartificiallyinflatethenumberofactiveisolates,asmentionedin
severalstudies[63,75,76]. Similarly,thepresenceofseveralantifungalcompounds—suchascyclicand
linearpeptidesanddiketopiperazines—hasbeenreportedintheclassicallactate-tryptone-yeastextract
mediumusedtogrowpropionibacteria[77]. Otherimportantpointsarethenatureandconcentration
ofsugarsandthebufferingcapacityofthemedium,whicharehighlyexpectedtoinfluencetheamount
oforganicacidsandthefinalpHofthemedium,thusmodifyingantifungalactivity. Somealternatives
totheuseofsyntheticlaboratorymediahavebeenrecentlyreported. Particularattentionhasbeen
paidtodesignmediamorecloselyrelatedtofoodsandusablein24-wellplatestoscreenbacterial
antifungalactivity. Thisapproachwasrecentlyusedwithawheatflourhydrolysateagarmedium
andwithyogurt[63,78]. Similarly,amethodusingamodelcheesedistributedin24-wellplateswas
also recently developed allowing for high-throughput antifungal activity screening of either LAB
fermentatesorLABstarters[79].
Whatevertheconsideredscreeningexperiments,itisimportanttotakeintoaccounttheconditions
prevailinginsitutodeveloprelevantscreeningapproaches. Actually,severalstudieshaveshownthat
strainsexhibitinginvitroantifungalactivitywerefarlessorevennolongeractivewhentestedinthe
foodproducts[63,80–82]. Whengrownonawheatflourmedium,20%ofthe270testedLABstrains
representingsixgenerascreenedwerefoundtoinhibitthefivefungaltargetstestedwhilenonewere
detectedwhengrowninMRSagarwithoutacetate[63].
Forallthesereasons,resultsofinvitroscreeningarehighlyheterogeneousintermsofproportion
of strains and nature of species harboring antifungal activity. To maximize the chances of finding
efficientstrainsinsitu,approachesbasedontheuseoffood-relatedmediaandscreeningconditions
shouldbefurtherdeveloped,inparticularatahighthroughputscaleforlargecollectionsofstrains.
Microorganisms2017,5,37 8of35
3.1.2. ValidationbyChallenge-TestintheFoodProducts
In all cases, in situ evaluation in the actual food products of the antifungal activity of the
microorganismsortheirmetabolitesselectedfrominvitroscreeningisessential,since,asstatedabove
(seealsochapterV),manystudiesreportdiscrepanciesbetweenresultsobservedinvitroandinsitu[83].
The developed approaches depend on the type of foods to be protected (raw material or
processed food, fermented or not), and on the antifungal microorganism used, to maximize
efficiencywithoutadverseeffectsonthesensorycharacteristicsofthefoodorontheenvironment.
Biopreservation/biocontrolcanbeachievedeither(i)byaddinganactiveingredientconstitutedof
afractioncontainingpurifiedmetabolites,acell-freesupernatant(CFS),afermentate;or(ii)byusing
cellsexpectedtogrowonorinthefoodproduct.
Concerning postharvest fruits, bacterial cells, yeast cells, and cell-free supernatants of molds
wereevaluatedbysprayingthemontothesurfaceofanintactfruitoronawound,beforeinoculation
of the fungal target [31,84–86]. Concerning fermented foods, antifungal bacteria can be added as
adjunct cultures during the process, along with the usual starters. This has been done in different
foodsincludingdairyproducts[87–89]andbakeryproductsforwhichadjunctstrainswereaddedin
thesourdough[90–93]. Alternatively,antifungalingredientscanbeincorporatedduringtheprocess
orsprayedoverthefood,asdoneforbakeryproducts[63]. Inthisstudy,only12outof69LABand
PABstrainsselectedfrominvitroscreeningonwheyflouragarshowedaneffectinsituafterhaving
sprayedthefermentedmediumonthesurfaceofthebakeryproducts,thusconfirmingthenecessityto
gobeyondinvitrotests.
Inmostcases,fungaltargetsareinoculatedandtheabilityoftestedbioprotectiveagentstorestrain
fungalgrowthisevaluated. Theirimpactonmycotoxinproductioncanalsobeinvestigated,asdone
forexamplefordeoxynivalenolproducedbyFusariumculmorumduringmalting[94]andaflatoxin
producedinvitrobyAspergillusflavusandAspergillusparasiticus[95]. Environmentalchallengetests
arealsoappliedinwhichtheactivityistestedtopreventenvironmentalfungalcontaminants,after
exposingtheproductstotheairbornemoldsoftheenvironment,forexampleinabakery[83]ordairy
environment[89]. Theinterestofappropriateinsitutestsagainstabroadrangeofcontaminantshas
recently beenillustratedbystudies usingantifungalbacteriain breadbioprotection.Amongstthe
threeLactobacillusstrainstestedinvitroandinsitu,thebestperformingstrainininsituenvironmental
challengetestswastheleastactiveinvitro[83]. Inanotherstudy,theantifungalstrainL.amylovorus
DSM19280wastestedincomparisontoanon-antifungalL.amylovorusstraintoprotectwheatand
quinoasourdoughbreadsandclearlyshowedthattheproductionofantifungalcompoundsvaried
accordingtotheflourtype[80].
3.2. AntifungalMicroorganisms
Alargenumberofmicroorganismshavebeentestedfortheirantifungalactivityagainstfood
spoilers. Thispartmainlyfocusesonthemicroorganismssuccessfullytestedasbioprotectivetoolsin
foodproducts.
Lactic acid bacteria (LAB) are by far the main microorganisms tested for application in dairy
and bakery productions, the two main food sectors studied for biopreservation against fungi.
Thesemicroroganismshavealsobeentestedinbrewing(maltingprocess),andinthemanufactureof
fermentedvegetablesortoprotectgrains,seeds,orfruits.Forfruitbioprotection,themaincandidates
tested are bacteria, especially from the Bacillus group, and various yeast species. For fermented
meats, yeast (D. hansenii) and Penicillium species have been tested against fungal spoilers.
Many of these antifungal species have the characteristics of ‘microbial weeds’—i.e., species able
todominatecommunitiesthatdevelopinopenmicrobialhabitats—aconceptrecentlyintroducedfor
microorganismsbyCrayetal. 2013[96]. Table1liststhepublishedliteratureonthissubjectoverthe
pastthreeyears.
Microorganisms2017,5,37 9of35
Table1.Activebioprotectiveculturesagainstfungalcontaminantsofpostharvestfruitsandprocessedandrawfoods.
AntifungalMicroorganisms
FoodField Group InSituTest SourceofMicroorganism ApplicationMethod ActivitySpectrum(Inhibited/Tested) Reference
(ActiveInSitu/TestedStrains)
bakery LAB LactobacillusbrevisITM18 yeast-leavenedbread sourdough CFSasingredient Aspergillusniger [97]
bakery LAB LactobacillusplantarumHD1 Koreandraftricewine kimchi CFS AspergillusfumigatusandPichiakudriavzevii [98]
bakery LAB LactobacillusamylovorusDSM19280(1/1) sourdoughquinoabread cerealisolate cellsinsourdough environmentalmolds [80]
SL778:fermentateas
bakery LAB LactobacillusplantarumCRL778 wheatbread homemadewheatdough environmentalmolds [99]
ingredient
Fusariumculmorum(only1testedinsitu),
LactobacillusplantarumUFG121(only1insitufrombest Penicilliumchrysogenum,Penicillium
bakery LAB oat-basedproduct food cellsinsourdough [69]
2/88invitro) expansum,Penicilliumroqueforti,and
Aspergillusflavus(5/7invitro)
bakery LAB LactobacillusamylovorusDSM19280(1/3) sourdoughwheatbread cerealisolate cellsasstarter Fusariumculmorum [83]
LactobacillusreuteriR29,LactobacillusbrevisR2, sourdoughofquinoaand
bakery LAB human,pork,andcereal cellsinsourdough environmentalmolds [90]
LactobacillusamylovorusDSM19280 ricebread
LactobacillusbulgaricusCECT4005,Lactobacillus
plantarumCECT749(activeinsitu2/6),Lactobacillus
Aspergillusparasiticus(onlyonetested
bakery LAB johnsoniiCECT289,LactobacillusrhamnosusCECT288, loafbread notdetailed cellsinsourdough [100]
insitu)andPenicilliumexpansum
LactobacillisruminisCECT1324,andBifidobacterium
bifidumCECT870T(6activeinvitro/16)
Leuconostoccitreum(5strains),Lactobacillussakei,
CladosporiumsphaerospermumandWallemia
Lactobacillusplantarum,LactobacillusspicheriO15, wholecultureassourdough
poundcakeandmilk milk,milkrollsourdough, sebionpoundcake;andEurotiumrepens,
bakery LAB&PAB Lactobacillusreuteri5529,LactobacillusbrevisLu35, ingredientformilkbread [63]
breadroll andothersnotdetailed Aspergillusniger,andPenicillium
PropionibacteriumacidipropioniciandPropionibacterium rollandsprayed
corylophilumonmilkbreadroll
freudenreichiiLSaci68(bysurface-spraying12LAB/69)
Debaryomyceshansenii,Kluyveromyceslactis,
LactobacillusharbinensisK.V9.3.1Np,Lactobacillus.
Kluyveromycesmarxianus,Penicillium
dairy LAB rhamnosusK.C8.3.1I,andLactobacillusparacasei yogurt cowandgoatmilk cellsasadjunctculture [78]
brevicompactum,Rhodotorulamucilaginosa,
K.C8.3.1Hc1(3/11)
andYarrowialipolytica(6/6)
dairy LAB LactobacilluscaseiAST18(1/1) yogurt Chinesedairyproducts cellsasadjunctculture Penicilliumsp.(1/1) [88]
Penicilliumsp.nov.DCS1541,Penicillium
dairy LAB LactobacillusparacaseiDCS302 yogurt notdetailed cellsasadjunctculture [101]
solitum(2/8)
dairy LAB LactobacillusharbinensisK.V9.3.1Np(1/2) yogurt cowmilk cellsasadjunctculture Yarrowialipolytica(1/1) [87]
pigfeces(isolatedby cellsasadjunctculture
dairy LAB LactobacillusreuteriINIAP57 semi-hardewemilkcheese Notevaluated [102]
Langa2003) supplementedwithglycerol
Penicilliumexpansum(1/1)and
dairy LAB LactobacillusamylovorusDSM19280(1/1) cheddarcheese cerealenvironment cellsasadjunctculture [89]
environmentalmolds
freshherbs,fruits, cellsasaddedtothe
dairy LAB 12strainsofLactobacillusplantarum(12/897) cottagecheese Penicilliumcommune [103]
andvegetables finishedproduct
L.rhamnosusA238,L.rhamnosusA119(2/5)The
associationofL.rhamnosusA238withB.animalissubsp. cellsaddedtothe
dairy LAB cottagecheese notdetailed Penicilliumchrysogenum(1/1) [104]
lactisA026,andL.rhamnosusA119withB.animalis finishedproduct
subsp.lactisA026
malting LAB LactobacillusbrevisR2∆(1/1) barleymaltextract porcineisolate cellsasstarter Notevaluated [105]
fermentation
Microorganisms2017,5,37 10of35
Table1.Cont.
AntifungalMicroorganisms
FoodField Group InSituTest SourceofMicroorganism ApplicationMethod ActivitySpectrum(Inhibited/Tested) Reference
(ActiveInSitu/TestedStrains)
LactobacillusbrevisR2∆andLactobacillusplantarum barleymaltextract(wort)
malting LAB porcineandbarleyisolate cellsasstarter Fusariumculmorum [106]
FST1.7(2/2) fermentation
malting LAB LactobacillusbrevisR2∆(1/1) barleyinmaltingprocess porcineisolate cellsasstartersandCFS Fusariumculmorumand [107]
Fusariumgraminearum
LactobacillusreuteriR29andLactobacillusamylovorus maltingprocess(steeping CFS(wortasgrowthmedia)
malting LAB humanandcerealisolates Fusariumculmorum [94]
DSM19280 andgermination) asthesteepingliquor
Pediococcusspp.A19(testedinsitu)Pediococcusspp.
fermented Aspergilluscarbonarius,Aspergillusniger,
LAB A21,LactobacillusplantarumB4496,Lactobacillusbrevis cocoa fermentingcocoa cellsasstarter [108]
vegetables andAspergillusochraceus
207,andLactobacillussanfranciscensisBB12(5/13)
Penicilliumexpansum(onlyonetestedin
fermented gari,fermentedcassava situ),Aspergillusflavus,Aspergillusniger,
LAB LactobacillusfermentumYML014 tomatopuree cells [109]
vegetables (starchyroot) Candidaalbicans,andZygosaccharomyces
rouxii(lowinhibitionofyeasts)
fermented LactobacillushelveticusKLDS1.8701(1/4alsoL.
LAB fermentedsoybeanmilk dairyproducts cellsasadjunctculture Penicilliumsp.(1/1) [81]
vegetables helveticus)
submergedinpurified
rottenjackfruit,guava,and A.niger,F.moniliforme,F.graminearum,
grain/seed LAB Lactococcussp.BSN307 wheatgrains volatileorganiccompound [110]
animalsfecalsamples F.chlamydosporum,andF.oxysporum
2,4-di-tert-butylphenol
wheatseeds(Triticum Aspergillusniger,Rhizopusstolonifer,Mucor
grain/seed LAB LactobacillusplantarumLR/14 notdetailed AMPLR14solution [111]
aestivumvar.HD2824) racemosus,andPenicilliumchrysogenum.
grain/seed LAB LactobacillusplantarumYML007(1/1400) soybean kimchi CFS Aspergillusniger [112]
kefirgrainscontainasymbioticconsortiumofLABand
LAB&
grain/seed yeasts(Lactobacillusplantarum,L.kefir,Lactococcuslactis arepa(corncakes) kefirgrains CFS Aspergillusflavus [113]
Fungi
subsp.lactis,SaccharomycesandAcetobacter)
LAB& Lactobacilluslactissubsp.lactisLABW1,LABW3,
fruit other LABW4,BurkholderiacenocepaciaVBC7and jackfruit rottenjackfruit cellssprayedoverthefruit Rhizopusstolonifer [114]
bacteria PseudomonaspoaeVBK1
Alternariabrassicicola(1testedinsitu),
CFScultivatedwithxylitol
LactobacillusparacaseiŁOCK0921(1testedinsitu) Alternariaalternata,Aspergillusniger,
fruit LAB wildcherries plantandhuman orgalactosyl-xylitoldirectly [115]
(1/(9invitro/60)) Fusariumlateritium,Geotrichumcandidum,
onwildcherries
andMucorhiemalis(6/8)
plants,fermentedwheat cellssprayedoverthefruit
LacticacidbacteriastrainsLCM5,LAB58,LAB13,
fruit LAB apple bran,pickles,and (woundedand Penicilliumexpansum [116]
andLAB43(4/6)
sauerkraut non-wounded)
Penicilliumroqueforti,Penicilliumcitrinum,
Chinesenaturally Penicilliumoxalicum,Aspergillusfumigatus,
fruit/dairy LAB LactobacillusplantarumTK9 citrus,applesandyogurt cells [117]
fermentedcongee Aspergillusflavus,and
Rhizopusnigricans(6/7)
Fusariumsporotrichioides,Fusarium
Other maize,sorghumand cerealssubmerged
grain/seed AcetobacternigricansAZT54(0/1) paddyfieldsoilsamples graminearum,Fusariumpoae,andFusarium [82]
bacteria wheatgrains insuspension
equiseti(4/10invitro)