Food fermentations by yeasts and filamentous fungi

Food fermentations
by yeasts and
filamentous fungi
Prof. Anna Maráz
Department of Microbiology and Biotechnology
Faculty of Food Science
Corvinus University of Budapest
ERASMUS IP Maribor 2011

Yeasts
Position of yeasts and moulds (filamentous
microfungi) in fungal taxonomy
~ 800 species
In food: ~ 50 species
Fungi (Eumycota)
Chytridiomycetes
Zygomycetes
Ascomycetes
Basidiomycetes
Deuteromycetes
(Fungi imperfecti)
Moulds
~ 80.000 species
In food: ~ 100 species

Main differences between yeasts and filamentous fungi
(moulds)
Characteristics
Vegetative cells
Growth rate (generation time)
Metabolism
Lytic enzymes (hydrolases)
Toxin production
Yeasts
Unicellular (budding and
pseudomycelia)
1-2 hours
Facultative
aerobic/anaerobic
Few
No
Moulds
Mycelia
6-48 hours
Aerobic
Many
Yes
Dimorfic fungi: ability for budding and pseudomycelia or true mycelia

Yeasts
- Cell morphology: ellipsoidal, cylindrical,
lemon-shaped, filamentous (with
pseudohyphae or septate hyphae)
- Metabolism: respiration and in some cases
ethanolic fermentation
Saccharomyces cerevisiae

Budding
Budscars
Saccharomyces cerevisae

Fission yeasts
e.g. Schizosaccharomyces pombe

Pseudomycelium formation
e.g. Candida albicans

Glycolysis
Piruvatdecarboxylase
Alcoholdehydrogenase
Glucose 2 pyruvate 2 acetaldehide 2 etanol
C 6H 12O 6
Ethanolic fermentation
CH 3-CO-COOH
CH 3-CHO
2 CO 2
2 NADH 2 2 NAD
C 6 H 12 O 6 = 2C 2 H 5 OH + 2CO 2
180 g glucose 92 g ethanol + 88 g (44,8 normal liter) CO 2
CH 3-CH 2-OH

End products of alcoholic fermentation
and pathways connected to it

Formation of higher alcohols
during the synthesis of amino acids

Tradition of food fermentations
• Ancient tribes: milk clotting, spontaneous fermentation of
beverages
• Sumerians B.C. 3800: beer from cereals
• Ancient Egypt: beer from cereals, dough for leavening bread
• Greek and Roman civilizations: culture of
wine, beer (Dionysos, Bacchus)
• Middle ages: beer, wine, cheese
production in monasteries

Recognition of microorganisms:
- Louis Pasteur: diseases of wine and beer caused by
microbes
- Christian Hansen: use of pure cultures of yeast for beer
fermentation
19. century: development of biochemistry:
- Fermentation in cell free extracts
- Recognition of enzymes and intermediers of fermentation
- The glycolytic sequence: Embden-Meyerhof pathway

20th century:
• Exploitation of microorganisms in the fermentation and
processing of foods:
- Bread, alcoholic beverages, dairy products
- Meat products, vegetables
• Development of starter cultures
• Industrial fermentations
- Natural antimicrobial substances
- Pharmaceuticals, fine chemicals, enzymes
21st century:
• Modern biotechnology
• Molecular biology and genetic engineering

Role of microorganisms in the production of
alcoholic beverages (examples)
________________________________________________________
Yeasts
Sacch. cerevisiae fermentation of wine, ale-beer
Sacch. pastorianus fermentation of lager beer
Hanseniaspora / Kloeckera spontaneous fermentation of wine
Schizosacch. pombe fermentation of rum
Moulds
Aspergillus oryzae koji preparation of sake
Bacteria
Oenococcus oenos malo-lactic fermentation of wine
Enterobacteria fermentation of lambic beer
Acetic acid bacteria flavour development in rum
Zymomonas mobilis fermentation of pulque
________________________________________________________

Raw ingradients
Pre-treatment
Boiling
Fermentation
Yeast recycling
Destillation
Maturation
Final alcohol content
(%, v/v)
Major alcoholic beverages – summary of production
Yes
No
Yes: weeks
3-6
Beer
Barley, adjuncts
(rice, wheat,
maize, etc.)
Malting, meshing
Yes (hops)
S. cerevisae
S. pastorianus
No
S. cerevisae
No
Yes
Yes: years
40-45
Whyisky
Barley (Malt
whisky
Barley, wheat
(Grain whisky)
Malting, meshing
Crushing,
macerati
on
No
S. cerevisae
S. bayanus
No
No
Yes: months,
years
8-14
Wine
Grapes
Spirits, liqueurs
Barley, maize,
molasses,
grapes, whey,
etc.
Variable,
dependent on
substrate
No
S. cerevisae
K. marxianus
(whey)
No
Yes
Varies
35-45
Graeme, 1998

BREWING

History of brewing in pictures
Brewery in the XVIth century Fermentation in open container
Modern brewery Fermentation in tanks

The brewing process
Malting- Barley soaked for 5-7 days to germinate and
activate amylases; then dried, crushed
Mashing - Malt mixed with water, adjuncts added, heated to hydrolyse
starch, then at 74 o C enzymes inactivated, the wort settled, filtered
Wort boiling- Hops extract solubilized, microbes inactivated
Fermentation - Brewer’s yeast added, 5-15 o C for 1-2 weeks
Aging - Young (‘green’) beer stored, lagered at 0 o C for weeks to
months; yeast settled
Finishing - Filtering, carbonation, sterilization, packaging

Process steps Main biochemical conversions
_____________________________________________________________________________________
Barley grains
Water → ↓
Malting ----- Development of enzymes (amylase, protease)
↓
Kilning ----- Stop respiration in germinating barley
↓
Milling
Water, adjuncts → ↓
Mashing ----- Enzymatic hydrolysis of barley
↓
Spent grains ← Wort separation
Hops → ↓
Wort boiling
↓
Spent hops ← Wort separation, cooling
Yeast → ↓
Fermentation ----- Production of ethanol, CO 2 , other metabolites
(eg. diacetyl)
↓
Yeast ← Beer clarification
↓
Maturation, lagering ----- Final flavor development
↓
Filtration
↓
Packaging
↓
Pasteurization
Flow chart of brewing

Typical changes in beer fermentation

Traditional use in brewing
Pitching yeast: Saccharomyces cerevisiae – lager beer
Sacharomyces pastorianus – ale beer
Advantages: converting carbohydrates to ethanol, wort to
beer, producing flavors, reducing contamination
Novel use before steeping:
Control of mycotoxin producing fusaria by
Debaryomyces hansenii
Lambic beer (gueuze) spontaneous, natural fermentation
Brettanomyces, Candida, etc, lactics, enterobacters
Weiβbier: top fermenting yeasts

Wine fermentation

Traditional use in wine making
Wine yeasts: Saccharomyces cerevisiae
Saccharomyces bayanus – low temperature
Saccharomyces uvarum – Botryotized grape
Advantages: rapid fermentation, ethanol tolerance,
absence of off-flavours
‘Wild’ yeasts are always present:
Hanseniaspora / Kloeckera start fermentation
Candida, Pichia, Metschnikowia contribute to some degree
in developing the ‘bouque’
Novel trends: use of mixed starters

Outline of wine fermentation
White wines Red wines
___________________________________________________
Grapes Grapes
Stemming, crushing Stemming, crushing
Settling, pressing Maceration with skin
Yeast fermentation Yeast fermentation
Clarification
Maturation, fining
Filtration
Bottling
Malo-lactic fermentation

Growth of yeast species during the
fermentation of wine
o S. cerevisiae • Hanseniaspora / Kloeckera ■ Candida spp

Other alcoholic beverages
Special types of wine
- Champagne and sparkling wines
- Sherry
- Sake
- Cider
Spirits (distilled alcoholic beverages)
- Whisky, rum, vodka, gin, tequila, etc

Indigenous fermented foods
Regions Products
Orient Soy
Indian Vegetables
African Mixed
American Fish
The indigenous fermented foods constitute a group of foods that are
produced in homes, villages, and small cottage industries
Some indigenous food fermentations such as soy sauce (shoyu), Japanese
miso, Indonesian tempe, Indonesian tape ke ten, Japanese sake, Indian
idli and dosai, and fish and shrimp sauces and pastes are
commercialized at large scale

Types of indigenous foods
1. Basic type of fermentations made by koji, in which
microorganisms are grown on a cereal-grain or legume
substrate to produce a crude enzyme concentrate, koji, that
can be used to hydrolyse components in particular
fermentation (e.g. soy sauce, miso, tempe, oncom, tape,
and rice wines such as sake);
2. Fermentations involving proteolysis of vegetable proteins by
microbial enzymes in the presence of salt and/or acid with
production of amino acid and peptide mixtures with a meatlike
flavour (e.g. soy sauce, Indonesian miso)
3. Fermentations involving enzymic hydrolysis of fish and
shrimp or other marine animals in the presence of relatively
high salt concentrations to produce meatflavoured sauces
and pastes

Types of indigenous foods (cont.)
4. Fermentations producing a meat-like texture in a cerealgrain-legume
substrate by means of fungal mycelium
that knits the particles together (e.g. Indonesian and
Malaysian tempe, Indonesian oncom)
5. Fermentation in which organic acids are major products
(this category includes Korean kimchi, African ogi, idli,
dosai, tape, and also tempe, in which acidification
occurs during the initial soaking of soybeans)
6. Fermentations in which ethanol is a major product (e.g.
rice wines, palm toddies, sugar cane wines, agave
pulque, pito beer, and tape ketan)

Examples of indigenous fermented beverages

Genetic improvement of yeast starters
– brewing yeasts
- Introduction of glucoamylase activity to
produce low alcoholic beer
- Elimination of phenolic off-flavor
- Reduction of vicinal diketone formation
- Introduction of killer activity / killer
resistance (also in wine yeast)

Examples of genetically modified brewer’s
yeasts
Transformed gene(s) Effect References
STA2 S.cer.var. diastaticus Meaden et al.1985
Glucoamylase - A.awamori dextrin fermenting, Cole et al.1988
α-amylase & glucoamylase higher ethanol yield Dohmen et al.1990
- Schwan. occidentalis Lancashire et al.1989
FLO1 S.cerevisiae better flocculation Watari et al.1991
Acetolactate decarboxylase reduced production Fuji et al.1990
diacetyl Shimizu et al.1989
MET25 sulfuhydrase reduced H 2 S Omura et al.1995
production

Genetic improvement of yeast starters –
wine yeasts
- Reduction of fusel alcohol production
- Reduction of H 2S production
- Introducing flocculation and reducing of
foaming
- Increasing yield of glycerol

Examples of genetically modofied wine
yeasts
Transformed gene(s) Effects References
Pectate lyase filtering,clarification Gonzales-Candelas
et al. 1995
malolactic genes (Lb.lactis) ML fermentation Volschenk et al.1997
GDP1 S. cerevisiae sensory quality Michnick et al.1997
K1 killer toxin S. cer. competitive advantage Boone et al. 1990
Resveratrol synthase (poplar) resveratrol Becker et al. 2003
& coenzyme-A ligase (grape) production

Approval of GM food yeasts
Baker’s yeast, maltose derepressed 1989
Brewer’s yeast, expressing STA gene 1993
Sake yeast, flavour enhanced 2002
Wine yeast, malolactic fermentation 2003

1,8
1,6
1,4
1,2
1
0,8
0,6
Effects of wine consumption
on the death risk from different deseases
Relative risk of death
halálozási kockázat, bázis =1
Risk of death Halálozási depending kockázat a on borfogyasztás wine consumption függvényében
(12 years, éves vizsgálat, 34000 34000 persons, főre, males 40-60 éves between francia 40-60, férfiak, Nancy
Nancy)
rákos Cancer betegség
egyéb Other okú betegségek
cardiovasculáris Cardio-vasculare desase betegségek
0 1-2 pohár 2-3 pohár 3-5 pohár 5-7 pohár 7-12 pohár > 12
Glass pohár of wine bor naponta / day
a rákos betegségek emésztőszervi és fül-orr-
gégészeti jellegűek

Negative effects of wine consumption
• Ethanol
– Toxic effects for central nervous system, liver, stomach
etc.
– Moderate consumption (linked to meal) is not harmful
• Females: 1,5 – 2 dl wine /day
• Males:2-3 dl wine /day
– „French paradox”
• Methanol
– high toxicity: consumption of 8-10 g causes serious
visual disturbances (lethal dose: 40-100 g)
– methanol content of Hungarian wines: 0.02 - 0.3 g/l

Brakedown of methanol in human
body
CH 3 – OH
CH 2 = O
HCOOH
Xantin-oxidáz-kataláz (XOK)
B 12 vitamine
Formic acid – high toxicity!

Negative effects of wine consumption (cont.)
• sulphur dioxide
– relatively low toxicity for humans (in solution)
– allergic reaction in some consumers (particularly
asthmatics): wheezing, flushing, tingling etc.
– its role in headache is possible but not fully proven
• biogenic amines (except serotonin)
– headache (red-wine induced headache symptom)
– allergic reactions
– decomposition of amines in human body by
monoamine-oxidase enzyme is inhibited by
ethanol!

The main biogenic amines of wines

MYCOTOXINS
?
The only known toxin that my be present in wine is
Ochratoxin-A (OTA)

OA ug/l
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
Kontroll Botrytis Penicillium Asp. ochraceus
OTA concentrations
measured in different wines
OA ug/l
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
Aszú base
(before
fermentation)
OTA production of some
mould on sterile grape musts
Aszú wine
(after
fermentation)
(pH 3,2; 20 o C, 2 weeks)
Irsai Olivér
Sauvignon
Chardonnay

Positive effects of wine
consumption

Correlation between the yearly wine consumption and
the death rate (/1000 persons) of cardio-vasculare
desases
Death / 100 persons
„French paradox”
Wine consumption Log L/person/year

Positive effects of wine consumption
(cont.)
• Resveratrol
– Chemical nature: stylbene (phenolic compound)
– Origin: phytoalexyne, produced by the plant (grape) in
stress situation, e.g. as a response to fungal infection
– Effects: Increase of the plasma level of high-density
lipoprotein (H.D.L.), maintenance of low level of “bad”
cholesterol (low density lipoprotein: L.D.L) -
prevention of arteriosclerosis and coronary heart
disease.
– Level in wines: 0 - 8 mg/l (red wines contain more
than white wines)

Cisresveratrol
R=sugar
Isomers of resveratrol
Transresveratrol
3,4,5trihidroxisztilbén-3-ß-mono-Dglükozid

Procyanidines: condensed katechines and leucoanthocyanines
Procianidindimer
Maintenance of good-cholesterol level in blood
Procianidintetramer

Positive effects of wine consumption (cont.)
• Minerals, organic acids
– positive nutritional effects
• Serotonin (biogenic amine):
– antidepressive effect
• Wine is poor vitamin source!

Conclusion
A glass of wine a day keeps the doctor away!

Thank you for your attention!