DESCRIPTION

The present invention relates to a composition for biofortification of food plants, to a method of biofortifying food plants using this composition and to the food plants thus fortified.

BACKGROUND OF THE INVENTION

Recently, an increasing number of studies have highlighted the importance of the quality and nutritional value of food resources in relation to the onset of some diseases.

Food plants have always been the main source of microelements and antioxidant compounds for human and animal nutrition. Scientific data indicate that diets rich in vegetables are associated with a lower risk of developing various degenerative diseases, such as tumors and cardiovascular diseases. The intrinsic lability of nutrients deriving from the processing and cooking of these foods and the variability of absorption, and their consumption by the human body, nonetheless determine the need for continuous availability, and hence consumption, of the microelements and the antioxidant compounds contained therein, to prevent subsequent deficiency of these nutrients.

Vitamins and microelements are essential compounds for a wide range of enzymatic reactions required in the metabolism of amino acids, lipids, carbohydrates and nucleic acids. Deficiencies of vitamins, and in particular of folates, can cause serious diseases or increase the risk of the onset of cardiovascular and neurodegenerative diseases and cancer. As human beings are unable to synthesize these nutritional elements, they must be obtained from food sources, with plants as the main source. The preservation of an adequate state of these nutritional elements is therefore a key issue in human public health strategies.

The term “folate” or “folates” refers to the vitamin in its natural form present in foods, while folic acid (monopteroylglutamic or pteroylmonoglutamic acid) is the oxidized form of the vitamin, and identifies the synthetic molecule.

The active form of folic acid is tetrahydrofolic acid, which is obtained through enzymatic reduction. Tetrahydrofolate (THF), and folates in general, are a soluble tripartite vitamin in group B, called vitamin B9, composed of pterin, p-aminobenzoate (p-ABA) and one or more glutamate residues.

The formula of p-aminobenzoic acid, or p-ABA, is indicated below:

The following diagram indicates the formula of THF in its various forms:

A carbon unit with varying degrees of oxidation, such as the formyl (C=OR), methylene (CH2) and methyl (CH3) groups, can be enzymatically attached at the positions N-5 or N-10 of THF, and the resulting folates substituted with carbon are enzymatically interconvertible and act as carbon donors in the complex biochemical reactions that affect the biosynthesis of nucleotides, the homeostasis of amino acids, the methylation of proteins, phospholipids and nucleic acids, and redox defense.

Folate is synthesized by plants and bacteria. In plants the three parts of the THF molecule are produced separately in plastids, mitochondria and cytosol, and then joined together, as shown in the following diagram:

Humans and animals are incapable of producing folates de novo and therefore for their intake they depend on food sources, in particular green leaves and plants. All natural forms of folate are unstable, in particular through oxidative scission promoted by light, which can produce significant losses of folates in fruit and vegetables after harvesting. Moreover, the natural forms of folate can be lost during processing and cooking. However, these forms can be stabilized in vivo by antioxidant compounds such as ascorbate and glutathione, and by the bond with specific proteins.

The main methods used to remedy the deficiency of folates and microelements have been the addition of chemically synthesized folic acid to cereal based products or voluntary integration through taking folic acid tablets. However, these strategies have potential disadvantages due to the fact that the folic acid is not a natural compound and has potential negative effects. Some research has recently also been dedicated to the development of genetically modified plants that contain a higher amount of folates compared to natural plants, but the cultivation and marketing of these plants has numerous limits, also due to regulatory questions.

As the metabolism of folates requires, as enzymatic cofactors, microelements such as selenium (Se) and zinc (Zn), the content of which is often insufficient in the natural ground, there is increasing interest in the search for new strategies to increase both natural folate and the content of microelements in food plants.

WO 2014/173936 A2 describes a method for promoting the growth of a plant and/or improving its resistance to stress, as well as protecting plants from the undesirable effects of herbicides, through the application of a derivative of benzoic acid, in particular p-ABA. WO 2014/173936 A2 provides some examples of the protective action of p-ABA against the harmful side effects of specific herbicides. There is also described the combined use of the derivatives of benzoic acid with one or more pesticides and possibly with surfactants. No mention is made to increase of folates.

Gorelova V. et al, Folates in Plants: Research Advances and Progress in Crop Biofortification, Front. Chem, 2017; 5;21, describes the role of folates in the synthesis of important biomolecules and mentions the increase of folates in plants by means of genetic engineering techniques aimed at increasing the precursors pterin and p-ABA. The authors report a poor success of this approach. In any case, these are complex and costly genetic engineering methods and which cause concern in the general public.

Therefore, there is the need to enrich food plants with folates and essential microelements with agronomic biofortification methods.

ABSTRACT OF THE INVENTION

Therefore, an object of the present invention is to provide a composition for biofortification of food plants adapted to increase the content of folates and of certain microelements required for folate metabolism.

Another object of the invention is to provide a method of biofortifying food plants that increases the content of folates and of certain microelements required for folate metabolism. A further object of the present invention is to provide food plants fortified with folates and microelements essential for folate metabolism.

Therefore, an aspect of the present invention relates to a composition for biofortification of food plants comprising p-amino benzoic acid (p-ABA), a selenium salt and a zinc salt in amounts effective for increasing the folate, selenium and zinc content in the food plant.

According to an aspect of the invention, said composition comprises an aqueous solution comprising: a) 50 ppm to 9000 ppm of p-ABA; b) 2 ppm to 120 ppm of a selenium salt; c) 10 ppm to 18000 ppm of a zinc salt.

Another aspect of the present invention relates to a method of biofortifying food plants comprising the administration of a composition comprising p-amino benzoic acid (p-ABA), a selenium salt, and a zinc salt in amounts effective to increase the folate, selenium, and zinc content of the plant by application to the leaf apparatus of the plant grown on soil or above ground.

A further aspect of the invention relates to a plant enriched in folate, selenium and zinc content by means of the biofortification method as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

- Fig. 1 shows the concentration of Se and Zn in plants grown according to the invention;

- Fig. 2 shows the concentration of 5 -methyltetrahydrofolate in plants grown according to the invention; and

- Fig. 3. shows the concentration of oligomers of inulin in plants grown according to the invention.

DESCRIPTION OF THE INVENTION

A first aspect of the invention relates to a composition for biofortification of food plants comprising p-amino benzoic acid (p-ABA), a selenium salt, and a zinc salt in amounts effective for increasing the folate, selenium, and zinc content of the food plant.

As already mentioned in the preamble, folates play an essential role in the complex biochemical reactions that affect biosynthesis of nucleotides, homeostasis of amino acids, methylation of proteins, phospholipids and nucleic acids, and redox defense. Moreover, the metabolism of folates requires, as enzymatic cofactors, microelements such as selenium (Se) and zinc (Zn), the content of which is often inadequate in the natural ground.

Therefore, the increase of the content of folates, selenium, and zinc in food plants is an important strategy to make these fundamental substances available to humans through diet. However, the scientific literature has not described a composition or a method adapted to produce a combined biofortification of folates with selenium and zinc, implemented in an effective and safe manner. In fact, the effectiveness of the composition and of the method is only obtained if enrichment of folates, Se and Zn is quantitatively significant, and safety is obtained if the final amount present in the plant is below the toxicity thresholds that Se and Zn can exhibit. Moreover, with regard to folates, it was not known whether the administration of p-ABA led to a significant increase of folates, in view of the fact that p-ABA is only one of the three structural components of the molecule of folate, the others being pterin and glutamate.

According to the present invention, food plants, hereinafter referred to also as vegetables, which can be fortified with agronomic methods are all broad leaf plants that contain folates. More particularly, these food plants comprise broad leaf vegetables containing folates and other edible vegetables, and in more detail varieties and cultivars of the species Lactuca sativa, Cichorium intybus, Cichorium endivia, Brassica oleracea, Brassica rapa, Brassica napus, Lepidium sativum, Raphanus sativus, Beta vulgaris, Cynara scolymus, Cynara cardunculus, Taraxacum officinale, Borago officinalis, Salsola soda, Asparagus officinalis, Valerianella locusta, Eruca sativa, Spinacia oleracea, Capparis spinosa, Allium cepa, Allium ascalonicum, Allium sativum, Allium porrum, Allium fistulosum, Allium schoenoprasum, Muscari comosum, Foeniculum vulgare, Anthriscus cerefolium, Anethum graveolens, Coriandrum sativum, Daucus carota, Apium graveolens, Ocimum basilicum, Petroselinum crispum, Petroselinum hortense, Scorzonera hispanica, Tragopogon porrifolius, Pastinaca sativa, Solanum lycopersicum, Solanum melongena, Solanum tuberosum, Capsicum annuum, frutescens, Capsicum chinense, Cucumis sativus, Cucurbita pepo, Cucurbita maxima, Cucurbita moschata, Lagenaria siceraria, Phaseolus vulgaris, Cicer arietinum, Lens culinaris, Lathyrus sativus, Lupinus albus, Vicia faba, Pisum sativum, Vigna unguiculata, Vigna angularis, Vigna radiata, Glycine max.

Among the salads the varieties chosen in the group comprising the varieties and cultivars of Lactuca sativa are preferred.

The biofortification of salads, and particularly of lettuce, is advantageous both due to the natural content of THF and to the fact that they can be consumed prevalently as fresh product, hence without losing bioactive substances due to cooking.

According to the present invention, the growing method of the aforesaid vegetables can be implemented on soil or above ground. With regard to above ground methods, hydroponic, aeroponic and substrate culture are preferred. On soil methods include both greenhouse and open field growing.

A first aspect of the invention relates to a composition for biofortification of food plants comprising p-amino benzoic acid (p-ABA), a selenium salt, and a zinc salt in amounts effective for increasing the folate, selenium, and zinc content of the food plant.

According to an aspect of the invention, the aforesaid composition comprises an aqueous solution comprising; a) 50 ppm to 9000 ppm of p-ABA, preferably 500 to 6000 ppm of p-ABA, more preferably 1500 to 3000 ppm of p-ABA; b) 2 ppm to 120 ppm of a selenium salt; preferably 30 to 90 ppm of a selenium salt, more preferably 50 to 70 ppm of a selenium salt; c) 10 ppm to 18000 ppm of a zinc salt; preferably 1500 to 12000 ppm of a zinc salt, more preferably 3000 to 6000 ppm of a zinc salt.

The role of p-ABA as precursor of folates and the importance of folates in the complex biochemical reactions of the human metabolism have been mentioned in the preamble and are known to the person skilled in the art.

Selenium (Se) is an essential micronutrient for humans and animals, as it performs a role as coenzyme in many cell functions. It is known to possess antioxidant, cardioprotective, proapoptotic, DNA repair and antitumor properties. Selenium is found in more than 20 selenoproteins and selenoenzymes, such as the redox enzyme glutathione peroxidase (GPX), which contributes to preventing oxidative cell damage, and thioredoxin reductase, which reduces the oxidized molecules in animals and in some plants.

Inorganic forms of Se such as selenates (Se ⁺⁶ ) and selenites (Se ⁺⁴ ), or organic forms such as selenomethionine and selenocysteine are found in the plant, but selenate is more easily transported by the roots and accumulates to a much greater degree in the leaves compared to selenites or to organic selenium. Selenates are mainly metabolized to selenomethionine, which is the main Se compound in plants. Although Se is not essential for the nutrition of plants, horticultural crops are the preferred source for the integration of Se as they contain organic Se forms that are available for humans.

In the composition of the invention Se is present preferably as alkali metal selenate, more preferably sodium selenate NaiSeCM. Zinc (Zn) is an essential microelement component of thousands of proteins in plants and is the second most abundant transition metal in organisms after iron (Fe). Its importance also for humans is due to its presence in over three hundred different metalloenzymes, without counting the hundreds more that transport and process Zn, and for its important role in activation of the transcription factors involved in gene expression, signal transduction, and mechanisms such as transcription, replication and apoptosis. Its low solubility in soils, in particular in those with a high pH, rather than the low total amount of Zn, is the main reason for its widespread deficiency in cultivated plants, and hence in populations, even in richer countries.

In the composition of the invention Zn is preferably present as inorganic salt, complex, chelate, more preferably zinc sulfate ZnSC .

Another aspect of the present invention relates to a method of biofortifying food plants comprising administering a composition comprising p-amino benzoic acid (p-ABA), a selenium salt, and a zinc salt in amounts effective for increasing the folate, selenium, and zinc content of the food plant, by application to the leaf apparatus of the plant grown on soil or above ground.

The combined administration to food plants of p-ABA in order to increase folates, and of Se and Zn as microelements important for their role in cell physiological processes, is therefore particularly advantageous.

The biofortification composition according to the invention can be applied by adding to the ground or applying to the leaves as spray. Application to the ground can be more complex as the different pH conditions and the composition of the soil can influence the availability of the substance administered.

Instead, foliar fertilization is more effective and safer as the substances applied to the leaves can enter the leaf through cuticular absorption or through the stomata and subsequent translocation to the sites of use.

The biofortification method according to the invention provides for the application of a volume of solution according to the invention from 100 to 800 liters per hectare (1/ha), preferably from 150 to 600 1/ha.

Consequently, with reference to the preferred range 150-600 1/ha, based on the different concentrations, the amount of substances applied are as follows: i. with the solution containing 50 ppm to 9000 ppm of p-ABA from 7.5 g/ha to 5400 g/ha di p-ABA are applied; ii. with the solution containing 2 ppm to 120 ppm of a selenium salt from 0.3 g/ha to 72 g/ha of selenium salt are applied; iii. with the solution containing 10 ppm to 18000 ppm of a zinc salt from 1.5 g/ha to 10800 g/ha of zinc salt are applied.

For the administration of large amounts of the components a), b) and c) of the composition the repeated application method is also possible. Therefore, for example, the application of an amount of p-ABA of 6000 or 9000 ppm can be implemented by administering a solution of 2000 and 3000 ppm respectively three times. Likewise, the application of an amount of ZnSO4 of 1500 or 12000 ppm can be implemented by administering a solution of 500 or 4000 ppm respectively three times. The same criteria are applied to other amounts in the same high ranges of the composition intervals indicated.

The solution of the composition of the components a), b) and c) is prepared by dissolving the components a), b) and c) in water, in the amounts desired.

The food plants suitable to be biofortified with the composition of the invention can be cultivated both on soil and above ground.

For cultivation above ground in hydroponic culture, the plants are typically nourished with Hoagland solution, which has the following composition: nitrogen 210 ppm, potassium 235 ppm, calcium 200 ppm, phosphorus 31 ppm, sulfur 64 ppm, magnesium 48 ppm, boron 0,5 ppm, iron 1 to 5 ppm, manganese 0.5 ppm, zinc 0.05 ppm, copper 0.02 ppm and molybdenum 0.01 ppm.

For cultivation on soil, normal agronomic techniques can be used, optionally with the use of fertilizers, as is known to the person skilled in the art.

It has been found that the application of the composition of the invention led to an enrichment in folates, selenium and zinc in the plants treated, without selective absorption competitive phenomena inhibiting the absorption of one or more of the compounds administered.

Unexpectedly, folate enrichment was obtained notwithstanding the fact that only one of the three precursors of folate, i.e., p-ABA was administered, while pterin and glutamate were not administered.

Moreover, an enrichment of numerous metabolites that form bioactive compounds useful not only for the metabolism of the plants but also for humans, increasing the nutritional content of the food plant, was found.

In particular, an enrichment of inulin in the plants treated was surprisingly found.

Inulins are the simplest group of linear fructans and, unlike starch, which is produced by many types of plants, are industrially extracted from chicory. Inulin comprises glucosyl portions that terminate the chain and of repetitive fructosyl units linked by β (2,1) bonds and are soluble in vivo while in vitro it is impossible to reproduce their degree of solubility. Due to the (2,1) bonds, inulin is not digested by the enzymes in the human digestive system, exhibiting benefits for health due to its functional properties: reduced caloric value, dietary fiber and prebiotic effects, hypoglycemic action and enhancement of calcium and bioavailability of magnesium. Moreover, it could be a good replacement for sugar in nutrition for diabetics, being 35% sweeter than sucrose.

Various studies have reported that prebiotics such as inulin stimulate the growth of intestinal bacteria that are beneficial for the host, such as lactobacilli and bifidobacteria which, by stimulating the immune system, decrease the level of pathogenic bacteria in the intestine and reduce the risk of atherosclerosis by reducing the synthesis of triglycerides and fatty acids in the liver, as well as lowering their levels in serum. It is also known that inulin is present in lettuce, which is capable of synthesizing it in the stem and in the roots in small amounts, but not in the leaves.

The examples set forth below show some embodiments of the invention and are provided by way of non-limiting example.

EXAMPLES

Materials and methods

The sodium selenate powder (Na2SeO4) and the p-ABA (vitamin Hl) were supplied by the firm ACEF S.p.A. (Italy), while the zinc sulfate (ZnSCU) by the firm Fabbrica Cooperativa Perfosfati Cerea S.C. (Italy).

The compositions to apply as foliar spray were prepared at ambient temperature in water at the required concentration, and were applied to the plants after 31 days of cultivation.

The food plants treated comprised lettuce of the variety gentilina grown on soil in a climatic chamber. The climatic chamber is a useful growth medium as it allows strict control on the plants, avoiding climatic variations. In particular, it allows comparison between plants treated with the new compositions of the invention and plants sprayed only with water.

Both the control plants and those treated with the composition of the invention were grown in a sandy-loam agricultural soil fertilized with NPK 6.12.18 (nitrogen/phosphorus/potassium) mineral fertilizers, as is known in the art, irrigated with drip system.

The management conditions of the climatic chamber were as follows: duration of the day of 12 hours, temperature of 22/17 °C (day/night), relative humidity of 55-65%. The sandy-loam soil cultivation substrate (taken from the same plot as a trial conducted outdoors) and fertilization with mineral fertilizer with a low chloride concentration NPK 6.12.18, total nitrogen 6.2%, ammonia nitrogen N-NH4 4,1%, urea nitrogen N-NH2 1.0% and nitric oxide N-NO2 1.2%; phosphorus pentoxide P2O5 12,4%, soluble phosphorus pentoxide 11.0%, potassium oxide K2O 18.2%, sulfur dioxide SO231.4% (supplied by FCP, Cerea, Italy).

As already mentioned, the control plants were sprayed with a foliar spray comprising only water.

The composition of the invention comprises an aqueous solution of: a) 2000 ppm of p-ABA; b) 60 ppm of sodium selenate; c) 4000 ppm of zinc sulphate.

The tests were conducted on three groups of 10 plants each, to offset the possible differences of phenotype. The amount of composition applied was equivalent to applying 300 1/ha in the open field, hence the following amounts of the single components were administered, expressed in grams per hectare; i. 600 g/ha of p-ABA; ii. 18 g/ha of selenium salt; iii. 1200 g/ha of zinc salt.

The plants were then collected and sampled choosing two leaves for each plant section, at the top, in the middle and at the bottom. The leaves were then transported to the laboratory after instant freezing using dry ice at controlled temperature.

Analysis of the microelements by ICP-MS (inductively coupled plasma mass spectrometry) High precision, high resolution, high sensitivity and very low detection limits are required to detect and quantify the trace elements present at ppm level in plants. The ICP-MS has all these features and allows simultaneous analysis of multielements and trace elements.

The samples for microelement analysis were first decontaminated by washing in a Tween-20 (1 g/L) Milli-Q water solution (Milli - Q water, Merck Millipore) and three rinses in Milli-Q water to remove dust and contamination transmitted from the soil, then stored at -80 °C before being lyophilized. Lyophilization was carried out with Freeze Dryer Modulyo (Edwards Vacuum, Milan, Italy) and the samples were stored in a dry place at ambient temperature until digestion.

The lyophilized samples were subjected to digestion in a micro wave oven (Mars 5, CEM, USA) according to different acid digestion mineralization protocols closed in polytetrafluoroethylene containers (PTFE, Teflon®) previously decontaminated with a 0.5% solution of HNO3. During the experiments, ultra pure deionized water (Milli - Q water, Merck Millipore) with resistivity of 18.2 Ω. cm-1 was used, both for all the dilutions and to rinse the microwave containers. All the reagents used to determine the microelements were analytical grade. An analytical balance was used to weigh the samples and to add the reagents before and after microwave treatment to assess the integrity of the seals and any loss of matter.

All the polypropylene (PP) consumption materials were decontaminated with a 0.5% solution of HNO.3 for at least 2 hours up to a maximum of 12 hours to avoid microelement contamination and overestimation.

Instrument settings (ICP / MS)

Microelement analysis was conducted using the ICP-MS (Inductively Coupled Plasma-Mass Spectrometry) system (Thermo Scientific X Series II, Fischer Scientific, Hampton, New Hampshire, USA) equipped with collision/reaction cell technology (CCT) and with an ASX 520 autosampler (Cetac Technologies, Omaha, NB, USA).

The digestion method applied was validated using certified reference material (CRM) from Trace Element in Spinach Leaves (SRM1570a, Sigma Aldrich, Milan, Italy).

The CRM 1570 A samples and the analytical blanks were also prepared in the same way and analyzed to detect any experimental losses or cross contaminations. Precision was assessed in terms of percentage coefficients of variation (CV%), which in turn were obtained by measuring the standard deviation relating to nine CRMs analyzed separately.

Metabolite analysis by UPLC/MS (Ultra Performance Liquid Chromatography/Mass Spectrometry)

Analysis of the bioactive molecules was analytically complex due to the large number of structural analogs, lability and low level in the samples of natural foods. UPLC/MS - qTOF (Time-of-Flight Mass Spectrometry) was used for a non- targeted metabolomic approach and a sensitive and simultaneous determination of the different folates in a particular matrix.

Extraction of whole metabolites from lettuce

The lettuce samples collected for bioactive compounds and analysis of the forms of folate were immediately frozen in liquid nitrogen and pulverized in a fine powder with liquid nitrogen in an Al 1 mill (IKA, Wilmington, NC, USA) and stored at -80 °C until analysis.

With the aim of not wasting significant metabolites and vitamins, whole metabolites were extracted using a protocol with the use of an extraction reagent prepared ad hoc. The extraction reagent, composed of methanol/water (MeOH/HzO, 80:20) and ascorbic acid 0.1% (p/v) to protect the folates during the procedure, was prepared fresh each time and left to extract the largest possible number of metabolites. An amount of 500 mg of pulverized lettuce was weighed in a dark Pyrex glass round bottomed flask where 5 volumes of reagent for cold extraction were added to each sample and placed in an ultrasonic bath with ice for 15 minutes. The extraction blends were then centrifuged at 4,500 rpm for 10 minutes at 4 °C and the supernatants were collected and stored in glass vial until analysis. All the chemical products used for extraction and analysis were of LC/MS grade of purity. All handling took place in dim lighting conditions.

Analysis of the forms of folate

Prior to analysis, the samples were diluted 1:2 with LC/MS grade H2O, filtered with PVDF 0.22 pm filter to be analyzed directly with UPLC/MS Xevo G2-XS Q-TOF (Waters, Milford, Massachusetts, USA), with ionization source of ESI (Electro Spray Ionization) type in positive (+) and negative (-) ionization mode. The standard and the mass-to-charge ratio (m/z) used to detect the folates were: m/z(+) 138.055 for 4-aminobenzoic acid (p-ABA) C7H7NO2, m/z(+) 442.147 for folic acid C19H19N7O6, purchased from Sigma Reagents (Sigma Aldrich, Milan, Italy), m/z(+) 474.173 for 5-formyl-5 6 7 8-tetrahydrofolic acid C20H21N7O7 (Ca), m/z(+) 460.194 for 5-methyl-5, 6, 7, 8-tetrahydrofolic acid C20H23N7O6 * 4H2O (Ca), m/z(+) 456.163 for 5,10-methylenetetrahydrofolic acid C20H22N7O6 (Cl) purchased from Schrick Laboratories (Schrick, Switzerland). All the compounds indicated were in any case detectable both in positive and in negative ionization mode, with the exception of p-ABA only detectable in positive ionization mode. All the standards were injected at concentrations of 1 ng/pl.

The reverse phase ultra performance liquid chromatography (UPLC) technique, Acquity I- class (Waters), was carried out in gradient conditions on a BEH C-18 column (100 mm x 2.1 mm; particle size 2.7 pm, Waters) at 30 °C with the FTN autosampler (Waters) refrigerated at 10 °C. The mobile phase comprised 0.1% of formic acid in water (solvent A) and 100% of acetonitrile (solvent B) and was pumped at a flow speed of 0.350 ml/min. The initial condition (99% A + 1% B) was isocratic for 1 minute. The proportion of B was increased linearly to 40% in 9 minutes, to 70% in 12 minutes, where it was maintained for 1 minute, followed by an immediate increase to 99% in 30 seconds. Then, for the remaining 6 minutes the elution was isocratic for 2 minutes to then return to 99% of solvent A in 10 seconds. Then, the mobile phase was adapted to its initial composition and maintained for 3 minutes for rebalancing. 5 micro liters were injected onto the column. The UPLC system was coupled with a Xevo G2 - XS mass spectrometer, equipped with electrospray ionization (ESI) source and quadrupole - Time of flight (qTOF). The parameters of the source were: capillary voltage 0.8 KV, cone voltage 30 V and source temperature 120 °C. Nitrogen was used both as nebulization gas, at a flow rate of 50 1/h, and as desolvation gas, at the temperature of 500 °C with a flow rate of 1000 1/h. The instrument was operated in positive and negative ESI mode with a collision energy of 35V and data were acquired every 0.3 seconds with a range from 50 to 2000 m/z.

Statistical analysis

The estimation data of the microelements were calculated starting from ICP (ion count per second) and expressed in pg/g of the dry weight. The spaces were used to estimate the limits of quantification (LOQ), represented by 3σ.

The forms of folate and the bioactive compounds were expressed as relative abundance of the peak area of each specific compound, in arbitrary units.

The matrix of the metabolomic data was prepared with the software Progenesis QI (Waters) and allowed the relative average of all the metabolites to be calculated.

An ample metabolite identification program was implemented, based on an “internal” mass library and for many metabolites putative identification was confirmed by the fragmentation pattern in ms/ms spectra and by comparison with authentic commercial standards.

The metabolites were explored differentially as follows: the abundant metabolites (Intensity> 2000 in the control samples) were selected and the abundance ratio of the metabolites between each of the treatment and control samples was calculated. A threshold of 1.5 was chosen arbitrarily to select the differentially accumulated metabolites in the samples treated, while the threshold of 0.67 was chosen arbitrarily for those metabolites that accumulated differentially in the control samples.

Moreover, a targeted approach in negative ionization mode was used for the three folates: folic acid, 5-formyl tetrahydrofolate and 5,10-methylene tetrahydrofolate, regardless of their differential accumulation in the samples.

All calculations were carried out using GraphPad Prism version 5 of the software (GraphPad Software, San Diego, USA). Analysis of the statistical data was carried out applying a t-test and were considered statistically significant data with p <0.05.

Results

ICP-MS analysis of the microelements of biofortified plants

The results on the profiles of the microelements in the plants treated with the biofortification composition of the invention, herein referred to as “Mix” (2000 ppm of p-ABA; 60 ppm of Na2SeO4; 4000 ppm of ZnSO4) are shown in Table 1:

Table 1

The “Ctrl” column gives the content of microelements of the lettuce sprayed only with water, hence the natural microelement content of the plant.

While the content of the other microelements was substantially stable after treatment with the composition Mix of the invention, with the exception of magnesium which exhibited an increase, the two microelements subjected to enrichment by means of biofortification exhibited significantly higher levels.

Fig. 1 shows the increase of the levels of Se and Zn alone, compared to the control plants treated only with water. The amounts of Se and Zn correspond to the values indicated in Table 1.

With regard to zinc, the lettuce was enriched by 47.5% compared to the untreated plant ((116.1 - 78.4): 78.4 x 100 = 47.5%).

With regard to selenium, the lettuce was enriched by 10 times compared to the untreated plant.

UPLC/MS analysis of the metabolites in the biofortified and control lettuce

Analysis of the forms of folate

In all the samples analyzed in positive ionization mode (expected m/z 460.194) 5-methyl- THF, detected m/z 460.193 and retention time 3.72 minutes was visible, and hence found. In the cultivation of lettuce in climatic chamber 5-methyl-THF was therefore present in an amount greater than 14.5% with foliar spraying of the composition “Mix” according to the invention compared to the control sample of lettuce “Ctrl”, sprayed only with water. The data are also shown in Fig. 2.

Analysis of the bioactive compounds

With the use of the software Progenesis QI it was possible to carry out, in negative ionization mode, non-targeted total analysis, from the raw data, of all the metabolites extracted in samples of biofortified and control lettuce. With this approach, 50 differentially accumulated metabolites were detected in the lettuce cultivated in climatic chamber.

It was thus possible to detect, moreover, that treatment with the composition of the invention “Mix” led to an increase in the pentose and hexose oligomers of inulin. Fig. 3 shows the significant increase of these oligomers.

Analogous results were obtained with above ground cultivation methods.

Therefore, it is evident that with the composition of the invention it is possible to obtain a folate enrichment in the plants treated, together with an enrichment of microelements such as Se and Zn, which play an important role in the metabolism of folates, and moreover to obtain an enrichment of other bioactive compounds such as inulin.