Screening, isolation and characterisation of antimicrobial/antifungal peptides produced by lactic acid bacteria isolated from wine

Morgan, Joanne (2003-03)

Thesis (MSc)--Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: Winemaking is an age-old tradition that dates back to as early as 6000 BC. In our modern era there are several insects and microorganisms that pose a threat to the grapevine, the environment and the final wine product. Farmers and winemakers are becoming aware of the threat and the fight against disease, spoilage and/or pathogenic microorganisms is on the rise. Currently, the natural environment is being altered through rural developments, pollution and disaster, which in turn is responsible for altering the natural micro flora. The result is a harsh battle between man and microorganism. The weapon used often against microorganisms is chemical preservatives, such as sulphur dioxide. These chemical preservatives change the nutritional value, quality and wholesomeness of the wine. Chemical preservatives suppress the quality of the wine with a reduction in wine consumption by the consumers. Until the 18th century, wine was regarded as a safe drink and prescribed by doctors. In the zo" century alcohol consumption became the focus point of some health campaigners. Medical science restored the good name of wine in the 1990s when it came to light that moderate red wine consumption may aid in preventing heart disease and assist in stress management. The only drawback that lowers consumption levels is the use of chemical preservatives. It is of utmost importance to place the focus on health issues and the development of natural preservation methods that are environmentally friendly and contributes to the overall wholesomeness of the wine. Due to these demands, the scientific community placed the focus of research projects on the development and enhancement of biopreservation methods, in order to minimise chemical preservation use. One of the most promising biocontrol agents is bacteriocins. These proteinaceous molecules produced by various lactic acid bacteria exert antimicrobial activity towards closely related organism. Research has shown that bacteriocins may aid in the prevention of wine-spoilage and enhance natural preservation techniques. Most of the research on biopreservation in food and beverages has been performed on the bacteriocins of LAB. No evidence could be found that indicated bacteriocin production by wine isolated LAB in South Africa. This study is therefore, of utmost importance and is considered to be novel pioneering work for the South African wine industry. The main objective of this study was to screen wine isolated LAB for the production of antimicrobial and/or antifungal compounds. This was followed by the isolation and characterisation of the produced bacteriocins. This study forms part of a greater project that focuses on wine preservation, under the auspices of the Institute for Wine Biotechnology.The research results in this study indicated the production of bacteriocins by wine isolated LAB of South African origin. It was found that numerous isolates exerted antimicrobial activity towards other wine associated LAB. The most predominant species that gave the highest activity was Lactobacillus brevis and Lactobacillus paracasei. Experimental results indicated that the bacteriocins produced by these two species were thermo-stable and active over a wide pH range, including the temperatures and pH values that reign in the South African wine environment. The antimicrobial activity was lost after treatment with proteolytic enzymes, such as proteinase K and lysozyme. The size, production and growth kinetic curves of the bacteriocins under investigation showed similar results that are comparable to other findings in the literature. Antifungal activity was detected against Botryfis cinerea that indicated limited inhibitory activity towards spore germination, but had no effect on hyphal growth. This study provides novel information regarding bacteriocin production by LAB isolated from the South African wine industry. The results indicate the suitability of these bacteriocins as possible biopreservatives in the wine environment. The proposed results obtained in this study will aid in the development of bacteriocinproducing, tailored made wine yeast or LAB that may in future, play vital roles in the winemaking process.

AFRIKAANSE OPSOMMING: Wynmaak is 'n eeu oue tradisie wat terugdateer tot so vroeg soos 6000 jaar v.C. In ons moderne eeu is daar verskeie insekte en mikro-organismes wat In bedreiging vir die wingerdstok, asook die omgewing en die finale wynproduk inhou. Boere en wynmakers word al hoe meer bewus van hierdie bedreiging, terwyl die stryd teen siektes, bederf en/of patogene mikro-organismes ook aan die toeneem is. Tans word die natuurlike omgewing deur landelike ontwikkeling, besoedeling en natuurlike rampe verander, wat op sy beurt weer verantwoordelik is vir die verandering van mikroflora. Die gevolg is 'n harde stryd tussen die mens en mikro-organismes. Die wapen wat gereeld ingespan word in die stryd teen mikro-organismes, is chemiese preserveermiddels, soos swaweidioksied. Hierdie chemiese preserveermiddels verander die voedingswaarde, kwaliteit en die voedsaamheid van die wyn. Dit onderdruk ook die gehalte van wyn, wat meebring dat minder wyn deur die verbruiker gedrink word. Tot en met die agtiende eeu is wyn deur dokters as 'n veilige drankie voorgeskryf. In die twintigste eeu het alkoholverbruik die fokuspunt van gesondheidskamvegters geword. In die 1990's het die mediese wetenskap wyn se goeie naam in ere herstel toe dit aan die lig gekom het dat In matige verbruik van rooiwyn moontlik hartsiektes kan voorkom en help om stres te beheer. Die enigste nadelige faktor wat verbruikersvlakke verlaag, is die gebruik van chemiese preserveermiddels. Dit is uiters noodsaaklik om die fokus op gesondheidskwessies te plaas en die ontwikkeling van natuurlike preserveermetodes wat omgewingsvriendelik is en tot die algehele voedsaamheid van wyn bydra. As gevolg van hierdie eise het wetenskaplikes die fokus geplaas op navorsingsprojekte vir die ontwikkeling en verbetering van biopreserveringsmetodes met die doelom die gebruik van chemiese preserveermiddels te verminder. Een van die belowendste biokontrolemiddels is bakteriosiene. Hierdie proteïenagtige molekule word deur verskeie melksuurbakterieë vervaardig en oefen anti-mikrobiese aktiwiteit teenoor nabyverwante organismes uit. Navorsing het getoon dat bakteriosiene moontlik kan help in die voorkoming van wynbederf en natuurlike preserveertegnieke kan verbeter. Die meeste van die navorsing op biopreservering in voedsel en drank is op die bakteriosiene van melksuurbakterieë uitgevoer. Geen bewys kon gevind word in Suid Afrika wat bakteriosienproduksie deur wyn-geïsoleerde melksuurbakterieë aangedui het nie. Hierdie studie is daarom baie belangrik en word as baanbreker werk vir die Suid Afrikaanse wynbedryf beskou. Die hoofdoel van hierdie studie was om wyn-geïsoleerde melksuurbakterieë vir die produksie van anti-mikrobiese en/of anti-fungiese substanse te toets. Dit is gevolg deur die isolasie en karakterisering van die geproduseerde bakteriosiene. Hierdie studie maak deel uit van 'n groter projek wat fokus op wynpreservering en wat onder leiding van die Instituut van Wynbiotegnologie uitgevoer word. Navorsingsresultate van hierdie studie dui op die produksie van bakteriosiene deur wyn-geïsoleerde melksuurbakterieë van Suid Afrikaanse oorsrong. Daar is gevind dat verskeie isolate anti-mikrobiese aktiwiteit teenoor ander wynverwante malksuurbakterieë uitgeoefen het. Die oorheersende spesie wat die hoogste aktiwiteit getoon het, was Lactobacillus brevis en Lactobacillus paracasei. Eksperimentele uitslae dui daarop dat die bakteriosiene wat deur hierdie twee spesies geproduseer word, termostabiel en aktief is oor 'n wye pH reeks, insluitende die temperature en pH-waardes wat in die Suid Afrikaanse wynomgewing voorkom. Die anti-mikrobiese aktiwiteit het verlore gegaan na behandeling met proteolitiese ensieme soos proteïnase K. Die groote, produksie en groeikinetika kurwes van die bakteriosiene wat ondersoek is, toon vergelykbare resultate met ander bevindings in die literatuur. Anti-fungiese aktiwiteit is opgemerk teen Botrytis cinerea, wat beperkte inhiberende aktiwiteit ten opsigte van spoorontkieming aangedui het, maar geen effek op hifegroei gehad nie. Hierdie studie verskaf nuwe inligting aangaande bakteriosienproduksie deur melksuurbakterieë wat van die Suid Afrikaanse wynomgewing geïsoleer is. Die resultate dui op die geskiktheid van hierdie bakteriosiene as moontlike biopreserveermiddels in die wynbedryf. Die voorgestelde resultate deur hierdie studie verkry sal help in die ontwikkeling van bakteriosien produserende, spesifiek vervaardigse wyngis of melksuurbakterieë, wat in die toekoms 'n baie belangrike rol in die wynmaakproses sal speel.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/53582
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