Beer spoiling mechanisms of Lactobacillus brevis

Govže, Mateja (author), Čadež, Neža (mentor), O'Sullivan, Tadhg (co-mentor)

Lactobacillus brevis is the most frequently detected lactic acid bacteria in spoiled beer. With phenotypic, genomic and transcriptomic approaches we investigated the beer spoiling mechanisms of L. brevis and determined potential key genes that enable bacterium to grow in beer. For that purpose two L. brevis strains MB124 and MB449 isolated from beer were selected. By sub-culturing at elevated temperature (37°C) and/or in the presence of antibiotic Novobiocin several strains with changed plasmid profiles and reduced/lost beer spoiling ability were isolated. The genomes of three plasmid variants isolated from MB124 and four from MB449 with reduced/lost beer spoiling ability and different hop-resistance gene content (horA, hitA in horC) were sequenced. With comparative genomic analysis of the variants and the parental strain we confirmed that all three hop-resistance genes play a role in hop resistance although their effects differed. When L. brevis MB449 lost all of the three hop-resistance genes it was not able to grow in beer. Further, we showed that genes located on scaffold 2 (putative plasmid) extended adaptation phase of the strain MB449 in beer. Transcriptomic analysis (RNA-seq) of the strain MB124 demonstrated that plasmid-associated gene horC plays a key role in adaptation to beer. Spoilage mechanisms of the strain MB124 determined by chemical and transcriptomic analysis during growth in beer showed that it switched from carbohydrate metabolism into amino (arginine) and organic acids (pyruvate, malate) metabolism.

• Language: English
• Keywords: beer, hop, Humulus lupulus, beer spoilage, lactic acid bacteria, Lactobacillus brevis, genome, genome sequencing, genome analysis, RNA sequencing, transcriptomic analysis, gene expression analysis, fenotypic analysis
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Govže]
• Year: 2017
• PID: 20.500.12556/RUL-95602
• UDC: 579.67:663.4:57.088.7
• COBISS.SI-ID: 4819064
• Publication date in RUL: 21.09.2017

Secondary language

• Language: Slovenian
• Title: Mehanizmi kvara piva mlečnokislinskih bakterij Lactobacillus brevis

Abstract

Lactobacillus brevis je najpogosteje prisotna mlečnokislinska bakterija v pokvarjenem pivu. Na podlagi fenotipskih, genomskih in transkriptomskih informacij smo raziskovali mehanizme kvara in potencialno opredelili ključne gene, ki omogočajo rast bakteriji L. brevis v pivu. V raziskavo smo vključili seva L. brevis MB124 in MB449 izolirana iz piva. Z gojenjem pri višji temperaturi (37°C) oz. v prisotnosti antibiotika novobiocina smo izolirali variante obeh sevov, ki so z izgubo plazmidov zmanjšale oz. izgubile sposobnost kvara. Trem variantam pridobljenim iz seva MB124 in štirim iz seva MB449, ki so imele spremenjeno kinetiko rasti v pivu in so izgubile vsaj enega izmed genov odpornosti proti hmelju (horA, hitA in horC), smo ugotovili nukleotidno zaporedje njihovih genomov. S primerjalno analizo genomov variant in divjih sevov smo opredelili gene, zaradi katerih se jim je spremenila rast v pivu. Analize so pokazale, da v primeru odsotnosti vseh treh genov odpornosti proti hmelju, sev MB449 ne more rasti v pivu. Pomembno vlogo pri preživetju seva MB449 v pivu je imel tudi domnevni plazmid 2 (scaffold 2), katerega izguba je podaljšala fazo prilagajanja rasti v pivu. Transkriptomska analiza (RNA-seq) seva MB124 je pokazala, da ima plazmidni gen horC najverjetneje ključno vlogo pri rasti tega seva v pivu. S kemijsko in transkriptomsko analizo rasti seva MB124 v pivu smo odkrili, da je sev med rastjo preklopil presnovo ogljikovih hidratov v presnovo amino kisline arginin in piruvične in jabolčne kisline.

• Keywords: pivo, hmelj, Humulus lupulus, kvar piva, mlečnokislinske bakterije, Lactobacillus brevis, genom, sekvenciranje genoma, genomska analiza, sekvenciranje RNK, transkriptomska analiza, analiza izražanja genov, fenotipska analiza