THE REFERENCE FROM BARLEY TO BEER

1
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BEER SPOILAGE ORGANISMS
BEER IMPACT

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Learning outcomes

o Microorganisms contaminating wort and beer

o Impact on beer quality of microorganism contamination

o Different method to detect biological contamination

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 Types of Organisms
o Of the micro-organisms from air, water and raw materials that enter the brewery,
some are particularly well adapted to this environment.
o Growth of these organisms on raw materials and during fermentation can release
metabolites that can seriously affect the stability and organoleptic properties of beer.
o Some bacteria and yeasts can proliferate in beer to produce hazes and off-flavours.

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Hygiene in brewing
The contaminating micro-organism is enemy N°2 in brewing

lactobacillus
Wild yeast

pediococus
The safety of consumer
Biogenic amines, nitrosamines
It is a danger for The business
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 Bacteria to spoilage beer
wort

packaging

Draught dispense

Raw material
Fermentation
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 Micro organisms type
o Bacteria:

Acetobacter, Gluconobacter, Lactobacillus, Pediococcus, Zymomonas,

Obesumbacterium (Hafnia) proteus; Enterobacter, Megasphaera, Bacillus.

o Wild yeast:

Brettanomyces, Hansenula, Candida, Kloeckera, Pichia, plus Saccharomyces.

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Impact of microbiological contamination in beer

safety
- No pathogens bacteria
 Pathogenies bacteria do not survive in beer
- alcohol content
- pH<4.5
- antimicrobial compounds from hop
- anaerobic environment

- Biogenic amine produce by bacteria

- mashing, fermentation
- Cadaverin, histamin, putrescin, …
- ATNC (carcinogenic) produce during mashing at low temperature by Bacillus

- Mycotoxins (carcinogenic) coming from malt, wheat, corn

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Biogenic amine in beer

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Biogenic amine evolution during au fermentation

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Schematic representation on nitrosamine production

o
r
fm
t
r
o
Wi
Na
r
tte ATNC (apparent total N-nitroso
w
)
1ae
tr
c
a
bti
r
eo
ar h
)
2os
p
compounds) should be less than 20
ppb.
d
l
i
w e
ys
at

r
t
i
Nt
ie Problems
o
wt
rm
an
ies
r
t
i
not
a
so
in a
e
yt
sai
mns
e o Water
o Hops
o Plant hygiene
N
T
As
'
C

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Beer survey

ATNC nb de bière
< 10 ppb 150
10-20 ppb 1
20-50 ppb 1
50 -100 pbb 1
> 100 ppb 2

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Impact of microbiological contamination in beer
Qualitive alteration

 haze, flake, turbidity

 Foam stability
 Over attenuation of beer
 Off flavor
- acidity (lactic, propionic, acetic): sour
- diacetyl: butter
- sulfur compounds : DMS, H2S, ...
- alcohol : propanol, isobutanol
- yeast
-…

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 Bacilli Bacilli
(rod-shaped) (chains)

Diplobacilli Tetrads
(pairs) (fours )

Shape of
bacteria Cocci
(spherical)
Sarcinae
(cubes of eight)

Diplocoqci
(pairs) Staphylococci
(bunch of grapes )

Streptococci
(chains)

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 Acetobacter & Gluconobacter
Size 0.6 - 0.8 μm x 1.0- 4.0 μm

Shape Pleiomorphic; Ellipsoidal to Rods

Can exist in Pairs & Form Chains
Reactions Gram -ve; Catalase +ve

Examples Acetobacter aceti; A. pasteurianus

Gluconobacter oxydans
Metabolism Obligate Aerobes
Microaerophilic capacity; do not produce
Nitrate
Risk Beer Dispense; Cask Beers

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Acetobacter & Gluconobacter - Spoilage

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Acetobacter & Gluconobacter - Control

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Impact of contamination by acetobacter

Forms skin layer on beer surface

Beer goes cloudy

Sharrp
vinegar

Vinegary / acidic flavour

Coming from acetic acid

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 Lactobacillus
Size 0.6-1.2 μm x 1.0-1.5 μm

Shape Rods
Formation of Chains
Reactions Gram +ve; Catalase -ve

Examples L. brevis, L. delbruckei, L. casei,

L. buchneri
Metabolism Aerotolerant Anaerobe
Stimulated by CO2
Risk Fermentation through to Product

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Lactobacillus acidophilus

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 Lactobacillus – Spoilage & Control

♦ Glucose, Maltose and Maltotriose fermented to

lactic acid (homofermentative pathway)

♦ Silky Turbidity

♦ Diacetyl synthesis

♦Hop resistant (not normally associated with Gram

Positive bacteria); acid tolerant

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Impact of Lactobacillus contamination

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 Pediococcus
Size 0.7 μm diameter

Shape Coccus (round)

Forms Tetrads
Reactions Gram +ve; Catalase -ve

Examples P. Damnosus

Metabolism Anaerobic
CO2 Required for Growth
Risk Fermentation onwards; can infect
at any stage (including yeast
slurries)

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Pediococcus damnosus

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 Pediococcus – Spoilage & Control

♦ Can cause Diacetyl and Rope (slime)

♦ Metabolises Maltose and Cellobiose

♦ Is susceptible to hop resin and acid washing.

♦ Primary source of contaminant is unknown!

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 Zymomonas
Size 1.0 -1.5 μm x 2.0-6.0 μm

Shape Short, Straight, Plump Rods; mainly in

pairs or clumps (classically “rosettes”)
Reactions Gram -ve

Examples Zymomonas mobilis

Metabolism Anaerobic (but can tolerate O2)

Ferments Glucose & Fructose, but not
Maltose
Risk Ale Breweries
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Zymomonas mobilis

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 Zymomonas – Spoilage & Control

♦ Grows very rapidly to produce H2S & Acetaldehyde

and turbidity

♦ Grows in presence of high Ethanol and can carry

out a full fermentation (up to 15% ethanol)

♦ Source is soil, water, old walls, cask washers

♦ Controlled by good hygiene / CIP

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 Enterobacteriaceae
Size 0.3 -1.0 μm x 1.0-6.0 μm

Shape Short, Straight Rods

Reactions Gram -ve

Examples Obesumbacterium proteus;

Enterobacterium agglomerans;
Klebsiella terigena
Metabolism Aerobic (and facultative anaerobes),
Are sensitive to ethanol and low pH and so do
not grow in beer, but grow rapidly in wort;
Produce DMS and other sulphur compounds;
Can reduce Nitrate.
Risk Wort and Early Fermentation
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 Enterobacteriaceae - Spoilage

♦ Can Produce DMS & other sulphur compounds

♦ Can Produce Diacetyl, 2.3 Butanediol, Acetoin

♦ Organic acids

♦ Phenolics

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Enterobacteriaceae - Control

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 Megasphaera
Size Large; up to 1.6 m diameter

Shape Coccus (round)

Reactions Gram –ve; Catalase -ve

Examples Megasphaera cerevisiae

Metabolism Strictly Anaerobic

Does not reduce Nitrate
Risk Wort Spoiler; Can contaminate sugars
and syrups.
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 Megasphaera - Spoilage

♦ Turbidity

♦ Short chain fatty acids (butyric, caproic,

valeric and
iso-valeric acids)

♦ H2S

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 Megasphaera - Control

♦ Can not grow at pH less than 4.1

♦ Growth suppressed by more than 3.5%

ethanol

♦ Oxygen

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 Pectinatus

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 Pectinatus - Spoilage

♦H2S

♦ Acetic acid

♦ Turbidity

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 Pectinatus - Control

♦ Growth at pH 4.5 – 6.0

♦ Growth slow at pH 4.0

♦ Oxygen

♦ Good hygiene in packaging

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 Bacillus

Shape Rods; forms Endospores

Reactions Gram +ve; Catalase +ve

Examples Bacillus coagulans

Metabolism Aerobic; Thermophilic

Can reduce Nitrate
Risk Non-Beer spoiler; can grow in hot, sweet
wort

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 Bacillus – Risks, Uses & Control

♦ Spores can survive wort boiling and produce

lactic acid in sweet wort;
♦ Can reduce Nitrate; risk of Nitrosamine
formation
♦ Several commercial enzymes are extracted
from Bacillus cultures.
♦ Controlled in brewing by:
• Hop acids
• Beer pH
• Ethanol

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Micrococcus / Staphylococcus

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 Beer spoilage bacteria
Gram negative bacteria (1)
Family Genus Major species Characteristics Beer spoilage
Acetic acid Acetobacter A. aceti Pleomorphic, catalase positive, strict Specific to aerobic or microaerophilic
bacteria A. pasteurianus aerobes, oxidises ethanol to carbon environments, i.e. dispense and draught
dioxwide and water (unpasteurised) products but not keg or
smallpack, forms a haze and surface film

Acetic acid Gluconobacter G. oxydans Pleomorphic, some motile with flagella, Specific to aerobic or microaerophillic
bacteria catalase positive, strict aerobes, oxidises environments, i.e. dispense and draught
ethanol to acetic acid products but not keg or smallpack, forms a
haze, surface film and viscous "ropiness"

Enterobacte- See below See below Typically rods, facultatively anaerobic, Generally (but not exclusively) limited to
riaceae (coli- diversity of fermentation products (organic wort and early fermentation, most common
forms) acids, butanedio, phenolics) generally is O.proteus – which (within the
sensitive to pH (< 4.4) and ethanol (> 2%) Enterobacteriaceae) best survives
fermentation

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Beer spoilage bacteria Gram negative bacteria (2)
Family Genus Major species Characteristics Beer spoilage
Enterobacteria- Obesumbacte- O. proteus Catalase positive, specific to brewery Grows in wort and during early
ceae rium (H.protea) environments, 'short fat rod', ethanol fermentation, produces sulphur off-
(coliforms) (syn.Hafnia) tolerant (<6%), found in yeast heads products (DMS), inhibits fermentation
and slurries and consequent danger rate and results in high beer pH,
of recycling contamination reduces nitrate to nitrite leading to
the formation of ATNCS
Enterobacteria- Citrobacter C. freundii Catalase positive, straight rod Occasional contaminant of pitched
ceae wort, can accelerate fermentation,
(coliforms) produces organic acids and DMS,
does not survive fermentation
Enterobacteria- Enterobacter E. agglomerans Rods Similar to O. proteus in associating
ceae (syn. Rahnella) (R. aquatilis) with yeast and in surviving
(coliforms) E. cloacae fermentation conditions, forms
diacetyl and DMS, increases initial
fermentation rate, final pH higher
Enterobacteria- Klebsiella K. terrigena Rods Forms phenolic off-flavours (4-vinil-
ceae K. aerogenes guaiacol) like some wild yeasts and
(coliforms) K. pneumoniae DMS

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 Beer spoilage bacteria Gram negative bacteria (3)
Family Genus Major species Characteristics Beer spoilage
Enterobacteria- Zymomonas Z. mobilis Rods, anaerobic but tolerate oxygen, Specific to ale fermentations/breweries
ceae (coliforms) catalase positive, ferment glucose (temperature) and glucose primed beers,
efficiently to ethanol, do not ferment off-flavours include hydrogen sulphide
maltose, very ethanol tolerant, grows best and acetaldéhyde
at 25-30°C
Pectinatus P. cerevisiiphilus Obligately anaerobic (consequently difficult Oxygen sensitivity restricts risk to low
to recover using conventional oxygen conditions in process or in
microbiological methods) curved rods package, off-flavours include hydrogen
(elongated in old cultures) sulphide (and other sulphur compounds),
acetaldehyde, propionic and other weak
acids, grows best at elevated pH (4.5-6)
with growth being weaker at pH 3.7-4

Megasphaera M. cerevisiae Obligately anaerobic, cocci, catalase Oxygen sensitivity generally restricts risk to
negative, difficult to detect low oxygen conditions in process or in
package, off-flavours (described as "foul",
"putrid" with a "faecal" aroma) include
hydrogen sulphide, butyric and other
short-chain fatty acids, cannot grow at pH
<4.1 or ethanol > 3.5-5.5%
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 Beer spoilage bacteria - Gram positive bacteria
Family Genus Major species Characteristics Beer spoilage
Lactic acid bacteria Lactobacillus L. brevis Aerotolerant anaerobes, catalase Potent beer spoiler growing optimally at pH 4-
L. damnosus negative, metabolism is either 5, forms 'silky' turbidity, acidity,
L. casei homofermentative (main product is occasionally viscous 'ropiness' and – notably
L. fermentum lactic acid) or heterofermentative – diacetyl, impact on beer in process and in
L. buchneri (products include lactic acid, acetic package.
L. delbrueckii acid, ethanol, and carbon dioxide Variable sensitivity to hop iso--acids-beer
L. lindneri spoilers 10 x more resistant than sensitive
strains
Lactic acid bacteria Pediococcus P. damnosus (syn. P. Aerotolerant anaerobes catalase Resistant to hop iso--acids and ethanol
cerevi-siae) (almost negative, cocci, occur as tetrads, (<10%, v/v), impact on beer in process and in
exclusively in homofermentative package.
breweries) Spoilage through diacetyl production
P. inopinatus ('sarcina sickness') acidity and haze
Bacillus B. coagulan Aerobic, catalase positive rods that Sensitive to hop iso--acids, not beer
produce resistant spores, thermophillic spoilers, found in hot brewing liquor and
sweet wort, form lactic acid in wort at 55-
70°C, implicated in the formation of ATNCS in
sweet wort.
Micrococcus M. Kristinae Catalase positive, typically strict Widely distributed in breweries, can survive in
M. varians aerobes although M. kristinae is a beer but rarely cause spoilage, M. kristinae
facultative anaerobe, cossi reported to spoil products ('fruity' aroma)
with
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ETHIOPIA pH, 2023
low bitterness. 44
 Wild Yeasts

o Wild Yeasts: not all are harmful nor cause spoilage, but their presence indicates
contamination.
o Several are aerobic (Pichia, Hansenula, Candida), but can grow to form films on the
surface of beer, plus hazes and off flavours.
o The more widespread wild yeasts are Saccharomyces sp. and can cause haze and
turbidity; S. diastaticus can utilise wort dextrins (by secreting amyloglucosidase),
resulting in over-attenuation.
o Some wild yeast (Brettanomyces, Candida) often produce a phenolic off-flavour, due to
the presence of the POF gene, which allows yeast to decarboxylate wort ferulic acid to
produce 4-vinyl guaiacol (used to produce German “weissbier”).
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 Wild Yeasts
o Any yeast not deleberately introducded

o Occupy similar niche to brewing strains

o Compete directly with brewing yeast

o Difficult to detect

o Robust

o Resistant to acid washing

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 Sources of Wild Yeasts

o Primings

o Hops

o Casks

o Pitching yeast

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 Types of Wild Yeasts
o Other types of brewing yeast (ale/lager)

o Variants

o Saccharomyces and non-saccharomyces

o Diastatic strains

o POF+ strains

o Killer yeast
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Photomicrograph of (A) wild yeast, and (B) brewing yeast
culture contaminated with wild yeast.

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Saccharomyces diastaticus

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Wild Yeast - Contamination

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Effects of Wild Yeasts on Process & Plant
Yeast Effect on wort or beer
Grows rapidly in the presence of air.
Pichia
Forms a film on the surface of the beer.
Candida Grows rapidly in the presence of air.
mycoderma Forms a film on the surface of the beer.
Continues to ferment all carbohydrates so there is
Saccharomyces
no control of attenuation.
diastaticus
Also causes off flavours.
Torulopsis Fails to sediment and causes hazes.

Very slow growing but it produces acid and causes

off flavours. Note that some beers are intentionally
Brettanomyces pitched with Brettanomyces cultures to produce
specific desired flavours, but these flavours are not
considered desirable in the majority of beers.
Grows rapidly in the presence of air.
Hansenula
Forms a film on the surface of the beer.
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Saccharomyces Wild Yeasts

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Non - Saccharomyces Wild Yeasts

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Wild Yeast – Contamination Effects

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Impact of Brettanomyces contamination

Yeast deposits in
beer

sour
Tart

Funky barnyard
Horse blanket
Vinegary
acerbic

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Other Wild Yeasts

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 Beer spoilage micro-organisms
Non-Saccharomyces wild yeast
Genus Major species Characteristics Beer spoilage
Brettanomyces B. anamalus Teleomorph (similar morphology, Notable for causing off-flavour in bottle
B. bruxellensis physiology) of Dekkera, oxygen conditioned beers, succeed
B. lambicus stimulates fermentation, ferment Saccharomyces in the spontaneous
glucose, rarely maltose but not fermentation of wort (lambic and
sucrose, produce acetic acid, nitrate gueuze)
reducing
Candida B. tropicalis Fermentation typically limited to Infection limited to the initial "aerobic"
C. boidinii glucose (C. tropicalis can ferment phase of fermentation or unpasteurised
C. vini (formally C. maltose) draught beers, reports that some
mycodema) strains can grow poorly anaerobically
Cryptococcus C. laurentii Unable to ferment but can assimilate a Can be found in beer in process or in
wide range of sugars, some strains package, survives but does not spoil
produce pigments
Dekkera D. bruxellensis Teleomorph of Brettanomyces forming Spoil unpasteurised draught beer
D. hansenii ascospores, oxygen stiumates
fermentation, ferment glucose, sucrose
and maltose (strain variable)
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 Beer spoilage microorganisms
Non-Saccharomyces wild yeast (2)
Genus Major species Characteristics Beer spoilage
Kluyveromyces K. marxianus Glucose is fermented vigorously, other wort Spoils soft drinks, fruit juices and high-sugar
sugars are variable as is lactose products, common contaminant of dairy
fermentation, thermotolerant (growth 37- products
43°C)
Pichia (including P. anomala Fermentation usually limited to glucose Infection limited to the initial "aerobic"phase of
Hansenula) P. fermantan fermentation, can spoil unpasteurised draught
P. membranifaciens beer, forms haze and surface films, P.
membranifaciens reported to give a
sauerkraut flavour
Rhodotorula R. glutinis Some strains are pigmented red, unable to Water borne, found in pitching yeast, can
R. mucilginosa ferment but can assimilate a wide range or survive but not spoil beer
T. delbrueckii sugars
Torulaspora T. delbrueckii Ferments glucose and variably maltose and Pitching yeast contaminant, can spoil
sucrose, phenotypically close to unpasteurised draught beer, capable of poor
Saccharomyces anaerobic growth
Zygosaccharomyces Z. bailii Ferments glucose and variably maltose and Infamous spoilage organism of soft drinks,
Z. rouxii sucrose, notably osmophillic fruit juices and high-sugar products
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DECTECTION

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 Sample point Sampling procedure Comment
Tap is sterilised, usually by Easy to operate. The tap is
heat. difficult to sterilise.
Sample tap in a tank
Sample is run into a sterile The tap needs to be cleaned
bottle. when the tank is cleaned.
The membrane is easily
A sterile needle is inserted
cleaned when the tank is
into the membrane.
Membrane sample point in a cleaned.
tank
Sample is run into a sterile The membrane needs to be
bottle. replaced regularly.
The valve body is arranged
so that the internal sampling
area can be sterilised before
use.

Aseptic sample valve, e.g. When the valve is closed it is

Keofit possible to pass sterilising
liquid through the top sample
point and out through the
bottom. This sterilises the

sampling
entire internal surface of the
valve.
A sample is continuously
Continuous sampling from a ‘dripped’ into a sterile bottle Representative of the whole
process line from a sample point in, for batch of beer.
example, a filter line.
Swabbing The plant is checked by
Access to plant not always
rubbing with a sterile swab,
possible, especially in large
normally wetted with saline
automated breweries.
solution.

The area being swabbed

needs to be of consistent area
Sometimes the saline is
and consistent location every
added after swabbing.
time that item of plant is
sampled.

The saline solution is

normally plated out using
agar growth media.
Samples of the final
Samples of the final drainings
drainings of the final rinse
Rinse samples of the final rinse water, or
water, orIFBM
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flush water are taken.
taken.
 Detection- 1
1. Pour Plates
o Applicable to any liquid sample; suitable for micro- loading of 1 to 500 cfu/ml.
o Sample is mixed into cooled molten agar medium and poured into sterile Petri dish to
solidify.
o Colonies are counted after incubation.

2. Spread Plates
o Applicable to any liquid sample; suitable for micro- loading of 5 to 5000 cfu/ml.
o Sample (0.1 ml?) is spread over the surface of a suitable agar gel and incubated.
o Colonies grow on the agar surface and are counted; if greater than 5000/ml = TNTC.

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Spread plate

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 Sample Handling - 2
3. Membrane Filtration
o Liquid samples containing too few organisms to be detected by conventional plating (less
than 1 cfu/ml).
o Membranes used are 47 – 50 mm in diameter; made of cellulose nitrate; 0.2 µm pore size;
o Membranes placed on surface of agar medium plates; incubated and colonies counted.
o Can also be used in Continuous Sampling Systems
4. Forcings
o Forcing test allows contamination to develop to a degree which enables them to be detected
as a haze or under a microscope.
o Forcing test is applicable to wort pre-pitch, bright beers and packaged beers.
o Principle is to incubate packaged beer or sterile-filled bottle sample at 27ºC for 3 weeks.

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Membrane filtration

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 Filtration on membrane

Preparation of the work plan Preparation of the work plan

Forceps flame sterilization

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 Incubation

o Controlled temperature are atmospheres are used to obtain either optimal growth
conditions for all microorganisms present or selective growth conditions for some of
them.
o 4 types of organisms can be distinguished, depending on the need for, or reaction to,
oxygen:
a) strict aerobes; require oxygen to grow;
b) microaerophiles; grow best at low oxygen levels;
c) facultative organisms; grow well in presence or absence of oxygen;
d) strict anaerobes; can not tolerate oxygen.

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 Incubation

1. Aerobic Incubation: used for cultivation of aerobic and facultative organisms;

o Applicable to film yeasts, moulds, water bacteria, aerobic
beer spoilers & to total count analysis applied to yeast,
wort and beer.

2. Anaerobic Incubation: used to cultivate anaerobes

selectively, such as Lactobacillus/ Pediococcus;
o Carried out in gas jars with gas generator kits to create an anaerobic atmosphere.

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Anaerobic jar

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 Detection and Quantification - 1
Examination
o Direct microscopy is not possible unless the level of contamination is extremely
high

o If visible haze is produced, it is possible to envisage >105 yeast cells/ml or 106 – 107
bacterial cells/ml.

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Classical microscope technics

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 Detection and Quantification - 2
Identification
o Various microorganisms can cause problems at various stages in brewing and should be
isolated and identified, to allow corrective action to be implemented.
o Selective media – encourage growth of specific organisms and suppress others; many are
available and the basis of many is the presence of an inhibitor to prevent certain groups of
organisms.
o For example:
• Cycloheximide (Actidione) – inhibits yeast, permits growth of bacteria.
• Tetracycline – inhibits bacteria, permits growth of yeast.
• Copper salts – inhibit brewing yeast, permit growth of “wild” yeast.
• Phenylethanol – inhibits Gram-negative bacteria.
• Vancomycin – inhibits non beer spoilage Gram-positive bacteria.
Non-specific growth media are useful to obtain an overall view of total hygiene.
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 Detection and Quantification - 3
Selective Media - examples include:
o WLD (Wallerstein Lboratory Differential Agar);
• a nutrient medium with a dye that changes colour when pH drops, thus detecting acid producing organisms,
plus cycloheximide to inhibit yeast; used to provide a general bacterial growth; blue dye turns yellow due to
acid production.
o Raka-Ray;
• used to isolate lactic acid bacteria; contains phenylethanol to inhibit growth of Gram- -ve bacteria, +
cycloheximide to inhibit yeast; incubated anaerobically to select lactics.
o YM (MYGP) + Copper agar;
• used to isolate non-brewing strains of Saccharomyces wild yeast; copper concentration inhibits brewing
yeasts.
o Lysine agar;
• used to isolate non-Saccharomyces wild yeast; contains lysine as the only source of N; Saccharomyces yeast
can not utilise lysine and so do not grow.
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Selective Media - 1

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Selective Media - 2

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Selective Media - 3

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 Detection and Quantification - 4

Gram Staining
o Differentiates between positive and negative bacteria;
o Depends on differences in cell wall structure;
o Gram-positive cells retain a crystal violet-iodine
complex after washing with ethanol or acetone;
o Gram-negative cells do not.

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Principle of Gram Staining for Bacteria

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Gram Stain Protocol

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Gram stain protocol

17/01/2023
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Gram Stain – Typical Result

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The potassium hydroxide test ‘KOH test’

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Gram Stain – Response

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 Detection and Quantification - 5

Confirmatory Tests
♦ Allow definitive identification of specific organisms,
although selective media are usually sufficient in most
cases.
♦ Example is API diagnostic kit.

Modern Approaches
♦ The search for a “Real-Time” test

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 ATP-meter
Cell of micro-organism Optical detection of the ATP molecule(source of energy for living
organisms)

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Bioluminescence

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Polymerase Chain Reaction - PCR

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 PCR Protocol

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IFBM | Reproduction prohibited | Advanced course in brewing technology | BGI ETHIOPIA | January 2023 89
IFBM | Reproduction prohibited | Advanced course in brewing technology | BGI ETHIOPIA | January 2023 90
 PCR

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 Immunological Methods

♦ Monoclonal Antibodies (Mabs)

♦ Mab Reactivity to Surface Antibodies for

Lactobacillus and Pediococcus

♦ Applications are limited only to a few

organisms

♦ Mabs are expensive to generate

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 Why Hygiene in brewery
Residues from
process Microorganisms

mineral
scale, beertstone Bacteria
Yeast

Organic compounds fungi

Sugar, lipid, proteins
Phages

Microbial Contaminations
Alteration of end product

Danger for the consumer (biogenic amine , ATNC)

And business
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CONTROLLING FACTORS

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 BEER PROCESS

Risk zone if temperature

< 50°C

Risk zones for

Microorganism
contamination

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Microorganism from malt to beer

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 Source of
Comment
contamination
Brewing water in wort will be boiled as part of
the process, but additions later in the process
Water (e.g. dilution water), cleaning final rinse water,
pre and post beer transfer purging water, must
all be sterile.
All naturally grown material harbours micro-
organisms. These are unlikely to be a problem
Brewing materials (malt,
where added/used before wort boiling as this
hops & adjuncts)
kills all flora and fauna present in the wort run
into the kettle(copper).
Storage of yeast may allow growth of other
micro-organisms already present in Raw material
the yeast slurry. This is a major problem
Pitching yeast because a contamination can proliferate
throughout the brewery unless controlled by
effective plant cleaning and possibly acid
washing.
The powders and liquids as supplied are
unlikely to be contaminated, but the additions
Additives (filter aid etc.) make-up and dosing process, and the water
used for mixing and purging can be a source
of contamination.
New bottles and cans may contain dust and
debris.

Returnable bottles, casks and kegs may be

Packages heavily contaminated when returned from the
trade because they contain small volumes of
beer, which may have been in the container for
a long time, allowing any contaminants time to
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grow on the remaining beer.
Environnement

Source of
Comment
contamination
Storage areas for raw materials will
encourage insects and other pests such as
Insects and other
rodents unless maintained in a hygienic
pests
condition. They will carry contamination
and must be kept away from open vessels
Contamination can be picked up from
Walls and floors poorly maintained walls and floors
especially in open vessels.

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 Source of
Comment
contamination
Wort chillers and wort mains are important
because of the nature of the wort, and high
levels of fouling experienced, particularly in the
heat exchangers, which can protect
Brewhouse plant microorganisms if cleaning is inadequate. The
wort is a rich medium for many microorganisms
to grown in, rich in sugars, proteins, minerals
and oxygen from the wort aeration/oxygenation
system.
Difficult to clean because of the residue left by
Fermenting vessels
the yeast and hops. FVs are a major source of
contamination because of the long time that the
beer is in contact with them.
Equipments
This can be a source of contamination because
Yeast handling plant
of the nature of the yeast itself and the
complexity of the plant makes it difficult to clean.
Easier to clean than FVs but there is
Maturation plant some yeast soil and again, the beer is in them
for a long time.
Difficult to clean because of the complex
Filtration plant
pipework and the design of the filter itself.
Easy to clean because the beer in the tanks is
Bright beer vessels bright and is likely to have a low level of
microbial contamination.
Difficult to clean because of the complex
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pipework and the design of the filler itself.
Process
Source of
Comment
contamination
Wort is a perfect medium for micro-organisms to grown in. It will be
Wort sterile after boiling, but is easily contaminated if brought into contact
with microorganisms.
Beer will support micro-organisms but in itself it is unlikely to be a
source of contamination. A more likely source is the plant. Beer
Beer in process
from other breweries may contain more, or different
contaminants, and should be treated with suspicion.
Beer recovered from surplus yeast can be a major source of
contamination because often the plant it has been processed in is not
maintained to such high hygiene standards as say FVs. Any recycling
operation needs special care because of the possibility of
Recovered beer perpetuating a contamination.

Beer recovered from packaging operations is also a major

source of contamination.
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 Infecting
Characteristics Effects on beer
microorganism
Grows along with the yeast in wort but Causes off flavours (“parsnips”) in
dies off early in the fermentation. a very slow fermentation.
Can result in increase in
nitrosamines.
Obesumbacteria. Presence indicates ineffective wort
(Hafnia) main or FV cleaning.

Very common in top-fermenting

yeasts but less common
in lager yeasts.
Infecting
Escherichia
Grows in wort or partially
fermented beer. Is an indicator of micro-
contamination of water supplied to
brewery.
Can produce DMSand diacetyl,
organism
They do not grow below pH 4.3. and flavours described as “herbal
phenolic.”
in wort
They are generally unable to grow
Enterobacter
in beer but grow rapidly in wort.
Some can metabolise nitrates to Can result in increase in
nitrites so increasing nitrosamines, nitrosamines.
particularly in lauter and mash tun
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residues
 Infecting microorganisms of piching yeast

Infecting microorganism Characteristics Effects on beer

Produces lactic
acid and diacetyl. Beer goes
Will also use any sugar left cloudy and tastes sour, and
Pediococcus
after fermentation. smells of sour milk or honey.
Beer may go glutinous and
“ropey”.
Produces lactic
Will also use any sugar left acid and diacetyl. Beer goes
Lactobacillus
after fermentation. cloudy and tastes sour, and
smells of sour milk or honey
Abnormal flavours
(possibly acetic acid) and cloudy
Wild yeast
beer. May form a film on beer
surface. Over attenuates.

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 Infecting microorganisms during fermentation
Infecting microorganism Characteristics Effects on beer
Produces lactic acid and diacetyl.
Beer goes cloudy and tastes
Will also use any sugar left after
Pediococcus sour, and smells of sour milk or
fermentation.
honey. Beer may go glutinous
and “ropey”.

Produces lactic acid and diacetyl.

Will also use any sugar left after Beer goes cloudy and tastes
Lactobacillus
fermentation. sour, and smells of sour milk or
honey
Abnormal flavours
Wild yeast (such as (possibly acetic acid) and cloudy
Brettanomyces) beer. May form a film on beer
surface. Over attenuates.

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Infecting microorganisms during maturation

Infecting microorganism Characteristics Effects on beer

Pediococcus Produces lactic acid and diacetyl. Beer
Will also use any sugar left goes cloudy and tastes sour, and smells
after fermentation. of sour milk or honey. Beer may go
glutinous and “ropey”.
Lactobacillus Produces lactic acid and diacetyl. Beer
Will also use any sugar left
goes cloudy and tastes sour, and smells
after fermentation.
of sour milk or honey.
Wild yeast Abnormal flavours (possibly acetic acid)
and cloudy beer. May form a film on
beer surface. Over attenuates.
Zymomonas Cloudy beer, bad egg aroma.

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 Infecting microorganism Characteristics Effects on beer
Produces lactic acid and diacetyl.
Beer goes cloudy and tastes sour,
Will also use any sugar left
Pediococcus and smells of sour milk or honey.
after fermentation.
Beer may go glutinous and

Infecting
“ropey”.

Produces lactic acid and diacetyl.

Will also use any sugar left
Lactobacillus Beer goes cloudy and tastes sour,
after fermentation.
and smells of sour milk or honey.
Produces acetic acid in presence of
microorganisms
of bright and
Most common in cask-
air, and forms a skin on the surface
Acetobacter conditioned beer. Grow very
of the beer. Beer goes cloudy and
quickly even at low pH.
tastes of vinegar.
Convert sugar
into alcohol, acetaldehydeand
Beer goes very cloudy and smells
packaged
hydrogen sulphide. Grow
Zymomonas
quickly in liquid sugars.
Sometimes associated with
of bad eggs. Beer may go
glutinous and ‘ropy’. beer
‘primed’ beers.
Obligate anaerobe that thrives
in extremely low DO levels
found in modern bright beers;
often associated throughout “Baby sick” and bad egg aromas.
Megasphaera
the brewery with biofilms. Cloudy beer.
Produces considerable
amounts of butyric and caproic
acids and hydrogen sulphide.
Obligate anaerobe. Produces
Bad egg aroma, vinegary. Cloudy
Pectinatus considerable amounts of | acetic
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beer.
acid and hydrogen sulphide.
 Prevention of Contamination
o Cleanliness is essential in the prevention of microbial infection.
o Wherever suitable nutrients (wort, sugar syrups, spent yeasts, etc) accumulate, micro-
organisms will proliferate.
o This is particularly important In poorly designed or installed pipework, valves, coolers and
other pieces of brewery equipment in which chronic contamination can occur and lead to the
continual infection of wort and beer.
o This problem should be eliminated by attention to recommended cleaning procedures.

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 Prevention of Contamination

♦ Hygiene is the Key to Product Quality

♦ Dirty Vessels / Mains are Primary Source of Product Microbial Loading

♦ Ineffective Cleaning can result in Biofilms

♦ Microbes in Biofilms are 100 x more difficult to Kill

♦ May NOT be Detected by Sampling

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 Prevention of Contamination
o Cleaned and sterilised plant should contain a sufficiently low level of debris that:
(a) no residual flavours from ingredients or absorbed detergent are imparted to the brew; and
(b) a low level of micro-organisms remains on the surface.

o This is generally achieved in a safe and economical way by combining a detergent and a
sanitiser.
o Common detergents include alkalies (e. g caustic soda), complex phosphate (e .g sodium
tripolyphosphate), organic compounds (e.g sodium gluconate, surfactants), and organic and
mineral acids.
o Steam is expensive and dangerous but is used for difficult equipment that is inaccessible.
Wet, rather than superheated steam is the most effective.

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Rules of good hygienic practice in the brewing industry
Theme Focus on Details include

Training in basic microbiology and hygiene

Motivation, supervision, selection, training
Personnel Training in the principles and theory of CIP
and personal hygiene
Housekeeping and pest control

Drainage, lighting, ventilation

Design criteria for internal and external
Building environnent Building security, maintenance and
areas and surfaces
cleanliness
Design criteria and materials of Layout, operation, maintenance and
Plant and equipment
construction cleanliness
Scope includes raw materials intake
Process control Importance of a quality assurance system
through storage to packaged product
Importance that the product released to Sampling and analysis plans,
Product
trade meets specification specifications
Responsibilities of "awareness" and
Legislation Appropriate references
"compliance" to legislative requirements

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Regulation
o Responsibility for the methods implemented
• to ensure product safety.
• Consideration of hazards related to:
– - The quality of the product
– - The health of the operators
– - The health of consumers.
o Provide any evidence to prove the good application and appreciate the effectiveness.

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 Hygiene
o Objective:
Identify the principles applied to plant design and methods of cleaning the surfaces
to maintain appropriate levels of hygiene

o Outcomes
− Define the key principles applied in the design of
process plant that impact on hygiene
− Identify the key factors that ensure appropriate
hygiene in the plants
− Identify the key factors in the design of effective
cleaning techniques

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 Definitions
o Physical cleanliness - visually clean.
o Chemical cleanliness – anything in contact with the cleaned surface
suffers no contamination. In practice, clean water will completely
wet the surface and drain as a continuous film without forming
rivulets or droplets.
o Detergent – a cleaning agent. By a combination of physical and
chemical processes, a detergent removes soil from a surface.
o Biocide or Disinfectant – an agent for destruction of micro-
organisms, but not necessarily 100%. In the food industries the
words Sanitizer, Sanitization are commonly used to imply both
combined cleaning and disinfection.
o Sterilant – an agent for complete destruction or removal of micro-
organisms, which is unlikely to be achieved by chemical means, and
so is often used wrongly as a synonym of disinfectant or sanitizer.
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 Definition
+ Hygienic o Sterilization
• All microorganisms were killed
• The surface is clean and sterile
o Disinfection
• Most microorganisms have been killed (except spores)
• A disinfected surface is clean but not sterile
o detergence
• 99.9% of microorganisms were killed, viruses and mold present
• Clean surface but not disinfected and not sterile
o Cleaning
• Microorganisms were not killed
- Hygienic • The surface is clean or dirty
• Clean surface may be contaminated
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Prevention of spoilage
▪ Most important factor is cleanliness:
▪ avoid rough internal surfaces, and elaborate pipe-work bends etc.

▪ Absolute sterility is not attainable but cleanliness can be

▪ CIP systems have much improved:

▪ rotating high pressure jets
▪ spray balls can be used

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 Purpose of Cleaning

o To remove permanently all the soil from the surface of the vessel (or pipe work) and leave
it in a condition suitable for use.
o A clean vessel is a permanent state – i.e. a vessel is either clean or dirty.

o Potential spoilage organisms will be present in residues.

•They can produce taints in the residue.

o Scale can similarly harbour spoilage organisms

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 Types of Soil and Scale
• Organic soil:
– Protein material
– Resins, carbohydrates Organic polymers
– Hop oils, hop resins
– Tannins, etc.

• Organic material derived yeast (protein, fats, sugar)

• Remove by dissolving
Caustic soda (sodium hydroxide) is very effective
• but reacts with C02 which can cause tanks to implode

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 Types of Soil and Scale
o Inorganic soil:
o mainly scale ( “beerstone” - calcium carbonate/ calcium oxalate )
o Mg2+,Ca2+ (oxalates, phosphates, sulphates, silicates, etc.)
o Other metal complexes (Mn2+,Zn2+, Fe2+, etc.)

▪ Should be removed by dissolving:

- Use acids ( nitric or phosphoric )

- Alkalis can be used but a sequestering agent is needed to strip

the calcium from stainless steel and these are expensive.

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Beerstone

Beerstone

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Biofilm

Growth of biofilms- differents steps

•Bacterial biofilms are structured clusters of cells

matrix coated bacteria polymeric and attached to
a surface.
•The biofilm protects the bacteria and their allows
you to survive in hostile environmental conditions.

1. Fixation of micro-organism on surface

2. Growth of bacteria and organic matrice (EPS)
3. Mature biofilm contaminating

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 Purpose of Sterilization

Is to remove and kill all micro-organisms on the

surface of the vessel, and remove any
organisms which may come into contact with
the product – wort or beer.

A vessel is only sterile for a period of time – i.e.

after sterilization – over time the vessel could
become contaminated.

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 Hygiene Factors
The underlying principles that determine the need for a
cleaning system are as follows:

• Define the factors affecting performance of

cleaning systems

• Determine the composition of the soils, scale or

biofilm accumulating on the surfaces of the plant

• Identify the microbiology and chemistry of cleaning

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 Four factors in effective cleaning: sinner circle

• Four factors involved in cleaning plant.

• An effective clean involves balancing these together.

• If you lessen one factor, you have to compensate
by increasing another.
For example, reduced time to clean, can be compensated by increasing the
temperature of the cleaning agent.

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 Hygiene Factors

• Mechanical factor

– Quantity of the soil to be removed from the surface

– Flow rates
– Turbulence
– Shear stress
– Pressures and pressure drops
– Water hammer prevention (lifts seats on valves allowing
CIP into product line)
– Efficiency of flow and pressure meters
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 Hygien factors
Mechanical factors
The effective influence of the movement of the fluid on the cleaning becomes
perceptible only from the moment when there is turbulence (see Reynolds number).

Reynolds number = Re = ρ.w.d

µ
ρ = density of liquid (kg/m 3)
w = speed (m s-1)
d = diameter of pipe (m)
µ = viscosity (Pa. s)

 Re <2000 laminar current

(parallel streams, single broadcast)

 2000 <Re <3000 transition current

 Re> 3000 turbulent current

(diffusion + transversal flows).
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 Mechanical factors

Cleaning CIP > closed circuit / piping .

Limit of turbulent regime

This table indicates for the different diameters of piping the minimum flow
rate to guarantee to ensure a turbulent regime

DIAMETER (mm) FLOW (m3/h) SPEED (m/s)

38 7 1,71

53 12 1,51

65 18 1,51

80 28 1,55

100 43 1,52

The turbulent regime is reached for a circulation speed> 1.9 m / s

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 Pipework design

Flow must be turbulent

Avoid deadlegs and non turbulent flow

•Smooth pipework bends are easier to clean.
•‘T’ pieces are impossible to clean.
•The flow of cleaning fluid should be fast enough to create turbulence
which gives a mechanical cleaning action.
•Flow rates of 2 metres per second minimum are required.
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T pieces are inevitable
o Make sure the flow is the right way

Dead legs should be avoided when designing pipework.

Where dead legs are unavoidable, the orientation should be in

the direction of flow.
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 Hygiene Factors
• Chemical factor

– Quantity and quality of the soil to be removed from the surface

– Initial concentration as measured and efficiency of conductivity
meter
– Topping up in the circuit
– Presence of solids in solution
– Dilution in circuit
– Neutralization e.g. CO2 by caustic
– Return to holding vessel or drain in multi use system

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 Hygiene Factors

• Temperature

– Initial temperature as measured

– Controlling in the circuit

– Cooling in the circuit e.g. pass over cold surface of

process vessels

– Return to holding vessel or drain in multi use system

– Efficiency of temperature control

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Efficiency of Soda as temperature

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 Hygiene Factors

• Time

– Time for complete process as measured

– Time to wash out or rinse the previous chemical or product
– Time for chemical to react on surface soil
– Time to drain the detergent in the bottom of the vessel
– Time to sequence CIP and CIR pumps
– Time to sequence opening and closing of automated valves
– Efficiency of time control

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 Detergents
▪ Detergents are formulated for specific tasks:

▪ Caustic Soda - dissolves organics, but dissolves aluminium, very hazardous, reacts
with CO2, does not rinse well

▪ Silicates - dissolves organics but less aggressive than caustic soda, good
dispersant

▪ Acids - dissolves scale, not affected by CO2 but need high pressure spray heads

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 How work detergent

Detergent

soil
microbes
Germs and soil in suspension in detergent

Rinsing

Elimination of soil and microbes during rinsing

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 Detergents
▪ Phosphates
Strong dispersing power - hold insoluble particles in
suspension, easily rinsed

▪ Wetting Agents
Teepol, reduce surface tension can cause foaming, easily
rinsed

▪ Sequestering Agents
EDTA, keep carbonate in solution by stripping calcium from
surfaces

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 Wetting agents
W
e
t
t
ing
ag
e
nt
s

'
Bead'o
fwa
t
er
si
tt
i
ng o
na
sur
fa
ce.

W a
t
erwi
tha
'
w e
t
t
ing
'age
n
t
add
ed.

• Water tends to form beads on surfaces especially if they are a bit greasy.
• A wetting agent like ‘teepol’ makes the water much better at wetting the
surface.
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 D i
sso l
v ing a gen t
s
Dissolving agents
(s (solvent)
olve nt
s )
Th
esame
soi
l
P
a
r
t
ic
l
eso
f
soi
l d
iss
ol
ved
i
na
l
i
qui
d

• A substance goes into solution when it is dissolved. For example sugar can
be dissolved in water and it becomes part of the liquid.
• Caustic soda is a very good solvent for detergent purposes. It will dissolve
most things even organic matter.
• Acids like nitric or phosphoric are also good at dissolving soil.
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 Dispersing agents

Soil on the The same soil

plant surface dispersed in a
liquid

• Solids are dispersed in a liquid when they are held in the liquid
and don’t settle out too quickly.
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 Sequestering agents
S
e
q
ue
s
t
er
i
nga
g
e
nt
s

B
ee
rs
t
one
o
nCl
ea
n
t
h
epl
an
t s
u
r
fac
e
s
ur
f
ac
e

• Beer stone is scale that forms on plant surfaces. It is worse in hard water
areas.
• It is very difficult to remove but sequestering agents like EDTA will help.
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 R
i
nsin g a ge
Rinsing agentsnt
s

D
e
ter
g
ent Cl
ea
n
a
d
her
i
ngt
osur
fac
e
p
l
an
ts
ur
fa
ce

• It is important that the detergent can be removed from the plant once its job
has been done.
• Caustic is difficult to remove without the addition of rinsing agents.
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 Choice of detergent
▪ High Temperature Equipment
▪ - mashing vessels and coppers
Must keep heating surfaces clear of soil and scale. Use hot caustic soda containing anti-foam

▪ Vessels Receiving Boiled Wort

▪ - tanks and whirlpools-
Acid wash or if caustic soda used must include sequestrant to remove scale

▪ Fermentation, Yeast Handling and Maturation Vessels-

▪ Requirement for sterility.
Cold caustic soda with wetting, rinsing agents and sequestrant; can use acids in rotation.

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 Choice of detergent
o Bright beer tanks
• Lower soiling - use acids to maintain CO2 atmosphere

o Pipework and packaging lines

• usually hot caustic soda with wetting, rinsing and sequestering

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 Choice of detergents
o Containers
o Bottles:
can have a very high level of organic soil, sometimes left on for a long time Hot
are cleaned with hot caustic soda with additives to help especially rinsing agents.
o Kegs:
caustic only if all population is steel
often breweries will have a mixture of steel and aluminum so they will use a non-caustic
alkali detergent with the usual additives.
o Casks:
depends on population; often cleaned with hot water jetting and steam sterilized
wooden casks are often cleaned with hot water but metal would be treated the same as
kegs

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 Cleaning Chemicals
Detergent Ingredients
• Caustic or Alkali Detergents • Acid detergent

– Caustic soda (base), sodium – Phosphoric acid/nitric acid

gluconate, sodium heptonate, blend (1.2:1 ratio) – base,
amino tris
(methylenephosphonic acid), polycarboxylates, wetting
EDTA, wetting agent, chlorine agents, 1-hydroxyethylidene –
source 1,1-diphosphonic acid

• Alkali detergent (Al Tanks)

– Sodium metasilicate,
tetrasodium phosphate,
sodium tripolyphosphates,
soda ash, wetting agent

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 Alcali detergent: ECOLAB
Products:

Alkalis are products that have a basic pH (between 7 and 14) in

aqueous solution.
Soda (sodium hydroxide)
Potash (potassium hydroxide)
• lipid
They are used to remove: • Proteins
• Sugars

-Mip EA > detergent , formulated product (potash + complexings antifoam at hot

temperature)
-Asepto FL-D > detergent disinfectant, (soda + chlorine)

-Soda + stabicic (hydrogen peroxide and anti foam)

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 Acid: ECOLAB
Products:

Acids are product with an acid pH (between 0 to 7) in aqueous

solution

Nitric acid
Phosphoric acid
Sulfamic acid
• scale
They are used to remove: • rust taint

-Horolith V> detergent , formulated product (Phospho-Nitric acid)

-Horolith OXY> detergent (phosphoric+ H2O2)

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Desinfection / Sterilisation
o Destruction of microorganisms to a previously defined goal

o Sterilization: destroy all

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 Choice of sterilant
▪ Often difficult to kill all microbes
▪ When we kill, we first have an exponential curve then asymptotic
▪ Never sure that a particular treatment killed all the microbes

Life cells /ml

Time
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Disinfection

Disinfection An essential second step

5% of
95% of microorganism m

Elimination during Elimination during

detergence disinfection

5% of
95% of risk
risk

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Disinfection

The power of multiplication

Multiplication of bacteria
in 24 hours

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 Sterilant

Sterilants work by creating a hostile environment for micro-organisms;

• Temperature
• pH
• Chemical or surface activity
• Oxidation

oThey should be
– effective at low concentrations against a wide range of contaminants
– Non toxic and easily rinsed
– Ideally they should be left on the surface hence residues should not effect beer quality

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 Sterilant
Heat, Radiation or Chemicals:
o Heat
• is very effective if sufficient time and temperature are used, but is expensive.
▪ Steam
▪ not very effective at low pressure in large vessels, good for mains; equipment like seals must be able
to stand the temperature.
• can carry undesirable odour
▪ Radiation
▪ only common treatment is continuous ultra violet sterilization of a water supply
▪ Chemicals
▪ simplest used to be ozone (O3) but it is corrosive, therefore now many others:

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 Use of Steam

o Saturated (wet) steam is very effective for sterilizing cleaned equipment only.
o Steam bakes some types of soil on to surfaces, making it more difficult to remove,
and may protect embedded micro-organisms.
o Steam kills all types of micro-organism, although to achieve sterility at atmospheric
pressure requires continuous steaming for 1½ hours.
o Steam is usually freely available in a brewery, but subsequent rinsing may be
required (depending on steam quality).
o Radiated heat from a vessel being steam sterilized could affect a nearby
fermentation if the vessels are close.

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Impact Temps / temperature

Hotchaude
Eau water Dry heat
Chaleur sèche Vapeur steam
Humid humide
Température (°C) temps Température (°C) temps Température (°C) temps
85 20 min 120 8 heures 100 20 heures
140 2,5 heures 110 2,5 heures
160 1 heure 115 50 min
170 40 min 120 20 min
180 20 min 121 15 min
125 6,5 min
130 2,5 min

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 Chemical sterilant
▪ Halogens
▪ chlorine and iodine
Effective, cheap, but dangerous and corrosive; will taint beer flavour if not rinsed
off.
Hypochlorite must be used in alkaline solution or chlorine gas is evolved!!
Not effective on heavy soil
Iodophors - release iodine used in acid solution.

▪ Quaternary Ammonium Compounds

Effective way to use ammonia, but must be rinsed
Very bad effect on foam

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 Chemical sterilant

▪ Peroxides
▪ produce free radical oxygen which kills the cell. Hydrogen peroxide is
safe but is not a very effective sterilant.
▪ Peracetic Acid
▪ used with hydrogen peroxide to improve stability,effective and no
adverse effect on flavour

▪ Formaldehyde
▪ very effective but very poisonous, unpleasant aroma

▪ Amphoteric surfactants
▪ good at getting into uneven surfaces, are effective, do not taint beer or
affect head, but expensive
IFBM | Reproduction prohibited | Advanced course in brewing technology | BGI ETHIOPIA | January 2023 155
 Choice of sterilant
▪ It is often difficult to kill all microbes
▪ Kill is at first exponential then asymptotic
▪ Never sure if a particular treatment has killed all

▪ Key Factors –
amount of soil, initial number of organisms, temperature and pH used for appropriate sterilant.

o Wort mains and beer mains can easily be sterilized with hot water or steam. They may be
sterilized with surfactants or peracetic acid.
o Beer vessels usually cannot be sterilized with heat so surfactants or peracetic acid can be
used.
o Packaging plant can easily be sterilized with hot water or steam. They may be sterilized with
surfactants or peracetic acid. IFBM | Reproduction prohibited | Advanced course in brewing technology | BGI ETHIOPIA | January 2023 156
 Disinfectant: ECOLAB

Product:

They are used to kill microorganism :

-Oxonia active > disinfectant (per acetic acid)

-Nodsan> disinfectant (triamin)
-Alcodes> disinfectant self drying homologated without
rinsing (alcohol + glutaraldehyde)
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 Safety hazards
o Can be dangerous to handle
o Keep people and chemicals separate
o Always aim to use the least aggressive
He
al
th a
nd
chemical which does the job S
a
fe
tyatWo
rk
Act
o COSHH regulations detail storage and
handling.
o Use protective clothing.
o Showers and eye baths

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Limestone

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Inside vessels

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Presence of beerstone inside the fermenter

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Pipe

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 CIP design
o Time and temperature of cleaning.
o Maintaining detergent strength.
o Detergent & rinse water flow rates & volumes.
o Effective scavenging between each segment of a cycle.
o Procedures for monitoring cleaning system capability and
performance.
o Advantages/disadvantages of single use and systems with
detergent/rinse recovery.
o Factors affecting cleaning frequency appropriate for different
types of plant.
o Design features.
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 Cleaning in place ( CIP )

D
e
li
ver
y
o Principles.
T
a
nk
• Programmes.
• Spray heads.
• Automation and monitoring. C
.I
.P.
s
yst
em R
e
tu
rn

P
i
pew
o
rk

D
e
li
ver
y

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 Cleaning in place ( CIP )
CIP Principles. D
e
liv
er
y
o CIP is the circulation of detergents, rinses
and sterilants through the plant to clean it T
a
nk
without dismantling.
o The chemicals can be recovered or C
.I
.P.
dumped after use in a total loss system. s
yst
emRe
t
urn
o Cleaning tanks with CIP uses a spray
head.
o Cleaning mains and other plant involves P
ip
ew
or
k
circulation.
o There is a delivery and a return system. D
e
liv
er
y

o The system can be automated.

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Cleaning in place ( CIP )

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Cleaning in place ( CIP )

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CLEANING PROGRAM

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Nature of equiement Residues to remove Objective of treatment Frequency of realization Method Products to use

Plan de nettoyage
Agglomerates of malt or Remove of agglomerates Presence of agglomerates in
Elimination Manuel Elimination
cereal silo

Malt silo If i presence of insects:

fumigation of empty & clean Attention (use only
If needed
silo . Treatment on 2 times if authorised products)
recurrence of insects
dry miller without aspiraion Dust of malt, varoius physically and visually Dismount and use a vaccuum manual brushing, vaccum
End of weekly production
system particles clean cleaner cleaner
dry miller with aspiraion Dust of malt, varoius physically and visually Dismount and use a vaccuum manual brushing, vaccum
At the revison of miller
system particles clean cleaner cleaner
various particles, starch, Caustic soda at 3,5% and
physically and visually Rinse, clean for 30 minutes
sucgars, proteins, tanins, End of weekly production 80°C (if pump can be
clean and rinse (CIP)
Humid miller scale cleaned at this temperature)
various particles, starch,
Mash vessel & cereal Visual abscence of organic Rinse, clean for 30 minutes Caustic soda at 1,5% and
sucgars, proteins, tanins, End of weekly production
cooker &mineral residues and rinse (CIP) 80°C
scale

various particles, starch,

Visual abscence of organic
sucgars, proteins, tanins, After each brewing cycle Rinser for 30 min Clean water
&mineral residues
scale

Recommandation: function of
brewing condition and water
Solution of caustic soda at
hardness, if des traces are
1,5%, 80°C with 0,5% of
visual , a cleaning at caustic Rinse, clean and rinse with
Lautern tun chlorine to remove s tanins .
soda with chlorine to clean water
Nitric acid at 2%, 65°C to
remove tannins; acid
remove mineral
cleaning to remove mineral if
necessary

In case of filtration problem

Hot water at 80°C and
or prologened stop, it is Clean with mechanical action
mechanical action (high
advisable to check the flase if necessary
pressure water)
funds
various particles, starch, Solution of caustic soda at
Visual abscence of organic
sucgars, proteins, tanins, After each brewing cycle Rinse and clean 1,5%, 80°C and rinse with
& mineral residues
scale clean water
In case of filtration problem Cleaning of cloth with high
pressure hot water
Water at 80°C
Membrane filter

Recommendation: for Rinse, disinfect, rinse with

productions with a prolonged clean water after prolonged
shutdown, the filter must be shutdown and when restarting Per acetic acid solution 0.3%
disinfected

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 Cleaning program
Nature of equipment Residues to remove Objective of treatment Frequency of realization Method Products to use

Depending on clogging and Rinse, clean for 20 minutes

Caustic soda at 1,5% and
various particles, starch, heating slopes (increase in and rinse with clean water
80°C
sucgars, proteins, tanins, Visual abscence of organic energy consumption) (CIP)
whitish deposits of minerals, & mineral residues
hop particles
Rinse, clean for 20 minutes
Caustic soda at 1,5% and
Kettle End of production and rinse with clean water
80°C
(CIP)
Add sodium hypochlorite 1.5% soda solution at 80 °C
Brownish deposits (tannins) Visual absence of organic & if required (0.5% vol / vol) to the soda with addition of 0.5% sodium
mineral residues solution hypochlorite
In weekly production Treatment with nitric acid at 1% nitric acid solution at 65 °
Whitish deposits
Visual absence of organic & (preventive treatment) 65 °C for 10 minutes, rinse C
mineral residues Depending on the hardness with potable water 5% nitric acid solution at 65 °
of the water, when required C

Rinse, clean for 30 minutes

Caustic soda at 1,5% and
Hop particles, tannins, haze End of weekly production and rinse with clean water
Visual absence of organic & 80°C
Decanter or whirlpool (CIP)
mineral residues
Proteins, tannins, mineral
deposits, hop particles

if continuous brewing, rinse

Proteins, tannins, mineral Microbiology and visually
Immediately after use with hot water at 90°C for 20 Hot water at 90°C
deposits, hop particles clean
min

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 Cleaning program

Nature of equipment Residues to remove Objective of treatment Frequency of realization Method Products to use
Rinse, clean and disinfect,
Cycle of 30 min (15 minutes
Proteins, tannins, mineral 1,5% soda solution at 80°C.
After each brewing cycle in each way ) and rinse with
deposits, hop particles Per acetic solution at 0,3%
sterile water (80°C or
Cooling and oxygenation microfiltered)

Recommendation: for
productions with a prolonged
Rinse, disinfect, rinse with
shutdown, equipment must
sterile water after a 0,3% per acetic solution.
be disinfected. IF water is
prolonged shutdown and at Once a week or month 1%
hard, nitric acid should be
the restart . Cycle of 30 min nitric acid at 65°C.
used (frequency: every week
(15 minutes in each way )
at one time every month
function of hardness of water)

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 Cleaning program
Nature of equipment Residues to remove Objective of treatment Frequency of realization Method Products to use
Visually and microbiology Rinse enought with cold 1,5% soda solution at 80°C
Yeast, sugars, proteins, clean After each use and again if water to remove yeast and
tannins, mineral deposits, no use for more than two totally CO2. Clean for 30
hop particles days minutes and rinse with sterile
potable water (CIP)
Fermenter
Before filling disinfected with per acetic 0.3% per acetic then rinse
solution then rinse with with sterile potable water
sterile potable water
If necessary at least once a Nitric acid solution for 30
Whitish deposits (beer stone) 2% nitric acid at 65°C
year minutes
Visually and microbiology Rinse enought with cold 1,5% soda solution at 80°C
Yeast, sugars, proteins, clean After each use and again if water to remove yeast and
tannins, mineral deposits, no use for more than two totally CO2. Clean for 30
hop particles days minutes and rinse with sterile
Maturation vessel
potable water (CIP)
Before filling disinfected with per acetic 0.3% per acetic then rinse
solution then rinse with with sterile potable water
sterile potable water
Proteins, mineral deposits Visually and microbiology Rinse 15 seconds and 1% nitric acid solution
clean remove water without CO2 (2
After each use and again if times), clean with nitric acid
Filtered beer vessel no use for more than two solution at 30°C for 20
days minutes, wash out, rinse 3
times 15 seconds with sterile
water (CIP)
All vessels under CO2 If CO2 is not removed from Cleaning by pulse of soda or See the supplier of cleaning
tanks, do not use soda to other cleaning solution solution to use and the
clean due to procedure to follow
depressurization of tank

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Nature of equipment Residues to remove Objective of treatment Frequency of realization Method Products to use
Wooden vessels If you want to keep indigenous Cleaning with hot water then Hot water at 55°C for 20 min.
microflora, no cleaning. steam sterilization Steam for 20 min

Secondary piece: valves of Visually and microbiology After each use with empty Brush the pieces then rinse Concentrated cleaning liquid
sampling, les joints, probe, clean tank and before cleaning for general maintenance
Yeast, proteins, mineral
serpentines, les gauges and without corrosion of metallic
deposits.
valves of fermenter or other surface
tanks
Beer cellulosic membrane Yeast et proteins Visually and microbiology
After each use Clean with hot water Water at 55°C for 20 min
filter with diatome clean
clean by circulation of hot Rinsing water at 55°C.
water for 10 minutes. Disinfection water at 80-
Before each use Disinfected with very hot 85°C
water for 30 minutes and
rinse with sterile water.

Yeast et proteins Visually and microbiology Rinse with water and remove
After each use
Candle or metallic plate filter clean totally CO2
Cleaning by solution 1,5% soda solution at 80°C
circulation circulate la
Before each use
solution for 20 minutes and
rinse (CIP)
Disinfected with 0.3% per 0.3% per acetic solution or
acetic or very hot water at very hot water at 90°C for 30
90°C for 30 minutes min
Candle or metallic plate filter If CO2 is not remove from the Pulse cleaning of cleaning See the supplier of cleaning
under CO2 filter, don do not use soda solution solution to use and the
solution for cleaning procedure to follow
efficiency and violent
depressurization of vessel.

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Nature of equipment Residues to remove Objective of treatment Frequency of realization Method Products to use
Yeast and proteins 1,5% soda solution at 65°C
first possibility : Rinse the
filter with water. Circulation of
soda solution for 20 minutes
and then rinse with sterile
water (if possible make a
link with bottling) of bottling
Second possibility: If 5% soda at 65°C
impossible to circulate
Visually and microbiology solution: Remove the
Sterile filter After each use
clean cartridge. Rinse with cold
water for 15 minutes. Put the
filter in soda solution for 24
hours maximum. Rinse à
water potable
What else the procedure, it 0,2% nitric acid solution (pH
had to put the filter in solution 1,85) at 30°C. 0,3 % per
of nitric acid and then acetic solution.
disinfected before use in
per acetic solution
Pipe and flexible Yeast, sugars, proteins, end of production 1,5% soda solution at 80°C.
mineral deposits Establish a loop between the
different ducts by connecting
to a pump.p. Rinse with
water, clean with soda
solution for 20 minutes and
rinse with sterile potable
water. the flow should be at
1,5 of beer flow to have a
turbulent flow (> 2 m/s). Be
Visually and microbiology careful of pipes that do not
clean withstand high temperatures.
Bottling (is barometric or Yeast, sugars, proteins, end of production Cleaning according supplier According supplier
atmospheric) and kegging mineral deposits recommendation. Some recommendations
pieces can be altered by
some chemical products and
conditions (temperature,
pressure and time)
Pasteurisation of bottle microorganisms, mineral during the production Treated water with bactericide, Bactericide, algaecide and
deposits and corrosion algaecide and anti-corrosive anti-corrosive compounds
compounds (according supplier
recommendation)

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