Department of

Food Science and Technology

Faculty of Agricultural Engeenering

ITP321 Food Fermentation Technology

Lecture Note #10

Fungal Fermentation

LILIS NURAIDA
Course learning outcome Lecture 9

• Upon successful completion of this course, the student will

have been able to explain:
• fermentation techniques,
• chemical and biochemical changes, and
• microorganisms commonly used for mold fermentation to
produce fermented foods, pigment and citric acid; mixed
fermentation of molds and other microorganism.

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Fungal fermentation
• Tempe
• Oncom
• Mold ripened Cheeses
• Citric acid

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Tempe
• Fermented soybean
o Other legumes could also be used
o Bean and the mold are protein source
• Used starter culture: laru or usar
o Main microorganisms Rhyzopus oligosporus
• Process:
o Soybean are boiled, soaked overnight, seed coast are
removed, split into halves, boiled, dried, cooled, inoculated.
o packed into banana leaves or perforated plastic bag and
then left to ferment for 2 days
 The mold grows through to knit together the beans and turn them into
tempe.
o Alternative: use of dehulled soybean

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Production method of tempe at household
industries
soybean Boiling soybean 1st Boiling step

Dehulling in traditional way

• They are Soaking overnight*
many Washing
production Dehulling using
methods dehulling machine
applied in Soaking overnight*
different Washing
Household 2nd boiling step in soaking water
tempe Innoculation (cassave solid waste-
industries based starter) Cooling at room T for 8 h
• Production Packaging in plastic bag
methods Innoculation with commercial starter
affects Incubation for
microbial Incubation in bulk covered with plastic bag for
population 48 h*) 36 h*)
in tempe. Cutting and packaging
Tempe

Tempe
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Packaging for tempe fermentation
• Banana leaf
• Perforated plastic bag
• Wooden
Container should:
– Permits access of sufficient O2 for growth of the mold
The access of oxygen is not sufficient to promote sporulation with
darkening of mycelium
– Control the temperature
– Retain the moisture during fermentation
– Avoid free contact of free water with the bean

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Fermentation process of tempe
• Hydration/Acid fermentation
o Acid fermentation take place during soaking
pH falls to 4.5-5.3
Does not affect the mold growth, but prevents the development of undesirable
bacteria that might spoil the bean
Lactic acid bacteria may involve during this step
o Acidification the bean lengthen the fermentation time before ammonia is deliberated
The mold in tempe fermentation is very proteolytic; deamination following hydrolisis
releases ammonia, cauisng pH to raise.
Above pH 7, sufficient free ammonia is released to kill the mold
Glucono delta lactone or soaking water from previous batch have been used to reduce
soaking period

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• Cooking:
oDestroy contaminating bacteria
oDestroy trypsin inhibitor
oRelease some of nutrient required by mold
• Draining
oAvoid excessive water that would favor bacterial growth and
spoil the bean during fermentation

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Biochemical changes during tempe
fermentation
 Changes in lipids
 Deliberating fatty acid during a 72 h at 37 C
 Changes in carbohydrate
 Principals sugar in soybean: sucrose, stachyose and raffinose
 Principal changes in carbohydrate are the rapid removal of
hexoses and the slow hydrolysis of stachyose
 Changes in amino acids
 Available lysine and methionine show a decrease during tempe
fermentation
 pH increase to 7.1, as the result of active proteolysis and
deamination of amino acids
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Characteristic of R. oligosporus
(NRRL strain 2710)
• Ability to grow rapidly at T 30-42 C
• Inability to ferment sucrose
• High proteolytic activity
• High lypolytic activity
• Production strong antioxidant
• Ability to produce typical tempe cake
• Ability to grow on wheat and other starch cereal substrate without
noticeable amounts of organic acid

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Vit B12 in Tempe
• Commercial tempe contains vit B12
• Tempe made with pure mold does not contain vit B12
• Responsible bacterium to produce vit B12 Klebsiella pneumonia
o Common microorganism on plant materials
o Probably present in tempe inoculum
o K. pneumoniae also known as opportunistic pathogens causing
pneumonia and liver abscess in human. However K. pneumoniae isolated
from tempe were genetically distinct from those of medical isolates.

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Klebsiella pneumonia in tempe

 It does not spoil tempe

 Grows at T 15-45 C; optimum T for production vit B12 at
37 C
 Growth of the bacteria does not interfere with growth of
tempe mold
 Growth at pH as low as 3.5
 Produce vit B12 150 ng/g soybean substrate

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 Microbial growth during tempe
fermentation

Molds
Yeasts
AS: After soaking
SDBR: Home Industry
1
WJB: Home Industry 2
applying second boiling
step before inoculation
Nurdini et al., 2015

Lactic Acid Bacteria LNU/FTSP– FFT321

Miscellaneous molds in tempe
 R. stolonifer,
 R. oryzae,
 R. arrhizus
 Mucor rouxii
 Mucor javanicus
 Trichosporon pullulans
 Aspergillus niger
 Fusarium

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Other microoganisms in tempe
 Soaking:
 Lactic acid bacteria

 Solid state fermentation:

 Lactic Acid Bacteria:

 Lactobacillus fermentum, Lactobacillus delbrueckii subsp. delbrueckii,

Lactobacillus delbrueckii subs. bulgaricus dan Lactobacillus mucosae
 Acetobacter

 Other bacteria:

 Klebsiella sp., Enterobacter sp. Enterococcus faecium dan Cronobacter sp

 Yeast

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 LAB in tempe

Stage Moreno et al. Nuraida et al. Efriwati et al. Nurdini et al.

(2002) (2008) (2008) (2014)*)
Log cfu/g
After soaking 7.8 9.0 6.0 5.3

After second <4 NA NA 1.8

boiling
Tempe 6.8 6.0 7.8 8.1

*)process applied second boiling step before innoculation

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Undesirable microorganism in tempe

• Tempe also contains undesirable microorganisms that can reduce

its quality and safety.
• Bacillus contamination and grows during tempe fermentation
produced unacceptable quality, a bitter taste.
• Bacillus sp. exhibits high proteolytic activity for metabolizing
protein into amino acids.
• Cronobacter sakazakii has been reported to be present in tempe.
(Seumahu, 2012)
• Artificially inoculated C. sakazakii in tempe at initial concentrations of 1
cfu/g and 3 cfu/g grew well during tempe fermentation without affecting the
mold growth, while tempe inoculated with B. cereus at 3 log CFU/g
resulted in spoiled ones (Dewanti et al. 2017).
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Non soybean tempe
• In many areas in Central Java, tempe is also made from other beans:
• Winged bean (Psophocarpus tetragonolobus),
• koro benguk (velvet bean, Mucuna pruriens),
• koro pedang (red sword bean, Canavalia ensiformis;
• white sword bean, Canavalia gladiata),
• beludru buncis (Phaseolus vulgaris),
• kacang polong merpati or kacang gude (pigeon pea, Cajanus cajan),
• lamtoro (banana shrubs Selong, Leucaena leucocephala), and
• okara (tofu/soy milk solid waste).

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Non soybean tempe
• When beans other than soybean are used to make tempe, its
name is derived from the raw material used:
• tempe koro benguk,
• tempe kecipir,
• tempe gembus (ampas tahu) and others
• As the characteristics of non-soy substrates are different than
soybean:
• some steps during the preparation of the raw materials requires
adjustment: duration of soaking and boiling, addition of carbohydrate
source

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 Tempe bongkrek:
 Made of coconut pressed cake
 Fermented by tempe starter

 Often contaminated with Pseudomonas (Bulkholderia)

cocovenenans
As contaminant producing bongkrek acid, very toxic

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Oncom
• Oncom made of peanut presscake or tofu solid
waste mixed with tapioca solid waste (onggok) as
additonal carbohydrate source
• Oncom eaten to a lesser extent than tempe
• Two type of oncom:
• Oncom hitam (Black Oncom):
• Fermented peanut presscake
• Oncom merah (Red Oncom):
• Tofu solid waste
• Typical to West Java
• They are eaten after they are fried, made as chips
or cooked with vegetables

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 Fermentation of Oncom

• Controlled fermentation with addition of starter culture

• Red Oncom (Oncom merah)
• The essential molds belong to Neurospora (N. crassa and N. sitophila)
• Yeast and other microorganism are present
• Bacteria likely to be responsible for the production of Vit B12 are also present
• Black oncom:
• Use similar starter as tempe: Rhizopus oligosporus
• During fermentation:
• Protein is hydrolised to smaller component, however total amino acid profiles was not very much
affected
• Lipid is hydrolised deliberating free fatty acid. Total fat content decreases.
• Carbohydrate is fermented to produce organic acid by bacteria present during fermentation
• Hydolyses of oligosaccharides increases digestibility

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 Mold ripened cheeses

• Primary starter: lactic acic bacteria, especially Lactococcus

lactis.
– Used to produce acid before addition of renin
• Secondary starter:
– Inoculated into curd before aging
– Blue cheese: Penicillium roquefortii
– Camembert: P. camemberti

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Mold ripened Cheese

• Blue-mold cheeses have Penicillium

roqueforti grown on the surface and
within the curd
• Produce protease, peptidase and lipases
• White-mold ripened cheese
• Camembert and Brie
• Spore of P. camemberti or P. caseicolum or
P. candidum grow on the surface
• Proteolysis and lipolysis

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Mold ripened Cheese
Cheese Classification Cheese Type Microorganisms involved
Soft, ripened 1-5 months Brie Lactococcus cremoris, Penicillium camemberti, Brevibacterium linens
Neufchatel Lactococcus diacetilactis, Lactococcus lactis, Geotrichum candidum
Camembert Lactococcus lactis, Lactococcus cremoris, Penicillium camemberti or
Geotrichum candidum or P. caseicolum
Semi soft, ripened 1-12 Blue Lactococcus lactis, Lactococcus cremoris, Penicillium roqueforti,
months Penicillium glaucum
Gorgonzola Lactococcus lactis, Lactococcus cremoris, Penicillium roqueforti,
Penicillium glaucum
Roquefort Lactococcus lactis, Lactococcus cremoris, Penicillium roqueforti,
Penicillium glaucum

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Blue Cheese
Homogenized, Rennet added, Curd collected in
Pasteurized, Incubated for Hoop, drained 16 h,
Starter added, firm set, Salted in brine
Incubated at 90 F cut, cooked at 100 F For 7 d
For 0.2% acidity Whey drained
increase
Spiked to let air to get inside
Stored at 40 F for Mold spores added,
curing for 3 m Stired at 50 F in high
Humidity for 4 week
Semi hard (46% moisture, 50% fat, mold ripened cheese
Made from whole milk (cow’s)
Crumbly body, mottled blue color and sharp lipolytic flavor
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Microbiology of Mold Ripened Cheese:
Blue Cheese
• Blue Cheese:
– Lac. cremoris and L. lactis and also Leuconostoc sp as
primary starters:
• Grow until curing and produce lactate, diacetyl, acetate,
CO2 and acetaldehyde from lactose
– Penicillium roqueforti as secondary starter:
• Grows during storage at 50 oF in high humidity giving the
mottled green appearance.
• Growth continue until curing

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Biochemistry of cheese ripening - 1

• Biochemical changes in cheese during ripening may be grouped into:

• primary (lipolysis, proteolysis and metabolism of residual lactose and of
lactate and citrate) and
• secondary (metabolism of fatty acids and of amino acids) events.
• Residual lactose is metabolized rapidly to lactate during the early stages of
ripening.
• Lactate is an important precursor for a series of reactions including
racemization, oxidation or secondary microbial metabolism.
• Citrate metabolism is of great importance in certain varieties.

McSweeney. 2004. International Journal of Dairy Technology. Vol 57, No 2/3

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Biochemistry of cheese ripening - 2

• Lipolysis in cheese is catalysed by lipases from various source.

• Endogenous enzyme in the milk and cheese microflora
• enzymes from rennet (rennet also contain lypase).
• Proteolysis is the most complex biochemical event that occurs during ripening
and is catalysed by:
• enzymes from residual coagulant/rennet,
• the milk (particularly plasmin) and
• proteinases and peptidases from lactic acid bacteria and other microorganisms (molds).
• Development of volatile flavor compounds:
• Secondary reactions from metabolites including from fatty acids and amino acids.

McSweeney. 2004. International Journal of Dairy Technology. Vol 57, No 2/3

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Citric acid
 Over 130.000 tons produced worldwide each year tropophase idiophase
used in foods and beverages
 Iron citrate as a source of iron preservative for
stored blood, tables, ointments, in detergents as a
replacement for polyphosphates
 A microbial fermentation for production of citric acid
developed in 1923
 Today >99% of the world’s output produced
microbially Aspergillus niger
 Submerged fermentation in large fermenters
 Sucrose as substrate, and citric acid produced
during idiophase
o During trophophase (growth phase) mycelium
produced and CO2 released
o During idiophase (stationary phase) glucose
and fructose are metabolized directly to citric
acid

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Production of citric acid
 Two stage fermentation:
 Growing medium:
To produce mycelium (tropophase)
4% sucrose plus buffer and mineral at pH 5.8
The mold was grown for 4 days
 Production medium:
10% sucrose plus mineral at pH 3.5
 Inhibit the growth of mycelium The mold was separated from
growing medium and re-inoculated into production medium
(idiophase)
Incubated at 30 oC for 1 week.

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 • Aconitase inhibited by
low pH, elimination of
iron.
TCA
cycle • Citric acid can be
accumulated by using
a mutant lacking an
enzyme of the cycle or
by inhibiting the flow of
the cycle

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Biochemical Basis of the Production of
Citric Acid
 Accumulation of citric acid could be done by:
 Mutation
 giving rise to mutant organisms which may only use part of a metabolic pathway, or
regulatory mutants; that is using a mutant lacking an enzyme of the cycle.
 Inhibiting the free-flow of the cycle through altering the environmental conditions:
 temperature, pH, medium composition (especially the elimination of ions and cofactors
considered essential for particular enzymes).
 The concentrations of iron, manganese, magnesium, zinc, and phosphate must be
limited.
 To ensure their removal the medium is treated with ferro-cyanide or by ion
exchange resins.

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Submerged Fermentation for Citric
Acid Production
 The fermentor is made of acid-resistant materials such as stainless steel.
 The carbohydrate sources: deionized molasses, sucrose or glucose.
 MgSO4, 7H2O and KH2PO4 at about 1% and 0.05-2% respectively
 The pH is never allowed higher than 3.5.
 Copper is used at up to 500 ppm as an antagonist of the enzyme aconitase
which requires iron.
 Mold needs oxygent but igh aeration is deleterious to citric acid production,
 mechanical agitation is not necessary
 Air may be bubbled through  Anti-foam is added.
 The fungus occurs as a uniform dispersal of pellets in the medium.
 The fermentation lasts for 5-14 days.

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Extraction of Citric Acid
• The broth is filtered until clear.
• Calcium citrate is precipitated by the addition of magnesium-free
Ca(OH)2.
• Calcium citrate is filtered and the filter cake is treated with sulfuric
acid to precipitate the calcium.
• The dilute solution containing citric acid is purified by treatment
with activated carbon and passing through ion exchange beds.
• The purified dilute acid is evaporated to yield crystals of citric acid.

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Discussion
1. Can you make tempe without soaking overnight? What are the
purposes of overnight soaking in fermentation of tempe?
2. Are there any other microorganism present in tempe or during tempe
fermentation? What are the effect of their presence? You can give
examples.
3. What are the requirements of packaging used for fermentation of
tempe?
4. What are the roles of mold in cheese fermentation?
5. What happen during cheese ripening?
6. How the micoroganism synthesise citrtic acid?
7. What are the critical factors for citric acid fermentation!

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 Thank you

Koordinator:
Prof. Dr. Lilis Nuraida, MSc.
Department Food Science and Technology
Faculty of Agricultural Engeenering
IPB University
lnuraida@gmail.com