WO1999051746A1 - souche de levure améliorée - Google Patents

souche de levure améliorée Download PDF

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Publication number
WO1999051746A1
WO1999051746A1 PCT/EP1999/002518 EP9902518W WO9951746A1 WO 1999051746 A1 WO1999051746 A1 WO 1999051746A1 EP 9902518 W EP9902518 W EP 9902518W WO 9951746 A1 WO9951746 A1 WO 9951746A1
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yeast
yeast cell
pdrl2
gene
cell according
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PCT/EP1999/002518
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English (en)
Inventor
Rutger Jan Van Rooijen
Peter Piper
Karl Kuchler
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Dsm N.V.
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Priority to AU34216/99A priority Critical patent/AU3421699A/en
Publication of WO1999051746A1 publication Critical patent/WO1999051746A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/047—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
    • C12C12/004—Genetically modified microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

  • the present invention relates to an improved yeast strain.
  • Weak acid preservatives are generally thougth to be safe anti-microbials and their use for the preservation of foods and beverages is widespread.
  • the use of the calcium salt of propionic acid for the prevention of fungal growth on bakery products for example is very common for the bakers in some parts of the world, such as the Far East and South America.
  • sulfite has been used for centuries for the sterilization of vessels used in wine making.
  • weak acids are in a dynamic, pH-dependent equilibrium between their undissociated molecules and anionic states.
  • Acidic pH favors the undissociated, uncharged state, a state in which weak acid preservatives exert much stronger antimicrobial action. This is probably because such action largely involves the uncharged acid diffusing through the plasma membrane into the cytosol .
  • the weak acid encounters a more neutral pH and consequently dissociates. This dissociation releases protons, resulting in acidification of the cytoplasm, which in turn leads to the inhibition of several metabolic processes [Krebs, H.A., et al , 1983, Biochem-J 214: 657-663].
  • a major drawback with respect to the use of weak acids as anti-fungal agents in bakery applications is that the leavening power of baker's yeast products decreases considerably in the presence of weak acids. As a - 2 - consequence, proofing times, i.e. the time for a prepared dough to leaven before baking, are considerably longer.
  • yeast cells weak acid preservatives characteristically cause an extended lag phase and cell stasis, rather than cell death.
  • the ability of certain yeast species to grow at low pH in the presence of weak (organic) acid food preservatives enables them to act as important agents of food spoilage and can cause considerable economic loss [Deak, T., 1991, Adv. Appl . Microbiol . 36: 179-278; Fleet, G. , 1992, Crit Rev Biotechnol 12: 1-44].
  • Saccharomyces cerevisiae will grow in the presence of up to 3 mM sorbic acid at pH 4.5, although the presence of the preservative causes both a considerable lag phase extension and a reduction of final biomass yield [Stratford, M. , and P.A. Anslow, 1996, FEMS-Microbiol- ett 142:53-58; Piper, P.W., et al . , 1997, Cell Stress Chap. 2: 12-24].
  • S . cerevisiae is sometimes identified as a food spoilage organism, other more weak acid tolerant and osmotolerant yeasts such as Zygosaccharomyces baillii are more frequently found to cause food spoilage. These yeast species are sometimes capable of adapting to growth in the presence of the highest levels of weak organic acids allowed in food preservation, even at pH values less than the pKas of these acids [Deak, 1991; Fleet, 1992] .
  • the present invention provides a baker's yeast, which has a comparable leavening power in applications with and without calcium-propionate, or other weak acids. Leavening power or gassing power can be determined in Test A and B or in Test A' and B' .
  • weak acids is meant an organic acid - 3 - with a pK A value of from 3 to 7. Examples of such weak acids are sorbate, propionate or benzoate.
  • Pdrl2 is a member of the ATP-binding cassette (ABC) protein superfamily and shares high homology with two previously identified ABC drug efflux pumps. Transcription of the PDR12 gene and expression of the Pdrl2 protein both increase in yeast cells in response to weak acid stress. Furthermore a pdrl2 deletion mutant of yeast is hypersensitive to weak acid stress and also shows impaired ability to carry out energy-dependent weak acid efflux. Additionally, constitutive (over) expression of the PDR12 gene in yeast cells confers increased resistance to weak acids on those cells. This evidence has led us to conclude that the PDR12 gene encodes a weak acid pump.
  • ABSC ATP-binding cassette
  • Constitutive expression of a gene means that the expression of the gene is not influenced by the - 4 - physiological conditions in which the host cell grows.
  • the PDR12 gene encodes a molecular pump enabling the cell to pump out weak acids that have entered the cell.
  • Yeast strains containing a gene constitutively expressing weak acid pump are preferably used for the commercial production of baker's yeast in a form such as for example compressed, cream, dry or instant dry yeast.
  • commercial production is meant production in a fermentor of more than lm 3 .
  • Such a baker's yeast typically shows advantageously a ratio of the gassing power in test A / test B or a ratio of the gassing power in test A' / test B' of at least 85%, preferably 90% and more preferably 100%.
  • constitutive expression of a gene encoding a weak acid pump can be obtained by replacing the natural promoter by a promoter of any gene that is constitutively expressed in yeast.
  • constitutive yeast promoters are described in Nacken et al [Gene 175 (1996) 253-260] and Mumberg et al . [Gene (1995) 156, 119-122] and indicate, for example, the ADHI or PmAI gene promotors .
  • constitutive expression can be obtained by mutagenesis of the natural promoter of the gene encoding the weak acid pump. This can be achieved by the treatment of yeast cells with mutagenic agents or oligonucleotides and selecting for mutants that constitutively express genes encoding weak acid pumps .
  • the natural promoter of a gene encoding a weak acid pump is replaced by a promoter that can be regulated in yeast.
  • this regulatable promoter can be induced or derepressed by the addition of certain compounds to the growth medium resulting in the - 5 - expression of the gene encoding a weak acid pump.
  • regulatable promoters may be used that are induced or derepressed in the absence of certain compoment in the growth medium.
  • one or more copies of a gene encoding a weak acid pump under control of its natural promoter may be introduced.
  • an industrial yeast strain will contain 2 to 3 copies (as a consequence of its aneuploidic nature) of the PDR 12 gene. By introducing extra copies the yeast strain becomes less sensitive to weak acids.
  • preferred (e.g. industrial) yeast strains are non-haploid strains. It will be appreciated that combinations of these embodiments can be made to obtain yeast strains that are resistant against weak acids.
  • yeast strains having one or more constitutively expressed gene(s) encoding a weak acid pump will most preferably be employed.
  • Such strains may be obtained via introduction of these genes into yeast cells on single- or multicopy plasmids, or via integration as a single copy or multiple copies into the genome of the host cell.
  • these strains may be obtained via selection after mutagenesis or classical breeding techniques.
  • FIG. 1 Purified plasma membrane fractions from sorbate-treated yeast cells show a highly induced (S) membrane protein
  • A Wild type - 6 - cells cultured overnight in pH 4.5 YPD in the absence (1) and presence (2) of 1 mM sorbate. About 40 ⁇ g total membrane protein per lane were separated by SDS PAGE through a 9% gel and stained with Coomassie Blue.
  • B About 8 ⁇ g of total plasma membrane proteins from wild type (3) and pdrl2 (4) cells cultured for 6 h in pH 4.5 YPD in the presence of 1 mM sorbate were analyzed by SDS-PAGE and silver-staining. The main 100 kDa band represents the Pmal plasma membrane H+-ATPase.
  • Figure 2 Primary sequence and predicted membrane topology of PDR12.
  • A Shaded boxes Pdrl2 represent peptide sequences obtained from microsequencing of the sorbate-induced protein
  • FIG. 1 Solid black lines represent the polypeptide chain. Putative transmembrane segments are shown as vertical black bars. The two conserved ABC domains are marked by the black ovals and "ATP”. Dotted oval balls indicate potential N-linked carbohydrate.
  • Figure 3 Northern analysis of total RNA from yeast cells grown on YPD pH 4.5 subjected to weak acid stress. Hybridization to radiolabeled probes specific for the genes indicated to the left of the figure panels was carried out by routine methods. An actin-specific probe (ACT1) served as a control for equal RNA loading. Lane 1: 20 ⁇ g total RNA from unstressed wild type (FY1679-28C) cells
  • Lane 2 20 ⁇ g total RNA from FY1679-28C cells challenged with 1 mM sorbate for 1 h
  • Lane 3 20 ⁇ g total RNA from FY1679-28C cells challenged with 9 mM sorbate for 1 h - 7 -
  • FIG. 4 Immunological detection of Pdrl2 in wild type and sorbate-treated cells.
  • A Total cell extracts of wild type and pdrl2 cells were immunoblotted using a polyclonal antiserum raised against a GST-Pdrl2 fusion protein.
  • B Cell extracts from untreated (-) and 9 mM sorbate-treated (+) pH 4.5 and pH 7.0
  • FY1769-28C cultures were analysed for PDR12 expression by immunoblotting .
  • the non-specific cross reaction at higher molecular mass serves as an internal control for equal protein loading in each lane.
  • Figure 5 Growth curves of wild type (A) and pdrl2 (B) cells in liquid pH 4.5 YPD medium containing 10 mM propionic acid.
  • Figure 6 Intracellular accumulation of [14C] benzoate by wild type (solid symbols) and pdrl2 cells (open symbols) before and after glucose addition marked by an vertical arrow (at 5 min) . Cells were either grown at pH 4.5 (A) or at pH 4.5, then pre-treated with 1 mM sorbate for 2h (B) as described in Material and Methods .
  • Figure 7 Physical maps of PDR12 expression plasmids PJ1PDR12 and pJ2 * PDR12.
  • the second sorbate-induced polypeptide had a mobility corresponding to a protein of about 170 kDa and was readily detectable by silver-staining (Fig. 1, B, lane 3) .
  • the band in the 130-190 kDa size range was blotted onto a PA/DF membrane, digested with lysyl-endopeptidase and the resulting peptides were resolved by HPLC.
  • four peptide sequences obtained from this digest four
  • KMVYFGDIGPNSETLLK were perfect matches to regions 287-300, 366-383, 838-859 and 1062-1078, respectively, of a large open reading frame (ORF) , YPL058c, present in the yeast Proteome Database (Fig. 2, A) .
  • YPL058c on chromosome XVI is predicted to encode the 1511 -residue protein Pdrl2 , a typical member of the ATP-binding cassette (ABC) protein superfamily [Kuchler, K. , and R. Egner, 1997, Unusual protein secretion and translocation pathways in yeast: implication of ABC transporters. Unusual Secretory Pathways: From Bacteria to Man. 49-85.
  • the predicted topology of the Pdrl2 transporter includes twelve predicted transmembrane-spanning ⁇ -helices and two highly conserved nucleotide binding domains, the hallmark domains of all ABC proteins (Fig. 2, B) .
  • Pdrl2 is highly homologous to two previously identified ABC drug efflux pumps, Snq2
  • a pdrl2 deletion strain was genetically constructed and the protein pattern plasma membrane fractions were analyzed after sorbate treatment in both wild type and isogenic pdrl2 cells.
  • Coomassie-stained gels (Fig. 1, A) showed that a sorbate-induced protein of 170 kDa (S) found in wild type cells (Fig. 1, A, B) was completely absent in pdrl2 cells. Based on these results, we investigated the effects of different stresses on the mRNA levels of three ABC transporter genes, PDR5, SNQ2 and PDR12.
  • the PDR12 open reading frame of 4533 bp potentially encodes a 1511-residue protein, with a predicted molecular mass of 171 kDa.
  • a polyclonal anti-Pdrl2 antiserum was raised in rabbits using a bacterially-expressed GST-Pdrl2 fusion protein as the antigen.
  • Total cell extracts were prepared from both wild type and isogenic pdrl2 cells and subjected to immunoblotting (Fig. 4, A).
  • a polypeptide band with an - 10 - expected molecular mass of 170 kDa was recognized by the antiserum in wild type cell extracts, whereas no protein in this molecular mass range was detectable in cell extracts from the pdrl2 deletion mutant (Fig. 4, A) .
  • Possible sorbate-mediated induction of PDR12 was also tested by immunoblotting. Cells from an overnight culture of wild type FY1679-28C were inoculated into fresh pH 4.5 and pH 7.0 YPD medium. Both cultures were then grown to an OD600 of 0.7-1.0, at which point sorbate was added to half of each culture to a final concentration of 9 mM.
  • a pdrl2 deletion strain is hypersensitive to propionic acid
  • Baker's yeast strains 221Ng- PDR12 ADH1 , 227Ng- PDR12 PMA1 , 237Ng -PDR12 AD111 and 237Ng -PDR12 P1SA1 that contain the PDR12 gene under control of one of the constitutive ADH1 or PMA1 promoters were cultivated in a down-scaled fed-batch production process. Part of the yeast was dried for the production of IDY products. The compressed yeast and IDY products were tested in gassing tests A (+ 0.3% calcium propionate) and B (- calcium propionate) for their resistance against calcium propionate. The Table below gives the normalized gassing values. The indicated values are given as the percentage of the gassing value of the wild-type strain in test B (- calcium-propionate) .
  • Transformants containing an integrated copy of the constitutive PDR12 gene in the chromosomal SIT2 locus - 14 were selected for their ability to grow on plates acetamide . Subsequently, transformants were grown on plates containing fluoracetamide to select for the pop-out of the amdS marker and E. coli DNA [EP 635574] . Correct genomic integration of constructs and proper looping-out was confirmed by PCR and Southern blot of genomic DNA isolated from several independent transformants exactly as described elsewhere [Mahe et al , 1996 Mol. Microbiol. 20: 109-117].
  • a glutathione-S-transferase (GST) -PDR12 gene fusion was constructed as follows. A 500 bp PCR fragment of PDR12 was generated from a genomic DNA template using the custom- made primers, PDR12- 8 : 5' -CGA-CTG-ACG-AAT-TCA-TTG-AGA-AAG-3 ' and PD.R12-528: 5 ' -CAT-TTC-ACC-GAA-TTC-AAC-GAC-ACC-3 ' . The PCR product was digested with EcoRI and cloned into the EcoRI site of plasmid pGEX-5X-l (Pharmacia) . The resulting plasmid pYM53 allowed for bacterial expression of the N-terminal 164 aa (amino acids 8-172) of Pdrl2 fused in frame to the C-terminus of GST.
  • the pdrl2 : :hisG-URA3-hisG deletion plasmid was constructed in two steps. First, the above mentioned 500 bp EcoRI fragment obtained by PCR with primers PDR12- 8 and PDR12-528 was inserted in the EcoRI site of plasmid pYM28, which contains the hisG-URA3-hisG element [Mahe, et al , 1996, Mol. Microbiol. 20: 109-117, Mahe et al , 1996, J. Biol. Chem. 271: 25167-25172], to give plasmid pYMI14.
  • the 3 '-end of the PDR12 gene was cloned as a 840 bp BamHI-XhoI fragment (generated by PCR using the primers PDR12- 31 : 5 ' -CGT-GCA-TCT-CAT-GCA-GG-3 ' and PDi?12-32 : - 15 -
  • plasmid pYM63 Xhol-cleaved pYMI14, to give plasmid pYM63.
  • PDR12 ADH1 and PDR12 PMA1 expression plasmids pJlP i?12 and pJ2 * PDR12 , respectively (shown in Fig. 7) the PDR12 gene was picked up by PCR using the following primers:
  • strains FY1679-28C and YYM19 were grown to late exponential phase at 30°C on YEPD medium containing no stress agent . Cultures were then diluted to an OD600 of 0.2 in YEPD (pH 4.5), followed by growth to an OD600 of 0.8. The culture was divided in two parts, with or without 10 mM of propionic acid, and the OD600 was monitored for 2 hours at 30°C.
  • the E.coli strain DH5 ⁇ carrying plasmid pYM53 was grown at 30°C to an OD600 of 0.7.
  • Expression of the GST-Pdrl2 fusion protein was induced by adding isopropyl 3-D-thiogalactopyranoside to a final concentration of 0.1 mM and the cells were grown for a further 4 h.
  • the cells were harvested by centrifugation, resuspended in 1/50 of the original culture volume of ice-cold phosphate-buffered saline (PBS) . Cell lysis was achieved by sonication on ice using a Bandelin Sonicator.
  • PBS ice-cold phosphate-buffered saline
  • the lysate was centrifuged at 10,000 x g to remove insoluble material. The supernatant then was incubated with 1 ml of a 50% (w/v) slurry of glutathione Sepharose 4B beads (Pharmacia) for 16 h at 4°C on a rotation mixer.
  • the glutathione-Sepharose beads were - 16 - washed 4 times with ice-cold PBS and the GST-Pdrl2 fusion protein was eluted by incubating the beads for 10 min with 2 ml of a solution of 5 mM reduced glutathione at room temperature.
  • yeast plasma membrane fractions were partially s purified and fractionated by one-dimensional SDS-PAGE exactly as described previously [Piper, 1997] .
  • Peptide microsequencing was performed on protein samples blotted onto PVDF membranes by routine laboratory methods [Harlow, 1988] .
  • o Protein extracts from whole yeast cells were isolated essentially as described elsewhere [Egner, R. , et al . , 1995, Mol. Cell. Biol. 15: 5879-5887]. Briefly, yeast cell extracts were prepared from exponentially growing cultures by lysing 1 OD600 of cells with 150 ⁇ l 1.85 M NaOH, 7.5% 5 mercaptoethanol for 10 min on ice.
  • Overnight FY1679-28C and pdrl2 cultures were diluted 100-fold in water, inoculated into two flasks with 100 ml pH 4.5 YPD and grown to an OD600 of 0.7-1.0. Each culture was then divided into two 50 ml portions and sorbic acid was added to a final concentration of 1 mM to one of these. After a further 2h incubation at 30°C, the cells were harvested, washed in ice-cold water and resuspended in 5.4 ml 20 mM sodium citrate pH 4.5 at room temperature.
  • RNA isolation, radiolabeling and Northern analysis Total yeast RNA was isolated, fractionated through agarose gels and hybridized to radiolabeled probes using standard methods [Piper, P., 1994, Measurement of Transcription. Molecular Genetics of Yeast: A Practical Approach] . DNA fragments were radiolabeled using a Megaprime Labeling Kit under conditions recommended by the manufacturer (Amersham) . The PDJ?12-specific probe ( +8 to +4787 region of PDR12) was amplified by PCR from total yeast genomic DNA with the primers PDR12- 8 and PDR12-32 using standard PCR conditions [Mahe et al , 1996, Mol. Microbiology 20, 109-117] .
  • the fedbatch cultivations using cane molasses and ammonia as substrates, were carried out essentially as described in patent EP0306107A2.
  • Cells were cultivated in 10 1 fermentors with a net volume of 6 1. During the fermentation, pH and temperature were maintained at desired values by automatic control.
  • the yeast obtained by this cultivation was concentrated and washed with tap water in a - 18 - laboratory nozzle centrifuge. Yeast creams were compressed to a dry matter content of approximately 35%.
  • the measured protein content (%N*6.25) varied between 40% and 50%. Drying of the compressed yeast was performed on a laboratory scale fluid bed dryer, consisting of a conical glass tube built on an air supply systm. Details of the drying procedure can be found in for instance US patent 3,843,800.
  • the dry matter content of the dried IDY yeast was 96.5%.
  • Test A determination of gassing power in the presence of calcium-propionate
  • IDY yeast 750 mg of dried IDY yeast is manually mixed with 62.5 grams of standard flour (e.g. IBIS flour Meneba) and incubated at 28°C for 10 min. Subsequently, 38.1 ml of a solution containing sodium-chloride (19.70 g/1) , sucrose (262.47 g/1) and calcium-propionate (4.92 g/1) is added and a dough is prepared in a Hook mixer by mixing for 6.00 minutes at 120 rpm. After preparation, the doughs are transferred to steel barrels that are equilibrated at 28°C. The barrels are closed, and after equilibration the carbon-dioxide production is measured at 28°C for 180 min by a pressure transducer in the closed barrel .
  • standard flour e.g. IBIS flour Meneba
  • Test A' is identical except that 2.5 grams of compressed yeast is used. - 19 -
  • Test B determination of gassing power in the absence of calciumpropionate
  • IDY yeast 750 mg of dried IDY yeast is manually mixed with 62.5 grams of standard flour (e.g. IBIS flour Meneba) and incubated at 28°C for 10 min. Subsequently, 38.1 ml of a solution containing sodium-chloride (19.70 g/1) and sucrose (262.47 g/1) is added and a dough is prepared in a Hook mixer by mixing for 6.00 minutes at 120 rpm. After preparation, the doughs are transferred to steel barrels that are equilibrated at 28°C. The barrels are closed, and after equilibration the carbon-dioxide production is measured at 28°C for 180 min by a pressure transducer in the closed barrel .
  • standard flour e.g. IBIS flour Meneba
  • Test B' is identical to test B except that 2.5 grams of compressed yeast is used.
  • the weak acid resistance can be calculated as the ratio between the results in test A and B times 100%.

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Abstract

L'invention concerne une souche de levure améliorée comprenant au moins un gène codant pour une pompe à acides faibles sous le contrôle d'un promoteur, le gène est exprimée de manière constitutive.
PCT/EP1999/002518 1998-04-07 1999-04-07 souche de levure améliorée WO1999051746A1 (fr)

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AU34216/99A AU3421699A (en) 1998-04-07 1999-04-07 Improved yeast strain

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EP98201094 1998-04-07
EP98201094.4 1998-04-07

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Cited By (4)

* Cited by examiner, † Cited by third party
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CN102409002A (zh) * 2011-02-18 2012-04-11 乐斯福公司 能够生产耐渗透压的并对弱有机酸具有内在耐受性的面包酵母的酿酒酵母菌株、其制备方法以及应用
WO2011151326A3 (fr) * 2010-05-31 2013-02-28 Vib Vzw Utilisation de transporteurs pour moduler la production d'arômes par la levure
US9085781B2 (en) 2012-07-23 2015-07-21 Edeniq, Inc. Acetate resistance in yeast based on introduction of a mutant HAA1 allele
CN112153904A (zh) * 2018-05-15 2020-12-29 乐斯福公司 用于禽类或用于反刍动物的益生菌

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CN109880709A (zh) * 2019-04-29 2019-06-14 宁夏贺兰山东麓庄园酒业有限公司 一种桃红葡萄酒的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4346115A (en) * 1976-12-24 1982-08-24 Lesaffre Et Cie Fermentation of acid-containing doughs
EP0645094A1 (fr) * 1993-09-24 1995-03-29 Dsm N.V. Amélioration de production de gaz et alcool des souches de levure

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4346115A (en) * 1976-12-24 1982-08-24 Lesaffre Et Cie Fermentation of acid-containing doughs
EP0645094A1 (fr) * 1993-09-24 1995-03-29 Dsm N.V. Amélioration de production de gaz et alcool des souches de levure

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BALZI, E., ET AL.: "PDR5,a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 3, 1994, pages 2206 - 2214, XP002088029 *
PIPER, P.W., ET AL.: "Hsp30, the integral plasma membrane heat shock protein of Saccharomyces cerevisiae, is a stress-inducible regulator of plasma membrane ATPase", CELL STRESS + CHAPERONES, vol. 2, 1997, pages 12 - 24, XP002088030 *
PIPER,P., ET AL.: "the Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast", THE EMBO JOURNAL, vol. 17, no. 15, August 1998 (1998-08-01), pages 4257 - 4265, XP002088031 *
SERVOS, J., ET AL.: "gene SNQ2 of Saccharomyces cerevisiae, which confers resistance to 4-nitroquinoline-N-oxide and other chemicals, encodes a 169kDa protein homologous to ATP-dependent permeases", MOLECULAR AND GENERAL GENETICS, vol. 236, 1993, pages 214 - 218, XP002088028 *
WINNET, E., ET AL.: "untitled", EMBL SEQUENCE DATA LIBRARY, 1 November 1996 (1996-11-01), heidelberg, germany, XP002088027 *

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