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International Biodeterioration & Biodegradation 62 (2008) 364–367

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ib iod

In-vitro antifungal activity of chilli extracts in combination withLactobacillus casei against common sapstain fungi

Tripti Singh*, Colleen ChittendenEnsis, Wood Processing, 49 Sala Street, Private Bag 3020, Rotorua, New Zealand

a r t i c l e i n f o

Article history:Received 17 August 2007Received in revised form 18 October 2007Accepted 18 October 2007Available online 17 June 2008

Keywords:ChilliCapsicumCapsaicinOleoresinsSapstain fungiLactobacillus casei

* Corresponding author. Tel.: þ64 7 343 5329; fax:E-mail address: tripti.singh@ensisjv.com (T. Singh

0964-8305/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.ibiod.2007.10.009

a b s t r a c t

The efficacy of chilli juice and/or chilli (capsicum) extract oleoresins as antisapstain agents was evaluatedagainst two common wood-discolouring fungi, Sphaeropsis sapinea and Leptographium procerum. Possiblesynergy between chilli juice and Lactobacillus casei as antisapstain agents was also assessed.Both the chilli juice and the capsicum oleoresin showed moderate antifungal activity. No growth of thetest fungi was observed on plates amended with 50% chilli juice after 3 weeks of incubation. In thepresence of 0.1% oleoresins, fungal biomass was reduced by more than half when compared withcontrols.The synergy between chilli and L. casei was apparent; the combination chilli/L. casei treatment systemafforded much better inhibition than chilli or L. casei alone. In the presence of 25% chilli juice with L. caseithe growth of test fungi was stopped.

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1. Introduction

In this era of increased concern about the safety of chemicalsused in wood protection, natural methods of wood protection andnatural preservatives are receiving increased attention.

Chilli has been used since ancient times not only for increasingthe flavour of foods, but for its preservation (Govindarajan, 1985)and medicinal properties (Govindarajan and Satyanarayana, 1991;Chowdhury et al., 1996). The antimicrobial properties of chilli havebeen attributed to the presence of some property of their extract,oleoresins (Meena and Sethi, 1994). Chilli peppers belong to thefamily Solanaceae, genus Capsicum and species annuum or fru-tescens. The name capsicum comes from the Greek kapto, whichmeans ‘‘to bite’’. The compound that is primarily responsible forthe pungency is capsaicin (8-methyl-n-vanillyl-6-nonenamide)and a group of similar substances called capsaicinoids, whichincludes dihydrocapsaicin and nordihydrocapsaicin (Poyrazogluet al., 2005).

For many chilli growers and merchandisers, who are in thebusiness of chill seed extraction, the remaining juice is a wasteproduct after seed extraction. In this study, the juice of Rocotospecies, which is a hot variety but not as hot as the Thai, Cayenne, orhabanero varieties, was used. For comparison, capsicum oleoresin

þ64 7 343 5507.).

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extracted from Habanero peppers was also included in this study.The capsicum oleoresin used was obtained from a commercialcompany (Nature shop, Sydney, Australia); the product containeda mixture of naturally occurring essential oil and resin fromhabanero peppers. One kilogram of capsicum oleoresin is equiva-lent to approximately 18–20 kg of habanero peppers.

It is known that capsicum and pepper extract-containing for-mulations exhibit properties that repel plant insects, birds, andtermites (Kumbhar et al., 2001). The numerous applications ofcapsicum include its use to discourage growth of living organisms,particularly for covering materials for underwater objects such asboat hulls or water intake pipes (United States Patent No. 5226380;Fischer, 1993). The covering materials include waterproof coatingssuch as adhesive or paint containing capsicum derivatives such asCayenne pepper or oleoresin, with the coating applied to the outersurface of the object to be protected, to repel marine organismsthat might otherwise attach themselves to the object. A recentU.S. Patent was filed by Neumann (2003) (Application No.20030056436) for developing a capsicum based seed coating forpreventing destruction of crop seeds and grains by insects. How-ever, in the literature, there is some discrepancy regarding theantimicrobial activity of chilli. Wilson et al. (1997) evaluated theantifungal activity of plant extract against Botrytis cinerea andfound that among 345 extracts analysed, 13 showed high levels ofactivity, with species of Allium and Capsicum predominating. Bycontrast when Thyagaraja and Hosono (1996) assayed the abilityof chilli, coriander, pepper, cumin, and asafetida to inhibit food

Fig. 1. Extent of S. sapinea growth (in colony diameter) after 3 weeks of incubation oncontrol plates and plates amended with different concentrations of raw, autoclaved, orfiltered chilli juice (n¼ 5; error bars refer to standard errors of the means).

T. Singh, C. Chittenden / International Biodeterioration & Biodegradation 62 (2008) 364–367 365

spoilage mould, only asafetida showed promising results in inhib-iting the fungal growth.

The objective of this study was to test the efficacy of Rocoto chillijuice and the extract of habanero chilli oleoresins against wood-degrading fungi with the view of establishing the role of chilliextract as a wood preservative. Lactobacillus casei were isolatedfrom chilli juice and the synergy between chilli and L. casei was alsoevaluated.

2. Materials and methods

2.1. Efficacy of chilli juice

Chilli juice was obtained after processing whole chilli in vegetable and citrusjuicer (Elegance, Zip). Pomace was discarded and the chilli juice was tested againsttwo sapstain fungi, Leptographium procerum (Kendrick) M.J. Wingfield and Sphaer-opsis sapinea (Fr.:Fr.) Dyko and Sutton, using nutrient medium containing 2% maltextract agar amended with different concentrations of chilli ranging from 0.1 to 50%in three different forms: (a) raw, (b) filtered, or (c) autoclaved. Since raw chilli juicehas a high microbial content, two sterilization methods – autoclaving and filtering –were used to remove these microbes from the raw juice.

Chilli amended and control plates were inoculated with individual test fungi. A5-mm core of fungal inoculum cut from the edge of an actively growing colony wastransferred onto the centre of petri dishes (hereafter referred to as plates). Allinoculated plates were incubated in a controlled environment chamber in the darkat 75% relative humidity and 25 �C.

Colony diameter, in millimeters, of test fungi growing on plates was recordedweekly for 3 weeks. At final assessment time (after 3 weeks), average colonydiameter was calculated for each test fungus and chilli concentration using fivereplicate plates.

2.2. Morphological analysis

Microscope slides were coated with a thin (500-ml) layer of 2% of water–agaramended with 25% filtered chilli . After the agar film had solidified, a small fungalplug of test fungi was inoculated at the centre of the agar film. Inoculated slides wereincubated at 25 �C and 75% RH in the dark. Two replicates were prepared. After 4days of incubation, differential interference contrast microscopy was used toobserve the difference in morphology and branching pattern of hyphae caused bythe treatments. The total number of branches was recorded within a 200-mmperipheral length of the mycelium. For each slide, five hyphal tips (peripheral zone)were selected at random and the mean branching frequency was calculated for eachconcentration. Images were taken for each hyphal element using a Zeiss Axioplan 2light microscope with a 10� or 20� objective.

2.3. Synergy between chilli juice and L. casei

L. casei was isolated from chilli juice, suggesting that this bacterium is able togrow in this juice. To establish an antifungal synergy between L. casei and chilli juice,the pure culture of this bacterium was obtained from ESR (Environmental Scienceand Research Ltd., New Zealand culture collection reference) culture collection andwas spread onto both adjusted and control plates before inoculation with the testfungi as described earlier. Weekly assessment was carried out for 3 weeks.

2.4. Efficacy of capsicum oleoresins

Two test fungi were grown on malt–agar (2% malt extract,1% agar) plates. After 7days of incubation, distilled water (2 ml) was added to each plate, and the surface ofthe spores/mycelium was lightly scratched with a wire loop to release hyphalfragments. The suspension was blended for 5 min, filtered (pore size 30 mm) andadjusted to a concentration of 106 propagules ml�1.

Erlenmeyer flasks containing yeast–malt liquid medium (0.2% yeast extract and1.5% malt extract) were prepared and then amended with either 0.1 or 1% oleoresinsbefore inoculating with 1 ml of inoculum of the test fungus. The total volume in theflasks was 100 ml. Three flasks per treatment were prepared as just described foreach of the fungi.

After 6 days of incubation, total fungal biomass was determined by filtering theculture broth through a pre-weighted glass micro-fiber filter (Whatman GF/C). Filtercontaining mycelia were dried at 105 �C overnight, cooled in a silica gel desiccator(to prevent moisture absorption), and re-weighed.

2.5. Statistical analysis

A two-way ANOVA was conducted and LSD tests were used to compare thedifferences between treatments and test fungi. Differences were considered to besignificant at P� 0.05.

3. Results and discussion

Overall, the results showed that both chilli juice and the oleo-resins capsicum have antifungal activity against S. sapinea and L.procerum. The antifungal activity of the chilli could be enhanced inan integrated approach by combining chilli juice with L. casei(P� 0.05).

3.1. Efficacy of chilli juice

The growth of S. sapinea was restricted on plates amended with35% Rocoto filtered chilli juice. However, when the juice wasautoclaved, it stopped the growth of S. sapinea at this concentra-tion, suggesting that the active compounds were not destroyedduring autoclaving but that part of the active component(s) mighthave been removed during filtering. The growth of S. sapinea wasstopped at the concentration of 50% for both filtered and autoclavedchilli juices (Fig. 1). However, unlike S. sapinea, the growth ofL. procerum was not inhibited even in the presence of 50% chillijuice (Fig. 2), indicating variation in the tolerance level of differentfungi.

Plates amended with raw chilli, i.e., chilli juice without anysterilization process, inhibited the growth of both test fungi (Figs. 1and 2), which could be attributed to large amounts of microor-ganisms present in the chilli juice (Karunaratne and Dharmawanse,1999). This retardation of fungal colonisation could be either just aneffect of population (Berger et al., 1996; Bowers and Locke, 2000)and therefore nutrient exhaustion on the growth medium, or aneffect of the production of secondary metabolites that may possessbioactivity during incubation (Chitarra et al., 2002; Schnurer andMagnusson, 2005). This was not determined in the present studybut may warrant further investigation. Production of highly activeantifungal compounds by soil- and plant-associated bacteria is welldocumented (Grimont and Grimont, 1992; Levenfors et al., 2004).Zulpa et al. (2003) also indicated the importance of the fungussensitivity to the different substances present in the extracellularproducts of common bacteria.

3.2. Morphological analysis

Microscopic analysis revealed that the morphology of both testfungi was severely compromised when exposed to chilli. Myceliawere much more ‘‘fluffy’’ and compact when compared with thecontrol (not shown) and microscopic examination showed that thehyphae that had been exposed to chilli had excessive branching.Compared to the control, hyphae grown on medium amended with25% filtered chilli juice had a significant (P� 0.05) increase in

Fig. 2. Extent of L. procerum growth (in colony diameter) after 3 weeks of incubationon control plates and plates amended with different concentrations of raw, autoclaved,or filtered chilli juice (n¼ 5; error bars refer to standard errors of the means).

Fig. 4. The growth (in colony diameter) of test fungi on control plates and platesamended with different concentrations of filtered chilli juice with and without L. caseiafter 3 weeks of incubation (n¼ 3; error bars refer to standard errors of the means).

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branching (Fig. 3). This increase in branch frequency at theperipheral region of the hyphae is a probable response to stressinduced by chilli. Protein secretion in filamentous fungi occursaround the apical and subapical region of the advancing hyphal tip(Gordon et al., 2000; Muller et al., 2002). It has been shown thathyper-branching is the result of at least one unprocessed protein,which could directly affect chitin syntheses (Muller et al., 2002).

3.3. Synergy between chilli juice and L. casei

After 3 weeks of incubation, compared to the control, plates thatwere amended with 0.1% chilli juice restricted the growth ofL. procerum (P� 0.05), but not S. sapinea (Fig. 4). The growth ofS. sapinea was only restricted when both chilli and L. casei wereused (P� 0.05); at 0.1%, filtered chilli with bacteria restricted colonygrowth to 30 mm compared to 85 mm (full plate diameter) forcontrols or either chilli or bacteria alone. This suggested an anti-fungal synergistic effect between chilli and L. casei. When 25% chilliwith L. casei was used, growth of both test fungi was completelyinhibited (Fig. 4).

Latobacilli species are known to produce antimicrobial pep-tides, heterogeneous antibiotic substances, lactic acid and otherorganic acids called bacteriocins, which are important in the bio-preservation of food (Stiles, 1996). A few studies have shown theantifungal activity of lactic acid bacteria against wood surfacecontaminant fungi; Yang and Clausen (2004) indicated anti-mouldactivity of two Lactobacillus bacteria. Zulpa et al. (2003) showedgrowth inhibition of a strain of S. sapinea by extracelluar productsfrom cyanobacteria and lactic acid bacteria. Feio et al. (2004)examined the antifungal activities of Bacillus subtilis againstphytopathogenic, sapstain, and mould fungi. The bioautographicmethod revealed the presence of two active closely related

Fig. 3. Total numbers of branches counted in 200 mm peripheral length of test fungalmycelium after exposure with 25% filtered chilli in comparison to control (n¼ 10; errorbars refer to standard errors of the means).

peptides responsible for antifungal activity, which is in agreementwith the earlier results of Katz and Demain (1977).

Although many lactic acid bacteria produce bacteriocins, ribo-somally synthesized peptides, or proteins (Nes et al., 1996), theseare generally only active against closely related bacterial speciesand there is little evidence that bacteriocins should have any effecton fungal growth (Jack et al., 1995). However, unlike past studiesdone on bacteriocins, very few reports exist on antifungal peptidesof lactic acid bacteria (Schnurer and Magnusson, 2005). Batish et al.(1989) suggested that the antifungal substance produced by Lac-tobacillus isolate was proteinaceous since activity disappears withproteinase treatment. A study done by Magnusson and Schnurer(2001) showed that a proteinaceous compound from Lactobacilluscoryniformis strain Si3 had an antifungal effect against severalmoulds and yeasts. The study also showed that when a liquid cul-ture of L. coryniformis was supplemented with ethanol, or formicacid or acetic acid, the total amount of antifungal peptide increased.

In this study, the actual mechanism of the synergy betweenchilli juice and bacteria is unknown. However, it is very likely thatchilli juice might have some of the acid components that worksynergistically in the production of antifungal peptides producedby bacteria. An attempt will be made to measure the acetic acid andother acid components present in Rocoto chilli juice and their effecton pH of the medium. It is well documented that the effect of aceticacid and propionic acid is often dependent on the decrease in pHcaused by lactic acid (Woolford, 1984; Eklund, 1989).

3.4. Efficacy of capsicum oleoresins

The effect of two different concentrations of oleoresins on fun-gal growth of test fungi growing in liquid broth is shown in Fig. 5. As

Fig. 5. Dry weight biomass (in grams) after incubation of test fungi for 6 days inoleoresin-amended nutrient broth (n¼ 3; error bars refer to standard errors of themeans).

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expected, after 6 days of incubation, fungal biomass decreased asthe level of oleoresins capsicum increased in the culture solution.At a concentration of 0.1% w/v of oleoresins, dry weight biomass ofboth sapstain fungi was significantly (P� 0.05) reduced whencompared with the control, by 58% and 66% for S. sapinea andL. procerum, respectively. There was little further reduction at 1%w/v of oleoresins.

This antifungal activity of oleoresins is in agreement with thework of Chowdhury et al. (1996), which showed the extract of chillito have antifungal activity against different dermatophytes andyeast pathogens.

4. Conclusions

On the basis of the current work, the following conclusions canbe drawn:

B Both chilli and extract of chilli oleoresins showed antifungalactivity against two selected sapstain fungi.

B Enhanced antifungal activity was observed in an integratedapproach, whereby chilli juice was combined with L. casei.

B With respect to applying chilli or extract of chilli as a benignapproach to sapstain control, it is necessary to analyse andcharacterise chilli and/or oleoresins. A study is currentlyunderway to determine the most appropriate fraction re-garding antifungal activity.

B In order to understand the mechanism involved in the synergybetween chilli and L. casei, a detailed understanding of thebioactive secondary metabolites of L. casei in combination withchilli is warranted.

Acknowledgments

The authors acknowledge Dr. Damiano Vesentini for helpfuldiscussion and assistance in sourcing the oleoresins. Also, theauthors wish to thank Mr. Alan Mackie for providing chilli juice.

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