**5. Protein removal by bentonite and potential alternatives**

Removal of the proteins remaining in finished wine before bottling is critical for wine protein stabilisation. In the wine industry, bentonite, a swelling 2:1 aluminosilicate clay, is usually added to wine to remove the proteins [85]. As proteins in wine are positively charged at wine pH and bentonite carries a net negative charge, wine proteins can be absorbed onto bentonite by cationic exchange [1, 85]. Hsu and Heatherbell [86] have shown that wine proteins with higher pI (5.8–8.0) and intermediate MW (32–45 kDa) are preferentially removed by bentonite fining, but a proportion of wine proteins, which have a MW range from 60 to 65 kDa and pI range from 4.1 to 8.0, are highly resistant to removal by bentonite fining. In that study, the authors also pointed out that the removal of proteins with lower pI (4.1–5.8) and lower MW (12.6 and 20–30 kDa) was necessary for protein stabilisation. In contrast to this conclusion, another study [56] showed that the amount of protein depletion correlated linearly with the level of bentonite addition, implying no bentonite selectivity based on isoelectric point. Different conclusions from these two studies might be partly attributed to the different methods used to separate and quantify the proteins [87].

Bentonite fining is effective in removing proteins to stabilise the wine, but the use of bentonite could also remove some important aroma and flavour compounds [88], as well as result in loss of wine as lees [89]. Thus, alternative treatments for protein stabilisation that can reduce or eliminate the use of bentonite are of great interest for winemakers. Potential bentonite alternatives that have been studied are summarised in **Table 2**.

Ultrafiltration is very effective in removing proteins in wine, and up to 90% of wine proteins can be retained using a 10 kDa molecular weight cut-off membrane; as a result, bentonite requirement is greatly reduced [90]. However, in addition to the high setup and running costs, ultrafiltration could also result in the loss of beneficial aroma compounds in wine [91]. Studies on Riesling and Gewürztraminer wines using ultrafiltration with membrane nominal MW cut-off from 10 to 50 kDa showed that ultrafiltration can significantly decrease the overall aroma intensity and fruity, floral, sweet and honey/caramel aromas, but also increase vegetative aroma [92]. Furthermore, a reduction in browning colour (A420 nm) and total phenolics was also observed in filtered Riesling and Gewürztraminer wines [93].

Short-term pasteurisation (90°C for 1 min) can reduce bentonite requirement up to 70% [94], but heating of juice could impart negative sensory implications into resultant wine [95]. A recent study reported that applying heating of juice at 75°C for 1 min in the presence of a heat-tolerant protease, aspergillopepsin (AGP), derived from fungus *Aspergillus niger*, before fermentation showed a significant reduction of PR proteins without damaging wine quality [96]. In general, thaumatin-like proteins and chitinases are highly resistant to proteases, but heating unfolds these proteins and thus they can be degraded by protease. Another promising protease BcAP8 (aspartic acid protease) from *Botrytis cinerea* has been proven to

**213**

**Table 2.**

*Pathogenesis-Related Proteins in Wine and White Wine Protein Stabilization*

and reduction of bentonite

Acceleration of protein denaturation in juice and thus lower bentonite requirement

proteins during flash pasteurisation

without heat denaturation

white wines at low addition rates without deleterious sensory

and protect wine from browning

**Advantages Disadvantages Reference**

High setup and running costs and possible loss of flavour compounds

Impart negative sensory implications into wine

Heating required for better efficiency, which may alter sensory properties of wine

Does not remove all PR

Long-term prevention of protein haze formation is

Interact with phenolics which may alter wine colour and texture

Removal of nanoparticles

Not a good settling agent [99, 100]

proteins

unknown

and the costs

[90, 91]

[94, 95]

[96]

[97]

[101]

[103]

[104, 105]

**Ultrafiltration**

**Heating**

**Enzymes**

**Polysaccharides**

be effective against grape chitinases during juice fermentation without the need for heating [97]. This protease could potentially reduce the bentonite requirement, but

**Nanomaterials**

Novel fining agents such as polysaccharides could be another potential class of bentonite alternatives [98]. Carrageenan, a food grade polysaccharide extracted from seaweeds, is effective in heat stabilising white wines at low addition rates (125–250 mg/L) without deleterious sensory impacts compared to bentonite treated wines [99, 100]. However, technical issues including frothing, slower filterability and risk of over-fining should be considered when applying carrageenan for protein stabilisation, particularly when it is used prior to or during fermentation. In addition, yeast mannoproteins, the highly glycosylated polypeptides present in yeast cell walls, have also been reported to have a protective effect on protein haze formation in wine [101]. Thus, mannoproteins extracted from purified yeast cell walls could be added into juice/wine to reduce the addition of bentonite, but further research is required to understand whether this protection against protein aggregation is suitable for long-term wine storage. Chitin [102] and chitosan [103], polysaccharides principally from *Aspergillus niger*, also have potential to remove haze-forming

it is less effective on the degradation of thaumatin-like proteins.

in removing proteins

*DOI: http://dx.doi.org/10.5772/intechopen.92445*

Ultrafiltration Efficient in protein removal

requirement

in wine

Aspartic acid protease Capable of degrading chitinases

Carrageenan Effective in heat stabilising

impacts

formation in wine

Mannoproteins Protective effect on protein haze

Chitin and chitosan Interact and remove PR proteins

Nanoparticles High surface area and efficiency

*Summary of bentonite alternatives for white wine protein stabilisation.*

Aspergillopepsin Very active towards wine

**Bentonite alternatives**

Short-time pasteurisation


*Pathogenesis-Related Proteins in Wine and White Wine Protein Stabilization DOI: http://dx.doi.org/10.5772/intechopen.92445*

#### **Table 2.**

*Chemistry and Biochemistry of Winemaking, Wine Stabilization and Aging*

**5. Protein removal by bentonite and potential alternatives**

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.

Removal of the proteins remaining in finished wine before bottling is critical for wine protein stabilisation. In the wine industry, bentonite, a swelling 2:1 aluminosilicate clay, is usually added to wine to remove the proteins [85]. As proteins in wine are positively charged at wine pH and bentonite carries a net negative charge, wine proteins can be absorbed onto bentonite by cationic exchange [1, 85]. Hsu and Heatherbell [86] have shown that wine proteins with higher pI (5.8–8.0) and intermediate MW (32–45 kDa) are preferentially removed by bentonite fining, but a proportion of wine proteins, which have a MW range from 60 to 65 kDa and pI range from 4.1 to 8.0, are highly resistant to removal by bentonite fining. In that study, the authors also pointed out that the removal of proteins with lower pI (4.1–5.8) and lower MW (12.6 and 20–30 kDa) was necessary for protein stabilisation. In contrast to this conclusion, another study [56] showed that the amount of protein depletion correlated linearly with the level of bentonite addition, implying no bentonite selectivity based on isoelectric point. Different conclusions from these two studies might be partly attributed to the different methods used to separate and

Bentonite fining is effective in removing proteins to stabilise the wine, but the use of bentonite could also remove some important aroma and flavour compounds [88], as well as result in loss of wine as lees [89]. Thus, alternative treatments for protein stabilisation that can reduce or eliminate the use of bentonite are of great interest for winemakers. Potential bentonite alternatives that have been studied are

Ultrafiltration is very effective in removing proteins in wine, and up to 90% of wine proteins can be retained using a 10 kDa molecular weight cut-off membrane; as a result, bentonite requirement is greatly reduced [90]. However, in addition to the high setup and running costs, ultrafiltration could also result in the loss of beneficial aroma compounds in wine [91]. Studies on Riesling and Gewürztraminer wines using ultrafiltration with membrane nominal MW cut-off from 10 to 50 kDa showed that ultrafiltration can significantly decrease the overall aroma intensity and fruity, floral, sweet and honey/caramel aromas, but also increase vegetative aroma [92]. Furthermore, a reduction in browning colour (A420 nm) and total phenolics was also observed in filtered Riesling and Gewürztraminer wines [93]. Short-term pasteurisation (90°C for 1 min) can reduce bentonite requirement up to 70% [94], but heating of juice could impart negative sensory implications into resultant wine [95]. A recent study reported that applying heating of juice at 75°C for 1 min in the presence of a heat-tolerant protease, aspergillopepsin (AGP), derived from fungus *Aspergillus niger*, before fermentation showed a significant reduction of PR proteins without damaging wine quality [96]. In general, thaumatin-like proteins and chitinases are highly resistant to proteases, but heating unfolds these proteins and thus they can be degraded by protease. Another promising protease BcAP8 (aspartic acid protease) from *Botrytis cinerea* has been proven to

**212**

quantify the proteins [87].

summarised in **Table 2**.

*Summary of bentonite alternatives for white wine protein stabilisation.*

be effective against grape chitinases during juice fermentation without the need for heating [97]. This protease could potentially reduce the bentonite requirement, but it is less effective on the degradation of thaumatin-like proteins.

Novel fining agents such as polysaccharides could be another potential class of bentonite alternatives [98]. Carrageenan, a food grade polysaccharide extracted from seaweeds, is effective in heat stabilising white wines at low addition rates (125–250 mg/L) without deleterious sensory impacts compared to bentonite treated wines [99, 100]. However, technical issues including frothing, slower filterability and risk of over-fining should be considered when applying carrageenan for protein stabilisation, particularly when it is used prior to or during fermentation. In addition, yeast mannoproteins, the highly glycosylated polypeptides present in yeast cell walls, have also been reported to have a protective effect on protein haze formation in wine [101]. Thus, mannoproteins extracted from purified yeast cell walls could be added into juice/wine to reduce the addition of bentonite, but further research is required to understand whether this protection against protein aggregation is suitable for long-term wine storage. Chitin [102] and chitosan [103], polysaccharides principally from *Aspergillus niger*, also have potential to remove haze-forming

proteins in wine, but the wine colour and texture could be affected as chitosan interacts with phenolics and organic acids in wine.

Nanotechnology is currently a very active research topic in food science. Recently some researchers also become interested in using nanomaterials to remove proteins in wine. Magnetic steel nanoparticles coated with acrylic acid have been tested and are highly efficient in attracting and thus removing haze-forming proteins [104]. Another study using mesoporous nanomaterials to fine Muscat Ottonel and Pedro Ximenez wines also confirmed the high efficiency of nanomaterials in removing haze-forming proteins, and the loss of aroma compounds in wine due to addition of nanomaterials was even less than bentonite [105]. Nanomaterials have shown great potential as bentonite alternative to remove proteins in wine, but the cost of coating the nanoparticles and the removal of nanoparticles from wine after the treatment are the main concerns for their wide application in the wine industry.
