Barley with very low levels of hordeins

Abstract

The present invention relates to methods of producing a food or malt-based beverage suitable for consumption by a subject with Coeliac's disease. In particular, the present invention relates to methods of producing a food or malt-based beverage with very low levels of hordeins. Also provided are barley plants which produce grain that can be used in the methods of the invention.

1 . A method of producing a food or malt-based beverage ingredient, or a food or a malt-based beverage, the method comprising (i) processing barley grain to produce malt, wort, flour or wholemeal, and/or (ii) mixing barley grain, or malt, wort, flour or wholemeal produced from said grain, with at least one other food or beverage ingredient, wherein the barley grain, malt, wort, flour or wholemeal comprises about 50 ppm or less hordeins, thereby producing the food or malt-based beverage ingredient, food or malt-based beverage. 
     
     
         2 . The method of  claim 1 , wherein the grain, malt, wort, flour or wholemeal comprises about 20 ppm or less, about 10 ppm or less, about 5 ppm or less, about 0.05 ppm to about 50 ppm, or about 0.05 ppm to about 20 ppm, about 0.05 ppm to about 10 ppm, about 0.05 ppm to about 5 ppm, about 0.1 ppm to about 5 ppm, about 3.9 ppm, or about 1.5 ppm, hordeins. 
     
     
         3 . The method of  claim 1  or  claim 2 , wherein the average weight of the grain is at least about 35 mg, at least about 39 mg, at least about 41 mg, at least about 47 mg, about 35 mg to about 60 mg, about 40 mg to about 60 mg, about 45 mg to about 60 mg, about 39.1 mg, about 41.8 mg or about 47.2 mg. 
     
     
         4 . The method according to any one of  claims 1  to  3 , wherein at least about 80%, at least about 90%, at least about 95%, about 80% to about 98%, or about 80% to about 93%, of the grain do not pass through a 2.8 mm sieve. 
     
     
         5 . The method according to any one of  claims 1  to  4 , wherein the grain is from a plant which has a harvest index of at least 40%, about 40% to about 60%, about 40% to about 55%, or about 40% to about 50%. 
     
     
         6 . The method according to any one of  claims 1  to  5 , wherein the grain has a length to thickness ratio of less than about 5, less than about 4, less than about 3.8, about 2 to about 5, or about 2.5 to about 3.8. 
     
     
         7 . The method according to any one of  claims 1  to  6 , wherein the flour or wholemeal produced from the grain comprises about 10 ppm or less, about 5 ppm or less, about 0.05 ppm to about 10 ppm, or about 0.05 ppm to about 5 ppm, about 3.9 ppm, or about 1.5 ppm, hordeins. 
     
     
         8 . The method according to any one of  claims 1  to  7 , wherein the malt or wort produced from the grain comprises less than about 50 ppm, or less than about 20 ppm, hordeins. 
     
     
         9 . The method according to any one of  claims 1  to  8 , wherein the grain, or malt, wort, flour or wholemeal produced from said grain, has a level of less than 10%, less than 5% or less than 2% of a wild-type level, or is lacking, one or more than one or all of:
 i) B-hordeins comprising a sequence of amino acids provided as SEQ ID NO:53, 
 ii) B-hordeins comprising a sequence of amino acids provided as SEQ ID NO:54, 
 iii) C-hordeins comprising a sequence of amino acids provided as SEQ ID NO:55, and 
 iv) D-hordeins comprising a sequence of amino acids provided as SEQ ID NO:56, 
 
       wherein each of the levels of less than 10%, less than 5% or less than 2% is relative to a wild-type barley grain, or malt, wort, flour or wholemeal produced from said grain, of the barley variety Bomi, Sloop, Baudin, Yagan, Hindmarsh, or Commander. 
     
     
         10 . The method of  claim 9 , wherein the B-hordeins are at least B1-hordein and B3-hordein. 
     
     
         11 . The method of  claim 9  or  claim 10 , wherein the grain, or malt, wort, flour or wholemeal produced from said grain, further has a level of less than 10%, less than 5% or less than 2% of a wild-type level, or is further lacking;
 i) γ-hordeins comprising a sequence of amino acids provided as SEQ ID NO:57, and/or 
 ii) avenin-like A proteins comprising a sequence of amino acids provided as SEQ ID NO:52, 
 
       wherein each of the levels of less than 10%, less than 5% or less than 2% is relative to a wild-type barley grain, or malt, wort, flour or wholemeal produced from said grain, of the barley variety Bomi, Sloop, Baudin, Yagan, Hindmarsh, or Commander. 
     
     
         12 . The method according to any one of  claims 1  to  11 , wherein the grain is homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted. 
     
     
         13 . The method according to any one of  claims 1  to  12 , wherein the grain is homozygous for a null allele of the gene encoding D-hordein at the Hor3 locus, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the null allele of the gene encoding D-hordein, the null allele preferably comprising a stop codon, splice site mutation, frame-shift mutation, insertion, deletion or encoding a truncated D-hordein, or where most or all of the D-hordein encoding gene has been deleted. 
     
     
         14 . The method of  claim 13 , wherein the truncated D-hordein has a stop codon at the triplet encoding amino acid number 150. 
     
     
         15 . The method according to any one of  claims 1  to  14 , wherein the grain is homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C-hordeins, or wherein the malt, wort, flour or wholemeal produced from said grain comprises DNA which comprises the allele at the Lys3 locus. 
     
     
         16 . The method according to any one of  claims 1  to  15 , wherein the grain, malt, wort, flour or wholemeal comprises about 1% or less, about 0.01% or less, about 0.007% or less, about 0.0027% or less, about 0.001% to about 1%, about 0.001% to about 0.01%, about 0.007%, or about 0.0027%, of the level of hordeins when compared to grain from a corresponding wild-type barley plant or malt, wort, flour or wholemeal produced in the same manner from grain from a corresponding wild-type barley plant. 
     
     
         17 . The method of  claim 16 , wherein the grain is from a plant which has at least 60%, at least 80%, at least 90%, about 60% to 100%, about 70% to 100%, about 80% to 100%, about 60%, about 70%, about 80%, or about 90% of the grain yield of the wild-type barley plant. 
     
     
         18 . The method of  claim 16  or  claim 17 , wherein the average weight of the grain is at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100%, of grain of the wild-type barley plant. 
     
     
         19 . The method according to any one of  claims 16  to  18 , wherein the grain, or malt, wort, flour or wholemeal produced from said grain, comprises about 10% or less, about 5% or less, about 2% or less, about 0.1% to about 10%, or about 0.1% to about 10%, of one or more than one or all of the following when compared to the corresponding wild-type barley plant:
 i) B-hordeins comprising a sequence of amino acids provided as SEQ ID NO:53, 
 ii) B-hordeins comprising a sequence of amino acids provided as SEQ ID NO:54, 
 iii) C-hordeins comprising a sequence of amino acids provided as SEQ ID NO:55, and 
 iv) D-hordeins comprising a sequence of amino acids provided as SEQ ID NO:56. 
 
     
     
         20 . The method of  claim 19 , wherein the B-hordeins are at least B1 and B3. 
     
     
         21 . The method according to any one of  claims 16  to  20 , wherein the grain, or malt, wort, flour or wiolemeal produced from said grain, which further comprises about 10% or less, about 5% or less, about 1% or less, about 0.1% to about 10%, or about 0.1% to about 10%, of the following when compared to the corresponding wild-type barley plant;
 i) γ-hordeins comprising a sequence of amino acids provided as SEQ ID NO:57, and/or 
 ii) avenin-like A proteins comprising a sequence of amino acids provided as SEQ ID NO:52. 
 
     
     
         22 . The method according to any one of  claims 16  to  21 , wherein the wild-type barley plant is Bomi, Sloop, Baudin, Yagan, Hindmarsh, or Commander. 
     
     
         23 . The method according to any one of  claims 1  to  22 , wherein the coeliac toxicity of flour produced from the grain is less than about 5%, or less than about 1%, of flour produced from grain of a corresponding wild-type barley plant. 
     
     
         24 . The method according to any one of  claims 1  to  23 , wherein the average grain weight is at least 1.05 fold, at least 1.1 fold, or 1.05 to 1.3 fold, higher than a grain which is
 i) homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, 
 ii) homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C hordeins, and 
 iii) homozygous for a wild type allele of D hordein encoding a full-length protein. 
 
     
     
         25 . The method according to any one of  claims 1  to  24 , wherein the grain is from a plant which has a grain yield which is least 1.20 fold, or at least 1.35 fold, or 1.2 to 1.5 fold, or 1.2 to 2.0 fold higher than the grain yield from a plant which is
 i) homozygous for an allele of the Hor2 locus where most or all of the B-hordein encoding genes have been deleted, 
 ii) homozygous for an allele at the Lys3 locus of barley which results in the grain lacking C hordeins, and 
 iii) homozygous for a wild type allele of D hordein encoding a full-length protein. 
 
     
     
         26 . The method according to any one of  claims 1  to  25 , wherein at least about 50% of the genome of the barley grain is identical to the genome of a barley cultivar Sloop, Hindmarsh, Oxford or Maratime. 
     
     
         27 . The method according to any one of  claims 1  to  26 , wherein the grain is from a non-transgenic plant. 
     
     
         28 . The method according to any one of  claims 1  to  27 , wherein the grain is from a transgenic plant. 
     
     
         29 . The method of  claim 28 , wherein the plant comprises a transgene which encodes a polynucleotide which down-regulates the production of at least one hordein in the grain. 
     
     
         30 . The method according to any one of  claims 1  to  29  which comprises producing flour or wholemeal from the grain. 
     
     
         31 . The method according to any one of  claims 1  to  29  which comprises producing malt from the grain. 
     
     
         32 . The method according to any one of  claims 1  to  31 , wherein the malt-based beverage is beer and the method comprises germinating the grain or cracked grain derived therefrom. 
     
     
         33 . The method of  claim 32  which further comprises fractionating dried germinated grain into two or more of an endosperm fraction, an endothelial layer fraction, a husk fraction, an acrospire fraction, and a malt rootlets fraction; and combining and blending predetermined amounts of two or more of the fractions. 
     
     
         34 . The method according to any one of  claims 1  to  33 , wherein at least about 50% of the grain germinates within 3 days following imbibition. 
     
     
         35 . The method according to any one of  claims 1  to  34 , wherein the food ingredient or malt-based beverage ingredient is flour, starch, malt, or wort, or wherein the food is leavened or unleavened breads, pasta, noodles, breakfast cereals, snack foods, cakes, pastries or foods containing flour-based sauces. 
     
     
         36 . The method according to any one of  claims 1  to  35 , wherein the malt-based beverage is beer or whiskey. 
     
     
         37 . The method according to any one of  claims 1  to  36 , wherein the food or malt-based beverage is for human consumption. 
     
     
         38 . The method according to any one of  claims 1  to  37 , wherein following consumption of the food or drink at least one symptom of coeliac's disease is not developed by a subject with said disease. 
     
     
         39 . A barley plant which produces grain comprising about 50 ppm or less hordeins. 
     
     
         40 . The barley plant of  claim 39 , wherein the grain comprises one or more of the features defined in  claims 2  to  29 . 
     
     
         41 . Grain of a barley plant of  claim 39  or  claim 40 . 
     
     
         42 . A method of producing barley grain, the method comprising:
 a) growing a barley plant of  claim 39  or  claim 40 ,   b) harvesting the grain, and   c) optionally processing the grain.   
     
     
         43 . The method of  claim 42  which comprises growing at least 10,000 plants in a field in an area of at least one hectare. 
     
     
         44 . A method of producing flour, wholemeal, starch, malt, wort or other product obtained from grain, the method comprising:
 a) obtaining grain of  claim 41 , and   b) processing the grain to produce the flour, wholemeal, starch, malt, wort or other product.   
     
     
         45 . A product produced from a barley plant of  claim 39  or  claim 40 , or grain of  claim 41 . 
     
     
         46 . The product of  claim 45 , wherein the product is a food ingredient, malt-based beverage ingredient, food product or malt-based beverage product. 
     
     
         47 . The product of  claim 46 , wherein the malt-based beverage product is beer or whiskey. 
     
     
         48 . The product of  claim 45 , wherein the product is a non-food product. 
     
     
         49 . The product of  claim 48 , wherein the non-food product is selected from the group consisting of: films, coatings, adhesives, building materials and packaging materials. 
     
     
         50 . A food or malt-based beverage produced using a method according to any one of  claims 1  to  38 . 
     
     
         51 . Beer comprising one or more barley grain proteins and less than 0.9 ppm hordeins. 
     
     
         52 . The beer of  claim 51  which comprises at least about 2% ethanol. 
     
     
         53 . Flour or wholemeal comprising one or more barley grain proteins and less than about 50 ppm, or less than about 20 ppm, hordeins. 
     
     
         54 . Malt or wort comprising one or more barley grain proteins and less than about 50 ppm, or less than about 20 ppm, hordeins. 
     
     
         55 . A method of identifying an allele of a barley D hordein gene which encodes a truncated D hordein, the method comprising
 i) obtaining a sample comprising nucleic acids from a barley plant, and   ii) analysing the sample for the presence or absence of a guanine residue at position 450 of the open reading frame encoding D hordein,   wherein the presence of the guanine indicates the D hordein gene encodes a truncated D hordein.   
     
     
         56 . The method of  claim 55 , wherein step b) comprises amplifying genomic DNA using the primers GGCAATACGAGCAGCAAAC (SEQ ID NO: 66) and CCTCTGTCCTGGTTGTTGTC (SEQ ID NO: 67), or a variant of one or both thereof, and contacting products of the amplification with the restriction enzyme KpnI, wherein the absence of cleavage indicates the D hordein gene encodes a truncated D hordein. 
     
     
         57 . A method of avoiding or reducing the incidence or severity of coeliac's disease in a subject, the method comprising orally administering to the subject a food or malt-based beverage according to any one of  claims 46 ,  47 ,  50  or  51  to  54 , or a grain according to  claim 41 , wherein the reduction of the incidence or severity of coeliac's disease is relative to when the subject is orally administered the same amount of a corresponding food or malt-based beverage made from wild-type barley grain. 
     
     
         58 . Use of a food or malt-based beverage according to any one of  claims 46 ,  47 ,  50  or  51  to  54 , or a grain according to  claim 41 , for the manufacture of a food or beverage for orally administering to a subject to avoid or reduce the incidence or severity of coeliac's disease.