Control of bacterial spoilage in wine with natural (biological) antimicrobial peptides produced by lactic acid bacteria


Project Number
MIC 02

Project title
Control of bacterial spoilage in wine with natural (biological) antimicrobial peptides produced by lactic acid bacteria

Project leader
Dicks, L M T

Institution
University of Stellenbosch

Team members
Van Reenen, C A
Bauer, R
Nel, H A
Balla, E
Van der Merwe, I

Project description
The production of antimicrobial proteins or peptides by the lactic acid bacteria Pediococcus, Lactobacillus, Leuconostoc and Carnobacterium are well known. In contrast to many of the preservatives used in the food industry, these antimicrobial peptides are non-toxic, active at relatively low concentrations, and tasteless. These characteristics, and the ongoing pressure in the food (and wine) industry to reduce the use of allergic and carcinogenic additives, led to a renewed interest in using these peptides as preservatives.

A total of 90 newly isolated strains were screened for activity against malo-lactic bacteria and members of the genera acetobacter and gluconobacter. From this screening test, one strain (designated BDN and later renamed as strain 423) was selected for further studies.

An antimicrobial peptide produced by strain 423 was purified, its amino acid sequence determined and DNA primers constructed. The DNA sequence of a fragment on one of the plasmids harboured by strain 423 revealed an operon-like structure similar to pediocin PA-1, pediocin AcH (produced by Pediococcus acidilactici) and coagulin (produced by Bacillus coagulants). Four open reading frames (ORFs) were identified, viz. plaA, plaB, plaC and plaD. plaA encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, as determined by nucleotide and protein sequencing, with a 19-amino acid N-terminal leader peptide.

A PCR-generated fragment was cloned into plasmid pPRL2, yielding plasmid pPLA423. Plasmid pPRL2 is the 6.11 kb single-copy yeast-E. coli shuttle vector YCplac111 (bla, LEU2), containing the yeast alcohol dehydrogenase I promoter (ADH1P), the terminator (ADH1T) sequences, and the mating pheromone a-factor 1 secretion signal (MFa1S) DNA insert. The SalI-SmaI ADHIP-MFa1S-bcnA-ADHIT DNA fragment from pPLA423 was cloned into the multiple cloning site of the multicopy yeast-E. coli shuttle vector YEp352, generating plasmid YEpPla423. Plasmid YEpPla423 was eventually introduced into Saccharomyces cerevisiae L5366h to yield transformant L5366h[YEpPla423].

Production of bacteriocin 423 by recombinant Saccharomyces cerevisiae cells was determined by overlaying colonies of yeast transformants on agar plates with soft agar containing Listeria monocytogenes cells. Preliminary studies with the peptide proved that it is stable in wine, i.e. in the presence of sulphur dioxide and alcohol. The next phase of the project involves the transformation of the gene construct to an industrial wine strain of Saccharomyces cerevisiae.

Presentation(s)
Nel, H A, Bauer, F F, Vandamme, E J, Hallemeersch, I and Dicks, L M T. 2001. Fermentation optimization of Pediococcus damnosus NCFB 1832 and the production of pediocin PD-1. Paper presented at the Nizo Dairy Conference on Food Microbes – From Knowledge to Application. Ede, The Netherlands.

Van Rheenen, C A, Chikindas, M L, Van Zyl, W H, and Dicks, L M T. 2001. Genetic characterization of bacteriocin 423 and expression in Saccharomyces cerevisiae. Paper presented at the Nizo Dairy Conference on Food Microbes – From Knowledge to Application. Ede, The Netherlands.

Bauer, F F, Nel, H A and Dicks, L M T. 2001. The control of lactic acid bacteria by a new bacteriocin, pediocin PD-1, in the production of wine. Paper presented at the 16th Congress of the Southern African Society for Plant Pathology. Durban, South Africa.

Bauer, F F and Dicks, L M T. 2001. The effect of various parameters on the antimicrobial activity of pediocin PD-1. Paper presented at the 16th Congress of the Southern African Society for Plant Pathology. Durban, South Africa.

Nel, H A, Bauer, F F, Vandamme, E J and Dicks, L M T. 2001. The influence of medium composition on the production of pediocin PD-1, an antimicrobial peptide active against malolactic bacteria. Paper presented at the 25th National Congress of the South African Society for Enology and Viticulture. 15-16 November, Somerset West, South Africa.

Bauer, F F, Nel, H A and Dicks, L M T. 2001. The use of an antimicrobial peptide, pediocin PD-1, in winemaking. Paper presented at the 25th National Congress of the South African Society for Enology and Viticulture. 15-16 November, Somerset West, South Africa.

Article
Nel, H A, Bauer, F F, Vandamme, E J, Dicks, L M T. 2001. Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1, Journal of Applied Microbiology, v. 91 (6) (p. 1131-1138)

Van Reenen, C A, Van Zyl, W H, Chikindas, M L, Dicks, L M T. 2003. Isolation, purification and partial characterisation of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum 423, International Journal of Food Microbiology, v. 81 (1) (p. 29-40)

Note
Published articles serve as Final Report.

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