EP1337139A2 - Modification de la teneur en nitrate et en acide oxalique d'une plante - Google Patents

Modification de la teneur en nitrate et en acide oxalique d'une plante

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Publication number
EP1337139A2
EP1337139A2 EP01979515A EP01979515A EP1337139A2 EP 1337139 A2 EP1337139 A2 EP 1337139A2 EP 01979515 A EP01979515 A EP 01979515A EP 01979515 A EP01979515 A EP 01979515A EP 1337139 A2 EP1337139 A2 EP 1337139A2
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EP
European Patent Office
Prior art keywords
plant
concentration
nitrate
reduced
oxalic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01979515A
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German (de)
English (en)
Other versions
EP1337139A4 (fr
Inventor
Corinne Johnson
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Cornell Research Foundation Inc
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Cornell Research Foundation Inc
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Application filed by Cornell Research Foundation Inc filed Critical Cornell Research Foundation Inc
Publication of EP1337139A2 publication Critical patent/EP1337139A2/fr
Publication of EP1337139A4 publication Critical patent/EP1337139A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general

Definitions

  • Oxalic acid is an organic acid present in algae, fungi, lichens, higher plants, and animals including humans (Oke, 1969). The role of oxalic acid is unclear and it is usually thought to be a useless end product of carbohydrate metabolism (Hodgkinson, 1977). Oxalic acid forms crystals with several minerals including (but not limited to) calcium, magnesium, potassium, sodium, iron, and zinc. The oxalic acid in spinach is primarily bound to calcium or potassium salts. Oxalic acid levels as high as 13% of the dry weight of spinach have been reported for some cultivars (Hodgkinson, 1977).
  • oxalic acid as a secondary metabolite of vitamin C (Hodgkinson, 1977).
  • Human urine always contains small levels of calcium oxalate (Oke, 1969).
  • Deposits of calcium oxalate crystals in the kidneys are a common form of kidney stone (Massey et al, 1993).
  • 90% of oxalic acid excreted in the urine comes from endogenous synthesis (Massey et al., 1993) and is a secondary metabolite of ascorbic acid (vitamin C) in both plants and animals.
  • the top 8 foods or plants known to increase urinary oxalate are identified by Massey et al ( 1993) as rhubarb, spinach, beets, nuts, chocolate, strawberries. wheat bran, and tea.
  • Other foods or plants containing oxalate include leafy green vegetables, asparagus, runner beans, beetroot, brussel sprouts, cabbage, carrots, cauliflower, celery, chives, lettuce, marrow, mushrooms, onions, parsley, green peas, potatoes, radishes, rhubarb, spinach, tomatoes, turnips, apples, apricots, ripe bananas, gooseberries, grapefruits, melons, oranges, peaches, pears, pineapples, plums, blueberries, raspberries, strawberries, arugula, beet greens, collard greens, kale, endive, bok choy, dandelion greens, escarole, cole, mache, mustard greens, radicchio,
  • Oxalic acid oxidase is an enzyme that catalyses the breakdown of oxalic acid into carbon dioxide and hydrogen peroxide. Oxygen is required for the reaction.
  • the formula for the reaction is:
  • the enzyme is present in spinach leaves, but is usually not active. Through studies on beet root extracts it was determined that the sole factor inhibiting oxalic acid oxidase was nitrate. Low concentration of nitrate in vitro (50 ⁇ M) is sufficient to inhibit the enzyme (Oke, 1969).
  • Oxalic acid oxidase catalyses the break down of oxalic acid into carbon dioxide and hydrogen peroxide in the presence of oxygen.
  • Oxalic acid oxidase has been studied in spinach leaves (Oke, 1969). Although present in the leaves, the oxidase is inhibited by even low levels of nitrate ( ⁇ - 50 M) (Oke, 1969). Nitrate may also play another role in increasing leaf oxalate.
  • Nitrate is ubiquitous in most fresh vegetables and accounts for approximately 80% of nitrate in the diet. Nitrate itself is not particularly toxic. Most nitrate is excreted from the body through the urine (Lee, 1970). However approximately 5% of consumed nitrate is converted to nitrite in the oral cavity, gastrointestinal tract, and liver. It is the reduced form of nitrate, nitrite, that is the primary cause for concern. High levels of nitrate consumed by infants can lead to methemoglobinaemia, or blue baby syndrome. The primary concern for healthy adults is the potential for post-consumption formation of carcinogenic N- nitrosamine compounds. Fresh spinach may contain up to 740 ppm nitrate, and reducing soluble nitrate and oxalate levels in vegetables would improve the nutritional value of these crops to humans or other animals.
  • the present invention provides methods for altering the concentration or level of soluble oxalic acid or oxalate in a plant, comprising growing the plant in a hydroponic nutrient medium which contains a nitrogen source, and then altering the content of the hydroponic medium prior to harvest of the plant so that the medium does not contain a nitrogen source, so that the concentration of oxalic acid or oxalate in the plant is altered.
  • the methods of the present invention may also be used to alter the concentration or level of soluble nitrate in a plant, and to alter the concentration or level of nitrate and soluble oxalic acid or oxalate in a plant. Further, the methods of the present invention may be used to alter the concentration or level of one or more nutrient quality indicators in a plant.
  • Figure 1 Oxalate and Nitrate concentration in spinach leaves treated with six pre-harvest draw-down method treatments.
  • Figure 2 Time-course pre-harvest draw down of nitrate and oxalate in spinach, cv Whitney transferred to RO water for five days.
  • FIG. 4 Comparison of spinach treated for 192 h (8 d) in RO water, RO water + tent, or control plants in a nitrate nutrient solution.
  • FIG. 5 Reduction in nitrate and oxalate in spinach cv Alrite transferred to RO water for 168 h (7 days). Spinach treated in RO water, RO water + tent, or control plants in a nitrate nutrient solution.
  • Figure 10 Combined nitrate concentration data points from four draw-down experiments show similarity in slope and shape of lines during draw- down.
  • Graph combines results from tap water, RO water, and spinach cultivars Whitney and Alrite.
  • Figure 12 During draw-down procedure, nitrate is used in vacuoles and plants can continue to grow (add dry weight).
  • Figure 13 Decline in three indicator vitamins with increasing duration of oxalate draw-down period: A) vitamin C, B) Folate, C) Beta- carotene.
  • FIG. 14 Oxalate concentration in spinach leaves as a function of nitrate concentration. Data from four nitrate draw-down experiments combined shows saturation of nitrate influence on oxalate concentration at 200 umoles/g DW in this set of experiments.
  • Controlled Environment Agriculture is a combination of horticultural and engineering techniques that optimize crop production, crop quality, and production efficiency (L.D. Albright, 1990, Environment Control for Animals and Plants).
  • hydroponic nutrient solution or medium is a water-based formulation containing essential nutrients for plant growth.
  • the solutions contain mineral elements and compounds, containing but not limited to nitrogen, phosphorous, potassium, calcium, magnesium, oxygen, sulphur, boron, chlorine, copper, iron, manganese, molybdenum, zinc, in addition to many minerals present in trace amounts.
  • the pH of the solution is controlled to allow solubility of the mineral elements and compounds in the solution. (Jones, 1997, Hydroponics, A practical Guide for the Soilless Grower).
  • the nitrogen concentration of the hydroponic nutrient solution or medium may range from, but is not limited to, slightly under 50 to more than 350 ppm.
  • the reduction in concentration of oxalic acid, nitrate, or nutrient quality indicators, as accomplished by the present invention may be allowed to continue as long as plants maintain a marketable or healthy appearance, or a dark-green appearance.
  • nutrient quality indicators includes vitamins and/or minerals in a plant that are important for human or animal nutrition, and includes, but is not limited to vitamin C, ascorbic acid, folate, folic acid, vitamin B9, ⁇ -carotene, a vitamin A precursor, lutein, calcium, iron, vitamin A, vitamin E, and other vitamins and minerals.
  • mature plant includes plants that have reached a sufficient stage of growth and contain the desired characteristics, so that they may be harvested for any useful purpose.
  • the term “mature plant” may describe a plant that has reached approximately 2-7 ounces of fresh weight after about 20-35 days from seeding.
  • the present invention may be useful in altering the concentrations of oxalic acid, oxalate, nitrate, or nutritional quality indicators of any plant species, including, but not limited to, plants that contain oxalic acid or nitrate, such as spinach, beets, nuts, chocolate, cacao, strawberries, wheat bran, tea, leafy green vegetables, asparagus, runner beans, beetroot, brussel sprouts, cabbage, carrots, cauliflower, celery, chives, duckweed, lettuce, marrow, mushrooms, onions, parsley, green peas, potatoes, radishes, rhubarb, spinach, tomatoes, turnips, apples, apricots, ripe bananas, gooseberries, grapefruits, melons, oranges, peaches, pears, pineapples, plums, blueberries, raspberries, strawberries, arugula, beet greens, collard greens, kale, endive, bok choy, dandelion greens, escarole
  • the present invention may also be useful in altering the concentrations of oxalic acid, oxalate, nitrate, or nutritional quality indicators of other plant species, including, but not limited to, corn, rapeseed, alfalfa , rice, rye, millet, pearl millet, proso , foxtail millet, finger millet, sunflower, safflower, wheat, soybean, tobacco, potato, peanuts, cotton, potato, cassava, coffee, coconut, pineapple, cocoa , banana, avocado, fig, guava , mango, olive, papaya , cashew, macadamia, almond, sugar beets, sugarcane, oats, duckweed, barley, vegetables, ornamentals, conifers, tomatoes, green beans, lima beans, peas, Cucumis (including cucumber, cantaloupe, and musk melon), azalea, hydrangea, hibiscus, roses, tulips, daff
  • Seeds were spread evenly on the blotter paper and distilled water was added to keep germinating seeds moistened. Seeds in germination boxes were then placed in a 5 °C cooler for 7 days while the imbibition and germination continued. Seed planting and hydroponic pond system seed holders EXPERIMENT I.
  • Floating polystyrene trays were prepared by cutting eleven 28 cm x 0.6 cm (11 x 0.25 in) strips out of each 0.3 m x 0.6 m (1 x 2 ft) tray. Thirty felt strips were cut from a bolt into rectangles 28 cm x 10 cm (1 1 x 4 in). Two pieces of felt were placed together to form cloth sandwich-strips and were fed through the polystyrene strip-holders. Felt-strip pairs were held in place by wedging a small piece of polystyrene at both ends of each felt-strip pair. The felt-strip seed holders wicked up water and nutrients from the pond solution and maintained a moist environment around the seeds and roots.
  • the foam boards were 2.5 cm (1 inch) thick and roots grew downward, between the two felt strips into the oxygenated hydroponic solution below. Seeds were placed with root oriented downward between two pieces of felt and held by friction fit over the hydroponic nutrient solution. Only seeds that had germinated during the one- week in the cooler were planted. Three seeds were planted between each felt strip pair. Seedling radicals were approximately 2 to 5 mm long at time of sowing.
  • Seeds were sown into reverse-osmosis (RO) water-leached rockwool flats.
  • the moistened seeded rockwool flats were covered and maintained at 18°C for 3 days after sowing without need for additional watering.
  • growth chamber temperature was increased to 22 °C and ebb and flow watering was initiated at a rate of twice per day.
  • Seedlings were watered automatically with a nutrient solution containing no ammonium and no chloride.
  • Cool white fluorescent lamps in the growth chamber were set to 25% from initiation of imbibition to age 3 days.
  • lights were increased to 50% maximum intensity.
  • Whitney seedlings in rockwool cubes were transferred from the growth chamber to polystyrene floaters in an 18 °C hydroponic pond.
  • Greenhouse temperature was controlled at 24 °C continuous.
  • Light control program was set to deliver 16 mols of light per day.
  • Supplemental light was provided by an array of high-pressure sodium lamps.
  • the pond solution nutrient medium (or hydroponic nutrient medium) recipe was adjusted to eliminate sources of ammonium and chloride, due to reported toxicity problems in spinach with these ions (Elia et al, 1999). Pond temperature was maintained at 18 °C. Nutrient solution pH and electrical conductivity (EC) were monitored and corrections made as needed. Set point for pH was 5.8. Nitric acid was used to decrease pH, and potassium hydroxide was used to increase pH. Electrical conductivity set point was 920 micro S for this particular nutrient solution. Daily water samples were taken and frozen for nitrate analysis. Nitrate concentration in the NO 3 solution ranged from 0.2 to 2.5 mM. Nitrate concentration in the urea pond ranged from 0 to 1.1 mM. Nitrate in the RO water pond ranged from 0 to 0.01 mM.
  • plants were transferred to one of three RO water-filled tanks for 5 days to draw down nitrate in cell vacuoles.
  • Plant from experiments V and VI used tap water as RO water line was not functioning in greenhouse (GH).
  • Tanks were not oxygenated, but were stirred regularly by hand to aerate.
  • Tanks were located in GH 15 C with photoperiod from 6 am to 10 pm, continuous HPS lighting. Greenhouse temperature during nitrate draw-down phase was 20 °C. There were three tanks used in the test, with six plants per tank.
  • EXPERIMENTS II-VI Six plants in each tank were divided in two. Three plants were placed in a CO 2 draw-down tent. Humidity in tent was controlled with a de-humidifier. A 4 liter open reservoir of 1 N KOH was placed inside the tent to assist in draw down of CO ⁇ . The other three plants in each tank remained outside the tents. Oxalate draw-down time was two days. Tanks were located in GH 15 °C with photoperiod from 6 am to 10 pm, continuous HPS lighting. Greenhouse temperature during oxalate draw-down phase was 20 °C.
  • plants were harvested by separating above-root portion from roots with a razor. Plants were placed in a 70 °C drying oven inside brown paper bags and dried for two days. Plants in the second tap water experiment were freeze dried rather than oven dried for additional nutrient analyses.
  • Dried plant samples were bulked by treatment and age at harvest and were ground on a Wiley Mill, using a #20 screen.
  • Soluble oxalate and nitrate were extracted together in the same supernatant.
  • a sample of ground tissue was weighed into a 15 ml tube.
  • Ten milliliters of HC1 (0.1 N) was added and mixed on a vortex mixer.
  • Test tubes were sealed and placed on a shaker for one hour at room temperature. Samples were centrifuged and 2 ml of supernatant removed and centrifuged again in 2.2 ml microcentrifuge tubes. A 0.5 ml portion was removed and diluted with 4.5 ml of 18 M ohm water.
  • freeze dried tissue samples from three time points during the second tap-water draw-down experiment were sent to ENI Laboratories (www. eurofins. com, Eurofins Scientific, Dayton, NJ) for accredited nutrient analysis.
  • the time points selected for analysis were Day 0 (starting point control), Day 5 (day nitrate reserves are depleted), and Day 8 (last day spinach had marketable appearance).
  • the number of samples and types of analysis were limited due to cost.
  • Three nutrients were selected as indicators of potential loss of nutrients with the pre-harvest nitrate/oxalate reduction technique.
  • the water soluble vitamin, vitamin C was selected as the first indicator because spinach contains ample amounts of vitamin C for measurement, and ascorbic acid decline with post-harvest food preparation is well documented.
  • Folic acid total vitamin B9 was selected because spinach is among the vegetables high in folic acid and this would be an important nutrient to preserve.
  • the vitamin A precursor, ⁇ -carotene was selected to represent fat- soluble vitamins.
  • oxalate was reduced from 1 120 to 633 ⁇ mol/ g DW (i.e., from 10 % to 6 % DW) in five days of RO water treatment.
  • Nitrate was reduced from 548 to 15 ⁇ mol/g DW (i.e., 3% to 0% DW) in five days (120 hours) ( Figure 2).
  • the experiment was extended to eight days in Experiment III, and a humidity-controlled tent treatment was added as an end-point for comparision with controls.
  • oxalate concentration was reduced from 1203 to 387 ⁇ mol/g DW (i.e., from 11% to 3% DW) in eight days (Figure 3).
  • Nitrate concentration was reduced from 449 to 0 ⁇ mol/g DW (i.e., 3% to 0° o DW) in eight days.
  • the RO water + tent treatment plants were not significantly different from the plants given the RO water treatment alone (Figure 4).
  • Alrite has a faster bolting tendency than Whitney.
  • Alrite leaf nitrate concentration was reduced from 299 to 0 umol/ g DW (i.e., from 2% to 0 % DW) and leaf oxalate concentration was reduced from 1181 to 432 ⁇ mol/g DW (i.e., from 10% to 4 % DW) after 7 days of the draw-down treatment in RO water (Figure 5).
  • Soluble leaf nitrate content was lower in Alrite (299 ⁇ mol/g DW) than in Whitney (449 ⁇ mol/g DW).
  • the lower concentration of nitrate in Alrite than in Whitney is consistant with findings in other experiments (e.g., Table 4.4) and may be a genetic difference in leaf nitrate concentration.
  • the results of the oxalate dra -down procedure were the same for Alrite as for Whitney.
  • the draw-down method was performed using tap-water instead of RO water to determine whether cost could be lowered by using tap water for the method.
  • Nitrate was 691 ⁇ mol/g DW (4% DW) in the control treatment, and 83 ⁇ mol/g DW (1 % DW) in the tap water treament.
  • Tap water contains nitrate, so it is not surprising that the tap water treated plants did not reach 0 nitrate in the same time that the RO water treated plants reached 0 nitrate concentration.
  • Oxalate was 945 ⁇ mol/g DW (8% DW) in the control plants and 552 ⁇ mol/g DW (5% DW) in tap-water treated plants (Figure 7).
  • Leaf nitrate concentration was reduced from 284 to 0 ⁇ mol/g DW (2 % to 0% DW).
  • Fresh tap water was added to the treatment ponds after to 120 h harvest (day 5).
  • Tap water contains nitrate, and the increase in pond-water nitrate due to adding fresh water resulted in leaf concentration increasing on day 6 (144 h) and then falling again within 2 days.
  • leaf oxalate concentration followed and increased one day after the nitrate peak (day 7) at 168 hours. It is notable that the increase in oxalate following the water- refresh did not shift the draw-down to a higher recovery level.
  • Figure 7 shows nitrate and oxalate leaf concentrations during the course of the tap-water drawdown method perfo ⁇ ned on spinach, cv Whitney. Also indicated are the three sampling dates for vitamin Analysis. The last marketable day is indicated, but oxalate concentration continued to fall until the last plants were removed on day 1 1 (264 h). Use of RO water was superior to tap when water-replenishment was needed due to presence of nitrate in tap water. Oxalic acid was reduced by one- half to two-thirds through the 7 to 8 day pre-harvest treatment. The reduction in oxalic acid paralleled nitrate reduction in time-course studies.
  • the slope of the line is approximately -3.3 and indicates that in the method developed here, oxalate is removed at a rate of 3.3 umol per hour per g DW. This is equivalent to a removal rate of approximately 0.02% oxalate/g DW per hour. It would be expected then, that 78.5 umol oxalate are removed per day per g DW (i.e., 0.49% DW per day)
  • Plant dry weight data from the final draw-down experiment demonstrates that the spinach plants continue to grow during the nitrate draw-down period, as the plants are using up nitrate stores in the vacuoles. When the nitrate reaches 0, plant growth stops ( Figure 12). Vitamin reduction with pre-harvest treatment
  • vitamin C mg/100 g DW
  • equation: [Beta-carotene (IU/100 g DW)] -1610.2*(Days of draw-down) +
  • the pre-harvest technique reduced soluble nitrate levels in the spinach leaves to undetectable levels by day 5 of the pre-harvest draw-down treatment.
  • Soluble oxalic acid also reduced by one-half to two thirds by the method. Reduction in oxalic acid occurred and paralleled the reduction in nitrate in all time-course studies. The leaf concentration of the three vitamin indicators also declined with increasing time in the oxalate draw-down treatment.

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  • Life Sciences & Earth Sciences (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Botany (AREA)
  • Ecology (AREA)
  • Forests & Forestry (AREA)
  • Environmental Sciences (AREA)
  • Cultivation Of Plants (AREA)
  • Hydroponics (AREA)

Abstract

La présente invention concerne des procédés permettant de modifier la teneur ou le niveau de nitrate, d'acide oxalique ou d'oxalate soluble, ou des indicateurs de la valeur nutritive d'une plante, consistant à faire pousser la plante dans un milieu nutritif hydroponique renfermant une source d'azote, puis à modifier la teneur du milieu hydroponique avant la récolte de la plante afin que le milieu ne contienne pas une source d'azote, de façon à modifier la teneur en acide oxalique ou en oxalate de la plante.
EP01979515A 2000-10-27 2001-10-05 Modification de la teneur en nitrate et en acide oxalique d'une plante Withdrawn EP1337139A4 (fr)

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US24405000P 2000-10-27 2000-10-27
US244050P 2000-10-27
PCT/US2001/031254 WO2002034755A2 (fr) 2000-10-27 2001-10-05 Modification de la teneur en nitrate et en acide oxalique d'une plante

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EP1337139A2 true EP1337139A2 (fr) 2003-08-27
EP1337139A4 EP1337139A4 (fr) 2004-12-29

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AU (1) AU2002211465A1 (fr)
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CN102763555A (zh) * 2012-08-08 2012-11-07 恩施土家族苗族自治州烟草物资供应公司 一种烟叶漂浮育苗方法

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CN102204445A (zh) * 2011-06-03 2011-10-05 汕头市微补植物营养科技有限公司 一种大棚西瓜施肥方法
CN102204445B (zh) * 2011-06-03 2012-07-25 汕头市微补植物营养科技有限公司 一种大棚西瓜施肥方法
CN102763555A (zh) * 2012-08-08 2012-11-07 恩施土家族苗族自治州烟草物资供应公司 一种烟叶漂浮育苗方法
CN102763555B (zh) * 2012-08-08 2013-12-25 恩施土家族苗族自治州烟草物资供应公司 一种烟叶漂浮育苗方法

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