Characterisation and identification of the active microbial consortium present in Kepi grains

Schoeman, Tersia (2001-12)

Thesis (MSc)--University of Stellenbosch, 2001.

Thesis

ENGLISH ABSTRACT: Kepi is an acidic, self-carbonated milk beverage that is produced by fermenting milk with grain-like structures that contain naturally occurring microbes, including lactic acid bacteria (LAB) and yeasts. The specific microbes present in the Kepi grains are responsible for an acidic-alcoholic fermentation of the milk and also contributes to the various health properties exhibited by Kepi. The combination of microbes in the Kepi grains can vary considerably depending on which type of milk is fermented, the method by which Kepi is produced, the origin of the grains and how the grains are stored. In this study, the impact of various environmental conditions including the different stages during Kepi production, grain origin, Iyophilisation and packaging in three different packaging materials, on the microbial community of Kepi grains were studied using selective growth media, morphology and biochemical characteristics. It was found that there was a general decrease in the microbial counts from laboratory produced Kepi grains, the longer Kepi was produced on a continuous basis. This decrease in microbial counts was also observed during the different stages of Kepi production. The average LAB counts obtained from laboratory produced grains decreased from 1.1 x 108 cfu.q" after 3 d of activation to 6.3 x 107 cfu.q' after 10 d of mass production to 9.7 x 106 cfu.q' after a further 30 d of normal Kepi production. The average yeast counts increased from no detectable yeasts after 3 d of activation to 5.7 x 107 cfu.q' after 10 d of mass production and then decreased again to 7.2 x 106 cfu.q' after 30 d of normal Kepi production. The combination of the isolates varied according to the method by which the Kepi grains were produced and the stress conditions that were applied. Laboratory produced Kepi grains contained the following LAB: Lactobacillus fermentum, Lb. brevis 3, Lb. p/antarum, Lb. de/brueckii subsp. de/brueckii, Lactococcus /actis subsp. /actis and Leuconostoc mesenteroides subsp. cremoris. The identified yeasts and mycelial fungi were a Zygosaccharomyces strain, Cryptococcus humico/us, Candida /ambica, C. krusei, C. kefyr and Geotrichum candidum. The influence of grain origin on the microbial content of Kepi grains was also investigated using samples of Kepi grains from eight different Southern African sources. The microbial counts of the various Kepi grain samples were found to vary from 6.0 x 105 cfu.q" to 1.7 x 108 cfu.q". Five Lactobacillus, two Leuconostoc, four Candida, one Saccharomyces and a Zygosaccharomyces strain were isolated from these grains, with each grain type having its own unique microbial combination. The microbial content of the Kepi grains that were Iyophilised, packaged in three different packaging materials and stored at room temperature for two months, was very similar. Lactobacillus delbrueckii subsp. delbrueckii was isolated from the Kepi grains packaged in "low density polyethylene film" (LOPE). The grains packaged in "oriented polyester film" (OPET) contained Lb. delbrueckii subsp. delbrueckii and Lb. brevis, while Lb. delbrueckii subsp. delbrueckii and Lb. curvatus was present in the grains packaged in "methallised oriented polyester film" (MOPET). The average microbial counts obtained from the Kepi grains packaged in OPET (2.7 x 106 cfu.q') were only slightly higher than that of the grains packaged in LOPE (1.2 x 106 cfu.q') and OPET (1.4 x 106 cfu.q'). It was concluded that packaging materials for Kepi grains should rather be evaluated on the quality of Kepi produced with the packaged grains than by the specific characteristics of the packaging materials. The enrichment of Kepi grains with propionibacteria was also evaluated. A polymerase chain reaction (PCR) based method, specifically designed for the rapid identification of propionibacteria, was developed and tested successfully. Using this technique it was concluded that propionibacteria were not a natural part of the Kepi beverage and grains as used in this study. However, during the enrichment of the grains with propionibacteria it was determined that a propionibacteria concentration of 1 x 108 cfu.rnt' was needed for successful PCR amplification results. The data obtained in this study clearly showed that the method by which Kepi is produced, the origin of Kepi grains and the method of Kepi grain preservation changes the relationship between the microbes constituting the grains to such an extent that a different microbial community is assembled. It was also concluded that traditional methods should be used together with newer methods in determining this microbial community.

AFRIKAANSE OPSOMMING: Kepi is 'n self-gekarboneerde, effens suur melkdrankie wat geproduseer word deur melk te fermenteer met korrels waarin mikrobes (melksuurbakterieë en giste) natuurlik voorkom. Die mikrobes in die Kepi korrels is verantwoordelik vir 'n suuralkoholiese fermentasie en dra verder by tot die verskeie gesondheidseienskappe wat Kepi besit. Die kombinasie van mikrobes in die Kepi korrels wissel afhangende van die tipe melk wat gebruik word, die metode waarvolgens Kepi gemaak word, die oorsprong van die korrels en hoe die korrels geberg word. In hierdie studie is die impak van verskeie omgewingskondisies insluitende die verskillende stadiums tydens Kepi produksie, korreloorsprong, vriesdroging en verpakking in drie verskillende verpakkingsmateriale, op die mikrobiese samestelling van Kepi korrels bepaal m.b.v. selektiewe groei media en morfologiese en biochemiese eienskappe. Dit is gevind dat daar 'n afname was in die mikrobiese tellings van laboratorium geproduseerde Kepi korrels hoe langer Kepi op 'n aaneenlopende basis geproduseer is. Die afname in mikrobiese tellings is ook waargeneem tydens die verskillende stadiums van Kepi produksie. Die gemiddelde melksuurbakterieë tellings van laboratorium geproduseerde korrels het afgeneem vanaf 1.1 x 108 kve.q' na 3 d van aktivering tot 6.3 x 107 kve.q" na 10 d van massakweking tot 9.7 x 106 kve.q" na 'n verdere 30 d van normale Kepi produksie. Die gemiddelde gis tellings het gestyg vanaf geen giste na 3 d van aktivering tot 5.7 x 107 kve.q" na 10 d van massakweking en het toe weer gedaal tot 7.2 x 106 kve.q' na 30 d van normale Kepi produksie. Die kombinasie van die isolate het gewissel na gelang van die metode waarop die Kepi korrels geproduseer is en die stres kondisies wat toegepas is. Laboratorium geproduseerde Kepi korrels het bestaan uit Lactobacillus fermentum, Lb. brevis 3, Lb. p/antarum, Lb. de/brueckii subsp. de/brueckii, Lactococcus /actis subsp. /actis 1en Leuconostoc mesenteroides subsp. cremoris. Die giste en misiliëre fungi wat geïs~leer is was 'n Zygosaccharomyces stam, Cryptococcus humico/us, Candida lambica, C. krusei, C. kefyr en Geotrichum candidum. Die invloed wat die oorsprong van Kepi korrels op die mikrobiese samestelling daarvan het, is bepaal m.b.v. Kepi korrels afkomstig van agt verskillende dele in Suidelike Afrika. Die mikrobiese tellings van die verskeie tipes Kepi korrels het gewissel vanaf 6.0 x 105 kve.q' tot 1.7 x 108 kve.q", Vyf Lactobacillus, twee Leuconostoc, vier Candida, een Saccharomyces en 'n Zygosaccharomyces is geïsoleer vanuit die korrels, waarvan elke tipe korrel sy eie unieke mikrobiese samestelling gehad het. Die mikrobiese samestelling van korrels wat gevriesdroog, verpak is in drie verskillende verpakkingsmateriale en by kamertemperatuur gestoor is vir twee maande, was baie eenders. Vanuit die Kepi korrels wat verpak is in "lae digtheid polietileen film" (LOPE) is Lb. delbrueckii subsp. teetis geïsoleer. Die korrels wat verpak is in "georienteerde poltester film" (OPET) het Lb. delbrueckii subsp. leetis en Lb. brevis besit, terwyl Lb. delbrueckii subsp. leetis en Lb. curvatus teenwoordig was in die korrels wat in "gemetileerde georienteerde poltester film" (MOPET) verpak is. Die gemiddelde mikrobiese tellings van die korrels wat verpak is in OPET (2.6 x 106 kve.q') was effens hoër as dié van die korrels wat verpak is in LOPE (1.2 x 106 kve.q") en MOPET (1.3 x 106 kve.q"). Dit is bepaal dat verpakkingsmateriale vir Kepi korrels eerder geevalueer moet word op die kwaliteit van die Kepi wat met die verpakte korrels geproduseer word, as op die spesifieke eienskappe van die verpakkingsmateriale. Die mikrobiese verryking van Kepi korrels met propionibakterieë is ook ondersoek. 'n Polimerase ketting reaksie (PKR) gebaseerde metode, spesifiek ontwerp vir die vinnige identifikasie van propionibakterieë, is ontwikkel en suksesvol getoets. Met hierdie tegniek is bepaal dat propionibakterieë nie 'n natuurlike deel is van die Kepi drankie en korrels soos gebruik in hierdie studie. Gedurende die verryking van Kepi korrels met propionibakterieë is dit egter ook bepaal dat 'n propionibakterieë konsentrasie van 1 x 108 kve.rnl' nodig is vir suksesvolle PKR amplifikasie resultate. Die data verkry in hierdie studie het duidelik gewys dat die metode van Kepi produksie, die oorsprong van Kepi korrels en die metode waarop Kepi korrels gepreserveer word, verander die verhouding tussen die mikrobes in die korrels tot so 'n mate dat 'n nuwe mikrobiese gemeenskap saamgestel word. Die gevolgtrekking is ook gemaak dat tradisionele metodes saam met nuwer metodes gebruik moet word in die bepaling van hierdie mikrobiese gemeenskap.

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