WO2002042442A2 - Hefestamm für den verzehr - Google Patents
Hefestamm für den verzehr Download PDFInfo
- Publication number
- WO2002042442A2 WO2002042442A2 PCT/EP2001/011887 EP0111887W WO0242442A2 WO 2002042442 A2 WO2002042442 A2 WO 2002042442A2 EP 0111887 W EP0111887 W EP 0111887W WO 0242442 A2 WO0242442 A2 WO 0242442A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- yeast
- seq
- primer
- primer pairs
- sequences
- Prior art date
Links
Classifications
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Definitions
- the present invention relates to a new yeast strain with improved properties for human or animal consumption and a method for characterizing yeast.
- the yeast Saccaromyces cerevisiae was used in the ancient times for the production of fermented drinks.
- the best known beverages fermented by yeast are beer and wine.
- the beer that is brewed according to the Purity Law issued in 1516 can be understood as beer in the strict sense. According to this, only barley, hops, water and Saccharomyces cerevisiae may be used to produce "beer".
- yeast strains used generally belong to the Saccharomyces genus, their physiological properties differ significantly from one another.
- Probiotics are living microorganisms for nutritional supplements with beneficial effects on the host organism e.g. B. by improving the intestinal, microbiological balance.
- Scientific and medical research has increasingly been carried out in recent years, emphasizing the essential importance of gastrointestinal microflora for the health and resistance of humans and animals.
- Probiotics should be living microorganisms that have certain antagonistic properties to the predominant intestinal flora so that they can integrate positively into the natural symbiosis. Yeasts used as probiotics can often fit into the biofilm of the intestinal surface and then form a protective barrier against potential pathogens. They prevent the multiplication and the settlement of bacteria ingested with the food. Duck clostridia Robacteria or Campylobacter, which can form enterotoxins, are limited in their growth by a healthy intestinal flora.
- Saccharomyces cerevisiae as an apathogenic germ, cannot multiply or permanently settle in the human host.
- Gram-positive germs such as lactobacilli, are synergistically promoted in their growth due to the vitamin content of the yeast.
- the immune system is stimulated by the antigenic properties of the yeast cells.
- the activities of intestine-associated disaccharidases such as saccharase, lactase and maltase are increased. Disaccharides are split and then reabsorbed.
- Enteropathogenic E.coli can be bound to yeast cells with their mannose-sensitive surface appendages so that they cannot attach to the intestinal wall.
- the lining of the intestinal epithelium by yeasts prevents pathogenic germs from attaching.
- the yeast's vitamin B content also has positive effects on the skin.
- Saccharomyces cerevisiae can prevent prophylactically the dangers of diarrhea. Diarrhea is particularly likely if the intestinal flora is reduced after antibiotics. Antibiotic-resistant E. coli can multiply and, as an indirect consequence of the suppression of gram-positive germs, penetrate more into the upper parts of the intestine. A precondition for their pathogenicity is adherence to the intestinal epithelium, which is made possible by certain microbial pathogenicity factors. An infection can have very different courses depending on the E.co// strain. In addition to bacterial enterocolitis and diarrhea, more severe forms of the disease, such as hemorrhagic uraemic syndrome, can also be caused. Another prophylactic application of S. cerevisiae is against Candida albicans mycoses. Oral intake of S. cerevisiae can reduce the intestinal Candida cell count.
- the present invention was based on the technical problem of specifying a new yeast strain which is suitable for human or animal consumption, with the least possible impairment of the taste of the yeast-containing agent. Another technical problem was to provide a method with which any strains of yeast can be clearly distinguished from one another.
- the first problem is solved according to the invention by a yeast strain, which in the molecular genetic characterization by means of PCR with primer pair 3, comprising SEQ ID No. 5 and SEQ ID No. 6, with primer pair 4 comprising SEQ ID No. 7 and SEQ ID No. 8, with the primer pair 7 comprising SEQ ID No. 13 and SEQ ID No. 14 and with the primer pair 9 comprising SEQ ID No. 17 and SEQ ID No. 18, the band pattern shown in FIG. 1 provides.
- the second problem is solved according to the invention by a method for identifying and characterizing yeast comprising the step: performing a PCR analysis of the genetic material of the yeast with at least one of the primer pairs 1 to 13, preferably with at least 4 different primer pairs, very particularly preferably with all Primer pairs 1 - 13.
- the yeast strain according to the invention can best be characterized and identified on the basis of its genetic fingerprint, which can easily be created by means of PCR and primer combinations according to the invention.
- Primer combinations 1 to 13 can be used in any combination with one another to produce the genetic fingerprint. The most detailed information is of course obtained when all 13 primer pairs are used to analyze the yeast genome. However, the combination of primer pairs 3, 4, 7 and 9 already provides a particularly characteristic pattern. Patterns created and combinations of primer pairs represent a clear genetic fingerprint of the respective yeast strain and even allow the differentiation of genetically very closely related yeast strains.
- a particularly preferred strain Sacharomyces BCD No. 14143 was deposited with the German Collection of Microorganisms and Cell Cultures GmbH under the accession number DSM 13850.
- Derivatives derived therefrom are also particularly preferred.
- a “derivative” is understood to mean a yeast strain which differs from the stored strain by one or more nucleotide exchanges, the essential characteristics being retained, in particular the genetic fingerprint as it is with a combination of primer pairs, for example the primer pairs 3, 4, 7 and 9, particularly preferably with all primer pairs 1-13.
- Primers with one of SEQ ID Nos. 1-26, sequences hybridizing therewith and sequences with at least 80% identity to these sequences are regarded as primers according to the invention.
- “Hybridizing” means that the hybridizing sequence has a degree of identity to the primer sequence in question that allows only the primer sequence to be recognized even in the presence of further sequences, it being within the range of skill in the art that is suitable for this Preferred hybridization conditions correspond to those of the PCR program specified in the examples when using a conventional PCR buffer.
- “80% identity” means that the same nucleotide is in the reference sequence at 8 out of 10 corresponding positions as in the given one Sequence selected from SEQ ID Nos. 1 - 26.
- the yeast according to the invention is particularly suitable for producing agents which are intended for ingestion by humans or animals.
- the addition of the yeast according to the invention leads to practically no taste impairment of the agent to be absorbed.
- the yeast according to the invention is particularly suitable as a component of a medicament, a probiotic, a fermentation drink, a suspension, an extract or a pastry.
- Beer, non-alcoholic beer or fermented fruit juices are particularly suitable as fermentation drinks.
- the yeast according to the invention is introduced into the agent to be administered in lyophilized form.
- the agent containing the yeast according to the invention is preferably used for the treatment of diarrhea, colitis, intestinal infections, candida infections or skin diseases.
- the primers according to the invention furthermore allow a method for identifying and characterizing yeasts, the method being able to identify any yeast on the basis of their genetic fingerprint, even if these yeasts are practically indistinguishable from one another by biochemical parameters.
- the genome of the yeast in question is subjected to a PCR analysis, at least one of the primer pairs 1 to 13 being used.
- the more primer pairs used in combination to analyze the genome in question the more precisely and in detail the genetic fingerprint will be obtained from the yeast in question. Therefore, if two predetermined yeasts are to be examined to determine whether they are identical or different from one another, their genome is examined using the primer pairs according to the invention, it being possible to use as many combinations of primer pairs until a significant difference in the band pattern is obtained.
- primer pairs 3, 4, 7 and 9 are primer pairs 3, 4, 7 and 9.
- the formulation may contain live or dead yeast lines in the administered product.
- yeast cells can be separated during the production process, so that only the fermented product or the product supplemented with yeast excretions is consumed.
- a preferred Sacc ⁇ a / Omyces strain can be added to a juice, beer or wine drink as a probiotic suspension.
- the suspension can also be incubated for some time to cause fermentation.
- the alcohol produced can be removed from a fermented drink, e.g. to produce a non-alcoholic beer or wine.
- Saccharomyces BCD can be administered as lyophilized yeast. In this case, it can be used against indications such as diarrhea, colitis, candida infections or general gastrointestinal disorders. It can also be used to improve skin quality.
- Saccharomyces BCD is in the addition to baking mixes for the production of bread, baked goods, steamed pasta or the like.
- Extracts of Saccharomyces BCD can be used for any of the applications previously described.
- individual components of the yeast can be administered in enriched or purified form.
- Saccharomyces BCD The characterization of Saccharomyces BCD using PCR is shown in FIG. 1. In each case, a closely related comparison strain (left) and Saccharomyces BCD (right) are plotted. The arrows indicate differences between the tribes. The primer pairs PP3, PP4, PP9, PP7 were used.
- Saccharomyces BCD is a strain of the genus Saccharomyces. On the basis of a molecular biological detection method, this can be clearly distinguished from other Saccharomyces strains, in particular from those of the species Saccharomyces cerevisiae.
- the yeast is grown for 2 days at 30 ° C in a medium with 2% glucose.
- the genomic DNA is isolated from each 2 ml overnight culture using a commercially available kit.
- a PCR is then set up as follows:
- primer pairs used in the PCR reactions are summarized in this table.
- a primer of each pair can be coupled to a dye for better identification.
- the band pattern is characterized by the number of bands per pair of primers and the size of the bands per pair of primers. Occasionally, an amplification product can also be completely missing. In combination with different pairs of primers, a two-dimensional band pattern is created that allows a direct comparison between different yeast strains. Table 2: Band patterns obtained with different primers for different yeasts
- the yeast 9, 10 ... 39 shows the band pattern which is obtained with the primer pairs (PP) 3, 4, 7 and 9, respectively. No 2 of the yeasts tested showed the same band pattern for all 4 primer pairs.
- the results show the separation of the PCR fragments on a sequencing gel, the separation being carried out under the following conditions: A DNA sequencer from LI-COR (Lincoln, USA) was used. The PCR samples (marked with the dye IRD 800) were separated by electrophoresis using an acrylamide gel which is 0.25 mm thick and 50 cm long for about 8 hours at 1500 V.
- A-M denotes identical band patterns; stands for two different yeasts with PP1 e.g. "E", the two yeasts have the same band pattern with this primer pair.
- the combination of these patterns characterizes the respective strains. It is remarkable that Saccharomyces cerev / s / ae strains can also be distinguished, their use as brewer's yeast, wine yeast etc. actually is identical.
- Saccharomyces BCD Saccharomyces BCD No. 14143
- DSM 13850 German Collection for Microorganisms
- the Saccharomyces BCD strain was inoculated 2% in DS medium for the preculture and incubated for 20 h at 28 ° C. and 120 rpm. At the time of harvest, the culture has an OD600 of approx. 25.
- Vitamins and trace elements have also been added to the medium.
- Flavor scale from 1 (very good) to 5 (not sufficient) (average of several test subjects). The judgment of 5-10 test persons served as a taste criterion. In the scale, a digestible kefir, comparable to a fresh yogurt, was rated as very good. The grade 1 (very good) was only given if the kefir had flavors that placed it well above the taste of other types of kefir. A still acceptable deviation in the digestibility of the ideal drink (too acidic, too much gas, slightly bitter aftertaste, etc.) was rated good (2), a strong deviation (kefir is not a pleasure to drink) was rated satisfactory (3). Inedible drinks and nausea-causing drinks would have received a sufficient (4) or insufficient (5) rating. Strains A and B come from the BioteCon Diagnostics strain collection. They are representative of many other yeast strains tested. Stability of the Ivophilized Saccharomyces BCD
- yeast Saccharomyces BCD For administration as a drug, it may be necessary to lyophilize the yeast Saccharomyces BCD.
- An experimental series is shown below, showing that the yeast is able to quantitatively survive the process of lyophilization. In particular, it is possible to lyophilize the strain starting from yeast milk.
- the subsequent fermentation conditions lead to high numbers of living organisms and enable a good survival rate in the subsequent processing.
- the amount of inoculation for pre-cultivation is 2.5%, ie the cultivation of a 5 m 3 fermenter results in the following setting: 3 I - 125 I -> 5 m 3 (main culture).
- the preculture stages are used as batch batches for 20 h at 30 ° C.
- the culture suspension (approx. 5 m 3 ) is first centrifuged and the culture supernatant is discarded. The biomass is then washed with drinking water. This washing step is made up with about 15% water based on the processed yeast suspension.
- the washed wet biomass is mixed with 20% protective medium (based on the dry substance).
- the protective medium is used in solid form as a powder in the case of yeast milk or as a 20% aqueous solution for resuspension of a firm yeast mass.
- the freezing process plays a crucial role in the vitality of the yeast cells. Slow freezing with a temperature change of 1 ° C / min leads to the optimal survival rate in the product. It is lyophilized at a temperature between -20 and -30 ° C. After lyophilization, the product contains at least 2 x 10 10 viable yeast cells and has a water content of ⁇ 5%. With a suspension of 1.5 g of Saccharomyces BCD in 5 ml of water, the eutectic point was determined to be -18 ° C.
- Saccharomyces BCD can be taken as a suspension for use as a probiotic.
- the yeast can be suspended in a drink. It is also possible to ferment the drink with the help of the yeast for a few days. Since alcohol is produced in this case, a probiotic beer or a probiotic wine can be produced in this way. In addition, the alcohol can be removed from these alcoholic beverages secondarily, so that probiotic alcohol-reduced or non-alcoholic fermented beverages are produced.
- Saccharomyces BCD can be lyophilized for administration as a drug. In this case, an uptake by humans or animals is possible in a high cell count. For example, after the administration of Saccharomyces BCD, a significant reduction in diarrhea was found in horses.
- Saccharomyces BCD as a component of baked foods, such as bread, cakes, pastries and the like. Extracts from this yeast can also be produced. In this case, individual components that are consumed can be enriched or isolated.
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2002220628A AU2002220628A1 (en) | 2000-11-24 | 2001-10-15 | Yeast strain for consumption |
EP01997547A EP1335992A2 (de) | 2000-11-24 | 2001-10-15 | Hefestamm für den verzehr |
US10/432,565 US20040076615A1 (en) | 2000-11-24 | 2001-10-15 | Novel yeast strain for consumption |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10058379.2 | 2000-11-24 | ||
DE10058379A DE10058379A1 (de) | 2000-11-24 | 2000-11-24 | Neuer Hefestamm für den Verzehr |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2002042442A2 true WO2002042442A2 (de) | 2002-05-30 |
WO2002042442A3 WO2002042442A3 (de) | 2002-09-19 |
Family
ID=7664520
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/011887 WO2002042442A2 (de) | 2000-11-24 | 2001-10-15 | Hefestamm für den verzehr |
Country Status (5)
Country | Link |
---|---|
US (1) | US20040076615A1 (de) |
EP (1) | EP1335992A2 (de) |
AU (1) | AU2002220628A1 (de) |
DE (1) | DE10058379A1 (de) |
WO (1) | WO2002042442A2 (de) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012159045A1 (en) | 2011-05-19 | 2012-11-22 | Baxter International Inc. | Detection of circulating adamts13-antibody complexes |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5677274A (en) * | 1993-02-12 | 1997-10-14 | The Government Of The United States As Represented By The Secretary Of The Department Of Health And Human Services | Anthrax toxin fusion proteins and related methods |
WO2000026370A2 (en) * | 1998-11-03 | 2000-05-11 | Mitokor | Adenine nucleotide translocator (ant) fusion proteins and uses thereof |
WO2001040499A2 (fr) * | 1999-12-01 | 2001-06-07 | Centre National De La Recherche Scientifique-Cnrs | Procedes de criblage cellulaire de composes aptes a moduler l'activite des complexes ubiquitine-ligases scf et leurs applications |
-
2000
- 2000-11-24 DE DE10058379A patent/DE10058379A1/de not_active Withdrawn
-
2001
- 2001-10-15 AU AU2002220628A patent/AU2002220628A1/en not_active Abandoned
- 2001-10-15 WO PCT/EP2001/011887 patent/WO2002042442A2/de not_active Application Discontinuation
- 2001-10-15 EP EP01997547A patent/EP1335992A2/de not_active Withdrawn
- 2001-10-15 US US10/432,565 patent/US20040076615A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5677274A (en) * | 1993-02-12 | 1997-10-14 | The Government Of The United States As Represented By The Secretary Of The Department Of Health And Human Services | Anthrax toxin fusion proteins and related methods |
WO2000026370A2 (en) * | 1998-11-03 | 2000-05-11 | Mitokor | Adenine nucleotide translocator (ant) fusion proteins and uses thereof |
WO2001040499A2 (fr) * | 1999-12-01 | 2001-06-07 | Centre National De La Recherche Scientifique-Cnrs | Procedes de criblage cellulaire de composes aptes a moduler l'activite des complexes ubiquitine-ligases scf et leurs applications |
Non-Patent Citations (2)
Title |
---|
HANNULA J ET AL: "Phenotypic and genotypic characterization of oral yeasts from Finland and the United States." ORAL MICROBIOLOGY AND IMMUNOLOGY, Bd. 12, Nr. 6, Dezember 1997 (1997-12), Seiten 358-365, XP001073708 ISSN: 0902-0055 * |
QUESADA M P ET AL: "Use of random amplified polymorphic DNA (RAPD-PCR) in the characterization of wine yeasts." AMERICAN JOURNAL OF ENOLOGY AND VITICULTURE, Bd. 46, Nr. 2, 1995, Seiten 204-208, XP001073911 ISSN: 0002-9254 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012159045A1 (en) | 2011-05-19 | 2012-11-22 | Baxter International Inc. | Detection of circulating adamts13-antibody complexes |
Also Published As
Publication number | Publication date |
---|---|
EP1335992A2 (de) | 2003-08-20 |
DE10058379A1 (de) | 2002-06-06 |
AU2002220628A1 (en) | 2002-06-03 |
WO2002042442A3 (de) | 2002-09-19 |
US20040076615A1 (en) | 2004-04-22 |
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