EP1910412A2 - Thaumatine obtenue a partir d'orge transgenique - Google Patents

Thaumatine obtenue a partir d'orge transgenique

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Publication number
EP1910412A2
EP1910412A2 EP06762900A EP06762900A EP1910412A2 EP 1910412 A2 EP1910412 A2 EP 1910412A2 EP 06762900 A EP06762900 A EP 06762900A EP 06762900 A EP06762900 A EP 06762900A EP 1910412 A2 EP1910412 A2 EP 1910412A2
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EP
European Patent Office
Prior art keywords
thaumatin
barley
transgenic plant
seq
malt
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German (de)
English (en)
Inventor
Rainer Stahl
Renate LÜHRS
Harald Dargatz
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SUMAR EHF
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Maltagen Forschung GmbH
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Publication of EP1910412A2 publication Critical patent/EP1910412A2/fr
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    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits

Definitions

  • the present invention relates to thaumatin from transgenic plants from the cultivated plant barley (Hordeum vulgare), extracts and products obtainable therefrom, in particular flour and malt products, together with food and feed for humans and animals.
  • Thaumatin (E959) is approved according to various directives in the EU (94/35 / EC, 95/2 / EC, 2003/115 / EC, 88/388 / EC), Switzerland, USA, Canada, Israel, Mexico, Japan , Korea, Singapore, Hong Kong, Australia, New Zealand and South Africa. Thaumatin is also classified as GRAS (Generally recognized as safe, FDA criterion). Thaumatin is also being tested by JEFCA (Joint Expert Committee on Food Additives of the FAO / WHO) and the European Safety Authority (EFSA) (Gibbs (supra), Faus I. (supra)).
  • JEFCA Joint Expert Committee on Food Additives of the FAO / WHO
  • EFSA European Safety Authority
  • Thaumatococcus daniellii six isoforms of the protein could be found, all of which are sweet (Thaumatin I, II, III, a, b, c).
  • Thaumatin I and II are the major forms, differing only in 4 amino acids, each consisting of 207 amino acids.
  • the three-dimensional structure of thaumatin I consists of three domains, with domains II and III characterized by disulfide-bridged regions.
  • thaumatin 8 forms disulfide bridges (from De Vos et al., Proc Natl Acad., USA (1985) 82: 1406-1409; Ogata et al., J. Mol. Biol.
  • thaumatin 2,000 to 3,000-fold higher (on a weight basis, on a molar basis than 100,000-fold) compared to sucrose, but the sweetness of thaumatin is perceived somewhat delayed.
  • thaumatin like other vegetable sweeteners, may have a licorice-like aftertaste (Faus I. (2000) Appl. Microbiol. Biotechnol. 53: 145-151).
  • thaumatin The heat stability of thaumatin depends on the matrix. Higginbotham reported that the sweetness of thaumatin I is retained after one hour of cooking and subsequent cooling (even at a pH below 5.5) (Higginbotham JD (1986) In: Nabors LO, Gelardi R. C, eds. Alternative Sweeteners, Marcel Dekker New York : 103-134). Thaumatin is definitely stable under pasteurizing conditions (Gibbs BF et al., Nutrion Research (1996), 16 (9): 1619-1630.) In purified form, the protein aggregates and loses its sweetening power at a temperature of 70 ° C and pH 7.0 (Kaneko R. and Kitabatake N., J. Agric. Food Chem. (1999) 47 (12): 4950-5). Thaumatin is stable in freeze-dried form, soluble in water and alcohol and therefore suitable for the production of beverages.
  • thaumatin In addition to sweetening power, thaumatin also has taste enhancing properties (Gibbs et al., Nutrition Research (1996) 16 (9): 1619-1630; It is therefore excellently suited to mask the sweetness of some sweeteners and to improve the mouthfeel. Thaumatin is added for the flavor rounding of sugar alcohols and sweetener mixtures (EP0681789, EP0847242, US6562392, US6555146, US Pat.
  • thaumatin is also used for pure taste enhancement of sauces and meat (Gibbs et al. (Supra), US6420527).
  • the shrub Thaumatococcus danielli from whose fruits thaumatin is extracted, occurs in the rainforests of West Africa.
  • the cultivation of this shrub is very difficult because the plants have to be shaded and pollinated by certain insects exclusively. In field cultivation, therefore, only a quarter of the plants produce fruits (Faus (supra)). Accordingly, attempts are repeatedly made to cultivate the tropical plant Thaumatococcus daniellii more efficiently.
  • thaumatin mainly fruits are used, which are collected in the forests.
  • the majority of the thaumatin 20% to 40% of the dry weight, is enriched in the seed coat of the fruits.
  • the creamy white seed coat is located on the apex of the seed and is surrounded by a transparent gel. The mechanical separation of this tissue from the seed is relatively expensive.
  • Thaumatin can be obtained from the fruits by a simple aqueous extraction (Van der WeI and Loewe, Eur. J. Biochem. (1972) 31: 221-5). By ultrafiltration or ion exchange chromatography can be carried out a further purification of thaumatin (US 4221704). The cleaning is made more difficult by the gel that surrounds the seed coat, since it swells up very much with aqueous extraction.
  • Various methods for facilitating the purification of thaumatin from Thaumatococcus danielli are known. By lyophilizing the fruit, e.g.
  • the seed coat is easier to remove, and with the aid of salts or acids swelling of the gel is prevented and thaumatin extracted more efficiently (US 4011206, US 4221704, EP0003911).
  • the conventional extraction and enrichment and isolation of thaumatin is therefore associated with high costs.
  • Flavoring of sweetener mixtures (eg in NATREEN: WO0106872, EP1198181) but also excellent for the sweetening of drinks (soft drinks, alcohol drinks, coffee and cocoa drinks), dairy products (yoghurts, desserts), sweets, chocolates, chewing gum and chewing tablets, etc
  • Thaumatin is also used as a flavoring in feed and as a flavor enhancer in sauces and diet (mainly "pet food") (Etheridge K. (1994) In: Witty: M., Higginbotham JD, eds. Thaumatin, Boca Raton: CRC Press, pp. 47-59, Trelie & Lyle: GB2185674, US6251464, EP0929233 , WO9803082, US59324438, EP0684312).
  • Fillers are used, which are sweetened with the help of sweeteners.
  • the sugar alcohols may alienate the consistency and taste of certain products (e.g., baked goods).
  • sugar alcohols contain calories, so that they are only partially suitable for the production of low-calorie products.
  • thaumatin As an endogenous product in food and feed for humans and animals.
  • Table 1 Overview of biotechnical processes for the production of thaumatin in various organisms
  • Monellin is also a protein from a tropical plant (Dioscoreophyllum comminsii), which gives an intense sweet taste.
  • a sweeter taste in the fruits can be achieved in tomato by the transformation with the monellin gene under the control of the fruit-specific E8 promoter.
  • a sweeter taste could be detected in all tissues of lettuce transformed with the monellin gene under the control of the constitutive CaMV35S promoter.
  • Potato Neither for a taste improvement, nor for the economic production of thaumatin, the potato is suitable. Due to the high sweetening power of thaumatin, the sweetness can be tasted at a concentration of 10 ⁇ 8 M, which corresponds to approx. 2 ⁇ g / 1. The yield in the potato is between 2 ⁇ g / kg and 2 mg / kg (Witty, Methods Enzymol. (1992) 216: 441-7).
  • tobacco receives numerous toxic ingredients that make obsolete the extraction of the sweetener for food and feed production.
  • transgenic plants from the cultivated plant barley (Hordeum vulgare) is selected as a bioreactor (hereinafter also: “transgenic plant of barley”).
  • brazzein Only the transformation of the gene for the sweet protein brazzein from the plant Pentadiplandra brazzeana in monocot maize is known. In corn, the yields of brazzein are about 4% of the soluble protein, which corresponds to about 1 g / kg Kornmaterial (Faus (2000) Appl. Microbiol Biotechnol 53: 145-15, Lamphear et al (2005) Plant Biotechnol Journal 3 (1): 103-114).
  • thaumatin can be expressed in the transgenic plants according to the invention from the crop barley in high yields while maintaining the sweetening power and sufficient stability (so-called "active thaumatin”) .
  • the yields obtained are more than 2 g / kg, 3 g / kg or more (grain material) of active thaumatin, which corresponds to a sweetening power of about 3-9 kg of sucrose.
  • the sweetness and stability of the endogenously expressed thaumatin can be obtained according to the invention by the correct folding of the thaumatin - ie the active thaumatin - in the barley transgenic plant according to the invention.
  • the invention therefore relates to a transgenic plant according to the invention from the cultivated plant barley (Hordeum vulgare) containing more than 2 g of thaumatin or active thaumatin or a protein having an activity for thaumatin in 1 kg of grain material. Therefore, the invention also relates, on the one hand, to suitable polynucleotides or nucleic acids or other analogs (eg T-DNA) which code for thaumatin or for a protein with an activity for thaumatin and which are suitable according to the invention for transforming barley by means of a vector to obtain such transgenic plants Barley. Suitable vectors are described by way of example in: Hellen and Mullineaux (2000) A guide to Agrobacterium binary TI vectors.
  • the invention therefore likewise relates to a transgenic plant according to the invention from the cultivated plant barley (Hordeum vulgare) having at least one polynucleotide which is stably integrated into the genome after its transformation and coding for thaumatin, in particular by means of promoters and, if appropriate, further auxiliary sequences (enhancers, terminators, signal sequences, etc.). for the controlled expression of thaumatin.
  • cultivated plant barley Hordeum vulgare
  • auxiliary sequences enhancers, terminators, signal sequences, etc.
  • thaumatin is specifically targeted into the endoplasmic reticulum (ER) of the endosperm of the grain material or seed (targeting), in particular for the purpose of achieving the correct folding of the thaumatin.
  • ER endoplasmic reticulum
  • protein disulfide isomerases play an essential role in the correct expression of the three-dimensional structure and thus the stability of the proteins.
  • the protein disulfide isomerases catalyze the rearrangement of the disulfide bridges upon protein folding (Wilkinson and Gilbert, Biochim, Biophys, Acta (2004) 1699: 35-44, Sitia and Molteni Sei, STKE (2004) 239: 27).
  • the significance of these enzymes for the stability and thus accumulation of thaumatin is demonstrated by the investigations in Aspergillus awamori. In this non-plant organism, e.g. by a defined overexpression of protein disulfide isomerase (PDI) a fivefold higher yield of thaumatin can be achieved (Moralejo et al., Mol. Genet. Genomics (2001) 266: 246-253).
  • PDI protein disulfide isomerase
  • Disulfide bridges and thus the correct, biologically active protein conformation, can not in principle form under the reducing conditions maintained inside the cell. In eukaryotic cells, therefore, the folding of the proteins and the formation of the disulfide bridge pattern takes place under partially oxidizing conditions in certain compartments, for example in the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • the protein disulfide Isomerases (PDI) play an essential role in the formation of the complex three-dimensional structures of the proteins (Boston et al., Plant Mol. Biol. (1996) 32: 191-222; Wilkinson and Gilbert, Biochim. Biophys. Acta (2004) 1699: 35-44).
  • polynucleotides according to the invention coding for thaumatin are transformed under the control of an endosperm-specific promoter.
  • endosperm-specific promoters are not, but are not limited to, promoters of storage proteins from barley, e.g. the hordein B, C, D, G promoters (hordein D, compare SEQ ID NO: 1, Förde et al.,
  • an endosperm-specific promoter is preferred, and protein disulfide isomerases are also present in the present invention to allow for the correct folding of thaumatin. Especially this combination allows the desired high yields in the endosperm of the transgenic plants according to the invention, particularly preferably barley.
  • the invention further relates to the use of signal sequences for the targeted targeting of thaumatin into the endoplasmic reticulum
  • ER ER of the endosperm cells and the correct accumulation in "protein bodies" of these endosperm cells, for which signal sequences of the storage proteins of the barley, eg Hordein-B, Hordein-C, Hordein-D, Hordein-G can preferably be used (Förde et al Nucleic Acids Res. (1985) 25: 7327-39;
  • the thaumatin-specific N- and C-terminal signal sequences also lead to targeted targeting into the ER and to the correct accumulation in the "protein bodies".
  • the signal sequences are processed correctly by barley-specific proteases.
  • Such signal sequences are, for example, not conclusive SEQ ID NO: 16-17 and their expression products SEQ ID NO: 18-19.
  • the invention relates to a polynucleotide according to the invention coding for thaumatin or for a protein with activity for thaumatin, which is the subject of a construct or an expression cassette containing promoters and optionally further auxiliary sequences for the controlled expression of thaumatin, wherein the promoter is preferably an endosperm-specific promoter (supra) as well as said signal sequences (supra) for targeting into the endoplasmic reticulum (ER) of the endosperm cells.
  • the promoter is preferably an endosperm-specific promoter (supra) as well as said signal sequences (supra) for targeting into the endoplasmic reticulum (ER) of the endosperm cells.
  • the invention also relates to a vector comprising such a construct or expression cassette according to the invention and a barley transgenic plant according to the invention which are produced by means of such a vector.
  • a construct or expression cassette are SEQ ID NO: 20-21.
  • transgenic plants are obtained from the cultivated plant barley (Hordeum vulgare), plant cells or protoplasts having at least one polynucleotide according to the invention which is stably integrated into the genome after its transformation. Therefore, the invention also relates to seed obtained from the transformed plants or transgenic plants.
  • the invention relates to grain material or seeds of a transgenic plant from the cultivated plant barley (Hordeum vulgare), which contains more than 2 g or more than 3 g of thaumatin in 1 kg of grain material.
  • thaumatin is a 207 amino acid protein sequence with 8 disulfide bridges, which occurs natively in isoforms I, II.
  • Known sequences are in particular SEQ ID NO: 22-24 as well as the coding sequences SEQ ID NO: 25-27.
  • proteins having an activity for thaumatin are in particular SEQ ID NO: 22-24 as well as the coding sequences SEQ ID NO: 25-27.
  • the native amino acid sequence of thaumatin I has been prepared in correct folding in a heterologous organism, or in a plant.
  • thaumatin II has preferably been heterologously expressed in the various non-monocotyledonous organisms (see Table 1).
  • Thaumatin I has hitherto been produced recombinantly only in the form of mutated variants (US Pat. No. 5,222,164, WO9005775, EP0540651, Lee et al, Biochemistry (1988) 27: 5101-5107).
  • thaumatin I has both higher sweetness (Masuda et al., Biotechnol. Bioeng. (2004) 85: 761-9) and higher thermostability (Higginbotham JD (1986) In: Nabors LO, Gelardi R. C, eds. Alternative Sweeteners, Marcel Dekker New York: 103-134).
  • the invention relates to a novel amino acid sequence according to SEQ ID NO: 28, wherein thaumatin I with the specific N and C terminus from a transgenic plant according to the invention, in particular barley, can be isolated.
  • the sequence presented is an active thaumatin.
  • the invention also relates to polynucleotides encoding the amino acid sequence according to SEQ ID NO: 28, in particular according to the DNA sequence SEQ ID NO: 29.
  • transgenic plant refers to plants produced by recombinant genetic engineering and / or microbiological processes and were not prepared by conventional breeding methods and contain at least one polynucleotide of the invention encoding thaumatin. Methods for producing transgenic plants have been described (Tingay S., McElroy D., Kalla R., Fieg S., Wang M., Thorton S. and Brettel R. (1997): Agrobacterium tumefaciens -mediated barley transformation.) Plant Journal 11, Wan Y. and Lemaux P. (1994): Generation of a large number of independently transformed fertile barley plants., Plant Physiol. 104; 37-48, Stahl R., H. Horvath, J.
  • T-DNA integration a mode of illegitimate recombination in plants; Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell M., Van Montagu M., Leemans J.; "Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants "; Nucleic Acids Res. 13: 4777-4788 (1985)).
  • the transgenic plants according to the invention from the cultivated plant barley can easily be malted to malt. By roasting, malt can obtain additional flavor components. Malt is already a component of various food and feed. Malt can take over the role of the filler for the use of thaumatin and is therefore excellent as a food or
  • Feed containing endogenous thaumatin suitable.
  • the invention also relates to a flour product or malt product, further extracts and powders, or a food and feed for humans and animals containing grain material from the barley transgenic plant according to the invention.
  • the invention further relates to food, low-calorie or dietetic foods consisting of beverages, beer, bread, coffee and cocoa drinks, confectionery, ice cream, ready meals, milk and yoghurt products, cereal products, sweets, chocolate, chewing gum and chewing tablets, biscuits, cakes, Sauces, dressing containing an extract or meal, malt product, extracts thereof, from a transgenic plant according to the invention from the cultivated plant barley (Hordeum vulgare) or from its grain material.
  • the malt powder from transgenic barley combines the properties of thaumatin (sweetening and flavor enhancement) with the pleasant taste of malt.
  • the powder is excellently suitable as an additive to both foods, e.g. Coffee and cocoa drinks, confectionery, ices, prepared meals, milk and yoghurt products,
  • thaumatin has a very high heat stability in malt, it is also very well suited as an additive for pastry and cake production. Due to the high sweetening power of thaumatin barley no further sweetener has to be added to the products.
  • the malt powder from the thaumatin barley can therefore be used excellently for the production of low-calorie and dietetic products.
  • the preparation of these various products is known to the person skilled in the art. For example, baking mixes of biscuits are added with up to 10% malt powder (100 g malt / kg baking mix). The addition of 100 g of thaumatin malt corresponds in terms of sweetening power to an addition of 300 g of sucrose / kg baking mixture.
  • malt-containing beverage powders e.g., NESTOMALT, EP1068807
  • malt-containing beverage powders e.g., NESTOMALT, EP1068807
  • the addition of sugar or sugar substitutes can preferably be completely dispensed with.
  • the thaumatin in the malt not only gives the sweetness but also a pleasant mouthfeel, which can not be mediated by other sweeteners or sugar alcohols.
  • Flour and malt from the transgenic barley according to the invention can be added directly to feed mixtures.
  • One ton of piglet feed is e.g. sweetened with 20-40 kg of lactose (e.g., ANILAC, ANIFIT).
  • lactose e.g., ANILAC, ANIFIT
  • lactose has only a 0.6-fold sweetening power.
  • a corresponding sweetening can be achieved by adding 1.5-2.5 kg of the inventive thaumatin malt, or flour per ton of piglet feed.
  • Flour and malt from the transgenic plants according to the invention can be added in particular as flavor enhancers in animal feed.
  • the inventive malt from transgenic barley containing thaumatin is suitable for the production of a Malt extract, malt syrup or instant malt powder.
  • a malt extract is suitable to guarantee a 100% solubility.
  • the strength of the malty taste can be varied by an extraction method (eg Owades JL: Preparation of a non-alcoholic malt beverage, US Pat. No. 5,151,557).
  • Extracts from the malt of thaumatin barley also provide an ideal basis for the preparation of savory, low-calorie or dietary drinks.
  • a watery extract combines the low-calorie sweetening and flavor-enhancing properties of thaumatin with the flavor of the malt extract.
  • a syrup or extract powder or by freeze-drying also an instant powder can be prepared by known methods under vacuum.
  • the obtained malt containing thaumatin is ideal for the production of tasty diet beers. Due to the sweetening power of thaumatin, the addition of sugar (up to 50 g / l) is not necessary in the production of malt beer. The taste of light beers, which have no more sweetness due to the complete fermentation of maltose, can be substantially improved by adding the thaumatin malt. Thaumatin malt thus enables the production of tasty, low-calorie beers according to the purity law.
  • Malt extract is not only the basis for the production of beer but is also used for the production of various "soft drinks” (eg US4001436, ÜS5120557, US5415885, US6534109). Since the maltose in the malt extract compared to sucrose, glucose or fructose has only a low sweetening power, additional sucrose, glucose, fructose or other sweeteners must be added to sweeten these beverages (eg US5120557, US5415885).
  • the sweetness of the malt extract is increased by the enzymatic conversion of maltose to glucose.
  • other sweeteners must be added to the beverage (e.g., US4001436, ÜS6534109).
  • the malt extract containing thaumatin from the transgenic barley according to the invention in contrast, has the intense sweetening power of thaumatin. Only small amounts of the syrup or instant malt powder must be added to the drinks to obtain the desired sweetness. An addition of other sweeteners is not necessary, so the extract, the
  • Syrup, powder or instant powder are ideal for making low-calorie, dietetic drinks.
  • the invention further relates to a flavor enhancer consisting of a food or feed according to the invention, which is added to any food or feed.
  • Example 1 Construction of Hordein Signal / Thaumatin Construct / Expression Cassette / Vector: The following work was performed according to standard methods as described in Manniatis et al. (supra) are described. The sequence for the mature thaumatin gene was amplified by PCR using extracted DNA (Maniatis et al.) From leaves of Thaumatococcus daniellii. For this, primers R1004 (SEQ ID No. 30) at the 5 'end and R1005 (SEQ ID No. 31) at the 3' end of the sequence for the mature thaumatin gene were used.
  • Plasmid preparation this plasmid (RS 607, Fig. Id) was transformed into AGL 1 cells, which in turn were used to transform immature barley embryos.
  • the barley transformation was carried out according to Tingay et al (supra).
  • Example 2 Construction of Thaumatin Signal / Thaumatin / C-terminal Thaumatin Signal Construct / Expression Cassette / Vector: The following work was performed according to standard methods as described in Manniatis et al. (supra) are described. Using PCR, the sequence for the thaumatin gene, including the sequences for the N- and C-terminal signal peptides, was amplified using extracted DNA (Maniatis et al., Supra) from leaves of Thaumatococcus daniellii. For this purpose, the
  • Example 3 Preparation and Analysis of Transgenic Barley Lines Per construct (607 with expression cassette (SEQ ID No. 20), 608 with expression cassette (SEQ ID No. 21), see Examples 1 and 2), in each case 50 independent, transgenic barley lines were regenerated and in the greenhouse cultivated to seed maturity. To select the lines with the highest yield Thaumatin was carried out single-grain analyzes of mature seeds.
  • Mature grains of the obtained transgenic barley lines were finely ground individually.
  • the resulting flour was suspended in 80 mM citrate buffer (pH 3.0) and incubated at 37 ° C. for 30 minutes.
  • the proteins thus extracted were obtained after centrifugation in the clear supernatant.
  • the extracts obtained were neutralized, precipitated with acetone after determination of the protein concentration and taken up in loading buffer and separated by SDS-PAGE.
  • Per line 3-4 grains were analyzed.
  • the first investigations on the expression of thaumatin I in the barley lines were carried out by means of Western Blot analyzes (FIG. 2a). For this purpose, the thaumatin-specific antibody from Abcam was used.
  • the purification of pure thaumatin from the transgenic barley according to the invention can largely follow the protocol of Töre & Lyle (US Pat. No. 4,011,206, US Pat. No. 4,222,004).
  • Fine flour of the grain material of the transgenic barley according to the invention is treated with water (acidified by the addition of phosphoric, citric, formic, ascorbic and the like acids, which in the Food range, to pH 2.7-3.0) in the ratio 1: 4 mixed.
  • the proteins thus extracted were obtained after centrifugation
  • the extracts obtained were desalted by means of a Sephadex G25 M gel filtration and buffered in 50 mM BICINE, pH 8, 8.
  • the thaumatin-containing protein mixture was then subjected to cation exchange chromatography, and the extracts were assayed for SP-Sepharose Fast Flow column applied ( Figure 2b) and a pre-elution with 7-8 column volumes
  • Example 5 Production of malting powder: The methods and the apparatuses and devices used therefor for the production of malt and mash are the subject of the study of the brewing industry and the person skilled in the art known. Reference is made in particular to "The technology of malting” by Schuster / Weinfurtner / Narziss, 6th ed., 1976, and to "demolition of the beer brewery” by Narziss, 4th ed., 1980. For a better understanding of the present invention, the malting of the prior art will be described in more detail below.
  • barley is processed into malt by using the process steps of weeding, germination, drying and hardening in the malting plant.
  • the grains absorb up to 48% of water.
  • the germinating grains are cooled to about 15 ° C tempered air with constant ventilation.
  • the malt grains have a water content of about
  • Germination is stopped by drying on the kiln. First, the water is removed at temperatures ⁇ 60 ° C to 12% and then sealed at about 85 ° C to 4.5% water content.
  • the matted malt with a water content of about 4.5% is storable.
  • One kilogram of malt contains 1-3 g, especially 2 g or more of active thaumatin, i. the sweetness of 1 kg of malt corresponds to about 3-9 kg of sucrose.
  • the last step in the malting business is to clean the matted malt.
  • the malt is either peeled or produced in a special grinding a fine low-spicy product, which is 60% soluble.
  • the malt powder from the transgenic barley according to the invention contains 1-3 kg, in particular 2 kg of thaumatin per ton Malt powder, ie one kilogram has a sweetness of 3-9 kg of sucrose.
  • the malt powder is ideal as an additive for the production of diet products. It can be used for the preparation of baking mixes, coffee, cocoa drinks, dairy and
  • chocolate products, sweets, etc. are used directly.
  • the desired sweetness can only be achieved by adding the malt powder, additional sweeteners are not necessary.
  • An extract is prepared by the mixture of malt and water, for example in the weight ratio 1: 5.
  • the water is brought to a pH between 2.7 and 3.0 by the addition of acids approved for food (citric acid, formic acid, phosphorous acid, etc.).
  • FIG. 1a 607-11, 607-12, 607-28, 607-29, various transgenic
  • FIG. 2b SDS-PAGE of various independent transgenic TO lines transformed with the construct 607 or 608, respectively.
  • Th extract from grains of a non-transgenic barley line mixed with 4.5 ⁇ g thaumatin I + II (Sigma)
  • FIG. 3a is a diagrammatic representation of FIG. 3a.

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Abstract

L'invention concerne des plantes transgéniques d'orge renfermant plus de 2 g de thaumatine par kilogramme de matière en grains, les extraits et les produits obtenus à partir de ces plantes, en particulier, les produits farinacés et les produits de malt, y compris les produits pour l'alimentation humaine et pour la nourriture animale.
EP06762900A 2005-07-30 2006-07-28 Thaumatine obtenue a partir d'orge transgenique Withdrawn EP1910412A2 (fr)

Applications Claiming Priority (2)

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DE102005035888A DE102005035888A1 (de) 2005-07-30 2005-07-30 Thaumatin aus transgenen monokotylen Pflanzen
PCT/EP2006/007525 WO2007014726A2 (fr) 2005-07-30 2006-07-28 Thaumatine obtenue a partir d'orge transgenique

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EP1910412A2 true EP1910412A2 (fr) 2008-04-16

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US (1) US20090031458A1 (fr)
EP (1) EP1910412A2 (fr)
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WO (1) WO2007014726A2 (fr)

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JP2010506129A (ja) * 2006-10-03 2010-02-25 ワイス エルエルシー 凍結乾燥法および装置
US10364880B2 (en) * 2017-01-05 2019-07-30 United Technologies Corporation Oil quieting direction control baffle
EP3576550A4 (fr) * 2017-02-01 2020-09-09 Manna Nutritional Group LLC Farine à haute teneur en fibres, haute teneur en protéines, faible teneur en glucides, liquide sucré, édulcorants, céréales et leurs procédés de production
IL257269A (en) * 2018-01-31 2018-04-09 Olender Tsviya Signaling is directed to the endoplasmic reticulum
CZ308160B6 (cs) * 2018-12-17 2020-01-29 Agra Group A.S. Sladidlo přírodního původu chuťovým profilem imitující cukr s ovocnými tóny s až 20násobnou sladivostí oproti cukru

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WO2005026366A1 (fr) * 2003-09-11 2005-03-24 Maltagen Forschung Gmbh Nouveau promoteur de plante specifique de l'endosperme pour plantes cultivees

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US5234834A (en) * 1987-06-19 1993-08-10 The Regents Of The University Of California Constructs for expression of monellin in plant cells
CA1341467C (fr) * 1988-07-29 2004-12-07 John C. Rogers Production de polypeptides d'interet commercial a l'aide d'endosperme genetiquement modifie
EP0428572A1 (fr) * 1988-07-29 1991-05-29 Washington University School Of Medicine Production de polypeptides pour usage commercial au moyen d'un tissu d'endosperme transforme genetiquement
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MORALEJO FRANCISCO-JOSE ET AL: "Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 65, no. 3, 1 March 1999 (1999-03-01), pages 1168 - 1174, XP002205864, ISSN: 0099-2240 *
WEBER UND KRISCHKE: "Isolierung von Thaumatin und HSA aus transgener Gerste", ZWISCHENBERICHT IGB, 1 March 2009 (2009-03-01), pages 1,2,12 - 13 *

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WO2007014726A3 (fr) 2007-06-07
DE102005035888A1 (de) 2007-02-01
US20090031458A1 (en) 2009-01-29
WO2007014726A2 (fr) 2007-02-08

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