Daniel McCulloch - IBD Asia Pacific Convention...

Daniel McCulloch

Craft Beer Micro – On a Budget

Asia Pacific Convention 2018 | Wellington, New Zealand Institute of Brewing and Distilling Asia Pacific Section 2

What’s covered

• The Lab - A location to prepare and review Microbiology

• HLP Media - Cheapest way to detect and identify absolute beer spoilers

• Tech and Prep - Aseptic sampling techniques & media prep

• We have growth - How to review a HLP + basic identification

• Help me Doc - What do you do with the results?

• Hide and seek - Possible locations for problems to occur from past experience in our brewery process


Where to Begin?


Start with a basic space that is free of dust, with limited air flo a d a ess to a si k. Make su e it s a sealed oo .

Equipment basics

• Microscope (actually spend money on this $500-$700).

• For the media, a heat stir plate ($150-$300) or Pressure cooker ($150), magnetic stir bars ($4 ea), Erlenmeyer flasks ($9 ea), Porta-Gaz Burner ($69), Nitrile gloves, 15ml tubes (500 for $102.20), Sterile pipette tips or an auto-pipettor ($150) & tips.

• Scales 0.01g ($300-$500),

• Reagent bottles & lids to hold media ($9.50) borosilicate glass 3.3


Incubator 30C ±2C

• Esky & Thermometer next to the Boiler / Hot Liquor Tank

• Heat Mat & Esky with a thermometer

• Chicken Egg Incubator ($30-$100)

• Buffet server ($89) with a temp controller ($39-$69)

• Pie Warmer converted with a temp controller & fan ($70+$55)

• Actual Incubator ($500-$8000) depending on size


Sample Ports - Basic

• Hard to clean. You can use a 70%+ ethanol and swab to do this. Next, flame the spout, and cool spout whilst taking the sample. Be aware that these sample ports can give false positive/negative results due to their design.

• If you have the plastic zwickle knob, they make a little flame guard that helps protect it and also increases the lifespan of the zwickle.


Sample Ports - Aseptic

• Accurate but expensive

• Needle aseptic require membranes approx. 18 jabs


>$500 $150

$400

Howzit Work?


Filled with ethanol

Withdrawing a sample from the production equipment through a closed circuit. The port has been sterilised and remains sterile as there is no exposure to the atmosphere during the sampling process.

Cleaning?


Expensive ports will have a CIP clip

Cheaper ports & you will need to think about adequate flow with port position

A Port that “shares the love”

- when only using one port on all tanks

• Do t dis o e t if you ha e the al e on a tank already!

• Keep sample port in a sanitiser bucket when not in use e.g PAA

• Always put back together in the bucket.

• Spray the valve connection on the tank and flame to sanitise.

• Always spray with 70%+ ethanol before use


Dip your toe approach

• Let's sta t ith HLP Hsu s La to a illus Pediococcus) Selective Media


Absolutely harmful bacteria

Lactobacillus spp. E.g; Lactobacillus brevis, Lactobacillus lindneri

Pediococcus spp. Pediococcus dammosus, Pediococcus cerevisiiphilus

But what does it cost?

• Cost break-down (*approx.):

• Brewpal (right) PCR for example $43 per test + equipment costs


Sterile Sample Taking


Sterile Work Space

• Approx. 30 centimetres to 50 centimetres radius


Tank Sampling


FV Take around 1.015 SG (- 2 SG) / 3.8 °P (- 0.5 °P) to ensure sample is well mixed for population Or with convection on chilling BBT On transfer Chill or CO2 carbonation

A Tank on chill risks sample taking of layering and not obtaining a well mixed sample of the population in tank, i.e. only a snapshot sample.

Sampling in the Cellar


Che k that it s a , ut ot goi g to u you! ASBC - Microbiological Control 1. Aseptic Sampling

Prep it, prep it real good

• Weigh out 100 ml water & 7g HLP with 0.7g (10%) agar to extend in waterbath.

• Cycloheximide (Actidione) stops protein biosynthesis and will affect you, ea PPE a d a P2 ask. You do t a t to do li es of this!! Read SDS &

work instruction.


Fry up

• Boil it up on a heat stir plate (2-3 minutes of boiling) with a magnetic stir bar

• Heat it up in a pressure cooker on steam setting for 21 minutes

• Microwave for 30 seconds, it only needs to boil.

• Do not fully close media bottle! Close it ¾ of the way or leave it ¼ open.


HLP only needs to boil.

Over cooking Cycloheximide can reduce it s effe ti e ess.

Sample to media

• Transfer 0.1ml (starting out) or 1ml (when your confident) into a 15ml Centrifuge tube

• Top up to 10ml with HLP


Prep +ve & -ve

• Invert your HLP tube twice and incubate at 30C

• Create a positive control by spiking a tube with 0.1ml of Lactobacillus plantarum

• Create a negative control by pouring HLP in a tube to 10ml

• Incubate for 2-7, 12 days


What’s under your chair?


??

Doc, hit me with the result!

• Basic, my positive control grew, my negative control did not. My sample grew.

• What is a CFU (colony forming unit) so how many dots in how many ml.


Let’s review what grew • Pick a colony, place on slide, put a cover slip on top, drop of immersion oil

and view at 1000x


Use your smartphone via eyepiece

• This is Pediococcus damnosus @ 1000x


Turn your flash off

Use your smartphone via eyepiece

• This is Lactobacillus & Wild Yeast taken through my smart phone @ 1000x


How far do you want to go? • Basic. Something has grown. • Take sniff, does it smell sour? • Taking a look under the microscope. Is it a

Rod, Tetrad, or Wild yeast? • Sometimes culture yeast (house yeast) can • cloud the HLP, but spoilers will still be evident • in the tube. • Taking a photo of the colony morphology. • Taking a photo via microscope • send to a colleague. • Use the Bible -> $285 AUD --------------------> • Send to external DNA lab (more for repeat ID) • Further ID - Gram stain, Catalase test, KOH, etc


If you do want to give HLP a go, have a chat to Hayden @ the Lallemand stand 38

Investigate a bit further • KOH - A viscous (stringy) reaction indicates the presence of Gram-negative

bacteria [Red in Gram Stain]. No reaction indicates Gram-positive Bacteria [Purple in Gram stain].


http://learn.chm.msu.edu/vibl/

content/oxidase.html

Potassium hydroxide 3%

Hydrogen peroxide 3%

•Catalase Test - EBC - 2.3.7 - Catalase Test •KOH Test - EBC - 2.3.6.2 KOH Method for Gram Differentiation •Gram Stain - ASBC - Microbiological Control 3. Differential Staining •EBC 2.3.9.1 - Gram Staining for Differentiation of Bacteria •Oxidase Test - EBC - 2.3.9.4 Oxidase Test

Use plastic or Wooden Loop. Metal will react with H2O2

The result

• 2 cells of Lactobacillus in 1ml of beer, over 5 days will cause sourness & Turbidity ** L. brevis for example pH 4-5 Brewery strains are typically insensitive to hops but ethanol becomes inhibitory at around 5 % and grows optimally at about 30 Celsius, strain dependant, PU >15 forms extremely long, parallel-walled single and double rods* Sour (Lactic acid)

• Pediococcus 4 cells per 1 ml of beer, 14 days until Diacetyl, etc ** Pediococcus damnosus cocci occur in pairs and tetrads through division in two planes, Diacetyl (Butter), Acidity, Hop tolerant >15 PU*


*ID source: Brewing Microbiology Third Edition, ** examples from past experience strain dependant

How long will it last? • This is never definitive. It depends on the beer style, the ABV content, the IBU & Dry hop

amount, CO2, Reduced Nutrients, Temperature, etc. Do the Rods or Tetrads have horA, horC, hitA (absolute beer spoilage strains) hop resistent ge es? You o t e a le to ID ia HLP edia, ut you a use a PCR like Brewpal for example.

• I would suggest if you have a core range of beers, then order in the Potential spoilers, dilute and inoculate into your already bottled beer and do some shelf life testing, tasting weekly, 1 month, 2 month, etc

• You need to create an in-house specification for your core range from shelf testing & experience over time (for example, do you sell P1 [2-16 cells per ml] kegs locally if they will be gone in a week?)

• Your best chance at reducing issues is to start testing and measuring


There are some great articles out there on shelf life;

IBD -> Isolation, Identification, and Characterisation of Beer-Spoilage Lactic Acid Bacteria from Microbrewed Beer from Victoria, Australia Garry Menz, Christian Andrighetto, Angiolella Lombardi, Viviana Corich, Peter Aldred and Frank Vriesekoop IBD -> 125th Anniversary Review: Bacteria in brewing: The good, the bad and the ugly - Authors Frank Vriesekoop,, Moritz Krahl, Barry Hucker, Garry Menz

Can we save it?

• Pasteurisation - depends on the cell load, if a cloudy beer will need la ifi atio / filt atio o eally high PU s.

• Sterile Filtration - requires Lenticular flow before Sterile filtration - loss of hop oils. Can try 0.65 micron membrane for hop oils opposed to 0.45 membrane

• Ditch it?

• Blend it? (not at all recommend, have heard some people do it)

• Dry Hop it, add SINAMAR® and call it a late hopped sour?


Polish a turd expression?

Sensitivity - How balls deep?

• 0.1 ml, then 1ml, then 50ml, then 100ml?

• Then enrichments or membrane filtration of samples.

• Centrifuge and enrich?

• Or Membrane filter and enrich the sample.

• Membrane > PCR enrich > PCR result?

• What s you house spe ifi atio fo Mi o Wo t, FV, BBT?

• e.g 5 cfu -1ml, 100ml? For what level of organism?

• Absolutely harmful bacteria versus Potentially harmful bacteria?


Example thresholds: http://www.brewingscience.com/PDF/BSI_brewers_lab_handbook.pdf - Page 3 Colour Atlas - p104

Where did it all go wrong?

• Follow the process steps Test every time the sweet liquid moves. Brewhouse -> Heat exchanger -> O2 dosing -> Wort Line -> FV CIP -> Yeast Pitching -> Filtration -> BBT CIP -> Package -> Rinse water

• pH Get your end of fermentation pH down to 4.2 (±0.1). Why? pH - below 4.2 is generally considered critical for inhibiting pathogens and toxin production simply because when the pH of beer is raised, more organisms can grow and spore formation risk can be heightened.


Initial Yeast Specification? Cone to cone?

Pitching Rate? (lazy brewer)


Yep pit hed it o e ….

Yeast Counting? Viability?


Most disliked brewing comment; Oh ust

be a happy ferment.. hu kle…

Transfer kit?


Sanitise Everything


When heat is used for disinfection, moist heat is far more effective than dry heat. L. brevis has been shown to withstand

more than 60 min at 80°C in dry conditions (Donhauser et al. 1991)

Soul Glo

Heat Exchanger - The Death Star!

• We hot caustic flush at the start of day and back flush hot water at the end. On Saturday, we do a full CIP. After caustic rinse, we leave Phos/Nitric acid in the heat exchanger over the weekend.

• Pop Top Samples An easy and cheap start to micro testing your heat exchanger. If it pops, plate it. If they are all popping and you can rule out sa pli g e o a d B e e CIP e o , the it s ti e to clean the heat exchanger.


If it pops your wort is fermenting

without your yeast

Spray Balls - Are they working?

• Gunk caught in them, too short in design leaving blind spots? Try adding beer DE Extract powder to man-way, PRV, Dry Hop port and Hot rinsing. We had 7 FV s ith too sho t Sp ay all le gth si e a ufa tu e!!


Do t al ays just t ust you manufacturer Question everything.

CIP Regime

• ATP test ($5)

• 3M petrifilm your rinse water e y si ple & does t e ui e i u atio .

• Titrate your chemicals (seriously a colour dropper, think fish tank pH) Is your caustic really 3% ?


Titrate

How many drops until clear? % is calculated with a simple formula

If e hit a y fail i a ta k e go th ough a uke it f o o it egi e; 1) Cold rinse of fittings and tank 2) Hot rinse tank 3) Sacrificial caustic clean if it foams do it again, ensure CO2 is removed for foaming 4) Phosphoric & Nitric acid clean (re-passivate stainless steel, micro control and beer stone removal) 5) Additional caustic clean if any bio-film protected by beer stone residues 6) Rinse 7) Sterilant

How is the micro-flora entering?

• Returned growlers, bottles, kegs

• Rental kegs are a massive beer STI transporter (you know that delicious B ett ee f o Vi ? Well it s o ight th oughout you e e y due to you incorrect cleaning of one keg)

• Grain from all countries, dust, spent grain storage, etc

• Shoes, food and equipment being stored within the brewery, that home brewers batch just dropped off for you to try...


Rinse & change

sterilant Buckets

regularly

Floors! & Equipment


Foam Party

• We do this twice a week at night.

• Also with a bigger clean down on Sat.

• Remember to keep records too!

• Wash away the foam that has killed the micro organisms, otherwise you are just leaving nutrient behind for the next generation.

• Environmental sampling with media plates to monitor and swab tests.


CO2 lines - Compressed Inoculation

• CIP them once a month.

• Make sure you attach one way valves.

• We CIP through a custom manifold once a month or when we hit a fail from our plate testing.


Autoclaved water & kit.

• Bubble through CO2 on low pressure for 20 minutes


Keg Sampling


Safety Deviation! Wear safety glasses As glass sample bottles can explode!

Thanks to Amanda from Rocks for showing us the design from Miller-Coors. If it s possi le to add a pigs-tail, I would for this design

Pipework


In the end it’s not that tough to start micro! • Is it as hard as brewing in a heat wave?


Pausing from Brewing in a Heat Wave in the lab, poo Be y… Brewing glove pillows! That lab air con though!

Recommended Reading


Brewing Specific Microbiology • Colour atlas and handbook of beverage biology -by Werner Back • Brewing Microbiology Third Edition -F.G. Priest, Iain Campbell • Yeast - Chris White & Jamil Zainasheff • Brewing Microbiology Managing Microbes, Ensuring Quality and

Valorising Waste. Editor Annie Hill. • Brewing Microbiology Bokulich and Bamforth

Quality Management • Quality Management -Essential planning for brewers - Mary

Pellettieri • Beer a Quality Perspective - Charles W. Bamforth • Standards of brewing - Charles W. Bamforth • Yeast - Jamil Zainasheff & Chris White

Brewing • Technology Brewing & Malting - Wolfgang Kunze • Brewing Yeast & Fermentation - Chris Boulton & David Quain

Yeast Propagation • The Yeast in the Brewery - AnnemÜller/Manger/Lietz

Methods of Analysis & Resources EBC analytica http://www.analytica-ebc.com/ ASBC http://www.ASBCnet.org MBAA http://www.mbaa.com/Pages/default.aspx

Contact info

• Email: [email protected]

• Insta: vonbrews

• More than happy to take questions via email or hat afte the talk. I ll e a ou d hi h e e sta d

has beer.

• Tha ks, hopefully I as t too u h of a pu ish!


Bogan Micro

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