Isolation and characterisation of the antimicrobial peptides produced by acetic acid bacteria

[摘要] ENGLISH ABSTRACT: Wine quality is greatly influenced by the number of microorganisms, which occurthroughout the winemaking process. Yeasts are responsible for the alcoholicfermentation, the lactic acid bacteria (LAB) are responsible for malolacticfermentation (MLF), while acetic acid bacteria (AAB) are responsible for convertingethanol to acetic acid. These microorganisms are present on the grapes and in thecellar and these consequently serve as gateways to the fermentation tanks wherethey will affect the wine quality. However, these microorganisms can be seen eitheras beneficial or as wine spoilage microorganisms, depending on the conditions thatprevail throughout the winemaking process. It is thus very important to prevent anyprocess that could lead to the lowering of the wine quality. In this regard, some of thefactors that should always be evaluated include the quality of the grapes, winemakingtechniques and quality control.One of the measures that have been implemented during winemaking to ensurethe microbial stability is the use of chemical preservatives. Sulphur dioxide (502) hasbeen, and is, used widely as primary preservative in winemaking. However, anever-increasing consumer resistance against the use of chemical preservatives hasdeveloped as it poses possible health risks and decreases the sensorial quality ofwine. An alternative approach to chemical preservation that has triggered numerousnew investigations, is biological preservation or biopreservation. This is the use of thenatural microbial flora and/or their antimicrobial products, such as bacteriocins, toinhibit or destroy the other sensitive microorganisms that are unwanted in the sameenvironment.Evidence in the wine industry has shown that bacterial spoilage still is a verycommon problem in many wineries. This bacterial spoilage can lead to, amongstother, two main problems, which are of great concern to winemakers. This includehigh levels of volatile acidity, resulting in the wine having a vinegary off-flavour, andsluggish/stuck fermentation, which is the result of compounds such as acetic- andother fatty acids that causes inhibition of the yeast's growth. With acetic acid beingthe common link in both cases, it became evident that investigations should beperformed on the main producer of acetic acid, namely AAB. As a result, AAB turnedout to be one of the main spoilage microorganisms associated with winemaking.Most of the research on biopreservation in the food and beverage industry hasbeen performed on the Gram-positive LAB. The fact that their spectrum of inhibitioncurrently excludes most Gram-negative bacteria, specifically AAB, indicated that AABshould be screened in search of possible antimicrobial compounds that could beapplied to control their cell numbers during winemaking. No evidence of antimicrobialaction amongst AAB could be found in literature, therefore this work was considerednovel.The main objectives of this study were to screen wine isolates of AAB for theproduction of antimicrobial compounds. This was followed by the isolation and preliminary characterisation of the antimicrobial substances produced. Variousattempts to optimise the production of the antimicrobial compounds and isolationprocedures, were also included. This study forms part of a larger researchprogramme that has been initiated at the Institute for Wine Biotechnology atStellenbosch University on the biopreservation in wine.Our results indicated that possible antimicrobial compounds of proteinaceousnature, produced by AAB isolated from wine, do exist. It was found that two differentspecies of AAB, namely Acetobacter aeeti and Gluconobacter frateurii, producedantimicrobial compounds that inhibited other species of AAB. Preliminary resultsindicated that these compounds are heat sensitive and stable in a wide pH range. Itwas also shown that after the action of proteolytic enzymes, such as proteinase Kand a-chemotrypsin, all inhibitory activity was lost. This study also revealed theexistence of the species Gluconobacter frateurii, which have not yet been associatedwith the winemaking environment.This study made a valuable contribution to the limited amount of information andunderstanding of AAB, not only in the wine environment, but also elsewhere. Theresults and findings of this research would serve as platform for further projects. Thismight soon lead to the development of antimicrobial substances or tailoredwine-yeasts with antimicrobial abilities, which can be applied during winemaking toassist the winemaker in combatting high cell numbers and subsequent spoilage byAAB.

[发布机构] Stellenbosch University