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Quality Assurance

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While the VBNC state has mostly only been studied in detail for bacteria, it has also been suggested that this state is also possible with eukaryotes (yeast). It has been reported that although there are many methods for detecting ''Brettanomyces'' in winemaking, there are cases when ''Brettanomyces'' is not found using culturing techniques, but years later have still infected wine. Agnolucci et al. (2010) found that sulfur dioxide induces the VBNC state in 7 ''Brettanomyces'' strains isolated from wine at concentrations of 0.2 mg/L (molecular SO<sub>2</sub>) for 5 out of the 7 strains to 0.4 mg/L for the other 2 strains after 24 hours of incubation in a synthetic wine medium that was supplemented with various amounts of sulfur dioxide. At 0.4 mg/L they found that all but one strain had 0% culturable cells, and 4-26% VBNC cells depending on the strain. At 0.8 mg/L, no strains had culturable cells, but they all had at least 4.6-17% VBNC cells (percent of the original number of cells before being exposed to the sulfur dioxide). Even at 1 mg/L of sulfur dioxide levels, there were 2.9-15% VBNC cells, depending on the strain (the ability for the ''Brettanomyces'' to remain viable at 1mg/L of sulfur dioxide might have also been due to the pH only being 3.5, and ethanol only being 13%). They also found that 2.1 mg/L was required to reduce the VBNC state of cells to zero after 55 days of incubation and limit the amount of ethyl phenols produced by ''Brettanomyces''. They reported that trypan blue was the best method for detecting VBNC cells <ref>[https://www.sciencedirect.com/science/article/pii/S0168160510003958?via%3Dihub Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis. Agnolucci M, Rea F, Sbrana C, Cristani C, Fracassetti D, Tirelli A, Nuti M. 2010. DOI: https://doi.org/10.1016/j.ijfoodmicro.2010.07.022.]</ref>.
The Agnolucci et al. (2010) study did not provide a method or data for resuscitating the VBNC ''Brettanomyces'' cells so that they can again divide and grow colonies, and this has been criticized because resuscitation of VBNC cells is considered an important aspect of strengthening the conclusion that the cells are indeed VBNC. Serpaggi et al. (2012) found similar results with using 0.8 mg/L of molecular SO<sub>2</sub>, which resulted in all ''Brettanomyces'' cells from one wine strain to not be culturable on YPD after being incubated in synthetic wine medium that was supplemented with SO<sub>2</sub> for 2 days, while the viability of the cells remained high for as long as 11 days (they did not check viability after 11 days, and the viability count remained constant from day 2 to day 11; viability was determined by staining with fluorescein diacetate which stains when certain metabolic esterase activity is present). They were able to resuscitate the cells by adding NaOH to the media to bring the pH up from 3.5 to 4.0 in order to effectively eliminate the molecular sulfur dioxide (molecular SO<sub>2</sub> is the only form of SO<sub>2</sub> that is significantly effective at inhibiting microbes, and it is only stable at very low pH's). They noted that VBNC cells were about 20% smaller in size than culturable cells. The cells in the VBNC state did not produce 4EG phenol but did produce a very small amount of 4EP phenol <ref>[https://www.ncbi.nlm.nih.gov/pubmed/22365358 Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces. Serpaggi V, Remize F, Recorbet G, Gaudot-Dumas E, Sequeira-Le Grand A, Alexandre H. 2012. DOI: 10.1016/j.fm.2011.12.020.]</ref>. It has also been demonstrated that the presence of minerals and vitamins, as well as p-coumaric acid, can assist in resuscitating so-called VBNC cells of ''Brettanomyces'' <ref>[https://www.mdpi.com/2306-5710/9/3/69 Chandra M, Branco P, Prista C, Malfeito-Ferreira M. Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensis. Beverages. 2023; 9(3):69. https://doi.org/10.3390/beverages9030069.]</ref>.
It is worth noting that the VBNC state in ''Brettanomyces'' has not been tested against a higher concentration of SO<sub>2</sub> (for example when wineries use much higher concentrations of SO<sub>2</sub> solutions as a sanitizer), other chemical sanitizers, and pasteurization-level temperatures. For example, it has been proposed that steaming barrels in order for them to reach 140°F (60°C) for 20 minutes is enough to sanitize them (see [[Barrel#Sanitizing|Barrel Sanitizing]] for more information). However, Nunes de Lima et al. (2020) inoculated wines with various strains of ''Brettanomyces'' after raising the pH of the wine to 3.8, which rendered the amount of free SO<sub>2</sub> in the wines completely ineffective, and many of the strains entered a VBNC state. This indicates that other unknown factors can induce a VBNC state in ''Brettanomyces''. It has been documented that other factors, such as nutrient starvation, extreme temperatures, osmotic pressure and oxygen, has caused a VBNC state in other microorganisms (VBNC has been mostly studied in bacteria) <ref name="Nunes de Lima 2020">[https://www.sciencedirect.com/science/article/abs/pii/S0740002020302069 Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines. Adriana Nunes de Lima, Rui Magalhães, Francisco Manuel Campos, José António Couto. 2020. DOI: https://doi.org/10.1016/j.fm.2020.103617.]</ref>.

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