c-ray
09-26-2008, 06:37 PM
who knows if this has any merit, but here it is regardless
from http://www.wipo.int/pctdb/en/wo.jsp?IA=WO2007085842&wo=2007085842&DISPLAY=DESC
WO 2007085842 20070802
PLANT TREATMENT METHOD AND MEANS THEREFORE
The present invention relates to a method for altering the level of phytochemicals in plant cells and/or plant tissue and means therefor. In particular, the invention relates to a method for altering the level of phytochemicals such as plant primary or secondary metabolites in harvested plant cells and/or plant tissue by applying wavelengths of light of selected wavelength and intensity thereto that are selected from wavelengths of light from the white light or visible spectrum and means therefor.
It is known that the application of light from the UV spectrum, such as UV-B and UV-C can help to increase the levels of for example 'essential oils' and secondary metabolites in whole plants. However, UV-B and UV-C is problematic to handle for humans and is heavily implicated in cancerous disease processes. As such, UV-B and UV-C light is considered potentially harmful to healthy mammalian tissue and is considered hazardous to use.
'Essential oils' are responsible in large part for the aromaticity associated with many plants, such as plants comprising perfumed flowers and herbs, such as culinary herbs. Essential oils consist mainly of terpenoids and can include such compounds as 1 ,8-cineole, limonene, linalool and β-ocimene. Other compounds which may be found in essential oils, that is, oils which are not terpenoids, can include phenyl-propanoid-derived compounds such as methyl chavicol, methyl cinnamate, eugenol, and methyl eugenol. Thus, the term 'essential oils' is used in a qualitative sense to encompass compounds as indicated herein which contribute to the aromaticity of plants such as perfumed ornamentals and culinary herbs.
Ultraviolet light (and specifically UV-B) is known to have effects on the levels of secondary compounds of the phenyl-propanoid pathway of plants via action on key regulatory enzymes such as phenylalaline ammonia-lyase (Kuhn, D.N. et al (1984) Proc. Natl. Acad. ScL, USA, 81, 1102-1106) and chalcone synthase (Batschauer, A. et al (1996) The Plant Journal 9, 63-69 and Christie, J. M. and Jenkins, G.I. (1996) The Plant Cell 8, 1555-1567). There are many published reports of UV-B stimulation of phenolic compounds, including surface flavonols and flavonoids (Cuadra, P. and Harborne, J. B. (1996) Zeitschrift fur Naturforschung 51c, 671-680 and Cuadra, P. et al (1997) Phytochemistry 45, 1377-1383), anthocyanins (Yatsuhashi, H. et al (1982) Plant Physiology 70, 735-741 and Oelmϋller, R. and Mohr, H. (1985). Proc. Natl. Acad. ScL, USA 82, 6124-6128) and betacyanins (Rudat, A. and Goring, H. (1995). J. Expl. Bot. 46, 129-134) and these compounds have been
implicated both in plant defence (Chappell, J. and Hahlbrock, K. (1984) Nature 311, 76-78 and Guevara, P. et a/ (1997) Phyton 60, 137-140) and as protection against UV-light (Lois, R. (1994) Planta 194, 498-503; Ziska, LH. et al (1992) Am. JnI. BoL 79, 863-871 and Fiusello, N. et al (1985) Allionia (Turin) 26, 79-88).
FR 3542567 describes the application of blue and/or red light to certain fruits, typically un- harvested fruits, at night for periods of long duration measured in days. Furthermore, it appears that the effect of such light was also ascertained on leaf discs incubated in a 0.1 mole sucrose solution in an incubator. The object of that invention appears to be to alter anthocyanin concentration in the skins of the fruits to make them appear more attractive to the consumer. There does not appear to be a mention of the actual level of light intensity that strikes the fruit surface, and neither does there appear to be a reference to any relationship between the light source(s) used and how far they should be from the fruit surfaces.
The source light intensity referred to in FR 3542567 is alleged to lie within the range of 1 to 200 microW/cm2 (from 100 microEinsteins up to 20,000 microEinsteins), depending on light wavelength used (e.g. blue light at 0.82 microW/cm2 (82 Einsteins); red light 1.19 microW/cm2 (119 microEinsteins) over a period of 114 hours (leaf discs); e.g. red light at 10 microW/cm 2 (1000 microEinsteins) and 20 microW/cm2 (2000 microEinsteins) on apple trees treated for 30 nights at 15 minutes per night; e.g. blue light and red light at about 100 microW/cm2 (10,000 microEinsteins) on apples for 4 hours between 22.00 hrs and 02.00hrs in the morning).
WO 2004/103060 describes the application of white light enriched with blue to harvested plant material that is capable of photosynthesis. However, that international application does not include a technical teaching to blue light being applied at a particular light intensity to the target plant material surface.
Although observations have been reported on the effects of certain bands of UV light and of infrared light in altering, typically increasing the levels of certain phytochemicals within plant cells, the available art appears to be silent on the effect of shining light from visible spectrum wavelengths of specified light intensity onto the plant cell surface or plant tissue surface.
A recognised problem that is associated with harvested vegetables or harvested vegetable
parts is that the levels of plant phytochemicals, such as plant secondary metabolites, starts to decrease almost immediately, post-harvest. For example, as harvested vegetables are processed for freezing and/or canning or are simply placed in refrigerators, such as domestic appliances or simply on open surfaces in a room for short periods for eating later by consumers, they lose much of their nutritional content in terms of the levels of phytochemicals found therein. Such phytochemicals include antioxidants such as vitamins, e.g. vitamins C and/or E, glucosinolat.es, such as sinigrin, sulphoraphane, 4- methylsulphinylbutyl glucosinolate, and/or 3 methyl - sulphinylpropyl glucosinolate, progoitrin and glucobrassicin, isothiocyanates, indoles (products of glucosinolate hydrolysis), glutathione, carotenoids such as beta-carotene, lycopene, and the xanthophyll carotenoids such as lutein and zeaxanthin, phenolics comprising the flavonoids such as the flavonols (e.g. quercetin, rutin), the flavans/tannins (such as the procyanidins comprising coumarin, proanthocyanidins, catechins, and anthocyanins), flavones (e.g. luteolin from artichokes), phytoestrogens such as coumestans, lignans, resveratrol, isoflavones e.g. genistein, daidzein, and glycitein, and resorcyclic acid lactones, and organosulphur compounds, phytosterols, terpenoids such as carnosol, rosmarinic acid, glycyrrhizin and saponins, and chlorophyll and chlorphyllin, sugars, and other food products such as anthocyanins, vanilla and other fruit and vegetable flavours and texture modifying agents and the like. Research indicates that the antioxidant properties of certain phytochemicals may help protect against the effects of ageing and chronic diseases, such as cancer and cardiovascular disease in mammals, and in particular in humans.
Phytochemicals can thus serve as pharmaceutical compounds per se in mammalian species, such as humans, or pharmaceutically active derivatives can be synthesised from other phytochemicals, such as intermediate compounds therefore, and able to be isolated from plants. Thus, phytochemicals that may be substantially pharmaceutically inactive may find a use in providing intermediates for the synthesis of active agents for the treatment of diseases such as cancers, and/or in pain management of mammals suffering from diseases, such as humans. Phytochemicals known to be useful in the design of and/or provision of pharmaceutically active compounds include vincristine and vinblastine from Catharanthus roseus, taxanes such as those described in US 5 665 576, for example, taxol (paclitaxel), baccatin III, 10-desacetylbaccatin III, 10-desacetyl taxol, xylosyl taxol, 7-epitaxol, 7- epibaccatin III, 10-desacetylcephalomannine, 7-epicephalomannine, taxotere, cephalomannine, xylosyl cephalomannine, taxagifine, 8-benxoyloxy taxagifine, 9-acetyloxy taxusin, 9-hydroxy taxusin, taiwanxam, taxane Ia, taxane Ib, taxane Ic, taxane Id, GMP
paclitaxel, 9-dihydro 13-acetylbaccatin III, and 10-desacetyl-7-epitaxol from plants of the family Taxaceae such as plants of the genera Amentotaxus, Austrotaxus, Pseudotaxus, Torreya and Taxus, for example from plants of the genus Taxus, such as T. brevifolia, T. baccata, T. x media (e.g. Taxus media hicksii, Taxus x media Rehder), T. wallichiana, T. Canadensis, T. cuspidata, T. floridiana, T. celebica, and T. x hunnewelliana, T. Canadensis, and tetrahydrocannabinol (THC) and cannabidiol (CBD) from cannabis plants such as Cannabis sativa, Cannabis indica, and Cannabis rudβraiis, and other pharmaceuticals such as genistein, diadzein, codeine, morphine, quinine, shikonin, ajmalacine, serpentine and the like.
It has now been observed that by exposing or directing certain wavelengths selected from those making up white light onto harvested plant material such as green plant parts or plant cells comprising chlorophyll the level of phytochemicals therein can be transiently increased. Such phytochemicals include primary and secondary metabolites as described herein and other phytochemicals for use as pharmaceuticals, for example, as alluded to herein. As a consequence, the level of desired plant phytochemicals, such as plant secondary metabolites e.g. antioxidants, can be increased in harvested plant material by the simple application of wavelengths of light for relatively short periods of time selected from those wavelengths or bands found in cold light, that is, visible light.
According to the present invention there is provided a method of altering the level of at least one phytochemical in a harvested plant cell comprising chlorophyll or in harvested plant tissue comprising chlorophyll, the said plant cell or said plant tissue being capable of photosynthesis or absorption of light energy, by shining only blue light from the visible spectrum onto the surface of the plant cell or the plant tissue wherein the light intensity of the blue light striking the said surface of the plant cell or said surface of the plant tissue is sufficient to initiate a biochemical process within the said plant cell or said plant tissue thereby altering the level of at least one phytochemical therein.
"Harvested plant tissue" may comprise harvested vegetable matter including cut plant parts such as broccoli florets, green beans, cabbage heads, harvested fruits such as apples, pears and other green or unripe fruits, such as unripe tomatoes, and may include any form of plastid capable of forming a plant phytochemical on application of at least blue light thereto. Examples of such plastids include etioplasts, chloroplasts, and chromoplasts
The level of blue light intensity that strikes the harvested plant cell or harvested plant tissue surface may be any that effects an alteration in the level of at least one phytochemical within the plant cell or plant tissue. The intensity of blue light striking the harvested plant cell or plant tissue may be at least 5 microEinsteins +/- 3 microEinsteins. The level of blue light intensity used in the method of the invention may lie in the range of from 5 microEinsteins +/- 3 microEinsteins up to 400 microEinsteins +/- 50 microEinsteins; from 5 microEinsteins +/- 3 microEinsteins up to 300 microEinsteins +/- 50 microEinsteins; from 5 microEinsteins +/- 3 microEinsteins up to 200 microEinsteins +/- 50 microEinsteins; from 50 microEinsteins +/- 10 microEinsteins up to 150 microEinsteins +/- 30 microEinsteins; about 100 microEinsteins +/- 20 microEinsteins; 200 microEinsteins +/- 50 microEinsteins; 250 microEinsteins +/- 50 microEinsteins and the like, depending on design. An example of the level of blue light in combination with red light, that is to say wherein the plant material is not exposed to any other light source other than blue and/or red light, is given in the examples hereinafter using refrigeration conditions of 0° C - 1° C, that is to say a temperature that may be used in a typical domestic refrigerator. The process of the invention whether it employs blue light alone or a combination of two wavelengths of light selected from only the red and blue visible spectrum may be employed at any temperature in the range of from -0.5° Centigrade to a higher ambient temperature in which the harvested plant cells remain capable of photosynthetic activity. A suitable temperature range in which the process of the invention may be employed is from -0.5° Centigrade to about 45° Centigrade and in one application of the process of the invention, it can be employed within a chilling temperature range typically found under domestic refrigeration conditions and commercial refrigeration conditions or other cooling conditions, such as from -0.5° Centigrade to 18° Centigrade, and preferably from about 1° Centigrade to about 16° Centigrade, and most preferably from about 1° Centigrade to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16° Centigrade. The skilled addressee will also appreciate that the method of the invention may be employed at a temperature in the range of from about + 8° Centigrade to about room temperature (+ 25° Centigrade). Typically, the process of the invention is performed on harvested plant material wherein the ambient relative humidity lies between 60% and 100%, such as 65% RH, 70% RH, 75% RH or 80% RH, The level of blue light intensity at the plant part surface may be augmented with white light from a second light source or where white light is not used, the second light source may provide red light, the combined level of light intensity striking the surface of the plant material from both of said light sources may be in the range of from 40 microEinsteins +/- 25 microEinsteins up to 3000 microEinsteins +/- 300 microEinsteins or more depending on design. Examples of ranges of combined light red and
blue light intensities that may be used in the present invention include 240 microEinsteins +/- 100 microEinsteins up to 2000 microEinsteins+/- 200 microEinsteins; 300 microEinsteins +/- 100 microEinsteins up to 1500 microEinsteins +/- 150 microEinsteins; 500 microEinsteins +/- 200 microEinsteins; 40 microEinsteins +/- 10 microEinsteins upto IOOmicroEinsteins +/- 25 microEinsteins; 15 microEinsteins +/- 5 microEinsteins up to 300 microEinsteins +/- 50 microEinsteins; 15 microEinsteins +/- 5 microEinsteins up to 200 microEinsteins +/- 20 microEinsteins; 15 microEinsteins +/- 5 microEinsteins up to 150 microEinsteins +/- 15 microEinsteins; 40 microEinsteins +/- 10 microEinsteins and the like. Naturally, the skilled addressee will appreciate that lower light intensities for red, blue, or red and blue combinations of light of from 30 microEinsteins +/- 10 microEinsteins to about 100 microEinsteins +/- 25 microEinsteins will be sufficient for use in refrigeration or other under cover applications such as domestic household goods, and the like.
The wavelength of blue light used may be selected from the range of from 41 Onm to 490nm such that the selected wavelength of blue light is, or wavelengths of blue light are, capable of altering the level of phytochemicals found in an harvested plant cell or in harvested plant tissue. Typically, the level of phytochemicals contained within harvested plant material is raised upon exposure to desired wavelengths of light over a suitable time interval and at a suitable light intensity according to the invention. Examples of blue light wavelength ranges and values used in the method of the invention include from 420nm - 480nm; from 435nm - 465nm; and 450nm +/- 15 nm. Thus, the skilled addressee will appreciate that the wavelength(s) of blue light used in the present invention on plant material such as harvested vegetables or green leaf matter or green plant cells in culture, such as moss cells e.g. cells of physcomitrella patens, according to the method of the invention, constitute wavelengths of blue light and do not include the violet or higher energy light wavelengths.
Additionally, the harvested plant material may be exposed to blue light from one light source in conjunction with white light (that is to say, light from the visible spectrum) from a second light source. This second source of white light may already be enriched with blue light, such as, in the case of conventional light emitting diodes (LEDs) which emit light having a bias towards blue light emission, and in the case of certain white halogen lights e.g. the General Electric Quartzline EHJ, 250W, 24V light. The first and/or second light source may also be further enriched with red light of a wavelength that lies in the range of from 600nm - 700 nm. The red light intensity of red light striking the target plant material as described herein typically lies in the range of from 1 to 200 microEinsteins +/- 50 microEinsteins. Examples of
the red light intensity striking the plant material surface include 5 microEinsteins +/- 2 microEinsteins upto 150 microEinsteins +/- 50 microEinsteins; 30 microEinsteins +/- 5 microEinsteins up to 150 microEinsteins +/- 50 microEinsteins; 25 microEinsteins +/- 10 microEinsteins up to 100 microEinsteins +/- 20 microEinsteins; and the like. The skilled addressee will appreciate that the actual intensity of light to be employed on the plant surface will depend on design and plant material used.
Furthermore, it is to be understood that the light wavelength or wavelengths employed in the present invention are selected from so-called 'cold light' wavelengths, that is, the light used in the present invention does not comprise UV wavelengths and does not constitute infrared wavelengths, both forms of which are potentially hazardous to use. In a preferred embodiment, the wavelengths or bands of light used lie in the range of from 420nm to 490nm for blue light; 400nm to 700 nm white light enriched with blue light as herein described; and/or 600 nm to 700 nm for red light or in any combination of light wavelengths therein, depending on design and the phytochemical of interest. Examples of the red wavelength used in the present invention may be selected from a wavelength within the range of from 600nm to 700nm; 620nm to 680 nm; 625nm to 670 nm; or at about 640nm +/- 15nm. Red or blue light or a combination of both red and blue light at any given energy ratio may be employed in the method of the invention. For instance, the energy ratio of Blue light : Red light may be selected from within the range of from 10:1 to 1 :10, 9:1 to 1 :9, 8:1 to 1 :8, 7:1 to 1 :7, 6:1 to 1 :6, and 5:1 to 1 :5, such as 5:2 to 2:5, 5:3 to 3:5, or 5:4 to 4:5. Other Blue light : Red light ratios may be selected from within the ranges 4:1 to 1 :4, 3:1 to 1:3, 2:1 to 1 :2, and 1:1 and any permutation within these ranges depending on design. The actual red, blue or blue:red light or red:blue light energy ratio selected may depend on species, age of plant parts, the phytochemical of interest and design. Typically, one unit of energy for blue light may be about 15 microEinsteins +/- 3 microEinsteins and one unit of energy for red light may be about 2 microEinsteins +/- 1 microEinsteins. From such approximations the light intensity of red light, or blue light, or blue light : red light ratio shone onto plant material such as leaf surfaces may be made. Naturally, the skilled addressee will appreciate that depending on the plant cells or plant tissue employed, the length of time that the plant cells or tissue is exposed to light of wavelengths outlined herein will alter with design. Suitably, the length of time that plant cells or plant tissue may be exposed to wavelengths used in the present invention for an effect on phytochemical levels to be observed is for a predetermined time interval. The time interval may be selected from a continuous time interval or a pulsed time interval. Typically, the time interval is a pulsed time interval of a
predetermined frequency that is spread over a time period that is longer in duration than the said pulsed time interval. The time period can be of any length of duration and can be up to 96 hours or more in duration. When a pulsed time interval is employed, the pulsed time interval may be of any length and may lie, for example in the range of from 1 second up to 120 minutes; 1 minute to 60 minutes; 5 minutes to 40 minutes; 10 to 30 minutes; 10 to 20 minutes; 15 minutes and the like depending on design, plant part species, and requirements. Naturally, the man skilled in the art will appreciate that there will be a time interval between light pulses during which the described light sources will not be shining onto the plant material of interest. Furthermore, the man skilled in the art will appreciate that the said time intervals between separate light pulses may be shorter in duration than the pulsed light interval, of the same duration as the pulsed light interval or of longer duration than the light pulse interval. Typically, the level of phytochemicals is elevated on the application of light to the plant tissue or plant cell culture over short time intervals as alluded to herein.
In a further variant in the operating of the method of the instant invention, the light from the said one or more light sources is shone onto the plant cell or plant tissue surface for a predetermined time interval for a continuous time interval. The continuous time interval can be of any length of time up to 96 hours or more in duration. Examples of continuous time intervals include 168 hours; 144 hours; 96 hours; and 72 hours and the like. Examples of ranges from which continuous intervals may be selected include 30 minutes to 96 hours; 30 minutes to 96 hours; 30 minutes to 48 hours; 30 minutes to 24 hours; 30 minutes to 12 hours; 30 minutes to 8 hours and the like. Naturally, the man skilled in the art will also appreciate that the number of minutes or hours will be selected depending on design, plant species and need.
In a further aspect the invention can be employed on any plant tissue that is capable of responding to exposure to, or irradiation with, wavelengths of light as outlined herein. Preferably, the plant tissue comprises tissue that is capable of photosynthesis and/or blue and red light adsorption. Plant material that can be used in the method of the invention includes all green vegetables and green seeds, e.g. peas, green beans, spinach, snow peas (mange tout) species from the Brassica oleracea such as broccoli, green cabbage, red cabbage, Brussels sprouts, kohlrabi, cauliflower, white cabbage, and the like, and all plant material, such as green plant material, for example, cells comprising chlorophyll, green stems, calyx, leaves, and the like that is able to respond to wavelengths of light as hereinbefore described. Other plant material that may be treated in accordance with
methods of the invention may be green material such as green needles derived from non- vegetable sources such as plants of the order Taxaceae as described herein, tea leaves, and of cells grown in plant cell cultures in bioreactors such as moss cells and tissues (e.g. protonema) from physcomitrella patens, and other plant cell cultures e.g. callus cell cultures, cultures of lemnospora species, algae or even somatic embryo clusters and fruits such as tomatoes, apples, grapes, unripe (green) bananas, mangoes, kiwi fruit, pineapples, and the like. Naturally, the man skilled in the art will appreciate that "fruit" is used in the context of the shopper at the supermarket or green grocer.
In a further embodiment, there is provided a method of raising the phytochemical content in live plant cells or plant tissue in an environment by exposing the said plant cells or tissue with light of at least a wavelength selected from light of wavelengths found in cold light from an artificial light source. Naturally, the skilled addressee will appreciate that light as described, herein and employed in the instant invention alters the phytochemical profile of a plant cell or plant tissue, such as a harvested tissue lies. Preferably, the combination of light sources includes red light of a wavelength that may be selected from a wavelength within the range of from 600nm - 700nm, preferably from 620nm - 680 nm, more preferably from 625nm - 670 nm, and generally at about 640nm +/- 15nm. Red or blue light or a combination of red and blue light, or a combination of red and/or blue light with white light at any selected energy ratio may be employed in the method of the invention. In a preferred embodiment, the said plant cells or plant tissue can be located under cover. 'Under cover' means that the cells or tissue is located under cover when exposed, for example, during a food processing step prior to further processing such as freezing or canning or heat treating or cooking as alluded to hereinbelow.
Where advantage is to be gained from heat shocking the harvested plant cells or harvested plant tissue, the method of the invention may be employed at a temperature within the range of from + 35 degrees Centigrade to about +45 degrees centigrade, for example, at +40, +41 , +42, +43, +44 or +45 degrees Centigrade, for a period of from a few seconds, for example 30 seconds up to a few minutes, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 minutes or more depending on plant tissue type and design. Naturally, the skilled addressee will appreciate that the heat shock temperature should be such that it does not deleteriously affect the general viability of the plant material that is subjected to a heat shock step.
In a further aspect, there is provided a method of harvesting plant cells or plant tissues under cover wherein the said plant cells or plant tissues are exposed with light as herein described from one or more artificial light sources.
Also included as an aspect of the present invention is harvested plant material or plant cells obtainable by a method according to the present invention and having altered levels of phytochemicals, typically elevated levels of phytochemicals when compared to plant material or plant cells that have not been exposed to light of wavelengths used in the method of the present invention.
'Cover' is to be understood as a general term and may be taken to mean a receptacle in which the plant material or plant cells may be placed, for example a closed container with a built-in light source therein, such as a refrigerator unit comprising an in-built light source that can be activated on demand for a predetermined time interval. Thus, for carrying out the method of the invention use can be made of cooling means, such as a conventional refrigerator comprising a light source capable of emitting blue light in the manner hereinbefore described. Alternatively, 'under cover' may be taken to mean a processing factory wherein harvested plant material is exposed to one or more light sources producing light of appropriate wavelength or wavelengths over a short period of time during the processing operation, such as canning, freezing plant material, or immediately prior to the cooking of foods for canning or for baby food manufacture e.g. purees and the like, and further processed foods such as soups, vegetable-based sauces and the like.
Thus as a further aspect of the invention there is provided a processed food obtainable by a food processing method by exposing live plant cells with light of wavelengths as herein described at light intensities as herein described. Suitable wavelengths of light are those described herein and these are applied for appropriate, predetermined time intervals as described herein. A still further aspect of the invention provides a food processing method comprising exposing live plant cells to light wavelengths as herein described from at least one artificial light source. Typically the wavelength(s) of the light is/are selected from wavelengths as herein described and is applied for a predetermined period of time sufficient to alter the phytochemical profile of the exposed plant cells and/or harvested plant tissue.
"Plant cells" also includes those plant parts or tissues which display an aromaticity which is detectable by the human olfactory senses when cut or harvested. Such plants may display
the aromaticity naturally, for example in the case of cut herbs, from the cut leaves. The plant cells or tissue or parts include members of the Labiatae, such as the broad-leafed herbs. Suitable examples of broad-leafed herbs include basil, oregano, sage, coriander, dill, marjoram and thyme. Other herbs, such as cut herbs that may benefit from being treated according to the present invention include chives, garlic, bay leaf, lemon balm, mint, lavender, parsley, the fennels, e.g. bronze fennel and common fennel, and the like. A more complete list of common herbs to which the invention can be applied is to be found in Taylors Guide to Herbs 1995, Eds. Buchanan R. & Tenebaum F. Houghton Mifflin Co. New York: the teaching of this guide reference is hereby incorporated into the teaching of the present specification. Naturally, the skilled addressee will appreciate that the said plant cells or plant parts are alive when exposed to light in accordance with the present invention and are capable of responding to the application of the cold light-derived light stimulus.
Plant cells or plant parts may be harvested at any stage of growth so long as the harvested plant cells or tissue are capable of responding to the application of light of wavelength and duration as outlined herein. In a preferred embodiment, the harvested plant cells or tissue of broad - leaf herbs can be exposed to wavelengths of light used in the present invention from the 3 to 4 leaf stage and most preferably in the case of culinary herbs such as basil, the 5- leaf stage. It is envisaged that plant cells and/or tissue such as culinary herbs and green vegetables are most usefully exposed as herein-described immediately before processing (e.g. freeze drying, adding to processed foods such as sauces, soups, canned goods and the like), that is to say after the harvesting of cuttings from such plants and/or the provision of young plants for processing e.g. as dried herbs. Dried herbs treated with light as outlined herein immediately post-harvest, for a short period of time, particularly those measured at the 5-leaf stage, are considered to display an increased aromaticity relative to controls which are not exposed to light as described herein.
The artificial light source or sources can be of any suitable conventional type, such as a light emitting diode or even a white light source comprising filters that let through light of the desired wavelength(s). The light source may be placed at any distance from the harvested material provided that the light energy used is sufficient to influence, for example to induce or saturate oxygen evolution at the photosystem Il reaction centre and/or to trigger, that is set off, a transient photo-oxidative stress and/or a moderate photosynthetic electron transport inhibition. Optimising of the light energy and light composition may be performed for example, by monitoring oxygen evolution and chlorophyll a fluorescence using
conventional methods (e.g. according to the instruction manual and software of Hansatech Instruments Ltd., King's Lynn, UK). It is preferable to locate the light source in a position which affords the greatest amounts of irradiation per square unit (e.g. cm2, m2 etc.) of the harvested plant material. Suitably, depending on the size of the covered area, for example that of a processing compartment in a processing factory, or of a refrigerator or other container such as a microwave oven or magnetron fitted with a suitable light source capable of being manually or automatically activated, for example, by employing a timing means and thereby emitting wavelengths of light as indicated herein and described herein. Alternatively, an independent container specifically designed for exposing plant parts or cells to light of wavelengths as described herein may be employed, in a further alternative, the number of light sources may be as little as one to a whole 'battery1 of light sources arranged in series and/or in parallel, for example, in a food processing factory setting, each light source being suitably distanced one from the other at appropriate intervals in such a manner as to effect exposure of the plant material to light of wavelengths as described herein which results in a significant alteration in the level of phytochemicals found therein, preferably an increase of desired phytochemicals.
In a further embodiment of the invention there is provided use of blue light from an artificial light source in a method of processing plant cells or harvested plant tissue under cover. Preferably, the blue light wavelength is selected from the wavelengths of light found as herein described. The blue light may be used in conjunction with other wavelengths of light as herein described.
In a further embodiment, there is provided use of at least blue light in a method as described herein for increasing the phytochemical content in harvested live plant material. In a preferment, the said plant material is located under cover.
In a further embodiment of the invention there is provided the use of plant parts exposed to blue light as described herein in the manufacture of human foodstuffs, such as frozen vegetables (e.g. spinach or plant parts from a Brassica species) or seeds (e.g. peas), bottled or canned condiments, for example sauces for meat, fish and poultry dishes, flavourings, for example tapenade, salad dressings, cooking oils such as olive oil, sunflower oil and the like, soups, pasta and cheeses.
As another application of the present invention, blue light or red light or a combination of red
and blue light may be employed in a greenhouse setting on growing plants. In Northern Hemisphere countries such as Holland, the Scandinavian countries, Belgium, Germany and the UK many varieties of ornamental plants, greenhouse produced lettuce, tomatoes and other salad vegetables are grown under cover. The lighting is supplied in the form of yellow light, typically from sodium lamps. However, such lighting systems lose a lot of energy as heat and do not mimic the blue, red or red and blue spectra of natural sunlight. By modulating the light intensity of blue and/or red light that is shone onto plants, it is possible to optimise the growing phase of the plant and to improve seed set, plant habit, and yield. Thus plants can be produced which are in optimum health and have a full complement of phytochemicals as alluded to herein. In a still further embodiment of the invention there is provided use of blue light and/or red light in improving seed set of plants grown under cover in a greenhouse or in a hydroponics growing system. Furthermore there is provided as another aspect of the invention use of blue light and/or red light in optimising the plant habit of plants grown in the greenhouse or in a hydroponics system. Such uses provide for more efficient production of plants that are grown under cover in the greenhouse, such as ornamentals, salad plants such as lettuces, tomatoes, capsicums and the like.
from http://www.wipo.int/pctdb/en/wo.jsp?IA=WO2007085842&wo=2007085842&DISPLAY=DESC
WO 2007085842 20070802
PLANT TREATMENT METHOD AND MEANS THEREFORE
The present invention relates to a method for altering the level of phytochemicals in plant cells and/or plant tissue and means therefor. In particular, the invention relates to a method for altering the level of phytochemicals such as plant primary or secondary metabolites in harvested plant cells and/or plant tissue by applying wavelengths of light of selected wavelength and intensity thereto that are selected from wavelengths of light from the white light or visible spectrum and means therefor.
It is known that the application of light from the UV spectrum, such as UV-B and UV-C can help to increase the levels of for example 'essential oils' and secondary metabolites in whole plants. However, UV-B and UV-C is problematic to handle for humans and is heavily implicated in cancerous disease processes. As such, UV-B and UV-C light is considered potentially harmful to healthy mammalian tissue and is considered hazardous to use.
'Essential oils' are responsible in large part for the aromaticity associated with many plants, such as plants comprising perfumed flowers and herbs, such as culinary herbs. Essential oils consist mainly of terpenoids and can include such compounds as 1 ,8-cineole, limonene, linalool and β-ocimene. Other compounds which may be found in essential oils, that is, oils which are not terpenoids, can include phenyl-propanoid-derived compounds such as methyl chavicol, methyl cinnamate, eugenol, and methyl eugenol. Thus, the term 'essential oils' is used in a qualitative sense to encompass compounds as indicated herein which contribute to the aromaticity of plants such as perfumed ornamentals and culinary herbs.
Ultraviolet light (and specifically UV-B) is known to have effects on the levels of secondary compounds of the phenyl-propanoid pathway of plants via action on key regulatory enzymes such as phenylalaline ammonia-lyase (Kuhn, D.N. et al (1984) Proc. Natl. Acad. ScL, USA, 81, 1102-1106) and chalcone synthase (Batschauer, A. et al (1996) The Plant Journal 9, 63-69 and Christie, J. M. and Jenkins, G.I. (1996) The Plant Cell 8, 1555-1567). There are many published reports of UV-B stimulation of phenolic compounds, including surface flavonols and flavonoids (Cuadra, P. and Harborne, J. B. (1996) Zeitschrift fur Naturforschung 51c, 671-680 and Cuadra, P. et al (1997) Phytochemistry 45, 1377-1383), anthocyanins (Yatsuhashi, H. et al (1982) Plant Physiology 70, 735-741 and Oelmϋller, R. and Mohr, H. (1985). Proc. Natl. Acad. ScL, USA 82, 6124-6128) and betacyanins (Rudat, A. and Goring, H. (1995). J. Expl. Bot. 46, 129-134) and these compounds have been
implicated both in plant defence (Chappell, J. and Hahlbrock, K. (1984) Nature 311, 76-78 and Guevara, P. et a/ (1997) Phyton 60, 137-140) and as protection against UV-light (Lois, R. (1994) Planta 194, 498-503; Ziska, LH. et al (1992) Am. JnI. BoL 79, 863-871 and Fiusello, N. et al (1985) Allionia (Turin) 26, 79-88).
FR 3542567 describes the application of blue and/or red light to certain fruits, typically un- harvested fruits, at night for periods of long duration measured in days. Furthermore, it appears that the effect of such light was also ascertained on leaf discs incubated in a 0.1 mole sucrose solution in an incubator. The object of that invention appears to be to alter anthocyanin concentration in the skins of the fruits to make them appear more attractive to the consumer. There does not appear to be a mention of the actual level of light intensity that strikes the fruit surface, and neither does there appear to be a reference to any relationship between the light source(s) used and how far they should be from the fruit surfaces.
The source light intensity referred to in FR 3542567 is alleged to lie within the range of 1 to 200 microW/cm2 (from 100 microEinsteins up to 20,000 microEinsteins), depending on light wavelength used (e.g. blue light at 0.82 microW/cm2 (82 Einsteins); red light 1.19 microW/cm2 (119 microEinsteins) over a period of 114 hours (leaf discs); e.g. red light at 10 microW/cm 2 (1000 microEinsteins) and 20 microW/cm2 (2000 microEinsteins) on apple trees treated for 30 nights at 15 minutes per night; e.g. blue light and red light at about 100 microW/cm2 (10,000 microEinsteins) on apples for 4 hours between 22.00 hrs and 02.00hrs in the morning).
WO 2004/103060 describes the application of white light enriched with blue to harvested plant material that is capable of photosynthesis. However, that international application does not include a technical teaching to blue light being applied at a particular light intensity to the target plant material surface.
Although observations have been reported on the effects of certain bands of UV light and of infrared light in altering, typically increasing the levels of certain phytochemicals within plant cells, the available art appears to be silent on the effect of shining light from visible spectrum wavelengths of specified light intensity onto the plant cell surface or plant tissue surface.
A recognised problem that is associated with harvested vegetables or harvested vegetable
parts is that the levels of plant phytochemicals, such as plant secondary metabolites, starts to decrease almost immediately, post-harvest. For example, as harvested vegetables are processed for freezing and/or canning or are simply placed in refrigerators, such as domestic appliances or simply on open surfaces in a room for short periods for eating later by consumers, they lose much of their nutritional content in terms of the levels of phytochemicals found therein. Such phytochemicals include antioxidants such as vitamins, e.g. vitamins C and/or E, glucosinolat.es, such as sinigrin, sulphoraphane, 4- methylsulphinylbutyl glucosinolate, and/or 3 methyl - sulphinylpropyl glucosinolate, progoitrin and glucobrassicin, isothiocyanates, indoles (products of glucosinolate hydrolysis), glutathione, carotenoids such as beta-carotene, lycopene, and the xanthophyll carotenoids such as lutein and zeaxanthin, phenolics comprising the flavonoids such as the flavonols (e.g. quercetin, rutin), the flavans/tannins (such as the procyanidins comprising coumarin, proanthocyanidins, catechins, and anthocyanins), flavones (e.g. luteolin from artichokes), phytoestrogens such as coumestans, lignans, resveratrol, isoflavones e.g. genistein, daidzein, and glycitein, and resorcyclic acid lactones, and organosulphur compounds, phytosterols, terpenoids such as carnosol, rosmarinic acid, glycyrrhizin and saponins, and chlorophyll and chlorphyllin, sugars, and other food products such as anthocyanins, vanilla and other fruit and vegetable flavours and texture modifying agents and the like. Research indicates that the antioxidant properties of certain phytochemicals may help protect against the effects of ageing and chronic diseases, such as cancer and cardiovascular disease in mammals, and in particular in humans.
Phytochemicals can thus serve as pharmaceutical compounds per se in mammalian species, such as humans, or pharmaceutically active derivatives can be synthesised from other phytochemicals, such as intermediate compounds therefore, and able to be isolated from plants. Thus, phytochemicals that may be substantially pharmaceutically inactive may find a use in providing intermediates for the synthesis of active agents for the treatment of diseases such as cancers, and/or in pain management of mammals suffering from diseases, such as humans. Phytochemicals known to be useful in the design of and/or provision of pharmaceutically active compounds include vincristine and vinblastine from Catharanthus roseus, taxanes such as those described in US 5 665 576, for example, taxol (paclitaxel), baccatin III, 10-desacetylbaccatin III, 10-desacetyl taxol, xylosyl taxol, 7-epitaxol, 7- epibaccatin III, 10-desacetylcephalomannine, 7-epicephalomannine, taxotere, cephalomannine, xylosyl cephalomannine, taxagifine, 8-benxoyloxy taxagifine, 9-acetyloxy taxusin, 9-hydroxy taxusin, taiwanxam, taxane Ia, taxane Ib, taxane Ic, taxane Id, GMP
paclitaxel, 9-dihydro 13-acetylbaccatin III, and 10-desacetyl-7-epitaxol from plants of the family Taxaceae such as plants of the genera Amentotaxus, Austrotaxus, Pseudotaxus, Torreya and Taxus, for example from plants of the genus Taxus, such as T. brevifolia, T. baccata, T. x media (e.g. Taxus media hicksii, Taxus x media Rehder), T. wallichiana, T. Canadensis, T. cuspidata, T. floridiana, T. celebica, and T. x hunnewelliana, T. Canadensis, and tetrahydrocannabinol (THC) and cannabidiol (CBD) from cannabis plants such as Cannabis sativa, Cannabis indica, and Cannabis rudβraiis, and other pharmaceuticals such as genistein, diadzein, codeine, morphine, quinine, shikonin, ajmalacine, serpentine and the like.
It has now been observed that by exposing or directing certain wavelengths selected from those making up white light onto harvested plant material such as green plant parts or plant cells comprising chlorophyll the level of phytochemicals therein can be transiently increased. Such phytochemicals include primary and secondary metabolites as described herein and other phytochemicals for use as pharmaceuticals, for example, as alluded to herein. As a consequence, the level of desired plant phytochemicals, such as plant secondary metabolites e.g. antioxidants, can be increased in harvested plant material by the simple application of wavelengths of light for relatively short periods of time selected from those wavelengths or bands found in cold light, that is, visible light.
According to the present invention there is provided a method of altering the level of at least one phytochemical in a harvested plant cell comprising chlorophyll or in harvested plant tissue comprising chlorophyll, the said plant cell or said plant tissue being capable of photosynthesis or absorption of light energy, by shining only blue light from the visible spectrum onto the surface of the plant cell or the plant tissue wherein the light intensity of the blue light striking the said surface of the plant cell or said surface of the plant tissue is sufficient to initiate a biochemical process within the said plant cell or said plant tissue thereby altering the level of at least one phytochemical therein.
"Harvested plant tissue" may comprise harvested vegetable matter including cut plant parts such as broccoli florets, green beans, cabbage heads, harvested fruits such as apples, pears and other green or unripe fruits, such as unripe tomatoes, and may include any form of plastid capable of forming a plant phytochemical on application of at least blue light thereto. Examples of such plastids include etioplasts, chloroplasts, and chromoplasts
The level of blue light intensity that strikes the harvested plant cell or harvested plant tissue surface may be any that effects an alteration in the level of at least one phytochemical within the plant cell or plant tissue. The intensity of blue light striking the harvested plant cell or plant tissue may be at least 5 microEinsteins +/- 3 microEinsteins. The level of blue light intensity used in the method of the invention may lie in the range of from 5 microEinsteins +/- 3 microEinsteins up to 400 microEinsteins +/- 50 microEinsteins; from 5 microEinsteins +/- 3 microEinsteins up to 300 microEinsteins +/- 50 microEinsteins; from 5 microEinsteins +/- 3 microEinsteins up to 200 microEinsteins +/- 50 microEinsteins; from 50 microEinsteins +/- 10 microEinsteins up to 150 microEinsteins +/- 30 microEinsteins; about 100 microEinsteins +/- 20 microEinsteins; 200 microEinsteins +/- 50 microEinsteins; 250 microEinsteins +/- 50 microEinsteins and the like, depending on design. An example of the level of blue light in combination with red light, that is to say wherein the plant material is not exposed to any other light source other than blue and/or red light, is given in the examples hereinafter using refrigeration conditions of 0° C - 1° C, that is to say a temperature that may be used in a typical domestic refrigerator. The process of the invention whether it employs blue light alone or a combination of two wavelengths of light selected from only the red and blue visible spectrum may be employed at any temperature in the range of from -0.5° Centigrade to a higher ambient temperature in which the harvested plant cells remain capable of photosynthetic activity. A suitable temperature range in which the process of the invention may be employed is from -0.5° Centigrade to about 45° Centigrade and in one application of the process of the invention, it can be employed within a chilling temperature range typically found under domestic refrigeration conditions and commercial refrigeration conditions or other cooling conditions, such as from -0.5° Centigrade to 18° Centigrade, and preferably from about 1° Centigrade to about 16° Centigrade, and most preferably from about 1° Centigrade to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16° Centigrade. The skilled addressee will also appreciate that the method of the invention may be employed at a temperature in the range of from about + 8° Centigrade to about room temperature (+ 25° Centigrade). Typically, the process of the invention is performed on harvested plant material wherein the ambient relative humidity lies between 60% and 100%, such as 65% RH, 70% RH, 75% RH or 80% RH, The level of blue light intensity at the plant part surface may be augmented with white light from a second light source or where white light is not used, the second light source may provide red light, the combined level of light intensity striking the surface of the plant material from both of said light sources may be in the range of from 40 microEinsteins +/- 25 microEinsteins up to 3000 microEinsteins +/- 300 microEinsteins or more depending on design. Examples of ranges of combined light red and
blue light intensities that may be used in the present invention include 240 microEinsteins +/- 100 microEinsteins up to 2000 microEinsteins+/- 200 microEinsteins; 300 microEinsteins +/- 100 microEinsteins up to 1500 microEinsteins +/- 150 microEinsteins; 500 microEinsteins +/- 200 microEinsteins; 40 microEinsteins +/- 10 microEinsteins upto IOOmicroEinsteins +/- 25 microEinsteins; 15 microEinsteins +/- 5 microEinsteins up to 300 microEinsteins +/- 50 microEinsteins; 15 microEinsteins +/- 5 microEinsteins up to 200 microEinsteins +/- 20 microEinsteins; 15 microEinsteins +/- 5 microEinsteins up to 150 microEinsteins +/- 15 microEinsteins; 40 microEinsteins +/- 10 microEinsteins and the like. Naturally, the skilled addressee will appreciate that lower light intensities for red, blue, or red and blue combinations of light of from 30 microEinsteins +/- 10 microEinsteins to about 100 microEinsteins +/- 25 microEinsteins will be sufficient for use in refrigeration or other under cover applications such as domestic household goods, and the like.
The wavelength of blue light used may be selected from the range of from 41 Onm to 490nm such that the selected wavelength of blue light is, or wavelengths of blue light are, capable of altering the level of phytochemicals found in an harvested plant cell or in harvested plant tissue. Typically, the level of phytochemicals contained within harvested plant material is raised upon exposure to desired wavelengths of light over a suitable time interval and at a suitable light intensity according to the invention. Examples of blue light wavelength ranges and values used in the method of the invention include from 420nm - 480nm; from 435nm - 465nm; and 450nm +/- 15 nm. Thus, the skilled addressee will appreciate that the wavelength(s) of blue light used in the present invention on plant material such as harvested vegetables or green leaf matter or green plant cells in culture, such as moss cells e.g. cells of physcomitrella patens, according to the method of the invention, constitute wavelengths of blue light and do not include the violet or higher energy light wavelengths.
Additionally, the harvested plant material may be exposed to blue light from one light source in conjunction with white light (that is to say, light from the visible spectrum) from a second light source. This second source of white light may already be enriched with blue light, such as, in the case of conventional light emitting diodes (LEDs) which emit light having a bias towards blue light emission, and in the case of certain white halogen lights e.g. the General Electric Quartzline EHJ, 250W, 24V light. The first and/or second light source may also be further enriched with red light of a wavelength that lies in the range of from 600nm - 700 nm. The red light intensity of red light striking the target plant material as described herein typically lies in the range of from 1 to 200 microEinsteins +/- 50 microEinsteins. Examples of
the red light intensity striking the plant material surface include 5 microEinsteins +/- 2 microEinsteins upto 150 microEinsteins +/- 50 microEinsteins; 30 microEinsteins +/- 5 microEinsteins up to 150 microEinsteins +/- 50 microEinsteins; 25 microEinsteins +/- 10 microEinsteins up to 100 microEinsteins +/- 20 microEinsteins; and the like. The skilled addressee will appreciate that the actual intensity of light to be employed on the plant surface will depend on design and plant material used.
Furthermore, it is to be understood that the light wavelength or wavelengths employed in the present invention are selected from so-called 'cold light' wavelengths, that is, the light used in the present invention does not comprise UV wavelengths and does not constitute infrared wavelengths, both forms of which are potentially hazardous to use. In a preferred embodiment, the wavelengths or bands of light used lie in the range of from 420nm to 490nm for blue light; 400nm to 700 nm white light enriched with blue light as herein described; and/or 600 nm to 700 nm for red light or in any combination of light wavelengths therein, depending on design and the phytochemical of interest. Examples of the red wavelength used in the present invention may be selected from a wavelength within the range of from 600nm to 700nm; 620nm to 680 nm; 625nm to 670 nm; or at about 640nm +/- 15nm. Red or blue light or a combination of both red and blue light at any given energy ratio may be employed in the method of the invention. For instance, the energy ratio of Blue light : Red light may be selected from within the range of from 10:1 to 1 :10, 9:1 to 1 :9, 8:1 to 1 :8, 7:1 to 1 :7, 6:1 to 1 :6, and 5:1 to 1 :5, such as 5:2 to 2:5, 5:3 to 3:5, or 5:4 to 4:5. Other Blue light : Red light ratios may be selected from within the ranges 4:1 to 1 :4, 3:1 to 1:3, 2:1 to 1 :2, and 1:1 and any permutation within these ranges depending on design. The actual red, blue or blue:red light or red:blue light energy ratio selected may depend on species, age of plant parts, the phytochemical of interest and design. Typically, one unit of energy for blue light may be about 15 microEinsteins +/- 3 microEinsteins and one unit of energy for red light may be about 2 microEinsteins +/- 1 microEinsteins. From such approximations the light intensity of red light, or blue light, or blue light : red light ratio shone onto plant material such as leaf surfaces may be made. Naturally, the skilled addressee will appreciate that depending on the plant cells or plant tissue employed, the length of time that the plant cells or tissue is exposed to light of wavelengths outlined herein will alter with design. Suitably, the length of time that plant cells or plant tissue may be exposed to wavelengths used in the present invention for an effect on phytochemical levels to be observed is for a predetermined time interval. The time interval may be selected from a continuous time interval or a pulsed time interval. Typically, the time interval is a pulsed time interval of a
predetermined frequency that is spread over a time period that is longer in duration than the said pulsed time interval. The time period can be of any length of duration and can be up to 96 hours or more in duration. When a pulsed time interval is employed, the pulsed time interval may be of any length and may lie, for example in the range of from 1 second up to 120 minutes; 1 minute to 60 minutes; 5 minutes to 40 minutes; 10 to 30 minutes; 10 to 20 minutes; 15 minutes and the like depending on design, plant part species, and requirements. Naturally, the man skilled in the art will appreciate that there will be a time interval between light pulses during which the described light sources will not be shining onto the plant material of interest. Furthermore, the man skilled in the art will appreciate that the said time intervals between separate light pulses may be shorter in duration than the pulsed light interval, of the same duration as the pulsed light interval or of longer duration than the light pulse interval. Typically, the level of phytochemicals is elevated on the application of light to the plant tissue or plant cell culture over short time intervals as alluded to herein.
In a further variant in the operating of the method of the instant invention, the light from the said one or more light sources is shone onto the plant cell or plant tissue surface for a predetermined time interval for a continuous time interval. The continuous time interval can be of any length of time up to 96 hours or more in duration. Examples of continuous time intervals include 168 hours; 144 hours; 96 hours; and 72 hours and the like. Examples of ranges from which continuous intervals may be selected include 30 minutes to 96 hours; 30 minutes to 96 hours; 30 minutes to 48 hours; 30 minutes to 24 hours; 30 minutes to 12 hours; 30 minutes to 8 hours and the like. Naturally, the man skilled in the art will also appreciate that the number of minutes or hours will be selected depending on design, plant species and need.
In a further aspect the invention can be employed on any plant tissue that is capable of responding to exposure to, or irradiation with, wavelengths of light as outlined herein. Preferably, the plant tissue comprises tissue that is capable of photosynthesis and/or blue and red light adsorption. Plant material that can be used in the method of the invention includes all green vegetables and green seeds, e.g. peas, green beans, spinach, snow peas (mange tout) species from the Brassica oleracea such as broccoli, green cabbage, red cabbage, Brussels sprouts, kohlrabi, cauliflower, white cabbage, and the like, and all plant material, such as green plant material, for example, cells comprising chlorophyll, green stems, calyx, leaves, and the like that is able to respond to wavelengths of light as hereinbefore described. Other plant material that may be treated in accordance with
methods of the invention may be green material such as green needles derived from non- vegetable sources such as plants of the order Taxaceae as described herein, tea leaves, and of cells grown in plant cell cultures in bioreactors such as moss cells and tissues (e.g. protonema) from physcomitrella patens, and other plant cell cultures e.g. callus cell cultures, cultures of lemnospora species, algae or even somatic embryo clusters and fruits such as tomatoes, apples, grapes, unripe (green) bananas, mangoes, kiwi fruit, pineapples, and the like. Naturally, the man skilled in the art will appreciate that "fruit" is used in the context of the shopper at the supermarket or green grocer.
In a further embodiment, there is provided a method of raising the phytochemical content in live plant cells or plant tissue in an environment by exposing the said plant cells or tissue with light of at least a wavelength selected from light of wavelengths found in cold light from an artificial light source. Naturally, the skilled addressee will appreciate that light as described, herein and employed in the instant invention alters the phytochemical profile of a plant cell or plant tissue, such as a harvested tissue lies. Preferably, the combination of light sources includes red light of a wavelength that may be selected from a wavelength within the range of from 600nm - 700nm, preferably from 620nm - 680 nm, more preferably from 625nm - 670 nm, and generally at about 640nm +/- 15nm. Red or blue light or a combination of red and blue light, or a combination of red and/or blue light with white light at any selected energy ratio may be employed in the method of the invention. In a preferred embodiment, the said plant cells or plant tissue can be located under cover. 'Under cover' means that the cells or tissue is located under cover when exposed, for example, during a food processing step prior to further processing such as freezing or canning or heat treating or cooking as alluded to hereinbelow.
Where advantage is to be gained from heat shocking the harvested plant cells or harvested plant tissue, the method of the invention may be employed at a temperature within the range of from + 35 degrees Centigrade to about +45 degrees centigrade, for example, at +40, +41 , +42, +43, +44 or +45 degrees Centigrade, for a period of from a few seconds, for example 30 seconds up to a few minutes, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 minutes or more depending on plant tissue type and design. Naturally, the skilled addressee will appreciate that the heat shock temperature should be such that it does not deleteriously affect the general viability of the plant material that is subjected to a heat shock step.
In a further aspect, there is provided a method of harvesting plant cells or plant tissues under cover wherein the said plant cells or plant tissues are exposed with light as herein described from one or more artificial light sources.
Also included as an aspect of the present invention is harvested plant material or plant cells obtainable by a method according to the present invention and having altered levels of phytochemicals, typically elevated levels of phytochemicals when compared to plant material or plant cells that have not been exposed to light of wavelengths used in the method of the present invention.
'Cover' is to be understood as a general term and may be taken to mean a receptacle in which the plant material or plant cells may be placed, for example a closed container with a built-in light source therein, such as a refrigerator unit comprising an in-built light source that can be activated on demand for a predetermined time interval. Thus, for carrying out the method of the invention use can be made of cooling means, such as a conventional refrigerator comprising a light source capable of emitting blue light in the manner hereinbefore described. Alternatively, 'under cover' may be taken to mean a processing factory wherein harvested plant material is exposed to one or more light sources producing light of appropriate wavelength or wavelengths over a short period of time during the processing operation, such as canning, freezing plant material, or immediately prior to the cooking of foods for canning or for baby food manufacture e.g. purees and the like, and further processed foods such as soups, vegetable-based sauces and the like.
Thus as a further aspect of the invention there is provided a processed food obtainable by a food processing method by exposing live plant cells with light of wavelengths as herein described at light intensities as herein described. Suitable wavelengths of light are those described herein and these are applied for appropriate, predetermined time intervals as described herein. A still further aspect of the invention provides a food processing method comprising exposing live plant cells to light wavelengths as herein described from at least one artificial light source. Typically the wavelength(s) of the light is/are selected from wavelengths as herein described and is applied for a predetermined period of time sufficient to alter the phytochemical profile of the exposed plant cells and/or harvested plant tissue.
"Plant cells" also includes those plant parts or tissues which display an aromaticity which is detectable by the human olfactory senses when cut or harvested. Such plants may display
the aromaticity naturally, for example in the case of cut herbs, from the cut leaves. The plant cells or tissue or parts include members of the Labiatae, such as the broad-leafed herbs. Suitable examples of broad-leafed herbs include basil, oregano, sage, coriander, dill, marjoram and thyme. Other herbs, such as cut herbs that may benefit from being treated according to the present invention include chives, garlic, bay leaf, lemon balm, mint, lavender, parsley, the fennels, e.g. bronze fennel and common fennel, and the like. A more complete list of common herbs to which the invention can be applied is to be found in Taylors Guide to Herbs 1995, Eds. Buchanan R. & Tenebaum F. Houghton Mifflin Co. New York: the teaching of this guide reference is hereby incorporated into the teaching of the present specification. Naturally, the skilled addressee will appreciate that the said plant cells or plant parts are alive when exposed to light in accordance with the present invention and are capable of responding to the application of the cold light-derived light stimulus.
Plant cells or plant parts may be harvested at any stage of growth so long as the harvested plant cells or tissue are capable of responding to the application of light of wavelength and duration as outlined herein. In a preferred embodiment, the harvested plant cells or tissue of broad - leaf herbs can be exposed to wavelengths of light used in the present invention from the 3 to 4 leaf stage and most preferably in the case of culinary herbs such as basil, the 5- leaf stage. It is envisaged that plant cells and/or tissue such as culinary herbs and green vegetables are most usefully exposed as herein-described immediately before processing (e.g. freeze drying, adding to processed foods such as sauces, soups, canned goods and the like), that is to say after the harvesting of cuttings from such plants and/or the provision of young plants for processing e.g. as dried herbs. Dried herbs treated with light as outlined herein immediately post-harvest, for a short period of time, particularly those measured at the 5-leaf stage, are considered to display an increased aromaticity relative to controls which are not exposed to light as described herein.
The artificial light source or sources can be of any suitable conventional type, such as a light emitting diode or even a white light source comprising filters that let through light of the desired wavelength(s). The light source may be placed at any distance from the harvested material provided that the light energy used is sufficient to influence, for example to induce or saturate oxygen evolution at the photosystem Il reaction centre and/or to trigger, that is set off, a transient photo-oxidative stress and/or a moderate photosynthetic electron transport inhibition. Optimising of the light energy and light composition may be performed for example, by monitoring oxygen evolution and chlorophyll a fluorescence using
conventional methods (e.g. according to the instruction manual and software of Hansatech Instruments Ltd., King's Lynn, UK). It is preferable to locate the light source in a position which affords the greatest amounts of irradiation per square unit (e.g. cm2, m2 etc.) of the harvested plant material. Suitably, depending on the size of the covered area, for example that of a processing compartment in a processing factory, or of a refrigerator or other container such as a microwave oven or magnetron fitted with a suitable light source capable of being manually or automatically activated, for example, by employing a timing means and thereby emitting wavelengths of light as indicated herein and described herein. Alternatively, an independent container specifically designed for exposing plant parts or cells to light of wavelengths as described herein may be employed, in a further alternative, the number of light sources may be as little as one to a whole 'battery1 of light sources arranged in series and/or in parallel, for example, in a food processing factory setting, each light source being suitably distanced one from the other at appropriate intervals in such a manner as to effect exposure of the plant material to light of wavelengths as described herein which results in a significant alteration in the level of phytochemicals found therein, preferably an increase of desired phytochemicals.
In a further embodiment of the invention there is provided use of blue light from an artificial light source in a method of processing plant cells or harvested plant tissue under cover. Preferably, the blue light wavelength is selected from the wavelengths of light found as herein described. The blue light may be used in conjunction with other wavelengths of light as herein described.
In a further embodiment, there is provided use of at least blue light in a method as described herein for increasing the phytochemical content in harvested live plant material. In a preferment, the said plant material is located under cover.
In a further embodiment of the invention there is provided the use of plant parts exposed to blue light as described herein in the manufacture of human foodstuffs, such as frozen vegetables (e.g. spinach or plant parts from a Brassica species) or seeds (e.g. peas), bottled or canned condiments, for example sauces for meat, fish and poultry dishes, flavourings, for example tapenade, salad dressings, cooking oils such as olive oil, sunflower oil and the like, soups, pasta and cheeses.
As another application of the present invention, blue light or red light or a combination of red
and blue light may be employed in a greenhouse setting on growing plants. In Northern Hemisphere countries such as Holland, the Scandinavian countries, Belgium, Germany and the UK many varieties of ornamental plants, greenhouse produced lettuce, tomatoes and other salad vegetables are grown under cover. The lighting is supplied in the form of yellow light, typically from sodium lamps. However, such lighting systems lose a lot of energy as heat and do not mimic the blue, red or red and blue spectra of natural sunlight. By modulating the light intensity of blue and/or red light that is shone onto plants, it is possible to optimise the growing phase of the plant and to improve seed set, plant habit, and yield. Thus plants can be produced which are in optimum health and have a full complement of phytochemicals as alluded to herein. In a still further embodiment of the invention there is provided use of blue light and/or red light in improving seed set of plants grown under cover in a greenhouse or in a hydroponics growing system. Furthermore there is provided as another aspect of the invention use of blue light and/or red light in optimising the plant habit of plants grown in the greenhouse or in a hydroponics system. Such uses provide for more efficient production of plants that are grown under cover in the greenhouse, such as ornamentals, salad plants such as lettuces, tomatoes, capsicums and the like.