Beer mix beverages: Spoilage yeasts / susceptible beverages?
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1 EBC Symposium 2014 Sensory aspects and technology of beer-mix beverages 8th of September 2014 Beer mix beverages: Spoilage yeasts / susceptible beverages? Mathias Hutzler & Maximilian Michel
2 Outline Beer mixed beverages microbiology? BMB yeast challenge test - setting BMB yeast challenge test - results Further developement: Product safety test + risk assessment rouble shooting tools media/pcr Mathias Hutzler Slide 2
3 Beer mixed beverages microbiology? Source: New products Substrate variety New recipes New habitats Microbiological safety? Mathias Hutzler Slide 3
4 Beer mixed beverages microbiology? Early state of BMB: Beer and lemonade Beer and coke Bitter units ph value Carbon dioxide Ethanol Current state of BMB: Beer and Fruit juice Energy drinks Coffee Liqueur ea Sweeteners Caffeine otaly different recipes Low alcohol Fruit residues New matrices.. Mathias Hutzler Slide 4
5 BMB yeast challenge test - setting? Individual spoilage potential?? Microbiologically relevant factors?? experimental setting?? Spoilage microorganisms (yeast/ bacteria)?? Analysis of spoilage results? Mathias Hutzler Slide 5
6 BMB yeast challenge test - setting Microbiologically relevant chemical parameters Fermentable sugars (f.s.(g/100ml)) Alcohol concentration (A. (% v/v)) Protein concentration (P. (g/100ml)) Free amino nitrogen (FAN (g/100ml)) Bitter units (BU) ph value (ph) R1 L1 S3 N1 F.S. g/100ml 4,15 0,15 3,21 7,16 A % v/v 2,39 2,76 3 0,82 P g/100ml 0,23 0,28 0,64 0,36 FAN g/100ml 4,46 6,51 17,88 8,87 BU 14,3 10,3 14,6 13,3 ph 3,77 4,00 4,11 3,66 CO 2 % w/w 0,53 0,58 0,51 0,46 Carbon dioxide concentration (CO 2 (% w/w)) Mathias Hutzler Slide 6
7 BMB yeast challenge test - setting Beer mixed beverages Main group Subgroup Sample designation Special features Number BMB containing lemonade Radler ( Shandys ) R1, R2 2 Beer + lemonade with fruit aroma L1 Sweetener based 1 L2, L3, L4, L5 4 BMB with caffeinecontaining lemonade C1 Sweetener based 1 C2, C3, C4, C5, C6 5 Various BMB Beer + spirits S1, S2, S3 3 Beer + energy drink E1 1 BMB with low alcohol concentration N1, N2 Juice portion 2 N3 1 Malt beer 1 1 Mathias Hutzler Slide 7
8 BMB yeast challenge test - setting Saccharomyces spoilage yeasts S. pastorianus (UM 34/70) 2 Brewing culture yeasts: S. pastorianus (bottom-fermenting /lager) UM 34/70 S. cerevisiae (top-fermenting/ wheat beer) UM 68 3 S. sensu lato foreign yeasts: S. castellii (S. cas.) (now: Kazachstania cas.) S. exiguus (S. exi.) (now: Kazachstania exi.) S. kluyveri (S.klu.) (now: Lanchacae klu.) 14 S. sensu stricto foreign yeasts: S. bayanus (2) (S. bay.) S. bayanus/uvarum (S. bay./uv.) S. cerevisiae (3) (S. c.) S. cerevisiae var. ellipsoideus (S. c. ell.) S. cerevisiae var. diastaticus (5) (S. c. dia.) S. paradoxus (S. para.) S. pastorianus (S. pas.) S. cerevisiae (UM 68) Mathias Hutzler Slide 8
9 BMB yeast challenge test - setting Non-Saccharomyces Spoilage yeasts 19 Non-S. foreign yeasts Candida boidinii (C. boi.) Candida sake (C. sa.) Candida tropicalis (C. tro.) Cryptococcus albidus (C. alb.) Debaryomyces hansenii (D. ha.) Dekkera bruxellensis (2) (D. bru.) Dekkera naardenensis (D. naa.) Hansenula anomala (H. ano.) Pichia membranaefaciens (P. mem.) Pichia anomala (P. ano.) Rhodutorula glutinis (R. glu.) Schizosaccharomyces pombe (S. pom.) orulaspora delbrueckii (. del.) Zygosaccharomyces bailii (Z. bai.) Zygosaccharomyces bisporus (Z. bis.) Zygosaccharomyces rouxii (Z. rou.) Mathias Hutzler Slide 9
10 BMB yeast challenge test - setting Analyzed spoilage results Haze Sensory changes Spoilage Bottle internal pressure Mathias Hutzler Slide 10
11 BMB yeast challenge test - setting Overview BMB Analysis of microbiologically relevant parameters Inoculation Potential spoilage yeasts Incubation Analysis haze sensory changes bottle internal pressure Mathias Hutzler Slide 11
12 BMB yeast challenge test - results Aim: Beverage specific spoilage potential pattern Generating a classification pattern Comparison of spoilage results to other BMB s possible Reliable results to adjust possible actions and produce save BMB s Mathias Hutzler Slide 12
13 BMB yeast challenge test - reference HUZLER M., WELLHOENER U., ENGE C., GEIGER E.: Beer-mixed beverages: dangerous spoilage yeasts, susceptible beverages?, Brauwelt international, 26, 4 (2008), Mathias Hutzler Slide 13
14 BMB yeast challenge test - results A. (% v/v) 2,39 2,43 2,76 3,11 3,11 2,92 2,72 2,66 2,99 2,46 3,20 2, ,78 3 2,51 0,82 0,83 1,02 0,16 BU 14,30 12,30 10,30 11,20 9,40 16,80 9,90 10,30 17,90 12,00 15,90 11,50 10,70 14,50 11,10 14,60 20,50 13,30 13,40 3,10 6,20 ph 3,77 3,53 4,00 3,51 3,47 3,71 3,82 4,15 3,69 3,84 3,78 3,61 3,56 3,36 3,69 4,11 3,09 3,66 3,72 3,21 4,81 CO2 (% w/w)) 0,53 0,51 0,58 0,55 0,55 0,52 0,53 0,56 0,56 0,52 0,50 0,53 0,52 0,56 0,50 0,51 0,51 0,46 0,44 0,60 0,53 f.s. (g/100ml) 4,15 4,72 0,15 3,62 3,38 5,34 2,52 0,13 3,56 4,72 4,22 3,06 5,38 2,53 3,37 3,21 5,92 7,16 6,56 7,23 8,66 P. (g/100ml) 0,23 0,23 0,28 0,31 0,30 0,28 0,29 0,32 0,29 0,36 0,36 0,34 0,28 0,25 0,42 0,64 0,26 0,36 0,37 0,07 0,44 FAN 4,46 3,06 6,51 7,53 5,61 5,15 5,96 4 5,98 7,58 5,25 6,19 4,65 1,05 7,37 17,88 4,63 8,87 8,28 1,53 13,49 (mg/100ml) UG 34/70 OG 68 S. bay. 1 S. bay. 2 S. bay. /uv. S. c. (1) S. c. (2) S. c. (3) S. c. ell. S. c. dia.(1) S. c. dia.(2) S. c. dia.(3) S. c. dia.(4) S. c. dia.(5) R1 R2 L1 L2 L3 L4 L5 C1 C2 C3 C4 C5 C6 S1 S2 S3 E1 N1 N2 N3 4,0 4,9 3,8 2,4 2,5 5,5 4,7-3,3 5,5 2,3 2,8 2,6 5,7 3,8 2,4 5,0-2,7 4,2 2,4 2,5 3,3 1,8 5, ,8 2,3 2,6 2,8 2,5 3,1 3,8 2,2 2,5 2,6 2,7 3,0 4,6 4,5 2,6 2,6 4,2-2,0 3,8-2,5 2, ,1 2,6 1,82 1,8 2,1 2,8 1, ,7 4,3 2, , ,3 2,0 3,6 4,3 2, ,9 4,2-4,5 5,4 2,9 3,5 3,4 2, ,8 2,2 2,35 2,6 3,1 4,3 5,0 4,8 3, ,5 3,3 3,3 3,6 3,3 5,4 5,6 5,0 4,7 4,0 5,6 2,5-2,2 2,4 3,1 1,8 2,6 2,8 4, ,1 2,9 2,6 3,4 2,75 M 2,4 4,4 4,8 3,1 4,4 2,5 1,8 Legend BMB with sweetener Bottle explosion Slight haze or individual particles + Haze, growth + with PCR +/- with PCR Haze, growth/ sensory changes / pressure in bar at 12 C (6bar =full scale) PCR spoilage yeast detection possible PCR spoilage yeast detection possible/some reactions inhibited Not analyzed (due to bacterial contamination/ pressure measurement not possible due to bottle shape) Mathias Hutzler Slide 14
15 BMB yeast challenge test - results A. (% v/v) 2,39 2,43 2,76 3,11 3,11 2,92 2,72 2,66 2,99 2,46 3,20 2, ,78 3 2,51 0,82 0,83 1,02 0,16 BU 14,30 12,30 10,30 11,20 9,40 16,80 9,90 10,30 17,90 12,00 15,90 11,50 10,70 14,50 11,10 14,60 20,50 13,30 13,40 3,10 6,20 ph 3,77 3,53 4,00 3,51 3,47 3,71 3,82 4,15 3,69 3,84 3,78 3,61 3,56 3,36 3,69 4,11 3,09 3,66 3,72 3,21 4,81 CO2 (% w/w)) 0,53 0,51 0,58 0,55 0,55 0,52 0,53 0,56 0,56 0,52 0,50 0,53 0,52 0,56 0,50 0,51 0,51 0,46 0,44 0,60 0,53 f.s. (g/100ml) 4,15 4,72 0,15 3,62 3,38 5,34 2,52 0,13 3,56 4,72 4,22 3,06 5,38 2,53 3,37 3,21 5,92 7,16 6,56 7,23 8,66 P. (g/100ml) 0,23 0,23 0,28 0,31 0,30 0,28 0,29 0,32 0,29 0,36 0,36 0,34 0,28 0,25 0,42 0,64 0,26 0,36 0,37 0,07 0,44 FAN (mg/100ml) 4,46 3,06 6,51 7,53 5,61 5,15 5,96 4 5,98 7,58 5,25 6,19 4,65 1,05 7,37 17,88 4,63 8,87 8,28 1,53 13,49 R1 R2 L1 L2 L3 L4 L5 C1 C2 C3 C4 C5 C6 S1 S2 S3 E1 N1 N2 N S. par. 3,27 3,7 3,1 2,9 S. past S. cas. 3, S. exi. 2, S. klu. C. boi C. sak C. tro C. alb D. han D. bru. (1) 2,4 2,06 D. bru. (2) D. naa. 2, H. ano P. mem P. ano R. glu S. pom. 2,1 2,4 2,4 2,2 2,0 2,1 1,8. del Z. bai Z. bis Z. rou PCR S.c./dia /- +/ ,3 -/+/ 4,0 4,8 3,2 4,9 M Legend BMB with sweetener Bottle explosion Slight haze or individual particles + Haze, growth + with PCR +/- with PCR Haze, growth/ sensory changes / pressure in bar at 12 C (6bar =full scale) PCR spoilage yeast detection possible PCR spoilage yeast detection possible/some reactions inhibited Not analyzed (due to bacterial contamination/ pressure measurement not possible due to bottle shape) Mathias Hutzler Slide 15
16 BMB yeast challenge test - results BMB Radler, containing lemonade (R1) Yeast Result Yeast Result UG 34/70 4,0 C. sak. OG 68 4,9 C. tro. S. bay. 1 3,8 C. alb. S. bay. 2 2,4 D. han. - S. bay. /uv. 2,5 D. bru. (1) S. c. (1) D. bru. (2) - S. c. (2) 5,5 D. naa. + S. c. (3) 4,7 H. ano. - S. c. ell. - P. mem. - S. c. dia.(1) P. ano. - S. c. dia.(2) R. glu. - S. c. dia.(3) S. pom. 2,1 S. c. dia.(4). del. - S. c. dia.(5) Z. bai. S. par. 3,27 Z. bis. S. past. Z. rou. S. cas. PCR S.c./dia. + S. exi. S. klu. - C. boi. - Beer + lemonade with added fruit aroma (sweetner based) (L1) Yeast Result Yeast Result UG 34/70 C. sak. OG 68 - C. tro. - S. bay. 1 C. alb. - S. bay. 2 - D. han. - S. bay. /uv. - D. bru. (1) 2,4 S. c. (1) 2,8 D. bru. (2) - S. c. (2) D. naa. + S. c. (3) H. ano. - S. c. ell. P. mem. - S. c. dia.(1) 2,3 P. ano. - S. c. dia.(2) R. glu. - S. c. dia.(3) 2,6 S. pom. - S. c. dia.(4) 2,8. del. - S. c. dia.(5) Z. bai. - S. par. Z. bis. - S. past. - Z. rou. - S. cas. PCR S.c./dia. + S. exi. - S. klu. C. boi. - - Legend BMB with sweetener Bottle explosion Slight haze or individual particles + Haze, growth + with PCR +/- with PCR Haze, growth/ sensory changes / pressure in bar at 12 C (6bar =full scale) PCR spoilage yeast detection possible PCR spoilage yeast detection possible/some reactions inhibited Not analyzed (due to bacterial contamination/ pressure measurement not possible due to bottle shape) Mathias Hutzler Slide 16
17 BMB Product safety test risk assessment Mathias Hutzler Slide 17
18 BMB Product safety test risk assessment Mathias Hutzler Slide 18
19 BMG1 UG BMG2 UG Standard BMG BMB Product safety test risk assessment BMB Microrganisms Beverage 1. Saccharomyces cerevisiae Saccharomyces pastorianus ssp. carlsbergensis Sacch. cerevisiae var. diastaticus Dekkera anomala Rhodotorula mucilaginosa Kazachstania exigua Mathias Hutzler Slide 19
20 BMB Product safety test risk assessment Mathias Hutzler Slide 20
21 BMB Product safety test risk assessment Mathias Hutzler Slide 21
22 BMB Product safety test risk assessment Reference: RIEDL R., KOOB J., HUZLER M., HACKL C., JACOB F.: Microbiologically sensitive beverages - a risk assessment system. Brauwelt international, 4 (2013), 31, Mathias Hutzler Slide 22
23 rouble-shooting tools media/ PCR orange lemonade sweetener beer mixed beverages radler caffeine-lemonade beer lemon-beer race detection/ identification of contamination Pre-enrichment media? Real-ime PCR Identification Saccharomyces cerevisiae Result: top-fermenting brewing/ bottom-fermenting yeast? wild yeast? fingerprint necessary for contamination localization? Mathias Hutzler Slide 23
24 hank you for your attention hanks to Prof. Jacob Co-Authors of related papers eam Research Center Weihenstephan Mathias Hutzler, Maximilian Michel Alte Akademie Freising-Weihenstephan elefon: +49 (0) 8161 / elefax: +49 (0) 8161 / [email protected] Mathias Hutzler Slide 24
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