EP3271312A1 - Use of proanthocyanidins for limiting denitrification - Google Patents

Abstract

The invention relates to the use of at least one proanthocyanidin for limiting denitrification, consisting in the reduction of NO3 in N2O et N2, caused by microorganisms particularly present in an environment, by bringing said at least one proanthocyanidin into contact with said microorganisms.

Description

 The present invention relates to the environmental field, and more specifically to a means of limiting the denitrificadon intervening in the environment, especially in the soil, and more particularly the crop,

^Nitrogen is a limiting factor for plant growth (LeBauer & ïreseder, 2008). The development of plants therefore strongly depends on the processes of the nitrogen cycle which condition the transformation of nitrogen in all its forms including the availability of NH and G3, which are the assimilable forms of nitrogen by plants.

The atmospheric nitrogen is fixed in the soils and transformed into ammonium (H4 or more precisely MH ⁺ ) by fixing bacteria, another source of Nf is the mineralization of the nitrogenous organic matter, then the nitrification corresponding to the transformation of the nitrogen. ammonium nitrate (NO3, or more precisely NO3 ^' ) followed by the reduction of O3 in N ₂ O and Hz occur. Various publications have investigated the influence of condensed tannins or terpenes on nitrogen mineralization and nitrification. Adamczyk Bartosz et al: "Esponse of oneself! C and transformations to condensed tannins and different organic Necoodensed tannin complexes, vol. 64, January 8, 2013, pages 163-170, discloses that in soil samples, the addition of condensed tannins decreases nitrification, suggesting their toxic effect. and / or precipitating proteins, Ba! dwin IT et al: "Protein binding phenolics and inhibition of nitrification in subalpine balsam fir soils", vol. 15 n ^O 4 1 January 1983, pages 419-423, studied the effects on nitrification of a methanolic extract of forest soils. It is concluded that the condensed tannins present in the extracts studied are the components exhibiting the most important nitrification inhibitory activity. Adamczyk Sylvvia and have: "Potential response of self! to processes ^of sterpenes, triterpenes and tannins: nitrification, growfh of microorganisms and precipitation of proteins' _t flight 67, March 24, 2013, pages 47-52, describes the activity of terpenes and tannic acid, as well as condensed tannins , as inhibitors of nitrification in soil suspensions with an excess of MH4-N. RL Bradley et al. "Changes to ore N cyciing and microblal communities in black spruce humus after additions of (MH4) 2S04 and condensed tannins extracted from Kalmia angustifolia and basam fir", August 1, 2000, pages 1227-1240 reports that condensed tannins decrease nitrogen mineralization, by binding and sequestering organic sources of nitrogen. On unfertilized humus, a decrease in nitrate concentration is observed,

Among the processes of the nitrogen cycle, denitrification, mostly carried out by so-called denitrifying bacteria, which consists of the reduction of NO3 (nitrate) to H ₂ Q and 2, is considered as the main biological pathway of loss of nitrogen. In European agro-ecosystems, these losses represent 59% of the total losses of the system (Genema et al., 2009). Indeed, the gaseous forms of nitrogen, N3O and N¾ generated by denitrification, are released in the atmosphere and thus made inaccessible to plants.

 In a conventional way in agriculture, nitrogen losses caused by denitrification and responsible for yield loss are offset by the addition of nitrogen fertilizers. However, this contribution increases the emission of Η * 0, a greenhouse gas 380 times more potent than CC¾.

 Some solutions, based on synthetic compounds, such as dimethylpyrazophosphate (DMPP), have been proposed to temporarily limit losses by inhibiting nitrilation. These compounds inhibit the activity of nitrifying bacteria in the soil and consequently limit the transformation of the soil. ammonium (NHU) to nitrate (NO3).

Root and rhizome extracts from Failopla spp., An invasive plant complex, have been described as reducing the denitrification process of soybeans (Bardon et al, 2014). Bardon et al 2014 identified in this publication a number of metabolites present in the tested extract of Faiiopia spp. including (+) ofcechin, {+} - epiatechin., piceatanoi glucoside, resveratroloside, iemodine, physion, resveratrol and pictreide and show a correlation between catechin concentration in the extracts and ί Inhibition of denitrifioation.

 Subsequently, the inventors of the present patent application have tested the effect of catechin, as well as all the compounds identified in this publication (epicatechin, resveratrol, picone, emodyn and physcion) on the denltrification of bacterial strains. denltrifsantes and found, in a totally unexpected way, that the catechine itself, added to the other molecules identified as mainly present in the extract, had no effect on denitrification,

 The inventors then demonstrated that other molecules, not yet identified in this publication, proanthocyanidins were responsible for the inhibition of the denervation observed with extracts of Faliopsa x bohemica. In addition, these compounds do not affect the activity of the enzymes of respiration, an important function for the growth of soil microorganisms.

 In this context, the present invention relates to the use of at least one proanthocyanidin to imitate denitrification caused by microorganisms by contacting said at least one proanthocyanidin with said microorganisms.

The microorganisms that cause the denitrifying activity that is reduced in the context of {nvention are most often present in a medium. When used according to (Invention, the microorganisms will then be present in the medium with the said at least one proanthocyanidin, in which case the proanthocyanidin will be placed in contact with the medium and, consequently, with the microorganisms to be killed. In this case, the contact between proanthocyanidin and microorganisms will be due to a proanthocyanidin diffusion phenomenon in the medium The medium (with which proanthocyanidin is brought into contact, called the treated medium) may be a solid medium, such as soil, and in particular crop soil, compost, manure, soil, compost, sewage sludge, sediment or a liquid medium, such as an aqueous medium such as river water, water wastewater, wastewater, without this list being exhaustive. Under [Invention, the contacting of at least one proanthocyanidlne with the medium of interest., Can be performed by any suitable technique ,, _", particularly by applying the proanthocyanidlne on said medium or by mixing the proanthocyanidin with said medium. Contacting time with the microorganisms to the effect of said microorganisms on ocyanidlnes prooant corresponds to the desired period for ^obtaining the effect limiting dénitrlficatlon.

In the context of the invention, one or more proanhocyanidine (s) are (are) used to limit the denitrlficatlon caused by microorganisms normally having the capacity to reduce O3 (nitrate present in the treated medium) in f¾0 and In particular, such microorganisms possess the genes coding for nitrate and nitrite reductases. Such microorganisms include fungi such as Cylindrocarpan tonkmense _? Fusanum oxysporum or bacteria of the Pseudomonas genera Badlius _f Burkholderia, Achromobacter, Ochmbadrum, Citmbac er Paraeoceus, Micrococcus, such as the species Pseudomonas brassicacearum, Pseudomonas stutzeii, aicaligenes Pseudomonas, Pseudomonas fiuoresœns, Burkholderia vietnamiensis, Achromobacter xyiomxfdans, Parawcws denitrihcans, Badilus oers, known to be denitrifying bacteria. These bacteria are most often present in soils,

The contacting with the microorganisms is carried out so as to obtain a reduction in the denlthfication caused by the microorganisms having a denitrifying activity, or in other words so as to at least partially inhibit the denitrifying activity of the microorganisms. In the context of the invention, such a limitation, reduction or inhibition is to be considered, with respect to the emission of 20 and ¾ and / or the denitrilfication (transformation of nitrate O3 or more precisely N <¼ ^" into N 3 and f ) which would be obtained in the absence of contacting the microorganisms concerned with said proanthocyanidin. To evaluate the percentage of inhibition of denlnflcation, the denitrlficatlon activity is obtained by measuring the emission of ½G -f H _2f d one hand in the presence and, on the other hand, the absence of proanthocyanldine, which allows to obtain a percent inhibition of the denitrification corresponding to (transmission ₂ 0 + H ₂ in the presence of proanthocyanidin) / (emission 2O + Nfe absence of proanthocyanldine) * 100 . In the context of (Invention, the inhibition of denitrification by proanthocyanidines can in particular reach a maximum level of 100%, and in the context of the invention will be obtained the% dinhsbitlon denitrification, generally from 20 to 100 %, preferably 50 to 100%, The technique described in Dambraville et al., 2006, and used in the examples, can be used to evaluate U ₂ -U 2 uptake. the amount of proanthocyanidin (s) used will be adjusted by those skilled in the art to achieve the desired effect.

 The proanthocyanidins, also known as proanthocyanidic tannins, condensed tannins, or proanthocyanidols, are fiavonoids, and more specifically polymers of hydroxyflavan-3-ol. The hydroxyflavan-5-ol monomers constituting the proanthocyanidines have the following structure (I). ):

in which R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ , which are identical or different, are chosen from hydrogen, hydroxyl, gallate, methyl or oside groups.

These monomers are therefore more or less hydroxylated on the rings A and 8, ranging from 0 to 3 hydroxyl groups. These monomers, hydroxyflavan-3-ol, may also be esterified with one or more gallate and / or glycosylated and / or methylated groups on the rings A, 8 and C There are two main types of proanthocyanidins, both of which can be used in the context of the invention, these two types being defined by the bonds between monomers: proanthocyanidins type A and proanthocyanidins type 8, proanthocyanidins type B which are preferred in the context of ^the invention, are polymers hydroxyffevan d ^f ~ 3 ~ Oi which two consecutive monomers are linked by a single covalent bond between two carbons, depending on the positions 4-6 or 4-8 (respective positions in both monomers), as shown in the formula corresponding to a dimer comprising a 4-8 linkage, followed by a 4-6 linkage:

 (II)

with R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ , R 1. R ² ', R ^y and R ^6', R ^r, R, R ^{3 ",} R ^4", R ⁵ "and R ^6" may be the same groups or different groups representing hydrogen, a hydroxyl group a gallate group, a methyl group or a saccharide group, The proanthocyanidins of type A are polymers of hydroxyflavan-3-ol of which two consecutive monomers are connected by two covalent bonds between two carbons, at position 2-7 and 4-8 (respective positions in the two monomers), illustrated in the case of a dimer by formula (III) below i

(III) with R ^1, R ² _f R ^3, R ⁴ R ^s and _{f ^6;} R ^r , R _f ^{4 '} , R ^5' and R ^{6 '} , which may be the same groups or different groups, representing hydrogen, a hydroxyl group, a gailate group, a methyl group or an osidic group.

 The proanthocyanidins which can be used in the context of the invention consist of 2 to n monomers (hydroxyflavan ~ 3 ~ ol) connected by covalent bonds previously described, according to all the possible variations. In particular, within the scope of the invention, one or more proanthocyanidins chosen from among the dimer, irimer and tetramer will be used.

The most well known proanthocyanidins are polymers of (- / +) - catechin, (7 ÷) epicatechin., (- / - f-) ~ gallocatechin and (- / ÷) - epigallocatechin. In the group of proanthocyanidins are found subgroups of molecules, such as, for example, prodelphinidines. (prodeiphinidoi) or procyanidins (procyanidol). Procyanidins correspond to polymers of (- / -) ~ cathehine and / or {- / - * ^■ } - epicatechin. The prodelphinidins of (- / -) ~ galfocatechin polymers and (~ / -f) -epigalfocaté hlne, Par {- · / +}, denotes {+) or {Isomer {-} or a mixture of these isomers »

Proanthocyanidins are natural products which can, in particular, be obtained and purified from extracts of Faifopia spp., And in particular Falhpia x bohemka as evidenced by the inventors, or else from Vitis vinifera seeds, Pteridium spp. VMdum spp. Schfûops / s spp ,, Vicia spp _v C / 5 spp., Quercus spp ,, many commercial proanthocyanldines are available, especially in LAF OT (Bordeaux, France), Organic Herb Inc. (Changsha , China) Nanjing Zelang Medical Technology Co., Ltd. (Jiangsu, China),

 In the context of [the invention, use will preferably be made of proanthocyanidins of type B, and even more preferably procyanidins of type 8, in particular in the form of dimer, trimer or tetramer,

 According to particular embodiments of the invention, said at least one proanthocyanidin used is not in the form of an extract of Fafiopia spp., Containing proanthocyanidins, and in particular Faiiopia x bohemica, and / or said at least one proanthocyanidin. is used, in the absence of cathehine and / or epicatechin,

 In the context of the invention, the proanthocyanidin (s) used may be applied to the medium containing the microorganisms whose denitrification effect is to be limited, or mixed with the latter, in the form of an aqueous solution or suspension or still in the form of a powder or granule,

Embodiments in which microorganisms are present in soil will now be described in more detail. In the context of the invention, at least one proanthocyanidin may, in particular, be used to promote plant growth on a culture soil, by reducing denitrification caused by microorganisms. Having a denitrifying activity, and in particular caused by denitrifying bacteria present in said soil Proanthocyanidins being natural products, their use in crops is in total respect with the environment Proanthocyanidins can therefore be used in organic farming.

 In the context of the use according to the invention on a soil, at least one proanthoeyanidine is applied to a soi, and in particular a culture soil, for example by deposition or spraying on the ground.

 In particular, said at least one proanthoeyanidine is used to limit denitrification caused by soil bacteria, such as Pseudo onas brassacacearum and / or Sac us cernas,

 In the case of a solution of proanthocyanidin (s), it may be applied by spraying or any other appropriate means. The soils that can be treated in the context of the invention include, inter alia, cultivation, market gardening, cereal growing, viticulture and more generally, soils where various plants of agronomic interest, including various plants or flowers , are grown.

 The effect of the proanthocyanidin (s) used on denitrification will be obtained over a wide range of temperatures, especially at a soil temperature of 0 to 5 ° C. and with an optimum effect at a moisture content greater than 50% of the field capacity.

 An effective amount of proanthocyanidin (s) to achieve the desired denitrification limitation will be applied to the soil. The amount applied will be adapted by those skilled in the art, in particular, depending on the formulation and the soil moisture content. . In particular, from 0.01 to 10 mg / g of dry sol, and preferably from 0.05 to 5 mg / g of dry sol, of proanthocyanidene (s) is applied. These amounts are applicable to solid media, other than the self, mentioned above, in which the microorganisms could be present.

The results presented in the context of the invention show that proanthocyanidins, and in particular type B procyanidins, make it possible to reduce the process of bacterial denitrification of soils. The results were obtained on strains of bacteria in culture, but also on ones of different natures (one of river edge and grassland soil) and containing communities of complex and different denitrifying bacteria,

 The use according to the invention thus makes it possible to limit nitrogen losses by denitrification and, consequently, greenhouse gas emissions, and also to reduce the need to use nitrogen fertilizers in agriculture. Nevertheless, the use of proanthocyaoidin (s) may be associated, in the context of the invention, with one or more other fertilizers (nitrogen or phosphated, for example).

 In the context of the invention, at least one proanfhocyanidine may be applied to a culture soil, to promote the growth of said culture against denitrification caused by microorganisms, and in particular bacteria, present in said soi. Said application may be made without the joint application of a nitrogen fertilizer.

 As regards now, the embodiments in which the microorganisms are present in a liquid medium, a composition in solid or liquid form will be integrated in said medium. An effective amount of the ocyanidine proant (s) to achieve the desired denitrification limitation will be incorporated into said medium. The amount incorporated will be adapted by those skilled in the art. By way of example, from 0.01 to 2.5 mg of proanthocyanidin (s) per milliliter of liquid medium, and preferably from 0.01 to 1 mg of proanthocyanidin (s) per milliliter of liquid medium will be incorporated.

 The examples below, with reference to the appended figures, illustrate the invention, but are not limiting in nature.

Fig. I: Percent inhibition (relative to control) on the denitrification by Pseudomons brassicacearum of a mixture of the major compounds present in the alkaline bohemia and the Faptapta x bo emica extract. The vertical bars correspond to the variations obtained on the different tests. The stars indicate the significant variations relative to the control (Tukey test, α <0.05). Figure 2; Chroroatograrnnie at 280 nm extract Faiiopia x bohemica showing proanthocyanidins and their monomers detected as well as their UV spectrum.

Figuras 3A to 3 €: ESi positive mass spectra of proanthocyanidins of type 8 found in Fallopila x bohemica (Dlmers (F gyre 3A) _f Trimers (Figure 38), Tetramers (Figure 3 £).

 Figure 4: Percentage inhibition of denitrification and respiration of a) Pseudomonas brassileacearum and b) E cereus as a function of concentration of purified proanthocyanidins »Stars indicate significant differences in means with control without proanthocyanidins (Tukey test a <0.05).

Figure S: Percentage inhibition of denitrification of river (Ainii) and grassland (Αΐηδ ^' ) soils as a function of commercial proanthocyanidin concentration. The stars indicate significant differences in means with control (Tukey test, <0.05).

Figure 6; ^Percentage Inhibition of denitrification prairie soil AINL i after 7 days of incubation (to field capacity) e presence of different amounts of commercial proanthocyanidins. The stars indicate significant differences in means with control (Tukey's test, a <0.05).

 F g re 7 t Percentage inhibition of potential nifrification in the soil of Ain il (triangle) and that of A! N6 (round) as a function of proanthocyanidin concentration. The bars indicate standard errors.

 L MATES IELS AND METHODS

 A) - BIOLOGICAL SOURCE OF PROANTHOCYANIDINES

 - PREPARATION OF EXTRACTS OF FALLY SPP,

Compounds were extracted from roots and rhizomes of the genotype F. x bohemica (Chrtek and Chrtkov). The rhizomes collected in the field were planted in pots in compost (lasmann 153 / sand, 80/20). cultured for six months in a greenhouse, under artificial light at 21 ° C The rhizomes and roots produced were harvested, cleaned with water and freeze-dried. Sixty-five grams of rhizomes and lyophilized roots ground using a TissueLyser it bead millet! (Qlagen Venlo, Netherlands) were suspended in 750 ml of water: methanol (50/50, v / v) and sonicated for 30 min. After filtration using a vacuum pump, the eluate was collected, and another extraction was carried out on the residue with 2 x 500 mL of pure methanol. All the eluates were then mixed and dried. these dry extracts were re ~ suspended in a water mixture; methanol (50/50, v / v) at a concentration of 10 mg ml ^-1 .

 - IDENTIFICATION OF PROANTHOCYANITIS CONTAINED IN THE EXTRACT OF E X 80 EMICA;

 The extract was analyzed using a UHPLC 1290 infinity series apparatus (Agitate Technologies, Santa Clara, USA) coupled to a G4212A DAD diode array UV detector (Agitate Technologies, Santa Clara, USA) and Q-TGF 6530 series mass spectrometer (Agllent Technologies, Santa Clara, USA),

Separation of compounds was performed on a column Porosheli 120 EC-C18 (2.7 _f 3.0 pm x 100 mm, Agllent Technologies, Santa Clara, USA) at a temperature of 60 ° C. The mobile phase was a linear gradient of 0.4% acetic acid in water (solvent A) and acetonitrile (solvent B). The mobile phase passed through the column at a flow rate of 0.6 ml / min. The linear gradient was from G to 1.5 min 2% solvent B; 1.5 to 22 min 2% to 17% solvent B; 22 to 41 min 17% to 1.00% solvent B; 41 to 41.5 min 100% solvent B, the system was previously balanced before each sample, the injection volume was ΙμΙ.

UV spectra were recorded between 190 and 600 nm and the 280 nm response was used for qualitative interpretations. The ESI source was optimized as follows: Positive Ionization mode, 80/2000 m / z spectra scan, 3.0 kV capillary voltage, 70 V fragmentor, Collision-induced dissociation (CID) collision ) was set at 20 eV. Nitrogen was used as a gas nebiller with a flow of 12 l / min and a temperature of 310 ° C to 40 psi. Proanthocyanidins were identified in the extract by UV, HS and MS / MS spectra analysis using the MassHunter Qualitative Analyst software (Agitant Technologies, Santa Clara, USA), the spectra obtained being shown in Part II B).

 - PURIFICATION OF PROANTHOCYANIDINES. FROM E x SOHEMICA

The proanthocyanidins contained in the E x bohe ica extract were purified using a Sephadex LH20 column. Sephadex LH 20 was previously equilibrated three times by wetting 25 g of Sephadex LH 20 with 100 ml of 80% ethanol and then removing the supernatant. The extract E x bohemica (100 ml) concentrated at 10 mg ml ^-1 then added to the equilibrated Sephadex LH and stirred for 3 min. Extracted Sephadex was loaded into a 10 ml glass buchner. Sephadex LH 20 was washed with ethanol. 95% until the absorbance of the water at 280 nro is close to zero. Sephadex LH 20 was then washed with 50% acetone until the Sephadex was completely washed. The 50% acetone eluate recovered was evaporated using a rotary evaporator. The dried proanthocyanidines, thus purified, were subsequently dissolved in 10 mg.mf ¹ in water, this solution being subsequently called purified proanthocyanidines. This solution contains the dimer, trimers and tetramers identified in FIGS. 3A and 38, but not the monomers catechin and epicatechin,

 s) - COMMERCIAL SOURCE OF PROANTHOCYA DIES

 It is proanthocyanidins purified from grape seeds (Bîotan, Laffort, Bordeaux, France). UHPLC analysis made it possible to verify that these proanthocyanidines were not mixed with catechin or with epicatechin,

 IT EFFECT OF PROANTHOCYANIDINES ON DENITRIDY:

 A) - TESTING THE EFFECT OF THE MAJORITY COMPOUNDS CONTAINED IN THE EXTRACTS OF E X BOHEMICA

The effect of catechin, epicatechin, picteide, resveratrol, remodin and physeion, identified by Bardon et al (2014), as the major components of the extracts of Failopia x bohemica, were tested in mixture on the denitrification activity of Pseudomonas brass / cacearvm, as described in the following paragraph. From pure commercial products (Slgma-Aldch, Saint Louis, USA), all these compounds were prepared as a mixture in a methanol solution / water (v / v, 50/50) at the concentration found in the extracts of Faliopia x bohemica The concentrations of these compounds exposed to bacteria in the medium are 0.0037 mg ml ^{* 1} catechin, 0.0057 mg mi ¹ epicatechin, 0.022 mg ml- ¹ pictide, 0.0007 mg of ^1- resverratrol, 0.0015 mg ml- ¹ emodin and 0.0047 mg ml- ¹ physcion),

 B> - BACTERIAL STRAINS DEN TiRiFI ANTES

^The effect of proanthocyanidins purified from F. x bohemica extracts was tested at concentrations of 0.1; 0.05; 0.01 or 0 mg.ml- ¹ on the denitrification and respiration of Pseudomanas brasscaœaru NFH421 and Badllus œreus A19, two denltriflant bacteria frequently found in soybeans, Four this, 5 ml of MB liquid growth medium with 20 mM NO.sub.2. , containing the different concentrations of proanthocyanidins, was inoculated at an optical density of (OD) ~ 0 L The denitrification measurements were carried out in plasma bottles sealed by a rubber stopper, by the assay of the accumulation of f¾Q This was done after replacing the air in the flasks with a mixture of helium and acetylene (He / Hs, 90/10). Each of the treatments was carried out in four replicates. to block the transformation of N ₂ 0 in _2f the measurement of f émissionG emission being then recognized as representative of emission of ₂ 0 and H ₂ (Dambreville et al., 2006).

Respiration measurements were performed in sealed plasma bottles by CO 2 accumulation assay _. without replacing the atmosphere of the flasks.

The N ₂ O and C0 ₂ emitted were measured every two hours for 48 hours using a microcatheter (pGO 30Q0 _f SRA instruments, Marcy tolle, France).

 C) - COMPLEX COMPLEXES OF SOIL IN SUSPENSION

In order to test the effect of proanthocyanidins on the deification of complex communities present in the soil _, proanthocyanidines commercial purified from grape seeds were used (Laffort, Bordeaux, France). Samples of two soils were collected at the Ainl sites (45 ° 57 ^ί 53 ^ίί , 5 ^Ό 15'25Έ) and ΑΙηβ (45 ° 48Ί5 "Ν, 5 ° 10 ^* 29" E) and sieved at 2 mm. the déûitrifîcatlon rate was measured emission% G every two hours for 30 hours under anaerobic conditions with a micro- cathanomètre (P6C-R30Û0 _f SRA instruments., Marcy l'Etoile, France). One gram of dry equivalent sol was placed in a 50mm plasma flask sealed with a rubber stopper. The atmosphere of the flask was replaced by a He / CiH mixture _? . mixture (90/10 v / v). The sols were suspended in 10 ml of 0.1M phosphate buffer (MaH 2 PG and NaHPO 3 pH 7.5) supplemented with glucose (0.5mg of C-glucose ¹ ), glutamic acid ( 0.5 mg of glutamic C-atide.mr ¹ ) and N £ ¾ (20 mM). The effect of proanthocyanidins on denitrification was measured for concentrations of 2.5; i; 0.5; 0.1; 0.05; 0.01 and 0 mg.ml ^* * in the end, adding water or proanthocyanidins to the soil suspensions. The soil suspensions were stirred at 28 ° C throughout the experiment. All treatments were performed in four replicates.

 D) - EFFECTS OF PROA THOCVA ÎDINES ON A COMPLEX FLOOR COMPLEX REPORTED IN MASS OF PROANTHOCYANIDINES IN RELATION TO A MASS OF DRY SOIL

A range of active proanthocyanidin concentrations on soil denitrification reported in g of dry soil was carried out. The soil used for this experiment is the so-called Ain IL. Ten grams of dry equivalent soil were incubated in a plasma bottle in the open air for? days at 28 ° C to field capacity, the equivalent of 10; 5; 1; 0.5; 0.1; 0.05; 0.01 and mg. g ^"1 of commercial proanthocyanidin solids were applied during humidification to the soil field capacity Each treatment was carried out in four replicas After 7 days, the denitrification measure of the treated soils was carried out by measuring emission of i¾u anaerobically every 30 min for 6 hours using a microcatharometer (pG € - 3000, SRA instruments, Marcy l'Etoile, France), the plasma bottles were sealed by a rubber stopper, l atmosphere of the flasks has been replaced by helium using a bench. The soils were supplemented with a solution of 0.5 ml containing the equivalent of 50 μgN-NCh.g- ¹ sol dry, 0.5 mg of C-aclde glutarnique.g ^"1 dry soil and 0.5 mg of C-glucose.g ^"1 self sec.

IIL RESULTS

 A) - EFFECT OF MAJORITY COMPOUNDS ON THE DENITRIFICATION OF

PSEUDQMQNÂS 8RA $ STCACEARUM

The dénitrlficatlon of Pseudamo s èrassicacearu is not Inhibited by first mixing the major compounds (catéchlne., Epicatechin ,, piceid, resveratrol, emodin and physcion) present in extracts Faiïopia x ùohemÎcs, whereas ^a marked inhibitory effect is obtained with the extract Failopia x ho emiœ for the same concentration of these compounds, as shown in Figure I. It thus appears that the inhibitory activity obtained does ^not involve these compounds ,.

 8) - IDENTIFICATION OF PROANTHOCYANIDINES CONTAINED IN THE EXTRACTS OF F. X BOHEMICÂ

Proanthocyanidins detected by UHPLC in ^extract F, x bo EMICA are Type B procyanldines ranging from d mother téfcramère of catéchlne and epicatechin with a absorbaoce maximaie at 278 nm and molecular weights of 579.15, 867.21 and 1155.27 g mol ^-1 , as shown in Figures 2 and 3 (the spectrum of Fig 3 corresponds to the dimer, the spectrum of Figure 38 to the trimer and the spectrum of Figure 3C to the tetramer).

 c) - EFFECT OF PURIFIED PROANTHOCYANIDINES ON DENETRAFICATIGN

BACTERIA DENIXR l FI AN T E S

It appears that the purified proanthocyanidins significantly inhibit dénitrlfication Pseudomonas brassicacearu and B cereus _t as the results shown in Figure 4, for the two bacterial strains ,, denitrification is affected up to 80%. The response to inhibition is dose-dependent between 0.01 and 2.5 mg. D) - EFFECT OF COMMERCIAL PROANTHOCYANIDINES ON SOIL DENITRIFICATION IN SUSPENSION

It has also been observed that commercial proanthocyanidins tested significantly inhibit the denitiation of the two suspended soils (Figure S). The inhibition is dose-dependent with a maximum ^of inhibition reached in 0.5 mg.mi ^"1 for soil Άίηί! And 1 nig. Mi ⁱ for self Ain6. The maximum inhibition was 85% for 80% for soil Αίηδ. The lowest significant effect measured was at a concentration of 0.01 mg.ml ¹ ,

 E) ~ EFFECTS OF PROANTHOCYANIDINES ON A COMPLEXITY OF SOIL COMPLEX IN THE MASS OF PROANTHOCYANIDINES IN RELATION TO A MASS OF

DRY SOIL

Commercial proanthocyanidins significantly inhibit denitrification Âînll soil after 7 days of contact with said proanthocyanidins (Figure S), the inhibition is dose-dependent with a maximum of 100% of inhibition reached 5 mg.g ^'1 dry soil. The smallest significant effect was at a concentration of 0.1 mg ^/ g ^, of dry soil,

 F) - Effect of proanthocynidines on the diet

The potential nitritlcafion of the two soil types (AM I. and Αίηβ) was measured in suspension (1 g of dry equivalent sol per 10 ml) without proanthocyanidins (control) and in the presence of commercial proanthocyanidins, concentrated to 0.5; 0.1; 0.05 and 0.01 g ^/ cm 3 in phosphate buffer solution at soil pH 7.5 (NaHiPC 2 and NaHPG ₂ , 12Η20) (to limit pH changes during incubation) supplemented with (NH SGs (SO g.mL ^"1). soils were placed in 150 ml flasks plasma aerobically blocked with psrafilm, flasks were set stirring at 140 rpm at 28 ^e C" Each conditions were realized in 4 replicas.

Nltrifscation potential measurement (PBN "nltrifylng enzymatic actlvlty"} was performed by assaying the production of NC¾ over time using an ionic ^dpf chromafographe Dionex DX 120 (Thermo Fisher Sclentific, Waltham, USA), in removing and filtering at 0.2 μm, 1 ml of suspension every 2 hours between 2 and 10 am The measurement of potential nitridation corresponds to the extent enzymatlque pool present in the sample initially, and is expressed by the production pg N- C .g ^"dry soil. fi ¹ between 2 and 10 hours.

The results are presented Figyre 7. These results show that in the same concentrations tested ia denitrification, proanthocyanidins ^do not have a significant effect on the nitrifeation.

Bibliographical references:

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Claims

1 - Use of at least one proanthocyanidlne to limit denitrification, consisting in the reduction of NO3 2O and N _2, caused by microorganisms, by contacting said at least proanthocyanidlne lesdlts with microorganisms,  2 ~ Use according to claim I characterized in that said at least one proanthocyanidin is a type 8 proanthocyanidin>  3 - Use according to claim 1 or 2 characterized in that said at least proanthocyanidine consists exclusively of monomers selected from cathehine and epicatenol.  4 - Use according to any one of claims 1 to 3 characterized in that said at least one proanthocyanidine is a dimer, trimer or tetramer.  5 - Use according to any one of claims 1 to 4 characterized in that the microorganisms are present in a medium in which said at least proanthocyanidine is: present.  6 ~ Use according to claim 5 characterized in that the medium is a sol and Tau at least one proanthocyanidine is applied to said soi, for example by deposition or spraying,  7 - Use according to claim 6 characterized in that from 0.01 to 10 mg / g of dry sol., And preferably from 0.5 to 5 mg / g of dry sol, of proanthocyanidine (s) is applied to said ground  S - Use according to any one of Claims 1 to 7, characterized in that the said at least one proanthocyanidin is used to limit the denmation caused by denitrifying bacteria, in particular chosen from Pseudomonas brassicaœarum, Pseudomonas stuizen, Pseudomonas afcigenes, Pseudomonas fufuosens, Butkholderia vietnamiensis, Acroxylosoxidans, Paracoccus denitriflcans, Badifus cereus, - Use according to any one of claims 1 to 8 characterized in that said at least one proanthocyanidin is not in the form of an extract of Fallopia spp. containing proanthocyanidins. 10 - Use according to any one of claims i to 9 characterized in that said at least proanthocyanidin is used in the absence of cathehine and / or epicatenol.