Recent Developments in Yeast/Bacterial Cultures and Diagnostics

1. Recent Developments in Yeast/Bacterial Cultures and Diagnostics K.C. Fugelsang, Professor Emeritus of Enology, California State University, Fresno

2. Yeast and Bacterial Starters • Before the development of commercial active dry yeast, winemakers wanting to use starter inocula were forced to propagate these from stock cultures maintained in the winery or purchase already-expanded liquid cultures from local independent laboratories. • The process involved transferring pure cultures from lab media to sterile juice and, over a period of several weeks, expanding the volume to a level of 1 to 5% (v/v) of the expected fermentation volume through a series of aseptic transfers. • Maintenance of pure cultures during multiple transfers required a significant amounts of time, resources and skill. Further, there was the risk of contamination. • In 1965, Red Star Yeast (Universal Foods Corporation) released the first commercial wine active dry yeast (WADY or ADY). • Today, the vast majority of winemakers employ one or more of the dozens of available ADY cultures. • Reported properties: • Alcohol resistance (up to 17% v/v) • Compatibility with MLF and indigenous (native) yeast

3. Yeast and Bacterial Starters • Low fermentation temperatures • Ester production • Low H2S production • Enhanced palate structure (mouth-feel). • Color and structure compatible (including enhanced release of polyphenol- reactive polysaccharides). • Enhanced fructophilic properties • Intensified varietal character (S. blanc and Pinot noir). Suppliers are now recommending pairing selected strains with specific cultivars. • Even with the advantages of commercial Saccharomyces cultures, the winemaking community still remains divided with regard to the philosophy and practice of using starters. • At one extreme are those that rely solely on yeasts and bacteria native to the winery (including Sacc sp.) and vineyard. • Others prefer to encourage the growth of some non–Saccyeasts early in alcoholic fermentation but eventually inoculate with a Sacc starter as desired flavor/aromatics/structure develop.

4. Yeast and Bacterial Starters • Still others use Sacc starters but at lower than recommended inoculum levels. • Although winemakers often have strain preferences for particular applications, the issue continues to be one of debate. • Research dating to the early 90s suggested that selected non-Sacc (“native”) species may contribute, in positive fashion, to quantitative/qualitative diversity of products and by-products. • Wines produced from selected co-fermentations were described as having improved structural (“mouth feel”) properties as well as enhanced aromatics (particularly ethyl esters and phenylethanol) and overall complexity compared with monocultures of Sacc controls. • However, there were practical problems associated with early attempts to utilize non-Sacc cultures. • Non-Saccharomycesmonocultures have limited fermentation capabilities in terms of both rate, particularly late in the process, and complete utilization of sugars (Rs <2 g/L).

5. Yeast and Bacterial Starters • Early attempts at commercialization of non-Sacc ADY were limited by viability issues during drying and rehydration. • Results from our own lab (and others) in the early 90s suggested that while use of non-Sacc. monoculture fermentations might not be feasible, their use in co- or mixed cultures appeared promising. • Strains of Torulaspora delbrueckii were among the first to be recognized as playing a positive role in sensory properties of sequential mixed-culture fermentations. • Inoculation strategy utilized initial addition of Torulaspora followed by Sacc at 48+ hours. • Today, Chr. Hansen produces mixed-culture starters that include Sacc. x T. delbrueckii, Sacc. x Kluyveromyces thermotolerans, Sacc. x K. thermotolerans x T. delbrueckii as well as a monoculture of Pichia kluyveri. • Management Concerns: • Non-Saccharomyces strains have a high demand for Yeast Assimilable Nitrogen (YAN). This is a leading cause of stuck/ protracted fermentation in native fermentations.

6. Malolactic Starters • Thus, a nutritional monitoring and supplementation program (utilizing balanced formulations) is strongly encouraged. • Microbiological and chemical monitoring should be increased. Malolactic Fermentation and LAB Starters: As was the case with yeast, before the availability of lyophilized commercial LAB cultures in the 80s, wineries relied on native microflora to induce MLF. • With the widespread use of wooden storage tanks and barrels, a ready source of “in-house” inoculum was commonly available. • Under these conditions, promotion of MLF was accomplished by maintaining a temperature of 21°C/70°F, not adding sulfites, and maintaining a pH greater than 3.2. • Given that MLF can occur during, immediately following, or up to several months after completion of alcoholic fermentation, there was always a significant risk of spoilage. • Moreover, spontaneous MLF by unidentified lactic acid bacteria led to unpredictable and/or undesirable flavor characteristics in wines.

7. Malolactic Starters • Because of this, we continue to advise winemakers to regularly monitor the wine microscopically in addition to routine QC tracking of malic and acetic acids. • Although some wineries continue the tradition of using native microflora, winemakers increasingly inoculate grape must or wine with LAB starter cultures to improve the success of MLF. • Although one may still elect to prepare their own starters from in-house isolates, numerous strain of O. oeni are available in lyophilized, frozen concentrates, and liquid forms. • Lyophilized starter cultures usually contain high populations of viable bacteria (>108 CFU/g) and are easy to ship and store. Keys to Successful MLF: • Starter Viable Cell Number: Minimum VCN 106/mL • Nutritional concerns: LAB are fastidious. Maximum viability and conversion is improved with the use of LAB-specific rehydration medium (for lyophilized preparation) and, subsequently in wine, LAB-specific nutritional supplement.

8. Malolactic Starters 3. Sulfur Dioxide: Best results are associated with delaying additions until MLF conversion is complete or at a point where further activity is unlikely. • Where MLF is required, low sulfite-producing (“ML-friendly”) yeast strains should be selected. • Depending upon lactic strain, tolerance of TSO2 from 30-70 mg/L is reported. • Wine Alcohol Content: Most commercialized strains can handle alcohol levels approaching 14% v/v. • Where levels are higher, there are a couple of strains that can be utilized. Consult suppliers for recommendations. • Wine pH: Most strains perform well to pH 3.1- 3.3. Growth is promoted at higher pH but such an environment also tend to support growth of spoilage species/strains. • Thus, well prepared, aggressive cultures are required at both extremes. 6. Temperature: Optimal 62-70oF. Temperatures below 50oF result in slow growth and, potentially, stuck MLF.

9. Malolactic Starters Diacetyl Production: Consumer interest in fruit-forward wines has driven culture suppliers to identify and propagate strains that either do not produce (or produce low levels of) diacetyl. • Such low diacetyl or “diacetyl-free” strains either do not utilize (or only partially utilize) citric acid precursor. • Since citric acid utilization is reduced, such strains also produce lower levels of acetic acid. • However, lower levels of diacetyl may also be achieved by initiating MLF during the course of alcoholic fermentation. Inoculation : When MLF starter cultures are used, the winemaker is faced with the decision as to the timing of bacterial inoculation: • Post- Alcoholic Fermentation (“Old World”): Driven by concerns regarding formation of acetic acid by LAB growing on grape sugars and potential antagonistic interactions between yeast and bacteria, many winemakers, traditionally, have opted to inoculate upon completion of alcoholic fermentation.

10. Malolactic Starters • Co-Inoculation (“New World”): Research, dating back to the 80s, suggests that early inoculation of LAB along with, or shortly after, yeast starters is best for inducing and rapid completion of MLF. • This approach relies on availability of nutritional stores needed by LAB that have not yet depleted by yeast growth. • Ethanol and SO2, known to be inhibitory to O. oeni are present in lower concentrations. • This approach also promotes fruit-forward styles. • CAUTION: Must/juice pH should be a consideration with co-inoculation. In cases where pH >3.5, growth of heterofermenters on grape sugars during alcoholic fermentation may increase the potential for VA.

11. Laboratory Identification of Microbes Qui suis-je? WhwW

12. Laboratory Identification of Microbes Goals: • Preemptive Monitoring: Assists in making decisions before they become critical. • Forensic Identification: After-the-fact characterization of causative microbe(s) involved in spoilage. Classic Methods: Phenotypic techniques have traditionally served as the basis for identification and enumeration of bacteria and yeasts. • Such methods include determining the fundamental physical (morphological) and biochemical properties of the viable microorganism including utilization of different forms of carbon and nitrogen, synthesis of specific metabolites, or presence of certain biochemical pathways. • However, most of these methods require significant amounts of the time for culturing microorganisms and therefore results can be delayed for periods of days to several weeks. Because of delays, the usefulness of the information to the winemaker can pass or be of limited value.

13. Laboratory Identification of Microbes • Further, growth on laboratory media may be differentially and unpredictably affected by residency period in wine. • The cumulative stress of alcohol x SO2 x pH can lead to a condition of greatly reduced metabolic activity whereby cells are alive but not replicating and, hence, not detected as part of the population upon plating. • The Viable Non-Culturable (VNC) condition has been reported widely and includes wine species such as Brettanomyces and Oenococcus sp. • Thus, classic culture methods may yield semi-specific results and, depending upon inherent ability to recover during the period of incubation, lead to potential false negatives. • Basic Microscopy: Regardless of any follow-up work, microscopic examination represents the important first step. • Advantages: Fast, relatively inexpensive and can facilitate separation into basic groups based upon size and morphology.

14. Laboratory Identification of Microbes • Disadvantages: requires minimum >104 cells/mL. • Note: Brettanomyces can destroy a wine at VCN <103/mL. Results can be subjective. • Morphology often varies with growth/incubation medium. • Additionally, monitoring viability requires differential staining. A useful tool for yeast but not bacteria. Real-Time Methods: • During the last 20 years, research in molecular biology and genetics has led to the development of techniques capable of detecting and characterizing microorganisms at low populations and in a fraction of the time (hours vs days/weeks) required for classic isolation and identification. • While identification based upon phenotypic expression requires that the organism be fully viable, identification based on genetic (molecular) methods do not have this requirement. • In fact, molecular methods have the ability to target both live cells as well as viable non-culturable (VNC).

15. Laboratory Identification of Microbes PROBE DETECTION SYSTEMS • Several proprietary fluorescence dye-based detection systems that characterize and quantify probe-bound nucleotide sequences have been developed and commercialized in recent years. • Such systems rely on oligonucleotide probes that incorporate fluorescent dyes that “illuminate” when a match between complimentary (microbially-derived) strands are made. • Systems are available to detect numerous species of LAB and AAB, as well as spoilage yeast and Saccharomyces. • So-called Scorpion® probes owe their name to the generalized similarity in appearance between the oligonucleotide probe and the tail (stinger) of a scorpion. • Scorpion® technology has recently found interest among commercial laboratories servicing the wine industry. • With Scorpion® probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide containing a fluorophore and a quencher.

16. Laboratory Identification of Microbes • Once the primer is hybridized to the target, the fluorophore and the quencher separate as the “hairpin loop” unfolds and hybridizes to the newly formed complementary sequence. This separation leads to an increase in the fluorescence emitted by the fluorophore that can be measured. Results of Gene-Based Versus Phenotypic Identification: • Turn-around-time: 12-24h versus 2+ weeks for identification based upon phenotype. • Microbial diversity: For most utilizing molecular methods for the first time, the “cast-of-characters” typically expands. Pediococci, lactobacilli and other species suddenly are being reported in wineries with “no previous history” based upon classic methods. • Specificity: Organism-specific DNA signature requires three exact DNA sequences, aligned and properly spaced, to detect the target organism. This specificity ensures accurate species identification for all of the wine spoilage microbes.

17. Laboratory Identification of Microbes • Information per Assay: Using multiplex assay, DNA arising from several species of bacterial/yeast can be amplified and identified in a single assay. • Population Density: Gene specific detection is far more sensitive at representing the total population than classic plating due, in part, to the VNC condition. Potentially, differences in viable cell density between classic plating and genetic detection can be large. Concerns: the primary concern with gene-based identification is whether (or not) results represent only viable cell DNA or are they inflated by environmental DNA or dead cells in the sample? The issue of false-positives hinges on stability of DNA in the environment. • Supporters argue that the environment of wine rapidly degrades microbial DNA to the point it would not be detected by gene probe technology. In this regard, studies by Descenzo (2011) indicate scorpion-based assay did not detect dead cells. • Others contend that extracellular DNA is more stable than presumed.

18. Concluding Thoughts… Given the array of yeast cultures (Saccharomyces monocultures, non-Sacc and mixed), lab or pilot -scale trials are recommended before general commercialized use. Nutritional analysis (YAN) should always be part of pre-fermentation monitoring program. Balanced nutritional formulations are recommended and should be added incrementally over the first-half of fermentation. Nutritional requirements and supplements for yeast are different than those for LAB. Specific supplements are available for each application. 3. A proactive program of microbiological monitoring should be implemented.