Sourdough Yeast Research Articles

Differentiation of sourdough yeast species by a novel species-specific PCR assay

The objective of this study was to develop species-specific primer pairs based on the internal transcribed spacer region (ITS) of the ribosome DNA for species identification of the frequently found sourdough yeast species. Species-specific primers were designed by the alignment of sourdough yeast ITS sequences, which were then employed for PCR using the template DNA of sourdough yeast strains. PCR primers were shown to be specific for the following species: Issatchenkia orientalis (Candida krusei), C. humilis, Kazachstaniaexigua (C. holmii), Pichia anomala and P. subpelliculosa. Therefore, we conclude that our novel species-specific primers could be used to rapidly and accurately identify the most frequent sourdough yeast species using a PCR-based assay.

Variations of internal pH in typical Italian sourdough yeasts during co-fermentation with lactobacilli

The effects of organic acids (lactic and acetic) and extracellular pH (pHex) on the intracellular pH (pHi) of Saccharomyces cerevisiae and Candida milleri during co-fermentation with lactobacilli were investigated by using Fluorescence-Ratio-Imaging-Microscopy (FRIM). Yeasts were grown in a system that partially mimics sourdough composition, using individual fermentation and combinations with lactic acid bacteria. Fermentations were carried out at 25 °C for 22 h at an initial pH of 5.3. The two yeast species grew equally well during the co-fermentations with lactobacilli. Our results reveal large differences in pHi values between the two yeast species, primarily in relation with pHex changes, while the concentration of organic acids did not seem to affect the pHi. Moreover, the pHi of S. cerevisiae seemed to be affected by maltose consumption. The pH gradient (difference between internal and external pH) of S. cerevisiae remained rather constant, ranging from 2.0 to 2.5. C. milleri instead exhibited a higher pHi, that remained constant throughout the experiments and was unaffected by pHex and/or sugar consumption. Thus, the pH gradient of C. milleri varied much more than that of S. cerevisiae, ranging from 2.3 to 3.8. Our results suggest that the two yeast species have different pHi regulation mechanisms.

Microbial re-inoculation reveals differences in the leavening power of sourdough yeast strains

A method based on microbial re-inoculation, or the so-called backslopping and subsequent proofing of rye bread dough simulating commercial one-stage sourdough process, was used for the screening of the leavening capacity of sourdough yeast strains. Two yeast strains were initially tested with seven Lactobacillus strains. Thereafter, 17 yeast strains, mostly of sourdough origin, were tested with a backslopping procedure with heterofermentative Lactobacillus brevis as an acidifying lactic acid bacteria (LAB). The highest leavening capacity was found in sourdoughs containing Candida milleri, in particular when it was accompanied by obligately homofermentative Lactobacillus acidophilus or facultatively heterofermentative Lactobacillus plantarum when it acted homofermentatively. The leavening capacity of the reference strain Saccharomyces cerevisiae was about half that of C. milleri in all sourdoughs tested. The re-inoculation procedure increased the differences found in the leavening capacity of the tested yeast strains during final proofing of rye bread dough. The backslopped sourdoughs containing a heterofermentative Lactobacillus strain were more suppressive than those containing a homofermentative strain. The highest leavening capacity was found in C. milleri strains. The use of one backslopping cycle before assaying the leavening capacity of a laboratory sourdough is recommended since it helps to differentiate between yeast strains to be tested for their leavening power in the final bread dough.

Effects of yeasts and bacteria on the levels of folates in rye sourdoughs

Fermentation of rye dough is often accompanied with an increase in folate content. In this study, three sourdough yeasts, Candida milleri CBS 8195, Saccharomyces cerevisiae TS 146, and Torulaspora delbrueckii TS 207; a control, baker's yeast S. cerevisiae ALKO 743; and four Lactobacillus spp., L. acidophilus TSB 262, L. brevis TSB 307, L. plantarum TSB 304, and L. sanfranciscensis TSB 299 originally isolated from rye sourdough were examined for their abilities to produce or consume folates. The microorganisms were grown in yeast extract–peptone– d-glucose medium as well as in small-scale fermentations that modelled the sourdough fermentation step used in rye baking. Total folate contents were determined using Lactobacillus rhamnosus (ATCC 7469) as the growth indicator organism. The microorganisms studied did not excrete folates into the media in significant amounts. Yeasts increased the folate contents of sterilised rye flour–water mixtures from 6.5 μg/100 g to between 15 and 23 μg/100 g after 19-h fermentation, whereas lactic acid bacteria decreased it to between 2.9 and 4.2 μg/100 g. Strains of Lactobacillus bulgaricus, L. casei, L. curvatus, L. fermentum, L. helveticus, Pediococcus spp., and Streptococcus thermophilus that were also tested gave folate contents after fermentation that varied between 2 and 10.4 μg/100 g. Although the four Lactobacillus spp. from sourdough consumed folates their effect on folate contents in co-cultivations was minimal. It was concluded that the increase of folate content during fermentation was mainly due to folate synthesis by yeasts. Fermentation of non-sterilised flour–water mixtures as such resulted in three-fold increases in the folate contents. Two folate producing bacteria were isolated from the non-sterilised flour and identified as Enterobacter cowanii and Pantoea agglomerans.

Cloning, sequencing and application of the LEU2 gene from the sour dough yeast Candida milleri

We have cloned by complementation in Saccharomyces cerevisiae and sequenced a LEU2 gene from the sour dough yeast Candida milleri CBS 8195 and studied its chromosomal location. The LEU2 coding sequence was 1092 nt long encoding a putative beta-isopropylmalate dehydrogenase protein of 363 amino acids. The nucleotide sequence in the coding region had 71.6% identity to S. cerevisiae LEU2 sequence. On the protein level, the identity of C. milleri Leu2p to S. cerevisiae Leu2p was 84.1%. The CmLEU2 DNA probe hybridized to one to three chromosomal bands and two or three BamHI restriction fragments in C. milleri but did not give any signal to chromosomes or restriction fragments of C. albicans, S. cerevisiae, S. exiguus or Torulaspora delbrueckii. Using CmLEU2 probe for DNA hybridization makes it easy to quickly identify C. milleri among other sour dough yeasts.

Growth characteristics and metabolic flux analysis of Candida milleri.

The growth characteristics of the sourdough yeast Candida milleri was studied in a carbon-limited aerobic chemostat culture on defined medium. The effect of glucose, xylose, and glucose-xylose mixture on metabolite production and on key enzyme activities was evaluated. Xylose as a sole carbon source was not metabolized by C. milleri. Glucose as a sole carbon source produced only biomass and carbon dioxide. When a glucose-xylose mixture (125:125 C-mM) was used as a carbon source, a small amount of xylose was consumed and a low concentration of xylitol was produced (7.20 C-mM). Enzymatic assays indicated that C. milleri does not possess xylitol dehydrogenase activity and its xylose reductase is exclusively NADPH-dependent. In glucose medium both NAD(+)- and NADP(+)-dependent aldehyde dehydrogenase activities were found, whereas in a glucose-xylose medium only NADP(+)-dependent aldehyde dehydrogenase activity was detected. The developed metabolic flux analysis corresponded well with the experimentally measured values of metabolite production, oxygen consumption (OUR), and carbon dioxide production (CER). Turnover number in generation and consumption of ATP, mitochondrial and cytosolic NADH, and cytosolic NADPH could be calculated and redox balance was achieved. Constraints were imposed on the flux estimates such that the directionality of irreversible reactions is not violated, and cofactor dependence of the measured enzyme activities were taken into account in constructing the metabolic flux network.

A Polyphasic Study on the Taxonomic Position of Industrial Sour Dough Yeasts

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The minimum growth temperature was 8 degrees C for C. milleri and also for most of sour dough yeasts.

Antibacterial activity of sourdough lactic acid bacteria: isolation of a bacteriocin-like inhibitory substance from Lactobacillus sanfranciscoC57

A total of 232 strains from nine species of Lactobacillusisolated from sourdoughs were screened for antagonistic activity against sourdough-related micro-organisms. Seventy-seven strains on agar medium and 52 in culture supernatants, re-adjusted to pH 6.5 and catalase-treated, showed antagonistic activity. The activities were species and strain specific showing different spectra of inhibition against sourdough lactobacilli. All the strains were inhibitory to Bacillus subtilisbut not against sourdough yeasts and moulds. Lactobacillus sanfranciscoand Lactobacillus plantarumstrains had the largest inhibitory spectrum. All the antimicrobial compounds produced from strains of different species contained a protein moiety and were differently sensitive to different proteinases. A bacteriocin-like inhibitory substance, which was heat-stable (100°C for 20 min), insensitive to lipase and α-amylase, of a protein nature, with an inhibitory spectrum centred about lactic acid bacteria and a bactericidal or bacteriolytic mode of action was isolated from Lb. sanfranciscoC57. The antimicrobial substance also inhibited Listeria monocytogenes, and was mainly produced in the stationary phase of growth and at pH 4.0–5.0. Lb. sanfranciscoC57 variants, which did not contain the native c. 17 kbp plasmid, maintained their antagonistic activity, therefore, the gene encoding for the bacteriocin-like inhibitory substance from Lb. sanfranciscoC57 is chromosomally located.

Flavour of sourdough wheat bread crumb

This study investigates the effect of adding sourdough to wheat bread dough on the production of flavour compounds in wheat bread crumb. The sourdoughs were fermented with starter cultures of lactic acid bacteria alone and in combination with sourdough yeasts. The volatile compounds in the bread crumb were isolated by a dynamic headspace technique and extraction analysis, analysed by gas chromatography (GC), and identified on the basis of GC retention times for reference compounds and mass spectrometry (MS). The chemical analyses were combined with sensory evaluation. The volume of the loaves increased significantly when the doughs had 5–20% sourdough added compared with the control bread (bread without sourdough). In the sourdough bread, the content of acetic acid, 2-methylpropanoic acid, and 3-methylbutanoic acid was generally higher, and loaves made with the addition of sourdoughs fermented withLactobacillus plantarum, L. delbrueckii, orL. sanfrancisco had a higher content of 2- or 3-methyl-1-butanol than control bread. Interactions were seen between the starter cultures and the sourdough yeasts, and the production of the following compounds was increased depending on the starter culture used and on the sourdough yeast: ethanol, 2-methylpropanol, 2/3-methyl-1-butanol, 2-phenylethanol, benzyl alcohol, acetic acid, 2-methylpropanoic acid, and 3-methylpropanoic acid. Bread made with an addition of 5% to 15% sourdough fermented withL. sanfrancisco had a pleasant, mild and sour odour and taste.L. plantarum bread had a strong, sour and unpleasant odour and a metallic sour taste with a sour aftertaste, but when the sourdough was also supplemented with the sourdough yeastSaccharomyces cerevisiae, the bread attained a more aromatic wheat bread flavour, which may be caused, in part, by a higher content of 2/3-methyl-1-butanol, 2-methylpropanoic acid, 3-methylbutanoic acid and 2-phenylethanol.

Volatile compounds in wheat sourdoughs produced by lactic acid bacteria and sourdough yeasts

The content of volatile compounds in wheat sourdoughs fermented with four different starter cultures of the genusLactobacillus was compared with those in chemically acidified doughs. Sourdoughs as well as chemically acidified doughs were also combined with four different sourdough yeasts to investigate the effect of adding yeasts on the production of volatiles. The volatile compounds in the doughs were isolated by a dynamic headspace technique, analysed by gas chromatography (GC), and identified on the basis of GC retention times for reference compounds and mass spectrometry. Few volatile compounds were identified in the chemically acidified doughs compared to the sourdoughs. The content of volatile compounds in the sourdoughs varied with the starter culture used. Ethanol and ethyl acetate were produced in the highest amounts in sourdoughs fermented with heterofermentative cultures, especiallyL. sanfrancisco. Apart from ethanol,n-hexanol was the dominating alcohol in all the sourdoughs. The content of other compounds was low. When sourdough yeasts were added to the sourdoughs, the number and amount of volatile compounds increased. The content of ethanol, 2-methyl-propanol and 2/3-methyl-1-butanol was highest in the sourdoughs with addedSaccharomyces cerevisiae, whereas the ethyl acetate content was highest in the sourdoughs with added strain A. The content of esters other than ethyl acetate was low. The carbonyls acetoin and diacetyl were produced in small amounts in the sourdoughs with the addition of strain A.

Electrofusion of an industrial Baker’s yeast strain with a sour dough yeast

A method for hybridization of yeast protoplasts ofSaccharomyces cerevisiae andCandida holmii by electrofusion was optimized. The hybrids were screened on maltose-acetate agar plates. The average fusion frequency was 1.1xl0-3. Two hybrids of 132 collected from selection plates were found to be stable over 15 sequential shake flask cultivations. However, the strains reverted during production of baker’s yeast in a laboratory scale process imitating the industrial process.

Growth Requirements of San Francisco Sour Dough Yeasts and Bakers' Yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.