13,590
edits
Changes
update to VBNC
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 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>[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>.
===Sensory Analysis===