Title of Invention

A PROCESS FOR REDUCING THE ACRYLAMIDE CONTENT OF HEAT-TREATED FOODS

Abstract !he present invention relates to a process for reducing the acrylamide content of heat-treated foods compared with corresponding heat-treated foods produced from non-genetically-modified plant material, comprising: (a) selecting genetically modified plant material originating from potato plants, wherein the genetic modification leads to a reduction in the content of soluble sugars, compared with corresponding non-genetically modified plant material from wild type plants, and wherein said genetic modification leads to a reduction in the activity of one or more endogenous RI proteins occurring in the plant cell compared with corresponding plant cells of wild type plants which have not been genetically modified; (b) processing the said plant material to give a food; and (c) heat-treating the food produced in process step b) at temperatures of at least 100°C.
Full Text

Process for reducing the acrylamide content of heat-treated foods
ie present invention relates to a process for reducing the acrylamide ntent of heat-treated foods, compared with conventional heat-treated foods.
recentiy, the Swedish National Food Administration (NFA) and scientists >m Stockholm University have published new research results according which, in various foods which are given a high heat treatment on preparation, acrylamide, a toxic and possibly carcinogenic substance, is formed. The NFA informed other national and international authorities and organizations in order to stimulate international collaboration and search, since acrylamide formation on heating foods is obviously a despread phenomenon. Then, in summer 2002, in Geneva, an expert consultation took place which had been convened jointly by the Food and agriculture Organization of the United Nations (FAO) and the World Health rganization (WHO) (WHO, FAO/WHO Consultation on the Heaith implications of Acrylamide in Food (Geneva, 25-27 June 2002).
he expert consultation discussed the following as essential end points of ie toxicological effects of acrylamide: neurotoxicity, reproductive toxicity, lutagenicity and carcinogenicity.
in particular, the expert consultation started from the position that the enotoxic potential of acrylamide and its metabolic product glycidamide lays an important role. In vivo, acrylamide is genotoxic in somatic cells nd in germ cells. It can therefore cause Inheritable damage at the level of ie genes and also the chromosomes. As is known, one of Its metabolic roducts is glycidamide, a chemically reactive epoxide, which can react



Under certain experimental conditions, acrylamide appears to form in vitro in the reaction of amino adds, in particular asparagine (Mottram et ai., Nature 419, (2002), 448; Stadler et ai.. Nature 419, (2002), 449) with sugars, for example fructose, galactose, lactose or sucrose (Stadler et ai.. Nature 419, (2002), 449).
The causes of the variability in acrylamide contents in heat-treated foods are not yet sufficiently understood (WHO. FAG/WHO Consultation on the Health Implications of Acrylamide in Food (Geneva, 25-27 June 2002)).
The international expert consultation convened by the FAO and the WHO recommended study of the relationship between processing conditions of foods and the formation of acrylamide, and also the optimization of processing conditions with the aim of minimizing acrylamide contents.
Processes for minimizing acrylamide contents in heat-treated foods have not yet been described to date in the prior art and are urgently required. The object therefore underlying the present invention is to provide processes which permit the production of heat-treated foods which, compared with conventional heat-treated foods, have a reduced acrylamide content.
This object is achieved by the provision of the embodiments described in the patent claims.
The present invention therefore relates to a process for reducing the acrylamide content of heat-treated foods compared with corresponding conventional heat-treated foods comprisirg . -
a) providing or selecting plant material which; compared with corresponding conventional plant material, has a reduced



"heat-treated foods". The preliminary stages, in particular potato slices, for producing the heat-treated foods may aiso be present in the precooked or blanched form or frozen form.
The term "heat-treated food*, in the context of the presenr invention, is :c be taken to mean any food which has been exposed to temperatures of > 10Q°C, preferably of 110°C to 230°C. in particular 120°G-20C3C. preferably of 15G°C-170°C. oarticulariy preferable 15QoC-130°C. The :er.^ "heat treatment", in the context of the present invention, is to be taken to mean any treatment which, under standard pressure conditions, leads to temperatures of above 100°C, in particular it is to he taken to mean deep-fat frying, grilling, frying, roasting, extruding, backing or microwave heating, autoclaving or parfrying.
The heat treatment time can differ depending on the food. The absolute acry/lamide contents"aiways increase wirh the heat treatment time. With the aid of the present invention it is now possible to lower the acryiamide content of a food which has been heat-treated at a defined temperature for a defined time by a defined method, compared with conventional heat-treated foods.
In the context of the present invention, especially with regard to potato chips and crisps, the heat treatment, when this is a deep-fat frying process, is carried out for 10 seconds to 8 minutes, preferably for 2 to 5 minutes, particularly preferably for 2 to 3 minutes, if the heat treatment is a baking process, the heat treatment is carried out. in the context of the present invention, for one to 120 minutes preferabiy for 5 to 30 minutes.
in the context of the present invention, especially with regard to the production of partially fried (parfried) potato chips, the heat treatment of



In the context of the present invention the English terms 'potato crisp" and "potato chips* are used instead of the synonymous American terms "potato chips" and "French fry".
The term "conventional heat-treated food", in the context of the present invention, is to be taken to mean a food which has been produced from conventional plant material. The term "corresponding conventional heat-treated food", in the context of the present invention, preferably relates to a heat-treated food which has been produced from conventional plant material which has been processed and heat-treated in the same manner as the plant material to be used according to the invention which, compared with corresponding conventional plant material, however, has a reduced content of soluble sugars and/or amino acids, owing to a genetic modification.
The term "plant materia!", in the context of the present invention, is to be taken to mean any material which consists of plants or comprises parts of plants. Preferably, the said parts of plants are harvested products of plants, for example tubers, fruits, seeds, onions, leaves and roots. The plant material can originate from any desired plant species, that is to say both monocotyledonous and also dicotyledonous plants. Preferably this is plant material from agricultural farmed plants, that is to say from plants which are cultivated by humans for purposes of nutrition or for technical, in particular, industrial, purposes. Particular preference is given to plant material from starchy plants (for example wheat, barley, oats, rye, potatoes, maize, rice, peas, manioc), in particular from potato plants.
The term "conventional plant material", in the context of the present invention, is to be taken to mean, in particular, plant material of corresponding non-genetically-modified plants, that is to say of plants

which do not have a genetic modification which leads to a reduction in the content of soluble sugars, in particular glucose and/or fructose, and/or to a reduction in the content of amino acids, in particular asparagine, compared with corresponding wild type plants. Conventional plant material, in the context of the present invention, however, can also originate from genetically modified plants which have been genetically modified in another aspect, but where the genetic modification does not lead to a reduction in the content of soluble sugars, in particular glucose and/or fructose, and/or to a reduction in the content of amino acids, in particular asparagine, compared with corresponding wild type plants.
The term "genetic modification" is defined hereinafter.
The term "soluble sugars", in the context of the present invention, is to be taken to mean any water-soluble sugars occurring in plant material, preferably the soluble sugars are hexoses, preferably reducing sugars, in particular fructose and/or glucose.
The term "reducing the content of soluble sugars" or "reduced content of soluble sugars", in the context of the present invention, is to be taken to mean reducing the content of soluble sugars, preferably to mean reducing the content of soluble sugars of the plant material, in particular fructose and/or glucose, by at least 10%, in particular by at least 15%, preferably by at least 20%, and particularly preferably by at least 40%, in particular by 50%-95%, preferably by 60%-90% compared with the content of soluble sugars, in particular fructose and/or glucose, of corresponding conventional heat-treated foods or of corresponding conventional plant material
The term "amino acid", in the context of the present invention, is to be

taken to mean any amino acid occurring in plant material, preferably alanine, arginine; aspartic acid, cysteine, glutamine, methionine, threonine and valine, more preferably asparagine.
The term "reducing the content of amino acids" or "reduced content of amino acids", in the context of the present invention, is to be taken to mean reducing the content of amino acids, preferably to mean reducing the content of amino acids of the plant material, in particular asparagine, by at least 10%, in particular by at least 15%, preferably by at least 20%, and particularly preferably by at least 40%, compared with the content of amino acids,, in particular asparagine, of corresponding conventional heat-treated foods or of corresponding conventional plant material.
The causes of the variability of acrylamide content in heat-treated foods are not yet adequately understood (WHO, FAO/WHO Consultation on the Health Implications of Acrylamide in Food (Geneva, 25-27 June 2002), so that to date no processes for minimizing acrylamide contents of heat-treated foods have been described. In particular, no processes were described which have the selection of particular plant materials as their basis.
It has now surprisingly been found that the choice of the starting plant material which is used to produce heat-treated foods has a critical effect on the acrylamide content of such foods. The invention teaches for the first time that the use of plant material which, compared with corresponding conventional plant material, has a reduced content of soluble sugars and/or amino acids permits the production of foods which, after heat treatment, have a lower acrylamide content than in the case of the use-of plant material having conventional contents of soluble sugars and/or amino acids. The present invention therefore teaches, to avoid the

formation of acrylamide in heart -treated foods, to use plant material which has a comparatively low content of soluble sugars and/or amino acids.
Methods of determining the content of sugars, in particular fructose and glucose, in plant material are known to those skilled in the art and are described, for example, in Muller-Rober et al. (Mol. Gen. Genet. 224, (1990), 136-146) and also in the text which follows. In context with the present invention, the determination of the content of glucose, fructose and/or sucrose is preferably performed as described below ("Determination of glucose, fructose and sucrose").
Methods of determining ;the content of amino acids, in particular asparagine, in plant material are known to those skilled in the art and are described, for example, in Cohen, Meys, Tarvin (1988), The pico-tag method: A Manual of advanced techniques for amino acid analysis, Millipore Corporation, Milford, Mass., USA. Preferred is the method described by Roessner et al. (Plant Physiology 127, (2001), 749-764).
In a further embodiment of the inventive process, the plant material used is characterized in that it is genetically modified, the genetic modification leading to a reduction in the content of soluble sugars, in particular glucose and/or fructose, compared with corresponding conventional plant material of wild type plants.
The "genetic modification", in the context of the present invention, can be any genetic modification which leads to a reduction in the content of soluble sugars, compared with corresponding conventional plant material of wild type plants.
In the context of the present invention, the genetic modification can be

caused by mutagenesis of one or more genes. The type of mutation is not critical for this, provided that rt leads to a reduction in the content of soluble sugars compared with corresponding conventional plant material of wild type plants.
The term "mutagenesis", in the context of the present invention, is to be taken to mean any type of mutation, for example deletions, point mutations (nucleotide replacements), insertions, inversions, gene conversions or chromosome translocations.
The mutation can be caused by the use of chemical agents or high-energy
radiation (for example X-ray, neutron, gamma or UV radiation).
Agents which can be used for causing chemically induced mutations and
the mutations resulting thereby by reaction of the corresponding mutagens
are described, for example, in Ehrenberg and Husain, 1981, (Mutation
Research 86, 1-113), Muller, 1972-(Biotogisches Zentralblatt 91 (1), 31-
48). The generation of rice mutants using gamma rays, ethyl
methanesulphonate (EMS), N-methyl-N-nitrourea or sodium azide (NaN3)
is described, for example, in Jauhar and Siddiq (1999, Indian Journal of
Genetics, 59 (1), 23-28), in Rao (1977, Cytologica 42, 443-450), Gupta
and Sharma (1990, Oryza 27, 217-219) and Satoh and Omura (1981,
Japanese Journal of Breeding 31 (3), 316-326). The generation of wheat
mutants using NaN3 and maleic hydrazide (1,2-dihydropyridazine-3t6-
dione) is described, by way of example, in Arora et al. (1992, Annals of
Biology 8 (1), 65-69). A review of the production of wheat mutants using
various types of higher-energy radiation and chemical agents is given in
Scarascia-Mugnozza et al. (1993, Mutation Breeding Review 10, 1-28).
Svec et al. (1998, Cereal Research Communications 26 (4), 391-396)
describe the use of N-ethyl-N-nitrourea for generating mutants in triticale.
The use of MMS and gamma radiation for generating millet mutants is

described in Shashidhara et at. (1990, Journal of Maharashtra Agricultural Universities 15 (11,20-23).
The production of mutants in plant species which principally reproduce vegetatively has been described, for example, for potatoes which produce a modified starch (Hovenkamp-Hermelink et al. (1987, Theoretical and Applied Genetics 75, 217-221), and for mint having an increased oil yield and modified oil quality (Dwivedi et al., 2000, Journal of Medicinal and Aromatic Plant Sciences 22,460-463).
All of these methods are suitable in principle for providing plant material which, owing to a genetic modification, has a reduced content of soluble sugars compared with corresponding conventional plant material of wild -type plants and is therefore suitable for use in the inventive process.
Mutations in the appropriate genes can be discovered using methods known to those skilled in the art. In particular, those which can be employed for this purpose are analyses based on hybridization with probes (Southern Blot), amplification by means of the polymerase chain reaction (PCR), sequencing relevant genomic sequences, and searching for individual nucleotide replacements. A method for identifying mutations on the basis of hybridization patterns is, for example, the search for restriction fragment length polymorphism (RFLP) (Nam et al., 1989, The Plant Cell 1, 699-705; Leister and Dean, 1993, The Plant Journal 4 (4), 745-750). A method based on PCR is, for example, the analysis of amplified fragment length polymorphism (AFLP) (Castiglioni et al., 1998, Genetics 149, 2039-2056; Meksem et at, 2001, Molecular Genetics and Genomics 265, 207-214; Meyer et al., 1998, Molecular and General Genetics 259, 150-160). The use of amplified fragments cleaved by restriction endonucleases (Cleaved Amplified Polymorphic Sequences, CAPS) can also be used for identifying mutations (Konieczny and



99, (2002), 7166-7171) and in the international patent applications
W098/27212, Wo00/77229, WO00/28052 and have the characteristics
below. Important characteristics of R1 proteins are i) their amino acid
sequence (see, for example, GenBank Acc, No, A61831. Yo9533); it) their
localization in the plastids of plant cells: iii) their ability to affect the degree
of phosphorylation of the starch in plants. Further, the term "R1 protein"
refers to a protein catalysing the phosphorylation of starch in a dikinase-
type reaction in which three substrates, an a-polyglucan. ATP and H2C
are converted into three products, an a-poiyglucan-P; AMP anc
orthophosphate (Ritte et aL, PNAS Vol. 99 No. 10, (2002). 7166-7171}. A
synonym, which is used in the more recent literature for the term "R1
protein", is the term MGWD protein" which is the abbreviation for 'alpha-
glucan water dikinase" (Blennow et al, Trends in Plant Science Vol. 7 No
10 (2002), 445-450). Therefore, with respect to the present invention, the
term aR1 protein" comprises also "GWD proteins".
For example, inhibiting the R1 gene coding for an R1 protein fron
potatoes leads, in transgenic potato plants, to a reduction in the phosphate
content of the starch which can be isolated from the potato tubers
Moreover, Lorberth et al show that the R1 protein from Solanun
tuberosum is able to phosphorylate bacteria! glycogen when th corresponding R1 cDNA is expressed in E. cofi (Lorberth et aL, Natun
Biotech. 16, (1998), 473^77).
Ritte et aL (Plant J. 21, (2000), 387-391) showed that the R1 protein fror Solanum tuberosum in potato plants reversibly binds to starch granules with the strength of the binding to the starch granule being dependent o the metabolic status of the plant In the form bound to starch granules, th protein in potato plants is principally present in (eaves whicn have bee grown in the dark. After .illuminating the leaves the protein, in contrast, i principally present in the soluble form which is not bound to the staro qranule.



Preferably, in the context of the present invention, these are acid invertases, which are also called vacuolar invertases, and have been described, for example, in Zrenner et ai. (Planta 198, (1996), 246-252).
Potato plants having decreased invertase activity have been described, for example, in Zrenner et al. (Planta 198, (1996), 246-252} and in Greiner et al. (Nature Biotechnology 17, (1999), 708-711).
in the context of the present invention, it is of particular importance that the reduction in invertase activity in transgenic potato plants, in particular in those which express a vacuolar invertase inhibitor from tobacco (Greiner et al., Nature Biotechnology 17, (1999), 708-711), leads to cold-stored potato tubers of these transgenic plants having a decreased content of soluble sugars, in particular fructose and giucose, compared with tubers of corresponding wild type plants which have net been genetically modified.
The term "reduction in activity", in the context of the present invention, means a reduction compared with corresponding non genetically-modified cells in the expression of endogenous genes which code for R1 or invertase proteins and/or a reduction of the amount of R1 protein or invertase protein in the cells of the plant materia! and/or a reduction in the enzymatic activity of the R1 or invertase proteins in the cells of the plant material.
The term "reduction in the activity of one or more endogenous R1 proteins occurring in the plant cell", in the context of the present invention, is to be taken to.mean a reduction in the expression of one or more endogenous genes which code for R1 proteins, and/or a reduction in the amount of R1 protein in the cells of the plant material and/or a reduction In the enzymatic



determined on the basis of an enzymatic assay described by Ritte et al. (PNAS 99, (20021, 7166-7171).
The reduction in enzymatic activity of the invertase protein can be determined by the method described by Greiner et al. (Nature Biotechnology 17, (1999), 708).
A reduction in the enzymatic activity of the R1 or invertase protein preferably means a reduction in activity compared with corresponding non genetically-modified cells by at least 50%, in particular by at least 60%, and preferably by .at least 70%.
A reduction in the enzymatic activity of the R1 protein preferably means a reduction in activity of R1 compared with R1 activity of corresponding non genetically-modified cells by at least 50%, in particular by at least 60%, and preferably by at least 70%.
A reduction in the enzymatic activity of the invertase protein preferably means a reduction in activity of the invertase protein compared with invertase activity of corresponding non genetically-modified cells by at least 50%, in particular by at least 60%, and preferably by at least 70%.
In a further embodiment of the inventive process, the genetic modification
is the introduction of one or more foreign nucleic acid moiecuies, the
presence and/or expression of which leads to the reduction in the activity
of one or more endogenous R1 proteins occurring in the piant cell
compared with corresponding plant cells of wild type plants which have not
been genetically modified. , .
In a further embodiment of the inventive process, the genetic modification

is the introduction of one or more foreign nucleic acid molecules, the presence and/or expression of which leads to the reduction in the activity of one or more endogenous invertase proteins occurring in the plant cell compared with corresponding plant ceils of wild type plants which have not been genetically modified.
The term "foreign nucleic acid molecule" or "foreign nucleic acid molecules", in the context of the present invention, is to be taken to mean a molecule which either does not occur naturally in corresponding plant cells, or which does not occur naturally in the plant cells in the specific spatial arrangement or which is localized at a site in the genome of the plant cell at which it does not naturally occur. Preferably, the foreign nucleic acid molecule is a recombinant molecule which consists of various elements, the combination or specific spatial arrangement of which does not occur naturally in plant cells.
In a further preferred embodiment of the inventive process, the foreign nucleic acid molecule is selected from the group consisting of
(a) DNA molecules which code for at least one antisense RNA causing a reduction in expression of endogenous genes which code for R1 proteins;
(b) DNA molecules which, via a cosuppression effect, lead to reduction of the expression of endogenous genes coding for R1 proteins;
(c) DNA molecules which code for at least one ribozyme which cleaves in a specific manner transcripts of endogenous genes coding for R1 proteins;
(d) nucleic acid molecules which are introduced by means of in vivo mutagenesis and lead to a mutation or insertion of a heterologous sequence in genes coding for endogenous R1 proteins, the mutation or insertion causing a reduction in the expression of the

said genes or the synthesis of inactive R1 proteins;
(e) DNA molecules which simultaneously code for at least one antisense RNA and at least one sense RNA, the said antisense RNA and the said sense RNA forming a double-stranded RNA molecule which causes a reduction in the expression of endogenous genes coding for R1 proteins;
(f) DNA molecules which contain transposons, the integration of the transposon sequences leading to a mutation or an insertion in endogenous genes coding for R1 proteins which causes a reduction in the expression of the said genes or the synthesis of inactive R1 proteins; and „ .
(g) T-DNA molecules which, via insertion in endogenous genes coding for R1 protein cause a reduction in the expression of genes coding for Rl protein or the synthesis of inactive R1 proteins.
a further preferred embodiment of the inventive process, the foreign cleic acid molecule is selected from the group consisting of
(a) DNA molecules which code for an invertase inhibitor.
(b) DNA molecules which code for at least one antisense RNA which causes a reduction in expression of endogenous genes coding for invertase proteins;
(c) DNA molecules which, via a cosuppression effect, lead to reduction of the expression of endogenous genes coding for invertase proteins;
(d) DNA molecules which code for at least one ribozyme which cleaves in a specific manner transcripts of endogenous genes coding for invertase proteins;
(e) nucleic acid molecules which are introduced by means of in-vivo mutagenesis and which lead to a mutation or an insertion of a heterologous sequence in endogenous genes coding for invertase

proteins, the mutation or insertion causing a reduction in the expression of the said genes or the synthesis of inactive Invertase proteins;
(f) DNA molecules which simultaneously code for at least one antisense RNA and at least one sense RNA, the said antisense RNA and the said ^sense RNA forming a double-stranded RNA molecule which causes a reduction in the expression of endogenous genes coding for invertase proteins;
(g) DNA molecules which contain transposons, the integration of the transposon sequences leading to a mutation or an insertion in endogenous genes coding for invertase proteins, which causes a reduction in the expression of the said genes or the synthesis of inactive invertase proteins; and
(h) T-DNA molecules which, via insertion in endogenous genes coding for invertase protein, cause a reduction in the expression of genes coding for invertase protein, or the synthesis of inactive invertase proteins.
To inhibit the gene expression by means of antisense or cosuppression techniques, for example, a DNA molecule can be used which comprises the entire sequence coding for an R1 protein or invertase protein and possibly existing flanking sequences, and also DNA molecules which comprise only part of the coding sequence, with these parts needing to be long enough to cause an antisense effect or cosuppression effect in the cells. Suitable sequences are generally sequences up to a minimum length of 15 bp, preferably a minimum length of 21 bp, preferably a length of 100-500 bp, and for an efficient antisense or cosuppression inhibition, particular preference is given to sequences having a length over 500 bp. These statements apply correspondingly to the inhibition of BE I gene expression.

For antisense or cosuppression approaches, it is also suitable to use DNA sequences which have a high degree of homology to the endogenous sequences in the plant cell which code for an R1 protein or invertase protein. The minimum homology should be greater than approximately 65%. The use of sequences having homologies of at least 90%, in particular between 95 and 100%, is to be preferred. These statements apply correspondingly to the inhibition of BE I gene expression.
In addition, to achieve an antisense or cosuppression effect, the use of introns is also conceivable, that is to say non-coding regions of genes which code for an R1 protein or invertase protein.
The use of intron sequences for inhibiting gene expression of genes which code for proteins of starch biosynthesis has been described, for example, in the international patent applications WO97/04112, WO97/04113, W098/37213, W098/37214. These statements apply correspondingly to the inhibition of BE I gene expression.
A person skilled in the art is familiar with how to achieve an antisense and cosuppression effect. The process of cosuppression inhibition has been described, for example, in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et alM (Curr. Top. Microbiol. Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top. Microbiol. Immunol 197 (1995), 43-46), Palaqui and Vaucheret (Plant Mol. Biol. 29 (1995), 149-159), Vaucheret et al., (Mol. Gen. Genet 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621).
The expression of ribozymes for reducing activity of certain enzymes in cells is also known to those skilled in the art and is described, for example, in EP-B1 0321201. The expression of ribozvmes in plant cells has been

described, for example, in Feyter et al. (Mot. Gen. Genet. 250, (1996),
329-338). ;
In addition, the reduction of the R1 or invertase activity in the plant cells of the plant material can also be achieved by ttin-vivo mutagenesis", in which, by transformation of cells, a hybrid RNA-DNA oligonucleotide ("chimeroplasf) is introduced into cells (Kipp, P.B. et ai., Poster Session at the 5th International Congress of Plant Molecular Biology, 21.-27. September 1997, Singapore; R. A. Dixon and C.J. Arntzen, Meeting report on "Metabolic Engineering in Transgenic Plants", Keystone Symposia, Copper Mountain, CO, USA, TIBTECH 15, (1997), 441-447; international patent application WO 9515972; Kren et ai., Hepatology 25, (1997), 1462-1468; Cole-Strauss et al.v Science 273, (1996), 1386-1389; Beetham et alM (1999), PNAS 96, 8774-8778).
A part of the DNA component of the RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an endogenous R1 or invertase gene, but, compared therewith, has a mutation or contains a heterologous region which is enclosed by the homologous regions. By base-pairing the homologous regions of the RNA-DNA oligonucleotide and of the endogenous nucleic acid molecule, followed by homologous recombination, the mutation or heterologous region present in the DNA component of the RNA-DNA oligonucleotide can be transferred into the genome of a plant cell. This leads to a reduction in activity of one or more R1 or invertase proteins. These statements apply correspondingly to the inhibition of BE I gene expression.
In addition, the R1 or invertase activity can also be reduced in the plant cells by the simultaneous expression of sense and antisense RNA molecules of the respective target gene to be repressed, preferably of the R1 or invertase gene.

This can be achieved, for example, by using chimeric constructs which contain inverted repeats of the respective target gene or parts of the target gene. In this case the chimeric constructs code for sense and antisense RNA molecules of the respective target gene. Sense and antisense RNA are synthesized in planta simultaneously as one RNA molecule, with sense and antisense RNA being separated from one another by a spacer and able to form a double-stranded RNA molecule. This technology is aiso called "RNAi technology".
It has been shown that introducing inverted repeat DNA constructs into the genome of plants is a highly efficient method for repressing the genes corresponding to the inverted repeat DNA constructs (Waterhouse et al.. Proc. Natl. Acad. Sci. USA 95, (1998), 13959-13964; Wang and Waterhouse, Plant Mol. Biol. 43, (2000), 67-82; Singh et ai., Biochemical Society Transactions Vol. 28 part 6 (2000), 925- 927; Liu et ai., Biochemical Society Transactions Vol. 28 part 6 (2000), 927-929); Smith et al., (Nature 407, (2000), 319-320; international patent application WO99/53050 A1). Sense and antisense sequences of the target gene or the target genes can also be expressed separately from one another by means of the same or different promoters (Nap, J-P et al, 5th Internationa! Congress of Plant Molecular Biology, Quebec, 18-24 June, 2000; Poster S7-27, Presentation Session S7). These statements apply correspondingly to the inhibition of BE I gene expression.
The reduction in R1 or invertase activity in the plant cells of the plant material can thus also be achieved by producing double-stranded RNA molecules of R1 or invertase genes. Preferably, for this purpose, inverted repeats of DNA molecules of R1 or invertase genes or cDNAs are introduced into the genome of plants, the DNA molecules to be transcribed (R1 or invertase genes or cDNAs or fragments of these genes or cDNAs) being under the control of a promoter which controls the expression of the

said DNA molecules. These statements apply correspondingly to the inhibition of BE I gene expression.
Furthermore it is known that the formation of double-stranded RNA molecules of promoter DNA molecules in plants in trans can lead to a methylation and a transcriptional inactivation of homologous copies of these promoters which are to be termed hereinafter target promoters (Mette et al., EMBO J. 19, (2000), 5194-5201).
Via the inactivation of the target promoter it is thus possible to reduce the gene expression of a certain target gene (for example R1 or invertase gene) which exists naturally under the control of this target promoter. That is to say the DNA molecules which comprise the target promoters of the genes to be repressed (target genes) are in this case, in contrast to the original function of promoters in plants, used not as control elements for the expression of genes or cDNAs, but are themselves used as transcribable DNA molecules.
To produce the double-stranded target promoter RNA molecules in planta which can be present there as RNA hairpin molecules, preferably, constructs are used which contain inverted repeats of the target promoter DNA molecules, the target promoter DNA molecules being under the control of a promoter which controls the gene expression of the said target promoter DNA molecules. These constructs are then introduced into the genome of plants. The expression of the "inverted repeats" of the said target promoter DNA molecules leads in planta to the formation of double-stranded target promoter RNA molecules (Mette et al., EMBO J. 19, (2000), 5194-5201). By this means the target promoter can be inactivated. The reduction in R1 or invertase activity in the plant cells can thus also be achieved by producing double-stranded RNA molecules of promoter sequences of R1 or invertase genes. Preferably, for this purpose, inverted repeats of promoter DNA molecules of R1 or invertase oromoters are

introduced into the genome of plants, the target promoter DNA molecules to be transcribed (R1 or invertase promoter) being under the control of a promoter which controls the expression of the said target promoter DNA molecules. These statements apply correspondingly to the inhibition of BE I gene expression.
In a further embodiment of the present invention, the foreign nucieic acid molecule is inserted transposons or what is called transfer DNA (T-DNA) into a gene coding for an R1 or invertase protein, the activity of the said proteins being reduced as a result in the relevant cell of the plant material. These statements apply correspondingly to the inhibition of BE I gene expression.
In principle, the plant material suitable for the inventive process can be produced not only using homologous, but also heterologous, transposons. the use of homologous transposons also being taken to mean those which are already naturally present in the plant genome. These statements apply correspondingly to the inhibition of BE I gene expression.
Modifying gene expression by means of transposons is known to these skilled in the art. A review of the use of endogenous and heterologous transposons as tools in plant biotechnology is given in Ramachandran and Sundaresan (2001, Plant Physiology and Biochemistry 39, 234-252). The possibility of identifying mutants in which specific genes have been inactivated by transposon insertion mutagenesis is described in a review by Maes et al. (1999, Trends in Plant Science 4 (3), 90-96). Producing rice mutants with the aid of endogenous transposons is described by Hirochika (2001: Current Opinion in Plant Biology 4, 113-122). The-identlficaticn of maize genes using endogenous retrotransposons is reported, for example, by Hanley et al. (2000, The Plant Journal 22 (4), 557-566). The possibility

of producing mutants using retrotransposons and methods for identifying mutants are described by Kumar and Hirochika (2001, Trends in Plant Science 6 (3), 127-134). The activity of heterologous transpcsons in different species has been described not only for dicotyledonous but also for monocotyledonous plants: e.g. for rice (Greco et ai., 2001. Plant Physiology 125, 1175-1177; Liu et ai., 1999, Molecular and General Genetics 262, 413-420; Hiroyuki et aL 1999, The Plant Journal 19 (5). 605-613; Jeon and Gynheung, 2001, Plant Science 161. 211-219). barley (2000, Koprek et ai., The Plant Journal 24 (2), 253-263) Arabidopsis thaliana (Aarts et ai., 1993, Nature 363, 715-717, Schmidt and Wiilmitzer, 1989, Molecular and General Genetics 220, 17-24; Aitmann et aL, 1GS2r Theoretical and Applied Genetics 84, 371-383; Tissier et ai., 1999. The Plant Cell 11, 1841-1852), tomato (Beizile and Yoder, 1992, The Plant Journal 2 (2), 173-179) and potatoes (Frey et al., 1989, Molecular and General Genetics 217, 172-177; Knapp et aL 1983, Molecular and General Genetics 213, 285-290).
T-DNA insertion mutagenesis is based on the fact that certain sections ;T-DNA) of Ti-plasmids from Agrobacterium can integrate into the genome of plant cells. The site of integration in the plant chromosome is not fixed here, that can be at any desired position, if the T-DNA integrates Into a section of the chromosome representing a gene function, this can lead to a modification of gene expression and thus also to a change in the activity of a protein coded for by the gene in question, in particular, the integration of a T-DNA into the coding region of a protein frequently leads to the corresponding protein no longer being able to be synthesized by the ceil in question, or not in active form. The use of T-DNA insertions for producing mutants is described, for example, for Arabidopsis thaliana (Krysan et ai.. 1999, The Plant Ceil 11, 2283-2290: Atipiroz-Leehan and Feidmann, 1997, Trends in genetics 13 (4), 152-156; Parinov and Sundaresan, 2000,

Current Opinion in Biotechnology 11, 157-161) and rice (Jeon and An, 2001, Plant Science 161, 211-219; Jeon et al., 2000, The Plant Journal 22 (6), 561-570). Methods for identifying mutants which have been produced using T-DNA insertion mutagenesis are described, inter aiia, by Young et ai.,(2001, Plant Physiology 125, 513-518), Parinov et al. (1999 The Plant cell 11, 2263-2270), Thomeycroft et ai. (2001. Journal of Experimental Botany 52, 1593-1601), and McKinney et al. [1995. The Plant Journal 8 (4),613-622).
T-DNA mutagenesis is suitable in principle for producing the plant material which can be used in the inventive process.
In a further embodiment of the inventive process, the genetic modification not only leads to a reduction in the activity of one or more endogenous R1 proteins occurring in the plant cell, but also at the same time to a reduction fn the activity of one or more endogenous branching enzymes of isoform I occurring in the plant cell (branching enzyme! = BEi protein), compared with corresponding non-genetically-modified plant cells of wild type plants.
The term WBEI protein", in the context of the present invention, is to be taken to mean a branching enzyme (= BE) of isoform !. Preferably the BEi protein originates from potato plants.
The naming of the isoforms here is based on the nomenclature proposed by Smith-White and Preiss (Smith-White and Preiss, Plant Mo! Biol. Rep. 12, (1994), 67-71, Larsson et al., Plant Moi Biol. 37, (1993), 505-511). This nomenclature starts from the position that ai! enzymes which have a higher homology (identity) at the amino acid level to the BEI protein from maize (GenBank Ace. No. D11081; Baba et al.,- Biochem. Biophys. Res. Commun. 181 (1), (1991), 87-94; Kim et ai. Gene 216. '1898), 233-243) than to the BEII protein from maize (Genbank Ace. No AF072725,

U65948), are called branching enzyme of isoform I, or for short BEI proteins.
Nucleic acid moiecules coding for UBE1 protein" have been described for numerous plants, for example for maize (Genbank Acc. Nc. D 11081, AF 072724), rice (Genbank Ace. No. D11082), peas (Genbank Ace. No. X80010) and potatoes. Various forms of the BE! gene and of the BE! protein from potatoes have been described, for example, in Khoshnoodi et al., Eur. J. Biochem. 242 (1), 148-155 (1996), Genbank Acc. Nc. Y 08786 and in Kossmann et al., Mol. Gen. Genet. 230, (1991), 39-44). In potato plants, the BEI gene is principally expressed in the tubers and scarcely at all in the leaves (Larsson et al., Plant Mol. Biol. 37, (1998). 505-511).
Regarding the genetic modification which leads to a reduction in the R1 activity, the statements made above apply. The genetic modification which leads to a reduction in the activity of the BEI protein I (branching enzyme I), can be the introduction of one or more foreign nucleic acid molecules, the presence and/or expression of which leads to the reduction in the activity of one or more endogenous BEi proteins of isoform 1 occurring in the plant cell compared with corresponding non-genetically-modified plant cells of wild type plants.
The term "reduction in the activity of one or more endogenous branching enzymes of isoform I occurring in the plant ceil"., in the context of the present invention, is to be taken to mean a reduction compared with corresponding non genetically-modified cells in the expression cf one or more endogenous genes which code for BEI proteins, and/or a reduction in the amount of BEI protein in the cells of the plant material and/or a reduction In ihe enzymatic activity of the BEI proteins in the cells of the plant material.

The reduction in expression can be determined, for exaple bv measuring the amount of transcripts coding for B£i protein, for example by Northern blot analysis or RT-PCR. A reduction preferably means a reduction in the amount of transcripts compared with the corresponding non genetically-modified ceils by at ieast 50%, in particular by at !east 70%, oreferabiy by at least 85%, and particularly preferably by at least 95%.
The reduction in the amount of BEI proteins which results in a recucec activity of this protein in the plant cells in question can be determined, for example, by immunological methods, such as Western bict analysis, ELISA (Enzyme Linked Immuno Sorbent Assay; or RIA (Radio Immune Assay). A reduction preferably means a reduction in the amount of 3Ei protein.compared with the corresponding non genetically-modified calls by at ieast 50%„ in particular by at least 70%, preferably by ai ieast 35%. and particularly preferably by at least 95%.
In a further preferred embodiment of the inventive process the foreign nucleic acid molecule which leads to the reduction in activity of one or more endogenous BEI proteins of isoform ! occurring in the plant call is selected from the group consisting of
(a) DNA molecules which code for at least one antisense RMA causing a reduction in the expression of endogenous genes which cede for BEi proteins;
(b) DNA molecules which, via a cosuppressicr affect, iead to reduction in the expression of endogenous genes which code for 8Ei proteins; . -
(c) DNA molecules which code for at ieast are ribczyme which cleaves in a specific manner transcripts of endogenous genes ceding for



With respect to the differing conceivable ways for producing genetic modifications which lead to a reduction in the amino acid content, in particular asparagine, the statements made in generai above apply in the context of the genetic modifications which !ead to a reduction in the content of sugars.
in a further embodiment of the inventive process; the genetic modification leads to a reduction in the activity of one or more endogenous asparagine synthetase proteins occurring in the plant cefi compared with corresponding plant cells of wild type plants which have not been genetically modified.
An "asparagine synthetase protein", in the context of the present invention, is to be taken to mean a protein which catalyses the conversion of aspartate to asparagine with the conversion of ATP to AMP and pyrophosphate, and of glutamine to glutamate. Sequence information for asparagine synthetases (asnl) has been described, for example, in Lam et al. (Plant Physiol. 106(4), (1994), 1347-1357).
Plants having decreased asparagine synthetase activity have, compared with corresponding wild type plants, reduced content of asparagine (Annua! Meeting of the American Society of Plant Biologists in Madison; Wl, USA, (1998), Molecular and transgenic studies of asparagine synthetase genes in Arabidopsis thaliana , Abstract Number 535).
With respect to the definition of the term "reduction in activity", the statements made above in connection with the R1 or invertase protein apply accordingly. The activity of asparagine synthetase can be determined, for example, by the method described by Romagni and Dayan

(Journal of Agricultural & Food Chemistry 48(5), (2000), 1692-1696).
In a further embodiment of the inventive process, the genetic modification is the introduction of one or more foreign nucleic acid molecules, the presence and/or expression of which leads to a reduction in the activity of one or more endogenous asparagine synthetase proteins occurring in the plant ceil, compared with corresponding plant cells from wild type plants which have not been genetically modified.
The term foreign nucleic acid molecule" here has the meaning already defined above.
In a further embodiment of the inventive process, the foreign nucleic acid molecule is selected from the group consisting of
(a) DNA molecules which code for at least one antisense RNA which causes a reduction th the expression of endogenous genes coding for asparagine synthetase proteins;
(b) DNA molecules which, via a cosuppression effect, lead to a reduction in the expression of endogenous genes coding for asparagine synthetase proteins;
(c) DNA molecules which code for at least one ribozyme which cleaves in a specific manner transcripts of endogenous genes coding for asparagine synthetase proteins;
(d) nucleic acid molecules introduced by in-vivo mutagenesis which lead to a mutation or insertion of a heterologous sequence into genes coding for endogenous asparagine synthetase protein, the mutation or insertion causing ai reduction in the expression of the said genes or the synthesis of inactive asparagine synthetase protein;
(e) DNA molecules which code simultaneous!v far at least one

antisense RNA and at least one sense RNA, the said antisense RNA and the said sense RNA forming a double-stranded RNA molecule which r causes a reduction in the expression of endogenous genes coding for asparagine synthetase proteins;
(f) DNA molecules which contain transposons, the integration of the _ transposon sequences leading to a mutation or an insertion in genes coding for endogenous asparagine synthetase proteins which causes a reduction in the expression of the said genes or the synthesis of inactive asparagine synthetase proteins; and
(g) T-DNA molecules which, via insertion in genes coding for endogenous asparagine synthetase protein, cause a reduction In* the expression of genes coding for asparagine synthetase protein or the synthesis of inactive asparagine synthetase proteins.
In this connection, the statements already made generally above in a different context on carrying out the genetic engineering approaches (antisense, cosuppression and ribozyme techniques, in-vivo mutagenesis, transposons, T-DNA insertion) apply accordingly to the genetic modification of asparagine synthetase activity.
In a further embodiment of the inventive process, the genetic modification leads to an increase in activity of an ADP-glucose pyrophosphatase protein, compared with corresponding plant cells of wild type plants which have not been genetically modified.
The ADP glucose pyrophosphorylase activity can be determined, for example, as described in M0Iler-R6ber et al. ( EMBO J. 11, (1992), 1229-1238).
An "ADP-glucose pyrophosphorylase protein", in the context of the present

invention, is to be taken to mean a protein which catalyses the conversion of glucose-1-phosnate and ATP into ADP-glucose and pyrophosphate.
In a further embodiment of the inventive process, the genetic modification is the introduction of one or more foreign nucleic acid molecules, the presence and/or expression of which leads to the increase in the activity of one or more ADP-glucose pyrophosphorylase proteins occurring in the plant cell compared with corresponding plant cells of wild type plants which have not been genetically modified.
The term "foreign nucleic acid molecule" here has the meaning already
*' » * *
defined above. S •
■ .*...■ *
Preferably, the foreign nucleic acid molecule codes for a deregulated ADP-glucose pyrophosphorylase, particularly preferably the ADP-glucose pyrophosphorylase from E. coli which is termed glgC16 and which leads, on expression in transgenic potato plants, to an increased starch synthesis rate. Cold-stored potato tubers of these plants show a significantly reduced accumulation of hexoses (Stark et al., Science 258, (1992), 287-292; Stark et al., Ann. NY Acad. Sci. 792, (1996), 26-37).
In a further embodiment, the present invention relates to the use of the above described plant material, which can be used in the inventive process for producing heat-treated foods which have a reduced acrylamide content compared with corresponding conventional heat-treated foods.
In a further embodiment, the present invention relates to the use of plant material which, compared with corresponding conventional plant material,

has a reduced content of soluble sugars and/or amino acids for producing heat-treated foods having a reduced acrylamide content.
In a further embodiment, the present invention relates to the use of the ^above described plant material which can be used in the inventive process for reducing the acrylamide content of heat-treated foods.
In a further embodiment, the present invention relates to a process for identifying plant material which is suitable for producing heat-treated foods having a reduced acrylamide content, which comprises:
a) determining the content of soluble sugars and/or amino acids in plant material which is suitable for producing heat-treated foods; and
b) selecting such plant material according to process step a) which, compared with corresponding conventional plant material, has a reduced content of soluble sugars and/or amino acids.
All of the publications and patents referred to in the specification are hereby incorporated by the reference in their entirety.

Methods:
Determination of glucose, fructose and sucrose:
To determine the contents of glucose, fructose and sucrose in potato tubers, small pieces (diameter approximately 10 mm) of potato tubers are frozen in liquid nitrogen and then extracted for one hour at 80°C in 0.5 ml of 80% (vol/vol.) ethanol After centrifugation (3 min, 3 000 rpm), the supernatant is withdrawn and the deposit is again extracted in 0.5 ml of 80% (vol/vol.) ethanol. This process is repeated. The combined supematants are used to determine the amount of soluble sugars. Soluble glucose, fructose and sucrose are determined quantitatively in an assay solution of the following composition: 100 mM imidazole/HCI (pH 6.9)
5 mM MgCI2 2 mM NAD*
1 mM ATP
200 pi of sample
2 units of glucose-6-phosphatedehydrogenase (from Leuconostoc
mesenteroides)
The assay solution is incubated at room temperature for 5 min. The sugars
are then determined using customary photometric methods by measuring
the absorption at 340 nm after the successive addition of
1 500 units of yeast hexokinase (to determine glucose)
2.5 units of yeast phosphogiucoisomerase (to determine fructose)
350 units of yeast (5-fructosidase (to determine sucrose) to a reaction
volume of 200 pL

The examples below illustrate the invention:
Example 1
Production of potato crisps and potato chips from potato tubers
To produce potato crisps and potato chips, ripe potato tubers of transgenic potato plants which have a decreased expression of the R1 gene (Lorberth et alM Nature Biotechnology 16, (1998), 473-477) and also potato tubers of potato plants which have a decreased R1 gene expression and in addition a decreased expression of branching* enzyme i gene (W097/11188) were used.
The crisps and chips were further processed immediately after harvest and also after storage at 4°C for differing times.
The tubers were peeled by hand and then sliced in a slicing machine {model Chef200( from Saro Emmerich, Germany) into slices for the production of crisps or cut using a punch (Weisser, Germany) to form chips.
The samples were deep-fat fried in a deep-fat fryer (Frita4, Franke, Frifri aro GmbH, Germany) for differing times using plant fat (Palmaja, Meylip mbH & Co. KG, Germany) at a temperature of 180°C.
The deep-fat fried products were then comminuted and analyzed for their acrylamide content This was detected using GC/MS or LC/MS-MS after derivatization (Epa method 8032a, U.S. Environmental Protection Agency). This ensures, in addition to a iow limited determination, a high selectivity of detection.

In the case of ail deep-fat fried samples, it was found that the acrylamide content in the transgenic potato tubers was reduced by at least 15% compared with the acrylamide content of the tubers of the corresponding wild type plants.
Example 2
Determination of the acrylamide content of potato crisps and chips produced from potato tubers having decreased R1- and branching enzyme I - gene expression.
The potato crisps and chips produced according to example 1 were analyzed for their acrylamide content
Non-genetically-modified plants are termed hereinafter wild type plants. The transgenic potato plants which have a decreased expression of the • R1 gene (Lorberth et al., Nature Biotechnology 16, (1998), 473-477) and in addition a decreased branching enzyme I gene expression (see international patent application WO 97/11188) are termed hereinafter 015VL001.
If freshly harvested tubers of potato plants are used for deep-fat frying at 180°C for 3 and 6 minutes, the potato crisps have the following acrylamide content:


Table 1: Percentage acrylamide content of crisps (produced from potato tubers directly after harvesting). The wild type was set at 100%.
The absolute acrylamide content increases greatly with increasing deep-fat frying time. This is the case not only for crisps from wild type tubers, but also for crisps from transgenic tubers. For both deep-fat frying times, the increase in acrylamide content in the crisps which were produced from the transgenic potato tubers, however, is significantly reduced compared with the crisps of wild type plants. At a deep-fat frying time of 3 min, the acrylamide content in the transgenic crisps is reduced by approximately 70% compared with the wild type crisps. At a deep-fat frying time of 6 min, there is a reduction in acrylamide formation in the transgenic crisps of approximately 50% compared with the wild type.
In a further experiment, potato tubers stored at 4°C were used for producing potato crisps. After harvest, the transgenic tubers and the associated wild type tubers were stored at 4°C for 56 days. Potato crisps and potato chips were produced and deep-fat fried at 180°C for differing times under the conditions described above:

Table 2: Percentage acrylamide content of crisps (produced from tubers stored at4°C). The wild type was set at 100%.

The absolute acryiamide content always greatly increases in the products from potatoes stored at 4°(X However, it is shown that the acryiamide content in the crisps made from transgenic potato tubers increases by approximately 70% less compared with the crisps made from corresponding wild type plants, both for a deep-fat frying time of 3 min, and for a deep-lat frying timeof 6 min.
In a further experiment, potato chips were produced from cold-stored potato tubers (stored at 4°C for 56 days) as described in example 1 and deep-fat fried. In contrast to the potato crisps, the potato chips were pre-fried for 30 seconds at 18G°C, laid out on kitchen paper, and cooled to room temperature and only then deep-fat fried for the specified time.

Table 3: Percentage acryiamide content of potato chips (produced from cold-stored tubers). The wild type was set at 100%.
The absolute acryiamide contents are lower in the potato chips compared with potato crisps. This is certainly primarily due to the smaller surface area of the potato chips compared with potato crisps per kg of potato. The percentage acryiamide contents, in this product also, show a reduction in the potato chips made from transgenic potato plants by approximately 50% at both deep-fat frying times compared with potato chips made from wild type tubers.

In the context of industrial production of crisps or potato chips, the sliced potatoes were blanched before deep-fat frying. The blanching can take place in a water or steam blancher. The blanchin&conditions are not fixed values, but vary very greatly depending on the quality of the potatoes used. During the blanching, soluble sugars are partly washed out. This causes more uniform browning of the potato products in deep-fat frying.
To demonstrate that the inventive process also leads to a reduction in the acrylamide formation in the potato products made from transgenic potato plants under changed process conditions, compared with the products from corresponding wild type tubers, the blanching was performed on a laboratory scale by washing the sliced potatoes with hot mains water.
For this purpose, approximately 200 g of potatoes (stored at 4°C for 56 days after harvest) were washed three times, each time using 5 litres of mains water at 45°C, each time for 1.5 minutes. The sliced potatoes were then dried on domestic paper and deep-fat fried at 180°C for 3 minutes as described above:

Table 4: Percentage acrylamide content of washed crisps (produced from potato tubers). The wild type was set at 100%.

The washing leads to a reduction in acrylamide formation in crisps which were produced from potato tubers of wild type plants by approximately 16% compared with unwashed potato crisps.
Compared with the "washed11 crisps which were produced from potato tubers of wild type plants, the "washed" crisps which were produced from potato tubers of the transgenic potato plants have an acrylamide formation which is decreased by approximately 80%.
In a further analysis, the contents of soluble sugars, in particular glucose and/or fructose, were determined cojnpared with the corresponding conventional plant material of wild type plants:
For this purpose the potato tubers were peeled and a sample having a diameter of approximately 0.5 cm sample was cut out using a cork borer (from Roth). From this sample a slice approximately 2 mm thick each time from the start, one quarter and. one half from 5 different* tubers in each case was combined in a reaction vessel and used to determine soluble sugars.
The determination of the contents of sugars, in particular fructose and glucose, of plant material is known to those skilled in the art and was carried out as described above.



Table 6: Comparison of the percentage soluble sugar contents of fresh harvested and stored tuber samples based on tubers of the corresponding wild type (100%).
Storage causes a sharp rise in the contents of soluble sugars in wild type plants. The tubers of line 015VL show, directly after harvest, contents of glucose and fructose reduced by approximately 30%-40% compared with wild type plants. After the above described cold storage, there is reduction of glucose or fructose of approximately 50% in the transgenic plants compared with the corresponding wild type plants.
If the content of glucose or fructose is correlated with the acrylamide content in crisps, it is seen that there is a linear correlation between the content of glucose or fructose in the potato tuber and the formation of acrylamide in the deep-fat fried product crisps.

It is has thus been shown for the first time that there is a correlation between the formation of acrylamide in heat-treated foods and the content of soluble sugars of the plant material used to produce the heat-treated food. The effect on reduction of acrylamide formation is much more pronounced than expected.
Example 3
Determination of the acrylamide content of potato crisps and potato chips produced from potato tubers having decreased R1-gene expression.
The potato crisps and potato chips produced according to example 1, which were produced from potato tubers having decreased R1 gene expression, were analyzed for their acrylamide content.
In this case, as already described in example 2, firstly tests were made of differing deep-fat frying times, and also samples which had been differently stored or washed were studied.
The results described in example 2 were confirmed, that is to say potato crisps and potato chips produced from potato tubers having decreased R1 gene expression, likewise show, under the conditions described in more detail in example 2, less acrylamide compared with corresponding products which were produced from potato tubers from corresponding non-genetically-modified wild type plants.

Production of differing varieties of transgenic potato plants having reduced R1 gene expression
To produce transgenic potato plants having reduced R1 gene expression, the T-DNA of plasmid IR5/29 was transferred to potato plants of cultivars Tomensa, Solara and Bintje, using agrobacteria, as described in Rocha-Sosa et al. (EMBO J. 8, (1989), 23-29).
Notes on vector IR5/29:
(R5-29 is a derivative of plasmid pGSV71 which contains, inter alia, the sequence of the promoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosa et al., (1989), see above) and the. complete R1-cDNA (Lorberth et a!. Nature Biotechnology 16, (1998), 473-477) in-the "sense" orientation to the promoter.
pGSV71 is a derivative of plasmid pGSV7, which is derived from the intermediate vector pGSV1. pGSV1 is a derivative of pGSC1700, the construction of which was described by Cornelissen and Vanderwiele ((1989), Nuclear transcriptional activity of the tobacco plastid psbA promoter. Nucleic Acids Research 17: 19-29). pGSV1 was obtained from pGSC1700 by deletion of the carbenicillin resistance gene and deletion of the T-DNA sequences of the TL-DNA region of plasmid pTiB6S3. pGSV7 contains the replication origin of plasmid pBR322 (Bolivar et al.t (1977), Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene, 2: 95-113) and the replication origin of the pseudomonas plasmid pVS1 (Itoh et al., (1984), Genetic and molecular characterization of-the Pseudomonas plasmid pVS1. Plasmid* 11:206-220).

pGSV7 also contains the selectable marker gene aadA from thel transposon Tn1331 from Klebsiella pneumoniae, which confers resistance to the antibiotics spectinomycin and streptomycin (Tolmasky, (1990), Sequencing and expression of aadA, bla, and tnpR from the multiresistance transposon Tn1331. Plasmid. 24 (3): 218-226; Tolmasky and Crosa, (1993), Genetic organization of antibiotic resistance genes (aac(6')-lb, aadA, and oxa9) in the multiresistance transposon Tn1331. Plasmid 29(1): 31-40).
The plasmid pGSV71 was obtained by cloning a chimeric bar gene between the border regions of pGSV7. The chimeric bar gene contains the promoter sequence of the cauliflower mosaic virus for initiating transcription (Odell et ah, (1985), Identification of DNA sequences required for activity of the Cauliflower Mosaic Virus 35S promoter. Nature 313: 810-812), the bar gene from Streptomyces hygroscopicus (Thompson et al.t (1987); Characterization of the herbicide resistance gene bar from Streptomyces hygroscopicus. The EMBO Journal, 6: 2519-2523) and the untranslated 3' region of the nopaline synthase gene of T-DNA of pTiT37 for termination of transcription and polyadenylation. The bar gene confers tolerance towards the herbicide glufosinate ammonium. The T-DNA contains the following elements in the order cited:
- the left border sequence of the TL-DNA from pTiB6S3 (Gielen et al., (1984), The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5. The EMBO J. 3:835-846).
the promoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosa et al., 1989, see above) in a sense orientation based on the left border sequence of the TL-DNA
the complete R1-cDNA (Lcrberth et alM (1998), see above} in a sense orientation based on the patanin promoter

the polyadenylation signal (3* end) of the octopine synthase gene (gene 3) of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., (1984), see above) in a sense orientation based on the left border sequence of the TL-DNA
the Taql fragment of the non-translated 3" end of the nopaline synthase gene (31 nos) from the T-DNA of plasmid pTiT37 (Depicker et al., (1982), Nopaline synthase: transcript mapping and DNA sequence. Journal of molecular and applied Genetics 1: 561-573) in an antisense orientation based on the left border sequence of the TL-DNA
the coding sequence of the phosphinothricin resistance gene (bar) from Streptomyces hygroscopicus (Thompson et al. (1987, see above) in an antisense orientation based on the left border sequence of the" TL-DNA. The two terminal codons at the 5' end of the bar wild type gene were replaced by the codons ATG and GAC. the P35S3 promoter region of cauliflower mosaic virus (Odell et al., (1985), see above) in an antisense orientation based on the left border sequence of the TL-DNA
the right border sequence of the TL-DNA from plasmid pTiB6S3 (Gielen et al., (1984), see above.
After the various potato cultivars had been transformed, Western biot analysis (Lorberth et al., Nature Biotechnology 16, (1998), 473-477) was used to identify, for each of the cultivars, various lines whose tubers had a markedly reduced amount of R1 protein, owing to a cosuppression effect.
The plants of cultivar Tomensa obtained by transformation using plasmid IR5/29 were termed 093IR plants, those of cultivar Solara were termed 095IR plants, and those of cultivar Brntje were termed 092IR plants.

Potato tubers from lines 0931R360, 095IR049 and 092IR002 were used to produce potato chips (example 5).
Example 5
Determination of the acrylamide content of potato chips which had been produced from different varieties of potato tubers having decreased Rl gene expression
» » Freshly harvested potato tubers of the plants produced according to example 4 were processed to potato chips in accordance with example 1 and pre-deep-fat fried according to example 2 for 30 seconds at 180°C, laid out on kitchen paper and cooled to room temperature and then deep-" fat fried at 180°C for 3 minutes.
The potato chips produced had the following acrylamide contents:


Tab. 1: Percentage acrylamide content of potato chips (produced from potato tubers directly after harvest): Each corresponding wild type was set at 100%.
The absolute acrylamide contents of the potato chips produced in part vary considerably between the cuitivars used. This is primarily due to the differing absolute values of soluble sugars. For instance potato chips from cultivar Solara, for example, exhibited not only the highest acrylamide contents but also the highest soluble sugar contents.
The relative acrylamide contents of the potato chips, however, showed for* all transgenic cuitivars used a considerable reduction in the amount of acrylamide by approximately 40-50% compared with potato chips which had been produced from wild type tubers.
In a further analysis, the contents of soluble sugars, in particular glucose, fructose and sucrose, were determined in the potato tubers from the various cuitivars:
For this purpose the potato tubers were peeled in accordance with example 2 and using a cork borer (from Roth), a sample of diameter approximately 0.5 mm was cut out From this cork borer sample, an approximately 2 mm thick slice was taken in each case from the start, one quarter and half way, from each of 5 different tubers and combined in a reaction vessel and used for determining soluble sugars.
The contents of sugars, in particular fructose and glucose, of plant material were determined as described above.

As mentioned above, the absolute values of soluble sugars vary greatly between the cultiyars studied.
Cuttivar Solara shows the highest glucose, fructose and sucrose contents. Tomensa shows the lowest glucose and fructose contents, and Bintje the lowest sucrose contents. Tubers from line 0931R360, directly after harvest, show glucose and sucrose contents reduced by approximately 30-40% compared with the corresponding wild type plants. Tubers of line 0951R049, directly after harvest, show glucose and fructose contents reduced by approximately 10-30% compared with wild type plants.
If the total glucose and/or fructose content is correlated with the acrylamide content in potato chips, it may be seen that there is a linear correlation between the glucose and/of fructose content and the formation of acrylamide in-the potato chips.
It was thus confirmed for various cultivars that the use -of potato plants having decreased R1 gene expression permits the production of heat-treated foods, in particular potato chips, which are distinguished by a markedly reduced acrylamide content compared with corresponding heat-treated foods which are produced from corresponding non-genetically-modified wild type plants.

Determination of the acrylamide content of potato chips produced from stored potato tubers of differing varieties of reduced R1 - gene expression
Potato tubers stored at 4°C for 73 days from the plants produced in accordance with Example 4 were processed to potato chips in accordance with Example 1 and deep-fat prefried at 180°C for 30 seconds in accordance with Example 2, placed on kitchen paper and cooled to room temperature, and then deep-fat fried at 180°C for 3 minutes.
The potato chips produced had the following acrylamide contents:

Table 1: Percentage acrylamide content in potato chips (produced from potato tubers stored at 4°C). The corresponding respective wild types were set at 100%.
The absolute acrylamide content in the products from potatoes stored at 4°C always increases markedly. However, it is found for the varieties listed here, also, that the acrylamide content in the potato chips from transgenic

potato tubers increases by approximately 30-45% less compared with the potato chips from corresponding wild type plants.
In a further experiment, potato tubers of the cultivar Cesiree having reduced Rl-gene expression (Lorberth et ai., Nature Biotechnology 15: (1998), 473-477) were stored at 4°C for 73 days. Potato chips and crisps were produced as described in Example 1. Crises were deep-fat fried for 3 minutes at 180°C as described in Example 2 in cenrast to the potato crisps, potato chips were deep-fat pre-friec at 18C=C for 3C seconds, placed on kitchen paper and cooled to room temperature and then deep-fat fried for 3 minutes.
The potato chips and crisps produced had the renewing acrylamide contents:
Potato chips ! Crisps ;
deep-fat frying time j deep-fat frying time y
3 min 13 min j ,
Wild type 100% |100% ,
009VL045 "56% 1^3% {
I I 1 !
Table 2: Percentage acryiarnide content of potato chips and crisps (produced from tubers stored at 4°C). The wild type was set at 100%.
The absolute acrylamide content in the products from potatoes stored at 4°C always increases markedly. However, - is aisc found in this experiment that the acrylamide content increases by approximately 70% less in the crisps made from transgenic potato tubers compared with the crises made from the'corresoondinq wild type plants.

In a further experiment, potato tubers of cultivar Desiree of reduced R1-gene expression"(Lorberth et al., Nature Biotechnology 16, (1998), 473-477) were stored at 8°C for 73 days. These stored potato tubers were processed into potato chips in accordance with Example 1 and deep-fat pre-fried for 30 seconds at 180°C in accordance with Example 2, placed on kitchen paper and cooled to room temperature and then deep-fat fried at 180°C for 3 minutes.
The potato chips produced had the following acrylamide contents;

Table 3: Percentage acrylamide content of potato chips (produced from tubers stored at 8°C). The wildtype was set at 100%.
The absolute acrylamide content in the products from potato tuber stored at 8°C does not increase as greatly as in the case of potato tubers stored at 4°C. However, in this experiment also, it is found that the acrylamide content in the potato chips from transgenic potato tubers increases by approximately 48% less compared with potato chips made from corresponding wild type plants.






fish and vegetables, bread coatings for nuts, tortilla chips, bread or cereal formulations, pre-cooked meals baby food.
6. Process according to one of Claim 1 tc 5. in which the plant material used is characterized in that it is genetically modified, the genetic modification leading to a reduction in the center.: of solucie sugars, compared with corresponding conventional plant: materal from wild type plants.
7. Process according to Claim 8, in which the said genetic modification leads to a reduction in the activity of one or more endogenous R1 proteins occurring in the plant ceil compared with, corresponding plant cells of wild type plants which have not been genetically modified.
3. Process according to one of Claims 6 or 7, In which the sale generic modification is the introduction of one cc more foreign nucleic acic molecules, the presence and/or expression of which leads to the reduction in the activity of one or more endogenous Rl proteins occurring in the plant ceil compared with corresponding piant ceiis of wild type plants which have not been genetically modified.
3. Process according to Claim 8. in which the said foreign nucleic acid molecules are selected from the group consisting of
(a) DNA molecules which code for at least one antisense RNA causing a reduction in expression of endogenous genes which code for R1 proteins;
(b) DNA molecules which, via a cosuppressicn effect, lead tc reduction of the expression of endogenous genes coding for R1 proteins;





12. The use of plant material according to one of Claims 1t 6,7, 8, 9 or 10 for producing heat-treated foods which, compared with corresponding conventional heat-treated foods, have a reduced acrylamide content.
13.The use according to Claim 12, in which the said acrylamide content is reduced by least 15% compared with the acrylamide content of corresponding conventional heat-treated foods.
14.The use according to Claims 12 or 13, in which the said heat-treated foods are selected from the group consisting of potato crisps, (potato) chips, parfried potato chips, mashed potato, biscuits, crackers, crisp bread, breakfast cereals, maize crisps (tacos), popcorn, bread crisps, wafers, salt sticks, coffee, bread, rolls, cakes, rice crisps, pizza and toast, in addition tortillas, croquettes, wedges, potato sticks, twisters, bread coatings for meat, fish and vegetables, bread coatings for nuts, tortilla chips, bread or cereal formulations, pre-cooked meals baby food.
15. The use of plant material according to one of Claims 1, 6, 7, 8, 9 or 10 for reducing the acrylamide content of heat-treated foods.
16. Process for identifying plant material which is suitable for producing heat-treated foods having a reduced acrylamide content, comprising:
a) determining the content of soluble sugars and/or amino acids - of the plant material which is suitable for producing heat-treated foods; and

b) selecting such plant material according "to process step a) which, compared with corresponding conventional plant material, has a reduced content of soluble sugars and/or amino acids.


Documents:

0827-chenp-2005 abstract-duplicate.pdf

0827-chenp-2005 claims-duplicate.pdf

0827-chenp-2005 description (complete)-duplicate.pdf

0827-chenp-2005-abstract.pdf

0827-chenp-2005-claims.pdf

0827-chenp-2005-correspondnece-others.pdf

0827-chenp-2005-correspondnece-po.pdf

0827-chenp-2005-description(complete).pdf

0827-chenp-2005-form 1.pdf

0827-chenp-2005-form 18.pdf

0827-chenp-2005-form 3.pdf

0827-chenp-2005-form 5.pdf

0827-chenp-2005-pct.pdf


Patent Number 228004
Indian Patent Application Number 827/CHENP/2005
PG Journal Number 10/2009
Publication Date 06-Mar-2009
Grant Date 27-Jan-2009
Date of Filing 04-May-2005
Name of Patentee BAYER CROPSCIENCE AG
Applicant Address ALFRED-NOBEL-STRASSE 50, 40789 MONHEIM,
Inventors:
# Inventor's Name Inventor's Address
1 SOYKA, Stephan Golzstrasse 19, 10781 Berlin,
2 FROHBERG, Claus Pilzwald 17, 14532 Kleinmachnow,
3 QUANZ, Martin Oppelner Strasse 34, 10997 Berlin,
4 ESSIGMANN, Bernd Menzelstrasse 26, 12157 Berlin,
PCT International Classification Number A23L 1/01
PCT International Application Number PCT/EP2003/012476
PCT International Filing date 2003-11-07
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 02025008.0 2002-11-08 EUROPEAN UNION
2 03090151.6 2003-05-21 EUROPEAN UNION
3 03003235.3 2003-02-21 EUROPEAN UNION