1. Tom Mascara Technical Manager Enzyme Solutions Pty Ltd

2. Yeast Cell Wall Fermenting Yeast

3. Yeast Cell Wall Protease Action and Fermenting Yeast

4. Proteases in Yeast Fermentations Intact Yeast do not secrete their own proteases Adding additional proteases may help to break down protein and provide more assimilable peptides and amino acids for fermenting yeasts

5. Nutrient Requirements for Wine Successful fermentation from juice to wine require adequate levels of available Nitrogen. The amount of nitrogen required is dependent on the yeast strain and the fermentation conditions. Requirement is for minimum of 220 mg/L of nitrogen (YAN) for complete fermentation, higher sugar juices require more. Lack of nitrogen can lead to stuck fermentations

6. Wine Industry Practice Recommended to measure Yeast Assimilable Nitrogen (YAN) Sum of NH4 and Free Amino Nitrogen measurements Di-ammonium Phosphate often used as NH4 Commercial fermentation supplements (Yeast Foods) are also available – mixtures of DAP, inactivated yeast, vitamins, minerals.

7. Are all Nitrogen sources the same? Inorganic Sources DAP Organic Sources From the grape Addition of inactivated yeast in fermentation aid Organic sources are recognized as the preferred source – DAP is considered to be Junk Food for yeasts. It can lower wine pH and produce unwanted flavours

8. Hypothesis: Can adding a protease enzyme also assist in wine production by completing the fermentation and decreasing the time?

9. Typical Fermenter Setup

10. Plot: Density vs. Time Density ( o Baume ) Time ( Hrs)

12. Statistical Analysis All the results were adjusted to a zero baseline by adding 0.8 Be The area under each treatment curve was estimated by the area of a trapezoid method, Single Factor ANOVA and Multiple Comparison Tests to determine if there was significant differences Results were significantly different at the P= 0.01 level (99% confidence)

13. Summary of Results All fermentations performed similarly until they reached ~0 Be (7-8 days). Untreated control ferments slowed and appeared to stop at -0.4 Be, indicating a slow or stuck fermentation at day 14 At 0 Be, all the protease treatments continued to ferment, finally settling at -0.8 Be (would be considered finished in wine industry) Reduced fermentation time with 10 & 20 mg/L finished at 11 days, 2 mg/L finished at 12 days. Importantly - No off-flavours with the proteases

14. Conclusions Addition of protease has ensured white wine fermentations are complete with reduced fermentation times The response was dose related, and 10 mg/L seems to be sufficient as 20 mg/L only offered minimal advantage. Gives a winemaker additional confidence and potential to increase Plant throughput by reducing their fermentation times These results have led to the commercial formulation and release of a yeast nutrient containing protease activity.