WILD YEASTSTRAINS. AmericanHomebrewersAssociation

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1 RESEARCH & EDUCATION FUND THEBREWING POTENTIALOFNEW WILD YEASTSTRAINS AmericanHomebrewersAssociation

2 ISOLATION OF NEW WILD YEAST STRAINS AND CHARACTERIZATION OF THEIR BREWING POTENTIAL By Michael Lentz STATEMENT OF PURPOSE Wild and spontaneously fermented beers are growing in popularity among homebrewers. Most of these beers are fermented by the use of either pure cultures of commercially available unconventional yeast and bacteria, or spontaneous fermentation using captured, mixed local microbes. Compared to standard ale and lager yeast, there are very limited strains available for wild ales. Spontaneous fermentation is unpredictable and can vary geographically as well as seasonally within a local environment. This project will determine the potential of pure strains of newly isolated wild yeast for use in the fermentation of unique beers. Yeasts with brewing potential are known to inhabit the skin of a wide variety of fruits and grains. New yeast strains from various sources will be isolated and characterized for physiological properties, growth in beer wort, and aroma/flavor characteristics. MATERIALS & METHODS Isolation. Yeasts were isolated by soaking a sterile swab in MYPG broth (5) and swabbing the surface of fruit. Alternatively, a sample of fruit or grain was swirled in a sample of the same broth using sterile forceps. Sources of yeast include pindo palm fruit (Florida), loquat (Florida), hackberry, (Pennsylvania), blackberry (Pennsylvania), and pale ale malt (Briess). After 4-7 days of growth at room temperature, samples were streaked onto MYPG agar plates for isolation. After 2-4 days growth, isolates were determined to be yeast or bacteria by colony appearance and microscopy. Yeast colonies were picked and grown in liquid MYPG medium for further purification and analysis. Samples were stored at - 80 C with 15% glycerol as a cryopreservative. A typical sample plate is shown in figure 1. Identification. Standard yeast polymerase chain reaction (PCR) primers ITS1 and ITS4 (4) were used to amplify a region of the ribosomal RNA genes from the isolates. Restriction enzymes were then used to digest the PCR products, which were resolved by agarose gel electrophoresis. Fragments were compared to published databases (1, 2) for species identification. For two isolates, the PCR product was sequenced and compared to sequences in the NCBI database. HOMEBREWERSASSOCIATION.ORG 1

3 Figure 1. Mixed colony growth on MYPG agar aft er st reaking from liquid cult ure. Alcohol tolerance. 100% ethanol was added to MYPG media to make final concentrations of zero- 12%. Water was used as a volume adjustment to keep the nutrient concentration consistent for all samples. 200 microliters of medium was distributed into wells of a 96- well culture plate in triplicate. Two microliters of pure yeast culture were added to each well and growth was determined visually after 4 days. Wort attenuation. Three different worts were prepared for determination of beer fermentation potential of the isolates. #1 and #2 were and O.G. worts prepared from Briess light dry malt extract, with an average analysis of 13% glucose, 48% maltose, 14% maltotriose, and 19% higher dextrins (Briess.com). #3 was from an O.G mash of malted barley without carbohydrate analysis. All gravities were determined by refractometer using a wort correction factor of For worts 1 and 3, 50 milliliters of wort in flasks was inoculated with yeast cells to a density of 1 million cells per milliliter. Flasks were foil covered and incubated with shaking at room temperature for four weeks. Wort #2 was from the beer analysis procedure described below. Final brix readings were taken with a refractometer and converted to specific gravity to determine real and apparent attenuation using published calculators (3). Not all strains were tested in all three worts. Beer analysis. 10 liters of wort was prepared from Briess light dry malt extract. Bittering hops were added to ~30 IBU (calculated). There were no flavor or aroma hops. After cooling and aerating with pure oxygen, 400 milliliters of wort was added to 500 milliliter PET bottle fermenters. 40 milliliters of starter culture were added to each wort sample and fermented at ~20 C (68 F) for four weeks with airlocks. Beer was carefully poured to a 12 ounce sanitized bottled, dextrose solution added to generate ~2.5 vol. CO2, and capped. Remaining beer was used to determine final gravity. After four weeks, carbonated beer was chilled and judged for general drinkability. RESULTS Yeast isolates were readily collected from every source sampled. Some sources (pindo palm fruit) yielded a variety of yeast species and bacteria, while other sources (hackberry, blackberry, loquat) had a lower diversity of microorganisms. Examples of isolated colonies and yeast cells are shown in figure 2. A total of 13 strains were selected for further analysis. HOMEBREWERSASSOCIATION.ORG 2

4 Figure 2. Samples of colony (top row) and cell (bottom row) morphologies. A, Bret t anomyces bruxellensis; B, Candida diversa; C, Issat chenkia t erricola; D, B. anomalus. Genetic analysis was used to identify yeast isolates using PCR and published rdna RFLP databases (1, 2). The identification of strains selected for further analysis is described in table 1. Of the 13 strains selected, six are members of the Brettanomyces genus, with B. bruxellensis and B. anomalus both represented. Four isolates are members of the Candida genus, two are Pichia kudriavzevii, and one was identified as Issatchenkia terricola. TABLE 1. Sources of yeast isolates, their identification based on rdna RFLP analysis, and brewing potential Strain Source Identification Beer Potential Bc01 Pindo palm (Butia capitata) Candida incommunis a Low Bc02 Pindo palm (B. capitata) Brettanomyces bruxellensis b Med Bc04 Pindo palm (B. capitata) Issatchenkia terricola a Low Bc07 Pindo palm (B. capitata) B. bruxellensis b Low Bc08 Pindo palm (B. capitata) C. diversa c Low Bc10 Pindo palm (B. capitata) C. diversa c Low Bc11 Pindo palm (B. capitata) B. bruxellensis b Med Rs01 Blackberry (Rubus sp.) B. anomalus b High Cs01 Hackberry (Celtis sp.) B. anomalus b High Hv01 Barley malt (Hordeum vulgare) Pichia kudriavzevii b Low Hv02 Barley malt (H. vulgare) P. kudriavzevii b Low Ej01 Loquat (Eriobotrya japonica) Candida sp. (tentative) b Med Ej02 Loquat (E. japonica) B. anomalus b High a Esteve- Zarzoso et al, 1999, Intl. J. System. Bacteriol. 49: 329. HOMEBREWERSASSOCIATION.ORG 3

5 b Pham et al, 2011, J. Inst. Brew. 117: 556. c This study, by sequence analysis. In order to succeed as a brewing strain, yeast must exhibit some degree of alcohol tolerance. This property was analyzed by growing yeast in microculture in the presence of varying concentrations of ethanol. Growth was assessed visually, and the results are shown in table 2. There was considerable variation among the different isolates for growth in ethanol. All of the Brettanomyces strains grew in 12% ethanol, the highest level tested. Other isolates varied in tolerance from poor growth above 4% ethanol, to efficient growth at 10-12%. TABLE 2. Growth at 48 hours in MYPG with indicated ethanol concentrations a Alc % Bc01 Bc02 Bc04 Bc07 Bc08 Bc10 Bc11 Rs01 Cs01 Hv01 Hv02 Ej01 Ej02 No Yeast % % % % % % % a -, no growth; +, minimal growth; ++, moderate growth; +++, robust growth Yeast that naturally reside on fruit have ready access to simple sugars as a food source. Beer wort is a complex mixture of simple sugars, maltose, and dextrins. Efficient attenuation of beer wort will be required if new strains are to function in a brewery environment. Yeast isolates were used to ferment three different worts. Fermentation was allowed to continue for four weeks, after which apparent attenuation was determined. There was considerable variation in fermentation potential among the different strains, as shown in figure 3. Surprisingly, there was even variation within a single isolate for attenuation of the three different worts. Several isolates consistently ferment to apparent attenuation of at least 70%, indicating potential as a brewing strain. HOMEBREWERSASSOCIATION.ORG 4

6 Figure 3. Attenuation of beer wort. Three different worts were fermented with the indicated strains for 4 weeks, after which apparent attenuation was determined.,1.040 O.G. wort; n1.048 O.G. wort; n1.056 O.G. wort. *, **; these samples were fermented with only one or two of the worts, as indicat ed. Yeast can be alcohol tolerant and efficient fermenters, but will not be of use to a brewer if the resulting beer smells or tastes bad! Wild yeast are well- known for producing many metabolic by- products, in particular phenolic compounds, that may have undesirable aroma and flavor properties. To assess potential for beer brewing, strains were used to ferment 400 milliliter test batches. Fermentation was allowed to proceed for four weeks, after which the beer was bottled, carbonated, and chilled. Beer characteristics were assessed by a panel of BJCP judges. Not surprisingly, many strains exhibited strong phenolic character of plastic, medicinal, and burnt/smoky. The strains that were shown to be poor fermenters exhibited the expected sweet, under attenuated worty character, in some cases with underlying fruity notes. Several of the strains produced beer with a complex, spicy, fruity, or funky character desired in wild ales. Due to the subjective nature of this analysis, the wild beers were categorized as having low, medium, or high potential as a brewing strain. The low performing group was dominated by undesirable phenolics of plastic, burnt rubber, Listerine, etc. These characters were less evident in the group with medium potential. The highest performing group had much lower levels of undesirable phenols, and had additional characters of spice, low smoke, and/or fruitiness. This data is included in table 1. DISCUSSION/CONCLUSIONS With proper resources, it is easy to isolate new strains of yeast from the environment. Based on the small sample size represented here, a small but significant portion of isolates are likely to HOMEBREWERSASSOCIATION.ORG 5

7 be useful and interesting to the commercial or home brewer. It was somewhat surprising how frequent Brettanomyces strains were isolated from diverse environments. It is also of interest that of the Brett isolates, none of the B. bruxellensis isolates produced drinkable beer, while all of the B. anomalus isolates could be used for brewing. Half gallon batches of beer were brewed with the three B. anomalus isolates, which were evaluated by the owners/brewers of Green Room Brewing in Jacksonville Beach, FL. Based on this analysis, we brewed a Florida Saison, with orange blossom honey, Florida orange, tangerine, and tangelo peel, and Florida grown lemongrass. A 30 gallon portion of this brew (figure 4) is fermenting with B. anomalus yeast isolated from Florida loquat fruit (strain Ej02). Yeast from this batch will be used to ferment a full seven barrel brew. Figure 5 shows a sample from the five gallon Saison that served to generate the starter for the Green Room Brewing Florida Saison. Figure 4. T ransf e r of 3 0 Figure 5. Saison bre we d w it h gallons of Saison wort t o a wild Bret t anomy ce s barre l f e rm e nt er. anomalus. I intend to continue this project with several goals in mind. I will continue to isolate and characterize new yeasts for their potential as brewing strains. I also plan to characterize strain variation in phenolic off- flavor (POF) gene expression. Finally, I will pursue avenues to make interesting strains available to the broader brewing community. ACKNOWLEDGEMENTS I am grateful to the AHA for their support of this project. Thanks to Rick and Travis for help with experimentation. I wish to thank Eric and the crew at Green Room Brewing for their support and their spirit of adventure. Thanks also to Eric, Billy, John, Jeff, and George for beer analysis. Sorry you had to suffer through a sea of phenolics to help find the few redeeming strains! Portions of this work are under review for publication in the Journal of the Institute of Brewing. HOMEBREWERSASSOCIATION.ORG 6

8 REFERENCES 1. Esteve- Zarzoso B, Belloch C, Uruburu F, Querol A Identification of yeasts by RFLP analysis of the 5.8S rrna gene and the two ribosomal internal transcribed spacers. Intl. J.Syst. Bacteriol. 49: Pham T, Wimalasena T, Box W, Koivuranta K, Storgards E, Smart K, Gibson B Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery 'Wild' Yeast Contaminants. J. Inst. Brew. 117: Terrill S Refractometer estimates of final gravity. In Terrill S (ed.), Seanterrill.com, vol White TJ, Bruns T, Lee S, Taylor JW Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics., p In Innis MA, Gelfand DH, Sninsky JJ, White TJ (ed.), PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., New York. 5. Yacobson C The Brettanomyces Project. Accessed December 9, HOMEBREWERSASSOCIATION.ORG 7