Changes

Diastatic strains of ''Saccharomyces cerevisiae'' can have a wide range of temperature tolerance. Strains that can form ascospores or vegetative cells can be more heat tolerant. One thesis paper reported it taking 9 minutes at 60°C to kill 90% of a strain of diastatic ''S. cerevisiae'' in ascospore form <ref>[https://www.sciencedirect.com/science/article/pii/S2214799322000170#bib0135 Spoilage yeasts in beer and beer products. Inge M Suiker, Han AB Wösten. Current Opinion in Food Science. Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. 02/19/2022.]</ref>.

Star San has only been officially tested by Five Star Chemicals against the pathogenic bacteria species ''E. coli'' and ''S. Aureus'', which is the minimum baseline required by the EPA to be labeled a "sanitizer" <ref>[https://www.homebrewersassociation.org/forum/index.php?topic=31537.msg409346#msg409346 Conn, Denny. American Homebrew Association forums. 04/08/2018. retrieved 07/09/2018.]</ref>. While we are not aware of any publicly available published studies on the efficacy of StarSan to kill yeast, several studies with other acid anionic sanitizers have confirmed that they are effective against yeast. Lee et al. (2007) found that an acid sanitizer very similar to Star San that uses citric acid instead of phosphate but the same surfactant (sodium dodecylbenzene sulfonate) took 5 minutes to kill ''Saccharomyces cerevisiae'', ''E. coli'', and ''Listeria innocua'' at room temperature (some species were killed faster than others with the ''E. coli'' actually being more resistant than the yeast), and one minute if the sanitizer was heated to 40°C on both metal and LDPE plastic (they compared the acid anionic sanitizer to 35% hydrogen peroxide, which killed all organisms with 15 seconds, indicating that this acid anionic sanitizer is effective at killing yeast, but it takes longer than a stronger chemical such as hydrogen peroxide). This study did not make mention of biofilms, however, the cultures were allowed to grow and dry overnight which could have allowed for biofilm formation <ref>[https://onlinelibrary.wiley.com/doi/full/10.1111/j.1750-3841.2007.00496.x Efficacy of Two Acidic Sanitizers for Microbial Reduction on Metal Cans and Low-Density Polyethylene Film Surfaces. J. LEE, M.J. GUPTA, J. LOPES, AND M.A. PASCALL. 2007.]</ref>. Five star Star Chemicals [httphttps://www.fivestarchemicalsbsgcraft.com/wp-contentResources/Craftbrewing/PDFs/uploadsProduct_Spec_and_Data_Sheets/Star-San-HB4Product_Spec_Sheets/StarSan_Tech.pdf also recommends 5 minutes of contact time with Star San]. Winniczuk et al. (1997) found that three phosphoric acid anionic sanitizers ("CS-100" and "CS-101-lf" by Chemical Systems of Florida, and "Clear-Clean" by Pelican Brand) were less effective at killing yeast than bacteria in the timeframe tested (1 minute contact time), but they were still effective at killing yeast at high concentrations (peracetic acid also required a higher concentration to kill yeast than bacteria). However, one of the acid anionic sanitizers tested was more effective than the other two, indicating that the chemical makeup of the particular acid anionic sanitizer has an impact on how effective it is as a sanitizer relative to other acid anionic sanitizers. Additionally, they found that peracetic acid, iodophor, and chlorine dioxide required less concentration than the acid anionic sanitizers to be effective (again, tested at 1 minute exposure time) <ref>[http://lp7lc5er8n.scholar.serialssolutions.com/?sid=google&auinit=PP&aulast=Winniczuk&atitle=Minimum+inhibitory+concentrations+of+antimicrobials+against+micro-organisms+related+to+citrus+juice&id=doi:10.1006/fmic.1997.0103&title=Food+microbiology&volume=14&issue=4&date=1997&spage=373&issn=0740-0020 Minimum inhibitory concentrations of antimicrobials against micro-organisms related to citrus juice. P.P Winniczuk, M.E Parish. 1997.]</ref>.

"Viable but nonculturable" ('''VBNC''') is a newly identified state for bacteria that are not able to grow or form colonies on typical growth media (i.e., lack of cell division), but they remain viable (alive) and retain a limited level of metabolic activity (reduced nutrient transport, respiration, and synthesis of compounds) while sometimes being able to regain their population when returned to a more ideal environment. The cells often exhibit dwarfing, and they can remain in this state without dying for 4-12+ months depending on the species. An appropriate viability test for a given species can be performed to show that the cells are not dead, even though they don't grow on typical growth media (for example, intracellular hydrolysis of CTC or reduction of INT as an indication of metabolic activity, by establishing the presence of an intact cytoplasmic membrane via BacLight® or propidium iodide, or by multi-parameter flow cytometry). Cells enter this state as a way to survive some sort of stress in their environment (for example, osmotic stress, too much oxygen exposure, exposure to white light, etc.). Treatments such as pasteurization in milk and chlorination of wastewater have also been shown to induce VBNC. A number of species have been found to be able to enter the VBNC state, including ''E. coli'', ''Lactobacillus plantarum'', ''L. lactis'', ''L. linderi'', ''L. casei'', ''L. plantarum'', ''L. paracollinocides'', ''L. acetotolerans'', and several species of ''Salmonella''. Early studies on VBNC microbes were not able to fully show that the resuscitation was truly from VBNC cells rather than a very small number of culturable cells, but later studies were able to show that some bacteria can be resuscitated from a VBNC state, although most bacteria that enter a VBNC state have not been shown to be able to be resuscitated <ref>[https://pdfs.semanticscholar.org/e661/934dca6bb0dbb31a8781f3193232b7b5a8a4.pdf The Viable but Nonculturable State in Bacteria. James D. Oliver. The Journal of Microbiology. 2005.]</ref><ref name="Liu_2018">[https://www.frontiersin.org/articles/10.3389/fmicb.2018.02076/full#B28 Induction and Recovery of the Viable but Nonculturable State of Hop-Resistance Lactobacillus brevis. Junyan Liu, Yang Deng, Thanapop Soteyome, Yanyan Li, Jianyu Su, Lin Li, Bing Li, Mark E. Shirtliff, Zhenbo Xu, and Brian M. Peters. Front. Microbiol. 2018. DOI: https://doi.org/10.3389/fmicb.2018.02076.]</ref>. The concept of VBNC cells is somewhat controversial in microbiology; some experts argue that there is no difference between so-called "VBNC" cells and persister cells <ref>[https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.15463 Song, S. and Wood, T.K. (2021), ‘Viable but non-culturable cells’ are dead. Environ Microbiol, 23: 2335-2338. https://doi.org/10.1111/1462-2920.15463.]</ref>. See also this [https://www.facebook.com/groups/MilkTheFunk/posts/6036200423074788/ MTF thread by Dr. Bryan Heit].

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 SO₂, 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 SO₂ 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 SO₂ is the only form of SO₂ 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>.