Raphanus with increased anthocyanin levels

Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part

Reexamination Certificate

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C800S260000, C800S266000, C800S268000, C800S298000, C435S410000, C435S420000

Reexamination Certificate

active

06686517

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to plants of the genus Raphanus that contain increased levels of anthocyanins, In particular the invention relates to edible Raphanus sprouts containing increased levels of anthocyanins, as well as to methods for their production.
BACKGROUND OF THE INVENTION
There is an increasing consumer demand for edible young vegetable plants, also referred to as sprouts. Sprouts may be produced by germinating seeds in either liquid medium and harvested before the cotyledons appear, as is e.g. done with alfalfa. Alternatively, seeds may be sown onto a water-soaked solid support, such as cellulose, and allowed to germinate and grow in e.g. small disposable containers until or beyond the cotyledons appear. Further growth of the plantlets may be arrested, e.g. by cooling the plantlets, usually before the plants reach a height of about 4 to 15 cm. The plantlets are then ready for consumption. Sprouts of e.g. cress, daikon (a type of radish sprout) and mustard grow this way are very popular and many other vegetables and herbs are nowadays also available in the form of sprouts. The popularity of vegetable sprouts may at least in part be explained by the notion that they are healthy. Sprouts are indeed known to be very rich in vitamins and minerals such as the vitamins C and B1, potassium, calcium, phosphorus, magnesium and iron, while at the same time they are low in calories. Daikon sprouts, i.e. Sprouts of
Raphanus sativa longipinatus
, are particularly popular in the U.S, and in Japan were they are usually sold as young two-leafed green plants. Unfortunately, daikon sprouts do not contain anthocyanins, a compound that could further contribute to the health promoting effect of these sprouts.
Anthocyanins are present in many plants of higher order where they are responsible for the red, violet, blue or bluish black colours of flowers and fits. They are heterocyclic 2-phenyl-chromenol multiring systems (see also Formula 1) of varying hydroxylation patterns and varying absorption spectra in the visible light range. The sugar-free aglycon components of anthocyanins are referred to as anthocyanidins. They are obtained easily by hydrolysis of the glycosides contained in common fruits.
More recently, anthocyanins have drawn attention for their health promoting effects (see e.g. WO 92/03146). E.g, it is known that anthocyanins can act as scavengers for oxygen radicals such as superoxide anion radical, hydrogen peroxide, hydroxyl radical, alkoxyl radicals, peroxyl radicals for singulett oxygen, and many other radicals. Anthocyanins have also been described as photobiological inhibitors that intervene as regulators and detoxifiers in sensitised photoreactions which take place through oxygen, thereby preventing the radical and radical chain reactions which damage cells and nucleic acids and protein molecules. Anthocyanins also protect against cell toxic and carcinogenic aldehydes such as e.g. 4-hydroxy-hexenal, 4-hydroxy-octenal, 4-hydroxy-nonenal, propanal, butanal, pentanal, hexanal, 2,4-hepta-dienal, malonic dialdehyde, and others. They even prevent the formation thereof within the framework of lipoperoxidative chain reactions. As such they may aid in the prevention of cancer or may delay the effects of ageing. Furthermore, they detoxify the acetaldehyde resulting from ethanol decomposition and the formaldehyde resulting from methanol decomposition.
Some species of Raphanus do produce anthocyanins, as is most notable from the red colour on the outside of the radish varieties as usually sold in Europe and the U.S. However, no Raphanus sprouts are available that contain appreciable levels of anthocyanins. Thus, it is an object of the present invention to provide for Raphanus plants containing increased levels of anthocyanins, in particular, it is an object of the present invention to provide for Raphanus plants, the sprouts of which contain increased levels of anthocyanins. Advantages of the anthocyanin containing sprouts of the invention over other consumable anthocyanin sources, such as e.g. fruits like blueberry or grapes, include (1) the much shorter cultivation time; (2) the relatively high concentration of anthocyanins in the sprouts allowing to consume only small amounts of the sprouts to meet a certain anthocyanin intake; and (3) the great variety a dishes and recipes in which the sprouts may be applied.
DESCRIPTION OF THE INVENTION
In a first aspect the invention relates to a plant of the genus Raphanus, whereby the plant upon germination of its seed produces a sprout that comprises one or more anthocyanins at a level of at least 100 nmol per gram fresh weight of sprout. A Raphanus sprout is herein defined as any developmental stage of a Raphanus plant ranging from a germinating seed to a plantlet that has a height of no more than 20, preferably no more than 17, 15, 14, 12 or 10 cm. Preferably, a Raphanus sprout is a Raphanus plant in a developmental stage beyond a germinating seed and preferably having no more than two leaves, i.e. the cotyledons. Further preferred embodiments of Raphanus sprout are herein defined below.
The Raphanus plants of the invention, or sprouts or turnips thereof, comprise anthocyanins at a level of preferably at least 100, 200, 400, 800, 1500, 3000, 4000, 5000 or 6000 nmol per gram of fresh weight plant material. Fresh plant material of the Raphanus plants of the invention has an anthocyanin content of preferably at least 100, 200, 400, 800, 1500, 2500, 3500 or 4500 ppm (parts per million). The anthocyanin levels or content may be determined photospectrometrically, using a malvine calibration curve to estimate the anthocyanin levels or contents of anthocyanins extracted from fresh plant material as described in Example 4. Preferably, these anthocyanin levels are present in sprouts of the Raphanus plants of the invention. It is to be understood that these anthocyanin levels relate to the total level of anthocyanins and thus may comprise the various glycosylation forms of the anthocyanins as well as anthocyanins with different anthocyanidin moieties as defined below. The anthocyanin levels are expressed per gram fresh weight of plant material, whereby preferably, if present, the roots have been removed by cutting prior to the determination of the fresh weight of the material to be extracted. Alternatively, the anthocyanin levels in the Raphanus plants of the invention may be defined by comparison to a reference plant of the invention such as the
Raphanus sativa
line V33, (i.e. ATCC No. PTA-3630). Thus, a Raphanus plants of the invention preferably has an anthocyanin content of preferably at least 2, 5, 10, 20, 50 or 75% of the anthocyanin content of the
Raphanus sativa
line V33, (i.e. ATCC No. PTA-3630), whereby tie anthocyanin content of both plants is determined in the same part(s) of the plants and/or at the same developmental stage of the plants and using tie same analytical technique.
The Raphanus plants of the invention comprise anthocyanins. Anthocyanin are herein defined as compounds having the following characteristics, (1) comprising a molecular structure as shown in Formula 1, having a 2-phenyl benzopyrylium cation (flavylium ion); (2) an intense red, pink, violet, or purple colour; (3) a strong shift in colour at high alkaline pH (towards green to yellow); (4) solubility in water. The anthocyanins contained in the Raphanus plants of the invention preferably comprise an anthocyanin having a anthocyanidin moiety with the structure of Formula 1, wherein R
1
is OH or OCH
3
, and wherein R
2
is H, OH, or OCH
3
. The anthocyanin may contain one or more glycosides attached to any of the hydroxyl groups of the anthocyanidin moiety, but not to each of the hydroxyl groups, whereby preferably at least the hydroxyl group in the 3 position is glycosylated. The Raphanus plants of the invention comprise anthocyanins that preferably have an absorbance maximum at a wavelength higher than 515, 520, 525, or 530 nm and preferably at wavelength less than 550, 545, 540 or 535 nm. The Raphanus plants of the invention pre

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