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    • 专利摘要:
    Bacillus amyloliquefaciens bacterial strain QVI5 (CECT 9371), microorganism of the group of Gram + bacteria, Bacillus genus, stimulant of secondary metabolism of phenolic compounds, improver of fruit extracts and raspberry leaf and strawberry on the enzymes related to the regulation of blood glucose (alpha glucosidase), hypertension (ACE-angiotensin-converting enzyme), and inflammation (cycloxygenase COX2). This strain has been isolated from the rhizosphere of Pinus pinea, on nutritive agar (PCA), and has been characterized from the morphological, biochemical and genetic point of view by sequencing the 16s gene. It can be used in order to improve the properties of the extracts with respect to their application on enzymes related to the metabolic syndrome, or in order to modify the secondary metabolism to improve the phenolic compounds in plant species of agronomic, pharmacological and food interest, and obtain more active ingredients and/or new foods with a content standardized in phenols. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2694593A1
    申请号:ES201730818
    申请日:2017-06-21
    公开日:2018-12-21
    发明作者:Enrique GUTIERREZ ALBANCHEZ；Francisco Javier GUTIERREZ MAÑERO；José Antonio LUCAS GARCÍA；Beatriz Ramos Solano
    申请人:Fundacion Universitaria San Pablo CEU；
    IPC主号:
    专利说明:

    Bacillus amyloliquefaciens QV15 stimulant of secondary metabolism ofphenolic compounds and the inhibitory capacity of the extracts of5 raspberry and strawberry on the enzymes related to the metabolic syndrome.
    The present invention relates to a strain of Bacillus amyloliquefaciens (QV15, internal laboratory code) for application in plants with the aim of improving the synthesis of phenolic compounds of secondary metabolism with interest
    10 agronomic, pharmacological and nutritional, specifically, improve the color in strawberry and raspberry fruits, in addition to improving the properties of raspberry and strawberry extracts on the inhibition effect on alpha glucosidase, ACE and COX2 for improvement of symptoms or prevention of the metabolic syndrome.
    15 This strain, which when it was isolated was assigned the internal reference L81, has been deposited for patent purposes in the Spanish Type Culture Collection (CECT), dated May 31, 2017, where it has been assigned the number 9371 The CECT is located in the research building of the University of Valencia, located on the Burjassot campus (DP 46100 - Valencia, Spain).
    20 This bacterial strain can serve as a basis for the preparation of different types of products that stimulate the secondary metabolism of plants. of agronomic, pharmacological and food interest, and obtain a greater quantity of active ingredients and / or new foods with a content standardized in phenols, such as fruits of the forest (strawberries and
    25 raspberries) with higher content of phenolic compounds, specifically anthocyanins, improving coloration and ºBrix. These products will improve the content of phenolic nature bioactives that may constitute active ingredients of various medicines, improve the quality of certain foods. In addition, it may be used to improve the properties of blackberry, raspberry and strawberry fruit extracts on the
    30 inhibition effect on alpha glucosidase and COX2 for improvement of metabolic syndrome.
    TECHNICAL FIELD.
    35 The invention falls within the fields of biotechnology, pharmacology and new foods.


    STATE OF THE ART.
    The mechanisms of action of the bacteria that promote plant growth can be summarized in two types: direct, when the metabolites produced alter the
    5 plant metabolism (hormonal activity, stimulation of mechanismsdefensive ..), and indirect, when they synthesize compounds that facilitate the uptake ormobilization of nutrients or prevent the growth of pathogenic microorganisms withoutinvolve the plant, without altering the metabolism of the plant.
    10 In this case, one of the direct mechanisms is of interest, that is, those that alter the metabolism of the plant. The plant has a secondary metabolism, highly inducible, related to the defense of the plant and adaptations to adverse situations, which it has to face. Within this secondary metabolism is the metabolism of phenolic compounds, which besides
    15 are related to the defense of the plant, are of interest for human health, both when they consume foods of plant origin that contain them naturally, and plant extracts dedicated to nutritional supplements. They are also important as a source of active ingredients for obtaining medicines. The source of carbon skeletons to nourish the secondary metabolism is the
    20 photosynthesis, and any mechanism that affects this process, will affect the health of the plant.
    Bacillus amyloliquefaciens belongs to the group of Gram + bacteria. The genus Bacillus is common among soil bacteria, and may be opportunistic pathogens in animals and plant pathogens. Following the taxonomy of the Bergeys Manual, edition
    March 25, 2001, this bacterium falls within the Bacteria Domain, Phylum Firmicutes, Bacilli Class, Order Bacillales, Family Bacillaceae, Genus Bacillus, Species B.amyloliquefaciens. The genus Bacillus is very common in the edaphic system, and has been described repeatedly as a protective bacterium against different plant diseases. Can produce non-fluorescent siderophores
    30 catechol, which, among other functions, act as molecules capable of capturing the iron of the medium for the metabolism of the microorganism The presence of Bacillus sp. in the rhizosphere of different plants it affects in a beneficial way its physiology indicating that it is very possible its selection at rhizospheric level.
    There are numerous references in the scientific literature that cite strains of the genus Bacillus as capable of carrying out numerous activities of interest in the


    field of biotechnology, agriculture, and plant pathology. In the field of biotechnology, there are studies i) on their role as biological indicators for sterilization, in biodefense studies, ii) on their influence on the primary metabolism of the plant, increasing its growth and production, iii) as agents of disinfection, iv) as antimicrobial agents for their ability to produce antimicrobial molecules of the polyketide or lipopeptide type. In the field of agriculture and phytopathology there are numerous references on the ability of Bacillus amyloliquefaciens to induce plant defenses; On the other hand, there are strains capable of producing chitinases and glucanases, directly protecting against Alternaria and Fusarium fungi. There are also references to strains of the genus Bacillus able to protect against saline stress and against the foliar pathogen Pseudomonas syringae DC3000 (Barriuso et al, 2008 Phytopathology), but none of Bacillus amyloliquefaciens. Finally, there is no strain of Bacillus or Bacillus amyloliquefaciens to modulate the biosynthesis pathway of phenylpropanoids, flavonoids and anthocyanins, increasing the concentration of polyphenols, specifically
    flavonols and anthocyanins.
    On the other hand, it is widely known that extracts of vegetable origin rich in flavonoids, anthocyanins and other phenolic compounds, have a high antioxidant power beneficial to health. Extracts from blueberry species have been cited frequently as healthy products because of their antioxidant capacity, neurodegenerative prevention, avoiding loss of bone mass, coronary prevention and anticancer effects. Berry extracts, both wild berries and commercial species, have also been related to hypoglycemic activity, inhibition of adipogenesis, improvement of risk factors for cardiovascular diseases, anti-inflammatory capacity and ability to induce satiety and counteract overweight. A recent article by Lila, M.A. (Functional Foods in Health and Disease, 2011, 2: 13-24 Page 13 of 24), discusses the impact of bioflavonoids from berries on metabolic syndrome biomarkers, 30 associated with conditions of diabetes, overweight or obesity and diseases
    cardiovascular
    Within this field of extracts of plant origin with beneficial health qualities, two other recent articles by prestigious researchers, 35 Sharma, Kumar (Journal of Diabetology, June 2011; 2: 4) and Kaume, Howard, Devareddy
    (J.10 Agric. Food Chem. 2012, 60, 5716-5727), reflect well the state of the art


    Closer to the object of the present invention, in the sense that the fruits of the blackberry (Rubus sp.var Loch Ness), due to their high levels in the aforementioned phenolic compounds, have been related, among other functions, to the hypoglycemic activity , and the ability to induce satiety and counteract overweight. Of
    In fact, the article by Sharma and Kumar, deals with the antidiabetic effect of extracts of fruits of Rubus ellipticus in mice with diabetes induced by alloxan, although it is noteworthy the absence of studies in healthy mice and normal animals.
    Well, following this line of research, the inventor team has
    10 managed to prepare methanolic extracts from strawberries and raspberries obtained from plants treated with Bacillus amyloliquefaciens QV15 during the entire production cycle. These extracts are perfectly characterized in their composition and antioxidant power and have been shown to have a greater ability to inhibit the enzymes alpha-glucosidase, ACE and COX2, which makes them potentially useful in the
    15 preparation of food preparations and drugs for the prevention and improvement of symptoms associated with metabolic syndrome.
    Performing a retrospective search of patents worldwide in the Spanish production database Invenet (OEPM) and in the international Worlwide, through the Esp @ cenet system, this conclusion drawn from the scientific literature consultation is confirmed: although that there is a similar reference with a strain of Pseudomonas fluorescens N21.4 (ES 2 336 758 B1), the lack of patent documents relating to the Bacillus amyloliquefaciens bacterial species that support the ability to stimulate secondary metabolism, specifically the route of
    25 phenylpropanoids, flavonoids and anthocyanins. In this way it is able to modify the metabolic profile of the plant, and, therefore, of the beneficial effects on health that the fruit or extracts prepared from this plant material may have.
    After the aforementioned search it was found that there are related patents
    30 with the strain of Bacillus amyloliquefaciens published worldwide, of which none is related to the ability to modulate the metabolic profile of the fruit at the level of phenolic compounds, in the route of flavonoids and anthocyanins, this global effect being the object of the present invention Most patents refer to crop protection through the bacterial production of enzymes
    35 (glucanases, chitinases, etc.), which exert their protective effect when released on the outside


    of the cell, at edaphic or foliar level (indirect mechanism), and does not imply the metabolism of the plant in this protection (direct mechanism).
    THE INVENTION.
    The object of the invention described herein and which, in view of the state of the prior art, is understood to comply with the conditions of novelty and inventive activity necessary to be able to be worthy of the patent right, is the isolation and characterization of the invention. bacterial strain Bacillus amyloliquefaciens QV15
    10 (CECT 9371), which is a microorganism from the group of Gram + bacteria, genus Bacillus, with the ability to modulate secondary metabolism, more specifically the metabolism of phenolic compounds, as well as improving the properties of plant extracts on enzymes related to the metabolic syndrome (COX2, ACE and alpha glucosidase).
    15 The physiological characteristics and the genetic analysis of this strain make it possible to identify it unequivocally, differentiating it from other Bacillus species.
    Once isolated and characterized, with internal reference code L81,
    20 performed various tests to highlight the potential of this bacterium. These were, production of auxins, degradation of 1-aminociclopropane-1carboxylate, solubilization of phosphate and production of siderophores and chitinases, resulting positive for the production of siderophores.
    25 Until now, consistent experiments have been carried out inoculating bacterial suspensions of Bacillus amyloliquefaciens QV15 on Arabidopsis thaliana, improving photosynthesis (φPSII / NPQ), without affecting the growth of the plant. In these experiments, an increase in SOD activity and a decrease in APX activity and the rest of ROS scavenging activities have been detected.
    30 with a moderate increase in the glucanase (PR2) and chitinase (PR3) activities of the plant. When these previously inoculated plants with QV15 are subjected to the shock with Xanthomonas campestris pv tomato, the plants resisted this attack much better, presenting a protection of 60%, associated to an increase of more than double that in the controls in activity PR2 and PR3 .


    On the other hand, field experiments have been carried out on plants of blackberry (Rubus var Loch Ness), raspberry (Rubus idaeus) and strawberry (Fragaria vesca) in production greenhouses. Applications have been made with QV15 at a radical level, from September to February, every 15 days, under production conditions against
    5 station. An increase in the synthesis of phenolic compounds is detected in all of them; In default, this increase is controlled at the level of transcription factors, stimulating the transcription of MYB6, and of certain genes in the flavonol biosynthesis pathway.
    10 These experiments carried out in different plant species show that Bacillus amyloliquefaciens QV15 is capable of modulating the secondary metabolism of plants, as well as improving the properties of raspberry and strawberry fruit extracts by improving the inhibition effect of alpha glucosidase enzymes, ACE and COX2 with respect to the non-inoculated controls and, therefore, this bacterium, or any
    15 molecule derived from it, can be used in any type of crop of agronomic interest, pharmacological or nutritional, agrarian or forestry, for the improvement of symptoms or prevention of metabolic syndrome.
    Bacillus amyloliquefaciens QV15 can be applied to blackberry plants (Rubus var.
    20 Loch Ness), raspberry (Rubus idaeus) and strawberry (Fragaria vesca) modifying the content of phenolic compounds in leaves and fruits, especially in derivatives of flavonols, anthocyanins and catechins. In mulberry leaves it acts at the level of transcription factors, as well as in certain genes of the biosynthesis pathway of flavonols and anthocyanins, and thereby modifies the metabolic profile (flavonols and derivatives) in leaves.
    25 In blackberry fruits, it also acts at the level of transcription factors and of certain genes in the biosynthesis pathway of flavonols and anthocyanins. This can be applied to any plant species that constitute red fruits or berries, such as strawberry, raspberry, blackberry, blueberry, or blueberry, or grape, in order to increase their content in phenolic compounds of pharmacological interest,
    30 nutrition, specifically in raspberries and strawberries to improve the anthocyanin content and, therefore, its coloration.
    The above-mentioned experiments on the use of Bacillus amyloliquefaciens QV15 as a stimulant of the secondary metabolism of phenolic compounds, specifically anthocyanins in strawberry and raspberry, and to improve the extracts on the action of the


    enzymes alpha glucosidase, ACE and COX2 are exposed at the end of this report, within the section embodiment.
    The purpose pursued, in short, with this invention and that constitutes
    5 the technical advantage provided with it, is to have a bacterium that stimulates thesecondary metabolism of phenolic compounds in plants of agronomic interest,pharmacological, nutritional, achieving a double effect: on the one hand, improvingcontent in anthocyanins in strawberries and raspberries, and therefore, their commercial value, andOn the other hand, obtain the vegetable raw material source of improved extracts with
    10 compared to controls not inoculated for their effect on the enzymes alphaglucosidase, ACE and COX2 and, therefore, can be used for the improvement of symptoms or prevention of metabolic syndrome.
    Consequently, with the present patent application, the use of the
    15 strain Bacillus amyloliquefaciens QV15, or any fraction thereof, for application in any type of plant species, forming part of any preparation, either individually or in combination with other organisms, in order to stimulate the secondary metabolism of phenolic compounds in plants of agronomic, pharmacological, nutritional interest and that at the same time improves the obtained extracts to
    20 from plant material treated with the strain, on the action of alpha glucosidase, ACE and COX2.
    FORM OF REALIZATION.
    25 Studying the rhizosphere of two Pinus species, selected for their forestry interest, we find the strain of the genus Bacillus that is reviewed here.
    The strain was isolated from the rhizosphere of a natural population of Pinus pinea L. During the isolation of bacteria carried out in the rhizosphere of two species of 30 pine (Pinus pinaster Aiton and Pinus pinea L.) and in the mycorrhizal fungus mycorrhizal associated with both, Lactarius deliciosus (Fries) SF Gray., In autumn of 2000, coinciding with the fruiting period of Lactarius deliciosus, in the Sierra de Aracena (Huelva). As a result of this sampling, 720 strains were collected, including Bacillus amyloliquefaciens (QV15, internal code of the
    35 laboratory). The isolation of said strain was carried out on nutritive agar (PCA).


    In the laboratory, this microorganism is maintained at a high survival rate in 20% glycerol in nutrient broth (Pronadisa) at -80ºC or in 15% glycerol in water at -20ºC and easily recovered in the culture medium used for Isolation in both solid phase and liquid phase at 28 ° C.
    For the characterization of the strains, different phenotypic characters that are detailed in this report were considered: (i) morphology of the colonies (ii) morphology of the cells, (iii) sequencing of the ribosomal DNA gene corresponding to the 16S subunit. iv) Genome sequencing
    10 Morphological, biochemical and genetic characteristics of Bacillus amyloliquefaciens QV15.
    The taxonomic characterization of Bacillus amyloliquefaciens was carried out
    By identifying the strain by partial sequencing of the 16S ribosomal DNA, its comparison with the existing sequences in the databases revealed a 100% homology with a strain of Bacillus amyloliquefaciens.
    Next, the morphology of the colonies is specified at 24 h of 20 incubation at 28 ° in agar for standard methods (PCA).
    TABLE 1
    QV15
    Size of the colony <1 mm •
    Shape circular
    Edge Smooth
    Transparency Do not
    Consistency Creamy
    Colour Dark yellow
    Growing in liquid medium (Lennox Pronadisa) the color of the medium changes to
    darker yellow from the exponential phase of growth to the stationary phase of
    increase.


    The morphological characters of Bacillus amyloliquefaciens QV15 at 24 h incubation at 28 ° in agar for standard methods (PCA) correspond to a sporulated Gram positive bacillus.
    5 Next, we proceeded to the genetic analysis of the strain for itsidentification, for them the following steps were followed:
    DNA extraction.
    For the extraction of the DNA, the colonies grew for 24 hours in Lennox (Pronadisa) at 28 ° C under agitation. After this time, the DNA was extracted
    of each bacterium with the Ultraclean ™ Microbial DNA isolation kit (MoBio, CA,
    USA), according to the manufacturer's instructions.
    15 Amplification of 16S rDNA.
    The 1500 bp corresponding to this region were amplified by PCR with the following primers: direct 5'-AGA GTT TGA TCC TGG CTC AG-3 'and reverse 5'AAG GAG GTG ATC CAG CCG CA-3' (Ulrike, 1989), in a reaction of 25 μL with 1X of 10X buffer, 2.5 mM MgCl2, 250 μM of each DNTP, 2.5 μM forward primer, 2.5
    20 μM reverse primer 1.25 units of DNA polymerase (AmpliTaq Applied) and 100 ng of bacterial DNA. The amplification was performed in a GeneAmp 2700 thermocycler (Applied Biosystems) with the following conditions: 95ºC 5 minutes, followed by 25 cycles of 94ºC 30 seconds, 65.5ºC 30 seconds and 72ºC 30 seconds, ending with 7 minutes at 72ºC.
    Display of gels.
    The PCR product was resolved on a 1% (w / v) agarose gel in TrisAcetate-EDTA buffer (1% TAE) with ethidium bromide (0.5 mg / mL) and visualized on a GelDoc2000 ™ image analyzer. 170-8126 (Biorad, CA, USA).
    DNA sequencing.
    Once the amplification was verified, the PCR product was purified with the UltraClean ™ PCR Kit Clean-up DNA purification (MoBio, CA, USA), sequenced


    UNIT OF GENOMIC SCIENTIFIC PARK OF MADRID-U.C.M. in an ABI PRIMS® 377 Sequencer DNA sequencer (Applied Biosystems, CA, USA).
    Computer analysis of the sequence.
    The sequences were aligned with the program Bioedit Sequence Aligment editor
    5.0.3. ®, were manually reviewed and corrected and analyzed by BLASTN 2.2.6 (Altschul et al., 1997) in the GeneBank EMBL and DDBJ (NCBI BLASTR website: http://www.ncbi.nlm.nih.gov /), resulting in the highest homology: Bacillus
    10 amyloliquefaciens QV15 16S ribosomal RNA gene, complete sequence, and the sequence is deposited in GeneBank with access number AY307364.1
    Bacillus amyloliquefaciens as an enhancer of adaptation to stress conditions and stimulating the primary and secondary metabolism of phenolic compounds.
    15 1st. Elicitation experiment in Arabidopsis thaliana seedlings by inoculating the bacterium twice at the root level, in the fourth and fifth weeks after germination of the seeds. Three days after the second inoculation, the pathogen (Xantomonas campestris) is inoculated at the foliar level by a spray of the
    20 pathogen. Photosynthesis was determined by fluorescence (Fo, Fv / Fm, FPSII, and NPQ), enzymatic activities (ROS scavenging cycle, and defense enzymes), and the relative index of disease. It was observed: i) an increase in the activity of SOD and a decrease in APX, ii) an increase in the activity of glucanases, chitinases, and cellulases, in the treatment with pathogenic QV15 +, while in QV15 they maintain
    25 an activity similar or inferior to the control, demonstrating a systemic defense induction, iii) a reduction in the symptomatology of the disease caused by the attack of the pathogen of 63.61%.
    2nd. Elicitation experiment on Rubus var Loch Ness plants applying the
    30 bacteria in the root.-A bacterial suspension of strain QV15 was inoculated into the root of Rubus var Loch Ness plants every two weeks (September 2014 to February 2015), since the transplant. Photosynthesis was determined by fluorescence (Fo, Fv / Fm, FPSII, and NPQ), enzymatic activities (ROS cycle, and defense enzymes), leaf bioactives (total phenols, flavonols and anthocyanins), chlorophylls, in two
    35 sampling moments (flowering and maximum fruiting); in maximum fructification the nutritional parameters (pH, ºBrix, and% citric acid) and bioactives were determined in


    fruits; the gene expression of the pathway of flavonoids and defense proteins in leaves and fruits collected in fructification has been studied; and finally measures have been taken with fruit extracts on the inhibition of enzymes related to the regulation of glucose (alpha-amylase and alpha-glucosidase), on hypertension (ACE), 5 and on inflammation (COX2), enzymes related to the metabolic syndrome. It was observed: i) at the level of photosynthesis FPSII increases and NPQ decreases ii) induction of SOD activity, decrease in APX, iii) increase in the activity and expression of defense proteins (glucanases, and chitinases), both in flowering and in fruiting, related to an increase in the defense against pathogens, 10 specifically against Mildiu, iv) at the level of bioactive leaves an increase in phenols is observed in flowering, although flavonols and anthocyanins are not altered, while in fruiting they decrease all in the leaves, v) increase in chlorophyll A, B and total both in flowering and fruiting, vi) in the fruits a decrease in phenols was observed, while flavonols and anthocyanins were not altered, indicating
    15 modification of secondary metabolism vii) in blackberries, an increase in antioxidant potential, viii) increased expression of flavonol and anthocyanin pathway genes CHS, F3H, DFR, LAR, and GST1 (in fruit and leaves) , data that coincide with the analysis of bioactives, and that suggest a deviation of the flavonols route towards the production of catechins in intermediate stages of maturation.
    20 The extracts were prepared from fresh blackberries and leaves of the "Loch Ness" variety. For the extracts used to measure the effect on alpha-glucosidase, ACE, and COX2, the berries were lyophilized first, then extracted with 80% methanol, centrifuged and the organic fraction was evaporated in vacuo. The extract with 20%
    25 water was characterized; while for the extracts used for the rest of the measurements an extraction with 80% methanol was carried out. So the measurements obtained for alpha-glucosidase, ACE, and COX2 would be in dry weight while for the rest in fresh weight.
    30 For the characterization of the extract, the following determinations were made:
    The total phenolic content of the extract is determined by the colorimetric method of Singleton V.L., Rossi J.A. (1965) Colorimetry of total phenolics with 35 phosphomolibdicphosphotungstic acid reagent. Am J Enol Vitic 10 16,144-158, which is based on the oxidation in basic medium of the hydroxyl groups of the phenols by the


    Folin-Ciocalteu reagent. The results are expressed as mg of gallic acid / g of extract. In this way, the extracts obtained following the processes detailed below have a minimum total phenolic content of 20 mg / g.
    5 The total flavanol content of the extract is determined by the methodcolorimetric analysis of aluminum chloride from Zishen et al. (1999) Zhishen J, MengchengT, Jianming W (1999) The determination of flavonoid contents in mulberry and theirscavenging effects on superoxide radicals. Food Chem 64: 555-559. doi: 2010.1016 / S0308-8146 (98) 00102-2 with modifications. It is expressed as mg
    10 equivalents of catechin per gram of fresh extract.
    The total anthocyanin content was determined by the differential pH method described by Giusti and Wrolstad (2001), Giusti MM, Wrolstad RE (2001) Anthocyanins Characterization and measurement with UV-visible spectroscopy. In: Wrolstad RE,
    15 Acree TE, An H, Decker EA, Penner MH, Reid DS, Schwartz SJ, Shoemaker CF, Sporns P, Wiley J (ed) Current Protocols in Food Analytical Chemistry. New York, pp F121-F129. doi: 10.1002 / 0471142913.faf0102s00, which evaluates the different absorbance of anthocyanins at different pH. The results are expressed as 30 mg of cyanidin-3-glucoside per gram of fresh extract.
    For the measurement of enzymatic activities related to defense and oxidative stress, the methods described in Garcıá-Limones, C., Hervás, A., Navas-Cortés, JA, Jiménez-Dıaz, RM, & Tena, M. ( 2002). Induction of
    An antioxidant enzyme system and other oxidative stress markers associated with 25 compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp. ciceris Physiological and Molecular Plant Pathology, 61 (6), 325-337. Lee, B.-R., Jung, W.J., Lee, B.-H., Avice, J.-C., Ourry, A., & Kim, T.H. (2008). Kinetics of drought-induced pathogenesis-related proteins and their physiological significance in white clover leaves. Physiology Plantarum, 132 (3), 329-337. 30 Saravanakumar, D., Lavanya, N., Muthumeena, B., Raguchander, T., Suresh, S., & Samiyappan, R. (2008). Pseudomonas fluorescens enhances resistance and natural enemy population in rice plants against leaffolder pest. Journal of Applied Entomology, 132 (6), 469-479. Xu, C., Natarajan, S., & Sullivan, J. H. (2008). Impact of solar ultraviolet-B radiation on the antioxidant defense system in soybean lines differing in
    35 flavonoid contents. Environmental and Experimental Botany, 63 (1-3), 39-48.


    For the measurements of chlorophylls, the method described in Harmut K. Lichtenthaler and Claus Buschmann (2001) was followed. Extraction of Photosynthetic Tissues: Chlorophylls an Carotenoids. Current Protocols in Food Analytical Chemistry F.4.2.1
    F.4.2.1.
    3º. Elicitation experiment on raspberry plants (Rubus idaeus var Adelita), inoculating the bacteria at the root level, every two weeks, from October 2015 to May 2016, analyzing the two production maximums of the raspberry (January and May). Photosynthesis was determined by fluorescence (Fo, Fv / Fm,
    10 FPSII, and NPQ), bioactive (flavonole phenols, and anthocyanins) in leaves and fruits, nutritional in fruits (pH, ºBrix, and% citric acid); and finally measures have been taken with the methanolic extracts of fruit on the inhibition of enzymes related to the regulation of glucose (alpha amylase and alpha glucosidase), hypertension (ACE) and on inflammation (COX2), enzymes related to the
    15 metabolic syndrome. It was observed in fruits: i) an increase of ºBrix, reduction in the amount of citric acid, as well as a decrease in pH, ii) increase in the amount of anthocyanins, phenols and flavonols, with respect to control, iii) increase in the ability of fruit extracts to inhibit alpha glucosidase, ACE and COX2. The content of anthocyanins, phenols and flavonols in fruits of inoculated plants and control
    20 appears in table 2; the IC50 of α-glucosidase, and the% inhibition of ACE and COX2 appear in Table 3.
    Table 2. Flavonole Anthocyanin Phenols (mg Gallic Equivalents (mg Equivalents (+) - (mg Equivalents / 100g fresh weight) catechin (195) / 100g cyanidin-3weight fresh) glucoside / 100g fresh weight) Winter Spring Winter Spring Winter Spring Control 154.17 ± 7. 389.47 ± 10. 6.06 ± 0.24 87.46 ± 1.18 9.39 ± 1.27 23.52 ± 1.5 70
    QV15 248.45 ± 6. 379.44 ± 7.4 11.28 ± 0.92 77.67 ± 1.18 4.62 ± 0.19 21.19 ± 1.76 45


    Table 3
    α Glucosidase (IC50, mg of dry extract / ml) % ACE inhibition [10 mg dry extract / ml]% COX2 inhibition [10 mg dry extract / ml]
    Winter Spring WinterSpring WinterSpring
    Control 3.80 ± 0.164.66 ± 0.0592.15 ± 0.0491.180.0229.59 ± 0.8729.02 ± 0.25
    QV15 3.32 ± 0.043.36 ± 0.2291.88 ± 0.0192.09 ± 0.0732.24 ± 0.8832.40 ± 0.49
    The raspberry extract was prepared from fresh raspberries from the
    5 variety "Adelita". For the extracts used to measure the effect on alpha
    glucosidase, ACE, and COX2, the blackberries were first lyophilized, then extracted with 80% methanol, centrifuged and the organic fraction was evaporated in vacuo. The extract
    with 20% water was characterized; 'while for the extracts used for the
    The rest of the measurements an extraction with 80% methanol was carried out. So the measures
    10 obtained for alpha-glucosidase, ACE, and COX2 would be in dry weight while for the rest in fresh weight.
    For the characterization of the extract the following determinations were made:
    The total phenolic content of the extract is determined by the colorimetric method of Singleton V.L., Rossi J.A. (1965) Colorimetry of total phenolics with phosphomolibicarphosphotungstic acid reagent. Am J Enol Vitic 10 16,144-158, which is based on the oxidation in basic medium of the hydroxyl groups of the phenols by the
    20 Folin-Ciocalteu reagent. The results are expressed as mg of gallic acid / g of extract. In this way, the extracts obtained following the processes detailed below have a minimum total phenolic content of 20 mg / g.
    The total flavanol content of the extract is determined by the method
    25 colorimetric of aluminum chloride from Zishen et al. (1999) Zhishen J, Mengcheng T, Jianming W (1999) The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 64: 555-559. doi: 20 10.1016 / S0308-8146 (98) 00102-2 with modifications. It is expressed as mg equivalents of catechin per gram of fresh extract.


    The total anthocyanin content was determined by the differential pH method described by Giusti and Wrolstad (2001), Giusti MM, Wrolstad RE (2001) Anthocyanins Characterization and measurement with UV-visible spectroscopy. In: Wrolstad RE, Acree TE, An H, Decker EA, Penner MH, Reid DS, Schwartz SJ, Shoemaker CF,
    5 Sporns P, Wiley J (ed) Current Protocols in Food Analytical Chemistry. New York, pp F121-F129. doi: 10.1002 / 0471142913.faf0102s00, which evaluates the different absorbance of anthocyanins at different pH. The results are expressed as 30 mg of cyanidin-3-glucoside per gram of fresh extract.
    10 4th. Elicitation experiment in strawberry plants (Fragaria vesca var Fortuna) inoculating after the transplant every two weeks at the root level throughout the plant cycle (January to May 2016). Photosynthesis was determined by fluorescence (Fo, Fv / Fm, FPSII, and NPQ) in the middle of the productive cycle (March 2016) and at the end (May 2016), the bioactive (flavonole, and anthocyanin phenols) and nutritional
    15 (pH, ºBrix, and% citric acid) in fruits at both times; and, finally, measurements have been made with the methanolic extracts of fruit on the inhibition of enzymes related to the regulation of glucose (alpha amylase and alpha glucosidase), hypertension (ACE) and on inflammation (COX2), enzymes related to the metabolic syndrome. It was observed: i) at the level of photosynthesis a decrease of Fo and
    20 NPQ, so it is understood that the plant is less stressed than the control and loses less energy from photosynthesis in the form of heat, so it will be used for the generation of primary or secondary metabolites. Ii) an increase in anthocyanins, iii) an increase of the ºBrix, iv) a decrease of the rotten fruit and increase in the fruit of first quality, v) increase of the properties of
    25 fruit extracts on alpha glucosidase, ACE and COX2. The content of anthocyanins, phenols and flavonols in fruits of inoculated plants and control appears in table 4; the IC50 of α-glucosidase, and the% inhibition of ACE and COX2 appear in table 5.
    Table 4. Flavonole Anthocyanin Phenols (mg Gallic Equivalents (mg Equivalents (+) - (mg Equivalents / 100g fresh weight) catechin (195) / 100g cyanidin-3weight fresh) glucoside / 100g fresh weight) Winter Spring Winter Spring Winter Spring Control 315.79 ± 6.86 188.24 ± 2.7 80.15 ± 0.37 38.40 ± 0.49 17.24 ± 1.62 21.19 ± 5.61


    4
    QV15 315.79 ± 22.4 202.50 ± 2.7 59.38 ± 1.35 29.30 ± 0.55 39.11 ± 0.19 20.28 ± 2.6 15
    Table 5. α Glucosidase (IC50, mg% ACE inhibition [10% COX2 inhibition of dry extract / ml) mg of dry extract / [10 mg of ml extract] dry / ml]
    Winter Spring Winter Spring Winter Spring Control 11.56 ± 1.05 8.27 ± 0.43 95.2 ± 0.06 99.06 ± 0.02 35.39 ± 0.21 33.76 ± 0.32 QV15 9.99 ± 0.1 10.04 ± 0.56 92.41 ± 0.01 91.98 ± 0.14 36.61 ± 1.15 34.12 ± 0.55
    5 The strawberry extract was prepared from fresh strawberries of the variety "Fortuna". For the extracts used to measure the effect on alpha-glucosidase, ACE, and COX2, the berries were lyophilized first, then extracted with 80% methanol, centrifuged and the organic fraction was evaporated in vacuo. The extract with 20%
    water was characterized; 'for the extracts used for the rest of the measurements
    10 an extraction with 80% methanol was carried out. So the measurements obtained for alpha-glucosidase, ACE, and COX2 would be in dry weight while for the rest in fresh weight.
    5th Elicitation experiment in strawberry plants (Fragaria vesca var Fortuna)
    15 inoculating after the transplant every two weeks at the radical level throughout the plant cycle (October to March 2017). Photosynthesis was determined by fluorescence (Fo, Fv / Fm, FPSII, and NPQ) in the middle of the production cycle (January 2017), and at the end of the cycle (March 2017) the bioactive (flavonole, and anthocyanin phenols) and nutritional (pH, ºBrix, and% citric acid) and the size of the fruit. It was observed: i) a
    20 increase in the amount of larger fruit after inoculations ii) an increase in anthocyanins, iii) an increase in flavonols, iv) a decrease in% citric acid. The strawberry extract was prepared from fresh strawberries of the variety
    "Fortune". An extraction with 80% methanol was carried out.


    INDUSTRIAL APPLICATION.
    Given the aforementioned properties of Bacillus amyloliquefaciens QV15 as a stimulator of secondary metabolism, this bacterial strain has a specific application in the agri-food, chemical and pharmaceutical industries, since it can be used as part of any preparation (individually or in combination with others). microorganisms) and making it come into contact (to the strain or any part of it) with the seed, the root or aerial system of the plants by any means available, in any plant species, or in any form of
    10 in vitro culture, to increase the concentration of secondary metabolites of phenolic nature with pharmacological and / or nutritional interest. In raspberry and strawberry, to improve the coloration in the production in counter-season, specifically due to the increase of anthocyanins; and to obtain improved extracts for their ability to inhibit the alpha glucosidase, ACE and COX2 enzymes.

    权利要求:
    Claims (6)
    [1]
    1. Bacillus amyloliquefaciens QV15 (CECT 9371), a microorganism from the group of Gram + bacteria, Bacillus genus, characterized by its ability to stimulate
    5 secondary metabolism of phenolic compounds of plant species,specifically flavonoids, anthocyanins, catechins and to produce an increase ofBrix.
    [2]
    2. Bacillus amyloliquefaciens QV15 (CECT 9371), according to claim 1,
    10 characterized by its ability to increase anthocyanin content in strawberry and raspberry fruits.
    [3]
    3. Bacillus amyloliquefaciens QV15 (CECT9371), a microorganism of the group of Gram + bacteria, Bacillus genus, characterized by its ability to improve
    15 properties of raspberry and strawberry fruit extracts as inhibitors of enzymes related to the metabolic syndrome: alpha glucosidase, blood glucose regulators, ACE, angiotensin-converting hypertension, and COX2, inflammation.
    4. Use of Bacillus amyloliquefaciens QV15 (CECT 9371), or any molecule derived therefrom, according to claims 1 to 3, for application in any type of crop of agronomic, pharmacological or nutritional, agrarian or forestry interest, in order to increase in bioactives and / or the improvement of leaf and / or fruit extracts on the effect of these on alpha glucosidase, ACE and COX2.
    [5]
    5. Use of Bacillus amyloliquefaciens QV15 (CECT 9371), or any molecule derived therefrom, according to claims 1 to 4, for application in any species of plants of the Rubus sp family.
    6. Use of Bacillus amyloliquefaciens QV15 (CECT 9371), or any molecule derived therefrom, according to claim 4 5, for its application in any plant species of those that constitute red fruits or berries, (such as strawberry, raspberry, mora, bilberry, or blueberry), or the constituents of grapes, in order to improve the organoleptic qualities and the coloring of the fruit, especially
    35 anthocyanins.

    [7]
    7. Use of Bacillus amyloliquefaciens QV15 (CECT 9371), or any molecule derived therefrom, according to claims 1 to 6, either the single strain or in combination with other organisms, forming part of any preparation, either individually
    or in combination with other organisms, and by any available means that puts the bacteria in contact with the seed, the root or aerial system of the plants.
    [8]
    8. Use of Bacillus amyloliquefaciens QV15 (CECT 9371), or any molecule derived therefrom, according to claims 1 to 6, either the single strain or in combination with other organisms, or forming part of any preparation, either
    10 individually or in combination with other organisms, and by any available means that puts the bacteria in contact with plant cells in any state of differentiation in in vitro culture.
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    同族专利:
    公开号 | 公开日
    EA201992374A1|2020-05-28|
    JP2020524522A|2020-08-20|
    MX2019015261A|2020-02-12|
    CA3067875A1|2018-12-27|
    CL2019003465A1|2020-05-29|
    TN2019000304A1|2021-05-07|
    PE20200714A1|2020-06-30|
    EP3643775A4|2021-03-31|
    ES2694593B2|2019-06-28|
    AU2018287094A1|2020-02-06|
    EP3643775A1|2020-04-29|
    US20210076684A1|2021-03-18|
    MA47663B1|2020-10-28|
    ZA202000371B|2021-05-26|
    MA47663A1|2020-05-29|
    WO2018234599A1|2018-12-27|
    CO2019012690A2|2020-01-17|
    BR112019026342A2|2020-07-21|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20160183537A1|2014-12-29|2016-06-30|Fmc Corporation|Bacillus amyloliquefaciens rti472 compositions and methods of use for benefiting plant growth and treating plant disease|
    ES2336758B1|2009-12-03|2011-04-06|Fundacion Universitaria San Pablo Ceu|PSEUDOMONAS FLUORESCENS N. 21.4 STIMULANT OF THE SECONDARY METABOLISM OF PHENOLIC COMPOUNDS.|
    KR20110120748A|2010-04-29|2011-11-04|충남대학교산학협력단|Bacillus amyloliquefaciens cp1 and contro method of strawberry anthracnose using the same|
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    JP2015181423A|2014-03-25|2015-10-22|株式会社アイエイアイ|Strain belonging to bacillus, microbiological agent, and plant cultivation method|
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    BR112019026342-0A| BR112019026342A2|2017-06-21|2018-05-22|bacillus amyloliquefaciens qv15 stimulating the secondary metabolism of phenolic compounds and the ability to inhibit raspberry and strawberry extracts on enzymes related to the metabolic syndrome|
    US16/602,882| US20210076684A1|2017-06-21|2018-05-22|Bacillus amyloliquefaciens QV15 secondary metabolism stimulant for the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts for enzymes related to metabolic syndrome|
    MA47663A| MA47663B1|2017-06-21|2018-05-22|Bacillus amyloliquefaciens qv15 secondary metabolism stimulator for secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extract for enzymes related to metabolic syndrome.|
    AU2018287094A| AU2018287094A1|2017-06-21|2018-05-22|Bacillus amyloliquefaciens QV15 stimulant for the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts for enzymes related to metabolic syndrome|
    CA3067875A| CA3067875A1|2017-06-21|2018-05-22|Bacillus amyloliquefaciens qv15 secondary metabolism stimulant for the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts for enzymes related to metabolic syndrome|
    EA201992374A| EA201992374A1|2017-06-21|2018-05-22|BACILLUS AMYLOLIQUEFACIENS QV15 - A SECONDARY METABOLISM STIMULATOR FOR SECOND METABOLISM OF PHENOLIC COMPOUNDS AND INHIBITING ABILITY OF EXTRACTS OF Raspberry and Strawberry Relative to Sorbent Enzymes|
    MX2019015261A| MX2019015261A|2017-06-21|2018-05-22|Bacillus amyloliquefaciens qv15 stimulant for the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts for enzymes related to metabolic syndrome.|
    JP2019571539A| JP2020524522A|2017-06-21|2018-05-22|Inhibitory ability of raspberry and strawberry extracts against secondary metabolic stimulator Bacillus amyloliquefaciens QV15 and metabolic syndrome-related enzymes for secondary metabolism of phenolic compounds|
    EP18820445.7A| EP3643775A4|2017-06-21|2018-05-22|Bacillus amyloliquefaciens qv15 stimulant for the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts for enzymes related to metabolic syndrome|
    TNP/2019/000304A| TN2019000304A1|2017-06-21|2018-05-22|Bacillus amyloliquefaciens qv15 stimulant for the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts for enzymes related to metabolic syndrome|
    PCT/ES2018/070369| WO2018234599A1|2017-06-21|2018-05-22|Bacillus amyloliquefaciens qv15 stimulant for the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts for enzymes related to metabolic syndrome|
    PE2019002272A| PE20200714A1|2017-06-21|2018-05-22|BACILLUS AMYLOLIQUEFACIENS QV15 STIMULANT OF SECONDARY METABOLISM OF PHENOLIC COMPOUNDS AND OF THE INHIBITING CAPACITY OF RASPBERRY AND STRAWBERRY EXTRACTS ON ENZYMES RELATED TO METABOLIC SYNDROME|
    CONC2019/0012690A| CO2019012690A2|2017-06-21|2019-11-14|Bacillus amyloliquefaciens qv15 stimulating the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts on enzymes related to metabolic syndrome|
    CL2019003465A| CL2019003465A1|2017-06-21|2019-11-27|Bacillus amyloliquefaciens qv15 stimulating the secondary metabolism of phenolic compounds and the inhibitory capacity of raspberry and strawberry extracts on the enzymes related to metabolic syndrome.|
    ZA2020/00371A| ZA202000371B|2017-06-21|2020-01-20|Bacillus amyloliquefaciens qv15 stimulant for the secondary metabolism of phenolic compounds and the inhibitory capacity of rasberry and strawberry extracts for enzymes related to metabolic syndrome|
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