ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 24 (2007) 393–402
www.elsevier.com/locate/fm

Rhizopus oligosporus and yeast co-cultivation during barley tempeh

fermentation—Nutritional impact and real-time PCR quantification
of fungal growth dynamics
Xin Mei Fenga,, Volkmar Passotha, Charlotte Eklund-Jonssonb,
Marie Larsson Almingerb, Johan Schnürera
a
Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Box 7025, SE-750 07 Uppsala, Sweden
b
Department of Chemical and Biological Engineering, Food Science, Chalmers University of Technology, SE-412 96 Göteborg, Sweden
Received 3 March 2006; received in revised form 2 June 2006; accepted 23 June 2006
Available online 30 August 2006

Abstract

Barley tempeh was produced by fermenting barley kernels with Rhizopus oligosporus. The potential of the yeasts Saccharomyces
cerevisiae (three strains), S. boulardii (one strain), Pichia anomala (one strain) and Kluyveromyces lactis (one strain) to grow together with
R. oligosporus during barley tempeh fermentation was evaluated. All yeast strains grew during the fermentation and even during cold
storage of tempeh (Po0:01). The growth of yeasts slightly increased the ergosterol contents, but did not influence amino acid contents
and compositions, and did not reduce phytate contents. Slight increases of vitamins B6 and niacinamide, and slight decreases of B1 and
biotin were observed. Quantification of fungal growth is difficult during mixed species fermentations because ergosterol is found in all
fungal species, and colony-forming-unit (cfu) estimations are not reliable for R. oligosporus and other sporulating fungi. Therefore, we
developed a quantitative real-time PCR method for individually quantifying S. cerevisiae and R. oligosporus growth in barley tempeh.
The PCR results were highly correlated with the ergosterol content of R. oligosporus and with the number of cfu of S. cerevisiae. Thus,
real-time PCR is a rapid and selective method to quantify yeasts and R. oligosporus during mixed species fermentation of inhomogenous
substrate such as barley tempeh.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Yeast; Rhizopus oligosporus; Nutrition; Barley tempeh; Real-time PCR

1. Introduction 1989; Sandström and Sandberg, 1992; Bohn et al., 2004).

Tempeh is a traditional Indonesian food prepared by
fermentation of dehulled and cooked soybeans with
moulds of the genus Rhizopus (mainly Rhizopus oligos-
porus) to a compact and sliceable cake (Shama and Hall,
1991). Tempeh can also be produced from cereal grains,
e.g. barley, through fermentation with R. oligosporus (Berg
et al., 2001; Feng et al., 2005). Phytate (myo-inositol
hexakisphosphate or IP6) is the principal storage form of
phosphorus in cereal grains and other plant materials
(Reddy et al., 1982). Minerals, such as iron, magnesium
and zinc, are strongly bound to phytate (Hallberg et al.,
Corresponding author. Tel.: +46 18 673212; fax: +46 18 673392.
E-mail address: xinmei.feng@mikrob.slu.se (X.M. Feng).
Humans and other monogastric animals lack enzymes in
the gastrointestinal tract to degrade phytate, and a diet rich
in phytate leads to a considerably impaired absorption of
dietary minerals (Hallberg et al., 1989; Sandström and
Sandberg, 1992; Bohn et al., 2004). Therefore, phytate
is regarded as a nutritional inhibitor. During the fermenta-
tion process, R. oligosporus produces phytase that degrades
phytate, and consequently increases the availability of
minerals in barley (Eklund-Jonsson et al., 2006). R.
oligosporus also produces ergosterol (provitamin D2) and
some vitamins (Wiesel et al., 1997).
Yeasts such as Saccharomyces cerevisiae have been used
as a single-cell protein source for animals and humans
(Daghir and Sell, 1982; Litchfield, 1983). They can produce
savoury flavours such as glutamic acid, peptides and

0740-0020/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2006.06.007
 ARTICLE IN PRESS
394 X.M. Feng et al. / Food Microbiology 24 (2007) 393–402
ribonucleotides from the degradation of cell protein and
RNA (Walker, 1999). They can also produce phytase to
degrade phytate (Andlid et al., 2004; Passoth et al., 2006),
ergosterol (Tan et al., 2003) and different vitamins
(Diplock et al., 1961; Fleet, 1990). Yeasts can inhibit other
fungi (Druvefors et al., 2002; Passoth and Schnürer, 2003),
and recently, it has been found that yeasts can also inhibit
pathogenic bacteria (Goerges et al., 2006; Leverentz et al.,
2006).
Yeasts can grow spontaneously in soybean tempeh
(Steinkraus et al., 1983; Samson et al., 1987). However,
their role in the tempeh fermentation is not well under-
stood. Many yeast species, e.g. Trichosporon beigelii,
Clavispora (Candida) lusitaniae, Candida maltosa, Candida
intermedia, Yarrowia lipolytica, Lodderomyces elongisporus,
Rhodotorula mucilaginosa, Candida sake, Hansenula fabia-
ni, Candida tropicalis, Candida parapsilosis, Pichia mem-
branaefaciens, Rhodotorula rubra, Candida rugosa, Candida
curvata, Hansenula anomola) are found in soybean tempeh
(Samson et al., 1987). Among them, T. beigelii was the
most frequently detected yeast species. Several reports have
suggested that T. beigelii can cause mycosis in humans and
animals (Gonzalez et al., 2001; Khandpur and Reddy,
2002; Youker et al., 2003). This yeast is widely spread in
soil and water (Han et al., 2000), and might be only an
incident contaminant in tempeh. Introducing known food-
grade yeasts to tempeh might improve the nutritional
quality and simultaneously reduce the risk of unwanted
yeasts, moulds and bacteria. However, the yeasts intro-
duced to tempeh must not restrict the growth of
R. oligosporus.
Previously, we have quantified R. oligosporus growth by
measuring hyphal lengths and ergosterol content when
lactic acid bacteria were introduced into barley tempeh
(Feng et al., 2005). Direct microscopy determinations of
hyphal lengths allows differential quantification of mycelial
and yeast biomass during co-cultivation experiments
(Björnberg and Schnürer, 1993). However, measuring of
hyphal length is a time-consuming method with a large
experimental error (Feng et al., 2005). Ergosterol, provi-
tamin D2, is produced by both yeasts and moulds (Pasanen
et al., 1999), and thus cannot be used to separately
determine the growth of yeasts and R. oligosporus in a
co-cultivation system. Traditionally fungi are quantified as
colony-forming-units (cfu) on selective substrates. How-
ever, for spore forming filamentous fungi such as R.
oligosporus, this method is not ideal (Schnürer, 1993).
Quantitative real-time PCR, using specific primers, might
overcome the problems of the conventional methods, and
has been used for rapid quantification of yeasts and moulds
contaminating yogurt and pasteurized food products
(Bleve et al., 2003).
In this study, we investigated whether yeasts can grow in
barley tempeh, and estimated the effects of yeast growth on
the nutritional characters of barley tempeh, such as
contents of amino acids, ergosterol and vitamins, as well
as on phytate reduction. To determine the effect of co-
cultivation with yeasts on R. oligosporus growth during
barley tempeh fermentation, we developed a selective real-
time PCR quantification method based on yeast and
R. oligosporus total DNA directly extracted from barley
tempeh.
2. Materials and methods
2.1. Microbial strains
R. oligosporus ATCC 64063 (Strain number J401,
maintained in the culture collection of the Department of
Microbiology, Swedish University of Agricultural Sciences,
Uppsala, Sweden) is an efficient strain for barley tempeh
fermentation (Berg et al., 2001). Fungal spores were
maintained on sterile silica grains in Eppendorf tubes at
2 1C. Six yeast strains from three species were used in this
study (Table 1). S. cerevisiae (S. boulardii) and Kluyver-
omyces lactis are regarded as food grade, or even probiotics
species (Breunig and Steensma, 2003; Galvao et al., 2005),
and may be especially appropriate for tempeh production.
Pichia anomala J121 was selected as it has strong growth
ability and inhibitory effects on growth of several moulds
on cereal grains (Druvefors et al., 2002).
2.2. Preparation of barley tempeh
To produce inocula for tempeh fermentation, R.
oligosporus spores were prepared by inoculating three to
five grains of spore-containing silica onto a 100 ml MEA
agar (Oxoid) slant in a 200 ml bottle with a loosely
tightened lid (for air access) and incubating at 30 1C. After
4–5 days, spores were harvested and washed three times
with NaCl solution (9 g/L) to remove the residual nutrients
of the medium. The spore concentration was assessed with
a haemocytometer and the spore suspension was stored at
2 1C (Feng et al., 2005). Yeast strains were grown in 5 ml of
YPD broth (10 g yeast extract, 20 g peptone,
20 g glucose L1) for 24 h. Hundred microlitres of the pre-
culture was inoculated into a new 5 ml YPD broth and
incubated for 24 h again. Yeast cells were washed twice
with NaCl solution, and re-suspended in 1 ml of NaCl
solution. The concentration of this cell suspension was
approx 108 cells/ml as estimated with a haemocytometer.
Commercially available pearled whole barley kernels
(Gourmet Korn, a mixture of different cultivars) were
obtained from Kungsörnen AB, Järna, Sweden. Barley
(300 g) was soaked in 500 ml tap water containing 0.12 M
lactic acid (VWR international AB, Stockholm) for 6 h at
room temperature. After draining, the barley was boiled
for 10 min in 750 ml tap water supplemented with 6 g NaCl.
The drained and cooled (42 1C) barley was inoculated with
R. oligosporus at approx 104 spores/g wet weight and yeasts
at approx 104 cfu/g. Barley inoculated with R. oligosporus
at approx 104 spores/g alone was used as a control. The
inoculated barley (60 g wet weight) was packed into
disposable, sterile petridishes (9  1.5 cm), which were
 ARTICLE IN PRESS
X.M. Feng et al. / Food Microbiology 24 (2007) 393–402 395

Table 1
Yeasts strains and their origin

Species Culture no.a Strain nos.b Origin References (selected)

Saccharomyces cerevisiae J122 ATCC 9763; CBS 2978; Distiller’s yeast, wild-type (Birol et al., 1998)
CBS 5900; DSM 1333; strain
NRRL Y-567
Saccharomyces cerevisiae J543 Baker’s yeast, Svenska
Jästfabrik AB, Sollentuna,
Sweden. Isolated 2004
Saccharomyces cerevisiae J191 Baker’s yeast, Svenska (Petersson and Schnürer,
Jästfabrik AB, Sollentuna, 1995)
Sweden
Saccharomyces boulardii J551 Precosa, AstraZeneca AB (Kuhle and Jespersen, 2003;
(subspecies of S. cerevisiae) Kotowska et al., 2005)
Pichia anomala J121 CBS 100487 From grains in airtight (Björnberg and Schnürer,
storage. 1993)
Kluyveromyces lactis J469 ATCC8585; CBS2359; Isolated from creamery, USA (Steensma et al., 1988; Picos
DSM70799; NRRLY- et al., 1993)
1140
a
Culture no. in the Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
b
ATCC, American Type Culture Collection, Manassas, VA, USA; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; DSM,
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; NRRL, ARS Culture Collection, Northern Regional
Research Laboratory, National Center for Agricultural Utilization Research, Peoria, IL, USA.

individually wrapped in plastic bags and incubated at 35 1C
in a ventilated growth cabinet (Binder, Tillquist instrument
AB, Stockholm) (Feng et al., 2005).
2.3. Analysis of yeast growth during tempeh fermentation
and storage
Twenty-gram-portions of barley tempeh were aseptically
removed from each petridish, mixed with 180 ml of sterile
peptone water (2 g/L in distilled water), homogenized in a
stomacher (Stomacher 400, Seward, Sweden) at normal
speed for 2 min, and 10-fold diluted in sterile peptone
water. Then 0.1 ml of adequate dilution was surface-spread
on DG18 agar (Oxoid) plates containing chloramphenicol
(Sigma) (1 g/L) to prevent bacterial growth. The agar plates
were incubated at 25 1C, and the cfu were counted after 3–4
days. Meanwhile, the pH values of the tempeh cakes were
measured in the first dilution.
2.4. Ergosterol analysis
Freeze-dried and milled samples (0.4–0.5 g) were ex-
tracted in 10 ml glass tubes with screw caps. Five-a
cholesterol (50 ml of 2 mg/ml) was used as an internal
standard. Each sample was mixed with 5 ml of ethanol/
methanol (60/40), five potassium hydroxide pellets (ap-
proximately 0.5 g) and 0.2 ml of pyrogallol solution (10 g/L
in ethanol). Saponification and extraction were made at
80 1C for 90 min. The samples were shaken vigorously
every 10 min. After cooling 4 ml toluene was mixed with the
sample. After addition of 2.5 ml distilled water the samples
were mixed and centrifuged. The supernatants were
transferred to new tubes, air-dried and dissolved in 0.5 ml
toluene. Two hundred microlitres of the solution were
transferred to GC vials. The samples were analysed
quantitatively with GC HP-5890 coupled with an HP-
7673 Auto sampler (Hewlett Packard, Waldbronn,
Germany). Two microlitres of the samples was auto-
injected. The injector temperature was 280 1C and the
column (HP Ultra-1, 50 m0.32 mm df 0.52 mm) was
programmed from 260 to 320 1C with gradual increase at
10 1C/min, keeping at the final temperature for 15 min. The
detector temperature was 320 1C.
The results were evaluated with Borwin Chromatogra-
phy software (Le Fontanil, France).
2.5. Analysis of phytate (myo-inositol hexakisphosphate)
Freeze-dried and milled samples (0.5 g) were extracted by
stirring with 0.5 M HCl (10 ml) for 3 h and then centri-
fuged. The supernatant was frozen at 20 1C overnight,
thawed and centrifuged through an ultracentrifugal filter
device (Micron YM-30, Millipore, Bedford MA, USA).
Phytate (myo-inositol hexakisphosphate or IP6) was
quantified by high performance ion chromatography
(HPIC) (Carlsson et al., 2001). Samples were run in
duplicates and phytate content was determined as
mmol phytate g1 dry matter.
2.6. Analysis of amino acid and vitamins
Amino acids and vitamins (vitamins B1, B6, Niacina-
mide and biotin) were analysed by AnalyCen Nordic AB,
Lidköping, Sweden. Amino acids were analysed according
to standard SS-EN ISO 13903:2005, except tryptophane
that was analysed according to EU standard (Eu Dir 2000/
45/EG part C). Vitamin B1 was analysed according to van
den Berg et al. (1996), B6 and niacin amide were estimated
 ARTICLE IN PRESS
396 X.M. Feng et al. / Food Microbiology 24 (2007) 393–402
by microbiological assay (Freed, 1966; Hashim and Fields,
1979), and biotin was analysed according to Indyk et al.
(2000) and Quant Biotin Kit User’s guide (Edition
December 2000, BiaCore, Uppsala, Sweden).
2.7. Extraction of R. oligosporus and S. cerevisiae total
DNA from barley tempeh
R. oligosporus and S. cerevisiae J543 were inoculated at
approx 104 spores (cfu)/g wet barley in pure- or co-culture
experiments. Triplicate samples were taken after 0, 9, 12, 17
and 20 h incubation at 35 1C. To extract total DNA from
the barley tempeh samples, 1 g of frozen barley tempeh
(wet weight) from each sample was added with 2 ml of lysis
buffer (50 mM Tris-Cl pH 8.0; 50 mM EDTA; 3% SDS;
2% of b-mercaptoethanol), 2 ng of EcoR1-linearized
pUC19 plasmid with ampR gene (as an external standard
for the subsequent real time PCR) and 2 g acid-washed
glass beads (0.45–0.5 mm in diameter, Sigma). The samples
were vortexed for 5  1 min at maximum speed (Vortex-
genie 2), and kept for 1 min on ice in between. Subse-
quently, they were incubated in a water bath at 65 1C for
10 min with gentle mixing after 5 min. One volume (2 ml) of
phenol/chloroform (1/1) was added and the solution was
mixed by inverting the tube for few times. The mixture was
centrifuged and 1 ml of the upper aqueous layer was
transferred to a new Eppendorf tube. The phenol/chloro-
form treatment was repeated until no white precipitate
occurred in the upper aqueous layer. Subsequently, the
DNA was precipitated by adding 0.1 volume of 3 M NaAc
(pH 5.2) and 1 volume of ice-cold 96% EtOH. The pellet
was washed with 100 ml of 70% EtOH, air-dried and
dissolved in 200 ml of TE buffer (10 mM Tris-Cl, pH 8.0
and 1 mM EDTA).
2.8. Real-time PCR
The selected target genes for specific detection were the
R. oligosporus chs1 gene (encoding for a chitin synthase,
EMBL accession number D10159), the S. cerevisiae PDA1
gene (encoding for the E1-alpha subunit of the pyruvate
dehydrogenase complex, EMBL accession number
X71664), and the ampR gene of the pUC19-plasmid
(encoding a beta-lactamase, EMBL-accession number
M77789). Primers for real time PCR were designed using
Table 2
Characteristics of primers for real-time PCR quantification of DNA
Gene Primer pairs
Roligchreal 1F 50 -GTCCCCTTCTTAAGCCCAATG-30
Roligchreal 1 R 50 -GCGACCACCAGGCTTTGTAC-30
ScPDArealF1 50 -GAAAGCCGTTCTGGCTGAAT-30
ScPDArealR1 50 -GCATGGAACCACCCTTACCA-30
AmprealtimeF1 50 -CCGGCAACAATTAATAGACTGGAT-30
AmprealtimeR1 50 -GGGCCGAGCGCAGAA-30
a
The primer melting temperature (Tm) was calculated by the Primer Express Software v2.0, Applied Biosystem, California, USA.
the Primer Expresss Software v2.0 (Applied Biosystems).
The sequences, amplicon length and Tm of the specific
primers are given in Table 2.
The primers were tested at different concentrations for
non-specific DNA-amplification (dimer-formation) by run-
ning real time PCR without any templates. For the
subsequent analysis, primers were used at the maximum
concentrations where no non-specific amplifications oc-
curred (Table 2). The primers were also tested with
template DNA from other microorganisms used in this
study, and no non-specific amplification was observed.
The amplification efficiencies (E) of the respective DNA
amplicons were empirically determined from the amplifica-
tion of serial dilutions of the respective target DNA
according to Wilhelm and Pingoud (2003):
E ¼ 10½1=slope ,
where the slope was calculated from the standard curve of
DNA concentrations vs. the CT values. The CT value is
defined as the cycle number at which the fluorescence
surpasses the noise threshold.
To produce target DNA for the amplification efficiency
determination, parts of the chs1 and PDA1-gene sequences
were amplified from respective genomic DNA using the
primer pairs Roligch1F1410 (50 -gttttagctgccatgggtgt-30 )
and Roligch1R1952 (50 -gaccaaagacggactccaaa-30 ), and
PDA306up (50 -atcacmtcttayagatg-30 ) and PDA849d (50 -
gacatrgartgrccacc-30 ), respectively. For ampR DNA,
EcoR1-linearized pUC19-plasmid was used.
To quantify S. cerevisiae and R. oligosporus DNA
extracted from tempeh, real-time PCR was performed with
the ABI Prisms 7000 Sequence Detection System from
Applied Biosystems using SYBR green I as detective dye.
The reaction mixture contained 1 ml of sample DNA, the
appropriate primer concentrations (Table 2), 12.5 ml of
2  SYBRs Green Master Mix (see user’s manual) and
sterile ultrafiltered dH2O to a final volume of 25 ml. Each
combination of sample DNA and primer pairs was
analysed in triplicates. The PCR was carried out by using
the default reaction parameters.
The log of the initial concentration of any amplified
DNA molecule is proportional to ECT, where E is the
amplification efficiency, and CT is the cycle number for
surpassing the noise threshold (see above). Thus, the real-
time PCR data are expressed as relative DNA copy
Concentration (nM) Tma (1C) Amplicon length (bp) Target gene
160 59 62 R. oligosporus chs 1 gene
160 59 62
160 58 68 S. cerevisiae PDA 1 gene
160 59 68
40 59 65 pUC19 ampR gene
40 59 65
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X.M. Feng et al. / Food Microbiology 24 (2007) 393–402 397

numbers (R) of the sample gene copy number related to the
external standard gene (ampR) copy number according to
the equation:
CTðStandardÞ
E Standard
R¼ .
E CTðSampleÞ
Sample
2.9. Statistical analysis
Differences in treatments were analysed using the One or
Two Way Repeated Measure ANOVA procedures in
SigmaPlot 9.0 (Systat Software Inc., Richmond, USA).
The Tukey test was used for post hoc pairwise comparisons
of levels.
3. Results
3.1. Growth of yeasts during co-cultivation with
R. oligosporus in barley tempeh
R. oligosporus was inoculated together with each yeast
strain (Table 1). The number of yeasts was determined as
cfu immediately after inoculation, after 20 h incubation at
35 1C, and after storage of the 20 h-fermented tempeh cakes
for 7 weeks at 2 1C. All yeasts inoculated at approx 4.0 log
units increased significantly (Po0:01) during the barley
tempeh fermentation and even during tempeh storage
(Table 3). During 20 h fermentation, the cfu of P. anomala
J121 increased by 3.4 log units, while the other five strains
increased between 2.6 and 2.9 log units. During storage of
tempeh at 2 1C, the cfu numbers of K. lactis J469 and
P. anomala J121 increased by 2.2 and 1.3 log units,
respectively, while the four S. cerevisiae/S. boulardii strains
increased between 0.6 and 0.8 log units. The pH increased
slightly from 4.6 immediately after inoculation to 4.8 after
fermentation in all treatments, including the control. This
implies that the growth of yeasts does not markedly
influence the pH values. The growth of yeasts at this
inoculation levels did not visibly affect the growth of R.
oligosporus. All tempeh cakes, whether co-cultivated with
yeasts or not, had firm texture and were sliceable, exhibited
sweet and fruity odour, and had a creamy white colour.
3.2. Ergosterol and vitamin production
Ergosterol is a component of the fungal cell membrane,
both in filamentous fungi and in yeasts. Thus, increased
ergosterol content was expected for R. oligosporus-yeast co-
fermentations. Tempeh fermented with R. oligosporus had
a significant (Po0:001) increase in the ergosterol contents
compared to non-fermented barley (Table 4). Co-cultiva-
tion with different yeasts further increased the ergosterol
content by 7.1–17.5 mg/g dry tempeh (Table 4). However,
the statistical significance of these differences could not be
proven. Calculating from the ergosterol contents and the
cfu numbers estimated in barley inoculated with S.
cerevisiae J543 alone, the ergosterol content of individual
cells of S. cerevisiae J543 was 3.4–6.6  107 mg/cfu. Thus,
the expected increase in the ergosterol content in tempeh
co-fermented with this yeast strain was in the range of
2.7–5.2 mg/g barley, which is close to the experimentally
determined value (9.9 mg/g) (Table 4). This, together with
the fact that a similar increase was observed for all yeast
co-fermentations, indicates that the detected increases of
ergosterol content in barley are due to the growth of yeasts.
The concentrations of some vitamins were also surveyed
for barley tempeh fermented with R. oligosporus and S.
cerevisiae J191 for 20 h. S. cerevisiae co-cultivation
increased vitamins B6 and niacinamide from 0.7 to 0.9
and 53.0 to 69.1 mg/kg dry tempeh, respectively. In
contrast, vitamin B1 decreased from 0.5 to 0.2 mg/kg dry
tempeh and biotin from 117.5 to 94.5 mg/kg dry tempeh.
3.3. Phytate content of tempeh made by co-fermentation of
R. oligosporus and yeasts
One of the aims of barley tempeh fermentation is to
reduce the content of phytate. Yeast can produce phytase,
and it was anticipated that the growth of R. oligosporus
together with yeasts would reduce phytate further. Barley
tempeh fermented with R. oligosporus alone significantly

Table 3 Table 4
Growth of yeasts during barley tempeh fermentation and during tempeh Ergosterol and phytate contents of barley and barley tempeh fermented
cold storage with R. oligosporus J401 alone or co-inoculated with S. cerevisiae J122,
J543 or J191, S. boulardii J551, P. anomala J121 or K. lactis J469
Strains Fermentation Storage at 2 1C
Treatment Ergosterol (mg/g dry matter) Phytate (mmol/g)
0 ha 20 ha 7 weeksb
Non-fermented barleya 0.570.3 2.770.2
S. cerevisiae J 122 4.070.4 6.970.1 7.770.0 J401b 57.571.7 1.970.1
S. cerevisiae J 543 4.070.4 6.970.1 7.570.0 J401+J 122b 67.777.2 1.970.1
S cerevisiae J 191 4.170.4 6.970.1 7.670.0 J401+J 543b 67.472.8 1.870.0
S. boulardii J 551 4.170.4 6.970.1 7.770.4 J401+J 191b 71.978.8 2.070.0
P. anomala J 121 4.070.4 7.470.2 8.770.0 J401+J 551b 75.072.4 2.070.0
K. lactis J 469 4.270.5 6.870.2 9.070.0 J401+J 121b 67.773.5 1.970.1
J401+J 469b 64.571.8 2.070.1
a
Data are given as log cfu/g dry tempeh (mean7SD, n ¼ 4, and
mean7deviation from mean values, bn ¼ 2). Mean7SD, an ¼ 6, and mean7deviation from mean value, bn ¼ 2.
 ARTICLE IN PRESS
398 X.M. Feng et al. / Food Microbiology 24 (2007) 393–402

(Po0:001) reduced the phytate contents by 28.3%
compared to non-fermented barley (Table 4). However,
yeast co-fermentations with R. oligosporus did not sig-
nificantly reduce the phytate contents further compared to
R. oligosporus fermentation alone (Table 4).
3.4. Total amino acids content in barley after
co-fermentation of R. oligosporus and yeasts
Amino acid contents were determined for tempeh cakes
fermented by R. oligosporus alone, or co-cultivated with
either S. cerevisiae J122, P. anomala J121 or K. lactis
J469 (Table 5). R. oligosporus fermentation alone or
co-fermentation with J122, J121 and J469 did not
considerably change the total and essential amino acids
contents compared to non-fermented barley (0 h, J401)
(Table 5).
3.5. Development of a rapid DNA extraction method for
yeast and R. oligosporus on barley tempeh
Extraction of microbial DNA from an inhomogenous
system like tempeh is difficult due to the big particles of
barley kernels. Moreover, ground barley kernels will
release a number of different compounds that may affect
PCR amplification (Rossen et al., 1992). Considering that
most of the mycelia (Varzakas, 1998) and the yeast cells are
likely in the outside of the kernels, we avoided to use
methods including grinding of barley kernels. Instead, we
tried an alkalic lysis-boiling (Olsson et al., 1999) and a
lysing buffer-heating method (Bilkova and Kralova, 1999).
The alkalic lysis-boiling method is efficient to extract DNA
from spores and hyphae of filamentous fungi (Olsson et al.,
1999), but in our hands it did not work for yeasts, as
evaluated by PCR amplification using specific primers. In
contrast, the lysing buffer-heating method was efficient
in extracting DNA from yeast, but not from R. oligosporus
in barley tempeh. Using the lysis buffer method as starting
point, we added a mechanical cell disruption step by
vortexing the barley samples with glass beads. This
modification resulted in a successful isolation of DNA
from both R. oligosporus and S. cerevisiae. Some para-
meters were further optimized. Incubating time at 65 1C
was reduced from 25 to 10 min without loosing extraction
efficiency. Vortexing barley samples for 5 min after adding
of the glass beads gave maximal extraction of DNA from
yeast cells, while 2–10 min of vortexing did not influence
the DNA extraction efficiency of R. oligosporus. This was
verified by estimating the relative DNA copy numbers with
real-time PCR. Therefore, vortexing for 5 min, followed by
incubation for 10 min at 65 1C was used for the DNA
extraction.

Table 5
Amino acid composition of barley and barley tempeh fermented with R. oligosporus alone or co-inoculated with S. cerevisiae J122, P. anomala J121 or
K. lactis J469

Item 0h 20 h

J401a J401a J401+J122b J401+J121b J401+J469b

Cystine 2.870.0 2.870.0 2.8 2.8 2.8

Methionine 1.870.0 1.870.0 1.8 1.8 1.8
Aspartic acid 5.770.2 5.770.2 5.5 6.0 6.0
Threonine 3.670.1 3.570.0 3.5 3.7 3.5
Serine 5.070.2 4.770.1 4.8 4.8 4.6
Glutamic acid 25.670.4 22.672.6 26.7 26.7 23.3
Proline 14.170.0 13.370.8 14.7 14.1 13.8
Glycine 3.970.0 3.870.1 3.9 3.9 3.9
Alanine 3.970.0 4.370.2 4.4 4.6 4.4
Valine 7.070.1 6.370.6 6.9 6.7 6.5
Isoleucine 4.670.0 4.570.1 4.6 4.6 4.6
Leucine 8.170.0 7.770.1 8.1 8.1 7.8
Tyrosine (calculated) 3.970.0 3.870.1 4.1 3.9 3.9
Phenylalanine 6.670.1 6.470.2 6.5 6.7 6.2
Histidine 2.570.0 2.470.1 2.3 2.5 2.3
Ornitine o0.2 o0.2 o0.2 o0.2 o0.2
Lysine 3.270.0 3.570.0 3.5 3.7 3.5
Arginine 5.170.0 4.870.0 4.8 5.1 4.8
Hydroxiproline o0.2 o0.2 o0.2 o0.2 o0.2
Tryptophane 1.570.0 1.470.0 1.5 1.4 1.4
Ammonia 1.870.0 1.870.0 1.8 1.8 1.8
Essential amino acids 37.470.1 36.071.3 37.2 37.8 36.2
Total amino acids 108.870.9 103.075.5 110.4 111.1 105.1

Data are given as g amino acid(s)/kg dry matter. Values represent mean values7deviation from mean (n ¼ 2)a or single determinations (n ¼ 1)b. The
measuring tolerance given by the analysing company is 8% for each amino acid except for tryptophane (10%).
 Essential amino acid.
 ARTICLE IN PRESS
X.M. Feng et al. / Food Microbiology 24 (2007) 393–402
3.6. Real-time PCR quantification of R. oligosporus and
S. cerevisiae and correlation to ergosterol contents and cfu
Different primer pairs were designed (Table 2) and the
amplification efficiencies were tested. The theoretical
maximum amplification efficiency (E value) of real-time
PCR of one amplification cycle is 2 (Wilhelm and Pingoud,
2003). The measured E value was 1.95 for the R.
oligosporus chs1 gene, 1.90 for the S. cerevisiae PDA1 gene
and 1.93 for the ampR gene.
The growth of R. oligosporus alone, or together with S.
cerevisiae J543, in barley tempeh was analysed by real-time
PCR. For a comparison, ergosterol contents were analysed
on all barley tempeh samples fermented by R. oligosporus
alone, and on some samples fermented by R. oligosporus
together with S. cerevisiae. For R. oligosporus grown alone,
both real-time PCR values and ergosterol contents
increased with time (Figs. 1 and 2). However, the ergosterol
content showed a lag phase in the beginning of the
fermentation (Fig. 1), while this lag phase was not detected
with the real-time PCR method (Fig. 2). Therefore there
was a relatively low correlation (r ¼ 0:83, n ¼ 5, Po0:1)
between the ergosterol contents and the real-time PCR
data. Removing the lag phase data values, a high degree of
correlation (r ¼ 0:99, n ¼ 4, Po0:01) was found. For R.
oligosporus co-inoculation with yeast, the R. oligosporus
growth was apparently decreased due to high inoculation
level of S. cerevisiae (more than 4.5 log cfu/g). The
ergosterol content in the co-cultivation tempeh (51.7 mg/g
dry tempeh) was also lower than that in the pure culture
fermentation of R. oligosporus (63.2 mg/g). However, no
significant differences were observed between the real-time
PCR values for R. oligosporus grown alone and grown
together with S. cerevisiae (Fig. 2).
Obviously, the correlation between ergosterol produc-
tion and genome copy number in R. oligosporus was
changed during co-cultivation with high number of yeast
cells. Because high nucleus copy numbers were found to be
connected with high branching frequency in Aspergillus
nidulans (Lin and Momany, 2004), we assumed a similar
2.50
2.00
Log ergosterol content
1.50
1.00
0.50
0.00
-0.50
-1 4 9 14
Time (h)
Fig. 1. Ergosterol content of barley tempeh fermented by Rhizopus
oligosporus during 20 h at 35 1C (n ¼ 3).
399
Log relative DNA copies of R. oligosporus
0.0
-1.0
-2.0
-3.0
-4.0
-5.0
-6.0
-1 4 9 14 19
Time (h)
Fig. 2. Real-time PCR quantification of Rhizopus oligosporus grown alone
(B) or together with S. cerevisiae J543 (’) during 20 h barley tempeh
fermentation at 35 1C (n ¼ 3).
phenomenon in R. oligosporus. To investigate whether
S. cerevisiae can increase branches on the hyphae of R.
oligosporus, we cultivated R. oligosporus and S. cerevisiae
together on malt extract agar (MEA) plates. On the control
plates without S. cerevisiae, R. oligosporus covered the
whole plates and sporulated within 24 h. In contrast, in the
mixed culture plates, R. oligosporus grew slower and did
not sporulate in the same time period. This slower growth
was accompanied by a high branching frequency of the
mycelia of R. oligosporus (Fig. 3). Therefore, the high DNA
copy numbers found in R. oligosporus in co-cultivation
with yeast may indicate an increased branching frequency
induced by the yeast. In this situation, real-time PCR
results might not accurately correlate to the biomass
production of the mould.
The growth of S. cerevisiae cultivated alone, or grown
together with R. oligosporus, was determined with real-time
PCR and as cfu. Both techniques gave values that increased
with time (Figs. 4 and 5). The real-time PCR values were
highly correlated (r ¼ 0:98, n ¼ 9, Po0:001) with the yeast
cfu numbers. However, in the real-time PCR analysis of the
late fermentation stage, there was a small but significant
(Po0:05) difference between cultivation with and without
R. oligosporus (Fig. 5), which was not observed in cfu
determination (Fig. 4). This might indicate that real-time
PCR quantification was more precise than cfu determina-
tion. Our results show that R. oligosporus has no or only
slight influence on the growth of S. cerevisiae (Figs. 4
and 5).
4. Discussion
Our results demonstrated that different yeasts could
grow together with R. oligosporus during barley tempeh
fermentation and increase the ergosterol content of
19 resulting barley tempeh cakes by 12–31%. Nevertheless,
the nutritional impact of yeast co-cultivation was lower
than expected. However, an improvement in nutritional
values may be obtained by selecting specific strains with
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400 X.M. Feng et al. / Food Microbiology 24 (2007) 393–402

Fig. 3. Effect of S. cerevisiae J543 on R. oligosporus hyphal morphology (400  ). (A) Control (100 ml of 104 spores/ml of R. oligosporus was spread on
MEA agar plates); (B) R. oligosporus cultivated together with S. cerevisiae J543 (100 ml of mixed suspension including 104 spores/ml of R. oligosporus and
105 cells/ml of S. cerevisiae J543 was spread on MEA agar plates).

7.5 roqueforti (Druvefors et al., 2002), but at this inoculation

level no inhibition on R. oligosporus growth was observed.
7.0
We also found that the other tested yeasts did not affect R.
Log cfu of S. cerevisiae

6.5 oligosporus growth at this inoculation level. However,

yeasts added at levels higher than 104 cfu/g or higher than
6.0 the R. oligosporus inoculation level inhibited the growth of
R. oligosporus. Yeasts, especially non-food grade yeasts,
5.5
such as T. beigelii, that frequently spontaneously grow in
5.0 soybean tempeh fermentations (Samson et al., 1987), may
be able to disturb tempeh production and even constitute a
4.5 hazard for human health. Therefore, it is desirable to
4.0 prevent the spontaneous growth of yeasts in tempeh
-1 4 9 14 19 fermentation. The optimal yeast strains for co-cultivation
Time (h) should be food-grade and have low inhibitory potential
towards R. oligosporus, but should also have a high
Fig. 4. Colony forming units (cfu) of S. cerevisiae J543 grown alone (&)
or together with R. oligosporus (~) in barley during 20 h fermentation at
competitive ability against other yeasts, moulds or bacteria.
35 1C (n ¼ 3). Yeast co-cultivation did not further reduce the phytate
content. The maximum phytate reduction obtained in the
present study was only about one third of the total content
Log relative DNA copies of S. cerevisiae

in raw material. Similar results were found by Sutardi and

-1.5 Buckle (1985) who studied phytate reduction in soybeans
fermented with different strains of R. oligosporus. How-
-2.5 ever, in a recent study, modifications of the grain surface
have been found to greatly improve phytate reduction, and
97% of the original phytate of whole barley kernels was
-3.5 degraded during tempeh fermentation (Eklund-Jonsson et
al., 2006). Thus, the non-degraded phytate in our study
-4.5 may be inaccessible for microbial degradation.
The real-time PCR results of S. cerevisiae were highly
(r ¼ 0:98, n ¼ 9, Po0:001) correlated with the cfu numbers
-5.5
-1 4 9 14 19 of S. cerevisiae. Quantification of yeasts by real-time PCR
Time (h) is much faster than by determination of cfu, and especially
useful in mixed culture systems. In contrast, quantification
Fig. 5. Real-time PCR quantification of S. cerevisiae J543 grown alone of filamentous fungi is a challenging task because the
(&) or together with R. oligosporus (E) in barley during 20 h
fermentation at 35 1C (n ¼ 3).
fungal colony consists of mycelia and single-celled spores.
This makes it difficult to define the filamentous fungal
growth by biomass, hyphal length, cell numbers or nucleus
specific performances (for instance with high folate copy numbers. Several methods have been established, but
production). every method has its drawbacks. For instance, the number
Inoculation of Pichia anomala at 104 cfu/g inhibits of cfu correlates rather with the number of spores than
several mycotoxigenic fungal species such as Penicillium with mycelial growth (Schnürer, 1993). Hyphal length
 ARTICLE IN PRESS
X.M. Feng et al. / Food Microbiology 24 (2007) 393–402
determination is time-consuming and prone to substantial
experimental error (Feng et al., 2005), while the ergosterol
content of the fungal membrane can vary with fungal age,
and can be affected by oxygen supply and light (Nout et al.,
1987). Furthermore, with the exception of cfu determina-
tion with further subculturing none of these methods are
species-specific. Here, we have described a selective real-
time PCR method for determining relative nucleus copy
numbers of filamentous fungi in the solid-state tempeh
fermentation. The real-time PCR results were in general
correlated with ergosterol contents of the tempeh. How-
ever, the lag phase observed by ergosterol determination
but not by real-time PCR determination suggests that
DNA replication may occur prior to hyphal growth during
the early stages of the fungal growth. Lin and Momany
(2004) demonstrated that a high proportion of nucleus
copy numbers correlates with high hyphal branches in
abnormal hyphal branch mutants of A. nidulans. Our
results indicate that high nucleus copy numbers in R.
oligosporus may correlate with increased formation of
hyphal branches that were induced by co-cultivation with
growth inhibitory microorganisms. This suggests that in
this special situation real-time PCR quantification of
filamentous fungi should be combined with other quanti-
fication methods.
5. Conclusion
A number of yeasts could grow together with R.
oligosporus during barley tempeh fermentation. The
inoculation level of yeasts at 4 log value did not signifi-
cantly affect the growth of R. oligosporus, which indicates
that yeasts can be introduced to barley tempeh to increase
its safety and nutritional values. Real-time PCR determi-
nation was demonstrated to be a rapid, reliable and
selective method for quantification of fungi in barley
tempeh fermentation although it in some cases requires
verification by other methods. It may also be possible to
apply in other solid-state food and feed fermentation by
choosing appropriate primer combinations.
Acknowledgements
This project was financed by VINNOVA (Swedish
Agency for Innovation Systems). The authors would also
like to thank M. Drabkova for helpful advices, N.G.
Carlsson for help with the analyses of ergosterol and
phytate, and Dr. M. Pell for statistical analysis.
References
Andlid, T.A., Veide, J., Sandberg, A.-S., 2004. Metabolism of extracellular
inositol hexaphosphate (phytate) by Saccharomyces cerevisiae. Int.
J. Food Microbiol. 97 (2), 157–169.
Berg, S., Olsson, J., Swanberg, M., Schnürer, J., Eriksson, A., 2001.
Method for the production of fermented cereal food products and
products thereof. World Intellectual Property Organization, Interna-
tional patent application number: PCT/SE02/00357.
401
Bilkova, K., Kralova, B., 1999. Izolace biomakromolekul, Fakulta
potravinarske a biochemicke technologie. Praha, Vydavatelstvi
VSCHT.
Birol, G., Doruker, P., Kirdar, B., Onsan, Z.I., Ulgen, K., 1998.
Mathematical description of ethanol fermentation by immobilised
Saccharomyces cerevisiae. Process Biochem. 33 (7), 763–771.
Björnberg, A., Schnürer, J., 1993. Inhibition of the growth of grain-
storage molds in vitro by the yeast Pichia anomala (Hansen)
Kurtzman. Can. J. Microbiol. 39 (6), 623–628.
Bleve, G., Rizzotti, L., Dellaglio, F., Torriani, S., 2003. Development of
reverse transcription (RT)-PCR and real-time RT-PCR assays for
rapid detection and quantification of viable yeasts and molds
contaminating yogurts and pasteurized food products. Appl. Environ.
Microbiol. 69 (7), 4116–4122.
Bohn, T., Davidsson, L., Walczyk, T., Hurrell, R.F., 2004. Phytic acid
added to white-wheat bread inhibits fractional apparent magnesium
absorption in humans. Am. J. Clin. Nutr. 79 (3), 418–423.
Breunig, K.D., Steensma, H.Y., 2003. Kluyveromyces lactis: genetics,
physiology, and application. In: de Winde, J.H. (Ed.), Functional
Genetics of Industrial Yeasts. Springer, Berlin, Heidelberg, New York,
pp. 171–205.
Carlsson, N.G., Bergman, E.L., Skoglund, E., Hasselblad, K., Sandberg,
A.S., 2001. Rapid analysis of inositol phosphates. J. Agric. Food
Chem. 49 (4), 1695–1701.
Daghir, N.J., Sell, J.L., 1982. Amino acid limitations of yeast single-cell
protein for growing chickens. Poultry Sci. 61 (2), 337–344.
Diplock, A.T., Green, J., Edwin, E.E., Bunyan, J., 1961. Tocopherol,
ubiquinones and ubichromenols in yeasts and mushrooms. Nature 189
(4766), 749–750.
Druvefors, U., Jonsson, N., Boysen, M.E., Schnürer, J., 2002. Efficacy of
the biocontrol yeast Pichia anomala during long-term storage of moist
feed grain under different oxygen and carbon dioxide regimens. FEMS
Yeast Res. 2 (3), 389–394.
Eklund-Jonsson, C., Sandberg, A.-S., Alminger, M., 2006. Reduction of
phytate content while preserving mineral content during whole grain
cereal tempe fermentation. J. Cereal Sci., in press, doi:10.1016/
j.ics.2006.05.005
Feng, X.M., Eriksson, A.R.B., Schnürer, J., 2005. Growth of lactic acid
bacteria and Rhizopus oligosporus during barley tempeh fermentation.
Int. J. Food Microbiol. 104 (3), 249–256.
Fleet, G.H., 1990. Yeasts in dairy-products. J. Appl. Bacteriol. 68 (3),
199–211.
Freed, M., 1966. Association of Vitamin Chemist. Interscience,
New York.
Galvao, K.N., Santos, J.E., Coscioni, A., Villasenor, M., Sischo, W.M.,
Berge, A.C., 2005. Effect of feeding live yeast products to calves
with failure of passive transfer on performance and patterns of
antibiotic resistance in fecal Escherichia coli. Reprod. Nutr. Dev. 45
(4), 427–440.
Goerges, S., Aigner, U., Silakowski, B., Scherer, S., 2006. Inhibition of
Listeria monocytogenes by food-borne yeasts. Appl. Environ. Micro-
biol. 72 (1), 313–318.
Gonzalez, R.N., Wilson, D.J., Sickles, S.A., Zurakowski, M.J., Wey-
brecht, P.M., Walsh, A.K., 2001. Outbreaks of clinical mastitis caused
by Trichosporon beigelii in dairy herds. J. Am. Vet. Med. Assoc. 218
(2), 238.
Hallberg, L., Brune, M., Rossander, L., 1989. Iron-absorption in man—
ascorbic-acid and dose-dependent inhibition by phytate. Am. J. Clin.
Nutr. 49 (1), 140–144.
Han, M.H., Choi, J.H., Sung, K.J., Moon, K.C., Koh, J.K., 2000.
Onychomycosis and Trichosporon beigelii in Korea. Int. J. Dermatol.
39 (4), 266–269.
Hashim, N.B., Fields, M.L., 1979. Germination and relative nutritive
value of corn meal and corn chips. J. Food Sci. 44 (3), 936–937.
Indyk, H.E., Evans, E.A., Caselunghe, M.C.B., Persson, B.S., Finglas,
P.M., Woollard, D.C., Filonzi, E.L., 2000. Determination of biotin
and folate in infant formula and milk by optical biosensor-based
immunoassay. J. AOAC Int. 83 (5), 1141–1148.
 ARTICLE IN PRESS
402 X.M. Feng et al. / Food Microbiology 24 (2007) 393–402
Khandpur, S., Reddy, B.S., 2002. Itraconazole therapy for white piedra
affecting scalp hair. J. Am. Acad. Dermatol. 47 (3), 415–418.
Kotowska, M., Albrecht, P., Szajewska, H., 2005. Saccharomyces
boulardii in the prevention of antibiotic-associated diarrhoea in
children: a randomized double-blind placebo-controlled trial. Aliment.
Pharmacol. Ther. 21 (5), 583–590.
Kuhle, A., Jespersen, L., 2003. The taxonomic position of Saccharomyces
boulardii as evaluated by sequence analysis of the D1/D2 domain of
26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial
cytochrome-c oxidase II gene. System. Appl. Microbiol. 26 (4),
564–571.
Leverentz, B., Conway, W.S., Janisiewicz, W., Abadias, M., Kurtzman,
C.P., Camp, M.J., 2006. Biocontrol of the food-borne pathogens
Listeria monocytogenes and Salmonella enterica serovar poona on
fresh-cut apples with naturally occurring bacterial and yeast antago-
nists. Appl. Environ. Microbiol. 72 (2), 1135–1140.
Lin, X.R., Momany, M., 2004. Identification and complementation of
abnormal hyphal branch mutants ahbA1 and ahbB1 in Aspergillus
nidulans. Fung. Genet. Biol. 41 (11), 998–1006.
Litchfield, J.H., 1983. Single-cell proteins. Science 219 (4585), 740–746.
Nout, M.J.R., Bonants van Laarhoven, T.M.G., Jongh, P.D., Koster,
P.G.D., 1987. Ergosterol content of Rhizopus oligosporus NRRL 5905
grown in liquid and solid substrates. Appl. Microbiol. Biotech. 26 (5),
456–461.
Olsson, J., Schnürer, J., Hagsholm Peddersen, L., Rossen, L., 1999. A
rapid and efficient method for DNA extraction from fungal spores and
mycelium for PCR-based detection. J. Food Mycol. 2 (1), 251–260.
Pasanen, A.L., Yli-Pietila, K., Pasanen, P., Kalliokoski, P., Tarhanen, J.,
1999. Ergosterol content in various fungal species and biocontami-
nated building materials. Appl. Environ. Microbiol. 65 (1), 138–142.
Passoth, V., Schnürer, J., 2003. Non-conventional yeasts in antifungal
application. In: de Winde, J.H. (Ed.), Functional Genetics of
Industrial Yeasts. Springer, Berlin, Heidelberg, pp. 297–329.
Passoth, V., Fredlund, E., Druvefors, U.Ä., Schnürer, J., 2006.
Biotechnology, physiology and genetics of the yeast Pichia anomala.
FEMS Yeast Res. 6, 3–13.
Petersson, S., Schnürer, J., 1995. Biocontrol of mold growth in high-
moisture wheat stored under airtight conditions by Pichia anomala,
Pichia guilliermondii, and Saccharomyces cerevisiae. Appl. Environ.
Microbiol. 61 (3), 1027–1032.
Picos, M.A.F., Torres, A.M.R., Ramil, E., Cerdan, M.E., Breunig, K.D.,
Hollenberg, C.P., Zitomer, R.S., 1993. Sequence of a cytochrome-c
gene from Kluyveromyces lactis and its upstream region. Yeast 9 (2),
201–204.
Reddy, N.R., Sathe, S.K., Salunkhe, D.K., 1982. Phytates in cereals and
legumes. Adv. Food Res. 28, 1–92.
Rossen, L., Norskov, P., Holmstrom, K., Rasmussen, O.F., 1992.
Inhibition of PCR by components of food samples, microbial
diagnostic assays and DNA-extraction solutions. Int. J. Food
Microbiol. 17 (1), 37–45.
Samson, R.A., Kooij, J.A.V., Boer, E.D., 1987. Microbiological quality of
commercial tempeh in the Netherlands. J. Food Protect. 50 (2), 92–94.
Sandström, B., Sandberg, A.S., 1992. Inhibitory effects of isolated inositol
phosphates on zinc absorption in humans. J. Trace Elem. Electrolytes
Health Dis. 6 (2), 99–103.
Schnürer, J., 1993. Comparison of methods for estimating the biomass of
three food-borne fungi with different growth patterns. Appl. Environ.
Microbiol. 59 (2), 552–555.
Shama, G., Hall, G., 1991. Tempeh foods. Eur. Food Drink Rev.
(Summer), 27–28, 31.
Steensma, H.Y., Dejongh, F.C.M., Linnekamp, M., 1988. The use of
electrophoretic karyotypes in the classification of yeasts—Kluyvero-
myces marxianus and Kluyveromyces lactis. Curr. Genet. 14 (4),
311–317.
Steinkraus, K.H., Cullen, R.E., Pederson, C.S., Nellis, L.F., Gavitt, B.K.,
1983. Indonesian tempeh and related fermentations. In: Steinkraus,
K.H., Cullen, R.E., Pederson, C.S., Nellis, L.F., Gavitt, B.K. (Eds.),
Handbook of Indigenous Fermented Foods. Marcel Dekker, New
York, pp. 1–94.
Sutardi, Buckle, K.A., 1985. Phytic acid changes in soybeans fermented by
traditional inoculum and six strains of Rhizopus oligosporus. J. Appl.
Bacteriol. 58 (6), 539–543.
Tan, T.W., Zhang, M., Gao, H., 2003. Ergosterol production by fed-batch
fermentation of Saccharomyces cerevisiae. Enzyme Microb. Technol.
33 (4), 366–370.
van den Berg, H., van Schaik, F., Finglas, P.M., de Froidmont-Gortz, I.,
1996. Third EU MAT intercomparison on methods for the determi-
nation of vitamins B-1, B-2 and B-6 in food. Food Chem. 57 (1),
101–108.
Varzakas, T., 1998. Rhizopus oligosporus mycelial penetration and
enzyme diffusion in soya bean tempe. Process Biochem. 33 (7),
741–747.
Walker, G.M., 1999. Yeast—Physiology and Biotechnology. Wiley,
Chichester.
Wiesel, I., Rehm, H.J., Bisping, B., 1997. Improvement of tempe
fermentations by application of mixed cultures consisting of Rhizopus
sp. and bacterial strains. Appl. Microbiol. Biotech. 47 (3), 218–225.
Wilhelm, J., Pingoud, A., 2003. Real-time polymerase chain reaction.
ChemBioChem 4 (11), 1120–1128.
Youker, S.R., Andreozzi, R.J., Appelbaum, P.C., Credito, K., Miller, J.J.,
2003. White piedra: further evidence of a synergistic infection. J. Am.
Acad. Dermatol. 49 (4), 746–749.