Patent Application
20080096260
The present invention relates to a method of preparing a heat-treated product with a low water content from raw material comprising carbohydrate, protein and water. It also relates to an asparaginase for use in the method
E. Tabeke et al. (J. AgriC. Food Chem., 2002, 50, 4998-5006) reported that acrylamide is formed during heating of starch-rich foods to high temperatures. The acrylamide formation has been ascribed to the Maillard reaction (D. S. Mottram et al., R. H. Stadtler et al., Nature, 419, 3 Oct. 2002, 448-449).
WO 00/56762 discloses expressed sequence tags (EST) from A. oryzae.
Kim, K.-W.; Kamerud, J. Q.; Livingston, D. M.; Roon, R. J., (1988) Asparaginase II of Saccharomyces cerevisiae. Characterization of the ASP3 gene. J. Biol. Chem. 263:11948, discloses the peptide sequence of an extra-cellular asparaginase
According to the invention, the formation of acrylamide during heat treatment of raw material comprising carbohydrate, protein and water is reduced by treating the raw material with an enzyme before the heat treatment. Accordingly, the invention provides a method of preparing a heat-treated product, comprising the sequential steps of:
a) providing a raw material which comprises carbohydrate, protein and water
b) treating the raw material with an enzyme, and
c) heat treating to reach a final water content below 35% by weight.
The enzyme is capable of reacting on asparagine or glutamine (optionally substituted) as a substrate or is a laccase or a peroxidase.
The invention also provides an asparaginase for use in the process and a polynucleotide encoding the asparaginase.
Raw Material and Enzyme Treatment
The raw material comprises carbohydrate, protein and water, typically in amounts of 10-90% or 20-50% carbohydrate of the total weight. The carbohydrate may consist mainly of starch, and it may include reducing sugars such as glucose, e.g. added as glucose syrup, honey or dry dextrose. The protein may include free amino acids such as asparagine and glutamine (optionally substituted).
The raw material may include tubers, potatoes, grains, oats, barley, corn (maize), wheat, nuts, fruits, dried fruit, bananas, sesame, rye and/or rice.
The raw material may be in the form of a dough comprising finely divided ingredients (e.g. flour) with water, The enzyme treatment may be done by mixing (kneading) the enzyme into the dough and optionally holding to let the enzyme act. The enzyme may be added in the form of an aqueous solution, a powder, a granulate or agglomerated powder. The dough may be formed into desired shapes, e.g. by sheeting, cutting and/or extrusion.
The raw material may also be in the form of intact vegetable pieces, e.g. slices or other pieces of potato, fruit or bananas, whole nuts, whole grains etc. The enzyme treatment may comprise immersing the vegetable pieces in an aqueous enzyme solution and optionally applying vacuum infusion. The intact pieces may optionally be blanched by immersion in hot water, e.g. at 70-100° C., either before or after the enzyme treatment.
The raw material may be grain intended for malting. e.g. malting barley or wheat. The enzyme treatment of the grain may be done before, during or after the malting (germination).
The raw material before heat treatment typically has a water content of 10-90% by weight and is typically weakly acidic. e.g. having a pH of 5-7.
Heat Treatment
The process of the invention involves a heat treatment at high temperature to reach a final water content (moisture content) in the product below 35% by weight, typically 1-20%, 1-10% or 2-5%. During the heat treatment, the temperature at the surface of the product may reach 110-220° C., e.g. 110-170° C. or 120-160° C.
The heat treatment may involve, frying, particularly deep frying in tri- and/or diglycerides (animal or vegetable oil or fat, optionally hydrogenated), e.g. at temperatures of 150-180° C. The heat treatment may also involve baking in hot air, e.g. at 160-310° C. or 200-250° C. for 2-10 minutes, or hot-plate heating. Further, the heat treatment may involve kilning of green malt.
Heat-Treated Product
The process of the invention may be used to produce a heat-treated product with low water content from raw material containing carbohydrate and protein, typically starchy food products fried or baked at high temperatures. The heat-treated product may be consumed directly as an edible product or may be used as an ingredient for further processing to prepare an edible or potable product.
Examples of products to be consumed directly are potato products, potato chips (crisps), French fries, hash browns, roast potatoes, breakfast cereals, crisp bread, muesli, biscuits, crackers, snack products, tortilla chips, roasted nuts, rice crackers (Japanese “senbei”), wafers, waffles, hot cakes, and pancakes.
Malt (e.g. caramelized malt or so-called chocolate malt) is generally further processed by mashing and brewing to make beer.
Enzyme Capable of Reacting with Asparagine or Glutamine (Optionally Substituted) as a Substrate
The enzyme may be capable of reacting with asparagine or glutamine which is optionally glycosylated or substituted with a peptide at the alpha-amino and/or the carboxyl position. The enzyme may be an asparaginase, a glutaminase, an L-amino acid oxidase, a glycosylasparaginase, a glycoamidase or a peptidoglutaminase.
The glutaminase (EC 3.5.1.2) may be derived from Escherichia coli. The L-amino acid oxidase (EC 1.4.3.2) capable of reacting with asparagine or glutamine (optionally glycosylated) as a substrate may be derived from Trichoderma harzianum (WO 94/25574). The glycosylasparaginase (EC 3.5.1.26, aspartylglucosaminidase, N4-(N-acetyl-beta-glucosaminyl)-Lasparagine amidase) may be derived from Flavobacterium meningoseptiutm. The glycoamidase (peptide N-glycosidase, EC 3.5.1.52) may be derived from Flavobacterium meningosepticum. The peptidoglutaminase may be peptidoglutaminase I or II (EC 3.5.1.43, EC 3.5.1.44).
The enzyme is used in an amount which is effective to reduce the amount of acrylamide in the final product. The amount may be in the range 0.1-100 mg enzyme protein per kg dry matter, particularly 1-10 mg/kg. Asparaginase may be added in an amount of 10-100 units per kg dry matter where one unit will liberate 1 micromole of ammonia from L-asparagine per min at pH 8.6 at 37° C.
Asparaginase
The asparaginase (EC 3.5.1.1) may be derived from Saccharomyces cerevisiae, Candia utilis, Escherichia coli, Aspergillus oryzae, Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, or Penicillium citrinum. It may have the amino acid sequence shown in SEQ ID NO: 2 (optionally truncated to residues 27-378, 30-378, 75-378 or 80-378), 4, 6, 8, 10, 12 or 13 or a sequence which is at least 90% (particularly at least 95%) identical to one of these. It may be produced by use of the genetic information in SEQ ID NO: 1, 3, 5, 7, 9 or 11, e.g., as described in an example.
Whitehead Institute, MIT Center for Genome Research, Fungal Genome Initiative has published A. nidulans release 1 and F. graminearum release 1 on the Internet at http://www-genome.wi.mit.edu/ftp/distribution/annotation/ under the Aspergillus Sequencing Project and the Fusarium graminearm Sequencing Project. Preliminary sequence data for Aspergillus fumigatus was published on The Institute for Genomic Research website at http://www-genome.wi.mit.edu/ftp/distribution/annotation/.
The inventors inserted the gene encoding the asparaginase from A. oryzae into E. coli and deposited the clone under the terms of the Budapest Treaty with the DSMZ—Deutsche Sammlung von Microorganismen and Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig. The deposit number was DSM 15960, deposited on 6 Oct. 2003.
Alignment and Identity
The enzyme and the nucleotide sequence of the invention may have homologies to the disclosed sequences of at least 90% or at least 95%, e.g. at least 98%.
For purposes of the present invention, alignments of sequences and calculation of identity scores were done using a Needleman-Wunsch alignment (i.e. global alignment), useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is −12 for proteins and −16 for DNA, while the penalty for additional residues in a gap is −2 for proteins and −4 for DNA. Alignment is from the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology, 183:63-98).
Laccase or Peroxidase
The laccase (EC 1.10.3.2) may be of plant or microbial origin, e.g. from bacteria or fungi (including filamentous fungi and yeasts). Examples include laccase from Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinus, e.g., C. cinereus, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radita, or Coriolus, e.g., C. hirsutus.
The peroxidase (EC 1.11.1.7) may be from plants (e.g. horseradish or soybean peroxidase) or microorganisms such as fungi or bacteria, e.g. Coprinus, in particular Coprinus cinereus f. microsporus (IFO 8371), or Coprinus macrorhizus, Pseudomonas, e.g. P. fluorescens (NRRL B-11), Streptoverticillium, e.g. S. verticillium ssp. verticillium (IFO 13864), Streptomyces, e.g. S. thermoviolaceus (CBS 278.66), Streptomyces, e.g. S. viridosporus (ATCC 39115). S. badius (ATCC 39117), S. phaeochromogenes (NRRL B-3559), Pseudomonas, e.g. P. pyrrocinia (ATCC 15958), Fusarium, e.g. F. oxysporum (DSM 2672) and Bacillus, e.g. B. stearothermophilus (ATCC 12978).
Oxidoreductase Capable of Reacting with a Reducing Sugar as a Substrate
The method of the invention may comprise treating the raw material with an oxidoreductase capable of reacting with a reducing sugar as a substrate. The oxidoreductase may be an oxidase or dehydrogenase capable of reacting with a reducing sugar as a substrate such as glucose and maltose.
The oxidase may be a glucose oxidase, a pyranose oxidase, a hexose oxidase, a galactose oxidase (EC 1.1.3.9) or a carbohydrate oxidase which has a higher activity on maltose than on glucose. The glucose oxidase (EC 1.1.3.4) may be derived from Aspergillus niger e.g. having the amino acid sequence described in U.S. Pat. No. 5,094,951. The hexose oxidase (EC 1.1.3.5) may be derived from algal species such as Iridophycus flaccidum, Chondrus crispus and Euthora cristata. The pyranose oxidase may be derived from Basidiomycete fungi, Peniophora gigantean, Aphyllophorales, Phanerochaete chrysosporium, Polyporus pinsitus, Bierkandera adusta or Phlebiopsis gigantean. The carbohydrate oxidase which has a higher activity on maltose than on glucose may be derived from Microdochium or Acremonium, e.g. from M. nivale (U.S. Pat. No. 6,165,761), A. strictum, A. fusidioides or A. potronii.
The dehydrogenase may be glucose dehydrogenase (EC 1.1.1.47, EC 1.1.99.10), galactose dehydrogenase (EC 1.1.1.48), D-aldohexose dehydrogenase (EC 1.1.1.118, EC 1.1.1.119), cellobiose dehydrogenase (EC 1.1.5.1, e.g. from Humicola insolens), fructose dehydrogenase (EC 1.1.99.11, EC 1.1.1.124, EC 1.1.99.11), aldehyde dehydrogenase (EC 1.2.1.3, EC 1.2.1.4, EC 1.2.1.5). Another example is glucose-fructose oxidoreductase (EC 11.1.99.28).
The oxidoreductase is used in an amount which is effective to reduce the amount of acrylamide in the final product. For glucose oxidase, the amount may be in the range 50-20,000 (e.g. 100-10,000 or 1,000-5,000) GODU/kg dry matter in the raw material. One GODU is the amount of enzyme which forms 1 μmol of hydrogen peroxide per minute at 30° C., pH 5.6 (acetate buffer) with glucose 16.2 g/l (90 mM) as substrate using 20 min. incubation time. For other enzymes, the dosage may be found similarly by analyzing with the appropriate substrate.
DAP2C-1
11 g MgSO4.7H2O
1 g KH2PO4
2 g Citric acid, monohydrate
30 g maltodextrin
6 g K3PO4.3H2O
0.5 g yeast extract
0.5 ml trace metals solution
1 ml Pluronic PE 6100 (BASF, Ludwigshafen, Germany)
Components are blended in one liter distilled water and portioned out to flasks, adding 250 mg CaCO3 to each 10 ml portion.
The medium is sterilized in an autoclave. After cooling the following is added to 1 liter of medium:
23 ml 50% w/v (NH4)2HPO4, filter sterilized
33 ml 20% lactic acid, filter sterilized
Trace Metals Solution
6.8 g ZnCl2
2.5 g CuSO4.5H2O
0.24 g NiCl2.6H2O
13.9 g FeSO4.7H2O
8.45 g MnSO4.H2O
3 g Citric acid, monohydrate
Components are blended in one liter distilled water.
Stock Solutions
50 mM Tris buffer, pH 8.6
189 mM L-Asparagine solution
1.5 M Trichloroacetic Acid (TCA)
Nessler's reagent, Aldrich Stock No. 34, 514-8 (Sigma-Aldrich, St. Louis, Mo. USA)
Asparaginase, Sigma Stock No A4887 (Sigma-Aldrich, St. Louis. Mo. USA)
Assay
Enzyme Reaction:
500 micro-l buffer
100 micro-l L-asparagine solution
350 micro-l water
are mixed and equilibrated to 37° C.
100 micro-l of enzyme solution is added and the reactions are incubated at 37° C. for 30 minutes.
The reactions are stopped by placing on ice and adding 50 micro-l of 1.5M TCA.
The samples are mixed and centrifuged for 2 minutes at 20,000 g
Measurement of Free Ammonium:
50 micro-l of the enzyme reaction is mixed with 100 micro-l of water and 50 micro-l of Nessler's reagent. The reaction is mixed and absorbance at 436 nm is measured after 1 minute.
Standard;
The asparaginase stock (Sigma A4887) is diluted 0.2, 0.5, 1, 1.5, 2, and 2.5 U/ml.
Libraries of cDNA of mRNA from Aspergillus oryzae were generated, sequenced and stored in a computer database as described in WO 00/56762.
The peptide sequence of asparaginase II from Saccharomyces cerevisiae (Kim, K.-W.; Kamerud, J. Q.; Livingston, D. M.; Roon, R. J., (1988) Asparaginase II of Saccharomyces cerevisiae. Characterization of the ASP3 gene. J. Biol. Chem. 263:11948), was compared to translations of the Aspergillus oryzae partial cDNA sequences using the TFASTXY program, version 3.2t07 (Pearson et al, Genomics (1997) 46:24-36). One translated A. oryzae sequence was identified as having 52% identity to yeast asparaginase II through a 165 amino acid overlap. The complete sequence of the cDNA insert of the corresponding clone (deposited as DSM 15960) was determined and is presented as SEQ ID NO: 1, and the peptide translated from this sequence, AoASP, is presented as SEQ ID NO: 2. This sequence was used to design primers for PCR amplification of the AoASP encoding-gene from DSM 15960, with appropriate restriction sites added to the primer ends to facilitate sub-cloning of the PCR product (primers AoASP7 and AoASP8, SEQ ID NOS: 14 and 15). PCR amplification was performed using Extensor Hi-Fidelity PCP Master Mix (ABgene, Surrey, U.K.) following the manufacturer's instructions and using an annealing temperature of 55° C. for the first 5 cycles and 65° C. for an additional 30 cycles and an extension time of 1.5 minutes.
The PCR fragment was restricted with BamHI and HindIII and cloned into the Aspergillus expression vector pMStr57 using standard techniques. The expression vector pMStr57 contains the same elements as pCaHj483 (WO 98/00529), with minor modifications made to the Aspergillus NA2 promoter as described for the vector pMT2188 in WO 01/12794, and has sequences for selection and propogation in E. coli, and selection and expression in Aspergillus. Specifically, selection in Aspergillus is facilitated by the amdS gene of Aspergillus nidulans, which allows the use of acetamide as a sore nitrogen source. Expression in Aspergillus is mediated by a modified neutral amylase II (NA2) promoter from Aspergillus niger which is fused to the 5′ leader sequence of the triose phosphate isomerase (tpi) encoding-gene from Aspergillus nidulans, and the terminator from the amyloglucosidase-encoding gene from Aspergillus niger. The asparaginase-encoding gene of the resulting Aspergillus expression construct, pMStr90, was sequenced and the sequence agreed completely with that determined previously for the insert of DSM 15960
The Aspergillus oryzae strain BECh2 (WO 00/39322) was transformed with pMStr90 using standard techniques (Christensen, T. et al., (1988), Biotechnology 6, 1419-1422). Transformants were cultured in DAP2C-1 medium shaken at 200 RPM at 30° C. and expression of AoASP was monitored by SDS-PAGE and by measuring enzyme activity.
Culture broth from the preceding example was centrifuged (20000× g, 20 min) and the supernatants were carefully decanted from the precipitates. The combined supernatants were filtered through a Seitz EKS plate in order to remove the rest of the Aspergillus host cells. The EKS filtrate was transferred to 10 mM Tris/HCl, pH 8 on a G25 sephadex column and applied to a Q sepharose HP column equilibrated in the same buffer. After washing the Q sepharose HP column extensively with the equilibration buffer, the asparaginase was eluted with a linear NaCl gradient (0-->0.5M) in the same buffer. Fractions from the column were analysed for asparaginase activity (using the pH 6.0 Universal buffer) and fractions with activity were pooled. Ammonium sulfate was added to the pool to 2.0M final concentration and the pool was applied to a Phenyl Toyopearl S column equilibrated in 20 mM succinic acid, 2.0M (NH4)2SO4, pH 6.0. After washing the Phenyl column extensively with the equilibration buffer, the enzyme was eluted with a linear (NH4)2SO4 gradient (2.0-->0M) in the same buffer. Fractions from the column were again analysed for asparaginase activity and active fractions were further analysed by SDS-PAGE. Fractions which was judged only to contain the asparaginase, were pooled as the purified preparation and was used for further characterization. The purified asparaginase was heterogeneously glycosylated judged from the coomassie stained SDS-PAGE gel and in addition N-terminal sequencing of the preparation revealed that the preparation contained different asparaginase forms, as four different N-terminal were found starting at amino acids A27, S30, G75 and A80 respectively of SEQ ID NO: 2. However the N-terminal sequencing also indicated that the purified preparation was relatively pure as no other N-terminal sequences were found by the analysis.
The purified asparaginase from the preceding example was used for characterization.
Asparaginase Assay
A coupled enzyme assay was used. Asparaginase was incubated with asparagine and the liberated ammonia was determined with an Ammonia kit from Boehringer Mannheim (cat. no. 1 112 732) based on glutamate dehydrogenase and NADH oxidation to NAD+ (can be measured as a decrease in A375). Hence the decrease in absorbance at 375 nm was taken as a measure of asparaginase activity.
450 micro-l asparagine substrate was placed on ice in an Eppendorf tube. 50 micro-l asparaginase sample (diluted in 0.01% Triton X-100) was added. The assay was initiated by transferring the Eppendorf tube to an Eppendorf thermomixer, which was set to the assay temperature. The tube was incubated for 15 minutes on the Eppendorf thermomixer at its highest shaking rate (1400 rpm). The incubation was stopped by transferring the tube back to the ice bath and adding 500 micro-l Stop reagent. The tube was vortexed and centrifuged shortly in an ice-cold centrifuge to precipitate the proteins in the tube. The amount of ammonia liberated by the enzyme was measured by the following procedure: 20 micro-l supernatant was transferred to a microtiter plate, 200 micro-l Ammonia reagent A was added and A375 was read (A375(initial)). Then 50 micro-l Ammonia reagent B was added and after 10 minutes at room temperature the plate was read again (A375(final)). A375(initial)−A375(final) was a measure of asparaginase activity. A buffer blind was included in the assay (instead of enzyme) and the decrease in A375 in the buffer blind was subtracted from the enzyme samples.
pH-Activity, pH-Stability, and Temperature-Activity of Asparaginase
The above asparaginase assay was used for obtaining the pH-activity profile, the pH-stability profile as well as the temperature-activity profile at pH 7.0. For the pH-stability profile the asparaginase was diluted 7× in the Universal buffers and incubated for 2 hours at 37° C. After incubation the asparaginase samples were transferred to neutral pH, before assay for residual activity, by dilution in the pH 7 Universal buffer.
The results for the: pH-activity profile at 37° C. were as follows, relative to the residual activity at after 2 hours at pH 7.0 and 5° C.:
The results for the pH-stability profile (residual activity after 2 hours at 37° C.) were as follows:
The results for the temperature activity profile (at pH 7.0) were as follows:
Other Characteristics
The relative molecular weight as determined by SDS-PAGE was seen as a broad band (a smear) at Mr=40-65 kDa.
N-terminal sequencing showed four different terminals, corresponding to residues 27-37, 30-40, 75-85 and 80-91 of SEQ ID NO: 2, respectively.
Penicillium citrinum was grown in MEX-1 medium (Medium B in WO 98/38288) in flasks shaken at 150 RPM at 26° C. for 3 and 4 days. Mycelium was harvested, a cDNA library constructed, and cDNAs encoding secreted peptides were selected and sequenced by the methods described in WO 03/044049. Comparison to known sequences by methods described in WO 03/044049 indicated that Penicillium sequence ZY132299 encoded an asparaginase. The complete sequence of the corresponding cDNA was determined and is presented as SEQ ID NO: 11, and the peptide translated from this sequence is presented as SEQ ID NO: 12.
Asparaginase from A. oryzae having the amino acid sequence shown in SEQ ID NO: 2 was prepared and purified as in Examples 1-2 and added at various dosages to potato chips made from 40 g of water, 52.2 g of dehydrated potato flakes, 5.8 g of potato starch and 2 g of salt.
The flour and dry ingredients were mixed for 30 sec. The salt and enzyme were dissolved in the water, and the solution was adjusted to 30° C. The solution was added to the flour. The dough was further mixed for 15 min. The mixed dough was placed in a closed plastic bag and allowed to rest for 15 min at room temperature.
The dough was then initially compressed for 60 sec in a dough press.
The dough was sheeted and folded in a noodle roller machine until an approx. 5-10 mm dough is obtained. The dough was then rolled around a rolling pin and allowed to rest for 30 min in a plastic bag at room temperature. The dough was sheeted further to a final sheet thickness of approx 1.2 mm.
The sheet was cut into squares of approx 3×5 cm.
The sheets were placed in a frying basket, placed in an oil bath and fried for 45 sec at 180° C. The noodle basket was held at a 45° angle until the oil stopped dripping. The products were removed from the basket and left to cool on dry absorbent paper.
The potato chips were homogenized and analyzed for acrylamide. The results were as follows:
The results demonstrate that the asparaginase treatment is effective to reduce the acrylamide content in potato chips, that the acrylamide reduction is clearly dosage dependent, and that the acrylamide content can be reduced to a very low level.
Potato chips were made as follows with addition of enzyme systems which are capable of reacting on asparagine, as indicated below.
Recipe:
Dough Procedure:
The potato flakes and potato starch are mixed for 30 sec in a mixer at speed 5. Salt and enzyme are dissolved in the water. The solution is adjusted to 30° C. +/−1° C. Stop mixer, add all of the salt/enzyme solution to flour. The dough is further mixed for 15 min.
Place mixed dough in plastic bag, close bag and allow the dough to rest for 15 min at room temperature.
The dough is then initially compressed for 60 sec in a dough press.
The dough is sheeted and folded in a noodle roller machine until an approx. 5-10 mm dough is obtained. The dough is then rolled around a rolling pin and the dough is allowed to rest for 30 min in a plastic bag at room temperature. The dough is sheeted further to a final sheet thickness of approx 1.2 mm.
Cut the sheet into squares of approx 3×5 cm.
Sheets are placed in a frying basket, placed in the oil bath and fried for 60 sec at 180° C. Hold the noodle basket at a 45° angle and let the product drain until oil stops dripping. Remove the products from the basket and leave them to cool on dry absorbent paper.
The results from acrylamide analysis were as follows:
The results demonstrate that all the tested enzyme systems are effective in reducing the acrylamide content of potato chips.
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2002 01547 | Oct 2002 | DK | national |
This application is a divisional of U.S. Ser. No. 10/530,931, which is a 35 U.S.C. 371 national application of PCT/DK2003/000684 filed Oct. 10, 2003, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no, PA 2002 01547 filed Oct. 11, 2002 and U.S. provisional application No. 60/421,897 filed Oct. 29, 2002, the contents of which are fully incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60421897 | Oct 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10530931 | Jun 2005 | US |
| Child | 11952593 | Dec 2007 | US |
