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Food Microbiology 25 (2008) 771– 777

Contents lists available at ScienceDirect

Food Microbiology

0740-00

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.com/locate/fm

Detection and identification of wild yeasts in Champus, a fermentedColombian maize beverage

Esteban Osorio-Cadavid a, Clemencia Chaves-Lopez b, Rosanna Tofalo b, Antonello Paparella b,Giovanna Suzzi b,�

a Departamento de Biologıa, Universidad del Valle, Calle 13 No. 100-00, Cali, Colombiab Dipartimento di Scienze degli Alimenti, Universita degli Studi di Teramo, Via C. Lerici, 1, 64023 Mosciano Stazione (TE), Italy

a r t i c l e i n f o

Article history:

Received 16 January 2008

Received in revised form

29 April 2008

Accepted 30 April 2008Available online 7 May 2008

Keywords:

Yeasts

Champus

5.8S-ITS region

D1/D2 domain

Aromatic compounds

20/$ - see front matter & 2008 Elsevier Ltd. A

016/j.fm.2008.04.014

esponding author. Tel.: +39 0861 266938; fax

ail address: gsuzzi@unite.it (G. Suzzi).

a b s t r a c t

The aim of this study was to identify and characterise the predominant yeasts in Champus, a traditional

Colombian cereal-based beverage with a low alcoholic content.

Samples of Champus from 20 production sites in the Cauca Valley region were analysed. A total of

235 yeast isolates were identified by conventional microbiological analyses and by polymerase chain

reaction-restriction fragment length polymorphism (PCR-RFLP) of ITS1-5.8S rDNA-ITS2. The dominant

species were: Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia fermentans, Pichia kluyveri var.

kluyveri, Zygosaccharomyces fermentati, Torulospora delbruekii, Galactomyces geotrichum and Hansenias-

pora spp. Model Champus systems were inoculated with single strains of some isolated sporogenus

species and the aromatic profiles were analysed by SPME. Analysis of data showed that Champus strains

produced high amounts of esters. The aromatic compounds produced by Saccharomyces and non-

Saccharomyces yeasts from Champus can exert a relevant influence on the sensory characteristics of the

fermented beverage. The Champus strains could thus represent an important source for new yeast

biotypes with potential industrial applications.

& 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Many indigenous cereal-based fermented foods are prepared indifferent parts of the world, such as Pito (Steinkraus, 1977), Sekete(Adegoke et al., 1995), Kwass (Pederson, 1979), Pozol (Wacher etal., 2000), Boza (Gotcheva et al., 2000) and Champus, amongothers. These beverages possess peculiar nutritional and sensoryproperties, derived from the fermentation of specific rawmaterials (Campbell-Platt, 1994). The preparation of manyindigenous or traditional fermented beverages is still a traditionalart in homes, villages and small-scale industries. Generally,natural cereal-based fermentations are carried out by yeasts,lactic acid bacteria and fungi, sometimes forming a complexmicrobiota acting in cooperation (Blandino et al., 2003).

Champus is a popular cereal-based low alcoholic beverage witha sweet, sour taste, widely consumed in rural and urban areas ofColombia and in some other South American countries, includingEcuador and Peru. Different cereals such as wheat, rye and maizeor their combinations can be used for Champus production,together with many other ingredients, for example pineapple or

ll rights reserved.

: +39 0861 266915.

‘‘lulo’’ (Solanum quitoense Lam.), ‘‘panela’’ (sugar cane) syrup,clove, cinnamon and orange tree leaves. These ingredients canvary according to different traditional recipes, in parti-cular cinnamon and cloves. The popularity of this drink is dueto its pleasant taste and flavour, but also due to its nutritionalproperties.

Colombian Champus is produced by boiling corn kernels formany hours in order to soften them; then, after cooling the grainsat room temperature, fruits are added together with ‘‘panela’’syrup (4–6%) and the other ingredients. The beverage is stored atabout 12–15 1C and consumed within 24–48 h. During this time,fermentation occurs and a low alcoholic beverage with 2.5–4.2%of alcohol content and pH values between 3.5 and 4.0, dependingon the quantities of fruit added, is obtained. Fermentation can alsobe enhanced by increasing temperature, obtaining a alcoholicbeverage called Mazato.

The majority of the microorganisms associated with cornkernels are destroyed by heat treatment during processing, whilefruits act as an inoculum of yeasts and lactic acid bacteria, whichferment Champus during its storage. Yeasts mainly degradecarbohydrates, while bacteria possess a proteolytic activity(Chavan and Kadam 1989). Species of lactic acid bacteria (LAB)and yeasts have also been found in the fermentation of KenyanBusa, Kaffir beer, Nigerian Ogi, Pito, Sekete (Adegoke et al., 1995;

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E. Osorio-Cadavid et al. / Food Microbiology 25 (2008) 771–777772

Beuchat, 1995; Pederson, 1979; Sanni, 1992; Steinkraus, 1977), andin Bulgarian Boza (Gotcheva et al., 2000). Studies of the yeastsinvolved during Champus production have never been performedbefore. Thus, the purpose of the present study was to determineand characterise the yeast populations during Champus fermen-tation, in samples collected from different areas of South WestColombia.

2. Materials and methods

2.1. Samples

Twenty samples of Champus, purchased from differentproducers of Cauca Valley Region (Colombia), were asepticallycollected in sterile bottles, maintained at 4–6 1C and analysed.Fig. 1 shows the traditional flow-chart for Champus manufacturing.

2.2. Yeast enumeration and isolation

Yeast counts were carried out in duplicate using a serialdilution method. Aliquots of 0.1 ml of Champus diluted sampleswere plated onto YEPD (1% yeast extract, 2% peptone and 2%dextrose) medium with chloramphenicol (50 mg l�1) and incu-bated at 28 1C for 2 days. Either 50% of the colonies were selectedor, if the plate contained less than 10 colonies, all of them wereselected, according to Harrigan and Lachance (1976). A randomnumber of each colony type was recovered. Isolates were purifiedby streak plating and subcultured onto YEPD medium forsubsequent identification. The purified isolates were maintainedat �80 1C in YEPD broth containing 20% (v/v) glycerol.

Addition of sugar cane molasses,clove, cinnamon and orange tree

Corn

Cooling at 12-15°C

Boiling for 2 hours

Cooling at 25-35°C

Addition of pulp fruit

Final product

Fig. 1. Flow-chart for Champus manufacturing.

2.3. Identification and phenotypic characterisation of yeasts

Isolates were submitted to conventional yeast identificationfollowing the taxonomic criteria described by Barnett et al.(2000). Yeast presumptive identification was performed by theircell morphology, mode of vegetative reproduction and physiolo-gical characterisation.

The ability to ferment glucose, maltose, sucrose, galactose andmelibiose as carbon sources was evaluated in Durham tubes,containing YEP with 2% of the appropriate sugars, at 25 1C for 7days. The capacity to grow at 37 and 40 1C was assessed after 2and 7 days in YEPD tubes, incubated in a thermostatic bath. The b-glucosidase activity was determined by replica plating the yeastonto arbutin agar (Sigma-Aldrich, Italy) after 3 and 7 days;positive colonies developed a dark brown colour. Pectinase,protease, cellulose, chitinase and starch degrading activities wereperformed according to Strauss et al. (2001).

2.4. Molecular yeast identification

Yeast cells were grown aerobically in YEPD at 28 1C. Totalgenomic DNA was extracted and purified from 7 ml cultures asdescribed by Querol et al. (1992). Identification of the isolates wasperformed by PCR-RFLP of ITS1-5.8S rDNA-ITS2 region asdescribed by Esteve-Zarzoso et al. (1999). For amplification ofthe ITS1-5.8S rDNA-ITS2 region, the primers used were ITS1 (50-TCC GTA GGT GAA CCT GCG G-30) and ITS4 (50-TCC TCC GCT TATTGA TAT GC-30). The PCR products were digested with 1U of HaeIII,HinfI and CfoI endonucleases (Roche Diagnostics, Mannheim,Germany) at 37 1C for 2 h.

The reactions were performed in an automatic thermal cycler(GeneAmpsPCR System 9700, Perkin-Elmer, Norwalk, CT, USA)under the following conditions: initial denaturation at 94 1C for5 min, 30 cycles of denaturation at 94 1C for 30 s, annealing at55 1C for 30 s, extension at 72 1C for 1 min, final extension at 72 1Cfor 10 min, holding at 4 1C. PCR products and their restrictionfragments were analysed on 1.5% and 2% agarose gel, respectively,in 1�TBE (89 mM Tris-borate, 2 mM EDTA pH 8) buffer. Gels werestained with ethidium bromide. Fragment lengths were estimatedby comparing them with a 1-kb plus DNA ladder (Invitrogen,Carlsbad, CA, USA) as size marker.

2.5. Sequencing of the D1/D2 domain of the large subunit (26S)

ribosomal DNA

Sequencing of the D1/D2 domain of the large subunit (26S)ribosomal DNA was performed for the major groups of theisolates. The analysis was performed according to Kurtzman andRobnett (1998). NL-1 (50-GCATATCAATAAGCGGAGGAAAAG-30)and NL-4 (50-GGTCCGTGTTTCAAGACGG-30) primers were usedfor the amplification of the D1/D2 domain. The reactions wereperformed in an automatic thermal cycler (GeneAmpsPCR System9700, Perkin-Elmer, Norwalk, CT, USA) under the followingconditions: initial denaturation at 94 1C for 5 min, 30 cycles ofdenaturation at 94 1C for 1 min, annealing at 55 1C for 30 s,extension at 72 1C for 1 min, final extension at 72 1C for 10 min,holding at 4 1C. Both D1/D2 domain and ITS1-5.8S rDNA-ITS2region were sequenced after PCR amplification. PCR products werepurified by the GFXTM PCR DNA and Gel Band Purification Kit(Amersham Biosciences AB, Uppsala, Sweden), following themanufacturer’s instructions, and delivered to C.R.I.B.I. (PaduaUniversity, Italy) for sequencing. The sequences were assembledand compared with the sequences reported in the GenBank usingthe basic local alignment search tool (BLAST) algorithm.

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E. Osorio-Cadavid et al. / Food Microbiology 25 (2008) 771–777 773

2.6. Frequency percentage analysis

Colonies randomly collected from the plates at the highestdilution give a high probability to pick up strains belonging to thedominant species (Pulvirenti et al., 2004). In order to studyspecies distribution in our samples, the method proposed bySolieri et al. (2006) has been performed. This method is based onthe evaluation of the number of times each species was detectedin the samples, without considering the strain number belongingto the species. In this way, it was possible to obtain the number ofpositive samples for each species and the correspondingfrequency, defined as the number of positive samples of a speciesdivided by the total number of samples expressed in percentage.

2.7. Volatile compounds production

In order to characterise the aromatic role of some isolatesinvolved in the Champus fermentation, strains of Saccharomyces

cerevisiae (9), Pichia fermentans (6) and Pichia kluyveri (8) wereinoculated in Champus model systems (CMS) containing 5% ofpanela syrup, 3% boiled corn, 3% of filtered lulo and pineapplejuice, in sterilised water. The model Champus systems wereinoculated with a final concentration of 106 cells ml�1 of 48 h pre-cultures grown in YEPD. Alcoholic fermentations were conductedin 250 ml Erlenmeyer-flasks at 25 1C for 48 h. Three fermentationswere carried out with each strain, and duplicates of each samplewere injected.

2.8. Solid phase microextraction– gas chromatography (SPME– GC)

analysis of volatile compounds (VOCs)

VOCs were analysed using gas-chromatography–mass spectro-metry coupled with a solid phase microextraction (GC–MS–SPME)technique. The samples were then equilibrated for 40 min at 45 1Cand volatiles were adsorbed on a fused silica fibre covered by75 mm carboxen/polydimethylsiloxane (CAR/PDMS), (Supelco,Steiheim, Germany). Adsorbed molecules were desorbed in thegas-chromatograph for 8 min. For peak detection, an Agilent

Table 1Some phenotypical characteristics of the yeasts strains isolated from Champus

Isolates Group Number of isolates Presence of

pseudomycelium

Hydrolysis of

Arbutin

Re

10

1 16 6a 1

2 15 1 1 5

3 12 3 1

4 13 2

5 14 3 5 6

6 11 4

7 15 4

8 12 2 4

9 13 1 4 5

10 11 1 3

11 10 2 3 4

12 9 2 4

13 11 2 1 1

14 10 3 1

15 12 1 1

16 10 3 1 2

17 9 1 2 2

18 12 4 2

19 10 2 2

20 9 3 2 5

Total 235 42 20 56

a positive strains.

Hewlett–Packard 6890 GC, equipped with a MS detector 5970MSD (Hewlett–Packard, Geneva, Switzerland), and a 50 m�0.32i.d. fused silica capillary column, coated with a 1.2mm poly-ethylenglycol film (Chrompack CP-Wax 52 CB, Middelburg, TheNetherlands) as stationary phase, were used. The conditions wereas follows: injection temperature, 250 1C; detector temperature,220 1C; carrier gas (He) flow rate, 1 ml min�1; splitting ratio, 1:20(v/v). The oven temperature was programmed as follows: 50 1C for2 min; from 50 to 65 1C, at 1 1C min�1; from 65 to 220 1C, at5 1C min�1, then holding for 22 min. Volatile peak identificationwas carried out by computer matching of mass spectral data withthose of the compounds contained in the Agilent Hewlett–PackardNIST 98 and Wiley vers. 6 mass spectral database.

The VOCs, expressed as percentages, were corrected consider-ing the pattern obtained in non-inoculated controls.

2.9. Statistical analysis

One-way analysis of variance and least significant difference(LSD) were used to statistically interpret mean differences inmean values, if any, at 95% and 99% accuracy level.

3. Results

Plate counts from 20 samples of Colombian Champus showed ayeast load of 7.271.2 log CFU ml�1. Table 1 shows the maincharacteristics of the isolate groups obtained from these samples.Among the isolates, 62 out of 235 were grown at 40 1C, 95 isolateswere able to grow in high osmotic conditions (50% glucose), 56were tolerant to 10% of NaCl and 23 were tolerant to 500 ppm ofcycloheximide. With regard to enzymatic activity, all strains werenot able to produce extracellular enzymes to degrade pectin,cellulose, chitin and starch, but 20 isolates were able to hydrolysearbutin.

Based on phenotypic characterisation, the isolates weregrouped into 13 groups by means of cluster analysis in order toassist their identification (data not shown).

sistance to Growth at 40 1C

% NaCl 500 ppm

cycloeximide

50% glucose

2 3

1 4 1

1 4

4 1

7 1

2 4 3

5 8

1 6 7

5 5

4 4

1 5 2

4 6

1 5 4

4 4

4 6 4

6 5 3

2 7

2 6 1

3 5

6 1

23 95 62

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Table 3Number of positive samples for each yeasts species

Species Positive samples Frequency (%)a

S. cerevisiae 13 65

Issatchenkia orientalis 14 70

Pichia fermentans 13 65

Pichia kluyveri var. kluyveri 3 30

Zygosaccharomyces fermentati 1 5

Torulospora delbruekii 6 30

Galactomyces geotritichum 9 45

Hanseniaspora spp. 5 25

a Referred to the total number of samples.

E. Osorio-Cadavid et al. / Food Microbiology 25 (2008) 771–777774

3.1. Yeast identification by molecular genetic methods

Species identification of 65 yeast, randomly chosen among the 13phenotypes, was performed by PCR-RFLP of ITS1-5.8S rDNA-ITS2.The PCR products digested with HaeIII, HinfI and CfoI endonucleaseswere analysed and their profiles, obtained by agarose gel electro-phoresis, were converted using Fingerprinting II software, to thesizes of the fragments generated. Eight different profiles wereobtained. Table 2 contains the lengths of the 5.8S-ITS regionamplified by PCR and the fragments obtained after digestion withthree restriction endonucleases. Six of eight groups were identifiedafter comparing the molecular mass of restriction products withthose previously described (Esteve-Zarzoso et al., 1999; Villa-Carvajal et al., 2006; de Llanos Frutos et al., 2004). Those six groupswere S. cerevisiae, Issatchenkia orientalis, P. fermentans, Zygosacchar-

omyces fermentati, Torulospora delbruekii and Hanseniaspora spp. Forthe most important yeast species, identification was furtherconfirmed by sequencing of the D1/D2 domain of the large subunit(26S) rDNA. Within each of these species, a representative isolatewas analysed. For all sequenced isolates, homologies from 99%to 100% were obtained with sequences in GenBank. The identifica-tion of groups I, II, III, V and VI was confirmed by comparison withthe sequences obtained from GenBank. The isolates in group IV wereidentified as P. fermentans/P. kluyveri var. kluyveri (homology of 99%)by comparison with the sequences obtainable from GeneBank. Thesetwo species, which have been reported to have considerablesimilarities, were differentiated by comparison of their restrictionfragments in this study (Table 2). The isolates clustered in group VIIwere identified by sequencing of D1/D2 domain as Galactomyces

geotrichum. To confirm the restriction profiles obtained, thesequences of ITS1-5.8S rDNA-ITS2 region were analysed withWebcutter 2.0 programme.

3.2. Frequency percentage analysis

Table 3 shows the frequency of positive samples of thedifferent species. I. orientalis, S. cerevisiae, P. fermentans were themost common species, being isolated in 70% and 60% samples,respectively, followed by G. geotrichum (45%), P. kluyveri var.kluyveri, T. delbruekii (30%) and Hanseniaspora spp. (15%). Thespecies Z. fermentati was found in one sample only.

3.3. VOC profiles of Champus-like beverages

To determine the VOC produced by the different yeast speciesduring fermentation, CMS were prepared with cereals and fruits,as described in Section 2, and inoculated with the single strains toperform fermentation. In general, the VOCs identified in themodel systems were characterised by a high proportion of furanicaldehydes as furfural, 5-methyl furfural, 2-furanaldehyde and

Table 2Lenghts of the PCR products and the restriction fragments obtained for the yeasts isola

Number of isolates Species Amplifie

I 12 S. cerevisiae 880

II 11 Issatchenkia orientalis 480

III 13 Pichia fermentans 450

IV 8 Pichia kluyveri var. kluyveri 450

V 1 Zygosaccharomyces fermentati 700

VI 11 Torulospora delbruekii 800

VII 9 Galactomyces geotritichum 400

VIII 3 Hanseniaspora spp. 750

hydroxymethyl furfural, which accounted for approximately 38%of the total aromatic compounds, followed by higher alcohols(12%), particularly isoamyl alcohol, 2-phenylethanol and 2,3-butandiol, and esters (19%) (Table 4).

In total, 27 VOCs in the inoculated CMS were identified bySPME/GC–MS and grouped according to chemical classes (Table 5).They included 6 alcohols, 14 esters, 3 acids and 3 aldehydes.

The aromatic profile of P. fermentans strains resulted to bemore complex with respect to the other yeast species (Table 5).Overall, 1-pentanol, butyl caprylate, phenyl acetic acid, isoamylcaprylate, diethyl succinate, ethyl nonanoate and differentiatedthe three species. In particular, P. kluyveri was characterised by theproduction of butyl caprylate, P. fermentans by 1-pentanol, phenylacetic acid and ethyl nonanoate. S. cerevisiae was insteadcharacterised by diethyl succinate and isoamyl caprylate produc-tion. Among the alcohols, 2-phenyl ethanol was the mostabundant, followed by isoamyl alcohol. Moreover, a large numberof the isolates were able to produce furfuril alcohol.

Table 6 shows the peak areas of the VOCs detected. In the headspace of the samples inoculated with P. fermentans strains highquantities of isoamyl acetate and ethyl caprylate were detected,whereas in those inoculated with S. cerevisiae strains significantquantities of ethyl caproate and amyl acetate were observed. Onthe other hand, the P. kluyveri strains produced lower concentra-tions of esters with respect to the other two species.

Aldehydes and acids were detected in small quantities in allthe species. In the samples inoculated with S. cerevisiae and P.

fermentans strains furfural, 5-methyl furfural and benzaldhydewere detected, while in those inoculated with P. kluyveri var.kluyveri strains only benzaldehyde was detected. The highestacetic acid producer was P. fermentans.

4. Discussion

Traditional fermented beverages prepared with the mostcommon varieties of cereals (such as rice, wheat, corn or

tes

d product Restriction fragments

Cfo I Hae III Hinf I

365+365 320+180+150 365+155

195+60 400 240+140

170+100 350+80 260+200

175+115 370+90 250+200

310+280+90 300+210+95 340+340

330+220+150+100 800 410+380

400 400 270+100

320+310+105 750 350+200+180

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Table 4Relative area (%) and peak area of volatile compounds detected in Champus model

system

Compounds Relative area (%) Peak area

Alcohols

Ethanol 0.0370.01a 3221b

Isoamylalcohol 2.7770.12 348595

Heptanol 0.2070.02 24800

2-Phenyl ethanol 3.3170.18 417699

2,3-butanediol 5.7770.15 727231

Esters2-phenylethylacetate 10.1570.56 1279283Ethyl acetate 2.4370.23 306266Ethyl butyrate 0.3570.12 44360

2-Butanoic acid methyl ester 1.2470.18 156446

Phenyl acetate 0.4870.15 60752

Pentanoic acid methyl ester 0.3870.10 47443

Capryl acetate 0.1770.02 21742

Ethyl octanoate 2.2370.15 281001

Ethyl decanoate 1.4470.32 181210

Aldehydes

1-Hexenal 1.2670.32 158271

Furfural 6.8170.27 8576895-Methyl Furfural 1.5270.02 1914492-Furanaldehyde 19.2070.45 2419216

Hydroxymethyl Furfural 9.3270.28 1174102

Others

Dimethyl sulfide 5.5570.12 699849

P-ethylphenol 6.4170.21 807543

p-ethylguaiacol 5.8370.25 734349

Acetic acid 6.3970.56 804812

n.ic 2.0470.15 264061

a Mean and standard deviation of three repetitions.b Mean of three repetitions, the coefficients of variability, ranged between 5%

and 10%.c n.i non-identified peaks.

Table 5Volatile compounds produced by P. kluyveri var. kluyveri, P. fermentans and S.

cerevisiae in Champus model system

Compounds P. kluvyeri

var. kluyveri

(8 strains)

P. fermentans

(6 strains)

S. cerevisiae

(9 strains)

Alcohols

Ethanol 11.8671.24 23.60713.51 48.17714.23

Isoamyl alcohol 0.2070.03 0.7570.36 4.4974.79

1-pentanol n.r 0.5570.28 n.r

Furfuryl alcohol 0.1070.05 0.2170.12 0.9370.65

2-phenyl ethanol 1.3570.28 3.7470.55 9.5974.81

1,3-Butanediol 0.2770.39 0.3170.55 n.r

Total 13.78 29.15 63.18

Esters

Ethyl acetate 11.4670.93 32.60716.80 n.r

Isoamyl acetate 21.77714.06 11.9975.70 n.r

Amyl acetate 0.7370.32 0.0270.01 3.1170.02

Hexyl acetate n.r 0.2070.08 n.r

Diethyl succinate n.r n.r 0.0370.03

2-phenylethyl

acetate

32.87711.01 18.4573.18 6.9973.74

Butyl caprylate 0.0170.02 n.r n.r

Ethyl caproate 17.7173.28 0.8070.44 14.1078.35

Ethyl capryate 0.2870.10 1.6270.94 4.1673.64

Ethyl nonanoate n.r 0.0870.02 n.r

Ethyl caprate n.r 0.4570.24 2.3470.23

Isoamyl caprylate n.r n.r 0.1170.11

Total 84.83 65.61 30.84

Acids

Acetic acid 0.7970.21 0.6970.18 1.4272.01

Phenylacetic acid n.r 0.3870.54

Caprylic acid n.r 3.7472.29 1.5170.61

Total 0.79 4.81 2.93

Aldehydes

5-methyl furfural n.r 0.4970.69 1.4872.09

Benzaldehyde 0.3970.53 0.2670.19 0.4270.27

Furfural n.r 0.2870.11 0.1770.32

Total 0.38 1.03 1.90

The results are means and standard deviations of two repetitions in three different

experiments.

n.r: non rilevable.

E. Osorio-Cadavid et al. / Food Microbiology 25 (2008) 771–777 775

sorghum) are well known in many parts of the world. Althoughthe microbiota of these products is quite complex and not wellknown, yeasts have been reported to be involved in severaldifferent types of indigenous fermented foods and beverages(Zulu et al., 1997; Torner et al., 1992; Gadaga et al., 2001).

Seven yeasts genera were isolated from Colombian Champussamples. The analysis of 35 samples showed that P. fermentans,

S. cerevisiae and I. orientalis were the dominant species. Thediversity of yeasts from Champus may be related to the rawmaterial used. The dominant yeast species were G. geotrichum

strictly oxidative, followed by the fermentative yeasts P. fermen-

tans, S. cerevisiae, T. delbruekii and I. orientalis.

The species isolated from Champus in this work have beenreported in other fermented cereal-based or starchy foods. In fact,S. cerevisiae has been reported to be the commonest yeast inindigenous fermented foods and beverages, where it has beenshown to be very important, especially in the fermentation ofcereals and alcoholic beverages (Jespersen, 2003). It also plays aleading role in the fermentation of maize dough that forms thebasis for a variety of different foods in Africa and South America,contributing to a large proportion of the daily intake (Romano etal., 2006). P. fermentans has been frequently isolated fromfermented and non-fermented beverages (Arias et al., 2002),whereas T. delbruekii and I. orientalis were isolated during coffeefermentation (Masoud et al., 2004). The latter species has alsobeen isolated from Boza, a Bulgarian cereal-based fermentedbeverage (Botes et al., 2007). T. delbrueckii, an osmotolerantspecies (Kreger-van Rij, 1984; Barnett et al., 2000), has beenisolated from Pito, a traditional African guinea corn beverage

(Sefa-Dedeh et al., 1999), and it is commonly associated withalcoholic fermentation of wine and champagne production (Heardand Fleet, 1993). G. geotrichum, responsible for the degradation offruit and vegetable juices, has been associated with spontaneousfermentations during the production of sour cassava starch inBrazil (Lacerda et al., 2005), and Pozol (Wacher et al., 2000),whereas P. kluyveri has never been reported in cereal-basedfermented beverages.

The sensory properties of traditional fermented beverages arethe result of the combined metabolic activity of single strains ormicrobial groups, together with the process characteristics. Infermented cereals, yeasts give a useful contribution to theimprovement of flavour and to product acceptability (Banigo etal., 1974; Odunfa and Adeyele, 1985). In the case of ColombianChampus, sugar cane concentration, types of fruit, temperature,use of spices and orange tree leaves have a relevant role in thedevelopment of the typical sensory profile. The yeast responsiblefor the Champus production can be distinguished into twogroups: yeasts with a high fermentative activity and low VOCproduction and yeasts with a low fermentative activity and astrong capability to form VOC such as esters.

In general, non-Saccharomyces strains from Champus and inparticular P. fermentans produced high levels of esters, especiallyethyl acetate, ethyl caprylate and 2-phenyl-ethyl acetate, whichare mostly responsible for the flowery and fruity aroma. During

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Table 6Volatile compounds (expressed as peak area) detected in Champus model

inoculated with P. kluyveri var. kluyveri, P. fermentans and S. cerevisiae

Compounds P. kluvyeri

var. kluyveri

(8 strains)

P. fermentans

(6 strains)

S. cerevisiae

(9 strains)

Alcohols

Ethanol 425651 1266221 5893040

Isoamyl alcohol 55829 606017 706099

1-pentanol n.r 322508 n.r

Furfuryl alcohol 13758 11885 193294

2-phenyl ethanol 372986 2789203 1731493

1,3-Butanediol 38914 96769 n.r

Total

Esters

Ethyl acetate 686992 9419065 n.r

Isoamyl acetate 3000612 5879291 n.r

Amyl acetate 100315 7967 378049

Hexyl acetate n.r 118297 n.r

Diethyl succinate n.r n.r 10263

2-phenyl ethyl

acetate

1040795 9658392 1597764

Butyl octanoate 1804 n.r n.r

Ethyl caproate 2441764 393320 2618409

Ethyl caprylate 4009 851958 773452

Ethyl nonanoate n.r 34376 n.r

Ethyl caprate n.r 537510 975726

Isoamyl caprylate n.r n.r 23647

Acids

Acetic acid 18951 820105 422156

Phenylacetic acid n.r 286181 n.r

Caprylic acid n.r n.r 449497

Aldehydes

5-methyl furfural n.r 288055 438719

Benzaldehyde 52232 112472 152231

Furfural n.r 22698 35431

The results are means and standard deviations of two repetitions in three different

experiments. The coefficients of variability, ranged between 5% and 10%.

n.r: non rilevable.

E. Osorio-Cadavid et al. / Food Microbiology 25 (2008) 771–777776

fermentation, the formation of esters is carried out by intracel-lular enzyme-catalysed reactions, and is largely dependent on thetype of carbon source and the variety of assimilable nitrogen usedin the medium, but also on the fermentation parameters anddissolved oxygen, as well as on the genus, species and strainsemployed (Verstrepen et al., 2003). The species belonging to thegenus Pichia, such as P. kluyveri, P. quercuum and P. heedii, havebeen previously described as having a strong double couplingactivity of acetyl-CoA formation and alcohol esterification,possibly due to alcohol acetil trasferase (AATase) activity (Oda etal., 1999; Oda and Otha, 1997).

In the non-fermented CMS, a high proportion of furanicaldehydes was determined. Furfural, a furanic aldehyde, has beenreported to have an inhibitory effect on the specific growth rate, aswell as on the fermentation rate of yeasts (Banerjee et al., 1981,Palmqvist and Hahn-Hagerdal, 1999), depending on its concentra-tion (Horvath et al., 2003). As furfural was not detected in all thefermented MCM, we can suggest that all the strains of P. kluyyveri,the three strains of P. fermentans and the two strains of S.

cerevisiae were able to convert furfural into furfuryl alcohol. Thelatter, it may be derived by a yeast-mediated reduction of furfurylaldehydes which were formed during thermal treatment. Thisreduction is vital for overcoming the toxic effects of compounds,such as furfural (Taherzadeh et al., 2000). These results suggest apossible in situ detoxification of inhibitors in the Champus.

On the other hand, in the present study, 4 strains of P.

fermentans, 6 of P. kluyvery and 2 of S. cerevisiae were positive tothe splitting of arbutin. The extracellular b-glucosidase activity in

Saccharomyces and non-Saccharomyces strains had been reportedin several studies (Fia et al., 2005; Manzanares et al., 2000; Riccioet al., 1999; Rosi et al., 1994). It is well known that b-glucosidasesare the key enzymes in the enzymatic release of aromaticcompounds from glycosidic precursors present in fruit juices,musts and wines (Palmeri and Spagna, 2007). In particular, b-glucosidase activity from yeasts has been associated with anincrease of linalool, benzyl alcohol and 2-phenylethanol levels inthe juices of several fruits, such as peach, cherry, strawberry,passion fruit, orange, apple and papaya (Gueguen et al., 1996). Ourresults showed that the yeast species and strains isolated fromColombian Champus, with their particular metabolic profiles,possessed a great biodiversity in VOC production, representing animportant source for new yeast biotypes with potential industrialapplications.

5. Conclusions

Valuable information regarding yeasts’ indigenous microflora,isolated during spontaneous Champus fermentation, was ob-tained. P. kluyveri var. kluyveri has not been previously reported infermented cereal-based beverages. In this study, many yeastisolates produced different quantities of esters, which areassociated with a pleasant aroma. Considering that it is importantbut difficult to evaluate the exact contribution of the yeasts to thechemical composition of the Champus, further investigationsmight clarify the specific contribution of the single species andthe mechanisms involved in Champus production. In any case, thedata obtained in this study might be useful for the selection ofyeasts with desirable characteristics for Champus fermentation.

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