The present invention relates to novel nucleic acid sequences which, upon expression in a procaryotic or eucaryotic cell, code for a polypeptide having an elevated specific xylose isomerase activity compared to the wildtype Thermus thermophilus xylose isomerase. They are selected from a) nucleic acid sequences shown in SEQ. ID. No 1; b) the complementary strand of the sequence defined in (a) above; c) nucleic acid sequences which hybridize to the sequences defined in (a) or (b) above; d) nucleic acid sequences which, but for the degeneracy of the genetic code, would hybridize to the sequences defined in (a), (b) or (c) above and which code for the same polypeptide as those defined in (a), (b) or (c) above. The present invention further provides a process of producing ethanol from xylose containing materials comprising contacting cells that express such nucleic acid sequences and novel modified xylose isomerases that advantageously can be applied in the production of fructose syrups.

This invention relates to nucleic acid sequences encoding novel modified xylose isomerases, to vectors carrying the sequences, to the polypeptides encoded by these sequences, to the production of a polypeptide encoded by these sequences and to the use of the isomerase in a process of ethanol production. The present invention further provides novel modified xylose isomerases that advantageously can be applied in the production of fructose syrups, in particular high fructose corn syrups.
D-xylose is a five-carbon sugar. It is present in nature, for example as xylan polymer in plant hemicellulose. As a carbon source, D-xylose has been reported to be utilised widely by bacteria and to a lesser extent by fungi. D-xylose is first converted to its keto isomer, D-xylulose, which is then phosphorylated to D-xylulose-5-phosphate, a normal pentose phosphate pathway intermediate.
There are two possible routes for the isomerisation of D-xylose: (a) direct conversion to D-xylulose by an isomerase; or (b) an oxidoreductive pathway, in which D-xylose is first reduced to xylitol and in a second reaction xylitol is oxidised to D-xylulose. The enzymes involved in this oxidoreductive pathway require coenzymes. The direct isomerisation is reported from bacteria and plant, while the oxidoreductive pathway is the main route for D-xylose to D-xylulose conversion in fungi.
Xylose isomerase catalyses the direct isomerisation of D-xylose to D-xylulose and vice versa, with the direction dependent on the relative concentration of the aldo and keto forms and the reaction conditions. Xylose isomerase also catalyses of interconversion of the 6carbon aldose sugar D-glucose and its keto isomer D-fructose. Thus, bacterial xylose isomerases are used industrially to produce D-fructose from D-glucose.
The economic use of lignocellulosic biomass as a renewable energy source is strongly dependent on the fermentation of xylose to ethanol. Since the best ethanol-producing organism, Saccharomyces cerevisiae is not able to utilise xylose, major efforts have been taken to develop recombinant strains possessing the necessary enzymes. The xylose reductase and xylitol dehydrogenase from Pichia stipitis were cloned and expressed in Saccharomyces cerevisiae. The resulting strains were able to ferment xylose, however the yields were far from the theoretical due to the xylitol excretion (Tantirungkij et al. (1993)).
Several attempts to circumvent this problem by introducing the alternative direct isomerisation pathway have been made. The xylose isomerases form two clusters based on the sequence homology, the actinomycetic genes showing less than 40% homology to the others. From the latter subgroup the Bacillus subtilis (Amore et al. (1989)) and the Escherichia coli (Sarthy et al. (1987)) xylose isomerases were expressed inactively in Saccharomyces cerevisiae, while introduction of the Clostridium thermosulfurogenes (Moes et al. (1996)) gene did not result in a protein that cross-reacted with a specific antibody. From the first subgroup the Actinoplanes missouriensis (Amore et al. (1989)) gene was cloned in yeast but the production of xylose isomerase failed due to an aberrant transcription start.
The Thermus thermophilus xylose isomerase only recently has been actively expressed in Saccharomyces cerevisiae (Walfridsson et al. (1996)). This gene is closely related to the actinomycetic genes showing approximately 65% homology. The enzyme, however, has only trace activity at 30xc2x0 C., because of its high (95xc2x0 C.) temperature optimum. The other important factor for the poor performance of the strain was the formation of xylitol, probably by the unspecific NADPH linked aldose reductase described by (Kuhn et al. (1995)), which is coded by the gene GRE3. The xylitol formation has a dual effect on the ethanol yield; it does not only lead to carbon loss, but it also competitively inhibits the xylose isomerase (Yamanaka (1969)). By increasing intracellular xylitol concentration the apparent affinity of the xylose isomerase towards xylose decreases and more xylose is channelled into xylitol, until the NADPH pool of the cell is depleted. In the presence of active xylose isomerase some xylitol can be formed also by the endogenous xylitol dehydrogenase described by (Richard et al. (1999)). To prevent the xylitol impairing the xylose isomerase and thereby blocking the xylose metabolism of the recombinant yeast, it is advantegous to have a xylose isomerase expressed which is less sensitive to the presence of xylitol.
In light of the previously described problems, there is a need to find a xylose isomerase being actively expressed in Saccharomyces cerevisiae and being more active at a broader range of pH values and temperatures.
In the present invention, a series of novel xylose isomerases have been isolated. They were modified from the Thermus thermophilus enzyme by random mutagenesis and identified as having elevated activity at mesophilic ( less than 40xc2x0 C.) temperatures, compared to the wild type enzyme. Expressed either in procaryotic or eucaryotic cells, the specific enzyme activity in the mutants is at least 10% higher than that of the wildtype enzyme. They are, therefore, expected to be more suitable for expression in yeast. The present invention thus comprises any isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity, than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2. The specific activity of the enzyme is to be determined as described in example 3, wherein one unit of xylose (glucose) isomerase activity is defined as the amount of crude enzyme needed to produce 1 xcexcmol of product per minute under the assay conditions. The specific activity (U minxe2x88x921 mgxe2x88x921) is herein determined from the activity and the protein concentration of the crude enzyme preparations.
The present invention further comprises any isolated nucleic acid sequence that is derived and shows at least one nucleic acid modification from the nucleic acid sequence shown in SEQ. ID. No. 2.
Furthermore, the mutants show a higher thermostability than the wildtype enzyme. One embodiment of the invention thus comprises an isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity at temperatures above 80xc2x0 C. such as 81xc2x0 C., 82xc2x0 C., 83xc2x0 C., 84xc2x0 C., 85xc2x0 C., 86xc2x0 C., 87xc2x0 C., 88xc2x0 C., 89xc2x0 C., 90xc2x0 C., 91xc2x0 C., 92xc2x0 C., 93xc2x0 C., 94xc2x0 C., 95xc2x0 C., 96xc2x0 C., 97xc2x0 C., 98xc2x0 C., 99xc2x0 C. and/or 100xc2x0 C., than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2.
In another embodiment of the invention, the mutants also show a higher acceptance for xylitol, which is an inhibitor to xylose isomerase, compared to the wildtype enzyme.
In a further embodiment of the invention, the mutated xylose isomerases have a broader pH range than the wildtype, even though it may have the same pH optimum at xcx9c7.0. The present invention therefor also comprises any isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity at acidic pH values i.e. lower than pH 7.5 such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.5 and/or pH 4.0 than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2.
In a preferred embodiment, the present invention provides:
An isolated nucleic acid sequence which codes upon expression in a procaryotic or eucaryotic host cell for a polypeptide having xylose isomerase activity at mesophilic temperatures, which nucleic acid sequence is selected from
a) nucleic acid sequences shown in SEQ. ID. No 1, wherein nnn at positions 1114-1116 stand for a codon coding for an amino acid other than glutamate, preferably a codon coding for glycine and nnn at positions 487-489 stand for a codon coding for any amino acids, preferably phenylalanine, more preferably leucine
b) the complementary strand of the sequence defined in (a) above;
c) nucleic add sequences which hybridise to the sequences defined in (a) or (b) above and the residues corresponding to the residues nnn and nnn in SEQ. ID. No 1 are as defined in (a) or (b) above;
d) nucleic acid sequences which, but for the degeneracy of the genetic code, would hybridise to the sequences defined in (a), (b) or (c) above and which code for the same polypeptide as those defined in (a), (b) or (c) above;
a polypeptide encoded by a nucleic acid sequence as defined above;
a vector comprising a nucleic acid sequence as defined above;
cells transformed or transfected with such a vector; a process of producing ethanol which comprises:
(i) contacting such transformed cells with a substrate that contains one or more carbon sources selected from xylose and polymerised xylose moieties;
(ii) culturing the said cells in conditions under which the isomerisation of D-xylose to D-xylulose occurs and under which the D-xylulose is further catabolised to ethanol; and
(iii) recovering the ethanol;
a process of producing a polypeptide as defined above comprising expressing a nucleic acid sequence as defined above in a cell as defined above;
a process of producing D-fructose which comprises:
(i) contacting such polypeptide with a substrate containing D-glucose;
(ii) incubating the substrate with the polypeptide in conditions under which the isomerisation of D-glucose to D-fructose occurs;
(iii) recovering the mixture of D-glucose and D-fructose
The nucleic acid sequences of the present invention are preferably DNA, though they may be RNA or even PNA. It will be obvious to those of skill in the art that, in RNA sequences according to the invention, the T residues shown in SEQ. ID. No. 1 will be replaced by U.
The nucleic acid sequences of the present invention include in the sequence shown in SEQ. ID. No. 1, wherein the nnn at the positions 1114-1116 stand for a nucleotide triplet, which upon expression codes for an amino acid other than glutamate, preferably glycine. Such nucleotide triplet is for example GGA. Similarly, the nnn at the positions 487-489 stand for a nucleotide triplet, which upon expression codes for any amino acid, preferably phenilalanine, more preferably leucine. Such a nucleotide triplet is for example CTC.
As apparent from the ove, the nucleic acid sequences of the present invention are not limited to the sequences of SEQ. ID. No 1. Rather, the sequences of the invention include sequences that are closely related to these sequences and that encode a polypeptide having xylose isomerase activity, xylose isomerase activity being the ability to catalyze the direct interconversion of xylose to xylulose. For example, four preferred sequences of such polynucleotides are shown in SEQ. ID. No. 3, 5, 7, and 9. These nucleic acid sequences may be prepared by altering SEQ. ID. No. 1 by any conventional method, or they may be isolated from an organism or made synthetically. Such alterations, isolations, or syntheses may be performed by any suitable method, for example by the methods of Sambrook et al. (Sambrook et al. (1989)).
For example, the sequences of the invention include sequences that are capable of selective hybridisation to those of SEQ. ID. No. 1; preferably those wherein the residues selectively hybridising to the residues marked as nnn in SEQ. ID. No. 1 are coding for an amino acid other than glutamate, preferably glycine; and which code for a polypeptide having xylose isomerase activity. Such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al. (1989)). xe2x80x9cHybridisationxe2x80x9d is a process in which a strand of nucleic acids joins with a complementary strand through base pairing. The degree of complementarity of two non-identical, but very similar strands, and their length influence the hybridisation conditions. In the present invention, the conditions for hybridisation are meant to be those of a stringent hybrisisation according to (Sambrook et al. (1989)).
Also the sequences of the invention include sequences that are different from those defined above because of the degeneracy of the genetic code and encode the same polypeptide. Degenerate code stands for a genetic code in which a particular amino acid can be coded by two or more different codons. Degeneracy occurs because of the fact that of the 64 possible base triplets, 3 are used to code the stop signals, and the other 61 are left to code for only 20 different amino acids.
Terms and abbreviations in this document have their normal meanings unless otherwise stated. All nucleic acid sequences are written in the direction from the 5xe2x80x2(stands for prime) end to the 3xe2x80x2 end also referred to as 5xe2x80x2 to 3xe2x80x2. All amino acid or protein sequences, unless otherwise designated, are written from the amino terminus (N-terminus) to the carboxy terminus (C-terminus).
The codon usage is the standard published and internationally used single or triple letter amino acid code.
The word xe2x80x9cbase pairxe2x80x9d or xe2x80x9cbpxe2x80x9d refers to DNA or RNA. The single letters A, C, T and G correspond to the 5-monophosphate forms of the deoxyribonucleosides (deoxy)adenine, (deoxy)cytidine, (deoxy)guanine and (deoxy)thymidine, respectively. Furthermore, the abbreviations U, C, G and T may correspond to the 5xe2x80x2-monophosphate forms of the ribonucleosides uracil, cytidine, guanine and thymine, respectively, occurring in RNA molecules. xe2x80x9cBase pairxe2x80x9d in double stranded DNA refers to a pairing of A with T or C with G. In a heteroduplex of RNA paired with DNA, base pair may refer to a pairing of A with U or C with G.
The term xe2x80x9cvectorzxe2x80x9d stands for a nucleic acid compound used for the transformation of cells. A vector contains a polynucleotide sequence corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transformed. Plasmids, viruses and bacteriophage are suitable vectors. Artificial vectors can be constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. The term vector also includes recombinant DNA cloning vectors and recombinant DNA expression vectors.
The term xe2x80x9cPromoterxe2x80x9d stands for a DNA sequence which directs transcription of DNA to RNA,
The term xe2x80x9cplasmidxe2x80x9d refers to an extrachromosomal (usually) self-replicating genetic element. Plasmids are commonly designated by a lower case xe2x80x9cpxe2x80x9d followed by letters or numbers. The plasmids used here are either commercially available, publicly available on an unrestricted basis or can be constructed from available plasmids following published procedures. In addition, equivalent plasmids to those described are known and are apparent to the skilled worker in this field.
xe2x80x9cTransformationxe2x80x9d means the introduction of DNA into an organism in a way that it can replicate itself, either as an extrachromosomal element or by chromosomal integration. Methods of transforming bacterial and eukaryotic hosts are well known. Many of these methods, such as nuclear injection, protoplast fusion or calcium treatment with calcium chloride are summarized in (Sambrook, et al. (1989)).
xe2x80x9cTransfectionxe2x80x9d is the operation of adding an expression vector to a host cell, whereby the cell itself takes up the DNA molecule and either integrates it into the chromosomes (stable transfection) or keeps it separate from the chromosomal DNA in the nucleus as a transient transcribed plasmid (transient transfection). Many different methods of transfection are known, for example calcium phosphate co-precipitation and electroporation. A successful transfection can be monitored by a specific site introduced in the transfected DNA molecule which can be recognised by an antibody. This DNA site can either be a specifically introduced xe2x80x9cflagxe2x80x9d or xe2x80x9ctagxe2x80x9d meaning a specific sequence which is easily recognised by an antibody. It could also be an intrinsic part of the expressed gene, recognised by a specific antibody. Another possibility would be the fusion to a fluorescent protein like eg GFP (green fluorescent protein).
The expressions xe2x80x9ccomplementaryxe2x80x9d and xe2x80x9ccomplementarityxe2x80x9d refer to the matter of base pairing of purines and pyrimidines that associate through hydrogen bonding in double stranded nucleic acid. The base pairs guanine and cytosine, adenine and thymidine as well as adenine and uracil are complementary.
xe2x80x9cIsolated amino acid sequencexe2x80x9d refers to any amino acid sequence, constructed or synthesised, which is locationally distinct from the naturally occurring sequence.
A xe2x80x9cprimerxe2x80x9d is a nucleic acid fragment, which functions as an initiating substrate for enzymatic or synthetic elongation.
The term xe2x80x9ca polypeptidexe2x80x9d in the present application is generally understood to mean a polypeptide of the invention, as shown for example in SEQ. ID. No. 4, 6, 8, or 10. It is also within the meaning of xe2x80x9ca polypeptidexe2x80x9d that several polypeptides can be used, i.e. in the present context xe2x80x9caxe2x80x9d means xe2x80x9cat least onexe2x80x9d unless explicitly indicated otherwise.
The sequence xe2x80x9cidentityxe2x80x9d can be calculated as                     (                              N            ref                    -                      N            dif                          )                  100              N      ref        ,
wherein Ndif is the total number of non-identical residues in the two sequences when aligned, and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCMTC (Ndif=2 and Nref=8). A gap is counted as non-identity of the specific residue(s), ie the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (Ndif=2 and Nref=8). Sequence identity can alternatively be calculated by the BLAST program, eg the BLASTP program (Pearson W. R and D. J. Lipman (1988))(www.ncbi.nlm.nih.gov/cgi-bin/BLAST). Alignment can be performed with the global align algorithm with default parameters as described by (Huang and Miller (1991)), available at http://www.ch.embnet.org/software/LALIGN_form.html.
One preferred embodiment of the invention thus comprises a nucleic acid sequence, which nucleic acid sequence is identical at least 85%, such as at least 90%, at least 95%, at least 99% or at least 100% to any of the sequences defined above.
A yeast expressing active xylose isomerase can be used in a process for producing ethanol from xylose containing materials. Suitable substrates for ethanol production include xylose itself and any compound that the cells can convert into xylose. For example, suitable substrates include polymers that contain xylose moieties. Thus, xylan, arabinoxylan and xyloglucan polymers, which comprise xylose moieties, are suitable substrates. Accordingly, hemicellulosic and lignocellulosic substrates, such as plant biomass, are suitable substrates.
In processes of ethanol production according to the present invention, xylose is typically released from xylan by enzymatic or chemical hydrolysis under acidic or basic conditions, or by heating or by a combination of these techniques. For example xylose can be released from xylan by a combination of acidic hydrolysis and heating under pressure. The yeasts typically used in ethanol production are not capable of enzymatically hydrolysing xylans to release xylose, although some yeasts are capable of doing so and of metabolising the xylose to ethanol. Such yeasts, when transformed with a vector according to the invention, are included within the scope of the invention.
Although the preferred processes of ethanol production according to the invention comprise contacting transformed or transfected cells with a suitable substrate, other processes of producing ethanol are also possible. For example, a polypeptide according to the invention may be added to a cellular fermentation broth in order to liberate xylose outside the cells, which is then taken up and metabolised by them.
In the present invention, ethanol will typically be produced by a fermentation broth that comprises yeast cells according to the invention, water, one or more sources of xylose and other nutrients. These nutrients preferably include a suitable nitrogen source, such as (NH4)2SO4. The presence of hexose sugars, such as glucose may enhance both ethanol formation productivity and growth. Also, the presence of Mn2 or Mg2 ions is desirable as one of these ions is necessary to the function of the mutant xylose isomerases. Further, the presence of low levels of oxygen is desirable, especially in fermentation broths containing Saccharomyces cerevisiae.
The process may be carried out at any temperature that facilities ethanol production, but temperatures from 15 to 40xc2x0 C. are preferred and temperatures from 30 to 35xc2x0 C. are particularly preferred. In one embodiment of the present invention, the novel xylose isomerases therefor show at least 10% elevated activity at temperatures ranging from 20-60xc2x0 C., such as in an especially preferred embodiment at 30-35xc2x0 C.
For the ethanol-producing processes of the invention, it is preferred that transformed or transfected yeast cells, especially those of S. cerevisiae are used. Recently a Saccharomyces cerevisiae strain was constructed with the GRE3 gene disrupted (WO99/54477). This strain shows reduced levels of xylitol formation, when it is metabolising xylose. In one preferred embodiment of the invention, cells of a S. cerevisiae strain according to the patent application (WO99/54477) are used. As the person skilled in the art will be aware of, though, this should not exclude the possible usage of other transfected yeast cells, such as a cell of a species selected from a list that should be regarded only as illustrative and not exclusive, consisting of Pichia sp., Candida sp., Shizosaccharomyces sp., Zygosaccharomyces sp. and Saccharomyces sp. Furthermore, other Saccharomyces families are embodied in the scope of the invention, for example; Saccharomyces cerevisisae, Saccharomyces bayanus and Saccharomyces carlsbergensis.
In the ethanol-producing processes of the invention, the substrate is wholly or partly converted to ethanol, which may be recovered by any suitable means known in the art. The xylose isomerases according to the invention are also suitable for industrial high fructose syrup production. In this process D-glucose, preferably in the form of starch hydrolysate, is partially converted to D-fructose. Typical process conditions are 60xc2x0 C., pH 7.5 with 45 ppm Mg2+ and 45% w/w D-glucose as substrate. The substrate is preferably obtained as starch hydrolysate, produced for example from wheat starch with the methods described in (Godfrey and Reichelt (1983)).
In a conventional industrial process, the aim is, in most cases, to obtain a sugar solution, in which 40 to 45% of the sugar is fructose. When the activity of the enzyme decreases with the ageing of the column, the desired level is maintained by reducing the flow rate.
Random Mutagenesis of the xylA Gene
The xylA gene of the Thermus thermophilus was amplified using polymerase chain reaction (PCR) from the pBXI plasmid (Walfridsson et at (1996)) as template. Three samples with the following (identical) composition were prepared:
The nucleic acid sequence of Primer 1 is shown in SEQ. ID. No.11, that of Primer 2 is shown in SEQ. ID. No. 12.
The samples were placed in a thermocycler (Perkin Elmer GeneAmp2400) and subjected to the following temperature program, where the 2nd segment was repeated 6 times:
Temperature program:
The samples were mixed and divided again into three aliquots to equalise the concentrations. The following reagents were added to these samples:
The temperature program described above was performed in the thermocycler mentioned above with a single 2.segment. Half of the samples (50 xcexcl) was removed and saved on xe2x88x9220xc2x0 C. (samples A1, B1 and C1). To the remaining 50 xcexcl samples the following reagents were added:
The temperature program described above was performed in the thermocycler mentioned above with only one 2.segment. Half of the samples (50 xcexcl) was removed and saved on xe2x88x9220xc2x0 C. (samples A2, B2 and C2).
With the remaining 50 xcexcl samples the addition of the reagents and the temperature program were performed again with the same conditions as above. 50 xcexcl of each sample were saved on xe2x88x9220xc2x0 C. (A3, B3 and C3). The rest of them were applied to agarose (2%) gel electrophoresis to check the reaction efficiency.
All of the nine samples (A1-3, B1-3, and C1-3) were purified with High Pure(trademark) PCR Product Purification Kit (Roche Diagnostics). With each samples the following reaction were set up:
The temperature program described above was performed in the thermocycler mentioned above with ten repeated 2.segment. The samples were purified as before and used for ligation experiments.
Cloning and Selection of Mutant xylA Genes
The mutant xylA genes obtained in the previous example were inserted into the general cloning vector pGEM-t Easy (Promega Corp.). 300 ng of vector (pGEM-t Easy) was incubated overnight at 4xc2x0 C. with 200 to 500 ng of insert in total volume of 10 xcexcl. A control reaction without insert was also conducted.
The ligation products were transformed into E. coli HB101 (xylA5), which is a xylose isomerase deficient strain. HB101 High Efficiency Competent Cells (Promega Corp.) were transformed according to the suppliers recommendations, and 3xc3x97100 xcexcl or 5xc3x97200 xcexcl were plated on LB-agar supplemented with ampicillin (50 xcexcl/xcexcl) and X-gal (20 xcexcg/xcexcl). Colonies where moved to MacConkey-agar plates with ampicillin and xylose (2%), either with toothpicks or by replica plating. The plates were incubated at 37xc2x0 C. overnight and thereafter incubated at different temperatures (37xc2x0 C. and 55xc2x0 C.) until the colour reaction, which indicates xylose utilisation (orange to red), was seen. On the MacConkey plates, E. coli DH5xcex1 (harbouring pUC18 for ampR) and E. coli HB101 (harbouring pGEM-t Easy for ampR) were used as positive and negative control, respectively. Note that the strain HB101 has Xylxe2x88x92, whereas the strain DH5xcex1 has Xyl+ phenotype.
All colonies similar colour reaction (xylose utilisation) to the positive control were spread on new MacConkey-amp-xylose plates, LB-amp plates and inoculated in liquid LB-amp (50 xcexc/xcexcl). Plasmid DNA from the liquid cultures were prepared using the CTAB method, and subjected to EcoRI digestion to establish if they contained the xy/A-insert. Plasmids with insert were then retransformed into E. coli HB101 to confirm their phenotype on MacConkey plates. Correct colonies were cultured as above and the plasmids purified with Nucleon mini plasmid DNA preparation kit (Amersham). These DNAs were used in the subsequent experiments and to determine the nucleic acid sequences of the mutated genes. Schematic representation one of the obtained plasmid is shown in FIG. 1.
Kinetic Characterisation of the Mutant Xylose Isomerases
E. coli HB101 haboring plods PUSM1010, PUSM1021, pUSM1024, pTJSM1026, and pUSMwt, with the mutated and the wildtype xylose isomerase genes respectively (as described in the previous two examples) were grown at 37xc2x0 C. in 50 ml LB media containing 100 xcexcl/ml ampicillin. The cells were harvested by centrifugation in the stationary phase of growth and washed once with ice cold distilled water. Washed cells were resusprended in 100 mM triethanolamine, pH 7.0, 0.5 mg/ml lysozyme (130000 U/mg), 0.25 mg/ml DNase, and 1 mM phenylmethylsulphonyl fluoride (PMSF) in dimethylsulfoxide (DMSO). The suspensions were kept at room temperature for one hour and then on ice for two hours before being put in a freezer at xe2x88x9220xc2x0 C. The cell lysates were thawed on ice, cell debris was removed by centrifugation (1300 rpm for 15 minutes at 4xc2x0 C.) and the supernatants were used as the crude enzyme preparations.
Protein concentration was determined with the Bradford method (Bradford (1976)) using the Pierce protein reagent with bovine serum albumin as standard.
The xylose isomerase was assayed by incubating 700 mM D-xylose with suitable amount of crude enzyme preparation in the presence of 10 mM MnCl2 and 100 mM triethanolamine, pH 7.0 at the desired temperature in 0.5 ml volume. Glucose isomerase activity was assayed under the same reaction conditions, except that D-glucose instead of xylose was used in the reaction mixture. The reaction was terminated after 10 minutes by adding 150 xcexcl 50% trichloroaceticacid. The reaction mixture was neutralized with 185 xcexcl 2 M Na2CO3. The formed xylulose or fructose was measured on a COBAS MIRA (Roche) automated spectrophotometer via enzymatic reduction with 0.04 or 0.5 units of Sorbitol Dehydrogenase (purchased from Roche), respectively in the presence of 0.15 mM NADH at pH 7.0, 37xc2x0 C. in 200 xcexcl reaction volume. The rate of disappearance of NADH was followed at 340 nm and the concentration of D-xylulose or D-fructose was calculated using calibration curves. One unit of xylose (glucose) isomerase activity was defined as the amount of crude enzyme needed to produce 1 xcexcmol of product per minute under the assay conditions. The specific activity (U minxe2x88x921 mgxe2x88x921) was determined from the activity and the protein concentration of the crude enzyme preparations. Comparison between the specific activities at 30xc2x0 C. is shown in FIG. 2 and Table 1. The change of the specific activity with the temperature for each enzyme is displayed in FIG. 3. The temperature optimum for the mutants were roughly the same as for the wild type enzyme except for mutant 1021, which had xcx9c10xc2x0 C. lower temperature optimum.
The kinetic parameters Vmax (xcexcmol minxe2x88x921 mgxe2x88x921) and Km (mM) were determined from Michaelis-Menten plots of specific activities at various substrate concentrations by nonlinear regression analysis with Sigma Plot (SSPS Inc.) software. Typically, duplicate measurements were used to determine the Km value at 6-10 concentrations of the substrate, with a concentration range covering the value of Km.
The inhibition constant for xylitol (Ki) was determined by incubating the crude enzyme preparations with different xylose concentrations (20-400 mM) at different fixed xylitol concentrations. The mutants had a higher Ki for xylitol, which is an inhibitor to xylose isomerase, compared to the wildtype enzyme.
The effect of pH on the activity of the enzymes was followed in the pH range 5-10 in a buffer prepared by mixing acetate, piperazine-N,Nxe2x80x2-bis-(2-ethanolsulphonic acid) (PIPES), N-2-hydroxyehylpiperazine-Nxe2x80x2-2-etanesulfnicacid (HEPES) and glycine at final concentration 50 mM each as described by (Dekker at al. (1992) Appl. Mirobiol.
Biotechnol. 36: 727-732). The pH was adjusted at 60xc2x0 C. with NaOH. The assay was performed at 60xc2x0 C. in mixture containing 700 mM D-xylose 10 mM MnCl2. Above pH 7.0 corrections were made for the chemical isomerisation of D-xylose. The change of the specific activity with the pH for each enzyme is shown in FIG. 4. The mutated xylose isomerases all had a broader pH range than the wildtype, but the same pH optimum xcx9c7.0. Thermal stability of wild type xylose isomerases and mutated xylose isomerases where tested by incubating metal free enzyme preparations at 70xc2x0 C. in 200 mM triethanolamine, pH7.0, 10 mM MnCl2. Residual activities of timed aliquots were recorded using xylose as substrate.
Table 2 illustrates a summary of the catalytic properties of the tested mutated xylose isomerases and the wild type xylose isomerase.
Furthermore, the effect of different bivalent cations (10 mM) on the activity of EDTA treated enzymes was investigated. The specific activity with 10 mM MnCl2 at 60xc2x0 C. was set to 100% for each enzyme. The results are shown in table 3.
Construction of Yeast Expression Vector
The vector pMA91 (Mellor et al. (1983)) was hydrolysed with the restriction enzyme HindIII (Roche) to obtain the yeast PGK1 promoter and terminator sequences. The digested DNA was electrophorised on 1.0 % agarose gel and the 1.8 kb fragment was cut out from the gel with a scalpel. The DNA fragment was recovered from the agarose slice with the QIAqiuck Gel extraction kit (QIAGEN). YEplac112 (Gietz and Sugino (1988)) was linearised with HindIII, digested with Shrimp Alkaline Phosphatase (Roche) and purified with QlAquick PCR purification kit (QIAGEN). The two DNA fragments were ligated using T4 DNA ligase (Gibco BRL) according to the supplier""s recommendations. Ligation products were transformed into E. coli HB101 and plated onto LB agar plates with 100 xcexcg/ml ampicillin. The plates were incubated overnight on 37xc2x0 C., 5 colonies were picked up and inoculated into 5 ml LB media with 100 xcexcg/ml ampicillin. The cultures were incubated overnight on 37xc2x0 C. with rocking. Plasmids were isolated with QIAprep Spin kit (QIAGEN) and used for restriction analysis. One isolate showing the correct HindIII digestion pattern was chosen and it was inoculated into 100 ml LB media with 100 xcexcg/ml ampicillin. The culture was grown overnight on 37xc2x0 C. with 200 rpm shaking. Large scale plasmid preparation was performed with QIAGEN MAXIPREP (QIAGEN) and the obtained plasmid (in the following referred as YEplacPGK) was used in the following experiment. pUSM1026 was transformed into E coli GM2163 (damxe2x88x92) and plated onto LB agar with 100 xcexcl/ml ampicillin and 10 xcexcg/ml chloramphenicol. A colony was inoculated into 5-10 ml LB with 100 xcexcg/ml ampicillin and 10 xcexcg/ml chloramphenicol. The culture was grown overnight on 37xc2x0 C. with rocking. Unmethylated plasmid was isolated from the culture with Quantum Prep plasmid miniprep kit (BIO-RAD). The plasmid was cut with BclI (Roche) on 50xc2x0 C. and applied to a 1.0% agarose gel. The 1.2 kb fragment was cleaved out with a scalpel and purified from the agarose gel with QlAquick Gel extraction kit (QIAGEN). 1-5 xcexcg of YEplacPGK was digested with BglII (Roche), dephosphorilated with Shrimp Alkaline Phosphatase (Roche) and purified with QlAquick PCR purification kit (QIAGEN). The two DNA fragments were mixed and ligated with T4 DNA ligase (Gibco BRL) under the conditions recommended by the manufacturer. The ligation mixture was transformed into E. coli and plated onto LBagar with 100 xcexcg/ml ampicillin. 8-10 colonies were picked and inoculated into 5-10 ml LB+100 xcexcg/ml ampicillin. After the cultures had grown overnight on 37xc2x0 C. with rocking, plasmids were isolated with Quantum Prep plasmid miniprep (BIO-RAD). The plasmids were sequenced to determine the direction of the insert compared to the promoter. One preparatum with the correct sequence was chosen to be used in the subsequent experiments and named M1026.
To construct a control plasmid similar to the one above, but containing the wild type xylA gene, the plasmid pBXI (Walfridsson et al. (1986)) was partially hydrolysed with HindIII (Roche). After separating the fragments on 0.7% agarose gel the 3.0 kb fragment was cut out and purified with QlAquick Gel extraction kit (QIAGEN). The fragment carrying the promoter-gene-terminator cassette was inserted into YEplac112, which was linearised with HindIII and dephosphorilated before. The ligation products were transformed into E. coli and plasmids were reisolated from 3 colonies, as described above. An isolate showing the expected restriction pattern with HindIII was used in the following experiments and named YEplacTthXI. The constructed yeast expression plasmids are shown in FIG. 5 (YEplacPGK, YEplacM1026) and FIG. 6. (YEplacTthXI).
Expression of the Mutant xylA in Saccharomyces cerevisiae
Plasmids YEplacPGK, YEplacM1026 and YEplacTthXI were transformed into Saccharomyces cerevisiae CEN.PK2-1D with the method of (Gietz and Woods (1994)). The transformants were plated onto SC-Trp plates and incubated on 30xc2x0 C. for 2 days. Transformants were inoculated into 5 ml SC-Trp media and were grown overnight on 30xc2x0 C. with rocking. The cells were harvested by centrifugation (10 minutes, 5000 g), suspended in distilled water and centrifuged again. The cell pellets were suspended in 500 xcexcl Y-PER reagent (Pierce) supplemented with 1 mM phenylmethylsulfonyl fluoride. The extraction was performed on room temperature for 30 minutes with shaking. After removing the cell debris by centrifugation for 5 minutes at 13000 g, the resulting supernatants were used for determining the xylose isomerase activity. The xylose isomerase assay and the protein concentration measurements were as described in Example 3.

Xylose isomerase with improved properties


The frozen dessert products of the present invention preferably comprise a base mixture of, on a percent solids (dry) basis, about 1% to about 12% high fructose corn syrup, about 5% to about 20% corn syrup having a DE of about 36, about 2% to about 22% sucrose, about 2% to about 6% maltodextrin, about 0.2% to about 5% egg white, about 1% to about 9% FRUITRIM(copyright) blend (comprising fruit juices and natural grain dextrins), about 0.2% to about 1.5% stabilizer, about 0% to about 0.12% emulsifier, about 0% to about 1.5% modified food starch, about 0% to about 5% milk fat, about 0% to about 10% milk solids non-fat (MSNF) with the balance being water, and on a volume basis, from about 20% to about 90% fruit puree. Flavor ingredients may be added. The method of production for the frozen dessert products comprises the steps of blending the above ingredients to form a base mixture, pasteurizing and homogenizing the base mixture, adding fruit puree to form a final mixture, extruding to a final density of about 4.5 to about 8 lbs/gallon, packaging the final mixture, and hardening the final mixture to a core temperature below xe2x88x9210 degrees F.

1. Field of the Invention
The present invention relates, in general, to frozen dessert products. More particularly, the present invention relates to formulations and methods for producing frozen products for dessert use that provide creamy smooth texture and high fruit delivery.
2. Statement of the Problem
Frozen dessert products enjoy great popularity. Many variations of frozen products have been developed for dessert use, including xe2x80x9csorbetxe2x80x9d-type products. Sorbet-type products are particularly popular and have a wide appeal. Distinguishing features of conventional sorbet products are that they are generally composed of a combination of sugar, frozen water, fruit, juice, and perhaps other flavors.
Others have expended considerable effort to develop a sorbet-type product, but such products are unable to deliver both a creamy texture and high fruit delivery. When fruit pieces and fruit puree are added to such products in order to deliver fruit, the usual texture achieved is cold and icy, rather than smooth and creamy.
The present invention is directed to frozen dessert products having a desirable creamy texture and providing high fruit delivery. In addition, the present invention provides for different embodiments of frozen dessert products. All of these characteristics are obtained by the present invention with simple and straightforward formulations. The use of such simple and straightforward formulations is unexpected and not predictable.
xe2x80x9cHigh fruit deliveryxe2x80x9d is defined for this description as the ability to provide in the product a strong fruit flavor and a high quantity of fruit in the form of fruit puree and/or fruit pieces.
The frozen dessert product of the present invention is formulated with ingredients that may provide fat, protein, carbohydrates, water, sweeteners, fruit taste, fruit, emulsifier, and stabilizer constituents in a combination that permits the attainment of the desirable properties described above. The attainment with these ingredients of these properties is deemed unexpected and not predictable.
The frozen dessert product of the present invention preferably comprises, on a percent solids (dry) basis, about 1% to about 12% high fructose corn syrup, about 5% to about 20% corn syrup having a DE of about 36, about 2% to about 22% sucrose, about 2% to about 6% maltodextrin, about 0.2% to about 5.0% egg white, about 1% to about 9% FRUITRIM(copyright) blend (comprising fruit juices and natural grain dextrins), and about 0.2% to about 1.5% stabilizer. In some embodiments, the frozen dessert product may comprise in addition to the above items, about 0% to about 0.12% emulsifier, about 0% to about 1.5% modified food starch, about 0% to about 10% yogurt culture, about 0% to about 5% milk fat, or about 0% to about 10% milk solids non-fat (MSNF). The product may also be fortified and/or enriched with vitamins, minerals, and fiber.
A method for production of the frozen dessert products is also provided by the present invention. In this method, a base mixture is prepared by placing into a mixer high fructose corn syrup, corn syrup (36 DE), sucrose, maltodextrin, egg white, FRUITRIM(copyright) blend, stabilizer, emulsifier (if used), modified food starch (if used), milk fat (if used), milk solids non-fat (if used), and vitamins (if used). The base mixture is agitated for a time to thoroughly mix the ingredients. The base mixture is then pasteurized and homogenized. After these steps, the base mixture is placed in a holding tank for flavoring with, for example, fruit puree, flavor ingredients, and/or fruit mixture. The base mixture and fruit puree or flavor ingredients are blended to form a final mixture and then passed through a scrape surface heat exchanger with final extrusion temperature from 18 degrees F to 23 degrees F. Fruit pieces and bulky flavor ingredients may be added prior to packaging using a conventional fruit feeder. Final product density ranges from about 4.5 to about 8 lbs/gallon. The final mixture is then packaged by conventional methods. The packaged mixture is then hardened for a predetermined period of time to a core temperature below xe2x88x9210 degrees F.
Accordingly, it is an object of the present invention to provide frozen dessert products having a desirable creamy texture. It is another object of the present invention to provide frozen dessert products that have a high fruit delivery. It is a further object of the present invention to provide methods for producing frozen dessert products having the above-identified properties.
These and other advantages, features, and objects of the present invention will be more readily understood in view of the following detailed description.
The present invention provides frozen dessert products that provide a creamy texture and high fruit delivery. Methods of production for the frozen dessert products are also disclosed herein. The frozen dessert products can be produced by the methods in a variety of flavors.
Throughout the specification and claims, percentages and ratios are by weight unless otherwise indicated and temperatures are in degrees Fahrenheit unless otherwise indicated.
The frozen dessert products of the present invention generally contain high fructose corn syrup, corn syrup having a DE of about 36, sucrose, maltodextrin, egg whites, FRUITRIM(copyright) blend (comprising fruit juices and natural grain dextrins), and stabilizers. In some embodiments, emulsifiers, milk solids non-fat, milk fat, modified food starch, and/or vitamins are present in the formulation. The amount and exact character of each ingredient will depend upon such factors as the character of the other ingredients and the desired nature of the end product. For example, the amount of fat is varied to provide regular and non-fat frozen dessert products. Furthermore, for example, egg whites are added in various amounts to provide a desired texture.
In an important embodiment, the composition of the present invention that can be used to prepare frozen dessert products generally has the following components as set forth in Table I.
Milk solids non-fat can be derived from cream, milk (including condensed milk, skim milk, and condensed skim milk), and non-fat dry milk. Milk solids non-fat aid in providing textural properties.
Dairy cream and milk are the preferred fat sources. It is to be understood that other fats can be used in the present invention.
The sweetener ingredients preferably are a combination of high fructose corn syrup, corn syrup, sucrose, maltodextrin, and FRUITRLM(copyright) blend that provide a desired level of sweetness and texture to the frozen dessert products. The non-high-fructose corn syrup preferably has a dextrose equivalent (DE) of about 36, which is in the low DE range of corn syrups. Corn syrups possessing a DE of about 24 to about 42 can be used in this invention. Corn syrup of 36 DE may be used in a highly preferred embodiment for ease of processability and satisfactory low-molecular-weight sugar composition.
The stabilizer ingredient may also contribute to the attainment of the desired features of the new frozen dessert products. The stabilizer ingredient is used to improve the ability of the products to withstand commercial shelf life and substantial heat shock without undue deterioration. The stabilizer ingredient may include stabilizers derived from plant gums and animal sources. Other stabilizers may be appropriate for use in the present invention, and such stabilizers will be evident to those skilled in the art and are meant to be included in this description of the frozen dessert products.
FRUITRIM(copyright) is the trademark for a liquid, carbohydrate-based blend formed from fruit juice(s) and dextrins from grain(s). FRUITRIM(copyright) blend is obtainable from Advanced Ingredients, Inc., 331 Capitola Avenue, Suite F, Capitola, Calif. 95010. The combination of mono- and disaccharides from the fruit juice(s) with selected medium and long-chain dextrins from grain creates a very effective, sweet-tasting ingredient with natural humectancy and low-water activity. FRUITRIM(copyright) blend comprises the following, in an approximate analysis: about 78% carbohydrate, said carbohydrate comprising about 50% to about 56% monosaccharides, about 12% to about 16% disaccharides, and about 6% to about 12% higher saccharides; about 18% to about 22% moisture; about 1.0% protein; about 0.7% ash, and about 0.25% fat. FRUITRIM(copyright) blend comprises about 78% to about 82% solids. The specific gravity of FRUITRIM(copyright) blend is about 11.8 lbs/gallon, and the viscosity is about 4,500 cps to about 22,000 cps at 25 degrees Centigrade and at 80xc2x12% solids. In a second type of analysis, FRUITRIM(copyright) blend comprises liquid carbohydrates as: from about 66% to about 74% monosaccharides, from about 12% to about 21% disaccharides, and from about 8% to about 16% other carbohydrates. Under the NLEA, the carbohydrates in FRUITRIM(copyright) blend would be divided into the following categories on labels: sugarsxe2x80x9476 g/100 g; total fiberxe2x80x940 g/100 g; other carbohydratesxe2x80x948 g/100 g.
Water is present in the new frozen dessert products as a dispersion medium for the other ingredients of the product. Water also influences the positive textural characteristics of the present invention. The portion of water in the product is determined on an xe2x80x9cas neededxe2x80x9d (q.s.) basis to provide the balance of the mixture with reference to a total weight of 100% after the weights of the other ingredients are taken into account. The portion of water that does freeze forms ice crystals that give the product rigidity as well as the refreshing taste perception characteristic of a frozen dessert. The portion of water in the products that remains unfrozen provides balanced texture for the product.
The above ingredients when combined as described in greater detail hereinbelow form a base mixture.
Flavor ingredients can be added to the base mixture if desired. Flavor ingredients are selected from known flavors according to the desired taste, taking into account the other ingredients. Flavor ingredients include, by way of illustration, fruit purees, fruit juices, as well as other flavor ingredients, for example, fruit flavors (such as concentrates or extracts), artificial flavors, yogurt culture, or ascorbic or citric acid. Fruit puree can be any type or combination of pureed fruits, for example, such as strawberry, banana, strawberry-banana, raspberry, peach, mixed berries, and the like. Fruit puree may also contain coloring, flavoring, and/or citric acid. Fruit puree is added as described below from about 20% to about 90% by volume, that is, for example, in one embodiment, when 20% by volume fruit puree is added to the base mix, the final product will contain about 80% by volume of base mix and about 20% by volume of fruit puree. Fruit pieces can be added from about 0% to about 12% by volume. Note, however, that the ingredient ranges specified herein, unless stated otherwise, are for an unflavored product. Those skilled in the art will comprehend the adjustments appropriate for the particular flavor ingredient used.
In addition to the foregoing ingredients, the frozen dessert products of the present invention can include other ingredients if desired, such as, for example, starches, emulsifiers, milk fat, and milk solids non-fat (MSNF). If starches are used in the formulations of the present invention, any modified food starch, such as, for example, corn starch, potato starch, rice starch, or tapioca starch, can be used. The starch used in the compositions of the present invention is preferably modified food starch. Modified starches, when used at low levels (less than about 1.5%), offer improved texture (creaminess) and improved shelf-life (stability) to the frozen dessert products. It is thought that starch molecules organize water in fashion similar to that of hydrocolloid (stabilizer) molecules by obstructing ice crystal growth and/or changing the morphology of ice crystals. Milk fat can be added to enhance creaminess, or other types of fats may be used for the same purpose.
In order to achieve the frozen dessert products of the present invention, a particular method is preferably used. In a preferred method of the present invention, a base mixture is formed by adding the ingredients in Table I above to a kettle provided with an agitating device. The preferred agitating device is a Lanco/Breddo(trademark) high shear mixer/liquefier.
The ingredients in Table I are added to the mixer, and agitation is commenced and continued for a certain period to form a base mixture. The base mixture is then pasteurized via vat under suitable time and temperature conditions, preferably at a temperature of about 165 degrees F for about 30 minutes or by high temperature processing (HTST) at about 180 degrees F to about 200 degrees F for about 30 seconds to about 180 seconds.
The pasteurized base mixture is then homogenized in a conventional two-stage homogenizer. In a preferred embodiment, homogenization comprises a first stage performed at a pressure of about 1,500 psi and a second stage following the first stage, with the second stage performed at about 500 psi.
After pasteurization and homogenization, the base mixture is placed in a holding tank. Fruit puree is then added to the flavoring tank in an amount as desired and blended with the base mixture to form a final mixture. If any other flavoring ingredients are desired to be added, they are then blended into the final mixture. In an alternative embodiment, the fruit puree and flavoring ingredients, if any, are added to the base mixture at the same time. After flavoring, the final mixture is passed through a scrape surface heat exchanger with a final extrusion temperature of from about 18 degrees F to about 23 degrees F. Fruit pieces and bulky flavors may be added prior to packaging using a conventional fruit feeder. For example, bulky flavors may comprise chocolate chips, candy bits, variegate, for example, such as fudge ribbon or chocolate ribbon or caramel ribbon, or nuts, all or any of which can be added to the final mixture before packaging. Final product density ranges from about 4.5 to about 8 lbs/gallon. The final mixture is then packaged using conventional methods. The packaged mixture is then hardened to a core temperature below about xe2x88x9210 degrees F.
Of importance to the present invention, the desired properties of a creamy texture and a high fruit delivery are obtained in the frozen dessert products of this invention with the use of the above-described ingredients in a straightforward combination and method of production. These results are deemed unexpected and advantageous.

Frozen dessert products and method of production


A foldable mechanism for a playpen includes a connection element having substantially an U-shaped cross section with two open side faces, one front face, and one rear face, the front and rear faces being provided with a U-shaped cutout and a plurality of first openings and a plurality of second openings; a press-down block having two sides each with a side groove having a narrow portion and a enlarged open end, front and rear faces each having a groove with two closed ends, an elongated through hole passing through the front and rear faces, and a hollow center with one closed end for receiving a spring, the press-down block being attached to the connection element by a first screw passing through the through hole and pressed against the spring, such that the press-down block can be pressed down, or biased, with respect to the connection element by an external force and moved back by the spring force; and a pair of hollow rods each having therein a U-shaped reed spring with protrusions on both outer faces of the reed spring, each rod being attached by a second screw to the connection element.

1. Field of the Invention
This invention relates to a foldable mechanism, and more specifically, to a foldable mechanism for a playpen.
2. Description of Prior Arts
A wide variety of playpens, have been designed, developed, and commercialized for many years. One of these designs is that the playpen may be folded for storage.
A well known foldable playpen, as shown in FIG. 1, uses a foldable structure 1, comprising a coupling element 2 having a substantially U-shaped cross section; a pair of hollow rods 3 each having therein an engaging element 4 which may extend or retract with respect to the rods through a spring 5, and pivotally screwed to the coupling element 2 by a screw 6; a fixed block 7 secured in the coupling element 2, having a cutout 8 for engaging with the engaging element 4; and a press-down element 9 positioned below and slidably connected to the fixed block 7 for disengaging the engagement between the engaging element 4 and the fixed block 7 when the press-down element 9 is pressed down, or biased, with respect to the fixed block 7 by an external force. When the playpen is in an unfolded condition, the rods 3 are in a horizontal orientation with the engaging element 4 being engaged with the cutout 8 of the fixed block 7. When the playpen is in a folded condition, the press-down element 9 is pressed to disengage the engaging element 4 from the fixed block 7, such that the rods 3 may pivot to a vertical orientation.
In the prior art stated above, the foldable structure uses many parts, which not only complicate the structure but also increase the cost thereof.
Further, in the prior art, most of the parts, such as the fixed block, the press-down element, and the engaging elements, are made of, for example, plastic, which will further increase the manufacturing cost.
Therefore, a foldable playpen that uses few parts and is less expensive to manufacture would be very desirable.


A portable electrical control and display device is described, having an indicator panel, a keyboard with at least one key pad, and a single housing designed as a single piece and made of a temperature-resistant material, which seals the control and display device in a water-tight manner.

1. Technical Field
This application relates to a control and display device and, in particular, to a protective covering for the device.
2. Description of Related Art
Devices, such as portable control and display devices, are generally known in a plurality of embodiments, for example, as measuring instruments. These instruments may be used in the food industry and in food processing plants such as, for example, slaughterhouses, refrigeration plants, restaurants, and the like. Instruments that may be used, for example, in the food industry and in areas related to food processing, may be required to comply with certain hygienic requirements and other regulatory requirements such as those set forth in national and international regulations. These requirements may relate to the cleanliness of the instruments, such as measuring instruments. The requirements may relate to, for example, prevention of contamination, such as, bacterial contamination, of the measuring instruments.
In order to avoid such contamination, the instruments may include protective devices. These protective devices are used for hermetically sealing the measuring instruments against water, dust, and similar contamination in a way so that the functionality and operability of the instrument are not affected by the protective device. One type of protective device includes flexible, mostly transparent, bag-type protective sheathings. These sheathings may, more or less, protect the instrument from water, dust, and the like. The functionality and operability of the instrument protected by the protective sheathing remain preserved thanks to the flexible sheathing. However, these flexible protective sheathings may be made of a relatively thin plastic material. The functionality of such protective sheathings may be reduced over time due to the rough operating conditions to which the measuring instruments may be exposed. Under extreme operating conditions, there is also the danger that the instrument may become damaged due to the effect of external forces, since the thin protective sheathing may be extremely sensitive to impact and forces. In other words, the protective sheathings may not be able to absorb such forces thereby possibly resulting in damage to the instrument.
Another disadvantage of these known protective sheathings is the fact that even the slightest damage to the protective sheathing surface may result in impairing the required water-tightness. This may subsequently irreversibly damage or destroy an electronic component that may be included in the instrument, and may be sensitive to moisture. Furthermore, once the instrument is in the protective sheathing, it cannot be attached to a bracket provided, and external components cannot be attached to the instrument.
German Patent Application 39 05 130 A1 describes an improved protective device for a measuring instrument. In contrast to the foregoing bag-type protective sheathing, German Patent Application 39 05 130 A1 describes a cover-like shield made of a non-deformable material used as a protective device. The shield covers the control and display elements arranged in the housing, thus protecting them. In order to attach the cover to the housing, fastening elements are provided. To ensure tightness, an integrated sealing lip is provided around the housing part of the measuring instrument. The shield that covers and seals the control and display elements is transparent at least in the areas of the above-named display and control elements.
As an alternative, it is also conceivable to design the protective device as a two-part protective housing (holster). An internal contour of the protective housing may be designed so that the measuring instrument can be placed inside with essentially no play, and the two protective housing parts held together by fastening elements.
Although the protective device designed as a non-deformable shield and the protective housing designed as a holster have a high degree of water-tightness when used with a control and display device, providing protection against the effect of outside forces, there may still remain problems and drawbacks.
Manufacturing costs may be substantially increased since the measuring instruments to be used in the aforementioned areas requires an additional protective housing. Even in this case, it is not possible to use a shield made of a very soft material, for example, a plastic, since such material does not have the rigidity required to obtain an effective seal or, alternatively may have this property only under certain conditions. Therefore, a two-component part having high rigidity may be used in order to obtain the required impact-absorbing soft surface. This may be, however, unacceptable due to the high cost involved.
Due to the sealing lip around and integrated in the housing part, a large surface is sealed. This may be very expensive especially when a tight seal is required. Therefore, often a plurality of support points, for example, screws or clamps, may be required and may result in an inability to obtain the required high degree of tightness. For example, screws must be perfectly tightened to obtain a tight seal in an effort to prevent gaps, and thus untightness, from occurring. In particular, when the screws are frequently removed from the measuring instruments, this gap-free seal becomes more and more difficult to guarantee.
Additionally, sealing forces and joining forces act in the same direction when the shield is mounted on the housing. Accordingly, higher sealing forces may be required to be applied for a good seal than in an alternative instance when the forces do not act in parallel. Undesirable tolerances and slack may be poorly compensated for, or not at all, when the shield is mounted on the measuring instruments due to the close geometric relationship between the shield and the measuring instrument.
Failure to use such a protective housing in the aforementioned cases for reasons of time or convenience or due to lack of tools for fastening the fastening means may occur, and the measuring instrument used may not be able to be cleaned in a convenient and, mainly, reliable manner in a cleaning machine.
An additional condition in handling the aforementioned measuring instruments is the high hygienic requirements. In the aforementioned fields of application of the instruments provided with such protective housings, different cleaning procedures are used. It has been found that cleaning procedures, for example, such as one using a wet but not previously used cleaning cloth, a disposable cloth, a cleaning agent (RIX), or a cleaning agent with subsequent alcohol disinfection, clean the instruments, more or less well. However, complete, one-hundred-percent cleaning cannot be assured with these cleaning procedures. Only when instruments are cleaned in a cleaning machine for 45 minutes at a temperature of 75 degrees Celcius is one hundred-percent cleaning effect achieved with both non-fatty and fatty contamination.
In order to reliably meet the required hygienic requirements, it is therefore desirable to subject the instruments to cleaning in a cleaning machine.
The invention provides a control and display device ensuring a relatively high degree of tightness, in particular, water-tightness. This is achieved according to the present invention by a control and display device which includes a single one-piece housing made of a temperature-resistant material, which seals the control and display device in a water-tight manner from the outside.
The present invention is based on the idea of integrating previously used multi-part additional protective housings into essentially one housing for a measuring instrument. This measuring instrument therefore includes a single, impact-resistant and water-tight housing, which may be a fixed component of this measuring instrument. Advantages of this measuring instrument, compared with known, two-part or multi-part, measuring instruments that require a water-tight housing (holster, shield) for use in wet environments, include low manufacturing costs.
An embodiment of the single-part housing is also very safe for the process concerning tightness since no screws are used that have to be perfectly tightened. Since the housing may be made of a temperature-resistant material and provide for a seal from the outside in a water-tight manner, it is suitable for cleaning with hot water of at least 75 degrees Celcius. Thus the housing may be cleaned in a cleaning machine in a simple manner, whereby the highest possible degree of disinfection is guaranteed.
The control and display device according to the present invention ensures not only the highest possible degree of water-tightness, but also very effective protection against impact and contamination is achieved at the same time.
Due to its elasticity and its shape, the housing fits the shape of the sleeve in an almost form-fitting manner. Thus no gap may occur between the housing and the sleeve, whereby the arrangement according to the present invention also provides for compliance with hygienic requirements.
In the case of a defective housing and/or a defective measuring instrument, the housing may be detached from the measuring instrument in a simple manner and, if necessary, replaced with a new housing. After checking and, if needed, repair, an instrument that gives the visual impression of being new may be provided by replacing the housing.
According to one embodiment of the present invention, the housing encloses a sleeve in which the circuit board, the indicator panel, the keyboard, or at least the contact-sensitive areas of the keyboard, are accommodated. This sleeve may be designed in the form of a box and may be closed on one or more sides by a cover. The sleeve has the function of supporting, as well as accommodating, the aforementioned elements of the measuring instrument. In another embodiment, the sleeve may be omitted, if, for example, the housing itself assumes the protective function. In this instance, the housing may have one or more support braces.
One advantage of the housing according to the present invention is that the housing may be mounted on the sleeve without tools while still achieving a high degree of tightness. This may be particularly advantageous if, for example, the measuring instrument used for on-site measurements cannot be cleaned. In this case, a plurality of clean, i.e., disinfected, housing sheathings may be used, for example, and substituted for the used housing sheathings.
Another advantage that may be achieved using the present invention is that the directions of the joining force and the sealing force may be perpendicular to one another. In order to achieve a high degree of tightness, relatively small sealing forces are sufficient for sealing. Since sealing occurs perpendicularly to the direction of joining with almost no force, no fastening elements such as screws or clamps are required here, which may reduce development and manufacturing time, sealing surfaces and therefore fastening material and sealing material costs.
The sealing device between sleeve and housing may be implemented using, for example, a peripheral groove or a peripheral ridge, applied to the sleeve or the housing so that the ridge engages in the groove when the sleeve and the housing are joined together. In this instance, a particularly good sealing effect may be achieved between the two parts by the use of an elastic material in conjunction with an at least slightly larger groove compared to the ridge. The sealing effect of the sealing device between the sleeve and the housing may be further enhanced by applying sealing lips on the groove and/or the ridge.
In another embodiment of the sealing device, a plug or a ring, advantageously reinforced by braces, is provided, which is pushed into the housing that is open on one side. This ring or plug may be attached to a cover, to the sleeve, or to the housing. This ring or plug may be have pointed pins, nubs, peripheral ribs, or wedges, which engage in the softer material of the jacket on the opposite side or press on the harder material by deformation to achieve a good sealing effect. In these cases, cylindrical rings or plugs, which taper slightly in the direction of insertion, may be used to achieve a sealing effect. The sealing device may also include a snap-in detection device to ensure reliable engagement of the ridge in the groove, or of the plug, or ring, in the housing, or the sleeve.
A more or less soft plastic, such as an elastomer, may be used as the housing material in accordance with the application and the need. This plastic material may have the required impact-absorbing rigidity required for sealing and protection, as well as the soft surface for the functionality of the keys to be pressed. The housing may be made, at least in part, of a TPU, TPE, or PVC material. An injection molded part that may be easily and inexpensively manufactured, may be used as the housing.
In using such materials, for example, soft PVC or thermoplastic TPU, a fully transparent housing may be used in an embodiment. However, an alternative embodiment may include a housing of a semi-transparent material. The use of transparent or semi-transparent materials may-result in ease of reading characters, for example, those included on the indicator panel, the keyboard, and/or additionally, markings on the sleeve. In an embodiment, the housing may also be manufactured of a non-transparent, colored material. In this case, a transparent protective sight glass may be provided in the area of the indicator panel.
The housing may be an elongated plastic hose having the same diameter as, or a slightly larger diameter than, the sleeve of the measuring instrument. In this case, assembly may be performed as by pulling the hose over the measuring instrument and providing it with suitable sealing devices at its open ends. The adhesive forces may be provided by the adhesion of the hose having a larger size to the sleeve. When a hose is used, advantageously little or no tool costs may be involved.
According to another embodiment of the present invention, the housing includes a number of push rods corresponding to the number of keys in the keyboard area. The push rods raise outward over the surface of the housing. The control area or the keyboard of the measuring instrument may also be formed by a housing section having a reduced wall thickness. In this case, the control area may be manufactured in one piece with the housing from the same material.
In another embodiment, recesses may be provided, on the side facing away from the control area or the keyboard. These recesses may be used for attaching the device to a corresponding bracket. To achieve increased rigidity in use and improved handling of the housing, in another embodiment of the device at least one reinforcing strip is provided, which is made of the same material as the housing. The reinforcing strip furthermore may provide increased protection of the circuit board located inside the sleeve against forces acting from the outside.
The sealing effect in the area of an opening may be ensured by matching the shape and size of the opening to the shape and size of the plug, so that the housing is in contact with the plug over the entire surface of the latter and thus in a water-tight manner. In another embodiment, a sealing lip is provided in the opening, which is in a linear peripheral contact with the plug passing through it, thus providing a water-tight seal. This variant has the advantage that the plug may be inserted in the opening using little force. The opening may be closed with an appropriate cap when no plug is used.


An oral composition comprises perlite, characterised in that it comprises from 0.01 to 0.9% by weight perlite.

1. Field of the Invention
This invention relates to an oral composition comprising low levels of perlite.
2. The Related Art
The inclusion of abrasives in oral compositions such as toothpaste is well known. The abrasive has a cleaning as well as a polishing/whitening benefit. The removal of tartar from the tooth surface is thought to reduce the incidence of caries.
Dental Practitioners recommend that tartar removal should be exercised professionally at least twice a year, preferably more but it is recognised that the build-up of tartar can be reduced by effective brushing of the teeth.
Typical abrasives used in toothpastes include silicas and chalk, however, the prior art also discloses the use of perlite in toothpaste formulations. EP-B1-0 268 763 (Hawe-Neos) describes a dental care medium for prophylactic dental hygiene comprising perlite and a synthetically produced precipitation silicic acid. U.S. Pat. No. 5,266,304 (Baffelli) describes dental care and cleaning composition in a water-free part containing at least 40% by weight of perlite as a sole and combined cleaning and polishing body. U.S. Pat. No. 5,597,553 (Baffelli) describes a toothpaste comprising as an abrasive, preferably as sole abrasive, expanded perlite, generally with a particle size in the range of 1 to 150 xcexcm, in particular with main fraction in the region 20 xcexcm. The perlite is comprises from 1 to not more than 15% by weight of the composition.
The abrasivity of a toothpaste is measured according to a protocol described in the journal of Dental Research (1976) 55(4), 563. This describes how the Relative Dental Abrasion (RDA) and Relative Enamel Abrasion (REA) are evaluated.
Ideally, a toothpaste will be capable of cleaning the teeth without wearing down the tooth enamel and dentine. So while it is necessary to have a cleaning efficacy a toothpaste with too high an RDA is undesirable. In Advanced Dental Research Vol 11, (4) pp576-579 is described a method for evaluating the Pellicle Cleaning Ratio (PCR) which is commonly used as a measure of cleaning.
The use of high quantities of perlite also has its disadvantages. Perlite is a natural product and while it is purified to a reasonable degree before incorporation into an oral care formulation it still comprises impurities. These impurities are usually of a grey colour and introduce speckling of the formulation. This is particularly noticeable where the product is a white paste. Again, this is seen as a significant consumer negative. A further disadvantage of high levels of perlite is that it behaves as a catalyst for the degradation of flavour ingredients, particularly in products where the pH is relatively high, e.g. in oral compositions comprising bicarbonate. This is a clear disadvantage for the consumer but is also unacceptable for the manufacturer as the cost of the flavour often accounts for the bulk of the raw materials costs. It is also known for high quantities of perlite to cause syneresis of typical dentifrice formulations.
We have surprisingly found that perlite can be incorporated as a cleaning agent in smaller amounts than is disclosed in the prior art, while still providing an effective cleaning benefit. Further the abrasive effect of the reduced levels of perlite also provide for a greater cleaning/polishing with respect to abrasivity than higher quantities of perlite, i.e. the PCR substantially maintained or reduced only partially, while the RDA is significantly reduced by using smaller amounts of perlite.
Accordingly, the invention provides an oral composition comprising from 0.01 to 0.9% by weight of perlite.
Preferably, the composition according to the invention comprises from 0.1 to 0.8% and especially from 0.3 to 0.7% by weight of perlite.
Perlite is a naturally occurring sodium/potassium/aluminium silicate and is available commercially from Seitzfilterwerke as Perlite C; from Elfatochem as Ceka Flo MA/P/2A/R; and from World Mineral as Europearl 475/900S/E50.
A preferred perlite comprises particles of average size below 300 and preferably below 200 xcexcm in diameter. It is understood that the size reference to diameter is approximate as the particles are of random shape and not necessarily spherical. If a sample comprises larger particles of perlite it is preferred that the sample is sieved through a suitable gauge before being included in the formulation.
The composition according to the invention may be any oral, non-food composition, e.g. toothpaste and may be in the form of a gel, paste, gum or any other suitable type.
The composition according to the invention may also comprise ingredients, which are common in dentifrices. Examples of such ingredients include:
antimicrobial agents, e.g. Triclosan, chlorhexidine, copper-, zinc- and stannous salts such as zinc citrate, zinc sulphate, zinc glycinate, sodium zinc citrate and stannous pyrophosphate, sanguinarine extract, metronidazole, quaternary ammonium compounds, such as cetylpyridinium chloride; bis-guanides, such as chlorhexidine digluconate, hexetidine, octenidine, alexidine; and halogenated bisphenolic compounds, such as 2,2xe2x80x2 methylenebis-(4-chloro-6-bromophenol);
anti-inflammatory agents such as ibuprofen, flurbiprofen, aspirin, indomethacin etc.;
anti-caries agents such as sodium-, calcium-, magnesium- and stannous fluoride, aminefluorides, disodium monofluorophosphate, sodium trimeta phosphate and casein;
plaque buffers such as urea, calcium lactate, calcium glycerophosphate and strontium polyacrylates;
vitamins such as Vitamin C;
plant extracts;
desensitising agents, e.g. potassium citrate, potassium chloride, potassium tartrate, potassium bicarbonate, potassium oxalate, potassium nitrate and strontium salts;
anti-calculus agents, e.g. hypophosphite-containing polymers, organic phosphonates and phosphocitrates etc.;
gum protection agents, e.g. vegetable oils such as sunflower oil, rape seed oil, soybean oil and safflower oil; silicone oil; and hydrocarbon oil. The gum protection agent may be an agent capable of improving the permeability barrier of the gums. A complete description of agents capable of improving the permeability barrier of the gum is found in our co-pending application GB 9810521;
biomolecules, e.g. bacteriocins, antibodies, enzymes, etc.;
flavours, e.g. peppermint and spearmint oils;
preservatives;
opacifying agents;
colouring agents;
pH-adjusting agents;
sweetening agents;
pharmaceutically acceptable carriers, e.g. starch, sucrose, water or water/alcohol systems etc.;
surfactants, such as anionic, nonionic, cationic and zwitterionic or amphoteric surfactants;
particulate abrasive materials such as silicas, aluminas, calcium carbonates, dicalciumphosphates, calcium pyrophosphates, hydroxyapatites, trimetaphosphates,
insoluble hexametaphosphates and so on, including agglomerated particulate abrasive materials;
humectants such as glycerol, sorbitol, propyleneglycol, xylitol, lactitol etc.;
binders and thickeners such as sodium carboxymethyl-cellulose, xanthan gum, gum arabic etc. as well as synthetic polymers such as polyacrylates and carboxyvinyl polymers such as Carbopol(copyright);
buffers and salts; and
other optional ingredients that may be included are e.g. bleaching agents such as peroxy compounds e.g. potassium peroxydiphosphate, effervescing systems such as sodium bicarbonate/citric acid systems, colour change systems, and so on.
The oral composition may also be in any of the product forms common in the art, e.g. dentifrice (gel or paste), lozenge, chewing gum etc.

Unilever Home and Personal Care USA, division of Conopco, Inc.


A cooling system for cooling of the squealer tip surface region of a high pressure turbine blade used in a gas turbine engine and a method for making a system for cooling of the squealer tip surface region of a high pressure turbine blade used in a gas turbine engine. The method comprises the steps of channeling apertures in a tip cap to a diameter of about 0.004xe2x80x3 to about 0.020xe2x80x3 to allow passage of cooling fluid from a cooling fluid source; applying a bond coat of about 0.0005xe2x80x3 to about 0.010xe2x80x3 in thickness to the tip cap such that the bond coat partially fills the channels; applying a porous TBC layer of at least about 0.003xe2x80x3 in thickness to the bond coat, such that the porous TBC fills the channels; applying a dense ceramic TBC layer over the porous layer; and, passing cooling fluid from a cooling fluid source through the channel into the porous TBC. The density of the dense TBC layer can be varied as needed to achieve desired cooling objectives. Because the channel exit is filled with porous TBC material, cooling fluid flows through the porous passageways in the porous TBC layer into the squealer tip. Although the passageways provide a plurality of tortuous routes, the increased density of the TBC in the dense ceramic layer provides a resistance to flow of the cooling fluid and effectively causes the cooling fluid to more efficiently spread through the TBC into the squealer tip before exiting into the gas stream at the outer surface.

This invention relates generally to gas turbine engines, and in particular, to a cooled flow path surface region of a turbine blade tip.
In gas turbine engines, for example, aircraft engines, air is drawn into the front of the engine, compressed by a shaft-mounted rotary-type compressor, and mixed with fuel. The mixture is burned, and the hot exhaust gases are passed through a turbine mounted on a shaft. The flow of gas turns the turbine, which turns the shaft and drives the compressor and fan. The hot exhaust gases flow from the back of the engine, driving it and the aircraft forward.
During operation of gas turbine engines, the temperatures of combustion gases may exceed 3,000xc2x0 F., considerably higher than the melting temperatures of the metal parts of the engine, which are in contact with these gases. Operation of these engines at gas temperatures that are above the melting temperatures of the metal components is a well established art, and depends in part on supplying a cooling air to the outer surfaces of the metal parts through various methods. The metal parts of these engines that are particularly subject to high temperatures, and thus require particular attention with respect to cooling, are metal parts forming combustors and parts located aft of the combustor including turbine blades, turbine vanes and exhaust nozzles.
The hotter the turbine inlet gases, the more efficient is the operation of the jet engine. There is thus an incentive to raise the turbine inlet gas temperature. However, the maximum temperature of the turbine inlet gases is normally limited by the materials used to fabricate the turbine vanes and turbine blades of the turbine. In current engines, the turbine vanes and blades are made of nickel-based superalloys, and can operate at metal surface temperatures of up to 2100xc2x0-2200xc2x0 F.
The metal temperatures can be maintained below melting levels with current cooling techniques by using a combination of improved cooling designs and insulating thermal barrier coatings (TBCs). For example, with regard to the metal blades and vanes employed in aircraft engines, some cooling is achieved through convection by providing passages for flow of cooling air internally within the blades so that heat may be removed from the metal structure of the blade by the cooling air. Such blades essentially have intricate serpentine passageways within structural metal forming the cooling circuits of the blade.
Small internal orifices have also been devised to direct this circulating cooling air directly against certain inner surfaces of the airfoil to obtain cooling of the inner surface by impingement of the cooling air against the surface, a process known as impingement cooling. In addition, an array of small holes extending from the hollow core through the blade shell can provide for bleeding cooling air through the blade shell to the outer surface where a film of such air can protect the blade from direct contact with the hot gases passing through the engines, a process known as film cooling.
In another approach, a TBC is applied to the turbine blade component, which forms an interface between the metallic component and the hot gases of combustion. The TBC includes a ceramic coating that is applied to the external surface of metal parts within engines to impede the transfer of heat from hot combustion gases to the metal parts, thus insulating the component from the hot combustion gas. This permits the combustion gas to be hotter than would otherwise be possible with the particular material and fabrication process of the component. TBCs have also been used in combination with film cooling techniques wherein an array of fine holes extends from the hollow core through the TBC to provide cooling air onto the outer surface of the TBC.
Certain designs of airfoil tips utilize film cooling techniques. Film cooling is achieved by passing cooling air through discrete film cooling holes, typically ranging from 0.015xe2x80x3 to about 0.030xe2x80x3 in hole diameters. The film cooling holes are typically drilled with laser or EDM or ES machining. Due to mechanical limitations, each film hole has an angle ranging from 20xc2x0 to 90xc2x0 relative to the external surface. Therefore, each film jet exits from the hole with a velocity component perpendicular to the surface. Because of this vertical velocity component and a complex aerodynamic flow circulation near the tip of a turbine blade, commonly referred to as the xe2x80x9csquealer tipxe2x80x9d, each film jet will have a tendency to lift or blow off from the external surface and mix with the hot exhaust gases, resulting in poor film cooling effectiveness in the area surrounding the squealer tip.
TBCs are well-known ceramic coatings, for example, yttrium-stabilized zirconia (YSZ). Ceramic TBCs usually do not adhere well directly to the superalloys used in the substrates. Therefore, an additional metallic layer called a bond coat is placed between the substrate and the thermal barrier coating. The bond coat may be made of a nickel-containing overlay alloy, such as a MCrAlX, or other compositions more resistant to environmental damage than the substrate, or alternatively, the bond coat may be a diffusion nickel aluminide or platinum aluminide, whose surface oxidizes to a protective aluminum oxide scale that provides improved adherence to the ceramic top coatings. The bond coat and the overlying TBC are frequently referred to as a thermal barrier coating system.
Multi layer coatings are known in the art. For example, U.S. Pat. No. 5,846,605 to Rickerby et al. is directed to a coating having a plurality of alternate layers having different structures that produce a plurality of interfaces. The interfaces provide paths of increased resistance to heat transfer to reduce thermal conductivity. A bond coat overlying a metallic substrate is bonded to a TBC. The TBC comprises a plurality of layers, each layer having columnar grains, the columnar grains in each layer extending substantially perpendicular to the interface between the bond coat and metallic substrate. The structure is columnar to ensure that the strain tolerance of the ceramic TBC is not impaired. The difference in structure of the layers is the result of variations in the microstructure and/or density/coarseness of the columnar grains of the ceramic.
U.S. Pat. No. 5,705,231 to Nissley et al. is directed to a segmented abradable ceramic coating system having enhanced abradability and erosion resistance. A segmented abradable ceramic coating is applied to a bond coat comprising three ceramic layers that are individually applied. There is a base coat foundation layer, a graded interlayer, and an abradable top layer. The coating is characterized by a plurality of vertical microcracks.
U.S. Pat. No. 4,503,130 to Bosshart et al. is directed to coatings having a low stress to strength ratio across the depth of the coating. Graded layers of metal/ceramic material having increasing ceramic composition are sequentially applied to the metal substrate under conditions of varied substrate temperature. Excessive stresses induced by differential strains between the layers is avoided. The effect of substrate temperature control and the differing coefficients of thermal expansion between materials of successive layers are matched to achieve the desired result.
U.S. Pat. No. 6,045,928 to Tsantrizos et al. is directed to a TBC comprising an MCrAlY bond coat and a dual constituent ceramic topcoat. The topcoat comprises a monolithic zirconia layer adjacent to the bond coat, a monolithic layer of calcia-silica representing the outer surface of the TBC and a graded interface between the two to achieve good adhesion between the two constituents to achieve an increased thickness of the topcoat, thereby, providing for a greater temperature drop across the TBC system. As used by Tsantrizos et al., monolithic refers to a uniform composition of a layer, while a graded interface refers to a layer having a changing composition from one monolithic composition to the other monolithic composition.
U.S. Pat. No. 4,576,874 to Spengler et al. is directed to a coating to increase resistance to spalling and corrosion. The coating is not intended to be a thermal barrier coating. A porous ceramic is applied over a MCrAlY bond coat and a dense ceramic is then applied over the porous ceramic. The porous portion is a transition zone to allow for differences in thermal expansion and provides little thermal insulation.
Improved environmental resistance to destructive oxidation and hot corrosion is desirable. In addition, the alloying elements of the bond coat interdiffuse with the substrate alloy, changing the composition of the protective outer layer so that the walls of the turbine airfoils are consumed. This loss of material reduces the load carrying capability of the airfoil, thereby limiting blade life. This interdiffusion can also reduce environmental resistance of the coating. This interdiffusion and its adverse effects can be reduced by controlling the temperature of the component in the region. of the bond coat/substrate interface.
Thus, there is an ongoing need for an improved thermal barrier coating system, especially surrounding the squealer tip, wherein the environmental resistance and long-term stability of the thermal barrier coating system is improved so that higher engine efficiencies can be achieved. The bond coat temperature limit is critical to the TBC""s life and has had an upper limit of about 2100xc2x0 F. Once the bond coat exceeds this temperature, the coating system will quickly deteriorate, due to high temperature mechanical deformation and oxidation, as well as interdiffusion of elements with the substrate alloy. The coating system can separate from the substrate exposing the underlying superalloy component to damage from the hot gasses.
In particular, the squealer tip is the most difficult location to cool in a turbine blade. The squealer tip is located away from the convection cooling in the center of the blade, and the complex aerodynamic flow field near the squealer tip makes film cooling very inefficient. This inefficient cooling results in tip deterioration much earlier than desired, and requires tip repairs after relatively short time in-service to recover the tip clearance for better turbine efficiency.
As described above, to be more effective in permitting the attainment of higher engine operating temperatures, a TBC requires active cooling on the backside of the region being cooled. A TBC has not been considered for use in the squealer tip region or airfoils partly because of physical constraints and partly because no backside cooling was available to take advantage of the capabilities of the TBC by removing heat, thereby preventing a build-up of temperature in this region. During the airfoil manufacturing process, to prevent application of TBC coating in this area, the squealer tip and cap have usually been masked during the TBC coating process. However, recently, in order to reduce the manufacturing costs associated with the time consuming process of masking, the TBC coating application has been extended to cover the squealer tip, thus avoiding the masking process. The consequence of the presence of the TBC in this region has been that the higher temperatures experienced in this region because of a lack of cooling causes the TBC to spall from the backside of the squealer tip region after several cycles of engine operation, resulting in the same general configuration that occurs when masking is performed to prevent the deposition of the TBC. However, since it has become routine to apply a TBC to this region, it would therefore be advantageous to take advantage of the presence of the TBC by further improving squealer tip cooling by intentionally incorporating an effective TBC coating system to extend squealer tip life, which can be accomplished by providing adequate cooling of the region.
What is needed are improved designs that will allow a turbine engine blade squealer tip to run at higher operating temperatures, thus improving engine performance without the need for additional cooling air for the blade. It is also desirable to have a system that can take advantage of the thermal insulation provided by TBC. The present invention fulfills this need, and further provides related advantages.
The present invention provides a method for cooling the squealer tip region of a high pressure turbine blade used in a gas turbine engine comprising the steps of channeling apertures in a substrate to a diameter sufficient to allow passage of cooling fluid from a cooling fluid source; applying a sufficiently thick bond coat to the substrate such that the bond coat partially fills the apertures; applying a porous TBC layer to the bond coat, such that the TBC partially or completely fills the apertures; and applying a dense ceramic layer that is more dense than the porous TBC layer on top of the porous TBC layer. Optionally, conventional TBC can be applied on the concave (pressure side) and convex (suction side) of the airfoil surface.
In this manner, cooling fluid passes from a cooling fluid source through a channel aperture adjacent to the squealer tip into the porous TBC. Because the channel aperture is filled with porous TBC material, cooling fluid flows through the porous passageways into the porous TBC layer, continuing to flow between the bond coat and the dense coat, exiting to the squealer tip or any locations inside the tip cavity. In this manner, cooling fluid is directed to the squealer tip, previously unobtainable using known methods.
The present invention further comprises both the cooled blade and squealer tip region formed by the foregoing methods and the blade and squealer tip with the ceramic layers for cooling the squealer tip.
One advantage of the present invention is that the passageways provide a plurality of tortuous routes, whereby the increased density of the TBC in the dense outer layer having reduced porosity provides a resistance to flow of the cooling fluid and effectively causes the cooling fluid to more efficiently spread through the porous TBC before exiting at the outer surface.
Another advantage of the present invention is that the multi-layered TBC system forms cooling paths which allow cooling air from the interior of the blade to flow from tip holes adjacent to the squealer tip into the porous ceramic layer flowing between the bond coat and the dense coat , thereby flowing to the tip or any locations inside the tip cavity, providing efficient active convection cooling for both the squealer tip substrate and the bond coat by allowing heat to be removed from the squealer tip.
By removing heat from this region, the integrity of the bond coat can be maintained at higher engine firing temperatures, resulting in a more efficient usage of cooling fluid than that of the prior art to achieve a higher turbine engine efficiency and performance while improving squealer tip service life.
Still another advantage of the present invention is that the cooling channel exit apertures, being at least partially filled with porous TBC, have more flow resistance than open apertures and, therefore, provide a more uniform cooling flow distribution compared to unfilled conduits that transfer the same amount of cooling fluid, resulting in more efficient heat transfer.
Other features and advantages of the present invention will be apparent from the following more detailed description of the preferred embodiment, taken in conjunction with the accompanying figures which illustrate, by way of example, the principles of the invention.

Cooled thermal barrier coating on a turbine blade tip