Enhancing the Wholesomeness of Wine


Project Number
IWBT 05-05

Project title
Enhancing the wholesomeness of wine

Project leader
Du Toit, M A

Institution
University of Stellenbosch. Department of Viticulture and Enology. Institute for Wine Biotechnology

Team members
Van Rensburg, P
Trollope, K
Rabe, J
Potgieter, N
Bauer, F F
Swiegers, H

Project description
The enhancement of wholesomeness of wine can be achieved by either increasing the concentration of substances that have been generally recognised as being beneficial, or by decreasing the concentration of compounds that are known to have a detrimental effect on human health.

Here we describe the progress made with projects that aim at increasing the concentration of resveratrol and L-carnitine, as well as decreasing the amounts of biogenic amines.

Introduction of the genes for resveratrol production into laboratory yeast and subsequent expression and functioning of the heterologous proteins do occur, as investigated in previous work. This project aims to stably integrate the genes, ultimately, into wine yeast in order to be able to produce resveratrol during fermentation of both red and white wines. The wholesomeness of wine will be enhanced, with associated health benefits

20 yeast strains isolated from vineyards and wine cellars were tested for ß-glucosidase activity. The strains exhibiting the highest enzyme activity were selected to investigate the effect of ethanol, glucose and sulphor dioxide on their ß-glucosidase activities. D. pseudopolymorphus, exhibiting high activity at higher glucose and sulphur dioxide concentrations and relative resistance to high ethanol values, appears to have an appropriate kinetic profile for use in the wine industry. We have now commenced the process of cloning the gene from D. pseudopolymorphus encoding ß-glucosidase using degenerate primers.

The biogenic amine-producing ability of 400 lactic acid bacteria (LAB) isolated from South African wine was determined, using a decarboxylase screening plate method. The potential to produce the biogenic amines histamine, tyramine, putrescine and cadaverine was investigated. Tyramine was the main amine formed by the lactic acid bacteria strains investigated. Putrescine was also produced. Lactobacillus hilgardii strains were shown to be mainly responsible for biogenic amine formation. Two Lactobacillus brevis strains also produced biogenic amines. None of the lactic acid bacteria produced histamine or cadaverine.

The yeast Saccharomyces cerevisiae does not biosynthetise carnitine. However, there are many yeast and fungi that do biosynthetise carnitine. The biochemical pathway for carnitine biosynthesis was first elucidated in the filamentous fungi Neurospora crassa. Trimethylated lysine is used as precursor and in a three step process is converted to gamma-butyrobetaine which is converted by the gamma-butyrobetaine hydroxylase to carnitine in the last step. Here we report on the cloning and expression of a genomic gene fragment of N. crassa coding for a gamma-butyrobetaine hydroxylase (BBH) and functional expression in yeast. In the carnitine dependent mutant strain, cit2, this gene could suppress the growth defect on non-fermentable carbon sources in the presence of gamma-butyrobetaine by endogenously producing L-carnitine. This is the first report of a carnitine biosynthetising S. cerevisiae. We also demonstrate a plate selection method for selecting carnitine producing microorganisms.

Presentation(s)
Swiegers, J H, Vaz, F M, Pretorius, I S, Wanders, R J and Bauer, F F. 2002. Carnitine biosynthesis in Neurospora crassa: Cloning of a cDNA coding for a trimethyllysine hydroxylase and functional expression in Saccharomyces cerevisiae. Paper presented at the Cape Biotechnology Forum. Somerset West, South Africa.

Swiegers, J H, Vaz, F M, Pretorius, I S, Wanders, R J and Bauer, F F. 2003. Ras regulates the carnitine shuttle bypass, the glyoxylate cycle, through suppression of cit2. Paper presented at the 21st International Conference on Yeast Genetics and Molecular Biology. 7-12 July, Goteborg, Sweden.

Du Toit, M A. 2003. Biopreservation and increasing the wholesomeness of wine. Presentation to the Lallemand Scientific Meeting: Beverages, Fermentation, Security and Health. San Francisco, California, United States.

Becker, J, Trollope, K, Van der Merwe, M J, Du Toit, M A, Lambrechts, M G, Vivier, M A and Pretorius, I S. 2004. Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the antioxidant resveratrol. Paper presented at the 13th Conference of the South African Society for Microbiology. 2-5 April, Stellenbosch, South Africa.

Downing, L, Du Toit, M A, Lambrechts, M G, Muller-Patterson, E, La Grange-Nel, K and Pretorius, I S. 2004. Biogenic amines in wine – the South African situation. Paper presented at the 13th Conference of the South African Society for Microbiology. 2-5 April, Stellenbosch, South Africa.

Downing, L, Patterson, H E, La Grange-Nel, K, Pretorius, I S, Lambrechts, M G and Du Toit, M A. 2004. Biogenic amines in wine – the South African situation. Paper presented at the 19th International ICFMH Symposium Food Micro. 12-16 September, Portoroza Slovenia.

Downing, L, Patterson, H E, La Grange-Nel, K, Pretorius, I S, Lambrechts, M G and Du Toit, M A. 2004. Biogenic amines in wine – the South African situation. Paper presented at the 12th Australian Wine Industry Technical Conference. Melbourne, Australia.

Downing, L, Patterson, H E, La Grange-Nel, K, Pretorius, I S, Lambrechts, M G and Du Toit, M A. 2004. Biogenic amines in wine – the South African situation. Paper presented at the 13th Conference of the South African Society for Microbiology. 2-5 April, Stellenbosch, South Africa.

Becker, J, Trollope, K, Van Der Merwe, M J, Du Toit, M A, Lambrechts, M G, Vivier, M A and Pretorius, I S. 2004. Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the antioxidant resveratrol. Paper presented at the 13th Conference of the South African Society for Microbiology. 2-5 April, Stellenbosch, South Africa.

Bauer, F F, Pretorius, I S and Swiegers, J H. 2004. Cloning and expression of Neurospora crassa carnitine biosynthesis genes in Saccharomyces cerevisiae. Paper presented at the 13th Conference of the South African Society for Microbiology. 2-5 April, Stellenbosch, South Africa.

Kroppenstedt, S, Van Der Merwe, M J, Pretorius, I S and Bauer, F F. 2004. Molecular characterisation of the three carnitine acetyltransferases in Saccharomyces cerevisiae. Paper presented at the13th Conference of the South African Society for Microbiology. 2-5 April, Stellenbosch, South Africa.

Du Toit, M A. 2004. Biogenic amines: A South African case study. Presentation to Cape WineLab, SA Wine Laboratories and Elsenburg. Stellenbosch, South Africa.

Du Toit, M A. 2004. Wine and the red cheeks – biogenic amines. Paper presented at the 8th Anchor Biotechnology Technical Symposium. Stellenbosch, South Africa.

Van Rensburg P, Stidwell, T, Lambrechts, M G, Cordero Otero, R R and Pretorius, I S. 2004. The application of ß-glucosidase producing recombinant wine yeast strains in wine production. Paper presented at the 18th Congress of the South African Genetics Society. 4-7 April, Stellenbosch. South Africa

Van Rensburg, P, Ubeda Iranzo, J F, Briones-Perez, A I, Potgieter, N, Arevalo Villena, M, Pretorius, I S and Cordero Otero, R R. 2004. Evaluation of ß-glucosidases from the grape native yeasts. Paper presented at the 13th Conference of the South African Society for Microbiology. 2-5 April, Stellenbosch, South Africa.

Du Toit, M A, Trollope, K M, Vivier, M A and Van Rensburg, P. 2005. Enhancing the wholesomeness of wine. Paper presented at the 18th Congress of the South African Association for Food Science and Technology (SAAFoST). 5-8 September, Stellenbosch, South Africa.

Du Toit, M A, Downing, L, Le Grange-Nel, K and Lambrechts, M G. 2005. What about biogenic amines, Ochratoxin A etc.? Paper presented at the International Conference of Health and Wine: Vindaba 2005. 13-16 September, Stellenbosch, South Africa.

Du Toit, M A. 2005. What about biogenic amines? Lake International Technologies: Information Day, Stellenbosch.

Du Toit, M A, Trollope, K M, Topley, E and Vivier, M A. 2006. Using Saccharomyces cerevisiae to make wine more healthy. Paper presented at the 20th International Conference of the International Committee on Food Microbiology and Hygiene: Food Micro 2006. 29 August – 2 September, Bologna, Italy.

Smit, A Y, Du Toit, W J and Du Toit, M A. 2005. The influence of wine parameters on the production of biogenic amines by lactic acid bacteria. Paper presented at the 3rd International Congress of the South African Society for Viticulture and Enology. 28-30 July, Cape Town, South Africa.

Trollope, K M, Du Toit, M A and Vivier, M A. 2005. Genetic engineering of Saccharomyces cerevisiae to increase resveratrol levels in wine. Paper presented at the 3rd International Congress of the South African Society for Viticulture and Enology. 28-30 July, Cape Town, South Africa

Ranwedzi, N, Smit, A, Van Rensburg, P and Cordero Otero, R R. 2005. Optimization of ß-glucosidase activity in recombinant Saccharomyces cerevisiae. Paper presented at the 3rd International Congress of the South African Society for Viticulture and Enology. 28-July, Cape Town, South Africa.

Trollope, K M, Du Toit, M A and Vivier, M A. 2006. Genetic engineering of Saccharomyces cerevisiae to increase resveratrol levels in wine. Paper presented at the 14th Conference of the South African Society for Microbiology. 9-12 April, Pretoria, South Africa.

Ranwedzi, N, Smit, A, Cordero Otero, R R and Van Rensburg, P. 2006. Optimizing ß-glucosidase enzyme production in wine yeast. Paper presented at the 14th Conference of the South African Society for Microbiology. 9-12 April, Pretoria, South Africa.

Smit, A, Du Toit, M A, Navascüs, E and Moreno-Arribas, M V 2007. Influence of winemaking practices on biogenic amine content in wines: A perspective of South African and Spanish winemaking conditions. Paper presented at the International Symposium of Microbiology and Integral Quality of Wine: Microbiology and Food Safety of Wine. Vilafranca del Penedes, Spain.

Article
Swiegers, J H, Vaz, F M, Pretorius, I S, Wanders, R J A, Bauer, F F. 2002. Carnitine biosynthesis in Neurospora crassa: Identification of a cDNA coding for N-trimethyllysine hydroxylase and its functionals expression in Saccharomyces cerevisiae, FEMS Microbiology Letters, v. 210 (1) (p. 19-23)

Cordero Otero, R R, Ubeda Iranzo, J F, Briones-Perez, A I, Potgieter, N, Arevalo Villena, M, Pretorius, I S, Van Rensburg, P. 2003. Characterisation of the ß-glucosidase activity produced by enological strains of non-Saccharomyces yeasts, Journal of Food Science, v. 68 (8) (p. 2564-2569)

Du Toit, M A. 2005. Biogeenamienproduksie in wyn, WineLand, Mnth Jun (p. 86)

Van Rensburg, P, Stidwell, T, Lambrechts, M G, Cordero Otero, R R, Pretorius, I S. 2005. Development and assessment of a recombinant Saccharomyces cerevisiae wine yeast producing two aroma-enhancing ß-glucosidase encoded by the Saccharomycopses fibuligeria BGL1 and BLG2 genes, Annals of Microbiology, v. 55 (1) (p. 33-42)

Franken, J, Kroppenstedt, S, Swiegers, J H, Bauer, F F. 2008. Carnitine and carnitine acetyltransferases in the yeast Saccharomyces cerevisiae: A role for carnitine in stress protection, Current Genetics, Mnth Jun v. 53 (6) (p. 347-360)

Paper
Downing, L, Patterton, H E, Le Grange-Nel, K, Lambrechts, M G, Pretorius, I S, Du Toit, M. 2005. Biogenic amines in wine: The South African situation, IN: Blair, R (ed). Proceedings of the 12th Australian Wine Industry Technical Conference. 24-29 July 2004,Melbourne, Australia. (p. 351) Australian Wine Research Institute, Adelaide, Australia.

FinalReport.pdf

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