Microbial Communities in Retail Draft Beers and the Biofilms They Produce
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bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456920; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Title: 2 Microbial communities in retail draft beers and the biofilms they produce 3 4 Authors: 5 Nikhil Bose, Daniel P. Auvil, Erica L. Moore, and Sean D. Moore 6 7 Affiliation: 8 Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida 9 Orlando, FL 32816 10 11 correspondence: 12 [email protected] 13 Keywords: 14 beer, bacteria, yeast, biofilm, Acetobacter, Lactobacillus, Fructilactobacillus 15 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456920; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 16 Abstract 17 In the beer brewing industry, microbial spoilage presents a consistent threat that 18 must be monitored and controlled to ensure the palatability of a finished product. Many 19 of the predominant beer spoilage microbes have been identified and characterized, but 20 the mechanisms of contamination and persistence remain an open area of study. Post- 21 production, many beers are distributed as kegs that are attached to draft delivery 22 systems in retail settings where ample opportunities for microbial spoilage are present. 23 As such, restaurants and bars can experience substantial costs and downtime for 24 cleaning when beer draft lines become heavily contaminated. Spoilage monitoring on the 25 retail side of the beer industry is often overlooked, yet this arena may represent one of 26 the largest threats to the profitability of a beer if its flavor profile becomes substantially 27 distorted. In this study, we sampled and cultured microbial communities found in beers 28 dispensed from a retail draft system to identify the contaminating bacteria and yeasts. 29 We also evaluated their capability to establish new biofilms in a controlled setting. 30 Among four tested beer types, we identified over a hundred different contaminant 31 bacteria and nearly twenty wild yeasts. The culturing experiments demonstrated that 32 most of these microbes were viable and capable of joining new biofilm communities. 33 From these data, we provide an important starting point for the efficient monitoring of 34 beer spoilage in draft systems and provide suggestions for cleaning protocol 35 improvements that can benefit the retail community. 36 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456920; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 37 Importance 38 Beer production, packaging, and service are each vulnerable to contamination by 39 microbes that metabolize beer chemicals and impart undesirable flavors, which can 40 result in the disposal of entire batches. Therefore, great effort is taken by brewmasters to 41 reduce and monitor contamination during production and packaging. A commonly 42 overlooked quality control stage of a beer supply chain is at the retail service end, where 43 beer kegs supply draft lines in bars and restaurants under non-sterile conditions. We 44 found that retail draft line contamination is rampant and that routine line cleaning 45 methods are insufficient to efficiently suppress beer spoilage. Thus, many customers 46 unknowingly experience spoiled versions of the beers they consume. This study 47 identified the bacteria and yeast that were resident in draft beer samples and also 48 assessed their abilities to colonize tubing material as members of stable biofilm 49 communities. 50 51 52 Introduction 53 Beer production involves controlled fermentation of plant sugar extracts in the 54 presence of flavoring compounds to generate desirable beverages. During the brewing 55 process, substantial effort is given to minimize exposure to spoilage microbes that 56 compete for resources and impart undesirable flavors. In addition to careful 57 fermentation, many finished beers are also filtered or pasteurized to further improve 58 product stability, sometimes at the cost of product flavor quality. Unfortunately, these 59 efforts go to waste if a beer is spoiled during the packaging, distribution, or dispensing 60 stages. In our study, we observed that routine draft line cleaning procedures are 61 insufficient to maintain beer quality in retail draft systems and that resilient microbial 62 biofilms persist that rapidly reestablish complex spoilage communities. To begin to 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456920; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 63 address this issue, we characterized microbial communities obtained from commercial 64 draft beers and monitored their populations after they established biofilms during 65 culturing. 66 In the nineteenth century, Louis Pasteur established that certain yeasts could be 67 isolated and used to produce wines and beers with consistent and desirable 68 characteristics (1). With those studies also came the discovery that beer and wine 69 spoilage was caused by different microbes that competed for food resources and 70 generated undesirable metabolites, such as lactic and acetic acid (1). Thus, the industry 71 of fermented beverage production rapidly shifted away from so called "wild" inoculations 72 and industry standards were put in place to carefully monitor and control the presence of 73 both desirable and undesirable microbes (2). In the last few decades, the craft beer 74 industry has revisited the use of alternative microbes and combinatorial culturing to 75 greatly expand the style range and flavor profiles. With some irony, one goal of these 76 efforts is to develop create products with scent and flavor complexities that match wild- 77 fermented ales and lambics (3, 4). Nevertheless, great care and expense is still applied 78 to minimize contamination by spoilage microbes and to ensure product stability (2, 5, 6). 79 Spoilage microbes enter the brewing process primarily from the addition of non- 80 sterile ingredients, air exposure, or contaminated equipment. Several spoilage microbes 81 are well known to the brewing community because they are commonly encountered and 82 present a consistent threat; among these lactic acid bacteria (LAB), acetic acid bacteria 83 (AAB), and wild yeasts represent dominant cohorts (6-10). Interestingly, these types of 84 microbes are also present as desirable members of the microbial communities found in 85 wild fermentations, wherein they can improve flavor balance and impart sour 86 characteristics as the beers are aged to maturity (11-13). In these aging processes, 87 groups of microbes overtake one another to dominate the community in a cascading 88 fashion, with each group consuming old metabolites and creating new ones. In addition, 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456920; this version posted August 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 89 members of a microbial community can exhibit synergistic or antagonistic relationships 90 with each other, which promotes unpredictable community restructuring depending on 91 the metabolic and combat capabilities of the founding members (14-18). The transitions 92 through community structures are a key feature that provides unique complexity to the 93 finished products. However, this type of conditioning process can be highly 94 unpredictable; even different strains of a microbial species can exhibit notably different 95 growth capabilities and differentially consume or release metabolites that alter beer 96 flavor (4, 12). 97 Historically, the identification of spoilage microbes relied on the ability to culture a 98 contaminant so that it could be subsequently characterized phenotypically and 99 biochemically (7). More recently, sensitive techniques to detect known spoilage 100 microbes have been developed that can reveal the presence and abundance of a 101 particular microbe’s genome that employ either image cytometry (19), polymerase chain 102 reactions (PCR) (8), bioluminescence (20), or molecular probing (21). While PCR is 103 excellent for characterizing microbes in post-production beer and for predicting shelf life, 104 it is unable to detect genes outside those that are targeted, so other potential spoilage 105 microbes go unnoticed. These limitations could largely be overcome using next- 106 generation DNA deep-sequencing to monitor mixed microbial communities because all 107 recoverable DNA can be interrogated and the abundance of non-culturable microbes 108 can also be established (22, 23). Unfortunately, the time and costs associated with 109 deep-sequencing are not compatible with routine beer production protocols. 110 Deep-sequencing has been applied to thoroughly evaluate the presence of 111 microbes and particular genes associated with spoilage in an active brewery (9). What 112 emerged from that study were mosaic maps of microbial communities that were 113 influenced both by location and nutrient availability in each brewery station. A main 114 conclusion from that investigation was that repeated exposure to the beer itself was 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.18.456920; this version posted August 19, 2021.