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Innovative Use of Non-Saccharomyces in Bio-Protection: T REPORT Innovative Use of Non-Saccharomyces in Bio-Protection: T. delbrueckii and M. pulcherrima Applied to a Machine Harvester Summary 1 Leticia Chacon-Rodriguez, Goals: 1 C.M. Lucy Joseph, organic wines, along with the need for improved worker safety. Winemakers 2 Bastien Nazaris, continue This to searchstudy addresses for alternatives the increasing to SO as demand an antioxidant for “natural” and antimicrobial and certified Joana Coulon,2 2 agent. This study compares the use of blended non-Saccharomyces cerevisiae Shaun Richardson,3 yeasts—Torulaspora delbrueckii (Td) and Metschnikowia pulcherrima (Mp)— and Daniel A. Dycus3* as antimicrobial agents to a standard addition of SO2 on Cabernet Sauvignon. Cite this article: Chacon-Rodriguez L, Joseph CML, California. In conjunction with this comparison study, a proof of concept pro- Nazaris B, Coulon J, Richardson S totypeThis fruit illustrates possesses the over use 10 of times a novel the spray normal method microbial for the flora application typically foundof these in and Dycus DA. 2020. Innovative non-Saccharomyces yeasts onto a grape machine harvester for bioprotection. use of non-Saccharomyces in bio- protection: T. delbrueckii and M. Key Findings: pulcherrima applied to a machine harvester. Catalyst 4:82-90. Research Winery: • Overall, the blended yeasts performed better than a standard addition of SO2 at controlling wine spoilage organisms on compromised fruit. • Organisms related to wine spoilage responded differently to Td/Mp than to SO2. The Td/Mp treatment exhibited an advantage over the SO2 treatment. The Td/Mp treatment appeared to work best against Zygosaccharomyces, Lactobacillus kunkeei, Hanseniaspora uvarum, and acetic acid bacteria. It was less effective against Pediococcus and other Lactobacillus species. 1Department of Viticulture and Enology, University of California, • Different stages of the trial fermentations were affected differently by Td/Mp 595 Hilgard Lane, Davis, CA 95616; and SO2. The Td/Mp populations performed best during prefermentation and 2 BioLaffort, 11 rue Aristide Bergès, the early stages of fermentation. 33270 Floirac, France; and 3Laffort USA, 1460 Cader Lane, Suite C, • Td/Mp showed an antagonistic effect on microorganisms responsible for wine Petaluma, CA 94954. spoilage. There were fewer microorganisms related to spoilage growing in *Corresponding author the three bioreactors with non-Saccharomyces species than in the bioreactors ([email protected]) acting as experimental controls with 60 mg/L SO2 added during processing. Acknowledgments: The authors • Td/Mp treatment increased the implantation capacity of S. cerevisiae acknowledge the contributions of Lorna Kreutz, Director of compared to the use of SO2. Using identical inoculation rates of S. cerevisiae, Winemaking, Foley Estates Vineyard we found more S. cerevisiae cells growing in the Td/Mp bioreactors than in the and Winery; ETS Laboratories; and bioreactors treated with SO2. Furthermore, we observed greater population Ferminkasi Inc. reduction and fewer cells/mL of S. cerevisiae at the end of fermentation. Manuscript submitted March 2020, revised May 2020, accepted Field Trial: June 2020 • A reduction in spoilage microorganisms occurred when using Td/Mp directly This is an open access article applied to the harvester. distributed under the CC BY license • Applying Td/Mp yeasts to the grape harvester reduced aromas related to vola- https://creativecommons.org/ licenses/by-nc/4.0/). tile acidity coming from the machine. By downloading and/or receiving this Impact and Significance: The use of Td/Mp yeasts provides an alternative to article, you agree to the Disclaimer SO for controlling the growth of organisms related to wine spoilage. Incorpo- of Warranties and Liability. The 2 full statement of the Disclaimers is rating these yeasts as a bioprotectant by applying them during the harvest and available at https://www.asevcatalyst. transport processes reduces the risk of detrimental microbial organisms in the org/content/proprietary-rights-notice- harvested fruit, juice, and wine. catalyst. If you do not agree to the Disclaimers, do not download and/or accept this article. Key words: Metschnikowia pulcherrima, microbial populations, Torulaspora doi: 10.5344/catalyst.2020.20003 delbrueckii, vineyard management, yeast ecology discovery into practice 4:2 (2020) page 82 A Publication of the American Society for Enology and Viticulture Innovative Use of Non-Saccharomyces in Bio-Protection – 83 Overview non-Saccharomyces species.10,11,12,13,14 Many of the mecha- nisms for increased mouthfeel result from an increase Winemakers continue to search for alternatives to SO2 in mannoprotein content from the cell.13 T. delbrueckii in winemaking. Many producers seek to reduce SO2 to take advantage of market opportunities in the “natural” is also known to metabolize sugar to produce alterna- - tive compounds such as glycerol or pyruvic acid via the 15 lines, to improve worker safety, and to utilize natural Crabtree effect. An additional study reveals an impact wine movement by following organic certification guide regarding the modulation of astringency resulting from 16 products for targeted applications. Winemakers current- T. delbrueckii fermentation. products known to have similar efficacy to synthetic The use of M. pulcherrima as a biological control agent ly use SO2 to control microbial growth. However, it is also a powerful antioxidant and inhibits browning reactions. is possible thanks to its ability to produce the natural antimicrobial compound pulcherrimin. This compound Because the properties of SO2 allow many different wine- 6 making applications,1 reducing or completely removing is an insoluble red pigment with antifungal activity. Pul- cherrimin has been shown to deplete iron in growth me- SO2 from winemaking requires an investigation of each processing step, beginning with grape harvesting and dia, which in a fermentation could result in inhibition of 17 transport. organisms requiring iron for growth. This mechanism Bioprotection is a relatively new term and emerging of iron depletion occurs via the precipitation of iron(III) concept in several food industries.2,3,4 In this study, the ions caused by an interaction with pulcherriminic acid, 6 term refers to a natural agent that controls the growth of a precursor of pulcherrimin secreted by M. pulcherrima. unwanted organisms through ecological processes such Several microorganisms exhibit inhibitory effects from pulcherrimin, including Candida tropicalis, Candida albi- blend of Torulaspora delbrueckii and Metschnikowia pul- cans, Brettanomyces/Dekkera, Hanseniaspora, Pichia, and cherrimaas competition. (Td/Mp This, 1:1 ratiostudy by examines cell count) the as efficacy a bioprotec of a- Botrytis cinerea. S. cerevisiae appears unaffected by this tion agent. This mixed culture is a commercial product antimicrobial activity.3 In addition, some strains of M. currently used by winemakers. The nature and impact pulcherrima produce a killer factor to suppress growth of these yeast species on winemaking is a current area of of killer-sensitive organisms. M. pulcherrima is also de- research at universities and developmental laboratories scribed as a biofungicide capable of reducing B. cinerea worldwide.2,5,6 on postharvest fruits via nutrient competition.18 This study investigates T. delbrueckii as a co-inoculum Uninoculated non-S. cerevisiae yeasts, often called for the bioprotection of grapes and juices. Some data sug- “wild” yeasts, are commonly associated with the pro- gest that the effectiveness of T. delbrueckii as a bioprotec- duction of ethyl acetate and negative sensory charac- tant depends on the matrix.21 Another study illustrates teristics.14 However, mixed cultures of T. delbrueckii that T. delbrueckii populations show much less severe and other yeasts are known to produce positive aromas losses in viability during the early stages of fermentation without the negatively associated sensory attributes.14 than other non-Saccharomyces species, making it an ideal M. pulcherrima is known to produce high concentrations choice as a competitive species for inoculation during of esters19,20,21,22,23 and - prefermentation.7,8 tivity, which catalyzes the release of varietal aromas.5 also possesses β-glucosidase ac In addition to its use for bioprotection, T. delbrueckii An additional study illustrates that co-inoculations of M. is known to enhance the aromatics of wine produced in pulcherrima with S. cerevisiae reduce the total amount of a cofermentation with Saccharomyces cerevisiae and is 4 Wines inoculated initially widely used in industry for this purpose.9 Evidence from with S. cerevisiae and M. pulcherrima show contributions the University of Bordeaux found that co-inoculation acetic acid in the final wine. - with T. delbrueckii and S. cerevisiae produced 54% less uct.12 volatile acidity and 60% less acetaldehyde than inocula- of 2-phenylethanolIn this study, we validatedand several the esters bioprotective in the final nature prod of tion with S. cerevisiae alone.10 Other studies show a posi- the mixed cultures at the University of California Davis tive sensory impact of cofermentation in both sequential Research Winery. The commercially available bioprotec- and simultaneous mixed cultures of T. delbrueckii and tion product is compared to a standard addition of SO2 on S. cerevisiae, most notably an increase in fruity aroma fruit possessing a high number of microbial organisms - related to wine spoilage. leasing volatile
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