The diversity of yeasts in wet processing of coffee, their role in mucilage degradation and interaction with ochratoxin A (OTA) producing Aspergillus ochraceus

Abstract

It was the main aim of this thesis to identify the yeast species predominant along the coffee processing chain from picking of cherries to fermentation and drying and investigate their role in coffee fermentation. For this purpose, coffee samples were collected from two coffee processing sites in Tanzania. Yeasts were identified by the classical conventional methods and by the culture independent technique Denaturating Gradient Gel Electrophoresis (DGGE). Furthermore, the ability of the yeasts to degrade pectin was investigated and the antagonist activity of the predominant yeasts during coffee fermentation against ochratoxin A (OTA) producing Aspergillus ochraceus were studied. The yeasts in coffee samples were determined by classical methods of cultivation and isolation. It was observed that the yeasts counts were in the range of 4.0 x 104 to 5.0 x 107 cfu / g along the coffee processing chain with an increase during fermentation. Further, the isolated yeasts were identified by genotyping using ITS-PCR and sequence analysis of the D1/D2 domain of the 26S rRNA gene. In addition, DGGE of PCR-amplified 26S rRNA gene was performed to detect yeasts directly from coffee samples without cultivation. Three predominant yeasts were detected by the classical methods of isolation and cultivation and by the DGGE technique. They include Pichia kluyveri, Pichia anomala and Hanseniaspora uvarum. The predominant yeast species during fermentation and drying was P. kluyveri. Pichia anomala was observed in high numbers at the beginning of fermentation and during drying of coffee beans. Hanseniaspora uvarum dominated during fermentation but decreased in numbers during drying. Kluyveromyces marxianus, Candida pseudointermedia, Issatchenkia orientalis, Pichia ohmeri and Torulaspora delbruekii, which were found in low numbers of 103 cfu / g or below during the different stages of processing were not detected by the DGGE method. On the other hand, Saccharomyces cerevisiae and Candida xestobii were only observed in the DGGE profile for a sample of drying beans i.e. they were not detected by cultivation. With regard to the predominant yeasts in coffee samples, a good agreement was observed between classical methods of isolation and cultivation and the DGGE profiles of the same coffee samples. Summary iii All strains of P. anomala, P. kluyveri and H. uvarum were found to have pectinolytic activity with P. anomala S16 and P. kluyveri S13Y4 showing the strongest activity. Enzymatic assays showed that the three yeast species were observed to secrete polygalacturonase (PG) but not pectin esterase (PE) or pectin lyase (PL). Pichia anomala S16 and P. kluyveri S13Y4 were found to produce higher amounts of PG when grown in coffee broth (CB) than in the yeast polygalacturonic acid (YPA) substrate. The maximum amounts of PG produced by P. anomala S16 and P. kluyveri S13Y4 were obtained at pH 6.0 and an incubation temperature of 30 oC, conditions close to coffee fermentation. For PG secreted by P. anomala S16, the optimum pH and temperature for the enzymatic activity were 5.5 and 40 oC. For PG produced by P. kluyveri S13Y4 the strongest activity was observed at pH 5.0 and 50 oC. To solve the problem of OTA contamination in coffee, the antifungal activity of P. anomala, P. kluyveri and H. uvarum against A. ochraceus was investigated. The three yeasts were observed to inhibit growth of A. ochraceus when co-cultured on malt yeast extract (MEA) and coffee agar (CA) with a significant higher inhibition on MEA than on CA. In addition, P. anomala and P. kluyveri were found to have a stronger effect on growth of A. ochraceus than H. uvarum. The three yeasts prevented spore germination of A. ochraceus in yeast glucose peptone (MYGP) broth. In yeast-free supernatant of MYGP broth after incubation periods of 24 and 48 h, spores of A. ochraceus did not germinate; after 72 h very short germ tubes were observed but further development of the germ tubes was inhibited. Furthermore, P. anomala, P. kluyveri and H. uvarum were able to prevent production of OTA by A. ochraceus when cocultured on MEA. On CA medium, P. anomala and P. kluyveri prevented A. ochraceus from producing OTA. However, OTA formation on CA was not affected by the presence of H. uvarum. For the understanding of the antifungal properties observed, the effects of volatile compounds produced by P. anomala, P. kluyveri and H. uvarum on growth of A. ochraceus and production of ochratoxin A (OTA) were studied. The main volatile compounds produced by the three yeasts were analyzed by gas chromatography-mass spectrometry (GC-MS). Exposure of A. ochraceus to the gaseous phase of malt yeast glucose peptone (MYGP) plates inoculated with P. anomala, P. kluyveri and H. uvarum inhibited fungal growth with the two Pichia spp. Summary iv showing the strongest effect. The main esters and alcohols produced by the three yeasts were ethyl acetate, isobutyl acetate, 2-phenyl ethyl acetate, ethyl propionate and isoamyl alcohol. The individual esters and alcohols were found to affect fungal growth. The most effective compound in inhibiting fungal growth was 2-phenyl ethyl acetate. Exposure of A. ochraceus to MYGP plates inoculated with P. anomala, P. kluyveri and H. uvarum prevented production of OTA. On CA plates, only the headspace of P. anomala and P. kluyveri prevented OTA production. A comparison of the effects of different headspace concentrations of the individual volatiles on OTA formation by A. ochraceus showed that 2-phenyl ethyl acetate was also the most effective compound in reduction of OTA. In conclusion, P. anomala and P. kluyveri were the predominant yeasts during coffee fermentation and drying and they are for the first time shown to have a role in degradation of mucilage surrounding the coffee beans. Furthermore, the antifungal effect of P. anomala and P. kluyveri against growth and OTA formation by A. ochraceus demonstrate the possibility of using the two yeasts as starter cultures in coffee fermentation. However, further in vivo studies i.e. use of yeasts as starter cultures should be carried out to confirm the inhibition of fungal growth and OTA formation. In vivo starter cultures should also be conducted to disclose any undesired effect on the quality of coffee beans in terms of colour and aroma.