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Alternative Brewing Organisms in Wort Fermentation for Novel Non-To-Low Alcoholic Beverages with Unique Flavour Profiles

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Alternative Brewing Organisms in Wort Fermentation for Novel Non-To-Low Alcoholic Beverages with Unique Flavour Profiles Alternative brewing organisms in wort fermentation for novel non-to-low alcoholic beverages with unique flavour profiles MASTER THESIS Mariana Machado Pires Canoso Integrated Master on Bioengineering – Biological Engineering Faculty of Engineering of University of Porto (FEUP), Portugal Supervisor (FEUP) Prof. F. Xavier Malcata Supervisor (Carlsberg Research Laboratory) Marta Mikš Gemma Buron-Moles June 2017 Never be afraid of going through the hot sand to reach the sea Mom ii Acknowledgments First of all, I would like to say the biggest thank you I can to Gemma and Marta. For Gemma, thanks for the HUGE support and teaching, for all your dedication and patient for my questions and mistakes, for always be ready to help, for spending so much time with me even when a bunch of other things were around… THANKS for caring so much about me and for being my non-Danish mum in Denmark for the past 4/5 months! For Marta, thanks for the advices, guidance and support, for the discussions and great learnings, for always giving your best and getting time for me in your super busy agenda. Without you both, for sure, this project would not be possible! Thanks to Kathrine for your constant good mood and friendship during the long days in the laboratory, for always helping me, for making my days funnier and more relaxed. For Jonas and Rocío, thanks for such a nice environment at the office, for all the laughs and chitchats, but also for the productive discussions and support. For Rosa and Jochen, I feel quite grateful for the chance of developing this project at Carlsberg Research Laboratory. Thanks to all those that made lunch time, cake club and Friday bar always so enjoyable and amusing. I would also like to express my gratitude to Prof. Malcata for accepting me under his academic guidance in this project - and in particular his careful review of this thesis, and also on behalf the MIB’s Direction for allowing me to take this great journey. For Catarina, João and Rita, also enormous thanks for all the excellent moments during the last years, for sure you contributed a lot to make it much better. Last but not least, thanks to Augusto for being the input of this great adventure and for being always present during this 5 year journey. Thanks to my family and friends for always believing in me and giving me their best support on everything. O Prof. Francisco Xavier Malcata, orientador desta dissertação, é membro integrado do LEPABE – Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia, financiado pelos Projetos (i) POCI-01-0145-FEDER- 006939 (Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia, UID/EQU/00511/2013) financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) e por fundos nacionais através da Fundação para a Ciência e a Tecnologia I.P., (ii) NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) e Programa Operacional Regional do Norte (NORTE2020) iii Abstract The non-alcoholic and clean label beverages are getting more and more important in the market, due to an increased interest in terms of health and nutrition. Food grade lactic acid bacteria (LAB) strains and non-conventional yeast were tested in attempts to find strains with high productivity of volatile organic compounds (VOCs), associated to a fresh and sour profile (without off-flavours) and a low or inexistent level of alcohol. All strains were inoculated in high fan glucose wort (HFGW); after preliminary sensory evaluation, the best candidates were selected, and also tested in maltose wort (MW). In the first screening, optical density (OD), pH, alcohol by volume (ABV) and VOCs concentration were measured; an aroma evaluation was also performed. In order to explain the correlation between all this data and the strains tested, as well as shed light onto their relative influence, cluster analysis (CA), principal component analysis (PCA) and partial least squares analysis (PLS) were performed. For the candidates, growth parameters (OD, pH and ABV) were measured, and a flavour (aroma and taste) evaluation was performed. In terms of results encompassing LAB, PCA explained 93% of the variance obtained in the VOCs and growth parameters (OD, pH and ABV) data, being acetaldehyde and 4-vinylguaiacol the principal components identified (83% and 10%, respectively). Comparisons between PCA results and the candidates for a role in flavour evaluation indicated that acetaldehyde might be the chief responsible VOC for the fresh and sour profile obtained. For non-conventional yeast, PCA explained 94% variance obtained in the VOCs and growth parameters data, being isoamyl alcohol and acetaldehyde the principal components identified (accounting for 63% and 31% of variability, respectively). Comparisons between the PCA results and the candidates to flavour evaluation showed that isoamyl alcohol and acetaldehyde were probably associated with the fresh and sour profile; however, more studies are suggested to take a more educated decision. Apart from Lactobacillus pentosus (Lb.25), Lactobacillus salivarius (Lb.56) and confidential yeast 2 (Y2) that presented a sweeter profile in MW, almost no sensory differences were noticed between the yeast and bacteria candidates, when fermented in HFGW and MW. For downstream processing of fermented MW, pasteurization was performed, yet no sensory differences were perceived between pasteurized and non-pasteurized samples. Different fermentation times (24 h and 7 d) were also tested for the MW fermented by non-conventional yeast, and differences in ABV level and aroma profile were noticed. iv Contents 1. Introduction ............................................................................................................................................. 1 2. State of Art ............................................................................................................................................... 2 2.1. Non-to-low alcoholic beer ................................................................................................................ 2 2.1.1. Background ............................................................................................................................... 2 2.1.2. Production processes ................................................................................................................ 3 2.2. Lactic acid bacteria ........................................................................................................................... 3 2.2.1. Taxonomy ................................................................................................................................. 4 2.2.1.1. Lactobacillus .................................................................................................................... 5 2.2.1.2. Pediococcus ...................................................................................................................... 5 2.2.1.3. Leuconostoc ...................................................................................................................... 5 2.2.1.4. Lactococcus ...................................................................................................................... 5 2.2.2. Growth and metabolism ............................................................................................................ 6 2.2.2.1. Alternative pathways ........................................................................................................ 8 2.2.2.2. Impact of media characteristics ........................................................................................ 8 2.2.3. Flavour ...................................................................................................................................... 9 2.3. Non-conventional yeasts ................................................................................................................. 12 2.3.1. Overview ................................................................................................................................ 12 2.3.2. Growth and metabolism .......................................................................................................... 13 2.3.3. Flavour .................................................................................................................................... 14 2.4. Aims of the project ......................................................................................................................... 15 3. Material and Methods .......................................................................................................................... 16 3.1. Media .............................................................................................................................................. 16 3.1.1. MRS medium .......................................................................................................................... 16 3.1.2. GM17 medium ........................................................................................................................ 16 3.1.3. YPD medium .......................................................................................................................... 16 3.1.4. High fan glucose wort ............................................................................................................
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