8/05/2017. Fermented beverages as an alternative for soft drinks. Water kefir. Water kefir

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1 Fermented beverages as an alternative for soft drinks Non- and low-alcoholic beverages with acetic acid bacteria as an overlooked contributor ir. Jonas De Roos Studiereeks Micro-Organismen: Part 1 - The Good & The Ugly Stadsbrouwerij De Koninck 11 mei 2017 Research Group of Industrial Microbiology and Food Biotechnology Prof. Dr. ir. Luc De Vuyst Prof. Dr. ir. Frédéric Leroy Prof. Dr. Stefan Weckx 2 3 1

2 Introduction: Food and beverage industry 4 Eurostat, UN COMTRADE, OECD Introduction: Beverage industry Alcoholic drinks Non-alcoholic drinks 5 Eurostat Introduction: Beverage industry Non-alcoholic beverages 6 Eurostat international 2

3 Introduction: Beverage industry Soft drinks Unhealthy perception Consumer trends evolve towards Sustainability Healthiness Naturalness Traditional, less sweet flavour and taste profiles Sugar-free soft drinks Sweeteners Stevia Natural fermentation processes as a healthy alternative? Kombucha 7 Introduction: Beverage industry Fermented beverages Alcoholic beverages < 18 % (v/v) ethanol Beer Wine Ciders % (v/v) ethanol Fortified wines > 35 % (v/v) ethanol Distilled alcoholic beverages Low-alcoholic beverages % (v/v) ethanol Milk kefir Non-alcoholic beverages < 0.5 % (v/v) ethanol Kombucha Yeast fermentation Mixed fermentation Bacterial fermentation 8 Belgisch Ministerie van Economische Zaken Introduction: Bionade production Malting Barley malt Grinding Brewing Extraction of fermentable carbohydrates Enzymatic activity Fermenting Gluconobacter oxydans Gluconic acid Juice pressing Heat treatment Mixing and bottling 9 Bionade 3

4 Introduction: Acetic acid bacteria Acetic acid bacteria (AAB) Gram-negative Ubiquitous Aerobic Associated with plants, flowers, and fruits Most prevalent genera Acetobacter Gluconobacter Gluconacetobacter Industrially used for Vinegar production Cellulose production Vitamin C production Acetic acid bacteria as an overlooked contributor Acetic acid bacteria are present in spontaneously or backslopped fermented foods Promising industrial potential for the beverage industry 10 Bartowsky and Henschke, 2008; Yamada and Yukphan, 2008; Potahkos et al., 2016 Introduction: Acetic acid bacteria Acetic acid fermentation has a heathy image Reducing carbohydrate calories Regulating food intake through the control of gut hormone expression Renaissance of traditional products Kombucha Lambic beer Increasing research towards these products including acetic acid bacteria fermentations 11 Hugenholtz, 2013 Introduction: Acetic acid bacteria Problems with traditional isolation methods such as plating Labour-intensive Viable but nonculturable (VBNC) state - When oxygen becomes available they can start to grow Spontaneous fermentation processes harbour a wide variety of microorganisms Recent solutions Optimised selective media in combination with high-throughput dereplication and identification methods - mdms selective agar medium - (GTG) 5 -PCR and AFLP fingerprinting of genomic DNA - MALDI-TOF MS analysis Culture-independent methods - Amplicon sequencing - Metagenomics - Metatranscriptomics 12 Bartowsky and Henschke, 2008;Cleenwerck & De Vos, 2008; Cleenwerck et al., 2009; De Vuyst et al., 2008; Papalexandratou et al., 2009, 2013; Li et al.,

5 13 14 Kombucha Occurs worldwide Indischer teepilz Tea fungus Fungus japonicus Theebier Manchurian mushroom Supposed to originate from Russia or China Confusion about the name Water, sugar, tea, and a kombucha culture Sugar as carbon source Black or green tea for nutrients - Amino acids - Vitamins - Minerals 15 Dufresne and Farnworth, 2000; Potakos et al.,

6 Kombucha Fermentation Symbiosis of bacteria and yeasts Open vessels 1-3 weeks Room temperature Kombucha culture Cellulose layer Broad spectrum of yeast species Lactic acid bacteria Acetic acid bacteria with Komagataeibacter xylinus as the most characteristic species 16 Dufresne and Farnworth 2000; Pothakos et al., 2016 Core microbiota Kombucha cultures grown in different microenvironments Amplicon sequencing with metabarcoding Bacterial component of the community is relatively stable - Komagataeibacter and Gluconobacter spp. - Less Acetobacter spp. Several yeast genera 17 Marsch et al., 2014; Reva et al., 2015; Pothakos et al., 2016 Combined microbiological and biochemical approach Radical scavenging D-saccharic acid-1,4-lactone content and caffeine degradation, change during the fermentation Some Gluconacetobacter species are very efficient in the production of D-saccharic acid 1,4-lactone from glucose Both the microbial community structure as well as the dynamics play an important role in the biochemistry of the fermentation 18 Yang et al., 2010; Chakravorty et al., 2016; Potahkos et al.,

7 Industrial potential Thriving industry Increasing market of specialty brews Fastest growing functional beverage category Kombucha Brewers International association Research and patents to optimise production yields 19 Nguyen et al., 2016; Pothakos et al.,

8 Occurs worldwide Ginger beer Aquakefir Tibicos California bees Ale nuts Japanese beer seeds Origin is unclear Derived from the leaves of the Opuntia cactus? Water, sugar, dried fruits, and kefir grains Sugar as carbon source Dried fruits for nutrients - Amino Acids - Vitamins - Minerals 22 Lutz, 1899, Laureys & De Vuyst, 2014 Fermentation Symbiosis of bacteria and yeasts Weck jar (microoxygenation) h Room temperature grains Brittle Translucent Cauliflower-shaped Consist of water and insoluble dextran Lactic acid bacteria Yeasts 23 Pidoux 1989, Laureys & De Vuyst., 2014 Core microbiota fermentation processes started with inocula from different origins Acetic acid bacteria - Not considered part of the core microbiota - Often isolated depending on the growth conditions 10 Liquor 10 Grains 8 8 Lactic acid bacteria (log CFU) Log CFU/ml 6 4 Log CFU/ml 6 4 Log Acetic (mdms) acid bacteria (log CFU) Yeasts (log CFU) Time (h) Time (h) 24 Gulitz et al., 2011, 2013; Laureys & De Vuyst, 2014; Stadie et al., 2013; Laureys et al.,

9 Core microbiota Specific lactic acid bacteria - Lactobacillus hilgardii: grain growth (dextran production) - L. casei/paracasei - L. nagelii Saccharomyces cerevisiae Bifidobacterium aquikefiri - Not considered part of the core microbiota 25 Gulitz et al., 2011, 2013; Laureys & De Vuyst, 2014; Stadie et al., 2013; Laureys et al., 2016 Combined microbiological and biochemical approach The water kefir grain inoculum both determines - Microbial species diversity - Metabolite concentrations Species diversity is impacted by - Backsloping time - Buffer capacity - Fermentation temperature - Oxygen availability - Nutrient concentrations Aerobic conditions - More acetic acid bacteria - More ethyl acetate - Less higher esters Liquor could also be used as alternative inoculum - Less metabolite production 26 Laureys & De Vuyst, 2017; Laureys et al., 2017 Industrial potential Mostly produced at household level Small-scale operations Trial and error mentality Research to optimise production yields 27 Laureys & De Vuyst, 2014, 2017; Laureys et al.,

10 8/05/ Lambic beer Local, traditional beer of the Senne valley Ten traditional breweries in Brussels region Two additional breweries in South West Flanders Belgian-style acidic ales: American coolship ales Oldest surviving commercial brewing style Lambic beer is the spontaneous fermentation of Barley malt Unmalted wheat Aged dry hops 30 Can proceed up to three years and is traditionally carried out in wooden casks (oak, chestnut) Verachtert & Iserentant, 1995; De Keersmaecker et al., 1996; ; Bokulich et al., 2012; Pothakos et al., 2016; Spitaels et al.,

11 Lambic beer production process Microbial community dynamics during spontaneous fermentation (core microbiota) Enterobacteriaceae phase Main fermentation phase: - Saccharomyces cerevisiae - Saccharomyces pastorianus Acidification phase - Pediococcus damnosus - Lactobacillus brevis - Transient occurrence of acetic acid bacteria Maturation phase - Brettanomyces bruxellensis 31 Van Oevelen et al., 1976, 1977; Verachtert & Iserentant, 1995; De Keersmaecker et al., 1996 Lambic beer production process Microbial community dynamics during spontaneous fermentation 32 De Keersmaecker et al., 1996 Gueuze beer Lambic beer is the basis for gueuze, kriek, faro, Gueuze is a refermentation in the bottle of a blend of Young lambic (typically 6 months to 1 year) Old lambic (1+ years) 08/05/2017 p De Keersmaecker et al., 1996; Spitaels et al.,

12 Core microbiota Enterobacteriaceae phase - Absent when wort is acidified Main fermentation phase - Saccharomyces cerevisiae - Saccharomyces pastorianus Acidification phase - Pediococcus damnosus Maturation phase - Brettanomyces bruxellensis 34 Spitaels et al., 2014, 2015 Core microbiota Enterobacteriaceae phase - Absent when wort is acidified Main fermentation phase - Saccharomyces cerevisiae - Saccharomyces pastorianus Acidification phase - Pediococcus damnosus Maturation phase - Brettanomyces bruxellensis 35 Spitaels et al., 2014, 2015 Acetic acid bacteria are found inconsistently Sampling method? Culturing methods? Two new AAB species were discovered Acetobacter lambici Gluconobacter cerevisiae 36 Spitaels et al., 2014,

13 Industrial potential Revival of both Gueuze and lambic beer Increasing market of specialty brews Gaining world wide attention Export of lambic and lambic-derived beers American coolship ales - Amplicon sequencing study (Bokulich et al., 2012) Lacking is a combined microbiological and biochemical approach 37 Pothakos et al., 2016; Spitaels et al.,

14 Conclusions Acetic acid bacteria are commonly isolated from fermented beverages Role often remains undetermined Lack of studies focussing on functionality Combined approach: biochemical analyses and high-throughput microbial analysis Metagenome analysis supplemented with metatranscriptomic data - Examples: Illeghems et al., 2012, 2015; Wu et al., 2017 Industrial potential Addition to soft drinks Healthy image Examples - Kombucha - Bionade 40 Acknowledgements Prof. Dr. ir. Luc De Vuyst Prof. Dr. Stefan Weckx Prof. Dr. Peter Vandamme IMDO team Artisan brewery