Diastaticus
Saccharomyces cerevisiae var. diastaticus has developed a bad reputation. It has been the culprit in highly publicized recalls, and poses a serious economic and safety risk to brewers. On the other hand, diastatic yeast can produce the gold standard of a style — such as saison. A yeast contaminant can be much harder to identify than a bacterial species because of the similarities to brewer’s yeast. The differentiation between specific strains can be difficult and often utilizes several techniques. We will explore this fascinating bug to find what makes it tick, how it can be managed, and how it can be embraced. Let’s start with the blueprint for all biology — the genetic code.
Genetic code
Brewer’s yeast makes ethanol and CO2by metabolizing sugars in the wort. The enzyme glucoamylase plays a major role in this conversion in many yeast species. The production and function of this enzyme is affected by more than one gene, making it a polygenic trait. Diastaticus has been defined by the presence of the STA genes. While the exact function of this family is not known, it is accepted that STA1 affects the export of glucoamylase. As with all polygenic traits there are other mechanisms at play, but a simplified view would show glucoamylase being released by STA1-positive yeast (diastaticus) and not released by “normal” brewer’s yeast. The additional and less restricted enzyme is able to break down starches and other high molecular weight compounds into simple sugars, which can be fermented. In an open system this will lead to high attenuation and, often, a thin mouthfeel. However in a closed system with available carbohydrates, the increased pressure can lead to bottle gushers, and sometimes exploding cans and bottles. The problem isn’t the diastatic yeast itself, the problem is either improper use of a known strain or a contamination event. Brewers would be better able to manage either situation with better tools. Large breweries utilize sophisticated molecular genetic techniques that are often out of the reach of smaller breweries. Even on a genetic level there are few differences, and they sometimes don’t tell the whole story.
STA1 isn’t exactly a fingerprint for diastatic yeast, as stated in the simplified example. Just carrying a gene does not guarantee that it is expressed to a level to cause problems. Other, possibly uncharacterized genes, act as the gas pedals and the brakes to regulate gene expression. An organism’s collection of unique genes is known as its genotype. To complicate matters further, genes and traits are not 1-to-1. In addition to polygenic traits, phenotypic genes control two or more often unrelated traits. The genetic code is read through this landscape into a physical organism. Humans and yeast have observable characteristics — hair color, phenol production, alcohol tolerance. The complete collection of these characteristics resulting from the interaction of its genotype with the environment. Just testing for the STA1 gene does not tell the whole story. Combining genotype and phenotype testing would be the best way to evaluate a strain for re-fermentation risk. However, defining diastaticus has been a challenge since day one.
Discovery
Diastatic yeast was first reported in 1943 in the comprehensive A System of Wort Analysis by Bishop and Whitely. Later published in the Institute of Brewing in London, this report covers topics from yeast flocculation to fermentation vessel design. Researchers noted an “exceptional” secondary yeast that performed uniquely in their attenuation studies. In a well controlled setting all other pure cultures reduced an original gravity (OG) of 1.025 to an average of 1.011 in 48 hours, whereas this “Sample #164” consistently fermented to below 1.005 in the same amount of time. The researchers hypothesized that this yeast was able to export diastase enzyme and break down starches and ferment them. They tested this by preparing growth media with starch as the sole carbon source, and were able to track fermentation. Bishop and Whitely hypothesized that other yeast could excrete the enzyme as well, but at varying levels. They noted that this could explain the conflicting reports at the time about the ability to ferment dextrins. Understanding some of the checks and balances written into the genetic code, the diastatic trait is a spectrum. This makes the jobs of brewery quality control teams even more difficult.
I worked with Dr. Matthew Farber of the University of Sciences brewing program in Philadelphia, Pennsylvania to survey many of the current techniques for identifying diastaticus in order to propose a recommendation to breweries. We wanted to cover assays that could fit into the quality control efforts of nano- to macro-scale breweries.
Methods
Yeast Strains — A collection of commercial yeast were used in this series of assays. Of the nine strains chosen for these experiments, eight had been known to contain the STA1 gene. Generally these were high-attenuating strains, meaning they fermented completely resulting in a dry beer. They tended to be Belgian-type strains and most were used in the brewing of saison styles. All known diastatic strains are POF (phenolic off flavor) positive. They carry a gene that causes them to produce 4-vinyl guaiacol or other phenolic molecules at a detectable level. Not all yeast chosen produce these molecules under normal brewing conditions. Controls for these experiments were two English ale strains that are not considered high attenuating or diastatic. Along with these commercial strains, four industry isolates were also used. These came from a contamination event at a brewery.
Medias — Selective media promotes the growth of certain microorganisms over others, and is an important tool in any quality lab. Newly developed Farber Phan Diastatic Media (FPDM) uses cupric sulfate as a selective agent. Although the biology is not well understood, diastatic yeast appear to tolerate levels of copper higher than control yeast. FPDM contains soluble starch as a food source for the diastatic yeast that are able to metabolize high molecular weight carbohydrates. Once the plates are chilled the starches become more opaque. If the yeast is able to metabolize them there will be a zone of clearing around the colony. See Chart 1 below for a result of our FPDM test on the nine selected commercial strains.
PCR Assay — Researchers and quality control personnel often use polymerase chain reaction (PCR) to identify a specific gene of interest. This technique works by amplifying a specific gene and detecting the number of copies. Philadelphia-based Invisible Sentinel brings this powerful molecular technology to the food and beverage industry. They have a line of products designed specifically for the needs of modern brewers. Their simple cassette format allows brewers to test samples for organisms including Pediococcus, Lactobacillus, and Saccharomyces diastaticus. We ran our yeast library on the brewSTAT platform for rapid PCR-based detection of the STA1 gene. We began by placing individual colonies of each strain into the pre-filled tubes. The tubes went through an amplification cycle of heating and cooling before being read. This test kit uses antibody-based cassettes to generate a readout. The samples were pipetted onto the sample well and left to develop for two minutes, then the switch was retracted to reveal the results (shown in Chart 2).
Sporulation Staining — Contaminants that can form spores pose a greater threat to producers, as they are able to evade conventional sanitation measures. Fixed cells were stained and observed under a microscope to determine spore-forming ability. In order to induce sporulation, colonies grown on yeast extract peptone dextrose (YPD) were streaked for isolation on sodium acetate agar plates. They were incubated at room temperature for three days, out of direct sunlight. A thin layer of cells were heat-fixed onto microscope slides using a Bunsen burner. The slides were placed over a beaker of hot water to keep them damp with steam as the stains were applied. Blotting paper was saturated with Malachite green and placed over the slides. The paper was removed and the slides were rinsed with water after ten minutes. A secondary stain of Safranin was applied using blotting paper for two minutes. The primary stain will stain all spores blue, and the secondary stain will stain all other cells pink. The slides were thoroughly rinsed off and observed at 400x magnification. Results shown on Chart 3, below.
Over-Attenuation Assay — The major concern over diastaticus is its ability to re-ferment packaged beer. In order to simulate this scenario in the lab, we introduced the strains being tested to finished beer and allowed them to ferment (if they could) for a week. The beer was filtered to remove any foreign microbes. The yeast were grown overnight in media, then in a 50/50 mixture of the broth and the beer in order to allow them to adjust to the new environment. Tubes with 10 mL of filtered beer were inoculated with the same amount of yeast and placed in the incubator for seven days. They were then moved to a 39 °F (4 ºC) refrigerator to simulate cold crashing to cause the yeast to fall out of solution. The density of each sample was determined using a densitometer. The data was normalized for evaporation using a control tube that went in the incubator and refrigerator with the other tubes but was not inoculated with any yeast. Each of the test strains were compared to a non-diastatic control English ale yeast. A strain is considered potentially able to over-attenuate when it ferments to three specific gravity points below the standard. The results from this test are shown in Chart 4.
Biofilms
When microorganisms find a suitable environment, with a comfortable temperature and consistent access to nutrients, they can establish communities called biofilms on surfaces. They produce a matrix for better adhesion and to facilitate the transfer of signaling molecules between cells. This microbial “slime” acts as a pseudo-organism with cells at different layers taking on different roles. Channels are formed to distribute nutrients deep into the biofilm and release waste products. Cells can detach from the biofilm and colonize new surfaces. These structures tend to form in areas with liquid flow and high nutrient levels. The biofilm allows the microbes to flourish off the nutrients while providing protection against the shear of the flow. Microbes in biofilms are more resistant to biocidal treatments. The matrix offers a physical barrier. Another major concern over biofilms is the presence of “persister” cells. These cells are in a dormant state and play a major role in the capacity of biofilms to survive and recover from disturbances.
There is evidence that in addition to spore-forming, diastatic yeast are better able to form biofilms. Cleaning protocols must be stringent to discourage biofilm formation and clear off any that starts to form. Equipment in a brewery makes the perfect environment for biofilms. Sweet wort and carbohydrate-loaded beer flows quickly through hundreds or thousands of feet of tubing and plumbing in an average-size brewery. Stainless steel is particularly vulnerable to befouling. Any scratches in a pipe or your fermenting bucket, or bad welds, extruded gaskets, and non-sanitary valves, fittings, or pumps could provide a community of microbes a hiding place from your cleaning procedure.
UK standards recommend the application of a caustic agent for 30 minutes to clean lines that are regularly cleaned and not heavily contaminated. In laboratory testing, thick biofilms required longer applications of 60 minutes with fresh cleaner passed through every ten minutes. Scientists are working to develop better biofilm removal agents, such as enzymes designed to degrade the matrix before cleaning. The best ways to manage biofilm growth are to take apart equipment regularly, use hot water and caustic
cleaners, and replace damaged and worn equipment.
Discussion
Not all of the strains that tested positive for the STA1 or grew on the selective media actually over-attenuated in finished beer. This could be due to the over-attenuation assay used, which attempts to approximate what happens in a packaged beer. The test for STA1 does not account for the understudied STA2 and STA3 genes. It is not clear how they affect the diastatic ability of the yeast, but this is an area that should be studied further.
Homebrewers should take reasonable precautions to avoid unwanted attenuation by diastatic yeast. Good cleaning and sanitization practices will greatly reduce the risk of any contamination event. Mature biofilms can be visible to the naked eye. Check equipment that is kept in a wet environment for a slimy residue. Take apart equipment regularly to thoroughly clean and allow to completely dry. Watch for unusual characteristics that are not to style if cross-contamination was possible. It is important to keep track of your gravity readings and know where you should be with your chosen yeast. Homebrewers do not need to invest in a laboratory with the capabilities to identify the contaminant species. You probably have already brewed with a diastatic strain and haven’t had any problems, but it is something to keep in mind if beers are not turning out as planned.
In the commercial brewing world, in facilities where diastaticus is a concern, we recommend a two-pronged approach to assessing potential contamination. Looking at the genotype is important, and a good place to start. Invisible Sentinel makes this relatively easy and larger breweries should make room in their lab budget for this technology. The selective media is an easy and cheap way to monitor the starch metabolizing phenotype. A good QC program rests on consistent monitoring. Raw ingredients (including yeast) should be tested as well as vessels on the cold side of the brewing process. A European study found that of 52 confirmed events, 48 originated in-house. Of those, 70% were traced back to the bottling/canning lines. This area of the brewery should receive additional cleaning after processing a beer fermented with diastatic yeast and should be closely monitored.
References:
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