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

Graduation date: 1995

Thesis (MSc Food Sc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Kefir is a fermented dairy beverage resulting from the fermentation of milk with reusable Kefir grains. The grains consist of a complex combination of lactic acid bacteria and yeasts in a symbiotic relationship, embedded in a polysaccharide matrix called kefiran. Various problems are experienced during the commercialisation of the ready-made Kefir beverage and, therefore, it is more advantageous to market the grains, enabling the consumer to produce the beverage at home. Kefir grains could be mass-cultured and then preserved by Iyohilisation for successful long-term storage and easy distribution, during commercialisation. The microbial balance of the Kefir grains changes during both mass-culturing and freeze-drying, which will have an influence on the sensory properties of the Kefir beverage produced. The aim of this study was the optimisation of the production of Kefir from mass-cultured grains and from freezedried mass-cultured grains respectively. The sensory characteristics of the fermented beverages produced from these mass-cultured and preserved grains were determined. Mass-cultured Kefir grains were activated and Kefir produced using nine methods with different activation times and temperatures, different grain:milk ratios (36, 72 and 108 g grains.l⁻¹) and with different heat-treated milks (pasteurised, double pasteerised and UHT). The best Kefir beverage was produced by activation of the grains at 22°C for two successive 24 h incubation periods, followed by Kefir production at 22°C for 18 h and a maturation period at 18°C for 6 h. The milk was replaced before every incubation period, excluding the maturation period, and the fermentation vessel was swirled five times at the start of fermentation and after 18 h. This method resulted in a sour beverage with a thick consistency and the characteristic effervescence and flavour of Kefir. The optimal grain:milk ratio was identified as 36 g grains.l⁻¹ and the best heat-treated milks for the production of Kefir beverage were UHT and double pasteurised milk. Mass-cultured Kefir grains were freeze-dried for 1, 2, 3 and 6 d and the moisture loss determined. Freeze-dried grains were rehydrated for 1, 2, 6, 12 and 18 h to determine the optimal rehydration time. A sensory analysis was performed to compare the properties of Kefir produced from mass-cultured grains (Me), freeze-dried mass-cultured grains that were rehydrated and activated (FDRA) and activated mass-cultured grains that were freeze-dried and rehydrated (AFDR). The chemical compositions of mass-cultured grains (MC), mass-cultured, freezedried grains (MCFD), mass-cultured, freeze-dried grains that were rehydrated and activated (FDRA) and activated mass-cultured grains that were freeze-dried and rehydrated (AFDR), were also investigated. The optimum time to freeze-dry grains was 2 d and to rehydrate freeze-dried gtains was 1 h. The sensory analysis indicated that Kefir beverages prepared from FDRA and AFDR grains did not differ significantly and were less fermented than Kefir produced from MC grains. It was concluded that Kefir with excellent sensory characteristics can be produced from mass-cultured grains. Freeze-drying is a better method to preserve Kefir grains than freezing due to mass loss during freezing and easier distribution and storage of freeze-dried grains. The supplementation of freeze-dried grains with additional lactic acid bacteria and yeast isolates should be investigated. / AFRIKAANSE OPSOMMING: Kefir is 'n gefermenteerde suiwelproduk wat geproduseer word deur die fermentasie van melk met herbruikbare Kefirkorrels. Die korrels bestaan uit 'n komplekse kombinasie van melksuurbakterië en giste en is ingebed in 'n polisakkaried matriks genaamd kefiran. Verskeie probleme word ondervind met die kommersialisering van die klaar voorbereide Kefirdrankie en dit is meer voordelig om die korrels te bemark. Dit sal die verbruiker daartoe in staat stel om self Kefir tuis te produseer. Kefirkorrels kan in massa gekweek word en dan gevriesdroog word om langtermyn storing en verspreiding te vergemaklik tydens kommersialisering. Die spesifieke mikrobiese balans van die Kefirkorrels word tydens massakweking en vriesdroging versteur. Dus sal hierdie twee prosesse 'n invloed hê op die sensoriese eienskappe van die Kefir drankie geproduseer. Die doel van hierdie studie was die optimisering van die produksie van Kefir vanaf massagekweekte korrels en gevriesdroogde massagekweekte korrels. Die sensoriese karakteristieke van die Kefir geproduseer met hierdie korrels is ondersoek. Massagekweekte Kefirkorrels is geaktifeer en Kefir is geproduseer met nege verskillende metodes met variasies in die tyd en temperatuur kombinasies, verskillende korrel:melk verhoudings (36, 72 en 108g korrels.l⁻¹) en verskillende hittebehandelde melke (gepasteuriseerd, dubbel gepasteuriseer en UHT). Die beste Kefirdrankie is geproduseer deur die aktivering van die korrels by 22°C vir twee 24 h inkubasieperiodes, gevolg deur Kefir produksie by 22°C vir 18 uur en 'n verouderingsperiode by 18°C vir 6 h. Die melk was voor elke inkubasieperiode vervang, uitsluitende die verouderingsperiode. Die fermentasie houer is vyf maal gedraai aan die begin van fermentasie en na 12 h. Hierdie metode het gelei tot 'n drankie wat suur was met 'n dik konsistensie en die karakteristieke vonkeling en geur van Kefir. Die optimale korrel:melk ratio is geidentifiseer as 36 9 korrels.l⁻¹ en die verkieslike hittebehandelde melke is dubbel gepasteuriseerde en UHT melk. Massagekweekte Kefirkorrels was vir 1, 2, 3 en 6 dae gévriesdroog en die massaverlies is bepaal. Gevriesdroog korrels is gerehidreer vir 1, 2, 6, 12 en 18 h om die optimale rehidrasietyd te bepaal. 'n Sensoriese analise is uitgevoer om die eienskappe te vergelyk van Kefir geproduseer van massagekweekte korrels (MC), gevriesdroogde massagekweekte korrels wat gerehidreer en geaktiveer is (FDRA) en geaktiveerde massagekweekte korrels wat gevriesdroog en gerehidreed is (AFDR). Die chemiese samestelling van massagekweekte korrels (MC), massagekweekte, gevriesdroogde korrels (MCFD), massagekweekte, gevriesdroogde korrels wat gerehidreer en geaktiveer is (FDRA) en geaktiveerde massagekweekte korrels wat gevriesdroog en gerehidreer is (AFDR), is bepaal. Die optimum tydperk vir vriesdroging van korrels was 2 d en vir rehidrasie van gevriesdroogde korrels was 1 h. Die sensoriese analise het aangedui dat Kefir wat van FDRA en AFDR korrels geproduseer is, nie betekenisvol van mekaar verskil het nie, maar minder gefennenteerd was as Kefir wat van Me korrels geproduseer is. Die gevolgtrekking is gemaak dat 'n Kefirdrankie met uitstekende eienskappe geproduseer kan word met massagekweekte korrels. Vriesdroging is 'n beter metode as bevriesing om Kefirkorrels te preserveer a.g.v die ver1iesvan massa tydens bevriesing en die vergemakliking van vervoer en verspreiding van gevriesdroogde korrels. Die aanvulling van gevriesdroogde korrels met addisionele melksuurbakterieêen giste moet nog ondersoek word.

Probiotics are viable nonpathogenic microbes that positively affect host health. Probiotics inhibit infection, activate immunity, and promote mucosal-barrier development. Many microbes have probiotic activity. Nonetheless, the selection of stable strains and their specific mechanism(s) of action are not fully elucidated. Bacteria from ‘Amabere amaruranu’ cultured milk from Kenya were isolated and identified by PCR sequence analysis of the 16S rRNA gene. Isolates were examined for stability to acid and bile, antimicrobial activity, mucin production, and degradation and sensitivity to antibiotics, hence their potential for probiotics. Lactobacillus isolates were acid unstable, bile-stable, nonmucinolytic, and presented antibacterial activity. L. rhamnosus cell fractions increased MUC4 and MUC3 expression in colon cells. Bacillus isolates were acid and bile stable, nonmucinolytic and lacked antimicrobial activity. In conclusion, Lactobacillus isolates that were nonmucinolytic, stable in bile, demonstrated antibacterial activity, sensitive to antibiotics, and stimulated increase MUC4 and MUC3 levels in colon cells could be potential probiotics.

This study was performed to test the in vitro effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu”, a traditional Kenyan cultured milk, on 3T3-L1 and Caco-2 cell lines. Cultures of fully mature 3T3-L1 adipocytes were treated with bacterial isolate cell extract (CE), filtered spent broth (FSB) from overnight bacterial culture, or with a PBS control. Expression levels of PPAR³1 and 2, C/EBP±, and ATGL proteins in 3T3-L1 cells were upregulated by FSB treatment. CE treatment did not affect protein expression levels. Expression of MTTP and SREBP-1c proteins in Caco-2 cells showed no change with either treatment. Optical density measurements from Oil-Red-O stained 3T3-L1 adipocytes increased from PBS control cells to 25μl/ml FSB treated cells; measurements were reduced by treatments above 25μl/ml FSB. In conclusion, filtered spent broth prepared from a culture of Lactobacillus rhamnosus, isolated from “amabere amaruranu” cultured milk showed PPAR³1 and 2, C/EBP±, and ATGL agonistic properties.

Thesis (MSc Food Sc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Maas is a traditional fermented milk drink of the indigenous people of Southern Africa and can thus be used to uplift the nutritional status of the South African population, especially for the lower income groups. Furthermore, the problem of lactose intolerance among the Black population can also be addressed by the consumption of Maas. The objective of this study was to screen mesophylic lactic acid bacterial strains (25 in total) from the University of Stellenbosch Food Science Culture Collection for suitable metabolite production and then to produce traditional Maas with a starter culture combination that produces a distinctive acid and traditional flavour. The representative 25 single lactic acid starter strains were identified as Lactococcus lactis subsp. leetis biovar diacetylactis (12 strains), L. leetis subsp. leetis (four strains) and L. leetis subsp. cremoris (nine strains). These strains were inoculated into pasteurised full cream milk and activated for 8 h at 22°C. Pasteurised full cream milk was then inoculated with each of the activated starter strains, incubated at 22°C for 16 h and assessed for acid production abilities (pH = 4.6) under controlled time-temperature conditions. The results of this study showed that nine of the single strains, L. lactis subsp. leetis biovar diacetylactis (S1, S2, S3 and S5), L. teetis subsp. lactis (S13, S15 and S16) and two L. leetis subsp. cremoris strains (S17 and S22), produced sufficient acid, rendering them suitable for the use as starters in the production of traditional Maas. A pH range of 4.3 - 5.1 was reached by the nine single strains after 16 h at 22°C. Two-strain starter combinations were then formed by combining the most suitable single L. leetis subsp. leetis biovar diacetylactis, L. lactis subsp. lactis and L. lactis subsp. cremoris strains, respectively. From the data, it was concluded that acceptable Maas could be produced with four two-strain combinations (S3S 17, S3S22, S5S17 and S5S22). This selection was again based on suitable acid and metabolite production, as well as on sensory evaluation of the final product. These four two-strain combinations produced sufficient acid to reach a pH in the 4.6 - 4.8 range, and showed a high metabolite concentration for the most suitable compounds and formed a thick, smooth and creamy body texture after 16 h at 22°C. Three-strain combinations formed between the two-strain starter combinations and L. leetis subsp. teetis strains (813, 815 and 816), were also evaluated. With these combinations a lack of a pronounced Maas flavour was found. Thus, it was decided to add aroma producing strains of the species Leuconostoc mesenteroides subsp. dextranicum (strain L1) and L. mesenteroides subsp. citrovorum (strain L2) to the three-strain combinations. Four culture combinations (A, B, C and D) were then formed by combining the selected Leuconostoc strains (L1 and L2) with the most suitable Lactococcus strains (83,817,813 and 822). These combinations produced sufficient acid to reach the pH 4.5 - 4.6 range after 14 h at 22°C. Acetaldehyde was the major flavour metabolite formed in the Maas made with these four combinations, with concentrations ranging between 26.6 - 89.3 mg.l ̄ ¹, while other flavour metabolites (ethanol, acetone, diacetyl and 2-butanone) were present at lower concentrations. It was found that three of the four culture combinations (A, C and D) were characterised by a superior, but delicate flavour and a typical characteristic Maas body texture. Fruit flavoured Maas was subsequently prepared with the three most suitable culture combinations (A, C and D) using 11 flavours and a sensory evaluation performed. The statistically evaluated data showed that the appearance, smoothness, flavour intensity, sweetness and overall acceptability were influenced by the type of fruit flavour and the culture combination. Fruit flavour 4 (banana) was the most preferred flavour. The sensory panellists also indicated that the culture combination C gave the best overall acceptability over a three week study period. Data on the shelf-life study of natural unflavoured Maas, prepared with the three culture combinations (A, C and D), showed that the Maas still had an acceptable appearance, taste and good microbiological quality after 15 d at refrigerated temperatures. / AFRIKAANSE OPSOMMING: Maas is 'n tradisionele gefermenteerde melkdrankie onder die inheemse bevolking van Suid-Afrika en kan gebruik word om die voedingstatus van die Suid-Afrikaanse bevolking te verhoog, veral vir die laer inkomste groepe. Bowendien, kan die probleem van laktose intoleransie onder die Swart gemeenskap ook aangespreek word deur die verbruik van Maas. Die doel van hierdie studie was om enkelstam mesofiliese melksuur bakterieë (25 in totaal) van die Universiteit van Stellenbosch Voedselwetenskap Kultuur Versameling te ondersoek vir geskikte metaboliet produksie en tradisionele Maas met 'n kenmerkende suurheid en tradisionele geur met 'n geskikte kultuur kombinasie te produseer. Die toonaangewende 25 enkelstamme is Lactococcus lactis subsp. leetis biovar diacetylactis (12 stamme), L. lactis subsp. lactis (vier stamme) en L. lactis subsp. cremoris (nege stamme). Hierdie stamme was in gepasteuriseerde volroom melk geïnokuleer en geaktiveer vir 8 h teen 22°C. 'n Inokulum van die onderskeie geaktiveerde stamme is hierna in gepasteuriseerde volroom melk geplaas, vir 16 h teen 22°C geïnkubeer en hul vermoë om suur te produseer (pH = 4.6) onder beheerde tyd-temperatuur kondisies is bepaal. Die resultaat van die studie het aangedui dat nege enkelstamme, naamlik L. leetis subsp. lactis biovar diacetylactis (S1, S2, S3 en S5), L. lactis subsp. leetis (S13, S15 en S16) en twee L. leetis subsp. cremoris (S 17 en S22), geskikte suurheidsvlakke vir die produksie van Maas bereik het. 'n pH vlak van 4.3 - 5.1 is na 16 h teen 22°C deur hierdie nege enkelstamme bereik. Twee-stam kombinasies is onderskeidelik gevorm tussen die geskikte enkel L. lactis subsp lactis biovar diacetylactis, L. lactis subsp. lactis en L. lactis subsp. cremoris stamme. Die gevolgtrekking gemaak uit die data, is dat aanvaarbare Maas voorberei kan word met vier van die twee-stam kombinasies (S3S17, S3S22, S5S17 en S5S22) op grond van suurvorming, metaboliet produksie en sensoriese evaluasie. Hierdie vier kombinasies het genoegsame suur geproduseer om 'n pH vlak van 4.6 - 4.8 bereik, hoë metaboliet konsentrasies geproduseer en 'n dik, gladde en romerige tekstuur aangeneem na 16 h teen 22°C. Drie-stam kombinasies is gevorm tussen die onderskeie twee-stam kombinasies en L. lactis subsp. lactis stamme (813,815 en 816) en ook geëvalueer. Die tekort aan 'n skerp Maas geur in die drie-stam kombinasies het daartoe gelei dat Leuconostoc mesenteroides subsp. dextranicum (stam L1) en L. mesenteroides subsp. citrovorum (stam L2) bygevoeg is. Vier kultuur kombinasies (A, B, C en D) is gevorm deur die geselekteerde Leuconostoc stamme (L1 en L2) te kombineer met die mees gepaste Lactococcus stamme (83, 817, 813 en 822). Hierdie kombinasies het genoegsame suur geproduseer wat 'n pH vlak van 4.5 - 4.6 na 14 h teen 22°C bereik het. In die Maas wat met bovermelde kombinasies gemaak is, was die asetaldehied die mees geproduseerde geur metaboliet teen konsentrasies van 26.6 - 89.3 mg.l ̄ ¹. Ander geur metaboliete (etanol, asetoon, diasetiel, 2-butanoon) is in laer konsentrasies geproduseer. Daar is gevind dat drie uit die vier kultuur kombinasies (A, C en D) 'n superieur, delikate geur wat 'n tipies karakteristiek van die Maas gehad het. Vrugte gegeurde Maas geproduseer met die drie kultuur kombinasies (A, C en D) deur 11 geursels te gebruik, is sensories geëvalueer. Die statistiese geëvalueerde data het getoon dat die voorkoms, gladheid, geur intensiteit, soetheid en die algehele aanvaarbaarheid beïnvloed is deur die tipe vrugte geursels en die kultuur kombinasies. Die vrugte geursel 4 (piesang) het voorkeur geniet. Die sensoriese paneellede het ook aangedui dat kultuur kombinasie C die algehele mees aanvaarbare Maas geproduseer het oor die studie periode van drie weke. Data van die rakleeftyd van die natuurlike ongegeurde Maas wat geproduseer is met die drie kultuur kombinasies (A, C en D) het aangedui dat die Maas na 15 d by yskas temperatuur steeds 'n aanvaarbare voorkoms, smaak en goeie mikrobiologiese kwaliteit gehad het.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Kepi is a refreshing, fermented dairy beverage that has been consumed for centuries and is traditionally made by incubating Kepi grains in milk. The Kepi grain is a complex starter culture consisting of a variety of lactic acid bacteria and yeasts. The successful marketing of the grains requires the effective preservation of the microbes present in the grains as well as an appropriate packaging that will retain the acidification activity of the preserved grains over an extended period of time. The aim of this study was to evaluate different preservation techniques and packaging materials in terms of their respective abilities to retain grain viability and activity over an extended storage period. Four different preservation techniques (freezing at -18°C, refrigeration at 4°C, air-drying and Iyophilisation) and three packaging materials including a low density polyethylene film (LOPE), an oriented polyester film (OPET) and a metallised oriented polyester film (MOPET), were evaluated. Activity tests were used to evaluate the impact of the preservation techniques in terms of the retainment of the acidification activity of the preserved grains, and the storage potential of the preserved and packaged grains. The activity tests included changes in pH, %TA, lactic acid production and lactose and volatile compound content over an 18 h fermentation period. In addition, the microbial viability of the packaged Iyophilised grains after two months of storage, was also investigated. Frozen and refrigerated grains showed the best retainment of the acidification activity over a 10-month storage period. Air-drying and Iyophilisation showed a good retention of the activity up to three months of storage, but the application of these techniques both resulted in a retarded initial acidification activity. After 10 months of storage, the air-dried and Iyophilised grains showed only a low acidification activity. No volatile compounds could be detected during the course of the fermentation period, due to the relative short fermentation period of 18 h. Overall, the best retainment of the fermentation activity was given by the LOPE and the OPET packaging films. However, the storage period had a considerable influence on the retention of the activity of the packaged Iyophilised grains. The viability study of the Iyophilised packaged Kepi grains after two months of storage showed leuconostocs and lactobacilli to be the prevalent microbes in the grains. Low microbial counts were obtained from the lactococciselecting medium for all three of the differently packaged Kepi grains, whereas no growth was observed on the media that selected for the propionibacteria and yeasts. The OPET packaging film provided the best preservation of the microbial composition. It was, therefore, concluded that all four preservation techniques would be suitable for the preservation of Kepi grains and the subsequent storage at room temperature for three months. However, for storage periods of 10 months or longer the use of freezing and refrigeration are recommended as most suitable preservation techniques. All three of the packaging materials proved to be suitable for the packaging and storage of the Iyophilised Kepi grains for periods of up to one month. However, for storage periods of two months or longer, the use of the OPET film for the packaging and retainment of the acidification activity of the Iyophilised grains, can be recommended. / AFRIKAANSE OPSOMMING: Kepi is 'n eeu-oue verfrissende, gefermenteerde suiweldrankie wat tradisioneel vervaardig word deur Kepikorrels in melk te inkubeer. Hierdie Kepikorrels bestaan uit 'n komplekse samestelling van hoofsaaklik melksuurbakteriee en giste. Die effektiewe preservering en verpakking van die korrels is belangrike voorvereistes vir die suksesvolle bemarking daarvan. Dis belangrik dat die preserverinq en die verpakking van die korrels 'n positiewe bydrae sal lewer tot die behoud van die fermentasie-aktiwiteit van die mikrobes in die korrels oar 'n verlengde opbergingsperiode. Die doel van hierdie studie was om die opbergingspotensiaal van verskillend gepreserveerde en -verpakte Kepikorrels te evalueer in terme van die behoud van die lewensvatbaarheid en fermentasie-aktiwiteit van die samestellende mikrobes. Vier verskillende preserveringstegnieke (bevriesing by -18°C, verkoeling by 4°C, lugdroging en vriesdroging) en drie verskillende tipes verpakkingsmateriale, nl. 'n "low density polyethylene film" (LOPE), 'n "oriented polyester film" (OPET) en 'n "metallised oriented polyester film" (MOPET) was qeevalueer. Aktiwiteitstoetsing was gebruik om die impak van die verskillende preserveringstegnieke en die verpakkingsmateriale op die behoud van die fermentasie-aktiwiteit van die Kepikorrels te ondersoek. Die verskillende aktiwitieitstoetse wat gedoen is, het die meting van die verandering in pH, %TA, melksuur- en laktosekonsentrasie oor 'n fermentasieperiode van 18 h ingesluit. Tesame met die aktiwitietstoetsing IS die lewensvatbaarheid van die gevriesdroogde, verpakte Kepikorrels na twee maande van opberging ook ondersoek. Die bevrore en verkoelde Kepikorrels het die beste behoud van aktiwitiet na 'n 10-maande opbergingsperiode getoon. Die gelugdroogde en gevriesdroogde korrels het 'n goeie behoud van aktiwiteit getoon vir 'n opbergingstydperk van tot drie maande, maar beide die lugdroging- en vriesdrogingstegnieke het 'n aanvanklik vertraagde fermentasie-aktiwitieit getoon. Na 'n : opbergingsperiode van 10 maande het beide die gelugdroogde en gevriesdroogde korrels egter 'n lae fermentasie-aktiwiteit getoon. As gevolg van 'n relatiewe kort fermentasieperiode van 18 h kon geen vlugtige komponente in die Kepimonsters gevind word nie. Die LDPE- en OPET-verpakkingsmateriale het die beste behoud van die fermentasie-aktiwiteit van die gevriesdroogde korrels getoon. Die opbergingsperiode het egter 'n aansienlike impak op die aktiwitietsbehoud van die korrels gehad. Die lewensvatbaarheidstudie het aangetoon dat Leuconostoc- en Lactobacillus-spesies die oorheersende mikrobes in die verpakte, gevriesdroogde Kepikorrels na 'n opbergingsperiode van twee maande was. Lae mikrobiese tellings vir al drie van die verpakkingsmateriale was gevind op die Lactococcusselekterende medium, en geen mikrobegroei kon op die giste- en propionibakteriee-selekteringsmedium waargeneem word nie. Die beste behoud van die mikrobiese samestelling in die verpakte, gevriesdroogde Kepikorrels was gevind vir die OPET-verpakkingsmateriaal. Die gevolgtrekking kan gemaak word dat al vier die preserveringstegnieke geskik is vir die preservering van die Kepikorrels en die daaropvolgende opberging van drie maande by kamertemperatuur. Vir opbergingsperiodes van 10 maande en langer word die gebruik van bevriesing en verkoeling aanbeveel as die mees geskikte preserveringstegnieke. AI drie verpakkingsmateriale kan gebruik word vir die verpakking en opberging van gevriesdroogde Kepikorrels vir 'n tydperk van een maand. Indien 'n opbergingsperiode van twee maande of langer verlang word, word die OPET-verpakkingsmateriaal aanbeveel vir die suksesvolle behoud van die fermentasie-aktiwiteit van die Kepikorrels.

Thesis (MSc Food Sc )--Stellenbsosch University, 2003. / ENGLISH ABSTRACT: Kepi is a fermented milk beverage that originated in Eastern Europe. Traditional Kepi is a lightly acidic, carbonated beverage, with a slight yeasty taste. The starter used to produce this beverage is an irregularly shaped, yellowish-white grain-like structure similar in appearance to a cauliflower floret. The characteristic flavour of Kepi is produced by a complex spectrum of microbial species that include species of yeasts, lactic acid bacteria, acetic acid bacteria and mycelial fungi. At the end of the fermentation process the grainy starter can be recovered and re-used, since the microbes can easily be recovered as a solid matrix. The microbes comprising Kepi grains have only been identified using classical identification techniques such as selective growth media, morphological, physiological and biochemical characteristics. In this study, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis was used to typify and identify the complex microbial consortium present in the Kepi grains. A part of the 168 ribosomal RNA (rRNA) gene from the microbial population in mass-cultured, traditionally cultured and Irish Kepi grains were amplified using 'Eubacterial' specific primers and a part of the 268 rRNA gene was amplified using yeast specific primers. The PCR fragments were resolved by DGGE, resulting in unique fingerprints for the Eubacteria and yeasts present in the different Kepi grain types. The traditionally cultured Kepi grains were found to incorporate the most Eubacteria and yeast species, while the mass-cultured Kepi grains contained the lowest number of Eubacteria and yeast species. The different Eubacteria and yeast species were identified by cloning the PCR products and sequencing the cloned inserts. The obtained DNA sequences were compared to sequences available on the NCBI website. 8ix lactobacilli were identified: Lb. crispatus (KC-4); three Lb. species (KC-36, KC-38 and KC-43); and two unculturable lactobacilli (KC-2 and KC-3). The yeasts were identified as Saccharomyces cerevisiae (KC-y18) and Candida lambica (KC-y1). Unidentified isolates from kefiran strings that could not be identified using traditional methods were also identified by cloning the PCR products and sequencing the cloned inserts. The four isolates were identified as Lb. kefiri (KGI-A), Lb. parakefiri (KGIB), Lb. gallina rum (KGI-D) and an unculturable Lactobacillus (KGI-5). The phylogenetic relationship between the identified lactobacilli and the lactobacilli commonly found in Kepi grains was determined. The identified lactobacilli were grouped together in a clade with a bootstrap support value of 84%. The clade also contained representatives of Lb. delbrueckii subsp. lactis, Lb. acidophilus, Lb. gallinarum, Lb. helveticus, Lb. crispatus, Lb. species and unculturable lactobacilli. The bands in the peR-based DGGE fingerprints of the Eubacteria and the yeasts were identified, and a DGGE marker was subsequently constructed for the rapid identification of the Eubacteria present in mass-cultured Kepi grains. The data obtained in this study clearly showed that Kepi grains that are cultured differently, as well as Kepi grains from different origins have unique peRbased DGGE banding patterns for both the Eubacteria and yeasts present in the grains. The complex microbial consortium comprising Kepi grains could be typified and identified using PeR-based DGGE, DNA cloning and sequencing. The identification of the members of the microbial consortium is of importance for the future commercialisation of the mass-cultured Kepi grains. / AFRIKAANSE OPSOMMING: Kepi is 'n gefermenteerde melkdrankie wat sy oorsprong het in Oos Europa. Tradisionele Kepi is 'n effens suur, gekarboneerde drankie wat effens na gis smaak. Die beginkultuur wat gebruik word om dié drankie te maak is 'n oneweredige, geel-wit korrelagtige struktuur wat baie lyk soos 'n blomkoolkoppie. Die karakteristieke smaak van Kepi word geproduseer deur 'n komplekse spektrum mikrobiese spesies wat giste, melksuur- en asynsuurbakterieë en ~. misillêre fungi insluit. Aan die einde van die fermentasieproses kan die korrelagtige beginkultuur herwin word en weer gebruik word, aangesien die mikrobes maklik herwin kan word as 'n soliede matriks. Die mikrobes waaruit Kepikorrels bestaan, is nog slegs met behulp van klassieke identifikasiemetodes soos selektiewe groeimedia, morfologiese, fisiologiese and biochemiese eienskappe geïdentifiseer. In hierdie studie is polimerase kettingreaksie (PKR)-gebaseerde denaturerende gradiënt jelelektroforese (DGGE) analise gebruik om die komplekse mikrobiologiese konsortium in die Kepikorrels te tipeer en te identifiseer. 'n Gedeelte van die 16S ribosomale RNS (rRNS) geen van die mikrobiologiese populasie in massagekweekte, tradisioneel gekweekte en Ierse Kepikorrels is geamplifiseer met 'Eubakferiële' spesifieke peilers en In gedeelte van die 26S rRNS geen is geamplifiseer met gis spesifieke peilers. Die PKR fragmente is onderskei deur DGGE, wat unieke vingerafdrukke vir die Eubakteriële- en gisspesies in die verskillende Kepikorrel tipes gelewer het. Die tradisioneel gekweekte Kepikorrels het die meeste Eubakteriële- en gisspesies geïnkorporeer, terwyl die Ierse Kepikorrels die minste Eubakteriële- en gisspesies geïnkorporeer het. Die verskillende Eubakteriële- en gisspesies is geïdentifiseer deur klonering van die PKR produkte en deur die gekloneerde insetsels se volgordes te bepaal. Die ONS volgordes is dan vergelyk met volgordes wat op die NCSI webwerf beskikbaar is. Ses lactobacilli is geïdentifiseer: Lb. ctispetus (KC-4); drie Lb. spesies (KC-36, KC-38 en KC-43); en twee onkultiveerbare lactobacilli (KC-2 en KC-3). Die giste is geïdentifiseer as Saccharomyces cerevisiae (KC-y18) en Candida lambica (KC-y1). Ongeïdentifiseerde isolate van kefiranstringe is ook geïdentifiseer deur klonering van die PKR produkte en deur die gekloneerde insetsels se volgorde te bepaal. Dié vier isolate is geïdentifiseer as Lb. kefiri (KGIA), Lb. parakefiri (KGI-B), Lb. gallina rum (KGI-D) en 'n onkultiveerbare Lactobacillus (KGI-5). Die filogenetiese verwantskap is bepaal tussen die geïdentifiseerde lactobacilli en lactobacilli wat geredelik in Kepikorrels gevind word. Die geïdentifiseerde lactobacilli was saam in 'n groep gegroepeer met 'n bootstrap waarde van 84%. Die groep het ook verteenwoordigers van Lb. delbrueckii subsp. lactis, Lb. acidophilus, Lb. gallina rum, Lb. helveticus, Lb. crispatus, Lb. species en 'n onkultiveerbare laktobacilli ingesluit. Die bande in die PKR-gebaseerde DGGE vingerafdrukke van die Eubakterieë en die giste is geïdentifiseer, en 'n DGGE merker is gemaak vir die vinnige identifikasie van die Eubakterieë wat in die massagekweekte Kepikorrels teenwoordig is. Die data wat in die studie verkry, is wys duidelik dat Kepikorrels wat op verskillende maniere gekweek is, en wat verskillende oorspronge het, unieke PKR-gebaseerde DGGE bandpatrone het vir beide die Eubakterieë en giste wat in die korrels teenwoordig is. Die komplekse mikrobiologiese konsortium waaruit Kepikorrels bestaan kon getipeer en geïdentifiseer word deur PKR-gebaseerde DGGE, klonering van DNS en volgordebepaling. Die identifikasie van lede van die mikrobiologiese konsortium is belangrik vir die toekomstige kommersialisasie van die massagekweekte Kepikorrels.

A study was conducted to isolate and identify bacteria from “amabere amaruranu” cultured-milk from Kenya and to evaluate the isolates’ potential to be used as probiotics. Isolates were identified using PCR sequence analysis of the 16S rRNA gene and the API® 50 identification system. Identified isolates included: Acetobacter tropicalis, Bacillus pumilus, Bacillus safensis, Lactobacillus paracasei, Lactobacillus rhamnosus, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus sciuri. Their potential as probiotics was evaluated using their ability to survive in acid-then-bile conditions, antibacterial activity (against Escherichia coli, Klebsialla pneumonia, Enterococcus faecalis, Pseudomonas aeruginosa and Enterobacter cloacae), mucin degradation activity, and sensitivity to antibiotics (ampicillin, bacitracin, chloramphenicol, erythromycin, kanamycin, penicillin, streptomycin and tetracycline). Lactobacillus isolates were tolerant to acid-then-bile conditions, non-mucinolytic and inhibited growth of indicator strains but only L. rhamnosus was sensitive to all test antibiotics. Bacillus isolates were tolerant to acid-then-bile conditions, non-mucinolytic, lacked antibacterial activity and only B. safensis was sensitive to all test antibiotics. Acetobacter tropicalis isolates were non-mucinolytic but were intolerant to acid-then-bile conditions. In conclusion, both Lactobacillus rhamnosus and Bacillus safensis isolates that showed tolerance to digestive tract conditions, were sensitive to antibiotics and were non-mucinolytic would be recommended for further consideration as candidate probiotics.

A study was conducted to isolate and identify bacteria from “amabere amaruranu” cultured-milk from Kenya and to evaluate the isolates’ potential to be used as probiotics. Isolates were identified using PCR sequence analysis of the 16S rRNA gene and the API® 50 identification system. Identified isolates included: Acetobacter tropicalis, Bacillus pumilus, Bacillus safensis, Lactobacillus paracasei, Lactobacillus rhamnosus, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus sciuri. Their potential as probiotics was evaluated using their ability to survive in acid-then-bile conditions, antibacterial activity (against Escherichia coli, Klebsialla pneumonia, Enterococcus faecalis, Pseudomonas aeruginosa and Enterobacter cloacae), mucin degradation activity, and sensitivity to antibiotics (ampicillin, bacitracin, chloramphenicol, erythromycin, kanamycin, penicillin, streptomycin and tetracycline). Lactobacillus isolates were tolerant to acid-then-bile conditions, non-mucinolytic and inhibited growth of indicator strains but only L. rhamnosus was sensitive to all test antibiotics. Bacillus isolates were tolerant to acid-then-bile conditions, non-mucinolytic, lacked antibacterial activity and only B. safensis was sensitive to all test antibiotics. Acetobacter tropicalis isolates were non-mucinolytic but were intolerant to acid-then-bile conditions. In conclusion, both Lactobacillus rhamnosus and Bacillus safensis isolates that showed tolerance to digestive tract conditions, were sensitive to antibiotics and were non-mucinolytic would be recommended for further consideration as candidate probiotics.