• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    Corn enriched in antioxidants to improve the nutritional quality of the egg. The present invention relates to a nutritive or nutraceutical composition comprising a part of a corn plant enriched in antioxidants or a derivative thereof and with the uses of said composition for obtaining eggs enriched in antioxidants. The invention also relates to the use of a part of a corn plant enriched in antioxidants or a derivative thereof for obtaining eggs enriched in antioxidants. The invention also relates to an egg and egg products derived from said egg obtained according to the uses of the invention. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2558953A1
    申请号:ES201531699
    申请日:2015-11-23
    公开日:2016-02-09
    发明作者:José Antonio MORENO MARTÍNEZ；Carmen NOGAREDA BURCH；Eduardo ANGULO ASENSIO；Paul Christou；Changfu Zhu；Teresa CAPELL
    申请人:CENTRE DE RECERCA EN AGROTECNOLOGIA-AGROTECNIO；Institucio Catalana de Recerca i Estudis Avancats ICREA；Universitat de Lleida；
    IPC主号:
    专利说明:

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    "Phytoeno", as used in the present invention, refers to an intermediate of 40 carbon atoms in the biosynthesis of carotenoids.
    "Beta carotene ketolase", as used in the present invention, refers to the
    The enzyme encodes the crtW gene and catalyzes the two stages of introduction of the oxo group into the beta-yonone ring of beta-carotene at both ends. The sequence coding for C. reinhardtii beta carotene ketolase has the sequence shown in SEQ ID NO: 14.
    "Beta carotene hydroxylase", as used in the present invention refers to the
    10 enzyme encoded by the crtZ gene and involved in the two stages of hydroxylation of the beta-yonone ring of beta carotene at both ends. The sequence that codes for the beta carotene hydroxylase of Brevidomonas sp. It has the sequence shown in SEQ ID NO: 15.
    "Lycopene epsilon cyclase", as used herein, refers to an enzyme that acts
    15 on the lycopene-all-trans carotenoid that has psi-terminal groups at both ends, in a first step it becomes δ-carotene; and then in ε-carotene.
    "RNAi" or interfering RNA ", as used in the present invention, refers to RNA molecules capable of silencing the expression of a gene. To that end, RNAi are
    20 typically double stranded oligonucleotides that are at least 30 base pairs in length, and more preferably comprise about 25, 24, 23, 22, 21, 20, 19, 18 or 17 base pairs of ribonucleic acids. The siRNA of lycopene epsilon cyclase has the sequence shown in SEQ ID NO: 16.
    "Crossing", as used in the present invention, refers to the sexual reproduction of two plants, resulting in offspring inheriting part of the genetic material of each parent. Relative organisms must be genetically compatible and can be of different varieties or of very close species. The crossing originally occurs under natural cross pollination between plants whose
    30 genetic constitution is different.
    12
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    Nutritious or nutraceutical is for animal consumption. The nutritive or nutraceutical composition may be in the form of a solution or as a solid, depending on the use and / or the mode of application and / or the mode of administration.
    It is understood that a part of a corn plant or a derivative thereof is found
    5 enriched in antioxidants, when it presents antioxidant levels higher than the levels of a reference sample or control sample. In particular, a part of a plant or derivative thereof can be considered to have higher levels of antioxidants when the levels are at least 1 time, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90
    10 times, 100 times or even more with respect to the reference sample or control sample. The person skilled in the art will understand that it is not necessary for an antioxidant-enriched plant to contain high levels of said antioxidants in all tissues of said plant, but it is sufficient that at least one plant tissue contains high levels of antioxidants so that the plant can be considered
    15 as "enriched in antioxidants." In that case, the reference sample to determine the degree of antioxidant enrichment is determined by comparison with antioxidant levels is a sample of the same tissue or part of a control plant. The level of a single type of antioxidant is also included by antioxidant level.
    A control or reference sample, as used in the present invention, may be the value that corresponds to the levels of antioxidants present in a part of a corn plant or derived therefrom that is in the wild, that is not genetically modified.
    The person skilled in the art knows various methods to determine whether a corn plant or
    The derivative thereof is enriched in antioxidants, for example by determining antioxidant levels, particularly carotenoids by HPLC (high performance liquid chromatography) or by determining the increase in antioxidant capacity.
    The antioxidant capacity can be determined by quantifying the ability of the antioxidant compounds to react with a given free radical,
    fifteen


    or determining the potential such compounds would have to reduce the complex formed between Fe (III) ions and the TPTZ reagent (2,4,6-tripyridyl-s-triazine).
    Among those tests that are based on measuring the ability of antioxidants to react with a free radical, the following are worth mentioning, the ORAC test (from
    5 English “Oxygen Radical Absorbance Capacity” or oxygen radical absorption capacity), TEAC test (Trolox Equivalent Antioxidant Capacity or Antioxidant Capacity as Trolox Equivalent) and DPPH Test (2,2-Diphenyl-1-picrilhydrazil).
    The nutritive or nutraceutical compositions of the invention comprise at least 0.01% of the part of a plant enriched in antioxidants or derived therefrom,
    10 more preferably at least 0.10%, 0.50%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% 20% , 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95%.
    In a particular embodiment, the nutritive or nutraceutical composition of the invention comprises at least 50 to 80% of a part of a corn plant enriched in antioxidants or a derivative thereof.
    In a more particular embodiment, the nutritive or nutraceutical composition of the invention comprises at least 60 to 70% of a part of an antioxidant-enriched corn plant or a derivative thereof, more particularly at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69.
    In another particular embodiment, the part of the corn plant is the endosperm, the seed or derivatives thereof. As an illustrative, non-limiting example, the derivative of the seed or endosperm can be a flour, semolina, corn oil, corn starch, cornstarch or corn syrup among others.
    The nutritive or nutraceutical composition according to the present invention can be
    25 prepare directly from the part of the plant enriched in antioxidants. In a preferred embodiment, the part of the plant is the endosperm or corn kernels, in which case said composition contains at least 40%, preferably more than 50% or more preferably more than 60%.
    16


    Alternatively, the part of the plant that is used to prepare the nutritive or nutraceutical composition according to the present invention can be subjected to a partial purification to obtain a derivative enriched in the antioxidant product. Thus, in the case that the antioxidant compounds are carotenoids and that the part of the plant that is used is the endosperm, the nutritive or nutraceutical composition contains an extract of corn or endosperm grains that can be found partially or totally purified so that the carotenoid content is higher than what appears in the part of the enriched corn plant. Corn derivatives that can be used in the present invention include cornmeal, corn bran, corn oil, starch
    10 corn, corn syrup, and the like.
    Thus, for example, according to the present invention, it is possible to extract the carotenoids present in the part of the corn plants or derivatives thereof to obtain a nutritive or nutraceutical composition.
    The person skilled in the art knows various methods to extract carotenoids from a part
    15 of a corn plant, as extracted from frozen endosperms and HPLC, as described in Naqvi S. et al., PNAS 2009, vol. 106, not 19.
    As one skilled in the art will understand, the nutritive or nutraceutical composition of the invention must comply with the country's food regulations and legislation.
    There are several known methods for preparing nutritional compositions and
    20 nutraceuticals widely known to those skilled in the art and which can be used in the present invention.
    The compositions of the invention or nutraceuticals may be in a liquid, semi-solid or solid form. In a particular embodiment the compositions or nutraceuticals of the invention are in solid form and more particularly it is a feed.
    The nutritional or nutraceutical compositions of the invention can be formulated with the usual excipients and adjuvants for oral compositions or food supplements, such as and without limitation, fatty components, aqueous components, humectants, preservatives, texturizing agents, flavors, aromas, antioxidants and dyes common in the food sector.
    17
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    zeaxanthin; γ-carotene, β-cryptoxanthin, lutein and zeaxanthin; γ-carotene, lycopene, lutein and zeaxanthin; α-carotene, α-cryptoxanthin, β-cryptoxanthin and lycopene; α-carotene, αcriptoxanthin, β-cryptoxanthin and lutein; α-carotene, α-cryptoxanthin, β-cryptoxanthin and zeaxanthin; α-carotene, α-cryptoxanthin, lycopene and lutein; α-carotene, α5 cryptoxanthin, lycopene and zeaxanthin; α-carotene, α-cryptoxanthin, lutein and zeaxanthin; α-carotene, β-cryptoxanthin, lycopene and lutein; α-carotene, β-cryptoxanthin, lycopene and zeaxanthin; α-carotene, β-cryptoxanthin, lutein and zeaxanthin; α-carotene, lycopene, lutein and zeaxanthin; α-cryptoxanthin, β-cryptoxanthin, lycopene and lutein; α-cryptoxanthin, βcriptoxanthin, lycopene and zeaxanthin; α-cryptoxanthin, β-cryptoxanthin, lutein and zeaxanthin;
    Α-cryptoxanthin, lycopene, lutein and zeaxanthin; or enriched in cryptoxanthin, lycopene, lutein and zeaxanthin.
    In another embodiment of the nutritive composition A or nutraceutical A of the invention, the corn plant or derivative thereof enriched in carotenoids is enriched in at least 15 carotenoids, by way of illustration it is enriched in β-carotene, γ carotene, α-carotene, α-cryptoxanthin and β-cryptoxanthin; β-carotene, γ-carotene, α-carotene, αcriptoxanthin and lycopene; β-carotene, γ-carotene, α-carotene, α-cryptoxanthin and lutein; β-carotene, γ-carotene, α-carotene, α-cryptoxanthin and zeaxanthin; β-carotene, γ-carotene, α-carotene, β-cryptoxanthin and lycopene; β-carotene, γ-carotene, α-carotene, β-cryptoxanthin and lutein; β-carotene, γ-carotene, α-carotene, β-cryptoxanthin and zeaxanthin; β-carotene, γ carotene, α-carotene, lycopene and lutein; β-carotene, γ-carotene, α-carotene, lycopene and zeaxanthin; β-carotene, γ-carotene, α-carotene, lutein and zeaxanthin; β-carotene, γ carotene, α-cryptoxanthin, β-cryptoxanthin and lycopene; β-carotene, γ-carotene, αcriptoxanthin, β-cryptoxanthin and lutein; β-carotene, γ-carotene, α-cryptoxanthin, β25 cryptoxanthin and zeaxanthin; β-carotene, γ-carotene, α-cryptoxanthin, lycopene and lutein; β-carotene, γ-carotene, α-cryptoxanthin, lycopene and zeaxanthin; β-carotene, γ-carotene, αcriptoxanthin, lutein and zeaxanthin; β-carotene, γ-carotene, β-cryptoxanthin, lycopene and lutein; β-carotene, γ-carotene, β-cryptoxanthin, lycopene and zeaxanthin; β-carotene, γ carotene, β-cryptoxanthin, lutein and zeaxanthin; β-carotene, γ-carotene, lycopene, lutein and zeaxanthin; β-carotene, α-carotene, α-cryptoxanthin, β-cryptoxanthin and lycopene; β-carotene, α-carotene, α-cryptoxanthin, β-cryptoxanthin and lutein; β-carotene, α-carotene, αcriptoxanthin, β-cryptoxanthin and zeaxanthin; β-carotene, α-carotene, α-cryptoxanthin, lycopene and lutein; β-carotene, α-carotene, α-cryptoxanthin, lycopene and zeaxanthin; β-carotene, α
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    One skilled in the art knows the appropriate techniques for the generation of transgenic plants. For a review of gene transfer to plants, including vectors, DNA transfer methods, etc., see, for example, Twyman RM, 2004, Handbook of Plant Biotechnology. John Wiley & Sons Inc., NY, pp 263-289, or Komari et al., 2004
    5 Handbook of Plant Biotechnology. John Wiley & Sons Inc., NY, pp 233-262.
    Briefly, such methods may include: 1) use of a live vector that carries the genetic material to the white cell by means of genetically modified viruses or the natural mechanism of infection of the soil bacterium Agrobacterium tumefaciens, 2)
    The use of protoplasts, which are plant cells that have been released from the cell wall, or 3) the biolistics that consists of bombarding the cells with microscopic metal particles coated with the DNA that is to be introduced.
    Specifically, for the generation of transgenic corn plants, the
    15 introduction of DNA into corn cells including electroporation, microinjection, microprojectile bombardment, liposome fusion, Agrobacterium-mediated transfer, macroinjection and exposure to naked DNA in solution.
    To make the gene contained in the recombinant DNA molecule be expressed in the
    20 levels and in the appropriate tissues, it is desirable, but not necessary for genes of plant origin, that is contained in an expression cassette that includes a promoter sequence of functional transcription in plants, the coding part of the gene of interest and a terminator of functional transcription in plants. Additionally, a sequence encoding a transit peptide that directs the enzyme to an enzyme can be included.
    25 specific cell compartment. The transit peptide and corresponding processing signals can be derived from any plant protein that is synthesized in the cytoplasm and translocated to the subcellular compartment of interest, either the plastid or the mitochondria. For example, sequences derived from genes encoding the small subunit of the cell may be used to locate plastids
    30 ribulose bisphosphate carboxylase or CAB proteins that are those that bind to chlorophylls a and b to formal light-harvesting antennas (Van den Broek et al Nature (London) 313, 358-363 (1985)).
    25
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    In an embodiment of the present invention when the corn plant expresses the transgene corresponding to the phytoen synthase with the sequence shown in SEQ ID NO: 1, said sequence additionally presents at the 3'-end the nopaline terminator sequence
    5 synthase with the sequence shown in SEQ ID NO: 9.
    In another particular embodiment, when the corn plant expresses the transgene corresponding to carotene desaturase has the sequence shown in SEQ ID NO: 2, said sequence additionally present at the 3'-terminus sequence of 10 ADP-glucose pyrophosphorylase SEQ ID NO : 10. In another embodiment, the carotene desaturase has the sequence shown in SEQ ID NO: 2, then the carotene desaturase is part of a fusion protein comprising in position N with respect to the carotene desaturase a sequence for protein targeting to the chloroplast, and additionally at the 3'-end it presents the ADP-glucose terminator sequence
    15 pyrophosphorylase SEQ ID NO: 10.
    The transgenes that can be used in the present invention can be placed under the control of regulatory elements that ensure expression in plant cells. In general, such regulatory elements comprise an active promoter in the cells of
    20 plants.
    Thus, promoters that can be used include promoters of the plant to be transformed, promoters of other plants or of any origin that are functional in the white plant and direct a specific constitutive, inducible or tissue expression. For example, promoters derived from the Ti plasmid of Agrobactertum tumefaciens can be used such as the promoters of octopine synthetase, nopahna synthetase or sgropin synthetase. In addition, other promoters such as the 35S promoter of cauliflower mosaic virus or those derived from geminivirus may be included. For a review of useful promoters, see Peremarti A. et al, Plant Mol Biol 73: 363–
    30 378
    Temporary, inducible or specific tissue expression can be achieved through the use of promoters or regulatory sequences that have the desired expression specificity.
    27
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    One skilled in the art knows various methods for determining the level of antioxidants as explained in detail above, in relation to a plant enriched in antioxidants.
    The compositions of the invention can be used to obtain antioxidant-enriched eggs from any female oviparous individual that is fed with said compositions.
    In a preferred embodiment the eggs are eggs of a poultry, more particularly of chicken. 10 Uses of a part of an antioxidant-enriched corn plant or a derivative thereof
    In another aspect, the invention relates to the use of a part of a corn plant
    15 enriched in antioxidants or a derivative thereof to obtain eggs enriched in antioxidants.
    In a particular embodiment of the use of the invention, the part of the corn plant is the endosperm, the seed or derivatives thereof.
    In another particular embodiment, the antioxidants that are enriched in a part of a corn plant or in a derivative thereof are carotenoids.
    In a more particular embodiment of the use of the invention, the part of the corn plant or
    The derivative thereof is enriched in carotenoids selected from the group consisting of β-carotene, γ-carotene, α-carotene, α-cryptoxanthin, β-cryptoxanthin, lycopene, lutein, zeaxanthin and combinations thereof (use A of the invention)
    In a particular embodiment of the use A of the invention, the part of the corn plant or
    The derivative thereof is enriched in only one of the carotenoids, or in 2, 3, 4, 5, 6, 7 or 8 selected from β-carotene, γ-carotene, α-carotene, α-cryptoxanthin, βcriptoxanthin, lycopene, lutein, zeaxanthin and combinations thereof.
    38
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    5'UTR region of the rice alcohol dehydrogenase gene has the sequence shown in SEQ ID NO: 17.
    In another particular embodiment, the eggs are eggs of a poultry, particularly 5 hen.
    All the previously described embodiments and limitations are equally applicable to this aspect. 10 Egg and egg products
    In another aspect, the invention relates to an egg and egg products derived from said egg obtained according to any use of the invention, specifically the use of the nutritive or nutraceutical composition A, B or C, or uses of the part of a corn plant
    15 enriched in antioxidants A, B or C.
    As the person skilled in the art will understand, the eggs of the invention are obtained after feeding a female bird with a nutritive or nutraceutical composition A, B or C or with a corn plant enriched with antioxidants A, B or C.
    20 All the previously described embodiments and limitations are equally applicable to this aspect.
    MATERIALS AND METHODS 25 Corn Transformation
    Corn
    30 Maize (Zea mays) variety M37W (white endosperm) was obtained from CSIR, Pretoria, South Africa. The NSL 30876 high oil line named R38 in this description was kindly provided by the USDA, ARS, NCRPIS, Iowa State University, Regional Plant Introduction Station, Ames, Iowa, United States, 50011
    43
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    Eggs with yolks within the color range accepted by the consumer. This effect is not achieved by conventional standard corn used in the same proportion in the diet.
    The use of developed corn varieties allows reducing the cost of manufactured feed by eliminating the cost of adding carotenes exogenously and simplifying the feed manufacturing process.
    The term "Sequence listing" in the sequence list refers to "Sequence list", "Artificial Sequence" to "Artificial sequence" and the term "DNA" refers to "DNA".
    52
    权利要求:
    Claims (1)
    [1]
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    68-Use according to claim 67 wherein the D-hordein promoter has the sequence shown in SEQ ID NO: 12, the LMW glutenin promoter has the sequence shown in SEQ ID NO: 11 and the gamma-zein promoter has the sequence shown in SEQ ID NO: 13.
    5
    69-Use according to any of claims 58, 59, 61-68 wherein if the transgene encoding a beta carotene ketolase has the sequence SEQ ID NO: 14 and / or the transgene encoding the beta carotene hydroxylase has the sequence SEQ ID NO: 15, then the transgene has a sequence at the 5´ end
    10 for the targeting of proteins to the chloroplast.
    70-Use according to claim 69 wherein the sequence for the targeting of proteins to the chloroplast is the sequence encoding the small subunit of the Ribulose 1.5 bisphosphate Carboxylase-Oxygenase.
    Use according to claim 70 wherein the sequence encoding the small subunit of Ribulose 1.5 bisphosphate Carboxylase-Oxygenase has the sequence shown in SEQ ID NO: 8.
    Use according to any of claims 58, 59, 61-71 wherein the transgene additionally comprises at the 3'-end the 5'UTR region of the rice alcohol dehydrogenase gene.
    73-Use according to claim 72 wherein the 5'UTR region of the rice alcohol dehydrogenase gene has the sequence shown in SEQ ID NO: 17.
    74-Use according to any of claims 40-73 wherein the eggs are eggs of a poultry.
    30- Use according to claim 74 wherein the poultry is chicken.
    76-Egg and egg products derived from said egg obtained according to any of claims 37-75.
    62
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    同族专利:
    公开号 | 公开日
    ES2558953B1|2016-11-18|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20060031963A1|2002-08-20|2006-02-09|Sungene Gmbh & Co. Kgaa|Method for the production of ketocarotinoids in flower petals on plants|
    US8872006B1|2009-06-23|2014-10-28|Pioneer Hi Bred International Inc|Maize variety inbred PH11V8|
    CN102321649A|2011-09-22|2012-01-18|天津大学|Lycium chinense miller lycopene beta-cyclase gene, recombinant vector containing gene, host cell and application|
    CN110423766B|2019-08-29|2021-06-29|南京大学|Lycopene beta-cyclase gene and coding protein and application thereof|
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