Moulds and Yeasts in the Dairy
Industry
Dairy Australia /NCDEA Webinar
16 July, 2014
Hubert Roginski
Spoilage of dairy products
• Bacteria
- psychrotrophic
- proteases
- lipases
- thermoduric
- spore-forming
• Moulds
• Yeasts
Moulds and Yeasts
• Very adaptable
• Grow in diverse environments
• Moulds of significance to dairy industry are
aerobic
• Yeasts can grow both aerobically and
anaerobically (“Pasteur effect”; 1857)
Approx. composition, pH, and aw of selected
dairy products (Frank, 2001)
Moulds – basic facts
• Eukaryotic cells
• Microscopic fungi that form visible mycelial
growth
• Mycelium: a mass of hyphae, unbranched or
branched filaments - tubular structures with a
rigid cell wall, external to cell membrane
• Hyphae may or may not be septate
• Hyphae grow mainly by apical (tip growth)
extension
Mould spores
• for reproduction
• in majority of moulds – conidia (2-5 μm in
diameter)
• in some genera – sporangiospores
• In some genera – ascospores (sexual spores)
• not resistant, unlike bacterial spores
-ascospores are more resistant to heat
and desiccation then conidia & similar spores
Mould spores
• M.s. become easily airborne,
contaminating air and all surfaces in
factory
• A Penicilium spp. may produce 100,000
conidia from a single colony (Hocking, 1997)
Growth of moulds
– Low aw requirements
– Broad range of pH
– Mesophilic and psychrotrophic
– Overwhelming majority are aerobic
Minimal aw required for growth at 25 °C
(Farkas, 2001)
Moulds in dairy products
• Moulds are ubiquitous in the dairy environment
• Beneficial – used in manufacture of mouldripened cheese varieties
• Detrimental
- Spoilage of dairy products
Cheese, yoghurt, butter, cream,
concentrated products
- Mycotoxin production
- Aspergillus spp. & Penicillium spp.
- Relatively low levels in dairy foods
Mould spoilage of cheese
The susceptibility to spoilage depends on (Sorhaug,
2011):
• sanitation during manufacture and ripening
• length and degree of ripening
• storage conditions (temperature, relative
humidity, type and extent of packaging)
• water activity (aw)
• composition
aw requirements for moulds isolated
from cheese (Hocking, 1997)
Most Penicillium spp. grow at a low aw and are
quite salt tolerant
P. glabrum, P. roqueforti and P. commune and some
other species can grow at aw as low as 0.80 at 25
°C
Cladosporium spp. are less tolerant
C. cladosporioides has a minimum aw of 0.86 at
25 °C
Cheese spoilage moulds – pH range
A wide pH range
P. roqueforti - pH 3.0 to 10.3
may grow even at a lower pH under ideal
conditions (Hocking, 1997)
Closely-related species of Penicillium spp. may
grow even at pH 2.0 (Pitt & Hocking, 1997; cited
after Hocking, 1997)
Moulds in cheese spoilage
• Penicillium spp., Cladosporium spp., Phoma
spp. and some other moulds can grow at 1–
5 °C.
• Some Penicillium spp., particularly P.
roqueforti, can grow in the presence of only
1% oxygen; CO2 at ≥40% can prevent their
growth (Sørhaug, 2011)
Moulds in cheese spoilage
• Other genera found in cheeses (Hocking &
Faedo, 1992):
Alternaria spp.
Cladosporium spp.
Eurotium spp.
Fusarium spp.
Mucor spp.
Moulds in commercial packages of cheese hard, semihard, semisoft (Sørhaug, 2011)
Origin of the data:
Australia, New Zealand
Argentina, South Africa
Norway, Turkey, Spain, Italy, Denmark, Greece,
France, Germany, Belgium, the Netherlands, the
Czech Republic, Switzerland, Malta, Portugal
(Azores), United Kingdom
United States, Costa Rica
Japan
Moulds in commercial packages of cheese hard, semihard, semisoft (Sørhaug, 2011)
• 50 to > 90% of the isolates were Penicillium spp.
• Dominant species:
P. commune, P. nalgiovense, and P. roqueforti
• Less often isolated:
P. brevicompactum, P. chrysogenum, P. citrinum,
P. cyclopium, P. expansum, P. glabrum, P.
granulatum, P. palitans,
P. solitum, P. verrucosum, P. viridicatum
Changing taxonomy of moulds
(an example: Penicillium spp.; Sørhaug, 2011)
Moulds isolated from cheese
Genera accounting for <10% of the total mould
isolates (Sørhaug, 2011):
• Aspergillus spp. (A. versicolor)
• Cephalosporium spp.
• Cladosporium spp.
• Geotrichum spp.
• Mucor spp.
• Scopulariopsis spp.
• Syncephalastrum spp.
Thread mould
• Develops during maturation
• Dark threads or spots on cheese surface
- usually near folds or creases in the plastic wrap
• Most common species (in Cheddar cheese, according
to Hocking, 1997):
- Cladosporium cladosporioides & C. herbarum
- Penicillium spp. – P. commune and P. glabrum most
common
- Phoma spp. also commonly found in maturing
Cheddar (Hocking & Faedo, 1992)
Thread mould
• The problem is most common in cheese made in
continuous block forming systems in production
of Cheddar and similar hard cheeses (Hocking &
Faedo, 1992)
• The mould can grow on the cheese surface
• Main cause: the whey that comes out of the
cheese during vacuum packaging (Hocking & Faedo,
1992)
- the whey is then entrapped in the creases or
folds of the plastic packaging
Fungi in vacuum-packaged Cheddar (4-12 months)
affected by thread mould (1) (Hocking & Faedo, 1992)
Fungi in vacuum-packaged Cheddar
affected by thread mould (2) (Hocking & Faedo, 1992)
Fungi in vacuum-packaged Cheddar
affected by thread mould (3) (Hocking & Faedo, 1992)
Fungi isolated from cheese factory air
(Hocking & Faedo, 1992)
Fungi isolated from the curd
(Hocking & Faedo, 1992)
Aspergillus flavus
• closely related to A. parasiticus, A. oryzae and A.
sojae (Dobson, 2011)
• does not grow at refrigeration temperature (min.
temp. 7 °C; for aflatoxin production: 15 °C)
• a known aflatoxin producer
• an opportunistic pathogen - aspergillosis in
immunocompromised people
• Cheddar and similar cheese varieties do not
provide a favourable environment for A. flavus
Fungi isolated from the whey
(Hocking & Faedo, 1992)
Fungi isolated from the continuous block former
(Hocking & Faedo, 1992)
Cheese thread mould – environmental
samples
(Hocking & Faedo, 1992)
• A number of sources of contamination are
possible
• Thread mould spoilage in vacuum
packaged cheese during maturation is
caused by a relatively small number of
fungal species
Thread mould in Cheddar (3-6 m) in Argentina
(Basilico et al., 2001)
Thread mould in retail packs
(Hocking, 1994)
• Penicillium spp. most common
- P. commune and P. roqueforti dominant
• Also found:
- other species of Penicillium
- C. cladosporioides
- Eurotium spp.
Growth of cheese spoilage moulds
• A good growth at low temperatures
• Many can grow near freezing or slightly
below
- Growth of C. cladosporioides reported at -10 °C;
-2 °C to -5 °C is a more realistic minimum (Hocking,
1997)
- All Penicillium spp. isolated from cheese grow
well at 1-5 °C, with maxima close to 30 °C or even
slightly above (Pitt & Hocking, 1997)
Mycoflora of Kaşar (a hard Turkish cheese)
(Aran & Eke, 1987)
Moulds in Kuflu cheese (mould-ripened, semihard Turkish cheese) (Hayaloglu & Kirbag, 2007)
Metabolites of cheese moulds (1)
• Mycotoxins
• Decarboxylation of sorbate to 1,3-pentadiene by
sorbate-resistant species of Penicillium spp. (e.g.,
P. roqueforti), causing a ‘kerosene’ taint in
cheese spreads
• Reduction of sorbate to 4-hexanoic acid and 4hexanol by other Penicillium spp.
Metabolites of cheese moulds (2)
• Bitter peptides in surface-ripened or blue-vein
cheeses, caused by strains of P. camemberti, P.
roqueforti and Geotrichum candidum
• Other off-flavours in cheese caused by moulds:
musty
mushroom
plastic
rancid
Mycotoxins produced in cheese by moulds
(Sørhaug, 2011)
Mycotoxins in culture extracts of P. verrucosum var.
cyclopium and P. roqueforti grown on YE Sucrose Agar
(Aran & Eke, 1987)
Mycotoxins of public health significance
• ochratoxin A and sterigmatocystin
• P. verrucosum and A. ochraceus, producers of
ochratoxin A are not frequent isolates from cheese
• Aspergillus versicolor
- a mesophile that does not grow below 10 °C
- can be controlled by modified atmosphere
packaging
- refrigerated ripening and storage of cheese plus
packaging to reduce oxygen prevent its growth and
production of sterigmatocystin (Sørhaug, 2011)
Minimising risks caused by mycotoxins
• Moulds grow on the surface of cheese
• The migration of mycotoxins into the
cheese is seldom greater than 2 cm
• Removing >2 cm layer of cheese would
remove the mycotoxins (Sørhaug, 2011)
Moulds – routes into dairy plants
• air – spores are airborne
• transport vehicles entering processing plant
• various ingredients, for example - fruit purees
for stirred yoghurt manufacture
• packaging materials
• personnel: clothing, footware, hair etc.
• all potential sources of moulds need to be
considered when determining the risk of
contamination
Mould control in plant environment
• Air quality control in:
- processing
- ripening
- and packaging areas
• Efficient air-filtration systems - essential in dairy
processing plants to reduce the numbers of mould
spores
• Direction of air flow and location of outlets
• Air quality: <50-100 m-3 moulds and yeasts is
recommended for air in cheese processing plants
• Compressed air: <50 m-3 moulds and yeasts
Mould control in plant environment
• Reduction of mould spores in the air by using
high-efficiency particle air (HEPA) filters in all
inlets
- They are designed to remove 90-99% of
particles ≥0.3 μm which reduces the numbers of
mould spores in the air
- Proper maintenance and regular filter
replacement are critical for good air quality
Mould control in plant environment
• The use of positive air pressure in critical areas
can significantly decrease the level of mould
contamination
• The air intake and exhaust systems in the plant
need to be adequately separated from each other
to prevent recontaminated air from returning into
the processing plant
Mould control in plant environment
• To improve air quality, “cleanroom technology” is
sometimes used in some cheese processing
plants, particularly in the ripening rooms
• Control of:
- air filtration and circulation
- movement of people who are dressed in
‘cleanroom’ attire
• Use of footbaths and airlocks before entering the
area
Mould control in plant environment
• Generally, ‘cleanrooms’ should have air in which
particles >0.5 mm are limited to 0.3 m-3 of air
• Humidifiers with sterilised and even
demineralised water are also used in these areas
• Control of the temperature also assists in
slowing the growth of moulds in cheeses
Mould control in plant environment
• Plant design – separating ‘clean’ from ‘dirty’ areas
• Clean storage of packaging materials
• Personnel movement – traffic through ‘clean’ areas
should be minimised
• Personnel hygiene – minimise manual handling of
product, wear hairnets, gloves etc.
• Packaging and distribution
- consider strength and flexibility of packaging film, its gas
permeability
- use the best gas mixture
(based on Hocking, 1997)
Mould control in plant environment
Cleaning schedules :
• Designating equipment and areas of the factory
• Cleaning protocols – specifying cleaning agents
and sanitisers
• Frequency of cleaning and sanitising
• Nominating the person responsible
(based on Hocking, 1997)
Mould control in plant environment
• Moulds can grow in moist environments found in
dairy plants and establish themselves on:
- ceilings
- floors
- walls
- floor drains
if these areas are not properly cleaned and
sanitised
Mould control in plant environment
• The choice of sanitisers is critical
• Quaternary ammonium compounds and chlorinebased sanitisers are more effective against mould
spores than either peracetic acid or peroxides
• If the mould spore count is still high, either:
- the sanitiser used is not effective, or
- the fog-generating equipment cannot produce
fog particle size small enough to be suspended in
the air for the recommended contact time with
the sanitiser
Antifungal effects of commercial sanitisers
(Koruklouglu et al., 2006)
• Commercial sanitisers:
- alcohol, peracetic acid, iodophors, aldehydes,
quaternary ammonium compounds (QAT, a, b and c),
and a chlorine-based agent
- different concentrations.
• The microorganisms tested:
- two moulds: Aspergillus niger (5 strains) and
Penicillium roqueforti (5 strains)
- six yeasts: Saccharomyces cerevisiae, Sacch. uvarum,
Kloeckera apiculata, Candida oleophila, Metschnikowia
fructicola, Schizosaccharomyces pombe
Antifungal effects of commercial sanitisers
(Koruklouglu et al., 2006)
• QAT (a) and QAT (c) were most effective against
all the microorganisms tested
• The chlorine-based disinfectant proved most
effective against the moulds at all
concentrations (0.5, 1.0, 1.5 and 2.0%).
• The efficacy of peracetic acid and alcohol-based
sanitisers was higher against the yeasts than
against the moulds tested
Effect of antifungal agents on survival of conidia
of Phoma glomerata (Basilico et al., 2001)
Effect of antifungal agents on the survival of
conidia of Phoma glomerata (Basilico et al., 2001)
• Sorbic acid and potassium sorbate were not
effective against P. glomerata
• MIC for Phoma spp. (Fente-Sampayo et al., 1995;
cited after Basilico et al., 2001):
750 ppm of potassium sorbate and 2.5 ppm
natamycin
Control of mould growth – new packaging
technologies
• Packaging material can be coated with antimycotic
agents:
- sorbates
- propionates
- natamycin
• Antimycotic agents can be incorporated directly into
the packaging material
• Excluding oxygen by the use of vacuum and modified
atmospheric packaging is also used to limit growth of
moulds on cheeses
Control of mould growth – new packaging
technologies
• Modified atmosphere packaging (MAP):
- the use of:
more than 50% carbon dioxide and
less than 0.5% oxygen
- will prevent spoilage moulds from growing on MAPpackaged cheeses
• Problems with MAP:
- package leakage and pinhole defects
- allow moulds to grow and cause spoilage
Growth of fungi and mycotoxin production on cheese
under modified atmospheres (Taniwaki et al., 2001)
• Species studied:
Mucor plumbeus, Fusarium oxysporum,
Byssochlamys fulva, B. nivea, Penicillium
commune, P. roqueforti, Aspergillus flavus and
Eurotium chevalieri
• Cheddar cheese
• Decreasing concentrations of O2 (5% to <0.5%
and increasing concentrations of CO2 (20–40%).
Fungi selected for the study by Taniwaki et al. (2001)
Growth of fungi on cheese under modified
atmospheres (Taniwaki et al., 2001)
• All fungi examined grew in atmospheres
containing 20% and 40% CO2 with 1% or 5% O2
• Growth was reduced by 20–80%, compared with
growth in air, depending on species
• At 20% or 40% CO2 with <0.5% O2, only B. nivea
showed growth (very slow)
Growth of B. nivea on Cheddar cheese
(Taniwaki et al., 2001)
Growth of fungi on cheese under modified
atmospheres (Taniwaki et al., 2001)
Mycotoxin production on cheese under
modified atmospheres (Taniwaki et al., 2001)
The formation of aflatoxins B1 and B2,
roquerfortine C and cyclopiazonic acid:
• greatly reduced
• not totally inhibited in these atmospheres.
Mycotoxin production on cheese under modified
atmospheres (Taniwaki et al., 2001)
Mould growth in yoghurt
(D. Cecchin, NCDEA, 2014)
Penicillium spp. in yoghurt
(J.Barlin, NCDEA, 2014)
Mould spoilage in yoghurt
• Mould genera that can grow on yoghurt surface:
Penicillium, Aspergillus, Mucor, Rhizopus, Alternaria,
Monilia and Absidia (Sørhaug, 2011)
• Moulds grow more slowly than yeast contaminants in
yoghurts
• Fruit purees added to yoghurt – usually the main source
of moulds and yeasts
- heat-resistant moulds often do not grow well at low
temperatures
- some genera (e.g., Mucor spp.) grow well at
refrigeration temperature
Talaromyces spp. (relatively heat-resistant) may be
present in fruit-flavoured yoghurt (Sørhaug, 2011)
Moulds in cultured milk products
The moulds Mucor spp. and Aureobasidium spp. as
well as the unpigmented algae Prototheca spp.
show yeastlike growth at submerged cultivation,
hence a risk of mistaking them for yeasts
Mucor spp. and Prototheca spp. produce large
quantities of carbon dioxide and are
opportunistic pathogens (see Mucor
circinelloides mentioned earlier)
Mucor circinelloides in yoghurt (USA), 2013
Mucor circinelloides in yoghurt (USA), 2013
Duke University article (mBio, 8 July, 2014):
• The U.S. FDA: yogurt products were
contaminated with Mucor circinelloides
• a mucoralean fungal pathogen
• >200 consumers complained of symptoms
incl. vomiting, nausea, and diarrhoea
• The manufacturer voluntarily withdrew the
affected yogurt products from the market
Mucor circinelloides in yoghurt (USA), 2013
Duke University authors:
“We successfully cultured an M. circinelloides
isolate and found that the isolate belongs to the
species M. circinelloides f. circinelloides, which is
often associated with human infections. In
murine and insect host models, the isolate was
virulent. While information disseminated in the
popular press would suggest this fungal
contaminant poses little or no risk to consumers,
our results show instead that it is capable of
causing significant infections in animals.”
Moulds in butter and dairy spreads
• Hydrolytic rancidity
Rhizopus spp.
Penicillium spp.
Cladosporium spp.
Aspergillus spp.
Less common now, owing to improved sanitation
and control of dairy plant air (Sørhaug, 2011)
Geotrichum candidum is often included with the moulds
causing butter spoilage, but this organism has been
clasisfied as a yeast for almost 30 years now (EliskasesLechner et al., 2011)
Moulds in butter and dairy spreads
Penicillium spp. and Cladosporium spp.
produce off-flavours
- including 2-methylisoborneol and
geosmin
- ‘earthy’ flavour
Mould spoilage of cream
• Penicillium spp. – in cream that is stored for
extended periods at refrigeration temperatures
• In addition, Geotrichum candidum and other
lipase-producing yeasts can grow on cream
containing added sucrose for sale to bakeries
because they produce lipases
Mould growth on 45% cream
(D. Cecchin, NCDEA, 2014)
Moulds in sweetened condensed milk
The main spoilage organisms in sweetened
condensed milk (with 42-45% sucrose):
Osmophilic, sucrose-fermenting yeasts and moulds
(Ledford, 1998)
Aspergillus spp. and Penicillium spp. can grow on the
surface:
- poor sanitation in the processing plant
- entry of mould spores and
- a large enough headspace in the can (Sørhaug,
2011)
Flavoured UHT milk
Fusarium oxysporum
found in flavoured UHT milk in Australia
• owing to the production of thick-walled
chlamydoconidia
• the ability to tolerate low oxygen tensions
(Sørhaug, 2011)
Chlamydoconidia/chlamydospores – thick-walled resting
spores produced by certain fungi from cells of hyphae.
Mould spoilage of other products
• Heat-resistant fungi, which produce ascospores,
do not normally spoil dairy products
but
• Byssochlamys nivea, Eupenicillium brefeldianum,
Neosartorya fischeri, and Talaromyces
avellaneus
have been reported as causes of spoilage in
products such as UHT custard and cream cheese
(Sørhaug, 2011)
Yeasts – basic facts
Yeasts
- not a taxon
- eukaryotic organisms defined by morphological and
physiological criteria
A ‘typical’ yeast:
- unicellular, saprophytic organism, which ferments
various carbohydrates
- reproduces asexually by budding
- some y. reproduce asexually by fission
- Sometimes - reproduction by ascospores
- Cells are larger than those of bacteria, on the average –
several μm
Yeasts in raw milk
• Total yeast count in milk is usually quite low
101-103 CFU/ml
• Main species:
Candida intermedia, Can. parapsilosis, Cryptococcus curvatus,
Debaryomyces hansenii, Galactomyces geotrichum,
Kluyveromyces marxianus, K. lactis, Pichia farinosa, Pic.
fermentans, Pic. membranaefaciens, Pic. anomala, Trichosporon
beigelii & Yarrowia lipolytica
• Yeasts are killed during pasteurisation but may survive,
e.g., in porous gaskets
• Recontamination can occur
Yeasts - effect of heat
• Pasteurization of milk destroys:
- vegetative cells of yeasts
- blastospores
- chlamydospores of Candida albicans & other
species
- ascospores of ascosporogenous yeasts
- arthrospores of the Endomycetaceae (e.g.,
Galactomyces geotrichum)
Yeasts - effect of heat
Zygosaccharomyces bailii (often found on
cheese, in fruit mixes and yoghurt)
• the most heatresistant yeast species
• the heat inactivation values
- vegetative cells: D1min = 56 °C, z = 4 °C
- ascospores: D1min= 64° C, z = 3 °C
Yeasts - effect of heat
• Under low water activity (aw) conditions, e.g.,
when yeast cells are pressed in porous gaskets,
heat may be less effective
• Areas in the processing plant that pose a risk of
recontamination:
heat exchangers
cooling water
filling equipment
air
packaging materials
Spoilage of milk and pH-neutral products
• Generally, in pasteurised milk and pHneutral
dairy products recontamination with yeasts is of
little importance
• The filling operation is not sterile, so the milk is
most frequently recontaminated with Gramnegative organisms
• These bacteria, and not yeasts, are responsible
for the shelflife limit of 7-14 days of pasteurised
milk
Products with severe heat treatment
history
• Various milk products are heattreated at 90-110
°C, for a short time
• Under such conditions, all bacteria as well as the
spores from thermophilic moulds are destroyed
• Yeasts can only contribute to or cause spoilage
through recontamination
Yeasts in cultured milk products
• Together with contamination with moulds,
contamination with yeasts is the largest
microbiological problem in these products
• Fruitcontaining fermented milk products spoil
quickly, owing to the high fructose and sucrose
content of the fruit preparations, which
promote yeast growth and fermentation
Yeasts in cultured milk products
• Cultured milk products (yoghurt, sour cream,
cottage cheese etc.) should be free of yeasts
- low pH of these products favours growth of
yeasts, e.g., yoghurt pH range is 3.8-4.5
- yeasts can still grow at 0 °C
- spoilage caused by fermentation (CO2
produced), proteolysis and lipolysis
- contamination with yeasts often creates the
largest microbiological problem in these products
Yeasts in cultured milk products
• Kefir, kumys – y. are part of the normal microflora
• Yeast spoilage in:
- Yoghurt, primarily fruit-flavoured yoghurt
- Butter and buttermilk
- Buttermilk should not contain >200 CFU/ml
Yeasts in yoghurt – importance of cold storage
The doubling time in fruit yoghurt for Sac.
cerevisiae, without shaking:
D30 °C = ~5 h
D20 °C = ~10 h
D10 °C = ~62 h
D4 °C = ~84h
for Gal. geotrichum: D30 °C = ~6 h
D20 °C = ~12 h, D10 °C = ~96h, D4 °C = ~7 days
(Büchl & Seiler, 2011)
Yeasts in cultured milk products
Symptoms of yeast spoilage:
- swelling of the cups
- changes in texture
- product discoloration
- offflavours, offtastes
- visible microbial colonies on the product surface
Yeasts in cultured milk products
• Fruit preparations are delivered to the
plant in large containers
• Even negligible contamination with yeasts
in these containers can lead to immense
losses
Yeasts in cultured milk products
The risk of spoilage by yeasts can be reduced by:
• filling temperature of the fruit mix of <15 °C
• chilled storage of the container
• the avoidance of a stepwise emptying of the
container
• high sugar concentration in the fruit preparation
• prompt processing
Yeasts in cultured milk products
• Large dairy plants increasingly produce the fruit
preparations themselves
• This reduces the risk of microbial contamination
of the product
• The fruit preparation is pumped directly from
the cooking boiler or tubular heat exchanger
through a cooler into the storage tank
Yeasts in cultured milk products
Geotrichum candidum ('white mould'
Galactomyces geotrichum) found in cultured
products is characteristic of milk
The xerophilic species Zygosaccharomyces spp.,
Citeromyces matritensis, Can. versatilis, Pichia
etchellsii, Pic. ciferrii and Pic. sorbitophila are
typical of fruit preparations
Yeasts in cultured milk products
The dominant yeast species from fruit
preparations and contaminated fruitcontaining
cultured milk products are:
Sac. cerevisiae, Pic. anomala, Pic. fabianii, Pic.
membranaefaciens, Hs. vineae, Hs. uvarum,
Debaryomyces hansenii (a typical cheese
yeast), Can. parapsilosis, Can. tropicalis, Can.
intermedia, Ts. delbrueckii and Cs. lusitaniae
pH-neutral fruit-containing products
• Yeasts are, next to bacilli and moulds, the most
common spoilage organisms in products such as
milk rice or milk pudding
• Additions based on fruit, cocoa, nuts, vanilla
husks, vitamin mixes or cereals
• All the yeast species that can be found in the
added preparations, in the dairybased
component, and in the dairy environment are
potential contaminants
pH-neutral fruit-containing products
• The common symptoms of spoilage:
- blowing of containers
- a change in the product consistency or in
aroma and flavour
pH-neutral fruit-containing products
• In most cases, the added preparations are the
cause of the spoilage, owing to the availability
of nutrients favouring yeast growth (glucose,
fructose, sucrose, organic acids)
• The occasional long storage time of the product
containers at 20 °C compounds the problem; in
such situations, a considerable increase in yeast
count can occur.
pH-neutral fruit-containing products
Yeasts pose no problems in:
• ‘underlaid’ products, where first the fruit puree
is filled in the cup and then the hot dairy
portion at 70-80 °C is added on top; no yeasts
are present in the milk portion
• toppings made from whipped cream or
vegetable oil foams, since these have been
heated and there is little chance for yeasts to
multiply there
Yeasts in dairy plants
• Yeast species identifed most commonly in the
dairy plant environment (foors, walls,
equipment):
Debaryomyces hansenii, Clavispora lusitaniae,
Rhodotorula spp., Cryptococcus spp., Can.
intermedia, Can. parapsilosis, Can. sorbophila,
K. marxianus, Ya. lipolytica, Issatchenkia spp.,
Trichosporon spp. and Gal. geotrichum
Yeasts in cheeses
• Quality defects in white cheeses (quark,
cottage cheese, cream cheese)
• Soft, semi-hard and hard cheeses
-in some cheeses – beneficial effects on
the desirable microflora, on flavour and
aroma
- defects of aroma, flavour & texture
(blowing of young cheese) in other
cheeses
Yeasts in cheeses
The yeast species most frequently isolated from
acid curd cheeses (quark, Gervais, cottage
cheese, cream cheese) are:
Galactomyces geotrichum, Kluyveromyces
marxianus, K. lactis, Pichia membranifaciens, P.
guilliermondii, Debaryomyces hansenii,
Trichosporon beigelii, Issatchenkia orientalis, and
Yarrowia lipolytica
(Büchl & Seiler, 2011)
Yeast-related defects in acid curd cheeses
The yeast count thresholds for defects:
slightly versus strongly ‘yeasty, fermenting, fruity,
old, musty, bitter’ are 104–105 and 105–106 cfu
ml-1, respectively
These effects are species-dependent
- Galactomyces geotrichum produces the strongest
effect , followed by Kluyveromyces spp., Pichia
membranifaciens, Saccharomyces cerevisiae,
Debaryomyces hansenii, Issatchenkia orientalis,
Yarrowia lipolytica, and Saccharomyces exiguus (Büchl &
Seiler, 2011)
Yeast-related defects in acid curd cheeses
A separator curd
• initially contaminated with 100 cfu g-1
• the first signs of sensory defects after 5–7 days
at 10 °C
• it was spoiled after 10 days
• a good product should have <100 cfu g-1 yeast
contaminants
(Büchl & Seiler, 2011)
Yeast-related defects in acid curd cheeses
• The generation times of yeasts in curd at:
2 °C - 100h
4 °C - 50h
6 °C - 20h
10 °C - 10 h
• Absence of yeasts - an important indicator of good
manufacturing practice (GMP)
• With the presence of yeasts the shelf life of
products at 10–6 C is limited to 10–15 days
(Büchl & Seiler, 2011)
Effects of yeasts on cheese
• Yeasts growing in brine cause its de-acidification,
creating the risk of growth of salt-tolerant pathogens,
such as Staphylococcus aureus
• There are large differences in the cheese counts
between the surface, middle and core layers of the
cheese, causing diferences in pH across the cheese
• In Taleggio cheese, after 35 days of ripening at 3-10 °C,
the pH in these three layers was 6.5, 5.5 and 5.2,
respectively
- In some countries, antifungal agents natamycin (pimaricin),
propionate, or sorbate are used to inhibit the growth of
yeasts on the surfaces of hard and semihard cheeses
(Büchl & Seiler, 2011)
Effects of yeasts on cheese
• The yeast count on the surface increases rapidly at
the start of maturation
- after 8–10 days it reaches a maximum of 106–109 cfu
g-1 or 108 cfu cm2
- this count decreases slightly during further ripening
- the softer the rind, the higher the initial yeast count
The predominant species:
Debaryomyces hansenii, Trichosporon beigelii, Yarrowia
lipolytica, Kluyveromyces marxianus, Candida zeylanoides,
C. catenulata, Torulapsora delbrueckii, and Galactomyces
geotrichum
(Büchl & Seiler, 2011)
Effects of yeasts on cheese
Populations of different genera and species are found,
depending on:
• milk quality
• water and salt content of the cheese
• production hygiene
• possible addition of yeast culture
• storage temperature
• stage of ripening
• competing flora
• location of cheese sampling
(Büchl & Seiler, 2011)
Effects of yeasts on cheese
Population of yeasts is also affected by:
• the geographic region
• manufacturer
• the range of products made on site
• production lot or batch
• age of the brine bath
• season of the year
• the methods of isolation, enumeration, and
identification of yeasts used
Debaryomyces hansenii is usually the prevailing species
(it is salt-tolerant, with an aw minimum of 0.85)
(Büchl & Seiler, 2011)
Osmotic tolerance of yeasts isolates
• Debaryomyces hansenii is usually the prevailing species
(it is salt-tolerant, with an aw minimum of 0.85)
- D. hansenii still shows growth to 0.3 OD after 100 h
incubation in a broth medium containing yeast extract,
malt extract, glucose, and 18% (w/v) NaCl
• Other species isolated from cheeses are:
Kluyveromyces marxianus (15% NaCl), Torulaspora
delbrueckii (14%), Yarrowia lipolytica (14%), Pichia
farinosa (14%), Candida versatilis (14%), Saccharomyces
unisporus (14%), Candida zeylanoides (13%), Candida
catenulata (13%), Saccharomyces cerevisiae (9%; aw
minimum 0.94), Galactomyces geotrichum (2%), and
Trichosporon beigelii (2%) (Büchl & Seiler, 2011)
Osmotic tolerance of yeasts isolates
For Debaryomyces hansenii
- the optimum for growth is between 0 and 11%
NaCl
- the inhibitory NaCl concentration is 24%
The xerotolerant Zygosaccharomyces spp. and
Citeromyces matritensis have a high tolerance to
low aw (minimum 0.65-0.60) in an environment
with high sugar concentrations, yet are relatively
sensitive to NaCl
(Büchl & Seiler, 2011)
Yeasts in vacuum-packaged Cheddar affected by
thread mould (Hocking & Faedo, 1992)
Health risks from yeasts
• Some yeast genera and species are facultative
pathogens
- Opportunistic pathogenic yeasts usually found in
milk from mastitic cows
- Infections in infants, seniors, pregnant women,
immunocompromised people, persons with
AIDS, diabetics and alcoholics
Health risks from yeasts
• An infection with Filobasidiella (a teleomorph of
Cryptococcus spp.) may lead to Cryptococcus
mycosis of:
- the brain
- lungs and the entire respiratory tract
- bone marrow
- kidneys
- digestive tract
- eyes
- skin
- central nervous system
- nails
Yeasts and Moulds – Detection and
Enumeration
• Traditional methods based on agar media:
- Malt extract agar
- Czapek yeast extract agar (CYA)
- Davis’s yeast salt agar
- Orange serum agar
- Oxytetracycline-glucose-yeast extract agar (OGYE)
Yeasts and Moulds – Detection and
Enumeration
• Methods based on agar media:
- Dichloran-rose bengal-chloramphenicol agar (DRBC)
- DG18 agar (dichloran 18% glycerol agar) – for
osmophilic (xerophilic) fungi (Harrigan, 1998)
- Creatine sucrose dichloran agar (CREAD),
developed for selection of mould species high in
lipids and protein.
- Moulds commonly detected on cheese, Penicillium spp.
and Aspergillus spp., grow well on this medium (Sørhaug,
2011).
Yeast colonies on DRBC
(J. Barlin, NCDEA, 2013)
Yeasts and Moulds – Detection and
Enumeration
3M Petrifilm plates
Save laboratory space and media
preparation time
Yeast & Moulds - Y&M Count Petrifilm Plate
3M Yeast and Mould Count Petrifilm Plate
3M Rapid Yeast & Mould Count Plate
• Result in 48 hours
• Included in the AOAC Research
Institute’s Performance Tested
MethodsSM (Certification #121301,
January 2014)
3M Rapid Yeast & Mould Count Plate
Geotrichum candidum in yoghurt
after 48 hrs, compared to a DRBC agar
plate after 5 days
Yeasts on Rapid Y & M Count Plate
Yeasts in kefir
(Chr. Higgs, NCDEA, 2014)
Yeast Count – effect of time & temp.
Moulds on Rapid Y & M Count Plate
Mould Count – effect of time & temp.
Yeasts and Moulds – Detection and
Enumeration
Instrumental methods:
• Immunochemical – based on antigenantibody recognition, e.g., enzyme-linked
immunosorbent assay (ELISA)
• Molecular techniques, based on recognition
of genetic material, e.g., polymerase chain
reaction (PCR)
Controlling yeasts and moulds in the product
Antagonism between some lactic acid bacteria and
moulds and yeasts
Chr. Hansen’s FreshQ cultures for applications in
fermented milk products
- selected results shown on the following slides
FreshQ®2 and 4 inhibit yeast
Example: Debaryomyces hansenii (50 cfu/g) in yogurt stored at 7ºC/45ºF
130
FreshQ®2 and 4 inhibit yeast
Example: Yarrowia lipolytica (50 cfu/g) in yogurt stored at 7ºC/45ºF
131
FreshQ®2 and 4 increase time to visible mould
Example: FreshQ® 2 in Greek yogurt 1,5% fat
Challenge test with high contamination level: 1000 spores/spot!
Number of days until first mould growth is visible (average of two cups):
Still no growth
on day 51
Penicillium commune
Aspergillus versicolor
P. palitans
P. crustosum
P. paneum
P. palitans
P. paneum
P. roqueforti
FreshQ®2 and 4 cause significant mould inhibition
Example: FreshQ® 2 in stirred yogurt 1,5% fat, 30 days storage @ 7ºC/45ºF
Challenge test with high contamination level: 1000 spores/spot!
Reference
FreshQ® 2
P. crustosum
133
A. versicolor
P. commune
P. brevicompactum
133
More challenging moulds are also inhibited by FreshQ®2 and 4
Example: P. palitans, P. crustosum and P. paneum inoculated at 1000 spores/cup
on stirred yoghurt 1,5% fat and stored for 28 days at 7ºC / 45ºF
Reference
FreshQ® 2
FreshQ® 4
P. palitans
P. crustosum
P. paneum
More challenging moulds are also inhibited by FreshQ®2 and 4
Example: Two different Rhizopus stolonifer isolates inoculated (500 spores
/cup) on stirred yoghurt 1,5% fat and stored for 36 days at 7C/45F
Reference
FreshQ® 2
FreshQ® 4
FreshQ®2 & 4 work over a broad temperature range
Challenge test with P. crustosum added at 1000 spores/cup and stored 36 days
7ºC/45ºF
12ºC/54ºF
22ºC/72ºF
Reference
FreshQ® 2
FreshQ® 4
136
FreshQ®4 was developed to better target also the ‘Zygomycetes’
moulds (i.e., Mucor spp. and Rhizopus spp.)
Example: FreshQ® inhibition of Mucor spp. (100 spores/cup) after 15 days storage at 22°C / 72°F
Growth rate of this mold is reduced substantially, with FreshQ®4 providing better suppression than
FreshQ®2
Reference
FreshQ®2
Reference
FreshQ®4
137
FreshQ® 2 & 4 have no impact on fermentation
time
138
FreshQ® 2 & 4 have no impact on fermentation
time
139
FreshQ® 2 & 4 have no impact on post acidification
Stirred yogurt 1,5 % fat, 3,5 % protein
140
FreshQ® 2 & 4 have no impact on post acidification
Stirred yogurt 1,5 % fat, 3,5 % protein
141
FreshQ® 2 & 4 have no impact on post acidification
Stirred yogurt 1,5 % fat, 3,5 % protein
142
FreshQ® 2 & 4 have no impact on post acidification
Stirred yogurt 1,5 % fat, 3,5 % protein
143
FreshQ® 2 & 4 have no impact on post acidification
Stirred yogurt 1,5 % fat, 3,5 % protein
144
FreshQ® 2 & 4 have no impact on post acidification
Stirred yogurt 1,5 % fat, 3,5 % protein
145
Texture is the same with FreshQ®
146
Texture is the same with FreshQ®
147
No negative flavour impact of FreshQ® 2 & 4
• In the development of FreshQ® 2 & 4 there has been great
focus on selecting strains with minimum impact on the
process and flavour of the yogurt
• In some cases it will be possible to detect a small difference in
the flavour, especially towards end of shelf life. In these cases
Chr. Hansen’s experience is that yogurt made with FreshQ® 2
& 4 tastes more fresh and appealing than the reference
• In conclusion FreshQ® 2 & 4 can have the effect of keeping you
product fresher in flavour, even in cases where there is no
detectable contamination in the reference product without
FreshQ® 2 & 4
148
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