Identifying and Selecting Wild Yeast Strains in Hard Cider

Cider making is a complex process that draws on centuries of tradition, artistry and science. In short, ripe apples are ground to a pomace and pressed to extract the juice. Fermentation can then be initiated spontaneously or through inoculating with commercial yeast. Once primary fermentation is complete the cider is racked to a second vessel to undergo a second fermentation and further age. Attention is paid to the sugar, acid and tannin levels of the apples and timing of harvesting. Measurements of brix, specific gravity and pH are among the most commonly tracked metrics throughout the fermentation process. Yeast nutrients, pectic enzymes, clarifying agents and other adjuncts may be used in the process but are not mandatory. The aging process is shaped to a large extent by the amount of oxygen contact with which the cider comes in contact; stainless steel prevents oxygen while plastic and oak barrels invite more contact.       

The pied de cuve method will yield a starting culture of yeast that is highly diverse and contains a large number of yeast strains- that may or may not yield successful hard ciders. This process will require outdoor temperatures in the range of 50 to 65 degrees and occur over a period of no more than 72 hours. Four liters of unfermented cider (must) will be transferred to a sanitized HDPE bucket. The bucket will be covered in a fine cheese cloth adhered by tape. The bucket will then be placed in the desired apple growing location, specifically in the understory of the tree. We will use this method at  Rogers Orchards, which uses EcoCertified spraying methods (including fungicides which inhibit native yeast colonies); the orchards of pomologist Matt Kaminisky, who maintains a no-spray heirloom apple orchard in Hadley, MA; and finally among the wild apples found growing in the mountains of Catskill, NY, foraged by colleagues Tim Graham, Anna Rosencranz and Dave Snyder at Left Bank Ciders.

To go from this highly diverse starting community to 10 yeast strains that can reliably yield delicious ciders, we will conduct selection experiments using sterile technique. 

We will first take the starting cultures from pied de cuve and inoculate them into YPD media with penicillin-streptomycin. YPD media consists of 1% Yeast Extract, 2% Peptone from animal tissue, 10% dextrose solution (20% glucose), and 2% penicillin-streptomycin solution. This media is autoclaved at 250℉ at high pressure to ensure that it is sterile of all outside bacteria and yeast. The dextrose solution is vacuum-filter sterilized and then added to the media, and penicillin-streptomycin is added to kill any bacteria. This will ensure that our strains do not produce acetic acid due to Acetobacter. After growing in 500 µL YPD media for 24 hours, we will freeze the initial pied de cuve communities by adding glycerol to make a 40% glycerol yeast solution in YPD and placing them in a laboratory grade -80℃ freezer. This freezing method will continue to be used in these methods as yeast can be stored indefinitely using this method and will stay alive in the freezer and can be revived at any time. 

We will then take the pied de cuve communities and put them through a series of replicated evolution experiments in 2 types of cider. These cider types will include the juice from a cider apple and the juice from a dessert apple, normally used as an eating apple, and we will use a 0.2 µm vacuum filter to sterilize the ciders. Each of the 6 starting pied de cuve communities will be inoculated into the ciders in 96 well plates, and will be replicated 88 times. There will be 8 wells in each plate that do not contain yeast as negative controls to detect any contamination. Replication will ensure that we do not lose advantageous yeast strains due to random chance. These yeast communities will be subject to selection pressures that select for high alcohol tolerance and low temperature tolerance. Low temperature tolerance will be selected for by subjecting the communities in every plate to 50℉. High alcohol tolerance will be selected for by adding 25 µL of ethanol to the cider to ensure we select for yeast that can produce at least 5% ABV. The experiments will continue for 2 weeks, the amount of time that Rogers Orchards keeps their yeast in fermented cider. 

After the selection experiments, each community should have a much smaller amount of yeast strains that have survived the selection process. We will take each community and use streaking on YPD agar plates to isolate yeast strains. YPD agar contains the same elements as YPD media, with an additional 1.6% agar added to make the media solidify. This streaking method will isolate single cells of yeast that will then replicate themselves to create a colony on the plate that is one yeast strain. From each replicated community, we will isolate the most abundant yeast strain, for a total of 528 possible yeast strains with traits advantageous for cider making in 2 types of cider, a traditional cider apple variety and a dessert apple variety, which will again be indefinitely frozen. 

The yeast strains isolated from the selection experiments will then go through a rigorous process of testing each strain in the fermentation process in the 2 cider varieties. Isolated strains will be inoculated into the 2 types of cider in 96 well plates and the oxygen will be blown off and replaced with an inert gas such as CO2 and covered with plastic adhesive film to stop oxygen access. We will then allow each yeast strain to ferment the cider and test for a few undesirable yeast traits including hydrogen sulfide production, acetaldehyde production, film production, and excessive yeast suspension. Hydrogen sulfide production will be tested using sulphite indole motility (SIM) medium which will blacken upon contact with hydrogen sulfide. Acetaldehyde production will be tested using an acetaldehyde assay kit. Film production will be detected by eye, and excessive yeast suspension will be quantified using the common OD600 measure, which is the optical density of each well at 600 nm fluorescence. This process will select out strains from the 528 possible strains and based on these measures we will select 10 strains that we will move forward for testing in the ciderhouse. 

We will sequence 10 yeast strains we isolate before we send them for testing in the ciderhouse, to ensure we do not select for yeast species that could cause undesirable cider such as the genus Brettanomyces. We will conduct bomb lysis DNA extractions to isolate the DNA from each strain. We will then conduct a PCR reaction using the primers for the ITS region to amplify the ITS region in each of these strains. Finally we will send these strains to Azenta sequencing to determine which species of yeast we have isolated. Based on which species we have isolated, we will use a primer pair that is species specific to then genotype each yeast strain and give it a name. 

These 10 yeast strains will be packaged in YPD media and taken to the ciderhouse to be pitched into ciders that will then be taste tested by the customers of Long View Ciderhouse.