Microorganisms in the brewery:

Transcription

1 Nebraska Grower and Brewery Conference January 5-6, 2017 Microorganisms in the brewery: From Acetobacter to Zymomonas and most everything in-between Bob Hutkins

2 The beer microbiome

3 The brewery microbiome

4 For microbiologists, each brew is a microbiology experiment 1. Medium preparation 2. Inoculation and incubation 3. Separate organisms from medium 4. Aseptic packaging

5 Why brewers should care about brewery microorganisms: 1. Safety 2. Compliance 3. Quality 4. Opportunity

6 Manufacturing Safety Accidents Physical, chemical and biological hazards Product Allergens Microbiological

7 Compliance GMPs, HACCP, FDA, and FSMA All food producers must register with the FDA This includes breweries

8 Compliance GMPs, HACCP, FDA, and FSMA All food producers must register with the FDA This includes breweries This means that breweries must have GMPs FSMA is also headed your way

9 Good Manufacturing Practices (GMPs) Good Manufacturing Practices for Craft Brewers (GMPCB), developed by the Brewers Association) GMPs consists of SOPs and other minimum requirements that all food manufacturers must meet to assure products are of high quality and do not pose any risk to the consumer or public. GMPs are prerequisite for HACCP GMP compliance is required (or else)

10 Good Manufacturing Practices ensure products are safe in the Brewery Why? ensure a clean and safe environment for employees ensure sanitary conditions are in place. reduce risks associated with pest infestations reduce risks associated with public health hazards reduce all other risks that could harm a business (including being shut down or action by civil suits)

11 Good Manufacturing Practices ensure products are safe in the Brewery Why not? ensure a clean and safe environment for employees ensure sanitary conditions are in place. reduce risks associated with pest infestations reduce risks associated with public health hazards reduce all other risks that could harm a business (including being shut down or action by civil suits)

12 Implementing Good Manufacturing Practices Use quality raw materials Perform lab tests that can be validated and/or trusted Establish SOPs Educate and train staff Recognize and account for deviations in product quality Create a good quality management system Package and label the product according to guidelines Meet (or exceed) sanitary and processing requirements

13 The beer and brewery microbiome Who is there, from where do they come, and what are they doing?

14 The beer and brewery microbiome 1. Who is there? yeast Saccharomyces non-saccharomyces Brettanomyces Kloeckera Pichia lactic acid bacteria Lactobacillus Pediococcus acetic acid bacteria Enterobacteriacae Staphylococci wild yeast and fungi Zymomonas

15 Lactic acid bacteria A C B 5.0 µm D E D A, Lactobacillus delbrueckii; B. Lactobacillus brevis; C, Pediococcus pentosaceus; D, Pediococcus damnosus; E, Lactobacillus helveticus

16 Genera of lactic acid bacteria Aerococcus Carnobacterium Enterococcus Lactobacillus Lactococcus Leuconostoc Oenococcus Pediococcus Streptococcus Tetragenococcus Vagococcus Weissella

17 Lactic acid bacteria important in fermented foods Lactobacillus Lactococcus Leuconostoc Oenococcus Pediococcus Streptococcus Tetragenococcus

18 Lactic acid bacteria important in beer Lactobacillus Pediococcus

19 Diagnostic characteristics of lactic acid bacteria Fermentative Low mol% G + C Non-sporeforming Gram positive rods and cocci Facultative anaerobes Catalase negative Non-motile Acid-tolerant

20 Practical characteristics of beer LAB Homofermentative (e.g., Pediococcus damnosus) Heterofermentative (e.g., Lactobacillus brevis) Hop tolerance Ethanol tolerance Produce diacetyl Produce biofilms Produce biogenic amines

21 The beer and brewery microbiome 2. From where do they come? ingredients malt hops water yeast adjuncts flavors environment air, water equipment personnel kettles, fermenters, tanks, fillers worker hands, clothing

22 The brewery microbiome

23 Source of microorganisms in the brewery Ingredients barley Fusarium toxins gushing malt hops yeast water air/gas LAB, fungi important during steeping and germination important for antimicrobial activity, not as a source of m/o source of starter strains, but also contaminants

24 Source of microorganisms in the brewery Equipment milling, mashing, lauter tun kettle and whirlpool separator fermentor maturation, bright tanks packaging

25 !!!!!!!!!!!!!!!!!!!!! Potential sources of microbiological contamination (!) during the brewing process. Vaughan et al., J. Inst. Brew. 111:

26 Abundance of micoorganisms in the brewery Source: Bokulich et al. elife 2015;4:e04634

27 Distribution of bacteria in the brewery Source: Bokulich et al. elife 2015;4:e04634

28 The beer and brewery microbiome 3. What are they doing? yeasts anaerobic maltose, other sugars ethanol + CO fermentation 2 glycerol diacetyl phenolics acetic acid acetaldehyde butanediol esters, higher alcohols amino acids H 2 S, SO 2, other sulfites

29 The beer and brewery microbiome 3b. What are they doing? bacteria maltose, other sugars anaerobic fermentation lactic acid diacetyl LAB aerobic oxidation acetic acid AAB amino acids biogenic amines LAB

30 Microbiological quality control in the brewery Same principles apply for beer as for any other perishable food Thus, for beer (and other fermented foods), it s a very thin line between fermented and spoiled

31 Spoilage versus Success: a matter of control Exert control by: Acidity and ph Temperature Moisture Salt Substrate availability

32 In beer, control is by: Low ph Temperature Ethanol Hops Absence of oxygen Substrate availability

33 Beer spoilage should be rare, because: low ph, high ethanol, iso-alpha acids beer is attenuated, few nutrients anaerobic, aerobes cannot grow kettle boil (kill step) stainless steel everywhere But: acid-, ethanol-, and hop-tolerant organisms exist in nature, including breweries wort is nutrient-rich exposure not insignificant (with plenty of opportunities for contamination

34 Beer spoilage organisms: from field to glass barley in field/ malting Aspergillus Fusarium Adapted from Vaughan et al., J. Inst. Brew. 111: mashing/ wort separation Pediocuccus Klebsiella Bacillus Citrobacter fermentation Pediococcus Selenomonas Zymophilus Obesumbacterium stabilization/ packaging Pediocuccus Lactobacillus Pectinatus Megasphaera Zymomonas Pectinatus Micrococcus Megasphaera Pediocuccus Lactobacillus finished beer Acetobacter Gluconacetobacter Pediocuccus Lactobacillus dispense

35 Controlling microbial defects in beer defect diacetyl lactic acid acetic acid sulfur haze cause Pedicococcus Lactobacillus lactic acid bacteria (LAB) acetic acid bacteria (AAB) LAB, AAB, others LAB, yeasts control cleaning sanitation GMP cleaning sanitation GMP cleaning sanitation GMP cleaning sanitation GMP cleaning sanitation GMP

36 Other strategies to prevent microbial defects mash acidification acid addition lactic acid fermentation

37 Viable counts of lactic acid bacteria during a typical lager mash LAB cfu per ml Vaughan et al., J. Inst. Brew. 111:

38 Other strategies to prevent microbial defects mash acidification fermentation packaging/distribution acid addition lactic acid fermentation alpha acid-rich hops pasteurization filtration

39 There are always new trouble-makers Pectinatus and Megasphaera strict anaerobes offensive rotten egg aroma extensive turbidity problematic in late stages of brewing Hard-to-culture LAB Viable But Not Culturable (VBNC) Hop-resistant LAB

40 Strategies for preventing microbial spoilage (works for food, beer, anything) 1. Keep out (asepsis) 2. Remove them 3. Prevent/delay growth 4. Kill/destroy 5. Combinations (Hurdles)

41 Hurdle Concept in Beer organism A organism B ethanol hops ph Eh T

42 Synergistic Effect of Hurdles ethanol hops ph Eh T ethanol hops ph Eh T

43 !!!!!!!!!!!!!!!! Filler!!!!! Potential sources of microbiological contamination (!) during the brewing process. Vaughan et al., J. Inst. Brew. 111:

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45 Contamination hotspots: Fitting into tight spots Beer Bacteria LAB AAB Yeast

46 The filling station is critically important, as this is where beer is especially vulnerable to contamination This short gap, both in space and time, is sufficient for all sorts of organisms to gain access

47 Surveying for microbes in the brewery 1. Microscopic 2. Cultural 3. Indirect 4. Molecular

48 Microscopy for counting: Easy, informative, inexpensive

49 Cultural Can be qualitative or quantitative Can be general purpose or group-specific These depend on the methods and media Selective v. Non-selective Detection v. Enumeration Detection v. Identification MRS UBA

50 Traditional Methods Also informative, but can take 3 5 days

51 Rapid, qualitative tests ATP Luminescence Can be used for rinse water or equipment (swabs) Easy, rapid, informative Nearly real-time Relative Light Units (RLU) can be scored as pass/fail

52 Source: Hill, A Brewing Microbiology: Managing Microbes, Ensuring Quality and Valorising Waste

53 Modern methods

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55 ID to species level

56 How to know, before it s too late, that you have a problem? This is the key reason for having a HACCP plan. HACCP, after all, is intended to prevent problems Thus, regular assessment of ingredients, equipment, and processing is the best way to minimize risks of contamination

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59 Consider the following scenarios: What is the tolerance for a mistake? Soldiers deployed to Afghanistan with three week rations Shuttle astronauts at the Space Station for 3 months Caterer to Olympic Athletes in Rio Infant food manufacturer New craft brewer with investors Common goal = zero defects

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61 Sanitation basics brewing = 90% cleaning + 10% paperwork HACCP Hygiene in the brewery choice of cleaners and sanitizers dealing with biofilms and chronic problems Assessment and testing

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64 Biofilms in the brewery Biofilms can form on equipment, even stainless steel Bacteria within biofilms are more resistant to heat, antimicrobials, and cleaning and sanitizing agents Biofilms will continue to shed bacteria into the food

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66 Lambic, coolhouse and sour beers Perhaps the original beer produced by humans Initiated by spontaneous or wild fermentation In Belgium, lambics are seasonal, fall to spring This is because the boiled wort needs to cool overnight to about 70 F in a shallow open vessel called a koelschip The U.S. version, called coolship ale, mimics the traditional lambic process

67 Microbiology of lambic-style beers Fermentation is a classic ecological succession One group makes conditions right for the next Four general distinct, but overlapping phases: 1. Enterobacteriacae 2. Primary alcoholic fermentation 3. Acid fermentation 2. Attenuation

68 ACA fermentation profile yeast bacteria ph plato Bokulich et al., PLOS ONE

69 Bacterial diversity of traditional spontaneously fermented lambic beer Spitaels et al., PLOS ONE

70 Yeast diversity of traditional spontaneously fermented lambic beer Spitaels et al., PLOS ONE

71 Brewhouse microbiota are responsible for fermentation of American Coolship Ale Bokulich et al., PLOS ONE

72 Questions