**4. Protein stability tests**

To avoid protein haze formation in bottled white wine, a protein stability test is usually conducted before bottling in the winery. If the wine is not protein stabilised, a range of bentonite fining trials will be carried out to determine the minimum required dosage of bentonite addition for protein stabilisation. The most common protein stability test is the heat test, which is a heating procedure to force protein haze formation. Wine samples are normally heated to 80°C for 6 h and then left to cool down to 4°C overnight. The turbidity in heated wine samples is measured by a nephelometer and expressed as nephelometer turbidity units (NTU). Turbidity measurement of less than 2.0 NTU is usually recommended. Different temperatures and durations of heating could have a great impact on the resulting haze formation [81]. A recent study suggests that the less severe condition of heating at 80°C for 2 h is more appropriate to predict bentonite requirement for wine stored in the short term to medium term [82]. The cooling temperature and time are also critical to the accuracy of heat test results. A recent study [83] investigated the influence of heating and cooling conditions on protein heat test results. In this study, white wines were heated at 80°C for a time ranging from 0.5 to 6.0 h and then cooled down for 0.5–18 h at 0, 4 or 20°C, respectively. The results indicated that heating at 80°C for 2 h and then cooled at 20°C for 3 h enabled the repeatable production of haze and bentonite requirement.

As traditional heat test is very time-consuming, near infrared (NIR) spectroscopy has been studied for its potential to predict protein stability with high efficiency [84]. Results from 111 white wines representing multiple regions and varieties in California showed that the turbidity of wine could be predicted from the short-wavelength NIR spectra, but further IR analysis on a large number of wines will be required for the application of NIR in the global wine industry, and the high cost of equipment may limit its widespread use.

There are also commercial reagents, i.e. Bentotest and Proteotest, available for winemakers to check the protein stability in white wine. In general, these commercial reagents are considered to be harsher than the heat test, and as a result, the addition of bentonite is normally more than the actual requirement for protein stabilisation. Other tests including trichloroacetic acid (TCA), tannin and ethanol tests were originally established for protein quantification by precipitation of proteins in wine, but they are rarely used in the winery for checking the protein stability as bentonite requirement can be significantly overestimated using those tests.
