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FOOD MICROBIOLOGY
Food Microbiology 24 (2007) 393402 www.elsevier.com/locate/fm

Rhizopus oligosporus and yeast co-cultivation during barley tempeh fermentationNutritional impact and real-time PCR quantication of fungal growth dynamics
Xin Mei Fenga,, Volkmar Passotha, Charlotte Eklund-Jonssonb, Marie Larsson Almingerb, Johan Schnurera
b a Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Box 7025, SE-750 07 Uppsala, Sweden Department of Chemical and Biological Engineering, Food Science, Chalmers University of Technology, SE-412 96 Go teborg, 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 inuence 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. Quantication of fungal growth is difcult 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 Tempeh is a traditional Indonesian food prepared by fermentation of dehulled and cooked soybeans with moulds of the genus Rhizopus (mainly Rhizopus oligosporus) 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). 0740-0020/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2006.06.007

1989; Sandstrom and Sandberg, 1992; Bohn et al., 2004). 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; Sandstrom and Sandberg, 1992; Bohn et al., 2004). Therefore, phytate is regarded as a nutritional inhibitor. During the fermentation 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; Litcheld, 1983). They can produce savoury avours such as glutamic acid, peptides and

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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 Schnurer, 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 understood. Many yeast species, e.g. Trichosporon beigelii, Clavispora (Candida) lusitaniae, Candida maltosa, Candida intermedia, Yarrowia lipolytica, Lodderomyces elongisporus, Rhodotorula mucilaginosa, Candida sake, Hansenula fabiani, Candida tropicalis, Candida parapsilosis, Pichia membranaefaciens, 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 foodgrade yeasts to tempeh might improve the nutritional quality and simultaneously reduce the risk of unwanted yeasts, moulds and bacteria. However, the yeasts introduced to tempeh must not restrict the growth of R. oligosporus. Previously, we have quantied 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 quantication of mycelial and yeast biomass during co-cultivation experiments (Bjornberg and Schnurer, 1993). However, measuring of hyphal length is a time-consuming method with a large experimental error (Feng et al., 2005). Ergosterol, provitamin 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 quantied as colony-forming-units (cfu) on selective substrates. However, for spore forming lamentous fungi such as R. oligosporus, this method is not ideal (Schnurer, 1993). Quantitative real-time PCR, using specic primers, might overcome the problems of the conventional methods, and has been used for rapid quantication 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 realtime PCR quantication 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 efcient 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 Kluyveromyces 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 ve 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 45 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 preculture 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 Kungsornen AB, Jarna, 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

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X.M. Feng et al. / Food Microbiology 24 (2007) 393402 Table 1 Yeasts strains and their origin Species Saccharomyces cerevisiae Culture no.a J122 Strain nos.b ATCC 9763; CBS 2978; CBS 5900; DSM 1333; NRRL Y-567 Origin Distillers yeast, wild-type strain Bakers yeast, Svenska Jastfabrik AB, Sollentuna, Sweden. Isolated 2004 Bakers yeast, Svenska Jastfabrik AB, Sollentuna, Sweden Precosa, AstraZeneca AB CBS 100487 ATCC8585; CBS2359; DSM70799; NRRLY1140 From grains in airtight storage. Isolated from creamery, USA References (selected) (Birol et al., 1998) 395

Saccharomyces cerevisiae

J543

Saccharomyces cerevisiae

J191

(Petersson and Schnurer, 1995) (Kuhle and Jespersen, 2003; Kotowska et al., 2005) (Bjornberg and Schnurer, 1993) (Steensma et al., 1988; Picos et al., 1993)

Saccharomyces boulardii (subspecies of S. cerevisiae) Pichia anomala Kluyveromyces lactis

J551 J121 J469

Culture no. in the Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden. 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.
b

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 34 days. Meanwhile, the pH values of the tempeh cakes were measured in the rst dilution. 2.4. Ergosterol analysis Freeze-dried and milled samples (0.40.5 g) were extracted 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), ve potassium hydroxide pellets (approximately 0.5 g) and 0.2 ml of pyrogallol solution (10 g/L in ethanol). Saponication 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 HP7673 Auto sampler (Hewlett Packard, Waldbronn, Germany). Two microlitres of the samples was autoinjected. 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 nal temperature for 15 min. The detector temperature was 320 1C. The results were evaluated with Borwin Chromatography 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 centrifuged. The supernatant was frozen at 20 1C overnight, thawed and centrifuged through an ultracentrifugal lter device (Micron YM-30, Millipore, Bedford MA, USA). Phytate (myo-inositol hexakisphosphate or IP6) was quantied 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, Niacinamide and biotin) were analysed by AnalyCen Nordic AB, Lidkoping, 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

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by microbiological assay (Freed, 1966; Hashim and Fields, 1979), and biotin was analysed according to Indyk et al. (2000) and Quant Biotin Kit Users 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 (0.450.5 mm in diameter, Sigma). The samples were vortexed for 5 1 min at maximum speed (Vortexgenie 2), and kept for 1 min on ice in between. Subsequently, 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/chloroform 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 specic 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 quantication of DNA Gene Roligchreal 1F Roligchreal 1 R ScPDArealF1 ScPDArealR1 AmprealtimeF1 AmprealtimeR1
a

the Primer Expresss Software v2.0 (Applied Biosystems). The sequences, amplicon length and Tm of the specic primers are given in Table 2. The primers were tested at different concentrations for non-specic DNA-amplication (dimer-formation) by running real time PCR without any templates. For the subsequent analysis, primers were used at the maximum concentrations where no non-specic amplications occurred (Table 2). The primers were also tested with template DNA from other microorganisms used in this study, and no non-specic amplication was observed. The amplication efciencies (E) of the respective DNA amplicons were empirically determined from the amplication of serial dilutions of the respective target DNA according to Wilhelm and Pingoud (2003): E 101=slope , where the slope was calculated from the standard curve of DNA concentrations vs. the CT values. The CT value is dened as the cycle number at which the uorescence surpasses the noise threshold. To produce target DNA for the amplication efciency determination, parts of the chs1 and PDA1-gene sequences were amplied 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 users manual) and sterile ultraltered dH2O to a nal 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 amplied DNA molecule is proportional to ECT, where E is the amplication efciency, and CT is the cycle number for surpassing the noise threshold (see above). Thus, the realtime PCR data are expressed as relative DNA copy

Primer pairs 50 -GTCCCCTTCTTAAGCCCAATG-30 50 -GCGACCACCAGGCTTTGTAC-30 50 -GAAAGCCGTTCTGGCTGAAT-30 50 -GCATGGAACCACCCTTACCA-30 50 -CCGGCAACAATTAATAGACTGGAT-30 50 -GGGCCGAGCGCAGAA-30

Concentration (nM) 160 160 160 160 40 40

Tma (1C) 59 59 58 59 59 59

Amplicon length (bp) 62 62 68 68 65 65

Target gene R. oligosporus chs 1 gene S. cerevisiae PDA 1 gene pUC19 ampR gene

The primer melting temperature (Tm) was calculated by the Primer Express Software v2.0, Applied Biosystem, California, USA.

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numbers (R) of the sample gene copy number related to the external standard gene (ampR) copy number according to the equation: R
CTStandard E Standard

yeasts or not, had rm texture and were sliceable, exhibited sweet and fruity odour, and had a creamy white colour. 3.2. Ergosterol and vitamin production

E CTSample Sample

. Ergosterol is a component of the fungal cell membrane, both in lamentous fungi and in yeasts. Thus, increased ergosterol content was expected for R. oligosporus-yeast cofermentations. Tempeh fermented with R. oligosporus had a signicant (Po0:001) increase in the ergosterol contents compared to non-fermented barley (Table 4). Co-cultivation with different yeasts further increased the ergosterol content by 7.117.5 mg/g dry tempeh (Table 4). However, the statistical signicance 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 3.46.6 107 mg/cfu. Thus, the expected increase in the ergosterol content in tempeh co-fermented with this yeast strain was in the range of 2.75.2 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 signicantly

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 signicantly (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 ve 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 inuence 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

Table 3 Growth of yeasts during barley tempeh fermentation and during tempeh cold storage Strains Fermentation 0 ha S. cerevisiae J 122 S. cerevisiae J 543 S cerevisiae J 191 S. boulardii J 551 P. anomala J 121 K. lactis J 469 4.070.4 4.070.4 4.170.4 4.170.4 4.070.4 4.270.5 20 ha 6.970.1 6.970.1 6.970.1 6.970.1 7.470.2 6.870.2 Storage at 2 1C

Table 4 Ergosterol and phytate contents of barley and barley tempeh fermented 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 Treatment Ergosterol (mg/g dry matter) 0.570.3 57.571.7 67.777.2 67.472.8 71.978.8 75.072.4 67.773.5 64.571.8 Phytate (mmol/g) 2.770.2 1.970.1 1.970.1 1.870.0 2.070.0 2.070.0 1.970.1 2.070.1

7 weeksb 7.770.0 7.570.0 7.670.0 7.770.4 8.770.0 9.070.0

a

Non-fermented barleya J401b J401+J 122b J401+J 543b J401+J 191b J401+J 551b J401+J 121b J401+J 469b

Data are given as log cfu/g dry tempeh (mean7SD, mean7deviation from mean values, bn 2).

n 4, and Mean7SD, an 6, and mean7deviation from mean value, bn 2.

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(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 signicantly 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 difcult due to the big particles of barley kernels. Moreover, ground barley kernels will release a number of different compounds that may affect PCR amplication (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 efcient to extract DNA from spores and hyphae of lamentous fungi (Olsson et al., 1999), but in our hands it did not work for yeasts, as evaluated by PCR amplication using specic primers. In contrast, the lysing buffer-heating method was efcient 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 modication resulted in a successful isolation of DNA from both R. oligosporus and S. cerevisiae. Some parameters were further optimized. Incubating time at 65 1C was reduced from 25 to 10 min without loosing extraction efciency. Vortexing barley samples for 5 min after adding of the glass beads gave maximal extraction of DNA from yeast cells, while 210 min of vortexing did not inuence the DNA extraction efciency of R. oligosporus. This was veried 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 J401a Cystine Methionine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine (calculated) Phenylalanine Histidine Ornitine Lysine Arginine Hydroxiproline Tryptophane Ammonia Essential amino acids Total amino acids 2.870.0 1.870.0 5.770.2 3.670.1 5.070.2 25.670.4 14.170.0 3.970.0 3.970.0 7.070.1 4.670.0 8.170.0 3.970.0 6.670.1 2.570.0 o0.2 3.270.0 5.170.0 o0.2 1.570.0 1.870.0 37.470.1 108.870.9 20 h J401a 2.870.0 1.870.0 5.770.2 3.570.0 4.770.1 22.672.6 13.370.8 3.870.1 4.370.2 6.370.6 4.570.1 7.770.1 3.870.1 6.470.2 2.470.1 o0.2 3.570.0 4.870.0 o0.2 1.470.0 1.870.0 36.071.3 103.075.5 J401+J122b 2.8 1.8 5.5 3.5 4.8 26.7 14.7 3.9 4.4 6.9 4.6 8.1 4.1 6.5 2.3 o0.2 3.5 4.8 o0.2 1.5 1.8 37.2 110.4 J401+J121b 2.8 1.8 6.0 3.7 4.8 26.7 14.1 3.9 4.6 6.7 4.6 8.1 3.9 6.7 2.5 o0.2 3.7 5.1 o0.2 1.4 1.8 37.8 111.1 J401+J469b 2.8 1.8 6.0 3.5 4.6 23.3 13.8 3.9 4.4 6.5 4.6 7.8 3.9 6.2 2.3 o0.2 3.5 4.8 o0.2 1.4 1.8 36.2 105.1

Data are given as g amino acid(s)/kg dry matter. Values represent mean values7deviation from mean (n 2)a or single determinations (n 1)b. The measuring tolerance given by the analysing company is 8% for each amino acid except for tryptophane (10%). Essential amino acid.

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3.6. Real-time PCR quantication of R. oligosporus and S. cerevisiae and correlation to ergosterol contents and cfu Different primer pairs were designed (Table 2) and the amplication efciencies were tested. The theoretical maximum amplication efciency (E value) of real-time PCR of one amplication 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 0:83, n 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 0:99, n 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 signicant 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 production 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

Log relative DNA copies of R. oligosporus

0.0 -1.0 -2.0 -3.0 -4.0 -5.0 -6.0 -1 4 9 Time (h) 14 19

Fig. 2. Real-time PCR quantication of Rhizopus oligosporus grown alone (B) or together with S. cerevisiae J543 () during 20 h barley tempeh fermentation at 35 1C (n 3).

2.50 Log ergosterol content 2.00 1.50 1.00 0.50 0.00 -0.50 -1 4 9 Time (h) 14 19

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 0:98, n 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 signicant (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 quantication was more precise than cfu determination. Our results show that R. oligosporus has no or only slight inuence 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 resulting barley tempeh cakes by 1231%. Nevertheless, the nutritional impact of yeast co-cultivation was lower than expected. However, an improvement in nutritional values may be obtained by selecting specic strains with

Fig. 1. Ergosterol content of barley tempeh fermented by Rhizopus oligosporus during 20 h at 35 1C (n 3).

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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 7.0 Log cfu of S. cerevisiae 6.5 6.0 5.5 5.0 4.5 4.0 -1 4 9 Time (h) 14 19

Fig. 4. Colony forming units (cfu) of S. cerevisiae J543 grown alone (&) or together with R. oligosporus (~) in barley during 20 h fermentation at 35 1C (n 3).

-1.5

-2.5

-3.5

-4.5

-5.5 -1 4 9 Time (h) 14 19

Fig. 5. Real-time PCR quantication of S. cerevisiae J543 grown alone (&) or together with R. oligosporus (E) in barley during 20 h fermentation at 35 1C (n 3).

specic performances (for instance with high folate production). Inoculation of Pichia anomala at 104 cfu/g inhibits several mycotoxigenic fungal species such as Penicillium

roqueforti (Druvefors et al., 2002), but at this inoculation level no inhibition on R. oligosporus growth was observed. We also found that the other tested yeasts did not affect R. oligosporus growth at this inoculation level. However, yeasts added at levels higher than 104 cfu/g or higher than the R. oligosporus inoculation level inhibited the growth of R. oligosporus. Yeasts, especially non-food grade yeasts, such as T. beigelii, that frequently spontaneously grow in soybean tempeh fermentations (Samson et al., 1987), may be able to disturb tempeh production and even constitute a hazard for human health. Therefore, it is desirable to prevent the spontaneous growth of yeasts in tempeh fermentation. The optimal yeast strains for co-cultivation should be food-grade and have low inhibitory potential towards R. oligosporus, but should also have a high competitive ability against other yeasts, moulds or bacteria. 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 in raw material. Similar results were found by Sutardi and Buckle (1985) who studied phytate reduction in soybeans fermented with different strains of R. oligosporus. However, in a recent study, modications of the grain surface have been found to greatly improve phytate reduction, and 97% of the original phytate of whole barley kernels was degraded during tempeh fermentation (Eklund-Jonsson et al., 2006). Thus, the non-degraded phytate in our study may be inaccessible for microbial degradation. The real-time PCR results of S. cerevisiae were highly (r 0:98, n 9, Po0:001) correlated with the cfu numbers of S. cerevisiae. Quantication of yeasts by real-time PCR is much faster than by determination of cfu, and especially useful in mixed culture systems. In contrast, quantication of lamentous fungi is a challenging task because the fungal colony consists of mycelia and single-celled spores. This makes it difcult to dene the lamentous fungal growth by biomass, hyphal length, cell numbers or nucleus copy numbers. Several methods have been established, but every method has its drawbacks. For instance, the number of cfu correlates rather with the number of spores than with mycelial growth (Schnurer, 1993). Hyphal length

Log relative DNA copies of S. cerevisiae

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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 determination with further subculturing none of these methods are species-specic. Here, we have described a selective realtime PCR method for determining relative nucleus copy numbers of lamentous fungi in the solid-state tempeh fermentation. The real-time PCR results were in general correlated with ergosterol contents of the tempeh. However, 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 quantication of lamentous fungi should be combined with other quantication 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 signicantly 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 determination was demonstrated to be a rapid, reliable and selective method for quantication of fungi in barley tempeh fermentation although it in some cases requires verication 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 nanced 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
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